U.S. patent application number 15/903337 was filed with the patent office on 2019-01-03 for phenyloxadiazole derivatives as pgds inhibitors.
The applicant listed for this patent is Sanofi. Invention is credited to Harpal S. GILL, Andrea HILLEGASS, George LEE, Christopher L. VANDEUSEN, Franz J. WEIBERTH.
Application Number | 20190000845 15/903337 |
Document ID | / |
Family ID | 43606440 |
Filed Date | 2019-01-03 |
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United States Patent
Application |
20190000845 |
Kind Code |
A1 |
VANDEUSEN; Christopher L. ;
et al. |
January 3, 2019 |
PHENYLOXADIAZOLE DERIVATIVES AS PGDS INHIBITORS
Abstract
This invention is directed to a compound of formula (I):
##STR00001## wherein R1, R2 and R3 are as defined herein, a
pharmaceutical composition comprising the compound, intermediates
and processes for making said compounds, and the use of the
compound to treat allergic and/or inflammatory disorders,
particularly disorders such as allergic rhinitis, asthma, chronic
obstructive pulmonary disease (COPD) and age-related macular
degeneration (AMD).
Inventors: |
VANDEUSEN; Christopher L.;
(East Windsor, NJ) ; WEIBERTH; Franz J.; (Ringoes,
NJ) ; GILL; Harpal S.; (Lambertville, NJ) ;
LEE; George; (Somerville, NJ) ; HILLEGASS;
Andrea; (Nazareth, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sanofi |
Paris |
|
FR |
|
|
Family ID: |
43606440 |
Appl. No.: |
15/903337 |
Filed: |
February 23, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15258892 |
Sep 7, 2016 |
9937175 |
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15903337 |
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13439511 |
Apr 4, 2012 |
9469627 |
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15258892 |
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PCT/US2010/051727 |
Oct 7, 2010 |
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13439511 |
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61249693 |
Oct 8, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/00 20180101;
C07D 217/06 20130101; A61P 11/02 20180101; A61P 29/00 20180101;
A61P 11/06 20180101; A61P 37/08 20180101; A61P 5/00 20180101; C07D
271/06 20130101; A61P 27/02 20180101; A61P 11/00 20180101; C07D
413/14 20130101; A61K 31/506 20130101 |
International
Class: |
A61K 31/506 20060101
A61K031/506; C07D 217/06 20060101 C07D217/06; C07D 271/06 20060101
C07D271/06; C07D 413/14 20060101 C07D413/14 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 26, 2010 |
FR |
1056094 |
Claims
1. A compound of formula (I): ##STR00027## wherein R1 is hydrogen
or C.sub.1-C.sub.6alkyl; R2 is hydrogen, halogen or
C.sub.1-C.sub.3alkyl; and R3 is hydroxyalkyl; or a pharmaceutically
acceptable salt thereof.
2-24. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to phenyloxadiazole
compounds, their preparation, pharmaceutical compositions
containing these compounds, and their pharmaceutical use in the
treatment of disease states capable of being modulated by the
inhibition of the prostaglandin D synthase.
BACKGROUND OF THE INVENTION
[0002] Allergic rhinitis, the most common atopic disease, has an
estimated prevalence ranging from about 5 to about 22 percent of
the general human population and is characterized by the symptoms
of sneezing, nasal discharge, and nasal congestion. These symptoms
are believed to be triggered by multiple mediators released from
mast cells and other inflammatory cells. Current therapies, such as
antihistamines, deal effectively with the sneezing and nasal
discharge, but have little effect on congestion, which is a key
symptom affecting the quality of life of patients.
[0003] Local allergen challenge in patients with allergic rhinitis,
bronchial asthma, allergic conjunctivitis and atopic dermatitis has
been shown to result in rapid elevation of prostaglandin D2
"(PGD2)" levels in nasal and bronchial lavage fluids, tears and
skin chamber fluids. PGD2 has many inflammatory actions, such as
increasing vascular permeability in the conjunctiva and skin,
increasing nasal airway resistance, airway narrowing and cosinophil
infiltration into the conjunctiva and trachea. PGD2 is the major
cyclooxygenase product of arachidonic acid produced from mast cells
on immunological challenge [Lewis, R A, Soter N A, Diamond P T,
Austen K F, Oates J A, Roberts L J II, Prostaglandin D2 generation
after activation of rat and human mast cells with anti-IgE, J.
Immunol. 129, 1627-1631, 1982]. Activated mast cells, a major
source of PGD2, are one of the key players in driving the allergic
response in conditions such as asthma, allergic rhinitis, allergic
conjunctivitis, allergic dermatitis and other diseases [Brightling
C E, Bradding P, Pavord I D, Wardlaw A J, New Insights into the
role of the mast cell in asthma, Clin. Exp. Allergy 33, 550-556,
2003].
[0004] In the presence of sulfhydryl compounds, PGD2 is formed by
the isomerization of PGH2, a common precursor of prostanoids, by
catalytic action of prostaglandin D synthase "(PGDS)". There are
two isoforms of the PGDS enzyme: L-PGDS; and H-PGDS. H-PGDS is a
cytosolic enzyme, which is distributed in the peripheral tissues,
and which is localized in the antigen-presenting cells, mast cells,
megakaryocytes, and Th2 lymphocytes. The action of the product PGD2
is mediated by G-protein coupled receptors: D prostaglandin "(DP)"
and crTH2. See (1) Prostaglandin D Synthase: Structure and
Function. T. Urade and O. Hayaishi, Vitamin and Hormones, 2000, 58,
89-120, (2) J. J. Murray, N. Engl. J. Med., 1986 Sep. 25;
315(13):800, and (3) Urade et. al, J. Immunology 168: 443-449,
2002.
[0005] Without wishing to be bound by theory, inhibiting the
formation of PGD2 should have an effect on nasal congestion and,
therefore, be of therapeutic benefit in allergic rhinitis. In
addition, we believe that a PGDS inhibitor should be of therapeutic
benefit in a number of other indications such as bronchial asthma,
age-related macular degeneration (AMD) an/or or chronic obstructive
pulmonary disease (COPD).
[0006] Age-related macular degeneration (AMD) is a degenerative and
progressive ocular disease that results in loss of fine, central
vision due to the degeneration of the macula. AMD is the most
common cause of blindness in Europe and the United States for
individuals over 50 years of age.
[0007] Chronic obstructive pulmonary disease (COPD) is a
progressive, inflammatory disease that involves chronic bronchitis
and emphysema. Symptoms include airflow limitation, excessive mucus
production, coughing, reduced exercise capacity and reduced quality
of life.
[0008] PGDS inhibitors have been reported. The compound, HQL-79, is
reported to be a weak PGDS inhibitor, and is antiasthmatic in
guinea pig and rat models (Matsusshita, et al., Jpn. J. Pharamcol.
78: 11, 1998). The compound Tranilast is described as a PGDS
inhibitor. (Inhibitory Effect of Tranilast on Prostaglandin D
Synthesase. K. Ikai, M. Jihara, K. Fujii, and Y. Urade. Biochemical
Pharmacology, 1989, 28, 2773-2676). The following published patent
applications also disclose PGDS inhibitors:
US2008/0207651A1 and US2008/0146569A1--pyridine and pyrimidine
carboxamides; JP2007-51121--pyrimidine carboxamides;
WO2007/007778--benzimidazole derivatives;
WO2008/122787--piperazine(thio)carboxamides; and
WO2005/094805--imine and amide derivatives.
SUMMARY OF THE INVENTION
[0009] The present invention is directed to a compound of formula
(I):
##STR00002##
wherein: R1 is hydrogen or C.sub.1-C.sub.6alkyl; R2 is hydrogen,
halogen or C.sub.1-C.sub.3alkyl; and R3 is hydroxyalkyl or a
pharmaceutically acceptable salt thereof.
[0010] Another aspect of the present invention is a pharmaceutical
composition comprising a pharmaceutically effective amount of a
compound according to formula (I) and a pharmaceutically acceptable
carrier.
[0011] Another aspect of the present invention is directed to a
method of treating allergic and/or inflammatory disorders,
particularly disorders such as allergic rhinitis, asthma, chronic
obstructive pulmonary disease (COPD) and/or age-related macular
degeneration (AMD) in a patient in need thereof by administering to
the patient a compound according to formula (I). Another aspect of
the invention is a process for preparing compounds of formula
(I).
DETAILED DESCRIPTION OF THE INVENTION
Definition of Terms
[0012] As used above, and throughout the description of the
invention, the following terms unless otherwise indicated, shall be
understood to have the following meanings:
[0013] "Alkyl" means straight or branched aliphatic hydrocarbon
having 1 to about 20 carbon atoms. Particular alkyl has 1 to about
12 carbon atoms. More particular alkyl is lower alkyl. Branched
means that one or more lower alkyl groups such as methyl, ethyl or
propyl are attached to a linear alkyl chain. "Lower alkyl" means 1
to about 4 carbon atoms in a linear alkyl chain that may be
straight or branched.
[0014] "Hydroxyalkyl" means OH-alkyl-. Particular hydroxyalkyl is
hydroxy(C.sub.1-C.sub.6)alkyl-. Exemplary hydroxyalkyl includes
1-hydroxy-1-methyl-ethyl.
[0015] "Compounds of the present invention", and equivalent
expressions, are meant to embrace compounds of Formula (I) as
hereinbefore described. Reference to intermediates, whether or not
they themselves are claimed, is meant to embrace their salts,
N-oxides and solvates, where the context so permits.
[0016] "Halo" or "halogen" means fluoro, chloro, bromo, or iodo.
Particular halo or halogen is fluoro or chloro.
[0017] "Patient" includes human and other mammals.
[0018] "Pharmaceutically acceptable salts" refers to the non-toxic,
inorganic and organic acid addition salts, and base addition salts,
of compounds of the present invention. These salts may be prepared
in situ during the final isolation and purification of the
compounds or by separately reacting the purified compound in its
free base form with a suitable organic or inorganic acid and
isolating the salt thus formed. In some cases, the compounds
themselves are capable of self-protonating basic sites on the
molecule and forming an internal amphoteric salt.
[0019] "Suitable couple reagent" refers to a reagent suitable for
reacting an amine with a carboxylic acid. Suitable coupling
reagents include, but are not limited to, DMTMM,
carbonyldiimidazole (CDI) and TBTU, DCC, phosphonium salts, and
uronium salts.
