There are no acknowledgments to mention here.

1. Pilot scale synthesis of compounds ACT051-2 and ACT051-3
<ol>
	<li>Synthesis of 2-bromo-5-tosyl-5H-pyrrolo[2,3-b]pyrazine (ACT051-2)
	<ol>
		<li>In a round-bottom flask, dissolve 50.0 g of the compound 2-bromo-5H-pyrrolo[2,3-b]pyrazine (ACT051-1; 0.25 M) in 15 mL of N,N-dimethylformamide (DMF; 3 V).</li>
		<li>Add 65.3 g of diisopropylethylamine (DIPEA; 0.51 M) to the reaction solution under nitrogen protection (provide a gas and moisture barrier) and cool the temperature to 0-5 &#176;C through a cool water bath. For nitrogen protection, pump the pressure of the reactor to -0.75--0.8 MPa, then pass N2 to balance the pressure at 0.1 MPa.</li>
		<li>Add 60.20 g of TsCl dissolved in 12 mL of DMF (0.32 M). Raise the temperature to 20-30 &#176;C through the warm water bath and stir for about 1 h. Add cool water (600.0 mL, 0-10 &#176;C) to the mixture and stir for another 1 h.</li>
		<li>Filter the product under a vacuum using a glass funnel with a sanding board padded with filter paper. Wash with water (200.0 mL) several times and dry using an electric thermostatic drying oven to obtain a pale-yellow solid (ACT051-2) with a yield of 78%.</li>
	</ol>
	</li>
	<li>Synthesis of tert-butyl (5-toluenesulfonyl-5H-pyrrole [2,3-b] pyrazine-2-yl) carbamate (ACT051-3)
	<ol>
		<li>Dissolve 176.11 g of ACT051-2 in 366.31 g of 1,4-dioxane in a three-port round-bottom flask.</li>
		<li>Add 65.75 g of tert-butyl carbamate, 138.21 g of granular potassium carbonate (2.0 eq), 11.57 g of xantphos (0.04 eq), and 2.25 g of Pd(OAc)2 (1.28% wt) to the solution.</li>
		<li>Heat the mixture to 105 &#176;C and stir for 7 h under a nitrogen atmosphere. Allow the mixture to cool to room temperature and filter the product with a Buchner funnel (the filter paper aperture is 80-120 &#181;m).</li>
		<li>Wash the filter residue with ethyl acetate (200 mL). Use a circulating water vacuum pump to concentrate the product under reduced pressure at 50-60 &#176;C with a pressure value of -0.095 MPa. Keep the pump working to maintain the pressure and obtain a dark brown oil.</li>
		<li>Purify the crude product with column chromatography, eluting with petroleum ether and ethyl acetate (V/V, 10/1) to obtain the target compound as a white solid with a yield of 93.5%.</li>
	</ol>
	</li>
</ol>
2. Pilot scale-up synthesis of compounds ACT051-2 and ACT051-3
<ol>
	<li>Pilot scale-up synthesis of 2-bromo-5-tosyl-5H-pyrrolo[2,3-b]pyrazine (ACT051-2)
	<ol>
		<li>Add 1.0 kg of ACT051-1 (5.05 M) and 1.305 kg of DIPEA (10.1 M) to a three-port round-bottom flask. Add 3 L of DMF (3 V) to the flask and dissolve the solid. Heat the reaction mixture to 35 &#176;C.</li>
		<li>Add 1.203 kg of TsCl (6.31 M) to the reaction solution and stir for 1 h. Stir the mixture until the completion of the reaction is confirmed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). In brief, take a small amount of reaction product and subject this to TLC monitoring. When the TLC shows that there is almost no raw material left, send the sample to the HPLC central control, and detect the reaction to raw material or the product ratio to be 0.1/99.9.</li>
		<li>Pour in 8.4 L of cool water and stir for another 0.5 h. Filter all liquids with a Buchner funnel (the filter paper aperture is 80-120 &#181;m) and rinse the crude product with 600 mL of water.</li>
		<li>Dry the resulting product at 70 &#176;C overnight using an electric thermostatic drying oven and obtain a product with a yield of 94.9%.</li>
	</ol>
	</li>
	<li>Pilot scale-up synthesis of tert-butyl (5-toluenesulfonyl-5H-pyrrole [2,3-b] pyrazine-2-yl) carbamate (ACT051-3)
	<ol>
		<li>Add 3.31 L of tert-amyl alcohol and 4.97 L of toluene (V/V, 2/3) to the reaction kettle.</li>
		<li>To the solution, add 1.66 kg of ACT051-2, 0.83 kg of tert-butyl carbamate, 1.301 kg of powdered potassium carbonate, 0.11 kg of xantphos, and 0.31 kg of DIPEA.</li>
		<li>Evacuate nitrogen three times, as in step 1.1.2, repeat this 3x, and add 10 g of Pd(OAc)2 (0.60% wt) to the reaction solution under nitrogen protection.</li>
		<li>Heat the reaction mixture to 90 &#176;C and stir for 4 h. Cool the mixture to 40 &#176;C or below.</li>
		<li>Filter the reaction solution with a Buchner funnel (the filter paper aperture is 80-120 &#181;m) using diatomite as a filter aid and washing the filter cake with toluene.</li>
		<li>Collect and concentrate the filtrate. Use a circulating water vacuum pump to concentrate the filtrate under reduced pressure at 50-60 &#176;C with a pressure value of -0.095 MPa. Keep the pump working to maintain the pressure.</li>
		<li>Add 300 mL of heptane and stir for 20 min. Filter the reaction solution again with a Buchner funnel (the filter paper aperture is 80-120 &#181;m) and rinse the crude product with heptane (50 mL, three times). Dry and obtain the product with a yield of 96.3%.</li>
	</ol>
	</li>
</ol>
3. Industrial production of compounds ACT051-2 and ACT051-3
<ol>
	<li>Industrial production of 2-bromo-5-tosyl-5H-pyrrolo[2,3-b]pyrazine (ACT051-2)
	<ol>
		<li>Confirm that the reactor is clean and free of water, and make sure the mixing device makes no noise when turning on and the bottom discharge valve of the reactor has been closed.</li>
		<li>Add 355.50 kg of DMF (3 V) into the 2,000 L enamel reactor and start to stir. Add 125.04 kg of ACT051-1 (1.0 eq) and 164.8 kg of DIPEA (2.0 eq) to the solution.</li>
		<li>Drop the temperature to 20-25 &#176;C under nitrogen protection. Add 150.22 kg of TsCl (1.25 eq) at 25-35 &#176;C in 10 batches within 3 h.</li>
		<li>Allow the mixture to remain at 25-35 &#176;C and stir for 2 h. Prepare 750.20 kg of cool water (3-4 &#176;C) in another 2,000 L reactor.</li>
		<li>Monitor the reaction by HPLC and confirm completion of the reaction when the remaining raw material is 0.5%.</li>
		<li>Add the cool water ( 3-4 &#176;C) to the reaction mixture and stir at 15-30 &#176;C for 1.5 h. Collect the filtrate and rinse with water (250-500 kg, 2-4 V) until it becomes neutral (tested using pH paper).</li>
		<li>Dry the product at 60-65 &#176;C for 30.5 h to obtain a light brown solid with a yield of 95.5%.</li>
	</ol>
	</li>
	<li>Industrial production of tert-butyl (5-toluenesulfonyl-5H-pyrrole [2,3-b] pyrazine-2-yl) carbamate (ACT051-3)
	<ol>
		<li>Confirm the reactor is clean and free of water, and make sure the mixing device is normal and the bottom discharge valve of the reactor has been closed.</li>
		<li>Add 340.10 kg of tert-amyl alcohol (2 V) and 552.50 kg of toluene (3 V) into a 2,000 L enamel reactor and start to stir.</li>
		<li>Add 212.40 kg of ACT051-2 (1.0 eq), 106.00 kg of tert-butyl carbamate (1.5 eq), 166.70 kg of powdered potassium carbonate (2.0 eq), 14.10 kg of xantphos (0.04 eq), 39.40 kg of DIPEA (0.5 eq), and 1.06 kg of Pd(OAc)2 (0.5% wt) to the solution.</li>
		<li>Replace the nitrogen four times, allow the mixture to heat to 85-95 &#176;C under nitrogen protection, and stir for 3 h. Monitor the reaction by HPLC and confirm completion of the reaction when the remaining raw material is 0.44%.</li>
		<li>Cool the reaction temperature to 20-30 &#176;C. Collect the solution in batches and place it in a 125 L plastic bucket for 30 min.</li>
