HYDROXYSTEROID COMPOUNDS. THEIR INTERMEDIATES.
PROCESS OF PREPARATION. COMPOSITION AND USES THEREOF
FIELD OF INVENTION
The present invention relates hydroxysteroid compounds and their intermediates,
composition comprising the same and method of preparation thereof and uses of
the compounds of the present invention.
BACKGROUND OF THE INVENTION
Mitochondria are the powerhouses of the cell that are responsible for generating
more than 90% of the energy needed by the body to sustain life and support
growth. When mitochondrial function fails, less energy is generated within the cell,
resulting in cell injury and ultimately cell death. Mitochondria are susceptible to
degradation due to oxygen radicals produced by their own metabolic processes.
Damaged mitochondria are later expelled by the cell. Their replacement by new
mitochondria is called mitochondrial biogenesis. The proliferation of mitochondria
or their hypertrophy to meet increased metabolic demand is also called
mitochondrial biogenesis. It is signified by the expression of additional
mitochondrial proteins, particularly those related to oxidative phosphorylation. The
capacity for mitochondrial biogenesis is significantly lost with age. Thus many
diseases of aging are associated with loss of mitochondria in various tissues, whose
specialized function is diminished in the context of diminished mitochondrial
function and/or number. Many disease states, such as those that have
neuromuscular disease symptoms, sarcopenia, muscular dystrophy, diabetes
mellitus, dementia, Parkinson's disease, Huntington's disease, amyotrophic lateral
sclerosis (ALS), obesity, hyperlipidemia, heart failure, lupus, and ocular conditions
such as age-related macular degeneration (AMD), are associated with progressive
mitochondrial loss in various tissues. A number of drugs and drug classes also have
an effect on mitochondrial function and biogenesis and can affect organ function
and even lead to organ degeneration or other side effects which are directly related
to the toxic effect of these drugs on the mitochondria.
Ischemic and ischemia/reperfusion injury are accompanied by decrease in
mitochondrial function and number, leading to apoptotic cell death, necrosis, and
functional organ deterioration in ischemic conditions such as myocardial infarction
and stroke. Despite considerable advances in the diagnosis and treatment of such
conditions, there remains a need for prophylactic and therapeutic approaches for
the treatment of these conditions.
Compounds that have functions on mitochondrial activity are currently limited and
there remains a need for novel compounds prophylactic and therapeutic approaches
for the treatment of these conditions associated with chronic mitochondrial
dysfunction and toxicity. Thus there is a need for compounds and treatments that
stimulate mitochondrial function in response to increased metabolic demand and
induce mitochondrial replication in response to agents or conditions that cause
depletion of mitochondria in one or more tissues. Reflecting this understanding,
the phrase "mitochondrial toxicity" as used herein refers to failure of the
mitochondria resulting from the administration of chemical compositions to a
subject.
Mitochondria are critical to cell function and the effects of mitochondrial disease
can be varied and can take on unique characteristics. The severity of the specific
defect may be great or small and often affect the operation of the mitochondria and
multiple tissues more severely, leading to multi-system diseases. Injury to, or
dysfunction of, skeletal muscle mitochondria generally results in muscle weakness
and atrophy, termed sarcopenia in severe states. In the case of generalized muscle
weakness, reduction in bone density can be generalized, one of the causes of the
bone disease known as osteoporosis. Depleted mitochondria in the heart can
eventuate in the symptoms of congestive heart failure and eventual death. Loss of
mitochondrial density in the brain is associated with neurodegeneration states such
as Huntington's disease, Alzheimer's disease, and Parkinson's disease. Generalized
loss of mitochondria including liver mitochondria can result in hyperlipidemia,
hypertension, and insulin resistance progression to Type 2 diabetes. Liver
mitochondria are injured by fructose uptake. Fructose, uric acid, and other agents
injurious to liver mitochondria can cause accumulation of intracellular lipids,
particularly triglycerides that contribute to the syndrome of hepatic steatosis, and
increased synthesis and export of triglycerides that contributes to systemic
hyperlipidemia, and ultimately obesity and insulin resistance.
Hydroxysteroids are hydroxylated compounds with a sterol structure and are
known to be produced in cells when the mitochondria are exposed to high levels of
endogenous ¾ ¾ which then acts via the mitochondrial enzyme, 11b-
hydroxylase, to hydroxylate a variety of steroids, including cholesterol,
pregnenolone, progesterone, and others. Hydroxylation can occur in numerous
positions, including the 7, 16, and 11 positions. These molecules, termed
hydroxysteroids, are then sulfated and secreted into the extracellular space, where
in the brain they modulate GABA-receptors and calcium channels on the plasma
membrane. No intracellular activity of hydroxylsteroids has previously been
described.
The present invention discloses novel hydroxyl steroids, their intermediates,
process for synthesis of hydroxysteroids and their intermediates and composition
comprising the same with their action on mitochondria.
OBJECT OF THE INVENTION
An object of the present invention is to provide novel steroidal compounds, a
process for synthesis of hydroxysteroids and their intermediates and compositions
comprising the same and effect of the said compounds on mitochondria.
Summary of the Invention
The present invention provides hydroxysteroids and their intermediates of formula
(I):
FORMULA I
or a salt thereof, wherein:
is independently either a single bond, a double bond or a cyclopropyl ring,
provided that adjacent double bonds are not allowed;
A , A2, A 3 are independently selected from the group comprising hydi
hydroxyl, hydroxymethyl, halogen, C1-C6 alkyl;
B and C are each independently selected from the group comprising hydrogen,
hydroxyl, halogen, -OR „ -COR ,-COOR ,OCOR , CH2OH, CH2OR , -CONHR ,
CONHR R7, , -C(OH)R R7, NHR , NHR5CONHR , - R NHCOOR 7, -NR R7,
C(0)heteroaryl, C(0)heterocyclyl, C1- 2 straight or branched chain alkyl, 5-6
membered heterocycloalkyl, 5-6 membered heteroaryl;
wherein the C1-C12 straight or branched chain alkyl or 5-6 membered
heterocycloalkyl or 5-6 membered heteroaryl; may be further optionally substituted
with one or more substituents selected from the group comprising halogen, C1-C6
alkyl, -OR , -COOR ,-CONHR , -OCOR , =NOH, NR R7, -NR COR7, 5-6
membered heterocycloalkyl or 5-6 membered heteroaryl; C1-C12 alkyl substituted
5-6 membered heterocycloalkyl or C1-C12 alkyl substituted 5-6 membered
heteroaryl;
or B and C may combine together to form =0, =NOR6, NHR6, 5-6 membered
heterocycloalkyl, or 5-6 membered heteroaryl;
wherein the said 5-6 membered heterocycloalkyl, 5-6 membered heteroaryl may
optionally contain one or more heteroatom;
wherein, the heteroatom may be O, N, S;
Ri, R2, and R4 are independently selected from the group comprising hydrogen,
deuterium, hydroxyl, halogen, =0, -OR , -NR R7, -COR , -COOR ,-OCOR ,-
CONR R7, -C alkyl, -Otert-butyldimethylsilyl;
R 3 is hydroxyl, carbonyl, OCOR ;
wherein R3 is in beta configuration;
R is selected from hydrogen, hydroxyl, halogen, OR ;
R and R7are each independently selected from the group comprising hydrogen,
halogen, hydroxyl, C1- 2 alkyl, -N¾, -(CH2)nNH2, 3-6 membered cycloalkyl, 4-6
membered heterocycloalkyl, 5-6 membered heteroaryl;
X is selected from CH, NH, NR , O or S;
n is 0 to 3.
The present invention discloses processes for preparation of the compounds of
formula I, compositions comprising the compounds of formula and use of the
compounds of the present invention in mitochondrial biogenesis and AMPkinase
activation.
The present invention also discloses the use of 11b-hydroxypregnenolone and 11b-
hydroxyprogestrone in mitochondrial biogenesis and AMP kinase activation.
BRIEF DESCRIPTION OF FIGURES
Figure l a depictsmitochondrial biogenesis and Figure l b depicts stimulation of
expression of mitochondrial transcription factors at 3 hours post exposure (I) by
1I b-hydroxypregnenolone;
Figure 2a depicts activation of AMP KINASE at 1 hour post-exposure by 11b-
hydroxypregnenolone and Figure 2b depicts activation APPLl and translocation to
the nucleus in 3 hr by I ΐ b-hydroxypregnenolone;
Figure 3a depicts stimulation of expression of mitochondrial transcription factors
at 3 hours post exposure (II) by I ΐ b-hydroxypregnenolone and Figure 3b depicts
increase of mitochondrial DNA content at 24 hr post exposure by 11b-
hydroxypregnenolone ;
Figure 4 depicts reduction of mitochondrial superoxide generation by 11b-
hydroxypregnenolone ;
Figure 5 depicts reduction of hepatic gluconeogenesis and stimulation of fat
oxidation by I ΐ b-hydroxypregnenolone.
DEFINITIONS
The term "alkyl" refers to a linear or branched saturated monovalent hydrocarbon,
wherein the alkylene may optionally be substituted as described herein. The term
"alkyl" also encompasses both linear and branched alkyl, unless otherwise
specified. In certain embodiments, the alkyl is a linear saturated monovalent
hydrocarbon that has the specified number of carbon atoms, or branched saturated
monovalent hydrocarbon of specified number of carbon atoms. As used herein,
linear CI- C6 and branched C3- C6 alkyl groups are also referred as "lower
alkyl. "Examples of alkyl groups include, but are not limited to, methyl, ethyl,
propyl (including allisomeric forms), w-propyl, isopropyl, butyl (including all
isomeric forms), «-butyl, isobutyl, sec-butyl, i-butyl, pentyl (including all isomeric
forms), and hexyl (including all isomeric forms).For example, CI - C6 alkyl refers
to a linear saturated monovalent hydrocarbon of 1 to 6 carbon atoms or a branched
saturated monovalent hydrocarbon of 3 to 6 carbon atoms.
The term "cycloalkyl" as used herein refers to a saturated or partially unsaturated,
monocyclic, fused bicyclic or bridged polycyclic ring assembly containing from
3 to 12 ring atoms, or the number of atoms indicated. Cycloalkyl can include any
number of carbons, such as C3_6, C4 _6, C _ , C3_s, C4_s, C _ , and C6 .8. Saturated
monocyclic cycloalkyl rings include, for example, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, and cyclooctyl. Saturated bicyclic and
polycyclic cycloalkyl rings include, for example, norbornane, [2.2.2]bicyclooctane,
decahydronaphthalene and adamantane. Cycloalkyl groups can also be partially
unsaturated, having one or more double bonds in the ring.
Representative cycloalkyl groups that are partially unsaturated include, but are not
limited to, cyclobutene, cyclopentene, cyclohexene, cyclohexadiene (1,3- and 1,4-
isomers), cycloheptene, cycloheptadiene, cyclooctene, cyclooctadiene (1,3-, 1,4-
and 1,5-isomers), norbornene, and norbornadiene. Unless otherwise stated,
cycloalkyl groups are unsubstituted. A "substituted cycloalkyl" group can be
substituted with one or more moieties selected from halo, hydroxy, amino,
alkylamino, nitro, cyano, and alkoxy.
The term "heteroaryl" refers to a monocyclic aromatic group and/or multicyclic
aromatic group that contain at least one aromatic ring, wherein at least one
aromatic ring contains one or more heteroatoms independently selected from 0, S,
and N. Each ring of a heteroaryl group may contain one or two O atoms, one or two
S atoms, and/or one to four N atoms, provided that the total number of heteroatoms
in each ring is four or less and each ring contains at least one carbon atom. In
certain embodiments, the heteroaryl has from 5 to 20, from 5 to 15, or from 5 to 10
ring atoms. Examples of monocyclic heteroaryl groups include, but are not limited
to, furanyl,imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxadiazolyl, oxazolyl,
pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, thiadiazolyl,
thiazolyl, thienyl, tetrazolyl, triazinyl,andtriazolyl. Examples of bicyclic heteroaryl
groups include, but are not limited to,benzofuranyl, benzimidazolyl,
benzoisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl,
benzothiophenyl, benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridinyl,
imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothienyl,
isoindolyl, isoquinolinyl, isothiazolyl, naphthyridinyl, oxazolopyridinyl,
phthalazinyl, pteridinyl, purinyl, pyridopyridyl, pyrrolopyridyl, quinolinyl,
quinoxalinyl, quinazolinyl, thiadiazolopyrimidyl, and thienopyridyl. Examples of
tricyclic heteroarylgroupsinclude, but are not limited to, acridinyl, benzindolyl,
carbazolyl, dibenzofuranyl, perimidinyl, phenanthrolinyl, phenanthridinyl,
phenarsazinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and xanthenyl. In certain
embodiments, heteroaryl may also be optionally substituted as described herein.
The term heteroaralkyl refers to an aralkyl group as defined above, in which one or
more (preferably 1, 2, 3 or 4) carbon atoms have been replaced by an oxygen,
nitrogen, silicon, selenium, phosphorus, boron or sulphur atom (preferably oxygen,
sulphur or nitrogen), that is to say groups that in accordance with the above
definitions contain both aryl or heteroaryl and alkyl, alkenyl, alkynyl and/or
heteroalkyl and/or cycloalkyl and/or heterocycloalkyl groups. A hetero-aralkyl
group preferably contains one or two aromatic ring systems ( 1 or 2 rings) with
from 5 or 6 to 10 carbon atoms and one or two alkyl, alkenyl and/or alkynyl groups
having from 1 or 2 to 6 carbon atoms and/or a cycloalkyl group having 5 or 6 ring
carbon atoms, with 1, 2, 3 or 4 of those carbon atoms having been replaced by
oxygen, sulphur or nitrogen atoms. Examples are aryl-heteroalkyl, arylheterocycloalkyl,
aryl-heterocycloalkenyl, arylalkyl-heterocycloalkyl, arylalkenylheterocycloalkyl,
arylalkynyl-heterocycloalkyl, arylalkyl-heterocycloalkenyl,
heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heteroaryl-heteroalkyl,
heteroarylcyclo- alkyl, heteroarylcycloalkenyl, heteroaryl-heterocycloalkyl,
heteroaryl-heterocycloalkenyl, heteroarylalkylcycloalkyl, _ g _ heteroarylalkylheterocycloalkenyl,
heteroaryl-heteroalkyl-cycloalkyl, heteroarylheteroalkylcycloalkenyl
and hetero-aryl-heteroalkyl-heterocycloalkyl groups, the
cyclic groups being saturated or mono-, di- or tri-unsaturated. Specific examples
are the tetrahydroisoquinolinyl, benzoyl, 2- or 3-ethylindolyl, 4-methylpyridino, 2-,
3- or 4-methoxyphenyl, 4-ethoxyphenyl and 2-, 3- or 4-carboxylphenylalkyl
groups.
The term "heterocycloalkyl" refers means a non-aromatic monocyclic or polycyclic
ring comprising carbon and hydrogen atoms and at least one heteroatom,
preferably, 1 to 4 heteroatoms selected from nitrogen, oxygen, and sulfur. A
heterocycloalkyl group can have one or more carbon-carbon double bonds or
carbon-heteroatoms double bonds in the ring as long as the ring is not rendered
aromatic by their presence. Examples of heterocycloalkyl groups include aziridinyl,
pyrrolidinyl, pyrrolidino, piperidinyl, piperidino, piperazinyl, piperazino,
morpholinyl, morpholino, thiomorpholinyl, thiomorpholino, tetrahydrofuranyl,
tetrahydrothiofuranyl, tetrahydropyranyl, and pyranyl. A heterocycloalkyl group
can be unsubstituted or substituted with one or two suitable substituents.
Preferably, the heterocycloalkyl group is a monocyclic or bicyclic ring, more
preferably, a monocyclic ring, wherein the ring comprises from 2 to 6 carbon atoms
and from 1 to 3 heteroatoms, referred to herein as (C.sub.l-
C.sub.6)heterocycloalkyl .
The term "non-aromatic", as used herein, refers to a cyclic moiety, that may be
unsaturated, but that does not have an aromatic character.
The term "substituted" refers to where hydrogen radical on a molecule has been
replaced by another atom radical, a functional group radical or a moiety radical;
these radicals being generally referred to as "substituents."
The use of terms "a" and "an" and "the" and similar references in the context of
describing the invention (especially in the context of the following claims) are to be
construed to cover both the singular and the plural, unless otherwise indicated
herein or clearly contraindicated by context.
The term "salt(s)", as employed herein, denotes acidic and/or basic salts formed
with inorganic and/or organic acids and bases. Zwitterions (internal or inner salts)
are included within the term "salt(s)" as used herein (and may be formed, for
example, where the R substituents comprise a basic moiety such as an amino
group). Also included herein are quaternary ammonium salts such as alkyl
ammonium salts. Pharmaceutically acceptable (i.e., non-toxic, physiologically
acceptable) salts are preferred.
