TW201446248A - Combination of an EGFR T790M inhibitor and an EGFR inhibitor for the treatment of non-small cell lung cancer - Google Patents

Abstract

This invention relates to a method of treating non-small cell lung cancer by administering a combination of an EGFR T790M inhibitor in combination with a low-dose amount of a panHER inhibitor. This invention also relates to a method of treating non-small cell lung cancer by administering a combination of an irreversible EGFR T790M inhibitor in combination with an EGFR inhibitor.

Description

Combination of epidermal growth factor receptor (EGFR) T790M inhibitor and EGFR inhibitor for the treatment of non-small cell lung cancer

The present invention relates to a method of treating non-small cell lung cancer by administering a combination of an EGFR T790M inhibitor in combination with a low dose of a panHER inhibitor. The invention also relates to a method of treating non-small cell lung cancer by administering a combination of an irreversible EGFR T790M inhibitor in combination with an EGFR inhibitor.

Non-small cell lung cancer is the leading cause of cancer deaths worldwide, with an estimated 1.4 million new cases diagnosed each year. In the most common form of non-small cell lung cancer, lung cancer, patients with mutations in the epidermal growth factor receptor (EGFR) constitute between 10-30% of the entire population. EGFR inhibitors are most effective in this group of patients, such as erlotinib or gefitinib (Paez et al. Science 2004; Lynch et al NEJM 2004; Pao et al. PNAS 2004). ). The most common activating mutation associated with a good response to these inhibitors is a deletion in exon 19. (eg, E746-A750) and point mutations in the activation loop (exon 21, particularly L858R). Additional somatic mutations that have been identified to a lesser extent to date include point mutations: G719S, G719C, G719A, L861 and small insertions in exon 20 (JNCI 2005 by Shigematsu et al; JCO 2003 by Fukuoka et al; Kris JAMA 2003; and Shepherd et al. (NEJM 2004).

Although these agents are effective in treating EGFR mutant subpopulations, most of the patients who initially responded developed resistance. The primary mechanism of resistance observed in approximately 50% of patients is attributed to the second mutation (T790M), which occurs on the gatekeeper gene sulphonic acid residues (Kosaka et al., CCR 2006; Balak et al. CCR 2006; and Engelman et al. Science 2007).

Improved therapies for the treatment of non-small cell lung cancer involve many unmet medical requirements and it is necessary to identify novel combinations to improve treatment outcomes.

Each of the specific examples described below can be combined with any other specific example described herein, which is inconsistent with the specific examples that are combined. Moreover, each of the specific examples described herein is intended to be within the scope of the pharmaceutically acceptable salts of the compounds described herein. Thus, the phrase 〝 or their pharmaceutically acceptable salts are encompassed by the description of all compounds described herein.

Some specific examples described herein are directed to a method of treating non-small cell lung cancer comprising administering an effective amount of an irreversible EGFR T790M inhibitor in combination with an effective amount of an EGFR inhibitor to a patient in need thereof.

In a further embodiment of the method of the invention, the irreversible EGFR T790M inhibitor is 1-{(3R,4R)-3-[({5-chloro-2-[(1-methyl-1H-pyrazole-)- 4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-ene- 1-ketone or a pharmaceutically acceptable salt thereof.

In some embodiments of the methods of the invention, the irreversible EGFR T790M inhibitor is N-methyl-N-[trans-3-({2-[(1-methyl-1H-pyrazol-4-yl))) Amino]-5-(pyridin-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)cyclobutyl]prop-2-enylamine or its pharmaceutically acceptable Accept the salt.

In a particular embodiment of the method of the invention, the irreversible EGFR T790M inhibitor is N-[trans-3-({5-chloro-2-[(1,3-dimethyl-1H-pyrazole-4) -amino)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)cyclobutyl]-N-methylprop-2-endecylamine or a pharmaceutically acceptable salt.

In a further embodiment of the method of the invention, the irreversible EGFR T790M inhibitor is 1-{(3R,4R)-3-[({5-chloro-2-[(3-methoxy-1-methyl) -1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl} Prop-2-en-1-one or a pharmaceutically acceptable salt thereof.

In a specific embodiment of the method of the invention, the EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, icotinib, vandetanib, pull Latatinib, neratinib, afatinib, pelitinib, dacomitinib, canertinib, cetux Monoclonal antibody (cetuximab) and panitumumab, or their pharmaceutically acceptable salts.

In a further embodiment of the method of the invention, the EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, iclotinib, vantinidin, lapatinib, Nilotinib, afatinib, piratinib, dacomtinib and carnitinib, or their pharmaceutically acceptable salts.

In some embodiments of the methods of the invention, the EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, afatinib, and dacomtinib, or their pharmaceuticals Acceptable salt.

In a particular embodiment of the method of the invention, the EGFR inhibitor is gefitinib or a pharmaceutically acceptable salt thereof.

In some embodiments of the methods of the invention, the EGFR inhibitor is erlotinib or a pharmaceutically acceptable salt thereof.

In a particular embodiment of the method of the invention, the EGFR inhibitor is afatinib or a pharmaceutically acceptable salt thereof.

In some embodiments of the methods of the invention, the EGFR inhibitor is dacomitinib or a pharmaceutically acceptable salt thereof.

In a further embodiment of the method of the invention, the EGFR inhibitor is a reversible EGFR inhibitor.

In a further embodiment of the method of the invention, the reversible EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, ectinib, vantinib and lapata Nie, or their pharmaceutically acceptable salts.

In a particular embodiment of the method of the invention, the reversible EGFR inhibitor is gefitinib or a pharmaceutically acceptable salt thereof.

In a further embodiment of the method of the invention, the reversible EGFR inhibitor is erlotinib or a pharmaceutically acceptable salt thereof.

In an additional embodiment of the methods of the invention, the EGFR inhibitor is an irreversible EGFR inhibitor.

In a specific embodiment of the method of the invention, the irreversible EGFR inhibitor is selected from the group consisting of: nerotinib, afatinib, piritinib, dacomitinib, and carnitinib, Or their pharmaceutically acceptable salts.

In a further embodiment of the method of the invention, the irreversible EGFR inhibitor is afatinib or a pharmaceutically acceptable salt thereof.

In some embodiments of the methods of the invention, the irreversible EGFR inhibitor is dacomitinib or a pharmaceutically acceptable salt thereof.

Some specific embodiments of the invention relate to a method of treating non-small cell lung cancer comprising administering an effective amount of an EGFR T790M inhibitor in combination with a panHER inhibitor to a patient in need thereof, wherein the panHER inhibitor is administered according to a non-standard clinical Dosing regimen is administered.

In a further embodiment of the method of the invention, the non-standard clinical dosing regimen is a non-standard clinical dose or a non-standard dosing schedule.

In some embodiments of the methods of the invention, the non-standard clinical dosing regimen is a low dose of a panHER inhibitor.

In a specific embodiment of the method of the invention, the non-standard clinical dosing regimen is an intermittent dosing regimen.

In a further embodiment of the method of the invention, the EGFR T790M inhibitor is selected from the group consisting of Go6976, PKC412, AP26113, HM61713, WZ4002, CO-1686 and TAS-2913, or their pharmaceuticals Acceptable salt.

In a further embodiment of the method of the invention, the EGFR T790M inhibitor is 1-{(3R,4R)-3-[({5-chloro-2-[(1-methyl-1H-pyrazole-4-) Amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-en-1- A ketone or a pharmaceutically acceptable salt thereof.

In a specific embodiment of the method of the invention, the EGFR T790M inhibitor is N-methyl-N-[trans-3-({2-[(1-methyl-1H-pyrazol-4-yl))amino] 5-(-pyridin-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)cyclobutyl]prop-2-enylamine or a pharmaceutically acceptable salt thereof .

In an additional embodiment of the method of the invention, the EGFR T790M inhibitor is N-[trans-3-({5-chloro-2-[(1,3-dimethyl-1H-pyrazol-4-yl) Amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)cyclobutyl]-N-methylprop-2-endecylamine or a pharmaceutically acceptable salt thereof.

In a further embodiment of the method of the invention, the EGFR T790M inhibitor is 1-{(3R,4R)-3-[({5-chloro-2-[(3-methoxy-1-methyl-1H) -pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}- 2-en-1-one or a pharmaceutically acceptable salt thereof.

In some embodiments of the methods of the invention, the panHER inhibitor is selected from the group consisting of lapatinib, nerotinib, and afar Tinidinib, piritinib, dacomtinib and carnitinib, or their pharmaceutically acceptable salts.

In a particular embodiment of the method of the invention, the panHER inhibitor is selected from the group consisting of lapatinib, nerotinib, afatinib, piratinib, dacomitinib and Carnitinib, or their pharmaceutically acceptable salts.

In a further embodiment of the method of the invention, the panHER inhibitor is afatinib or a pharmaceutically acceptable salt thereof.

In a particular embodiment of the method of the invention, the panHER inhibitor is dacomitinib or a pharmaceutically acceptable salt thereof.

In a further embodiment of the method of the invention, the panHER inhibitor is an irreversible EGFR inhibitor.

In a particular embodiment of the method of the invention, the irreversible panHER inhibitor is selected from the group consisting of: nerotinib, afatinib, piritinib, dacomitinib and carnitine Nie, or their pharmaceutically acceptable salts.

In a particular embodiment of the method of the invention, the irreversible panHER inhibitor is afatinib or a pharmaceutically acceptable salt thereof.

In some embodiments of the methods of the invention, the irreversible panHER inhibitor is dacomitinib or a pharmaceutically acceptable salt thereof.

A specific embodiment of the invention pertains to a method of treating non-small cell lung cancer comprising administering a combination of a synergistic amount of an EGFR T790M inhibitor and an EGFR inhibitor to a patient in need thereof.

In some embodiments of the methods of the invention, EGFR T790M inhibition The agent is selected from the group consisting of Go6976, PKC412, AP26113, HM61713, WZ4002, CO-1686 and TAS-2913, or their pharmaceutically acceptable salts.

In a further embodiment of the method of the invention, the EGFR T790M inhibitor is 1-{(3R,4R)-3-[({5-chloro-2-[(1-methyl-1H-pyrazole-4-) Amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-en-1- A ketone or a pharmaceutically acceptable salt thereof.

In a specific embodiment of the method of the invention, the EGFR T790M inhibitor is N-methyl-N-[trans-3-({2-[(1-methyl-1H-pyrazol-4-yl))amino] 5-(-pyridin-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)cyclobutyl]prop-2-enylamine or a pharmaceutically acceptable salt thereof .

In a specific embodiment of the method of the invention, the EGFR T790M inhibitor is N-[trans-3-({5-chloro-2-[(1,3-dimethyl-1H-pyrazol-4-yl) Amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)cyclobutyl]-N-methylprop-2-endecylamine or a pharmaceutically acceptable salt thereof.

In some embodiments of the methods of the invention, the EGFR T790M inhibitor is 1-{(3R,4R)-3-[({5-chloro-2-[(3-methoxy-1-methyl-1H-) Pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}propane-2 -en-1-one or a pharmaceutically acceptable salt thereof.

In a further embodiment of the method of the invention, the EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, iclotinib, vantinidin, lapatinib, Narotinib, afatinib, skin Ritinib, dacomitinib, carnitinib, cetuximab, and panituzumab, or their pharmaceutically acceptable salts.

In some embodiments of the methods of the invention, the EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, iclotinib, vantinib, lapatinib, nai Rotorinib, afatinib, piritinib, dacomitinib and carnitinib, or their pharmaceutically acceptable salts.

In a particular embodiment of the method of the invention, the EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, afatinib and dacomitinib, or their pharmaceuticals Acceptable salt.

In some embodiments of the methods of the invention, the EGFR inhibitor is gefitinib or a pharmaceutically acceptable salt thereof.

In a particular embodiment of the method of the invention, the EGFR inhibitor is erlotinib or a pharmaceutically acceptable salt thereof.

In some embodiments of the methods of the invention, the EGFR inhibitor is a panHER inhibitor.

In a further embodiment of the method of the invention, the panHER inhibitor is selected from the group consisting of lapatinib, nerotinib, afatinib, piritinib, dacotinib, and Carnitinib, or their pharmaceutically acceptable salts.

In a specific embodiment of the method of the invention, the panHER inhibitor is selected from the group consisting of lapatinib, nerotinib, afatinib, piritinib, dacomitinib and canaine Tinidil, or their pharmaceutically acceptable salts.

In some embodiments of the methods of the invention, the panHER inhibitor is Afatinib or a pharmaceutically acceptable salt thereof.

In an additional embodiment of the method of the invention, the panHER inhibitor is dacomitinib or a pharmaceutically acceptable salt thereof.

In an additional embodiment of the methods of the invention, the panHER inhibitor is an irreversible EGFR inhibitor.

In some embodiments of the methods of the invention, the irreversible panHER inhibitor is selected from the group consisting of: nerotinib, afatinib, piritinib, dacomitinib, and carnitinib. , or their pharmaceutically acceptable salts.

In a particular embodiment of the method of the invention, the irreversible panHER inhibitor is afatinib or a pharmaceutically acceptable salt thereof.

In a particular embodiment of the method of the invention, the irreversible panHER inhibitor is dacomitinib or a pharmaceutically acceptable salt thereof.

Some specific examples of the invention relate to a synergistic combination of: (a) an EGFR T790M inhibitor; and (b) an EGFR inhibitor, wherein component (a) and component (b) have a synergistic effect.

In some embodiments of the combinations of the invention, the EGFR T790M inhibitor is selected from the group consisting of Go6976, PKC412, AP26113, HM61713, WZ4002, CO-1686, and TAS-2913, or their medicinal properties Accept the salt.

In a further embodiment of the combination of the invention, the EGFR T790M inhibitor is 1-{(3R,4R)-3-[({5-chloro-2-[(1-methyl-1H-pyrazole-4-) Amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-en-1- Ketone or its pharmaceutically acceptable salt.

In a specific embodiment of the combination of the invention, the EGFR T790M inhibitor is N-methyl-N-[trans-3-({2-[(1-methyl-1H-pyrazol-4-yl)amino)] 5-(-pyridin-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)cyclobutyl]prop-2-enylamine or a pharmaceutically acceptable salt thereof .

In an additional embodiment of the combination of the invention, the EGFR T790M inhibitor is N-[trans-3-({5-chloro-2-[(1,3-dimethyl-1H-pyrazol-4-yl) Amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)cyclobutyl]-N-methylprop-2-endecylamine or a pharmaceutically acceptable salt thereof.

In some embodiments of the combinations of the invention, the EGFR T790M inhibitor is 1-{(3R,4R)-3-[({5-chloro-2-[(3-methoxy-1-methyl-1H-) Pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}propane-2 -en-1-one or a pharmaceutically acceptable salt thereof.

In some embodiments of the combinations of the invention, the EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, iclotinib, vantinib, lapatinib, nai Rotorinib, afatinib, piratinib, dacomtinib, carnitinib, cetuximab, and panituzumab, or their pharmaceutically acceptable salts.

In a specific embodiment of the combination of the invention, the EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, ectinib, validinib, lapatinib, narnau Tinidil, afatinib, piritinib, dacomtinib and carnitinib, or their pharmaceutically acceptable salts.

In a specific embodiment of the combination of the invention, the EGFR inhibitor is selected from Groups of gefitinib, erlotinib, afatinib and dacomtinib, or their pharmaceutically acceptable salts.

In a particular embodiment of the combination of the invention, the EGFR inhibitor is gefitinib or a pharmaceutically acceptable salt thereof.

In a further embodiment of the combination of the invention, the EGFR inhibitor is erlotinib or a pharmaceutically acceptable salt thereof.

In some embodiments of the combinations of the invention, the EGFR inhibitor is a panHER inhibitor.

In a further embodiment of the combination of the invention, the panHER inhibitor is selected from the group consisting of lapatinib, nerotinib, afatinib, piritinib, dacotinib, and Carnitinib, or their pharmaceutically acceptable salts.

In an additional embodiment of the combination of the invention, the panHER inhibitor is selected from the group consisting of lapatinib, nerotinib, afatinib, piritinib, dacotinib, and Carnitinib, or their pharmaceutically acceptable salts.

In a particular embodiment of the combination of the invention, the panHER inhibitor is afatinib or a pharmaceutically acceptable salt thereof.

In some embodiments of the combination of the invention, the panHER inhibitor is dacomitinib or a pharmaceutically acceptable salt thereof.

In a specific embodiment of the combination of the invention, the panHER inhibitor is an irreversible EGFR inhibitor.

In an additional embodiment of the combination of the invention, the irreversible panHER inhibitor is selected from the group consisting of: Nicotine, afatinib, piritinib, dacomtinib and carnitinib, or their pharmaceutically acceptable salts.

In some embodiments of the combinations of the invention, the irreversible panHER inhibitor is afatinib or a pharmaceutically acceptable salt thereof.

In a particular embodiment of the combination of the invention, the irreversible panHER inhibitor is dacomitinib or a pharmaceutically acceptable salt thereof.

Figure 1 shows the C>T EGFR T790M mutation identified by Sanger sequencing in RPC9 and 3, and the percentages represent the castPCR quantified EGFR T790M dual gene relative to the total EGFR dual gene.

Figure 2 shows dose response curves of PC9 and RPC9 strains 3 and 6 treated with various concentrations of dacomtinib (Figure 2A) or erlotinib (Figure 2B) in a cell viability assay.

Figure 3 shows the dose response curve in the RPC9 colony 6 cell survival assay. Figure 3A shows dose response curves for Compound A (〝 compound A〞) and Dacomtinib (〝daco〞), alone and in combination. Figure 3B shows dose response curves for Compound A and erlotinib (ererlo), alone and in combination. Figure 3C shows the percent inhibition of Compound A as a single agent and in combination with dacomitinib and erlotinib at selected concentrations.

Figure 4 shows the dose response curve in the RPC9 colony 6 cell survival assay. Figure 4A shows dose response curves for Compound B (〝 compound B〞) and Dacomtinib (〝daco〞), alone and in combination. Figure 4B shows Dose response curves for Compound B and erlotinib (ererlo) are shown separately and in combination. Figure 4C shows the percent inhibition of Compound B as a single agent and in combination with dacomitinib and erlotinib at selected concentrations.

Figure 5 shows the dose response curve in the RPC9 colony 6 cell survival assay. Figure 5A shows the dose response curve for Compound A (〝 compound A〞) alone and in combination with dacomitinib (〝daco〞). Figure 5B shows the dose response curve for Compound A alone and in combination with gefitinib. Figure 5C shows the dose response curve for Compound A alone and in combination with afatinib (〝afat(R)). Figure 5D shows the percent inhibition of Compound A as a single agent and in combination with dacomitinib, gefitinib and afatinib at selected concentrations.

Figure 6 shows the dose response curve in the RPC9 colony 6 cell survival assay. Figure 6A shows the dose response curve for Compound B (〝 compound B〞) alone and in combination with dacomitinib (〝daco〞). Figure 6B shows the dose response curve for Compound B alone and in combination with gefitinib. Figure 6C shows the dose response curve for Compound B alone and in combination with afatinib (〝afat(R)). Figure 6D shows the percent inhibition of Compound B as a single agent and in combination with dacomitinib, gefitinib and afatinib at selected concentrations.

Figure 7 shows Western immunoblots of the degree of phosphorylation of EGFR, AKT and ERK in RPC9 colony 6 cells. GAPDH was included as a protein loading control. Figure 7A shows RPC9 colony 6 cells treated with a combination of DMSO, dacomtinib, Compound A or Dacomtinib + Compound A (〝 compound A〞). Figure 7B shows RPC9 colony 6 cells treated with a combination of DMSO, erlotinib, Compound A or erlotinib + Compound A (〝 compound A〞).