		<li>Filter the reaction solution with a chemical suction filter barrel (the filter bag aperture is 10-15 &#181;m) using diatomite as a filter aid and washing the filter cake with toluene (185.20-370.40 kg, 1-2 V).</li>
		<li>Collect the filtrate in an enamel vessel and use a circulating water vacuum pump under reduced pressure at 55-65 &#176;C for 10 h with a pressure value of -0.095 MPa. Keep the pump working to maintain the pressure and obtain the product compound as a viscous liquid.</li>
		<li>Evaporate the product twice by pumping toluene and continuing the reaction. Concentrate the final product for 4 h. Purify the resulting product with column chromatography, eluting with heptane and ethyl acetate (V/V, 10/1-3/1), to obtain a solid with a yield of 98.5%.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Kerschbaumer, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Points+to+consider+for+the+treatment+of+immune-mediated+inflammatory+diseases+with+Janus+kinase+inhibitors:+a+systematic+literature+research.">Points to consider for the treatment of immune-mediated inflammatory diseases with Janus kinase inhibitors: a systematic literature research.</a> RMD Open. 6 (3), e001374 (2020).</li><li>Fragoulis, G. E., Brock, J., Basu, N., McInnes, I. B., Siebert, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+for+JAK+inhibitors+in+the+treatment+of+immune-+mediated+rheumatic+and+related+conditions.">The role for JAK inhibitors in the treatment of immune- mediated rheumatic and related conditions.</a> The Journal of Allergy and Clinical Immunology. 148 (4), 941-952 (2021).</li><li>Shaw, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P220+Long-term+safety+profile+of+upadacitinib+in+patients+with+rheumatoid+arthritis,+psoriatic+arthritis,+or+ankylosing+spondylitis.">P220 Long-term safety profile of upadacitinib in patients with rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis.</a> Rheumatology. 61, 133 (2022).</li><li>Keeling, S., Maksymowych, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JAK+inhibitors,+psoriatic+arthritis,+and+axial+spondyloarthritis:+a+critical+review+of+clinical+trials.">JAK inhibitors, psoriatic arthritis, and axial spondyloarthritis: a critical review of clinical trials.</a> Expert Review of Clinical Immunology. 17 (7), 701-715 (2021).</li><li>Fleischmann, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+efficacy+of+elsubrutinib+or+upadacitinib+alone+or+in+combination+(ABBV-599)+in+patients+with+rheumatoid+arthritis+and+inadequate+response+or+intolerance+to+biological+therapies:+a+multicentre,+double-blind,+randomised,+controlled,+phase+2+trial.">Safety and efficacy of elsubrutinib or upadacitinib alone or in combination (ABBV-599) in patients with rheumatoid arthritis and inadequate response or intolerance to biological therapies: a multicentre, double-blind, randomised, controlled, phase 2 trial.</a> The Lancet Rheumatology. 4 (6), e395-e406 (2022).</li><li>Stamatis, P., Bogdanos, D. P., Sakka, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upadacitinib+tartrate+in+rheumatoid+arthritis.">Upadacitinib tartrate in rheumatoid arthritis.</a> Drugs of Today. 56 (11), 723-732 (2020).</li><li>Rubbert-Roth, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trial+of+upadacitinib+or+abatacept+in+rheumatoid+arthritis.">Trial of upadacitinib or abatacept in rheumatoid arthritis.</a> The New England Journal of Medicine. 383 (16), 1511-1521 (2020).</li><li>Asfour, L., Getsos Colla, T., Moussa, A., Sinclair, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+chronic+alopecia+areata+and+severe+atopic+dermatitis+successfully+treated+with+upadacitinib.">Concurrent chronic alopecia areata and severe atopic dermatitis successfully treated with upadacitinib.</a> International Journal of Dermatology. 61 (11), e416-e417 (2022).</li><li>Cantelli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Upadacitinib+improved+alopecia+areata+in+a+patient+with+atopic+dermatitis:+A+case+report.">Upadacitinib improved alopecia areata in a patient with atopic dermatitis: A case report.</a> Dermatologic Therapy. 35 (4), e15346 (2022).</li><li>Traves, P. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=JAK+selectivity+and+the+implications+for+clinical+inhibition+of+pharmacodynamic+cytokine+signalling+by+filgotinib,+upadacitinib,+tofacitinib+and+baricitinib.">JAK selectivity and the implications for clinical inhibition of pharmacodynamic cytokine signalling by filgotinib, upadacitinib, tofacitinib and baricitinib.</a> Annals of the Rheumatic Diseases. 80 (7), 865-875 (2021).</li><li>Rozema, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+scalable+enantioselective+synthesis+of+JAK+inhibitor+upadacitinib.">Development of a scalable enantioselective synthesis of JAK inhibitor upadacitinib.</a> Organic Process Research &amp; Development. 26 (3), 949-962 (2022).</li><li>Wynn, J. P., Hanchar, R., Kleff, S., Senyk, D., Tiedje, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biobased+technology+commercialization:+the+importance+of+lab+to+pilot+scale-up.">Biobased technology commercialization: the importance of lab to pilot scale-up.</a> Metabolic Engineering for Bioprocess Commercialization. , 101-119 (2016).</li><li>Tang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Influenza virus replication inhibitor and use thereof. , (2020).</li><li>Ren, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitors+of+influenza+virus+replication+and+uses+thereof.">Inhibitors of influenza virus replication and uses thereof.</a> Center for Biotechnology Information. , (2020).</li><li>Van Epps, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+synthesis+of+tricyclic+cores+for+kinase+inhibition.">Design and synthesis of tricyclic cores for kinase inhibition.</a> Bioorganic &amp; Medicinal Chemistry Letters. 23 (3), 693-698 (2013).</li><li>Paul, F., Patt, J., Hartwig, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palladium-catalyzed+formation+of+carbon-nitrogen+bonds.+Reaction+intermediates+and+catalyst+improvements+in+the+hetero+cross-coupling+of+aryl+halides+and+tin+amides.">Palladium-catalyzed formation of carbon-nitrogen bonds. Reaction intermediates and catalyst improvements in the hetero cross-coupling of aryl halides and tin amides.</a> Journal of the American Chemical Society. 116 (13), 5969-5970 (1994).</li><li>Zhou, T., Ji, C. L., Hong, X., Szostak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palladium-catalyzed+decarbonylative+Suzuki-Miyaura+cross-coupling+of+amides+by+carbon-nitrogen+bond+activation.">Palladium-catalyzed decarbonylative Suzuki-Miyaura cross-coupling of amides by carbon-nitrogen bond activation.</a> Chemical Science. 10 (42), 9865-9871 (2019).</li><li>Sain, S., Jain, S., Srivastava, M., Vishwakarma, R., Dwivedi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+palladium-catalyzed+cross-coupling+reactions+in+organic+synthesis.">Application of palladium-catalyzed cross-coupling reactions in organic synthesis.</a> Current Organic Synthesis. 16 (8), 1105-1142 (2019).</li><li>Takale, B. S., Kong, F. Y., Thakore, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+applications+of+Pd-catalyzed+Suzuki-Miyaura+and+Buchwald-Hartwig+couplings+in+pharmaceutical+process+chemistry.">Recent applications of Pd-catalyzed Suzuki-Miyaura and Buchwald-Hartwig couplings in pharmaceutical process chemistry.</a> Organics. 3 (1), 1-21 (2021).</li></ol>