[0020] Exemplary acid addition salts include the hydrobromide,
hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,
oxalate, valerate, oleate, palmitate, stearate, laurate, borate,
benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,
succinate, tartrate, naphthylate, mesylate, glucoheptonate,
lactiobionate, sulfamates, malonates, salicylates, propionates,
methylene-bis- -hydroxynaphthoates, gentisates, isethionates,
di-p-toluoyltartrates, methanesulfonates, ethanesulfonates,
benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates and
laurylsulfonate salts. See, for example S. M. Berge, et al.,
"Pharmaceutical Salts," J. Pharm. Sci., 66, 1-19 (1977) that is
incorporated herein by reference. Base addition salts can also be
prepared by separately reacting the purified compound in its acid
form with a suitable organic or inorganic base and isolating the
salt thus formed. Base addition salts include pharmaceutically
acceptable metal and amine salts. Suitable metal salts include the
sodium, potassium, calcium, barium, zinc, magnesium, and aluminum
salts. A particular base addition salt is sodium salt or potassium
salt. Suitable inorganic base addition salts are prepared from
metal bases which include sodium hydride, sodium hydroxide,
potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium
hydroxide, magnesium hydroxide, and zinc hydroxide. Suitable amine
base addition salts are prepared from amines which have sufficient
basicity to form a stable salt, and particularly include those
amines which are frequently used in medicinal chemistry because of
their low toxicity and acceptability for medical use. Ammonia,
ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine,
choline, N,N'-dibenzylethylenediamine, chloroprocaine,
diethanolamine, procaine, N-benzylphenethylamine, diethylamine,
piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium
hydroxide, triethylamine, dibenzylamine, ephenamine,
dehydroabietylamine, N-ethylpiperidine, benzylamine,
tetramethylammonium, tetraethylammonium, methylamine,
dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g.,
lysine and arginine, and dicyclohexylamine.
[0021] A particular embodiment of the invention is a compound of
formula (I) wherein:
R1 is hydrogen; R2 is hydrogen; and R3 is hydroxyalkyl; or a
pharmaceutically acceptable salt thereof.
[0022] Another particular embodiment of the invention is a compound
of formula (I) wherein:
R1 is C.sub.1-C.sub.6alkyl; R2 is hydrogen; and R3 is hydroxyalkyl;
or a pharmaceutically acceptable salt thereof.
[0023] Another particular embodiment of the invention is a compound
of formula (I), which is: [0024]
2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
3-5-(1-hydroxy-1methyl-ethyl)[1,2,4]oxadiazol-3-yl]benzyl amide;
[0025] 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
((S)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-eth-
yl)-amide; or [0026] 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
((R)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-eth-
yl)-amide; or a pharmaceutically acceptable salt thereof.
[0027] It is to be understood that this invention covers all
appropriate combinations of the particular embodiments referred
thereto.
[0028] The present invention also includes within its scope a
pharmaceutical composition comprising a pharmaceutically effective
amount of a compound of the invention, in admixture with a
pharmaceutically acceptable carrier.
[0029] Compounds of the present invention are PGDS inhibitors and
thus, are useful for treating allergic and/or inflammatory
disorders, particularly disorders such as allergic rhinitis,
asthma, chronic obstructive pulmonary disease (COPD), chronic
rhinosinusitus (CRS), and age-related macular degeneration (AMD).
Accordingly, another invention herein is directed to a method of
treating a patient suffering from allergic rhinitis, asthma,
chronic obstructive pulmonary disease (COPD) and/or age-related
macular degeneration (AMD) comprising administering to the patient
a pharmaceutically effective amount of compound of formula (I).
[0030] In addition to the indications and disorders recited above,
PGDS inhibitors, including compounds of formula I, are useful for
the treatment of PGD2 mediated diseases including DP1, DP2, TP
& PPAR gamma associated diseases. Such diseases and disorders
include the following:
1) Skin diseases including atopic dermatitis, chronic urticaria,
flushing Proc Natl Acad Sci U.S.A. 2006 Apr. 25; 103(17):6682-7);
2) Allergic diseases of the digestive system such as eosinophilic
eosophagitis; 3) Neurodegenerative diseases such as Alzheimer's and
Krabbes disease (The Journal of Neuroscience, Apr. 19, 2006,
26(16):4383-4393); 4) Muscle diseases such as Duchenne Muscular
Dystrophy and Polymyositis (American Journal of Pathology. 2009;
174:1735-1744); 5) Conditions associated with increased eosinophils
or Eosinophilic Syndrome; 6) Diseases of the eye such as Uveitis,
Graves Ophthalmopathy, allergic conjunctivitis and glaucoma; 7)
Vascular injury associated with diabetes such as diabetic
retinopathy or with metabolic syndrome (Diabetes Res Clin Pract.
2007 June; 76(3):358-67); and 8) Bone diseases such as rheumatoid
arthritis and ostcoarthritis (J Rheumatol 2006; 33:1167-75).
[0031] References herein directed to treating should be understood
to include prophylactic therapy to inhibit PGDS, as well as to
treat an established acute or chronic or physiological conditions
associated with PGDS to essentially cure a patient suffering
therefrom, or ameliorate the physiological conditions associated
therewith. Physiological conditions discussed herein include some,
but not all, of the possible clinical situations where an
anti-allergic rhinitis and/or asthma treatment is warranted. Those
experienced in this field are well aware of the circumstances
requiring treatment.
[0032] In practice, the compound of the present invention may be
administered in pharmaceutically acceptable dosage form to humans
and other mammals by topical or systemic administration, including
oral, inhalational, rectal, nasal, buccal, sublingual, vaginal,
colonic, parenteral (including subcutaneous, intramuscular,
intravenous, intradermal, intrathecal and epidural), intracisternal
and intraperitoneal. It will be appreciated that the particular
route may vary with for example the physiological condition of the
recipient.
[0033] "Pharmaceutically acceptable dosage forms" refers to dosage
forms of the compound of the invention, and includes, for example,
tablets, dragees, powders, elixirs, syrups, liquid preparations,
including suspensions, sprays, inhalants tablets, lozenges,
emulsions, solutions, granules, capsules and suppositories, as well
as liquid preparations for injections, including liposome
preparations. Techniques and formulations generally may be found in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., latest edition.
[0034] A particular aspect of the invention provides for the
compound of the invention to be administered in the form of a
pharmaceutical composition.
[0035] Pharmaceutically acceptable carriers include at least one
component selected from the group comprising pharmaceutically
acceptable carriers, diluents, coatings, adjuvants, excipients, or
vehicles, such as preserving agents, fillers, disintegrating
agents, wetting agents, emulsifying agents, emulsion stabilizing
agents, suspending agents, isotonic agents, sweetening agents,
flavoring agents, perfuming agents, coloring agents, antibacterial
agents, antifungal agents, other therapeutic agents, lubricating
agents, adsorption delaying or promoting agents, and dispensing
agents, depending on the nature of the mode of administration and
dosage forms.
[0036] Exemplary suspending agents include ethoxylated isostearyl
alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite,
agar-agar and tragacanth, or mixtures of these substances.
[0037] Exemplary antibacterial and antifungal agents for the
prevention of the action of microorganisms include parabens,
chlorobutanol, phenol, sorbic acid, and the like.
[0038] Exemplary isotonic agents include sugars, sodium chloride,
and the like.
[0039] Exemplary adsorption delaying agents to prolong absorption
include aluminum monostearate and gelatin.
[0040] Exemplary adsorption promoting agents to enhance absorption
include dimethyl sulfoxide and related analogs.
[0041] Exemplary diluents, solvents, vehicles, solubilizing agents,
emulsifiers and emulsion stabilizers, include water, chloroform,
sucrose, ethanol, isopropyl alcohol, ethyl carbonate, ethyl
acetate, benzyl alcohol, tetrahydrofurfuryl alcohol, benzyl
benzoate, polyols, propylene glycol, 1,3-butylene glycol, glycerol,
polyethylene glycols, dimethylformamide, Tween.RTM. 60, Span.RTM.
60, cetostearyl alcohol, myristyl alcohol, glyceryl mono-stearate
and sodium lauryl sulfate, fatty acid esters of sorbitan, vegetable
oils (such as cottonseed oil, groundnut oil, olive oil, castor oil
and sesame oil) and injectable organic esters such as ethyl oleate,
and the like, or suitable mixtures of these substances.
[0042] Exemplary excipients include lactose, milk sugar, sodium
citrate, calcium carbonate and dicalcium phosphate.
[0043] Exemplary disintegrating agents include starch, alginic
acids and certain complex silicates. Exemplary lubricants include
magnesium stearate, sodium lauryl sulfate, talc, as well as high
molecular weight polyethylene glycols.
[0044] The choice of pharmaceutical acceptable carrier is generally
determined in accordance with the chemical properties of the active
compound such as solubility, the particular mode of administration
and the provisions to be observed in pharmaceutical practice.
[0045] Pharmaceutical compositions of the present invention
suitable for oral administration may be presented as discrete units
such as a solid dosage form, such as capsules, cachets or tablets
each containing a predetermined amount of the active ingredient, or
as a powder or granules; as a liquid dosage form such as a solution
or a suspension in an aqueous liquid or a non-aqueous liquid, or as
an oil-in-water liquid emulsion or a water-in-oil liquid emulsion.
The active ingredient may also be presented as a bolus, electuary
or paste.
[0046] "Solid dosage form" means the dosage form of the compound of
the invention is solid form, for example capsules, tablets, pills,
powders, dragees or granules. In such solid dosage forms, the
compound of the invention is admixed with at least one inert
customary excipient (or carrier) such as sodium citrate or
dicalcium phosphate or: (a) fillers or extenders, as for example,
starches, lactose, sucrose, glucose, mannitol and silicic acid, (b)
binders, as for example, carboxymethylcellulose, alginates,
gelatin, polyvinylpyrrolidone, sucrose and acacia, (c) humectants,
as for example, glycerol, (d) disintegrating agents, as for
example, agar-agar, calcium carbonate, potato or tapioca starch,
alginic acid, certain complex silicates and sodium carbonate, (e)
solution retarders, as for example paraffin, (f) absorption
accelerators, as for example, quaternary ammonium compounds, (g)
wetting agents, as for example, cetyl alcohol and glycerol
monostearate, (h) adsorbents, as for example, kaolin and bentonite,
(i) lubricants, as for example, talc, calcium stearate, magnesium
stearate, solid polyethylene glycols, sodium lauryl sulfate, (j)
opacifying agents, (k) buffering agents, and agents which release
the compound of the invention in a certain part of the intestinal
tract in a delayed manner.
