PYRAZOLE DERIVATIVES USEFUL AS ALDOSTERONE SYNTHASE INHIBITORS
The present invention relates to compounds and salts useful for inhibiting
aldosterone synthase and pharmaceutical compositions thereof.
Aldosterone synthase is the rate limiting enzyme for the production of
aldosterone. Elevated plasma aldosterone levels have been associated with progressive
renal disease leading to chronic kidney disease. Infusion of aldosterone in rats has been
observed to produce kidney fibrosis and elevated proteinuria - a marker for kidney
damage. Animal models of kidney disease have shown that aldosterone synthase
inhibitors are useful for the treatment of kidney disease.
The main causes of kidney disease are diabetes leading to diabetic nephropathy
and hypertension. Aldosterone synthase inhibitors have also been shown to be useful for
the treatment of resistant hypertension particularly when combined with angiotensin
converting enzyme (ACE) inhibitors or angiotensin receptor blockers (ARBs). Studies
have also shown significantly elevated levels of aldosterone in patients with congestive
heart failure (CHF). Aldosterone blockade has been shown to improve survival in
patients with CHF.
Aldosterone synthase inhibitors have been disclosed in, for example, U.S. Patent
5,0 7 5 and European Patent Publication number 0 165 904. Aldosterone synthase
inhibitor compounds have the potential to also inhibit production of Cort isol testosterone
and/or estradiol. Inhibition of Cortisol, testosterone and/or estradiol is an undesired side
effect of current aldosterone synthase inhibitors. Thus, there is a need to discover new
aldosterone synthase inhibitors. Additionally, the need also exists to discover new
aldosterone synthase inhibitors that selectively inhibit production of aldosterone
compared to the inhibition of Cortisol, testosterone and/or estradiol production.
The present invention provides alternate aldosterone synthase inhibitors. Also, the
present invention provides compounds that may selectively inhibit aldosterone production
compared to production of Cortisol, testosterone and/or estradiol.
The present invention provides a compound of the formula:
wherein
n is 0, 1, or 2;
m is 1 or 2;
R and R2 are independently selected from hydrogen, - C , ; - ;
R3 s hydrogen, -CN, - F -CI, -CH , -OCH , or -CF 3;
R4 is at each instance independently selected from -F, C , -Br, -CH 3, -OCH 3, -CF 3 and
-CN;
or a pharmaceutically acceptable salt thereof.
The present invention provides a method of treating chronic kidney disease
comprising administering an effective amount of a compound of the invention or a
pharmaceutically acceptable salt thereof to a patient in need thereof.
The present invention also provides a method of treating diabetic nephropathy
comprising administering an effective amount of a compound of the invention or a
pharmaceutically acceptable sa t thereof to a patient in need thereof.
The present invention further provides a method of treating congestive heart
failure or hypertension comprising administering an effective amount of a compound of
the invention or a pharmaceutically acceptable salt thereof to a patient in need thereof.
The present invention additionally provides a pharmaceutical composition
comprising a compound of the invention or a pharmaceutically acceptable salt thereof,
and one or more pharmaceutically acceptable carriers, diluents or excipients. In a
particular embodiment, the composition further comprises one or more other therapeutic
agents.
Further, the present invention also provides compounds of the invention or
pharmaceutically acceptable salts thereof for use in therapy, in particular for the treatment
of chronic kidney disease, diabetic nephropathy, congestive heart failure, or hypertension.
Furthermore, the present invention provides compounds of the invention or
pharmaceutically acceptable salts thereof for use in the treatment of chronic kidney
disease. The present invention also provides compounds of the invention or
pharmaceutically acceptable salts thereof for use in the treatment of diabetic nephropathy.
The present invention further provides compounds of the invention or pharmaceutically
acceptable salts thereof for use in the treatment of congestive heart failure and
hypertension.
Even further, the present invention pro vides the use of a compound of the
invention or a pharmaceutically acceptable salt thereof for the manufacture of a
medicament for the treatment of chronic kidney disease, diabetic nephropathy, congestive
heart failure, or hypertension.
The present invention a so provides a method of treating chronic kidney disease
comprising co-administering effective amounts of a compound of the invention an d one
or more of an angiotensin-converting enzyme inhibitor (ACE inhibitor), an angiotensin
receptor blocker (ARB), or a mineralocorticoid receptor (MR antagonist. ACE inhibitors
include benazepril (marketed in the XJ.S. as Lotensin®), captopril (marketed in the U.S. as
Capoten®), enalapril/enaiaprilat (marketed in the U.S. as Vasotec® oral and injectable),
fosinopril (marketed in the U.S. as Monopril®), lisinopril (marketed in the U.S. as
Zestril® and Prinivil®), moexipril (marketed in the U.S. as Univasc®), perindopri!
(marketed in the U.S. as Aceon®), quinapril (marketed in the U.S. as Accupril®),
ramiprii (marketed in the U.S. as Altace®), and trandolaprii (marketed in the U.S. as
Mavik®). ARBs include candesartan (marketed in the U.S. as Atacand®), irbesartan
(marketed in the U.S. as Avapro©), olmesartan (marketed in the U.S. as Benicar ®),
losartan (marketed in the U.S. as Cozaar ®), valsartan (marketed in the U.S. as Diovan®),
telmisartan (marketed in the U.S. as Micardis ®), and eprosartan (marketed in the U.S. as
Teveten ©). The present invention further provides a method of treating diabetes or
hypertension or congestive heart failure comprising co-administering effective amounts
of a compound of the invention and a diuretic. Diuretics include torsemide (marketed in
the U.S. as Demadex®), furosemide (marketed in t e U.S. as Lasix®), bumetanide
(marketed in the U.S. as Bumex ®), ethacrynic acid (marketed in the U.S. as Edecrin®),
torsemide (marketed n the U.S. as Demadex ®), amiloride. (marketed in the U.S. as
Midamor®}, acetazoiamide (marketed in the U.S. as Diamox®), pamabrom (marketed in
the U.S. as Aqua-Ban®), mannitol (marketed in the U.S. as Aridol® or Osmitrol®),
traimterene (marketed in the U.S. as Dyrenium®), spironolactone (marketed in the U.S.
as Aldactone®), amiloride (marketed in the U.S. as Midamor®), indapamide (marketed in
the U.S. as Lozo!®), hydrochlorothiazide (marketed in the U.S. as HydroDIURIL®),
metolazone (marketed in the U.S. as Zaroxolyn® or Mykrox®), methylclothiazide
(marketed in the U.S. as Aquatensen® or Enduron®), hydrocholorthiazide (marketed in
the U.S. as Aquazide H® or Esidrix® or Microzide®}, chlorothiazide (marketed in the
U.S. as Diuril®), bendroflumethiazide (marketed in the U.S. as Naturetin®), polythiazide
(marketed in the U.S. as Renese®), hydroflumethiazide (marketed in the U.S. as
Saluron®), and chlorthalidone (marketed in the U.S. as Thalitone®). For a complete
listing also see, e.g., Physician 's Desk Reference, 2012 Edition, PDR Network (201 )
Figure 1 is a spectrogram of a representative X-ray powder diffraction (XRD)
pattern for 4-[(4R)-6,6-dimethyl-4,5-dihydro-lH-eyclopenta[c]pyrazol-4-yl]benzonitrile
hemihydrates. The XRD spectrogram was obtained as described in the Example l a
below. Figure 2 is a spectrogram of a representative XRD pattern for 4-[(4R)-6,6~
dimethyl~4,5-dil ydro- lH-cyelopenta[c]pyrazol-4-yl]benzonitrile; phosphoric acid. The
XRD spectrogram was obtained as described in the Example 1b below.
The term "nitrogen protecting group" is taken to mean a moiety that is stable to
projected reaction conditions and yet may be selectively removed by reagents an
reaction conditions compatible with the regenerated amine. Such groups are well known
by the skilled artisan and are described in the literature. See, e.g., Greene and VVuts,
Protective Groups in Organic Synthesis, Third Edition, Chapter 7, John Wiley and Sons
Inc., (1999).
The skilled artisan will appreciate that compounds of the invention can exist in
tautomeric forms, as depicted below in I and 1(a). When any reference in this application
to one of the specific tautomers of the compounds of the invention is given, it is
understood to encompass both tautomeric forms and all mixtures thereof.
1(a)
The skilled artisan will appreciate that compounds of the invention are comprised
a core that contains at least one chiral center, represented by in 1(b) below:
Although the present invention contemplates all individual enantiomers, as well as
mixtures of the enantiomers of said compounds including racemates, the compounds with
the absolute configuration as illustrated in 1(c) below are preferred compounds of the
invention.
(c)
Isomers of compounds of the invention are labeled as isomer 1, isomer 2, etc.,
begi ing with the first to e ute (lower retention time) from the chromatographic
separation method employed and disclosed herein.
Additionally, the skilled artisan will appreciate that additional chiral centers may
be created in the compounds of the invention by the selection of certain variables. The
present invention contemplates all individual enantiomers or diastereomers, as well as
mixtures of the enantiomers and diastereomers of said compounds including racemates.
The skilled artisan will also appreciate that the Cahn-Ingold-Prelog (R) or (S)
designations for all chiral centers will vary depending upon the substitution patterns of
the particular compound. The single enantiomers or diastereomers may be prepared
beginning with chiral reagents or by stereoselective or stereospecific synthetic techniques.
Alternatively, the single enantiomers or diastereomers may be isolated from mixtures by
standard chiral chromatographic or crystallization techniques at any convenient point in
the synthesis of compounds of the invention.
The compounds of the present invention are cyclopentylpyrazoles or
teirahydroindazoles, and accordingly, react with any of a number of inorganic and organic
acids to form pharmaceutically acceptable acid addition salts. Pharmaceutically
acceptable salts and common methodology for preparing them are well known in the art.
See, e.g., P. Stahl, etal. Handbook of Pharmaceutical Salts: Properties, Selection and
Use, (VCHA/Wiley-VCH, 2002); S.M. Berge, et aί , "Pharmaceutical Salts," Journal of
Pharmaceutical Sciences, Vol. 66, No. 1, Januar 1977. Preferred pharmaceutically
acceptable salts of the invention are those formed with hydrochloric acid or phosphoric
acid.
The compounds of the present invention are preferably formulated as
pharmaceutical compositions administered by a variety of routes. Most preferably, such
compounds are for oral administration. Such pharmaceutical compositions and processes
for preparing same are well known in the art. See, e.g., Remington: The Science and
Practice of Pharmacy (A. Gennaro, et. al, eds., 19"' ed., Mack Publishing Co., 1995).
The compounds of the present invention are generally effective over a wide
dosage range. For example, dosages per day normally fall within the range of about 0.0
to about 30 mg/kg of body weight. n some instances dosage levels beiow the iow er limit
of the aforesaid range may be more than adequate, while in other cases still larger doses
may be employed without causing any harmful side effect, and therefore the above
dosage range is not intended to limit the scope of the invention in any way. t will be
understood that the amount of the compound actually administered will be determined by
a physician, in the light of the relevant circumstances, including the condition to be
treated, the chosen route of administration, the actual compound or compounds
administered the age, weight, and response of the individual patient and the severity of
the patient's symptoms.
As used herein the terms "treating" and "treat" mean slowing or delaying the
progression of a disease, such as kidney disease, in a patient in need thereof. Diabetes
and hypertension are the two main causes of chronic kidney diseases. In many patients
with chronic kidney disease, diabetes and hypertension co-exist. Diabetic patients with
chronic kidney disease (diabetic nephropathy) are likely to have accelerated progress to
ESRD. They are also at high risk of mortality, mostly due to cardiovascular complications
such as heart disease. Chronic kidney disease is classified based on glomerular filtration
rates wherein filtration rates decrease from Stage I though stage 5 or ESRD. For review
of aldosterone synthase literature see, for example, See J . Am. Soc. Nephrol 14, 2395-
2401 (2003); Kidney Inter-national, 21, 98-101 (1982); and Circulation , 3087-3094
(2005). As used herein the term "chronic kidney disease" refers to kidney disease that
persists for more than three months.
As used herein, the phrase "effective amount" means an amount of a compound of
the invention that is sufficient to treat in one or more doses a condition or detrimental
effect thereof herein described or an amount of a compound of the invention tha is
sufficient to inhibit aldosterone synthase to achieve the objectives of the invention.
As used herein, the phrase "co-administering" means the administration of a
compound of the invention and another compound described herein, separately ,
simultaneously or sequentially over a period of time as determined by a qualified care
giver.
As used herein, "patient" refers to a mammal, preferably human.
Although all of the exemplified compounds of the invention are useful aldosterone
synthase inhibitors, certain classes of compounds are preferred. The following
paragraphs describe such preferred classes:
a) R and R are both -CH 3;
b) R an d R2 are both hydrogen;
c) R is -CH 3 and R - is hydrogen;
d) m is ;
e) m is 2;
f) R is -CN;
g) R s -F;
h ) R is -Cl;
i) R is - C 3;
j ) s nyctrogen;
) is ) ;
1) n is ;
m) n is 2;
n) R4 is -F;
o) R4 s -Cl;
p) R4 is -Br;
q) R4 is -CH 3;
r) R4 is -CN;
s) the R4 substituent is in the meta position relative to R ;
t) R3 is -CN an d R4 is -F;
u) R3 is -CN an d R4 is C ;
v) R is - F or -Cl and R4 is -F, -Cl, or -Br;
w) 3 is -Cl and R4 is -F;
x) R3 is -Cl and R4 is -C ;
y) R3 is - F and R4 is -F;
z) R3 is - F and R4 is -Cl;
aa) The compound of the present invention is a free base;
bb) The compound of the present invention is the hydrochloride salt;
cc) T e compound of the present invention is the phosphate salt.
