Patent Application
20120270875
The present invention relates to organic compounds useful for therapy and/or prophylaxis in a mammal, and in particular to LFA-1 antagonists and dual LFA-1/MAC-1 antagonists useful for treating inflammatory diseases and disorders.
In an embodiment of the present invention, provided are compounds of general formula (I):
wherein:
or absent if R1 is
R3 and R4, independently or each other, are H, methyl, trifluoromethyl or ethyl;
R5 is lower alkyl, cycloalkyl, lower alkyl-cycloalkyl, heterocycloalkyl, isoquinoline, quinoline, adamantane, NR7R8, OR9, unsubstituted heteroaryl, heteroaryl substituted with phenyl, unsubstituted phenyl or phenyl substituted with hydroxy or methyl;
R6 is hydrogen, lower alkyl, alkoxy, cycloalkyl, aryl, heteroaryl,
R7 and R8, independently of each other, are hydrogen, lower alkyl, cycloalkyl, aryl or heteroaryl;
R9 is lower alkyl, cycloalkyl, phenyl or heteroaryl; and
R10, R11, R12 and R13, independently of each other, are hydrogen or lower alkyl,
or a pharmaceutically acceptable salt thereof.
In a further embodiment of the invention, provided is a pharmaceutical composition comprising a therapeutically effective amount of a compound according to formula (I) and a therapeutically inert carrier.
In a still further embodiment of the invention, provided is a method for the treatment or prophylaxis of asthma or COPD, which method comprises the step of administering an effective amount of a compound according to formula (I) to a patient in need thereof.
All documents cited to or relied upon below are expressly incorporated herein by reference.
Asthmatic and COPD symptoms such as restricted breathing and airway inflammation can be linked to the migration and activation of leukocytes into the lung. LFA-1 on neutrophils and MAC-1 on macrophages are major receptors that upon activation promote leukocyte infiltration and activation into the lung. Therefore, LFA-1 antagonists and dual LFA-1/MAC-1 antagonists are desirable therapeutics for the treatment of inflammatory diseases and disorders.
Provided herein are LFA-1 antagonist and dual LFA-1/MAC-1 antagonist compounds. The compounds of the invention are useful for the treatment of inflammatory diseases and disorders such as, for example, asthma and COPD.
It is to be understood that the terminology employed herein is for the purpose of describing particular embodiments, and is not intended to be limiting. Further, although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.
As used herein, the term “alkyl”, alone or in combination with other groups, refers to a branched or straight-chain monovalent saturated aliphatic hydrocarbon radical of one to twenty carbon atoms, preferably one to sixteen carbon atoms, more preferably one to ten carbon atoms.
The term “cycloalkyl” refers to a monovalent mono- or polycarbocyclic radical of three to ten, preferably three to six carbon atoms. This term is further exemplified by radicals such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, norbornyl, adamantyl, indanyl and the like. In a preferred embodiment, the “cycloalkyl” moieties can optionally be substituted with one, two, three or four substituents, with the understanding that said substituents are not, in turn, substituted further. Each substituent can independently be, alkyl, alkoxy, halogen, amino, hydroxyl or oxygen (O═) unless otherwise specifically indicated. Examples of cycloalkyl moieties include, but are not limited to, optionally substituted cyclopropyl, optionally substituted cyclobutyl, optionally substituted cyclopentyl, optionally substituted cyclopentenyl, optionally substituted cyclohexyl, optionally substituted cyclohexylene, optionally substituted cycloheptyl, and the like or those which are specifically exemplified herein.
The term “heterocycloalkyl” denotes a mono- or polycyclic alkyl ring, wherein one, two or three of the carbon ring atoms is replaced by a heteroatom such as N, O or S. Examples of heterocycloalkyl groups include, but are not limited to, morpholinyl, thiomorpholinyl, piperazinyl, piperidinyl, pyrrolidinyl, tetrahydropyranyl, tetrahydrofuranyl, 1,3-dioxanyl and the like. The heterocycloalkyl groups may be unsubstituted or substituted and attachment may be through their carbon frame or through their heteroatom(s) where appropriate, with the understanding that said substituents are not, in turn, substituted further.
The term “lower alkyl”, alone or in combination with other groups, refers to a branched or straight-chain alkyl radical of one to nine carbon atoms, preferably one to six carbon atoms, more preferably one to four carbon atoms. This term is further exemplified by radicals such as methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, n-pentyl, 3-methylbutyl, n-hexyl, 2-ethylbutyl and the like.
The term “aryl” refers to an aromatic mono- or polycarbocyclic radical of 6 to 12 carbon atoms having at least one aromatic ring. Examples of such groups include, but are not limited to, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthalene, 1,2-dihydronaphthalene, indanyl, 1H-indenyl and the like.
The term “heteroaryl,” refers to an aromatic mono- or polycyclic radical of 5 to 12 atoms having at least one aromatic ring containing one, two, or three ring heteroatoms selected from N, O, and S, with the remaining ring atoms being C. One or two ring carbon atoms of the heteroaryl group may be replaced with a carbonyl group.
The alkyl, lower alkyl, aryl and heteroaryl groups described above may be substituted independently with one, two, or three substituents, with the understanding that said substituents are not, in turn, substituted further. These substituents may optionally form a ring with the heteroaryl group to which they are connected. Substituents may include, for example: carbon-containing groups such as alkyl, aryl, arylalkyl (e.g. substituted and unsubstituted phenyl, substituted and unsubstituted benzyl); halogen atoms and halogen-containing groups such as haloalkyl (e.g. trifluoromethyl); oxygen-containing groups such as alcohols (e.g. hydroxyl, hydroxyalkyl, aryl(hydroxyl)alkyl), ethers (e.g. alkoxy, aryloxy, alkoxyalkyl, aryloxyalkyl), aldehydes (e.g. carboxaldehyde), ketones (e.g. alkylcarbonyl, alkylcarbonylalkyl, arylcarbonyl, arylalkylcarbonyl, arycarbonylalkyl), acids (e.g. carboxy, carboxyalkyl), acid derivatives such as esters (e.g. alkoxycarbonyl, alkoxycarbonylalkyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl), amides (e.g. aminocarbonyl, mono- or di-alkylaminocarbonyl, aminocarbonylalkyl, mono- or di-alkylaminocarbonylalkyl, arylaminocarbonyl), carbamates (e.g. alkoxycarbonylamino, aryloxycarbonylamino, aminocarbonyloxy, mono- or di-alkylaminocarbonyloxy, arylminocarbonloxy) and ureas (e.g. mono- or di-alkylaminocarbonylamino or arylaminocarbonylamino); nitrogen-containing groups such as amines (e.g. amino, mono- or di-alkylamino, aminoalkyl, mono- or di-alkylaminoalkyl), azides, nitriles (e.g. cyano, cyanoalkyl), nitro; sulfur-containing groups such as thiols, thioethers, sulfoxides and sulfones (e.g. alkylthio, alkylsulfinyl, alkylsulfonyl, alkylthioalkyl, alkylsulfinylalkyl, alkylsulfonylalkyl, arylthio, arysulfinyl, arysulfonyl, arythioalkyl, arylsulfinylalkyl, arylsulfonylalkyl); and heterocyclic groups containing one or more heteroatoms, (e.g. thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, aziridinyl, azetidinyl, pyrrolidinyl, pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl, pyridazinyl, piperidyl, hexahydroazepinyl, piperazinyl, morpholinyl, thianaphthyl, benzofuranyl, isobenzofuranyl, indolyl, oxyindolyl, isoindolyl, indazolyl, indolinyl, 7-azaindolyl, benzopyranyl, coumarinyl, isocoumarinyl, quinolinyl, isoquinolinyl, naphthridinyl, cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl, quinoxalinyl, chromenyl, chromanyl, isochromanyl, phthalazinyl, benzothiazoyl and carbolinyl).
As used herein, the term “alkoxy” means alkyl-O—; and “alkoyl” means alkyl-CO—. Alkoxy substituent groups or alkoxy-containing substituent groups may be substituted by, for example, one or more alkyl groups, with the understanding that said substituents are not, in turn, substituted further.
As used herein, the term “halogen” means a fluorine, chlorine, bromine or iodine radical, preferably a fluorine, chlorine or bromine radical, and more preferably a fluorine or chlorine radical.
Compounds of formula I can have one or more asymmetric carbon atoms and can exist in the form of optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, optically pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates. The optically active forms can be obtained for example by resolution of the racemates, by asymmetric synthesis or asymmetric chromatography (chromatography with a chiral adsorbents or eluant). The invention embraces all of these forms.
As used herein, the term “pharmaceutically acceptable salt” means any pharmaceutically acceptable salt of the compound of formula (I). Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases. Such acids include, for example, acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, dichloroacetic, formic, fumaric, gluconic, glutamic, hippuric, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, oxalic, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, oxalic, p-toluenesulfonic and the like. Particularly preferred are fumaric, hydrochloric, hydrobromic, phosphoric, succinic, sulfuric and methanesulfonic acids. Acceptable base salts include alkali metal (e.g. sodium, potassium), alkaline earth metal (e.g. calcium, magnesium) and aluminum salts.
In the practice of the method of the present invention, an effective amount of any one of the compounds of this invention or a combination of any of the compounds of this invention or a pharmaceutically acceptable salt thereof, is administered via any of the usual and acceptable methods known in the art, either singly or in combination. The compounds or compositions can thus be administered orally (e.g., buccal cavity), sublingually, parenterally (e.g., intramuscularly, intravenously, or subcutaneously), rectally (e.g., by suppositories or washings), transdermally (e.g., skin electroporation) or by inhalation (e.g., by aerosol), and in the form or solid, liquid or gaseous dosages, including tablets and suspensions. The administration can be conducted in a single unit dosage form with continuous therapy or in a single dose therapy ad libitum. The therapeutic composition can also be in the form of an oil emulsion or dispersion in conjunction with a lipophilic salt such as pamoic acid, or in the form of a biodegradable sustained-release composition for subcutaneous or intramuscular administration.
Useful pharmaceutical carriers for the preparation of the compositions hereof, can be solids, liquids or gases. Thus, the compositions can take the form of tablets, pills, capsules, suppositories, powders, enterically coated or other protected formulations (e.g. binding on ion-exchange resins or packaging in lipid-protein vesicles), sustained release formulations, solutions, suspensions, elixirs, aerosols, and the like. The carrier can be selected from the various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water, saline, aqueous dextrose, and glycols are preferred liquid carriers, particularly (when isotonic with the blood) for injectable solutions. For example, formulations for intravenous administration comprise sterile aqueous solutions of the active ingredient(s) which are prepared by dissolving solid active ingredient(s) in water to produce an aqueous solution, and rendering the solution sterile. Suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, talc, gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like. The compositions may be subjected to conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers and the like. Suitable pharmaceutical carriers and their formulation are described in Remington's Pharmaceutical Sciences by E. W. Martin. Such compositions will, in any event, contain an effective amount of the active compound together with a suitable carrier so as to prepare the proper dosage form for proper administration to the recipient.
The dose of a compound of the present invention depends on a number of factors, such as, for example, the manner of administration, the age and the body weight of the subject, and the condition of the subject to be treated, and ultimately will be decided by the attending physician or veterinarian. Such an amount of the active compound as determined by the attending physician or veterinarian is referred to herein, and in the claims, as a “therapeutically effective amount”. For example, the dose of a compound of the present invention is typically in the range of about 1 to about 1000 mg per day. Preferably, the therapeutically effective amount is in an amount of from about 1 mg to about 500 mg per day.
It will be appreciated, that the compounds of general formula I in this invention may be derivatized at functional groups to provide derivatives which are capable of conversion back to the parent compound in vivo. Physiologically acceptable and metabolically labile derivatives, which are capable of producing the parent compounds of general formula I in vivo are also within the scope of this invention.
The present invention provides novel compounds of general formula (I):
wherein:
or absent if R1 is
R3 and R4, independently or each other, are H, methyl, trifluoromethyl or ethyl;
R5 is lower alkyl, cycloalkyl, lower alkyl-cycloalkyl, heterocycloalkyl, isoquinoline, quinoline, adamantane, NR7R8, OR9, unsubstituted heteroaryl, heteroaryl substituted with phenyl, unsubstituted phenyl or phenyl substituted with hydroxy or methyl;
R6 is hydrogen, lower alkyl, alkoxy, cycloalkyl, aryl, heteroaryl,
R7 and R8, independently of each other, are hydrogen, lower alkyl, cycloalkyl, aryl or heteroaryl;
R9 is lower alkyl, cycloalkyl, phenyl or heteroaryl; and
R10, R11, R12 and R13, independently of each other, are hydrogen or lower alkyl,
or a pharmaceutically acceptable salt thereof.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R1 is
In another embodiment of the invention, provided is a compound according to formula (I), wherein R1 is phenyl, mono- or bi-substituted independently with hydroxy, halogen, lower alkyl, alkoxy, —OC(O)CH3 or —OC(O)CH(CH3)2.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R1 is heteroaryl, mono- or bi-substituted with hydroxy.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R2 is absent.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R3 is methyl.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R4 is methyl.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R5 is lower alkyl, —C(O)CH3, cycloalkyl, lower alkyl-cycloalkyl, heterocycloalkyl, isoquinoline, quinoline, adamantane or NR7R8, OR9.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R5 is unsubstituted heteroaryl, heteroaryl substituted with phenyl, unsubstituted phenyl or phenyl substituted with hydroxy or methyl.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R5 is adamantane, chlorothiophene, pyrazine, thiazole, thiophene, —C(O)CH3, thiazole-phenyl, pyridine, dimethyl-thiazole, isoquinoline, pyridine-thiazole, methyl-thiophene, methyl-pyrazine or ethyl-thiophene.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R6 is hydrogen or lower alkyl.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R6 is methyl.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R6 is:
In another embodiment of the invention, provided is a compound according to formula (I), wherein R7 is hydrogen or lower alkyl.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R8 is hydrogen or lower alkyl.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R9 is lower alkyl or phenyl.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R10 is hydrogen or methyl.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R11 is hydrogen or methyl.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R12 is hydrogen or methyl.
In another embodiment of the invention, provided is a compound according to formula (I), wherein R13 is hydrogen or methyl.
and forming a compound of formula (I):
or a pharmaceutically acceptable salt thereof.
or absent if R1 is
R3 and R4, independently or each other, can be H or lower alkyl;
R5 can be lower alkyl, —C(O)CH3, cycloalkyl, lower alkyl-cycloalkyl, heterocycloalkyl, isoquinoline, quinoline, adamantane, NR7R8, OR9, substituted or unsubstituted heteroaryl or substituted or unsubstituted phenyl;
R6 can be hydrogen, lower alkyl, alkoxy, cycloalkyl, aryl, heteroaryl, —(CH2)2O(CH2)2OCH2CH3—,
R7 and R8, independently of each other, can be hydrogen, lower alkyl, cycloalkyl, aryl or heteroaryl;
R9 can be lower alkyl, cycloalkyl, phenyl or heteroaryl; and
R10, R11, R12 and R13, independently of each other, can be hydrogen, lower alkyl or alkoxy.
A solution of 2-[3-(3-methoxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (2.42 g, 7.05 mmol) DCM (50 ml) was cooled in an ice-water bath and BBr3/DCM (1M, 14.1 ml, 14.1 mmol) was added dropwise. The resulting solution was allowed to warm up to room temperature and stirred at room temperature for 2 h. The solution was quenched with ice water and extracted with DCM. The organic layers were combined, washed with water and brine, dried over MgSP4, filtered, and concentrated to afford the desired product (2 g, 86% yield). MS m/e 330.1 (M+H+).
A mixture of 3-(methoxy-phenyl)-propylamine hydrochloride (254 mg, 1.26 mmol), 2-chloro-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (288 g, 1.26 mmol) and KOAc (495 mg, 5.04 mmol) in ethanol (10 ml) was microwaved at 120° C. for 1 h. The reaction mixture was cooled to room temperature, quenched with 20% NaHCO3 and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified flash chromatography with 30% EtOAC in hexane to afford the desired product (303.3 mg, 67% yield). MS m/e 358.0 (M+H+).
A solution of 4-ethyl-2-[3-(3-methoxy-phenyl)-propylamino]-6-methyl-pyrimidine-5-carboxylic acid ethyl ester (303.3 mg, 0.85 mmol) in DCM (5 ml) was cooled in an ice-water bath and BBr3/DCM (1M, 1.7 ml, 1.7 mmol) was added dropwise. The resulting solution was allowed to warm up to room temperature and stirred for a further 2 h. The solution was quenched with ice water and extracted with DCM. The organic layers were combined, washed with water and brine, dried over MgSO4, filtered, and concentrated to afford the desired product (243.8 mg, (83% yield). MS m/e 344.0 (M+H+).
