Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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COMPOSITIONS AND METHODS FOR INHIBITING CATHEPSIN PROTEASES
Cross Reference to Related Applications
[0001] This application claims the benefit of U.S. provisional application
serial no.
60/862,500, filed October 23, 2006, which is incorporated herein by reference
in its entirety.
Background Art
[0002] Most cathepsins are endopeptidases belonging to the family of papain-
like cysteine
proteases. Cathepsins are generally highly concentrated in lysosomal and
endosomal
compartments and play a role in a broad array of physiological processes.
Among them are non-
specific functions such as degradation of both internalized and cellular
proteins, as well as more
specialized functions in the processing of enzymes and hormones. Various
diseases are linked
to the overexpression of certain proteases. For example, inhibition of
specific cathepsin
proteases is believed to have therapeutic implications on pathological
conditions associated with
cellular homeostasis, apoptosis, tumor invasion and metastasis, bone
resorption and antigen
presentation.
[0003] Most small molecule inhibitors of proteases, including those for
cathepsins, consist of
a peptidomimetic that recognizes specific pockets in the enzyme and defines
substrate
selectivity, and an electrophilic "warhead" that makes important contacts to
the catalytic
domain. The nature of the electrophilic group determines the classification of
these small
molecules into reversible and irreversible inhibitors. Several electrophilic
groups have been
routinely employed in the inhibition of proteases by covalent interaction of
enzyme with
inhibitors. For example, aldehydes, semicarbazones, nitriles and ketones
usually lead to
reversible inhibition, whereas halomethylketones, epoxides and Michael
acceptors are among
the functionalities used for irreversible inhibition.
[0004] To date, relatively few protease inhibitors have successfully
progressed through
clinical trials. Major issues involve non-druglike properties of the peptidic
part of the inhibitor
and/or the lack of selectivity, often attributed to non-specific interactions
of the electrophilic
functionality with endogenous nucleophiles in vivo. In order to circumvent
these drawbacks
commonly seen with the classical approach of substrate-based drug design,
novel molecular
concepts need to be explored.
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Disclosure of the Invention
[0005] The invention provides compounds and pharmaceutical compositions
thereof, which
may be useful as inhibitors for cathepsin proteases.
[0006] In one aspect, the present invention provides compounds of Formula (1):
X
0
N
(R'), (1)
or pharmaceutically acceptable salts thereof, wherein:
XisOorS;
Rl is ORZ, halo, (CR2)õR3, nitro, cyano, amino, amido, sulfonamide, or an
optionally
substituted Cl_6alkyl, C2_6 alkenyl, or C3_6 alkynyl;
RZ is H, (CRZ)õR3, or an optionally substituted Cl_6alkyl, C2_6 alkenyl or
C3_6 alkynyl;
R3 is an optionally substituted aryl, heteroaryl, carbocyclic ring or
heterocyclic ring;
m is 1-3; and
n is 0-4.
[0007] In one embodiment, m is 1. For example, Rl is halo or OR2, wherein R2
may be an
optionally substituted phenyl, benzyl or C1_6 alkyl. In some examples,
compounds having
Formula (1) have a cis stereoconformation. In other examples, compounds having
Formula (1)
have a trans stereoconformation.
[0008] In another aspect, the invention provides pharmaceutical compositions
comprising
compounds having Formula (1) and a pharmaceutically acceptable excipient.
[0009] In yet another aspect, the invention provides methods for inhibiting a
cathepsin
protease, comprising administering to a system or a subject in need thereof, a
therapeutically
effective amount of a compound of Formula (1), or pharmaceutically acceptable
salts or
pharmaceutical compositions thereof, thereby inhibiting said cathepsin
protease.
[0010] The present invention also provides methods for treating a condition or
disease
mediated by cathepsin protease activity, comprising administering to a system
or a subject in
need thereof, a therapeutically effective amount of a compound of Formula (1),
or
pharmaceutically acceptable salts or pharmaceutical compositions thereof,
thereby treating said
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cathepsin protease-mediated condition or disease. In one embodiment, the
invention provides
methods for treating a condition or disease mediated by papain-like cathepsin
protease,
including but not limited to cellular homeostasis, apoptosis, tumor invasion
and metastasis, bone
resorption and antigen presentation. For example, the compounds of the
invention may be used
to treat osteoporosis, arthritis, asthma, auto-immune disease and tumors.
[0011] In yet another aspect, the invention provides the use of compounds
having Formula
(1) for the manufacture of a medicament for treating a disease mediated by
cathepsin protease,
more particularly papain-like cathepsin protease.
[0012] In the above methods for using the compounds of the invention, a
compound having
Formula (1) may be administered to a system comprising cells or tissues. In
other embodiments,
a compound having Formula (1) may be administered to a human or animal
subject.
Definitions
[0013] "Alkyl" refers to a moiety and as a structural element of other groups,
for example
halo-substituted-alkyl and alkoxy, and may be straight-chained or branched. An
optionally
substituted alkyl, alkenyl or alkynyl as used herein may be optionally
halogenated (e.g., CF3), or
may have one or more carbons that is substituted or replaced with a
heteroatom, such as NR, 0
or S (e.g., -OCHzCHzO-, alkylthiols, thioalkoxy, alkylamines, etc).
[0014] "Aryl" refers to a monocyclic or fused bicyclic aromatic ring
containing carbon
atoms. For example, aryl may be phenyl or naphthyl. "Arylene" means a divalent
radical
derived from an aryl group.
[0015] "Heteroaryl" as used herein is as defined for aryl above, where one or
more of the
ring members are a heteroatom. Examples of heteroaryls include but are not
limited to pyridyl,
indolyl, indazolyl, quinoxalinyl, quinolinyl, benzofuranyl, benzopyranyl,
benzothiopyranyl,
benzo[1,3]dioxole, imidazolyl, benzo-imidazolyl, pyrimidinyl, furanyl,
oxazolyl, isoxazolyl,
triazolyl, tetrazolyl, pyrazolyl, thienyl, etc.
[0016] A "carbocyclic ring" as used herein refers to a saturated or partially
unsaturated,
monocyclic, fused bicyclic or bridged polycyclic ring containing carbon atoms,
which may
optionally be substituted, for example, with =0. Examples of carbocyclic rings
include but are
not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,
cyclopropylene, cyclohexanone,
etc.
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[0017] A "heterocyclic ring" as used herein is as defined for a carbocyclic
ring above,
wherein one or more ring carbons is a heteroatom. For example, a heterocyclic
ring may contain
N, 0, S, -N=, -S-, -S(O), -S(O)Z-, or -NR- wherein R may be hydrogen, Cl4alkyl
or a protecting
group. Examples of heterocyclic rings include but are not limited to
morpholino, pyrrolidinyl,
pyrrolidinyl-2-one, piperazinyl, piperidinyl, piperidinylone, 1,4-dioxa-8-aza-
spiro[4.5]dec-8-yl,
etc.
[0018] Unless otherwise indicated, when a substituent is deemed to be
"optionally
substituted," it is meant that the substituent is a group that may be
substituted with one or more
group(s) individually and independently selected from, for example, an
optionally halogenated
alkyl, alkenyl, alkynyl, alkoxy, alkylamine, alkylthio, alkynyl, amide, amino,
including mono-
and di-substituted amino groups, aryl, aryloxy, arylthio, carbonyl,
carbocyclic, cyano,
cycloalkyl, halogen, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl,
heterocyclic, hydroxy,
isocyanato, isothiocyanato, mercapto, nitro, 0-carbamyl, N-carbamyl, 0-
thiocarbamyl,
N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, 0-
carboxy,
perhaloalkyl, perfluoroalkyl, silyl, sulfonyl, thiocarbonyl, thiocyanato,
trihalomethanesulfonyl,
and the protected compounds thereof. The protecting groups that may form the
protected
compounds of the above substituents are known to those of skill in the art and
may be found in
references such as Greene and Wuts, Protective Groups in Organic Synthesis, 3d
Ed., John
Wiley & Sons, New York, NY, 1999, and Kocienski, Protective Groups, Thieme
Verlag, New
York, NY, 1994, which are incorporated herein by reference in their entirety.
