WO2022241975A1 - Methods for treating cancers associated with egfr mutation - Google Patents

Methods for treating cancers associated with egfr mutation Download PDF

Info

Publication number
WO2022241975A1
WO2022241975A1 PCT/CN2021/117497 CN2021117497W WO2022241975A1 WO 2022241975 A1 WO2022241975 A1 WO 2022241975A1 CN 2021117497 W CN2021117497 W CN 2021117497W WO 2022241975 A1 WO2022241975 A1 WO 2022241975A1
Authority
WO
WIPO (PCT)
Prior art keywords
unsubstituted
halogenated
alkyl
substituted
cancer
Prior art date
Application number
PCT/CN2021/117497
Other languages
French (fr)
Inventor
Guo Li
Xi Xia
Qiangang ZHENG
Jidong ZHU
Lin Du
Hao ZHUGE
Wenqi CUI
Original Assignee
Etern Biopharma (Shanghai) Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Etern Biopharma (Shanghai) Co., Ltd. filed Critical Etern Biopharma (Shanghai) Co., Ltd.
Publication of WO2022241975A1 publication Critical patent/WO2022241975A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the present invention generally relates to the diagnosis and treatment of EGFR-related disease.
  • the present invention relates to the methods for treating cancer patients with an insertion mutation at exon 20 of the EGFR gene using SHP2 inhibitors.
  • the epidermal growth factor receptor also named ErbB-1 and Her1, is a receptor tyrosine kinase belonging to the ErbB receptor family.
  • EGFR is activated by binding to its ligands in the epidermal growth factor family, including epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) .
  • EGF epidermal growth factor
  • TGF alpha transforming growth factor alpha
  • EGFR Upon activated by its growth factor ligands, EGFR undergoes a transition from an inactive monomeric form to an active homodimer form or to an active heterodimer form in which EGFR pairs with another member of the ErbB receptor family, such as ErbB-2/Her2/neu.
  • EGFR dimerization stimulates EGFR’s intrinsic intracellular protein-tyrosine kinase activity, resulting in the autophosphorylation of several tyrosine (Y) residues in the C-terminal domain of EGFR, including Y992, Y1045, Y1068 and Y1173.
  • This autophosphorylation elicits downstream activation of several proteins with phosphotyrosine-binding SH2 domain.
  • These downstream signaling proteins initiate several signal transduction cascades, principally the MAPK, Akt and JNK pathways, leading to DNA synthesis and the modulation of cell phenotypes such as cell migration, adhesion and proliferation.
  • Mutations of EGFR gene have been associated with a number of cancers, including lung adenocarcinoma, anal cancers, glioblastoma, and epithelial tumors of the head and neck. These mutations typically lead to the overexpression or constant activation of EGFR, which results in uncontrolled cell proliferation.
  • EGFRi tyrosine kinase inhibitors
  • monoclonal antibodies such as gefitinib, erlotinib, afatinib, osimertinib, and icotinib for lung cancer, and cetuximab for colon cancer.
  • EGFR T790M mutation accounts for over 50%of acquired resistance to 1 st and 2 nd generation EGFR-TKIs.
  • frame insertion mutations in EGFR exon 20 demonstrate intrinsic resistance to clinically achievable doses of current EGFR TKIs, including third-generation inhibitors. Therefore, there is an urgent need to develop new methods that can be used to treat EGFR-driven drug-resistant cancers.
  • the present disclosure provides a method for treating a subject having an EGFR-related disease or condition, wherein an insertion mutation with exon 20 of the EGFR gene is detected in a biological sample of the subject.
  • the method comprises administering to the subject a therapeutic effective amount of an SHP2 inhibitor.
  • the present disclosure provides a method for identifying a subject with an EGFR-related disease or condition who is likely to be responsive to treatment with an SHP2 inhibitor, said method comprising (a) providing a biological sample of the subject; (b) detecting presence or absence of an insertion mutation at exon 20 of the EGFR gene in the biological sample; and (c) identifying the subject as being likely to be responsive to treatment with the SHP2 inhibitor if the insertion mutation at exon 20 of the EGFR gene is detected in the biological sample.
  • the insertion mutation of the EGFR gene is an in-frame insertion mutation.
  • the insertion mutation of the EGFR gene is selected from the group consisting of A763_Y764insFQEA, A763_Y764insFQQA, A767_V769dupASV, V769_D770insASV, D770_N771insGL, D770_N771insGT, D770_N771insNPG, D770_N771insSVD, E762Q_insFQEA, H773_V774insH, H773_V774insH, H773_V774insNPH, M766_A767insAI, M766_A767insASV, N771_H773dupNPH, P772_H773insYNP, P772_V774insPHV, S768_770dupSVD, V
  • the insertion mutation is detected using a sequencing assay, an amplification assay, or a hybridization assay. In certain embodiments, the insertion mutation is detected using high-throughput sequencing (or next generation sequencing, NGS) method.
  • the EGFR-related disease or condition is cancer.
  • the cancer is selected from the groups consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC) ) , glioblastoma, conventional glioblastoma multiforme, uterus cancer (e.g., infiltrating renal pelvis and ureter urothelial carcinoma, endometrial endometrioid adenocarcinoma) , bladder cancer (e.g., bladder urothelial carcinoma) , melanoma, renal cancer, liver cancer, myeloma, prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, thyroid cancer, hematological cancer, leukemia and non-Hodgkin’s lymphoma.
  • the cancer is resistant to an EGFR tyrosine kinase inhibitor.
  • the cancer is metastatic.
  • the biological sample is tissue or blood. In certain embodiments, the biological sample comprises a cancer cell or DNA from a cancer cell.
  • a second mutation in a gene in the RTK/RAS/MAPK signaling pathway is further detected in the biological sample of the subject.
  • the gene is EGFR, KRAS or BRAF.
  • the method further comprises administering to the subject a second therapeutic agent.
  • the second therapeutic agent is an EGFR inhibitor, a KRAS inhibitor, a BRAF inhibitor, a MEK inhibitor or a CDK inhibitor.
  • the EGFR inhibitor is Gefitinib, Erlotinib, Afatinib, Dacomitinib, Osimertinib, TAK-788, Poziotinib, JNJ-61186372, Almonertinib (HS-10296) , Amivantamab (JNJ-372) , furmonertinib (AST2818) , or DZD9008.
  • the KRAS inhibitor is AMG510, or MRTX849.
  • the BRAF inhibitor is Vemurafenib, Dabrafenib, Encorafenib or Sorafenib.
  • the MEK inhibitor is Trametinib, Cobimetinib, Selumetinib, Pimasertib or Binimetinib.
  • the CDK inhibitor is Palbociclib, Ribociclib or Abemaciclib.
  • the second therapeutic agent is an immune checkpoint inhibitor, such as PD-1/PD-L1 antagonist.
  • FIG. 1 shows the effect of test compounds on proliferation of ex vivo cultured LU0387 cells.
  • FIG. 2 shows the combinational effect of Compound A and TAK-788 on proliferation of ex vivo cultured LU0387 cells.
  • FIG. 3 shows the antitumor activity of test compounds in LU0387 PDX model.
  • administering means taking, providing or delivering a compound or composition to a desired site for biological action.
  • methods for administering include but are not limited to oral route, transduodenal route, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion) , local administration, and transrectal administration.
  • parenteral injection including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion
  • local administration and transrectal administration.
  • administration techniques that can be used for the compounds and methods as described herein, such as those discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington’s, Pharmaceutical Sciences (current edition) , Mack Publishing Co., Easton, Pa.
  • the compounds and compositions discussed herein are administered orally.
  • biological sample means a sample obtained from a biological source.
  • biological sample include, without limitation, bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal flushing fluids, plural fluid, ascitic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchioalveolar lavage fluid, etc., and tissues, such as biopsy tissue (e.g. biopsied bone tissue, bone marrow, breast tissue, gastrointestinal tract tissue, lung tissue, liver tissue, prostate tissue, brain tissue, nerve tissue, meningeal tissue, renal tissue, endometrial tissue, cervical dittuse, lymph node tissue, muscle tissue, or skin tissue) , a paraffin embedded tissue.
  • biopsy tissue e.g. biopsied bone tissue, bone marrow, breast tissue, gastrointestinal tract tissue, lung tissue, liver tissue, prostate tissue, brain tissue, nerve tissue, meningeal tissue, renal tissue, endometrial tissue, cervical dittuse, lymph node tissue, muscle
  • the biological sample can comprise cancer cells.
  • the biological sample is a fresh or archived sample obtained from a tumor, e.g., by a tumor biopsy or fine needle aspirate.
  • the biological sample also can be any biological fluid containing cancer cells. The collection of a biological sample from a subject is performed in accordance with the standard protocol generally followed by hospital or clinics, such as during a biopsy.
  • cancer refers to any diseases involving an abnormal cell growth and includes all stages and all forms of the disease that affects any tissue, organ or cell in the body.
  • the term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and cancers of all stages and grades including pre-and post-metastatic cancers.
  • cancers can be categorized according to the tissue or organ from which the cancer is located or originated and morphology of cancerous tissues and cells.
  • cancer types include, acute lymphoblastic leukemia (ALL) , acute myeloid leukemia, adrenocortical carcinoma, anal cancer, astrocytoma, childhood cerebellar or cerebral, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumor, brain cancer, breast cancer, Burkitt's lymphoma, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, emphysema, endometrial cancer, ependymoma, esophageal cancer, Ewing family of tumors, Ewing's sarcoma, gastric (stomach) cancer, glioma, head and neck cancer, heart cancer, Hodgkin lymphoma, islet cell carcinoma (endocrine pancreas) , Kaposi sarcoma, kidney cancer (renal lymphoma
  • complementarity refers to the ability of a nucleic acid to form hydrogen bond (s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary) .
  • detecting, ” “determining, ” “assessing, ” and “measuring” can be used interchangeably and refer to both quantitative and semi-quantitative determinations. Where either a quantitative and semi-quantitative determination is intended, the phrase “determining a level” of a polynucleotide or polypeptide of interest or “detecting” a polynucleotide or polypeptide of interest can be used.
  • hybridizing refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions.
  • stringent conditions refers to hybridization and wash conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences in a mixed population (e.g., a cell lysate or DNA preparation from a tissue biopsy) .
  • a stringent condition in the context of nucleic acid hybridization are sequence dependent, and are different under different environmental parameters.
  • An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on an array or on a filter in a Southern or Northern blot is 42°C. using standard hybridization solutions (see, e.g., Sambrook and Russell Molecular Cloning: A Laboratory Manual (3rd ed. ) Vol. 1-3 (2001) Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY) .
  • An example of highly stringent wash conditions is 0.15 M NaCl at 72°C for about 15 minutes.
  • An example of stringent wash conditions is a 0.2 ⁇ SSC wash at 65°C for 15 minutes. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal.
  • An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is l ⁇ SSC at 45°C for 15 minutes.
  • An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4 ⁇ SSC to 6 ⁇ SSC at 40°C for 15 minutes.
  • nucleic acid and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA) , transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers.
  • the nucleic acid molecule may be linear or circular.
  • “pharmaceutical composition” refers to a formulation of a compound and a medium generally accepted in the art for delivering a biologically active compound to a subject (e.g., human) .
  • the medium includes a pharmaceutically acceptable carrier.
  • the purpose of the pharmaceutical composition is to promote the administration to living organisms, which facilitates the absorption of active ingredients and thus exerts biological activity.
  • beneficial response can be expressed in terms of a number of clinical parameters, including loss of detectable tumor (complete response) , decrease in tumor size and/or cancer cell number (partial response) , tumor growth arrest (stable disease) , enhancement of anti-tumor immune response, possibly resulting in regression or rejection of the tumor; relief, to some extent, of one or more symptoms associated with the tumor; increase in the length of survival following treatment; and/or decreased mortality at a given point of time following treatment. Continued increase in tumor size and/or cancer cell number and/or tumor metastasis is indicative of lack of beneficial response to treatment, and therefore decreased responsiveness.
  • SHP2 refers to “Src homology-2 domain-containing protein tyrosine phosphatase-2” , also called SH-PTP2, SH-PTP3, Syp, PTP1D, PTP2C, SAP-2 or PTPN11.
  • the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) .
  • a human includes pre and post-natal forms.
  • a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • the term “subject” is used herein interchangeably with “individual” or “patient. ”
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • terapéuticaally effective amount refers to the amount of at least one medicament or compound sufficient to relieve one or more symptoms of the disease or disease being treated to a certain extent after administration. The result may be the reduction and/or remission of signs, symptoms or causes, or any other desired changes in the biological system.
  • a “therapeutically effective amount” for treatment is the amount of a composition comprising a compound as disclosed herein required to provide a clinically significant disease relief effect. Techniques such as dose escalation tests may be used to determine the effective amount suitable for any individual case.
  • treatment “treat” or “treating” a disease or condition as used herein include the following meanings: (i) prevention of the occurrence of the disease or condition in a subject, especially when the subject is susceptible to the disease or condition but has not been diagnosed with the disease or condition; (ii) suppression of the disease or condition, that is, inhibition of the development of the disease or condition; (iii) alleviation of the disease or condition, that is, abatement of the status of the disease or condition; or (iv) relief of the symptoms caused by the disease or condition. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
  • the methods provided in this disclosure relate to the discovery that certain SHP2 inhibitors can be used to treat EGFR-related diseases such as cancer, in particular, cancer associated with an insertion mutation at exon 20 of the EGFR gene.
  • SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase-2) , also named SH-PTP2, SH-PTP3, Syp, PTP1D, PTP2C, SAP-2 or PTPN11 is a tyrosine kinase phosphatase that plays an important role in cell signaling and is a target for the treatment of major diseases such as diabetes, autoimmune diseases and cancers.
  • SHP2 is mutated or highly expressed in various diseases, such as Noonan Syndrome, Leopard Syndrome, juvenile myelomonocytic leukemia, neuroblastoma, melanoma, acute myeloid leukemia, breast cancer, esophageal cancer, lung cancer, colon cancer, head cancer, squamous cell carcinoma of head and neck, gastric cancer, anaplastic large cell lymphoma, and glioblastoma, etc.
  • Molecular biological studies show that SHP2 is involved in multiple tumor cell signaling pathways, such as MAPK, JAK/STAT, and PI3K/Akt, etc.
  • SHP2 is also responsible for the signal transduction of PD-1/PD-L1 immunosuppressive pathway. As a result, inhibition of SHP2 activity has been proposed to reverse immunosuppression in tumor microenvironment.
  • SHP2 consists of two N-terminal Src Homolgy-2 domains (N-SH2 and C-SH2) and a protein tyrosine phosphatase catalytic domain (PTP) .
  • N-SH2 combines with PTP to form a ring structure, which hinders the binding of PTP to substrate, thus inhibiting the enzyme catalytic activity.
  • PTP catalytic domain is released to exhibit phosphatase activity.
  • the SHP2 inhibitor used in the methods disclosed herein is an allosteric inhibitor in the non-catalytic region, such as the compound disclosed in WO2015107493A1, WO2016203404A1, WO2016203406A1, WO2017216706A1, WO2017211303A1, CN201710062495, WO2018136265A1, and WO2018057884.
  • the SHP2 inhibitor used in the methods disclosed herein is a compound disclosed in Chinese Patent Application No. 201811314910.1 or WO2020094018, the disclosure of which is incorporated herein via reference.
  • the SHP2 inhibitor is a compound of Formula Ib:
  • R 4 is selected from H, halogen, substituted or unsubstituted amino, substituted or unsubstituted C 1 -C 6 alkyl, or substituted or unsubstituted C 1 -C 6 alkoxyl;
  • X 4 and X 5 are independently selected from C or N, and X 4 and X 5 are not N simultaneously;
  • ring D is selected from substituted or unsubstituted 4 to 8-membered heterocyclyl, substituted or unsubstituted 4 to 8-membered carbocyclic group, substituted or unsubstituted C 5-10 aryl, or substituted or unsubstituted 5 to 10-membered heteroaryl; wherein the heterocyclyl or heteroaryl comprises 1-3 heteroatoms selected from: N, O, S or P;
  • any of the above term “substituted” refers to one or more hydrogen atoms on the group are substituted by a substituent selected from: -D, halogen, -OH, -NO 2 , -NH 2 , -N (unsubstituted or halogenated C 1 -C 6 alkyl) 2 , -CN, unsubstituted or halogenated C 1 -C8 alkyl, unsubstituted or halogenated C 1 -C 8 alkoxyl, unsubstituted or halogenated C 1 -C 8 alkoxyl-C 1 -C 8 alkyl, unsubstituted or halogenated C 3 -C 8 cycloalkyl, unsubstituted or halogenated C 3 -C 8 cycloalkyl-C 1 -C 8 alkyl, unsubstituted or halogenated C 1 -C 6 alkyl carbonyl, unsubstit
  • the SHP2 inhibitor is a compound of Formula I:
  • X 1 and X 2 are independently selected from a bond, O, CR a R b or NR c ;
  • X 3 is selected from a bond, CR a R b , NR c , S or O;
  • X 4 is selected from N or CR c ; and R a , R b and R c are independently selected from H, D, halogen, substituted or unsubstituted C 1-6 alkyl, or substituted or unsubstituted C 1-6 alkoxyl;
  • R 1 , R 2 , R 3 , R 4 and R 7 are independently selected from H, D, -OH, halogen, substituted or unsubstituted amino, substituted or unsubstituted C 1-6 alkyl, or substituted or unsubstituted C 1-6 alkoxyl; and R 1 , R 2 , R 3 , R 4 and R 7 cannot be -OH or -NH 2 simultaneously;
  • ring A is selected from substituted or unsubstituted C 4-8 cyclic hydrocarbyl, substituted or unsubstituted 4 to 8-membered heterocyclyl, substituted or unsubstituted C 5-10 aryl, or substituted or unsubstituted 5 to 10-membered heteroaryl, wherein the heterocyclyl or heteroaryl comprises 1-3 heteroatoms selected from the following atoms: N, O, S or P;
  • ring C is selected from substituted or unsubstituted C 4-8 cyclic hydrocarbyl, substituted or unsubstituted 5 to 6-membered monocyclic heterocyclyl, substituted or unsubstituted 8 to 10-membered bicyclic heterocyclyl, substituted or unsubstituted C 5-10 monocyclic or bicyclic aryl, substituted or unsubstituted 5 to 6-membered monocyclic heteroaryl, or substituted or unsubstituted 8 to 10-membered bicyclic heteroaryl, wherein the heterocyclyl or heteroaryl comprises 1-4 heteroatoms selected from the following atoms: N, O, S or P;
  • R 5 and R 6 are independently selected from H, D, -OH, halogen, cyano, substituted or unsubstituted amino, substituted or unsubstituted C 1-6 alkyl, or substituted or unsubstituted C 1-6 alkoxyl;
  • n is any integer from 0 to 3;
  • the “substituted” refers to one or more hydrogen atoms on the group are substituted by a substituent selected from the following substituents: halogen, -OH, -NO 2 , -NH 2 , -NH (unsubstituted or halogenated C 1-6 alkyl) , -N (unsubstituted or halogenated C 1-6 alkyl) 2 , - CN, unsubstituted or halogenated C 1-8 alkyl, unsubstituted or halogenated C 1-8 alkoxyl, unsubstituted or halogenated C 1-8 alkoxyl-C 1-8 alkyl, unsubstituted or halogenated C 3-8 cycloalkyl-C 1-8 alkyl, unsubstituted or halogenated C 1-6 alkyl carbonyl, unsubstituted or halogenated C 1-6 alkoxyl carbonyl, hydroxamic acid group, unsub
  • one of X 1 and X 2 is CH 2 , and the other is a bond.
  • X 3 is S.
  • X 4 is selected from N or CH.
  • R 1 , R 2 , R 3 , R 4 and R 7 are independently selected from H, D, -OH, -F, -Cl, -Br, -NH 2 , -NHC 1-3 alkyl, methyl, ethyl, propyl, isopropyl, butyl, methoxy, ethoxy, propoxy or isopropoxy; C 1-3 alkyl substituted by halogen, -NH 2 , -OH, C 1-3 alkyl or C 1-3 alkoxyl; or C 1-3 alkoxyl substituted by halogen, -NH 2 , -OH, C 1-3 alkyl or C 1-3 alkoxyl.
  • R 5 and R 6 are independently selected from H, -OH, -F, -Cl, -Br, -CN, -NH 2 , -NHC 1-3 alkyl, methyl, ethyl, propyl, isopropyl, butyl, methoxy, ethoxy, propoxy or isopropoxy; C 1-3 alkyl substituted by halogen, -NH 2 , -OH, C 1-3 alkyl or C 1-3 alkoxyl; or C 1-3 alkoxyl substituted by halogen, -NH 2 , -OH, C 1-3 alkyl or C 1-3 alkoxyl.
  • the substituent is selected from –F, -Cl, -Br, -OH, -NO 2 , -NH 2 , -NH (C 1-6 alkyl) , -N (C 1-6 alkyl) 2 , -CN, C 1-6 alkyl, C 1-4 alkoxyl, C 1-4 alkoxyl-C 1-6 alkyl, C 3-8 cycloalkyl-C 1-8 alkyl, C 1-6 alkyl carbonyl, C 1-6 alkoxyl carbonyl, C 1-6 alkyl thiol, -S (O) 2 N (C 1-6 alkyl) 2 , -S (O) 2 C 1-6 alkyl, -N (C 1-6 alkyl) S (O) 2 N (C 1-6 alkyl) 2 , -S (O) N (C 1-6 alkyl) 2 , -S (O) (C 1-6 alkyl) , -N (C 1-6 alkyl) (C
  • the substituent is selected from –F, -Cl, -Br, -OH, -NO 2 , -NH 2 , -NH (C 1-3 alkyl) , -N (C 1-3 alkyl) 2 , -CN, C 1-3 alkyl, C 1-3 alkoxyl, C 1-3 alkyl carbonyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclooctyl, pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, thiomorpholinyl, phenyl, naphthyl, anthracyl, phenanthryl, fluorenyl, thiophenyl, imidazolyl,
  • the substituent is selected from –F, -Cl, -Br, -OH, -NO 2 , -NH 2 , -NH (C 1-3 alkyl) , -N (C 1-3 alkyl) 2 , -CN, methyl, ethyl, propyl, isopropyl, butyl, methoxy, ethoxy, propoxy, isopropoxy or phenyl.
  • one or more of the hydrogens are substituted by D in the position selected from: H on a ring atom other than a heteroatom on ring A, H on an amino-substituted ring atom on ring B, H on a ring atom other than a heteroatom on ring C, H on position 7 of ring E, and H on position 2 or 3 of ring F.
  • the number of D substitution is 1-4.
  • the ring C is selected from any of the following groups:
  • X 5 , X 6 , X 7 , X 8 and X 9 are independently selected from N or CR d ; and at most 3 of X 5 , X 6 , X 7 , X 8 and X 9 are N simultaneously;
  • X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 and X 17 are independently selected from N or CR d ; and at most 5 of X 10 , X 11 , X 12 , X 13 , X 14 , X 15 , X 16 and X 17 are N simultaneously;
  • X 18 , X 19 , X 20 and X 21 are independently selected from N or CR d , and at most 3 of X 18 , X 19 , X 20 and X 21 are N simultaneously;
  • R 6 and R 8 are independently selected from H, -NH 2 , -CN, -OH, -NO 2 , halogen, unsubstituted or halogenated C 1-6 alkyl, or unsubstituted or halogenated C 1-6 alkoxyl; and
  • R d is selected from H, halogen, unsubstituted or halogenated C 1-6 alkyl, or unsubstituted or halogenated C 1-6 alkoxyl.
  • the ring C is selected from any of the following groups:
  • X 5 , X 6 , X 7 , X 8 and X 9 are N, the others are CR d ;
  • X 18 , X 19 , X 20 and X 21 are N, the others are CR d ;
  • R 6 is selected from H, -NH 2 , -CN, -OH, -NO 2 , -F, -Cl, -Br, methyl, ethyl, propyl, isopropyl, butyl, methoxy, ethoxy, propoxy, isopropoxy, fluorinated or brominated C 1-3 alkyl, fluorinated or brominated C 1-3 alkoxyl; and
  • R d is selected from H, -F, -Cl, -Br, methyl, ethyl, propyl, isopropyl, butyl, methoxy, ethoxy, propoxy, isopropoxy, fluorinated or brominated C 1-3 alkyl, or fluorinated or brominated C 1-3 alkoxyl.
  • the ring C is selected from any of the following groups:
  • the H on position 5 and/or 6 of the pyridine ring is substituted by D, one or more of the H on position 4, 5 and 6 of the benzene ring is substituted by D.
  • the ring A is selected from substituted or unsubstituted C 4-6 cyclic hydrocarbyl, substituted or unsubstituted 4 to 6-membered heterocyclyl, substituted or unsubstituted C 5-6 aryl, or substituted or unsubstituted 5 to 6-membered heteroaryl, wherein the heterocyclyl or heteroaryl comprises 1-3 N atoms.
  • the ring A is selected from any of the following groups:
  • the ring A is selected from any of the following groups:
  • one or more H on the ring atoms other than the heteroatom or F-substituted atom are substituted by D.
  • halogen refers to fluorine, chlorine, bromine or iodien.
  • Haldroxyl refers to -OH group.
  • Haldroxyl alkyl refers to an alkyl as defined below substituted by hydroxyl (-OH) .
  • Niryl refers to -NO 2 .
  • Cyano refers to -CN.
  • Amino refers to -NH 2 .
  • substituted amino refers to an amino substituted by one or two of the alkyl, alkyl carbonyl, aryl alkyl, heteroaryl alkyl as defined below, for example, substituted amino may be monoalkyl amino, dialkyl amino, alkyl acylamino, aryl alkyl amino, heteroaryl alkyl.
  • Carboxyl refers to -COOH.
  • alkyl refers to a fully saturated straight or branched hydrocarbon chain group, consisting only of carbon atoms and hydrogen atoms, for example, comprising 1 to 12 (preferably 1 to 8, more preferably 1 to 6) carbon atoms, and connected to the rest of the molecule by a single bond.
  • alkyl includes, but is not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-amyl, 2-methylbutyl, 2, 2-dimethylpropyl, n-hexyl, heptyl, 2-methylhexyl, 3-methylhexyl, octyl, nonyl and decyl, etc.
  • the term “alkyl” refers to an alkyl group containing 1 to 8 carbon atoms.
  • alkenyl refers to a straight or branched hydrocarbon chain group, consisting only of carbon atoms and hydrogen atoms, for example, comprising 2 to 20 (preferably 2 to 10, more preferably 2 to 6) carbon atoms, comprising at least one double bond, and connected to the rest of the molecule by a single bond, Examples, include but are not limited to, vinyl, propenyl, allyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-1, 4-dienyl, etc.
  • cyclic hydrocarbyl refers to a stable non-aromatic monocyclic or polycyclic hydrocarbyl (such as alkyl, alkenyl or alkynyl) consisting only of carbon atoms and hydrogen atoms, which may comprise fused ring system, bridged ring system or spiro ring system, comprise 3 to 15 carbon atoms, preferably comprise 3 to 10 carbon atoms, more preferably comprise 3 to 8 carbon atoms, for example, comprise 3, 4, 5, 6, 7 or 8 carbon atoms, and which is saturated or unsaturated and may be connected to the rest of the molecule via any suitable carbon atom by a single bond.
  • cyclic hydrocarbyl include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclooctyl, 1H-indenyl, 2, 3-dihydroindenyl, 1, 2, 3, 4-tetrahydro-naphthyl, 5, 6, 7, 8-tetrahydro-naphthyl, 8, 9-dihydro-7H-benzocycloheptene-6-yl, 6, 7, 8, 9-tetrahydro-5H-benzocycloheptenyl, 5, 6, 7, 8, 9, 10-hexahydro-benzocyclooctenyl, fluorenyl, bicyclo
  • heterocyclyl refers to a stable 3 to 20-membered non-aromatic cyclic group consisting of 2 to14 carbon atoms (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms) and 1 to 6 heteroatoms selected from nitrogen, phosphorus, oxygen or sulfur.
  • a heterocyclyl may be a monocyclic ring system, a dicyclic ring system, a tricyclic ring system or a ring system with more rings, and may comprise fused ring system, bridged ring system or spiro ring system; the nitrogen, phosphorus or sulfur atoms in the heterocyclyl may be optionally oxidized; the nitrogen atoms in the heterocyclyl may be optionally quaternized; and the heterocyclyl may be partially or fully saturated.
  • the heterocyclyl may be connected to the rest of the molecule via a carbon atom or a heteroatom by a single bond.
  • heterocyclyl In a heterocyclyl containing fused ring, one or more rings may be aryl or heteroaryl as defined below, provided that the connection point between the group and the rest of the molecule is a non-aromatic ring atom.
  • heterocyclyl is preferably a stable 4 to 11-membered non-aromatic monocyclic, dicyclic, bridged or spiro ring group comprising 1 to 3 heteroatoms selected from nitrogen, oxygen or sulfur, more preferably a stable 4 to 8-membered non-aromatic monocyclic, dicyclic, bridged or spiro ring group comprising 1 to 3 heteroatoms selected from nitrogen, oxygen or sulfur.
  • heterocyclyls include but are not limited to: pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, thiomorpholinyl, 2, 7-diaza-spiro [3.5] nonane-7-yl, 2-oxa-6-aza-spiro [3.3] heptane-6-yl, 2, 5-diaza-bicyclo [2.2.1] heptane-2-yl, azacyclobutanyl, pyranyl, tetrahydropyranyl, thiopyranyl, tetrahydrofuranyl, oxazinyl, dioxolanyl, tetrahydroisoquinolinyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, quinolizinyl, thiazolidinyl, isothiazolidinyl, isoxazolidinyl, di
  • aryl refers to a conjugated hydrocarbon ring system group comprising 6 to 18 carbon atoms (preferably comprising 6 to 10 carbon atoms, for example, 6, 7, 8, 9 or 10 carbon atoms) .
  • aryl may be a monocyclic ring system, a dicyclic ring system, a tricyclic ring system or a ring system with more rings, and may be fused with the cyclic hydrocarbyl or heterocyclyl as defined above, provided that the aryl and the rest of the molecule are connected via an atom on the aromatic ring by a single bond.
  • Exemplary aryls include but are not limited to phenyl, naphthyl, anthracyl, phenanthrenyl, fluorenyl, 2, 3-dihydro-1H-isoindolyl, 2-benzoxazolinone, 2H-1, 4-benzoxazin-3 (4H) -one-7-yl, etc.
  • aryl alkyl refers to an alkyl as defined above which is substituted by an aryl as defined above.
  • heteroaryl refers to a 5 to 16-membered conjugated ring system group comprising 1 to 15 carbon atoms (preferably comprising 1 to 10 carbon atoms, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms) and 1 to 6 heteroatoms selected from nitrogen, oxygen or sulfur.
  • a heteroaryl may be a monocyclic ring system, a dicyclic ring system, a tricyclic ring system or a ring system with more rings, and may be fused with the cycloalkyl or heterocyclyl, provided that the heteroaryl and the rest of the molecule are connected via an atom on the aromatic ring by a single bond.
  • the nitrogen, carbon or sulfur atoms in the heteroaryl may be optionally oxidized; the nitrogen atoms in the heteroaryl may be optionally quaternized.
  • heteroaryl is preferably a stable 5 to 12-membered aromatic group comprising 1 to 5 heteroatoms selected from nitrogen, oxygen or sulfur, and is more preferably a stable 5 to 10-membered aromatic group comprising 1 to 4 heteroatoms selected from nitrogen, oxygen or sulfur, or a 5 to 6-membered aromatic group comprising 1 to 3 heteroatoms selected from nitrogen, oxygen or sulfur.
  • heteroaryls include but are not limited to thiophenyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, benzimidazolyl, benzopyrazolyl, indolyl, furanyl, pyrrolyl, triazolyl, tetrazolyl, triazinyl, indolizinyl, isoindolyl, indazolyl, isoindazolyl, purinyl, quinolinyl, isoquinolinyl, diazanaphthalenyl, naphthyridinyl, quinoxalinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl, phenanthrolinyl, acridinyl,
  • heteroaryl alkyl refers to an alkyl as defined above which is substituted by heteroaryl as defined above.
  • “optional” or “optionally” means that the subsequently described event or condition may or may not occur, and such description includes both occurrence and non-occurrence of the event or condition.
  • “optionally substituted aryl” means an aryl being substituted or not being substituted, and such description includes both substituted aryl and unsubstituted aryl.
  • the “optional” substituent employed in the claims and description of the disclosure is selected from alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, cyano, nitro, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cyclic hydrocarbyl, or optional substituted heterocyclyl.
  • Stepisomer refers to a compound composed of the same atoms, bonded by the same bonds, but having different three-dimensional structures. The present disclosure will cover various stereoisomers and mixtures thereof.
  • Tautomer refers to an isomer formed by the transfer of proton from one atom of a molecule to another atom of the same molecule. All tautomeric forms of a compound of the disclosure will also be included within the scope of the disclosure.
  • the compound of the disclosure may comprise one or more chiral carbon atoms, and thus may produce enantiomers, diastereomers, and other stereoisomeric forms.
  • Each chiral carbon atom can be defined as (R) -or (s) -based on stereochemistry.
  • the disclosure is intended to include all possible isomers, as well as racemes and optically pure forms thereof.
  • a compound of the disclosure can be prepared by using raceme, diastereomer or enantiomer as raw material or intermediate.
  • the optically active isomers can be prepared by using chiral synthons or chiral reagents, or separated by conventional techniques, such as crystallization and chiral chromatography.
  • the disclosure also includes all suitable isotopic variations of the compounds of the present disclosure or pharmaceutically acceptable salts thereof.
  • Isotopic variations of the compounds of the present disclosure or pharmaceutically acceptable salts thereof are defined as those in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass often found in nature.
  • Isotopes that can be incorporated into the compounds of the present disclosure and their pharmaceutically acceptable salts thereof include but are not limited to H, C, N and O, for example, 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 17 O, 18 O, 35 S, 18 F, 36 Cl and 125 I.
  • Suitable isotopic variations of the compounds or pharmaceutically acceptable salts thereof of the present disclosure may be prepared by conventional techniques using appropriate isotopic variants of suitable reagents.
  • the compound has a structure selected from:
  • the compound has a structure selected from:
  • the isotopic substitution of the isotope-substituted derivative of the compound herein relates to atom comprises but not limited to hydrogen, carbon, nitrogen, oxygen, fluorine, phosphorus, chlorine or iodine; and preferably is 2 H, 3 H, 11 C, 13 C, 14 C, 15 N, 17 O, 18 O, 18 F, 31 P, 32 P, 35 S, 36 Cl or 125 I.
  • the compound has a structure of also named as Compound A.
  • the compound has a structure of also named as Compound B.
  • the compound has a structure of also named as Compound C.
  • the SHP2 inhibitor provided herein is capable of inhibiting proliferation of tumor cells bearing one or more EGFR exon 20 insertion mutation at a maximal inhibition percentage significantly higher than that of reference compound TAK-788.
  • the inhibition of proliferation is determined by a cell viability assay comparable to that disclosed in Example 3 as provided herein.
  • TAK-788 is a small-molecule tyrosine kinase inhibitor (TKI) designed to selectively target EGFR and human EGFR 2 (HER2) exon 20 insertion mutations.
  • TKI small-molecule tyrosine kinase inhibitor
  • maximal inhibition percentage refers to the highest (i.e. plateau of) inhibition percentage achievable by a compound to inhibit proliferation of the tumor cells.
  • the percentage of inhibition on proliferation increases with the increase in concentration of the compound (e.g., an SHP2 inhibitor or an EGFR inhibitor) , however, it could reach a plateau where no more inhibition can be achieved despite of further increase in the concentration of the compound.
  • the higher the maximal inhibition percentage the more effective of the inhibition.
  • the maximal inhibition percentage may vary to some degree depending on different assays, such as cell viability assay and other suitable assays.
  • the SHP2 inhibitor provided herein have maximal inhibition percentage that is significantly higher (e.g. at least 30%higher, 40%higher, 50%higher, 60%higher, 70%higher, 80%higher, 90%higher, or 100%higher) than that of the reference compound TAK-788.
  • the assay conditions can be similar to those provide in Example 3 of the present disclosure.
  • the SHP2 inhibitor can be formulated with a pharmaceutically acceptable carrier.
  • the carrier when present, can be blended with the SHP2 inhibitor in any suitable amounts, such as an amount of from 5%to 95%by weight of carrier, based on the total volume or weight of SHP2 inhibitor and the carrier.
  • the amount of carrier can be in a range having a lower limit of any of 5%, 10%, 12%, 15%, 20%, 25%, 28%, 30%, 40%, 50%, 60%, 70%or 75%, and an upper limit, higher than the lower limit, of any of 20%, 22%, 25%, 28%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 95%.
  • the amount of carrier in a specific embodiment may be determined based on considerations of the specific dose form, relative amounts of SHP2 inhibitor, the total weight of the composition including the carrier, the physical and chemical properties of the carrier, and other factors, as known to those of ordinary skill in the formulation art.
  • the SHP2 inhibitor may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, the SHP2 inhibitor may be administered in conjunction with other treatments. The SHP2 inhibitor may be encapsulated or otherwise protected against gastric or other secretions, if desired.
  • a suitable, non-limiting example of a dosage of the SHP2 inhibitor disclosed herein is from about 1 mg/kg to about 2400 mg/kg per day, such as from about 1 mg/kg to about 1200 mg/kg per day, 75 mg/kg per day to about 300 mg/kg per day, including from about 1 mg/kg to about 100 mg/kg per day.
  • Other representative dosages of such agents include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg, 2200 mg/kg, and 2300 mg/kg per day.
  • the dosage of the SHP2 inhibitor in human is about 400 mg/day given every 12 hours. In some embodiments, the dosage of the SHP2 inhibitor in human ranges 300-500 mg/day, 100-600 mg/day or 25-1000 mg/day.
  • the effective dose of SHP2 inhibitor disclosed herein may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
  • EGFR has been associated with many human diseases including cancer, inflammatory disease, wound healing and fibrosis.
  • mutations of EGFR gene have been associated with a number of cancers, including lung adenocarcinoma, anal cancers, glioblastoma, and epithelial tumors of the head and neck. These mutations typically lead to the overexpression or constant activation of EGFR, which results in uncontrolled cell proliferation.
  • the cancer to be treated with the methods disclosed herein includes all cancer types disclosed herein.
  • the cancer to be treated with the methods disclosed herein includes Noonan Syndrome, Leopard Syndrome, juvenile myelomonocytic leukemia, neuroblastoma, melanoma, acute myeloid leukemia, breast cancer, esophageal cancer, lung cancer, colon cancer, head cancer, squamous cell carcinoma of head and neck, gastric cancer, anaplastic large cell lymphoma or glioblastoma.
  • the cancer to be treated with the methods disclosed herein includes lung cancer (e.g., non-small cell lung cancer (NSCLC) ) , glioblastoma, conventional glioblastoma multiforme, uterus cancer (e.g., infiltrating renal pelvis and ureter urothelial carcinoma, endometrial endometrioid adenocarcinoma) , bladder cancer (e.g., bladder urothelial carcinoma) , melanoma, renal cancer, liver cancer, myeloma, prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, thyroid cancer, hematological cancer, leukemia and non-Hodgkin’s lymphoma.
  • lung cancer e.g., non-small cell lung cancer (NSCLC)
  • glioblastoma e.g., conventional glioblastoma multiforme
  • uterus cancer e.g., infiltrating renal pelvis and ureter
  • the cancer comprises an EGFR exon 20 insertion mutation.
  • Exon 20 of EGFR gene encompasses nucleotides that translate into amino acid residues at positions 762 to 823 of EGFR protein, which contains a C-helix (residues 762-766) and the loop following C-helix (residues 767-774) .
  • the amino acid position of EGFR protein is based on the standard amino acid sequence of EGFR found in the sequence shown in accession number P00533-1 in the SwissProt database, or in the sequence shown in accession number NP_005219.2 in the NCBI database, which is also provided here as SEQ ID NO: 1.
  • Insertion mutations could induce ligand-independent EGFR pathway activation and promote tumorigenesis.
  • in frame insertion refers to an insertion of one or more amino acid-encoding codons in a gene that does not alter the normal reading frame of the gene.
  • the in frame insertion within exon 20 of EGFR gene, as described herein, can be at any place within the exon 20 of EGFR gene, and the insertion can introduce any number (e.g., 1, 2, 3, 4, or 5) of amino acid residues.
  • the insertion is within the C-helix (i.e. residues 761-766 of EGFR) .
  • the insertion is within the loop following C-helix (i.e., residues 767-775 of EGFR) .
  • the insertion mutation within exon 20 of EGFR gene promotes active conformation of EGFR or destabilizes inactive confirmation of EGFR.
  • the insertion mutation within exon 20 of EGFR gene enhances tyrosine kinase activity of EGFR in the absence of an EGFR ligand.
  • the EGFR exon 20 insertion mutation includes a mutation such as but not limited to any of the mutations described in Yasuda, et al., 2013, Sci. Transl. Med. 5 (216) : 216ra177; doi: 10.1126/scitranslmed. 3007205, and Arcila, et al., 2013, Mol. Cancer Ther. 12: 220; each of which is incorporated herein in its entirety by reference.
  • the exon 20 insertion mutation of the EGFR gene is selected from the group consisting of A763_Y764insFQEA, A763_Y764insFQQA, A767_V769dupASV, V769_D770insASV, D770_N771insGL, D770_N771insGT, D770_N771insNPG, D770_N771insSVD, E762Q_insFQEA, H773_V774insH, H773_V774insH, H773_V774insNPH, M766_A767insAI, M766_A767insASV, N771_H773dupNPH, P772_H773insYNP, P772_V774insPHV, S768_770dupSVD, V769_D770insASV, Y764_V765insHH
  • the cancer is resistant to an EGFR inhibitor.
  • An EGFR inhibitor refers to a molecule capable of inhibiting tyrosine kinase activity of EGFR, and may be a small molecule compound or a biological macromolecule such as an antibody or an antibody fragment or the like.
  • Exemplary small molecule EGFR inhibitors include, but are not limited to, first generation EGFR inhibitors such as gefitinib, erlotinib, and icotinib; second generation EGFR inhibitors such as afatinib and dacomitinib; and third-generation EGFR inhibitors such as osimertinib (AZD9291) , olmutinib, josartinib, rociletinib, naquotinib, lazertinib and the like.
  • Exemplary macromolecular EGFR inhibitors include, but are not limited to, cetuximab, panitumumab, nimotuzumab, and necitumumab.
  • the cancer is resistant to an EGFR inhibitor selected from: gefitinib, erlotinib, icotinib, afatinib, dacomitinib, lapatinib, osimertinib (AZD9291) , soloartinib, rociletinib, naquotinib, vandetanib, neratinib, pelitinib, canertinib, brigatinib, PKC412, Go6976, mavelertinib, olmutinib, WZ4002, TAS2913, cetuximab, panitumumab, nimotuzumab, necitumumab avitinib, HS-10296 and TQB3804.
  • the cancer is resistant to osimertinib.
  • the cancer is resistant to EGFR tyrosine kinase inhibitor TAK-788, osimertinib, Poziotinib, Almonertinib (HS-10296) , Amivantamab (JNJ-372) (see, Neijssen et al, J. Biol. Chem. (2021) 296: 100641) , furmonertinib (AST2818) , or DZD9008 (see, Yan Xu et al, abstract 3081 of Proceedings: AACR Annual Meeting 2019; March 29-April 3, 2019; Atlanta, GA) .
  • TAK-788 also named mobocertinib and AP32788, which has the following structure, is a selective inhibitor of EGFR.
  • Poziotinib has a chemical structure shown below:
  • Almonertinib (HS-10296) has a chemical structure shown below:
  • Furmonertinib (AST2818) has a chemical structure shown below:
  • Osimertinib which has the following structure, is a third-generation EGFR tyrosine kinase inhibitor.
  • Drug resistance as used herein refers to being refractory or non-responsive to a therapeutic agent, such as an EGFR inhibitor.
  • a therapeutic agent such as an EGFR inhibitor.
  • the number of tumor cells is increased despite of being treated with a therapeutic agent.
  • the cancer resistant to EGFR inhibitor is lung cancer (e.g., small cell lung cancer or non-small cell lung cancer) .
  • the cancer is resistant to a chemotherapy.
  • chemotherapy include such as cisplatin, cyclophosphamide, chlorambucil, busulfan, melphalan, carmustine, streptozotocin, triethylenemelamine, mitomycin C, methotrexate, etoposide, 6-mercaptopurine (6MP) , 6-thiocguanine (6TG) , cytarabine (Ara-C) , 5- fluorouracil (5-FU) , capecitabine, dacarbazine (DTIC) , actinomycin D, doxorubicin (DXR) , daunorubicin (daunomycin) , bleomycin, mithramycin, vincristine (VCR) , vinblastine, paclitaxel and pactitaxel derivatives, the cytostatic agents, dexamethasone (DEX) , prednisone, hydroxyurea, aspara
  • arnifostine dactinomycin, mechlorethamine (nitrogen mustard) , streptozocin, cyclophosphamide, lornustine (CCNU) , doxorubicin lipo, gemcitabine, daunorubicin lipo, procarbazine, mitomycin, docetaxel, aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan) , 10-hydroxy 7-ethyl-camptothecin (SN38) , floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon alpha, interferon beta, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plic
  • the cancer is metastatic.
  • the presence or absence of insertion mutation within exon 20 of the EGFR gene in a biological sample can be detected using proper methods known in the art including, without limitation, sequencing assay, amplification assay and hybridization assay.
  • any biological sample suitable for conducting the methods provided herein can be obtained from the subject including, without limitation, bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal flushing fluids, plural fluid, ascitic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchioalveolar lavage fluid, etc., and tissues, such as biopsy tissue (e.g. biopsied bone tissue, bone marrow, breast tissue, gastrointestinal tract tissue, lung tissue, liver tissue, prostate tissue, brain tissue, nerve tissue, meningeal tissue, renal tissue, endometrial tissue, cervical dittuse, lymph node tissue, muscle tissue, or skin tissue) .
  • bodily fluid such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal flushing fluids
  • plural fluid ascitic fluid
  • cerebrospinal fluid saliva
  • sweat tears
  • sputum bronchioalveolar lavage fluid
  • tissues such as biopsy tissue (e
  • the biological sample is processed to isolate or extract cancer cell (such as circulating tumor cell) before detecting the presence or absence of the insertion mutation.
  • cancer cells can be separated by immunomagnetic separation technology such as that available from Immunicon (Huntingdon Valley, Pa. ) .
  • nucleic acid e.g., DNA or RNA is isolated from the biological sample before detecting the presence or absence of the insertion mutation.
