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WO2024148008A1 - Treatment of neuroendocrine prostate cancer - Google Patents

Treatment of neuroendocrine prostate cancer Download PDF

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Publication number
WO2024148008A1
WO2024148008A1 PCT/US2024/010041 US2024010041W WO2024148008A1 WO 2024148008 A1 WO2024148008 A1 WO 2024148008A1 US 2024010041 W US2024010041 W US 2024010041W WO 2024148008 A1 WO2024148008 A1 WO 2024148008A1
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WIPO (PCT)
Prior art keywords
compound
subject
acceptable salt
pharmaceutically acceptable
agents
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PCT/US2024/010041
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French (fr)
Inventor
Anneleen Daemen
Melissa R. JUNTTILA
Lori S. Friedman
Natalie YUEN
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Oric Pharmaceuticals Inc
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Oric Pharmaceuticals Inc
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Publication of WO2024148008A1 publication Critical patent/WO2024148008A1/en
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    • 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
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Prostate cancer is the second leading cause of cancer-related death in men in the US. Androgen deprivation therapy (ADT) and blockade are commonly used to treat prostate cancer. However, relapse occurs with subsequent progression to metastatic castration-resistant prostate cancer (mCRPC) after treatment with androgen biosynthesis inhibitors or androgen receptor (AR) antagonists through multiple acquired resistance mechanisms. While some mechanisms of resistance are androgen receptor (AR) dependent, and can be treated with other hormonal therapies, like AR antagonists such as enzalutamide, other mechanisms of resistance occur by trans-differentiation of adenocarcinoma cells into neuroendocrine prostate cancer (NEPC).
  • ADT Androgen deprivation therapy
  • mCRPC metastatic castration-resistant prostate cancer
  • AR androgen receptor
  • NEPC neuroendocrine prostate cancer
  • NEPC is distinct from prostate adenocarcinoma and is an aggressive form of prostate cancer that is resistant to current therapies used in the context of advanced prostate adenocarcinoma.
  • NEPC displays high proliferative rates and tumor dissemination to visceral organs such as lung and liver.
  • NEPC is a significant problem with recent evidence indicating that approximately twenty percent of subjects with progressive hormone-resistant prostate cancer suffer from NEPC, and data demonstrates that subjects determined to suffer from NEPC have a median time of survival of less than a year. Thus, there remains a need to develop new methods of treating subjects determined to have NEPC.
  • Z is O or S
  • X is O, CR 5 , CR 5 OH, or C(R 5 )2, wherein: when X is O, - is a single bond; when X is C(R 5 )2, - is a single bond; when X is CR 5 OH, - is a single bond; or when X is CR 5 , - is a double bond;
  • R 1 is aryl, heteroaryl, L-cycloalkyl, -N(R 5 )heterocyclyl, or L-heterocyclyl, wherein the aryl, the heteroaryl or the cyclyl portion of the L-cycloalkyl, -N(R 5 )heterocyclyl, or L-heterocyclyl is optionally substituted with one or more R 4 ;
  • R 2 is cyano, -COOR 5 , -C(O)N(R 5 )2, or -C(O)N(R 5 )2 wherein each R 5 taken together with the nitrogen atom to which they are attached form a 5 - 8 membered heterocyclic ring optionally substituted with one or more R 4 ; each R 3 is independently C1-C3 alkyl or halogen; each R 4 is independently oxo, cyano, halogen, -POsiCi-C, alkyl):.
  • L is a bond or C1-C4 alkylene
  • Y 1 is a bond, -C(O)-, or -NHC(O)-;
  • Y 2 is a bond, -S-, -SO-, -SO 2 -, or -NR 5 SO 2 -, each R 5 is hydrogen or C1-C3 alkyl;
  • R 6 is hydrogen, C1-C3 alkyl, halogen, haloalkyl, hydroxyalkyl, or heteroalkyl; each R 7 is oxo, cyano, hydroxyl, alkoxy, halogen, haloalkyl, hydroxyalkyl, heteroalkyl, cycloalkyl, -L-N(R 5 )2, Ci-Ce alkyl or -Y’-heterocyclyl; and n is 1 or 2.
  • n 1
  • R 2 is cyano. In other embodiments, R 2 is - COOR 5 or -C(O)N(R 5 )2. In other embodiments, R 2 is -COOR 5 . In other embodiments, R 2 is -C(O)N(R 5 )2. [0007] In other embodiments are provided the methods disclosed herein, wherein in the compounds of Formula (I) , or a pharmaceutically acceptable salt thereof, R 3 is halogen. In some embodiments R 3 is fluorine.
  • X is C(R 5 )2 and - is a single bond.
  • X is CR 5 and - is a double bond.
  • X is O and - is a single bond.
  • R 1 is aryl optionally substituted with one or more R 4 .
  • the aryl is phenyl optionally substituted with one or more R 4 .
  • the phenyl is substituted with one, two or three R 4 .
  • the one, two or three R 4 are each independently halogen, -PCffC i-C alkyl)2, hydroxyl, hydroxyalkyl, aralkyl, haloalkyl, - COOR 5 , -Y’-Ci-Ce alkyl, Y 2 -CI-C 6 alkyl, -L-N(R 5 ) 2 , -O-L-N(R 5 ) 2 , -C(CF 3 )N(R 5 ) 2 , -Y’-N(R 5 ) 2 , -Y 2 -N(R 5 ) 2 , Y 2 -haloalkyl, -L-heteroaryl, -L-heterocyclyl, or -Y 1 -heterocyclyl, wherein the heterocyclyl portion of the - L-heterocyclyl or -Y 1 -heterocyclyl is optionally substituted with one or more R 7
  • R 4 is -Y’-Ci-Ce alkyl and Y 1 is a bond and the Ci-Ce alkyl is methyl, ethyl, isopropyl, butyl or pentyl.
  • R 4 is -Y 2 -Ci-Ce alkyl and Y 2 is a -SO2- and the Ci-Ce alkyl is methyl.
  • R 4 is -Y 2 -haloalkyl and Y 2 is -S- or -SO2- and the haloalkyl is trifluoromethyl.
  • R 4 is -L-N(R 5 )2 and L is a bond and each R 5 is hydrogen, each R 5 is methyl or one R 5 is methyl and one R 5 is hydrogen.
  • R 4 is -L-N(R 5 )2 and L is methylene or ethylene and each R 5 is hydrogen, each R 5 is methyl or one R 5 is methyl and one R 5 is hydrogen.
  • R 4 is -Y’-N(R 5 )2, Y 1 is -C(O)- and each R 5 independently is hydrogen, each R 5 is independently methyl or one R 5 is methyl and one R 5 is hydrogen.
  • R 4 is -Y 2 -N(R 5 )2, Y 2 is -SO2- and each R 5 independently is hydrogen, each R 5 is methyl or one R 5 is methyl and one R 5 is independently hydrogen.
  • R 4 is -Y 1 -heterocyclyl and Y 1 is -C(O)- and the heterocyclyl portion of the L- heterocyclyl is piperazinyl or 4-methyl-piperazinyl.
  • R 4 is -L-heterocyclyl and L is a bond and the heterocyclyl portion of the L-heterocyclyl is azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, or 3/. 2 -azabicyclo
  • R 4 is -L-heterocyclyl, wherein L is a methylene and the heterocyclyl portion of the L- heterocyclyl is azetidinyl, oxetanyl, pyrrolidinyl piperidinyl, each optionally substituted with one or more R 7 selected from C1-C3 alkyl, alkoxy, hydroxyl and halogen.
  • R 4 is -Y 1 - heterocyclyl and Y 1 is -C(O)- and the heterocyclyl portion of the Y’-heterocyclyl is morpholinyl optionally substituted with one or more C1-C3 alkyl.
  • R 4 is -L-heteroaryl optionally substituted with one or more R 7 .
  • the -L-heteroaryl is tetrazolyl.
  • R 4 is -POsfCi-Cs alkyl)2.
  • R 4 is -COOR 5 .
  • R 4 is hydroxyalkyl.
  • R 4 is -O-L-N(R 5 )2.
  • R 4 is aralkyl.
  • heteroaryl is pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazinyl, pyridyl, pyridinyl-2-one, pyrazinyl, pyridazinyl, pyrimidinyl, isoxazolyl, isoindolinyl, naphthyridinyl, 1,2,3,4-tetrahydroisoquinolinyl, or 5,6-dihydro-4H-pyrrolo[l,2-b]pyrazolyl, each optionally substituted with one or more R 4 .
  • the heteroaryl is substituted with one or more R 4 ; wherein each R 4 is independently cyano, halogen, -Y’-Ci-Ce alkyl, -Y 2 -Ci-Ce alkyl, alkoxy, hydroxyalkyl, heteroalkyl, haloalkyl, -L-cycloalkyl, -L-N(R 5 )2, -Y'-N(R 5 )2, -L-heteroaryl, -L-heterocyclyl, or -Y 1 - heterocyclyl, wherein the heteroaryl of the -L-heteroaryl or the heterocyclyl portion of the L-heterocyclyl, or Y 1 -heterocyclyl is optionally substituted with one or more R 7 .
  • the heteroaryl is pyrazolyl optionally substituted with one R 4 independently selected from hydroxyalkyl, heteroalkyl, haloalkyl, -Y’-Ci-Ce alkyl, -L-N(R 5 )2, L-heterocyclyl or L-heteroaryl, wherein the heteroaryl of the L- heteroaryl or the heterocyclyl portion of the L-heterocyclyl is optionally substituted with one or more R 7 .
  • R 4 is -L-heteroaryl and L is methylene wherein the heteroaryl is pyridyl optional substituted with one or more R 7 .
  • R 4 is -L-heterocyclyl optionally substituted with one or more R 7 where L is a bond and the heterocyclyl portion of the L-heterocyclyl is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, piperazinyl or 4-methylpiperazinyl.
  • R 4 is -L-heterocyclyl optionally substituted with one or more R 7 where L is methylene and the heterocyclyl portion of the L-heterocyclyl is azetidinyl, oxetanyl, pyrrolidinyl, pyrrolidinone, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, piperazinyl or 4-methylpiperazinyl.
  • R 4 is -L-N(R 5 )2 where L is methylene and each R 5 is independently hydrogen, each R 5 is independently C1-C3 alkyl or one R 5 is C1-C3 alkyl and one R 5 is hydrogen.
  • R 4 is -Y’-Ci-Ce alkyl where Y 1 is a bond and the Ci-Ce alkyl is methyl, ethyl or isopropyl.
  • the heteroaryl is pyrazolyl optionally substituted with two R 4 groups each independently selected from hydroxyalkyl, heteroalkyl, haloalkyl, and -Y’-Ci-Ce alkyl.
  • heteroaryl is pyridyl optionally substituted with one R 4 independently selected from cyano, halogen, alkoxy, hydroxyalkyl, heteroalkyl, haloalkyl, -Y’-Ci-Ce alkyl, -L-N(R 5 )2, -Y’-N(R 5 )2, -L-cycloalkyl, or -L- heterocyclyl optionally substituted with one or more R 7 .
  • R 1 is -L-cycloalkyl optionally substituted with one or more R 4 .
  • R 1 is -L-heterocyclyl optionally substituted with one or more R 4 .
  • L is a bond and the heterocyclyl is piperidinyl or tetrahydropyranyl.
  • n is 2.
  • prostate cancer in a subject, wherein the prostate cancer has been determined to comprise neuroendocrine prostate cancer, comprising administering to the subject a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:
  • Formula (I) is Compound (Compound 1), or a pharmaceutically acceptable salt thereof.
  • Formula (I) is Compound (Compound 3), or a pharmaceutically acceptable salt thereof.
  • XRPD x-ray powder diffraction
  • crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) pattern at 9.8° ⁇ 0.2° 2-theta, 15.2° ⁇ 0.2° 2-theta, and 17.7° ⁇ 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of about 172 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 205 °C to about 210 °C.
  • XRPD x-ray powder diffraction
  • crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 206 °C to about 210 °C, or from about 207 °C to about 210 °C, or from about 208 °C to about 210 °C, or from about 209 °C to about 210 °C.
  • the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 1% upon heating the sample from about 25 °C to a temperature prior to melting.
  • the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 1% upon heating the sample from about 25 °C to about 380 °C.
  • crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) pattern at 5.5° ⁇ 0.2° 2-theta, 8.6° ⁇ 0.2° 2-theta, 15.9° ⁇ 0.2° 2-theta, 19.9° ⁇ 0.2° 2-theta, and 24.1° ⁇ 0.2° 2-theta.
  • XRPD x-ray powder diffraction
  • crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) pattern at 10.6° ⁇ 0.2° 2-theta, 11.0° ⁇ 0.2° 2-theta, 15.4° ⁇ 0.2° 2- theta, 21.0° ⁇ 0.2° 2-theta, and 26.3° ⁇ 0.2° 2-theta.
  • XRPD x-ray powder diffraction
  • crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 203 °C to about 208 °C, or from about 203 °C to about 206 °C, or from about 203 °C to about 205 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 2% upon heating the sample from about 25 °C to about 380 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 2% upon heating the sample from about 25 °C to about 210 °C.
  • crystalline form of Compound 4 exhibits a peak in an x-ray powder diffraction (XRPD) pattern at 7.7° ⁇ 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further a peak in an x-ray powder diffraction (XRPD) pattern at 15.4° ⁇ 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further a peak in an x-ray powder diffraction (XRPD) pattern at 19.2° ⁇ 0.2° 2-theta.
  • XRPD x-ray powder diffraction
  • crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 203 °C to about 208 °C, or from about 203 °C to about 206 °C, or from about 203 °C to about 205 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 2% upon heating the sample from about 25 °C to about 380 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 2% upon heating the sample from about 25 °C to about 210 °C.
  • crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) pattern at 5.5° ⁇ 0.2° 2-theta, 8.6° ⁇ 0.2° 2- theta, 15.9° ⁇ 0.2° 2-theta, 19.9° ⁇ 0.2° 2-theta, and 24.1° ⁇ 0.2° 2-theta.
  • XRPD x-ray powder diffraction
  • crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) pattern at 10.6° ⁇ 0.2° 2-theta, 11.0° ⁇ 0.2° 2-theta, 21.0° ⁇ 0.2° 2-theta, and 26.3° ⁇ 0.2° 2-theta.
  • XRPD x-ray powder diffraction
  • the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 203 °C to about 210 °C.
  • the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 206 °C to about 210 °C.
  • crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 203 °C to about 208 °C, or from about 203 °C to about 206 °C, or from about 203 °C to about 205 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 2% upon heating the sample from about 25 °C to about 380 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 2% upon heating the sample from about 25 °C to about 210 °C.
  • crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) patern at 5.5° ⁇ 0.2° 2-theta, 8.6° ⁇ 0.2° 2- theta, 15.9° ⁇ 0.2° 2-theta, 19.9° ⁇ 0.2° 2-theta, and 24.1° ⁇ 0.2° 2-theta.
  • XRPD x-ray powder diffraction
  • crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) patern at 10.6° ⁇ 0.2° 2-theta, 11.0° ⁇ 0.2° 2-theta, 21.0° ⁇ 0.2° 2-theta, and 26.3° ⁇ 0.2° 2-theta.
  • XRPD x-ray powder diffraction
  • the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry patern of from about 203 °C to about 210 °C.
  • the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry patern of from about 206 °C to about 210 °C.
  • crystalline form of Compound 4 exhibits peaks in an x-ray powder diffraction (XRPD) patern at 9.6° ⁇ 0.2° 2-theta, 5.7° ⁇ 0.2° 2-theta, 19.7° ⁇ 0.2° 2-theta, and 22.0° ⁇ 0.2° 2-theta , and (b) less than about 10% degradation when the crystalline form is stored at 25 °C and 60% relative humidity for at least 7 days.
  • XRPD x-ray powder diffraction
  • crystalline form of Compound 4 exhibits less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% degradation when the crystalline form is stored at 25 °C and 60% relative humidity for at least 7 days.
  • crystalline form of Compound 4 exhibits (a) peaks in an x-ray powder diffraction (XRPD) pattern at 7.7° ⁇ 0.2° 2-theta, 13.7° ⁇ 0.2° 2-theta, and 19.2° ⁇ 0.2° 2-theta, and (b) less than about 10% degradation when the crystalline form is stored at 25 °C and 60% relative humidity for at least 7 days.
  • XRPD x-ray powder diffraction
  • crystalline form of Compound 4 exhibits less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% degradation when the crystalline form is stored at 25 °C and 60% relative humidity for at least 7 days.
  • crystalline form of Compound 4 exhibits less than about 10% degradation when the crystalline forms are stored at 40 °C and 75% relative humidity for at least 7 days. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% degradation when the crystalline form is stored at 40 °C and 75% relative humidity for at least 7 days.
  • crystalline form of Compound 4 exhibits (a) a peak in an x-ray powder diffraction (XRPD) pattern at 8. 1° ⁇ 0.2° 2-theta, and (b) less than about 10% degradation when the crystalline form is stored at 40 °C and 75% relative humidity for at least 7 days.
  • XRPD x-ray powder diffraction
  • crystalline form of Compound 4 exhibits (a) peaks in an x- ray powder diffraction (XRPD) pattern at 9.6° ⁇ 0.2° 2-theta, 5.7° ⁇ 0.2° 2-theta, 19.7° ⁇ 0.2° 2-theta, and 22.0° ⁇ 0.2° 2-theta , and (b) less than about 10% degradation when the crystalline form is stored at 40 °C and 75% relative humidity for at least 7 days.
  • XRPD x- ray powder diffraction
  • crystalline form of Compound 4 exhibits less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% degradation when the crystalline form is stored at 60 °C for at least one week.
  • Formula (I) is Compound 12: (Compound 12), or a pharmaceutically acceptable salt thereof.
  • the methods disclosed herein wherein the subject has been administered one or more first agents prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the one or more first agents is selected from (a) luteinizing hormone -releasing hormone (LHRH) agonists, (b) luteinizing hormone-releasing hormone (LHRH) antagonists, (c) androgen receptor inhibitors, (d) inhibitors of cytochrome P450 17A1, and/or (e) antiandrogens.
  • the neuroendocrine prostate cancer in the subject has been determined to be progressing following the administration to the subject of the one or more first agents.
  • the neuroendocrine prostate cancer in the subject has been determined to comprise visceral metastases or lung metastases.
  • the visceral metastases comprise liver metastases.
  • the neuroendocrine prostate cancer in the subject has been determined to comprise bulky lymphadenopathy or a pelvic mass.
  • the pelvic mass is 5 cm or more in size.
  • the neuroendocrine prostate cancer in the subject has been determined to comprise bone metastases.
  • the neuroendocrine prostate cancer in the subject has been determined to comprise 20 or more bone metastases.
  • the bone metastases are lytic bone metastases.
  • the methods disclosed herein wherein the subject has been administered one or more first agents prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the one or more first agents is a luteinizing hormone-releasing hormone (LHRH) agonist.
  • the luteinizing hormone-releasing hormone (LHRH) agonist is selected from goserelin, histrelin, leuprolide, and triptorelin.
  • the luteinizing hormone -releasing hormone (LHRH) agonist is goserelin.
  • the luteinizing hormone -releasing hormone (LHRH) agonist is histrelin.
  • the luteinizing hormone -releasing hormone (LHRH) agonist is leuprolide.
  • the luteinizing hormone- releasing hormone (LHRH) agonist is triptorelin.
  • the methods disclosed herein wherein the subject has been administered one or more first agents prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the one or more first agents is an inhibitor of cytochrome P450 17A1.
  • the one or more inhibitors of cytochrome P450 17A1 is abiraterone acetate.
  • a biological sample obtained from the subject has been determined to exhibit one or more biological markers prior to the administration of the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the one or more biological markers are selected from synaptophysin (SYP), chromogranin A (CGA), insulinoma-associated protein 1 (INSMI), syntaxin 1 (STX1), ISL LIM homeobox 1 (ISL1), INSM transcriptional repressor 1 (INSMI), secretagogin (SECG), neural cell adhesion molecule 1 (NCAM1;
  • the biological sample obtained from the subject has been determined to exhibit synaptophysin (SYP) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • the biological sample obtained from the subject has been determined to exhibit chromogranin A (CGA) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • the biological sample obtained from the subject has been determined to exhibit insulinoma-associated protein 1 (INSMI) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • INSMI insulinoma-associated protein 1
  • the biological sample obtained from the subject has been determined to exhibit secretagogin (SECG) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • the biological sample obtained from the subject has been determined to exhibit neural cell adhesion molecule 1 (NCAM1; CD56) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • the biological sample obtained from the subject has been determined to exhibit neuronspecific enolase (NSE) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • the biological sample obtained from the subject has been determined to exhibit an amount of the one or more biological markers that exceeds 2.5 times the upper limit of normal. In some embodiments, the biological sample obtained from the subject has been determined to exhibit an amount of the one or more biological markers that exceeds 2.75 times the upper limit of normal. In some embodiments, the biological sample obtained from the subject has been determined to exhibit an amount of the one or more biological markers that exceeds 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times the upper limit of normal.
  • the biological sample obtained from the subject is blood, and the biological sample has been determined to exhibit an amount of chromogranin A (CGA) that exceeds 101 ng/mL prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • CGA chromogranin A
  • more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or more than 90% of cells in the biologic sample demonstrate staining for syntaxin 1 (STX1) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • STX1 syntaxin 1
  • the one or more additional therapeutic agents are selected from chemotherapeutic agents.
  • the chemotherapeutic agents are selected from actinomycin, azacytidine, azathioprine, bleomycin, bortezomib, chlorambucil, cyclophosphamide, daunorubicin, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, idarubicin, irinotecan, lurbinectedin, mechlorethamine, mitoxantrone, teniposide, topotecan, valrubicin, vemurafenib, vinblastine, vincristine, and vindesine.
  • the chemotherapeutic agent is actinomycin. In some embodiments, the chemotherapeutic agent is azacytidine. In some embodiments, the chemotherapeutic agent is azathioprine. In some embodiments, the chemotherapeutic agent is bleomycin. In some embodiments, the chemotherapeutic agent is bortezomib. In some embodiments, the chemotherapeutic agent is chlorambucil. In some embodiments, the chemotherapeutic agent is cyclophosphamide. In some embodiments, the chemotherapeutic agent is daunorubicin. In some embodiments, the chemotherapeutic agent is doxifluridine.
  • the chemotherapeutic agent is teniposide. In some embodiments, the chemotherapeutic agent is topotecan. In some embodiments, the chemotherapeutic agent is valrubicin. In some embodiments, the chemotherapeutic agent is vemurafenib. In some embodiments, the chemotherapeutic agent is vinblastine. In some embodiments, the chemotherapeutic agent is vincristine. In some embodiments, the chemotherapeutic agent is vindesine.
  • the one or more additional therapeutic agents are selected from antimetabolites.
  • the one or more antimetabolites are selected from azacytidine, 6- mercaptopurine, capecitabine, hydroxyurea, cladribine, pralatrexate, thioguanine, decitabine, clofarabine, nelarabine, fludarabine, 5 -fluorouracil, gemcitabine, cytarabine, pemetrexed, and methotrexate, cytarabine (Ara-C), floxuridine, fludarabine, pentostatin, and trifluridine/tipiracil combination.
  • the antimetabolite is 5 -fluorouracil. In some embodiments, the antimetabolite is gemcitabine. In some embodiments, the antimetabolite is cytarabine. In some embodiments, the antimetabolite is pemetrexed. In some embodiments, the antimetabolite is and methotrexate. In some embodiments, the antimetabolite is cytarabine (Ara-C). In some embodiments, the antimetabolite is floxuridine. In some embodiments, the antimetabolite is fludarabine. In some embodiments, the antimetabolite is pentostatin. In some embodiments, the antimetabolite is a trifluridine/tipiracil combination.
  • the platinum -based agent is triplatin tetranitrate. In further embodiments, the platinum-based agent is pheanthriplatin. In further embodiments, the platinum -based agent is picoplatin. In further embodiments, the platinum -based agent is satraplatin.
  • the one or more additional therapeutic agents are selected from N- terminal domain inhibitors of androgen receptor.
  • the N-terminal domain inhibitor of androgen receptor is selected from EPI-001, EPI-002 (ralaniten), EPI-506, and EPI-7386.
  • the N-terminal domain inhibitor of androgen receptor is EPI-001.
  • the N-terminal domain inhibitor of androgen receptor is EPI-002 (ralaniten).
  • the N- terminal domain inhibitor of androgen receptor is EPI-506.
  • the N-terminal domain inhibitor of androgen receptor is EPI-7386.
  • the methods disclosed herein wherein the one or more additional therapeutic agents are selected from poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors.
  • PARP poly(adenosine diphosphate-ribose) polymerase
  • the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors are selected from olaparib, niraparib, rucaparib, talazopari, veliparib, pamiparib, CEP-9722, and E7016.
  • the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is olaparib.
  • the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is niraparib. In some embodiments, the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is rucaparib. In some embodiments, the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is talazopari. In some embodiments, the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is veliparib. In some embodiments, the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is pamiparib.
  • the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is CEP-9722. In some embodiments, the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is E7016.
  • the methods disclosed herein wherein the one or more additional therapeutic agents are selected from inhibitors of CYP17.
  • the inhibitor of CYP17 is galeterone.
  • aralkyl and arylalkyl as used herein mean an aryl group covalently linked to an alkylene group wherein the moiety is linked to another group via the alkyl moiety.
  • An exemplary aralkyl group is -(Ci-C6)alkyl(Ce-Cio)aryl, including, without limitation, benzyl, phenethyl, and naphthylmethyl.
  • heteroaralkyl groups include pyridylmethyl, pyridylethyl, pyrrolylmethyl, pyrrolylethyl, imidazolylmethyl, imidazolylethyl, thiazolylmethyl, thiazolylethyl, benzimidazolylmethyl, benzimidazolylethyl quinazolinylmethyl, quinolinylmethyl, quinolinylethyl, benzofuranylmethyl, indolinylethyl isoquinolinylmethyl, isoinodylmethyl, cinnolinylmethyl, and benzothiophenylethyl.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active agents may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the proportions of a co-solvent system may be suitably varied without destroying its solubility and toxicity characteristics.
  • identity of the co-solvent components may be varied: for example, other low-toxicity non-polar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may be substituted for dextrose.
  • the pharmaceutical compositions also may comprise suitable solid- or gel-phase carriers or excipients. These carriers and excipients may provide marked improvement in the bioavailability of poorly soluble drugs. Examples of such carriers or excipients include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • the pharmaceutically acceptable formulations of the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, that may be used to practice the methods disclosed herein may contain a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in an amount of from about 0.5 w/w % to about 95 w/w %, or from about 1 w/w % to about 95 w/w %, or from about 1 w/w % to about 75 w/w %, or from about 5 w/w % to about 75 w/w %, or from about 10 w/w % to about 75 w/w %, or from about 10 w/w % to about 50 w/w %.
  • the compound of Formula (I), or a pharmaceutically acceptable salt thereof is administered in an amount between about 150 mg to about 350 mg per day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount between about 150 mg to about 300 mg per day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount between about 160 mg to about 300 mg per day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount of about 160 mg per day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount of about 200 mg per day.