[0019] The terms "co-administration" or "combined administration" or the like
as used
herein are meant to encompass administration of the selected therapeutic
agents to a single
patient, and are intended to include treatment regimens in which the agents
are not necessarily
administered by the same route of administration or at the same time.
[0020] The term "pharmaceutical combination" as used herein refers to a
product obtained
from mixing or combining active ingredients, and includes both fixed and non-
fixed
combinations of the active ingredients. The term "fixed combination" means
that the active
ingredients, e.g. a compound of Formula (1) and a co-agent, are both
administered to a patient
simultaneously in the form of a single entity or dosage. The term "non-fixed
combination"
means that the active ingredients, e.g. a compound of Formula (1) and a co-
agent, are both
administered to a patient as separate entities either simultaneously,
concurrently or sequentially
with no specific time limits, wherein such administration provides
therapeutically effective
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levels of the active ingredients in the body of the patient. The latter also
applies to cocktail
therapy, e.g. the administration of three or more active ingredients.
[0021] The term "therapeutically effective amount" means the amount of the
subject
compound that will elicit a biological or medical response in a cell, tissue,
organ, system, animal
or human that is being sought by the researcher, veterinarian, medical doctor
or other clinician.
[0022] The term "administration" and or "administering" of the subject
compound should be
understood to mean as providing a compound of the invention including a pro-
drug of a
compound of the invention to the individual in need of treatment.
Details Description of the Invention
[0023] The invention provides compounds and compositions for inhibiting
cathepsin
proteases, more particularly papain-like cathepsin proteases. The present
invention also
provides methods for treating a condition or disease mediated by cathepsin
protease activity,
particularly a condition or disease mediated by papain-like cathepsin
protease, comprising
administering to a system or a subject in need thereof, a therapeutically
effective amount of a
compound of Formula (1), or pharmaceutically acceptable salts or
pharmaceutical compositions
thereof, thereby treating said papain-like cathepsin protease-mediated
condition or disease.
[0024] Papain-like cysteine proteases have been identified as key proteolytic
activities in
degenerative, invasive, and immune system related disorders. (Lecaille et al.,
Chem. Rev. 2002,
102:4459-4488; Br6mme et al., Curr. Pharm. Des. 2002, 8:1639-1658). For
example, cathepsin
K is the major bone-degrading activity in osteoclasts, and its selective
inhibition may be
beneficial for treating osteoporosis and certain forms of arthritis. Cathepsin
S plays an
important role in MHC class II dependent antigen presentation; inhibition of
cathepsin S
significantly decreases the response to antigens, rendering cathepsin S as a
drug target for
asthma and certain auto-immune diseases. (Riese et al., J. Clin. Invest. 1998,
101:2351-2363).
Cathepsins also have been implicated in tumor invasion and metastasis, and in
tumors of the
central nervous systems, such as astrocytoma, glioblastoma and meningioma.
(Berquin et al.,
Perspect. Drug Discovery Design 1995, 2:371-388; Levicar et al., J.
Neurooncol. 2002, 58:21-
32).
[0025] In one aspect, the compounds of the invention have Formula (1):
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X
0
Z
(R'),, (1)
or pharmaceutically acceptable salts thereof, wherein:
XisOorS;
Rl is ORZ, halo, (CR2)õR3, nitro, cyano, amino, amido, sulfonamide, or an
optionally
substituted Cl_6alkyl, C2_6 alkenyl, or C3_6 alkynyl;
RZ is H, (CRZ)õR3, or an optionally substituted Cl_6alkyl, C2_6 alkenyl or
C3_6 alkynyl;
R3 is an optionally substituted aryl, heteroaryl, carbocyclic ring or
heterocyclic ring;
m is 1-3; and
n is 0-4.
[0026] In one embodiment, the compounds of the invention may be used to
inhibit papain-
like cathepsin proteases. Table 1 shows the apparent inhibition constants
(K;(app), M) of
various compounds of the invention for inhibition of various cathepsins Stre1=
relative
stereoconformation.
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x
~-O
N
OR
entry X Strel R CatB CatS CatC CatL CatK CatH CatX CatF CatV
8 0 trans 0.046 0.473 1.727 1.270 2.438 >30 0.553 5.908 3.317
12 0 cis 0.001 0.016 0.018 0.022 0.038 2.653 0.197 0.331 0.131
13 S cis 0.519 0.696 0.606 0.994 1.833 >100 7.149 >10 5.166
14 0 cis 0.005 0.017 0.029 0.026 0.104 6.591 0.326 0.487 0.158
15 0 cis 0.002 0.174 0.403 0.263 0.511 >10 0.547 3.760 1.289
16 0 cis 0.008 0.298 0.191 0.230 1.267 >10 1.102 1.826 0.894
19 0 trans OR = Br 0.158 4.553 >10 5.816 >30 >100 >10 >10 >10
6 >100 >100 >100 >100 >100 >100 >100 >100 >100
11 >100 >100 >100 >100 >100 >100 >100 >100 >100
21 >100 >100 >100 >100 >100 >100 >100 >100 >100
Table 1
[0027] Cyclic carbamates 8 and 12-16 showed inhibitory activity across the
board of papain-
like cathepsin proteases. Within this protease family, the apparent K; values
are lowest for
cathepsin B, followed by cathepsins S, C, L, and K. Moderate activities are
seen against
cathepsins V, X, F and H. The compounds are shown to be inactive against other
families of
cysteine proteases such as caspases and against members of the serine protease
family such as
hepsin, thrombin, MT-SP1 or trypsin.
[0028] Generally, there is a drop in K; values when comparing the trans to the
cis configured
stereoisomers (cf. 8 vs 12). Analog 12, which contains the more hydrophobic
dimethylphenyl
group, seemed to be consistently more active in the cathepsin panel when
compared to the
phenyl substituted analog 14. The thiocarbamate 13 has substantially less
inhibitory effects
when compared to the oxo-carbamates. The alkyl-substituted analogs 15 (R =
benzyl) and 16 (R
= isopropyl) generally displayed about 10-fold higher K;-values than 12 or 14.
An exception is
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the low nanomolar activity of the benzyl substituted analog 15 against
Cathepsin B, representing
the most selective inhibitor for Cathepsin B in the series (100-fold).
[0029] Although the mechanism is not necessary to practice the invention, the
mechanism of
inhibition by the bicyclic carbamates of the invention is examined in a
dialysis study. Briefly,
cathepsin B is first incubated at 37 C with 0.2 M of compound 12 (200x K;)
for complete
suppression of catalytic activity (2.9 rfu/sec for inhibitor treated versus
121 rfu/sec for vehicle
control). Then the mixtures are dialyzed extensively at 4 C to remove unbound
inhibitor before
the cathepsin activities are re-measured at 37 C (51 rfu/sec for inhibitor
treated versus 71 rfu/sec
for vehicle control). The apparent recovery of cathepsin B activity in
compound 12 treated
sample after dialysis suggests that compound 12 is a partially reversible
inhibitor. However, a
careful examination of the progress curves revealed that the substrate
conversion is accelerated
over the course of the assay (indicated by the dotted arrows). This rate
increase may likely
caused by a temperature sensitive release of covalently bound compound 12 from
the active site,
since the substrate conversion of the control is not affected by the
incubation temperature.