The authors have nothing to disclose.

The reaction conditions of synthesis, including reaction temperature, time, the selection of reaction reagents, and the ratio of materials, affect the feasibility of the reaction, yields, purity, and production cost, especially for scale-up production.
In the laboratory synthesis of ACT051-2, TsCl in liquid form (dissolved in DMF; step 1.1.3) can be used to in the reaction; however, it is not suitable for pilot scale-up synthesis or industrial production, as using liquid TsCl for this reaction increases the amount of solvent in the reaction system, resulting in more waste fluid. As a result, we chose solid TsCl for the reaction in the pilot scale-up and industrial production (steps 2.1.2 and 3.1.3 of the protocol, respectively) and obtained good experimental results, keeping in mind the concept of environmental protection and green chemistry (Table 1).
In addition, it was found in the pilot scale-up experiments that adding a large amount of TsCl at a low temperature makes the reaction system too viscous to be stirred, resulting in the risk of encapsulation of the raw material. To address this risk, the temperature was increased from 0-5 &#176;C to 25-35 &#176;C (steps 2.1.1 or 3.1.4 of the protocol), sufficient stirring was obtained for the solid TsCl added, the reaction solution system had good fluidity, and the reaction proceeded smoothly (Table 2).
Moreover, a large amount of water (15 mL/g ACT051-1) was required for the post-treatment experiments of ACT051-2, which was not suitable for the scaling up production experiments and generated more waste solution. Therefore, from the perspective of environmental protection, the optimal amount of water used for post-treatment was immediately investigated in the exploration of the production process conditions. As shown in Table 3, when the amount of post-treatment water was reduced to 6 mL/g ACT051-1, the yield of the experiment showed a little impact, but could greatly improve the efficiency and reduce the generation of waste liquid.
In exploring the industrial production process route of the intermediate ACT051-3, we conducted a large number of process optimization experiments and solved many scaling problems. In the synthesis of ACT051-3, ACT051-2 underwent a Buchwald-Hartwig coupling reaction with tert-butyl carbamate catalyzed by Pd(OAc)2 and xanthphos to give the compound ACT051-3. Pd(0) is an active species of palladium commonly used in coupling responses like Suzuki and Buchwald16,17,18,19. However, Pd(II) is commonly used to catalyze the reaction. As a result, we used an amine compound (DIPEA) to reduce Pd(II) to Pd(0), leaving the catalytic system of the entire reaction in a Pd(0) and Pd(II) catalytic cycle. With the addition of DIPEA, the amount of the expensive catalyst Pd(OAc)2 was greatly reduced from 1.28% to 0.5% (Table 4), which greatly reduced the cost of scale-up production and further strengthened the rationality and feasibility of the method.
The long reaction time brings about a large number of side reactions and generates impurities, which is not conducive to scaling up production. Through extensive laboratory studies, it was found that changing the state of potassium carbonate from granular to powder (steps 1.2.2 or 2.2.2 of the protocol) resulted in an appropriate reduction of the reaction time, which is more favorable for industrial production (Table 5). In addition, considering that the 1,4-dioxane used in the small-scale pilot experiment was more difficult to concentrate (step 1.2.1 of the protocol), we considered the combination of toluene and tert-butanol (steps 2.2.1 or 3.2.2 of the protocol; V/V, 2/3), which are better concentrated solvents, as reaction solvents for the industrial production. The results showed that the reaction obtained a great promotion rate, reduced the reaction time, and improved the efficiency of the reaction (see Table 6).
In conclusion, after repeated exploration of the experimental conditions, the optimal process scale-up conditions for compounds ACT051-2 and ACT051-3 were finally obtained, and the process flow diagram of this scale-up production is shown in Figure 2. The results of the scaled-up production showed that the whole process was stable, and the product yield was normal (steps 3.1.7 or 3.2.8 of the protocol).
The industrial production route obtained in this study is a new route available, and the feasibility of this process route in future industrial production was also confirmed. In addition, the results obtained in this study provide some technical research basis for future research of the industrial production route of compounds ACT051-2 and ACT051-3.
However, there is still room for optimization and improvement of the process route, such as the amount of TsCl (1.25 eq), which is excessive in the synthesis of ACT051-2 and can be further reduced. In addition, in the synthesis of ACT051-3, only toluene can be used as the reaction solvent to facilitate post-processing, and the amount of catalyst Pd(OAc)2 may continue to decrease. The above technical issues can be further studied and explored in future work to better scale-up the production of the synthetic compound ACT051-3.

