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WO2014140989A2 - Combinaison d'un inhibiteur de l'egfr t790m et d'un inhibiteur de l'egfr dans le traitement d'un cancer du poumon non à petites cellules - Google Patents

Combinaison d'un inhibiteur de l'egfr t790m et d'un inhibiteur de l'egfr dans le traitement d'un cancer du poumon non à petites cellules Download PDF

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Publication number
WO2014140989A2
WO2014140989A2 PCT/IB2014/059401 IB2014059401W WO2014140989A2 WO 2014140989 A2 WO2014140989 A2 WO 2014140989A2 IB 2014059401 W IB2014059401 W IB 2014059401W WO 2014140989 A2 WO2014140989 A2 WO 2014140989A2
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WIPO (PCT)
Prior art keywords
egfr
inhibitor
compound
dacomitinib
pharmaceutically acceptable
Prior art date
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Ceased
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PCT/IB2014/059401
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English (en)
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WO2014140989A3 (fr
Inventor
Zelanna Iris GOLDBERG
John Charles Kath
Stephen Paul Letrent
Scott Lawrence Weinrich
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pfizer Corp Belgium
Pfizer Corp SRL
Original Assignee
Pfizer Corp Belgium
Pfizer Corp SRL
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Priority to RU2015137596A priority Critical patent/RU2015137596A/ru
Priority to KR1020157024916A priority patent/KR20150119210A/ko
Priority to CN201480014630.2A priority patent/CN105073116A/zh
Priority to EP14710651.2A priority patent/EP2968336A2/fr
Priority to MX2015012106A priority patent/MX2015012106A/es
Priority to SG11201506531WA priority patent/SG11201506531WA/en
Priority to BR112015023020A priority patent/BR112015023020A2/pt
Priority to CA2904797A priority patent/CA2904797A1/fr
Application filed by Pfizer Corp Belgium, Pfizer Corp SRL filed Critical Pfizer Corp Belgium
Priority to AU2014229468A priority patent/AU2014229468A1/en
Publication of WO2014140989A2 publication Critical patent/WO2014140989A2/fr
Publication of WO2014140989A3 publication Critical patent/WO2014140989A3/fr
Priority to IL240730A priority patent/IL240730A0/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4709—Non-condensed quinolines 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
    • A61K31/506—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
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • 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
    • A61K31/535—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
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/553—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having at least one nitrogen and one oxygen as ring hetero atoms, e.g. loxapine, staurosporine
    • 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
    • A61K31/66—Phosphorus compounds
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to a method of treating non-small cell lung cancer by administering a combination of an EGFR T790M inhibitor in combination with a low-dose amount of a panHER inhibitor.
  • This invention also relates to a method of treating non- small cell lung cancer by administering a combination of an irreversible EGFR T790M inhibitor in combination with an EGFR inhibitor.
  • Non-small cell lung cancer is the leading cause of cancer death worldwide, with an estimated 1 .4 million new cases diagnosed each year.
  • lung adenocarcinoma which is the most common form of non-small cell lung cancer
  • EGFR inhibitors such as erlotinib or gefitinib can be most effective (Paez et al. Science 2004; Lynch et al. NEJM 2004; Pao et al, PNAS 2004).
  • the most common activating mutations associated with good response to these inhibitors are deletions within exon 19 (e.g.
  • E746-A750 and point mutations in the activation loop (exon 21 , in particular, L858R). Additional somatic mutations identified to date but to a lesser extent include point mutations: G719S, G719C, G719A, L861 and small insertions in Exon 20 (Shigematsu ei a/ JNCI 2005; Fukuoka et al. JCO 2003; Kris et al JAMA 2003 and Shepherd et al NEJM 2004).
  • Some embodiments described herein relate to a method of treating non-small cell lung cancer comprising administering to a patient in need thereof an effective amount of an irreversible EGFR T790M inhibitor in combination with an effective amount of an EGFR inhibitor.
  • the irreversible EGFR T790M inhibitor is 1 - ⁇ (3R,4R)-3-[( ⁇ 5-chloro-2-[(1 -methyl-1 H-pyrazol-4-yl)amino]- 7/-/-pyrrolo[2,3-d]pyrimidin-4-yl ⁇ oxy)methyl]-4-methoxypyrrolidin-1 -yl ⁇ prop-2-en-1 -one, or a pharmaceutically acceptable salt thereof.
  • the irreversible EGFR T790M inhibitor is /V-methyl-/V-[irans-3-( ⁇ 2-[(1 -methyl-1 /-/-pyrazol-4-yl)amino]-5- (pyridin-2-yl)-7/-/-pyrrolo[2,3-cf]pyrimidin-4-yl ⁇ oxy)cyclobutyl]prop-2-enamide, or a pharmaceutically acceptable salt thereof.
  • the irreversible thermoplastic material in certain embodiments of the method of the present invention, the irreversible thermoplastic material
  • EGFR T790M inhibitor is /V-[frans-3-( ⁇ 5-chloro-2-[(1 ,3-dimethyl-1 /-/-pyrazol-4-yl)amino]- 7/-/-pyrrolo[2,3-d]pyrimidin-4-yl ⁇ amino)cyclobutyl]-/V-methylprop-2-enamide, or a pharmaceutically acceptable salt thereof.
  • the irreversible EGFR T790M inhibitor is 1 - ⁇ (3R,4R)-3-[( ⁇ 5-chloro-2-[(3-methoxy-1 -methyl-1 H-pyrazol-4- yl)amino]-7/-/-pyrrolo[2,3-cf]pyrimidin-4-yl ⁇ oxy)methyl]-4-methoxypyrrolidin-1 -yl ⁇ prop-2- en-1 -one, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, icotinib, vandetanib, lapatinib, neratinib, afatinib, pelitinib, dacomitinib, canertinib, cetuximab and panitumumab, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is selected from the group consisting of gefitinib, erlotinib, icotinib, vandetanib, lapatinib, neratinib, afatinib, pelitinib, dacomitinib, and canertinib, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is selected from the group consisting of gefitinib, eriotinib, afatinib, and dacomitinib, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is gefitinib, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is eriotinib, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is afatinib, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is dacomitinib, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is a reversible EGFR inhibitor.
  • the reversible EGFR inhibitor is selected from the group consisting of gefitinib, eriotinib, icotinib, vandetanib, and lapatinib, or a pharmaceutically acceptable salt thereof.
  • the reversible EGFR inhibitor is gefitinib, or a pharmaceutically acceptable salt thereof.
  • the reversible EGFR inhibitor is eriotinib, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is an irreversible EGFR inhibitor.
  • the irreversible EGFR inhibitor is selected from the group consisting of neratinib, afatinib, pelitinib, dacomitinib, and canertinib, or a pharmaceutically acceptable salt thereof.
  • the irreversible EGFR inhibitor is afatinib, or a pharmaceutically acceptable salt thereof.
  • the irreversible thermoplastic material in some embodiments of the method of the present invention, the irreversible thermoplastic material
  • EGFR inhibitor is dacomitinib, or a pharmaceutically acceptable salt thereof.
  • Some embodiments of the present invention relate to a method of treating non- small cell lung cancer comprising administering to a patient in need thereof an effective amount of an EGFR T790M inhibitor in combination with a panHER inhibitor, wherein the panHER inhibitor is administered according to a non-standard clinical dosing regimen.
  • the non-standard clinical dosing regimen is a non-standard clinical dose or a non-standard dosing schedule.
  • the non-standard clinical dosing regimen is a low-dose amount of the panHER inhibitor.
  • the non-standard clinical dosing regimen is an intermittent dosing regimen.
  • the EGFR T790M inhibitor is selected from the group consisting of Go6976, PKC412, AP261 13, HM61713, WZ4002, CO-1686 and TAS-2913, or a pharmaceutically acceptable salt thereof.
  • the EGFR T790M inhibitor is 1 - ⁇ (3R,4R)-3-[( ⁇ 5-chloro-2-[(1 -methyl-1 H-pyrazol-4-yl)amino]-7/-/-pyrrolo[2, 3- d]pyrimidin-4-yl ⁇ oxy)methyl]-4-methoxypyrrolidin-1 -yl ⁇ prop-2-en-1 -one, or a
  • the EGFR T790M inhibitor is /V-methyl-/V-[frans-3-( ⁇ 2-[(1 -methyl-1 H-pyrazol-4-yl)amino]-5-(pyridin-2-yl)-7/-/- pyrrolo[2,3-d]pyrimidin-4-yl ⁇ oxy)cyclobutyl]prop-2-enamide, or a pharmaceutically acceptable salt thereof.
  • the EGFR T790M inhibitor is /V-[irans-3-( ⁇ 5-chloro-2-[(1 ,3-dimethyl-1 /-/-pyrazol-4-yl)amino]-7/-/- pyrrolo[2,3-d]pyrimidin-4-yl ⁇ amino)cyclobutyl]-/V-methylprop-2-enamide, or a
  • the EGFR T790M inhibitor is 1 - ⁇ (3R,4R)-3-[( ⁇ 5-chloro-2-[(3-methoxy-1 -methyl-1 /-/-pyrazol-4-yl)amino]-7/-/- pyrrolo[2,3-d]pyrimidin-4-yl ⁇ oxy)methyl]-4-methoxypyrrolidin-1 -yl ⁇ prop-2-en-1 -one, or a pharmaceutically acceptable salt thereof.
