WO2009014972A1 - Methods of affecting gastrointestinal transit and gastric emptying, and compounds useful therein - Google Patents

Methods of affecting gastrointestinal transit and gastric emptying, and compounds useful therein Download PDF

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Publication number
WO2009014972A1
WO2009014972A1 PCT/US2008/070254 US2008070254W WO2009014972A1 WO 2009014972 A1 WO2009014972 A1 WO 2009014972A1 US 2008070254 W US2008070254 W US 2008070254W WO 2009014972 A1 WO2009014972 A1 WO 2009014972A1
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Prior art keywords
alkyl
amino
mmol
aryl
optionally substituted
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PCT/US2008/070254
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French (fr)
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Qingyun Liu
Brian Zambrowicz
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Lexicon Pharmaceuticals, Inc.
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Priority to AU2008279426A priority Critical patent/AU2008279426A1/en
Priority to EP08826540A priority patent/EP2178536A1/en
Priority to CA2694443A priority patent/CA2694443A1/en
Priority to BRPI0813835A priority patent/BRPI0813835A2/en
Priority to JP2010518297A priority patent/JP2010534662A/en
Priority to CN200880100490A priority patent/CN101801385A/en
Publication of WO2009014972A1 publication Critical patent/WO2009014972A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to methods of affecting gastric transit and gastric emptying, and to compounds and compositions useful therein.
  • the neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] is involved in multiple central nervous facets of mood control and in regulating sleep, anxiety, alcoholism, drug abuse, food intake, and sexual behavior. It has also been implicated in the regulation of vascular tone, gut motility and cell-mediated immune responses. Walther, D. J., et al., Science 299:76 (2003). 5-HT also plays a role in clotting and hemostasis: platelets — which cannot themselves make 5-HT — take up large amounts of peripheral 5-HT. Goodman & Gilman's The Pharmacological Basis of Therapeutics, 10 th ed., p. 274-5 (McGraw-Hill, 2001).
  • Serotonin is synthesized in two steps from the amino acid tryptophan. Goodman & Gilman's, p. 270. The first step is rate-limiting, and is catalyzed by the enzyme tryptophan hydroxylase (TPH), which has two known isoforms: TPHl, which is expressed in the periphery, and TPH2, which is expressed primarily in the brain. Walther, D. J., et al., Science 299:76 (2003).
  • TPH tryptophan hydroxylase
  • the principle route by which serotonin is removed from the body involves the enzyme monoamine oxidase (MAO), which converts the compound to 5-hydroxyindole acetaldehyde, which is then converted to 5-hydroxyindole acetic acid (5 -HIAA) by the enzyme aldehyde dehydrogenase.
  • MAO monoamine oxidase
  • 5 -HIAA 5-hydroxyindole acetic acid
  • mice reportedly expressed normal amounts of serotonin in classical serotonergic brain regions, but largely lacked serotonin in the periphery. Id. In another, the knockout mice exhibited abnormal cardiac activity, which was attributed to a lack of peripheral serotonin. Cote, F., et al, PNAS 100(23): 13525-13530 (2003). Because serotonin is involved in so many biochemical processes, drugs that affect serotonin levels or affect serotonin receptors are often attended by adverse effects. For example, parenteral injection of the TPH inhibitor p-chlorophenylalanine (p-CPA) to rats reportedly decreased their gastrointestinal motility. Sailer, C. F., Strieker, E.
  • p-CPA TPH inhibitor
  • This invention is directed, in part, to methods of affecting gastrointestinal transit and gastric emptying, which comprise inhibiting peripheral tryptophan hydroxylase (TPH) in patients in need thereof, without substantially affecting their brain 5 -HT levels.
  • TPH peripheral tryptophan hydroxylase
  • the TPH is inhibited by administering to the patient an effective amount of a compound of formula I:
  • A is optionally substituted cycloalkyl, aryl, or heterocycle
  • Figure 1 shows the effect of oral administration of a potent TPHl inhibitor on the gastrointestinal (GI) motility of rats.