Upatinib has become a globally popular Janus kinase 1 (JAK1) inhibitor for treating immune disorders in recent years1,2. This drug has demonstrated significant therapeutic effects on psoriaticarthritis (PsA)3,4, rheumatoid arthritis (RA)5,6,7, and atopic dermatitis (AD)8,9. Additionally, due to its high selectivity10, Upatinib has a wide range of clinical applications. Tert-butyl (5-tosyl-5h-pyrolo [2,3-b] pyrazin-2-yl) carbamate (ACT051-3) is an important intermediate of Upatinib. Its main structural components are the pyrrole ring and the pyrazine ring, which can be used in the preparation of new nitrogen-containing tricyclic kinase inhibitors for the treatment of immune and tumor diseases11.
Pilot scale-up is a medium-sized scale-up (50x-100x) of the process route and conditions determined by the laboratory pilot study, followed by process testing, industrial investigation, and optimization to determine the best industrial production and operating conditions12.
At present, the lab synthesis routes for this intermediate compound (ACT051-3) have been reported, but they have only been performed on a small scale due to problems of low yield, complex reactions, and high equipment requirements, which still have a lot of room to be optimized11,13,14,15. However, no process route has been reported for pilot scale-up and industrial production of the intermediate compound ACT051-3 at present.
Therefore, in this study, we investigated the pilot scale-up and production route of the compound ACT051-3, with reference to the better reported laboratory synthetic routes. Compared with the original laboratory synthesis route, many appropriate adjustments and improvements were made to the reaction conditions, and other factors that may affect the reaction results were investigated. Finally, the most suitable process parameters for the optimal route were identified, and we obtained a process route that is simple to operate, low cost, and environmentally friendly, which is suitable for the pilot scale-up and production of ACT051-3.

<table><tbody><tr><td>2-bromo-5H-pyrrolo[2,3-b]pyrazine</td><td>Nanjing Cook Biotechnology Co., Ltd.</td><td>19120110</td><td></td></tr><tr><td>1,4-dioxane</td><td>Liaoning cook Biotechnology Co., Ltd</td><td>General Reagent</td><td></td></tr><tr><td>1H NMR</td><td></td><td>Bruker AVIII 500</td><td></td></tr><tr><td>37% chloride acid molecular grade</td><td>NEON</td><td>02618 NEON</td><td></td></tr><tr><td>4-toluenesulfonyl chloride (TsCl)</td><td>Nanjing Cook Biotechnology Co., Ltd.</td><td>AR A2010137</td><td></td></tr><tr><td>Anti-Chicken IgY (H+L), highly cross-adsorbed, CF&amp;nbsp;488A antibody produced in donkey</td><td>Sigma-Aldrich</td><td>SAB4600031</td><td></td></tr><tr><td>Anti-mouse IgG (H+L), F(ab&amp;prime;)2</td><td>Sigma-Aldrich</td><td>SAB4600388</td><td></td></tr><tr><td>BD FACSCanto II</td><td>BD Biosciences</td><td>BF-FACSC2</td><td></td></tr><tr><td>BD FACSDiva CS&amp;amp;T research beads (CS&amp;amp;T research beads)</td><td>BD Biosciences</td><td>655050</td><td></td></tr><tr><td>BD FACSDiva software 7.0</td><td>BD Biosciences</td><td>655677</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A4503</td><td></td></tr><tr><td>Centrifuge 5702 R</td><td>Eppendorf</td><td>Z606936</td><td></td></tr><tr><td>Circulating water vacuum pump</td><td>Guangzhou Zhiyan Instrument Co., Ltd</td><td>SHZ-D(&lt;img alt=&quot;Equation 1&quot; src=&quot;/files/ftp_upload/64514/64514eq01.jpg&quot; style=&quot;margin: auto;&quot;/&gt;)</td><td></td></tr><tr><td>CML latex, 4% w/v</td><td>Invitrogen</td><td>C37253</td><td></td></tr><tr><td>Diatomite</td><td>Guangzhou Qishuo Chemical Co., Ltd.</td><td>/</td><td></td></tr><tr><td>Double cone rotary vacuum dryer</td><td>Jiangsu Yang-Yang Chemical Equipment Plant Inc</td><td>SZE-500T</td><td></td></tr><tr><td>enamel kettle</td><td>Jiangsu Yang-Yang Chemical Equipment Plant Inc</td><td>CS-03-002</td><td>1000L / 2000L</td></tr><tr><td>heptane</td><td>Nanjing Cook Biotechnology Co., Ltd.</td><td>General Reagent</td><td></td></tr><tr><td>HPLC</td><td>Guangzhou aoyi Technology Trading Co., Ltd</td><td>LC-2030C 3D</td><td></td></tr><tr><td>Large scale rotary evaporators</td><td>Guangzhou Xingshuo Instrument Co.,Ltd.</td><td>RE-2002</td><td></td></tr><tr><td>Low temperature and constant temperature stirring reaction bath</td><td>Guangzhou Yuhua Instrument Co., Ltd</td><td>XHDHJF-3005</td><td></td></tr><tr><td>Low temperature coolant circulating pump</td><td>Guangzhou Jincheng Scientific Instrument Co., Ltd</td><td>XHDLSB-5/25</td><td></td></tr><tr><td>Megafuge 8R</td><td>Thermo Scientific</td><td>TS-HM8R</td><td></td></tr><tr><td>N, N-Diisopropyl ethylamine (DIPEA)</td><td>Apicci Pharm</td><td>General Reagent</td><td></td></tr><tr><td>N-dimethylformamide (DMF)</td><td>Guangzhou bell Biotechnology Co., Ltd</td><td>General Reagent</td><td></td></tr><tr><td>Octanoid acid</td><td>Sigma-Aldrich</td><td>O3907</td><td></td></tr><tr><td>Pd(OAc)2</td><td>Xi&#039;an Catalyst New Materials Co.,ltd.</td><td>200704</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>1003335620</td><td></td></tr><tr><td>Potassium carbonate (K2CO3)</td><td>Guangzhou Zhonghua Trade Co.,Ltd.</td><td>General Reagent</td><td></td></tr><tr><td>Tert amyl alcohol</td><td>Nanjing Cook Biotechnology Co., Ltd.</td><td>General Reagent</td><td></td></tr><tr><td>tert-Butyl carbamate</td><td>Nanjing Cook Biotechnology Co., Ltd.</td><td>General Reagent</td><td></td></tr><tr><td>Thermo Mixer Heat/Cool</td><td>KASVI</td><td>K80-120R</td><td></td></tr><tr><td>toluene</td><td>Liaoning cook Biotechnology Co., Ltd</td><td>General Reagent</td><td></td></tr><tr><td>Vacuum drying oven</td><td>Guangzhou Yuhua Instrument Co., Ltd</td><td>DZF-6090</td><td></td></tr><tr><td>Water</td><td>/</td><td>/</td><td></td></tr><tr><td>Xantphos</td><td>Liaoning cook Biotechnology Co., Ltd</td><td>Asp20-44892</td><td></td></tr></tbody></table>

