WO2010149666A1 - Methods for treating neoplasia - Google Patents

Methods for treating neoplasia Download PDF

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Publication number
WO2010149666A1
WO2010149666A1 PCT/EP2010/058843 EP2010058843W WO2010149666A1 WO 2010149666 A1 WO2010149666 A1 WO 2010149666A1 EP 2010058843 W EP2010058843 W EP 2010058843W WO 2010149666 A1 WO2010149666 A1 WO 2010149666A1
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Prior art keywords
compound
amino
aminopropyl
alkyl
optionally substituted
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PCT/EP2010/058843
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French (fr)
Inventor
Konstantinos Alevizopoulos
Christos Stournaras
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Medexis S.A.
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Publication of WO2010149666A1 publication Critical patent/WO2010149666A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • 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
    • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
    • A61K31/566—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol having an oxo group in position 17, e.g. estrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • neoplasia Approximately one million Americans are diagnosed with neoplasia every year, and about half a million people in the United States die of the disease annually. Although improvements in neoplasia detection, diagnosis, and treatment have increased the survival rate for many types of neoplasia, only about 60 percent of people diagnosed with neoplasia are alive five years after treatment, making neoplasia the second leading cause of death in the United States. One of the reasons for this poor long term survival rate is that many patients develop multidrug resistant neoplasias. After several cycles of chemotherapy, some tumor cells become resistant not only to the agent used in the chemotherapy, but also to compounds with different structures and mechanisms of action.
  • ATP binding cassette superfamily of transporter proteins acts as an energy-dependent drug efflux pump and alterations in these transporter proteins are associated with the development of multi-drug resistant neoplasias.
  • the activity of this family of proteins prevents the intracellular accumulation of a broad range of cytotoxic drugs.
  • the invention features methods for the treatment of neoplasias, including multidrug resistant neoplasias.
  • the invention provides a method of treating or preventing neoplasia in a subject in need thereof.
  • the method comprises administering to the subject a therapeutically effective amount of a compound of Formula (I)
  • R 1 is H or -OH
  • R 8 is H, alkyl, or -NO 2 ;
  • R 9 is H or formyl
  • R 10 for each occurrence, is the same or different and is H or halogen
  • R 11 is H or alkyl
  • R 12 is alkyl or phenyl
  • R 13 is alkyl optionally substituted by a 4 or 6-membered heterocyclic ring, a 5 or 6- membered heteroaryl, amino(Ci_ 4 )alkoxyl, (Ci_ 4 )alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; aminoalkyl optionally substituted by one or more alkyl; or aminoacyl optionally substituted by alkyl;
  • R 14 is aminoalkyl optionally substituted by one or more alkyl or acetyl; alkyl optionally substituted by guanidinyl or a 4- to 6-membered heterocyclic ring; or a 4- to 6-membered heterocyclic ring optionally substituted by alkyl;
  • R 15 and R 16 are aminoalkyl optionally substituted by one or more alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
  • R 14 is amino(Ci_C 4 )alkyl, that is further optionally substituted by one or more groups.
  • R 14 is selected from the group consisting of aminoethyl, 2-aminopropyl, 2-amino-2-methylpropyl, 2-amino- dimethylethyl, methylaminoethyl, methylaminopropyl, aminopropyl, aminobutyl, dimethylaminoethyl, and acetylaminoethyl.
  • R 14 is that is further substituted by a 4- or 6-membered heterocylic ring or by a guanidinyl group.
  • R 14 include pyrrolidinyl- substituted methyl and guanidinylethyl.
  • Embodiments of the methods of the invention also provide compounds of Formula (I) wherein R 14 is a 4- to 6-membered heterocyclic ring, that is optionally substituted by (Ci-
  • R 14 is azetidinyl, pyrrolidinyl, or piperidinyl, wherein the azetidinyl, pyrrolidinyl, or piperidinyl group is further optionally substituted by a methyl group.
  • the invention also provides a method of treating or preventing neoplasia, which comprises administering to a subject in need thereof compounds of Formula (I) wherein A is CH(OR 13 ).
  • R 13 is amino(Ci_C 4 )alkyl, which is optionally substituted by one or more Certain examples of R 13 include dimethylaminopropyl, dimethylaminoethyl, ethylaminobutyl, and dimethylaminobutyl.
  • R 13 is ethyl or propyl, and that the ethyl or propyl group is further substituted by a substituent selected from the group consisting of pyrrolidinyl, piperidinyl, imidazolyl, dimethylamino-ethoxyl, pyrrolidinyl-ethoxyl, and 4-methylpiperazinyl.
  • R 15 include aminoethyl and dimethylaminoethyl.
  • R 16 can be, but is not limited to, aminopropyl.
  • the methods of the invention also include administering to a subject identified as in need thereof compounds of Formula (I) wherein one of R 2 and R 3 is H, the other is -OH.
  • Examples of R 14 include, but are not limited to, aminoethyl, methylaminopropyl and pyrrolidinyl.
  • Another embodiment provides compounds of Formula (I) wherein A is CH(OR 13 ).
  • R 13 can be, but is not limited to, aminoethyl, methylaminoethyl and dimethylaminopropyl.
  • R 14 is amino(Ci_C 4 )alkyl that is optionally substituted by one or more (Ci-C 4 )alkyl groups.
  • R 14 is a 4- to 6-membered heterocyclic ring, that is optionally substituted by (Ci-C/Oalkyl.
  • R 14 examples include, but are not limited to, aminoethyl, aminopropyl, methylaminopropyl, 2-aminopropyl, methylaminoethyl, 3-amino- 2-methylpropyl, and pyrrolidinyl.
  • R 13 can be, but is not limited to, aminopropionyl, 3-aminobutanionyl, or 3-amino-2- methylpropionyl.
  • R 15 can be, but is not limited to, aminopropyl and aminoethyl.
  • R 16 examples include, but are not limited to, aminopropyl.
  • One embodiment provides compounds of Formula (I) wherein A is CH-S(O)R 16 .
  • R 16 include, but are not limited to, aminopropyl.
  • R 14 is pyrrolidinyl or aminoethyl.
  • the R 14 group can be, but is not limited to, aminoethyl, 2-aminopropyl, 2-amino-dimethylethyl, and 3- amino-2-methylpropyl.
  • A is CH-S(O)R 16 .
  • R 16 can be, but is not limited to, aminopropyl.
  • Certain embodiments of the methods of the invention include administering to the subject compounds of Formula (I) wherein one of R 2 and R 3 is H, the other is methyl.
  • the R 14 group can be, but is not limited to, aminoethyl, aminopropyl, methylaminoethyl, methylaminopropyl, 3-amino-2- methylpropyl, and 2-aminopropyl.
  • the compounds of Formula (I) have A being CH(OR 13 ).
  • the R 13 group can be, but is not limited to, aminopropionyl, 3- aminobutanionyl, and amino-2-methylpropionyl.
  • A is CH- SR 16 or CH-S(O)R 16 , wherein R 16 can be, but is not limited to, aminopropyl.
  • inventions include administering to the subject compounds of Formula (I) wherein one of R 2 and R 3 is H and the other is CH 2 OH, CH 2 CH 2 OH or CH 2 OCH 3 .
  • One embodiment provides that A is CH-S(O)R 16 .
  • R 16 is aminopropyl.
  • the methods of the invention comprise administering to the subject compounds of Formula (I) wherein one of R 2 and R 3 is H, and the other is -C(O)NH 2 , -ONO 2 , formylamino, ethynyl, or methoxycarbonyl.
  • the compounds of Formula (I) have A being CH-S(O)R 16 , wherein R 16 can be, but is not limited to, aminopropyl.
  • This R 14 group can be, but is not limited to, aminoethyl or pyrrolidinyl.
  • Other embodiments of the invention also provide compounds of Formula (I) wherein one of R 4 and R 5 is H, and the other is -OH.
  • compounds of Formula (I) have both of R and R being H.
  • the compounds of Formula (I) have A being CH- S(O)R 16 .
  • One example of the R 16 group is aminopropyl .
  • the methods of the invention also include administering compounds of Formula (I) wherein one of R 2 and R 3 is H, the other is -CH 2 OH.
  • the R 14 group include, but are not limited to, aminopropyl, aminoethyl, methylaminopropyl, amino-2 -methylpropyl, 2-amino- dimethylethyl, and pyrrolidinyl.
  • Another embodiment provides compounds of Formula (I) wherein A is CH(OR 13 ).
  • the R 13 group can be, but is not limited to, aminopropionyl, 3- aminobutanionyl, or amino-2-methylpropionyl.
  • the compounds of Formula (I) can also have A as CH-SR 16 or CH-S(O)R 16 .
  • An example of the R 16 group is aminopropyl.
  • R 2 and R 3 is H, and the other is -CH 2 OH.
  • Another embodiment provides that both of R and R are H.
  • Examples of the R 14 group include aminoethyl, aminopropyl, methylaminoethyl, methylaminopropyl, 2-aminopropyl, amino-2-methylpropyl, 2-amino-dimethylethyl, and pyrrolidinyl.
  • Another embodiment provides compounds of Formula (I) wherein A is CH(OR 13 ).
  • the R 13 group can be, but is not limited to, aminopropionyl, 3-aminobutanionyl, or amino-2- methylpropionyl.
  • R 15 can be, but is not limited to, aminopropyl or aminoethyl.
  • Other embodiments also provide that A is CH-SR 16 or CH-S(O)R 16 .
  • One example of the R 16 group is aminopropyl.
  • One embodiment provides compounds of Formula (I) wherein both of R and R are H.
  • R 14 is 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2-methylpropyl, 2-amino-dimethylethyl, or aminoethyl.
  • A is CH(OR 13 ).
  • R 13 can be, but is not limited to, aminopropionyl, 3-aminobutanionyl, or 3-amino-2-methylpropionyl.
  • R 15 can be, but is not limited to, aminopropyl or aminoethyl.
  • Other embodiments provide that A is CH-SR 16 or CHS(O)R 16 .
  • One example of the R 16 group is aminopropyl.
  • the methods of the invention also include administering compounds of Formula (I) wherein one of R 4 and R 5 is H and the other is methyl.
  • the compounds of Formula (I) have both of R 2 and R 3 as H.
  • R 14 is selected from the group consisting of 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2-methylpropyl, 2- amino-dimethylethyl, and aminoethyl.
  • A is CH(OR 13 ).
  • R 13 group examples include aminopropionyl, 3-aminobutanionyl, and amino-2-methylpropionyl.
  • R 15 can be, but is not limited to, aminopropyl or aminoethyl.
  • A is CH-SR 16 or CH-S(O)R 16 .
  • One example of the R 16 group is aminopropyl.
  • the compounds have both of R and R as H.
  • the compounds of Formula (I) can also have one of R 4 and R 5 as H, and the other being -C(O)-NH 2 , -C(O)-OCH 3 , -CH 2 OH, ethynyl, formylamino, -CH 2 OCH 3 , or -ONO 2 .
  • both of R and R are H.
  • Another embodiment provides that the compounds of Formula (I) have A as CH-S(O)R 16 .
  • One example of the R 16 group is aminopropyl.
  • R 4 and R 5 together with the carbon to which they are attached form a cyclopropyl ring.
  • the compounds have both of R and R as H.
  • R 14 can be, but is not limited to, pyrrolidinyl or aminoethyl.
  • the methods of the invention comprise administering compounds of Formula (I) wherein R 1 is -OH.
  • R 13 group can be, but is not limited to, aminopropionyl, 3-aminobutanionyl, or amino-2-methylpropionyl.
  • R 15 is aminopropyl or aminoethyl.
  • A is CH-SR 16 or CH-S(O)R 16 .
  • One example of the R 16 group is aminopropyl.
  • the methods of the invention include administering compounds of Formula (I) wherein R and R together with the carbon to which they are attached form
  • R 14 examples include, but are not limited to, 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2-methylpropyl, 2-amino-dimethylethyl, and aminoethyl.
  • A is CH(OR 13 ).
  • R 13 is aminopropionyl, 3- aminobutanionyl, or 3 -amino-2-methylpropionyl.
  • A is CH- SR 16 or CH-S(O)R 16 .
  • One example of the R 16 group is aminopropyl.
  • Embodiments of the invention also provide compounds of Formula (I) wherein one of R and R is H and the other is methyl.
  • the preceding R 14 group can be, but is not limited to, 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2-methylpropyl, and aminoethyl.
  • Another embodiment provides the compounds wherein A is CH(OR 13 ). Examples of the R 13 group include aminopropionyl, 3-aminobutanionyl, and amino-2-methylpropionyl.
  • R 15 can be, but is limited to, aminopropyl or aminoethyl.
  • R 16 is aminopropyl.
  • R 2 and R 3 are H, and the other is -C(O)-NH 2 , -C(O)-OCH 3 , -CH 2 OH, ethynyl, formylamino, -CH 2 OCH 3 , or -NO 2 .
  • A is -CH-S(O)R 16 .
  • R 16 is aminopropyl.
  • One embodiment provides the compounds of Formula (I) wherein A is -CH-S(O)R 16 . In one embodiment, the preceding R 16 is aminopropyl.
  • R 14 can be, but is not limited to, 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2-methylpropyl, 2-amino-dimethylethyl, or aminoethyl.
  • the preceding R 13 group can be, but is not limited to, aminopropionyl, 3-aminobutanionyl, or amino-2-methylpropionyl.
  • R 15 can be, but is not limited to, aminopropyl or aminoethyl, and one example of R 16 is aminopropyl.
  • Embodiments of the invention also provide compounds of Formula (I) wherein one of R 4 and R 5 is H and the other is methyl. In some instances, the compounds have both of R 2 and R 3 being H.
  • R 14 include 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, 3-amino-2- methylpropyl, 2-amino-dimethylethyl, and aminoethyl.
  • R 13 is aminopropionyl, 3-aminobutanionyl, or amino-2-methylpropionyl.
  • R 15 is aminopropyl or aminoethyl.
  • R 16 is aminopropyl.
  • the invention also provides compounds of Formula (I) wherein R and R are both H.
  • one of R 4 and R 5 is H, and the other is -C(O)NH 2 , -ONO 2 , formylamino, ethynyl, -CH 2 OH, -CH 2 -O-CH 3 , or methoxycarbonyl.
  • A is CH-S(O)R 16 , while one example of R 16 is aminopropyl.
  • the invention provides that one of R 6 and R 7 is H and the other is - OH, and R 1 is H.
  • Certain embodiments provide the compounds of Formula (I) wherein one of R 2 and R 3 is H, and the other is -OH, and both of R 4 and R 5 are H.
  • A is CH(OR 13 ), while R 13 is selected from the group consisting of methylaminopropyl, dimethylaminopropyl, dimethylaminobutyl, aminopropyl, dimethylaminoethyl, aminoethyl, pyrrolidinylethyl, 2-(2- (dimethylamino)ethoxy) ethyl, 2-(2-(pyrrolidin-l-yl)ethoxy)ethyl, 2-guanidinylethyl, and 3- guanidinylpropyl.
  • R 15 can be, but is not limited to, aminoethyl or dimethylaminoethyl.
  • R 14 is aminoalkyl.
  • R 13 is aminoalkyl.
  • the invention also provides methods of treating or preventing neoplasia in a subject in need thereof.
  • the methods include administering to the subject a compound of Formula (Ia)
  • R 1 is H or -OH
  • R 4 and R 5 are H, and the other is H or -OR 8 ;
  • R 8 is H or optionally substituted RR 1133 iiss ((CCii--CC 44 ))aallkkyyll ooppttiioonnaallllyy ssuubbssttiittuutteedd bbyy a a 4 or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, amino(Ci_ 4 )alkoxyl, (Ci_ 4 )alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; amino(Ci-C 4 )alkyl optionally substituted by one or more (Ci-C 4 )alkyl; or amino(Ci-C 4 )acyl optionally substituted by (Ci- C 4 )alkyl; and
  • R 14 is an amino(Ci-C 4 )alkyl optionally substituted by one or more or a 4- to 6-membered heterocyclic ring optionally substituted by and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
  • One embodiment provides a compound of Formula (Ia) wherein R 1 is H.
  • Another embodiment provides a compound of Formula (Ia) wherein R 4 and R 5 are H.
  • One exemplified R 14 is an amino(Ci-C 4 )alkyl that is optionally substituted by one or more (Ci- C 4 )alkyl.
  • Another exemplified R 14 is a 4- to 6-membered heterocyclic ring that is optionally substituted by (C r C 4 )alkyl.
  • A is CH(OR 13 ).
  • R 13 is (Ci- C 4 )alkyl optionally substituted by a 4 or 6-membered heterocyclic ring.
  • R 13 is 2- (pyrrolidin- 1 yl) ethyl.
  • Certain compounds for use in accordance with the methods of the invention include (1 OR, 13S)-3-(2-aminoethoxyimino)-l 0, 13-dimethyldodecahydro-lH- cyclopenta[a]phenanthrene-6, 17(1 OH, 14H)-dione (1) :
  • the methods of the invention also include administering to a subject in need thereof a compound of Formula (Ib):
  • R 1 is H or -OH
  • X is C(R 10 ) 2 or N-OR u ;
  • One of R 4 and R 5 is H, and the other is H or -OR 8 ;
  • R 8 is H or substituted or unsubstituted R 10 , for each occurrence, is the same or different and is H or halogen;
  • R u is H or (Ci_C 4 )alkyl; and
  • R 14 is an amino(Ci_C 4 )alkyl optionally substituted by one or more or a 4- to
  • R 15 and R 16 are amino(Ci_C 4 )alkyl optionally substituted by one or more (Ci-C 4 )alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
  • One embodiment provides a compound of Formula (Ib) wherein R 1 is H. In another embodiment, R 1 is OH.
  • Another embodiment provides a compound of Formula (Ib) wherein X is C(R 10 ) 2 .
  • R 10 is H.
  • X is N-OR 11 .
  • One example of the R 11 group is H.
  • Another example of R 11 is (d_C 4 )alkyl.
  • R 14 is an amino(Ci_ 4 )alkyl that is optionally substituted by one or more
  • R 14 is a 4- to 6-membered heterocyclic ring.
  • R 15 is an ammo(C M )alkyl.
  • A is CH-SR 16 . In another embodiment, A is CH-S(O)R 16 . Certain instances provide that R 16 is an amino(Ci_ 4 )alkyl.
  • Certain compounds of Formula (Ib) for use in accordance with the methods of the invention include compounds as follows: a) ( 1 OR, 13 S)-3 -(2-aminoethoxyimino)- 10,13 -dimethyl-6-methylenetetradecahydro- 1 H- cyclopenta[a]phenanthren-17(2H)-one (3):
  • the methods of the invention also provide administering to a subject in need thereof a compound of Formula (Ic): wherein
  • R 1 is H or -OH
  • R 2 and R 3 are H, and the other is optionally substituted by hydroxyl or methoxy;
  • R 4 and R 5 are H and the other is -OR 8 ; or
  • R 8 is H or substituted or unsubstituted (Ci_C 4 )alkyl
  • R 14 is an amino(Ci_C 4 )alkyl optionally substituted by one or more or a 4- to 6-membered heterocyclic ring optionally substituted by and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
  • R 14 in Formula (Ic) is an amino(Ci_C 4 )alkyl that is optionally substituted by one or more
  • R 14 is a 4- to 6-membered heterocyclic ring optionally substituted by
  • Certain compounds of Formula (Ic) for use in accordance with the methods of the invention include i) (6S, 1 OR, 13 S)-3-(2-aminoethoxyimino)-6-(hydroxymethyl)- 10, 13-dimethyldodecahydro- 1 H- cyclopenta[a]phenanthrene-7, 17(2H,8H)-dione (9): ii) (6S, 1 OR, 13S)-6-(hydroxymethyl)-l 0, 13-dimethyl-3-(pyrrolidin-3-yloxyimino)dodecahydro- lH-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione (10):
  • the methods of the invention also provide administering to a subject in need thereof a compound of Formula (Id):
  • R 1 is H or -OH
  • R 4 and R 5 taken together with the carbon atom to which they are attached form C(R 10 ) 2 ;
  • R 10 for each occurrence, is the same or different and is H or halogen
  • R 14 is an amino(Ci_C 4 )alkyl optionally substituted by one or more or a 4- to 6-membered heterocyclic ring optionally substituted by (Ci_C 4 )alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
  • One embodiment provides a compound of Formula (Id) wherein R 1 is H. Another embodiment provides a compound of Formula (Id) wherein R 10 is H. Another embodiment provides that R 10 is halogen. Certain instances provide compounds of Formula (Id) wherein R 14 is an amino(Ci_C 4 )alkyl that is optionally substituted by one or more In other instances, R 14 is a 4- to 6-membered heterocyclic ring that is optionally substituted by (Ci- C 4 )alkyl.
  • Certain compounds of Formula (Id) used in accordance with the methods of the invention include: aa) ( 1 OS, 13 S)-3 -(2-aminoethoxyimino)- 10, 13 -dimethyl-7-methylenetetradecahydro- 1 H- cyclopenta[a]phenanthren-17(2H)-one (13):
  • the methods of the invention also provide administering to a subject in need thereof a compound of Formula (Ie):
  • R 1 is H or -OH;
  • R 13 is (Ci_C 4 )alkyl optionally substituted by a 4 or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, amino(Ci_ 4 )alkoxyl, (Ci_ 4 )alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; amino(Ci_C 4 )alkyl optionally substituted by one or more (Ci_C 4 )alkyl; or amino(Ci_C 4 )acyl optionally substituted by (Ci- C 4 )alkyl;
  • R 14 is an amino(Ci_C 4 )alkyl optionally substituted by one or more or a 4- to 6-membered heterocyclic ring optionally substituted by
  • R 15 and R 16 are amino(Ci_C 4 )alkyl optionally substituted by one or tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
  • One embodiment provides a compound of Formula (Ie) wherein R 1 is H.
  • R 15 is amino(Ci_C 4 )alkyl.
  • R 15 is amino(Ci_C 4 )alkyl that is substituted by one or more
  • Certain compounds of Formula (Ie) for use in accordance with the invention include 1) 6,17-dihydroxy- 10,13 -dimethylhexadecahydro- 1 H-cyclopenta[a]phenanthrene-3 - carbaldehyde O-2-aminoethyl oxime (16):
  • the invention provides methods for reducing the growth, proliferation, or survival of a neoplastic cell, the method comprising contacting the cell with an effective amount of a compound of this invention.
  • the compound reduces the growth, proliferation, or survival of a neoplastic cell.
  • the neoplastic cell is derived from a tissue selected from the group consisting of lung, breast, CNS, colon, prostate, ovary, pancreas, kidney and melanoma.
  • the cell expresses MDR-I or P-glycoprotein.
  • Yet another aspect of this invention provides a method of inducing cell death in a neoplastic cell. The method includes contacting the cell with a therapeutically effective amount of a compound of this invention, thereby inducing cell death.
  • the cell is in a subject. Another embodiment provides that the cell death is apoptotic cell death.
  • a further aspect of this invention provides a method of preventing or treating a neoplasia in a subject. This method includes administering to the subject a therapeutically effective amount of a compound of this invention.
  • the subject recited in the methods of the invention is a mammal. In another embodiment, the subject is a human patient.
  • the methods of the invention reduce the growth or proliferation of a neoplasia in a subject.
  • the neoplasia recited in the methods of the invention can be, but is not limited to, a lung, breast, CNS, colon, prostate, ovary, pancreas, kidney or skin cancer.
  • the neoplasia is resistant to one or more therapeutic agents.
  • the neoplasia is multidrug resistant. Certain embodiments provide that the neoplasia has alterations in the expression or activity of an ABC transporter, tubulin, or topoisomerase polypeptide or polynucleotide. In other embodiments, the neoplasia has an increase in the expression or activity of MDRl or P-glycoprotein.
  • the invention provides a method for the treatment of a subject having a multidrug resistant or refractory neoplasia.
  • This method includes administering to the subject a therapeutically effective amount of a compound of this invention and a pharmaceutically acceptable excipient.
  • the subject is a human patient.
  • the method reduces the growth or proliferation of the neoplasia.
  • the method induces the death of a neoplastic cell.
  • the neoplasia is resistant to one or more therapeutic agents.
  • the neoplasia has alterations in the expression or activity of an ABC transporter, tubulin, or topoisomerase polypeptide or polynucleotide.
  • Other embodiments provide that the neoplasia has an increase in the expression or activity of MDRl or P- glycoprotein.
  • the method may further comprise administering a compound selected from the group consisting of vinca alkaloids, anthracyc lines, epipodophyllotoxins, taxanes, antibiotics, antimicrotubule drugs, protein synthesis inhibitors, toxic peptides, topoisomerase inhibitors, DNA intercalators, anti-mitotics, platinum-based chemotherapeutic agents.
  • the method may also treat a patient having end-stage disease.
  • This invention also provides a composition for the treatment of a neoplasia.
  • the composition includes a therapeutically effective amount of a compound of the invention and a pharmaceutically acceptable excipient.
  • the composition may further include a therapeutically effective amount of a chemotherapeutic compound.
  • a further aspect of this invention provides a packaged pharmaceutical for the treatment of neoplasia.
  • the packaged pharmaceutical includes a therapeutically effective amount of a compound of the invention, and written instructions for administration of the compound.
  • the invention provides a method of preventing or treating a neoplasia (e.g., a membrane androgen positive solid tumor or hematological malignancy) in a subject. The method involves administering to the subject (e.g., mouse, dog, human) a therapeutically effective amount of a Na + K + ATPase inhibitor that inhibits ligand binding to a membrane androgen receptor, thereby preventing or treating the neoplasia.
  • a neoplasia e.g., a membrane androgen positive solid tumor or hematological malignancy
  • the method involves administering to the subject (e.g., mouse, dog, human) a therapeutically effective amount of a Na + K + ATPase inhibitor that inhibits ligand
  • the Na + K + ATPase inhibitor binds a Na + K + ATPase and inhibits Na + K + ATPase activity. In another embodiment, the Na + K + ATPase inhibitor binds to a membrane androgen receptor and competitively inhibits ligand binding to the receptor. In another embodiment, the Na + K + ATPase inhibitor induces cell death in a neoplastic cell of the neoplasia. In still other embodiments, the neoplasia is a prostate cancer, breast cancer, or colon cancer. In yet another aspect, the invention provides a method for treating or preventing prostate cancer in a subject.
  • the method involves administering to the subject an effective amount of a compound capable of binding and inhibiting a Na + K + ATPase, and further capable of competitively inhibiting ligand binding to the membrane androgen receptor on a prostate cancer cell.
  • the method induces cell death (e.g., apoptosis) in a cell of the prostate cancer.
  • the compound binds the membrane androgen receptor.
  • the compound is istaroxime.
  • the invention provides a composition for the treatment or prevention of a neoplasia, the composition containing a therapeutically effective amount of a compound capable of binding and inhibiting a Na + K + ATPase, and further capable of competitively inhibiting ligand binding to the membrane androgen receptor on a neoplastic cell and a pharmaceutically acceptable excipient, where the therapeutically effective amount is sufficient to induce cell death in a neoplastic cell.
  • the invention provides a packaged pharmaceutical for the treatment of neoplasia involving a therapeutically effective amount of a compound capable of binding and inhibiting a Na + K + ATPase, and further capable of competitively inhibiting ligand binding to the membrane androgen receptor on a prostate cancer cell, and written instructions for administration of the compound for use in treating the neoplasia.
  • the invention provides a method of preventing or treating a neoplasia in a subject, the method involving administering to the subject a therapeutically effective amount of a Na + K + ATPase inhibitor that binds to a membrane androgen receptor, thereby preventing or treating the neoplasia.
  • the Na + K + ATPase inhibitor induces cell death in a neoplastic cell of the neoplasia.
  • the invention provides a method for treating or preventing prostate cancer in a subject, the method involving administering to the subject an effective amount of a Na + K + ATPase inhibitor that binds to a membrane androgen receptor on a prostate cancer cell.
  • the method induces cell death in a cell of the prostate cancer.
  • the compound is istaroxime.
  • the invention provides a composition for the treatment or prevention of a neoplasia, the composition containing a therapeutically effective amount of a Na + K + ATPaSe inhibitor that binds to a membrane androgen receptor on a neoplastic cell and a pharmaceutically acceptable excipient, where the therapeutically effective amount is sufficient to induce cell death in a neoplastic cell.
  • the invention provides a packaged pharmaceutical for the treatment of neoplasia involving a therapeutically effective amount of a Na + K + ATPase inhibitor that binds to a membrane androgen receptor on a prostate cancer cell, and written instructions for administration of the compound for use in treating the neoplasia.
  • the invention further provides methods for treating neoplasia.
  • Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.
  • Figures 1A-1C are graphs showing that the compound 1/istaroxime inhibited the proliferation and viability of colon cancer cell line HCTl 16, and prostate cancer cell lines PC3 and DU145. Istaroxime's inhibitory properties were not shared by Na + K + ATPase inhibitor, rostafuroxin.
  • Figures 2A, 2B and 2C present in vitro anti-cancer data obtain with SRB and MTT assays in 22 cancer lines including multi drug resistant cell lines.
  • Figures 3A-3C are graphs presenting data on the anti-tumor activity of compounds 1 and 4 in PC-3 prostate xenografts ( Figures 3 A, B) and of compound 4 in A549 lung xenografts ( Figure 3C) respectively.
  • Figures 4 A and 4B are photomicrographs showing that compound 1 (istaroxime) and compound 4, but not rostafuroxin, abolish binding of Testosterone-HSA (testosterone 3-(O- carboxymethyl)oxime: human serum albumin) conjugates and fluorescein isothiocyanate (testosterone-HSA-FITC) conjugates to the membrane androgen receptor.
  • Testosterone-HSA testosterone 3-(O- carboxymethyl)oxime: human serum albumin
  • fluorescein isothiocyanate testosterone-HSA-FITC
  • the invention features methods comprising compounds delineated herein that are useful for the treatment of neoplasia.
  • the compounds of the invention are useful for the treatment of multidrug resistant neoplasia.
  • the invention is based, at least in part, on the discovery that compounds of the invention including istaroxime, (£",Z)-3-(2-Aminoethoxyimino)androstane-6,17-dione, have potent antineoplastic activity in vitro.
  • the compounds of the invention reduced the viability and/or cell proliferation of twenty two different cell lines representative of lung, breast, CNS, colon, prostate, ovary, pancreas, kidney and melanoma neoplasias, including a multidrug resistant cell line. Furthermore, compounds of the invention exhibited stong anti-tumor action in prostate and lung cancer xenografts in vivo.
  • administration includes routes of introducing a compound(s) to a subject to perform their intended function.
  • routes of administration include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal), oral, inhalation, rectal and transdermal.
  • the pharmaceutical preparations are, of course, given by forms suitable for each administration route. For example, these preparations are administered in tablets or capsule form, by injection, inhalation, topical by lotion or ointment; and rectal by suppositories. Oral administration is preferred.
  • the injection can be bolus or can be continuous infusion.
  • the compound can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally effect its ability to perform its intended function.
  • the compound can be administered alone, or in conjunction with either another agent as described above (e.g. another chemotherapeutic agent) or with a pharmaceutically-acceptable carrier, or both.
  • the compound can be administered prior to the administration of the other agent, simultaneously with the agent, or after the administration of the agent.
  • the compound can also be administered in a proform which is converted into its active metabolite, or more active metabolite in vivo.
  • alkyl refers to the radical of saturated aliphatic groups, including straight- chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C 1 -C30 for straight chain, C3-C30 for branched chain), preferably 26 or fewer, and more preferably 20 or fewer.
  • preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 3, 4, 5, 6 or 7 carbons in the ring structure.
  • alkyl as used throughout the specification and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoro
  • alkylaryl is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
  • alkyl also includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six, and most preferably from one to four carbon atoms in its backbone structure, which may be straight or branched-chain.
  • lower alkyl groups include methyl, ethyl, n-propyl, i-propyl, tert-butyl, hexyl, heptyl, octyl and so forth.
  • the term "lower alkyl” includes a straight chain alkyl having 4 or fewer carbon atoms in its backbone, e.g., C 1 -C 4 alkyl.
  • alkoxy refers to an alkyl or a cycloalkyl group which is linked to another moiety though an oxygen atom. Alkoxy groups can be optionally substituted with one or more substituents.
  • alkoxyalkyl refers to alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or sulfur atoms.
  • alkenyl and alkynyl refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond, respectively.
  • the invention contemplates cyano and propargyl groups.
  • ameliorate means to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • alteration refers to a change (increase or decrease) in a parameter as detected by standard art known methods, such as those described herein.
  • aryl refers to the radical of aryl groups, including 5- and 6-membered single- ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like.
  • aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles," “heteroaryls” or “heteroaromatics.”
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, s
  • carcinoma refers to a malignant tumor of potentially unlimited growth that expands locally by invasion and systemically by metastasis.
  • carcinoma is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary.
  • carcinosarcomas e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues.
  • An "adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
  • chiral refers to molecules which have the property of non-superimposability of the mirror image partner, while the term “achiral” refers to molecules which are superimposable on their mirror image partner.
  • Detect refers to identifying the presence, absence or amount of the object to be detected.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • diastereomers refers to stereoisomers with two or more centers of dissymmetry and whose molecules are not mirror images of one another.
  • an effective amount refers to the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient.
  • the effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.
  • a therapeutically effective amount of a compound delineated herein may range from about 0.1 ⁇ g to 20 milligram per kilogram of body weight per day (mg/kg/day) (e.g., O.l ⁇ g/kg to 2mg/kg, 0.3-3 ⁇ g/kg, 0.18-0.54mg/kg). In other embodiments, the amount varies from about 0.1 mg/kg/day to about 100 mg/kg/day. In still other embodiments, the amount varies from about 0.001 ⁇ g to about 100 ⁇ g/kg (e.g., of body weight).
  • mg/kg/day milligram per kilogram of body weight per day
  • the amount varies from about 0.1 mg/kg/day to about 100 mg/kg/day.
  • the amount varies from about 0.001 ⁇ g to about 100 ⁇ g/kg (e.g., of body weight).
  • compounds of the invention are administered in a dose of about 0.1-200mg/kg/day to a mouse, preferably 1-100 mg/kg, more preferably 10-60 mg/kg.
  • a dog receives 0.1-10 mg/kg of compound.
  • a human subject receives 0.1 ⁇ g/kg to 5mg/kg parenteral compound per day.
  • 0.1-lO ⁇ g/kg compound is administered to a human subject.
  • 0.1-1 mg/kg total per day is administered to a human subject.
  • treatment of a subject with a therapeutically effective amount of a compound delineated herein can include a single treatment or, preferably, can include a series of treatments.
  • a subject is treated with a compound delineated herein in the range of between about 0.1 ⁇ g to 20 milligram per kilogram of body weight per day (mg/kg/day) (e.g., 0.1 ⁇ g/kg to lOmg/kg, 0.1-lO ⁇ g/kg, 0.1-lmg/kg).
  • the amount varies from about 0.1 mg/kg/day to about 100 mg/kg/day. In still other embodiments, the amount varies from about 0.001 ⁇ g to about 100 ⁇ g/kg (e.g., of body weight).
  • the dosage is administered one time per day, two times per day, or one time per week. Treatment is carried out for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of a compound delineated herein used for treatment may increase or decrease over the course of a particular treatment.
  • enantiomers refers to two stereoisomers of a compound which are non- superimposable mirror images of one another.
  • An equimolar mixture of two enantiomers is called a “racemic mixture” or a “racemate.”
  • halogen designates -F, -Cl, -Br or -I.
  • haloalkyl is intended to include alkyl groups as defined above that are mono-
  • hydroxyl means -OH.
  • heteroatom as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
  • heteroaryl refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-4 ring heteroatoms if monocyclic, 1- 6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S, and the remainder ring atoms being carbon. Heteroaryl groups may be optionally substituted with one or more substituents.
  • heteroaryl groups include, but are not limited to, pyridyl, furanyl, benzodioxolyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl thiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, triazolyl, thiadiazolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxadiazolyl, and indolyl.
  • heterocyclic refers to organic compounds that contain at least at least one atom other than carbon (e.g., S, O, N) within a ring structure.
  • the ring structure in these organic compounds can be either aromatic or non-aromatic.
  • heterocyclic moeities include, are not limited to, pyridine, pyrimidine, pyrrolidine, furan, tetrahydrofuran, tetrahydrothiophene, and dioxane.
  • isomers or “stereoisomers” refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
  • multidrug resistant refers to a reduced susceptibility to one or more chemotherapeutic agents.
  • P-glycoprotein polypeptide refers to a protein having at least about 85% or more amino acid identity to NCBI Accession No. CAA41558 or a fragment thereof that has ABC transporter activity.
  • MDRl polynucleotide refers to a nucleic acid sequence encoding a P- glycoprotein polypeptide.
  • neoplastic refers to those cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth.
  • a neoplastic disease state may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state.
  • the term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.
  • “Pathologic hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non- pathologic hyperproliferative cells include proliferation of cells associated with wound repair.
  • the language “inhibiting the growth” of the neoplasm includes the slowing, interrupting, arresting or stopping its growth and metastases and does not necessarily indicate a total elimination of the neoplastic growth.
  • modulate refers to increases or decreases in a parameter in response to exposure to a compound of the invention.
  • the common medical meaning of the term “neoplasia” refers to "new cell growth” that results as a loss of responsiveness to normal growth controls, e.g. to neoplastic cell growth.
  • a “hyperplasia” refers to cells undergoing an abnormally high rate of growth. However, as used herein, the term neoplasia generally refers to cells experiencing abnormal cell growth rates. Neoplasias include “tumors,” which may be either benign, premalignant or malignant.
  • obtaining as in “obtaining compound” is intended to include purchasing, synthesizing or otherwise acquiring the compound.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • polycyclyl or “polycyclic radical” refer to the radical of two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings.
  • Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl,
  • polymorph refers to solid crystalline forms of a compound of the present invention or complex thereof. Different polymorphs of the same compound can exhibit different physical, chemical and/or spectroscopic properties. Different physical properties include, but are not limited to stability (e.g., to heat or light), compressibility and density (important in formulation and product manufacturing), and dissolution rates (which can affect bioavailability).
  • Differences in stability can result from changes in chemical reactivity (e.g., differential oxidation, such that a dosage form discolors more rapidly when comprised of one polymorph than when comprised of another polymorph) or mechanical characteristics (e.g., tablets crumble on storage as a kinetically favored polymorph converts to thermodynamically more stable polymorph) or both (e.g., tablets of one polymorph are more susceptible to breakdown at high humidity).
  • changes in chemical reactivity e.g., differential oxidation, such that a dosage form discolors more rapidly when comprised of one polymorph than when comprised of another polymorph
  • mechanical characteristics e.g., tablets crumble on storage as a kinetically favored polymorph converts to thermodynamically more stable polymorph
  • both e.g., tablets of one polymorph are more susceptible to breakdown at high humidity.
  • Different physical properties of polymorphs can affect their processing.
  • prodrug includes compounds with moieties which can be metabolized in vivo. Generally, the prodrugs are metabolized in vivo by esterases or by other mechanisms to active drugs. Examples of prodrugs and their uses are well known in the art (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. ScL 66:1-19).
  • the prodrugs can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Hydroxyl groups can be converted into esters via treatment with a carboxylic acid.
  • prodrug moieties include substituted and unsubstituted, branch or unbranched lower alkyl ester moieties, ⁇ e.g., propionoic acid esters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl esters ⁇ e.g., dimethylaminoethyl ester), acylamino lower alkyl esters ⁇ e.g., acetyloxymethyl ester), acyloxy lower alkyl esters ⁇ e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters ⁇ e.g., benzyl ester), substituted ⁇ e.g., with methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkyl amides, and hydroxy
  • a prophylactically effective anti-neoplastic amount of a compound refers to an amount of a compound delineated herein or otherwise described herein which is effective, upon single or multiple dose administration to the patient, in preventing or delaying the occurrence of the onset of a neoplastic disease state.
  • reference is meant a standard or control condition.
  • subject includes organisms which are capable of suffering from a neoplasia or who could otherwise benefit from the administration of a compound of the invention, such as human and non-human animals.
  • Preferred human animals include human patients suffering from or prone to suffering from a neoplasia, as described herein.
  • non-human animals of the invention includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, and non-mammals, such as non-human primates, also sheep, dog, cow, chickens, amphibians, and reptiles.
  • sulfhydryl or "thiol” means -SH.
  • systemic administration means the administration of a compound(s), drug or other material, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • tautomers refers to isomers of organic molecules that readily interconvert by tautomerization, in which a hydrogen atom or proton migrates in the reaction, accompanied in some occasions by a switch of a single bond and an adjacent double bond.
  • the invention provides a number of targets that are useful for the development of highly specific drugs to treat or prevent a disorder characterized by the methods delineated herein.
  • the methods of the invention provide a facile means to identify therapies that are safe for use in subjects.
  • the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high- volume throughput, high sensitivity, and low complexity.
  • Istaroxime Istaroxime (PST 2744) or (£",Z)-3-(2-Aminoethoxyimino)androstane-6,17-dione is a Na + /K + -ATPase inhibitor.
  • the Na + /K + -ATPase, or Na + /K + pump is a complex of integral membrane proteins that actively transports sodium and potassium ions across the cell plasma membrane.
  • the enzyme functions as a receptor for cardiac glycosides, such as ouabain, digoxin, marinobufagenin and others (reviewed in Mijatovic T et al., Biochim Biophys Acta. 2007; 1776:32-57).
  • Istaroxime is chemically unrelated to cardiac glycosides, which also display Na + /K + - ATPase inhibitory activity.
  • Cardiac glycosides comprise steroidal and glycoside moieties, and have a lactone ring at the Cl 7 position.
  • istaroxime is an aglycone; it has no lactone ring at C 17, but it does have an aminoethoxy-imino moiety at C3. Despite these distinguishing structural features, istaroxime effectively inhibits Na + /K + -ATPase.
  • Istaroxime had an IC 5 O of 0.2 ⁇ M when tested in vitro on purified dog kidney ATPase as compared to 0.4 ⁇ M for the cardiac glycoside digoxin (De Munari S et al., J Med Chem. 2003 Aug 14;46(17):3644-54). Istaroxime has both inotropic and lusitropic properties. Its inotropic potency expressed as ED 80 was 4.84 ⁇ mol/kg when tested in vivo by slow intravenous infusion in anesthetized guinea pigs (compared to 0.41 of digoxin).
  • the inotropic effects of istaroxime are due to its ability to inhibit Na + /K + - ATPase at the sarcolemma, leading to an increase in cytosolic calcium and thus improved contractility.
  • the lusitropic effects are related to istaroxime's ability to stimulate sarcoplasmic reticulum calcium ATPase 2 (SERCA2), leading to rapid sequestration of cytosolic calcium into the sarcoplasmic reticulum (SR) during diastole and thereby promoting myocardial relaxation (Mattera GG et al., Am J Cardiol. 2007 Jan 22;99(2A):33A-40A).
  • Rostafuroxin (PST 2238) or 17 ⁇ -(3-furyl)-5 ⁇ -androstane-3 ⁇ ,14 ⁇ ,17 ⁇ -triol, is a steroidal compound that is chemically unrelated to both cardiac glycosides and istaroxime.