  • the asterisk identifies data wherein p ⁇ 0.01 when compared with vehicle control using the t test or one-way ANOVA test.
  • Figure 2 shows the effect of oral administration of a potent TPHl inhibitor on the gastric emptying of rats.
  • the asterisk identifies data wherein p ⁇ 0.01 when compared with vehicle control using the t test or one-way ANOVA test.
  • Figure 3 shows the effect of oral administration of a potent TPHl inhibitor on the blood and proximal colon levels of 5-HT of the rats for which data is presented in figures 1 and 2. In both cases, p ⁇ 0.0001 using one-way ANOVA.
  • This invention is based, in part, on the discovery of compounds that are potent inhibitors of TPH (e.g., TPHl). When administered to mammals, preferred compounds of the invention reduce peripheral serotonin levels.
  • TPH potent inhibitors of TPH
  • alkenyl means a straight chain, branched and/or cyclic hydrocarbon having from 2 to 20 (e.g., 2 to 10 or 2 to 6) carbon atoms, and including at least one carbon-carbon double bond.
  • alkenyl moieties include vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-l-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1-heptenyl, 2- heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1- decenyl, 2-decenyl and 3-decenyl.
  • alkyl means a straight chain, branched and/or cyclic (“cycloalkyl”) hydrocarbon having from 1 to 20 (e.g., 1 to 10 or 1 to 4) carbon atoms. Alkyl moieties having from 1 to 4 carbons are referred to as "lower alkyl.” Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl and dodecyl.
  • Cycloalkyl moieties may be monocyclic or multicyclic, and examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl. Additional examples of alkyl moieties have linear, branched and/or cyclic portions (e.g., l-ethyl-4-methyl- cyclohexyl).
  • alkyl includes saturated hydrocarbons as well as alkenyl and alkynyl moieties.
  • alkoxy means an -O-alkyl group. Examples of alkoxy groups include -OCH 3 , -OCH 2 CH 3 , -O(CH 2 ) 2 CH 3 , -O(CH 2 ) 3 CH 3 , -O(CH 2 ) 4 CH 3 , and -O(CH 2 ) 5 CH 3 .
  • alkylaryl or "alkyl-aryl” means an alkyl moiety bound to an aryl moiety.
  • alkylheteroaryl or “alkyl-heteroaryl” means an alkyl moiety bound to a heteroaryl moiety.
  • alkylheterocycle or “alkyl-heterocycle” means an alkyl moiety bound to a heterocycle moiety.
  • alkynyl means a straight chain, branched or cyclic hydrocarbon having from 2 to 20 (e.g., 2 to 20 or 2 to 6) carbon atoms, and including at least one carbon-carbon triple bond.
  • alkynyl moieties include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-l-butynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl and 9-decynyl.
  • aryl means an aromatic ring or an aromatic or partially aromatic ring system composed of carbon and hydrogen atoms.
  • An aryl moiety may comprise multiple rings bound or fused together.
  • aryl moieties include anthracenyl, azulenyl, biphenyl, fluorenyl, indan, indenyl, naphthyl, phenanthrenyl, phenyl, 1,2,3,4-tetrahydro-naphthalene, and to IyI.
  • arylalkyl or “aryl-alkyl” means an aryl moiety bound to an alkyl moiety.
  • biohydrolyzable amide means an amide, ester, carbamate, carbonate, ureido, or phosphate, respectively, of a compound that either: 1) does not interfere with the biological activity of the compound but can confer upon that compound advantageous properties in vivo, such as uptake, duration of action, or onset of action; or 2) is biologically inactive but is converted in vivo to the biologically active compound.
  • biohydrolyzable esters include lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters, and choline esters.
  • biohydrolyzable amides include lower alkyl amides, ⁇ -amino acid amides, alkoxyacyl amides, and alkylaminoalkyl-carbonyl amides.