[0047] A tablet may be made by compression or molding, optionally
with one or more accessory ingredients. Compressed tablets may be
prepared by compressing in a suitable machine the active ingredient
in a free-flowing form such as a powder or granules, optionally
mixed with a binder, lubricant, inert diluent, preservative,
surface active or dispersing agent. Excipients such as lactose,
sodium citrate, calcium carbonate, dicalcium phosphate and
disintegrating agents such as starch, alginic acids and certain
complex silicates combined with lubricants such as magnesium
stearate, sodium lauryl sulfate and talc may be used. A mixture of
the powdered compounds moistened with an inert liquid diluent may
be molded in a suitable machine to make molded tablets. The tablets
may optionally be coated or scored and may be formulated so as to
provide slow or controlled release of the active ingredient
therein.
[0048] Solid compositions 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.
[0049] If desired, and for more effective distribution, the
compound can be microencapsulated in, or attached to, a slow
release or targeted delivery systems such as a biocompatible,
biodegradable polymer matrices (e.g., poly(d,l-lactide
co-glycolide)), liposomes, and microspheres and subcutaneously or
intramuscularly injected by a technique called subcutaneous or
intramuscular depot to provide continuous slow release of the
compound(s) for a period of 2 weeks or longer. The compounds may be
sterilized, for example, by filtration through a bacteria-retaining
filter, or by incorporating sterilizing agents in the form of
sterile solid compositions that can be dissolved in sterile water,
or some other sterile injectable medium immediately before use.
[0050] "Liquid dosage form" means the dose of the active compound
to be administered to the patient is in liquid form, for example,
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups and elixirs. In addition to the active compound, the liquid
dosage forms may contain inert diluents commonly used in the art,
such solvents, solubilizing agents and emulsifiers.
[0051] When aqueous suspensions are used they can contain
emulsifying agents or agents which facilitate suspension.
[0052] Pharmaceutical compositions suitable for topical
administration mean formulations that are in a form suitable to be
administered topically to a patient. The formulation may be
presented as a topical ointment, salves, powders, sprays and
inhalants, gels (water or alcohol based), creams, as is generally
known in the art, or incorporated into a matrix base for
application in a patch, which would allow a controlled release of
compound through the transdermal barrier. When formulated in an
ointment, the active ingredients may be employed with either a
paraffinic or a water-miscible ointment base. Alternatively, the
active ingredients may be formulated in a cream with an
oil-in-water cream base. Formulations suitable for topical
administration in the eye include eye drops wherein the active
ingredient is dissolved or suspended in a suitable carrier,
especially an aqueous solvent for the active ingredient.
Formulations suitable for topical administration in the mouth
include lozenges comprising the active ingredient in a flavored
basis, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert basis such as gelatin
and glycerin, or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0053] The oily phase of the emulsion pharmaceutical composition
may be constituted from known ingredients, in a known manner. While
the phase may comprise merely an emulsifier (otherwise known as an
emulgent), it desirably comprises a mixture of at least one
emulsifier with a fat or an oil or with both a fat and an oil. In a
particular embodiment, a hydrophilic emulsifier is included
together with a lipophilic emulsifier that acts as a stabilizer.
Together, the emulsifier(s) with, or without, stabilizer(s) make up
the emulsifying wax, and together with the oil and fat make up the
emulsifying ointment base which forms the oily dispersed phase of
the cream formulations.
[0054] If desired, the aqueous phase of the cream base may include,
for example, a least 30% w/w of a polyhydric alcohol, i.e. an
alcohol having two or more hydroxyl groups such as, propylene
glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and
polyethylene glycol (including PEG 400) and mixtures thereof. The
topical formulations may desirably include a compound that enhances
absorption, or penetration of the active ingredient through the
skin, or other affected areas.
[0055] The choice of suitable oils or fats for a composition is
based on achieving the desired properties. Thus a cream should
particularly be a non-greasy, non-staining and washable product
with suitable consistency to avoid leakage from tubes or other
containers. Straight or branched chain, mono- or dibasic alkyl
esters such as di-isopropyl myristate, decyl oleate, isopropyl
palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of
branched chain esters known as Crodamol CAP may be used. These may
be used alone or in combination depending on the properties
required. Alternatively, high melting point lipids such as white
soft paraffin and/or liquid paraffin or other mineral oils can be
used.
[0056] Pharmaceutical compositions suitable for rectal or vaginal
administrations mean formulations that are in a form suitable to be
administered rectally or vaginally to a patient and containing at
least one compound of the invention. Suppositories are a particular
form for such formulations that can be prepared by mixing the
compounds of this invention with suitable non-irritating excipients
or carriers such as cocoa butter, polyethylene glycol or a
suppository wax, which are solid at ordinary temperatures but
liquid at body temperature and therefore, melt in the rectum or
vaginal cavity and release the active component.
[0057] Pharmaceutical composition administered by injection may be
by transmuscular, intravenous, intraperitoneal, and/or subcutaneous
injection. The compositions of the present invention are formulated
in liquid solutions, in particular in physiologically compatible
buffers such as Hank's solution or Ringer's solution. In addition,
the compositions may be formulated in solid form and redissolved or
suspended immediately prior to use. Lyophilized forms are also
included. The formulations are sterile and include emulsions,
suspensions, aqueous and non-aqueous injection solutions, which may
contain suspending agents and thickening agents and anti-oxidants,
buffers, bacteriostats and solutes which render the formulation
isotonic, and have a suitably adjusted pH, with the blood of the
intended recipient.
[0058] Pharmaceutical composition of the present invention suitable
for nasal or inhalational administration means compositions that
are in a form suitable to be administered nasally or by inhalation
to a patient. The composition may contain a carrier, in a powder
form, having a particle size for example in the range 1 to 500
microns (including particle sizes in a range between 20 and 500
microns in increments of 5 microns such as 30 microns, 35 microns,
etc.). Suitable compositions wherein the carrier is a liquid, for
administration as for example a nasal spray or as nasal drops,
include aqueous or oily solutions of the active ingredient.
Compositions suitable for aerosol administration may be prepared
according to conventional methods and may be delivered with other
therapeutic agents. Inhalational therapy is readily administered by
metered dose inhalers or any suitable dry powder inhaler, such as
the Eclipse, Spinhaler.RTM., or Ultrahaler.RTM. as described in
patent application WO2004/026380, and U.S. Pat. No. 5,176,132.
[0059] Actual dosage levels of active ingredient(s) in the
compositions of the invention may be varied so as to obtain an
amount of active ingredient(s) that is (are) effective to obtain a
desired therapeutic response for a particular composition and
method of administration for a patient. A selected dosage level for
any particular patient therefore depends upon a variety of factors
including the desired therapeutic effect, on the route of
administration, on the desired duration of treatment, the etiology
and severity of the disease, the patient's condition, weight, sex,
diet and age, the type and potency of each active ingredient, rates
of absorption, metabolism and/or excretion and other factors.
[0060] Total daily dose of the compound of this invention
administered to a patient in single or divided doses may be in
amounts, for example, of from about 0.001 to about 100 mg/kg body
weight daily and particularly 0.01 to 10 mg/kg/day. For example, in
an adult, the doses are generally from about 0.01 to about 100,
particularly about 0.01 to about 10, mg/kg body weight per day by
inhalation, from about 0.01 to about 100, particularly 0.1 to 70,
more especially 0.5 to 10, mg/kg body weight per day by oral
administration, and from about 0.01 to about 50, particularly 0.01
to 10, mg/kg body weight per day by intravenous administration.
[0061] The percentage of active ingredient in a composition may be
varied, though it should constitute a proportion such that a
suitable dosage shall be obtained. Dosage unit compositions may
contain such amounts or such submultiples thereof as may be used to
make up the daily dose. Obviously, several unit dosage forms may be
administered at about the same time. A dosage may be administered
as frequently as necessary in order to obtain the desired
therapeutic effect. Some patients may respond rapidly to a higher
or lower dose and may find much lower maintenance doses adequate.
For other patients, it may be necessary to have long-term
treatments at the rate of 1 to 4 doses per day, in accordance with
the physiological requirements of each particular patient. It goes
without saying that, for other patients, it will be necessary to
prescribe not more than one or two doses per day.
[0062] The formulations can be prepared in unit dosage form by any
of the methods well known in the art of pharmacy. Such methods
include the step of bringing into association the pharmaceutically
active ingredient with the carrier that constitutes one or more
accessory ingredients. In general the formulations are prepared by
uniformly and intimately bringing into association the active
ingredient with liquid carriers or finely divided solid carriers or
both, and then, if necessary, shaping the product.
[0063] The formulations may be presented in unit-dose or multi-dose
containers, for example sealed ampoules and vials with elastomeric
stoppers, and may be stored in a freeze-dried (lyophilized)
condition requiring only the addition of the sterile liquid
carrier, for example water for injections, immediately prior to
use. Extemporaneous injection solutions and suspensions may be
prepared from sterile powders, granules and tablets of the kind
previously described.
[0064] Compounds of the invention may be prepared by the
application or adaptation of known methods, by which is a meant
method used heretofore or described in the literature, for example
those described by R. C. Larock in Comprehensive Organic
Transformations, VCH publishers, 1989.
[0065] In the reactions described hereinafter it may be necessary
to protect reactive functional groups, for example hydroxy, amino,
imino, thio or carboxy groups, where these are desired in the final
product, to avoid their unwanted participation in the reactions.
Conventional protecting groups may be used in accordance with
standard practice, for examples see T. W. Greene and P. G. M. Wuts,
Protecting Groups in Organic Synthesis, 3rd edition, John Wiley
& Sons, Inc., 1999.
[0066] A compound of formula (I) may be prepared (as shown in
Scheme I below) by reacting a amine of type XI with a
pyridylpyrimidinyl carboxylic acid (preparation shown in Scheme II)
in the presence of a dehydrating coupling reagent, such as DMTMM,
in a variety of solvents including but not limited to DMF. Suitable
couple reagents include, but are not limited to, DMTMM,
carbonyldiimidazole (CDI) and TBTU, DCC, phosphonium salts, and
uronium salts. A compound of formula (I) may also be prepared (as
shown in Scheme Ia below) by direct coupling of an amine of type XI
with a pyridylpyrimidinyl ester (preparation shown in Scheme II) in
the presence of 0.1 to 1.0 equivalents of
1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD). The reaction can be
performed in the absence of solvent, or in the presence of added
solvents, including, but not limited to ethers, esters, aromatic
hydrocarbons. The use of strong bases other than TBD, including,
but not limited to DBU and tetramethyl guanidine, also give
product. The amine XI may be prepared through a process as detailed
in Scheme III. A benzylic bromide VII may be reacted with
Di-tert-butyl iminodicarboxylate in the presence of bases including
but not limited to cesium carbonate in a variety of solvents
including but not limited to DMF to yield compounds VIII. These
compounds of type VIII may then be reacted with hydroxylamine (in
the presence of bases including but not limited to triethylamine in
the cases where salts of hydroxylamine are used, such as
hydroxylamine hydrochloride) in a variety of solvents including but
not limited to methanol to yield an amidoxime IX. The amidoxime may
be reacted with a compound containing a carboxy functionality
including but not limited to a methyl carboxylate in the presence
of a base including but not limited to potassium carbonate either
in the presence or absence of a solvent including but not limited
to toluene (in certain cases the carboxy functionality may serve as
a solvent for the reaction) to yield an oxadiazole X. The
oxadiazole X may then be exposed to acidic conditions including but
not limited to hydrogen chloride in methanol to yield an amine XI.