  • the panHER inhibitor is selected from the group consisting of lapatinib, neratinib, afatinib, pelitinib, dacomitinib, and canertinib, or a pharmaceutically acceptable salt thereof. In certain embodiments of the method of the present invention, the panHER inhibitor is selected from the group consisting of lapatinib, neratinib, afatinib, pelitinib, dacomitinib, and canertinib, or a pharmaceutically acceptable salt thereof.
  • the panHER inhibitor is afatinib, or a pharmaceutically acceptable salt thereof.
  • the panHER inhibitor is dacomitinib, or a pharmaceutically acceptable salt thereof.
  • the panHER inhibitor is an irreversible EGFR inhibitor.
  • the irreversible panHER inhibitor is selected from the group consisting of neratinib, afatinib, pelitinib, dacomitinib, and canertinib, or a pharmaceutically acceptable salt thereof.
  • the irreversible panHER inhibitor is afatinib, or a pharmaceutically acceptable salt thereof.
  • the irreversible panHER inhibitor is dacomitinib, or a pharmaceutically acceptable salt thereof.
  • Certain embodiments of the present invention relate to a method of treating non- small cell lung cancer comprising administering to a patient in need thereof a synergistic amount of an EGFR T790M inhibitor in combination with an EGFR inhibitor.
  • the EGFR T790M inhibitor is selected from the group consisting of Go6976, PKC412, AP261 13, HM61713, WZ4002, CO-1686 and TAS-2913, or a pharmaceutically acceptable salt thereof.
  • the EGFR T790M inhibitor is 1 - ⁇ (3R,4R)-3-[( ⁇ 5-chloro-2-[(1 -methyl-1 H-pyrazol-4-yl)amino]-7/-/-pyrrolo[2, 3- d]pyrimidin-4-yl ⁇ oxy)methyl]-4-methoxypyrrolidin-1 -yl ⁇ prop-2-en-1 -one, or a
  • the EGFR T790M inhibitor is /V-methyl-/V-[frans-3-( ⁇ 2-[(1 -methyl-1 /-/-pyrazol-4-yl)amino]-5-(pyridin-2-yl)-7/-/- pyrrolo[2,3-d]pyrimidin-4-yl ⁇ oxy)cyclobutyl]prop-2-enamide, or a pharmaceutically acceptable salt thereof.
  • the EGFR T790M inhibitor is /V-[frans-3-( ⁇ 5-chloro-2-[(1 ,3-dimethyl-1 H-pyrazol-4-yl)amino]-7/-/-pyrrolo[2,3- c
  • the EGFR T790M inhibitor is 1 - ⁇ (3R,4R)-3-[( ⁇ 5-chloro-2-[(3-methoxy-1 -methyl-1 H-pyrazol-4-yl)amino]-7H- pyrrolo[2,3-d]pyrimidin-4-yl ⁇ oxy)methyl]-4-methoxypyrrolidin-1 -yl ⁇ prop-2-en-1 -one, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is selected from the group consisting of gefitinib, eriotinib, icotinib, vandetanib, lapatinib, neratinib, afatinib, pelitinib, dacomitinib, canertinib, cetuximab and
  • panitumumab or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is selected from the group consisting of gefitinib, eriotinib, icotinib, vandetanib, lapatinib, neratinib, afatinib, pelitinib, dacomitinib, and canertinib, or a pharmaceutically
  • the EGFR inhibitor is selected from the group consisting of gefitinib, eriotinib, afatinib, and dacomitinib, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is gefitinib, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is eriotinib, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is a panHER inhibitor.
  • the panHER inhibitor is selected from the group consisting of lapatinib, neratinib, afatinib, pelitinib, dacomitinib, and canertinib, or a pharmaceutically acceptable salt thereof.
  • the panHER inhibitor is selected from the group consisting of lapatinib, neratinib, afatinib, pelitinib, dacomitinib, and canertinib, or a pharmaceutically acceptable salt thereof.
  • the panHER inhibitor is afatinib, or a pharmaceutically acceptable salt thereof.
  • the panHER inhibitor is dacomitinib, or a pharmaceutically acceptable salt thereof. In additional embodiments of the method of the present invention, the panHER inhibitor is an irreversible EGFR inhibitor.
  • the irreversible panHER inhibitor is selected from the group consisting of neratinib, afatinib, pelitinib, dacomitinib, and canertinib, or a pharmaceutically acceptable salt thereof.
  • the irreversible panHER inhibitor is afatinib, or a pharmaceutically acceptable salt thereof.
  • the irreversible panHER inhibitor is dacomitinib, or a pharmaceutically acceptable salt thereof.
  • component (a) an EGFR T790M inhibitor; and (b) an EGFR inhibitor, wherein component (a) and component (b) are synergistic.
  • the EGFR T790M inhibitor is selected from the group consisting of Go6976, PKC412, AP261 13, HM61713, WZ4002, CO-1686 and TAS-2913, or a pharmaceutically acceptable salt thereof.
  • the EGFR T790M inhibitor is 1 - ⁇ (3R,4R)-3-[( ⁇ 5-chloro-2-[(1 -methyl-1 H-pyrazol-4-yl)amino]-7H- pyrrolo[2,3-d]pyrimidin-4-yl ⁇ oxy)methyl]-4-methoxypyrrolidin-1 -yl ⁇ prop-2-en-1 -one, or a pharmaceutically acceptable salt thereof.
  • the EGFR T790M inhibitor is /V-methyl-/V-[frans-3-( ⁇ 2-[(1 -methyl-1 /-/-pyrazol-4-yl)amino]-5-(pyridin-2-yl)-7/-/- pyrrolo[2,3-d]pyrimidin-4-yl ⁇ oxy)cyclobutyl]prop-2-enamide, or a pharmaceutically acceptable salt thereof.
  • the EGFR is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N
  • T790M inhibitor is /V-[irans-3-( ⁇ 5-chloro-2-[(1 ,3-dimethyl-1 /-/-pyrazol-4-yl)amino]-7/-/- pyrrolo[2,3-d]pyrimidin-4-yl ⁇ amino)cyclobutyl]-/V-methylprop-2-enamide, or a
  • the EGFR T790M inhibitor is 1 - ⁇ (3R,4R)-3-[( ⁇ 5-chloro-2-[(3-methoxy-1 -methyl-1 /-/-pyrazol-4- yl)amino]-7/-/-pyrrolo[2,3-cf]pyrimidin-4-yl ⁇ oxy)methyl]-4-methoxypyrrolidin-1 -yl ⁇ prop-2- en-1 -one, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is selected from the group consisting of gefitinib, eriotinib, icotinib, vandetanib, lapatinib, neratinib, afatinib, pelitinib, dacomitinib, canertinib, cetuximab and
  • panitumumab or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is selected from the group consisting of gefitinib, eriotinib, icotinib, vandetanib, lapatinib, neratinib, afatinib, pelitinib, dacomitinib, and canertinib, or a pharmaceutically
  • the EGFR inhibitor is selected from the group consisting of gefitinib, eriotinib, afatinib, and dacomitinib, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is gefitinib, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is eriotinib, or a pharmaceutically acceptable salt thereof.
  • the EGFR inhibitor is a panHER inhibitor.
  • the panHER inhibitor is selected from the group consisting of lapatinib, neratinib, afatinib, pelitinib, dacomitinib, and canertinib, or a pharmaceutically acceptable salt thereof.
  • the panHER inhibitor is selected from the group consisting of lapatinib, neratinib, afatinib, pelitinib, dacomitinib, and canertinib, or a pharmaceutically acceptable salt thereof.
  • the panHER inhibitor is afatinib, or a pharmaceutically acceptable salt thereof.
  • the panHER inhibitor is dacomitinib, or a pharmaceutically acceptable salt thereof.
  • the panHER inhibitor is an irreversible EGFR inhibitor.
  • the irreversible panHER inhibitor is selected from the group consisting of neratinib, afatinib, pelitinib, dacomitinib, and canertinib, or a pharmaceutically acceptable salt thereof. In some embodiments of the combination of the present invention, the irreversible panHER inhibitor is afatinib, or a pharmaceutically acceptable salt thereof.
  • the irreversible panHER inhibitor is dacomitinib, or a pharmaceutically acceptable salt thereof.
  • Figure 1 shows that Sanger sequencing identified a C>T EGFR T790M mutation in RPC9 clone 3 and clone 6, with the percentages shown representing castPCR quantified EGFR T790M alleles with respect to the total EGFR alleles.
  • Figure 2 shows dose response curves in cell viability assays for PC9 and RPC9 clones 3 and 6 that were treated with various concentration of dacomitinib (Figure 2A) or erlotinib ( Figure 2B).
  • Figure 3 shows dose response curves in an RPC9 clone 6 cell viability assay.
  • Figure 3A shows dose response curves of Compound A ("compd A”) and dacomitinib (“daco”) alone and in combination.