Figure 8 shows the Western immunological dot method based on the combination of DMSO, dacomtinib, compound A or dacomitinib (〝daco〞) + compound A (〝 compound A〞). The result of the specific gravity measurement of the belt (Fig. 7A). Inhibition of pEGFR Y1068 (Fig. 8A), pAKT S473 (Fig. 8B) and pERK T202/Y204 (Fig. 8C) was determined by comparison to a DMSO control.

Figure 9 shows the Western immunological dot method based on RPC9 colony 6 cells treated with a combination of DMSO, erlotinib, compound A or errozinib + compound A (〝 compound A〞). The result of the specific gravity measurement of the belt (Fig. 7B). Inhibition of pEGFR Y1068 (Figure 9A), pAKT S473 (Figure 9B), and pERK T202/Y204 (Figure 9C) was determined by comparison to the DMSO control.

Figure 10 shows Western immunological dot method for the degree of phosphorylation of EGFR, AKT and ERK in RPC9 colony 6 cells. GAPDH was included as a protein loading control. Figure 10A shows RPC9 colony 6 cells treated with a combination of DMSO, dacomtinib, compound B or dacomtinib + compound B (indole compound B). Figure 10B shows RPC9 colony 6 cells treated with a combination of DMSO, erlotinib, Compound B or erlotinib + Compound B (〝 compound B〞).

Figure 11 shows the Western immunological dot method based on RPC9 colony 6 cells treated with a combination of DMSO, dacomtinib, compound B or dacomitinib (〝daco〞) + compound B (〝 compound B〞). Belt 10A) The results of the specific gravity measurement method. Inhibition of pEGFR Y1068 (Fig. 11A), pAKT S473 (Fig. 11B) and pERK T202/Y204 (Fig. 11C) was determined by comparison to a DMSO control.

Figure 12 shows the Western immunological dot method based on RPC9 colony 6 cells treated with a combination of DMSO, erlotinib, compound B or errozinib + compound B (〝 compound B〞). The result of the specific gravity measurement of the belt (Fig. 10B). Inhibition of pEGFR Y1068 (Fig. 12A), pAKT S473 (Fig. 12B) and pERK T202/Y204 (Fig. 12C) was determined by comparison to a DMSO control.

Figure 13 is a graphical representation of the results of a xenograft pattern of SCID mice bearing RPC9 6 tumors, randomized daily to oral vehicle, dacomitinib, Compound A or Dacomtinib. (〝daco〞) + compound A (〝 compound A〞) treatment. Figure 13A is a graph showing tumor volume, which is measured 3 times per week and is represented by a graph of the standard error of the mean and mean values. Fig. 13B is a graph showing the weight of each group, which is recorded daily and the percentage change is represented by a graph of the standard error of the mean and the mean.

Figure 14 is a graphical representation of the results of a xenograft pattern of SCID mice bearing RPC9 colon 6 tumors, randomized daily to oral vehicle, dacomitinib, compound B or dacomitinib. (〝daco〞) + compound B (〝 compound B〞) treatment. Figure 14A is a graph showing tumor volume, which is measured 3 times per week and is represented by a graph of the standard error of the mean and mean values. Figure 14B is a graph showing the weight of each group, which is recorded daily and the percentage change is represented by a graph of the standard error of the mean and mean.

Figure 15 is a diagram showing SCID mice bearing RPC9 colon 6 tumors. As a result of the xenograft mode, mice were randomized daily and treated with buccal, dacomitinib, compound B or dacomitinib (〝daco〞) + compound B (〝 compound B〞). Figure 15A is a graph showing the tumor volume of a single agent treatment group. Figure 15B is a graph showing the tumor volume of the combined treatment group.

Figure 16 is a graphical representation of the results of a xenograft pattern of SCID mice bearing RPC9 6 tumors, randomized daily to oral, erlotinib, Compound A or erlotinib. (〝erlo〞) + compound A (〝 compound A〞) treatment. Figure 16A is a graph showing tumor volume, which is measured 3 times per week and is represented by a graph of the standard error of the mean and mean values. Fig. 16B is a graph showing the weight of each group, which is recorded daily and the percentage change is represented by a graph of the standard error of the mean and the mean.

Detailed description of the invention

The epidermal growth factor receptor (HER/EGFR) family of members/receptors of the human epidermal growth factor receptor includes EGFR/HER-1, HER2/neu/erbB-2, HER3/erbB-3, and HER4/erbB-4.

EGFR inhibitors potently inhibit the common activating mutations of EGFR (L858R and delE746-A750). Common activating mutations are also known as single mutant or single mutant forms. Examples of EGFR inhibitors include gefitinib, erlotinib, iclotinib, fultetanil, lapatinib, nerotinib, afatinib, piritinib, dacomitinib And carnitinib. The monoclonal antibody antibody of EGFR is also an EGFR inhibitor as defined in the present invention. Such as cetuximab and panituzumab.

The EGFR inhibitor can be a reversible or irreversible inhibitor. A reversible inhibitor of the tyrosine kinase domain of the EFGR molecule attaches to the receptor and periodically detaches from the receptor. Gefitinib, erlotinib, iclotinib, vantinidin and lapatinib are examples of reversible EGFR inhibitors. An irreversible inhibitor of the tyrosine kinase domain of the EFGR molecule irreversibly binds to EGFR. Nilotinib, afatinib, rititinib, dacomitinib and carnitinib are examples of irreversible EGFR inhibitors.

An EGFR inhibitor is an inhibitor of at least one member of the HER family. Gefitinib, erlotinib, iclotinib and vantinidin are selective EGFR/HER-1 tyrosine kinase inhibitors (TKI). Cetuximab and panituzumab are monoclonal antibodies specific for EGFR/HER-1.

Pan-HER inhibitors are agents that block diverse members of the HER family. Lapatinib, nerotinib, afatinib, piratinib, dacomitinib, and carnitinib are examples of pan-HER inhibitors. Lapatinib, nerotinib, afatinib and piritinib inhibit EGFR and HER2 members of the HER family. Dacomitinib and carnitinib inhibit EGFR, HER2 and HER4 members of the HER family.

EGFR T790M inhibitors potently inhibit common activating mutations (L858R and delE746-A750) and gatekeeper gene mutations (T790M). The EGFR T790M inhibitors of the invention preferentially inhibit the double mutant forms of EGFR (L858R/T790M and delE746-A750/T790M) more than single mutants (L858R and delE746-A750). Examples of EGFR T790M inhibitors include Go6976, PKC412, AP26113, HM61713, WZ4002, CO-1686 and TAS-2913.

The EGFR T790M inhibitor may be a reversible or irreversible inhibitor. Go6976, PKC412 and AP26113 are examples of reversible EGFR T790M inhibitors. HM61713, WZ4002, CO-1686 and TAS-2913 are examples of irreversible EGFR T790M inhibitors.

The EGFR T790M inhibitor of the present invention also includes 1-{(3R,4R)-3-[({5-chloro-2-[(1-methyl-1H-pyrazol-4-yl)amino]-7H -pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-en-1-one (〝A) , N-methyl-N-[trans-3-({2-[(1-methyl-1H-pyrazol-4-yl)amino]-5-(pyridin-2-yl)-7H- Pyrrolo[2,3-d]pyrimidin-4-yl}oxy)cyclobutyl]prop-2-enylamine (〝 compound B〞), N-[trans-3-({5-chloro-) 2-[(1,3-Dimethyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}amino)cyclobutyl]- N-methylprop-2-enoxime (indole compound C〞), and 1-{(3R,4R)-3-[({5-chloro-2-[(3-methoxy-1-methyl) -1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl }prop-2-en-1-one (anthracene compound D), or a pharmaceutically acceptable salt thereof. Compound A, Compound B, Compound C, and Compound D are examples of irreversible EGFR T790M inhibitors.

The following abbreviations may be used herein: Ac (ethionyl); APCI (atomic pressure chemical ionization); Boc (third butoxycarbonyl); Boc ₂ O (di-tert-butyl dicarbonate); Brett Phos palladium ring ( Chloro[2-(dicyclohexylphosphino)-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl][2-(2-amine Base ethyl)phenyl]palladium(II)); DCC (1,3-dicyclohexylcarbodiimide); DCM (dichloromethane); Deoxo-Fluor ^® (bis-bis(2-methoxy) Ethylethyl)aminothio); DIAD (diisopropyl azodicarboxylate); DIEA (diisopropylethylamine); DIPEA (N,N-diisopropylethylamine); DMAP (4- Dimethylaminopyridine); DMEM (Dulbecco's modified Eagle's medium); DMF (dimethylformamide); DMSO (dimethyl hydrazine); DPPA (diphenyl phosphorazidate); EGTA ([ethylene bis(oxyethylene)]tetraacetic acid); eq (equivalent); Et (ethyl); EtOH (ethanol) EtOAc (ethyl acetate); Et ₂ O (diethyl ether); FBS (fetal calf serum); HATU (2-(7-aza-1H-benzotriazol-1-yl)-1, 1,3,3-tetramethylurea); HEPES(4-(2-hydroxyethyl)-1-piperidyl Ethanesulfonic acid); HMDS (bis(trimethylsulfonyl)amine, also known as hexamethyldiazepine or hexamethyldioxane); HOAc (acetic acid); HPLC (high performance liquid phase) Chromatography); iPr (isopropyl); iPrMgCl (isopropyl magnesium chloride); iPrOH (isopropanol); KHMDS (bis(trimethylsulfonyl) potassium amide); LAH (lithium aluminum hydride) LCMS (liquid chromatography-mass spectrometry); LiHMDS (lithium bis(trimethylsulfonyl) amination); Me (methyl); MeOH (methanol); MeCN (acetonitrile); MTBE (methyl third Butyl ether); N (standard); N/A (not obtained); NaHMDS (sodium bis(trimethylsulfonyl) amination); N/D (not determined); NIS (N-iodosuccinimide) NMM (N-methylmorpholine); NMR (nuclear magnetic resonance); Pd ₂ (dba) ₃ (parade (diphenyleneacetone) dipalladium (0)); PG (protecting group); Ph (phenyl ); PhI(OAc) ₂ (iodobenzene diacetate); PMSF (phenyl methylsulfonium fluoride); psi (pounds per square inch); Rf (retention factor); RPMI (Roswell Park Memorial Institute ( Roswell Park Memorial Institute)) rt (room temperature); sat. (saturated); SCX (strong cation exchange); SEM (2-(trimethylsulfonyl)ethoxymethyl); SEM-Cl (2- (trimethylsulfonyl)ethoxymethyl chloride); SFC (supercritical Fluid Chromatography); TBAF (tetrabutylammonium fluoride); TBDPS (t-butyldiphenylsulfonyl); TBS (t-butyldimethylhydrazino); t-BuXPhos palladium ring (chlorine [ 2-di-t-butylphosphino)-2',4',6'-triisopropyl-1,1'-biphenyl][2-(2-aminoethyl)phenyl)]palladium(II) ; TFA (trifluoroacetate); THF (tetrahydrofuran); TLC (thin layer chromatography); toluene (methylbenzene); toluenesulfonyl (p-toluenesulfonyl); and Xantphos (4,5- Bis(diphenylphosphino)-9,9-dimethyldibenzopyran).

Some specific examples pertain to pharmaceutically acceptable salts of the compounds described herein. The pharmaceutically acceptable salts of the compounds described herein include the acid addition salts and base addition salts thereof.

Some specific examples are also directed to pharmaceutically acceptable acid addition salts of the compounds described herein. Suitable acid addition salts are formed from acids which form non-toxic salts. Non-limiting examples of suitable acid addition salts (i.e., salts containing a pharmacologically acceptable anion) include, but are not limited to, acetate, acid citrate, adipate, aspartate, benzoic acid Salt, besylate, bicarbonate/carbonate, hydrogen sulfate/sulfate, hydrogen tartrate, borate, camphorsulfonate, citrate, cyclohexylamine sulfonate, ethanedisulfonate, Ethane sulfonate, ethane sulfonate, formate, fumarate, glucoheptonic acid Salt, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, hydroxy Ethane sulfonate, lactate, malate, maleate, malonate, methanesulfonate, methanesulfonate, methyl sulfate, naphthate, 2-naphthalene sulfonate, Nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, glucose diphosphate , stearates, succinates, tannins, tartrates, p-toluenesulfonates, tosylates, trifluoroacetates, and xinofoate salts.

Additional specific examples pertain to the base addition salts of the compounds described herein. Suitable base addition salts are formed from the base which forms a non-toxic salt. Non-limiting examples of suitable basic salts include aluminum, arginine, benzathine, calcium, choline, diethylamine, diethanolamine, glycine, lysine, magnesium, meglumine, ethanolamine , potassium, sodium, tromethamine and zinc salts.

The compounds described herein which are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids which can be used in the preparation of the pharmaceutically acceptable acid addition salts of such basic compounds as described herein are those which form non-toxic acid addition salts, for example salts containing a pharmacologically acceptable anion, such as a salt. Acid salt, hydrobromide, hydroiodide, nitrate, sulfate, hydrogen sulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citric acid Salt, acid citrate, tartrate, pantothenate, hydrogen tartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, Glucose Aldehyde, gluconate, formate, benzoate, glutamate, methane sulfonate, ethane sulfonate, besylate, p-toluene sulfonate and dihydroxynaphthalene An acid salt [i.e., 1,1 '-methylene-bis-(2-hydroxy-3-naphthoic acid)] salt. The compounds described herein, including basic moieties such as amine groups, can form pharmaceutically acceptable salts with various amino acids other than those described above.

Chemical bases which can be used as reagents to prepare pharmaceutically acceptable base salts of those compounds in which the compounds described herein are acidic in nature are those which form non-toxic base salts with such compounds. Such non-toxic basic salts include, but are not limited to, those derived from such pharmacologically acceptable cations, such as alkali metal cations (eg, potassium and sodium) and alkaline earth metal cations (eg, calcium and magnesium), Ammonium or water soluble amine addition salts (such as N-methylglucamine-(meglumine) and lower alkanolammonium), and other basic salts of pharmaceutically acceptable organic amines.

The compounds of the specific examples described herein include all stereoisomers (eg, cis and trans isomers) of the compounds described herein and all optical isomers (eg, R and S mirror isomers), and Racemic mixtures of isomers, non-image isomer mixtures, and other mixtures. While all stereoisomers are encompassed within the scope of our patent application, those skilled in the art will recognize that particular stereoisomers may be preferred.

In some embodiments, the compounds described herein can exist in several tautomeric forms, including the enol and imine forms, and the keto and enamine forms, as well as geometric isomers and mixtures thereof. All such tautomeric forms are included within the scope of the specific examples of the invention. Tautomers are present as a mixture of tautomeric groups in solution. In solid form, usually in one form Tautomers predominate. Even though it is possible to illustrate with one tautomer, specific examples of the invention include all tautomers of the compounds of the invention.

Specific examples of the invention also include atropisomers of the compounds described herein. Atropisomer refers to a compound that can be separated into a rotationally hindered isomer.

It is also possible to form half salts of acids and bases, such as hemisulfate and hemicalcium salts.

For a review of suitable salts, see Handbook of Pharmaceutical Salts: Properties, Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002). Methods for making pharmaceutically acceptable salts of the compounds described herein are known to those skilled in the art.

The term oxime solvate as used herein describes a molecular complex comprising a compound described herein and one or more pharmaceutically acceptable solvent molecules (eg, ethanol).

The compounds described herein may also exist in unsolvated as well as solvated forms. Thus, some specific examples pertain to hydrates and solvates of the compounds described herein.

Compounds described herein containing one or more asymmetric carbon atoms may exist in two or more stereoisomers. When the compounds described herein contain an alkenyl group or an alkenyl group, cis/trans (or Z/E) geometric isomers are possible. When the structural isomers can be mutually converted via low energy, tautomerism (tautomerism) can occur. This may be in the form of a proton tautomerism in a compound described herein containing, for example, an imido group, a keto group or a thiol group, or in a compound containing an aromatic moiety. Also known as the form of valence tautomerism. A single compound may exhibit more than one type of isomerism.

Included within the scope of specific examples of the invention are all stereoisomers, geometric isomers and tautomeric forms of the compounds described herein, including compounds exhibiting more than one type of isomerism, and one or more thereof mixture. Also included are acid addition or base salts wherein the counterion is optically active, such as d-lactate or l-isoamine; or a racemate such as dl-tartrate or dl-arginine.

The cis/trans isomers can be separated by conventional techniques well known to those skilled in the art, such as by chromatography and fractional crystallization.

Conventional techniques for the preparation/isolation of individual mirror image isomers include the palm synthesis from a suitable optically pure precursor or the use of, for example, palm oil high pressure liquid chromatography (HPLC) for the racemate (or salt or derivative). Analysis of the object of the object.

Alternatively, the racemate (or racemic precursor) can be combined with a suitable optically active compound (eg, an alcohol, or a base or acid in the case where the compound described herein contains an acidic or basic moiety, such as 1-phenylethylamine or tartaric acid) reaction. The resulting non-image-isomerized mixture can be separated by chromatography and/or fractional crystallization, and one or both of the non-image isomers are converted to the corresponding ones in a manner well known to those skilled in the art. Pure mirror image isomer.

The term "treating" as used herein means reversing, alleviating, inhibiting the progression of a condition or condition to which the term applies or the progression of one or more symptoms of such condition or condition, or preventing a condition or condition or One or more symptoms of a condition or condition, unless otherwise indicated. As made in this article The term "treatment" as used herein refers to the act of treating the treatment as described above, unless otherwise indicated.

Patients to be treated in accordance with the present invention include any warm-blooded animal such as, but not limited to, humans, monkeys or other low-vision primates, horses, dogs, rabbits, guinea pigs or mice. For example, the patient is a human. Those skilled in the art can easily identify individual patients who are suffering from non-small cell lung cancer and need treatment.

The term "additional enthalpy" means that the combination of two compounds or a targeted agent results in the sum of the individual agents. The term "synergy" or "synergy" as used herein means that the combined result of the two agents is greater than the sum of the agents. The amount of synergy is the combined amount of the two agents that cause a synergistic effect.

The optimal range of the effect and the absolute dose range of the components of the effect can be clearly measured by administering different ranges of w/w ratios and doses to the patient in need of treatment to determine one or two Synergistic interaction between components. The complexity and cost of conducting clinical studies on human patients makes it impractical to use this form of testing as the primary mode of synergy. However, synergistic observations of in-tube or in vivo modes predict effects in humans and other species, and exist in vitro or in vivo modes as described herein for measuring synergistic effects, and may also be used The results of the studies were applied to pharmacokinetic/pharmacodynamic methods to predict the range of effective dose to plasma concentration ratios, as well as the absolute doses and plasma concentrations necessary in humans and other species.

In a specific embodiment, the method of the invention relates to a method of treating non-small cell lung cancer comprising administering an effective amount of an EGFR T790M inhibitor A combination of panHER inhibitors is administered to a patient in need thereof, wherein the panHER inhibitor is administered according to a non-standard clinical dosing regimen in an amount sufficient to achieve a synergistic effect. In this particular example, the synergistic combination of the methods of the invention with a targeted therapeutic agent is, in particular, an EGFR T790M inhibitor and a panHER inhibitor.

In a specific embodiment, the method of the present invention relates to a method of treating non-small cell lung cancer comprising administering an effective amount of a combination of an EGFR T790M inhibitor and a low dose of a panHER inhibitor to a patient in need thereof in an amount sufficient to achieve a synergistic effect . In this particular example, the synergistic combination of the methods of the invention with a targeted therapeutic agent is, in particular, an EGFR T790M inhibitor and a panHER inhibitor.