[0030] The potential adduct formation between cathepsin B and inhibitor
(compound 12) is
analyzed by mass spectrometry following tryptic/chymotryptic digestion. Two
active site
fragments of the test enzyme (cathepsin B) are identified as I9EIRDQGSCGSC*W30
and
ZZDQGSCGSC*W30 from the untreated cathepsin B sample. For inhibitor treated
cathepsin B,
both fragments gained a mass of 247, corresponding to a single covalent
conjugation of 12 to the
catalytic cysteine residues (C). The stability of this adduct is sensitive to
temperature increase.
Compared to the control that is kept on ice, a 2-hour incubation at 37 C
promoted the
dissociation of this enzyme-inhibitor adduct, resulting in a 4-fold increase
in abundance of a
metabolite. The multiple reaction monitoring (MRM) characteristics of the
metabolite are
identical to that of the suggested dissociation product 11.
[0031] In summary, the invention provides compounds of Formula (1) that are
selective for
the cathepsin family, with subtype selectivities following the order B > S>
C,L,K > V,X,F,H. A
preference for hydrophobic R-groups in cis configuration relative to the
carbamate oxygen is
observed, with apparent K; values for Cathepsin B in the single digit
nanomolar range. The
carbamate functionality in the scaffold is substantially destabilized, and
offers a weak point for
nucleophilic attack by the active site cysteine thiol. The bicycle
subsequently undergoes ring-
opening and covalently binds to the catalytic cysteine, leading to inhibition
of the enzyme. This
hypothesis is in accordance with the mass spectrometric analysis of digested
enzyme. After
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incubation with compound 12, the fragments which include the catalytic
cysteine covalently
carry the inhibitor. The non-linear rate recovery seen in dialysis studies and
characterization of
metabolites via MRM suggest that the inhibitor slowly gets hydrolyzed off the
enzyme as its
ring-opened synthetic precursor 11 at ambient temperature.
Administration and Pharmaceutical Compositions
[0032] In general, compounds of the invention will be administered in
therapeutically
effective amounts via any of the usual and acceptable modes known in the art,
either singly or in
combination with one or more therapeutic agents. A therapeutically effective
amount may vary
widely depending on the severity of the disease, the age and relative health
of the subject, the
potency of the compound used and other factors. In general, satisfactory
results are indicated to
be obtained systemically at daily dosages of from about 0.03 to 2.5 mg/kg per
body weight. An
indicated daily dosage in the larger mammal, e.g. humans, is in the range from
about 0.5 mg to
about 100 mg, conveniently administered, e.g. in divided doses up to four
times a day or in
retard form. Suitable unit dosage forms for oral administration comprise from
ca. 1 to 50 mg
active ingredient.
[0033] Compounds of the invention may be administered as pharmaceutical
compositions by
any conventional route, in particular enterally, e.g., orally, e.g., in the
form of tablets or
capsules, or parenterally, e.g., in the form of injectable solutions or
suspensions, topically, e.g.,
in the form of lotions, gels, ointments or creams, or in a nasal or
suppository form.
[0034] Pharmaceutical compositions comprising a compound of the present
invention in free
form or in a pharmaceutically acceptable salt form in association with at
least one
pharmaceutically acceptable carrier or diluent may be manufactured in a
conventional manner
by mixing, granulating or coating methods. For example, oral compositions may
be tablets or
gelatin capsules comprising the active ingredient together with a) diluents,
e.g., lactose,
dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b)
lubricants, e.g., silica, talcum,
stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for
tablets, together with
c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin,
tragacanth, methylcellulose,
sodium carboxymethylcellulose and or polyvinylpyrrolidone; and if desired, d)
disintegrants,
e.g., starches, agar, alginic acid or its sodium salt, or effervescent
mixtures; and/or e) absorbents,
colorants, flavors and sweeteners. Injectable compositions may be aqueous
isotonic solutions or
suspensions, and suppositories may be prepared from fatty emulsions or
suspensions.
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[0035] The compositions may be sterilized and/or contain adjuvants, such as
preserving,
stabilizing, wetting or emulsifying agents, solution promoters, salts for
regulating the osmotic
pressure and/or buffers. In addition, they may also contain other
therapeutically valuable
substances. Suitable formulations for transdermal applications include an
effective amount of a
compound of the present invention with a carrier. A carrier may include
absorbable
pharmacologically acceptable solvents to assist passage through the skin of
the host. For
example, transdermal devices are in the form of a bandage comprising a backing
member, a
reservoir containing the compound optionally with carriers, optionally a rate
controlling barrier
to deliver the compound to the skin of the host at a controlled and
predetermined rate over a
prolonged period of time, and means to secure the device to the skin. Matrix
transdermal
formulations may also be used. Suitable formulations for topical application,
e.g., to the skin
and eyes, may be aqueous solutions, ointments, creams or gels well-known in
the art. Such may
contain solubilizers, stabilizers, tonicity enhancing agents, buffers and
preservatives.
[0036] Compounds of the invention may be administered in therapeutically
effective
amounts in combination with one or more therapeutic agents (pharmaceutical
combinations).
Where the compounds of the invention are administered in conjunction with
other therapies,
dosages of the co-administered compounds will of course vary depending on the
type of co-drug
employed, on the specific drug employed, on the condition being treated and so
forth.
[0037] For example, the compounds of the invention may be used in combination
with a
chemotherapeutic agent to treat a cell proliferative disorder and tumors.
Examples of
chemotherapeutic agents which may be used in the compositions and methods of
the invention
include but are not limited to anthracyclines, alkylating agents (e.g.,
mitomycin C), alkyl
sulfonates, aziridines, ethylenimines, methylmelamines, nitrogen mustards,
nitrosoureas,
antibiotics, antimetabolites, folic acid analogs (e.g., dihydrofolate
reductase inhibitors such as
methotrexate), purine analogs, pyrimidine analogs, enzymes, podophyllotoxins,
platinum-
containing agents, interferons, and interleukins. Particular examples of known
chemotherapeutic agents which may be used in the compositions and methods of
the invention
include, but are not limited to, busulfan, improsulfan, piposulfan, benzodepa,
carboquone,
meturedepa, uredepa, altretamine, triethylenemelamine,
triethylenephosphoramide,
triethylenethiophosphoramide, trimethylolomelamine, chlorambucil,
chlornaphazine,
cyclophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine
oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil
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mustard, carmustine, chlorozotocin, fotemustine, lomustine, nimustine,
ranimustine,
dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman,
aclacinomycins, actinomycin
F(1), anthramycin, azaserine, bleomycin, cactinomycin, carubicin,
carzinophilin, chromomycin,
dactinomycin, daunorubicin, daunomycin, 6-diazo-5-oxo-l-norleucine,
doxorubicin, epirubicin,
mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin,
plicamycin,
porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, ubenimex,
zinostatin,
zorubicin, denopterin, methotrexate, pteropterin, trimetrexate, fludarabine, 6-
mercaptopurine,
thiamiprine, thioguanine, ancitabine, azacitidine, 6-azauridine, carmofur,
cytarabine,
dideoxyuridine, doxifluridine, enocitabine, floxuridine, fluorouracil,
tegafur, L-asparaginase,
pulmozyme, aceglatone, aldophosphamide glycoside, aminolevulinic acid,
amsacrine,
bestrabucil, bisantrene, carboplatin, cisplatin, defofamide, demecolcine,
diaziquone, elfornithine,
elliptinium acetate, etoglucid, etoposide, flutamide, gallium nitrate,
hydroxyurea, interferon-
alpha, interferon-beta, interferon-gamma, interleukin-2, lentinan, lonidamine,
mitoguazone,
mitoxantrone, mopidamol, nitracrine, pentostatin, phenamet, pirarubicin,
podophyllinic acid, 2-
ethylhydrazide, procarbazine, razoxane, sizofiran, spirogermanium, paclitaxel,
tamoxifen,
teniposide, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine,
urethane, vinblastine,
vincristine, and vindesine.