  • the compound of Formula (I), or a pharmaceutically acceptable salt thereof is administered to a subject in need thereof in 28-day cycles. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof in multiple 28-day cycles. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof for at least one 28-day cycle. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof on each day of each 28-day cycle.
  • the methods described herein comprise administering the compositions and formulations comprising the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, in combination with one or more additional therapeutic agents, to the subject or subject in need thereof in multiple cycles repeated on a regular schedule with periods of rest in between each cycle.
  • treatment given for one week followed by three weeks of rest is one treatment cycle.
  • the length of a treatment cycle depends on the treatment being given. In some embodiments, the length of a treatment cycle ranges from two to six weeks. In some embodiments, the length of a treatment cycle ranges from three to six weeks. In some embodiments, the length of a treatment cycle ranges from three to four weeks.
  • the length of a treatment cycle is three weeks (or 21 days). In some embodiments, the length of a treatment cycle is four weeks (28 days). In some embodiments, the length of a treatment cycle is 56 days. In some embodiments, a treatment cycle lasts one, two, three, or four weeks. In some embodiments, a treatment cycle lasts three weeks. In some embodiments, a treatment cycle lasts four weeks. The number of treatment doses scheduled within each cycle also varies depending on the drugs being given.
  • the amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and/or pharmaceutical formulations comprising them that corresponds to such an amount varies depending upon factors such as the particular salt or form, disease condition and its severity, the identity (e.g., age, weight, sex) of the subject or host in need of treatment, but can nevertheless be determined according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the liquid formulation type, the condition being treated, and the subject or host being treated.
  • the GnRH antagonist is degarelix. In some embodiments, the GnRH antagonist is elagolix. In some embodiments, the GnRH antagonist is ganirelix. In some embodiments, the GnRH antagonist is linzagolix. In some embodiments, the GnRH antagonist is relugolix.
  • the prostate cancer in the subject has progressed after having been administered at least one androgen receptor antagonist.
  • the at least one androgen receptor antagonist is selected from abiraterone, enzalutamide, apalutamide, and darolutamide.
  • the prostate cancer in the subject has progressed after having been administered abiraterone.
  • the prostate cancer in the subject has progressed after having been administered enzalutamide.
  • the prostate cancer in the subject has progressed after having been administered apalutamide.
  • the prostate cancer in the subject has progressed after having been administered darolutamide.
  • prostate cancer in the subject exhibits evidence of progressive disease by the Prostate Cancer Working Group 3 (PCWG3) criteria, comprising one or more of (a) 2 or more rising levels of prostate specific antigen (PSA) a minimum of one week apart with the latest result being at least 2.0 ng/mL, (b) 1.0 ng/mL PSA rise, (c) confirmation of 2 new bone lesions on last systemic therapy, and (d) soft tissue progression according to RECIST 1.1 guidelines.
  • PCWG3 Prostate Cancer Working Group 3
  • ANC ANC >1500 cells/mm 3 (1.5 x 103 cells/mm 3 );
  • platelets >100,000 /pL (100 x 109 /L);
  • hemoglobin >9.0 g/dL (90 g/L);
  • AST SGOT
  • ALT ALT
  • bilirubin ⁇ 1.5 x ULN;
  • QTcF QTcF ⁇ 470 msec.
  • [00161] Disclosed herein are methods comprising: (a) providing a biologic sample obtained from a subject having prostate cancer; (b) assaying to detect in the biologic sample obtained from the subject a presence or absence of a biomarker; (c) detecting the presence or absence of the biomarker in the biologic sample using the methods described herein; and (d) administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, if the biomarker is present in the biological sample.
  • the presence of one or more biomarkers described herein indicate the prostate cancer in the subject is neuroendocrine prostate cancer.
  • a biomarker such as synaptophysin (SYP), chromogranin A (CGA), insulinoma-associated protein 1 (INSMI), syntaxin 1 (STX1), ISL LIM homeobox 1 (ISL1), INSM transcriptional repressor 1 (INSM1), secretagogin (SECG), neural cell adhesion molecule 1 (NCAM1; CD56), neuron-specific enolase (NSE), and gastrin-releasing peptide (GRP).
  • a biological sample obtained from the subject has been determined to exhibit a biomarker that may comprise one or more alterations in one or more genes selected from TP53, RBI, and PTEN.
  • the presence, absence, or level, of such biomarkers may be measured, collectively or individually, in a biological sample obtained from a subject, such as a sample of a solid tumor, such as a neuroendocrine prostate cancer, or from a sample of a relevant biological fluid, such as a blood sample.
  • a biological sample obtained from a subject such as a sample of a solid tumor, such as a neuroendocrine prostate cancer, or from a sample of a relevant biological fluid, such as a blood sample.
  • the one or more biomarkers are detected in plasma or serum that is derived from a blood sample obtained from the subject.
  • the methods of detection disclosed herein are useful for predicting a therapeutic response to a therapy described herein (e.g., the administration to a subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof), monitor the treatment using the therapy of, and treating with the therapy, a proliferative disease or condition described herein in a subject.
  • a therapy described herein e.g., the administration to a subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof
  • the expression of a biomarker in a biological sample from a subject is measured by use of immunohistochemistry (IHC) assays.
  • a biomarker such as synaptophysin (SYP), chromogranin A (CGA), insulinoma-associated protein 1 (INSMI), syntaxin 1 (STX1), ISL LIM homeobox 1 (ISL1), INSM transcriptional repressor 1 (INSMI), secretagogin (SECG), neural cell adhesion molecule 1 (NCAM1; CD56), neuron-specific enolase (NSE), and gastrin-releasing peptide (GRP), is measured by use of immunohistochemistry (IHC) assays.
  • immunohistochemistry (IHC) assays are commercially available, or may be developed and utilized according to methods known to those having ordinary skill in the art.
  • Immunohistochemistry techniques utilize an antibody to probe and visualize cellular antigens in situ, generally by chromogenic or fluorescent methods.
  • antibodies or antisera, polyclonal antisera, or monoclonal antibodies specific for each marker are used to detect expression.
  • the antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase.
  • unlabeled primary antibody is used in conjunction with a labeled secondary antibody, comprising antisera, polyclonal antisera, or a monoclonal antibody specific for the primary antibody.
  • Immunohistochemistry protocols and kits are well known in the art and are commercially available.
  • Two general methods of IHC are generally available; direct and indirect assays.
  • binding of antibody to the target antigen is determined directly.
  • This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme -labeled primary antibody, which can be visualized without further antibody interaction.
  • a labeled reagent such as a fluorescent tag or an enzyme -labeled primary antibody, which can be visualized without further antibody interaction.
  • unconjugated primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody.
  • a chromagenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.
  • the primary and/or secondary antibody used for immunohistochemistry typically will be labeled with a detectable moiety.
  • Numerous labels are available which can be generally grouped into the following categories.
  • the antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991) for example and radioactivity can be measured using scintillation counting.
  • colloidal gold particles are colloidal gold particles.
  • the enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques.
  • the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically.
  • the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above.
  • the chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor.
  • Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat.
  • luciferin 2,3- dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, P-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • HRPO horseradish peroxidase
  • alkaline phosphatase P-galactosidase
  • glucoamylase lysozyme
  • saccharide oxidases e.g., glucose oxidase, galactose oxidase, and glucose-6-
  • DAB 3,3 -Diaminobenzidine
  • AP alkaline phosphatase
  • P-D-Gal P-D-galactosidase
  • a chromogenic substrate e.g., p-nitrophenyl-p-D-galactosidase
  • Anorogenic substrate e.g., 4-methylumbelliferyl-P-D-galactosidase
  • the label is indirectly conjugated with the antibody.
  • the antibody can be conjugated with biotin and any of the four broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner.
  • the antibody is conjugated with a small hapten and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody.
  • indirect conjugation of the label with the antibody can be achieved.
  • Biological samples obtained from subjects comprising tissue samples may be prepared according to protocols commonly used in the art.
  • sections of paraffin-embedded cells or tissues are obtained by (1) preserving tissue in fixative, (2) dehydrating the fixed tissue, (3) infiltrating the tissue with fixative, (4) orienting the tissue such that the cut surface accurately represents the tissue, (5) embedding the tissue in paraffin (making a paraffin block), (6) cutting tissue paraffin block with a microtome in sections of 4-5 picometers, and (7) mounting sections onto slides.
  • the slides may then be read by a pathologist or the like assessing for the presence or absence of a biomarker, or of abnormal or normal cells or a specific cell type and provides the loci of the cell types of interest.
  • a pathologist or the like would review the slides and identify normal cells and abnormal cells (such as abnormal or tumor cells).
  • Any means of defining the loci of the cells of interest may be used (e.g., coordinates on an X-Y axis.
  • tissue section prior to, during or following IHC may be desired.
  • epitope retrieval methods such as heating the tissue sample in citrate buffer may be carried out [see, e.g., Leong et al. Appl.
  • tissue section is exposed to primary antibody for a sufficient period of time and under suitable conditions such that the primary antibody binds to the target protein antigen in the tissue sample. Appropriate conditions for achieving this can be determined by routine experimentation.
  • the label is an enzymatic label (e.g. HRPO) which catalyzes a chemical alteration of the chromogenic substrate such as 3,3 ’-diaminobenzidine chromogen.
  • the enzymatic label is conjugated to antibody which binds specifically to the primary antibody (e.g. the primary antibody is rabbit polyclonal antibody and secondary antibody is goat anti -rabbit antibody). Specimens thus prepared may be mounted and coverslipped. Slide evaluation is then determined, e.g. using a microscope.
  • IHC may be combined with morphological staining, either prior to or thereafter.
  • the sections mounted on slides may be stained with a morphological stain for evaluation.
  • the morphological stain to be used provides for accurate morphological evaluation of a tissue section.
  • the section may be stained with one or more dyes each of which distinctly stains different cellular components.
  • hematoxylin is use for staining cellular nucleic of the slides. Hematoxylin is widely available. An example of a suitable hematoxylin is Hematoxylin II (Ventana). When lighter blue nuclei are desired, a bluing reagent may be used following hematoxylin staining.
  • staining may be optimized for a given tissue by increasing or decreasing the length of time the slides remain in the dye.
  • the tissue section may be analyzed by standard techniques of microscopy. Generally, a pathologist or the like assesses the tissue for the presence of abnormal or normal cells or a specific cell type and provides the loci of the cell types of interest. Thus, for example, a pathologist or the like would review the slides and identify normal cells and abnormal cells (such as abnormal or tumor cells). Any means of defining the loci of the cells of interest may be used (e.g., coordinates on an X-Y axis).
  • the presence, or an absence, and/or a level of expression of the biomarker is detected in the sample obtained from a subject by analyzing the genetic material in the sample.
  • the genetic material is obtained from blood, serum, plasma, sweat, hair, tears, urine, and other techniques known by one of skill in the art.
  • the sample comprises circulating tumor RNA (ctRNA).
  • the sample comprises peripheral blood mononuclear cells (PBMCs).
  • the sample comprises circulating tumor cells (CTCs).
  • the genetic material is obtained from a tumor biopsy or liquid biopsy.
  • a tumor biopsy comprises a formalin-fixed paraffin embedded biopsy, a fresh frozen biopsy, a fresh biopsy, or a frozen biopsy.
  • a liquid biopsy comprises PBMCs, circulating tumor RNA, plasma cell-free RNA, or circulating tumor cells (CTCs). Tumor and liquid biopsies can undergo additional analytic processing for sample dissociation, cell sorting, and enrichment of cell populations of interest.
  • methods of detecting a presence, absence, or level of a biomarker in a biologic sample obtained from the subject involve detecting a nucleic acid sequence.
  • the nucleic acid sequence comprises deoxyribonucleic acid (DNA), such as in the case of detecting complementary DNA (cDNA) of an mRNA transcript.
  • the nucleic acid sequence comprises a denatured DNA molecule or fragment thereof.
  • the nucleic acid sequence comprises DNA selected from: genomic DNA, viral DNA, mitochondrial DNA, plasmid DNA, amplified DNA, circular DNA, circulating DNA, cell-free DNA, or exosomal DNA.
  • the DNA is single -stranded DNA (ssDNA), double -stranded DNA, denaturing double -stranded DNA, synthetic DNA, and combinations thereof.
  • the circular DNA may be cleaved or fragmented.
  • the nucleic acid sequence comprises ribonucleic acid (RNA).
  • the nucleic acid sequence comprises fragmented RNA.
  • the nucleic acid sequence comprises partially degraded RNA.
  • the nucleic acid sequence comprises a microRNA or portion thereof.
  • the nucleic acid sequence comprises an RNA molecule or a fragmented RNA molecule (RNA fragments) selected from: a microRNA (miRNA), a pre-miRNA, a pri-miRNA, a mRNA, a pre-mRNA, a viral RNA, a viroid RNA, a virusoid RNA, circular RNA (circRNA), a ribosomal RNA (rRNA), a transfer RNA (tRNA), a pre-tRNA, a long non-coding RNA (IncRNA), a small nuclear RNA (snRNA), a circulating RNA, a cell- free RNA, an exosomal RNA, a vector-expressed RNA, an RNA transcript, a synthetic RNA, and combinations thereof.
  • miRNA microRNA
  • pre-miRNA pre-miRNA
  • a pri-miRNA a RNA
  • mRNA a pre-mRNA
  • a pri-miRNA a
  • a biomarker is detected by subjecting a sample obtained from the subject to a nucleic acid-based detection assay.
  • the nucleic acid-based detection assay comprises quantitative polymerase chain reaction (qPCR), reverse transcription PCT (RT-qPCR), gel electrophoresis (including for e.g., Northern or Southern blot), immunohistochemistry (IHC), immunofluorescence (IF), in situ hybridization (ISH) such as fluorescent in situ hybridization (FISH), cytochemistry, microarray, or sequencing.
  • the sequencing technique comprises next generation sequencing.
  • the methods involve a hybridization assay such as Anorogenic qPCR (e.g., TaqManTM, SYBR green, SYBR green I, SYBR green II, SYBR gold, ethidium bromide, methylene blue, Pyronin Y, DAPI, acridine orange, Blue View or phycoerythrin), which involves a nucleic acid amplification reaction with a specific primer pair, and hybridization of the amplified nucleic acid probes comprising a detectable moiety or molecule that is specific to a target nucleic acid sequence.
  • a number of amplification cycles for detecting a target nucleic acid in a qPCR assay is about 5 to about 30 cycles.
  • the number of amplification cycles for detecting a target nucleic acid is at least about 5 cycles. In some instances, the number of amplification cycles for detecting a target nucleic acid is at most about 30 cycles. In some instances, the number of amplification cycles for detecting a target nucleic acid is about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 30, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 15 to about 20, about 15 to about 25, about 15 to about 30, about 20 to about 25, about 20 to about 30, or about 25 to about 30 cycles.
  • the probe may be a hydrolysable probe comprising a Auorophore and quencher that is hydrolyzed by DNA polymerase when hybridized to a target nucleic acid.
  • the presence of a target nucleic acid is determined when the number of amplification cycles to reach a threshold value is less than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 cycles.
  • hybridization may occur at standard hybridization temperatures, e.g., between about 35 °C and about 65 °C in a standard PCR buffer.
  • An additional exemplary nucleic acid -based detection assay comprises the use of nucleic acid probes conjugated or otherwise immobilized on a bead, multi -well plate, or other substrate, wherein the nucleic acid probes are configured to hybridize with a target nucleic acid sequence.
  • the nucleic acid probe is specific to one or more of a polynucleotide sequence that encodes a relevant biomarker as disclosed herein.
  • the nucleic acid probe specific to a biomarker comprises a nucleic acid probe sequence sufficiently complementary to the polynucleotide sequence that encodes the relevant biomarker protein.
  • the probe comprises a transcribed polynucleotide sequence (e.g., RNA, cDNA).
  • the nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length and sufficient to specifically hybridize under standard hybridization conditions to the target nucleic acid sequence.
  • the target nucleic acid sequence is immobilized on a solid surface and contacted with a probe, for example by running the isolated target nucleic acid sequence on an agarose gel and transferring the target nucleic acid sequence from the gel to a membrane, such as nitrocellulose.
  • the probe(s) are immobilized on a solid surface, for example, in an Affymetrix gene chip array, and the probe(s) are contacted with the target nucleic acid sequence.
  • the term “probe” with regards to nucleic acids refers to any nucleic acid molecule that is capable of selectively binding to a specifically intended target nucleic acid sequence.
  • probes are specifically designed to be labeled, for example, with a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, or other labels or tags that are known in the art.
  • the fluorescent label comprises a fluorophore.
  • the fluorophore is an aromatic or heteroaromatic compound.
  • the fluorophore is a pyrene, anthracene, naphthalene, acridine, stilbene, benzoxazole, indole, benzindole, oxazole, thiazole, benzothiazole, canine, carbocyanine, salicylate, anthranilate, xanthenes dye, coumarin.
  • xanthene dyes include, e.g., fluorescein and rhodamine dyes.
  • Fluorescein and rhodamine dyes include, but are not limited to 6-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N; N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX).
  • Suitable fluorescent probes also include the naphthylamine dyes that have an amino group in the alpha or beta position.
  • naphthylamino compounds include l-dimethylaminonaphthyl-5 -sulfonate, l-anilino-8-naphthalene sulfonate, and 2-p-toluidinyl-6- naphthalene sulfonate, 5 -(2 '-aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS).
  • Detection of the mRNA involves amplification of the subject’s nucleic acid by the polymerase chain reaction (PCR).
  • the PCR assay involves use of a pair of primers capable of amplifying at least about 10 contiguous nucleobases within a nucleic acid sequence, thereby amplifying the one or more gene products in the biomarker.
  • Anorogenic quantitative PCR quantitation is based on amount of Huorescence signals (TaqMan and SYBR green).
  • the nucleic acid probe is conjugated to a detectable molecule.
  • the detectable molecule may be a Huorophore.
  • the nucleic acid probe may also be conjugated to a quencher.
  • the assay for detecting the presence or absence of mRNA encoding a relevant biomarker comprises reverse -transcribing the relevant mRNA molecule to produce a corresponding complementary DNA (cDNA) molecule.
  • the assay further comprises contacting the cDNA molecule with a nucleic acid probe comprising a nucleic acid sequence that is complementary to a nucleic acid sequence of the cDNA molecule.
  • the assay comprises detecting a double-stranded hybridization product between the nucleic acid probe and the cDNA molecule.
  • the hybridization product is further amplified using a pair of primers.
  • this technique utilizes a column or resin based nucleic acid purification scheme such as those commonly sold commercially, one non-limiting example would be the GenElute Bacterial Genomic DNA Kit available from Sigma Aldrich.
  • the nucleic acid is stored in water, Tris buffer, or Tris-EDTA buffer before subsequent analysis.
  • the nucleic acid material is extracted in water. In some cases, extraction does not comprise nucleic acid purification.
  • a decrease in the quantity of ctRNA suggests the solid tumor is shrinking and treatment with a compound of Formula (I), or a pharmaceutically acceptable salt thereof, is effective.
  • a lack of ctRNA in the bloodstream indicates that the cancer has not returned after treatment with a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • a subject having neuroendocrine prostate cancer comprising: (a) providing a biologic sample obtained from a subject having prostate cancer; (b) assaying to detect in the biologic sample obtained from the subject a presence or absence of a biomarker; (c) detecting the presence or absence of the biomarker in the biologic sample using the methods described herein; and (d) administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, if the biomarker is present in the biological sample.
  • expression of a biomarker is based on the expression level of the biomarker deviating from a reference expression level.
  • the expression level is high, relative to the reference expression level. In some embodiments, the expression level is low, relative to the reference expression level. In some embodiments, the reference expression level is derived from an individual, or a group of individuals, that do not have cancer. In some embodiments, the reference expression level is derived from an individual, or a group of individuals, that have cancer that does not therapeutically respond to the compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the expression level deviates from the reference expression level by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
  • the determination of expression or the presence of a biomarker is defined based on the percentage of cells that stain weakly, moderately, or strongly for the relevant biomarker, with the threshold defining the minimal percentage of cells that are required to stain positive at the various intensity levels (>a% of prostate tumor cells stain weakly, >b% of prostate tumor cells stain moderately, >c% of prostate tumor cells stain strongly, or a combination thereof).
  • a label is on or associated with the container.
  • a label is on a container when letters, numbers or other characters forming the label are attached, molded, or etched into the container itself, a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert.
  • a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.
  • X is O, CR 5 , CR 5 OH, or C(R 5 )2, wherein: when X is O, - is a single bond; when X is C(R 5 )2, - is a single bond; when X is CR 5 OH, - is a single bond; or when X is CR 5 , - is a double bond;
  • R 2 is cyano, -COOR 5 , -C(O)N(R 5 )2, or -C(O)N(R 5 )2 wherein each R 5 taken together with the nitrogen atom to which they are attached form a 5 - 8 membered heterocyclic ring optionally substituted with one or more R 4 ; each R 3 is independently C1-C3 alkyl or halogen; each R 4 is independently oxo, cyano, halogen, -POXCi-C alkyl)2, hydroxyl, alkoxy, hydroxyalkyl, heteroalkyl, aralkyl, haloalkyl, -COOR 5 , -Y 2 -haloalkyl, -Y’-Ci-Ce alkyl, -Y 2 -Ci-Ce alkyl, -L- cycloalkyl, -L-heteroaryl, -L-heterocyclyl, -Y'-hetero
  • Embodiment 6 The method of any of embodiments 1-4, wherein R 2 is -COOR 5 .
  • Embodiment 14 The method of embodiment 13, wherein the aryl is phenyl optionally substituted with one or more R 4 .
  • Embodiment 17 The method of embodiment 16, wherein R 4 is -Y'-Ci-Ce alkyl and Y 1 is a bond and the Ci-Ce alkyl is methyl, ethyl, isopropyl, butyl, or pentyl.
  • Embodiment 18 The method of embodiment 16, wherein R 4 is -Y 2 -Ci-Ce alkyl and Y 2 is a -SO 2 - and the Ci-Ce alkyl is methyl.
  • Embodiment 19 The method of embodiment 16, wherein R 4 is -Y 2 -haloalkyl and Y 2 is -S- or -SO 2 - and the haloalkyl is trifluoromethyl.
  • Embodiment 20 The method of embodiment 16, wherein R 4 is -L-N(R 5 )2 and L is a bond and each R 5 is hydrogen, each R 5 is methyl or one R 5 is methyl and one R 5 is hydrogen.
  • Embodiment 21 The method of embodiment 16, wherein R 4 is -L-N(R 5 )2 and L is methylene or ethylene and each R 5 is hydrogen, each R 5 is methyl or one R 5 is methyl and one R 5 is hydrogen.
  • Embodiment 22 The method of embodiment 16, wherein R 4 is -Y'-N(R 5 )2, Y 1 is -C(O)- and each R 5 independently is hydrogen, each R 5 is independently methyl or one R 5 is methyl and one R 5 is hydrogen.
  • Embodiment 23 The method of embodiment 16, wherein R 4 is -Y 2 -N(R 5 )2, Y 2 is -SO2- and each R 5 independently is hydrogen, each R 5 is methyl or one R 5 is methyl and one R 5 is independently hydrogen.
  • Embodiment 24 The method of embodiment 16, wherein R 4 is -Y 1 -heterocyclyl and Y 1 is -C(O)- and the heterocyclyl portion of the L-heterocyclyl is piperazinyl or 4-methyl-piperazinyl.
  • Embodiment 25 The method of embodiment 16, wherein R 4 is -L-heterocyclyl and L is a bond and the heterocyclyl portion of the L-heterocyclyl is azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, or 3/. 2 -azabicyclo
  • Embodiment 26 The method of embodiment 16, wherein R 4 is -L-heterocyclyl, wherein L is a methylene and the heterocyclyl portion of the L-heterocyclyl is azetidinyl, oxetanyl, pyrrolidinyl piperidinyl, each optionally substituted with one or more R 7 selected from C1-C3 alkyl, alkoxy, hydroxyl and halogen.
  • Embodiment 27 The method of embodiment 16, wherein R 4 is -Y 1 -heterocyclyl and Y 1 is -C(O)- and the heterocyclyl portion of the Y 1 -heterocyclyl is morpholinyl optionally substituted with one or more C1-C3 alkyl.
  • Embodiment 28 The method of embodiment 16, wherein R 4 is -L-heteroaryl optionally substituted with one or more R 7 .
  • Embodiment 29 The method of embodiment 28, wherein the -L-heteroaryl is tetrazolyl.
  • Embodiment 31 The method of embodiment 16, wherein R 4 is -COOR 5 .
  • Embodiment 32 The method of embodiment 16, wherein R 4 is hydroxyalkyl.
  • Embodiment 34 The method of embodiment 16, wherein R 4 is aralkyl.
  • Embodiment 36 The method of embodiment 35, wherein the heteroaryl is pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazinyl, pyridyl, pyridinyl-2-one, pyrazinyl, pyridazinyl, pyrimidinyl, isoxazolyl, isoindolinyl, naphthyridinyl, 1,2,3,4-tetrahydroisoquinolinyl, or 5,6-dihydro-4H-pyrrolo[l,2- b]pyrazolyl, each optionally substituted with one or more R 4 .
  • the heteroaryl is pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazinyl, pyridyl, pyridinyl-2-one, pyrazinyl, pyridazinyl
  • Embodiment 38 The method of embodiment 37, wherein the heteroaryl is pyrazolyl optionally substituted with one R 4 independently selected from hydroxyalkyl, heteroalkyl, haloalkyl, -Y’-Ci-Ce alkyl, - L-N(R 5 )2, L-heterocyclyl or L-heteroaryl, wherein the heteroaryl of the L-heteroaryl or the heterocyclyl portion of the L-heterocyclyl is optionally substituted with one or more R 7 .
  • R 4 independently selected from hydroxyalkyl, heteroalkyl, haloalkyl, -Y’-Ci-Ce alkyl, - L-N(R 5 )2, L-heterocyclyl or L-heteroaryl, wherein the heteroaryl of the L-heteroaryl or the heterocyclyl portion of the L-heterocyclyl is optionally substituted with one or more R 7 .
  • Embodiment 40 The method of embodiment 38, wherein R 4 is -L-heterocyclyl optionally substituted with one or more R 7 where L is a bond and the heterocyclyl portion of the L-heterocyclyl is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, piperazinyl, or 4-methylpiperazinyl.
  • Embodiment 41 The method of embodiment 38, wherein R 4 is -L-heterocyclyl optionally substituted with one or more R 7 where L is methylene and the heterocyclyl portion of the L-heterocyclyl is azetidinyl, oxetanyl, pyrrolidinyl, pyrrolidinone, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, piperazinyl, or 4-methylpiperazinyl.
  • Embodiment 43 The method of embodiment 38, wherein R 4 is -Y'-Ci-Ce alkyl where Y 1 is a bond and the Ci-Ce alkyl is methyl, ethyl, or isopropyl.
  • Embodiment 50 A method of treating prostate cancer in a subject, wherein the prostate cancer has been determined to comprise neuroendocrine prostate cancer, comprising administering to the subject a
  • Embodiment 52 The method of embodiment 51, wherein the compound is: a pharmaceutically acceptable salt thereof.