In another embodiment, the method of the present invention relates to a method of treating non-small cell lung cancer comprising administering an effective amount of an irreversible EGFR T790M inhibitor in combination with an effective amount of an EGFR inhibitor to a patient in need thereof, The amount is sufficient to achieve a synergistic effect. In this particular example, the method of the invention is directed to a synergistic combination of targeted therapeutic agents, particularly an irreversible EGFR T790M inhibitor and an EGFR inhibitor.

As used herein, an effective dose refers to an amount of a substance, agent, compound or composition sufficient to prevent or inhibit the progression of tumor cell growth or cancer metastasis in a combination of the invention. The therapeutic or pharmacological effectiveness of the dosage and administration regimen can also be characterized by the ability to induce, enhance, maintain, or prolong remission in a patient undergoing a particular tumor.

As used herein, a non-standard clinical dosing regimen refers to the administration of a single mutant form that effectively inhibits EGFR (L858R and delE746-A750). A regime of a substance, agent, compound or composition, but which is different from the amount or dose typically used in clinical settings. Non-standard clinical dosing regimens include non-standard clinical doses or non-standard dosing schedules.

A low dose guanidine as used herein refers to an amount or dose of a substance, agent, compound or composition effective to inhibit a single mutant form of EGFR (L858R and delE746-A750), but which is typically used in clinical settings. The amount or dose of a lower dose or dose.

Those skilled in the art will be able to determine the appropriate amount or dosage of each compound for administration to a patient in the combination of the invention according to known methods, taking into account factors such as age, weight, general Health, the compound administered, the route of administration, the nature and progression of non-small cell lung cancer requiring treatment, and other agents present.

Implementation of the methods of the invention can be accomplished via a variety of administration protocols. The compounds of the combination of the invention may be administered intermittently, in parallel or sequentially. If necessary, a repeated dosing regimen can be performed to achieve the desired reduction or reduction of cancer cells. In a particular embodiment, the compounds of the combination of the invention can be administered in an intermittent dosing regimen.

Administration of a compound of the combination of the invention can be accomplished by any method that can deliver the compound to the site of action. Such methods include oral, intraduodenal, parenteral (including intravenous, subcutaneous, intramuscular, intravascular or perfusion), topical and rectal administration.

The compounds of the methods or combinations of the invention may be formulated prior to administration. The formulation is preferably adapted to a particular mode of administration. These compounds can be used in this technology The pharmaceutically acceptable carriers are known to be formulated and can be administered in a wide variety of dosage forms known in the art. In making the pharmaceutical compositions of the present invention, the active ingredient is usually mixed with apharmaceutically acceptable carrier or diluted in a carrier or enclosed in a carrier. Such carriers include, but are not limited to, solid diluents or fillers, excipients, sterile aqueous vehicles, and various non-toxic organic solvents. Unit dosage form or pharmaceutical composition includes tablets, capsules (such as gelatin capsules), pills, powders, granules, aqueous and non-aqueous oral solutions and suspensions, diamond ingots, ingots, hard candy tablets, sprays, creams , ointments, suppositories, jelly gels, gels, pastes, lotions, ointments, injectable solutions, elixirs, syrups, and parenteral solutions in containers suitable for subdividing into individual doses.

Parenteral formulations include pharmaceutically acceptable aqueous or non-aqueous solutions, dispersions, suspensions, emulsions, and sterile powders for their preparation. Examples of the carrier include water, ethanol, polyol (propylene glycol, polyethylene glycol), vegetable oil, and injectable organic esters such as ethyl oleate. Fluidity can be maintained by the use of an envelope such as lecithin, an surfactant or by maintaining an appropriate particle size. Exemplary parenteral administration forms include solutions or suspensions of the compounds of the invention in sterile aqueous solutions (for example, aqueous propylene glycol or aqueous dextrose). These dosage forms can be suitably buffered if necessary.

In addition, lubricants such as magnesium stearate, sodium lauryl sulfate and talc are often used for the purpose of tableting. A similar type of solid composition can also be used in soft and hard-filled gelatin capsules. Thus, preferred materials include lactose or milk sugar and high molecular weight polyethylene glycols. When it is desired to use an aqueous suspension and hydrazine Oral administration of the active compound with various sweeteners or flavoring agents, coloring substances or dyes and, if necessary, emulsifiers or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerol or the like Combination) combination.

Methods of preparing various pharmaceutical compositions having a particular amount of active compound are known or apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences (1975), 15th edition of Mack Publishing Company, Easter, Pa.

The invention also relates to a kit comprising a therapeutic agent of the combination of the invention and a written indication of administration of a therapeutic agent. In one embodiment, the written instructions formulate and define a mode of administration of the therapeutic agent, such as a simultaneous or sequential administration of a therapeutic agent of the invention.

The examples and processes provided below further illustrate and exemplify the compounds described herein and methods of preparing such compounds. The scope of the specific examples described herein is not limited in any way by the following examples and methods. In the examples below, the molecular system with a single palm center is present as a racemic mixture unless otherwise noted. Those molecules having two or more palmitic centers are present as a racemic mixture of non-image isomers unless otherwise noted. Single mirror isomers/non-image isomers can be obtained by methods known to those skilled in the art.

In the examples shown, the salt form was occasionally isolated due to mobile phase additives during chromatographic purification based on HPLC. In these examples, salts such as formates, trifluoroacetate, and acetate were separated and tested without further processing. It should be recognized that those skilled in the art can use standard methods (such as using ion exchange columns or using mild aqueous bases). A simple alkaline extraction is carried out to achieve a free base form.

The compounds described herein can generally be prepared by methods known in the art of chemistry, particularly in light of the teachings herein. The specific methods used to make the compounds described herein are provided as further examples of specific properties and are exemplified in the Reaction Schemes and Experimental Sections provided below.

Example Example 1:1-{(3R,4R)-3-[5-chloro-2-(1-methyl-1H-pyrazol-4-ylamino)-7H-pyrrolo[2,3-d Pyrimidin-4-yloxymethyl]-4-methoxy-pyrrolidin-1-yl}propenone trifluoroacetate (also known as 〝1-{(3R,4R)-3-[({5 -Chloro-2-[(1-methyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4 -Methoxypyrrolidin-1-yl}prop-2-en-1-one trifluoroacetate hydrazine and hydrazine 1-((3R,4R)-3-(((5-chloro-2-((1) -methyl-1H-pyrazol-4-yl)amino)-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy)methyl)-4-methoxypyrrolidin-1 -Based)prop-2-en-1-one trifluoroacetate 〞) (manufactured by hydrazine compound A 〞 trifluoroacetate)

Step 1: (3S,4R)-1-Benzyl-4-methoxy-pyrrolidine-3-carboxylic acid A Ester production method

N-(methoxymethyl)-N-(trimethylmethylidenemethyl)-benzylamine (204 g, 2 eq.) was added dropwise to 0 ° C in 2-Me-THF (600 mL). A solution of (E)-3-methoxy-methyl acrylate (50 g, 430.6 mmol) in TFA (6.7 mL). After the addition, the reaction was allowed to warm to room temperature and stirred for 2 hours. The reaction was transferred to a separatory funnel and saturated NaHCO _3, saturated NaCl solution, then dried over Na ₂ SO ₄ and the solvent removed, leaving the crude racemic product became a yellow oil, which on SiO ₂ (10 Purification of %-35% EtOAc / heptanes afforded mp. The mirror image isomer was isolated using a palmitic-SFC (Chiralpak AD-H 4.6 x 250 mm column, 4% MeOH with 0.1% diethylamine, 140 bar, 3.0 mL/min) to give the desired single product. It was confirmed by comparison with known standards (34 g, 31.7% yield). Specific optical rotation [α] _D ²⁷ = +23.8 ° (C = 1.3, MeOH). ^{1 H NMR (400MHz, DMSO-} d6) δ ppm 2.55-2.63 (m, 2H) 2.69 (dd, J = 9.95,6.42Hz, 1H) 2.82-2.88 (m, 1H) 2.90-2.96 (m, 1H) 3.23 (s, 3H) 3.51-3.63 (m, 2H) 3.66 (s, 3H) 4.07-4.12 (m, 1H) 7.22-7.39 (m, 5H). m/z (APCI+) of (C ₁₄ H ₁₉ NO ₃ ) was 250.0 (M+H) ⁺ .

Step 2: Preparation of (3S,4R)-4-methoxy-pyrrolidine-1,3-dicarboxylic acid 1-t-butyl ester 3-methyl ester

A solution of (3S,4R)-1-benzyl-3-methoxy-pyrrolidine-3-carboxylic acid methyl ester (35 g, 140.4 mmol) in ethanol (500 mL) was flushed with nitrogen. Pd(OH) ₂ (2 g, 0.1 eq.) was then added and the mixture was stirred overnight at about 15 psi under a hydrogen atmosphere (via a hydrogen balloon). The reaction was then filtered through celite and di-t-butyl dicarbonate (30.9 g, 1 eq.) was slowly added to the obtained filtrate with stirring. After 1 hour, the reaction was concentrated and the crude material was purified eluting EtOAc EtOAc EtOAc EtOAc Analyze it. The product was partially combined and concentrated to give title compound (35.81 g, 98%). ^{1 H NMR (400MHz, DMSO-} d6) δ ppm 1.39 (s, 9H) 3.17 (br.s., 1H) 3.23-3.28 (m, 4H) 3.35-3.53 (m, 3H) 3.65 (s, 3H) 4.06 (d, J = 4.78 Hz, 1H). The m/z (APCI+) of the product of Boc (C ₇ H ₁₃ NO ₃ ) was subtracted from 160.1 (M+H) ⁺ . Specific optical rotation: [a] D = -12.5 degrees (C = 0.87, MeOH).

Step 3: Preparation of (3R,4R)-3-hydroxymethyl-4-methoxy-pyrrolidine-1-carboxylic acid tert-butyl ester

Lithium borohydride (12.7 g, 4 equivalents) was added in portions to (3S,4R)-4-methoxy-pyrrolidine-1,3-dicarboxylic acid 1-t-butyl ester in THF (600 mL) In a solution of 3-methyl ester (35.81 g, 138.1 mmol), the reaction was then heated to 60 ° C for 4 hours. The reaction was quenched with water at 0 ° C and extracted with EtOAc. The organic layer was washed with saturated NaCl and dried over Na ₂ SO _4. The solvent was removed and the residue purified via SiO ₂ plug tube: Purification (3 EtOAc of 1 / heptane) to give the title compound as a clear oil (29.35 g, 92% of yield). ¹ H NMR (400MHz, CHLOROFORM -d) δ ppm 1.46 (s, 9H) 2.37-2.47 (m, 1H) 3.19 (dd, J = 11.08,5.29Hz, 1H) 3.33 (d, J = 4.03Hz, 4H) 3.50- 3.66 (m, 4H) 3.77-3.83 (m, 1H). The m/z (APCI+) of the product (C ₆ H ₁₃ NO ₂ ) minus Boc was 132.2 (M+H) ⁺ . Specific optical rotation: [a] D = +9.3 degrees (C = 0.86, MeOH).

Step 4: (3R,4R)-3-[5-Chloro-2-(1-methyl-1H-pyrazol-4-ylamino)-7H-pyrrolo[2,3-d]pyrimidine-4 Process for preparing -butyloxymethyl]-4-methoxy-pyrrolidine-1-carboxylic acid tert-butyl ester

Method A: (using microwave heating)

Potassium terp-butoxide (25% w/w in toluene, 1.6 mL, 3.5 mmol) was added to a microwave vial of 2, 4, 5 - 3 in 1,4-dioxane (15 mL) Chloro-7H-pyrrolo[2,3-d]pyrimidine (904 mg, 4.1 mmol) and (3R,4R)-3-hydroxymethyl-4-methoxy-pyrrolidine-1-carboxylic acid A solution of tributyl ester (940 mg, 4.1 mmol). The resulting solution was stirred at ambient temperature for 15 minutes. LCMS showed quantitative formation of (3R,4R)-3-(2,5-dichloro-7H-pyrrolo[2,3-d]pyrimidin-4-yloxymethyl)-4-methoxy-pyrrolidine 1-carboxylic acid tert-butyl ester. 1-Methyl-1H-pyrazol-4-ylamine (474 mg, 4.9 mmol) and t-BuXPhos palladium ring (110 mg, 0.04 mol equivalent) were added to the obtained reaction solution. The reaction mixture was stirred and heated to 100 ° C for 45 minutes using a microwave of normal absorption grade. The reaction mixture was filtered through EtOAc <RTI ID=0.0> The crude material was purified by flash chromatography eluting eluting elut elut ^{1 H NMR (400MHz, DMSO-} d6) δ ppm 11.50 (br.s., 1H) 9.06 (s, 1H) 7.85 (s, 1H) 7.52 (s, 1H) 7.05 (d, J = 2.27Hz, 1H) 4.30-4.53(m,2H)3.86-3.96(m,1H)3.80(s,3H)3.55-3.68(m,1H)3.43-3.53(m,1H)3.24-3.31(m,3H)2.71(br. s., 1H) 1.39 (br.s., 9H). The product of Boc minus C ₁₆ H ₂₀ ClN ₇ O _{2 has an} m/z (APCI+) of 378.1 (M+H) ⁺ with a Cl isotope pattern.

Method B: Using heat to heat

Potassium terp-butoxide (25% w/w in toluene, 80 mL, 167 mmol) was added to a 2, 4, 5- in 1,4-dioxane (100 mL) in a round bottom flask. Trichloro-7H-pyrrolo[2,3-d]pyrimidine (9.28 g, 41.7 mmol) and (3R,4R)-3-hydroxymethyl-4-methoxy-pyrrolidine-1-carboxylic acid A solution of the third butyl ester (9.65 grams, 41.7 millimoles). The resulting reaction solution was stirred at ambient temperature for 30 minutes. LCMS showed quantitative formation of (3R,4R)-3-(2,5-dichloro-7H-pyrrolo[2,3-d]pyrimidin-4-yloxymethyl)-4-methoxy-pyrrolidine 1-carboxylic acid tert-butyl ester. 1-Methyl-1H-pyrazol-4-ylamine (4.86 g, 50.1 mmol) and t-BuXPhos palladium ring (1.1 g, 1.67 mmol, 0.04 mol equivalent) were added to the obtained reaction solution. . The reaction mixture was stirred and heated to 90 ° C in an oil bath for 1 hour. The reaction mixture was then filtered through EtOAc (EtOAc)EtOAcEtOAcEtOAc. The filtrate was evaporated and purified EtOAc EtOAcqqqqqq ^{1 H NMR (400MHz, DMSO-} d6) δ ppm 11.51 (br.s., 1H) 9.07 (s, 1H) 7.86 (s, 1H) 7.52 (s, 1H) 7.06 (d, J = 2.20Hz, 1H) 4.31-4.54(m,2H)3.92(br.s.,1H)3.80(s,3H)3.55-3.68(m,1H)3.44-3,55(m,1H)3.30(d,J=18.34Hz, 3H) 2.72 (br.s., 1H) 1.39 (br.s., 9H). The M/z (APCI+) of C ₂₁ H ₂₈ ClN ₇ O ₄ was 378.2 (M+H) ⁺ with a Cl isotope pattern.

Step 5: [5-Chloro-4-((3R,4R)-4-methoxy-pyrrolidin-3-ylmethoxy)-7H-pyrrolo[2,3-d]pyrimidin-2-yl Method for preparing -(1-methyl-1H-pyrazol-4-yl)-amine trifluoroacetate

Add TFA (10.1 mL, 208 mmol) to (3R,4R)-3-[5-chloro-2-(1-methyl-1H-pyrazole-4) in DCM (60 mL) -ylamino)-7H-pyrrolo[2,3-d]pyrimidin-4-yloxymethyl]-4-methoxy-pyrrolidine-1-carboxylic acid tert-butyl ester (12.40 g, 26 m The solution was stirred and the resulting solution was stirred at ambient temperature for 2.5 hours. The volatiles were removed and diethyl ether (150 mL) was then evaporated. The resulting suspension was stirred for 2 hours then filtered to give a light pink solid. This was washed with diethyl ether (30 mL). ^{1 H NMR (400MHz, DMSO-} d6) δ ppm 11.56 (br.s., 1H) 9.09 (s, 3H) 7.85 (s, 1H) 7.54 (s, 1H) 7.09 (d, J = 2.32Hz, 1H) 4.48 (d, J = 6.48 Hz, 2H) 4.11 (br.s., 1H) 3.81 (s, 3H) 3.46-3.60 (m, 1H) 3.35-3.45 (m, 2H) 3.32 (s, 3H) 3.15 ( Dq, J = 12.01, 6.02 Hz, 1H) 2.88 (m, J = 6.42, 6.42 Hz, 1H). The m/z (APCI+) of the parent molecule C ₁₆ H ₂₀ ClN ₇ O ₂ was 378.2 (M+H) ⁺ with a Cl isotope pattern.

Step 6: 1-{(3R,4R)-3-[5-Chloro-2-(1-methyl-1H-pyrazol-4-ylamino)-7H-pyrrolo[2,3-d] Process for preparing pyrimidin-4-yloxymethyl]-4-methoxy-pyrrolidin-1-yl}propenone trifluoroacetate

[5-Chloro-4-((3R,4R)-4-methoxy-pyrrolidin-3-ylmethoxy)-7H-pyrrolo[2,3-d]pyrimidin-2-yl]- (l-methyl -1H- pyrazol-4-yl) - amine (15.0 g (2TFA salt), 24.7 mmol) the mixture, ethyl acetate (200 mL) and saturated aqueous NaHCO ₃ (100 ml) at Stir at 0 ° C for 10 minutes. Propylene hydrazine chloride (2.3 ml, 29 mmol, 1.1 mol equivalent) was added dropwise and the resulting mixture was stirred at ambient temperature for 30 min. Ethyl acetate (150 mL) was added and the organic layer was separated. The aqueous layer with ethyl acetate (150 mL), combined organic layers were dried over Na ₂ SO ₄ and evaporated to give a solid which is SFC (ZymorSPHER HAP 5μ 21.2 × 150 mm column, to CO ₂ in The title compound (8.3 g, 78% yield) was obtained as a white solid. ¹ H NMR (400 MHz, DMSO-d6) δ </ RTI></RTI></RTI></RTI></RTI></RTI></RTI></RTI></RTI></RTI></RTI></RTI></RTI><RTIgt; 10.27, 1.34 Hz, 1H) 6.14 (dd, J = 16.75, 2.32 Hz, 1H) 5.68 (dt, J = 10.27, 2.32 Hz, 1H) 4.44 (d, J = 6.24 Hz, 2H) 3.82-4.09 (m, 2H) 3.80 (s, 3H) 3.57-3.76 (m, 2H) 3.47 - 3.54 (m, 1H) 3.31 (d, J = 4.65 Hz, 3H) 2.67 - 2.92 (m, 1H). The m/z (APCI+) of the parent molecule C ₁₉ H ₂₂ ClN ₇ O ₃ was 431.9 (M+H) ⁺ with a Cl isotope pattern.