In the cases where R1 alkyl substitution is desired in amine XI,
these amines may be prepared according to Scheme IV (either in
enantioenriched or racemic form) using the tert-butyl sulfinamide
methodology developed by Ellman.
##STR00003##
##STR00004##
##STR00005##
##STR00006##
[0067] It will be appreciated that compounds of the present
invention may contain asymmetric centers. These asymmetric centers
may independently be in either the R or S configuration. It will be
apparent to those skilled in the art that certain compounds of the
invention may also exhibit geometrical isomerism. It is to be
understood that the present invention includes individual
geometrical isomers and stereoisomers and mixtures thereof,
including racemic mixtures, of compounds of Formula (I)
hereinabove. Such isomers can be separated from their mixtures, by
the application or adaptation of known methods, for example
chromatographic techniques and recrystallization techniques, or
they are separately prepared from the appropriate isomers of their
intermediates.
[0068] The compounds of the invention, their methods or preparation
and their biological activity will appear more clearly from the
examination of the following examples that are presented as an
illustration only and are not to be considered as limiting the
invention in its scope. Compounds of the invention are identified,
for example, by the following analytical methods.
[0069] Mass Spectra (MS) are recorded using a Micromass LCT mass
spectrometer. The method is positive electrospray ionization,
scanning mass m/z from 100 to 1000.
[0070] 300 MHz .sup.1H nuclear magnetic resonance spectra (.sup.1H
NMR) are recorded at ambient temperature using a Varian Mercury
(300 MHz) spectrometer with an ASW 5 mm probe. In the .sup.1H NMR
chemical shifts (.delta.) are indicated in parts per million (ppm)
with reference to tetramethylsilane (TMS) as the internal
standard.
[0071] As used in the examples and preparations that follow, as
well as the rest of the application, the terms used therein shall
have the meanings indicated: "kg"=kilograms, "g"=grams,
"mg"=milligrams, "g"=micrograms, "mol"=moles, "mmol"=millimoles,
"M"=molar, "mM"=millimolar, "M"=micromolar, "nM"=nanomolar,
"L"=liters, "mL" or "ml"=milliliters, "L"=microliters, ".degree.
C."=degrees Celsius, "mp" or "m.p."=melting point, "bp" or
"b.p."=boiling point, "mm of Hg"=pressure in millimeters of
mercury, "cm"=centimeters, "nm"=nanometers, "abs."=absolute,
"conc."=concentrated, "c"=concentration in g/mL, "rt"=room
temperature, "TLC"=thin layer chromatography, "HPLC"=high
performance liquid chromatography, "i.p."=intraperitoneally,
"i.v."=intravenously, "s"=singlet, "d"=doublet; "t"=triplet;
"q"=quartet; "m"=multiplet, "dd"=doublet of doublets; "br"=broad,
"LC"=liquid chromatograph, "MS"=mass spectrograph,
"ESI/MS"=electrospray ionization/mass spectrograph,
"R.sub.T"=retention time, "M"=molecular ion, "PSI"=pounds per
square inch, "DMSO"=dimethyl sulfoxide,
"DMF"=N,N-dimethylformamide, "DCM"=dichloromethane,
"HCl"=hydrochloric acid, "SPA"=Scintillation Proximity Assay,
"EtOAc"=ethyl acetate, "PBS"=Phosphate Buffered Saline,
"IUPAC"=International Union of Pure and Applied Chemistry,
"MHz"=megahertz, "MeOH"=methanol, "N"=normality.
"THF"=tetrahydrofuran. "min"=minute(s), "N.sub.2"=nitrogen gas,
"MeCN" or "CH.sub.3CN"=acetonitrile, "Et.sub.2O"=ethyl ether,
"TFA"=trifluoroacetic acid, ".about."=approximately,
"MgSO.sub.4"=magnesium sulfate, "Na.sub.2SO.sub.4"=sodium sulfate,
"NaHCO.sub.3"=sodium bicarbonate, "Na.sub.2CO.sub.3"=sodium
carbonate, "MCPBA"=3-Chloroperoxybenzoic acid,
"NMP"=N-methylpyrrolidone, "PS-DCC"=polymer
supported-dicyclohexylcarbodiimide, "LiOH"=Lithium hydroxide,
"PS-trisamine"=polymer supported-trisamine, "PGH2"=prostaglandin
H2, "PGD2"=prostaglandin D2; "PGE2"=prostaglandin E2,
"hPGDS"=Hematopoietic PGD2 Synthase, "GSH"=glutathione (reduced),
"EIA"=Enzyme immunoassay, "KH.sub.2PO.sub.4"=potassium phosphate,
monobasic, "K.sub.2HPO.sub.4"=potassium phosphate, dibasic,
"FeCl.sub.2"=ferrous chloride, "MOX"=methoxylamine; "EtOH"=ethanol,
"DMSO"=dimethylsulfoxide, "Ag.sub.2O"=silver(I) oxide,
"HATU"=O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate, "HOAt"=1-hydroxy-7-azabenzotriazole,
"DIPEA"=N,N-diisopropylethylamine,
"HOTT"=S-(1-Oxido-2-pyridyl)-N,N,N,N'-tetramethylthiuronium
hexafluorophosphate,
"HCTU"=N,N,N',N'-tetramethyl-O-(6-chloro-1H-benzotriazol-1-yl)uronium
hexafluorophosphate, "PyBrOP"=bromo-tris-pyrrolidinophosphonium
hexafluorophosphate, "LiAlH4"=lithium aluminum hydride,
"PyAOP"=(7-azabenzotriazol-1-yloxy)-tripyrrolidinophosphonium
hexafluorophosphate,
"TBTU"=O-benzotriazol-1-yl-N,N,N,N,-tetramethyluronium
tetrafluoroborate, "NaHMDS"=sodium bis(trimethylsilyl)amide,
"NMP"=N-methyl-2-pyrrolidinone, "HOSA"=hydroxylamine-O-sulfonic
acid,
"DMTMM"=4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride, "TMSN.sub.3"=trimethylsilyl azide,
"TBAF"=tetrabutylammonium fluoride, "TFAA"=trifluoro acetic
anhydride.
EXAMPLES
[0072] Following procedures similar to those described in the above
examples, the following compounds are made:
Example 1
2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-benzylamide
##STR00007##
[0073] Step 1
##STR00008##
[0075] 3-Bromomethyl-benzonitrile (42.9 g, 219 mmol, 1 equivalent)
is combined with Di-tert-butyl iminodicarboxylate (50 g, 230.13
mmol, 1.05 equivalents) and cesium carbonate (74.98 g, 230.13 mmol,
1.05 equivalents) in N,N-dimethyl formamide (DMF) (230 mL). The
reaction is stirred at room temperature overnight and then
partitioned between diethyl ether (500 mL) and water (1 L). The
aqueous layer is extracted with an additional portion of diethyl
ether (250 mL) and the combined ether layers are washed with brine
(2.times.200 mL). The organic layer is then dried (MgSO.sub.4),
filtered, and reduced in vacuo to yield oil that slowly
crystallized to give 2-[(3-cyanophenyl)methyl]-imidodicarbonic acid
1,3-bis(1,1-dimethylethyl) ester (72 g, 99%). MS: 333 (M+H), 355
(M+Na).
[0076] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=1.47 (s, 18H),
4.79 (s, 2H), 7.42 (t, 1H), 7.54-7.60 (m, 3H).
Step 2
##STR00009##
[0078] Hydroxylamine hydrochloride (23.43 g, 375 mmol, 2.5
equivalents) is added to a solution of
2-[(3-cyanophenyl)methyl]-imidodicarbonic acid
1,3-bis(1,1-dimethylethyl) ester (50 g, 150 mmol, 1 equivalent) in
methanol (450 mL) and the mixture is chilled in an ice water bath.
Triethylamine (37.87 g, 375 mmol, 2.5 equivalents) is added and the
reaction mixture is allowed to stir overnight, warming up to room
temperature slowly as the bath thaws. The reaction is then reduced
in vacuo and the residue partitioned between ethyl acetate (1 L)
and water (500 mL). The water layer is extracted with an additional
portion of ethyl acetate (200 mL) and the combined organic layers
are washed with brine (200 mL), dried over sodium sulfate, and
filtered. At this point, heptane and toluene (100 mL each) are
added and the reaction reduced in vacuo to yield
2-[[3-[(hydroxyamino) iminomethyl]phenyl]methyl]-imidodicarbonic
acid 1,3-bis(1,1-dimethylethyl) ester as a clear gel (54.7 g
(>99%), that is used directly without further purification.
Step 3
##STR00010##
[0080] Potassium carbonate (4.35 g, 31.46 mmol, 1.15 equivalents)
is added to a flask charged with 2-[[3-[(hydroxyamino)
iminomethyl]phenyl]methyl]-imidodicarbonic acid
1,3-bis(1,1-dimethylethyl) ester (10 g, 27.36 mmol, 1 equivalent)
from step 2 in Toluene (30 mL), followed by
2-Hydroxy-2-methyl-propionic acid methyl ester (3.716 g, 31.46
mmol, 1.15 equivalents). The reaction is heated to reflux. After 48
hours the reaction is partitioned between EtOAc (300 mL) and water
(200 mL). The EtOAc is washed with Brine (100 mL), dried over
sodium sulfate, filtered and then reduced in vacuo to yield a
residue that is taken on directly.
[0081] A solution of 4N HCl in dioxane (60 mL) is added to an ice
chilled mixture of the residue (27 mmol) from the previous reaction
in p-dioxane (60 mL). The ice water bath is removed and the
reaction is allowed to warm to room temperature. After 6 hours, the
reaction is diluted with diethyl ether (200 mL). The white solid is
collected via filtration, washed with diethyl ether (.about.50 mL)
and then dried in vacuo to yield
3-[5-(1-Hydroxy-1-methyl-ethyl)-[1,2,4]oxadiazol-3-yl]-benzyl-amine
hydrochloride (5.84 g, 79% over two steps). MS: 234 (M+H). .sup.1H
NMR (300 MHz, DMSO): .delta.=1.626 (s, 6H), 4.13-4.15 (d, 2H), 6.11
(bs, 1H), 7.62 (t, 1H), 7.72 (d, 1H), 8.02 (d, 1H), 8.15 (s, 1H),
8.45 (bs, 3H).