A preferred embodiment of the compounds of the present invention relates to
compounds of the invention of the following formula,
wherein n is 0, 1, or 2; R and R2 are independently selected from hydrogen, -CH 3, and
-CH CH ; R3 is hydrogen. --CN, -F, -CI, or -CF ; R4 is at each instance independently
selected from -F, -CI, -Br, -CH 3, -CF ,and -CN; or a pharmaceutically acceptable salt
thereof.
Another preferred embodiment of the compounds of the present invention relates
to compounds of the invention of the following formula,
wherein n is 0 or ; R1 and R are independently selected from hydrogen and -CH 3;
R is hydrogen, -CN, halo (CI), -OCH 3, -CH 3; R4 is at each instance independently
selected from halo (F), -CH , and -QCH ; or a pharmaceutically acceptable salt thereof.
A further preferred embodiment of the compounds of the present invention relates
to compounds of the invention wherein n is 0 or 1; m is 1 or 2; R1 and R2 are
independently selected from hydrogen. -CH 3, and -CH 2C 3; R is -CN; is at each
instance independently selected from -F, -CI, -Br. -CH , and -OCH 3; or a
pharmaceutically acceptable sa t thereof.
An additional prefeiTed embodiment of the compounds of the present invention
relates to compounds of the invention wherein n is 0 or ; m is : R and R are
independently selected from hydrogen, -CH 3, and -CH C 3; R is -CN; R4 is -F, -CI, or
-Br; or a pharmaceutically acceptable salt thereof.
Another preferred embodiment of the compounds of the present invention relates
to compounds of the invention wherein n is 0 or 1; is 2; R and R2 are independently
selected from hydrogen and -CH 3; R3 is -CN; R4 is -F, -CI, -Br, -CH 3, or -OCH ; or a
pharmaceutically acceptable salt thereof.
A further preferred embodiment of the compounds of the present invention relates
to compounds of the invention wherein n is 1; m is 1 or 2; R and R2 are independently
selected from hydrogen and -CH 3; is -CN; R is -F, -CI, -Br, or -C 3; or a
pharmaceutically acceptable salt thereof.
An additional preferred embodiment of the compounds of the present invention
relates to compounds of the invention wherein is 1; m is 1; R and R2 are - C 3; R3 is
-CN; R4 is -F, -CI, or -Br; or a pharmaceutically acceptable salt thereof.
Another preferred embodiment of the compounds of the present invention relates
to compounds of the invention wherein n is 1; m is 2; R1 and R are independentlyselected
from hydrogen an -CH 3; is -CN; R is -F, -CI, -Br, or - C 3; or a
pharmaceutically acceptable salt thereof.
A further preferred embodiment of the compounds of the present invention relates
to compounds of the invention wherein n is ; m is 1 or 2; R and R2 are -CH 3; R3 is
-CN; R4 is -F; or a pharmaceutically acceptable salt thereof.
An additional preferred embodiment of the compounds of the present invention
relates to compounds of the invention wherein n is ; m is 2; R and R are -CH ; R3 is
-CN; R4 is -F; or a pharmaceutically acceptable salt thereof.
Another preferred embodiment of the compounds of the present invention relates
to compounds of the invention wherein n is 0, ί , or 2; m is 1 or 2; R1 and R2 are
independently selected from hydrogen and -CH ; R3 is - F or -CI; R4 is a each instance
independently selected from -F. -CI, -Br, -CH , and -CF ; or a pharmaceutically
acceptable salt thereof.
An additional preferred embodiment of the compounds of the present invention
relates to compounds of the invention wherein n is 0, 1, or 2; m is 1; R and R are
independently selected from hydrogen and -CH 3; R3 is - F or -CI; R4 is at each instance
independently selected from -F, -CI, -Br, -CH3, and -CF ; or a pharmaceutically
acceptable salt thereof.
A further preferred embodiment of the compounds of the present invention relates
to compounds of the invention wherein n is 0, 1, or 2; m is 2; R and R2 are independently
selected from hydrogen and -CH 3; RJ is - F or - C ; R is at each instance independently
selected from -F, -CI, -Br, -CH3, and -~CF ; or a pharmaceutically acceptable salt
thereof.
Another preferred embodiment of the compounds of the present invention relates
to compounds of the invention wherein n is 0, 1, or 2; m is 1; R and R are independently
selected from hydrogen and -C ; R ' is hydrogen; R4 is at each instance independently
selected from -F, -CI, -Br, -C¾, and -CN; or a pharmaceutically acceptable salt thereof.
A further preferred embodiment of the compounds of the present invention relates
to compounds of the invention wherein m is 1: R an d R - are -~CH3; V is -CN; n is 0 or
1; R4 is -F, or a pharmaceutically acceptable salt thereof.
An especially preferred embodiment of compounds of the present invention is 4-
(6,6-dimethyl-4,5-dihydro- lH-cyclopenta[c]pyrazol-4-yi)benzonitrile:
or a pharmaceutically acceptable salt thereof.
Another especially preferred embodiment of compounds of the present invention is 4-
[(4R)-(6,6-dimethyi-4,5-dihydro- lH-cyc enta[c]pyrazol-4-yl)]benzonitrile:
or a pharmaceutically acceptable salt thereof.
A further especially preferred compound of the present invention is 4-[(4R)-(6,6-
dimethyl-4,5-dihydro-lH-cyclopenta[c]pyrazol-4-yl)]benzonitrile.
Another especially preferred compound of the present invention is 4-(6,6-
(¾methyl-4,5-dihydro-lH-cyclopenta[c]pyrazol-4-yl)-3-fluoro-beiizonitri1e:
or a pharmaceutically acceptable salt thereof.
A further especially preferred compound of the present invention is 4-(6,6-
dimeliiyl-4,5-dihydro-lH-cyclopenta[c3pyrazol-4-yl)-3-fluoro-benzonitrile, Isomer 1, or a
pharmaceutically acceptable salt thereof.
An additional especially preferred compound of the present invention is 4-(6,6-
dimethyl-4,5-dihydro-lH-cyclopenta[c]pyrazol-4-yl)-3-fluoro-benzonitrile.
An even further especially preferred compound of the present invention is 4-(6,6-
dimethyl-4,5-dihydro-lH-cyclopenta[c]pyrazo1-4-yl)-3-fluoro-beiizonitri1e, Isomer 1.
The compounds of the invention are inhibitors of aldosterone synthase. Thus, the
present invention provides a method of inhibiting aldosterone synthase that comprises
administering to a patient in need of said treatment an aldosterone synthase -inhibiting
amount of a compound of the present invention. It is preferred tha t the patient to be
treated by the administration of the compounds of the present invention is a mammal
preferably human.
As inhibitors of aldosterone synthase, the compounds of the present invention are
believed to be useful for the treatment of chronic kidney disease, diabetic neuropathy,
congestive heart failure, and hypertension.
Irs a preferred embodiment, the present invention provides a method of treating
chronic kidney disease comprising administering a compound of the invention to a patient
in need thereof.
In another preferred embodiment, a compound of the present invention pro vides a
method for treating diabetic nephropathy, said method comprising administering an
effective amount of a compound of the invention or a pharmaceutically acceptable salt
thereof to a patient in need thereof.
In yet another preferred embodiment the present invention provides a method for
treating chronic kidney disease comprising co-administering a compound of the invention
and an ACE inhibitor to a patient in need thereof.
In an additional preferred embodiment the present invention provides a method
for treating chronic kidney diseases comprising co-administering a compound of the
invention an d an ARB to a patient in need thereof.
The compounds of the present invention, or salts thereof, may be prepared by a
variet of procedures known in the art, some of which are illustrated in the Schemes,
Preparations, and Examples below. The specific synthetic steps for each of the routes
described may be combined in different ways, or in conjunction with steps from different
schemes, to prepare compounds of the present invention, or salts thereof. The products of
each step in the schemes below can be recovered by conventional methods, including
extraction, evaporation, precipitation, chromatography, filtration, trituration, or
crystallization.
Certain stereochemical centers have been left unspecified and certain substituents
have been eliminated in the following schemes for the sake of clarity and are not intended
to limit the teaching of the schemes in any way. Furthermore, individual isomers,
enantiomers, or diastereomers may be separated at any convenient point in the synthesis
of compounds of the present invention by methods such as chiral chromatography.
Additionally, the intermediates described in the following schemes contain a number of
nitrogen protecting groups. The variable protecting group may be the same or different in
each occurrence depending on the particular reaction conditions and the particular
transformations to be performed. The protection and deprotection conditions are wel
known to the skilled artisan and are described in the literature. See, e.g., Greene and
W s, Protective Groups in Organic Synthesis, Third Edition, Chapter 7, John Wiley and
Sons Inc., (1999).
The abbreviations used herein are defined according to Aldrichimica Acta, Vol.
17, No. 1, 1984. Other abbreviations are defined as follows: "AC " refers to
acetonitrile; "BGNAR" refers to 2,2 '-bis(dipheny Iphosphino)- , l'-binaphthalene;
"Bredereck's reagent" refers to r -butoxy bis(dimethyiamino)methane; 2C 2(COD)
refers to cyclooctadiene rhodium chloride dimer; "CMV" refers to cytomegalovirus;
"DCM" refers to dichloromethane; "DMEA" refers to dimeihylethylamine; "DMF" refers
to dimethylformamide; "DMF-DMA" refers to dimethylformamide-dimethyl acetal or
l ,l dimethoxy N N dimethyl methanamine; "DMEM" refers to Dulbecco's Modified
Eagle's Medium; "DMSO" refers to dimethylsuifoxide; "DOC" refers to 1-
deoxycordcosterone; "EtOAc" refers to ethyl acetate; "EtOH" refers to ethy alcohol or
ethanol; "Ex" refers to example; "FA" refers to formic acid; "flash chromatography"
refers to purification over silica gel; "HEC" refers to hydroxy ethyl cellulose; "IS" refers
to internal standard; "IPA" refers to isopropyl alcohol or isopropanol; "LiHMDS" refers
to lithium bis(trimethyfsilyl)amide; "MeOH" refers to methyl alcohol or methanol;
"MTBE" refers to methyl t-butyi ether; "NMP" refers to N-methylpyrrolidone;
"PdAllyiCl" refers to -Allylpalladi m(II) chloride dimer; "Pd (dba)3" refers to
tris(dibenzylideneacetone) dipalladium; "Phosphine ligand" refers to ά -tertbuty3(
2',4'.6'-triisopiOpylbiphenyl-2-y3)phosphine; "Prep" refers to preparation; "SFC"
refers to supercritical fluid chromatography; "TFA" refers to trifluoroacetic acid; "THF"
refers to tetrahydrofuran; "TUP" refers to tetrahydropyranyl; "Tr" refers to retention time
an "Tosyl" refers to toluenesulfonyl.
In the schemes below, all substituents unless otherwise indicated, are as
previously defined. The reagents and starting materials are generally readily available to
one of ordinary skill in the art. Others may be made by standard techniques of organic
and heterocyclic chemistry which are analogous to the syntheses of known structurallysimilar
compounds and the procedures described in the Preparations and Examples which
follow including any novel procedures.
Scheme I
Scheme I depicts the alleylation of an appropriate compound of formula (1) with
an aryl boronic acid (2) in a 1,4 Michael Addition to give compound (3) which can be
treated with Bredereck's reagent an hydrazine to give compounds of (I). The variable
groups are as previously defined.
For example, the skilled artisan will recognize that there are a variety of
conditions useful for facilitating! ,4 Michael Additions. Accordingly, a suitable catalyst
such as bis(l,5-cyclooctadienerhodium chloride), bis(norbomadiene)rhodium (I)
tetrafluoroborate, or antimony trichloride with a palladium (II) catalyst such as palladium
acetate can be used along with the appropriate aryl boronic acid (2). A suitable
phosphine reagent such as racemic-2,2'-bis(diphenylphosphino)-l ,1-binap yl or the
chirai binap (S)-(-)-2 2'-bis(diphenylphosphino)-l , -binaphthyl can be used to attempt a
chiral addition. An appropriate inorganic base such as sodium acetate, potassium acetate,
potassium carbonate, or an organic base such as triethylamine with an appropriate solvent
such as THF with isopropyl alcohol, dioxane and water or an acid such as acetic acid can
be used to facilitate the reaction to give a compound of formula (3, Step 1). The ketone
can then be alkylated with dimethylformamide-dimethyl acetal or -bu oxy
bis(dimethylamino)methane (Bredereck's reagent) to give the dimethylaminomethylene
adduct, compound (4. Step 2) which can then be cyclized to form the pyrazole using
hydrazine, hydrazine hydrate, or hydrazine hydrochloride to give compounds of the
present invention in Step 4. Alternatively, a compound of formula (3) can be taken
directly to compounds of the present invention without isolation of the intermediate
compound (4) by reaction with Bredereck's reagent and then reaction in situ with the
hydrazine reagent to give compounds of the present invention. Step 3.
Alternatively, when R and R2 are both hydrogen a gem dialkyl functionality can
be installed using an iodo-alkyl reagent in the presence of a base, such as lithium
hexamethyldisilylazide.