  • Rostafuroxin is an aglycone that has a furyl instead of a lactone ring at C 17, and a hydroxy- moiety at C3.
  • Rostafuroxin belongs to a new class of antihypertensive agents that reduce blood pressure and prevent hypertension-related organ complications by selectively correcting the molecular and functional alterations of the Na + /K + pump (e.g., alterations induced by genetic (i.e., Adducin) and/or hormonal (ouabain) mechanisms) without affecting the normal physiological mechanisms of blood pressure control.
  • Rostafuroxin inhibited purified dog kidney Na+/K+-ATPase with an IC 5 O of 25 ⁇ M, whereas at low K+ concentration (0.5 mM) the IC 5 owas 1.5 ⁇ M.
  • rostafuroxin antagonizes ouabain triggering of the Src-epidermal growth factor receptor (EGFR)-dependent signaling pathway leading to renal Na+/K+-ATPase, and ERK tyrosine phosphorylation and activation.
  • EGFR Src-epidermal growth factor receptor
  • ERK tyrosine phosphorylation and activation In the vasculature, rostafuroxin normalizes the increased myogenic tone caused by nanomolar levels of ouabain. Rostafuroxin has a very high safety ratio.
  • the invention provides a compound of Formula (I)
  • R 1 is H or -OH
  • R 8 is H, alkyl, or -NO 2 ;
  • R 9 is H or formyl
  • R 10 for each occurrence, is the same or different and is H or halogen;
  • R 11 is H or alkyl;
  • R 12 is alkyl or phenyl
  • R 13 is alkyl optionally substituted by a 4 or 6-membered heterocyclic ring, a 5 or 6- membered heteroaryl, amino(Ci_ 4 )alkoxyl, (Ci_ 4 )alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; aminoalkyl optionally substituted by one or more alkyl; or aminoacyl optionally substituted by alkyl;
  • R 14 is aminoalkyl optionally substituted by one or more alkyl or acetyl; alkyl optionally substituted by guanidinyl or a 4- to 6-membered heterocyclic ring; or a 4- to 6-membered heterocyclic ring optionally substituted by alkyl;
  • R 15 and R 16 are aminoalkyl optionally substituted by one or more alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
  • One embodiment of of the methods of the invention provides compounds of Formula (I) wherein R 1 is H. Another embodiment provides that R 1 is -OH.
  • one of R 6 and R 7 is H, and the other is -OH.
  • one of R 6 and R 7 is H, and the other is that is unsubstituted or substituted with a hydroxyl or an amino group. In another embodiment, one of R 6 and R 7 is H, and the other is substituted with an amino group.
  • one of R 6 and R 7 is H, and the other is -OC(O)R 12 .
  • R 12 is In another embodiment, R 12 is phenyl.
  • both of R 4 and R 5 are H. In another embodiment, one of R 4 and R 5 is H, and the other is -OR 8 . In one embodiment, R 8 is H. Another embodiment provides that R 8 is Yet another embodiment provides that R 8 is -NO 2 .
  • one of R 4 and R 5 is H, and the other is that is unsubstituted or substituted with hydroxyl or In certain embodiment, one of R 4 and R 5 is H, and the other is methyl. In one embodiment, one of R 4 and R 5 is H, and the other is -CH 2 OH or -CH 2 OCH 3 .
  • Embodiments of the invention also include compounds of Formula (I) wherein, R 4 and R 5 is H, and the other is -C(O)-NH 2 , -C(O)-O(C r C 4 )alkyl, -NHR 9 or (C 2 -C 4 )alkynyl. Certain embodiments provide that one of R 4 and R 5 is H, and the other is -C(O)-NH 2 , -C(O)-OCH 3 , formylamino or ethynyl.
  • R 11 is H or One embodiment provides that R 11 is methyl.
  • R 4 and R 5 together with the carbon to which they are attached form a cyclopropyl ring.
  • R 4 and R 5 together with the carbon to which they are attached form C N-OH.
  • both of R and R are H.
  • Another embodiment of the invention provides that compounds of Formula (I) wherein one of R 2 and R 3 is H, and the other is -OR 8 . Certain embodiments provide that one of R 2 and R 3 is H, and the other is -OH, -O(C r C 4 )alkyl, or -ONO 2 . One embodiment provides that one of R and R is -OH, and the other is that is u mnssuubbssttiittuutteedd oorr ssuubbstituted with hydroxyl. In one embodiment, one of R 2 and R 3 is -OH, and the other is -CH 2 OH.
  • one of R 2 and R 3 is H, and the other is that is unsubstituted or substituted by hydroxyl.
  • Another embodiment provides that one of R and R is H, and the other is that is unsubstituted or substituted by Embodiments of the invention provide that one of R and R is H, and the other is methyl, CH 2 OH, CH 2 CH 2 OH or CH 2 OCH 3 .
  • One embodiment of the invention provides compounds of Formula (I), wherein one of R 2 and R 3 is H, and the other is -C(O)-NH 2 , -C(O)-O(C r C 4 )alkyl,-NHR 9 or (C 2 -C 4 )alkynyl.
  • Embodiments include compounds of the invention wherein, one of R and R is OH, and the other is -C(O)-NH 2 , -C(O)OCH 3 , -NH 2 , formylamino or ethynyl.
  • R 14 is amino(Ci-C 4 )alkyl that is unsubstituted or substituted by one or more or acetyl. Certain embodiments provide that R 14 is aminoethyl, 2- aminopropyl, 2-amino-2-methylpropyl, 3-amino-2-methylpropyl, 2-amino-dimethylethyl, methylaminoethyl, methylaminopropyl, aminopropyl, aminobutyl, dimethylaminoethyl, or acetylaminoethyl.
  • R 14 is (Ci-C/Oalkyl that is unsubstituted or substituted by guanidinyl or a 4- to 6-membered heterocyclic ring.
  • R 14 is methyl or ethyl, that is further substituted with pyrrolidinyl or guanidinyl.
  • R 14 is a 4- to 6-membered heterocyclic ring that is unsubstituted or substituted by (Ci-C 4 )alkyl. Certain embodiments provide that R 14 is azetidinyl, pyrrolidinyl, piperazinyl, or piperidinyl. Other embodiments provide that R 14 is a methyl-susbstituted pyrrolidinyl or piperazinyl group.
  • A is CH(OR 13 ).
  • R 13 is (Ci-C 4 )alkyl that is unsubstituted or substituted by a 4 or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, amino(Ci_ 4 )alkoxyl, (Ci_ 4 )alkoxyl substituted by a 4- or 6- membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl.
  • R 13 is ethyl or propyl, that is further substituted by pyrrolidinyl, piperidinyl, imidazolyl, dimethylamino-ethoxyl, pyrrolidinyl- ethoxyl, or 4-methylpiperazinyl.
  • R 13 is amino(Ci_ 4 )alkyl that is unsubstituted or substituted by one or more (Ci_ 4 )alkyl. Certain embodiments provide that R 13 is dimethylaminopropyl, dimethylaminoethyl, ethylaminobutyl, or dimethylaminobutyl. In one embodiment, R 13 is amino(Ci_ 4 )acyl that is unsubstituted or substituted by one or more (Ci-C 4 )alkyl. In one embodiment, R 13 is amino(Ci_ 4 )acyl that is substituted by methyl. Certain embodiments provide that R 13 is aminopropionyl, 3-aminobutanionyl, or 3-amino-2-methylpropionyl.
  • A is CH-SR 16 , wherein R 16 is amino(Ci_ 4 )alkyl that is unsubstituted or substituted by one or more (Ci-C 4 )alkyl.
  • R 16 is aminopropyl.
  • A is CH-S(O)R 16 , wherein R 16 is amino(Ci_ 4 )alkyl that is unsubstituted or substituted by one or more (Ci-C 4 )alkyl.
  • R 16 is aminopropyl.
  • the invention provides a compound having Formula (Ia):
  • R 1 is H or -OH
  • R 4 and R 5 are H, and the other is H or -OR 8 ;
  • R 8 is H or optionally substituted (Ci-C 4 )alkyl
  • R 13 is (Ci-C 4 )alkyl optionally substituted by a 4 or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, amino(Ci_C 4 )alkoxyl, (Ci_C 4 )alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; amino(Ci-C 4 )alkyl optionally substituted by one or more (Ci-C 4 )alkyl; or amino(Ci-C 4 )acyl optionally substituted by (Ci- C 4 )alkyl; and R 14 is an amino(Ci-C 4 )alkyl optionally substituted by one or more or a 4- to 6-membered heterocyclic ring optionally substituted by (Ci_C 4 )alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides,
  • R 1 is H. In another embodiment, R 1 is -OH.
  • R 14 is an amino(Ci-C 4 )alkyl that is unsubstituted or substituted by one or more
  • R 14 is a 4- to 6-membered heterocyclic ring that is unsubstituted or substituted by (Ci-C 4 )alkyl.
  • R 14 include aminoethyl and pyrrolidinyl.
  • A is CH(OR 13 ).
  • R 13 is (Ci- C/i)alkyl that is unsubstituted or substituted by a 4 or 6-membered heterocyclic ring.
  • An example of R 13 is 2-(pyrrolidin-l-yl)ethyl.
  • Another aspect of the invention provides a compound of Formula (Ib)
  • R 1 is H or -OH
  • R 4 and R 5 are H, and the other is H or -OR 8 ;
  • R 8 is H or substituted or unsubstituted (Ci-C 4 )alkyl
  • R 10 for each occurrence, is the same or different and is H or halogen
  • R 11 is H or (Ci-C 4 )alkyl
  • R 14 is an amino(Ci-C 4 )alkyl optionally substituted by one or more (Ci-C 4 )alkyl; or a 4- to 6-membered heterocyclic ring optionally substituted by (Ci-C 4 )alkyl;
  • R 15 and R 16 are amino(Ci-C 4 )alkyl optionally substituted by one or more (Ci-C 4 )alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
  • R 1 is H.
  • R 1 is OH.
  • X is C(R 10 ) 2 .
  • Certain embodiments provide compounds of Formula (Ib) wherein R 10 is H.
  • X is N-OR 11 .
  • R 11 is H.
  • R 11 is
  • R 14 is an amino(Ci-C 4 )alkyl that is unsubstituted or substituted by one or more (Ci-C 4 )alkyl.
  • R 14 is a 4- to 6- membered heterocyclic ring that is unsubstituted or substituted by (Ci-C/Oalkyl.
  • Examples of R 14 include aminoethyl and pyrrolidinyl.
  • R 15 is amino(Ci-C 4 )alkyl.
  • Certain embodiments of the methods of the invention provide compounds of Formula (Ib) wherein A is CH-SR 16 . Another embodiment provides that A is CH-S(O)R 16 . Instances provide that R 16 is amino(Ci-C 4 )alkyl.
  • the invention provides a compound of Formula (Ic)
  • R 1 is H or -OH;
  • C One of R 2 and R 3 is H, and the other is optionally substituted by hydroxyl or methoxy;
  • R 4 and R 5 are H and the other is -OR 8 ; or
  • R 8 is H or substituted or unsubstituted (Ci_C 4 )alkyl; and R 14 is an amino(Ci_C 4 )alkyl optionally substituted by one or more or a 4- to 6-membered heterocyclic ring optionally substituted by (Ci_C 4 )alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
  • R 1 is H. Another embodiment provides that R 1 is -OH.
  • One embodiment provides compounds of Formula (Ic) wherein one of R 2 and R 3 is H and the other is that is substituted by hydroxyl or methoxy. Certain embodiments provide that one of R 2 and R 3 is H and the other is -CH 2 OH.
  • one of R 4 and R 5 is H and the other is -OR 8 , wherein R 8 is H, or substituted or unsubstituted (Ci-C/Oalkyl.
  • R 14 is an amino(Cr C/i)alkyl that is unsubstituted or substituted by one or more
  • R 14 is a 4- to 6-membered heterocyclic ring that is unsubstituted or substituted by one or more
  • Examples of R 14 include aminoethyl and pyrrolidinyl.
  • Another aspect of this invention provides compounds of Formula (Id)
  • R 1 is H or -OH
  • R 4 and R 5 taken together with the carbon atom to which they are attached form C(R 10 ) 2 ;
  • R 10 for each occurrence, is the same or different and is H or halogen
  • R 14 is an amino(Ci-C 4 )alkyl optionally substituted by one or more or a 4- to 6-membered heterocyclic ring optionally substituted by and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
  • R 1 is H. In another embodiment, R 1 is -OH.
  • R 10 is H. In another embodiment, R 10 is halogen, preferably, F.
  • R 14 is an amino(Ci-C 4 )alkyl that is unsubstituted or substituted by one or more (Ci-C 4 )alkyl. In another embodiment, R 14 is a 4- to 6-membered heterocyclic ring that is unsubstituted or substituted by (Ci-C 4 )alkyl. Examples of R 14 include aminoethyl and pyrrolidinyl.
  • the methods of the invention include administering a compound of Formula (Ie):
  • R 1 is H or -OH
  • R 13 is (Ci-C 4 )alkyl optionally substituted by a 4 or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, amino(Ci-C 4 )alkoxyl, (Ci-C 4 )alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; amino(Ci-C 4 )alkyl optionally substituted by one or more (Ci-C 4 )alkyl; or amino(Ci-C 4 )acyl optionally substituted by (Ci- C 4 )alkyl;
  • R 14 is an amino(Ci-C 4 )alkyl optionally substituted by one or more (Ci-C 4 )alkyl; or a 4- to 6-membered heterocyclic ring optionally substituted by (Ci-C 4 )alkyl;
  • R 15 and R 16 are amino(Ci-C 4 )alkyl optionally substituted by one or more (Ci-C 4 )alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
  • R 1 is H.
  • the compounds used in accordance with the methods of the invention include the compounds shown in Table 1 as follows:
  • Specific compounds of the invention include the following compounds, the structures of which are shown in Table 2: (1 OR, 13 S)-3 -(2-aminoethoxyimino)- 10,13 -dimethyldodecahydro- 1 Hcyclopenta[a]phenanthrene-
  • the structures of some of the compounds of the invention include asymmetric carbon atoms. Accordingly, the isomers arising from such asymmetry ⁇ e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and/or by stereochemically controlled synthesis.
  • Naturally occurring or synthetic isomers can be separated in several ways known in the art. Methods for separating a racemic mixture of two enantiomers include chromatography using a chiral stationary phase (see, e.g., , "Chiral Liquid Chromatography,” WJ. Lough, Ed. Chapman and Hall, New York (1989)). Enantiomers can also be separated by classical resolution techniques. For example, formation of diastereomeric salts and fractional crystallization can be used to separate enantiomers.
  • the diastereomeric salts can be formed by addition of enantiomerically pure chiral bases, such as brucine, quinine, ephedrine, strychnine, and the like.
  • diastereomeric esters can be formed with enantiomerically pure chiral alcohols, such as menthol, followed by separation of the diastereomeric esters and hydrolysis to yield the free, enantiomerically enriched carboxylic acid.
  • the invention also provides methods for treating a subject for a neoplasia by administering to the subject an effective amount of a compound of formula (I) or otherwise described herein.
  • the subject is a mammal, in particular a human.
  • compounds are administered in combination with a pharmaceutically diluent or acceptable carrier.
  • the compound can be administered using a pharmaceutically acceptable formulation.
  • the pharmaceutically-acceptable carrier provides sustained delivery of the compound to a subject for at least four weeks after administration to the subject.
  • the compound is administered orally. In other embodiments, the compound is administered intravenously. In yet other embodiments, the compound is administered topically. In still other embodiments, the compound is administered topically or parenterally.
  • dosages may vary depending on the particular indication, route of administration and subject, the compounds are typically administered at a concentration of about 0.1 ⁇ g to 20 milligram per kilogram of body weight per day (mg/kg/day) (e.g., 0.1 ⁇ g/kg to 2mg/kg, 0.3-3 ⁇ g/kg, 0.18-0.54mg/kg). In other embodiments, the amount varies from about 0.1 mg/kg/day to about 100 mg/kg/day. In still other embodiments, the amount varies from about 0.001 ⁇ g to about 100 ⁇ g/kg (e.g., of body weight).
  • a therapeutically effective amount or a prophylactically effective amount of a compound described herein can readily by one skilled in the art.
  • the dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated and the particular compound being employed.
  • a number of factors are considered, including, but not limited to: the specific hyperplastic/neoplastic cell involved; pharmacodynamic characteristics of the particular agent and its mode and route of administration; the desired time course of treatment; the species of mammal; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the kind of concurrent treatment (i.e., the interaction of the compounds of the invention with other co-administered therapeutics); and other relevant circumstances.
  • U.S. Patent 5,427,916, for example describes method for
  • a therapeutically effective amount and a prophylactically effective amount of a compound of the invention is expected to vary from about about 0.1 ⁇ g to 20 milligram per kilogram of body weight per day (mg/kg/day) (e.g., O.l ⁇ g/kg to lOmg/kg, 0.1-10 ⁇ g/kg, 0.1-lmg/kg). In other embodiments, the amount varies from about 0.1 mg/kg/day to about 100 mg/kg/day.
  • the amount varies from about 0.001 ⁇ g to about 100 ⁇ g/kg (e.g., of body weight).
  • Compounds which are determined to be effective for the prevention or treatment of neoplasias in animals may also be useful in treatment of neoplasias in humans.
  • Those skilled in the art of treating neoplasias in humans will know, based upon the data obtained in animal studies, the dosage and route of administration of the compound to humans. In general, the dosage and route of administration in humans is expected to be similar to that in animals.
  • Another aspect of the invention comprises obtaining the compound of the invention.
  • the invention features methods for inhibiting the proliferation, growth, or viability of a neoplastic cell by contacting the cells with a compound of formula (I) or otherwise described herein.
  • the method includes a step of contacting a neoplastic cell with an effective amount of a compound of the invention.
  • the present method can be performed on cells in culture, e.g., in vitro or ex vivo, or can be performed on cells present in an animal subject, e.g., as part of an in vivo therapeutic protocol.
  • the therapeutic regimen can be carried out on a human or other subject.
  • the compounds of the invention or otherwise described herein can be tested initially in vitro for their inhibitory effects on the proliferation or survival of neoplastic cells.
  • cell lines that can be used are lung cancer cell lines (e.g., H460, EKVX, A549), breast cancer cell lines (e.g., MCF7, T47D), CNS cancer cell lines (e.g., SF268, U251, SF295), colon cancer cell lines (e.g., HCTl 16, HCT15), prostate (e.g., PC3, DU145), ovarian cancer cell lines (e.g., IGROVl, 0VCAR5, 0VCAR3, NCI-ADRRES), pancreatic cancer cell lines (e.g., SU8686), renal cancer cell lines (e.g., CAKI), and melanoma cancer cell lines (e.g., LOXIMVI, SKMEL28, MB435, UACC62).
  • lung cancer cell lines e.g., H460, EKV
  • the antineoplastic activity of compounds of the invention can be tested in vivo using various animal models known in the art. For example, xenographs of human neoplastic cells or cell lines, such as PC-3 or A549 cells, are injected into immunodef ⁇ cient mice (e.g., nude or SCID) mice. Compounds of the invention are then administered to the mice and the growth and/or metastasis of the tumor is compared in mice treated with a compound of the invention relative to untreated control mice. Agents that reduce the growth or metastasis of a tumor or increase mice survival are identified as useful in the methods of the invention.
  • immunodef ⁇ cient mice e.g., nude or SCID mice
  • Neoplasia growth is typically uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells.
  • Neoplasias can affect a variety of cell types, tissues, or organs, including but not limited to an organ selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestines, kidney, liver, lung, lymph node, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and vagina, or a tissue or cell type thereof.
  • Neoplasias include cancers, such as acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute monocytic leukemia, acute myeloblasts leukemia, acute myelocytic leukemia, acute myelomonocytic leukemia, acute promyelocytic leukemia, acute erythroleukemia, adenocarcinoma, angiosarcoma, astrocytoma, basal cell carcinoma, bile duct carcinoma, bladder carcinoma, brain cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, colon cancer, colon carcinoma, craniopharyngioma, cystadenocarcinoma, embryonal carcinoma, endotheliosarcoma, ependymoma, epithelial carcinoma, Ewing'
  • Multidrug Resistant Neoplasias As exemplified by results obtained with istaroxime, compounds 2 and 4, neoplasias that are resistant or refractory to anti-neoplastic therapies are likely to be susceptible to treatment with the compounds delineated herein. Neoplasias that display resistance to a wide variety of chemotherapeutic agents are described as multidrug resistant. Multidrug resistant neoplasias are characterized by their ability to resist treatment with compounds having diverse structures and mechanisms of action. Multidrug resistance is generally related to alterations in a family of proteins known as ATP -binding cassette (ABC) transporters.
  • ABSC ATP -binding cassette
  • Multidrug resistant neoplasias often display increased expression of ATP-binding cassette (ABC) transporters, which function as ATP-dependent efflux pumps. These pumps actively transport a wide array of anti-cancer and cytotoxic drugs out of the cell.
  • ABC ATP-binding cassette
  • P-gp P-glycoprotein transporters
  • MDR3 genes in human the MRP subfamily, and bile salt export protein (ABCBl 1; Cancer Res (1998) 58, 4160-4167), MDR-3 (Nature Rev Cancer (2002) 2, 48-58), lung resistance protein (LRP) and breast cancer resistant protein (BCRP).
  • BCRP breast cancer resistant protein
  • compounds of the invention are particularly useful for neoplasias showing alterations in the activity or expression of MDRl, MDR2, or P-gP).
  • the drug resistance of the tumor is mediated through the overexpression of P-gp.
  • Therapeutic agents to which resistance is conferred via the action of P-gp include, but are not limited to: vinca alkaloids (e.g., vinblastine), the anthracyclines (e.g., adriamycin, doxorubicin), the epipodophyllotoxins (e.g., etoposide), taxanes (e.g., paclitaxel, docetaxel), antibiotics (e.g., actinomycin D and gramicidin D), antimicrotubule drugs (e.g., colchicine), protein synthesis inhibitors (e.g., puromycin), toxic peptides (e.g., valinomycin), topoisomerase Inhibitors (e.g., topotecan), DNA intercalators (e.g., ethidium bromide) and anti-mitotics. See WO 99/20791.
  • the methods and pharmaceutical compositions of the present invention are useful for treating tumors resistant to any one or more of above- listed
  • the methods of the invention are useful for treating resistant or refractory neoplasias, where the resistance is conferred by an alteration in a topoisomerase (e.g., topoisomerase II), protein kinase C and specific glutathione transferase enzyme.
  • Methods of the invention are also useful for the treatment of neoplasias showing resistance to taxanes (e.g., paclitaxel and docetaxel). Such resistance is typically mediated by alterations in tubulin.
  • compounds delineated herein are useful for treating neoplasias that are refectory to platinum-based chemotherapeutic agents, including carboplatin, cisplatin, oxaliplatin, iproplatin, tetraplatin, lobaplatin, DCP, PLD-147, JMl 18, JM216, JM335, and satraplatin.
  • platinum-based chemotherapeutic agents also include the platinum complexes disclosed in EP 0147926, U.S. Pat. No. 5,072,011, U.S. Pat. Nos.
  • the methods and pharmaceutical compositions of the invention are generally useful for treating resistant and/or refractory neoplasias to any one or more of drugs known in the art or described herein.
  • the methods and compositions of the invention are useful for the treatment of patients having end-stage disease, which includes patients for whom no effective therapeutic regimen exists or patients identified as having less than about 3, 6, 9 or 12 months to live.
  • the compounds of the invention are administered in combination with any other standard anti-neoplasia therapy or conventional chemotherapeutic agent, such as an alkylating agent; such methods are known to the skilled artisan and described in Remington's Pharmaceutical Sciences by E. W. Martin.
  • agents of the invention e.g., istaroxime
  • any conventional antineoplastic therapy including but not limited to, surgery, radiation therapy, or chemotherapy.
  • chemotherapeutic agents include, but are not limited to, abiraterone, alemtuzumab, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, bicalutamide, busulfan, capecitabine, carboplatin, carmustine, celecoxib, chlorambucil, 2- chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, Cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, estramustine phosphate, etodolac, etoposide, exemestane, floxuridine, fludarabine, 5-fluorouracil, flutamide, formestane, gemcitabine, gentuzumab, goserelin, hexamethyl
  • a combination of the invention comprises any one or more of the following: vinca alkaloids (e.g., vinblastine), taxanes (e.g., paclitaxel, docetaxel), epothilones (e.g., ixabepilone), antifolates (e.g., Methotrexate), purine analogs (e.g., fludarabine), pyrimidine analogs (e.g., gemcitabine), DNA intercalators (e.g., ethidium bromide), topoisomerase Inhibitors (e.g., topotecan), alkylating agents (e.g., carmustine, bendamustine), platinum-based agents (e.g., cisplatin, oxaliplatin), receptor antagonists (e.g, atrasentan), hormone agents (e.g.
  • vinca alkaloids e.g., vinblastine
  • taxanes e.g., paclitaxel, do
  • anti-androgens aromatase inhibitors
  • anthracyclines e.g., adriamycin, doxorubicin
  • epipodophyllotoxins e.g., etoposide
  • antibiotics e.g., actinomycin D and gramicidin D
  • antimicrotubule drugs e.g., colchicine
  • protein synthesis inhibitors e.g., puromycin
  • toxic peptides e.g., valinomycin
  • enzyme inhibitors e.g. CDK inhibitors
  • compositions for the treatment of a neoplasia comprising an effective amount a compound of the invention and a pharmaceutically acceptable carrier.
  • compositions of the invention comprise a compound described herein in combination with a conventional chemotherapeutic agent.
  • such compositions are labeled for the treatment of cancer.
  • the effective amount is effective to reduce the growth, proliferation, or survival of a neoplastic cell or to otherwise treat or prevent a neoplasia in a subject, as described herein.
  • the compound is administered to the subject using a pharmaceutically-acceptable formulation.
  • these pharmaceutical compositions are suitable for oral or parenteral administration to a subject.
  • the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non- aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
  • the subject for example, drenches (aqueous or non- aqueous solutions
  • the methods of the invention further include administering to a subject a therapeutically effective amount of a compound in combination with a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable refers to those compounds of the invention, compositions containing such compounds, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable excipient includes pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, solvent or encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • a liquid or solid filler such as a liquid or solid filler, diluent, carrier, solvent or encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydrox
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulf ⁇ te, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulf ⁇ te, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (
  • compositions containing a compound(s) include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration.
  • the compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
  • compositions include the step of bringing into association a compound(s) with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a compound with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • compositions of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound(s) as an active ingredient.
  • a compound may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example,
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical- formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of the compound(s) include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsif ⁇ ers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • the oral compositions can include adjuvants, such as wetting
  • Suspensions in addition to the active compound(s) may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compound(s) with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
  • compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of a compound(s) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound(s) may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to compound(s) of the present invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound(s), excipients, such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • the compound(s) can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound.
  • a nonaqueous (e.g., fluorocarbon propellant) suspension could be used.
  • Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound.
  • an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically-acceptable carriers and stabilizers.
  • the carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (T weens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids, such as glycine, buffers, salts, sugars or sugar alcohols.
  • Aerosols generally are prepared from isotonic solutions.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound(s) to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the active ingredient across the skin.
  • the rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active ingredient in a polymer matrix or gel.
  • Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
  • compositions of this invention suitable for parenteral administration comprise one or more compound(s) in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • microorganisms Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions.
  • isotonic agents such as sugars, sodium chloride, and the like into the compositions.
  • prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally-administered drag form is accomplished by dissolving or suspending the drag in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of compound(s) in biodegradable polymers, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • biodegradable polymers such as polylactide-polyglycolide.
  • Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • the compound(s) When the compound(s) are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to
  • the compound(s), which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels and time course of administration of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • An exemplary dose range is from about about 0.1 ⁇ g to 20 milligram per kilogram of body weight per day (mg/kg/day) (e.g., O.l ⁇ g/kg to 10mg/kg, 0.1-lO ⁇ g/kg, 0.1-1 mg/kg).
  • the amount varies from about 0.1 mg/kg/day to about 100 mg/kg/day.
  • the amount varies from about 0.001 ⁇ g to about 100 ⁇ g/kg (e.g., of body weight). Ranges intermediate to the above-recited values are also intended to be part of the invention.
  • kits for the treatment or prevention of neoplasia includes a therapeutic or prophylactic composition containing an effective amount of a compound of the invention in unit dosage form.
  • a compound of the invention is provided in combination with a conventional chemotherapeutic agent.
  • the kit comprises a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • a compound of the invention is provided together with instructions for administering the compound to a subject having or at risk of developing neoplasia.
  • the instructions will generally include information about the use of the composition for the treatment or prevention of neoplasia.
  • the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of ischemia or symptoms thereof; precautions; warnings; indications; counter- indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • the combined organic extracts were dried over NaCl and Na 2 SC> 4 , filtered, and evaporated to dryness.
  • the crude product was crystallized from EtOAc/MeOH (2/1, 10 mL/g) to give a first crop as a white solid (12.5 g).
  • the mother liquors were evaporated and the residue crystallized from EtOAc/MeOH (2/1, 10 mL/g) to give a second crop as a white solid (6.4 g).
  • the procedure was repeated to give a third crop as a white solid as compound al (2.1 g; 75% overall yield).
  • the HCl salt of Compound (5) was prepared as described in Example 1 for preparation of compound (1): starting from 6-(E)- hydroxyiminoandrostane-3,17-dione (compound (c2)) (60 mg) and 2-aminoethoxyamine dihydrochloride (Intermediate (I))(28 mg).
  • the crude product was crystallized from acetone to give the title compound as a white solid (20 mg, 26%).
  • H NMR 600 MHz, CDCl 3 ) ⁇ 4.17 (bs, 2H), 3.71 (m, IH), 3.48 (m, IH), 3.20 (m, 3H), 2.50 - 1.10 (m, 18H), 0.88 (s, 6H).
  • 6(E)-Methoxyiminoandrostane-3,17-dione (compound (c4)) was prepared in 75% yield from 3,3:17,17-bis(ethylendioxy)-6(E)-methoxyiminoandrostane (compound (c3)) by the above procedure for the preparation of 6-methyleneandrostane-3, 17-dione (compound (b6)).
  • the combined organic extracts were washed with H 2 O, dried over Na 2 S ⁇ 4 and evaporated to dryness.
  • the HCl salt of Compound (6) was prepared in 30% yield as described in Example 1 for the preparation of compound (1), starting from 6-(E)- methoxyiminoandrostane-3,17-dione
  • HCl salt of Compound (8) Preparation of HCl salt of Compound (8)
  • the HCl salt of the title compound (Compound (8)) was prepared according to the procedure described in Example 1 for the preparation of compound (1) and starting from 5a- hydroxy-6-methylenandrostane-3,17-dione (compound d5) (200 mg) and 2-aminoethoxyamine dihydrochloride (Intermediate (I)) (94 mg).
  • the combined organic layers were washed with brine, dried over Na 2 SC ⁇ and the solvent evaporated to dryness.
  • the solid obtained was washed with EtOAc/Et 2 O. After drying under vacuum overnight HCl salt of Compound (8) was obtained (120 mg, 46%).
  • the fumaric acid salt of the title compound (Compound (9)) was prepared in 62% yield according to the procedure described in Example 1 for the preparation of compound (1), starting from 6 ⁇ -hydroxymethylandrostane-3,7,17-trione (compound e4) and 2-aminoethoxyamine- dihydrochloride (Intermediate (I)).
  • the combined organic extracts were dried over Na 2 S ⁇ 4 , filtered and evaporated to dryness.
  • the residue was purified by flash chromatography (SiO 2 , DCM/MeOH/26% NH 4 OH 90/10/1 ) to afford the title compound as free amine. To the concentrated fractions a stoichiometric amount of fumaric acid in MeOH was added.
  • HCl salt of the title compound (Compound (H)) was prepared according to the procedure described in Example 2, and starting from 6 ⁇ -hydroxymethyl-7 ⁇ -hydroxyandrostane- 3,17-dione (compound fl) (49 mg) and 3-(R)-pyrrolidinyloxyamine dihydrochloride (Intermediate (II)) (25 mg). After 2 hours at room temperature, NaCl was added and stirred for 15 min. The mixture was extracted with THF (3x) and the combined organic phases were washed with brine, dried over Na 2 SO/), and filtered.
  • 3,3:17,17-Bis(ethylendioxy)-7-methyleneandrostane was prepared in 62% yield from 3,3:17,17-bis(ethylendioxy)androstane-7-one (compound h4) by the procedure described above in Example 4 for the preparation of 3,3 : 17, 17-bis(ethylendioxy)-6- methyleneandrostane (compound b5).
  • l H NMR 600 MHz, CDCl 3 ): ⁇ 4.67 (s, IH), 4.60 (s, IH), 3.96-.3.60 (m, 8H), 2.02-0.75 (m, 20H), 0.94 (s, 3H), 0.86 (s, 3H).
  • n-Pentane was distilled off and after heating at 80 0 C for 18 h the mixture was quenched with H 2 O and extracted with CH 2 CL. (3x). The combined organic extracts were dried over Na 2 S ⁇ 4 , and evaporated to dryness. The residue was purified by flash chromatography (Si ⁇ 2 , cyclohexane/Et 2 O 70/30) to give 3,3:17,17-bis(ethylendioxy)-6-difluoro- methyleneandrostane (compound h7) (231 mg, 85%).
  • 6-Difluoromethyleneandrostane-3,17-dione (compound h8) was prepared in 99% yield from 3,3:17,17- bis(ethylendioxy)-6-difluoromethyleneandrostane (compound h7) (by the procedure described above in Example 4 for the preparation of 6-methyleneandrostane-3, 17- dione (compound b6).
  • the combined organic extracts were washed with H 2 O, dried over Na 2 SO 4 and evaporated to dryness.
  • 1 H NMR 300 MHz, acetone-d 6 ): ⁇ 2.85-0.95 (m, 20H), 1.12 (s, 3H), 0.88 (s, 3H).
  • Na+/K+-ATPase inhibitory activity of the different compounds Compound 1 (Istaroxime) inhibited the Na + /K + -ATPase with an IC 5 O of 0.2 ⁇ M when tested in vitro on purified dog kidney ATPase (De Munari S et al., J Med Chem. 2003 Aug 14;46(17):3644-54).
  • rostafuroxin inhibited purified dog kidney Na+/K+-ATPase with an IC 5 O of 25 ⁇ M.
  • the IC 5 O was 1.5 ⁇ M (Ferrari P et al, J Pharmacol Exp Ther. 1998 Apr;285(l):83-94).
  • Table 3 summarizes the Na+/K+- ATPase inhibitory activity of Compound Nos. 1-21. Ouabain and digoxin, two well known cardiotonic Na+/K+-ATPase inhibitors were used as positive controls.
  • Compound 1 (Istaroxime) inhibited the proliferation and survival of neoplastic cells
  • Istaroxime was a potent inhibitor of cell proliferation and viability.
  • the proliferation/viability of all cell lines was completely abolished in the presence of ouabain, digoxin and istaroxime.
  • rostafuroxin a well established Na + K + - ATPase inhibitor, showed no effect in vitro.
  • Na + K + - ATPase inhibitors are not equally effective in inhibiting cell proliferation and viability.
  • Na + K + - ATPase inhibitors have selective anti-proliferative / growth inhibitory actions.
  • FIGS 2A and 2B summarize results characterizing the effects of Compounds 1-21 and rostafuroxin on GI50, which measures the cell growth inhibitory power of the compound, TGI, which measures cytostatic effect (TGI) and LC50, which signifies cytotoxic effect.
  • TGI cell growth inhibitory power of the compound
  • TGI cytostatic effect
  • LC50 cytotoxic effect
  • SRB sulforhodamine B
  • SRB provides a colorimetric measure of a compound's anti-cancer activity.
  • SRB assays were performed with compounds of the invention and rostafuroxin in 22 different cell lines, which represent nine different tumor panels (lung, breast, CNS, colon, prostate, ovary, pancreas, kidney and melanoma). Ouabain and digoxin were used as controls.
  • Paclitaxel was added as a non- Na + K + - ATPase inhibitor control.
  • NCI- ADR-RES NCI- ADR-RES cells express high levels of MDRl and P-glycoprotein. Consistent with the results reported above, compounds 1 , 2 and 4 showed significant anti-tumor activity in this line. Most importantly, compounds 1 and 2 exhibited comparable GI50 values in multi-drug resistant NCI-ADR-RES cells, as well as in non-resistant MCF-7 cells. These results indicate that compounds of the invention are just as effective in cells that are multidrug resistant as they are in other cancers. These results further indicate that compounds of the invention are effective chemotherapeutic agents for the treatment of refractory and/or multidrug resistant tumors in subjects.
  • FIG. 3 presents data on the anti-tumor activity of compound 1 (istaroxime) and compound 4 in PC-3 prostate xenografts as well as in A549 lung xenografts (compound 4 only).
  • compound 4 For what concerns toxic effects of the compounds, one drug related death occurred with compound 4 at the highest dose (25mgk; dpi 34). Overall, compound 4 induced body weight loss which remained within acceptable limits (weight loss was less than 15% of the initial body weight). Notably, in the same experiment, 2 paclitaxel-treated animals died at dpi 16 and 27 in the corresponding group (paclitaxel used as a positive control).
  • mAR membrane androgen receptor
  • the membrane androgen receptor is capable of transmitting rapid (non-genomic) androgen signals resulting in robust actin cytoskeleton re- organization in membrane androgen expressing cells (Papadopoulou et al, IUBMB Life. 2009 Jan;61(l):56-61).
  • mAR may comprise a membrane-bound form of the classical, pro-oncogenic intracellular androgen receptor (AR).
  • testosterone-albumin conjugates have been shown to possess significant anti-cancer activity in various in vitro and in vivo models, independent of the status of the classical androgen receptor (reviewed in Papadopoulou et al, IUBMB Life. 2009 Jan;61(l):56-61, see also PCT/IB03/02785). Similar results have been reported for dihydro-testosterone bovine serum albumin (DHT-BSA) conjugates in C6 cells (Gatson et al, Endocrinology 2006, 147:2028-2034).
  • DHT-BSA dihydro-testosterone bovine serum albumin
  • Testosterone-HSA-FITC (TAC-FITC) conjugates showed clear membrane staining. This staining was absent from control (HSA-FITC) treated cells.
  • Cells pre-treated with testosterone 3-(O- carboxymethyl)oxime (Testo-CMO; the steroid moiety present in TAC-FITC conjugates) were devoid of membrane staining, which indicates efficient competition for binding to the membrane androgen receptor.
  • istaroxime pre-treated cells Figure 4A
  • compound 4 pre- treated cells Figure 4B
  • the other Na + K + ATPase inhibitor used in this experiment, rostafuroxin ( Figure 4A) failed to block binding of TAC-FITC to mAR.
  • Testosterone-HSA-FITC 39.2 mg of HSA-FITC conjugate respectively.
  • All cell lines were obtained from the American Type Culture Collection (Manassas, VA) or the National Cancer Institute, NIH (Bethesda, MD, USA) and were adapted to grow in the commerially available culture media RPMI 1640, which was supplemented with 25 mM HEPES, 2 mM L-Glutamine, 5-10% fetal bovine serum and antibiotics in a 5% CO 2 humidified atmosphere (100%) at 37°C.
  • NCI-ADR-RES cells have been classified as ovarian cancer cells by the National Cancer Institute.
  • MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide
  • the medium was aspirated and MTT dissolved in RPMI 1640 w/o phenol red was added to each well to a final concentration of 0.1 mg/ml (HCTl 16) or 0.25 mg/ml (PC3, DU145 cells).
  • HCTl 16 cells two hours incubation in the dark (37°C, 5% CO 2 ) the supernatant was discarded.
  • the converted dye blue formazan crystals was solubilized by adding 200 ⁇ l dimethylsulfoxide to each well. Absorbance was measured at 550 ran with reference at 655 ran using a spectrophotometer with the respective filters. All assays were performed in triplicate.
  • IC50 values of the compounds of the invention were incubated with 5 concentrations of each compound (typically, 10OuM, lOuM, IuM, 10OnM, 1O nM) for forty weight hours before processing the samples as described above. IC50 values were then calculated using the Origin® program software (OriginLab, Northampton, MA). Note that calculated IC50 values based on the MTT assay may differ from GI50 values calculated with the SRB assay (see below) due to inherent difference in assay methodologies.
  • human tumor cells from different cancer panels were seeded into 96 well plates in 100 ⁇ L (plating densities ranging from 5-40,000 cells/well depending on the doubling time of individual cell lines) in serum-containing media for twenty- four hours prior to the addition of the compounds to be assayed. After 24 hours, one plate of each cell line was fixed in situ with trichloroacetic acid (TCA). The fixed slides were used to determine the cell population for each cell line at the time of drug addition (Tz). Each compound to be tested was solubilized in DMSO and the desired concentration was then added to the medium and diluted serially 1 :2, 1 :4 or 1 :10 to provide a total often drug concentrations plus control (in a final volume of 200 ⁇ L).
  • TCA trichloroacetic acid
  • the starting dose before any dilution was 100 uM for all compounds.
  • each culture was incubated for forty-eight hours.
  • the assay was terminated with the addition of cold TCA.
  • the supernatant was discarded, and the plates were washed with tap water and air dried.
  • Sulforhodamine B (SRB) solution was then added to each well. After staining, the bound stain was solubilized and the absorbance was read on an automated plate reader at a wavelength of 515 nm.
  • the LC50 concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning
  • PC-3 cells prostate adenocarcinoma, grade IV
  • A549 cells lung carcinoma
  • Optimal DT/DC value was used as a measure of drug activity. Losses of weight, neurological disorders and behavioral and dietary changes were also recorded as indicators of toxicity (side effects). The experiment was terminated when tumor size in untreated animals reached a volume of about 1000-1500 mm 3 . All animals were treated according to Greek laws (2015/92), guidelines of the European Union and the European council (86/609 and ETS123, respectively), and Compliance with Standards for Human Care and Use of Laboratory Animals, NIH, USA (Assurance No. A5736-01). PC-3 experiments
  • mice were randomly divided into 2 groups of 6 animals as described below:
  • Group B 40 mgk Compound 1 treated animals (WFI)
  • Istaroxime was administered intraperitoneallly at the following dpis: dl4, dl5, dl6, dl7, d21, d22, d23 and d24.
