Novel pyrrolopyrimidine compounds as inhibitors of protein kinases

ES 2 618 007 T3

DESCRIPTION

New Pyrrolopyrimidine Compounds as Protein Kinase Inhibitors

Cross reference to related requests

This application claims the priority benefit of US Provisional Application No. 61 / 680,231, filed August 6, 2012, entitled NOVEL EGFR MODULATORS AND USES THEREOF ”, US Provisional Application No. 61 /814,147, filed April 19, 2013, entitled NOVEL PYRROLOPYRIMIDINE COMPOUNDS AS INHIBITORS OF PROTEIN KINASES, ”US Patent Application. No. 13 / 843,554, filed March 15, 2013, titled NOVEL EGFR MODULATORS AND USES THEREOF ”and US Patent Application No. 13 / 917,514, filed June 13, 2013, titled NOVEL EGFR MODULATORS AND USES THEREOF. This application, in certain respects, refers to US Provisional Application No. 61 / 586,718, filed on January 13, 2012, entitled "Heterocyclic Compounds and Uses as Anticancer Agents" and to US Patent Application. . N ° 13 / 740,182, filed on January 12, 2013, entitled HETEROCYCLIC COMPOUNDS AND USES AS ANTICANCER AGENTS.

Technical field

The field of this invention is pharmaceutical compounds, compositions and methods, especially when related to compositions and methods for the treatment of proliferative disorders and other diseases related to dysregulation of a kinase (such as, but not limited to, EGFR (including HER ), Alk, PDGFR, BLK, BMX / ETK, BTK, FLT3 (D835Y), ITK, JAK1, JAK2, JAK3, TEC and TXK) and / or the respective routes.

Previous technique

Protein kinases are a group of enzymes that regulate various important biological processes including cell growth, proliferation, survival, invasion and differentiation, organ formation, tissue repair and regeneration, etc. Protein kinases exert their physiological functions through the catalysis of protein phosphorylation and thereby modulate cellular activities. Because protein kinases have strong effects on cells, their activities are highly regulated. Kinases are activated or deactivated by phosphorylation (sometimes by autophosphorylation), by binding activator proteins or inhibitory proteins, or small molecules, or by controlling their location in cells relative to their substrates. Dysfunctions in kinase activities, arising from genetic abnormalities or environmental factors, are known to be associated with many diseases. Several serious disease states, including cancer and chronic inflammation, are associated with the stimulation of intracellular signaling, and since kinases positively relay signaling events, their inhibition offers a powerful way to inhibit or control signal transduction cascades.

The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / cneu ( ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4). EGFR is the cell surface receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. Mutations that affect EGFR expression or activity could result in cancer. EGFR is upregulated in most types of solid tumors, ie, lung cancer, breast cancer, and brain tumor. Mutations, amplifications, or misregulation of EGFR or family members are estimated to be involved in approximately 30% of all epithelial cancers. Therapeutic approaches have been developed based on the inhibition of EGFR either by an antibody drug or by a small molecular inhibitory drug, such as gefitinib and erlotinib. In the case of non-small cell lung cancer, gefitinib and erlotinib have shown benefit in approximately 10-40% of patients. However, acquired resistance to gefitinib or erlotinib after a period of treatment becomes a major clinical problem. Research has confirmed that a major reason for the resistance developed is due to the presence of a new mutation in T790M, which is the EGFR “gatekeeper”. Subsequently, inhibitors that can defeat this T790M have been developed and showed advantages in clinical experiment, such as BIBW2992. However, these T790M targeting EGFR inhibitors still have relative inhibitory activity towards wild type EGFR, limiting clinical application. There is a need to further develop a more effective type of EGFR inhibitor that targets substantially the mutation and not substantially the wild-type protein.

Other protein kinases that are useful targets for small molecule pharmaceuticals include lymphoid tyrosine kinase B (BLK), Janus kinase 1 (JAK1), bone marrow kinase on the X chromosome (BMX / ETK), Bruton's tyrosine kinase (BTK ), Janus kinase 2 (JAK2), Janus kinase 3 (JAK3), hepatocellular carcinoma expressed tyrosine kinase (TEC), resting lymphocyte kinase (TXK, also known as RLK), FMS-like tyrosine kinase 3 (FLT3 ) and FLT3 (D835Y).

Summary

The present invention is directed to certain pyrrolopyrimidine derivatives and pharmaceutical compositions and to these compounds and compositions for use in the treatment of proliferative and other disorders.

ES 2 618 007 T3

The present disclosure provides a compound of Formula (VIII):

<img file="ES2618007T3_D0001.tif" />

in which

X¹ is O, NH, S, CH₂oCF₂;

R¹ and R² are independently selected from hydrogen, halo, C 1-6 alkyl, and Ci-ei haloalkyl

R³ selected from halo, hydroxyl, C1-6 alkyl, C1-6 alkoxy, cyano, and nitro;

n is a number from zero to 4;

R⁴ selected from hydrogen, C1-6 alkyl, C3-7 cycloalkyl, and -NR²²R²³;

wherein the alkyl or cycloalkyl is unsubstituted or is substituted with hydroxyl or amino; and each of R²² and R²³ is independently selected from hydrogen and C1-6 alkyl or R²² and R²³ they can be linked to form a 3- to 10-membered ring;

R⁵ is selected from hydrogen and Cie alkyl;

R⁶ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁷ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁸ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

Q is CR⁹ or N;

where R⁹ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R¹¹ is selected from hydrogen and Cie alkyl;

R¹² is selected from hydrogen and Cie alkyl;

R¹³ is selected from hydrogen, C1-6 alkyl, C1-6 acyl, SC&gt; 2-alkyl (Ci-6), C3-7 cycloalkyl, and Ce-2o aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, C 1-6 alkoxy or halo; Y

-NR¹⁸R¹⁹ that)

<img file="ES2618007T3_D0002.tif" />

where R¹⁰ is selected from hydrogen and Cie alkyl;

R¹⁵ is unsubstituted methyl or is unsubstituted or hydroxy, methoxy or halo substituted C2-4 alkyl; and m is 1 or 2;

or is (b) where R¹⁹ and R⁹ taken together they form a 5 or 6 membered heteroaryl ring optionally substituted with Cis alkyl which is unsubstituted or substituted with amino, hydroxyl or halo; and R¹⁸ is hydrogen or C1-6 alkyl or is absent to satisfy the valence of the heteroaryl ring;

ES 2 618 007 T3

R¹⁰-N Ν-ί with the condition that none of R⁶ or R⁷ be methoxy when NR¹⁸R¹⁹ either ¡or a pharmaceutically acceptable salt thereof.

The present disclosure provides a compound of Formula (la) and (Ib):

R¹ OR

<img file="ES2618007T3_D0003.tif" />

in which

R¹ and R² are independently selected from hydrogen, halo, Cie alkyl, and Ci-e¡ haloalkyl

R³ selected from halo, hydroxyl, Cie alkyl, Cie alkoxy, cyano, and nitro;

n is a number from zero to 4;

R⁴ selected from hydrogen, Cie alkyl, C3-7 cycloalkyl, and -NR²²R²³;

wherein the alkyl or cycloalkyl is unsubstituted or is substituted with hydroxyl or amine; and each of R²² and R²³ is independently selected from hydrogen and C1-6 alkyl or R²² and R²³ they can be linked to form a 3- to 10-membered ring;

R⁵ is selected from hydrogen and Ci-e¡ alkyl

R⁶ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁷ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁸ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

Q is CR⁹ or N;

R⁹ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R¹⁰ selected from hydrogen and Ci-alkyl is one or two;

Ring A is an aromatic ring;

ES 2 618 007 T3

R²⁰ and R²¹ are independently selected from hydrogen and Ci-e alkyl; wherein the alkyl is unsubstituted or is substituted with amino, hydroxyl, or halo; where R²¹ may be absent as needed to satisfy valence;

R¹¹ is selected from hydrogen and Cie alkyl;

R¹² is selected from hydrogen and Cie alkyl; Y

R¹³ is selected from hydrogen, Cie alkyl, Cie acyl, SC&gt; 2-Cie alkyl, C3-7 cycloalkyl, and Ce-20 aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, C1-6 alkoxy, or halo;

or a pharmaceutically acceptable salt thereof.

The present disclosure provides a compound of Formula (II):

<img file="ES2618007T3_D0004.tif" />

in which

R¹ and R² independently selected from hydrogen, halo, C1-6 alkyl, and Cie haloalkyl;

R³ selected from halo, hydroxyl, C1-6 alkyl, C1-6 alkoxy, cyano, and nitro;

n is a number from zero to 4;

R⁴ selected from hydrogen, C1-6 alkyl, C3-7 cycloalkyl, and -NR²²R²³;

wherein the alkyl or cycloalkyl is unsubstituted or is substituted with hydroxyl or amino; and each of R²² and R²³ is independently selected from hydrogen and C1-6 alkyl or R²² and R²³ they can be linked to form a 3- to 10-membered ring;

R⁵ is selected from hydrogen and Cie alkyl;

R⁶ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁷ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁸ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

Q is CR⁹oN;

R⁹ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R¹⁰ is selected from hydrogen and Cie alkyl;

R¹¹ is selected from hydrogen and Cie alkyl;

R¹² is selected from hydrogen and Cie alkyl; Y

R¹³ is selected from hydrogen, C1-6 alkyl, and Ce-20 aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, C1-6 alkoxy, or halo; or a pharmaceutically acceptable salt thereof.

The present disclosure provides a compound of Formula (III):

ES 2 618 007 T3

<img file="ES2618007T3_D0005.tif" />

where

R¹ and R² independently selected from hydrogen, halo, Cie alkyl, and Ci-e haloalkyl;

R³ selected from halo, hydroxyl, Cie alkyl, Cie alkoxy, clane, and nitro;

n is a number from zero to 4;

R⁴ selected from hydrogen, Cie alkyl, C 3-7 cycloalkyl, and -NR²²R²³;

wherein the alkyl or cycloalkyl is unsubstituted or substituted with hydroxyl or amino; and where each of R²² and R²³ is independently selected from hydrogen and C1-6 alkyl or R²² and R²³ they can be linked to form a 3- to 10-membered ring;

R⁵ selected from hydrogen and Ci-e alkyl;

R⁶ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁷ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁸ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁹ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R¹⁰ selected from hydrogen and Ci-e alkyl;

R¹¹ selected from hydrogen and Ci-e alkyl;

R¹² selected from hydrogen and Ci-e alkyl; Y

R¹³ is selected from hydrogen, C1-6 alkyl, and Ce-20 aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, C1-6 alkoxy, or halo; or a pharmaceutically acceptable salt thereof.

The present disclosure provides a compound of Formula (IV):

<img file="ES2618007T3_D0006.tif" />

where

R¹ and R² are independently selected from hydrogen, halo, C 1-6 alkyl, and Ci-e¡ haloalkyl R³ selected from halo, hydroxyl, C1-6 alkyl, C1-6 alkoxy, cyano, and nitro;

ES 2 618 007 T3 n is a number from zero to 4;

R⁴ selected from hydrogen, Cie alkyl, C3-7 cycloalkyl, and -NR²²R²³;

wherein the alkyl or cycloalkyl is unsubstituted or is substituted with hydroxyl or amine; and where each of R²² and R²³ is independently selected from hydrogen and C1-6 alkyl or R²² and R²³ they can be linked to form a 3- to 10-membered ring;

R⁵ selected from hydrogen and Ci-e alkyl;

R⁶ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁷ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁸ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R¹⁰ selected from hydrogen and Ci-e alkyl;

R¹¹ selected from hydrogen and Ci-e alkyl;

R¹² selected from hydrogen and Ci-e alkyl; Y

R¹³ is selected from hydrogen, C1-6 alkyl, and Ce-20 aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, C1-6 alkoxy, or halo; or a pharmaceutically acceptable salt thereof.

The present disclosure provides a compound of Formula (V):

<img file="ES2618007T3_D0007.tif" />

where

R¹ and R² independently selected from hydrogen, halo, C1-6 alkyl, and C1-e haloalkyl;

R⁶ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁷ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁸ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

Q is CR⁹oN;

R⁹ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R¹⁰ selected from hydrogen and Ci-e alkyl;

R¹¹ selected from hydrogen and Ci-e alkyl;

R¹² selected from hydrogen and Ci-e alkyl; Y

R¹³ is selected from hydrogen, C1-6 alkyl, and Ce-20 aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, C1-6 alkoxy, or halo; or a pharmaceutically acceptable salt thereof.

The present disclosure provides a compound of Formula (VI):

ES 2 618 007 T3

<img file="ES2618007T3_D0008.tif" />

in which

R¹ and R² Independently selected from hydrogen, halo, Cie alkyl and Ci-e haloalkyl;

R⁸ selected from hydrogen, halo, Cie alkyl, Cie haloalkyl, Cie alkoxy, Cie haloalkoxy, hydroxyl, cyano, and nitro;

Q is CR⁹ or N;

R⁹ selected from hydrogen, halo, Cie alkyl, Cie haloalkyl, Cie alkoxy, Cie haloalkoxy, hydroxyl, cyano, and nitro;

R¹⁰ is selected from hydrogen and Cie alkyl; Y

R¹³ is selected from hydrogen, Cie alkyl, and Ce-20 aryl, wherein each alkyl or aryl is unsubstituted or is substituted with hydroxyl, Cie alkoxy, or halo; or a pharmaceutically acceptable salt thereof.

In certain embodiments, the present disclosure provides a compound of Formula (Vil) as described below.

In certain embodiments, the compound of Formula (I) - (VIII) is a compound selected from the species described or exemplified in the detailed description below.

In a further aspect, the present disclosure provides a pharmaceutical composition comprising at least one compound of Formula (I) - (HIV) or a pharmaceutically acceptable salt thereof. The pharmaceutical compositions may further comprise a pharmaceutically acceptable carrier or excipient. The present disclosure also provides a compound of Formula (I) - (HIV) or a pharmaceutically acceptable salt thereof for use as a medicament.

In another aspect, the present disclosure provides a compound of Formula (I) - (VHI) or a pharmaceutically acceptable salt thereof for use in therapy. In another aspect, the present disclosure provides a compound of Formula (I) - (HIV) or a pharmaceutically acceptable salt thereof for use in treating a proliferative disorder. In another aspect, the present disclosure provides the use of a compound of Formula (I) - (VHI) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a proliferative disorder.

With the compounds presented herein, a condition that is associated with EGFR inhibitory activity that targets mutated EGFR but not wild-type EGFR can be treated. The compounds of the invention can be administered to a subject in need of such treatment by administering an effective amount of at least one compound of Formula (I) - (VHI) or a pharmaceutically acceptable salt thereof. In this regard, the mutated EGFR may comprise a T790M mutation. The present disclosure provides the use of a compound of Formula (I) (VIII) in the preparation of a medicament for the treatment of such diseases and medical conditions and the use of such compounds and salts for the treatment of such diseases and medical conditions.

With the compounds according to the invention, mutated EGFR can be inhibited in a cell, wherein the cell is contacted with an effective amount of at least one compound of Formula (I) - (VHI) or a salt thereof, and / or with at least one pharmaceutical composition of the embodiments, wherein the contact is in vitro, ex vivo, or in vivo. In this regard, the mutated EGFR may comprise a T790M mutation.

With the compounds according to the invention a disease or medical condition associated with a kinase inhibitory activity can be treated, wherein a subject in need of such treatment is administered an effective amount of at least one compound of Formula (I) - (VHI ) or a pharmaceutically acceptable salt thereof, wherein the kinase can be selected from the group consisting of EGFR, EGFR (T790M), BLK, BMX / ETK, BTK, JAK2, JAK3, TEC, TXK, FLT3 and FLT3 (D835Y) . A compound of Formula (I) - (VIII) can also be used in the preparation of a medicament for the treatment of such diseases and medical conditions and the compounds and salts can be used for the treatment of such diseases and medical conditions. With the compounds according to the invention, a

ES 2 618 007 T3 kinase mutated in a cell, wherein the cell is contacted with an effective amount of at least one compound of Formula (I) - (VIII) or a salt thereof, and / or with at least one pharmaceutical composition of the embodiments, wherein the contact is in vitro, ex vivo, or in vivo. In this regard, the mutated kinase may be FLT3 with a D835Y mutation.

One of ordinary skill in the art will recognize that compounds of Formula (II) - (VI) are compounds of Formula (I) and that compounds of Formula (I) - (VII) are compounds of Formula (VIII).

Additional embodiments, features, and advantages of the invention will be apparent from the following detailed description and through practice of the invention.

Brief description of the drawings

Figure 1 shows SDS-PAGE of certain H1975 lung cancer cell effectors treated with varying concentrations of Compound 3.

Figure 2 shows immunoblots of certain effectors in Compound 3-treated tumors at various time intervals.

Figure 3 shows a graph of changes in the body weight of mice in the different groups in the NCIH1975 model.

Figure 4 shows a graph of changes in the body weight of mice in the different groups in the HCC827 model.

Figure 5 shows a graph of changes in the body weight of mice in the different groups in the A431 model.

Figure 6 shows a graph of the tumor volume of mice in the different groups in the NCI-H1975 model.

Figure 7 shows a graph of the tumor volume of mice in the different groups in the HCC827 model.

Figure 8 shows a graph of the tumor volume of mice in the different groups in the A431 model.

Figures 9A-9F show graphs of SDS-PAGE (9A, 9C, 9E) and inhibition (9B, 9D, 9F) of EGFR Tyr1068 phosphorylation and downstream signaling in H1975 lung cancer cells treated with varying concentrations of Compound 3 (9A, 9B), Gefitinib (9C, 9D) and WZ4002 (9E, 9F).

Figures 10A-10F show graphs of SDS-PAGE (10A, 10C, 10E) and inhibition (10B, 10D, 10F) of EGFR Tyr1068 phosphorylation and downstream signaling in EGFR HCC-827 mutant cells treated with concentrations Compound 3 (10A, 10B), Gefitinib (10C, 10D) and WZ4002 (10E, 10F) variables.

Figures 11A-11F show graphs of SDS-PAGE (11A, 11C, 11E) and inhibition (11B, 11D, 11F) of EGFR Tyr1068 phosphorylation and downstream signaling in A431 cells expressing WT EGFR that were treated with varying concentrations of Compound 3 (11A, 11B), Gefitinib (11C, 11D) and WZ4002 (11E, 11F).

Figure 12 shows the inhibition of EGFR phosphorylation in H1975 tumor tissues when treated with a single dose of Compound 3 at 12.5, 50 and 200 mg / kg.

Figure 13 shows the inhibition of EGFR phosphorylation in H1975 tumor tissues when treated with eight doses of Compound 3 at 12.5 and 50 mg / kg, compared to Gefitinib at 100 mg / kg.

Figure 14 shows the results of a cell-based pulsatile monitoring assay demonstrating that Compound 3 is an irreversible inhibitor of H1975 cell proliferation with the EGFR T790M mutation.

Figure 15A shows the IC50 titer curve for Compound 3 against BLK. Figure 15B shows the IC50 titration curve for staurosporine against BLK.

Figure 16 shows the IC50 titration curve for Compound 3 against BMX / ETK.

Figure 17 shows the IC50 titration curve for Compound 3 against BTK.

Figure 18 shows the IC50 titer curve for Compound 3 against FLT3 (D835Y).

Figure 19 shows the IC50 titer curve for Compound 3 against ITK.

Figure 20 shows the IC50 titer curve for Compound 3 against JAK2.

Figure 21 shows the IC50 titer curve for Compound 3 against JAK3.

Figure 22 shows the IC50 titer curve for Compound 3 against TEC.

ES 2 618 007 T3

Figure 23 shows the IC50 titer curve for Compound 3 against TXK.

Detailed description

The present invention is directed to certain pyrrolopyrimidine derivatives, pharmaceutical compositions, and these compounds and compositions for use in the treatment of proliferative disorders. These compounds described herein exhibit anti-tumor, anti-cancer, anti-inflammatory, anti-infective, and anti-proliferative activity. In some embodiments, the compounds have been shown to possess anticancer activity in cell-based assays described herein using various cancer cell lines, which demonstrate highly effective EGFR inhibitory activity that targets the mutation substantially and not substantially. wild protein. In some embodiments, the mutated EGFR comprises a T790M mutation. Accordingly, the compounds and compositions comprising the compounds of the embodiments are useful for treating conditions characterized by those mutated cancer cells. In certain cases, the compounds are useful in treating sarcoma, squamous cancer, fibrosarcoma, cervical cancer, gastric carcinoma, skin cancer, leukemia, lymphoma, lung cancer, non-small cell lung cancer, colon cancer, CNS cancer. , melanoma, ovarian cancer, kidney cancer, prostate cancer, breast cancer, liver cancer, head and neck cancers, and pancreatic cancer.

In other embodiments, the compounds have been shown to have activity against a range of protein kinases, including EGFR, EGFR (T790M), BLK, BMX / ETK, BTK, JAK2, JAK3, TEC, TXK, FLT3, and FLT3 (D835Y). In certain cases, the compounds are useful for treating cancers, tumors, inflammatory diseases, autoimmune diseases, or immunologically related diseases. In other embodiments, such diseases are mediated by at least one kinase selected from BTK, JAK3, ITK, and BMX. In other embodiments, cancers, tumors, inflammatory diseases, autoimmune diseases, or immunologically mediated diseases are mediated by abnormally activated B lymphocytes, T lymphocytes, or both. In other embodiments, the inflammatory diseases, autoimmune diseases or immunologically mediated diseases are arthritis, rheumatoid arthritis, spondyloarthropathy, gouty arthritis, osteoarthritis, juvenile arthritis, other arthritic conditions, lupus, systemic lupus erythematosus (SLE), a skin related disease, psoriasis, dry eye, eczema, dermatitis, atopic dermatitis, pain, a lung disorder, lung inflammation, Adult respiratory distress syndrome (ARDS), pulmonary sarcoidosis, chronic pulmonary inflammatory disease, chronic obstructive pulmonary disease (COPD), cardiovascular disease, atherosclerosis, myocardial infarction, congestive heart failure, cardiac reperfusion injury, inflammatory enteropathy, Crohn's disease , ulcerative colitis, irritable bowel syndrome, asthma, Sjogren's syndrome, autoimmune thyroid disease, urticaria (cnidosis), multiple sclerosis, scleroderma, organ transplant rejection, heteroplastic graft, idiopathic thrombocytopenic purpura (ITP), Parkinson's disease, Alzheimer's disease, diseases associated with diabetes, inflammation, pelvic inflammatory disease, allergic rhinitis, allergic bronchitis, allergic sinusitis, leukemia, lymphoma, B-cell lymphoma, T-cell lymphoma, myeloma, acute lymphoid leukemia (ALL), chronic lymphoid leukemia (CLL), acute myeloid leukemia (AML), Chronic myeloid leukemia (CML), hairy cell leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), diffuse large B-cell lymphoma, follicular lymphoma, sarcoma, squamous cell cancer, fibrosarcoma, cancer cervical cancer, gastric carcinoma, skin cancer, leukemia, lymphoma, lung cancer, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, breast cancer, liver cancer, head and neck cancers, or pancreatic cancer. In other embodiments, the diseases are autoimmune disease or transplant-induced inflammatory disorders including, but not limited to, allogeneic transplantation, graft-versus-host disease, or autoimmune diabetes.

Before the present invention is further described, it is to be understood that this invention is not limited to the particular embodiments described, as these, of course, can vary. It is further to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

It should be noted that as used herein and in the appended claims, the singular forms a ", a" and the include plural references unless the context clearly dictates otherwise. It should also be noted that claims may be drafted to exclude any optional elements. As such, this discussion is intended to serve as an antecedent basis for the use of exclusive terminology such as "only", only and the like in connection with the mention of elements of the claims or the use of a negative limitation.

As used herein, the terms including (n) ", containing (n)", and comprising (n) are used in their open non-limiting sense.

To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term approximately. It is understood that, whether the term approximately is used explicitly or not, each quantity given herein refers to the actual value given and is also understood to refer to the approximation to this value given that it would be reasonably inferred based on normal experience in the specialty, including equivalents and approximations due to experimental and / or measurement conditions for this given value. Whenever a return is given as a percentage, such return refers to a mass of the entity for which the return is given relative to the maximum amount of the same entity that could be obtained under the conditions

ES 2 618 007 T3 particular stoichiometric. Concentrations given as percentages refer to mass ratios, unless otherwise indicated.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning that is commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

Except where otherwise indicated, the methods and techniques of the present embodiments are generally performed according to conventional methods well known in the art and which are described in various general and more specific references that are cited and discussed throughout the present. descriptive memory. See, p. eg, Loudon, Organic Chemistry, Fourth Edition, New York: Oxford University Press, 2002; Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms and Structure, Fifth Edition, Wiley-Interscience, 2001.

The nomenclature used herein to name the compounds in question is illustrated in the Examples herein. This nomenclature has generally been derived using commercially available AutoNom software (MDL, San Leandro, Calif.).

It is appreciated that certain features of the invention, which, for clarity, are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which, for brevity, are described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments dealing with the chemical groups represented by the variables are specifically encompassed by the present invention and are disclosed herein as if each and every combination were disclosed individually and explicitly, to the extent that these Combinations encompass compounds that are stable compounds (ie, compounds that can be isolated, characterized, and tested for biological activity). Furthermore, all subcombinations of chemical groups listed in the embodiments that describe these variables are also specifically encompassed by the present invention and are disclosed herein as if each and every such subcombination of chemical groups were disclosed herein individually and explicitly.

Chemical terms

The term "alkyl" refers to a straight or branched chain alkyl group having 1 to 12 carbon atoms in the chain. Examples of alkyl groups include methyl (Me), ethyl (Et), n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tere-butyl (tBu), pentyl, isopentyl, tert-pentyl, hexyl, isohexyl, and groups that in light of normal experience in the art and the teachings provided herein they would be considered equivalent to any one of the foregoing examples.

The term "alkoxy" refers to an alkyl group as defined above, attached to an oxygen atom. The alkoxy group is connected to the parent structure through the oxygen atom.

The term "amino" refers to a group -NH₂ or a mono- or dialkylamino group.

The term "cycloalkyl" refers to a saturated or partially saturated, monocyclic, fused polycyclic, bridged polycyclic, or spiro-type polycyclic carbocycle having 3 to 12 ring atoms per carbocycle. Illustrative examples of cycloalkyl groups include the following entities, in the form of appropriately linked moieties:

<img file="ES2618007T3_D0009.tif" />

The term heteroaryl refers to a monocyclic, fused bicyclic, or fused polycyclic aromatic heterocycle (ring structure having ring atoms selected from carbon atoms and up to four heteroatoms selected from nitrogen, oxygen, and sulfur) having 3 to 12 ring atoms per heterocycle. Illustrative examples of heteroaryl groups include the following entities, in the form of appropriately linked moieties:

ES 2 618 007 T3 .0. X_XS. /^N\ XAXA

O. O, G, G. G, G, GG, 1

H

XNN

TO,

<img file="ES2618007T3_D0010.tif" />

<img file="ES2618007T3_D0011.tif" />

//

<img file="ES2618007T3_D0012.tif" />

<img file="ES2618007T3_D0013.tif" />

<img file="ES2618007T3_D0014.tif" />

The term halogen represents chlorine, fluorine, bromine or iodine. The term halo represents chlorine, fluoro, bromine, or iodine. The term "haloalkyl" means an alkyl as defined above, substituted with one or more halogen atoms. The term "haloalkoxy" means an alkoxy as defined above, substituted with one or more halogen atoms.

The term "acyl" refers to an RC (O) - group of 1 to 10 carbon atoms of a linear, branched or cyclic configuration or a combination thereof, linked to the parent structure through a carbonyl functionality. This group can be saturated or unsaturated and aliphatic or aromatic.

The term cyano refers to the group -CN.

The term nitro refers to the group -NO2.

The term "hydroxyl" refers to the group -OH.

Those skilled in the art will recognize that the species listed or illustrated above are not exhaustive and that additional species may also be selected within the scope of these defined terms.

The term "substituted" means that the specified group or moiety bears one or more substituents. The term "unsubstituted" means that the specified group does not bear substituents. The term "optionally substituted" means that the specified group is unsubstituted or is substituted with one or more substituents. When the term "substituted" is used to describe a structural system, it is understood that the substitution is present in any position of the system allowed by valence.

Any formula depicted herein is intended to represent a compound of that structural formula as well as certain variations or forms. For example, a formula given herein is intended to include a racemic form or one or more enantiomeric, diastereoisomeric or geometric isomers or a mixture thereof. Additionally, any formula given herein is intended to refer to a hydrate, solvate or polymorph of this compound or a mixture thereof.

Any formula given herein is also intended to represent unlabeled as well as isotopically-labeled forms of the compounds. Isotopically labeled compounds have structures represented by the formulas given herein except that one or more atoms are replaced by an atom having a selected atomic mass or mass number. Examples of isotopes that can be incorporated into the compounds of embodiments include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine, chlorine, and iodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸OR, ¹⁷OR, ³¹P, ³²P, ³⁵Yes, ¹⁸F, ³⁶CI and ¹²⁵l, respectively. These isotopically-labeled compounds are useful in metabolic studies (preferably with¹⁴C), reaction kinetics studies (with, for example ²H o ³H), detection or imaging techniques [such as positron emission tomography (PET) or digital single photon emission tomography (SPECT)] including drug or substrate tissue distribution assays or in the radioactive treatment of patients. In particular, a compound marked with¹⁸F or ¹¹C may be particularly preferred for PET or SPECT studies. On the other hand, substitution with heavier isotopes such as deuterium (i.e.²H) may provide certain therapeutic benefits resulting from increased metabolic stability, for example increased in vivo half-life or reduced dosage requirements. The isotopically-labeled compounds of the embodiments and the prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and the preparations described below by substituting a readily available isotopically-labeled reagent for a non-isotopically-labeled reagent. .