  • Various methods of extraction are suitable for isolating the DNA or RNA from cells or tissues, such as phenol and chloroform extraction, and various other methods as described in, for example, Ausubel et al., Current Protocols of Molecular Biology (1997) John Wiley &Sons, and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3 rd ed. (2001) .
  • kits can also be used to isolate DNA and/or RNA, including for example, the NucliSens extraction kit (Biomerieux, Marcy l'Etoile, France) , QIAamp TM mini blood kit, Agencourt Genfind TM , mini columns (Qiagen) , RNA mini kit (Thermo Fisher Scientific) , and Eppendorf Phase Lock Gels TM .
  • NucliSens extraction kit Biomerieux, Marcy l'Etoile, France
  • QIAamp TM mini blood kit Agencourt Genfind TM , mini columns (Qiagen)
  • RNA mini kit Thermo Fisher Scientific
  • Eppendorf Phase Lock Gels TM Eppendorf Phase Lock Gels TM .
  • a skilled person can readily extract or isolate RNA or DNA following the manufacturer’s protocol.
  • Sequencing methods known in the art can be used to the detection of insertion mutation within the exon20 of the EGFR gene.
  • sequencing methods can be categorized to traditional or classical methods and high throughput sequencing (next generation sequencing (NGS) ) .
  • NGS next generation sequencing
  • Traditional sequencing methods include Maxam-Gilbert sequencing (also known as chemical sequencing) and Sanger sequencing (also known as chain-termination methods) .
  • High throughput sequencing involves sequencing-by-synthesis, sequencing-by-ligation, and ultra-deep sequencing (such as described in Marguiles et al., Nature 437 (7057) : 376-80 (2005) ) .
  • Sequence-by-synthesis involves synthesizing a complementary strand of the target nucleic acid by incorporating labeled nucleotide or nucleotide analog in a polymerase amplification. Immediately after or upon successful incorporation of a label nucleotide, a signal of the label is measured and the identity of the nucleotide is recorded.
  • sequence-by-synthesis may be performed on a solid surface (or a microarray or a chip) using fold-back PCR and anchored primers.
  • Target nucleic acid fragments can be attached to the solid surface by hybridizing to the anchored primers, and bridge amplified. This technology is used, for example, in the sequencing platform.
  • Pyrosequencing involves hybridizing the target nucleic acid regions to a primer and extending the new strand by sequentially incorporating deoxynucleotide triphosphates corresponding to the bases A, C, G, and T (U) in the presence of a polymerase. Each base incorporation is accompanied by release of pyrophosphate, converted to ATP by sulfurylase, which drives synthesis of oxyluciferin and the release of visible light. Since pyrophosphate release is equimolar with the number of incorporated bases, the light given off is proportional to the number of nucleotides adding in any one step. The process is repeated until the entire sequence is determined.
  • a nucleic acid amplification assay involves copying a target nucleic acid (e.g. DNA or RNA) , thereby increasing the number of copies of the amplified nucleic acid sequence. Amplification may be exponential or linear. Exemplary nucleic acid amplification methods include, but are not limited to, amplification using the polymerase chain reaction ( "PCR" , see U.S.
  • RT-PCR reverse transcriptase polymerase chain reaction
  • the nucleic acid amplification assay is a PCR-based method. PCR is initiated with a pair of primers that hybridize to the target nucleic acid sequence to be amplified, followed by elongation of the primer by polymerase which synthesizes the new strand using the target nucleic acid sequence as a template and dNTPs as building blocks. Then the new strand and the target strand are denatured to allow primers to bind for the next cycle of extension and synthesis. After multiple amplification cycles, the total number of copies of the target nucleic acid sequence can increase exponentially.
  • intercalating agents that produce a signal when intercalated in double stranded DNA may be used.
  • exemplary agents include SYBR GREEN TM and SYBR GOLD TM . Since these agents are not template-specific, it is assumed that the signal is generated based on template-specific amplification. This can be confirmed by monitoring signal as a function of temperature because melting point of template sequences will generally be much higher than, for example, primer-dimers, etc.
  • a detectably labeled primer or a detectably labeled probe can be used, to allow detection of the insertion mutation corresponding to that primer or probe.
  • multiple labeled primers or labeled probes with different detectable labels can be used to allow simultaneous detection of multiple insertion mutation.
  • Nucleic acid hybridization assays use probes to hybridize to the target nucleic acid, thereby allowing detection of the target nucleic acid.
  • Non-limiting examples of hybridization assay include Northern blotting, Southern blotting, in situ hybridization, microarray analysis, and multiplexed hybridization-based assays.
  • the probes for hybridization assay are detectably labeled.
  • the nucleic acid-based probes for hybridization assay are unlabeled. Such unlabeled probes can be immobilized on a solid support such as a microarray, and can hybridize to the target nucleic acid molecules which are detectably labeled.
  • hybridization assays can be performed by isolating the nucleic acids (e.g. RNA or DNA) , separating the nucleic acids (e.g. by gel electrophoresis) followed by transfer of the separated nucleic acid on suitable membrane filters (e.g. nitrocellulose filters) , where the probes hybridize to the target nucleic acids and allows detection.
  • suitable membrane filters e.g. nitrocellulose filters
  • the hybridization of the probe and the target nucleic acid can be detected or measured by methods known in the art. For example, autoradiographic detection of hybridization can be performed by exposing hybridized filters to photographic film.
  • hybridization assays can be performed on microarrays.
  • Microarrays provide a method for the simultaneous measurement of the levels of large numbers of target nucleic acid molecules.
  • the target nucleic acids can be RNA, DNA, cDNA reverse transcribed from mRNA, or chromosomal DNA.
  • the target nucleic acids can be allowed to hybridize to a microarray comprising a substrate having multiple immobilized nucleic acid probes arrayed at a density of up to several million probes per square centimeter of the substrate surface.
  • the RNA or DNA in the sample is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative levels of the RNA or DNA. See, U.S. Patent Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and 6,344,316.
  • arrays may be peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Patent Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992.
  • Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an all-inclusive device.
  • Useful microarrays are also commercially available, for example, microarrays from Affymetrix, from Nano String Technologies, QuantiGene 2.0 Multiplex Assay from Panomics.
  • the method of the present disclosure further involves administering to the subject an anti-cancer agent other than a SHP2 inhibitor.
  • the administration of the second anti-cancer agent depends on the detection of a second mutation in the cancer.
  • the method provided in the present disclosure involves detecting a second mutation in a gene in the RTK/RAS/MAPK signaling pathway in the biological sample of the subject.
  • the gene is EGFR, KRAS or BRAF.
  • the RTK/RAS/MAPK signaling pathway is a chain of protein in the cell that communicates a signal from a receptor tyrosine kinase on the surface of the cell to the DNA in the nucleus of the cell.
  • the RTK/RAS/MAPK signaling pathway has been associated with uncontrolled growth in many cancers.
  • the method of present disclosure further comprises administering to the subject a second therapeutic agent.
  • the second therapeutic agent is an EGFR tyrosine kinase inhibitor, a KRAS inhibitor, a BRAF inhibitor, a MEK inhibitor or a CDK inhibitor.
  • the EGFR tyrosine kinase inhibitor is Gefitinib, Erlotinib, Afatinib, Dacomitinib or Osimertinib.
  • the KRAS inhibitor is AMG510 or MRTX849.
  • the BRAF inhibitor is Vemurafenib, Dabrafenib, Encorafenib or Sorafenib.
  • the MEK inhibitor is Trametinib, Cobimetinib, Selumetinib, Pimasertib or Binimetinib.
  • the CDK inhibitor is Palbociclib, Ribociclib or Abemaciclib.
  • Additional anti-cancer agents that can be used in combination with the SHP2 inhibitor disclosed herein include, without limitation: alkylating agents or agents with an alkylating action, such as cyclophosphamide (CTX; e.g. ) , chlorambucil (CHL; e.g. ) , cisplatin (CisP; e.g. ) busulfan (e.g. ) , melphalan, carmustine (BCNU) , streptozotocin, triethylenemelamine (TEM) , mitomycin C, and the like; anti-metabolites, such as methotrexate (MTX) , etoposide (VP16; e.g.
  • CHL chlorambucil
  • CisP cisplatin
  • TEM triethylenemelamine
  • TEM triethylenemelamine
  • mitomycin C and the like
  • anti-metabolites such as methotrexate (MTX) , etoposide (VP16;
  • 6-mercaptopurine (6MP) 6-mercaptopurine
  • 6-thiocguanine (6TG)
  • cytarabine Ara-C
  • 5-fluorouracil 5-FU
  • capecitabine e.g.
  • dacarbazine DTIC
  • antibiotics such as actinomycin D, doxorubicin (DXR; e.g.
  • daunorubicin (daunomycin) , bleomycin, mithramycin and the like; alkaloids, such as vinca alkaloids such as vincristine (VCR) , vinblastine, and the like; and other antitumor agents, such as paclitaxel (e.g. ) and pactitaxel derivatives, the cytostatic agents, glucocorticoids such as dexamethasone (DEX; e.g.
  • DEX dexamethasone
  • corticosteroids such as prednisone, nucleoside enzyme inhibitors such as hydroxyurea, amino acid depleting enzymes such as asparaginase, leucovorin, folinic acid, raltitrexed, and other folic acid derivatives, and similar, diverse antitumor agents.
  • the following agents may also be used as additional agents: arnifostine (e.g. ) , dactinomycin, mechlorethamine (nitrogen mustard) , streptozocin, cyclophosphamide, lornustine (CCNU) , doxorubicin lipo (e.g. ) , gemcitabine (e.g.
  • daunorubicin lipo e.g.
  • procarbazine mitomycin
  • docetaxel e.g.
  • aldesleukin carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan) , 10-hydroxy 7-ethyl-camptothecin (SN38) , floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon alpha, interferon beta, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinore
  • the anti-cancer agent used in combination with the SHP2 inhibitor is an anti-hormonal agent.
  • anti-hormonal agent includes natural or synthetic organic or peptide compounds that act to regulate or inhibit hormone action on tumors.
  • Anti-hormonal agents include, for example: steroid receptor antagonists, anti-estrogens such as tamoxifen, raloxifene, aromatase inhibiting 4 (5) -imidazoles, other aromatase inhibitors, 42-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (e.g.
  • anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above; agonists and/or antagonists of glycoprotein hormones such as follicle stimulating hormone (FSH) , thyroid stimulating hormone (TSH) , and luteinizing hormone (LH) and LHRH (leuteinizing hormone-releasing hormone) ; the LHRH agonist goserelin acetate, commercially available as (AstraZeneca) ; the LHRH antagonist D-alaninamide N-acetyl-3- (2-naphthalenyl) -D-alanyl-4-chloro-D-phenylalanyl-3- (3-pyridinyl) -D-alanyl-L-seryl-N6- (3-pyridinylcarbonyl) -L-lysyl-N6- (3-pyridinyl-N
  • non-steroidal anti-androgen nilutamide (5, 5-dimethyl-3- [4-nitro-3- (trifluoromethyl-4′-nitrophenyl) -4, 4-dimethyl-imidazolidine-dione)
  • antagonists for other non-permissive receptors such as antagonists for RAR, RXR, TR, VDR, and the like.
  • the anti-cancer agent used in combination with the SHP2 inhibitor is an angiogenesis inhibitor.
  • Anti-angiogenic agents include, for example: VEGFR inhibitors, such as SU-5416 and SU-6668 (Sugen Inc. of South San Francisco, Calif., USA) , or as described in, for example International Application Nos. WO 99/24440, WO 99/62890, WO 95/21613, WO 99/61422, WO 98/50356, WO 99/10349, WO 97/32856, WO 97/22596, WO 98/54093, WO 98/02438, WO 99/16755, and WO 98/02437, and U.S. Pat. Nos.
  • VEGF inhibitors such as IM862 (Cytran Inc. of Kirkland, Wash., USA) ; angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colo. ) and Chiron (Emeryville, Calif. ) ; and antibodies to VEGF, such as bevacizumab (e.g. Avastin TM , Genentech, South San Francisco, Calif.
  • a recombinant humanized antibody to VEGF a recombinant humanized antibody to VEGF
  • integrin receptor antagonists and integrin antagonists such as to ⁇ v ⁇ 3 , ⁇ v ⁇ 5 and ⁇ v ⁇ 6 integrins, and subtypes thereof, e.g. cilengitide (EMD 121974)
  • EMD 121974 cilengitide
  • anti-integrin antibodies such as for example ⁇ v ⁇ 3 specific humanized antibodies (e.g. ); factors such as IFN-alpha (U.S. Pat. Nos. 41530,901, 4,503,035, and 5,231,176) ; angiostatin and plasminogen fragments (e.g.
  • kringle 14, kringle 5, kringle 1-3 (O'Reilly, M.S. et al. (1994) Cell 79: 315-328; Cao et al. (1996) J. Biol. Chem. 271: 29461-29467; Cao et al. (1997) J. Biol. Chem. 272: 22924-22928) ; endostatin (O'Reilly, M. S. et al. (1997) Cell 88: 277; and International Patent Publication No. WO 97/15666) ; thrombospondin (TSP-1; Frazier, (1991) Curr. Opin. Cell Biol.
  • PF4 platelet factor 4
  • plasminogen activator/urokinase inhibitors plasminogen activator/urokinase inhibitors
  • urokinase receptor antagonists heparinases
  • fumagillin analogs such as TNP-4701
  • suramin and suramin analogs angiostatic steroids
  • bFGF antagonists flk-1 and flt-1 antagonists
  • anti-angiogenesis agents such as MMP-2 (matrix-metalloprotienase 2) inhibitors and MMP-9 (matrix-metalloprotienase 9) inhibitors.
  • MMP-2 matrix-metalloprotienase 2 inhibitors
  • MMP-2 and MMP-9 inhibitors are those that have little or no activity inhibiting MMP-1. More preferred, are those that selectively inhibit MMP-2 and/or MMP-9 relative to the other matrix-metalloproteinases (i.e. MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13) .
  • MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13 matrix-metalloproteinases
  • the anti-cancer agent used in combination with the SHP2 inhibitor is a tumor cell pro-apoptotic or apoptosis-stimulating agent.
  • the anti-cancer agent used in combination with the SHP2 inhibitor is a signal transduction inhibitor.
  • Signal transduction inhibitors include, for example: erbB2 receptor inhibitors, such as organic molecules, or antibodies that bind to the erbB2 receptor, for example, trastuzumab (e.g. ) ; inhibitors of other protein tyrosine-kinases, e.g. imatinib (e.g. ) ; ras inhibitors; raf inhibitors; MEK inhibitors; mTOR inhibitors; cyclin dependent kinase inhibitors; protein kinase C inhibitors; and PDK-1 inhibitors (see Dancey, J. and Sausville, E. A. (2003) Nature Rev.
  • Drug Discovery 2 92-313, for a description of several examples of such inhibitors, and their use in clinical trials for the treatment of cancer) ; GW-282974 (Glaxo Wellcome plc) ; monoclonal antibodies such as AR-209 (Aronex Pharmaceuticals Inc. of The Woodlands, Tex., USA) and 2B-1 (Chiron) ; and erbB2 inhibitors such as those described in International Publication Nos. WO 98/02434, WO 99/35146, WO 99/35132, WO 98/02437, WO 97/13760, and WO 95/19970, and U.S. Pat. Nos. 5,587,458, 5,877,305, 6,465,449 and 6,541,481.
  • the anti-cancer agent used in combination with the SHP2 inhibitor is a cancer immunotherapy agent, such as an antibody specifically binding to an immune checkpoint.
  • Immune checkpoints include, for example: A2AR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, BTLA, CD48, CD160, CD244, CTLA-4, ICOS, LAG-3, LILRB1, LILRB2, LILRB4, OX40, PD-1, PD-L1, PD-L2, SIRPalpha (CD47) , TIGIT, TIM-3, TIM-1, TIM-4, and VISTA.
  • the anti-cancer agent used in combination with the SHP2 inhibitor is a PD-1/PD-L1 antagonist.
  • the anti-cancer agent used in combination with the SHP2 inhibitor is an anti-proliferative agent.
  • Anti-proliferative agents include, for example: Inhibitors of the enzyme farnesyl protein transferase and inhibitors of the receptor tyrosine kinase PDGFR, including the compounds disclosed and claimed in U.S. Pat. Nos. 6,080,769, 6,194,438, 6,258,824, 6,586,447, 6,071,935, 6,495,564, 6,150,377, 6,596,735 and 6,479,513, and International Patent Publication WO 01/40217.
  • the SHP2 inhibitor and/or the second anti-cancer agent is administered at a reduced amount relative to the amount otherwise required to be therapeutically effective for single agent treatment.
  • the combination of the SHP2 inhibitor and the second anti-cancer agent can provide a significantly higher therapeutic effect than otherwise achievable for single agent treatment.
  • Step 1 4-cyanopiperidine-1-carboxylic acid tert-butyl ester (1.05g, 5mmol) and THF (20mL) were successively added to a dry 100mL flask. Under the protection of nitrogen, the mixture was cooled to -78 °C, and then 2M of LDA (3.3mL, 6.5mmol) was slowly added to the reaction mixture. The reaction mixture was allowed to react for 1 hour, and then 3-bromo-2- (bromomethyl) pyridine (1.24g, 5mmol) was added thereto, and then the reaction mixture was allowed to continue to react for 2 hours.
  • LDA 3.3mL, 6.5mmol
  • Step 3 5-oxo-5, 7-dihydrospiro [cyclopenta [b] pyridine-6, 4'-piperidine] -1'-carboxylic acid tert-butyl ester (A2-2, 0.302g, 1mmol) , tetraethyl titanate (1.37g, 6mmol) and (R) - (+) -tert-butylsulfinamide (0.480g, 4mmol) were successively added to a dry 100mL single-necked flask and the mixture was stirred under heating and reflux for 15 hours.
  • Step 4 (R, Z) -5- ( (tert-butylsulfinyl) imino) -5, 7-dihydrospiro [cyclopenta [b] pyridine-6, 4'-piperidine] -1'-carboxylic acid tert-butyl ester (A2-3, 0.60g, 1.48mmol) and ethanol (20mL) were successively added to a dry 100mL single-necked flask, then sodium borodeuteride (0.186g, 4.44mmol) was added. The resulting mixture was stirred for 2 hours at room temperature. TLC and LCMS indicated that the reaction was completed. Acetic acid (1mL) was slowly added to quench the reaction.
  • Step 5 To a solution of (S) -5- ( (R) -tert-butylsulfonamido) -dihydrospiro [cyclopenta [b] pyridine-6, 4'-piperidine] -1'-carboxylic acid tert-butyl ester -5-D (A2-4, 0.816g, 2.0mmol) in dichloromethane (10mL) was added trifluoroacetic acid (3 mL) . The resulting mixture was stirred at room temperature for 2 hours. TLC and LCMS indicated that the reaction was completed.
  • Step 1 To a solution of 5-chloro-8-iodoimidazo [1, 2-c] pyrimidine (B1, 0.359g, 1.29 mmol) and (R) -N- ( (S) -5, 7-dihydrospiro [cyclopenta [b] pyridine-6, 4’ -piperidine] -5-yl-5-D) -2-methylpropane-2-sulfinamide (A2 0.400g, 1.29mmol) in acetonitrile (10mL) was added N, N-Diisopropylethylamine DIPEA (0.500g, 3.87mmol) . The mixture was stirred for 3 hours at 70 °C.
  • Step 2 A mixture of Pd 2 (dba) 3 (50 mg, 0.05 mmol) , DIPEA (140 mg, 1.09 mmol) , Xantphos (63 mg, 0.11 mmol) , B2 (300mg, 0.54mmol) , sodium 2-amino-3-chloropyridine-4-mercaptan (C1, 110 mg, 0.60 mmol) and 1, 4-dioxane (10mL) was stirred at 105°C for 12 hours under nitrogen atmosphere. To the reaction mixture was added ethyl acetate (100mL) and saturated brine and then the resulted mixture was stirred at room temperature for 10 minutes. The organic layer was separated.
  • Step 3 To a solution of (R) -N- ( (S) -1'- (8- ( (2-amino-3-chloropyridin-4-yl) thio) imidazolo [1, 2-c] pyrimidin-5-yl) -5, 7-dihydrospiro [cyclopenta [b] pyridin-6, 4'-piperidin] -5-yl- 5-D) -2-methylpropane-2-sulfinamide (B3, 150mg, 0.26mmol) in methanol (8mL) was added Hydrochloric acid (1, 4-dioxane solution, 4M, 5mL) . The mixture was stirred for 2 hours. TLC and LCMS indicated that the reaction was completed.
  • This example illustrates the activity of exemplary compounds in EGFR exon 20ins NSCLC patient-derived organoids.
  • TAK-788 is a small-molecule tyrosine kinase inhibitor (TKI) designed to selectively target EGFR and human EGFR 2 (HER2) exon 20 insertion mutations.
  • TKI tyrosine kinase inhibitor
  • Osimertinib is a third generation irreversible and mutant-selective EGFR inhibitor.
  • Compound A, B and C surprisingly exhibited potent anti-proliferative activity in five EGFR exon 20ins NSCLC PDOs, among which three were also tested with Osimertinib, and exhibited apparent drug-resistance.
  • Compound A surprisingly showed competitive EC 50 and even better Top Inhibition Rate than TAK-788, which directly binds to and inhibits the exon 20ins-mutated EGFR.
  • Compounds A, B and C are also tested in comparison with additional agents reported to be effective to treat tumors bearing EGFR exon 20 insertions, including, for example, Gefitinib, Erlotinib, Afatinib, Dacomitinib, JNJ-61186372, Poziotinib, Almonertinib (HS-10296) , Amivantamab (JNJ-372) , furmonertinib (AST2818) , and DZD9008. Compounds A, B and C show better efficacy than these compounds in inhibiting tumor cells bearing EGFR exon 20 insertions.
  • This example illustrates the activity of exemplary compounds in ex vivo cultured EGFR exon 20ins PDX-derived primary cells.
  • Compound A potently suppressed proliferation of LU0387 cells, with comparable activity to TAK-788, and was much more potent than Osimertinib and Cisplatin in head-to-head comparisons.
  • This example illustrates the combinational effect of Compound A and TAK-788 in ex vivo cultured EGFR exon 20ins PDX-derived primary cells.
  • Combination effect was tested between Compound A and TAK-788 using a dose matrix assay in LU0387 EGFR exon 20ins-bearing PDX ex vivo cell culture.
  • Cell suspensions of the ex vivo cultured LU0387 were seeded into 96-well plates and incubated for 24 h, then treated with increasing concentrations of each drug to generate a 6 ⁇ 6 dose matrix. After incubation for 7 days, cell viability was assessed by CellTiter-Glo assay kit (Promega) according to the manufacturer’s instructions.
  • Compound A exhibits synergistic effect with TAK-788 at inhibiting proliferation of EGFR exon 20ins-bearing LU0387 primary cells in 2D ex vivo culture system.
  • This example illustrates the in vivo efficacy of exemplary compounds in EGFR exon 20ins NSCLC PDX model.
  • mice were subcutaneously transplanted with tumor fragments (2–3 mm 3 ) derived from an EGFR exon 20ins-bearing NSCLC PDX, LU0387 (H773_V774insNPH) .
  • the mice were randomized into sub-groups when tumor volume reached to 100 ⁇ 200 mm 3 , followed by oral administration (QD for 28days) of Compound A, TAK-788 or combination of Compound A and TAK-788, respectively.
  • Tumor volumes and body weights were measured twice a week through the treatment and continued for additional 19 days after drug withdrawal.
  • Compound A demonstrated strong in vivo anti-tumor activity in a dose-dependent manner in LU0387 PDX model (See FIG. 3) .
  • the head-to-head comparison study suggested Compound A had superior activity to TAK-788, as Compound A at 5 mg/kg exhibited comparable activity with TAK-788 at 10 mg/kg, and Compound A at 15 mg/kg induced significant tumor regression.
  • Compound A showed more sustainable tumor suppression after drug withdrawal.
  • Compound A at 5 mg/kg exhibited profound synergistic effect with TAK-788 at 10 mg/kg, as significant and persistent tumor regressions were observed. All of the treatment were tolerated in this study.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Hematology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present provides a method for treating a subject having an EGFR-related disease or condition, wherein an insertion mutation within exon 20 of the EGFR gene is detected in a biological sample of the subject. In some embodiments, the method comprises administering to the subject a therapeutic effective amount of a pharmaceutical composition comprising a SHP2 inhibitor.

