Note: Descriptions are shown in the official language in which they were submitted.
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HETEROCYCLIC SULFONAMIDE DERIVATIVES
Cross-Reference to related Applications
[0001] This application claims the benefit of priority to patent
application
1634/DEL/11 filed in India on 09 June 2011. The full disclosure of this
application is
incorporated herein by reference in its entirety and for all purposes.
Field of the Invention
[0002] The invention relates to heterocyclic sulfonamide compounds and
pharmaceutical compositions thereof, in particular heterocyclic sulfonamide
compounds
that are specific inhibitors of kinase activity of MEK. The invention also
relates to the use
of the compounds and compositions thereof in the management of
hyperproliferative
diseases like cancer and inflammation.
Background of the Invention
[0003] Hyperproliferative diseases like cancer and inflammation are
receiving
a lot of attention from the scientific community and there is a strong desire
to discover
compounds that provide therapeutic benefits with regard to treating
hyperproliferative
diseases. In this regard efforts have been made to identify and target
specific mechanisms
which play a role in proliferating the diseases.
[0004] One target of interest is the over-activation of mitogen-
activated
protein (MAP) kinase cascade which is known to play an important role in cell
proliferation and differentiation. This pathway can be activated when a growth
factor
binds to its receptor tyrosine kinase. This interaction promotes RAS
association with
RAF and initiates a phosphorylation cascade through MEK (MAP kinase) to ERK.
Inhibition of this pathway is known to be beneficial in treating
hyperproliferative
diseases. MEK is an attractive therapeutic target because the only known
substrates for
MEK phosphorylation are the MAP kinases, ERK1 and ERK2. Constitutive
activation of
MEK/ERK was been found in pancreatic, colon, lung, kidney and ovarian primary
tumor
samples.
[0005] Phosphorylation of MEK appears to increase its affinity and its
catalytic activity toward ERK as well as is affinity for ATP. This invention
describes
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compounds that inhibit MEK activity by modulation of ATP binding, association
of MEK
with ERK by mechanisms that are competitive, and/or allosteric and/or
uncompetitive.
[0006] Activation of MEK has been demonstrated in many disease models
thus suggesting that inhibition of MEK could have potential therapeutic
benefit in various
diseases such as Pain (see, e.g., Evidence of efficacy in pain models
described in J.
Neurosci. 22:478, 2002; Acta Pharmacol Sin. 26:789 2005; Expert Opin Ther
Targets.
9:699, 2005; and Mol. Pain. 2:2, 2006): Stroke (see, e.g., Evidence of
efficacy in stroke
models significant neuroprotection against ischemic brain injury by inhibition
of the
MEK described in J. Pharmacol. Exp. Ther. 304:172, 2003; and Brain Res.
996:55, 2004);
Diabetes (see, e.g., Evidence in diabetic complications described in Am. J.
Physiol.
Renal.286, F120 2004); Inflammation (see e.g., Evidence of efficacy in
inflammation
models described in Biochem Biophy. Res. Com. 268:647, 2000); and Arthritis
(see, e.g,
Evidence of efficacy in experimental osteoarthritis and arthritis as described
in J. Clin.
Invest. 116:163. 2006).
[0007] Although inhibition of MEK has been shown to have potential
therapeutic benefit in several studies, there still remains a need to find
compounds having
commercial application.
Summary of the Invention
[0008] The invention provides the compounds (S)-N-(4,5-difluoro-6-((2-
fluoro-4-iodophenyl)amino)benzofuran-7-y1)-1-(2,3-dihydroxypropyl)cyclopropane-
1-
sulfonamide and (R)-N-(4,5-difluoro-64(2-fluoro-4-iodophenyl)amino)benzofuran-
7-y1)-
1-(2,3-dihydroxypropyl)cyclopropane-l-sulfonamide:
Ficzr-irjt. OH 1:61 ii::_5_ JOH
SO2 NH
H
F SO2
NH H F
N N
\O 0 0 \O 0 0
F I F I
F F
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[0009] These compounds of the invention exist as isomeric
configurations
with chiral centers. Both isomeric forms are contemplated to be within the
scope of the
present invention. Each compound can be prepared as single isomers and/or
separated
into single isomers by techniques known to those skilled in the art.
Therefore, the
compounds of the present invention can be used in their single isomer or
isomeric form.
Also contemplated are the pharmaceutically acceptable salts thereof.
[0010] In another aspect of the present invention, a pharmaceutical
composition is provided which comprises the compounds described above, or a
pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable
excipient.
Definitions
[0011] Unless specified otherwise, the term "compound of the present
invention" refers to (S)-N-(4,5-difluoro-64(2-fluoro-4-
iodophenyl)amino)benzofuran-7-
y1)-1-(2,3-dihydroxypropyl)cyclopropane-l-sulfonamide or (R)-N-(4,5-difluoro-
64(2-
fluoro-4-iodophenyl)amino)benzofuran-7-y1)-1-(2,3-dihydroxypropyl)cyclopropane-
1-
sulfonamide, and salts thereof, as well as all isotopically labeled compounds
(including
deuterium substitutions), and inherently formed moieties (e.g., polymorphs,
solvates
and/or hydrates).
Description of Preferred Embodiments
[0012] The present invention provides compounds and pharmaceutical
compositions thereof that are useful in the treatment of diseases, conditions
and/or
disorders modulated by the inhibition of kinase activity of MEK.
[0013] Compounds of the present invention may be synthesized by
synthetic
routes that include processes analogous to those well-known in the chemical
arts,
particularly in light of the description contained herein. The starting
materials are
generally available from commercial sources such as Aldrich Chemicals
(Milwaukee,
Wis.) or are readily prepared using methods well known to those skilled in the
art (e.g.,
prepared by methods generally described in Louis F. Fieser and Mary Fieser,
Reagents
for Organic Synthesis, v. 1-19, Wiley, New York (1967-1999 ed.), or Beilsteins
Handbuch der organischen Chemie, 4, Aufl. ed. Springer-Verlag, Berlin,
including
supplements (also available via the Beilstein online database)).
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[0014] For a detailed description of the individual reaction steps, see
the
Examples section below. Those skilled in the art will appreciate that other
synthetic
routes may be used to synthesize the inventive compound. Although specific
starting
materials and reagents are described, those of skill in the art will
appreciate that other
starting materials and reagents can be easily substituted to provide a variety
of derivatives
and/or reaction conditions.
[0015] Compounds of the invention are capable of forming salts with
bases, in
particular pharmaceutically acceptable bases such as those well known in the
art; suitable
such salts include metal salts, particularly alkali metal or alkaline earth
metal salts such as
sodium, potassium, magnesium or calcium salts, or salts with ammonia or
pharmaceutically acceptable organic amines or heterocyclic bases such as
ethanolamines,
benzylamines or pyridine. These salts may be prepared by known salt-forming
procedures.
[0016] The present invention includes isotopically-labeled or -enriched
compounds of the present invention. Representative examples of isotopes
suitable for
inclusion in the compounds of the invention include isotopes of hydrogen, such
as 2H and
3H, carbon, such as 11C, 13C and 14C, chlorine, such as 36C1, fluorine, such
as 18F, iodine,
such as 123I and 125I, nitrogen, such as 13N and 15N, oxygen, such as 150, 170
and 180,
phosphorus, such as 32P, and sulphur, such as 35S.
[0017] Substitution with heavier isotopes such as deuterium, i.e. 2H,
may
afford certain therapeutic advantages resulting from greater metabolic
stability, for
example, increased in vivo half-life or reduced dosage requirements, and hence
may be
preferred in some circumstances.
[0018] Isotopically-labeled compounds of the present invention can
generally
be prepared by conventional techniques known to those skilled in the art or by
processes
analogous to those described in the accompanying Examples and Preparations
Sections
using an appropriate isotopically-labeled reagent in place of the non-labeled
reagent
previously employed.
[0019] The compounds of the present invention may exist in unsolvated
as
well as solvated forms with pharmaceutically acceptable solvents such as
water, ethanol,
and the like, and it is intended that the invention embrace both solvated and
unsolvated
forms. For purposes of the present invention, solvates (including hydrates)
are considered
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pharmaceutical compositions, e.g., a compound of the present invention in
combination
with an excipient, wherein the excipient is a solvent.
[0020] The present invention also relates to a pharmaceutical
composition
comprising a compound of the present invention and a pharmaceutically
acceptable
excipient.
[0021] Suitable excipients generally include corn starch, potato
starch, tapioca
starch, starch paste, pre-gelatinized starch, sugars, gelatin, natural gums,
synthetic gums,
sodium alginate, alginic acid, tragacanth, guar gum, cellulose, ethyl
cellulose, cellulose
acetate, carboxymethyl cellulose calcium, sodium carboxymethylcellulose,
methyl
cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose,
magnesium
aluminum silicate, polyvinyl pyrrolidone, talc, calcium carbonate, powdered
cellulose,
dextrates, kaolin, mannitol, silicic acid, sorbitol, agar-agar, sodium
carbonate,
croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch
glycolate,
clays, sodium stearate, calcium stearate, magnesium stearate, stearic acid,
mineral oil,
light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other
glycols, sodium
lauryl sulfate, hydrogenated vegetable oil, peanut oil, cottonseed oil,
sunflower oil,
sesame oil, olive oil, corn oil, soybean oil, zinc stearate, sodium oleate,
ethyl oleate, ethyl
laureate, silica, and combinations thereof.
[0022] A typical formulation is prepared by mixing a compound of the
present
invention and a carrier, diluent or excipient. Suitable carriers, diluents and
excipients are
well known to those skilled in the art and include materials such as
carbohydrates, waxes,
water soluble and/or swellable polymers, hydrophilic or hydrophobic materials,
gelatin,
oils, solvents, water, and the like. The particular carrier, diluent or
excipient used will
depend upon the means and purpose for which the compound of the present
invention is
being applied. Solvents are generally selected based on solvents recognized by
persons
skilled in the art as safe (GRAS) to be administered to a mammal. In general,
safe
solvents are non-toxic aqueous solvents such as water and other non-toxic
solvents that
are soluble or miscible in water. Suitable aqueous solvents include water,
ethanol,
propylene glycol, polyethylene glycols (e.g., PEG400, PEG300), etc. and
mixtures
thereof. The formulations may also include one or more buffers, stabilizing
agents,
surfactants, wetting agents, lubricating agents, emulsifiers, suspending
agents,
preservatives, antioxidants, opaquing agents, glidants, processing aids,
colorants,
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sweeteners, perfuming agents, flavoring agents and other known additives to
provide an
elegant presentation of the drug (i.e., a compound of the present invention or
pharmaceutical composition thereof) or aid in the manufacturing of the
pharmaceutical
product (i.e., medicament).
[0023] The formulations may be prepared using conventional dissolution
and
mixing procedures. For example, the bulk drug substance (i.e., compound of the
present
invention or stabilized form of the compound (e.g., complex with a
cyclodextrin
derivative or other known complexation agent)) is dissolved in a suitable
solvent in the
presence of one or more of the excipients. The compound of the present
invention is
typically formulated into pharmaceutical dosage forms to provide an easily
controllable
dosage of the drug and to give the patient an elegant and easily handleable
product.
[0024] The composition is generally formulated into various dosage
forms
selected from a group comprising tablet, troches, lozenges, aqueous or oily
suspensions,
ointment, patch, gel, lotion, dentifrice, capsule, emulsion, creams, spray,
drops,
dispersible powders or granules, emulsion in hard or soft gel capsules, syrups
and elixirs.
[0025] The pharmaceutical composition (or formulation) for application
may
be packaged in a variety of ways depending upon the method used for
administering the
drug. Generally, an article for distribution includes a container having
deposited therein
the pharmaceutical formulation in an appropriate form. Suitable containers are
well-
known to those skilled in the art and include materials such as bottles
(plastic and glass),
sachets, ampoules, plastic bags, metal cylinders, and the like. The container
may also
include a tamper-proof assemblage to prevent indiscreet access to the contents
of the
package. In addition, the container has deposited thereon a label that
describes the
contents of the container. The label may also include appropriate warnings.
[0026] The compounds of the present invention are useful as both
prophylactic
and therapeutic treatments for diseases or conditions related to the
hyperactivity of MEK,
as well as diseases or conditions modulated by the Raf/Ras/Mek pathway.
[0027] Thus, as a further aspect, the invention relates to a method for
treating
a disease or condition related to the hyperactivity of MEK, or a disease or
condition
modulated by the MEK cascade, comprising administration of an effective
therapeutic
amount of a compound of the present invention.
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[0028] As a further aspect, the invention relates to a method for
treating
proliferative diseases, such as cancer, comprising administration of an
effective amount
of a compound of the present invention.