The nomenclature C, j with j&gt; i, when applied herein to a class of substituents, is understood to refer to embodiments for which each and every carbon member number of i, j including i and j. By way of example, the term C1.3 independently refers to embodiments

ES 2 618 007 T3 having one carbon member (Ci), embodiments having two carbon members (C2) and embodiments having three carbon members (C3).

Any dis-substituent mentioned herein is understood to encompass the various binding possibilities when more than one such possibility is allowed. For example, reference to the disubstituent -AB-, when A / B, refers herein to this disubstituent with A linked to a first substituted member and B linked to a second substituted member and also refers to this disubstituent with A linked to the second substituted member and B linked to the first substituted member.

The present disclosure provides pharmaceutically acceptable salts of the compounds represented by Formulas (I) - (VIII), preferably of those described above and of the specific compounds exemplified herein and pharmaceutical compositions comprising these salts and methods of using these salts. .

A "pharmaceutically acceptable salt" is understood to mean a salt of a free acid or base of a compound represented herein that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to the subject. See, generally, SM Berge, et al., "Pharmaceutical Salts", J. Pharm. Sci., 1977, 66, 119. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of subjects without excessive toxicity, irritation or allergic response. A compound described herein may possess a sufficiently acidic group, a sufficiently basic group, both types of functional groups or more than one of each type and accordingly react with a number of inorganic or organic bases and inorganic and organic acids, to form a pharmaceutically acceptable salt.

Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, heptanes, caprilates, acrylates, formates, caprilates, acrylates, formates, caprilates, acrylates, formates, caprylates. propriolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-1,4-dioates, hexyne-1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulphonates, methylsulphonates, propylsulphonates, besylates, xylenesulphonates, naphthalene-1-sulphonates, naphthalene-2-sulphonates, phenylacetates, phenylbutylates, phenylautyrates, hydroxybutyrates, γ-lactates mandates.

For a compound of Formulas (I) - (VIII) containing a basic nitrogen, a pharmaceutically acceptable salt can be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, nitric acid, boric acid, phosphoric acid and the like or with an organic acid, such as acetic acid, phenylacetic acid, propionic acid, stearic acid, lactic acid, ascorbic acid, maleic acid, hydroximaleic acid, isethionic acid, succinic acid, valeric acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, oleic acid, palmitic acid, lauric acid , a pyranosidylic acid, such as glucuronic or galacturonic acid, an α-hydroxy acid, such as mandelic acid, citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid, 2-acetoxybenzoic acid, naphthoic acid or cinnamic acid, a sulfonic acid, such as lauryl sulfonic acid, p-toluenesulfonic acid, methanesulfonic acid or ethanesulfonic acid or any compatible mixture of acids such as those given as examples herein and any other acids and mixtures thereof that are considered equivalent or acceptable substitutes in light of the normal level of expertise in this technology. In certain embodiments, the pharmaceutically acceptable salt is the HCl salt, maleic acid salt, HBr salt, hydroxybutanedioic acid salt, fumaric acid salt, lactic acid salt, tartaric acid salt, or methanesulfonic acid salt.

The present disclosure provides pharmaceutically acceptable prodrugs of the compounds of Formulas (I) (VIII) and their use in therapeutic treatment. The term "prodrug" means a precursor of an indicated compound which, after administration to a subject, yields the compound in vivo through a chemical or physiological procedure such as solvolysis or enzymatic cleavage or under physiological conditions (e.g. eg, a prodrug on being brought to physiological pH becomes the compound of Formulas (I) - (VIII)). A "pharmaceutically acceptable prodrug" is a prodrug that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to the subject. Illustrative procedures for the selection and preparation of suitable prodrug derivatives are described, for example, in Design of Prodrugs, ed. H. Bundgaard, Elsevier, 1985.

The present disclosure provides pharmaceutically active metabolites of compounds of Formulas (I) - (VIII) and uses of these metabolites in the methods of the embodiments. A "pharmaceutically active metabolite" means a pharmacologically active product of metabolism in the body of a compound of Formulas (I) - (VIII) or a salt thereof. Prodrugs and active metabolites of a compound can be determined using standard techniques known or available in the art. See, p. eg, Bertolini et al., J. Med. Chem. 1997, 40, 2011-2016; Shan et al., J. Pharm. Sci. 1997, 86 (7), 765-767; Bagshawe, Drug Dev. Res. 1995, 34, 220-230; Bodor, Adv. Drug Res. 1984, 13, 255-331; Bundgaard, Design of Prodrugs (Elsevier Press, 1985); and Larsen, Design and Application of Prodrugs, Drug Design and Development (Krogsgaard-Larsen et al., eds., Harwood Academic Publishers, 1991).

Representative realizations

Formula (VIII)

ES 2 618 007 T3

The present disclosure provides a compound of Formula (VIII). In some embodiments, X is O or NH. In other embodiments, X¹ it is CH2 or CF2. In still other embodiments, X¹ that.

In some embodiments of Formula (VIII), -NR¹⁸R¹⁹ it is

<img file="ES2618007T3_D0015.tif" />

N- In other embodiments, -NR¹⁸R¹⁹ is r¹⁵-n (% - NH ^r”-ⁿTO ^m . In some embodiments, R¹⁵ it is methyl, hydroxylate, methoxylate, or fluoroetyl. In other embodiments, R¹⁵ it is fluoroetllo. In some embodiments, m is 1. In other embodiments, m is 2.

In some embodiments, R⁹ and R¹⁹ taken together they form an optionally substituted 5- or 6-membered heteroaryl ring. In some embodiments, R¹⁹ and R⁹ together they form a 5- or 6-membered ring optionally substituted with C1-6 alkyl that is unsubstituted or substituted with amine. In some embodiments, the heteroaryl ring is substituted with dimethyl amylnomethyl. In other embodiments, R⁹ and R¹⁹ taken together they form pyrrole or pyridine. In some embodiments, R¹ is aimeuiaminoetllo.

<img file="ES2618007T3_D0016.tif" />

In some embodiments, R⁶ it is methoxy. In other embodiments, R⁷ it is methoxy. In certain cases, R⁷ it is hydrogen or methoxy.

In some embodiments of Formula (VIII), each variable herein is defined as described below for any one of Formulas (I) - (VII) or embodiments thereof. In particular, certain embodiments of Formula (VIII) are as defined for each variable for Formula (I) below and such definitions are incorporated herein by reference.

Formula (I)

The present disclosure provides a compound of Formula (la) and (Ib):

R¹ OR

<img file="ES2618007T3_D0017.tif" />

(the),

R¹ OR

<img file="ES2618007T3_D0018.tif" />

(Ib) in which

R¹ and R² are independently selected from hydrogen, halo, C1-6 alkyl and Cie haloalkyl; R³ selected from halo, hydroxyl, C1-6 alkyl, C1-6 alkoxy, clane, and nitro; n is a number from zero to 4;

R⁴ selected from hydrogen, C 1-6 alkyl, C 3-7 cycloalkyl, and -NR²²R²³;

wherein the alkyl or cycloalkyl is unsubstituted or is substituted with hydroxyl or amine; Y

ES 2 618 007 T3

2. 3 22 23 each of R and R is independently selected from hydrogen and Ci-6 alkyl or R and R may be linked to form a 3 to 10 membered ring;

R⁵ selected from hydrogen and C1-6 alkyl;

R⁶ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁷ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁸ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

Q is CR⁹or N;

where R⁹ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R¹⁰ selected from hydrogen and C1-6 alkyl;

a is one or two;

Ring A is an aromatic ring;

R²⁰ and R²¹ independently selected from hydrogen and C1-6 alkyl; wherein the alkyl is unsubstituted or is substituted with amino, hydroxyl, or halo; where R²¹ may not be present to satisfy valence;

R¹¹ selected from hydrogen and C1-6 alkyl;

R¹² selected from hydrogen and C1-6 alkyl; Y

R¹³ is selected from hydrogen, C1-6 alkyl, C1-6 acyl, SÜ2-C1-6 alkyl, C3-7 cycloalkyl, and C6-20 aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, alkoxy C1-6 or halo;

or a pharmaceutically acceptable salt thereof.

Formula (I) is understood to refer to Formula (Ia) and Formula (Ib).

In Formula (I), R¹ and R² they are independently selected from hydrogen, halo, C1-6 alkyl and C1-6 haloalkyl. In certain cases, R¹ it is hydrogen. In certain cases, R¹ is C1-6 alkyl. In certain cases, R¹ it is methyl or ethyl. In certain cases, R² it is hydrogen. In certain cases, R² is C1-6 alkyl. In certain cases, R² it is methyl or ethyl. In certain cases, R¹ and R² they are each hydrogen.

In Formula (I), n is a number from zero to 4. In certain cases, n is zero. In certain cases, n is one. In certain cases, n is 2. In certain cases, n is 3. In certain cases, n is 4.

In Formula (I), R³ it is selected from halo, hydroxyl, C1-6 alkyl, C1-6 alkoxy, cyano, and nitro. In certain cases, R³ it's halo. In certain cases, R³ is hydroxyl. In certain cases, R³ is C1-6 alkyl. In certain cases, R³ is C1-6 alkoxy. In certain cases, R³ is cyano. In certain cases, R³ it is nitro.

In Formula (I), R⁴ selected from hydrogen, C1-6 alkyl, C3-7 cycloalkyl, and -NR²²R²³; wherein the alkyl or cycloalkyl is unsubstituted or is substituted with hydroxyl or amino; and where each of R²² and R²³ is independently selected from hydrogen and C1-6 alkyl or R²² and R²¹ they can be linked to form a 3- to 10-membered ring. In certain cases, R⁴ it is hydrogen. In certain cases, R⁴ is C1-6 alkyl. In certain cases, R⁴ is C 3-7 cycloalkyl. In certain cases, R⁴ is -NR²²R²³.

In certain cases, R⁴ is unsubstituted C1-6 alkyl. In certain cases, R⁴ is C1-6 alkyl which is substituted with hydroxyl. In certain cases, R⁴ is C1-3 alkyl which is substituted with hydroxyl. In certain cases, R⁴ is C1-6 alkyl which is substituted with amino. In certain cases, R⁴ is C1-6 alkyl which is substituted with -NH2. In certain cases, R⁴ is C1-6 alkyl which is substituted with -N (CH3) 2. In certain cases, R⁴ is C1-3 alkyl which is substituted with -NH2. In certain cases, R⁴ is C1-3 alkyl which is substituted with -N (CH3) 2.

In certain cases, R⁴ is unsubstituted C 3-7 cycloalkyl. In certain cases, R⁴ is unsubstituted C3 cycloalkyl. In certain cases, R⁴ is unsubstituted C4 cycloalkyl. In certain cases, R⁴ is unsubstituted C5-6 cycloalkyl. In certain cases, R⁴ is unsubstituted C7 cycloalkyl.

22 23 22 23

In certain cases, R is -NR R, where each of R and R is independently selected from hydrogen and 22 23 22 23 C1-6 alkyl or R and R can be linked to form a 3- to 10-membered ring. In certain cases, R and R are 22 23 22 23 22 hydrogen. In certain cases, R and R are C1-6 alkyl. In certain cases, R and R are C1-3 alkyl. In certain cases, R and R²³ they are methyl.

ES 2 618 007 T3

In certain cases,, ιΑκ

R²² Y

R can be linked to form a 3 to 10 membered ring, so that R is, where w is a number from 1 to 8. In certain cases, R²² and R²³ they can be linked to form a 3-membered ring. In certain cases, R²² and R²³ they can be linked to form a 4-membered ring. In certain cases, R²² and R²³ they can be linked to form a 5-membered ring. In certain cases, R²² and R²³ they can be linked to form a 6-membered ring. In certain cases, R²² and R²³ they can be linked to form a 7-membered ring.

In Formula (I), R⁵ is selected from hydrogen and Cie alkyl. In certain cases, R⁵ it is hydrogen. In certain cases, R⁵is Cie alkyl. In certain cases, R⁵ is methyl. In certain cases, R⁵ is ethyl. In certain cases, R⁵ is C1.3 alkyl. In certain cases, R⁵ is C4-6 alkyl

In Formula (I), R⁶ is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, clane and nitro. In certain cases, R⁶ it is hydrogen. In certain cases, R⁶ it's halo. In certain cases, R⁶ it is fluoro. In certain cases, R⁶ it is chlorine. In certain cases, R⁶ it is bromine. In certain cases, R⁶ is C1-6 alkyl. In certain cases, R⁶ is C1-6 haloalkyl. In certain cases, R⁶ is C2-6 alkoxyIn certain cases, R⁶ is C1-6 haloalkoxy. In certain cases, R⁶ is hydroxyl. In certain cases, R⁶ it is clano. In certain cases, R⁶ it is nitro.

In Formula (I), R⁷ is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, clane and nitro. In certain cases, R⁷ it is hydrogen. In certain cases, R⁷ it's halo. In certain cases, R⁷ it is fluoro. In certain cases, R⁷ it is chlorine. In certain cases, R⁷ it is bromine. In certain cases, R⁷ is C1-6 alkyl. In certain cases, R⁷ is C1-6 haloalkyl. In certain cases, R⁷ is C2-6 alkoxyIn certain cases, R⁷ is C1-6 haloalkoxy. In certain cases, R⁷ is hydroxyl. In certain cases, R⁷ it is clano. In certain cases, R⁷ it is nitro.

In Formula (I), R is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, clane and nitro. In certain cases, R⁸ it is hydrogen. In certain cases, R⁸ it's halo. In certain cases, R⁸ it is fluoro. In certain cases, R is chlorine. In certain cases, R is bromine. In certain cases, R is C1-6 alkyl. In certain cases, R is C1-6 haloalkyl. In certain cases, R⁸ is C1-6 alkoxy. In certain cases, R⁸ is C1-6 haloalkoxy. In certain cases, R⁸ is hydroxyl. In certain cases, R⁸ it is clano. In certain cases, R⁸ it is nitro.

In Formula (I), Q is CR⁹ or N. In certain cases, Q is CR⁹. In certain cases, Q is N.

In Formula (I), R⁹ is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, clane and nitro. In certain cases, R⁹ it is hydrogen. In certain cases, R⁹ it's halo. In certain cases, R⁹ it is fluoro. In certain cases, R⁹ it is chlorine. In certain cases, R⁹ it is bromine. In certain cases, R⁹ is C1-6 alkyl. In certain cases, R⁹ is C1-6 haloalkyl. In certain cases, R⁹ is C1-6 alkoxy. In certain cases, R⁹ is C1-6 haloalkoxy. In certain cases, R⁹ is hydroxyl. In certain cases, R⁹ it is clano. In certain cases, R⁹ it is nitro. In certain cases, R⁹ it is hydrogen or fluoro.

In Formula (I), R¹⁰ is selected from hydrogen and C1-6 alkyl. In certain cases, R¹⁰ it is hydrogen. In certain cases, 10 '' 10^{J 1} 10 10

R is C1-6 alkyl. In certain cases, R is methyl. In certain cases, R is ethyl. In certain cases, R is C1.3 alkyl. In certain cases, R¹⁰ is C4-6 alkyl

In Formula (I), a is one or two and Ring A is an aromatic ring. In certain cases, a is one, as shown:

R²¹ R⁸ R⁸

<img file="ES2618007T3_D0019.tif" />

. In certain cases, a is two, as shown:

In Formula (I), R²⁰ and R²¹ Independently selected from hydrogen and Cie alkyl; wherein the alkyl is unsubstituted or is substituted with amine, hydroxyl, or halo; where R^2T may not be present to satisfy the

twenty-one 111 2q I heard Valencia. In certain cases, R and R are independently hydrogen. In certain cases, R and R are independently unsubstituted C1-6 alkyl. In certain cases, R²⁰ and R²¹ They are independently C1-6 alkyl, substituted with amine. In certain cases, R²⁰ and R²¹ are Independently C1-6 alkyl, substituted with -NR²⁴R²⁵, on

25 ^J * ¹ 20 21 where R and R are independently selected from hydrogen and C1-6 alkyl. In certain cases, R and R are independently C1-6 alkyl, substituted with -NR²⁴R²⁵, where R²⁴ and R²⁵ are selected Regardless of ^{1 1} 20 21 ^{J 1} 24 25 hydrogen and C1.3 alkyl. In certain cases, R and R are independently C1-6 alkyl, substituted with -NR R, where R²⁴ and R²⁵ Independently selected from hydrogen and methyl. In certain cases, R²⁰ and R²¹ are independently C1.3 alkyl, substituted with -NR²⁴R²⁵, where R²⁴ and R²⁵ Independently selected from hydrogen and methyl. In certain cases, R²⁰ and R²¹ They are Independently C1-6 alkyl, substituted with hydroxyl. In certain cases, R²⁰ and R²¹ are C1-6 alkyl, substituted with halo.

In Formula (I), R¹¹ is selected from hydrogen and C1-6 alkyl. In certain cases, R¹¹ it is hydrogen. In certain cases, R¹¹ is C1-6 alkyl. In certain cases, R¹¹ is methyl. In certain cases, R¹¹ is ethyl. In certain cases, R¹¹ is C1.3 alkyl. In certain cases, R¹¹ is C4-6 alkyl

ES 2 618 007 T3

In Formula (I), R¹² is selected from hydrogen and Cie alkyl. In certain cases, R¹² it is hydrogen. In certain cases, R¹² is Cie alkyl. In certain cases, R¹² is methyl. In certain cases, R¹² is ethyl. In certain cases, R¹² is C1.3 alkyl. In certain cases, R¹² is C4-6 alkyl

In Formula (I), R¹³ is selected from hydrogen, C1-6 alkyl, C1-6 acyl, S02-alkyl (Ci_6), C3-7 cycloalkyl, and Ce-20 aryl, wherein each alkyl or aryl is unsubstituted or is substituted with hydroxyl, alkoxy C1-6 or halo. In certain cases, R¹³it is hydrogen. In certain cases, R¹³ is C1-6 alkyl. In certain cases, R¹³ is acllo C1-6. In certain cases, R¹³ is SO2alkyl (Ci_6). In certain cases, R¹³ is C 3-7 cycloalkyl. In certain cases, R¹³ is C6-20 arylIn certain cases, R¹³ is C1-6 alkyl substituted with hydroxyl or halo.

In certain cases, R¹³ is unsubstituted C1-6 alkyl. In certain cases, R¹³ is C1-6 alkyl which is substituted with hydroxyl. In certain cases, R¹³ is - (CH2)_mOH, where m is a number from one to 3. In certain cases, R¹³ is -CH2OH. In certain cases, R¹³ is C1-6 alkyl which is substituted with C1-6 alkoxy.

In certain cases, R¹³ is C1-6 alkyl which is substituted with halo. In certain cases, R¹³ is - (CH2) mX, where m is a number from one to 3 and X is halo. In certain cases, R¹³ it is C1-6 alkyl which is substituted with fluoro. In certain cases, R¹³is - (CH2) mF, where m is a number from one to 3. In certain cases, R¹³ is - (CFh ^ F.

In certain cases, R¹³ is acllo C1-6. In certain cases, R¹³ is acllo C1. In certain cases, R¹³ is acllo C2. In certain cases, R¹³ is acllo C3. In certain cases, R¹³ is acllo C4-6

In certain cases, R¹³ is S02-alkyl (Ci_6). In certain cases, R¹³ is S02-alkyl (Ci). In certain cases, R¹³ is SO2alkyl (C2). In certain cases, R¹³ is S02-alkyl (C3). In certain cases, R¹³ is S02-alkyl (C4-6).

In certain cases, R¹³ is C 3-7 cycloalkyl. In certain cases, R¹³ is unsubstituted C3 cycloalkyl. In certain cases, R¹³ is unsubstituted C4 cycloalkyl. In certain cases, R¹³ is unsubstituted C5-6 cycloalkyl. In certain cases, R¹³ is unsubstituted C7 cycloalkyl.

In certain cases, R¹³ is unsubstituted Ce-20 aryl. In certain cases, R¹³ is Ce-20 aryl which is substituted with hydroxyl. In certain cases, R¹³ is Ce-20 aryl which is substituted with C1-6 alkoxy. In certain cases, R¹³ is Ce-20 aryl which is substituted with halo.

In certain cases, R⁶, R⁷, R⁸ and R⁹ they are hydrogen. In certain cases, R⁶, R⁷ and R⁸ are hydrogen and R⁹ it's halo. In certain cases, R⁶, R⁷ and R⁸ are hydrogen and R⁹ it is fluoro.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹⁰ is methyl. In certain cases, R⁶, R⁷, R⁸ they are hydrogen; R⁹ is halo; and R¹⁰ is methyl. In certain cases, R⁶, R⁷, R⁸ they are hydrogen; R⁹ is fluoro; and R¹⁰ is methyl.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹³ it is hydrogen. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is halo; and R¹³ it is hydrogen. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is fluoro; and R¹³ it is hydrogen.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹³ is -CH2OH. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is halo; and R¹³ is -CH2OH. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is fluoro; and R¹³ is -CH2OH.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹³ is - (CH2) mF, where m is a number from one to 3. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is halo; and R¹³ is - (CH2) mF, where m is a number from one to 3. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is fluoro; and R¹³ is - (CH2)_mF, where m is a number from one to 3.

Formula (II)

The present disclosure provides a compound of Formula (II):

<img file="ES2618007T3_D0020.tif" />

in which

R¹ and R² are independently selected from hydrogen, halo, C1-6 alkyl and Cie haloalkyl;

ES 2 618 007 T3

R³ selected from halo, hydroxyl, Ci-6 alkyl, Ci-6 alkoxy, cyano, and nitro;

n is a number from zero to 4;

R⁴ selected from hydrogen, C1-6 alkyl, C3-7 cycloalkyl, and -NR²²R²³;

wherein the alkyl or cycloalkyl is unsubstituted or is substituted with hydroxyl or amino; Y

2. 3 22 21 each of R and R is independently selected from hydrogen and C 1-6 alkyl or R and R may be linked to form a 3 to 10 membered ring;

R⁵ selected from hydrogen and C1-6 alkyl;

R⁶ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁷ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁸ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

Q is CR⁹or N;

where R⁹ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R¹⁰ selected from hydrogen and C1-6 alkyl;

R¹¹ selected from hydrogen and C1-6 alkyl;

R¹² selected from hydrogen and C1-6 alkyl; Y

R¹³ is selected from hydrogen, C1-6 alkyl, and C6-20 aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, C1-6 alkoxy, or halo; or a pharmaceutically acceptable salt thereof.

In Formula (II), R¹ and R² they are independently selected from hydrogen, halo, C1-6 alkyl and C1-6 haloalkyl. In certain cases, R¹ it is hydrogen. In certain cases, R¹ is C1-6 alkyl. In certain cases, R¹ it is methyl or ethyl. In certain cases, R² it is hydrogen. In certain cases, R² is C1-6 alkyl. In certain cases, R² it is methyl or ethyl. In certain cases, R¹ and R² they are hydrogen.

In Formula (II), n is a number from zero to 4. In certain cases, n is zero. In certain cases, n is one. In certain cases, n is 2. In certain cases, n is 3. In certain cases, n is 4.

In Formula (II), R³ it is selected from halo, hydroxyl, C1-6 alkyl, C1-6 alkoxy, cyano, and nitro. In certain cases, R³ it's halo. In certain cases, R³ is hydroxyl. In certain cases, R³ is C1-6 alkyl. In certain cases, R³ is C1-6 alkoxy. In certain cases, R³ is cyano. In certain cases, R³ it is nitro.

In Formula (II), R⁴ selected from hydrogen, C1-6 alkyl, C3-7 cycloalkyl, and -NR²²R²³; wherein the alkyl or cycloalkyl is unsubstituted or is substituted with hydroxyl or amino; and where each of R²² and R²³ is independently selected from hydrogen and C1-6 alkyl or R²² and R²¹ they can be linked to form a 3- to 10-membered ring. In certain cases, R⁴ it is hydrogen. In certain cases, R⁴ is C1-6 alkyl. In certain cases, R⁴ is C 3-7 cycloalkyl. In certain cases, R⁴ is -NR²²R²³.

In certain cases, R⁴ is unsubstituted C1-6 alkyl. In certain cases, R⁴ is C1-6 alkyl which is substituted with hydroxyl. In certain cases, R⁴ is C1-3 alkyl which is substituted with hydroxyl. In certain cases, R⁴ is C1-6 alkyl which is substituted with amino. In certain cases, R⁴ is C1-6 alkyl which is substituted with -NH2. In certain cases, R⁴ is C1-6 alkyl which is substituted with -N (CH3) 2. In certain cases, R⁴ is C1-3 alkyl which is substituted with -NH2. In certain cases, R⁴ is C1-3 alkyl which is substituted with -N (CH3) 2.

In certain cases, R⁴ is unsubstituted C 3-7 cycloalkyl. In certain cases, R⁴ is unsubstituted C3 cycloalkyl. In certain cases, R⁴ is unsubstituted C4 cycloalkyl. In certain cases, R⁴ is unsubstituted C5-6 cycloalkyl. In certain cases, R⁴ is C7 unsubstituted cycloalkyl.

22 23 22 23

In certain cases, R is -NR R where each of R and R is independently selected from hydrogen and 22 23 22 23 C1-6 alkyl or R and R may be linked to form a 3 to 10 membered ring. In certain cases, R and R are 22 23 22 23 22 hydrogen. In certain cases, R and R are C1-6 alkyl. In certain cases, R and R are C1-3 alkyl. In certain cases, R and R²³ they are methyl.

ES 2 618 007 T3

In certain cases, R²² and R²³ may be linked to form a 3 to 10 membered ring, so that R⁴ that is, where w is a number from 1 to 8. In certain cases, R²² and R²³ they can be linked to form a 3-membered ring. In certain cases, R²² and R²³ they can be linked to form a 4-membered ring. In certain cases, R²²and R²³ they can be linked to form a 5-membered ring. In certain cases, R²² and R²³ they can be linked to form a 6-membered ring. In certain cases, R²² and R²³ they can be linked to form a 7-membered ring.

In Formula (II), R⁵ is selected from hydrogen and Cie alkyl. In certain cases, R⁵ it is hydrogen. In certain cases, R⁵is Cie alkyl. In certain cases, R⁵ is methyl. In certain cases, R⁵ is ethyl. In certain cases, R⁵ is C1.3 alkyl. In certain cases, R⁵ is C4-6 alkyl

In Formula (II), R⁶ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁶ it is hydrogen. In certain cases, R⁶ it's halo. In certain cases, R⁶ it is fluoro. In certain cases, R⁶ it is chlorine. In certain cases, R⁶ it is bromine. In certain cases, R⁶ is C1-6 alkyl. In certain cases R⁶is C1-6 haloalkyl. In certain cases, R⁶ is C2-6 alkoxyIn certain cases, R⁶ is C1-6 haloalkoxy. In certain cases, R ° is hydroxyl. In certain cases, R⁶ is cyano. In certain cases, R⁶ it is nitro.

In Formula (II), R⁷ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁷ it is hydrogen. In certain cases, R⁷ it's halo. In certain cases, R⁷ it is fluoro. In certain cases, R⁷ it is chlorine. In certain cases, R⁷ it is bromine. In certain cases, R⁷ is C1-6 alkyl. In certain cases R⁷is C1-6 haloalkyl. In certain cases, R⁷ is C2-6 alkoxyIn certain cases, R⁷ is C1-6 haloalkoxy. In certain cases, R⁷ is hydroxyl. In certain cases, R⁷ is cyano. In certain cases, R⁷ it is nitro.

In Formula (II), R⁸ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁸ it is hydrogen. In certain cases, R⁸ it's halo. In certain cases, R⁸ it is fluoro. In certain cases, R is chlorine. In certain cases, R is bromine. In certain cases, R is C1-6 alkyl. In certain cases R is C1-6 haloalkyl. In certain cases, R⁸ is C1-6 alkoxy. In certain cases, R⁸ is C1-6 haloalkoxy. In certain cases, R⁸ is hydroxyl. In certain cases, R⁸ is cyano. In certain cases, R⁸ it is nitro.

In Formula (II), Q is CR⁹ or N. In certain cases, Q is CR⁹. In certain cases, Q is N.

In Formula (II), R⁹ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁹ it is hydrogen. In certain cases, R⁹ it's halo. In certain cases, R⁹ it is fluoro. In certain cases, R⁹ it is chlorine. In certain cases, R⁹ it is bromine. In certain cases, R⁹ is C1-6 alkyl. In certain cases R⁹is C1-6 haloalkyl. In certain cases, R⁹ is C1-6 alkoxy. In certain cases, R⁹ is C1-6 haloalkoxy. In certain cases, R⁹ is hydroxyl. In certain cases, R⁹ is cyano. In certain cases, R⁹ it is nitro.

In Formula (II), R¹⁰ is selected from hydrogen and C1-6 alkyl. In certain cases, R¹⁰ it is hydrogen. In certain cases, 10 '' 10^{J 1} 10 10

R is C1-6 alkyl. In certain cases, R is methyl. In certain cases, R is ethyl. In certain cases, R is C1.3 alkyl. In certain cases, R¹⁰ is C4-6 alkyl

In Formula (II), R¹¹ is selected from hydrogen and C1-6 alkyl. In certain cases, R¹¹ it is hydrogen. In certain cases, R¹¹ is 1-6 alkyl. In certain cases, R¹¹ is methyl. In certain cases, R¹¹ is ethyl. In certain cases, R¹¹ is C1.3 alkyl. In certain cases, R¹¹ is C4-6 alkyl

In Formula (II), R¹² is selected from hydrogen and C1-6 alkyl. In certain cases, R¹² it is hydrogen. In certain cases, R¹² is 1-6 alkyl. In certain cases, R¹² is methyl. In certain cases, R¹² is ethyl. In certain cases, R¹² is C1.3 alkyl. In certain cases, R¹² is C4-6 alkyl

In Formula (II), R¹³ is selected from hydrogen, C1-6 alkyl, and Ce-20 aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, C1-6 alkoxy, or halo. In certain cases, R¹³ it is hydrogen. In certain cases, R¹³ is C1-6 alkyl. In certain cases, R¹³ is C6-20 aryl

In certain cases, R¹³ is unsubstituted C1-6 alkyl. In certain cases, R¹³ is C1-6 alkyl which is substituted with hydroxyl. In certain cases, R¹³ is - (CH2)_mOH, where m is a number from one to 3. In certain cases, R¹³ is -CH2OH. In certain cases, R¹³ is C1-6 alkyl which is substituted with C1-6 alkoxy.