A mixture of 4-ethyl-2-[3-(3-hydroxy-phenyl)-propylamino]-6-methyl-pyrimidine-5-carboxylic acid ethyl ester (244 mg, 0.71 mmol) and LiOH.H2O (745 mg, 17.8 mmol) in dioxane/water (10 ml/10 ml) was heated to 90° C. for 6 h. The solution was cooled to room temperature, treated with aqKHSO3 to bring the pH to 2-4 and extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and evaporated to give the title compound (219 mg, 98% yield). MS m/e 315.9 (M+H+).
In a microwave reaction vessel, was charged with 3-(methoxy-phenyl)-propylamine hydrochloride (135 mg, 0.67 mmol), 2-chloro-4,6-diethyl-pyrimidine-5-carboxylic acid ethyl ester (108 mg, 0.45 mmol), potassium acetate (177 mg, 1.8 mmol) and ethanol (4 ml). The mixture was heated to 150° C. for 1 h in a microwave oven. The mixture was diluted with EtOAc (150 ml), washed with 20% aqNaHCO3, water and brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography with 30% EtOAc in hexane to afford the desired product (45 mg, 27% yield). MS m/e 372.0 (M+H+).
A solution of 4,6-diethyl-2-[3-(3-methoxy-phenyl)-propylamino]-pyrimidine-5-carboxylic acid ethyl ester (45 mg, 0.121 mmol) in DCM (1 ml) was cooled in an ice-water bath and BBr3/DCM (1M, 0.24 ml, 0.24 mmol) was added dropwise. The resulting solution was allowed to warm up to room temperature and stirred for a further 2 h. The solution was quenched with ice water and extracted with DCM. The organic layers were combined, washed with water and brine, dried over MgSO4, filtered, and concentrated to give the desired product (30 mg, 69% yield). MS m/e 357.9 (M+H+).
A mixture of 4,6-diethyl-2-[3-(3-hydroxy-phenyl)-propylamino]-pyrimidine-5-carboxylic acid ethyl ester (30 mg, 0.084 mmol), and LiOH.H2O (88 mg, 2.1 mmol), in dioxane/water (2 ml/2 ml) was heated to 90° C. overnight. The solution was cooled to room temperature, treated with aqKHSO4 to bring the pH to 2-4 and extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and concentrated to give the title compound (26 mg, 94% yield). MS m/e 329.9 (M+H+).
To a solution of 5-(3-allyloxy-phenyl)-pentanoic acid ethyl ester (356 mg, 1.35 mmol) in xylenes (15 ml) was added Lawesson's reagent (655 mg, 1.62 mmol). The mixture was refluxed for 3 h. After cooling to room temperature, a precipitate was removed by filtration and the solution was concentrated to dryness. The crude residue was purified by flash chromatography with a 5-10% EtOAc in hexane gradient to afford the corresponding thioester (92 mg, 24% yield). This thioester was dissolved in MeOH (2 ml) and treated with ammonia (7N in MeOH, 462 ul, 3.23 mmol) and ammonium chloride (173 mg, 3.23 mmol). The mixture was refluxed for 40 min, cooled to room temperature, quenched with 1N NaOH and extracted with EtOAc. The organic layer was concentrated under reduced pressure to afford the desired amidine as a yellow paste (67 mg, 89% yield).
A solution of methyl acetoacetate (10.8 ml, 0.1 mmol), trimethyl orthoformate (15.2 ml, 0.12 mmol), pyridine (0.8 ml, 0.01 mmol) and acetic acid (0.6 ml, 0.01 mmol) in toluene (30 ml) was stirred at 80° C. for 18 h. The mixture was concentrated under reduced pressure to afford crude keto ester B as an orange oil. This crude reagent (400 ul) was dissolved in methanol (1 ml) and the amidine A (0.29 mmol, 67 mg) was added in one portion. The mixture was refluxed for 26 h, cooled to room temperature and concentrated under reduced pressure. The crude residue was purified by flash chromatography with a 10-40% EtOAc in hexane gradient to afford 2-[4-(3-Allyloxy-phenyl)-butyl]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester. This ester was then hydrolyzed with LiOH.H2O (0.35 mmol, 15 mg) in dioxane/H2O (2 ml/0.5 ml) at reflux for 2 h. The mixture was quenched with pH=3 buffer and extracted with EtOAc. The layers were separated; the organic layer was dried over Na2SO4, filtered and concentrated to afford the desired acid as a white solid (67 mg, 65% overall yield from amidine A).
The pyrimidinone B (5.96 g, 30.4 mmol) and the mesylate A (4.95 g, 20.26 mmol) were dissolved in dry DMF (100 ml) containing Cs2CO3 (13.2 g, 40 mmol) and heated to 75° C. for 6 h. The mixture was cooled to room temperature, poured into water and extracted with EtOAc. The combined organic layers were successively washed with 1N HCl, water and brine, then dried over MgSO4, filtered and concentrated under reduced pressure. The crude was purified by flash chromatography (10% EtOAc/Hexanes) to afford the desired product as a yellow oil (5.6 g, 54% yield)
A solution of 2-[3-(3-methoxy-phenyl)-propoxy]-pyrimidine-5-carboxylic acid ethyl ester (2.5 g, 7.3 mmol) in dry DCM (25 ml) was cooled to 0° C. in a ice bath and BBr3/DCM (1M, 14.6 ml, 14.6 ml) was added dropwise at 0° C. After 5 min, the cooling bath was removed and the mixture was allowed to warm up to room temperature and was stirred for 2 h. The mixture was quenched with water and extracted with DCM. The combined organic layers were washed with saturated aqueous NaHCO3 and brine, then dried over MgSO4, filtered and concentrated under reduced pressure to afford a yellow oil (2.3 g). This material was dissolved in MeOH (25 ml) and treated with 1N NaOH (15 ml) at 70° C. for 8 h. The mixture was concentrated under reduced pressure and partitioned between an aqueous buffer (pH=5) and EtOAc. The organic phases was washed with water and brine, then dried over MgSO4, filtered and concentrated under reduced pressure to afford the desired acid as a beige solid (1.25 g, 57% yield).
A mixture of 3′-methoxy-biphenyl-2-ylmethyl-amine hydrochloride salt (73 mg, 0.29 mmol), 2-chloro-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (42 mg, 0.19 mmol) and KOAc (66 mg, 0.66 mmol) in ethanol (3 ml) was microwaved at 120° C. for 1 h. The reaction mixture was cooled to room temperature, quenched with 1NHCl and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated to afford the desired product (80 mg, 100% yield).
A similar procedure was used to make the following two intermediates:
A solution of 2-[(3′-methoxy-biphenyl-2-ylmethyl)-amino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (80 mg, 0.19 mmol) in dry DCM (2 ml) was cooled to 0° C. in a ice bath, and BBr3/DCM (1M, 380 ul, 0.38 mmol) was added dropwise at 0° C. The reaction mixture was stirred at 0° C. for 1 h, then quenched with water and extracted with DCM. The combined organic layers were washed with water and brine, then dried over Na2SO4, filtered and concentrated under pressure to afford the de-methylated compound which was not purified. This material was dissolved in H2O/dioxane (1.5 ml/1.5 ml) and treated with LiOH.H2O (80 mg, 1.9 mmol)) at 60° C. overnight. The mixture was quenched with concentrated HCl to pH=1, concentrated under reduced pressure to ½ its volume, then partitioned between brine and EtOAc. The combined organic phases were dried over Na2SO4, filtered and concentrated under reduced pressure to afford the desired acid (75 mg, 100% overall yield).
A similar procedure was used to make the following two intermediates:
A mixture of 2-amino-6-methoxy-1,2,3,4-tetrahydro-naphthalene hydrochloride (87 mg, 0.43 mmol), 2-chloro-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (71 mg, 0.33 mmol) and KOAc (150 mg, 1.5 mmol) in ethanol (3 ml) was microwaved at 120° C. for 1 h. The reaction mixture was cooled to room temperature, quenched with 1NHCl and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated. The crude residue was purified by flash chromatography with a 5-60% EtOAC in hexane gradient to afford the desired product (69 mg, 60% yield).
A similar procedure was used to make the following intermediate:
A solution of 2-(6-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (36 mg, 0.1 mmol) in dry DCM (2 ml) was cooled to 0° C. in a ice bath, and BBr3/DCM (1M, 200 ul, 0.2 mmol) was added in one portion at 0° C. The reaction mixture was stirred at 0° C. for 1 h, then quenched with ice/water and extracted with DCM. The combined organic layers were dried over Na2SO4, filtered and concentrated under pressure to afford the de-methylated compound which was not purified. This material was dissolved in H2O/dioxane (1.0 ml/1.0 ml) and treated with LiOH.H2O (42 mg, 1.0 mmol)) at 60° C. overnight. The mixture was quenched with 1NHCl/brine (1:1) and extracted with EtOAc. The combined organic phases were dried over Na2SO4, filtered and concentrated under reduced pressure to afford the desired acid (30 mg, 100% overall yield).
A solution of 2-(8-methoxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (261 mg, 0.73 mmol) in dry DCM (7 ml) was cooled to 0° C. in a ice bath, and BBr3/DCM (1M, 1.5 ml, 1.47 mmol) was added in one portion at 0° C. The reaction mixture was stirred at 0° C. for 1 h, then quenched with ice/water and extracted with DCM. The combined organic layers were dried over Na2SO4, filtered and concentrated under pressure to afford the de-methylated compound which was not purified. This material was dissolved in H2O/dioxane (5.0 ml/5.0 ml) and treated with LiOH.H2O (300 mg, 7.3 mmol)) at 60° C. overnight. The mixture was quenched with 1NHCl/brine (1:1) and extracted with EtOAc. The combined organic phases were dried over Na2SO4, filtered and concentrated under reduced pressure to afford the desired acid (166 mg, 72% overall yield).
A microwave reaction vessel was charged with 2-chloro-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (208 mg, 0.857 mmol), 4-(2-amino-ethyl)-phenol hydrochloride (223 mg, 1.286 mmol), potassium acetate (336 mg, 3.43 mmol), and ethanol (4 ml), then heated to 150° C. for 1 h in a microwave oven. The mixture was cooled to room temperature and quenched with cold water. The precipitate was collected by filtration, washed with water and dried over sodium sulfate to afford the desired product (285 mg, 100% yield). MS m/e 315.9 (M+H+).
A solution of 2-[2-(4-hydroxy-phenyl)-ethylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (285 mg, 0.90 mmol) in dioxane (10 ml) was treated with a solution of lithium hydroxide monohydrate (948 mg, 22.59 mmol) in water (10 ml). The mixture was stirred at 90° C. for 12 h, then cooled to room temperature and quenched with aqueous potassium hydrogen sulfate to adjust the pH to ˜2-4. The resulting solution was extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and concentrated to give the title compound (298 mg, 100% yield). MS m/e 287.9 (M+H+).
A solution of 2-chloro-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (1 g, 4.66 mmol) and propargylamine (0.45 ml, 7.0 mmol) in EtOH (10 ml) was microwaved at 160° C. for 1 h. The mixture was cooled to room temperature, quenched with 10% aqK2CO3 and extracted with EtOAc. The combined organic extracts were washed water and brine, dried over Na2SO4, filtered and concentrated to give the product as a yellow solid which was not further purified (1.05 g, 92% yield). MS m/e 234.0 (M+H+).
To a solution of 4,6-dimethyl-2-prop-2-ynylamino-pyrimidine-5-carboxylic acid ethyl ester (4.79 g, 20.53 mmol) in dioxane (100 ml) was added a solution of lithium hydroxide monohydrate (21.53 g, 513.25 mmol) in water (100 ml). The mixture was stirred at 90° C. for 6 h, then cooled to room temperature and quenched with a solution of KHSO4 (80 g, 588 mmol) in water (1000 ml) to bring the pH to 2-4. The mixture was extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, and concentrated to give a the desired product as a white solid (3 g, 71% yield). MS m/e 205.9 (M+H+).
A solution of 4,6-dimethyl-2-prop-2-ynylamino-pyrimidine-5-carboxylic acid (1 g, 4.3 mmol), 4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (0.75 ml, 5.16 mmol), bis(cyclopentadienyl)zirconium(IV) chloride hydride (11 mg, 0.43 mmol), and triethylamine (0.6 ml, 0.43 mmol) in THF (10 ml) was microwaved at 150° C. for 20 min Excess 4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (1.5 ml, 10.32 mmol) was added and the solution was microwaved again at 150° C. for 20 h. The mixture was directly loaded onto a silica gel column and purified by flash chromatography with 40% EtOAc in hexane to give the desired product (1.19 g, 77% yield). MS m/e 362.0 (M+H+).
A microwave reaction vessel was charged with 4,6-dimethyl-2-[(E)-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-allylamino]-pyrimidine-5-carboxylic acid ethyl ester (300 mg, 0.83 mmol), 5-bromo-2-methyl-phenol (155 mg, 0.83 mmol), [1,1′-bis (diphenylphosphinoferrocene)dichloropalladium(II) DCM complex (34 mg, 0.042 mmol), potassium carbonate (229 mg, 1.66 mmol), DMF (6 ml), and water (1 ml). The mixture was degassed and flushed with nitrogen, followed by heating to 135° C. for 15 min in a microwave oven. The mixture was partitioned between EtOAc and water. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography with 30% EtOAc in hexane to give the desired product (145 mg, 51% yield). MS m/e 342.0 (M+H+).
A solution of 2-[(E)-3-(3-hydroxy-4-methyl-phenyl)-allylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (145 mg, 0.42 mmol) in EtOH (80 ml) was passed through H-Cube with 10% Pd/C small cartridge at 10 bar, room temperature and 1 ml per min. Solvent was removed by evaporation and the crude product was used directly in the next step. MS m/e 344.0 (M+H+).
A mixture of 2-[3-(3-hydroxy-4-methyl-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester, lithium hydroxide monohydrate (440 mg, 10.5 mmol), in dioxane/water (25 ml/25 ml) was stirred at 95° C. for 4 h, then cooled to room temperature and quenched with aqueous potassium hydrogen sulfate to pH 2-4. The resulting solution was extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and evaporated to give the title compound (110 mg, 83% yield) MS m/e 316.0 (M+H+).
A microwave reaction vessel was charged with 4,6-dimethyl-2-[(E)-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-allylamino]-pyrimidine-5-carboxylic acid ethyl ester (300 mg, 0.83 mmol), 3-bromo-phenol (172 mg, 1.0 mmol), [1,1′-bis(diphenylphosphinoferrocene)-dichloropalladium(II) DCM complex (68 mg, 0.083 mmol), potassium carbonate (229 mg, 1.6 mmol), DMF (4 ml), and water (0.5 ml). The mixture was degassed and flushed with nitrogen, followed by heating to 140° C. for 10 min in a microwave oven. The mixture was directly loaded onto column. Eluting with a 0-50% EtOAc in hexane gradient provided pure product (136.4 mg, 50% yield). MS m/e 328.0 (M+H+).
A mixture of 2-[(E)-3-(3-hydroxy-phenyl)-allylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (136 mg, 0.41 mmol), lithium hydroxide monohydrate (430 mg, 10.25 mmol), dioxane (10 ml) and water (10 ml) was heated to 90° C. for 4 h, then cooled to room temperature and quenched with aqueous potassium hydrogen sulfate to pH 2-4. The resulting solution was extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and evaporated to give the title compound (111 mg, 90% yield). MS m/e 299.9 (M+H+).
A microwave reaction vessel was charged with 4,6-dimethyl-2-[(E)-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-allylamino]-pyrimidine-5-carboxylic acid ethyl ester (250 mg, 0.69 mmol), 6-benzyloxy-2-bromopyridine (183 mg, 0.69 mmol), [1,1′-bis(diphenylphosphinoferrocene)-dichloropalladium(II) DCM complex (28.2 mg, 0.035 mmol), potassium carbonate (190 mg, 1.38 mmol), DMF (4 ml), and water (1 ml). The mixture was degassed and flushed with nitrogen, followed by heating to 135° C. for 15 min in a microwave oven. The mixture was directly loaded onto column. Eluting with 30% EtOAc in hexane provided the desired product (140 mg, 48% yield). MS m/e 419.0 (M+H+).