[0029] Examples of cancers include but are not limited to:
angiosarcoma,
fibrosarcoma, rhabdomyosarcoma, liposarcoma, myxoma, rhabdomyoma, fibroma,
lipoma, teratoma; bronchogenic carcinoma, squamous cell carcinoma,
undifferentiated
small cell carcinoma, undifferentiated large cell carcinoma, alveolar
(bronchiolar)
carcinoma, bronchial adenoma, lymphoma, chondromatous hanlartoma,
inesothelioma,
esophageal squamous cell carcinoma, leiomyosarcoma, leiomyosarcoma, ductal
adenocarcinoma, insulinorna, glucagonoma, gastrinoma, vipoma, stomach and
small
bowel carcinoid tumors, adenocarcinoma, Karposi's sarcoma, leiomyoma,
hemangioma,
lipoma, neurofibroma, fibroma, tubular adenoma, villous adenoma, hamartoma,
Wilm's
tumor [nephroblastoma, leukemia, bladder and urethra squamous cell carcinoma,
transitional cell carcinoma, adenocarcinoma, seminoma, teratoma, embryonal
carcinoma,
teratocareinoma, choriocarcinoma, interstitial cell carcinoma, fibroadenoma,
adenomatoid
tumors, hepatoma (hepatocellular carcinoma), cholangiocarcinoma,
hepatoblastoma,
hepatocellular adenoma, hemangioma, osteogenic sarcoma (osteosarcoma),
malignant
fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lyinphoma
(reticulum
cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma,
osteochronfroma
(osteocartilaginous exostoses), benign chondroma, chondroblastoma,
chondromyxofibroma, osteoid osteoma and giant cell tumors, osteoma, granuloma,
xanthoma, osteitis defornians, meningioma, meningiosarcoma, gliomatosis,
astrocytoma,
medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma
multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors,
spinal
cord neurofibroma, meningioma, glioma, endometrial carcinoma, cervical
carcinoma,
pre-tumor cervical dysplasia, ovarian carcinoma, serous cystadenocarcinoma,
mucinous
cystadenocarcinoma, granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,
dysgerminoma, malignant teratoma, intraepithelial carcinoma, adenocarcinoma,
melanoma), vaginal clear cell carcinoma, botryoid sarcoma (embryonal
rhabdomyosarcoma), fallopian tube carcinoma, acute and chronic myeloid
leukemia,
acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative
diseases, multiple myeloma, myelodysplastic syndrome, Hodgkin's disease, non-
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Hodgkin's lymphoma, malignant lymphoma, malignant melanoma, basal cell
carcinoma,
moles, dysplastic nevi, angioma, dermatofibroma, keloids, psoriasis, and
neuroblastoma.
[0030] The present invention includes a method of treating cancer
selected
from non-small cell lung carcinoma, pancreatic carcinoma, bladder carcinoma,
colon
carcinoma, myeloid disorders, breast cancer, prostate cancer, thyroid cancer,
melanoma,
adenomas and carcinomas of the ovary, eye, liver, biliary tract, and nervous
system and
advanced solid tumors with KRAS, NRAS and BRAF mutations, comprising
administering to a subject in need of such treatment an effective amount of a
compound
of the invention.
[0031] The compounds of the present invention may also be useful in the
treatment of other diseases or conditions related to the hyperactivity of MEK.
Thus, as a
further aspect, the invention relates to a method of treatment of a disorder
selected from:
xenograft (cellos), skin, limb, organ or bone marrow transplant) rejection;
osteoarthritis;
rheumatoid arthritis; cystic fibrosis; complications of diabetes (including
diabetic
retinopathy and diabetic nephropathy); hepatomegaly; cardiomegaly; stroke
(such as
acute focal ischemic stroke and global cerebral ischemia); heart failure;
septic shock;
asthma; chronic obstructive pulmonary disorder; Alzheimer's disease; and
chronic or
neuropathic pain.
[0032] The term "chronic pain" for purposes of the present invention
includes,
but is not limited to, idiopathic pain, and pain associated with chronic
alcoholism, vitamin
deficiency, uremia, or hypothyroidism. Chronic pain is associated with
numerous
conditions including, but not limited to, inflammation, and post-operative
pain.
[0033] As used herein, the term "neuropathic pain" is associated with
numerous conditions which include, but are not limited to, inflammation,
postoperative
pain, phantom limb pain, burn pain, gout, trigeminal neuralgia, acute herpetic
and
postherpetic pain, causalgia, diabetic neuropathy, plexus avulsion, neuroma,
vasculitis,
viral infection, crush injury, constriction injury, tissue injury, limb
amputation, and nerve
injury between the peripheral nervous system and the central nervous system.
[0034] Compounds of the present invention may also be useful as
antiviral
agents for treating viral infections such as HIV, hepatitis (B) virus (HBV)
human
papilloma virus (HPV), cytomegalovirus (CMV], and Epstein-Barr virus (EBV).
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[0035] Compounds of the present invention may also be useful in the
treatment of restenosis, psoriasis, allergic contact dermatitis, autoimmune
disease,
atherosclerosis and inflammatory bowel diseases, e.g. Crohn's disease and
ulcerative
colitis.
[0036] A MEK inhibitor of the present invention may be usefully
combined
with another pharmacologically active compound (additional therapeutic agent),
or with
two or more other pharmacologically active compounds, particularly in the
treatment of
cancer. For example, a compound of the present invention, as defined above,
may be
administered simultaneously, sequentially or separately in combination with
one or more
additional therapeutic agents selected from an anticancer drug (or
chemotherapy agent), a
pain medication, an antiemetic, an antidepressant or an anti-inflammatory
agent.
Chemotherapy agents include, for example, mitotic inhibitors such as a taxane,
a vinca
alkaloid, paclitaxel, docetaxel, vincristine, vinblastine, vinorelbine or
vinflunine, and
other anticancer agents, e.g. cisplatin, 5-fluorouracil or 5-fluoro-2-4(1
H,3H)-
pyrimidinedione (5FU), flutamide or gemcitabine.
[0037] Such combinations may offer significant advantages, including
synergistic activity, in therapy.
[0038] A compound of the present invention may also be used to
advantage in
combination with other antiproliferative compounds. Such antiproliferative
compounds
include, but are not limited to aromatase inhibitors; antiestrogens;
topoisomerase I
inhibitors; topoisomerase II inhibitors; microtubule active compounds;
alkylating
compounds; histone deacetylase inhibitors, such as LBH589; compounds which
induce
cell differentiation processes; cyclooxygenase inhibitors; MMP inhibitors;
mTOR
inhibitors, such as RAD001; antineoplastic antimetabolites; platin compounds;
compounds targeting/decreasing a protein or lipid kinase activity and further
anti-
angiogenic compounds; compounds which target, decrease or inhibit the activity
of a
protein or lipid phosphatase; gonadorelin agonists; anti-androgens; methionine
aminopeptidase inhibitors; bisphosphonates; biological response modifiers;
antiproliferative antibodies; heparanase inhibitors; inhibitors of Ras
oncogenic isoforms;
telomerase inhibitors; proteasome inhibitors; compounds used in the treatment
of
hematologic malignancies; compounds which target, decrease or inhibit the
activity of
Flt-3, such as PKC412; Hsp90 inhibitors such as 17-AAG (17-allylamino-gelda-
namycin,
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NSC330507), 17-DMAG (17-dimethylaminoethylamino-17-demethoxy-geldana-mycin,
NSC707545), IPI-504, CNF1010, CNF2024, CNF1010 from Conforma Therapeutics and
AUY922; temozolomide (TEMODAL); kinesin spindle protein inhibitors, such as
SB715992 or SB743921 from GlaxoSmithKline, or pentamidine/chlorpromazine from
CombinatoRx; PI3K inhibitors, such as BEZ235; RAF inhibitors, such as RAF265
and
LGX818; EDG binders, antileukemia compounds, ribonucleotide reductase
inhibitors, S-
adenosylmethionine decarboxylase inhibitors, antiproliferative anti-bodies or
other
chemotherapeutic compounds. Further, alternatively or in addition they may be
used in
combination with other tumor treatment approaches, including surgery, ionizing
radiation,
photodynamic therapy, implants, e.g. with corticosteroids, hormones, or they
may be used
as radiosensitizers. Also, in anti-inflammatory and/or antiproliferative
treatment,
combination with anti-inflammatory drugs is included. Combination is also
possible with
antihistamine drug substances, bronchodilatatory drugs, NSAID or antagonists
of
chemokine receptors.
[0039] The term "aromatase inhibitor" as used herein relates to a
compound
which inhibits the estrogen production, i.e. the conversion of the substrates
androstenedione and testosterone to estrone and estradiol, respectively. The
term
includes, but is not limited to steroids, especially atame-stane, exemestane
and formestane
and, in part-icular, non-steroids, especially aminoglutethimide, roglethimide,
pyridoglutethimide, trilostane, testolactone, ketokonazole, vorozole,
fadrozole,
anastrozole and letrozole. Exemestane can be administered, e.g., in the form
as it is
marketed, e.g. under the trademark AROMASIN. Formestane can be administered,
e.g.,
in the form as it is marketed, e.g. under the trademark LENTARON. Fadrozole
can be
administered, e.g., in the form as it is marketed, e.g. un-der the trademark
AFEMA.
Anastrozole can be administered, e.g., in the form as it is marketed, e.g.
under the
trademark ARIMIDEX. Letrozole can be administered, e.g., in the form as it is
marketed,
e.g. under the trademark FEMARA or FEMAR. Amino glutethimide can be
administered, e.g., in the form as it is marketed, e.g. under the trademark,
ORIMETEN.
A combination of the invention comprising a chemo-therapeutic agent which is
an
aromatase inhibitor is particularly useful for the treatment of hormone
receptor positive
tumors, e.g., breast tumors.
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[0040] The term "anti-estrogen" as used herein relates to a compound
which
antagonizes the effect of estrogens at the estrogen receptor level. The term
includes, but
is not limited to tamoxifen, fulvestrant, raloxifene and raloxifene
hydrochloride.
Tamoxifen can be administered, e.g., in the form as it is marketed, e.g. under
the
trademark NOLVADEX. Ralo-xifene hydrochloride can be administered, e.g., in
the form
as it is marketed, e.g. under the trademark EVISTA. Fulvestrant can be
formulated as
disclosed in US 4,659,516 or it can be administered, e.g., in the form as it
is marketed,
e.g. under the trademark FASLODEX. A combination of the invention comprising a
chemotherapeutic agent which is an anti-estrogen is particularly useful for
the treatment
of estrogen receptor positive tumors, e.g. breast tumors.
[0041] The term "anti-androgen" as used herein relates to any substance
which
is capable of in-hibiting the biological effects of androgenic hormones and
includes, but is
not limited to, bicalutamide (CASODEX), which can be formulated, e.g. as
disclosed in
US 4,636,505.
[0042] The term "gonadorelin agonist" as used herein includes, but is
not
limited to abarelix, goserelin and goserelin acetate. Goserelin is disclosed
in US
4,100,274 and can be administered, e.g., in the form as it is marketed, e.g.
under the
trademark ZOLADEX. Abarelix can be formulated, e.g. as disclosed in US
5,843,901.
[0043] The term "topoisomerase I inhibitor" as used herein includes,
but is not
limited to topotecan, gimatecan, irinotecan, camptothecin and its analogues, 9-
nitrocamptothecin and the macromolecular camptothecin conjugate PNU-166148
(compound Al in W099/ 17804). Irinotecan can be administered, e.g. in the form
as it is
marketed, e.g. under the trademark CAMPTOSAR. Topotecan can be administered,
e.g.,
in the form as it is marketed, e.g. under the trademark HYCAMTIN.
[0044] The term "topoisomerase II inhibitor" as used herein includes,
but is
not limited to the an-thracyclines such as doxorubicin (including liposomal
formulation,
e.g. CAELYX), daunorubicin, epirubicin, idarubicin and nemorubicin, the
anthraquinones
mitoxantrone and losoxantrone, and the podophillotoxines etoposide and
teniposide.
Etoposide can be administered, e.g. in the form as it is marketed, e.g. under
the trademark
ETOPOPHOS. Teniposide can be administered, e.g. in the form as it is marketed,
e.g.
under the trademark VM 26-BRISTOL. Doxorubicin can be administered, e.g. in
the
form as it is marketed, e.g. under the trademark ADRIBLASTIN or ADRIAMYCIN.
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Epirubicin can be administered, e.g. in the form as it is marketed, e.g. under
the
trademark FARMORUBICIN. Idarubicin can be administered, e.g. in the form as it
is
marketed, e.g. under the trademark ZAVEDOS. Mitoxantrone can be administered,
e.g.
in the form as it is marketed, e.g. under the trademark NOVANTRON.