  • Embodiment 53 The method of embodiment 51, wherein the compound is: a pharmaceutically acceptable salt thereof.
  • Embodiment 54 The method of embodiment 51, wherein the compound is: or a pharmaceutically acceptable salt thereof.
  • Embodiment 55 The method of embodiment 51, wherein the compound is: a pharmaceutically acceptable salt thereof.
  • Embodiment 59 The method of embodiment 51, wherein the compound is: a pharmaceutically acceptable salt thereof.
  • Embodiment 60 The method of embodiment 51, wherein the compound is: or a pharmaceutically acceptable salt thereof.
  • Embodiment 61 The method of embodiment 51 , wherein the compound is: a pharmaceutically acceptable salt thereof.
  • Embodiment 62 The method of embodiment 51, wherein the compound is: a pharmaceutically acceptable salt thereof.
  • Embodiment 64 The method of any one of embodiments 1-63, wherein the subject has been administered one or more first agents prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and wherein the one or more first agents is selected from the group consisting of (a) luteinizing hormone -releasing hormone (LHRH) agonists, (b) luteinizing hormone -releasing hormone (LHRH) antagonists, (c) androgen receptor inhibitors, (d) inhibitors of cytochrome P450 17A1, and (e) antiandrogens.
  • LHRH luteinizing hormone -releasing hormone
  • LHRH luteinizing hormone -releasing hormone
  • LHRH luteinizing hormone -releasing hormone
  • LHRH luteinizing hormone -releasing hormone
  • LHRH luteinizing hormone -releasing hormone
  • c androgen receptor inhibitors
  • inhibitors of cytochrome P450 17A1 inhibitors of cytochrome P450 17A1
  • Embodiment 67 The method of embodiment 64, wherein (a) the neuroendocrine prostate cancer in the subject has been determined to be progressing, and (b) testosterone levels in the subject have been determined to be equal to or less than 50 mg/mL, following the administration to the subject of the one or more first agents.
  • Embodiment 68 The method of embodiment 64, wherein (a) the neuroendocrine prostate cancer in the subject has been determined to be radiographically progressing, and (b) testosterone levels in the subject have been determined to be 50 mg/mL or less following the administration to the subject of the one or more first agents.
  • Embodiment 74 The method of embodiment 73, wherein the neuroendocrine prostate cancer in the subject has been determined to comprise 20 or more bone metastases.
  • Embodiment 81 The method of embodiment 80, wherein the androgen receptor inhibitor is selected from enzalutamide, apalutamide, and darolutamide.
  • Embodiment 85 The method of any one of embodiments 64-75, wherein the one or more first agents is an inhibitor of cytochrome P450 17A1.
  • Embodiment 86 The method of embodiment 85, wherein the one or more inhibitors of cytochrome P450 17A1 is abiraterone acetate.
  • Embodiment 95 The method of embodiment 89 or 90, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for synaptophysin (SYP).
  • IHC immunohistochemical stain for synaptophysin
  • Embodiment 112 The method of any one of embodiments 1-111, wherein the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to the subject in an amount between about 0.01 mg/kg per day to about 300 mg/kg per day.
  • Embodiment 113 The method of embodiment 112, wherein the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to the subject in an amount between about 0. 1 mg/kg per day to about 100 mg/kg per day.
  • Embodiment 114 The method of any one of embodiments 1-113, further comprising administering to the subject one or more additional therapeutic agents.
  • Embodiment 118 The method of embodiment 115, wherein the one or more additional therapeutic agents are selected from mitotic inhibitors.
  • Embodiment 133 The method of embodiment 132, wherein the one or more heat shock protein 90 (HSP90) inhibitors are selected from tanespimycin, luminespib, alvespimycin, ganetespib, BIIB021, onalespib, geldanamycin, NVP-BEP800, SNX-2112 (PF-04928473), PF-04929113 (SNX-5422), KW-2478, XL888, TAS-116, VER-50589, CH5138303, VER-49009, NMS-E973, zelavespib (PU-H71), and HSP990 (NVP-HSP990).
  • HSP90 heat shock protein 90
  • Embodiment 141 The method of embodiment 140, wherein the anti-PD-Ll agents are selected from atezolizumab, avelumab, durvalumab, MPDL3280A (RG7446), MDX-1105 (BMS-936559), BMS- 935559, MSB0010718C, and MEDI4736.
  • the anti-PD-Ll agents are selected from atezolizumab, avelumab, durvalumab, MPDL3280A (RG7446), MDX-1105 (BMS-936559), BMS- 935559, MSB0010718C, and MEDI4736.
  • Embodiment 142 The method of embodiment 115 wherein the one or more additional therapeutic agents are selected from anti-CTLA-4 agents.
  • Embodiment 144 The method of embodiment 114, wherein the one or more additional therapeutic agents are selected from surgery, radiation, and prostate -specific membrane antigen (PSMA) targeted agents.
  • PSMA prostate -specific membrane antigen
  • Example 1 Prostate cancer cell line NCI-H660 represents neuroendocrine prostate cancer
  • RT-qPCR reactions were set up in triplicate using 3 pL of 3 ng/ pL of RNA in 7 pL of pre-mixed SensiFASTTM SYBRNo-ROX One-Step Kit reagents (BIOLINE# BIO-98005). The RT-qPCR reactions were run on a Bio-Rad CFX384 Real-Time PCR System and data was plotted with GraphPad Prism 8 software.
  • RT-qPCR was used to measure the expression levels of androgen receptor (AR), synaptophysin (SYP), and chromogranin A (CGA) in prostate cancer cell lines including VCaP, LNCaP, CWR22PC, 22Rvl, LREX’, LAPC4, H660, PC3 and DU145 (FIG. 1A, FIG. IB, FIG. 1C).
  • AR androgen receptor
  • SYP synaptophysin
  • CGA chromogranin A
  • Example 2 Administration of Compound 4 to NCI-H660 tumor-bearing male athymic nude mice [00343] NCI-H660 (ATCC® CRL-5813TM) androgen-independent neuroendocrine prostate tumor cells were inoculated into right flanks of 90 intact male athymic nude mice.
  • RNA sequencing was performed on the H660 samples obtained from mice treated with 100 mg/kg of Compound 4 or vehicle from this Example 2. Extracted RNA was process into libraries for RNA-sequencing at Q2 Solutions-EA Genomics (North Carolina, USA). RNA-sequencing data was aligned using Kallisto 0.46.1 (Bray et al, Nature Biotechnology 2016) against a combined human Gencode v38 and mouse Gencode vM27 reference of the protein coding transcripts (Frankish et al, Nucleic Acids Research 2019).
  • ASCL1 and MYCL have been associated with lineage trans-differentiation in prostate cancer (see, for example, Davies et al, Cancer Discovery 2023).
  • Example 6 Thermal gravimetric analyses and differential scanning calorimetry analyses of Form 1 and Form 2 of Compound 4

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Abstract

Disclosed herein are methods of treating neuroendocrine prostate cancer in a subject by administrating to the subject a compound of Formula (I)

Description

TREATMENT OF NEUROENDOCRINE PROSTATE CANCER
CROSS-REFERENCE
[0001] This application claims the benefit of U. S. Provisional Application Serial No. 63/478,296 filed January 3, 2023 and U. S. Provisional Application Serial No. 63/518,146 filed August 8, 2023; which are hereby incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Prostate cancer is the second leading cause of cancer-related death in men in the US. Androgen deprivation therapy (ADT) and blockade are commonly used to treat prostate cancer. However, relapse occurs with subsequent progression to metastatic castration-resistant prostate cancer (mCRPC) after treatment with androgen biosynthesis inhibitors or androgen receptor (AR) antagonists through multiple acquired resistance mechanisms. While some mechanisms of resistance are androgen receptor (AR) dependent, and can be treated with other hormonal therapies, like AR antagonists such as enzalutamide, other mechanisms of resistance occur by trans-differentiation of adenocarcinoma cells into neuroendocrine prostate cancer (NEPC). Rarely does NEPC arise de novo, the majority of cases emerge following relapse of prostate adenocarcinoma (PCa) with androgen -deprivation therapy (ADT) (Palmgren 2007; Yuan 2007; Lotan 2011). Molecular mechanisms controlling the emergence of lethal NEPC remain unclear, although nonclinical reports suggest that PRC2 activity contributed to the transition from PCa to NEPC; moreover, PRC2 activity as well as EZH2 expression are elevated in NEPC patient tumors (Beltran 2016; Ku 2017). Thus, inhibiting PRC2 activity could potentially be a treatment option to treat subjects determined to suffer from NEPC. Clinically, NEPC is distinct from prostate adenocarcinoma and is an aggressive form of prostate cancer that is resistant to current therapies used in the context of advanced prostate adenocarcinoma. In addition, NEPC displays high proliferative rates and tumor dissemination to visceral organs such as lung and liver. NEPC is a significant problem with recent evidence indicating that approximately twenty percent of subjects with progressive hormone-resistant prostate cancer suffer from NEPC, and data demonstrates that subjects determined to suffer from NEPC have a median time of survival of less than a year. Thus, there remains a need to develop new methods of treating subjects determined to have NEPC.
SUMMARY OF THE INVENTION
[0003] Provided herein are methods treating prostate cancer in a subject, wherein the prostate cancer has been determined to comprise neuroendocrine prostate cancer, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I)
Figure imgf000004_0001
Formula (I) or a pharmaceutically acceptable salt thereof: wherein:
- represents a single or a double bond;
Z is O or S;
X is O, CR5, CR5OH, or C(R5)2, wherein: when X is O, - is a single bond; when X is C(R5)2, - is a single bond; when X is CR5OH, - is a single bond; or when X is CR5, - is a double bond;
R1 is aryl, heteroaryl, L-cycloalkyl, -N(R5)heterocyclyl, or L-heterocyclyl, wherein the aryl, the heteroaryl or the cyclyl portion of the L-cycloalkyl, -N(R5)heterocyclyl, or L-heterocyclyl is optionally substituted with one or more R4;
R2 is cyano, -COOR5, -C(O)N(R5)2, or -C(O)N(R5)2 wherein each R5 taken together with the nitrogen atom to which they are attached form a 5 - 8 membered heterocyclic ring optionally substituted with one or more R4; each R3 is independently C1-C3 alkyl or halogen; each R4 is independently oxo, cyano, halogen, -POsiCi-C, alkyl):. hydroxyl, alkoxy, hydroxyalkyl, heteroalkyl, aralkyl, haloalkyl, -COOR5, -Y2-haloalkyl, -Y’-Ci-Ce alkyl, -Y2-Ci-Ce alkyl, -L-cycloalkyl, -L-heteroaryl, -L-heterocyclyl, -Y'-heterocyclyl, -Y2-heterocyclyl, -L-N(R5)2, - O-L-N(R5)2, -C(CF3)N(R5)2, -Y’-N(R5)2, -Y2-N(R5)2 wherein the ring portion of the aralkyl, -L- cycloalkyl, -L-heteroaryl, -L-heterocyclyl or -Y’-heterocyclyl is optionally substituted with one or more R7;
L is a bond or C1-C4 alkylene;
Y1 is a bond, -C(O)-, or -NHC(O)-;
Y2 is a bond, -S-, -SO-, -SO2-, or -NR5SO2-, each R5 is hydrogen or C1-C3 alkyl;
R6 is hydrogen, C1-C3 alkyl, halogen, haloalkyl, hydroxyalkyl, or heteroalkyl; each R7 is oxo, cyano, hydroxyl, alkoxy, halogen, haloalkyl, hydroxyalkyl, heteroalkyl, cycloalkyl, -L-N(R5)2, Ci-Ce alkyl or -Y’-heterocyclyl; and n is 1 or 2.
[0004] In other embodiments are provided the methods disclosed herein, wherein in the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, Z is O. In other embodiments are provided the methods disclosed herein, wherein in the compounds of Formula (I) , or a pharmaceutically acceptable salt thereof, Z is S.
[0005] In other embodiments are provided the methods disclosed herein, wherein in the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, n is 1.
[0006] In other embodiments are provided the methods disclosed herein, wherein in the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, R2 is cyano. In other embodiments, R2 is - COOR5 or -C(O)N(R5)2. In other embodiments, R2 is -COOR5. In other embodiments, R2 is -C(O)N(R5)2. [0007] In other embodiments are provided the methods disclosed herein, wherein in the compounds of Formula (I) , or a pharmaceutically acceptable salt thereof, R3 is halogen. In some embodiments R3 is fluorine.
[0008] In other embodiments are provided the methods disclosed herein, wherein in the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, X is C(R5)2 and - is a single bond. In other embodiments, X is CR5 and - is a double bond. In other embodiments, X is O and - is a single bond.
[0009] In other embodiments are provided the methods disclosed herein, wherein in the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is aryl optionally substituted with one or more R4. In some embodiments the aryl is phenyl optionally substituted with one or more R4. In other embodiments, the phenyl is substituted with one, two or three R4. In some embodiments the one, two or three R4 are each independently halogen, -PCffC i-C alkyl)2, hydroxyl, hydroxyalkyl, aralkyl, haloalkyl, - COOR5, -Y’-Ci-Ce alkyl, Y2-CI-C6 alkyl, -L-N(R5)2, -O-L-N(R5)2, -C(CF3)N(R5)2, -Y’-N(R5)2, -Y2-N(R5)2, Y2 -haloalkyl, -L-heteroaryl, -L-heterocyclyl, or -Y1 -heterocyclyl, wherein the heterocyclyl portion of the - L-heterocyclyl or -Y1 -heterocyclyl is optionally substituted with one or more R7. In other embodiments, R4 is -Y’-Ci-Ce alkyl and Y1 is a bond and the Ci-Ce alkyl is methyl, ethyl, isopropyl, butyl or pentyl. In still other embodiments, R4 is -Y2-Ci-Ce alkyl and Y2 is a -SO2- and the Ci-Ce alkyl is methyl. In further embodiments, R4 is -Y2 -haloalkyl and Y2 is -S- or -SO2- and the haloalkyl is trifluoromethyl. In further embodiments, R4 is -L-N(R5)2 and L is a bond and each R5 is hydrogen, each R5 is methyl or one R5 is methyl and one R5 is hydrogen. In other embodiments, R4 is -L-N(R5)2 and L is methylene or ethylene and each R5 is hydrogen, each R5 is methyl or one R5 is methyl and one R5 is hydrogen. In further embodiments, R4 is -Y’-N(R5)2, Y1 is -C(O)- and each R5 independently is hydrogen, each R5 is independently methyl or one R5 is methyl and one R5 is hydrogen. In other embodiments, R4 is -Y2-N(R5)2, Y2 is -SO2- and each R5 independently is hydrogen, each R5 is methyl or one R5 is methyl and one R5 is independently hydrogen. In still other embodiments, R4 is -Y1 -heterocyclyl and Y1 is -C(O)- and the heterocyclyl portion of the L- heterocyclyl is piperazinyl or 4-methyl-piperazinyl. In other embodiments, R4 is -L-heterocyclyl and L is a bond and the heterocyclyl portion of the L-heterocyclyl is azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, or 3/.2-azabicyclo|3.1 ,0]hexanyl, each optionally substituted with one or more R7 selected from oxo, C1-C3 alkyl, alkoxy, hydroxyl, and halogen. In still further embodiments, R4 is -L-heterocyclyl, wherein L is a methylene and the heterocyclyl portion of the L- heterocyclyl is azetidinyl, oxetanyl, pyrrolidinyl piperidinyl, each optionally substituted with one or more R7 selected from C1-C3 alkyl, alkoxy, hydroxyl and halogen. Further embodiments provide R4 is -Y1- heterocyclyl and Y1 is -C(O)- and the heterocyclyl portion of the Y’-heterocyclyl is morpholinyl optionally substituted with one or more C1-C3 alkyl. In further embodiments, R4 is -L-heteroaryl optionally substituted with one or more R7. In still further embodiments, the -L-heteroaryl is tetrazolyl. Further embodiments provide R4 is -POsfCi-Cs alkyl)2. In other embodiments, R4 is -COOR5. Other embodiments provide R4 is hydroxyalkyl. In yet other embodiments, R4 is -O-L-N(R5)2. Further embodiments provide R4 is aralkyl. [0010] In other embodiments are provided the methods disclosed herein, wherein in the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is heteroaryl optionally substituted with one or more R4. In further embodiments, heteroaryl is pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazinyl, pyridyl, pyridinyl-2-one, pyrazinyl, pyridazinyl, pyrimidinyl, isoxazolyl, isoindolinyl, naphthyridinyl, 1,2,3,4-tetrahydroisoquinolinyl, or 5,6-dihydro-4H-pyrrolo[l,2-b]pyrazolyl, each optionally substituted with one or more R4. In further embodiments, the heteroaryl is substituted with one or more R4; wherein each R4 is independently cyano, halogen, -Y’-Ci-Ce alkyl, -Y2-Ci-Ce alkyl, alkoxy, hydroxyalkyl, heteroalkyl, haloalkyl, -L-cycloalkyl, -L-N(R5)2, -Y'-N(R5)2, -L-heteroaryl, -L-heterocyclyl, or -Y1- heterocyclyl, wherein the heteroaryl of the -L-heteroaryl or the heterocyclyl portion of the L-heterocyclyl, or Y1 -heterocyclyl is optionally substituted with one or more R7. In still further embodiments, the heteroaryl is pyrazolyl optionally substituted with one R4 independently selected from hydroxyalkyl, heteroalkyl, haloalkyl, -Y’-Ci-Ce alkyl, -L-N(R5)2, L-heterocyclyl or L-heteroaryl, wherein the heteroaryl of the L- heteroaryl or the heterocyclyl portion of the L-heterocyclyl is optionally substituted with one or more R7. Further embodiments provide R4 is -L-heteroaryl and L is methylene wherein the heteroaryl is pyridyl optional substituted with one or more R7. In yet further embodiments, R4 is -L-heterocyclyl optionally substituted with one or more R7 where L is a bond and the heterocyclyl portion of the L-heterocyclyl is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, piperazinyl or 4-methylpiperazinyl. Still further embodiments provide R4 is -L-heterocyclyl optionally substituted with one or more R7 where L is methylene and the heterocyclyl portion of the L-heterocyclyl is azetidinyl, oxetanyl, pyrrolidinyl, pyrrolidinone, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, piperazinyl or 4-methylpiperazinyl. In further embodiments, R4 is -L-N(R5)2 where L is methylene and each R5 is independently hydrogen, each R5 is independently C1-C3 alkyl or one R5 is C1-C3 alkyl and one R5 is hydrogen. In yet other embodiments, R4 is -Y’-Ci-Ce alkyl where Y1 is a bond and the Ci-Ce alkyl is methyl, ethyl or isopropyl. In further embodiments, the heteroaryl is pyrazolyl optionally substituted with two R4 groups each independently selected from hydroxyalkyl, heteroalkyl, haloalkyl, and -Y’-Ci-Ce alkyl. Still other embodiments provide the heteroaryl is pyridyl optionally substituted with one R4 independently selected from cyano, halogen, alkoxy, hydroxyalkyl, heteroalkyl, haloalkyl, -Y’-Ci-Ce alkyl, -L-N(R5)2, -Y’-N(R5)2, -L-cycloalkyl, or -L- heterocyclyl optionally substituted with one or more R7.
[0011] In other embodiments are provided the methods disclosed herein, wherein in the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is -L-cycloalkyl optionally substituted with one or more R4.
[0012] In other embodiments are provided the methods disclosed herein, wherein in the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, R1 is -L-heterocyclyl optionally substituted with one or more R4. In some embodiments, L is a bond and the heterocyclyl is piperidinyl or tetrahydropyranyl. [0013] In other embodiments are provided the methods disclosed herein, wherein in the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, n is 2.
[0014] Also provided herein are methods of treating prostate cancer in a subject, wherein the prostate cancer has been determined to comprise neuroendocrine prostate cancer, comprising administering to the subject a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:
Figure imgf000007_0001
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
Figure imgf000011_0001
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
Figure imgf000019_0001
[0015] Further provided herein are methods of treating prostate cancer in a subject, wherein the prostate cancer has been determined to comprise neuroendocrine prostate cancer, comprising administering to the subject a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt thereof, selected from the group consisting of:
Figure imgf000019_0002
Figure imgf000020_0001
pharmaceutically acceptable salt thereof.
[0016] In other embodiments are provided the methods disclosed herein, wherein the compound of
Formula (I) is Compound
Figure imgf000020_0002
(Compound 1), or a pharmaceutically acceptable salt thereof.
[0017] In other embodiments are provided the methods disclosed herein, wherein the compound of
Figure imgf000020_0003
[0018] In other embodiments are provided the methods disclosed herein, wherein the compound of
Formula (I) is Compound
Figure imgf000021_0001
(Compound 3), or a pharmaceutically acceptable salt thereof.
[0019] In other embodiments are provided the methods disclosed herein, wherein the compound of
Figure imgf000021_0003
[0020] Also provided herein are methods of treating prostate cancer in a subject, wherein the prostate cancer has been determined to comprise neuroendocrine prostate cancer, comprising administering to the subject a therapeutically effective amount of Compound
Figure imgf000021_0002
(Compound 4), or a pharmaceutically acceptable salt thereof, wherein Compound 4 is in crystalline form. Also provided herein are such methods wherein the crystalline form of Compound 4 is an anhydrous form. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in an x-ray powder diffraction (XRPD) pattern at 8.1° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a further peak in an x-ray powder diffraction (XRPD) pattern at 9.6° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) pattern at 5.7° ± 0.2° 2-theta, 19.7° ± 0.2° 2- theta, and 22.0° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) pattern at 9.8° ± 0.2° 2-theta, 15.2° ± 0.2° 2-theta, and 17.7° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of about 172 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 205 °C to about 210 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 206 °C to about 210 °C, or from about 207 °C to about 210 °C, or from about 208 °C to about 210 °C, or from about 209 °C to about 210 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 1% upon heating the sample from about 25 °C to a temperature prior to melting. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 1% upon heating the sample from about 25 °C to about 380 °C.
[0021] Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in an x-ray powder diffraction (XRPD) pattern at 7.7° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) pattern at 13.7° ± 0.2° 2-theta and 19.2° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) pattern at 5.5° ± 0.2° 2-theta, 8.6° ± 0.2° 2-theta, 15.9° ± 0.2° 2-theta, 19.9° ± 0.2° 2-theta, and 24.1° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) pattern at 10.6° ± 0.2° 2-theta, 11.0° ± 0.2° 2-theta, 15.4° ± 0.2° 2- theta, 21.0° ± 0.2° 2-theta, and 26.3° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 203 °C to about 210 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 203 °C to about 208 °C, or from about 203 °C to about 206 °C, or from about 203 °C to about 205 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 2% upon heating the sample from about 25 °C to about 380 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 2% upon heating the sample from about 25 °C to about 210 °C. [0022] Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in an x-ray powder diffraction (XRPD) pattern at 7.7° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further a peak in an x-ray powder diffraction (XRPD) pattern at 15.4° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further a peak in an x-ray powder diffraction (XRPD) pattern at 19.2° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a further peak in an x-ray powder diffraction (XRPD) pattern at 13.7° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) pattern at 5.5° ± 0.2° 2-theta, 8.6° ± 0.2° 2-theta, 15.9° ± 0.2° 2-theta, 19.9° ± 0.2° 2-theta, and 24.1° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) pattern at 10.6° ± 0.2° 2-theta, 11.0° ± 0.2° 2-theta, 21.0° ± 0.2° 2-theta, and 26.3° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 203 °C to about 210 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 206 °C to about 210 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 203 °C to about 208 °C, or from about 203 °C to about 206 °C, or from about 203 °C to about 205 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 2% upon heating the sample from about 25 °C to about 380 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 2% upon heating the sample from about 25 °C to about 210 °C.
[0023] Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits peaks in an x-ray powder diffraction (XRPD) pattern at 7.7° ± 0.2° 2-theta and 15.4° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in an x-ray powder diffraction (XRPD) pattern at 19.2° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a further peak in an x-ray powder diffraction (XRPD) pattern at 13.7° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) pattern at 5.5° ± 0.2° 2-theta, 8.6° ± 0.2° 2- theta, 15.9° ± 0.2° 2-theta, 19.9° ± 0.2° 2-theta, and 24.1° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) pattern at 10.6° ± 0.2° 2-theta, 11.0° ± 0.2° 2-theta, 21.0° ± 0.2° 2-theta, and 26.3° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 203 °C to about 210 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 206 °C to about 210 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry pattern of from about 203 °C to about 208 °C, or from about 203 °C to about 206 °C, or from about 203 °C to about 205 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 2% upon heating the sample from about 25 °C to about 380 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 2% upon heating the sample from about 25 °C to about 210 °C.
[0024] Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits peaks in an x-ray powder diffraction (XRPD) pattern at 7.7° ± 0.2° 2-theta and 19.2° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in an x-ray powder diffraction (XRPD) patern at 15.4° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a further peak in an x-ray powder diffraction (XRPD) patern at 13.7° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) patern at 5.5° ± 0.2° 2-theta, 8.6° ± 0.2° 2- theta, 15.9° ± 0.2° 2-theta, 19.9° ± 0.2° 2-theta, and 24.1° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits further peaks in an x-ray powder diffraction (XRPD) patern at 10.6° ± 0.2° 2-theta, 11.0° ± 0.2° 2-theta, 21.0° ± 0.2° 2-theta, and 26.3° ± 0.2° 2-theta. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry patern of from about 203 °C to about 210 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry patern of from about 206 °C to about 210 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a peak in a differential scanning calorimetry patern of from about 203 °C to about 208 °C, or from about 203 °C to about 206 °C, or from about 203 °C to about 205 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 2% upon heating the sample from about 25 °C to about 380 °C. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits a loss in mass in a thermal gravimetric analysis of less than about 2% upon heating the sample from about 25 °C to about 210 °C.
[0025] Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits less than about 10% degradation when stored at 25 °C and 60% relative humidity for at least 7 days. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% degradation when the crystalline form is stored at 25 °C and 60% relative humidity for at least 7 days.
[0026] Also provided herein are such methods wherein the crystalline form of Compound 4 (a) exhibits a peak in an x-ray powder diffraction (XRPD) patern at 8. 1° ± 0.2° 2-theta, and (b) exhibits less than about 10% degradation when the crystalline form is stored at 25 °C and 60% relative humidity for at least 7 days. Also provided herein are such methods wherein the crystalline form of Compound 4 (a) exhibits peaks in an x-ray powder diffraction (XRPD) patern at 9.6° ± 0.2° 2-theta, 5.7° ± 0.2° 2-theta, 19.7° ± 0.2° 2-theta, and 22.0° ± 0.2° 2-theta , and (b) less than about 10% degradation when the crystalline form is stored at 25 °C and 60% relative humidity for at least 7 days. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% degradation when the crystalline form is stored at 25 °C and 60% relative humidity for at least 7 days.