  • mice were randomly divided into 4 groups of 6 animals as described below:
  • Group B paclitaxel 20mgk (Cremophor/EtOH/WFI)
  • Group C 25 mgk Compound 4 treated animals (WFI)
  • Group D 17mgk Compound 4 treated animals (WFI)
  • Compound 4 was administered intraperitoneallly at the following dpis: Group B: dl3, dl4, dl5, d20, d21, d22, d27, d28, d29 Group C: once daily from dl3 to d30
  • Paclitaxel was administered intraperitoneallly at dpi 13, 17, 21, 24, 28
  • mice In order to develop A549 xenografts, female mice 6-8 weeks old were inoculated bilateral at the two axilla regions at day 0. Mice were observed for the development of tumors every two days by eye and palpation. At day 20 post inoculation (dpi), mean tumor volume was measured at 117.6 mm 3 . Subsequenbtly, mice were randomly divided into groups of 6 animals as described below: Group A: untreated animals
  • Group B paclitaxel 18 mgk (Cremophor/EtOH/WFI)
  • Group D 15 mgk Compound 4 treated animals (WFI)
  • Group E 10 mgk Compound 4 treated animals (WFI)
  • Compound 4 was administered intraperitoneallly at the following dpis:
  • Group B d20, d21, d22, d23, d24, d27, d28, d34, d35, d38, d41, d42, d45, d48, d49, d50, d51, d52
  • Group C d20, d21, d22, d23, d24, d27, d28, d29, d34, d35, d36, d37, d38, d41, d42, d43, d44, d45, d48, d49, d50, d51, d52
  • Group D [(QDx5;2) x5] starting at dpi20
  • Paclitaxel was administered intraperitoneallly at dpi 21, 24, 28, 31, 35, 38, 42, 45, 49, 52
  • LNCaP or DUl 45 prostate cancer cells were cultured on 0.1 % gelatin-coated glass coverslips in RPMI 1640 medium supplemented with 2 mM L-
  • Glutamine, 10 % Fetal Bovine Serum and 1% Penicillin/Streptomycin for at least 48 hours until reaching 70% confluency.
  • cells were incubated with 40 ⁇ M testosterone-H SA-FITC for 1 hour at room temperature. The cells were then washed three times with phosphate buffered saline (PBS) and fixed with 3.7% formaldehyde in PBS for 5 minutes at room temperature.
  • PBS phosphate buffered saline
  • Human serum albumin-FITC was used as a control for background staining. After permeabilization with ice-cold acetone for 4 minutes at room temperature, cell nuclei were stained with DAPI.
  • Coverslips were mounted on slides by using the Slow Fade/Antifade Reagent (Molecular Probes) and studied under a LEICA DMLB microscope, equipped with the appropriate fluorescence filters and a Leica DC 300F camera. Specimens were analyzed using the Leica FW4000 computer program.

Abstract

The invention features compositions and methods useful for the treatment of neoplasia. In particular embodiments, the compounds of the invention are useful for the treatment of multidrug resistant neoplasia.

Description

METHODS FOR TREATING NEOPLASIA
RELATED APPLICATION This application claims priority to U.S. provisional application Ser. No. 61/219,327, filed June 22, 2009, and the subject matter described herein is related to the subject matter described in U.S. provisional application Ser. No. 61/240,540, filed September 8, 2009. The entire disclosures of these applications are incorporated herein by this reference.
BACKGROUND OF THE INVENTION
Approximately one million Americans are diagnosed with neoplasia every year, and about half a million people in the United States die of the disease annually. Although improvements in neoplasia detection, diagnosis, and treatment have increased the survival rate for many types of neoplasia, only about 60 percent of people diagnosed with neoplasia are alive five years after treatment, making neoplasia the second leading cause of death in the United States. One of the reasons for this poor long term survival rate is that many patients develop multidrug resistant neoplasias. After several cycles of chemotherapy, some tumor cells become resistant not only to the agent used in the chemotherapy, but also to compounds with different structures and mechanisms of action. It is believed that the ATP binding cassette superfamily of transporter proteins acts as an energy- dependent drug efflux pump and alterations in these transporter proteins are associated with the development of multi-drug resistant neoplasias. The activity of this family of proteins prevents the intracellular accumulation of a broad range of cytotoxic drugs.
SUMMARY OF THE INVENTION
As described below, the invention features methods for the treatment of neoplasias, including multidrug resistant neoplasias.
In one aspect, the invention provides a method of treating or preventing neoplasia in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of a compound of Formula (I)
Figure imgf000003_0001
wherein:
R1 is H or -OH;
One of R2 and R3 is H or OH, and the other is H; -OR8; alkyl optionally substituted with hydroxyl or alkoxy; -C(O)-NH2; -C(O)-O-alkyl; -NHR9; alkynyl; or R2 and R3 together with the carbon to which they are attached form C=O, C=C(R10)2, or C=N-OR11;
One of R4 and R5 is H, and the other is H; -OR8; alkyl optionally substituted with hydroxyl or alkoxy; -C(O)-NH2; -C(O)-O-alkyl; -NHR9; alkynyl; or R4 and R5 together with the carbon to which they are attached form C=O, C=C(R10)2, C=N-OR11, or a cyclopropyl ring; One of R6 and R7 is H, and the other is -OH; alkoxy optionally substituted with a hydroxyl or an amino group; optionally substituted heterocyclic or heteroaryl; or -OC(O)R12; or R6 and R7 together with the carbon atom to which they are attached form C=O;
R8 is H, alkyl, or -NO2;
R9 is H or formyl; R10, for each occurrence, is the same or different and is H or halogen;
R11 is H or alkyl;
R12 is alkyl or phenyl;
A is CH(OR13), C=N-OR14, CH-CH=N-OR15, C=CH-CH=N-OR15, CH-CH=CH-R15, CH-SR16, or CH-S(O)R16; R13 is alkyl optionally substituted by a 4 or 6-membered heterocyclic ring, a 5 or 6- membered heteroaryl, amino(Ci_4)alkoxyl, (Ci_4)alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; aminoalkyl optionally substituted by one or more alkyl; or aminoacyl optionally substituted by alkyl;
R14 is aminoalkyl optionally substituted by one or more alkyl or acetyl; alkyl optionally substituted by guanidinyl or a 4- to 6-membered heterocyclic ring; or a 4- to 6-membered heterocyclic ring optionally substituted by alkyl;
R15 and R16, each independently, are aminoalkyl optionally substituted by one or more alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
One embodiment of the methods of the invention provides compounds of Formula (I) wherein R6 and R7 together with the carbon to which they are attached form C=O. Another embodiment provides compounds of Formula (I) wherein R1 is H. Another embodiment provides administering to the subject compounds of Formula (I) wherein R4 and R5 are both H. Certain embodiments provide administering compounds of Formula (I) wherein R and R3 together with the carbon to which they are attached form C=O.
In one embodiment, the method of the invention comprises administering compounds of Formula (I) wherein A is C=N-OR14.
Certain embodiments provide that R14 is amino(Ci_C4)alkyl, that is further optionally substituted by one or more
Figure imgf000004_0001
groups. In some instances, R14 is selected from the group consisting of aminoethyl, 2-aminopropyl, 2-amino-2-methylpropyl, 2-amino- dimethylethyl, methylaminoethyl, methylaminopropyl, aminopropyl, aminobutyl, dimethylaminoethyl, and acetylaminoethyl.
Another embodiment provides that R14 is
Figure imgf000004_0002
that is further substituted by a 4- or 6-membered heterocylic ring or by a guanidinyl group. Examples of R14 include pyrrolidinyl- substituted methyl and guanidinylethyl.
Embodiments of the methods of the invention also provide compounds of Formula (I) wherein R14 is a 4- to 6-membered heterocyclic ring, that is optionally substituted by (Ci-
C4)alkyl. In certain embodiments, R14 is azetidinyl, pyrrolidinyl, or piperidinyl, wherein the azetidinyl, pyrrolidinyl, or piperidinyl group is further optionally substituted by a methyl group.
The invention also provides a method of treating or preventing neoplasia, which comprises administering to a subject in need thereof compounds of Formula (I) wherein A is CH(OR13). In one embodiment, R13 is amino(Ci_C4)alkyl, which is optionally substituted by one or more Certain examples of R13 include dimethylaminopropyl, dimethylaminoethyl, ethylaminobutyl, and dimethylaminobutyl.
In another embodiment, R13 is ethyl or propyl, and that the ethyl or propyl group is further substituted by a substituent selected from the group consisting of pyrrolidinyl, piperidinyl, imidazolyl, dimethylamino-ethoxyl, pyrrolidinyl-ethoxyl, and 4-methylpiperazinyl.
Certain embodiments of the invention provide compounds of Formula (I) wherein A is CH-CH=N-OR15 OrC=CH-CH=N-OR15. Examples of R15 include aminoethyl and dimethylaminoethyl.
Other embodiments of the methods of the invention provide compounds of Formula (I), wherein A is CH-S(O)R16. R16 can be, but is not limited to, aminopropyl. The methods of the invention also include administering to a subject identified as in need thereof compounds of Formula (I) wherein one of R2 and R3 is H, the other is -OH. One embodiment provides compounds of Formula (I) wherein A is C=N-OR14. Examples of R14 include, but are not limited to, aminoethyl, methylaminopropyl and pyrrolidinyl. Another embodiment provides compounds of Formula (I) wherein A is CH(OR13).
The aforementioned R13 can be, but is not limited to, aminoethyl, methylaminoethyl and dimethylaminopropyl.
Other embodiments of the invention provide administering to the subject compounds of Formula (I) wherein R and R together with the carbon to which they are attached form C=CH2. One embodiment provides compounds of Formula (I) wherein A is C=N-OR14. Certain embodiments provide that R14 is amino(Ci_C4)alkyl that is optionally substituted by one or more (Ci-C4)alkyl groups. Another embodiment provides that R14 is a 4- to 6-membered heterocyclic ring, that is optionally substituted by (Ci-C/Oalkyl. Examples of R14 include, but are not limited to, aminoethyl, aminopropyl, methylaminopropyl, 2-aminopropyl, methylaminoethyl, 3-amino- 2-methylpropyl, and pyrrolidinyl.
Another embodiment provides compounds of Formula (I) wherein A is CH(OR13). And R13 can be, but is not limited to, aminopropionyl, 3-aminobutanionyl, or 3-amino-2- methylpropionyl.
Embodiments of the methods of the invention also provide compounds of Formula (I) wherein A is CH-CH=CH-R15. And R15 can be, but is not limited to, aminopropyl and aminoethyl.
Other embodiments provide compounds of Formula (I) wherein A is CH-SR16. Examples of R16 include, but are not limited to, aminopropyl.
The methods of the invention also include administering to the subject compounds of Formula (I) wherein R2 and R3 together with the carbon to which they are attached form C=CF2. One embodiment provides compounds of Formula (I) wherein A is CH-S(O)R16. Examples of R16 include, but are not limited to, aminopropyl. In another embodiment, the compounds of Formula (I) have A being C=N-OR14. In some embodiments, R14 is pyrrolidinyl or aminoethyl.
Another embodiment of the invention provides that the methods comprise administering compounds of Formula (I) wherein R2 and R3 together with the carbon to which they are attached form C=N-OR11. Certain embodiments provide that R11 is H or methyl.
In one embodiment, the compounds of Formula (I) have A being C=N-OR14. The R14 group can be, but is not limited to, aminoethyl, 2-aminopropyl, 2-amino-dimethylethyl, and 3- amino-2-methylpropyl. Another embodiment provides that A is CH-S(O)R16. And R16 can be, but is not limited to, aminopropyl. Other embodiments also provide that A is CH-CH=CH-R15, of which R15 can be, but is not limited to, aminopropyl. Certain embodiments of the methods of the invention include administering to the subject compounds of Formula (I) wherein one of R2 and R3 is H, the other is methyl. In one embodiment, the compounds of Formula (I) have A being C=N-OR14. The R14 group can be, but is not limited to, aminoethyl, aminopropyl, methylaminoethyl, methylaminopropyl, 3-amino-2- methylpropyl, and 2-aminopropyl. In another embodiment, the compounds of Formula (I) have A being CH(OR13). The R13 group can be, but is not limited to, aminopropionyl, 3- aminobutanionyl, and amino-2-methylpropionyl. Another embodiment provides that A is CH- SR16 or CH-S(O)R16, wherein R16 can be, but is not limited to, aminopropyl. Embodiments of the invention also provide compounds of Formula (I) wherein A is CH-CH=CH-R15. Examples of R15 include aminopropyl and aminoethyl.
Other embodiments of the invention include administering to the subject compounds of Formula (I) wherein one of R2 and R3 is H and the other is CH2OH, CH2CH2OH or CH2OCH3. One embodiment provides that A is CH-S(O)R16. In certain instances, R16 is aminopropyl.
In one embodiment, the methods of the invention comprise administering to the subject compounds of Formula (I) wherein one of R2 and R3 is H, and the other is -C(O)NH2, -ONO2, formylamino, ethynyl, or methoxycarbonyl. In one embodiment, the compounds of Formula (I) have A being CH-S(O)R16, wherein R16 can be, but is not limited to, aminopropyl. In another embodiment, A is C=N-OR14, when one of R2 and R3 is H and the other is ethynyl, formylamino, or methoxycarbonyl. This R14 group can be, but is not limited to, aminoethyl or pyrrolidinyl. Other embodiments of the invention also provide compounds of Formula (I) wherein one of R4 and R5 is H, and the other is -OH. In one embodiment, compounds of Formula (I) have both of R and R being H. In certain instances, the compounds of Formula (I) have A being CH- S(O)R16. One example of the R16 group is aminopropyl .
The methods of the invention also include administering compounds of Formula (I) wherein one of R2 and R3 is H, the other is -CH2OH. One of the embodiments provides compounds of Formula (I) wherein A is C=N-OR14. Examples of the R14 group include, but are not limited to, aminopropyl, aminoethyl, methylaminopropyl, amino-2 -methylpropyl, 2-amino- dimethylethyl, and pyrrolidinyl. Another embodiment provides compounds of Formula (I) wherein A is CH(OR13). The R13 group can be, but is not limited to, aminopropionyl, 3- aminobutanionyl, or amino-2-methylpropionyl. Further, the compounds of Formula (I) can also have A as CH-SR16 or CH-S(O)R16. An example of the R16 group is aminopropyl. In another embodiment, A can also be CH-CH=CH-R15, while examples of R15 include aminopropyl and aminoethyl.
Another embodiment of the invention also provides compounds of Formula (I) wherein R4 and R5 together with the carbon to which they are attached form C=O. One embodiment provides that one of R2 and R3 is H, and the other is -CH2OH. Another embodiment provides that both of R and R are H. Certain embodiments provide compounds of Formula (I) wherein A is C=N-OR14. Examples of the R14 group include aminoethyl, aminopropyl, methylaminoethyl, methylaminopropyl, 2-aminopropyl, amino-2-methylpropyl, 2-amino-dimethylethyl, and pyrrolidinyl. Another embodiment provides compounds of Formula (I) wherein A is CH(OR13). The R13 group can be, but is not limited to, aminopropionyl, 3-aminobutanionyl, or amino-2- methylpropionyl. In another embodiment, A is CH-CH=CH-R15, while R15 can be, but is not limited to, aminopropyl or aminoethyl. Other embodiments also provide that A is CH-SR16 or CH-S(O)R16. One example of the R16 group is aminopropyl.
Further, the methods of the invention include administering compounds of Formula (I) wherein R4 and R5 together with the carbon to which they are attached form C=CF2 or C=CH2. One embodiment provides compounds of Formula (I) wherein both of R and R are H. Certain embodiments provide compounds of Formula (I) wherein A is C=N-OR14. In some instances, R14 is 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2-methylpropyl, 2-amino-dimethylethyl, or aminoethyl. In another embodiment, A is CH(OR13). And R13 can be, but is not limited to, aminopropionyl, 3-aminobutanionyl, or 3-amino-2-methylpropionyl.
Another embodiment provides that A is CH-CH=CH-R15, and R15 can be, but is not limited to, aminopropyl or aminoethyl. Other embodiments provide that A is CH-SR16 or CHS(O)R16. One example of the R16 group is aminopropyl.
The methods of the invention also include administering compounds of Formula (I) wherein one of R4 and R5 is H and the other is methyl. In one embodiment, the compounds of Formula (I) have both of R2 and R3 as H. One embodiment provides the compounds of Formula (I) wherein A is C=N-OR14. In certain embodiments, R14 is selected from the group consisting of 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2-methylpropyl, 2- amino-dimethylethyl, and aminoethyl. In another embodiment, A is CH(OR13). Examples of the R13 group include aminopropionyl, 3-aminobutanionyl, and amino-2-methylpropionyl. Another embodiment provides that A is CH-CH=CH-R15. And R15 can be, but is not limited to, aminopropyl or aminoethyl. In other embodiments, A is CH-SR16 or CH-S(O)R16. One example of the R16 group is aminopropyl.
The invention also provides compounds of Formula (I) wherein R4 and R5 together with the carbon to which they are attached form C=NOH. In one embodiment, the compounds have both of R and R as H. In another embodiment, the compounds of Formula (I) have A as C=N- OR14, and R14 can be, but is not limited to, aminoethyl or pyrrolidinyl.
In certain embodiments, the compounds of Formula (I) can also have one of R4 and R5 as H, and the other being -C(O)-NH2, -C(O)-OCH3, -CH2OH, ethynyl, formylamino, -CH2OCH3, or -ONO2. In one embodiment, both of R and R are H. Another embodiment provides that the compounds of Formula (I) have A as CH-S(O)R16. One example of the R16 group is aminopropyl.
Other embodiments provide compounds of Formula (I) wherein R4 and R5 together with the carbon to which they are attached form a cyclopropyl ring. In one embodiment, the compounds have both of R and R as H. Some instances provide the compounds of Formula (I) wherein A is C=N-OR14. And R14 can be, but is not limited to, pyrrolidinyl or aminoethyl.
In one embodiment, the methods of the invention comprise administering compounds of Formula (I) wherein R1 is -OH.
One embodiment provides compounds of Formula (I) wherein R4 and R5 are both H. Certain embodiments provide the compounds of Formula (I) wherein R2 and R3 together with the carbon to which they are attached form C=CH2. In one embodiment, the compounds of Formula (I) have A as C=N-OR14. Certain examples of R14 include 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2-methylpropyl, aminoethyl, 2-amino- dimethylethyl, and pyrrolidinyl. In one embodiment, A is CH(OR13). The preceding R13 group can be, but is not limited to, aminopropionyl, 3-aminobutanionyl, or amino-2-methylpropionyl. Another embodiment provides that A is CH-CH=CH-R15. In certain instances, R15 is aminopropyl or aminoethyl. In other embodiments, A is CH-SR16 or CH-S(O)R16. One example of the R16 group is aminopropyl.
In another embodiment, the methods of the invention include administering compounds of Formula (I) wherein R and R together with the carbon to which they are attached form
C=CF2 or C=N-OR11. In one embodiment, R11 is H. In another embodiment, R11 is CH3. Certain embodiments provide the compounds wherein A is CH-SR16 or CH-S(O)R16. In some instances, R16 is aminopropyl. In another embodiment, A is C=N-OR14. And R14 can be, but is not limited to, aminoethyl or pyrrolidinyl. Certain embodiments of the invention provide compounds of Formula (I) wherein both of R2 and R3 are H .Certain embodiments provide the compounds wherein A is C=N-OR14. Examples of R14 include, but are not limited to, 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2-methylpropyl, 2-amino-dimethylethyl, and aminoethyl. One embodiment provides that A is CH(OR13). In some instances, R13 is aminopropionyl, 3- aminobutanionyl, or 3 -amino-2-methylpropionyl. Another embodiment provides that A is CH- SR16 or CH-S(O)R16. One example of the R16 group is aminopropyl. Certain embodiments also provide that A is CH-CH=CH-R15. Examples of the R15 group include aminopropyl and aminoethyl.
Embodiments of the invention also provide compounds of Formula (I) wherein one of R and R is H and the other is methyl. One embodiment provides the compounds wherein A is C=N-OR14. The preceding R14 group can be, but is not limited to, 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2-methylpropyl, and aminoethyl. Another embodiment provides the compounds wherein A is CH(OR13). Examples of the R13 group include aminopropionyl, 3-aminobutanionyl, and amino-2-methylpropionyl. Certain embodiments provide the compounds of Formula (I) wherein A is CH-CH=CH-R15, -CH-SR16 or -CH-S(O)R16. R15 can be, but is limited to, aminopropyl or aminoethyl. And one example of the R16 is aminopropyl.
Other embodiments of the invention also provide compounds of Formula (I) wherein one of R2 and R3 is H, and the other is -C(O)-NH2, -C(O)-OCH3, -CH2OH, ethynyl, formylamino, -CH2OCH3, or -NO2. One embodiment provides the compounds wherein A is -CH-S(O)R16. One example of R16 is aminopropyl.
In certain embodiments, the invention provides compounds of Formula (I) wherein R and R3 are both H. Some instances provide the compounds wherein R4 and R5 together with the carbon to which they are attached form C=O, C=CH2, difluoromethylene or C=N-OR11. In one embodiment, R11 is H. In another embodiment, R11 is methyl. One embodiment provides the compounds of Formula (I) wherein A is -CH-S(O)R16. In one embodiment, the preceding R16 is aminopropyl.
Other instances provide the compounds of Formula (I) wherein when R4 and R5 together with the carbon to which they are attached form C=CH2. In one embodiment, the compounds have A as C=N-OR14. And R14 can be, but is not limited to, 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2-methylpropyl, 2-amino-dimethylethyl, or aminoethyl.
In one embodiment, when R4 and R5 together with the carbon to which they are attached form C=CH2, A is CH(OR13). The preceding R13 group can be, but is not limited to, aminopropionyl, 3-aminobutanionyl, or amino-2-methylpropionyl. In another embodiment, when R4 and R5 together with the carbon to which they are attached form C=CH2, A is CH-
CH=CH-R15 or -CH-SR16, while R15 can be, but is not limited to, aminopropyl or aminoethyl, and one example of R16 is aminopropyl.
Embodiments of the invention also provide compounds of Formula (I) wherein one of R4 and R5 is H and the other is methyl. In some instances, the compounds have both of R2 and R3 being H. One embodiment provides the compounds wherein A is C=N-OR14. Examples of R14 include 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, 3-amino-2- methylpropyl, 2-amino-dimethylethyl, and aminoethyl. Another embodiment provides the compounds wherein A is CH(OR13), CH-CH=CH-R15, CH-SR16 or CH-S(O)R16. In one embodiment, R13 is aminopropionyl, 3-aminobutanionyl, or amino-2-methylpropionyl. In a separate embodiment, R15 is aminopropyl or aminoethyl. And one example of R16 is aminopropyl. The invention also provides compounds of Formula (I) wherein R and R are both H. One embodiment provides that one of R4 and R5 is H, and the other is -C(O)NH2, -ONO2, formylamino, ethynyl, -CH2OH, -CH2-O-CH3, or methoxycarbonyl. Another embodiment provides that A is CH-S(O)R16, while one example of R16 is aminopropyl.
In one embodiment, the invention provides that one of R6 and R7 is H and the other is - OH, and R1 is H. Certain embodiments provide the compounds of Formula (I) wherein one of R2 and R3 is H, and the other is -OH, and both of R4 and R5 are H. One embodiment provides the compounds wherein A is C=N-OR14, while R14 can be, but is not limited to, dimethylaminoethyl, aminopropyl, aminoethyl, 2-aminopropyl, methylaminoethyl, methylaminopropyl, or 3-amino-2- methylpropyl. One embodiment provides that A is CH(OR13), while R13 is selected from the group consisting of methylaminopropyl, dimethylaminopropyl, dimethylaminobutyl, aminopropyl, dimethylaminoethyl, aminoethyl, pyrrolidinylethyl, 2-(2- (dimethylamino)ethoxy) ethyl, 2-(2-(pyrrolidin-l-yl)ethoxy)ethyl, 2-guanidinylethyl, and 3- guanidinylpropyl. Another embodiment provides that A is CH-CH=N-OR15 or C=CH-CH=N- OR15, while R15 can be, but is not limited to, aminoethyl or dimethylaminoethyl. Other embodiments provide the compounds of Formula (I) wherein R and R together with the carbon to which they are attached to form C=O. In one embodiment, A is C=N-OR14 or CH(OR13). One instance provides that R14 is aminoalkyl. In another instance, R13 is aminoalkyl.
The invention also provides methods of treating or preventing neoplasia in a subject in need thereof. The methods include administering to the subject a compound of Formula (Ia)
Figure imgf000010_0001
wherein
A is C=N-OR14 or CH(OR13);
R1 is H or -OH;
One of R4 and R5 is H, and the other is H or -OR8;
R8 is H or optionally substituted RR1133 iiss ((CCii--CC44))aallkkyyll ooppttiioonnaallllyy ssuubbssttiittuutteedd bbyy a a 4 or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, amino(Ci_4)alkoxyl, (Ci_4)alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; amino(Ci-C4)alkyl optionally substituted by one or more (Ci-C4)alkyl; or amino(Ci-C4)acyl optionally substituted by (Ci- C4)alkyl; and
R14 is an amino(Ci-C4)alkyl optionally substituted by one or more
Figure imgf000011_0001
or a 4- to 6-membered heterocyclic ring optionally substituted by
Figure imgf000011_0002
and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
One embodiment provides a compound of Formula (Ia) wherein R1 is H. Another embodiment provides a compound of Formula (Ia) wherein R4 and R5 are H.
One embodiment provides a compound of Formula (Ia) wherein A is C=N-OR14. One exemplified R14 is an amino(Ci-C4)alkyl that is optionally substituted by one or more (Ci- C4)alkyl. Another exemplified R14 is a 4- to 6-membered heterocyclic ring that is optionally substituted by (CrC4)alkyl.
In another embodiment, A is CH(OR13). Certain instances provide that R13 is (Ci- C4)alkyl optionally substituted by a 4 or 6-membered heterocyclic ring. One example of R13 is 2- (pyrrolidin- 1 yl) ethyl.
Certain compounds for use in accordance with the methods of the invention include (1 OR, 13S)-3-(2-aminoethoxyimino)-l 0, 13-dimethyldodecahydro-lH- cyclopenta[a]phenanthrene-6, 17(1 OH, 14H)-dione (1) :
Figure imgf000011_0003
(10R,13S)-10,13-dimethyl-3-((R)-pyrrolidin-3-yloxyimino)dodecahydro-lH- cyclopenta[a]phenanthrene-6, 17(1 OH, 14H)-dione (2) :
Figure imgf000012_0001
(3S,5S,10R,13S)-10,13-dimethyl-3-(2-(pyrrolidin-l-yl)ethoxy)dodecahydro-lH- cyclopenta[a]phenanthrene-6, 17(1 OH, 14H)-dione (15) :
Figure imgf000012_0002
The methods of the invention also include administering to a subject in need thereof a compound of Formula (Ib):
Figure imgf000012_0003
wherein
A is C=N-OR14, CH-CH=CH-R15, CH-SR16 or CH-S(O)R16;
R1 is H or -OH;
X is C(R10)2 or N-ORu; One of R4 and R5 is H, and the other is H or -OR8; R8 is H or substituted or unsubstituted R10, for each occurrence, is the same or different and is H or halogen; Ru is H or (Ci_C4)alkyl; and R14 is an amino(Ci_C4)alkyl optionally substituted by one or more
Figure imgf000013_0001
or a 4- to
6-membered heterocyclic ring optionally substituted by (Ci_C4)alkyl;
R15 and R16, each independently, are amino(Ci_C4)alkyl optionally substituted by one or more (Ci-C4)alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
One embodiment provides a compound of Formula (Ib) wherein R1 is H. In another embodiment, R1 is OH.
Another embodiment provides a compound of Formula (Ib) wherein X is C(R10)2. In certain instances, R10 is H. In another embodiment, X is N-OR11. One example of the R11 group is H. Another example of R11 is (d_C4)alkyl.
Another embodiment of the invention provides a compound of Formula (Ib) wherein A is C=N-OR14. In one instance, R14 is an amino(Ci_4)alkyl that is optionally substituted by one or more In another instance, R14 is a 4- to 6-membered heterocyclic ring.
One embodiment of the invention provides a compound of Formula (Ib) wherein A is CH-CH=CH-R15. In one instance, R15 is an ammo(CM)alkyl.
In one embodiment, A is CH-SR16. In another embodiment, A is CH-S(O)R16. Certain instances provide that R16 is an amino(Ci_4)alkyl.
Certain compounds of Formula (Ib) for use in accordance with the methods of the invention include compounds as follows: a) ( 1 OR, 13 S)-3 -(2-aminoethoxyimino)- 10,13 -dimethyl-6-methylenetetradecahydro- 1 H- cyclopenta[a]phenanthren-17(2H)-one (3):
Figure imgf000013_0002
b) ( 1 OR, 13S)-IO513 -dimethyl-6-methylene-3 -(pyrrolidin-3 -yloxyimino)tetradecahydro- IH- cyclopenta[a]phenanthren-17(2H)-one (4):
Figure imgf000014_0001
c) ( 1 OR, 13 S)-3 -(2-aminoethoxyimino)-6-(hydroxyimino)- 10,13 -dimethyltetradecahydro- IH- cyclopenta[a]phenanthren-17(2H)-one (5):
Figure imgf000014_0002
d) ( 1 OR, 13 S)-3 -(2-aminoethoxyimino)-6-(methoxyimino)- 10,13 -dimethyltetradecahydro- IH- cyclopenta[a]phenanthren-17(2H)-one (6):
Figure imgf000014_0003
e) (5 S, 1 OR, 13 S)-5-hydroxy- 10,13 -dimethyl-6-methylene-3 -(pyrrolidin-3 -yloxyimino)tetra- decahydro- 1 H-cyclopenta[a]phenanthren- 17(2H)-one (7):
Figure imgf000015_0001
f) (5S, 1 OR, 13S)-3-(2-aminoethoxyimino)-5-hydroxy-l 0, 13-dimethyl-6-methylenetetra- decahydro- 1 H-cyclopenta[a]phenanthren- 17(2H)-one (8) :
Figure imgf000015_0002
g) (5S, 1 OR, 13 S)-3-(5-aminopent- 1 -enyl)-6-(hydroxyimino)- 10, 13-dimethyltetradecahydro- 1 H- cyclopenta[a]- phenanthren-17(2H)-one (18):
Figure imgf000015_0003
h) (3R,5R, 1 OR, 13 S)-3 -(3 -Aminopropylthio)- 10,13 -dimethyl-6-methylenetetradecahydro- 1 H- cyclopenta- [a]phenanthren-17(2H)-one (19):
Figure imgf000016_0001
i) (5R, 1 OR, 13 S)-3 -(3 -aminopropylsulfmyl)- 10, 13 -dimethyl-ό-methylenetetradecahydro- 1 H- cyclopenta[a]phenanthren-17(2H)-one (20):
Figure imgf000016_0002
Figure imgf000016_0003
( 1 OR, 13 S)-3- ((R)-pyrrolidin-3-yloxyimino)-6-(methoxyimino)- 10,13 dimethyltetradecahydro-lH-cyclopenta[a]phenanthren-17(2H)-one (21)
The methods of the invention also provide administering to a subject in need thereof a compound of Formula (Ic):
Figure imgf000017_0001
wherein
R1 is H or -OH;
One of R2 and R3 is H, and the other is
Figure imgf000017_0002
optionally substituted by hydroxyl or methoxy;
One of R4 and R5 is H and the other is -OR8; or
R4 and R5 together with the carbon to which they are attached form C=O;
R8 is H or substituted or unsubstituted (Ci_C4)alkyl; and
R14 is an amino(Ci_C4)alkyl optionally substituted by one or more
Figure imgf000017_0003
or a 4- to 6-membered heterocyclic ring optionally substituted by
Figure imgf000017_0004
and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
One embodiment provides a compound of Formula (Ic) wherein R1 is H. Another embodiment provides a compound of Formula (Ic) wherein one of R and R is H and the other is -CH2OH. Another embodiment provides a compound of Formula (Ic) wherein R4 and R5 together with the carbon to which they are attached form C=O. Certain embodiments also provide compounds of Formula (Ic) wherein one of R4 and R5 is H and the other is -OR8. In one embodiment, R14 in Formula (Ic) is an amino(Ci_C4)alkyl that is optionally substituted by one or more In another embodiment, R14 is a 4- to 6-membered heterocyclic ring optionally substituted by
Figure imgf000017_0005
Certain compounds of Formula (Ic) for use in accordance with the methods of the invention include i) (6S, 1 OR, 13 S)-3-(2-aminoethoxyimino)-6-(hydroxymethyl)- 10, 13-dimethyldodecahydro- 1 H- cyclopenta[a]phenanthrene-7, 17(2H,8H)-dione (9):
Figure imgf000018_0001
ii) (6S, 1 OR, 13S)-6-(hydroxymethyl)-l 0, 13-dimethyl-3-(pyrrolidin-3-yloxyimino)dodecahydro- lH-cyclopenta[a]phenanthrene-7,17(2H,8H)-dione (10):
Figure imgf000018_0002
iii) (6S,7S, 1 OR, 13S)-7-hydroxy-6-(hydroxymethyl)-l 0, 13-dimethyl-3-(pyrrolidin-3- yloxyimino)tetradecahydro- 1 H-cyclopenta[a]phenanthren- 17(2H)-one (11):
Figure imgf000018_0003
iv) (6S,7S, 1 OR, 13S)-3-(2-aminoethoxyimino)-7-hydroxy-6-(hydroxymethyl)-l 0, 13- dimethyltetradecahydro-lH-cyclopenta[a]phenanthren-17(2H)-one (12):
Figure imgf000019_0001
The methods of the invention also provide administering to a subject in need thereof a compound of Formula (Id):
Figure imgf000019_0002
wherein
R1 is H or -OH;
R4 and R5 taken together with the carbon atom to which they are attached form C(R10)2 ;
R10, for each occurrence, is the same or different and is H or halogen; and
R14 is an amino(Ci_C4)alkyl optionally substituted by one or more
Figure imgf000019_0003
or a 4- to 6-membered heterocyclic ring optionally substituted by (Ci_C4)alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
One embodiment provides a compound of Formula (Id) wherein R1 is H. Another embodiment provides a compound of Formula (Id) wherein R10 is H. Another embodiment provides that R10 is halogen. Certain instances provide compounds of Formula (Id) wherein R14 is an amino(Ci_C4)alkyl that is optionally substituted by one or more
Figure imgf000019_0004
In other instances, R14 is a 4- to 6-membered heterocyclic ring that is optionally substituted by (Ci- C4)alkyl. Certain compounds of Formula (Id) used in accordance with the methods of the invention include: aa) ( 1 OS, 13 S)-3 -(2-aminoethoxyimino)- 10, 13 -dimethyl-7-methylenetetradecahydro- 1 H- cyclopenta[a]phenanthren-17(2H)-one (13):
Figure imgf000020_0001
bb) (10S,13S)-3-(2-aminoethoxyimino)-7-(difluoromethylene)-10,13-dimethyltetradecahydro- 1 H-cyclopenta[a]phenanthren- 17(2H)-one (14) :
Figure imgf000020_0002
The methods of the invention also provide administering to a subject in need thereof a compound of Formula (Ie):
Figure imgf000020_0003
wherein
R1 is H or -OH; A is CH(OR13), C=N-OR14, CH-CH=N-OR15, C=CH-CH=N-OR15, CH-CH=CH-R15, CH-SR16, or CH-S(O)R16;
R13 is (Ci_C4)alkyl optionally substituted by a 4 or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, amino(Ci_4)alkoxyl, (Ci_4)alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; amino(Ci_C4)alkyl optionally substituted by one or more (Ci_C4)alkyl; or amino(Ci_C4)acyl optionally substituted by (Ci- C4)alkyl;
R14 is an amino(Ci_C4)alkyl optionally substituted by one or more
Figure imgf000021_0001
or a 4- to 6-membered heterocyclic ring optionally substituted by
Figure imgf000021_0002
R15 and R16, each independently, are amino(Ci_C4)alkyl optionally substituted by one or
Figure imgf000021_0003
tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
One embodiment provides a compound of Formula (Ie) wherein R1 is H. One embodiment provides a compound of Formula (Ie) wherein A is CH-CH=N-OR15. Another embodiment provides that A is C=CH-CH=N-OR15. In one instance, R15 is amino(Ci_C4)alkyl. In another instance, R15 is amino(Ci_C4)alkyl that is substituted by one or more
Figure imgf000021_0004
Certain compounds of Formula (Ie) for use in accordance with the invention include 1) 6,17-dihydroxy- 10,13 -dimethylhexadecahydro- 1 H-cyclopenta[a]phenanthrene-3 - carbaldehyde O-2-aminoethyl oxime (16):
Figure imgf000021_0005
2) 2-((5S,6S,10R,13S,17S)-6,17-dihydroxy-10,13-dimethylhexahydro-lH- cyclopenta[a]phenanthren-3(2H,4H, 1 OH, 12H, 13H, 14H, 15H, 16H, 17H)-ylidene)acetaldehyde O- 2-(dimethylamino)ethyl oxime (17):
Figure imgf000022_0001
In another aspect, the invention provides methods for reducing the growth, proliferation, or survival of a neoplastic cell, the method comprising contacting the cell with an effective amount of a compound of this invention. The compound reduces the growth, proliferation, or survival of a neoplastic cell.
In one embodiment, the neoplastic cell is derived from a tissue selected from the group consisting of lung, breast, CNS, colon, prostate, ovary, pancreas, kidney and melanoma. In a separate embodiment, the cell expresses MDR-I or P-glycoprotein. Yet another aspect of this invention provides a method of inducing cell death in a neoplastic cell. The method includes contacting the cell with a therapeutically effective amount of a compound of this invention, thereby inducing cell death. In one embodiment, the cell is in a subject. Another embodiment provides that the cell death is apoptotic cell death.
A further aspect of this invention provides a method of preventing or treating a neoplasia in a subject. This method includes administering to the subject a therapeutically effective amount of a compound of this invention.
In one embodiment, the subject recited in the methods of the invention is a mammal. In another embodiment, the subject is a human patient.
In certain embodiments, the methods of the invention reduce the growth or proliferation of a neoplasia in a subject. The neoplasia recited in the methods of the invention can be, but is not limited to, a lung, breast, CNS, colon, prostate, ovary, pancreas, kidney or skin cancer.
In some instances, the neoplasia is resistant to one or more therapeutic agents. In one embodiment, the neoplasia is multidrug resistant. Certain embodiments provide that the neoplasia has alterations in the expression or activity of an ABC transporter, tubulin, or topoisomerase polypeptide or polynucleotide. In other embodiments, the neoplasia has an increase in the expression or activity of MDRl or P-glycoprotein.
In another aspect, the invention provides a method for the treatment of a subject having a multidrug resistant or refractory neoplasia. This method includes administering to the subject a therapeutically effective amount of a compound of this invention and a pharmaceutically acceptable excipient. In one embodiment, the subject is a human patient. In a separate embodiment, the method reduces the growth or proliferation of the neoplasia. In another embodiment, the method induces the death of a neoplastic cell.
In one embodiment, the neoplasia is resistant to one or more therapeutic agents. In another embodiment, the neoplasia has alterations in the expression or activity of an ABC transporter, tubulin, or topoisomerase polypeptide or polynucleotide. Other embodiments provide that the neoplasia has an increase in the expression or activity of MDRl or P- glycoprotein.
The method may further comprise administering a compound selected from the group consisting of vinca alkaloids, anthracyc lines, epipodophyllotoxins, taxanes, antibiotics, antimicrotubule drugs, protein synthesis inhibitors, toxic peptides, topoisomerase inhibitors, DNA intercalators, anti-mitotics, platinum-based chemotherapeutic agents. The method may also treat a patient having end-stage disease. This invention also provides a composition for the treatment of a neoplasia. The composition includes a therapeutically effective amount of a compound of the invention and a pharmaceutically acceptable excipient. The composition may further include a therapeutically effective amount of a chemotherapeutic compound.
A further aspect of this invention provides a packaged pharmaceutical for the treatment of neoplasia. The packaged pharmaceutical includes a therapeutically effective amount of a compound of the invention, and written instructions for administration of the compound. In another aspect, the invention provides a method of preventing or treating a neoplasia (e.g., a membrane androgen positive solid tumor or hematological malignancy) in a subject. The method involves administering to the subject (e.g., mouse, dog, human) a therapeutically effective amount of a Na+K+ ATPase inhibitor that inhibits ligand binding to a membrane androgen receptor, thereby preventing or treating the neoplasia. In one embodiment, the Na+K+ ATPase inhibitor binds a Na+K+ ATPase and inhibits Na+K+ ATPase activity. In another embodiment, the Na+K+ ATPase inhibitor binds to a membrane androgen receptor and competitively inhibits ligand binding to the receptor. In another embodiment, the Na+K+ ATPase inhibitor induces cell death in a neoplastic cell of the neoplasia. In still other embodiments, the neoplasia is a prostate cancer, breast cancer, or colon cancer. In yet another aspect, the invention provides a method for treating or preventing prostate cancer in a subject. The method involves administering to the subject an effective amount of a compound capable of binding and inhibiting a Na+K+ ATPase, and further capable of competitively inhibiting ligand binding to the membrane androgen receptor on a prostate cancer cell. In one embodiment, the method induces cell death (e.g., apoptosis) in a cell of the prostate cancer. In another embodiment, the compound binds the membrane androgen receptor. In still another embodiment, the compound is istaroxime. In yet another aspect, the invention provides a composition for the treatment or prevention of a neoplasia, the composition containing a therapeutically effective amount of a compound capable of binding and inhibiting a Na+K+ ATPase, and further capable of competitively inhibiting ligand binding to the membrane androgen receptor on a neoplastic cell and a pharmaceutically acceptable excipient, where the therapeutically effective amount is sufficient to induce cell death in a neoplastic cell.
In yet another aspect, the invention provides a packaged pharmaceutical for the treatment of neoplasia involving a therapeutically effective amount of a compound capable of binding and inhibiting a Na+K+ ATPase, and further capable of competitively inhibiting ligand binding to the membrane androgen receptor on a prostate cancer cell, and written instructions for administration of the compound for use in treating the neoplasia.
In yet another aspect, the invention provides a method of preventing or treating a neoplasia in a subject, the method involving administering to the subject a therapeutically effective amount of a Na+K+ ATPase inhibitor that binds to a membrane androgen receptor, thereby preventing or treating the neoplasia. In one embodiment, the Na+K+ ATPase inhibitor induces cell death in a neoplastic cell of the neoplasia.
In still another aspect, the invention provides a method for treating or preventing prostate cancer in a subject, the method involving administering to the subject an effective amount of a Na+K+ ATPase inhibitor that binds to a membrane androgen receptor on a prostate cancer cell. In one embodiment, the method induces cell death in a cell of the prostate cancer. In another embodiment, the compound is istaroxime.
In still another aspect, the invention provides a composition for the treatment or prevention of a neoplasia, the composition containing a therapeutically effective amount of a Na+K+ATPaSe inhibitor that binds to a membrane androgen receptor on a neoplastic cell and a pharmaceutically acceptable excipient, where the therapeutically effective amount is sufficient to induce cell death in a neoplastic cell.
In still another aspect, the invention provides a packaged pharmaceutical for the treatment of neoplasia involving a therapeutically effective amount of a Na+K+ ATPase inhibitor that binds to a membrane androgen receptor on a prostate cancer cell, and written instructions for administration of the compound for use in treating the neoplasia.
The invention further provides methods for treating neoplasia. Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A-1C are graphs showing that the compound 1/istaroxime inhibited the proliferation and viability of colon cancer cell line HCTl 16, and prostate cancer cell lines PC3 and DU145. Istaroxime's inhibitory properties were not shared by Na+K+ ATPase inhibitor, rostafuroxin.
