Everything about Crizotinib powder


  1.What is Crizotinib powder?
  2.How does Crizotinib powder works?
  3. Crizotinib powder Materials and methods
  4.Approvals and indications for Crizotinib powder
  5. Clinical trials for Crizotinib powder
  6.The Conclusion of Crizotinib powder
  7. Where to Buy Crizotinib powder?



Crizotinib powder video




I.Crizotinib powder basic Characters:


Name: Crizotinib powder
CAS: 877399-52-5
Molecular Formula: C21H22Cl2FN5O
Molecular Weight: 450.343
Melt Point: 197-203°C
Storage Temp: RT
Color: white to pale-yellow powder



1.What is Crizotinib powder?aasraw


Crizotinib powder(trade name Xalkori, Pfizer) is an anti-cancer drug acting as an ALK (anaplastic lymphoma kinase) and ROS1 (c-ros oncogene 1) inhibitor, approved for treatment of some non-small cell lung carcinoma (NSCLC) in the US and some other countries, and undergoing clinical trials testing its safety and efficacy in anaplastic large cell lymphoma, neuroblastoma, and other advanced solid tumors in both adults and children.

Crizotinib powder is an anti-cancer agent acting as anaplastic lymphoma kinase inhibitor and recently approved for treatment of non-small cell lung carcinoma.The retrosynthetic analysis of this molecule clearly takes our eyes to two major building blocks, as described in Scheme 1, being the racemic 1-(2,6-dichloro-3-fluorophenyl)ethanol a very interesting target for biotransformations. Indeed, some research groups have already reported their results on biocatalytic or biotransformation approaches for arriving on the desired intermediate.



2.How does Crizotinib powder works?aasraw


Crizotinib powder has an aminopyridine structure, and functions as a protein kinase inhibitor by competitive binding within the ATP-binding pocket of target kinases. About 4% of patients with non-small cell lung carcinoma have a chromosomal rearrangement that generates a fusion gene between EML4 (‘echinoderm microtubule-associated protein-like 4’) and ALK (‘anaplastic lymphoma kinase’), which results in constitutive kinase activity that contributes to carcinogenesis and seems to drive the malignant phenotype.The kinase activity of the fusion protein is inhibited by Crizotinib powder. Patients with this gene fusion are typically younger non-smokers who do not have mutations in either the epidermal growth factor receptor gene (EGFR) or in the K-Ras gene. The number of new cases of ALK-fusion NSLC is about 9,000 per year in the U.S. and about 45,000 worldwide.

ALK mutations are thought to be important in driving the malignant phenotype in about 15% of cases of neuroblastoma, a rare form of peripheral nervous system cancer that occurs almost exclusively in very young children.

Crizotinib powder inhibits the c-Met/Hepatocyte growth factor receptor (HGFR) tyrosine kinase, which is involved in the oncogenesis of a number of other histological forms of malignant neoplasms.

Crizotinib powder is currently thought to exert its effects through modulation of the growth, migration, and invasion of malignant cells. Other studies suggest that Crizotinib powder might also act via inhibition of angiogenesis in malignant tumors.

In order to get chiral 1-phenylethanol derivative, Liang and co-workers have used several microorganism for efficient reduction the ketone towards intermediate. Other groups tried to increase the efficiency and selectivity towards the chiral intermediate by using similar approaches. A fit-for-purpose route published by Koning and co-workers from Pfizer revels the strategy for producing the racemic alcohol by traditional NaBH4 reduction, followed by acetylation, resolution mediated by PLE and Mitsunobu inversion towards the required enantiomer.

Lipases are always the first option when thinking about obtaining highly enantiomeric pure chiral alcohols by kinetic resolutions or dynamic kinetic resolution (DKR) methods. Furthermore, lipases can be combined with racemization catalysts, namely palladium, iridium, vanadium or ruthenium complexes complexes. Unfortunately, to the best of our knowledge, no reports of lipases used on the kinetic or DKR of 1-(2,6-dichloro-3-fluorophenyl)ethanol were found.

