U.S. patent application number 13/621396 was filed with the patent office on 2013-01-17 for aminopyrazole triazolothiadiazole inhibitors of c-met protein kinase.
This patent application is currently assigned to Vertex Pharmaceuticals Incorporated. The applicant listed for this patent is David Lauffer, Pan Li, Kira McGinty. Invention is credited to David Lauffer, Pan Li, Kira McGinty.
Application Number | 20130018072 13/621396 |
Document ID | / |
Family ID | 42370912 |
Filed Date | 2013-01-17 |
United States Patent
Application |
20130018072 |
Kind Code |
A1 |
Lauffer; David ; et
al. |
January 17, 2013 |
AMINOPYRAZOLE TRIAZOLOTHIADIAZOLE INHIBITORS OF C-MET PROTEIN
KINASE
Abstract
The present invention relates to compounds of formula I, which
is useful in the inhibition of c-Met protein kinase. The invention
also provides pharmaceutically acceptable compositions comprising
compounds of formula I and methods of using the compositions in the
treatment of proliferative disorders. ##STR00001##
Inventors: |
Lauffer; David; (Stow,
MA) ; Li; Pan; (Lexington, MA) ; McGinty;
Kira; (Schenectady, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Lauffer; David
Li; Pan
McGinty; Kira |
Stow
Lexington
Schenectady |
MA
MA
NY |
US
US
US |
|
|
Assignee: |
Vertex Pharmaceuticals
Incorporated
Cambridge
MA
|
Family ID: |
42370912 |
Appl. No.: |
13/621396 |
Filed: |
September 17, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
12788608 |
May 27, 2010 |
8269012 |
|
|
13621396 |
|
|
|
|
61181786 |
May 28, 2009 |
|
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Current U.S.
Class: |
514/314 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 43/00 20180101; A61P 9/10 20180101; A61P 35/00 20180101; A61P
35/04 20180101; C07D 513/04 20130101 |
Class at
Publication: |
514/314 |
International
Class: |
A61K 31/4709 20060101
A61K031/4709; A61P 35/00 20060101 A61P035/00; A61P 35/04 20060101
A61P035/04 |
Claims
1-13. (canceled)
14. A method of treating or lessening the severity of a metastatic
cancer in a patient comprising administering the following
compound: ##STR00014## or a pharmaceutical composition comprising
said compound, in an amount sufficient to treat or lessen the
severity of said proliferative disorder in said patient.
14. A method of treating or lessening the severity of a
proliferative disorder selected from glioblastoma; a gastric
carcinoma; or a cancer selected from colon, breast, prostate,
brain, liver, pancreatic or lung cancer in a patient comprising
administering the following compound: ##STR00015## or a
pharmaceutical composition comprising said compound, in an amount
sufficient to treat or lessen the severity of said proliferative
disorder in said patient.
15. The method according to claim 14, wherein said disorder is
hepatocellular carcinoma.
Description
[0001] This present application claims the benefit, under 35 U.S.C.
.sctn.119, to U.S. Provisional Application No. 61/181,786, filed
May 28, 2009 the entire disclosure of which is incorporated herein
by reference.
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to selective inhibitors of
c-Met. The invention also provides pharmaceutically acceptable
compositions comprising a c-Met inhibitor and methods of using the
compositions in the treatment of various proliferative
disorders.
BACKGROUND OF THE INVENTION
[0003] Hepatocyte growth factor (HGF), also known as scatter
factor, is a multi-functional growth factor that enhances
transformation and tumor development by inducing mitogenesis and
cell motility. Further, HGF promotes metastasis by stimulating cell
motility and invasion through various signaling pathways. In order
to produce cellular effects, HGF must bind to its receptor, c-Met,
a receptor tyrosine kinase. c-Met, a widely expressed heterodimeric
protein comprising of a 50 kilodalton (kDa) .alpha.-subunit and a
145 kDa alpha-subunit (Maggiora et al., J. Cell Physiol.,
173:183-186, 1997), is overexpressed in a significant percentage of
human cancers and is amplified during the transition between
primary tumors and metastasis. The various cancers in which c-Met
overexpression is implicated include, but are not limited to,
gastric adenocarcinoma, renal cancer, small cell lung carcinoma,
colorectal cancer, prostate cancer, brain cancer, liver cancer,
pancreatic cancer, and breast cancer. c-Met is also implicated in
atherosclerosis and lung fibrosis.
[0004] Accordingly, there is a great need to develop compounds
useful as inhibitors of c-Met protein kinase receptor. In
particular, preferred compounds should have high affinity to the
c-Met receptor and show functional activity as antagonists, while
showing little affinity for other kinase receptors or for targets
known to be associated with adverse effects.
SUMMARY OF THE INVENTION
[0005] It has been found that
3-(quinolin-6-yl)methyl-N-(1H-pyrrol-3-yl)-[1,2,4]triazolo[3,4-b][1,3,4]t-
hiadiazol-6-amines are effective in the inhibition of c-Met.
[0006] Accordingly, the invention features a compound having the
formula:
##STR00002##
or a pharmaceutically acceptable salt thereof, wherein each or
R.sup.1, R.sup.2, R.sup.3, R.sup.4, and R.sup.5 is as defined
elsewhere herein.
[0007] The invention also provides pharmaceutical compositions that
include a compound of formula I and a pharmaceutically acceptable
carrier, adjuvant, or vehicle. In addition, the invention provides
methods of treating or lessening the severity of a proliferative
disease, condition, or disorder in a patient that includes the step
of administering to the patient a therapeutically effective dose of
a compound of formula I, or a pharmaceutical composition
thereof.
DETAILED DESCRIPTION OF THE INVENTION
Definitions and General Terminology
[0008] As used herein, the following definitions shall apply unless
otherwise indicated. For purposes of this invention, the chemical
elements are identified in accordance with the Periodic Table of
the Elements, CAS version, and the Handbook of Chemistry and
Physics, 75.sup.th Ed. 1994. Additionally, general principles of
organic chemistry are described in "Organic Chemistry," Thomas
Sorrell, University Science Books, Sausalito: 1999, and "March's
Advanced Organic Chemistry," 5.sup.th Ed., Smith, M. B. and March,
J., eds. John Wiley & Sons, New York: 2001, the entire contents
of which are hereby incorporated by reference.
Description of the Compound of the Invention
[0009] In a first aspect, the invention features the following
compounds of formula I:
##STR00003##
or a pharmaceutically acceptable salt thereof, wherein [0010]
R.sup.1 is C.sub.1-3 aliphatic; [0011] R.sup.2 is hydrogen, fluoro,
or methyl; [0012] R.sup.3 is hydrogen, fluoro, or methyl; [0013]
each R.sup.4 is, independently, hydrogen or fluoro; and [0014]
R.sup.5 is hydrogen, chloro, cyclopropyl, or C.sub.1-4 aliphatic,
optionally substituted with 1-3 fluorine atoms.
[0015] In one embodiment R.sup.2 is methyl and R.sup.3 is hydrogen.
In another embodiment, R.sup.2 is hydrogen and R.sup.3 is
methyl.
[0016] In another embodiment, each of R.sup.2 and R.sup.3 is
fluoro.
[0017] In another embodiment of the compounds of the invention,
R.sup.4 is hydrogen.
[0018] In another embodiment of the compounds of the invention,
R.sup.1 is methyl and R.sup.5 is hydrogen.
[0019] In a further embodiment, R.sup.1 is methyl, each of R.sup.2
and R.sup.3 is fluorine, and each of R.sup.4 and R.sup.5 is
hydrogen.
[0020] In another embodiment, the compound is a hydrochloride
salt.
[0021] Compounds of formula I include the following:
##STR00004## ##STR00005##
[0022] In another aspect, the invention features a pharmaceutical
composition comprising a compound of formula I, or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier, adjuvant or vehicle. In one embodiment, the
composition includes an additional chemotherapeutic or
anti-proliferative agent, an anti-inflammatory agent, an agent for
treating atherosclerosis, or an agent for treating lung
fibrosis.
[0023] In another aspect, the invention features a method of
treating or lessening the severity of a proliferative disorder in a
patient comprising administering a compound of formula I in an
amount sufficient to treat or lessen the severity of a
proliferative disorder in said patient. In one embodiment, the
proliferative disorder is metastatic cancer. In another embodiment,
the proliferative disorder is a glioblastoma; hepatocellular
carcinoma, a gastric carcinoma; or a cancer selected from colon,
breast, prostate, brain, liver, pancreatic or lung cancer.
[0024] In another embodiment, the proliferative disorder is a
metastatic cancer.
Compositions, Formulations, and Administration of Compounds of the
Invention
[0025] In another aspect, the invention provides a composition
comprising a compound of formula I or a pharmaceutically acceptable
derivative thereof and a pharmaceutically acceptable carrier,
adjuvant, or vehicle. In one embodiment, the amount of compound in
a composition of this invention is such that is effective to
measurably inhibit c-Met in a biological sample or in a patient.
Preferably the composition of this invention is formulated for
administration to a patient in need of such composition. Most
preferably, the composition of this invention is formulated for
oral administration to a patient.
[0026] The term "patient", as used herein, means an animal,
preferably a mammal, and most preferably a human.
[0027] It will also be appreciated that the compounds of formula I
can exist in free form for treatment, or where appropriate, as a
pharmaceutically acceptable derivative thereof. According to the
present invention, a pharmaceutically acceptable derivative
includes, but is not limited to, pharmaceutically acceptable
prodrugs, salts, esters, salts of such esters, or any other adduct
or derivative which upon administration to a patient in need is
capable of providing, directly or indirectly, a compound of formula
I as otherwise described herein, or a metabolite or residue
thereof.
[0028] As used herein, the term "pharmaceutically acceptable salt"
refers to those salts which are, within the scope of sound medical
judgment, suitable for use in contact with the tissues of humans
and lower animals without undue toxicity, irritation, allergic
response and the like.
[0029] Pharmaceutically acceptable salts are well known in the art.