Figures 2A, 2B and 2C present in vitro anti-cancer data obtain with SRB and MTT assays in 22 cancer lines including multi drug resistant cell lines.
Figures 3A-3C are graphs presenting data on the anti-tumor activity of compounds 1 and 4 in PC-3 prostate xenografts (Figures 3 A, B) and of compound 4 in A549 lung xenografts (Figure 3C) respectively.
Figures 4 A and 4B are photomicrographs showing that compound 1 (istaroxime) and compound 4, but not rostafuroxin, abolish binding of Testosterone-HSA (testosterone 3-(O- carboxymethyl)oxime: human serum albumin) conjugates and fluorescein isothiocyanate (testosterone-HSA-FITC) conjugates to the membrane androgen receptor.
DETAILED DESCRIPTION OF THE INVENTION
The invention features methods comprising compounds delineated herein that are useful for the treatment of neoplasia. In particular embodiments, the compounds of the invention are useful for the treatment of multidrug resistant neoplasia. The invention is based, at least in part, on the discovery that compounds of the invention including istaroxime, (£",Z)-3-(2-Aminoethoxyimino)androstane-6,17-dione, have potent antineoplastic activity in vitro. As shown herein below, the compounds of the invention reduced the viability and/or cell proliferation of twenty two different cell lines representative of lung, breast, CNS, colon, prostate, ovary, pancreas, kidney and melanoma neoplasias, including a multidrug resistant cell line. Furthermore, compounds of the invention exhibited stong anti-tumor action in prostate and lung cancer xenografts in vivo.
Definitions
Before a further description of the present invention, and in order that the invention may be more readily understood, certain terms are first defined and collected here for convenience.
The term "administration" or "administering" includes routes of introducing a compound(s) to a subject to perform their intended function. Examples of routes of administration that can be used include injection (subcutaneous, intravenous, parenterally, intraperitoneally, intrathecal), oral, inhalation, rectal and transdermal. The pharmaceutical preparations are, of course, given by forms suitable for each administration route. For example, these preparations are administered in tablets or capsule form, by injection, inhalation, topical by lotion or ointment; and rectal by suppositories. Oral administration is preferred. The injection can be bolus or can be continuous infusion. Depending on the route of administration, the compound can be coated with or disposed in a selected material to protect it from natural conditions which may detrimentally effect its ability to perform its intended function. The compound can be administered alone, or in conjunction with either another agent as described above (e.g. another chemotherapeutic agent) or with a pharmaceutically-acceptable carrier, or both. The compound can be administered prior to the administration of the other agent, simultaneously with the agent, or after the administration of the agent. Furthermore, the compound can also be administered in a proform which is converted into its active metabolite, or more active metabolite in vivo.
The term "alkyl" refers to the radical of saturated aliphatic groups, including straight- chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The term alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen, sulfur or phosphorous atoms. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C3-C30 for branched chain), preferably 26 or fewer, and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 3, 4, 5, 6 or 7 carbons in the ring structure.
Moreover, the term alkyl as used throughout the specification and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls," the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. Cycloalkyls can be further substituted, e.g., with the substituents described above. An "alkylaryl" moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). The term "alkyl" also includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively. Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six, and most preferably from one to four carbon atoms in its backbone structure, which may be straight or branched-chain. Examples of lower alkyl groups include methyl, ethyl, n-propyl, i-propyl, tert-butyl, hexyl, heptyl, octyl and so forth. In preferred embodiment, the term "lower alkyl" includes a straight chain alkyl having 4 or fewer carbon atoms in its backbone, e.g., C1-C4 alkyl.
The term "alkoxy," as used herein, refers to an alkyl or a cycloalkyl group which is linked to another moiety though an oxygen atom. Alkoxy groups can be optionally substituted with one or more substituents.
The terms "alkoxyalkyl," "polyaminoalkyl" and "thioalkoxyalkyl" refer to alkyl groups, as described above, which further include oxygen, nitrogen or sulfur atoms replacing one or more carbons of the hydrocarbon backbone, e.g., oxygen, nitrogen or sulfur atoms.
The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond, respectively. For example, the invention contemplates cyano and propargyl groups.
The term "ameliorate" means to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease. The term "alteration" refers to a change (increase or decrease) in a parameter as detected by standard art known methods, such as those described herein.
The term "aryl" refers to the radical of aryl groups, including 5- and 6-membered single- ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, benzoxazole, benzothiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Aryl groups also include polycyclic fused aromatic groups such as naphthyl, quinolyl, indolyl, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles," "heteroaryls" or "heteroaromatics." The aromatic ring can be substituted at one or more ring positions with such substituents as described above, as for example, halogen, hydroxyl, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Aryl groups can also be fused or bridged with alicyclic or heterocyclic rings which are not aromatic so as to form a polycycle (e.g., tetralin).
The term "cancer" refers to a malignant tumor of potentially unlimited growth that expands locally by invasion and systemically by metastasis. The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
The term "chiral" refers to molecules which have the property of non-superimposability of the mirror image partner, while the term "achiral" refers to molecules which are superimposable on their mirror image partner.
In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
"Detect" refers to identifying the presence, absence or amount of the object to be detected. By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
The term "diastereomers" refers to stereoisomers with two or more centers of dissymmetry and whose molecules are not mirror images of one another.
The term "effective amount" refers to the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. A therapeutically effective amount of a compound delineated herein (i.e., an effective dosage) may range from about 0.1 μg to 20 milligram per kilogram of body weight per day (mg/kg/day) (e.g., O.lμg/kg to 2mg/kg, 0.3-3μg/kg, 0.18-0.54mg/kg). In other embodiments, the amount varies from about 0.1 mg/kg/day to about 100 mg/kg/day. In still other embodiments, the amount varies from about 0.001 μg to about 100 μg/kg (e.g., of body weight). One of skill in the art can readily extrapolate from dosages shown to be effective in in vivo testing to dosages that are likely to be effective in humans. In one embodiment, compounds of the invention are administered in a dose of about 0.1-200mg/kg/day to a mouse, preferably 1-100 mg/kg, more preferably 10-60 mg/kg. In another embodiment, a dog receives 0.1-10 mg/kg of compound. In another embodiment, a human subject receives 0.1 μg/kg to 5mg/kg parenteral compound per day. In yet another embodiment, 0.1-lOμg/kg compound is administered to a human subject. In still another embodiment, 0.1-1 mg/kg total per day is administered to a human subject. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a compound delineated herein can include a single treatment or, preferably, can include a series of treatments. In one example, a subject is treated with a compound delineated herein in the range of between about 0.1 μg to 20 milligram per kilogram of body weight per day (mg/kg/day) (e.g., 0.1 μg/kg to lOmg/kg, 0.1-lOμg/kg, 0.1-lmg/kg). In other embodiments, the amount varies from about 0.1 mg/kg/day to about 100 mg/kg/day. In still other embodiments, the amount varies from about 0.001 μg to about 100 μg/kg (e.g., of body weight). If desired, the dosage is administered one time per day, two times per day, or one time per week. Treatment is carried out for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of a compound delineated herein used for treatment may increase or decrease over the course of a particular treatment.
The term "enantiomers" refers to two stereoisomers of a compound which are non- superimposable mirror images of one another. An equimolar mixture of two enantiomers is called a "racemic mixture" or a "racemate."
The term "halogen" designates -F, -Cl, -Br or -I. The term "haloalkyl" is intended to include alkyl groups as defined above that are mono-
, di- or polysubstituted by halogen, e.g., fluoromethyl and trifluoromethyl. The term "hydroxyl" means -OH.
The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus. The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-4 ring heteroatoms if monocyclic, 1- 6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S, and the remainder ring atoms being carbon. Heteroaryl groups may be optionally substituted with one or more substituents. Examples of heteroaryl groups include, but are not limited to, pyridyl, furanyl, benzodioxolyl, thienyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl thiazolyl, isoxazolyl, quinolinyl, pyrazolyl, isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, triazolyl, thiadiazolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzofuryl, indolizinyl, imidazopyridyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzothiadiazolyl, benzoxadiazolyl, and indolyl.
The term "heterocyclic" as used herein, refers to organic compounds that contain at least at least one atom other than carbon (e.g., S, O, N) within a ring structure. The ring structure in these organic compounds can be either aromatic or non-aromatic. Some examples of heterocyclic moeities include, are not limited to, pyridine, pyrimidine, pyrrolidine, furan, tetrahydrofuran, tetrahydrothiophene, and dioxane.
The term "isomers" or "stereoisomers" refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.
The term "multidrug resistant" refers to a reduced susceptibility to one or more chemotherapeutic agents.
The term "P-glycoprotein polypeptide" refers to a protein having at least about 85% or more amino acid identity to NCBI Accession No. CAA41558 or a fragment thereof that has ABC transporter activity.
The term "MDRl polynucleotide" refers to a nucleic acid sequence encoding a P- glycoprotein polypeptide.
The term "neoplastic" refers to those cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. A neoplastic disease state may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. "Pathologic hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non- pathologic hyperproliferative cells include proliferation of cells associated with wound repair.
The language "inhibiting the growth" of the neoplasm includes the slowing, interrupting, arresting or stopping its growth and metastases and does not necessarily indicate a total elimination of the neoplastic growth. The term "modulate" refers to increases or decreases in a parameter in response to exposure to a compound of the invention. The common medical meaning of the term "neoplasia" refers to "new cell growth" that results as a loss of responsiveness to normal growth controls, e.g. to neoplastic cell growth. A "hyperplasia" refers to cells undergoing an abnormally high rate of growth. However, as used herein, the term neoplasia generally refers to cells experiencing abnormal cell growth rates. Neoplasias include "tumors," which may be either benign, premalignant or malignant.
The term "obtaining" as in "obtaining compound" is intended to include purchasing, synthesizing or otherwise acquiring the compound.
The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
The terms "polycyclyl" or "polycyclic radical" refer to the radical of two or more cyclic rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings. Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkyl, alkylaryl, or an aromatic or heteroaromatic moiety.
The term "polymorph" as used herein, refers to solid crystalline forms of a compound of the present invention or complex thereof. Different polymorphs of the same compound can exhibit different physical, chemical and/or spectroscopic properties. Different physical properties include, but are not limited to stability (e.g., to heat or light), compressibility and density (important in formulation and product manufacturing), and dissolution rates (which can affect bioavailability). Differences in stability can result from changes in chemical reactivity (e.g., differential oxidation, such that a dosage form discolors more rapidly when comprised of one polymorph than when comprised of another polymorph) or mechanical characteristics (e.g., tablets crumble on storage as a kinetically favored polymorph converts to thermodynamically more stable polymorph) or both (e.g., tablets of one polymorph are more susceptible to breakdown at high humidity). Different physical properties of polymorphs can affect their processing.
The term "prodrug" includes compounds with moieties which can be metabolized in vivo. Generally, the prodrugs are metabolized in vivo by esterases or by other mechanisms to active drugs. Examples of prodrugs and their uses are well known in the art (See, e.g., Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. ScL 66:1-19). The prodrugs can be prepared in situ during the final isolation and purification of the compounds, or by separately reacting the purified compound in its free acid form or hydroxyl with a suitable esterifying agent. Hydroxyl groups can be converted into esters via treatment with a carboxylic acid. Examples of prodrug moieties include substituted and unsubstituted, branch or unbranched lower alkyl ester moieties, {e.g., propionoic acid esters), lower alkenyl esters, di-lower alkyl-amino lower-alkyl esters {e.g., dimethylaminoethyl ester), acylamino lower alkyl esters {e.g., acetyloxymethyl ester), acyloxy lower alkyl esters {e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters {e.g., benzyl ester), substituted {e.g., with methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alkyl amides, di-lower alkyl amides, and hydroxy amides. Preferred prodrug moieties are propionoic acid esters and acyl esters. Prodrugs which are converted to active forms through other mechanisms in vivo are also included.
The language "a prophylactically effective anti-neoplastic amount" of a compound refers to an amount of a compound delineated herein or otherwise described herein which is effective, upon single or multiple dose administration to the patient, in preventing or delaying the occurrence of the onset of a neoplastic disease state.
Furthermore the indication of stereochemistry across a carbon-carbon double bond is also opposite from the general chemical field in that "Z" refers to what is often referred to as a "cis" (same side) conformation whereas "E" refers to what is often referred to as a "trans" (opposite side) conformation. Both configurations, cis/trans and/or Z/E are encompassed by the compounds of the present invention.
With respect to the nomenclature of a chiral center, the terms "d" and "1" configuration are as defined by the IUPAC Recommendations. As to the use of the terms, diastereomer, racemate, epimer and enantiomer, these will be used in their normal context to describe the stereochemistry of preparations.
By "reference" is meant a standard or control condition.
The term "subject" includes organisms which are capable of suffering from a neoplasia or who could otherwise benefit from the administration of a compound of the invention, such as human and non-human animals. Preferred human animals include human patients suffering from or prone to suffering from a neoplasia, as described herein. The term "non-human animals" of the invention includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, and non-mammals, such as non-human primates, also sheep, dog, cow, chickens, amphibians, and reptiles.
The term "sulfhydryl" or "thiol" means -SH.
The phrases "systemic administration," "administered systemically", "peripheral administration" and "administered peripherally" as used herein mean the administration of a compound(s), drug or other material, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
As used herein, the term "tautomers" refers to isomers of organic molecules that readily interconvert by tautomerization, in which a hydrogen atom or proton migrates in the reaction, accompanied in some occasions by a switch of a single bond and an adjacent double bond.
*****************
The invention provides a number of targets that are useful for the development of highly specific drugs to treat or prevent a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high- volume throughput, high sensitivity, and low complexity.
Istaroxime Istaroxime (PST 2744) or (£",Z)-3-(2-Aminoethoxyimino)androstane-6,17-dione is a Na+/K+-ATPase inhibitor. The Na+/K+-ATPase, or Na+/K+ pump is a complex of integral membrane proteins that actively transports sodium and potassium ions across the cell plasma membrane. In addition to pumping ions across the membrane, the enzyme functions as a receptor for cardiac glycosides, such as ouabain, digoxin, marinobufagenin and others (reviewed in Mijatovic T et al., Biochim Biophys Acta. 2007; 1776:32-57).
Istaroxime is chemically unrelated to cardiac glycosides, which also display Na+/K+- ATPase inhibitory activity. Cardiac glycosides comprise steroidal and glycoside moieties, and have a lactone ring at the Cl 7 position. In contrast, istaroxime is an aglycone; it has no lactone ring at C 17, but it does have an aminoethoxy-imino moiety at C3. Despite these distinguishing structural features, istaroxime effectively inhibits Na+/K+-ATPase.
Istaroxime had an IC5O of 0.2 μM when tested in vitro on purified dog kidney ATPase as compared to 0.4 μM for the cardiac glycoside digoxin (De Munari S et al., J Med Chem. 2003 Aug 14;46(17):3644-54). Istaroxime has both inotropic and lusitropic properties. Its inotropic potency expressed as ED80 was 4.84 μmol/kg when tested in vivo by slow intravenous infusion in anesthetized guinea pigs (compared to 0.41 of digoxin). The inotropic effects of istaroxime are due to its ability to inhibit Na+/K+- ATPase at the sarcolemma, leading to an increase in cytosolic calcium and thus improved contractility. The lusitropic effects are related to istaroxime's ability to stimulate sarcoplasmic reticulum calcium ATPase 2 (SERCA2), leading to rapid sequestration of cytosolic calcium into the sarcoplasmic reticulum (SR) during diastole and thereby promoting myocardial relaxation (Mattera GG et al., Am J Cardiol. 2007 Jan 22;99(2A):33A-40A).
This mechanism also contributes to increased contractility as the greater calcium uptake in the sarcoplasmic reticulum allows the release of a greater amount of calcium at the subsequent systole with increased contractility. Intravenous administration of istaroxime in dogs, showed that the positive inotropic activity of this compound is combined with a favourable hemodynamic profile (no effect on heart rate, reduction of peripheral resistance), absence of alterations of cardiac rhythm and conduction system, fast/ high reversibility of action and improved safety index (De Munari S et al., J Med Chem. 2003 Aug 14;46(17):3644-54). Istaroxime is currently being tested in clinical trials for Acute Decompensated Heart Failure and Stable Heart Failure. Methods for synthesizing istaroxime are known in the art and are described herein. For example, European Patent No. EPl 156058 describes a process for the preparation of istaroxime and istaroxime analogues. International Application Nos. WO 2007/118830 and WO 2007/118832 describe the process for the preparation of istaroxime analogues and their use in the treatment of cardiovascular diseases. As reported herein, istaroxime has now been shown to be useful for the treatment of neoplasia.
Rostafuroxin
Rostafuroxin (PST 2238) or 17β-(3-furyl)-5β-androstane-3β,14β,17α-triol, is a steroidal compound that is chemically unrelated to both cardiac glycosides and istaroxime. Rostafuroxin is an aglycone that has a furyl instead of a lactone ring at C 17, and a hydroxy- moiety at C3. Rostafuroxin belongs to a new class of antihypertensive agents that reduce blood pressure and prevent hypertension-related organ complications by selectively correcting the molecular and functional alterations of the Na+/K+ pump (e.g., alterations induced by genetic (i.e., Adducin) and/or hormonal (ouabain) mechanisms) without affecting the normal physiological mechanisms of blood pressure control.
Rostafuroxin inhibited purified dog kidney Na+/K+-ATPase with an IC5O of 25 μM, whereas at low K+ concentration (0.5 mM) the IC5owas 1.5 μM. In the kidney, rostafuroxin antagonizes ouabain triggering of the Src-epidermal growth factor receptor (EGFR)-dependent signaling pathway leading to renal Na+/K+-ATPase, and ERK tyrosine phosphorylation and activation. In the vasculature, rostafuroxin normalizes the increased myogenic tone caused by nanomolar levels of ouabain. Rostafuroxin has a very high safety ratio. It also fails to interact with other pathways involved in blood pressure regulation. It appears to be both well tolerated and effective for the control of hypertension in patients (Ferrari P et al., Am J Physiol Regul Integr Comp Physiol. 2006 Mar;290(3):R529-35).
Methods for synthesizing rostafuroxin are described in EP Patent No. EP0583578, which describes rostafuroxin and its derivatives, and methods for the preparation of these compounds, as well as pharmaceutical compositions comprising them. International Patent Application No. WO 2007/060206 describes methods for the preparation of rostafuroxin, as well as pharmaceutical compositions comprising rostafuroxin.
Compounds of the Invention
Compounds useful for the treatment of neoplasia, in accordance with the methods described herein, include those described in US Patent No. 5,914,324, entitled "6-Hydroxy and 6-oxo-androstane derivatives active on the cardiovascular system and pharmaceutical compositions containing same" to Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., in PCT Publication No. WO/2007/118830, entitled "Azaheterocyclyl Derivatives of Androstanes and Androstenes as Medicaments for Cardiovascular Disorders", to Sigma-Tau Industrie Farmaceutiche Riunite S.p.A., in PCT Publication No. WO/2007/118832, entitled "Amino Derivatives of Androstanes and Androstenes as Medicaments for Cardiovascular Disorders," and in European Patent Application 0825197, all of which are incorporated herein by reference in their entirety.
In one aspect, the invention provides a compound of Formula (I)
Figure imgf000035_0001
wherein:
R1 is H or -OH;
One of R2 and R3 is H or OH, and the other is H; -OR8; alkyl optionally substituted with hydroxyl or alkoxy; -C(O)-NH2; -C(O)-O-alkyl; -NHR9; alkynyl; or R2 and R3 together with the carbon to which they are attached form C=O, C=C(R10)2, or C=N-OR11; One of R4 and R5 is H, and the other is H; -OR8; alkyl optionally substituted with hydroxyl or alkoxy; -C(O)-NH2; -C(O)-O-alkyl; -NHR9; alkynyl; or R4 and R5 together with the carbon to which they are attached form C=O, C=C(R10)2, C=N-OR11, or a cyclopropyl ring;
One of R6 and R7 is H, and the other is -OH; alkoxy optionally substituted with a hydroxyl or an amino group; optionally subsituted heterocyclic or heteroaryl; or -OC(O)R12; or R6 and R7 together with the carbon atom to which they are attached form C=O;
R8 is H, alkyl, or -NO2;
R9 is H or formyl;
R10, for each occurrence, is the same or different and is H or halogen; R11 is H or alkyl;
R12 is alkyl or phenyl;
A is CH(OR13), C=N-OR14, CH-CH=N-OR15, C=CH-CH=N-OR15, CH-CH=CH-R15, CH-SR16, or CH-S(O)R16;
R13 is alkyl optionally substituted by a 4 or 6-membered heterocyclic ring, a 5 or 6- membered heteroaryl, amino(Ci_4)alkoxyl, (Ci_4)alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; aminoalkyl optionally substituted by one or more alkyl; or aminoacyl optionally substituted by alkyl;
R14 is aminoalkyl optionally substituted by one or more alkyl or acetyl; alkyl optionally substituted by guanidinyl or a 4- to 6-membered heterocyclic ring; or a 4- to 6-membered heterocyclic ring optionally substituted by alkyl;
R15 and R16, each independently, are aminoalkyl optionally substituted by one or more alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof. One embodiment of of the methods of the invention provides compounds of Formula (I) wherein R1 is H. Another embodiment provides that R1 is -OH.
In one embodiment, R6 and R7 together with the carbon to which they are attached form C=O. In another embodiment, one of R6 and R7 is H, and the other is -OH.
In one embodiment, one of R6 and R7 is H, and the other is
Figure imgf000036_0001
that is unsubstituted or substituted with a hydroxyl or an amino group. In another embodiment, one of R6 and R7 is H, and the other is
Figure imgf000036_0002
substituted with an amino group.
In one embodiment, one of R6 and R7 is H, and the other is -OC(O)R12. In one embodiment, R12 is
Figure imgf000036_0003
In another embodiment, R12 is phenyl.
In one embodiment, both of R4 and R5 are H. In another embodiment, one of R4 and R5 is H, and the other is -OR8. In one embodiment, R8 is H. Another embodiment provides that R8 is
Figure imgf000037_0001
Yet another embodiment provides that R8 is -NO2.
In one embodiment, one of R4 and R5 is H, and the other is
Figure imgf000037_0002
that is unsubstituted or substituted with hydroxyl or
Figure imgf000037_0003
In certain embodiment, one of R4 and R5 is H, and the other is methyl. In one embodiment, one of R4 and R5 is H, and the other is -CH2OH or -CH2OCH3.
Embodiments of the invention also include compounds of Formula (I) wherein, R4 and R5 is H, and the other is -C(O)-NH2, -C(O)-O(CrC4)alkyl, -NHR9 or (C2-C4)alkynyl. Certain embodiments provide that one of R4 and R5 is H, and the other is -C(O)-NH2, -C(O)-OCH3, formylamino or ethynyl.
In one embodiment, the invention provides compounds of Formula (I) wherein R4 and R5 together with the carbon to which they are attached form C=O. Certain embodiments of the invention provide compounds wherein, R4 and R5 together with the carbon to which they are attached form C=C(R10)2, and R10 is, for each occurrence independently, H or halogen. One embodiment provides that R10 is F.
In one embodiment, R4 and R5 together with the carbon to which they are attached form C=N-OR11. In certain embodiments, R11 is H or
Figure imgf000037_0004
One embodiment provides that R11 is methyl. In another embodiment, R4 and R5 together with the carbon to which they are attached form a cyclopropyl ring.
One embodiment provides that R4 and R5 together with the carbon to which they are attached form C=N-OH. Another embodiment provides that R4 and R5 together with the carbon to which they are
Figure imgf000037_0005
In one embodiment, R4 and R5 together with the carbon to which they are attached form C=N-OCH3.
The invention also provides compounds of Formula (I) wherein, R and R together with the carbon to which they are attached form C=O. Another embodiment provides that R2 and R3 together with the carbon to which they are attached form C=C(R10)2, wherein R10 is each independently H or halogen (preferably, F). In another embodiment, R2 and R3 together with the carbon to which they are attached form C=N-OR11. One embodiment provides that R11 is H. In another embodiment, R11 is (Ci- C/i)alkyl. Certain embodiment provides that R11 is methyl.
In one embodiment, both of R and R are H.
Another embodiment of the invention provides that compounds of Formula (I) wherein one of R2 and R3 is H, and the other is -OR8. Certain embodiments provide that one of R2 and R3 is H, and the other is -OH, -O(CrC4)alkyl, or -ONO2. One embodiment provides that one of R and R is -OH, and the other is
Figure imgf000038_0001
that is u mnssuubbssttiittuutteedd oorr ssuubbstituted with hydroxyl. In one embodiment, one of R2 and R3 is -OH, and the other is -CH2OH.
In one embodiment, one of R2 and R3 is H, and the other is
Figure imgf000038_0002
that is unsubstituted or substituted by hydroxyl. Another embodiment provides that one of R and R is H, and the other is
Figure imgf000038_0003
that is unsubstituted or substituted by
Figure imgf000038_0004
Embodiments of the invention provide that one of R and R is H, and the other is methyl, CH2OH, CH2CH2OH or CH2OCH3.
One embodiment of the invention provides compounds of Formula (I), wherein one of R2 and R3 is H, and the other is -C(O)-NH2, -C(O)-O(CrC4)alkyl,-NHR9 or (C2-C4)alkynyl. Embodiments include compounds of the invention wherein, one of R and R is OH, and the other is -C(O)-NH2, -C(O)OCH3, -NH2, formylamino or ethynyl.
One embodiment of the invention includes compounds of Formula (I) wherein A is C=N-OR14. In one embodiment, R14 is amino(Ci-C4)alkyl that is unsubstituted or substituted by one or more
Figure imgf000038_0005
or acetyl. Certain embodiments provide that R14 is aminoethyl, 2- aminopropyl, 2-amino-2-methylpropyl, 3-amino-2-methylpropyl, 2-amino-dimethylethyl, methylaminoethyl, methylaminopropyl, aminopropyl, aminobutyl, dimethylaminoethyl, or acetylaminoethyl.
In another embodiment, R14 is (Ci-C/Oalkyl that is unsubstituted or substituted by guanidinyl or a 4- to 6-membered heterocyclic ring. Embodiments of the invention include that R14 is methyl or ethyl, that is further substituted with pyrrolidinyl or guanidinyl.
In one embodiment, R14 is a 4- to 6-membered heterocyclic ring that is unsubstituted or substituted by (Ci-C4)alkyl. Certain embodiments provide that R14 is azetidinyl, pyrrolidinyl, piperazinyl, or piperidinyl. Other embodiments provide that R14 is a methyl-susbstituted pyrrolidinyl or piperazinyl group.
In another embodiment of the invention, A is CH(OR13). Certain embodiments provide that R13 is (Ci-C4)alkyl that is unsubstituted or substituted by a 4 or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, amino(Ci_4)alkoxyl, (Ci_4)alkoxyl substituted by a 4- or 6- membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl. In one embodiment, R13 is ethyl or propyl, that is further substituted by pyrrolidinyl, piperidinyl, imidazolyl, dimethylamino-ethoxyl, pyrrolidinyl- ethoxyl, or 4-methylpiperazinyl.
In another embodiment, R13 is amino(Ci_4)alkyl that is unsubstituted or substituted by one or more (Ci_4)alkyl. Certain embodiments provide that R13 is dimethylaminopropyl, dimethylaminoethyl, ethylaminobutyl, or dimethylaminobutyl. In one embodiment, R13 is amino(Ci_4)acyl that is unsubstituted or substituted by one or more (Ci-C4)alkyl. In one embodiment, R13 is amino(Ci_4)acyl that is substituted by methyl. Certain embodiments provide that R13 is aminopropionyl, 3-aminobutanionyl, or 3-amino-2-methylpropionyl.
One embodiment of the invention also provides that A is CH-CH=N-OR15, wherein R15 is amino(Ci_4)alkyl that is unsubstituted or substituted by one or more
Figure imgf000039_0001
Certain embodiments provide that R15 is aminoethyl or dimethylaminoethyl
Another embodiment provides that A is C=CH-CH=N-OR15, wherein R15 is amino(Ci_ 4)alkyl that is unsubstituted or substituted by one or more (Ci-C4)alkyl. Certain embodiments provide that R15 is aminoethyl or dimethylaminoethyl.
In one embodiment, A is CH-CH=CH-R15, wherein R15 is amino(Ci_4)alkyl that is unsubstituted or substituted by one or more (Ci-C4)alkyl. Certain embodiments provide that R15 is aminopropyl or aminoethyl.
In one embodiment, A is CH-SR16, wherein R16 is amino(Ci_4)alkyl that is unsubstituted or substituted by one or more (Ci-C4)alkyl. One example of R16 is aminopropyl.
And in another embodiment, A is CH-S(O)R16, wherein R16 is amino(Ci_4)alkyl that is unsubstituted or substituted by one or more (Ci-C4)alkyl. One example of R16 is aminopropyl.
In one aspect, the invention provides a compound having Formula (Ia):
Figure imgf000039_0002
wherein
A is C=N-OR14 or CH(OR13);
R1 is H or -OH;
One of R4 and R5 is H, and the other is H or -OR8;
R8 is H or optionally substituted (Ci-C4)alkyl;
R13 is (Ci-C4)alkyl optionally substituted by a 4 or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, amino(Ci_C4)alkoxyl, (Ci_C4)alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; amino(Ci-C4)alkyl optionally substituted by one or more (Ci-C4)alkyl; or amino(Ci-C4)acyl optionally substituted by (Ci- C4)alkyl; and R14 is an amino(Ci-C4)alkyl optionally substituted by one or more
Figure imgf000040_0001
or a 4- to 6-membered heterocyclic ring optionally substituted by (Ci_C4)alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
In one embodiment, R1 is H. In another embodiment, R1 is -OH.
One embodiment provides compounds of Formula (Ia) wherein both of R4 and R5 are H. Another embodiment provides compounds wherein A is C=N-OR14. In one instance, R14 is an amino(Ci-C4)alkyl that is unsubstituted or substituted by one or more
Figure imgf000040_0002
Another embodiment provides that R14 is a 4- to 6-membered heterocyclic ring that is unsubstituted or substituted by (Ci-C4)alkyl. Examples of R14 include aminoethyl and pyrrolidinyl.
In certain embodiments, A is CH(OR13). One embodiment provides that R13 is (Ci- C/i)alkyl that is unsubstituted or substituted by a 4 or 6-membered heterocyclic ring. An example of R13 is 2-(pyrrolidin-l-yl)ethyl.
Another aspect of the invention provides a compound of Formula (Ib)
Figure imgf000040_0003
wherein
A is C=N-OR14, CH-CH=CH-R15, CH-SR16 or CH-S(O)R16;
R1 is H or -OH;
X is C(R10^ Or N-OR11J
One of R4 and R5 is H, and the other is H or -OR8;
R8 is H or substituted or unsubstituted (Ci-C4)alkyl;
R10, for each occurrence, is the same or different and is H or halogen;
R11 is H or (Ci-C4)alkyl; and
R14 is an amino(Ci-C4)alkyl optionally substituted by one or more (Ci-C4)alkyl; or a 4- to 6-membered heterocyclic ring optionally substituted by (Ci-C4)alkyl;
R15 and R16, each independently, are amino(Ci-C4)alkyl optionally substituted by one or more (Ci-C4)alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
One embodiment provides the compounds of Formula (Ib), wherein R1 is H. In another embodiment, R1 is OH.
In one embodiment, X is C(R10)2. Certain embodiments provide compounds of Formula (Ib) wherein R10 is H.
In another embodiment, X is N-OR11. One embodiment provides that R11 is H. In another embodiment, R11 is
Figure imgf000041_0001
One embodiment of the methods of the invention provides compounds of Formula (Ib) wherein A is C=N-OR14. One instance provides that R14 is an amino(Ci-C4)alkyl that is unsubstituted or substituted by one or more (Ci-C4)alkyl. In another instance, R14 is a 4- to 6- membered heterocyclic ring that is unsubstituted or substituted by (Ci-C/Oalkyl. Examples of R14 include aminoethyl and pyrrolidinyl.
Another embodiment of the methods of the invention provides compounds of Formula (Ib) wherein A is CH-CH=CH-R15. One instance provides that R15 is amino(Ci-C4)alkyl.
Certain embodiments of the methods of the invention provide compounds of Formula (Ib) wherein A is CH-SR16. Another embodiment provides that A is CH-S(O)R16. Instances provide that R16 is amino(Ci-C4)alkyl.
In another aspect, the invention provides a compound of Formula (Ic)
Figure imgf000041_0002
wherein
R1 is H or -OH; C One of R2 and R3 is H, and the other is
Figure imgf000041_0003
optionally substituted by hydroxyl or methoxy;
One of R4 and R5 is H and the other is -OR8; or
R4 and R5 together with the carbon to which they are attached form C=O;
R8 is H or substituted or unsubstituted (Ci_C4)alkyl; and R14 is an amino(Ci_C4)alkyl optionally substituted by one or more
Figure imgf000042_0001
or a 4- to 6-membered heterocyclic ring optionally substituted by (Ci_C4)alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
In one embodiment, R1 is H. Another embodiment provides that R1 is -OH.
One embodiment provides compounds of Formula (Ic) wherein one of R2 and R3 is H and the other is
Figure imgf000042_0002
that is substituted by hydroxyl or methoxy. Certain embodiments provide that one of R2 and R3 is H and the other is -CH2OH.
In another embodiment, R4 and R5 together with the carbon to which they are attached form C=O. In one embodiment, one of R4 and R5 is H and the other is -OR8, wherein R8 is H, or substituted or unsubstituted (Ci-C/Oalkyl.
One embodiment provides compounds of Formula (Ic) wherein R14 is an amino(Cr C/i)alkyl that is unsubstituted or substituted by one or more
Figure imgf000042_0003
In another embodiment, R14 is a 4- to 6-membered heterocyclic ring that is unsubstituted or substituted by one or more
Figure imgf000042_0004
Examples of R14 include aminoethyl and pyrrolidinyl.
Another aspect of this invention provides compounds of Formula (Id)
Figure imgf000042_0005
wherein R1 is H or -OH;
R4 and R5 taken together with the carbon atom to which they are attached form C(R10)2 ;
R10, for each occurrence, is the same or different and is H or halogen; and
R14 is an amino(Ci-C4)alkyl optionally substituted by one or more
Figure imgf000042_0006
or a 4- to 6-membered heterocyclic ring optionally substituted by
Figure imgf000042_0007
and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
In one embodiment, R1 is H. In another embodiment, R1 is -OH.
In certain embodiments, R10 is H. In another embodiment, R10 is halogen, preferably, F.
In one embodiment, R14 is an amino(Ci-C4)alkyl that is unsubstituted or substituted by one or more (Ci-C4)alkyl. In another embodiment, R14 is a 4- to 6-membered heterocyclic ring that is unsubstituted or substituted by (Ci-C4)alkyl. Examples of R14 include aminoethyl and pyrrolidinyl.
In another aspect, the methods of the invention include administering a compound of Formula (Ie):
Figure imgf000043_0001
wherein
R1 is H or -OH;
A is CH(OR13), C=N-OR14, CH-CH=N-OR15, C=CH-CH=N-OR15, CH-CH=CH-R15, CH-SR16, or CH-S(O)R16;
R13 is (Ci-C4)alkyl optionally substituted by a 4 or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, amino(Ci-C4)alkoxyl, (Ci-C4)alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; amino(Ci-C4)alkyl optionally substituted by one or more (Ci-C4)alkyl; or amino(Ci-C4)acyl optionally substituted by (Ci- C4)alkyl;
R14 is an amino(Ci-C4)alkyl optionally substituted by one or more (Ci-C4)alkyl; or a 4- to 6-membered heterocyclic ring optionally substituted by (Ci-C4)alkyl;
R15 and R16, each independently, are amino(Ci-C4)alkyl optionally substituted by one or more (Ci-C4)alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
In one embodiment, R1 is H.
In one embodiment, A is CH-CH=N-OR15. In another embodiment, A is C=CH-CH=N- OR15. In certain embodiments, R15 is an amino(Ci-C4)alkyl. In another embodiment, R15 is amino(Ci-C4)alkyl substituted by one or more (Ci-C4)alkyl.
The compounds used in accordance with the methods of the invention include the compounds shown in Table 1 as follows:
Table 1
Figure imgf000044_0001
Figure imgf000045_0001
44
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Specific compounds of the invention include the following compounds, the structures of which are shown in Table 2: (1 OR, 13 S)-3 -(2-aminoethoxyimino)- 10,13 -dimethyldodecahydro- 1 Hcyclopenta[a]phenanthrene-
6,17(10H,14H)-dione (l)
( 1 OR, 13 S)- 10, 13 -dimethyl-3 -((R)-pyrrolidin-3 -yloxyimino)dodecahydro- 1 H-cyclopenta
[a]phenanthrene-6, 17(1 OH, 14H)-dione (2)
( 1 OR, 13 S)-3 -(2-aminoethoxyimino)- 10,13 -dimethyl-6-methylenetetradecahydro- IH- cyclopenta[a]phenanthren-17(2H)-one (3)
( 1 OR, 13S)-IO513 -dimethyl-6-methylene-3 -(pyrrolidin-3 -yloxyimino)tetradecahydro- IH- cyclopenta[a]phenanthren- 17(2H)-one (4)
( 1 OR, 13 S)-3 -(2-aminoethoxyimino)-6-(hydroxyimino)- 10,13 -dimethyltetradecahydro- 1 H- cyclopenta[a]phenanthren- 17(2H)-one (5) (1 OR, 13 S)-3 -(2-aminoethoxyimino)-6-(methoxyimino)- 10, 13 -dimethyltetradecahydro- 1 H- cyclopenta[a]phenanthren- 17(2H)-one (6)
(5S, 1 OR, 13S)-5-hydroxy-l 0, 13-dimethyl-6-methylene-3-(pyrrolidin-3-yloxyimino) tetradecahydro- 1 H-cyclopenta[a]phenanthren- 17(2H)-one (7)
(5S, 1 OR, 13S)-3-(2-aminoethoxyimino)-5-hydroxy-l 0, 13-dimethyl-6-methylenetetradecahydro- lH-cyclopenta[a]phenanthren-17(2H)-one (8)
(6S, 1 OR, 13S)-3-(2-aminoethoxyimino)-6-(hydroxymethyl)- 10, 13 -dimethyldodecahydro- 1 H- cyclopenta[a]phenanthrene-7, 17(2H,8H)-dione (9)
(6S, 1 OR, 13 S)-6-(hydroxymethyl)- 10,13 -dimethyl-3 -(pyrrolidin-3 -yloxyimino)dodecahydro- 1 H- cyclopenta[a]phenanthrene-7, 17(2H,8H)-dione (10) (6S,7S, 1 OR, 13S)-7-hydroxy-6-(hydroxymethyl)-l 0, 13-dimethyl-3-(pyrrolidin-3-yloxyimino) tetradecahydro- 1 H-cyclopenta[a]phenanthren- 17(2H)-one (11)
(6S,7S, 1 OR, 13S)-3-(2-aminoethoxyimino)-7-hydroxy-6-(hydroxymethyl)-l 0, 13- dimethyltetradecahydro- 1 H-cyclopenta[a]phenanthren- 17(2H)-one (12) ( 1 OS, 13 S)-3 -(2-aminoethoxyimino)- 10,13 -dimethyl-7-methylenetetradecahydro- IH- cyclopenta[a]phenanthren-17(2H)-one (13)
( 1 OS, 13 S)-3 -(2-aminoethoxyimino)-7-(difluoromethylene)- 10,13 -dimethyltetradecahydro- 1 H- cyclopenta[a]phenanthren- 17(2H)-one (14)
(3 S,5S, 1 OR, 13S)- 10, 13-Dimethyl-3 -(2-(pyrrolidin- 1 -yl)ethoxy)dodecahydro- 1 H- cyclopenta[a]phenanthrene-6,17(10H,14H)-dione (15)
(Z)-6, 17-Dihydroxy- 10,13 -dimethylhexadecahydro- 1 H-cyclopenta[a]phenanthrene-3 - carbaldehyde O-2-aminoethyl oxime (16)
2-((5S,6S, 1 OR, 13 S, 17S)-6, 17-dihydroxy- 10, 13-dimethylhexahydro- 1 H-cyclopenta-
[a]phenanthren-3 (2H,4H, 1 OH, 12H, 13H, 14H, 15H, 16H, 17H)-ylidene)acetaldehyde 0-2- (dimethylamino) ethyl oxime (17)
(5S, 1 OR, 13S)-3-(5-Aminopent- 1 -enyl)-6-(hydroxyimino)- 10, 13 -dimethyltetradecahydro- 1 H- cyclopenta[a]phenanthren- 17(2H)-one (18)
(3R,5R, 1 OR, 13 S)-3 -(3 - Aminopropylthio)- 10,13 -dimethyl-6-methylenetetradecahydro- 1 H- cyclopenta[a]phenanthren- 17(2H)-one (19) (5R, 1 OR, 13 S)-3 -(3 -aminopropylsulfmyl)- 10,13 -dimethyl-6-methylenetetradecahydro- 1 H- cyclopenta[a]phenanthren- 17(2H)-one (20)
( 1 OR, 13 S)-3-((R)-pyrrolidin-3-yloxyimino)-6-(methoxyimino)- 10,13 dimethyltetradecahydro- lH-cyclopenta[a]phenanthren-17(2H)-one (21)
Table 2
Figure imgf000054_0001
Figure imgf000055_0001
The structures of some of the compounds of the invention include asymmetric carbon atoms. Accordingly, the isomers arising from such asymmetry {e.g., all enantiomers and diastereomers) are included within the scope of this invention, unless indicated otherwise. Such isomers can be obtained in substantially pure form by classical separation techniques and/or by stereochemically controlled synthesis.
Naturally occurring or synthetic isomers can be separated in several ways known in the art. Methods for separating a racemic mixture of two enantiomers include chromatography using a chiral stationary phase (see, e.g., , "Chiral Liquid Chromatography," WJ. Lough, Ed. Chapman and Hall, New York (1989)). Enantiomers can also be separated by classical resolution techniques. For example, formation of diastereomeric salts and fractional crystallization can be used to separate enantiomers. For the separation of enantiomers of carboxylic acids, the diastereomeric salts can be formed by addition of enantiomerically pure chiral bases, such as brucine, quinine, ephedrine, strychnine, and the like. Alternatively, diastereomeric esters can be formed with enantiomerically pure chiral alcohols, such as menthol, followed by separation of the diastereomeric esters and hydrolysis to yield the free, enantiomerically enriched carboxylic acid. For separation of the optical isomers of amino compounds, addition of chiral carboxylic or sulfonic acids, such as camphorsulfonic acid, tartaric acid, mandelic acid, or lactic acid can result in formation of the diastereomeric salts.
Uses of the Compounds of the Invention
The invention also provides methods for treating a subject for a neoplasia by administering to the subject an effective amount of a compound of formula (I) or otherwise described herein. In certain embodiments, the subject is a mammal, in particular a human.