A mixture of 2-[(E)-3-(6-benzyloxy-pyridin-2-yl)-allylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (140 mg, 0.33 mmol), and lithium hydroxide monohydrate (351 mg, 8.36 mmol), in dioxane/water (10 ml/10 ml) was stirred at 90° C. for 9 h, then cooled to room temperature and quenched with aqueous potassium hydrogen sulfate to pH 2-4. The resulting solution was extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and concentrated to give the title compound (111 mg, 86% yield). MS m/e 391.0 (M+H+).
To a solution of 2-fluoro-3-methoxy-phenyl boronic acid (219 mg, 1.29 mmol) and 4,6-dimethyl-2-ynylamino-pyrimidine-5-carboxylic acid ethyl ester (200 mg, 0.87 mmol) in anhydrous DCM (15 ml) was added [1,1′-bis(diphenylphosphinoferrocene)-dichloropalladium(II) DCM complex (71 mg, 0.087 mmol), potassium carbonate (60.1 mg, 4.25 mmol) and silver (II) oxide (504 mg, 2.18 mmol). The mixture was stirred at room temperature for 3 days, then directly loaded onto a silicagel column. Eluting with 30% EtOAc in hexane provided the desired product (22 mg, 7% yield).
A solution of 2-[3-(2-Fluoro-3-methoxy-phenyl)-prop-2-ynylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (95 mg, 0.27 mmol) in EtOH (200 ml) was passed through H-Cube with a 10% Pd/C small cartridge at 40 bar, 40° C. and 1 ml per min. Solvent was then removed by evaporation to give the desired compound (90 mg, 94% yield). MS m/e 362.0 (M+H+).
To a solution of 2-[3-(2-fluoro-3-methoxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (90 mg, 0.249 mmol) in anhydrous DCM was added at 0° C. BBr3/DCM (1M, 0.5 ml, 0.5 mmol). The mixture was stirred at room temperature for 2 h, then quenched with ice water. The mixture was diluted with DCM. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and evaporated to give the desired product (70 mg, 81% yield). MS m/e 347.9 (M+H+).
A mixture of 2-[3-(2-fluoro-3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (70 mg, 0.202 mmol), and lithium hydroxide monohydrate (21 mg, 5.037 mmol) in dioxane/water (3 ml/3 ml) was stirred at 90° C. for 18 h, cooled to room temperature and quenched with aqueous potassium hydrogen sulfate to adjust the pH to 2-4. The mixture was extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and concentrated to give the title compound (60 mg, 93% yield). MS m/e 319.9 (M+H+).
A microwave reaction vessel was charged with 4,6-dimethyl-2-[(E)-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-allylamino]-pyrimidine-5-carboxylic acid ethyl ester (300 mg, 0.83 mmol), 3-bromo-6-fluoro-phenol (340 mg, 1.66 mmol), dichloro-bis(triphenylphosphine)palladium (II) (38 mg, 0.042 mmol), 1,2,3,4,5-pentaphenyl-1′-(di-tert-butyl-phosphino)-ferrocene (58 mg, 0.083 mmol), cesium fluoride (388 mg, 2.49 mmol), and THF (20 ml). The mixture was degassed, flushed with nitrogen and heated to 120° C. for 20 min in a microwave oven. The mixture was directly loaded onto a silicagel column Eluting with a 0-30% EtOAc in hexane gradient provided pure product (139 mg, 47% yield). MS m/e 359.9 (M+H+).
A solution of 2-[(E)-3-(2-fluoro5-methoxy-phenyl)-allylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (139 mg, 0.39 mmol) in EtOH (250 ml) was passed through H-Cube with 10% Pd/C small cartridge at 20 bar, room temperature and 1 ml per min. Solvent was removed by evaporation to give the desired product (123 mg, 88% yield). MS m/e 361.9 (M+H+).
A solution of 2-[3-(2-fluoro-5-methoxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (123 mg, 0.34 mmol) in anhydrous DCM was treated at 0° C. with BBr3/DCM (1M, 0.7 ml, 0.7 mmol). The mixture was allowed to warm up to room temperature and stirred for 2 h, then quenched with ice water. The mixture was extracted with DCM. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and concentrated to give the desired product (72 mg, 61% yield). MS m/e 347.9 (M+H+).
A mixture of 2-[3-(2-fluoro-5-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (72 mg, 0.21 mmol), and lithium hydroxide monohydrate (217 mg, 5.25 mmol) in dioxane/water (5 ml/5 ml) was stirred at 90° C. overnight, then cooled to room temperature and quenched with aqueous potassium hydrogen sulfate to adjust the pH to 2-4. The mixture was extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and concentrated to give the title compound, (62.5 mg, 93% yield). MS m/e 319.9 (M+H+).
A mixture of 4,6-dimethyl-2-[(E)-3-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)-allylamino]-pyrimidine-5-carboxylic acid ethyl ester (150 mg, 0.415 mmol), 3,5-dibromo-4-methyl-phenol (221 mg, 0.83 mmol), dichloro-bis(triphenylphosphine)palladium (II) (19 mg, 0.021 mmol), 1,2,3,4,5-pentaphenyl-1′-(di-tert-butyl-phosphino)-ferrocene (29 mg, 0.042 mmol), and cesium fluoride (189 mg, 1.245 mmol) in THF (10 ml) was stirred at room temperature for 24 h. The mixture was directly loaded onto a silicagel column Eluting with a 0-30% EtOAc in hexane gradient afforded the desired product (94 mg, 54% yield). MS m/e 421.7 (M+H+).
A solution of 2-[(E)-3-(3-bromo-5-hydroxy-2-methyl-phenyl)-allylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (94 mg, 0.22 mmol) in EtOH (250 ml) was passed through H-Cube with 10% Pd/C small cartridge at 1 atmosphere, room temperature and 1 ml per min. After concentration, the residue was purified by flash chromatography with a 0-50% EtOAc in hexane gradient to afford the desired product (19 mg, 25% yield). MS m/e 344.0 (M+H+).
A solution of 2-[3-(2-methyl-5-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (19 mg, 0.055 mmol) in dioxane (3 ml) was treated with a solution of lithium hydroxide monohydrate (58 mg, 1.38 mmol) in water (3 ml). The mixture was stirred at 90° C. for 4 h, then cooled to room temperature and quenched with aqueous potassium hydrogen sulfate to adjust the pH to ˜2-4. The resulting solution was extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and evaporated to give the title compound (15.8 mg, 91% yield). MS m/e 315.9 (M+H+).
A microwave reaction vessel was charged with 3-(methoxy-phenyl)-propylamine hydrochloride (404 mg, 2.0 mmol), 2-chloro-4-methyl-pyrimidine-5-carboxylic acid ethyl ester (400 mg, 2.0 mmol), potassium acetate (785 mg, 8.0 mmol) and ethanol (10 ml), then heated to 150° C. for 1 h in a microwave oven. The mixture was cooled to room temperature and extracted with EtOAc. The organic phase was washed with 20% sodium bicarbonate, water and brine, then dried over sodium sulfate, filtered, and evaporated. The residue was purified by flash chromatography with 30% EtOAc in hexane to afford the desired compound (568.4 mg, 86% yield). MS m/e 330.0 (M+H+).
A solution of 2-[3-(3-methoxy-phenyl)-propylamino]-4-methyl-pyrimidine-5-carboxylic acid ethyl ester (568 mg, 1.72 mmol) in anhydrous DCM (10 ml) was treated at 0° C. with BBr3/DCM (1M, 3.4 ml, 3.4 mmol). The mixture was allowed to warm up to room temperature and stirred for 2 h, then quenched with ice water. The organic layer was separated and the aqueous layer was extracted by DCM. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and concentrated to give the desired product (530.6 mg, 98% yield). MS m/e 315.9 (M+H+).
A mixture of 2-[3-(3-hydroxy-phenyl)-propylamino]-4-methyl-pyrimidine-5-carboxylic acid ethyl ester (530 mg, 1.68 mmol), and lithium hydroxide monohydrate (1.76 g, 42 mmol), in dioxane/water (5 ml/5 ml) was stirred at 60° C. overnight, then cooled to room temperature and quenched with aqueous potassium hydrogen sulfate to adjust the pH to ˜2-4. The resulting solution was extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and evaporated to give the title compound (415 mg, 86% yield). MS m/e 287.9 (M+H+).
A microwave reaction vessel was charged with 3-(methoxy-phenyl)-propylamine (1.03 g, 6.24 mmol), 2-chloro-4-trifluoromethyl-pyrimidine-5-carboxylic acid methyl ester (1 g, 4.16 mmol), potassium acetate (1.22 g, 12.48 mmol) and methanol (10 ml). The mixture was heated to 150° C. for 1 h in a microwave oven. The mixture was cooled to room temperature and extracted with EtOAc. The organic phase washed with water and brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography with 25% EtOAc in hexane to afford the desired product (917 mg, 60% yield). MS m/e 369.9 (M+H+).
A solution of 2-[3-(3-methoxy-phenyl)-propylamino]-4-trifluoromethyl-pyrimidine-5-carboxylic acid ethyl ester (917 mg, 2.48 mmol) in anhydrous DCM (10 ml) was treated at 0° C. with BBr3/DCM (1M, 5 ml, 5 mmol). The mixture was allowed to warm up to room temperature and stirred for 2 h, then quenched with ice water. The organic layer was separated and the aqueous layer was extracted by DCM. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and concentrated to give the desired product (0.9 g, 100% yield). MS m/e 355.9 (M+H+).
A mixture of 2-[3-(3-hydroxy-phenyl)-propylamino]-4-trifluoromethyl-pyrimidine-5-carboxylic acid methyl ester (0.9 g, 2.53 mmol), and lithium hydroxide monohydrate (2.13 g, 50.66 mmol), in dioxane/water (20 ml/20 ml) was stirred at 0° C. overnight, then cooled to room temperature and quenched with aqueous potassium hydrogen sulfate to adjust the pH to ˜2-4. The resulting solution was extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and evaporated to give the title compound (770 mg, 89% yield). MS m/e 341.9 (M+H+).
A mixture of 3-(1H-indazol-4-yl)-propylamine dihydrochloride (1.61 g, 7.5 mmol), 2-chloro-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (2.23 g, 9.0 mmol) and EtOH (30 ml) was heated in a microwave reactor at 160° C. for 1.0 h, diluted with EtOAc (200 ml), washed with brine (200 ml) and re-extracted with EtOAc (200 ml). The combined organic layers were washed with brine, dried over sodium sulfate, filtered, concentrated in the presence of silica gel and chromatographed (10-80% EtOAc in hexane) to give the desired product (2.28 g, 81% yield).
To a solution of 2-[3-(3-(1H-indazol-4-yl)-propylamino)-4,6-dimethyl-pyrimidine-5-carboxylic acid ethyl ester (1.79 g, 5.06 mmol) in dioxane (35 ml) was added a solution of lithium hydroxide (4.24 g, 101.2 mmol) in water (45 ml). The mixture was heated to reflux for 2 hours, cooled to room temperature, acidified with 2N KHSO4, concentrated, filtered, and the precipitate was washed with water to afford the title compound (1.64 g, 99% yield).
To a solution of 2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid (1.76 g, 5.84 mmol) in anhydrous DMF (60 ml) was added Et3N (2.5 ml, 7.0 mmol), HBTU (2.66 g, 7.01 mmol), HOBT (0.95 g, 7.01 mmol), and H-DAP(Boc)OMe hydrochloride (1.79 g, 7.01 mmol). The mixture was stirred at room temperature for 3 h, diluted with brine (200 ml) and extracted with ethyl acetate. The combined organic layers were washed with 1:1 saturated sodium bicarbonate/brine and brine, then dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by flash chromatography with a 40-100% EtOAc in hexane gradient to give the title compound (2.66 g, 91% yield). MS m/e 501.9 (M+H+).
To a solution of (S)-3-tert-Butoxycarbonylamino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (2.66 g, 5.30 mmol) in MeOH (10 ml) was added 4.0 M HCl in dioxane (20 mL). After one hour the mixture was concentrated and azeotroped with MeOH. The product was triturated with ether, filtered, and washed with ether to afford the title compound (2.16 g, 93% yield). MS m/e 401.9 (M+H+).
To a solution of 2-[3-(3-hydroxy-4-methyl-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid (110 mg, 0.35 mmol) in anhydrous DMF (5 ml) was added triethylamine (0.15 ml, 1.05 mmol), HBTU (137 mg, 0.36 mmol), HOBT (49 mg, 0.36 mmol), and H-DAP(Boc)OMe hydrochloride (92 mg, 0.36 mmol). The mixture was stirred at room temperature for 2 h, followed by direct loading onto a column. Eluting with a 0-80% EtOAc in hexane gradient afforded the title compound contaminated with DMF (250 mg). MS m/e 516.1 (M+H+).
To a solution of 2-[(E)-3-(3-hydroxy-phenyl)-allylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid (111 mg, 0.37 mmol) in anhydrous DMF (3 ml) was added at room temperature triethylamine (0.15 ml, 1.11 mmol), HBTU (144 mg, 0.38 mmol), HOBT (51 mg, 0.38 mmol), and H-DAP(Boc)OMe hydrochloride (96.3 mg, 0.3 mmol). The mixture was stirred at room temperature overnight, followed by direct loading onto a silicagel column. Eluting with a 40-100% EtOAc in hexane gradient provided the desired compound contaminated with DMF (274.3 mg). MS m/e 500.0 (M+H+).
To a solution of 2-[3-(2-fluoro-3-hydroxyl-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid (60 mg, 0.19 mmol) in anhydrous DMF (4 ml) was added at room temperature triethylamine (0.13 ml, 1.05 mmol), HBTU (143 mg, 0.38 mmol), HOBT (51 mg, 0.38 mmol), and H-DAP(Boc)OMe hydrochloride (96 mg, 0.38 mmol). The mixture was stirred at room temperature for 18 h, then quenched with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and evaporated. The residue was purified by flash chromatography with a 40-100% EtOAc on hexane gradient to yield the desired product (94 mg, 96% yield). MS m/e 520.0 (M+H+).
To a solution of 2-[3-(2-methyl-5-hydroxyl-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid (15.8 mg, 0.05 mmol) in anhydrous DMF (2 ml) was added triethylamine (25 mg, 0.25 mmol), HBTU (37.9 mg, 0.1 mmol), HOBT (13.5 mg, 0.10 mmol), and H-DAP(Boc)OMe hydrochloride (25.5 mg, 0.10 mmol). The mixture was stirred at room temperature for 3 h, quenched with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with a 40-100% EtOAc in hexane gradient to give the desired product (26 mg, 100% yield). MS m/e 516.0 (M+H+).
To a solution of 2-[3-(2-fluoro-5-hydroxyl-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid (62.5 mg, 0.196 mmol) in anhydrous DMF (4 ml) was added triethylamine (0.14 ml, 0.98 mmol), HBTU (149 mg, 0.39 mmol), HOBT (53 mg, 0.39 mmol), and H-DAP(Boc)OMe hydrochloride (100 mg, 0.39 mmol). The mixture was stirred at room temperature over the weekend, then quenched with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and evaporated. The residue was purified by flash chromatography with a 40-100% EtOAc in hexane gradient to afford the desired product (62 mg, 61% yield). MS m/e 520.0 (M+H+).
To a solution of 2-[3-(3-hydroxyl-phenyl)-propylamino]-pyrimidine-5-carboxylic acid (900 mg, 3.29 mmol) in anhydrous DMF (10 ml) was added triethylamine (1.4 ml, 9.87 mmol), HBTU (1.5 g, 3.85 mmol), HOBT (534 mg, 3.95 mmol), and H-DAP(Boc)OMe hydrochloride (864 mg, 3.39 mmol). The mixture was stirred at room temperature overnight, then quenched with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and evaporated. The residue was purified by flash chromatography with a 0-90% EtOAc in hexane gradient to give the desired product (820 mg, 53% yield). MS m/e 474.0 (M+H+).
To a solution of 2-[3-(3-hydroxyl-phenyl)-propylamino]-4-methyl-pyrimidine-5-carboxylic acid (415 mg, 1.444 mmol) in anhydrous DMF (5 mL) was added triethylamine (0.6 ml, 4.33 mmol), HBTU (559 mg, 1.47 mmol), HOBT (199 mg, 1.47 mmol), and H-DAP(Boc)OMe hydrochloride (375 mg, 1.47 mmol). The mixture was stirred at room temperature overnight, then quenched with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and evaporated. The residue was purified by flash chromatography with a 0-80% EtOAc in hexane gradient to give the title compound (482 mg, 68% yield). MS m/e 488.0 (M+H+).
