CATHEPSIN S INHIBITOR COMPOUNDS
The present invention is directed toward inhibitor compounds of the proteolytic
enzyme cathepsin S and methods of treatment comprising administration thereof.
Specifically, the present invention is directed toward stereoisomers. More specifically,
the present invention provides potent, selective and reversible stereoisomer inhibitor
compounds having two chiral centers.
Cathepsin S is a lysosomal cysteine protease. It belongs to a larger family of
cathepsins, including L, B, K, V and F. Selectivity to cathepsin S may avoid undesired
consequences such as side effects. Cathepsin S is produced by inflammatory cells such as
dendritic cells, B lymphocytes and macrophages. It is involved in the pathology of
several conditions including atherosclerosis and abdominal aortic aneurysm (AAA) (J.
Clin. Invest. 1999, 104(9), 1191-1 \91)(Am. J. Path. 2007, 170(3), 809-817).
The endothelial cells of the arterial wall may malfunction due to several factors
that lead to plaque formation and buildup in the arterial wall including high levels of
cholesterol, stress, overall health and genetics. This malfunction leads to the production
and recruitment of inflammation cells from the blood that penetrate the arterial wall to
protect from damage. These inflammation cells ultimately produce cathepsin S. An
effect of cathepsin S is to degrade the extracellular matrix proteins such as elastin and
collagen that make up the arterial wall. Although extracellular remodeling of the cells is
ongoing to repair the damaged arterial wall, if too much proteolytic degradation of the
matrix occurs, compared to deposition of matrix proteins, an imbalance may lead to
instability of plaques formed within the arterial wall. Too much plaque instability could
result in plaque rupture and potentially thrombotic -related events. Thus, inhibition of
cathepsin S provides a means for treating atherosclerosis.
Occasionally, the extracellular matrix of the abdominal aorta may also be
weakened by the excess degradation leading to a condition known as AAA. Currently,
AAA is the tenth leading cause of death in men greater than 55 years old. There is no
known approved medication treatment indicated for AAA. Inhibition of cathepsin S
provides an option for addressing this unmet medical need.
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Furthermore, Cathepsin S is implicated in autoimmune disorders which may
include rheumatoid arthritis, lupus and psoriasis, through its intracellular trafficking
involvement in the initiation of an immune response (Immunity, 2001, 15, 909-9 \9)(Eur.
J. Immunol. 2005, 35, 2552-2562). Specifically, cathepsin S cleaves lip 10 (plO) in B
lymphocyte and dendritic cells to generate CLIP (class-II associated Ii-peptide). This
allows loading of a peptide fragment, e.g., self-antigen, and subsequent presentation of
the class II Major Histocompatability Complex (MHC) molecules on the cell surface of
the antigen presenting cells. Subsequent activation of immature T cells results thereby
generating an autoimmune response. Thus, inhibition of cathepsin S blocks plO
processing and surface presentation of T cell antigens, thereby providing a means for
treating autoimmune related disorders such as rheumatoid arthritis, psoriasis, and lupus.
Accordingly the present invention provides a compound of Formula (I):
wherein Z is -CH2-, -CH2CH2-, -OCH2-, -CH2CH2CH2-, or-OCH 2CH2-;
R1 is H, F, or CI;
R2 is H, methyl, ethyl, propyl, or isopropyl;
or a pharmaceutically acceptable salt thereof.
An aspect of the present invention provides a pharmaceutical composition
comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof and
a pharmaceutically acceptable diluent or carrier.
Another aspect of the present invention provides methods for treatment of AAA
by administering a therapeutically effective amount of a compound or pharmaceutically
acceptable salt thereof of the present invention or a pharmaceutical composition
comprising a compound or pharmaceutically acceptable salt thereof of the present
invention. A further aspect of the present invention provides methods for treatment of
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AAA in a mammal such as a human, dog, cat, cow, horse, sheep, or monkey by
administering a therapeutically effective amount of a compound or pharmaceutically
acceptable salt thereof of the present invention or a pharmaceutical composition
comprising a compound or pharmaceutically acceptable salt thereof of the present
invention. A further aspect of the present invention provides methods for treatment of
AAA in a human whose aortic diameter is greater than the normal diameter of
approximately 3 cm but less than approximately 5 cm and surgical or endovascular repair
is not required, by administering a therapeutically effective amount of a compound or
pharmaceutically acceptable salt thereof of the present invention or a pharmaceutical
composition comprising a compound or pharmaceutically acceptable salt thereof of the
present invention. Yet a further aspect of the present invention provides methods for
treatment of AAA in a human whose aortic diameter is greater than approximately 5 cm
but surgical or endovascular repair is not a treatment option by administering a
therapeutically effective amount of a compound or pharmaceutically acceptable salt
thereof of the present invention or a pharmaceutical composition comprising a compound
or pharmaceutically acceptable salt thereof of the present invention.
Another aspect of the present invention provides methods for treatment of plaque
instability in a mammal such as a human, dog, cat, cow, horse, sheep, or monkey by
administering a therapeutically effective amount of a compound or pharmaceutically
acceptable salt thereof of the present invention or a pharmaceutical composition
comprising a compound or pharmaceutically acceptable salt thereof of the present
invention.
A further aspect of the present invention provides methods for treatment of
atherosclerosis in a mammal such as a human, dog, cat, cow, horse, sheep, or monkey by
administering a therapeutically effective amount of a compound or pharmaceutically
acceptable salt thereof of the present invention or a pharmaceutical composition
comprising a compound or pharmaceutically acceptable salt thereof of the present
invention.
Yet another aspect of the present invention provides methods for treatment of
autoimmune disorders in a mammal such as a human, dog, cat, cow, horse, sheep, or
monkey in need thereof by administering a therapeutically effective amount of a
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compound or pharmaceutically acceptable salt thereof of the present invention or a
pharmaceutical composition comprising a compound or pharmaceutically acceptable salt
thereof of the present invention. A further aspect of the present invention provides
methods for treatment of psoriasis, rheumatoid arthritis, or lupus in a human by
administering a therapeutically effective amount of a compound or pharmaceutically
acceptable salt thereof of the present invention or a pharmaceutical composition
comprising a compound or pharmaceutically acceptable salt thereof of the present
invention.
A further aspect of the present invention provides a compound or
pharmaceutically acceptable salt thereof for use in therapy. Another aspect of the present
invention provides a compound or pharmaceutically acceptable salt thereof for use in the
treatment of abdominal aortic aneurysm, plaque instability, atherosclerosis, or
autoimmune disorders such as rheumatoid arthritis, psoriasis, and lupus. Yet another
aspect of the present invention is the use of a compound or pharmaceutically acceptable
thereof for the manufacture of a medicament for the treatment of abdominal aortic
aneurysm, plaque instability, atherosclerosis, or autoimmune disorders such as
rheumatoid arthritis, psoriasis, and lupus.
Another aspect of the present invention provides a pharmaceutical composition
comprising a compound or pharmaceutically acceptable salt thereof of the present
invention in combination with one or more pharmaceutically acceptable carriers, diluents,
or excipients, and optionally one or more other therapeutic agents.
Yet another aspect of the present invention provides a compound of the present
invention, or a pharmaceutically acceptable salt thereof, for use in the treatment of
abdominal aortic aneurysm, plaque instability, atherosclerosis, rheumatoid arthritis,
psoriasis, and lupus.
Yet another aspect of the present invention provides the use of a compound of the
present invention, or a pharmaceutically acceptable salt thereof, for the manufacture of a
medicament for the treatment of abdominal aortic aneurysm, plaque instability,
atherosclerosis, rheumatoid arthritis, psoriasis, and lupus.
Embodiments of the inhibitor compounds and methods for treatment comprising
administration thereof in the present invention include any combination of Ri, R2 and Z
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as described above. Specifically, an embodiment of the present invention is directed
toward a compound of Formula (I) or pharmaceutically acceptable salt thereof where Ri
is H or F. More specifically, an embodiment of the present invention is directed toward a
compound of Formula (I) or pharmaceutically acceptable salt thereof where Ri is F.
Another embodiment of the present invention is a compound of Formula (I) or
pharmaceutically acceptable salt thereof where R2 is methyl or ethyl. Specifically, an
embodiment of the present invention is a compound of Formula (I) or pharmaceutically
acceptable salt thereof where R2 is methyl.
Yet another embodiment of the present invention is directed to a compound of
Formula (I) or a pharmaceutically acceptable salt thereof where Z is -CH2-, -CH2CH2-,
-OCH2-, -CH2CH2CH2-, or -OCH 2CH2- . Specifically, an embodiment of the present
invention is a compound of Formula (I) or pharmaceutically acceptable salt thereof where
Z is -CH2CH2- or -OCH2- . Further embodiments of the present invention consist of a
combination where Ri is H or F, R2 is methyl or ethyl, and Z is -CH2CH2- or -OCH2-.
Preferred compounds of the present invention as exemplified hereafter are:
[(3R,4S)-4-[(4-Fluorobenzoyl)amino]-6-[4-(oxetan-3-yl)piperazin-l-yl]chroman-3-yl] Nmethylcarbamate,
[(lS,2S)-l-[(4-Fluorobenzoyl)amino]-7-[4-(oxetan-3-yl)piperazin-l-yl]tetralin-2-yl] Nmethylcarbamate,
[( 1S,2S)- 1-[(4-Fluorobenzoyl)amino] -6- [4-(oxetan-3-yl)piperazin- 1-yl] indan-2-yl] Nmethylcarbamate,
[(8S,9S)-9-[(4-fluorobenzoyl)amino]-2-[4-(oxetan-3-yl)piperazin-l-yl]-6,7,8,9-
tetrahydro-5H-benzo[7]annulen-8-yl] N-methylcarbamate, and
[(4S,5S)-5-[(4-fluorobenzoyl)amino]-7-[4-(oxetan-3-yl)piperazin- l-yl]-2, 3,4,5-
tetrahydro-l-benzoxepin-4-yl] N-methylcarbamate.
More preferred compounds of the present invention are:
[(3R,4S)-4-[(4-Fluorobenzoyl)amino]-6-[4-(oxetan-3-yl)piperazin-l-yl]chroman-3-yl] Nmethylcarbamate,
[(lS,2S)-l-[(4-Fluorobenzoyl)amino]-7-[4-(oxetan-3-yl)piperazin-l-yl]tetralin-2-yl] Nmethylcarbamate,
and
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[(8S,9S)-9-[(4-Fluorobenzoyl)amino]-2-[4-(oxetan-3-yl)piperazin-l-yl]-6,7,8,9-
tetrahydro-5H-benzo[7]annulen-8-yl] N-methylcarbamate.