In our continuous effort towards the development of biocatalytic protocols for the synthesis of intermediates for API synthesis, herein, we report our studies on the kinetic and DKR of 1-(2,6-dichloro-3-fluorophenyl)ethanol, an important intermediate on Crizotinib powder synthesis.




3.Crizotinib powder Materials and methodsaasraw


Name: Crizotinib powder
CAS: 877399-52-5
Molecular Formula: C21H22Cl2FN5O
Molecular Weight: 450.343
Melt Point: 197-203°C
Storage Temp: RT
Color: white to pale-yellow powder


1-(2,6-Dichloro-3-fluoro-phenyl)-ethanol and isopropenyl acetate purchased from Sigma-Aldrich. Candida antarctica lipase B (CAL B) purchased from Novozymes. All other materials were at least reagent-grade.

  • Kinetic resolution reactions

The reaction mixture was composed by a 3 mL solution of 0.048 mmol 1-(2,6-dichloro-3-fluoro-phenyl)-ethanol, 0.096 mmol acyl donor and 60 mg of enzyme (20% w/v) in solvent. The screening reactions were performed in silicon carbide plates at temperatures of 60–80 °C, under magnetic stirring to obtain 1-(2,6-dichloro-3-fluorophenyl)ethyl acetate (1H NMR (300 MHz, CDCl3) δ 7.26 (dd, J = 8.3, 5.6 Hz, 5H), 7.03 (t, J = 8.5 Hz, 3H), 6.39 (q, J = 7.1 Hz, 3H), 2.09 (s, 3H), 1.66 (d, J = 6.9 Hz, 3H). In order to screen the influence of the different reaction medium’s components, enzymes, solvent, acyl donor and temperature were evaluated. A 20 μL aliquot was transferred to a new vial and 980 μL of ethyl acetate was added and analyzed in the GC-FID.

  • Dynamic kinetic resolution (DKR) reactions in continuous flow

Continuous flow experiments were performed using an Asia Flow System (Syrris) which included a syringe pump, a heater and a glass column. The glass column was packed with immobilized CAL B and VOSO4.XH2O alternately. A 10 mL-packed-bed reactor with 4 layers of enzyme (each layer containing 500 mg of the catalyst) and 3 layers of VOSO4.XH2O (each layer containing 500 mg of the catalyst) separated by thin cotton layers was obtained. The bed-packed reactor was heated at 80 °C and perfused with isooctane at a flow rate of 1.0 mL.min?1. Continuous flow reactions were carried out by pumping a isooctane solution (15 mL) of 1-(2,6-dichloro-3-fluoro-phenyl)-ethanol (0.1 M) and vinyl acetate as acyl donor (0.1 M) through the bed-packed reactor at flow rates of 0.1, 0.5 and 1.0 mL.min?1, which correspond to residence times of 100, 50 and 10 min, respectively. Due to the long reaction time in batch, the solution was recycled in the continuous flow to optimize the conversion results. Aliquots of 20 μL were collected from the output of the reactor after pumping the reactional solution for two times the residence time and after 21 h. Afterwards, the aliquots were diluted with ethyl acetate to 1.0 mL solution and analyzed by GC-FID.

  • Chromatography analysis

Kinetic Resolution and Dynamic Kinetic Resolution
1-(2,6-Dichloro-3-fluorophenyl)-ethanol GC-FID: (Shimadzu CG2010 – chiral capillary column Gamadex – 225) 8 μL samples were injected at 117 °C. The oven was heated at 2 °C/min to 150 °C then maintained for 5 min and at 30 °C/min to 200 °C and then maintained for 1 min, with a 3.0 mL/min flow, and a Split injection mode (Ratio: 20:1).