Alternative Example 1:1-{(3R,4R)-3-[({5-Chloro-2-[(1-methyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[ 2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-en-1-one (also known as 〝1-((3R) , 4R)-3-(((5-chloro-2-((1-methyl-1H-pyrazol-4-yl)amino)-7H-pyrrolo[2,3-d]pyrimidine-4- Method for preparing oxy)methyl)-4-methoxypyrrolidin-1-yl)prop-2-en-1-one oxime)

Step 1: Preparation of (3,4-trans)-1-benzyl-3-methoxypyrrolidin-3-carboxylic acid methyl ester

Trans-methyl-3-methoxyacrylate (500 ml, 540 g, 4.65 mol) with benzylmethylmethoxymethyltrimethyldecylamine (595 ml, 552.1 g, 2.3 mol) The mixture was mixed with magnetic stirring under a nitrogen atmosphere. TFA (2.7 mL, 4.14 grams, 36.3 millimoles) was added to this mixture which resulted in an exotherm to about 95 °C in 30 seconds. The resulting mixture was then heated under reflux for 1 hour (note: at the beginning, the reflux temperature was about 104 ° C, and after 1 hour it dropped to about 90 ° C). Three batches were added on this scale plus another batch using 325 ml of benzylmethylmethoxymethyltrimethyldecylamine compound. The two of the batches were combined and poured into 2N HCl (5 liters). The mixture was extracted with EtOAc (3 liters and 2 liters). Cooling on ice, the aqueous layer came to pH 9 by the addition of 50% NaOH (aqueous). The aqueous layer was extracted with EtOAc (2.5 liters, 1.5 liters and 1 liter). The combined organic layers were washed with brine (3 liters) washed and dried over Na ₂ SO _4. The remaining batches are organized in the same way. All the organic layers were filtered and EtOAcqqqqqq After batch purification by bulb-to-bulb distillation (0.1 mbar, 100 ° C - 145 ° C), the title compound (yield: 69%) was obtained as a yellow oil.

Step 2: Preparation of (3,4-trans)-4-methoxypyrrolidine-3-carboxylic acid methyl ester

Methyl (3,4-trans)-1-benzyl-4-methoxypyrrolidin-3-carboxylate (463.3 g, 1858 mmol) was dissolved in iPrOH (2 liter). 20% Pd(OH) ₂ /C (50 g, 37% moisture, Aldrich) was added to this solution and the mixture was stirred vigorously. An H ₂ pressure of 11.8 bar was applied and refilled several times until ¹ H-NMR showed complete conversion. The mixture was filtered through celite and the celite was rinsed with iPrOH. The filtrate was concentrated in vacuo to give the title compound ( 266 g, Used as is in the next step.

Step 3: Preparation of (2R,3R)-2,3-bis(benzyloxy)-3-carboxypropionic acid (3R,4S)-3-methoxy-4-(methoxycarbonyl)pyrrole

A solution of methyl (3,4-trans)-4-methoxypyrrolidin-3-carboxylate (480 g, 2.84 mol) in ethanol (1 liter) was added to ethanol (5 In liters of O, O-dibenyl-L-tartaric acid (1 kg, 2.79 mol) in a warm solution. A clear solution was inoculated and allowed to crystallize overnight. The resulting solid was separated and washed with ethanol. The enriched material was recrystallized five times from ethanol (2 times 5 liters, 4 liters, 3.5 liters, and 3 liters) to supply 216 grams (15% yield) of salt with 98% of the mirror image isomer Excess (using the following conditions, Rt 12.18 minutes).

Palm purity determination:

Sample preparation: 5 mg of salt was mixed with DCM (1.5 mL) and 2N NaOH (0.2 mL). The DCM layer was dried and analyzed by gas chromatography.

Column: Agilent Cyclosil B; 30 meters × 250 cm × 0.25 cm

Temperature: 90 ° C (0 minutes) to 5 ° C / minute to 180 ° C (4 minutes). The total operating time is 22 minutes.

Injection temperature: 250 ° C

Detector: 250 ° C; FID

Injection volume: 1.0 microliter

Split ratio: 25:1

Column flow rate: 2.2 ml / min (H2)

Step 4: Preparation of (3S,4R)-4-methoxypyrrolidine-1,3-dicarboxylic acid 1-t-butyl 3-methyl ester

The in DCM and saturated NaHCO ₃ (2R, 3R) -2,3- bis (benzyloxy) -3-carboxy-propionic acid (3R, 4S) -3- methoxy-4- (methoxycarbonyl A mixture of pyrrole ingots (216 grams, 420 millimoles) was mechanically stirred and Boc ₂ O (119 grams, 546 millimoles) was added in portions. The mixture was stirred overnight at room temperature and the layers were separated. The aqueous phase was extracted with DCM and the combined organic layers were washed with brine, dried and concentrated. This was fed 138 grams of crude title product (85% yield) mixed with 33% Boc ₂ O. Used directly in the next step.

Step 5: Preparation of (3R,4R)-3-(hydroxymethyl)-4-methoxypyrrolidine-1-carboxylic acid tert-butyl ester

(3S,4R)-4-methoxypyrrolidinium-1,3-dicarboxylic acid 1-tert-butyl 3-methyl ester (138 g, containing Boc ₂ O, about 2:1, ~0.35 mol ) Dissolved in 2 liters of THF. The solution was mechanically stirred and cooled to -78 °C. A lithium aluminum hydride solution (2.4 M in THF, 200 mL, 0.5 mol) was added dropwise over 30 min maintaining the temperature below -70 °C. The temperature is then allowed to rise to -30 °C. A saturated solution of sodium potassium tartrate tetrahydrate (aqueous, 100 ml) was slowly added to terminate the reaction. A solid material via Bed Na ₂ SO ₄ and filtered. The filtrate was concentrated in vacuo to give the title compound <RTI ID=0.0>(</RTI><RTIgt; ¹ H-NMR (300MHz, CDCl3): ppm 3.80 (q, J = 5.1 Hz, 1H), 3.62 (d, J = 6.2 Hz, 2H), 3.56 (m, 1H), 3.52 (d, J = 7.4 Hz) , 1H), 3.40-3.30 (m, 1H), 3.36 (s, 3H), 2.42 (m, 1H), 1.46 (s, 9H). m/z (GCMS) for C ₁₁ H ₂₁ NO ₄ was 231.2 (M) ⁺ . m/z (APCI+) of C ₁₁ H ₂₁ NO ₄ was 132.0 (M+H) ⁺ . Specific optical rotation: [a] D = +11.6 degrees (c 0.77, MeOH). Chiral purity determination: Chiralpak AD-H 21.2 × 250mm 5u column, which is 12% MeOH: 88% CO ₂ mobile analysis of compatibility, and kept at 120 bar at 35 ℃. Flow rate of 62 ml/min. 3.36 minutes of Rt.

Step 6: (3R,4R)-3-[({5-chloro-2-[(1-methyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d Preparation method of pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidine-1-carboxylic acid tert-butyl ester

Potassium tert-butoxide (25% w/w in toluene, 80 mL, 167 mmol) was added to 2,4,5- in 1,4-dioxane (100 mL) in a round bottom flask. Trichloro-7H-pyrrolo[2,3-d]pyrimidine (9.28 g, 41.7 mmol) and (3R,4R)-3-(hydroxymethyl)-4-methoxypyrrolidin-1-carboxylate A solution of acid tert-butyl ester (9.65 grams, 41.7 millimoles). The resulting reaction solution was stirred at ambient temperature for 30 minutes. LCMS showed quantitatively formed intermediate (3R,4R)-3-{[(2,5-dichloro-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy]methyl}-4 - methoxypyrrolidine-1-carboxylic acid tert-butyl ester. 1-methyl-1H-pyrazol-4-ylamine (4.86 g, 50.1 mmol), t-BuXPhos palladium ring (1.1 g, 1.67 mmol, 0.04 mol equivalent) was added to the above reaction solution And the reaction mixture was stirred in an oil bath and heated to 90 ° C for 1 hour. LCMS showed the reaction was completed. The reaction mixture was filtered through EtOAc (EtOAc)EtOAc. The combined filtrate was evaporated to remove the volatiles to give a dark residue. The residue was dissolved in ethyl acetate (300 mL) and filtered th The filtrate was evaporated and purified EtOAc EtOAcqqqqqq ^{1 H NMR (400MHz, DMSO-} d6) δ ppm 11.51 (br.s., 1H) 9.07 (s, 1H) 7.86 (s, 1H) 7.52 (s, 1H) 7.06 (d, J = 2.20Hz, 1H) 4.31-4.54(m,2H)3.92(br.s.,1H)3.80(s,3H)3.55-3.68(m,1H)3.44-3.55(m,1H)3.30(d,J=18.34Hz,3H) 2.72 (br.s., 1H) 1.39 (br.s., 9H). The M/z (APCI+) of C ₂₁ H ₂₈ ClN ₇ O ₄ was 378.2 (M+H) ⁺ with a Cl isotope pattern. Optical rotation: [a] d = -8.3 degrees (c = 0.24, MeOH).

Step 7: (3R,4R)-3-[({5-chloro-2-[(1-methyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d Method for preparing pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrole indium trifluoroacetate

Add TFA (10.1 mL, 208 mmol) to (3R,4R)-3-[({5-chloro-2-[(1-methyl-1H-py) in DCM (60 mL)) Zin-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidine-1-carboxylic acid tert-butyl ester (12.40 g, 26 mmol) solution and the resulting solution was stirred at ambient temperature for 2.5 hours. The volatiles were removed to give a residue and diethyl ether (150 ml) was added. The resulting suspension was stirred for 2 h and EtOAc (EtOAc m.) ^{1 H NMR (400MHz, DMSO-} d6) δ ppm 11.56 (br.s., 1H) 9.09 (s, 3H) 7.85 (s, 1H) 7.54 (s, 1H) 7.09 (d, J = 2.32Hz, 1H) 4.48 (d, J = 6.48 Hz, 2H) 4.11 (br.s., 1H) 3.81 (s, 3H) 3.46-3.60 (m, 1H) 3.35-3.45 (m, 2H) 3.32 (s, 3H) 3.15 ( Dq, J = 12.01, 6.02 Hz, 1H) 2.88 (m, J = 6.42, 6.42 Hz, 1H). The m/z (APCI+) of the parent molecule C ₁₆ H ₂₀ ClN ₇ O ₂ was 378.2 (M+H) ⁺ with a Cl isotope pattern. Optical rotation: [a] d = -4.1 degrees (c = 0.24, MeOH).

Step 8: 1-{(3R,4R)-3-[5-Chloro-2-(1-methyl-1H-pyrazol-4-ylamino)-7H-pyrrolo[2,3-d] Process for producing pyrimidin-4-yloxymethyl]-4-methoxy-pyrrolidin-1-yl}prop-2-en-1-one

(3R,4R)-3-[({5-chloro-2-[(1-methyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidine 4-yl} oxy) methyl] -4-methoxy-pyrrolo ingot trifluoroacetate (15.0 g, 24.7 mmol), ethyl acetate (200 mL) and saturated aqueous NaHCO ₃ (100 ml) The mixture was stirred at 0 ° C for 10 minutes. Propylene hydrazine chloride (2.3 ml, 29 mmol, 1.1 mol equivalent) was added dropwise and the resulting mixture was stirred at ambient temperature for 30 min. Ethyl acetate (150 ml) and the organic layer was separated; the aqueous layer with ethyl acetate (150 mL), the combined organic layers were dried Na ₂ SO ₄ and evaporated to give a solid which was acetic acid via Purification by gradient chromatography of 0-50% ethanol in the ester afforded a white solid. This solid was then recrystallized from ethanol (1 g of crude material in 10 ml of ethanol) and seeded with seed crystals by heating with hot glue. Upon cooling, the title compound (7.47 g, 70%) was obtained as white solid. ^{1 H NMR (400MHz, DMSO-} d6) δ ppm 11.50 (br.s., 1H) 9.06 (s, 1H) 7.85 (s, 1H) 7.51 (s, 1H) 7.04 (d, J = 2.32Hz, 1H) 6.58 (ddd, J=16.78, 10.30, 1.16 Hz, 1H) 6.13 (dd, J = 16.81, 2.38 Hz, 1H) 5.67 (dt, J = 10.3, 2.23 Hz, 1H) 4.43 (d, J = 6.24 Hz, 2H)3.95-4.05(m,1H)3.68-3.85(m,4H)3.56-3.66(m,2H)3.44-3.53(m,1H)3.30(d,J=4.65Hz,3H)2.68-2.90(m , 1H). The m/z (APCI+) of the parent molecule C ₁₉ H ₂₂ ClN ₇ O ₃ is 432.1 (M+H) ⁺ with a Cl isotope pattern. Palm purity assay: Whelk-O1 (R, R) 4.6 x 250 mm column, 30% EtOH at 140 bar, 3 ml/min. Rt = ~ 8.8 minutes, peak 1, > 99% ee. Optical rotation: [a] D22 = -3.1 degrees (c 0.14, EtOH). Elemental analysis: Theoretical value: C, 52.84; H, 5.13; Cl, 8.21.; N, 22.70. Found: C, 52.45; H, 5.38; Cl, 7.91; N, 22.02.

Example 2: N-methyl-N-[trans-3-({2-[(1-methyl-1H-pyrazol-4-yl))amino]-5-(pyridin-2-yl) Method for preparing -7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)cyclobutyl]prop-2-enylamine (indole compound B〞)

Step 1: Preparation of 2,4-dichloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine

N-iodosuccinimide (62.8 grams, 279 millimoles, 1.05 equivalents) was added to 2,4-dichloro in DMF (266 mL, 1.0 M) at such a rate that the internal temperature was maintained below 50 °C. A solution of -7H-pyrrolo[2,3-d]pyrimidine (50.0 g, 266 mmol, 1.00 equivalent). The reaction mixture was stirred vigorously and cooled in an ambient temperature bath over 1.5 h. The reaction mixture was diluted with ice water (1.5 liter) and the obtained precipitate was separated by filtration. The precipitate was washed with EtOAc (EtOAc) (EtOAc). ¹ H NMR (400 MHz, DMSO-d.). δ. m/z (APCI+) of C ₆ H ₂ Cl ₂ IN ₃ was 313.9 (M+H) ⁺ .

Step 2: Preparation of 2,4-dichloro-5-iodo-7-{[2-(trimethylsulfonyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine

2-(Trimethylhydrazinyl)ethoxymethyl chloride (59.5 ml, 337 mmol, 1.30 equivalent) was added dropwise to 2,4-dichloro in THF (600 mL) over 5 min. -5-Iodo-7H-pyrrolo[2,3-d]pyrimidine (81.4 g, 259 mmol, 1.00 equiv) and cooling of diisopropylethylamine (105 ml, 596 mmol, 2.30 equivalent) (0 ° C) in solution. The reaction mixture was allowed to stir at 0 ° C for 3 hours. At this point the reaction mixture was filtered and the filtrate was concentrated in vacuo. The resulting thick oil was diluted with EtOAc (400 mL), washed sequentially with saturated aqueous NH ₄ Cl (2 × 200 mL) and brine (2 × 200 mL), dried over Na ₂ SO ₄ dried and concentrated in vacuo. The material was dissolved in a minimum of DCM (50 mL) and diluted with EtOAc (EtOAc). This solution was concentrated to a total volume of 150 ml which accelerated the wet milling of the title compound. The mixture was filtered to give the title compound <RTI ID=0.0>( </RTI></RTI><RTIgt; ). The two fractions were combined to give the desired product (110.6 g) as an off white solid. _{^{1 H NMR (400MHz, CDCl 3}} ) δ ppm 7.50 (s, 1H), 5.57 (s, 2H), 3.61 (t, J = 8.0Hz, 2H), 0.95 (t, J = 8.0Hz, 2H), 0.01 (s, 9H). m/z (APCI+) of C ₁₂ H ₁₆ Cl ₂ IN ₃ OSi was 444.0 (M+H) ⁺ .

Step 3: 2,4-Dichloro-5-(pyridin-2-yl)-7-{[2-(trimethylindolyl)ethoxy]methyl}-7H-pyrrolo[2,3- d] pyrimidine method

The i-PrMgCl solution (47.3 ml, 94.6 mmol, 1.40 equiv, 1.00 M THF) was added dropwise to 2,4-dichloro-5-iodo-7- in THF (350 mL) over 4 min. Cooling (-78 ° C) of {[2-(trimethylsulfonyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine (30.0 g, 68.0 mmol, 1.00 equivalent) In solution. The reaction mixture was stirred at -78 °C for 2 hours and then freshly prepared with ZnBr ₂ (24.7 g, 110 mmol, 1.62 eq, dried at 130 ° C) in THF (100 mL). 15 minutes. The mixture was stirred at -78 ° C for an additional 1 hour, then warmed to ambient temperature and stirred for an additional 0.5 h. The reaction mixture at this point to Pd (PPh _{3) 4} (3.94 g, 3.38 mmol, 0.05 equiv.) And 2-iodo-pyridine (10.8 mL, 101 mole, 1.50 eq.) And heated to 65 deg.] C over 10 hours . Upon cooling to ambient temperature, the reaction mixture was concentrated to ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ The layers were separated and the aqueous extracted with EtOAc EtOAc. The combined organics brine (300 mL), dried (Na ₂ SO ₄₎ and concentrated in vacuo. The oil was purified by flash chromatography eluting with EtOAc EtOAc (EtOAc) Light brown solid. _{^{1 H NMR (400MHz, CDCl 3}} ) δ ppm 8.76-8.63 (m, 1H), 7.83-7.77 (m, 1H), 7.74 (s, 1H), 7.67 (d, J = 7.8Hz, 1H), 7.35- 7.29 (m, 1H), 5.68 (s, 2H), 3.61 (dd, J = 7.6, 8.9 Hz, 2H), 1.03-0.92 (m, 2H), -0.01 (s, 9H). m/z (APCI+) of C ₁₇ H ₂₀ Cl ₂ N ₄ OSi was 395.1 (M+H) ⁺ .

Step 4: Preparation of (trans-3-{[t-butyl(dimethyl)decyl)oxy}cyclobutyl)-carbamic acid tert-butyl ester

Benzyl (cis-3-hydroxycyclobutyl)aminecarboxylic acid in THF (1.0 liter) (62.0 g, 330 mmol, 1.00 equivalent) and 4-nitrobenzoic acid (60.8 g, 360 mil) Mohr, 1.10 equivalents of cooled (0 ° C) solution in the order of PPh ₃ (130 g, 490 mol, 1.48 equivalents) and diethyl azodicarboxylate (86.3 g, 490 mmol, 1.48 equivalents) deal with. After the addition, the reaction mixture was refluxed for 4 days, cooled to ambient temperature and concentrated in vacuo. The residue was recrystallized from i-PrOH to give a white solid (63 g).

K ₂ CO ₃ (51.6 g, 370 mmol) was added to a solution of the above-obtained 4-nitrobenzoate (63 g) in MeOH (1.0 liter) and H ₂ O (200 mL) . The resulting mixture was refluxed for 2 hours, cooled to ambient temperature and filtered. The filtrate was concentrated in vacuo and partitioned between EtOAc and aqueous 10% Na ₂ CO _3. The resulting organic layer was washed with brine and concentrated to give a white solid (31.0 g).

TBSCl (91.0 g, 600 mmol, 1.50 equivalent) was added to a solution of the above-obtained alcohol (75.0 g, 400 mmol, 1.00 equivalent) in pyridine (1.0 liter). The reaction mixture was stirred at ambient temperature for 2 hours and then concentrated in vacuo. The residue was purified by flash chromatography eluting eluting elut elut ^{1 H NMR (400MHz, DMSO-} d6) δ ppm 7.13 (d, 1H) 4.38-4.47 (m, 1H) 3.90 (br.s., 1H) 2.00-2.16 (m, 4H) 1.36 (s, 9H) 0.81 -0.89 (m, 9H) - 0.01 - 0.01 (m, 6H). The m/z (APCI+) of C ₁₀ H ₂₃ NOSi is 202.1 (M-Boc+H) ⁺ .