To a solution of 2-[3-(3-hydroxyl-phenyl)-propylamino]-4-trifluoromethyl-pyrimidine-5-carboxylic acid (770 mg, 2.26 mmol) in anhydrous DMF (10 ml) was added triethylamine (0.95 ml, 6.78 mmol), HBTU (1.03 g, 2.71 mmol), HOBT (366 mg, 2.71 mmol), and H-DAP(Boc)OMe hydrochloride (591 mg, 2.32 mmol). The mixture was stirred at room temperature overnight, then quenched with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and evaporated. The residue was purified by flash chromatography with a 0-60% EtOAc in hexane gradient to give the desired product (970 mg, 79% yield). MS m/e 441.9 (M+H+-Boc).
To a solution of 4-ethyl-2-[3-(3-hydroxy-phenyl)-propylamino]-6-methyl-pyrimidine-5-carboxylic acid (219 mg, 0.694 mmol) in anhydrous DMF (10 ml) was added triethylamine (0.3 ml, 2.082 mmol), HBTU (268 mg, 0.708 mmol), HOBT (96 mg, 0.708 mmol), and H-DAP(Boc)OMe hydrochloride (180 mg, 0.708 mmol). The mixture was stirred at room temperature overnight, then quenched with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and evaporated. The residue was purified by flash chromatography with a 0-80% EtOAc in hexane gradient to give the title compound (265 mg, 74% yield). MS m/e 516.0 (M+H+-Boc).
To a solution of 4,6-diethyl-2-[3-(3-hydroxy-phenyl)-propylamino]-pyrimidine-5-carboxylic acid (26 mg, 0.08 mmol) in anhydrous DMF (2 ml) was added triethylamine (40 mg, 0.4 mmol), HBTU (33 mg, 0.09 mmol), HOBT (12 mg, 0.09 mmol), and H-DAP(Boc)OMe hydrochloride (22 mg, 0.09 mmol). The mixture was stirred at room temperature overnight, then quenched with water and extracted with EtOAc. The organic layer was separated, washed with brine, dried over sodium sulfate, filtered and evaporated. The residue was purified by flash chromatography with a 0-80% EtOAc in hexane gradient to give the title compound (21.6 mg, 52% yield). MS m/e 530.0 (M+H+).
To a solution of 2-[3-(1H-indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid (2.11 g, 6.45 mmol) in anhydrous DMF (60 ml) was added triethylamine (2.7 ml, 7.74 mmol), HBTU (2.94 g, 7.74 mmol), HOBT (1.05 g, 7.74 mmol), and H-DAP(Boc)OMe hydrochloride (1.97 g, 7.74 mmol). The mixture was stirred at room temperature for 18 h, diluted with brine and extracted with EtOAc. The combined organic layers were washed with brine, saturated sodium bicarbonate and brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (50-100% EtOAc in hexane) to give the desired product (2.98 g, 88% yield).
A solution of (S)-3-tert-Butoxycarbonylamino-2-({2-[3-(1H-indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (0.114 g, 0.22 mmol) in MeOH (2 ml) was treated with 4.0 M HCl in dioxane (5 ml). After one hour at room temperature, the mixture was concentrated and azeotroped with MeOH. The product was triturated with ether, filtered, and washed with excess ether to afford the title compound (HCl salt) (98 mg, 98% yield).
To a solution of 2-[3-(3-hydroxy-phenyl)-propoxy]-pyrimidine-5-carboxylic acid (10.25 g, 4.14 mmol) and H-DAP(BOC)-OMe.HCl (1.06 g, 4.14 mmol) in DMF (20 ml) were successively added at room temperature triethylamine (1.49 ml, 10.76 mmol), and HBTU (2.04 g, 5.4 mmol). The mixture was stirred at room temperature for 2 h then quenched with water and extracted with EtOAc. The organic layer was washed with saturated aqueous NaHCO3 and brine, then dried over Na2SO4, filtered and concentrated. The crude product was purified by flash chromatography with a 20-75% EtOAC in hexane gradient to afford the desired product (1.7 g, 82% yield).
The starting BOC amine (1.7 g, 3.3 mmol) was treated with 4N HCl in dioxane (20 ml) at room temperature for 45 hr and concentrated to dryness to afford a foam. This material was suspended in dry ether and kept at room temperature overnight. The solvent was removed under reduced pressure and the resulting white solid was dried in a vacuum desiccator to afford the HCl salt of the amine as a white solid (1.6 g, 100% yield).
(S)-3-amino-2-({2-[(3′-hydroxy-biphenyl-2-ylmethyl)-amino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride:
To a solution of 2-[(3′-hydroxy-biphenyl-2-ylmethyl)-amino]-4,6-dimethyl-pyrimidine-5-carboxylic acid (75 mg, 0.2 mmol) and H-DAP(BOC)-OMe.HCl (77 mg, 0.3 mmol) in DMF (2 ml) were successively added at room temperature triethylamine (112 ul, 0.8 mmol), HOBT (30 mg, 0.2 mmol) and HBTU (114 mg, 0.3 mmol). The mixture was stirred at room temperature overnight then quenched with 1NHCl and extracted with EtOAc. The organic layer was washed with pH=8 buffer and brine, then dried over Na2SO4, filtered and concentrated. The crude product was purified by flash chromatography with a 40-100% EtOAC in hexane gradient to afford the desired product (75 mg, 68% yield). This material was dissolved in methanol (1.5 ml) and treated with 4NHCl in dioxane (0.5 ml) at room temperature for 3.5 h. The reaction mixture was concentrated under reduced pressure to afford the desired amine HCl salt (71 mg, 100% yield).
A similar procedure was used to make the following two intermediates:
To a solution of 2-(6-hydroxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-4,6-dimethyl-pyrimidine-5-carboxylic acid (36 mg, 0.1 mmol) and H-DAP(BOC)-OMe.HCl (33 mg, 0.15 mmol) in DMF (1.5 ml) were successively added at room temperature triethylamine (60 ul, 0.4 mmol), HOBT (15 mg, 0.1 mmol) and HBTU (60 mg, 0.15 mmol). The mixture was stirred at room temperature overnight then quenched with 1NHCl and extracted with EtOAc. The organic layer was washed with brine, then dried over Na2SO4, filtered and concentrated. The crude product was purified by flash chromatography with a 50-100% EtOAC in hexane gradient to afford the desired product. This material was dissolved in methanol (1.0 ml) and treated with 4NHCl in dioxane (0.3 ml) at room temperature for 2.5 h. The reaction mixture was concentrated under reduced pressure to afford the desired amine HCl salt (20 mg, 40% overall yield).
To a solution of 2-(2-(8-hydroxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-4,6-dimethyl-pyrimidine-5-carboxylic acid (0.166 g, 0.53 mmol), Et3N (0.22 ml, 1.59 mmol), and H-DAP(Boc)OMe hydrochloride (0.16 g, 0.64 mmol), in anhydrous DMF (4 ml) was added a solution of HBTU (0.24 g, 0.64 mmol) and HOBT (0.086 g, 0.64 mmol) in DMF (4 ml). The mixture was stirred at room temperature overnight, diluted with brine and extracted with EtOAc. The combined organic layers were washed with 1:1 satNaHCO3/brine and brine, then dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude residue was purified by flash chromatography with a 10-100% EtOAc in hexane gradient to give the title compound (188 mg, 69% yield). MS m/e 514.0 (M+H+).
To a solution of (S)-3-tert-butoxycarbonylamino-2-{[2-(8-hydroxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-4,6-dimethyl-pyrimidine-5-carbonyl]-amino}-propionic acid methyl ester (188 mg, 0.367 mmol) in MeOH (1 ml) was added 4.0 M HCl ind (4 ml). After one hour the mixture was concentrated and azeotroped with MeOH to afford the title compound (209 mg, 100% yield). MS m/e 414.0 (M+H+).
To a solution of 4,6-dimethyl-2-prop-2-ynylamino-pyrimidine-5-carboxylic acid (3 g, 14.62 mmol) in DMF (120 ml) was added triethylamine (10.2 ml, 73.1 mmol), HBTU (6.65 g, 17.54 mmol), HOBT (2.4 g, 17.54 mmol), and H-DAP(Boc)OMe hydrochloride (4.47 g, 17.54 mmol). The mixture was stirred at room temperature overnight then quenched with 1NHCl and extracted with EtOAc. The organic layer was washed with brine, then dried over Na2SO4, filtered and concentrated. The crude product was purified by flash chromatography with a 40-100% EtOAC in hexane gradient to afford the desired product as a white solid (4.62 g, 78% yield). MS m/e 406.1 (M+H+).
A solution of (S)-3-tert-butoxycarbonylamino-2-[(4,6-dimethyl-2-prop-2-ynylamino-pyrimidine-5-carbonyl)-amino]-propionic acid methyl ester (4.62 g, 11.39 mmol) in DCM (35 ml) was cooled to 0° C. and 30% TFA/DCM (15 ml) was added dropwise. The solution was stirred at room temperature for 1 h. The solvent and excess TFA were removed by evaporation under vacuum. The residue was dissolved in DMF (50 ml), and treated with DIEA (19.8 ml, 113 mmol) at 0° C. Thiophene-2-carboxylic acid (1.75 g, 13.67 mmol), HBTU (5.2 g, 13.67 mmol) and HOBT (1.85 g, 13.67 mmol) were added and the mixture was stirred at room temperature over the weekend. The mixture was partitioned between EtOAc and water. The layers were separated and the organic layer was washed with brine, dried over Na2SO4, filtered and concentrated. The residue was purified by flash chromatography with a 40-100% EtOAC in hexane gradient to afford the desired product as a white solid (4 g, 85% yield). MS m/e 415.9 (M+H+).
To a solution of 2-[(E)-3-(6-benzoxy-pyridin-2-yl)-allylamino]-4,6-dimethyl-pyrimidine-5-carboxylic acid (111 mg, 0.284 mmol) in anhydrous DMF (5 ml) was added triethylamine (0.12 ml, 0.85 mmol), HBTU (110 mg, 0.29 mmol), HOBT (39 mg, 0.29 mmol), and H-DAP(Boc)OMe hydrochloride (74 mg, 0.29 mmol). The mixture was stirred at room temperature overnight, then directly loaded onto a silicagel column. Eluting with a 30-100% EtOAc in hexane gradient afforded the desired product (122 mg, 73% yield). MS m/e 591.0 (M+H+).
To a solution of 2-[4-(3-Allyloxy-phenyl)-butyl]-4,6-dimethyl-pyrimidine-5-carboxylic acid (64 mg, 0.188 mmol) and H-DAP(BOC)-OMe.HCl (72 mg, 0.282 mmol) in DMF (2 ml) were successively added at room temperature triethylamine (78 ul, 0.56 mmol), HOBT (47 mg, 0.34 mmol) and HBTU (130 mg, 0.39 mmol). The mixture was stirred at room temperature for 2 h then quenched with pH=3 buffer and extracted with EtOAc. The organic layer was washed with saturated aqueous NaHCO3 and brine, then dried over Na2SO4, filtered and concentrated. The crude product was purified by flash chromatography with a 10-60% EtOAC in hexane gradient to afford the desired diamino propionic acid derivative (34 mg, 34% yield). This material was treated with 4N HCl in dioxane (1.5 ml) for 1 h and concentrated to dryness to afford the free amine as the HCl salt that was then taken up in DMF (1 ml). Thiophene-2-carboxylic acid (14 mg, 0.10 mmol) was added, followed by triethylamine (30 ul, 0.21 mmol), HOBT (12 mg, 0.08 mmol) and HBTU (32 mg, 0.08 mmol). The mixture was stirred at room temperature for 1 h 30 min, quenched with pH=3 buffer and extracted with EtOAc. The organic layer was washed with saturated aqueous NaHCO3 and brine, then dried over Na2SO4, filtered and concentrated. The crude product was purified by flash chromatography with a 50-100% EtOAC in hexane gradient to afford the desired thiophene amide (10 mg, 26% yield from the BOC derivative). This material was dissolved in DCM (300 ul) and treated for 40 min at room temperature with PhSiH3 (25 ul, 0.18 mmol) in the presence of a catalytic amount of Pd[PPh3]4 (2.5 mg, 0.002 mmol). The mixture was quenched with pH=3 buffer and extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4, filtered and concentrated. The crude product was purified by flash chromatography with a 50-100% EtOAC in hexane gradient to afford the desired phenol (10 mg, 100% yield). The ester was then hydrolyzed with LiOH.H2O (0.35 mmol, 15 mg) in THF/H2O (0.2 ml/0.2 ml) at room temperature for ½ h. The mixture was quenched by adding 220 ul of 1N KHSO4 then partitioned between pH=3 buffer and EtOAc. The layers were separated. The organic layer was dried over Na2SO4, filtered and concentrated to afford the desired acid (3 mg, 34% yield, 90% pure). MS m/e 497.0 (MH+).
To a solution of thiophene-2-carboxylic acid (0.46 g, 3.6 mmol) and HBTU (1.78 g, 4.7 mmol) in DMA (20 ml) were successively added at room temperature triethylamine (1.0 ml, 7.2 mmol) and (S)-3-amino-2-{2-[3-(3-hydroxy-phenyl)-propoxy]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino-propionic acid methyl ester (1.6 g, 3.6 mmol). The mixture was stirred at room temperature for 18 h then quenched with water and extracted with EtOAc. The organic layer was washed with saturated aqueous NaHCO3 and brine, then dried over MgSO4, filtered and concentrated. The crude product was purified by flash chromatography with a 20-100% EtOAC in hexane gradient to afford the desired product (1.29 g, 70% yield).
A solution of (S)-2-({2-[3-(3-hydroxy-phenyl)-propoxy]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (1.29 g, 2.52 mmol) in methanol (15 ml) was treated with 2NNaOH (10 ml, 20 mmol) and the reaction mixture was stirred at room temperature for 2.5 h. The volume of the reaction mixture was reduced to a third and water was added. This aqueous solution was stirred at room temperature and the pH was adjusted to 5.0 with 1NHCl to induce precipitation of the desired acid. The off-white precipitate was collected by filtration, washed with water and dried in a vacuum desiccator to give the product as a beige foam (0.84 g, 65% yield). MS m/e 499.0 (MH+).
To a solution of (S)-3-amino-2-({2-[3-(1H-indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (0.05 g, 30.1 mmol) and triethylamine (70 ul, 0.5 mmol) in DMF (1 ml) was added Ac2O (50 ul. 0.5 mmol). The mixture was stirred at room temperature for 2 h then quenched with 1NHCl and stirred at room temperature overnight. The phases were separated. The desired product was present in the acidic aqueous layer. This layer was made basic by adding NaOH pellets. This promoted the saponification of the acid. The aqueous layer was concentrated under reduced pressure and the residue was taken up in pH=2 buffer. The solution was concentrated and the resulting white solid was triturated with DMF/dioxane (1:10), then filtered. The filtrate was concentrated under reduced pressure to afford the desired acid (18 mg, 40% yield). MS m/e 454.3 (WO.
To a solution of thiophene-2-carboxylic acid (0.028 g, 0.21 mmol), HBTU (0.1 g, 0.26 mmol) and HOBT (0.035 g, 0.26 mmol) in DMF (5 ml) were successively added at room temperature triethylamine (0.09 ml, 0.26 mmol) and (S)-3-amino-2-({2-[3-(1H-indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (0.1 g, 0.21 mmol). The mixture was stirred at room temperature for 18 h then quenched with water and extracted with EtOAc. The organic layer was washed with saturated aqueous NaHCO3 and brine, then dried over MgSO4, filtered and concentrated. The crude product was purified by flash chromatography with a 50-100% EtOAC in hexane gradient to afford the desired product (0.059 g, 52% yield).
A solution of (S)-2-({2-[3-(1H-indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (0.059 g, 0.11 mmol) in THF/water (4 ml/5 ml)) was treated with LiOH.H2O (0.023 g, 0.55 mmol) and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was quenched by adding 1N KHSO4 (0.55 ml) and water. The white precipitate was collected by filtration, washed with water then suspended in acetonitrile/water and lyophilized. White solid (0.056 g, 98% yield). MS m/e 522.1 (MH+).
To a solution of thiophene-2-carboxylic acid (0.24 g, 1.85 mmol), HBTU (0.84 g, 2.22 mmol) and HOBT (0.30 g, 0.22 mmol) in DMF (40 ml) were added at room temperature triethylamine (0.56 ml, 5.55 mmol) and (S)-3-amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (0.81 g, 1.85 mmol). The mixture was stirred at room temperature for 1 h then quenched with water/brine and extracted with EtOAc. The organic layer was washed with brine, then dried over Na2SO4, filtered and concentrated. The crude product was purified by flash chromatography with a 40-100% EtOAC in hexane gradient to afford the desired product (0.59 g, 62% yield).