In certain cases, R¹³ is C1-6 alkyl which is substituted with halo. In certain cases, R¹³ is - (CH2) mX, where m is a number from one to 3 and X is halo. In certain cases, R¹³ it is C1-6 alkyl which is substituted with fluoro. In certain cases, R¹³is - (CH2) mF, where m is a number from one to 3. In certain cases, R¹³ is - (CFh ^ F.

In certain cases, R¹³ is unsubstituted Ce-20 aryl. In certain cases, R¹³ is Ce-20 aryl which is substituted with hydroxyl. In certain cases, R¹³ is Ce-20 aryl which is substituted with C1-6 alkoxy. In certain cases, R¹³ is Ce-20 aryl which is substituted with halo.

In certain cases, R⁶, R⁷, R⁸ and R⁹ they are hydrogen. In certain cases, R⁶, R⁷ and R⁸ are hydrogen and R⁹ it's halo. In certain cases, R⁶, R⁷ and R⁸ are hydrogen and R⁹ it is fluoro.

ES 2 618 007 T3

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹⁰ is methyl. In certain cases, R⁶, R⁷, R⁸ they are hydrogen; R⁹ is halo; and R¹⁰ is methyl. In certain cases, R⁶, R⁷, R⁸ they are hydrogen; R⁹ is fluoro; and R¹⁰ is methyl.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹³ it is hydrogen. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is halo; and R¹³ it is hydrogen. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is fluoro; and R¹³ it is hydrogen.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹³ is -CH2OH. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is halo; and R¹³ is -CH2OH. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is fluoro; and R¹³ is -CH2OH.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹³ is - (CH2)_mF, where m is a number from one to 3. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is halo; and R¹³ is - (CH2)_mF, where m is a number from one to 3. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is fluoro; and R¹³ is - (CH2)_mF, where m is a number from one to 3.

Formula (lll)

The present disclosure provides a compound of Formula (III):

<img file="ES2618007T3_D0021.tif" />

in which

R¹ and R² independently selected from hydrogen, halo, C1-6 alkyl, and C1-e haloalkyl;

R³ selected from halo, hydroxyl, C1-6 alkyl, C1-6 alkoxy, cyano, and nitro;

n is a number from zero to 4;

R⁴ selected from hydrogen, C1-6 alkyl, C3-7 cycloalkyl, and -NR²²R²³;

wherein the alkyl or cycloalkyl is unsubstituted or is substituted with hydroxyl or amino; and where each of R²² and R²³ is independently selected from hydrogen and C1-6 alkyl or R²² and R²³ they can be linked to form a 3- to 10-membered ring;

R⁵ selected from hydrogen and Ci-e alkyl;

R⁶ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁷ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁸ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁹ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R¹⁰ selected from hydrogen and Ci-e alkyl;

R¹¹ selected from hydrogen and Ci-e alkyl;

R¹² selected from hydrogen and Ci-e alkyl; Y

R¹³ is selected from hydrogen, C 1-6 alkyl, and Ce-2 aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, C 1-6 alkoxy, or halo; or a pharmaceutically acceptable salt thereof.

In Formula (lll), R¹ and R² they are independently selected from hydrogen, halo, C1-6 alkyl and C1-6 haloalkyl. In certain cases, R¹ it is hydrogen. In certain cases, R¹ is C1-6 alkyl. In certain cases, R¹ it is methyl or ethyl. In certain cases, R² it is hydrogen. In certain cases, R² is C1-6 alkyl. In certain cases, R² it is methyl or ethyl. In certain cases, R¹ and R² they are hydrogen.

ES 2 618 007 T3

In Formula (III), n is a number from zero to 4. In certain cases, n is zero. In certain cases, n is one. In certain cases, n is 2. In certain cases, n is 3. In certain cases, n is 4.

In Formula (III), R³ it is selected from halo, hydroxyl, Cie alkyl, Cie alkoxy, cyano, and nitro. In certain cases, R³ it's halo. In certain cases, R³ is hydroxyl. In certain cases, R³ is Cie alkyl. In certain cases, R³ is Cie alkoxy. In certain cases, R³ is cyano. In certain cases, R³ it is nitro.

In Formula (III), R⁴ selected from hydrogen, Cie alkyl, C3-7 cycloalkyl, and -NR²²R²³; wherein the alkyl or cycloalkyl is unsubstituted or is substituted with hydroxyl or amino; and in each of R²² and R²³ is independently selected from hydrogen and C1-6 alkyl or R²² and R²¹ they can be linked to form a 3- to 10-membered ring. In certain cases, R⁴ it is hydrogen. In certain cases, R⁴ is C1-6 alkyl. In certain cases, R⁴ is C 3-7 cycloalkyl. In certain cases, R⁴ is -NR²²R²³.

In certain cases, R⁴ is unsubstituted C1-6 alkyl. In certain cases, R⁴ is C1-6 alkyl which is substituted with hydroxyl. In certain cases, R⁴ is C1.3 alkyl which is substituted with hydroxyl. In certain cases, R⁴ is C1-6 alkyl which is substituted with amino. In certain cases, R⁴ is C1-6 alkyl which is substituted with -NH2. In certain cases, R⁴ is C1-6 alkyl which is substituted with -N (CH3) 2- In certain cases, R⁴ is C1.3 alkyl which is substituted with -NH2. In certain cases, R⁴ is C1.3 alkyl which is substituted with -N (CH3) 2 ·.

In certain cases, R⁴ is unsubstituted C 3-7 cycloalkyl. In certain cases, R⁴ is unsubstituted C3 cycloalkyl. In certain cases, R⁴ is cycloalkyl C₄ not replaced. In certain cases, R⁴ is unsubstituted C5-6 cycloalkyl. In certain cases, R⁴ is C7 unsubstituted cycloalkyl.

In certain cases, R⁴ is -NR²²R²³ where each of R²² and R²³ are independently selected from hydrogen and C1-6 alkyl or R²² and R²³ they can be linked to form a 3- to 10-membered ring. In certain cases, R²² and R²³ they are hydrogen. In certain cases, R²² and R²³ are C1-6 alkyl. In certain cases, R²² and R²³ they are C1.3 alkyl. In certain cases, R²²and R²³ they are methyl.

In certain cases, R²² and R²³ may be linked to form a 3 to 10 membered ring, so that R⁴ that is, where w is a number from 1 to 8. In certain cases, R²² and R²³ they can be linked to form a 3-membered ring. In certain cases, R²² and R²³ they can be linked to form a 4-membered ring. In certain cases, R²² and R²³ they can be linked to form a 5-membered ring. In certain cases, R²² and R²³ they can be linked to form a 6-membered ring. In certain cases, R²² and R²³ they can be linked to form a 7-membered ring.

In Formula (III), R⁵ is selected from hydrogen and C1-6 alkyl. In certain cases, R⁵ it is hydrogen. In certain cases, R⁵is C1-6 alkyl. In certain cases, R⁵ is methyl. In certain cases, R⁵ is ethyl. In certain cases, R⁵ is C1.3 alkyl. In certain cases, R⁵ is C alkyl₄-6 In Formula (III), R⁶ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁶ it is hydrogen. In certain cases, R⁶ it's halo. In certain cases, R⁶ it is fluoro. In certain cases, R⁶ it is chlorine. In certain cases, R⁶ it is bromine. In certain cases, R⁶ is C1-6 alkyl. In certain cases, R⁶is C1-6 haloalkyl. In certain cases, R⁶ is C2-6 alkoxyIn certain cases, R⁶ is C1-6 haloalkoxy. In certain cases, R⁶ is hydroxyl. In certain cases, R⁶ is cyano. In certain cases, R⁶ it is nitro.

In Formula (III), R⁷ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁷ it is hydrogen. In certain cases, R⁷ it's halo. In certain cases, R⁷ it is fluoro. In certain cases, R⁷ it is chlorine. In certain cases, R⁷ it is bromine. In certain cases, R⁷ is C1-6 alkyl. In certain cases R⁷is C1-6 haloalkyl. In certain cases, R⁷ is C2-6 alkoxyIn certain cases, R⁷ is C1-6 haloalkoxy. In certain cases, R⁷ is hydroxyl. In certain cases, R⁷ is cyano. In certain cases, R⁷ it is nitro.

In Formula (III), R³ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R³ it is hydrogen. In certain cases, R³ it's halo. In certain cases, R³ it is fluoro. In certain cases, R is chlorine. In certain cases, R is bromine. In certain cases, R is C1-6 alkyl. In certain cases R is C1-6 haloalkyl. In certain cases, R³ is C1-6 alkoxy. In certain cases, R³ is C1-6 haloalkoxy. In certain cases, R³ is hydroxyl. In certain cases, R³ is cyano. In certain cases, R³ it is nitro.

In Formula (III), R⁹ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁹ it is hydrogen. In certain cases, R⁹ it's halo. In certain cases, R⁹ it is fluoro. In certain cases, R⁹ it is chlorine. In certain cases, R⁹ it is bromine. In certain cases, R⁹ is C1-6 alkyl. In certain cases R⁹is C1-6 haloalkyl. In certain cases, R⁹ is C1-6 alkoxy. In certain cases, R⁹ is C1-6 haloalkoxy. In certain cases, R⁹ is hydroxyl. In certain cases, R⁹ is cyano. In certain cases, R⁹ it is nitro.

In Formula (III), R¹⁰ is selected from hydrogen and C1-6 alkyl. In certain cases, R¹⁰ it is hydrogen. In certain cases, 10 '' 10^{J 1} 10 10

R is C1-6 alkyl. In certain cases, R is methyl. In certain cases, R is ethyl. In certain cases, R is C1.3 alkyl. In certain cases, R¹⁰ is C alkyl₄-621

ES 2 618 007 T3

In Formula (III), R¹¹ is selected from hydrogen and Cie alkyl. In certain cases, R¹¹ it is hydrogen. In certain cases, R¹¹ is Cie alkyl. In certain cases, R¹¹ is methyl. In certain cases, R¹¹ is ethyl. In certain cases, R¹¹ is C1.3 alkyl. In certain cases, R¹¹ is C4-6 alkyl

In Formula (III), R¹² is selected from hydrogen and C1-6 alkyl. In certain cases, R¹² it is hydrogen. In certain cases, R¹² is 1-6 alkyl. In certain cases, R¹² is methyl. In certain cases, R¹² is ethyl. In certain cases, R¹² is C1.3 alkyl. In certain cases, R¹² is C4-6 alkyl

In Formula (III), R¹³ is selected from hydrogen, C1-6 alkyl, and Ce-20 aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, C1-6 alkoxy, or halo. In certain cases, R³ it is hydrogen. In certain cases, R¹³ is C1-6 alkyl. In certain cases, R¹³ is C6-20 aryl

In certain cases, R¹³ is unsubstituted C1-6 alkyl. In certain cases, R¹³ is C1-6 alkyl which is substituted with hydroxyl. In certain cases, R¹³ is - (CH2)_mOH, where m is a number from one to 3. In certain cases, R¹³ is -CH2OH. In certain cases, R¹³ is C1-6 alkyl which is substituted with C1-6 alkoxy.

In certain cases, R¹³ is C1-6 alkyl which is substituted with halo. In certain cases, R¹³ is - (CH2) mX, where m is a number from one to 3 and X is halo. In certain cases, R¹³ it is C1-6 alkyl which is substituted with fluoro. In certain cases, R¹³is - (CH2)_mF, where m is a number from one to 3. In certain cases, R¹³ is - (CH₂)₂F.

In certain cases, R¹³ is unsubstituted Ce-20 aryl. In certain cases, R¹³ is Ce-20 aryl which is substituted with hydroxyl. In certain cases, R¹³ is Ce-20 aryl which is substituted with C1-6 alkoxy. In certain cases, R¹³ is Ce-20 aryl which is substituted with halo.

In certain cases, R⁶, R⁷, R⁸ and R⁹ they are hydrogen. In certain cases, R⁶, R⁷ and R⁸ are hydrogen and R⁹ it's halo. In certain cases, R⁶, R⁷ and R⁸ are hydrogen and R⁹ it is fluoro.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹⁰ is methyl. In certain cases, R⁶, R⁷, R⁸ they are hydrogen; R⁹ is halo; and R¹⁰ is methyl. In certain cases, R⁶, R⁷, R⁸ they are hydrogen; R⁹ is fluoro; and R¹⁰ is methyl.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹³ it is hydrogen. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is halo; and R¹³ it is hydrogen. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is fluoro; and R¹³ it is hydrogen.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹³ is -CH2OH. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is halo; and R¹³ is -CH2OH. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is fluoro; and R¹³ is -CH₂OH.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹³ is - (CH₂)_mF, where m is a number from one to 3. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is halo; and R¹³ is - (CH₂)_mF, where m is a number from one to 3. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is fluoro; and R¹³ is - (CH₂)_mF, where m is a number from one to 3.

Formula (IV)

The present disclosure provides a compound of Formula (IV):

<img file="ES2618007T3_D0022.tif" />

in which

R¹ and R² are independently selected from hydrogen, halo, C1-6 alkyl, and Cie haloalkyl; R³ selected from halo, hydroxyl, C1-6 alkyl, C1-6 alkoxy, cyano, and nitro; n is a number from zero to 4;

R⁴ selected from hydrogen, C1-6 alkyl, C3-7 cycloalkyl, and -NR²²R²³;

wherein the alkyl or cycloalkyl is unsubstituted or is substituted with hydroxyl or amino; Y

ES 2 618 007 T3 where each of R²² and R²³ is independently selected from hydrogen and Cie alkyl or R²² and R²³ they can be linked to form a 3- to 10-membered ring;

R⁵ is selected from hydrogen and Cie alkyl;

R⁶ selected from hydrogen, halo, Cie alkyl, Cie haloalkyl, C alkoxy₂-6, haloalkoxy Cie, hydroxyl, cyano and nitro;

R⁷ selected from hydrogen, halo, Cie alkyl, Cie haloalkyl, C alkoxy₂.e, haloalkoxy Cie, hydroxyl, cyano, and nitro;

R³ selected from hydrogen, halo, Cie alkyl, Cie haloalkyl, Cie alkoxy, Cie haloalkoxy, hydroxyl, cyano, and nitro;

R¹⁰ is selected from hydrogen and Cie alkyl;

R¹¹ is selected from hydrogen and Cie alkyl;

R¹² is selected from hydrogen and Cie alkyl; Y

R¹³ is selected from hydrogen, Cie alkyl and aryl Οβ-₂ο, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, Cie alkoxy, or halo; or a pharmaceutically acceptable salt thereof.

In Formula (IV), R¹ and R² are independently selected from hydrogen, halo, Cie alkyl, and Cie haloalkyl. In certain cases, R¹ it is hydrogen. In certain cases, R¹ is Cie alkyl. In certain cases, R¹ it is methyl or ethyl. In certain cases, R² it is hydrogen. In certain cases, R² is Cie alkyl. In certain cases, R² it is methyl or ethyl. In certain cases, R¹ and R² they are hydrogen.

In Formula (IV), n is a number from zero to 4. In certain cases, n is zero. In certain cases, n is one. In certain cases, n is 2. In certain cases, n is 3. In certain cases, n is 4.

In Formula (IV), R³ it is selected from halo, hydroxyl, Cie alkyl, Cie alkoxy, cyano, and nitro. In certain cases, R³ it's halo. In certain cases, R³ is hydroxyl. In certain cases, R³ is Cie alkyl. In certain cases, R³ is Cie alkoxy. In certain cases, R³ is cyano. In certain cases, R³ it is nitro.

In Formula (IV), R⁴ selected from hydrogen, Cie alkyl, C3-7 cycloalkyl, and -NR²²R²³; wherein the alkyl or cycloalkyl is unsubstituted or is substituted with hydroxyl or amino; and where each of R²² and R²³ is independently selected from hydrogen and C1-6 alkyl or R²² and R²¹ they can be linked to form a 3- to 10-membered ring. In certain cases, R⁴ it is hydrogen. In certain cases, R⁴ is C1-6 alkyl. In certain cases, R⁴ is C 3-7 cycloalkyl. In certain cases, R⁴ is -NR²²R²³.

In certain cases, R⁴ is unsubstituted C1-6 alkyl. In certain cases, R⁴ is C1-6 alkyl which is substituted with hydroxyl. In certain cases, R⁴ is C1.3 alkyl which is substituted with hydroxyl. In certain cases, R⁴ is C1-6 alkyl which is substituted with amino. In certain cases, R⁴ is C1-6 alkyl which is substituted with -NH2. In certain cases, R⁴ is C1-6 alkyl which is substituted with -N (CH3) 2. In certain cases, R⁴ is C1.3 alkyl which is substituted with -NH2. In certain cases, R⁴ is C1.3 alkyl which is substituted with -N (CH3) 2.

In certain cases, R⁴ is unsubstituted C 3-7 cycloalkyl. In certain cases, R⁴ is unsubstituted C3 cycloalkyl. In certain cases, R⁴ is unsubstituted C4 cycloalkyl. In certain cases, R⁴ is unsubstituted C5-6 cycloalkyl. In certain cases, R⁴ is unsubstituted C7 cycloalkyl.

In certain cases, R⁴ is -NR²²R²³ where each of R²² and R²³ is independently selected from hydrogen and C1-6 alkyl or R²² and R²³ they can be linked to form a 3- to 10-membered ring. In certain cases, R²² and R²³ they are hydrogen. In certain cases, R²² and R²³ they are C1-6 alkyl. In certain cases, R²² and R²³ they are C1.3 alkyl. In certain cases, R²²and R²³ they are methyl.

In certain cases, R²² and R²³ may be linked to form a 3 to 10 membered ring, so that R⁴ be

<img file="ES2618007T3_D0023.tif" />

, where w is a number from 1 to 8. In certain cases, R²² and R²³ they can be linked to form a 3-membered ring. In certain cases, R²² and R²³ they can be linked to form a 4-membered ring. In certain cases, R²² and R²³ they can be linked to form a 5-membered ring. In certain cases, R²² and R²³ they can be linked to form a 6-membered ring. In certain cases, R²² and R²³ they can be linked to form a 7-membered ring.

In Formula (IV), R⁵ is selected from hydrogen and C1-6 alkyl. In certain cases, R⁵ it is hydrogen. In certain cases, R⁵is C1-6 alkyl. In certain cases, R⁵ is methyl. In certain cases, R⁵ is ethyl. In certain cases, R⁵ is C1.3 alkyl. In certain cases, R⁵ is C4-6 alkyl

In Formula (IV), R⁶ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁶ it is hydrogen. In certain cases, R⁶ it's halo. In certain cases, R⁶ it is fluoro.

ES 2 618 007 T3

In certain cases, R⁶ it is chlorine. In certain cases, R⁶ it is bromine. In certain cases, R⁶ is Cie alkyl. In certain cases R⁶is haloalkyl Cie. In certain cases, R⁶ is C2-6 alkoxyIn certain cases, R⁶ is C1-6 haloalkoxy. In certain cases, R ° is hydroxyl. In certain cases, R⁶ is cyano. In certain cases, R⁶ it is nitro.

In Formula (IV), R⁷ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁷ it is hydrogen. In certain cases, R⁷ it's halo. In certain cases, R⁷ it is fluoro. In certain cases, R⁷ it is chlorine. In certain cases, R⁷ it is bromine. In certain cases, R⁷ is C1-6 alkyl. In certain cases R⁷is C1-6 haloalkyl. In certain cases, R⁷ is C2-6 alkoxyIn certain cases, R⁷ is C1-6 haloalkoxy. In certain cases, R⁷ is hydroxyl. In certain cases, R⁷ is cyano. In certain cases, R⁷ it is nitro.

In Formula (IV), R⁸ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁸ it is hydrogen. In certain cases, R⁸ it's halo. In certain cases, R⁸ it is fluoro. In certain cases, R is chlorine. In certain cases, R is bromine. In certain cases, R is C1-6 alkyl. In certain cases R is C1-6 haloalkyl. In certain cases, R⁸ is C1-6 alkoxy. In certain cases, R⁸ is C1-6 haloalkoxy. In certain cases, R⁸ is hydroxyl. In certain cases, R⁸ is cyano. In certain cases, R⁸ it is nitro.

In Formula (IV), R¹⁰ is selected from hydrogen and C1-6 alkyl. In certain cases, R¹⁰ it is hydrogen. In certain cases, 10 '' 10^{J 1} 10 10

R is C1-6 alkyl. In certain cases , R is methyl. In certain cases, R is ethyl. In certain cases, R is C1.3 alkyl. In certain cases, R¹⁰ is C4-6 alkyl In Formula (IV), R¹¹ is selected from hydrogen and C1-6 alkyl. In certain cases, R¹² it is hydrogen. In certain cases, R¹¹ is 1-6 alkyl. In certain cases, R¹¹ is methyl. In certain cases, R¹¹ is ethyl. In certain cases, R¹¹ is C1.3 alkyl. In certain cases, R¹¹ is C4-6 alkyl In Formula (IV), R¹² is selected from hydrogen and C1-6 alkyl. In certain cases, R¹² it is hydrogen. In certain cases, R¹² is 1-6 alkyl. In certain cases, R¹² is methyl. In certain cases, R¹² is ethyl. In certain cases, R¹² is C1.3 alkyl. In certain cases, R¹² is C alkyl₄-6 In Formula (IV), R¹³ is selected from hydrogen, C 1-6 alkyl, and Ce-2 aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, C 1-6 alkoxy, or halo. In certain cases, R³ it is hydrogen. In certain cases, R¹³ is C1-6 alkyl. In certain cases, R¹³ is C6-20 aryl

In certain cases, R¹³ is unsubstituted C1-6 alkyl. In certain cases, R¹³ is C1-6 alkyl which is substituted with hydroxyl. In certain cases, R¹³ is - (CH2)_mOH, where m is a number from one to 3. In certain cases, R¹³ is -CH2OH. In certain cases, R¹³ is C1-6 alkyl which is substituted with C1-6 alkoxy.

In certain cases, R¹³ is C1-6 alkyl which is substituted with halo. In certain cases, R¹³ is - (CH2) mX, where m is a number from one to 3 and X is halo. In certain cases, R¹³ it is C1-6 alkyl which is substituted with fluoro. In certain cases, R¹³is - (CH2) mF, where m is a number from one to 3. In certain cases, R¹³ is - (CFh ^ F.

In certain cases, R¹³ is unsubstituted Ce-20 aryl. In certain cases, R¹³ is Ce-20 aryl which is substituted with hydroxyl. In certain cases, R¹³ is Ce-20 aryl which is substituted with C1-6 alkoxy. In certain cases, R¹³ is Ce-20 aryl which is substituted with halo.

In certain cases, R⁶, R⁷ and R⁸ they are hydrogen. In certain cases, R⁶, R⁷ and R⁸ are hydrogen and R¹⁰ is methyl. In certain cases, R⁶, R⁷ and R⁸ are hydrogen and R¹³ it is hydrogen. In certain cases, R⁶, R⁷ and R⁸ are hydrogen and R¹³ is -CH2OH. In certain cases, R⁶, R⁷ and R⁸ are hydrogen and R¹³ it is hydrogen. In certain cases, R⁶, R⁷ and R ^ are hydrogen and R¹³ is (CH2)_mF, where m is a number from one to 3.

Formula (V)

The present disclosure provides a compound of Formula (V):

<img file="ES2618007T3_D0024.tif" />

in which

ES 2 618 007 T3

R¹ and R² independently selected from hydrogen, halo, Ci-6 alkyl and Ci_e haloalkyl;

R⁶ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁷ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R⁸ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

Q is CR⁹or N;

R⁹ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R¹⁰ selected from hydrogen and C1-6 alkyl;

R¹¹ selected from hydrogen and C1-6 alkyl;

R¹² selected from hydrogen and C1-6 alkyl;

R¹³ is selected from hydrogen, C1-6 alkyl, and C6-20 aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, C1-6 alkoxy, or halo; or a pharmaceutically acceptable salt thereof.

In Formula (V), R¹ and R² they are independently selected from hydrogen, halo, C1-6 alkyl and C1-6 haloalkyl. In certain cases, R¹ it is hydrogen. In certain cases, R¹ is C1-6 alkyl. In certain cases, R¹ it is methyl or ethyl. In certain cases, R² it is hydrogen. In certain cases, R² is C1-6 alkyl. In certain cases, R² it is methyl or ethyl. In certain cases, R¹ and R² they are hydrogen.

In Formula (V), R⁶ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁶ it is hydrogen. In certain cases, R⁶ it's halo. In certain cases, R⁶ it is fluoro. In certain cases, R⁶ it is chlorine. In certain cases, R⁶ it is bromine. In certain cases, R⁶ is C1-6 alkyl. In certain cases, R⁶is C1-6 haloalkyl. In certain cases, R⁶ is C2-6 alkoxy. In certain cases, R⁶ is C1-6 haloalkoxy. In certain cases, R⁶ is hydroxyl. In certain cases, R⁶ is cyano. In certain cases, R⁶ it is nitro.

In Formula (V), R⁷ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C2-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁷ it is hydrogen. In certain cases, R⁷ it's halo. In certain cases, R⁷ it is fluoro. In certain cases, R⁷ it is chlorine. In certain cases, R⁷ it is bromine. In certain cases, R⁷ is C1-6 alkyl. In certain cases, R⁷is C1-6 haloalkyl. In certain cases, R⁷ is C2-6 alkoxy. In certain cases, R⁷ is C1-6 haloalkoxy. In certain cases, R⁷ is hydroxyl. In certain cases, R⁷ is cyano. In certain cases, R⁷ it is nitro.

In Formula (V), R⁸ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁸ it is hydrogen. In certain cases, R⁸ it's halo. In certain cases, R⁸ it is fluoro. In certain cases, R⁸ it is chlorine. In certain cases, R⁸ it is bromine. In certain cases, R⁸ is C1-6 alkyl. In certain cases, R⁸is C1-6 haloalkyl. In certain cases, R⁸ is C1-6 alkoxy. In certain cases, R⁸ is C1-6 haloalkoxy. In certain cases, R⁸ is hydroxyl. In certain cases, R⁸ is cyano. In certain cases, R⁸ it is nitro.

In Formula (V), Q is CR⁹ or N. In certain cases, Q is CR⁹. In certain cases, Q is N.

In Formula (V), R⁹ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁹ it is hydrogen. In certain cases, R⁹ it's halo. In certain cases, R⁹ it is fluoro. In certain cases, R⁹ it is chlorine. In certain cases, R⁹ it is bromine. In certain cases, R⁹ is C1-6 alkyl. In certain cases, R⁹is C1-6 haloalkyl. In certain cases, R⁹ is C1-6 alkoxy. In certain cases, R⁹ is C1-6 haloalkoxy. In certain cases, R⁹ is hydroxyl. In certain cases, R⁹ is cyano. In certain cases, R⁹ it is nitro.

In Formula (V), R¹⁰ is selected from hydrogen and C1-6 alkyl. In certain cases, R¹⁰ it is hydrogen. In certain cases, 10 10 10 10

R is C1-6 alkyl. In certain cases, R is methyl. In certain cases, R is ethyl. In certain cases, R is C1-3 alkyl. In certain cases, R¹⁰ is C4-6 alkyl.

In Formula (V), R¹¹ is selected from hydrogen and C1-6 alkyl. In certain cases, R¹¹ it is hydrogen. In certain cases, 11 11 11 11

R is C1-6 alkyl. In certain cases, R is methyl. In certain cases, R is ethyl. In certain cases, R is C1-3 alkyl. In certain cases, R¹¹ is C4-6 alkyl.

In Formula (V), R¹² is selected from hydrogen and C1-6 alkyl. In certain cases, R¹² it is hydrogen. In certain cases, R¹² is C1-6 alkyl. In certain cases, R¹² is methyl. In certain cases, R¹² is ethyl. In certain cases, R¹² is C1-3 alkyl. In certain cases, R¹² is C4-6 alkyl.

In Formula (V), R¹³ is selected from hydrogen, C1-6 alkyl and C6-20 aryl, wherein each alkyl or aryl is unsubstituted or is substituted with hydroxyl, C1-6 alkoxy, or halo. In certain cases, R¹³ it is hydrogen. In certain cases, R¹³ is C1-6 alkyl. In certain cases, R¹³ is C6-20 aryl.

ES 2 618 007 T3

In certain cases, R¹³ is unsubstituted Cie alkyl. In certain cases, R¹³ is Cie alkyl which is substituted with hydroxyl. In certain cases, R¹³ is - (CHhjmOH, where m is a number from one to 3. In certain cases, R¹³ is -CH2OH. In certain cases, R¹³ is C1-6 alkyl which is substituted with C1-6 alkoxy.

In certain cases, R¹³ is C1-6 alkyl which is substituted with halo. In certain cases, R¹³ is - (CHhjmX, where m is a number from one to 3 and X is halo. In certain cases, R¹³ it is C1-6 alkyl which is substituted with fluoro. In certain cases, R¹³is - (CH2)_mF, where m is a number from one to 3. In certain cases, R¹³ is - (CEL ^ F.