Most preferred compound of the present invention is:
[(3R,4S)-4-[(4-Fluorobenzoyl)amino]-6-[4-(oxetan-3-yl)piperazin-l-yl]chroman-3-yl] Nmethylcarbamate.
As used above and throughout the specification of the invention, the following
terms, unless otherwise indicated will have the following meaning:
The term "abdominal aortic aneurysm" (or "AAA") as used herein shall mean a
localized dilation or bulge of the abdominal aorta in a mammal causing the size of at least
a segment of the abdominal aorta to exceed the size of an otherwise considered normal
state. The abdominal aorta may be measured and compared in terms of any measurement
dimension including but not limited to luminal diameter, luminal perimeter, and luminal
area. The means for measurement and diagnosis may be through the use of ultrasound,
CT scan, or other imaging techniques. For example, AAA is present in a human when the
aortic diameter is greater than its normal diameter, approximately 3 cm. If the aortic
diameter is however more than approximately 5 cm, then immediate surgical or
endovascular repair (stent or graft) is the standard of care to prevent rupture and potential
fatality. If however such treatment is unavailable or not an option due to any reason, e.g.,
age, then this population may also be treated using the present invention.
The term "in need thereof as used herein shall mean having or being diagnosed
with a condition, e.g., atherosclerosis, AAA, autoimmune disorder such as psoriasis,
lupus or rheumatoid arthritis, that requires treatment.
The term "mammal" as used herein shall mean a human or nonhuman mammal
such as a dog, cat, cow, horse, sheep, or monkey.
The term "pharmaceutically acceptable salt thereof refers to salts of the
compounds of the present invention. Examples and methods for their preparation are well
within the knowledge of those skilled in the art. See, for example, Stahl et ah,
"Handbook of Pharmaceutical Salts: Properties, Selection and Use," VCHA/Wiley-VCH,
2002.
The term "therapeutically effective amount" refers to the amount or dose of a
compound of Formula (I) or composition comprising a compound of Formula (I) to
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achieve treatment. A therapeutically effective amount can be readily determined by the
attending physician, as one skilled in the art, by considering a number of factors known to
a person skilled in the art such as, for example, weight, height, age, general health of the
patient, severity of the condition, mode of administration, dosing regimen, etc.
The term "treatment" as used herein shall mean slowing the rate or progression of
a disease state. It may also include halting the disease state. The term may further
include not only halting the disease but also reducing any disease state that already has
occurred. For example, in the context of AAA, the term "treatment" may mean slowing
of the expansion rate of an abdominal aortic aneurysm. It may also include stopping the
expansion of the abdominal aortic aneurysm. Furthermore, it may include reducing any
expansion that has already occurred.
The designation " " refers to a bond that protrudes forward out of the plane of
the page.
The designation " "" " refers to a bond that protrudes backward out of the plane
of the page.
The terms "-OCH 2-" and "-OCH 2CH2-" within the definition of "Z" are
understood to mean that the oxygen is adjacent to the fused benzo ring.
The compounds of the present invention are preferably formulated as
pharmaceutical compositions. Examples and methods for their preparation are well
within the knowledge of those skilled in the art. See, for example, Remington: The
Science and Practice of Pharmacy (A. Gennaro, et al, eds. 19th ed., Mack Publishing Co.,
1995).
The compounds of Formula I, or salts thereof, may be prepared by a variety of
procedures known in the art, as well as those described in the Schemes, Preparations, and
Examples below. The specific synthetic steps for each of the routes described may be
combined in different ways to prepare compounds of Formula I, or pharmaceutically
acceptable salts thereof.
The following preparations and examples further illustrate the invention and
represent typical synthesis of the compounds of Formula (I), including any novel
compounds, as described generally above. The reagents and starting materials are readily
available to, or may be readily synthesized by, one of ordinary skill in the art. It should
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be understood that the Preparations and Examples are set forth by way of illustration and
not limitation, and that various modifications may be made by one of ordinary skill in the
art.
The R or S configuration of compounds of the invention may be determined by
standard techniques such as X-ray analysis and correlation with chiral-HPLC retention
time. The naming of the following Preparations and Examples is generally done using
the IUPAC naming feature in Symyx Isentris® version 3.2.3.
As used herein, the following terms have the meanings indicated: "AcOH" refers
to acetic acid; "BCA" refers to bicinchoninic acid; "b.i.d." refers to two times a day;
"brine" refers to saturated aqueous NaCl solution; "cat." refers to a catalytic amount;
"CD74" refers to the invariant chain (Ii); "CDI" refers to , -carbonyldiimidazole;
"DMAP" refers to 4-dimethylaminopyridine; "DMSO" refers to dimethyl sulfoxide;
"DTT" refers to dithiothreitol; "EDTA" refers to ethylenediaminetetraacetic acid;
"EtOH" refers to ethanol; "hr." refers to hour(s); "IC50" refers to the concentration of an
agent which produces 50% of the maximal inhibitory response possible for that agent or,
alternatively, to the concentration of an agent which produces 50% displacement of
ligand binding to the receptor; "IMAC" refers to Immobilized Metal Affinity
Chromatography; "IPA" refers to isopropyl alcohol; "LC ES/MS" refers to liquid
chromatography electrospray mass spectrometry; "MCPBA" refers to metachloroperoxybenzoic
acid; "MeOH" refers to methanol; "min." refers to minute(s);
"NBS" refers to N-bromosuccinimide "NMP" refers to N-methylpyrrolidine; "/"
refers to not available; "plO" refers to a fragment of the invariant chain CD74; "PBS"
refers to phosphate buffered saline; "PWBC" refers to peripheral white blood cells;
"PvFU" refers to relative fluorescence units; "SFC" refers to supercritical fluid
chromatography; "STAB" refers to sodium triacetoxyborohydride; "SDS" refers to
sodium dodecyl sulfate; "THF" refers to tetrahydrofuran; "t-boc" or "boc" refers to tertbutoxycarbonyl;
"TRITON® X-100" refers to polyethylene glycol p-(l, 1,3,3-
tetramethylbutyl)-phenyl ether.
The substituents, unless otherwise indicated, are as previously defined.
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Scheme 1
chiral
(3)
Formation of intermediate (3) can be carried out in accordance with reactions as
depicted in Scheme 1.
In Scheme 1, Step A, 6-bromo-2H-chromene is treated with NBS to form a
bromohydrin (1). Preferred conditions use a solvent mixture of DMSO/water at about 0 -
50 °C, but more preferably at room temperature.
In Step B, the bromohydrin (1) is treated with ammonium hydroxide to provide
the amino alcohol (2). For example, the bromohydrin is reacted with ammonium
hydroxide in a solvent mixture of THF and EtOH at room temperature to 60 °C for 4 to
24 hr.
In Scheme , Step C, the racemic amino alcohol is resolved into its 3R, 4S and 3S,
4R enantiomers to provide the chiral amino alcohol (3). Methods for resolution are
commonly known to those skilled in the art and include crystallization as a salt of a chiral
acid, or separation of the enantiomers by chiral chromatography.
6-Bromo-2H-chromene is commercially available or can be prepared by methods
commonly known in the art. For example 6-bromo-4-chromanone can be reduced to the
alcohol and subsequently eliminated to obtain 6-bromo-2H-chromene.
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Scheme 2
chiral
(7)
In Scheme 2 is depicted the synthesis for the intermediate of formula (7).
In Scheme 2, Step A, a cyclic alkene of formula (4) is oxidized with MCPBA to
obtain an epoxide of formula (5). The reaction is performed in a biphasic mixture of a
halogenated solvent, such as dichloromethane and an aqueous base, such as aqueous
sodium bicarbonate. The MCPBA is added in portions at -10 to 10 °C and the reaction
allowed to warm to room temperature with stirring for 1 to 8 hr. Additional MCPBA is
added if needed.
In Step B, an epoxide of formula (5) is treated with ammonia to form a racemic
amino alcohol of formula (6). Preferred conditions use a sealed vessel with an inert
solvent, such as THF, with the addition of ammonia in MeOH. The reaction is heated at
50 to 100 °C for 1 to 4 days, adding additional ammonia if necessary.
In Scheme 2, Step C a racemic amino alcohol (6) is resolved to a chiral amino
alcohol of formula (7) as previously described in Scheme 1, Step C. For example, 1-
amino-7-bromo-tetralin-2-ol (Y = -CH2-) is resolved by crystallization with (+)-di-l,4-
toluoyl-D -tartaric acid. The free amine can be obtained by treatment of the salt with
aqueous base to obtain the (IS, 2S)-amino alcohol wherein Y = -C¾-. Alternatively, the
racemic material (6) can be carried through to final products and the enantiomers
separated by chiral chromatography.
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It will be understood by one skilled in the art that the cyclic alkenes of formula (4)
can readily be obtained either commercially or by methods known in the literature. For
example, the corresponding ketone can be reduced to the alcohol and subsequently
eliminated to the alkene.
Scheme 3
(11)
Formation of intermediate (11) can be carried out in accordance with reactions as
depicted in Scheme 3.
In Scheme 3, Step A, the amino indane (9) is obtained from the nitro indane (8) by
hydrogenation over 5% palladium on carbon according to procedures contained in the
literature for the 1R,2R enantiomer (US 7,326,731 B2).
In Step B, the amino indane (9) is converted to the iodoindane (10) using a
Sandmeyer reaction. The diazonium salt is formed in situ in a solvent such as acetonitrile
using p-toluenesulfonic acid and an aqueous solution of sodium nitrite. The diazonium
salt is subsequently treated with an aqueous solution of potassium iodide at a temperature
of 0 to 30 °C for 0.5 to 6 h to provide the iodoindane (10).