For example, S. M. Berge et al., describe pharmaceutically
acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19,
1977, which is incorporated herein by reference. Pharmaceutically
acceptable salts of compounds of formula I include those derived
from suitable inorganic and organic acids and bases. Examples of
pharmaceutically acceptable, nontoxic acid addition salts are salts
of an amino group formed with inorganic acids such as hydrochloric
acid, hydrobromic acid, phosphoric acid, sulfuric acid and
perchloric acid or with organic acids such as acetic acid, oxalic
acid, maleic acid, tartaric acid, citric acid, succinic acid or
malonic acid or by using other methods used in the art such as ion
exchange. Other pharmaceutically acceptable salts include adipate,
alginate, ascorbate, aspartate, benzenesulfonate, benzoate,
bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptonate,
glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate,
hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate,
laurate, lauryl sulfate, malate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oleate, oxalate, palmitate, pamoate, pectinate, persulfate,
3-phenylpropionate, phosphate, picrate, pivalate, propionate,
stearate, succinate, sulfate, tartrate, thiocyanate,
p-toluenesulfonate, undecanoate, valerate salts, and the like.
Salts derived from appropriate bases include alkali metal, alkaline
earth metal, ammonium and N.sup.+(C .sub.1-4 alkyl).sub.4
salts.
[0030] As described above, the pharmaceutically acceptable
compositions of the present invention additionally comprise a
pharmaceutically acceptable carrier, adjuvant, or vehicle, which,
as used herein, includes any and all solvents, diluents, or other
liquid vehicle, dispersion or suspension aids, surface active
agents, isotonic agents, thickening or emulsifying agents,
preservatives, solid binders, lubricants and the like, as suited to
the particular dosage form desired. In Remington: The Science and
Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy,
Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia
of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,
1988-1999, Marcel Dekker, New York, the contents of each of which
is incorporated by reference herein, are disclosed various carriers
used in formulating pharmaceutically acceptable compositions and
known techniques for the preparation thereof. Except insofar as any
conventional carrier medium is incompatible with a compound of
formula I, such as by producing any undesirable biological effect
or otherwise interacting in a deleterious manner with any other
component(s) of the pharmaceutically acceptable composition, its
use is contemplated to be within the scope of this invention.
[0031] Some examples of materials which can serve as
pharmaceutically acceptable carriers include, but are not limited
to, ion exchangers, alumina, aluminum stearate, lecithin, serum
proteins, such as human serum albumin, buffer substances such as
phosphates, glycine, sorbic acid, or potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes, such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, wool fat, sugars such
as lactose, glucose and sucrose; starches such as corn starch and
potato starch; cellulose and its derivatives such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients such as cocoa
butter and suppository waxes; oils such as peanut oil, cottonseed
oil; safflower oil; sesame oil; olive oil; corn oil and soybean
oil; glycols; such a propylene glycol or polyethylene glycol;
esters such as ethyl oleate and ethyl laurate; agar; buffering
agents such as magnesium hydroxide and aluminum hydroxide; alginic
acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol, and phosphate buffer solutions, as well as other non-toxic
compatible lubricants such as sodium lauryl sulfate and magnesium
stearate, as well as coloring agents, releasing agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives
and antioxidants can also be present in the composition, according
to the judgment of the formulator.
[0032] The compositions of the present invention may be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intraocular,
intrahepatic, intralesional and intracranial injection or infusion
techniques. Preferably, the compositions are administered orally,
intraperitoneally or intravenously. Sterile injectable forms of the
compositions of this invention may be aqueous or oleaginous
suspension. These suspensions may be formulated according to
techniques known in the art using suitable dispersing or wetting
agents and suspending agents. The sterile injectable preparation
may also be a sterile injectable solution or suspension in a
non-toxic parenterally acceptable diluent or solvent, for example
as a solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium.
[0033] For this purpose, any bland fixed oil may be employed
including synthetic mono- or diglycerides. Fatty acids, such as
oleic acid and its glyceride derivatives are useful in the
preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant, such as carboxymethyl cellulose or similar dispersing
agents that are commonly used in the formulation of
pharmaceutically acceptable dosage forms including emulsions and
suspensions. Other commonly used surfactants, such as Tweens, Spans
and other emulsifying agents or bioavailability enhancers which are
commonly used in the manufacture of pharmaceutically acceptable
solid, liquid, or other dosage forms may also be used for the
purposes of formulation.
[0034] The pharmaceutically acceptable compositions of this
invention may be orally administered in any orally acceptable
dosage form including, but not limited to, capsules, tablets,
aqueous suspensions or solutions. In the case of tablets for oral
use, carriers commonly used include lactose and corn starch.
Lubricating agents, such as magnesium stearate, are also typically
added. For oral administration in a capsule form, useful diluents
include lactose and dried cornstarch. When aqueous suspensions are
required for oral use, the active ingredient is combined with
emulsifying and suspending agents. If desired, certain sweetening,
flavoring or coloring agents may also be added.
[0035] Alternatively, the pharmaceutically acceptable compositions
of this invention may be administered in the form of suppositories
for rectal administration. These can be prepared by mixing the
agent with a suitable non-irritating excipient that is solid at
room temperature but liquid at rectal temperature and therefore
will melt in the rectum to release the drug. Such materials include
cocoa butter, beeswax and polyethylene glycols.
[0036] The pharmaceutically acceptable compositions of this
invention may also be administered topically, especially when the
target of treatment includes areas or organs readily accessible by
topical application, including diseases of the eye, the skin, or
the lower intestinal tract. Suitable topical formulations are
readily prepared for each of these areas or organs.
[0037] Topical application for the lower intestinal tract can be
effected in a rectal suppository formulation (see above) or in a
suitable enema formulation. Topically-transdermal patches may also
be used.
[0038] For topical applications, the pharmaceutically acceptable
compositions may be formulated in a suitable ointment containing
the active component suspended or dissolved in one or more
carriers. Carriers for topical administration of compounds of
formula I include, but are not limited to, mineral oil, liquid
petrolatum, white petrolatum, propylene glycol, polyoxyethylene,
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutically acceptable compositions can be
formulated in a suitable lotion or cream containing the active
components suspended or dissolved in one or more pharmaceutically
acceptable carriers. Suitable carriers include, but are not limited
to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl
esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and
water.
[0039] For ophthalmic use, the pharmaceutically acceptable
compositions may be formulated, e.g., as micronized suspensions in
isotonic, pH adjusted sterile saline or other aqueous solution, or,
preferably, as solutions in isotonic, pH adjusted sterile saline or
other aqueous solution, either with or without a preservative such
as benzylalkonium chloride. Alternatively, for ophthalmic uses, the
pharmaceutically acceptable compositions may be formulated in an
ointment such as petrolatum. The pharmaceutically acceptable
compositions of this invention may also be administered by nasal
aerosol or inhalation. Such compositions are prepared according to
techniques well-known in the art of pharmaceutical formulation and
may be prepared as solutions in saline, employing benzyl alcohol or
other suitable preservatives, absorption promoters to enhance
bioavailability, fluorocarbons, and/or other conventional
solubilizing or dispersing agents.
[0040] Most preferably, the pharmaceutically acceptable
compositions of this invention are formulated for oral
administration.
[0041] Liquid dosage forms for oral administration include, but are
not limited to, pharmaceutically acceptable emulsions,
microemulsions, solutions, suspensions, syrups and elixirs. In
addition to the active compounds, the liquid dosage forms may
contain inert diluents commonly used in the art such as, for
example, water or other solvents, solubilizing agents and
emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, and perfuming agents.
[0042] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0043] The injectable formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0044] In order to prolong the effect of a compound of formula I,
it is often desirable to slow the absorption of this compound from
subcutaneous or intramuscular injection. This may be accomplished
by the use of a liquid suspension of crystalline or amorphous
material with poor water solubility. The rate of absorption of a
compound of formula I then depends upon its rate of dissolution
that, in turn, may depend upon crystal size and crystalline form.
Alternatively, dissolving or suspending a compound of formula I in
an oil vehicle accomplishes delayed absorption of a parenterally
administered compound form. Injectable depot forms are made by
forming microencapsule matrices of a compound of formula I in
biodegradable polymers such as polylactide-polyglycolide. Depending
upon the ratio of compound to polymer and the nature of the
particular polymer employed, the rate of compound release can be
controlled. Examples of other biodegradable polymers include
poly(orthoesters) and poly(anhydrides). Depot injectable
formulations are also prepared by entrapping a compound of formula
I in liposomes or microemulsions that are compatible with body
tissues.
[0045] Compositions for rectal or vaginal administration are
preferably suppositories which can be prepared by mixing a compound
of formula I with suitable non-irritating excipients or carriers
such as cocoa butter, polyethylene glycol or a suppository wax
which are solid at ambient temperature but liquid at body
temperature and therefore melt in the rectum or vaginal cavity and
release the active compound.
[0046] Solid dosage forms for oral administration include capsules,
tablets, pills, powders, and granules. In such solid dosage forms,
the active compound is mixed with at least one inert,
pharmaceutically acceptable excipient or carrier such as sodium
citrate or dicalcium phosphate and/or a) fillers or extenders such
as starches, lactose, sucrose, glucose, mannitol, and silicic acid,
b) binders such as, for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants
such as glycerol, d) disintegrating agents such as agar-agar,
calcium carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate, e) solution retarding agents such
as paraffin, f) absorption accelerators such as quaternary ammonium
compounds, g) wetting agents such as, for example, cetyl alcohol
and glycerol monostearate, h) absorbents such as kaolin and
bentonite clay, and i) lubricants such as talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof. In the case of capsules, tablets and
pills, the dosage form may also comprise buffering agents.
[0047] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like. The solid dosage forms of
tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings and other
coatings well known in the pharmaceutical formulating art. They may
optionally contain opacifying agents and can also be of a
composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract,
optionally, in a delayed manner. Examples of embedding compositions
that can be used include polymeric substances and waxes. Solid
compositions of a similar type may also be employed as fillers in
soft and hard-filled gelatin capsules using such excipients as
lactose or milk sugar as well as high molecular weight polethylene
glycols and the like.
[0048] The active compounds can also be in micro-encapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, dragees, capsules, pills, and granules can be prepared
with coatings and shells such as enteric coatings, release
controlling coatings and other coatings well known in the
pharmaceutical formulating art. In such solid dosage forms the
active compound may be admixed with at least one inert diluent such
as sucrose, lactose or starch. Such dosage forms may also comprise,
as is normal practice, additional substances other than inert
diluents, e.g., tableting lubricants and other tableting aids such
a magnesium stearate and microcrystalline cellulose. In the case of
capsules, tablets and pills, the dosage forms may also comprise
buffering agents. They may optionally contain opacifying agents and
can also be of a composition that they release the active
ingredient(s) only, or preferentially, in a certain part of the
intestinal tract, optionally, in a delayed manner. Examples of
embedding compositions that can be used include polymeric
substances and waxes.