A solution of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (0.59 g, 1.15 mmol) in THF/water (20/30 ml) was treated with LiOH.H2O (0.24 g, 5.77 mmol) and the reaction mixture was stirred at room temperature for 1.5 h. The mixture was quenched with 1N KHSO4 (6 ml) and concentrated to about half its volume. The white precipitate was collected by suction filtration, washed with water and air-dried.
(0.54 g, 94% yield). MS m/e 497.9 (MH+).
To a solution of (S)-3-amino-2-({2-[(3′-hydroxy-biphenyl-2-ylmethyl)-amino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester, HCl salt (71 mg, 0.16 mmol) and thiophene-2-carboxylic acid (22 mg, 0.17 mmol) in DMF (2 ml), were successively added at room temperature triethylamine (66 ul, 0.47 mmol), HOBT (24 mg, 0.16 mmol) and HBTU (66 mg, 0.17 mmol). The mixture was stirred at room temperature for 0.5 h then quenched with 1NHCl and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated. The crude product was purified by flash chromatography with a 50-100% EtOAC in hexane gradient to afford the desired product (47 mg, 62% yield).
A similar procedure was used to make the following two examples:
A solution of (S)-2-({2-[(3′-hydroxy-biphenyl-2-ylmethyl)-amino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (47 mg, 0.08 mmol) in H2O/dioxane (1 ml/1 ml) was treated with LiOH.H2O (35 mg, 0.84 mmol) at room temperature for 1.5 h. The reaction mixture was quenched with 1NHCl (1.5 ml) and concentrated under reduced pressure. The residue was partitioned between pH=2 buffer and EtOAc. The organic phase was dried over Na2SO4, filtered and concentrated to afford the desired acid (43 mg, 100% yield). MS m/e 545.9 (MH+).
A similar procedure was used to make the following two examples:
To a solution of (S)-3-amino-2-({2-[6-hydroxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (18 mg, 0.04 mmol) and thiophene-2-carboxylic acid (6 mg, 0.05 mmol) in DMF (0.5 ml), were successively added at room temperature triethylamine (18 ul, 0.13 mmol), HOBT (7 mg, 0.04 mmol) and HBTU (17 mg, 0.05 mmol). The mixture was stirred at room temperature for 1 h then quenched with 1NHCl and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated. The crude product was purified by flash chromatography with a 50-100% EtOAC in hexane gradient to afford the desired product (12 mg, 60% yield).
A similar procedure was used to prepare the following analog:
A solution of (S) 2-(2-[6-hydroxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino]-4,6-dimethyl-pyrimidine-5-carbonyl-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (24 mg, 0.04 mmol) in H2O/dioxane (1 ml/1 ml) was treated with LiOH.H2O (17 mg, 0.40 mmol) at room temperature for 3 h. The reaction mixture was quenched with 1NHCl and concentrated under reduced pressure. The residue was partitioned between pH=2 buffer and EtOAc/MeOH (10/1). The organic phase was dried over Na2SO4, filtered and concentrated. The residue was purified by reverse phase HPLC to afford the desired acid as white solid (5 mg, 25% yield). MS m/e 509.9 (MH+).
A similar procedure was used to prepare the following analog:
A reaction vessel containing 3-iodo-phenol (39.7 mg, 0.18 mmol), DIEA (1 ml), dichlorobis(triphenylphosphine)palladium (II) (4.2 mg, 0.006 mmol), copper (I) iodide (2.3 mg, 0.012 mmol) and DMF (1 ml) was sealed, degassed, and flushed with nitrogen. The mixture was stirred at room temperature for 30 min. A solution of (S)-2-[(4,6-dimethyl-2-prop-2-ynylamino-pyrimidine-5-carbonyl)-amino]-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (50 mg, 0.1 mmol) and DIEA (0.5 ml) in DMF (0.5 ml) was added at room temperature. The reaction mixture was stirred at room temperature for 18 h. The mixture was extracted with EtOAc. The combined organic extracts were washed with water and brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with a 40-100% EtOAc in hexane gradient to afford the desired product as a yellow solid (29 mg, 48% yield). MS m/e 508.0 (M+H+).
A mixture of (S)-2-({2-[3-(3-hydroxy-phenyl)-prop-2-ynylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (552.6 mg, 1.09 mmol), and Lindlar's catalyst (350 mg) in EtOAc (100 ml) was subjected to hydrogenation via Parr Shaker Type Apparatus at 40 psi and room temperature for 8 h. The solution was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by flash chromatography with 10% MeOH in DCM to give the expected product as a white solid (352.5 mg, 63% yield). MS m/e 510.0 (M+H+).
A reaction vessel containing 4-bromo-indazole (71 mg, 0.36 mmol), DIEA (1 ml), dichlorobis(triphenylphosphine)palladium (II) (8.4 mg, 0.012 mmol), copper (I) iodide (4.4 mg, 0.024 mmol) and DMF (1 ml) was sealed, degassed, and flushed with nitrogen. The mixture was stirred at room temperature for 30 min. A solution of (S)-2-[(4,6-dimethyl-2-prop-2-ynylamino-pyrimidine-5-carbonyl)-amino]-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (100 mg, 0.24 mmol) and DIEA (0.5 ml) in DMF (0.5 ml) was added at room temperature. The reaction mixture was stirred at 50° C. for 18 h. The mixture was cooled to room temperature and extracted with EtOAc. The combined organic extracts were washed with water and brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with 20% MeOH in DCM to give pure product (37.3 mg, 29% yield). MS m/e 532.0 (M+H+).
A reaction vessel containing 6-bromoindazole (141.9 mg, 0.72 mmol), Et3N (1.5 ml), dichlorobis(triphenylphosphine)palladium (II) (16.8 mg, 0.024 mmol), copper (I) iodide (9.2 mg, 0.048 mmol) and DMF (2 ml) was sealed, degassed and flushed with nitrogen. The mixture was stirred at room temperature for 30 min. A solution of (S)-2-[(4,6-dimethyl-2-prop-2-ynylamino-pyrimidine-5-carbonyl)-amino]-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (200 mg, 0.48 mmol) in DMF (2 ml) was added at room temperature. The reaction mixture was stirred at 47° C. for 24 h. The mixture was cooled to room temperature and extracted with EtOAc. The combined organic extracts were washed with water and brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with a 40-100% EtOAc in hexane gradient to give pure product (99.2 mg, 37% yield). MS m/e 532.0 (M+H+).
A solution of (S)-2-({2-[3-(1H-indazol-6-yl)-prop-2-ynylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (85 mg, 0.16 mmol) in MeOH (75 ml) was passed through H-Cube with 5% Pd/C small cartridge at 1 atmosphere, room temperature and 1 ml per min. Solvent was then removed by evaporation and the residue was purified by flash chromatography with 10% MeOH in DCM to give the desired product (65 mg, 76% yield). MS m/e 536.0 (M+H+).
A reaction vessel containing 3-fluoro-5-bromophenol (69 mg, 0.36 mmol), Et3N (1.5 ml), dichlorobis(triphenylphosphine)palladium (II) (8.4 mg, 0.012 mmol) and copper (I) iodide (4.6 mg, 0.024 mmol) in DMF (1 ml) was sealed, degassed, and flushed with nitrogen. The mixture was stirred at room temperature for 30 min. A solution of (S)-2[(4,6-dimethyl-2-prop-2-ynylamino-pyrimidine-5-carbonyl)-amino]-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (100 mg, 0.24 mmol) in DMF (1 mL) was added at room temperature. The reaction mixture was stirred at room temperature over the weekend. The mixture was extracted with EtOAc. The combined organic extracts were washed with water and brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with 10% MeOH in DCM to give pure product (51 mg, 40% yield). MS m/e 525.9 (M+H+).
A solution of (S)-2-({2-[3-(3-fluoro-5-hydroxy-phenyl)-prop-2-ynylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]propionic acid methyl ester (67.7 mg, 0.13 mmol) in MeOH (50 ml) was passed through H-Cube with 5% Pd/C small cartridge at 1 atmosphere, room temperature and 1 ml per min. Solvent was then removed by evaporation and the residue was purified by flash chromatography with 90% EtOAc in hexane to give pure product (18.2 mg, 26% yield). MS m/e 530.0 (M+H+).
A reaction vessel containing 2-fluoro-5-bromophenol (138 mg, 0.72 mmol), Et3N (1.5 ml), dichlorobis(triphenylphosphine) palladium (II) (16.8 mg, 0.024 mmol) and copper (I) iodide (9.2 mg, 0.048 mmol) in DMF (2 ml) was sealed, degassed, and flushed with nitrogen. The mixture was stirred at room temperature for 30 min. A solution of (S)-2[(4,6-dimethyl-2-prop-2-ynylamino-pyrimidine-5-carbonyl)-amino]-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (200 mg, 0.48 mmol) in DMF (2 ml) was added at room temperature. The reaction mixture was stirred at 47° C. for 18 h. The mixture was extracted with EtOAc. The combined organic extracts were washed with water and brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with 10% MeOH in DCM to give pure product (42 mg, 17% yield). MS m/e 526.0 (M+H+).
A solution of (S)-2-({2-[3-(2-fluoro-5-hydroxy-phenyl)-prop-2-ynylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]propionic acid methyl ester (42 mg, 0.08 mmol) in MeOH (50 ml) was passed through H-Cube with 5% Pd/C small cartridge at 1 atmosphere, room temperature and 1 ml per min. Solvent was then removed by evaporation and the residue was purified by flash chromatography with 5% MeOH in DCM to give pure product (42 mg, 99% yield). MS m/e 530.0 (M+H+).
A reaction vessel containing 3-iodo-2-methyl-phenol (253 mg, 0.72 mmol), Et3N (2 ml) dichlorobis(triphenylphosphine)palladium (II) (25.3 mg, 0.036 mmol) and copper (I) iodide (13.5 mg, 0.072 mmol) in DMF (2 ml) was sealed, degassed, and flushed with nitrogen. The mixture was stirred at room temperature for 30 min A solution of (S)-2-[(4,6-dimethyl-2-prop-2-ynylamino-pyrimidine-5-carbonyl)-amino]-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (300 mg, 0.72 mmol) and Et3N (1 ml) in DMF (1 ml) was added at room temperature. The reaction mixture was stirred at room temperature for 18 h. The solution was purified directly by preparative HPLC under neutral condition to give pure product (370 mg, 48% yield). MS m/e 521.9 (M+H+).
A solution of (S)-2-({2-[3-(3-hydroxy-2-methyl-phenyl)-prop-2-ynylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]propionic acid methyl ester (370 mg, 0.71 mmol) in MeOH (200 ml) was passed through H-Cube with 10% Pd/C small cartridge at 30 bar, 30° C. and 1 ml per min twice. The solvent was then removed by evaporation and the residue was purified by flash chromatography with 100% EtOAc to afford the desired product (197 mg, 53% yield). MS m/e 525.9 (M+H+).
A reaction vessel containing 1-chloro-2-iodo-4-methoxy-benzene (388 mg, 1.44 mmol), Et3N (2 ml), dichlorobis(triphenylphosphine)palladium (II) (25.3 mg, 0.036 mmol) and copper (I) iodide (13.5 mg, 0.072 mmol) in DMF (2 ml) was sealed, degassed, and flushed with nitrogen. The mixture was stirred at room temperature for 30 min. A solution of (S)-2-[(4,6-dimethyl-2-prop-2-ynylamino-pyrimidine-5-carbonyl)-amino]-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (300 mg, 0.72 mmol) and Et3N (1 ml) in DMF (1 ml) was added at room temperature. The reaction mixture was stirred at room temperature for 18 h. The mixture was extracted with EtOAc. The combined organic extracts were washed with water and brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with 30% EtOAc in hexane to afford the title compound (284 mg, 71% yield). MS m/e 555.8 (M+H+).
A solution of (S)-2-({2-[3-(2-chloro-5-methoxy-phenyl)-prop-2-ynylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]propionic acid methyl ester (284 mg, 0.51 mmol) in MeOH (300 ml) was passed through H-Cube with 5% Pt/C sulfided small cartridge at 40 bar, room temperature and 1 ml per min three times. Solvent was then removed by evaporation to give pure product (18.1 mg, 63% yield). MS m/e 559.8 (M+H+).
A reaction vessel containing 4-iodo-phenol (212 mg, 0.963 mmol), Et3N (3 ml), dichlorobis(triphenylphosphine)palladium (II) (1 mg, 0.024 mmol), copper (I) iodide (9.2 mg, 0.048 mmol) and (S)-2-[(4,6-dimethyl-2-prop-2-ynylamino-pyrimidine-5-carbonyl)-amino]-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (20 mg, 0.48 mmol) in DMF (3 ml) was sealed, degassed, flushed with nitrogen and stirred at room temperature overnight. The mixture was extracted with EtOAc. The combined organic extracts were washed with water and brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with 10% MeOH in EtOAc to afford title compound (81 mg, 33% yield). MS m/e 507.9 (M+H+).
A solution of (S)-2-({2-[3-(4-hydroxy-phenyl)-prop-2-ynylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]propionic acid methyl ester (81 mg, 0.16 mmol) in MeOH (300 ml) was passed through H-Cube with 10% Pd/C sulfided small cartridge at 20 bar, room temperature and 1 ml per min. Solvent was then removed by evaporation to give pure product (36 mg, 44% yield). MS m/e 511.9 (M+H+).
A solution of HBTU (48 mg, 0.127 mmol) and HOBT (17 mg, 0.126 mmol) in anhydrous DMF (2 ml) was added to a mixture of (S)-3-Amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (50 mg, 0.114 mmol), 2-phenyl-1,3-thiazole-4-carboxylic acid (24 mg, 0.117 mmol) and triethylamine (0.07 ml, 0.502 mmol) in anhydrous DMF (1 ml). The reaction mixture was stirred at room temperature for 1.25 h, diluted with brine, and extracted with ethyl acetate. The combined organic layers were washed with 1/1 aqueous saturated sodium bicarbonate solution/brine and brine, then dried over MgSO4, filtered, concentrated and chromatographed (ethyl acetate to 9/1 ethyl acetate/methanol) to give the desired product as a colorless oil (35.5 mg, 52.8%). MS m/e 589 (M+H+).
A similar procedure was used to afford the following 6 examples:
A solution of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(2-phenyl-thiazole-4-carbonyl)-amino]-propionic acid methyl ester (35.5 mg, 0.060 mmol) in THF (0.7 ml) was treated with a solution of lithium hydroxide monohydrate (25 mg, 0.596 mmol) in water (2 ml) and the reaction mixture was stirred at room temperature for 1.5 h. The reaction solution was acidified to pH ca. 3 with aqueous 1N KHSO4, concentrated to remove THF, and diluted with water. The precipitate was collected by filtration and air dried to afford the desired product as a white solid (27.6 mg, 79.6% yield). MS m/e 575.0 (M+H+)
A similar procedure was used to afford the following 6 examples:
To a solution of (S)-3-tert-butoxycarbonylamino-2-({2-[3-(3-hydroxy-4-methyl-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (180 mg, 0.35 mmol) in DCM (35 ml) was added dropwise 30% TFA/CH2Cl2 (10 ml) at 0° C. The solution was stirred at room temperature for 3 h. Solvent and excess TFA were removed by vacuum evaporation. The residue was dissolved in DMF (3 ml) and triethylamine (1 ml, 7 mmol) was added dropwise at 0° C. followed by thiophene-2-carboxylic acid (53.8 mg, 0.42 mmol), HBTU (59 mg, 0.42 mmol) and HOBT (57 mg, 0.42 mmol). The mixture was stirred at room temperature for 12 h. The mixture was extracted with EtOAc. The combined organic extracts were washed with water and brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with 10% MeOH in DCM to give the desired product (84.7 mg, 46% yield). MS m/e 526.0 (M+H+).
A solution of (S)-3-tert-butoxycarbonylamino-2-({2-[(E)-3-(3-hydroxy-phenyl)-allylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (183 mg, 0.37 mmol) was treated with 50% TFA in DCM (4 ml) at room temperature for 2 h. The solution was stirred at room temperature for 3 h. Solvent and excess TFA were removed by vacuum evaporation at 30-35° C. The residue was dissolved in anhydrous DMF (3 ml) and treated with triethylamine (1 ml, 7. mmol) at 0° C. Thiophene-2-carboxylic acid (56.9 mg, 0.4 mmol), HBTU (168 mg, 0.44 mmol) and HOBT (60 mg, 0.44 mmol) were added at room temperature and the mixture was stirred at room temperature over the weekend. The mixture was extracted with EtOAc. The combined organic extracts were washed with water and brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with a 50-100% EtOAc in hexane gradient to give the desired product (45 mg, 24% yield). MS m/e 509.9 (M+H+).