In Step C, the boc protected iodoindane (10) is taken to the iodo amino indane
( 11) under acidic conditions, such as HCl or trifluoroacetic acid. Methods for introducing
and removing nitrogen protecting groups are well known in the art (see, e.g., Greene and
Wuts, Protective Groups in Organic Synthesis , 3rd Ed., John Wiley and Sons, New York,
(1999)). Preferred conditions use HCl in dioxane at 0 to 25 °C for 0.5 to 4 h.
rert-butyl-N-[(lS,2S)-6-amino-2-hydroxy-indan-l-yl]carbamate (8) can be
prepared by methods known in the art. For example, racemic-trans-l-amino-6-
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nitroindan-2-ol can be obtained in six steps from indene (Adv. Synth. Catal. 2005, 347,
255-265). The racemic material is resolved and isolated as the 1R,2R and 1S,2S salts of
(+)-L-mandelic acid. The 1S,2S salt is freed to lS,2S-£raws-l-amino-6-nitroindan-2-ol
and the amine subsequently protected as the -butyl carbamate to obtain material in
greater than 97% ee as analyzed by chiral HPLC.
Scheme 4
Formation of compounds of the invention of Formula I can be carried out in
accordance with reactions as depicted in Scheme 4.
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In Scheme 4, Step A, the amino alcohol of formula (12) (either chiral or racemic)
is acylated to obtain an amide of formula (13). Various acylation methods are well
known in the art using either a carboxylic acid or an acid chloride. Preferred conditions
use an appropriate benzoyl chloride in a solvent mixture of THF and aqueous sodium
bicarbonate at a temperature of 0 to 25 °C for 1 to 8 hr. If the starting amino alcohol is
used as the salt of a chiral acid, sufficient base is used to generate the free amine.
In Step B, the bromo or iodo amide of formula (13) is coupled with a protected 2-
oxo-piperazine using copper (I) iodide and a ligand, such as sym-dimethylethylene
diamine to provide an oxopiperazine of formula (14). The reaction is preferably
performed in a sealed vessel, under an inert atmosphere, in the presence of an inorganic
base such as potassium carbonate. The reaction is run in an inert solvent, such as NMP at
a temperature of 80 to 150 °C.
In Scheme 4, Step C, the oxopiperazine is selectively reduced in the presence of
the benzamide to provide the piperazine of formula (15). The reaction is accomplished
using a reducing agent such as borane-dimethyl sulfide complex in an inert solvent such
as THF at a temperature of 0 to 40 °C for one to four hours. Additional borane-dimethyl
sulfide complex may be used to drive the reaction to completion.
Alternatively, in Step D, the piperazine is reacted directly with the iodo or bromo
amide (13) using N-tert-butoxycarbonylpiperazine in a coupling reaction. The reaction
proceeds in the presence of a Pd catalyst, such as allylpalladium(II) chloride dimer and a
ligand such as tri-tert-butylphosphonium tetrafluoroborate. A base is used, such as
sodium tert-butoxide in an inert solvent such as DMSO at temperature of 20 to 120 °C for
0.5 to 8 hr. to provide the piperazine of formula (15).
In Step E, the boc protecting group is removed to give the unprotected piperazine
of formula (16). Acidic conditions for removal of boc groups, such as HC1 in dioxane,
are well known in the art.
Subsequently, in Step F, the unprotected amine (16) is reacted in a reductive
amination with 3-oxetanone to provide an oxetanyl piperazine of formula (17). Various
methods for accomplishing reductive aminations are well known in the art. Preferred
conditions use a reducing agent, such as sodium triacetoxy borohydride, in an inert
solvent such as acetonitrile. The reaction is carried out at 0 to 40 °C for about 0.5 to 4 hr.
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Additional sodium triacetoxy borohydride and 3-oxetanone may be added to drive the
reaction to completion. Alternatively, the reaction can be accomplished using a reducing
agent such as sodium cyanoborohydride, in a solvent mixture such as MeOH and glacial
acetic acid, in the presence of molecular sieves. The reaction proceeds at a temperature of
0 to 60 °C for about 1 to 24 hr.
In Scheme 4, Step G, the alcohol of the oxetanyl piperazine (17) is converted to
the carbamate of Formula I . There are various means available to the skilled artisan for
synthesizing carbamates such as triphosgene/amine, 4-nitrophenyl chloroformate/amine,
CDI/amine or directly with an appropriately substituted isocyanate. The preferred
method for compounds of the invention wherein Z = -CH2-, makes use of the appropriate
isocyanate (0=C=N-R 2) . For example, the alcohol (17) in an aprotic solvent such as
dichloromethane, dioxane, or preferably THF, in the presence of an organic base, such as
DMAP or preferably 4-pyrrolidinopyridine, is treated with methyl isocyanate. The
reaction is run in a sealed vessel at 20 to 70 °C for about 2 to 16 hr. Compounds of the
invention wherein the carbamate moiety is a primary carbamate (R2 = H) can be
synthesized using CDI/ammonia, sodium cyanate, or chlorosulfonyl isocyanate. Other
methods make use of trichloroacetyl isocyanate in an aprotic solvent. Subsequently, the
trichloroacetyl group is cleaved using neutral AI2O or an organic acid such as p-toluene
sulfonic acid. A preferred method for synthesis of the carbamate of Formula I makes use
of CDI in an inert solvent such as THF at 0 to 25 °C for 4 - 20 hr. to form the imidazole
N-carboxylic ester in situ. Subsequent reaction with an appropriate amine (H2NR2)
provides the carbamate of Formula I .
It will be recognized by one skilled in the art that the intermediates described
above can be carried along as racemic mixtures and resolved as a final step, preferably by
chiral HPLC, to provide enantiomerically pure compound.
Preparation 1
6-Bromochroman-4-ol
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To a suspension of 7-bromo-3,4-dihydronaphthalen-l(2H)-one (3.95 kg, 17.4 mol)
in ethanol (20 L) at room temperature add sodium borohydride (221 g, 5.84 mol) in
portions over 3 hr. Concentrate the clear red solution. Add the residue into ice (about 3
kg) to form a suspension and then slowly add ice-cooled 0.5 M HCl (10 L) into the above
suspension with stirring. Extract the mixture with methyl tert-butyl ether (20 L and 5 L).
Wash the combined organic phase with saturated aqueous sodium bicarbonate (10 L) and
brine (2 10 L), concentrate the solvent under reduced pressure and dry the wet solid in
air overnight to obtain the title compound as a pale yellow solid (4. 10 kg, quantitative).
LC-ES/MS m/z 2 11 [M- H-20 +H]+.
Prepare the alcohols in the table below by essentially following the procedure
described in Preparation 1 using the appropriate ketone.
Preparation 5
6-Bromo-2H-chromene
-16-
To a solution of 6-bromochroman-4-ol (3.98 kg, 17.4 mol) in toluene (18 L) add
-toluenesulfonic acid monohydrate (95 g, 499.42 mmol) at room temperature. Attach a
Dean-Stark trap and reflux for 3 hr. and 50 min. Allow the mixture to cool to 50 °C in air
and pour into saturated aqueous sodium bicarbonate (10 L) and ice (about 3 kg) with
stirring. Separate the two layers. Back extract the aqueous layer with methyl -butyl
ether (4 L). Wash the organic layer with saturated sodium bicarbonate (10 L) and brine
( 1 10 L), and evaporate the solvent under reduced pressure to obtain the title compound
as a brown oil (4.90 kg, >100%, contains toluene). LCMS m/z ( Br) 209 [M+H]+.
Prepare the alkenes in the table below by essentially following the procedure
described in Preparation 5 using the appropriate alcohol.
Preparation 9
Racemic-£raws-3,6-dibromochroman-4-ol
(±)
To a solution of DMSO (16 L) and water (2.5 L) at room temperature add 6-
bromo-2H-chromene (4.90 kg, 23.2 mol). Add NBS (3.47 kg, 19.5 mol) in portions over
3.5 hr. Pour the mixture into methyl tert-butyl ether (12 L) and water (18 L), separate the
-17-
two layers. Wash the organic layer with saturated sodium bicarbonate (7 L), water (8 L)
and brine ( 8 L). Back extract the aqueous layer with methyl tert-butyl ether (4 L) and
evaporate the solvent of the combined organic layers under reduced pressure to obtain the
title compound as a reddish brown solid (5.10 kg, 71%).
Preparation 10
(3R,4S)-4-Amino-6-bromo-chroman-3-ol
To a solution of racemic-/raws-3,6-dibromochroman-4-ol (5.10 kg, 16.6 mol) in
tetrahydrofuran (5.0 L) and ethanol (5.0 L) at room temperature, add ammonium
hydroxide (13.0 L) in one portion and slowly heat to 43 °C over 2 hr. and continue
heating for 14 hr. Concentrate the mixture under reduced pressure to remove about 9 L of
solvent. Add methyl tert-butyl ether (10 L) to the residual slurry and stir the mixture for
3 hr. at 25 °C. Collect the precipitate by filtration, wash with water (2 x 2 L) and then
methyl tert-butyl ether (3 x 1.5 L) to obtain a wet solid. Dry in air at room temperature
overnight to obtain the title compound as a pale yellow solid (2.78 kg, 69%). LC-ES/MS
m/z ( Br) 244 [M+H]+. Separate the enantiomers by purifying in 100 g portions on a 11
x 33 cm CHIRALPAK® AD, 20 column eluting with 100% methanol with 0.2%
dimethylethylamine (steady state recycle purification) to obtain the 3S,4R enantiomer
(isomer 1, 1362.5 g, 97.4% ee) and the title compound (isomer 2, 1323.7 g, 97.5% ee).
Conditions for analytical chiral HPLC analysis: 4.6 x 150 mm CHIRALPAK® AD-H
5 column, 100% methanol with 0.2% dimethylethylamine, flow rate 0.5 mL/min.,
isomer 1 TR = 4.69 min, isomer 2 TR = 6.27 min.
Preparation 11
Racemic-c/s-6-bromo- 1a,2,3,7b-tetrahydronaphtho[ l,2-b]oxirene
-18-
To a solution of 6-bromo-l,2-dihydronaphthalene (250 g, 1.20 mol) in
dichloromethane (3.75 L) add saturated aqueous sodium bicarbonate (1250 mL) and cool
to 0-5 °C. Add MCPBA (77%, 275 g, 1.23 mol, 1.03 eq) in portions. Stir the mixture
mechanically at 0-5 °C for 1.5 hr. and allow to warm to room temperature. Stir at room
temperature for approximately 1.5 hr. and add additional MCPBA (77%, 15.0 g, 67
mmol, 0.05 eq) at room temperature and stir for approximately one hour. Dilute the
mixture with dichloromethane (3750 mL) and separate the layers. Wash the organic layer
with saturated aqueous sodium bicarbonate (3 L). Mix a 10% aqueous sodium bisulfite
solution (3750 mL) with aqueous saturated sodium bicarbonate (1250 mL) to prepare a
solution with a final pH of approximately 7. Add this solution to the dichloromethane
layer and stir at room temperature for about 10 min. Separate the layers, wash the organic
layer with saturated aqueous sodium bicarbonate (3 L) and brine (3 L). Concentrate in
vacuo to obtain the title compound as an oil (263 g, 98%). GC/MS m/z ( Br/ 1Br)
224/226 [M+].