In accordance with the methods of the invention, compounds are administered in combination with a pharmaceutically diluent or acceptable carrier. In one embodiment, the compound can be administered using a pharmaceutically acceptable formulation. In advantageous embodiments, the pharmaceutically-acceptable carrier provides sustained delivery of the compound to a subject for at least four weeks after administration to the subject.
In certain embodiments, the compound is administered orally. In other embodiments, the compound is administered intravenously. In yet other embodiments, the compound is administered topically. In still other embodiments, the compound is administered topically or parenterally. Although dosages may vary depending on the particular indication, route of administration and subject, the compounds are typically administered at a concentration of about 0.1 μg to 20 milligram per kilogram of body weight per day (mg/kg/day) (e.g., 0.1 μg/kg to 2mg/kg, 0.3-3μg/kg, 0.18-0.54mg/kg). In other embodiments, the amount varies from about 0.1 mg/kg/day to about 100 mg/kg/day. In still other embodiments, the amount varies from about 0.001 μg to about 100 μg/kg (e.g., of body weight).
Determination of a therapeutically effective amount or a prophylactically effective amount of a compound described herein can readily by one skilled in the art. The dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated and the particular compound being employed. In determining the therapeutically effective amount or dose, and the prophylactically effective amount or dose, a number of factors are considered, including, but not limited to: the specific hyperplastic/neoplastic cell involved; pharmacodynamic characteristics of the particular agent and its mode and route of administration; the desired time course of treatment; the species of mammal; its size, age, and general health; the specific disease involved; the degree of or involvement or the severity of the disease; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the kind of concurrent treatment (i.e., the interaction of the compounds of the invention with other co-administered therapeutics); and other relevant circumstances. U.S. Patent 5,427,916, for example, describes method for predicting the effectiveness of antineoplastic therapy in individual patients, and illustrates certain methods which can be used in conjunction with the treatment protocols of the instant invention.
Treatment can be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage should be increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired. A therapeutically effective amount and a prophylactically effective amount of a compound of the invention is expected to vary from about about 0.1 μg to 20 milligram per kilogram of body weight per day (mg/kg/day) (e.g., O.lμg/kg to lOmg/kg, 0.1-10μg/kg, 0.1-lmg/kg). In other embodiments, the amount varies from about 0.1 mg/kg/day to about 100 mg/kg/day. In still other embodiments, the amount varies from about 0.001 μg to about 100 μg/kg (e.g., of body weight). Compounds which are determined to be effective for the prevention or treatment of neoplasias in animals, e.g., dogs, rodents, may also be useful in treatment of neoplasias in humans. Those skilled in the art of treating neoplasias in humans will know, based upon the data obtained in animal studies, the dosage and route of administration of the compound to humans. In general, the dosage and route of administration in humans is expected to be similar to that in animals.
The identification of those patients who are in need of prophylactic treatment for hyperplastic/neoplastic disease states is well within the ability and knowledge of one skilled in the art. Certain of the methods for identification of patients who are at risk of developing neoplastic disease states which can be treated by the subject method are appreciated in the medical arts, such as family history of the development of a particular disease state and the presence of risk factors associated with the development of that disease state in the subject patient. A clinician skilled in the art can readily identify such candidate patients, by the use of, for example, clinical tests, physical examination and medical/family history.
Another aspect of the invention comprises obtaining the compound of the invention.
Neoplasia
The invention features methods for inhibiting the proliferation, growth, or viability of a neoplastic cell by contacting the cells with a compound of formula (I) or otherwise described herein. In general, the method includes a step of contacting a neoplastic cell with an effective amount of a compound of the invention. The present method can be performed on cells in culture, e.g., in vitro or ex vivo, or can be performed on cells present in an animal subject, e.g., as part of an in vivo therapeutic protocol. The therapeutic regimen can be carried out on a human or other subject.
The compounds of the invention or otherwise described herein can be tested initially in vitro for their inhibitory effects on the proliferation or survival of neoplastic cells. Examples of cell lines that can be used are lung cancer cell lines (e.g., H460, EKVX, A549), breast cancer cell lines (e.g., MCF7, T47D), CNS cancer cell lines (e.g., SF268, U251, SF295), colon cancer cell lines (e.g., HCTl 16, HCT15), prostate (e.g., PC3, DU145), ovarian cancer cell lines (e.g., IGROVl, 0VCAR5, 0VCAR3, NCI-ADRRES), pancreatic cancer cell lines (e.g., SU8686), renal cancer cell lines (e.g., CAKI), and melanoma cancer cell lines (e.g., LOXIMVI, SKMEL28, MB435, UACC62). Alternatively, the antineoplastic activity of compounds of the invention can be tested in vivo using various animal models known in the art. For example, xenographs of human neoplastic cells or cell lines, such as PC-3 or A549 cells, are injected into immunodefϊcient mice (e.g., nude or SCID) mice. Compounds of the invention are then administered to the mice and the growth and/or metastasis of the tumor is compared in mice treated with a compound of the invention relative to untreated control mice. Agents that reduce the growth or metastasis of a tumor or increase mice survival are identified as useful in the methods of the invention.
The methods discussed herein can be used to inhibit the proliferation of virtually any neoplastic cell. Neoplasia growth is typically uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells. Neoplasias can affect a variety of cell types, tissues, or organs, including but not limited to an organ selected from the group consisting of bladder, bone, brain, breast, cartilage, glia, esophagus, fallopian tube, gallbladder, heart, intestines, kidney, liver, lung, lymph node, nervous tissue, ovaries, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, and vagina, or a tissue or cell type thereof. Neoplasias include cancers, such as acoustic neuroma, acute leukemia, acute lymphocytic leukemia, acute monocytic leukemia, acute myeloblasts leukemia, acute myelocytic leukemia, acute myelomonocytic leukemia, acute promyelocytic leukemia, acute erythroleukemia, adenocarcinoma, angiosarcoma, astrocytoma, basal cell carcinoma, bile duct carcinoma, bladder carcinoma, brain cancer, breast cancer, bronchogenic carcinoma, cervical cancer, chondrosarcoma, chordoma, choriocarcinoma, chronic leukemia, chronic lymphocytic leukemia, chronic myelocytic leukemia, colon cancer, colon carcinoma, craniopharyngioma, cystadenocarcinoma, embryonal carcinoma, endotheliosarcoma, ependymoma, epithelial carcinoma, Ewing's tumor, glioma, heavy chain disease, hemangioblastoma, hepatoma, Hodgkin's disease, large cell carcinoma, leiomyosarcoma, liposarcoma, lung cancer, lung carcinoma, lymphangioendotheliosarcoma, lymphangiosarcoma, macroglobulinemia, medullary carcinoma, medulloblastoma, melanoma, meningioma, mesothelioma, myxosarcoma, neuroblastoma, non-Hodgkin's disease, oligodendroglioma, osteogenic sarcoma, ovarian cancer, pancreatic cancer, papillary adenocarcinomas, papillary carcinoma, pinealoma, polycythemia vera, prostate cancer, rhabdomyosarcoma, renal cell carcinoma, retinoblastoma, schwannoma, sebaceous gland carcinoma, seminoma, small cell lung carcinoma, squamous cell carcinoma, sweat gland carcinoma, synovioma, testicular cancer, uterine cancer, Waldenstrom's fibrosarcoma, and Wilm's tumor.
Multidrug Resistant Neoplasias As exemplified by results obtained with istaroxime, compounds 2 and 4, neoplasias that are resistant or refractory to anti-neoplastic therapies are likely to be susceptible to treatment with the compounds delineated herein. Neoplasias that display resistance to a wide variety of chemotherapeutic agents are described as multidrug resistant. Multidrug resistant neoplasias are characterized by their ability to resist treatment with compounds having diverse structures and mechanisms of action. Multidrug resistance is generally related to alterations in a family of proteins known as ATP -binding cassette (ABC) transporters.
Multidrug resistant neoplasias often display increased expression of ATP-binding cassette (ABC) transporters, which function as ATP-dependent efflux pumps. These pumps actively transport a wide array of anti-cancer and cytotoxic drugs out of the cell. In mammals, the superfamily of ABC transporters includes P-glycoprotein (P-gp) transporters (MDRl and
MDR3 genes in human), the MRP subfamily, and bile salt export protein (ABCBl 1; Cancer Res (1998) 58, 4160-4167), MDR-3 (Nature Rev Cancer (2002) 2, 48-58), lung resistance protein (LRP) and breast cancer resistant protein (BCRP). These proteins can recognize and efflux numerous substrates with unrelated chemical structures, including many chemotherapeutics. Other causes of multidrug resistance have been attributed to changes in topoisomerase II, protein kinase C and specific glutathione transferase enzymes.
In particular embodiments, compounds of the invention (e.g., istaroxime, compounds 2 and 4) are particularly useful for neoplasias showing alterations in the activity or expression of MDRl, MDR2, or P-gP). In particular embodiments, the drug resistance of the tumor is mediated through the overexpression of P-gp. Numerous mechanisms can lead to over expression of P-gp, including amplification of the MDR-I gene (Anticancer Res (2002) 22, 2199-2203), increased transcription of the MDR-I gene (J Clin Invest (1995) 95, 2205-2214; Cancer Lett (1999) 146, 195-199; Clin Cancer Res (1999) 5, 3445-3453; Anticancer Res (2002) 22, 2199-2203), by mutations in the MDR-I gene (Cell (1988) 53, 519-529; Proc Natl Acad Sci USA (1991) 88, 7289-7293; Proc Natl Acad Sci USA (1992) 89, 4564-4568) and chromosomal rearrangements involving the MDR-I gene (J Clin Invest (1997) 99, 1947-1957). Therapeutic agents to which resistance is conferred via the action of P-gp include, but are not limited to: vinca alkaloids (e.g., vinblastine), the anthracyclines (e.g., adriamycin, doxorubicin), the epipodophyllotoxins (e.g., etoposide), taxanes (e.g., paclitaxel, docetaxel), antibiotics (e.g., actinomycin D and gramicidin D), antimicrotubule drugs (e.g., colchicine), protein synthesis inhibitors (e.g., puromycin), toxic peptides (e.g., valinomycin), topoisomerase Inhibitors (e.g., topotecan), DNA intercalators (e.g., ethidium bromide) and anti-mitotics. See WO 99/20791. The methods and pharmaceutical compositions of the present invention are useful for treating tumors resistant to any one or more of above- listed drugs.
In still other embodiments, the methods of the invention are useful for treating resistant or refractory neoplasias, where the resistance is conferred by an alteration in a topoisomerase (e.g., topoisomerase II), protein kinase C and specific glutathione transferase enzyme. Methods of the invention are also useful for the treatment of neoplasias showing resistance to taxanes (e.g., paclitaxel and docetaxel). Such resistance is typically mediated by alterations in tubulin. In other embodiments, compounds delineated herein are useful for treating neoplasias that are refectory to platinum-based chemotherapeutic agents, including carboplatin, cisplatin, oxaliplatin, iproplatin, tetraplatin, lobaplatin, DCP, PLD-147, JMl 18, JM216, JM335, and satraplatin. Such platinum-based chemotherapeutic agents also include the platinum complexes disclosed in EP 0147926, U.S. Pat. No. 5,072,011, U.S. Pat. Nos. 5,244,919, 5,519,155, 6,503,943 (LA- 12/PLD- 147), 6350737, and WO 01/064696 (DCP). In sum, the methods and pharmaceutical compositions of the invention are generally useful for treating resistant and/or refractory neoplasias to any one or more of drugs known in the art or described herein. In particular embodiments, the methods and compositions of the invention are useful for the treatment of patients having end-stage disease, which includes patients for whom no effective therapeutic regimen exists or patients identified as having less than about 3, 6, 9 or 12 months to live.
Combination Therapies
In certain embodiments, the compounds of the invention are administered in combination with any other standard anti-neoplasia therapy or conventional chemotherapeutic agent, such as an alkylating agent; such methods are known to the skilled artisan and described in Remington's Pharmaceutical Sciences by E. W. Martin. For example, if desired, agents of the invention (e.g., istaroxime) are administered in combination with any conventional antineoplastic therapy, including but not limited to, surgery, radiation therapy, or chemotherapy. Conventional chemotherapeutic agents include, but are not limited to, abiraterone, alemtuzumab, altretamine, aminoglutethimide, amsacrine, anastrozole, azacitidine, bleomycin, bicalutamide, busulfan, capecitabine, carboplatin, carmustine, celecoxib, chlorambucil, 2- chlorodeoxyadenosine, cisplatin, colchicine, cyclophosphamide, cytarabine, Cytoxan, dacarbazine, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubicin, estramustine phosphate, etodolac, etoposide, exemestane, floxuridine, fludarabine, 5-fluorouracil, flutamide, formestane, gemcitabine, gentuzumab, goserelin, hexamethylmelamine, hydroxyurea, hypericin, ifosfamide, imatinib, interferon, irinotecan, letrozole, leuporelin, lomustine, mechlorethamine, melphalen, mercaptopurine, 6-mercaptopurine, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, paclitaxel, pentostatin, procarbazine, raltitrexed, rituximab, rofecoxib, streptozocin, tamoxifen, temozolomide, teniposide, 6-thioguanine, topotecan, toremofme, trastuzumab, vinblastine, vincristine, vindesine, and vinorelbine. In particular embodiments, a combination of the invention comprises any one or more of the following: vinca alkaloids (e.g., vinblastine), taxanes (e.g., paclitaxel, docetaxel), epothilones (e.g., ixabepilone), antifolates (e.g., Methotrexate), purine analogs (e.g., fludarabine), pyrimidine analogs (e.g., gemcitabine), DNA intercalators (e.g., ethidium bromide), topoisomerase Inhibitors (e.g., topotecan), alkylating agents (e.g., carmustine, bendamustine), platinum-based agents (e.g., cisplatin, oxaliplatin), receptor antagonists (e.g, atrasentan), hormone agents (e.g. anti-androgens, aromatase inhibitors), anthracyclines (e.g., adriamycin, doxorubicin), epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., actinomycin D and gramicidin D), antimicrotubule drugs (e.g., colchicine), protein synthesis inhibitors (e.g., puromycin), toxic peptides (e.g., valinomycin), enzyme inhibitors (e.g. CDK inhibitors) and anti-mitotics.
Pharmaceutical Compositions
The invention also provides pharmaceutical compositions for the treatment of a neoplasia, comprising an effective amount a compound of the invention and a pharmaceutically acceptable carrier. In particular embodiments, compositions of the invention comprise a compound described herein in combination with a conventional chemotherapeutic agent. In still other embodiments, such compositions are labeled for the treatment of cancer. In a further embodiment, the effective amount is effective to reduce the growth, proliferation, or survival of a neoplastic cell or to otherwise treat or prevent a neoplasia in a subject, as described herein.
In an embodiment, the compound is administered to the subject using a pharmaceutically-acceptable formulation. In certain embodiments, these pharmaceutical compositions are suitable for oral or parenteral administration to a subject. In still other embodiments, as described in detail below, the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non- aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound. In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human.
The methods of the invention further include administering to a subject a therapeutically effective amount of a compound in combination with a pharmaceutically acceptable excipient. The phrase "pharmaceutically acceptable" refers to those compounds of the invention, compositions containing such compounds, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase "pharmaceutically-acceptable excipient" includes pharmaceutically- acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, solvent or encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen- free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions. Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfϊte, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. Compositions containing a compound(s) include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration. The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
Methods of preparing these compositions include the step of bringing into association a compound(s) with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
Compositions of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in- water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound(s) as an active ingredient. A compound may also be administered as a bolus, electuary or paste.
In solid dosage forms of the invention for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.
The tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical- formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
Liquid dosage forms for oral administration of the compound(s) include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifϊers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
Suspensions, in addition to the active compound(s) may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
Pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compound(s) with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
Compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
Dosage forms for the topical or transdermal administration of a compound(s) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound(s) may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
The ointments, pastes, creams and gels may contain, in addition to compound(s) of the present invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. Powders and sprays can contain, in addition to a compound(s), excipients, such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
The compound(s) can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound.
Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically-acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (T weens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids, such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions. Transdermal patches have the added advantage of providing controlled delivery of a compound(s) to the body. Such dosage forms can be made by dissolving or dispersing the agent in the proper medium. Absorption enhancers can also be used to increase the flux of the active ingredient across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the active ingredient in a polymer matrix or gel. Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this invention.
Pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compound(s) in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drag form is accomplished by dissolving or suspending the drag in an oil vehicle.
Injectable depot forms are made by forming microencapsule matrices of compound(s) in biodegradable polymers, such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
When the compound(s) are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to
99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically-acceptable carrier.
Regardless of the route of administration selected, the compound(s), which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
Actual dosage levels and time course of administration of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. An exemplary dose range is from about about 0.1 μg to 20 milligram per kilogram of body weight per day (mg/kg/day) (e.g., O.lμg/kg to 10mg/kg, 0.1-lOμg/kg, 0.1-1 mg/kg). In other embodiments, the amount varies from about 0.1 mg/kg/day to about 100 mg/kg/day. In still other embodiments, the amount varies from about 0.001 μg to about 100 μg/kg (e.g., of body weight). Ranges intermediate to the above-recited values are also intended to be part of the invention.
Kits
The invention provides kits for the treatment or prevention of neoplasia. In one embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of a compound of the invention in unit dosage form. In some embodiments, a compound of the invention is provided in combination with a conventional chemotherapeutic agent. In other embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
If desired a compound of the invention is provided together with instructions for administering the compound to a subject having or at risk of developing neoplasia. The instructions will generally include information about the use of the composition for the treatment or prevention of neoplasia. In other embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of ischemia or symptoms thereof; precautions; warnings; indications; counter- indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase
Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.
EXEMPLIFICATION OF THE INVENTION
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.
I. CHEMICAL EXAMPLES - SYNTHESIS AND METHODS OF PREPARATION
Compounds of the invention can be synthesized by methods described in this section, the examples, and the chemical literature. A. Preparation of Intermediates
2-Aminoethoxy-amine dihydrochloride (Intermediate (I))
Figure imgf000069_0001
1) NH2NH2, EtOH ,0,
H7N' SNH7 2HCl
2) 6N HC1
Intermediate (I)
Yield 75 °/ Scheme for Preparation of Intermediate (I)
Intermediate (I) was prepared according to the scheme described above. The detailed reaction procedures were described in M. C. Pankaskie et ah, An Improved Synthetic Route to Aminoxypropylamine (APA) and Related Homologs , Synthetic Communications vol 19 (3&4), pages 339-344 (1989).
3(Λ)-Pyrrolidinyloxyamine dihydrochloride (Intermediate (II))
Figure imgf000069_0002
N-terf-butoxycarbonyl-(S)-pyrrolidinol
Figure imgf000069_0003
B
Scheme for Preparation of Intermediate (II)
To a solution of N-tert-butoxycarbonyl-(S)-pyrrolidinol (10.0 g) and triethylamine (8.2 mL) in CH2CI2 (150 mL) at 0° C, methanesulfonyl chloride (4.34 mL) was added. After stirring at room temperature for 3 h, the reaction mixture was poured into ice/water and extracted with CH2CI2. The organic phase was washed with 5% aqueous NaHCθ3, water, brine, dried and evaporated to dryness to give an oil which solidified after standing overnight in the refrigerator. The solid was triturated with Et2O to give N-tert-butoxycarbonyl-(S)-3-pyrrolidinyl methansulfonate (13.0 g, 92%) (Compound A) as a light yellow solid. 1H-NMR (300 MHz, DMSO-de, ppm from TMS): δ 5.23 (IH, m), 3.60-3.10 (4H, m), 3.23 (3H, s), 2.11 (2H, m), 1.39 (9H, s).
To a suspension of KOH powder (4.86 g) in DMSO (250 mL) under vigorous stirring, benzophenone oxime (7.86 g) was added. After stirring at room temperature for 30 min, a solution of N-tert-butoxycarbonyl-(S)-3-pyrrolidinyl methansulfonate (10 g) (Compound A) in DMSO (70 mL) was added. After 18 h at room temperature the reaction was poured into iced water (900 mL) and extracted with Et2O. The combined organic layers were washed with water, brine, dried and the solvent evaporated. Benzophenone O- [(R)-3-pyrrolidinyl] oxime (Compound B) was obtained (13.0 g, 96%) as a white solid and used without purification in the next step. l H-NMR (300 MHz, DMSO-d6, ppm from TMS): δ 7.50-7.20 (1OH, m), 4.84 (IH, m), 3.50-3.00 (4H, m), 2.01 (2H, m), 1.38 (9H, s); 13C-NMR (75 MHz, CDCl3): 157.7, 154.6, 136.5, 133.1, 129.4, 129.3, 128.8, 128.2, 128.0, 127.9, 79.2, 51.2, 44.2, 28.5.
Compound B (13.0 g) was suspended in 6N HCl (250 mL) and the mixture was refluxed for 2 h. After cooling, the reaction was extracted with Et2O. The aqueous layer was evaporated to give a crude brown solid which was treated with 0.34 g of activated carbon in absolute EtOH (255 mL) at reflux for 2h. The solid obtained after evaporation was crystallized with 96% EtOH (40 mL) to give the 3(R)-pyrrolidinyloxyamine dihydrochloride (Intermediate (II)) (2.98 g, 72%), as an off white solid. l H- NMR (300 MHz, DMSO-dg, ppm from TMS): δ 11.22 (3H, bb), 9.74 (IH, bb), 9.54 (IH, bb), 4.98 (IH, m), 3.60-3.00 (4H, m), 2.40-2.00 (2H, m).
B. Preparation of Compounds of The Invention
The invention is further illustrated by the following examples which in no way should be construed as being further limiting.
EXAMPLE 1
Synthesis of HCl salt of (1 OR, 13S)-3-(2-aminoethoxyimino)-l 0, 13-dimethyldodecahydro- lHcyclopenta[a]phenanthrene-6,17(10H,14H)-dione (Compound (I))
Figure imgf000071_0001
HCl salt of Compound (1)
Preparation of HCl salt of Compound (1)
To a stirred solution of dehydroepiandrosterone (30.0 g, 104 mmol) ("DHEA") in THF (450 mL) at -100C under N2 was added IM BH3-THF complex in THF (260 mL, 260 mmol). After completing the addition, the mixture was stirred at room temperature for 3h. H2O (500 mL) was cautiously added dropwise followed by NaBO3*4H2O (31.4 g, 204 mmol). After stirring at room temperature overnight, the mixture was filtered. The solid was washed with THF and then discarded. The two liquid phases were separated and the aqueous layer was saturated with NaCl and extracted with THF (3 x 200 mL). The combined organic extracts were dried over NaCl and Na2SC>4, filtered, and evaporated to dryness. The crude product was crystallized from EtOAc/MeOH (2/1, 10 mL/g) to give a first crop as a white solid (12.5 g). The mother liquors were evaporated and the residue crystallized from EtOAc/MeOH (2/1, 10 mL/g) to give a second crop as a white solid (6.4 g). The procedure was repeated to give a third crop as a white solid as compound al (2.1 g; 75% overall yield). 1H NMR (600 MHz, CDCl3): δ 3.56 (m, IH), 3.46 (m, IH), 3.30 (m, IH), 2.16 (m, IH), 2.00 - 0.90 (m, 17H), 0.83 (s, 3H), 0.81 (m, IH), 0.71 (s, 3H), 0.66 (m, IH); mp 232-234 0C.
To a solution of compound al (21.0 g, 68 mmol) in acetone (380 mL) was added an excess of Jones reagent (83.5mL) dropwise, maintaining the temperature below 40 0C. 5min after completion of the addition, z'-PrOH (10 mL) was added and, after further 10 min, the suspension was filtered and the filtrate evaporated to dryness. The residue was treated with H2O
(300 mL) and extracted with EtOAc (3 x 100 mL). The combined organic extracts were washed with H2O (100 mL), 5% aqueous NaHCO3 solution (100 mL), H2O (100 mL), dried over Na2SO4 and evap o rate d to dryne s s to g ive ( 5 S , 1 O R, 1 3 S )-10,13-dimethyldecahydro-lH- cyclopenta[a]phen-anthrene-3,6,17(2H,10H,14H)-trione (compound a2) as a white solid (15.3 g, 74%); a sample was crystallized from i-PrOH to give a white solid. 1H NMR (600 MHz, CDCl3): δ 2.70 - 1.30 (m, 20H), 0.99 (s, 3H), 0.91 (s, 3H); mp 195-197 0C. (lit: 10b 196-196.5 0C).
To a stirred solution of compound a2 (1.83 g) in THF (40 mL), a solution of the 2- aminoethoxyamine dihydrochloride (Intermediate (I), 894_mg) in H2O (19 mL) was rapidly added dropwise. After 1.5 h, NaCl (45 mmol) was added and the mixture was stirred for 10 min. The phases were separated and the aqueous phase was extracted with THF (3x). The combined organic extracts were dried over Na2SO/), filtered, and evaporated to give an oily residue. The crude product was dissolved in CH2Cl2 (0.15 M) and washed with a saturated aqueous solution of NaCl (3x). The organic layer was dried again over Na2Sθ4 and evaporated to dryness. The crude product was crystallized from MeOH/EtOAc to afford the HCl salt of (1OR, 13S)-3-(2- aminoethoxyimino)- 10, 13 -dimethyldodecahydro- 1 Hcyclopenta[a]phenanthrene-6, 17(1 OH, 14H)- dione (HCl salt of compound (I)) in 56% yield as a white solid, mp 220-225 0C (dec). 1H-NMR (600 MHz, DMSO-dg): δ 9.38 (2H, bs), 4.73 (IH, m), 3.35 - 2.90 (5H, m), 2.60 - 1.15 (21H, m), 0.79 and 0.76 and 0.76 (s and s and s, 6H). 13C-NMR (75 MHz, DMSO-dg): 219.5, 209.8, 209.6, 161.0, 160.9, 80.1, 80.0, 57.0, 55.9, 52.6, 52.4, 51.0, 49.6, 49.5, 47.9, 44.9, 43.7, 41.5, 37.8, 37.1, 37.0, 35.6, 31.4, 30.3, 26.9, 21.6, 20.9, 20.7, 13.9, 12.6, 12.5.
EXAMPLE 2
Synthesis of HCl salt of (1 OR, 13S)-10, 13-dimethyl-3-((R)-pyrrolidin-3-yloxyimino)dodecahydro- IH-cyclopenta [a] phenanthrene-6, 17(1 OH, 14H)-dione (Compound (2))
Figure imgf000072_0001
Preparation of HCl salt of Compound (2)
To a solution of 3-(R)-pyrrolidinyloxyamine dihydrochloride (Intermediate (II), 0.58g), and Na2HPO4 12H2O (2.38 g) in water (10 mL), a solution of (5S,10R,13S)-10,13-dimethyl- decahydro-lH-cyclopenta[a]phen-anthrene-3,6,17(2H,10H,14H)-trione (Compound a2) (1 g) in THF (20 mL) was added. After 2 hours at room temperature, NaCl (0.99 g) was added and stirred for 15 min. The mixture was extracted with THF (2 x 12.5 mL) and the combined organic phases were washed with brine (3 x 19 mL), dried over Na2 SO4 and evaporated to dryness. The HCl salt of compound (2) was obtained as a white solid after washing of the residue with EtOAc, and filtration (1.00 g, 72%). 1H NMR (600 MHz, DMSO-d6): δ 9.38 (2H, bs), 4.73 (IH, m), 3.35 - 2.90 (5H, m), 2.60 - 1.15 (21H, m), 0.79 (3H, s), 0.76 (3H, s).
EXAMPLE 3
Synthesis of HCl salt of (10R,l 3S)-3-(2-aminoethoxyimino)-10,l 3-dimethyl-6- methylenetetradecahydro-lH-cyclopenta[a]phenanthren-l 7(2H)-one (Compound (3))
Figure imgf000073_0001
Preparation of HCl salt of Compound (3)
To a stirred solution of androstane-3/3,6α,17/3-triol (bl) (2.6 g, 8.43 mmol) in acetone/H2O/pyridine (256/52/1.4 mL), NBS (freshly crystallized from H2O; 6 g, 33.72 mmol) was added. After stirring at room temperature for 18 h, the solution was quenched with cone. HCl (0.8 mL) to pH =3 and, after 10 min, NaOH (small beads, 1 g, 0.025 mmol) was added to pH= 8. The organic solvent was evaporated and the aqueous phase was extracted with EtOAc (4 x 50 mL). The combined organic extracts were dried over Na2Sθ4 and evaporated to dryness to give a brown solid. The crude product was triturated with H2O (2 x 50 mL) and filtered. After drying under vacuum at 400C, 6α-hydroxyandrostane-3,17-dione (compound b2) (2.22 g, 86%) was obtained as an off-white solid. 1H NMR (600 MHz, CD3OD): δ 3.45 (m, IH), 2.61 (m, IH), 2.55 - 1.20 (m, 17H), 1.08 (s, 3H), 0.99 (m, IH), 0.89 (s, 3H), 0.85 (m, IH).
A solution of compound b2 (2.2 g, 7.22 mmol), ethylene glycol (14.9 mL, 267 mmol) and CSA (50 mg, 0.216 mmol) in cyclohexane (204 mL) was stirred at reflux for 12 h with a Dean-Stark trap. After cooling to room temperature, the mixture was neutralized with 5% aqueous NaHCO3 solution. The organic layer was separated and washed with brine (2 x 40 mL), dried over Na2SO4 and evaporated to dryness to give 3,3: 17,17-bis(ethylendioxy)androstan-6α- ol (compound b3) as a white solid (2.4 g, 85%). 1H NMR (600 MHz, CD3OD): δ 3.96 - 3.81 (m, 8H), 3.37 (m, IH), 2.05 - 1.20 (m, 18H), 1.08 (s, 3H), 0.88 (m, IH), 0.82 (s, 3H), 0.78 (m, IH). A solution of compound b3 (2.10 g, 5.36 mmol) and Dess-Martin periodinane (4.50 g, 10.6 mmol) in DCM (360 mL) was stirred at room temperature for 3 h and then quenched by addition of aqueous NaHCO3ZNaS2O3 solution (20 ml/60 ml). After stirring for 10 min, the mixture was filtered from celite and the cake was washed with DCM . The layers were separated, and the aqueous phase was extracted with DCM (3x). The combined organic extracts were washed with aqueous NaHCO3 and brine respectively, dried over Na2SO4 and evaporated to dryness to give 3,3:17,17-bis(ethylendioxy)androstane-6-one (compound b4) as a white foam (2.09 g, 99%). 1H NMR (600 MHz, CDCl3): δ 3.90 - 3.78 (m, 8H), 2.44 (m, IH), 2.25 (m, IH), 1.92 - 1.20 (m, 18H), 0.77 (s, 3H), 0.70 (s, 3H).
To a stirred suspension of methyltriphenylphosphonium bromide (8.03 g) in dry THF (61 mL) cooled at 0 0C under Ar, potassium tert-butoxide (2.44 g) was added. After stirring for 10 min, a solution of 3,3:17,17- bis(ethylendioxy)androstane-6-one (compound b4) (2.08 g) in dry THF (6OmL) was added dropwise at room temperature over 0.5 h. After 1.5 h at room temperature, the mixture was quenched by addition of 5% NaH2PO4 aqueous solution and extracted with Et2O (2 x 6OmL). The combined organic extracts were washed with 5% NaH2PO4 aqueous solution, brine, dried over Na2SO4 and evaporated to dryness. The residue was purified by flash chromatography (SiO2, 15% Ethyl Acetate / Petroleum Ether) to give 3,3:17,17- bis(ethylendioxy)-6-methyleneandrostane (compound b5) (1.84 g, 89 %). 1H NMR (600 MHz, CDCl3): δ 4.69 (m, IH), 4.39 (m, IH), 3.96 - 3.81 (m, 8H), 2.27 (m, IH), 2.03 (m, IH), 1.97 (m, IH), 1.81 - 1.20 (m, 15H), 0.96 (m, IH), 0.86 (m, IH), 0.82 (s, 3H), 0.68 (s, 3H).
A solution of 3,3:17,17-bis(ethylendioxy)-6-methyleneandrostane (compound b5) (1.84 g) and pTSA " H2O (4.93 g) in acetone (213 mL) was stirred at room temperature for 4.5 h. The solution was neutralized by addition of 5% aqueous NaHCO3 and acetone was evaporated. The aqueous suspension was extracted with CH2Cl2 (3x). The combined organic extracts were washed with H2O, dried over Na2SO4 and evaporated to dryness to give 6-methyleneandrostane- 3,17-dione (compound (b6)) (1.28 g) in 90% yield. 1H NMR (600 MHz, CDCl3): δ 4.85 (s, IH), 4.47 (s, IH), 2.51 - 1.20 (m, 19H), 0.99 (m, IH), 0.91 (s, 3H), 0.88 (s, 3H).
Prepared in 90% yield as described in Example 1 for compound (1) starting from 6- methyleneandrostane-3,17-dione (compound b6) and 2-aminoethoxyamine dihydrochloride (Intermediate (I)). The crude product was triturated with Et2O to yield the HCl salt of the title compound (Compound (3)). 1H NMR (600 MHz, CDCl3) δ 8.47 (bs, 3H), 4.83 (m, IH), 4.59 and 4.51 (s and s, IH), 4.26 (m, IH), 3.33 (bs, 2H), 3.20 (m, IH), 2.50 - 1.23 (m, 19H), 0.99 (m, IH), 0.86 (s, 3H), 0.80 (s, 3H). MS (ESI) : 359 [M-Cl]+ EXAMPLE 4
Synthesis of HCl salt of (10R,l 3S)-IO713-dimethyl-6-methylene-3-(pyrrolidin-3- yloxyimino)tetradecahydro-lH-cyclopenta[a]phenanthren-l 7(2H)-one (Compound (4))
(4)
Figure imgf000075_0001
Preparation of HCl salt of Compound (4)
The HCl salt of Compound (4) was prepared in 75% yield as described in Example 2 starting from 6-methyleneandrostane-3,17-dione (compound b6) and 3-(R)- pyrrolidinyloxy- amine dihydrochloride (Intermediate (II)). 13C-NMR (75 MHz, DMSOd6): 219.9, 161.7, 161.4,
148.7, 148.5,107.1, 80.0, 79.9, 53.9, 53.7, 50.8, 49.8, 49.6, 47.6, 43.9, 36.8, 36.7, 35.7, 31.6,
30.3, 27.6, 24.0, 21.7, 20.7, 13.9, 11.9, 11.8.; MS (ESI): 385.1 [M-Cl]+
EXAMPLE 5
Synthesis of HCl salt of (1 OR, 13S)-3-(2-aminoethoxyimino)-6-(hydroxyimino)-l 0, 13- dimethyltetradecahydro-lH-cyclopentafaJphenanthren-17(2H)-one (Compound (5))
Figure imgf000076_0001
HCl Salt of Compound (5)
Preparation of HCl salt of Compound (5)
To a stirred solution of 3,3:17,17-bis(ethylendioxy)androstan-6-one (compound (b4))
(0.054 g, 0.138 mmol) in THF (22 mL) a solution Of NH2OH . HCl (0.016 g, 0.24 mmol), Na2HPO4.12H2O (0.084 g, 0.24 mmol) in H2O (0.36 mL) was added. After stirring overnight at room temperature, NaCl was added and the mixture was extracted with EtOAc (2x). The combined organic extracts were washed with brine, dried over Na2SO4 , filtered and evaporated to dryness to give 3,3:17,17-bis(ethylendioxy)-6(£")-hydroxyimino-androstane (compound (cl)
(0.055 g, 98%). 1H NMR (600 MHz, CDCl3): δ 9.00 (bs, IH), 3.95 - 3.80 (m, 8H), 3.33 (m, IH), 2.24 (m, IH), 1.97 (m, IH), 1.80 - 1.00 (m, 17H), 0.81 (s, 3H), 0.73 (s, 3H).
3,3:17,17-bis(ethylendioxy)-6(E)-hydroxyiminoandrostane (cl) (O.lg, 0.247mmol) was disolved in 1,4-dioxane (2.5 mL) and a 5% aqueous H2SO4 solution (1.2 mL) was added dropwise. The resulting mixture was stirred at room temperature, until consumption of the starting material, monitored by TLC. The solution was then neutralized by addition of a saturated NaHCO3 solution, and the extraction was done with EtOAc. The organic phase was washed with water and brine, dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by flash chromatography (SiO2, 25% Acetone / Petroleum ether) to yield 0.050 g (64%) of the desired product (c2) as a white solid.
1H NMR (600 MHz, CDCl3): δ 9.00 (s, IH), 3.48 (m, IH), 2.61- 1.20 (m, 18H), 1.08 (m, IH), 0.95 (s, 3H), 0.86 (s, 3H).
The HCl salt of Compound (5) was prepared as described in Example 1 for preparation of compound (1): starting from 6-(E)- hydroxyiminoandrostane-3,17-dione (compound (c2)) (60 mg) and 2-aminoethoxyamine dihydrochloride (Intermediate (I))(28 mg). The crude product was crystallized from acetone to give the title compound as a white solid (20 mg, 26%). H NMR (600 MHz, CDCl3) δ 4.17 (bs, 2H), 3.71 (m, IH), 3.48 (m, IH), 3.20 (m, 3H), 2.50 - 1.10 (m, 18H), 0.88 (s, 6H).
EXAMPLE 6
Synthesis of HCl salt of (1 OR, 13S)-3-(2-aminoethoxyimino)-6-(methoxyimino)-l 0, 13- dimethyltetradecahydro-lH-cyclopenta[a]phenanthren-l 7(2H)-one (Compound (6))
Figure imgf000077_0001
HCl salt of Compound (6)
Preparation of HCl salt of Compound (6) To a stirred solution of 3,3 : 17, 17-bis(ethylendioxy)androstan-6-one (compound (b4))
(0.32 g) in pyridine (6.4 mL) NH2OCH3 ΗC1 (0.125 g) was added. After stirring overnight at room temperature, the solution was evaporated and the residue was treated with H2O and extracted with CH2Cl2 (2x). The combined organic extracts were washed with brine, dried over
Na2Sθ4, filtered and evaporated to dryness to give 3,3: 17,17-bis(ethylendioxy)-6(E)- methoxyimino- androstane (compound (c3)) (0.344 g, 100%). 1H NMR (600 MHz, CDCl3): δ
3.98 - 3.82 (m, 8H), 3.78 (s, 3H), 3.21 (m, IH), 1.97 (m, IH), 1.83 - 0.83 (m, 18H), 0.82 (s, 3H),
0.75 (s, 3H).
6(E)-Methoxyiminoandrostane-3,17-dione (compound (c4)) was prepared in 75% yield from 3,3:17,17-bis(ethylendioxy)-6(E)-methoxyiminoandrostane (compound (c3)) by the above procedure for the preparation of 6-methyleneandrostane-3, 17-dione (compound (b6)). The combined organic extracts were washed with H2O, dried over Na2Sθ4 and evaporated to dryness.
1H NMR (600 MHz, CDCl3) δ 3.82 (s, 3H), 3.37 (m, IH), 2.62 - 1.04 (m, 19H), 0.97 (s, 3H),
0.88 (s, 3H).
The HCl salt of Compound (6) was prepared in 30% yield as described in Example 1 for the preparation of compound (1), starting from 6-(E)- methoxyiminoandrostane-3,17-dione
(compound (c4)) and 2-aminoethoxyamine dihydrochloride (Intermediate (I))ZH NMR (600 MHz, CDCl3) δ 8.41 (bb, 3H), 4.25 (m, 2H), 3.83 (s, 1.5H), 3.82 (s, 1.5H), 3.34 (m, 3H), 3.20 (m, IH), 2.25 - 0.99 (m, 18H), 0.85 (s, 3H), 0.84 (s, 3H); 13C-NMR (75 MHz, CDCl3): 220.2, 162.0, 161.9, 157.4, 157.0, 68.3, 65.8, 65.0, 61.5, 61.3, 53.9, 51.5, 51.4, 50.5, 48.0, 47.9, 40.7, 40.4, 39.5, 39.3, 35.1, 31.3, 31.2, 21.7, 21.7, 20.7, 13.9, 13.8, 11.9, 11.8.
EXAMPLE 7
Synthesis of HCl salt of (5S,10R,13S)-5-hydroxy-10,13-dimethyl-6-methylene-3-(pyrrolidin-3- yloxyimino) tetradecahydro-1 H-cyclopenta[a] phenanthren-17(2H)-one (Compound (7))
Figure imgf000078_0001
Preparation of HCl salt of Compound (7)
To a stirred solution of 3jβ-hydroxyandrost-5-en-17-one (DHEA) (0.81 g) in CH2Cl2 (7.4 mL) cooled at 0 0C, a solution of mCPBA (0.77 mg) in CH2Cl2 (14 mL) was added dropwise. After 0.5h at 0 0C and 0.5 h at room temperature, a 10% Na2SO3 aqueous solution was added. The mixture was neutralized by addition of 5% aqueous NaHCO3 solution and extracted with CH2Cl2 (3 x 100 mL). The combined organic extracts were washed with H2O, dried over Na2Sθ4 , and evaporated to dryness to give a mixture: 3yβ-hydroxy-5α,6α-epoxyandrostan-17-one (compound dl(a)) and 3yβ-hydroxy-5yβ,6yβ-epoxyandrostan-17-one (compound dl(b)) as a white foam (1/1 mixture; 1.24 g, 97%). 1H NMR (300 MHz, acetone-d6): 3/?-hydroxy-5a,6a- epoxyandrostan-17-one (compound dl(a)) δ 3.26 (d, IH), 2.96 (d, IH), 2.70-1.12 (m, 18H), 1.36 (s, 3H), 0.83 (s, 3H); 3jS-hydroxy-5jS,6jS-epoxyandrostan-17-one (compound dl(b)): δ 2.98 (d, IH), 2.93 (d, IH), 2.71-1.13 (m, 18H), 1.06 (s, 3H), 0.84 (s, 3H).
To a solution of a 1/1 mixture of 3/Miydroxy-5α,6α-epoxyandrostan-17- one (compound dl(a)) and 3/Miydroxy-5yβ,6yβ-epoxyandrostane-17-one (compound dl(b)) (2.10 g, 6.90 mmol) in acetone (38 mL), Jones reagent (8.35 mL) was added dropwise, maintaining the temperature below 40 0C. 5 min after completion of the addition, i-PrOH (10 mL) was added and, after further 10 min, the suspension was filtered and the filtrate evaporated to dryness. The residue was treated with H2O (300 mL) and extracted with EtOAc (3 x 100 mL). The combined organic extracts were washed with H2O (100 mL), 5% aqueous NaHCO3 solution (100 mL), H2O (100 mL), dried over Na2Sθ4 and evaporated to dryness to give 5α-hydroxyandrostane- 3,6,17-trione (compound d2) as a white solid (1.65 g, 75%). l H NMR (300 MHz, acetone-de): δ 5.00 (s, IH),
2.85 (m, 2H), 2.45-1.25 (m, 17H), 1.06 (s, 3H), 0.88 (s, 3H).