The term "pharmaceutically acceptable salts" refers to the acid addition salt
compound formed with a suitable acid selected from an inorganic acid such as
hydrochloric acid, hydrobromic acid; or an organic acid such as benzene sulfonic
acid, maleic acid, oxalic acid, fumaric acid, succinic acid, p-toluenesulfonic acid
and malic acid.
The term "hydrate" as used herein designates a crystalline molecular compound in
which water molecules are incorporated into the crystal lattice. Generally speaking,
a hydrate thus designates a crystalline form of a molecular compound, whereby the
only further molecules incorporated into the crystal lattice are water molecules.
The term "stereoisomer' s" refers to at least two compounds having the same
molecular formula and connectivity of atoms, but having a different arrangement of
atoms in a three-dimensional space. In view of the present disclosure, a
stereoisomer can be, for example, an enantiomer, a diastereomer, or a meso
compound.
The term "prophylactic" as used herein refers variously to medicaments, amounts
or quantities, methods, uses and effects, etc., that prevent and/or aid in preventing
infections.
The term "therapeutic" as used herein refers to preventing, ameliorating, treating,
improving, or curing a disease or condition.
The term "about," as used herein, is intended to qualify the numerical values which
it modifies, denoting such a value as variable within a margin of error. When no
particular margin of error, such as a standard deviation to a mean value given in a
chart or table of data, is recited, the term "about" should be understood to mean
that range which would encompass the recited value and the range which would be
included by rounding up or down to that figure as well, taking into account
significant figures.
The term "disease" as used herein is intended to be generally synonymous, and is
used interchangeably with, the terms "disorder," "syndrome," and "condition" (as
in medical condition), in that all reflect an abnormal condition of the human or
animal body or of one of its parts that impairs normal functioning, is typically
manifested by distinguishing signs and symptoms, and causes the human or animal
to have a reduced duration or quality of life.
The term "muscular diseases" refers to diseases associated with impaired skeletal
muscle or cardiac muscle cell number or function.
The term "combination therapy" means the administration of two or more
therapeutic agents to treat a therapeutic condition or disorder described in the
present disclosure. Such administration encompasses co-administration of these
therapeutic agents in a substantially simultaneous manner, such as in a single
capsule having a fixed ratio of active ingredients or in multiple, separate capsules
for each active ingredient. In addition, such administration also encompasses use of
each type of therapeutic agent in a sequential manner. In either case, the treatment
regimen will provide beneficial effects of the drug combination in treating the
conditions or disorders described herein. In certain embodiments, a combination of
compounds is administered such that the clearance half-life of each compound
from the body overlaps at least partially with one another. For example, a first
pharmaceutical has a clearance half-life of 1 hour and is administered at time=0,
and a second pharmaceutical has a clearance half-life of 1 hour and is administered
at time=45 minutes.
The phrase "therapeutically effective" is intended to qualify the amount of active
ingredients used in the treatment of a disease or disorder or on the effecting of a
clinical endpoint.
The term "therapeutically acceptable" refers to those compounds (or salts,
prodrugs, tautomers, zwitterionic forms, etc.) which are suitable for use in contact
with the tissues of patients without undue toxicity, irritation, and allergic response,
are commensurate with a reasonable benefit/risk ratio, and are effective for their
intended use.
As used herein, reference to "treatment" of a patient is intended to include
prophylaxis. Treatment may also be preemptive in nature, i.e., it may include
prevention of disease. Prevention of a disease may involve complete protection
from disease, for example as in the case of prevention of infection with a pathogen,
or may involve prevention of disease progression. For example, prevention of a
disease may not mean complete foreclosure of any effect related to the diseases at
any level, but instead may mean prevention of the symptoms of a disease to a
clinically significant or detectable level. Prevention of diseases may also mean
prevention of progression of a disease to a later stage of the disease.
The term "patient" is generally synonymous with the term "subject" and includes
all mammals including humans. Examples of patients include humans, livestock
such as cows, goats, sheep, pigs, and rabbits, and companion animals such as dogs,
cats, rabbits, and horses. Preferably, the patient is a human.
Compounds and compositions disclosed herein are administered in an "effective
amount." This term is defined hereinafter. Unless dictated otherwise, explicitly or
otherwise, an "effective amount" is not limited to a minimal amount sufficient to
ameliorate a condition, or to an amount that results in an optimal or a maximal
amelioration of the condition. In the case when two or more compounds are
administered together, an effective amount of one such compound may not be, in
and of itself, be an effective amount, but may be an effective amount when used
together with additional compounds.
While the phrase "administered together" as used herein may refer to the provision
of chemical compositions in the same pharmaceutical composition, the phrase as
used herein is not intended to imply that this must be so. Rather, two or more
chemical compositions are "administered together" if the for the clearances of
each composition from the body overlaps at least partially with one another. For
example, if a first pharmaceutical has a Ti 2 for clearance of 1 hour and is
administered at time=0, and a second pharmaceutical has a for clearance of 1
hour and is administered at time=45 minutes, such pharmaceuticals are considered
administered together. Conversely, if the second drug is administered at time=2
hours, such pharmaceuticals are not considered administered together.
Detailed description of the Invention
A. Compounds of the present invention
The present invention provides hydroxysteroids and their intermediates of formula
(I):
FORMULA I
or a salt thereof, wherein:
is independently either a single bond, a double bond or a cyclopropyl ring,
provided that adjacent double bonds are not allowed;
A , A2, A 3 are independently selected from the group comprising hydi
hydroxyl, hydroxymethyl, halogen, C1-C6 alkyl;
B and C are each independently selected from the group comprising hydrogen,
hydroxyl, halogen, -OR „ -COR ,-COOR ,OCOR , CH2OH, CH2OR , -CONHR ,
CONHR R7, , -C(OH)R R7, NHR , NHR5CONHR , - R NHCOOR7, -NR R7,
C(0)heteroaryl, C(0)heterocyclyl, C1-C12 straight or branched chain alkyl, 5-6
membered heterocycloalkyl, 5-6 membered heteroaryl;
wherein the C1-C12 straight or branched chain alkyl or 5-6 membered
heterocycloalkyl or 5-6 membered heteroaryl; may be further optionally substituted
with one or more substituents selected from the group comprising halogen, C1-C6
alkyl, -OR , -COOR ,-CONHR , -OCOR , =NOH, NR R7, -NR COR7, 5-6
membered heterocycloalkyl or 5-6 membered heteroaryl; C1-C12 alkyl substituted
5-6 membered heterocycloalkyl or C1-C12 alkyl substituted 5-6 membered
heteroaryl;
or B and C may combine together to form =0, =NOR , NHR , 5-6 membered
heterocycloalkyl, or 5-6 membered heteroaryl;
wherein the said 5-6 membered heterocycloalkyl, 5-6 membered heteroaryl may
optionally contain one or more heteroatom;
wherein, the heteroatom may be O, N, S;
Ri, R2, and R4 are independently selected from the group comprising hydrogen,
deuterium, hydroxyl, halogen, =0, -OR , -NR R7, -COR , -COOR ,-OCOR ,-
CONR R7, -C alkyl, -Otert-butyldimethylsilyl;
R 3 is hydroxyl, carbonyl, OCOR6;
wherein R 3 is in beta configuration;
R is selected from hydrogen, hydroxyl, halogen, OR ;
R and R7are each independently selected from the group comprising hydrogen,
halogen, hydroxyl, C1- 2 alkyl, -NH2, -(CH2)n H2, 3-6 membered cycloalkyl, 4-6
membered heterocycloalkyl, 5-6 membered heteroaryl;
X is selected from CH, NH, NR , O or S;
n is 0 to 3.
The present invention provides hydroxysteroids and their intermediates of formula
(II):
FORMULA II
or a salt thereof, wherein:
is independently either a single bond, a double bond or a cyclopropyl ring,
provided that adjacent double bonds are not allowed;
A , A2, are independently selected from the group comprising hydrogen, hydroxyl,
hydroxymethyl, halogen, -C alkyl;
B and C are each independently selected from the group comprising hydrogen,
hydroxyl, halogen, -OR „ -COR , -COOR ,OCOR , CH2OH, CH2OR , -CONHR ,
CONHR R7, , -C(OH)R R7, NHR , NHR5CONHR , - R NHCOOR7, -NR R7,
C(0)heteroaryl, C(0)heterocyclyl, C1-C12 straight or branched chain alkyl, 5-6
membered heterocycloalkyl, 5-6 membered heteroaryl;
wherein the C1-C12 straight or branched chain alkyl or 5-6 membered
heterocycloalkyl or 5-6 membered heteroaryl; may be further optionally substituted
with one or more substituents selected from the group comprising halogen, C1-C6
alkyl, -OR , -COOR ,-CONHR , -OCOR , =NOH, NR R7, -NR COR7, 5-6
membered heterocycloalkyl or 5-6 membered heteroaryl; C1-C12 alkyl substituted
5-6 membered heterocycloalkyl or -C12 alkyl substituted 5-6 membered
heteroaryl;
or B and C may combine together to form =0, =NOR , NHR , 5-6 membered
heterocycloalkyl, or 5-6 membered heteroaryl;
wherein the said 5-6 membered heterocycloalkyl, 5-6 membered heteroaryl may
optionally contain one or more heteroatom;
wherein, the heteroatom may be O, N, S;
R and R 4 are independently selected from the group comprising hydrogen,
deuterium, hydroxyl, halogen, =0, -OR , -NR R7, -COR , -COOR ,-OCOR ,-
CONR R7, -C alkyl, -Otert-butyldimethylsilyl;
R and R7are each independently selected from the group comprising hydrogen,
halogen, hydroxyl, alkyl, -NH2, -(CH2)nNH2, 3-6 membered cycloalkyl, 4-6
membered heterocycloalkyl, 5-6 membered heteroaryl;
n is 0 to 3.
The present invention provides hydroxysteroids and their intermediates of formula
(III):
FORMULA III
or a salt thereof, wherein:
A and A2 are independently selected from the group comprising hydrogen,
hydroxyl, hydroxymethyl, halogen, C1-C6 alkyl;
B and C are each independently selected from the group comprising hydrogen,
hydroxyl, halogen, -OR „ -COR ,-COOR ,OCOR , CH2OH, CH2OR , -CONHR ,
CONHR R7, , -C(OH)R R7, NHR , NHR5CONHR , - R NHCOOR7, -NR R7,
C(0 )heteroaryl, C(0 )heterocyclyl, C1-C12 straight or branched chain alkyl, 5-6
membered heterocycloalkyl, 5-6 membered heteroaryl;
wherein the C1-C12 straight or branched chain alkyl or 5-6 membered
heterocycloalkyl or 5-6 membered heteroaryl; may be further optionally substituted
with one or more substituents selected from the group comprising halogen, C1-C6
alkyl, -OR , -COOR ,-CONHR , -OCOR , =NOH, NR R7, -NR COR7, 5-6
membered heterocycloalkyl or 5-6 membered heteroaryl; C1-C12 alkyl substituted
5-6 membered heterocycloalkyl or C1-C12 alkyl substituted 5-6 membered
heteroaryl;
or B and C may combine together to form =0, =NOR , NHR , 5-6 membered
heterocycloalkyl, or 5-6 membered heteroaryl;
wherein the said 5-6 membered heterocycloalkyl, 5-6 membered heteroaryl may
optionally contain one or more heteroatom;
wherein, the heteroatom may be O, N, S;
R4 is independently selected from the group comprising hydrogen, deuterium,
hydroxyl, halogen, =0, -OR , -NR R7, -COR , -COOR6,-OCOR ,-CONR R7, -
C(, alkyl, -Otert-butyldimethylsilyl;
R and R7 are each independently selected from the group comprising hydrogen,
halogen, hydroxyl, C1-C12 alkyl, -N¾, -(CH2)nNH2, 3-6 membered cycloalkyl, 4-6
membered heterocycloalkyl, 5-6 membered heteroaryl;
n is 0 to 3.
diates of formula
FORMULA IV
or a salt thereof, wherein:
Al and A2 are independently selected from the group comprising hydi
hydroxyl, hydroxymethyl, halogen, C1-C6 alkyl;
B and C are each independently selected from the group comprising hydrogen,
hydroxyl, halogen, -OR „ -COR ,-COOR ,OCOR , CH2OH, CH2OR , -CONHR ,
CONHR R7, , -C(OH)R R7, NHR , NHR5CONHR , - R NHCOOR7, -NR R7,
C(0)heteroaryl, C(0)heterocyclyl, C1-C12 straight or branched chain alkyl, 5-6
membered heterocycloalkyl, 5-6 membered heteroaryl;
wherein the C1-C12 straight or branched chain alkyl or 5-6 membered
heterocycloalkyl or 5-6 membered heteroaryl; may be further optionally substituted
with one or more substituents selected from the group comprising halogen, C1-C6
alkyl, -OR , -COOR ,-CONHR , -OCOR , =NOH, NR R7, -NR COR7, 5-6
membered heterocycloalkyl or 5-6 membered heteroaryl; C1-C12 alkyl substituted
5-6 membered heterocycloalkyl or C1-C12 alkyl substituted 5-6 membered
heteroaryl;
or B and C may combine together to form =0, =NOR , NHR , 5-6 membered
heterocycloalkyl, or 5-6 membered heteroaryl;
wherein the said 5-6 membered heterocycloalkyl, 5-6 membered heteroaryl may
optionally contain one or more heteroatom;
wherein, the heteroatom may be O, N, S;
R4 is independently selected from the group comprising hydrogen, deuterium,
hydroxyl, halogen, =0, -OR , -NR R7, -COR , -COOR ,-OCOR ,-CONR R7, -
C(, alkyl, -Otert-butyldimethylsilyl;
R and R7 are each independently selected from the group comprising hydrogen,
halogen, hydroxyl, C1-C12 alkyl, -NH2, -(CH2)nNH2, 3-6 membered cycloalkyl, 4-6
membered heterocycloalkyl, 5-6 membered heteroaryl;
n is 0 to 3.
The compounds of the present invention may be illustrated but not limited to the
examples as provided in Table 1.