[0038] Synergistic effects may also occur with other immunomodulatory or anti-
inflammatory substances, for example when used in combination with
cyclosporin, rapamycin,
or ascomycin, or immunosuppressant analogues thereof, for example cyclosporin
A(CsA),
cyclosporin G, FK-506, rapamycin, or comparable compounds, corticosteroids,
cyclophosphamide, azathioprine, methotrexate, brequinar, leflunomide,
mizoribine,
mycophenolic acid, mycophenolate mofetil, 15-deoxyspergualin,
immunosuppressant
antibodies, especially monoclonal antibodies for leukocyte receptors, for
example MHC, CD2,
CD3, CD4, CD7, CD25, CD28, B7, CD45, CD58 or their ligands, or other
immunomodulatory
compounds, such as CTLA41g.
[0039] The invention also provides for a pharmaceutical combinations, e.g. a
kit, comprising
a) a first agent which is a compound of the invention as disclosed herein, in
free form or in
pharmaceutically acceptable salt form, and b) at least one co-agent. The kit
may comprise
instructions for its administration.
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Processes for Making Compounds of the Invention
[0040] The compounds of the present invention may be prepared according to
Scheme 1.
Cbz H Cbz Cbz Cbz
N a),1) N a)' b) N C) N
01,' - U ," Q 3- aoH + 'O R
OH O =
OH 2 3 OR OH
17 4 5
k) g), h), i) d) d)
12 N=HBr N N H
C-~ c1O H (~~O " 'OR
OH H
gr OR OR O H
18 11 6 7
e), f) e), f) e), f)
O~O O~O OO OO
X~ N~OR ~~
~ OR
Br OR
19 12 8 9
5l'-l Br O~
I), m O
21
Scheme 1
[0041] In Scheme 1, the reagents and conditions are: (a) CbzCl, 0.5 M
Na2CO3/dioxane 5:2,
rt, 5h; (b) mCPBA, DCM, 0 C-rt, 7h; (c) ROH, 2 M NaOH/MeCN 1:4, reflux, 48h;
(d) 1 atm
H2, Pd/C (cat), EtOH, rt, 3h; (e) triphosgene, NEt3, DCM, 0 C, lh; (f) NaH,
rt, 18h; (g) PPh3,
DEAD, p-nitrobenzoic acid, THF, 50 C, 48h; (h) NaOH, MeOH, rt, lh; (i) 1 atm
H2, Pd/C (cat),
EtOH, rt, 3h; (j) 0S04, NMO, citric acid, tBuOH/H20 1:1, 50 C, 12h; (k) HBr,
HOAc, 0 C-rt,
3h; (1) 2,3-dimethylphenol, K2C03, MeCN, 60 C, 12h; (m) MeNH2, MeOH, 50 C, 2h;
(n) CDI,
DMAP, benzene, reflux, 4h. Compounds 3-21 are racemic mixtures.
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[0042] According to synthetic procedures described by Paioni (U.S. patent no.
4,160,837,
incorporated herein by reference in its entirety), the tetrahydropyridine 2 is
converted to the
racemic Cbz-protected epoxide 3 in two steps. Opening of the epoxide 3 (ROH =
2,3-
dimethylphenol) under basic aqueous conditions gives a mixture of
regioisomers. As expected
for a favored trans-diaxial ring-opening, the ratio of 4-aryloxy piperidines 4
versus 3-
aryloxypiperidines 5 is about 5:1. The regioisomers are chromatographically
separated and
hydrogenolytically deprotected to give the open forms 6 and 7. The cyclization
of 6 requires a
two step procedure. First the piperidine-nitrogen is selectively carbonylated
to the
corresponding chloroformate using triphosgene and triethylamine, then the 3-
hydroxy group is
deprotonated using sodium hydride, which leads to spontaneous intramolecular
cyclization to
give racemic trans-4-(2,3-dimethyl-phenoxy)-6-oxa-l-aza-bicyclo[3.2.1]octan-7-
one 8.
Attempts to cyclize the chloroformate intermediate of 7 to the bicycle 9 are
unsuccessful,
probably due to the unfavorable boat conformation of the piperidine ring
required for
intramolecular ring closure.
[0043] The Mitsunobu reaction using p-nitrobenzoic acid as the nucleophile is
employed to
invert the stereocenter at position 3 of compound 4. Alternatively, the
inversion may be
accomplished by an oxidation/reduction sequence using Pyr=SO3 and Red-Al.
Saponification of
the intermediate 10 in methanolic base followed by deprotection gives
piperidine 11.
Intramolecular cyclization analogous to the above described procedure gives
cis-4-(2,3-
dimethyl-phenoxy)-6-oxa-l-aza-bicyclo[3.2.1]octan-7-one 12.
[0044] Both stereoisomers 8 and 12 are isolated as white, stable solids. The
compounds are
also stable in aqueous basic or neutral solutions, while spontaneous ring
opening is observed
under fairly acidic conditions (pH<2). Analogs 13-16 (Table 1, supra) are
synthesized in a
similar fashion to the synthesis of 12. In the case of analog 13, thiophosgene
is used instead of
phosgene in the cyclization step; for analogs 15 and 16, the corresponding
alcohol
(benzylalcohol for 15, isopropanol for 16) is used as solvent together with
NaH in the opening of
epoxide 3. An alternative synthetic route via dihydroxylation leads to the
protected
tetrahydropyridine 17. Selective cyclization and alkylation of the deprotected
diol failed to give
the desired bicyclic carbamate analogs. Treatment of the epoxide 3 with HBr
selectively gives
bromide 18 which could be cyclized to the carbamate 19, but any attempts to
displace the
bromide were unsuccessful. The control compound 21 is synthesized in three
steps starting from
epibromohydrin 20.
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[0045] Furthermore, compounds of the present invention may be obtained in the
free form,
or as a salt thereof if salt forming groups are present, or as esters if ester
forming groups are
present. Compounds of the present invention that have acidic groups may be
converted into
salts with pharmaceutically acceptable bases, e.g., an aqueous alkali metal
hydroxide,
advantageously in the presence of an ethereal or alcoholic solvent, such as a
lower alkanol.
Resulting salts may be converted into the free compounds, e.g., by treatment
with acids. These
or other salts may also be used for purification of the compounds obtained.
Ammonium salts
may be obtained by reaction with the appropriate amine, e.g., diethylamine,
and the like.
[0046] In certain aspects, compounds of the present invention having basic
groups may be
converted into acid addition salts, especially pharmaceutically acceptable
salts. These may be
formed, for example, with inorganic acids such as mineral acids (e.g.,
sulfuric acid, a phosphoric
or hydrohalic acid); organic carboxylic acids (e.g., C1-C4 alkyl carboxylic
acids such as acetic
acid, which may be unsubstituted or substituted by halogen; saturated or
unsaturated
dicarboxylic acids, such as oxalic, succinic, maleic or fumaric acid;
hydroxycarboxylic acids,
such as glycolic, lactic, malic, tartaric or citric acid); amino acids, such
as aspartic or glutamic
acid); organic sulfonic acids (e.g., C1-C4 alkylsulfonic acids such as
methanesulfonic acid); or
arylsulfonic acids which may be unsubstituted or substituted (for example, by
halogen).
Preferred may be salts formed with hydrochloric acid, methanesulfonic acid and
maleic acid.