[0049] Dosage forms for topical or transdermal administration of a
compound of formula I include ointments, pastes, creams, lotions,
gels, powders, solutions, sprays, inhalants or patches. The active
component is admixed under sterile conditions with a
pharmaceutically acceptable carrier and any needed preservatives or
buffers as may be required. Ophthalmic formulation, eardrops, and
eye drops are also contemplated as being within the scope of this
invention. Additionally, the present invention contemplates the use
of transdermal patches, which have the added advantage of providing
controlled delivery of a compound of formula I to the body. Such
dosage forms can be made by dissolving or dispensing a compound of
formula I in the proper medium. Absorption enhancers can also be
used to increase the flux of a compound of formula I across the
skin. The rate can be controlled by either providing a rate
controlling membrane or by dispersing a compound of formula I in a
polymer matrix or gel.
[0050] Compounds of formula I are preferably formulated in dosage
unit form for ease of administration and uniformity of dosage. The
expression "dosage unit form" as used herein refers to a physically
discrete unit of agent appropriate for the patient to be treated.
It will be understood, however, that the total daily usage of a
compound of formula I and compositions comprising a compound of
formula I will be decided by the attending physician within the
scope of sound medical judgment. The specific effective dose level
for any particular patient or organism will depend upon a variety
of factors including the disorder being treated and the severity of
the disorder; the activity of the specific compound employed; the
specific composition employed; the age, body weight, general
health, sex and diet of the patient; the time of administration,
route of administration, and rate of excretion of the specific
compound employed; the duration of the treatment; drugs used in
combination or coincidental with the specific compound employed,
and like factors well known in the medical arts.
[0051] The amount of a compound of formula I that may be combined
with the carrier materials to produce a composition in a single
dosage form will vary depending upon the host treated, the
particular mode of administration. Preferably, the compositions
should be formulated so that a dosage of from 0.01 to 100 mg/kg
body weight/day of the inhibitor can be administered to a patient
receiving these compositions. In one example, compositions are
formulated such that the dosage of a compound of formula I can be
from 3 to 30 mg/kg body weight/day. In another example,
compositions are formulated such that the dosage of a compound of
formula I can be from 5 to 60 mg/kg body weight/day.
[0052] Depending upon the particular condition, or disease, to be
treated or prevented, additional therapeutic agents, which are
normally administered to treat or prevent that condition, may also
be present in the compositions of this invention. As used herein,
additional therapeutic agents that are normally administered to
treat or prevent a particular disease, or condition, are known as
"appropriate for the disease, or condition, being treated."
Examples of additional therapeutic agents are provided infra.
[0053] The amount of additional therapeutic agent present in the
compositions of this invention will be no more than the amount that
would normally be administered in a composition comprising that
therapeutic agent as the only active agent. Preferably the amount
of additional therapeutic agent in the presently disclosed
compositions will range from about 50% to 100% of the amount
normally present in a composition comprising that agent as the only
therapeutically active agent.
Uses of Compounds of Formula I and Compositions Comprising
Compounds of Formula I
[0054] According to one embodiment, the invention relates to a
method of inhibiting c-Met protein kinase activity in a biological
sample comprising the step of contacting said biological sample
with a compound of formula I, or a composition comprising said
compound. The term "biological sample," as used herein, means a
sample outside a living organism and includes, without limitation,
cell cultures or extracts thereof biopsied material obtained from a
mammal or extracts thereof and blood, saliva, urine, feces, semen,
tears, or other body fluids or extracts thereof. Inhibition of
kinase activity in a biological sample is useful for a variety of
purposes known to one of skill in the art. Examples of such
purposes include, but are not limited to, biological specimen
storage and biological assays. In one embodiment, the method of
inhibiting kinase activity in a biological sample is limited to
non-therapeutic methods.
[0055] The term "c-Met" is synonymous with "c-MET," "cMet", "MET",
"Met" or other designations known to one skilled in the art.
[0056] According to another embodiment, the invention relates to a
method of inhibiting c-Met kinase activity in a patient comprising
the step of administering to said patient a compound of formula I,
or a composition comprising said compound.
[0057] The term "c-Met-mediated disease" or "c-Met-mediated
condition", as used herein, means any disease state or other
deleterious condition in which c-Met is known to play a role. The
terms "c-Met-mediated disease" or "c-Met-mediated condition" also
mean those diseases or conditions that are alleviated by treatment
with a c-Met inhibitor. Such conditions include, without
limitation, renal, gastric, colon, brain, breast, prostate, liver,
pancreatic, or lung cancer, glioblastoma, atherosclerosis, or lung
fibrosis.
[0058] In one aspect, the present invention features a method
treating a proliferative disorder in a patient comprising the step
of administering to the patient a therapeutically effective dose of
a compound of formula I or a composition comprising a compound of
formula I.
[0059] According to one embodiment, the proliferative disorder is
cancer, such as, for example, renal, gastric, colon, brain, breast,
liver, prostate, and lung cancer, or a glioblastoma.
[0060] In another embodiment, the present invention relates to a
method of treating or lessening the severity of hepatocellular
carcinoma in a patient in need thereof, comprising administering to
said patient a compound of formula I or composition thereof.
[0061] In another embodiment, the proliferative disorder is
polycythemia vera, essential thrombocythemia, chronic idiopathic
myelofibrosis, myeloid metaplasia with myelofibrosis, chronic
myeloid leukemia (CML), chronic myelomonocytic leukemia, chronic
eosinophilic leukemia, hypereosinophilic syndrome, systematic mast
cell disease, atypical CML, or juvenile myelomonocytic
leukemia.
[0062] In another embodiment, the proliferative disorder is
atherosclerosis or lung fibrosis.
[0063] Another aspect of the present invention relates to a method
of inhibiting tumor metastasis in a patient in need thereof,
comprising administering to said patient a compound of formula I or
a composition thereof.
[0064] Depending upon the particular condition, or disease, to be
treated, additional therapeutic agents that are normally
administered to treat that condition may also be present in the
compositions of this invention. As used herein, additional
therapeutic agents that are normally administered to treat a
particular disease, or condition, are known as "appropriate for the
disease, or condition, being treated".
[0065] In one embodiment, chemotherapeutic agents or other
anti-proliferative agents may be combined with a compound of
formula I to treat proliferative diseases and cancer. Examples of
known chemotherapeutic agents include, but are not limited to,
alkylating agents, such as, for example, cyclophosphamide,
lomustine, busulfan procarbazine, ifosfamide, altretamine,
melphalan, estramustine phosphate, hexamethylmelamine,
mechlorethamine, thiotepa, streptozocin, chlorambucil,
temozolomide, dacarbazine, semustine, or carmustine; platinum
agents, such as, for example, cisplatin, carboplatinum,
oxaliplatin, ZD-0473 (AnorMED), spiroplatinum, lobaplatin
(Aeterna), carboxyphthalatoplatinum, satraplatin (Johnson Matthey),
tetraplatin BBR-3464, (Hoffmann-La Roche), ormiplatin, SM-11355
(Sumitomo), iproplatin, or AP-5280 (Access); antimetabolites, such
as, for example, azacytidine, tomudex, gemcitabine, trimetrexate,
capecitabine, deoxycoformycin, 5-fluorouracil, fludarabine,
floxuridine, pentostatin, 2-chlorodeoxyadenosine, raltitrexed,
6-mercaptopurine, hydroxyurea, 6-thioguanine, decitabine
(SuperGen), cytarabin, clofarabine (Bioenvision), 2-fluorodeoxy
cytidine, irofulven (MGI Pharma), methotrexate, DMDC (Hoffmann-La
Roche), idatrexate, or ethynylcytidine (Taiho); topoisomerase
inhibitors, such as, for example, amsacrine, rubitecan (SuperGen),
epirubicin, exatecan mesylate (Daiichi), etoposide, quinamed
(ChemGenex), teniposide, mitoxantrone, gimatecan (Sigma-Tau),
irinotecan (CPT-11), diflomotecan (Beaufour-Ipsen),
7-ethyl-10-hydroxy-camptothecin, TAS-103 (Taiho), topotecan,
elsamitrucin (Spectrum), dexrazoxanet (TopoTarget), J-107088 (Merck
& Co), pixantrone (Novuspharma), BNP-1350 (BioNumerik),
rebeccamycin analogue (Exelixis), CKD-602 (Chong Kun Dang),
BBR-3576 (Novuspharma), or KW-2170 (Kyowa Hakko); antitumor
antibiotics, such as, for example, dactinomycin (actinomycin D),
amonafide, doxorubicin (adriamycin), azonafide, deoxyrubicin,
anthrapyrazole, valrubicin, oxantrazole, daunorubicin (daunomycin),
losoxantrone, epirubicin, bleomycin, sulfate (blenoxane),
therarubicin, bleomycinic acid, idarubicin, bleomycin A,
rubidazone, bleomycin B, plicamycin, mitomycin C, porfiromycin,
MEN-10755 (Menarini), cyanomorpholinodoxorubicin, GPX-100 (Gem
Pharmaceuticals), or mitoxantrone (novantrone), antimitotic agents,
such as, for example, paclitaxel, SB 408075 (GlaxoSmithKline),
docetaxel, E7010 (Abbott), colchicines, PG-TXL (Cell Therapeutics),
vinblastine, IDN 5109 (Bayer), vincristine A, 105972 (Abbott),
vinorelbine, A 204197 (Abbott), vindesine, LU 223651 (BASF),
dolastatin 10 (NCI), D 24851 (ASTAMedica), rhizoxin (Fujisawa),
ER-86526 (Eisai), mivobulin (Warner-Lambert), combretastatin A4
(BMS), cemadotin (BASF), isohomohalichondrin-B (PharmaMar), RPR
109881A (Aventis), ZD 6126 (AstraZeneca), TXD 258 (Aventis),
PEG-paclitaxel (Enzon,) epothilone B (Novartis), AZ10992 (Asahi), T
900607 (Tularik), IDN-5109 (Indena), T 138067 (Tularik), AVLB
(Prescient NeuroPharma), cryptophycin 52 (Eli Lilly), azaepothilone
B (BMS), vinflunine (Fabre), BNP-7787 (BioNumerik), auristatin PE
(Teikoku Hormone), CA-4 prodrug (OXiGENE), BMS 247550 (BMS),
dolastatin-10 (NIH), BMS 184476 (BMS), CA-4 (OXiGENE), BMS 188797
(BMS), or taxoprexin (Protarga); aromatase inhibitors, such as, for
example, aminoglutethimide, exemestane, letrozole, atamestane
(BioMedicines), anastrazole, YM-511 (Yamanouchi), or formestane;
thymidylate synthase inhibitors, such as, for example, pemetrexed
(Eli Lilly), nolatrexed (Eximias), ZD-9331 (BTG), or CoFactor.TM.