  • Figure 3B shows dose response curves of
  • Figure 3C shows the percent of inhibition at selected concentrations of Compound A as a single agent and in combination with dacomitinib and erlotinib.
  • Figure 4 shows dose response curves in an RPC9 clone 6 cell viability assay.
  • Figure 4A shows dose response curves of Compound B ("compd B") and dacomitinib (“daco”) alone and in combination.
  • Figure 4B shows dose response curves of
  • Figure 4C shows the percent of inhibition at selected concentrations of Compound B as a single agent and in combination with dacomitinib and erlotinib.
  • Figure 5 shows dose response curves in an RPC9 clone 6 cell viability assay.
  • Figure 5A shows dose response curves of Compound A ("compd A”) alone and in combination with dacomitinib ("daco”).
  • Figure 5B shows dose response curves of Compound A alone and in combination with gefitinib ("gefi”).
  • Figure 5C shows dose response curves of Compound A alone and in combination with afatinib ("afat”).
  • Figure 5D shows the percent of inhibition at selected concentrations of Compound A as a single agent and in combination with dacomitinib, gefitinib and afatinib.
  • Figure 6 shows dose response curves in an RPC9 clone 6 cell viability assay.
  • Figure 6A shows dose response curves of Compound B ("compd B") alone and in combination with dacomitinib ("daco”).
  • Figure 6B shows dose response curves of Compound B alone and in combination with gefitinib ("gefi”).
  • Figure 6C shows dose response curves of Compound B alone and in combination with afatinib ("afat”).
  • Figure 4D shows the percent of inhibition at selected concentrations of Compound B as a single agent and in combination with dacomitinib, gefitinib and afatinib.
  • Figure 7 shows a Western immunoblot of the phosphorylation levels of EGFR, AKT, and ERK in RPC9 clone 6 cells. GAPDH was included as a protein loading control.
  • Figure 7A shows the RPC9 clone 6 cells treated with DMSO, dacomitinib, Compound A or a combination of dacomitinib + Compound A ("Compd A").
  • Figure 7B shows the RPC9 clone 6 cells treated with DMSO, erlotinib, Compound A or a combination of erlotinib + Compound A ("Compd A").
  • Figure 8 shows the densitometry results on the bands of the Western immunoblot (Figure 7A) of the RPC9 clone 6 cells treated with DMSO, dacomitinib, Compound A or a combination of dacomitinib ("Daco") + Compound A ("Compd A”).
  • Inhibition of pEGFR Y1068 ( Figure 8A), pAKT S473 ( Figure 8B), and pERK T202/Y204 ( Figure 8C) was determined by comparison to the DMSO control.
  • Figure 9 shows the densitometry results on the bands of the Western immunoblot (Figure 7B) of the RPC9 clone 6 cells treated with DMSO, erlotinib, Compound A or a combination of erlotinib ("Erlo") + Compound A ("Compd A”).
  • Inhibition of pEGFR Y1068 ( Figure 9A), pAKT S473 ( Figure 9B), and pERK T202/Y204 ( Figure 9C) was determined by comparison to the DMSO control.
  • Figure 10 shows a Western immunoblot of the phosphorylation levels of EGFR, AKT, and ERK in RPC9 clone 6 cells. GAPDH was included as a protein loading control.
  • Figure 10A shows the RPC9 clone 6 cells treated with DMSO, dacomitinib, Compound B or a combination of dacomitinib + Compound B ("Compd B").
  • Figure 10B shows the RPC9 clone 6 cells treated with DMSO, erlotinib, Compound B or a combination of erlotinib + Compound B ("Compd B").
  • Figure 1 1 shows the densitometry results on the bands of the Western
  • T202/Y204 ( Figure 12C) was determined by comparison to the DMSO control.
  • Figure 13 graphs the results of the xenograft model with RPC9 clone 6 tumor bearing SCID mice, which were randomized, daily and orally treated with vehicle, dacomitinib, Compound A, or dacomitinib ("Daco") + Compound A ("Compd A").
  • Figure 13A graphs the tumor volumes, which were measured 3 times per week and graphed with mean and standard error of the mean.
  • Figure 13B graphs the body weight of each group, which was recorded daily and percentage changes were graphed with mean and standard error of the mean.
  • Figure 14 graphs the results of the xenograft model with RPC9 clone 6 tumor bearing SCID mice, which were randomized, daily and orally treated with vehicle, dacomitinib, Compound B, or dacomitinib ("Daco") + Compound B (Compd B").
  • Figure 14A graphs the tumor volumes, which were measured 3 times per week and graphed with mean and standard error of the mean.
  • Figure 14B graphs the body weight of each group, which was recorded daily and percentage changes were graphed with mean and standard error of the mean.
  • Figure 15 graphs the results of the xenograft model with RPC9 clone 6 tumor bearing SCID mice, which were randomized, daily and orally treated with vehicle, dacomitinib, Compound B, or dacomitinib ("Daco") + Compound B ("Compd B").
  • Figure 15A graphs the tumor volumes of single agent treatment groups.
  • Figure 15B graphs the tumor volumes of combination treatment groups.
  • Figure 16 graphs the results of the xenograft model with RPC9 clone 6 tumor bearing SCID mice, which were randomized, daily and orally treated with vehicle, erlotinib, Compound A, or erlotinib ("Erlo") + Compound A (Compd A").
  • Figure 16A graphs the tumor volumes, which were measured 3 times per week and graphed with mean and standard error of the mean.
  • Figure 16B graphs the body weight of each group, which was recorded daily and percentage changes were graphed with mean and standard error of the mean.
  • the members of the human epidermal growth factor receptor/epidermal growth factor receptor (HER/EGFR) family of receptors include EGFR/HER-1 , HER2/neu/erbB- 2, HER3/erbB-3 and HER4/erbB-4.
  • EGFR inhibitors effectively inhibit the common activating mutations (L858R and delE746-A750) of EGFR.
  • the common activating mutations are also referred to as single mutants or single mutant forms.
  • Examples of EGFR inhibitors include gefitinib, erlotinib, icotinib, vandetanib, lapatinib, neratinib, afatinib, pelitinib, dacomitinib and canertinib.
  • Monoclonal antibody inhibitors of EGFR such as cetuximab and
  • panitumumab are also EGFR inhibitors, as defined in the present invention.
  • Inhibitors of EGFR may be reversible or irreversible inhibitors.
  • Reversible inhibitors of the tyrosine kinase domain of the EFGR molecule attach to and periodically detach from the receptor.
  • Gefitinib, erlotinib, icotinib, vandetanib and lapatinib are examples of reversible EGFR inhibitors.
  • Irreversible inhibitors of the tyrosine kinase domain of the EFGR molecule bind to EGFR irreversibly.
  • Neratinib, afatinib, pelitinib, dacomitinib and canertinib are examples of irreversible EGFR inhibitors.
  • EGFR inhibitors are inhibitors of at least one member of the HER family.
  • Gefitinib, erlotinib, icotinib and vandetanib are selective EGFR/HER-1 tyrosine kinase inhibitors (TKI).
  • Cetuximab and panitumumab are monoclonal antibodies specific to EGFR/HER-1.
  • a pan-HER inhibitor is an agent that block multiple members of the HER family.
  • Lapatinib, neratinib, afatinib, pelitinib, dacomitinib and canertinib are examples of pan- HER inhibitors.
  • Lapatinib, neratinib, afatinib and pelitinib inhibit the EGFR and HER2 members of the HER family.
  • Dacomitinib and canertinib inhibit the EGFR, HER2, and HER4 members of the HER family.
  • the EGFR T790M inhibitors of the present invention preferentially inhibit the double mutant forms of EGFR (L858R/T790M and delE746-A750/T790M) over the single mutants (L858R and delE746-A750).
  • Examples of EGFR T790M inhibitors include Go6976, PKC412, AP261 13, HM61713, WZ4002, CO-1686 and TAS-2913.
  • Inhibitors of EGFR T790M may be reversible or irreversible inhibitors.
  • Go6976, PKC412 and AP261 13 are examples of reversible EGFR T790M inhibitors.
  • HM61713, WZ4002, CO-1686 and TAS-2913 are examples irreversible EGFR T790M inhibitors.
  • EGFR T790M inhibitors of the present invention also include 1 - ⁇ (3R,4R)-3-[( ⁇ 5- chloro-2-[(1 -methyl-1 H-pyrazol-4-yl)amino]-7H-pyrrolo[2,3-c ]pyrimidin-4-yl ⁇ oxy)methyl]- 4-methoxypyrrolidin-1 -yl ⁇ prop-2-en-1 -one ("Compound A”), /V-methyl-/ ⁇ /-[irans-3-( ⁇ 2-[(1 - methyl-1 /-/-pyrazol-4-yl)amino]-5-(pyridin-2-yl)-7/-/-pyrrolo[2,3-d]pyrimidin-4- yl ⁇ oxy)cyclobutyl]prop-2-enamide (“Compound B”), /V-[irans-3-( ⁇ 5-chloro-2-[(1 ,3- dimethyl-1 H-pyrazol-4-yl)amino]-7H
  • DIPEA diisopropylethylamine
  • DMAP dimethylaminopyridine
  • DMEM Dulbecco's modified Eagle's medium
  • hexafluorophosphate hexafluorophosphate
  • HEPES 4-(2-hydroxyethyl)-1 -piperazineethanesulfonic acid
  • HMDS bis(trimethylsilyl)amine, which is also known as hexamethyldisilazane or hexamethyldisiloxane
  • HOAc acetic acid
  • HPLC high-performance liquid
  • Phl(OAc) 2 iodobenzene diacetate
  • PMSF phenylmethylsulfonyl fluoride
  • psi pounds per square inch
  • Rf retention factor
  • RPMI Roswell Park Memorial Institute
  • rt room temperature
  • compositions described herein relate to the pharmaceutically acceptable salts of the compounds described herein.