Prepare the epoxides in the table below by essentially following the procedure
described in Preparation 11 using the appropriate alkene.
Preparation 14
Racemic-£raws-l-amino-7-bromo-tetralin-2-ol
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In a 2 L pressure reactor that is equipped with a mechanical stirrer dissolve
racemic-c/s-6-bromo-la,2,3,7b-tetrahydronaphtho[l,2-b]oxirene (67.0 g, 298 mmol) in
tetrahydrofuran (200 mL). Add 7 M ammonia in methanol (350 mL), seal the reactor and
stir at 70 °C for 2 days. Add additional 7 M ammonia in methanol (140 mL), seal the
reactor and stir at 70 °C for 1 day. Concentrate the reaction slurry in vacuo to obtain a
residue (68.1 g). Add diethyl ether (760 mL) and stir the slurry at room temperature for
approximately 5 hr. Collect the solid by filtration, rinse with diethyl ether (2 x 215 mL)
and dry overnight to obtain the title compound (58.1 g, 81%). GC/MS m/z ( Br/ 1Br)
224/226 [M-NH2]+.
Prepare the aminoalcohols in the table below by essentially following the
procedure described in Preparation 14 using the appropriate epoxide.
LC-ES/MS
Prep Chemical name Structure m/z
( Br/ 1Br)
Racemic-£raws-5-amino-3- OH 256/258
15 bromo-6,7,8,9-tetrahydro-
[M+H]+
5H-benzo[7]annulen-6-ol
Racemic-£raws-5-amino-7- OH 258/260
16 bromo-2,3,4,5-tetrahydro-lbenzoxepin-
4-ol
[M+H]+
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Preparation 17
(l S,2S)- l-Amino-7-bromo- l,2,3,4-tetrahydronaphthalen-2-ol (2S,3 S)- 2,3-bis(4-
methylbenzoyloxy)succinate
Add (+)-di- l ,4-toluoyl-D-tartaric acid (2.30 kg, 5.95 mol) in one portion to a
solution of racQmic-trans- -amino-7-bromo- 1,2,3,4-tetrahydronaphthalen-2-ol ( 1.40 kg,
5.78 mol) in acetonitrile (16.0 L) and water (3 .50 L). Heat the thick suspension to reflux
(77 °C) under nitrogen for 30 min. The stirrer does not start to stir until about 46 °C.
Turn off the heat to the oil bath and allow the mixture to cool to room temperature with
stirring. After 5 hr., filter the thick slurry through a Buchner funnel and rinse with
acetonitrile (2 L). Continue to filter for one hour until no more solvent is collected. Dry
the wet cake in air at room temperature overnight to obtain the desired salt as a yellow
solid ( 1.70 kg). Add the solid into acetonitrile (15 L) and water (3 .3 L) and heat the
resulting thick slurry to reflux (77 °C) under nitrogen with stirring for 30 min. Turn off
the heat to the oil bath and allow to cool to room temperature with stirring. After 5 hr.,
filter the thick slurry through a Buchner funnel and rinse with acetonitrile (2 L). Continue
to filter for 30 min. until no more solvent is collected. Dry the wet cake at room
temperature for 48 h to obtain the desired salt as a pale yellow solid (800 g, 22%). LCES/
MS m/z ( Br) 242 [M+H]+ for the free base. 99.0% ee. Analytical conditions for
enantiomeric excess determination by supercritical fluid chromatography (SFC):
Column: CHIRALPAK® AD-H (0.46 x 250 mm, 5 ), carbon dioxide flow rate: 2.55
mL/min., co-solvent: methanol with 0.1% diethyl amine, co-solvent flow rate: 0.45
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niL/min., backpressure: 150 bar, column temperature: 40.9 °C. Isomer 1 T
0.5% area and isomer 2 (title compound) TR = 8.46 min., 99.5% area.
Preparation 18
tert-Butyl N-[(lS,2S)-6-amino-2-hydroxy-indan-l-yl]carbamate
Obtain tert-butylN-[(lS,2S)-2-hydroxy-6-nitro-indan-l-yl]carbamate of >97% ee
by essentially following literature procedures (Kozhushkov, S.I. et. al. Adv. Synth. Catal.
2005, 347, 255-265). Reduce the nitro group using the same procedure as described for
the 1R, 2R enantiomer (US 7,326,73 1 B2) to obtain the title compound.
Preparation 19
tert-Butyl N-[(l S,2S)-2-hydroxy-6-iodo-indan- 1-yl]carbamate
Cool a suspension of tert-butyl N-[(lS,2S)-6-amino-2-hydroxy-indan-lyljcarbamate
(25.0 g, 94.6 mmol) in acetonitrile (800 mL) in an ice bath for 30 min and
add p-toluenesulfonic acid monohydrate (53.97 g, 283.7 mmol) in one portion. Add a
solution of sodium nitrite (13.05 g, 189.2 mmol) in water (30 mL) to the mixture in
portions for 5 min and stir for 30 min. Add a solution of potassium iodide (39.25 g, 236.5
mmol) in water (50 mL) dropwise to the mixture over 10 min. and stir for 20 min. in an
ice bath. Allow the mixture to warm up to room temperature and stir for 1.5 hr. Make
the dark red solution alkaline with 10% aqueous sodium carbonate. Concentrate the
resulting mixture in vacuo to remove the acetonitrile. Collect the brown precipitate by
filtration and dry in a vacuum oven at 50 °C overnight to obtain the title compound (33.9
g, 96%). LC-ES/MS m/z 320 [M+H]+.
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Preparation 20
N-[(3R,4S)-6-Bromo-3-hydroxy-chroman-4-yl]-4-fluoro-benzamide
To a suspension of (3R,4S)-4-amino-6-bromo-chroman-3-ol (692 g, 2.84) and
sodium bicarbonate (476 g, 5.67 mol) in tetrahydrofuran (3.0 L) and water (3.0 L) at
17 °C add 4-fluorobenzoyl chloride (369 mL, 3.12 mol) dropwise via addition funnel over
15 min. and stir for 2 hr. Add water (2 L) and methyl tert-butyl ether (3.5 L) and stir for
5 min. Separate the organic phase and wash it with water (2 L) and brine (2 L). Dry the
organic portion over anhydrous magnesium sulfate and concentrate in vacuo to obtain a
pale yellow solid (2.3 kg). Triturate the solid in heptane (5 L) at 25 °C for 3 h. Collect
by filtration and dry in an oven at 50 °C for 12 hr. to obtain the title compound (975 g,
94%) as a white solid. LC-ES/MS m/z ( Br/ 1Br) 366/368 [M+H]+.
Prepare the amides in the table below by essentially following the procedure
described in Preparation 20 using the appropriate racemic or enantiopure aminoalcohol
free base or salt. When using the salt of the aminoalcohol (Preparation 21) use saturated
sodium bicarbonate as the base.
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Preparation 24
4-Fluoro-N-[(lS,2S)-2-hydroxy-6-iodo-indan-l-yl]benzamide
To a solution of tert-butyl N-[(lS,2S)-2-hydroxy-6-iodo-indan-l-yl]carbamate
(39.6 mmol, 14.9 g) in 1,4-dioxane (200 mL), add a solution of 4 M hydrogen chloride in
dioxane (100 mL) at room temperature and stir for 2 hr. Concentrate the resulting
suspension in vacuo and dry in a vacuum oven for 48 hr. Dissolve the residue in THF
(25 mL) and cool in an ice bath for 30 min. Add 5 M aqueous sodium hydroxide (17.4
mL, 87.2 mmol) and 4-fluorobenzoyl chloride (3.78 mL, 40.4 mmol) dropwise. Warm
the solution to room temperature and stir for one hour. Concentrate the black solution in
vacuo and dilute with water (50 mL). Collect the solid by filtration and dry at 40 °C in a
vacuum oven overnight to obtain the title compound (14.4 g, 91%). LC-ES/MS m/z 398
[M+H]+.
Preparation 25
-Butyl 4-[(2S,3S)-3-[(4-fluorobenzoyl)amino]-2-hydroxy-indan-5-yl]-3-
piperazine- 1-carboxylate
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Purge a mixture of 4-fluoro-N-[(lS,2S)-2-hydroxy-6-iodo-indan-l-yl]benzamide
(8.70 g, 21.9 mmol), 4-N-boc-2-oxo-piperazine (4.61 g, 22.3 mmol), and potassium
carbonate (6. 12 g, 43.8) in N-methylpyrrolidone (100 mL) with nitrogen gas. Add copper
(I) iodide (2.11 g, 11.0 mmol) and sym-dimethylethylene diamine (2.34 mL, 21.9 mmol).
Seal the flask with a septum and stir at 100 °C for 6 hr. Cool the mixture to room
temperature and dilute with ethyl acetate (100 mL). Wash the organic portion with water
(3 100 mL), then brine, and then dry over sodium sulfate, filter, and concentrate to
dryness. Purify the residue by column chromatography (120 g silica,2 to 5%
methanol/chloroform) to obtain the title compound (7.63 g, 74%). LC-ES/MS m/z 470
[M+H]+.
Preparation 26
-Butyl 4-[(3S,4S)-4-[(4-fluorobenzoyl)amino]-3-hydroxy-tetralin-6-yl]-3-
piperazine- 1-carboxylate
Prepare the title compound using N-[(lS,2S)-7-bromo-2-hydroxy-tetralin-l-yl]-4-
fluoro-benzamide by essentially following Preparation 25 for tert-butyl 4-[(2S,3S)-3-[(4-
fluorobenzoyl)amino]-2-hydroxy-indan-5-yl]-3-oxo-piperazine-l-carboxylate. LCES/
MS m/z 484 [M+H]+.