  • biohydrolyzable carbamates include lower alkylamines, substituted ethylenediamines, aminoacids, hydroxy alkylamines, heterocyclic and heteroaromatic amines, and polyether amines.
  • disease or disorder mediated by peripheral serotonin and “disease and disorder mediated by peripheral serotonin” mean a disease and/or disorder having one or more symptoms, the severity of which are affected by peripheral serotonin levels.
  • heteroalkyl refers to an alkyl moiety (e.g., linear, branched or cyclic) in which at least one of its carbon atoms has been replaced with a heteroatom (e.g., N, O or S).
  • heteroaryl means an aryl moiety wherein at least one of its carbon atoms has been replaced with a heteroatom (e.g., N, O or S).
  • heteroatom e.g., N, O or S.
  • examples include acridinyl, benzimidazolyl, benzofuranyl, benzoisothiazolyl, benzoisoxazolyl, benzoquinazolinyl, benzothiazolyl, benzoxazolyl, furyl, imidazolyl, indolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, phthalazinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolinyl, tetrazolyl, thiazolyl, and tri
  • heterocycle refers to an aromatic, partially aromatic or non-aromatic monocyclic or polycyclic ring or ring system comprised of carbon, hydrogen and at least one heteroatom (e.g., N, O or S).
  • a heterocycle may comprise multiple (i.e., two or more) rings fused or bound together.
  • Heterocycles include heteroaryls.
  • heterocyclealkyl refers to a heterocycle moiety bound to an alkyl moiety.
  • heterocycloalkyl refers to a non-aromatic heterocycle.
  • heterocycloalkylalkyl or “heterocycloalkyl- alkyl” refers to a heterocycloalkyl moiety bound to an alkyl moiety.
  • the terms “manage,” “managing” and “management” encompass preventing the recurrence of the specified disease or disorder, or of one or more of its symptoms, in a patient who has already suffered from the disease or disorder, and/or lengthening the time that a patient who has suffered from the disease or disorder remains in remission.
  • the terms encompass modulating the threshold, development and/or duration of the disease or disorder, or changing the way that a patient responds to the disease or disorder.
  • pharmaceutically acceptable salts refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases.
  • suitable pharmaceutically acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, N 5 N'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine.
  • Suitable non-toxic acids include inorganic and organic acids such as acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic, phosphoric, propionic, salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonic acid.
  • inorganic and organic acids such as acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethe
  • non-toxic acids include hydrochloric, hydrobromic, phosphoric, sulfuric, and methanesulfonic acids.
  • specific salts thus include hydrochloride and mesylate salts.
  • Others are well-known in the art. See, e.g., Remington' s Pharmaceutical Sciences, 18 ed. (Mack Publishing, Easton PA: 1990) and Remington: The Science and Practice of Pharmacy, 19 th ed. (Mack Publishing, Easton PA: 1995).
  • the term "potent TPHl inhibitor” is a compound that has a TPH 1 IC 50 of less than about 10 ⁇ M.
  • the terms “prevent,” “preventing” and “prevention” contemplate an action that occurs before a patient begins to suffer from the specified disease or disorder, which inhibits or reduces the severity of the disease or disorder, or of one or more of its symptoms.
  • the terms encompass prophylaxis.
  • prodrug encompasses pharmaceutically acceptable esters, carbonates, thiocarbonates, N-acyl derivatives, N-acyloxyalkyl derivatives, quaternary derivatives of tertiary amines, N-Mannich bases, Schiff bases, amino acid conjugates, phosphate esters, metal salts and sulfonate esters of compounds disclosed herein.
  • prodrugs include compounds that comprise a biohydrolyzable moiety ⁇ e.g., a biohydrolyzable amide, biohydrolyzable carbamate, biohydrolyzable carbonate, biohydrolyzable ester, biohydrolyzable phosphate, or biohydrolyzable ureide analog).