[0045] The term "microtubule active compound" relates to microtubule
stabilizing, microtubule destabilizing compounds and microtublin
polymerization
inhibitors including, but not limited to taxanes, e.g. paclitaxel and
docetaxel, vinca
alkaloids, e.g., vinblastine, especially vinblastine sulfate, vincristine
especially vincristine
sulfate, and vinorelbine, discodermolides, cochicine and epothilones and
derivatives
thereof, e.g. epothilone B or D or derivatives thereof. Paclitaxel may be
administered e.g.
in the form as it is marketed, e.g. TAXOL. Docetaxel can be administered,
e.g., in the
form as it is marketed, e.g. under the trademark TAXOTERE. Vinblastine sulfate
can be
administered, e.g., in the form as it is marketed, e.g. under the trademark
VINBLASTIN
R.P. Vincristine sulfate can be administered, e.g., in the form as it is
marketed, e.g. under
the trademark FARMISTIN. Discodermolide can be obtained, e.g., as disclosed in
US
5,010,099. Also included are Epothilone derivatives which are disclosed in WO
98/10121, US 6,194,181, WO 98/25929, WO 98/08849, WO 99/43653, WO 98/22461
and WO 00/31247. Especially preferred are Epothilone A and/or B.
[0046] The term "alkylating compound" as used herein includes, but is
not
limited to, cyclophosphamide, ifosfamide, melphalan or nitrosourea (BCNU or
Gliadel).
Cyclophosphamide can be administered, e.g., in the form as it is marketed,
e.g. under the
trademark CYCLOSTIN. Ifosfamide can be administered, e.g., in the form as it
is
marketed, e.g., under the trademark HOLOXAN.
[0047] The term "histone deacetylase inhibitors" or "HDAC inhibitors"
relates
to compounds which inhibit the histone deacetylase and which possess
antiproliferative
activity. This includes compounds such as sodium butyrate, LDH589 disclosed in
WO
02/22577, especially N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indo1-3-yflethyl]-
amino]methyl] pheny1]-2E-2-propenamide, N-hydroxy-3-[4-[[[2-(2-methy1-1H-indo1-
3-
y1)-ethyl] -amino] methyl]pheny1]-2E-2-propenamide and pharmaceutically
acceptable
salts thereof, especially the lactate salt. It further especially includes
suberoylanilide
hydroxamic acid (SAHA), M5275, FK228 (formerly FR901228), trichostatin A and
compounds disclosed in US 6,552,065, in particular, N-hydroxy-344-[[[2-(2-
methy1-1H-
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indo1-3-y1)-ethylFamino]-methyl]phenyl]-2E-2-propenamide, or a
pharmaceutically
acceptable salt thereof.
[0048] The term "antineoplastic antimetabolite" includes, but is not
limited to,
5-Fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylating
compounds, such
as 5-azacy-ti-dine and decitabine, methotrexate and edatrexate, and folic acid
antagonists
such as pemetrexed. Capecitabine can be administe-red, e.g., in the form as it
is marketed,
e.g. under the trademark XELODA. Gemcitabine can be administered, e.g., in the
form as it
is marketed, e.g. under the trademark GEMZAR.
[0049] The term "platin compound" as used herein includes, but is not
limited
to, carboplatin, cis-platin, cisplatinum and oxaliplatin. Carboplatin can be
administered,
e.g., in the form as it is marketed, e.g. under the trademark CARBOPLAT.
Oxaliplatin
can be administered, e.g., in the form as it is marketed, e.g. under the
trademark
ELOXATIN.
[0050] The term "compounds targeting/decreasing a protein or lipid
kinase
activity"; or a "protein or lipid phosphatase activity"; or "further anti-
angiogenic
compounds" as used herein includes, but is not limited to, protein tyrosine
kinase and/or
serine and/or threonine kinase inhibitors or lipid kinase inhibitors, e.g.,
[0051] a) compounds targeting, decreasing or inhibiting the activity of
the
platelet-derived growth factor-receptors (PDGFR), such as compounds which
target,
decrease or inhibit the activity of PDGFR, especially compounds which inhibit
the PDGF
receptor, e.g. a N-phenyl-2-pyrimidine-amine derivative, e.g. imatinib, SU101,
SU6668
and GFB-111;
[0052] b) compounds targeting, decreasing or inhibiting the activity of
the
fibroblast growth factor-receptors (FGFR);
[0053] c) compounds targeting, decreasing or inhibiting the activity of
the
insulin-like growth factor receptor I (IGF-IR), such as compounds which
target, decrease
or inhibit the activity of IGF-IR, especially compounds which inhibit the
kinase activity
of IGF-I receptor, such as those compounds disclosed in WO 02/092599, or
antibodies
that target the extracellular domain of IGF-I receptor or its growth factors;
[0054] d) compounds targeting, decreasing or inhibiting the activity of
the
Trk receptor tyrosine kinase family, or ephrin B4 inhibitors;
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[0055] e) compounds targeting, decreasing or inhibiting the activity of
the
Axl receptor tyrosine kinase family;
[0056] f) compounds targeting, decreasing or inhibiting the activity of
the Ret
receptor tyrosine kinase;
[0057] g) compounds targeting, decreasing or inhibiting the activity of
the
Kit/SCFR receptor tyrosine kinase, i.e C-kit receptor tyrosine kinases - (part
of the
PDGFR family), such as compounds which target, decrease or inhibit the
activity of the c-
Kit receptor tyrosine kinase family, especially compounds which inhibit the c-
Kit
receptor, e.g. imatinib;
[0058] h) compounds targeting, decreasing or inhibiting the activity of
members of the c-Abl family, their gene-fusion products (e.g. BCR-Abl kinase)
and
mutants, such as com-pounds which target decrease or inhibit the activity of c-
AbI family
members and their gene fusion products, e.g. a N-pheny1-2-pyrimidine-amine
derivative,
e.g. imatinib or nilotinib (AMN107); PD180970; AG957; NSC 680410; PD173955
from
ParkeDavis; or dasatinib (BMS-354825);
[0059] i) compounds targeting, decreasing or inhibiting the activity of
members of the protein kinase C (PKC) and Raf family of serine/threonine
kinases,
members of the MEK, SRC, JAK, FAK, PDK1, PKB/Akt, and Ras/MAPK family
members, and/or members of the cyclin-dependent kinase family (CDK) and are
especially those staurosporine derivatives disclosed in US 5,093,330, e.g.
midostaurin;
examples of further compounds include e.g. UCN-01, safingol, BAY 43-9006,
Bryostatin
1, Perifosine; Ilmofosine; RO 318220 and RO 320432; GO 6976; Isis 3521;
LY333531/LY379196; isochinoline compounds such as those disclosed in WO
00/09495;
FTIs; BEZ235 (a P13K inhibitor) or AT7519 (CDK inhibitor);
[0060] j) compounds targeting, decreasing or inhibiting the activity of
protein-
tyrosine kinase inhibitors, such as compounds which target, decrease or
inhibit the
activity of protein-tyrosine kinase inhibitors include imatinib mesylate
(GLEEVEC) or
tyrphostin. A tyrphostin is preferably a low molecular weight (mw<1500)
compound, or
a pharmaceutically acceptable salt thereof, especially a compound selected
from the
benzylidenemalonitrile class or the S-arylbenzenemalonirile or bisubstrate
quinoline class
of compounds, more especially any compound selected from the group consisting
of
Tyrphostin A23/RG-50810; AG 99; Tyrphostin AG 213; Tyrphostin AG 1748;
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Tyrphostin AG 490; Tyrphostin B44; Tyrphostin B44 (+) enantiomer; Tyrphostin
AG
555; AG 494; Tyrphostin AG 556, AG957 and adaphostin (4- I[(2,5-
dihydroxyphenyl)methyl]aminol -benzoic acid adamantyl ester; NSC 680410,
adaphostin);
[0061] k) compounds targeting, decreasing or inhibiting the activity of
the
epidermal growth factor family of receptor tyrosine kinases (EGFR, ErbB2,
ErbB3,
ErbB4 as homo- or heterodimers) and their mutants, such as compounds which
target,
decrease or inhibit the activity of the epidermal growth factor receptor
family are
especially compounds, proteins or antibodies which inhibit members of the EGF
receptor
tyrosine kinase family, e.g. EGF receptor, ErbB2, ErbB3 and ErbB4 or bind to
EGF or
EGF related ligands, and are in particular those compounds, proteins or
monoclonal
antibodies generically and specifically disclosed in WO 97/02266, e.g. the
compound of
ex. 39, or in EP 0 564 409, WO 99/03854, EP 0520722, EP 0 566 226, EP 0 787
722, EP
0 837 063, US 5,747,498, WO 98/10767, WO 97/30034, WO 97/49688, WO 97/38983
and, especially, WO 96/30347 (e.g. compound known as CP 358774), WO 96/33980
(e.g.
compound ZD 1839) and WO 95/03283 (e.g. compound ZM105180); e.g. trastuzumab
(Herceptin), cetuximab (Erbitux), Iressa, Tarceva, OSI-774, CI-1033, EKB-569,
GW-
2016, E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 or E7.6.3, and 7H-pyrrolo-[2,3-
d]pyrimidine derivatives which are disclosed in WO 03/013541; and
[0062] 1) compounds targeting, decreasing or inhibiting the activity of
the c-
Met receptor, such as compounds which target, decrease or inhibit the activity
of c-Met,
especially compounds which inhibit the kinase activity of c-Met receptor, or
antibodies
that target the extracellular domain of c-Met or bind to HGF.
[0063] Further anti-angiogenic compounds include compounds having
another
mechanism for their activity, e.g. unrelated to protein or lipid kinase
inhibition e.g.
thalidomide (THALOMID) and TNP-470.
[0064] Compounds which target, decrease or inhibit the activity of a
protein or
lipid phosphatase are e.g., inhibitors of phosphatase 1, phosphatase 2A, or
CDC25, e.g.
okadaic acid or a derivative thereof.
[0065] Compounds which induce cell differentiation processes are e.g.
retinoic
acid, or tocopherol or tocotrienol.
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[0066] The term cyclooxygenase inhibitor as used herein includes, but
is not
limited to, e.g. Cox-2 inhibitors, 5-alkyl substituted 2-arylaminophenylacetic
acid and
derivatives, such as celecoxib (CELEBREX), rofecoxib (VIOXX), etoricoxib,
valdecoxib or a 5-alkyl-2-arylaminophenylacetic acid, e.g. 5-methy1-2-(2'-
chloro-6'-
fluoroanilino)phenyl acetic acid, lumiracoxib.
[0067] The term "bisphosphonates" as used herein includes, but is not
limited
to, etridonic, clodronic, tiludronic, pamidronic, alendronic, ibandronic,
risedronic and
zoledronic acid. "Etridonic acid" can be administered, e.g., in the form as it
is marketed,
e.g. under the trademark DIDRONEL. "Clodronic acid" can be administered, e.g.,
in the
form as it is marketed, e.g. under the trademark BONEFOS. "Tiludronic acid"
can be
administered, e.g., in the form as it is marketed, e.g. under the trademark
SKELID.
"Pamidronic acid" can be administered, e.g. in the form as it is marketed,
e.g. under the
trademark AREDIA. "Alendronic acid" can be administered, e.g., in the form as
it is
marketed, e.g. under the trademark FOSAMAX. "Ibandronic acid" can be
administered,
e.g., in the form as it is marketed, e.g. under the trademark BONDRANAT.
"Risedronic
acid" can be administered, e.g., in the form as it is marketed, e.g. under the
trademark
ACTONEL. "Zoledronic acid" can be administered, e.g. in the form as it is
marketed,
e.g. under the trademark ZOMETA.
[0068] The term "mTOR inhibitors" relates to compounds which inhibit
the
mammalian target of rapamycin (mTOR) and which possess antiproliferative
activity
such as sirolimus (Rapamune), everolimus (Certicanb), CCI-779 and ABT578.
[0069] The term "heparanase inhibitor" as used herein refers to
compounds
which target, decrease or inhibit heparin sulfate degradation. The term
includes, but is
not limited to, PI-88.
[0070] The term "biological response modifier" as used herein refers to
a
lymphokine or interferons, e.g. interferon.
[0071] The term "inhibitor of Ras oncogenic isoforms", e.g. H-Ras, K-
Ras, or
N-Ras, as used herein refers to compounds which target, decrease or inhibit
the oncogenic
activity of Ras e.g. a "farnesyl transferase inhibitor" e.g. L-744832, DK8G557
or
R115777 (Zarnestra).