Scheme I
Deprotection
Dehydration
Alternatively, the appropriately protected tetrahydroindazol-4-one as a mixture of
regioisomers of pyrazole can be separated chromatographically or reacted as a mixture.
The pyrazole protecting group can reside on either nitrogen atom and was arbitrarily
assigned. One skilled in the art will recognize the position of the pyrazole protecting can
be interchanged without affecting the outcome of the subsequent reactions shown. "PG"
is a protecting group for the amino group. Such protecting groups are well known and
appreciated in the art. Compound 5 can be alkylated with a Grignard reagent which is
prepared by the treatment of the appropriately substituted 4-iodo phenyl analog with
isopropylmagnesium chloride or bromide in an appropriate solvent such as THF to give
the 5,6~dihydro-2H-indazol-4-ol (6, Step 5). In one embodiment, the carbinols (6) can be
dehydrated an d d -protected in a single transformation to give the alkene (7, Step 6) by
treatment with an acid such as HQ or TFA in an alcoholic solvent such as methanol.
Compound (7) can then be reduced by hydrogenation to give compound (8, Step 7).
Alternatively, the sequence can be accomplished by step-wise by treatment of (6) with
FA in DCM, the resulting olefin reduced by hydrogenation using catalysts such as
Pt(OH) 2 and de-protected with a base such as KOH in MeOH to give compound (8).
Scheme III
Scheme 111 depicts the alkylation of an appropriate compound of formula (9) with
an aryl boronic acid to give compound ( 0) that after hydrogeiiation and deprotection
gives a compound of the present invention. One skilled in the art can recognize the order
of Step 10 can be transposed to give a compound of (8 .
For example, the protected etrahydroindazol-4-one regioisomers (5) ca be
separated chromatographically or reacted as a mixture with a suitable base such as
LiHMDS in a polar aprotic solvent such as THF and the resulting enolate quenched with
N-phenylbis(trifluoromethanesulfonamide) to give the eno rrifiate (9, Step 8) as a
mixture of regioisomers that can be separated chromatographically or reacted as a
mixture. Compound 9 can be reacted with an appropriate boronic acid (2) under Suzuki-
Miyaura cross coupling conditions to give compound 10, Step 9. The skilled artisan will
recognize that there are a variety of conditions useful for facilitating such cross-coupling
reactions. Accordingly, a suitable palladium reagent includes
tetrakis(triphenylphosphine)palladium(0), bis(triphenyiphosphine)paliadium(Il) chloride,
tris(dibenzylideneacetone)dipalladium (0) with tricyclohexylphosphine, ( , -
bis(diphenyfphosphino)ferrocene)pa1ladium(II) chloride, or palladium(IX) acetate. A
suitable base includes cesium carbonate, sodium carbonate, potassium carbonate, or
potassium phosphate tribasic monohvdrate. Compound (10) can be hydrog enated and the
resulting product de-protected to give a compound of Formula (I, Step 10). One skilled in
the art can recognize that the final two transformations can be transposed to give a
compound of (8).
Preparations and Examples
The following preparations and examples further illustrate the invention.
Unless noted to the contrary, the compounds illustrated herein are named and
numbered using Symyx® Draw version 3.2 (Symyx Solutions, Inc.) or IUPACNAME
ACDLABS.
Preparation 1
5,5-Dimethylcyclopent-2-en-l-one
2,2-Dimethyleyclopentanone (50.0 g, 445.75 mmoi) is added to a solution of ally!
diethyl phosphate (165.4 g, 846.92 mmol) in t-amyl alcohol (557 mL). Potassium
carbonate (75.5 g, 537.12 mmol), and Pd(OAc)2 (5 g, 22.29 mmol) are added and the
mixture is heated at 80 °C for 12 hours. The mixture is cooled to ambient temperature,
diluted with acetone, (500 mL) filtered through diatomaceous earth, and concentrated to
dryness. The crude material is distilled under vacuum (60 °C a 20 mm Hg) to give the
title compound (30 g, 61%, 272 34 mmol). ' M (300.13 MHZ, CD C ) d 7.62-7.59
(m, l). 6.12 (dt, j 5.9, 2.1 Hz, l l). 2.54 (t, J 2.4 Hz, 2H), 1.10 (s, 6H).
Preparation 2
(4,5-Dif!uoro-2-methyl-phenyl)boronic acid
-Butyl lithium (4.6 mL, .5 mmol} is added to the mixture of l-bronio-4,5-
difluoro-2-methylbenzene (2.0 g, 9.6 mmol) and trimethyl borate (1.5 g, .5 mmol) in
anhydrous tetrahydrofuran (30 mL), drop wise, at -78 C over one hour, under an
atmosphere of argon. The reaction mixture is stirred for another hour at the same
temperature, quenched and acidified with 1 N HC1. The resulting mixture is extracted
with ethyl acetate(3x). The combined organic layer is washed with brine, dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo.
Preparation 3
(+/-)-4-(3,3-Dimethyl-4-oxo-cyc3opentyl)benzoiiitrile
Bis(l,5-cyelooctadienerliodiimi chloride) (1.09 g, 2.18 mmol), racemic-2,2'-
bis(diphenyiphosphino)-l , -binaphthyi (3.05 g, 4.90 mmol) are added to tetrahydrofuran
(72 mL) and the mixture is stirred under a nitrogen atmosphere for 30 minutes. This
solution is added to a mixture of 4-cyanoplienylboronic acid (80.04 g, 544.68 mmol), 5,5-
dimethylcyclopent-2-en-l-one (24 g, 217.87 mmol), potassium carbonate (40.65 g,
294.13 mmol), tetrahydrofuran (144 mL), and isopropyl alcohol (16.7 mL) at 60 °C. The
mixture is stirred at 60 C for hours an d then concentrated to dryness. The crude
mixture is poured into water (500 mL) and is extracted with ethyl acetate (2 x 500 mL).
The organic extracts are dried over MgSCU, filtered through silica gel, and concentrated
to dryness. The residue is purified by silica gel flash chromatography, eluting with 6 :
hexane : ethyl acetate to give the title compound (4 g, 88%, 192.24 mmol). NMR
(300.13 MHz, CDCl,) d 7.65-7.62 ( , 2H), 7.37 (d, J 8.1 Hz, 2H), 3.56-3.43 ( , I I).
2.86-2.76 (rn, H), 2.42-2.32 (m, 1H), 2.32-2.24 (m, . 1.86 (t, J 2.4 Hz, . 1. 7
(s, 3H), 1.15 (s, 3H).
Preparation 4
(+/-)-3-(3,4-Difluorophenyl)cyclopentanone
To 3,4-difluorophenylboronic acid (9.47g. 60 mmol), cyclopent-2-enone (4.93g,
60 mmol), antimony trichloride (1.3 g, 6 mmol). sodium acetate. (9.84g, 120 mmol) and
palladium acetate ( .35g, 6 mmol) are added to acetic acid (300 ml..) under an argon
atmosphere. The reaction is stirred at room temperature overnight. The reaction mixture
is filtered and poured into water. The material is extracted with ethyl acetate (3x),
washed with saturated sodium bicarbonate (3x), brine (3x), dried with sodium sulfate,
filtered, and concentrated in vacuo. The material is purified using silica gel
chromatography eluting with 0% ethyl acetate in pet ether to give the title compound as
a yellow oil (6.9g, 58%). T-iNMR (300 MHz, CDC13) d 7.14-6.95 (m, 3H), 3.41-3.37 (tn,
Hi). 2.64-2.61 (m, i l l). 2.46-2.23 (m, 4H), 1 93-1.84 . 1H).
The following compounds are prepared essentially as described for (+/-)-3-(3,4-
difl orop enyl)cyc openta one.
Table 1

Preparation 26
(+/~)~(2Z)-3-(3,4-Difluorophenyl)-2-((cUmetiiylamino)methylene)cyclopentanone
To (+/-)-3-(3, 4-difluorophenyl)cyclopentanone (6.9 g, 35 mmol) is added N,Ndimethyiformamide
dimethylacetal (80 mL) and stirred at 80 C overnight. The mixture
is cooled to room temperature and concentrated in vacuo. The residue is purified by silica
gel flash chromatography, eluting with 2% methanol in chloroform, to give the crude
mixture as a yellow oil (8.5g, 96%). LC/MS (M+H) 234.
The following compounds are prepared essentially as described for (+/-)-(2Z)-3-
(3,4-difluorophenyl)-2-((dimethylamino)methylene)cyclopentanone.
Table 2
4,4-dimethyl-3-oxocyelopers
ty l]benzoni tri e
(+/-)-(2Z)-3-(4-
Chlorophenyl)-2-
34 250
((dimethylamino)methylene)c
Vclopentanone
(+/-)-4-[(2Z)-2-
(Dimethylaminomethylene)-
35 241
3-oxocvc3
opentyl]benzoiiitril e
(+/-)-(2Z)-3-(4-
Fluorophenyl)-2-
36 234
((dimethy3amino)methy3ene)c
vclopentanone
(+/-)-4-[(2Z)-2-
(dime thy1aminome thy1ene)-
37 269
4,4-dimethyl-3-oxocyclopentyljbenzoni
tri le
a. After the reaction is concerstrated in vacuo, the residue is diluted with ethyl acetate
and water. The organic phase is separated and the aqueous is extracted three
times with ethyl acetate. The organic layers are combined and washed with brine,
dried over sodium sulfate, and concentrated in vacuo. Flash chromatography
eluting with 50% ethyl acetate an d pet ether, followed by 2%
methanoi/dichloromethane gives the title compound
b. Same work up as in (a). Flash chromatography eluting with 50% ethyl acetate and
pet ether, followed by 4% methanoi/dichloromethane gives the title compound
c. The reaction is carried out with butoxy -N, N , N', N '-tetramethylmethanedi amine
in toluene and heated to 60 °C, overnight. The work up is described in (a) and the
chromatography utilizes 2% methanoi/dichloromethane.
Preparation 38
(+/-)-4-[(2Z)-2-(Dimethylaminomethylene)-4,4-diethyl-3-oxo-cyclopentyl]benzonitrile
Lithium liexamethyidisilylazide (40 L, 40 mmol) is added to a solution of (+/-)-
4-[(2Z)-2-(dimethylaminomethylene)-3-oxo-cyclopentyl]benzonitrile (0.96 g, 4 mmol) in
anhydrous tetrahydrofuran (100 mL), drop wise at -30 °C, under an argon atmosphere,
a d stirred for one hour. At the same temperature, iodoethane (12.48 g, 80 mmol) is
added to the mixture drop wise, and the reaction mixture is allowed to warm to room
temperature and stirred overnight. The reaction is quenched by adding saturated
ammonium chloride (100 mL). The resulting mixture is extracted with ethyl acetate
(3x100 mL). The combined organic layer is dried over anhydrous sodium sulfate and
concentrated in vacuo to give the crude product as a black solid (0.99 g, 83.4%). This
used in the next step without further purification. ES MS (m/z) 297 (M+l).
The following compound is prepared essentially as described for (-'7-)-4-[(2Z)-2-
(dimethyiaminomethyiene)-4,4-die l y -3-oxo-cyciopen y ]benzonitrile.
Table 3
Preparation 40
(3S)-3-(3-Chiorophenyl)cyclopentanone
Bis(norbornadiene)rhodi m (1) tetrafiuoro borate (0.20 g, 0.53 mmol) an d (S)--(--)-
2,2'-Bis(diphenylphosphino)-l,l'-binaphthyl (0.30 g, 0.48 mmol) are dissolved 1,4-
dioxane ( 8 mL). The solution is degassed with nitrogen for 2 hours. 3-
Chlorophenylboronic acid (4.95 g, 3 .67 mmol) is dissolved in dioxane (24 ml.) and
water (6 mL). The mixture is degassed for another 2 hours. The two solutions are
combined and stirred for two hours under a nitrogen stream. 2-Cyclopentenone (2.0 g,
24.36 mmol) and triethylamine (2.1 m , 15.07 mmol) are added to the reaction
sequentially, via syringe. This is stirred until completion under a stream of nitrogen. The
reaction is filtered through a pad of diatomaceous earth and concentrated in vacuo. The
residue is purified via silica gel flash chromatography, eluting with 20% ethyl acetate in
exane to give the title compound (4.23 g, 89.2%) as a clear, yellow oil. i v! (400
MHz, CDC13) d 1.9 (IH, M), 2.25 (2H, M), 2.45 (2H, M), 2.65 (IH, M), 3.35 (IH, M),
7.1 ( H, D), 7.25 (3H, M).
The following compounds are prepared essentially as described for (3S)-3-(3-
chlorophenyl)cyclopentanone.