Step 5: Preparation of (trans-3-{[t-butyl(dimethyl)decyl)oxy}cyclobutyl)-methylaminecarboxylic acid tert-butyl ester

Add NaH (60% dispersion in oil, 22.2 g, 550 mmol, 1.50 equiv) to THF (1.0 liter) in portions (trans-3-{[t-butyl (dimethyl) a solution of decyl]oxy}cyclobutyl)aminecarboxylic acid tert-butyl ester (111 g, 370 mmol, 1.00 equivalent). After the addition, the reaction mixture was stirred for additional 0.5 h, cooled (0 ° C) and was taken dropwise with methyl iodide (38.2 mL, 615 mM, 1.66 eq.). After an additional 5 hours at ambient temperature, the reaction mixture was taken from EtOAc EtOAc m. 84% yield). ^{1 H NMR (400MHz, DMSO-} d6) δ ppm 4.64 (br.s., 1H) 4.29-4.37 (m, 1H) 2.73 (s, 3H) 2.31-2.43 (m, 2H) 1.97-2.08 (m, 2H ) 1.37 (s, 9H) 0.86 (s, 9H) - 0.01 - 0.05 (m, 6H). m/z (APCI+) of C ₁₁ H ₂₅ NOSi was 216.2 (M-Boc+H) ⁺ .

Step 6: Preparation of (trans-3-hydroxycyclobutyl)methylaminecarboxylic acid tert-butyl ester

TBAF (930 ml, 930 mmol, 1.55 equivalents, 1 M in THF) was added to THF (1.0 liter) (trans-3-{[t-butyl(dimethyl)decyl)oxy A solution of butyl butyl)methylaminecarboxylic acid tert-butyl ester (195 g, 600 mmol, 1.00 equivalent). The reaction mixture was stirred at ambient temperature for 3 hours and then concentrated in vacuo. The resulting residue was partitioned between ₄ Cl (500 mL) EtOAc (1.0 L) and saturated aqueous NH. The resulting organic layer was concentrated with EtOAc EtOAc EtOAc. ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm 4.78 (s, 1H), 4.41-4.38 (m, 1H), 2.82 (s, 3H), 2.41-2.38 (m, 2H), 2.23-2.20 (m, 2H) ), 1.47 (s, 9H). The m/z (ESI+) of C ₁₀ H ₁₈ NO ₃ was 146.1 (M- ^t Bu+H) ⁺ .

Step 7: Methyl {trans-3-[(2-[(1-methyl-1H-pyrazol-4-yl)amino]-5-(pyridin-2-yl)-7-{[2 Process for preparing -(trimethyldecyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy]cyclobutyl}aminecarboxylic acid tert-butyl ester

(trans-3-hydroxycyclobutyl)methylamine in THF (100 ml) was added in three portions of potassium bis(trimethylsulfonyl)amide (12.4 g, 62.3 mmol, 1.12 eq.) A solution of tert-butyl formate (12.9 grams, 64.0 millimoles, 1.15 equivalents) in a cooled solution. After the addition, the alkoxide solution was allowed to warm to ambient temperature over 0.5 hours. 2,4-Dichloro-5-(pyridin-2-yl)-7-{[2-(trimethyldecyl)ethoxy]methyl}-7H-pyrrolo[2,3-d] Pyrimidine (22.0 grams, 55.6 millimoles, 1.00 equivalents) and THF (300 mL) were charged to another flask and cooled (0 ° C). This solution was treated with an alkoxide solution via a catheter for 5 minutes and then at 0 °C for an additional 0.5 hours. The reaction mixture was then diluted with brine (100 mL) EtOAc. The layers were separated and aqueous was extracted with EtOAc EtOAc. Then the organic layers were washed with brine (200 mL), dried (Na ₂ SO ₄₎ and concentrated in vacuo. The resulting viscous oil (31.0 g), which was not further purified, was used in the next step.

1-Methyl-1H-pyrazol-4-amine (7.57 g, 77.9 mmol, 1.40 eq.), Pd ₂ dba ₃ (2.68 g, 2.78 mmol, 0.05 eq.), 4,5-bis ( Diphenylphosphino)-9,9-dimethyldibenzopyran (3.25 g, 5.56 mmol, 0.10 equivalent) and Cs ₂ CO ₃ (45.8 g, 139 mmol, 2.5 equivalent) were added to 1 , a solution of the above obtained oil (31.0 g) in 4-dioxane (300 ml). The reaction mixture was then flushed with a stream of nitrogen for 20 minutes and heated at 105 ° C with vigorous stirring for 10 hours. The reaction mixture was diluted with EtOAc (EtOAc)EtOAc. The residue was purified by flash chromatography eluting eluting elut elut elut ^{1 H NMR (400MHz, DMSO-} d6) δ ppm 9.17 (s, 1H), 8.56 (d, J = 4.3Hz, 1H), 8.14 (d, J = 7.9Hz, 1H), 7.93 (br.s., 1H), 7.87-7.79 (m, 1H), 7.69 (s, 1H), 7.55 (s, 1H), 7.23 (dd, J = 5.0, 7.2 Hz, 1H), 5.57 (br.s., 2H), 5.47 (br.s., 1H), 4.84-4.66 (m, 1H), 3.82 (s, 3 H), 3.63-3.53 (m, 2H), 2.84 (s, 3 H), 2.75-2.62 (m, 2H), 2.47-2.34 (m, 2H), 1.38 (s, 9H), 0.86 (t, J = 8.0 Hz, 2H), -0.11 (s, 9H). m/z (APCI+) of C ₃₁ H ₄₄ N ₈ O ₄ Si was 621.3 (M+H) ⁺ .

Step 8: 3-Chloro-N-methyl-N-{trans-3-[(2-[(1-methyl-1H-pyrazol-4-yl)amino]-5-(pyridine-2 -yl)-7-{[2-(trimethylindolyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy]cyclobutyl}propyl Method for preparing guanamine

TFA (70.0 ml, 914 mmol, 22.7 eq.) was added dropwise to the methyl {trans-3-[(2-[(1-methyl-1H-pyrazole) in MeCN (600 mL)) 4-yl)amino]-5-(pyridin-2-yl)-7-{[2-(trimethylindolyl)ethoxy]methyl}-7H-pyrrolo[2,3-d A solution of pyridyl-4-yl)oxy]cyclobutyl}carbamic acid tert-butyl ester (25.0 g, 40.3 mmol, 1.00 equiv) in a cooled (0 ° C) solution. The reaction mixture was stirred at 0 °C and then allowed to warm to ambient temperature overnight. The reaction mixture was then cooled (0 ° C), adjusted to pH = 8 with aqueous NaOH and the phases were separated. The organic phase (3 × 300 ml) and the aqueous phase was extracted with EtOAc and the combined in saline (2 × 200 ml), washed, dried (Na ₂ SO ₄₎ and concentrated in vacuo. The resulting residue was partially purified by flash chromatography eluting with a gradient of 2-7% MeOH in DCM to afford 4-{[trans-3-(methylamino)cyclobutyl as a yellow gum. ]oxy}-N-(1-methyl-1H-pyrazol-4-yl)-5-pyridin-2-yl-7-{[2-(trimethylindolyl)ethoxy]methyl }-7H-pyrrolo[2,3-d]pyrimidin-2-amine, which was used directly in the next step.

Add 3-chloropropionyl chloride (8.25 ml, 86.4 mmol, 1.51 eq.) to 4-{[trans-3-(methylamino)cyclobutyl in DCM (500 mL). ]oxy}-N-(1-methyl-1H-pyrazol-4-yl)-5-pyridin-2-yl-7-{[2-(trimethylindolyl)ethoxy]methyl A solution of &lt;7H-pyrrolo[2,3-d]pyrimidin-2-amine and DIPEA (16.0 mL, 91.9 mmol, 1.61 eq) was then stirred at ambient temperature for one hour. The reaction mixture was then H ₂ O ₍₂ × 100 ml) and brine (100 mL), dried (Na ₂ SO ₄₎ and concentrated in vacuo. The resulting gum was triturated with EtOAc (EtOAc)EtOAc. ^{1 H NMR (400MHz, DMSO-} d6) δ ppm 9.10-9.25 (m, 1H) 8.51-8.61 (m, 1H) 8.10-8.25 (m, 1H) 7.92-8.05 (m, 1H) 7.82-7.91 (m, 1H) 7.67-7.76(m,1H)7.47-7.60(m,1H)7.17-7.30(m,1H)5.55-5.64(m,2H)5.40-5.54(m,1H)4.63-5.31(m,1H) 3.83(s,3 H)3.74-3.81(m,2H)3.53-3.66(m,2H)2.92-3.08(m,3H)2.81-2.89(m,2H)2.58-2.79(m,2H)2.29-2.46 (m, 2H) 0.75-0.93 (m, 2H) - 10.10 (s, 9H). m/z (APCI+) of C ₂₉ H ₃₉ ClN ₈ O ₃ Si was 611.2 (M+H) ⁺ .

Step 9: N-Methyl-N-[trans-3-({2-[(1-methyl-1H-pyrazol-4-yl)amino]-5-(pyridin-2-yl)- Process for preparing 7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)cyclobutyl]prop-2-enylamine

Add HCl solution (250 mL, 4M in 1,4-dioxane) to 3-chloro-N-methyl-N-{trans-3- in DCM/iPrOH (9:1, 250 mL) [(2-[(1-methyl-1H-pyrazol-4-yl)amino]-5-(pyridine- 2-yl)-7-{[2-(trimethylindolyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)oxy]cyclobutyl} A solution of propiamine (24.0 grams, 39.2 millimoles, 1.00 equivalents). The reaction mixture was stirred overnight at ambient temperature and concentrated in vacuo to give 3-chloro-N-[trans--3-({7-(hydroxymethyl)-2-[(1-methyl-1H-) Pyrazol-4-yl)amino]-5-pyridin-2-yl-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)cyclobutyl]-N-methylpropionate The amine was used in the next step without further purification.

The intermediate from the previous step was dissolved in 1,4-dioxane (80 ml) and concentrated aqueous NH ₄ OH (50 mL). The reaction mixture was stirred at ambient temperature for 3 hours and then concentrated in vacuo. The residue was triturated with EtOAc (EtOAc) (EtOAc)EtOAc. -methyl-1H-pyrazol-4-yl)amino]-5-pyridin-2-yl-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)cyclobutyl]propyl The guanamine was used in the next step without further purification.

K ₂ CO ₃ (21.4 g, 155 mmol, 3.95 eq.) was added to 3-chloro-N-methyl-N-[trans-3-({2-[(1)) in EtOH (400 mL) -methyl-1H-pyrazol-4-yl)amino]-5-pyridin-2-yl-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)cyclobutyl]propyl The solution of the guanamine was stirred overnight at ambient temperature. The reaction mixture was filtered to EtOAc (EtOAc)EtOAc. MTBE (200 mL) was added to precipitate a crude product which was collected by filtration. The filtrate was concentrated and purified by flash chromatography eluting with a gradient of 2-EtOAc MeOH in DCM to afford an additional portion of crude product. The combined crude material via the used 5% MeCN to H ₂ O (0.225% HCOOH) in H ₂ O (0.225% HCOOH) in 25% MeCN performing a gradient elution of YMC-Actus Triact C18 (150 mm × 30 Purification by reverse phase chromatography on a mp EtOAc (m.).

The sequentially added to H ₂ O the title compound (200 mL) formate (5.14 g) in a saturated aqueous solution of NaHCO ₃ (100 mL) and EtOAc (200 mL). The layers were separated and aqueous was extracted with EtOAc EtOAc. The combined organics were dried (Na ₂ SO ₄₎ and concentrated in vacuo to give the title compound as an amorphous solid. A portion of this amorphous solid (~3 g) was dissolved in EtOAc (40 mL). This solution was inoculated with ~5 mg of crystalline product and allowed to stir overnight at ambient temperature. The crystallization flask was cooled (0 ° C) for 1 hour to promote further crystallization. The product was collected by EtOAc (EtOAc) (EtOAcjjjjj Mp = 204.9 °C. ¹ H NMR (400 MHz, DMSO-d6, 30 ° C) δ ppm 11.65 (s, 1H), 8.93 (s, 1H), 8.54 (d, J = 3.9 Hz, 1H), 8.14 (d, J = 8.1 Hz, 1H), 7.86 (s, 1H), 7.82 (t, J = 7.3 Hz, 1H), 7.53 (s, 1H), 7.52 (s, 1H), 7.20 (ddd, J = 1.0, 4.9, 7.4 Hz, 1H) ), 6.71 (br.s., 1H), 6.08 (br.s., 1H), 5.66 (br.s., 1H), 5.53 (br.s., 1H), 5.31-4.79 (m, 1H) , 3.82 (s, 3 H), 3.15-2.93 (m, 3 H), 2.76 (br.s., 2H), 2.46 (br.s., 2H). ¹ H NMR (400 MHz, DMSO-d6, 80 ° C) δ ppm 11.41 (br.s., 1H), 8.59 (s, 1H), 8.55-8.52 (m, 1H), 8.13 (d, J = 8.1 Hz, 1H), 7.84 (s, 1H), 7.80 (dt, J = 1.9, 7.7 Hz, 1H), 7.55 (s, 1H), 7.50 (s, 1H), 7.18 (ddd, J = 1.0, 4.8, 7.4 Hz) , 1H), 6.66 (dd, J = 10.6, 16.8 Hz, 1H), 6.05 (dd, J = 2.3, 16.8 Hz, 1H), 5.63 (dd, J = 2.3, 10.5 Hz, 1H), 5.59-5.53 ( m, 1H), 5.00 (t, J = 8.0 Hz, 1H), 3.82 (s, 3H), 3.04 (s, 3H), 2.82 - 2.71 (m, 2H), 2.55 - 2.45 (m, 2H). m/z (APCI+) of C ₂₃ H ₂₄ N ₈ O ₂ was 445.2 (M+H) ⁺ . Elemental analysis: found: C, 61.96; H, 5.50; N, 24.93. C ₂₃ H ₂₄ N ₈ O ₂ + 0.038 EtOAc + 0.030 eq. EtOH requires C, 62.06; H, 5.49; N, 24.95.

Example 3: N-[trans-3-({5-chloro-2-[(1,3-dimethyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[2, 3-d]pyrimidin-4-yl}amino)cyclobutyl]-N-methylprop-2-enylamine (〝 compound C〞)

Step 1: Method for preparing methyl (cis-3-[1-methyl-1-(trimethylsulfonyl)ethoxy]cyclobutyl}carbamic acid tert-butyl ester

TBSCl (12.7 g, 84 mmol, 1.50 eq.) was added to a solution of &lt;RTI ID=0.0&gt; Equivalent) in solution. After the addition, the mixture was stirred at ambient temperature for 3 hours. TLC (petroleum ether / EtOAc = 3 / 1) showed complete consumption of starting material. The reaction mixture was concentrated and EtOAc EtOAc m. The combined organic layers were concentrated to give the crude crude material: cis-3-[1-methyl-1-(trimethylsulfonyl)ethoxy]cyclobutyl}aminecarboxylic acid tert-butyl ester, which Take Used in the next step without further purification.

The crude material {cis-3-[1-methyl-1-(trimethylsulfonyl)ethoxy]cyclobutyl}carbamic acid tert-butyl ester was diluted in THF (300 mL) to NaH ( 60%, 3.36 grams, 84 millimoles, 1.50 equivalents were batch processed and stirred at room temperature for 30 minutes. The mixture was cooled to 0 ° C and treated with methyl iodide (23.9 g, 168 mmol, 3.0 eq.). After the addition, the reaction mixture was stirred at ambient temperature for 5 hours. TLC (petroleum ether / EtOAc = 10/1) showed that starting material was consumed completely. The reaction mixture was concentrated and purified with EtOAc EtOAc EtOAc EtOAc

Step 2: Preparation of (cis-3-hydroxycyclobutyl)methylaminecarboxylic acid tert-butyl ester

TBAF (22.0 grams, 84 millimoles, 1.50 equivalents) was added in portions to the methyl {cis-3-[1-methyl-1-(trimethylsulfonyl) ethoxylate in THF (200 mL) A solution of tert-butyl butyl carbamate (18 g, 56 mmol, 1.0 eq.). After the addition, the mixture is Stir at ambient temperature for 3 hours. TLC (petroleum ether: EtOAc = 2:1) showed complete consumption of starting material. The reaction mixture was concentrated and EtOAc EtOAcqqqqqqq

Step 3: Preparation of (trans-3-aminocyclobutyl)methylaminecarboxylic acid tert-butyl ester

MsCl (14.1 g, 0.123 mol, 1.38 equiv) was added dropwise to a solution of (cis-3-hydroxycyclobutyl)methylaminecarboxylic acid tert-butyl ester (18.0) in DCM (300 mL) over 30 min. Gram, 0.089 moles, 1.0 eq.) and triethylamine (37 mL, 0.267 mol, 3.0 eq.) in a vigorously stirred cooled (-30 ° C) solution. The reaction mixture was then allowed to warm to ambient temperature and stirred for 1 hour. The reaction mixture was diluted with water (100 mL) and DCM (EtOAc). The organic phase was separated, washed with water (2 × 100 mL), saturated aqueous NH ₄ Cl (3 × 100 ml) and brine (100 mL) washed, dried over anhydrous Na ₂ SO ₄ and concentrated to give become a crude product as a yellow solid Cis-methanesulfonic acid-3-[(tatabutoxycarbonyl)(methyl)amino]cyclobutyl ester, which was used in the next step without further purification.

The crude cis-methanesulfonic acid-3-[(tatabutoxycarbonyl)(methyl)amino]cyclobutyl ester obtained above was dissolved in DMF (250 mL) and NaN ₃ (28.77 g) , 0.44 mol, 5 equivalents). The resulting mixture was then heated to 70 ° C and stirred overnight. After cooling, water (1500 mL) and EtOAc (300 mL) were added to the mixture. The phases were separated and the aqueous extracted with EtOAc EtOAc. The combined organic phases with saturated aqueous NaHCO ₃ (2 × 100 mL), water (2 × 200 ml) and brine (100 mL) washed, dried over anhydrous Na ₂ SO ₄ and evaporated to give crude material (trans-3 -Tributyl butyl azide cyclobutyl)methylaminecarboxylate, which was used directly in the next step.

Above crude acid tert-butyl ester was methylamine (trans-3-azido-cyclobutyl) in MeOH (100 mL) Saturated ₃ NH MeOH (200 mL) was added via syringe to a hydrogen atmosphere under In a mixture with Pd/C (2.5 g). The resulting mixture was stirred at ambient temperature for 36 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. This crude material was purified by column chromatography eluting from EtOAc/EtOAc (EtOAc)

Step 4: Preparation of 2,4,5-trichloro-7-{[2-(trimethylsulfonyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine

N-chloroammonium imine (44.5 g, 332 mmol, 1.05 equivalent) was added to DMF (1800 mL) at ambient temperature as in Example 2, 2,4-dichloro-prepared in Step 1. A solution of 7-{[2-(trimethylsulfonyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine (100.0 g, 316 mmol, 1.0 eq.). The resulting mixture was then stirred at 80 ° C for 3 hours. The reaction mixture was then cooled to ambient temperature and poured into ice water (3 liters). The white precipitate formed was collected and dried in vacuo to give titled compound (99.7 g, 90% yield). ¹ H NMR (400 MHz, CDCl ₃ ) δ ppm = 7.35 (s, 1H), 5.58 (s, 2H), 3.60-3.49 (m, 2H), 1.00-0.89 (m, 2H), -0.02 (s, 9H) ). m/z (APCI+) of C ₁₂ H ₁₆ Cl ₃ N ₃ OSi was 352.0 (M+H) ⁺ .