  • Pharmaceutically acceptable salts of the compounds described herein include the acid addition and base addition salts thereof.
  • Suitable acid addition salts are formed from acids which form non-toxic salts.
  • suitable acid addition salts i.e., salts containing pharmacologically acceptable anions, include, but are not limited to, the acetate, acid citrate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, bitartrate, borate, camsylate, citrate, cyclamate, edisylate, esylate, ethanesulfonate, formate, fumarate, gluceptate, gluconate, glucuronate,
  • methanesulfonate methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, p-toluenesulfonate, tosylate, trifluoroacetate and xinofoate salts.
  • Suitable base addition salts are formed from bases which form non-toxic salts.
  • suitable base salts include the aluminium, arginine,
  • benzathine calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.
  • the compounds described herein that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids.
  • compounds described herein are those that form non-toxic acid addition salts, e.g., salts containing pharmacologically acceptable anions, such as the hydrochloride,
  • the compounds described herein that include a basic moiety, such as an amino group may form pharmaceutically acceptable salts with various amino acids, in addition to the acids mentioned above.
  • the chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of those compounds of the compounds described herein that are acidic in nature are those that form non-toxic base salts with such compounds.
  • Such non-toxic base salts include, but are not limited to those derived from such
  • pharmacologically acceptable cations such as alkali metal cations (e.g., potassium and sodium) and alkaline earth metal cations (e.g., calcium and magnesium), ammonium or water-soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines.
  • the compounds of the embodiments described herein include all stereoisomers (e.g., cis and trans isomers) and all optical isomers of compounds described herein (e.g., R and S enantiomers), as well as racemic, diastereomeric and other mixtures of such isomers. While all stereoisomers are encompassed within the scope of our claims, one skilled in the art will recognize that particular stereoisomers may be preferred.
  • the compounds described herein can exist in several tautomeric forms, including the enol and imine form, and the keto and enamine form and geometric isomers and mixtures thereof. All such tautomeric forms are included within the scope of the present embodiments. Tautomers exist as mixtures of a tautomeric set in solution. In solid form, usually one tautomer predominates. Even though one tautomer may be described, the present embodiments includes all tautomers of the present compounds.
  • the present embodiments also include atropisomers of the compounds described herein.
  • Atropisomers refer to compounds that can be separated into rotationally restricted isomers.
  • Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts.
  • solvate is used herein to describe a molecular complex comprising a compound described herein and one or more pharmaceutically acceptable solvent molecules, for example, ethanol.
  • the compounds described herein may also exist in unsolvated and solvated forms. Accordingly, some embodiments relate to the hydrates and solvates of the compounds described herein.
  • tautomeric isomerism ('tautomerism') can occur.
  • This can take the form of proton tautomerism in compounds described herein containing, for example, an imino, keto, or oxime group, or so-called valence tautomerism in compounds which contain an aromatic moiety.
  • a single compound may exhibit more than one type of isomerism.
  • Cis/trans isomers may be separated by conventional techniques well known to those skilled in the art, for example, chromatography and fractional crystallisation.
  • Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC).
  • the racemate (or a racemic precursor) may be reacted with a suitable optically active compound, for example, an alcohol, or, in the case where a compound described herein contains an acidic or basic moiety, a base or acid such as 1 -phenylethylamine or tartaric acid.
  • a suitable optically active compound for example, an alcohol, or, in the case where a compound described herein contains an acidic or basic moiety, a base or acid such as 1 -phenylethylamine or tartaric acid.
  • the resulting diastereomeric mixture may be separated by chromatography and/or fractional crystallization and one or both of the diastereoisomers converted to the corresponding pure enantiomer(s) by means well known to a skilled person.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.
  • treatment refers to the act of treating as “treating” is defined immediately above.
  • a patient to be treated according to this invention includes any warm-blooded animal, such as, but not limited to human, monkey or other lower-order primate, horse, dog, rabbit, guinea pig, or mouse.
  • the patient is human.
  • Those skilled in the medical art are readily able to identify individual patients who are afflicted with non- small cell lung cancer and who are in need of treatment.
  • additive means that the result of the combination of the two compounds or targeted agents is the sum of each agent individually.
  • additive means that the result of the combination of the two compounds or targeted agents is the sum of each agent individually.
  • “synergistic” are used to mean that the result of the combination of the two agents is more than the sum of each agent together.
  • a “synergistic amount” is an amount of the combination of the two agents that result in a synergistic effect.
  • the optimum range for the effect and absolute dose ranges of each component for the effect may be definitively measured by administration of the components over different w/w ratio ranges and doses to patients in need of treatment.
  • the complexity and cost of carrying out clinical studies on patients renders impractical the use of this form of testing as a primary model for synergy.
  • the method of the invention is related to a method of treating non-small cell lung cancer comprising administering to a patient in need thereof an effective amount of an EGFR T790M inhibitor in combination with a panHER inhibitor, where the panHER inhibitor is administered according to a non-standard clinical dosing regimen, in amounts sufficient to achieve synergistic effects.
  • the method of the invention is related to a synergistic combination of targeted therapeutic agents, specifically an EGFR T790M inhibitor and a panHER inhibitor.
  • the method of the invention is related to a method of treating non-small cell lung cancer comprising administering to a patient in need thereof an effective amount of an EGFR T790M inhibitor in combination with a low-dose amount of a panHER inhibitor, in amounts sufficient to achieve synergistic effects.
  • the method of the invention is related to a synergistic combination of targeted therapeutic agents, specifically an EGFR T790M inhibitor and a panHER inhibitor.
  • the method of the invention is related to a method of treating non-small cell lung cancer comprising administering to a patient in need thereof an effective amount of an irreversible EGFR T790M inhibitor in combination with an effective amount of an EGFR inhibitor, in amounts sufficient to achieve synergistic effects.
  • the method of the invention is related to a synergistic combination of targeted therapeutic agents, specifically an irreversible EGFR T790M inhibitor and an EGFR inhibitor.
  • an "effective" amount refers to an amount of a substance, agent, compound, or composition that is sufficient to prevent or inhibit the growth of tumor cells or the progression of cancer metastasis in the combination of the present invention.
  • Therapeutic or pharmacological effectiveness of the doses and administration regimens may also be characterized as the ability to induce, enhance, maintain or prolong remission in patients experiencing specific tumors.
  • a “non-standard clinical dosing regimen,” as used herein, refers to a regimen for administering a substance, agent, compound, or composition, which effectively inhibits the single mutant forms (L858R and delE746-A750) of EGFR, but which is different than the amount or dose typically used in a clinical setting.
  • a “non-standard clinical dosing regimen” includes a “non-standard clinical dose” or a "non-standard dosing schedule”.
  • a “low-dose amount”, as used herein, refers to an amount or dose of a
  • the practice of the method of this invention may be accomplished through various administration regimens.
  • the compounds of the combination of the present invention can be administered intermittently, concurrently or sequentially. Repetition of the
  • administration regimens may be conducted as necessary to achieve the desired reduction or diminution of cancer cells.
  • the compounds of the combination of the present invention can be administered in an intermittent dosing regimen.
  • Administration of the compounds of the combination of the present invention can be effected by any method that enables delivery of the compounds to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion), topical, and rectal administration.
  • the compounds of the method or combination of the present invention may be formulated prior to administration.
  • the formulation will preferably be adapted to the particular mode of administration.
  • These compounds may be formulated with
  • compositions of the present invention the active ingredient will usually be mixed with a pharmaceutically acceptable carrier as known in the art and administered in a wide variety of dosage forms as known in the art.
  • the active ingredient will usually be mixed with a pharmaceutically acceptable carrier as known in the art and administered in a wide variety of dosage forms as known in the art.
  • the active ingredient will usually be mixed with a pharmaceutically acceptable carrier as known in the art and administered in a wide variety of dosage forms as known in the art.
  • Such carriers include, but are not limited to, solid diluents or fillers, excipients, sterile aqueous media and various non-toxic organic solvents. Dosage unit forms or
  • compositions include tablets, capsules, such as gelatin capsules, pills, powders, granules, aqueous and nonaqueous oral solutions and suspensions, lozenges, troches, hard candies, sprays, creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, injectable solutions, elixirs, syrups, and parenteral solutions packaged in containers adapted for subdivision into individual doses.
  • Parenteral formulations include pharmaceutically acceptable aqueous or nonaqueous solutions, dispersion, suspensions, emulsions, and sterile powders for the preparation thereof.