Table 1: Illustrative compounds of present invention

(4aR,5S,6aS,7S)-5-hydroxy-4a,6adimethyl-
7-(2-methyl- 1,3-dioxolan-2-
1025 yl)-4,4a,4b,5,6,6a,7,8,9,9a,9b,10-
dodecahydro-lH-indeno[5,4-f]quinolin-
2(3H)-one H
(4aR,5S,6aS,7S)-5-hydroxy-4a,5,6atrimethyl-
7-(2-methyl- 1,3-dioxolan-2-
1026 yl)-4,4a,4b,5,6,6a,7,8,9,9a,9b,10-
dodecahydro-lH-indeno[5,4-f]quinolin-
2(3H)-one H
(4aR,5S,6aS,7S)-5-hydroxy-4a,6adimethyl-
2-oxo-
1027 2,3,4,4a,4b,5,6,6a,7,8,9,9a,9b,10-
tetradecahydro- 1H-indeno [5,4-
H
f]quinoline-7-carboxylic acid
(HS,13S)-17-(l-hydroxyethyl)-13-
methyl-7,8,9,11, 12,13,14,15,16,17-
1028
decahydro-6Hcyclopenta[
a]phenanthrene-3 ,11-diol
l-((HS,13S)-3,ll-dihydroxy-13-
methyl-7,8,9,11, 12,13,14,15,16,17-
1029 decahydro-6Hcyclopenta[
a]phenanthren-17-
yl)ethanone
(HS,13S)-17-(l-hydroxyethyl)-3-
methoxy- 13-methyl-
1030
7,8,9,11, 12,13, 14,15, 16,17-decahydro-
6H-cyclopenta[a]phenanthren- 11-ol
(llS,13S)-3-((tertbutyldimethylsilyl)
oxy)- 13-methyl- 17-
1031 (2-methyl- 1,3-dioxolan-2-yl) -
7,8,9,11, 12,13, 14,15, 16,17-decahydro-
6H-cyclopenta[a]phenanthren- 11-ol
(llS,13S)-13-methyl-17-(2-methyl-l,3-
dioxolan-2-yl)-
1032 7,8,9,11, 12,13, 14,15, 16,17-decahydro-
6H-cyclopenta[a]phenanthrene-3 ,11-
diol
(10R,llS,13S,17S)-ll,17-dihydroxy-
10,13-dimethyl-
1033 6,7,8,9,10,11,12,13,14,15,16,17-
dodecahydro-3Hcyclopenta[
a]phenanthren-3-one
(10R,llS,13S)-ll-hydroxy-10,13-
dimethyl-7,8,9,10,11, 12,13,14,15,16-
1034 decahydro-3Hcyclopenta[
a]phenanthrene-3,17(6H)-
dione
(HS,13S,17S)-4,ll-dihydroxy-l,13-
dimethyl-7,8,9,11, 12,13,14,15,16,17-
1035 decahydro-6Hcyclopenta[
a]phenanthrene-17-
carboxylic acid OH
(llS,13S,17S)-13-methyl-
7,8,9,11, 12,13, 14,15, 16,17-decahydro-
1036
6H-cyclopenta[a]phenanthrene-3, 11,17-
triol

((llS,13S,17S)-4,ll-dihydroxy-l,13-
dimethyl-7,8,9,11, 12,13,14,15,16,17-
1047 decahydro-6Hcyclopenta[
a]phenanthren-17-
yl)(piperazin- 1-yl)methanone OH
(llS,13S,17S)-17-(hydroxymethyl)- -OH
1,13-dimethyl-
1048 7,8,9,11, 12,13, 14,15, 16,17-decahydro-
6H-cyclopenta[a]phenanthrene-4, 11-
diol OH
(11S,13S,17S)- 11-hydroxy-4-methoxy-
1,13-dimethyl-
1049 7,8,9,11, 12,13, 14,15, 16,17-decahydro-
6H-cyclopenta[a]phenanthrene-17-
carboxylic acid
(11S,13S,17S)-17-
((dimethylamino)methyl)- 1,13-
1050 dimethyl-7,8,9,11, 12,13,14,15,16,17-
decahydro-6Hcyclopenta[
a]phenanthrene-4, 11-diol OH
1-((11S,13S)-11 -hydroxy-3-methoxy-
13-methyl-7,8,9,ll, 12,13,14,15,16,17-
1051 decahydro-6Hcyclopenta[
a]phenanthren-17-
yl)ethanone
(E)-l-((llS,13S)-3,ll-dihydroxy-13-
methyl-7,8,9,11, 12,13,14,15,16,17-
1052 decahydro-6Hcyclopenta[
a]phenanthren-17-
yl)ethanone oxime

(4aR,5S,6aS,7S)-5-hydroxy-4a,6adimethyl-
2-oxo-
1065 2,3,4,4a,4b,5,6,6a,7,8,9,9a,9b,10-
tetradecahydro- 1H-indeno [5,4-
f]quinoline-7-carboxylic acid
The compounds of the present invention include:
i . (10R,11S,13S,17S)-2,3,6,7,8,9,10,1 1,12,13, 14,15,16,17-tetradecahydro-llhydroxy-
10,13-dimethyl-3-oxo-lH-cyclopenta[a]phenanthrene-17-
carboxylic acid
ii. (3S,8S,9S,10S,llS,13S,14S,17S)-hexadecahydro-3,ll-dihydroxy-N,10,13-
trimethyl-lH-cyclopenta[a]phenanthrene-17-carboxamide
iii. (3S,8S,9S,10S,llS,13S,14S,17S)-N-(2-aminoethyl)-hexadecahydro-3,lldihydroxy-
10, 13-dimethyl- lH-cyclopenta[a]phenanthrene- 17-carboxamide
iv. (3S,5R,6R,10R,llS,13S,17S)-hexadecahydro-6-methoxy-10,13-dimethyl-
17-(2-methyl- 1,3-dioxolan-2-yl)- 1H-cyclopenta[a]phenanthrene-3 ,5,11-triol
v. (4aR,5S,6aS)-5-hydroxy-4a,6a-dimethyl-4,4a,4b,5,6,6a,9,9a,9b,10-
decahydro-lH-indeno[5,4-f]quinoline-2,7(3H,8H)-dione(4a'R,5'S,6a'S)-5'-
hydroxy-4a ,,5,,6a,-trimethyl-3 ,,4,,4a,,4b,,5,,6,,6a,,8,,9,,9a,,9b,,10'-
dodecahydrospiro[ [1,3] dioxolane-2,7'-indeno[5,4-f] quinolin] -2'(1'H)-one
vi. (4aR,5S,6aS)-4,4a,4b,5,6,6a,9,9a,9b,10-decahydro-5-hydroxy-4a,6adimethyl-
1H-indeno[5 ,4-fj quinoline-2,7(3H, 8H)-dione
vii. (4aR,5S,6aS)-7-acetyl-4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-5-
hydroxy-4a,5,6a-trimethyl-lH-indeno[5,4-f]quinolin-2(3H)-one
viii. (11S)-7,8,9,11, 12,13,14,15, 16,17-decahydro-3,ll-dihydroxy-6Hcyclopenta[
a]phenanthrene-17-carboxylic acid
ix. (4aR,6aS)-2,3,4,4a,4b,5,6,6a,7,8,9,9a,9b,10-tetradecahydro-4a,6a-dimethyl-
2,5-dioxo- 1H-indeno [5,4-fjquinoline-7-carboxylic acid
x. (17S)-17-acetyl-7,8, 13,15, 16,17-hexahydro-3-hydroxy-l-methyl-6Hcyclopenta
[a]phenanthren-l l(9H,12H,14H)-one
xi. (13S,17S)-17-acetyl-7,8,13,15,16,17-hexahydro-3-hydroxy-13-methyl-6Hcyclopenta[
a]phenanthren-ll(9H,12H,14H)-one
xii. (10R,llS,13S,17S)-ll-hydroxy-10,13-dimethyl-3-oxo-
6,7,8,9,10,11, 12,13, 14,15, 16,17-dodecahydro-3Hcyclopenta[
a]phenanthrene-17-carboxylic acid
xiii. (10R,llS,13S,17S)-ll-hydroxy-N,N,10,13-tetramethyl-3-oxo-
6,7,8,9,10,11, 12,13, 14,15, 16,17-dodecahydro-3Hcyclopenta[
a]phenanthrene-17-carboxamide
xiv. (10R,llS,13S,17S)-17-acetyl-ll-hydroxy-10,13-dimethyl-
6,7,8,9,10,11, 12,13, 14,15, 16,17-dodecahydro-3Hcyclopenta[
a]phenanthren-3-one
xv. (10R,llS,13S,17S)-ll-hydroxy-17-((R)-l-hydroxyethyl)-10,13-dimethyl-
6,7,8,9,10,11, 12,13, 14,15, 16,17-dodecahydro-3Hcyclopenta[
a]phenanthren-3-one
xvi. (4a'R,5'S,6a'S)-5'-hydroxy-4a',6a'-dimethyl-
' ' a' b' ' ' a' ' ' a' b O'-dodecahydros iro dioxolane '-
indeno [5,4-f] quinolin] -2'(1Ή )-one
xvii. (4aR,5R,6aS)-5-hydroxy-4a,5,6a-trimethyl-4,4a,4b,5,6,6a,9,9a,9b,10-
decahydro-lH-indeno[5,4-f]quinoline-2,7(3H,8H)-dione
xviii. (4aR,5S,6aS)-5,7-dihydroxy-4a,6a-dimethyl-4,4a,4b,5,6,6a,7,8,9,9a,9b,10-
dodecahydro-lH-indeno[5,4-f]quinolin-2(3H)-one
xix. (4aR,5S,6aS)-7-fluoro-5-hydroxy-4a,6a-dimethyl-
4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-lH-indeno[5,4-f]quinolin-
2(3H)-one
xx. (4aR,5S,6aS,Z)-5-hydroxy-7-(hydroxyimino)-4a,6a-dimethyl-
4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-lH-indeno[5,4-f]quinolin-
2(3H)-one
xxi. (4aR,5S,6aS)-5,7-dihydroxy-l,4a,6a-trimethyltetradecahydro-lHindeno[
5 ,4-f] quinolin-2(3H)-one
xxii. (4aR,5S,6aS)-5,7-dihydroxy-l,4a,6a-trimethyl-
4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-lH-indeno[5,4-fJquinolin-
2(3H)-one
xxiii. (4aR,5S,6aS)-7-amino-5-hydroxy-4a,6a-dimethyltetradecahydro-lHindeno[
5 ,4-f] quinolin-2(3H)-one
xxiv. (4aR,5S,6aS,7S)-5-hydroxy-4a,6a-dimethyl-7-(2-methyl-l,3-dioxolan-2-
yl)-4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-lH-indeno[5,4-f]quinolin-
2(3H)-one
xxv. (4aR,5S,6aS ,7S)-5-hydroxy-4a,5,6a-trimethyl-7-(2-methyl-l,3-dioxolan-2-
yl)-4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-lH-indeno[5,4-f]quinolin-
2(3H)-one
xxvi. (4aR,5S,6aS,7S)-5-hydroxy-4a,6a-dimethyl-2-oxo-
2,3,4,4a,4b,5,6,6a,7,8,9,9a,9b,10-tetradecahydro-lH-indeno[5,4-
f]quinoline-7-carboxylic acid
xxvii. (llS,13S)-17-(l-hydroxyethyl)-13-methyl-7,8,9,ll, 12,13,14,15,16,17-
decahydro-6H-cyclopenta[a]phenanthrene-3,ll-diol
xxviii. l-((llS,13S)-3,ll-dihydroxy-13-methyl-7,8,9,ll, 12,13,14,15,16,17-
decahydro-6H-cyclopenta[a]phenanthren-17-yl)ethanone
xxix. ( 1lS,13S)-17-(l-hydroxyethyl)-3-methoxy-13-methyl-
7,8,9,11, 12,13, 14,15, 16, 17-decahydro-6H-cyclopenta[a]phenanthren- 11-ol
xxx. (llS,13S)-3-((tert-butyldimethylsilyl)oxy)-13-methyl-17-(2-methyl-l,3-
dioxolan-2-yl)-7,8,9,ll, 12,13, 14,15,16,17-decahydro-6Hcyclopenta[
a]phenanthren-l l-ol
xxxi. (llS,13S)-13-methyl-17-(2-methyl-l,3-dioxolan-2-yl)-
7,8,9,ll,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,l 1-
diol
xxxii. (10R,llS,13S,17S)-ll,17-dihydroxy-10,13-dimethyl-
6,7,8,9,10,11, 12,13, 14,15, 16,17-dodecahydro-3Hcyclopenta[
a]phenanthren-3-one
xxxiii. (10R,llS,13S)-ll-hydroxy-10,13-dimethyl-7,8,9,10,l 1,12,13,14,15,16-
decahydro-3H-cyclopenta[a]phenanthrene-3,17(6H)-dione
xxxiv. (llS,13S,17S)-4,ll-dihydroxy-l,13-dimethyl-7,8,9,l 1,12,13,14,15,16,17-
decahydro-6H-cyclopenta[a]phenanthrene-17-carboxylic acid
xxxv. (llS,13S,17S)-13-methyl-7,8,9,ll,12,13,14,15,16,17-decahydro-6Hcyclopenta[
a]phenanthrene-3 ,11,17-triol
xxxvi. (4aR,5S,6aS,7S)-7-acetyl-5-hydroxy-4a,6a-dimethyl-
4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-lH-indeno[5,4-f]quinolin-
2(3H)-one
xxxvii. (4aR,5S,6aS,7S)-5-hydroxy-7-(2-hydroxypropan-2-yl)-4a,6a-dimethyl-
4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-lH-indeno[5,4-f]quinolin-
2(3H)-one
xxxviii. l-((8S,9S,10R,llS,13S,14S)-3,ll-dihydroxy-10,13-dimethyl-
2,3,4,7,8,9,10,11, 12,13, 14,15,16,17-tetradecahydro-lHcyclopenta[
a] phenanthren- 17-yl)ethanone
xxxix. (8S,9S,10R,llS,13S,14S)-17-acetyl-ll-hydroxy-10,13-dimethyl-
6,7,8,9,10,11, 12,13, 14,15, 16,17-dodecahydro-lHcyclopenta[
a]phenanthren-3(2H)-one
xl. (10R,llS,13S,17S)-ll-hydroxy-10,13-dimethyl-3-oxo-
6,7,8,9,10,11, 12,13, 14,15, 16,17-dodecahydro-3Hcyclopenta[
a]phenanthrene-17-carboxamide
xli. (10R,llS,13S,17S)-ll-hydroxy-17-(hydroxymethyl)-10,13-dimethyl-
6,7,8,9,10,11, 12,13, 14,15, 16,17-dodecahydro-3Hcyclopenta[
a]phenanthren-3-one
xlii. (10R, 11S,13S, 17S)- 17-((dimethylamino)methyl)- 11-hydroxy- 10, 13-
dimethyl-6,7,8,9,10,11, 12,13, 14,15, 16,17-dodecahydro-3Hcyclopenta[
a]phenanthren-3-one
xliii. (llS,13S,17S)-methyl 4,ll-dihydroxy-l,13-dimethyl-
7,8,9,1 1,12, 13, 14, 15,16, 17-decahydro-6H-cyclopenta[a]phenanthrene-17-
carboxylate
xliv. (llS,13S,17S)-4,ll-dihydroxy-l,13-dimethyl-7,8,9,l 1,12,13,14,15,16,17-
decahydro-6H-cyclopenta[a]phenanthrene-17-carboxamide
xlv. (11S,13S, 17S)-4, 11-dihydroxy-N,N,1,13-tetramethyl-
7,8,9,1 1,12, 13, 14, 15,16, 17-decahydro-6H-cyclopenta[a]phenanthrene-17-
carboxamide
xlvi. ((llS,13S,17S)-4,ll-dihydroxy-l,13-dimethyl-7,8,9,ll, 12,13,14,15,16,17-
decahydro-6H-cyclopenta[a]phenanthren-17-yl)(piperazin-l-yl)methanone
xlvii. (11S,13S, 17S)- 17-(hydroxymethyl)- 1,13-dimethyl-
7,8,9,1 1,12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthrene-4,l 1-
diol
xlviii. (11S,13S,17S)-11 -hydroxy-4-methoxy- 1,13-dimethyl-
7,8,9,1 1,12, 13, 14, 15,16, 17-decahydro-6H-cyclopenta[a]phenanthrene-17-
carboxylic acid
xlix. (11S,13S, 17S)- 17-((dimethylamino)methyl)- 1,13-dimethyl-
7,8,9,1 1,12, 13, 14, 15, 16, 17-decahydro-6H-cyclopenta[a]phenanthrene-4,l 1-
diol
1. 1-((1 1S,13S)- 11-hydroxy-3-methoxy- 13-methyl-
7,8,9,11, 12,13, 14,15, 16, 17-decahydro-6H-cyclopenta[a]phenanthren- 17-
yl)ethanone
li. (E)-l-((llS,13S)-3,ll-dihydroxy-13-methyl-7,8,9,ll, 12,13,14,15,16,17-
decahydro-6H-cyclopenta[a]phenanthren-17-yl)ethanone oxime
lii. (E)-l-((llS,13S)-3,ll-dihydroxy-13-methyl-7,8,9,ll, 12,13,14,15,16,17-
decahydro-6H-cyclopenta[a]phenanthren-17-yl)ethanone oxime
liii. ( 1lS,13S)-17-(2-hydroxypropan-2-yl)-13-methyl-
7,8,9,ll,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthrene-3,l 1-
diol
liv. (llS,13R)-17-ethyl-13-methyl-7,8,9,ll,12,13,14,15,16,17-decahydro-6Hcyclopenta[
a]phenanthrene-3 ,11-diol
lv. (8S,9S,10R,llS,13S,14S,Z)-ll-hydroxy-17-(hydroxyimino)-10,13-
dimethyl-6,7,8,9,10,11, 12,13, 14,15, 16,17-dodecahydro-3Hcyclopenta[
a]phenanthren-3-one
lvi. (8S,9S,10R,llS,13S,14S)-17-amino-ll-hydroxy-10,13-dimethyl-
6,7,8,9,10,11, 12,13, 14,15, 16,17-dodecahydro-3Hcyclopenta[
a]phenanthren-3-one
lvii. (8S,9S ,1OR, 11S,13S,14S)- 11-hydroxy- 10, 13-dimethyl-
6,7,8,9,10,11, 12,13, 14,15, 16,17-dodecahydro-3Hcyclopenta[
a]phenanthren-3-one
lviii. (8S,9S ,1OR, 11S,13S,14S,17S)- 11,17-dihydroxy- 10,13,17-trimethyl-
6,7,8,9,10,11, 12,13, 14,15, 16,17-dodecahydro-3Hcyclopenta[
a]phenanthren-3-one
lix. (llR,13S,17S)-ll,13,17-trimethyl-7,8,9,ll,12,13,14,15,16,17-decahydro-
6H-cyclopenta[a]phenanthrene-3,ll,17-triol
lx. (llS,13S)-13-methyl-7,8,9,ll,12,13,14,15,16,17-decahydro-6Hcyclopenta[
a]phenanthrene-3 ,11-diol
lxi. (llS,13S,17S)-17-amino-13-methyl-7,8,9,ll,12,13,14,15,16,17-decahydro-
6H-cyclopenta[a]phenanthrene-3 ,11-diol
lxii. (US, 13S,Z)-3,ll-dihydroxy-13-methyl-7,8,9,ll, 12,13, 15, 16-octahydro-
6H-cyclopenta[a]phenanthren- 17(14H)-one oxime
lxiii. (4aR,5S,6aS,7S)-5-hydroxy-4a,6a-dimethyl-7-(2-methyl-l,3-dioxolan-2-
yl)-4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-lH-indeno[5,4-f]quinolin-
2(3H)-one
lxiv. (4aR,5S,6aS,7S)-5-hydroxy-4a,6a-dimethyl-2-oxo-
2,3,4,4a,4b,5,6,6a,7,8,9,9a,9b,10-tetradecahydro-lH-indeno[5,4-
fjquinoline-7-carboxylic acid
The present invention includes within its scope the salts and isomers. Compounds
of the present invention after being novel may in some cases form salts which are
also within the scope of this invention. The term "salt(s)", as employed herein,
denotes acidic and/or basic salts formed with inorganic and/or organic acids and
bases. Zwitterions (internal or inner salts) are included within the term "salt(s)" as
used herein (and may be formed, for example, where the R substituents comprise a
basic moiety such as an amino group). Also included herein are quaternary
ammonium salts such as alkyl ammonium salts. Pharmaceutically acceptable (i.e.,
non-toxic, physiologically acceptable) salts are preferred.