[0027] Also provided herein are such methods wherein the crystalline form of Compound 4 (a) exhibits a peak in an x-ray powder diffraction (XRPD) patern at 7.7° ± 0.2° 2-theta, and (b) exhibits less than about 10% degradation when the crystalline form is stored at 25 °C and 60% relative humidity for at least 7 days. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits (a) peaks in an x-ray powder diffraction (XRPD) pattern at 7.7° ± 0.2° 2-theta, 13.7° ± 0.2° 2-theta, and 19.2° ± 0.2° 2-theta, and (b) less than about 10% degradation when the crystalline form is stored at 25 °C and 60% relative humidity for at least 7 days. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% degradation when the crystalline form is stored at 25 °C and 60% relative humidity for at least 7 days.
[0028] Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits less than about 10% degradation when the crystalline forms are stored at 40 °C and 75% relative humidity for at least 7 days. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% degradation when the crystalline form is stored at 40 °C and 75% relative humidity for at least 7 days.
[0029] Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits (a) a peak in an x-ray powder diffraction (XRPD) pattern at 8. 1° ± 0.2° 2-theta, and (b) less than about 10% degradation when the crystalline form is stored at 40 °C and 75% relative humidity for at least 7 days. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits (a) peaks in an x- ray powder diffraction (XRPD) pattern at 9.6° ± 0.2° 2-theta, 5.7° ± 0.2° 2-theta, 19.7° ± 0.2° 2-theta, and 22.0° ± 0.2° 2-theta , and (b) less than about 10% degradation when the crystalline form is stored at 40 °C and 75% relative humidity for at least 7 days. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% degradation when the crystalline form is stored at 40 °C and 75% relative humidity for at least 7 days.
[0030] Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits (a) a peak in an x-ray powder diffraction (XRPD) pattern at 7.7° ± 0.2° 2-theta, and (b) less than about 10% degradation when the crystalline form is stored at 40 °C and 75% relative humidity for at least 7 days. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits (a) peaks in an x- ray powder diffraction (XRPD) pattern at 7.7° ± 0.2° 2-theta, 13.7° ± 0.2° 2-theta, and 19.2° ± 0.2° 2-theta, and (b) less than about 10% degradation when the crystalline form is stored at 40 °C and 75% relative humidity for at least 7 days. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% degradation when the crystalline form is stored at 40 °C and 75% relative humidity for at least 7 days. [0031] Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits less than about 10% degradation when the crystalline form is stored at 60 °C for at least one week. Also provided herein are such methods wherein the crystalline form of Compound 4 exhibits less than about 1%, or less than about 2%, or less than about 3%, or less than about 4%, or less than about 5%, or less than about 6%, or less than about 7%, or less than about 8%, or less than about 9% degradation when the crystalline form is stored at 60 °C for at least one week.
[0032] In other embodiments are provided the methods disclosed herein, wherein the compound of
Figure imgf000026_0001
[0033] In other embodiments are provided the methods disclosed herein, wherein the compound of
Figure imgf000026_0002
[0034] In other embodiments are provided the methods disclosed herein, wherein the compound of
Figure imgf000026_0003
[0035] In other embodiments are provided the methods disclosed herein, wherein the compound of
Figure imgf000027_0001
(Compound 8), or a pharmaceutically acceptable salt thereof.
[0036] In other embodiments are provided the methods disclosed herein, wherein the compound of
Formula (I) is Compound
Figure imgf000027_0002
(Compound 9), or a pharmaceutically acceptable salt thereof.
[0037] In other embodiments are provided the methods disclosed herein, wherein the compound of
Figure imgf000027_0003
[0038] In other embodiments are provided the methods disclosed herein, wherein the compound of
Figure imgf000027_0004
[0039] In other embodiments are provided the methods disclosed herein, wherein the compound of
Formula (I) is Compound 12:
Figure imgf000028_0001
(Compound 12), or a pharmaceutically acceptable salt thereof.
[0040] In other embodiments are provided the methods disclosed herein, wherein the subject has been administered one or more first agents prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the one or more first agents is selected from (a) luteinizing hormone -releasing hormone (LHRH) agonists, (b) luteinizing hormone-releasing hormone (LHRH) antagonists, (c) androgen receptor inhibitors, (d) inhibitors of cytochrome P450 17A1, and/or (e) antiandrogens. In other embodiments are provided the methods disclosed herein, wherein the neuroendocrine prostate cancer in the subject has been determined to be progressing following the administration to the subject of the one or more first agents. In some embodiments, the neuroendocrine prostate cancer in the subject has been determined radiographically to be progressing following the administration to the subject of the one or more first agents. In other embodiments (a) the neuroendocrine prostate cancer in the subject has been determined to be progressing, and (b) testosterone levels in the subject have been determined to be equal to or less than 50 mg/mL, following the administration to the subject of the one or more first agents. In further embodiments (a) the neuroendocrine prostate cancer in the subject has been determined to be radiographically progressing, and (b) testosterone levels in the subject have been determined to be 50 mg/mL or less following the administration to the subject of the one or more first agents.
[0041] In other embodiments are provided the methods disclosed herein, the neuroendocrine prostate cancer in the subject has been determined to comprise visceral metastases or lung metastases. In some embodiments, the visceral metastases comprise liver metastases. In further embodiments, the neuroendocrine prostate cancer in the subject has been determined to comprise bulky lymphadenopathy or a pelvic mass. In some embodiments, the pelvic mass is 5 cm or more in size. In other embodiments, the neuroendocrine prostate cancer in the subject has been determined to comprise bone metastases. In some embodiments, the neuroendocrine prostate cancer in the subject has been determined to comprise 20 or more bone metastases. In further embodiments, the bone metastases are lytic bone metastases.
[0042] In other embodiments are provided the methods disclosed herein, wherein the subject has been administered one or more first agents prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the one or more first agents is a luteinizing hormone-releasing hormone (LHRH) agonist. In some embodiments, the luteinizing hormone-releasing hormone (LHRH) agonist is selected from goserelin, histrelin, leuprolide, and triptorelin. In one embodiment, the luteinizing hormone -releasing hormone (LHRH) agonist is goserelin. In one embodiment, the luteinizing hormone -releasing hormone (LHRH) agonist is histrelin. In one embodiment, the luteinizing hormone -releasing hormone (LHRH) agonist is leuprolide. In one embodiment, the luteinizing hormone- releasing hormone (LHRH) agonist is triptorelin.
[0043] In other embodiments are provided the methods disclosed herein, wherein the subject has been administered one or more first agents prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the one or more first agents is a luteinizing hormone -releasing hormone (LHRH) antagonist. In some embodiments, the luteinizing hormone -releasing hormone (LHRH) antagonist is selected from degarelix and relugolix. In one embodiment, the luteinizing hormone -releasing hormone (LHRH) antagonist is degarelix. In one embodiment, the luteinizing hormone- releasing hormone (LHRH) antagonist is relugolix.
[0044] In other embodiments are provided the methods disclosed herein, wherein the subject has been administered one or more first agents prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the one or more first agents is an androgen receptor inhibitor. In some embodiments, the androgen receptor inhibitor is selected from enzalutamide, apalutamide, and darolutamide. In one embodiment, the androgen receptor inhibitor is enzalutamide. In one embodiment, the androgen receptor inhibitor is apalutamide. In one embodiment, the androgen receptor inhibitor is darolutamide.
[0045] In other embodiments are provided the methods disclosed herein, wherein the subject has been administered one or more first agents prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the one or more first agents is an inhibitor of cytochrome P450 17A1. In one embodiment, the one or more inhibitors of cytochrome P450 17A1 is abiraterone acetate.
[0046] In other embodiments are provided the methods disclosed herein, wherein the subject has been administered one or more first agents prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the one or more first agents is an antiandrogen. In some embodiments, the antiandrogen is selected from egestrol, bicalutamide, flutamide, and nilutamide. In one embodiment, the antiandrogen is egestrol. In one embodiment, the antiandrogen is bicalutamide. In one embodiment, the antiandrogen is egestrol flutamide. In one embodiment, the antiandrogen is nilutamide.
[0047] In other embodiments are provided the methods disclosed herein, wherein a biological sample obtained from the subject has been determined to exhibit one or more biological markers prior to the administration of the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, wherein the one or more biological markers are selected from synaptophysin (SYP), chromogranin A (CGA), insulinoma-associated protein 1 (INSMI), syntaxin 1 (STX1), ISL LIM homeobox 1 (ISL1), INSM transcriptional repressor 1 (INSMI), secretagogin (SECG), neural cell adhesion molecule 1 (NCAM1;
CD56), neuron-specific enolase (NSE), and gastrin-releasing peptide (GRP). In some embodiments, the biological sample obtained from the subject has been determined to exhibit synaptophysin (SYP) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the biological sample obtained from the subject has been determined to exhibit chromogranin A (CGA) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the biological sample obtained from the subject has been determined to exhibit insulinoma-associated protein 1 (INSMI) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the biological sample obtained from the subject has been determined to exhibit syntaxin 1 (STX1) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the biological sample obtained from the subject has been determined to exhibit ISL LIM homeobox 1 (ISL1) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the biological sample obtained from the subject has been determined to exhibit INSM transcriptional repressor 1 (INSMI) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the biological sample obtained from the subject has been determined to exhibit secretagogin (SECG) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the biological sample obtained from the subject has been determined to exhibit neural cell adhesion molecule 1 (NCAM1; CD56) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the biological sample obtained from the subject has been determined to exhibit neuronspecific enolase (NSE) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the biological sample obtained from the subject has been determined to exhibit gastrin -releasing peptide (GRP) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the biological sample obtained from the subject is tissue or blood. In some embodiments, the presence or absence of one or more biomarkers is determined in serum or plasma derived from a blood sample taken from the subject. In some embodiments, the biological sample is tissue. In some embodiments, the tissue is from a biopsy of the prostate cancer. In some embodiments, the biological sample is blood. In some embodiments, the biological sample obtained from the subject has been determined to exhibit an amount of the one or more biological markers that exceeds the upper limit of normal. In some embodiments, the biological sample obtained from the subject has been determined to exhibit an amount of the one or more biological markers that exceeds 1.25 times the upper limit of normal. In some embodiments, the biological sample obtained from the subject has been determined to exhibit an amount of the one or more biological markers that exceeds 1.5 times the upper limit of normal. In some embodiments, the biological sample obtained from the subject has been determined to exhibit an amount of the one or more biological markers that exceeds 1.75 times the upper limit of normal. In some embodiments, the biological sample obtained from the subject has been determined to exhibit an amount of the one or more biological markers that exceeds 2 times the upper limit of normal. In some embodiments, the biological sample obtained from the subject has been determined to exhibit an amount of the one or more biological markers that exceeds 2.25 times the upper limit of normal. In some embodiments, the biological sample obtained from the subject has been determined to exhibit an amount of the one or more biological markers that exceeds 2.5 times the upper limit of normal. In some embodiments, the biological sample obtained from the subject has been determined to exhibit an amount of the one or more biological markers that exceeds 2.75 times the upper limit of normal. In some embodiments, the biological sample obtained from the subject has been determined to exhibit an amount of the one or more biological markers that exceeds 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times the upper limit of normal.
[0048] In other embodiments are provided the methods disclosed herein, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for synaptophysin (SYP) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
[0049] In other embodiments are provided the methods disclosed herein, wherein the biological sample obtained from the subject is blood, and the biological sample has been determined to exhibit an amount of chromogranin A (CGA) that exceeds 101 ng/mL prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
[0050] In other embodiments are provided the methods disclosed herein, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for chromogranin A (CGA) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
[0051] In other embodiments are provided the methods disclosed herein, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for insulinoma-associated protein 1 (INSMI) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
[0052] In other embodiments are provided the methods disclosed herein, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for syntaxin 1 (STX1) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45%, more than 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, or more than 90% of cells in the biologic sample demonstrate staining for syntaxin 1 (STX1) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
[0053] In other embodiments are provided the methods disclosed herein, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for ISL LIM homeobox 1 (ISL1) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
[0054] In other embodiments are provided the methods disclosed herein, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for INSM transcriptional repressor 1 (INSMI) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
[0055] In other embodiments are provided the methods disclosed herein, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for secretagogin (SECG) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
[0056] In other embodiments are provided the methods disclosed herein, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for neural cell adhesion molecule 1 (NCAM1; CD56) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. [0057] In other embodiments are provided the methods disclosed herein, wherein the biological sample obtained from the subject is blood, and the biological sample has been determined to exhibit an amount of neuron-specific enolase (NSE) that exceeds 17.6 ng/mL prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
[0058] In other embodiments are provided the methods disclosed herein, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for gastrin -releasing peptide (GRP) prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
[0059] In other embodiments are provided the methods disclosed, wherein a biological sample obtained from the subject has been determined to exhibit one or more alterations in one or more genes selected from TP53, RBI, and PTEN prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, a biological sample obtained from the subject has been determined to exhibit one or more alterations in TP53 prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, a biological sample obtained from the subject has been determined to exhibit one or more alterations in RB 1 prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, a biological sample obtained from the subject has been determined to exhibit one or more alterations in PTEN prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the one or more alterations in one or more genes selected from TP53, RBI, and PTEN is determined by use of immunohistochemical staining. In some embodiments, the one or more alterations in one or more genes selected from TP53, RBI, and PTEN is determined by use of DNA sequencing. [0060] In some embodiments (a) the one or more genes are selected from RBI and PTEN, and (b) 10% or fewer epithelial cells obtained from the biological sample are stained by the immunohistochemical staining. In some embodiments, the number of epithelial cells obtained from the biological sample that are stained by the immunohistochemical staining are determined by observation at least 25 times, at least 50 times, at least 75 times, at least 100 times, at least 125 times, at least 150 times, at least 175 times, at least 200 times, at least 225 times, at least 250 times, at least 275 times, or at least 300 times magnification.
[0061] In some embodiments (a) the one or more genes is TP53, and (b) 10% or more epithelial cells obtained from the biological sample are stained by the immunohistochemical staining. In some embodiments, the number of epithelial cells obtained from the biological sample that are stained by the immunohistochemical staining are determined by observation at least 25 times, at least 50 times, at least 75 times, at least 100 times, at least 125 times, at least 150 times, at least 175 times, at least 200 times, at least 225 times, at least 250 times, at least 275 times, or at least 300 times magnification.
[0062] In some embodiments, the methods disclosed herein rely on immunohistochemical staining of a biological sample obtained from a subject, wherein the biological sample comprises tissue, and wherein the tissue comprises cells. In some embodiments, the number of cells obtained from a biological sample comprising tissue that are stained by the immunohistochemical staining are determined by observation at least 25 times, at least 50 times, at least 75 times, at least 100 times, at least 125 times, at least 150 times, at least 175 times, at least 200 times, at least 225 times, at least 250 times, at least 275 times, or at least 300 times magnification.
[0063] In some embodiments, the one or more alterations in one or more genes selected from TP53, RBI, and PTEN is determined by use of DNA sequencing, and wherein the one more alterations comprise one or more of (a) nonsynonymous missense mutations, (b) stop-gain mutations, (c) frameshift insertions, (d) frameshift deletions, (e) non-frameshift insertions, (f) non-frameshift deletions, and/or (e) copy number losses. In some embodiments, the one or more alterations in one or more genes selected from TP53, RBI, and PTEN is determined by use of DNA sequencing, and the one more alterations comprise one or more nonsynonymous missense mutations. In some embodiments, the one or more alterations in one or more genes selected from TP53, RBI, and PTEN is determined by use of DNA sequencing, and the one more alterations comprise one or more stop-gain mutations. In some embodiments, the one or more alterations in one or more genes selected from TP53, RBI, and PTEN is determined by use of DNA sequencing, and the one more alterations comprise one or more frameshift insertions. In some embodiments, the one or more alterations in one or more genes selected from TP53, RBI, and PTEN is determined by use of DNA sequencing, and the one more alterations comprise one or more frameshift deletions. In some embodiments, the one or more alterations in one or more genes selected from TP53, RBI, and PTEN is determined by use of DNA sequencing, and the one more alterations comprise one or more non-frameshift insertions. In some embodiments, the one or more alterations in one or more genes selected from TP53, RBI, and PTEN is determined by use of DNA sequencing, and the one more alterations comprise one or more non -frameshift deletions. In some embodiments, the one or more alterations in one or more genes selected from TP53, RB1, and PTEN is determined by use of DNA sequencing, and the one more alterations comprise one or more copy number losses.
[0064] In other embodiments are provided the methods disclosed herein, wherein one or more cells comprising the prostate cancer in the subject have been determined to exhibit one or more of (a) small cell carcinoma, (b) scanty cytoplasm, (c) darkly-stained nuclei with homogenous chromatin pattern, (d) moderate amounts of cytoplasm, (e) centrally located, round and regular nuclei with fine, granular and homogeneous chromatin, (f) the absence of mitosis and necrosis, (g) mixed histology comprising adenocarcinoma and small cell components, and (h) growth in sheets prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, one or more cells comprising the prostate cancer exhibits small cell carcinoma. In some embodiments, one or more cells comprising the prostate cancer exhibits scanty cytoplasm. In some embodiments, one or more cells comprising the prostate cancer exhibits darkly-stained nuclei with homogenous chromatin pattern. In some embodiments, one or more cells comprising the prostate cancer exhibits moderate amounts of cytoplasm. In some embodiments, one or more cells comprising the prostate cancer exhibits centrally located, round and regular nuclei with fine, granular, and homogeneous chromatin. In some embodiments, one or more cells comprising the prostate cancer exhibits the absence of mitosis and necrosis. In some embodiments, one or more cells comprising the prostate cancer exhibits mixed histology comprising adenocarcinoma and small cell components. In some embodiments, one or more cells comprising the prostate cancer exhibits growth in sheets.
[0065] Also provided are the methods disclosed herein, wherein the methods further comprise administering to the subject one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are selected from chemotherapeutic agents, mitotic inhibitors, antimetabolites, platinum-based agents, N-terminal domain inhibitors of androgen receptor, poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors, inhibitors of CYP17, inhibitors of androgen receptor protein expression, heat shock protein 90 (HSP90) inhibitors, bromodomain and extra-terminal domain family (BET) inhibitors, androgen receptor degraders, anti-PD-1 agents, anti-PD-Ll agents, and anti- CTLA-4 agents, or combinations thereof.
[0066] In some embodiments, the one or more additional therapeutic agents are selected from chemotherapeutic agents. In some embodiments, the chemotherapeutic agents are selected from actinomycin, azacytidine, azathioprine, bleomycin, bortezomib, chlorambucil, cyclophosphamide, daunorubicin, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, idarubicin, irinotecan, lurbinectedin, mechlorethamine, mitoxantrone, teniposide, topotecan, valrubicin, vemurafenib, vinblastine, vincristine, and vindesine. In some embodiments, the chemotherapeutic agent is actinomycin. In some embodiments, the chemotherapeutic agent is azacytidine. In some embodiments, the chemotherapeutic agent is azathioprine. In some embodiments, the chemotherapeutic agent is bleomycin. In some embodiments, the chemotherapeutic agent is bortezomib. In some embodiments, the chemotherapeutic agent is chlorambucil. In some embodiments, the chemotherapeutic agent is cyclophosphamide. In some embodiments, the chemotherapeutic agent is daunorubicin. In some embodiments, the chemotherapeutic agent is doxifluridine. In some embodiments, the chemotherapeutic agent is doxorubicin. In some embodiments, the chemotherapeutic agent is epirubicin. In some embodiments, the chemotherapeutic agent is epothilone. In some embodiments, the chemotherapeutic agent is etoposide. In some embodiments, the chemotherapeutic agent is idarubicin. In some embodiments, the chemotherapeutic agent is irinotecan. In some embodiments, the chemotherapeutic agent is lurbinectedin. In some embodiments, the chemotherapeutic agent is mechlorethamine. In some embodiments, the chemotherapeutic agent is mitoxantrone. In some embodiments, the chemotherapeutic agent is teniposide. In some embodiments, the chemotherapeutic agent is topotecan. In some embodiments, the chemotherapeutic agent is valrubicin. In some embodiments, the chemotherapeutic agent is vemurafenib. In some embodiments, the chemotherapeutic agent is vinblastine. In some embodiments, the chemotherapeutic agent is vincristine. In some embodiments, the chemotherapeutic agent is vindesine.
[0067] In other embodiments are provided the methods disclosed herein, wherein the one or more additional therapeutic agents are selected from mitotic inhibitors. In some embodiments, the mitotic inhibitors are selected from paclitaxel, docetaxel, cabazitaxel, tesetaxel, and nab -paclitaxel. In some embodiments, the mitotic inhibitor is paclitaxel. In some embodiments, the mitotic inhibitor is docetaxel. In some embodiments, the mitotic inhibitor is cabazitaxel. In some embodiments, the mitotic inhibitor is tesetaxel. In some embodiments, the mitotic inhibitor is nab-paclitaxel.
[0068] In yet other embodiments, the one or more additional therapeutic agents are selected from antimetabolites. In some embodiments, the one or more antimetabolites are selected from azacytidine, 6- mercaptopurine, capecitabine, hydroxyurea, cladribine, pralatrexate, thioguanine, decitabine, clofarabine, nelarabine, fludarabine, 5 -fluorouracil, gemcitabine, cytarabine, pemetrexed, and methotrexate, cytarabine (Ara-C), floxuridine, fludarabine, pentostatin, and trifluridine/tipiracil combination. In some embodiments, the antimetabolite is azacytidine. In some embodiments, the antimetabolite is 6-mercaptopurine. In some embodiments, the antimetabolite is capecitabine. In some embodiments, the antimetabolite is hydroxyurea. In some embodiments, the antimetabolite is cladribine. In some embodiments, the antimetabolite is pralatrexate. In some embodiments, the antimetabolite is thioguanine. In some embodiments, the antimetabolite is decitabine. In some embodiments, the antimetabolite is clofarabine. In some embodiments, the antimetabolite is nelarabine. In some embodiments, the antimetabolite is fludarabine. In some embodiments, the antimetabolite is 5 -fluorouracil. In some embodiments, the antimetabolite is gemcitabine. In some embodiments, the antimetabolite is cytarabine. In some embodiments, the antimetabolite is pemetrexed. In some embodiments, the antimetabolite is and methotrexate. In some embodiments, the antimetabolite is cytarabine (Ara-C). In some embodiments, the antimetabolite is floxuridine. In some embodiments, the antimetabolite is fludarabine. In some embodiments, the antimetabolite is pentostatin. In some embodiments, the antimetabolite is a trifluridine/tipiracil combination.
[0069] In some embodiments, the one or more additional therapeutic agents are selected from platinumbased agents. In some embodiments, the platinum -based agents are selected from cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, triplatin tetranitrate, pheanthriplatin, picoplatin, and satraplatin. In further embodiments, the platinum-based agent is cisplatin. In further embodiments, the platinum-based agent is carboplatin. In further embodiments, the platinum-based agent is oxaliplatin. In further embodiments, the platinum-based agent is nedaplatin. In further embodiments, the platinum -based agent is lobaplatin. In further embodiments, the platinum -based agent is triplatin tetranitrate. In further embodiments, the platinum-based agent is pheanthriplatin. In further embodiments, the platinum -based agent is picoplatin. In further embodiments, the platinum -based agent is satraplatin.
[0070] In still further embodiments, the one or more additional therapeutic agents are selected from N- terminal domain inhibitors of androgen receptor. In some embodiments, the N-terminal domain inhibitor of androgen receptor is selected from EPI-001, EPI-002 (ralaniten), EPI-506, and EPI-7386. In some embodiments, the N-terminal domain inhibitor of androgen receptor is EPI-001. In some embodiments, the N-terminal domain inhibitor of androgen receptor is EPI-002 (ralaniten). In some embodiments, the N- terminal domain inhibitor of androgen receptor is EPI-506. In some embodiments, the N-terminal domain inhibitor of androgen receptor is EPI-7386.
[0071] In other embodiments are provided the methods disclosed herein, wherein the one or more additional therapeutic agents are selected from poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors. In some embodiments, the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors are selected from olaparib, niraparib, rucaparib, talazopari, veliparib, pamiparib, CEP-9722, and E7016. In some embodiments, the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is olaparib. In some embodiments, the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is niraparib. In some embodiments, the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is rucaparib. In some embodiments, the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is talazopari. In some embodiments, the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is veliparib. In some embodiments, the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is pamiparib. In some embodiments, the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is CEP-9722. In some embodiments, the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitor is E7016.
[0072] In other embodiments are provided the methods disclosed herein, wherein the one or more additional therapeutic agents are selected from inhibitors of CYP17. In one embodiment, the inhibitor of CYP17 is galeterone.
[0073] In other embodiments are provided the methods disclosed herein, wherein the one or more additional therapeutic agents are selected from inhibitors of androgen receptor protein expression. In some embodiments, the inhibitor of androgen receptor protein expression is niclosamide or galeterone. In some embodiments, the inhibitor of androgen receptor protein expression is niclosamide. In some embodiments, the inhibitor of androgen receptor protein expression is galeterone.
[0074] In other embodiments are provided the methods disclosed herein, wherein the one or more additional therapeutic agents are selected from one or more heat shock protein 90 (HSP90) inhibitors. In some embodiments, the one or more heat shock protein 90 (HSP90) inhibitors are selected from tanespimycin, luminespib, alvespimycin, ganetespib, BIIB021, onalespib, geldanamycin, NVP-BEP800, SNX-2112 (PF-04928473), PF-04929113 (SNX-5422), KW-2478, XL888, TAS-116, VER-50589, CH5138303, VER-49009, NMS-E973, zelavespib (PU-H71), and HSP990 (NVP-HSP990). In some embodiments, the heat shock protein 90 (HSP90) inhibitor is tanespimycin. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is luminespib. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is alvespimycin. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is ganetespib. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is BIIB021. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is onalespib. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is geldanamycin. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is NVP-BEP800. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is SNX-2112 (PF- 04928473). In some embodiments, the heat shock protein 90 (HSP90) inhibitor is PF-04929113 (SNX- 5422). In some embodiments, the heat shock protein 90 (HSP90) inhibitor is KW-2478. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is XL888. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is TAS-116. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is VER-50589. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is CH5138303. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is VER-49009. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is NMS-E973. In some embodiments, the heat shock protein 90 (HSP90) inhibitor is zelavespib (PU-H71). In some embodiments, the heat shock protein 90 (HSP90) inhibitor is HSP990 (NVP-HSP990).
[0075] In other embodiments are provided the methods disclosed herein, wherein the one or more additional therapeutic agents are selected from one or more bromodomain and extra-terminal domain family (BET) inhibitors. In some embodiments, the bromodomain and extra-terminal domain family (BET) inhibitor is selected from JQ1, 1-BET 151 (GSK1210151A), I-BET 762 (GSK525762), GSK778 (iBET- BD1), GSK046 (iBET-BD2), OTX-015, TEN-010, CPI-203, CPI-0610, olinone, RVX-208, ABBV-744, LY294002, AZD5153, MT-1, MS645, MS417, SJ432, RVX-208, ABBV-075 (mivebresib), BMS-986158, PLX51107, INCB054329, INCB057643, FT-1101, CC-90010, and ODM-207. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is JQ1. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is I-BET 151 (GSK1210151A). In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is I-BET 762 (GSK525762). In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is GSK778 (iBET-BDl). In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is GSK046 (iBET-BD2). In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is OTX-015. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is TEN-010. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is CPI -203. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is CPI-0610. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is olinone. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is RVX-208. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is ABBV-744. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is LY294002. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is AZD5153. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is MT-1. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is MS645. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is MS417. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is SJ432. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is RVX-208. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is ABBV-075 (mivebresib). In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is BMS-986158. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is PLX51107. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is INCB054329. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is INCB057643. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is FT-1101. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is CC- 90010. In one embodiment, the bromodomain and extra-terminal domain family (BET) inhibitor is ODM- 207.