(S)-2-({2-[(E)-3-(6-benzyloxy-pyridin-2-yl)-allylamino]-4,-6-dimethyl-pyrimidine-5-carbonylamino-propionic acid methyl ester (122 mg, 0.21 mmol) was treated with 30% TFA/CH2Cl2 (5 ml) at room temperature for 2 h. Solvent and excess TFA were removed by vacuum evaporation. The residue was dissolved in anhydrous DMF (3 ml) and triethylamine (0.59 ml, 4.2 mmol) was added dropwise at 0° C. Thiophene-2-carboxylic acid (32 mg, 0.25 mmol), HBTU (95 mg, 0.25 mmol) and HOBT (34 mg, 0.25 mmol) were successively added at room temperature and the mixture was stirred for 12 h. The mixture was extracted with EtOAc. The combined organic extracts were washed with water and brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with a 50-100% EtOAc in hexane gradient to give the desired product (92 mg, 73% yield). MS m/e 601.0 (M+H+).
A solution of (S)-2-({2-[(E)-3-(6-benzyloxy-pyridin-2-yl)-allylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (92 mg, 0.153 mmol) and lithium hydroxide monohydrate (32 mg, 0.77 mmol) in dioxane/water (10 ml/10 ml) was stirred at room temperature overnight, then quenched with aqueous potassium hydrogen sulfate to pH 2-4. The mixture was extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, and filtered to give the desired product in EtOAc. This solution was not concentrated but was used directly in the next step. MS m/e 587.0 (M+H+).
A solution of (S)-2-({2-[(E)-3-(6-benzyloxy-pyridin-2-yl)-allyamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (about 0.1 mmol) in ethyl acetate (about 20 ml) was passed two times through H-Cube with 10% Pd/C small cartridge at 70 bar and 50° C., at a rate of 1 ml/min. The solution was concentrated and the residue was purified by preparative HPLC under neutral condition to give the desired product (7.7 mg). MS m/e 498.9 (M+H+).
(S)-3-tert-Butoxycarbonylamino-2-({2-[3-(2-fluoro-3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (94 mg, 0.18 mmol) was treated with 50% TFA/CH2Cl2 (3 ml) at room temperature for 2 h. Solvent and excess TFA were removed by vacuum evaporation. The residue was dissolved in anhydrous DMF (1 ml) and triethylamine (0.5 ml, 3.62 mmol) was added at 0° C. Thiophene-2-carboxylic acid (46.4 mg, 0.36 mmol), HBTU (65 mg, 0.36 mmol) and HOBT (49 mg, 0.36 mmol), were then added at room temperature and the mixture was stirred for 12 h. The reaction mixture was quenched with water and extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and evaporated. The residue was purified by flash chromatography with a 40-100% EtOAc in hexane gradient to give the title compound (47 mg, 49% yield). MS m/e 529.8 (M+H+).
(S)-3-tert-butoxycarbonylamino-2-({2-[3-(2-fluoro-5-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (62 mg, 0.12 mmol) in MeOH (0.5 ml) was treated with 4NHO/dioxane (2 ml) at room temperature for 1.5 h. The solution was concentrated to dryness and the residue was dissolved in anhydrous DMF (3 ml). Triethylamine (0.1 ml, 0.714 mmol), thiophene-2-carboxylic acid (30.6 mg, 0.24 mmol), HBTU (65 mg, 0.36 mmol) and HOBT (49 mg, 0.36 mmol) were successively added at room temperature and the mixture was stirred for 12 h. The reaction mixture was quenched with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with a 40-100% EtOAc in hexane gradient to give the title compound (43.5 mg, 69% yield). MS m/e 529.8 (M+H+).
(S)-3-tert-Butoxycarbonylamino-2-({2-[3-(2-methyl-5-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (26.3 mg, 0.12 mmol) in MeOH (0.5 ml) was treated with 4NHCl/dioxane (2 ml) at room temperature for 1.5 h. The solution was concentrated to dryness and the residue was dissolved in anhydrous DMF (2 ml). Triethylamine (31 mg, 0.306 mmol), thiophene-2-carboxylic acid (13 mg, 0.102 mmol), HBTU (39 mg, 0.102 mmol) and HOBT (14 mg, 0.102 mmol) were successively added at room temperature and the mixture was stirred for 2 h. The reaction mixture was quenched with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with a 40-100% EtOAc in hexane gradient to give the title compound (18 mg, 67% yield). MS m/e 526.0 (M+H+).
(S)-3-tert-butoxycarbonylamino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (820 mg, 1.73 mmol) was treated with 30% TFA in DCM (50 ml) at room temperature for 2 h. The solution was concentrated to dryness and the residue was dissolved in anhydrous DMF (20 ml). Triethylamine (4.8 ml, 34.6 mmol), thiophene-2-carboxylic acid (266 mg, 2.08 mmol), HBTU (790 mg, 2.08 mmol) and HOBT (281 mg, 2.08 mmol) were successively added at room temperature and the mixture was stirred overnight. The reaction mixture was quenched with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with a 40-100% EtOAc in hexane gradient to give the desired product (220 mg, 26% yield). MS m/e 483.9 (M+H+).
(S)-3-tert-Butoxycarbonylamino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4-methyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (482 mg, 0.99 mmol) was treated with 50% TFA in DCM (5 ml) at room temperature for 2 h The solution was concentrated to dryness and the residue was dissolved in anhydrous DMF (5 ml). Triethylamine (2.8 ml, 19.8 mmol), thiophene-2-carboxylic acid (152 mg, 1.19 mmol), HBTU (452 mg, 1.19 mmol) and HOBT (161 mg, 1.19 mmol) were successively added at room temperature and the mixture was stirred overnight. The reaction mixture was quenched with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with a 40-100% EtOAc in hexane gradient to give the desired product (319 mg, 65% yield). MS m/e 497.9 (M+H+).
(S)-3-tert-butoxycarbonylamino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4-trifluoromethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (970 mg, 1.79 mmol) was treated at room temperature with 30% TFA in DCM (50 ml) for 2 h. The solution was concentrated to dryness and the residue was dissolved in anhydrous DMF (20 ml). Triethylamine (5 ml, 35.8 mmol), thiophene-2-carboxylic acid (275.5 mg, 2.15 mmol), HBTU (816 mg, 2.15 mmol) and HOBT (290 mg, 2.15 mmol were successively added at room temperature and the mixture was stirred overnight. The reaction mixture was quenched with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with a 40-100% EtOAc in hexane gradient to give the title compound (450 mg, 46% yield). MS m/e 552.0 (M+H+).
A solution of (S)-3-tert-butoxycarbonylamino-2-({4-ethyl-2-[3-(3-hydroxy-phenyl) propylamino]-6-methyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (265 mg, 0.514 mmol) was treated at room temperature with 50% TFA in DCM (5 ml) for 2 h. The solution was concentrated to dryness and the residue was dissolved in anhydrous DMF (5 ml). Triethylamine (1.4 ml, 10.28 mmol), thiophene-2-carboxylic acid (79 mg, 0.617 mmol), HBTU (234 mg, 0.617 mmol) and HOBT (83 mg, 0.617 mmol) were successively added at room temperature and the mixture was stirred overnight. The reaction mixture was quenched with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with a 40-100% EtOAc in hexane gradient to give the desired product (221 mg, 82% yield). MS m/e 526.0 (M+H+).
(S)-3-tert-butoxycarbonylamino-2-({4,6-diethyl-2-[3-(3-hydroxy-phenyl) propylamino]-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (22 mg, 0.04 mmol) was treated at room temperature with 50% TFA in DCM (2 ml) for 2 h. The solution was concentrated to dryness and the residue was dissolved in anhydrous DMF (2 ml). Triethylamine (84 mg, 0.8 mmol), thiophene-2-carboxylic acid (6.4 mg, 0.05 mmol), HBTU (9 mg, 0.05 mmol) and HOBT (7 mg, 0.05 mmol) were successively added at room temperature and the mixture was stirred overnight. The reaction mixture was quenched with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with a 40-100% EtOAc in hexane gradient to give the desired product (9.4 mg, 42% yield). MS m/e 540.0 (M+H+).
A solution of (S)-2-({2-[3-(3-hydroxy-phenyl)-prop-2ynylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]propionic acid methyl ester (15 mg, 0.03 mmol) in dioxane (1 ml) was treated at room temperature with a solution of lithium hydroxide monohydrate (6.3 mg, 0.15 mmol) in water (1 ml). The mixture was stirred at room temperature overnight, then quenched with aqueous potassium hydrogen sulfate to adjust the pH to ˜2-4 and extracted with EtOAc. The organic extracts were combined, washed with brine, dried over sodium sulfate, filtered and evaporated to give the title compound (14 mg, 95% yield). MS m/e 493.9 (M+H+).
A similar procedure was used to afford the following 6 examples:
(S)-2-({2-[3-(1H-indazol-4-yl)-prop-2ynylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]propionic acid methyl ester (37.3 mg, 0.07 mmol) afforded the product (21.5 mg, 59% yield). MS m/e 518.0 (M+H+).
(S)-2-({2-[3-(1H-indazol-6-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]propionic acid methyl ester (65 mg, 0.12 mmol) afforded the product (35 mg, 56% yield). MS m/e 522.0 (M+H+).
(S)-2-({2-[3-(3-fluoro-5-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]propionic acid methyl ester (18.2 mg, 0.034 mmol afforded the product (9.7 mg, 55% yield). MS m/e 516.0 (M+H+).
(S)-2-({2-[3-(4-fluoro-3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]propionic acid methyl ester (42 mg, 0.079 mmol) afforded the product (8.4 mg, 21% yield). MS m/e 516.0 (M+H+).
(S)-2-({2-[3-(3-hydroxy-2-methyl-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]propionic acid methyl ester (197 mg, 0.375 mmol) afforded the product (59 mg, 31% yield). MS m/e 511.9 (M+H+).
(S)-2-({2-[3-(3-hydroxy-4-methyl-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]propionic acid methyl ester (84.7 mg, 0.16 mmol) afforded the product (66 mg, 81% yield). MS m/e 512.0 (M+H+).
A solution of (S)-2-({2-[3-(2-chloro-5-methoxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]propionic acid methyl ester (18.1 mg, 0.323 mmol) in anhydrous DCM (10 ml) was treated at 0° C. with BBr3/DCM (1M, 1.4 ml, 1.4 mmol). The mixture was allowed to warm up to room temperature and stirred for 2 h, then quenched with ice water. The mixture was evaporated to dryness and the residue was dissolved in methanol (5 ml). A solution of lithium hydroxide monohydrate (68 mg, 1.62 mmol) in water (5 ml) was added and the mixture was stirred at room temperature for 2 h then concentrated to dryness. The residue was dissolved in THF (10 ml) and treated with aqueous potassium hydrogen sulfate to pH 2-4. THF was then removed by evaporation and the mixture was diluted with cold water. The solid was collected by filtration and washed with water and cold methanol to afford the title compound (125 mg, 73% yield). MS m/e 531.9 (M+H+).
A solution of (S)-2-({2-[3-(4-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (36 mg, 0.07 mmol) in THF (5 ml) was treated at room temperature with a solution of lithium hydroxide monohydrate (100 mg, 2.38 mmol) in water (5 ml). The mixture was stirred at room temperature overnight, then quenched with aqueous potassium hydrogen sulfate to adjust the pH to ˜2-4. THF was removed by evaporation and the mixture was diluted with cold water. The white precipitate was collected by filtration, washed with water and dried to afford pure product (36 mg, 100% yield). MS m/e 497.9 (M+H+).
A similar procedure was used to afford the following 10 examples:
(S)-2-({2-[3-(2-fluoro-3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]propionic acid methyl ester (47 mg, 0.089 mmol) afforded the product (17 mg, 37% yield). MS m/e 515.8 (M+H+).
(S)-2-({2-[3-(2-fluoro-5-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (43.5 mg, 0.082 mmol) afforded the desired product (41 mg, 100% yield). MS m/e 515.8 (M+H+).
(S)-2-({2-[3-(2-methyl-5-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (18 mg, 0.034 mmol) afforded the desired product (17 mg, 100% yield). MS m/e 511.8 (M+H+).
(S)-2-({2-[(Z)-3-(hydroxy-phenyl)-allylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (352.5 mg, 0.69 mmol) afforded the desired product (141 mg, 41% yield). MS m/e 495.9 (M+H+).
(S)-2-({2-[(E)-3-(hydroxy-phenyl)-allylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (45 mg, 0.09 mmol) afforded the desired product (29.5 mg, 66% yield). MS m/e 495.9 (M+H+).
(S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (220 mg, 0.47 mmol) afforded the product, (16 mg, 7% yield). MS m/e 469.9 (M+H+).
(S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4-methyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (319 mg, 0.64 mmol) afforded the product (221.8 mg, 72% yield). MS m/e 483.9 (M+H+).
(S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4-trifluoromethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (450 mg, 0.816 mmol) afforded the product (305 mg, 70% yield). MS m/e 538.0 (M+H+).
(S)-2-({4-Ethyl-2-[3-(3-hydroxy-phenyl)-propylamino]-6-methyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (221 mg, 0.42 mmol) afforded the product (62 mg, 29% yield). MS m/e 511.9 (M+H+).
(S)-2-({4,6-diethyl-2-[3-(3-hydroxy-phenyl)-propylamino]-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (9.4 mg, 0.02 mmol) afforded the product (9 mg, 100% yield). MS m/e 526.0 (M+H+).
To a mixture of (S)-3-Amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (77 mg, 0.175 mmol), 3,5-dihydroxybenzoic acid (27 mg, 0.175 mmol), and triethylamine (53 mg, 0.525 mmol) in DMF (4 mL) was added a solution of HBTU (80 mg, 0.21 mmol) and HOBT (28 mg, 0.21 mmol) in DMF (2 mL). The mixture was stirred at room temperature 0.75 h, diluted with EtOAc, washed with brine, dried over sodium sulfate, filtered and concentrated to give the title compound (82.1 mg, 87% yield). MS m/e 538.1 (M+H+).
To a solution (S)-3-(3,5-Dihydroxy-benzoylamino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (82.1 mg, 0.153 mmol) in THF (5 mL) was added a solution of lithium hydroxide monohydrate (51 mg, 1.22 mmol) in water (6 mL). The mixture was then stirred at room temperature overnight. The mixture was then diluted with 0.5 N, extracted with EtOAc. The extracts were combined, washed with water and brine, dried over sodium sulfate, filtered, and purified by reverse phase HPLC to give the desired compound (2.0 mg, 2.5% yield) MS m/e 524.2 (M+H+).
To a mixture of (S)-3-Amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (65 mg, 0.152 mmol), 3-hydroxybenzoic acid (21 mg, 0.152 mmol), and triethylamine (46 mg, 0.456 mmol) in DMF (3 mL) was added a solution of HBTU (69 mg, 0.182 mmol) and HOBT (25 mg, 0.182 mmol) in DMF (1 mL). The mixture was stirred at room temperature 1 h, diluted with brine and extracted with EtOAc. The combined organic layers were washed with brine, saturated sodium bicarbonate, and brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (40-100% EtOAc in hexane) to afford the desired compound (42 mg, 53% yield) MS m/e 521.9 (M+H+).
To a solution (S)-3-(3-Hydroxy-benzoylamino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (42 mg, 0.081 mmol) in THF (3 mL) was added a solution of lithium hydroxide monohydrate (17 mg, 0.41 mmol) in water (4 mL). The mixture was then stirred at room temperature 2 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, concentrated, filtered, and the precipitate washed with water to afford title compound (30.1 mg, 74% yield). MS m/e 508.3 (M+H+).
To a mixture of (S)-3-Amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (75 mg, 0.17 mmol), thiophene-3-carboxylic acid (24 mg, 0.19 mmol), and triethylamine (52 mg, 0.51 mmol) in DMF (2 ml) was added a solution of HBTU (7 mg, 0.19 mmol) and HOBT (26 mg, 0.19 mmol) in DMF (2 ml). The mixture was stirred at room temperature overnight, diluted with brine and extracted with EtOAc. The combined organic layers were washed with 1:1 brine-saturated sodium bicarbonate, and brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the title compound (66 mg, 76% yield). MS m/e 511.9 (M+H+).