scaled-up preparation of an intermediate of upatinib, act051-3

Upatinib, a Janus kinase inhibitor drug, was developed by a biotech company to treat immune diseases. The compound tert-butyl (5-toluenesulfonyl-5h-pyrrole [2,3-b] pyrazine-2-yl) carbamate (ACT051-3) is an important intermediate of Upatinib. To date, the steady industrial production of this intermediate compound (ACT051-3) has not been reported. In this study, we described the specific synthesis method and process of the compound ACT051-3 in terms of laboratory synthesis, pilot scale-up, and industrial production. During the exploration of the process route for ACT051-3, many appropriate adjustments and improvements were made to the reaction conditions, finally leading to the successful development of the optimal industrial production process for ACT051-3. The reaction time was nearly doubled by changing the state of the potassium carbonate involved in the reaction, which greatly improved the reaction efficiency. Additionally, by introducing N,N-diisopropylethylamine (DIPEA) to the reaction, the amount of the expensive catalyst Pd(OAc)2 was reduced 2.5-fold, significantly lowering production costs, confirming the feasibility of this process route and the industrial production of ACT051-3, and satisfying market demand for this important intermediate.

This study provides the scaled-up synthesis process for the important intermediate ACT051-3 of Upatinib (Figure 1 and Figure 2). The protocol section (steps 1-3) specifically shows the gram-grade synthesis, pilot-scale kilogram-grade synthesis, and scale-up production step of the compound ACT051-2 and the intermediate ACT051-3.
In the course of exploring the optimal route for the compound ACT051-2, as shown in Table 1, it was found that solid TsCl was more involved in the reaction than liquid TsCl (dissolved in DMF, step 3.1 of the protocol) and significantly reduced the amount of DMF by nearly threefold. In addition, the product yield was increased from 97.49% to 98.44% by increasing the temperature of the mixed solution when TsCl was added from 0-5 &#176;C to 23-35 &#176;C (shown in Table 2). Further, experiments on the consumption of post-treatment water were conducted. As shown in Table 3, after a 2.5-fold reduction in water consumption (from 15 mL/g ACT051-2 to 6 mL/g ACT051-2), the reaction yield decreased by 2.5%, but the waste solution generation was reduced, and the reaction efficiency was significantly improved.
A series of experimental conditions were developed to obtain the optimized process route for the intermediate ACT051-3. As shown in Table 4, by introducing DIPEA into the reaction and replacing the reaction solvent with tert-amyl alcohol/toluene (V/V, 2/3), the amount of Pd(OAc)2 was reduced 2.5-fold (from 1.28% wt to 0.5% wt), which significantly reduced the production cost and further improved the feasibility of scaling up the production. Additionally, by changing the state of K2CO3 involved in the reaction, the reaction time was reduced from 7 h to 3.5 h, which greatly improved the reaction efficiency (as shown in Table 5). Additionally, by switching from tert-amyl alcohol/1,4-dioxane (V/V, 1/4) to tert-amyl alcohol/toluene (V/V, 2/3), the reaction time was shortened to 3 h, the product peak area increased from 84.22% to 88.52%, and the time it took for the product to concentrate was significantly shortened, all of which improved the reaction&#39;s efficiency (see Table 6).
Both compounds ACT051-2 and ACT051-3 were chemically characterized by proton nuclear magnetic resonance (1H NMR), HPLC, and high-resolution mass spectrometry. The analysis methods (HPLC, 1H NMR, and electrospray ionization [ESI] spectroscopy) of ACT051-2 and ACT051-3 can be found in the supporting work (Supplementary Table 1, Supplementary Figure 1, Supplementary Figure 2, Supplementary Figure 3, Supplementary Figure 4, Supplementary Figure 5, and Supplementary Figure 6). The characterization data for ACT051-2 and ACT051-3 are reported below:
2-bromo-5-tosyl-5H-pyrrolo[2,3-b]pyrazine (ACT051-2): 
1H NMR (500 MHz, DMSO-d6)&#948;8.59 (s,1H), 8.37 (d, J = 4.1 Hz, 1H), 8.00 (d, J = 8.2 Hz, 2H), 7.46 (d, J = 8.2 Hz, 2H), 7.02 (d, J = 4.0 Hz, 1H), 3.29 (d, J = 11.9 Hz, 3H). ESI: m/z calculated for C13H10BrN3O2S [M] + 352.21, found to be 352.00.
Tert-butyl (5-toluenesulfonyl-5H-pyrrole [2,3-b] pyrazine-2-yl) carbamate (ACT051-3): 
1H NMR (500 MHz, CDCl3) &#948;8.98 (s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.84 (d, J = 4.1 Hz, 1H), 7.21 (s, 1H), 7.19-7.17 (m, 1H), 6.53 (d, J = 4.1 Hz, 1H), 2.30 (s, 3H), 1.45 (s, 9H). ESI: m/z calculated for C18H20N4O4S [M+H] + 389.12, found to be389.15.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64514/64514fig01.jpg" /> 
Figure 1: Synthesis route of the intermediate ACT051-3. (A) The reaction route and conditions of ACT051-3 before optimization: i) DMF, DIPEA, TsCl; ii) xantphos, Pd(OAc)2, K2CO3, tert-amyl alcohol/1,4-dioxane (V/V, 1/4); (B) The reaction route and conditions of ACT051-3 after optimization: i) DMF, DIPEA, TsCl; ii) xantphos, Pd(OAc)2, K2CO3, DIPEA, tert-amyl alcohol/toluene (V/V, 2/3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64514/64514fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64514/64514fig02.jpg" /> 
Figure 2: The process flow diagram of compounds ACT051-2 and ACT051-3 in scale-up production. (A) Process flow diagram of ACT051-2 in scale-up production. (B) Process flow diagram of ACT051-3 in scale-up production. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64514/64514fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Number</td>
			<td>State of TsCl</td>
			<td>V(DMF) (Laboratory test)</td>
			<td>V(DMF) (Pilot scale up)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>dissolve in DMF</td>
			<td>8.5 V</td>
			<td>54 V</td>
		</tr>
		<tr>
			<td>2</td>
			<td>solid</td>
			<td>3.0 V</td>
			<td>18 V</td>
		</tr>
	</tbody>
</table>