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Preparation 27
-Butyl 4-[(2S,3S)-3-[(4-fluorobenzoyl)amino]-2-hydroxy-indan-5-yl]piperazi
carboxylate
Cool a solution of tert-butyl 4-[(2S,3S)-3-[(4-fluorobenzoyl)amino]-2-hydroxyindan-
5-yl]-3-oxo-piperazine-l -carboxylate (8.85 g, 18.9 mmol) in anhydrous
tetrahydrofuran (40 mL) in an ice bath for 30 min. and add borane-dimethyl sulfide
complex (2 M, 37.7 mL, 75.40 mmol) under nitrogen. Warm the mixture to room
temperature and stir for 1.5 hr. Add a second portion of borane-dimethyl sulfide complex
(2 M, 9.42 mL, 18.9 mmol) at room temperature and stir for one hour. Cool in an ice bath
for 10 min and carefully add water dropwise, followed by addition of saturated sodium
bicarbonate solution (50 mL). Dilute the resulting suspension with ethyl acetate
(150 mL). Wash the organic portion with brine, dry over sodium sulfate, and concentrate
in vacuo. Purify the crude product by flash chromatography (120 g silica, 40 to 50%
ethyl acetate/hexanes) to obtain the title compound (6.8 g, 79%). LC-ES/MS m/z 456
[M+H]+.
Preparation 28
-Butyl 4-[(3S,4S)-4-[(4-fluorobenzoyl)amino]-3-hydroxy-tetralin-6-yl]piperazi
carboxylate
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Prepare the title compound using tert-butyl 4-[(3S,4S)-4-[(4-
fluorobenzoyl)amino]-3-hydroxy-tetralin-6-yl]-3-oxo-piperazine-l-carboxylate by
essentially following Preparation 27 for tert-butyl 4-[(2S,3S)-3-[(4-
fluorobenzoyl)amino]-2-hydroxy-indan-5-yl]piperazine-l-carboxylate. LC-ES/MS m/z
470 [M+H]+.
Preparation 29
4-Fluoro-N-[(lS,2S)-2-hydroxy-7-piperazin-l-yl-tetralin-l-yl]benzamide hydrochloride
To a solution of tert-butyl 4-[(3S,4S)-4-[(4-fluorobenzoyl)amino]-3-hydroxytetralin-
6-yl]piperazine-l-carboxylate (19.4 g, 41.3 mmol) in 1,4-dioxane (430 mL) add
4 M hydrogen chloride in dioxane (135 mL) at room temperature. Stir the resulting slurry
mechanically at 50 °C for 13 hr. Concentrate the reaction in vacuo to obtain the title
compound (24.8 g, quantitative). LC-ES/MS m/z 370 [M+H]+.
Preparation 30
4-Fluoro-N-[(3R,4S)-3-hydroxy-6-piperazin-l-yl-chroman-4-yl]benzamide hydrochloride
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Degas (bubble nitrogen through the mixture) a suspension of tri- -
butylphosphonium tetrafluoroborate (16.0 g, 54.6 mmol) and allylpalladium(II) chloride
dimer (5.07 g, 27.3 mmol) in DMSO (1.40 L) with stirring at room temperature. Add Ntert-
butoxycarbonylpiperazine (3 15 g, 1.64 mol, 97% pure) and N-[(3R,4S)-6-bromo-3-
hydroxy-chroman-4-yl]-4-fluoro-benzamide (200.0 g, 546 mmol). Stir the mixture for 15
min under nitrogen and then heat at 80 °C. Add sodium tert-butoxide (179 g, 1.80 mol)
and heat at 93-98 °C for 45 min. Pour the mixture over a solution of 1M phosphoric acid
(2.73 L, 2.73 mol, adjusted to pH = 2.3 with 50% sodium hydroxide) and ethyl acetate
(800 mL) and stir for 10 min. Separate the layers and extract the aqueous layer with ethyl
acetate (3 x 300 mL). Wash the combined organic layer with semi-saturated brine (2 x
500 mL). Treat the organic layer with activated carbon (20 g) and silica gel (200 g) and
stir for 30 min. Filter through a pad of diatomaceous earth and concentrate under reduced
pressure to obtain a dark oil. Dissolve the oil in methanol (800 mL), add 4 M hydrogen
chloride in dioxane (410 mL), and stir the solution at 40 °C for 1 hr. Evaporate the
solvent in vacuo, suspend the residue in acetonitrile (1.6 L) and stir at room temperature
for 1 hr. Collect the solid by filtration under nitrogen to avoid hydration of the salt.
Wash the filtrate with acetonitrile (500 mL) and methyl tert-butyl ether ( 1 L), and dry
under vacuum to obtain the title compound (262 g, 73%). LC-ES/MS m/z 372 [M+H]+.
Prepare the amines in the table below by essentially following the procedure
described in Preparation 30 using the appropriate racemic or enantiopure bromide.
LCPrep
Chemical name Structure ES/MS
m/z
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Preparation 33
4-Fluoro-N-[(3R,4S)-3-hydroxy-6-[4-(oxetan-3-yl)piperazin-l-yl]chroman-4-
yljbenzamide
Add sodium triacetoxyborohydride (20 g, 94.4 mmol) to a slurry of 4-fluoro-N-
[(3R,4S)-3-hydroxy-6-piperazin-l-yl-chroman-4-yl]benzamide hydrochloride (32.0 g,
78.5 mmol) and 3-oxetanone (6.87 g, 98.1 mmol) in acetonitrile (300 mL). Stir the slurry
for 20 min. and add more sodium triacetoxyborohydride (20 g, 94.4 mmol) in one portion.
Stir the brown mixture at 28 °C for approximately 2 hr. Add additional sodium
triacetoxyborohydride (10 g, 47.2 mmol) and stir the mixture for 30 min. Add 3-
oxetanone (2 g, 28.6 mmol) and after 20 min add additional 3-oxetanone ( 1 g,
14.3 mmol). Stir for 30 min., pour the mixture into a saturated aqueous sodium
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bicarbonate solution ( 1 L) and add dichloromethane ( 1 L). Separate the organic layer and
concentrate in vacuo to obtain a pale grey solid. Purify the solid by silica gel column
chromatography eluting with 3 to 6% isopropanol/dichloromethane to obtain the title
compound as a white solid (19.2 g, 73%). LC-ES/MS m/z 428 [M+H]+.
Prepare the oxetanes in the table below by essentially following the procedure
described in Preparation 33 using the appropriate racemic or enantiopure amine.
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Preparation 37
4-Fluoro-N-[(lS,2S)-2-hydroxy-6-[4-(oxetan-3-yl)piperazin-l-yl]indan-l-yl]benzamide
To a solution of -butyl 4-[(2S,3S)-3-[(4-fluorobenzoyl)amino]-2-hydroxyindan-
5-yl]piperazine-l-carboxylate (300 mg, 0.659 mmol) in 1,4-dioxane (5.0 mL), add
4 M hydrogen chloride in dioxane (5.0 mL) at room temperature and stir for 1 hr.
Concentrate the suspension in vacuo. Add to the residue saturated aqueous sodium
bicarbonate (50 mL) and extract with ethyl acetate (3 x 50 mL). Wash the combined
organic layers with brine, dry over sodium sulfate, and concentrate in vacuo. Dissolve
the residue in methanol (20 mL) and add 3-oxetanone (142 mg, 1.98 mmol), glacial acetic
acid (170 , 2.96 mmol), and crushed activated 4A molecular sieves. Stir the resulting
suspension at 50 °C for 1 hr. Add sodium cyanoborohydride (13 1 mg, 1.98 mmol) at
room temperature and stir overnight. Add a saturated sodium bicarbonate solution (20
mL) and stir for 20 min. Extract with dichloromethane (3 x 50 mL) and wash the
combined organic layers with water and brine. Dry the organic portion over sodium
sulfate, filter, and concentrate to obtain the title compound (220 mg, 81%). LC-ES/MS
m/z 412 [M+H]+.
Preparation 38
Racemic-/raws-3,6-dibromochroman-4-ol
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Heat a solution of 6-bromochroman-4-ol (25.0 g, 109.1 mmol) in toluene (500
mL) to 100 °C. Add p-toluenesulfonic acid monohydrate (1.1 g, 5.5 mmol) and, using a
Dean-Stark trap to collect water, stir at 100 °C for 2 hr. Remove the heat source and
quench the reaction mixture with ice cold 0.5N NaOH (250 mL). Further cool the
reaction mixture to 5-10 °C, and then separate the layers. Wash the organic layer with
water (25 mL) and concentrate the organics under reduced pressure to approximately half
their original volume. Add DMSO (100 mL) and continue to concentrate under reduced
pressure until no more toluene distills.
Cool the DMSO solution of the intermediate olefin to 0-5 °C, add water (17 mL)
and re-cool to 0-5 °C. Add N-bromosuccinimide (21.7 g, 120.0 mmol) in 5 portions over
30 minutes. Stir at 0-5 °C for 1 hr. Dilute the reaction mixture with ethyl acetate (100
mL) and water (50 mL), stir 10 min., and separate the layers. Back extract the aqueous
layer with ethyl acetate (100 mL). Wash the combined organics with 1:1 brine/water (2 x
50 mL), dry over sodium sulfate and concentrate under reduced pressure to obtain the title
compound as a white solid (33.0 g, 99%). 1H NMR (DMSO- ) 4.26 (m, 1H), 4.37 (m,
2H), 4.65 (m, 1H), 6.27 (d, 1H), 6.80 (d, 1H), 7.35 (dd, 1H), 7.46 (bd, 1H). GC/MS m/z
( Br/ 1Br) 308/310 [M+].
Preparation 39
Racemic-/raws-4-amino-6-bromo-chroman-3-ol
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To a solution of racemic-£raws-3,6-dibromochroman-4-ol (30.0 g, 97.4 mmol) in
isopropyl alcohol (300 mL) at ambient temperature (500 mL), add ammonium hydroxide
(28-30% aq., 150 mL, 2.2 mol). Stir slowly for 15 hr. Filter the reaction mixture and
dilute the filtrate with water (200 mL). Concentrate the resulting solution under reduced
pressure to one half of its original weight. Add water (100 mL), stir the mixture at
ambient temperature for 10 min., then cool to 0-5 °C and stir for an additional 30 min.
Collect the precipitate by filtration and wash the solids with water (25 mL) and heptanes
(25 mL). Vacuum dry the material at 40 °C to afford the title compound as a pale yellow
solid (21.2 g, 89.2%). H NMR (DMSO-i¾) 1.96 (bs, 2H), 3.53 (m, 2H), 3.87 (dd, 1H),
4.11 (dd, 1H), 5.15 (bs, 1H), 6.66 (d, 1H), 7.19 (dd, 1H), 7.48 (dd, 1H). LC-ES/MS m/z
( Br) 244 [M+H].