Description

METHODS FOR TREATING CANCERS ASSOCIATED WITH EGFR MUTATION FIELD OF THE INVENTION
The present invention generally relates to the diagnosis and treatment of EGFR-related disease. In particular, the present invention relates to the methods for treating cancer patients with an insertion mutation at exon 20 of the EGFR gene using SHP2 inhibitors.
BACKGROUND
The epidermal growth factor receptor (EGFR) , also named ErbB-1 and Her1, is a receptor tyrosine kinase belonging to the ErbB receptor family. EGFR is activated by binding to its ligands in the epidermal growth factor family, including epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) . Upon activated by its growth factor ligands, EGFR undergoes a transition from an inactive monomeric form to an active homodimer form or to an active heterodimer form in which EGFR pairs with another member of the ErbB receptor family, such as ErbB-2/Her2/neu. EGFR dimerization stimulates EGFR’s intrinsic intracellular protein-tyrosine kinase activity, resulting in the autophosphorylation of several tyrosine (Y) residues in the C-terminal domain of EGFR, including Y992, Y1045, Y1068 and Y1173. This autophosphorylation elicits downstream activation of several proteins with phosphotyrosine-binding SH2 domain. These downstream signaling proteins initiate several signal transduction cascades, principally the MAPK, Akt and JNK pathways, leading to DNA synthesis and the modulation of cell phenotypes such as cell migration, adhesion and proliferation.
Mutations of EGFR gene have been associated with a number of cancers, including lung adenocarcinoma, anal cancers, glioblastoma, and epithelial tumors of the head and neck. These mutations typically lead to the overexpression or constant activation of EGFR, which results in uncontrolled cell proliferation. The identification of EGFR as an oncogene has led to the development of anticancer therapeutics that inhibit EGFR (EGFRi, including tyrosine kinase inhibitors (TKIs) and monoclonal antibodies) , such as gefitinib, erlotinib, afatinib, osimertinib, and icotinib for lung cancer, and cetuximab for colon cancer.
However, many patients treated with EGFRi develop resistance to the treatment. For example, the EGFR T790M mutation accounts for over 50%of acquired resistance to 1 st and 2 nd generation EGFR-TKIs. Further, in frame insertion mutations in EGFR exon 20 demonstrate intrinsic resistance to clinically achievable doses of current EGFR TKIs, including third-generation inhibitors. Therefore, there is an urgent need to develop new methods that can be used to treat EGFR-driven drug-resistant cancers.
SUMMARY
In one aspect, the present disclosure provides a method for treating a subject having an EGFR-related disease or condition, wherein an insertion mutation with exon 20 of the EGFR gene is detected in a biological sample of the subject. In some embodiments, the method comprises administering to the subject a therapeutic effective amount of an SHP2 inhibitor.
In another aspect, the present disclosure provides a method for identifying a subject with an EGFR-related disease or condition who is likely to be responsive to treatment with an SHP2 inhibitor, said method comprising (a) providing a biological sample of the subject; (b) detecting presence or absence of an insertion mutation at exon 20 of the EGFR gene in the biological sample; and (c) identifying the subject as being likely to be responsive to treatment with the SHP2 inhibitor if the insertion mutation at exon 20 of the EGFR gene is detected in the biological sample.
In certain embodiments, the insertion mutation of the EGFR gene is an in-frame insertion mutation. In certain embodiments, the insertion mutation of the EGFR gene is selected from the group consisting of A763_Y764insFQEA, A763_Y764insFQQA, A767_V769dupASV, V769_D770insASV, D770_N771insGL, D770_N771insGT, D770_N771insNPG, D770_N771insSVD, E762Q_insFQEA, H773_V774insH, H773_V774insH, H773_V774insNPH, M766_A767insAI, M766_A767insASV, N771_H773dupNPH, P772_H773insYNP, P772_V774insPHV, S768_770dupSVD, V769_D770insASV, Y764_V765insHH, and delD770insGY.
In certain embodiments, the insertion mutation is detected using a sequencing assay, an amplification assay, or a hybridization assay. In certain embodiments, the insertion mutation is detected using high-throughput sequencing (or next generation sequencing, NGS) method.
In certain embodiments, the EGFR-related disease or condition is cancer. In certain embodiments, the cancer is selected from the groups consisting of lung cancer (e.g., non-small cell lung cancer (NSCLC) ) , glioblastoma, conventional glioblastoma multiforme, uterus cancer (e.g., infiltrating renal pelvis and ureter urothelial carcinoma, endometrial endometrioid adenocarcinoma) , bladder cancer (e.g., bladder urothelial carcinoma) , melanoma, renal cancer, liver cancer, myeloma, prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, thyroid cancer, hematological cancer, leukemia and non-Hodgkin’s lymphoma. In certain embodiments, the cancer is resistant to an EGFR tyrosine kinase inhibitor. In certain embodiments, the cancer is metastatic.
In certain embodiments, the biological sample is tissue or blood. In certain embodiments, the biological sample comprises a cancer cell or DNA from a cancer cell.
In certain embodiments, a second mutation in a gene in the RTK/RAS/MAPK signaling pathway is further detected in the biological sample of the subject. In certain embodiments, the gene is EGFR, KRAS or BRAF.
In certain embodiments, the method further comprises administering to the subject a second therapeutic agent. In certain embodiments, the second therapeutic agent is an EGFR inhibitor, a KRAS inhibitor, a BRAF inhibitor, a MEK inhibitor or a CDK inhibitor. In certain embodiments, the EGFR inhibitor is Gefitinib, Erlotinib, Afatinib, Dacomitinib, Osimertinib, TAK-788, Poziotinib, JNJ-61186372, Almonertinib (HS-10296) , Amivantamab (JNJ-372) , furmonertinib (AST2818) , or DZD9008. In certain embodiments, the KRAS inhibitor is AMG510, or MRTX849. In certain embodiments, the BRAF inhibitor is Vemurafenib, Dabrafenib, Encorafenib or Sorafenib. In certain embodiments, the MEK inhibitor is Trametinib, Cobimetinib, Selumetinib, Pimasertib or Binimetinib. In certain embodiments, the CDK inhibitor is Palbociclib, Ribociclib or Abemaciclib. In certain embodiments, the second therapeutic agent is an immune checkpoint inhibitor, such as PD-1/PD-L1 antagonist.
DESCRIPTION OF DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1 shows the effect of test compounds on proliferation of ex vivo cultured LU0387 cells.
FIG. 2 shows the combinational effect of Compound A and TAK-788 on proliferation of ex vivo cultured LU0387 cells.
FIG. 3 shows the antitumor activity of test compounds in LU0387 PDX model.
DETAILED DESCRIPTION OF THE INVENTION
Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for  the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.
Definitions
The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art, notations and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the biological and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over the definition of the term as generally understood in the art.
As used herein, the singular forms “a” , “an” and “the” include plural references unless the context clearly dictates otherwise.
As used herein, the term “administering” means taking, providing or delivering a compound or composition to a desired site for biological action. These methods for administering include but are not limited to oral route, transduodenal route, parenteral  injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intraarterial injection or infusion) , local administration, and transrectal administration. One skilled in the art is familiar with the administration techniques that can be used for the compounds and methods as described herein, such as those discussed in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington’s, Pharmaceutical Sciences (current edition) , Mack Publishing Co., Easton, Pa. In a preferred embodiment, the compounds and compositions discussed herein are administered orally.
The term “biological sample” means a sample obtained from a biological source. Examples of biological sample include, without limitation, bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal flushing fluids, plural fluid, ascitic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchioalveolar lavage fluid, etc., and tissues, such as biopsy tissue (e.g. biopsied bone tissue, bone marrow, breast tissue, gastrointestinal tract tissue, lung tissue, liver tissue, prostate tissue, brain tissue, nerve tissue, meningeal tissue, renal tissue, endometrial tissue, cervical dittuse, lymph node tissue, muscle tissue, or skin tissue) , a paraffin embedded tissue. In certain embodiments, the biological sample can comprise cancer cells. In some embodiments, the biological sample is a fresh or archived sample obtained from a tumor, e.g., by a tumor biopsy or fine needle aspirate. The biological sample also can be any biological fluid containing cancer cells. The collection of a biological sample from a subject is performed in accordance with the standard protocol generally followed by hospital or clinics, such as during a biopsy.
As used herein, the term “cancer” refers to any diseases involving an abnormal cell growth and includes all stages and all forms of the disease that affects any tissue, organ or cell in the body. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and cancers of all stages and grades including pre-and post-metastatic cancers. In general, cancers can be categorized according to the tissue or organ from which the cancer is located or originated and morphology of cancerous tissues and cells. As used herein, cancer types include, acute lymphoblastic leukemia (ALL) , acute myeloid leukemia, adrenocortical carcinoma, anal cancer, astrocytoma, childhood cerebellar or cerebral, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumor, brain cancer, breast cancer, Burkitt's lymphoma, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, colon cancer, emphysema, endometrial cancer, ependymoma, esophageal cancer, Ewing family of tumors, Ewing's sarcoma, gastric (stomach) cancer, glioma, head and neck cancer, heart cancer, Hodgkin lymphoma, islet cell carcinoma  (endocrine pancreas) , Kaposi sarcoma, kidney cancer (renal cell cancer) , laryngeal cancer, leukemia, liver cancer, lung cancer, medulloblastoma, melanoma, neuroblastoma, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer) , retinoblastoma, , skin cancer, stomach cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thyroid cancer, vaginal cancer, visual pathway and hypothalamic glioma.
The term “complementarity” refers to the ability of a nucleic acid to form hydrogen bond (s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100%complementary) .
It is noted that in this disclosure, terms such as “comprises” , “comprised” , “comprising” , “contains” , “containing” and the like have the meaning attributed in United States Patent law; they are inclusive or open-ended and do not exclude additional, un-recited elements or method steps.
The terms “detecting, ” “determining, ” “assessing, ” and “measuring” can be used interchangeably and refer to both quantitative and semi-quantitative determinations. Where either a quantitative and semi-quantitative determination is intended, the phrase “determining a level” of a polynucleotide or polypeptide of interest or “detecting” a polynucleotide or polypeptide of interest can be used.
The term “hybridizing” refers to the binding, duplexing, or hybridizing of a nucleic acid molecule preferentially to a particular nucleotide sequence under stringent conditions. The term “stringent conditions” refers to hybridization and wash conditions under which a probe will hybridize preferentially to its target subsequence, and to a lesser extent to, or not at all to, other sequences in a mixed population (e.g., a cell lysate or DNA preparation from a tissue biopsy) . A stringent condition in the context of nucleic acid hybridization are sequence dependent, and are different under different environmental parameters. An extensive guide to the hybridization of nucleic acids is found in, e.g., Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes part I, Ch. 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays, ” (1993) Elsevier, N.Y. Generally, highly stringent hybridization and wash conditions are selected to be about 5℃ lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature  (under defined ionic strength and pH) at which 50%of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on an array or on a filter in a Southern or Northern blot is 42℃. using standard hybridization solutions (see, e.g., Sambrook and Russell Molecular Cloning: A Laboratory Manual (3rd ed. ) Vol. 1-3 (2001) Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY) . An example of highly stringent wash conditions is 0.15 M NaCl at 72℃ for about 15 minutes. An example of stringent wash conditions is a 0.2×SSC wash at 65℃ for 15 minutes. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is l×SSC at 45℃ for 15 minutes. An example of a low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4×SSC to 6×SSC at 40℃ for 15 minutes.
The term “nucleic acid” and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function, known or unknown. Non-limiting examples of polynucleotides include a gene, a gene fragment, exons, introns, messenger RNA (mRNA) , transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence, nucleic acid probes, and primers. The nucleic acid molecule may be linear or circular.
In the disclosure, “pharmaceutical composition” refers to a formulation of a compound and a medium generally accepted in the art for delivering a biologically active compound to a subject (e.g., human) . The medium includes a pharmaceutically acceptable carrier. The purpose of the pharmaceutical composition is to promote the administration to living organisms, which facilitates the absorption of active ingredients and thus exerts biological activity.
The term “responsive” or “responsiveness” as used in the context of a patient’s response to a cancer therapy, are used interchangeably and refer to a beneficial patient response to a treatment as opposed to unfavorable responses, i.e. adverse events. In a patient, beneficial response can be expressed in terms of a number of clinical parameters, including loss of detectable tumor (complete response) , decrease in tumor size and/or cancer cell  number (partial response) , tumor growth arrest (stable disease) , enhancement of anti-tumor immune response, possibly resulting in regression or rejection of the tumor; relief, to some extent, of one or more symptoms associated with the tumor; increase in the length of survival following treatment; and/or decreased mortality at a given point of time following treatment. Continued increase in tumor size and/or cancer cell number and/or tumor metastasis is indicative of lack of beneficial response to treatment, and therefore decreased responsiveness.
As used herein, “SHP2” refers to “Src homology-2 domain-containing protein tyrosine phosphatase-2” , also called SH-PTP2, SH-PTP3, Syp, PTP1D, PTP2C, SAP-2 or PTPN11.
As used herein, the term “subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate) . A human includes pre and post-natal forms. In many embodiments, a subject is a human being. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. The term “subject” is used herein interchangeably with “individual” or “patient. ” A subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
The term “therapeutically effective amount” “effective amount” or “pharmaceutically effective amount” as used herein refers to the amount of at least one medicament or compound sufficient to relieve one or more symptoms of the disease or disease being treated to a certain extent after administration. The result may be the reduction and/or remission of signs, symptoms or causes, or any other desired changes in the biological system. For example, a “therapeutically effective amount” for treatment is the amount of a composition comprising a compound as disclosed herein required to provide a clinically significant disease relief effect. Techniques such as dose escalation tests may be used to determine the effective amount suitable for any individual case.
The term “treatment” “treat” or “treating” a disease or condition as used herein include the following meanings: (i) prevention of the occurrence of the disease or condition in a subject, especially when the subject is susceptible to the disease or condition but has not been diagnosed with the disease or condition; (ii) suppression of the disease or condition, that is, inhibition of the development of the disease or condition; (iii) alleviation of the disease or condition, that is, abatement of the status of the disease or condition; or (iv) relief of the symptoms caused by the disease or condition. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.
The SHP2 Inhibitor
The methods provided in this disclosure, in one aspect, relate to the discovery that certain SHP2 inhibitors can be used to treat EGFR-related diseases such as cancer, in particular, cancer associated with an insertion mutation at exon 20 of the EGFR gene.
SHP2 (Src homology-2 domain-containing protein tyrosine phosphatase-2) , also named SH-PTP2, SH-PTP3, Syp, PTP1D, PTP2C, SAP-2 or PTPN11 is a tyrosine kinase phosphatase that plays an important role in cell signaling and is a target for the treatment of major diseases such as diabetes, autoimmune diseases and cancers. SHP2 is mutated or highly expressed in various diseases, such as Noonan Syndrome, Leopard Syndrome, juvenile myelomonocytic leukemia, neuroblastoma, melanoma, acute myeloid leukemia, breast cancer, esophageal cancer, lung cancer, colon cancer, head cancer, squamous cell carcinoma of head and neck, gastric cancer, anaplastic large cell lymphoma, and glioblastoma, etc. Molecular biological studies show that SHP2 is involved in multiple tumor cell signaling pathways, such as MAPK, JAK/STAT, and PI3K/Akt, etc. In addition, SHP2 is also responsible for the signal transduction of PD-1/PD-L1 immunosuppressive pathway. As a result, inhibition of SHP2 activity has been proposed to reverse immunosuppression in tumor microenvironment.
SHP2 consists of two N-terminal Src Homolgy-2 domains (N-SH2 and C-SH2) and a protein tyrosine phosphatase catalytic domain (PTP) . In the self-inhibiting state, N-SH2 combines with PTP to form a ring structure, which hinders the binding of PTP to substrate, thus inhibiting the enzyme catalytic activity. When the tyrosine of an upstream receptor protein is phosphorylated and binds to N-SH2, PTP catalytic domain is released to exhibit phosphatase activity.
In certain embodiments, the SHP2 inhibitor used in the methods disclosed herein is an allosteric inhibitor in the non-catalytic region, such as the compound disclosed in WO2015107493A1, WO2016203404A1, WO2016203406A1, WO2017216706A1, WO2017211303A1, CN201710062495, WO2018136265A1, and WO2018057884.
In preferred embodiments, the SHP2 inhibitor used in the methods disclosed herein is a compound disclosed in Chinese Patent Application No. 201811314910.1 or WO2020094018, the disclosure of which is incorporated herein via reference.
In certain embodiments, the SHP2 inhibitor is a compound of Formula Ib:
Figure PCTCN2021117497-appb-000001
or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereoisomer, tautomer, solvate, isotope-substituted derivative, polymorph, prodrug or metabolite thereof, wherein,
R 4 is selected from H, halogen, substituted or unsubstituted amino, substituted or unsubstituted C 1-C 6 alkyl, or substituted or unsubstituted C 1-C 6 alkoxyl;
X 4 and X 5 are independently selected from C or N, and X 4 and X 5 are not N simultaneously;
ring D is selected from substituted or unsubstituted 4 to 8-membered heterocyclyl, substituted or unsubstituted 4 to 8-membered carbocyclic group, substituted or unsubstituted C 5-10 aryl, or substituted or unsubstituted 5 to 10-membered heteroaryl; wherein the heterocyclyl or heteroaryl comprises 1-3 heteroatoms selected from: N, O, S or P;
wherein any of the above term “substituted” refers to one or more hydrogen atoms on the group are substituted by a substituent selected from: -D, halogen, -OH, -NO 2, -NH 2, -N (unsubstituted or halogenated C 1-C 6 alkyl)  2, -CN, unsubstituted or halogenated C 1-C8 alkyl, unsubstituted or halogenated C 1-C 8 alkoxyl, unsubstituted or halogenated C 1-C 8 alkoxyl-C 1-C 8 alkyl, unsubstituted or halogenated C 3-C 8 cycloalkyl, unsubstituted or halogenated C 3-C 8 cycloalkyl-C 1-C 8 alkyl, unsubstituted or halogenated C 1-C 6 alkyl carbonyl, unsubstituted or halogenated C 1-C 6 alkoxyl carbonyl, hydroxamic acid group, unsubstituted or halogenated C 1-C 6 alkyl thiol, -S (O)  2N (unsubstituted or halogenated C 1-C 6 alkyl)  2, -S (O)  2 unsubstituted or halogenated C 1-C 6 alkyl, -N (unsubstituted or halogenated C 1-C 6 alkyl) S (O)  2N (unsubstituted or halogenated C 1-C 6 alkyl)  2, -S (O) N (unsubstituted or halogenated C 1-C 6 alkyl)  2, -S (O) (unsubstituted or halogenated C1-C6 alkyl) , -N (unsubstituted or halogenated C 1-C 6 alkyl) S (O) N (unsubstituted or halogenated C 1-C 6 alkyl)  2, -N (unsubstituted or halogenated C 1-C 6 alkyl) S (O) (unsubstituted or halogenated C 1-C 6 alkyl) , unsubstituted or halogenated 5 to 8-membered aryl, unsubstituted or halogenated 5 to 8-membered heteroaryl, or unsubstituted or halogenated 4 to 8-membered saturated heterocyclyl or carbocyclic group; wherein the heteroaryl comprise 1-4 heteroatoms selected from: N, O or S, the heterocyclyl comprise 1-4 heteroatoms selected from: N, O or S.
In certain embodiments, the SHP2 inhibitor is a compound of Formula I:
Figure PCTCN2021117497-appb-000002
or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereoisomer, tautomer, solvate, isotope-substituted derivative, polymorph, prodrug or metabolite thereof, wherein:
X 1 and X 2 are independently selected from a bond, O, CR aR b or NR c;
X 3 is selected from a bond, CR aR b, NR c, S or O;
X 4 is selected from N or CR c; and R a, R b and R c are independently selected from H, D, halogen, substituted or unsubstituted C 1-6 alkyl, or substituted or unsubstituted C 1-6 alkoxyl;
R 1, R 2, R 3, R 4 and R 7 are independently selected from H, D, -OH, halogen, substituted or unsubstituted amino, substituted or unsubstituted C 1-6 alkyl, or substituted or unsubstituted C 1-6 alkoxyl; and R 1, R 2, R 3, R 4 and R 7 cannot be -OH or -NH 2 simultaneously;
ring A is selected from substituted or unsubstituted C 4-8 cyclic hydrocarbyl, substituted or unsubstituted 4 to 8-membered heterocyclyl, substituted or unsubstituted C 5-10 aryl, or substituted or unsubstituted 5 to 10-membered heteroaryl, wherein the heterocyclyl or heteroaryl comprises 1-3 heteroatoms selected from the following atoms: N, O, S or P;
ring C is selected from substituted or unsubstituted C 4-8 cyclic hydrocarbyl, substituted or unsubstituted 5 to 6-membered monocyclic heterocyclyl, substituted or unsubstituted 8 to 10-membered bicyclic heterocyclyl, substituted or unsubstituted C 5-10 monocyclic or bicyclic aryl, substituted or unsubstituted 5 to 6-membered monocyclic heteroaryl, or substituted or unsubstituted 8 to 10-membered bicyclic heteroaryl, wherein the heterocyclyl or heteroaryl comprises 1-4 heteroatoms selected from the following atoms: N, O, S or P;
R 5 and R 6 are independently selected from H, D, -OH, halogen, cyano, substituted or unsubstituted amino, substituted or unsubstituted C 1-6 alkyl, or substituted or unsubstituted C 1-6 alkoxyl;
n is any integer from 0 to 3; and
the “substituted” refers to one or more hydrogen atoms on the group are substituted by a substituent selected from the following substituents: halogen, -OH, -NO 2, -NH 2, -NH (unsubstituted or halogenated C 1-6 alkyl) , -N (unsubstituted or halogenated C 1-6 alkyl)  2, - CN, unsubstituted or halogenated C 1-8 alkyl, unsubstituted or halogenated C 1-8 alkoxyl, unsubstituted or halogenated C 1-8 alkoxyl-C 1-8 alkyl, unsubstituted or halogenated C 3-8 cycloalkyl-C 1-8 alkyl, unsubstituted or halogenated C 1-6 alkyl carbonyl, unsubstituted or halogenated C 1-6 alkoxyl carbonyl, hydroxamic acid group, unsubstituted or halogenated C 1-6 alkyl thiol, -S (O)  2N (unsubstituted or halogenated C 1-6 alkyl)  2, -S (O)  2 unsubstituted or halogenated C 1-6 alkyl, -N (unsubstituted or halogenated C 1-6 alkyl) S (O)  2N (unsubstituted or halogenated C 1-6 alkyl)  2, -S (O) N (unsubstituted or halogenated C 1-6 alkyl)  2, -S (O) (unsubstituted or halogenated C 1-6 alkyl) , -N (unsubstituted or halogenated C 1-6 alkyl) S (O) N (unsubstituted or halogenated C 1-6 alkyl)  2, -N (unsubstituted or halogenated C 1-6 alkyl) S (O) (unsubstituted or halogenated C 1-6 alkyl) , unsubstituted or halogenated C 5-10 aryl, unsubstituted or halogenated 5 to 10-membered heteroaryl, unsubstituted or halogenated C 4-8 cyclic hydrocarbyl, or unsubstituted or halogenated 4 to 8-membered heterocyclyl, wherein the heterocyclyl and heteroaryl comprise 1-4 heteroatoms selected from the following atoms: N, O or S;
wherein one or more hydrogens in the ring A, ring B, ring C, ring D, ring E and/or ring F are optionally substituted by D.
As a preferred embodiment, one of X 1 and X 2 is CH 2, and the other is a bond.
As a preferred embodiment, X 3 is S.
As a preferred embodiment, X 4 is selected from N or CH.
As a preferred embodiment, R 1, R 2, R 3, R 4 and R 7 are independently selected from H, D, -OH, -F, -Cl, -Br, -NH 2, -NHC 1-3 alkyl, methyl, ethyl, propyl, isopropyl, butyl, methoxy, ethoxy, propoxy or isopropoxy; C 1-3 alkyl substituted by halogen, -NH 2, -OH, C 1-3 alkyl or C 1-3 alkoxyl; or C 1-3 alkoxyl substituted by halogen, -NH 2, -OH, C 1-3 alkyl or C 1-3 alkoxyl.
As a preferred embodiment, R 5 and R 6 are independently selected from H, -OH, -F, -Cl, -Br, -CN, -NH 2, -NHC 1-3 alkyl, methyl, ethyl, propyl, isopropyl, butyl, methoxy, ethoxy, propoxy or isopropoxy; C 1-3 alkyl substituted by halogen, -NH 2, -OH, C 1-3 alkyl or C 1-3 alkoxyl; or C 1-3 alkoxyl substituted by halogen, -NH 2, -OH, C 1-3 alkyl or C 1-3 alkoxyl.
As a preferred embodiment, the substituent is selected from –F, -Cl, -Br, -OH, -NO 2, -NH 2, -NH (C 1-6 alkyl) , -N (C 1-6 alkyl)  2, -CN, C 1-6 alkyl, C 1-4 alkoxyl, C 1-4 alkoxyl-C 1-6 alkyl, C 3-8 cycloalkyl-C 1-8 alkyl, C 1-6 alkyl carbonyl, C 1-6 alkoxyl carbonyl, C 1-6 alkyl thiol, -S (O)  2N (C 1-6 alkyl)  2, -S (O)  2 C 1-6 alkyl, -N (C 1-6 alkyl) S (O)  2N (C 1-6 alkyl)  2, -S (O) N (C 1-6 alkyl)  2, -S (O) (C 1-6 alkyl) , -N (C 1-6 alkyl) S (O) N (C 1-6 alkyl)  2, -N (C 1-6 alkyl) S (O) (C 1-6 alkyl) , substituted or unsubstituted C 5-10 aryl, substituted or unsubstituted 5 to 10-membered heteroaryl,  substituted or unsubstituted C 4-8 cyclic hydrocarbyl, or substituted or unsubstituted 4 to 8-membered heterocyclyl, wherein the heterocyclyl and heteroaryl comprise 1-4 heteroatoms selected from the following atoms: N, O or S.
As a preferred embodiment, the substituent is selected from –F, -Cl, -Br, -OH, -NO 2, -NH 2, -NH (C 1-3 alkyl) , -N (C 1-3 alkyl)  2, -CN, C 1-3 alkyl, C 1-3 alkoxyl, C 1-3 alkyl carbonyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclooctyl, pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, thiomorpholinyl, phenyl, naphthyl, anthracyl, phenanthryl, fluorenyl, thiophenyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, benzimidazolyl, benzopyrazolyl, indolyl, furanyl, pyrrolyl, triazolyl, tetrazolyl, triazinyl, indolizinyl, isoindolyl, indazolyl, isoindazolyl, purinyl, quinolinyl or isoquinolinyl.
As a preferred embodiment, the substituent is selected from –F, -Cl, -Br, -OH, -NO 2, -NH 2, -NH (C 1-3 alkyl) , -N (C 1-3 alkyl)  2, -CN, methyl, ethyl, propyl, isopropyl, butyl, methoxy, ethoxy, propoxy, isopropoxy or phenyl.
As a preferred embodiment, one or more of the hydrogens are substituted by D in the position selected from: H on a ring atom other than a heteroatom on ring A, H on an amino-substituted ring atom on ring B, H on a ring atom other than a heteroatom on ring C, H on position 7 of ring E, and H on  position  2 or 3 of ring F. Preferably, the number of D substitution is 1-4.
As a preferred embodiment, the ring C is selected from any of the following groups:
Figure PCTCN2021117497-appb-000003
wherein:
X 5, X 6, X 7, X 8 and X 9 are independently selected from N or CR d; and at most 3 of X 5, X 6, X 7, X 8 and X 9 are N simultaneously;
X 10, X 11, X 12, X 13, X 14, X 15, X 16 and X 17 are independently selected from N or CR d; and at most 5 of X 10, X 11, X 12, X 13, X 14, X 15, X 16 and X 17 are N simultaneously;
X 18, X 19, X 20 and X 21 are independently selected from N or CR d, and at most 3 of X 18, X 19, X 20 and X 21 are N simultaneously;
R 6 and R 8 are independently selected from H, -NH 2, -CN, -OH, -NO 2, halogen, unsubstituted or halogenated C 1-6 alkyl, or unsubstituted or halogenated C 1-6 alkoxyl; and
R d is selected from H, halogen, unsubstituted or halogenated C 1-6 alkyl, or unsubstituted or halogenated C 1-6 alkoxyl.
As a preferred embodiment, the ring C is selected from any of the following groups:
Figure PCTCN2021117497-appb-000004
wherein:
0, 1 or 2 of X 5, X 6, X 7, X 8 and X 9 are N, the others are CR d;
0, 1 or 2 of X 18, X 19, X 20 and X 21 are N, the others are CR d;
R 6 is selected from H, -NH 2, -CN, -OH, -NO 2, -F, -Cl, -Br, methyl, ethyl, propyl, isopropyl, butyl, methoxy, ethoxy, propoxy, isopropoxy, fluorinated or brominated C 1-3 alkyl, fluorinated or brominated C 1-3 alkoxyl; and
R d is selected from H, -F, -Cl, -Br, methyl, ethyl, propyl, isopropyl, butyl, methoxy, ethoxy, propoxy, isopropoxy, fluorinated or brominated C 1-3 alkyl, or fluorinated or brominated C 1-3 alkoxyl.
As a preferred embodiment, the ring C is selected from any of the following groups:
Figure PCTCN2021117497-appb-000005
optionally, the H on position 5 and/or 6 of the pyridine ring is substituted by D, one or more of the H on  position  4, 5 and 6 of the benzene ring is substituted by D.
As a preferred embodiment, the ring A is selected from substituted or unsubstituted C 4-6 cyclic hydrocarbyl, substituted or unsubstituted 4 to 6-membered heterocyclyl, substituted or unsubstituted C 5-6 aryl, or substituted or unsubstituted 5 to 6-membered heteroaryl, wherein the heterocyclyl or heteroaryl comprises 1-3 N atoms.
As a preferred embodiment, the ring A is selected from any of the following groups:
Figure PCTCN2021117497-appb-000006
As a preferred embodiment, the ring A is selected from any of the following groups:
Figure PCTCN2021117497-appb-000007
As a preferred embodiment, one or more H on the ring atoms other than the heteroatom or F-substituted atom are substituted by D.
In the disclosure, the term “halogen” refers to fluorine, chlorine, bromine or iodien.
“Hydroxyl” refers to -OH group.
“Hydroxyl alkyl” refers to an alkyl as defined below substituted by hydroxyl (-OH) .
“Carbonyl” refers to -C (=O) -group.
“Nitryl” refers to -NO 2.
“Cyano” refers to -CN.
“Amino” refers to -NH 2.