Table 1: Effect of different forms of TsCl on the synthetic compound ACT051-2. The different states of TsCl include liquid TsCl (dissolved in DMF) and solid TsCl. Experimental results show that solid TsCl is more conducive to industrial production.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Number</td>
			<td>Temperature (&#176;C)</td>
			<td>Condition of mix</td>
			<td>Whether has a clarification process</td>
			<td>Yield</td>
			<td>Purity</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0-5</td>
			<td>viscous</td>
			<td>No</td>
			<td>97.49%</td>
			<td>96.85%</td>
		</tr>
		<tr>
			<td>2</td>
			<td>25-35</td>
			<td>good mixing</td>
			<td>Yes</td>
			<td>98.44%</td>
			<td>96.99%</td>
		</tr>
	</tbody>
</table>
Table 2: Effect of adding TsCl at different temperatures on the synthesis of ACT051-2. Adding TsCl to the reaction at 0-5 &#176;C or 23-35 &#176;C.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Number</td>
			<td>Water consumption</td>
			<td>Yield</td>
			<td>Purity</td>
		</tr>
		<tr>
			<td>1</td>
			<td>15 mL / g ACT051-1</td>
			<td>97.49%</td>
			<td>96.85%</td>
		</tr>
		<tr>
			<td>2</td>
			<td>6 mL / g ACT051-1</td>
			<td>94.90%</td>
			<td>97.69%</td>
		</tr>
		<tr>
			<td>3</td>
			<td>9 mL / g ACT051-1</td>
			<td>95.07%</td>
			<td>96.71%</td>
		</tr>
	</tbody>
</table>
Table 3: Effect of different post-treatment water consumption on the synthesis of ACT051-2. Try the different post-treatment water consumptions, including 15 mL/g ACT051-1, 9 mL/g ACT051-1, and 6 mL/g ACT051-1. The optimal conditions were achieved at a post-treatment water volume of 6 mL/g ACT051-2.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Number</td>
			<td>Equivalent of DIEPA</td>
			<td>Equivalent of K2CO3</td>
			<td>Equivalent of Pd(OAc)2</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0.0 eq</td>
			<td>3.0 eq</td>
			<td>1.28% wt</td>
		</tr>
		<tr>
			<td>2</td>
			<td>2.0 eq</td>
			<td>2.0 eq</td>
			<td>0.60% wt</td>
		</tr>
		<tr>
			<td>3</td>
			<td>1.0 eq</td>
			<td>2.0 eq</td>
			<td>0.60% wt</td>
		</tr>
		<tr>
			<td>4</td>
			<td>0.5 eq</td>
			<td>2.0 eq</td>
			<td>0.60% wt</td>
		</tr>
	</tbody>
</table>
Table 4: Effect of adding DIPEA to the reaction for the synthesis of ACT051-3. Exploring the effect of DIPEA addition or not on the reaction. The results showed that the introduction of DIPEA reduced the amount of Pd(OAc)2 by a factor of 2.5 (from 1.28% wt to 0.5% wt).
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Number</td>
			<td>State of K2CO3</td>
			<td>Equivalent</td>
			<td>Reaction time (h)</td>
		</tr>
		<tr>
			<td>1</td>
			<td>solid</td>
			<td>2.0 eq</td>
			<td>7</td>
		</tr>
		<tr>
			<td>2</td>
			<td>powdered</td>
			<td>2.0 eq</td>
			<td>3.5</td>
		</tr>
	</tbody>
</table>
Table 5: Effect of different states of K2CO3 on the reaction of the synthetic compound ACT051-3. Select potassium carbonate in granular or powder form to participate in the reaction.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Number</td>
			<td>Dosage of Pd(OAc)2</td>
			<td>Reaction solvent</td>
			<td>V/V</td>
			<td>Reaction time / h</td>
			<td>Product&#160; peak area/ %</td>
		</tr>
		<tr>
			<td>1</td>
			<td>0.60% wt</td>
			<td>tert amyl alcohol / 1,4-dioxane</td>
			<td>1-4</td>
			<td>3.5</td>
			<td>84.22</td>
		</tr>
		<tr>
			<td>2</td>
			<td>0.60% wt</td>
			<td>tert amyl alcohol / 1,4-dioxane</td>
			<td>2-3</td>
			<td>3.5</td>
			<td>83.34</td>
		</tr>
		<tr>
			<td>3</td>
			<td>0.60% wt</td>
			<td>tert amyl alcohol / Toluene</td>
			<td>2-3</td>
			<td>3</td>
			<td>88.52</td>
		</tr>
		<tr>
			<td>4</td>
			<td>0.50% wt</td>
			<td>tert amyl alcohol / Toluene</td>
			<td>2-3</td>
			<td>2.25</td>
			<td>87.11</td>
		</tr>
	</tbody>
</table>
Table 6: Effect of different reaction solvents on the reaction of the synthetic compound ACT051-3. Tert-amyl alcohol/1,4-dioxane (V/V, 1/4) and tert-amyl alcohol/toluene (V/V, 2/3) are selected as reaction resolvents.
Supplementary Table 1: Analytical method of compounds ACT051-2 and ACT051-3. Specific chromatographic conditions for the analysis of compounds ACT051-2 and ACT051-3, including the instrument, method name, liquid phase column, mobile phase, column temperature, current speed, and wavelength. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64514/Supplementary table 1_RE.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Figure 1: The high-performance liquid chromatograms of ACT051-2. The results for the data were detected by HPLC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64514/Supplementary Figure 1_RE.png" target="_blank">Please click here to download this File.</a>
Supplementary Figure 2: The high-performance liquid chromatograms of ACT051-3. The results for the data were detected by HPLC. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64514/Supplementary Figure 2_RE.png" target="_blank">Please click here to download this File.</a>
Supplementary Figure 3: MS spectrum of ACT051-2. The results for the data were detected by ESI spectroscopy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64514/Supplementary Figure 3_RE.png" target="_blank">Please click here to download this File.</a>
Supplementary Figure 4: MS spectrum of ACT051-3. The results for the data were detected by ESI spectroscopy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64514/Supplementary Figure 4_RE.png" target="_blank">Please click here to download this File.</a>
Supplementary Figure 5: 1H NMR spectrum of ACT051-2. The results for the data were analyzed using MestReNova. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64514/Supplementary Figure 5_RE.png" target="_blank">Please click here to download this File.</a>
Supplementary Figure 6: 1H NMR spectrum of ACT051-3. The results for the data were analyzed using MestReNova. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64514/Supplementary Figure 6_RE.png" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Scaled-Up Preparation of an Intermediate of Upatinib, ACT051-3 at JoVE.com