[0072] The term "telomerase inhibitor" as used herein refers to
compounds
which target, decrease or inhibit the activity of telomerase. Compounds which
target,
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decrease or inhibit the activity of telomerase are especially compounds which
inhibit the
telomerase receptor, e.g. telomestatin.
[0073] The term "methionine aminopeptidase inhibitor" as used herein
refers
to compounds which target, decrease or inhibit the activity of methionine
aminopeptidase.
Compounds which target, decrease or inhibit the activity of methionine
aminopeptidase
are e.g. bengamide or a derivative thereof.
[0074] The term "proteasome inhibitor" as used herein refers to
compounds
which target, decrease or inhibit the activity of the proteasome. Compounds
which target,
decrease or inhibit the activity of the proteasome include e.g. Bortezomid
(Velcade) and
MLN 341.
[0075] The term "matrix metalloproteinase inhibitor" or ("MMP"
inhibitor) as
used herein includes, but is not limited to, collagen peptidomimetic and
nonpeptidomimetic inhibitors, tetracycline derivatives, e.g. hydroxamate
peptidomimetic
inhibitor batimastat and its orally bioavailable analogue marimastat (BB-
2516),
prinomastat (AG3340), metastat (NSC 683551) BMS-279251, BAY 12-9566, TAA211,
MMI270B or AAJ996.
[0076] The term "compounds used in the treatment of hematologic
malignancies" as used herein includes, but is not limited to, FMS-like
tyrosine kinase
inhibitors e.g. compounds targeting, decreasing or inhibiting the activity of
FMS-like
tyrosine kinase receptors (Flt-3R); interferon, 1-b-D-arabinofuransylcytosine
(ara-c) and
bisulfan; and ALK inhibitors e.g. compounds which target, decrease or inhibit
anaplastic
lymphoma kinase.
[0077] Compounds which target, decrease or inhibit the activity of FMS-
like
tyrosine kinase receptors (Flt-3R) are especially compounds, proteins or
antibodies which
inhibit members of the Flt-3R receptor kinase family, e.g. PKC412, TKI258,
midostaurin,
a staurosporine derivative, SU11248 and MLN518.
[0078] The term "HSP90 inhibitors" as used herein includes, but is not
limited
to, compounds targeting, decreasing or inhibiting the intrinsic ATPase
activity of HSP90;
degrading, targeting, decreasing or inhibiting the HSP90 client proteins via
the ubiquitin
proteosome pathway. Compounds targeting, decreasing or inhibiting the
intrinsic ATPase
activity of HSP90 are especially compounds, proteins or antibodies which
inhibit the
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ATPase activity of HSP90 e.g., 17-allylamino,17-demethoxygeldanamycin (17AAG),
a
geldanamycin derivative; other geldanamycin related compounds, and radicicol.
[0079] The term "antiproliferative antibodies" as used herein includes,
but is
not limited to, trastuzumab (Herceptin), Trastuzumab-DM1,erbitux, bevacizumab
(Avastin), rituximab (Rituxan), PR064553 (anti-CD40) and 2C4 Antibody. By
antibodies is meant e.g. intact monoclonal antibodies, polyclonal antibodies,
multispe-cific antibodies formed from at least 2 intact antibodies, and
antibodies
fragments so long as they exhibit the desired biological activity.
[0080] For the treatment of acute myeloid leukemia (AML), compounds of
formula (I) can be used in combination with standard leukemia therapies,
especially in
combination with therapies used for the treatment of AML. In particular,
compounds of
formula (I) can be administered in combination with, e.g., famesyl transferase
inhibitors
and/or other drugs useful for the treatment of AML, such as Daunorubicin,
Adriamycin,
Ara-C, VP-16, Teniposide, Mitoxantrone, Idarubicin, Carboplatinum and PKC412.
[0081] The term "antileukemic compounds" includes, for example, Ara-C,
a
pyrimidine analog, which is the 2-alpha-hydroxy ribose (arabinoside)
derivative of
deoxycytidine. Also included is the purine analog of hypoxanthine, 6-
mercaptopurine (6-
MP) and fludarabine phosphate.
[0082] Somatostatin receptor antagonists as used herein refers to
compounds
which target, treat or inhibit the somatostatin receptor such as octreotide,
and S0M230
(pasireotide).
[0083] Tumor cell damaging approaches refer to approaches such as
ionizing
radiation. The term "ionizing radiation" referred to above and hereinafter
means ionizing
radiation that occurs as either electromagnetic rays (such as X-rays and gamma
rays) or
particles (such as alpha and beta particles). Ionizing radiation is provided
in, but not
limited to, radiation therapy and is known in the art. See Hellman, Principles
of
Radiation Therapy, Cancer, in Principles and Practice of Oncology, Devita et
al., Eds.,
4th Edition, Vol. 1, pp. 248-275 (1993).
[0084] The term "EDG binders" as used herein refers a class of
immunosuppressants that modulates lymphocyte recirculation, such as FTY720.
[0085] The term "ribonucleotide reductase inhibitors" refers to
pyrimidine or
purine nucleoside analogs including, but not limited to, fludarabine and/or
cytosine
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arabinoside (ara-C), 6-thioguanine, 5-fluorouracil, cladribine, 6-
mercaptopurine
(especially in combination with ara-C against ALL) and/or pentostatin.
Ribonucleotide
reductase inhibitors are especially hydroxyurea or 2-hydroxy-1H-isoindole-1,3-
dione
derivatives, such as PL-1, PL-2, PL-3, PL-4, PL-5, PL-6, PL-7 or PL-8
mentioned in
Nandy et al., Acta Oncologica, Vol. 33, No. 8, pp. 953-961 (1994).
[0086] The term "S-adenosylmethionine decarboxylase inhibitors" as used
herein includes, but is not limited to the compounds disclosed in US
5,461,076.
[0087] Also included are in particular those compounds, proteins or
monoclonal antibodies of VEGF disclosed in WO 98/35958, e.g. 1-(4-
chloroanilino)-4-
(4-pyridylmethyl)phthalazine or a pharmaceutically acceptable salt thereof,
e.g. the
succinate, or in WO 00/09495, WO 00/27820, WO 00/59509, WO 98/11223, WO
00/27819 and EP 0 769 947; those as described by Prewett et al, Cancer Res,
Vol. 59, pp.
5209-5218 (1999); Yuan et al., Proc Natl Acad Sci U S A, Vol. 93, pp. 14765-
14770
(1996); Zhu et al., Cancer Res, Vol. 58, pp. 3209-3214 (1998); and Mordenti et
al.,
Toxicol Pathol, Vol. 27, No. 1, pp. 14-21 (1999); in WO 00/37502 and WO
94/10202;
ANGIOSTATIN, described by O'Reilly et al., Cell, Vol. 79, pp. 315-328 (1994);
ENDOSTATIN, described by O'Reilly et al., Cell, Vol. 88, pp. 277-285 (1997);
anthranilic acid amides; ZD4190; ZD6474; SU5416; SU6668; bevacizumab; or anti-
VEGF antibodies or anti-VEGF receptor antibodies, e.g. rhuMAb and RHUFab, VEGF
aptamer e.g. Macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgG1
antibody,
Angiozyme (RPI 4610) and Bevacizumab (Avastin).
[0088] Photodynamic therapy as used herein refers to therapy which uses
certain chemicals known as photosensitizing compounds to treat or prevent
cancers.
Examples of photodynamic therapy includes treatment with compounds, such as
e.g.
VISUDYNE and porfimer sodium.
[0089] Angiostatic steroids as used herein refers to compounds which
block or
inhibit angiogenesis, such as, e.g., anecortave, triamcinolone,
hydrocortisone, 11--
epihydrocotisol, cortexolone, 17-hydroxyprogesterone, corticosterone,
desoxycorticosterone, testosterone, estrone and dexamethasone.
[0090] Implants containing corticosteroids refers to compounds, such as
e.g.
fluocinolone, dexamethasone.
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[0091] "Other chemotherapeutic compounds" include, but are not limited
to,
plant alkaloids, hormonal compounds and antagonists; biological response
modifiers,
preferably lymphokines or interferons; antisense oligonucleotides or
oligonucleotide
derivatives; shRNA or siRNA; or miscellaneous compounds or compounds with
other or
unknown mechanism of action.
[0092] The structure of the active compounds identified by code nos.,
generic
or trade names may be taken from the actual edition of the standard compendium
"The
Merck Index" or from databases, e.g. Patents International (e.g. IMS World
Publications).
[0093] None of the quotations of references made within the present
disclosure is to be understood as an admission that the references cited are
prior art that
would negatively affect the patentability of the present invention.
[0094] The compounds of the present invention may also be administered
simultaneously, separately or sequentially in combination with one or more
other suitable
active agents selected from the following classes of agents: Anti IL-1 agents,
e.g:
Anakinra; anti cytokine and anti-cytokine receptor agents, e.g. anti IL-6 R
Ab, anti IL-15
Ab, anti IL-17 Ab, anti IL-12 Ab; B-cell and T-cell modulating drugs, e.g.
anti CD20 Ab;
CTL4-Ig, disease-modifying anti-rheumatic agents (DMARDs), e.g. methotrexate,
leflunamide, sulfasalazine; gold salts, penicillamine, hydroxychloroquine and
chloroquine, azathioprine, glucocorticoids and non-steroidal anti-
inflammatories
(NSAIDs), e.g. cyclooxygenase inhibitors, selective COX-2 inhibitors, agents
which
modulate migration of immune cells, e.g. chemokine receptor antagonists,
modulators of
adhesion molecules, e.g. inhibitors of LFA-1, VLA-4.
[0095] The pharmaceutical composition or combination of the present
invention can be in unit dosage of about 1-1000 mg of active ingredient(s) for
a subject of
about 50-70 kg, or about 1-500 mg or about 1-250 mg or about 1-150 mg or about
0.5-
100 mg, or about 1-50 mg of active ingredients. In general, suitable daily
dosages for
oral administration are from about 0.1 to about 10 mg/kg. However, it will be
understood
by those of skill in the art that the therapeutically effective dosage of a
compound, the
pharmaceutical composition, or the combinations thereof, is dependent on the
species of
the subject, the body weight, age and individual condition, the disorder or
disease or the
severity thereof being treated. A physician, clinician or veterinarian of
ordinary skill can
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readily determine the effective amount of each of the active ingredients
necessary to
prevent, treat or inhibit the progress of the disorder or disease.
[0096] The above-cited dosage properties are demonstrable in vitro and
in vivo
tests using advantageously mammals, e.g., mice, rats, dogs, monkeys or
isolated organs,
tissues and preparations thereof. The compounds of the present invention can
be applied
in vitro in the form of solutions, e.g., aqueous solutions, and in vivo either
enterally,
parenterally, advantageously intravenously, e.g., as a suspension or in
aqueous solution.
The dosage in vitro may range between about 10-3 molar and 10-9 molar
concentrations.
A therapeutically effective amount in vivo may range depending on the route of
administration, between about 0.1-500 mg/kg, or between about 1-100 mg/kg.
[0097] In general, a therapeutically effective amount of a compound of
the
present invention is administered to a patient in need of treatment. The term
"a
therapeutically effective amount" of a compound of the present invention
refers to an
amount of the compound of the present invention that will elicit the
biological or medical
response of a subject, for example, reduction or inhibition of an enzyme or a
protein
activity, or ameliorate symptoms, alleviate conditions, slow or delay disease
progression,
or prevent a disease, etc.
[0098] In yet another embodiment, a method for treating cancer in a
subject is
provided which comprises administering to a mammal in need of such treatment
an
effective amount of a compound of the present invention.
[0099] As used herein, the term "subject" refers to an animal.
Typically the
animal is a mammal. A subject also refers to for example, primates (e.g.,
humans, male
or female), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish,
birds and the
like. In certain embodiments, the subject is a primate. Preferably, the
subject is a human.
[00100] As used herein, the term "inhibit", "inhibition" or "inhibiting"
refers to
the reduction or suppression of a given condition, symptom, or disorder, or
disease, or a
significant decrease in the baseline activity of a biological activity or
process.
[00101] As used herein, the term "treat", "treating" or "treatment" of
any
disease or disorder, refers (i) to ameliorating the disease or disorder (i.e.,
slowing or
arresting or reducing the development of the disease or at least one of the
clinical
symptoms thereof); (ii) to alleviating or ameliorating at least one physical
parameter
including those which may not be discernible by the patient; or (iii) to
preventing or
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delaying the onset or development or progression of the disease or disorder.
In general,
the term "treating" or "treatment" describes the management and care of a
patient for the
purpose of combating the disease, condition, or disorder and includes the
administration
of a compound of the present invention to prevent the onset of the symptoms or
complications, alleviating the symptoms or complications, or eliminating the
disease,
condition or disorder.