  • carriers include water, ethanol, polyols (propylene glycol, polyethylene glycol), vegetable oils, and injectable organic esters such as ethyl oleate. Fluidity can be maintained by the use of a coating such as lecithin, a surfactant, or maintaining appropriate particle size.
  • Exemplary parenteral administration forms include solutions or suspensions of the compounds of the invention in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired.
  • lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tableting purposes.
  • Solid compositions of a similar type may also be employed in soft and hard filled gelatin capsules.
  • Preferred materials, therefor, include lactose or milk sugar and high molecular weight polyethylene glycols.
  • the active compound therein may be combined with various sweetening or flavoring agents, coloring matters or dyes and, if desired, emulsifying agents or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof.
  • the invention also relates to a kit comprising the therapeutic agents of the combination of the present invention and written instructions for administration of the therapeutic agents.
  • the written instructions elaborate and qualify the modes of administration of the therapeutic agents, for example, for simultaneous or sequential administration of the therapeutic agents of the present invention.
  • salt forms were occasionally isolated as a consequence of the mobile phase additives during HPLC based chromatographic purification.
  • salts such as formate, trifluorooacetate and acetate were isolated and tested without further processing. It will be recognized that one of ordinary skill in the art will be able to realize the free base form by standard methodology (such as using ion exchange columns, or performing simple basic extractions using a mild aqueous base).
  • the compounds described herein may be prepared by processes known in the chemical arts, particularly in light of the description contained herein.
  • Example 1 Preparation of 1 -f(3 4 ?)-3-r5-chloro-2-(1 -methyl-1 H-pyrazol-4- ylamino)-7H-pyrrolor2,3-dlpyrimidin-4-yloxymethvn-4-methoxy ⁇ yrrolidin-1 - vPpropenone trifluoroacetate (also known as "1 -((3 ?,4 ?)-3- ⁇ ((5- ⁇ -2- ⁇ (1 - methyl-1 H-pyrazol-4-yl)amino1-7H-pyrrolor2,3-cnpyrimidin-4-yl)oxy)methvn-4- methoxypyrrolidin-1 -yl)prop-2-en-1 -one trifluoroacetate" and "1 -((3R,4R)-3-(((5- chloro-2-((1 -methyl-1 H-pyrazol-4-yl)amino)-7H-pyrrolor2,3-d
  • Step 1 Preparation of (3S,4R)-1 -benzyl-4-methoxy-pyrrolidine-3-carboxylic acid methyl ester
  • Step 2 Preparation of (3S,4R)-4-methoxy-pyrrolidine-1 ,3-dicarboxylic acid 1 -fe/f- butyl ester 3-methyl ester
  • Step 3 Preparation of (3R4R)-3-hvdroxymethyl-4-methoxy-pyrrolidine-1 - carboxylic acid fe/f-butyl ester
  • Lithium borohydride (12.7 g, 4 eq) was added portionwise to a solution of
  • Step 4 Preparation of (3R,4R)-3-[5-chloro-2-(1 -methyl-1 /-/-pyrazol-4-ylamino)- 7/-/-pyrrolo[2,3-dlpyrimidin-4-yloxymethyl1-4-methoxy-pyrrolidine-1 -carboxylic acid tert- butyl ester
  • Method A (using microwave heating)
  • Method B using thermal heating To a solution 2,4,5-tnchloro-7/-/-pyrrolo[2,3-d]pynmidine (9.28 g, 41 .7 mmol) and (3R,4R)-3-hydroxymethyl-4-methoxy-pyrrolidine-1 -carboxylic acid fe/f-butyl ester (9.65 g, 41 .7 mmol) in 1 ,4-dioxane (100 mL) in a round bottom flask was added potassium fe/f-pentoxide (25 % w/w in toluene, 80 mL, 167 mmol). The resulting reaction solution was stirred at ambient temperature for 30 min.
  • Step 5 Preparation of [5-chloro-4-((3 4R)-4-methoxy-pyrrolidin-3-ylmethoxy)- 7/-/-pyrrolo[2,3-dlpyrimidin-2-yl1-(1 -methyl-1 /-/-pyrazol-4-yl)-amine trifluoroacetate
  • Step 6 Preparation of 1 - ⁇ (3R4R)-3-r5-chloro-2-(1 -methyl-1 /-/-pyrazol-4-ylamino)- 7H-pyrrolo[2,3-dlpyrimidin-4-yloxymethyl1-4-methoxy-pyrrolidin-1 -yl)propenone trifluoroacetate
  • Step 1 Preparation of methyl (3,4-frans)-1 -benzyl-4-methoxypyrrolidine-3- carboxylate
  • methoxymethyltrimethylsilylamine (595 ml_, 552.1 g, 2.3 mol) were mixed. To this mixture was added TFA (2.7 ml_, 4.14 g, 36.3 mmol) which resulted in an exotherm to approximately 95 °C in 30 seconds. The resulting mixture was then heated at reflux for 1 hr (note: at the beginning the reflux temperature was approximately at 104 °C and after 1 hr it had dropped to approximately at 90 °C). Three batches of this scale plus another batch using 325 ml_ benzyl methoxymethyltrimethylsilylamine compound were performed. Two of these batches were combined and poured into 2 N HCI (5 L). The mixture was extracted with EtOAc (3 L and 2 L).
  • Step 2 Preparation of met -frans)-4-methoxypyrrolidine-3-carboxylate
  • Methyl (3,4-frans)-1 -benzyl-4-methoxypyrrolidine-3-carboxylate (463.3 g, 1858 mmol) was dissolved in iPrOH (2 L). To this solution was added 20 % Pd(OH) 2 /C (50 g, 37 % moist, Aldrich) and the mixture was stirred vigorously. A pressure of 1 1 .8 bar H 2 was applied and refilling was done several times until 1 H-NMR showed complete conversion. The mixture was filtered through Celite and the Celite was rinsed with iPrOH. The filtrate was concentrated in vacuo to give the title compound as a dark yellow liquid (266 g, 90 % yield). Used as is in next step.
  • Sample preparation 5 mg salt was mixed with DCM (1.5 ml_) and 2 N NaOH (0.2 ml_). The DCM layer was dried and analysed by gas chromatography.
  • Step 4 Preparation of 1 -fe/f-butyl 3-methyl (3S,4R)-4-methoxypyrrolidine-1 ,3- dicarboxylate
  • Step 5 Preparation of fe/f-butyl (3 4R)-3-(hvdroxymethyl)-4-methoxypyrrolidine-
  • Step 6 Preparation of fe/f-butyl (3R,4R)-3-r( ⁇ 5-chloro-2-r(1 -methyl-1 /-/-pyrazol-4- yl)amino1-7/-/-pyrrolo[2,3-c lpyrimidin-4-yl)oxy)methyl1-4-methoxypyrrolidine-1 - carboxylate
  • Step 7 Preparation of (3 4ffl-3-rf(5-chloro-2-rf 1 -methyl-1 H-pyrazol-4-yl)amino1- 7/-/-pyrrolo[2,3-dlpyrimidin-4-yl)oxy)methyl1-4-methoxypyrrolidinium trifluoroacetate
  • Step 8 Preparation of 1 - ⁇ (3R4R)-3-r5-chloro-2-(1 -methyl-1 /-/-pyrazol-4-ylamino)- 7/-/-pyrrolo[2,3-dlpyrimidin-4-yloxymethyl1-4-methoxy-pyrrolidin-1 -yl)prop2-en-1 -one
  • Step 1 Preparation of 2,4-dichloro-5-iodo-7/-/-pyrrolo[2,3-c lpynmidine
  • Step 2 Preparation of 2,4-dichloro-5-iodo-7- ⁇ [2-(trimethylsilyl)ethoxy1methyl)-7/-/- pyrrolo[2,3-dlpyrimidine
  • the resulting thick oil was diluted with EtOAc (400 mL), washed sequentially with sat. aqueous NH 4 CI (2 x 200 mL) and brine (2 x 200 mL), dried over Na 2 S0 4 and concentrated in vacuo.
  • the resulting material was dissolved in a minimum volume of DCM (50 mL) and diluted with heptane (200 mL). This solution was
  • reaction mixture was stirred at -78 °C for 2 h and then treated with a freshly prepared solution of ZnBr 2 (24.7 g, 1 10 mmol, 1 .62 equiv, dried at 130 °C) in THF (100 mL) in a dropwise manner over 15 min.
  • the mixture was stirred at -78 °C for an additional 1 hr then warmed to ambient temperature and stirred for an additional 0.5 hrs.
  • the reaction mixture was treated with Pd(PPh 3 ) 4 (3.94 g, 3.38 mmol, 0.05 equiv) and 2-iodopyridine (10.8 mL, 101 mol, 1 .50 equiv) and heated to 65 °C for 10 hrs.