A solution of 5α-hydroxyandrostane-3,6,17-trione (compound d2) (0.67 g) and CSA (5 mg) in cyclohexane (50 ml) and 2-methyl-2-ethyl-l,3-dioxolane (3 mL) was stirred at 85 0C for 18h. After cooling to room temperature, the mixture was neutralized with 5% aqueous NaHCO3 solution. The organic layer was separated and washed with brine, dried over Na2Sθ4 and evaporated to dryness The residue was purified by flash chromatography (SiO2 , cyclohexane/acetone/CH2Cl280/10/10) to give 3,3:17,17-bis(ethylendioxy)-5α- hydroxyandrostan-6-one (compound d3) (0.59 g, 69%). 1H NMR (600 MHz, acetone-d6): δ 4.30 (s, IH), 4.04-3.78 (m, 8H), 2.68 (t, J=12 Hz, IH), 2.10-1.17 (m, 18H), 0.80 (s, 3H), 0.75 (s, 3H).
To a stirred suspension of methyltriphenylphosphonium bromide (1.76 g) in dry THF (30 mL) cooled at 0 0C under Ar, potassium tert- butoxide (520 mg) was added. After stirring for 10 min, a solution of 3,3:17,17-bis(ethylendioxy)-5α-hydroxyandrostan-6-one (compound d3) (250 mg) in dry THF (10 mL) was added dropwise at room temperature over 0.5 h. After 1,5 h at room temperature, the mixture was quenched by addition of 5% NaH2Pθ4 aqueous solution and extracted with EtOAc (2 x 60 mL). The combined organic extracts were washed with 5% NaH2Pθ4 aqueous solution, brine, dried over Na2Sθ4 and evaporated to dryness. The residue was purified by flash chromatography (SiO2, petroleum ether/ethyl acetate 90/10) to give 3,3:17,17-bis(ethylendioxy)-5α- hydroxy-6-methyleneandrostane (compound d4) (200 mg, 81%). 1H NMR (600 MHz, CDCl3): δ 4.68 (s, IH), 4.38 (s, IH), 3.98-3.83 (m, 8H), 2.18-1.05 (m, 20H), 0.81 (s, 3H), 0.68 (s, 3H).
5α-Hydroxy-6-methyleneandrostane-3,17-dione (compound d5) was prepared in 100% yield from 3,3:17,17- bis(ethylendioxy)-5α-hydroxy-6-methyleneandrostane (compound d4) by the above procedure for the preparation of compound (b6). The combined organic extracts were washed with H2O, dried over Na2SO4 and evaporated to dryness. l H NMR (600 MHz, CDCl3): δ
4.86 (s, IH), 4.48 (s, IH), 2.90-1.10 (m, 20H), 0.93 (s, 3H), 0.89 (s, 3H). Following the procedure described in Example 2 and starting from 5α-hydroxy-6- methyleneandrostane-3,17-dione (compound d5) (100 mg) and 3-(R)-pyrrolidinyloxyamine dihydrochloride (Intermediate (II), 55 mg), the HCl salt of compound (7) was obtained (80 mg, 58%) as a white powder. l H NMR (600 MHz, CDCl3): δ 10.09 (bs, IH), 9.67 (bs, IH), 4.84 (bs, IH), 4.58 (bs, IH), 4.52 (bs, IH), 3.52-3.10 (6H, m), 3.12 (m, IH), 2.52-1.00 (18H, m), 0.86 (3H, s), 0.80 (3H, s) ; 13C NMR (75 MHz, CDCl3): 221.0, 162.6, 149.7, 108.7, 96.5, 79.9, 50.9, 47.9, 45.6, 41.2, 36.3, 36.1, 36.0, 35.9, 31.8, 31.7, 31.5, 27.4, 27.1, 21.7, 20.7, 14.5, 13.8
EXAMPLE 8 Synthesis of HCl salt of (5S, 1 OR, 13S)-3-(2-aminoethoxyimino)-5-hydroxy-l 0, 13-dimethyl-6- methylenetetradecahydro-lH-cyclopentafaJphenanthren-l 7(2H)-one (Compound (8))
Figure imgf000080_0001
HCl salt of Compound (8) Preparation of HCl salt of Compound (8) The HCl salt of the title compound (Compound (8)) was prepared according to the procedure described in Example 1 for the preparation of compound (1) and starting from 5a- hydroxy-6-methylenandrostane-3,17-dione (compound d5) (200 mg) and 2-aminoethoxyamine dihydrochloride (Intermediate (I)) (94 mg). The combined organic layers were washed with brine, dried over Na2SC^ and the solvent evaporated to dryness. The solid obtained was washed with EtOAc/Et2O. After drying under vacuum overnight HCl salt of Compound (8) was obtained (120 mg, 46%). 1H NMR (SOO MHZ, CDCl3): δ 8.11 (bs, 3H), 4.88 (m, IH), 4.71 (m, IH), 4.66 (m, 2H), 3.20 (m, 4H), 2.61-1.11 (m, 18H), 0.91 (s, 3H), 0.85 (s, 3H).
EXAMPLE 9 Synthesis of fumaricacid salt of (6S, 1 OR, 13S)-3-(2-aminoethoxyimino)-6-(hydroxymethyl)-
10,13-dimethyldodecahydro-lH-cyclopenta[aJphenanthrene- 7, 17(2H, 8H)-dione (Compound
(9))
Figure imgf000081_0001
Fumaπc salt o compound (9)
Preparation of fumaric acid salt of Compound (9)
A solution of 3,3:17,17-bis(ethylendioxy)-androstan-7-one (compound el) (1.18 g) in THF (13 mL) at -78 0C, was added dropwise to a solution of chlorotrimethylsilane (3.1 mL) and LDA (13.1 mL, 1.8M in THF) in dry THF (13 mL) at -78° C under argon., in 30 minutes. After 2 h at the same temperature, TEA (6.1 mL) was added followed, after 30 min, by addition of solid NaHCθ3 and finally by extraction with EtOAc (3 x). The combined organic extracts were washed with brine (3 x), dried over Na2SC^ and evaporated to dryness. The residue was purified by flash chromatography (SiO2, petroleum ether/EtOAc 9/1) to afford 3,3:17,17- bis(ethylendioxy)-7-trimethylsilyloxy-androst-6-ene (compound e2), 1.08 g (78%). 1H NMR (300 MHz, CDCl3): δ 4.37 (s, IH), 3.90-3.70 (8H, m), 2.20-2.05 (IH, m), 1.90-0.90 (17H, m), 0.88 (3H, s), 0.76 (3H, s), 0.16 (9H, s).
To a solution of 2,6-diphenylphenol (1.9 g) in dry DCM (25 mL), trimethylaluminium (2 mL, 2M in hexane) was added. After Ih the temperature was brought to 0 0C and a solution of trioxane (115 mg) in dry DCM (0.5 mL) was added. After Ih the mixture was cooled to -78 0C and a solution of 3,3:17,17-bis(ethylendioxy)-7-trimethylsilyloxy-androst-6-ene (compound e2) (0.6 g) in dry DCM (8 mL) was added. After stirring overnight at -20 0C, the reaction was quenched by addition OfNaHCO3 saturated aqueous solution. The mixture was filtered on a celite pad and washed with DCM. The filtrate was washed with water, dried over Na2Sθ4 and evaporated to small volume. TBAF (1.4 mL, IM in THF) was added and the mixture stirred at room temperature for 1.5 h. The solution was washed with water, dried over Na2Sθ4 and evaporated to dryness. The residue was purified by flash chromatography (SiO2 , n- hexane/EtOAc 30/70) to give 3,3:17,17-bis(ethylendioxy)-6α-hydroxymethylandrostane-7-one (compound e3) (500 mg, 92%). l H NMR (600 MHz, CDCl3): δ 3.98-3.80 (m, 8H), 3.79 (m, IH), 3.62 (m, IH), 2.61 (t, J = 7.2 Hz , 0.5H), 2.38 (m, 2H), 2.21 (m, IH), 1.96-1.10 (m, 16H), 1.13 (3H, s), 0.84 (3H, s) ; 13 C NMR (75 MHz, CDC13):214.5, 118.5, 108.6, 65.2, 64.5, 64.3, 64.1, 59.3, 55.1, 51.9, 50.1, 46.4, 45.5, 43.0, 35.9, 35.3, 34.1, 34.0, 30.6, 29.6, 23.5, 21.4,14.4. A solution of 3,3 : 17, 17-bis(ethylendioxy)-6α-hydroxymethylandrostane-7-one (compound e3) (300 mg) and pTSA«H2O (558 mg) in acetone (30 mL) was stirred at room temperature for 12h. The solution was neutralized by addition of 5% aqueous NaHCθ3 and acetone was evaporated. The aqueous suspension was extracted with CH2CI2 (3 x). The combined organic extracts were washed with brine, dried over Na2Sθ4 and evaporated to dryness to afford 6α-hydroxymethylandrostane-3,7,17-trione (compound e4) (180 mg, 76%) after purification by flash chromatography (SiO2, cyclohexane/EtOAc 40/60). 1H NMR (600 MHz, CDCl3): δ 3.84 (m, IH), 3.60 (m, IH), 2.62 (t, J= 7.2 Hz, IH), 2.56-1.30 (m, 18H), 1.32 (s, 3H), 0.88 (s, 3H). The fumaric acid salt of the title compound (Compound (9)) was prepared in 62% yield according to the procedure described in Example 1 for the preparation of compound (1), starting from 6α-hydroxymethylandrostane-3,7,17-trione (compound e4) and 2-aminoethoxyamine- dihydrochloride (Intermediate (I)). The combined organic extracts were dried over Na2Sθ4, filtered and evaporated to dryness. The residue was purified by flash chromatography (SiO2, DCM/MeOH/26% NH4OH 90/10/1 ) to afford the title compound as free amine. To the concentrated fractions a stoichiometric amount of fumaric acid in MeOH was added. After addition of a 1/1 mixture of EtOAc/Et2O, the precipitate was filtered to give the fumaric acid salt of the title compound (Compound (9)), 96 mg, as white solid. l H NMR (600 MHz, CD3OD): δ 6.73 (s, 2H), 4.17 (m, 2H), 3.93 (dd, 0.5H), 3.86 (dd, 0.5H), 3.65 (m, IH), 3.23-3.17 (m, 2H), 2.73 (t, IH), 2.56-0.97 (m, 18H), 1.28 (s, 1.5H), 1.27 (s, 3H), 0.89 (s, 3H).
EXAMPLE 10
Synthesis of fumaric acid salt of (6S, 1 OR, 13S)-6-(hydroxymethyl)-l 0, 13-dimethyl-3-(pyrrolidin-
3-yloxyimino)dodecahydro-lH-cyclopenta[a]phenanthrene-7, 17(2H, 8H)-dione (Compound (W))
Figure imgf000082_0001
Fumaric salt o compound (10)
Preparation of fumaric acid salt of Compound (10) The fumaric acid salt of the title compound (Compound (1O)) was prepared in 67% yield according to the procedure described in Example 2 and starting from 6α- hydroxymethyl- androstane-3,7,17-trione (compound e4) and 3-(R)-pyrrolidinyloxyamine dihydrochloride (Intermediate (II)). The crude product was purified by flash chromatography (Siθ2, CHCI3 /MeOH/26% NH4OH 90/10/1). To the concentrated fractions a stoichiometric amount of fumaric acid in MeOH was added. After addition of a 1/1 mixture of EtOAc/Et2O, the precipitate was filtered to give the fumaric salt of the title compound (Compound (10)) as white solid. 1H NMR (600 MHz, CD3OD): δ 6.66 (s, 2H), 3.93 (dd, J = 11.6, 2.3 Hz, IH), 3.86 (dd, J = 11.6, 2.3 Hz, IH), 3.66 (ddd, J= 4.7,10.4, 11.6 Hz, 2H), 3.54 (bs, IH), 3.52 (bs, IH), 3.36 (m, 4H), 2.72 (t, J = 11.3 Hz, IH), 2.54-1.50 (m, 20H), 1.27 (s, 3H), 0.88 (s, 3H) ; 13C NMR (75 MHz, CD3OD)221.4, 211.2, 170.2, 160.9, 160.8, 134.9, 79.8, 57.0, 54.7, 52.4, 36.4, 34.9, 30.5, 26.7, 22.2, 20.9, 20.4, 12.8, 10.8, 10.7.
EXAMPLE 11
Synthesis of HCl salt of (6S, 7S,10R,13S)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyl-3- (pyrrolidin-3-yloxyimino) tetradecahydro-1 H-cyclopenta[a] phenanthren-17(2H)-one
(Compound (H))
Figure imgf000083_0001
Preparation of HCl salt of Compound (11)
To a stirred solution of 3,3:17,17-bis(ethylendioxy)-6α-hydroxymethylandrostane-7-one (compound e3) (200 mg) in MeOH (10 mL), NaBH4 (27 mg) was added at 0 0C. The temperature was raised to rt. After 1 h the mixture was quenched by addition of 5% NaH2PO4 and extracted with CH2Cl2. The combined organic extracts were washed with brine, dried over Na2SO4 and evaporated to dryness. The residue was dissolved in dioxane (2.5 mL) and IN HCl (0.8 mL) was added and the resulting mixture stirred at room temperature for 1 h. After evaporation to dryness, the residue was purified by flash chromatography (Siθ2, n- hexane/CH2Cl2/acetone 50/25/25) to give 6α-hydroxymethyl-7α-hydroxyandrostane-3,17-dione (compound fl) 49 mg in 25% yield . 1H NMR (300 MHz, CDCl3): δ 4.04 (t, J= 2.3 Hz, IH), 3.91 (dd, J = 2.6, 12.3 Hz , IH), 3.75 (dd, J= 1.3, 12.3 Hz, IH), 2.52-1.10 (m, 19H), 1.01 (s, 3H), 0.87 (s, 3H).
The HCl salt of the title compound (Compound (H)) was prepared according to the procedure described in Example 2, and starting from 6α-hydroxymethyl-7α-hydroxyandrostane- 3,17-dione (compound fl) (49 mg) and 3-(R)-pyrrolidinyloxyamine dihydrochloride (Intermediate (II)) (25 mg). After 2 hours at room temperature, NaCl was added and stirred for 15 min. The mixture was extracted with THF (3x) and the combined organic phases were washed with brine, dried over Na2SO/), and filtered. The solid precipitated from the filtrate was purified by flash chromatography (SiO2, CH2Cl2/Me0H/26% NH4OH 90/10/0.1) to give the HCl salt of the title compound (Compound (H)) in 72% yield. l H NMR (600 MHz, CD3OD): δ 4.03 (t, J=I.8 Hz, IH), 3.72 (m, 2H), 3.53 (d, J=12.8 Hz, IH), 3.33 (m, 5H), 3.15 (dd, J=3.4, 14.5 Hz, IH), 2.45-1.18 (19H, m), 0.97 (3H, s), 0.87 (3H, s).
EXAMPLE 12
Synthesis ofHCl salt of(6S, 7S,10R,13S)-3-(2-aminoethoxyimino)-7-hydroxy-6-(hydroxymethyl)- 10, 13-dimethyltetradecahydro-l H-cyclopenta[a] phenanthren-17(2H)-one (Compound (12))
Figure imgf000084_0001
fl
HCl salt of Compound (12) Preparation of HCl salt of Compound (12)
The HCl salt of the title compound (Compound (12)) was prepared according to the procedure described in Example 1 for the preparation of compound (1), starting from 6a- hydroxymethyl-7α-hydroxyandrostane-3,17-dione (compound fl) and 2-aminoethoxyamine dihydrochloride (Intermediate (I)). 1H-NMR (400 MHz, CD3OD) δ 4.20-4.17 (m, 2H), 4.05 (m, IH), 3.74-3.70 (m, 2H), 3.24-3.21 (m, 2H), 2.48-1.10 (m, 21H), 1.00 (s, 3H), 0.89 (s, 3H). EXAMPLE 13
Synthesis of HCl salt of (1 OS, 13S)-3-(2-aminoethoxyimino)-l 0, l3-dimethyl-7- methylenetetradecahydro-lH-cyclopentafaJphenanthren-l 7(2H)-one (13))
Figure imgf000085_0002
Figure imgf000085_0001
Figure imgf000085_0003
HCl salt of Compound (13)
Preparation of HCl salt of Compound (13)
4-Androstene-3,17-dione (compound hi) (2.0 g, 6.9 mmol), p-TsOH (0.1 g, 0.52 mmol), ethylene glycol (14mL) and toluene (100 mL) were refluxed under argon in a flask equipped with a Dean-Stark trap. After 24 h, the mixture was cooled to r.t, diluted with Et2O (25mL) and washed with saturated NaHCθ3 solution (10 mL) and water (3x25 mL). The organic portion was dried (Na2SOzI) and evaporated to dryness, giving yellow solids that were washed with MeOH to give diketal 3,3:17,17- bis(ethylendioxy)-5-androstene (compound h2) as a white powder (1.3 g, 62%). 1H NMR (300 MHz, CDCl3): δ 5.33 (m, IH), 4.03-3.73 (m, 8H), 2.55 (m, IH), 2.11 (m, IH), 2.07-1.93 (m, 2H), 1.88-0.98 (m, 15H), 1.01 (s, 3H), 0.84 (s, 3H). To a slurry of CrO3 (8.47 g, 84.7 mmol) in CH2Cl2 (110 mL) at (-20 to -30 0C) 3,5- dimethylpyrazole (8.15 g, 84.7 mmol) was added and the mixture was stirred at this temperature for 1.5h. 3,3:17,17-bis(ethylendioxy)-5-androstene (compound h2) (1.27 g, 3.39 mmol) was added and the thick, dark mixture was stirred for 4.5 h. The cooling bath was removed, water (9 mL) was added and the mixture was allowed to warm to rt. The mixture was suction- filtered through celite and the filtrate was washed with water (3x200 mL), dried (Na2SO/() and evaporated to dryness. The resulting residue was purified by column chromatography (4:1 CH2Cl2/Et0Ac) to give 3,3:17,17- bis(ethylendioxy)-5-androstene-7-one (compound h3) (570 mg, 46%) as a white solid. 1H NMR (600MHz, CDCl3, stabilized): δ 5.67 (m, IH), 4.07-3.77 (m, 8H), 2.67 (d, J=16.0 Hz, IH), 2.47 (m, IH), 2.33 (m, IH), 2.24 (t, J=I LO Hz, IH), 2.05-1.35 (m, 13H), 1.19 (s, 3H), 0.87 (s, 3H).
A mixture of 3,3 : 17, 17-bis(ethylendioxy)-5-androsten-7-one (compound h3) (550 mg) and 10% Pd/C (50 mg) in EtOAc (14 mL) was stirred under H2 at atm pressure for 24 h. The mixture was filtered through Celite and the filtrate evaporated to dryness to afford 3,3:17,17- bis(ethylen-dioxy)androstane-7-one (compound h4) (550 mg, 100% yield). 1H NMR (600 MHz, CDCl3): δ 3.96-3.70 (m, 8H), 2.54-1.10 (m, 20H), 1.07 (s, 3H), 0.83 (s, 3H).
3,3:17,17-Bis(ethylendioxy)-7-methyleneandrostane (compound h5) was prepared in 62% yield from 3,3:17,17-bis(ethylendioxy)androstane-7-one (compound h4) by the procedure described above in Example 4 for the preparation of 3,3 : 17, 17-bis(ethylendioxy)-6- methyleneandrostane (compound b5). l H NMR (600 MHz, CDCl3): δ 4.67 (s, IH), 4.60 (s, IH), 3.96-.3.60 (m, 8H), 2.02-0.75 (m, 20H), 0.94 (s, 3H), 0.86 (s, 3H).
7-Methyleneandrostane-3,17-dione (compound h6) was prepared in 45% yield from 3,3:17,17- bis(ethylendioxy)-7-methyleneandrostane (compound h5) by the procedure described above in Example 4 for the preparation of 6-methyleneandrostane-3, 17-dione (compound b6). 1H NMR (600 MHz, CDCl3): δ 4.79 (s, IH), 4.68 (s, IH), 2.53-0.88 (m, 20H), 1.16 (s, 3H), 0.93 (s, 3H).
The HCl salt of the title compound (Compound (13)) was prepared in 97% yield according to the procedure described in Example 1 for the preparation of compound (1), starting from 7-methyleneandrostane-3,l 7-dione (compound h6) and 2-aminoethoxyamine dihydro- chloride. The crude product was triturated with EtOAc. l H NMR (600 MHz, CDCl3): δ 8.43 (br, 3H), 4.76 (m, IH), 4.64 (m, IH), 4.24 (m, 2H), 3.47 (m, 0.5H), 3.32 (m, 2H), 3.17 (m, 0.5H), 2.99 (m, 0.5), 2.48-0.73 (m, 19H), 1.02 (s, 3H), 0.89 (s, 3H).
EXAMPLE 14
Synthesis of HCl salt of (1 OS, 13S)-3-(2-aminoethoxyimino)-7-(difluoromethylene)-l 0, 13- (14))
Figure imgf000087_0001
HCl salt of Compound (14) Preparation of HCl salt of Compound (14)
To a stirred solution of diethyl difluoromethylenephosphonate (0.33 mL) in DME (2.87 mL) in n-pentane (0.55 mL) at -78 0C, 1.9 M pentane solution of tert-butyllithium (1.08 mL) was added dropwise under argon. After 15 min at the same temperature a solution of 3,3:17,17- bis(ethylendioxy)androstane-6-one (compound h4) (250 mg) in DME (2.25 mL) and n-pentane (0.625 mL) was added dropwise. The mixture was stirred at -78°C for further 30 min and warmed up to room temperature. n-Pentane was distilled off and after heating at 80 0C for 18 h the mixture was quenched with H2O and extracted with CH2CL. (3x). The combined organic extracts were dried over Na2Sθ4, and evaporated to dryness. The residue was purified by flash chromatography (Siθ2, cyclohexane/Et2O 70/30) to give 3,3:17,17-bis(ethylendioxy)-6-difluoro- methyleneandrostane (compound h7) (231 mg, 85%). 1H NMR (300 MHz, acetone-d6): δ 3.85 (m, 8H), 2.52-0.80 (m, 20H), 0.97 (s, 3H), 0.84 (s, 3H). 19F NMR (282 MHz, acetone-d6): δ - 91.70 (A, J = 66.95, 5.91 Hz, IF), -92.43 (d, J = 66.82 Hz, IF).
6-Difluoromethyleneandrostane-3,17-dione (compound h8) was prepared in 99% yield from 3,3:17,17- bis(ethylendioxy)-6-difluoromethyleneandrostane (compound h7) (by the procedure described above in Example 4 for the preparation of 6-methyleneandrostane-3, 17- dione (compound b6). The combined organic extracts were washed with H2O, dried over Na2SO4 and evaporated to dryness. 1H NMR (300 MHz, acetone-d6): δ 2.85-0.95 (m, 20H), 1.12 (s, 3H), 0.88 (s, 3H). 19F NMR (282 MHz, acetone-dg): δ -91.70 (dd, J = 65.46, 5.53 Hz, IF), - 92.43 (d, J = 65.76 Hz, IF). The HCl salt of the title compound (Compound (14)) was prepared according to the procedure described in Example 1 for the preparation of compound (1), starting from 6- difluoromethyleneandrostane-3,17-dione (compound h8) (78 mg) and 2-aminoethoxyamine dihydrochloride (Intermediate (I)) (27 mg). The title compound was obtained by recrystallization from EtOAc/Et2O 1 :2, followed by filtration and drying under vacuum (60 mg, 75%) as a white solid. 1H NMR (600 MHz, CDCl3): δ 8.42 (3H, bs), 4.27 (2H, m), 3.36 (bs, 2H), 3.21 (d, J = 15.5 Hz, 0.5H), 3.05 (d, J = 15.5 Hz, 0.5H), 2.42-0.95 (22H , m), 1.03 (3H, s), 0.88 (3H, s). 19F NMR (282 MHz, CDCl3): δ -91.78 (d, J = 66.6 Hz, 0.5F), -91.94 (d, J = 66.6Hz, 0.5F), -92.81 (d, J = 66.2 Hz, IF); 13C NMR (75 MHz, CDCl3): δ 219.98, 219.93, 161.1, 160.9, 149.8 (dd, J = 279.0, 288.0 Hz), 91.5 (m), 68.4, 68.3, 53.6, 48.2, 48.0, 47.9, 46.9, 45.5, 45.4, 40.2, 40.1, 37.6, 37.5, 36.6, 35.5, 33.7, 30.5, 29.9 (m), 27.6, 27.3, 23.8, 23.7, 21.2, 20.3, 20.2, 13.1, 11.3, 11.1
EXAMPLE 15
Synthesis of (3S,5S,10R,13S)-10,13-dimethyl-3-(2-(pyrrolidin-l-yl)ethoxy)dodecahydro-lH- cyclopenta[a]phenanthrene-6,17(10H,14H)-dione (Compound (15))
Figure imgf000088_0001
aa3 aa4
Dess Martin peπodinane
Figure imgf000088_0003
Figure imgf000088_0002
Preparation of Compound (15)
To a solution of DHEA (3 g, 10.40 mmol) in dry THF (32 mL) at 0 0C, under Ar atmosphere, imidazole (2.12 g, 31.2 mmol) and I2 (7.92 g, 31.2 mmol) were added. After stirring for 30 minutes at 0 0C, TBDMSCl (1.74 g, 11.44 mmol) was added and the mixture was stirred at room temperature for 2.5 hours. The solvent was evaporated under reduced pressure and the dark residue was dissolved in EtOAc and washed with aq. Na2S2θ3. The organic layer was dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The residue was purified by flash chromatography (SiO2, cyclohexane/ EtOAc 9/1) to 3/J-tert- butyldimethylsilyloxy-androst-5-en-17-one (aal) 4.04 g (97%) as a white solid. 1 H-NMR (300 MHz, CDCl3) δ 5.34 (d, J= 5.1 Hz, IH), 3.48 (m, IH), 2.46 (m, IH), 2.33-0.88 (m, 18H), 1.02 (s, 3H),0.89 (s, 9H), 0.88 (s, 3H), 0.06 (s, 6H).
BH3THF (IM in THF, 3.1 mL, 3.10 mmol) was added at -10 0C to a solution of (aal) (500 mg, 1.24 mmol) in anhydrous tetrahydrofuran (55 mL) under argon atmosphere. The mixture was stirred at room temperature for 2 hours. Then H2O (60 mL) was carefully added, followed by the addition of NaBO3.4H2O (382 mg, 2.48 mmol) and the mixture was stirred overnight. The mixture was filtered and the white solid was washed with cold THF and was discarded. To the filtrate NaCl was added for the separation of the layers and the aqueous layer was washed with THF (x3) and was dried over anhydrous sodium sulphate and evaporated to dryness. The residue was purified by flash chromatography (cyclohexane/EtOAc 9/1) to afford 468 mg (89%) of 3/3-tert-butyldimethylsilyloxy-5α-androstan-6α,17/3-diol (aa2) as a white solid. l H-NMR (300 MHz, CDCl3) δ 3.64 (t, J= 8.4 Hz, IH), 3.52 (m, IH), 3.40 (m, IH), 2.12- 0.60 (m, 20H), 0.88 (s, 9H), 0.82 (s, 3H), 0.72 (s, 3H), 0.05 (s, 6H). A solution of 3/3-tert-butyldimethylsilyloxy-5 α-androstan-6α, 17/3-diol (aa2) (400 mg,
0.95 mmol), chloromethyl methyl ether (530 μL, 7.0 mmol) and di-isopropylethylamine (4.8 mL, 27.6 mmol) in dry dichloromethane (20 mL) was stirred for Ih at 0° C and overnight at room temperature. Citric acid (10% water solution, 50 mL) was added, the organic layer separated, washed with water and brine dried over Na2Sθ4, filtrated and evaporated to dryness under reduced pressure. The residue was purified by chromatography (hexane/ethyl acetate 85/15) to give 420 mg (87%) of 3β-tert-butyldimethylsilyloxy-6α,17β-dimethoxymethyl-5α- androstane (aa3) as a white solid. l H-NMR (300 MHz, CDCl3) δ 4.71 (d, J= 6.85 Hz, IH), 4.61 (AB system, 2H), 4.54 (d, J= 6.9 Hz, IH), 3.54-3.48 (m, 2H), 3.34 (s, 3H), 3.33 (s, 3H), 3.28 (dt, J= 4.4, 10.5 Hz, IH), 2.18-0.58 (m, 20H), 0.87 (s, 9H), 0.82 (s, 3H), 0.75 (s, 3H), 0.05 (s, 6H).
To a solution of 3/3-tert-butyldimethylsilyloxy-6α,17/3-dimethoxymethyl-5α-androstane (aa3) (420 mg, 0.82 mmol) in dry THF (10 mL) a solution of TBAF in THF (1.89 mL, 1.89 mmol) was added and the reaction mixture was stirred at room temperature overnight. Dichloromethane was added, the mixture was extracted with water and brine, the organic layer was separated, dried over Na2SO4, filtrated and evaporated to dryness under reduced pressure. The residue was purified by chromatography (hexane/ethyl acetate 65/35) to give 330 mg (100%) of 6α,17/3-dimethoxymethyl-5α-androstan-3/3-ol (aa4) as an amorphous solid. l H- NMR (300 MHz, CDCl3) δ 4.63 (d, J= 6.85 Hz, IH), 4.53 (AB system, 2H), 4.46 (d, J= 6.9 Hz, IH), 3.61-3.50 (m, 2H), 3.42 (s, 3H), 3.27 (s, 3H), 3.27-3.26 (m, IH), 2.50 (bs, IH), 2.24- 0.62 (m, 21H), 0.75 (s, 3H), 0.68 (s, 3H). A solution of 6a ,17/3 -di-methoxymethyl-5a-androstan-3/3-ol (aa4) (450 mg, 1.13 mmol) in dry DMF (10 mL) was added to a solution of sodium hydride (91 mg, 2.27 mmol, 60 % dispersion in oil) in dry DMF (5 mL), the reaction mixture was heated at 100 0C for 3 h, then cooled to room temperature and a solution ofN-(2-chloroethyl)-pyrrolidine (2.15 g, 16.1 mmol) in dry DMF (5 mL) was added dropwise. The reaction mixture was heated at 80 0C for 3 d and then diluted with brine and ethyl acetate. The organic layer was separated and washed with IN HCl (x3). The collected aqueous phase was neutralised with potassium hydroxide 4N and extracted with chloroform to give, after workup, a brown-yellowish oil which was purified by column chromatography (dichloromethane/methanol/ammonia 90/10/1) to give 65 mg (12%) of the product as brownish flakes. 1H-NMR (600 MHz, CDCl3) δ 4.69 (d, J = 6.85 Hz, IH), 4.59
(AB system, 2H), 4.51 (d, J = 8.2 Hz, IH), 3.99 (m, 2H), 3.49 (, t, J= 6.9 Hz, IH), 3.33 (m, IH), 3.31 (s, 6H), 3.25 (m, IH), 2.87 (m, 2H), 2.23 (m, 4H), 2.11-0.62 (m 24H), 0.78 (s, 3H), 0.72 (s, 3H). The intermediate was dissolved in acetonitrile (4.25 mL) and water (0.75 mL), p- toluensulfonic acid was added to pH=0.9 and the reaction mixture stirred overnight at room temperature and for 4 h at 60 0C. After usual work-up the residue was purified by flash chromatography (Siθ2, chloroform/methanol/ ammonia 90/10/1) to give 35 mg (65%) of 3/3 -(2- N -pyrrohdinylethoxy)-5α-androstan-6α,17/3-diol (aa5). 1H-NMR (600 MHz, CDCl3) δ 3.88 (IH, d), 3.57 (2H, m), 3.31 (3H, m), 3.00 (2H, bs), 2.17-0.62 (30H, m).
To a solution of of 3/3 -(2-N-pyrroh'dinylethoxy)-5α-androstan-6α,17/3-diol (aa5) (30 mg, 0.074 mmol) in dichloromethane (3 mL) at 0 0C, Dess Martin periodinane (126 mg, 0.296 mmol) was added and the reaction mixture was stirred at room temperature for 2 h. A solution of NaHCO3/Na2S2θ3 1/3 (5 mL) was added and the mixture was stirred until the organic layer became completely transparent. The organic phase was washed with brine, dried over Na2SO/), filtrated and evaporated to dryness under reduced pressure. The residue was purified by chromatography (chloroform/methanol/ammonia 90/10/1) to give 17 mg (57%) of 3/3 -(2-N - pyrrolidinylethoxy)-5 α-androstan-6, 17-dione (Compound (15)) as brownish flakes. 1H-NMR (600 MHz, CDCl3) δ 3.72 (m, 2H, ), 3.34 (m, IH), 3.11 ( m, 2H), 2.70 (m, 4H,), 2.42-1.20 (m, 24H,), 0.84 (s, 3H), 0.73 (s, 3H).
EXAMPLE 16
Synthesis of the oxalate salt of (Z)-6, 17-Dihydroxy-l 0, 13-dimethylhexadecahydro-l H- cyclopentafaJphenanthrene-3-carbaldehyde O-2-aminoethyl oxime (Compound (16))
Figure imgf000091_0001
aa4 bb1
Figure imgf000091_0002
bb2 bb3α:bb3β 1 : 3.5 OC
Figure imgf000091_0003
H oxalate salt of Compound (16)
Preparation of oxalate salt of Compound (16)
To a solution of 6α,17/3-di-methoxymethyl-5α-androstan-3/3-ol (aa4) (600 mg) in dichloromethane (90 mL)at O 0C was added Dess-Martin periodinane (1.1 g) and the mixture was stirred at room temperature for 2 hours. Subsequently, the reaction mixture was quenched with a mixture of sat. NaHCθ3/Na2S2θ3 1/3 (40 mL) and was stirred for 15 minutes. The mixture was then filtered through a Celite pad and the filtrate was washed with sat. NaHCθ3 and brine. The combined organic layers were dried over anhydrous sodium sulphate and evaporated to dryness, to give 570 mg (96%) of 6α,17/3-di-methoxymethyl-5α-androstan-3/3-one (bbl) as a white solid. l H-NMR (300 MHz, CDCl3) δ 4.71 (d, J= 6.85 Hz, IH), 4.62 (AB system, 2H), 4.50 (d, J= 8.2 Hz, IH), 3.54 (t, J= 8.1 Hz, IH), 3.43-3.38 (m, IH), 3.35 (s, 6H), 2.79-2.68 (m, IH), 2.40-0.73 (m, 20H), 1.03 (s, 3H), 0.79 (s, 3H).
A solution of 6α,17β-di-methoxymethyl-5α-androstan-3β-one (bbl) (570 mg) in THF/H2O/HC1 6N (2.4/3.5/8.5 mL) was heated at 55 0C and stirred until completion of the reaction. The mixture was then poured into brine and extracted with EtOAc (5x10 mL). The combined organic layers were dried over sodium sulfate and evaporated to dryness under reduced pressure. The residue was purified by flash chromatography (SiO2, dichloromethane/methanol 95/5) to give 6α,17/3-dihydroxyandrostan-3-one (compound bb2) (280 mg, 65%), as a white solid. l H-NMR (300 MHz, CDCl3) δ 3.66 (t, J= 8.4 Hz, IH), 3.49 (m, 2H), 2.76-2.69 (m, IH), 2.47-0.73 (m, 20H), 1.04 (s, 3H), 0.76 (s, 3H). Sodium hydride (60% in mineral oil, 250 mg) was carefully washed with n-hexane, under nitrogen atmosphere. Then anhydrous dimethylsulfoxide (60 mL) and trimethylsulfoxonium iodide (1.4 g) were added and the reaction mixture was stirred to complete dissolution. A solution of 3-oxo-5α-androstan-6α,17/3 -diol (compound bb2) (280 mg) in dry DMSO (13 mL) was added and the reaction mixture was stirred at room temperature for 2 hrs, then poured into 200 mL of ice and water and extracted with ethyl acetate to give, after usual workup, 3-spiro-oxirane-5α-androstan-6α,17β-diol (290 mg); 1 H-NMR (300 MHz, CDCl3): 3.65 (IH, J= 8.7 Hz, t), 3.38 (IH, m), 2.68-2.63 (2H, m), 2.12-0.73 (m, 22H), 0.86 (s, 3H), 0.74 (s, 3H). Freshly distilled boron trifluoride diethylether complex (145 μL) was added to a solution of crude 3-spiro-oxirane-5α-androstan-6α,17/3 -diol (290 mg) in anhydrous tetrahydrofuran (11 mL) at -30° C, under nitrogen atmosphere. After 4 hours, the reaction mixture was poured into disodium hydrogen phosphate (5%), extracted with chloroform (3x20 mL), the combined organic layers were washed with brine, separated and dried over anhydrous sodium sulfate to give, after evaporation to dryness, 400 mg of crude 3-formyl-5α-androstan-6α,17/3-diol
(compounds bb3α and bb3β) as an amorphous white solid (3α-formyl ββ -formyl = 1/3.5 determined by 1 H-NMR). 1 H-NMR (300 MHz, CDCl3): 9.69 (IH, s), 9.66 (IH, s), 3.75 (IH, m), 3.68-3.62 (IH, m), 3.41 (IH, m), 2.26-0.73 (25H, m). The two isomers were isolated by column chromatography (chloroform/methanol 97/3) to give 170 mg (74%) of the 3β isomer (bb3β) 1 H- NMR (300 MHz, CDCl3): 9.66 (IH, s), 3.75 (IH, m), 3.68-3.62 (IH, m), 3.41 (IH, m), 2.26-0.73 (19H, m), 0.79 (s, 3H), 0.74 (s, 3H)
A solution of 3β-formyl-5α-androstan-6α,17/3-diol (compound bb3β) (170 mg) in dioxane/water (2/1, 22 mL) was treated with sodium hydroxide (0.1 N) to pH = 13-14 and stirred for 15 minutes, then a solution O-(2-aminoethyl)-hydroxylamine dihydrochloride (120 mg) in water (3 mL) was poured in. After 3 hr, the organic solvent was distilled off, water was added and the aqueous solution was extracted with chloroform. (4x20 mL). The combined organic layers ere washed with brine, dried over sodium sulfate and evaporated to dryness under reduced pressure. The residue was purified by flash chromatography (SiO2, chloroform/methanol/ammonia 95/5/1) to give (Z)-6,17-dihydroxy-10,13- dimethylhexadecahydro-lH-cyclopenta[a]phenanthrene-3-carbaldehyde 0-2-aminoethyl oxime (Compound 16), which was dissolved in ethyl acetate/methanol 70/30 and treated with oxalic acid to give 35 mg of the oxalate salt of Compound (16) as a white solid. 1 H NMR (600 MHz, CD3OD): δ 7.45 (d, J = 6 Hz, IH), 4.19 (m, 2H), 3.58 (t, J= 8.4 Hz, IH), 3.35-3.31 (m, IH), 3.22 (m, 2H), 2.18-0.71 (m, 27H), 0.84 (s, 3H), 0.73 (s, 3H); . 13 C NMR (75 MHz, CD3OD) 156.5, 80.9, 68.75, 68.72, 54.1, 52.6, 50.8, 42.7, 40.7, 38.8, 38.7, 37.9, 36.5, 36.2, 34.2, 29.2, 26.3, 25.1, 22.9, 20.2, 12.3, 10.3. EXAMPLE 17
Synthesis of2-((5S, 6SJ0R, 13S, 17S)-6, 17-dιhydroxy-10,13-dιmethylhexahydro-lH-cyclopenta-
[a]phenanthren-3(2H, 4HJ OH, 12H,13H,14H,15H,16H, 17H)-ylιdene)acetaldehyde 0-2- (dimethylamino) ethyl oxime (Compound (17))
Figure imgf000093_0001
Preparation of oxalate salt of Compound (17) Trimethyl phosphonoacetate (0.4 mL) was added to a mixture of sodium hydride (60%,
88 mg) in anhydrous tetrahydrofuran (12 mL), and the mixture was stirred at room temperature under nitrogen atmosphere for 15 min, then 3-oxo-5α-androstan-6α,17/3-diol (bbl) (420 mg) dissolved in anhydrous tetrahydrofuran (5 mL) was added. After 4 hr the reaction was quenched with sodium dihydrogen phosphate (5%). The mixture was extracted several times with ethyl acetate, the combined organic layers were dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure.The crude residue was purified by chromatography (Siθ2; n- hexane/ethyl acetate 50/50) to give 122 mg of (£)-3-methoxycarbonylmethylen-5α-androstan- 6α,17 /3-diol (ccla) and 232 mg of (Z)-3-methoxycarbonylmethylen-5α -androstan-6α,17/3-diol as amorphous white solids (cclb). (£")-3-methoxycarbonylmethylen-5α-androstan-6α,17/3-diol (ccla): 1H-NMR (300 MHz, CDCl3,): 5.66 (IH, bs), 2m, 1H3.68 (3H, s), 3.65 (IH, t, J=8.8
Hz), 3.46 (IH, ddd, J =4.2, 10.7, 10.8 Hz), 2.57-2.53 (IH, m), 2.17-0.66 (18H, m), 0.94 (s, 3H), 0.74 (s, 3H); (Z)-3-methoxycarbonylmethylen-5α-androstan-6α,17β-diol (cclb): 1H-NMR (300 MHz, CDCl3,): 5.64 (IH, bs), 4.16-4.08 (IH, m), 3.68 (3H, s), 3.65 (IH, t, ./=8.6 Hz), 3.47 (IH, ddd, J =4.9, 10.9, 11.0 Hz), 2.41-2.29 (m, IH), 2.18-0.66 (19H, m), 0.94 (s, 3H), 0.74 (s, 3H). Diisobutylaluminum hydride (IM in DCM, 1.7 mL) was added at -78° C to a solution of
(£")-3-methoxycarbonylmethylen-5α-androstan-6α,17/3-diol (ccla) (120 mg) in anhydrous tetrahydrofuran (4.3 mL) under argon atmosphere. The mixture was stirred overnight, then it was quenched (-78° C) with HCl (0.1N, to pH = 3) and extracted with ethyl acetate. The organic layers were dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure. The residue was purified by flash chromatography (Siθ2, n-hexane/chloroform/acetone 10/45/45) to give 116 mg (99%) of (£>3-(2-hydroxyethylen)- 5a -androstan-6α,17β-diol (cc2a). 1H NMR (300 MHz, CDCl3): δ 5.43 (t, J= 6.7 Hz, IH), 4.14 (d, J = 6.4 Hz, 2H), 3.65 (t, J= 8.3 Hz, IH), 3.46 (m, IH), 2.48 (m, 2H), 2.10-0.60 (19H, m), 0.90 (s, 3H), 0.74 (s, 3H).