Preparation 40
3R, 4S)-4-amino-6-bromo-chroman-3-ol
Heat a mixture of racemic-£raws-4-amino-6-bromochroman-3-ol (21.1 g,
86.4 mmol), D-(+)-camphoric acid (17.3 g, 86.4 mmol), acetonitrile (633 mL) and water
(47.6 mL) to 70-75 °C and stirr for 10 min. The solution is allowed to cool to ambient
temperature over 4 hr. After 24 hr. at ambient temperature, the mixture is filtered. Rinse
the solids with 7% H20 in acetonitrile (2 x 20 mL). Heat the filtrate to 50 °C and add IN
NaOH (216.1 mL). Concentrate the resulting mixture under reduced pressure to remove
the acetonitrile. Add ¾ 0 (21 1 mL) and heat the solution to 70 °C. Cool to ambient
temperature and collect the precipitate by filtration. Wash with water (25 mL) and
vacuum dry at 50 °C overnight to obtain the title compound as an off-white solid (9.8 g,
46.4% of 50% theoretical weight, 98.7% ee).
Combine crude (3R,4S)-4-amino-6-bromochroman-3-ol (1.5 g, 6.15 mmol),
acetonitrile (12.0 mL) and water (3.0 mL) and heat to 70-75 °C. Stir the solution for
30 min. and allow the solution to cool to ambient temperature and then stir an additional 1
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hr. Cool the mixture to 0-5 °C and stir 30 min. Filter the solids and wash with cold 4 :1
acetonitrile: ¾ 0 (10 mL). Vacuum dry the solids at 50 °C overnight to obtain the
purified title compound as a white solid ( 1.1 g, 74.6%, 99.7% ee). 1H NMR (DMSO-rfe)
1.96 (bs, 2H), 3.53 (m, 2H), 3.87 (dd, IH), 4.1 1 (dd, IH), 5.15 (bs, IH), 6.66 (d, IH),
7.19 (dd, IH), 7.48 (dd, IH). LC-ES/MS m/z ( Br) 244 [M+H]. Conditions for
analytical chiral HPLC analysis: ChiralPak AD-H column (150x4.6mm, 4.6 micron);
flow rate 0.6 mL/min; wavelength 290 nm; eluent 100% MeOH + 0.2% v/v dimethylethylamine;
run time 10 min; column temperature 30 °C; isocratic.
Preparation 41
4-Fluoro-N-[(3R,4S)-3-hydroxy-6-(piperazin-l-yl) chroman-4-yl]benzamide
To a suspension of (3R, 4S)-4-amino-6-bromo-chroman-3-ol (10.0 g, 41.0 mmol)
in THF (50 mL) at ambient temperature, add sodium bicarbonate (5.2 g, 61.4 mmol) and
water (50 mL). Stir 5 min. and cool the mixture to 0-5 °C. Add 4-fluoro-benzoyl
chloride (6.5 g, 41.0 mmol) drop-wise via an addition funnel over 10 min. Stir the
mixture at 0-5 °C for 30 min., allow the mixture to warm to 15-20 °C and stir for an
additional 2 hr. Dilute the mixture with brine (50 mL) and THF (50 mL) and stir for 20
min. Separate the layers and concentrate the organic layer to one half its original volume.
Repeat the addition of THF (50 mL) and concentration to one half volume three times or
until the Karl Fisher water analysis of the solution is <0. 1%. Add DMSO (100 mL) and
continue concentrating the mixture under reduced pressure at 50 °C until THF no longer
distills.
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Dilute the resulting DMSO solution of the amide intermediate with DMSO
(200 mL) and toluene (45 mL) and de-gassed for 30 min. using a sub-surface sparge of
nitrogen gas. Add piperazine (16.6 g, 192.7 mmol) and de-gas the solution for 30 min.
Heat the solution to 40 °C and add allylpalladium(II) chloride dimer (0.72 g, 2 mmol) and
tri-tert-butyl-phosphonium tetrafluoroborate (1. 19 g, 4 mmol) under a nitrogen blanket.
Stir the mixture 10 min and add sodium tert-butoxide (13.0 g, 135.3 mmol) under a
nitrogen blanket. Raise the internal temperature to 70 °C and stir the mixture for 4 hr.
Cool the reactor contents to 20 °C and add water (75 mL) over 15 min. Adjust the pH of
the mixture to 6-7 using 6N HC1 and add ethyl acetate (150 mL). Stir reactor contents for
30 min. and then separate the layers. Back extract the aqueous layer with ethyl acetate
(2 x 150 mL). Discard the ethyl acetate extracts and adjust the pH of the aqueous layer to
11-12 using NaOH (40% aq.). Extract the aqueous layer with ethyl acetate (3 x 150 mL).
Combine the organic extracts and concentrate under reduced pressure to approximately
one half original volume. Add activated carbon (1.5g) and heat the mixture to 50-55 °C.
Stir at 50-55 °C for 1 hr., cool the mixture to 20 °C and filter to remove solids. Wash the
filtrate with brine (2 x 300 mL) and concentrate the organics under reduced pressure to
approximately 2 volumes (based on original starting material). Cool the mixture to
0-5 °C and stir for an additional 3 hr. Isolate the solids by filtration, wash with methyl
tert-butyl ether (30 mL) and vacuum dry the solids at 50 °C. The title compound is
recovered as an off-white solid (7.9 g, 52%). ¾ NMR (DMSO- ) 2.40-2.80 (bm, 8H),
3.65-3.95 (bm, 2H), 4.13 (d, 1H), 4.95 (m, 1H), 5.35 (bs, 1H), 6.63 (dd, 1H), 6.69 (d, 1H),
6.80 (d, 1H), 7.27 (m, 2H), 7.96 (m, 2H), 8.72 (bd, 1H). LC-ES/MS m/z 372 [M+H].
Preparation 42
4-Fluoro-N-[(3R,4S)-3-hydroxy-6-[4-(oxetan-3-yl) piperazin-l-yl] chroman-4-
yljbenzamide
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Combine 4-Fluoro-N-[(3R,4S)-3-hydroxy-6-(piperazin-l-yl) chroman-4-
yljbenzamide (8.2 g, 22.0 mmol) and 3-oxetanone (2.4 g, 33.0 mmol) in THF (164 mL).
Add acetic acid (1.3 mL, 22.0 mmol) and heat the resulting mixture to 35-40 °C. After
1 hr., add sodium triacetoxyborohydride (8.4 g, 39.6 mmol) and stir at 35-40 °C for 4 hr.
Cool the reaction mixture to 15 °C and add 2N NaOH (81.6 mL) over 30 min. Add brine
(81.6 mL) and stir the mixture for 30 min. Separate the layers and dilute the organic layer
with 2N NaOH (81.6 mL) and brine (81.6 mL). Stir the resulting mixture for 30 min.
Separate the layers and remove the aqueous layer. To the organic layer, add activated
carbon (1.0 g), raise the temperature to 50-55 °C and stir lhr. Filter the mixture to
remove solids and concentrate the filtrate under reduced pressure to approximately one
tenth of the original volume. Add ethyl acetate (164 mL) and concentrate under reduced
pressure to approximately one fourth original volume. Repeat this process twice with
ethyl acetate (2 x 164 mL). Concentrate the organics to approximately 3 volumes (based
on starting material), raise the internal temperature to 70-75 °C, and stir for 2 hr. Cool the
mixture to 10 °C, add heptane (82 mL) over 15 min and stir for an additional 2 hr. Isolate
the technical grade title intermediate by filtration and vacuum dry the solids at 50-55 C
(10.1 g recovered).
Purification: Suspend technical grade 4-fluoro-N-[(3R,4S)-3-hydroxy-6-[4-
(oxetan-3-yl) piperazin-l-yl] chroman-4-yl]benzamide (10.0 g, 23.4 mmol) in acetonitrile
(700 mL) and heat the mixture to reflux. Stir at reflux for 2 hr., cool to 50-55 °C and add
activated carbon (1.0 g). Stir at 50-55 °C for 2 hr. and filter to remove solids.
Concentrate the filtrate under reduced pressure to approximately 5 volumes based on the
starting technical grade material. Raise the internal temperature to 75-80 °C and stir for 1
hr. Cool the mixture to 20 °C and stir for an additional 4 hr. Isolate the title compound
by filtration to afford a white solid (6.8 g, 72%). ¾ NMR (DMSO-i¾) 2.33 (m, 4H),
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2.94 (m, 4H), 3.40 (m, 1H), 3.85 (m, 1H), 3.93 (d, 1H), 4.14 (d, 1H), 4.40 (dd, 2H), 4.49
(dd, 2H), 4.94 (m, 1H), 5.34 (d, 1H), 6.65 (d, 1H), 6.70 (d, 1H), 6.84 (dd, 1H), 7.27 (m,
2H), 7.97 (m, 2H), 8.75 (bd, 1H). LC-ES/MS m/z 428 [M+H].
Example 1
[(3 R,4S)-4-[(4-Fluorobenzoyl)amino] -6- [4-(oxetan-3-yl)piperazin- 1-yl]chroman-3 -yl] Nmethylcarbamate
To a solution of 4-fluoro-N-[(3R,4S)-3-hydroxy-6-[4-(oxetan-3-yl)piperazin-lyl]
chroman-4-yl]benzamide (72.0 g, 168 mmol) in THF (648 mL) add 1,1'-
carbonyldiimidazole (35.5 g, 219 mmol) and stir the solution at room temperature
overnight. Cool the mixture to 5 °C and add a solution of 2 M methylamine in THF
(210 mL, 421 mmol, 2.5 eq) dropwise over a period of 15 min. Stir for 30 min. and pour
over a mixture of water (720 mL) and methyl tert-butyl ether (216 mL). Stir the mixture
for 30 min. and decant the phases. Extract the aqueous phase with dichloromethane (3 x
216 mL). Wash the combined organic phases with brine (3 x 100 mL), dry over
anhydrous sodium sulfate, and concentrate in vacuo. Suspend the residue in ethyl acetate
(720 mL), heat at reflux for 2 hr. and cool to 10 °C. Collect the solid by filtration and dry
under vacuum overnight to obtain the title compound (64.0 g, 78%) as a white solid. LCES/
MS m/z 485 [M+H]+.