(BioKeys); DNA antagonists, such as, for example, trabectedin
(PharmaMar), mafosfamide (Baxter International), glufosfamide
(Baxter International), apaziquone (Spectrum Pharmaceuticals),
albumin +.sup.32P (Isotope Solutions), O6 benzyl guanine
(Paligent), thymectacin (NewBiotics), or edotreotide (Novartis);
farnesyltransferase inhibitors, such as, for example, arglabin
(NuOncology Labs), tipifarnib (Johnson & Johnson), lonafarnib
(Schering-Plough), perillyl alcohol (DOR BioPharma), or BAY-43-9006
(Bayer); Pump inhibitors, such as, for example, CBT-1 (CBA Pharma),
zosuquidar trihydrochloride (Eli Lilly), tariquidar (Xenova),
biricodar dicitrate (Vertex), or MS-209 (Schering AG); Histone
acetyltransferase inhibitors, such as, for example, tacedinaline
(Pfizer), pivaloyloxymethyl butyrate (Titan), SAHA (Aton Pharma),
depsipeptide (Fujisawa), or MS-275 (Schering AG); Metalloproteinase
inhibitors, such as, for example, Neovastat (Aeterna Laboratories),
CMT-3 (CollaGenex), marimastat (British Biotech), or BMS-275291
(Celltech); ribonucleoside reductase inhibitors, such as, for
example, gallium maltolate (Titan), tezacitabine (Aventis),
triapine (Vion), or didox (Molecules for Health); TNF alpha
agonists/antagonists, such as, for example, virulizin (Lorus
Therapeutics), revimid (Celgene), CDC-394 (Celgene), entanercept
(Immunex Corp.), infliximab (Centocor, Inc.), or adalimumab (Abbott
Laboratories); endothelin A receptor antagonists, such as, for
example, atrasentan (Abbott) YM-598 (Yamanouchi) or ZD-4054
(AstraZeneca); retinoic acid receptor agonists, such as, for
example, fenretinide (Johnson & Johnson) alitretinoin (Ligand)
or LGD-1550 (Ligand); immuno-modulators, such as, for example,
interferon dexosome therapy (Anosys), oncophage (Antigenics),
pentrix (Australian Cancer Technology), GMK (Progenics), ISF-154
(Tragen), adenocarcinoma vaccine (Biomira), cancer vaccine
(Intercell), CTP-37 (AVI BioPharma), norelin (Biostar), IRX-2
(Immuno-Rx), BLP-25 (Biomira), PEP-005 (Peplin Biotech), MGV
(Progenics), synchrovax vaccines (CTL Immuno), beta-alethine
(Dovetail), melanoma vaccine (CTL Immuno), CLL therapy (Vasogen),
or p21 RAS vaccine (GemVax); hormonal and antihormonal agents, such
as, for example, estrogens, prednisone, conjugated estrogens,
methylprednisolone, ethinyl estradiol, prednisolone,
chlortrianisen, aminoglutethimide, idenestrol, leuprolide,
hydroxyprogesterone caproate, goserelin, medroxyprogesterone,
leuporelin, testosterone, bicalutamide, testosterone propionate,
fluoxymesterone, flutamide, methyltestosterone, octreotide,
diethylstilbestrol, nilutamide, megestrol, mitotane, tamoxifen,
P-04 (Novogen), toremofine, 2-methoxyestradiol (EntreMed),
dexamethasone, or arzoxifene (Eli Lilly); photodynamic agents, such
as, for example, talaporfin (Light Sciences),
Pd-bacteriopheophorbide (Yeda), Theralux (Theratechnologies),
lutetium texaphyrin (Pharmacyclics), motexafin gadolinium
(Pharmacyclics), or hypericin; and tyrosine kinase inhibitors, such
as, for example, imatinib (Novartis), kahalide F (PharmaMar),
leflunomide (Sugen/Pharmacia), CEP-701 (Cephalon), ZD1839
(AstraZeneca), CEP-751 (Cephalon), erlotinib (Oncogene Science),
MLN518 (Millenium), canertinib (Pfizer), PKC412 (Novartis),
squalamine (Genaera), phenoxodiol, SU5416 (Pharmacia), trastuzumab
(Genentech), SU6668 (Pharmacia), C225 (ImClone), ZD4190
(AstraZeneca), rhu-Mab (Genentech), ZD6474 (AstraZeneca), MDX-H210
(Medarex), vatalanib (Novartis), 2C4 (Genentech), PKI166
(Novartis), MDX-447 (Medarex), GW2016 (GlaxoSmithKline), ABX-EGF
(Abgenix), EKB-509 (Wyeth), IMC-1C11 (ImClone), or EKB-569
(Wyeth).
[0066] In a further embodiment, the additional therapeutic agent is
not metabolized by more than 90% by Cytochrome P.sub.450 3A4
(CYP3A4).
[0067] Those additional agents may be administered separately from
a compound of formula I-containing composition, as part of a
multiple dosage regimen. Alternatively, those agents may be part of
a single dosage form, mixed together with a compound of formula I
in a single composition. If administered as part of a multiple
dosage regime, the two active agents may be submitted
simultaneously, sequentially or within a period of time from one
another normally within five hours from one another.
[0068] The amount of both, a compound of formula I and the
additional therapeutic agent (in those compositions which comprise
an additional therapeutic agent as described above)) that may be
combined with the carrier materials to produce a single dosage form
will vary depending upon the host treated and the particular mode
of administration. Preferably, the compositions of this invention
should be formulated so that a dosage of between 0.01-100 mg/kg
body weight/day of a compound of formula I can be administered. In
one example, compositions are formulated such that the dosage of a
compound of formula I can be from 3 to 30 mg/kg body weight/day. In
another example, compositions are formulated such that the dosage
of a compound of formula I can be from 5 to 60 mg/kg body
weight/day.
[0069] In those compositions that comprise an additional
therapeutic agent, that additional therapeutic agent and a compound
of formula I may act synergistically. Therefore, the amount of
additional therapeutic agent in such compositions will be less than
that required in a monotherapy utilizing only that therapeutic
agent. In such compositions a dosage of between 0.01-100 mg/kg body
weight/day of the additional therapeutic agent can be
administered.
[0070] The amount of additional therapeutic agent present in the
compositions of this invention will be no more than the amount that
would normally be administered in a composition comprising that
therapeutic agent as the only active agent. Preferably the amount
of additional therapeutic agent in the presently disclosed
compositions will range from about 50% to 100% of the amount
normally present in a composition comprising that agent as the only
therapeutically active agent.
[0071] Compounds of formula I, or pharmaceutical compositions
thereof, may also be incorporated into compositions for coating an
implantable medical device, such as prostheses, artificial valves,
vascular grafts, stents and catheters. Vascular stents, for
example, have been used to overcome restenosis (re-narrowing of the
vessel wall after injury). However, patients using stents or other
implantable devices risk clot formation or platelet activation.
These unwanted effects may be prevented or mitigated by pre-coating
the device with a pharmaceutically acceptable composition
comprising a kinase inhibitor. Suitable coatings and the general
preparation of coated implantable devices are described in U.S.
Pat. Nos. 6,099,562; 5,886,026; and 5,304,121. The coatings are
typically biocompatible polymeric materials such as a hydrogel
polymer, polymethyldisiloxane, polycaprolactone, polyethylene
glycol, polylactic acid, ethylene vinyl acetate, and mixtures
thereof. The coatings may optionally be further covered by a
suitable topcoat of fluorosilicone, polysaccarides, polyethylene
glycol, phospholipids or combinations thereof to impart controlled
release characteristics in the composition. Implantable devices
coated with a compound of formula I are another embodiment of the
present invention.
Preparation of Compounds of Formula I
[0072] In order that the invention described herein may be more
fully understood, the following examples are set forth. It should
be understood that these examples are for illustrative purposes
only and are not to be construed as limiting this invention in any
manner.
[0073] As used herein, other abbreviations, symbols and conventions
are consistent with those used in the contemporary scientific
literature. See, e.g., Janet S. Dodd, ed., The ACS Style Guide: A
Manual for Authors and Editors, 2nd Ed., Washington, D.C.: American
Chemical Society, 1997, herein incorporated in its entirety by
reference. The following definitions describe terms and
abbreviations used herein: [0074] Brine a saturated solution of
NaCl in water [0075] BSA bovine serum albumin [0076] DMSO
dimethylsulfoxide [0077] ESMS electrospray mass spectrometry [0078]
EtOAc ethyl acetate [0079] EtOH ethyl alcohol [0080] HPLC high
performance liquid chromatography [0081] LCMS liquid
chromatography-mass spectrometry [0082] Me methyl [0083] MeOH
methanol [0084] MTBE methyl t-butylether [0085] Ph phenyl [0086] RT
room temperature [0087] TCA trichloroacetic acid [0088] THF
tetrahydrofuran [0089] TFA trifluoacetic acid
EXAMPLE 1
Compounds of Formula II
[0090] Compounds 1001 and 1002 were purchased from Okeanos Tech,
Beijing, China (Catalog Nos. OK-J-05024 and OK-J-05025,
respectively).
##STR00006##
[0091] Compound 1004 was prepared as shown in Scheme 1.
Accordingly, as shown in step 1-i, to a suspension of NaH (60% in
mineral oil, 8.47 g, 212 mmol) in DMSO at 0.degree. C. (260 mL) was
slowly added diethyl 2-methylmalonate (Compound 1005, 29.5 g, 169.4
mmol). The mixture was stirred at 0.degree. C. for 2 hours and
3,4,5-trifluoronitrobenzene (25.0 g, 141.2 mmol) was added. The
resulting mixture was warmed to RT and stirred for 12 hours. The
reaction mixture was poured into saturated aq. NH.sub.4Cl solution
and the precipitate was collected by filtration. After washing with
water 3 times, the resulting diethyl
2-(2,6-difluoro-4-nitrophenyl)-2-methylmalonate (Compound 1006
[R.dbd.CH.sub.3], 44.5 g, 95% yield) was dried under reduced
pressure and used as is in the next reaction.