Table 4
Preparation 45
4-(4~Chloro-2-fluoro-phenyl)-2,2-dimethyl-cyclopentanone
A solution of 5,5-di ethylcycloper t-2-er - l -or e (42.0 g, 343.15 mmol), 4-chloro-
2-fluorophenylboronic acid (94 47 g, 514.72 mmol), sodium acetate (56.30 g, 686.30
mmol), acetic acid (1130 mL), Pd(OAc)2 (7.70 g, 34.31 mmol), and antimony trichloride
(7.83 g, 34.31 mmol) are stirred at ambient temperature for 16 hours. The solvent is
evaporated to low volume and the residual acetic acid is removed using toluene. MTBE
(500 mL) is added and the resulting precipitate is filtered off and discarded. The MTBE
solution is washed with water (500 mL), aq. a C (2x300 mL), dried over MgS0 4,
filtered, and concentrated to dryness. The residue is purified by silica gel flash
chromatography, eluting with hexane to hexane 0% MTBE to give the title compound
(72 g, 87%). ¾ NMR (300. 16 MHz, CDC13) d 7.2 1-7.06 (m, 3H), 3.67-3.59 (in, H),
2.8 1-2,72 ( , 1H), 2.41 -2.3 1 (m, ). 2.2 1 (ddd, J 12.6, 6.3, 2.2 Hz, IH), 1.89 •; !. J
12.3 Hz, . 1.159 (s, 3H), 1.137 (s, 3H).
Preparation 46
4-(3,3-Dimethyl-4-oxo-cyclopenryl)~3-fluoro-benzonitrile
Di-ieri-but>' i(2',4',6'-tr iisopropylbipheny]-2-yl)phosphme ( 1 75 g, 4. mmol) is
added to a solution of 4-(4-chloro-2-fluoro-phenyl)-2,2-dimethyl-cyelopentanone (33 g,
137. 10 mmol), zinc cyanide (9.66 g, 82.26 mmol), and N-methylpyrrolidone (148.50 mL)
at 125 °C and the mixture is stirred for 15 minutes. -Al]ylpalladium(II) chloride dimer
(0.76 g, 4. 1 mmol) is added to the solution and the mixture is stirred for 30 minutes.
Diatomaceous earth ( 5 g) is added and the mixture is cooled to rt. The mixture is
filtered through diatomaceous earth and washed with MTBE (450 mL). Water (450 mL)
is added and the mixiure is extracted with MTBE ( 150 mL). The mixture is washed with
brine, dr ied over MgS0 4, filtered, and concentrated to dryness to give the title compound
(34 g, 97%). T-I NMR (300. 6 MHz, CDCI3) d 7.47-7.33 ( , 3H), 3.76-3.63 (m, IH),
2.83-2.76 (m, H), 2.43-2.33 (m, ) . 2.25 (dd, J 6.3, 2.6 Hz, . 1.9 (t, J 2.3 Hz,
i l l). 1.168 (s, 3H), 1.147 (s, 3H).
Preparation 47
l-(p-Tolylsulfonyl)-6,7-dihydro-5H-indazol-4-one and 2-(p-Tolylsulfonyl)-6,7-dihydro-
5H-indazol-4-one
2,5,6,7-Tetrahydro-indazol-4-one (12.8 g, 9 1.2 mmol) is added to
dichloromethane (500 mL) and triethylamine (25.4 mL, 182.4 mmol). p-Toluenesulfony]
chloride ( 17.74 g, 9 .2 mmol) is then added and the mixture is stirred for 6 hours at
room temperature. The pH of the dark solution is adjusted to pH 3 with 1.0 N HC1, and
the mixture is transferred to a separator}' funnel. The organics are washed with water,
brine, dried over sodium sulfate, filtered, and evaporated to dryness. The residue is
purified by silica gel chromatography eluting with : hexanes/ethyl acetate to give the
title compounds (9.0 g, 34%) as a 1.5:1 ratio of regioisomers. ES/MS m z 291 (M+H).
Preparation 48
and 4-[4~
ydroxy 2- /:?-to y 8ui nyl)
4-Iodobenzonitrile (6.21 g, 26.84 mmol) is added to THF (40 0 mL) an d cooled to
0 °C. Isopropylmagnesium chloride (16. mL, 32.1 mmol) is added and stirred at 0 °C
under nitrogen atmosphere for 60 minutes. A 1.5:1 mixture of l-(p-tolylsulfonyl)-6,7-
dihydro-5H-indazol-4-one an 2-(p-toiylsulfonyl)-6,7-dihydro-5H-indazo]-4-one (6.24 g,
21.5 mmol) is dissolved in THF, and this solution is added to the anion drop-wise at 0 °C
and then allowed to warm to room temperature. The tan solution is quenched with HCl
(3.0 mL, 1N) and concentrated to dryness. The residue is purified by silica gel
chromatography eluting with 60% hexanes/ethyl acetate to give the title compounds.
This reaction is run a second time at the same scale and products of both rans combined
to give the title compounds as a mixture of regioisomers (8.98 g, 53%}. ES/MS m/z 394
(M+H).
The following compounds are prepared essentially by the method of 4-[4-
hydroxy-l-(p-tolylsulfonyl)-6,7-dihydro-5H-indazol-4-yl]benzonitrile and 4-[4-hydroxy-
2-( -toIylsulfony])-6,7-dihydro-5H-indazo -4-yl]benzonitrile using the appropriate a d
halide.
Table 5
anisole (jo-tolylsulfonyl)-6,7- - 8)
(2.0 eq) dibydro-5H-indazol-4-ol
and 4-(4-
methoxyphenyl)- -(ptoly
s fony1) -6,7-
dibydro-5H-indazol-4-ol
4-(4-C lorophenyi)- -(
4- tolylsulfonyl)-6,7-
Chlorophen dibydro-5H-mdazol-4-ol
5 1 ylmangnesi And 4-(4-chlorophenyl)- 405, 404
um Bromide 2-(p-toly3sulfonyi)-6,7-
(2.0 eq) dihydro-5H-indazol-4-ol
Preparation 52
4-(6,7-Dihydro-2H-indazol-4-yl)benzoniiriie
A mixture of 4~[4~hydroxy-l-(p-tolylsulfonyl)-6,7-dihydro-5H-indazol-4~
yljbenzonitrile and 4-[4-hydroxy-2-(p-tolylsulfonyl)-6,7-dihydro-5H-indazol-4-
yljbenzonitrile (8.56 g, 21.7 mmol) is added to 4.0 M HCl in dioxane (20.0 mL, 80.0
mmol), lieated to 80 °C for 2.0 3 and concentrated to dryness. T3ie residue is disso3ved in
dichloromethane/water, separated and the organics are dried over 2S0 4, filtered, and
evaporated. The residue is purified by silica gel chromatography 80% ethyl
acetate/hexanes to give the title compound (3.21 g, 67%). ES/MS m/z 222 (M+H), 220
(M-H).
The following compounds are prepared essentially by the method of 4-(6,
dihydro-2H-indazol-4-yl)benzonitrile.
Table 6
Preparation 55
4-(4-C loropheny )-2-( -tolyls lfonyl)-6,7-di ydroindazole
4-(4-Chlorophenyl)-2-(p-tolylsulfonyl)-6,7-dihydiO-5H-indazol-4-ol
(0.76 g, 1.89 mmol) is added to dichlorome thane (20.0 m l ) . trietliylsilane (6.04 m l . 37.7
mmol}, an d TFA (0. 6 L 2.07 mmol) and stirred at room temperature for 2.0 . The
reaction is quenched with saturated NaHC(¾, separated, the organics washed with brine
dried over a2S0 4, filtered, and evaporated to dryness to give the title compound (0.622
g, 86%). M (CDC 3) d 2.42 (s, 3H), 2.55 (m, 2H), 2.82 (t, 2 1 . 5.98 (t, 1H), 7.37-
7.3 1 (tn, 6H), 7.8 1 (s, 1H), 7.87 (d, 2H).
Preparation 56
4-(4-Chlorophenyl)~2-(p-tolylsulfonyl)-4,5,6,7-tetrahydromdazole
4-(4-Chlorophenyl)-2-(j?-toly1sulfonyi)-6,7-dihydroindazole (0.62 g, 1.89 mmol)
is added to ethanol (20.0 ml.) and ethyl acetate (10 ml.). Platinum (IV) oxide (0.22 g) is
added and the reaction is stirred under 40 psi of hydrogen at room temperature for 6.0 h .
The mixture is filtered through a plug of diatornaceous earth and evaporated under
reduced pressure to give the title compound (0.503 g, 80%). LC/MS m/z 389 (M+H), Tr
= 2.704 min.
Preparation 57
l -Tetrahydropyran-2-y3-6,7-dihydro-5H-indazo3-4-one and 2-Tetrahydropyran-2-yl-6,7-
dihydro-5H-indazol-4-one
2,5,6,7-Tetrahydro-indazo3-4-one (US2009/1 80 Al ) (5.0 g, 36.7 mmol) is
added to a solution of diliydropyran (3.4 g 40.4 mmo3) i C' 1•( i • ( 00 mL) and the
mixture is treated with -toluensuifonic acid (0. 1 g, 0.58 mmol) and stirred at room
temperature for 3 days. Saturated NaHC0 3 solution is added, and the contents are
transferred to a separator)' funnel. The organics are washed with brine, dried over
N32S04, filtered an d concentrated to dryness. The residue is purified by silica gel
chromatography eluting with CH C 1 to give the title compounds (5.32 g, 66%) as a
mixture of regioisomers. ¾ NMR (300 MHz, CDC13) d 8.04 (s, III), 7.90 (s, III), 5.35-
5.32 ( , ), 4.1 1-4.01 (m, 1H), 3.73-3.66 ( , 1H), 2.96-2.91 (m, 1H), 2.87-2.84 ( ,
1H), 2.51-2.47 (m, 2H), 2.19-2.02 (m, 4H), 1.72-1.61 (m, 4H).
Preparation 58
( 1-Tetrahydropyran-2-yl-6,7-dihy droindazol-4-y 1) trifluoromethanesulfonate
a d (2-Tetrahydropyran-2-yl-6,7-diliydroindazol-4-y1) trifluoromethanesulfonate
l-Tetrahydropyran-2-y3-6,7-dihydro-5H-indazol-4-one and 2-tetrahydropyran-2-
y]-6,7-dihydro-5H-indazol-4-one (2.03 g, 9.22 mraol) are added to T F (100 m ), the
solution is cooled to -78 °C, and treated with LiHMDS (10.14 mL, 10.14 mmol). After
stirring for hour, a solution of N-phenyibis(triiluouromethanesulfonimide) (3.68 g,
10.14 mmol) in THF (20 mL) is added drop wise at -78 °C, and allowed to warm to room
temperature over 17 hours. The reaction is quenched with saturated NH4CI, diluted with
diethyl ether, and the organics are washed with 0. N HCl, brine, dried over Na SC<4.
filtered, and concentrated to dryness. The residue is purified by silica gel
chromatography eluting with 85: 5 hexanes/ethyl acetate to give the title compounds (2 3
g, 51%) as a 3:1 mixture of regioisomers. ES/MS m/z 352 (M+H), 269 (M-THP).
Preparation 59
3-Methyl-4-(l-teti¾hydropyran-2-yl-6,7-dihydxoindazol-4-yl)benzonirrile or 3-Methyl-4-
(2-ietrahydropyran-2-yl-6,7-dihydroindazol-4-yl)beiizonitrile
( -Tetrahydropyran-2-yl-6,7-dihydroindazol-4-yl) trifluoromethanesulfonate, (2-
tetrahydropyran-2-yl-6,7-dihydroindazol-4-yl) trifluoromethanesulfonate
(1.0 g, 2.84 mmol) and (4-cyano-2-methylplienyl)boronic acid (0 502 g, 3.12 mmol) are
added to dioxane (80.0 mL) and a2C0 (0.601 mg, 5.68 mmol, 2.0 M) and de-gassed
with a stream of nitrogen. The solution is treated with
tetrakis(h-iphenylphosphine)palfadium (0.33 g , 0 28 mmol) and heated to 80 °C under
nitrogen for 17 hours. The mixture is cooled to ambient temperature and. filtered through
a plug of diatomaceous earth. The filtrate is diluted with ethyl acetate and the layers are
separated. The organics are washed with saturated. NaHC0 3, brine, dried over a2S0 4,
filtered, and concentrated to dryness. The residue is purified by silica gel
chromatography eluting with 9:1 hexanes/ethyl acetate to 4:1 hexanes/ethyl acetate to
give o e of the title compounds (0 492 g, 55%) as a single regioisomer. ES/MS m z 320
(M+H), 236 (M-THP).
Tl e following compound s prepared essentially by the method of 3-methyl-4-(ltetrahydropyran-
2-yl-6,7-dihydroindazol-4-yl)benzonitrile or 3-methyl-4-(2-
tetrahydTopyran-2-yl-6,7-dihydroindazol-4-yl)benzonitrile using the appropriate boronic
acid.
Table 7
Preparation 6
3-Methyl-4-(l-tetrahydropyran-2-yl^ or 3-
Methyl-4-(2-tetfahydropyran-2-yl-4,5,6,7-tetrahydroindazol-4-yl)benzonitrile
3-Methyl-4~(1-tetrahydropyran-2 -yl~6,7-dihydroindazol-4-yl)benzonitrile or 3-
methyl-4-(2-tetfahydropyran-2-yl-6,7-dihydroindazol-4-yl)benzomrrile (0.492 g, 1.54
mmol) and 5 % Pd/C wt w % (0.15 g) is added to EtOH (20.0 mL) and the mixture is
stirred under 45-35 psi of hydrogen for 72 hours. The mixture is filtered through a plug
of diatomaceous earth and concentrated to dryness. The residue is purified by silica gel
chromatography eluting with 7:3 hexanes/ethyl acetate to give one of the title compounds
(0. 62 g, 33%) as a single regioisomer. ES/MS m/z 322 (M+H).