  • the reaction mixture Upon cooling to ambient temperature, the reaction mixture was concentrated to a volume of ⁇ 200 mL, diluted with water (600 mL), sat. aqueous sodium potassium tartrate (100 mL) and EtOAc (400 mL). The layers were separated and the aqueous layer was extracted with EtOAc (4 x 300 mL). The combined organics were washed with brine (300 mL), dried (Na 2 SO 4 ), and concentrated in vacuo. The resulting oil was purified via flash chromatography eluting with a gradient of 0 - 30 % EtOAc in heptane to provide the title compound (23.5 g, 87 % yield) as an oil that converted on standing to a light tan solid.
  • Step 4 Preparation of fe/f-butyl (frans-3- ⁇ [te/f-butyl(dimethyl)silyl1oxy)cvclobutyl)- carbamate
  • Step 5 Preparation of fe/f-butyl (frans-3- ⁇ [fe/f-butyl(dimethyl)silvnoxy)cvclobutyl)- methylcarbamate
  • Step 6 Preparation of fe/f-butyl (frans-3-hydroxycvclobutyl)methylcarbamate OH
  • Step 7 Preparation of fe/f-butyl methyl ⁇ frans-3-[(2-[(1 -methyl-1 /-/-pyrazol-4- yl)amino1-5-(pyridin-2-yl)-7- ⁇ [2-(trimethylsilyl)ethoxy1methyl)-7/-/-pyrrolo[2,3-dlpyrimidin- 4-yl)oxy1cvclobutyl)carbamate
  • Step 8 Preparation of 3-chloro-/V-methyl-/V- ⁇ frans-3-r(2-r(1 -methyl-1 /-/-pyrazol-4- yl)amino1-5-(pyridin-2-yl)-7- ⁇ [2-(trimethylsilyl)ethoxy1methyl)-7/-/-pyrrolo[2,3-dlpyrimidin- 4-yl)oxy1cvclobutyl)propanamide
  • reaction mixture was stirred at 0 °C and then allowed to warm to ambient temperature overnight.
  • the aqueous phase was extracted with EtOAc (3 x 300 mL), and the combined organic phases were washed with brine (2 x 200 mL), dried (Na 2 S0 4 ), and concentrated in vacuo.
  • the resulting residue was partially purified by via flash chromatography eluting with a gradient of 2 - 7 % MeOH in DCM to provide 4- ⁇ [frans-3-(methylamino)cyclobutyl]oxy ⁇ -/V-(1 -methyl-1 /-/-pyrazol-4-yl)-5-pyridin- 2-yl-7- ⁇ [2-(trimethylsilyl)ethoxy]methyl ⁇ -7/-/-pyrrolo[2,3-d]pyrimidin-2-amine as a yellow gum, which was used directly in the next step.
  • Step 9 Preparation of /V-methyl-/V-[frans-3-( ⁇ 2-[(1 -methyl-1 /-/-pyrazol-4-yl)amino1- 5-(pyridin-2-yl)-7/-/-pyrrolo[2,3-dlpyrimidin-4-yl)oxy)cvclobutyllprop-2-enamide
  • the intermediate from the previous step was dissolved in 1 ,4-dioxane (80 mL) and concentrated aqueous NH 4 OH (50 mL). The reaction mixture was stirred at ambient temperature for 3 hrs and then concentrated in vacuo.
  • Step 1 Preparation of fe/f-butyl methyl ⁇ c/s-3-[1 -methyl-1 - (trimethylsilvDethoxylcyclobutvDcarbamate
  • Step 2 Preparation of fe/f-butyl (c/s-3-hvdroxycvclobutyl)methylcarbamate
  • Step 3 Preparation of fe/f-butyl (frans-3-aminocvclobutyl)methylcarbamate
  • Step 5 Preparation of 1 ,3-dimethyl-1 /-/-pyrazol-4-amine
  • Step 6 Preparation of fe/f-butyl ⁇ frans-3-r(2,5-dichloro-7- ⁇ [2- (trimethylsilyl)ethoxy1methyl)-7/-/-pyrrolo[2,3-dlpyrimidin-4- vDaminolcvclobutvDmethylcarbamate ⁇
  • Step 7 Preparation of fe/f-butyl ⁇ frans-3-[(5-chloro-2-[(1 ,3-dimethyl-1 /-/-pyrazol-4- yl)amino1-7- ⁇ [2-(trimethylsilyl)ethoxy1methyl)-7/-/-pyrrolo[2,3-dlpyrimidin-4- yl)amino1cvclobutyl)methylcarbamate
  • Step 8 Preparation of /V-[frans-3-( ⁇ 5-chloro-2-[(1 ,3-dimethyl-1 /-/-pyrazol-4-yl)amino1-7/-/- Pyrrolo[2,3-dlpyrimidin-4-yl)amino)cvclobutyl1-/V-methylprop-2-enamide
  • Step 2 Preparation of frans-ethyl-1 -benzyl-4-( ⁇ [tertbutyl(dimethyl)silyl1oxy)methyl) pyrrolidine-3-carboxylate
  • Step 3 Preparation of trans- -fe/f-butyl 3-ethyl-4-( ⁇ [fe/f- butyl(dimethyl)silylloxy)methyl) pyrrolidine-1 ,3-dicarboxylate
  • Step 4 Preparation of frans-te f-butyl-3-( ⁇ [te f-butyl(dimethyl)silyl1oxy)methyl)-4- (hydroxymethyl)pyrrolidine-l -carboxylate
  • Step 5 Preparation of frans-te f-butyl-3-( ⁇ [te f-butyl(dimethyl)silyl1oxy)methyl)-4-
  • Tetrabutylammonium iodide (0.1 10 g, 0.28 mmol), 50 % aqueous NaOH (20 mL) and dimethyl sulfate (0.325 mL, 3.41 mmol) were added to a solution of irans-ie f-butyl- 3-( ⁇ [ie/f-butyl(dimethyl)silyl]oxy ⁇ methyl)-4-(hydroxymethyl)pyrrolidine-1 -carboxylate (0.982 g, 2.84 mmol) in CH 2 CI 2 (20 mL). The reaction was stirred at rt overnight.
  • Step 6 Preparation of frans-fe/f-butyl-3-(hvdroxymethyl)-4- (methoxymethyl)pyrrolidine-l -carboxylate
  • Step 2 Preparation of fe/f-butyl (3-methylidenecvclobutyl)carbamate
  • Step 5 Preparation of fe/f-butyl ⁇ c/s-3-[1 -methyl-1 -
  • Step 6 Preparation of fe/f-butyl methyl ⁇ c/s-3-[1 -methyl-1 -
  • Step 8 Preparation of c/s-3-[(fe/f-butoxycarbonyl)(methyl)amino1cvclobutyl methanesulfonate
  • Triethylamine (4.14 mL, 29.79 mmol) was added into the solution of fe/f-butyl (c/s-3-hydroxycyclobutyl)methylcarbamate (2.0 g, 9.93 mmol) in CH2CI2 (30 mL) and the resulting mixture was cooled to -30 °C upon vigorous stirring.
  • the reaction mixture was then washed with water (two x 10 mL), aq. NH 4 CI (10 mL), brine (10 mL), dried over anhydrous Na 2 SO 4 and concentrated to give the title compound (2.5 g, 91 % yield) as yellow solid, which was used for next step directly.
  • Step 9 Preparation of fe/f-butyl (frans-3-azidocvclobutyl)methylcarbamate
  • Tyr1068 Sandwich ELISA Kit is a solid phase sandwich enzyme-linked immunosorbent assay (ELISA) that detects endogenous levels of phospho-EGF Receptor (Tyr1068) protein.
  • ELISA enzyme-linked immunosorbent assay
  • the following cell lines were evaluated in this assay: A549 (EGFR wildtype, endogenous), NCI-H1975 (EGFR L858R+T790M, endogenous),
  • NIH3T3/EGFR_wildtype NIH3T3/EGFR L858R+T790M and PC9-DRH (EGFR delE746- A750 +T790M).
  • NIH/3T3 parental, A549, and NCI-H1975 cells were purchased from the American Type Culture Collection (Manassas, VA). All cells were cultured according to ATCC recommendations. A549 cells were grown in RPMI media (Invitrogen, Carlsbad) supplemented with 10 % FBS (Sigma, St Louis, MO), and with 1 % Penn/Strep
  • NCI-H1975 cells were grown in RPMI (Invitrogen) supplemented with 10% FBS (Sigma), and with 1 % Penn/Strep (Invitrogen).
  • NIH/3T3 cells were grown in DMEM (Invitrogen) supplemented with 10 % newborn calf serum (Invitrogen), and NIH3T3/EGFR mutant cells were grown in complete media with 5 pg/mL puromycin (Invitrogen).
  • PC9-DRH cells were generated and cultured as described in Example 6.
  • Plasmids (pLPCX) with various EGFR constructs were made by GenScript (Piscataway, NJ), and stable pools of NIH/3T3 cells expressing these constructs were made at Pfizer La Jolla.
  • Cells were plated in complete culture media (50 L/well) on the bottom of clear tissue culture treated microtiter plates (#3595, Corning Inc, Corning, NY) and allowed to adhere overnight at 37 °C, 5 % CO2. Cells were seeded at the following concentrations: (A549: 40,000/well, NCI-H1975: 40,000/well, NIH3T3: 20,000/well, PC9-DRH:
  • Protease Inhibitor Cocktail Tablet (1 tablet/10 mL, #1 1836170001 , Roche, Indianapolis, IN), and PhosSTOP Phosphatase Inhibitor Cocktail Tablet (1 tablet/10 ml_, #04906837001 , Roche) in pure water.