[0092] As shown in step 1-ii, to a solution of diethyl
2-(2,6-difluoro-4-nitro-phenyl)-2-methylmalonate (44.5 g, 135 mmol)
in MeOH was added Pd/C (10%, 4.0 g) under an atmosphere of
nitrogen. The atmosphere was replaced with H.sub.2 and the mixture
hydrogenated at 50 psi for 3 days. The atmosphere was replaced with
nitrogen, the mixture filtered through diatomaceous earth, and the
volatiles removed under reduced pressure. The resulting diethyl
2-(4-amino-2,6-difluorophenyl)-2-methylmalonate (Compound 1007
[R.dbd.CH.sub.3], 40.5 g, 99% yield) was dried under reduced
pressure and used as is in the next reaction.
[0093] As shown in step 1-iii, to a solution of diethyl
2-(4-amino-2,6-difluorophenyl)-2-methylmalonate (40.0 g, 132.8
mmol) in methanol (200 mL) was added 6M NaOH (110.7 mL, 664.0
mmol). The mixture was heated at 100.degree. C. for 4 hours, cooled
to 0.degree. C., and acidified with conc. HCl until a pH of 3 was
obtained. The mixture was warmed to RT and stirred for 3 hours. The
resulting precipitate was collected by filtration, washed with
water, and dried under high vacuum at 50.degree. C. for 20 hours to
provide 2-(4-amino-2,6-difluorophenyl)propanoic acid (Compound 1008
[R.dbd.CH.sub.3], 22 g, 84% yield): .sup.1H NMR (300.0 MHz, DMSO)
.delta. 12.25 (brs, 1H), 6.16 (d, J=10.8 Hz, 2H), 5.58 (s, 2H),
3.74 (q, J=7.2 Hz, 1H) and 1.28 (d, J=7.2 Hz, 3H) ppm.
[0094] As shown in step 1-iv, a mixture of
2-(4-amino-2,6-difluorophenyl)propanoic acid (19.0 g, 94.45 mmol),
glycerol (35.83 g, 28.41 mL, 389.1 mmol), nitrobenzene (7.209 g,
6.028 mL, 58.56 mmol) and concentrated sulfuric acid (30.57 g,
16.61 mL, 311.7 mmol) was heated gently. After cessation of the
initial vigorous reaction, the mixture was heated to 170.degree. C.
for 16 hours. After cooling, the volatiles were removed under
reduced pressure, the residue dissolved in MeOH (150 mL), 150 mL of
6N NaOH were added, and the mixture was heated at 110.degree. C.
for 3 hours. After cooling to RT, the mixture was acidified with
concentrated HCl to a pH of 3. The resulting dark precipitate was
collected by filtration and washed with water. The precipitate was
taken up in ethanol and thionyl chloride (11.24 g, 6.891 mL, 94.45
mmol) was carefully added dropwise. After addition was complete,
the mixture was heated at 50.degree. C. for 20 hours. After cooling
to RT, the volatiles were removed under reduced pressure and the
residue was dissolved in a mixture of sat'd NaHCO.sub.3 and DCM.
The layers were separated and the aqueous layer extracted with DCM.
The combined organics were dried over MgSO4, reduced in volume
under reduced pressure, and subjected to medium-pressure silica gel
chromatography (0% EtOAc/Hexanes to 30% in 36 minutes) to provide
methyl 2-(5,7-difluoroquinolin-6-yl)propanoate (14.0 g, 56% yield
for two steps). The methyl ester (5.0 g) was saponified by taking
it up in methanol (30 mL), treating the resulting solution with
NaOH (16.58 mL of 6 M, 99.50 mmol), and stirring at RT for 20
hours. After careful acidification with conc. HCl to a pH of 2, the
resulting precipitate was collected by filtration and dried under
high vacuum to provide 2-(5,7-difluoroquinolin-6-yl)propanoic acid,
which was used as is in subsequent reactions. Compound 1003 can be
prepared by the same procedure as used in the preparation of
Compound 1004 by replacing--diethyl 2-methylmalonate with diethyl
malonate.
##STR00007##
EXAMPLE 2
Preparation of Compounds of Formula III
[0095] Compounds of formula III, wherein R.sup.2 and R.sup.3 are
hydrogen or methyl, can be prepared as shown in Scheme 2.
Accordingly, as shown in step 2-i of Scheme 2, the appropriately
substituted quinoline acetic acid of formula II (248.5 mmol, 1.0
equivalent) and 1,3-diaminothiourea (273.4 mmol, 1.1 equivalents)
is suspended in a mixture of tetramethylene sulfone (sulfolane, 38
mL) and water (57 mL). Methane sulfonic acid (546.7 mmol, 2.2
equivalents) is added to the mixture, whereupon all solids
dissolve. The reaction mixture is slowly warmed to 90.degree. C.
and the reaction heated at 90.degree. C. for 40 hours. The reaction
mixture is cooled in an ice bath and water (75 mL) is added,
followed by the careful addition of saturated sodium bicarbonate
(500 mL) until a pH 8 is achieved. The resulting precipitate is
collected by vacuum filtration, washed with water, saturated sodium
bicarbonate, water, and methyl t-butyl ether, respectively. The
product is dried in a vacuum oven at 55.degree. C. to afford a
compound of formula III.
##STR00008##
EXAMPLE 3
Preparation of
5-(difluoro(quinolin-6-yl)methyl)-4-(iminotriphenylphosphorano)-4H-1,2,4--
triazole-3-thiol (Compound 1011)
[0096] Compounds of Formula III, wherein each of R.sup.2 and
R.sup.3 is fluoro and R.sup.4 is hydrogen, can be prepared as shown
in Scheme 3. Accordingly, as shown in step 3-i, to a mixture of
6-iodoquinoline (10.0 g, 39.21 mmol, purchased from Hangzhou
Trylead Chemical Technology Co., Ltd., China) and copper
(nanopowder) (9.964 g, 156.8 mmol) in DMSO (150 mL) was added ethyl
2-bromo-2,2-difluoro-acetate (10.35 g, 50.97 mmol). The mixture was
heated at 60.degree. C. for 6h, during which time the mixture
turned from red copper suspension into a dark red near-homogenous
solution. After cooling to room temperature, the mixture was
diluted with ethyl acetate (300 mL) and aq saturated NH.sub.4Cl
solution (450 mL). After stirring for 30 minutes, the organic layer
was separated, washed with water, washed with brine, and dried over
magnisium sulfate. Removal of the volatiles under reduced pressure
gave crude product as red liquid. Purification by medium pressure
silica gel chromatography (DCM/ethyl acetate: 100% to 30% in 25
min.) gave ethyl 2,2-difluoro-2-(quinolin-6-yl)acetate (Compound
1009, 51% yield): .sup.1H NMR (300.0 MHz, CDCl.sub.3) d 9.04-9.03
(m, 1H), 8.29-8.21 (m, 2H), 8.15 (s, 1H), 7.93 (dd, J=2.1, 8.9 Hz,
1H), 7.52 (q, J=4.2 Hz, 1H), 4.35 (q, J=7.1 Hz, 2H) and 1.34 (t,
J=7.1 Hz, 3H) ppm.
[0097] As shown in step 3-ii in Scheme 3, Compound 1009 (10.0 g,
39.80 mmol) was dissolved in ethanol (100 mL), hydrazine (7.65 g,
7.50 mL, 239 mmol) was added, and the reaction mixture was stirred
at room temperature for 10 minutes. After pouring the mixture into
2N HCl solution, the aqueous mixture was washed twice with DCM and
the pH was adjusted to 8 while bubbling nitrogen gas through the
solution. The resulting aqueous solution was exhaustively extracted
with DCM (10.times.) and the combined organics dried over MgSO4,
filtered, and the volatiles removed under reduced pressure to
provide 2,2-difluoro-2-(quinolin-6-yl)acetohydrazide as a yellow
solid (Compound 1010, 91% yield). This compound that was used
directly without further purification.
[0098] As shown in step 3-iii of Scheme 3, Compound 1010 (3.55 g,
14.97 mmol) in EtOH (71 mL) was treated with potassium hydroxide
(924 mg, 16.5 mmol) and the reaction mixture gently warmed to
achieve homogeneity. Carbon disulfide (1.38 g, 1.09 mL, 18.2 mmol)
was added and the mixture stirred at 90.degree. C. for 4 hours, at
which time intermediate compound
5-(difluoro(quinolin-6-yl)methyl)-1,3,4-oxadiazole-2-thiol, sodium
salt was formed. To the refluxing solution was added hydrazine
(4.80 g, 4.70 mL, 150 mmol), followed by addition of 3A molecular
sieves (3 g). After refluxing for 2 hours, the sieves were removed
by filtration and washed with EtOH. The combined organics were
cooled to 0.degree. C. in an ice bath and treated with conc. HCl
under an atmosphere of nitrogen until a pH of 6.5 was achieved. The
precipitate was removed by filtration and the filtrate refluxed for
4 hours, using a Dean-Stark trap to collect any excess water. The
volatiles were removed under reduced pressure, the residue taken up
to water, and the pH adjusted to 6.5. The resulting solid was
collected by filtration, washed with water, and dried to produce
5-(difluoro(quinolin-6-yl)methyl)-4-amino-4H-1,2,4-triazole-3-thi-
ol (Compound 1011, 61% yield): .sup.1H NMR (300.0 MHz, DMSO)
.delta. 14.28 (s, 1H), 9.03-9.02 (m, 1H), 8.56 (d, J=8.0 Hz, 1H),
8.31 (s, 1H), 8.16 (d, J=8.8 Hz, 1H), 7.90 (dd, J=1.9, 8.8 Hz, 1H),
7.65 (q, J=4.2 Hz, 1H) and 5.69 (s, 2H) ppm.
##STR00009##
EXAMPLE 4
Compounds of Formula IV
[0099] 4-Isothiocyanato-1-methyl-1H-pyrazole (Compound 1012),
4-isothiocyanato-1,3-dimethyl-1H-pyrazole (Compound 1013), and
1-ethyl-4-isothiocyanato-1H-pyrazole (Compound 1014) were prepared
from 1-methyl-1H-pyrazol-4-amine, 1,3-dimethyl-1H-pyrazol-4-amine
(from Matrix Chemical Co.), and 1-ethyl-1H-pyrazol-4-amine (from
Oakwood Products), respectively, by reacting the pyrazolamine with
thiophosgene at 0.degree. C. in the presence of pyridine.
##STR00010##
EXAMPLE 5
Preparation of Compounds of Formula I
[0100] Compounds of formula I can be prepared as shown in Scheme 5.