The following compound is prepared essentially by the method of 3-methyl-4-(ltetrahydropyran-
2-yl-4,5,6,7-tetrahydroindazol-4-yl)benzonitrile
or 3-methyl-4-(2-tet ahydropyran-2-y]-4,5,6,7-tetrahydroindazol-4-y])benzonitrile
Table 8
Preparation 63
2-Fluoro-4-( 1-tetrahydropyran-2-yl-6,7-dihydroindazo3-4-y l)benzonitriie and 2-Fluoro-4-
(2-tetrahydropyran-2-yi-6,7-dihydroindazol-4-yl)benzoiiitri]e
(l -Tetrahydropyran-2-yl~6,7-dihyd oindazol-4-yl) trifluoromethanesulfonate, (2-
tetfahydropyran-2-yl-6,7-dihydroindazol-4-yl) trifluoromethanesulfonate
(0.201 g, 0.57 mmol) and (4-cyano-3-fluorophenyl)boronic acid (0.103 g, 0.63 mmol) are
added to dioxane (7.0 m ,) and Na C0 3 (0.12 mg, 1.14 mmol, 2.0 M), and the mixture is
degassed with a stream of nitrogen. The solution is treated with
teirakis(triphenylphosphine)palladium (0.07 g, 0.06 mmol) and heated to 80 °C under
nitrogen for 6 hours, and an additional 72 hours at room temperature. The reaction is
quenched with water, diluted with ethyl acetate, the organic layer is separated, washed
with saturated NaHCO , brine, dried over a S04, filtered, and concentrated to dryness.
The residue, is taken on to the next reaction without additional purification (0.231 g,
125%).
Preparation 64
4-(6,7-Dihydro-lH-mdazol-4-yl)-2-fluoro-benzonitrile
2-Fluoro-4-( 1-tetrahydropyran~2~yl-6,7-dihydroindazol-4-yl)benzonitrile and 2-
fluoro-4-(2-tetrahydropyran-2-yl-6,7-dihydroindazol-4-yl)benzonirrile (0.23 g, 0.7 1
mmo ) and 2SO4 (0.08 mL, 1.43 mmol) are added to CH3CN (5.0 mL) and the solution
is stirred at room temperature for 6.0 hours. Aqueous Na2C0 3 is added to basify the
reaction, which is then diluted with ethyl acetate, the layers separated, and the aqueous
back extracted with ethyl acetate (?>x). The organics are combined, dried over a2S0 ,
filtered, and concentrated to dryness. The residue is purified by silica gel flash
chromatography eluting with 99:1 C 2Cl2/MeOH to give the title compound (0.102 g,
60%}. ES/MS m z 240 (M+H).
Preparation 65
(+/-)-(trans)- 4-(4-Methyl-3-oxo-cyclohexyl)benzonitrile
and
Preparation 66
(+/-)-(ci s)-4-(4 -Me thy1-3~oxo-cyclohexyl)benzoni trile
Bis(l,5-cyclooctadienerhodium chloride) (0.06 g, 0.12 mmol), racemic-2,2'-
bis(diphenylphosphino)-l ,1'-bmaphthyl (0. 8 g, 0.29 mmol) are added to tetrahydrofuran
(40 mL) and the mixture is stirred under a nitrogen atmosphere for 30 minutes. This
solution is added to a mixture of 4-cyanophenylboronic acid (2.3 g, 5.69 mmol), 6-
methylcyclohex-2-en-l-one (Journal of Organic Chemistry, 1980 45(10), 1852-1863)
(1.28 g, 1.62 mmol), potassium carbonate (2.19 g, 15.69 mmol), a d isopropyl alcohol
(1.1 mL) at 60 °C. The mixture is stirred at 60 C for 16 hours and then concentrated to
dryness. The crude mixture is poured into water (10 mL) and is extracted with ethyl
acetate (2 x 20 mL). The organic extracts are dried over MgSC , filtered through silica
gel, an d concentrated to dryness. The residue is purified by silica gel flash
chromatography, eluting with 20% EtOAc/ hexane to give (trans)-4-(4-methyl-3-oxocyclohexyl)
benzonitrile (0.55 g, 22%) as the first eluting isomer and (cis)- 4-(4-methyl-3-
oxo-cyclohexyl)benzoni trile (0.55 g, 22%) as the second eluting isomer. ES/MS m/z 2 4
i v :•.
Preparation 67
(+/-)-(eis/trans)- 4-[7-Meihyl-4,5,6,7-tetrahydro-2H-indazol-4-yl]benzonitrile
(cis)-4-(4-Methyl-3-oxo-cyclohexy3)benzoniirile (0.55 g, 2 58 mmol) is added to
toluene (10 0 mL) an i rt-butoxybis(dimtlieylamino)methane (0.67 mL, 3.22 mmol) an
stirred at 120 C for 16 hours. The mixture is cooled to room temperature and
concentrated in vacuo. The residue is added to MeOH (10.0 mL) and hydrazine (0.07 mL,
2.32 mmol) and stirred at 80 °C for 1.0 hour. The mixture is cooled to room temperature
and concentrated in vacuo. The residue is purified by silica gel chromatography (35%-
55% EtOAc/hexanes) to give (+/-)-(eis/trans)- 4-[7-methyl-4,5,6,7-tetrahydro-2Hindazol-
4-yl]benzonitrile (0.360 g, 58%). ES/MS m/z 238 ( i ! .
Preparation 68
(+/-)-4-(4,4~Dimethyl~3-oxo-cyclohexyl)-3-methyl-benzonitrile
4-Cyano-2-methylphenylboronic acid (0.81 g, 5 mmol), 6,6-dimethylcyclohex -2-
enone (Canadian Journal of Chemistry, 1981, 59, 2096-2115) (0.55 g, 5 mmol), SbC
(0.11 g, 0 5 mmol), sodium acetate (0 82 g, 10 mmol), and palladium acetate (0.11 g, 0.5
mmol) are added to acetic acid (30 ml.) under an atmosphere of argon. The reaction
mixture is stirred at room temperature for three days. The mixture is filtered and the
filtrate is poured into water (150 mL) The organic phase is separated and the aqueous
phase is extracted with ethyl acetate (3 100 mL). The combined organic layers are
washed with saturated sodium bicarbonate solution (3x100 mL), brine (3x50 mL), dried
over anhydrous sodium sulfate, and concentrated in vacuo. The residue is purified by
silica gel column chromatography eluting with pet ether: EtOAc = 0 :1 to give the title
compound as a light yello solid (0.6 g, 50%}. GC/MS 241 (M+1).
The following compounds are prepared essentially by the method of (+/-)-4-(4,4-
dimethyl -3 -oxo-cyclohexyl)-3-methyl~benzonitrile.
Table 9
(+/-)-4~(4,4-Dimethyl-3 -
70 oxo-cyclohexyl)-2-fluorobenzonitrile
a.1!-! MR (300 MHz, IX ) d 7.60-7.56 (tn, 1H), 7.14-7.07(tn, 2H), 3.08-3.00 (m,
lH),2.73-2.67 (m, 1H), 2.51-2.46 (m, 1H), 2.03-1.60 (m, 4H), 1.25 (s, 3H), 1.12 (s, 3H).
Preparation 7
(+/-)-4-(4-Methoxyphenyl)-4,5,6,7- etral ydro- lH-indazole
4-(4~Methoxyphenyl)-6,7-dihydro-lH~indazole (0.141 g 0.62 mmol) is dissolved
in EtOH (10 mL) and THF (4 mL) and 5% Pd/C (0.090 g) is added. The mixture is
stirred under hydrogen (40 psi) for 4 hrs. The mixture is filtered through diatomaceous
earth and the filtrate is evaporated to dryness. The residue is purified by silica gel
cliromatography eluting with 98:2 CH Cl :MeOH to give the title compound (0.121g,
85%). ES/MS m/z 229 (M+i).
Example 1
4-[(4R)-(6,6-Dimethyl-4,5-dihydro-lH-cyclopenta[c]pyrazol-4-yl)]benzonitrile
DMF-DMA (46.26 g, 388.22 mmol) is added to (+/-) 4-(3,3-dimethyl-4-oxocyclopentyl)
benzonitrile (46 g, 213.52 mmol) and the mixture is stirred at 100 °C for 6
hours. Excess DMF-DMA is removed by vacuum. Isopropyl alcohol (248 mL) is added
followed by hydrazine monohydrate (10.69 g, 324.61 mmol} and acetic acid ( 11.12 mL).
The mixture is heated at 80 °C for 2 hours. The mixture is cooled to ambient
temperature and the solvent is evaporated to dryness. Water (50 mL) is added and the
mixture is extracted with DCM (3 50 mL). The organic extracts are dried over MgS0 4,
filtered, and concentrated to dryness. The residue is purified by silica gel flash
chromatography, eluting with 1: 1 hexane-ethyl acetate. The mixture of enantiomers is
puri fied by chiral HPLC Chiralpak AD using 40% P A / 60% n-hexane (2% DMEA),
column size 20 mhi, 8 X 25 cm, flow rate of 300 niL/min, UV detection 254 nm, and
loading of 5 g/5 min. The R enantiomer (isomer 1) is obtained by collecting the fraction
eluting at 5.53 minutes.
The enantiomer is farther purified by silica gel flash chromatography eluting
with 4:1 hexane-acetone to give the title compound (9.2 g, 38.77 mmol) as a yello solid.
ES/MS (m/z) 238(M+1), NM (300.16 MHz, CDC13) d 7.60-7.58 (m, 2H), 7.36 (d, J=
8.2 Hz, 2H), 7 (s, H), 4 36 (t, J 8.0 Hz, . 2 74 (dd, J 7.7, 2.6 Hz, HI;, 2.23-
2.16 (m, H), 1.40 (s, 3H), 1.35 (s,
Example a
4-[(4R)-6,6-dimethyl-4,5-dihydro-lH~cyclopenta[c]p>Tazol-4-yl]benzonitrile
hemihydrate
Suspend 4-[(4R)-(6,6-dimethyl-4,5-dihydro- -cyclopenta[c]pyrazol-4-
yl)]benzonitriie (45 mg, 0.190 mmol) in water ( 1 mL) and slurry at ambient temperature
for 1 hour. The solids are vacuum filtered and air dried to give the title compound (35 mg,
76%).
X-Ray Powder Diffraction
The XRD patterns of crystalline solids are obtained on a Bruker D4 Endeavor Xray
powder diffractometer, equipped with a CuKa source (l 1.54060 A) and a Vantec
detector, operating at 35 kV and 50 m.A. The sample is scanned between 4 and 40° in 2Q,
with a step size of 0.0087° in 2Qand a scan rate of 0.5 seconds/step, and with 0.6 mm
divergence, 5.28 m fixed anti-scatter, and 9.5 mm detector slits. The dry powder is
packed on a quartz sample holder and a smooth surface is obtained using a glass slide. It
is well known in the crystallography art that, for any given crystal form, the relative
intensities of the diffraction peaks may vary due to preferred orientation resulting from
factors such as crystal morphology and habit. Where the effects of preferred orientation
are present, peak intensities are altered, but the characteristic peak positions of the
polymorph are unchanged. See, e.g. , The J . . Pharmacopeia 33 National Formulary 28
Chapter < 941> Characterization of Crystalline Solids by X-ray Powder Diffraction
(XRPD) Official October 1, 2010-February 1, 20 . Furthermore, it is also well known
in the crystallography art that for any given crystal form the angular pea positions may
vary slightly. For example, peak positions can shift due to a variation in the temperature
or humidity at which a sample is analyzed, sample displacement, or the presence or
absence of an internal standard. In the present case, a peak position variability of ± 0.2 in
2Qwill take into account these potential variations without hindering the unequivocal
identification of the indicated crystal form. Confirmation of a crystal form may be made
based on any unique combination of distinguishing peaks (in units of ° 2Q), typically the
more prominent peaks. 'The crystal form diffraction patterns, collected at ambient
temperature and relative humidity, are adjusted based on N ST 675 standard peaks at 8.85
and 26.77 degrees 2-theta.
A prepared sample of the title compound is characterized by an XRD pattern
using Cu a radiation as having diffraction peaks (2-theta values) as described in Table 1
below. Specifically the pattern contains a peak at 23.75 in combination with one or more
of the peaks selected from the group consisting of 12.19, 15.53, 17.23, 17.78 and 20.61
with a tolerance for the diffraction angles of 0.2 degrees.
Peak Angle (2-Theta °) Intensity (%)
1 6.06 25
12.19 65
3 15.53 7
4 15.77 33
5 17.23 58
6 17.78 95
18.31 20
8 19.00 24
9 20.61 98
10 21.62 26
11 22.31 45
12 23.75 100
13 24.55 19
14 25.01 2 1
15 26.09 27
1 26.41 44
17 27.96 43
Example lb
4-[(4R)-6,6-Dimethy]-4,5-dihydro-lH-cyckjpenta[c]p>TazoI-4-yl]bOTzonilrile; phosph
acid
Dissolve 4-[(4R)-(6,6-dimeiriyl-4,5-dihydro-lH-cyclopenta[c]pyrazo3-4-
y1)]benzonitrile (445 n g) i isopropyl acetate ( 1 ml. ) To this mixture, is added 5 M
phosphoric acid ( 50 m ., 1.2 eq) drop wise. Localized rapid crystallization is noted and
brief sonication in a water bath precipitated a large plug of bright white solids. This plug
is broken up by adding isopropyl acetate (3 mL) to give a loose slurry. The solids are
then vacuum filtered and air dried to give the title compound (585 mg, 93%).