Step 5: Preparation of 1,3-dimethyl-1H-pyrazole-4-amine

A solution of 1,3-dimethyl-1H-pyrazole-4-amine hydrochloride (800 mg) in MeOH (15 mL) was used as a hydroxide resin (Bio Rad AG 1-X2 resin, catalog #143 -1255) Treatment until pH~8 is obtained. The mixture was stirred for 15 minutes. The resin was filtered off and washed with several portions of MeOH. The filtrate was concentrated under reduced pressure to give title crystall This material was used without further purification. ¹ H NMR (400 MHz, DMSO-d6) δ </ RTI></RTI></RTI></RTI></RTI></RTI></RTI></RTI></RTI></RTI><RTIgt; m/z (APCI+) of C ₅ H ₉ N ₃ is 112.1 (M+H) ⁺ .

Step 6: {trans-3-[(2,5-Dichloro-7-{[2-(trimethyldecyl)ethoxy]methyl}-7H-pyrrolo[2,3-d] Method for preparing pyridin-4-yl)amino]cyclobutyl}methylaminecarboxylic acid tert-butyl ester

(trans-3-aminocyclobutyl)methylaminecarboxylic acid tert-butyl ester (1020 mg, 5.1 mmol, 1.2 equivalents), 2,4,5- in MeCN (21.0 mL, 0.2 M) Trichloro-7-{[2-(trimethylindenyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidine (1500 mg, 4.253 mmol, 1.0 eq.) with DIPEA A mixture of (2.12 ml, 12.8 mmol, 3.0 equivalents) was heated at 80 ° C for 5.5 hours. The reaction mixture was diluted with water and extracted with EtOAc. The organic layer was dried over Na ₂ SO ₄ dried and concentrated. The residue was purified by flash chromatography eluting elut elut elut elut elut ¹ H NMR (400 MHz, DMSO-d6) δ ppm = 7.54 (s, 1H) 7.05 (d, J = 5.99 Hz, 1H) 5.40 (s, 2H) 4.74 (br.s., 1H) 4.41-4.54 (m ,1H)3.45-3.54(m,2H)2.83(s,3H)2.52-2.63(m,2H)2.28-2.42(m,2H)1.40(s,9H)0.78-0.89(m,2H)-0.08( s, 9H). m/z (APCI+) of C ₂₂ H ₃₅ Cl ₂ N ₅ O ₃ Si was 516.2 (M+H) ⁺ .

Step 7: {trans-3-[(5-chloro-2-[(1,3-dimethyl-1H-pyrazol-4-yl)amino]-7-{[2-(trimethyl) Method for preparing decyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}methylaminecarboxylic acid tert-butyl ester

Pd ₂ (dba) ₃ (393 mg, 0.425 mmol, 0.1 eq.), Xantphos (259 mg, 0.425 mmol, 0.1 eq.) and Cs ₂ CO ₃ (4160 mg, 12.8 mmol, 3.0 eq.) Charged with 1,3-dimethyl-1H-pyrazole-4-amine (567 mg, 5.10 mmol, 1.2 eq.) and {trans-3-[(2,5-dichloro-7-{ [2-(Trimethyldecyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}methylaminecarboxylic acid tert-butyl ester (2197 mg, 4.253 mmol, 1.00 equivalent) in a flask. Add 1,4-dioxane (42 mL, 0.1 M) and heat the mixture to 105 °C over 18 h. The reaction was cooled to room temperature and filtered through a pad of Celite. The filtrate was concentrated and the residue was purified EtOAcjjjjjjjjj ^{1 H NMR (400MHz, DMSO-} d6) δ ppm = 8.03 (s, 1H) 7.87 (s, 1H) 7.10 (s, 1H) 6.33 (d, J = 6.24Hz, 1H) 5.35 (s, 2H) 4.67 ( Br.s.,1H)4.55(br.s.,1H)3.68-3.75(m,3H)3.45-3.53 (m,2H)2.85(s,3H)2.52-2.60(m,2H)2.29-2.38( m, 2H) 2.12 (s, 3H) 1.40 (s, 9H) 0.78 - 0.88 (m, 2H) - 10.10 (s, 9H). m/z (APCI+) of C ₂₇ H ₄₃ ClN ₈ O ₃ Si was 591.3 (M+H) ⁺ .

Step 8: N-[trans-3-({5-chloro-2-[(1,3-dimethyl-1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3 -d]pyrimidin-4-yl}amino)cyclobutyl]-N-methylprop-2-enzamide

Adding TFA (31 ml) to {trans-3-[(1,3-dimethyl-1H-pyrazol-4-yl)amino) in DCM (42 mL) ]-7-{[2-(Trimethyldecyl)ethoxy]methyl}-7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}methylamine A solution of tributyl acrylate (1950 mg, 3.30 mmol, 1.0 eq.) in a cooled (0 ° C) solution. The reaction mixture was allowed to come to room temperature and stirred for a further 16 hours. The reaction mixture was then diluted with toluene (30 mL) and concentrated to dry. The resulting crude residue was dissolved and was stirred at ambient temperature for 3 hours in 1,4-dioxane (20 ml) and concentrated aqueous NH ₄ OH (20 mL). The reaction mixture was then evaporated to dryness. The resulting crude solid was partitioned in EtOAc (140 mL) and saturated aqueous Na and with Bing Xixi chloride (0.420 ml, 5.19 mmol, 1.58 eq.) Was stirred vigorously for 1 hour between ₂ CO ₃ (140 mL). The layers were separated and the aqueous extracted with EtOAc EtOAc. The combined organic layers were dried (Na ₂ SO _4), diluted with toluene (30 mL) and evaporated to dryness. The resulting solid was purified using a ZymorSpher HAP 150×21.2 mm column eluting with 20-40% EtOH @ 4%/min, 140 bar, 55 ml/min to afford the title compound (950 mg, 69 % yield). ^{1 H NMR (400MHz, DMSO-} d6,26 ℃) δ ppm = 11.16 (br.s., 1H), 7.80 (br.s., 1H), 7.77 (s, 1H), 6.88 (d, J = 2.4 Hz, 1H), 6.74 (dd, J = 10.5, 16.7 Hz, 1H), 6.32 (br.s., 1H), 6.07 (d, J = 15.9 Hz, 1H), 5.66 (d, J = 10.4 Hz, 1H), 5.30-4.75 (m, 1H), 4.59 (br.s., 1H), 3.71 (s, 3H), 3.15-2.88 (m, 3H), 2.62 (br.s., 2H), 2.40 ( Br.s., 2H), 2.08 (s, 3H). ¹ H NMR (400 MHz, DMSO-d6, 80 ° C) δ ppm = 10.93 (br.s., 1H), 7.73 (s, 1H), 7.41 (br.s., 1H), 6.82 (d, J = 2.2) Hz, 1H), 6.68 (dd, J = 10.5, 16.9 Hz, 1H), 6.16 (d, J = 5.9 Hz, 1H), 6.05 (dd, J = 2.4, 16.8 Hz, 1H), 5.63 (dd, J =2.4,10.5 Hz,1H), 4.94 (t, J=8.0 Hz, 1H), 4.60 (dd, J=4.0, 8.7 Hz, 1H), 3.72 (s, 3H), 3.04 (s, 3H), 2.72 -2.57 (m, 2H), 2.41 (ddd, J = 4.5, 8.9, 13.6 Hz, 2H), 2.10 (s, 3H). m/z (APCI+) of C ₁₉ H ₂₃ ClN ₈ O ₃ was 415.1 (M+H) ⁺ .

Example 4: 1-{(3R,4R)-3-[({5-chloro-2-[(3-methoxy-1-methyl-1H-pyrazol-4-yl)amino]- 7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-en-1-one (〝 compound D〞) Method of production

Prepared in a similar manner to Example 1, substituting 3-methoxy-1-methyl-1H-pyrazole-4-amine for 1-methyl-1H-pyrazol-4-amine and other non-critical Replace.

Important intermediate experiment procedure Process 1: Preparation of 3-(hydroxymethyl)-4-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

Step 1: Method for preparing (2E)-4-{[t-butyl(dimethyl)indenyl]oxy}but-2-enoic acid ethyl ester

The DIEA (2.75 mL, 16.6 mmol) and LiCl (5.54 g, 129 mmol) was added to CH ₃ CN third silicon oxy-butyldimethyl acetaldehyde (3.22 g (40 mL), 18.5 A solution of millimolar) and diethyl phosphinoacetate (4.66 g, 22.2 mmol) and the mixture was stirred at room temperature for 24 hours. The mixture was quenched with EtOAc (EtOAc)EtOAc. The organic layer was dried and concentrated MgSO _4. The residue was purified by flash chromatography eluting elut elut elut ¹ H NMR (400 MHz, chloroform-d) δ ppm 6.91 (dt, J = 15.42, 3.49 Hz, 1H) 6.01 (dt, J = 15.61, 2.27 Hz, 1H) 4.25 (dd, J = 3.27, 2.27 Hz, 2H) 4.12 (q, J = 7.22 Hz, 2H) 1.21 (t, J = 7.18 Hz, 3H) 0.84 (s, 9 H) 0.00 (s, 6 H).

Step 2: Preparation of trans-1-benzyl-4-({[t-butyl(dimethyl)indenyl]oxy}methyl)pyrrolidine-3-carboxylic acid ethyl ester

The TFA (0.280 mL, 3.64 mmol) was added to CH ₂ Cl ₂ at 0 ℃ (2E) -4 (30 mL) - {[tert-butyl (dimethyl) of silicon-based] oxy} Ethyl but-2-enoate (3.27 g, 13.4 mmol) and N-benzyl-1-methoxy-N-((trimethylmethyl)methyl)methylamine (4.14 g, 17.5) In millimoles). The reaction was stirred at room temperature overnight. The mixture was quenched with water (50 mL)EtOAcEtOAc. The combined organic layers were dried with MgSO ₄ and evaporated. The residue was purified by flash chromatography eluting elut elut elut elut elut elut ¹ H NMR (400MHz, CHLOROFORM -d) δ ppm 7.08-7.41 (m, 5H), 4.10 (q, J = 7.13Hz, 2H), 3.42-3.73 (m, 4H), 2.37-2.90 (m, 6H) , 1.22 (t, J = 7.05 Hz, 3H), 0.84 (s, 9H), 0.00 (d, J = 1.26 Hz, 6H).

Step 3: trans-3-ethyl-4-({[t-butyl(dimethyl)decyl)oxy}methyl)pyrrolidine-1,3-dicarboxylic acid-1-third Ester production method

Add Pd(OH) ₂ (300 mg) and Boc ₂ O (1.90 g, 8.61 mmol) to trans-1-benzyl-4-({[t-butyl) in EtOH (40 mL) A solution of ethyl (dimethyl)indolyl]oxy}methyl)pyrrolidine-3-carboxylate (trans mixture) (3.25 g, 8.61 mmol). The mixture was stirred overnight under _{H 2 (50psi, 50 ℃)} . The mixture was filtered through celite and the filtrate was concentrated. The residue was purified by flash chromatography eluting elut elut elut elut elut elut ¹ H NMR (400MHz, CHLOROFORM -d) δ ppm 4.13-4.25 (m, 2H) 3.65 (m, 5H) 3.14-3.29 (m, 1H) 2.84-3.00 (m, 1H) 2.47-2.70 (m, 1H) 1.46 (s, 9H) 1.27 (td, J = 7.11, 2.64 Hz, 3 H) 0.85 - 0.92 (m, 9H) 0.05 (s, 6 H).

Step 4: Preparation of trans-3-({[t-butyl(dimethyl)indenyl]oxy}methyl)-4-(hydroxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

Addition of LiBH ₄ (911 mg, 39.7 mmol) to trans-3-ethyl-4-({[t-butyl(dimethyl)indenyl)oxy} in THF (25 mL) A solution of pyrrolidine-1,3-dicarboxylic acid-1-tert-butyl ester (3.08 g, 7.95 mmol). The mixture was heated to reflux for 3 hours. The reaction mixture was cooled to room temperature then was quenched with water (15 mL) and stirred at room temperature for one hour. The mixture was diluted with water (60 mL) andEtOAcEtOAc The combined organic layers were washed with brine, dried over Na ₂ CH ₄ The crude product was purified by flash chromatography eluting elut elut elut ¹ H NMR (400 MHz, chloroform-d) δ ppm 3.73 (m, 1H), 3.61 (m, 2H), 3.52 (m, 2H), 3.45 (m, 1H), 2.90 - 3.09 (m, 2H), 2.04 - 2.32 (m, 2H), 1.46 (s, 9H), 0.92 (s, 9H), 0.10 (d, J = 1.01 Hz, 6H).

Step 5: trans-3-({[t-butyl(dimethyl)indenyl]oxy}methyl)-4-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester Method of production

The tetrabutylammonium iodide (0.110 g, 0.28 mmol), 50% aqueous NaOH (20 mL) and dimethyl sulfate (0.325 ml, 3.41 mmol) was added to CH ₂ Cl ₂ (20 ml) Trans-3-({[t-butyl(dimethyl)indenyl]oxy}methyl)-4-(hydroxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester (0.982 g, 2.84 millimoles) in solution. The reaction was stirred at room temperature overnight. TLC showed some starting material remaining, so additional dimethyl sulfate (0.150 mL) was added to the reaction mixture and stirred at room temperature for 3 hours. The ₃ OH (30 mL) was added aqueous NH to the reaction mixture and stirred at room temperature for 1 hour. The mixture was diluted with water (20 mL) and with CH ₂ Cl _{2 (2} × 30 mL). The organic layer was dried and concentrated MgSO _4. The residue was purified by flash chromatography eluting elut elut elut elut elut elut ¹ H NMR (400MHz, CHLOROFORM -d) δ ppm 3.60-3.70 (m, 1H), 3.55 (br.s., 2H), 3.37-3.48 (m, 1H), 3.34 (m, 4H), 3.05-3.23 (m, 2H), 2.22-2.40 (m, 1H), 2.07-2.21 (m, 1H), 1.43-1.49 (m, 9H), 0.89 (s, 9H), 0.05 (s, 6H).

Step 6: Preparation of trans-3-(hydroxymethyl)-4-(methoxymethyl)pyrrolidine-1-carboxylic acid tert-butyl ester

TBAF (1.0 M in THF, 2.45 mL, 2.45 mmol) was added to THF (5 [t-butyl (dimethyl) decyl) oxy) in THF (5 mL) A solution of tert-butyl 4-(methoxymethyl)pyrrolidine-1-carboxylate (290 mg, 0.81 mmol). The mixture was stirred at room temperature for 1 hour. The mixture was quenched with water and extracted with EtOAc. The organic layer was dried and concentrated MgSO _4. The crude product, which was not purified, was used in the subsequent step.

Process 2: Preparation of (trans-3-aminocyclobutyl)methylaminecarboxylic acid tert-butyl ester

Step 1: Method for preparing 3-methylenecyclobutanecarboxylic acid

Potassium hydroxide (264 grams, 4.7 moles) was added to a solution of 3-methylenecyclobutanecarbonitrile (110 grams, 1.18 moles) in ethanol (500 ml) and water (500 ml) and The resulting mixture was refluxed overnight. The ethanol was removed under reduced pressure, then the solution was cooled to below 10 °C and acidified to pH 1 with concentrated HCl. The mixture was extracted with EtOAc (EtOAc (EtOAc)EtOAc.

Step 2: Preparation of (3-methylenecyclobutyl)aminecarboxylic acid tert-butyl ester

The DPPA (574 g, 1.41 mole) was added dropwise to tert-butanol (1 liter) of 3-methyl cyclobutane carboxylic acid (132 g, 1.17 mole), and Et ₃ N (178 g, The solution was 1.76 moles and the resulting mixture was refluxed overnight. The mixture was then quenched with water (100 mL). After removal of tert-butanol, the residue was treated with saturated NH ₄ Cl (500 mL) and the resulting solid precipitate was collected, washed with saturated NH ₄ Cl and saturated NaHCO _3, to give the title compound as a white solid (165 g , 77% yield).

Step 3: Preparation of (3-ketocyclobutyl)aminecarboxylic acid tert-butyl ester

Add O ₃ at -78 ° C to a solution of (3-methylenecyclobutyl)aminecarboxylic acid tert-butyl ester (165 g, 0.91 Mo) in CH ₂ Cl ₂ (1000 mL) and MeOH (1000 mL) In the solution of the ear, until the solution turns blue. TLC (petroleum ether: EtOAc = 10:1) showed complete consumption of starting material. Nitrogen was then bubbled through the reaction to remove excess O _3, and then the mixture is quenched to Me ₂ S (200 mL) and stirred for 1 hour. The solution was concentrated to give a residue which is washed with water and saturated NaHCO _3, to give the title compound as a white solid (118 g, 70% of yield).

Step 4: Preparation of (cis-3-hydroxycyclobutyl)aminecarboxylic acid tert-butyl ester

A solution of lithium tri-t-butyl borohydride (648 ml, 1 M) in THF was added dropwise over 1.5 hours to (3-ketocyclobutyl)aminecarboxylic acid in THF (2000 mL). A solution of tributyl ester (100 grams, 54 millimoles). The resulting solution was allowed to warm to room temperature and stirred for an additional 1 hour. TLC (petroleum ether: EtOAc = 2:1) showed complete consumption of starting material. The reaction in aqueous suspension of NH ₄ Cl. Water (1000 mL) and EtOAc (2000 mL) were added to the mixture. The organic layer was separated, dried over MgSO ₄ dried and concentrated to give the crude material which was of from 10: purified by column chromatography EtOAc in operation, to supply the title compound as a white solid (62: 1 to 1: 2 petroleum ether of Grams, 61% yield).

Step 5: Preparation method of {cis-3-[1-methyl-1-(trimethyldecyl)ethoxy]cyclobutyl}carbamic acid tert-butyl ester

TBSCl (159 grams, 1.056 moles) was added to a solution of (cis-3-hydroxycyclobutyl)aminecarboxylic acid tert-butyl ester (62 grams, 0.33 moles) in pyridine (1 liter). After the addition, the mixture was stirred overnight at ambient temperature. TLC (petroleum ether: EtOAc = 2:1) showed complete consumption of starting material. The reaction was then concentrated, diluted with EtOAc (1 liter), the organic layer was separated, and water (3 × 300 ml) and brine (200 mL) washed and dried over MgSO _4, filtered and concentrated to dryness to give the crude title compound ( 108 g) which was used directly in the next step without further purification.

Step 6: Preparation of methyl (cis-3-[1-methyl-1-(trimethylsulfonyl)ethoxy]cyclobutyl}carbamic acid tert-butyl ester

Add NaH (60% in oil, 39.6 g, 0.99 mol) to the crude material in THF (1 liter) (cis-3-[1-methyl-1-(trimethylmethyl)) A solution of butyl ethoxy]cyclobutyl}carbamic acid tert-butyl ester (108 g) was added and the resulting mixture was stirred at room temperature for 30 min. The mixture was then cooled to 0 ° C and methyl iodide (140.58 g, 0.99 m) was added dropwise. After the addition, the mixture was stirred from 0 ° C to room temperature overnight. The mixture was suspended with saturated NH ₄ Cl, water (200 mL) and extracted with EtOAc. The organic layer was washed brine, dried over Na ₂ SO _4, then evaporated to give crude product, which was purified by silica gel chromatography technique, afforded the title compound as an oil (68.9 g, 87% of yield).

Step 7: Preparation of (cis-3-hydroxycyclobutyl)methylaminecarboxylic acid tert-butyl ester

TBAF (62 g, 0.24 mol) was added in portions to methyl {cis-3-[1-methyl-1-(trimethyldecyl)ethoxy]cyclobutane in pyridine (800 mL) a solution of tert-butyl carbamate (68.9 g, 0.217 mol). After the addition, the mixture was stirred at room temperature for 2 hours. The mixture was evaporated to dryness, the residue was dissolved in 1000 ml of ethyl acetate and a concentrated NH ₄ Cl (3 × 200 mL). The organic layer was dried over Na ₂ SO _4, filtered and concentrated to give the crude product which was purified from column chromatography EtOAc 1/20 to 1/5 of the operation / petroleum ether, to be supplied to the title compound as a white solid (26.3 grams, 60% yield).