All stereoisomer' s of the present compounds, such as those which may exist due to
asymmetric carbons on the R substituents of the compound, including enantiomeric
and diastereomeric forms, are contemplated within the scope of this invention.The
Compounds of the present invention may be present in their enantiomeric pure
forms or their mixtures.
B. Salts and Isomers and Counter Ions
The present invention includes within its scope the salts and isomers. Compounds
of the present invention after being novel may in some cases form salts which are
also within the scope of this invention. The term "salt(s)", as employed herein,
denotes acidic and/or basic salts formed with inorganic and/or organic acids and
bases. Zwitterions (internal or inner salts) are included within the term "salt(s)" as
used herein (and may be formed, for example, where the R substituents comprise a
basic moiety such as an amino group). Pharmaceutically acceptable (i.e., non-toxic,
physiologically acceptable) salts are preferred.All stereoisomer' s of the present
compounds, such as those which may exist due to asymmetric carbons on the R
substituents of the compound, including enantiomeric and diastereomeric forms,
are contemplated within the scope of this invention. The Compounds of the present
invention may be present in their enantiomeric pure forms or their mixtures.
C. Composition of the present invention
The invention thus provides the use of the novel compounds as defined herein for
use in human or veterinary medicine. The compound for use as a pharmaceutical
may be presented as a pharmaceutical composition. The invention therefore
provides in a further aspect a pharmaceutical composition comprising the novel
compounds of the invention along with pharmaceutically acceptable
excipients/carriers thereof and optionally other therapeutic and/or prophylactic
ingredients. The excipients/carriers must be "acceptable" in the sense of being
compatible with the other ingredients of the composition and not deleterious to the
recipient thereof. Suitably the pharmaceutical composition will be in an appropriate
formulation.
The pharmaceutical formulations may be any formulation and include those
suitable for oral, intranasal, or parenteral (including intramuscular and intravenous)
administration. The formulations may, where appropriate, be conveniently
presented in discrete dosage units and may be prepared by any of the methods well
known in the art of pharmacy. All methods include the step of bringing into
association the active compound with liquid carriers or finely divided solid carriers
or both, and then, if necessary, shaping the product into the desired formulation.
For these purposes the compounds of the present invention may be administered
orally, topically, intranasally, parenterally, by inhalation spray or rectally in dosage
unit formulations containing conventional non-toxic pharmaceutically acceptable
carriers, adjuvants and vehicles. The term parenteral as used herein includes
subcutaneous injections, intravenous, intramuscular, intrasteral injection or
infusion techniques. In addition to the treatment of warm-blooded animals such as
mice, rats, horses, dogs, cats, etc., the compounds of the invention are effective in
the treatment of humans.
D. USE OF COMPOUNDS OF THE PRESENT INVENTION
The compounds of the present invention are used in diseases and disorders
pertaining to mitochondrial biogenesis. The mitochondrial biogenesis-mediated
disease is selected from the group comprising skeletal or cardiac muscle diseases
associated with ischemia, or impaired or inadequate blood flow, diseases associated
with genetic disorders that directly or indirectly affect the number, structure, or
function of mitochondria, diseases associated with impaired neurological function
associated with decreased mitochondrial number or function, diseases associated
with loss of number, loss of function, or loss of correct, optimally efficient internal
organization of skeletal muscle cells or cardiac muscle cells, metabolic diseases,
and conditions associated with liver cell injury and altered fatty acid metabolism.
The mitochondrial biogenesis-mediated disease is selected from the group
comprising acute coronary syndrome, myocardial infarction, angina, renal injury,
renal ischemia, diseases of the aorta and its branches, injuries arising from medical
interventions, atherosclerosis, trauma, diabetes, hyperlipidemia, vascular stenosis,
peripheral arterial disease, vasculopathy, and vasculitis.The mitochondrial
biogenesis-mediated disease is selected from the group consisting of Friedreich's
ataxia, muscular dystrophy, Duchenne muscular dystrophy, Becker muscular
dystrophy, limb girdle muscular dystrophy, congenital muscular dystrophy,
facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy,
oculopharyngeal muscular dystrophy, distal muscular dystrophy, spinal muscular
atrophy and Emery-Dreifuss muscular dystrophy. Huntington's disease,
parkinson's disease, alzheimer's disease, and amyotrophic lateral sclerosis. The
mitochondrial biogenesis-mediated disease is selected from the group comprising
sarcopenia, congestive heart failure, aging, myocarditis, myositis, polymyalgia
rheumatic, polymyositis, HIV, cancer and/or the side effects of chemotherapy
targeting the cancer, malnutrition, aging, inborn errors of metabolism, trauma, and
stroke or other types of neurological impairment like hepatic steatosis, hepatic
fibrosis, cirrhosis, and hepatocyte or stellate cell injury.
The compounds of the present invention may be used in treating or preventing the
adverse effects of administration of compounds which exhibit mitochondrial
toxicity
The compounds of the present invention are capable of improving muscle structure
or function; improving mitochondrial effects associated with exercise; enhancing
the capacity for exercise in those limited by age, inactivity, diet, or diseases;
enhancing muscle health and function in response to exercise; enhancing muscle
health and function in the clinical setting of restricted capacity for exercise;
enhancing recovery of muscles from vigorous activity or from injury associated
with vigorous or sustained activity.
The compounds of the present invention are capable of enhancing sports
performance and endurance, building muscle shape and strength, or facilitating
recovery from the muscle related side effects of training or competition.
E. Dose of the compounds of the present invention
In further embodiments, the methods disclosed herein comprise the administration
of compounds of the disclosure in a total daily dose of about 0.001 mg/kg/dose to
about 10 mg/kg/dose, alternately from about 0.3 mg/kg/dose to about 3
mg/kg/dose. In another embodiment the dose range is from about 0.1 to about 1
mg/kg/day. Generally between about 0.01 mg and about 0.1 gram per day can be
administered; alternately between about 2.5 mg and about 200 mg can be
administered. The dose may be administered in as many divided doses as is
convenient.
In further embodiments, the desired concentration is maintained for at least 30
minutes, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or more.
In yet further embodiments, the desired concentration is achieved at least once
during each 12-hour period over at least 24 hours, 48 hours, 72 hours, 1 week, one
month, or more; or at least once during each 24-hour period over at least 48 hours,
72 hours, 1 week, one month, or more. In order to maintain a desired concentration
for a desired time, multiple doses of one or more compounds may be employed.
The dosing interval may be determined based on the clearance half-life for each
compound of interest from the body.
It is envisaged within the scope of the present invention that the compounds may
be administered in combination with other therapeutic agents. The compounds may
be administered together or sequentially.
Without being limited by theory, it is submitted that the novel compounds of the
present invention exhibit substantially different pharmacokinetic and
pharmacodynamic profiles. The invention is described in detail herein below with
respect to the following examples which are provided merely for illustration.
However, these examples may not be construed to restrict the scope of the
invention. Any embodiments that may be apparent to a person skilled in the art are
deemed to fall within the scope of present invention.
Example 1Preparation of compounds of the present invention
Step l :Compound[l] (500mg, 1.38mmol) was taken in ACN (200ml) and
trimethyl silyl iodide (1.17ml, 8.28mmol) was added dropwise. The reaction
mixture was stirred at room temperature for 20 min. After completion, the reaction
was quenched by 10% sodium thiosulfate (50ml), washed with saturated NaHCC 3
and extracted with ethyl acetate. Organic layer was separated; dried over Na2S0 4
and evaporated under reduced pressure to obtain [2] as light yellow solid (450 mg,
94%).MS (ESI) m/z (M++l) 347.
Step 2: To a stirred solution of [2] (1.0 g, 2.89mmol) in acetone, a water (50ml)
solution of NaI0 4 (2.47g, 11.5mmol) was added and reaction mixture was stirrerd
for 2 hr at rt. Reaction was quenched with water (25ml), extracted with EtOAc.
Organic layer was separated, dried over Na2S04 and evaporated under reduced
pressure to obtain [1001] as white solid (850 mg, 88%), MS (ESI) m/z (M++l) 333.
Step 3 : A solution of compound [1001] (l.Og, 3.01mmol) in dioxane (50ml) was
treated with TBDMSCl (0.022g, 0.152mmol) followed by DDQ (0.90g, 3.95mmol)
at 0°C. The mixture was allowed to stirred at RT for 2 hr. Reaction mixture was
diluted with ethyl acetate and washed with saturated sodium carbonate solution and
brine, dried over sodium sulfate, concentrated and purified by silica gel column
chromatography eluting with 3% MeOH:DCM to give compound [1013] as a
light yellow solid (0.61g, 61% yield). MS (ESI) m/z (M++1) 331.
Step 4: The compound [1013] (0.5g, 1.51 mmol) was taken in pyridine (15mL) and
zinc dust (19.6 g, 30.2 mmol) was added to it, followed by addition of water (0.5
ml ) . The reaction mixturewas heated at 115°C for 90 mins and cooled, filtered,
washed with 0.1 N HCl and extracted with DCM (2 X lOOmL). The organic layer
was dried over Na2S04, concentrated and purified by silica gel column
chromatography by eluting with 40% ethyl acetate: hexane to give compound [24]
as a white solid to obtain product [1035] as off white solid. Yield (0.38g, 76%
yield).MS (ESI) m/z (M++1)331.
Step-5 : A solution of [1013]( 0.2 mg, 0.606 mmol) in DMF (3 ml),EDC.HCl
(0.17 mg, 0.909 mmol) was added and mixture was stirred at room temperature for
30 min. Dimethyl amine 2M solution in THF (1.5 ml, 3.03 mmol) was added
followed by N-methyl morpholine (0.15 g , 1.51 mmol) and stirring was continued
for 18 hrs at room temperature. The reaction mixture was quenched with brine (50
ml), extracted with ethyl acetate (3 X 100 ml). The combined organic extract was
dried (Na2S0 4) concentrated to obtain crude which was purified by column
chromatography (silica gel 100-200 mesh) to obtain compound [1014] as off white
solid (0.125g , 58% yield).MS (ESI) m/z (M++l)358.
A few illustrated examples of this series synthesized based on example 1 include
1014, 1041-1050
Example 2 (a):Preparation of compounds of the present invention
[31] |32]
Step 1: A solution of compound [30] (l.Og, 3.04mmol) in dioxane (50ml) was
treated with TBDMSC1 (0.022g, 0.152mmol) followed by DDQ (0.90g, 3.95mmol)
at 0°C. The mixture was allowed to stirred at RT for 2 hr. Reaction mixture was
diluted with ethyl acetate and washed with saturated sodium carbonate solution and
brine, dried over sodium sulfate and purified by silica gel column chromatography
eluting with 40% ethyl acetate: hexane to give compound [30a] as a white solid
(0.45g, 45% yield). MS (ESI) m/z (M++l)325.
Step 2: A solution of compound [30a] ( 1 g, 3.08 mmol) in Dry THF (5 ml) was
taken. To this PCI5 (1.15 g, 5.55 mmol) was added at -80°C and stirred at 30mins.
After completion, 10% NaOH solution was added to adjust pH upto 7 and organic
layer was extracted with ethyl acetate. Organic phase was dried over sodium sulfate
and was evaporated under reduced pressure to get crude residue. The crude residue
was purified by silica gel column chromatography by eluting with 15-20% ethyl
acetate: hexane to obtain brown solid[31]. (0.80 g, 77% yield). MS (ESI) m/z
(M++l)313.
Step 3: The compound [31] (0.5g, 1.60 mmol) was taken in pyridine (15mL) and
Zn dust (20.8 g, 32 mmol) was added to it followed by addition of. 0.5 ml ofwater.
The reaction mixture was heated at 115°C for 90 mins for completion. Reaction
mixture was cooled, filtered, washed with 0.1 N HC1 and extracted with DCM (2 X
lOOmL). The organic layer was dried over Na2S0 4, concentrated to obtain product
[32] as off white solid. (0.35g, 73% yield), MS (ESI) m/z (M++l)297.
Example 2b:Preparation of compounds of the present invention
Step 1: A solution of compound [32] (l.Og, 3.3mmol) in DCM (20ml) was treated
with imidazole (0.6g, 9.9 mmol) followed by addition ofTBDMSCl (0.99 g,
6.6mmol) at RT.The reaction mass was heated at 40°C for 4 hrs. The Reaction
mixture was quenched with water and extracted with ethyl acetate. Organic phase
was washed with saturated sodium carbonate solution and brine, dried over sodium
sulfate and evaporated under reduced pressure to get crude product. The crude
product was subjected to purification over silica gel column chromatography using
2 - 3% ethyl acetate: hexane as eluent to give compound [33] as a white solid (1.2
g, 87% yield). MS (ESI) m/z (M++l)411.
Step 2: A mixture of [33] (0.5 mg, 1.21 mmol), p-toluenesulfonic acid
monohydrate (20 mg), and ethylene glycol (3ml) in benzene (100ml) was refluxed
with azeotropic removal of water for 4 hr. The mixture was diluted with ethyl
acetate, washed with saturated sodium bicarbonate solution and brine, dried over
sodium sulfate and concentrated by rotovap to obtain compound [34] as a light
yellow solid (130mg) with 87% yield.MS (ESI) m/z (M+H+)455.
Step 3: A solution of compound [34] ( 1 g, 2.44 mmol) in Dry THF (5 ml) was
taken. To this BH3.DMS (2.3ml, 24.4 mmol) was added at RT and reaction mass
was heated at 40°C for 3 hrs. After completion, 2 ml water was added at -30°C for
quenching followed by drop wise addition of 10% NaOH solution and H 2 ¾as a
mixture ( 1:1; 20 ml). The reaction mass was extracted with ethyl acetate. Organic
layer was dried over sodium sulfate and concentrated. The crude residue was
purified by silica gel column chromatography using 60 % ethyl acetate: hexane as
eluent to obtain yellow solid[35]. (0.6 g, 52% yield). MS (ESI) nt/z (M+- 1). 473
Step 4: A solution of compound [35] (0.5g, 1.16 mmol) in DCM (20ml) was
treated with Dess martin periodinane ( 1.97 g, 4.65 mmol) at RT for 20 hr. Reaction
mixture was diluted with DCM and washed with saturated sodium carbonate
solution and brine, dried over sodium sulfate and purified by silica gel column
chromatography using 30% ethyl acetate in hexane as eluent to give compound
[36] as a white solid (0.3 1g, 62% yield). MS (ESI) nt/z (M++l). 471
Step 5: A solution of compound [36] (0.2 g, 0.47 mmol) in ethanol (50 ml) was
added sodium borohydride (0.034g, 0.93 mmol) at 0°C. The reaction was stirred in
an ice bath for 8 hr. The reaction was quenched with water (5ml) and concentrated
under vacuum until solid appears. The solid residue was purified by silica gel
column chromatography (2% MeOH:DCM as an eluent) to give compound [1031]
as a white solid (0. 15g, Yield 75%). MS (ESI) nt/z (M++l).473
Step 6: A solution of compound [1031] (0.2g, 0.423mmol) in THF (10ml) was
treated with TBAF 1M solution in THF (0.85 ml, 0.847 mmol) at 0°C. The mixture
was allowed to stirred at RT for 1 hr. Reaction mixture was diluted with ethyl
acetate and washed with saturated sodium carbonate solution and brine, dried over
sodium sulfate and purified by silica gel column chromatography eluting with 40%
Ethyl acetate in Hexane to give compound [1032] as a light yellow solid (0. 125g,
82% yield).MS (ESI) m/z (M++l)359
Step 7 A solution of compound [1032] (0. 1 g, 0.21mmol) in acetone (7 ml) and
water (3 ml) was treated with concentrated sulfuric acid (0.5 ml) at 0°C. The
mixture was allowed to stirred for 2hr. Reaction mixture was diluted with ethyl
acetate and washed with saturated sodium carbonate solution and brine, dried over
sodium sulfate and purified purified by silica gel column chromatography eluting
with 40% Ethyl acetate :Haxene to give compound [1029] as a white solid
(0.05mg, 75% yield).MS (ESI) m/z (M++l) 315.