“Substituted amino” refers to an amino substituted by one or two of the alkyl, alkyl carbonyl, aryl alkyl, heteroaryl alkyl as defined below, for example, substituted amino may be monoalkyl amino, dialkyl amino, alkyl acylamino, aryl alkyl amino, heteroaryl alkyl.
“Carboxyl” refers to -COOH.
In the disclosure, as a group or part of other groups (for example, used in groups such as halogenated (such as fluorinated, chlorinated, brominated or iodinated) alkyl) , the term “alkyl” refers to a fully saturated straight or branched hydrocarbon chain group, consisting only of carbon atoms and hydrogen atoms, for example, comprising 1 to 12 (preferably 1 to 8, more preferably 1 to 6) carbon atoms, and connected to the rest of the molecule by a single bond. For example, “alkyl” includes, but is not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-amyl, 2-methylbutyl, 2, 2-dimethylpropyl, n-hexyl, heptyl, 2-methylhexyl, 3-methylhexyl, octyl, nonyl and decyl, etc.  In the case of the present disclosure, the term “alkyl” refers to an alkyl group containing 1 to 8 carbon atoms.
In the disclosure, as a group or part of other groups, the term “alkenyl” refers to a straight or branched hydrocarbon chain group, consisting only of carbon atoms and hydrogen atoms, for example, comprising 2 to 20 (preferably 2 to 10, more preferably 2 to 6) carbon atoms, comprising at least one double bond, and connected to the rest of the molecule by a single bond, Examples, include but are not limited to, vinyl, propenyl, allyl, but-1-enyl, but-2-enyl, pent-1-enyl, pent-1, 4-dienyl, etc.
In the disclosure, as a group or part of other groups, the term “cyclic hydrocarbyl” refers to a stable non-aromatic monocyclic or polycyclic hydrocarbyl (such as alkyl, alkenyl or alkynyl) consisting only of carbon atoms and hydrogen atoms, which may comprise fused ring system, bridged ring system or spiro ring system, comprise 3 to 15 carbon atoms, preferably comprise 3 to 10 carbon atoms, more preferably comprise 3 to 8 carbon atoms, for example, comprise 3, 4, 5, 6, 7 or 8 carbon atoms, and which is saturated or unsaturated and may be connected to the rest of the molecule via any suitable carbon atom by a single bond. Unless otherwise specifically indicated in the description, the carbon atoms in the cyclic hydrocarbyl may be optionally oxidized. Embodiments of cyclic hydrocarbyl include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cyclooctyl, 1H-indenyl, 2, 3-dihydroindenyl, 1, 2, 3, 4-tetrahydro-naphthyl, 5, 6, 7, 8-tetrahydro-naphthyl, 8, 9-dihydro-7H-benzocycloheptene-6-yl, 6, 7, 8, 9-tetrahydro-5H-benzocycloheptenyl, 5, 6, 7, 8, 9, 10-hexahydro-benzocyclooctenyl, fluorenyl, bicyclo [2.2.1] heptyl, 7, 7-dimethyl-bicyclo [2.2.1] heptyl, bicyclo [2.2.1] heptenyl, bicyclo [2.2.2] octyl, bicyclo [3.1.1] heptyl, bicyclo [3.2.1] octyl, bicyclo [2.2.2] octenyl, bicyclo [3.2.1] octenyl, adamantyl, octahydro-4, 7-methylene-1H-indenyl and octahydro-2, 5-methylene-dicyclopentadienyl, etc.
In the disclosure, as a group or part of other groups, the term “heterocyclyl” refers to a stable 3 to 20-membered non-aromatic cyclic group consisting of 2 to14 carbon atoms (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 carbon atoms) and 1 to 6 heteroatoms selected from nitrogen, phosphorus, oxygen or sulfur. Unless otherwise specifically indicated in the description, a heterocyclyl may be a monocyclic ring system, a dicyclic ring system, a tricyclic ring system or a ring system with more rings, and may comprise fused ring system, bridged ring system or spiro ring system; the nitrogen, phosphorus or sulfur atoms in the heterocyclyl may be optionally oxidized; the nitrogen atoms in the heterocyclyl may be optionally quaternized; and the heterocyclyl may be  partially or fully saturated. The heterocyclyl may be connected to the rest of the molecule via a carbon atom or a heteroatom by a single bond. In a heterocyclyl containing fused ring, one or more rings may be aryl or heteroaryl as defined below, provided that the connection point between the group and the rest of the molecule is a non-aromatic ring atom. For the purpose of the present disclosure, heterocyclyl is preferably a stable 4 to 11-membered non-aromatic monocyclic, dicyclic, bridged or spiro ring group comprising 1 to 3 heteroatoms selected from nitrogen, oxygen or sulfur, more preferably a stable 4 to 8-membered non-aromatic monocyclic, dicyclic, bridged or spiro ring group comprising 1 to 3 heteroatoms selected from nitrogen, oxygen or sulfur. Exemplary heterocyclyls include but are not limited to: pyrrolidinyl, morpholinyl, piperazinyl, homopiperazinyl, piperidinyl, thiomorpholinyl, 2, 7-diaza-spiro [3.5] nonane-7-yl, 2-oxa-6-aza-spiro [3.3] heptane-6-yl, 2, 5-diaza-bicyclo [2.2.1] heptane-2-yl, azacyclobutanyl, pyranyl, tetrahydropyranyl, thiopyranyl, tetrahydrofuranyl, oxazinyl, dioxolanyl, tetrahydroisoquinolinyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, quinolizinyl, thiazolidinyl, isothiazolidinyl, isoxazolidinyl, dihydroindolyl, octahydroindolyl, octahydroisoindolyl, pyrrolidinyl, pyrazolidinyl, phthalimido, etc.
In the disclosure, as a group or part of other groups, the term “aryl” refers to a conjugated hydrocarbon ring system group comprising 6 to 18 carbon atoms (preferably comprising 6 to 10 carbon atoms, for example, 6, 7, 8, 9 or 10 carbon atoms) . For the purpose of the present disclosure, aryl may be a monocyclic ring system, a dicyclic ring system, a tricyclic ring system or a ring system with more rings, and may be fused with the cyclic hydrocarbyl or heterocyclyl as defined above, provided that the aryl and the rest of the molecule are connected via an atom on the aromatic ring by a single bond. Exemplary aryls include but are not limited to phenyl, naphthyl, anthracyl, phenanthrenyl, fluorenyl, 2, 3-dihydro-1H-isoindolyl, 2-benzoxazolinone, 2H-1, 4-benzoxazin-3 (4H) -one-7-yl, etc.
In the disclosure, the term “aryl alkyl” refers to an alkyl as defined above which is substituted by an aryl as defined above.
In the disclosure, as a group or part of other groups, the term “heteroaryl” refers to a 5 to 16-membered conjugated ring system group comprising 1 to 15 carbon atoms (preferably comprising 1 to 10 carbon atoms, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms) and 1 to 6 heteroatoms selected from nitrogen, oxygen or sulfur. Unless otherwise specifically indicated in the description, a heteroaryl may be a monocyclic ring system, a dicyclic ring system, a tricyclic ring system or a ring system with more rings, and may be fused with the cycloalkyl or heterocyclyl, provided that the heteroaryl and the rest of the  molecule are connected via an atom on the aromatic ring by a single bond. The nitrogen, carbon or sulfur atoms in the heteroaryl may be optionally oxidized; the nitrogen atoms in the heteroaryl may be optionally quaternized. For the purpose of the disclosure, heteroaryl is preferably a stable 5 to 12-membered aromatic group comprising 1 to 5 heteroatoms selected from nitrogen, oxygen or sulfur, and is more preferably a stable 5 to 10-membered aromatic group comprising 1 to 4 heteroatoms selected from nitrogen, oxygen or sulfur, or a 5 to 6-membered aromatic group comprising 1 to 3 heteroatoms selected from nitrogen, oxygen or sulfur. Exemplary heteroaryls include but are not limited to thiophenyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl, oxadiazolyl, isoxazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, benzimidazolyl, benzopyrazolyl, indolyl, furanyl, pyrrolyl, triazolyl, tetrazolyl, triazinyl, indolizinyl, isoindolyl, indazolyl, isoindazolyl, purinyl, quinolinyl, isoquinolinyl, diazanaphthalenyl, naphthyridinyl, quinoxalinyl, pteridinyl, carbazolyl, carbolinyl, phenanthridinyl, phenanthrolinyl, acridinyl, phenazinyl, isothiazolyl, benzothiazolyl, benzothiophenyl, oxatriazolyl, cinnolinyl, quinazolyl, phenylthio, indolizinyl, phenanthrolinyl, isoxazolyl, phenoxazinyl, phenothiazinyl, 4, 5, 6, 7-tetrahydrobenzo [b] thiophenyl, naphthopyridinyl, [1, 2, 4] triazolo [4, 3-b] pyridazine, [1, 2, 4] triazolo [4, 3-a] pyrazine, [1, 2, 4] triazolo [4, 3-c] pyrimidine, [1, 2, 4] triazolo [4, 3-a] pyridine, imidazo [1, 2-a] pyridine, imidazo [1, 2-b] pyridazine, imidazo [1, 2-a] pyrazine, etc.
In the disclosure, the term “heteroaryl alkyl” refers to an alkyl as defined above which is substituted by heteroaryl as defined above.
In the disclosure, “optional” or “optionally” means that the subsequently described event or condition may or may not occur, and such description includes both occurrence and non-occurrence of the event or condition. For example, “optionally substituted aryl” means an aryl being substituted or not being substituted, and such description includes both substituted aryl and unsubstituted aryl. The “optional” substituent employed in the claims and description of the disclosure is selected from alkyl, alkenyl, alkynyl, halogen, haloalkyl, haloalkenyl, haloalkynyl, cyano, nitro, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cyclic hydrocarbyl, or optional substituted heterocyclyl.
“Stereisomer” refers to a compound composed of the same atoms, bonded by the same bonds, but having different three-dimensional structures. The present disclosure will cover various stereoisomers and mixtures thereof.
When a compound of the present disclosure contains an olefinic double bond, unless specified otherwise, the compound of the present disclosure is intended to include E-and Z-geometric isomers.
“Tautomer” refers to an isomer formed by the transfer of proton from one atom of a molecule to another atom of the same molecule. All tautomeric forms of a compound of the disclosure will also be included within the scope of the disclosure.
The compound of the disclosure may comprise one or more chiral carbon atoms, and thus may produce enantiomers, diastereomers, and other stereoisomeric forms. Each chiral carbon atom can be defined as (R) -or (s) -based on stereochemistry. The disclosure is intended to include all possible isomers, as well as racemes and optically pure forms thereof. A compound of the disclosure can be prepared by using raceme, diastereomer or enantiomer as raw material or intermediate. The optically active isomers can be prepared by using chiral synthons or chiral reagents, or separated by conventional techniques, such as crystallization and chiral chromatography.
Conventional techniques for preparing/separating individual isomers include chiral synthesis from suitable optically pure precursors, or separation of racemes (or racemates of salts or derivatives) using, for example, chiral high performance liquid chromatography, for example, see Gerald Gübitz and Martin G. Schmid (Eds. ) , Chiral Separations, Methods and Protocols, Methods in Molecular Biology, Vol. 243, 2004; A.M. Stalcup, Chiral Separations, Annu. Rev. Anal. Chem. 3: 341-63, 2010; Fumiss et al. (eds. ) , VOGEL’S ENCYCLOPEDIA OF PRACTICAL ORGANIC CHEMISTRY 5. sup. TH ED., Longman Scientific and Technical Ltd., Essex, 1991, 809-816; Heller, Acc. Chem. Res. 1990, 23, 128.
The disclosure also includes all suitable isotopic variations of the compounds of the present disclosure or pharmaceutically acceptable salts thereof. Isotopic variations of the compounds of the present disclosure or pharmaceutically acceptable salts thereof are defined as those in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass often found in nature. Isotopes that can be incorporated into the compounds of the present disclosure and their pharmaceutically acceptable salts thereof include but are not limited to H, C, N and O, for example,  2H,  3H,  11C,  13C,  14C,  15N,  17O,  18O,  35S,  18F,  36Cl and  125I. Suitable isotopic variations of the compounds or pharmaceutically acceptable salts thereof of the present  disclosure may be prepared by conventional techniques using appropriate isotopic variants of suitable reagents.
In preferred embodiments, the compound has a structure selected from:
Figure PCTCN2021117497-appb-000008
Figure PCTCN2021117497-appb-000009
In preferred embodiments, the compound has a structure selected from:
Figure PCTCN2021117497-appb-000010
As a preferred embodiment, the isotopic substitution of the isotope-substituted derivative of the compound herein relates to atom comprises but not limited to hydrogen, carbon, nitrogen, oxygen, fluorine, phosphorus, chlorine or iodine; and preferably is  2H,  3H,  11C,  13C,  14C,  15N,  17O,  18O,  18F,  31P,  32P,  35S,  36Cl or  125I.
In preferred embodiments, the compound has a structure of 
Figure PCTCN2021117497-appb-000011
also named as Compound A.
In preferred embodiments, the compound has a structure of 
Figure PCTCN2021117497-appb-000012
also named as Compound B.
In preferred embodiments, the compound has a structure of 
Figure PCTCN2021117497-appb-000013
also named as Compound C.
In certain embodiments, the SHP2 inhibitor provided herein is capable of inhibiting proliferation of tumor cells bearing one or more EGFR exon 20 insertion mutation at a maximal inhibition percentage significantly higher than that of reference compound TAK-788. In certain embodiments, the inhibition of proliferation is determined by a cell viability assay comparable to that disclosed in Example 3 as provided herein. TAK-788 is a small-molecule tyrosine kinase inhibitor (TKI) designed to selectively target EGFR and human EGFR 2 (HER2) exon 20 insertion mutations.
The term “maximal inhibition percentage” refers to the highest (i.e. plateau of) inhibition percentage achievable by a compound to inhibit proliferation of the tumor cells. In general, the percentage of inhibition on proliferation increases with the increase in concentration of the compound (e.g., an SHP2 inhibitor or an EGFR inhibitor) , however, it could reach a plateau where no more inhibition can be achieved despite of further increase in the concentration of the compound. The higher the maximal inhibition percentage, the more effective of the inhibition. The maximal inhibition percentage may vary to some degree depending on different assays, such as cell viability assay and other suitable assays.
In certain embodiments, the SHP2 inhibitor provided herein have maximal inhibition percentage that is significantly higher (e.g. at least 30%higher, 40%higher, 50%higher, 60%higher, 70%higher, 80%higher, 90%higher, or 100%higher) than that of the reference compound TAK-788. The assay conditions can be similar to those provide in Example 3 of the present disclosure.
In certain embodiments, the SHP2 inhibitor can be formulated with a pharmaceutically acceptable carrier. The carrier, when present, can be blended with the SHP2 inhibitor in any suitable amounts, such as an amount of from 5%to 95%by weight of carrier, based on the total volume or weight of SHP2 inhibitor and the carrier. In some embodiments, the amount of carrier can be in a range having a lower limit of any of 5%, 10%, 12%, 15%, 20%, 25%, 28%, 30%, 40%, 50%, 60%, 70%or 75%, and an upper limit, higher  than the lower limit, of any of 20%, 22%, 25%, 28%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, and 95%. The amount of carrier in a specific embodiment may be determined based on considerations of the specific dose form, relative amounts of SHP2 inhibitor, the total weight of the composition including the carrier, the physical and chemical properties of the carrier, and other factors, as known to those of ordinary skill in the formulation art.
The SHP2 inhibitor may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, the SHP2 inhibitor may be administered in conjunction with other treatments. The SHP2 inhibitor may be encapsulated or otherwise protected against gastric or other secretions, if desired.
A suitable, non-limiting example of a dosage of the SHP2 inhibitor disclosed herein is from about 1 mg/kg to about 2400 mg/kg per day, such as from about 1 mg/kg to about 1200 mg/kg per day, 75 mg/kg per day to about 300 mg/kg per day, including from about 1 mg/kg to about 100 mg/kg per day. Other representative dosages of such agents include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg, 2200 mg/kg, and 2300 mg/kg per day. In some embodiments, the dosage of the SHP2 inhibitor in human is about 400 mg/day given every 12 hours. In some embodiments, the dosage of the SHP2 inhibitor in human ranges 300-500 mg/day, 100-600 mg/day or 25-1000 mg/day. The effective dose of SHP2 inhibitor disclosed herein may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.
EGFR-related Diseases and EGFR Exon20 Insertion
EGFR has been associated with many human diseases including cancer, inflammatory disease, wound healing and fibrosis. In particular, mutations of EGFR gene have been associated with a number of cancers, including lung adenocarcinoma, anal cancers, glioblastoma, and epithelial tumors of the head and neck. These mutations typically lead to  the overexpression or constant activation of EGFR, which results in uncontrolled cell proliferation.
In certain embodiments, the cancer to be treated with the methods disclosed herein includes all cancer types disclosed herein.
In certain embodiments, the cancer to be treated with the methods disclosed herein includes Noonan Syndrome, Leopard Syndrome, juvenile myelomonocytic leukemia, neuroblastoma, melanoma, acute myeloid leukemia, breast cancer, esophageal cancer, lung cancer, colon cancer, head cancer, squamous cell carcinoma of head and neck, gastric cancer, anaplastic large cell lymphoma or glioblastoma.
In certain embodiments, the cancer to be treated with the methods disclosed herein includes lung cancer (e.g., non-small cell lung cancer (NSCLC) ) , glioblastoma, conventional glioblastoma multiforme, uterus cancer (e.g., infiltrating renal pelvis and ureter urothelial carcinoma, endometrial endometrioid adenocarcinoma) , bladder cancer (e.g., bladder urothelial carcinoma) , melanoma, renal cancer, liver cancer, myeloma, prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, thyroid cancer, hematological cancer, leukemia and non-Hodgkin’s lymphoma.
In certain embodiments, the cancer comprises an EGFR exon 20 insertion mutation.
Among the mutations of EGFR gene that are associated with cancers, insertion mutations, e.g., in frame insertions, within the exon 20 of EGFR gene have drawn particular attention because cancers bearing such mutations demonstrate intrinsic resistance to clinically achievable doses of current EGFR TKIs, including third-generation inhibitors. As a result, many efforts have been made to develop therapeutics to treat EGFR-exon20insertion-driven drug-resistant cancers.
Exon 20 of EGFR gene encompasses nucleotides that translate into amino acid residues at positions 762 to 823 of EGFR protein, which contains a C-helix (residues 762-766) and the loop following C-helix (residues 767-774) . The amino acid position of EGFR protein is based on the standard amino acid sequence of EGFR found in the sequence shown in accession number P00533-1 in the SwissProt database, or in the sequence shown in accession number NP_005219.2 in the NCBI database, which is also provided here as SEQ ID NO: 1.
Figure PCTCN2021117497-appb-000014
Figure PCTCN2021117497-appb-000015
Insertion mutations, particularly in frame insertions, could induce ligand-independent EGFR pathway activation and promote tumorigenesis. As used herein, “in frame insertion” refers to an insertion of one or more amino acid-encoding codons in a gene that does not alter the normal reading frame of the gene. The in frame insertion within exon 20 of EGFR gene, as described herein, can be at any place within the exon 20 of EGFR gene, and the insertion can introduce any number (e.g., 1, 2, 3, 4, or 5) of amino acid residues. In some embodiments, the insertion is within the C-helix (i.e. residues 761-766 of EGFR) . In some embodiments, the insertion is within the loop following C-helix (i.e., residues 767-775 of EGFR) . In some embodiments, the insertion mutation within exon 20 of EGFR gene promotes active conformation of EGFR or destabilizes inactive confirmation of EGFR. In some embodiments, the insertion mutation within exon 20 of EGFR gene enhances tyrosine kinase activity of EGFR in the absence of an EGFR ligand.
In certain embodiments, the EGFR exon 20 insertion mutation includes a mutation such as but not limited to any of the mutations described in Yasuda, et al., 2013, Sci. Transl. Med. 5 (216) : 216ra177; doi: 10.1126/scitranslmed. 3007205, and Arcila, et al., 2013, Mol. Cancer Ther. 12: 220; each of which is incorporated herein in its entirety by reference.
In certain embodiments, the exon 20 insertion mutation of the EGFR gene is selected from the group consisting of A763_Y764insFQEA, A763_Y764insFQQA, A767_V769dupASV, V769_D770insASV, D770_N771insGL, D770_N771insGT, D770_N771insNPG, D770_N771insSVD, E762Q_insFQEA, H773_V774insH, H773_V774insH, H773_V774insNPH, M766_A767insAI, M766_A767insASV, N771_H773dupNPH, P772_H773insYNP, P772_V774insPHV, S768_770dupSVD, V769_D770insASV, Y764_V765insHH, and delD770insGY.
In certain embodiments, the cancer is resistant to an EGFR inhibitor. An EGFR inhibitor refers to a molecule capable of inhibiting tyrosine kinase activity of EGFR,  and may be a small molecule compound or a biological macromolecule such as an antibody or an antibody fragment or the like.
Exemplary small molecule EGFR inhibitors include, but are not limited to, first generation EGFR inhibitors such as gefitinib, erlotinib, and icotinib; second generation EGFR inhibitors such as afatinib and dacomitinib; and third-generation EGFR inhibitors such as osimertinib (AZD9291) , olmutinib, nazartinib, rociletinib, naquotinib, lazertinib and the like. Exemplary macromolecular EGFR inhibitors include, but are not limited to, cetuximab, panitumumab, nimotuzumab, and necitumumab. In some embodiments, the cancer is resistant to an EGFR inhibitor selected from: gefitinib, erlotinib, icotinib, afatinib, dacomitinib, lapatinib, osimertinib (AZD9291) , nazartinib, rociletinib, naquotinib, vandetanib, neratinib, pelitinib, canertinib, brigatinib, PKC412, Go6976, mavelertinib, olmutinib, WZ4002, TAS2913, cetuximab, panitumumab, nimotuzumab, necitumumab avitinib, HS-10296 and TQB3804. In some embodiments, the cancer is resistant to osimertinib.
In certain embodiments, the cancer is resistant to EGFR tyrosine kinase inhibitor TAK-788, osimertinib, Poziotinib, Almonertinib (HS-10296) , Amivantamab (JNJ-372) (see, Neijssen et al, J. Biol. Chem. (2021) 296: 100641) , furmonertinib (AST2818) , or DZD9008 (see, Yan Xu et al, abstract 3081 of Proceedings: AACR Annual Meeting 2019; March 29-April 3, 2019; Atlanta, GA) .
TAK-788, also named mobocertinib and AP32788, which has the following structure, is a selective inhibitor of EGFR.
Figure PCTCN2021117497-appb-000016
Poziotinib has a chemical structure shown below:
Figure PCTCN2021117497-appb-000017
Almonertinib (HS-10296) has a chemical structure shown below:
Figure PCTCN2021117497-appb-000018
Furmonertinib (AST2818) has a chemical structure shown below:
Figure PCTCN2021117497-appb-000019
It has been reported that tumors bearing EGFR exon 20 insertions exhibited relatively good response to TAK-788 (Hirose et al. Lung Cancer, 152: 135-142 (2021) ) .
Osimertinib, which has the following structure, is a third-generation EGFR tyrosine kinase inhibitor.
Figure PCTCN2021117497-appb-000020
Studies have shown that osimertinib was active in certain NSCLC patients bearing EGFR exon 20 insertion mutation (Fang et al., BMC Cancer, 19: 595 (2019) ) .
Drug resistance as used herein refers to being refractory or non-responsive to a therapeutic agent, such as an EGFR inhibitor. For example, the number of tumor cells is increased despite of being treated with a therapeutic agent. In certain embodiments, the cancer resistant to EGFR inhibitor is lung cancer (e.g., small cell lung cancer or non-small cell lung cancer) .
In certain embodiments, the cancer is resistant to a chemotherapy. Examples of chemotherapy include such as cisplatin, cyclophosphamide, chlorambucil, busulfan, melphalan, carmustine, streptozotocin, triethylenemelamine, mitomycin C, methotrexate, etoposide, 6-mercaptopurine (6MP) , 6-thiocguanine (6TG) , cytarabine (Ara-C) , 5- fluorouracil (5-FU) , capecitabine, dacarbazine (DTIC) , actinomycin D, doxorubicin (DXR) , daunorubicin (daunomycin) , bleomycin, mithramycin, vincristine (VCR) , vinblastine, paclitaxel and pactitaxel derivatives, the cytostatic agents, dexamethasone (DEX) , prednisone, hydroxyurea, asparaginase, leucovorin, folinic acid, raltitrexed, . arnifostine, dactinomycin, mechlorethamine (nitrogen mustard) , streptozocin, cyclophosphamide, lornustine (CCNU) , doxorubicin lipo, gemcitabine, daunorubicin lipo, procarbazine, mitomycin, docetaxel, aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan) , 10-hydroxy 7-ethyl-camptothecin (SN38) , floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon alpha, interferon beta, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, and chlorambucil. In certain embodiments, the cancer is resistant to cisplatin.
In certain embodiments, the cancer is metastatic.
Methods of Detecting Insertion Mutation
The presence or absence of insertion mutation within exon 20 of the EGFR gene in a biological sample can be detected using proper methods known in the art including, without limitation, sequencing assay, amplification assay and hybridization assay.
Sample Preparation
Any biological sample suitable for conducting the methods provided herein can be obtained from the subject including, without limitation, bodily fluid, such as blood, plasma, serum, urine, vaginal fluid, uterine or vaginal flushing fluids, plural fluid, ascitic fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchioalveolar lavage fluid, etc., and tissues, such as biopsy tissue (e.g. biopsied bone tissue, bone marrow, breast tissue, gastrointestinal tract tissue, lung tissue, liver tissue, prostate tissue, brain tissue, nerve tissue, meningeal tissue, renal tissue, endometrial tissue, cervical dittuse, lymph node tissue, muscle tissue, or skin tissue) .
In certain embodiments, the biological sample is processed to isolate or extract cancer cell (such as circulating tumor cell) before detecting the presence or absence of the insertion mutation. For example, the cancer cells can be separated by immunomagnetic separation technology such as that available from Immunicon (Huntingdon Valley, Pa. ) .
In certain embodiments, nucleic acid, e.g., DNA or RNA is isolated from the biological sample before detecting the presence or absence of the insertion mutation. Various methods of extraction are suitable for isolating the DNA or RNA from cells or tissues, such as phenol and chloroform extraction, and various other methods as described in, for example,  Ausubel et al., Current Protocols of Molecular Biology (1997) John Wiley &Sons, and Sambrook and Russell, Molecular Cloning: A Laboratory Manual 3 rd ed. (2001) .
Commercially available kits can also be used to isolate DNA and/or RNA, including for example, the NucliSens extraction kit (Biomerieux, Marcy l'Etoile, France) , QIAamp TM mini blood kit, Agencourt Genfind TM, 
Figure PCTCN2021117497-appb-000021
mini columns (Qiagen) , 
Figure PCTCN2021117497-appb-000022
RNA mini kit (Thermo Fisher Scientific) , and Eppendorf Phase Lock Gels  TM. A skilled person can readily extract or isolate RNA or DNA following the manufacturer’s protocol.
Sequencing methods
Sequencing methods known in the art can be used to the detection of insertion mutation within the exon20 of the EGFR gene. In general, sequencing methods can be categorized to traditional or classical methods and high throughput sequencing (next generation sequencing (NGS) ) . Traditional sequencing methods include Maxam-Gilbert sequencing (also known as chemical sequencing) and Sanger sequencing (also known as chain-termination methods) .
High throughput sequencing, or next generation sequencing, by using methods distinguished from traditional methods, such as Sanger sequencing, is highly scalable and able to sequence the entire genome or transcriptome at once. High throughput sequencing involves sequencing-by-synthesis, sequencing-by-ligation, and ultra-deep sequencing (such as described in Marguiles et al., Nature 437 (7057) : 376-80 (2005) ) . Sequence-by-synthesis involves synthesizing a complementary strand of the target nucleic acid by incorporating labeled nucleotide or nucleotide analog in a polymerase amplification. Immediately after or upon successful incorporation of a label nucleotide, a signal of the label is measured and the identity of the nucleotide is recorded. The detectable label on the incorporated nucleotide is removed before the incorporation, detection and identification steps are repeated. Examples of sequence-by-synthesis methods are known in the art, and are described for example in U.S. Pat. No. 7,056,676, U.S. Pat. No. 8,802,368 and U.S. Pat. No. 7,169,560, the contents of which are incorporated herein by reference. Sequencing-by-synthesis may be performed on a solid surface (or a microarray or a chip) using fold-back PCR and anchored primers. Target nucleic acid fragments can be attached to the solid surface by hybridizing to the anchored primers, and bridge amplified. This technology is used, for example, in the
Figure PCTCN2021117497-appb-000023
sequencing platform.
Pyrosequencing involves hybridizing the target nucleic acid regions to a primer and extending the new strand by sequentially incorporating deoxynucleotide  triphosphates corresponding to the bases A, C, G, and T (U) in the presence of a polymerase. Each base incorporation is accompanied by release of pyrophosphate, converted to ATP by sulfurylase, which drives synthesis of oxyluciferin and the release of visible light. Since pyrophosphate release is equimolar with the number of incorporated bases, the light given off is proportional to the number of nucleotides adding in any one step. The process is repeated until the entire sequence is determined.
Amplification assay
A nucleic acid amplification assay involves copying a target nucleic acid (e.g. DNA or RNA) , thereby increasing the number of copies of the amplified nucleic acid sequence. Amplification may be exponential or linear. Exemplary nucleic acid amplification methods include, but are not limited to, amplification using the polymerase chain reaction ( "PCR" , see U.S. Patents 4,683,195 and 4,683,202; PCR Protocols: A Guide To Methods And Applications (Innis et al., eds, 1990) ) , reverse transcriptase polymerase chain reaction (RT-PCR) , quantitative real-time PCR (qRT-PCR) ; quantitative PCR, such as
Figure PCTCN2021117497-appb-000024
nested PCR, ligase chain reaction (See Abravaya, K., et al., Nucleic Acids Research, 23: 675-682, (1995) , branched DNA signal amplification (see, Urdea, M.S., et al., AIDS, 7 (suppl 2): S11-S14, (1993) , amplifiable RNA reporters, Q-beta replication (see Lizardi et al., Biotechnology (1988) 6: 1197) , transcription-based amplification (see, Kwoh et al., Proc. Natl. Acad. Sci. USA (1989) 86: 1173-1177) , boomerang DNA amplification, strand displacement activation, cycling probe technology, self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA (1990) 87: 1874-1878) , rolling circle replication (U.S. Patent No. 5,854,033) , isothermal nucleic acid sequence based amplification (NASBA) , and serial analysis of gene expression (SAGE) .