Scaled-Up Preparation of an Intermediate of Upatinib, ACT051-3

Here, we present a protocol for the scaled-up synthesis of the intermediate tert-butyl (5-toluenesulfonyl-5h-pyrrole [2,3-b] pyrazine-2-yl) carbamate (ACT051-3) of Upatinib.

Upatinib, a Janus kinase inhibitor drug, was developed by a biotech company to treat immune diseases. The compound tert-butyl ...

scaled-up-preparation-of-an-intermediate-of-upatinib-act051-3

Nanyang Technological University

Research

JoVE Journal

Chemistry

980 Views.  Aneo Chem-Tech Co., Ltd. Hier wordt een protocol gepresenteerd voor de opgeschaalde synthese van een tussenproduct van upatinib, ACT051-3. Deze methode is eenvoudig te bedienen, goedkoop en milieuvriendelijk. Bereid om te beginnen ACT051-2 door 50 gram van de verbinding 2-broom-5-tosyl-5H-pyrrolo[2, 3-b]pyrazine op te lossen in 15 milliliter N, N-dimethylformamide of DMF in een kolf met ronde bodem.Voeg 65,3 gram diisopropylethylamine toe aan de reactieoplossing onder stikstofbescherming en koel de temperatuur...

Video: Scaled-Up Preparation of an Intermediate of Upatinib, ACT051-3

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in most countries. The discovery of "oncogenic driver mutations," such as epidermal growth factor receptor (EGFR)-activating mutations, and subsequent development of molecular targeted agents of EGFR tyrosine kinase inhibitors (TKIs) (gefitinib, erlotinib, afatinib, dacomitinib, and osimertinib) have dramatically altered lung cancer treatment in recent decades. However, these drugs are still not effective in patients with non-small cell lung cancer (NSCLC) carrying EGFR-activating mutations. Following acquired resistance, the systemic progression of NSCLC remains a significant obstacle in treating patients with EGFR mutation-positive NSCLC. Here, we present a stepwise dose escalation method for establishing three independent acquired afatinib-resistant cell lines from NSCLC PC-9 cells harboring EGFR-activating mutations of 15-base pair deletions in EGFR exon 19. Methods for characterizing the three independent afatinib-resistance cell lines are briefly presented. The acquired resistance mechanisms to EGFR TKIs are heterogeneous. Therefore, multiple cell lines with acquired resistance to EGFR-TKIs must be examined. Ten to twelve months are required to obtain cell lines with acquired resistance using this stepwise dose escalation approach. The discovery of novel acquired resistance mechanisms will contribute to the development of more effective and safe therapeutic strategies.

A method for establishing afatinib-resistance cell lines from lung adenocarcinoma PC-9 cells was developed, and resistant cells were characterized. The resistant cells can be used to investigate epidermal growth factor receptor tyrosine kinase inhibitor-resistance mechanisms, applicable for patients with non-small cell lung cancer.

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in ...

Cancer Research

Chemical Inactivation of the E3 Ubiquitin Ligase Cereblon by Pomalidomide-based Homo-PROTACs

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all FDA approved drugs for the treatment of multiple myeloma, induce ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3) via the cereblon (CRBN) E3 ubiquitin ligase for proteasomal degradation. IMiDs have recently been utilized for the generation of bifunctional proteolysis targeting chimeras (PROTACs) to target other proteins for ubiquitination and proteasomal degradation by the CRBN E3 ligase. We designed and synthesized pomalidomide-based homobifunctional PROTACs and analyzed their ability to induce self-directed ubiquitination and degradation of CRBN. Here, CRBN serves as both, the E3 ubiquitin ligase and the target at the same time. The homo-PROTAC compound 8 degrades CRBN with a high potency with only minimal remaining effects on IKZF1 and IKZF3. CRBN inactivation by compound 8 had no effect on cell viability and proliferation of different multiple myeloma cell lines. This homo-PROTAC abrogates the effects of IMiDs in multiple myeloma cells. Therefore, our homodimeric pomalidomide-based compounds may help to identify CRBN&#8216;s endogenous substrates and physiological functions and investigate the molecular mechanism of IMiDs.

This work describes the synthesis and characterization of a pomalidomide-based, bifunctional homo-PROTAC as a novel approach to induce ubiquitination and degradation of the E3 ubiquitin ligase cereblon (CRBN), the target of thalidomide analogs.

The immunomodulatory drugs (IMiDs) thalidomide and its analogs, lenalidomide and pomalidomide, all FDA approved drugs for the treatment of multiple ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

Optimizing HCC Organoid Formation for Target Identification and Drug Development

Development and Optimization of A Human Hepatocellular Carcinoma Patient-Derived Organoid Model for Potential Target Identification and Drug Discovery

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents necessitate further research into its pathogenesis and treatment. Organoids, a novel model that closely resembles native tumor tissue and can be cultured in vitro, have garnered significant interest in recent years, with numerous reports on the development of organoid models for liver cancer. In this study, we have successfully optimized the procedure and established a culture protocol that enables the formation of larger-sized HCC organoids with stable passaging and culture conditions. We have comprehensively outlined each step of the procedure, covering the entire process of HCC tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation, and provided detailed precautions in this paper. These organoids exhibit genetic similarity to the original HCC tissues and can be utilized for diverse applications, including the identification of potential therapeutic targets for tumors and subsequent drug development.

Author Spotlight: Investigating Liver Cancer Pathogenesis Using Patient-Derived Organoids 

We provide a comprehensive overview and refinement of existing protocols for hepatocellular carcinoma (HCC) organoid formation, encompassing all stages of organoid cultivation. This system serves as a valuable model for the identification of potential therapeutic targets and the assessment of drug candidate effectiveness.