Step 4
##STR00011##
[0083] N-methyl morpholine (NMM) (1.12 g, 11.12 mmol, 1 equivalent)
is added to a mixture of 2-Pyridin-2-yl-pyrimidine-5-carboxylic
acid (2.24 g, 11.12 mmol, 1 equivalent) and
3-[5-(1-Hydroxy-1-methyl-ethyl)-[1,2,4]oxadiazol-3-yl]-benzyl-amine
hydrochloride (3 g, 11.12 mmol, 1 equivalent) in DMF (50 mL). After
stirring at room temperature for 5 minutes,
4-(4,6-Dimethoxy-[1,3,5]triazin-2-yl)-4-methyl-morpholin-4-ium
chloride (DMTMM) (3.08 g, 11.12 mmol, 1 equivalent) is added and
the reaction stirred at room temperature, for 3 hours.
[0084] The reaction is diluted into ice water (500 mL) and the
suspension is extracted with EtOAc (2.times.300 mL). The combined
ethyl acetate layers are washed with brine (2.times.100 mL), dried
over sodium sulfate, and reduced in vacuo to give crude product
which is recrystallized using ethyl acetate/ethanol to yield
2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-benzylamide
as white crystalline solid (1.95 g, 42%) Note: Yields are variable
depending on the purity of the coupling partners and solvents used
for recrystallization. MS: 417 (M+H). .sup.1H NMR (300 MHz, DMSO):
.delta.=1.62 (s, 6H), 4.65 (d, 2H), 6.08 (s, 1H), 7.54-7.63 (m,
3H), 7.93 (d, 1H), 7.99-8.04 (m, 2H), 8.45 (d, 1H), 8.79 (d, 1H),
9.37 (s, 2H), 9.57 (t, 1H).
[0085] Alternatively, the coupling may be achieved using CDI
(carbonyldiimidazole) or TBTU. The coupling shown below may be done
in, for example, DMF and/or THF.
##STR00012##
[0086] To a 5 L jacketed reactor was added 68.89 g of the
carboxylic acid and about 346 ml DMF. To this slurry was added 74.9
g of the CDI at 22.+-.2.degree. C. The amine (79.87 g) was
dissolved in about 69 mL DMF and added over 8 minutes. This turned
the thick slurry into a clear yellow/brown solution. The
temperature increased to 35.degree. C. Heptane (202 ml) was added
followed by water (596 ml) slowly over 20 minutes. During water
addition, the temperature increased from 22 to 33.degree. C. As the
reaction mixture was stirred, crystals began to form. Water (5.15
L) was added. The reaction mixture was filtered on a 185 mm
diameter Buchner and washed with 2.times.750 mL water. The cake was
collected and dried under vacuum (45.degree. C., 100 mbar pressure,
nitrogen flush) to yield 122.15 g of product.
[0087] HPLC method: Eclipse XDB phenyl column, 3.5 micron,
4.6.times.150 mm, detection at 254 nm, gradient: started at
5:95:0.1% ACN/water/TFA then ramped over 8 min to 70:30:0.1%
ACN/water/TFA, held 4.5 min; product retention time: 6.5 min.
[0088] Alternatively, the coupling may proceed via the acid
chloride as shown below.
##STR00013##
[0089] A 100 mL 3 neck round bottom flask equipped with magnetic
stirring, temperature controller, and Firestone Valve (N2) was
charged with
2-[3-(3-aminomethylphenyl)-[1,2,4]-oxadiazol-5-yl]-propan-2-ol free
base (600 mg, 2.57 mmole, 1 eq), NMP (5 mL) and triethylamine (2.25
mL). 2-Pyridin-2-yl-pyrimidine-5-carbonyl chloride HCl (0.7 g, 2.7
mmole, ca 96% acid) was added. The reaction was quenched after
about 2.5 hours by adding toluene (5 mL) and water (5.times.10 mL).
The reaction was filtered and the cake was washed with toluene and
water to yield a solid (0.85 g, 79% yield).
[0090] .sup.1H NMR (300 MHz, d.sub.6-DMSO): .delta.=1.61 (s, 6H),
4.64 (d, 2H), 6.08 (s, 1H), 7.6 (m, 3H), 7.95 (d, 1H), 8.04 (m,
2H), 8.45 (d, 1H), 8.8 (d, 1H), 9.37 (s, 1H), 9.57 (t, 1H)
Example 1a
Alternative Synthesis for
3-[5-(1-Hydroxy-1-methyl-ethyl)-[1,2,4]oxadiazol-3-yl]-benzyl-amine
hydrochloride
##STR00014##
[0091] Scheme V--Step 1
[0092] A 5-L jacketed glass reactor equipped with an overhead
mechanical stirrer, a thermocouple probe and a nitrogen purge was
charged at 20-25.degree. C. with 3-cyanobenzaldehyde (100.0 g,
0.763 mol, 1.0 eq.) and ethanol (200 proof) (394.5 g, 500 mL, 5 v/w
parts). To the suspension was charged via addition funnel, a
solution of hydroxylamine hydrochloride (159.0 g, 2.288 mole, 3.0
eq.) in water (250 mL, 2.5 parts) over a period of 30-45 min while
maintaining temperature of 20-25.degree. C. The addition funnel was
rinsed with water (20 mL) and the rinse was added to the reactor.
After addition of ca 45 mL of NH.sub.2OH.HCl solution, the solid
dissolved to provide a clear solution. Within 10 min the solution
turned cloudy and a solid crystallized to provide a suspension. The
solid is believed to be the oxime resulting from addition of
hydroxylamine to the aldehyde function. The suspension was stirred
at 20-25.degree. C. for 1 h. To the suspension was charged via a
addition funnel, a solution of sodium carbonate (121.25 g, 1.144
mole, 1.5 eq,) in water (390 mL, 3.9 parts) over a period of
1.5-2.0 h while maintaining a temperature of 20-22.degree. C. The
addition funnel was rinsed with water (20 mL) and the rinse was
added to the reactor. Evolution of CO.sub.2 was observed. The
suspension was heated to 29-30.degree. C. and stirred at
29-30.degree. C. for 24 h. Water (1.32 L, 13.2 parts) was charged
to the reactor over 45-60 min while maintaining a temperature of
30-32.degree. C. The suspension was heated to and held at
76-78.degree. C. for 30-60 min to get a clear solution. The
solution was cooled to 55-60.degree. C. over 90 min. Product
crystallized at 55-60.degree. C. The suspension was stirred at
55-60.degree. C. for 60 min. The suspension was cooled to
20-22.degree. C. over 8-12 h. The suspension was cooled to
2-5.degree. C. and stirred at 2-5.degree. C. for 4 h. The
suspension was filtered (Buchner funnel, 14.5 cm o.d.) and the cake
was washed with water (250 mL, 2.5 parts). The cake was dried under
suction for 5 h. The cake was transferred to a drying dish and
dried under vacuum (25-50 torr, 50.degree. C., N.sub.2) for 60 h to
provide 127.33 g (93.2% yield) of product as a white crystalline
solid with a purity of 99.9% (HPLC).
[0093] HPLC Method: Zorbax Eclipse XDB C8 column, 5 micron,
4.6.times.150 mm, 25.degree. C., detection at 240 nm, gradient:
5:95:0.1 CH.sub.3CN/H.sub.2O/TFA isocratic 2 min then ramped over
16 min to 90:10:0.1 CH.sub.3CN/H.sub.2O/TFA; product retention
time: 3.6-4.4 min (three peaks)
Scheme V--Step 2
[0094] A 5-L jacketed glass reactor equipped with an overhead
mechanical stirrer, a thermocouple probe and a nitrogen purge was
charged at 22-27.degree. C. with
(N-hydroxy-3-hydroxyiminomethyl)benzamidine) (100.0 g, 0.558 mol,
1.0 eq.) and 1-methyl-2-pyrrolidinone (NMP) (267.3 g, 260 mL, 2.6
v/w parts). To the suspension was charged via addition funnel
methyl 2-hydroxyisobutyrate (197.8 g, 1.674 mole, 3.0 eq.) over
15-30 min while maintaining temperature of 25-27.degree. C. The
mixture was stirred at 25-27.degree. C. for 30-45 min to get a
clear solution. To the solution was charged via an addition funnel
25 w % sodium methoxide solution in methanol (361.7 g, 1.674 mole,
3.0 eq.) over 30-60 min while maintaining a temperature of
25-27.degree. C. The solution was heated at 29-30.degree. C. for 7
hours. After 30-45 min at 29-30.degree. C., the solution turned
into a suspension. Water (1.8 L, 18 parts) was charged via an
addition funnel over 30-60 min while maintaining a temperature of
22-25.degree. C. The suspension dissolved to provide a clear
solution with a pH 12.2 (pH meter). The pH of the solution was
adjusted to 5.0 by charging hydrochloric acid (37.1 w %) (77.4 g,
0.787 mole, 1.4 eq) over 30-45 min while maintaining a temperature
of 22-25.degree. C. The product crystallized upon acidification
with hydrochloric acid. After cooling to 5-10.degree. C. and
stirring at 5-10.degree. C. for 2 h, the suspension was filtered
(Buchner funnel, 27.5 cm i.d.), and the cake was washed with water
(700 mL, 7 parts) and dried under suction for 7 h. The cake was
transferred to a drying dish and dried under vacuum (25-50 torr,
50.degree. C., N2) for 20-24 h to provide 132.0 g (95.6% yield) of
product as a white crystalline solid with a purity of 99.7%
(HPLC).
[0095] HPLC Method: Zorbax Eclipse XDB C8 column, 5 micron,
4.6.times.150 mm, 25.degree. C., detection at 240 nm, gradient:
5:95:0.1 CH.sub.3CN/H.sub.2O/TFA isocratic 2 min then ramped over
16 min to 90:10:0.1 CH.sub.3CN/H.sub.2O/TFA; product retention
time: 10.8 min
Scheme V--Step 3
[0096] A 5-L jacketed glass reactor equipped with an overhead
mechanical stirrer, a thermocouple probe and a nitrogen purge was
charged at 20-25.degree. C. with
3-[5-(1-hydroxy-1-methylethyl)-[1,2,4]oxadiazol-3-yl]benzaldehyde
oxime (100.0 g, 0.404 mol, 1.0 eq.) and glacial acetic acid (1888.2
g, 1.8 L, 18 v/w parts). The suspension was heated to 28-30.degree.