In certain embodiments, the nucleic acid amplification assay is a PCR-based method. PCR is initiated with a pair of primers that hybridize to the target nucleic acid sequence to be amplified, followed by elongation of the primer by polymerase which synthesizes the new strand using the target nucleic acid sequence as a template and dNTPs as building blocks. Then the new strand and the target strand are denatured to allow primers to bind for the next cycle of extension and synthesis. After multiple amplification cycles, the total number of copies of the target nucleic acid sequence can increase exponentially.
In certain embodiments, intercalating agents that produce a signal when intercalated in double stranded DNA may be used. Exemplary agents include SYBR GREEN TM and SYBR GOLD TM. Since these agents are not template-specific, it is assumed that the signal is generated based on template-specific amplification. This can be confirmed  by monitoring signal as a function of temperature because melting point of template sequences will generally be much higher than, for example, primer-dimers, etc.
In certain embodiments, a detectably labeled primer or a detectably labeled probe can be used, to allow detection of the insertion mutation corresponding to that primer or probe. In certain embodiments, multiple labeled primers or labeled probes with different detectable labels can be used to allow simultaneous detection of multiple insertion mutation.
Hybridization assay
Nucleic acid hybridization assays use probes to hybridize to the target nucleic acid, thereby allowing detection of the target nucleic acid. Non-limiting examples of hybridization assay include Northern blotting, Southern blotting, in situ hybridization, microarray analysis, and multiplexed hybridization-based assays.
In certain embodiments, the probes for hybridization assay are detectably labeled. In certain embodiments, the nucleic acid-based probes for hybridization assay are unlabeled. Such unlabeled probes can be immobilized on a solid support such as a microarray, and can hybridize to the target nucleic acid molecules which are detectably labeled.
In certain embodiments, hybridization assays can be performed by isolating the nucleic acids (e.g. RNA or DNA) , separating the nucleic acids (e.g. by gel electrophoresis) followed by transfer of the separated nucleic acid on suitable membrane filters (e.g. nitrocellulose filters) , where the probes hybridize to the target nucleic acids and allows detection. See, for example, Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7. The hybridization of the probe and the target nucleic acid can be detected or measured by methods known in the art. For example, autoradiographic detection of hybridization can be performed by exposing hybridized filters to photographic film.
In some embodiments, hybridization assays can be performed on microarrays. Microarrays provide a method for the simultaneous measurement of the levels of large numbers of target nucleic acid molecules. The target nucleic acids can be RNA, DNA, cDNA reverse transcribed from mRNA, or chromosomal DNA. The target nucleic acids can be allowed to hybridize to a microarray comprising a substrate having multiple immobilized nucleic acid probes arrayed at a density of up to several million probes per square centimeter of the substrate surface. The RNA or DNA in the sample is hybridized to complementary probes on the array and then detected by laser scanning. Hybridization intensities for each probe on the array are determined and converted to a quantitative value representing relative  levels of the RNA or DNA. See, U.S. Patent Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, and 6,344,316.
Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Patent No. 5,384,261. Although a planar array surface is often employed the array may be fabricated on a surface of virtually any shape or even a multiplicity of surfaces. Arrays may be peptides or nucleic acids on beads, gels, polymeric surfaces, fibers such as fiber optics, glass or any other appropriate substrate, see U.S. Patent Nos. 5,770,358, 5,789,162, 5,708,153, 6,040,193 and 5,800,992. Arrays may be packaged in such a manner as to allow for diagnostics or other manipulation of an all-inclusive device. Useful microarrays are also commercially available, for example, microarrays from Affymetrix, from Nano String Technologies, QuantiGene 2.0 Multiplex Assay from Panomics.
Combinatory Treatment with a SHP2 Inhibitor
In certain embodiments, the method of the present disclosure further involves administering to the subject an anti-cancer agent other than a SHP2 inhibitor. In certain embodiments, the administration of the second anti-cancer agent depends on the detection of a second mutation in the cancer.
In certain embodiments, the method provided in the present disclosure involves detecting a second mutation in a gene in the RTK/RAS/MAPK signaling pathway in the biological sample of the subject. In certain embodiments, the gene is EGFR, KRAS or BRAF. The RTK/RAS/MAPK signaling pathway is a chain of protein in the cell that communicates a signal from a receptor tyrosine kinase on the surface of the cell to the DNA in the nucleus of the cell. The RTK/RAS/MAPK signaling pathway has been associated with uncontrolled growth in many cancers.
In certain embodiments, after the mutation in the RTK/RAS/MAPK signaling pathway is detected, the method of present disclosure further comprises administering to the subject a second therapeutic agent. In certain embodiments, the second therapeutic agent is an EGFR tyrosine kinase inhibitor, a KRAS inhibitor, a BRAF inhibitor, a MEK inhibitor or a CDK inhibitor. In certain embodiments, the EGFR tyrosine kinase inhibitor is Gefitinib, Erlotinib, Afatinib, Dacomitinib or Osimertinib. In certain embodiments, the KRAS inhibitor is AMG510 or MRTX849. In certain embodiments, the BRAF inhibitor is Vemurafenib, Dabrafenib, Encorafenib or Sorafenib. In certain embodiments, the MEK inhibitor is  Trametinib, Cobimetinib, Selumetinib, Pimasertib or Binimetinib. In certain embodiments, the CDK inhibitor is Palbociclib, Ribociclib or Abemaciclib.
Additional anti-cancer agents that can be used in combination with the SHP2 inhibitor disclosed herein include, without limitation: alkylating agents or agents with an alkylating action, such as cyclophosphamide (CTX; e.g. 
Figure PCTCN2021117497-appb-000025
) , chlorambucil (CHL; e.g. 
Figure PCTCN2021117497-appb-000026
) , cisplatin (CisP; e.g. 
Figure PCTCN2021117497-appb-000027
) busulfan (e.g. 
Figure PCTCN2021117497-appb-000028
) , melphalan, carmustine (BCNU) , streptozotocin, triethylenemelamine (TEM) , mitomycin C, and the like; anti-metabolites, such as methotrexate (MTX) , etoposide (VP16; e.g. 
Figure PCTCN2021117497-appb-000029
) , 6-mercaptopurine (6MP) , 6-thiocguanine (6TG) , cytarabine (Ara-C) , 5-fluorouracil (5-FU) , capecitabine (e.g. 
Figure PCTCN2021117497-appb-000030
) , dacarbazine (DTIC) , and the like; antibiotics, such as actinomycin D, doxorubicin (DXR; e.g. 
Figure PCTCN2021117497-appb-000031
) , daunorubicin (daunomycin) , bleomycin, mithramycin and the like; alkaloids, such as vinca alkaloids such as vincristine (VCR) , vinblastine, and the like; and other antitumor agents, such as paclitaxel (e.g. 
Figure PCTCN2021117497-appb-000032
) and pactitaxel derivatives, the cytostatic agents, glucocorticoids such as dexamethasone (DEX; e.g. 
Figure PCTCN2021117497-appb-000033
) and corticosteroids such as prednisone, nucleoside enzyme inhibitors such as hydroxyurea, amino acid depleting enzymes such as asparaginase, leucovorin, folinic acid, raltitrexed, and other folic acid derivatives, and similar, diverse antitumor agents. The following agents may also be used as additional agents: arnifostine (e.g. 
Figure PCTCN2021117497-appb-000034
) , dactinomycin, mechlorethamine (nitrogen mustard) , streptozocin, cyclophosphamide, lornustine (CCNU) , doxorubicin lipo (e.g. 
Figure PCTCN2021117497-appb-000035
) , gemcitabine (e.g. 
Figure PCTCN2021117497-appb-000036
) , daunorubicin lipo (e.g. 
Figure PCTCN2021117497-appb-000037
) , procarbazine, mitomycin, docetaxel (e.g. 
Figure PCTCN2021117497-appb-000038
) , aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan) , 10-hydroxy 7-ethyl-camptothecin (SN38) , floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon alpha, interferon beta, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, and chlorambucil.
In certain embodiments, the anti-cancer agent used in combination with the SHP2 inhibitor is an anti-hormonal agent. As used herein, the term “anti-hormonal agent” includes natural or synthetic organic or peptide compounds that act to regulate or inhibit hormone action on tumors. Anti-hormonal agents include, for example: steroid receptor antagonists, anti-estrogens such as tamoxifen, raloxifene, aromatase inhibiting 4 (5) -imidazoles, other aromatase inhibitors, 42-hydroxytamoxifen, trioxifene, keoxifene, LY 117018, onapristone, and toremifene (e.g. 
Figure PCTCN2021117497-appb-000039
) ; anti-androgens such as flutamide,  nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above; agonists and/or antagonists of glycoprotein hormones such as follicle stimulating hormone (FSH) , thyroid stimulating hormone (TSH) , and luteinizing hormone (LH) and LHRH (leuteinizing hormone-releasing hormone) ; the LHRH agonist goserelin acetate, commercially available as
Figure PCTCN2021117497-appb-000040
 (AstraZeneca) ; the LHRH antagonist D-alaninamide N-acetyl-3- (2-naphthalenyl) -D-alanyl-4-chloro-D-phenylalanyl-3- (3-pyridinyl) -D-alanyl-L-seryl-N6- (3-pyridinylcarbonyl) -L-lysyl-N6- (3-pyridinylcarbonyl) -D-lysyl-L-leucyl-N6- (1-methylethyl) -L-lysyl-L-proline (e.g
Figure PCTCN2021117497-appb-000041
Ares-Serono) ; the LHRH antagonist ganirelix acetate; the steroidal anti-androgens cyproterone acetate (CPA) and megestrol acetate, commercially available as
Figure PCTCN2021117497-appb-000042
 (Bristol-Myers Oncology) ; the nonsteroidal anti-androgen flutamide (2-methyl-N- [4, 20-nitro-3- (trifluoromethyl) phenylpropanamide) , commercially available as
Figure PCTCN2021117497-appb-000043
 (Schering Corp. ) ; the non-steroidal anti-androgen nilutamide, (5, 5-dimethyl-3- [4-nitro-3- (trifluoromethyl-4′-nitrophenyl) -4, 4-dimethyl-imidazolidine-dione) ; and antagonists for other non-permissive receptors, such as antagonists for RAR, RXR, TR, VDR, and the like.
In certain embodiments, the anti-cancer agent used in combination with the SHP2 inhibitor is an angiogenesis inhibitor. Anti-angiogenic agents include, for example: VEGFR inhibitors, such as SU-5416 and SU-6668 (Sugen Inc. of South San Francisco, Calif., USA) , or as described in, for example International Application Nos. WO 99/24440, WO 99/62890, WO 95/21613, WO 99/61422, WO 98/50356, WO 99/10349, WO 97/32856, WO 97/22596, WO 98/54093, WO 98/02438, WO 99/16755, and WO 98/02437, and U.S. Pat. Nos. 5,883,113, 5,886,020, 5,792,783, 5,834,504 and 6,235,764; VEGF inhibitors such as IM862 (Cytran Inc. of Kirkland, Wash., USA) ; angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colo. ) and Chiron (Emeryville, Calif. ) ; and antibodies to VEGF, such as bevacizumab (e.g. Avastin TM, Genentech, South San Francisco, Calif. ) , a recombinant humanized antibody to VEGF; integrin receptor antagonists and integrin antagonists, such as to α vβ 3, α vβ 5 and α vβ 6 integrins, and subtypes thereof, e.g. cilengitide (EMD 121974) , or the anti-integrin antibodies, such as for example α vβ 3 specific humanized antibodies (e.g. 
Figure PCTCN2021117497-appb-000044
); factors such as IFN-alpha (U.S. Pat. Nos. 41530,901, 4,503,035, and 5,231,176) ; angiostatin and plasminogen fragments (e.g. kringle 14, kringle 5, kringle 1-3 (O'Reilly, M.S. et al. (1994) Cell 79: 315-328; Cao et al. (1996) J. Biol. Chem. 271: 29461-29467; Cao et al. (1997) J. Biol. Chem. 272: 22924-22928) ; endostatin (O'Reilly, M. S. et al. (1997) Cell 88: 277; and International Patent Publication No. WO 97/15666) ; thrombospondin (TSP-1; Frazier, (1991) Curr. Opin. Cell Biol. 3: 792) ; platelet factor 4 (PF4) ; plasminogen  activator/urokinase inhibitors; urokinase receptor antagonists; heparinases; fumagillin analogs such as TNP-4701; suramin and suramin analogs; angiostatic steroids; bFGF antagonists; flk-1 and flt-1 antagonists; anti-angiogenesis agents such as MMP-2 (matrix-metalloprotienase 2) inhibitors and MMP-9 (matrix-metalloprotienase 9) inhibitors. Examples of useful matrix metalloproteinase inhibitors are described in International Patent Publication Nos. WO 96/33172, WO 96/27583, WO 98/07697, WO 98/03516, WO 98/34918, WO 98/34915, WO 98/33768, WO 98/30566, WO 90/05719, WO 99/52910, WO 99/52889, WO 99/29667, and WO 99/07675, European Patent Publication Nos. 818, 442, 780, 386, 1, 004, 578, 606, 046, and 931, 788; Great Britain Patent Publication No. 9912961, and U.S. Pat. Nos. 5,863,949 and 5,861,510. Preferred MMP-2 and MMP-9 inhibitors are those that have little or no activity inhibiting MMP-1. More preferred, are those that selectively inhibit MMP-2 and/or MMP-9 relative to the other matrix-metalloproteinases (i.e. MMP-1, MMP-3, MMP-4, MMP-5, MMP-6, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12, and MMP-13) .
In certain embodiments, the anti-cancer agent used in combination with the SHP2 inhibitor is a tumor cell pro-apoptotic or apoptosis-stimulating agent.
In certain embodiments, the anti-cancer agent used in combination with the SHP2 inhibitor is a signal transduction inhibitor. Signal transduction inhibitors include, for example: erbB2 receptor inhibitors, such as organic molecules, or antibodies that bind to the erbB2 receptor, for example, trastuzumab (e.g. 
Figure PCTCN2021117497-appb-000045
) ; inhibitors of other protein tyrosine-kinases, e.g. imatinib (e.g. 
Figure PCTCN2021117497-appb-000046
) ; ras inhibitors; raf inhibitors; MEK inhibitors; mTOR inhibitors; cyclin dependent kinase inhibitors; protein kinase C inhibitors; and PDK-1 inhibitors (see Dancey, J. and Sausville, E. A. (2003) Nature Rev. Drug Discovery 2: 92-313, for a description of several examples of such inhibitors, and their use in clinical trials for the treatment of cancer) ; GW-282974 (Glaxo Wellcome plc) ; monoclonal antibodies such as AR-209 (Aronex Pharmaceuticals Inc. of The Woodlands, Tex., USA) and 2B-1 (Chiron) ; and erbB2 inhibitors such as those described in International Publication Nos. WO 98/02434, WO 99/35146, WO 99/35132, WO 98/02437, WO 97/13760, and WO 95/19970, and U.S. Pat. Nos. 5,587,458, 5,877,305, 6,465,449 and 6,541,481.
In certain embodiments, the anti-cancer agent used in combination with the SHP2 inhibitor is a cancer immunotherapy agent, such as an antibody specifically binding to an immune checkpoint. Immune checkpoints include, for example: A2AR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, BTLA, CD48, CD160, CD244, CTLA-4, ICOS, LAG-3, LILRB1, LILRB2, LILRB4, OX40, PD-1, PD-L1, PD-L2, SIRPalpha (CD47) , TIGIT, TIM-3,  TIM-1, TIM-4, and VISTA. In certain embodiments, the anti-cancer agent used in combination with the SHP2 inhibitor is a PD-1/PD-L1 antagonist.
In certain embodiments, the anti-cancer agent used in combination with the SHP2 inhibitor is an anti-proliferative agent. Anti-proliferative agents include, for example: Inhibitors of the enzyme farnesyl protein transferase and inhibitors of the receptor tyrosine kinase PDGFR, including the compounds disclosed and claimed in U.S. Pat. Nos. 6,080,769, 6,194,438, 6,258,824, 6,586,447, 6,071,935, 6,495,564, 6,150,377, 6,596,735 and 6,479,513, and International Patent Publication WO 01/40217.
In certain embodiments, the SHP2 inhibitor and/or the second anti-cancer agent is administered at a reduced amount relative to the amount otherwise required to be therapeutically effective for single agent treatment. In certain embodiments, the combination of the SHP2 inhibitor and the second anti-cancer agent can provide a significantly higher therapeutic effect than otherwise achievable for single agent treatment.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. All specific compositions, materials, and methods described below, in whole or in part, fall within the scope of the present invention. These specific compositions, materials, and methods are not intended to limit the invention, but merely to illustrate specific embodiments falling within the scope of the invention. One skilled in the art may develop equivalent compositions, materials, and methods without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.
Example 1 Synthesis of intermediate A2
Synthesis of intermediate A2: R-N- ( (S) -5, 7-dihydrospiro [cyclopenta [b] pyridine-6, 4’ -piperidine] -5-yl-5-D) -2-methylpropane-2-sulfinamide
Figure PCTCN2021117497-appb-000047
Figure PCTCN2021117497-appb-000048
Step 1: 4-cyanopiperidine-1-carboxylic acid tert-butyl ester (1.05g, 5mmol) and THF (20mL) were successively added to a dry 100mL flask. Under the protection of nitrogen, the mixture was cooled to -78 ℃, and then 2M of LDA (3.3mL, 6.5mmol) was slowly added to the reaction mixture. The reaction mixture was allowed to react for 1 hour, and then 3-bromo-2- (bromomethyl) pyridine (1.24g, 5mmol) was added thereto, and then the reaction mixture was allowed to continue to react for 2 hours. After the reaction, saturated ammonium chloride solution (15ml) was added to quench the reaction, the resulting mixture was extracted with ethyl acetate (3×30ml) , the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate and filtered, and the residue obtained by concentration under reduced pressure was purified by silica gel chromatography (0 to 30%gradient of ethyl acetate/petroleum ether) to obtain 4- ( (3-bromopyridine-2-yl) methyl) -4-cyanopiperidine-1'-carboxylic acid tert-butyl ester (A2-1, 1.40g, yield: 75%) as a white solid.
Step 2: Under the protection of nitrogen, 4- ( (3-bromopyridine-2-yl) methyl) -4-cyanopiperidine-1'-carboxylic acid tert-butyl ester (A2-1, 379mg, 1mmol) , trimethylamine (404mg, 4mmol) , bis (di-tert-butyl (4-dimethylaminophenyl) phosphine) dichloropalladium (II) Pd (AmPhos)  2Cl 2 (71mg, 0.1mmol) and DMA: H 2O=10: 1 (6mL) were successively added to a dry 25mL single-necked flask, and then the mixture was stirred at 130 ℃ for 18 hours. After the reaction was completed, the obtained residue was filtered and the residue obtained by concentration under reduced pressure was purified by silica gel chromatography (0 to 50%gradient of ethyl acetate: petroleum ether) to obtain 5-oxo-5, 7-dihydrospiro [cyclopenta [b] pyridine-6, 4'-piperidine] -1'-carboxylic acid tert-butyl ester (A2-2, 180mg, yield: 60%) as a yellow solid.
LCMS: m/z 303.1 [M+H]  +.
Step 3: 5-oxo-5, 7-dihydrospiro [cyclopenta [b] pyridine-6, 4'-piperidine] -1'-carboxylic acid tert-butyl ester (A2-2, 0.302g, 1mmol) , tetraethyl titanate (1.37g, 6mmol) and (R) - (+) -tert-butylsulfinamide (0.480g, 4mmol) were successively added to a dry 100mL single-necked flask and the mixture was stirred under heating and reflux for 15 hours. After the reaction system was cooled to room temperature, saturated brine (15 mL) was added to the reaction residue, after which the resulting mixture was stirred for 15 minutes and then  filtered through diatomite. The aqueous mixture was extracted with ethyl acetate (3x300mL) . The organic phase was dried over Na 2SO 4 and filtered, and volatiles were removed under reduced pressure. The resulting residue was purified by silica gel chromatography (0 to 50%gradient of ethyl acetate: petroleum ether) to obtain (R, Z) -5- ( (tert-butylsulfinyl) imino) -5, 7-dihydrospiro [cyclopenta [b] pyridine-6, 4'-piperidine] -1'-carboxylic acid tert-butyl ester (A2-3, 0.333g, yield: 82%) as a yellow solid.
LCMS: m/z 406.1 [M+H]  +.
Step 4: (R, Z) -5- ( (tert-butylsulfinyl) imino) -5, 7-dihydrospiro [cyclopenta [b] pyridine-6, 4'-piperidine] -1'-carboxylic acid tert-butyl ester (A2-3, 0.60g, 1.48mmol) and ethanol (20mL) were successively added to a dry 100mL single-necked flask, then sodium borodeuteride (0.186g, 4.44mmol) was added. The resulting mixture was stirred for 2 hours at room temperature. TLC and LCMS indicated that the reaction was completed. Acetic acid (1mL) was slowly added to quench the reaction. The solution was added ethyl acetate (100mL) and saturated brine and the resulted mixture was stirred at room temperature for 1 hour. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (100mL) . The organic phases were combined, washed with saturated brine, dried, filtered, concentrated and purified by silica gel column chromatography (petroleum ether: ethyl acetate= 1: 1 ) to obtain (S) -5- ( (R) -tert-butylsulfonamido) -dihydrospiro [cyclopenta [b] pyridine-6, 4'-piperidine] -1'-carboxylic acid tert-butyl ester-5-D (A2-4, 0.400g, yield: 66.2%) as a yellow oil.
LCMS: m/z: 409.5 [M+H]  + .
Step 5: To a solution of (S) -5- ( (R) -tert-butylsulfonamido) -dihydrospiro [cyclopenta [b] pyridine-6, 4'-piperidine] -1'-carboxylic acid tert-butyl ester -5-D (A2-4, 0.816g, 2.0mmol) in dichloromethane (10mL) was added trifluoroacetic acid (3 mL) . The resulting mixture was stirred at room temperature for 2 hours. TLC and LCMS indicated that the reaction was completed. The mixture was concentrated to dryness under reduced pressure to obtain (R) -N- ( (S) -5, 7-dihydrospiro [cyclopenta [b] pyridine-6, 4’ -piperidine] -5-yl-5-D) -2-methylpropane-2-sulfinamide (A2) as a white solid which was used directly in next step without further purification.
LCMS: m/z: 309.5 [M+H]  + .
Example 2: Synthesis of compound B
(S) -1'- (8- ( (2-amino-3-chloropyridin-4-yl) thio) imidazo [1, 2-c] pyrimidin-5-yl) -5, 7-dihydrospiro [cyclopenta [b] pyridine-6, 4'-piperidine] -5-D-5-amine
Figure PCTCN2021117497-appb-000049
Step 1: To a solution of 5-chloro-8-iodoimidazo [1, 2-c] pyrimidine (B1, 0.359g, 1.29 mmol) and (R) -N- ( (S) -5, 7-dihydrospiro [cyclopenta [b] pyridine-6, 4’ -piperidine] -5-yl-5-D) -2-methylpropane-2-sulfinamide (A2 0.400g, 1.29mmol) in acetonitrile (10mL) was added N, N-Diisopropylethylamine DIPEA (0.500g, 3.87mmol) . The mixture was stirred for 3 hours at 70 ℃. To the reaction mixture was added ethyl acetate (100mL) and saturated brine. The resulted mixture was stirred at room temperature for 1 hour. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (100mL) . The organic phases were combined, washed with saturated brine, dried, filtered and concentrated to dryness. The residue was purified by silica gel chromatography (dichloromethane: methanol =20: 1) to obtain B2 (0.610g, yield: 85.8%) as a yellow solid.
LCMS: m/z: 552.1 [M+H]  +
Step 2: A mixture of Pd 2 (dba)  3 (50 mg, 0.05 mmol) , DIPEA (140 mg, 1.09 mmol) , Xantphos (63 mg, 0.11 mmol) , B2 (300mg, 0.54mmol) , sodium 2-amino-3-chloropyridine-4-mercaptan (C1, 110 mg, 0.60 mmol) and 1, 4-dioxane (10mL) was stirred at 105℃ for 12 hours under nitrogen atmosphere. To the reaction mixture was added ethyl acetate (100mL) and saturated brine and then the resulted mixture was stirred at room temperature for 10 minutes. The organic layer was separated. The aqueous layer was extracted with ethyl acetate (100mL) . The organic phases were combined, washed with saturated brine, dried, filtered and concentrated to dryness. The residue was purified by silica gel chromatography (dichloromethane: methanol =20: 1) to obtain (R) -N- ( (S) -1'- (8- ( (2-amino-3-chloropyridin-4-yl) thio) imidazolo [1, 2-c] pyrimidin-5-yl) -5, 7-dihydrospiro [cyclopenta [b] pyridin-6, 4'-piperidin] -5-yl-5-D) -2-methylpropane-2-sulfinamide (B3, 210mg, 66.1%) .
LCMS: m/z: 584.2 [M+H]  + .
Step 3: To a solution of (R) -N- ( (S) -1'- (8- ( (2-amino-3-chloropyridin-4-yl) thio) imidazolo [1, 2-c] pyrimidin-5-yl) -5, 7-dihydrospiro [cyclopenta [b] pyridin-6, 4'-piperidin] -5-yl- 5-D) -2-methylpropane-2-sulfinamide (B3, 150mg, 0.26mmol) in methanol (8mL) was added Hydrochloric acid (1, 4-dioxane solution, 4M, 5mL) . The mixture was stirred for 2 hours. TLC and LCMS indicated that the reaction was completed. The mixture was concentrated to dryness under reduced pressure. To the residue was added Ethyl acetate (30 mL) and saturated sodium bicarbonate solution.. The organic layer was separated and the aqueous layer was extracted with ethyl acetate (100mL) . The organic phases were combined, washed with saturated brine, dried, filtered, concentrated to dryness under reduced pressure. The residue was purified by silica gel chromatography to obtain (S) -1'- (8- ( (2-amino-3-chloropyridin-4-yl) thio) imidazo [1, 2-c] pyrimidin-5-yl) -5, 7-dihydrospiro [cyclopenta [b] pyridine-6, 4'-piperidine] -5-D-5-amine (compound B, 100mg, yield: 81.0%) .
1H NMR (400 MHz, DMSO-d 6) : 8.36 (d, J = 4.8 Hz, 1H) , 8.03 (s, 1H) , 7.83 d, J = 1.2 Hz, ) , 7.74 (d, J = 8.0 Hz, 1H) , 7.58 (s, 1H) , 7.54 (d, J =5.6 Hz, 1H) , 7.23-7.20 (dd, J = 7.6, 5.2 Hz, 1H, 1H) , 6.34 (s, 2H) , 5.80 (d, J =5.6 Hz, 1H) , 3.97-3.94 (dd, J = 11.6, 7.6 Hz, 2H) , 3.39-3.37 (d, J = 13.6 Hz, 2H) , 3.15 (d, J =16.4 Hz, 1H ) , 2.86 (d, J =16.4 Hz, 1H ) , 2.03-1.94 (m, 2H) , 1.64 (d, J =13.6 Hz, 1H ) , 1.36 (d, J =13.6 Hz, 1H ) .
LCMS: m/z: 480.1 [M+H]  + .
Example 3
This example illustrates the activity of exemplary compounds in EGFR exon 20ins NSCLC patient-derived organoids.
Effects of Compound A, B and C on cell proliferation were evaluated in several NSCLC patient-derived organoids (PDOs) bearing EGFR exon 20 insertion mutations. Organoids were dissociated into single cells using TrypLE Express and resuspended in 50%Matrigel growth medium on ice. The single cell suspensions were then dispensed into 96-well plates (50 μL/well) in duplicate, added with 50μL GAS-Ad-ES medium. Drugs (Compound A, Compound B, Compound C, TAK-788, Osimertinib) were added in 3-fold serial dilutions 48 h after organoid plating. Following drug exposure for five days, cell viability was determined using CellTiter-Glo 3D assay kit (Promega) , according to the manufacturer’s instructions. Assay data were normalized to DMSO values, and EC 50 values and Top Inhibition rates (%) were determined using a four-parameter concentration–response model in GraphPad Prism 7. TAK-788 is a small-molecule tyrosine kinase inhibitor (TKI) designed to selectively target EGFR and human EGFR 2 (HER2) exon 20 insertion mutations. Osimertinib is a third generation irreversible and mutant-selective EGFR inhibitor.
As shown in Table 1, Compound A, B and C surprisingly exhibited potent anti-proliferative activity in five EGFR exon 20ins NSCLC PDOs, among which three were also tested with Osimertinib, and exhibited apparent drug-resistance. Besides, Compound A surprisingly showed competitive EC 50 and even better Top Inhibition Rate than TAK-788, which directly binds to and inhibits the exon 20ins-mutated EGFR.
Table 1. Inhibitory parameters of test compounds in NSCLC PDOs
Figure PCTCN2021117497-appb-000050
n.d.: not determined
n.a.: not appropriate
Compounds A, B and C are also tested in comparison with additional agents reported to be effective to treat tumors bearing EGFR exon 20 insertions, including, for example, Gefitinib, Erlotinib, Afatinib, Dacomitinib, JNJ-61186372, Poziotinib, Almonertinib (HS-10296) , Amivantamab (JNJ-372) , furmonertinib (AST2818) , and DZD9008. Compounds A, B and C show better efficacy than these compounds in inhibiting tumor cells bearing EGFR exon 20 insertions.
Example 4
This example illustrates the activity of exemplary compounds in ex vivo cultured EGFR exon 20ins PDX-derived primary cells.
Activity of Compound A was tested using 2D ex vivo cultured primary cells derived from LU0387 lung cancer patient-derived xenograft (PDX) , which harbors an oncogenic exon 20 insertion mutation (H773_V774insNPH) in EGFR. Cell suspensions of the ex vivo cultured LU0387 were seeded into 96-well plates and incubated for 24 h for attachment, followed by compound treatment for additional 7 days. Cell viability was determined using CellTiter-Glo assay kit (Promega) according to the manufacturer’s instructions. Assay data were normalized to DMSO values, and EC 50 values were determined using a four-parameter concentration–response model in GraphPad Prism 7.
As demonstrated in FIG. 1 and Table 2, Compound A potently suppressed proliferation of LU0387 cells, with comparable activity to TAK-788, and was much more potent than Osimertinib and Cisplatin in head-to-head comparisons.
Table 2. EC 50 of test compounds in ex vivo cultured LU0387 cells
Figure PCTCN2021117497-appb-000051
Example 5
This example illustrates the combinational effect of Compound A and TAK-788 in ex vivo cultured EGFR exon 20ins PDX-derived primary cells.
Combination effect was tested between Compound A and TAK-788 using a dose matrix assay in LU0387 EGFR exon 20ins-bearing PDX ex vivo cell culture. Cell suspensions of the ex vivo cultured LU0387 were seeded into 96-well plates and incubated for 24 h, then treated with increasing concentrations of each drug to generate a 6×6 dose matrix. After incubation for 7 days, cell viability was assessed by CellTiter-Glo assay kit (Promega) according to the manufacturer’s instructions. Data were normalized to DMSO values and synergy was determined by the LOEWE model using Combenefit software, which generated a score for each dose combination: a positive score indicates synergy, a score of 0 indicates additive, and a negative score indicates antagonism (FIG 2) .
Compound A exhibits synergistic effect with TAK-788 at inhibiting proliferation of EGFR exon 20ins-bearing LU0387 primary cells in 2D ex vivo culture system.
Example 6
This example illustrates the in vivo efficacy of exemplary compounds in EGFR exon 20ins NSCLC PDX model.
BALB/c nude mice were subcutaneously transplanted with tumor fragments (2–3 mm 3) derived from an EGFR exon 20ins-bearing NSCLC PDX, LU0387 (H773_V774insNPH) . The mice were randomized into sub-groups when tumor volume reached to 100～200 mm 3, followed by oral administration (QD for 28days) of Compound A, TAK-788 or combination of Compound A and TAK-788, respectively. Tumor volumes and  body weights were measured twice a week through the treatment and continued for additional 19 days after drug withdrawal.
Compound A demonstrated strong in vivo anti-tumor activity in a dose-dependent manner in LU0387 PDX model (See FIG. 3) . The head-to-head comparison study suggested Compound A had superior activity to TAK-788, as Compound A at 5 mg/kg exhibited comparable activity with TAK-788 at 10 mg/kg, and Compound A at 15 mg/kg induced significant tumor regression. Besides, Compound A showed more sustainable tumor suppression after drug withdrawal. Notably, Compound A at 5 mg/kg exhibited profound synergistic effect with TAK-788 at 10 mg/kg, as significant and persistent tumor regressions were observed. All of the treatment were tolerated in this study.
These results for the first time reveal the dependency of EGFR exon 20ins-driven cancers on SHP2, and treatment with SHP2 inhibitors as monotherapy or combinational partner could potently suppress the in vivo tumor growth, with competitive or even superior efficacy to EGFR inhibitors.
Taken together, Compound A and its analogs surprisingly exhibited potent anti-proliferative activity against EGFR exon 20ins NSCLC PDOs and superior in vivo efficacy to EGFR inhibitors.