[0047] In view of the close relationship between the free compounds and the
compounds in
the form of their salts or esters, whenever a compound is referred to in this
context, a
corresponding salt or ester is also intended, provided such is possible or
appropriate under the
circumstances. The compounds, including their salts, may also be obtained in
the form of their
hydrates, or include other solvents used for their crystallization.
[0048] The compounds of the present invention that comprise free hydroxyl
groups may also
exist in the form of pharmaceutically acceptable, physiologically cleavable
esters, and as such
may be included within the scope of the invention. Such pharmaceutically
acceptable esters
may be preferably prodrug ester derivatives, such being convertible by
solvolysis or cleavage
under physiological conditions to the corresponding compounds of the present
invention which
comprise free hydroxyl groups. Suitable pharmaceutically acceptable prodrug
esters may be
those derived from a carboxylic acid, a carbonic acid monoester or a carbamic
acid, preferably
esters derived from an optionally substituted lower alkanoic acid or an
arylcarboxylic acid.
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[0049] As will be apparent to one of skill in the art, certain compounds of
the present
invention may possess asymmetric carbon atoms (optical centers) or double
bonds; the
racemates, diastereomers, enantiomers, geometric isomers and individual
isomers may be all
intended to be encompassed within the scope of the present invention.
[0050] Detailed examples of the synthesis of a compound of Formula (1) can be
found in the
Examples, infra, to illustrate, but not limit the invention.
Example 1
trans-4-(2,3-Dimethyl-phenoxy)-6-oxa-l-aza-bicyclo[3.2.1loctan-7-one (8)
3,6-dihydro-2H-pyridine-l-carboxylic acid benzyl ester (2)
[0051] 1,2,3,6-Tetrahydropyridine (1.1 mL, 12 mmol) is dissolved in dioxane
(10 mL). An
aqueous solution of 0.5 M sodium carbonate (25 mL, 12.5 mmol) is added
followed by benzyl
chloroformate (1.8 mL, 12 mmol). The mixture is stirred for 3 h at rt, then
diluted with ethyl
acetate (50 mL). The organic layer is separated, washed with water and dried
over magnesium
sulfate. The solvents are removed in vacuo to give 3,6-dihydro-2H-pyridine-l-
carboxylic acid
benzyl ester as a colorless liquid. 1H-NMR (400 MHz, CDC13) S= 7.40-7.33 (m,
5H), 5.85 (m,
1H), 5.68 (m, 1H), 5.19 (s, 2H), 4.00 (t, J = 2 Hz, 2H), 3.60 (t, J 5.7 Hz,
2H), 1.72 (m, 2H).
MS calcd. for C13H16N02 (M+H+) 218.1, found 218.3.
7-oxa-3-aza-bicyclo[4.1.Olheptane-3-carboxylic acid benzyl ester (3)
[0052] The 3,6-Dihydro-2H-pyridine-1-carboxylic acid benzyl ester 2 (2.6 g, 12
mmol) is
dissolved in dichloromethane (100 mL). m-Chloroperbenzoic acid (4.5 g, 18
mmol) dissolved in
dichloomethane (40 mL) is added dropwise over 20 min. The mixture is stirred
at rt for 7 h, then
the solution is washed with 1 M aqueous sodium carbonate three times and once
with brine. The
organic phase is separated, dried (MgS04) and concentrated in vacuo to give
the racemic
epoxide 7-oxa-3-aza-bicyclo[4.1.0]heptane-3-carboxylic acid benzyl ester (3)
as a colorless oil.
'H-NMR (400 MHz, CDC13) S= 7.40-7.32 (m, 5H), 5.14 (s, 2H), 3.99 (m, 1H), 3.80
(m, 1H),
3.53 (m, 1H), 3.31 (m, 1H), 3.24 (m, 1H), 2.10 (m, 1H), 1.96 (m, 1H). MS
calcd. for C13H16N03
(M+H+) 234.1, found 234.3.
CA 02667816 2009-04-23
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trans -4-(2,3 -Dimethyl-phenoxy) -3 -h. d~y_piperidine-l-carboxylic acid
benzyl ester (4)
and trans-3-(2,3-dimethyl-phenoxy) -4-h. d~y_piperidine-l-carboxylic acid
benzyl ester (5)
[0053] 2,3-Dimethylphenol and 7-oxa-3-aza-bicyclo[4.1.0]heptane-3-carboxylic
acid benzyl
ester (3) (1.0 g, 4.3 mmol) are dissolved in acetonitrile (20 mL). 2 N aqueous
sodium hydroxide
(5 mL) is added and the mixture is heated to reflux for 48 h. The acetonitrile
is removed in
vacuo, and the aqueous remainder is diluted with water (20 mL) and extracted
twice with
dichloromethane (50 mL). The combined organic layers are washed with water and
brine, dried
over MgSO4 and concentrated. The crude products are separated by flash
chromatography
(hexanes/ethyl acetate gradient) to give trans-4-(2,3-dimethyl-phenoxy)-3-
hydroxy-piperidine-l-
carboxylic acid benzyl ester 4 and the regioisomer trans-3-(2,3-dimethyl-
phenoxy)-4-hydroxy-
piperidine-l-carboxylic acid benzyl ester 5 as white solids. 4: 1H-NMR (400
MHz, CDC13) S=
7.58-7.50 (m, 5H), 7.24 (t, J = 7.9 Hz, 1H), 7.01 (d, J = 7.5 Hz, 1H), 6.97
(d, J = 8.2 Hz, 1H),
5.35 (s, 2H), 4.44 (m, 1H), 4.26 (m, 1H), 4.08 (m, 1H), 4.01 (m, 1H), 3.50 (m,
2H), 2.47 (s, 3H),
2.35 (s, 3H), 2.28 (m, 1H), 1.85 (m, 1H). MS calcd. for C21H26N04 (M+H+)
356.2, found 356.3.
5: 1H-NMR (400 MHz, CDC13) S= 7.53-7.46 (m, 5H), 7.15 (t, br, 1H), 7.01 (d, J
= 7.5 Hz, 1H),
6.95 (d, br, 1H), 5.35 (br, 2H), 4.28-4.15 (m, 4H), 3.28-3.22 (m, 2H), 2.46
(s, 3H), 2.34 (m, 1H),
2.33 (s, 3H), 1.84 (m, 1H). MS calcd. for C21H26N04 (M+H+) 356.2, found 356.3.
trans -4-(2,3 -Dimethyl-phenoxy) -piperidin-3-ol (6)
[0054] trans-4-(2,3-Dimethyl-phenoxy)-3-hydroxy-piperidine-l-carboxylic acid
benzyl ester
4 (700 mg, 2.0 mmol) is dissolved in EtOH (10 mL) and a catalytic amount of
palladium (10%
on charcoal) is added. After stirring for 3 h at rt under 1 atm hydrogen, the
mixture is filtered
over CELITEO and washed with EtOH. The filtrate is concentrated in vacuo to
give the
piperidinol 6 as a white solid: 1H-NMR (400 MHz, MeOD) S= 7.04 (t, J = 7.9 Hz,
1H), 6.85 (d,
J = 6.0 Hz, 1H), 6.80 (d, J = 7.7 Hz, 1H), 4.38 (m, 1H), 3.96 (m, 1H), 3.34
(m, 1H), 3.16 (m,
1H), 2.97 (m, 2H), 2.28 (s, 3H), 2.27 (m, 1H), 2.21 (s, 3H), 1.83 (m, 1H). MS
calcd. for
C13H2ON02 (M+H+) 222.1, found 222.3.
trans-4-(2,3-Dimethyl-phenoxy)-6-oxa-l-aza-bicyclo[3.2.1loctan-7-one (8)
[0055] The piperidinol 6 (50 mg, 0.23 mmol) is dissolved in DCM (10 mL).