As shown in step 5-i of Scheme 5, a compound of formula III (453.3
mmol, 1.00 equivalent) and a compound of formula IV together in
pyridine are heated at 110.degree. C. for 15 hrs. After cooling to
room temperature, the reaction mixture is poured into 1N HCl
solution, the precipiate collected by filtration, washed with
water, and purified by medium pressure silica gel chromtagraphy.
When desirable, racemic mixtures of compounds can be separated into
their respective enantiomers by supercritical fluid chromatography
using a ChiralPak.RTM. AD-H column (20 mm.times.250 mm, 5 micron
column) or a ChiralCel.RTM. OJ-H column (20 mm.times.250 mm, 5
micron column), eluting with an appropriate MeOH (0.1%
DEA)/CO.sub.2 ratio at an appropriate flow rate.
##STR00011##
EXAMPLE 6
Alternative Preparation of Compounds of Formula I--Preparation of
3-(difluoro(quinolin-6-yl)methyl)-N-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]tri-
azolo[3,4-b][1,3,4]thiadiazol-6-amine (Compound 3)
[0101] Compounds of formula I can be also prepared as by reacting
4-(iminotriphenylphosphorano)-4H-1,2,4-triazole-3-thiols with
isocyanates. Accordingly, as shown in step 6-i of Scheme 6,
6-iodoquinoline (750 g, 2.94 mol) was loaded into a nitrogen-purged
22 L round bottom flask equipped with a mechanical stirrer,
temperature probe, temperature readout, nitrogen inlet line, and a
cooling bath. Anhydrous THF (5.25 L) was added and the resulting
solution cooled to -27.degree. C. using iPrOH/dry ice bath.
i-PrMgCl.LiCl (2.45 L, 1.3 M in THF, 1.1 eq) was added over 1 hour
17 minutes via an addition funnel, maintaining the temperature
between -26.degree. C. and -29.degree. C. The reaction mixture was
then stirred for 2.5 hours with the temperature maintained between
-20.degree. C. and -29.degree. C. The brown slurry was cooled to
-53.degree. C. over 25 min using an i-PrOH/dry ice bath and diethyl
oxalate (469 g, 0.44 L, 1.1 eq) was added over 1 hour 15 minutes
via an addition funnel, maintaining the temperature between
-51.degree. C. and -53.degree. C. The resulting dark solution was
allowed to warm up to RT overnight (.about.18 hours) to produce a
mustard-colored slurry. A solution of ammonium chloride (500 g,
9.35 mol, 3.18 eq) in water (4.5 L) was prepared and cooled to
10.degree. C. using an ice bath. The reaction mixture was
transferred into the ammonium chloride solution over 37 minutes via
a transfer line by pulling a slight vacuum on the 22 L flask
containing the stirring ammonium chloride solution. Once transfer
was completed, the ice bath was removed, EtOAc (3.75 L) was added,
and stirring was initiated. After about 15 min, stirring was
stopped and layers were allowed to separate. The aqueous phase
(pH=8) was extracted with EtOAc (3.75 L). The two organic layers
were combined and washed with NaCl solution (112 g in 2.5 L water).
The organic phase was concentrated under vacuum at 25.degree. C. to
provide an oil (763 g), which was purified by silica gel
chromatography (7:1 to 1:1 hexane/EtOAc). Fractions containing pure
produce were combined and concentrated under vacuum to yield ethyl
2-oxo-2-(quinolin-6-yl)acetate as a brown oil (Compound 1015, 503
g, 74.5% yield): .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 1.40
(t, 3H), 4.51 (q, 2H), 7.71 (dd, 1H), 8.21 (d, 1H), 8.24 (dd, 1H),
8.68 (dd,1H), 8.77 (dd, 1H), 9.11 (dd, 1H).
[0102] As shown in step 6-ii of Scheme 6, Compound 1015 (282 g,
1.230 mol) and DCM (2.82 L) were combined in a 12 L nitrogen-purged
round bottom flask equipped with a mechanical stirrer, nitrogen
inlet, temperature probe, and room temperature water bath. To the
resulting solution was added bis-(2-methoxyethyl)aminosulfur
trifluoride (DeoxoFluor.TM., 615 g, 0.50 L, 2.26 eq) over 45
minutes via an addition funnel. Absolute EtOH (12.8 g, 15 mL, 0.21
eq) was added via syringe in portions over 3 minutes and the
reaction allowed to stir overnight at ambient temperature.
In-process samples were taken, worked-up, and analyzed by
.sup.1H-NMR in order to monitor the progress of the reaction.
Typical starting material to product molar ratio after the first
ethanol addition was about 2:3. Accordingly, additional EtOH
portions (12.3 g, 0.2 eq) were sequentially added via syringe with
periods of 10 to 20 hours between additions until the observed
starting material content was lower than 10%. A quench solution was
prepared by mixing sodium bicarbonate (827 g, 8 equiv.) in water
(8.3 L) and cooling to to 13 .degree. C. in an ice bath. The
reaction mixture was transferred into the sodium bicarbonate quench
solution over 0.5 hour via a transfer line by pulling vacuum on the
22 L flask containing the stirring sodium bicarbonate solution.
Vigorous gas evolution was observed. The temperature was maintained
between 10.degree. C.-13.degree. C., during the quench, after which
time the ice bath was removed and the mixture stirred for 2 hours
at 12.degree. C.-15 .degree. C. The DCM layer was separated and the
aqueous layer extracted with DCM (2.times.1 L). The DCM layers were
combined and concentrated at 26.degree. C. under vacuum to give 349
g of crude oil which was purified by silica chromatography (7:1 to
4:1 hexane/EtOAc). Fractions containing pure product were combined
and concentrated to give an oil, which was taken up in 2.times.180
mL abs. EtOH and concentrated by rotary evaporation to yield ethyl
2,2-difluoro-2-(quinolin-6-yl)acetate as an oil (Compound 1009, 164
g, 53% yield): .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 1.24 (t,
3H), 4.35 (q, 2H), 7.67 (dd, 1H), 7.91 (dd, 1H), 8.20 (d, 1H), 8.37
(s,1H), 8.60 (d,1H), 9.05 (dd,1H); .sup.19F NMR (470 MHz,
DMSO-d.sub.6) .delta. -101.2.
[0103] As shown in step 6-iii of Scheme 6, to a 1 L round-bottomed
flask equipped with a stir bar and thermocouple was added Compound
1009 (164 g, 633.9 mmol) and EtOH (398 mL). The yellow solution was
cooled to 0.degree. C. using an ice/water bath. Sodium hydroxide
(570.5 mL of 2 M aqueous solution, 1.141 mol) was added slowly over
1 hour to the reaction mixture whilst maintaining the internal
temperature below 20.degree. C. The ice/water bath was removed and
the mixture stirred at room temp for 2 hours. The reaction mixture
was concentrated in vacuo and the yellow solid dried in a vacuum
oven (50.degree. C., 20-25 mm Hg, N.sub.2 sweep) to give sodium
2,2-difluoro-2-(quinolin-6-yl)acetate (Compound 1016, 156.0 g, 99%
yield): .sup.1H NMR (500 MHz, DMSO-d.sub.6) .delta. 7.50-7.55 (dd,
1H), 7.90-7.85 (dd, 1H), 8.10-8.15 (d, 1H), 8.10 (s, 1H), 8.40-8.45
(d, 1H), 8.95-8.90 (dd, 1H); .sup.19F NMR (470 MHz, DMSO-d.sub.6)
.delta. -98.15.
[0104] As shown in step 6-iv of Scheme 6, to a 3 L round-bottom
flask equipped with a heating mantel, reflux condenser,
thermocouple, mechanical stirrer, and purged with N.sub.2 was added
Compound 1016 (98.6 g, 326.4 mmol), 1,3-dimethyl-2-imidazolidinone
(1.607 L), and pyridine (38.73 g, 39.60 mL, 489.6 mmol). 50%
propanephosphonic acid anhydride (T3P.RTM.) in
2-methyltetrahydrofuran (415.4 g, 652.8 mmol) was added in a single
portion and a 15-20.degree. C. exotherm was observed. The reaction
mixture was heated to 70.degree. C. for 1 hour, at which time
thiocarbohydrazine (53.03 g, 489.6 mmol) was added in one portion.
The reaction mixture was stirred for an additional 3 hours and then
an additional portion of 50% T3P in 2-MeTHF (207.7 g, 326.4 mmol)
was added, followed by stirring at 70.degree. C. overnight. The
reaction mixture was cooled to room temperature. In a separate
flask, a solution of sodium bicarbonate (219.3 g, 2.611 mol) in
water (2.41 L) was cooled using an ice/water bath. The reaction
mixture was slowly added to the quench solution via cannula over 45
minutes, during which time foaming and precipitation of the product
were observed. The solution was stirred at 5.degree. C. for an
additional hour at pH=7. The resulting solids were collected by
suction filtration and the cake washed with water (3.2 L) and MTBE
(3.2 L). The white solid was dried in a vacuum oven (50.degree. C.,
20-25 mm Hg) to give
4-amino-5-(difluoro(quinolin-6-yl)methyl)-4H-1,2,4-triazole-3-thiol
(Compound 1011, 57 g, 58% yield): .sup.1H NMR (500 MHz,
DMSO-d.sub.6) .delta. 5.70-5.65 (s, 2H), 7.50-7.55 (dd, 1H),
7.90-7.85 (dd, 1H), 8.10-8.15 (d, 1H), 8.10 (s, 1H), 8.40-8.45 (d,
1H), 8.95-8.90 (dd, 1H), 14.3-14.25 (s, 1H); .sup.19F NMR (470 MHz,
DMSO-d.sub.6) .delta. -92.50.
[0105] As shown in step 6-v of Scheme 6, triphenylphosphine (17.66
g, 67.35 mmol), 1,1,1,2,2,2-hexachloroethane (15.94 g, 67.35 mmol),
Compound 1011 (13.37 g, 44.90 mmol) were combined in a 500 mL round
bottom flask fitted with a mechanical stirrer, thermocouple, under
an atmosphere of nitrogen. Anhydrous acetonitrile (461.0 mL) was
added followed by the addition of Et.sub.3N (14.09 g, 19.41 mL,
139.2 mmol) to the stirred mixture whilst maintaining the
temperature between 21.4-25.1.degree. C. The reaction mixture
became a clear solution and then became a slurry once the product
formed (within about 2 minutes). Water (808.9 mg, 808.9 .mu.L,
44.90 mmol) was then added followed by the addition of MeOH (14.39
g, 18.19 mL, 449.0 mmol) and the reaction then stirred for
additional 45 min. The solid was collected by filtration and the
cake washed with CH.sub.3CN (132 mL). The cake was dried in a
vacuum oven at 45.degree. C. with a nitrogen bleed to produce
5-(difluoro(quinolin-6-yl)methyl)-4-(iminotriphenylphosphorano)-4H-1,2,4--
triazole-3-thiol (Compound 1017, 25.57 g, 98.8% yield) as a beige
solid: .sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 7.51-7.42 (m,
6H), 7.70-7.56 (m, 12H), 8.11 (d, 1H), 8.16 (m, 1H), 8.49 (dd, 1H),
9;03 (dd, 1H), 13.64 (br s, 1H); .sup.19F NMR (376 MHz,
DMSO-d.sub.6) .delta. -91.77; .sup.31P NMR (162 MHz, DMSO-d.sub.6)
.delta. 19.71.