X-Ray Powder Diffraction
The XRD patterns of crystalline solids are prepared as described in Example 1a.
The title compound is characterized by an XRD pattern using CuKa radiation as
having diffraction peaks (2-theta values) as described in Table 1 below. Specifically the
pattern contains a peak at 5.56 in combination with one or more of the peaks selected
from the group consisting of 12.69, 16.97, 18.25, 19.39 and 22.92 with a tolerance for the
diffraction angles of 0.2 degrees.
Example 2
4-[(4S)-6,6-dimethyl-4,5-dihydro-lH-cyclopenta[c]pyrazol-4-yl]benzonitrile
Example 2 is prepared essentially by the method described for Example 1 by
collecting the traction eluting at 10.25 min. The collected fraction is further purified by
silica gel chromatography with 80% exa e and 20% acetone to give the title compound
(2 g).
Example 3
-i-/ ) 4-(4-Chlorophenyl)-6,6-dm^
(+/-)-4-(4-Chlorophenyl)-2.2-dimeihyl-cyclopentanone (0.250 g, 1.12 mmol) is
dissolved n isopropanol (5mL) and stirred. ¾r/-Butoxybis(dimethylamino)methane (0.33
ml., .57 mmol) is added drop wise to the reaction. The reaction is heated in a sealed vial
at 100 °C for 12 hours, cooled to room temperature, and concentrated to dryness. The
residue is diluted with isopropanol (5 mL). Hydrazine hydrate (0. mL, 2.25 mmol) is
added to the reaction and heated to 100 °C in a sealed vial for 5 hours. The reaction is
concentrated in vacuo. The residue is purified by silica gel flash chromatography eluting
with 20% ethyl acetate in hexanes to obtain the title compound (0.035 g, 3%) as a
yellow film. ES/MS (m/z) 247.0 (M+l).
The following Examples are prepared essentially as described for (+/-)-4-(4-
Chlorophenyl)-6,6-dimethyl-4,5 -dihydro- H-cyclopenta [cjpyrazole.
Table
Example 14
(+/-)-4-(3,4~Difluorophenyl)- 1,4,5,6-tetrahydrocyclopenta[c]pyrazole
To the mixture of (+/-)-(2Z)-3-(3,4-difiuorophenyl)-2-
((dimethylamino)methylene)cyclopentanone (8 5 g, 34 mmol) in ethanol (200 mL), is
added hydrazine hydrate (15 mL) and the mixture is heated to 80 °C, overnight. The
mixture is cooled to room temperature and concentrated in vacuo. The residue is purified
by preparative HPLC using a CXTH instrument with a DAISO 0 8 250 x 50 mm
column a 9 mL injection, a flow rate of 70 niL/min, a wavelength of 2 14 nm and a
mobile phase of 10-80% acetonitrile in 0.1% TFA/H20 to give the title compound as a
white solid (2. 1 , 28%). ES/MS m/z 22 (M+H).
The following Examples are prepared essentially as described for (+/-)-4-(3,4-
difi oropr enyl)-l ,4,5,6-tetrahydrocyclopeiUa[c]pyrazo3e.
Table

a. Upon completion an d concentration in vacuo, the residue is diluted with saturated
sodium bicarbonate solution and extracted with ethyl acetate (3x). The organics
are combined, washed with brine, dried over sodium sulfate, and concentrated in
vacuo. This is purified with silica ge chromatography, eluting with 2:1 pe
ether: ethyl acetate. The crude product is then subjected to the above mentioned
prep PLC conditions, and acidified with HC1 in ethyl acetate, to give a white
solid as the title compound.
b. A catalytic amount of acetic acid is used. The reaction is run at room temperature
for twelve hours.
c. Hydrazine hydrochloride is used instead of hydrazine hydrate.
Example 27
4-(3,4-Difluorophenyl)- 1,4,5,6-tetrahydrocyclopenta[c]pyrazole, isomer 1
The racemic material is subjected to chiral chromatography using Chiralcel® OJH
4 6 x 150 mm column with 20% 3A EtQH:80% C0 2, a flow rate of 5.0 mL/min at UV
of 230 am to give the pure enamiomer, isomer 1. This is then re-purified with silica gel
chromatography eluting with a step gradient, from 25% - 50% ethyl acetate/toluene, to
give the title compound (0. 121 g, 6. %). ES/MS (m/z) 22 .2 (M+1).
The following Examples are prepared essentially as described for 4-(3,4-
difl orop enyl)-l ,4,5,6-tetrahydrocyclopenta[c]pyrazo e isomer 1.
Table 12
H
2-Fluoro-4-(6,6-dimethy]-l,4,5,6-
tetxahydrocyclopenta[c]pyrazol-4- 256
yl)benzonitrile, isomer
N
H
4-(4-Cl lorop enyl)- l 4,5,6
teti¾hydrocyclopenta[c]pyrazole, 2 9
isomer 1°
CI
HN
4-(l ,4,5,6-
Tetrahydrocyclopenta[c]pyrazol- 210
4-yl)benzonitrile, isomer
N
4 (4 Chloropheny )-6.6-dimethy1-
4,5-dihydro-lH- 247
cyclopenta[c]pyrazole, isomer
H
4-(3-Chlorophyenyl)-6,6-
dimeihyl- 1,4,5,6- 247
tetraliydrocyclopenia[c]pyrazole.
isomer c
4-(4-Chloro-3-fluorophenyl)-6,6-
dimethyl- 1,4,5,6- 265
tetrahydrocyclopenta[c]pyrazole,
isomer "
H
4-(4-Chioro-2-fluorophenyl)-6,6-
dimethyl- 1,4,5,6-
F 265
tetrahydrocyclopenta[c]pyrazole,
isomer
CI
H
4-(4-Chloro-2-fl orophenyl)-6,6- _
dimethyl- 1,4,5,6- 265
F tetrahydrocyclopenta[e]pyrazole,
isomer 21
CI
HN
-(4-Fluorophenyl)-6,6-dimethyl-
1,4,5,6- 231
tetrahydrocyclopenta[c]pyrazole,
isomer
F
H
(2-Chloropheny )-6,6-dimethy1-
1,4,5,6- 247
tetrahydrocyclopenta[c]pyrazole,
isomer 21
b. Chiralcel® OJ-H column, 20% IPA(0.2% isopropyl amme/C0 2, 5 mL/min, 225
nM
c . Chiralpak® AD-H column, 0.2% DMEA/methanol, 30 mL/min, 225 nM.
d. Chiracel® OJ-H column, 20%> ) i 1CX 5 mL/min, 225 nM.
e. Chiralpak® C column, 30% IPA C0 2, 5 mL/min, 230 nM. After chiral
chromatography, the product is ehromatographed in a step gradient with 1-3%
MeOH/chloroform, a 2 ' chromatography with 2% MeOH/chloroform and
crystallized with ether to give the final product.
f . Chiralpak® AD-H column, 100% MeOH, 30 mL/min, 225 nM.
g. Chiralpak® AD-H column, 100% MeOH, 30 mL/min, 225 nM.
h. Chiralpak® AD-H column, 15% MeOH/c 0 2, 70 mL/min, 225 nM.
i. Chiralpak® OD-H column, 10% MeOH CO;-. 70 mL/min, 225 nM.
k . Chiralpak® AD-H column, 00% ethanoi, 18 mL/min, 225 nM.
1. Chiralpak®' AD-H column, 20% IPA/CO2, 70 mL/min, 225 nM.
m. Chiralpak® AD-H column, 20% ethanol/C0 2, 70 mL/min, 225 nM.
n. Chiralpak® AD-H column, 4/1 ethanol/ACN, 25 mL/min, 225 nM.
Example 43
(4 )-4-(3-ChlorophenyI)- l ,4,5,6-te ahydrocyclopenta[ ]pyrazo e
5:2
(3S)-3-(3-Chlorophenyl)cyclopentanone (1.5 g, 7.71 mmol) is dissolved in
isopropanoi (20 mL) and stirred. tert-Butoxybis(dimethylamino)methane (1.91 mL, 9.25
mmo ) is added drop wise to the reaction. The reaction is heated to 25 °C for 12 hours.
It is then cooled to room temperature and concentrated to dryness. The residue is diluted
with isopropanoi (20 mL). Hydrazine hydrate (0.37 mL, 1.56 mmol} is added to the
reaction and the reaction is heated to 00 °C for 5 hours. The reaction is concentrated in
vacuo and the residue is purified via silica gel chromatography, eluting with 20% ethyl
acetate in hexanes to obtain the title product (1.099 g) as a yellow film. This material is
further purified via chiral chromatography, employing a Chiralpak AD-H column, eluting
with 20% raethanol/C0 2, a flow rate of 70 mL/min, and UV detection at 225 tiM. The
title compound (0.442g, 26.2%) is isolated as a clear oil. ES/MS (m/z) 219.0 (M+l).
The following Examples are prepared essentially as described for (4R)-4-(3-
chloropheiiyl)~l,4,5,6-tetrahydrocyclopenta[c]pyrazole.
Table 13
a. Ch ra pa AD-H co umn, 100% MeOH, 30 mL m n, 225 nM.
Example 48
(+/-)-4-(6,6-Dimethyl-4,5-dibydro-lH-cyd^
Methanamine, -dirrseihoxy-N.N-dirrseihyl- (42.05 g, 352.84 mmol) is added to
4~(3,3-dimethyl-4~oxo-cyclopentyl)-3-fluoro-benzonitrile (34 g, 17.61 mmol) and the
mixture is stirred at 90 C for 16 hours. The mixture s cooled to ambient temperature
a d the excess DMF-DMA is evaporated to dryness. To the crude residue is added:
isopropyl alcohol (163.20 ml.), hydrazine monohydrate ( 1.78 g. 235 22 mmol), an
acetic acid (20.22 mL, 352.84 mmol) and the mixture is stirred at 70 °C for 2 hours. The
mixture is cooled to ambient temperature and the solvent evaporated to dryness. The
mixture is poured into water (200 mL) and is extracted with MTBE (2x200 mL). The
mixture is washed with brine, dried over MgSC , filtered,, and concentrated to dryness.
The residue is purified by silica gel flash chromatography, eluting with hexane and 10%
PA to give the title compound (30 g, 99%). ES/MS (m/z) 256 (M+l), H NMR (300.16
MHz, DM80) d 12.62-12.60 (m, 1H), 7.82 (dd, J= 1 5, 10.2 Hz, H), 7 62 (dd, J= 1.5, 8.1
Hz, 1H), 7.42-7.34 (m, IH), 4.52 (t, J= 7.7 Hz, IH), 2.73 (d, . 1.8 Hz, 1H), 2.07 (dd, j
8.1, 11.7 Hz, IH), 1.25 (s, 6H).
Example 49
4-(6,6-Dimethyl-4,5-dihydro- lH-cyclo ol-4-yl)-3-fl oro-benzonitri le, isomer
Racemic 4-(6,6-dimethyl-4,5-dihydro-lH-cyxlopenta[c]pyrazo3-4-yl)-3-iluorobenzonitrile
(14.4 g, 56.41 mmol) is purified by chiral supercritical fluid chromatography
(SFC) o a Chirafcel® OD-H column using C0 2 (100 bar) an MeOH with 0 2% DMEA,
column size 5 mh , 2*25 cm, flow rate of 65 niL/min, LJV detection 215 nm, and loading
of 60 rag/injection (each 5.1 min) to give the title compound, RT = 2.4 min, (5.5 g, 38%)
as a yellow solid. ES/MS (m/z) 256 (M+l), !R (300.16 MHz, de-DMSO) d 12.62-
12.60 (m, ), 7.82 (dd, J= 1.5, 10.2 Hz, H), 7.62 (dd, J= 1.5, 8.1 Hz, H), 7.42-7.34
(m, IH), 4.52 (t, J= 7.7 Hz, IH), 2.73 (d, J 1.8 Hz, IH), 2.07 (dd, j 8.1, 1.7 Hz, II I).
1.25 (s, 6H).
Example 50
4-(6,6-Dimethyl-4,5-dihydro-lH-cyd^ isomer
4-(6,6-Dimethyl-4,5-dj y dTO- lH-cyc enta[c]pyrazol-4-y])-3-fl oro-benzonitri l
isomer 2 is isolated using the described chiral chromatography conditions for isomer .
ES/MS (m/z) 256 (M+l).
Example 5
(+/-)-4-[6,6-Dimet yl-4,5-dihydro- lH-cycto penta[c]pyrazo -4-yl]benzonirrile
hydrochloride
To (+/-)-4-[(2Z)-2-(dimethylaniinomethylene)-4,4-diinethyl-3-oxocyclopentyl]
benzonitrile(3.2g, 20 mmo3) in ethanol (100 mL) and acetic acid (4 drops) is
added hydrazine hydrochloride (4.17g, 60 mmol) and the reaction is heated to 80 °C for
three hours. The reaction is then cooled to room temperature and concentrated in vacuo.
The residue is partitioned between ethyl acetate and saturated sodium bicarbonate
solution. The layers are separated and the organic is washed with brine, dried over
sodium sulfate, filtered, and concentrated in vacuo. The residue is purified by silica ge3
flash chromatography eluting with :2 ethyl acetate;pet ether. The resulting material is
treated with HCl in ethyl acetate to provide the title compound (2.3 g, 70%). ES/MS
(m/z) 238.2 (M+l).