  • media was flicked off and cells were washed once with ice-cold 1 mM Na 3 V0 4 in PBS (100 ⁇ / ⁇ , Invitrogen). The wash was then flicked off and ice-cold lysis buffer was added to the cells (50 ⁇ _ ⁇ ⁇ ). The plate was shaken for 20-30 min at 4 °C to completely lyse the cells.
  • Sample diluent (50 ⁇ / ⁇ ) was added to the ELISA plate, and the lysate (50 ⁇ _) was diluted into the sample diluent in each well of the ELISA plate. Plates were sealed and incubated overnight at 4 °C with shaking. The next day, wells were washed four times with 1 x Wash Buffer; plates were taped on lint-free paper after the final wash prior to adding Add Detection Antibody (green, 100 ⁇ / ⁇ ) to each well and incubating for 1 hr at 37 °C. After incubation, wells were washed as described. HRP-Linked secondary antibody (red, 100 ⁇ / ⁇ ) was added to each well and incubated for 30 min at 37 °C.
  • the results of the pEGFR Y1068 ELISA assays for the compounds tested are listed in Table 2.
  • the pEGFR ELISA IC 50 data shown in Table 2 for T790M_L858R is for 3T3 cell lines, unless otherwise indicated.
  • PC9 cells In parental PC9 cells, the EGFR delE746-A750 mutant allele is amplified and no wild-type EGFR allele can be detected.
  • Parental PC9 cells were utilized in the generation of the RPC9 cells. PC9 cells were cultured at 37 °C with 5 % C0 2 in RPMI 1640 medium supplemented with 10 % heat inactivated FBS. To generate EGFR inhibitor resistant cell lines, PC9 cells were initially treated with 0.5 nM dacomitinib. Once cells grew up to 90 % of confluence, they were split and the drug concentration was escalated by two-fold. After six weeks of such treatment, PC9 cells could grow in 2 nM dacomitinib. Single cell clones were generated and ten were selected for futher characterization. Those resistant cells were maintained in growth medium containing 2 ⁇ erlotinib, and were named RPC9 for Resistant PC9.
  • Step 3 Cell viability ICso determination
  • 3000 RPC9 cells per well were seeded in 90 ⁇ _ of growth medium in duplicate wells of a 96 well plate (Corning). 24 hours later, cells were treated with dacomitinib or erlotinib in an 1 1 point titration of 3-fold dilution in 10 ⁇ growth medium. The highest final concentration was 10 ⁇ . After 72 hours of treatment, cells were analyzed via CTG assay (Promega) following manufacture's instructions.
  • Step 4 Characterization of RPC9 cells harboring EGFR T790M mutation
  • RPC9 cells which harbor EGFR T790M mutation and are resistant to EGFR inhibitors such as dacomitinib and erlotinib were generated.
  • the RPC9 cells contain a mixture of both single mutant (EGFR delE746-A750) and double mutant (EGFR delE746-A750 and T790M) EGFR alleles, since the EGFR T790M allele constitutes about 10 % of the total EGFR alleles in RPC9 cells.
  • PC9-DRH cells dacomitinib resistant high
  • T790M T790M
  • the RPC9 cell pool resistant to 2 nM dacomitinib was further challenged with increasing concentrations of dacomitinib from 2 nM to 2 ⁇ in 8 weeks, as described in Step 1 .
  • PC9-DRH cells were maintained in growth medium, as described in Step 1 , containing 2 ⁇ dacomitinib.
  • PC9-DRH cells were analyzed, as described in Steps 2, 3, and 4.
  • PC9-DRH cells contain 70 % of their EGFR alleles as double mutant EGFR delE746-A750 and T790M.
  • IC50 1 ,651 nM
  • erlotinib IC50 >10,000 nM
  • gefitinib IC 50 >10,000 nM.
  • Example 7 RPC9 Cell Viability utilizing EGFR T790M inhibitors alone or in combination with dacomitinib or erlotinib
  • 3000 RPC9 cells, as prepared in Example 6, per well were seeded in 90 ⁇ _ of growth medium into duplicate wells of a 96 well plate (Corning). 24 hours later, cells were treated with one of the EGFR T790M inhibitors, Compound A, Compound B, Compound C or Compound D in an 1 1 point titration of three-fold dilution with or without either 4 nM dacomitinib or 300 nM erlotinib in 10 ⁇ growth medium. The highest final concentration was 10 ⁇ of Compound A, Compound B, Compound C or Compound D. After 72 hours of treatment, cells were analyzed via CTG assay (Promega) following manufacture's instruction.
  • the free plasma concentration at steady-state from the standard clinical dosing regimen of dacomitinib and erlotinib are 4 nM and 300 nM, respectively. At those concentrations, dacomitinib and erlotinib completely inhibited parental PC9 cell viability ( Figure 2A and 2B). Neither drug significantly inhibited RPC9 cell viability at the same concentrations ( Figure 2A and 2B).
  • the inhibition of viability in RPC9 clone 6 cells was potentiated by a combination of Compound A with either dacomitinib or erlotinib ( Figures 3A and 3B).
  • the viability IC50 of Compound A was 17 nM when combined with 4 nM dacomitinib and 15 nM when combined with 300 nM erlotinib (Table 3).
  • the viability IC 50 s for Compound A in combination decrease over 1 1 fold compared with that of Compound A treatment alone.
  • RPC9 clone 6 cells were treated with Compound B, dacomitinib and erlotinib also sensitized RPC9 clone 6 to Compound B ( Figures 4A and 4B).
  • the IC50 of Compound B was 4 nM in combination with dacomitinib and 5 nM in combination with erlotinib (Table 3).
  • the viability IC50S decreased by 9.5 fold and 7.6 fold compared with that of Compound B treatment alone.
  • the projected human exposure for Compound A is 190 nM at which concentration Compound A alone inhibited cell viability about 40 %.
  • the same concentration of Compound A achieved maximal inhibition (83 %) ( Figure 3A and 3B).
  • the projected human exposure is 90 nM at which concentration
  • compounds which specifically target the single mutant form of EGFR such as dacomitinib and erlotinib, used at their clinically relevant concentrations potentiated compounds which preferentially inhibit the double mutant form of EGFR, such as Compound A, Compound B, Compound C and Compound D, in clinically relevant models that harbor both double mutant and single mutant forms of EGFR.
  • Example 8 RPC9 Clone 6 Cell Viability utilizing EGFR T790M inhibitors alone or in combination with dacomitinib, gefitinib, or afatinib
  • cells were treated with one of the EGFR T790M inhibitors, Compound A or Compound B, with or without either 4 nM dacomitinib, 20 nM gefitinib or 20 nM afatinib.
  • the free plasma concentration at steady-state from the standard clinical dosing regimen of dacomitinib is 4 nM.
  • the free plasma concentration at steady-state from the standard clinical dosing regimen of gefitinib and afatinib is 20 nM.
  • the viability IC 5 oS of Compound A decreased 19 fold and 14 fold when combined with gefitinib and afatinib, respectively.
  • the IC50S of Compound B decreased 10 fold and 8 fold when combined with gefitinib and afatinib, respectively.
  • RPC9 clone 6 cells were generated and subcloned as described in Example 6. Cells were cultured in RPMI with 10 % FBS and were maintained under selective pressure (2 nM dacomitinib). For experiments, selective pressure was removed, cells plated onto 10 cm dishes and incubated overnight (37 °C, 5 % CO2) to achieve 70-80 % confluence for treatments.
  • Dacomitinib, erlotinib, Compound A, and Compound B were dissolved in 100 % DMSO. Dacomitinib (4 nM) and erlotinib (300 nM) were used at their free plasma exposure from standard clinical dosing regimen. Compound A and
  • Compound B were used at a range starting below the target modulation IC50 value and up to the predicted clinical free plasma exposure for each compound, respectively.
  • Cells were treated with dacomitinib or erlotinib and/or a titration of Compound A or Compound B, or with control (DMSO). Treatment was applied for 6 hours; at the end of the incubation period, cell pellets were collected and frozen until ready for analysis.
  • Immunoblottinci Cell pellets were treated with lysis buffer (150 mM NaCI, 1.5 mM MgCI 2 , 50 mM HEPES, 10 % glycerol, 1 mM EGTA, 1 % Triton ® X-100, 0.5 % NP-40) supplemented with 1 mM Na 3 VO 4 , 1 mM PMSF, 1 mM NaF, 1 mM ⁇ -glycerophosphate, protease inhibitor cocktail (Roche, Indianapolis, IN), and phosphatase inhibitor cocktail (Roche). Protein concentration of cell lysates was determined using the BCA Protein Assay (Pierce/Thermo Fisher Scientific, Rockford, IL) per the manufacturer's
  • Protein (10 g) was resolved by SDS-PAGE and transferred onto
  • nitrocellulose membranes Bio-Rad CriterionTM System, Hercules, CA. Blots were probed with primary antibodies to detect proteins of interest.