C. and stirred till a clear solution was obtained (30-45 min). The
solution was cooled to 22-24.degree. C. and zinc dust (105.8 g,
1.618 mole, 4.0 eq.) was added via an addition funnel over 90-120
min while maintaining a temperature of 22-26.degree. C. Note: Zinc
dust addition was exothermic. The suspension was stirred at
24-26.degree. C. for 2-3 hours. The suspension was filtered under
N.sub.2 (an inverted funnel with N.sub.2 supply) through Celite (40
g). The solids were washed with EtOH (200 proof)/H.sub.2O (1/1,
894.5 g, 1 L, 10 parts) and EtOH (200 proof) (250 mL, 197.3 g, 2.5
parts). The filtrate was transferred to a 5-L reactor and
concentrated under reduced pressure (45-50 torr, 44-47.degree. C.,
jacket temperature 50-55.degree. C.) to a volume of ca. 350 mL (3.5
parts). The vacuum was broken with N.sub.2 and the reactor was
cooled to 22.degree. C. The mixture was a thick suspension. Toluene
(2162.5 g, 2.5 L, 25 parts) was charged to the reactor and
suspension was concentrated under reduced pressure (70-75 torr,
42-47.degree. C., jacket temperature 50-55.degree. C.) to a volume
of ca 350 mL (3.5 parts). Vacuum was broken with N.sub.2 and the
reactor was charged with toluene (129.8 g, 150 mL, 1.5 parts) at
22.degree. C. The suspension was stirred at 22.degree. C. for 15-20
minutes and the phases were allowed to separate. The upper layer is
mainly toluene and the lower layer contains the acetate salt of
desired product.
[0097] HPLC Method: Zorbax Eclipse XDB C8 column, 5 micron,
4.6.times.150 mm, 25.degree. C., detection at 240 nm, gradient:
5:95:0.1 CH.sub.3CN/H.sub.2O/TFA isocratic 2 min then ramped over
16 min to 90:10:0.1 CH.sub.3CN/H.sub.2O/TFA; product retention
time: 7.9 min
Scheme V--Step 4a
[0098] 2-MeTHF (1290.0 g, 1.5 L, 15 parts) was added to the
reactor. Aqueous ammonium hydroxide (29.5 w %) (353.8 g, 400 mL, 4
parts) was charged via an addition funnel over 30-45 min while
maintaining a temperature of 20-25.degree. C. The mixture was
stirred at 22-25.degree. C. for 30-45 min and the phases were
allowed to separate. The pH of the aqueous phase should be basic
(pH observed 10.9). The organic phase was washed with 15.3 w %
aqueous sodium chloride (2.times.442.1 g, 2.times.400 mL, 2.times.4
parts). Note: 15.3 w % aq. NaCl was prepared by dissolving NaCl
(180 g) in water (1000 g). The organic phase was concentrated under
reduced pressure (100-110 torr, 30-34.degree. C., jacket
temperature 35-40.degree. C.) to a volume of ca 900 mL (9 parts).
Vacuum was broken with N.sub.2 and the solution was filtered to
remove a small amount of NaCl (ca 400 mg). The funnel was rinsed
with 2-MeTHF (86.0 g, 100 mL, 1 part) to provide a solution of
2-[3-(3-aminomethylphenyl)-[1,2,4]-oxadiazol-5-yl]-propan-2-ol free
base in 2-MeTHF/toluene (899.0 g, 1 L, 10 parts). Assay (w/w) of
the solution provided the product (83.61 g, 9.3 w %) in 88.7% yield
with a purity of 95.1 A % (HPLC); 2-MeTHF 68.7 w % and toluene 21.2
w %.
[0099] HPLC Method: Zorbax Eclipse XDB C8 column, 5 micron,
4.6.times.150 mm, 25.degree. C., detection at 240 nm, gradient:
5:95:0.1 CH.sub.3CN/H.sub.2O/TFA isocratic 2 min then ramped over
16 min to 90:10:0.1 CH.sub.3CN/H.sub.2O/TFA; product retention
time: 7.8 min
Scheme V--Step 4
[0100] A 5 L reactor equipped with mechanical stirrer, thermocouple
probe and N2 inlet was charged with THF (1.5 L) and
2-[3-(3-aminomethylphenyl)-[1,2,4]-oxadiazol-5-yl]-propan-2-ol AcOH
(111.27 g). The solution turned into a suspension. A solution of
Na.sub.2CO.sub.3 (85.73 g) in water (600 ml) was added slowly with
cooling (thermocouple at 15.degree. C.). The reaction was stirred
at room temperature for about 10 minutes. Di-tert-butyl dicarbonate
(97.1 g) in THF (90 mL) was added via a dropping funnel over about
12 minutes with cooling (thermocouple set to 15.degree. C.). The
reaction mixture was warmed (thermocouple set to 22.degree. C.).
The mixture separates into two distinct layers after first
appearing as a suspension and then becomes a suspension again.
Ethyl acetate (750 mL) was added and the suspension was stirred for
15 minutes at room temperature. Celite (545 (25 g) was added to the
reactor and the mixture was stirred for 15 minutes. The slurry was
transferred to a 4 L Erlenmeyer flask. It was filtered through
Celite 545 (sintered glass funnel, Kimax 2000 mL-125 C charged with
100 g of Celite 545). The Celite/zinc salts were washed with ethyl
acetate (500 mL). The organic layer was collected and washed with
1/1 H2O/sat. aq NaCl (2.times.500 mL), pH of aqueous layer 5-7. The
filtrate was charged to a clean reactor and the reactor was fitted
with a one-piece distillation apparatus (P=250 torr, .DELTA.p=5
torr, thermocouple set to 40.degree. C.). When the volume of liquid
in reactor was about 250 mL, the pressure was equalized with N2 and
the reaction was cooled (thermocouple set to 22.degree. C.). The
reactor was charged with ethyl acetate (1500 mL). Resumed
distillation (P=180-200 torr, .DELTA.p=5 torr, thermocouple set to
50.degree. C.) till volume of solution in the reactor was ca 500
mL. The pressure was equalized with N2 and the reaction was cooled
(thermocouple set to 22.degree. C.). Yield of
{3-[5-(1-hydroxy-1-methylethyl)-[1,2,4]oxadiazol-3-yl]-benzyl-carbamic
acid tert-butyl ester=126.46 g (quant., solution in ethyl acetate).
The solution was used in step 5
[0101] HPLC Method: Zorbax Eclipse XDB C8 column, 5 micron,
4.6.times.150 mm, 25.degree. C., detection at 240 nm, gradient:
5:95:0.1 CH.sub.3CN/H.sub.2O/TFA isocratic 2 min then ramped over
16 min to 90:10:0.1 CH.sub.3CN/H.sub.2O/TFA; product retention
time: 13.8 min
Scheme V--Step 5
[0102] A 5 L reactor equipped with a mechanical stirrer,
thermocouple and N2 inlet was charged with
{3-[5-(1-hydroxy-1-methylethyl)-[1,2,4]oxadiazol-3-yl]-benzyl-carbamic
acid tert-butyl ester (126.46 g) as solution in ethyl acetate (from
step 4). The solution was cooled (3-15.degree. C.). Added HCl gas
(102 g) from a lecture bottle over 30 minutes. The reaction was
warmed to 15.degree. C. over 45 minutes and a slurry was formed.
This slurry was transferred to an Erlenmeyer flask (1 L). The
contents were then filtered using a Buchner funnel. The cake was
rinsed with ethyl acetate (350 mL) and suction dried. The solid was
then transferred to a drying dish and dried (0.9'' Hg, 35 C, N2) to
yield 83.52 g of a solid (76.6% overall yield steps 3-5).
[0103] HPLC Method: Zorbax Eclipse XDB C8 column, 5 micron,
4.6.times.150 mm, 25.degree. C., detection at 240 nm, gradient:
5:95:0.1 CH.sub.3CN/H.sub.2O/TFA isocratic 2 min then ramped over
16 min to 90:10:0.1 CH.sub.3CN/H.sub.2O/TFA; product retention
time: 8.0 min
Example 1b
##STR00015##
[0105] A reactor with stirring and a nitrogen blanket was charged
with 2-Me-THF (5 mL), the ester (500 mg), the benzylamine (545 mg)
and 1,5,7-triazabicyclo[4,4,0]dec-5-ene (TBD) (97.5 mg, 0.3 eq) to
yield a yellowish suspension. The reactor was positioned into a
heating block that was preheated to 79.degree. C. The reaction was
stirred for about 3 hours, then was removed from the block, allowed
to cool to room temperature, then placed in an ice bath, stirred 15
minutes, and filtered. The reactor and cake were rinsed with 1 mL
cold 2-Me-THF. The white caked was rinsed with 5.times.2 mL water
at room temperature and suction dried for 1.5 hours. The white
solid (0.77 g) was transferred to an oven and heated at 70.degree.
C. (N2, 45 mbar) overnight. Yield: 750 mg, 77%.
[0106] Alternative work up: After a reaction employing 2-Me-THF (4
mL), the ester (300 mg), the benzylamine (327 mg) and
1,5,7-triazabicyclo[4,4,0]dec-5-ene (TBD) (58.5 mg, 0.3 eq) was
complete, the mixture was partitioned with 2 mL of water and
cooled. The organic phase was separated, diluted with 2 mL of
2-Me-THF, then was washed with 5 mL of water. The combine aqueous
phase was extracted with 2 mL of 2-Me-THF. The combined organic
phase was concentrated and dried. Yield: 0.57 g, 97%.
[0107] HPLC method: Eclipse XDB C8 column, 5 micron, 4.6.times.150
mm, 35.degree. C., detection at 270 nm, gradient: 5:95:0.1%
ACN/water/TFA held 5 min then ramped over 7 min to 50:50:0.1%
ACN/water/TFA, held 3 min: product retention time: 12.9 min.
Example 2
2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
((S)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-eth-
yl)-amide
##STR00016##
[0108] Step 1
##STR00017##
[0110] 3-Acetylbenzonitrile (5 g, 34.4 mmol) is added to a flask
containing (R)-(+)-2-Methyl-2-propanesulfinamide (3.48 g, 28.7
mmol) and titanium (IV) ethoxide (13.1 g, 57.4 mmol) in THF (70 mL)
and the reaction mixture heated at 75.degree. C. overnight. The
reaction mixture is cooled (-48.degree. C.) and L-Selectride (1M
solution in THF, 57.4 mL) added dropwise over 1 hour. The reaction
stirred for 2 hrs and allowed to warm to room temperature. The
reaction is then cooled to 0.degree. C. and methanol (3 mL) added.