Prepare the carbamate in the table below by essentially following the procedure
described in Example 1 using the appropriate enantiopure alcohol.
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Example 3
[(8S,9S)-9-[(4-Fluorobenzoyl)amino]-2-[4-(oxetan-3-yl)piperazin-l-yl]
tetrahydro-5H-benzo[7]annulen-8-yl] N-methylcarbamate
Chiral
Prepare the racemic carbamate using racemic-?ra«s-4-fluoro-N-[8-hydroxy-2-[4-
(oxetan-3-yl)piperazin-l-yl]-6,7,8,9-tetrahydro-5H-benzo[7]annulen-9-yl]benzamide by
essentially following the procedure for Example 1, above. LC-ES/MS m/z 497 [M+H]+.
Dissolve racemic-?ra«s-9-[(4-fluorobenzoyl)amino]-2-[4-(oxetan-3-yl)piperazin-l-yl]-
6,7,8,9-tetrahydro-5H-benzo[7]annulen-8-yl] N-methylcarbamate (1.03 g) in
dichloromethane (8 mL) and methanol (3 mL). Separate the enantiomers in 300 
portions by SFC on a CHIRALPAK® AD-H column (2. 1 x 25 cm, 5 ) . Mobile phase:
30% isopropanol with 0.2% isopropylamine/carbon dioxide. Flow rate: 70 mL/min.
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Detection: 225 nm. Obtain the title compound as isomer 1 (333 mg, 99% ee) and the 8R,
9R enantiomer as isomer 2 (359 mg, 97.5% ee). Determine enantiomeric excess by SFC
on a CHIRALPAK® AD-H (4.6 x 150 mm, 5 ) column using 30%> isopropanol with
0.2% isopropylamine/carbon dioxide. Flow rate: 5 mL/min. Detection: 225 nm. Isomer
1 (title compound) T = 1.76 min. Isomer 2 TR = 2.30 min.
Example 4
[(4S,5S)-5-[(4-Fluorobenzoyl)amino]-7-[4-(oxetan-3-yl)piperazin-l-yl]-2,3,4,5-
tetrahydro- 1-benzoxepin-4-yl] N-methylcarbamate
Prepare the racemic carbamate using racemic-?ra«s-4-fluoro-N-[4-hydroxy-7-[4-
(oxetan-3-yl)piperazin-l-yl]-2,3,4,5-tetrahydro-l-benzoxepin-5-yl]benzamide by
essentially following the procedure for Example 1, above. LC-ES/MS m/z 499 [M+H]+.
Dissolve racemic-?ra«s-[5-[(4-fluorobenzoyl)amino]-7-[4-(oxetan-3-yl)piperazin-l-yl]-
2,3,4,5-tetrahydro-l-benzoxepin-4-yl] N-methylcarbamate (100 mg) in methanol (3 mL)
and filter off insoluble solids. Separate the enantiomers in 1.5 mL portions (2 injections)
by chromatography on a CHIRALPAK® AD-H column (3 x 25 cm, 5 ) . Mobile
phase: 95% ethanol/5% acetonitrile. Flow rate: 18 mL/min. Detection: 225 nm.
Obtain the title compound as isomer 1 (42 mg, 99% ee) and the 4R,5R enantiomer as
isomer 2 (40 mg, 99% ee). Determine enantiomeric excess by HPLC on a
CHIRALPAK® AD-H (4.6 x 150 mm, 5 ) column using 95% ethanol/5% acetonitrile
with 0.2% dimethylethylamine. Flow rate: 1.0 mL/min. Detection: 225 nm. Isomer 1
(title compound) TR = 3.58 min. Isomer 2 TR= 5. 13 min.
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Example 5
[(lS,2S)-l-[(4-Fluorobenzoyl)amino]-6-[4-(oxetan-3-yl)piperazin-l-yl]indan-2-yl] Nmethylcarbamate
To a solution of 4-fluoro-N-[(lS,2S)-2-hydroxy-6-[4-(oxetan-3-yl)piperazin-lyl]
indan-l-yl]benzamide (220 mg, 0.535 mmol) in tetrahydrofuran (3.0 mL) add 4-
pyrrolidinopyridine (16 mg, 0.107 mmol) and seal in a pressure vial. Add methyl
isocyanate (97 , 1.60 mmol) by syringe to the mixture and stir at 60 °C overnight.
Cool the resulting suspension in an ice bath, collect the white solid by filtration, and dry
in a vacuum oven at 40 °C for 3 hr. to obtain the title compound (200 mg, 80%). LCES/
MS m/z 469 [M+H]+.
Example 6
[(3 R,4S)-4-[(4-Fluorobenzoyl)amino] -6- [4-(oxetan-3-yl)piperazin- 1-yl]chroman-3 -yl] Nmethylcarbamate
Suspend 4-fluoro-N- [(3R,4S)-3-hydroxy-6- [4-(oxetan-3 -yl) piperazin- 1-yl]
chroman-4 yl]benzamide (10.0 g, 23.4 mmol) in THF (200 mL) and add 1,1
carbonyldiimidazole (4.9 g, 30.4 mmol). Stir at ambient temperature for 3 hr. Cool the
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reaction mixture to 5 °C and add 2 M methyl amine (in THF, 2 1 mL, 42.1 mmol) over
15 min. Stir the resulting mixture at 5-0 °C for 3 hr. Add 2 N NaOH (100 mL) and stir
the mixture for lhr. at 10-30 °C. Separate the layers and wash the organics with 2 N
NaOH (4 x 100 mL). Add water (200 mL) and concentrate the mixture under reduced
pressure to approximately twenty volumes (based on starting material). Add THF (200
mL) and stir at ambient temperature for 1 hr. Filter the mixture and transfer the product
wetcake back to a clean reaction vessel. Add THF (200 mL) and water (200 mL) and stir
at ambient temperature for 1 hr. Isolate the technical grade title compound by filtration
and vaccum dry at 80 °C (9.0g, 80%).
Example 7
Crystallization/Solid Form Conversion of [(3R,4S)-4-[(4-Fluorobenzoyl)amino]-6-[4-
(oxetan-3 -yl)piperazin- 1-yl]chroman-3 -yl] N-methylcarbamate
Suspend technical grade [(3R,4S)-4-[(4-Fluorobenzoyl)amino]-6-[4-(oxetan-3-
yl)piperazin-l-yl]chroman-3-yl] N-methylcarbamate (50 g, 103.2 mmol) in THF (1500
mL) under a nitrogen atmosphere. Heat the mixture to 50-60 °C and stir 1 hr. Add
activated carbon (5.0 g) and continue stirring at 50-60 °C for 1 hr. Cool mixture to 40-50
°C and filter to remove solids. Concentrate the filtrate under reduced pressure to
approximately 5 volumes (based on starting material). Add acetonitrile (1000 mL) and
concentrate under reduced pressure to approximately 5 volumes. Repeat four times to
remove all THF and then concentrate the mixture to 10-15 volumes. Heat the mixture to
70-75 °C and stir for 18 hr. Cool the mixture to 15 °C and stir 4 hr. Isolate the white
crystals by filtration and vacuum dry at 55-60 °C to afford the title compound (42 g,
84%). H NMR (DMSO-i/6) 2.32 (m, 4H), 2.53 (d, 3H), 2.96 (m, 4H), 3.39 (m, 1H),
4.11-4.25 (m, 2H), 4.39 (dd, 2H), 4.51 (dd, 2H), 4.86 (m, 1H), 5.04 (m, 1H), 6.70-6.75
(m, 2H), 6.85 (dd, 1H), 7.17 (m, 1H)7.28 (m, 2H), 7.96 (m, 2H), 8.90 (bd, 1H). LCES/
MS m/z 485 [M+H]. Chiral assay 99.9% ee.
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Exemplified compounds of the present invention are tested in the following in
vitro and in vivo assays.
In vitro
Potency assays for human cathepsin S and mouse cathepsin S and selectivity
assays for human cathepsins L, B, K, V, and F are described below. The experimental
conditions and/or materials of each assay differ slightly and therefore such differences are
noted either directly within the general assay conditions or coded according to the
respective enzyme assay using designations l a through l g in Table 1 immediately
following the general assay conditions.
Test compounds are prepared in DMSO to make up a lOmM stock solution. The
stock solution is serially diluted in DMSO to obtain a ten-point dilution curve with a final
compound concentration range l a in a 96-well round-bottom plate before conducting the
in vitro enzymatic assay. Compounds are further diluted l b in assay buffer (used for
entire assay) described immediately hereafter:
Human cathepsins S L B F and mouse cathepsin S: 50 mM sodium phosphate
(pH 6.5) containing 2.5 mM DTT and 2.5 mM EDTA plus 0.01% TRITON® X-
100
Human cathepsins K and V: 100 mM sodium acetate (pH 5.5) containing 100 mM
NaCl, 2.5 mM DTT and 2.5 mM EDTA plus 0.01% TRITON® X-100
Ten of each dilution is added to each well of row A through H of a
corresponding low protein binding half area black plate (Costar 3694). Amount l c of
substrate, benzyloxycarbonyl-L-leucyl-L-arginine-4-methyl-coumaryl-7-amide (Peptide
Institute), prepared in assay buffer, is added to each well of the plate for a final
concentration Id. Amount l e of the respective enzyme, described immediately hereafter,
prepared in assay buffer is added to each well of the plate containing substrate and test
compound resulting in a final concentration I f to initiate the reaction.
Human cathepsins S L K and V: Obtained from Calbiochem.
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Mouse cathepsin S: Briefly, mouse cathepsin S is cloned in baculovirus using a
mCathepsin S-pAN51(T760) construct containing a histidine tag. IMAC is then
employed to purify the active protein.
Human cathepsin F: Briefly, human recombinant cathepsin F enzyme is prepared
in house as follows. Procathepsin F is cloned in 293E cells using the CathepsinFpAN60
(T-2188) construct containing a histidine tag. IMAC is then employed to
purify the protein. The procathepsin F is digested using pepsin and repurified
using Mono S column chromatography resulting in purified activated cathepsin F.
The mixture is briefly shaken at low speed on a plate mixer. The RFU of the
mixture is recorded using an Envision 2103 Multilabel Reader at excitation wavelength
355 nm and emission wavelength 460 nm for 0.1 sec after an incubation time l g at room
temperature. RFU are plotted versus inhibitor concentration and a curve is fitted with a
four-parameter logistic equation to obtain IC50 values using Activity Base (ver. 7.3.2.1).