[0076] In other embodiments are provided the methods disclosed herein, wherein the one or more additional therapeutic agents are selected from androgen receptor degraders. In some embodiments, the androgen receptor degraders are selected from ARV-110, ARV-330, SARD279, SARD033, ARCC-4, UT- 34, ARD-111, ARD-86, ARD-77, ARD-69, ARD-61, LX-1, or LX-2, or a pharmaceutically acceptable salt thereof. In one embodiment, the androgen receptor degrader is ARV-110. In one embodiment, the androgen receptor degrader is ARV-330. In one embodiment, the androgen receptor degrader is SARD279. In one embodiment, the androgen receptor degrader is SARD033. In one embodiment, the androgen receptor degrader is ARCC-4. In one embodiment, the androgen receptor degrader is UT-34. In one embodiment, the androgen receptor degrader is ARD-111. In one embodiment, the androgen receptor degrader is ARD-86. In one embodiment, the androgen receptor degrader is ARD-77. In one embodiment, the androgen receptor degrader is ARD-69. In one embodiment, the androgen receptor degrader is ARD-61. In one embodiment, the androgen receptor degrader is LX-1. In one embodiment, the androgen receptor degrader is LX-2.
[0077] In other embodiments are provided the methods disclosed herein, wherein the one or more additional therapeutic agents are selected from anti-PD-1 agents. In some embodiments, the anti-PD-1 agents are selected from pembrolizumab, nivolumab, cemiplimab, partalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001), dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, and AMP-514 (MEDI0680). In some embodiments, the anti-PD-1 agent is pembrolizumab. In some embodiments, the anti-PD-1 agent is nivolumab. In some embodiments, the anti-PD-1 agent is cemiplimab. In some embodiments, the anti-PD-1 agent is partalizumab (PDR001). In some embodiments, the anti-PD-1 agent is camrelizumab (SHR1210). In some embodiments, the anti-PD-1 agent is sintilimab (IBI308). In some embodiments, the anti-PD-1 agent is tislelizumab (BGB-A317). In some embodiments, the anti-PD-1 agent is toripalimab (JS 001). In some embodiments, the anti-PD-1 agent is dostarlimab (TSR-042, WBP-285). In some embodiments, the anti- PD-1 agent is INCMGA00012 (MGA012). In some embodiments, the anti-PD-1 agent is AMP-224. In some embodiments, the anti-PD-1 agent is AMP-514 (MEDI0680).
[0078] In other embodiments are provided the methods disclosed herein, wherein the one or more additional therapeutic agents are selected from anti-PD-Ll agents. In some embodiments, the anti-PD-Ll agents are selected from atezolizumab, avelumab, durvalumab, MPDL3280A (RG7446), MDX-1105 (BMS-936559), BMS-935559, MSB0010718C, and MEDI4736. In some embodiments, the anti-PD-Ll agent is atezolizumab. In some embodiments, the anti-PD-Ll agent is avelumab. In some embodiments, the anti-PD-Ll agent is durvalumab. In some embodiments, the anti-PD-Ll agent is MPDL3280A (RG7446). In some embodiments, the anti-PD-Ll agent is MDX-1105 (BMS-936559). In some embodiments, the anti- PD-Ll agent is BMS-935559. In some embodiments, the anti-PD-Ll agent is MSB0010718C. In some embodiments, the anti-PD-Ll agent is MEDI4736.
[0079] In other embodiments are provided the methods disclosed herein, wherein the one or more additional therapeutic agents are selected from anti-CTLA-4 agents. In some embodiments, the anti-CTLA- 4 agents are selected from ipilimumab and tremelimumab. In some embodiments, the anti-CTLA-4 agent is ipilimumab. In some embodiments, the anti-CTLA-4 agent is tremelimumab.
[0080] T In other embodiments are provided the methods disclosed herein, wherein the one or more additional therapeutic agents are selected from surgery, radiation, and prostate-specific membrane antigen (PSMA) targeted agents.
INCORPORATION BY REFERENCE
[0081] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE FIGURES
[0082] FIG. 1A depicts the mRNA level of AR in prostate cancer cell lines measured as described in Example 1.
[0083] FIG. IB depicts the mRNA level of neuroendocrine marker synaptophysin (SYP) in prostate cancer cell lines measured as described in Example 1.
[0084] FIG. 1C depicts the mRNA level of neuroendocrine marker chromogranin A (CGA, CHGA) in prostate cancer cell lines measured as described in Example 1.
[0085] FIG. 2A depicts tumor volume following days of treatment in NCI-H660 tumor-bearing male athymic nude mice following administration of vehicle or Compound 4, as described in Example 2.
[0086] FIG. 2B depicts the expression of ASCL1 gene in samples obtained from NCI-H660 tumor-bearing male athymic nude mice following administration of vehicle or Compound 4, as described in Example 2. [0087] FIG. 2C depicts the expression of MYCL gene in samples obtained from NCI-H660 tumor-bearing male athymic nude mice following administration of vehicle or Compound 4, as described in Example 2.
DETAILED DESCRIPTION OF THE INVENTION
[0088] As used in the specification and appended claims, unless specified to the contrary, the following terms have the meaning indicated below.
[0089] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an agent” includes a plurality of such agents, and reference to “the cell” includes reference to one or more cells (or to a plurality of cells) and equivalents thereof known to those skilled in the art, and so forth. When ranges are used herein for physical properties, such as molecular weight, or chemical properties, such as chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included. The term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range, in some instances, will vary between 1% and 15% of the stated number or numerical range. The term “comprising” (and related terms such as “comprise” or “comprises” or “having” or “including”) is not intended to exclude that in other certain embodiments, for example, an embodiment of any composition of matter, composition, method, or process, or the like, described herein, “consist of’ or “consist essentially of’ the described features.
[0090] “Administering” when used in conjunction with a therapeutic, including the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and/or the one or more additional therapeutic agents, means to administer a therapeutic systemically or locally, as directly into or onto a target tissue, or to administer a therapeutic to a subject whereby the therapeutic positively impacts the tissue to which it is targeted. Thus, as used herein, the term “administering”, when used in conjunction with a composition described herein, can include, but is not limited to, providing a composition into or onto the target tissue; providing a composition systemically to a subject by, e.g., oral administration whereby the therapeutic reaches the target tissue or cells. “Administering” a composition may be accomplished by injection, topical administration, and oral administration or by other methods alone or in combination with other known techniques.
[0091] The terms “determine,” “determined,” and “determining,” and the like, as used herein mean that it has been established that a pre-condition in a subject exists, or a condition precedent with respect to a subject has been satisfied, prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof. For example, it is specifically contemplated herein that a subject having prostate cancer is eligible for treatment by administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and, optionally, one or more additional therapeutic agents described herein, if it has been established that the prostate cancer in the subject comprises neuroendocrine prostate cancer. In some embodiments, the prostate cancer in the subject has been determined to comprise neuroendocrine prostate cancer by use of one or more markers. In other embodiments, the prostate cancer in the subject has been determined to comprise neuroendocrine prostate cancer if prostate cancer in the subject has been treated with one or more first agents, such as those selected from the group consisting of (a) luteinizing hormone -releasing hormone (LHRH) agonists, (b) luteinizing hormone -releasing hormone (LHRH) antagonists, (c) androgen receptor inhibitors, (d) inhibitors of cytochrome P450 17A1, and (e) antiandrogens, and the prostate cancer progresses following treatment with one or more of such first agents. Progression of prostate cancer in a subject can be determined by methods known to those having skill in the art, such as the use of radiological methods.
[0092] The term “animal” as used herein includes, but is not limited to, humans and non-human vertebrates such as wild, domestic and farm animals. As used herein, the terms “subject,” “subject” and “individual” are intended to include living organisms in which certain conditions as described herein can occur.
Examples include humans, monkeys, cows, sheep, goats, dogs, cats, mice, rats, and transgenic species thereof. In a preferred embodiment, the subject is a primate. In certain embodiments, the primate or subject is a human. In certain instances, the human is an adult. In certain instances, the human is child. In further instances, the human is under the age of 12 years. In certain instances, the human is elderly. In other instances, the human is 60 years of age or older. Other examples of subjects include experimental animals such as mice, rats, dogs, cats, goats, sheep, pigs, and cows. The experimental animal can be an animal model for a disorder, e.g., a transgenic mouse with hypertensive pathology.
[0093] The term “antiandrogen,” as used herein, means agents that counteract the effects of androgens in subjects. Antiandrogens include agents that act as androgen biosynthesis inhibitors, such as agents that inhibit 17 a-hydroxylase/C17,20-lyase (CYP17). Antiandrogens also include agents that inhibit a subject’s ability to utilize androgens by interacting with the androgen receptor, such as by competitively inhibiting androgen binding to androgen receptors, including by binding directly to the ligand-binding domain of the androgen receptor. Antiandrogens may also inhibit nuclear translocation of androgen receptors and their interaction with DNA as an antagonist, and impeding androgen receptor-mediated transcription. Antiandrogens also include AR degraders as described herein.
[0094] By “pharmaceutically acceptable”, is meant the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
[0095] The term “pharmaceutical composition” means a composition comprising at least one active ingredient, whereby the composition is amenable to investigation for a specified, efficacious outcome in a mammal (for example, without limitation, a human). Those of ordinary skill in the art will understand and appreciate the techniques appropriate for determining whether an active ingredient has a desired efficacious outcome based upon the needs of the artisan.
[0096] The term “progressing” as used herein refers to prostate cancer in a subject being resistant or no longer responsive to the treatment administered (e.g., an antiandrogen). A determination of whether a cancer, or one or more cells comprising a cancer, in a subject have become resistant to a specific treatment modality can be made by methods to known to those of ordinary skill in the art. For example, responsiveness, or non-responsiveness, as the case may be, of a cancer in a subject, or one or more cells comprising the cancer in a subject, can be assessed by measuring prostate-specific antigen (PSA) levels (by, for example, reference to Prostate Cancer Working Group 3 (PCWG3) criteria), increases or decreases in tumor size (by use of radiological methods, for example), use of Response Evaluation Criteria in Solid Tumors (RECIST response) (see, for example, Schwartz, et. al., Eur. J. Cancer, July 2016, vol. 62, pp. 132- 137, for a description of RECIST vl.l), duration of response, or progression-free survival.
[0097] As used herein, the term “therapeutic” means an agent utilized to treat, combat, ameliorate, prevent, or improve an unwanted condition or disease of a subject.
[0098] A “therapeutically effective amount” or “effective amount” as used herein refers to the amount of active compound or pharmaceutical agent that elicits a biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following: (1) preventing the disease; for example, preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease, (2) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), and (3) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) .
[0099] The terms “treat,” “treated,” “treatment,” or “treating” as used herein refers to both therapeutic treatment in some embodiments and prophylactic or preventative measures in other embodiments, wherein the object is to prevent or slow (lessen) an undesired physiological condition, disorder, or disease, or to obtain beneficial or desired clinical results. For the purposes described herein, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of the condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delay in onset or slowing of the progression of the condition, disorder or disease; amelioration of the condition, disorder or disease state; and remission (whether partial or total), whether detectable or undetectable, or enhancement or improvement of the condition, disorder or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. A prophylactic benefit of treatment includes prevention of a condition, retarding the progress of a condition, stabilization of a condition, or decreasing the likelihood of occurrence of a condition.
[00100] For simplicity, chemical moieties are defined and referred to throughout primarily as univalent chemical moieties (e.g., alkyl, aryl, etc.). Nevertheless, such terms may also be used to convey corresponding multivalent moieties under the appropriate structural circumstances clear to those skilled in the art. For example, while an “alkyl” moiety generally refers to a monovalent radical (e.g. CH3-CH2-), in certain circumstances a bivalent linking moiety can be “alkyl,” in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., -CH2-CH2-), which is equivalent to the term “alkylene.” (Similarly, in circumstances in which a divalent moiety is required and is stated as being “aryl,” those skilled in the art will understand that the term “aryl” refers to the corresponding divalent moiety, arylene.) All atoms are understood to have their normal number of valences for bond formation (i.e., 4 for carbon, 3 for N, 2 for O, and 2, 4, or 6 for S, depending on the oxidation state of the S).
[00101] The term “amino” as used herein refers to -NH2.
[00102] The term “acetyl” as used herein refers to “-C(O)CH3.
[00103] The term “acyl” as used herein refers to an alkylcarbonyl or arylcarbonyl substituent wherein the alkyl and aryl portions are as defined herein.
[00104] The term “alkyl” as used herein refers to straight and branched chain aliphatic groups having from 1 to 12 carbon atoms. As such, “alkyl” encompasses Ci, C2, C3, C4, C5, Ce, C7, Cs, C>, Cw, Cn and C12 groups. Examples of alkyl groups include, without limitation, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl.
[00105] The term “alkenyl” as used herein means an unsaturated straight or branched chain aliphatic group with one or more carbon-carbon double bonds, having from 2 to 12 carbon atoms. As such, “alkenyl” encompasses C2, C3, C4, C5, Ce, C7, Cs, C>, C10, Cn and C12 groups. Examples of alkenyl groups include, without limitation, ethenyl, propenyl, butenyl, pentenyl, and hexenyl.
[00106] The term “alkynyl” as used herein means an unsaturated straight or branched chain aliphatic group with one or more carbon-carbon triple bonds, having from 2 to 12 carbon atoms. As such, “alkynyl” encompasses C2, C3, C4, C5, Ce, C7, Cs, C>, C10, Cn and C12 groups. Examples of alkynyl groups include, without limitation, ethynyl, propynyl, butynyl, pentynyl, and hexynyl.
[00107] The terms “alkylene,” “alkenylene,” and “alkynylene” as used herein mean an alkyl, alkenyl, or alkynyl group, as defined hereinabove, that is positioned between and serves to connect two other chemical groups. Examples of alkylene groups include, without limitation, methylene, ethylene, propylene, and butylene. Exemplary alkenylene groups include, without limitation, ethenylene, propenylene, and butenylene. Exemplary alkynylene groups include, without limitation, ethynylene, propynylene, and butynylene.
[00108] The term “alkoxy” as used herein refers to -OCi-Ce alkyl.
[00109] The term “cycloalkyl” as used herein as employed herein is a saturated and partially unsaturated cyclic hydrocarbon group having 3 to 12 carbons. As such, “cycloalkyl” includes C3, C4, C5, Ce, C7, Cs, C>, C10, Ci 1 and C12 cyclic hydrocarbon groups. Examples of cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. [00110] The term “heteroalkyl” as used herein refers to an alkyl group, as defined hereinabove, wherein one or more carbon atoms in the chain are independently replaced by O, S, or NRX, wherein Rx is hydrogen or C1-C3 alkyl. Examples of heteroalkyl groups include methoxymethyl, methoxyethyl and methoxypropyl. [00111] The term “aryl” as used herein means a Ce-Ci4 aromatic moiety comprising one to three aromatic rings. As such, “aryl” includes Ce, Cio, C13, and C14 cyclic hydrocarbon groups. An exemplary aryl group is a Ce-Cio aryl group. Particular aryl groups include, without limitation, phenyl, naphthyl, anthracenyl, and fluorenyl.
[00112] The terms “aralkyl” and “arylalkyl” as used herein mean an aryl group covalently linked to an alkylene group wherein the moiety is linked to another group via the alkyl moiety. An exemplary aralkyl group is -(Ci-C6)alkyl(Ce-Cio)aryl, including, without limitation, benzyl, phenethyl, and naphthylmethyl. [00113] The terms “heterocyclyl” and “heterocyclic” as used herein mean a mono- or bicyclic (fused or spiro) ring structure having from 3 to 12 atoms, (3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 atoms), for example 4 to 8 atoms, wherein one or more ring atoms are independently -C(O)-, N, NR5, O, or S, and the remainder of the ring atoms are quaternary or carbonyl carbons. Examples of heterocyclic groups include, without limitation, epoxy, oxiranyl, oxetanyl, azetidinyl, aziridinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, piperazinyl, imidazolidinyl, thiazolidinyl, thiatanyl, dithianyl, trithianyl, azathianyl, oxathianyl, dioxolanyl, oxazolidinyl, oxazolidinonyl, decahydroquinolinyl, piperidonyl, 4- piperidonyl, thiomorpholinyl, dimethyl -morpholinyl, and morpholinyl. Specifically excluded from the scope of this term are compounds having adjacent ring O and/or S atoms.
[00114] As used herein, term “L-heterocyclyl” as used herein means a heterocyclyl group covalently linked to another group via an alkylene linker L, where L is C1-C4 alkylene.
[00115] The term “heteroaryl” as used herein means a group having 5 to 14 ring atoms, preferably 5, 6, 10, 13 or 14 ring atoms comprising an aromatic heterocyclic ring (e.g., having 6, 10, or 14 71 electrons shared in a cyclic array), and having, in addition to carbon atoms, from one to three heteroatoms that are each independently N, O, or S. “Heteroaryl” also includes fused multicyclic (e.g., bicyclic) ring systems in which one or more of the fused rings is non-aromatic, provided that at least one ring is aromatic and at least one ring contains an N, O, or S ring atom.
[00116] Examples of heteroaryl groups include acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzo[d]oxazol-2(3H)-one, 2H-benzo[b][l,4]oxazin-3(4H)-one, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, furanyl, furazanyl, imidazolinyl, imidazolyl, IH-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5- oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxazolidinyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-l,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5- thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl, and xanthenyl.
[00117] The terms “L-heteroaryl”, “heteroaralkyl” and “heteroarylalkyl” as used herein mean a group comprising a heteroaryl group covalently linked to another group via an alkylene linker. Examples of heteroalkyl groups comprise a Ci-Ce alkyl group and a heteroaryl group having 5, 6, 9, or 10 ring atoms. Examples of heteroaralkyl groups include pyridylmethyl, pyridylethyl, pyrrolylmethyl, pyrrolylethyl, imidazolylmethyl, imidazolylethyl, thiazolylmethyl, thiazolylethyl, benzimidazolylmethyl, benzimidazolylethyl quinazolinylmethyl, quinolinylmethyl, quinolinylethyl, benzofuranylmethyl, indolinylethyl isoquinolinylmethyl, isoinodylmethyl, cinnolinylmethyl, and benzothiophenylethyl. Specifically excluded from the scope of this term are compounds having adjacent ring O and/or S atoms. [00118] The terms “arylene,” “heteroarylene,” and “heterocyclylene” as used herein mean an bivalent aryl, heteroaryl, or heterocyclyl group, respectively, as defined hereinabove, that is positioned between and serves to connect two other chemical groups.
[00119] As employed herein, when a moiety (e.g., cycloalkyl, aryl, heteroaryl, heterocyclyl, urea, etc.) is described as “optionally substituted” without expressly stating the substituents it is meant that the group optionally has from one to four, preferably from one to three, more preferably one or two, non -hydrogen substituents.
[00120] The terms “halogen” and “halo” as used herein mean chlorine, bromine, fluorine, or iodine. [00121] The term “haloalkyl” as used herein means an alkyl chain in which one or more hydrogens have been replaced by a halogen. Exemplary haloalkyls are trifluoromethyl, difluoromethyl, fluorochloromethyl, chloromethyl, and fluoromethyl.
[00122] The term “hydroxy alkyl” as used herein means an alkyl chain, as defined herein, wherein at least one hydrogen of the alkyl chain has been replaced by hydroxyl.
[00123] The compounds of Formula (I), or pharmaceutically acceptable salts thereof, may be prepared using commercially available reagents and intermediates in the synthetic methods and reaction schemes described herein, those described in United States Patent No. 11,091,495, or may be prepared using other reagents and conventional methods well known to those skilled in the art. The contents of United States Patent No.
11,091,495 are hereby incorporated by reference forthat purpose.
[00124] For instance, intermediates for compounds and compounds of formula (I) of the present invention may be prepared according to General Reaction Schemes I or II: General Reaction Scheme I
Figure imgf000046_0001
[00125] In General Reaction Scheme I, R2 -ester substituted imidazo[l, 2 -c] pyrimidine A is coupled to R3 optionally substituted intermediate amine B by nucleophilic substitution to yield Intermediate C. A boronic acid derivative (Y)-R1 D is coupled via a Suzuki reaction with halogen substituted Intermediate C in the presence of a suitable base, e.g., sodium carbonate, and the R2 ester is converted to the acid by saponification with NaOH to generate intermediate acid E. The acid is converted to the corresponding amide, which is dehydrated to form title compound nitrile G.
General Reaction Scheme II
Figure imgf000046_0002
[00126] In General Reaction Scheme II, halogenated Intermediate C containing a suitable R2 reactant, e.g., an ester, in the presence of a suitable base is converted to acid intermediate by saponification, then treated with NH4CI in the presence of HATU to form the amide which is subsequently dehydrated to form nitrile Intermediate H. R1 is coupled to Intermediate H via a Suzuki reaction using boronic acid derivative (Y) in the presence of base. The nitrile group of R1 -containing Intermediate G is hydrolyzed in the presence of acid and water to afford title compound amide F.
[00127] In some embodiments are provided methods of treatment of prostate cancer in a subject, comprising administering to the subject a pharmaceutically acceptable salts of the compounds of Formula (I). The desired salt may be prepared by any suitable method available in the art, for example, treatment of the free base with an inorganic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, or with an organic acid, such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, a pyranosidyl acid, such as glucuronic acid or galacturonic acid, an alpha-hydroxy acid, such as citric acid or tartaric acid, an amino acid, such as aspartic acid or glutamic acid, an aromatic acid, such as benzoic acid or cinnamic acid, a sulfonic acid, such as p-toluenesulfonic acid or ethanesulfonic acid, or the like. It is specifically contemplated herein that references to the compounds of Formula (I), also refer in the alternative to pharmaceutically acceptable salts of compounds of Formula (I).
[00128] If the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is a solid, it is understood by those skilled in the art that the compounds or salts thereof may exist in different crystal or polymorphic forms, all of which are intended to be within the scope of the present invention and specified formulas.
[00129] Also provided herein are uses of isotopically-labeled compounds of Formula (I), or a pharmaceutically acceptable thereof, wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as 2H and 3H, carbon, such as nC, 13C and 14C, chlorine, such as 36C1, fluorine, such as 18F, iodine, such as 123I and 125I, nitrogen, such as 13N and 15N, oxygen, such as 150, 17O and 18O, phosphorus, such as 32P, and sulfur, such as 35 S. Certain isotopically-labeled compounds of the invention, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium (3H) and carbon-14 (14C) are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. Substitution with heavier isotopes such as deuterium, 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. Substitution with positron emitting isotopes, such as nC, 18F, 15O and 13N, can be useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy. Isotopically- labeled compounds of Formula (I), or a pharmaceutically acceptable salt thereof, can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described herein, using an appropriate isotopically-labeled reagent in place of the non-labeled reagent otherwise employed. [00130] In one aspect, the compositions described herein comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, are used for the treatment of neuroendocrine prostate cancer in subjects. Such compositions may be prepared in pharmaceutically acceptable dosage forms for administration to subjects. Pharmaceutically acceptable dosage forms include, for example, liquids, suspensions, powders for reconstitution, tablets, pills, sachets, or capsules of hard or soft gelatin (See, e.g., Remington: The Science and Practice of Pharmacy (Gennaro, 21st Ed. Mack Pub. Co., Easton, PA (2005)). The compounds of Formula (I), or a pharmaceutically acceptable salt thereof, may be formulated into pharmaceutical compositions as described below in any pharmaceutical form recognizable to the skilled artisan as being suitable. Pharmaceutical compositions of the invention comprise a therapeutically effective amount of at least one compound of Formula (I), or a pharmaceutically acceptable salt thereof, and an inert, pharmaceutically acceptable carrier or diluent.
[00131] The pharmaceutical carriers employed may be either solid or liquid. Exemplary solid carriers are lactose, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, and the like. Exemplary liquid carriers are syrup, peanut oil, olive oil, water, and the like. Similarly, the compositions may include time-delay or time-release material known in the art, such as glyceryl monostearate or glyceryl distearate alone or with a wax, ethylcellulose, hydroxypropylmethylcellulose, methylmethacrylate or the like. Further additives or excipients may be added to achieve the desired formulation properties. For example, a bioavailability enhancer, such as Labrasol, Gelucire or the like, or formulator, such as CMC (carboxymethylcellulose), PG (propyleneglycol), or PEG (polyethyleneglycol), may be added. Gelucire, a semi-solid vehicle that protects active ingredients from light, moisture, and oxidation, may be added, e.g., when preparing a capsule formulation.
[00132] If a solid carrier is used, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form, or formed into a troche or lozenge. The amount of solid carrier may vary, but generally will be from about 25 mg to about 1 g. If a liquid carrier is used, the preparation may be in the form of syrup, emulsion, soft gelatin capsule, sterile injectable solution or suspension in an ampoule or vial or non-aqueous liquid suspension. If a semi-solid carrier is used, the preparation may be in the form of hard and soft gelatin capsule formulations. The inventive compositions are prepared in unit-dosage form appropriate for the mode of administration, e.g. parenteral or oral administration.
[00133] To obtain a stable water-soluble dose form, a compound of Formula (I), or a pharmaceutically acceptable salt thereof, may be dissolved in an aqueous solution of an organic or inorganic acid, such as a 0.3 M solution of succinic acid or citric acid. If a soluble salt form is not available, the compound, or a pharmaceutically acceptable salt thereof, may be dissolved in a suitable co-solvent or combinations of cosolvents. Examples of suitable co-solvents include alcohol, propylene glycol, polyethylene glycol 300, polysorbate 80, glycerin and the like in concentrations ranging from 0 to 60% of the total volume. In an exemplary embodiment, a compound of Formula (I), or a pharmaceutically acceptable salt thereof, is dissolved in DMSO and diluted with water. The composition may also be in the form of a solution of a salt form of the active ingredient in an appropriate aqueous vehicle such as water or isotonic saline or dextrose solution.
[00134] Proper formulation is dependent upon the route of administration selected. For injection, a compound of Formula (I), or a pharmaceutically acceptable salt thereof, may be formulated into aqueous solutions, preferably in physiologically compatible buffers such as Hanks solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
[00135] For oral administration, the compounds can be formulated by combining the active compounds with pharmaceutically acceptable carriers known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained using a solid excipient in admixture with the active ingredient (agent), optionally grinding the resulting mixture, and processing the mixture of granules after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients include: fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; and cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
[00136] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, polyvinyl pyrrolidone, Carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active agents.
[00137] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
[00138] For administration intranasally or by inhalation, a compound of Formula (I), or a pharmaceutically acceptable salt thereof, may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of gelatin for use in an inhaler or insufflator and the like may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
[00139] The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit-dosage form, e.g., in ampoules or in multi -dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
[00140] Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active agents may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
[00141] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.