A mixture of (£")-3-(2-hydroxyethylen)-5α-androstan-6α,17/3-diol (cc2a) (115 mg) and manganese dioxide (558 mg) in dioxane (11 mL) was stirred at room temperature for 60 hrs, then it was filtered on a celite pad. The organic solution was dried over sodium sulfate and evaporated to dryness under reduced pressure, to give 112 mg (99%) of (£)-3-formylmethylen- 5α-androstan-6α,17β-diol (cc3a) l H NMR (600 MHz, CDCl3): δ 9.99 (d, IH, J = 8.2 Hz), 5.86 (d, J = 8.3 Hz, IH), 4.91-4.89 (m, IH), 3.65 (t, J= 8.3 Hz, IH), 3.48 (m, IH), 3.28 (m, IH), 2.64 (m, IH), 2.20-0.6 (24H, m), 0.96 (s, 3H), 0.74 (s, 3H). Using the procedure described for the synthesis (£)-3-formylmethylen-5α-androstan-
6 α, 17/3-diol (cc3 a) and starting from (Z)-3 -methoxycarbonylmethylen-5 α-androstan-6 a, 17/3- diol (cclb) it was possible to prepare (Z)-3-formylmethylen-5α-androstan-6α,17/3-diol (cc3b). 1H NMR (600 MHz, CDCl3): δ 10.03 (bs, IH), 5.83 (bs, IH), 3.72 (t, J= 8.3 Hz, IH), 3.48 (m, IH), 3.28 (m, IH), 2.50-0.6 (27H, m), 0.95 (s, 3H), 0.74 (s, 3H). A solution of O-(2-N,N-dimethylaminoethyl)-hydroxylamine dihydrochloride (73 mg) in 0.2 mL of IN NaOH/dioxane (2:1) was slowly dropped into a solution of (£)-3- formylmethylen-5α-androstan-6α,17/3 -diol (cc3a) (110 mg) in dioxane (2.6 mL) and the mixture was stirred at room temperature for 1 hour. NaOH (IN) was added to pH =10 and the mixture was extracted with chloroform (3x10 mL). The combined organic layers were dried over sodium sulfate and evaporated to dryness under reduced pressure. The residue (92 mg) was purified by flash chromatography (Siθ2, chloroform/methanol/ammonia 95/5/0.1) to give 55 mg of Compound (17a), which was dissolved in ethyl acetate/methanol 7/3 and treated with stoichiometric amount of oxalic acid (16 mg) to give 45 mg of oxalate salt of compound 17a as a white solid. 1 H-NMR (300 MHz, CD3OD, ): 8.20 (0.7H, m), 7.48 (m, 0.3H), 6.42 (m, 0.3H), 5.86 (d, J= 9.4 Hz, 0.7H), 4.38-4.33 (m, 2H), 4.14-4.12 (m, 0.5H), 3.68-3.66 (m, 0.5H), 3.57 (t, J= 8.4, IH), 3.46 (m, 2H), 2.94 (s, 6H), 2.73-0.66 (m, 24H), 0.97 (s, 3H), 0.74 (s, 3H).
A solution (1.7 M) of O-(2-N,N-dimethylaminoethyl)-hydroxylamine dihydrochloride (32 mg) in IN NaOH/dioxane 5/2, was slowly dropped into a solution of (Z)-3-formylmethylen- 5α-androstan-6α,17β -diol (cc3b) (32 mg) in dioxane (0.3 mL). After 1 h was added 10 ml of H2O and the mixture was extracted with chloroform (3x20 mL). The combined organic layers were dried over sodium sulfate and evaporated to dryness under reduced pressure. The residue (78 mg) was purified by flash chromatography (Siθ2, chloroform/methanol/ammonia 95/5/0.1) to give 70 mg of compound (17b), as a pale white powder. 1H NMR (600 MHz, CDCl3): δ 8.20 (d, J = 10.4 Hz, IH), 5.86 (d, J = 9.4 Hz, IH), 4.38-4.33 (m, 2H), 4.14-4.12 (m, 0.5H), 3.68-3.66 (m, 0.5H), 3.57 (t, J= 8.4, IH), 3.46 (m, 2H), 2.94 (s, 6H), 2.73-0.66 (m, 24H), 0.97 (s, 3H), 0.74 (s, 3H).
EXAMPLE 18
Synthesis of 5-((5S, 1 OR, 13S,E)-6-(hydroxyιmιno)-l 0, 13-dιmethyl-l 7-oxohexadecahydro-lH- cyclopenta[a]phenanthren-3-yl)pent-4-en-l-amιnιum 3-carboxyacrylate (thefumanc acid salt of Compound (18))
Figure imgf000095_0001
ddla ( J|l) ddlb(3α) 3 1
Figure imgf000095_0002
Preparation of Salt of Compound (18)
Sodium hydride (50% in mineral oil, 640 mg) was carefully washed with n-hexane, under nitrogen atmosphere. Then anhydrous dimethylsulf oxide (160 mL) and trimethylsolfoxonium iodide (2.9 g) were added and the reaction mixture was stirred to complete dissolution. 5α-androstan-3,6,17-trione (a2) (600 mg) was added and the reaction mixture stirred at room temperature for 2 hrs, then poured into ice and water and extracted with ethyl acetate to give, after usual workup, 3-spiro-oxirane-5α-androstan-6,17-dione. Freshly distilled boron trifluoride diethylether complex (300 μL) was added to a solution of crude 3-spiro- oxirane-5α-androstan-6,17-dione (590 mg) in anhydrous tetrahydrofuran (25 mL) at -30° C, under nitrogen atmosphere. After 70 minutes, the reaction mixture was poured into disodium hydrogen phosphate (5%), extracted with chloroform (3x50 mL), the combined organic layers were separated and dried over anhydrous sodium sulfate. The residue was purified by flash chromatography (SiO2; CH2Cl2:Et0Ac 9:1). to give, 3/?- formylandrostane-6, 17-dione (ddla) (370 mg, 62%) and of 3α-formylandrostane-6,17-dione (ddlb) (120 mg, 20%). 1H-NMR (300 MHz, DMSOd6, ppm from TMS): /?-isomer: δ 9.57 (IH, d), 2.45-1.10 (21H, m), 0.78 (3H, s), 0.63 (3H, s); a- isomer: δ 9.56 (IH, bs), 2.60-0.95 (21H, m), 0.76 (3H, s), 0.60 (3H, s). Potassium hydride (20% in mineral oil, 245 mg) was carefully washed with Et2O, under nitrogen atmosphere. At -78° C anhydrous THF (4 mL), 1,1,1,3,3,3-hexamethyldisilazane (200 mg) and (4-azidobutyl)triphenylphosphonium bromide (537 mg) were added. After complete dissolution, 3/?- formylandrostane-6, 17-dione (ddla) (350 mg) in THF (8 mL) was added and the reaction mixture was allowed to raise from -78° C to room temperature over 5 hrs. The mixture was poured into 5% aqueous NaHCθ3 and extracted with EtOAc. The combined organic layers were dried, evaporated to dryness, and after purification by flash chromatography (SiO2; hexane:CH2Cl2:acetone 70:15:15) gave (Z) 3β-(5- azidopent-l-enyl)androstane-6,17-dione (dd2a) (215 mg, 50%). 1H-NMR (300 MHz, DMSO-dg, ppm from TMS): δ 5.31 (2H, m), 3.34 (2H, t, J= 6.64 Hz),), 2.50-1.15 (25H, m), 0.87 (3H, s), 0.76 (3H, s). To a solution of (Z) 3jβ-(5-azidopent-l-enyl)androstane-6,l 7-dione (dd2a) (145 mg), in
THF (7 mL), triphenylphosphine (100 mg) and water (11 μL) were added and stirred at room temperature for 2 days. The crude product obtained after evaporation was purified by flash chromatography (SiO2; CH2Cl2MeOH 9:1 then CH2Cl2:Me0H:NH3 7:3:0.3). The solvent evaporated to reduced volume to remove ammonia, then IN hydrochloric acid was added: a precipitate was obtained and filtered to give (Z) 3yβ-(5-aminopent-l-enyl)androstane-6,l 7-dione hydrochloride (dd3a) (115 mg, 85%). 1H-NMR (300 MHz, DMSO-dg, ppm from TMS): δ 7.78 (3H, bb), 5.25 (2H, m), 2.73 (2H, m), 2.55-1.05 (25H, m), 0.78 (3H, s), 0.67 (3H, s).
To a solution of (4-azidobutyl)triphenylphosphonium bromide (750 mg) in THF (5 mL), lithium bis(trimethylsilyl)amide (IM in THF, 1.7 mL) was added at -5° C and the reaction mixture was stirred to complete dissolution. 3α-Formylandrostane-6,17-dione (ddlb) 490 mg) was added and the reaction mixture allowed to rise from -78° C to room temperature in 5 hrs. The mixture was poured into 5% aqueous NaHCO3 and extracted with EtOAc. The combined organic layers were dried over Na2Sθ4 and evaporated to dryness. The residue was purified by flash chromatography (SiO2; hexane:CH2Cl2:acetone 70:15:15) to give (Z) 3α-(5-azidopent-l- enyl)androstane-6, 17-dione (dd2b) (265 mg, 43%). 1H-NMR (300 MHz, acetone-dg, ppm from TMS): δ 5.79 (IH, m), 5.34 (IH, m), 3.35 (2H, t), 2.85 (IH, m), 2.60-1.20 (24H, m), 0.87 (3H, s), 0.79 (3H, s).
To a solution of (Z) 3α-(5-azidopent-l-enyl)androstane-6,l 7-dione (dd2b) (250 mg) in THF (12 mL), triphenylphosphine (172 mg) and water (19 μL) were added and stirred at room temperature for 2 days. The crude product obtained after evaporation was purified by flash chromatography (SiO2; CH2Cl2MeOH 9:1 then CH2Cl2:Me0H:NH3 7:3:0.3) to afford (Z) 3α-(5- aminopent-l-enyl)androstane-6,17-dione (dd3b) as a white solid (220 mg, 860Zo)-1H-NMR (300 MHz, DMSOd6, ppm from TMS): δ 7.87 (3H, bb), 5.74 (IH, m), 5.26 (IH, m), 2.74 (3H, m), 2.55-1.10 (24H, m), 0.78 (3H, s), 0.69 (3H, s).
To a stirred solution of (Z) 3/3-(5-aminopent-l-enyl)androstane-6,17-dione (dd3a) (250 mg) in THF (6 mL), a solution of NH2OH-HCl (24,6 mg), Na2HPO4 12 H2O (0.48 g) in H2O (2.4 mL) was rapidly added drop wise. After 1.5 h, NaCl (0.37 g) was added and the mixture stirred for 10 min. The phases were separated and the aqueous phase was extracted with TBF (2 x). The combined organic extracts were dried over Na2SO4, filtered and evaporated to dryness. The crude product was purified by flash chromatography (SiO2, CH2Cl2:Me0H 9:1 then CH2Cl2:Me0H:NH3 90: 10: I s. To the concentrated fractions a stoichiometric amount of fumarie acid in MeOH was added. After addition of EtOAc, the precipitate was filtered to give 103 mg (40 %) of the carboxyacrylate of Compound (18a) as a white solid. 1H-NMR (300 MHz, DMSO- dg, ppm from TMS): δ 10.36 (IH, s), 7.80 (3H, bb), 6.35 (2H, s), 5.25 (2H, m), 3.28 (IH, m), 2.73 (2H, m), 2.55-1.05 (25H, m), 0.78 (3H, s), 0.67 (3H, s). To a stirred solution of (Z) 3α-(5-aminopent-l-enyl)androstane-6,17-dione (dd3b) (250 mg) in THF (6 mL), a solution of NH2OH-HCl (24,6 mg), Na2HPO4 12 H2O (0.48 g) in H2O (2.4 mL) was rapidly added drop wise. After 1.5 h, NaCl (0.37 g) was added and the mixture stirred for 10 min. The phases were separated and the aqueous phase was extracted with THF (2 Xi1 The combined organic extracts were dried over Na2SO4, filtered and evaporated to dryness. The crude product was purified by flash chromatography (SiO2, CH2Cl2:Me0H 9:1 then
CH2Cl2 :MeOH:NH3 90:10:1). To the concentrated fractions a stoichiometric amount of fumarie acid in MeOII was added. After addition of EtOAc, the precipitate was filtered to give 88 mg (34 %) of the carboxyacrylate of Compound (18b) as a white solid. 1H- NMR (300 MHz, DMSO-dg, ppm from TMS): δ 10.36 (IH, s), 8.00 (3H, bb), 6.35 (2H, s), 5.77 (IH, m), 5.27 (IH, m), 3.29 (IH, m), 2.74 (3H, m), 2.54-1.09 (24H, m), 0.77 (3H, s), 0.69 (3H, s).
EXAMPLE 19
Synthesis of 3-((3R,5R,l OR, 13S)-10,13-dimethyl-6-methylene-17-oxohexadecahydro-lH- cyclopenta[a]phenanthren-3-ylthio)propan-l-aminium (Z)-3-carboxyacrylate (the carboxyacrylate of Compound (19))
Figure imgf000098_0001
Preparation of the carboxyacrylate of Compound (19)
To a solution of triphenylphosphine (2.38 g) in THF (140 mL) cooled at 0° C, diisopropyl azodicarboxylate (1.79 mL) was added dropwise. After stirring for 30 minutes, thioacetic acid (0.65 mL) and androstane-3yβ,6α,17yβ-triol (compound al) (2.00 g) were added. After 2 hrs at 0 0C and overnight at room temperature EtOAc was added. The mixture was washed with water and the organic layer evaporated to dryness. The crude product was purified by flash chromatography (Siθ2, cyclohexane: EtOAc 55:45) to give 3α-acetylthioandrostane- 6α,17β-diol (compound eel) (1.60 g, 66%). 1H-NMR (300 MHz, DMSO-dg, ppm from TMS): δ 4.42 (IH, bb), 4.28 (IH, bb), 3.91 (IH, bb), 3.42 (IH, m), 3.11 (IH, m), 2.28 (3H, s), 2.00-0.80 (2OH, m), 0.74 (3H, s), 0.60 (3H, s).
To a stirred suspension of 3α-acetylthioandrostane-6α,17yβ-diol (compound eel) (1.40 g) in CH2Cl2 (50 mL), NMNO (1.37 g), TPAP (68 mg) and powdered molecular sieves 4A (2.1 g) were added at room temperature. After 2 hrs NMNO (0.7 g), TPAP (34 mg) and molecular sieves 4A (1 g) were added again and the reaction was stirred for further 1.5 hrs. The crude product was purified by flash chromatography (SiO2, cyclohexane:EtOAc 7:3) to give 3a- acetylthioandrostane-6,17-dione (compound ee2) (1.07 g, 76%). 1H-NMR (300 MHz, acetone- dg, ppm from TMS): δ 3.99 (IH, bb), 2.55-1.20 (23H, m), 0.86 (3H, s), 0.79 (3H, s).
To a stirred solution of 3α-acetylthioandrostane-6,17-dione (compound ee2) (600 mg) in THF (8 mL) cooled at -50° C, a solution of ylide prepared from methyltriphenylphosphonium bromide (1.47 g) in THF dry (8 mL) at -50 0C and potassium tert-butoxide (484 mg), was added. After 2 h the temperature was raised to room temperature. The mixture was quenched by addition of 5% NaH2PO4 aqueous solution and extracted with EtOAc (2 x 60 mL). The combined organic extracts were washed with 5% NaH2PO4 aqueous solution, brine, dried over Na2SC>4, and evaporated to dryness. The residue was purified by flash chromatography (n- hexane/EtOAc 9/1) to give 3α-acetylthio-6-methyleneandrostan-17-one (compound ee3) (210 mg, 35% yield) and Sα-mercapto-ό-methyleneandrostan-lT-one (compound ee4) (208 mg, 35 % yield). 1H-NMR (300 MHz, DMSO-de, ppm from TMS): 3α-acetylthio-6-methyleneandrostane- 17-one (compound ee3): δ 4.73 (IH, m), 4.39 (IH, m), 3.96 (IH, m), 2.44-0.84 (2OH, m), 2.29 (3H, s), 0.75 (3H, s), 0.66 (3H, s); Sα-mercapto-ό-methyleneandrostane-π-one (compound ee4): δ 4.73 (IH, m), 4.38 (IH, m), 3.57 (IH, m), 2.52 (IH, d), 2.45- 0.95 (2OH, m), 0.76 (3H, s), 0.63 (3H, s).
To a solution of 3α-acetylthio-6-methyleneandrostane-17-one (compound ee3) (210 mg) in MeOH (3 mL), IN NaOH (0.6 mL) was added. After Ih at room temperature 5% NaH2PO4 aqueous solution was added and the mixture extracted with Et2O (2 x 20 mL). The combined organic extracts were washed with brine, dried over Na2SO4 and evaporated to dryness to give 3α-mercapto-6-methyleneandrostane-17-one (compound ee4) (185 mg, 100%).
To a stirred solution of 3α-mercapto-6-methyleneandrostane-17-one (compound ee4) (100 mg) in dry DMF (1.5 mL), NaH 60% in oil (13 mg) was added at 00C. After 5 min. a solution of N-trifluoroacetamidopropylbromide (147 mg) in DMF (0.5 mL) was added dropwise over 30 min. at room temperature. After 2 hrs, a 5% NaH2PO4 solution was added. The phases were separated and the aqueous phase was extracted with EtOAc. The organic layers were washed with brine, dried over Na2SO4 and evaporated to dryness. The crude product was purified by flash chromatography (SiO2, n-hexane/EtOAc 75/25) to give 3α-(3-trifluoro- acetamidopropylthio)-6-methyleneandrostane-17-one (compound ee5) (104 mg, 70%) 1H-NMR (300 MHz, DMSO-dg, ppm from TMS): δ 9.43 (IH, bb), 4.72 (IH, m), 4.41 (IH, m), 3.24 (3H, m), 2.50-0.86 (24H, m), 0.75 (3H, s), 0.65 (3H, s).
To a solution of 3α-(3-N-trifluoroacetamidopropylthio)-6-methylene-androstane-17-one (compound ee5) (85 mg) in MeOH/H2O 8/2 (5 mL), Ambersep 900 OH (1.4 g) was added and the resulting mixture was stirred overnight at room temperature. The resin was filtered and the solvent evaporated to small volume. Fumaric acid (20 mg) was added and the solution evaporated to dryness. The resulting solid was washed with Et2O, filtered and dessiccated to give the carboxyacrylate of Compound (19) (80 mg, 90% yield) as a white powder. 1H-NMR (300 MHz, DMSO-dg, ppm from TMS): δ 9.50 (4H, bb), 6.38 (2H, s), 4.73 (IH, m), 4.41 (IH, m), 3.24 (IH, m), 2.81 (2H, m), 2.56-0.84 (24H, m), 0.75 (3H, s), 0.65 (3H, s). EXAMPLE 20
Synthesis of 3-((S,R)-((3R,5R,10R,l 3S)-IOJ 3-dimethyl-6-methylene-l 7-oxohexadecahydro-lH- cyclopenta[a]phenanthren-3-yl)sulfinyl)propan-l-aminium (Z)-3-carboxyacrylate & (the
Figure imgf000100_0001
Preparation of the carboxyacrylate of Compound (20a & b)
To a solution of 3α-(3-trifluoroacetamidopropylthio)-6- methyleneandrostane-17-one (ee5) (286 mg) in dry CH3CN (14 mL), NMO (213 mg) and molecular sieves (4 A, 280 mg) were added followed by the addition of TPAP (10.6 mg). After Ih at room temperature the mixture was evaporated to dryness. The residue was purified by flash chromatography (Siθ2, n- hexane/acetone 65/35) to give 3α-[(S)-3 trifluoroacetamidopropylsulfmyl]-6-methylene- androstane-17-one (Compound ee6a) (100 mg, 34% yield) and 3α-[(R)-3-trifluoroacetamido- propylsulfmyl]-6-methyleneandrostane-17- one (Compound ee6b) (70 mg, 24% yield).
Compound ee6a: 1H-NMR (300 MHz, DMSOd6, ppm from TMS): δ 9.43 (IH, bb), 4.72 (IH, m), 4.41 (IH, m), 3.24 (3H, m), 2.50-0.86 (24H, m), 0.75 (3H, s), 0.65 (3H, s).
Compound ee6b: 1H-NMR (300 MHz, DMSO- dg, ppm from TMS): δ 9.43 (IH, bb), 4.70 (IH, m), 4.41 (IH, m), 3.24 (3H, m), 2.50-0.86 (24H, m), 0.75 (3H, s), 0.65 (3H, s).
To a solution of 3α-[(S)-3-trifluoroacetamidopropylsulfonyl]-6- methyleneandrostane- 17-one (Compound ee6a) (100 mg) or 3α-[(R)-3-trifluoroacetamidopropylsulfonyl]-6-methylene -androstane-17-one (Compound ee6a) (70 mg) in MeOH/H2O 8/2 (5 mL) or (3.5 mL), respectively, Ambersep 900 OH (1.4 g) or (0.98g), respectively was added and the resulting mixture was stirred overnight at room temperature. The resin was filtered and the solvent evaporated to small volume. Fumaric acid (20 mg) or (14 mg), respectively was added and the solution evaporated to dryness. The resulting solid was washed with Et2O, filtered and desiccated to give compound 20a (94 mg, 90% yield). 1H-NMR (300 MHz, DMSO-de, ppm from TMS): δ 7.98 (3H, bb), 6.42 (2H, s), 4.72 (IH, m), 4.41 (IH, m), 3.24 (3H, m), 2.50-0.86 (24H, m), 0.75 (3H, s), 0.65 (3H, s) or compound 20b (85 mg, 90% yield). 1H-NMR (300 MHz, DMSO-dg, ppm from TMS): δ 7.98 (3H, bb), 6.42 (2H, s), 4.70 (IH, m), 4.41 (IH, m), 3.24 (3H, m), 2.50-0.86 (24H, m), 0.75 (3H, s), 0.65 (3H, s).
EXAMPLE 21
Synthesis of oxalic salt of (1 OR, 13S)-3—((R)-pyrrolidin-3-yloxyimino)-6-(methoxyimino)-l 0, 13- dimethyltetradecahydro-lH-cyclopentafaJphenanthren-17(2H)-one (Compound (21))
21
Figure imgf000101_0001
To a stirred solution of 6-(£")-methoxyiminoandrostane-3,17-dione (compound (c4), see synthesis of Compound (6) above) (90 mg, 0.27 mmol)) in THF (1.7 mL), a solution of 3-(R)-pyrrolidinyloxyamine dihydrochloride (Intermediate (II), 52 mg, 0.30 mmol) in H2O (0.75 mL) was added dropwise. After 1.5 h the reaction was complete, NaCl (114 mg, 1.95 mmol) was added, and the reaction mixture was stirred for an additional 15 min. The aqueous layer was extracted with THF (x2) and the combined organic phases were dried over Na2SO4, were filtered and evaporated under reduced pressure. The residue was purified by flash column chromatography using CHCl3/MeOH/NH3 94/6/1 as elution solvent, to afford 106 mg (95%) of compound 21. 1H-NMR (600 MHz, CDCl3) δ 8.83 (IH, bs), 4.77 (2H, m), 3.81 (3H, s), 3.55-3.28 (5H, m), 3.12 (IH, m), 2.47-0.93 (19H, m), 0.83 (3H, s), 0.82 (3H, s). The pure product was dissolved in MeOH (0.3 rnL), and a stoichiometric amount of oxalic acid was, added. The oxalic salt of compound 21 was precipitated by addition Of Et2O (5 mL), and the white solid was filtered and dried in vacuo (97 mg).
II. BIOLOGICAL EXAMPLES
EXAMPLE 22
Na+/K+-ATPase inhibitory activity of the different compounds Compound 1 (Istaroxime) inhibited the Na+/K+-ATPase with an IC5O of 0.2 μM when tested in vitro on purified dog kidney ATPase (De Munari S et al., J Med Chem. 2003 Aug 14;46(17):3644-54). In contrast, rostafuroxin inhibited purified dog kidney Na+/K+-ATPase with an IC5O of 25 μM. At low K+ concentration (0.5 mM) the IC5O was 1.5 μM (Ferrari P et al, J Pharmacol Exp Ther. 1998 Apr;285(l):83-94). Table 3 summarizes the Na+/K+- ATPase inhibitory activity of Compound Nos. 1-21. Ouabain and digoxin, two well known cardiotonic Na+/K+-ATPase inhibitors were used as positive controls.
Table 3: Na+/K+- ATPase inhibitory activity of tested compounds
Figure imgf000102_0001
nd: not done All compounds tested inhibited the activity of the Na+K+ ATPase. Compounds 3, 4, 11 and 21 were the strongest Na+K+ ATPase inhibitors with activity below 10OnM. The inhibitory activity of ouabain was 96 nM, whereas digoxin had an IC5O of 204 nM.
EXAMPLE 23
Compound 1 (Istaroxime) inhibited the proliferation and survival of neoplastic cells
The effect of istaroxime and rostafuroxin on the proliferation and viability of cancer cell lines was tested in two prostate cancer cell lines, PC3 (Figure IA) and DUl 45 (Figure IB) and in one colon cancer cell line, HCTl 16 (Figure 1C). Each of the cultures was exposed to 33μM of istaroxime and rostafuroxin and results were analysed using a 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) proliferation/viability assay. This colorimetric assay uses the reduction of MTT to formazan as a measure of metabolic activity. Absorbance results are shown in Figures IA- 1C. Untreated cells cultured in the presence of serum were used as a negative control. The effects of ouabain and digoxin, two well known cardiotonic steroid inhibitors of the Na+K+- ATPase, were assayed as positive controls.
Istaroxime was a potent inhibitor of cell proliferation and viability. The proliferation/viability of all cell lines was completely abolished in the presence of ouabain, digoxin and istaroxime. Surprisingly, rostafuroxin, a well established Na+K+- ATPase inhibitor, showed no effect in vitro. These results show that Na+K+- ATPase inhibitors are not equally effective in inhibiting cell proliferation and viability. On the contrary, Na+K+- ATPase inhibitors have selective anti-proliferative / growth inhibitory actions. Without wishing to be bound by theory, it is likely that their activity in inhibiting the Na+K+- ATPase is not the sole mechanism that accounts for their inhibitory effect on neoplastic cell growth. It is likely that other functional determinants of different Na+K+- ATPase inhibitors contribute to the anti-proliferative effects observed.
EXAMPLE 24
Compounds of the invention had potent anti-cancer activity in 22 different cancer cell lines
Figures 2A and 2B summarize results characterizing the effects of Compounds 1-21 and rostafuroxin on GI50, which measures the cell growth inhibitory power of the compound, TGI, which measures cytostatic effect (TGI) and LC50, which signifies cytotoxic effect. Each of these parameters was calculated in a sulforhodamine B (SRB) assay. SRB provides a colorimetric measure of a compound's anti-cancer activity. SRB assays were performed with compounds of the invention and rostafuroxin in 22 different cell lines, which represent nine different tumor panels (lung, breast, CNS, colon, prostate, ovary, pancreas, kidney and melanoma). Ouabain and digoxin were used as controls. Paclitaxel was added as a non- Na+K+- ATPase inhibitor control.
The results obtained with SRB showed that compounds of the invention have potent anti-cancer activity in multiple cell lines. Some of the compounds exhibited single-digit micromolar GI50 action (e.g. compound 1/istaroxime) whereas others (e.g compounds 2, 4, 7 and 10) had on average GI50 values lower than 10OnM. In contrast, rostafuroxin did not show any significant anti-tumor activity in any of the cell lines tested. Ouabain and digoxin had GI50 activity also at the nanomolar level, which is similar to the activity of paclitaxel. When comparing LC50 values, compounds of the invention (e.g compound 4 and 10) were much stronger than ouabain, digoxin or even paclitaxel. Quantitative MTT assays were also performed in two cell lines (DU145/prostate and
CAKI/kidney) as validation of the results obtained with SRB. The calculated IC5O results obtained are presented in Figure 2C. These results are consistent with the SRB results and demonstrate the in vivo anti-cancer action of the compounds of the invention.
EXAMPLE 25
Compounds 1, 2 and 4 of the invention are effective against multidrug resistant neoplasia
To further evaluate the ability of compounds 1, 2 and 4 of the invention to inhibit tumor cell growth, their GI50/TGI/LC50 values were calculated in a drug resistant cell line, NCI- ADR- RES. NCI-ADR-RES cells express high levels of MDRl and P-glycoprotein. Consistent with the results reported above, compounds 1 , 2 and 4 showed significant anti-tumor activity in this line. Most importantly, compounds 1 and 2 exhibited comparable GI50 values in multi-drug resistant NCI-ADR-RES cells, as well as in non-resistant MCF-7 cells. These results indicate that compounds of the invention are just as effective in cells that are multidrug resistant as they are in other cancers. These results further indicate that compounds of the invention are effective chemotherapeutic agents for the treatment of refractory and/or multidrug resistant tumors in subjects.
EXAMPLE 26
Compounds of the invention show anti-tumor activity in human prostate and lung xenografts Figure 3 presents data on the anti-tumor activity of compound 1 (istaroxime) and compound 4 in PC-3 prostate xenografts as well as in A549 lung xenografts (compound 4 only). In PC-3 xenograft experiments, compound 1 inhibited tumor growth with an optimal DT/DC of 52.5 % at dpi 23 (p=0.002, see Figure 3A), where DT = T - Do and DC = C - Do (Do is the average tumor volume at the beginning of the treatment; T and C are the volumes of treated and untreated tumors, respectively, at a specified day. Apart from one death occurring suddenly at day 23 (after administration of 6 doses of the drug) istaroxime was not toxic under the experimental conditions used (as defined by body weight measurements).
In a separate PC-3 xenograft experiment (Figure 3B), compound 4 showed strong antitumor action with a DT/DC value of 10.77% with the highest dose (25mgk) at day 16 (p=8E-08) and a DT/DC value of 31.41 % for the 17mgk dose at dpi 23 (p=2E-09). Paclitaxel, used as a positive control, had a DT/TC value of 5.02% at dpi 27 (P=2E-05). Confirming these results, post mortem analysis on excised tumors showed a statistical significant tumor growth inhibition of compound 4 and a suppression of bone lymph node and front axilla metastases in comparison to the control group. For what concerns toxic effects of the compounds, one drug related death occurred with compound 4 at the highest dose (25mgk; dpi 34). Overall, compound 4 induced body weight loss which remained within acceptable limits (weight loss was less than 15% of the initial body weight). Notably, in the same experiment, 2 paclitaxel-treated animals died at dpi 16 and 27 in the corresponding group (paclitaxel used as a positive control).
In A549 xenograft experiments (Figure 3C), compound 4 showed the best DT/DC value at day 29 with the highest (20mgk) dose (-42.01% , p= 2,3E-08), whereas the two other doses, 15 and lOmgk, also showed significant activity with DT/DC values of -59,37% and -52,9% respectively at day 24 (p=5,4E-06 and 3E-05 respectively). Paclitaxel (used as a positive control), had a DT/DC of -46,68% at day 24 with a p=2.8-05. Throughout the A549 experiment, no drug related deaths or significant weight changes were recorded suggesting that the compounds were not toxic at the doses tested.
Overall, these results suggest that compounds of the invention have in vivo anti-tumor activity in animal models of cancer.
EXAMPLE 27
Compounds 1 {Istaroxime) and 4, but not rostqfuroxin, bind the membrane androgen receptor The membrane androgen receptor (mAR) is capable of transmitting rapid (non-genomic) androgen signals resulting in robust actin cytoskeleton re- organization in membrane androgen expressing cells (Papadopoulou et al, IUBMB Life. 2009 Jan;61(l):56-61). Some have speculated that mAR may comprise a membrane-bound form of the classical, pro-oncogenic intracellular androgen receptor (AR). Recent results point to the existence of a novel receptor that is structurally and functionally distinct from the classical androgen receptor (reviewed in Papadopoulou et al, IUBMB Life. 2009 Jan;61(l):56-61). For example, mAR-induced effects are observed even in the presence of anti-androgens and/or in cell lines deficient in the classical intracellular androgen receptor. In addition, membrane androgen receptors comprising testosterone-albumin conjugates are capable of inducing cell death -rather than proliferation- in AR-positive as well as AR-deficient cells. Consequently, testosterone-albumin conjugates have been shown to possess significant anti-cancer activity in various in vitro and in vivo models, independent of the status of the classical androgen receptor (reviewed in Papadopoulou et al, IUBMB Life. 2009 Jan;61(l):56-61, see also PCT/IB03/02785). Similar results have been reported for dihydro-testosterone bovine serum albumin (DHT-BSA) conjugates in C6 cells (Gatson et al, Endocrinology 2006, 147:2028-2034).
To identify compounds capable of binding to the membrane androgen receptor, and since the identity of the mAR is not yet known, compounds having the typical A-D ring structure of steroids were randomly screened in a fluorescent-based assay performed in LNCaP or DUl 45 cells for their capacity to preclude binding of a fluorescent mAR ligand (testosterone-HSA-FITC conjugates) to its receptor. To determine whether Na+K+ ATPase inhibitors, such as compounds 1 and 4 or rostafuroxin, were capable of blocking access of the conjugates to mAR, these compounds were added to the cells prior to incubation with testosterone-HSA-FITC conjugates. The results of these experiments are presented in Figures 4A and B. Testosterone-HSA-FITC (TAC-FITC) conjugates showed clear membrane staining. This staining was absent from control (HSA-FITC) treated cells. Cells pre-treated with testosterone 3-(O- carboxymethyl)oxime (Testo-CMO; the steroid moiety present in TAC-FITC conjugates) were devoid of membrane staining, which indicates efficient competition for binding to the membrane androgen receptor. Interestingly, istaroxime pre-treated cells (Figure 4A) or compound 4 pre- treated cells (Figure 4B) were also devoid of membrane fluorescence indicating that istaroxime precludes binding of TAC-FITC to the membrane androgen receptor. In contrast, the other Na+K+ ATPase inhibitor used in this experiment, rostafuroxin (Figure 4A), failed to block binding of TAC-FITC to mAR.
Taken together these results indicate that compounds 1 and 4, but not rostafuroxin, are capable of binding to the membrane androgen receptor. These results clearly indicate that specific Na+K+ ATPase inhibitors likely function as membrane androgen receptor ligands. Since mAR is functionally implicated in cancer death by apoptosis, it is likely that the anti-cancer action of different Na+K+ ATPase inhibitors depends not only on their capacity to bind and inhibit Na+K+ ATPase, but also on their ability to bind to the membrane androgen receptor. Interestingly, testosterone, a natural mAR ligand does not inhibit Na+K+ ATPase (Farr et al 2002, Biochemistry, 2002, 41 (4), 1137-1148).
The results reported herein were obtained using the following methods and materials.
Reagents Ouabain, digoxin, paclitaxel, Testosterone-HSA (testosterone 3-(O- carboxymethyl)oxime: human serum albumin) conjugates and fluorescein isothiocyanate (FITC) were purchased from Sigma (St.Louis, MO). Rostafuroxin was synthesized as reported in European Patent EP0583578.
Synthesis of fluorescent testosterone-HSA-FITC (TAC) and HSA-FITC conjugates To label testosterone-HSA or HSA conjugates with FITC, a freshly prepared solution containing 6 mg of fluorescein isothiocyanate in 3 ml of 0.1M sodium carbonate buffer at pH 9.5 and O0C was added with stirring to 60 mg of a testosterone-human serum albumin conjugate (HSA) or HSA dissolved in 12 ml buffer and stirring was continued overnight at 4 0C. The reaction mixture was placed in a dialysis tubing and dialyzed for five days against 2 liters of 10 mM ammonium bicarbonate NH4HCO3 at 40C. Fresh dialysis solution was provided at 24 hour intervals. After dialysis, the sample was examined by thin layer chromatography to preclude the existence of free un-reacted testosterone or FITC and was subsequently lyophilized to dryness yielding 40.2 mg of Testosterone-HSA-FITC and 39.2 mg of HSA-FITC conjugate respectively. To determine the relative conjugation ratios of HSA to FITC in each case, 0.3 mg of Testosterone-HSA-FITC, HSA-FITC conjugate or FITC were dissolved in 2 ml of 0.05 M Tris buffer pH = 8.4 and the absorbance at 252, 280 and 495 nm was measured. Based on the spectrophotometer results, testosterone-HSA-FITC conjugates contained 8.7 moles FITC per mole HSA, whereas HSA-FITC conjugates contained 7.62 moles FITC per mole HSA.
Cell lines
All cell lines were obtained from the American Type Culture Collection (Manassas, VA) or the National Cancer Institute, NIH (Bethesda, MD, USA) and were adapted to grow in the commerially available culture media RPMI 1640, which was supplemented with 25 mM HEPES, 2 mM L-Glutamine, 5-10% fetal bovine serum and antibiotics in a 5% CO2 humidified atmosphere (100%) at 37°C.
NCI-ADR-RES cells
NCI-ADR-RES cells have been classified as ovarian cancer cells by the National Cancer Institute.
MTT proliferation/viability assays
Cell proliferation/viability was assessed by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide] assay, which is commercially available (Sigma, St.Louis, MO). Cells were cultured in 96-well plates for twenty-four hours (7-10.000 cells/well) and incubated with 33 uM of Istaroxime, Rostafuroxin, Ouabain, and digoxin in serum-containing medium (or left untreated) for seventy-two hours. At the end of incubation, the medium was aspirated and MTT dissolved in RPMI 1640 w/o phenol red was added to each well to a final concentration of 0.1 mg/ml (HCTl 16) or 0.25 mg/ml (PC3, DU145 cells). After four hours (HCTl 16 cells: two hours) incubation in the dark (37°C, 5% CO2) the supernatant was discarded. The converted dye (blue formazan crystals) was solubilized by adding 200 μl dimethylsulfoxide to each well. Absorbance was measured at 550 ran with reference at 655 ran using a spectrophotometer with the respective filters. All assays were performed in triplicate. To determine IC50 values of the compounds of the invention, cells were incubated with 5 concentrations of each compound (typically, 10OuM, lOuM, IuM, 10OnM, 1O nM) for forty weight hours before processing the samples as described above. IC50 values were then calculated using the Origin® program software (OriginLab, Northampton, MA). Note that calculated IC50 values based on the MTT assay may differ from GI50 values calculated with the SRB assay (see below) due to inherent difference in assay methodologies.
Sulphorhodamine B (SRB) assays
SRB assays were performed according to standard National Cancer Institute Guidelines.
Briefly, human tumor cells from different cancer panels were seeded into 96 well plates in 100 μL (plating densities ranging from 5-40,000 cells/well depending on the doubling time of individual cell lines) in serum-containing media for twenty- four hours prior to the addition of the compounds to be assayed. After 24 hours, one plate of each cell line was fixed in situ with trichloroacetic acid (TCA). The fixed slides were used to determine the cell population for each cell line at the time of drug addition (Tz). Each compound to be tested was solubilized in DMSO and the desired concentration was then added to the medium and diluted serially 1 :2, 1 :4 or 1 :10 to provide a total often drug concentrations plus control (in a final volume of 200 μL).
The starting dose before any dilution was 100 uM for all compounds.
Following drug addition, each culture was incubated for forty-eight hours. The assay was terminated with the addition of cold TCA. The supernatant was discarded, and the plates were washed with tap water and air dried. Sulforhodamine B (SRB) solution was then added to each well. After staining, the bound stain was solubilized and the absorbance was read on an automated plate reader at a wavelength of 515 nm.
Using the absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the ten concentration levels (Ti)], the percentage growth was calculated at each of the drug concentrations levels. Percentage growth inhibition was calculated as:
[(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti>/=Tz [(Ti-Tz)/Tz] x 100 for concentrations for which Ti<Tz. Three dose response parameters were calculated for each experimental agent. Growth inhibition of 50 % (GI50) was calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50, which was the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment was calculated from [(Ti-Tz)/Tz] x 100 = -50.
Values were calculated for each of these three parameters when the level of activity was reached; however, if the effect was not reached or was exceeded, the value for that parameter was expressed as greater or less than the maximum or minimum concentration tested.
Xenograft Studies
To generate xenografts in mice, exponentially growing cultures of approximately 106 PC-3 cells (prostate adenocarcinoma, grade IV) or A549 cells (lung carcinoma) were injected subcutaneous Iy according to the British practice of bilateral trocar implants at the axillary region of 6-8 weeks old male (PC-3 cells) or female (A549 cells) Nod/Scid mice. The two tumor cell inoculums were added together for each mouse, thus producing a tumor burden per mouse value for data analysis. The advantage of the British system is reduced mouse-to-mouse variability and, thus, the ability to decrease the total number of animals per group, with five or six being sufficient for accurate data analysis of tumor response and evaluation of toxicities.
After having randomized the animals in groups, treatments started when the average tumor volume had reached about 100-200 mm . Tumor volume was calculated according to the formula [(axb2)/2], where a=length and b=width of the tumor as measured with a vernier's caliper (measurements were performed twice a week). All administrations were intraperitoneal. Treated animals received a single injection according to the schedule described for each study (see below). In addition to tumor volume, the parameter, %DT/DC was calculated, where DT = T - Do and DC = C - Do (Do is the average tumor volume at the beginning of the treatment; T and C are the volumes of treated and untreated tumors, respectively, at a specified day). Optimal DT/DC value was used as a measure of drug activity. Losses of weight, neurological disorders and behavioral and dietary changes were also recorded as indicators of toxicity (side effects). The experiment was terminated when tumor size in untreated animals reached a volume of about 1000-1500 mm3. All animals were treated according to Greek laws (2015/92), guidelines of the European Union and the European council (86/609 and ETS123, respectively), and Compliance with Standards for Human Care and Use of Laboratory Animals, NIH, USA (Assurance No. A5736-01). PC-3 experiments
In order to develop PC-3 xenografts, male mice 6-8 weeks old were inoculated bilateral at the two axilla regions at day 0. Mice were observed for the development of tumors every two days by eye and palpation. In the first experiment in PC-3 xenografts performed with istaroxime, mean tumor volume was measured at 176±83 mm at day 13 post inoculation (dpi),.
Subsequenbtly, mice were randomly divided into 2 groups of 6 animals as described below:
Group A: untreated animals
Group B: 40 mgk Compound 1 treated animals (WFI)
Where:
- WFI means Water For Injection
- mgk means mg/kg
Istaroxime was administered intraperitoneallly at the following dpis: dl4, dl5, dl6, dl7, d21, d22, d23 and d24.