In certain cases, R¹³ is unsubstituted C6-20 aryl. In certain cases, R¹³ is C6-20 aryl which is substituted with hydroxyl. In certain cases, R¹³ is C6-20 aryl which is substituted with C1-6 alkoxy. In certain cases, R¹³ is C6-20 aryl which is substituted with halo.

In certain cases, R⁶, R⁷, R⁸ and R⁹ they are hydrogen. In certain cases, R⁶, R⁷ and R⁸ are hydrogen and R⁹ it's halo. In certain cases, R⁶, R⁷ and R⁸ are hydrogen and R⁹ it is fluoro.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹⁰ is methyl. In certain cases, R⁶, R⁷, R⁸ they are hydrogen; R⁹ is halo; and R¹⁰ is methyl. In certain cases, R⁶, R⁷, R⁸ they are hydrogen; R⁹ is fluoro; and R¹⁰ is methyl.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹³ it is hydrogen. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is halo; and R¹³ it is hydrogen. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is fluoro; and R¹³ it is hydrogen.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹³ is -CH2OH. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is halo; and R¹³ is -CH2OH. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is fluoro; and R¹³ is -CH2OH.

In certain cases, R⁶, R⁷, R⁸ and R⁹ are hydrogen and R¹³ is - (CH2)_mF, where m is a number from one to 3. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is halo; and R¹³ is - (CH2)_mF, where m is a number from one to 3. In certain cases, R⁶, R⁷ and R⁸ they are hydrogen; R⁹ is fluoro; and R¹³ is - (CH2)_mF, where m is a number from one to 3.

Formula (Vl)

The present disclosure provides a compound of Formula (Vl):

<img file="ES2618007T3_D0025.tif" />

in which

R¹ and R² independently selected from hydrogen, halo, C1-6 alkyl, and Cie haloalkyl;

R⁸ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

Q is CR⁹ or N;

R⁹ selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro;

R¹⁰ is selected from hydrogen and Cie alkyl; Y

R¹³ is selected from hydrogen, C1-6 alkyl, and C6-20 aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, C1-6 alkoxy, or halo; or a pharmaceutically acceptable salt thereof.

In Formula (Vl), R¹ and R² they are independently selected from hydrogen, halo, C1-6 alkyl and C1-6 haloalkyl. In certain cases, R¹ it is hydrogen. In certain cases, R¹ is C1-6 alkyl. In certain cases, R¹ it is methyl or ethyl. In certain cases, R² it is hydrogen. In certain cases, R² is C1-6 alkyl. In certain cases, R² it is methyl or ethyl. In certain cases, R¹ and R² they are hydrogen.

In Formula (Vl), R⁸ It is selected from hydrogen, halo, C1-6 alkyl, C1-6 haloalkyl, C1-6 alkoxy, C1-6 haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁸ it is hydrogen. In certain cases, R⁸ it's halo. In certain cases, R⁸ it is fluoro. In certain cases, R is chlorine. In certain cases, R is bromine. In certain cases, R is C1-6 alkyl. In certain cases, R

ES 2 618 007 T3 is Cie. In certain cases, R⁸ is Cie alkoxy. In certain cases, R⁸ is haloalkoxy Cie. In certain cases, R⁸ is hydroxyl. In certain cases, R⁸ is cyano. In certain cases, R⁸ it is nitro.

In Formula (VI), Q is CR⁹ or N. In certain cases, Q is CR⁹. In certain cases, Q is N.

In Formula (VI), R⁹ selected from hydrogen, halo, Cie alkyl, Cie haloalkyl, Cie alkoxy, Cie haloalkoxy, hydroxyl, cyano, and nitro. In certain cases, R⁹ it is hydrogen. In certain cases, R⁹ it's halo. In certain cases, R⁹ it is fluoro. In certain cases, R⁹ it is chlorine. In certain cases, R⁹ it is bromine. In certain cases, R⁹ is Cie alkyl. In certain cases R⁹is haloalkyl Cie. In certain cases, R⁹ is Cie alkoxy. In certain cases, R⁹ is haloalkoxy Cie. In certain cases, R⁹ is hydroxyl. In certain cases, R⁹ is cyano. In certain cases, R⁹ it is nitro.

In Formula (VI), R¹⁰ is selected from hydrogen and Cie alkyl. In certain cases, R¹⁰ it is hydrogen. In certain cases, '' 10^J * 10 10

R is Cie alkyl. In certain cases, R is methyl. In certain cases, R is ethyl. In certain cases, R is C1.3 alkyl. In certain cases, R¹⁰ is C4-6 alkyl

In Formula (VI), R¹³ is selected from hydrogen, C1-6 alkyl, and Ce-20 aryl, wherein each alkyl or aryl is unsubstituted or substituted with hydroxyl, C1-6 alkoxy, or halo. In certain cases, R³ it is hydrogen. In certain cases, R¹³ is C1-6 alkyl. In certain cases, R¹³ is C6-20 aryl

In certain cases, R¹³ is unsubstituted C1-6 alkyl. In certain cases, R¹³ is C1-6 alkyl which is substituted with hydroxyl. In certain cases, R¹³ is - (CH2)_mOH, where m is a number from one to 3. In certain cases, R¹³ is -CH2OH. In certain cases, R¹³ is C1-6 alkyl which is substituted with C1-6 alkoxy.

In certain cases, R¹³ is C1-6 alkyl which is substituted with halo. In certain cases, R¹³ is - (CH2) mX, where m is a number from one to 3 and X is halo. In certain cases, R¹³ it is C1-6 alkyl which is substituted with fluoro. In certain cases, R¹³is - (CH2) mF, where m is a number from one to 3. In certain cases, R¹³ is - (Ch ^ F.

In certain cases, R¹³ is unsubstituted Ce-20 aryl. In certain cases, R¹³ is Ce-20 aryl which is substituted with hydroxyl. In certain cases, R¹³ is Ce-20 aryl which is substituted with Ci_e alkoxy. In certain cases, R¹³ is Ce-20 aryl which is substituted with halo.

In certain cases, R⁸ and R⁹ they are hydrogen. In certain cases, R⁸ is hydrogen and R⁹ it's halo. In certain cases, R⁸ is hydrogen and R⁹ it is fluoro.

In certain cases, R⁸ and R⁹ are hydrogen and R¹⁰ is methyl. In certain cases, R⁸ is hydrogen; R⁹ is halo; and R¹⁰ is methyl. In certain cases, R⁸ is hydrogen; R⁹ is fluoro; and R¹⁰ is methyl.

In certain cases, R⁸ and R⁹ are hydrogen and R¹³ it is hydrogen. In certain cases, R⁸ is hydrogen; R⁹ is halo; and R¹³ it is hydrogen. In certain cases, R⁸ is hydrogen; R⁹ is fluoro; and R¹³ it is hydrogen.

In certain cases, R⁸ and R⁹ are hydrogen and R¹³ is -CH2OH. In certain cases, R⁸ is hydrogen; R⁹ is halo; and R¹³ is CH2OH. In certain cases, R⁸ is hydrogen; R⁹ is fluoro; and R¹³ is -CH2OH.

In certain cases, R⁸ and R⁹ are hydrogen and R¹³ is - (CH2) mF, where m is a number from one to 3. In certain cases, R⁸is hydrogen; R⁹ is halo; and R¹³ is - (CH2) mF, where m is a number from one to 3. In certain cases, R⁸ is hydrogen; R⁹ is fluoro; and R¹³ is - (CH2)_mF, where m is a number from one to 3.

Formula (Vil)

The present disclosure provides a compound of Formula (Vil):

<img file="ES2618007T3_D0026.tif" />

in which

R¹ and R² independently selected from hydrogen, halo, C1-6 alkyl, and C1-e haloalkyl;

ES 2 618 007 T3

R⁸ selected from halo, Ci-e alkyl, Ci-e haloalkyl, Ci-e alkoxy, Ci-e haloalkoxy, hydroxyl, cyano, and nitro;

R¹⁰ is Ci-e alkyl; Y

R¹³ it is hydrogen or Ci-ei alkyl or a pharmaceutically acceptable salt thereof.

In some embodiments of Formula (Vil), R¹ and R² they are each hydrogen. In other embodiments, R⁸ it is halo, methyl, methoxy, or cyano. In still other embodiments, R⁸ it's halo. In still other embodiments, R⁸ it is fluoro. In some embodiments, R¹⁰ it is methyl, ethyl or isopropyl. In other embodiments, R¹⁰ is methyl. In some embodiments, R ~ is hydrogen, methyl, ethyl, or isopropyl. In other embodiments, R¹³ it is hydrogen.

In some embodiments of compounds of Formulas (I) - (VI), R⁶ and R⁷ they can also be methoxy, provided that neither R⁶ nor R⁷ let R be methoxy¹⁰ let it be methyl. In other embodiments of Formulas (l) - (VII), the group

<img file="ES2618007T3_D0027.tif" />

N-í is replaced by ^r15_n ^^ nh, where my R¹⁵ are defined herein.

Formula XX

In some embodiments, a compound of the invention is a compound of Formula XX:

<img file="ES2618007T3_D0028.tif" />

and NN r34 H (XX) in which:

R³⁰ and R³¹ are each independently H or C1-4 alkyl;

R³² is H and R³³ it is _R36__N^\_;

or R³² and R³³ taken together with the nitrogen to which they are bound form

<img file="ES2618007T3_D0029.tif" />

N-ien where R³⁶ is H, C1-4 alkyl or C1-4 haloalkyl; and R³⁷ is H or C1-4 alkyl;

Q is CH, CF, or N;

R³⁴ is H or methoxy;

R³⁵ is H, -CH2OH or -CH2CH2F;

<img file="ES2618007T3_D0030.tif" />

Nf, and R³⁷ is methyl; or a salt

32 33/37 where R is not methoxy when Q is CH, -NR R is -, and R is methyl; or or pharmaceutically acceptable thereof.

31 32 33

In some embodiments of Formula (XX), R and R are both H. In some embodiments R is H and R is

X '. In some embodiments, R³⁶ is -CH2CH2F. In other embodiments, R³² and R³³ taken together with the _R3⁷-n^Z nitrogen to which they are bound form '' -. In still other embodiments, R³⁷ is methyl. In other embodiments, Q is N and R³⁴ is H. In still other embodiments, Q is CH and R³⁴ it is methoxy. In still other embodiments, Q is CF and R³⁴ is H. In other embodiments, R³⁵ it's H.

ES 2 618 007 T3

In certain embodiments, the present disclosure provides a compound selected from:

<td>Compound</td><td>Structure</td><td>Chemical name</td>

<td> 1</td><td>0 ^^ NH OR Η H</td><td>N- (3 - ((2 - ((4- (4-metllplperac¡n-1 -¡l) phen¡l) amino) -7H-p¡rrolo [2,3-d] p¡r¡ m¡d¡n-4¡l) oxl) fen¡l) acr¡lam¡da</td>

<td> 2</td><td>OR Λ Η Η</td><td>N- (3 - ((2 - ((6- (4-metllplperac¡n-1 -ll) plr¡d ¡n-3-ll) amino) -7H-p¡rrolo [2,3d] p R¡m¡dln-4-¡l) ox¡) fen¡l) acr¡lam¡da</td>

<td> 3</td><td>0 ^ ΝΗ ό '' Ν '© -^Νγ © Η H</td><td>N- (3 - ((2 - ((3-fluoro-4- (4-metllp¡perac¡n-1 -ll) phen¡l) amino) -7H-plrrolo [2,3d] p¡r ¡M¡dln-4-¡l) ox¡) fen¡l) acr¡lam¡da</td>

<td> 4</td><td>Ο ^ ΝΗ Λ^ΧΝ © ^ ''^NV ©^Η ) ΗΟ</td><td>N- (3 - ((7- (hydrox¡met¡l) -2 - ((4- (4-methylp¡perac¡n-1-¡l) phen¡l) am¡no) - 7Hplrrolo [2,3-d] plr¡m¡d ¡n-4-ll) ox¡) phen¡l) acrylam¡da</td>

<td> 5</td><td>0 ^ ΝΗ Λ^Η ) ΗΟ</td><td>N- (3 - ((7- (h¡drox¡met¡l) -2 - ((6- (4-met¡lp¡perac¡n-1-¡l) p¡r¡d¡n-3 -¡L) am¡no) -7Hplrrolo [2,3-d] plr¡m¡d ¡n-4-¡l) ox¡) phen¡l) acrllam¡da</td>

<td> 6</td><td>0 ^ ΝΗ Λ -Ν © ^^Νη ^nxSa fAA_nTO_n^_n^;^Η ) ΗΟ</td><td>N- (3 - ((2 - ((3-fluoro-4- (4-metllp¡perac¡n-1 -ll) phen¡l) am¡no) -7- (hydrox¡met¡l) -7Hp¡rrolo [2,3-d] p¡r¡m¡dln-4-¡l) ox¡) phenl) acrylamide; Y</td>

ES 2 618 007 T3

<td>Compound</td><td>Structure</td><td>Chemical name</td>

<td> 7</td><td>z OR SA</td><td>N- (3 - ((7- (2-fluoroethyl) -2 - ((4- (4-methylpiperazin-1-yl) phenyl) amino) -7H-pyrrolo [2,3- d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

and pharmaceutically acceptable salts thereof.

In certain embodiments, the present disclosure provides a compound selected from:

<td>Compound</td><td>Structure</td><td>Chemical name</td>

<td> 8</td><td>0 NH Λ H ^H</td><td>N- (3 - ((2 - ((1- (2- (dimethylamino) ethyl) -1 H-indol-5-yl) amino) -7H-pyrrolo [2,3djpyrimid in-4-yl) oxy) phenyl ) acrylamide</td>

<td> 9</td><td>OR NH 6 Η H</td><td>N- (3 - ((2 - ((2 - ((dimethylamino) methyl) quinolin-6-yl) amino) -7H-pyrrolo [2,3djpyrimid in-4-yl) oxy) phenyl) acrylamide</td>

<td> 10</td><td>0 Λ H H</td><td>N- (3 - ((5-cyclopropyl-2 - ((4- (4-methylpiperazin-1-yl) phenyl) amino) -7Hpyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

<td> 11</td><td>OR N ^ | 0-0 γ JÚ3 H H</td><td>N- (3 - ((5-cyclopropyl-2 - ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) 7H-pyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

ES 2 618 007 T3

<td>Compound</td><td>Structure</td><td>Chemical name</td>

<td> 12</td><td>0 Η H</td><td>N- (3 - ((2 - ((4- (4-methylpiperazin-1-yl) phenyl) amino) -5- (pyrrolidin-1-yl) -7H- pyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

<td> 13</td><td>0 'n ^ l Óo 0 f - '^ AA H H</td><td>N- (3 - ((2 - ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) -5- (pyrrolidin-1- yl) -7H-pyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

<td> 14</td><td>0 IS OH Η H</td><td>N- (3 - ((5- (2-hydroxyethyl) -2 - ((4- (4-methylpiperazin-1-yl) phenyl) amino) -7H- pyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

<td> 15</td><td>0 ^ = y ^ NH (TS oh Ά xM&gt; Η H</td><td>N- (3 - ((2 - ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) -5- (2-hydroxyethyl) - 7H-pyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

<td> 16</td><td>í &lt;5 ~ ° i 0 Q /</td><td>N- (3 - ((5 - ((dimethylamino) methyl) -2 - ((4- (4-methylpiperazin-1-yl) phenyl) amino) - 7H-pyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

<td> 17</td><td>or θ-O _rN^υΠ λΧ ^ ' fA-A-AA! Η H</td><td>N- (3 - ((5 - ((d-methylamino) methyl) -2 - ((3-fluoro-4- (4-methylpiperazin-1 yl) phenyl) amino) -7H-pyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

ES 2 618 007 T3

<td>Compound</td><td>Structure</td><td>Chemical name</td>

<td> 18</td><td>0 ^ A_nhOX r Ai xA H H</td><td>N- (3 - ((5- (dimethylamino) -2 - ((4- (4-methylpiperazin-1-yl) phenyl) amino) -7H- pyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

<td> 19</td><td>0 N_H-Ν-μ A, _vΑίχΑ fX-XAXn H ^H</td><td>N- (3 - ((5- (dimethylamino) -2 - ((3-fluoro-4- (4-methylpiperazin-1 yl) phenyl) amino) -7H-pyrrolo [2,3-d] pyrimidin-4- yl) oxy) phenyl) acrylamide</td>

<td> 20</td><td>0 ^ Άη fS xx α AixA Η H</td><td>N- (3 - ((5- (2- (dimethylamino) ethyl) -2 - ((4- (4-methylpiperazin-1-yl) phenyl) amino) - 7H-pyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

<td> 21</td><td>0 iS \ - for AA fV% Ai^ton Η H</td><td>N- (3 - ((5- (2- (dimethylamino) ethyl) -2 - ((3-fluoro-4- (4-methylpiperazin-1 yl) phenyl) amino) -7H-pyrrolo [2,3-d ] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

<td> 22</td><td>0 Α'μμ '«-Μ A AxA H H</td><td>N- (3 - ((5- (aciridin-1-yl) -2 - ((4- (4-methylpiperazin-1-yl) phenyl) amino) -7H- pyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

<td> 23</td><td>0 Λ N ~ - | Ά Γ, Ai iA f-XX-nXiA H H</td><td>N- (3 - ((5- (aciridin-1-yl) -2 - ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) - 7H-pyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

ES 2 618 007 T3

<td>Compound</td><td>Structure</td><td>Chemical name</td>

<td> 24</td><td>or ^ - ^ NH 'p | P Η H</td><td>N- (3 - ((5- (acetydin-1 -yl) -2 - ((4- (4-methylpiperazin-1 -yl) phenyl) amino) -7Hpyrrolo [2,3-d] pyrimidin-4 -yl) oxy) phenyl) acrylamide</td>

<td> 25</td><td>OR Λ H ^H</td><td>N- (3 - ((5- (acetidin-1-yl) -2 - ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) 7H-pyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

<td> 26</td><td>0 Ó-J (I) H H</td><td>N- (3 - ((2 - ((4- (4-methylpiperazin-1 -yl) phenyl) amino) -5- (piperidin-1 -yl) -7Hpyrrolo [2,3-d] pyrimidin-4 -yl) oxy) phenyl) acrylamide</td>

<td> 27</td><td>OR &gt; ~ Ί 0-J 0 H ^H</td><td>N- (3 - ((2 - ((3-fluoro-4- (4-methylpiperazin-1 -yl) phenyl) amino) -5- (piperidin-1 yl) -7H-pyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

<td> 28</td><td>0 X ^ "Xl xJ0&gt;</td><td>N- (3 - ((7-cyclopropyl-2 - ((4- (4-methylpiperazin-1-¡l) phenyl) amino) -7Hpyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

<td> 29</td><td>0 == ^ NH to₀^H l</td><td>N- (3 - ((7-cyclopropyl-2 - ((3-fluoro-4- (4-methylpiperazin-1-yl) phenyl) amino) 7H-pyrrolo [2,3-d] pyrimidin-4-yl) oxy) phenyl) acrylamide</td>

ES 2 618 007 T3

<td>Compound</td><td>Structure</td><td>Chemical name</td>

<td> 30</td><td>0 X &lt;Λ °</td><td>N- (3 - ((2 - ((4- (4-methylpiperazin-1 -¡l) phenl) amine) -7- (methlsulfonl) -7Hp¡rrolo [2,3- d] p¡r¡m¡d¡n-4-¡l) ox¡) fen¡l) acr¡lam¡da</td>

<td> 31</td><td>0 ^ Ynh X^H or-^S\°</td><td>N- (3 - ((2 - ((3-fluoro-4- (4-methylp¡perac¡n-1-¡l) phen¡l) am¡no) -7- (methylsulfon¡l) -7H-p¡rrolo [2,3-d] p¡r¡m¡d¡n-4-¡l) ox¡) phen¡l) acrylam¡da</td>

<td> 32</td><td>0 'Ή! ¿L_or^ XlxX^H or&gt; ~</td><td>N- (3 - ((7-acet¡l-2 - ((4- (4-meth¡lp¡perac¡n-1-¡l) phen¡l) am¡no) -7H-p¡rrolo [ 2,3djpyrimidin-4-yl) ox¡) phenyl) acrylamide</td>

<td> 33</td><td>0 ^ Y'-NH X XOIaX &lt;E</td><td>N- (3 - ((7-acetyl-2 - ((3-fluoro-4- (4-methylp¡perac¡n-1 -¡l) phen¡l) amine) -7Hp¡rrolo [ 2,3-d] p¡r¡m¡d¡n-4-¡l) ox¡) fen¡l) acr¡lam¡da</td>

<td> 34</td><td>0 ^ NH • ^¿1x • ~^£'\ VX OCH₃ⁿ</td><td>N- (3- (2- (4- (1- (2-fluoroethyl) acetyl-3-lalamno) -2-methoxyphenolamine) -7H- p¡rrolo [2,3-d] p¡r¡m¡d¡n-4-¡lox¡) fen¡l) acr¡lam¡da</td>

<td> 35</td><td>OR * ^ νη " to_N„ , ΧΪΙ 1¼^F Y ^ -VA-a OCH₃ⁿ</td><td>N- (3- (2- (4- (1- (2-fluoroethyl) acetyl-d¡n-3-¡lam¡no) -2-methoxyphen¡lam¡no) -7Hp¡rrolo [ 2,3-d] p¡r¡m¡d¡n-4-¡lam¡no) fen¡l) acrylam¡da; Y</td>

ES 2 618 007 T3

<td>Compound</td><td colspan="2">Structure</td><td>Chemical name</td>

<td> 36</td><td> 0</td><td></td><td>N- (3 - ((2 - ((2- (p¡per¡d¡n-1-¡lmet¡l) qulnol¡n-6-¡l) am¡no) -7H-p¡rrolo [2 , 3djplrlmld ¡n-4-ll) ox¡) fen¡l) acr¡lam¡da</td>

<td></td><td>Λ</td><td></td><td></td>

<td></td><td>I</td><td></td><td></td>

<td></td><td>nA U UUaJ H</td><td>3 H</td><td></td>

and pharmaceutically acceptable salts thereof.

In certain embodiments, the present disclosure provides Compound 3, N- (3 - ((2 - ((3-fluoro-4- (4-methylpiperacin-1 ¡l) phenl) amine) -7H-p¡rrolo [2,3-d] p¡r¡m¡d ¡n-4-ll) ox¡) phenl) acrylam,

<img file="ES2618007T3_D0031.tif" />

and pharmaceutically acceptable salts thereof. In certain embodiments, the present disclosure provides the maleate salt of Compound 3, N- (3 - ((2 - ((3-fluoro-4- (4-methylp¡peracin-1-¡l) phen L) amino) -7H-p¡rrolo [2,3-d] p¡r¡m¡d¡n-4¡l) ox¡) phenl) acrllam¡da. In certain embodiments, the present disclosure provides the hydrochloride salt of Compound 3, N- (3 - ((2 - ((3-fluoro-4- (4-methylpiperacin-1-l) fenll) am ¡No) -7H-p¡rrolo [2,3-d] p¡r¡m¡d¡n-4-¡l) ox¡) phenl) acrllam¡da.

The disclosed pharmaceutical compositions can be formulated as a pharmaceutically acceptable salt of a disclosed compound. Pharmaceutically acceptable salts are non-toxic salts of a free base form of a compound that possesses the desired pharmacological activity of the free base. These salts can be derived from inorganic or organic acids. Non-limiting examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, proponates, decanoates, caprllates, acrylates, cautlates, formates , heptanoates, proplolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butylene-1,4-dloates, hexylene-1,6-dloates, benzoates, chlorobenzoates, methylbenzoates, dlnltrobenzoates, hydroxlbenzoates, methoxlbenzoates, phthalates, sulphonates, methylsulphonates, propllsulphonates, besllates, xllenesulphonates, naphthalene-1sulphonates, naphthalene-2-sulphonates, and phenylutylpropyl glulates, phenyloxylated, and phenylated glutathrolates mandates. Lists of other suitable pharmaceutically acceptable salts are found in Remington's Pharmaceutical Sciences, 17^to Edition, Mack Publishing Company, Easton, Pa., 1985.

Pharmaceutical compositions

For treatment purposes, pharmaceutical compositions comprising the compounds described herein may further comprise one or more pharmaceutically acceptable excipients. A pharmaceutically acceptable excipient is a substance that is non-toxic and otherwise biologically acceptable for administration to a subject. These excipients facilitate the administration of the compounds described herein and are compatible with the active ingredient. Examples of pharmaceutically acceptable excipients include stabilizers, lubricants, surfactants, thinners, antioxidants, binders, coloring agents, bulking agents, emulsifiers or flavor modifying agents. In preferred embodiments, the pharmaceutical compositions according to the embodiments are sterile compositions. The pharmaceutical compositions can be prepared using combination techniques known or made available to those of skill in the art.

Sterile compositions are within the present disclosure, including compositions that are in accordance with national and local regulations governing these compositions.

The pharmaceutical compositions and compounds described herein can be formulated as solutions, emulsions, suspensions or dispersions in suitable pharmaceutical solvents or carriers or as pills, tablets, lozenges, suppositories, sachets, lozenges, granules, powders, powders for reconstitution or capsules. together with solid carriers according to conventional methods known in the art for the preparation of various dosage forms. The pharmaceutical compositions of the embodiments can be administered via a route of

ES 2 618 007 T3 suitable administration, such as oral, parenteral, rectal, nasal, topical or ocular routes or by inhalation. Preferably, the compositions are formulated for intravenous or oral administration.

For oral administration, the compounds of the embodiments can be provided in a solid form, such as a tablet or capsule, or as a solution, emulsion, or suspension. To prepare oral compositions, compounds of embodiments can be formulated to give a dosage of, e.g. eg, about 0.01 to about 50 mg / kg per day or about 0.05 to about 20 mg / kg per day or about 0.1 to about 10 mg / kg per day. Oral tablets may include the active ingredient (s) in admixture with compatible pharmaceutically acceptable excipients such as diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents, and preserving agents. Suitable inert fillers include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, and the like. Exemplary liquid oral excipients include ethanol, glycerol, water, and the like. Starch, polyvinylpyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose, and alginic acid are exemplary disintegrating agents. The binding agents can include starch and gelatin. The lubricating agent, if present, can be magnesium stearate, stearic acid, or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption from the gastrointestinal tract or they may be enteric coated.

Capsules for oral administration include hard and soft gelatin capsules. To prepare hard gelatin capsules, the active ingredient (s) can be mixed with a solid, semi-solid or liquid diluent. Soft gelatin capsules can be prepared by mixing the active ingredient with water, an oil, such as peanut oil or olive oil, liquid paraffin, a mixture of mono- and di-glycerides of short chain fatty acids, polyethylene glycol 400 or propylene glycol.

Liquids for oral administration may be in the form of suspensions, solutions, emulsions or syrups or may be lyophilized or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid compositions may optionally contain: pharmaceutically acceptable excipients such as suspending agents (eg, sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel, and the like); non-aqueous vehicles, e.g. eg, oil (eg, almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol, or water; preservatives (eg, methyl or propyl p-hydroxybenzoate or sorbic acid); wetting agents such as lecithin; and, if desired, flavoring or coloring agents.

The compositions can be formulated for rectal administration as a suppository. For parenteral use, including intravenous, intramuscular, intraperitoneal, intranasal, or subcutaneous routes, the compounds of the embodiments may be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity or in a parenterally acceptable oil. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. These forms can be presented in unit dose form such as ampoules or disposable injection devices, in multiple dose forms such as vials from which the appropriate dose can be drawn, or in a solid form or a pre-concentrate that can be used. to prepare an injectable formulation. Illustrative infusion doses range from about 1 to 1,000 pg / kg / minute of agent mixed with a pharmaceutical carrier over a period ranging from several minutes to several days.

For nasal, inhaled, or oral administration, the pharmaceutical compositions can be administered using, for example, an aerosol formulation that also contains a suitable carrier.

For topical applications, the compounds of the embodiments are preferably formulated as creams or ointments or a similar vehicle suitable for topical administration. For topical administration, the compounds of the embodiments can be mixed with a pharmaceutical carrier in a concentration of from about 0.1% to about 10% of drug relative to vehicle. Another mode of administering the compounds of the embodiments may use a patch formulation to effect transdermal delivery.

In certain embodiments, the present disclosure provides a pharmaceutical composition comprising a compound of Formulas (I) - (VIII) and methylcellulose. In certain embodiments, the methyl cellulose is in a suspension of about 0.1, 0.2, 0.3, 0.4, or 0.5 to about 1%. In certain embodiments, the methylcellulose is in a suspension of about 0.1 to about 0.5, 0.6, 0.7, 0.8, 0.9, or 1%. In certain embodiments, the methylcellulose is in a suspension of about 0.1 to about 1%. In certain embodiments, the methylcellulose is in a suspension of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.8, or 1%. . In certain embodiments, the methylcellulose is in a suspension of about 0.5%.

As used herein, the terms "treat" or "treatment" encompass both preventive and curative treatment. Preventive treatment is understood to indicate a delay in the development of a disease, a symptom of a disease or medical condition, a suppression of symptoms that may appear, or a reduction in the risk of the development or recurrence of a disease or a symptom. Curative treatment includes reducing the severity of or suppressing the worsening of an existing disease, symptom, or condition. Thus, treatment includes improving or preventing the worsening of existing disease symptoms, preventing them from occurring

ES 2 618 007 T3 additional symptoms, improve or prevent underlying systemic causes of symptoms, inhibit disorder or disease, eg. eg, interrupting the development of the disorder or disease, alleviating the disorder or disease, causing regression of the disorder or disease, alleviating a condition caused by the disease or disorder, or stopping symptoms of the disease or disorder.