[00142] In addition to the formulations described above, a compound of Formula (I), or a pharmaceutically acceptable salt thereof, may also be formulated as a depot preparation. Such long -acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion-exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. A pharmaceutical carrier for hydrophobic compounds is a co-solvent system comprising benzyl alcohol, a non-polar surfactant, a water-miscible organic polymer, and an aqueous phase. The co-solvent system may be a VPD co-solvent system. VPD is a solution of 3% w/v benzyl alcohol, 8% w/v of the non-polar surfactant polysorbate 80, and 65% w/v polyethylene glycol 300, made up to volume in absolute ethanol. The VPD co-solvent system (VPD: 5W) contains VPD diluted 1: 1 with a 5% dextrose in water solution. This co-solvent system dissolves hydrophobic compounds well, and itself produces low toxicity upon systemic administration. The proportions of a co-solvent system may be suitably varied without destroying its solubility and toxicity characteristics. Furthermore, the identity of the co-solvent components may be varied: for example, other low-toxicity non-polar surfactants may be used instead of polysorbate 80; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g. polyvinyl pyrrolidone; and other sugars or polysaccharides may be substituted for dextrose.
[00143] Alternatively, other delivery systems for hydrophobic pharmaceutical compounds may be employed. Liposomes and emulsions are known examples of delivery vehicles or carriers for hydrophobic drugs. Certain organic solvents such as dimethylsulfoxide (DMSO) also may be employed, although usually at the cost of greater toxicity due to the toxic nature of DMSO. Additionally, the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent. Various sustained-release materials have been established and are known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days. Depending on the chemical nature and the biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.
[00144] The pharmaceutical compositions also may comprise suitable solid- or gel-phase carriers or excipients. These carriers and excipients may provide marked improvement in the bioavailability of poorly soluble drugs. Examples of such carriers or excipients include calcium carbonate, calcium phosphate, sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
[00145] Further, the pharmaceutical composition may be incorporated into a skin patch for delivery of the drug directly onto the skin.
[00146] Additionally, the pharmaceutically acceptable formulations of the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, that may be used to practice the methods disclosed herein may contain a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in an amount of from about 0.5 w/w % to about 95 w/w %, or from about 1 w/w % to about 95 w/w %, or from about 1 w/w % to about 75 w/w %, or from about 5 w/w % to about 75 w/w %, or from about 10 w/w % to about 75 w/w %, or from about 10 w/w % to about 50 w/w %.
[00147] It will be appreciated that the actual dosages of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, to be administered to a subject in need thereof, will vary according to the particular agent being used, the particular composition formulated, the mode of administration, and the particular site, host, and disease being treated. Those skilled in the art using conventional dosage -determination tests in view of the experimental data for a given compound may ascertain optimal dosages for a given set of conditions. For oral administration, an exemplary daily dose generally employed will be from about 0.001 to about 1000 mg/kg of body weight, with courses of treatment repeated at appropriate intervals. In some embodiments are provided the methods disclosed herein, wherein the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to the subject in an amount between about 0.01 mg/kg per day to about 300 mg/kg per day. In other embodiments are provided the methods disclosed herein, wherein the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to the subject in an amount between about 0.1 mg/kg per day to about 100 mg/kg per day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount between about 10 mg to 500 mg per day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount between about 100 mg to about 400 mg per day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount between about 150 mg to about 350 mg per day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount between about 150 mg to about 300 mg per day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount between about 160 mg to about 300 mg per day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount of about 160 mg per day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount of about 200 mg per day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount of about 240 mg per day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount of about 280 mg per day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered in an amount of about 320 mg per day.
[00148] Furthermore, the pharmaceutically acceptable formulations of the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, that may be used to practice the methods disclosed herein may contain a compound of Formula (I), or a pharmaceutically acceptable salt thereof, in an amount of about 10 mg to about 2000 mg, or from about 10 mg to about 1500 mg, or from about 10 mg to about 1000 mg, or from about 10 mg to about 750 mg, or from about 10 mg to about 500 mg, or from about 25 mg to about 500 mg, or from about 50 mg to about 500 mg, or from about 100 mg to about 500 mg.
[00149] In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof once a day. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof twice a day. the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof three times a day.
[00150] In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof in 28-day cycles. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof in multiple 28-day cycles. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof for at least one 28-day cycle. In some embodiments, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to a subject in need thereof on each day of each 28-day cycle.
[00151] In some instances, the methods described herein comprise administering the compositions and formulations comprising the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, in combination with one or more additional therapeutic agents, to the subject or subject in need thereof in multiple cycles repeated on a regular schedule with periods of rest in between each cycle. For example, in some instances, treatment given for one week followed by three weeks of rest is one treatment cycle. The length of a treatment cycle depends on the treatment being given. In some embodiments, the length of a treatment cycle ranges from two to six weeks. In some embodiments, the length of a treatment cycle ranges from three to six weeks. In some embodiments, the length of a treatment cycle ranges from three to four weeks. In some embodiments, the length of a treatment cycle is three weeks (or 21 days). In some embodiments, the length of a treatment cycle is four weeks (28 days). In some embodiments, the length of a treatment cycle is 56 days. In some embodiments, a treatment cycle lasts one, two, three, or four weeks. In some embodiments, a treatment cycle lasts three weeks. In some embodiments, a treatment cycle lasts four weeks. The number of treatment doses scheduled within each cycle also varies depending on the drugs being given.
[00152] Dosages of compositions described herein can be determined by any suitable method. Maximum tolerated doses (MTD) and maximum response doses (MRD) for the compounds of Formula (I), or a pharmaceutically acceptable salt thereof, and the additional therapeutic agents when administered to the subject, can be determined via established animal and human experimental protocols as well as in the examples described herein. For example, toxicity and therapeutic efficacy of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and the additional therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD50 and ED50. The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in a human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. Additional relative dosages, represented as a percent of maximal response or of maximum tolerated dose, are readily obtained via the protocols.
[00153] In some embodiments, the amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, and/or pharmaceutical formulations comprising them that corresponds to such an amount varies depending upon factors such as the particular salt or form, disease condition and its severity, the identity (e.g., age, weight, sex) of the subject or host in need of treatment, but can nevertheless be determined according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the liquid formulation type, the condition being treated, and the subject or host being treated.
[00154] Also provided herein are such methods, wherein the subject is 18 years old or older. Also provided herein are such methods, wherein the subject has undergone a bilateral orchiectomy. Also provided herein are such methods, wherein the subject has been administered a GnRH analogue or antagonist prior to the administration of the compound of Formula (I). Also provided herein are such methods, wherein the GnRH antagonist is selected from abarelix, cetrorelix, degarelix, elagolix, ganirelix, linzagolix, and relugolix. In some embodiments, the GnRH antagonist is abarelix. In some embodiments, the GnRH antagonist is cetrorelix. In some embodiments, the GnRH antagonist is degarelix. In some embodiments, the GnRH antagonist is elagolix. In some embodiments, the GnRH antagonist is ganirelix. In some embodiments, the GnRH antagonist is linzagolix. In some embodiments, the GnRH antagonist is relugolix.
[00155] Also provided herein are such methods, wherein the prostate cancer in the subject has progressed after having been administered at least one androgen receptor antagonist. In some embodiments, the at least one androgen receptor antagonist is selected from abiraterone, enzalutamide, apalutamide, and darolutamide. In some embodiments, the prostate cancer in the subject has progressed after having been administered abiraterone. In some embodiments, the prostate cancer in the subject has progressed after having been administered enzalutamide. In some embodiments, the prostate cancer in the subject has progressed after having been administered apalutamide. In some embodiments, the prostate cancer in the subject has progressed after having been administered darolutamide.
[00156] Also provided herein are such methods, wherein the subject has not received more than 2 chemotherapy regimens prior to the administration to the subject of the compound of Formula (I).
[00157] Also provided herein are such methods, wherein prior to the administration to the subject of the compound of Formula (I), the prostate cancer in the subject exhibits evidence of progressive disease by the Prostate Cancer Working Group 3 (PCWG3) criteria, comprising one or more of (a) 2 or more rising levels of prostate specific antigen (PSA) a minimum of one week apart with the latest result being at least 2.0 ng/mL, (b) 1.0 ng/mL PSA rise, (c) confirmation of 2 new bone lesions on last systemic therapy, and (d) soft tissue progression according to RECIST 1.1 guidelines.
[00158] Also provided herein are such methods, wherein prior to the administration to the subject of the compound of Formula (I), the prostate cancer in the subject exhibits evaluable disease according to RECIST 1.1 guidelines.
[00159] Also provided herein are such methods, wherein prior to the administration to the subject of the compound of Formula (I), the subject exhibits an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.
[00160] Also provided herein are such methods, wherein prior to the administration to the subject of the compound of Formula (I), the subject exhibits adequate organ function defined by one or more of: (a) ANC >1500 cells/mm3 (1.5 x 103 cells/mm3); (b) platelets >100,000 /pL (100 x 109 /L); (c) hemoglobin >9.0 g/dL (90 g/L); (d) AST (SGOT) or ALT (SGPT) <2.5 x ULN, <5.0 x ULN for patients with liver metastases; (e) bilirubin <1.5 x ULN; (f) estimated glomerular filtration rate >60 mL/min; and (g) QTcF <470 msec.
Methods of detecting biomarkers
[00161] Disclosed herein are methods comprising: (a) providing a biologic sample obtained from a subject having prostate cancer; (b) assaying to detect in the biologic sample obtained from the subject a presence or absence of a biomarker; (c) detecting the presence or absence of the biomarker in the biologic sample using the methods described herein; and (d) administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, if the biomarker is present in the biological sample. In some embodiments, the presence of one or more biomarkers described herein indicate the prostate cancer in the subject is neuroendocrine prostate cancer.
[00162] Disclosed herein are such methods that involve detecting the presence, absence, or level, of a biomarker, such as synaptophysin (SYP), chromogranin A (CGA), insulinoma-associated protein 1 (INSMI), syntaxin 1 (STX1), ISL LIM homeobox 1 (ISL1), INSM transcriptional repressor 1 (INSM1), secretagogin (SECG), neural cell adhesion molecule 1 (NCAM1; CD56), neuron-specific enolase (NSE), and gastrin-releasing peptide (GRP). In other embodiments, a biological sample obtained from the subject has been determined to exhibit a biomarker that may comprise one or more alterations in one or more genes selected from TP53, RBI, and PTEN.
[00163] The presence, absence, or level, of such biomarkers may be measured, collectively or individually, in a biological sample obtained from a subject, such as a sample of a solid tumor, such as a neuroendocrine prostate cancer, or from a sample of a relevant biological fluid, such as a blood sample. In some instances, the one or more biomarkers are detected in plasma or serum that is derived from a blood sample obtained from the subject. In some instances, the methods of detection disclosed herein are useful for predicting a therapeutic response to a therapy described herein (e.g., the administration to a subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof), monitor the treatment using the therapy of, and treating with the therapy, a proliferative disease or condition described herein in a subject.
[00164] In some embodiments, the expression of a biomarker in a biological sample from a subject, such as synaptophysin (SYP), chromogranin A (CGA), insulinoma-associated protein 1 (INSMI), syntaxin 1 (STX1), ISL LIM homeobox 1 (ISL1), INSM transcriptional repressor 1 (INSMI), secretagogin (SECG), neural cell adhesion molecule 1 (NCAM1; CD56), neuron-specific enolase (NSE), and gastrin-releasing peptide (GRP), is measured by use of immunohistochemistry (IHC) assays. Such immunohistochemistry (IHC) assays are commercially available, or may be developed and utilized according to methods known to those having ordinary skill in the art.
[00165] Immunohistochemistry techniques utilize an antibody to probe and visualize cellular antigens in situ, generally by chromogenic or fluorescent methods. In such techniques, antibodies or antisera, polyclonal antisera, or monoclonal antibodies specific for each marker are used to detect expression. The antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibody is used in conjunction with a labeled secondary antibody, comprising antisera, polyclonal antisera, or a monoclonal antibody specific for the primary antibody. Immunohistochemistry protocols and kits are well known in the art and are commercially available.
[00166] Two general methods of IHC are generally available; direct and indirect assays. According to the first assay, binding of antibody to the target antigen is determined directly. This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme -labeled primary antibody, which can be visualized without further antibody interaction. In a typical indirect assay, unconjugated primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody. Where the secondary antibody is conjugated to an enzymatic label, a chromagenic or fluorogenic substrate is added to provide visualization of the antigen. Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody. The primary and/or secondary antibody used for immunohistochemistry typically will be labeled with a detectable moiety. Numerous labels are available which can be generally grouped into the following categories. First, are radioisotopes, such as 35S, 14C, 1251, 3H, and 131I. The antibody can be labeled with the radioisotope using the techniques described in Current Protocols in Immunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, New York, N.Y., Pubs. (1991) for example and radioactivity can be measured using scintillation counting. Next, are colloidal gold particles. Third are fluorescent labels including, but are not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, Lissamine, umbelliferone, phycocrytherin, phycocyanin, or commercially available fluorophores such SPECTRUM ORANGE® and SPECTRUM GREEN® and/or derivatives of any one or more of the above. The fluorescent labels can be conjugated to the antibody using the techniques disclosed in Current Protocols in Immunology, supra, for example. Fluorescence can be quantified using a fluorimeter. Fourth are various enzyme-substrate labels are available and U.S. Pat. No. 4,275,149 provides a review of some of these. The enzyme generally catalyzes a chemical alteration of the chromogenic substrate that can be measured using various techniques. For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically. Alternatively, the enzyme may alter the fluorescence or chemiluminescence of the substrate. Techniques for quantifying a change in fluorescence are described above. The chemiluminescent substrate becomes electronically excited by a chemical reaction and may then emit light which can be measured (using a chemiluminometer, for example) or donates energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456), luciferin, 2,3- dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, P-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al. Methods for the Preparation of Enzyme -Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (ed J. Langone & H. Van Vunakis), Academic press, New York, 73: 147-166 (1981). Examples of enzyme -substrate combinations include, for example (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, wherein the hydrogen peroxidase oxidizes a dye precursor [e.g., orthophenylene diamine (OPD) or 3,3’,5,5’-tetramethyl benzidine hydrochloride (TMB)]. 3,3 -Diaminobenzidine (DAB) may also be used to visualize the HRP -labeled antibody; (ii) alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and (iii) P-D-galactosidase (P-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-p-D-galactosidase) or Anorogenic substrate (e.g., 4-methylumbelliferyl-P-D-galactosidase). Numerous other enzyme-substrate combinations are available to those skilled in the art. These methods are generally described in U.S. Pat. Nos. 4,275,149 and 4,318,980. Sometimes, the label is indirectly conjugated with the antibody. The skilled artisan will be aware of various techniques for achieving this. For example, the antibody can be conjugated with biotin and any of the four broad categories of labels mentioned above can be conjugated with avidin, or vice versa. Biotin binds selectively to avidin and thus, the label can be conjugated with the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten and one of the different types of labels mentioned above is conjugated with an anti-hapten antibody. Thus, indirect conjugation of the label with the antibody can be achieved.
[00167] Biological samples obtained from subjects comprising tissue samples may be prepared according to protocols commonly used in the art. Typically, sections of paraffin-embedded cells or tissues are obtained by (1) preserving tissue in fixative, (2) dehydrating the fixed tissue, (3) infiltrating the tissue with fixative, (4) orienting the tissue such that the cut surface accurately represents the tissue, (5) embedding the tissue in paraffin (making a paraffin block), (6) cutting tissue paraffin block with a microtome in sections of 4-5 picometers, and (7) mounting sections onto slides. The slides may then be read by a pathologist or the like assessing for the presence or absence of a biomarker, or of abnormal or normal cells or a specific cell type and provides the loci of the cell types of interest. Thus, for example, a pathologist or the like would review the slides and identify normal cells and abnormal cells (such as abnormal or tumor cells). Any means of defining the loci of the cells of interest may be used (e.g., coordinates on an X-Y axis.
[00168] Aside from the sample preparation procedures discussed above, further treatment of the tissue section prior to, during or following IHC may be desired. For example, epitope retrieval methods, such as heating the tissue sample in citrate buffer may be carried out [see, e.g., Leong et al. Appl.
Immunohistochem. 4(3):201 (1996)]. Following an optional blocking step, the tissue section is exposed to primary antibody for a sufficient period of time and under suitable conditions such that the primary antibody binds to the target protein antigen in the tissue sample. Appropriate conditions for achieving this can be determined by routine experimentation.
[00169] The extent of binding of antibody to the sample is determined by using any one of the detectable labels discussed above. For example, the label is an enzymatic label (e.g. HRPO) which catalyzes a chemical alteration of the chromogenic substrate such as 3,3 ’-diaminobenzidine chromogen. Preferably the enzymatic label is conjugated to antibody which binds specifically to the primary antibody (e.g. the primary antibody is rabbit polyclonal antibody and secondary antibody is goat anti -rabbit antibody). Specimens thus prepared may be mounted and coverslipped. Slide evaluation is then determined, e.g. using a microscope. [00170] IHC may be combined with morphological staining, either prior to or thereafter. After deparaffinization, the sections mounted on slides may be stained with a morphological stain for evaluation. The morphological stain to be used provides for accurate morphological evaluation of a tissue section. The section may be stained with one or more dyes each of which distinctly stains different cellular components. In one embodiment, hematoxylin is use for staining cellular nucleic of the slides. Hematoxylin is widely available. An example of a suitable hematoxylin is Hematoxylin II (Ventana). When lighter blue nuclei are desired, a bluing reagent may be used following hematoxylin staining. One of skill in the art will appreciate that staining may be optimized for a given tissue by increasing or decreasing the length of time the slides remain in the dye.
[00171] Automated systems for slide preparation and IHC processing are available commercially. The Ventana® BenchMark XT system is an example of such an automated system. [00172] After staining, the tissue section may be analyzed by standard techniques of microscopy. Generally, a pathologist or the like assesses the tissue for the presence of abnormal or normal cells or a specific cell type and provides the loci of the cell types of interest. Thus, for example, a pathologist or the like would review the slides and identify normal cells and abnormal cells (such as abnormal or tumor cells). Any means of defining the loci of the cells of interest may be used (e.g., coordinates on an X-Y axis).
[00173] In some embodiments, the presence, or an absence, and/or a level of expression of the biomarker is detected in the sample obtained from a subject by analyzing the genetic material in the sample. In some embodiments, the genetic material is obtained from blood, serum, plasma, sweat, hair, tears, urine, and other techniques known by one of skill in the art. In some embodiments the sample comprises circulating tumor RNA (ctRNA). In some embodiments the sample comprises peripheral blood mononuclear cells (PBMCs). In some embodiments the sample comprises circulating tumor cells (CTCs). In some cases, the genetic material is obtained from a tumor biopsy or liquid biopsy. In some embodiments, a tumor biopsy comprises a formalin-fixed paraffin embedded biopsy, a fresh frozen biopsy, a fresh biopsy, or a frozen biopsy. In some embodiments, a liquid biopsy comprises PBMCs, circulating tumor RNA, plasma cell-free RNA, or circulating tumor cells (CTCs). Tumor and liquid biopsies can undergo additional analytic processing for sample dissociation, cell sorting, and enrichment of cell populations of interest.
[00174] In some embodiments, methods of detecting a presence, absence, or level of a biomarker in a biologic sample obtained from the subject involve detecting a nucleic acid sequence. In some cases, the nucleic acid sequence comprises deoxyribonucleic acid (DNA), such as in the case of detecting complementary DNA (cDNA) of an mRNA transcript. In some instances, the nucleic acid sequence comprises a denatured DNA molecule or fragment thereof. In some instances, the nucleic acid sequence comprises DNA selected from: genomic DNA, viral DNA, mitochondrial DNA, plasmid DNA, amplified DNA, circular DNA, circulating DNA, cell-free DNA, or exosomal DNA. In some instances, the DNA is single -stranded DNA (ssDNA), double -stranded DNA, denaturing double -stranded DNA, synthetic DNA, and combinations thereof. The circular DNA may be cleaved or fragmented. In some instances, the nucleic acid sequence comprises ribonucleic acid (RNA). In some instances, the nucleic acid sequence comprises fragmented RNA. In some instances, the nucleic acid sequence comprises partially degraded RNA. In some instances, the nucleic acid sequence comprises a microRNA or portion thereof. In some instances, the nucleic acid sequence comprises an RNA molecule or a fragmented RNA molecule (RNA fragments) selected from: a microRNA (miRNA), a pre-miRNA, a pri-miRNA, a mRNA, a pre-mRNA, a viral RNA, a viroid RNA, a virusoid RNA, circular RNA (circRNA), a ribosomal RNA (rRNA), a transfer RNA (tRNA), a pre-tRNA, a long non-coding RNA (IncRNA), a small nuclear RNA (snRNA), a circulating RNA, a cell- free RNA, an exosomal RNA, a vector-expressed RNA, an RNA transcript, a synthetic RNA, and combinations thereof.
[00175] Disclosed herein, in some embodiments, a biomarker is detected by subjecting a sample obtained from the subject to a nucleic acid-based detection assay. In some instances, the nucleic acid-based detection assay comprises quantitative polymerase chain reaction (qPCR), reverse transcription PCT (RT-qPCR), gel electrophoresis (including for e.g., Northern or Southern blot), immunohistochemistry (IHC), immunofluorescence (IF), in situ hybridization (ISH) such as fluorescent in situ hybridization (FISH), cytochemistry, microarray, or sequencing. In some embodiments, the sequencing technique comprises next generation sequencing. In some embodiments, the methods involve a hybridization assay such as Anorogenic qPCR (e.g., TaqMan™, SYBR green, SYBR green I, SYBR green II, SYBR gold, ethidium bromide, methylene blue, Pyronin Y, DAPI, acridine orange, Blue View or phycoerythrin), which involves a nucleic acid amplification reaction with a specific primer pair, and hybridization of the amplified nucleic acid probes comprising a detectable moiety or molecule that is specific to a target nucleic acid sequence. In some instances, a number of amplification cycles for detecting a target nucleic acid in a qPCR assay is about 5 to about 30 cycles. In some instances, the number of amplification cycles for detecting a target nucleic acid is at least about 5 cycles. In some instances, the number of amplification cycles for detecting a target nucleic acid is at most about 30 cycles. In some instances, the number of amplification cycles for detecting a target nucleic acid is about 5 to about 10, about 5 to about 15, about 5 to about 20, about 5 to about 25, about 5 to about 30, about 10 to about 15, about 10 to about 20, about 10 to about 25, about 10 to about 30, about 15 to about 20, about 15 to about 25, about 15 to about 30, about 20 to about 25, about 20 to about 30, or about 25 to about 30 cycles. For TaqMan™ methods, the probe may be a hydrolysable probe comprising a Auorophore and quencher that is hydrolyzed by DNA polymerase when hybridized to a target nucleic acid. In some cases, the presence of a target nucleic acid is determined when the number of amplification cycles to reach a threshold value is less than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 cycles. In some instances, hybridization may occur at standard hybridization temperatures, e.g., between about 35 °C and about 65 °C in a standard PCR buffer.
[00176] An additional exemplary nucleic acid -based detection assay comprises the use of nucleic acid probes conjugated or otherwise immobilized on a bead, multi -well plate, or other substrate, wherein the nucleic acid probes are configured to hybridize with a target nucleic acid sequence. In some instances, the nucleic acid probe is specific to one or more of a polynucleotide sequence that encodes a relevant biomarker as disclosed herein. In some instances, the nucleic acid probe specific to a biomarker comprises a nucleic acid probe sequence sufficiently complementary to the polynucleotide sequence that encodes the relevant biomarker protein. In some instances, the probe comprises a transcribed polynucleotide sequence (e.g., RNA, cDNA). In some embodiments, the nucleic acid probe can be, for example, a full-length cDNA, or a portion thereof, such as an oligonucleotide of at least about 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length and sufficient to specifically hybridize under standard hybridization conditions to the target nucleic acid sequence. In some embodiments, the target nucleic acid sequence is immobilized on a solid surface and contacted with a probe, for example by running the isolated target nucleic acid sequence on an agarose gel and transferring the target nucleic acid sequence from the gel to a membrane, such as nitrocellulose. In some embodiments, the probe(s) are immobilized on a solid surface, for example, in an Affymetrix gene chip array, and the probe(s) are contacted with the target nucleic acid sequence. [00177] In some embodiments, the term “probe” with regards to nucleic acids, refers to any nucleic acid molecule that is capable of selectively binding to a specifically intended target nucleic acid sequence. In some instances, probes are specifically designed to be labeled, for example, with a radioactive label, a fluorescent label, an enzyme, a chemiluminescent tag, a colorimetric tag, or other labels or tags that are known in the art. In some instances, the fluorescent label comprises a fluorophore. In some instances, the fluorophore is an aromatic or heteroaromatic compound. In some instances, the fluorophore is a pyrene, anthracene, naphthalene, acridine, stilbene, benzoxazole, indole, benzindole, oxazole, thiazole, benzothiazole, canine, carbocyanine, salicylate, anthranilate, xanthenes dye, coumarin. Exemplary xanthene dyes include, e.g., fluorescein and rhodamine dyes. Fluorescein and rhodamine dyes include, but are not limited to 6-carboxyfluorescein (FAM), 2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N; N'-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX). Suitable fluorescent probes also include the naphthylamine dyes that have an amino group in the alpha or beta position. For example, naphthylamino compounds include l-dimethylaminonaphthyl-5 -sulfonate, l-anilino-8-naphthalene sulfonate, and 2-p-toluidinyl-6- naphthalene sulfonate, 5 -(2 '-aminoethyl)aminonaphthalene-l -sulfonic acid (EDANS). Exemplary coumarins include, e.g., 3-phenyl-7-isocyanatocoumarin; acridines, such as 9-isothiocyanatoacridine and acridine orange; N-(p-(2-benzoxazolyl)phenyl) maleimide; cyanines, such as, e.g., indodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5), indodicarbocyanine 5.5 (Cy5.5), 3-(-carboxy-pentyl)-3'-ethyl-5,5'- dimethyloxacarbocyanine (CyA); 1H, 5H, 11H, 15H-Xantheno[2,3, 4-ij: 5,6, 7-i'j']diquinolizin-18-ium, 9- [2 (or 4)-[[[6-[2,5-dioxo-l-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4 (or 2)-sulfophenyl]-2,3, 6,7, 12,13, 16,17-octahydro-inner salt (TR or Texas Red); or BODIPYTM dyes. In some cases, the probe comprises FAM as the dye label.
[00178] In some embodiments, detecting the one or more biomarkers comprises sequencing genetic material obtained from a sample from the subject. Sequencing can be performed with any appropriate sequencing technology, including but not limited to single-molecule real-time (SMRT) sequencing, Polony sequencing, sequencing by ligation, reversible terminator sequencing, proton detection sequencing, ion semiconductor sequencing, nanopore sequencing, electronic sequencing, pyrosequencing, Maxam -Gilbert sequencing, chain termination (e.g., Sanger) sequencing, +S sequencing, or sequencing by synthesis. Sequencing methods also include next-generation sequencing, e.g., modem sequencing technologies such as Illumina sequencing (e.g., Solexa), Roche 454 sequencing, Ion torrent sequencing, and SOLiD sequencing. In some cases, next-generation sequencing involves high-throughput sequencing methods. Additional sequencing methods available to one of skill in the art may also be employed.