[0106] As shown in step 6-vi of Scheme 6, to a 2 L, 4-necked,
round-bottomed flask fitted with overhead stirrer, thermocouple,
reflux condenser, and nitrogen bubbler was added
1-methylpyrazole-4-carboxylic acid (27.33 g, 216.7 mmol). Toluene
(600 mL) and triethylamine (30.70 g, 42.29 mL, 303.4 mmol) were
added at 20.1.degree. C. with no observed temperature increase. The
resulting white slurry became a colorless solution after heating to
103.degree. C. Diphenylphsophoryl azide (DPPA, 61.48 g, 48.14 mL,
216.7 mmol) was added over a period of 30 minutes, keeping the
temperature at between 103.1 and 107.degree. C. Heating was
discontinued and allowed to cool to room temperature. The resulting
4-isocyanato-1-methyl-1H-pyrazole was not isolated and instead to
it was added Compound 1017 (120 g, 216.7 mmol) in one portion at
room temperature. Analytical HPLC analysis immediately after the
addition showed 51.2% conversion of the starting material to
Compound 3. Starting with 216.7 mmol of
1-methylpyrazole-4-carboxylic acid, additional
4-isocyanato-1-methyl-1H-pyrazole was prepared as indicated above
in a separate flask. After cooling to room temperature, this
reaction mixture was transferred to the first reaction mixture via
cannula. HPLC analysis indicated 100% conversion after addition.
EtOAc (240 mL) was added to the reaction mixture and a white
precipitate formed. The reaction was stirred for 30 minutes and the
solid collected by suction filtration. The cake [(comprising
1-(3-(difluoro(quinolin-6-yl)methyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadia-
zol-6-yl)-1,3-bis(1-methyl-1H-pyrazol-4-yl)urea (Compound 1018) as
a by-product] was washed with EtOAc (600.0 mL). The filtrate was
concentrated in vacuo by rotary evaporation at 35.degree. C. to
give 272.4 g of a brown oil. The oil was dried under high vacuum
and was purified by column chromatography, using an 8:1 ratio of
SiO.sub.2 to crude oil and eluting with a gradient of 1% to 5% EtOH
in DCM to produce
3-(difluoro(quinolin-6-yl)methyl)-N-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]tri-
azolo[3,4-b][1,3,4]thiadiazol-6-amine (Compound 3, 111 g), which
was further purified by crystallization. Accordingly, a 33.5 g
portion of this material to 250 mL 3-neck round bottom flask fitted
with mechanical stirrer and nitrogen bubbler. The solid was an
orange-tan color. A total of 135 mL of CH.sub.3CN was added to give
a thick slurry. After 2.5 hours, the solids were collected by
suction filtration after 3 hours. The wet cake was washed with
CH.sub.3CN (67 mL) to give 13.9 g of wet solid. Vacuum drying was
carried out (43.degree. C., 20-25 in Hg, N.sub.2 sweep) over 15.5
hours to give 10.25 g of pure Compound 3 (>99.9% purity by HPLC
analysis, <0.1% PPh.sub.3O). The MeCN filtrate was treated with
equal amount of water. A solid precipitated and the slurry was
stirred for 2 hours. The solid was collected by suction filtration.
The wet cake was washed with 35 mL water. The cake was dried
(43.degree. C., 20-25 in Hg, N.sub.2 sweep) to give 9.5 g of a
solid material, which was treated with CH.sub.3CN as above to
provide an additional 4.78 g of pure Compound 3 (total=15.03 g,
57.7% overall yield from Compound 1017). The yield can be increased
further by aminolyzing the urea side product (Compound 1018) with
NH.sub.3/MeOH to recover additional Compound 3.
##STR00012## ##STR00013##
[0107] Analytical data for Compounds 1-8 are shown in Table 1.
TABLE-US-00001 TABLE 1 Physical Characterization of Compounds of
Formula I ESMS .sup.1H NMR (300 MHz, unless indicated otherwise),
Cmpd. No. (M + H) NMR peaks given as .delta. values in ppm 1 377.17
(methanol-d.sub.4) .delta. 8.81 (dd, J = 1.7, 4.3 Hz, 1H), 8.37
(dd, J = 0.9, 8.4 Hz, 1H), 8.04-7.98 (m, 2H), 7.79 (dd, J = 2.1,
8.8 Hz, 1H), 7.55 (dd, J = 1.9, 2.4 Hz, 1H), 7.52 (d, J = 4.3 Hz,
1H), 7.42 (d, J = 0.7 Hz, 1H), 4.86-4.80 (m, 1H), 3.79 (s, 3H) and
1.94 (d, J = 7.2 Hz, 3H) 2 377.17 (methanol-d.sub.4) .delta. 8.81
(dd, J = 1.7, 4.3 Hz, 1H), 8.37 (dd, J = 0.9, 8.4 Hz, 1H),
8.04-7.98 (m, 2H), 7.79 (dd, J = 2.1, 8.8 Hz, 1H), 7.55 (dd, J =
1.9, 2.4 Hz, 1H), 7.52 (d, J = 4.3 Hz, 1H), 7.42 (d, J = 0.7 Hz,
1H), 4.86-4.80 (m, 1H), 3.79 (s, 3H) and 1.94 (d, J = 7.2 Hz, 3H) 3
399.06 (DMSO-d.sub.6) .delta. 10.86 (s, 1H), 9.18 (dd, J = 1.5, 4.5
Hz, 1H), 8.83 (d, J = 8.3 Hz, 1H), 8.61 (s, 1H), 8.31 (d, J = 8.9
Hz, 1H), 8.11 (dd, J = 2.0, 8.9 Hz, 1H), 7.85 (dd, J = 4.6, 8.3 Hz,
1H), 7.66 (s, 1H), 7.43 (d, J = 0.4 Hz, 1H) and 3.79 (s, 3H) 4
413.21 (DMSO-d.sub.6) .delta. 10.20 (br, 1H), 9.04 (d, J = 2.9 Hz,
1H), 8.58 (d, J = 8.8 Hz, 1H), 8.45 (s, 1H), 8.20 (d, J = 8.6 Hz,
1H), 7.97 (dd, J = 1.9, 8.9 Hz, 1H), 7.67 (q, J = 4.2 Hz, 1H), 7.56
(s, 1H), 3.68 (s, 3H) and 2.07 (s, 3H) 5 391.24 (methanol-d.sub.4)
.delta. 9.25 (d, J = 6.3 Hz, 2H), 8.48 (s, 1H), 8.32 (s, 1H), 8.15
(dd, J = 6.3, 7.6 Hz, 1H), 8.14 (s, 1H), 7.80 (s, 1H), 7.51 (s,
1H), 5.21 (q, J = 7.2 Hz, 1H), 4.16 (q, J = 7.3 Hz, 2H), 2.03 (d, J
= 7.2 Hz, 3H) and 1.42 (t, J = 7.3 Hz, 3H) 6 391.24
(methanol-d.sub.4) .delta. 9.25 (d, J = 6.3 Hz, 2H), 8.48 (s, 1H),
8.32 (s, 1H), 8.15 (dd, J = 6.3, 7.6 Hz, 1H), 8.14 (s, 1H), 7.80
(s, 1H), 7.51 (s, 1H), 5.21 (q, J = 7.2 Hz, 1H), 4.16 (q, J = 7.3
Hz, 2H), 2.03 (d, J = 7.2 Hz, 3H) and 1.42 (t, J = 7.3 Hz, 3H) 7
391.20 (DMSO-d.sub.6) .delta. 9.91 (s, 1H), 8.93-8.92 (m, 1H), 8.47
(d, J = 7.9 Hz, 1H), 7.80-7.75 (m, 1H), 7.62-7.58 (m, 2H), 4.78 (q,
J = 7.5 Hz, 1H), 3.69 (s, 3H), 2.03 (s, 3H) and 1.83 (d, J = 7.2
Hz, 3H) ppm 8 391.20 (DMSO-d.sub.6) .delta. 9.91 (s, 1H), 8.93-8.92
(m, 1H), 8.47 (d, J = 7.9 Hz, 1H), 7.80-7.75 (m, 1H), 7.62-7.58 (m,
2H), 4.78 (q, J = 7.5 Hz, 1H), 3.69 (s, 3H), 2.03 (s, 3H) and 1.83
(d, J = 7.2 Hz, 3H) ppm
Biological Assay of Compounds of Formula I
EXAMPLE 3
c-Met Kinase Inhibition Assay
[0108] The compounds of the invention were screened for their
ability to inhibit c-Met kinase using a standard radiometric assay.
Briefly, in this kinase assay the transfer of the terminal
.sup.33P-phosphate in .sup.33P-ATP to substrate polyE4Y is
interrogated. The assay was carried out in 96-well plates to a
final volume of 100 .mu.L per well containing 0.5 nM c-Met, 100 mM
HEPES (pH 7.5), 10 mM MgCl.sub.2, 25 mM NaCl, 0.01% BSA, 1 mM DTT,
0.5 mg/mL polyE4Y, and 35 .mu.M ATP. Accordingly, compounds of the
invention were dissolved in DMSO to make 10 mM initial stock
solutions. Serial dilutions in DMSO were then made to obtain the
final solutions for the assay. A 1.5 .mu.L aliquot of DMSO or
inhibitor in DMSO was added to each well, followed by the addition
of .sup.33P-ATP, and finally the addition of c-Met and polyE4Y
(obtained from Sigma). After 20 min, the reaction was quenched with
50 .mu.L of 30% trichloroacetic acid (TCA) containing 4 mM ATP. The
reaction mixture was transferred to the 0.66 mm GF filter plates
(Corning) and washed three times with 5% TCA. Following the
addition of 50 .mu.L of Ultimate Gold.TM. high efficiency
scintillant (Packard Bioscience), the samples were counted in a
Packard TopCount NXT Microplate Scintillation and Luminescence
Counter (Packard BioScience). The K.sub.i values were calculated
using Microsoft Excel Solver macros to fit the data to the kinetic
model for competitive tight-binding inhibition. Each of Compounds 1
through 8 had a K.sub.i value for the inhibition of c-Met of less
than 200 nM.