Step 8: A solution of compound [1029] (0.1 g, 0.317 mmol) in ethanol (50 ml) was
added sodium borohydride (0.024g, 0.634 mmol) at 0°C. The reaction was stirred
in an ice bath for 4 hr. The reaction was quenched with water (5ml) and
concentrated under vacuum until solid appears. The solid residue was purified by
silica gel column chromatography (2% MeOH:DCM as an eluent) to give
compound [1028] as a white solid (0.080g, Yield 80%). MS (ESI) m/z (M++l)319.
A few illustrated examples of this series synthesized based on example 2b include
1028, 1051-1055
Step 1: A solution of compound [32] (l.Og, 3.3mmol) in DMF:Acetone (20ml) was
treated with Mel (1.4 g, 9.9 mmol) and K2C0 3 (1.36 g, 9.9 mmol) and catalytic
amount of 18-crown -6 at RT. The reaction mass was heated at 40°C for 14 hrs and
progress of the reaction was monitored by TLC. The reaction mixture was cooled
to room temperature and filtered. The filtrate was concentrated under reduced
pressure to evaporate excess of the solvent. Residual mass was dissolved in water
and extracted with ethyl acetate. Organic layer was washed with water and brine,
dried over sodium sulfate and purified by silica gel column chromatography using
with 2 - 3% ethyl acetate in hexane as eluents to give compound [37] as a white
solid (1.2 g, 87% yield), MS (ESI) m/z (M++l)311.
Step 2: Compound[38] was synthesized using the same procedure as mentioned in
example 2b. Yield 54 % .MS (ESI) m/z (M++l) 331.
Step 3: Compound[39] was synthesized using the same procedure as mentioned in
example 2b. Yield 65 %. MS (ESI) m/z (M++l) 327.
Step 4: Compound [1030]was synthesized using the same procedure as mentioned
in example 2b. Yield 70 %. MS (ESI) m/z (M++l) 331.
Step 1: A solution of chromium trioxide (3.3 g, 33.3 mmol) in water (4 mL) was
added dropwise to a solution of [1040] (10 g, 30.3 mmol) in acetic acid (300 ml).
The reaction was stirred for 1.5 hr and quenched by the addition of methanol (30
mL), stirring for 0.5 hr. The mixture was concentrated by rotovap at 70 °C,
dissolved in ethyl acetate/methanol, washed with saturated sodium bicarbonate and
brine, dried over sodium sulfate and concentrated to give an off white solid[41]
(9.0 g). 90% yield). MS (ESI) m/z (M++l) 329.
Step 2: A solution of compound [41] (l.Og, 3.04mmol) in dioxane (50ml) was
treated with TBDMSC1 (0.022g, 0.152mmol) followed by DDQ (0.90g, 3.95mmol)
at 0°C. The mixture was allowed to stirred at 40°C for 20 hr. Reaction mixture was
diluted with ethyl acetate and washed with saturated sodium carbonate solution and
brine, dried over sodium sulfate and purified by silica gel column chromatography
eluting with 2 - 3% MeOH:DCM to give compound [42] as a light yellow solid
(0.45g, 45% yield). MS (ESI) m/z (M++l)327.
Step 3: Compound[43] was synthesized using the same procedure as mentioned in
example 2b. Yield 78 % .MS (ESI) m/z (M++l) 371.
Step 4: Compound [44]was synthesized using the same procedure as mentioned in
example 2b. Yield 78 % .MS (ESI) m/z (M++l) 373.
Step 5: Compound[45] was synthesized using the same procedure as mentioned in
example 2b. Yield 78 % .MS (ESI) m/z (M++l) 329.
Step 6: Compound[1016] was synthesized using the same procedure as mentioned
in example 2b. Yield 78 % .MS (ESI) m/z (M++l) 331.
Example 5Preparation of compounds of the present invention
Step 1 : A solution of compound [50](0.21 mg, 0.6 mmol) in THF (4 ml) and
methanol (2 ml) was treated with a hot solution of sodium metaperiodate (383 mg,
1.8 mmol) in water and stirred for 1 hr at RT. The mixture was diluted with water
(10 ml) and concentrated by rotovap until a precipitate forms which is filtered,
washed with water and dried under hard vacuum to give compound [10] as a white
solid (0.15 mg). Yield 86 % .MS (ESI) m/z (M-l) 303.
Step 2 : Synthetic procedure is same as inexample 2 to provide [1034]. Yield 80 %
.MS (ESI) m/z (M++1) 301.
Step 3 : Synthetic procedure is same as that of example 2 to provide [1033]. Yield
75 % .MS (ESI) m/z (M++l) 303.
Some illustrated examples of this series synthesized based on example 5 include
1033,1056-1059
[62]
Step 1: Compound was synthesized using the same procedure as mentioned in
example 2a to provide [1034]. Yield 75 % .MS (ESI) m/z (M++l) 301.
Step 2: Compound was synthesized using the same procedure as mentioned in
example 2a to yield [61]. Yield 80 % .MS (ESI) m/z (M++l) 283.
Step 3: Compound was synthesized using the same procedure as mentioned in
example 2a to result in [62]. Yield 78 % .MS (ESI) m/z (M++l) 269.
Example 6b:
Step 1: Compound was synthesized using the same procedure as mentioned in
example 2b to provide [64]. Yield 70 % .MS (ESI) m/z (M++1) 383.
Step 2: Compound was synthesized using the same procedure as mentioned in
example 2b to provide [65]. Yield 56 % .MS (ESI) m/z (M++l) 403.
Step 3: Compound was synthesized using the same procedure as mentioned in
example 2b to provide [66]. Yield 7 1 % .MS (ESI) m/z (M++l) 399.
Step 4: Compound was synthesized using the same procedure as mentioned in
example 2b to result in [67]. Yield 78 % .MS (ESI) m/z (M++l) 403.
Step 5: A solution of compound [67] (O.lg, 0.248mmol) in THF (10ml) was
treated with TBAF 1M solution in THF (0.5 ml, 0.497mmol) at 0°C. The mixture
was allowed to stirred at RT for 1 hr. Reaction mixture was diluted with ethyl
acetate and washed with saturated sodium carbonate solution and brine, dried over
sodium sulfate and purified by silica gel column chromatography eluting with 40%
ethyl acetate inhexane to give compound [1036] as a light yellow solid (0.05g,
70% yield).MS (ESI) m/z (M++l) 289.
A few illustrated examples of this series synthesized based on example 6a & 6b
include 1060-1063
Example 7a: Preparation of compounds of the present invention
Step 1: A solution of hydrocortisone [1] (2.0 g, 5.52mmol) in EtOH-CH2C12
(40ml, 1:1) was treated with NaBH4 (84mg, 2.20mmol). After 2 hr, acetone (10ml)
was added followed by water 910ml) and NaI04 (2.95g, 13.8mmol). The solution
was stirred at room temperature overnight followed by the addition of water
(200ml), extraction with CHC13 and flushing through a silica gel plug. After
evaporation of the solvent, 1.5g of ketone [10] was obtained as a white solid with
90% yield.MS (ESI) m/z (M+H+)303.
Step 2: A solution of chromium trioxide (0.364 g, 3.64mmol) in water (lmL) was
added drop wise to a solution of compound [10] ( 1 g, 3.31mmol) in acetic acid
(30ml). The reaction was stirred for 1.5 hr and quenched by the addition of
methanol (30mL) while stirring for 0.5 hr. The mixture was concentrated by
rotovap at 70°C, dissolved in ethyl acetate/methanol, washed with saturated sodium
bicarbonate and brine, dried over sodium sulfate and concentrated to give
compound [11] as an off white solid (0.9g) with 90% yield.MS (ESI) m z (M+H+)
301.
Step 3: To a solution of [11] (0.9g, 3.0mmol) in tert-butanol (10ml), anhydrous
sodium carbonate (0.509g, 4.8mmol) and water (1ml) were added and the
reaction mixture was refluxed. A solution of sodium periodate (5.3g, 24.9mmol)
and potassium permanganate (66 mg, 0.42mmol) in water (14ml) was added
dropwise to the refluxed reaction mixture and refluxing was maintainedfor an
additional 3hr. The precipitate thus formed was filtered, washed with water. The
filtrate was acidified with 5M HC1 and extracted with dichloromethane. The
organic phase was dried with anhydrous Na2S04, filtered and filtrate was
evaporated. The crude product was purified by silica gel column chromatography
(MeOH:DCM 3:1) to obtained compound [12] as a yellow solid (320mg) with
33% of yield.MS (ESI) m/z (M+H+)321.
Step 4: To a mixture of compound [12] (320mg, l.Ommol) in acetic acid (4ml) was
added ammonium acetate (231mg, 3.0mmol) and refluxed for 4hr. The reaction
mixture was evaporated to remove acetic acid and washed with water, neutralized
with saturated bicarbonate solution and extracted with dichloromethane. The
organic layer was separated and dried over anhydrous Na2S04, filtered and filtrate
was evaporated under vacuum to obtained crude product. The crude product was
purified by silica gel column chromatography (2% MeOH: DCM as an eluent) to
give [13] as a light yellow solid (130mg) with 43% yield. MS (ESI) m/z
(M+H+)302.
Step 5: A mixture of [13] (130mg, 0.43mmol), p-toluenesulfonic acid monohydrate
(13mg), and ethylene glycol (1.3ml) in benzene (100ml) was refluxed with
azeotropic removal of water for 8 hr. The mixture was diluted with ethyl acetate,
washed with saturated sodium bicarbonate solution and brine, dried over sodium
sulfate and concentrated by rotovap to obtain compound [14] as a light yellow solid
(130mg) with 87% yield.MS (ESI) m/z (M+H+)346.
Step6: A solution of compound [14] (50mg, 0.14mmol) in 7:3 ethanol/THF (10
ml) was added sodium borohydride (llmg, 0.28mmol) at 0°C. The reaction was
stirred in an ice bath for 8 hr. The reaction was quenched with water (5ml) and
concentrated under vacuum until solid appears. The solid residue was purified by
silica gel column chromatography (2% MeOH:DCM as an eluent) to give
compound [1006] as a white solid (40mg, Yield 80%). MS (ESI) m/z (M+H+)348.
Step7: A solution of compound [15] (40 mg, O.llmmol) in acetone (7 ml) and
water (3 ml) was treated with concentrated sulfuric acid (0.1 ml) at 0°C. The
mixture was allowed to stirred for 2hr. Reaction mixture was diluted with ethyl
acetate and washed with saturated sodium carbonate solution and brine, dried over
sodium sulfate and purified purified by silica gel column chromatography eluting
with 2- 3% MeOH: DCM to give compound [1005] as a white solid (17mg, 50%
yield). MS (ESI) m/z (M+H+) 304.
Step 8: A solution of compound [14] (40mg, 0.13mmol) in ethanol (10 ml) was
added sodium borohydride (lOmg, 0.26mmol) at 0°C. The reaction was stirred in
an ice bath for 8 hr. The reaction was quenched with water (5ml) and concentrated
under vacuum until solid appears. The solid residue was purified by silica gel
column chromatography (2% MeOH:DCM as an eluent) to give compound [1008]
as a white solid (30mg, Yield 75%). MS (ESI) m/z (M++l)306.
Step9: To a stirred solution of compound [14] (50mg, 0.14mmol) in dry THF
(10ml) was added MeLi dropwise (0.06ml, 0.18mmol) at 0°C and allowed for 6hr
at room temperature. The reaction mixture was quenched with saturated solution of
ammonium chloride and extracted with ethyl acetate. Organic phase dried over
sodium sulfate, filter and filtrate was evaporated. The crude residue was purified by
silica gel column chromatography eluting with 2%-3% MeOH: DCM to give
compound [1006] as a light yellow solid (40mg, 76% yield).MS (ESI) m/z (M+H+)
362
SteplO: A solution of compound [1006] (40 mg, O.llmmol) in acetone (7ml) and
water (3ml) was treated with concentrated sulfuric acid (0.1ml) at OoC. The
mixture was allowed to stirred for 2hr. Reaction mixture was diluted with ethyl
acetate and washed with saturated sodium carbonate solution and brine, dried over
sodium sulfate and purified purified by silica gel column chromatography eluting
with 2% - 3% MeOH:DCM to give compound [1007] as a light yellow solid
(18mg, 51% yield). MS (ESI) m/z (M+H+) 318.
Example 7bPreparation of compounds of the present invention
Stepl: To a solution of [16] (3g, 9.0mmol) in tert-butanol (30ml), anhydrous
sodium carbonate (1.65 g, 28.09 mmol) and water (3 ml) was added and the
reaction mixture was refluxed. Solutions of sodium periodate (17.1, 50.0mmol) and
potassium permagnate (0.2 lg, 4.2mmol) in water (30ml) was dropwise added to
refluxed reaction mixture. The reaction mass was refluxed for an additional 3hr.
The precipitate was filtered washed with water and the filtrate was acidified with
5M HC1 and extracted with dichloromethane. The organic phase was dried with
anhydrous Na2S04, filtered and filtrate was evaporated. The crude product was
purified by silica gel column chromatography (MeOH:DCM 3:1) to obtain
compound [17] as a yellow solid (1.8g) with 56% of yield.MS (ESI) m z
(M+l)349.
Step2: To a mixture of compound [17] (1.8mg, 5.17mmol) in acetic acid (5ml) was
added ammonium acetate (1.19g, 15.5mmol) and refluxed at 130°C for 4 hr. The
reaction mixture was evaporated to remove acetic acid and washed with water,
neutralized with saturated and extracted with dichloromethane. The organic portion
dried over Na2S0 4, filter and filtrate was evaporated under vacuum. The crude
product was purified by silica gel column chromatography (2% MeOH: DCM as a
eluent) to give compound [18] as a light yellow solid (1.3 g) with 76 % yield. MS
(ESI) m/z (M+l) 330.
Step3: A mixture of Compound [18] (0. 300 g, 0.91mmol), p-toluenesulfonic acid
monohydrate (13mg), and ethylene glycol (2.5ml) in benzene (250ml) was refluxed
with azeotropic removal of water for 8 hr. The mixture was diluted with ethyl
acetate, washed with saturated sodium bicarbonate solution and brine, dried over
sodium sulfate and concentrated under vacuum to obtain compound [19] as a light
yellow solid (0.336g) with 99% yield.MS (ESI) m/z (M+l)374.
Step4: To a stirred solution of compound [19] (O.lg, 0.268mmol) in dry THF
(100ml) was added MeMgBr dropwise (0.134ml, 0.40mmol) at 0°C and allowed
for 6 hr at room temperature. The reaction mixture was quenched with saturated
solution of ammonium chloride and extracted with ethyl acetate. Organic phase
dried over sodium sulfate, filter and filtrate was evaporated. The crude residue was
purified by silica gel column chromatography eluting with 2-3% MeOH: DCM to
give compound [1026] as a light yellow solid (0.50g, 48 % yield).MS (ESI) m/z
(M+H+)390
Step5: A solution of compound [1026] (0.05g, 0.128mmol) in acetone (10ml) was
treated with concentrated sulfuric acid (0.05ml) at 0°C. The mixture was allowed to
stirred for 2 hr. Reaction mixture was diluted with ethyl acetate and washed with
saturated sodium carbonate solution and brine, dried over sodium sulfate and
purified purified by silica gel column chromatography eluting with 2-3%
MeOH:DCM to give compound [1008] as a light yellow solid (20 mg, 45% yield).
MS (ESI) m/z (M+H+)346.
Step 6: Compound [1025]was synthesized using the same procedure as mentioned
in step 6 example7a .Yield 80%. MS (ESI) m/z (M+l)376.
Step 7: Compound [1037]was synthesized using the same procedure as mentioned
in step 5 example7b. Yield 70%. MS (ESI) m/z (M+l)332.
Step 8: Compound [1038]was synthesized using the same procedure as mentioned
in step 4 example7b. Yield 75%. MS (ESI) m/z (M+l)348.
Example 7c Preparation of compounds of the present invention
Step- 1 : To a stirring solution of compound [13] (0.4 gm, 1.32 mmol) in EtOH (10
ml), NaBH4 (0.05 gm, 1.32 mmol) was added portion wise. Reaction was stirred
for additional 1 hour and progress of the reaction was monitored by TLC. After
completion of reaction, 10 ml of acetone was added followed by addition of water
(20 ml). Organic layer was extracted with ethyl acetate. Organic layer was dried
over sodium sulfate and evaporated to get crude compound [70](yield 90%), MS
(ESI) m/z (M+l) 304.
Step- 2:Compound [70] (0.065 mg, 0.165) was dissolved in DCM, followed by
addition of DAST (0.04 mg, 0.247) and stirred for one hour. Reaction was
monitored by TLC and was diluted with water after completion. Organic layer was
extracted with ethyl acetate and dried over sodium sulfate followed by evaporation
under reduced pressure to get crude compound [71] (yield 75%), MS(ESI) m/z
(M+l) 306.