[00179] In some instances, a number of nucleotides that are sequenced are at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 300, 400, 500, 2000, 4000, 6000, 8000, 10000, 20000, 50000, 100000, or more than 100000 nucleotides. In some instances, the number of nucleotides sequenced is in a range of about 1 to about 100000 nucleotides, about 1 to about 10000 nucleotides, about 1 to about 1000 nucleotides, about 1 to about 500 nucleotides, about 1 to about 300 nucleotides, about 1 to about 200 nucleotides, about 1 to about 100 nucleotides, about 5 to about 100000 nucleotides, about 5 to about 10000 nucleotides, about 5 to about 1000 nucleotides, about 5 to about 500 nucleotides, about 5 to about 300 nucleotides, about 5 to about 200 nucleotides, about 5 to about 100 nucleotides, about 10 to about 100000 nucleotides, about 10 to about 10000 nucleotides, about 10 to about 1000 nucleotides, about 10 to about 500 nucleotides, about 10 to about 300 nucleotides, about 10 to about 200 nucleotides, about 10 to about 100 nucleotides, about 20 to about 100000 nucleotides, about 20 to about 10000 nucleotides, about 20 to about 1000 nucleotides, about 20 to about 500 nucleotides, about 20 to about 300 nucleotides, about 20 to about 200 nucleotides, about 20 to about 100 nucleotides, about 30 to about 100000 nucleotides, about 30 to about 10000 nucleotides, about 30 to about 1000 nucleotides, about 30 to about 500 nucleotides, about 30 to about 300 nucleotides, about 30 to about 200 nucleotides, about 30 to about 100 nucleotides, about 50 to about 100000 nucleotides, about 50 to about 10000 nucleotides, about 50 to about 1000 nucleotides, about 50 to about 500 nucleotides, about 50 to about 300 nucleotides, about 50 to about 200 nucleotides, or about 50 to about 100 nucleotides.
[00180] In some cases, a hybridization assay, such as those described herein, is used to detect the mRNA encoding the biomarker in the sample. Exemplary probe sequences that are hybridizable to a target nucleic acid sequence comprise at least 10, but no more than 100 contiguous nucleotides comprising the relevant sequence. In some cases, RNA sequencing (RNAseq) is used to detect the mRNA encoding the relevant biomarker protein.
[00181] Detection of the mRNA, in some cases, involves amplification of the subject’s nucleic acid by the polymerase chain reaction (PCR). In some embodiments, the PCR assay involves use of a pair of primers capable of amplifying at least about 10 contiguous nucleobases within a nucleic acid sequence, thereby amplifying the one or more gene products in the biomarker. In Anorogenic quantitative PCR, quantitation is based on amount of Huorescence signals (TaqMan and SYBR green). In some embodiments, the nucleic acid probe is conjugated to a detectable molecule. The detectable molecule may be a Huorophore. The nucleic acid probe may also be conjugated to a quencher.
[00182] In some embodiments, the assay for detecting the presence or absence of mRNA encoding a relevant biomarker comprises reverse -transcribing the relevant mRNA molecule to produce a corresponding complementary DNA (cDNA) molecule. In some embodiments, the assay further comprises contacting the cDNA molecule with a nucleic acid probe comprising a nucleic acid sequence that is complementary to a nucleic acid sequence of the cDNA molecule. In some embodiments, the assay comprises detecting a double-stranded hybridization product between the nucleic acid probe and the cDNA molecule. In some embodiments, the hybridization product is further amplified using a pair of primers. In some embodiments, the primers comprises a first primer with a nucleic acid sequence comprising at least 10 but not more than 50 contiguous nucleic acids within a relevant nucleic acid sequence that binds to a top strand of the doublestranded hybridization product; and a second primer with a nucleic acid sequence comprising at least 10 but not more than 50 contiguous nucleic acids within a nucleic acid sequence that is reverse complement to the relevant nucleic acid sequence that binds to a bottom strand of the double -stranded hybridization product. [00183] Disclosed herein, in some embodiments, are methods comprising preparing a complementary DNA (cDNA) library. In some embodiments, the cDNA library is sequenced using suitable sequence methodologies disclosed herein. In some embodiments, the cDNA library is labeled, a plurality of nucleic acid probes is generated, and fixed to an immobile surface (such as a microarray). In some embodiments, the plurality of nucleic acid probes is capable of hybridizing to at least about 10 contiguous nucleotides of the two or more genes in a sample obtained from the subject. In some embodiments, detecting the presence of or absence of a biomarker includes detecting a high or a low level of expression of one or more genes as compared to a reference level.
[00184] Disclosed herein, in some embodiments, genetic material is extracted from a biologic sample obtained from a subject, e.g., a sample of blood, serum, or tissue. In certain embodiments where nucleic acids are extracted, the nucleic acids are extracted using any technique that does not interfere with subsequent analysis. In certain embodiments, this technique uses alcohol precipitation using ethanol, methanol, or isopropyl alcohol. In certain embodiments, this technique uses phenol, chloroform, or any combination thereof. In certain embodiments, this technique uses cesium chloride. In certain embodiments, this technique uses sodium, potassium or ammonium acetate or any other salt commonly used to precipitate DNA. In certain embodiments, this technique utilizes a column or resin based nucleic acid purification scheme such as those commonly sold commercially, one non-limiting example would be the GenElute Bacterial Genomic DNA Kit available from Sigma Aldrich. In certain embodiments, after extraction the nucleic acid is stored in water, Tris buffer, or Tris-EDTA buffer before subsequent analysis. In an exemplary embodiment, the nucleic acid material is extracted in water. In some cases, extraction does not comprise nucleic acid purification. In certain embodiments, RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland). [00185] In some aspects, circulating tumor RNA (ctRNA) is used to assess the expression levels of RNA molecules, shed by the tumor into the blood stream. In some embodiments, detection of ctRNA is useful, for example, for detecting and diagnosing a tumor. Because tumor DNA and RNA has acquired multiple genetic mutations, leading to tumor development, ctRNA are not an exact match to the individual’s DNA and RNA, respectively. Finding DNA and RNA with genetic differences aids in tumor detection.
Diagnosing the type of tumor using ctRNA can reduce the need for getting a sample of the tumor tissue (tumor biopsy), which can be challenging when a tumor is difficult to access, such as a tumor in the brain or lung.
[00186] In some embodiments, a decrease in the quantity of ctRNA suggests the solid tumor is shrinking and treatment with a compound of Formula (I), or a pharmaceutically acceptable salt thereof, is effective. In some embodiments, a lack of ctRNA in the bloodstream indicates that the cancer has not returned after treatment with a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
[00187] Described herein are methods of assessing genetic alterations by ctRNA profiling. In some embodiments, the genomic profiling is performed after each treatment cycle with a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the gene mutations indicate that the cancer is becoming resistant to the treatment with a compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the lack of gene mutations indicate that the cancer is not becoming resistant to the treatment with a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
[00188] In some embodiments, the expression of a biomarker is measured by immunofluorescence (IF) assays. In some embodiments, the expression of a biomarker is measured by in situ hybridization (ISH) assays. In some embodiments, the expression of a biomarker transcript levels are measured using assays such as quantitative polymerase chain reaction (qPCR), microarray, and RNA sequencing, or assays commercially available from companies such as Fluidigm and Nanostring.
[00189] Disclosed herein are methods of treating a subject having neuroendocrine prostate cancer, comprising: (a) providing a biologic sample obtained from a subject having prostate cancer; (b) assaying to detect in the biologic sample obtained from the subject a presence or absence of a biomarker; (c) detecting the presence or absence of the biomarker in the biologic sample using the methods described herein; and (d) administering to the subject a therapeutically effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, if the biomarker is present in the biological sample. In some embodiments, expression of a biomarker is based on the expression level of the biomarker deviating from a reference expression level. In some embodiments, the expression level is high, relative to the reference expression level. In some embodiments, the expression level is low, relative to the reference expression level. In some embodiments, the reference expression level is derived from an individual, or a group of individuals, that do not have cancer. In some embodiments, the reference expression level is derived from an individual, or a group of individuals, that have cancer that does not therapeutically respond to the compound of Formula (I), or a pharmaceutically acceptable salt thereof. In some embodiments, the expression level deviates from the reference expression level by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.
[00190] In some embodiments, the determination of expression or the presence of a biomarker is defined based on the percentage of cells that stain weakly, moderately, or strongly for the relevant biomarker, with the threshold defining the minimal percentage of cells that are required to stain positive at the various intensity levels (>a% of prostate tumor cells stain weakly, >b% of prostate tumor cells stain moderately, >c% of prostate tumor cells stain strongly, or a combination thereof). In some embodiments, the one or more of the cells comprising the prostate cancer has been determined to express a biomarker when > about 10%, > about 15%, > about 20%, > about 25%, > about 30%, > about 35%, > about 40%, > about 45%, > about 50%, > about 55%, > about 60%, > about 65%, > about 70%, > about 75%, > about 80%, > about 85%, > about 90%, or > about 95% of the prostate tumor cells stain weakly for the biomarker; when > about 10%, > about 15%, > about 20%, > about 25%, > about 30%, > about 35%, > about 40%, > about 45%, > about 50%, > about 55%, > about 60%, > about 65%, > about 70%, > about 75%, > about 80%, > about 85%, > about 90%, or > about 95% of the prostate tumor cells stain moderately for the biomarker; when > about 10%, > about 15%, > about 20%, > about 25%, > about 30%, > about 35%, > about 40%, > about 45%, > about 50%, > about 55%, > about 60%, > about 65%, > about 70%, > about 75%, > about 80%, > about 85%, > about 90%, or > about 95% of the prostate tumor cells stain strongly for the biomarker; or any combinations thereof.
Kits and articles of manufacture
[00191] Disclosed herein, in certain embodiments, are kits and articles of manufacture for use with one or more methods and compositions described herein. Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In one embodiment, the containers are formed from a variety of materials such as glass or plastic.
[00192] A kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.
[00193] In one embodiment, a label is on or associated with the container. In one embodiment, a label is on a container when letters, numbers or other characters forming the label are attached, molded, or etched into the container itself, a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In one embodiment, a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.
[00194] In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein. The pack, for example, contains metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for drugs, or the approved product insert. In one embodiment, compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
[00195] Disclosed herein is a kit comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, for use in treating neuroendocrine prostate cancer in a subject in need thereof and a package insert comprising instructions for measuring the expression of a biomarker described herein in one or more of the cells comprising the neuroendocrine prostate cancer and using the compound of Formula (I), or a pharmaceutically acceptable salt thereof, if one or more of the cells comprising the neuroendocrine prostate cancer has been determined to express the biomarker. Numbered Embodiments
[00196] Embodiment 1: A method of treating prostate cancer in a subject, wherein the prostate cancer has been determined to comprise neuroendocrine prostate cancer, comprising administering to the subject a therapeutically effective amount of a compound of Formula (I)
Figure imgf000065_0001
Formula (I) or a pharmaceutically acceptable salt thereof: wherein:
- represents a single or a double bond;
Z is O or S;
X is O, CR5, CR5OH, or C(R5)2, wherein: when X is O, - is a single bond; when X is C(R5)2, - is a single bond; when X is CR5OH, - is a single bond; or when X is CR5, - is a double bond;
R1 is aryl, heteroaryl, L-cycloalkyl, -N(R5)heterocyclyl, or L-heterocyclyl, wherein the aryl, the heteroaryl or the cyclyl portion of the L-cycloalkyl, -N(R5)heterocyclyl, or L-heterocyclyl is optionally substituted with one or more R4;
R2 is cyano, -COOR5, -C(O)N(R5)2, or -C(O)N(R5)2 wherein each R5 taken together with the nitrogen atom to which they are attached form a 5 - 8 membered heterocyclic ring optionally substituted with one or more R4; each R3 is independently C1-C3 alkyl or halogen; each R4 is independently oxo, cyano, halogen, -POXCi-C alkyl)2, hydroxyl, alkoxy, hydroxyalkyl, heteroalkyl, aralkyl, haloalkyl, -COOR5, -Y2-haloalkyl, -Y’-Ci-Ce alkyl, -Y2-Ci-Ce alkyl, -L- cycloalkyl, -L-heteroaryl, -L-heterocyclyl, -Y'-heterocyclyl, -Y2-heterocyclyl, -L-N(R5)2, -O-L- N(R5)2, -C(CF3)N(R5)2, -Y’-N(R5)2, or -Y2-N(R5)2, wherein the ring portion of the aralkyl, -L- cycloalkyl, -L-heteroaryl, -L-heterocyclyl or -Y’-heterocyclyl is optionally substituted with one or more R7;
L is a bond or C1-C4 alkylene;
Y1 is a bond, -C(O)-, or -NHC(O)-; Y2 is a bond, -S-, -SO-, -SO2-, or -NR5SO2-, each R5 is hydrogen or C1-C3 alkyl;
R6 is hydrogen, C1-C3 alkyl, halogen, haloalkyl, hydroxyalkyl, or heteroalkyl; each R7 is oxo, cyano, hydroxyl, alkoxy, halogen, haloalkyl, hydroxyalkyl, heteroalkyl, cycloalkyl, -L-N(R5)2, Ci-Ce alkyl, or -Y’-heterocyclyl; and n is 1 or 2.
[00197] Embodiment 2: The method of embodiment 1, wherein Z is O.
[00198] Embodiment 3: The method of embodiment 1, wherein Z is S.
[00199] Embodiment 4: The method of embodiment 2 or 3, wherein n is 1.
[00200] Embodiment 5: The method of any of embodiments 1-4, wherein R2 is cyano.
[00201] Embodiment 6: The method of any of embodiments 1-4, wherein R2 is -COOR5.
[00202] Embodiment 7: The method of any of embodiments 1-4, wherein R2 is -C(O)N(R5)2.
[00203] Embodiment 8: The method of any of embodiments 1-7, wherein R3 is halogen.
[00204] Embodiment 9: The method of embodiment 8, wherein the halogen is fluorine.
[00205] Embodiment 10: The method of any of embodiments 1-9, wherein X is C(R5)2 and - is a single bond.
[00206] Embodiment 11: The method of any of embodiments 1-9, wherein X is CR5 and - is a double bond.
[00207] Embodiment 12: The method of any of embodiments 1-9, wherein X is O and - is a single bond.
[00208] Embodiment 13: The method of any of embodiments 1-12 wherein R1 is aryl optionally substituted with one or more R4.
[00209] Embodiment 14: The method of embodiment 13, wherein the aryl is phenyl optionally substituted with one or more R4.
[00210] Embodiment 15: The method of embodiment 14, wherein the phenyl is substituted with one, two or three R4.
[00211] Embodiment 16: The method of embodiment 15, wherein the one, two or three R4 are each independently halogen, -POdC i-C alkyl)2, hydroxyl, hydroxyalkyl, aralkyl, haloalkyl, -COOR5, -Y’-Ci-Ce alkyl, Y2-CI-C6 alkyl, -L-N(R5)2, -O-L-N(R5)2, -C(CF3)N(R5)2, -Y’-N(R5)2, -Y2-N(R5)2, Y2-haloalkyl, -L- heteroaryl, -L-heterocyclyl, or -Y'-heterocyclyl, wherein the heterocyclyl portion of the -L-heterocyclyl or - Y’-heterocyclyl is optionally substituted with one or more R7.
[00212] Embodiment 17: The method of embodiment 16, wherein R4 is -Y'-Ci-Ce alkyl and Y1 is a bond and the Ci-Ce alkyl is methyl, ethyl, isopropyl, butyl, or pentyl.
[00213] Embodiment 18: The method of embodiment 16, wherein R4 is -Y2-Ci-Ce alkyl and Y2 is a -SO2- and the Ci-Ce alkyl is methyl.
[00214] Embodiment 19: The method of embodiment 16, wherein R4 is -Y2 -haloalkyl and Y2 is -S- or -SO2- and the haloalkyl is trifluoromethyl. [00215] Embodiment 20: The method of embodiment 16, wherein R4 is -L-N(R5)2 and L is a bond and each R5 is hydrogen, each R5 is methyl or one R5 is methyl and one R5 is hydrogen.
[00216] Embodiment 21 : The method of embodiment 16, wherein R4 is -L-N(R5)2 and L is methylene or ethylene and each R5 is hydrogen, each R5 is methyl or one R5 is methyl and one R5 is hydrogen.
[00217] Embodiment 22: The method of embodiment 16, wherein R4 is -Y'-N(R5)2, Y1 is -C(O)- and each R5 independently is hydrogen, each R5 is independently methyl or one R5 is methyl and one R5 is hydrogen. [00218] Embodiment 23: The method of embodiment 16, wherein R4 is -Y2-N(R5)2, Y2 is -SO2- and each R5 independently is hydrogen, each R5 is methyl or one R5 is methyl and one R5 is independently hydrogen. [00219] Embodiment 24: The method of embodiment 16, wherein R4 is -Y1 -heterocyclyl and Y1 is -C(O)- and the heterocyclyl portion of the L-heterocyclyl is piperazinyl or 4-methyl-piperazinyl.
[00220] Embodiment 25: The method of embodiment 16, wherein R4 is -L-heterocyclyl and L is a bond and the heterocyclyl portion of the L-heterocyclyl is azetidinyl, oxetanyl, pyrrolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, or 3/.2-azabicyclo|3. 1 ,0|hcxanyl. each optionally substituted with one or more R7 selected from oxo, C1-C3 alkyl, alkoxy, hydroxyl and halogen.
[00221] Embodiment 26: The method of embodiment 16, wherein R4 is -L-heterocyclyl, wherein L is a methylene and the heterocyclyl portion of the L-heterocyclyl is azetidinyl, oxetanyl, pyrrolidinyl piperidinyl, each optionally substituted with one or more R7 selected from C1-C3 alkyl, alkoxy, hydroxyl and halogen.
[00222] Embodiment 27: The method of embodiment 16, wherein R4 is -Y1 -heterocyclyl and Y1 is -C(O)- and the heterocyclyl portion of the Y1 -heterocyclyl is morpholinyl optionally substituted with one or more C1-C3 alkyl.
[00223] Embodiment 28: The method of embodiment 16, wherein R4 is -L-heteroaryl optionally substituted with one or more R7.
[00224] Embodiment 29: The method of embodiment 28, wherein the -L-heteroaryl is tetrazolyl.
[00225] Embodiment 30: The method of embodiment 16, wherein R4 is -PCfiC i-C, alkyl)2.
[00226] Embodiment 31 : The method of embodiment 16, wherein R4 is -COOR5.
[00227] Embodiment 32: The method of embodiment 16, wherein R4 is hydroxyalkyl.
[00228] Embodiment 33: The method of embodiment 16, wherein R4 is -O-L-N(R5)2.
[00229] Embodiment 34: The method of embodiment 16, wherein R4 is aralkyl.
[00230] Embodiment 35: The method of any of embodiments 1-12, wherein R1 is heteroaryl optionally substituted with one or more R4.
[00231] Embodiment 36: The method of embodiment 35, wherein the heteroaryl is pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, triazinyl, pyridyl, pyridinyl-2-one, pyrazinyl, pyridazinyl, pyrimidinyl, isoxazolyl, isoindolinyl, naphthyridinyl, 1,2,3,4-tetrahydroisoquinolinyl, or 5,6-dihydro-4H-pyrrolo[l,2- b]pyrazolyl, each optionally substituted with one or more R4.
[00232] Embodiment 37: The method of embodiment 36, wherein the heteroaryl is substituted with one or more R4; wherein each R4 is independently cyano, halogen, -Y’-Ci-Ce alkyl, -Y2-Ci-Ce alkyl, alkoxy, hydroxyalkyl, heteroalkyl, haloalkyl, -L-cycloalkyl, -L-N(R5)2, -Y'-N(R5)2, -L-heteroaryl, -L-heterocyclyl, or -Y1 -heterocyclyl, wherein the heteroaryl of the -L-heteroaryl or the heterocyclyl portion of the L- heterocyclyl, or Y1 -heterocyclyl is optionally substituted with one or more R7.
[00233] Embodiment 38: The method of embodiment 37, wherein the heteroaryl is pyrazolyl optionally substituted with one R4 independently selected from hydroxyalkyl, heteroalkyl, haloalkyl, -Y’-Ci-Ce alkyl, - L-N(R5)2, L-heterocyclyl or L-heteroaryl, wherein the heteroaryl of the L-heteroaryl or the heterocyclyl portion of the L-heterocyclyl is optionally substituted with one or more R7.
[00234] Embodiment 39: The method of embodiment 38, wherein R4 is -L-heteroaryl and L is methylene wherein the heteroaryl is pyridyl optional substituted with one or more R7.
[00235] Embodiment 40: The method of embodiment 38, wherein R4 is -L-heterocyclyl optionally substituted with one or more R7 where L is a bond and the heterocyclyl portion of the L-heterocyclyl is oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, piperazinyl, or 4-methylpiperazinyl.
[00236] Embodiment 41 : The method of embodiment 38, wherein R4 is -L-heterocyclyl optionally substituted with one or more R7 where L is methylene and the heterocyclyl portion of the L-heterocyclyl is azetidinyl, oxetanyl, pyrrolidinyl, pyrrolidinone, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, piperazinyl, or 4-methylpiperazinyl.
[00237] Embodiment 42: The method of embodiment 38, wherein R4 is -L-N(R5)2 where L is methylene and each R5 is independently hydrogen, each R5 is independently C1-C3 alkyl or one R5 is C1-C3 alkyl and one R5 is hydrogen.
[00238] Embodiment 43: The method of embodiment 38, wherein R4 is -Y'-Ci-Ce alkyl where Y1 is a bond and the Ci-Ce alkyl is methyl, ethyl, or isopropyl.
[00239] Embodiment 44: The method of embodiment 38, wherein the heteroaryl is pyrazolyl optionally substituted with two R4 groups each independently selected from hydroxyalkyl, heteroalkyl, haloalkyl, and - Y’-Ci-Ce alkyl.
[00240] Embodiment 45: The method of embodiment 36, wherein the heteroaryl is pyridyl optionally substituted with one R4 independently selected from cyano, halogen, alkoxy, hydroxyalkyl, heteroalkyl, haloalkyl, -Y’-Ci-Ce alkyl, -L-N(R5)2, -Y’-N(R5)2, -L-cycloalkyl, or -L-heterocyclyl optionally substituted with one or more R7.
[00241] Embodiment 46: The method of any of embodiments 1-12, wherein R1 is -L-cycloalkyl optionally substituted with one or more R4.
[00242] Embodiment 47: The method of any of embodiments 1-12, wherein R1 is -L-heterocyclyl optionally substituted with one or more R4.
[00243] Embodiment 48: The method of embodiment 47, wherein L is a bond and the heterocyclyl is piperidinyl or tetrahydropyranyl.
[00244] Embodiment 49: The method of any of embodiments 1-3, wherein n is 2.
[00245] Embodiment 50: A method of treating prostate cancer in a subject, wherein the prostate cancer has been determined to comprise neuroendocrine prostate cancer, comprising administering to the subject a
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
[00246] Embodiment 51 : A method of treating prostate cancer in a subject, wherein the prostate cancer has been determined to comprise neuroendocrine prostate cancer, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:
Figure imgf000081_0002
Figure imgf000081_0003
Figure imgf000082_0001
Figure imgf000082_0002
pharmaceutically acceptable salt thereof.
[00247] Embodiment 52: The method of embodiment 51, wherein the compound is:
Figure imgf000082_0003
a pharmaceutically acceptable salt thereof.
[00248] Embodiment 53 : The method of embodiment 51, wherein the compound is:
Figure imgf000082_0004
a pharmaceutically acceptable salt thereof. [00249] Embodiment 54: The method of embodiment 51, wherein the compound is:
Figure imgf000083_0001
or a pharmaceutically acceptable salt thereof.
[00250] Embodiment 55: The method of embodiment 51, wherein the compound is:
Figure imgf000083_0002
a pharmaceutically acceptable salt thereof.
[00251] Embodiment 56: The method of embodiment 51, wherein the compound is:
Figure imgf000083_0003
a pharmaceutically acceptable salt thereof.
[00252] Embodiment 57: The method of embodiment 51, wherein the compound is:
Figure imgf000083_0004
a pharmaceutically acceptable salt thereof. [00253] Embodiment 58: The method of embodiment 51, wherein the compound is:
Figure imgf000084_0001
a pharmaceutically acceptable salt thereof.
[00254] Embodiment 59: The method of embodiment 51, wherein the compound is:
Figure imgf000084_0002
a pharmaceutically acceptable salt thereof.
[00255] Embodiment 60: The method of embodiment 51, wherein the compound is:
Figure imgf000084_0003
or a pharmaceutically acceptable salt thereof.
[00256] Embodiment 61 : The method of embodiment 51 , wherein the compound is:
Figure imgf000084_0004
a pharmaceutically acceptable salt thereof. [00257] Embodiment 62: The method of embodiment 51, wherein the compound is:
Figure imgf000085_0001
a pharmaceutically acceptable salt thereof.
[00258] Embodiment 63: The method of embodiment 51, wherein the compound is:
Figure imgf000085_0002
or a pharmaceutically acceptable salt thereof.
[00259] Embodiment 64: The method of any one of embodiments 1-63, wherein the subject has been administered one or more first agents prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and wherein the one or more first agents is selected from the group consisting of (a) luteinizing hormone -releasing hormone (LHRH) agonists, (b) luteinizing hormone -releasing hormone (LHRH) antagonists, (c) androgen receptor inhibitors, (d) inhibitors of cytochrome P450 17A1, and (e) antiandrogens.
[00260] Embodiment 65 : The method of embodiment 64, wherein the neuroendocrine prostate cancer in the subject has been determined to be progressing following the administration to the subject of the one or more first agents.
[00261] Embodiment 66: The method of embodiment 64, wherein the neuroendocrine prostate cancer in the subject has been determined radiographically to be progressing following the administration to the subject of the one or more first agents.
[00262] Embodiment 67 : The method of embodiment 64, wherein (a) the neuroendocrine prostate cancer in the subject has been determined to be progressing, and (b) testosterone levels in the subject have been determined to be equal to or less than 50 mg/mL, following the administration to the subject of the one or more first agents.
[00263] Embodiment 68: The method of embodiment 64, wherein (a) the neuroendocrine prostate cancer in the subject has been determined to be radiographically progressing, and (b) testosterone levels in the subject have been determined to be 50 mg/mL or less following the administration to the subject of the one or more first agents.
[00264] Embodiment 69: The method of any one of embodiments 1-68, wherein the neuroendocrine prostate cancer in the subject has been determined to comprise visceral metastases or lung metastases. [00265] Embodiment 70: The method of embodiment 69, wherein the visceral metastases comprise liver metastases.
[00266] Embodiment 71: The method of any one of embodiments 1-68, wherein the neuroendocrine prostate cancer in the subject has been determined to comprise bulky lymphadenopathy or a pelvic mass. [00267] Embodiment 72: The method of embodiment 71, wherein the pelvic mass is 5 cm or more in size. [00268] Embodiment 73: The method of any one of embodiments 1-68, wherein the neuroendocrine prostate cancer in the subject has been determined to comprise bone metastases.