  • EGFR, pEGFR Y1068, AKT, pAKT S473, ERK, and pERK T202/204 antibodies were purchased from Cell Signaling Technology, Inc (Danvers, MA).
  • GAPDH antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
  • membranes were visualized by chemiluminescence (Pierce/Thermo Fisher Scientific) and densitometry was performed on the FluorChem Q Imaging System (ProteinSimple, Santa Clara, CA).
  • inhibition of pAKT appeared to be greater than additive at the lowest concentration of Compound A (18 % inhibition with eriotinib, 3 % inhibition with 10 nM Compound A versus 48 % with eriotinib + 10 nM Compound A; Figure 7B, 9B) and additive at the next highest dose (18 % inhibition with eriotinib, 31 % inhibition with 30 nM Compound A versus 49 % with eriotinib + 30 nM Compound A; ( Figure 7B, 9B), but achieved no greater than 50 % inhibition even at the higher doses.
  • Example 10 EGFR T790M inhibitors in combination with dacomitinib or erlotinib in the RPC9 clone 6 (dacomitinib and erlotinib resistant) Xenograft Model
  • RPC9 clone 6 cells were generated from parental PC9 cells as described in Example 6.
  • Parental PC9 cells contain EGFR delE746-A750 and are sensitive to the treatments of dacomitinib and eriotinib.
  • RPC9 clone 6 cell line was one of the selected resistant clones generated by dose-escalation treatment with dacomitinib ("daco").
  • RPC9 clone 6 cells contain approximately 10 % EGFR delE746-A750 and T790M and 90 % EGFR delE746-A750 alleles. Therefore, in the in vitro assays, RPC9 clone 6 was resistant to dacomitinib/erlotinib single agent treatments due to the EGFR delE746-A750 and T790M allele as well as resistant to Compound A ("compd A”) and Compound B (“compd B”) single agent treatments due to the EGFR delE746-A750 allele.
  • mice Four- to six-week-old SCID beige female mice were obtained from Charles River lab and maintained in pressurized ventilated caging at the Pfizer La Jolla animal facility. All studies were approved by Pfizer Institutional Animal Care and Use Committees. Tumors were established by subcutaneously injecting 5x10 6 RPC9 clone 6 cells suspended 1 : 1 (v/v) with reconstituted basement membrane (Matrigel, BD Biosciences). For tumor growth inhibition (TGI) studies, mice with established tumors of ⁇ 300 mm 3 were selected and randomized, then treated with EGFR T790M inhibitors as single agent or in combination with dacomitinib or eriotinib using the indicated doses and regimens.
  • TGI tumor growth inhibition
  • Tumor dimensions were measured with vernier calipers and tumor volumes were calculated using the formula of ⁇ /6 x larger diameter x (smaller diameter) 2 .
  • Tumor growth inhibition percentage (TGI %) was calculated as 100 x (1 -AT/AC).
  • Tumor regression percentage was calculated as 100 x ( ⁇ - ⁇ /starting tumor size).
  • Compound A was formulated in spray dried dispersion suspension in 0.5 % Methocel/20mM Tris Buffer at pH 7.4.
  • Compound B was formulated in in-situ lactate salt solution with 0.5 % Methocel.
  • Dacomitinib was formulated in 0.1 M lactic acid solution at pH 4.5.
  • Eriotinib was formulated in 40 % Captisol ® . All drugs were formulated and dosed at the concentration of 10 mL/kg. Tumor bearing mice were orally and daily administrated with indicated treatments; body weight and health observation were recorded daily. Results:
  • mice in the single and combination treatment groups of Compound A at 200 mg/kg and 50 mg/kg continued to receive treatments until the study day 61 .
  • the tumor growth inhibition and tumor regression were calculated and illustrated in Table 4. Results indicate that (1 ) combination of compound A with dacomitinib generated complete tumor regression at both dose ranges, (2) tumors in the single agent treatment groups of compound A at both 200 mg/kg and 50 mg/kg progressed in a dose-dependent manner which mimics in vitro resistance probably driven by EGFR delE746-A750 allele, and (3) tumors in the single treatment group of dacomitinib also progressed which mimics the in vitro resistance probably driven by EGFR delE746-A750 and T790M allele in this RPC9 clone 6 xenograft model.
  • the treatment period was further extended to assess the in vivo resistance by single agent treatment and combination treatment.
  • the single treatment group of dacomitinib continued to progress and was terminated at day 74 when tumor size reached above 1200 mm 3 .
  • the single treatment group of compound A at 200 mg/kg continued to progress and was terminated at day 95 when tumor size reached above 1400 mm 3 . Therefore, tumors in the single treatment groups of either dacomitinib or compound A continued to progress and demonstrated the in vivo resistance similar to the in vitro characteristics.
  • Table 5 Tumor Growth Inhibition and Regression in Study II.
  • RPC9 clone 6 tumor-bearing mice were randomized and treated with either single agent of Compound A or eriotinib, or Compound A in combination with eriotinib.
  • Eriotinib at 25 mg/kg gave an average unbound drug concentration of 300 nM in mouse plasma, which matches the average clinical exposure.
  • the body weight change percentages were plotted in Figure 16B, and indicated that all dose groups of this study were well tolerated with body weight loss less than 10 %.
  • Compound A was dosed at 400 mg/kg, 200 mg/kg, and 50 mg/kg as single agent or in combination with eriotinib at 25 mg/kg as indicated in the Figure 16A.
  • the tumor growth inhibition and tumor regression were calculated and illustrated in Table 6.
  • RPC9 clone 6 xenograft model which harbors both EGFR delE746-A750 and EGFR delE746-A750/T790M alleles exhibited tumor progression when treated with compound A, compound B, dacomitinib, or erlotinib as single agent treatment.
  • the model showed complete regression when treated with high doses of compound A with clinical relevant dose of dacomitinib or erlotinib as combination therapy, as well as demonstrated dose-dependent tumor regression when treated with low doses of compound B in combination with dacomitinib.

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Abstract

La présente invention concerne une méthode de traitement d'un cancer du poumon non à petites cellules par l'administration d'une combinaison d'un inhibiteur de l'EGFR T790M en association avec une faible dose d'un inhibiteur du panHER. L'invention a également trait à une méthode de traitement d'un cancer des poumons non à petites cellules par l'administration d'une combinaison d'un inhibiteur irréversible de l'EGFR T790M en association avec un inhibiteur de l'EGFR.
PCT/IB2014/059401 2013-03-14 2014-03-03 Combinaison d'un inhibiteur de l'egfr t790m et d'un inhibiteur de l'egfr dans le traitement d'un cancer du poumon non à petites cellules Ceased WO2014140989A2 (fr)

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BR112015023020A BR112015023020A2 (pt) 2013-03-14 2014-03-03 combinação de inibidor de egfr t790m e inibidor de egfr para o tratamento de câncer pulmonar de células não-pequenas
CN201480014630.2A CN105073116A (zh) 2013-03-14 2014-03-03 治疗非小细胞肺癌的egfr t790m 抑制剂与egfr 抑制剂的组合
EP14710651.2A EP2968336A2 (fr) 2013-03-14 2014-03-03 Combinaison d'un inhibiteur de l'egfr t790m et d'un inhibiteur de l'egfr dans le traitement d'un cancer du poumon non à petites cellules
MX2015012106A MX2015012106A (es) 2013-03-14 2014-03-03 Combinacion de un inhibidor de egfr t790m y un inhibidor de egfr para el tratamiento del cancer pulmonar de celulas no pequeñas.
SG11201506531WA SG11201506531WA (en) 2013-03-14 2014-03-03 Combination of an egfr t790m inhibitor and an egfr inhibitor for the treatment of non-small cell lung cancer
RU2015137596A RU2015137596A (ru) 2013-03-14 2014-03-03 Комбинация ингибитора EGFR T790М и ингибитора EGFR для лечения немелкоклеточного рака легкого
KR1020157024916A KR20150119210A (ko) 2013-03-14 2014-03-03 비소세포 폐암의 치료를 위한 egfr t790m 억제제와 egfr 억제제의 조합
CA2904797A CA2904797A1 (fr) 2013-03-14 2014-03-03 Combinaison d'un inhibiteur de l'egfr t790m et d'un inhibiteur de l'egfr dans le traitement d'un cancer du poumon non a petites cellules
AU2014229468A AU2014229468A1 (en) 2013-03-14 2014-03-03 Combination of an EGFR T790m inhibitor and an EGFR inhibitor for the treatment of non-small cell lung cancer
IL240730A IL240730A0 (en) 2013-03-14 2015-08-20 A combination of t790m egfr inhibitor and egfr inhibitor, for the treatment of non-small cell lung cancer

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BR112015023020A2 (pt) 2017-07-18
SG11201506531WA (en) 2015-09-29
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MX2015012106A (es) 2016-01-12
WO2014140989A3 (fr) 2014-12-04
JP2014177456A (ja) 2014-09-25
AU2014229468A1 (en) 2015-09-03
KR20150119210A (ko) 2015-10-23
CA2904797A1 (fr) 2014-09-18

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