Step-3: To a solution of compound [71](0.5 gm, 1.65 mmol) in EtOH (10 ml),
NaBH4 (0.31 gm, 8.27 mmol) was added and reaction mixture was heated at 40°C
for 18 hrs. Reaction was monitored by TLC and quenched with 10 ml of acetone
followed by a dilution with water. Organic layer was extracted with ethyl acetate
and dried over sodium sulfate followed by evaporation to get crude compound
[1020]as white solid (yield 80%), MS (ESI) m/z (M+l)308
Step:4 Compound [1019]was synthesized using the same procedure as mentioned
in step 3 example7c at elevated temperature. Yield 80 MS (ESI) m/z (M+1)306
Step:-5 Compound [1023](50 mg) was dissolved in EtOH andPd/C (5 mg) was
added. Reaction was stirred under hydrogen pressure for 3 hrs to
completion.Progress of the reaction was monitored with TLC. After completion
reaction masswas filtered over celite layerand excess of solvent was evaporated
under reduced pressure to get compound 1023 (yield 80%).MS (ESI) m/z (M+l)
308
Step 6: To a solution of compound [1023] (0.2 gm, 0.651mmol) in THF, was
added LAH (85 mg, 2.28 mmol) and reaction mixture was stirred for 12 hrs.
Progress of the reaction was monitored by TLC. Reaction was diluted with water
and extracted with ethyl acetate. Organic layer was dried over NaS04 and
evaporated to get compound [1023-1']- Yield 85%.MS (ESI) m/z (M+l) 294
Example 7dPreparation of compounds of the present invention
Step:-lTo a solution of compound [13] (0.1 gm, 0.33 mmol) in DMF (2ml), was
added NaH (O.Olgm, 0.33mm) at 0°C. Reaction mixture was stirred for 20 min at
same temp,followed by addition of Mel (0.05 ml,0.33mmol). Reaction mass was
stirred for additional 2-3 hrs at RT and progress of the reaction wasmonitored by
TLC. After completion, reaction was quenched with water and extracted with ethyl
acetate (100 X2). Organic layer was dried over anhy sodium sulfate, and
evaporated under vacuum, to get compound [74] (yield 90%).
MS (ESI) (M+l)316.
Step 2: Compound [1022] was synthesized using the same procedure as mentioned
in example 2b. Yield 80% . MS (ESI) m/z (M+l) 320.
Step 3:Compound [13] (0.5g, 1.65 mmol) was taken in MeOH :water (20 mL).
Hydroxylamine hydrochloride (0.24 g, 3.46 mmol) was added to this solution
followed by addition of sodium acetate (0.29 g, 3.63). The mixture was heated at
60°C for 2 hrs. The solvent was evaporated and crude mass was taken in water (50
ml), extracted with ethyl acetate (3 X 100 ml). The organic layer was separated,
dried over anhydrous Na2S0 4, filtered and concentrated to obtain compound as off
white solid (0.2 g, 95%).MS (ESI) m/z (M+l)317.
Step 4: Compound 1021 was synthesized using the same procedure as mentioned
in step 4 example 7c. Yield 80%. MS (ESI) m/z (M+l)319.
Step-5: The compoundl021 (0.2 g. 0.627 mmol) was taken in MeOH (50ml). Pd/C
(0.030 g) was added and reaction was stirred at room temperature under H2 atm
(60 psi, Parr shaker). After completion, the excess of solvent was filtered,
concentrated to obtain compound [1024] as light brown solid (0.17g, 88%).MS
(ESI) m/z (M+l)307.
Example 7e:Preparation of compounds of the present invention
Step 1: The compound [22] was synthesized in same procedure as in example 6
above; Yield 69 %, MS (ESI) m/z (M-l)349.
Step 2:The compound [1010] was synthesized in same procedure as in example 6;
Yield 31%, MS (ESI) m/z (M+l)334.
Step 4: Compound [1027]was synthesized using the same procedure as mentioned
in step 4 example 7c. Yield 76%; MS (ESI) m/z (M+1)334.
A few illustrative examples of this series synthesized based on example 7a, 7b and
7c include 1005, 1008,1017, 1018, 1020 - 1023, 1026, 1064, 1065
Example 8: Preparation of compounds of the present invention
Step 1: Compound [22] was synthesized in same procedure as in example 6 above;
Yield 69 %, MS (ESI) m/z (M-l)351.
Step 2: Compound [1010] was synthesized in same procedure as in example 6;
Yield 31%, MS (ESI) m/z (M+l)332.
ation of compounds of the resent invention
[1040] [41 ]
Step 1: A solution of chromium trioxide (3.3 g, 33.3 mmol) in water (4 mL) was
added dropwise to a solution of [1040](10 g, 30.3 mmol) in acetic acid (300 ml).
The reaction was stirred for 1.5 hr and quenched by the addition of methanol (30
mL), stirring for 0.5 hr. The mixture was concentrated by rotovap at 70 °C,
dissolved in ethyl acetate/methanol, washed with saturated sodium bicarbonate and
brine, dried over sodium sulfate and concentrated to give 17-acetyl-10,13-
dimethyl-l,6,7,8,9,10,12,13,14,15,16,17-dodecahydro-2Hcyclopenta[
a]phenanthrene-3,ll-dione as an off white solid (9.0 g).
Step 2
A solution of 70% formic acid (10 ul) in ethyl acetate (10 ml) is added to a solution
of acetic anhydride (4.8 mL) in ethyl acetate (30 ml) and the final volume is
brought up to 50 ml. [41] (500 mg, 1.5 mmol) was added and the reaction was
stirred for 7 min then washed with saturated sodium bicarbonate, dried over sodium
sulfate, and concentrated by rotovap at 70 °C. The resulting oil was dissolved in
methanol (10 ml), treated with pyridine ( 1 ml) and concentrated by rotovap to an
oil which was dissolved in ethyl acetate, washed with 10% citric acid, saturated
sodium bicarbonate, and brine, dried over sodium sulfate and concentrated to give
the acetate diene as an oil which solidified on standing. A mixture of
toluenesulfonic acid hydrate (110 mg) and ethylene glycol (2 ml) in benzene (25
ml) was refluxed with removal of water for 0.5 hr. A solution of the acetate diene
in benzene (12 ml) was added and the reaction was refluxed for 1.5 hr. The mixture
was diluted with ethyl acetate, washed with saturated sodium bicarbonate and
brine, and dried over sodium sulfate. The crude product was purified by silica gel
chromatography eluting with 20% to 30% ethyl acetate/hexanes to give acetic acid
[85]as a white crystalline solid (420 mg).66% yield, and MS (ESI) m z (M+l) 415.
Step 3
A chilled solution of acetic acid [85] (1.59 g) in 7:3 ethanol/THF (125 ml) was
added dropwise to an ice cold suspension of sodium borohydride (2.5 g) in 80%
ethanol (50 ml). The reaction was stirred in an ice bath for 8 hr then overnight
allowing the ice bath to melt slowly. The reaction was quenched with 10% acetic
acid and concentrated by rotovapuntil solid forms. The solid is filtered, washed
with water and dried to constant weight to give [86] as a white solid (797 mg). A
second crop is obtained by further concentrating the mother liquor (228 mg). MS
(ES-API) m/z (M+H+)377.8.
Step 4:
[86] (570 mg) and pyridinium p-toluenesulfonate (60 mg) were dissolved in 95%
acetone (40 ml) and stirred overnight at RT. The reaction mixture was concentrated
by rotovap until a solid appeared, filtered, washed with water and dried to constant
weight to give as a white solid[1039] (319 mg). The mother liquor was diluted
with ethyl acetate, washed with citric acid and brine, dried over sodium sulfate and
concentrated to give a second crop as a white solid (140 mg). 91% yield, MS (ESI)
m/z (M+l)353.
Example 10: Preparation of compounds of the present invention
Step 1:A solution of chromium trioxide (3.3 g, 33.3 mrnol) in water (4 mL) was
added dropwise to a solution of [90](10 g, 30.3 mmol) in acetic acid (300 ml). The
reaction was stirred for 1.5 hr and quenched by the addition of methanol (30 mL),
stirring for 0.5 hr. The mixture was concentrated by rotovap at 70 °C, dissolved in
ethyl acetate/methanol, washed with saturated sodium bicarbonate and brine, dried
over sodium sulfate and concentrated to give [41]as an off white solid (9.0 g).
Step 2: A mixture of [41](400 mg), toluenesulfonic acid hydrate (110 mg) and
ethylene glycol (2 ml) in benzene (25 ml) was refluxed with removal of water for
0.5 hr. A solution of the acetate diene in benzene (12 ml) was added and the
reaction was refluxed for 1.5 hr. The mixture was diluted with ethyl acetate,
washed with saturated sodium bicarbonate and brine, and dried over sodium
sulfate. The crude product was purified by silica gel chromatography eluting with
20% to 30% ethyl acetate/hexanes to give pure product [91] as a white crystalline
solid (420 mg). M/Z 417
Step 3:A chilled solution of [91] (0.59 g) in 7:3 ethanl/THF (125 ml) was added
dropwise to an ice cold suspension of sodium borohydride (2.5 g) in 80% ethanol
(50 ml). The reaction was stirred in an ice bath for 8 hr then overnight allowing the
ice bath to melt slowly. The reaction was quenched with 10% acetic acid and
concentrated by rotovap until a solid forms. The solid is filtered, washed with water
and dried to constant weight to give pure [92] as a white solid (497 mg). MS(ESAPI)
m/z (M+H+)419.
Step 4 : A solution of compound [92] (0.25g) in acetone (10ml) was treated with
concentrated sulfuric acid (0.05ml) at 0°C. The mixture was allowed to stirred for 2
hr. Reaction mixture was diluted with ethyl acetate and washed with saturated
sodium carbonate solution and brine, dried over sodium sulfate and purified
purified by silica gel column chromatography eluting with 2-3% MeOH:DCM to
give [1040]as a light yellow solid (0.180 mg). MS (ESI) m/z (M+H+)331
Example 11: Biological testing of the compounds of the present invention.
The compounds of the present invention were tested for their activity on AMP
KINASE. The activity on AMP KINASE was evaluated by quantitative fluorescent
immunoenzymatic assay of AMP KINASE phosphorylation status in cultured cells.
The 5-AMP-activated protein kinase (AMP KINASE) is a key sensor of
intracellular energy balance. AMP KINASE is activated in response to an increase
in the AMP/ATP ratio which can be caused by a number of factors such as muscle
contraction, starvation, or hypoxia. AMP KINASE is a heterotrimeric protein
complex comprising of a- (63 kDa), b- (38 kDa) and g - (38 kDa) subunits. For each
subunit, isoforms have been identified (al, a2, bΐ , b2, g ΐ , g2, g3) which
theoretically allow the formation of 12 different proteins. The a-subunit contains a
serine/threonine kinase domain and the regulatory subunits contain binding sites
for AMP and ATP (g -subunit) and for glycogen (b-subunit). AMP KINASE is
activated by phosphorylation on Thr-172 within the catalytic domain. AMP binding
results in a 2 to 5-fold increase in AMP KINASE activity compared to the basal
level. Binding of AMP to the g -subunit causes allosteric activation of the kinase
and induces a conformational change in the kinase domain that protects AMP
KINASE from dephosphorylation of Thr-172.
BioAssay Systems' cell-based ELISA measure phosphorylated AMP KINASE in
whole cells and normalizes the signal to the total protein content. The antibody
recognizes both a-subunits and, thus, can be used for cells from all tissues (human,
mouse, rat). This simple and efficient assay eliminates the need for cell lysate
preparation and can be used to study AMP KINASE regulation in short-term and
long-term assays. In this assay, cells grown in 96-well plates are fixed and
permeabilized in the wells. AMP KINASE phosphorylation (pAMPK) is measured
using a fluorescent ELISA followed by total protein measurement in each well.
A few illustrative compounds of the present invention were tested at IOhM and the
AMPKinase activation of the compounds is listed at Table2:
Table 2: AMP KINASE activation of the compounds of the present invention
Biological data
From Table 2, it may be seen that the compounds of the present invention are
capable of activating AMPKinase,
Example 12: Biological activity of [1039] and [1040] in 3-(4,5-Dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
6,000 BCAEC were seeded into clear 96-well flat bottom plates at 100 mΐ per well.
After starvation, cells were treated for 48 hours with the corresponding compounds.
Compounds were changed every 12 and 24 hours (with 100 mΐ per well of fresh
medium). Following the treatment medium was aspirated from plates, and washed
once with 100 mΐ HBSS, subsequently 100 mΐ per well 0.5 mg/ml MTT in phenol
red-free DMEM was added. Plates were incubated for 1 hour at 37 oC, and this was
followed by aspiration of MTT solution, addition of 100 mΐ per well DMSO to
dissolve formazan crystals, and incubation at 37°C for 15 minutes. Mitochondrial
function was quantified by absorbance was measured at 540 nm with background
substraction at 670 nm using. After MTT absorbance measure, media was removed
and cells quantify in each well as described elsewhere (Oliver et al, 1989) with
some modifications. In brief, 100 mΐ of 10 % formol saline to each well was added,
and incubated at 4°C for 24 hours. Next, formol solution was removed from the
wells and 100 mΐ of filtered 0.1 % (w/v) Methylene Blue in 0.01 M borate buffer
(pH 8.5) was added to each well. After 30 minutes, excess dye was removed and
washed off 3 times with 0.01 M borate buffer (pH 8.5). The stained dye on the cells
was eluted by the addition of 100 mΐ of 1:1 (v/v) ethanol and 0.1 M-HC1 in each
well. The plates were then gently shaken and incubated for 15 minutes under a
bench rocker. Absorbance was measured at 650 nm. The MTT/Methylene Blue
ratio was used to quantify the mitochondrial function. Oliver MH, Harrison NK,
Bishop JE, Cole PJ, Lauren GJ. A rapid and convenient assay for counting cells
cultured in microwell plates: application for assessment of growth factors. J Cell
Sci. 1989,3:513-8.
Slot Blot Method. Following treatment, bovine aortic endothelial cells grown in 12
well plates are scrapped in 50 mΐ of lysis buffer (1% Triton X-100, 20 mm Tris, 140
mm NaCl, 2 mm EDTA, and 0.1% SDS) with protease and phosphatase inhibitor
cocktails (P2714 and P2850, Sigma-Aldrich) supplemented with 0.15 mm PMSF, 5
mm Na3V0 4 and 3 mm NaF. Homogenates are passed through an insulin syringe
five times, sonicated for 30 min at 4°C and centrifuged (12,000 g) for 10 min. The
total protein content is measured in the supernatant using the Bradford method. A
total of 10-20 mg of protein are loaded into a pre-assembled slot blot apparatus to
be transferred under vacuum onto polyvinyl membranes, incubated for 1 h in
blocking solution (5% non-fat dry milk in TBS plus 0.1% Tween 20 (TBS-T)),
followed by a overnight incubation at 4° C with primary antibodies (oxidative
phosphorylation complex I or GAPDH). Primary antibodies are diluted in TBS-T
plus 5% non-fat dry milk. Membranes are washed (3 X for 5 min) in TBS-T and
incubated 1 h at room temperature in the presence of HRP-conjugated secondary
antibodies diluted in blocking solution. Membranes are again washed 3 times in
TBS-T, and the immunoblots developed using an ECL Plus detection kit
(Amersham-GE). Band intensities are digitally quantified using ImageJ software
(http://www.nih.gov). Complex I values are normalized for loading differences
using GAPDH after stripping and reprobing of the membrane. All Western blot
images to be obtained will be within the linear range of X-ray film to ensure
computer-assisted densitometry accuracy. The result is provided at Table 3.
Table 3: Biological Activity of [1039]
*NT - Not Tested
Example 13: AMP KINASE activation of the compounds [1039] and [1040]
AMP KINASE activation potential of the compounds was evaluated using cell
based ELISA.Hepatoma (Hep G2) liver cells were maintained in a T 75 culture
flask-containing 25 mM DMEM+10% fetal calf serum. The cells were maintained
in a T 75 culture flask-containing medium (DMEM+10% fetal calf serum). On
reaching a confluence of 70 to 80%, the cells were seeded in a 96 well plate at a
density of 40,000 cells per well in 25mM DMEM+10% FCS medium. The plates
were then incubated at 37° C. with 5% C ¾ for 24 hours. Various concentrations of
drugs were prepared in DMSO and diluted to required concentration with the
medium and incubated at 37° C. with 5% C ¾ for 30 min and lh for Epicatechin
analogs and 11-BHP analogs respectively. Metformin was used as positive control
.Cells were fixed with 4% formaldehyde for 30 minutes at room temperature and
washed three times with PBS containing 0.1% Triton X-100. Endogenous
peroxidase was quenched with 1 % ¾ ¾ in PBS-T (0.1% Tween 20) for 30
minutes and washed three times in PBS-T. Cells were blocked with 1% BSA in
PBS-T for 1 hour. The cells were incubated with 1:1000 dilution primary antibody
(Phospho-AMRKa (Thrl72) Rabbit mAb, Cell Signaling in PBS-T containing 5%
BSA at 4° C overnight. The cells were then washed three times with PBS-T for 5
minutes and incubated with 1:1000 dilution secondary antibody (Anti-rabbit IgG,
HRP-linked Antibody, Cell Signaling) in PBS-T with 1% BSA for 1 hour at RT.