[00102] As used herein, a subject is "in need of' a treatment if such
subject
would benefit biologically, medically or in quality of life from such
treatment.
[00103] Another aspect of the invention is a product comprising a
compound of
the present invention and at least one other therapeutic agent (or
pharmaceutical agent) as
a combined preparation for simultaneous, separate or sequential use in therapy
to enhance
apoptosis.
[00104] In the combination therapies of the invention, the compound of
the
present invention and the other therapeutic agent may be manufactured and/or
formulated
by the same or different manufacturers. Moreover, the compound of the present
invention and the other therapeutic (or pharmaceutical agent) may be brought
together
into a combination therapy: (i) prior to release of the combination product to
physicians
(e.g. in the case of a kit comprising the compound of the invention and the
other
therapeutic agent); (ii) by the physician themselves (or under the guidance of
the
physician) shortly before administration; (iii) in the patient themselves,
e.g. during
sequential administration of the compound of the invention and the other
therapeutic
agent.
[00105] Accordingly, the invention provides the use of a compound of the
present invention for treating a disease or condition by inhibiting the MAP
kinase
pathway, wherein the medicament is prepared for administration with another
therapeutic
agent. The invention also provides for the use of another therapeutic agent,
wherein the
medicament is administered as a combination of a compound of the present
invention
with the other therapeutic agent.
[00106] Embodiments of the present invention are illustrated by the
following
Examples. It is to be understood, however, that the embodiments of the
invention are not
limited to the specific details of these Examples, as other variations thereof
will be
known, or apparent in light of the instant disclosure, to one of ordinary
skill in the art.
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Process for Making Compounds of the Invention
[00107] The present invention also includes processes for the
preparation of
compounds of the invention. In the reactions described, it can be necessary to
protect
reactive functional groups, where these are desired in the final product, to
avoid their
unwanted participation in the reactions. Conventional protecting groups can be
used in
accordance with standard practice, for example, see T.W. Greene and P. G. M.
Wuts in
"Protective Groups in Organic Chemistry", John Wiley and Sons, 1991.
[00108] Individual enantiomers of the invention can be obtained from
chiral
separation of a racemate or can be individually synthesized from optically
pure reagents
using reaction scheme I:
Reaction Scheme I:
23
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0
HN-4 F
N
0 4 * 1
\
0
0 F F
SO2CI (2) OSO2C1
olo
0 (3a)
/
N1/441(3b)
0
0 0
Ohe....7 Oe...../A
140
SO2, F 40 so2 ,p F
N N
\ \
F F
(4a) F
\ 0 0
0/ , Oh.e.....
op s02. F 100 SO2 F F
NH H .NH H
N N
0 . 111P 1 0 so # ,
. \
F F
HO (5a) F (5b) F HO
HOhe......
HOe.......7A
SO2 F SO2 F
.NH H 'NH H
N N
0 op * , 0 op * ,
\ \
F F
F F
(S) enantiomer R-enantiomer
[00109] A compound of formula 4a can be prepared by reacting a compound
of
formula 2 with a compound of formula 3a in the presence of a suitable amine
(for
example, triethylamine, N 7N-Dii sopropy I ethyl arnin e, Triisopropylamine,
or the like), a
suitable catalyst (for example, N,N-dimethylpyridin-4-amine, or the like) and
a suitable
solvent (for example, DCM, 1,2-dichloromethane, toluene, tetrahydrofuranyl, or
the like).
The reaction proceeds at a temperature of about 25 C and can take up to about
12 hours to
complete.
24
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[00110] A compound of formula 5a can be prepared from a compound of
formula 4a in the presence of a suitable base (for example, potassium
trimethylsilanoate,
or the like). The reaction proceeds at a temperature of about 25 C and can
take up to
about 12 hours to complete.
[00111] The S-enantiomer can be prepared from a compound of formula 5a
in
the presence of a suitable Lewis acid (for example, trichlorborane, boron
trifluoride,
boron tribromide, or the like) and a suitable solvent (for example,
dichloromethane, 1,2-
dichloromethane, or the like). The reaction proceeds at a temperature of about
25 C and
can take up to about 1 hour to complete.
[00112] A compound of formula 4b can be prepared by reacting a compound
of
formula 2 with a compound of formula 3b in the presence of a suitable amine
(for
example, triethylamine, N,Nr-Diisopropylethylamine, Triisopropylamine or the
like), a
suitable catalyst (for example, N,N-dimethylpyridin-4-amine or the like) and a
suitable
solvent (for example, DCM, 1,2-dichloromethane, toluene, tetrahydrofuran, or
the like).
The reaction proceeds at a temperature of about 25 C and can take up to about
12 hours to
complete.
[00113] A compound of formula 5b can be prepared from a compound of
formula 4b in the presence of a suitable base (for example, potassium
trimethylsilanoate,
or the like). The reaction proceeds at a temperature of about 25 C and can
take up to
about 12 hours to complete.
[00114] The R-enantiomer can be prepared from a compound of formula 5b
in
the presence of a suitable Lewis acid(for example, trichlorborane boron
trifluoride, boron
tribromide, or the like) and a suitable solvent (for example, dichloromethane,
1,2-
dichloromethane, or the like). The reaction proceeds at a temperature of about
25 C and
can take up to about 1 hour to complete.
EXAMPLES
[00115] The following abbreviations used herein below have the
corresponding
meanings: TEA (Triethylamine); EA (Ethyl acetate); MCC (Microcrystalline
cellulose);
DMAP (4-Dimethylaminopyridine); DCM (Dichloromethane); THF (Tetrahydrofuran);
DMF (Dimethylformamide); LHMDS (lithium bis(trimethylsilyl)amide); CDI (1,1-
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Carbonyldiimidazole); PTSA (p-toluene sulfonic acid); RT (room temperature);
TLC
(thin layer chromatography); NMR (nuclear magnetic resonance); LC-MS (liquid
chromatography- mass spectrometry); and HPLC (high pressure liquid
chromatography
or high performance liquid chromatography).
Preparation of Key Intermediates
Preparation of (2,3,5-trifluoro-N-(2-fluoro-4-iodopheny1)-6-nitroaniline
F
H
F 0 Ns
F I
F
[00116] 1.0M LHMDS in hexane (153mL, 153mmol) was added drop wise to a
solution of 2-fluoro-4-iodoaniline (30.0g, 128mmol) in dry THF (600mL) at -78
C over a
period of 30 minutes and the resulting mixture was stirred at -78 C for a
further 30
minutes. This was followed by the addition of 2,3,4,6-tetrafluoronitrobenzene
(25g,
128mmol) in dry THF (150mL) and stirring was continued for a further 1 hour at
20-
40 C. The reaction was monitored by TLC (10% ethyl acetate in hexane). The
reaction
mixture was quenched with 2N HC1(100mL), concentrated and the concentrate was
partitioned between water (500mL) and ethyl acetate (300mL). The aqueous layer
was
washed with ethyl acetate (2x200mL). The combined organic phase was washed
with
water, brine solution, dried over anhydrous Na2SO4 and concentrated to afford
38g of the
crude product. Purification by column chromatography on silica gel (0-5% ethyl
acetate
in hexane) afforded 31g of the product (58.8% yield). LCMS: 95.5%, m/z = 410.9
(M-1).
[00117] For large scale synthesis, 1,2,3,5-tetrafluoro-4-nitrobenzene
was
prepared by precooling a solution of HNO3 (990 g) in H2SO4 (973 mL) and adding
it into
a cold solution of 1,2,3,5-tetrafluorobenzene (973.1 g) in H2SO4 (2920 mL) at
a
temperature between 0 C and 10 C over a 1.5 hour period. After the addition,
the yellow
solution was stirred at a temperature between 0 C and 10 C for 1 hour until
analysis
showed the reaction had gone to completion. The yellow solution was added in
portions
of water (9730 g) below 25 C followed by the addition of DCM (9730 mL) to
extract the
26
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aqueous layer. The DCM layer was then washed with water (10 L) twice. DCM was
then concentrated to give a yellow oil. The oil was distilled under pressure
(2.5 mbar) at
70 C to -75 C resulting in a yellow oil product (98% pure; 72.4% yield).
[00118] LiHMDS (1732 mL) was added to a solution of aniline in 2.5 L of
THF
at between -72 C and -65 C over 30 minutes. The resultant suspension was
stirred at -
70 C for 30 minutes. A solution of nitrobenzene in 300 mL of THF was then
added
dropwise into the above suspension at between -75 C and -70 C over 1 hour and
stirred at
this temperature for a further 15 minutes. An additional 100mL of LiHMDS was
added at
-70 C and stirred at this temperature for 15 minutes. HPLC analysis revealed
23.4% of
aniline, 6.6% nitrobenzene and 68.3% product. The solution was concentrated at
35 C.
The resultant oil was then dissolved in 3L of DCM followed by the addition of
2.5 L of
water below 30 C. HC1 (500 mL) was added (exothermic) and the solution was
stirred
for 15 minutes before separating. 2.5 L of water and 500 mL of HC1 was added
to the
DCM layer and the wash was repeated 3 times. The DCM layers were concentrated
to
dryness and the batches added together to give about 1.4 kg of crude product.
The solid
was added to 20 L of hot heptanes solution (72 C) and stirred for 1 hour. 200
g of active
charcoal was added with stirring for 20 minutes. The suspension was hot
filtered through
MCC (2 kg) and rinsed with 5 L of hot heptanes (75 C). The combined solution
was then
concentrated to 15 L and held overnight. The suspension was filtered to give
777 g of
orange solid with 99% purity. The mother liquor was concentrated to about 5 L
and
stirred at room temperature and filtered to give an additional 120 g of orange
solid. The
overall yield was 46.8%. 1H NMR (DMSO-d6, 400 MHz): 6 8.80 (s, 1H), 7.60-7.67
(m,
2H), 7.41 (d, 1H), 6.86 (t, 1H).
Preparation of 3-(2,2-diethoxyethoxy)-5,6-difluoro-N-(2-fluoro-4-iodopheny1)-2-
nitroaniline
No2 H F
0 la N is
Et0 OEti F I
F
27
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[00119] 2,2-Diethoxy-ethanol (0.209g, 1.2135mmo1) was added to a cooled
suspension of NaH (0.034g, 1.456mmol) in THF (5mL) at 0 C and the resulting
mixture
was stirred for 30 minutes at RT. (2-Fluoro-4-iodo-pheny1)-(2,3,5-trifluoro-6-
nitro-
pheny1)-amine (0.5g, 1.2135mmo1) in THF (10mL) was added slowly to the
reaction
mass at 0 C and stirring was continued for a further 15 minutes. The reaction
mass was
stirred overnight at room temperature. The reaction was monitored by TLC (20%
ethyl
acetate in hexane). The reaction mass was concentrated under reduced pressure
and the
concentrate was extracted with ethyl acetate. The organic layer was washed
with water,
brine solution, dried over sodium sulphate and concentrated under reduced
pressure to
afford the crude compound. Purification by column chromatography on silica gel
(15%
ethyl acetate in hexane) afforded 0.3g of the product (47% yield). Two by
products are
isolated in this reaction. 1H NMR (CDC13, 300 MHz): 6 7.42 (d, 1H), 7.35 (d,
1H), 6.90
(bs, 1H), 6.58-6.68 (m, 2H), 4.58 (t, 1H), 4.15 (d, 2H), 3.51-3.80 (m, 4H),
1.22 (t, 6H).
[00120] For large scale synthesis, 2,2-diethoxyethanol was added
dropwise into
a suspension of 60% sodium hydride in 5 L of THF at 1-2 C over 30 minutes and
then
stirred at 0-5 C for 1.5 hours. A solution of 750 g of 2,3,5-trifluoro-N-(2-
fluoro-4-
iodopheny1)-6-nitroaniline in 2.25 L of THF was then added dropwise into the
above
suspension at 0-5 C. After the addition, the purple solution was stirred at
room
temperature for 2 days and 17 hours. HPLC analysis showed 4.1% remaining of
2,3,5-
trifluoro-N-(2-fluoro-4-iodopheny1)-6-nitroaniline. 150 mL of water was added
very
slowly (gas generated) into the pre-cooled solution at 10 C. The solution was
then
concentrated to dryness. The resultant oil was then suspended in 5 L of
heptanes and
seeded with pure 3-(2,2-diethoxyethoxy)-5,6-difluoro-N-(2-fluoro-4-iodopheny1)-
2-
nitroaniline crystal and stirred for 30 minutes. After 1 hour of stirring, the
precipitate was
filtered and dried to yield 520 g (54.3%) of product with 96.4% purity.