One of ordinary skill in the art can modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration. In particular, the compounds can be modified to make them more soluble in water or other vehicle. This is also entirely within the normal skill of the art to modify the route of administration and the dosage regimen of a particular compound in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect in a patient.

The term "subject" refers to a mammalian patient in need of this treatment, such as a human.

The compounds can be administered to a subject in need of treatment for a proliferative disorder. An example of a proliferative disorder is cancer. In certain cases, the compounds are useful in treating sarcoma, squamous cancer, fibrosarcoma, cervical cancer, gastric carcinoma, skin cancer, leukemia, lymphoma, lung cancer, non-small cell lung cancer, colon cancer, CNS cancer. , melanoma, ovarian cancer, kidney cancer, prostate cancer, breast cancer, liver cancer, head and neck cancers, and pancreatic cancer.

The compounds can also be administered to a subject in need of treatment for a disease or medical condition that is mediated by a protein kinase selected from the group consisting of EGFR, EGFR (T790M), BLK, BMX / ETK, BTK, JAK1, JAK2, JAK3, TEC, TXK, FLT3 and FLT3 (D835Y). In certain cases, the compounds are useful for treating cancers, tumors, inflammatory diseases, autoimmune diseases, or immunologically related diseases. In other embodiments, these diseases are mediated by at least one kinase selected from BTK, JAK3, ITK, and BMX. In other embodiments, cancers, tumors, inflammatory diseases, autoimmune diseases, or immunologically mediated diseases are mediated by abnormally activated B lymphocytes, T lymphocytes, or both. In other embodiments, inflammatory diseases, autoimmune diseases, or immunologically mediated diseases are arthritis, rheumatoid arthritis, spondyloarthropathy, gouty arthritis, osteoarthritis, juvenile arthritis, other arthritic conditions, lupus, systemic lupus erythematosus (SLE), a disease related to skin, psoriasis, eczema, dermatitis, atopic dermatitis, pain, a lung disorder, lung inflammation, Adult respiratory distress syndrome (ARDS), pulmonary sarcoidosis, chronic pulmonary inflammatory disease, chronic obstructive pulmonary disease (COPD), cardiovascular disease, atherosclerosis, myocardial infarction, congestive heart failure, cardiac reperfusion injury, inflammatory enteropathy, Crohn's disease , ulcerative colitis, irritable bowel syndrome, asthma, Sjogren's syndrome, autoimmune thyroid disease, urticaria (cnidosis), multiple sclerosis, scleroderma, organ transplant rejection, heteroplastic graft, idiopathic thrombocytopenic purpura (ITP), Parkinson's disease, Alzheimer's disease, diseases associated with diabetes, inflammation, pelvic inflammatory disease, allergic rhinitis, allergic bronchitis, allergic sinusitis, leukemia, lymphoma, B-cell lymphoma, T-cell lymphoma, myeloma, acute lymphoid leukemia (ALL), chronic lymphoid leukemia (CLL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hairy cell leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), diffuse large B-cell lymphoma, follicular lymphoma, sarcoma, squamous cell cancer, fibrosarcoma, cancer cervical cancer, gastric carcinoma, skin cancer, leukemia, lymphoma, lung cancer, non-small cell lung cancer, colon cancer, CNS cancer, melanoma, ovarian cancer, kidney cancer, prostate cancer, breast cancer, liver cancer, head and neck cancers, or pancreatic cancer. In other embodiments, the diseases are autoimmune disease or transplant-induced inflammatory disorders including, but not limited to, allogeneic transplantation, graft-versus-host disease, or autoimmune diabetes.

In one aspect, the compounds and pharmaceutical compositions of the embodiments can be administered to a subject in need of treatment for a condition associated with EGFR inhibitory activity that targets substantially mutated EGFR but not substantially wild-type EGFR. In some embodiments, the mutated EGFR comprises a T790M mutation. The present disclosure provides the use of a compound of Formulas (I) - (VIII) in the preparation of a medicament for the treatment of such conditions and the use of these compounds and salts for the treatment of these conditions. In other aspects, the compounds and pharmaceutical compositions can be administered to a subject in need of treatment for a condition associated with an FLT3 inhibitory activity that targets substantially mutated FLT3 but not substantially wild-type FLT3. In some embodiments, the mutated FLT3 comprises a D835Y mutation. The present disclosure provides the use of a compound of Formulas (I) - (VIII) in the preparation of a medicament for the treatment of these conditions and the use of these compounds and salts for the treatment of these conditions.

In another aspect, the present disclosure provides a method of inhibiting mutated EGFR in a cell which comprises contacting the cell with an effective amount of at least one compound of Formulas (I) - (VIII) or a salt thereof, and / or with at least one pharmaceutical composition of the embodiments, wherein the contact is in vitro, ex vivo or in vivo. In some embodiments, the mutated EGFR comprises a T790M mutation. In another aspect, the present disclosure provides a method of inhibiting mutated FLT3 in a cell which comprises contacting the cell with an effective amount of at least one compound of Formulas (I) - (VIII) or a salt thereof, and / or with al

ES 2 618 007 T3 less a pharmaceutical composition comprising said compound or salt, wherein the contact is in vitro, ex vivo or in vivo.

In the inhibitory methods of the embodiments, an "effective amount" means an amount sufficient to inhibit the targeted receptor, e.g. eg, mutated EGFR but not wild-type EGFR or mutated FLT3 but not wild-type FLT3. In some embodiments, the mutated EGFR comprises a T790M mutation. In other embodiments, the mutated FLT3 comprises a D835Y mutation. Measurement of the degree of inhibition can be carried out by standard analytical methods such as those described below. This modulation is useful in a variety of settings, including in vitro assays. Other settings include ex vivo and in vivo.

In the methods of treatment according to the embodiments, an effective amount means an amount or dose sufficient to generally produce the desired therapeutic benefit in subjects in need of this treatment. The effective amounts or doses of the compounds of the embodiments can be determined by standard methods, such as modeling, dose escalation, or clinical experiments, taking into account common factors, e.g. eg, the mode or route of administration or delivery of the drug, the pharmacokinetics of the agent, the severity and course of the infection, the state of health, the condition and weight of the subject, and the judgment of the responsible physician. An exemplary dose is in the range of about 1 pg to 2 mg of active agent per kilogram of the subject's body weight per day, preferably about 0.05 to 100 mg / kg / day or about 1 to 35 mg / kg / day. day or about 0.1 to 10 mg / kg / day. The total dosage can be given in individual or divided dosage units (eg, twice a day, three times a day, four times a day).

Once improvement in the patient's condition has occurred, the dose can be adjusted for preventive or maintenance treatment. For example, the dosage or the frequency of administration or both can be reduced as a function of symptoms, to a level where the desired therapeutic or prophylactic effect is maintained. Of course, if symptoms have alleviated to an appropriate level, treatment can be stopped. However, patients may require long-term intermittent treatment with any recurrence of symptoms. Patients may also require long-term chronic treatment.

Drug combinations

The methods of the embodiments comprise administering an effective amount of at least one compound of Formula (I) - (VIII) or the embodiments thereof; optionally the compound can be administered in combination with one or more additional therapeutic agents, particularly therapeutic agents known to be useful for treating a proliferative disorder or cancer affecting a subject. In some embodiments, the one or more therapeutic agents are selected from anticancer agents (such as cellular signal transduction inhibitors, mitosis inhibitors, alkylating agents, antimetabolites, intercalating anticancer agents, topoisomerase inhibitors, immunotherapeutic agents, or anti-hormonal agents). , steroidal drugs, methotrexates, leflunomides, anti-TNFa agents, calcineurin inhibitors, and antihistamine drugs.

The additional active ingredients may be administered in a separate pharmaceutical composition from a compound of the embodiments or they may be included with a compound of the embodiments in a single pharmaceutical composition. Additional active ingredients can be administered simultaneously with, before or after administration of a compound of the embodiments.

Chemical synthesis

Exemplary chemical entities useful in the methods of the embodiments will now be described by reference to illustrative synthetic schemes for subsequent general preparation and the specific examples that follow. Those of skill will appreciate that, to obtain the various compounds herein, the starting materials can be suitably selected so that the desired substituents are ultimately carried through the reaction scheme with or without protection as appropriate to give the desired product. Alternatively, it may be necessary or desirable to employ, in place of the ultimately desired substituent, a suitable group that can be carried through the reaction scheme and replaced as appropriate with the desired substituent. On the other hand, one of skill in the art will appreciate that the transformations shown in the diagrams below can be performed in any order that is compatible with the functionality of the particular pendant groups. Each of the reactions represented in the general schemes is preferably carried out at a temperature of about 0 ° C to the reflux temperature of the organic solvent used. Unless otherwise specified, variables are as defined above with reference to Formula (I). One of ordinary skill in the art will also appreciate that the methods described in these exemplary schemes are also applicable to the preparation of compounds of Formula (VIII), as well as compounds of Formulas (II) - (VII).

A representative synthesis for the compounds in question is shown in Scheme 1.

ES 2 618 007 T3

Scheme 1

<img file="ES2618007T3_D0032.tif" />

Formula (the)

In Scheme 1, the variables are as defined herein. As discussed below, X^2nd and X^2bthey comprise an exit group. Raw materials can be obtained from commercial sources or through well-established synthetic procedures.

Referring to Scheme 1, the reaction of Compound 1-A with Compound 1-B through a nucleophilic reaction forms Compound 1-C. In Compound 1-A, the hydroxyl group is a nucleophile that can provide the ether bond in Compound 1-C. The nucleophilic can react in a nucleophilic substitution in which the nucleophilic displaces a leaving group from the other reactant. In alternative embodiments, analogs of 1-A are used to access compounds in which X¹ is NH or S. In Compound 1-B, X^2nd comprises an outlet group. Examples of leaving groups include, but are not limited to, halo, trphlate, fluorosulfonate, tosylate, or mesylate.

Following reference to Scheme 1, reaction of Compound 1-C with Compound 1-D under Buchwald-Hartwlg cross-coupling reaction conditions provides Compound 1-E. In Compound 1-D, the amine group is a nucleophile that can provide the amine bond in Compound 1-E. The nucleophilic can react in a nucleophilic aromatic substitution in which the nucleophilic displaces a leaving group from the other reactant. In Compound 1-C, X^2b comprises an outlet group. Examples of leaving groups include, but are not limited to, halo, trphlate, fluorosulfonate, tosylate, or mesylate.

Following reference to Scheme 1, the nitro group in Compound 1-E is reduced to give an amine group in Compound 1-F. The reduction of the nitro group can be carried out using an acid catalyst and a metal or using a metal catalyst under hydrogen gas. In the acid catalyzed reaction, iron, zinc, lithium, sodium or tin metal (typically tin chloride) can be used as the metal and inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid or phosphoric acid; organic carboxylic acids such as acetic acid or trifluoroacetic acid; acidic amine salts such as ammonium chloride. In addition, in the reduction using a metal catalyst under hydrogen gas, palladium, nickel, platinum, ruthenium or rhodium can be used as the metal catalyst.

Following reference to Scheme 1, the addition of Compound 1-F gives a compound of Formula (I). In the amidation reaction, Compound 1-F reacts with an acryloyl derivative comprising a leaving group. Examples of leaving groups include, but are not limited to, halo, trphlate, fluorosulfonate, tosylate, or mesylate. An amidation reaction can be carried out in a solvent, such as dimethylformamide or dichloromethane, in the presence of a base, such as triethylamine or dusopropyllamine. The amidation reaction can be carried out using a coupling agent, such as, for example, dlclclohexllcarbodilda (DCC), 1-ethyl-3- (3-dimethylamnopropll) carbodimida ( EDC) or N- [dimethylamine-1H-1,2,3-tri-azole [4,5-b] -p¡r¡d¡n-1-¡lmeth¡len] -N -metal-methanamino (HATU) together with 1-hydroxl-1 Hbenzotrlazole (HOBT).

In certain embodiments, a representative synthesis for the compounds in question is shown in Scheme 2.

ES 2 618 007 T3

Scheme 2

<img file="ES2618007T3_D0033.tif" />

Compound 8

In certain embodiments, a representative synthesis for the compounds in question is shown in Scheme 3. Scheme 3

<img file="ES2618007T3_D0034.tif" />

Compound 36

In certain embodiments, a representative synthesis for the compounds in question is shown in Scheme 4.

Scheme 4

ES 2 618 007 T3

<img file="ES2618007T3_D0035.tif" />

Compound 13

In certain embodiments, a representative synthesis for the compounds in question is shown in Scheme 5. Scheme 5

<img file="ES2618007T3_D0036.tif" />

Cu (OA)₂ pyridine

90 °,&gt; 36h

<img file="ES2618007T3_D0037.tif" />

{ NOT₂N

<img file="ES2618007T3_D0038.tif" />

jCc

<img file="ES2618007T3_D0039.tif" />

Compounds in which X¹ es NH are prepared according to Scheme 5-1, as shown for Exemplary Compound 35.

ES 2 618 007 T3

Scheme 5-1

<img file="ES2618007T3_D0040.tif" />

Compound 35

In accordance with this and as described in more detail herein, the present disclosure provides a process for preparing a compound of the present disclosure, the process involves: reacting a compound of Formula

<img file="ES2618007T3_D0041.tif" />

ES 2 618 007 T3

In accordance with this and as described in more detail herein, the present disclosure provides a process for preparing a compound of the present disclosure, the process involves: reacting a compound of Formula

<img file="ES2618007T3_D0042.tif" />

Based on this and as described in more detail herein, the present disclosure provides a process for preparing a compound of the present disclosure, the process involves:

<img file="ES2618007T3_D0043.tif" />

conducting an amidation reaction with an acryloyl derivative comprising a leaving group;

thereby producing a compound of Formula (I).

Based on this and as described in more detail herein, the present disclosure provides a process for preparing a compound of the present disclosure, the process involves:

<img file="ES2618007T3_D0044.tif" />

conducting an amidation reaction with an acryloyl derivative comprising a leaving group; thereby producing a compound of Formula (VIII).

ES 2 618 007 T3

In certain cases, the above procedures further involve the step of forming a salt of a compound of the present disclosure. The embodiments are directed to the other procedures described herein; and to the product prepared by any of the procedures described herein.

Examples

The following examples are offered to illustrate but not to limit the invention.

Example 1: Synthesis of Compounds 1 and 4:

Scheme 6

N

-,TO

Λ ^SEMCI - ι

NaH.THF 'N' ^{w N} H o ° cat.a

Faith NH₄CI

ElOH / water, reflux

HN

TO

6-B

TO

Η V-, Compound 4

SEM

<img file="ES2618007T3_D0045.tif" />

Η

6-E

J

SEM νη₃Η₂ο

MeOH

HN

OL

Oh

H ^H

Compound 1

<img file="ES2618007T3_D0046.tif" />

DCM. 0 ° C, 1 h

<img file="ES2618007T3_D0047.tif" />

A synthesis of N- (3 - ((7- (hydroxymethyl) -2 - ((4- (4-methylpiperazin-1-yl) phenyl) amino) -7H-pyrrolo [2,3-d] pyrimidin-4-yl) oxl) phenyl) acrylamide (Compound 4) and N- (3 - ((2 - ((4- (4-methylpiperazin-1-yl) phenyl) amino) -7H-pyrrolo [2,3-d] pyrimidin-410 yl) oxy) phenyl) acrylamide (Compound 1) and its intermediates are shown in Scheme 6 and described below.

Synthesis of 2,4-dichloro-7 - ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d] pyrimidine (Compound 6-B):

Cl

Cl

N

Λ

<img file="ES2618007T3_D0048.tif" />

SEM

Sodium hydride (60%, 46.7 mg, 3.06 mmol) was added to a mixture of Compound 2-A (575 mg, 3.06 mmol) and 2- (trimethylsilyl) ethoxymethyl chloride (561 mg, 3 , 37 mmol) in tetrahydrofuran (5 ml) at 0 ° C with stirring. The reaction mixture was allowed to warm to room temperature and stirred for 3 hours before quenching with water (5 ml). The mixture was extracted with ethyl acetate (10 ml x 3). The organic layers were combined, washed with brine, dried over

Na2SC&gt; 4 and filtered. The filtrate was concentrated and the crude material was purified by column chromatography (PE / EA = 20/1) to give Compound 6-B (520 mg, 53.4% ​​yield, M + H⁺= 319.27) as a light yellow solid.

Synthesis of 2-chloro-4- (3-nitrophenoxy) -7 - ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d] pyrimidine (Compound 6-C):

ES 2 618 007 T3

<img file="ES2618007T3_D0049.tif" />

K2CO3 (173.7 mg, 1.26 mmol) was added to a mixture of Compound 6-B (200 mg, 0.628 mmol) and 3-nitrophenol (96.2 g, 0.691 mmol) in dimethylformamide (2 ml). The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was then filtered. The filtrate was diluted with water and then extracted with ethyl acetate. The organic layer was washed with water, brine, and dried over Na₂SC&gt; 4. After filtration and removal of volatiles in vacuo, the crude product was purified by flash column chromatography (PE / EA = 20/1) to provide Compound 6-C (200 mg, yield 75.6 %, M + H⁺ = 421.92) as a white solid.

Synthesis of N- (4- (4-methylpiperazin-1-yl) phenyl) -4- (3-nitrophenoxy) -7 - ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-2amine (Compound 6-D):

<img file="ES2618007T3_D0050.tif" />

A mixture of Compound 6-C (150 mg, 0.356 mmol), 4- (4-methylpiperazino) aniline (70 mg, 0.356 mmol), tris (dibenzylideneacetone) dipalladium (36 mg, 0.0356 mmol), dicyclohexyl (2 ' 4 ', 6'-triisopropylbiphenyl-2-yl) phosphine (100 mg, 0.214 mmol) and potassium carbonate (197 mg, 1.424 mmol) in tert-butanol (8 ml) was stirred under argon at 80 ° C overnight . After cooling to room temperature, the reaction mixture was filtered through a pad of Celite. The Celite plug was washed with methanol and the filtrate was concentrated under reduced pressure. The residue was purified by flash column chromatography (DCM / MeOH = 20/1) to give Compound 6-D (180 mg, M + H⁺ = 576.23) as a yellow solid.

Synthesis of 4- (3-aminophenoxy) -N- (4- (4-methylpiperazin-1 -yl) phenyl) -7 - ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d ] pyrimidin-2amine (Compound 6-E):

<img file="ES2618007T3_D0051.tif" />

Compound 6-D (180 mg, 0.312 mmol) was dissolved in ethanol (6 ml) and water (2 ml) was added. Next, iron powder (90 mg, 1.61 mmol) and ammonium chloride (230 mg, 4.3 mmol) were added and the resulting mixture was heated under reflux for 3 hours. The reaction mixture was cooled to room temperature and filtered through a pad of Celite. The ethanol was removed in vacuo and the resulting residue was made basic with sodium bicarbonate and extracted with ethyl acetate. The organic layer was separated and dried using anhydrous sodium sulfate, concentrated, and purified by flash chromatography with 20: 1 dichloromethane-methanol to provide Compound 6-E (170 mg, M + H⁺= 546) as a white solid.

Synthesis of N- (3- (2- (4- (4-methylpiperazin-1-yl) phenylamino) -7 - ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3-d] pyrimid in-4yloxy) phenyl) acrylamide (Compound 6-F)

ES 2 618 007 T3

<img file="ES2618007T3_D0052.tif" />

Acryloyl chloride (33.8 mg, 0.374 mmol) was added dropwise to a solution of Compound 6-E (170 mg, 0.312 mmol) and diisopropylethylamine (55 mg, 0.426 mmol) in methylene chloride (3 ml) to 0 ° C. The reaction mixture was stirred for 1 hour. Water was added to quench the reaction. The organic layer was washed with water, brine, and dried over Na2SC&gt; 4. After filtration, the removal of volatiles was carried out under vacuum. The crude product was purified by flash chromatography (DCM / MeOH = 20/1) to provide Compound 6-F (125 mg, 66.9% yield, M + H⁺ = 600.8) as a white solid.

Synthesis of N- (3- (7- (hydroxymethyl) -2- (4- (4-methylpiperazin-1-yl) phenylamino) -7H-pyrrolo [2,3-d] pyrimidin-4-yloxy) phenyl) acrylamide (Compound 4)

<img file="ES2618007T3_D0053.tif" />

Compound 6-F (125 mg, 0.208 mmol) in methylene chloride (3 ml) and trifluoroacetic acid (1 ml) was stirred at room temperature for 3 hours. Verification by thin layer chromatography indicated that all raw material had been consumed. Then, saturated aqueous NaHCC&gt; 3 was added to the reaction mixture at 0 ° C. The reaction mixture was extracted with methylene chloride. The organic layer was washed with water, brine, dried over Na2SC&gt; 4 and filtered. The filtrate was concentrated and the crude material was purified by column chromatography (DCM / MeOH = 20/1) to give Compound 4 (70 mg, 71.5% yield, M + H⁺= 500.5) as a white solid.

Synthesis of N- (3- (2- (4- (4-methylpiperazin-1-yl) phenylamino) -7H-pyrrolo [2,3-d] pyrimidin-4-yloxy) phenyl) acrylamide (Compound 1)

<img file="ES2618007T3_D0054.tif" />

A solution of Compound 4 (100 mg, 0.2 mmol) in methanol (2 ml) was saturated with ammonia. The reaction mixture was stirred overnight at room temperature. Verification by LC-MS indicated that all raw material had been consumed. The solvent was concentrated and the crude material was purified by column chromatography (DCM / MeOH = 20/1) to give Compound 1 (60 mg, 63.8% yield, M + H⁺= 470.5) as a light yellow solid. Example 2: Synthesis of Compounds 2 and 5:

ES 2 618 007 T3

Scheme 7

<img file="ES2618007T3_D0055.tif" />

<img file="ES2618007T3_D0056.tif" />

<img file="ES2618007T3_D0057.tif" />

N

SEM

DCM, 0 ° C, 1 h

DIEA, CH₂= CHCOCI

<img file="ES2618007T3_D0058.tif" />

<img file="ES2618007T3_D0059.tif" />

<img file="ES2618007T3_D0060.tif" />

HN

OUN

H ^H

Compound 2

A synthesis of N- (3- (7- (hydroxymethyl) -2- (6- (4-methylpiperazin-1-yl) pyridin-3-ylamino) -7H-pyrrolo [2,3-d] pyrimidin 4yloxy) phenyl) acrylamide (Compound 5) and N- (3- (2- (6- (4-methylpiperazin-1-yl) pyridin-3-ylamino) -7H-pyrrolo [2,3-d] pyrimidin -45 yloxy) phenyl) acrylamide (Compound 2) and its intermediates are shown in Scheme 7 and described below.

Synthesis of N- (6- (4-methylpiperazin-1 -yl) pyridin-3-yl) -4- (3-nitrophenoxy) -7 - ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2 , 3-d] pyrimidin2-amine (Compound 7-B):

<img file="ES2618007T3_D0061.tif" />

Compound 7-B (62% yield from 3, M + H⁺= 577.3) was prepared according to the procedure of Compound 6-D using 3-amino-6- (4-methyl-1-piperazinyl) pyridine hydrochloride instead of 4- (4-methylpiperazino) aniline.

Synthesis of 4- (3-aminophenoxy) -N- (6- (4-methylpiperazin-1-yl) pyridin-3-yl) -7 - ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [ 2,3d] pyrimidin-2-amine (Compound 7-C):

<img file="ES2618007T3_D0062.tif" />

Compound 7-C (80% yield from Compound 7-B, M + H⁺= 547.3) was prepared according to the procedure of Compound 6-E.

Synthesis of N- (3- (2- (6- (4-methylpiperazin-1-yl) pyridin-3-ylamino) -7 - ((2- (trimethylsilyl) ethoxy) methyl) -7H-pyrrolo [2,3 -d] pyrimidine-4yloxy) phenyl) acrylamide (Compound 7-D)

ES 2 618 007 T3

<img file="ES2618007T3_D0063.tif" />

Compound 7-D (67% yield from Compound 7-C, M + H⁺= 601.3) was prepared according to the procedure of Compound 6-F.

Synthesis of N- (3- (7- (hydroxymethyl) -2- (6- (4-methylpiperazin-1-yl) pyridin-3-ylamino) -7H-pyrrolo [2,3-d] pyrimidin-45 yloxy) phenyl) acrylamide (Compound 5):

<img file="ES2618007T3_D0064.tif" />

Compound 7-E (70% yield from Compound 5, M + H⁺= 501.6) was prepared according to the procedure of Compound 4.

Synthesis of N- (3- (2- (6- (4-methylpiperazin-1-yl) pyridin-3-ylamino) -7H-pyrrolo [2,3-d] pyrimidin-4-yloxy) phenyl) acrylamide 10 ( Compound 2):

<img file="ES2618007T3_D0065.tif" />

Compound 2 (62% yield from Compound 5, M + H⁺= 471.5) was prepared according to the procedure of Compound 1.

Example 3: Synthesis of Compounds 3 and 6:

ES 2 618 007 T3

<img file="ES2618007T3_D0066.tif" />

Scheme 8

<img file="ES2618007T3_D0067.tif" />

N

SEM

DIEA, CH₂= CHCOCI

DCM, 0 ° C, 1 h

<img file="ES2618007T3_D0068.tif" />

Compound 6

ΝΗ3Ή2Ο

MeOH

Oh

<img file="ES2618007T3_D0069.tif" />

A synthesis of N- (3- (2- (3-fluoro-4- (4-methylpiperazin-1-yl) phenylamino) -7- (hydroxymethyl) -7H-pyrrolo [2,3-d] pyrimidin-4yloxy ) phenyl) acrylamide (Compound 6) and N- (3- (2- (3-fluoro-4- (4-methylpiperazin-1-yl) phenylamino) -7H-pyrrolo [2,3-d] pyrimidin-45 yloxy ) phenyl) acrylamide (Compound 3) and its intermediates are shown in Scheme 8 and described below.

Synthesis of N- (3-fluoro-4- (4-methylp¡perac¡n-1-¡l) phen¡l) -4- (3-nitrophenox¡) -7 - ((2- ( tr¡met¡ls¡l¡l) ethox¡) methyl) -7H-p¡rrolo [2,3d] plr¡mld¡n-2-amine (Compound 8-B)

<img file="ES2618007T3_D0070.tif" />

Compound 8-B (% yield from Compound 8-A, M + H⁺= 594.3) was prepared according to the procedure of Compound 6-D using 3-fluoro-4- (4-methoperazine-1-ll) anne in place of 4- (4-methoperazine) an¡ line.

Synthesis of 4- (3-amlnofenoxl) -N- (3-fluoro-4- (4-methylp¡perac¡n-1 -ll) phen¡l) -7 - ((2- (tr¡met¡ ls¡l¡l) ethox¡) methyl) -7H-p¡rrolo [2,3d] plr¡mld¡n-2-amine (Compound 8-C):

<img file="ES2618007T3_D0071.tif" />

Compound 8-C (85% yield from Compound 8-B, M + H⁺= 564.3) was prepared according to the procedure of Compound 6-E.

Synthesis of N- (3- (2- (3-fluoro-4- (4-metllplperac¡n-1 -¡l) phenylamine) -7 - ((2- (tr¡met¡ls¡l ¡L) ethoxy¡) methyl) -7H-p¡rrolo [2,3-d] p¡r¡m¡d¡n-4¡loxl) phen¡l) acrylam¡da (Compound 8- D)

ES 2 618 007 T3

<img file="ES2618007T3_D0072.tif" />

Compound 8-D (75% yield from Compound 8-C, M + H⁺= 618.3) was prepared according to the procedure for Compound 6-F.

Synthesis of N- (3- (2- (3-fluoro-4- (4-methylpiperazin-1-yl) phenylamino) -7- (hydroxymethyl) -7H-pyrrolo [2,3-d] pyrimidin-45-yloxy ) phenyl) acrylamide (Compound 6):

OR

<img file="ES2618007T3_D0073.tif" />

Compound 6 (78% yield from Compound 8-D, M + H⁺= 518.6) was prepared according to the procedure of Compound 4.

Synthesis of N- (3- (2- (3-fluoro-4- (4-methylpiperazin-1-yl) phenylamino) -7H-pyrrolo [2,3-d] pyrimidin-4-yloxy) phenyl) acrylamide 10 ( Compound 3):

<img file="ES2618007T3_D0074.tif" />

Compound 3 (83% yield from Compound 6, M + H⁺= 488.5) was prepared according to the procedure of Compound 1.

Example 4: N- (3- (7- (2-fluoroethyl) -2- (4- (4-methylpiperazin-1-yl) phenylamino) -7H-pyrrolo [2,3-d] pyrimid-in-4-yloxy ) phenyl) acrylamide 15 (Compound 7)

ES 2 618 007 T3

Scheme 9

<img file="ES2618007T3_D0075.tif" />

A synthesis of N- (3- (7- (2-fluoroethyl) -2- (4- (4-methylpiperazin-1-yl) phenylamino) -7H-pyrrolo [2,3-d] pyrimidin-4yloxy) phenyl) Acrylamide (Compound 7) and its intermediates are shown in Scheme 9 and described below.

Synthesis of 2,4-dichloro-7- (2-fluoroethyl) -7H-pyrrolo [2,3-d] pyrimidine (Compound 9-B)

<img file="ES2618007T3_D0076.tif" />

Sodium hydride (60%, 424 mg, 10.6 mmol) was added to a mixture of Compound 9-A (1 g, 5.3 mmol) and BrCH₂CH₂F (1.519 g, 11.9 mmol) in acetonitrile (10 ml) at room temperature. The reaction mixture was stirred for 4 hours before quenching with water and then extracted with ethyl acetate. The organic layer was washed with brine, dried over Na₂SO4 and leaked. The filtrate was concentrated and the crude material was purified by column chromatography (PE / EA = 20/1) to give Compound 9-B (1.1 g, 90% yield, M + H⁺= 234.0) as a light yellow solid.