Cells were washed three times with PBS-T for 5 minutes The cells were incubated
with 100 mΐ TMB substrate solution for 30minutes and the reaction was stopped
with 100 mΐ of 2N H2S0 4. Then the plate was read at 450 nM using ELISA plate
reader and absorbance recorded.% activity was calculated using DMSO control as
100%. The result is provided at Table 4.
Table 4: Activation of AMP KINASE after 30 mins, over baseline of 100.
Example 14: Effects of [1039] on human coronary artery endothelial cell
mitochondrial oxidative phosphorylation complex I protein levels.
Cells were grown in Dulbecco's modified Eagle's media supplemented with fetal
bovine serum (10%) to -90% confluence. Cells were treated with either vehicle
(water) or incremental doses of the agent (dissolved in water) for a total of 48 h
(treatment was repeated at the 24 h time point). At the end of the experiment, cells
were lysed in Western blot sample buffer. A fixed amount of protein (25
micrograms) was loaded onto a polyacrylamide gel and run for 1 h to allow for the
electrophoretic separation of proteins. The gel was then transferred onto a
polyvinyl membrane for incubation with blocking buffer prior to exposure to the
lry antibody (anti-human complex I). Following a 1 h incubation at room
temperature the membrane was washed and then exposed to the 2ry antibody for 1
h. after a second set of washes the membrane was incubated with an enhancedchemiluminescence
kit solution followed by exposure to X-ray film. The image
obtain was used to quantitative changes in complex I protein levels as a function of
compound dose. To normalize for loading differences the membrane was reincubated
(as above) with the subunit 6 ribosomal protein (S6RP) and exposed to
X-ray film. Changes in values for complex I levels are normalized to differences in
S6RP levels. All data are normalized to the values noted for cells treated with
vehicle only (=100%). The results are presented at Figure 1 a. From the figure, it
can be clearly seen that compound [1039] causes mitochondrial biogenesis.
The OHP (another depiction of [1039]) effects on mRNA levels of genes involved
in mitochondrial biogenesis PPARGCla -PGCla-, TFAM, and subunits 1 and 2 of
cytochrome oxidase. HepG2 cells were exposed to increasing amounts of OHP (in
uM) for 3 hours and then harvested for mRNA isolation (RNeasy kit, QIAGEN).
mRNA were converted to cDNA (iScript, Bio-Rad) and amount of gene expression
analyzed by real time PCR employing specific primers and using actin levels as
the housekeeping gene. Data was plotted with graphpad Prism4 and analyze by
ANOVA with ad hoc Tukey comparison between all columns (*P<0.05 and
**P<0.01). The results are presented at Figure lb. From the figure, it can be
clearly seen that compound [1039] expresses mitochondrial transcription factors as
early as 3 hours of exposure.
Example 15: Activation of AMP Kinase by [1039]
OHP effects on AMPKinase activation in HepG2 cells. HepG2 cells were exposed
to increasing amounts of OHP (in uM) for 1 hour and then harvested for protein
isolation employing a triton X-100 based lysis buffer containing phosphatase
inhibitors (Phosstop, Roche). Protein levels were quantified (BCA assay) and 40
ug of protein loaded for detection of total AMPK, phosphorylated (active) AMPK,
total and phosphorylated acetyl CoA carboxylase (ACC, AMPK target gene) and
actin loading control. (RNeasy kit, QIAGEN). OHP effects on APPLl nuclear
translocation. HepG2 cells were exposed to increasing amounts of OHP (in uM) for
1 hour and nuclear and cytosolic fractions isolated (Biovision nuclear/cytosolic
fractionation kit) and total protein quantified (BCA assay).40 ug of protein loaded
for detection of total APPLl, phosphorylated (active) AMPK, tubulin (a marker of
the cytosolic fraction) and lamin (a marker of the nuclear fraction). The results are
presented at Figure 2a and 2b. From the figures, it can be seen that [1039]
activates AMPkinase, even after 1 hour of exposure and activates APPLl and
causes its translocation to the nucleus.
Example 16: Activity of [1039] in mitochondria
OHP effects on mRNA levels of genes involved in mitochondrial biogenesis Nrf1
and TFB2M. HepG2 cells were exposed to increasing amounts of OHP (in uM) for
3 hours and then harvested for mRNA isolation (RNeasy kit, QIAGEN). mRNA
were converted to cDNA (iScript, Bio-Rad) and amount of gene expression
analyzed by real time PCR employing specific primers and using actin levels as the
housekeeping gene. HepG2 cells were exposed to increasing amounts of OHP (in
uM) for 24 hours and then harvested for DNA isolation (Promega, Wizard DNA
Isolation). Amount of DNA obtained quantified and analysis of mitochondrial
DNA (a marker of mitochondrial mass) determined by measuring the number of
copies of a mitochondrial gene codified by mitochondrial DNA (Cytochrome C)
versus the number of copies of a mitochondrial gene codified by nuclear DNA
(pyruvate dehydrogenase). Data was plotted with graphpad Prism4 and analyze by
ANOVA with ad hoc Tukey comparison between all columns (*P<0.05 and
**P<0.01). The results are depicted at Figures 3a and 3b. From the results, it can
be seen that compound [1039] stimulates the expression of mitochondrial
transcription factors and increases the DNA content of mitochondria.
Example 17: Effect of [1039] on mitochondrial oxidative stress
HepG2 cells were exposed to vehicle or OHP (10 nM and mitochondrail
superoxide production determined by confocal microscopy using the mitosox
determination kit (molecular probes), intensity data was plotted withgraphpad
Prism4 and analyze by ANOVA with ad hoc Tukey comparison between all
columns. See Figure 4. From figure 4, it is clear that [1039] reduces superoxide
generation in mitochondria.
Example 18: Effect of [1039] on hepatic gluconeogenesis and fat oxidation.
HepG2 cells were exposed to serum-free media for 3 hours and then exposed to
gluconeogenic factors (lactate and pyruvate 10:1 ratio) and stimulators (cAMP) in
the presenceof increasing concentrations of OHP (in nM). Intracellular glucose
production -a marker of de novo gluconeogenesis- was determined enzimatically
(biovision glucose determination kit), as well as intracellular beta-hydroxybutyrate
levels (a marker of fat oxidation) and triglycerides (substrate for fat oxidation).
Data was plotted withgraphpad Prism4 and analyze by ANOVA with ad hoc Tukey
comparison between all columns (*P<0.05 and **P<0.01). The results are depicted
at Figure 5. From Figure 5, It can be clearly seen that [1039] reduces hepatic
gluconeogenesis and stimulates oxidation of fat.

CLAIMS:
1. Hydroxysteroid compounds and their intermediates of formula (I):
or a salt thereof,wherein:
is independently either a single bond, a double bond or a cyclopropyl ring,
provided that adjacent double bonds are not allowed;
Ai, A2 A3 are independently selected from the group comprising hydrogen, hydroxyl,
hydroxymethyl, halogen, Ci-C alkyl.
B and C are each independently selected from the group comprising hydrogen, hydroxyl,
halogen, -OR6„ -COR6,-COOR6,OCOR6, CH2OH, CH2OR6, -CONHR6, CONHR6R , , -
C(OH)R R , NHR , NHR5CONHR , - R NHCOOR , -NR6R , C(0)heteroaryl,
C(0)heterocyclyl, Ci-Ci 2 straight or branched chain alkyl, 5-6 membered
heterocycloalkyl, 5-6 membered heteroaryl;
wherein the Ci-Ci 2 straight or branched chain alkyl or 5-6 membered heterocycloalkyl or
5-6 membered heteroaryl; may be further optionally substituted with one or more
substituents selected from the group comprising halogen, Ci-C alkyl, -OR , -COOR ,-
CONHR6, -OCOR6, =NOH, NR6R , -NR6COR , 5-6 membered heterocycloalkyl or 5-6
membered heteroaryl; Ci-Ci 2 alkyl substituted 5-6 membered heterocycloalkyl or Ci-Ci 2
alkyl substituted 5-6 membered heteroaryl;
or B and C may combine together to form =0, =NOR , NHR , 5-6 membered
heterocycloalkyl, or 5-6 membered heteroaryl;
wherein the said 5-6 membered heterocycloalkyl, 5-6 membered heteroaryl may
optionally contain one or more heteroatom;
wherein, the heteroatom may be O, N, S;
Ri, R2, and R 4 are independently selected from the group comprising hydrogen,
deuterium, hydroxyl, halogen, =0, -OR6, -NR6R , -COR6, -COOR6,-OCOR6,-CONR6R ,
Ci-C6 alkyl, -Otert-butyldimethylsilyl;
R3 is hydroxyl, carbonyl, OCOR6;
wherein R 3 is in beta configuration
R 5 is selected from hydrogen, hydroxyl, halogen, OR ;
R and R7are each independently selected from the group comprising hydrogen, halogen,
hydroxyl, C1-C12 alkyl, -NH2, -(CH2) NH2, 3-6 membered cycloalkyl, 4-6 membered
heterocycloalkyl, 5-6 membered heteroaryl;
X is selected from CH, NH, NR6, O or S.
n is 0 to 3.
2. Compounds as claims in Claim 1 of formula (II):
FORMULA II
or a salt thereof, wherein:
is independently either a single bond, a double bond or a cyclopropyl ring,
provided that adjacent double bonds are not allowed;
Ai, A2 are independently selected from the group comprising hydrogen, hydroxyl,
hydroxymethyl, halogen, Ci-C alkyl.
B and C are each independently selected from the group comprising hydrogen, hydroxyl,
halogen, -OR6„ -COR6,-COOR6,OCOR6, CH2OH, CH2OR6, -CONHR6, CONHR6R , , -
C(OH)R6R , NHR6, NHR5CONHR6, - R6NHCOOR , -NR6R , C(0)heteroaryl,
C(0)heterocyclyl, Ci-Ci 2 straight or branched chain alkyl, 5-6 membered
heterocycloalkyl, 5-6 membered heteroaryl;
wherein the Ci-Ci 2 straight or branched chain alkyl or 5-6 membered heterocycloalkyl or
5-6 membered heteroaryl; may be further optionally substituted with one or more
substituents selected from the group comprising halogen, Ci-C alkyl, -OR , -COOR ,-
CONHR6, -OCOR6, =NOH, NR6R , -NR6COR , 5-6 membered heterocycloalkyl or 5-6
membered heteroaryl; Ci-Ci 2 alkyl substituted 5-6 membered heterocycloalkyl or Ci-Ci 2
alkyl substituted 5-6 membered heteroaryl;
or B and C may combine together to form =0, =NOR , NHR , 5-6 membered
heterocycloalkyl, or 5-6 membered heteroaryl;
wherein the said 5-6 membered heterocycloalkyl, 5-6 membered heteroaryl may
optionally contain one or more heteroatom;
wherein, the heteroatom may be O, N, S;
Ri and R 4 are independently selected from the group comprising hydrogen, deuterium,
hydroxyl, halogen, =0, -OR6, -NR6R , -COR6, -COOR6,-OCOR6,-CONR6R , Ci-C6
alkyl, -Otert-butyldimethylsilyl;
R and R7are each independently selected from the group comprising hydrogen, halogen,
hydroxyl, C1-C12 alkyl, -NH2, -(CH2)nNH2, 3-6 membered cycloalkyl, 4-6 membered
heterocycloalkyl, 5-6 membered heteroaryl;
n is 0 to 3.
3. Compounds as claims in Claim 1, of formula (III):
FORMULA III
or a salt thereof, wherein:
Ai and A2 are independently selected from the group comprising hydrogen, hydroxyl,
hydroxymethyl, halogen, Ci-C alkyl;
B and C are each independently selected from the group comprising hydrogen, hydroxyl,
halogen, -OR6„ -COR6, -COOR6,OCOR6, CH2OH, CH2OR6, -CONHR , CONHR R , , -
C(OH)R R , NHR , NHR5CONHR , - R NHCOOR , -NR6R , C(0)heteroaryl,
C(0)heterocyclyl, Ci-Ci 2 straight or branched chain alkyl, 5-6 membered
heterocycloalkyl, 5-6 membered heteroaryl;
wherein the Ci-Ci 2 straight or branched chain alkyl or 5-6 membered heterocycloalkyl or
5-6 membered heteroaryl; may be further optionally substituted with one or more
substituents selected from the group comprising halogen, Ci-C alkyl, -OR , -COOR ,-
CONHR6, -OCOR6, =NOH, NR6R , -NR6COR , 5-6 membered heterocycloalkyl or 5-6
membered heteroaryl; Ci-Ci 2 alkyl substituted 5-6 membered heterocycloalkyl or Ci-Ci 2
alkyl substituted 5-6 membered heteroaryl;
or B and C may combine together to form =0, =NOR , NHR , 5-6 membered
heterocycloalkyl, or 5-6 membered heteroaryl;
wherein the said 5-6 membered heterocycloalkyl, 5-6 membered heteroaryl may
optionally contain one or more heteroatom;
wherein, the heteroatom may be O, N, S;
R4 is independently selected from the group comprising hydrogen, deuterium, hydroxyl,
halogen, =0, -OR6, -NR6R , -COR6, -COOR6,-OCOR6,-CONR6R , Ci-C6 alkyl, -Otertbutyldimethylsilyl;
R and R7 are each independently selected from the group comprising hydrogen, halogen,
hydroxyl, Ci-Ci 2 alkyl, -NH2, -(CH2)nNH2, 3-6 membered cycloalkyl, 4-6 membered
heterocycloalkyl, 5-6 membered heteroaryl;
n is 0 to 3.
4. Compounds as claims in Claim 1, of formula (IV):
FORMULA IV
or a salt thereof, wherein:
Al and A are independently selected from the group comprising hydrogen, hydroxyl,
hydroxymethyl, halogen, Ci-C alkyl;
B and C are each independently selected from the group comprising hydrogen, hydroxyl,
halogen, -OR6„ -COR6,-COOR6,OCOR6, CH2OH, CH2OR6, -CONHR6, CONHR6R , , -
C(OH)R R , NHR , NHR5CONHR , - R NHCOOR , -NR6R , C(0)heteroaryl,
C(0)heterocyclyl, Ci-Ci 2 straight or branched chain alkyl, 5-6 membered
heterocycloalkyl, 5-6 membered heteroaryl;
wherein the Ci-Ci 2 straight or branched chain alkyl or 5-6 membered heterocycloalkyl or
5-6 membered heteroaryl; may be further optionally substituted with one or more
substituents selected from the group comprising halogen, Ci-C alkyl, -OR , -COOR ,-
CONHR6, -OCOR6, =NOH, NR6R , -NR6COR , 5-6 membered heterocycloalkyl or 5-6
membered heteroaryl; Ci-Ci 2 alkyl substituted 5-6 membered heterocycloalkyl or Ci-Ci 2
alkyl substituted 5-6 membered heteroaryl;
or B and C may combine together to form =0, =NOR , NHR , 5-6 membered
heterocycloalkyl, or 5-6 membered heteroaryl;
wherein the said 5-6 membered heterocycloalkyl, 5-6 membered heteroaryl may
optionally contain one or more heteroatom;
wherein, the heteroatom may be O, N, S;
R4 is independently selected from the group comprising hydrogen, deuterium, hydroxyl,
halogen, =0, -OR6, -NR6R , -COR6, -COOR6,-OCOR6,-CONR6R , Ci-C6 alkyl, -Otertbutyldimethylsilyl;
R and R7 are each independently selected from the group comprising hydrogen, halogen,
hydroxyl, C1-C12 alkyl, -NH2, -(CH2)nNH2, 3-6 membered cycloalkyl, 4-6 membered
heterocycloalkyl, 5-6 membered heteroaryl;
n is 0 to 3.