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents ...

Assessing Specificity of Anticancer Drugs In Vitro

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key element is the quantitative determination of a tumor-specific genetic alteration in relation to a universal sequence using a dual-probe digital PCR assay and the subsequent calculation of the proportion of tumor cells. The assay is carried out on a culture containing tumor cells of an established line and spiked-in non-tumor cells. The mixed culture is treated with a test drug at various concentrations. After the treatment, DNA is prepared directly from the survived adhesive cells in wells of 96-well plates using a simple and inexpensive method, and subjected to a dual-probe digital PCR assay for measuring a tumor-specific genetic alteration and a reference universal sequence. In the present demonstration, a heterozygous deletion of the NF1 gene is used as the tumor-specific genetic alteration and a RPP30 gene as the reference gene. Using the ratio NF1/RPP30, the proportion of tumor cells was calculated. Since the dose-dependent change of the proportion of tumor cells provides an in vitro indication for specificity of the drug, this genetic and cell-based in vitro assay will likely have application potential in drug discovery. Furthermore, for personalized cancer-care, this genetic- and cell-based tool may contribute to optimizing adjuvant chemotherapy by means of testing efficacy and specificity of candidate drugs using primary cultures of individual tumors.

Assessing Specificity of Anticancer Drugs In Vitro

The goal of this protocol is to assess specificity of anticancer drugs in vitro using mixed cultures containing both tumor and non-tumor cells.

A procedure for assessing specificity of anticancer drugs in vitro using cultures containing both tumor and non-tumor cells is demonstrated. The key ...

Medicine

Developing and Testing Methylated Nano-Structured Dipeptides that Inhibit Src Kinase Activity In Vitro for Anti-Cancer Applications

Here, with the aim of developing a novel anti-cancer treatment, seven dipeptides were designed that contained methylated tryptophan and/or methylated arginine and were produced using Fmoc solid-phase peptide synthesis. Overexpression of the Src tyrosine kinase enzyme has been implicated in the development of different cancers. Dipeptides containing unnatural amino acids such as methylated arginine (RCH3), dimethylated arginine (R(CH3)2), and/or methylated tryptophan (WCH3) residues have earlier been shown to inhibit Src kinase. In this study, three such dipeptides, W-RCH3, WCH3-RCH3, and W-R(CH3)2, were tested using acellular assays and were found to have IC50 values (the concentration at which 50% inhibition occurs) of 510 nM, 916 nM, and 1 &#181;M, respectively. These values were comparable to those obtained for cyclic penta- to nano-W-R peptides ([W-R]5-[W-R]9) synthesized in previous studies. However, the unmethylated versions of the dipeptides did not show any inhibitory activity against Src kinase. All of these dipeptides (50 &#181;M) did not show any cytotoxicity after incubation up to 72 h with three different cancer cell lines, including leukemia (CCRF-CEM), breast adenocarcinoma (MDA-MB-231), and ovarian adenocarcinoma (SK-OV-3) cell lines, indicating the limited permeability of the peptides through the cell membrane. Therefore, further study is needed to improve the permeability of these peptides for anticancer applications, such as by using a peptide carrier or additional peptide functionalization. In summary, this study provides a protocol to synthesize and test peptides that inhibit Src kinase activity, and thus possess promising anticancer ability, as demonstrated using acellular and cellular assays.

Developing and Testing Methylated Nano-Structured Dipeptides that Inhibit Src Kinase Activity In Vitro for Anti-Cancer Applications

Presented here is a protocol for designing and testing dipeptides that can inhibit Src kinase enzyme activity using acellular and cellular assays for anti-cancer applications. The peptides formulated here (W-RCH3, WCH3-RCH3, and W-R(CH3)2)&#160;inhibited Src kinase with IC50 values of 510 nM, 916 nM, and 1 &#181;M, respectively.

Here, with the aim of developing a novel anti-cancer treatment, seven dipeptides were designed that contained methylated tryptophan and/or methylated ...

Generation of Human Kidney Tubuloids from Tissue and Urine

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high efficiency. Normal kidney tubuloids recapitulate many aspects of their tissue of origin. They represent distinct nephron segments - most notably of the proximal tubule, loop of Henle, distal tubules, and collecting duct - and can be used to study normal kidney physiology. Furthermore, tubuloid technology facilitates disease modeling, e.g., for infectious diseases as well as for cancer. Obtaining kidney epithelial cells for tubuloid generation is, however, dependent on leftover surgical material (such as partial) nephrectomies) or needle biopsies. The ability to grow tubuloids from urine would provide an alternative, less invasive source of healthy kidney epithelial cells. It has been previously shown that tubuloid cultures can be successfully generated from only a few milliliters of freshly collected urine. This article describes the protocols to generate and propagate ASC-derived human kidney tubuloid cultures from tissue and urine samples.

Human kidney tubuloid cultures represent a valuable in vitro model to study kidney physiology and disease. Tubuloids can be established from kidney tissue (healthy and diseased) as well as urine, the latter representing an easily obtainable and less invasive source of research material.

Adult stem cell (ASC)-derived human kidney epithelial organoids, or tubuloids, can be established from healthy and diseased kidney epithelium with high ...

Biology

Identifying Kinase Inhibitors that Modulate the Thymocyte Response to Strong TCR Signals

Source: Chen, E. W., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58946">Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries.</a> J. Vis. Exp. (2019).
In this video, we describe a method to identify the small-molecule kinase inhibitors that modulate the apoptosis of self-reactive CD4+CD8+ double-positive immature thymocytes. Apoptosis is induced in the double-positive thymocytes by activating them with anti-CD3- and anti-CD28-coated magnetic beads; this is followed by a small-molecule inhibitor treatment and flow cytometry analysis to detect if the inhibitors modulate the apoptotic marker expression.

Source: Chen, E. W., et al. Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries. J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Immunology

Immune Response

Host-Pathogen Interaction

Dose Escalation: A Method for Developing Drug Resistance in Cancer Cells

Source: Yamaoka T. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/59473/establishment-characterization-three-afatinib-resistant-lung">Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib.</a> J. Vis. Exp., 2019
The video describes the development of drug resistance in cancer cells by exposing cells to escalating doses of a drug after calculating IC50 values of the drug previously with MTT assay. The sample protocol describes the step-by-step process of developing drug-resistant cells.

Source: Yamaoka T. et al., Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing ...

Scaled-Up Preparation of an Intermediate of Upatinib, ACT051-3

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