[00269] Embodiment 74: The method of embodiment 73, wherein the neuroendocrine prostate cancer in the subject has been determined to comprise 20 or more bone metastases.
[00270] Embodiment 75: The method of embodiment 73 or 74, wherein the bone metastases are lytic bone metastases.
[00271] Embodiment 76: The method of any one of embodiments 64-75, wherein the one or more first agents is a luteinizing hormone -releasing hormone (LHRH) agonist.
[00272] Embodiment 77: The method of embodiment 76, wherein the luteinizing hormone-releasing hormone (LHRH) agonist is selected from goserelin, histrelin, leuprolide, and triptorelin.
[00273] Embodiment 78: The method of any one of embodiments 64-75, wherein the one or more first agents is a luteinizing hormone-releasing hormone (LHRH) antagonist.
[00274] Embodiment 79: The method of embodiment 78, wherein the luteinizing hormone-releasing hormone (LHRH) antagonist is selected from degarelix and relugolix.
[00275] Embodiment 80: The method of any one of embodiments 64-75, wherein the one or more first agents is an androgen receptor inhibitor.
[00276] Embodiment 81: The method of embodiment 80, wherein the androgen receptor inhibitor is selected from enzalutamide, apalutamide, and darolutamide.
[00277] Embodiment 82: The method of embodiment 81, wherein the androgen receptor inhibitor is enzalutamide.
[00278] Embodiment 83: The method of embodiment 81, wherein the androgen receptor inhibitor is apalutamide.
[00279] Embodiment 84: The method of embodiment 81, wherein the androgen receptor inhibitor is darolutamide.
[00280] Embodiment 85: The method of any one of embodiments 64-75, wherein the one or more first agents is an inhibitor of cytochrome P450 17A1.
[00281] Embodiment 86: The method of embodiment 85, wherein the one or more inhibitors of cytochrome P450 17A1 is abiraterone acetate.
[00282] Embodiment 87: The method of any one of embodiments 64-75, wherein the one or more first agents is an antiandrogen.
[00283] Embodiment 88: The method of embodiment 87, wherein the antiandrogen is selected from egestrol, bicalutamide, flutamide, and nilutamide. [00284] Embodiment 89: The method of any one of embodiments 1-88, wherein a biological sample obtained from the subject has been determined to exhibit one or more biological markers selected from synaptophysin (SYP), chromogranin A (CGA), insulinoma-associated protein 1 (INSMI), syntaxin 1 (STX1), ISL LIM homeobox 1 (ISL1), INSM transcriptional repressor 1 (INSMI), secretagogin (SECG), neural cell adhesion molecule 1 (NCAM1; CD56), neuron-specific enolase (NSE), and gastrin-releasing peptide (GRP).
[00285] Embodiment 90: The method of embodiment 89, wherein the biological sample obtained from the subject is tissue or blood.
[00286] Embodiment 91: The method of embodiment 89 or 90, wherein the biological sample is tissue.
[00287] Embodiment 92: The method of embodiment 91, wherein the tissue is from a biopsy of the prostate cancer.
[00288] Embodiment 93: The method of embodiment 89 or 90, wherein the biological sample is blood. [00289] Embodiment 94: The method of any one of embodiments 89-93, wherein the biological sample obtained from the subject has been determined to exhibit an amount of the one or more biological markers that exceeds the upper limit of normal.
[00290] Embodiment 95: The method of embodiment 89 or 90, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for synaptophysin (SYP).
[00291] Embodiment 96: The method of embodiment 89 or 90, wherein the biological sample obtained from the subject is blood, and the biological sample has been determined to exhibit an amount of chromogranin A (CGA) that exceeds 101 ng/mL.
[00292] Embodiment 97: The method of embodiment 89 or 90, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for insulinoma-associated protein 1 (INSM1).
[00293] Embodiment 98: The method of embodiment 89 or 90, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for syntaxin 1 (STX1).
[00294] Embodiment 99: The method of embodiment 89 or 90, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for ISL LIM homeobox 1 (ISL 1) .
[00295] Embodiment 100: The method of embodiment 89 or 90, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for INSM transcriptional repressor 1 (INSM1).
[00296] Embodiment 101: The method of embodiment 89 or 90, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for secretagogin (SECG). [00297] Embodiment 102: The method of embodiment 89 or 90, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for neural cell adhesion molecule 1 (NCAM1; CD56).
[00298] Embodiment 103: The method of any one of embodiments 89-93, wherein the biological sample obtained from the subject is blood, and the biological sample has been determined to exhibit an amount of neuron-specific enolase (NSE) that exceeds 17.6 ng/mL.
[00299] Embodiment 104: The method of embodiment 89 or 90, wherein the biological sample obtained from the subject is tissue, and the biological sample has been determined to exhibit a positive immunohistochemical (IHC) stain for gastrin -releasing peptide (GRP).
[00300] Embodiment 105: The method of any one of embodiments 1-104, wherein a biological sample obtained from the subject has been determined to exhibit one or more alterations in one or more genes selected from TP53, RBI, and PTEN.
[00301] Embodiment 106: The method of embodiment 105, wherein the one or more alterations in one or more genes selected from TP53, RBI, and PTEN is determined by use of immunohistochemical staining. [00302] Embodiment 107: The method of embodiment 106, wherein (a) the one or more genes are selected from RBI and PTEN, and (b) 10% or fewer epithelial cells obtained from the biological sample are stained by the immunohistochemical staining.
[00303] Embodiment 108: The method of embodiment 106, wherein (a) the one or more genes is TP53, and (b) 10% or more epithelial cells obtained from the biological sample are stained by the immunohistochemical staining.
[00304] Embodiment 109: The method of embodiment 105, wherein the one or more alterations in one or more genes selected from TP53, RBI, and PTEN is determined by use of DNA sequencing.
[00305] Embodiment 110: The method of embodiment 109, wherein the one more alterations comprise one or more of (a) nonsynonymous missense mutations, (b) stop-gain mutations, (c) frameshift insertions, (d) frameshift deletions, (e) non-frameshift insertions, (f) non-frameshift deletions, and/or (e) copy number losses.
[00306] Embodiment 111: The method of any one of embodiments 1-110, wherein one or more cells comprising the prostate cancer exhibits one or more of (a) small cell carcinoma, (b) scanty cytoplasm, (c) darkly-stained nuclei with homogenous chromatin pattern, (d) moderate amounts of cytoplasm, (e) centrally located, round and regular nuclei with fine, granular, and homogeneous chromatin, (f) the absence of mitosis and necrosis, (g) mixed histology comprising adenocarcinoma and small cell components, and (h) growth in sheets.
[00307] Embodiment 112: The method of any one of embodiments 1-111, wherein the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to the subject in an amount between about 0.01 mg/kg per day to about 300 mg/kg per day. [00308] Embodiment 113: The method of embodiment 112, wherein the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is administered to the subject in an amount between about 0. 1 mg/kg per day to about 100 mg/kg per day.
[00309] Embodiment 114: The method of any one of embodiments 1-113, further comprising administering to the subject one or more additional therapeutic agents.
[00310] Embodiment 115: The method of embodiment 114, wherein the one or more additional therapeutic agents are selected from chemotherapeutic agents, mitotic inhibitors, antimetabolites, platinum -based agents, N-terminal domain inhibitors of androgen receptor, poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors, inhibitors of CYP17, inhibitors of androgen receptor protein expression, heat shock protein 90 (HSP90) inhibitors, bromodomain and extra-terminal domain family (BET) inhibitors, androgen receptor degraders, anti-PD-1 agents, anti-PD-Ll agents, and anti-CTLA-4 agents, or combinations thereof. [00311] Embodiment 116: The method of embodiment 115, wherein the one or more additional therapeutic agents are selected from chemotherapeutic agents.
[00312] Embodiment 117: The method of embodiment 116, wherein the chemotherapeutic agents are selected from actinomycin, azacytidine, azathioprine, bleomycin, bortezomib, chlorambucil, cyclophosphamide, daunorubicin, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, idarubicin, irinotecan, lurbinectedin, mechlorethamine, mitoxantrone, teniposide, topotecan, valrubicin, vemurafenib, vinblastine, vincristine, and vindesine.
[00313] Embodiment 118: The method of embodiment 115, wherein the one or more additional therapeutic agents are selected from mitotic inhibitors.
[00314] Embodiment 119: The method of embodiment 118, wherein the mitotic inhibitors are selected from paclitaxel, docetaxel, cabazitaxel, tesetaxel, and nab -paclitaxel.
[00315] Embodiment 120: The method of embodiment 115, wherein the one or more additional therapeutic agents are selected from antimetabolites.
[00316] Embodiment 121: The method of embodiment 120, wherein the antimetabolites are selected from 6-mercaptopurine, capecitabine, hydroxyurea, cladribine, pralatrexate, thioguanine, decitabine, clofarabine, nelarabine, fludarabine, 5 -fluorouracil, gemcitabine, cytarabine, pemetrexed, and methotrexate.
[00317] Embodiment 122: The method of embodiment 115, wherein the one or more additional therapeutic agents are selected from platinum -based agents.
[00318] Embodiment 123: The method of embodiment 122, wherein the platinum-based agents are selected from cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, triplatin tetranitrate, pheanthriplatin, picoplatin, and satraplatin.
[00319] Embodiment 124: The method of embodiment 115, wherein the one or more additional therapeutic agents are selected from N-terminal domain inhibitors of androgen receptor.
[00320] Embodiment 125: The method of embodiment 124, wherein the N-terminal domain inhibitor of androgen receptor is selected from EPI-001, EPI-002 (ralaniten), EPI-506, and EPI-7386. [00321] Embodiment 126: The method of embodiment 115, wherein the one or more additional therapeutic agents are selected from poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors.
[00322] Embodiment 127: The method of embodiment 126, wherein the poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors are selected from olaparib, niraparib, rucaparib, talazopari, veliparib, pamiparib, CEP-9722, and E7016.
[00323] Embodiment 128: The method of embodiment 115, wherein the one or more additional therapeutic agents are selected from inhibitors of CYP17.
[00324] Embodiment 129: The method of embodiment 128, wherein the inhibitor of CYP17 is galeterone. [00325] Embodiment 130: The method of embodiment 115, wherein the one or more additional therapeutic agents are selected from inhibitors of androgen receptor protein expression.
[00326] Embodiment 131: The method of embodiment 130, wherein the inhibitor of androgen receptor protein expression is niclosamide or galeterone.
[00327] Embodiment 132: The method of embodiment 115, wherein the one or more additional therapeutic agents are selected from one or more heat shock protein 90 (HSP90) inhibitors.
[00328] Embodiment 133: The method of embodiment 132, wherein the one or more heat shock protein 90 (HSP90) inhibitors are selected from tanespimycin, luminespib, alvespimycin, ganetespib, BIIB021, onalespib, geldanamycin, NVP-BEP800, SNX-2112 (PF-04928473), PF-04929113 (SNX-5422), KW-2478, XL888, TAS-116, VER-50589, CH5138303, VER-49009, NMS-E973, zelavespib (PU-H71), and HSP990 (NVP-HSP990).
[00329] Embodiment 134: The method of embodiment 115, wherein the one or more additional therapeutic agents are selected from bromodomain and extra-terminal domain family (BET) inhibitors.
[00330] Embodiment 135: The method of embodiment 134, wherein the bromodomain and extra-terminal domain family (BET) inhibitor is selected from JQ1, 1-BET 151 (GSK1210151A), I-BET 762 (GSK525762), GSK778 (iBET-BDl), GSK046 (iBET-BD2), OTX-015, TEN-010, CPI-203, CPI-0610, olinone, RVX-208, ABBV-744, LY294002, AZD5153, MT-1, MS645, MS417, SJ432, RVX-208, ABBV- 075 (mivebresib), BMS-986158, PLX51107, INCB054329, INCB057643, FT-1101, CC-90010, and ODM- 207.
[00331] Embodiment 136: The method of embodiment 115, wherein the one or more additional therapeutic agents are selected from androgen receptor degraders.
[00332] Embodiment 137: The method of embodiment 136, wherein the androgen receptor degraders are selected from ARV-110, ARV-330, SARD279, SARD033, ARCC-4, UT-34, ARD-111, ARD-86, ARD-77, ARD-69, ARD-61, LX-1, and LX-2, or a pharmaceutically acceptable salt thereof.
[00333] Embodiment 138: The method of embodiment 115, wherein the one or more additional therapeutic agents are selected from anti-PD-1 agents.
[00334] Embodiment 139: The method of embodiment 138, wherein the anti-PD-1 agents are selected from pembrolizumab, nivolumab, cemiplimab, partalizumab (PDR001), camrelizumab (SHR1210), sintilimab (IBI308), tislelizumab (BGB-A317), toripalimab (JS 001), dostarlimab (TSR-042, WBP-285), INCMGA00012 (MGA012), AMP-224, and AMP-514 (MEDI0680).
[00335] Embodiment 140: The method of embodiment 115, wherein the one or more additional therapeutic agents are selected from anti-PD-Ll agents.
[00336] Embodiment 141: The method of embodiment 140, wherein the anti-PD-Ll agents are selected from atezolizumab, avelumab, durvalumab, MPDL3280A (RG7446), MDX-1105 (BMS-936559), BMS- 935559, MSB0010718C, and MEDI4736.
[00337] Embodiment 142: The method of embodiment 115 wherein the one or more additional therapeutic agents are selected from anti-CTLA-4 agents.
[00338] Embodiment 143: The method of embodiment 142, wherein the anti-CTLA-4 agents are selected from ipilimumab and tremelimumab.
[00339] Embodiment 144: The method of embodiment 114, wherein the one or more additional therapeutic agents are selected from surgery, radiation, and prostate -specific membrane antigen (PSMA) targeted agents.
EXAMPLES
Example 1: Prostate cancer cell line NCI-H660 represents neuroendocrine prostate cancer
[00340] To select a prostate cancer model representative of neuroendocrine prostate cancer (NEPC), nine prostate cancer models were characterized in vitro. LNCaP (ATCC®), CWR22PC (procured from Dr. Charles Sawyer’s lab, MSKCC), 22Rvl (ATCC®), LAPC4 (ATCC®), LREX’ (procured from Dr. Charles Sawyer’s lab, MSKCC), NCI-H660 (also referred to as H660; ATCC®), PC3 (ATCC®), and DU145 (ATCC®) cells were maintained in RPMI-1640 media (Coming #14-040-CM), and VCaP (ATCC®) cells were maintained in DMEM media (Coming #15-017-CM) supplemented with 10% fetal bovine serum (FBS) (OmegaScientific #FB11) containing high testosterone, 1% penicillin-streptomycin (Coming #30- 002-CI), 1% L-glutamine (Coming #25 -005 -CI), and 1% antibiotic -antimycotic (Coming #30-004-CI). [00341] Adherent cells or cell pellets harvested for mRNA analysis were first washed once with phosphate buffered saline (PBS) and then resuspended in 600 pL of RLT lysis buffer (Qiagen Inc.) supplemented with 1% of beta-mercaptoethanol. RNA was extracted by QIAcube using the RNeasy Mini QIAcube kit (Qiagen#74116) according to the manufacturer’s instructions. The extracted RNA samples were then quantified using a Nanodrop 8000 Spectrophotometer. RT-qPCR reactions were set up in triplicate using 3 pL of 3 ng/ pL of RNA in 7 pL of pre-mixed SensiFAST™ SYBRNo-ROX One-Step Kit reagents (BIOLINE# BIO-98005). The RT-qPCR reactions were run on a Bio-Rad CFX384 Real-Time PCR System and data was plotted with GraphPad Prism 8 software.
[00342] Nine in vitro prostate cancer models were characterized for neuroendocrine markers. RT-qPCR was used to measure the expression levels of androgen receptor (AR), synaptophysin (SYP), and chromogranin A (CGA) in prostate cancer cell lines including VCaP, LNCaP, CWR22PC, 22Rvl, LREX’, LAPC4, H660, PC3 and DU145 (FIG. 1A, FIG. IB, FIG. 1C). Among these lines, NCI-H660 lacked expression of AR (FIG. 1A) and showed the highest mRNA levels for neuroendocrine markers SYP (FIG. IB) and CGA (FIG. 1C). The NCI-H660 model was therefore selected as a representative androgen-independent NEPC model system.
Example 2: Administration of Compound 4 to NCI-H660 tumor-bearing male athymic nude mice [00343] NCI-H660 (ATCC® CRL-5813™) androgen-independent neuroendocrine prostate tumor cells were inoculated into right flanks of 90 intact male athymic nude mice. When tumor volumes reached 150-250 mm3 in volume, 35 tumor-bearing mice were selected and randomly assigned into three groups with 11-12 mice in each group: (a) the first group (n = 12) was orally administered vehicle for 20 days, (b) the second group (n = 11) was orally administered Compound 4 once per day at a dose of 100 mg/kg for 20 days, and (c) the third group (n = 12) was orally administered Compound 4 once per day at a dose of 200 mg/kg for 20 days. The tumor volume in each mouse in each group was then measured twice a week following the administration of vehicle or Compound 4. FIG. 2 A shows the average tumor volume for the mice (± SEM) in each group. Compound 4 demonstrated significant tumor growth inhibition as a monotherapy. Compared to vehicle control, 20-day daily oral administrations of Compound 4 at 100 and 200 mg/kg once daily inhibited tumor growth by 70.9% and 78.5%, respectively (FIG. 2A, t=test, p <0.05).
[00344] To identify which genes were affected in the mice treated with Compound 4 compared to those treated with vehicle, RNA sequencing was performed on the H660 samples obtained from mice treated with 100 mg/kg of Compound 4 or vehicle from this Example 2. Extracted RNA was process into libraries for RNA-sequencing at Q2 Solutions-EA Genomics (North Carolina, USA). RNA-sequencing data was aligned using Kallisto 0.46.1 (Bray et al, Nature Biotechnology 2016) against a combined human Gencode v38 and mouse Gencode vM27 reference of the protein coding transcripts (Frankish et al, Nucleic Acids Research 2019). Transcript-level counts were summarized at the gene level using tximport 1.26.1 (Soneson et al, FlOOORes 2015) in Bioconductor 3.16 (Huber et al, Nat Methods 2015). Differential expression analysis was performed with DESeq2 1.38.3 (Love et al, Genome Biology 2014) using the default modeling parameters and calculating p-values through the results function with parameters “IfcThreshold = log2(1.2)” & “altHypothesis = 'greaterAbs' .” Among the genes that exhibited an absolute log2 fold change greater than 1 and an adjusted p-value less than 0.01 were ASCL1 (FIG. 2B) and MYCL (FIG. 2C), which showed a strong decrease in expression in the mice treated with Compound 4 compared to the mice treated with vehicle. ASCL1 and MYCL have been associated with lineage trans-differentiation in prostate cancer (see, for example, Davies et al, Cancer Discovery 2023).
Example 3: Preparation of crystalline Form 1 of Compound 4
[00345] 150 pL of methanol was added to 50 mg of the free base of Compound 4 and the resulting slurry was stirred at room temperature for one day. The resulting solids were vacuum filtered and dried under ambient condition overnight to afford Form 1 of Compound 4.
Example 4A: Preparation of crystalline Form 2 of Compound 4
[00346] 400 mg of the free base of Compound 4 was dissolved in 1.5 mL of 2 -methyltetrahydrofuran at 50 °C, to which was added 1.5 mL n-heptane at about 47 °C, and the resulting mixture was cooled 10 °C. The resulting solids were vacuum filtered and allowed to air dry overnight under ambient conditions to afford Form 2 of Compound 4.
Example 4B: Preparation of crystalline Form 2 of Compound 4
[00347] A quantity of the free base of Compound 4 was dissolved in 2 -methyltetrahydrofuran (10 volumes) and then distilled to 3 volumes. The temperature of the solution was adjusted to about 25 °C and the resulting slurry was stirred for greater than 30 minutes. To the slurry was added n-heptane (7 volumes) over 2 hours and the resulting mixture was stirred for greater than 4 hours. The resulting solid was filtered, the filter cake was washed with 30% 2 -methyltetrahydrofuran /heptane (2 volumes) and dried in a vacuum oven to provide Form 2 of Compound 4.
Example 5: X-ray powder diffraction (XRPD) analysis of Form 1 and Form 2 of Compound 4
[00348] XRPD analyses of crystalline polymorphic forms of Compound 4 were performed using Panalytical X’pert3 X-ray powder diffractometer. Samples were spread on the middle of a zero -background Si holder. The 2-theta position was calibrated against a Panalytical Si reference standard disc. The parameters used for the analyses are set forth in Table 1.
Table 1
Figure imgf000093_0001
[00349] Polymorphic Form 1 of Compound 4 was analyzed by XRPD as set forth above and exhibited the peaks set forth in Table 2. The error associated with each °2 -theta position was determined to be ± 0.2° theta.
Table 2
Figure imgf000093_0002
Figure imgf000094_0001
[00350] Polymorphic Form 2 of Compound 4 was analyzed by XRPD as set forth above and exhibited the peaks set forth in Table 3. The error associated with each °2 -theta position was determined to be ± 0.2 °- theta.
Table 3
Figure imgf000094_0002
Example 6: Thermal gravimetric analyses and differential scanning calorimetry analyses of Form 1 and Form 2 of Compound 4
[00351] Thermal gravimetric analysis (TGA) data were collected using TA Discovery TGA 550 TGA from TA Instruments, and differential scanning calorimetry (DSC) analyses were performed using a TA Q2000 DSC from TA Instruments using the parameters set forth in Table 4.
Table 4
Figure imgf000094_0003
Figure imgf000095_0001
[00352] A thermal gravimetric analysis (TGA) of a sample of Form 1 of Compound 4, when conducted under the conditions set forth in Table 4, exhibited a weight loss of about 1% upon heating the sample from room temperature to about the onset of melting (about 207 °C). A differential scanning calorimetry (DSC) analysis of Form 1 of Compound 4, when conducted under the conditions set forth in Table 4, exhibited peaks at between about 170 °C and 172 °C, and between about 207 °C and 208 °C.
[00353] A thermal gravimetric analysis (TGA) of a sample of Form 2 of Compound 4, when conducted under the conditions set forth in Table 4, exhibited a weight loss of about 2% upon heating the sample from room temperature to about the onset of melting (about 204 °C). A differential scanning calorimetry (DSC) analysis of Form 2 of Compound 4, when conducted under the conditions set forth in Table 4, exhibited a peak between about 203 °C and 204 °C.
Example 7: Preparation of crystalline Form 2 of Compound 4
[00354] A reactor was evacuated and charged with nitrogen to atmospheric pressure. The reactor was then charged with a solution of Compound 4 (approximately 2.41 kg as determined by solution assay using HPLC) in 2 -methyltetrahydrofuran (2-MeTHF, 36 kg, 15 volumes) and the batch was concentrated to a batch volume of about 5 L (about 2 volumes) via distillation under reduced pressure. The resulting solution was adjusted to about 25 °C and then n-heptane (0.4 kg, 0.2 volumes) was added in portions over a period of about 3 hours. The resulting solution was then seeded with Compound 4 Form 2 (9 g, 0.4 wt%), the resulting mixture was stirred for about 1.3 hours, and then additional n-heptane (24 kg, 10 volumes) was added over about 6 hours. The resulting slurry was stirred for about 4.25 hours at 25 °C and then fdtered. The reactor was then rinsed with n-heptane (5.8 kg, 2.5 V), and this mixture was rinsed forward to the fdter cake, which was deliquored and the solids were dried under reduced pressure at 40 °C and 50 °C for 19 hours to provide 2.48 kg of Form 2 of Compound 4.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A method of treating prostate cancer in a subject, wherein the prostate cancer has been determined to comprise neuroendocrine prostate cancer, comprising administering to the subject a therapeutically effective amount of a compound selected from the group consisting of:
Figure imgf000096_0001
pharmaceutically acceptable salt thereof.
The method of claim 1, wherein the compound is:
Figure imgf000096_0002
acceptable salt thereof.
The method of claim 1, wherein the compound is:
Figure imgf000097_0001
pharmaceutically acceptable salt thereof.
The method of claim 1, wherein the compound is:
Figure imgf000097_0002
pharmaceutically acceptable salt thereof.
The method of claim 1, wherein the compound is:
Figure imgf000097_0003
pharmaceutically acceptable salt thereof.
The method of claim 1, wherein the compound is:
Figure imgf000097_0004
pharmaceutically acceptable salt thereof.
The method of claim 1, wherein the compound is:
Figure imgf000098_0001
acceptable salt thereof.
The method of claim 1, wherein the compound is:
Figure imgf000098_0002
acceptable salt thereof.
The method of claim 1, wherein the compound is:
Figure imgf000098_0003
acceptable salt thereof.
The method of claim 1, wherein the compound is:
Figure imgf000098_0004
acceptable salt thereof.
11. The method of claim 1, wherein the compound is:
Figure imgf000099_0001
pharmaceutically acceptable salt thereof.
The method of claim 1, wherein the compound is:
Figure imgf000099_0002
pharmaceutically acceptable salt thereof.
The method of claim 1, wherein the compound is:
Figure imgf000099_0003
pharmaceutically acceptable salt thereof.
14. The method of any one of claims 1-13, wherein the subject has been administered one or more first agents prior to the administration to the subject of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and wherein the one or more first agents is selected from the group consisting of (a) luteinizing hormone -releasing hormone (LHRH) agonists, (b) luteinizing hormone -releasing hormone (LHRH) antagonists, (c) androgen receptor inhibitors, (d) inhibitors of cytochrome P450 17A1, and (e) antiandrogens.
15. The method of claim 14, wherein the neuroendocrine prostate cancer in the subject has been determined to be progressing following the administration to the subject of the one or more first agents.
16. The method of claim 14 or 15, wherein the luteinizing hormone -releasing hormone (LHRH) agonist is selected from goserelin, histrelin, leuprolide, and triptorelin.
17. The method of claim 14 or 15, wherein the luteinizing hormone -releasing hormone (LHRH) antagonist is selected from degarelix and relugolix.
18. The method of claim 14 or 15, wherein the androgen receptor inhibitor is selected from enzalutamide, apalutamide, and darolutamide.
19. The method of claim 14 or 15, wherein the inhibitor of cytochrome P450 17A1 is abiraterone acetate.
20. The method of claim 14 or 15, wherein the antiandrogen is selected from egestrol, bicalutamide, flutamide, and nilutamide.
21. The method of any one of claims 1 to 20, further comprising administering to the subject one or more additional therapeutic agents.
22. The method of claim 21, wherein the one or more additional therapeutic agents are selected from chemotherapeutic agents, mitotic inhibitors, antimetabolites, platinum -based agents, N-terminal domain inhibitors of androgen receptor, poly(adenosine diphosphate -ribose) polymerase (PARP) inhibitors, inhibitors of CYP17, inhibitors of androgen receptor protein expression, heat shock protein 90 (HSP90) inhibitors, bromodomain and extra-terminal domain family (BET) inhibitors, androgen receptor degraders, anti-PD-1 agents, anti-PD-Ll agents, and anti-CTLA-4 agents, or combinations thereof.
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