For Human cathepsin F, a Packard Fusion Alpha Microplate Reader (0.5 sec
reading/well) is used to measure RFU and GraphPad Prism 4.03 software is used to plot
RFU versus inhibitor concentration.
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Table 1. In vitro assay experimental conditions
Following a protocol essentially as described above, exemplified compounds
display IC 0 in the human cathepsin S and mouse cathepsin S enzyme inhibitor assay of
less than 800 nM and 400 nM, respectively. Particularly, the compounds of Examples 1
and 2 display IC50S in the human cathepsin S enzyme inhibitor assay of about 6.4 nM and
10.2 nM respectively, and in the mouse cathepsin S enzyme inhibitor assay of about 1.7
nM and 3.9 nM, respectively, thus demonstrating that certain compounds within the scope
of the present invention are potent inhibitors of human and mouse cathepsin S.
For human cathepsins L, B, K, and V enzyme inhibitor assays, the compound of
Example 1 displays IC50S of about >167 , 5.2 , >100 , and 33 , respectively,
and Example 2 of about >167 , 94 , >100 , and >100 , respectively. For
human cathepsin F, the compound of Example 1 displays no inhibition up to 30 .
These results demonstrate that certain compounds within the scope of the present
invention are selective inhibitors of cathepsin S.
In vivo
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CaCh-induced AAA animal efficacy model
The AAA animal efficacy model using CaC¾ induction to study the effect
of cathepsin S inhibitors on AAA (J. Clin. Invest, 2002, 110(5), 625-632) is
modified as described below.
Wild-type male 129SvEv mice (10 weeks old) from Taconic (Cambridge
City, Indiana) divided into six groups for Example 1 and five groups for Example
2 with each group containing 12 mice are used. Groups 1-5 for Examples 1 and 2
are administered respectively a vehicle solution 1% NATROSOL®
(hydroxyethylcellulose)/0.25% TWEEN® 80 (polysorbate 80)/0.05% Antifoam-
1510® (Dow Corning) and 1, 3, 10, or 30 mg/kg of test compound in vehicle
solution, by oral gavage b.i.d. for 4 weeks. Group 6 (in the study of Example 1)
representing a sham group (0.9% saline applied to the aorta instead of CaCi2 and
dosed with vehicle) is included to establish a baseline. The first dose is given one
day prior to surgery (p.m.) and the second dose is given the morning of surgery.
Animals do not receive a p.m. dose on the day of surgery. B.i.d. dosing (a.m. and
p.m.) is continued the day after surgery for 28 days.
On the day of surgery, animals received analgesia (BUPRENEX®, 0.1
mg/kg) subcutaneously 10 min. pre-operatively and 3 hr. post-operatively. Mice
are anesthetized by inhalation of 2% isoflurane and a laparotomy is performed.
The abdominal aorta is exposed by retracting the bowel laterally with a surgical
retractor and leaving the bowel in the abdominal cavity. The abdominal aorta
from the level of the renal arteries to the iliac bifurcation is isolated from the
inferior vena cava and surrounding connective tissues using micro-surgical
techniques. Once isolated, the region of interest of the abdominal aorta is
wrapped with a premeasured sterile cotton gauze soaked in 0.25 M aqueous CaCl2
solution. In sham control animals, 0.9% saline is substituted for CaCl2. After 7
min., the gauze is removed and a second CaCi2 soaked gauze (or 0.9% saline
soaked gauze in sham animals) is applied. Following a second 7 minute period,
the gauze is removed, the aorta is rinsed with 0.9% saline and the abdomen is
closed. The animals are returned to general housing where they are housed
individually with ad lib access to standard rodent diet (Purina 2014) and water.
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After 4 weeks of dosing as described above, the aortic luminal perimeter,
area and diameter of the aortic segments that were wrapped with gauze are
determined by ultrasound (Biosound Ultrasound - 7.5 MHz) and statistically
analyzed with JMP® 7 software (Cary, North Carolina). Percentage reductions of
AAA determined by measurement of the aortic luminal perimeter (which
represents in this instance a more accurate measurement of the abdominal aorta
due to the irregular geometry associated with the aortic segment being measured)
are shown below in Table 1 and are represented as means ± standard deviation.
For Example 1, vehicle group is compared to sham group, and testing compound
is compared to vehicle. For Example 2, testing compound is compared to vehicle.
Table 2. In Vivo Percentage (%) Reduction of AAA
Following a protocol essentially as described above, the compounds of
Examples 1 and 2 reduce the aortic luminal perimeter in a dose-dependent
manner, and therefore demonstrate that certain compounds within the scope of the
invention reduce AAA.
plO accumulation assay
Wild-type male C57B6 or 129SvEv mice (10 weeks old) from Taconic
(Cambridge City, Indiana) divided into four groups are used, with each group
having 3 mice. Groups 1-4 are administered respectively a vehicle solution 1%
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NATROSOL® (hydroxyethylcellulose)/0.25% TWEEN® 80 (polysorbate
80)/0.05% Antifoam-1510 (Dow Corning) and 3, 10, or 30 mg/kg of test
compound in vehicle solution by oral gavage. After 4 hr., whole blood (300-500
) is drawn from each mouse. The step of removing red blood cells is
performed by adding one mL Flow Cytometry Lysing Solution™ (Santa Cruz
Biotechnology, Inc.), keeping at room temperature for 10 min., centrifuging at
4000 rpm for 5 min. and then discarding the supernatant. This step is repeated
once to remove the red blood cells completely. The PWBC pellet is resuspended
with 50 CytoBuster™ Reagent (Novagen) and sonicated at high power for
5-10 sec. The protein concentrations are determined using a BCA assay (Anal.
Biochem. 1988, 175, 231-237/
The p 10 amount is determined using a Western blot. Samples (0.5 g l
protein concentration) are denatured at 96 °C for 5 min. and 20 ΐ/well of samples
(^g/well) are loaded to 4-12% NUPAGE® NOVEX® Bis-Tris Midi-gel
(Invitrogen). Gels are run at 120 V for 60 min. with the NUPAGE® MES SDS
running buffer (Invitrogen). Proteins are transferred to 0.2 nitrocellulose
(BIO-RAD) at 100 V for 30 min. with NUPAGE® Transfer Buffer (Invitrogen)
and 20% methanol (EMD). Blot is briefly rinsed with PBS and blocked in 10 mL
in ODYSSEY® Blocking Buffer (LI-COR Biosciences) at room temperature for
60 min. For plO, the blot is incubated with primary antibody against mouse CD74
( 1 g/ml of rat anti-CD74 antibody)(BD Bioscience) in blocking buffer at 4 °C
overnight. For tubulin control, the blot is incubated with rabbit anti-beta tubulin
pAb (0.2 g/ml)(Abcam) in blocking buffer at 4 °C overnight. The blot is washed
with washing buffer (DPBS from HyClone) with 0.01% polysorbate 20 four times
for 10 min. each. The blot is then incubated with secondary antibody at room
temperature for 60 min. For plO, ALEXA FLUOR® 680 goat anti rat IgG
(1:5000 dilution)(Invitrogen) is used. For tubulin, ALEXA FLUOR® 680 goat
anti rabbit IgG (1:5000 dilution)(Invitrogen). The blot is again washed as
described above and placed in PBS for scanning.
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The images are captured by scanning on an ODYSSEY® Infrared Imager
(LI-COR Biosciences). The plO amount is analyzed with ODYSSEY® software
and normalized with the tubulin amount. The data are statistically analyzed with
JMP® 7 software (Cary, North Carolina). Relative plO accumulations (fold
increase) to the vehicle are shown in Table 3. Values are represented as means ±
standard deviation.
Table 3. In vivo relative plO accumulation in PWBC
Following the above protocol, the compounds of Examples 1 and 2 dose
dependently increase plO in PWBC, demonstrating that certain comounds within
the scope of the invention block antigen presentation.
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WE CLAIM:
1. A compound of Formula (I)
wherein,
Z is -CH2-, -CH2CH2-, -OCH2-, -CH2CH2CH2-, or -OCH 2CH2-;
R1 is H, F, or CI;
R2 is H, methyl, ethyl, propyl, or isopropyl;
or a pharmaceutically acceptable salt thereof.
2. The compound according to claim 1, wherein Ri is H or F, or a
pharmaceutically acceptable salt thereof.
3. The compound according to claims 1 or 2, wherein Ri is F, or a
pharmaceutically acceptable salt thereof.
4. The compound according to any one of claims 1to 3, wherein R2 is methyl or
ethyl, or a pharmaceutically acceptable salt thereof.
5. The compound according to any one of claims 1to 4, wherein R2 is methyl, or
a pharmaceutically acceptable salt thereof.
6. The compound according to any one of claims 1to 5, wherein Z is -CH2CH2-
or -OCH2-, or a pharmaceutically acceptable salt thereof.
7. The compound according to claim 1, having the formula:
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or a pharmaceutically acceptable salt thereof.
8. The compound according to claim 1, having the formula:
or a pharmaceutically acceptable salt thereof.
9. A pharmaceutical composition comprising a compound according to any one
of claims 1 to 8, and a pharmaceutically acceptable diluent or carrier.
10. A method for treatment of abdominal aortic aneurysm, plaque instability,
atherosclerosis, rheumatoid arthritis, psoriasis, or lupus in a mammal in need thereof,
comprising administering to said mammal a therapeutically effective amount of a
compound or pharmaceutically acceptable salt thereof according to any one of claims 1-8.
11. A method for treatment of abdominal aortic aneurysm, plaque instability,
atherosclerosis, rheumatoid arthritis, psoriasis, or lupus in a mammal in need thereof,
comprising administering to said mammal a therapeutically effective amount of a
pharmaceutical composition according to claim 9.
12. A method according to claims 10 or 11, wherein said mammal is a human.
13. A compound according to any one of claims 1-8, or a pharmaceutically
acceptable salt thereof, for use in therapy.
14. A compound according to any one of claims 1-8, or a pharmaceutically
acceptable salt thereof, for use in the treatment of abdominal aortic aneurysm, plaque
instability, atherosclerosis, rheumatoid arthritis, psoriasis, or lupus.
15. The use of a compound as claimed in any one of claims 1 to 8, or a
pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the
treatment of abdominal aortic aneurysm, plaque instability, atherosclerosis, rheumatoid
arthritis, psoriasis, or lupus.