EXAMPLE 4
Inhibition c-Met Activity in Snu5 Gastric Carcinoma Cells
[0109] Compounds of formula I were also screened for their ability
to inhibit the Luciferase-induced signal in an engineered Snu5 cell
line. Snu5 [obtained from American Type Culture Collection (Catalog
number CRL-5973)] is a human gastric carcinoma known to overexpress
c-Met, which is constitutively active. The cell line was transduced
with the retrovirus, pCLPCX, which contains a genetic construct
consisting of 6.times. AP1 promoter response elements and a
luciferase gene having a C-terminal PEST sequence (proteolytic
signal from mouse ornithine decarboxylase, which reduces the
half-life of the luciferase). The constitutively active c-Met
activates cellular pathways (principally MAP kinase), resulting in
AP-1-induced transcription of luciferase-PEST and translation into
the final product, the activity of which is quantifiable as a
chemiluminescent readout upon the addition of luciferin (Steady-Glo
from Promega.). Residual luminescence is strongly correlated to the
inhibition of c-Met. A stable cell line was obtained by selecting
the new cell line (Snu5-AP1-Luc-Pest) with puromycin. The cells
were grown in complete media [Iscove's media (Invitrogen)
containing 10% fetal bovine serum (FBS, Hyclone) and
penicillin/gentamycin (Invitrogen)]. Compounds of the invention
were dissolved in DMSO to make 10 mM initial stock solutions.
Serial dilutions in DMSO were then made and transferred to complete
medium to make a 10.times. solution. The Snu5-AP1-Luc-Pest cells
were counted and diluted to 200,000-cells/mL solution. The cells
(90 .mu.L) were added to each well in a 96-well black with clear
bottom plate (Costar). Then 10 .mu.L of the 10.times. compound
solution was added to the cells in triplicate. The plates were
incubated in a 37.degree. C./5% CO.sub.2 incubator. After 6 hours,
50 .mu.L of the Steady-Glo reagent (Promega) was added to each well
and placed on a plate shaker for 5 minutes to ensure that the cells
were completely lysed. The plate was read on a 1450 Microbeta
Liquid Scintillation and Luminescence Counter (Perkin-Elmer). Each
of Compounds 1 through 8 had an IC.sub.50 value for the inhibition
c-Met activity in Snu5 gastric carcinoma cells of less than 200
nM.
EXAMPLE 5
Inhibition of Tumor Growth in a Mouse Model
[0110] Compound 3 was investigated for its ability to inhibit tumor
growth of subcutaneously implanted SNU-5 gastric cancer cells in
severe combined immunodeficient (SCID) mice. SNU-5 cells (CRL-5973,
American Type Culture Collection, Manassas, Va.) were cultured in
ISCOVE's Modified Dulbecco's Medium (IMDM) (Invitrogen, Carlsbad,
Calif.) supplemented with 10% fetal bovine serum (FBS) (Hyclone,
Logan, Utah), 100 units/mL of penicillin, 100 mg/mL of streptomycin
(Invitrogen, Carlsbad, Calif.), and 2 mM L-glutamine. Cells were
cultured for fewer than 4 passages prior to implantation. Female
SCID mice (Fox Chase SCID, CB-17, mice weighing 17 to 19 g obtained
from Charles River Laboratories, Wilmington, Mass.) were injected
subcutaneously (s.c.) with 5.times.10.sup.6 SNU-5 cells into the
right dorsal axillary region on Day 0. Treatments were initiated on
Day 25 when the average tumor volume reached approximately 358
mm.sup.3.
[0111] Compound 3, formulated in a vehicle containing 30% (w/v)
Propylene Glycol and 10% Solutol (Sigma-Aldrich, St Louis, Mo.) as
a suspended homogenous form, was administered orally (p.o.) once
daily (QD) at total daily doses of 3, 10, and 30 mg/kg/day for 14
days. Tumor volumes (calculated using the ellipsoid formula,
(length.times.width.sup.2)/2, where length and width represented
the largest and smallest dimensions of the tumor, respectively)
were recorded for two weeks after the initiation of treatment. The
study was terminated 38 days after tumor implantation. Average
tumor volumes are presented in Table 2. Tumor weights at the
termination of the study are presented in Table 3.
TABLE-US-00002 TABLE 2 SNU-5 tumor volumes* Day 25 Day 28 Day 31
Day 35 Day 38 Vehicle control 357.6 .+-. 36.7 487.1 .+-. 45.8 578.4
.+-. 66.0 753.2 .+-. 77.9 937.1 .+-. 101.0 Compound 3, 359.5 .+-.
35.1 281.3 .+-. 28.7 256.5 .+-. 23.6 255.7 .+-. 21.1 273.4 .+-.
24.2 30 mg/kg/day Compound 3, 358.0 .+-. 17.1 354.2 .+-. 21.7 381.8
.+-. 25.6 406.6 .+-. 23.4 453.9 .+-. 27.3 10 mg/kg/day Compound 3,
356.1 .+-. 24.5 432.6 .+-. 31.2 511.9 .+-. 36.3 587.8 .+-. 39.5
670.4 .+-. 46.2 3 mg/kg/day *tumor volume measurements are in
mm.sup.3 and are reported as mean .+-. standard error
TABLE-US-00003 TABLE 3 SNU-5 tumor weights at study termination
Vehicle, Compound 3 Compound 3 Compound 3 Animal ID 10 mL/kg 30
mg/kg/day 10 mg/kg/day 3 mg/kg/day 1 863 350 275 508 2 838 327 305
368 3 896 150 371 679 4 974 246 309 596 5 857 180 319 619 6 1607
173 476 505 7 760 260 358 525 8 629 420 469 485 9 896 250 279 605
10 1151 156 660 655 11 418 851 12 400 770 13 387 405 14 410 790 15
349 938 Mean 947.1 251.2 385.7 619.9 SD 268.0 91.0 98.2 163.5 SE
84.7 28.8 25.4 42.2
[0112] As shown in Tables 2 and 3, Compound 3 demonstrated
significant and dose-dependent anti-tumor activity at all three
dose levels tested. A dose of 30 mg/kg/day resulted in tumor
regression of -23.9% (P<0.001) by tumor volume analysis. Tumors
harvested from the 3, 10, and 30 mg/kg/day VRT-846198 treatment
groups were significantly smaller than those harvested from the
vehicle control group, with percent weight reductions of 34.5%,
59.3%, and 73.5%, respectively (all P<0.002).
EXAMPLE 6
Inhibition of Tumor Metastasis in Mouse Model
[0113] Compound 3 was investigated for its ability to inhibit the
metastasis of subcutaneously implanted tumors to the lungs of
severe combined immunodeficient (SCID) mice. Accordingly, A549
cells (A549HGF-1m1115, transfected with hepatocyte growth factor,
luciferase, and green fluorescent protein) were cultured in
RPMI1640 medium (Invitrogen, Carlsbad, Calif.) supplemented with
10% fetal bovine serum (FBS) (Hyclone, Logan, Utah), 100 units/mL
of penicillin, 100 mg/mL of streptomycin (Invitrogen, Carlsbad,
Calif.), and 2 mM L-glutamine for fewer than 4 passages prior to
implantation.
3-(Difluoro(quinolin-6-yl)methyl)-N-(1-methyl-1H-pyrazol-4-yl)-[1,2,4]tri-
azolo[3,4-b][1,3,4]thiadiazol-6-amine (Compound 3) was formulated
in a vehicle containing 0.5% (w/v) methylcellulose (Sigma-Aldrich,
St Louis, Mo.) and 0.1% (v/v) Tween 80.TM. as a dissolved
homogenous form, which was prepared fresh each day and administered
to mice via oral gavage at a dosing volume of 10 mL/kg.
[0114] Female SCID mice were injected subcutaneously (s.c.) with
5.times.10.sup.6 A549HGF cells into the right dorsal axillary
region on Day 0. Treatments were initiated on the same day by oral
administration (p.o.) of Compound 3 once daily (QD) at total daily
doses of 30 and 60 mg/kg/day for 22 days. Ectopic tumor
measurements were recorded twice a week for 3 weeks after the
initiation of treatment. Compound 3 was found to result in no
significant change in primary A549 tumor cell growth at the site of
implantation for mice dosed at 30 or 60 mg/kg/day vs. the tumor
cell growth in mice dosed with vehicle alone.
[0115] In order to evaluate the anti-metastatic potential of
Compound 3, at the termination of the study all animal lung tissues
were harvested and lysed by homogenization for ex vivo
quantification via luciferase luminescence. Table 4 illustrates the
tumor cell content in lung tissue at study termination and the data
therein indicate that Compound 3 significantly inhibits the
formation of lung metastases in mice treated with Compound 3 at 60
mg/kg/day (average fluorescent count of 6672.3.+-.1986.1 SEM)
compared to vehicle controls (average fluorescent count of
23531.5.+-.8278.2 SEM, p<0.02).
TABLE-US-00004 TABLE 4 Luminescence of homogenized lung tissue in
SCID mice treated with Compound 3 vs. control animals treated with
only vehicle Vehicle, Compound 3 Compound 3 10 mL/kg 30 mg/kg/day
60 mg/kg/day Animal ID (counts) (counts) (counts) 1 6610 16300 4860
2 2980 5640 2470 3 1850 3890 2170 4 4300 5270 1480 5 21300 2270
2540 6 53200 2620 19300 7 9670 17200 21100 8 22600 21300 3800 9
26700 6430 16500 10 13300 4340 1440 11 112000 65400 2620 12 19300
2230 2920 13 12100 5540 Mean 23531.5 12740.8 6672.3 standard
deviation 29884.3 17828.3 7169.7 standard error 8278.2 5146.7
1986.1
[0116] All publications and patents cited in this specification are
herein incorporated by reference as if each individual publication
or patent were specifically and individually indicated to be
incorporated by reference. Although the foregoing invention has
been described in some detail by way of illustration and example
for purposes of clarity or understanding, it will be readily
apparent to those of ordinary skill in the art in light of the
teachings of this invention that certain changes and modifications
may be made thereto without departing from the spirit or scope of
the appended claims.
* * * * *