Step 8: Preparation of cis-methanesulfonic acid 3-[(tatabutoxycarbonyl)(methyl)amino]cyclobutyl ester

Triethylamine (4.14 ml, 29.79 mmol) was added to CH ₂ Cl ₂ (cis-3-hydroxy-cyclobutyl) methylamine acid tert-butyl ester (30 mL) (2.0 g, 9.93 mmol The solution of the molars was cooled to -30 °C with vigorous stirring. Methanesulfonium chloride (1.36 grams, 11.91 millimoles) was added dropwise over 10 minutes. The mixture is then allowed to warm to room temperature and stirred for 1 hour, until TLC analysis _{(MeOH / CH 2 Cl 2 =} 1/15) until the reaction was complete display. Then the reaction mixture with water (2 × 10 mL), aqueous NH ₄ Cl (10 mL), brine (10 mL) washed, dried over anhydrous Na ₂ SO ₄ dried and concentrated to give the title compound as a yellow solid (2.5 g , 91% yield), which was used directly in the next step.

Step 9: Preparation of (trans-3-azidocyclobutyl)methylaminecarboxylic acid tert-butyl ester

Cis-methanesulfonic acid 3 - [(tert-butoxy carbonyl) (methyl) amino] cyclobutyl ester (2.5 g, 8.94 mmol) was dissolved in DMF (25 mL) and was added NaN ₃ ( 2.84 grams, 43.69 millimoles). The resulting mixture was then heated to 70 ° C and stirred overnight. After cooling, water (150 mL) was added andEtOAc was evaporated The combined organic phases with water (3 × 20 ml) and brine (20 mL), washed, dried over anhydrous Na ₂ SO _4, then concentrated in vacuo to give the title compound as a yellow liquid (1.8 g, 89% of yield Rate) which was used without further purification.

Step 10: Preparation of (trans-3-aminocyclobutyl)methylaminecarboxylic acid tert-butyl ester

In MeOH (5 mL) (trans-3-azido-cyclobutyl) methylamine carboxylic acid NH ₃ (g) / MeOH (saturated, 50 ml) was added to a hydrogen atmosphere (hydrogen balloon) via syringe under A mixture of a third butyl ester (1.8 grams, 7.95 millimoles) and Pd/C (200 mg). The resulting mixture was stirred at room temperature for 3 h until TLC (EtOAc: petroleum ether = 1:2). The &lt;RTI ID=0.0&gt;&gt;&gt;&gt;&gt;&gt;&gt;

Biological embodiment Example 5: pEGFR Y1068 ELISA assay

To describe the effect of EGFR T790M inhibitors in cells with different EGFR mutation status, EGFR at Tyr1068 was determined in cells with wild-type EGFR or EGFR double mutants (L858R+T790M, EGFR delE746-A750+T790M) Phosphorylation inhibition. Department of EGFR phosphorylation of Y1068 phosphorylation by PathScan ^® -EGF receptor (Try1068) sandwich ELISA kit ^{(# 7240, Cell Signaling Technology ®} , Danvers, MA) measurements. The PathScan ^® Phosphorylation-EGF Receptor (Try1068) sandwich ELISA kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects the endogenous content of the phosphorylated-EGF receptor (Tyr1068) protein. The following cell lines were evaluated in this assay: A549 (EGFR wild type, endogenous), NCI-H1975 (EGFR L858R+T790M, endogenous), NIH3T3/EGFR_wild type, NIH3T3/EGFR L858R+T790M and PC9-DRH (EGFR del E746-A750+T790M). NIH/3T3 parental cells, A549 and NCI-H1975 cell lines were purchased from the American Type Culture Collection (Manassas, VA). All cell lines were cultured according to ATCC recommendations. The A549 cell line was grown in RPMI medium (Invitrogen, Carlsbad) supplemented with 10% FBS (Sigma, St Louis, MO) and 1% Penn/Strep (Invitrogen). The NCI-H1975 cell line was grown in RPMI (Invitrogen) supplemented with 10% FBS (Sigma) and 1% Penn/Strep (Invitrogen). The NIH/3T3 cell line was grown in DMEM (Invitrogen) supplemented with 10% newborn calf serum (Invitrogen), and NIH3T3/EGFR mutant cells were grown in complete medium with 5 μg/ml puromycin (Invitrogen). . The PC9-DRH cell line was produced and cultured as described in Example 6. Cytoplasts (pLPCX) with various EGFR constructs were made by GenScript (Piscataway, NJ), and stable pools of NIH/3T3 cells expressing these constructs were made in Pfizer La Jolla. Cell coverage in complete medium (50 μl/well) at the bottom of tissue culture treated clear microtiter plate (#3595, Corning Inc, Corning, NY) and allowed at 37 ° C, 5% CO ₂ Attached overnight. Cells were seeded at the following concentrations: (A549: 40,000/well, NCI-H1975: 40,000/well, NIH3T3: 20,000/well, PC9-DRH: 50,000/well). The next day, a compound dilution tray was prepared in a 96-well transparent V-bottom 0.5 ml polypropylene blocking disk (#3956, Corning, Inc). Each cell was not evaluated for all cell lines. Each compound evaluated was prepared as a DMSO stock solution (10 mM). Compounds on each plate were tested repeatedly on an 11-point serial dilution curve (1:3 dilution). Compound treatment solution (50 microliters) was added to the cell tray from the compound dilution dish. The highest concentration of compound was 1 or 10 [mu]M (final concentration) with a final concentration of 0.3% DMSO (#D-5879, Sigma). The plates were then incubated for 2 hours at 37 ° C, 5% CO ₂ . For the NIH3T3/wild-type assay, cells were serum starved for 24 hours prior to compound treatment; cell lines were treated as described in serum-free medium and then EGF (100 ng/ml, Calbiochem/EMD Chemicals, Gibbstown, NJ) ) Stimulate for 10 minutes. For the A549/wild-type assay, cells were plated in whole serum (10%) medium for 24 hours prior to compound treatment; cell lines were treated as described in whole serum medium followed by EGF (40 ng/ml/hunger) The medium, Invitrogen) was stimulated for 10 minutes. Prepare ice-cold lysis buffer (1x cell lysis buffer in pure water (#9803, Cell Signaling Technology), 1 mM sodium orthovanadate (Na ₃ VO ₄ , #96508, Sigma), 1 mM immediately before the end of the incubation. Phenylmethane sulfonium fluoride (PMSF, 52332, CalBiochem/EMD Chemicals), fully small EDTA-free protease inhibitor mixed lozenge (1 tablet/10 ml, #11836170001, Roche, Indianapolis, IN) and PhosSTOP phosphatase inhibition Mixing tablets (1 tablet/10 ml, #04906837001, Roche)). At the end of 2 hours, the medium was gently shaken off and the cells were washed once with ice cold 1 mM Na ₃ VO ₄ (100 μL/well, Invitrogen) in PBS. The wash solution was then gently shaken off and ice-cold lysis buffer (50 μl/well) was added to the cells. The plate was shaken at 4 ° C for 20-30 minutes to completely dissolve the cells. Sample diluent (50 [mu]l/well) was added to the ELISA plate and the lysate (50 [mu]L) was diluted into the sample diluent in each well of the ELISA plate. The plates were sealed and incubated overnight at 4 °C with shaking. The next day, the wells were washed four times with 1x wash buffer; after the final wash, the plates were wrapped in lint-free paper, then Add detection antibody (green, 100 μl/slot) was added to each well and Incubate at 37 ° C for 1 hour. After incubation, the slots were cleaned as described. Secondary antibodies (red, 100 [mu]l/well) linked to HRP were added to each well and incubated for 30 minutes at 37 °C. After incubation, the slots were cleaned as described. TMB substrate (100 microliters/well) was added to each well and the plates were incubated for 10 minutes at 37 °C or for 30 minutes at the highest room temperature. At the end of the incubation, a stop solution (100 [mu]l/slot) was added to each well and the plate was shaken and shaken for a few seconds. The absorbance at 450 nm was read on a PerkinElmer EnVision Excite Multilabel reader method for detecting absorbance or on a Molecular Devices SpectraMax ³⁸⁴ reader for detecting absorbance within 30 minutes after the addition of the stop solution. The data was analyzed using a four-parameter fit in Microsoft Excel.

The results of the pEGFR Y1068 ELISA assay of the tested compounds are shown in Table 2. In the data table ₅₀ of FIG. 2 T790M_L858R pEGFR ELISA IC 3T3 cell lines of data, unless other indicated.

Example 6: Production and Characterization of RPC9 and PC9-DRH Cells Step 1: Production of RPC9 cells from PC9 cells

In the PC9 parental cells, the EGFR delE746-A750 mutant pair gene was amplified and the wild type EGFR dual gene was not detectable. RPC9 cells were produced using PC9 parental cell cells. PC9 cells were incubated in RPMI 1640 medium supplemented with 10% heat-inactivated FBS at 37 ° C, 5% CO ₂ . To generate EGFR inhibitor resistant cell lines, PC9 cells were initially treated with 0.5 nM dacomtinib. Once the cells were grown to 90% confluency, the cells were split and the drug concentration was doubled. After 6 weeks of treatment with this, PC9 cells were able to grow in 2 nM dacomtinib. A single cell line was produced and 10 further characterizations were selected. Those resistant cells were maintained in growth medium containing 2 μM erlotinib and resistant PC9 was referred to as RPC9.

Step 2: CastPCR analysis

Genomic DNA was extracted from the RPC9 cell line according to the manufacturer's instructions using the Qiagen DNA mini-set and accepted by the manufacturer (ABI) according to the cast PCR (introduction group: Hs00000106_wt and Hs00000105). Data was analyzed via the ABI Mutation Detector software.

Step 3: Cell viability IC ₅₀ determination

3000 RPC9 cells per well were seeded in 90 microliters of growth medium in replicate wells of a 96-well plate (Corning). After 24 hours, cells were treated with dacomitinib or erlotinib in a 10 microliter growth medium in a 3-fold dilution of 11-point titration. The highest final concentration was 10 μM. After 72 hours of treatment, the cell lines were analyzed via CTG assay (Promega) according to the manufacturer's instructions.

Step 4: Characterization of RPC9 cells with the EGFR T790M mutation

Of the 10 colonies generated in step 1, Sanger sequencing identified a C>T mutation in EGFR exon 20, which corresponds to a clinically relevant T790M mutation. The sequences of representative strains, RPC9 strain 3 and strain 6 are shown in Figure 1. Further confirmation by cast PCR revealed 10.2% and 11.9% of the EGFR-pair genes carrying the EGFR T790M mutation in RPC9 plants 3 and 6, respectively (Fig. 1). PC9 cells are very sensitive to dacomitinib (Figure 2A). Even the lowest concentration (0.17 nM) of dacomtinib showed a 96% PC9 cell viability (Fig. 2A). It is not possible to calculate the IC ₅₀ from the dose response curve. RPC9 clones are 3 and 6 to 73 and the lines of the IC ₅₀ 64nM and more resistant (FIG. 2A). When the clones are RPC9 3 and 6 when erlotinib treatment, RPC9 clones are 3 and 6 in cell survival assay PC9 cells displayed increased IC ₅₀ (FIG. 2B) compared to more than 200 times.

Thus, RPC9 cells bearing the EGFR T790M mutation and resistance to EGFR inhibitors such as dacomtinib and erlotinib are produced. RPC9 cells contain a mixture of single mutant (EGFR del E746-A750) and double mutant (EGFR del E746-A750 and T790M) EGFR dual genes, as the EGFR T790M dual gene constitutes approximately 10% of the total EGFR dual gene in RPC9 cells.

Step 5: Production and Characterization of PC9-DRH Cells

In addition to RPC9 cells, PC9-DRH cells (DRH = T790M highly resistant to dacomitinib) were also produced. The RPC9 cell pool, which is highly resistant to 2 nM dacomitinib, was further challenged with daconitini at a concentration increased from 2 nM to 2 μM within 8 weeks, as described in step 1. PC9-DRH cells were maintained in growth medium containing 2 μM dacomtinib as described in step 1. PC9-DRH cells were analyzed as described in steps 2, 3 and 4. PC9-DRH cells contain 70% of their EGFR dual genes as double mutants EGFR del E746-A750 and T790M. RPC9 cells and similar, PC9-DRH-Com cells to erlotinib (IC ₅₀ = 1,651nM), erlotinib (IC _50> 10,000nM) and gefitinib (IC _50> 10,000nM) resistant. When used in the pEGFR Y1068 ELISA assay as described in Example 5, 2 [mu]M dacomtinib was removed from the growth medium and cells were allowed to grow for 36 hours prior to use in ELISA assays.

Example 7: RPC9 cell viability using EGFR T790M inhibitor alone or in combination with dacomitinib or erlotinib

3000 RPC9 cells prepared as in Example 6 per well were seeded in 90 microliters of growth medium in replicate wells of a 96-well plate (Corning). After 24 hours, cells were either EGFR T790M inhibitor, Compound A, Compound B, Compound C or Compound D with or without 4 nM Dacomtinib or 300 nM Erlotinib in 10 μl growth medium Treated in a 3-fold dilution in 11-point titration. The highest final concentration is 10 μM of Compound A, Compound B, Compound C or Compound D. After 72 hours of treatment, the cell lines were analyzed via CTG assay (Promega) according to the manufacturer's instructions.

The steady state plasma-free concentrations of the standard clinical dosing regimen from dacomitinib and erlotinib were 4 nM and 300 nM, respectively. At these concentrations, dacomitinib and erlotinib completely inhibited PC9 parental cell viability (Figures 2A and 2B). None of the drugs significantly inhibited RPC9 cell viability at the same concentration (Figures 2A and 2B).

The combination of Compound A with dacomitinib or erlotinib may inhibit survival in RPC9 colony 6 cells (Figures 3A and 3B). A compound of survival IC ₅₀ when the go-4nM combination with imatinib 17nM and 300nM erlotinib 15nM when erlotinib combination (Table 3). Compared to Compound A in combination with Compound A alone IC ₅₀ decreased survival rate of 11 times. Similarly, when RPC9 colony 6 cells were treated with Compound B, dacomitinib and erlotinib also made RPC9 strain 6 sensitive to Compound B (Figures 4A and 4B). IC ₅₀ Compound B of survival when the go-to 4nM and erlotinib in combination when in combination with erlotinib is erlotinib of 5 nM (Table 3). Compared to Compound B treatment alone reduced survival IC ₅₀ 9.5 fold and 7.6-fold. Importantly, human exposure to Compound A was predicted to be 190 nM, and Compound A at this concentration alone inhibited cell viability by about 40%. The same concentration of Compound A reached the maximum inhibition (83%) when combined with dacomitinib or erlotinib (Figures 3A and 3B). Similarly, the human exposure to Compound B was 90 nM, and Compound B at this concentration alone achieved 64% inhibition. The combination may further achieve up to 84% inhibition (Figures 4A and 4B). Therefore, the combination of dacomitinib or erlotinib enhances the survival rate of Compound A and Compound B. In addition to Compound A and Compound B, Compound C and Compound D also had synergistic effects with clinically relevant concentrations of dacomitinib and erlotinib (Table 3).

Conclusions Compounds that specifically target single-mutant forms of EGFR, such as dacomitinib and erlotinib, use their clinically relevant concentrations to make compounds such as Compound A, Compound B, Compound C, and Compounds D) It is possible to preferentially inhibit double-mutant forms of EGFR in a clinically relevant pattern of EGFR with both double and single mutant forms.

Example 8: Survival rate of RPC9 6 cells using EGFR T790M inhibitor alone or in combination with dacomtinib, gefitinib or afatinib

Using the method of Example 7, cells were treated with one of EGFR T790M inhibitor, Compound A or Compound B with or without 4 nM dacomtinib, 20 nM gefitinib or 20 nM afatinib.

The steady state plasma-free concentration of the standard clinical dosing regimen from dacomitinib was 4 nM. The standard plasma-free concentration of the standard clinical dosing regimen from gefitinib and afatinib was 20 nM.

The combination of Compound A with dacomitinib, gefitinib or afatinib may inhibit survival in RPC9 colony 6 cells (Figures 5A, 5B and 5C). Similarly, when RPC9 colony 6 cells were treated with Compound B, dacomitinib, gefitinib or afatinib also made RPC9 strain 6 sensitive to Compound B (Figures 6A, 6B and 6C). Thus, each of Compound A and Compound B had a synergistic effect with clinically relevant concentrations of dacomitinib, gefitinib, and afatinib (Figures 5 and 6). Similar to the embodiment discussed in Example 7, respectively, when gefitinib and when Nigeria afatinib combination, the survival rate of the IC ₅₀ Compound A 19-fold and 14-fold decrease. When Method A respectively and erlotinib in combination with gefitinib, IC ₅₀ Compound B is reduced 10-fold and 8-fold.

The conclusion is that compounds that specifically target a single mutant form of EGFR (such as dacomitinib, gefitinib or afatinib) use their clinically relevant concentrations to bring compounds (such as Compound A and Compound B) double The double mutant form of EGFR may be preferentially inhibited in clinically relevant patterns of EGFR in both mutant and single mutant forms.

Example 9: Combination of EGFR T790M inhibitor and dacomitinib or erlotinib in a dual gene mixed mode method

Cell culture: RPC9 colony 6 cells were generated and subcultured as described in Example 6. Cells were cultured in RPMI with 10% FBS and maintained at the selected pressure (2 nM dacomitini). The selection pressure used for the experiments was removed and the cells were plated on a 10 cm dish and incubated overnight (37 ° C, 5% CO ₂ ) to achieve a 70-80% fusion rate for treatment.

Treatment: Dacontinib, erlotinib, Compound A and Compound B were dissolved in 100% DMSO. Dacomitinib (4 nM) and erlotinib (300 nM) were used with no plasma exposure from standard clinical dosing regimens. Compound A and Compound B were adjusted to various clinical compound and the IC ₅₀ values of the start up is lower than predicted from the subject's plasma exposure amount in the range of no use. Cells were treated with dacomitinib or erlotinib and/or titrated with Compound A or Compound B, or treated with control (DMSO). The treatment was administered for 6 hours; at the end of the incubation period, the cell pellet was collected and frozen until ready for analysis.