  • Prodrugs of compounds disclosed herein are readily envisioned and prepared by those of ordinary skill in the art. See, e.g., Design of Prodrugs, Bundgaard, A. Ed., Elseview, 1985; Bundgaard, H., “Design and Application of Prodrugs," A Textbook of Drug Design and Development, Krosgaard-Larsen and H. Bundgaard, Ed., 1991, Chapter 5, p. 113-191; and Bundgaard, H., Advanced Drug Delivery Review, 1992, 8, 1-38.
  • a prophylactically effective amount of a compound is an amount sufficient to prevent a disease or condition, or one or more symptoms associated with the disease or condition, or prevent its recurrence.
  • a prophylactically effective amount of a compound is an amount of therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the disease.
  • the term “prophylactically effective amount” can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
  • protecting group when used to refer to part of a molecule subjected to a chemical reaction, means a chemical moiety that is not reactive under the conditions of that chemical reaction, and which may be removed to provide a moiety that is reactive under those conditions.
  • Protecting groups are well known in the art. See, e.g., Greene, T.W. and Wuts, P.G.M., Protective Groups in Organic Synthesis (3 rd ed., John Wiley & Sons: 1999); Larock, R.C., Comprehensive Organic Transformations (2 nd ed., John Wiley & Sons: 1999). Some examples include benzyl, diphenylmethyl, trityl, Cbz, Boc, Fmoc, methoxycarbonyl, ethoxycarbonyl, and pthalimido.
  • pseudohalogen refers to a polyatomic anion that resembles a halide ion in its acid-base, substitution, and redox chemistry, generally has low basicity, and forms a free radical under atom transfer radical polymerization conditions.
  • pseudohalogens include azide ions, cyanide, cyanate, thiocyanate, thiosulfate, sulfonates, and sulfonyl halides.
  • selective TPHl inhibitor is a compound that has a TPH2_IC 50 that is at least about 10 times greater than its TPH 1 IC 50 .
  • the terms “serotonin-mediated disease,” “serotonin- mediated disorder” and “serotonin-mediated disease or disorder” refer to a disease or disorder having one or more symptoms that are attributable to increased levels of peripheral 5- hydroxytryptamine (5-HT).
  • the term “stereomerically enriched composition of a compound refers to a mixture of the named compound and its stereoisomer(s) that contains more of the named compound than its stereoisomer(s).
  • a stereoisomerically enriched composition of (S)-butan-2-ol encompasses mixtures of (S)-butan-2-ol and (R)- butan-2-ol in ratios of, e.g., about 60/40, 70/30, 80/20, 90/10, 95/5, and 98/2.
  • stereomerically pure means a composition that comprises one stereoisomer of a compound and is substantially free of other stereoisomers of that compound.
  • a stereomerically pure composition of a compound having one stereocenter will be substantially free of the opposite stereoisomer of the compound.
  • a stereomerically pure composition of a compound having two stereocenters will be substantially free of other diastereomers of the compound.
  • a typical stereomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound, or greater than about 99% by weight of one stereoisomer of the compound and less than about 1% by weight of the other stereoisomers of the compound.
  • substituted when used to describe a chemical structure or moiety, refers to a derivative of that structure or moiety wherein one or more of its hydrogen atoms is substituted with an atom, chemical moiety or functional group such as, but not limited to, alcohol, aldehylde, alkoxy, alkanoyloxy, alkoxycarbonyl, alkenyl, alkyl (e.g.
  • a “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease or condition, or to delay or minimize one or more symptoms associated with the disease or condition.
  • a therapeutically effective amount of a compound is an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment or management of the disease or condition.
  • the term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.
  • TPH1_IC 5O is the IC50 of a compound for
  • TPHl as determined using the in vitro inhibition assay described in the Examples, below.
  • TPH2_IC 50 is the IC50 of a compound for TPH2 as determined using the in vitro inhibition assay described in the Examples, below.
  • treat contemplate an action that occurs while a patient is suffering from the specified disease or disorder, which reduces the severity of the disease or disorder, or one or more of its symptoms, or retards or slows the progression of the disease or disorder.