To a solution (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid methyl ester (66.1 mg, 0.129 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (54 mg, 1.29 mmol) in water (4 ml). The mixture was then stirred at room temperature for 2 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water, filtered, and the precipitate washed with water to afford the title compound (38.5 mg, 60% yield). MS m/e 498.1 (M+H+).
To a mixture of (S)-3-Amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (75 mg, 0.17 mmol), 5-methyl-2-thiophenecarboxylic acid (27 mg, 0.19 mmol), and triethylamine (52 mg, 0.51 mmol) in DMF (2 ml) was added a solution of HBTU (72 mg, 0.19 mmol) and HOBT (26 mg, 0.19 mmol) in DMF (2 ml). The mixture was stirred at room temperature overnight, diluted with brine and extracted with EtOAc. The combined organic layers were washed with 1:1 brine-saturated sodium bicarbonate and brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the desired product (70.3 mg, 78% yield). MS m/e 525.8 (M+H+).
To a solution (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(5-methyl-thiophene-2-carbonyl)-amino]-propionic acid methyl ester (70.3 mg, 0.13 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (56 mg, 1.34 mmol) in water (4 ml). The mixture was then stirred at room temperature for 1.5 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water, filtered, and the precipitate was washed with water to afford the title compound (45.3 mg, 67% yield). MS m/e 511.9 (M+H+).
To a mixture of (S)-3-Amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (75 mg, 0.17 mmol), 4-methyl-2-thiophenecarboxylic acid (27 mg, 0.19 mmol), and triethylamine (52 mg, 0.51 mmol) in DMF (2 ml) was added a solution of HBTU (72 mg, 0.19 mmol) and HOBT (26 mg, 0.19 mmol) in DMF (2 ml). The mixture was stirred at room temperature overnight, diluted with brine, and extracted with EtOAc. The combined organic layers were washed with 1:1 brine-saturated sodium bicarbonate), and brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the desired product (59.6 mg, 66% yield. MS m/e 525.9 (M+H+).
To a solution (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(4-methyl-thiophene-2-carbonyl)-amino]-propionic acid methyl ester (59.6 mg, 0.113 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (47 mg, 1.13 mmol) in water (4 ml). The mixture was then stirred at room temperature for 1.5 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water, filtered, and the precipitate was washed with water to afford the title compound (46.3 mg, 82% yield). MS m/e 511.9 (M+H+).
To a mixture of (S)-3-Amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (75 mg, 0.17 mmol), 3-methyl-2-thiophenecarboxylic acid (24 mg, 0.17 mmol), and triethylamine (52 mg, 0.51 mmol) in DMF (2 ml) was added a solution of HBTU (72 mg, 0.19 mmol) and HOBT (26 mg, 0.19 mmol) in DMF (2 ml). The mixture was stirred at room temperature overnight, diluted with brine (15 ml) and extracted with EtOAc. The combined organic layers were washed with 1:1 brine-saturated sodium bicarbonate, and brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (40-100% EtOAc in hexane) to give the title compound (63.6 mg, 71% yield). MS m/e 525.9 (M+H+).
To a solution (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(3-methyl-thiophene-2-carbonyl)-amino]-propionic acid methyl ester (46.3 mg, 0.088 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (37 mg, 0.88 mmol) in water (4 ml). The mixture was stirred at room temperature for 1 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water, filtered, and the precipitate was washed with water to afford the title compound (45.6 mg, 100% yield). MS m/e 511.9 (M+H+).
To a mixture of (S)-3-Amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (75 mg, 0.17 mmol), pyrazine-2-carboxylic acid (21 mg, 0.17 mmol), and triethylamine (52 mg, 0.51 mmol) in DMF (2 ml) was added a solution of HBTU (72 mg, 0.19 mmol) and HOBT (26 mg, 0.19 mmol) in DMF (2 ml). The mixture was stirred at room temperature overnight, diluted with brine and extracted with EtOAc. The combined organic layers were washed with 1:1 brine-saturated sodium bicarbonate and brine, then dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (50-100% EtOAc in hexane) to give the desired product (37.4 mg, 44% yield). MS m/e 508.0 (M+H+).
To a solution (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(pyrazine-2-carbonyl)-amino]-propionic acid methyl ester (61.2 mg, 0.117 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (49 mg, 1.17 mmol) in water (4 ml). The mixture was then stirred at room temperature for 1 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water, filtered, and the precipitate was washed with water to afford the title compound (44.1 mg, 80% yield). MS m/e 509.9 (M+H+).
To a mixture of (S)-3-Amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (75 mg, 0.17 mmol), 5-Chlorothiophene-2-carboxylic acid (28 mg, 0.17 mmol), and triethylamine (52 mg, 0.51 mmol) in DMF (2 ml) was added a solution of HBTU (72 mg, 0.19 mmol) and HOBT (26 mg, 0.19 mmol) in DMF (2 ml). The mixture was stirred at room temperature overnight, diluted with brine and extracted with EtOAc. The combined organic layers were washed with 1:1 brine-saturated sodium bicarbonate and brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the desired compound (62.2 mg, 67% yield). MS m/e 545.9 (M+H+).
To a solution (S)-3-[(5-Chloro-thiophene-2-carbonyl)-amino]-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (62.2 mg, 0.11 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (48 mg, 1.14 mmol) in water (4 ml). The mixture was stirred at room temperature for 1.5 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water, filtered, and the precipitate was washed with water to afford the title compound (45.7 mg, 79% yield). MS m/e 532.0 (M+H+).
To a mixture of (S)-3-Amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (75 mg, 0.17 mmol), m-toluic acid (23 mg, 0.17 mmol), and triethylamine (52 mg, 0.51 mmol) in DMF (2 ml) was added a solution of HBTU (72 mg, 0.19 mmol) and HOBT (26 mg, 0.19 mmol) in DMF (2 ml). The mixture was stirred at room temperature overnight, diluted with brine and extracted with EtOAc. The combined organic layers were washed with 1:1 brine-saturated sodium bicarbonate and brine, then dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the desired compound (67.5 mg, 77% yield). MS m/e 520.0 (M+H+).
To a solution (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-(3-methyl-benzoylamino)-propionic acid methyl ester (67.5 mg, 0.13 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (55 mg, 1.17 mmol) in water (4 ml). The mixture was then stirred at room temperature for 1 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water, filtered, and the precipitate was washed with water to afford the title compound (52.3 mg, 80% yield). MS m/e 505.9 (M+H+).
To a mixture of (S)-3-Amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (50 mg, 0.114 mmol), 1-adamantanecarboxylic acid (21 mg, 0.114 mmol), and triethylamine (35 mg, 0.34 mmol) in DMF (1.5 ml) was added a solution of HBTU (48 mg, 0.126 mmol) and HOBT (17 mg, 0.126 mmol) in DMF (1.5 ml). The mixture was stirred at room temperature for 2 h, diluted with brine and extracted with EtOAc. The combined organic layers were washed with 1:1 brine-saturated sodium bicarbonate and brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the desired product (45.8 mg, 73% yield). MS m/e 564.0 (M+H+).
To a solution (S)-3-[(adamantane-1-carbonyl)-amino]-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (67.5 mg, 0.13 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (55 mg, 1.17 mmol) in water (4 ml). The mixture was then stirred at room temperature for 1 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water, filtered, and the precipitate was washed with water to afford the title compound (52.3 mg, 80% yield). MS m/e 505.9 (M+H+).
To a mixture of (S)-3-amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (50 mg, 0.114 mmol), 5-methyl piperazinecarboxylic acid (16 mg, 0.114 mmol), and triethylamine (35 mg, 0.34 mmol) in DMF (1.5 ml) was added a solution of HBTU (48 mg, 0.126 mmol) and HOBT (17 mg, 0.126 mmol) in DMF (1.5 ml). The mixture was stirred at room temperature for 2.5 h, diluted with brine and extracted with EtOAc. The combined organic layers were washed with 1:1 brine-saturated sodium bicarbonate and brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (40-100% EtOAc in hexane) to give the title compound (32.9 mg, 59% yield). MS m/e 522.0 (M+H+).
To a solution (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(5-methyl-pyrazine-2-carbonyl)-amino]-propionic acid methyl ester (32.9 mg, 0.063 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (26 mg, 0.63 mmol) in water (4 ml). The mixture was then stirred at room temperature for 1 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water, filtered, and the precipitate was washed with water to afford the title compound (25.4 mg, 78% yield). MS m/e 507.9 (M+H+).
To a mixture of (S)-3-amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (50 mg, 0.114 mmol), 3-chlorothiophenecarboxylic acid (19 mg, 0.114 mmol), and triethylamine (35 mg, 0.34 mmol) in DMF (1.5 ml) was added a solution of HBTU (48 mg, 0.126 mmol) and HOBT (17 mg, 0.126 mmol) in DMF (1.5 ml). The mixture was stirred at room temperature overnight, diluted with brine and extracted with EtOAc. The combined organic layers were washed with 1:1 brine-saturated sodium bicarbonate and brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the title compound (45.8 mg, 92% yield). MS m/e 545.9 (M+H+).
To a solution (S)-3-[(3-chloro-thiophene-2-carbonyl)-amino]-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (45.8 mg, 0.084 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (35 mg, 0.84 mmol) in water (4 ml). The mixture was then stirred at room temperature for 1 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water, filtered, and the precipitate was washed with water to afford the title compound (28.1 mg, 66% yield). MS m/e 531.9 (M+H+).
To a mixture of (S)-3-amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (53 mg, 0.121 mmol), 5-ethylthiophenecarboxylic acid (19 mg, 0.121 mmol), and triethylamine (37 mg, 0.363 mmol) in DMF (1.5 ml) was added a solution of HBTU (50 mg, 0.133 mmol) and HOBT (18 mg, 0.133 mmol) in DMF (1.5 ml). The mixture was stirred at room temperature overnight, diluted with brine and extracted with EtOAc. The combined organic layers were washed with 1:1 brine-saturated sodium bicarbonate and brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the desired product (36.8 mg, 58% yield). MS m/e 539.8 (M+H+).
To a solution (S)-3-[(5-ethyl-thiophene-2-carbonyl)-amino]-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (36.8 mg, 0.068 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (28 mg, 0.68 mmol) in water (4 ml). The mixture was stirred at room temperature 1.5 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water, filtered, and the precipitate was washed with water to afford the title compound (23.9 mg, 67% yield). MS m/e 525.9 (M+H+).
A mixture of (S)-3-amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (50 mg, 0.114 mmol), phenyl isocyanate (45 mg, 0.38 mmol), triethylamine (38 mg, 0.38 mmol) and DMF (3 ml) was stirred at room temperature overnight, diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (0-100% EtOAc in hexane) to give the desired product (36.8 mg, 53% yield)
To a solution of (S)-2-({4,6-Dimethyl-2-[3-(3-phenyl carbamoyloxy-phenyl)-propylamino]-pyrimidine-5-carbonyl}-amino)-3-(3-phenyl-ureido)-propionic acid methyl ester (36.8 mg, 0.058 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (24 mg, 0.575 mmol) in water (4 ml). The mixture was stirred at room temperature for 1.5 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water, filtered, and the precipitate was washed with water to afford title compound, (18.8 mg, 62% yield). MS m/e 507.9 (M+H+).
A mixture of (S)-3-amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (50 mg, 0.114 mmol), methyl chloroformate (32 mg, 0.342 mmol), triethylamine (58 mg, 0.57 mmol) and DMF (3 ml) was stirred at room temperature overnight, diluted with brine and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (40-100% EtOAc in hexane) to give the title compound (30.9 mg, 57% yield)
To a solution of (S)-2-({2-[3-(3-Hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-methoxycarbonylamino-propionic acid methyl ester (30.9 mg, 0.067 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (28 mg, 0.67 mmol) in water (4 ml). The mixture was stirred at room temperature for 2 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water and extracted with EtOAc.
The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to afford the desired product (17.3 mg, 58% yield). MS m/e 445.8 (M+H+).
A mixture of (S)-3-amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (50 mg, 0.114 mmol), benzyl chloroformate (21 mg, 0.125 mmol), triethylamine (38 mg, 0.38 mmol) and DMF (3 ml) was stirred at room temperature overnight, diluted with brine and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the desired product (12.6 mg, 21% yield).
To a solution of (S)-3-benzyloxycarbonylamino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (12.6 mg, 0.024 mmol) in THF (2 ml) was added a solution of lithium hydroxide monohydrate (10 mg, 0.235 mmol) in water (3 ml). The mixture was stirred at room temperature for 2 h, treated with aqueous potassium hydrogen to pH 2-4, diluted with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to afford the title compound (5.7 mg, 46% yield). MS m/e 521.9 (M+H+).
A solution of (S)-3-amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (53 mg, 0.121 mmol), dimethylcarbamyl chloride (16 mg, 0.145 mmol), triethylamine (40 mg, 0.40 mmol) in DMF (2.5 ml) was stirred at room temperature overnight, diluted with brine and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (0-10% MeOH in EtOAc) to give the desired product (35.7 mg, 63% yield).
To a solution of (S)-3-(3,3-dimethyl-ureido)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (35.7 mg, 0.024 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (32 mg, 0.755 mmol) in water (4 ml). The mixture was stirred at room temperature 1 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated to afford the desired product (10.2 mg, 91% yield). MS m/e 459.0 (M+H+).
A mixture of (S)-3-Amino-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester hydrochloride (52 mg, 0.119 mmol), 3,4-Dihydroquinolin-1(2H)-carbonyl chloride (28 mg, 0.142 mmol), Triethylamine (40 mg, 0.40 mmol) and DMF (2.5 ml) was stirred at room temperature overnight, diluted with brine and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (40-100% EtOAc in hexane) to give the desired product (37.2 mg, 56% yield). MS m/e 561.0 (M+H+).
To a solution of (S)-3-[(3,4-dihydro-2H-quinoline-1-carbonyl)-amino]-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-propionic acid methyl ester (37.2 mg, 0.066 mmol) in THF (3 ml) was added a solution of lithium hydroxide monohydrate (28 mg, 0.66 mmol) in water (4 ml). The mixture was stirred at room temperature for 1 h, treated with aqueous potassium hydrogen sulfate to pH 2-4, diluted with water, filtered and the precipitate was washed with water to afford the desired product (13.0 mg, 36% yield). MS m/e 547.0 (M+H+).
To a mixture of (S)-3-Amino-2-{[2-(8-hydroxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-4,6-dimethyl-pyrimidine-5-carbonyl]-amino}-propionic acid methyl ester hydrochloride (50 mg, 0.111 mmol), Pyrazine-2-carboxylic acid (14 mg, 0.111 mmol), and Triethylamine (34 mg, 0.333 mmol) in DMF (1.5 mL) was added a solution of HBTU (46 mg, 0.122 mmol) and HOBT (16 mg, 0.122 mmol) in DMF (1.5 mL). The mixture was stirred at room temperature 2 h, diluted with brine (20 mL) and extracted with EtOAc (2×15 mL). The combined organic layers were washed with 1:1 brine-saturated sodium bicarbonate (2×20 mL), and brine (15 mL), dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give 24.7 mg title compound. MS m/e 520.0 (M+H+).
To a solution of (S)-2-{[2-(8-Hydroxy-1,2,3,4-tetrahydro-naphthalen-2-ylamino)-4,6-dimethyl-pyrimidine-5-carbonyl]-amino}-3-[(pyrazine-2-carbonyl)-amino]-propionic acid methyl ester (24.7 mg, 0.048 mmol) in THF (2 mL) was added a solution of lithium hydroxide monohydrate (20 mg, 0.48 mmol) in water (3 mL). The mixture was then stirred at room temperature 1 h, treated with potassium hydrogen sulfate aqueous solution to pH 2-4, diluted with water (20 mL), extracted with EtOAc (3.15 mL). The organic layers were combined, and washed with water (2×20 mL) to afford 8.7 mg title compound. MS m/e 505.9 (M+H+).
A mixture of (S)-2-({2-[3-(3-Hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (20 mg, 0.04 mmol), thionyl chloride (150 μl), and neopentyl alcohol (35 mg, 0.4 mmol) in dioxane (0.5 ml) was heated to 100° C. in a sealed tube overnight. The mixture was diluted with EtOAc, washed with water and brine, then dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the desired product (14.9 mg, 66% yield). MS m/e 568.1 (M+H+).