Triphosgene (45
mg, 0.15 mmol) is added and the mixture is cooled to 0 C. Then triethylamine
(96 L, 0.69
mmol) is added slowly in increments over 50 min. Sodium hydride (30 mg, 0.75
mmol) is
16
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added, and the mixture is warmed to rt and stirred at rt for 18h. The mixture
is washed with 0.25
M phosphate buffer (pH 6.2), the organic layer is separated, dried (MgSO4) and
concentrated.
The crude product is purified on reverse phase HPLC (MeCN/H20 gradient, no
acidic additives)
to give 8 as a white solid: 1H-NMR (400 MHz, CDC13) S= 7.06 (t, J = 7.9 Hz,
1H), 6.85 (d, J =
7.5 Hz, 1H), 6.66 (d, J = 8.2 Hz, 1H), 4.80 (t, J = 4.3 Hz, 1H), 4.73 (t, J =
4.3 Hz, 1H), 3.70 (d, J
= 11.9 Hz, 1H), 3.47-3.38 (m, 2H), 3.30 (m, 1H), 2.31 (m, 1H), 2.29 (s, 3H),
2.19 (s, 3H), 2.00
(m, 1H). MS calcd. for C14H18NO3 (M+H+) 248.1, found 248.3.
Example 2
trans-3-(2,3-Dimethyl-phenoxy)-piperidin-4-ol (7)
[0056] Trans-3-(2,3-Dimethyl-phenoxy)-piperidin-4-ol (7) is obtained as a
white solid
following the procedure for the piperidinol 6 in Example 1, replacing 4 with 5
as the reagent.
IH-NMR (400 MHz, CDC13) S= 7.03 (t, J = 7.9 Hz, 1H), 6.82 (d, J = 6.2 Hz, 1H),
6.81 (d, J
7.5 Hz, 1H), 4.08 (m, 1H), 3.88 (m, 1H), 3.34 (m, 1H), 3.12 (m, 1H), 2.73 (m,
1H), 2.59 (m,
1H), 2.27 (s, 3H), 2.16 (s, 3H), 2.12 (m, 1H), 1.64 (m, 1H). MS calcd. for
C13H20N02 (M+H+)
222.1, found 222.3.
Example 3
cis-4-(2,3-Dimethyl-phenoxy)-6-oxa-l-aza-bicyclo[3.2.1loctan-7-one (12)
[0057] The benzyl carbamate 4 (0.93 g, 2.6 mmol) is dissolved in THF (50 mL)
together
with triphenylphosphine (2.07 g, 7.9 mmol) and p-nitrobenzoic acid (1.32 g,
7.9 mmol). The
mixture is cooled to 0 C, then DEAD (1.24 mL, 7.9 mmol) is added slowly. The
mixture is
stirred at 50 C for 48h, concentrated and purified by flash chromatography
(hexanes/ethyl
acetate gradient) to yield cis-4-(2,3-Dimethyl-phenoxy)-3-(4-nitro-benzoyloxy)-
piperidine-l-
carboxylic acid benzyl ester as a white solid: 1H-NMR (400 MHz, CDC13) S= 8.21
(d, J 8.8
Hz, 2H), 8.03 (d, J = 8.0 Hz, 2H), 7.37-7.24 (m, 5H), 6.99 (t, J = 7.8 Hz,
1H), 6.79 (d, J 7.4
Hz, 1H), 6.73 (d, J = 8.2 Hz, 1H), 5.36 (m, 1H), 5.14 (m, 1H), 5.03 (m, 1H),
4.69 (m, 1H), 4.16
(m, 1H), 3.96-3.46 (m, 3H), 2.24 (s, 3H), 2.19 (m, 1H), 2.05 (s, 3H), 2.02 (m,
1H). MS calcd.
for C28H29N207 (M+H+) 505.2, found 505.3.
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cis -4-(2,3 -Dimethyl-phenoxy) -3 -h. d~y_piperidine-l-carboxylic acid benzyl
ester (10)
[0058] Powdered NaOH (0.4 g, 10 mmol) is dissolved in MeOH (20 mL). cis-4-(2,3-
Dimethyl-phenoxy)-3-(4-nitro-benzoyloxy)-piperidine-1-carboxylic acid benzyl
ester (0.5 g, 1
mmol) is added and the mixture is stirred for lh at rt. The mixture is
concentrated, DCM is
added and the organic layer is washed with water twice. The organic layer is
dried over MgSO4
and concentrated to give cis-4-(2,3-dimethyl-phenoxy)-3-hydroxy-piperidine-l-
carboxylic acid
benzyl ester as a white solid: 1H-NMR (400 MHz, CDC13) S= 7.39-7.28 (m, 5H),
7.05 (t, J = 7.9
Hz, 1H), 6.84 (d, J = 7.5 Hz, 1H), 6.77 (d, J = 8.2 Hz, 1H), 5.17 (s, 2H),
4.55 (m, 1H), 3.94 (m,
1H), 3.80 (m, 1H), 3.65 (m, 1H), 3.52 (m, 2H), 2.29 (s, 3H), 2.19 (s, 3H),
2.09 (m, 1H), 1.74 (m,
1H). MS calcd. for C21H26N04 (M+H+) 356.2, found 356.3.
cis -4-(2,3 -Dimethyl-phenoxy) -piperidin-3 -ol (11)
[0059] cis-4-(2,3-Dimethyl-phenoxy)-3-hydroxy-piperidine-l-carboxylic acid
benzyl ester
(300 mg, 0.85 mmol) is dissolved in EtOH (20 mL) and a catalytic amount of
palladium (10%
on charcoal) is added. After stirring for 3 h at rt under 1 atm hydrogen, the
mixture is filtered
over CELITEO and washed with EtOH. The filtrate is concentrated in vacuo to
give cis-4-(2,3-
Dimethyl-phenoxy)-piperidin-3-ol (11) as a white solid: 1H-NMR (400 MHz, MeOD)
S= 6.98
(t, J = 7.9 Hz, 1H), 6.82 (d, J = 8.2 Hz, 1H), 6.74 (d, J = 7.5 Hz, 1H), 4.48
(m, 1H), 3.90 (m,
1H), 3.04 (m, 1H), 2.91 (m, 1H), 2.83 (m, 1H), 2.64 (m, 1H), 2.24 (s, 3H),
2.20 (s, 3H), 1.98 (m,
1H), 1.70 (m, 1H). MS calcd. for C13H20N02 (M+H+) 222.1, found 222.3.
cis-4-(2,3-Dimethyl-phenoxy)-6-oxa-l-aza-bicyclo[3.2.1loctan-7-one (12)
[0060] cis-4-(2,3-Dimethyl-phenoxy)-piperidin-3-ol 11 (50 mg, 0.23 mmol) is
dissolved in
DCM (10 mL). Triphosgene (45 mg, 0.15 mmol) is added and the mixture is cooled
to 0 C.