Brine (150 mL) is added with stirring and the suspension filtered
through Celite. The crude material is extracted with ethyl acetate,
dried (MgSO.sub.4), filtered and evaporated under vacuum. The cruse
is purified by column chromatography eluting with heptane-ethyl
acetate to give
N-[(1S)-1-(3-cyanophenyl)ethyl]-2-methyl-[S(R)]-2-propanesulfinamide
(78%)
[0111] MS: 251 (M+H)
[0112] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=1.22 (s, 9H),
1.54 (d, 3H), 3.36 (bs, 1H), 4.55-4.7 (m, 1H), 7.43 (d, 1H), 7.46
(d, 1H), 7.56-7.6 (m, 2H), 7.64 (s, 1H).
Step 2
##STR00018##
[0113]
N-Hydroxy-3-[(S)-1-(2-methyl-propane-2-sulfinylamino)-ethyl]-benzam-
idine
[0114] Hydroxylamine hydrochloride (3.43 g, 55 mmol) and methanol
(70 mL) are added to a flask containing
N-[(1S)-1-(3-cyanophenyl)ethyl]-2-methyl-[S(R)]-2-propanesulfinamide
(5.5 g, 22 mmol) and the suspension cooled in an ice water bath.
Triethyl amine (5.55 g, 55 mmol) is added to the flask and the
reaction mixture is allowed to warm to room temperature over night.
The reaction mixture evaporated under reduced pressure and the
crude partitioned between water and DCM. Organic layer separated,
dried (Na.sub.2SO.sub.4) and evaporated under reduced pressure to
give
N-Hydroxy-3-[(S)-1-(2-methyl-propane-2-sulfinylamino)-ethyl]-benzamidine
(5.48 g).
[0115] MS: 284 (M+H). .sup.1H NMR (300 MHz, CDCl.sub.3):
.delta.=1.21 (s, 9H), 1.52 (s, 3H), 3.33 (s, 1H), 3.77 (bs, 1H),
4.59-4.61 (m, 1H), 4.88 (1H, bs), 7.35-7.37 (m, 2H), 7.50-7.52 (m,
1H), 7.64 (s, 1H)
Step 3
##STR00019##
[0116] 2-Methyl-propane-2-sulfinic acid
((S)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-eth-
yl)-amide
[0117] Methyl 2-Hydroxy-2-methyl-propionate (20 mL) and
K.sub.2CO.sub.3 (806 mg, 5.8 mmol) are added to a flask containing
N-Hydroxy-3-[(S)-1-(2-methyl-propane-2-sulfinylamino)-ethyl]-benzamidine
(1.5 g, 5.3 mmol) and heated under reflux for 6 hrs. The reaction
mixture is evaporated under reduced pressure and partitioned
between water and ethyl acetate. The organic layer separated, dried
(Na.sub.2SO.sub.4) and purified by flash column chromatography
eluting with heptane-ethyl acetate mixture to give
2-Methyl-propane-2-sulfinic acid
((S)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-eth-
yl)-amide (1.05 g).
[0118] MS: 352 (M+H).
[0119] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=1.22 (s, 9H),
1.58 (d, 3H), 1.75 (s, 6H), 3.48 (bs, 1H), 4.65 (m, 1H), 7.45-7.47
(m, 2H), 8.01 (m, 1H), 8.08 (s, 1H)
Step 4
##STR00020##
[0120]
2-{3-[3-((S)-1-Amino-ethyl)-phenyl]-1,2,4-oxadiazol-5-yl}-propan-2--
ol Hydrochloride
[0121] Hydrogen chloride in p-dioxane (4N, 1.42 mL) is added to a
cooled solution of 2-Methyl-propane-2-sulfinic acid
((S)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl-phenyl}-ethy-
l)-amide (1 g, 2.85 mmol) in methanol (3 mL) at 0.degree. C. and
stirred for 20 min. Diethyl ether (30 mL) is added, decanted, and
the residue is washed with another aliquot of diethyl ether. The
residue is dried in vacuo to yield 2-f
3-[3-((S)-1-Amino-ethyl)-phenyl]-1,2,4-oxadiazol-5-yl]-propan-2-ol
Hydrochloride (560 mg).
[0122] MS: 231 (ES+, --OH ionized)
[0123] .sup.1H NMR (300 MHz, DMSO): .delta.=1.55 (d, 3H), 1.63 (s,
6H), 4.53-4.57 (m, 1H), 6.1 (bs, 1H), 7.64 (t, 1H), 7.76 (d, 1H),
8.01 (d, 1H), 8.15 (s, 1H), 8.56 (bs, 2H)
Step 5
[0124] N-methyl morpholine (NMM) (196 mg, 1.94 mmol) is added to a
mixture of 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid (390 mg,
1.94 mmol) and
2-[3-[3-((S)-1-Amino-ethyl)-phenyl]-1,2,4-oxadiazol-5-yl]-propan-2-ol
Hydrochloride (550 mg, 1.94 mmol) in DMF (20 mL). After stirring at
room temperature for 5 minutes,
4-(4,6-Dimethoxy-[1,3,5]triazin-2-yl)-4-methyl-morpholin-4-ium
chloride (DMTMM) (537 mg, 1.94 mmol) is added and the reaction
stirred at room temperature for 2 hours. The reaction mixture
poured on to ice water and the suspension is extracted with EtOAc
(7.times.100 mL). The combined ethyl acetate layer is washed with
brine (50 mL), dried over sodium sulfate, and reduced in vacuo to
give crude product which is purified by HPLC (C18 column) eluting
with acetonitrile-water mixture to give
2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
((S)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-eth-
yl)-amide as amorphous glass (650 mg, 78%).
[0125] MS: 431 (M+H).
[0126] .sup.1H NMR (300 MHz, DMSO): .delta.=1.58 (d, 3H), 1.62 (s,
6H), 5.3 (m, 1H), 7.56 (t, 1H), 7.7 (d, 1H), 7.92 (m, 2H), 8.08 (s,
1H), 8.43 (t, 1H), 8.72 (d, 1H), 8.9 (d, 1H), 9.47 (s, 2H), 9.59
(d, 1H).
[0127] [.alpha.].sub.d (Methanol)=+57.2.degree.
Example 3
2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
((R)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-eth-
yl)-amide
##STR00021##
[0128] Step 1
##STR00022##
[0130] Potassium hydrogen sulfate (13.6 g, 100 mmol) is added to a
mixture of 3-Formylbenzonitrile (7.21 g, 55 mmol) and
(S)-(+)-2-Methyl-2-propanesulfinamide (6.06 g, 50 mmol) in toluene
(500 mL) and heated at 45.degree. C. for 2 days. The reaction
mixture is filtered, the filtrate evaporated under reduced pressure
and purified by column chromatography eluting with ethyl
acetate-heptane mixture to give,
N-[(3-cyanophenyl)methylene]-2-methyl-, [S(S)]-2-Propanesulfinamide
(9.65 g)
[0131] MS: 235 (M+H).
[0132] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=1.29 (s, 91H),
7.62 (t, 1H), 7.79 (d, 1H), 8.04 (d, 1H), 8.17 (bs, 1H), 8.60 (s,
1H).
Step 2
##STR00023##
[0134] Methyl magnesium bromide (34.3 mL of 3M solution in diethyl
ether, 102.9 mmol) is added over 30 minutes to a solution of
N-[(3-cyanophenyl)methylene]-2-methyl-, [S(S)]-2-Propanesulfinamide
(9.65 g, 41.18 mmol) in DCM (200 mL) at -45.degree. C. and stirred
at that temperature for 4 hrs. The cooling bath is then removed,
allowed to warm up to -10.degree. C. and quenched with saturated
NaHCO.sub.3 (250 mL). The organic layer is separated and the
aqueous layer is extracted with more DCM (100 mL). The organic
extracts are combined, dried (Na.sub.2SO.sub.4) and evaporated
under reduced pressure to give 2-Methyl-propane-2-sulfinic acid
[(R)-1-(3-cyano-phenyl)-ethyl]-amide as the major product.
[0135] MS: 251 (M+H).
[0136] .sup.1H NMR (300 MHz, CDCl.sub.3): .delta.=1.22 (s, 9H),
1.54 (d, 3H), 3.35 (s, 1H), 4.56-4.65 (m, 1H), 7.42-7.48 (m, 1H),
7.56-7.59 (m, 2H), 7.64 (s, 1H).
Step 3
##STR00024##
[0138] Hydrogen chloride (4N in p-dioxane, 21 mL) is added to a
solution of 2-Methyl-propane-2-sulfinic acid
[(R)-1-(3-cyano-phenyl)-ethyl]-amide (10.29 g, 41.1 mmol) in
methanol (21 mL) and stirred at room temperature for 40 minutes.
Reaction mixture is then evaporated under reduced pressure and the
crude triturated with diethyl ether to give an off white solid
which is crystallized from Methyl t-butyl ether and ethanol mixture
to give 3-((R)-1-Amino-ethyl)-benzonitrile hydrochloride as the
major product.
[0139] MS: 147 (M+H).
[0140] .sup.1H NMR (300 MHz, DMSO): .delta.=1.53 (d, 3H), 4.45-4.52
(m, 1H), 7.65 (t, 1H), 7.84-7.91 (m, 2H), 8.03 (s, 1H), 8.67 (bs,
3H).
Step 4
##STR00025##
[0141] N-methyl morpholine (NMM) (1.01 g, 10 mmol) is added to a
mixture of 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid (2 g, 10
mmol) and 3-((R)-1-Amino-ethyl)-benzonitrile hydrochloride (1.82 g,
10 mmol) in DMF (50 mL). After stirring at room temperature for 10
minutes,
4-(4,6-Dimethoxy-[1,3,5]triazin-2-yl)-4-methyl-morpholin-4-ium
chloride (DMTMM) (10 mmol) is added and the reaction stirred
overnight at room temperature. The reaction mixture is partitioned
between water (500 mL) and ethyl acetate (300 mL) and the aqueous
layer is extracted with more ethyl acetate (100 mL). Combined ethyl
acetate extracts is washed with saturated NaHCO.sub.3 (100 mL), and
brine (100 mL). The organic layer is dried (Na.sub.2SO.sub.4),
filtered and then evaporated under reduced pressure to give
2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
[(R)-1-(3-cyano-phenyl)-ethyl]-amide as the major product (3.2 g)
which is taken directly into the next reaction (amidoxime
formation).