Immunoblots: Cell pellets were supplemented with 1 mM Na ₃ VO ₄ , 1 mM PMSF, 1 mM NaF, 1 mM β-glycerophosphate, protease inhibitor cocktail (Roche, Indianapolis, IN) and phosphatase inhibitor cocktail (Roche) lysis buffer _{(150mM NaCl, 1.5mM MgCl 2,} 50mM HEPES, 10% ^{glycerol, 1mM EGTA, 1% Triton ®} X-100,0.5% NP-40) process. The protein concentration of the cell lysate was determined using a BCA Protein Assay (Pierce/Thermo Fisher Scientific, Rockford, IL) according to the manufacturer's instructions. The protein (10 micrograms) decomposition by SDS-PAGE and transferred to a nitrocellulose membrane ^{(Bio-Rad Criterion TM System,} Hercules, CA) on. The dots are probed with primary antibodies to detect proteins of interest. EGFR, pEGFR Y1068, AKT, pAKT S473, ERK and pERK T202/204 anti-systems were purchased from Cell Signaling Technology, Inc (Danvers, MA). The GAPDH anti-system was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). After incubation with secondary antibodies, the films were visualized by chemiluminescence (Pierce/Thermo Fisher Scientific) and the specific gravity method was performed on a FluorChem Q Imaging System (Protein Simple, Santa Clara, CA).

result

Cells treated with both Dartinib and Compound A showed reduced pEGFR signal, which was consistent with the greater effect of the combination at the two lowest doses relative to single agent therapy (77% of dacomitinib) Inhibition, inhibition of 24% of 10 nM Compound A versus 91% of Dacomtinib + 10 nM Compound A; inhibition of 30% of Compound A with 30% relative to Compound A with Dacomtinib + 30 nM 99% inhibition) (Fig. 7A, 8A). These combinations showed greater inhibition at the lowest dose than the additive pERK signal (34% inhibition with dacomitini, to 10 27% inhibition of nM Compound A relative to 100% inhibition with dacontinib + 10 nM Compound A); and at the highest dose followed by an additive effect (34% inhibition with dacomitini, 64% inhibition of 30 nM Compound A relative to 99% inhibition with dacomitinib + 30 nM Compound A) (Figure 7A, 8C). The higher dose of single agent therapy resulted in greater inhibition of pEGFR and pERK signaling; the additive calculation was limited by the maximum inhibition observed in the assay. In contrast, inhibition of pAKT appeared to be additive at the lowest concentration (37% inhibition with dacomitini, 22% inhibition with 10 nM compound A versus 64% with dacomitini + 10 nM compound A) Inhibition of %); however, inhibition of greater than 60-65% was not achieved, even at higher doses (Fig. 7A, 8B).

Cells treated with both erlotinib and Compound A showed a reduced pEGFR signal at three lowest concentrations of Compound A than the single agent therapy (61% inhibition with erlotinib, 41% inhibition of 10 nM Compound A relative to 86% inhibition with erlotinib + 10 nM Compound A; 27% inhibition with 30 nM Compound A versus 91% with erlotinib + 30 nM Compound A Inhibition; 16% inhibition of 100 nM Compound A versus 95% inhibition with erlotinib + 100 nM Compound A) (Figure 7B, 9A). These treatments showed greater inhibition of the combined pERK signal at the two lowest doses (31% inhibition with erlotinib, 38% inhibition with 10 nM compound A versus erlotinib + 10 nM) 100% inhibition of Compound A; inhibition of 54% inhibition of 30 nM Compound A relative to 100% inhibition with erlotinib + 30 nM Compound A; Figure 7B, 9C). The higher dose of single agent therapy leads to The greater pEGFR and pERK signal inhibition; the additive calculation is limited by the maximum inhibition observed in the assay. In contrast, inhibition of pAKT appears to be greater than the additive value at the lowest concentration of Compound A (18% inhibition with erlotinib, 3% inhibition with 10 nM Compound A versus erlotinib + 10 nM compound) 48% inhibition of A; Figure 7B, 9B); and at the highest dose followed by the additive value (18% inhibition with erlotinib, 31% inhibition with 30 nM compound A versus erlot 49% inhibition of Ni+30 nM Compound A; Figures 7B, 9B), but not greater than 50% inhibition, even at higher doses.

Cells treated with both Dartinib and Compound B showed a reduced effect of the pEGFR signal at the two lowest doses of Compound B compared to the single agent therapy (54% inhibition with dacomitinib, Inhibition of 46% inhibition of 3 nM Compound B versus 81% inhibition with Dacomtinib + 3 nM Compound B; inhibition of 17% with 10 nM Compound B versus 90% with Dacomtinib + 10 nM Compound B Inhibition), and at the highest dose followed by an additive value (54% inhibition with dacomitini, 33% inhibition with 30 nM Compound B versus 90% with Dacomtinib + 30 nM Compound B) Inhibition) (Fig. 10A, 11A). These same cells showed additive pERK signaling inhibition at the lowest dose (57% inhibition with dacomitinib, 55% inhibition with 3 nM compound B versus Dkontinib + 3nM compound B) 100% inhibition; Figures 10A, 11C). The higher dose of single agent therapy resulted in greater inhibition of pEGFR and pERK signaling; the additive calculation was limited by the maximum inhibition observed in the assay. In contrast, inhibition of pAKT does not achieve greater than 72% inhibition, even in At higher doses, and not at all or greater than the additive results (Figures 10A, 11B).

Cells treated with both erlotinib and Compound B showed a greater additive effect of reduced pEGFR signal at all concentrations of Compound B than with single agent therapy (12% inhibition with erlotinib, 3 nM 2% inhibition of Compound B relative to 74% inhibition with erlotinib + 3 nM Compound B; inhibition of 8% with 10 nM Compound B versus 66% with erlotinib + 10 nM Compound B; Inhibition of 22% of Compound B at 30 nM relative to inhibition of 82% of erlotinib + 30 nM of Compound B; inhibition of 60% of 100 nM of Compound B relative to 84% of erlotinib + 10 nM of Compound B Inhibition) (Fig. 10B, 12A). These treatments showed greater inhibition of the combined pERK signal at the lowest dose of Compound B (41% inhibition with erlotinib, 39% inhibition with 3 nM Compound B versus erlotinib + 99% inhibition of 3 nM Compound B; Figure 10B, 12C). The higher dose of single agent therapy resulted in greater inhibition of pEGFR and pERK signaling; the additive calculation was limited by the maximum inhibition observed in the assay. In contrast, inhibition of pAKT appears to be greater than the additive value at the lowest concentration of Compound B (29% inhibition with erlotinib, 15% inhibition with 3 nM Compound B versus erlotinib + 3 nM compound) 54% inhibition of B); however, no greater than 62% inhibition was achieved, even at higher doses (Fig. 10B, 12B).

Example 10: EGFR T790M inhibitor in DPC9 colon 6 (Dacontinib and erlotinib resistance) xenograft mode with dacomitinib or erro Combination of tilini background:

The RPC9 colon 6 cell line was generated from PC9 parental cells as described in Example 6. PC9 parental cells contain EGFR del E746-A750 and are sensitive to treatment with dacomitinib and erlotinib. The RPC9 colon 6 cell line is one of the selected resistant strains produced by increasing the dose of dacomitinib (〝daco〞). RPC9 colon 6 cells contain approximately 10% EGFR del E746-A750 and T790M and 90% EGFR del E746-A750 dual gene. Therefore, in the in vitro assay, RPC9 6 is resistant to Dacotinib/Errotidine single agent treatment due to EGFR delE746-A750 and T790M dual genes, and due to EGFR delE746-A750 dual gene The single agent treatment of Compound A (〝 compound A〞) and Compound B (〝 compound B〞) is resistant. The combination of Compound A or Compound B with clinically relevant concentrations of dacomitinib or erlotinib produces a synergistic effect on cell viability via synergistic inhibition of the EGFR signaling pathway. Therefore, an in vivo animal study was conducted to evaluate whether the combination of Compound A or Compound B with dacomitinib or erlotinib can produce a synergistic antitumor effect in the RPC9 Colon 6 xenograft mode.

method:

4- to 6-week old SCID beige female mice were obtained from Charles River lab and stored in pressurized and ventilated cages at the Pfizer La Jolla animal facility. All studies were approved by the Pfizer Institutional Animal Care and Use Committees. Tumor lines were established by subcutaneous injection of recombinant basal membrane (Matrigel, BD Biosciences) via 1:1 (v/v) suspension of 5 x 10 ⁶ RPC9 colon 6 cells. For tumor growth inhibition (TGI) studies, mice with tumors established up to ~300 mm3 were selected and arbitrarily arranged, followed by instructions for use as a single agent or EGFR T790M inhibitor in combination with dacomitinib or erlotinib The dosage and treatment of the solution. Tumor size was calculated using a vernier caliper and tumor volume using a formula of π/6 x larger diameter x (smaller diameter) ² . The percentage of tumor growth inhibition (TGI%) was calculated as 100 × (1 - ΔT / ΔC). The percentage of tumor regression was calculated as 100 x (1 - ΔT / initial tumor size).

Compound A was formulated into a spray dried dispersion suspension in 0.5% Methocel/pH 7.4 in 20 mM Tris buffer. Compound B was formulated in the form of a lactate solution on the spot with 0.5% Methocel. Dacomtinib was formulated into a 0.1 M lactic acid solution at pH 4.5. The erlotinib is formulated into 40% Captisol ^® . All drugs were formulated and administered at a concentration of 10 ml/kg. Mice with tumors were treated with oral and daily dosing instructions; body weight and health observations were recorded daily.

result: Study I: Combination of Compound A and Dacomtinib

In this study, mice bearing RPC9 colony 6 tumors were arbitrarily arranged and treated with a single dose of Compound A or Dacomtinib, or a combination of Compound A and Dacomtinib. 5 mg/kg of dacomitini gave an average unbound drug concentration of 4 nM in mouse plasma, which was consistent with 45 mg/ Mean clinical exposure of clinical doses in kg/day. The percentage change in body weight is plotted in Figure 13B and indicates that all dose groups in this study are well tolerated with less than 10% body weight loss. Compound A was administered as a single agent at 500 mg/kg, 200 mg/kg, and 50 mg/kg or in combination with 5 mg/kg of dartinib, as shown in Figure 13A. On the 39th day of the study, when the tumor size of the vehicle group reached an average of 1200 cubic millimeters, the single agent treatment of dacomitini produced tumor growth arrest and the single agent treatment of compound A produced dose-dependent tumor growth inhibition, as shown in the figure. Illustrated in 13A and Table 4. The combination of Compound A and dacomitinib in all dose ranges produced complete tumor regression, as exemplified in Table 4.

To further assess the combined effect on tumor regression, mice in the single and combination treatment groups of Compound A at 200 mg/kg and 50 mg/kg continued to receive treatment until day 61 of the study. Tumor growth inhibition and tumor regression were calculated and are illustrated in Table 4. The results indicate that (1) Compound A and dacomitini combine to achieve complete tumor regression in a combination of dose ranges, and (2) tumors in a single-agent treatment group of Compound A at 200 mg/kg and 50 mg/kg. Progression in a dose-dependent manner, which exhibits in vitro resistance that may be driven by the EGFR del E746-A750 dual gene, and (3) tumor progression in the single-agent treatment group of dacomitini, which is presented here Intra-tube resistance driven by EGFR del E746-A750 and T790M dual genes in the RPC9 Resistant 6 xenograft mode.

The treatment period is further extended to assess in vivo resistance of single agent treatments and combination treatments. The monotherapy group of dacomitini continued and was swollen The 74th day when the tumor size reached 1200 cubic millimeters or more was terminated. Similarly, a single dose treatment group of 200 mg/kg of Compound A was continued and terminated on day 95 when the tumor size reached above 1400 cubic millimeters. Thus, tumors in the single-agent treatment group of dacomitrine or Compound A continued to progress and demonstrated that in vivo resistance was similar to in vitro characteristics. The combination of dacomitinib with Compound A in the 50 mg/kg group achieved 100% TGI, and the combination of dacomitinib and 200 mg/kg Compound A maintained tumor regression until the end of study at day 120 As shown in Figure 13A and Table 4.

Study II: Combination of Compound B and Dacomtinib

In this study, mice bearing RPC9 6 tumors were arbitrarily arranged and treated with a single dose of Compound B or Dacomtinib, or a combination of Compound B and Dacomtinib. Dacomtinib is administered at 5 mg/kg and 1.5 mg/kg. Compound B is 50 mg/kg, 15 mg/kg and Administration at 5 mg/kg is shown in Figure 14A. The percentage change in body weight is plotted in Figure 14B and indicates that all dose groups of this study are well tolerated with less than 10% body weight loss. On day 36 of the study, when the tumor size of the vehicle group reached an average of 1000 cubic millimeters, the single agent treatment of dacomitini produced dose-dependent tumor growth inhibition, as exemplified in Figure 15A. Treatment with a single dose of 5 mg/kg and 15 mg/kg of Compound B was not significantly effective due to very low doses, whereas treatment with a single dose of 50 mg/kg of Compound B resulted in 47% of TGI, such as This is shown in Figure 15A. The combination of Compound B with dacomitinib produced a dose-dependent tumor regression, as shown in Figure 15B. Tumor growth inhibition and regression were calculated and are illustrated in Table 5.

Study III: Combination of Compound A and Erlotinib

In this study, mice bearing RPC9 colony 6 tumors were arbitrarily arranged and treated with a single dose of Compound A or erlotinib, or Compound A and A. Combination treatment with rotinib. 25 mg/kg erlotinib gave an average unbound drug concentration of 300 nM in mouse plasma, which met the mean clinical exposure. The percent change in body weight is plotted in Figure 16B and indicates that all dose groups in this study are well tolerated with less than 10% body weight loss. Compound A was administered as a single agent at 400 mg/kg, 200 mg/kg, and 50 mg/kg or in combination with 25 mg/kg erlotinib, as shown in Figure 16A. On the 45th day of the study, when the tumor size of the vehicle group reached more than 1500 cubic millimeters, the single agent treatment of erlotinib produced 51% of tumor growth inhibition and the single agent treatment of compound A produced dose-dependent tumor growth inhibition. As illustrated in Figure 16A and Table 6. Combinations of Compound A with erlotinib in all dose ranges produced tumor regression as exemplified in Table 6.

To further assess the combined effect on tumor regression, mice in the single and combination treatment groups of Compound A at 200 mg/kg and 50 mg/kg continued to receive treatment until the 73rd day of the study. Tumor growth inhibition and tumor regression were calculated and are illustrated in Table 6. The results of Study I in combination with dacomitinib were similar, and the results from this study also showed that the combination of Compound A and erlotinib produced tumor regression in both dose ranges, while Compound A or erlotinib alone Tumors in the treatment group continued to progress, indicating that in vivo resistance was driven by del or del/T790M dual genes.

Thus, it was concluded that Compound A binds to a combination of dacomitinib or erlotinib to achieve synergistic anti-tumor activity to induce tumor regression in a resistant RPC9 colony 6 xenograft mode.

in conclusion:

When treated with Compound A, Compound B, Dacomtinib or Erlotinib as a single agent, the RPC9 6 xenograft pattern with both EGFR delE746-A750 and EGFR delE746-A750/T790M dual genes was shown. The tumor progressed. When treated with a high dose of Compound A and a clinically relevant dose of dacomitinib or erlotinib as a combination therapy, this pattern shows complete regression, and when treated with a combination of low doses of Compound B and dacomitinib, This pattern demonstrates dose-dependent tumor regression. Therefore, current preclinical animal studies have successfully demonstrated that the combination strategy of EGFR T790M selective inhibitors with dacomitinib or erlotinib is based on a possible clinically translatable strategy to develop in the primary EGFR T790M clinical candidate in both NSCLC patients with both sexual and acquired EGFR mutations.

Claims (27)

An irreversible EGFR T790M inhibitor in combination with an epidermal growth factor receptor (EGFR) inhibitor for the preparation of a medicament for the treatment of non-small cell lung cancer in a patient. According to the use of the first aspect of the patent application, wherein the irreversible EGFR T790M inhibitor is 1-{(3R,4R)-3-[({5-chloro-2-[(1-methyl-1H-pyrazole) 4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-ene 1-one or a pharmaceutically acceptable salt thereof. The use according to claim 1 or 2, wherein the EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, and icotinib ), vandetanib, lapatinib, neratinib, afatinib, pelitinib, dacomitinib, Canertinib, cetuximab, and panitumumab, or their pharmaceutically acceptable salts. The use according to claim 1 or 2, wherein the EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, afatinib and dacomtinib, or Such as pharmaceutically acceptable salts. The use according to claim 1 or 2, wherein the EGFR inhibitor is erlotinib or a pharmaceutically acceptable salt thereof. The use according to claim 1 or 2, wherein the EGFR inhibitor is dacomitinib or a pharmaceutically acceptable salt thereof. Use of an EGFR T790M inhibitor in combination with a panHER inhibitor for the preparation of a medicament for the treatment of non-small cell lung cancer in a patient, wherein the panHER inhibitor is administered according to a non-standard clinical dosing regimen (dosing regimen) Give. The use according to item 7 of the scope of the patent application, wherein the non-standard clinical dosing regimen is a non-standard clinical dose or a non-standard dosing schedule. The use according to item 7 of the scope of the patent application, wherein the non-standard clinical dosing regimen is a low dose of a panHER inhibitor. The use according to item 7 of the scope of the patent application, wherein the non-standard clinical dosing regimen is an intermittent dosing regimen. The use according to any one of claims 7 to 10, wherein the EGFR T790M inhibitor is selected from the group consisting of Go6976, PKC412, AP26113, HM61713, WZ4002, CO-1686 and TAS-2913 , or their pharmaceutically acceptable salts. The use according to any one of claims 7 to 10, wherein the EGFR T790M inhibitor is 1-{(3R,4R)-3-[({5-chloro-2-[(1-methyl-)- 1H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}-propyl 2-En-1-one or a pharmaceutically acceptable salt thereof. The use according to any one of claims 7 to 10, wherein the panHER inhibitor is selected from the group consisting of lapatinib, nerotinib, afatinib, pirito Nikon, Dacomtinib and Kanai Tinidil, or their pharmaceutically acceptable salts. The use according to any one of claims 7 to 10, wherein the panHER inhibitor is afatinib or a pharmaceutically acceptable salt thereof. The use according to any one of claims 7 to 10, wherein the panHER inhibitor is dacomitinib or a pharmaceutically acceptable salt thereof. An EGFR T790M inhibitor in combination with an EGFR inhibitor for the preparation of a medicament for the treatment of non-small cell lung cancer in a patient. The use according to claim 16 wherein the EGFR T790M inhibitor is selected from the group consisting of Go6976, PKC412, AP26113, HM61713, WZ4002, CO-1686 and TAS-2913, or a pharmaceutical thereof Acceptable salt. According to the use of claim 16, wherein the EGFR T790M inhibitor is 1-{(3R,4R)-3-[({5-chloro-2-[(1-methyl-1H-pyrazole-4) -yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-ene-1 a ketone or a pharmaceutically acceptable salt thereof. The use according to any one of claims 16 to 18, wherein the EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, iclotinib, and van derita Nipa, lapatinib, nerotinib, afatinib, piratinib, dacomtinib, carnitinib, cetuximab and panituzumab, or their medicine Acceptable salt. The use according to any one of claims 16 to 18, wherein the EGFR inhibitor is selected from the group consisting of: Nie, erlotinib, afatinib and dacomtinib, or their pharmaceutically acceptable salts. The use according to any one of claims 16 to 18, wherein the EGFR inhibitor is erlotinib or a pharmaceutically acceptable salt thereof. A synergistic combination of: (a) an EGFR T790M inhibitor; and (b) an EGFR inhibitor, wherein the component (a) and the component (b) have a synergistic effect. According to the combination of claim 22, wherein the EGFR T790M inhibitor is selected from the group consisting of Go6976, PKC412, AP26113, HM61713, WZ4002, CO-1686 and TAS-2913, or their pharmaceuticals Acceptable salt. According to the combination of claim 22, wherein the EGFR T790M inhibitor is 1-{(3R,4R)-3-[({5-chloro-2-[(1-methyl-1H-pyrazole-4) -yl)amino]-7H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)methyl]-4-methoxypyrrolidin-1-yl}prop-2-ene-1 a ketone or a pharmaceutically acceptable salt thereof. The combination according to any one of claims 22-24, wherein the EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, iclotinib, and van derita Nipa, lapatinib, nerotinib, afatinib, piratinib, dacomtinib, carnitinib, cetuximab and panituzumab, or their medicine Acceptable salt. The combination according to any one of claims 22-24, wherein the EGFR inhibitor is selected from the group consisting of: Nie, erlotinib, afatinib and dacomtinib, or their pharmaceutically acceptable salts. The combination according to any one of claims 22-24, wherein the EGFR inhibitor is erlotinib or a pharmaceutically acceptable salt thereof.