Further product
was available in the mother liquor. 1H NMR (CDC13, 400 MHz): 6 7.42 (dd, 1H),
7.34 (d,
1H), 6.87 (bs, 1H), 6.56-6.67 (m, 2H), 4.79 (t, 1H), 4.06 (d, 2H), 3.74-3.82
(m, 2H), 3.59-
3.66 (m, 2H); 1.24(t, 6H).
f4,5-difluoro-7-nitro-benzofuran-6-y1)-(2-fluoro-4-iodo-pheny1)-amine:
28
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NO2 H F
\O 0 N 40
F I
F
[00121] [3-(2,2-Diethoxy-ethoxy)-5,6-difluoro-2-nitro-pheny1]-(2-fluoro-
4-
iodo-pheny1)-amine (1g, 1.9011mmol) was dissolved in glacial acetic acid
(10mL) and
concentrated under reduced pressure. The residue obtained was dissolved in dry
DCM
(10mL) and cooled to 0 C. This was followed by the addition of BF3.etherate
(2.04g,
14.476mmo1). The reaction was stirred for 12-16 hours at 20-40 C. The reaction
was
monitored by TLC (10% ethyl acetate in hexane). The reaction was quenched with
2N
NaOH solution (15mL), extracted with ethyl acetate (3x30mL) and the combined
organic
extracts were dried over sodium sulphate, concentrated under reduced pressure
to afford
the crude compound. Purification by column chromatography on silica gel (5%
ethyl
acetate in hexane) afforded 0.260g of the product (31% yield). 1H NMR (CDC13,
300
MHz): 6 8.95 (bs, 1H), 7.77 (d, 1H), 7.38-7.50 (1dd, id, 2H), 6.99 (d, 1H),
6.70-6.82 (m,
1H).
[00122] For scale up, a solution of 3-(2,2-diethoxyethoxy)-5,6-difluoro-
N-(2-
fluoro-4-iodopheny1)-2-nitroaniline (75g) in 300 mL of DCM was added quickly
into a
solution of BF3.ET20 in 100mL of DCM at 28-32 C with further stirring for 15
minutes.
After about 2 hours, analysis showed complete conversion. The solution was
then
quenched by 1N NaOH (dropwise) at a temperature below 30 C to pH 10 before
separating. DCM was then washed with water twice to a pH of about 7 and
concentrated
to dryness. 210 mL of MBTE was then added. The suspension was stirred for 30
minutes, filtered and dried to give 43 g of an orange solid. 1H NMR (CDC13,
400 MHz): 6
8.80 (bs, 1H), 7.75 (d, 1H), 7.48 (dd, 1H), 7.41(d; 1H); 6.97 (d, 1H), 6.75-
6.80 (m, 1H).
4,5-Difluoro-N6-(2-fluoro-4-iodo-phenyl)-benzofuran-6,7-diamine:
29
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NH2 H F
\O 0 N ilo
F I
F
[00123] Concentrated HC1(1mL) was added to a solution of (4,5-difluoro-7-
nitro-benzofuran-6-y1)-(2-fluoro-4-iodo-pheny1)-amine (0.260g, 0.599mmo1) in
THF
(5mL) at 0 C. This was followed by the addition of zinc dust (0.179g,
5.99mmol) at 0 C.
The reaction was stirred for 1 hour at 20-40 C. The reaction was monitored by
TLC (20%
ethyl acetate in hexane). The reaction was concentrated under reduced pressure
and the
concentrate was extracted with ethyl acetate (50mL). The organic layer was
washed with
water, brine solution, dried over sodium sulphate and concentrated under
reduced
pressure to afford 0.240g of the crude compound which was used for the next
step
without further purification. 1H NMR (CDC13, 300 MHz): 6 7.53 (d, 1H), 7.49
(dd, 1H),
7.20 (d, 1H), 6.00 (d, 1H), 6.20 (t, 1H), 5.42 (bs, 1H), 4.10 (bs, 2H).
[00124] For scale up synthesis, a solution of (4,5-difluoro-7-nitro-
benzofuran-
6-y1)-(2-fluoro-4-iodo-pheny1)-amine (165g) and NH4C1 in 2.5 L of THF and 2.5
L of
water was cooled to between 0 C and 5 C. Zinc dust was added (portionwise) at
a
temperature of less than 25 C (exothermic during first half of addition) over
30 minutes.
The addition of zinc dust resulted in a color change from yellow to dark
purple and to
pale yellow. Complete conversion was achieved with the addition of NH4C1 (50
g) and
zinc (60 g), followed by vigorous stirring for 20 minutes and a further 1.5
hours. Purity
was 89.5%. The suspension was filtered and rinsed with 1 L of water and 1.5 L
of THF.
The combined solution was concentrated to remove THF (87.7% purity). 3 L of EA
was
added with stirring for 30 minutes. The layers were separated; the EA layer
was washed
with 2 L of water twice and then dried of MgSO4. The EA was filtered and
concentrated
to dryness. The crude was used in the next step without further purification.
1H NMR
(CDC13, 400 MHz): 6 8.06 (d, 1H), 7.46 (dd, 1H), 7.40 (bs, 1H), 7.19 (d, 1H),
7.07 (d,
1H), 6.11(t; 1H); 5.33 (bs, 1H).
4,5-Difluoro-3-(2-fluoro-4-iodopheny1)-1H-benzofuro[6,7-d]imidazol-2(3H)-one:
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H N
N
0 0 .
I
F
F
[00125] CDI (0.144g, 0.891mmol) was added to a solution of 4,5-difluoro-
N6-
(2-fluoro-4-iodo-pheny1)-benzofuran-6,7-diamine (0.240g, 0.5940mmo1) in dry
DCM
(5mL). The reaction mass was stirred for 12-16 hours at 20-40 C. The reaction
was
monitored by TLC (30% ethyl acetate in hexane). The reaction mass was
concentrated
under reduced pressure and the concentrate was extracted with ethyl acetate.
The organic
layer was washed with water, brine solution, dried over sodium sulphate and
concentrated
under reduced pressure to afford the crude compound. Purification by column
chromatography on silica gel (30% ethyl acetate in hexane) afforded 0.180g of
the
product (70% yield). 1H NMR (DMSO-d6, 300 MHz): 6 12.15 (bs, 1H), 8.12 (d,
1H), 7.95
(dd, 1H), 7.79 (d, 1H), 7.50 (t, 1H), 7.21 (d, 1H).
[00126] For scale up synthesis, CDI(92.4g, 570mmol) was added to a
solution
of 4,5-difluoro-N6-(2-fluoro-4-iodo-pheny1)-benzofuran-6,7-diamine(153.6g,
380.1mmol) in 1500 mL of DCM at 0 C. The solution was then warmed to room
temperature and stirred for approximately 48 hours. Analysis showed 0.3%
remaining of
4,5-difluoro-N6-(2-fluoro-4-iodo-phenyl)-benzofuran-6,7-diamine with 86.1%
purity.
The solution was filtered to give crude gray solid with 96.4% purity. The
crude was
dissolved in 1.5 L of THF and then water (1.5 L) was added. The suspension was
then
concentrated to 1.7 L and filtered to give 87 g of product with 98.0% purity
(53.2%
yield). 1H NMR (DMSO-d6, 400 MHz): 6 12.29 (bs, 1H), 8.09 (d, 1H), 7.94 (dd,
1H),
7.76 (d, 1H), 7.47 (t, 1H), 7.19 (d, 1H).
1-(1-Allylcyclopropylsulfony1)-4,5-difluoro-3-(2-fluoro-4-iodopheny1)-1H-
benzofuro[6,7-d]imidazol-2(3H)-one:
31
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N---4c
N =\O
(I-34a)
[00127] TEA (0.2611g, 2.581mmol) was added to a solution of 4,5-difluoro-
3-
(2-fluoro-4-iodopheny1)-1H-benzofuro[6,7-d]imidazol-2(3H)-one0.37g,
0.8604mmo1) in
dry DCM (20mL) at 0 C. This was followed by the addition of 1-allyl-
cyclopropanesulfonyl chloride (0.229g, 1.89 mmol) and catalytic amount of DMAP
(10mg). The reaction mass was stirred for 12 hours at 20-40 C. The reaction
was
monitored by TLC (20% ethyl acetate in hexane). The reaction mass was diluted
with
DCM (50mL) and partitioned between water and DCM. The organic layer was washed
with water, brine solution and concentrated under reduced pressure to afford
the crude
product. Purification by column chromatography on silica gel (20% ethyl
acetate in
hexane) afforded 0.228g of the product (46% yield). 1H NMR (CDC13, 300 MHz): 6
7.71
(dd, 3H), 7.30 (t, 1H), 7.00 (s, 1H), 5.56-5.57 (m, 1H), 4.90 (t, 2H), 2.70-
2.80 (q, 2H),
1.90-2.05 (m, 2H), 1.10- 1.19 (m, 2H). LCMS: 98.85%, miz = 574.4 (M+1). HPLC:
97.1%.
1-Allyl-N-(4,5-difluoro-6-(2-fluoro-4-iodophenylamino)benzofuran-7-
yl)cyclopropane-1-
sulfonamide:
iP'NH
FOI
0
\O N
32
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[00128] Potassium trimethyl silanolate (0.105g, 0.82mmol) was added to a
solution of 1-(1-allylcyclopropylsulfony1)-4,5-difluoro-3-(2-fluoro-4-
iodopheny1)-1H-
benzofuro[6,7-d]imidazol-2(3H)-one (0.230g, 0.4108mmol) in THF (5mL) at 0 C.
The
reaction mass was stirred for 4 hours at 20-40 C. The reaction was monitored
by TLC
(10% ethyl acetate in hexane). The reaction mass was concentrated under
reduced
pressure and the concentrate was extracted with ethyl acetate. The organic
layer was
washed with water, brine solution, dried over sodium sulphate and concentrated
under
reduced pressure to afford the crude compound. Purification by column
chromatography
on silica gel (10% ethyl acetate in hexane) afforded 0.177g of the product
(78% yield).
1H NMR (CDC13, 300 MHz): 6 7.54 (d, 1H), 7.40 (dd, 1H), 7.25 (s, 1H), 7.05
(bs, 1H),
6.99 (d, 1H), 6.32-6.39 (m, 1H), 6.22 (s, 1H), 5.65-5.75 (m, 1H), 5.19 (s,
1H), 5.10 (d,
1H), 2.88 (d, 2H), 1.15 (t, 2H), 0.75 (t, 2H). LCMS: 96.32%, m/z = 548.8
(M+1).
HPLC: 97.19%.
isopropyl cyclopropanesulfonate
0
\o
K
[00129] IPA (11 L) was added to pyridine (1850 g; 1890 mL) at 20 C with
stirring for 1 hour. The solution was cooled to between -10 C and 2 C and
cyclopropane
sulfonic chloride (1100 g) was added dropwise followed by stirring at this
temperature for
70 hours. NMR analysis showed 85% of sulfonic chloride was consumed in the
reaction.
The reaction was cooled to 0 C and NaOH (312 g) in water (600 mL) was added
dropwise. The resultant product was concentrated to dryness and 45 C and
diluted with
Et0Ac (2750 mL), MTBE (1375 mL). The solution was stirred for 10 minutes and
filtered. Heptane (1375 mL) was added, filtered and the filtrate was
concentrated to
dryness giving 1080 g of product as a red liquid (84% yield). 1H NMR (CDC13,
400
MHz): 6 4.86 (m, 1H), 2.83 (m, 1H), 1.33 (d, 6H), 0.98-1.10(m, 4H).
(S)-2-((benzyloxy)methyl)oxirane
33
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0
1, ___________________________________ \
0
*
[00130] A slurry of NaH (267 g) and NaI (26.5 g) in DMF (3000 mL) was
cooled to between -15 C and -10 C with ice-brine mixture. A solution of
glycidol (480
g) in DMF (1000 mL) was added with stirring for 10 minutes. BnC1 (745.5 g) in
DMF
(800 mL) was added (slightly exothermic) and the reaction was warmed to room
temperature followed by stirring for approximately 16 hours. The solution was
stirred
for a further time period until HPLC analysis showed less than 1% of BnC1
remained.
The reaction was cooled to -5 C and water (4 L) was added. MBTE (5L) was added
with
stirring for 30 minutes. The organic and aqueous layers were separated and two
additions
of MBTE (5 L; 3.5 L) were used to extract the aqueous layer. The combined
organic
layer was washed with brine (1.8 L (x2)) and dried over Na2SO4 for 1 hour. The
organic
layer was concentrated to dryness, distilled under vacuum (5mm Hg, 85 C) to
give the
product as a yellow liquid (760 g).1H NMR (CDC13, 400 MHz): 6 7.27-7.37 (m,
5H), 4.51
(s, 2H), 3.74 (dd, 1H), 3.29 (dd, 1H), 3.14 (m, 1H), 2.72 (dd, 1H), 2.55 (dd,
1H).