  • Results and discussions

Based on our previous experience on kinetic resolutions of sec-phenylethanol derivatives, we have learned along these years that ortho-substituted aryl alcohols are less reactive for the kinetic resolution. Since 1-(2,6-dichloro-3-fluorophenyl)ethanol (2) contains substituents on both ortho positions, we were expecting long reaction times for this transformation. We have started by screening the influence of acyl donor on the reaction outcome when using commercial immobilized CAL B (N435) at 60 °C and cyclohexane as solvent for 10 days (Table 1).

Acyl donor evaluation for kinetic resolution of 1-(2,6-dichloro-3-fluorophenyl)ethanol (2) mediated by immobilized CAL-B.


Entry    Acyl Donor (1 eq)    Conv. (%)    e.e.prod (%)    E

  • 1    Isopropenyl acetate        44             99    >200
  • 2    Ethyl acetate                   32             99    >200
  • 3    Vinyl acetate                   26             95    54


Reaction conditions: 1-(2,6-dichloro-3-fluorophenyl)-ethanol (2) (0.048 mmol, 10mg), isopropenyl acetate or vinyl acetate or ethyl acetate (1eqv) as acyl donor, and 60mg (20%w/v) of CAL B Novozym 435 (N435) in cyclohexane (3 mL) for 10 days at 60 °C, measured by GC-FID method and enantioselectivity was measured based on (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol and the corresponding ester retention times.




4.Approvals and indications for Crizotinib powderaasraw


On August 26, 2011, the U.S. Food and Drug Administration approved Crizotinib powder (Xalkori) to treat certain late-stage (locally advanced or metastatic) non-small cell lung cancers that express the abnormal anaplastic lymphoma kinase (ALK) gene. Approval required a companion molecular test for the EML4-ALK fusion. In March 2016, the U.S. Food and Drug Administration approved Crizotinib powder in ROS1-positive non-small cell lung cancer.

As expected, isopropenyl acetate lead to the best results on the kinetic resolution of 1-(2,6-dichloro-3-fluorophenyl)ethanol (2), arriving on the desired acetylated R-enantiomer in good conversions and high selectivity (Table 1, entry 1). Vinyl acetate was probably too reactive under the designed reaction conditions, leading to a decrease of enantiomeric ratio. A blank experiment was performed in order to evaluate the uncatalyzed reaction and small amounts of acetylated alcohol could be obtained (<10%). Regarding ethyl acetate, surprisingly, it presented interesting selectivity with moderate conversion (Table 1, entries 3 and 2 respectively). Despite the fact that Table 1 present the results for the kinetic resolution after 10 days, the reactions were monitored in the first 3 days every 12 h and after the 3rd day, every 24 h. What could be seen is the conversion improvement along the days until 10 days of reaction. Further reaction time has also been evaluated (12 days) but no improvement on conversion was observed.

In order to try to reduce the reaction time, one of the strategies was to evaluate the effect of higher proportions of acyl donor but unfortunately with values higher then two equivalents the enantiomeric ratio dropped from >200 to 31, showing that the chemical esterification is taking place. Another attempt was to screen other lipases but no better results were found when compared to the commercial immobilized CAL B (N435) (Table S1 – Supporting information).

In order to try to reduce the long reaction time obtained for this specific substrate, different temperatures and solvents were evaluated.

Solvent screening for kinetic resolution of 1-(2,6-dichloro-3-fluorophenyl)ethanol, using isopropenyl acetate mediated by immobilized CAL B.


Entry    T (°C)    Solvent    Time (Days)    Conv. (%)    e.e.prod (%)    E

  • 1       60                ACN                    5               2              99    >200
  • 2       60               Acetone              5              n.d.           n.d.    n.d.
  • 3       60               MTBE                  5              n.d.           n.d.    n.d.
  • 4       60              Toluene               5              11              99    >200
  • 5       60               Hexane               6              n.d.           n.d.    n.d.
  • 6       60              Heptane              6             39              99    >200
  • 8       70              Isooctane             5              45             99    >200
  • 9       75              Isooctane             5              40             99    >200
  • 10     80             Isooctane              5             44             99    >200


Reaction conditions: 1-(2,6-dichloro-3-fluorophenyl)ethanol (2) (0.048 mmol, 10mg), isopropenyl acetate (2eqv) as acyl donor, and 60mg (20%w/w) Novozym 435 in different solvents (3 mL, ACN – acetonitrile, MTBE – methyl ter-butyl ether), measured by GC-FID method and enantioselectivity was measured based on (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol and the corresponding ester retention times.