Step 5
##STR00026##
[0143] Hydroxylamine hydrochloride (1.52 g, 24.2 mmol) is added to
a cooled solution of 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
[(R)-1-(3-cyano-phenyl)-ethyl]-amide (3.2 g, 9.7 mmol) in methanol
(40 mL) and the suspension cooled in an ice water bath. Triethyl
amine (2.44 g, 24.2 mmol) is added to the flask and the reaction
mixture is allowed to warm to room temperature over night. The
reaction mixture evaporated under reduced pressure and the crude
partitioned between water and ethyl acetate. Organic layer is
separated, dried (Na.sub.2SO.sub.4) and evaporated under reduced
pressure. Toluene (50 mL) and CHCl.sub.3 (50 mL) are added and
evaporated under reduced pressure to give
2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
{(R)-1-[3-(N-hydroxycarbamimidoyl)-phenyl]-ethyl}-amide (3 g) as
the major product.
[0144] MS: 363 (M+H).
[0145] .sup.1H NMR (300 MHz, DMSO): .delta.=1.54 (d, 3H), 5.18-5.27
(m, 1H), 5.80 (bs, 2H), 7.35 (t, 1H), 7.44 (d, 1H), 7.54-7.60 (m,
2H), 7.74 (s, 1H), 8.45 (d, 1H), 8.79 (d, 1H), 9.26 (d, 1H), 9.35
(s, 2H), 9.60 (s, 1H).
Step 6
2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
((R)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-eth-
yl)-amide
[0146] Methyl 2-hydroxy-2-methyl-propionate (2 mL) and
K.sub.2CO.sub.3 (219 mg, 1.59 mmol) are added to a microwave vial
containing 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
{(R)-1-[3-(N-hydroxycarbamimidoyl)-phenyl]-ethyl}-amide (0.5 g,
1.38 mmol) and heated at 180.degree. C. in a microwave for 10
minutes. The reaction mixture is evaporated under reduced pressure
and purified by reverse phase HPLC to give
2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
((R)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-eth-
yl)-amide as the major compound (110 mg).
[0147] MS: 431 (M+H).
[0148] .sup.1H NMR (300 MHz, DMSO): .delta.=1.58 (d, 3H), 1.61 (s,
6H), 5.27-5.31 (m, 1H), 6.08 (s, 1H), 7.53-7.60 (m, 2H), 7.67 (d,
1H), 7.91 (d, 1H), 8.02 (t, 1H), 8.08 (s, 1H), 8.45 (d, 1H), 8.79
(d, 1H), 9.35-9.39 (m, 3H).
[0149] 2-Pyridin-2-yl-pyrimidine-5-carboxylic acid
((R)-1-{3-[5-(1-hydroxy-1-methyl-ethyl)-1,2,4-oxadiazol-3-yl]-phenyl}-eth-
yl)-amide can also be prepared by following procedures similar to
those of Example 2 but substituting
2-{3-[3-((R)-1-Amino-ethyl)-phenyl]-1,2,4-oxadiazol-5-yl}-propan-2-ol
Hydrochloride for
2-{3-[3-((S)-1-Amino-ethyl)-phenyl]-1,2,4-oxadiazol-5-yl}-propan-2-ol
Hydrochloride.
In Vitro Assay Protocols to Identify Inhibitors of Hematopoietic
PGD2 Synthase
[0150] The compounds of the present invention can be tested for
enzymatic inhibiting activity against PGD2 Synthase according to
either one of the following assays.
Assay 1: Fluorescence Polarization Assay
[0151] As described in PCT publication WO 2004/016223, Example
II.
Assay 2: Enzyme Immunoassay (EIA) Method
I. Assay Solutions
[0152] a. Preparation of 0.1M K.sub.2HPO.sub.4/KH.sub.2PO.sub.4
buffer (pH 7.4) [0153] Prepare 0.1 M KH.sub.2PO.sub.4 from 1M
KH.sub.2PO.sub.4 (Sigma, Cat# P-8709) [0154] Prepare 0.1 M
K.sub.2HPO.sub.4 from powder of K.sub.2HPO.sub.4 (Fisher,
BP363-500) [0155] Mix 0.1 M K.sub.2HPO.sub.4 with 0.1 M
KH.sub.2PO.sub.4 to adjust pH to 7.4. [0156] b. Preparation of 0.5%
.gamma.-globulin [0157] Add 0.1 g of .gamma.-globulin (Sigma, Cat#
G-5009) to 20 mL 0.1 M K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 [0158]
buffer (pH 7.4) and make 1-mL/vial aliquots and store in
-80.degree. C. [0159] c. Preparation of 100 mM GSH [0160] Add 307
mg of GSH (Sigma, Cat# G-6529) to 10 mL 0.1 M
K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 [0161] buffer (pH 7.4) and store
at -80.degree. C. [0162] d. Preparation of Reaction buffer: [0163]
198 mL of 0.1M K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 buffer (pH 7.4)
[0164] 2 mM GSH--Prepared from 100 mM GSH [0165] 0.4 g Glycerol
[0166] 2 mL of 0.5% .gamma.-globulin [0167] Add 0.4 g of glycerol
and 2 mL of 0.5% .gamma.-globulin to 198 mL of 0.1 M
K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 [0168] buffer (pH7.4). [0169] Add
0.4 mL of 100 mM GSH to 19.6 mL reaction buffer before the assay
(enough for two 96- [0170] well plates). [0171] e. Preparation of
FeCl.sub.2/citric acid stopping solution: (8 mg/mL FeCl.sub.2, 0.1
M citric acid) [0172] Add 40 mg fresh FeCl.sub.2 (IGN, Cat#158046)
to 5 mL 0.1 M citric acid (Sigma, Cat#C0759). [0173] f. Preparation
of MOX reagent: [0174] 10% EtOH--Add 1 mL of EtOH to 9 mL of ultra
pure H.sub.2O [0175] Dissolve 0.1 g of methoxylamine (Cayman,
Cat#400036/) in 10% EtOH (10 mL). [0176] Add 0.82 g of sodium
acetate (Cayman, Cat#400037) to MOX solution and dissolve.
II. Materials and Method
[0176] [0177] Dimethylsulfoxide (DMSO; Sigma; Cat# D2650) [0178]
Prostaglandin D2-MOX express EIA kit (Caymen Chemical, Catalog No.
500151)
[0179] Before the assay, cool down 10 mL of acetone in
polypropylene tubes and empty 96 well plates in ice. All the
procedures except compound dilution are performed on ice.
III. Compound Dilution
[0180] 1. Dilute compound in DMSO
TABLE-US-00001 [0180] Vol of DMSO stock solution Compound
concentration (.mu.L) DMSO (.mu.L) (mM) 4 .mu.L of 10 mM 6 .mu.L 4
3 .mu.L of 4 mM 6 .mu.L 1.3333 3 .mu.L of 1.33 mM 6 .mu.L 0.4444 3
.mu.L of 0.44 mM 6 .mu.L 0.1481 3 .mu.L of 0.148 mM 6 .mu.L 0.0494
3 .mu.L of 0.049 mM 6 .mu.L 0.0165 3 .mu.L of 0.016 mM 6 .mu.L
0.0055
[0181] 2. Dilute 2 .mu.L of each above concentration of compound to
38 .mu.L of reaction buffer in 96-well plates and mix.
IV. Enzyme and Substrate Solution Preparation
[0181] [0182] 1. Preparation of 0.39 ng/.mu.L enzyme solution (0.35
ng/.mu.L at final after compound addition). [0183] Mix 4 .mu.L of 4
mg/mL human h-PGDS with 396 .mu.L of reaction buffer (to give
enzyme concentration 40 .mu.g/mL). Add 46.8 .mu.L of 40 .mu.g/mL
h-PGDS to 4.753 mL of reaction buffer to give a total volume of 4.8
mL [0184] 2. Preparation of Substrate Solution (PGH2): Add 0.375 mL
of 0.1 mg/mL of PGH2 to 1.625 mL acetone.
V. Enzyme Reaction:
[0184] [0185] 1. Add 60 .mu.L of enzyme solution to compound well
and positive control (without compound) in U-bottom polypropylene
plate on ice. [0186] 2. Add 60 .mu.L of reaction buffer and 6.6
.mu.L of 5% DMSO in reaction buffer into negative control wells in
the plate. [0187] 3. Add 6.6 .mu.L of diluted compound in reaction
buffer to the compound wells and mix. [0188] 4. Add 6.6 .mu.L of 5%
DMSO in reaction buffer to the positive control well. [0189] 5.
Incubate the plate in ice for at least 30 min. [0190] 6. Add 20
.mu.L of substrate (PGH2) solution to compound, negative and
positive control wells in the U-bottom 96 well plate on ice. [0191]
7. Dry the plate in cold room for about 25-28 min. [0192] 8.
Pipette 45 .mu.L of enzyme solution (above) into 96-wells with
dried PGH2 and mix 3 times. Incubate on the ice for 1 min. [0193]
9. Add 45 .mu.L of FeCl.sub.2 solution into each wells and mix.
[0194] 10. Add 90 .mu.L of MOX solution and mix. [0195] 11.
Incubate for 30 min at 60.degree. C. [0196] 12. Dilute the samples
2500.times. with EIA buffer.
VI. EIA Assay
[0196] [0197] Perform the assay according to the procedure in EIA
kit provided by Cayman. Total PGD2 levels (pg/mL) were determined
in the samples by EIA kits (Cayman Chemical, Catalog No.
500151)
[0198] Calculate amount of PGD2 as below [0199] Calculated %
Positive control according to the equation below;
[0199] % Positive control=(Compound value-Negative
control)/(Positive value-Negative control value).times.100.
% Positive control = ( Compound value - Negative control ) (
Positive value - Negative control value ) .times. 100 ##EQU00001##
[0200] Compound value=PGD2 levels (pg/mL) obtained from the
standard curve in EIA assay for the samples with compound [0201]
Negative control value=PGD2 levels (pg/mL) obtained from the
standard curve in EIA assay for the samples without enzyme [0202]
Positive control value=PGD2 levels (pg/mL) obtained from the
standard curve in EIA assay for the samples with enzyme but without
compound [0203] IC.sub.50s are determined by excel fit to get the x
value when y=1/2Ymax using 4 parameter logistic model for the
IC.sub.50 curves.
Results
[0204] Compounds within the scope of the invention produce 50%
inhibition in the Fluorescence Polarization Assay or the EIA assay
at concentrations within the range of about 1 nanomolar to about 30
micromolar, particularly about 1 nanomolar to about 1 micromolar,
and more particularly about 1 nanomolar to about 100 nanomolar.
TABLE-US-00002 hPGDS EIA IC50 Example nM solidsol .mu.M 1 12 135.9
2 11 854.9 3 26 29.4
[0205] The present invention may be embodied in other specific
forms without departing from the spirit or essential attributes
thereof.
* * * * *