(R)-isopropyl 1-(3-(benzyloxy)-2-hydroxypropyl)cyclopropane-1-sulfonate
)------
0 OH
\
0=S
ii
0-2C--
0
4110
[00131] A solution of (S)-2-((benzyloxy)methyl)oxirane (507 g), HMPA
(450
mL) and THF (4 L) was cooled with a dry ice-acetone bath to a temperature
between -
70 C and -60 C. n-BuLi (1500 mL; 2.4 M) was added, dropwise, over 75 minutes.
THF
34
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(450 g; 500 mL) was added to the reaction mixture, the temperature was raised
to -20 C
gradually and the mixture was stirred for approximately 16 hours until HPLC
analysis
showed the reaction had gone to completion. The reaction mixture was cooled to
-30 C
and water (300 mL) and concentrated aqueous HC1 (300 mL) were added. The
solution
was concentrated to dryness. Water (2 L) was added and the product extracted
twice with
Et0Ac (2.5L (x2)). The combined organic layers were washed with water (2.5 L),
brine
(2 L), dried over Na2SO4 (500 g) and filtered. The filtrate was concentrated
to dryness (at
45 C) giving crude product as a red liquid (712 g; 75% yield).
(R)-isopropyl 1-(2,3-bis(benzyloxy)propyl)cyclopropane-1-sulfonate
0
\r"---
0
\
0=s
ii 0 --sics
0
0*
[00132] Under a Nitrogen atmosphere was mixed NAH (91.5 g) in DMF (2 L)
and (R)-isopropyl 1-(3-(benzyloxy)-2-hydroxypropyl)cyclopropane-1-sulfonate
(715 g) in
DMF (1 L) at a temperature between -5 C and 5 C for 140 minutes. After
stirring for a
further 5 minutes, NaI (5.1 g) was added. After BnC1 (289.5 g) in DMF (300 mL)
was
added gradually, the reaction mixture was warmed slowly to room temperature
and stirred
for approximately 16 hours until HPLC analysis showed the reaction was
complete. The
reaction mixture was cooled to -5 C and water (4 L) was added followed by MTBE
(2 L
(x2)) with stirring for 20 minutes. The organic layer was separated, washed
with water
(2 L (x2)), brine (2 L), dried over Na2SO4 and concentrated to give 830g of
product.
(R)-1-(2,3-bis(benzyloxy)propyl)cyclopropane-l-sulfonic acid
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HO 0
\
0
0=S--2C
II
0
41Ik
[00133] A mixture of (R)-isopropyl 1-(2,3-
bis(benzyloxy)propyl)cyclopropane-
1-sulfonate (460 g) and KSCN (170 g) in THF 2.2 L) and water (2.2 L) at 25 C
was
refluxed for approximately 24 hours at 90 C until HPLC analysis showed that
the reaction
had gone to completion. The reaction mixture was concentrated to about 2L at
40 C and
extracted with MTBE (1.5 L (x3)). The organic layer was discarded and the
aqueous
layer was neutralized to pH 14 with KOH (30 g)in water (100 mL)and cooled to -
5 C.
The mixture was neutralized to a pH of between 2 and 3 with concentrated HC1
(205 mL)
and extracted with ETOAc (2 L (x2)). The combined organic layers was dried
over
Na2SO4 for approximately 16 hours, filtered and concentrated to dryness to
give the
product as a deep red oil (265 g).
(R)-1-(2,3-bis(benzyloxy)propyl)cyclopropane-l-sulfonyl chloride
0
CI 0
\
0=S
ll
0-2C1
0
4111
[00134] A mixture of (R)-1-(2,3-bis(benzyloxy)propyl)cyclopropane-l-
sulfonic
acid (630 g) and DMF (30 mL) in SOC12 (1.5 L) was refluxed for 2 hours until
HPLC
analysis showed the reaction had gone to completion. The reaction mixture was
36
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concentrated to dryness and the residue purified on silica gel chromoatography
with
eluent: heptane for 5 minutes, then IPAC: heptanes = 1:30; heptanes 1:10
resulting in the
product as a red oil (298.5 g; 52% yield; 95% purity). 1H NMR (CDC13, 400
MHz): 6 7.34
(m, 10H), 4.68 (d, 1H), 4.56 (bs, 1H), 4.53 (d, 1H), 4.16 (m, 1H), 3.54-3.62
(m, 2H), 2.63
(dd, 1H), 2.01 (q, 1H), 1.52-1.81 (m, 3H), 1.16 (m, 1H), 2.22 (bs, 1H), 1.75
(t, 2H), 1.38-
1.40 (m, 2H).
Example 1
1-(2,3-Dihydroxy-propy1)-cyclopropanesulfonic acid [4,5-difluoro-6-(2-fluoro-4-
iodo-
phenylamino)-benzofuran-7-yl]amide:
OH
F/3, IrOyi
SO2 NH
F
H
N
\O 0 0
F I
F
[00135] N-methyl morpholine oxide (0.035g, 0.3041mmol) was added to a
solution of 1-allyl-cyclopropane sulfonic acid [4,5-difluoro-6-(2-fluoro-4-
iodo-
phenylamino)-benzofuran-7-y1]-amide (0.167g, 0.3041mmol) in THF (5mL). This
was
followed by the addition of osmium tetroxide (0.0077g, 0.03041mmol) in water
(1mL).
The reaction mass was stirred for 16 hours at 30-40 C. The reaction was
monitored by
TLC (10% methanol in chloroform). The reaction mass was partitioned between
ethyl
acetate (50mL) and water. The organic layer was washed with water (3x50mL),
brine
solution and concentrated under reduced pressure to afford the crude product.
Purification
by column chromatography on silica gel (5% methanol in chloroform) afforded
0.090g of
the product (50% yield). 1H NMR (CDC13, 300 MHz): 6 7.69 (d, 2H), 7.40 (dd,
1H), 7.25
(s, 1H), 6.99 (d, 1H), 6.98 (bs, 1H), 6.38-6.40 (m, 1H), 4.25 (bs, 1H), 3.62
(dd, 2H), 3.32
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(d, 1H), 2.55 (q, 1H), 2.22 (bs, 1H), 1.75 (t, 2H), 1.38-1.40 (m, 2H). LCMS:
99.49%, m/z
= 582.9 (M+1). HPLC: 95.29%.
[00136] For preparation of the enantiomers, the synthesis of 1-(2,3-
dihydroxy-
propy1)-cyclopropanesulfonic acid [4,5-difluoro-6-(2-fluoro-4-iodo-
phenylamino)-
benzofuran-7-yl]amide (Example 1) was scaled as follows: N-methyl morpholine
oxide
(0.32g, 2.7272mmo1) was added to a solution of 1-allyl-cyclopropane sulfonic
acid [4,5-
difluoro-6-(2-fluoro-4-iodo-phenylamino)benzofuran-7-yl]amide (1.5g,
2.7272mmo1) in
THF (50mL). This was followed by the addition of osmium tetroxide (0.695g,
0.2737mmo1) in water (5mL). The reaction mass was stirred overnight at room
temperature. The reaction was monitored by TLC (10% methanol in chloroform).
The
reaction mass was partitioned between ethyl acetate (100mL) and water (50mL).
The
organic layer was washed with water (3x75mL), brine solution and concentrated
under
reduced pressure to afford the crude product. Purification by column
chromatography on
silica gel (5% methanol in chloroform) afforded 0.9g (56%, HPLC purity, 85%)
of the
product of racemic mixture which was further purified by preparative HPLC to
afford the
pure racemic compound 0.7g (HPLC purity >98%).
[00137] The individual enantiomers were obtained by chiral column
chromatography. 0.420g of racemic 1-(2,3-dihydroxy-propy1)-
cyclopropanesulfonic acid
[4,5-difluoro-6-(2-fluoro-4-iodo-phenylamino)-benzofuran-7-yl]amide was
applied to a
Chiralpak0 AD-HTm/02 semi prep column at 25 C. The mobile phase was composed
of
80% hexane/10% IPA/10% Me0H with a 1 mL/minute flow rate. The diluent was IPA.
The first eluted enantiomer, Example 1B, had a retention time of 10.971
minutes (0.185g,
44%) and the second eluted enantiomer, Example 1A, had a retention time 14.961
minutes (0.175g, 41%).
[00138] Alternatively, racemic 1-(2,3-dihydroxy-propy1)-
cyclopropanesulfonic
acid [4,5-difluoro-6-(2-fluoro-4-iodo-phenylamino)-benzofuran-7-yl]amide was
applied
to a Chiralpak0 AD-HTm/03 semi prep column at 25 C. The mobile phase was
composed
of 80% hexane/20% IPA with a 1 mL/minute flow rate. The diluent was ethanol.
The
first eluted enantiomer, Example 1A, had a retention time of 10.465 minutes
(49%) and
the second eluted enantiomer, Example 1B, had a retention time 13.535 minutes
(46%).
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[00139] The enantiomers were further purified by preparative HPLC to
give
Examples lA and 1B with HPLC purity > 99%.
[00140] The following synthetic description shows how (R)-N-(4,5-
difluoro-6-
((2-fluoro-4-iodophenyl)amino)benzofuran-7-y1)-1-(2,3-
dihydroxypropyl)cyclopropane-
1-sulfonamide was prepared:
(R)-N-(4,5-difluoro-642-fluoro-4-iodophenyl)amino)benzofuran-7-y1)-1-(2,3-
dihydroxypropyl)cyclopropane-l-sulfonamide
OH
SO2 NH
F
H
N
\O 0 0
F I
F
[00141] For large scale synthesis, a suspension of 4,5-difluoro-3-(2-
fluoro-4-
iodopheny1)-1H-benzofuro[6,7-d]imidazol-2(3H)-one (7 g; 16.275mmol), (R)-1-
(2,3-
bis(benzyloxy)propy1)-cyclopropane-l-sulfonyl chloride (9.641 g), TEA (4.94 g)
and a
catalytic amount of DMAP (0.596 g) in DCM (75 mL) was stirred at room
temperature
for approximately 24 to 96 hours until HPLC analysis showed the reaction had
gone to
completion. The solution was then added to 1N HC1 (60 mL) and stirred for 15
minutes
and then separated. The DCM layer was dried over MgSO4 and concentrated to
dryness.
The resultant (R)-1-(1-(2,3-bis(benzyloxy)propyl)cyclopropoxy)-4,5-difluoro-3-
(2-fluoro-
4-iodopheny1)-1H-benzofuro[6,7-d]imidazol-2(3H)-one compound with sulfur
monoxide
(1:1) was used in the following step without further purification.
[00142] Potassium trimethyl silanolate (165.5g, 1.29mo1) was added to a
solution of (R)-1-(1-(2,3-bis(benzyloxy)propyl)cyclopropoxy)-4,5-difluoro-3-(2-
fluoro-4-
iodopheny1)-1H-benzofuro[6,7-d]imidazol-2(3H)-one compound with sulfur
monoxide
(1:1) (251.2 g, 318.5mmol) in THF (2.5L) at 0 C. The solution was warmed up to
room
temperature and stirred for 1-2 hours until HPLC analysis showed the reaction
had gone
to completion. Water (1.5L) was added to the solution and the separated THF
layer was
39
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concentrated to dryness. MTBE (2L) was added to dissolve the oil and then
washed with
2N aqueous NaOH solution (1L) followed by washing with water (1.5L). Active
charcoal
(50g) was added into the MTBE solution for decolorization purpose and after
the
decolorization, the solution was concentrated to ¨400mL. Heptane (1.2L) was
added and
the suspension was seeded with a pure (R)-1-(2,3-bis(benzyloxy)propy1)-N-(4,5-
difluoro-
64(2-fluoro-4-iodophenyl)amino)benzofuran-7-y0cyclopropane-1-sulfonamide
crystal.
The suspension was stirred at room temperature overnight and filtered, the
white solid
was dried to give 165g of product with a 96% purity (68% overall yield).