As observed on Table 2, among all solvents evaluated for the desired resolution, isooctane presented the best results in terms of reaction time. Under the conditions studied, at 70 °C, isooctane could lead to 45% of conversion (42% yield) after 5 days, a 50% reduction on reaction time (Table 2, entry 7). Other solvents as acetone, MTBE, acetonitrile (ACN), toluene and hexane, have failed or lead to very poor conversions. At higher temperatures, no improvement on reaction conversion could be observed. An exception is heptane, which could lead to similar results to isooctane, but the later can lead us to a better stability for the immobilized enzyme.

Despite the good results obtained under kinetic resolution conditions, 50% of the product is waste or need to be racemized in order to be used again in another campaign.

Based on the results obtained for the kinetic resolution of 1-(2,6-dichloro-3-fluorophenyl)ethanol (2), we decided to move forward in order to develop a method for the DKR which should deliver the desired acetylated R-enantiomer of 1-(2,6-dichloro-3-fluorophenyl)ethanol (2) as main product. This strategy is based on the fact that after DKR, a simple hydrolysis leads to the desired enantiomer, which can be coupled with the side chain through standard nucleophilic aromatic substitution procedure. Two different racemization catalysts were tested for this purpose, vanadyl sulfate19–25 and Shvo’s ruthenium catalyst.26–30

The reactions were carried out at 70 °C, using isopropenyl acetate as acyl donor and the conversion analyzed by GC-FID daily during 5 days. It is important to note that short chain acyl donors are not the best choice for DKR mediated by vanadium catalysts, as already reported by other groups.20,22 Despite that, we decided to keep isopropenyl acetate since we already know that the kinetic resolution should be the rate limiting step of our process and a long chain acyl donor, as vinyl decanoate, could increase even more the reaction time.31 The results obtained for the dynamic kinetic resolution are presented on Table 3.

DKR of 1-(2,6-dichloro-3-fluorophenyl)ethanol (2) mediated by immobilized CAL-B and different racemization catalysts under batch conditions.


Entry    Racemization Catalyst    Conv. (%)    e.e.prod (%)    Selectivity (%)

  • 1    VOSO4            30           98              >99
  • 2    Shvo                35            92             >99


Reaction conditions:1-(2,6-dichloro-3-fluoro-phenyl)-ethanol (0,1 M), isopropenyl acetate (0,1 M) as acyl donor, in isooctane (15 mL) were carried by mixing all reagents and catalysts on a batch reactor. Determined by GC-FID and enantioselectivity was measured based on (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol and the corresponding ester retention times.




5.Clinical trials for Crizotinib powderaasraw


Crizotinib powder caused tumors to shrink or stabilize in 90% of 82 patients carrying the ALK fusion gene. Tumors shrank at least 30% in 57% of people treated. Most had adenocarcinoma, and had never smoked or were former smokers. They had undergone treatment with an average of three other drugs prior to receiving Crizotinib powder, and only 10% were expected to respond to standard therapy. They were given 250 mg Crizotinib powder twice daily for a median duration of six months. Approximately 50% of these patients suffered at least one side effect, such as nausea, vomiting, or diarrhea. Some responses to Crizotinib powder have lasted up to 15 months.

A phase 3 trial, PROFILE 1007, compares Crizotinib powder to standard second line chemotherapy (pemetrexed or taxotere) in the treatment of ALK-positive NSCLC. Additionally, a phase 2 trial, PROFILE 1005, studies patients meeting similar criteria who have received more than one line of prior chemotherapy.

Crizotinib powder is also being tested in clinical trials of advanced disseminated anaplastic large-cell lymphoma, and neuroblastoma.