Claims (31)

  1. A method for treating a subject having an EGFR-related disease or condition, wherein an insertion mutation within exon 20 of the EGFR gene is detected in a biological sample of the subject, the method comprising:
    administering to the subject a therapeutic effective amount of a pharmaceutical composition comprising a SHP2 inhibitor.
  2. The method of claim 1, wherein the SHP2 inhibitor is a compound of formula Ib
    Figure PCTCN2021117497-appb-100001
    or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereoisomer, tautomer, solvate, isotope-substituted derivative, polymorph, prodrug or metabolite thereof, wherein,
    R 4 is selected from H, halogen, substituted or unsubstituted amino, substituted or unsubstituted C 1-C 6 alkyl, or substituted or unsubstituted C 1-C 6 alkoxyl;
    X 4 and X 5 are independently selected from C or N, and X 4 and X 5 are not N simultaneously;
    ring D is selected from substituted or unsubstituted 4 to 8-membered heterocyclyl, substituted or unsubstituted 4 to 8-membered carbocyclic group, substituted or unsubstituted C 5-10 aryl, or substituted or unsubstituted 5 to 10-membered heteroaryl; wherein the heterocyclyl or heteroaryl comprises 1-3 heteroatoms selected from: N, O, S or P;
    wherein any of the above term “substituted” refers to one or more hydrogen atoms on the group are substituted by a substituent selected from: -D, halogen, -OH, -NO 2, -NH 2, -N(unsubstituted or halogenated C 1-C 6 alkyl)  2, -CN, unsubstituted or halogenated C 1-C8 alkyl, unsubstituted or halogenated C 1-C 8 alkoxyl, unsubstituted or halogenated C 1-C 8 alkoxyl-C 1-C 8 alkyl, unsubstituted or halogenated C 3-C 8 cycloalkyl, unsubstituted or halogenated C 3-C 8 cycloalkyl-C 1-C 8 alkyl, unsubstituted or halogenated C 1-C 6 alkyl carbonyl, unsubstituted or halogenated C 1-C 6 alkoxyl carbonyl, hydroxamic acid group, unsubstituted or halogenated C 1-C 6 alkyl thiol, -S (O)  2N (unsubstituted or halogenated C 1-C 6 alkyl)  2, -S (O)  2 unsubstituted or halogenated C 1-C 6 alkyl, -N (unsubstituted or halogenated C 1-C 6 alkyl) S (O)  2N (unsubstituted  or halogenated C 1-C 6 alkyl)  2, -S (O) N (unsubstituted or halogenated C 1-C 6 alkyl)  2, -S (O) (unsubstituted or halogenated C1-C6 alkyl) , -N (unsubstituted or halogenated C 1-C 6 alkyl) S (O) N (unsubstituted or halogenated C 1-C 6 alkyl)  2, -N (unsubstituted or halogenated C 1-C 6 alkyl) S (O) (unsubstituted or halogenated C 1-C 6 alkyl) , unsubstituted or halogenated 5 to 8-membered aryl, unsubstituted or halogenated 5 to 8-membered heteroaryl, or unsubstituted or halogenated 4 to 8-membered saturated heterocyclyl or carbocyclic group; wherein the heteroaryl comprise 1-4 heteroatoms selected from: N, O or S, the heterocyclyl comprise 1-4 heteroatoms selected from: N, O or S.
  3. The method of claim 1, wherein the SHP2 inhibitor is a compound of formula I
    Figure PCTCN2021117497-appb-100002
    or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereoisomer, tautomer, solvate, isotope-substituted derivative, polymorph, prodrug or metabolite thereof, wherein,
    X 1 and X 2 are independently selected from a bond, O, CR aR b or NR c;
    X 3 is selected from a bond, CR aR b, NR c, S or O;
    X 4 is selected from N or CR c; and R a, R b and R c are independently selected from H, D, halogen, substituted or unsubstituted C 1-6 alkyl, or substituted or unsubstituted C 1-6 alkoxyl;
    R 1, R 2, R 3, R 4 and R 7 are independently selected from H, D, -OH, halogen, substituted or unsubstituted amino, substituted or unsubstituted C 1-6 alkyl, or substituted or unsubstituted C 1-6 alkoxyl; and R 1, R 2, R 3, R 4 and R 7 cannot be -OH or -NH 2 simultaneously;
    ring A is selected from substituted or unsubstituted C 4-8 cyclic hydrocarbyl, substituted or unsubstituted 4 to 8-membered heterocyclyl, substituted or unsubstituted C 5-10 aryl, or substituted or unsubstituted 5 to 10-membered heteroaryl, wherein the heterocyclyl or heteroaryl comprises 1-3 heteroatoms selected from: N, O, S or P;
    ring C is selected from substituted or unsubstituted C 4-8 cyclic hydrocarbyl, substituted or unsubstituted 5 to 6-membered monocyclic heterocyclyl, substituted or unsubstituted 8 to 10-membered bicyclic heterocyclyl, substituted or unsubstituted C 5-10 monocyclic or bicyclic  aryl, substituted or unsubstituted 5 to 6-membered monocyclic heteroaryl, or substituted or unsubstituted 8 to 10-membered bicyclic heteroaryl, wherein the heterocyclyl or heteroaryl comprises 1-4 heteroatoms selected from: N, O, S or P;
    R 5 and R 6 are independently selected from H, D, -OH, halogen, cyano, substituted or unsubstituted amino, substituted or unsubstituted C 1-6 alkyl, or substituted or unsubstituted C 1-6 alkoxyl;
    n is any integer from 0 to 3; and
    wherein the term “substituted” refers to one or more hydrogen atoms on the group are substituted by a substituent selected from: halogen, -OH, -NO 2, -NH 2, -NH (unsubstituted or halogenated C 1-6 alkyl) , -N (unsubstituted or halogenated C 1-6 alkyl)  2, -CN, unsubstituted or halogenated C 1-8 alkyl, unsubstituted or halogenated C 1-8 alkoxyl, unsubstituted or halogenated C 1-8 alkoxyl-C 1-8 alkyl, unsubstituted or halogenated C 3-8 cycloalkyl-C 1-8 alkyl, unsubstituted or halogenated C 1-6 alkyl carbonyl, unsubstituted or halogenated C 1-6 alkoxyl carbonyl, hydroxamic acid group, unsubstituted or halogenated C 1-6 alkyl thiol, -S (O)  2N (unsubstituted or halogenated C 1-6 alkyl)  2, -S (O)  2 unsubstituted or halogenated C 1-6 alkyl, -N (unsubstituted or halogenated C 1-6 alkyl) S (O)  2N (unsubstituted or halogenated C 1-6 alkyl)  2, -S (O) N (unsubstituted or halogenated C 1-6 alkyl)  2, -S (O) (unsubstituted or halogenated C 1-6 alkyl) , -N (unsubstituted or halogenated C 1-6 alkyl) S (O) N (unsubstituted or halogenated C 1-6 alkyl)  2, -N (unsubstituted or halogenated C 1-6 alkyl) S (O) (unsubstituted or halogenated C 1- 6 alkyl) , unsubstituted or halogenated C 5-10 aryl, unsubstituted or halogenated 5 to 10-membered heteroaryl, unsubstituted or halogenated C 4-8 cyclic hydrocarbyl, or unsubstituted or halogenated 4 to 8-membered heterocyclyl, wherein the heterocyclyl and heteroaryl comprise 1-4 heteroatoms selected from: N, O or S, wherein one or more hydrogens in the ring A, ring B, ring C, ring D, ring E and/or ring F are optionally substituted by D.
  4. The method of claim 3, wherein the compound has a structure selected from:
    Figure PCTCN2021117497-appb-100003
    Figure PCTCN2021117497-appb-100004
  5. The method of claim 2 or 3, wherein the isotope-substituted derivative comprises an isotope of an atom selected from the group consisting of hydrogen, carbon, nitrogen, oxygen, phosphorus, 
    Figure PCTCN2021117497-appb-100005
    chlorine or iodine; and
    Figure PCTCN2021117497-appb-100006
    preferably is  2H,  3H,  11C,  13C,  14C,  15N,  18F,  31P,  32P,  35S,  36Cl or  125I.
  6. The method of any of claims 1 to 5, wherein the compound has a structure of
    Figure PCTCN2021117497-appb-100007
  7. The method of claim 1, wherein the insertion mutation within exon 20 of the EGFR gene is an in-frame insertion mutation.
  8. The method of claim 7, wherein the insertion mutation within exon 20 of the EGFR gene is selected from the group consisting of A763_Y764insFQEA, A763_Y764insFQQA, A767_V769dupASV, V769_D770insASV, D770_N771insGL, D770_N771insGT, D770_N771insNPG, D770_N771insSVD, E762Q_insFQEA, H773_V774insH, H773_V774insH, H773_V774insNPH, M766_A767insAI, M766_A767insASV, N771_H773dupNPH, P772_H773insYNP, P772_V774insPHV, S768_770dupSVD, V769_D770insASV, Y764_V765insHH, and delD770insGY.
  9. The method of claim 1, wherein the insertion mutation is detected using a sequencing assay, an amplification assay, or a hybridization assay.
  10. The method of claim 1, wherein the biological sample is tissue or blood.
  11. The method of claim 1, wherein the biological sample comprises a cancer cell or DNA from a cancer cell.
  12. The method of claim 1, wherein the disease or condition is cancer.
  13. The method of any one of claims 12, wherein the cancer is selected from the groups consisting of lung cancer, glioblastoma, uterus cancer, bladder cancer, melanoma, renal cancer, liver cancer, myeloma, prostate cancer, breast cancer, colorectal cancer, pancreatic cancer, thyroid cancer, hematological cancer, leukemia and non-Hodgkin’s lymphoma.
  14. The method of claim 12, wherein the cancer is non-small cell lung cancer (NSCLC) , conventional glioblastoma multiforme, glioblastoma, infiltrating renal pelvis and ureter  urothelial carcinoma, and endometrial endometrioid adenocarcinoma, or bladder urothelial carcinoma.
  15. The method of claim 12, wherein the cancer is resistant to an EGFR tyrosine kinase inhibitor.
  16. The method of claim 12, wherein the cancer is metastatic.
  17. The method of claim 1, wherein a second mutation in a gene in the RTK/RAS/MAPK signaling pathway is further detected in the biological sample of the subject.
  18. The method of claim 17, wherein the gene is EGFR, KRAS or BRAF.
  19. The method of claim 1, further comprising administering to the subject a second therapeutic agent.
  20. The method of claim 19, wherein the second therapeutic agent is an EGFR tyrosine kinase inhibitor, a KRAS inhibitor, a BRAF inhibitor, a MEK inhibitor, a CDK inhibitor, or an immune checkpoint inhibitor (e.g. PD-1/PD-L1 antagonist) .
  21. The method of claim 19, wherein the EGFR tyrosine kinase inhibitor is Gefitinib, Erlotinib, Afatinib, Dacomitinib, Osimertinib, TAK-788, Poziotinib, JNJ-61186372, Almonertinib (HS-10296) , Amivantamab (JNJ-372) , furmonertinib (AST2818) , or DZD9008.
  22. The method of claim 19, wherein the KRAS inhibitor is AMG510.
  23. The method of claim 19, wherein the BRAF inhibitor is Vemurafenib, Dabrafenib, Encorafenib or Sorafenib.
  24. The method of claim 19, wherein the MEK inhibitor is Trametinib, Cobimetinib, Selumetinib, Pimasertib or Binimetinib.
  25. The method of claim 19, wherein the CDK inhibitor is Palbociclib, Ribociclib or Abemaciclib.
  26. A method of identifying a subject with an EGFR-related disease or condition who is likely to be responsive to treatment with an SHP2 inhibitor, wherein the method comprises:
    (a) providing a biological sample of the subject;
    (b) detecting presence or absence of an insertion mutation at exon 20 of the EGFR gene in the biological sample,
    (c) identifying the subject as being likely to be responsive to treatment with the SHP2 inhibitor if the insertion mutation at exon 20 of the EGFR gene is detected in the biological sample.
  27. The method of claim 26, wherein the biological sample is tissue or blood.
  28. The method of claim 26, wherein the disease or condition is cancer.
  29. The method of claim 28, wherein the biological sample comprises a cancer cell or DNA from a cancer cell.
  30. The method of any one of claims 26-29, wherein the SHP2 inhibitor is a compound of formula Ib
    Figure PCTCN2021117497-appb-100008
    or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereoisomer, tautomer, solvate, isotope-substituted derivative, polymorph, prodrug or metabolite thereof, wherein,
    R 4 is selected from H, halogen, substituted or unsubstituted amino, substituted or unsubstituted C 1-C 6 alkyl, or substituted or unsubstituted C 1-C 6 alkoxyl;
    X 4 and X 5 are independently selected from C or N, and X 4 and X 5 are not N simultaneously;
    ring D is selected from substituted or unsubstituted 4 to 8-membered heterocyclyl, substituted or unsubstituted 4 to 8-membered carbocyclic group, substituted or unsubstituted  C 5-10 aryl, or substituted or unsubstituted 5 to 10-membered heteroaryl; wherein the heterocyclyl or heteroaryl comprises 1-3 heteroatoms selected from: N, O, S or P;
    wherein any of the above term “substituted” refers to one or more hydrogen atoms on the group are substituted by a substituent selected from: -D, halogen, -OH, -NO 2, -NH 2, -N(unsubstituted or halogenated C 1-C 6 alkyl)  2, -CN, unsubstituted or halogenated C 1-C8 alkyl, unsubstituted or halogenated C 1-C 8 alkoxyl, unsubstituted or halogenated C 1-C 8 alkoxyl-C 1-C 8 alkyl, unsubstituted or halogenated C 3-C 8 cycloalkyl, unsubstituted or halogenated C 3-C 8 cycloalkyl-C 1-C 8 alkyl, unsubstituted or halogenated C 1-C 6 alkyl carbonyl, unsubstituted or halogenated C 1-C 6 alkoxyl carbonyl, hydroxamic acid group, unsubstituted or halogenated C 1-C 6 alkyl thiol, -S (O)  2N (unsubstituted or halogenated C 1-C 6 alkyl)  2, -S (O)  2 unsubstituted or halogenated C 1-C 6 alkyl, -N (unsubstituted or halogenated C 1-C 6 alkyl) S (O)  2N (unsubstituted or halogenated C 1-C 6 alkyl)  2, -S (O) N (unsubstituted or halogenated C 1-C 6 alkyl)  2, -S (O) (unsubstituted or halogenated C1-C6 alkyl) , -N (unsubstituted or halogenated C 1-C 6 alkyl) S (O) N (unsubstituted or halogenated C 1-C 6 alkyl)  2, -N (unsubstituted or halogenated C 1-C 6 alkyl) S (O) (unsubstituted or halogenated C 1-C 6 alkyl) , unsubstituted or halogenated 5 to 8-membered aryl, unsubstituted or halogenated 5 to 8-membered heteroaryl, or unsubstituted or halogenated 4 to 8-membered saturated heterocyclyl or carbocyclic group; wherein the heteroaryl comprise 1-4 heteroatoms selected from: N, O or S, the heterocyclyl comprise 1-4 heteroatoms selected from: N, O or S.
  31. The method of any one of claims 26-29, wherein the SHP2 inhibitor is a compound having a structure of formula I,
    Figure PCTCN2021117497-appb-100009
    or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereoisomer, tautomer, solvate, isotope-substituted derivative, polymorph, prodrug or metabolite thereof, wherein,
    X 1 and X 2 are independently selected from a bond, O, CR aR b or NR c;
    X 3 is selected from a bond, CR aR b, NR c, S or O;
    X 4 is selected from N or CR c; and R a, R b and R c are independently selected from H, D, halogen, substituted or unsubstituted C 1-6 alkyl, or substituted or unsubstituted C 1-6 alkoxyl;
    R 1, R 2, R 3, R 4 and R 7 are independently selected from H, D, -OH, halogen, substituted or unsubstituted amino, substituted or unsubstituted C 1-6 alkyl, or substituted or unsubstituted C 1-6 alkoxyl; and R 1, R 2, R 3, R 4 and R 7 cannot be -OH or -NH 2 simultaneously;
    ring A is selected from substituted or unsubstituted C 4-8 cyclic hydrocarbyl, substituted or unsubstituted 4 to 8-membered heterocyclyl, substituted or unsubstituted C 5-10 aryl, or substituted or unsubstituted 5 to 10-membered heteroaryl, wherein the heterocyclyl or heteroaryl comprises 1-3 heteroatoms selected from: N, O, S or P;
    ring C is selected from substituted or unsubstituted C 4-8 cyclic hydrocarbyl, substituted or unsubstituted 5 to 6-membered monocyclic heterocyclyl, substituted or unsubstituted 8 to 10-membered bicyclic heterocyclyl, substituted or unsubstituted C 5-10 monocyclic or bicyclic aryl, substituted or unsubstituted 5 to 6-membered monocyclic heteroaryl, or substituted or unsubstituted 8 to 10-membered bicyclic heteroaryl, wherein the heterocyclyl or heteroaryl comprises 1-4 heteroatoms selected from: N, O, S or P;
    R 5 and R 6 are independently selected from H, D, -OH, halogen, cyano, substituted or unsubstituted amino, substituted or unsubstituted C 1-6 alkyl, or substituted or unsubstituted C 1-6 alkoxyl;
    n is any integer from 0 to 3; and
    wherein the term “substituted” refers to one or more hydrogen atoms on the group are substituted by a substituent selected from: halogen, -OH, -NO 2, -NH 2, -NH (unsubstituted or halogenated C 1-6 alkyl) , -N (unsubstituted or halogenated C 1-6 alkyl)  2, -CN, unsubstituted or halogenated C 1-8 alkyl, unsubstituted or halogenated C 1-8 alkoxyl, unsubstituted or halogenated C 1-8 alkoxyl-C 1-8 alkyl, unsubstituted or halogenated C 3-8 cycloalkyl-C 1-8 alkyl, unsubstituted or halogenated C 1-6 alkyl carbonyl, unsubstituted or halogenated C 1-6 alkoxyl carbonyl, hydroxamic acid group, unsubstituted or halogenated C 1-6 alkyl thiol, -S (O)  2N (unsubstituted or halogenated C 1-6 alkyl)  2, -S (O)  2 unsubstituted or halogenated C 1-6 alkyl, -N (unsubstituted or halogenated C 1-6 alkyl) S (O)  2N (unsubstituted or halogenated C 1-6 alkyl)  2, -S (O) N (unsubstituted or halogenated C 1-6 alkyl)  2, -S (O) (unsubstituted or halogenated C 1-6 alkyl) , -N (unsubstituted or halogenated C 1-6 alkyl) S (O) N (unsubstituted or halogenated C 1-6 alkyl)  2, -N (unsubstituted or halogenated C 1-6 alkyl) S (O) (unsubstituted or halogenated C 1- 6 alkyl) , unsubstituted or halogenated C 5-10 aryl, unsubstituted or halogenated 5 to 10-membered heteroaryl, unsubstituted or halogenated C 4-8 cyclic hydrocarbyl, or unsubstituted or halogenated 4 to 8-membered heterocyclyl, wherein the heterocyclyl and heteroaryl  comprise 1-4 heteroatoms selected from: N, O or S, wherein one or more hydrogens in the ring A, ring B, ring C, ring D, ring E and/or ring F are optionally substituted by D.
PCT/CN2021/117497 2021-05-20 2021-09-09 Methods for treating cancers associated with egfr mutation WO2022241975A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CNPCT/CN2021/094974 2021-05-20
CN2021094974 2021-05-20