Synthesis of 2-chloro-7- (2-fluoroethyl) -4- (3-nitrophenoxy) -7H-pyrrolo [2,3-d] pyrimidine (Compound 9-C)

<img file="ES2618007T3_D0077.tif" />

Compound 9-C (82% yield from Compound 9-B, M + H⁺= 337.0) was prepared according to the procedure of Compound 6-C.

Synthesis of 7- (2-fluoroethyl) -N- (4- (4-methylpiperazin-1 -yl) phenyl) -4- (3-nitrophenoxy) -7H-pyrrolo [2,3-d] pyrimidin 2-amine (Compound 9-D)

ES 2 618 007 T3

<img file="ES2618007T3_D0078.tif" />

F

Compound 9-D (73% yield from Compound 9-C, M + H⁺= 492.2) was prepared according to the procedure for Compound 6-D.

Synthesis of 4- (3-aminophenoxy) -7- (2-fluoroethyl) -N- (4- (4-methylpiperazin-1-yl) phenyl) -7H-pyrrolo [2,3-d] pyrimidin-2-amine 5 (Compound 9-E)

<img file="ES2618007T3_D0079.tif" />

Compound 9-E (81% yield from Compound 9-D, M + H⁺= 462.2) was prepared according to the procedure of Compound 6-E.

Synthesis of N- (3- (7- (2-fluoroethyl) -2- (4- (4-methylpiperazin-1-yl) phenylamino) -7H-pyrrolo [2,3-d] pyrimidin-4-yloxy) phenyl ) acrylamide 10 (Compound 7)

<img file="ES2618007T3_D0080.tif" />

Compound 7 (77% yield from Compound 9-E, M + H⁺= 516.6) was prepared according to the procedure of Compound 6-F.

Example 5: Synthesis of N- (3- (2- (4- (1- (2-fluoroethyl) acetidin-3-ylamino) -2-methoxyphenylamino) -7H-pyrrolo [2,3-d] pyrimidin-415 yloxy ) phenyl) acrylamide (Compound 34)

<img file="ES2618007T3_D0081.tif" />

ES 2 618 007 T3

Synthesis of tert-butyl 3- (3-methoxy-4-nitrophenylamino) acetidine-1-carboxylate

<img file="ES2618007T3_D0082.tif" />

4-Fluoro-2-methoxy-1-nitrobenzene (4.086 g) and tert-butyl 3-aminoacetidine-1-carboxylate (4.4 g), triethylamine (9.6 ml) and dimethylsulfoxide (20 ml) were charged to a 100 ml 3 necked round bottom flask equipped with a reflux condenser. The reaction mixture was heated at 95 ° C for 8 hours. The reaction mixture was poured into water (200 ml) and extracted with ethyl acetate (50 ml x 3). The organic layer was washed with brine (50 ml x 2), dried over sodium sulfate and fully concentrated under reduced pressure at 40 ° C to give the title compound (9 g) which was used without further purification.

Synthesis of N- (3-methoxy-4-nitrophenyl) acetidin-3-amine

NOc

<img file="ES2618007T3_D0083.tif" />

Or me

HN

<img file="ES2618007T3_D0084.tif" />

TFA (18 ml) was added at room temperature to tert-butyl 3- (3-methoxy-4-nitrophenylamino) acetidine-1-carboxylate (9 g). The reaction mixture was stirred for 15 min at room temperature and then concentrated under reduced pressure at 40 ° C to give the title compound as the TFA salt (7.24 g).

Synthesis of 1- (2-fluoroethyl) -N- (3-methoxy-4-nitrophenyl) acetidin-3-amine

<img file="ES2618007T3_D0085.tif" />

CS2CO3 (12 g) and 1,2-bromofluoroethane (1.5 g) in DMF (30 ml) were added to N- (3-methoxy-4-nitrophenyl) acetidin-3-amine (3 g). The reaction mixture was heated at 50 ° C for 8 h. The reaction mixture was poured into water and extracted into ethyl acetate (100 ml x3). The organic layer was washed with brine (100 ml x2), dried over sodium sulfate, and concentrated under reduced pressure. The crude material was purified by column chromatography (DCM / MeOH = 50/1 as elution) to give the title compound (1.35 g, 51% yield over 3 steps) as a yellow solid.

Synthesis of N1- (1- (2-fluoroethyl) acetidin-3-yl) -3-methoxybenzene-1,4-diamine

<img file="ES2618007T3_D0086.tif" />

A solution of 1- (2-fluoroethyl) -N- (3-methoxy-4-nitrophenyl) acetidin-3-amine (2.6 g) and Pd / C (1 g) in 1,4-dioxane (50 ml ) was hydrogenated for 4 hours at room temperature. The reaction mixture was filtered through diatomaceous earth, washing with MeOH. The filtrate was concentrated and purified by column chromatography (DCM / MeOH = 50/1 as elution) to provide the title compound (1.57 g, 68% yield, M + H⁺ = 240,2).

Pivalate synthesis of (2- (4- (1- (2-fluoroethyl) acetidin-3-ylamino) -2-methoxyphenylamino) -4- (3-nitrophenoxy) -7H-pyrrolo [2,3d] pyrimidin-7- yl) methyl (Compound 34-A)

ES 2 618 007 T3

<img file="ES2618007T3_D0087.tif" />

A mixture of N1- (1- (2-fluoroethyl) acetidin-3-yl) -3-methoxybenzene-1,4-diamine (870 mg, 3.64 mmol) and (2-chloro4- (3-nltrofenox) pivalate ¡) -7H-plrrolo [2,3-d] p¡r¡m¡d¡n-7-¡l) methyl (1.55 g, 3.83 mmol), potassium carbonate (1.35 g, 9 , 77 mmol), tris (dibenzylideneacetone) dipalladium (173 mg, 0.19 mmol) and dicyclohexyl (2 ', 4', 6'-triisopropylbiphenyl-2-yl) phosphine (222 mg, 0.47 mmol), a magnetite and f-BuOH (35 mL) was heated to reflux and stirred under nitrogen for 2 h. The mixture was cooled to 40 ~ 50 ° C and filtered through diatomaceous earth, washing with ethyl acetate (50 ml). The filtrate was concentrated under reduced pressure. The crude material was purified by column chromatography (DCM / MeOH = 50/1 as elution) to give the title compound (1.7 g, 74% yield, M + H⁺= 608.3) as a light yellow solid.

Synthesis of N1- (4- (3-aminophenoxy) -7H-pyrrolo [2,3-d] pyrimidin-2-yl) -N4- (1- (2-fluoroethyl) acetidin-3-yl) -2-methoxybenzene -1.4 diamine

<img file="ES2618007T3_D0088.tif" />

A pivalate mixture of (2- (4- (1- (2-fluoroethyl) acetidin-3-ylamino) -2-methoxyphenylamino) -4- (3-nitrophenoxy) -7H-pyrrolo [2,3d] pyrimidin-7 -yl) methyl (530 mg, 0.87 mmol), ΝΗ2ΝΗ2Ή2Ο (98%, 2.5 ml), Pd / C (110 mg), a magnetite and MeOH (10 ml) stirred at reflux temperature overnight . The mixture was cooled to room temperature and filtered through diatomaceous earth, washing with MeOH (20 ml). The filtrate was concentrated under reduced pressure. NaHCO3 (aq.) Was added and the mixture was extracted with ethyl acetate (30 ml x 3). The combined organic layers were concentrated under reduced pressure. The crude material was purified by column chromatography (DCM / MeOH = 40/1 as elution) to give the title compound (125 mg, 31% yield, M + H⁺= 464.2) as a white solid.

Synthesis of N- (3- (2- (4- (1- (2-fluoroethyl) acetidin-3-ylamino) -2-methoxyphenylamino) -7H-pyrrolo [2,3-d] pyrimidin-4yloxy) phenyl) acrylamide (Compound 34)

<img file="ES2618007T3_D0089.tif" />

A 50 ml round bottom flask with a magnetite was charged with N1- (4- (3-aminophenoxy) -7H-pyrrolo [2,3-d] pyrimidin2-yl) -N4- (1- (2-fluoroethyl) acetidin-3-yl) -2-methoxybenzene-1,4-diamine (125 mg, 0.27 mmol), diisopropylethylamine (43 mg,

0.33 mmol) and DCM (20 ml). The mixture was cooled with an ice bath until the temperature was below 0 ° C and a solution of acryloyl chloride (33 mg, 0.33 mmol) in THF ( 2 ml). The title compound is isolated and purified by preparative HPLC or preparative LC / MS or by other standard purification techniques. In some experiments, the title compound was isolated and purified by preparative LC / MS.

Compound 34 was also synthesized using a later alternative synthetic route:

ES 2 618 007 T3

<img file="ES2618007T3_D0090.tif" />

The synthesis of compound 34-B:

Fe powder (0.3 g) and NH4CI (0.5 g) in EtOH (60 ml) were added to compound 34-A (0.6 g) in a 100 ml round bottom flask. The reaction mixture was stirred at 90 ~ 100 ° C for 3 ~ 4 h. At this point, the reaction was complete. Indicated by TLC (DCM: MeOH = 8: 1). The reaction mixture was filtered through cellta and washed further with MeOH (-60 ml). The combined filtrate was concentrated under reduced pressure. The residue (oil) was dissolved in ethyl acetate (100 ml), washed with brine (50 ml x2) and dried over sodium sulfate. The organic layer was concentrated under reduced pressure. The obtained crude material was further purified with column chromatography (DCM: MeOH = 30: 1) to give the desired product 34-B (420 mg, M + H⁺= 578,5).

The synthesis of compound 34-C:

Acrylic acid (41 mg) and EDCI (176 mg) in DCM (30 ml) were added to compound 34-B (288 mg) in a 100 ml round bottom flask. The reaction mixture was stirred at 0 ° C (ice bath) for 1-1.5 h. At this point, TLC (DCM: MeOH = 7: 1) showed completion of the reaction. A small amount of water (0.5 ml) was added to quench the reaction. The reaction mixture was concentrated under reduced pressure. The obtained residue was dissolved in ethyl acetate (30 ml). The organic layer was washed with brine (10 ml x2), dried over sodium sulfate and concentrated under reduced pressure to give the crude product, which was further purified with column chromatography (DCM: MeOH = 30: 1) to give desired product 34-C (79 mg, M + H⁺=632,5).

The synthesis of compound 34:

Aqueous NaOH solution (2.5 mol / L) was added to compound 34-C (79 mg) in MeOH (15 ml) in a 100 ml round bottom flask. The reaction mixture was stirred at 0 ° C (ice bath) for 4-5 h. At this point, LC-MS indicated completion of the reaction. The reaction mixture was poured into water (100 ml), extracted with ethyl acetate (50 ml x3). The organic layer was washed with brine (30 ml x2), dried over sodium sulfate, and concentrated under reduced pressure. The crude material was further purified with column chromatography (DCM: MeOH = 25: 1) to give the desired product 34 (32 mg, M + H⁺= 464,5).

Additional exemplary compounds not shown in these synthetic examples are prepared from appropriate starting materials using methods analogous to those described in the preceding schemes and examples.

Biological example A:

Cell-based in vitro screening using a real-time electronic cell detection system (RT-CES)

ES 2 618 007 T3

Some tests and examples demonstrating the anticancer effects of the compounds of the embodiments are described below.

The anticancer activities for cancer cells of the pyrrolopyrimidine compounds of the embodiments with certain molecular targets, ie, EGFR (epidermal growth factor receptor), were investigated. The anticancer efficacy of pyrrolopyrimidine compounds can be preliminarily screened in vitro using a pool of EGFR cancer cell lines using a Real Time Electronic Cell Detection System (RT-CES) from ACEA Biosciences, Inc. (or the xCELLigence system from Roche Applied Sciences / ACEA Biosciences Inc.), which provides dynamic information on cellular responses after exposure to an anticancer agent.

The details of this electronic cellular detection technology, called Real Time Electronic Cell Detection (RT-CES®) and associated devices, systems and methods are described in patent number 7,732,127; Patent Number 7,192,752; Patent Number 7,459,303; patent number 7,468,255; Patent Number 7,470,533; US Patent No. 7,560,269; United States Provisional Application No. 60 / 397,749, filed July 20, 2002; United States Provisional Application No. 60 / 435,400, filed December 20, 2002; US Provisional Application No. 60 / 469,572, filed May 9, 2003, PCT Application No. PCT / US03 / 22557, filed July 18, 2003; PCT application number PCT / US03 / 22537, filed July 18, 2003; PCT application number PCT / US04 / 37696, filed November 12, 2004; PCT application number PCT / US05 / 04481, filed February 9, 2005; US Patent Application No. 10 / 705,447, filed November 10, 2003; US Patent Application No. 10 / 705,615, filed November 10, 2003; US Patent Application No. 10 / 987,732, filed November 12, 2004; US Patent Application No. 11 / 055,639, filed February 9, 2005, each of which is incorporated by reference. Additional details of the RT-CES technology are further disclosed in US Provisional Application No. 60 / 519,567, filed November 12, 2003, and US Provisional Application No. 60 / 542,927, filed February 9, 2004, United States Provisional Application Number 60 / 548,713, filed February 27, 2004; United States Provisional Application Number 60 / 598,608, filed August 4, 2004; United States provisional application number 60 / 598,609, filed on August 4, 2004; US Provisional Application No. 60 / 613,749, filed September 27, 2004; US Provisional Application No. 60 / 613,872, filed September 27, 2004; United States provisional application number 60 / 614,601, filed on September 29, 2004; United States Provisional Application No. 60 / 630,071, filed November 22, 2004; United States provisional application number 60 / 630,131, filed on November 22, 2004.

For cell-substrate or cell-electrode impedance measurement using RT-CES technology, microelectrodes having appropriate geometries are fabricated on the bottom of the microtiter plate or similar device, facing into the wells. The cells are introduced into the wells of the devices and make contact with and bind to the surfaces of the electrodes. The presence, absence or change of properties of the cells affects the electronic and ionic passage over the sensing surfaces of the electrode. Measuring the impedance between electrodes provides information about the biological state of the cells present on the detectors. When there are changes in the biological state of cells, analog electronic read signals are automatically measured in real time and converted into digital signals for processing and analysis.

In an RT-CES system, a cell index is automatically derived and provided based on the measured electrode impedance values. The cell index obtained for a given well reflects: 1) how many cells bind to the electrode surfaces in this well; and 2) how the cells in the well bind to the electrode surfaces in this well. Thus, the more cells of the same type under similar physiological conditions bind to the electrode surfaces, the higher the cell index. And, the better the cells bind to the electrode surfaces (eg, the cells are spread further to have larger contact areas or the cells bind more strongly to the electrode surfaces), the higher the cell index. The present inventors have found that cMet addictive cell lines would produce a transient impedance response profile when treated with positive control EGFR (epidermal growth factor receptor) inhibitors.

Through the RT-CES system, it has been observed that the pyrrolopyrimidine compounds described in the previous examples produce a cellular response impedance profile in the RT-CES system similar to that generated by positive control inhibitors. Furthermore, these compounds have been shown to inhibit EGFR (epidermal growth factor receptor) -induced cell migration in various cell lines. Furthermore, these compounds have been shown to have no or negligible effects when used to treat non-cMet addictive cancer cell lines.

The RT-CES system (or the xCELLigence RTCA system) comprises three components, an electronic detector analyzer, a device station, and 16X or 96X microtitre plate devices (ie E-Plate 16 or E-Plate 96). The series of microelectrode detectors were fabricated from glass slides using lithographic microfabrication methods and the electrode-containing slides are mounted in plastic trays to form electrode-containing wells. Each 16X (or 96X) microtiter plate device used in the RT-CES system comprises up to 16 (or 96) such electrode-containing wells. The device station receives the 16X or 96X microtiter plate devices and is capable of electronically switching any one of the wells to the

ES 2 618 007 T3 detector analyzer for impedance measurement. During operation, the devices with cells grown in the wells are placed in a device station (xCELLIgence RTCA SP station or RT-CES SP station) that is located inside an incubator. Electrical cables connect the device station to the detector analyzer (xCELLigence RTCA analyzer or RT-CES analyzer). Under the control of the RT-CES or xCELLigence RTCA software, the detector analyzer can automatically select the wells to be measured and continuously perform impedance measurements. The impedance data from the analyzer is transferred to a computer, analyzed and processed by the Integrated software.

The impedance measured between electrodes in an individual well depends on the geometry of the electrodes, the ionic concentration in the well, and whether cells are attached to the electrodes. In the absence of cells, the impedance of the electrodes is mainly determined by the ionic environment both at the electrode / solution interface and in the solution mass. In the presence of cells, cells bound to the sensing surfaces of the electrodes will alter the local ionic environment at the electrode / solution interface, leading to an increase in impedance. The more cells on the electrodes, the greater the increase in cell-electrode impedance. On the other hand, the impedance change also depends on the morphology of the cell and the degree to which the cells bind to the electrodes.

To quantify the state of cells based on the measured cell-electrode impedance, a parameter called Cell Index is derived, according to

IC = max ———-— 1

J where R_b(f) and R_Cei. (f) are the frequency-dependent electrode resistances (a component of impedance) with no cells or with cells present, respectively. N is the number of frequency points at which impedance is measured. Thus, the Cell index is a quantitative average of the state of the cells in a well containing electrode. Under the same physiological conditions, more cells bound to the electrodes lead to a Rcéi value. (t) higher, which leads to a higher value for the Cell index. On the other hand, for the same number of cells present in the well, a change in cell state such as morphology will lead to a change in the index.

Mobile. For example, an increase in cell adhesion or cell extension leads to a greater cell-electrode contact area that will lead to an increase in R_Chim. (t) and thus a higher value for the Cellular index. The Cell Index can also be calculated using a different formula than the one described here. Other methods for calculating Cell Index based on impedance measurement can be found in patent number 7,732,127; Patent Number 7,192,752; Patent Number 7,459,303; Patent Number 7,468,255; Patent Number 7,470,533; patent number

7,560,269 from the United States; PCT application number PCT / US04 / 37696, filed November 12, 2004, PCT application number PCT / US05 / 04481, filed February 9, 2005, US patent application number 10 / 987,732, filed November 12, 2004 and U.S. Patent Application No. 11 / 055,639, filed February 9, 2005.

Biological example B-1

Bioactivity of pyrrolopyrimidine compounds on EGFR mutated cell lines Material and methods Cell culture and reagents

All cell lines were obtained from the American Type Culture Collection and kept at 37 ° C with 5% CO2 in medium supplemented with 10% fetal bovine serum and 1% L-glutamine-penicillin-streptomycin. The cells

H1975 and HCC827 were cultured with RPMI 1640 medium. A431 cells were maintained in Dulbecco's modification of Eagle's medium. EGF, EGF (R&amp;D) inhibitors were resuspended and stored according to manufacturer's instructions.

Cell growth and proliferation inhibition assay

Cell proliferation was analyzed by a WST assay (Roche, Indianapolis, IN) following the manufacturer's instructions. H1975, HCC827, and A431 cells were seeded at 3,000, 3,000, and 4,000 cells per well in 96-well plates and, after a 24 hour incubation, cells were treated with test compounds for 72 hours. Cell viability was analyzed by incubating cells with WST-1 reagent for 2 hours and then with absorbance measurement at a wavelength of 450 nm. Data was calculated using GraphPad Prism version 4.0. IC50 values ​​were fitted using a non-linear regression model with a sigmoid dose response.

Western transfer

H1975 and A431 cells were seeded on 6-well plates at a concentration of 1 * 10® cells per well. After 24 hours of growth in serum-containing medium, cells were incubated in serum-free medium for 1 hour and then treated with the test compound for 2 hours. A431 cells are

ES 2 618 007 T3 stimulated with 30 ng / ml EGF during the last 20 minutes of compound treatment. Western blots were performed on whole cell extracts using antibodies to phospho-specific EGFR (pY1068), EGFR, total phospho-Akt (Ser-473), total Akt, phospho-ERK1 / 2 (pT202 / pY204) and total ERK1 / 2. (Cell Signaling Technology).

Tumor sections were flash frozen in liquid nitrogen for protein isolation and EGFR signal transduction was assessed by Western blotting with primary antibodies including the following: phospho-specific EGFR (pY1068), total EGFR, phospho-Akt (Ser-473 ), Total Akt, phospho-ERK1 / 2 (pT202 / pY204) and total ERK1 / 2.

ELISA assay

H1975 and A431 cells were seeded in each well of a 96-well plate at a density of 4&gt; &lt;10⁴ cells per well. After 24 hours of growth in serum-containing medium, cells were treated with test compound in serum-free medium for 2 hours. A431 cells were stimulated with 30 ng / ml EGF during the last 15 minutes of compound treatment. Cells were washed with ice cold PBS prior to extraction with 100 µl per well of cell lysis buffer. EGFR phosphorylation was measured using a sandwich ELISA with the pair of antibodies for phospho-specific EGFR (pY1068) and total EGFR.

Results

Compound 3 inhibits the proliferation of EGFR mutant cells

The following compounds were tested.

or

Compound 3 or

<img file="ES2618007T3_D0091.tif" />

och₃

Compound A .cl och₃

Compound B (WZ4002)

Ν '

Gefitinib

Sensitivity of cancer cell lines expressing EGFR WT, Exon 19 Del, L858R / T790M and delE746-A750 to Compound 3, Compound B and gefitinib. Cell proliferation assays were performed with increasing concentrations of the compounds over 72 hours using WST. IC50 values ​​were determined using GraphPad software.

Compound 3 inhibits the proliferation of H1975 to T790M positive cells more potently than gefitinib.

ES 2 618 007 T3

Table 1

<td>Compound</td><td>Cell H1975 (T790M / L858R)</td><td>Cell A431 (WT)</td><td>HCC827 cell (delE746-A750)</td>

<td>Compound 3</td><td>0.61 μΜ</td><td>10.8 μΜ</td><td>0.019 μΜ</td>

<td>Compound B</td><td>1.1 μΜ</td><td>4.5 μΜ</td><td>0.013 μΜ</td>

<td>Gefitinib</td><td>&gt; 10 μΜ</td><td>ND</td><td>0.024 μΜ</td>

Compound 3 inhibits EGFR phosphorylation in H1975 cells

Inhibition of EGFR phosphorylation and proliferation in Compound 3-treated H1975 cells. H1975 and A431 cells were incubated with various concentrations of Compound 3 or Compound B for 2 hours and whole cell extracts were directly harvested and tested for to pEGFR by ELISA. IC50 values ​​were determined using GraphPad software.

Table 2

<td>Compound</td><td>Cell H1975 (T790M / L858R)</td><td>Cell A431 (WT)</td>

<td>Compound 3</td><td>0.031 μΜ</td><td>12.7 μΜ</td>

<td>Compound B</td><td>0.063 μΜ</td><td>8.9 μΜ</td>

Compound 3 inhibits the EGFR signaling pathway in H1975 cells

Exponentially growing H1975 lung cancer cells were treated with Compound 3 at the indicated concentrations for 2 hours in serum-free medium. As shown in Figure 1, whole cell extracts were resolved by SDS-PAGE before transferring onto nitrocellulose membranes. Inhibition of EGFR phosphorylation leads to inhibition of its downstream effectors ρ-Akt and p-ERK. All antibodies were obtained from Cell Signaling.

Compound 3 inhibits the EGFR signaling pathway in H1975 tumors

Compound 3 was administered orally (PO) at 100 mg / kg and tumors were harvested at 1.4, 8, 18 and 25 hours after the individual dose. As shown in Figure 2, immunoblots were screened for pEGFR, total EGFR, pAkt, total Akt, p-ERK, and total ERK. Compound 3 inhibited EGFR phosphorylation in a time-dependent manner and inhibition at EGFR leads to inhibition of its downstream effectors ρ-Akt and p-ERK. Comparison between Compound 3 and Compound A

1. WST result

Table 3

<td>Compound</td><td>Cell H1975 (T790M / L858R)</td><td>Cell A431 (WT)</td><td>HCC827 cell (delE746-A750)</td>

<td>Compound 3</td><td>0.73 μΜ</td><td>0.62 μΜ</td><td>0.011 μΜ</td>

<td>Compounds A</td><td>1.63 μΜ</td><td>4.17 μΜ</td><td>0.023 μΜ</td>

two. ELISA result

Table 4

<td>Compound</td><td>Cell H1975 (T790M / L858R)</td><td>Cell A431 (WT)</td><td>H1975 cell (T790M / L858R) stimulation with EGF</td>

<td>Compound 3</td><td>0.0032 μΜ</td><td>0.4737 μΜ</td><td>0.025 μΜ</td>

<td>Compound A</td><td>0.0088 μΜ</td><td>1.0270 μΜ</td><td>0.091 μΜ</td>

Biological example B-2 25 Cell culture and reagents

All cell lines were obtained from the American Type Culture Collection and kept at 37 ° C with 5% CO2, in medium supplemented with 10% fetal bovine serum and 1% L-glutamine-penicillin-streptomycin. H1975 and HCC827 cells were cultured with RPMI 1640 medium. A431 cells were maintained in Dulbecco's Modification

ES 2 618 007 T3 of Eagle's medium. GTL-16 cells, T47D cells, and BxPC3 cells were cultured with RPMI 1640 medium. NIH-3T3 cells, H460 cells, and HepG2 cells were cultured with Dulbecco's Modification of Eagle's medium. A549 cells were cultured with F-12K nutrient mixing medium. H295R were cultured with DMEM: F12 medium. Reagent WST-1 was obtained from Roche. EGF (R&amp;D), EGF inhibitors were resuspended and stored according to manufacturer's instructions.

Cell growth and proliferation inhibition assay

Cell proliferation was examined by a WST assay (Roche, Indianapolis, IN) according to the manufacturer's instructions. H1975, HCC-827, and A431 cells were seeded at 3,000, 3,000, and 4,000 cells per well on 96-well plates and, after a 24 hr incubation, the cells were treated with test compounds for 72 hr. NIH-3T3 cells, A549 cells, H295R cells, GTL-16 cells, H460 cells, HepG2 cells, Hela cells, T47D cells, and BxPC3 cells were seeded at 2,000, 2,000, 5,000, 5,000, 2,500, 5,000, 2,000, 5,000 and 5,000 cells per well on 96-well plates. Cell viability was tested by incubating cells with WST-1 reagent for 3 h. Absorbance was measured at OD450-620 using the Beckman DTX880. Data was calculated using GraphPad Prism version 4.0. IC50's were adjusted using a non-linear regression model with a sigmoid dose response.

ELISA assays

H1975, HCC-827, and A431 cells were seeded on a 96-well plate at a density of 40,000, 40,000, and 60,000 cells per well, respectively. After 24 h of growth in serum-containing medium, cells were treated with test compound in serum-free medium for 2 h. A431 cells were stimulated with 50 ng / ml EGF during the last 15 min of compound treatment. Cells were washed with ice cold PBS prior to extraction with 100 µl per well of cell lysis buffer. EGFR phosphorylation was measured using a sandwich ELISA with the pair of antibodies for phospho-specific EGFR (pY1068) and total EGFR. Data was calculated using GraphPad Prism version 4.0. IC50's were adjusted using a non-linear regression model with a sigmoid dose response.

Western transfer

Cells H1975, HCC-827 and A431 were seeded on 6-well plates at a concentration of 1 × 10⁶ cells per well. After 24 h of growth in serum-containing medium, cells were incubated in serum-free medium for 1 h and then treated with test compound for 2 h. A431 cells were stimulated with 30 ng / ml eGf during the last 20 min of compound treatment. Western blots were performed on whole cell extracts using antibodies to phospho-specific EGFR (pY1068), total EGFR, phospho-Akt (Ser-473), total Akt, phospho-ERK1 / 2 (pT202 / pY204) and total ERK1 / 2. (Cell Signaling Technology). The density of the transfer band was acquired using Image J software and the IC50 of EGFR Tyr1068 phosphorylation was adjusted using a non-linear regression model using GraphPad Prism version 4.0.

Compound 3 was administered orally at the indicated dose (12.5, 50, 200 mg / kg) and gefitinib (GF) was administered orally at 100 mg / kg. Tumor tissues were harvested at 1, 4, 8, and 24 h on Day 1 and after the individual dose or harvested on Day 8 and after 8 consecutive doses (12.5, 50 mg / kg). Tumor sections were flash frozen in liquid nitrogen for protein isolation and EGFR signal transduction was assessed by Western blotting with primary antibodies including the following: phospho-specific EGFR (pY1068), total EGFR.

Cell-based pulsed follow-up assay to examine compound irreversibility

H1975s were seeded at 3,000 cells per well in the RTCA system (xCELLigence SP instrument, ACEA Biosciences). After one day of culture, cells were treated with compound of compound 3, WZ4002 at a concentration of 10 pM for 22 h and then removed compared to drugs maintained over time. Approximately 60 hours after recovery, the cells were subjected to a measurement of viability with WST.

Results

Compound 3 inhibited the proliferation of cancer cells harboring EGFR mutation

Compound 3 achieved the inhibition of the proliferation of H1975 cells (T790M / L858R) with the IC50 at 91 ± 60nM and with the IC50 at 19 ± 8 nM for HCC827 cells (Del E746-A750), while the sensitivity to A431 cells (WT) is much lower (IC50 = 2113 ± 1660 nM). In contrast, gefitinib, the first generation EGFR inhibitor, exhibited sensitivity to A431 cells, but had no activity on the proliferation of cells harboring the T790M mutation (IC50&gt; 20uM).