In the second PC-3 experiment performed with compound 4, mean tumor volume was measured at 176.7 mm at dpi 13. Subsequenbtly, mice were randomly divided into 4 groups of 6 animals as described below:
Group A: untreated animals
Group B: paclitaxel 20mgk (Cremophor/EtOH/WFI) Group C: 25 mgk Compound 4 treated animals (WFI) Group D: 17mgk Compound 4 treated animals (WFI)
Compound 4 was administered intraperitoneallly at the following dpis: Group B: dl3, dl4, dl5, d20, d21, d22, d27, d28, d29 Group C: once daily from dl3 to d30
Paclitaxel was administered intraperitoneallly at dpi 13, 17, 21, 24, 28
A549 experiment
In order to develop A549 xenografts, female mice 6-8 weeks old were inoculated bilateral at the two axilla regions at day 0. Mice were observed for the development of tumors every two days by eye and palpation. At day 20 post inoculation (dpi), mean tumor volume was measured at 117.6 mm3. Subsequenbtly, mice were randomly divided into groups of 6 animals as described below: Group A: untreated animals
Group B: paclitaxel 18 mgk (Cremophor/EtOH/WFI)
Group C: 20 mgk Compound 4 treated animals (WFI)
Group D: 15 mgk Compound 4 treated animals (WFI) Group E: 10 mgk Compound 4 treated animals (WFI)
Compound 4 was administered intraperitoneallly at the following dpis:
Group B: d20, d21, d22, d23, d24, d27, d28, d34, d35, d38, d41, d42, d45, d48, d49, d50, d51, d52 Group C: d20, d21, d22, d23, d24, d27, d28, d29, d34, d35, d36, d37, d38, d41, d42, d43, d44, d45, d48, d49, d50, d51, d52 Group D: [(QDx5;2) x5] starting at dpi20
Paclitaxel was administered intraperitoneallly at dpi 21, 24, 28, 31, 35, 38, 42, 45, 49, 52
Detection of membrane androgen receptors
The detection of membrane androgen receptors using fluorescent testosterone-serum albumin conjugates was performed as described by Kampa, M et al, 2002, Faseb J 16:1429- 1431, with minor modifications. Briefly, LNCaP or DUl 45 prostate cancer cells were cultured on 0.1 % gelatin-coated glass coverslips in RPMI 1640 medium supplemented with 2 mM L-
Glutamine, 10 % Fetal Bovine Serum and 1% Penicillin/Streptomycin for at least 48 hours until reaching 70% confluency. After a 24 hour serum starvation period, cells were incubated with 40 μM testosterone-H SA-FITC for 1 hour at room temperature. The cells were then washed three times with phosphate buffered saline (PBS) and fixed with 3.7% formaldehyde in PBS for 5 minutes at room temperature. Human serum albumin-FITC was used as a control for background staining. After permeabilization with ice-cold acetone for 4 minutes at room temperature, cell nuclei were stained with DAPI. Coverslips were mounted on slides by using the Slow Fade/Antifade Reagent (Molecular Probes) and studied under a LEICA DMLB microscope, equipped with the appropriate fluorescence filters and a Leica DC 300F camera. Specimens were analyzed using the Leica FW4000 computer program.
Membrane androgen receptor competition assays
To determine the capacity of a given compound to preclude binding of fluorescent testosterone-HSA conjugates (TAC) to the membrane androgen receptor, starved LNCaP or DU145 cells on coverslips prepared as described above were pre-treated with 40 μM of the indicated drug for 30 minutes prior to the addition of testosterone-HS A-FITC. At the end of the incubation period, cells were washed twice with PBS and 40 μM of testosterone-HS A-FITC was added. Specimens were prepared and analyzed as described above. Compounds binding to the membrane androgen receptor preclude binding of fluorescent testosterone-HSA conjugates to their target and abolish membrane-specific fluorescence.
Other Embodiments
From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.
Ill

Claims

What is claimed is:
1. A method of treating or preventing neoplasia in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a compound of Formula (I)
Figure imgf000113_0001
wherein:
R1 is H or -OH;
One of R2 and R3 is H or OH, and the other is H; -OR8; alkyl optionally substituted with hydroxyl or alkoxy; -C(O)-NH2; -C(O)-O-alkyl; -NHR9; alkynyl; or R2 and R3 together with the carbon to which they are attached form C=O, C=C(R10)2, or C=N-OR11;
One of R4 and R5 is H, and the other is H; -OR8; alkyl optionally substituted with hydroxyl or alkoxy; -C(O)-NH2; -C(O)-O-alkyl; -NHR9; alkynyl; or R4 and R5 together with the carbon to which they are attached form C=O, C=C(R10)2, C=N-OR11, or a cyclopropyl ring;
One of R6 and R7 is H, and the other is -OH; alkoxy optionally substituted with a hydroxyl or an amino group; optionally subsituted heterocyclic or heteroaryl; or -OC(O)R12; or R6 and R7 together with the carbon atom to which they are attached form C=O;
R8 is H, alkyl, or -NO2;
R9 is H or formyl;
R10, for each occurrence, is the same or different and is H or halogen;
R11 is H or alkyl;
R12 is alkyl or phenyl;
A is CH(OR13), C=N-OR14, CH-CH=N-OR15, C=CH-CH=N-OR15, CH-CH=CH-R15,
CH-SR , 1160, or CH-S(O)R 1160;
R13 is alkyl optionally substituted by a 4 or 6-membered heterocyclic ring, a 5 or 6- membered heteroaryl, amino(Ci.4)alkoxyl, (Ci_4)alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; aminoalkyl optionally substituted by one or more alkyl; or aminoacyl optionally substituted by alkyl; R14 is aminoalkyl optionally substituted by one or more alkyl or acetyl; alkyl optionally substituted by guanidinyl or a 4- to 6-membered heterocyclic ring; or a 4- to 6-membered heterocyclic ring optionally substituted by alkyl;
R15 and R16, each independently, are aminoalkyl optionally substituted by one or more alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
2. The method of claim 1 , wherein R6 and R7 together with the carbon to which they are attached form C=O.
3. The method of claim 2, wherein R1 is H.
4. The method of claim 3, wherein R4 and R5 are both H.
5. The method of claim 4, wherein R2 and R3 together with the carbon to which they are attached form C=O.
6. The method of claim 5, wherein A is C=N-OR14.
7. The method of claim 6, wherein R14 is amino(Ci_C4)alkyl, that is further optionally substituted by one or more (Ci-C4)alkyl groups.
8. The method of claim 7, wherein R14 is selected from the group consisting of aminoethyl,
2-aminopropyl, 2-amino-2-methylpropyl, 2-amino-dimethylethyl, methylaminoethyl, methylaminopropyl, aminopropyl, aminobutyl, dimethylaminoethyl, and acetylaminoethyl.
9. The method of claim 6, wherein R14 is
Figure imgf000114_0001
that is further substituted by a 4- or
6-membered heterocylic ring or by a guanidinyl group.
10. The method of claim 9, wherein R14 is pyrrolidinyl-substituted methyl or guanidinylethyl.
11. The method of claim 6, wherein R14 is a 4- to 6-membered heterocyclic ring optionally substituted by (d_C4)alkyl.
12. The method of claim 11, wherein R14 is azetidinyl, pyrrolidinyl, or piperidinyl, wherein the azetidinyl, pyrrolidinyl, or piperidinyl group is further optionally substituted by a methyl group.
13. The method of claim 5, wherein A is CH(OR13).
14. The method of claim 13, wherein R13 is amino(Ci_C4)alkyl, which is optionally substituted by one or more
Figure imgf000115_0001
15. The method of claim 14, wherein R13 is selected from the group consisting of dimethylaminopropyl, dimethylaminoethyl, ethylaminobutyl, and dimethylaminobutyl.
16. The method of claim 13, wherein R13 is ethyl or propyl, provided that said ethyl or propyl group is further substituted by a substituent selected from the group consisting of pyrrolidinyl, piperidinyl, imidazolyl, dimethylamino-ethoxyl, pyrrolidinyl-ethoxyl, and 4- methylpiperazinyl.
17. The method of claim 5, wherein A is CH-CH=N-OR15 or C=CH-CH=N-OR15, and R15 is aminoethyl or dimethylaminoethyl.
18. The method of claim 5, wherein A is CH-S(O)R16, wherein R16 is aminopropyl.
19. The method of claim 4, wherein one of R2 and R3 is H, the other is -OH.
20. The method of claim 19, wherein A is C=N-OR14.
21. The method of claim 20, wherein R14 is aminoethyl, methylaminopropyl or pyrrolidinyl.
22. The method of claim 19, wherein A is CH(OR13).
23. The method of claim 22, wherein R13 is aminoethyl, methylaminoethyl or dimethylaminopropyl.
24. The method of claim 4, wherein R2 and R3 together with the carbon to which they are attached form C=CH2.
25. The method of claim 24, wherein A is C=N-OR14.
26. The method of claim 25, wherein R14 is amino(Ci_C4)alkyl that is optionally substituted by one or more (Ci-C4)alkyl groups, or a 4- to 6-membered heterocyclic ring optionally substituted by (CrC4)alkyl.
27. The method of claim 26, wherein R14 is selected from the group consisting of aminoethyl, aminopropyl, methylaminopropyl, 2-aminopropyl, methylaminoethyl, 3-amino-2- methylpropyl, and pyrrolidinyl.
28. The method of claim 24, wherein A is CH(OR13).
29. The method of claim 28, wherein R13 is aminopropionyl, 3-aminobutanionyl, or 3- amino-2-methylpropionyl.
30. The method of claim 24, wherein A is CH-CH=CH-R15, and R15 is aminopropyl or aminoethyl.
31. The method of claim 24, wherein A is CH-SR16 or CH-S(O)R16, and R16 is aminopropyl.
32. The method of claim 4, wherein R and R together with the carbon to which they are attached form C=CF2.
33. The method of claim 32, wherein A is CH-S(O)R16 and R16 is aminopropyl.
34. The method of claim 32, wherein A is C=N-OR14 and R14 is pyrrolidinyl or aminoethyl.
35. The method of claim 4, wherein R and R together with the carbon to which they are attached form C=N-OR11, wherein R11 is H or methyl.
36. The method of claim 35, wherein A is C=N-OR14, and R14 is selected from the group consisting of aminoethyl, 2-aminopropyl, 2-amino-dimethylethyl, and 3-amino-2-methylpropyl.
37. The method of claim 35, wherein A is CH-S(O)R16 and R16 is aminopropyl.
38. The method of claim 35, wherein A is CH-CH=CH-R15 and R15 is aminopropyl.
39. The method of claim 4, wherein one of R2 and R3 is H, the other is methyl.
40. The method of claim 39, wherein A is C=N-OR14.
41. The method of claim 40, wherein R14 is selected from the group consisting of aminoethyl, aminopropyl, methylaminoethyl, methylaminopropyl, 3-amino-2-methylpropyl, and 2-aminopropyl.
42. The method of claim 39, wherein A is CH(OR13), and R13 is selected from the group consisting of aminopropionyl, 3-aminobutanionyl, and amino-2-methylpropionyl.
43. The method of claim 39, wherein A is CH-SR16 or CH-S(O)R16, and R16 is aminopropyl.
44. The method of claim 39, wherein A is CH-CH=CH-R15, and R15 is aminopropyl or aminoethyl.
45. The method of claim 4, wherein one of R and R is H and the other is CH2OH, CH2CH2OH or CH2OCH3, A is CH-S(O)R16, and R16 is aminopropyl.
46. The method of claim 4, wherein one of R and R is H, and the other is selected from the group consisting Of-C(O)NH2, -ONO2, formylamino, ethynyl, and methoxycarbonyl.
47. The method of claim 46, wherein A is CH-S(O)R16 and R16 is aminopropyl.
48. The method of claim 4, wherein when one of R2 and R3 is H and the other is ethynyl, formylamino, or methoxycarbonyl, A is C=N-OR14.
49. The method of claim 48, wherein R14 is aminoethyl or pyrrolidinyl.
50. The method of claim 3, wherein one of R4 and R5 is H, and the other is -OH.
51. The method of claim 50, wherein both of R2 and R3 are H, A is CH-S(O)R16, and R16 is aminopropyl .
52. The method of claim 50, wherein one of R and R is H, the other is -CH2OH.
53. The method of 52, wherein A is C=N-OR14, and R14 is selected from the group consisting of aminopropyl, aminoethyl, methylaminopropyl, amino-2-methylpropyl, 2-amino- dimethylethyl, and pyrrolidinyl.
54. The method of claim 52, wherein A is CH(OR13), and R13 is aminopropionyl, 3- aminobutanionyl, or amino-2-methylpropionyl.
55. The method of claim 52, wherein A is CH-SR16 or CH-S(O)R16, and R16 is aminopropyl.
56. The method of claim 52, wherein A is CH-CH=CH-R15, and R15 is aminopropyl or aminoethyl.
57. The method of claim 3, wherein R4 and R5 together with the carbon to which they are attached form C=O.
58. The method of claim 57, wherein one of R2 and R3 is H, and the other is -CH2OH; or both of R2 and R3 are H.
59. The method of claim 58, wherein A is C=N-OR14, and R14 is selected from the group consisting of aminoethyl, aminopropyl, methylaminoethyl, methylaminopropyl, 2-aminopropyl, amino-2-methylpropyl, 2-amino- 1,1 -dimethylethyl, and pyrrolidinyl.
60. The method of claim 58, wherein A is CH(OR13), and R13 is aminopropionyl, 3- aminobutanionyl, or amino-2-methylpropionyl.
61. The method of claim 58, wherein A is CH-CH=CH-R15, and R15 is aminopropyl or aminoethyl.
62. The method of claim 58, wherein A is CH-SR16 or CH-S(O)R16, and R16 is aminopropyl.
63. The method of claim 3 , wherein R4 and R5 together with the carbon to which they are attached form C=CF2 or C=CH2, both of R2 and R3 are H.
64. The method of claim 63, wherein A is C=N-OR14, and R14 is selected from the group consisting of 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2- methylpropyl, 2-amino-dimethylethyl, and aminoethyl.
65. The method of claim 63, wherein A is CH(OR13), and R13 is selected from the group consisting of aminopropionyl, 3-aminobutanionyl, and 3 -amino-2-methylpropionyl.
66. The method of claim 63, wherein A is CH-CH=CH-R15 and R15 is aminopropyl or aminoethyl.
67. The method of claim 63, wherein A is CH-SR16 or CHS(O)R16 and R16 is aminopropyl.
68. The method of claim 3, wherein one of R4 and R5 is H and the other is methyl, and both of R2 and R3 are H.
69. The method of claim 68, wherein A is C=N-OR14, and R14 is selected from the group consisting of 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2- methylpropyl, 2-amino-dimethylethyl, and aminoethyl.
70. The method of claim 68, wherein A is CH(OR13), and R13 is selected from the group consisting of aminopropionyl, 3-aminobutanionyl, and amino-2-methylpropionyl.
71. The method of claim 68, wherein A is CH-CH=CH-R15, and R15 is aminopropyl or aminoethyl.
72. The method of claim 68, wherein A is CH-SR16 or CH-S(O)R16, and R16 is aminopropyl.
73. The method of claim 3 , wherein R4 and R5 together with the carbon to which they are attached form C=NOH, both of R2 and R3 are H, A is C=N-OR14, and R14 is aminoethyl or pyrrolidinyl.
74. The method of claim 3, wherein one of R4 and R5 is H and the other is selected from the group consisting Of -C(O)-NH2, -C(O)-OCH3, -CH2OH, ethynyl, formylamino, -CH2OCH3, and -
ONO2, and both of R2 and R3 are H.
75. The method of claim 74, wherein A is CH-S(O)R16 and R16 is aminopropyl.
76. The method of claim 3, wherein R4 and R5 together with the carbon to which they are attached form a cyclopropyl ring, both of R2 and R3 are H, A is C=N-OR14, and R14 is pyrrolidinyl or aminoethyl.
77. The method of claim 2, wherein R1 is -OH.
78. The method of claim 77, wherein R4 and R5 are both H.
79. The method of claim 78, wherein R2 and R3 together with the carbon to which they are attached form C=CH2.
80. The method of claim 79, wherein A is C=N-OR14, and R14 is selected from the group consisting of 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2- methylpropyl, aminoethyl, 2-amino-dimethylethyl, and pyrrolidinyl.
81. The method of claim 79, wherein A is CH(OR13), and R13 is selected from the group consisting of aminopropionyl, 3-aminobutanionyl, and amino-2-methylpropionyl.
82. The method of claim 79, wherein A is CH-CH=CH-R15, and R15 is aminopropyl or aminoethyl.
83. The method of claim 79, wherein A is CH-SR16 or CH-S(O)R16, and R16 is aminopropyl.
84. The method of claim 78, wherein R and R together with the carbon to which they are attached form C=CF2 or C=N-OR11 and R11 is H or CH3.
85. The method of claim 84, wherein A is CH-SR16 or CH-S(O)R16, and R16 is aminopropyl.
86. The method of claim 84, wherein A is C=N-OR14, and R14 is aminoethyl or pyrrolidinyl.
87. The method of claim 78, wherein both of R and R are H.
88. The method of claim 87, wherein A is C=N-OR14, and R14 is selected from the group consisting of 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2- methylpropyl, 2-amino-dimethylethyl, and aminoethyl.
89. The method of claim 87, wherein A is CH(OR13), and R13 is selected from the group consisting of aminopropionyl, 3-aminobutanionyl, and 3-amino-2-methylpropionyl.
90. The method of claim 87, wherein A is CH-SR16 or CH-S(O)R16, and R16 is aminopropyl.
91. The method of claim 87, wherein A is CH-CH=CH-R15, and R15 is aminopropyl or aminoethyl.
92. The method of claim 78, wherein one of R and R is H and the other is methyl.
93. The method of claim 92, wherein A is C=N-OR14, and R14 is selected from the group consisting of 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2- methylpropyl, and aminoethyl.
94. The method of claim 92, wherein A is CH(OR13), and R13 is selected from the group consisting of aminopropionyl, 3-aminobutanionyl, and amino-2-methylpropionyl.
95. The method of claim 92, wherein A is CH-CH=CH-R15, -CH-SR16 or -CH-S(O)R16, provided that R15 is aminopropyl or aminoethyl, and R16 is aminopropyl.
96. The method of claim 78, wherein one of R2 and R3 is H, and the other is selected from the group consisting Of -C(O)-NH2, -C(O)-OCH3, -CH2OH, ethynyl, formylamino, -CH2OCH3, and -ONO2.
97. The method of claim 96, wherein A is -CH-S(O)R16, and R16 is aminopropyl.
98. The method of claim 77, wherein R2 and R3 are both H, R4 and R5 together with the carbon to which they are attached form C=O, C=CH2, difluoromethylene or C=N-OR11, wherein R11 is H or methyl .
99. The method of claim 98, wherein A is -CH-S(O)R16, and R16 is aminopropyl.
100. The method of claim 98, wherein when R4 and R5 together with the carbon to which they are attached form C=CH2, A is C=N-OR14, and R14 is selected from the group consisting of 2- aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, amino-2-methylpropyl, 2- amino-dimethylethyl, and aminoethyl.
101. The method of claim 98, wherein when R and R together with the carbon to which they are attached form C=CH2, A is CH(OR13), and R13 is selected from the group consisting of aminopropionyl, 3-aminobutanionyl, and amino-2-methylpropionyl.
102. The method of claim 98, wherein when R4 and R5 together with the carbon to which they are attached form C=CH2, A is CH-CH=CH-R15 or -CH-SR16, R15 is aminopropyl or aminoethyl, and R16 is aminopropyl.
103. The method of claim 77, wherein one of R4 and R5 is H and the other is methyl, and R2 and R3 are both H.
104. The method of claim 103, wherein A is C=N-OR14, and R14 is selected from the group consisting of 2-aminopropyl, aminopropyl, methylaminoethyl, methylaminopropyl, 3-amino-2- methylpropyl, 2-amino-dimethylethyl, and aminoethyl.
105. The method of claim 103, wherein A is CH(OR13), CH-CH=CH-R15, CH-SR16 or CH- S(O)R16, wherein R13 is selected from the group consisting of aminopropionyl, 3- aminobutanionyl, and amino-2-methylpropionyl; R15 is aminopropyl or aminoethyl; and R16 is aminopropyl.
106. The method of claim 77, wherein R2 and R3 are both H, one of R4 and R5 is H, and the other is selected from the group consisting Of-C(O)NH2, -ONO2, formylamino, ethynyl, - CH2OH, -CH2-O-CH3, and methoxycarbonyl.
107. The method of claim 106, wherein A is CH-S(O)R16 and R16 is aminopropyl.
108. The method of claim 1 , wherein one of R6 and R7 is H and the other is -OH, and R1 is H.
109. The method of claim 108, wherein one of R2 and R3 is H, and the other is -OH, and both of R4 and R5 are H.
110. The method of claim 109, wherein A is C=N-OR14, and R14 is dimethylaminoethyl, aminopropyl, aminoethyl, 2-aminopropyl, methylaminoethyl, methylaminopropyl, or 3-amino-2- methylpropyl.
111. The method of claim 109, wherein A is CH(OR13), and R13 is selected from the group consisting of methylaminopropyl, dimethylaminopropyl, dimethylaminobutyl, aminopropyl, dimethylaminoethyl, aminoethyl, pyrrolidinylethyl, 2-(2-(dimethylamino)ethoxy)ethyl, 2-(2- (pyrrolidin-l-yl)ethoxy) ethyl, 2-guanidinylethyl, and 3-guanidinylpropyl.
112. The method of claim 109, wherein A is CH-CH=N-OR15 or C=CH-CH=N-OR15, and R15 is aminoethyl or dimethylaminoethyl.
113. The method of claim 108, wherein R and R together with the carbon to which they are attached to form C=O.
114. The method of claim 113, wherein A is C=N-OR14 or CH(OR13), wherein R14 is aminoalkyl, R13 is aminoalkyl.
115. The method of claim 1, wherein said compound is of Formula (Ia)
Figure imgf000122_0001
wherein
A is C=N-OR14 or CH(OR13);
R1 is H or -OH;
One of R4 and R5 is H, and the other is H or -OR8;
R8 is H or optionally substituted
Figure imgf000122_0002
R13 is (Ci-C4)alkyl optionally substituted by a 4 or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, amino(Ci_4)alkoxyl, (Ci_4)alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; amino(Ci-C4)alkyl optionally substituted by one or more (Ci-C4)alkyl; or ammo(Ci-C4)acyl optionally substituted by (Ci- C4)alkyl; and
R14 is an amino(Ci-C4)alkyl optionally substituted by one or more (Ci-C4)alkyl; or a 4- to 6-membered heterocyclic ring optionally substituted by
Figure imgf000122_0003
and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
116. The method of claim 115, wherein R1 is H.
117. The method of claim 115, wherein R4 and R5 are H.
118. The method of claim 115, wherein A is C=N-OR 14
119. The method of claim 118, wherein R14 is an amino(Ci-C4)alkyl that is optionally substituted by (CrC4)alkyl.
120. The method of claim 118, wherein R14 is a 4- to 6-membered heterocyclic ring that is optionally substituted by (Ci-C4)alkyl.
121. The method of claim 115, wherein A is CH(OR13).
122. The method of claim 121, wherein R13 is
Figure imgf000123_0001
optionally substituted by a 4 or 6- membered heterocyclic ring.
123. The method of claim 122, wherein R13 is 2-(pyrrolidin- 1 -yl)ethyl.
124. The method of claim 115, wherein said compound of Formula (Ia) is (10R,13S)-3-(2- aminoethoxyimino)- 10, 13 -dimethyldodecahydro- 1 Hcyclopenta[a]phenanthrene-6, 17(1 OH, 14H)- dione (1):
Figure imgf000123_0002
125. The method of claim 115, wherein said compound of Formula (Ia) is (10R,13S)-10,13- dimethyl-3-((R)-pyrrolidin-3-yloxyimino)dodecahydro-lH-cyclopenta[a]phenanthrene-
6,17(10H,14H)-dione (2):
Figure imgf000124_0001
126. The method of claim 115, wherein said compound of Formula (Ia) is (3S,5S,10R,13S)- 10, 13-dimethyl-3-(2-(pyrrolidin- 1 -yl)ethoxy)dodecahydro- 1 H-cyclopenta[a]phenanthrene- 6,17(10H,14H)-dione (15):
Figure imgf000124_0002
127. The method of claim 1, wherein said compound is of Formula (Ib)
Figure imgf000124_0003
wherein
16
A is C=N-OR14, CH-CH=CH-R13, CH-SR16 or CH-S(O)R , 1166;. R1 is H or -OH; X is C(R10)2 or N-ORu;
One of R4 and R5 is H, and the other is H or -OR8;
R8 is H or substituted or unsubstituted
R10, for each occurrence, is the same or different and is H or halogen; R11 is H or (d_C4)alkyl; and
R14 is an amino(Ci_C4)alkyl optionally substituted by one or more
Figure imgf000125_0001
or a 4- to 6-membered heterocyclic ring optionally substituted by
Figure imgf000125_0002
R15 and R16, each independently, are ammo(Ci_C4)alkyl optionally substituted by one or more (Ci-C/Oalkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
128. The method of claim 127, wherein R1 is H.
129. The method of claim 127, wherein R1 is OH.
130. The method of claim 127, wherein X is C(R10)2.
131. The method of claim 130, wherein R10 is H.
132. The method of claim 127, wherein X is N-OR11.
133. The method of claim 132, wherein R11 is H.
134. The method of claim 132, wherein R11 is (d_C4)alkyl.
135. The method of claim 127, wherein A is C=N-OR14.
136. The method of claim 135, wherein R14 is amino(Ci_4)alkyl that is optionally substituted by one or more (Ci-C4)alkyl.
137. The method of claim 135, wherein R14 is a 4- to 6-membered heterocyclic ring.
138. The method of claim 127, wherein A is CH-CH=CH-R15.
139. The method of claim 138, wherein R15 is amino(Ci_4)alkyl.
140. The method of claim 127, wherein A is CH-SR16 or CH-S(O)R16.
141. The method of claim 140, wherein R16 is amino(Ci_4)alkyl.
142. The method of claim 127, wherein said compound of Formula (Ib) is (10R,13S)-3-(2- aminoethoxyimino)- 10, 13 -dimethyl-6-methylenetetradecahydro- 1 H-cyclopenta[a]phenanthren- 17(2H)-one (3):
Figure imgf000126_0001
143. The method of claim 127, wherein said compound of Formula (Ib) is (10R,13S)-10,13- dimethyl-6-methylene-3 -(pyrrolidin-3 -yloxyimino)tetradecahydro- 1 H- cyclopenta[a]phenanthren-17(2H)-one (4):
Figure imgf000126_0002
144. The method of claim 127, wherein said compound of Formula (Ib) is (10R,13S)-3-(2- aminoethoxyimino)-6-(hydroxyimino)- 10, 13-dimethyltetradecahydro- 1 H- cyclopenta[a]phenanthren-17(2H)-one (5):
Figure imgf000127_0001
145. The method of claim 127, wherein said compound of Formula (Ib) is (10R,13S)-3-(2- aminoethoxyimino)-6-(methoxyimino)- 10,13 -dimethyltetradecahydro- 1 H- cyclopenta[a]phenanthren-17(2H)-one (6):
Figure imgf000127_0002
146. The method of claim 127, wherein said compound of Formula (Ib) is (5S,10R,13S)-5- hydroxy- 10,13 -dimethyl-6-methylene-3 -(pyrrolidin-3 -yloxyimino)tetradecahydro- 1 H- cyclopenta[a]phenanthren-17(2H)-one (7):
Figure imgf000128_0001
147. The method of claim 127, wherein said compound of Formula (Ib) is (5S,10R,13S)-3-(2- aminoethoxyimino)-5-hydroxy- 10, 1 S-dimethyl-ό-methylenetetradecahydro- 1 H- cyclopenta[a]phenanthren-17(2H)-one (8):
Figure imgf000128_0002
148. The method of claim 127, wherein the compound of Formula (Ib) is (5S,10R,13S)-3-(5- aminopent- 1 -enyl)-6-(hydroxyimino)- 10, 13-dimethyltetradecahydro- 1 H-cyclopenta[a]- phenanthren-17(2H)-one (18):
Figure imgf000128_0003
149. The method of claim 127, wherein the compound of Formula (Ib) is (3R,5R,10R,13S)- 3 -(3 - Aminopropylthio)- 10,13 -dimethyl-6-methylenetetradecahydro- 1 H-cyclopenta- [a]phenanthren-17(2H)-one (19):
Figure imgf000129_0001
150. The method of claim 127, wherein the compound of Formula (Ib) is (5R,10R,13S)-3-(3- aminopropylsulfmyl)- 10, 1 S-dimethyl-ό-methylenetetradecahydro- 1 H-cyclopenta[a]phenanthren- 17(2H)-one (20):
Figure imgf000129_0002
151. The method of claim 127, wherein the compound of Formula (Ib) is (10R,13S)-3— ((R)- pyrrolidin-3-yloxyimino)-6-(methoxyimino)- 10,13 dimethyltetradecahydro- 1 H- cyclopenta[a]phenanthren- 17(2H)-one (21)
Figure imgf000129_0003
152. The method of claim 1, wherein said compound is of Formula (Ic)
Figure imgf000130_0001
wherein
R1 is H or -OH; One of R and R is H, and the other is
Figure imgf000130_0002
optionally substituted by hydroxyl or methoxy;
One of R4 and R5 is H and the other is -OR8; or
R4 and R5 together with the carbon to which they are attached form C=O;
R8 is H or substituted or unsubstituted
Figure imgf000130_0003
and R14 is an amino(Ci_C4)alkyl optionally substituted by one or more
Figure imgf000130_0004
or a 4- to
6-membered heterocyclic ring optionally substituted by (Ci_C4)alkyl; and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
153. The method of claim 152, wherein R1 is H.
154. The method of claim 152, wherein one of R2 and R3 is H and the other is -CH2OH.
155. The method of claim 152, wherein R4 and R5 together with the carbon to which they are attached form C=O.
156. The method of claim 152, wherein one of R4 and R5 is H and the other is -OR8;.
157. The method of claim 152, wherein R14 is an amino(Ci_C4)alkyl optionally substituted by
Figure imgf000130_0005
158. The method of claim 152, wherein R14 is a 4- to 6-membered heterocyclic ring optionally substituted by
Figure imgf000130_0006
159. The method of claim 152, wherein said compound of Formula (Ic) is (6S,10R,13S)-3-(2- aminoethoxyimino)-6-(hydroxymethyl)- 10, 13-dimethyldodecahydro- 1 H- cyclopenta[a]phenanthrene-7, 17(2H,8H)-dione (9):
Figure imgf000131_0001
160. The method of claim 152, wherein said compound of Formula (Ic) is (6S,10R,13S)-6- (hydroxymethyl)- 10,13 -dimethyl- 3 -(pyrrolidin-3 -yloxyimino)dodecahydro- 1 H- cyclopenta[a]phenanthrene-7,17(2H,8H)-dione (10):
Figure imgf000131_0002
161. The method of claim 152, wherein said compound of Formula (Ic) is (6S,7S,10R,13S)- 7-hydroxy-6-(hydroxymethyl)- 10,13 -dimethyl-3 -(pyrrolidin-3 -yloxyimino)tetradecahydro- 1 H- cyclopenta[a]phenanthren-17(2H)-one (11):
Figure imgf000132_0001
162. The method of claim 152, wherein said compound of Formula (Ic) is (6S,7S,10R,13S)- 3-(2-aminoethoxyimino)-7-hydroxy-6-(hydroxymethyl)-10,13-dimethyltetradecahydro-lH- cyclopenta[a]phenanthren-17(2H)-one (12):
Figure imgf000132_0002
163. The method of claim 1, wherein said compound is of Formula (Id)
Figure imgf000132_0003
wherein
R1 is H or -OH;
R4 and R5 taken together with the carbon atom to which they are attached form C(R10)2 ; R10, for each occurrence, is the same or different and is H or halogen; and R14 is an amino(Ci_C4)alkyl optionally substituted by one or more
Figure imgf000133_0001
or a 4- to 6-membered heterocyclic ring optionally substituted by and tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
164. The method of claim 163, wherein R1 is H.
165. The method of claim 163, wherein R10 is H.
166. The method of claim 163, wherein R10 is halogen.
167. The method of claim 163, wherein R14 is an amino(Ci_C4)alkyl optionally substituted by
Figure imgf000133_0002
168. The method of claim 163, wherein R14 is a 4- to 6-membered heterocyclic ring that is optionally substituted by (CpC/Oalkyl.
169. The method of claim 163, wherein said compound of Formula (Id) is (10S,13S)-3-(2- aminoethoxyimino)- 10, 1 S-dimethyl^-methylenetetradecahydro- 1 H-cyclopenta[a]phenanthren- 17(2H)-one (13):
Figure imgf000133_0003
170. The method of claim 163, wherein said compound of Formula (Id) is (10S,13S)-3-(2- aminoethoxyimino)-7-(difluoromethylene)- 10,13 -dimethyltetradecahydro- 1 H- cyclopenta[a]phenanthren-17(2H)-one (14):
Figure imgf000134_0001
171. The method of claim 1, wherein said compound is of Formula (Ie)
Figure imgf000134_0002
wherein
R1 is H or -OH;
A is CH(OR13), C=N-OR14, CH-CH=N-OR15, C=CH-CH=N-OR15, CH-CH=CH-R15,
CH-SR16, or CH-S(O)R16; R13 is (Ci.C/Oalkyl optionally substituted by a 4 or 6-membered heterocyclic ring, a 5 or
6-membered heteroaryl, amino(Ci_4)alkoxyl, (Ci_4)alkoxyl substituted by a 4- or 6-membered heterocyclic ring, a 5 or 6-membered heteroaryl, or guanidinyl; amino(Ci_C4)alkyl optionally substituted by one or more (Ci_C4)alkyl; or amino(Ci_C4)acyl optionally substituted by (Ci-
C4)alkyl; R14 is an amino(Ci_C4)alkyl optionally substituted by one or more
Figure imgf000134_0003
or a 4- to
6-membered heterocyclic ring optionally substituted by
Figure imgf000134_0004
R15 and R16, each independently, are ammo(Ci_C4)alkyl optionally substituted by one or
Figure imgf000134_0005
tautomers, stereoisomers, Z and E isomers, optical isomers, N-oxides, hydrates, polymorphs, pharmaceutically acceptable esters, salts, prodrugs and/or isotopic derivatives thereof.
172. The method of claim 171, wherein R1 is H.
173. The method of claim 171, wherein A is CH-CH=N-OR15 or C=CH-CH=N-OR15.
174. The method of claim 173, wherein R15 is amino(Ci_C4)alkyl.
175. The method of claim 173, wherein R15 is amino(Ci_C4)alkyl substituted by one or more (Ci_C4)alkyl.
176. The method of claim 171, wherein said compound of Formula (Ie) is 6, 17-dihydroxy- 10,13 -dimethylhexadecahydro- 1 H-cyclopenta[a]phenanthrene-3 -carbaldehyde O-2-aminoethyl oxime (16):
Figure imgf000135_0001
177. The method of claim 171, wherein said compound of Formula (Ie) is 2- ((5S,6S, 1 OR, 13S, 17S)-6, 17-dihydroxy- 10, 13-dimethylhexahydro- 1 H-cyclopenta[a]phenanthren- 3(2H,4H, 1 OH, 12H, 13H, 14H, 15H, 16H, 17H)-ylidene)acetaldehyde O-2-(dimethylamino)ethyl oxime (17):
Figure imgf000135_0002
178. A method for reducing the growth, proliferation, or survival of a neoplastic cell, the method comprising contacting the cell with an effective amount of a compound of any of claims 1-177, wherein the compound reduces the growth, proliferation, or survival of a neoplastic cell.
179. The method of claim 178, wherein the compound is selected as binding to a membrane androgen receptor or as competing with an endogenous ligand for binding to said receptor.
180. The method of claim 178, wherein said neoplasia is a solid tumor or hematological cancer.
181. The method of claim 178, wherein the neoplastic cell is derived from a tissue selected from the group consisting of lung, breast, CNS, colon, prostate, ovary, pancreas, kidney and melanoma.
182. The method of claim 178, wherein the cell expresses MDR-I or P-glycoprotein.
183. A method of inducing cell death in a neoplastic cell, the method comprising contacting the cell with a therapeutically effective amount of a compound recited in any one of claims 1- 177, thereby inducing cell death.
184. The method of claim 183, wherein the cell is in a subject.
185. The method of claim 183, wherein the cell death is apoptotic cell death.
186. A method of preventing or treating a neoplasia in a subject, the method comprising administering to the subject a therapeutically effective amount of a compound recited in any one of claims 1-177.
187. The method of any one of claims 1-177, 178 or 184, wherein the subject is a mammal.
188. The method of any one of claims 1-177, 178 or 184, wherein the subject is a human patient.
189. The method of any one of claims 1-177, 178 or 184, wherein the method reduces the growth or proliferation of a neoplasia in a subject.
190. The method of any one of claims 1-177, 178 or 184, wherein the neoplasia is a lung, breast, CNS, colon, prostate, ovary, pancreas, kidney or skin cancer.
RECTIFIED SHEET (RULE 91) ISA/EP
191. The method of any one of claims 1-190, wherein the neoplasia is resistant to one or more therapeutic agents.
192. The method of claim 191, wherein the neoplasia is multidrug resistant.
193. The method of claim 192, wherein the neoplasia has alterations in the expression or activity of an ABC transporter, tubulin, or topoisomerase polypeptide or polynucleotide.
194. The method of claim 192, wherein the neoplasia has an increase in the expression or activity of MDRl or P-glycoprotein.
195. A method for the treatment of a subject having a multidrug resistant or refractory neoplasia, the method comprising administering to the subject a therapeutically effective amount of a compound recited in any one of claims 1-177 and a pharmaceutically acceptable excipient.
196. The method of claim 195, wherein the subject is a human patient.
197. The method of claim 195, wherein the method reduces the growth or proliferation of the neoplasia.
198. The method of claim 195, wherein the method induces the death of a neoplastic cell.
199. The method of claim 195, wherein the neoplasia is resistant to one or more therapeutic agents.
200. The method of claim 195, wherein the neoplasia has alterations in the expression or activity of an ABC transporter, tubulin, or topoisomerase polypeptide or polynucleotide.
201. The method of claim 195, wherein the neoplasia has an increase in the expression or activity of MDRl or P-glycoprotein.
202. The method of claim 195, wherein the method further comprises administering a compound selected from the group consisting of vinca alkaloids, taxanes, epothilones, antifolates, purine analogs, pyrimidine analogs, DNA intercalators, topoisomerase inhibitors, topotecan, alkylating agents, platinum-based agents, receptor antagonists, hormone agents, anthracyclines, epipodophyllotoxins, antibiotics, antimicrotubule drugs, protein synthesis inhibitors, toxic peptides, enzyme inhibitors and anti-mitotics.
203. The method of claim 195, wherein the method treats a patient having end-stage disease.
204. A composition for the treatment of a neoplasia, the composition comprising a therapeutically effective amount of a compound recited in any one of claims 1-177 and a pharmaceutically acceptable excipient.
205. The composition of claim 204, further comprising a therapeutically effective amount of a taxane.
206. A packaged pharmaceutical for the treatment of neoplasia comprising a therapeutically effective amount of a compound recited in any of claims 1-177, and written instructions for administration of the compound.
207. A method of preventing or treating a neoplasia in a subject, the method comprising administering to the subject a therapeutically effective amount of a Na+K+ ATPase inhibitor that inhibits ligand binding to a membrane androgen receptor, thereby preventing or treating said neoplasia.
208. The method of claim 207, wherein the Na+K+ ATPase inhibitor binds a Na+K+ ATPase and inhibits Na+K+ ATPase activity.
209. The method of claim 207, wherein the Na+K+ ATPase inhibitor binds to a membrane androgen receptor and competitively inhibits ligand binding to said receptor.
210. The method of claim 207, wherein the Na+K+ ATPase inhibitor induces cell death in a neoplastic cell of said neoplasia.
211. The method of claim 207, wherein the neoplasia is a membrane androgen positive solid tumor or hematological malignancy.
212. The method of claim 207, wherein the neoplasia is a prostate cancer, breast cancer, or colon cancer.
213. A method for treating or preventing prostate cancer in a subject, the method comprising administering to said subject an effective amount of a compound capable of binding and inhibiting a Na+K+ ATPase, and further capable of competitively inhibiting ligand binding to the membrane androgen receptor on a prostate cancer cell.
214. The method of claim 213, wherein the method induces cell death in a cell of said prostate cancer.
215. The method of claim 214, wherein the cell death is by apoptosis.
216. The method of claim 213, wherein the compound binds the membrane androgen receptor.
217. The method of any of claims 207-213, wherein the compound is istaroxime.
218. A composition for the treatment or prevention of a neoplasia, the composition comprising a therapeutically effective amount of a compound capable of binding and inhibiting a Na+K+ ATPase, and further capable of competitively inhibiting ligand binding to the membrane androgen receptor on a neoplastic cell and a pharmaceutically acceptable excipient, wherein the therapeutically effective amount is sufficient to induce cell death in a neoplastic cell.
219. A packaged pharmaceutical for the treatment of neoplasia comprising a therapeutically effective amount of a compound capable of binding and inhibiting a Na+K+ ATPase, and further capable of competitively inhibiting ligand binding to the membrane androgen receptor on a prostate cancer cell, and written instructions for administration of the compound for use in treating said neoplasia.
220. A method of preventing or treating a neoplasia in a subject, the method comprising administering to the subject a therapeutically effective amount of a Na+K+ ATPase inhibitor that binds to a membrane androgen receptor, thereby preventing or treating said neoplasia.
221. The method of claim 220, wherein the Na+K+ ATPase inhibitor induces cell death in a neoplastic cell of said neoplasia.
222. The method of claim 220, wherein the neoplasia is prostate cancer.
223. A method for treating or preventing prostate cancer in a subject, the method comprising administering to said subject an effective amount of a Na+K+ ATPase inhibitor that binds to a membrane androgen receptor on a prostate cancer cell.
224. The method of claim 223, wherein the method induces cell death in a cell of said prostate cancer.
225. The method of any of claims 223, wherein the compound is istaroxime.
226. A composition for the treatment or prevention of a neoplasia, the composition comprising a therapeutically effective amount of a Na+K+ ATPase inhibitor that binds to a membrane androgen receptor on a neoplastic cell and a pharmaceutically acceptable excipient, wherein the therapeutically effective amount is sufficient to induce cell death in a neoplastic cell.
227. A packaged pharmaceutical for the treatment of neoplasia comprising a therapeutically effective amount of a Na+K+ ATPase inhibitor that binds to a membrane androgen receptor on a prostate cancer cell, and written instructions for administration of the compound for use in treating said neoplasia.
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CN109503691A (en) * 2018-12-05 2019-03-22 华中药业股份有限公司 A kind of synthetic method of 5 α-androstane -3,17- diketone
CN110878013A (en) * 2019-12-13 2020-03-13 西安瑞联新材料股份有限公司 Synthesis method of (trans ) -4-vinyl-4' - [ (E) -1-propenyl ] -bicyclohexane
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CN103360457A (en) * 2013-07-31 2013-10-23 中国生命药物治疗有限公司 Androstanedione derivative crystal form and preparation method therefor
CN103360457B (en) * 2013-07-31 2015-10-07 中国生命药物治疗有限公司 A kind of androstanedione derivative crystal formation and preparation method thereof
CN109438538A (en) * 2018-12-05 2019-03-08 华中药业股份有限公司 A kind of synthetic method of -17 beta-hydroxy -3- ketone of -17 Alpha-Methyl of Stanozolol intermediate androstane
CN109503691A (en) * 2018-12-05 2019-03-22 华中药业股份有限公司 A kind of synthetic method of 5 α-androstane -3,17- diketone
CN110878013A (en) * 2019-12-13 2020-03-13 西安瑞联新材料股份有限公司 Synthesis method of (trans ) -4-vinyl-4' - [ (E) -1-propenyl ] -bicyclohexane
CN110878013B (en) * 2019-12-13 2023-04-28 西安瑞联新材料股份有限公司 Synthesis method of (trans ) -4-vinyl-4' - [ (E) -1-propenyl ] -bicyclohexane
WO2022159678A1 (en) * 2021-01-22 2022-07-28 Windtree Therapeutics, Inc. Intravenous istaroxime for the treatment of acute heart failure

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