Example 52
(+/-)-4-[4,5,6,7-Tetrahydro-2H-mdazo3-4-yl]benzonitrile
4-(6,7-dihydro-2H-mdazol-4-yl)benzonitrile (3.21 g, 14.5 mmol) is added to THF
( 0 ml.,), MeOH ( 0 m .) , a d 5 % Pd/C (0.2 g) and hydrogenated under a balloon of 2
at room temperature for two hours. The mixture is filtered through a pad of
diatomaceous earth and evaporated to dryness. The residue is purified by silica gel flash
chromatography eluting with 50% -70% ethyl acetate/hexanes, to give the title compound
(3.15 g, 97%).
Example 53
4-[(4R)-4,5,6,7-Tetrahydro-2H-indazol-4-yl]beiizonitrile
The racemic mixture is purified by chiral chromatography (Chiralpak AD-H, 0.46
xl5 cm 100% MeOH/0.2% DMEA, 0.6 mL/min, 250 nm) to give the title compound
(1.19 g, 38%) as the second eluting isomer Tr = 3 2 1 min ES MS m/z 224 (M+H).
ES/MS (m/z) 224.0 (M+l).
The following Examples are prepared essentially by the method of 4-[(4R)-
4,5,6,7-tetrahydro-2H-indazol-4-yl]benzonitrile.
Table 14
ES/MS (m/z)
Ex# Chemical Name Structure
(M+l)
Chiral chromatography conditions: (4 6 150 mm. Chiralcel® OJ-H, 100%
MeOH, 0.2% DMEA, 1.0 mL/min, 225 nm, 2nd eluting enantiomer, Tr 3.726
min)
Chiral chromatography conditions: (4.6x150 mm, Chiralcel® AD-H, 100%
EtOH, 0.2% DMEA, 1.0 mL/min, 225 nm, 2nd eluting enantiomer, Tr 3.289
mm).
Example 56
4-(4-Chlorophenyl)-4,5,6,7-tetrahydro-2H-indazole isomer 2
4-(4-Chioropheny])-2-(p4olylsulfonyl)-4,5,6,7-tetrahydromdazoie (0.4 g, 1.04
mmol) is added to a solution of KOH (0.29 g, 5.2 mmol) in MeOH (25 ) and the
solution is heated to 65 °C for 2 hours. The solution is cooled to ambient temperature, the
solvent removed under reduced pressure and the resultmg solid diluted with water. HCl
is added to pH 4, the mixture is extracted with ethyl acetate, the layers are separated and
the aqueous layer is re-extracted with ethyl acetate. The organic layers are combined,
dried over Na2S 4 , filtered, and coriceiiirated to dryness. The residue is purified by chiral
chromatography (Chiralpak AD-H, 4.6x150 mm, 100% EtOH 0 2% DMEA, 225 mm, Tr
= 3.3 8 mm) to give the title compound (93 mg, 38%) as the second eluting isomer.
ES/MS m/z 234 (M+H).
Example 57
3-Methyl-4-(4,5,6,7-tetrahydro-lH-indazol-4-yl)benzomtrile-isomer
3-Methyl-4-(l-tetrahydropyran~2~yl-4,5,6,7-tetTahydroindazol-4-yl)benzonitrile
or 3-methyl-4-(2-tetrahydiOpyran-2-yl-4,5,6,7-tetrahydiOindazol-4-yl)benzonitnle
(0.16 g, 0.5 mmol) and 2S0 4 (0. mL, 1.99 mmol) are added to CH3C (5.0 m ) and
the solution is stirred at room temperature for 24 hours. Aqueous Na C0 3 is added until
basic pH. This is diluted with ethyl acetate the layers are separated, and the aqueous
layer back extracted with ethyl acetate (3x). The orgariics are combined, dried over
Na2S0 4, filtered, and concentrated to dryness. The residue is purified by silica gel flash
chromatography eluting with 6:4 hexanes/ethyl acetate to give the racemic mixture (0.088
g, 73%). The single enantiomer is obtained by chiral chromatography (Chiralpak AD-H,
4.6 50 mm, 100% EtOH 0.2% DMEA, 225 mm, Tr 4.168 min) to give the title
compound (0.032 g, 27%) as the second eluting isomer ES/MS m/z 238 (M+H).
The following Example is prepared essentially by the method of 3~methyl-4
(4,5,6,7-tetraliydro-lH-indazoi-4-yl)benzonitrife isomer 2.
Table 15
ES/MS (m/z)
Ex# Chemical Name Structure
(M+l)
2-Methoxy-4-(4,5,6,7-
tetrahydro- H-indazol-4- 254
58
Yl)benzonitrile, isomer 2
Purification of enantiomers by eh ra chromatography (Chiralcel® AD-H,
4.6 :150 mm, 100% MeOH, 0.2% DMEA, 1.0 niL/min, 225 nm, 2nd e!utin
enaniiomer, Tr = 3.608 min)
ixample 59
(-H/-)-2-Fluofo-4-(4,5,6,7-tetrahydiO-lH-indazol-4-yl)benzonitrile
4-(6,7-Dihydro- lH-indazol-4-yl)~2-flr oro-benzonitrile (0.102 g, 0.43 mmoi) 5 %
Pd/C vvt/wt% (0.04 g) is added to MeOH (5.0 mL) and the mixture is stirred under 45-35
psi of hydrogen for 3 hours. The mixture is filtered through a plug of diatomaceous earth
a d concentrated to dryness. The residue is purified by silica gel chromatography eluting
with 1:1 hexanes/ethyl acetate to give the title compound (0.021 g, 18%). ES/MS m z
242 (M+H).
Example 60
(+/-)-(trans)- 447-Methyl-4,5,6,7-tetrahydro~2H-mdazo{-4--yl]benzonitrile
(+/-)-(trans)-4-(4-Methyl-3-oxo-cyclohexyl)benzonitrile (0.55 g, 2.58 mmol) is
added to toluene (10 0 mL) an d r ,'-butoxybis(dirntlieylarnfflo)rnethane (0.67 mL, 3.22
mmols) and stirred at 120 C for 16 hours. The mixture is cooled to room temperature an d
concentrated in vacuo. The residue is added to MeOH (10.0 mL) and hydrazine (0.07 mL,
2.32 mmol) a d stirred at 80 °C tor 1.0 hour. The mixture is cooled to room temperature
and concentrated in vacuo. The residue is purified by silica gel chromatography (35%-
55% EtOAc/hexanes) to give (+/-)-(trans)-4-[7-methyl-4,5,6J-tetrahydro-2H-indazol-4-
y]]benzonitriie (0.18 g, 29%).
Example 6
4-[7-Methyl~4,5,6,7-tetrahydro-2H-indazol-4-yl]beiizonitrile-isomer 2
and
Example 62
4~[7-Methyl~4,5,6,7-tetrahydro-2H-indazol-4-yl]beiizonitrile-isomer 4
H
(+/-)-(cis/trans)- 4-[7-Methyl-4,5 6,7-tetrahydfo-2H-indazol-4-yl]benzonitrile
(0.36 g, .52 mmol) and (^7-)-(trans)-4-[7-methyl-4,5,6,7-tetrariydro-2H-mdazol-4-
y1]beiizonitrile (0.18 g, 0.76 mmol) are combined and the single enantiomers are obtained
by chiral chromatography (Chiraipak AD-H, 4.6><150 mm, 100% EtOH 0.2% DMEA,
225 mm) to give (cis)- 4-[7-methyl-4,5,6,7-tetrahydro-2H-mdazol-4-yl]benzonitrileisomer
2, (0.05 g, 27%) as the second eluting isomer. ES/MS m/z 238 (M+H), T = 2 988
min. (trans)- 4-[7-Methyl-4,5,6,7-ten ahydro-2 -indazol-4-yl]benzonitrile-isomer 4,
Example 6 1 (0.07 g, 19%). ES/MS m/z 238 (M+H).
Example 63
(+/'-)-4-(7,7-Dimethy3-2,4,5,6-tetxahydroindazol-4-y3)-3-methy]-benzomtri
hydrochloride
4-(4,4-Dimethyl-3-oxo-cyclohexyl)-3-methyl-benzonitrile (0.6 g. 2.49 mmol) in
N, N-dimethylfonnamide dimethylacetal (50 mL) is stirred at 90 C for two days. The
mixture is cooled to room temperature and concentrated in vacuo. The residue is diluted
with ethyl acetate (50 mL) and water (50 mL). The organic phase is separated and the
aqueous phase is extracted with ethyl acetate (3x50 mL). The combined organic phase is
washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The
residue is purified by silica gel flash chromatography, e ing with pet ether: EtOAc :1
to MeOH: DCM =1: 30 to give 4-[(2Z)-2-(dime%laminomethylene)-4,4-dimethyl-3-
oxo-cyclohexyl]-3-niethyl-benzonitrile (0.2 g, 27%).
Hydrazine hydrochloride (0.14 g, 2 mmol) is added to 4-[(2Z)-2-
(dimemyiaminomethylene)-4,4-dimethyl-3-oxo-cyclohexyl]-3-methyl-benzordtrile (0.2 g,
0.67 mmol) in ethanol (30 mL), then acetic acid (two drops) is added to the mixture. After
the addition is complete, the reaction mixture is stirred at 80 C for 3 h. The mixture is
cooled to room temperature and concentrated in vacuo. The residue is diluted with ethyl
acetate (50 mL) and saturated sodium bicarbonate solution (50 mL). The organic phase is
separated and the aqueous phase extracted with ethyl acetate (3x50 mL). The combined
organic phase is washed with brine, dried over anhydrous sodium sulfate, and
concentrated in vacuo. The residue is purified by silica gel column chromatography
(eluted with pet ethenEtOAc = 2:1 to 1:1) to give the title compound. The product is
added to HCT/ethyl acetate and concentrated in vacuo to give the HCI salt of the title
compound (0. 2 g, 67%). ES/MS 266 (M+H).
Example 64
4-(7,7-Dimethyl-2,4,5,6-terrahydiOindazol-4-yl)-3-methyl-benzonitrile, isomer 2
The single enantiomer is obtained by chiral chromatography (Chiralpak AD-H,
4.6 50 mm, 3:2 MeOH/CH3CN 0 2% isopropylamine, 225 mm, .0 mL/min, Tr 3.503
min) to give the title compound (0 038 g, 3 %) as the second eluting isomer. ES/MS m/z
266 (M+H).
The following Examples are prepared essentially by the method of 3-methyl-4-
(4,5,6,7-tetraliydro-lH-indazol-4-yl)benzonitrife.
a. 'urifieation of enantiomers by chiral chromatography (Chiralcel® AD-H,
4.6 <150 mm, 00% MeOH, 0.2% isopropyl amine, 1.0 mL/min, 225 nm, 2nd
eluting enantiomer, Tr = 4.175 min).
b. Step -X se ½r t-butoxybis(diniethylamino)niethane ( 1.0 eq) in toluene at 120 °C
for 26 hours.
Reagents employed in the following assays are readily available from commercial
sources or can be readily synthesized by one skilled in the art. Comparator compounds
used herein are fadrozole and LCI699. Fadrozole is an aromatase inhibitor marketed by
Novartis Corporation in Japan for the treatment of breast cancer under the trade name
AFEMA® (trademark of Novartis Corporation); (vww.righthealih.com/topic/Padrozoie
i i d May 26, 20 ! }. LCI 699 is an investigational drug being developed by Novartis
Corporation (Thompson Reuters Pharma Drug Report LCI699, (©Thompson Reuters
20 ) . Structural representations for fadrozole and LC1699 are as shown below.
Fadrozole LCI 699
Aldosterone Synthase inhibitor Assay
Chinese hamster fibroblast cells (V79, ATCC™) constitute vely expressing human
cypl 1B2 are established by transfection with a mammalian expression vector harboring
the huma cypl lB2 cD A under CMV promoter and a neomycin antibiotic resistant
gene for selection in mammalian cells. V79 cells are transfected in T225 cm2 flasks with
the lipofectamine transfection reagent and the human cy l 1B2 cDNA. Positive clones
are selected with the selection antibiotic geneticin at 1 mg/mL.
Aldosterone production from transfected cells is initiated by the addition of 1 mM
DOC in the medium. After 24 hours incubation, 100 L ceil culture medium is collected
and aldosterone concentration in the medium is determined using a liquid
chromatography-mass spectrometry (LC-MS) method. The medium is first extracted
using a Beckman Coulter FX liquid handling system (modified for use of organic
solvents) with a 96-tip head to add an internal standard (IS) solution to each well (10 mL•
of 100 ng/mL d7-aldosterone, (C/D/N Isotopes, Inc. Quebec, Canada}, in 15%
ACN/water). The wells are then extracted 3* with EtOAc (150 m ) using the FX,
combining the organic layers in a new 96-well plate. The solvent is dried in a Gene Vac
HT-4 or under nitrogen. The FX is then used to reconstitute the samples in %
ACN/water (60 mί ) and the plates are heat-sealed. The LC-MS method employs an
Agilent LC with a binary pump to produce a gradient of water and ACN, each containing
0.1% formic acid,, with a flow rate of 1 mL/min over a Betasil 2.1 x 10 mm C18 column.