ES 2 618 007 T3

<td>Compound</td><td>Cell H1975 (T790M / L858R)</td><td>Cell A431 (WT)</td><td>HCC827 cell (delE746-A750)</td>

<td>Compound 3</td><td>91 ± 60 nM</td><td>2113 ± 1660 nM</td><td>19 ± 8 nM</td>

<td>WZ4002</td><td>1905 ± 732 nM</td><td>4393 ± 617 nM</td><td>35 ± 12 nM</td>

<td>Gefitinib</td><td>&gt; 20,000 nM</td><td>523 ± 115 nM</td><td>9 ± 1 nM</td>

Compound 3 significantly reduced EGFR Tyr1068 phosphorylation in EGFR mutant cells

H1975 and A431 cells were incubated with various concentrations of Compound 3 or WZ4002 for 2 h and whole cell extracts were directly harvested and tested for pEGFR by ELISA. IC50 values ​​were determined using GraphPad software.

Cell-based ELISA assays verified that compound 3 significantly reduced EGFR Tyr1068 phosphorylation in EGFR mutant cell lines, while gefitinib showed phosphorylation inhibition to a much lesser degree.

<td>Compound</td><td>Cell H1975 (T790M / L858R)</td><td>Cell A431 (WT)</td>

<td>compound 3</td><td>4 ± 2 nM</td><td>650 ± 63 nM</td>

<td>WZ4002</td><td>32 ± 12 nM</td><td>970 ± 340 nM</td>

As shown in the table below, Compound 3 significantly reduced EGFR Tyr1068 phosphorylation and downstream signaling in EGFR mutant cells and is less efficient in the wild-type EGFR expressing cell line.

<td rowspan="2">EGFR genotype</td><td rowspan="2">Cellphone line</td><td colspan="3">IC50 (nM) by WB (phospho Tyr1068)</td>

<td>compound 3</td><td>Gefitinib</td><td>WZ4002</td>

<td>T790M / L858R</td><td>H1975</td><td> 4,4</td><td> 860</td><td> 21</td>

<td>DelE746-A750</td><td>HCC-827</td><td> 9,8</td><td> 5,4</td><td> 58</td>

<td>Wild</td><td>A431</td><td> 288</td><td> 1,6</td><td> 53</td>

<td colspan="2">Selectivity (A431 / H1975)</td><td>65X</td><td>0.002X</td><td>2.5X</td>

As shown in Figures 9A and 9B, Compound 3 inhibited EGFR Tyr1068 phosphorylation and downstream signaling in EGFR H1975 mutant cells. Comparative data for gefitinib and WZ4002 are shown in Figures 9C-9F.

As shown in Figures 10A and 10B, Compound 3 inhibited EGFR Tyr1068 phosphorylation and downstream signaling in EGFR HCC-827 mutant cells. Comparative data for gefitinib and WZ4002 are shown in Figures 10C-10F.

As shown in Figures 11A and 11B, Compound 3 was less effective in inhibiting EGFR Tyr1068 phosphorylation and downstream signaling in EGFR WT expressing A431 cells. Comparative data for gefitinib and WZ4002 are shown in Figures 11C-11F.

Compound 3 inhibited EGFR phosphorylation in H1975 tumors.

Compound 3 significantly inhibited EGFR phosphorylation in H1975 tumor tissues, at the three dosages of 12.5, 50 and 200 mg / kg. Inhibition of EGFR phosphorylation by Compound 3 was dose and time dependent. In contrast, inhibition of EGFR phosphorylation was not detected for gefitinib at the 100 mg / kg dosage.

As shown in Figure 12, Compound 3 inhibits EGFR phosphorylation in H1975 tumor tissues with a single dose of Compound 3.

As shown in Figure 13, Compound 3 inhibits EGFR phosphorylation in H1975 tumor tissues after 8 consecutive doses of Compound 3.

As shown in Figure 14, Compound 3 Irreversibly inhibited the proliferation of H1975 cells harboring the EGFR T790M mutation. The reversibility of compound 3 was examined by a cell-based pulsatile monitoring assay. As shown in Figure 14, by deleting compound 3 after a 22 hr treatment, the inhibition of H1975 proliferation was maintained (7.8 ± 1.3%) up to 60 hr. In contrast, the recovery of

ES 2 618 007 T3 treatment with WZ4002 was at 27 ± 10%. The results of this study demonstrated that compound 3 is an Irreversible EGFR Inhibitor and exhibited strong binding property relative to WZ4002.

Viability compared to vehicle control (%)

<td></td><td>compound 3 during 22 h of treatment</td><td>WZ4002 for 22 h of treatment</td>

<td>H1975 cell viability (%)</td><td> 7,8±1,3</td><td> 27±10</td>

1. WST result

<td>Compound</td><td>Cell H1975 (T790M / L858R)</td><td>Cell A431 (WT)</td><td>HCC827 cell (delE746-A750)</td>

<td>compound 3</td><td>0.19 uM</td><td>2.03 uM</td><td>0.011 uM</td>

<td>compound A</td><td>1.16 uM</td><td>11.82 uM *</td><td>0.023 uM</td>

* Note: The A431 value for Compound A was previously incorrectly reported as 9.14 uM.

two. ELISA result

<td>Compound</td><td>Cell H1975 (T790M / L858R)</td><td>Cell A431 (WT) EGF stimulation</td><td>Cell H1975 (T790M / L858R)</td>

<td>compound 3</td><td>0.0032 uM</td><td>0.4737 uM</td><td>0.025 uM</td>

<td>compound A</td><td>0.0088 uM</td><td>1.0270 uM</td><td>0.091 uM</td>

Biological example B-3

Using a new H1975 cell line and the ELISA protocol described above in Biological Example B-2, the following results were obtained:

ELISA assay

<td></td><td>IC50 (uM)</td><td>IC50 (uM)</td><td></td><td></td><td></td>

<td></td><td>H1975</td><td>Hcc827</td><td>IC50 (uM)</td><td></td><td></td>

<td></td><td>T790M / L858R</td><td>(delE746-A750)</td><td>A431WT</td><td>Selectivity</td><td>Selectivity</td>

<td></td><td>(without</td><td>(without</td><td>(stimulation</td><td>A431 / H1975</td><td>A431 / Hcc827</td>

<td></td><td>stimulation</td><td>stimulation</td><td>with EGF)</td><td></td><td></td>

<td></td><td>with EGF)</td><td>with EGF)</td><td></td><td></td><td></td>

<td></td><td> 0,0018</td><td> 0,0076</td><td> 0,1233</td><td> 68,5000</td><td> 16,2237</td>

<td>Comp. 3</td><td> 0,0016</td><td> 0,0072</td><td> 0,1300</td><td> 81,2500</td><td> 18,0556</td>

<td></td><td> 0,0010</td><td> 0,0072</td><td> 0,0901</td><td> 90,1000</td><td> 12,5139</td>

<td rowspan="2">Avg ± SD</td><td> 0,0015 ±</td><td rowspan="2"> 0,0073 ± 0,0002</td><td> 0,1145 ±</td><td> 79,9500 ±</td><td> 15,5977 ±</td>

<td> 0,0004</td><td> 0,0174</td><td> 10,8585</td><td> 2,8233</td>

<td></td><td> 0,0299</td><td> 0,0419</td><td> 0,6184</td><td> 20,6823</td><td> 14,7589</td>

<td>Comp. B (WZ4002)</td><td> 0,0257</td><td> 0,0385</td><td> 0,6592</td><td> 25,6498</td><td> 17,1221</td>

<td></td><td> 0,0212</td><td> 0,0391</td><td> 0,6097</td><td> 28,7594</td><td> 15,5934</td>

<td>Avg ± SD</td><td> 0,0256 ±</td><td rowspan="2"> 0,0399 ±0,0018</td><td> 0,6291±</td><td> 25,0305</td><td> 15,8248</td>

<td></td><td> 0,0043</td><td> 0,0264</td><td> ±4,0740</td><td> ±1,1984</td>

<td>P values ​​(Comp. 3 and Comp. B)</td><td> 0,0007</td><td> 0,000007</td><td> 0,000013</td><td> 0,0012</td><td> 0,9042</td>

<td></td><td> 0,0145</td><td> 0,0411</td><td> 0,5641</td><td> 38,9034</td><td> 13,7251</td>

<td>Comp. TO</td><td> 0,0159</td><td> 0,0395</td><td> 0,4794</td><td> 30,1509</td><td> 12,1367</td>

<td></td><td> 0,0040</td><td> 0,0305</td><td> 0,2884</td><td> 72,1000</td><td> 9,4557</td>

<td>Avg ± SD</td><td> 0,0115 ±</td><td> 0,00370 ±</td><td> 0,4440 ±</td><td> 47,0515 ±</td><td> 11,7725 ±</td>

<td></td><td> 0,0065</td><td> 0,0057</td><td> 0,1412</td><td> 22,1297</td><td> 2,1578</td>

<td>P * values ​​(Comp. 3 and Comp. TO)</td><td> 0,0565</td><td> 0,00085</td><td> 0,01618</td><td> 0,0819</td><td> 0,13569</td>

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Using a new H1975 cell line and the WST protocol described above (in Biological Examples B-2 with the presence of 5% fetal bovine serum), the following results were obtained:

WST Assay (Cell Viability Assay)

<td></td><td>IC50 (uM) H1975 T790M / L858R</td><td>IC50 (uM) Hcc827 (delE746-A750)</td><td>IC50 (uM) A431WT</td><td>Selectivity A431 / H1975</td><td>Selectivity A431 / Hcc827</td>

<td></td><td> 0,019</td><td> 0,003</td><td> 0,692</td><td> 36,42</td><td> 230,67</td>

<td>Comp. 3</td><td> 0,018</td><td> 0,008</td><td> 0,526</td><td> 29,22</td><td> 65,75</td>

<td></td><td> 0,019</td><td> 0,005</td><td> 0,694</td><td> 36,53</td><td> 138,80</td>

<td>Avg ± SD</td><td> 0,019 ±0,001</td><td> 0,005± 0,002</td><td> 0,637± 0,096</td><td> 34,057 ± 4,187</td><td> 145,072 ± 82,637</td>

<td></td><td> 0,053</td><td> 0,007</td><td> 0,901</td><td> 17,00</td><td> 128,71</td>

<td>Comp. B (WZ4002)</td><td> 0,080</td><td> 0,021</td><td> 1,027</td><td> 12,84</td><td> 48,90</td>

<td></td><td> 0,106</td><td> 0,013</td><td> 1,025</td><td> 9,67</td><td> 78,85</td>

<td>Avg ± SD</td><td> 0,080 ± 0,026</td><td> 0,014 ±0,007</td><td> 0,984± 0,072</td><td> 13,169± 3,676</td><td> 85,488± 40,317</td>

<td>P values ​​(Comp. 3 and Comp. B)</td><td> 0,0163</td><td> 0,1251</td><td> 0,0075</td><td> 0,0029</td><td> 0,3245</td>

<td></td><td> 0,106</td><td> 0,028</td><td> 2,914</td><td> 27,491</td><td> 104,071</td>

<td>Comp. TO</td><td> 0,111</td><td> 0,059</td><td> 2,617</td><td> 23,577</td><td> 44,356</td>

<td></td><td> 0,067</td><td> 0,035</td><td> 2,809</td><td> 41,925</td><td> 80,257</td>

<td>Avg ± SD</td><td> 0,095± 0,024</td><td> 0,041± 0,016</td><td> 2,780 ±0,151</td><td> 30,998 ± 9,664</td><td> 76,228 ± 30,061</td>

<td>P * values ​​(Comp. 3 and Comp. TO)</td><td> 0,0055</td><td> 0,0205</td><td>3.18539E-05</td><td> 0,6414</td><td> 0,2466</td>

* p values ​​1) &lt;0.05 (marked in bold) mean significant difference; 2) 0.05-0.15 means 5 difference / improvement Important but not significant; 3)&gt; 0.15 means no significant difference.

Based on the experimental data and p-values ​​in the two tables above, Compound 3 demonstrated significantly greater potency than Compounds A and B in both the WST and ELISA assays with ten (10) p-values ​​&lt;0, 05 and two (2) p values ​​between 0.05 - 0.15. Regarding selectivity, Compound 3 showed a general trend of higher selectivity than Compounds A and B in both the WST and ELISA tests with two (2) values ​​of p &lt;0.05, two (2) values ​​of p between 0.05-0.15 and four (4) p values ​​above 0.15. Biological example C

Evaluation of the efficacy of Compound 3 in the treatment of H1975, HCC827 and A431 Xenolngrafted mouse models

This example evaluates the efficacy of Compound 3 in the treatment of xenolngraft tumor models of human non-small cell lung adenocarclnoma NCI-H1975 (L858R / T790M), human lung adenocarclnoma

HCC827 (L858R) and human cutaneous epldermolde carcinoma A431 (WT) in athletic mice. Gefitinib, a first generation reversible EGFR tyrosine kinase inhibitor, was used as a positive control in these three xenolngraft tumor models in mice.

Experimental design and dosing plan 20 The experimental design and dosing plan are shown below

Table 5: Model NCI-H1975

<td>Group</td><td>n</td><td>Treatment</td><td>Dose (mg / kg)</td><td>Volume of dosage (μΙ / g)</td><td>Way of dosage</td><td>Solvent</td><td>Days of dosage</td><td>Plan</td>

<td> 1</td><td> 8</td><td>Vehicle</td><td> -</td><td> 16,7</td><td>PO</td><td>System of</td><td> 14</td><td>QD</td>

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<td>Group</td><td>n</td><td>Treatment</td><td>Dose (mg / kg)</td><td>Volume of dosage (pl / g)</td><td>Way of dosage</td><td>Solvent</td><td>Days of dosage</td><td>Plan</td>

<td></td><td></td><td></td><td></td><td></td><td></td><td>PEG</td><td></td><td></td>

<td> 2</td><td> 8</td><td>Compound 3</td><td> 25</td><td> 10</td><td>PO</td><td>PEG system</td><td> 14</td><td>QD</td>

<td> 3</td><td> 8</td><td>Compound 3</td><td> 50</td><td> 10</td><td>PO</td><td>PEG system</td><td> 14</td><td>QD</td>

<td> 4</td><td> 8</td><td>Compound 3</td><td> 100</td><td> 16,7</td><td>PO</td><td>PEG system</td><td> 14</td><td>QD</td>

<td> 5</td><td> 8</td><td>Gefltlnlb</td><td> 100</td><td> 10</td><td>PO</td><td>1% Tween80</td><td> 14</td><td>QD</td>

Table 6: HCC827 Model

<td>Group</td><td>n</td><td>Treatment</td><td>Dose (mg / kg)</td><td>Volume of dosage (pl / g)</td><td>Way of dosage</td><td>Solvent</td><td>Days of dosage</td><td>Plan</td>

<td> 1</td><td> 8</td><td>Vehicle</td><td> -</td><td> 10</td><td>PO</td><td> -</td><td> 35</td><td>QD</td>

<td> 2</td><td> 8</td><td>Compound 3</td><td> 50</td><td> 10</td><td>PO</td><td>PEG system</td><td> 35</td><td>QD</td>

<td> 3</td><td> 8</td><td>Compound 3</td><td> 50</td><td> 10</td><td>PO</td><td>MC 0.5%</td><td> 35</td><td>QD</td>

<td> 4</td><td> 8</td><td>Gefltlnlb</td><td> 100</td><td> 10</td><td>PO</td><td>1% Tween80</td><td> 7</td><td>QD</td>

Table 7: A431 model

<td>Group</td><td>n</td><td>Treatment</td><td>Dose (mg / kg)</td><td>Volume of dosage (μΙ / g)</td><td>Way of dosage</td><td>Solvent</td><td>Days of dosage</td><td>Plan</td>

<td> 1</td><td> 8</td><td>Vehicle</td><td> -</td><td> 16,7</td><td>PO</td><td>PEG system</td><td> 14</td><td>QD</td>

<td> 2</td><td> 8</td><td>Compound 3</td><td> 100</td><td> 16,7</td><td>PO</td><td>PEG system</td><td> 14</td><td>QD</td>

<td> 3</td><td> 8</td><td>Gefltlnlb</td><td> 100</td><td> 10</td><td>PO</td><td>1% Tween80</td><td> 14</td><td>QD</td>

Note: n: number of animals; Dosing volume: adjust the dosing volume based on body weight; PEG system: PEG200: alcohol: 5% dextrose = 4: 1: 5; The treatment plan was adjusted if body weight loss&gt; 15%.

Confinement of animals

Animals

Details about the animals are shown below.

Species: Mouse

Breed: atypical Nu / Nu

Age: 7-8 weeks

Gender: Female

Body weight: 20-25 g

Animal supplier: Vital Rlver Laboratories, Beljlng, China

Confinement conditions

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Mice were kept in individual ventilated cages at constant temperature and humidity with 4 animals in each cage at ACEA Bioscience Hangzhou Inc.

- Temperature: approximately 20-26 ° C.

- Humidity approximately 40-70%.

IVC cages: Made of polycarbonate. The size is 300mm x 180mm x 150mm. The bedding material is corn on the cob, which is changed twice a week.

Diet: The animals had free access to dry granulated food sterilized by irradiation throughout the study period.

Water: The animals had free access to sterile drinking water.

Cage identification: The identification labels for each cage contained the following information: number of animals, sex, breed, date of receipt, treatment, study number, group number, and treatment start date.

Identification of the animals: The animals were marked by an ear cut.

Experimental methods and procedures

Cell culture

The tumor cells NCI-H1975, HCC827 and A431 were maintained in vitro as a monolayer culture in medium supplemented with 10% fetal bovine serum, 100 U / ml penicillin and 100 pg / ml streptomycin at 37 ° C in an atmosphere 5% CO2 in air as recommended by ATCC. Tumor cells were normally subcultured twice a week by trypsin-EDTA treatment. Cells growing in an exponential growth phase were collected and counted for tumor inoculation.

Tumor inoculation

Each mouse was inoculated subcutaneously on the right flank with H1975 cells (5 x 10⁶), HCC827 (5 x 10⁶) and A431 (5 x 10⁶), respectively, in 0.2 ml of tumor growth medium. Treatments were started when the tumor size reached approximately 200-250 mm³. The test items were administered to the mice according to the predetermined regimen as shown in the experimental design table.

Observations

All procedures related to the handling, care and treatment of animals in this study were carried out according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC ). At the time of normal verification, the animals were checked for any effects of tumor growth and treatments on normal behavior such as mobility, consumption of feed and water (by observation), increase / loss of body weight (the weights were measured twice a week), eye / capillary matting and any other abnormal effects. Death and observed clinical signs were recorded based on the numbers of animals within each subgroup. Animals that were observed to be in a state of continuous deterioration were euthanized before death or before reaching a comatose state.

Tumor measurement and endpoints

The primary endpoint was to see if tumor growth could be delayed or the mice could be cured. Tumor size was measured twice a week in two dimensions using a caliper and the volume was expressed in mm³ using the formula: V = 0.5 axb² where a and b are the long and short diameters of the tumor, respectively. Tumor size was then used for calculations of both CT and T / C values. CT was calculated with T as the median time (in days) required for the tumors in the treatment group to reach a predetermined size (eg, 1,000 mm³) and C as the median time (in days) for the control group tumors to reach the same size. The T / C value (in percent) was an indication of antitumor efficacy; T and C were the mean volume of the treated and control groups, respectively, on a given day. Tumor weight was measured at the end of the study. The T / C value (in percent) was calculated where T and C were the mean tumor weights of the treated and control groups, respectively.

Statistic analysis

Summary statistics, including the mean and standard error of the mean (SEM), are provided for the volume of tumors in each group at each time point.

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A statistical analysis of the difference in tumor volume and tumor weight between groups was performed on the data obtained at the best therapeutic time after the final dose (day 15 after tumor inoculation).

One-way ANOVA was performed to compare tumor volume and tumor weight between groups, and when a significant F statistic (a ratio of treatment variance to error variance) was obtained, comparisons between groups were performed with LSD. and the Games-Howell test. All data was analyzed using SPSS

16.0. p &lt;0.05 was considered to be statistically significant.

Results

5.1. Body weights

The results of body weight changes in tumor bearing mice for the NCI-H1975, HCC827, and A431 models are shown in Figures 3, Figure 4, and Figure 5, respectively.

The body weights of the mice in different groups of the tumor bearing mice at the end of the treatment on NCI-H1975, HCC827 and A431 are shown in Table 8, Table 9 and Table 10 respectively.

Table 8. The body weights of the mice in the different groups on the NCI-H1975 model

<td>Treatment</td><td>Mice weight (g)^to day 23 (14)</td><td>^p</td>

<td>Vehicle</td><td> 22,99 ± 0,26</td><td> -</td>

<td>Compound 3 25 mg / kg po qd</td><td> 22,28± 0,55</td><td> 0,364</td>

<td>Compound 3 50 mg / kg po qd</td><td> 22,73 ± 0,33</td><td> 0,737</td>

<td>Compound 3 100 mg / kg po qd</td><td> 22,53± 0,66</td><td> 0,555</td>

<td>Gefitinib 100 mg / kg po qd</td><td> 20,43±0,71</td><td> 0,002</td>

Table 9. The body weights of the mice in the different groups on the HCC827 model

<td>Treatment</td><td>Mice weight (g)^to on the 25th (14)</td><td>^p</td>

<td>Vehicle</td><td> 23,83±0,71</td><td> -</td>

<td>Compound 3 (PEG) 50 mg / kg po qd</td><td> 23,26 ± 0,47</td><td> 0,523</td>

<td>Compound 3 (MC) 50 mg / kg po qd</td><td> 23,54± 0,67</td><td> 0,743</td>

<td>Gefitinib 100 mg / kg po qd</td><td> 23,70 ± 0,43</td><td> 0,887</td>

Table 10. The body weights of the mice in the different groups on the A431 model

<td>Treatment</td><td>Mice weight (g)^to on the 25th (14)</td><td>^p</td>

<td>Vehicle</td><td> 25,64 ± 0,53</td><td> -</td>

<td>Compound 3 100 mg / kg po qd</td><td> 25,66± 0,72</td><td> 0,979</td>

<td>Gefitinib 100 mg / kg po qd</td><td> 21,56 ± 0,51</td><td> 0,000</td>

Note: a. Mean ± SEM

Tumor volumes

The tumor sizes of the different groups at different times on NCI-H1975, HCC827, and A431 are shown in Table 11, Table 12, and Table 13, respectively.

Table 11. Tumor volumes in the different treatment groups in the NCI-H1975 model

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<td colspan="6">Tumor volume (mm³)^to</td>

<td>Days</td><td>Vehicle, PO, QD</td><td>Compound 3, PO, 25mpk</td><td>QD Compound 3, PO, QD 50mpk</td><td>Compound 3, PO, QD 100mpk</td><td>Gefitinib, PO, QD 100mpk</td>

<td> 9</td><td> 215,01± 20,88</td><td> 219,91 ± 22,33</td><td> 215,95± 21,58</td><td> 220,64± 22,95</td><td> 215,95 ±22,36</td>

<td> 12</td><td> 387,98 ±45,76</td><td> 284,09± 32,64</td><td> 255,85± 34,44</td><td> 181,40 ±21,15</td><td> 379,15 ±46,00</td>

<td> 16</td><td> 828,95 ± 58,76</td><td> 393,95± 42,09</td><td> 268,23± 47,77</td><td> 180,18 ±26,25</td><td> 737,84± 80,06</td>

<td> 19</td><td> 1425,22± 101,9</td><td> 514,88 ± 55,57</td><td> 346,01 ± 62,50</td><td> 207,28 ±42,54</td><td> 1195,5 ±67,91</td>

<td> 23</td><td> 2169,9 ± 170,8</td><td> 670,36 ±54,19</td><td> 373,01 ±63,35</td><td> 232,25 ± 37,11</td><td> 1702,5± 101,8</td>

Table 12. Tumor volumes in the different treatment groups in the HCC827 model

<td colspan="5">Tumor volume (mm³)^to</td>

<td>Days</td><td>Vehicle, PO, QD Vehicle</td><td>Compound 3, PO, QD 50mpk (PEG)</td><td>Compound 3, PO, QD 50mpk (0.5% MC)</td><td>Gefitinib, PO, QD 100mpk</td>

<td> 14</td><td> 215,94± 25,70</td><td> 211,90 ±23,00</td><td> 211,14 ±25,11</td><td> 212,28 ±26,35</td>

<td> 18</td><td> 291,15 ±24,42</td><td> 188,72 ±28,03</td><td> 216,63 ±27,69</td><td> 59,55 ±25,20</td>

<td> 21</td><td> 353,24 ± 25,64</td><td> 136,96 ±16,40</td><td> 245,14 ±33,44</td><td> 4,61± 3,16</td>

<td> 25</td><td> 453,43± 24,72</td><td> 95,73 ±15,38</td><td> 216,42 ±28,06</td><td> 1,25 ± 1,25</td>

<td> 28</td><td> 519,39 ±22,26</td><td> 111,96 ±22,05</td><td> 231,08 ±30,81</td><td> 1,25 ± 1,25</td>

<td> 32</td><td> 638,78± 32,70</td><td> 82,28 ±24,08</td><td> 277,59 ±42,02</td><td> 1,25 ± 1,25</td>

<td> 35</td><td> 762,43 ±47,22</td><td> 67,63 ±24,22</td><td> 293,64 ±43,98</td><td> 1,88 ±1,32</td>

<td> 39</td><td> 1092,53 ± 99,28</td><td> 69,44 ±30,35</td><td> 328,53 ±43,51</td><td> 1,88 ±1,32</td>

<td> 42</td><td> 1324,76± 141,54</td><td> 79,71 ±28,86</td><td> 302,31 ±35,83</td><td> 10,95 ± 6,13</td>

<td> 46</td><td> 1736,94 ±217,03</td><td> 84,26 ±35,62</td><td> 284,44 ±27,00</td><td> 23,71± 11,84</td>

<td> 49</td><td> 1920,11 ± 256,36</td><td> 77,59 ±42,07</td><td> 299,28 ±31,79</td><td> 41,00± 20,52</td>

Table 13. Tumor volumes in the different treatment groups in the A431 model

<td colspan="4">Tumor volume (mm³)^to</td>

<td>Days</td><td>Vehicle, PO, QD</td><td>Compound 3, PO, QD 100mpk</td><td>Gefitinib, PO, QD 100mpk</td>

<td> 11</td><td> 241,34± 28,69</td><td> 240,95 ±26,46</td><td> 239,83 ±23,30</td>

<td> 14</td><td> 472,09 ±71,50</td><td> 399,68 ± 42,62</td><td> 203,74 ±22,97</td>

<td> 18</td><td> 860,82 ± 120,62</td><td> 867,62 ±70,54</td><td> 139,70± 26,94</td>

<td> 21</td><td> 1211,0± 157,77</td><td> 1166,1 ±94,08</td><td> 139,70 ±22,07</td>

<td> 25</td><td> 1666,6 ± 233,36</td><td> 1627,7 ±146,0</td><td> 154,79± 32,62</td>

Note: a. Mean ± SEM

Inhibition of tumor growth

The inhibition of tumor growth in the NCI-H1975, HCC827, and A431 models is summarized in Table 14, Table 15, and Table 16, respectively.

Table 14. Effect of compounds in the treatment of the H1975 xenograft tumor model Treatment Tumor size (mm³)^to on day 23 (14) T / C (%) TC (days) at 300 mm³ p

<td>Vehicle</td><td> 2170± 171</td><td> -</td><td> -</td><td> -</td>

<td>Compound 3</td><td></td><td></td><td></td><td> 0,000</td>

<td>25 mg / kg po qd</td><td> 670± 54</td><td> 28,5%</td><td> 2,11</td><td></td>

<td>Compound 3</td><td></td><td></td><td></td><td> 0,000</td>

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<td>Treatment</td><td colspan="3">Tumor size (mm³)^to on day 23 (14) T / C (%) TC (days) at 300 mm³ p</td>

<td>50 mg / kg po qd</td><td> 373 ± 63</td><td> 16,0%</td><td> 6,76</td>

<td>Compound 3</td><td></td><td></td><td> 0,000</td>

<td>100 mg / kg po qd</td><td> 232± 37</td><td> 9,9%</td><td> &gt;14</td>

<td>Gefitinib</td><td></td><td></td><td> 0,345</td>

<td>100 mg / kg po qd</td><td> 1702 ±102</td><td> 77,4%</td><td> 0,08</td>

Table 15. Effect of compounds in the treatment of the HCC827 xenograft tumor model

<td>Treatment</td><td colspan="4">Tumor size (mm³)^to on day 49 (35) T / C (%) TRR^b on day 49 (35) TC (days) at 300mm³ P</td>

<td>Vehicle</td><td> 1920± 256</td><td> -</td><td> -</td><td> --</td>

<td>Compound 3 (PEG)</td><td></td><td></td><td></td><td> 0,001</td>

<td>50 mg / kg po qd</td><td> 78 ± 42</td><td> 2,8%</td><td> 71,4%</td><td> &gt;35</td>

<td>Compound 3 (MC)</td><td></td><td></td><td></td><td> 0,002</td>

<td>50 mg / kg po qd</td><td> 299± 32</td><td> 14,3%</td><td> -45,0%</td><td> 17,3</td>

<td>Gefitinib</td><td></td><td></td><td></td><td> 0,001</td>

<td>100 mg / kg po qd</td><td> 41± 21</td><td> 2,4%</td><td> 75,7%</td><td> &gt;35</td>

Table 16. Effect of compounds in the treatment of the A341 xenograft tumor model

<td>Treatment</td><td colspan="3">Tumor size (mm³)^to on day 25 (14) T / C (%) TC (days) at 300mm³ p</td>

<td>Vehicle</td><td> 1667± 233</td><td> -</td><td> --</td>

<td>Compound 3</td><td></td><td></td><td> 0,999</td>

<td>100 mg / kg po qd</td><td> 1628 ± 146</td><td> 98,3%</td><td> 0,35</td>

<td>Gefitinib</td><td></td><td></td><td> 0,001</td>

<td>100 mg / kg po qd</td><td> 154± 33</td><td> 8,7%</td><td> &gt;14</td>

Note: a. Mean ± SEM

b. Grade of tumor regression (%) = (1-tumor volume from post-treatment / tumor volume from 5 pre-treatment) * 100%

Tumor growth curve

The tumor growth curve in different groups of the mice having tumors in the NCI-H1975, HCC827 and A431 models are shown in Figure 6, Figure 7 and Figure 8, respectively.