Then triethylamine (96 L, 0.69 mmol) is added slowly in increments over 50
min. After
warming to rt, sodium hydride (30 mg, 0.75 mmol) is added, and the mixture is
stirred at rt
overnight. The mixture is diluted with DCM, washed with water, and the organic
layer is
separated, dried (MgS04) and concentrated. The crude product is purified on
reverse phase
HPLC (MeCN/H20 gradient, no acidic additives) to give 12 as a white solid: 1H-
NMR (400
MHz, CDC13) S= 7.03 (t, J = 7.9 Hz, 1H), 6.85 (d, J = 7.5 Hz, 1H), 6.72 (d, J
= 8.2 Hz, 1H),
4.87 (d, J = 5.1 Hz, 1H), 4.43 (dd, J = 6.7 Hz, J = 9.5 Hz, 1H), 3.56 (dd, J =
5.1 Hz, J = 12.2 Hz,
1H), 3.47 (dd, J = 7.3 Hz, J = 14.1 Hz, 1H), 3.47 (dt, J = 4.9 Hz, J = 13.7
Hz, 1H), 2.89 (d, J
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12.1 Hz, 1H), 2.35 (m, 1H), 2.28 (s, 3H), 2.22 (m, 1H), 2.18 (s, 3H). MS
calcd. for C14H18NO3
(M+H+) 248.1, found 248.3.
Example 4
cis-4-(2,3-Dimethyl-phenoxy)-6-oxa-l-aza-bicyclo[3.2.lloctane-7-thione (13)
[0061] Following the procedure for the bicyclic carbamate 12, replacing
triphosgene with
thiophosgene, the title compound 13 is prepared as a white solid: 1H-NMR (400
MHz, CDC13) S
= 7.04 (t, J = 7.9 Hz, 1H), 6.86 (d, J = 7.5 Hz, 1H), 6.72 (d, J = 8.2 Hz,
1H), 5.06 (d, J = 5.3 Hz,
1H), 4.39 (t, J = 8.3 Hz, 1H), 3.61 (m, 2H), 3.13 (m, 1H), 3.05 (d, J = 12.1
Hz, 1H), 2.31 (m,
1H), 2.28 (s, 3H), 2.20 (s, 3H), 2.15 (m, 1H). MS calcd. for C14H18N02S (M+H+)
264.1, found
264.3.
Example 5
cis-4-Phenoxy-6-oxa-l-aza-bicyclo[3.2.1loctan-7-one (14)
[0062] Following the procedure for the bicyclic carbamate 12, replacing 2,3-
dimethylphenol
with phenol, the title compound 14 is prepared as a white solid: 1H-NMR (400
MHz, CDC13) S=
7.34-7.28 (m, 2H), 7.05-6.94 (m, 3H), 4.93 (d, J = 4.6 Hz, 1H), 4.50 (m, 1H),
3.63 (m, 1H), 3.51
(m, 1H), 2.99 (m, 1H), 2.94 (d, J = 11.9 Hz, 1H), 2.39 (m, 1H), 2.20 (m, 1H).
MS calcd. for
C12H14N03 (M+H+) 220.1, found 220.3.
Example 6
cis-4-Benzyloxy-6-oxa-l-aza-bicyclo[3.2.1loctan-7-one (15)
[0063] The epoxide 3 (1.0 g, 4.3 mmol) is dissolved in benzyl alcohol (5 mL).
Sodium
hydride (0.86 g, 21.5 mmol) is added and the mixture is stirred at 50 C
overnight. Then the
solvent is removed in vacuo and the remainder is purified by flash
chromatography
(hexanes/ethyl acetate gradient) to yield 3-hydroxy-4-isobutoxy-piperidine-1-
carboxylic acid
benzyl ester. Following the procedure for the bicyclic carbamate 12, the title
compound 15 is
prepared as a white solid: 1H-NMR (400 MHz, CDC13) S= 7.38-7.32 (m, 5H), 4.82
(d, J = 6.1
Hz, 1H), 4.61 (s, 2H), 3.67 (m, 1H), 3.52 (m, 1H), 3.41 (m, 1H), 2.86 (m, 1H),
2.78 (d, J = 12.1
Hz, 1H), 2.21 (m, 1H), 2.04 (m, 1H). MS calcd. for C13H16N03 (M+H+) 234.1,
found 234.3.
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Example 7
cis-4-Isobutoxy-6-oxa-l-aza-bicyclo[3.2.1loctan-7-one (16)
[0064] Following the procedure for the bicyclic carbamate 15, benzyl alcohol
with
isobutanol, the title compound 16 is prepared as a white solid: 1H-NMR (400
MHz, CDC13) S=
4.83 (d, J = 5.1 Hz, 1H), 3.56 (m, 2H), 3.43 (m, 1H), 3.26 (m, 2H), 2.89 (m,
1H), 2.81 (d, J =
12.0 Hz, 1H), 2.21 (m, 1H), 2.00 (m, 1H), 1.84 (m, 1H), 0.92 (d, J = 6.7 Hz,
6H). MS calcd. for
CioH18N03 (M+H+) 200.1, found 200.3.
Example 8
trans-4-Bromo-6-oxa-l-aza-bicyclo[3.2.1loctan-7-one (19)
[0065] 7-oxa-3-aza-bicyclo[4.1.0]heptane-3-carboxylic acid benzyl ester (3)
(1.0 g, 4.3
mmol) is dissolved in DCM (32 mL). The solution is cooled to 0 C, then
hydrobromic acid
(30% in acetic acid, 8 mL, 30 mmol) is added dropwise and the mixture is
stirred for 3h at rt.
The product precipitated and is filtered to give 4-bromo-piperidin-3-ol
hydrobromide (18) as a
white solid: 1H-NMR (400 MHz, MeOD) S= 4.46 (m, 1H), 4.25 (m, 1H), 3.77 (m,
1H), 3.51 (m,
1H), 3.36 (m, 2H), 2.88 (m, 1H), 2.28 (m, 1H). MS calcd. for CsHiiBrNO (M+H+)
180.0, found
180.3.
[0066] 4-bromo-piperidin-3-ol hydrobromide (18) (100 mg, 0.4 mmol) and
triphosgene (80
mg, 0.27 mmol) are suspended in DCM (20 mL) and cooled to 0 C. Then
triethylamine (332
L, 2.4 mmol) is added slowly in increments over 60 min. After warming to rt,
the mixture is
stirred at rt overnight. Purification by flash chromatography (hexanes/ethyl
acetate gradient) to
yield 19 as a white solid: 1H-NMR (400 MHz, CDC13) S= 4.71 (t, J = 4.2 Hz,
1H), 4.42 (m, 1H),
3.76 (d, J = 12.3 Hz, 1H), 3.46-3.24 (m, 3H), 2.75 (m, 1H), 2.07 (m, 1H). MS
calcd. for
C6H9BrNO2 (M+H+) 206.0, found 206.3.
Example 9
5-(2,3-Dimethyl-phenoxymethyl)-3-methyl-oxazolidin-2-one (21)
[0067] Epibromohydrin 20 (0.7 mL, 8.2 mmol), 2,3-dimethylphenol (1.0 g, 8.2
mmol) and
K2C03 (1.13 g, 8.2 mmol) are suspended in MeCN (25 mL) and stirred at 60 C
overnight. The
mixture is concentrated, the remainder diluted with DCM and washed with water
twice. The
organic layer is dried, concentrated and purified by flash chromatography
(hexanes/ethyl acetate
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gradient) to yield 2-(2,3-dimethyl-phenoxymethyl)-oxirane as a colorless oil:
1H-NMR (400
MHz, CDC13) 8= 7.05 (t, J = 7.9 Hz, 1H), 6.81 (d, J = 7.5 Hz, 1H), 6.70 (d, J
= 8.2 Hz, 1H),
4.21 (dd, J = 3.1 Hz, J = 11.0 Hz, 1H), 3.97 (dd, J = 5.4 Hz, J = 11.0 Hz,
1H), 3.38 (m, 1H), 2.91
(t, J = 4.5 Hz, 1H), 2.79 (dd, J = 2.7 Hz, J = 5.0 Hz, 1H), 2.28 (s, 3H), 2.18
(s, 3H). MS calcd.
for CiiHis0z (M+H+) 179.1, found 179.1.