5. The compounds as claimed in 1, selected from the group comprising :
i. (10R,1 IS, 13S,17S)-2,3,6,7,8,9,10,1 1,12,13, 14,15,16,17-tetradecahydro-l 1-hydroxy-
10,13-dimethyl-3-oxo-lH-cyclopenta[a]phenanthrene-17-carboxylic acid;
ii. (3S,8S,9S,10S,l lS,13S,14S,17S)-hexadecahydro-3,ll-dihydroxy-N,10,13-trimethyl-
1H-cyclopenta[ a]phenanthrene- 17-carboxamide ;
iii. (3S,8S,9S,10S,l lS,13S,14S,17S)-N-(2-aminoethyl)-hexadecahydro-3,ll-dihydroxy-
10,13-dimethyl- 1H-cyclopenta[a]phenanthrene- 17-carboxamide ;
iv. (3S,5R,6R,10R,llS,13S,17S)-hexadecahydro-6-methoxy-10,13-dimethyl-17-(2-
methyl- 1,3-dioxolan-2-yl)- 1H-cyclopenta[a]phenanthrene-3 ,5, 11-triol;
v. (4aR,5S,6aS)-5-hydroxy-4a,6a-dimethyl-4,4a,4b,5,6,6a,9,9a,9b,10-decahydro-lHindeno
[5,4-f] quinoline-2,7(3H, 8H)-dione ;
vi. (4a'R,5'S,6a'S)-5'-hydroxy-4a',5',6a'-trimethyl-3',4',4a',4b',5',6',6a',8',9',9a',9b',10'-
dodecahydrospiro[[l,3]dioxolane-2,7'-indeno[5,4-f]quinolin]-2'(l'H)-one;
vii. (4aR,5S,6aS)-4,4a,4b,5,6,6a,9,9a,9b,10-decahydro-5-hydroxy-4a,6a-dimethyl-lHindeno
[5,4-f] quinoline-2,7(3H, 8H)-dione ;
viii. (4aR,5S,6aS)-7-acetyl-4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-5-hydroxy-4a,5,6atrimethyl-
lH-indeno[5,4-f]quinolin-2(3H)-one;
ix. ( l lS)-7,8,9,ll,12,13,14,15,16,17-decahydro-3,ll-dihydroxy-6Hcyclopenta[
a]phenanthrene-17-carboxylic acid;
x. (4aR,6aS)-2,3,4,4a,4b,5,6,6a,7,8,9,9a,9b,10-tetradecahydro-4a,6a-dimethyl-2,5-
dioxo-lH-indeno[5,4-f]quinoline-7-carboxylic acid;
xi. (17S)-17-acetyl-7,8,13,15,16,17-hexahydro-3-hydroxy-l-methyl-6H-cyclopenta
[a]phenanthren- 11(9H, 12H, 14H)-one;
xii. (13S,17S)-17-acetyl-7,8,13,15,16,17-hexahydro-3-hydroxy-13-methyl-6Hcyclopenta[
a]phenanthren-ll(9H,12H,14H)-one;
xiii. (10R,11S,13S,17S)-11 -hydroxy- 10,13-dimethyl-3-oxo-
6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-
carboxylic acid;
xiv. (1OR, 11S,13S,17S)-11-hydroxy-N,N, 10,1 3-tetramethyl-3-oxo-
6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-
carboxamide;
xv. (1OR, 11S,13S,17S)-17-acetyl- 11-hydroxy- 10, 13-dimethyl-
6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one;
xvi. (1OR, 11S,13S,17S)-11-hydroxy- 17-((R) -1-hydroxyethyl)- 10,13-dimethyl -
6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one;
xvii. (4a'R,5'S,6a'S)-5'-hydroxy-4a',6a'-dimethyl-3',4',4a',4b',5',6',6a',8',9',9a',9b',10'-
dodecahydrospiro[[l,3]dioxolane-2,7'-indeno[5,4-f]quinolin]-2'(l'H)-one;
xviii. (4aR,5R,6aS)-5-hydroxy-4a,5,6a-trimethyl-4,4a,4b,5,6,6a,9,9a,9b,10-decahydro-lHindeno
[5,4-f] quinoline-2,7(3H, 8H)-dione ;
xix. (4aR,5S,6aS)-5,7-dihydroxy-4a,6a-dimethyl-4,4a,4b,5,6,6a,7,8,9,9a,9b,10-
dodecahydro-lH-indeno[5,4-f]quinolin-2(3H)-one;
xx. (4aR,5S,6aS)-7-fluoro-5-hydroxy-4a,6a-dimethyl-4,4a,4b,5,6,6a,7,8,9,9a,9b,10-
dodecahydro-lH-indeno[5,4-f]quinolin-2(3H)-one;
xxi. (4aR,5S,6aS,Z)-5-hydroxy-7-(hydroxyimino)-4a,6a-dimethyl-
4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-lH-indeno[5,4-f]quinolin-2(3H)-one;
xxii. (4aR,5S,6aS)-5,7-dihydroxy-l,4a,6a-trimethyl-4,4a,4b,5,6,6a,7,8,9,9a,9b,10-
dodecahydro-lH-indeno[5,4-f]quinolin-2(3H)-one;
xxiii. (4aR,5S,6aS)-5,7-dihydroxy-4a,6a-dimethyltetradecahydro-lH-indeno[5,4-
f]quinolin-2(3H)-one;
xxiv. (4aR,5S,6aS)-7-amino-5-hydroxy-4a,6a-dimethyltetradecahydro-lH-indeno[5,4-
f]quinolin-2(3H)-one;
xxv. (4aR,5S,6aS,7S)-5-hydroxy-4a,6a-dimethyl-7-(2-methyl-l,3-dioxolan-2-yl)-
4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-lH-indeno[5,4-f]quinolin-2(3H)-one;
xxvi. (4aR,5S,6aS,7S)-5-hydroxy-4a,5,6a-trimethyl-7-(2-methyl-l,3-dioxolan-2-yl)-
4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-lH-indeno[5,4-f]quinolin-2(3H)-one;
xxvii. (4aR,5S,6aS,7S)-5-hydroxy-4a,6a-dimethyl-2-oxo-2,3,4,4a,4b,5,6,6a,7,8,9,9a,9b,10-
tetradecahydro-lH-indeno[5,4-f]quinoline-7-carboxylic acid;
xxviii. ( l lS,13S)-17-(l-hydroxyethyl)-13-methyl-7,8,9,l l,12,13,14,15,16,17-decahydro-6Hcyclopenta[
a]phenanthrene-3, 11-diol;
xxix. 1-((1 IS, 13S)-3,ll-dihydroxy-13-methyl-7,8,9,l 1,12,13,14,15, 16,17-decahydro-6Hcyclopenta[
a]phenanthren- 17-yl)ethanone ;
xxx. ( l lS,13S)-17-(l-hydroxyethyl)-3-methoxy-13-methyl-7,8,9,l 1,12,13,14,15,16,17-
decahydro-6H-cyclopenta[a]phenanthren- 11-ol;
xxxi. (11S,13S)-3-((tert-butyldimethylsilyl)oxy)- 13-methyl- 17-(2-methyl- 1,3-dioxolan-2-
yl)-7, 8,9, 11, 12, 13, 14,15, 16,1 7-decahydro-6H-cyclopenta[a]phenanthren- 11-ol ;
xxxii. ( l lS,13S)-13-methyl-17-(2-methyl-l,3-dioxolan-2-yl)-7,8,9,l 1,12,13,14,15, 16,17-
decahydro-6H-cyclopenta[a]phenanthrene-3,l 1-diol;
xxxiii. (10R,11S,13S,17S)-11,1 7-dihydroxy- 10, 13-dimethyl-
6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one;
xxxiv. (10R,1 IS, 13S)-ll-hydroxy-10,13-dimethyl-7,8,9,10,l 1,12,13, 14,15,16-decahydro-
3H-cyclopenta[a]phenanthrene-3,17(6H)-dione;
xxxv. ( l lS,13S,17S)-4,ll-dihydroxy-l,13-dimethyl-7,8,9,l l,12,13,14,15,16,17-decahydro-
6H-cyclopenta[a]phenanthrene- 17-carboxylic acid;
xxxvi. ( l lS,13S,17S)-13-methyl-7,8,9,l 1,12,13, 14,15,16,17-decahydro-6Hcyclopenta[
a]phenanthrene-3,ll,17-triol;
xxxvii. (4aR,5S,6aS,7S)-7-acetyl-5-hydroxy-4a,6a-dimethyl-4,4a,4b,5,6,6a,7,8,9,9a,9b,10-
dodecahydro-lH-indeno[5,4-f]quinolin-2(3H)-one;
xxxviii. (4aR,5S,6aS,7S)-5-hydroxy-7-(2-hydroxypropan-2-yl)-4a,6a-dimethyl-
4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-lH-indeno[5,4-f]quinolin-2(3H)-one;
xxxix. l-((8S,9S,10R,llS,13S,14S)-3,l l-dihydroxy-10,13-dimethyl-
2,3,4,7,8,9,10,ll,12,13,14,15,16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-17-
yl)ethanone;
xl. (8S,9S,10R,llS,13S,14S)-17-acetyl-ll-hydroxy-10,13-dimethyl-
6,7,8,9,10,1 1,12, 13, 14,15, 16, 17-dodecahydro-lH-cyclopenta[a]phenanthren-3(2H)-
one;
xli. (1OR, 11S,13S,17S)-11-hydroxy- 10,13-dimethyl-3-oxo-
6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthrene-17-
carboxamide;
xlii. (10R,11S,13S,17S)-11 -hydroxy- 17-(hydroxymethyl)- 10,1 3-dimethyl-
6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one;
xliii. (10R,11S,13S,17S)- 17-((dimethylamino)methyl)- 11-hydroxy- 10, 13-dimethyl-
6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one;
xliv. ( l lS,13S,17S)-methyl4,l l-dihydroxy-l,13-dimethyl-7,8,9,l 1,12,13,14,15, 16,17-
decahydro-6H-cyclopenta[a]phenanthrene-17-carboxylate;
xlv. ( l lS,13S,17S)-4,ll-dihydroxy-l,13-dimethyl-7,8,9,l l,12,13,14,15,16,17-decahydro-
6H-cyclopenta[ a]phenanthrene- 17-carboxamide ;
xlvi. ( 1lS,13S,17S)-4,ll-dihydroxy-N,N,l,13-tetramethyl-7,8,9,l 1,12,13,14,15,16,17-
decahydro-6H-cyclopenta[a]phenanthrene- 17-carboxamide;
xlvii. ((l lS,13S,17S)-4,ll-dihydroxy-l,13-dimethyl-7,8,9,l 1,12,13, 14,15,16,17-
decahydro-6H-cyclopenta[a]phenanthren-17-yl)(piperazin-l-yl)methanone;
xlviii. ( l lS,13S,17S)-17-(hydroxymethyl)-l,13-dimethyl-7,8,9,l 1,12,13,14,15,16,17-
decahydro-6H-cyclopenta[a]phenanthrene-4, 11-diol;
xlix. ( l lS,13S,17S)-l l-hydroxy-4-methoxy-l,13-dimethyl-7,8,9,l 1,12,13,14,15, 16,17-
decahydro-6H-cyclopenta[a]phenanthrene-17-carboxylic acid;
1. ( 1lS,13S,17S)-17-((dimethylamino)methyl)-l,13-dimethyl-
7,8,9,11,12,13,14,15,16,1 7-decahydro-6H-cyclopenta[a]phenanthrene-4, 11-diol ;
li. 1-((1 IS, 13S)-ll-hydroxy-3-methoxy-13-methyl-7,8,9,l 1,12,13,14,15, 16,17-
decahydro-6H-cyclopenta[a]phenanthren-17-yl)ethanone;
lii. (E)-l-((llS,13S)-3,l l-dihydroxy-13-methyl-7,8,9,l 1,12,13,14,15, 16,17-decahydro-
6H-cyclopenta[a]phenanthren-17-yl)ethanone oxime;
liii. (E)-l-((llS,13S)-3,l l-dihydroxy-13-methyl-7,8,9,l 1,12,13,14,15, 16,17-decahydro-
6H-cyclopenta[a]phenanthren-17-yl)ethanone oxime;
liv. ( l lS,13S)-17-(2-hydroxypropan-2-yl)-13-methyl-7,8,9,l 1,12,13,14,15, 16,17-
decahydro-6H-cyclopenta[a]phenanthrene-3,ll-diol;
lv. ( l lS,13R)-17-ethyl-13-methyl-7,8,9,ll,12,13,14,15,16,17-decahydro-6Hcyclopenta[
a]phenanthrene-3, 11-diol;
lvi. (8S,9S,10R,llS,13S,14S,Z)-ll-hydroxy-17-(hydroxyimino)-10,13-dimethyl-
6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one;
lvii. (8S,9S, 10R, 11S,13S, 14S)- 17-amino- 11-hydroxy- 10, 13-dimethyl-
6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one;
lviii. (8S,9S, 10R, 11S,13S, 14S)- 11-hydroxy- 10, 13-dimethyl-
6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one;
lix. (8S,9S, 10R, 11S,13S, 14S, 17S)- 11,17-dihydroxy- 10, 13,17-trimethyl-
6,7,8,9,10,ll,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-3-one;
lx. ( l lR,13S,17S)-ll,13,17-trimethyl-7,8,9,ll,12,13,14,15,16,17-decahydro-6Hcyclopenta[
a]phenanthrene-3,ll,17-triol;
lxi. ( 1lS,13S)-13-methyl-7,8,9,l 1,12,13,14,15, 16,17-decahydro-6Hcyclopenta[
a]phenanthrene-3, 11-diol;
lxii. ( l lS,13S,17S)-17-amino-13-methyl-7,8,9,ll,12,13,14,15,16,17-decahydro-6Hcyclopenta[
a]phenanthrene-3, 11-diol;
lxiii. (11S,13S,Z)-3 ,11-dihydroxy- 13-methyl-7 ,8,9,11,12,13,15,1 6-octahydro-6Hcyclopenta[
a]phenanthren- 17(14H)-one oxime ;
lxiv. (4aR,5S,6aS,7S)-5-hydroxy-4a,6a-dimethyl-7-(2-methyl-l,3-dioxolan-2-yl)-
4,4a,4b,5,6,6a,7,8,9,9a,9b,10-dodecahydro-lH-indeno[5,4-f]quinolin-2(3H)-one;
lxv. (4aR,5S,6aS,7S)-5-hydroxy-4a,6a-dimethyl-2-oxo-2,3,4,4a,4b,5,6,6a,7,8,9,9a,9b,10-
tetradecahydro-lH-indeno[5,4-f]quinoline-7-carboxylic acid.
6. Pharamceutically acceptable salts and steroisomers of the compounds as claimed in claim
5.
7. A pharmaceutical composition comprising the compounds as claimed in claim 1.
8. Use of the compounds as claimed in claim 1 for diseases and disorders pertaining to
mitochondrial biogenesis, selected from the group comprising skeletal or cardiac muscle
diseases associated with ischemia, or impaired or inadequate blood flow, diseases
associated with genetic disorders that directly or indirectly affect the number, structure,
or function of mitochondria, diseases associated with impaired neurological function
associated with decreased mitochondrial number or function, diseases associated with
loss of number, loss of function, or loss of correct, optimally efficient internal
organization of skeletal muscle cells or cardiac muscle cells, metabolic diseases, and
conditions associated with liver cell injury and altered fatty acid metabolism.
9. Use of the compounds as claimed in claim 1 for mitochondrial biogenesis-mediated
disease selected from the group comprising acute coronary syndrome, myocardial
infarction, angina, renal injury, renal ischemia, diseases of the aorta and its branches,
injuries arising from medical interventions, atherosclerosis, trauma, diabetes,
hyperlipidemia, vascular stenosis, peripheral arterial disease, vasculopathy, and
vasculitis, Friedreich's ataxia, muscular dystrophy, Duchenne muscular dystrophy,
Becker muscular dystrophy, limb girdle muscular dystrophy, congenital muscular
dystrophy, facioscapulohumeral muscular dystrophy, myotonic muscular dystrophy,
oculopharyngeal muscular dystrophy, distal muscular dystrophy, spinal muscular atrophy
and Emery-Dreifuss muscular dystrophy. Huntington's disease, parkinson's disease,
alzheimer's disease, and amyotrophic lateral sclerosis, sarcopenia, congestive heart
failure, aging, myocarditis, myositis, polymyalgia rheumatic, polymyositis, HIV, cancer
and/or the side effects of chemotherapy targeting the cancer, malnutrition, aging, inborn
errors of metabolism, trauma, and stroke or other types of neurological impairment like
hepatic steatosis, hepatic fibrosis, cirrhosis, and hepatocyte or stellate cell injury.
10. Use of the compounds as claimed in claim 1 for treating or preventing the adverse effects
of administration of compounds which exhibit mitochondrial toxicity
11. Use of the compounds as claimed in claim 1 for improving muscle structure or function;
improving mitochondrial effects associated with exercise; enhancing the capacity for
exercise in those limited by age, inactivity, diet, or diseases; enhancing muscle health and
function in response to exercise; enhancing muscle health and function in the clinical
setting of restricted capacity for exercise; enhancing recovery of muscles from vigorous
activity or from injury associated with vigorous or sustained activity.
12. Use of the compounds as claimed in claim 1 for enhancing sports performance and
endurance, building muscle shape and strength, or facilitating recovery from the muscle
related side effects of training or competition.