Tumor weights

The tumor weights of the mice in the different groups in the NCI-H1975, HCC827 and A431 models are shown in Table 17, Table 18 and Table 19, respectively.

Table 17. The antitumor activity of the compounds in the treatment of the model of NCI-H1975

<td>Treatment</td><td>Tumor weight (g) on ​​day 23 (14)</td><td>_Gi^today 23 (14)</td><td>^p</td>

<td>Vehicle</td><td> 1,99 ± 0,16</td><td> -</td><td> -</td>

<td>Compound 3 25 mg / kg po qd</td><td> 0,70± 0,04</td><td> 65,0%</td><td> 0,001</td>

<td>Compound 3 50 mg / kg po qd</td><td> 0,34 ± 0,08</td><td> 82,8%</td><td> 0,000</td>

<td>Gefitinib 100 mg / kg po qd</td><td> 1,69 ± 0,11</td><td> 15,1%</td><td> 0,717</td>

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Table 18. The antitumor activity of the compounds in the treatment of the HCC827 model

<td>Treatment</td><td>Tumor weight (g) on ​​day 49 (35 )</td><td>Gl ^today 49 (35)</td><td>P</td>

<td>Vehicle</td><td> 1,94± 0,32</td><td> -</td><td> -</td>

<td>Compound 3 (PEG) 50 mg / kg po qd</td><td> 0,06 ± 0,02</td><td> 96,7%</td><td> .004</td>

<td>Compound 3 (MC) 50 mg / kg po qd</td><td> 0,26 ± 0,04</td><td> 86,5%</td><td> .008</td>

<td>Gefitinib 100 mg / kg po qd</td><td> 0,03± 0,02</td><td> 98,3%</td><td> .004</td>

Table 19. The antitumor activity of the compounds in the treatment of the A431 model

<td>Treatment</td><td>Tumor weight (g) on ​​day 23 (14)</td><td>Gl ^today 23 (14)</td><td>P</td>

<td>Vehicle</td><td> 1,67± 0,29</td><td> -</td><td> -</td>

<td>Compound 3 100 mg / kg po qd</td><td> 1,60 ± 0,22</td><td> 4,0%</td><td> 0,817</td>

<td>Gefitinib 100 mg / kg po qd</td><td> 0,13± 0,03</td><td> 92,5%</td><td> 0,000</td>

a: Gl (Degree of Inhibition) = (PTcontroi - PT_Trat_totime) / PT_Controi ^x 100%

Biological example D

Synthesis of the maleate salt and the hydrochloride salt of compound 3 and pharmacokinetic study

The synthesis of the maleate and hydrochloride salts from the free base of Compound 3 is shown below:

<img file="ES2618007T3_D0092.tif" />

Ethanol / water (5:95)

HCl or maleic acid

<img file="ES2618007T3_D0093.tif" />

Maleic acid (1.2 eq.) Or HCl (2.2 eq.) Was added dropwise to the free base of Compound 3 (2 g) in ethanol / water (5:95, 22 ml) at 40 ° C . After the solid dissolved, the solution was cooled to room temperature and left overnight. The resulting crystals (light yellow or off-white) were collected, washed with cold water, and dried overnight (more than 85% yield).

Pharmacokinetic studies in rats with Compound 3 in free base, maleate salt and HCl salt forms:

A comparative pharmacokinetic study was performed on female rats (Zhe Jiang AMS) using Compound 3 in free base, maleate salt and HCl salt forms. Detailed study conditions along with experimental results are shown in Table 20 below:

Table 20

<td>Compound</td><td>Formulation</td><td>Dose (mg / kg)</td><td>Via</td><td>F%</td><td>AUCfinal (ng / ml * h)</td><td> 1¹/₂ (h)</td><td>Cmax (po) or Co (¡v) (ng / ml)</td>

<td>Compound 3 free base</td><td>PEG200O5W (50:50, v / v)</td><td> 4,7</td><td>¡.V.</td><td>n / a</td><td> 2195+ 140,2</td><td> 1,6±0,04</td><td> 4102,1± 675,8</td>

<td>Compound 3 free base</td><td>0.5% MC</td><td> 30,56</td><td>po</td><td> 10,7± 2,6</td><td> 1526,8± 364,6</td><td> 2,4±0,6</td><td> 242,0± 37,6</td>

<td>HCl salt</td><td>0.5% MC</td><td> 40,88</td><td>po</td><td> 30,6±</td><td> 5853,8±</td><td> 2,4±1,0</td><td> 1259,3± 359,0</td>

ES 2 618 007 T3

<td>Compound</td><td>Formulation</td><td>Dose (mg / kg)</td><td>Via</td><td>F%</td><td>AUCflnal (ng / ml * h)</td><td> 1¹/₂ (h)</td><td>Cmax (po) 0 Co (¡v) (ng / ml)</td>

<td>Compound 3</td><td></td><td></td><td></td><td> 8,2</td><td> 1565,4</td><td></td><td></td>

<td>Compound 3 maleate salt</td><td>0.5% MC</td><td> 42,00</td><td>po</td><td> 32,7± 9,8</td><td> 6412,2± 1917,8</td><td> 2,3±0,4</td><td> 1540,0± 528,5</td>

<td>Compound 3 free base</td><td>PEG200O5W (50:50, v / v)</td><td> 37,6</td><td>po</td><td> 23,0± 10,8</td><td> 4041,0± 1892,3</td><td> 2,8±0,3</td><td> 1263,3± 270,2</td>

The results show that in the same 0.5% methyl cellulose (MC) formulation, the salt forms (both the maleate and HCl salt) had about 3-fold better blodlsponsibility compared to the free base form.

Biological Example E

Compound 3 was tested against a range of other protein kinases.

Description of the trial. In vitro profiling of protein kinases was performed using the HotSpot assay platform. Briefly, specific kNase / substrate pairs along with required cofactors were prepared in reaction buffer. Compounds were contributed to the reaction, followed 15-20 minutes later by the addition of a mixture of ATP (Slgma, St. Louls MO) and³³P ATP (Perkln Elmer, Waltham MA) to a final concentration of 10 µΜ. Reactions were carried out at room temperature for 120 min, followed by dropping the reaction mixtures onto P81 Ion Exchange filter paper (Whatman Inc., Plscataway, NJ). Unbound phosphate was removed by intensive washing of the filters in 0.75% phosphoric acid. After subtraction of the background derived from control reactions containing Inactive enzyme, the kinase activity data was expressed as the percentage of kinase activity remaining in test samples compared to reactions in vehicle (dlmethyl sulfoxide). IC50 values ​​and curve fits were obtained using Prlsm (GraphPad Software).

Reaction conditions: Buffer conditions: 20 mM Hepes (pH 7.5), MgCl₂10 mM, 1 mM EGTA, 0.02% Brlj35, 0.02 mg / ml BSA, 0.1 mM Na3VO4, 2 mM DTT, and 1% DMSO. ATP concentration: 10 μΜ. Reaction time: 2 hours. Compound 3 was tested at 10 concentrations as follows with a comparison to the common kinase inhibitor staurosporine reference compound. Compounds were tested in a 10 point IC50 mode with a threefold dilution starting from 10 uM. The Control Compound was tested at a 10 point IC50 with a threefold dilution starting from 20 uM. All kinase reactions were performed at 10 uM ATP. Test concentrations are given below in molar units (M).

<td>Compound 3</td><td>Staurosporlna control</td>

<td>1.00E-05</td><td>2.00E-05</td>

<td>3.33E-06</td><td>6.67E-06</td>

<td>1.11E-06</td><td>2.22E-06</td>

<td>3.70E-07</td><td>7.41 E-07</td>

<td>1.23E-07</td><td>2.47E-07</td>

<td>4.12E-08</td><td>8.23E-08</td>

<td>1.37E-08</td><td>2.74E-08</td>

<td>4.57E-09</td><td>9.14E-09</td>

<td>1.52E-09</td><td>3.05E-09</td>

<td>5.08E-10</td><td>1.02E-09</td>

The following kinase enzymes were tested:

<td>Name</td><td>Symbol HUGO</td><td>Substratum general</td><td>Registration number Genbank</td><td>Protein registration number</td><td>Clone</td><td>Mutation</td><td>Expression</td><td>Hashtag</td>

<td>BLK</td><td>BLK</td><td>pEY</td><td>NP_001706</td><td>P51451</td><td>length complete</td><td> -</td><td>Insect</td><td>His N- termlnal</td>

<td>BMX / ETK</td><td>BMX</td><td>pEY</td><td>NP 001712</td><td>P51813</td><td>length complete</td><td> -</td><td>baculovlrus in cells of Sf21 insect</td><td>His C- termlnal</td>

ES 2 618 007 T3

<td>Name</td><td>Symbol HUGO</td><td>Substratum general</td><td>Registration number Genbank</td><td>Protein registration number</td><td>Clone</td><td>Mutation</td><td>Expression</td><td>Hashtag</td>

<td>BTK</td><td>BTK</td><td>pEY</td><td>NP_000052</td><td>Q06187</td><td>length complete</td><td> -</td><td>Insect</td><td>with His6 N- tag terminal</td>

<td>ITK</td><td>ITK</td><td>MBP</td><td>NP_005537</td><td>Q08881</td><td>length complete</td><td> -</td><td>Insect</td><td>GST N- terminal</td>

<td>JAK2</td><td>JAK2</td><td>pEY</td><td>NP_004963</td><td> 060674</td><td>aa 8091132 + g</td><td> -</td><td>Insect</td><td>GST N- terminal</td>

<td>JAK3</td><td>JAK3</td><td>JAK3tide</td><td>NP_000206</td><td>P52333</td><td>aa 7811124</td><td> -</td><td>Insect</td><td>GST N- terminal</td>

<td>TEC</td><td>TEC</td><td>pEY + Mn</td><td>NP_003206</td><td>P42680</td><td>length complete</td><td> -</td><td>baculoviral insect cell</td><td>His C-terminal tag</td>

<td>TXK</td><td>TXK</td><td>ABLtide</td><td>NP_003319,2</td><td>P42681</td><td>length complete</td><td> -</td><td>Insect</td><td>hashtag GST N- terminal</td>

<td>FLT3 (D835Y)</td><td>FLT3</td><td>Abltide</td><td>NP_004110</td><td>P36888</td><td>aa 564958</td><td>D835Y</td><td>baculoviral insect cell</td><td>hashtag His6 C- terminal</td>

Results: IC50 values ​​for each of the kinase targets are summarized as follows:

<td></td><td colspan="2">Compound IC50 (M)</td>

<td>Kinases</td><td>Compound 3</td><td>Staurosporine</td>

<td>BLK</td><td>2.34E-10</td><td>1.22E-09</td>

<td>BMX / ETK</td><td>3.45E-10</td><td>2.19E-09</td>

<td>BTK</td><td>3.99E-10</td><td>5.78E-09</td>

<td>FLT3 (D835Y)</td><td>1.09E-08</td><td>1.63E-11</td>

<td>ITK</td><td>5.17E-10</td><td>8.96E-09</td>

<td>JAK2</td><td>5.01 E-07</td><td>&lt;1.00E-9</td>

<td>JAK3</td><td>9.11E-11</td><td>2.99E-11</td>

<td>TEC</td><td>6.69E-10</td><td>2.68E-08</td>

<td>TXK</td><td>6.98E-10</td><td>1.28E-08</td>

Lists of IC50 value and raw data for each individual targeted enzyme are provided below.

The percentage of activity is a relative value compared to the buffer solution only. The DMSO is listed as a reference. The IC50 value for Compound 3 for BLK was 0.23 nM. The corresponding curves for Compound 3 are shown in Figures 15A and 15B, respectively.

% of activity

<td>Conc. (M)</td><td>Compound 3</td><td>Staurosporine</td><td>Conc. Estaurosp. (M)</td>

<td>1.00E-05</td><td> -0,41</td><td> 0,57</td><td>2.00E-05</td>

<td>3.33E-06</td><td> 2,22</td><td> 2,47</td><td>6.67E-06</td>

<td>1.11E-06</td><td> 4,39</td><td> 1,46</td><td>2.22E-06</td>

<td>3.70E-07</td><td> 1,49</td><td> -1,03</td><td>7.41 E-07</td>

<td>1.23E-07</td><td> -0,50</td><td> -2,23</td><td>2.47E-07</td>

<td>4.12E-08</td><td> -3,83</td><td> -1,99</td><td>8.23E-08</td>

<td>1.37E-08</td><td> -0,37</td><td> 1,34</td><td>2.74E-08</td>

ES 2 618 007 T3

<td>Conc. (M)</td><td>Compound 3</td><td>Staurosporlna</td><td>Cone, estaurosp. (M)</td>

<td>4.57E-09</td><td> 1,07</td><td> 10,32</td><td>9.14E-09</td>

<td>1.52E-09</td><td> 14,53</td><td> 27,25</td><td>3.05E-09</td>

<td>5.08E-10</td><td> 29,73</td><td> 53,08</td><td>1.02E-09</td>

<td>DMSO</td><td> 102,02</td><td> 97,98</td><td>DMSO</td>

The IC50 for Compound 3 for BMX / ETK was 0.35 nM. The curve for Compound 3 is shown in Figure 16.

% of activity

<td>Conc. (M)</td><td>Compound 3</td><td>Staurosporlna</td><td>Cone, estaurosp. (M)</td>

<td>1.00E-05</td><td> 1,44</td><td> 9,22</td><td>2.00E-05</td>

<td>3.33E-06</td><td> -4,22</td><td> -4,43</td><td>6.67E-06</td>

<td>1.11E-06</td><td> -0,82</td><td> -1,61</td><td>2.22E-06</td>

<td>3.70E-07</td><td> -3,51</td><td> -0,71</td><td>7.41 E-07</td>

<td>1.23E-07</td><td> -0,30</td><td> 1,44</td><td>2.47E-07</td>

<td>4.12E-08</td><td> 1,70</td><td> 8,90</td><td>8.23E-08</td>

<td>1.37E-08</td><td> 1,03</td><td> 18,68</td><td>2.74E-08</td>

<td>4.57E-09</td><td> 7,02</td><td> 24,52</td><td>9.14E-09</td>

<td>1.52E-09</td><td> 9,87</td><td> 39,02</td><td>3.05E-09</td>

<td>5.08E-10</td><td> 37,60</td><td> 64,45</td><td>1.02E-09</td>

<td>DMSO</td><td> 98,60</td><td> 96,41</td><td>DMSO</td>

The IC50 for Compound 3 for BTK was 0.40 nM. The curve for Compound 3 is shown in Figure 17.

% of activity

<td>Conc. (M)</td><td>Compound 3</td><td>Staurosporlna</td><td>Cone, estaurosp. (M)</td>

<td>1.00E-05</td><td> 3,65</td><td> 1,93</td><td>2.00E-05</td>

<td>3.33E-06</td><td> 0,69</td><td> -0,14</td><td>6.67E-06</td>

<td>1.11 E-06</td><td> -0,26</td><td> 0,21</td><td>2.22E-06</td>

<td>3.70E-07</td><td> 3,46</td><td> 3,51</td><td>7.41 E-07</td>

<td>1.23E-07</td><td> 1,71</td><td> 6,52</td><td>2.47E-07</td>

<td>4.12E-08</td><td> 1,89</td><td> 10,70</td><td>8.23E-08</td>

<td>1.37E-08</td><td> 4,18</td><td> 20,50</td><td>2.74E-08</td>

<td>4.57E-09</td><td> 3,32</td><td> 41,55</td><td>9.14E-09</td>

<td>1.52E-09</td><td> 11,43</td><td> 60,75</td><td>3.05E-09</td>

<td>5.08E-10</td><td> 40,24</td><td> 81,36</td><td>1.02E-09</td>

<td>DMSO</td><td> 96,43</td><td> 98,79</td><td>DMSO</td>

The IC50 for Compound 3 for FLT3 (D835Y) was 10.90 nM. The curve for Compound 3 is shown in Figure 18.

% of activity

<td>Conc. (M)</td><td>Compound 3</td><td>Staurosporlna</td><td>Cone, estaurosp. (M)</td>

<td>1.00E-07</td><td> 11,32</td><td> -1,65</td><td>1.00E-07</td>

<td>3.33E-08</td><td> 27,78</td><td> 0,98</td><td>3.33E-08</td>

ES 2 618 007 T3

<td>Conc. (M)</td><td>Compound 3</td><td>Staurosporine</td><td>Conc. Estaurosp. (M)</td>

<td>1.11E-08</td><td> 49,45</td><td> 0,31</td><td>1.11 E-08</td>

<td>3.70E-09</td><td> 70,12</td><td> 2,34</td><td>3.70E-09</td>

<td>1.23E-09</td><td> 92,41</td><td> -0,82</td><td>1.23E-09</td>

<td>4.12E-10</td><td> 99,20</td><td> 3,17</td><td>4.12E-10</td>

<td>1.37E-10</td><td> 104,37</td><td> 2,10</td><td>1.37E-10</td>

<td>4.57E-11</td><td> 94,42</td><td> 20,65</td><td>4.57E-11</td>

<td>1.52E-11</td><td> 97,21</td><td> 53,79</td><td>1.52E-11</td>

<td>5.08E-12</td><td> 102,50</td><td> 85,41</td><td>5.08E-12</td>

<td>DMSO</td><td> 99,21</td><td> 100,79</td><td>DMSO</td>

The IC50 for Compound 3 for ITK was 0.52 nM. The curve for Compound 3 is shown in Figure 19.

% of activity

<td>Conc. (M)</td><td>Compound 3</td><td>Staurosporine</td><td>Conc. Estaurosp. (M)</td>

<td>1.00E-05</td><td> 0,79</td><td> 0,35</td><td>2.00E-05</td>

<td>3.33E-06</td><td> 0,01</td><td> 0,60</td><td>6.67E-06</td>

<td>1.11E-06</td><td> 1,27</td><td> -0,12</td><td>2.22E-06</td>

<td>3.70E-07</td><td> 1,16</td><td> 1,90</td><td>7.41 E-07</td>

<td>1.23E-07</td><td> 0,90</td><td> 4,54</td><td>2.47E-07</td>

<td>4.12E-08</td><td> 1,70</td><td> 9,04</td><td>8.23E-08</td>

<td>1.37E-08</td><td> 1,34</td><td> 19,24</td><td>2.74E-08</td>

<td>4.57E-09</td><td> 2,39</td><td> 49,31</td><td>9.14E-09</td>

<td>1.52E-09</td><td> 4,41</td><td> 76,22</td><td>3.05E-09</td>

<td>5.08E-10</td><td> 52,22</td><td> 95,37</td><td>1.02E-09</td>

<td>DMSO</td><td> 101,99</td><td> 97,19</td><td>DMSO</td>

The IC50 for Compound 3 for JAK2 was 501 nM. The curve for Compound 3 is shown in Figure 20.

% of activity

<td>Conc. (M)</td><td>Compound 3</td><td>Staurosporine</td><td>Conc. Estaurosp. (M)</td>

<td>1.00E-05</td><td> -2,83</td><td> -5,43</td><td>2.00E-05</td>

<td>3.33E-06</td><td> 9,17</td><td> -8,65</td><td>6.67E-06</td>

<td>1.11 E-06</td><td> 23,15</td><td> -6,44</td><td>2.22E-06</td>

<td>3.70E-07</td><td> 60,18</td><td> -6,42</td><td>7.41 E-07</td>

<td>1.23E-07</td><td> 94,11</td><td> -6,76</td><td>2.47E-07</td>

<td>4.12E-08</td><td> 100,48</td><td> -7,25</td><td>8.23E-08</td>

<td>1.37E-08</td><td> 99,88</td><td> -3,41</td><td>2.74E-08</td>

<td>4.57E-09</td><td> 98,95</td><td> -0,60</td><td>9.14E-09</td>

<td>1.52E-09</td><td> 103,16</td><td> 5,51</td><td>3.05E-09</td>

<td>5.08E-10</td><td> 98,40</td><td> 18,77</td><td>1.02E-09</td>

<td>DMSO</td><td> 103,16</td><td> 100,65</td><td>DMSO</td>

To examine the inhibition of JAK3, Compound 3 was evaluated as in the other tests, but starting from a concentration of 100 nM. The dilution factor was triple as in the other tests, resulting in an interval

ES 2 618 007 T3 test concentration from 100 nM to 5.08 pM. The IC50 for Compound 3 for JAK3 was 91.1 pM. The curve for Compound 3 is shown in Figure 21.

% of activity

<td>Conc. (M)</td><td>Compound 3</td><td>Staurosporlna</td><td>Conc. Estaurosp. (M)</td>

<td>1.00E-07</td><td> -0,21</td><td> -0,79</td><td>1.00E-07</td>

<td>3.33E-08</td><td> 0,22</td><td> -0,88</td><td>3.33E-08</td>

<td>1.11E-08</td><td> -1,35</td><td> -0,51</td><td>1.11 E-08</td>

<td>3.70E-09</td><td> -0,10</td><td> 0,09</td><td>3.70E-09</td>

<td>1.23E-09</td><td> 1,25</td><td> -1,86</td><td>1.23E-09</td>

<td>4.12E-10</td><td> 9,43</td><td> 0,36</td><td>4.12E-10</td>

<td>1.37E-10</td><td> 35,13</td><td> 4,48</td><td>1.37E-10</td>

<td>4.57E-11</td><td> 72,27</td><td> 41,45</td><td>4.57E-11</td>

<td>1.52E-11</td><td> 89,22</td><td> 70,04</td><td>1.52E-11</td>

<td>5.08E-12</td><td> 92,29</td><td> 88,93</td><td>5.08E-12</td>

<td>DMSO</td><td> 102,06</td><td> 102,66</td><td>DMSO</td>

The IC50 for Compound 3 for TEC was 0.67 nM. The curve for Compound 3 is shown in Figure 22.

% of activity

<td>Conc. (M)</td><td>Compound 3</td><td>Staurosporlna</td><td>Conc. Estaurosp. (M)</td>

<td>1.00E-05</td><td> 2,69</td><td> 1,43</td><td>2.00E-05</td>

<td>3.33E-06</td><td> 7,10</td><td> 3,40</td><td>6.67E-06</td>

<td>1.11E-06</td><td> 5,72</td><td> 3,35</td><td>2.22E-06</td>

<td>3.70E-07</td><td> 11,05</td><td> 8,71</td><td>7.41 E-07</td>

<td>1.23E-07</td><td> 14,88</td><td> 18,31</td><td>2.47E-07</td>

<td>4.12E-08</td><td> 19,39</td><td> 31,27</td><td>8.23E-08</td>

<td>1.37E-08</td><td> 20,14</td><td> 47,73</td><td>2.74E-08</td>

<td>4.57E-09</td><td> 22,53</td><td> 70,74</td><td>9.14E-09</td>

<td>1.52E-09</td><td> 32,07</td><td> 79,41</td><td>3.05E-09</td>

<td>5.08E-10</td><td> 72,24</td><td> 93,91</td><td>1.02E-09</td>

<td>DMSO</td><td> 99,73</td><td> 99,19</td><td>DMSO</td>

The IC50 for Compound 3 for TXK was 0.70 nM. The curve for Compound 3 is shown in Figure 23.

% of activity

<td>Conc. (M)</td><td>Compound 3</td><td>Staurosporlna</td><td>Conc. Estaurosp. (M)</td>

<td>1.00E-05</td><td> -0,05</td><td> -0,60</td><td>2.00E-05</td>

<td>3.33E-06</td><td> -1,39</td><td> -0,74</td><td>6.67E-06</td>

<td>1.11 E-06</td><td> 4,24</td><td> 2,69</td><td>2.22E-06</td>

<td>3.70E-07</td><td> -2,01</td><td> 1,66</td><td>7.41 E-07</td>

<td>1.23E-07</td><td> 3,26</td><td> 9,25</td><td>2.47E-07</td>

<td>4.12E-08</td><td> 2,76</td><td> 17,37</td><td>8.23E-08</td>

<td>1.37E-08</td><td> 0,31</td><td> 32,52</td><td>2.74E-08</td>

<td>4.57E-09</td><td> 4,11</td><td> 58,10</td><td>9.14E-09</td>

ES 2 618 007 T3

<td>Conc. (M)</td><td>Compound 3</td><td>Staurosporine</td><td>Conc. Estaurosp. (M)</td>

<td>1.52E-09</td><td> 23,68</td><td> 79,12</td><td>3.05E-09</td>

<td>5.08E-10</td><td> 58,85</td><td> 95,83</td><td>1.02E-09</td>

<td>DMSO</td><td> 96,09</td><td> 100,00</td><td>DMSO</td>

Data for other compounds

1. ELISA assay (EGFR):

Using the protocol described in Biological Example B-2, the following compounds were also tested in the ELISA (EGFR) assay. The results are shown in Table 21 below.

Table 21

ELISA assay (EGFR)

<td>Comp./EGFR</td><td>IC50 (uM) H1975 T790M / L858R (without EGF stimulation)</td><td>IC50 (uM) A431 WT (EGF stimulation)</td><td>Selectivity A431 / H1975</td>

<td>Comp. 1</td><td> 0,0063</td><td> 0,6470</td><td> 102,70</td>

<td>Comp. two</td><td> 0,0010</td><td> 0,3000</td><td> 300,00</td>

<td>Comp. 4</td><td> 0,0670</td><td> 10,9000</td><td> 162,69</td>

<td>Comp. 5</td><td> 0,2430</td><td> 27,5000</td><td> 113,17</td>

<td>Comp. 6</td><td> 0,0398</td><td> 9,2700</td><td> 232,91</td>

<td>Comp. 7</td><td> 12,4100</td><td> &gt;20</td><td>N / A</td>

<td>Comp. 3. 4</td><td> 0,0217</td><td> 2,0170</td><td> 92,95</td>

two. Other Kinases - Enzyme Assays

Using the protocol described in Biological Example E, the following compounds were also tested against other kinases (BTK, Jak1, Jak2, and Jak3) (Table 22 below).

Table 22

<td rowspan="2">Comp. \ Kinases</td><td colspan="4">IC50 Enzyme Assays</td>

<td>BTK</td><td>JAK3</td><td>JAK2</td><td>JAK1</td>

<td>Comp. 1</td><td>5.79E-10</td><td>3.66E-11</td><td>5.70E-07</td><td>3.22E-06</td>

<td>Comp. two</td><td>3.62E-10</td><td>3.65E-11</td><td>2.06E-06</td><td>1.40E-05</td>

<td>Comp. 3</td><td>3.99E-10</td><td>9.11E-11</td><td>5.01 E-07</td><td>3.27E-06</td>

<td>Control staurosporine</td><td>4.97E-09</td><td>1.92E-11</td><td>1.96E-10</td><td>3.65E-10</td>

Other kinases (BTK and JAK3) - Cell-based assays

The protocol for the ClariCELL ™ JAK3 and BTK assays is described below:

• HEK293 human embryonic kidney cells were transiently transfected with either human wt BTK, human wt JAK3, or human JAK3 without kinase activity (for JAK3 negative controls). For BTK negative controls, wt BTK transfection plus 1 uM ibrutinib treatment was used.

· Cells were distributed in 96-well plates at approximately 8,000 cells / well for BTK or 20,000 cells per well for JAK3.

• Eight triple serial dilutions (or double for tofacitinib) were prepared for each 100% DMSO compound.

• The compounds were then diluted in water to 10x the final test concentration and 6% DMSO.

• Compounds were added to cells in 96-well plates (10-fold dilution in tissue culture medium) for a final concentration of 1x compound and 0.6% DMSO.

• Cells were incubated with compound at 37 ° C for 2 hours.

ES 2 618 007 T3 • Cells were subjected to ilsls and the Used was transferred to an ELISA plate that had previously been coated with antibody to capture the substrate (either human BTK or human JAK3).

• Plates were washed and then incubated with antibody to detect total tyrosine phosphorylation *.

• Plates were washed and then Incubated with HRP-labeled secondary antibody.

'HRP substrate was added and absorbance was read at 450nm.

• * Note that because a pananthphosphotrosine antibody was used, there is a possibility that tyrosine kinase activities could be measured in addition to BTK or JAK3.

The test results for BTK cell-based assays are shown in Table 23 below.

Table 23

<td rowspan="2">Comp. \ Clnasas</td><td>Cell-Based Assays (CAI) ICso (pM)</td>

<td rowspan="2">BTK 0.012 0.059</td>

<td>Comp. two</td>

<td></td><td> 0,026</td>

<td>Comp. 3</td><td> 0,036</td>

<td></td><td> 0,059</td>

<td></td><td> 0,013</td>

<td>Control (Ibrutinlb)</td><td> 0,013 0,067 0,012</td>

Test results for JAK3 cell-based assays are shown in Table 24 below.

Table 24

<td rowspan="2">Comp. \ Clnasas</td><td>Cell-Based Assays (CAI) IC50 (pM)</td>

<td rowspan="2">JAK3 0.20 0.36</td>

<td>Comp. two</td>

<td>Comp. 3</td><td> 0,23 0,28 0,25 0,36</td>

<td>Control (Tofacitlnib)</td><td> 2,3 2,3 1,2 3,7</td>