A mixture of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (50 mg, 0.1 mmol) and thionyl chloride (22 μL) in dioxane (1 ml) was stirred at room temperature 2 h. 1-butanol (23 mg, 0.31 mmol) was added. After 1 h, the mixture was diluted with EtOAc, washed with brine, then dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (20-100% EtOAc in hexane) to give the title compound (31.3 mg, 56% yield). MS m/e 554.3 (M+H+).
A mixture of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (150 mg, 0.3 mmol), chloropropylmorpholine (0.15 g, 0.90 mmol), sodium iodide (0.13 g, 0.90 mmol) and triethylamine (0.091 g, 0.90 mmol) in DMF (2 ml) was microwaved at 120° C. for 20 minutes. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (0-20% MeOH in EtOAc) to give the title compound 141.8 mg. MS m/e 625.2 (M+H+).
A mixture of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (50 mg, 0.1 mmol) and thionyl chloride (241) in dioxane (1 ml) was stirred at room temperature 2 h. 4-(2-hydroxyethyl)morpholine (41 mg, 0.31 mmol) and triethylamine (31 mg, 0.31 mmol) was added. After 4 h, the mixture was diluted with pH 9 Buffer and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (0-20% MeOH in EtOAc) to give the desired product (11.1 mg, 18% yield). MS m/e 611.1 (M+H+).
A mixture of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (50 mg, 0.1 mmol) and thionyl chloride (241) in dioxane (1 ml) was stirred at room temperature for 90 minutes. 1-propanol (19 mg, 0.31 mmol) was added. After 1.5 h, the mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the title compound (27.6 mg, 52% yield). MS m/e 540.0 (M+H+).
A mixture of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (50 mg, 0.1 mmol) and thionyl chloride (241) in dioxane (1 ml) was stirred at room temperature for 2 h. 2-methyl-1-propanol (23 mg, 0.31 mmol) was added. After 4 h, the mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the desired product (33.4 mg, 61% yield). MS m/e 553.9 (M+H+).
A mixture of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (50 mg, 0.1 mmol) and thionyl chloride (22 μl) in dioxane (1 ml) was stirred at room temperature for 2 h. 2-propanol (19 mg, 0.31 mmol) was added. After 18 h, the mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the title compound (26.7 mg, 49% yield). MS m/e 540.2 (M+H+).
A mixture of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.70 g, 1.4 mmol) and thionyl chloride (0.40 g) in dioxane (14 ml) was stirred at room temperature for 2 h. 3-pentanol (110 mg, 0.31 mmol) was added. After 18 h, the mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the desired product (302.1 mg, 38% yield). MS m/e 568.1 (M+H+).
A mixture of (S)-2-({2-[3-(3-Hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.75 g, 1.51 mmol) and thionyl chloride (0.54 g, 4.52 mmol) in dioxane (15 ml) was stirred at room temperature 2 h. Cyclopentanol (0.39 g, 4.52 mmol) was added. After 1.5 h, the mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the desired product (250 mg, 29% yield). MS m/e 566.1 (M+H+).
A mixture of (S)-2-({2-[3-(3-Hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (200 mg, 0.40 mmol), 2-chlorodimethylacetamide (0.15 g, 1.2 mmol), sodium iodide (0.18 g, 1.2 mmol) and triethylamine (0.12 g, 1.2 mmol) in DMF (2 ml) was microwaved at 120° C. for 20 minutes. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (100% EtOAc) to give the title compound (172.2 mg, 74% yield). MS m/e 583.2 (M+H+).
A mixture of (S)-2-({2-[3-(3-Hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.63 g, 1.26 mmol), 1-bromo-3-ethoxy-propane (0.84 g, 5.03 mmol), sodium iodide (0.75 g, 5.03 mmol) and triethylamine (0.51 g, 5.03 mmol) in DMF (4 ml) was microwaved at 120° C. for 20 minutes. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the desired product (280.4 mg, 38% yield). MS m/e 584.0 (M+H+).
A mixture of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (50 mg, 0.1 mmol) and thionyl chloride (46 μl) in dioxane (1 ml) was stirred at room temperature for 2 h. Tetrahydro-4-pyranol (0.20 g, 2.0 mmol) was added and the mixture was microwaved at 90° C. for 20 minutes. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the desired product (26.0 mg, 45% yield). MS m/e 582.2 (M+H+).
A solution of (S)-2-({2-[3-(hydroxyl-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (50 mg, 0.1 mmol), 2,2-dimethyl-propionic acid chloromethyl ester (15.1 mg, 0.1 mmol), and triethylamine (10 mg, 0.1 mmol) in anhydrous DMF (3 ml) was stirred at 80° C. overnight. The reaction mixture was cooled to room temperature, quenched with water and extracted with EtOAc. The organic layer was washed with water and brine, dried over sodium sulfate, filtered and concentrated. The residue was purified by flash chromatography with 10% MeOH in DCM to give the desired product (31 mg, 51% yield) pure product. MS m/e 612.1 (M+H+).
A mixture of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.20 g, 0.40 mmol), 2,2-dimethyl-propionic acid 1-chloro-ethyl ester (0.33 g, 2.0 mmol), sodium iodide (0.30 g, 2.0 mmol) and triethylamine (0.20 g, 2.0 mmol) in DMF (2.5 ml) was microwaved at 150° C. for 30 minutes. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (0-70% EtOAc in hexane) to give the desired product (143.3 mg, 57% yield). MS m/e 626.2 (M+H+).
A mixture of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.20 g, 0.40 mmol), Isobutyric acid 1-chloro-ethyl ester (0.18 g, 1.2 mmol), sodium iodide (0.18 g, 1.2 mmol) and triethylamine (0.12 g, 1.2 mmol) in DMF (2.5 ml) was microwaved at 120° C. for 20 minutes. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (20-100% EtOAc in hexane) to give the desired compound (103.3 mg, 42% yield). MS m/e 612.2 (M+H+).
A mixture of (S)-2-({2-[3-(3-Hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.20 g, 0.40 mmol), 1-(2-bromoethoxy)-2-ethoxyethane (0.32 g, 1.6 mmol), sodium iodide (0.24 g, 1.6 mmol) and triethylamine (0.16 g, 1.6 mmol) in DMF (3 ml) was microwaved at 120° C. for 20 minutes. The mixture was diluted with EtOAc (50 ml), washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (50-100% EtOAc in hexane) to give the title compound (135.4 mg, 55% yield). MS m/e 614.2 (M+H+).
A mixture of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.20 g, 0.40 mmol), 2-bromoethyl ethyl ether (0.31 g, 2.0 mmol), sodium iodide (0.30 g, 2.0 mmol) and triethylamine (0.20 g, 2.0 mmol) in DMF (2.5 ml) was microwaved at 120° C. for 20 minutes. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the title compound (124.1 mg, 54% yield). MS m/e 570.2 (M+H+).
A mixture of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid 3-ethoxy-propyl ester (0.05 g, 0.086 mmol), acetic anhydride (0.25 ml) and pyridine (0.25 ml) was stirred at room temperature for 3 h, diluted with water, and extracted with EtOAc. The organic layer was washed with water, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (20-100% EtOAc in hexane) to give the desired product (41.1 mg, 78% yield). MS m/e 626.0 (M+H+).
A mixture of (S)-2-({2-[3-(3-hydroxy-phenyl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid 3-ethoxy-propyl ester (0.154 g, 0.26 mmol), isobutyric anhydride (0.6 ml) and pyridine (0.6 ml) was stirred at room temperature for 1 h, diluted with EtOAc, washed with water, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (20-100% EtOAc in hexane) to give the desired product (141.3 mg, 83% yield). MS m/e 654.1 (M+H+).
A mixture of (S)-2-({2-[3-(1H-indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.05 g, 0.095 mmol), iodobutane (0.021 g, 0.114 mmol), and potassium carbonate (0.016 g, 0.114 mmol) in DMF (1 ml) was stirred at room temperature overnight. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (50-100% EtOAc in hexane) to give the desired product (38.2 mg, 58% yield). MS m/e 578.2 (M+H+).
A mixture of (S)-2-({2-[3-(1H-Indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.075 g, 0.143 mmol), ethyl iodide (0.027 g, 0.173 mmol), and potassium carbonate (0.024 g, 0.173 mmol) in DMF (1.5 ml) was stirred at room temperature overnight. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (50-100% EtOAc in hexane) to give the title compound (62.9 mg, 82% yield). MS m/e 550.2 (M+H+).
A mixture of (S)-2-({2-[3-(1H-Indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.05 g, 0.095 mmol), cyclopentyl iodide (0.022 g, 0.114 mmol), and potassium carbonate (0.016 g, 0.114 mmol) in DMF (1 ml) was stirred at room temperature overnight. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (50-100% EtOAc in hexane) to give the desired product (33.6 mg, 60% yield). MS m/e 590.1 (M+H+).
A mixture of (S)-2-({2-[3-(1H-indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.10 g, 0.19 mmol), 1-iodo-2-methylpropane (0.106 g, 0.58 mmol), and triethylamine (0.059 g, 0.58 mmol) in DMF (2 ml) was microwaved at 150° C. for 20 minutes. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (30-100% EtOAc in hexane) to give the title compound (82.7 mg, 75% yield). MS m/e 578.1 (M+H+).
A mixture of (S)-2-({2-[3-(1H-indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.05 g, 0.095 mmol), 3-bromopentane (0.022 g, 0.143 mmol), and potassium carbonate (0.020 g, 0.143 mmol) in DMF (1 ml) was stirred at room temperature overnight. 3-bromopentane (0.022 g, 0.143 mmol), and potassium carbonate (0.020 g, 0.143 mmol) were added and the reaction mixture was stirred at 50° C. for 5 h, then cooled to room temperature and stirred for 2 days. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (50-100% EtOAc in hexane) to give the title compound (28.3 mg, 50% yield). MS m/e 592.2 (M+H+).
A mixture of (S)-2-({2-[3-(1H-Indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.05 g, 0.095 mmol), 2-chlorodimethyl acetamide (0.017 g, 0.143 mmol), sodium iodide (0.021 g, 0.143 mmol), and potassium carbonate (0.020 g, 0.143 mmol) in DMF (1 ml) was stirred at room temperature overnight. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (100% EtOAc) to give the desired product (38.8 mg, 67% yield). MS m/e 607.1 (M+H+).
A mixture of (S)-2-({2-[3-(1H-indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.05 g, 0.095 mmol), 2-iodopropane (0.019 g, 0.114 mmol), and potassium carbonate (0.016 g, 0.114 mmol) in DMF (1 ml) was stirred at room temperature overnight. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (50-100% EtOAc in hexane) to give the desired product (32.8 mg, 62% yield). MS m/e 564.2 (M+H+).
A mixture of (S)-2-({2-[3-(1H-indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.05 g, 0.095 mmol), 1-iodopropane (0.019 g, 0.114 mmol), and potassium carbonate (0.016 g, 0.114 mmol) in DMF (1 ml) was stirred at room temperature overnight. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (40-100% EtOAc in hexane) to give the desired product (58.1 mg, 100% yield). MS m/e 564.1 (M+H+).
A mixture of (S)-2-({2-[3-(1H-Indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.05 g, 0.095 mmol), 1-iodopentane (0.023 g, 0.114 mmol), and potassium carbonate (0.016 g, 0.114 mmol) in DMF (1 ml) was stirred at room temperature overnight. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (40-100% EtOAc in hexane) to give the title compound (38.3 mg, 68% yield). MS m/e 592.2 (M+H+).
A mixture of (S)-2-({2-[3-(1H-indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.10 g, 0.19 mmol), chloropropylmorpholine (0.094 g, 0.58 mmol), sodium iodide (0.087 g, 0.58 mmol), and triethylamine (0.059 g, 0.58 mmol) in DMF (2 ml) was microwaved at 120° C. for 20 minutes. The mixture was diluted with EtOAc, washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (0-20% MeOH in EtOAc) to give the title compound (102.3 mg, 83% yield). MS m/e 649.3 (M+H+).
A mixture of (S)-2-({2-[3-(1H-indazol-4-yl)-propylamino]-4,6-dimethyl-pyrimidine-5-carbonyl}-amino)-3-[(thiophene-2-carbonyl)-amino]-propionic acid (0.10 g, 0.19 mmol), 1-bromo-3-ethoxy-propane (0.16 g, 0.96 mmol), sodium iodide (0.14 g, 0.96 mmol), and triethylamine (0.097 g, 0.96 mmol) in DMF (2 ml) was microwaved at 120° C. for 20 minutes. The mixture was diluted with brine and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered, concentrated in the presence of silica gel and chromatographed (50-100% EtOAc in hexane) to give the desired product (84.6 mg, 73% yield). MS m/e 608.2 (M+H+).
sLFA-1/ICAM-1 ELISA and Mac-1/ICAM-1 ELISA
Plates were coated with either 50 μl/well of 2.0 ug/ml solution of sLFA-1 or Mac-1 receptor in divalent cation buffer (1 mM MnCl2, 0.14M NaCl, 20 mM HEPES pH 7.2) at 4° C. overnight. Two hundred fifty μl of blocking buffer (1% BSA in divalent cation buffer) was added to each well 1 hour at 37° C. Plates were washed 3 times with wash buffer (TBS/0.05% Tween-20/1 mM MnCl2). The compound to be tested was solubilized in DMSO. A series of 1:3 dilutions were performed to achieve a concentration range of 0.45 nM-3 uM. Fifty μl of binding buffer (0.5% BSA in divalent cation buffer)/1% DMSO and 50 μl of the solutions to be tested were added to the appropriate wells and incubated for 1 hour. Fifty μl of 5dICAM-Fc (27 ng/ml) was added to the appropriate wells and 50 μl binding buffer was added to non-specific binding wells and incubated for 2 hours and washed. One hundred μl of 1:4000 HRP-goat anti-huIgG was added to each well and incubated for 1 hour and washed. One hundred μl of 1:1 TMB solution was added to each well and developed for 20 min at room temperature. Color development was stopped by adding 100 μl H3PO4 to each well. Absorbance was measured at 450 nm.
Human Mixed Lymphocyte Reaction (hMLR)
PBMC's were isolated from two healthy donors by Ficoll gradient. Cells were resuspended with 4 mL assay media and counted with a Coulter Counter. Both cell populations were resuspended to 1×107/mL. Stimulator cells were irradiated for 2.5 min (2000 RAD) in cesium irradiator. 5×105 cells were added to duplicate wells of the plate. Included were quadruplicate wells receiving responder cells alone and stimulator cells alone. The compound to be tested was solubilized in DMSO. A series of 1:3 dilutions were performed to achieve a concentration range of 0.45 nM-3 uM. One hundred μl of the solutions to be tested were added to the cell plate (100 μl of 2% DMSO/media added to control wells). Plates were then incubated for 2.5 days at 37° C. with 5% CO2. Plates were pulsed with 50 μl/well 3H-thymidine at 0.5 uCi/well then incubated for 6 hours at 37° C. with 5% CO2. Cells were harvested using Cell Harvester (TomTec) and counted on TopCount (Perkin Elmer).
Mouse Mixed Lymphocyte Reaction (mMLR)
Spleens were removed from C57B1/6 and Balb/c mice and placed in Hanks Balanced Salt Solution (HBSS). Red blood cells were removed with ACK lysing buffer and washed two times in assay media. Cells were resuspended with 4 mL assay media and counted with a Coulter Counter. Both cell populations were resuspended to 1×107/mL. Stimulator cells were irradiated for 2.5 min (2000 RAD) in cesium irradiator. 5×105 cells were added to duplicate wells of the plate. Included were quadruplicate wells receiving responder cells alone and stimulator cells alone. The compound to be tested was solubilized in DMSO. A series of 1:3 dilutions were performed to achieve a concentration range of 0.45 nM-3 uM. One hundred μl of the solutions to be tested were added to the cell plate (100 μl of 2% DMSO/media added to control wells). Plates were then incubated for 2.5 days at 37° C. with 5% CO2. Plates were pulsed with 50 μl/well 3H-thymidine at 0.5 uCi/well then incubated for 6 hours at 37° C. with 5% CO2. Cells were harvested using Cell Harvester (TomTec) and counted on TopCount (Perkin Elmer).
Table 1 provides the in vitro activity for representative LFA-1 antagonists and dual LFA-1/Mac-1 antagonists in the Examples:
It is to be understood that the invention is not limited to the particular embodiments of the invention described above, as variations of the particular embodiments may be made and still fall within the scope of the appended claims.
This application claims the benefit of U.S. Provisional Application No. 61/453,229, filed Mar. 16, 2011, which is hereby incorporated by reference in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 61453229 | Mar 2011 | US |