1-(2,3-dimethyl-phenoxY)-3-methylamino-propan-2-ol
[0068] 2-(2,3-Dimethyl-phenoxymethyl)-oxirane (0.3 g, 1.7 mmol) is dissolved
in MeOH (2
mL). Methylamine (40% in H20, 0.3 mL, 3.4 mmol) is added and the mixture is
stirred at 50 C
for 2 h. The solvents are removed in vacuo and the remainder is purified on
reverse phase HPLC
(MeCN/H20 gradient) to give 1-(2,3-dimethyl-phenoxy)-3-methylamino-propan-2-ol
as a
colorless oil: 1H-NMR (400 MHz, CDC13) 8= 7.02 (t, J = 7.9 Hz, 1H), 6.80 (d, J
= 7.5 Hz, 1H),
6.61 (d, J = 8.1 Hz, 1H), 4.42 (m, 1H), 3.98 (m, 2H), 3.26 (m, 2H), 2.80 (br,
3H), 2.24 (s, 3H),
2.10 (s, 3H). MS calcd. for C12H2ON02 (M+H+) 210.1, found 210.1.
5-(2,3-Dimethyl-phenoxymethyl)-3-methyl-oxazolidin-2-one (21)
[0069] 1-(2,3-Dimethyl-phenoxy)-3-methylamino-propan-2-ol (180 mg, 0.86 mmol)
is
dissolved in benzene (10 mL). Carbonyl diimidazole (167 mg, 1.03 mmol) is
added followed by
a catalytic amount of DMAP. The mixture is heated to reflux for 4 h. The
solvent is removed in
vacuo, and the remainder is purified on reverse phase HPLC (MeCN/H20 gradient)
to give 21 as
a colorless oil: 1H-NMR (400 MHz, CDC13) 8= 7.05 (t, J = 7.9 Hz, 1H), 6.82 (d,
J = 7.5 Hz,
1H), 6.67 (d, J = 8.2 Hz, 1H), 4.84 (m, 1H), 4.11 (d, J = 4.5 Hz, 2H), 3.74
(t, J = 8.7 Hz, 1H),
3.58 (t, J = 5.7 Hz, J = 8.6 Hz, 1H), 2.94 (s, 3H), 2.27 (s, 3H), 2.12 (s,
3H). MS calcd. for
C13H18N03 (M+H+) 236.1, found 236.1.
Example 10
Mass Spectrometric Analysis of Cathepsin B Digests
[0070] LC-MS analysis of samples is performed using an LCQ Deca XP Plus mass
spectrometer modified with a home-built nanospray source configured for online
desalting as
described in Licklider et al., Anal. Chem. 74:3076-3083 (2002). The peptide
digests are loaded
onto a 100 m i.d. precolumn packed with 2 cm of Monitor, 5 m, C18 (Column
Engineering,
Ontario, Canada), and desalted for 5 min at 5 L/min with 0.1 M HOAc. After
desalting, the
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precolumn is placed in line with a 75 m i.d. pulled tip (5 m opening) packed
with 8 cm of the
same packing material, and the ACN concentration is increased from 0% to 50%
over 90 min.
The column is then washed with 95% ACN before returning to 0%. The flow from
the HPLC
pump is passively split prior to the precolumn to achieve 250 nL/min. MS-MS
are acquired in a
data-dependent scanning mode with one full scan followed by three MS-MS scans
on the three
most intense precursor ions. The dynamic exclusion of previously selected
precursors is set to 1
min. Tandem MS data are analyzed with TurboSequest (Thermo Electron). A custom
database
containing 8 proteins is searched using the following parameters: variable
methionine oxidation,
carboxamidomethyl adduct on cysteine, and carbamate adduct on cysteine (+247)
or histidine
(+247).
Example 11
Peptides identified by LC-MS/MS from inhibitor treated cathepsin B
[0071] Cathepsin B is digested with trypsin and chymotrypsin. Peptides are
identified by
nano-LC-MS/MS as described in the methods section. "A" denotes
carboxymethylated cysteines
and "#" indicates modification by the strained carbamate compound. When
controlled for the
amount of injected material, the unmodified peptide is present at an amount
118-fold less than
the modified peptide. As determined by Q-Tof analysis of the intact protein,
there is an
additional cleavage of the pro-enzyme upon auto-activation to yield the active
enzyme with
alternative N-terminal sequence starting points. The cleavage at position -4
relative to the
expected major cleavage product is noted in Table 2.
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Sequence range Peptide Identified
-4-5 EDLK LPASF
1-5 LPASF
9-21 EQW PQC"PTIKEI R
19-30 EIRDQGSC"GSC#W
22-30 DQGSC"GSC#W
22-30 DQGSC"GSC"W
31-41 AFGAVEAISDR
33-41 GAVEAISDR
131-140 IC"EPGYSPTY
166-174 KNGPVEGAF
189-202 QHVTGEMMGGHAIR
203-214 ILGWGVENGTPY
215-221 WLVANSW
222-232 NTDWGDNGFFK
233-252 ILRGQDHC"GI ESEVVAGI PR
235-252 RGQDHC"GIESEVVAGIPR
236-252 GQDHC"GIESEVVAGIPR
Table 2
Example 12
Temperature Effects on Bicyclic Carbamate Adduct
[0072] Samples of cathepsin B are inhibited with compound 12 followed by
overnight
dialysis in order to remove any non-cathepsin bound compound. Dialyzed samples
are then
analyzed by LC-MS for the presence of the dissociation product 11. Prior to LC-
MS analysis,
the samples are incubated for 2 h on ice or at 37 C. The temperature effect on
carbamate
inhibited cathepsin B samples is evaluated using LC-MS with multiple reaction
monitoring
(MRM) on an Applied Biosystems/MDS SCIEX 4000 Q TRAP. MRM transitions for the
open
form of the carbamate are determined by infusion of a synthetic standard.
Collision energy and
exit cell potential are individually optimized for each of the selected
transitions. Cathepsin B
samples are loaded on a Phenomenex Luna C5 column (30 x 2mm) in 2% ACN, 0.1M
HOAc
and eluted using a 5 min linear gradient to 70% ACN, 0.1M HOAc and flow rate
of 300 L/min.
Effluent from the column is introduced into the 4000 QTRAP using a TurboV ion
source.
Source parameters are: Cur, 10; IS, 4500; TEM, 450; GS1, 30; GS2, 15.
[0073] Cathepsin B is inhibited with compound 12 and dialyzed overnight.
Compound 11 is
detected using LC-MS with MRM post dialysis (panel A) and after incubation for
2 hours on ice
(panel B) or at 37C (panel C). MRM transitions are chosen from collisionally
activated
dissociation of a synthetic standard of compound 11 (inset). Five MRM
transitions are
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monitored for the detection of compound 11 (222.1 to 100.0, 71.1, 69.0, 81.9,
and 55.0) and
only the most intense transition is graphed for clarity (222.1 to 100.0).
Example 13
Effect of Temperature on the Stability of the Strained Carbamate-Cathepsin B
Adduct
[0074] The presence of a metabolite of compound 12 is monitored by MRM. MRM
transitions are selected and optimized using a synthetic analog of a
metabolite (compound 11).
The abundance of the compound 11 increased four-fold after incubation at 37 C
for 2 hrs.
Temperature Time Metabolite
Signal Intensity
0 0 900
0 2hrs 1000
37 2hrs 4000
[0075] It is understood that the examples and embodiments described herein are
for
illustrative purposes only and that various modifications or changes in light
thereof will be
suggested to persons skilled in the art and are to be included within the
spirit and purview of this
application and scope of the appended claims. All publications, patents, and
patent applications
cited herein are hereby incorporated by reference for all purposes.
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