In February 2016 the J-ALEX phase III study comparing alectinib with Crizotinib powder ALK-positive metastatic NSCLC was terminated early because an interim analysis showed that progression-free survival was longer with alectinib. These results were confirmed in a 2017 analysis.

As can be observed on Table 3, the racemization protocol performed with Shvo or vanadyl sulphate catalyst, lead to moderate conversions towards the desired chiral alcohol. It is important to note that under the reaction conditions studied, Shvo’s catalyst did not performed better then VOSO4, even presenting a decreasing on enantiomeric excess of the product, showing that VOSO4 could be an interesting and cheaper alternative for further improvements.19–25,32–34

Following our recent studies on the use of VOSO4 for DKR under continuous-flow conditions 31 we applied the reactions conditions already optimized for sec-phenylethanol to our substrate 1-(2,6-dichloro-3-fluorophenyl)ethanol. In this way, the reaction was performed in closed loop system due to the high reaction time needed for the kinetic resolution step. Although we did not observe any incompatibility between CAL B and VOSO4,19–25,32–34 as previously reported by other authors, a continuous flow packed-bed reactor was designed so that four layers of the immobilized enzyme and three layers of VOSO4 were alternately disposed along a glass column and physically separated by a thin cotton layer. The results obtained under different reaction times and flow rates are presented below (Table 4).


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Continuous-flow DKR of 1-(2,6-dichloro-3-fluorophenyl)ethanol.


Entry    Flow Rate (mL.min?1)    Reaction Time (days)    Conv. (%)    e.e.prod (%)

  • 1              1.0               1                  28              98
  • 2              3                 38                98
  • 3              5                  44               98
  • 4              0.5              1                  32              98
  • 5              3                  47               96
  • 6              5                  57               96
  • 7              0.1              1                  33              88
  • 8              3                  47               84
  • 9              5                  47               82


Reaction conditions:1-(2,6-dichloro-3-fluoro-phenyl)-ethanol (2) (0,1 M), isopropenyl acetate (0,1 M) as acyl donor, in isooctane (15 mL) were carried out by pumping through the bed-packed reactor at different flow rates. The glass column was packed with immobilized N435 and VOSO4.XH2O alternately so that a 10 mL-packed-bed reactor with 4 layers of enzyme (each layer containing 500 mg of the catalyst) and 3 layers of VOSO4.XH2O (each layer containing 500 mg of the catalyst) separated by thin cotton layers. The bed-packed reactor was heated at 80 °C. Determined by GC-FID and enantioselectivity measured based on (S)-1-(2,6-dichloro-3-fluorophenyl)ethanol and the corresponding ester retention times.

The results presented on Table 4 show that under continuous-flow conditions the DKR mediated by Novozyme 435 and VOSO4, is more efficient in terms of conversion and reaction time when compared to the batch set-up, as observed in entry 6 (Table 4). No further improvement on reaction conversion could be observed increasing the reaction time, which seems to be a limitation in this specific case. Increasing the residence time by using low flow rates do not lead to better conversion and a small decrease on enantiomeric excess of the product could be observed (entries 7–9, Table 4). At 0.5 mL.min?1 a significant improve on reaction conversion could be obtained when compared to the batch process, it is important to note that despite the reaction has been performed for 5 days, the actual time of contact between reagents and catalysts is much less since it works on a closed loop system. Another advantage of DKR is the fact that the remaining starting material is racemic been able to be re-used in a different campaign without any further purification.



6.The Conclusion of Crizotinib powderaasraw


In conclusion we have developed different approaches towards production of chiral 1-(2,6-dichloro-3-fluorophenyl)ethanol (2), an important intermediate for Crizotinib powder (1) synthesis. Kinetic resolution was accomplished with very good conversions and high selectivity while dynamic kinetic resolution, also under batch conditions afforded poor results of conversions with good enenatiomeric excess of the product. Further improvement was obtained translating the dynamic kinetic resolution to a continuous-flow protocol where conversion could be improved with still good selectivity.



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