Publications (1)

Publication Number Publication Date
WO2022241975A1 true WO2022241975A1 (en) 2022-11-24

Family

ID=84141094

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/117497 WO2022241975A1 (en) 2021-05-20 2021-09-09 Methods for treating cancers associated with egfr mutation

Country Status (1)

Country Link
WO (1) WO2022241975A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023172940A1 (en) 2022-03-08 2023-09-14 Revolution Medicines, Inc. Methods for treating immune refractory lung cancer
WO2023240263A1 (en) 2022-06-10 2023-12-14 Revolution Medicines, Inc. Macrocyclic ras inhibitors

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020094018A1 (en) * 2018-11-06 2020-05-14 上海奕拓医药科技有限责任公司 Spiro aromatic ring compound and application thereof
WO2020108590A1 (en) * 2018-11-30 2020-06-04 上海拓界生物医药科技有限公司 Pyrimidine and five-membered nitrogen heterocycle derivative, preparation method therefor, and medical uses thereof
WO2020156243A1 (en) * 2019-01-31 2020-08-06 贝达药业股份有限公司 Shp2 inhibitor and application thereof
CN111704611A (en) * 2019-07-25 2020-09-25 上海凌达生物医药有限公司 Aryl spiro SHP2 inhibitor compound, preparation method and application
WO2020259679A1 (en) * 2019-06-28 2020-12-30 上海拓界生物医药科技有限公司 Pyrimidine five-membered nitrogen heterocyclic derivative, preparation method thereof and pharmaceutical use thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020094018A1 (en) * 2018-11-06 2020-05-14 上海奕拓医药科技有限责任公司 Spiro aromatic ring compound and application thereof
WO2020108590A1 (en) * 2018-11-30 2020-06-04 上海拓界生物医药科技有限公司 Pyrimidine and five-membered nitrogen heterocycle derivative, preparation method therefor, and medical uses thereof
WO2020156243A1 (en) * 2019-01-31 2020-08-06 贝达药业股份有限公司 Shp2 inhibitor and application thereof
WO2020259679A1 (en) * 2019-06-28 2020-12-30 上海拓界生物医药科技有限公司 Pyrimidine five-membered nitrogen heterocyclic derivative, preparation method thereof and pharmaceutical use thereof
CN111704611A (en) * 2019-07-25 2020-09-25 上海凌达生物医药有限公司 Aryl spiro SHP2 inhibitor compound, preparation method and application

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HALLIDAY, P.R. ET AL.: "Emerging Targeted Therapies for the Treatment of Non-small Cell Lung Cancer", CURRENT ONCOLOGY REPORTS, vol. 21, no. 3, 26 February 2019 (2019-02-26), XP036745561, ISSN: 1523-3790, DOI: 10.1007/s11912-019-0770-x *
SUN, YUTING ET AL.: "Allosteric SHP2 Inhibitor, IACS-13909, Overcomes EGFR- Dependent and EGFR-Independent Resistance Mechanisms toward Osimertinib", CANCER RES., vol. 80, no. 21, 14 September 2020 (2020-09-14), pages 4840 - 4853, XP093007501, ISSN: 0008-5472, DOI: 10.1158/0008-5472.CAN-20-1634 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023172940A1 (en) 2022-03-08 2023-09-14 Revolution Medicines, Inc. Methods for treating immune refractory lung cancer
WO2023240263A1 (en) 2022-06-10 2023-12-14 Revolution Medicines, Inc. Macrocyclic ras inhibitors

Similar Documents

Publication Publication Date Title
EP3216786B1 (en) Pyrimidine compounds, preparation method therefor and pharmaceutical uses thereof
WO2022241975A1 (en) Methods for treating cancers associated with egfr mutation
CN113015724A (en) Improved synthesis of key intermediates of KRAS G12C inhibitor compounds
EP4157837A1 (en) Annulated 2-amino-3-cyano thiophenes and derivatives for the treatment of cancer
JP2021526133A (en) KRAS G12C inhibitor for treating cancer
AU2020364007A1 (en) Bicyclic amines as CDK2 inhibitors
JP5291345B2 (en) Cancer treatment
JP6507234B2 (en) Pyrazole carboxamide compounds for use in the treatment of disorders mediated by bruton tyrosine kinase (BTK)
TW201720816A (en) Inhibitors of CXCR2
BRPI0922225A2 (en) umidazopyrazine syk inhibitors
CN103664878A (en) Hetero-aromatic ring and derivative type tyrosine kinase inhibitor thereof
KR20180052623A (en) The novel compound
US11981671B2 (en) Bicyclic pyrazolyl amines as CDK2 inhibitors
US9682982B2 (en) Inhibitors of adapter associated kinase 1, compositions comprising them, and methods of their use
EP3585789A1 (en) Inhibitors of bruton's tyrosine kinase
EP4100409A1 (en) [1,3]diazino[5,4-d]pyrimidines as her2 inhibitors
US20230192706A1 (en) Bicyclic amines as cdk2 inhibitors
WO2023099612A1 (en) Annulated 2-amino-3-cyano thiophenes and derivatives for the treatment of cancer
WO2020244654A1 (en) Method for treating cancer patients using c-met inhibitor
CN107406418B (en) 4, 5-dihydroimidazole derivatives and their use as histone demethylase (KDM2B) inhibitors
US11976073B2 (en) Bicyclic amines as CDK2 inhibitors
WO2016091916A1 (en) Pyrazolylaminopurines as itk inhibitors
CN114073704B (en) Use of fluorine-containing heterocyclic derivative with macrocyclic structure
TW201504239A (en) Protein tyrosine kinase inhibitor and application thereof
WO2023240189A1 (en) Tetrahydropyrido 3,4-d pyrimidine derivatives as kras inhibitors

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21940424

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21940424

Country of ref document: EP

Kind code of ref document: A1