A 25 mΐ aliquot of the sample is injected and a gradient from 20-100% ACN+0.1% formic
acid (FA) in i mm is initiated. Aldosterone eluies at 0.7 min. Starting conditions are
held for 1 minute to re-equilibrate the column. An AB 4000 tandem mass spectrometer
is used for MS/MS analysis in the negative ion mode. The MS/MS method monitors two
multiple reaction monitoring (MRM) transitions for aldosterone (359.19/331.09 &
359.19/188.8) and two for the IS (367.19/339.3 & 367.19/194.1). The area under the
peak from each transition is added together to comprise the signal from aldosterone and
IS, respectively. The ratio of these areas is compared to the controls from each plate to
give a % inhibition value for each sample. The detection limit for aldosterone is 40
pg/mL.
To determine the inhibition of aldosterone production by a test compound, V79-
hcypl 1B2 cells are seeded in a 96-well plate at 20,000 cells per well. DOC and various
concentrations of test compounds in 1:3 dilution increments are added to the cell culture
medium. After 24 hours incubation, 100 mΐ of cel medium are collected and aldosterone
concentration determined as described above. Data are fit to a 4 pararneter-fit logistics
curve to determine IC5 values.
Tl e Examples of the invention demonstrate potent aldosterone synthase inhibition
with IC50S of about <0.900 mM. Representative compounds are shown in Table 1.
Table 17*
*These data are the results from separate experiments. The above data expressed as a
geometric means show that Examples of the invention are potent aldosterone synthase
inhibitors i vitro.
Inhibition of Aldosterone Synthase in Rats
The effect of compounds on aldosterone production in rats is assessed using the
rat sodium-deficiency diet model. Studies are conducted using male Sprague Dawiey
rats, aged 6-7 weeks, and approximately 175-190 grams (Harlan Laboratories,
Indianapolis, IN, USA). Rats are singly housed under normal fight cycle (12 hours fight
and 12 hours dark) and received diet (Harlan Teklad 90228 Sodium Deficient Diet) and
water ad libitum. Rats are randomized by body weight and placed on Teklad 90228 for 7
days. On Day 7 rats are orally dosed at 10 mL/kg with vehicle (1% hydroxy ethyl
cellulose (HEC) / 0.25% TweenSO / 0.05% antifoam (AF), or Acacia 10% w/v / Antifoam
1510-US 0.05% v/v deionized water (DiW)). positive control ( 1 mg/ g, Fadrozole), or
test compound. At 3 hours post dose rats are bled (-0.5 mL) from the ocular orbit under
isoflurane anesthesia. At 6 hours post-dose the rats are euthanized with C0 2 and bled by
cardiac puncture. Blood samples are clotted at least 30 minutes and serum is prepared
and stored at approximately -80 °C until assayed. Aldosterone, steroids, and compound
exposure are analyzed by mass spectroscopy.
The effect of Examples 1 and 49 on aldosterone production in the rat Na-deficient
diet model is illustrated in Table 8 below.
Table 8
The data show that the Examples 1 and 49 inhibit aldosterone production in vivo
Cortisol Inhibition Assay
Chinese hamster fibroblast cells (V79) constitute vely expressing human cy 1B1
are established by transfeciion with a mammalian expression vector harboring the human
cyp B cDNA under CMV promoter and a neomycin antibiotic resistant gene for
selection in mammalian cells. V79 cells are transfected in T225 cm2 flasks with the
lipofectamine transfection reagent and the human cypl 1B cDNA. Positive clones are
selected with the selection antibiotic geneticin at 1 mg/mL. Cortisol production from
transfected cells is initiated by the addition of 1 mM 1 -deoxycortisol in the medium.
After 24 hours incubation, culture medium is collected and Cortisol concentration in the
medium is determined using a liquid ehromatography-mass spectrometry (LC-MS)
method. The cell media (100 m ) is transferred to a new deep-well 96-weli plate. A
Beckman Coulter FX liquid handling system (modified for use with organic solvents)
with a 96-tip head is used to add a IS solution to each wel (10 m of 200 ng/mL d4-
Cortisol). The wells are then extracted 3 with EtOAc (300 m ) using the FX. combining
the organic layers in a ne deep-well 96-well plate. The solvent is then dried in a
GeneVac HT-4 or under nitrogen. The FX is then used to reconstitute the samples in
50% MeOH/water ( 00 mΐ) and the plates are heat-sealed.
An HPLC with two pumps produces a gradient of water (containing 0.1% formic
acid) and MeOH with a flow rate of 0.6 mL/min over an Xbridge Shield P 18, 3.5
micron, 2.1 x 50 mm column with a 2 0 mm guard column of the same material in a 5
micron particle size. A 40 m aliquot of the sample is injected and a gradient from 20-
100% MeOH in 0.95 min is initiated. Cortisol elutes at 0 8 min. Starting conditions are
then held for 1 minute to re-equilibrate the column. An AB QTRAP 4000 tandem mass
spectrometer is used for MS/MS analysis in the positive ion mode. The MS/MS methods
monitor transitions for the Cortisol and IS at 363.0/ 12 0 and 367.3/121 .0 respectively.
These are respectively integrated to give the peak areas. The cortisol/IS area-ratio is used
to determine Cortisol concentration by comparison to a standard curve. The detection
limit for Cortisol is 1 ng/mL.
To determine the inhibition of Cortisol production by a test compound, V79-
human cyp B cells are seeded in a 96-weil plate at 20,000 cells per well. -
Deoxycortisol and various concentrations of test compounds in 1:3 dilution increments
are added to the cell culture medium. After 24 hours incubation, 100 m.1 of cell medium
are collected and Cortisol concentration determined as described above. Data are fit to a 4
parameter-fit logistics curve to determine IC50 values.
The Examples of the invention demonstrate modest potency in inhibiting Cortisol
production from V79-hcyp l Bl cells compared to comparator compounds as shown in
Table . The relative selectivity of inhibiting aldosterone production versus that of
inhibiting Cortisol production is calculated using the equation: Selectivity Ratio
ICsoChcypi 1B1) / IC 0(hcypl 1 2) .
Table 19*
* These data are the results from separate experiments. These data demonstrate that
Examples 1 an d 49 exhibit greater selectivity in the inhibition of aldosterone relative to
Cortisol inhibition than the comparator compounds.
Testosterone and Estradiol Production Assay
The human adrenocarcinoma cell line H295R is used to monitor the production of
testosterone and estradiol in vitro. Ceils seeded in 96-well plate at 50 000 cells per well
and cultured in DMEM medium supplemented with 2.5% Nuserum. Various
concentrations of tes compounds in 1:3 dilution increments are added to the cell culture
medium. After incubation for 48 hours, 00 mΐ culture medium is collected and d5-
estradiol and d3-testosterone are added as ISs for estradiol and testosterone respectively.
A equal volume of sodium carbonate/sodium bicarbonate buffer (0.5 mo3/L, pH
9.4) is added to the samples followed by freshly prepared dansyl chloride solution (50 m ,
20 mg/mL). Samples are mixed and incubated for 60 min at 60 °C. The samples are then
extracted 3x with EtOAc (300 mί ) using the FX, combining the organic layers in a new
deep-well 96-well plate. The solvent is then dried in a GeneVac HT-4 or under nitrogen.
The FX is used to reconstitute the samples in 50% MeOH/water (100 mΐ) and the plates
are heat-sealed.
A HPLC with two pumps produces a gradient of water (containing 0. % formic
acid) and MeOH with a flow rate of 0.6 mL/'min over an Xbridge Shield RP18, 3.5
micron 2.1 x 50 mm column with a 2.1x10 mm guard column of the same material in a 5
micron particle size. A 40 mΐ aliquot of the sample is injected and a gradient from 20-
100% MeOH in 0.95 min is initiated. An ABT QTRAP 4000 tandem mass spectrometer
is used for MS/MS analysis in the positive ion mode. The MS/MS methods monitor
transitions for testosterone (289/97), estradiol (506.3/171.0), and their respective ISs
292/109 and5 .3/ 71.0. These peaks are separately integrated to give the peak areas.
The area ratios of testosterone/18 and estradiol/IS are used to determine testosterone and
estradiol concentrations by comparison to their respective standard curves. The detection
limits for testosterone and estradiol are 0.1 ng/mL and 0.01 ng/mL respectively.
Examples 1 and 49 demonstrate weak inhibition of testosterone and estradiol
production from H295R cells. The results are shown in Table 20 along with the relative
selectivity for aldosterone compared to testosterone or estradiol for each compound.
Table 20*
Tests were not performed simultaneously for Examples of Table 20 and comparator
compounds.
Cynomolgus Monkey Aldosterone Inhibition Assay
Chinese hamster fibroblast cells (V79) constitutively expressing cynomolgus
monkey cyp B2 is established by transfection with a mammalian expression vector
harboring the cynomolgus monkey c p l B2 cDNA. This cell line was used to measure
the activity of compounds in inhibiting aldosterone production from cynomolgus monkey
enzyme. Cell culture condition and aldosterone detection method is performed following
the same protocol described in the "Aldosterone inhibition assay". Example 1 and
Example 49 display relative IC50 values of 0.00246 and 0.00 1 mM i the cynomolgus
monkey aldosterone inhibition assay respectively (11 = 1).
Cynomolgus Monkey Cortisol Inhibition Assay
Chinese hamster fibroblast cells (V79) constitutively expressing cynomolgus
monkey cypl B is established by transfection with a mammalian expression vector
harboring the cynomolgus monkey cy B cD A. This ce l line was used to measure
the activity of compounds in inhibiting aldosterone production from cynomolgus monkey
enzyme. Cell culture condition and Cortisol detection method is performed following the
same protocol described in the "Cortisol inhibition assay " . Example 1 and Example 49
display relative IC50 values of 0.209 and 0.579 mM in the cyiiomolgus monkey Cortisol
inhibition assay respectively (n = 1).
We claim:
1. A compound of the formula:
wherein
n is 0, i , or
m is 1 or 2;
R and R2 are independently selected from hydrogen, -C 3, and -CH 2C 3;
R3 is hydrogen, - C , -F, - C , -CH 3, ' or -CF 3;
R is at each instance independently selected from -F, -CI, -Br, -CH 3,
( . !] . and -CN;
or a pharmaceutically acceptable sa t thereof.
A compound of Claim 1 of the formula:
wherein
n is 0, 1, or 2;
R and R2 are independently selected from hydrogen, -CH 3, and -CH 2CH3;
R is hydrogen, -CN, -F, -CI, or -CF 3;
R is at each instance independently selected from -F. -CI, -Br, - C , -CF
and -CN;
or a pharmaceutically acceptable salt thereof.
A compound of Claim 1 of the formula:
wherein
n is 0 or ;
R1 and R2 are independently selected from hydrogen and -CH 3;
R is hydrogen, -CN, -CI, -OCH 3 or-CH ;
R4 is at each instance independently selected from -F, -CH 3, and -OCH 3;
or a pharmaceutically acceptable salt thereof.
A compound of any of Claims 1 or 2 wherein m is 1; R and R2 are -CH 3; R
is -CN ; n is 0 or ; R4 is -F, or a pharmaceutically acceptable salt thereof.
A compound of any of Claims 1 to 2 or 4 wherein the compound
dime hy l-4,5-dihydro- lH-cyclopen a[c]pyrdzo -4-yl)benzonitril :
or a pharmaceutically acceptable salt thereof.
A compound of Claim 5 wherein the compound is (6,6-dimetliyl-4,5-
diliydro - H-cyelopenta [c]pyrazol-4 yl)]benzonitril
or a pharmaceutically acceptable sal thereof.
A compound of Claim 6 wherein the compound is 4-[(4R)-(6,6-dimethyl-4,5-
dihydro-lH-cyc1openta[c]pyrazol-4-yl)]benzonitrile:
A compound of any of Claims 1 to 2 or 4 wherein the compound is 4-(6,6-
dimethyl-4,5-dihydro-ni-cyclopeiita[c]pyrazo3-4-yl)-3-fluoro-benzonitrile:
or a pharmaceutically acceptable salt thereof.
9. A compound of Claim 8 wherein the compound is 4-(6,6-dimethyi-4,5-
dihydro-l -cyclopenta[c]pyrazol-4 yl)-3-fl oro-benzonitrile:
0 . A pharmaceutical composition comprising a compound according to any of
Claims 1 to 9, or a pharmaceutically acceptable salt thereof, and one or more
pharmaceutically acceptable carriers diluents, or excipients.
1. A method for treating chronic kidney disease comprising administering an
effective amount of a compound of any of Claims 1 to 9, or a
pharmaceutically acceptable salt thereof to a patient in need thereof.
12. A method for treating diabetic nephropathy comprising administering an
effective amount of a compound of any of Claims 1 to 9, or a
pharmaceutically acceptable salt thereof to a patient in need thereof.
13. A compound of any of Claims 1 to 9, or a pharmaceutically acceptable salt
thereof, for use in therapy.
14. A compound of any of Claims 1 to 9, or a pharmaceutically acceptable salt
thereof, for use in the treatment of chronic kidney disease.
15. A compound of Claim 14, or a pharmaceutically acceptable salt thereof, for
use in the treatment of diabetic nephropathy.
16. A compound of any of Claims 1 to 9 or a pharmaceutically acceptable salt
thereof, for use in the manufacture of a medicament for the treatment of
chronic kidney disease.
17. A compound of any of Claims 1 to 9, or a pharmaceutically acceptable salt
thereof, for use in the manufacture of a medicament for the treatment of
diabetic nephropathy.