[00143] To a solution of (R)-1-(2,3-bis(benzyloxy)propy1)-N-(4,5-
difluoro-6-
((2-fluoro-4-iodophenyl)amino)benzofuran-7-yl)cyclopropane-l-sulfonamide
(140g,
183.6mmol) in DCM (700 mL) was added 1M BC13 solution in DCM (1.8L, 1.84mo1)
at -
65--70 C. The mixture was stirred at -65 ¨ -70 C for 30 minutes until HPLC
analysis
showed the reaction had gone to completion. The cold solution was added to 1N
HC1
solution (1.8 L) at below 15 C, stirred for 15mins and separated. The DCM
layer was
washed with water (2L), the brine (1.5L). DCM layer was concentrated to ¨250mL
and
filtered to give white solid. The solid was dissolved in isobutyl acetate
(240mL) and
heptane (500mL) was added. The suspension was stirred overnight, filtered and
dried to
give 70g of (R)-N-(4,5-difluoro-642-fluoro-4-iodophenyl)amino)benzofuran-7-y1)-
1-
f2,3-dihydroxypropyl)cyclopropane-l-sulfonamide with 98.4% purity (65.5%
yield,
ee>99%).
[00144] Comparing the retention time under the same HPLC conditions (80%
hexane/10% IPA/10% Me0H), the individually synthesized R-enantiomer has a
retention time of 10.43 minutes, corresponding to the position of Example 1B.
PHARMACOLOGICAL DATA
[00145] The inhibitory properties of compounds of present invention may
be
demonstrated using any one of the following test procedures:
[00146] A BRAF-MEK-ERK cascade assay is used to evaluate the effects of
these compounds as inhibitors of the MAP kinase pathway. An enzymatic cascade
assay
is set up using recombinant human activated BRAF (V599E) kinase (Cat No. 14-
557),
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human full length MEK1 kinase (Cat No. 14-706) and human full length active
MAP
Kinase 2/ERK2 (Cat No. 14-536) enzymes procured from Upstate. TR-FRET (Time
resolved fluorescence resonance energy transfer) detection technology is used
for the read
out. The assay buffer solution contains 50 mM Tris pH 7.5, 10 mM MgC12 , 1 mM
DTT,
0.01 % Tween 20, 0.1 nM activated BRAF, 2 nM inactive MEK1,10 nM inactive
ERK2,
100 iiiM ATP and 500 nM long chain biotin-peptide substrate (LCB-
FFKNIVTPRTPPP)
in a 384 well format. The kinase reaction is stopped after 90 minutes with 10
mM EDTA
and Lance detection mix (2 nM Eu-labeled phospho-serine/threonine antibody
(Cat.
No.AD0176-Perkin Elmer), 20 nM SA-APC (Cat No. CR130-100-Perkin Elmer) is
added. The TR-FRET signal (Excitation at 340 nm, Emission at 615 nm and 665
nm) is
read with 50 iLis delay time on a Victor3 V fluorimeter. The data is
calculated using the
ratio of readings at 665nm to 615 nm. The final concentration of DMSO is 2.5 %
in the
assay. Compounds are screened at 10 iiiM concentration with pre-incubation of
the
enzymes in the presence of test compound for 45 minutes.
[00147] Each individual IC50 is determined using a 10 point dose
response
curve generated by GraphPad Prism software Version 4 (San Diego, California,
USA)
using non linear regression curve fit for sigmoidal dose response (variable
slope).
[00148] In this cascade assay, the IC5Os of Examples 1, lA and 1B were
2.1+0.58 nM, 2+0.25 nM and 1.6+0.1 nM respectively (mean + SEM, n=3).
[00149] An in-vitro MAP kinase assay is set up using activated MAP
kinase
2/ERK2 (Cat. No.14-550) obtained from Upstate. TR-FRET detection technology is
used
for the read out.
[00150] The assay buffer solution contains 50 mM Tris pH 7.5, 10 mM
MgC12 ,
1 mM DTT, 0.01 % Tween 20, 1 nM activated ERK2, 100 iiiM ATP and 500 nM long
chain biotin-peptide substrate (LCB- FFKNIVTPRTPPP) in a 384 well format. The
kinase reaction is stopped after 90 minutes with 10 mM EDTA and Lance
detection mix
(2 nM Eu-labeled phospho-serine/threonine antibody (Cat.No. AD0176-Perkin
Elmer), 20
nM SA-APC (Cat. No. CR130-100-Perkin Elmer) is added. The TR-FRET signal
(excitation at 340 nm, emission at 615 nm and 665 nm) is read with 50 iLis
delay time on
Victor3 V fluorimeter. The data is calculated using the ratio of readings at
665nm to 615
nm. The final concentration of DMSO is 2.5 % in the assay. Compounds are
screened at
41
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M concentration with pre-incubation of the enzymes in the presence of test
compound for 45 minutes.
[00151] The radioactive filter binding assay is standardized using
recombinant
human activated BRAF (V599E) kinase (Cat No. 14-557) and kinase dead MEK1
(K97R)
( Cat No. 14-737) procured from Upstate. The incorporation of 32P into MEK1
(K97R)
by BRAF (V599E) is measured with final assay buffer conditions of 50 mM Tris
pH 7.5,
10 mM MgC12 , 1 mM DTT, 100 mM sucrose, 100 M sodium orthovanadate,5 M ATP
and 2 Ci [-y 32P] ATP and 500 mg MEK1 Kinase dead substrate. The enzymatic
reaction is stopped after 120 minutes with 8N HC1 (hydrochloric acid) and 1 mM
ATP.
The solution is spotted on P81 filter paper and washed 4 times with 0.75 %
orthophosphoric acid and lastly with acetone. The dried P81 filter papers are
read in a
Micro-beta Trilux scintillation counter. The final concentration of DMSO is 1
% in the
assay. Compounds are screened at 10 M concentration with pre-incubation of
the
enzymes in the presence of test compound for 45 minutes.
[00152] These assays described above are fully detailed in Han, Shulin,
et. al.,
Bioorganic & Medicinal Chemistry Letters (2005) 15, 5467-5473, and in Yeh, et.
al., Clin
Cancer Res (2007) 13 (5), 1576-1583.
[00153] The cell viability assay in A375 cells is set up in a 96-well
plate format
using XTT. XTT is a yellow tetrazolium salt that is cleaved to an orange
formazan dye
by the mitochondria of metabolically active cells. The procedure allows for
rapid
determination in a microtitre plate, to give reproducible and sensitive
results.
[00154] A375 cells are grown in DMEM media containing 10% FBS and 1mM
sodium pyruvate. Cells are trypsinized and seeded at 1000 cells/well. After
allowing the
cells to adhere overnight, compound is added to the wells at the following
final
concentrations: 10, 3, 1, 0.3, 0.1, 0.03, 0.01, 0.001, and 0.0001 M. The
assay is set up in
triplicates for each concentration. DMSO concentrations are kept at 0.5%
/well. Three
days after compound addition, the XTT assay is performed. Wells are washed
once with
PBS. 100 L of DMEM media without phenol red or FBS is added to each well. A
working solution of XTT containing lmg/m1 XTT and 100 L of PMS (stock
concentration 0.383 mg/ml) per 5m1 is prepared. 50 L of the working solution
of XTT
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is added to each well. Absorbance of the plate is read at 465nm using a
Spectramax 190
(Molecular Devices). The absorbance from wells with media and XTT alone, but
without
cells is considered the blank and subtracted from readings from all wells.
[00155] The cell viability assay is further described in Scudiero, et.
al., Cancer
Research (1988) 48, 4827-4833; Weislow, et. al., J. Natl. Cancer Institute,
(1989) 81,
577-586; and Roehm, et. al., J. Immunol.Methods [1991]142:257-265.
[00156] Percentage viability is calculated considering the blank
subtracted
value from wells treated with DMSO alone as 100% viable. GI50 values are
calculated
using Graphpad Prism, using non-linear regression curve fit for sigmoidal dose
response
(variable slope). Compounds of the invention were evaluated in this cell
viability assay.
Example lA has a GI50 of 13.4 nM and Example 1B has a GI50 of 29.6 nM, while
Example 1 has a GI50 of 17.6 nM.
A375 P-Erk (In-Cell-Western):
[00157] Human melanoma A375 cells were seeded at 50,000 cells per well
in
100 1 growth medium in Costar 96 well black clear bottom plates and placed at
37
C/5% CO2 over night. Test compounds were diluted in DMSO to generate a
concentration
curve. A 5 mM stock was used for the highest concentration at 500 times; to
yield a final
concentration of 10 M with 3-fold dilutions down to 0.0001 M. 1 1 of diluted
compound was added to 500 1 cell culture media and mixed well. Media was
removed
from cells and 200 1 of media containing compound was added. Cells were
treated for 3
hrs with compound at 37 C, 5% CO2.
[00158] After the compound incubation, cells were washed once with PBS
(Mg++,Ca++) and fixed in 4% paraformaldehyde/PBS for 1 hr at room temperature.
Following fixation, cells were washed three times with PBS/0.1% TritonX-
100(PBST),
and then blocked with 5% skimmed milk/PBST, for 1-2 hr. 50 L per well primary
antibody was added (rabbit-anti-phospho-ERK1/2) at 1:500 in 5% skimmed
milk/PBST
and incubated overnight at 4 C. Cells were washed four times with 100 1DELFIA
wash
buffer and 50 L per well secondary antibody was added (DELFIA-EU-N1-labeled
anti-
rabbit antibody) at 1:3000 in DELFIA assay buffer and incubated for 2 hr at
room
temperature in the dark (covered). Cells were washed 4X with 100 1DELFIA wash
buffer. 50 L per well Wallac-DELFIA enhancement solution was added. Plates
were
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shaken at room temperature for 20 mm and then read on the Perkin Elmer
Victor3v reader
on the Europium setting (emission/excitation of 615/340 nm).
[00159] EC50 values were calculated using DMSO diluent values as 0%
inhibition and counts of the highest tested concentration of the reference
inhibitor as
100% inhibition. All the concentrations along with DMSO were done in
triplicates. The
EC50 of Example lA is 6.4 nM compared with 15.6nM for Example 1B and 12.2nM
for
Example 1.
Pharmacokinetics in athymic nude rats
[00160] For pharmacokinetics studies, the following parameters were
calculated by non-compartmental regression analysis using Winnonlin 5.0
software
(Pharsight, Mountain View, CA, USA): half life in plasma (tv2term); plasma
clearance
(CL); plasma maximum concentration (Cmax); plasma area-under-the-concentration-
time-curve (AUC); and percent oral bioavailability (F%).
[00161] Pharmacokinetics parameters in rat for some compounds of the
invention are given in the table below:
i.v. (0.5 mg/kg) p.o. (3 mg/kg)
Compound AUC Cl T1/2 Vd Cmax AUC %F
iug.hr/mL mL/hr/kg hr mL/kg iug/mL
iug.hr/mL
Example-1 70+9 7.6+0.9 15.6+1.7 170+6 9.8+0.7 365+45 87
Example- 126+3 4.0+0.1 19.9+0.8 110+6 25.6+0.7 994+30 132
lA
Example- 100+14 5.2+0.7 17.1+1.8 125+6 25.2+2.7 752+84 126
1B
[00162] Examples lA and 1B show improved PK-parameters compared to
Example 1 (one-way analysis of variance).
A375 B-RafV600E human melanoma model in rat ¨ PK-PD experiment:
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[00163] A375 cells were thawed using a 37 C water bath. Cells were
transferred to a tube containing 10mls of warm DMEM medium. The tube was
centrifuged for 5 minutes at 1200 rpm and the supernatant was discarded. The
cell pellet
was re-suspended and transferred to a 75 cm2 tissue culture flask containing
15mls of
medium and cultured at 37 C in a 5% CO2 incubator.
[00164] On the day of the cells implantation, cells were harvested
(about 85%
confluent), and re-suspended in cold medium containing 4 mg/ml of matrigel.
This cell
suspension was injected, subcutaneously, to athymic-nude pre-irradiated (500
rads) rats.
Ten million cells (injection volume, 200 L) were injected, subcutaneously,
into the right
flank region of the rats 24 hours after irradiation. Tumor bearing rats were
randomized
when the tumor volume reached approximately 500 mm3 after about 15 to 20 days.
Three
rats were used for each time point.
[00165] Rats were treated orally with a single dose of 10, 30 and 60
mg/kg p.o.
of Example lA and 30 mg/kg of Example 1B. Plasma and tumor samples were taken
at
4, 12, 24 and 36 hours, post dosing. The mRNA expression levels of two direct
target
genes of MEK substrate P-Erk (DUSP6 and SPRY4) and an indirect target (BMF)
can be
measured. Upon treatment with MEK inhibitors, these genes have been shown to
be
regulated in a dose-dependent manner in tumor cell lines grown in vitro and in
vivo. The
tumor samples were pulverized, extracted and studied for expression of the
transcription
factor DUSP6 using a real time quantitative PCR.
[00166] Surprisingly, a two-way analysis of variance showed that Example-
1A
was significantly more effective than Example-1B at lowering levels of DUSP-6
at 12h
and 24h (p=0.003 and p=0.006, respectively).