  • one or more adjectives immediately preceding a series of nouns is to be construed as applying to each of the nouns.
  • the phrase "optionally substituted alky, aryl, or heteroaryl” has the same meaning as "optionally substituted alky, optionally substituted aryl, or optionally substituted heteroaryl.”
  • a chemical moiety that forms part of a larger compound may be described herein using a name commonly accorded it when it exists as a single molecule or a name commonly accorded its radical.
  • the terms "pyridine” and “pyridyl” are accorded the same meaning when used to describe a moiety attached to other chemical moieties.
  • stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or the portion of the structure is to be interpreted as encompassing all stereoisomers of it.
  • names of compounds having one or more chiral centers that do not specify the stereochemistry of those centers encompass pure stereoisomers and mixtures thereof.
  • any atom shown in a drawing with unsatisfied valences is assumed to be attached to enough hydrogen atoms to satisfy the valences.
  • chemical bonds depicted with one solid line parallel to one dashed line encompass both single and double (e.g. , aromatic) bonds, if valences permit.
  • Particular methods of this invention comprise the use of potent TPHl inhibitors.
  • potent TPHl inhibitors are disclosed herein and in U.S. patent application nos. 11/638,677 and 60/874,596, both filed December 12, 2006. These compounds are significantly more potent than p-chlorophenylalanine, which has a TPHl IC 5 O of about 93 ⁇ M.
  • A is optionally substituted cycloalkyl, aryl, or heterocycle
  • A is optionally substituted cycloalkyl, aryl, or heterocycle
  • particular compounds include those wherein A is optionally substituted cycloalkyl (e.g. , 6-membered and 5-membered).
  • A is optionally substituted aryl (e.g., phenyl or naphthyl).
  • A is optionally substituted heterocycle (e.g., 6-membered and 5-membered). Examples of 6-membered heterocycles include pyridine, pyridazine, pyrimidine, pyrazine, and triazine.
  • 5-membered heterocycles include pyrrole, imidazole, triazole, thiazole, thiophene, and furan.
  • A is aromatic. In others, A is not aromatic.
  • A is an optionally substituted bicyclic moiety (e.g., indole, iso-indole, pyrrolo-pyridine, or napthylene).
  • each of Ai and A 2 is independently a monocyclic optionally substituted cycloalkyl, aryl, or heterocycle.
  • Compounds encompassed by this formula include those wherein Ai and/or A 2 is optionally substituted cycloalkyl (e.g., 6-membered and 5-membered).
  • Ai and/or A 2 is optionally substituted aryl (e.g., phenyl or naphthyl).
  • Ai and/or A 2 is optionally substituted heterocycle (e.g., 6-membered and 5-membered).
  • 6- membered heterocycles include pyridine, pyridazine, pyrimidine, pyrazine, and triazine.
  • 5-membered heterocycles include pyrrole, imidazole, triazole, thiazole, thiophene, and furan.
  • Ai and/or A 2 is aromatic. In others, Ai and/or A 2 is not aromatic.
  • D is optionally substituted aryl (e.g., phenyl or naphthyl).
  • D is optionally substituted heterocycle (e.g., 6-membered and 5-membered).
  • 6-membered heterocycles include pyridine, pyridazine, pyrimidine, pyrazine, and triazine.
  • 5- membered heterocycles include pyrrole, imidazole, triazole, thiazole, thiophene, and furan.
  • D is aromatic.
  • D is not aromatic.
  • D is an optionally substituted bicyclic moiety (e.g. , indole, iso-indole, pyrrolo-pyridine, or napthylene).
  • E is optionally substituted aryl (e.g., phenyl or naphthyl).
  • E is optionally substituted heterocycle (e.g., 6-membered and 5-membered).
  • 6- membered heterocycles include pyridine, pyridazine, pyrimidine, pyrazine, and triazine.
  • 5-membered heterocycles include pyrrole, imidazole, triazole, thiazole, thiophene, and furan.
  • E is aromatic.
  • E is not aromatic.
  • E is an optionally substituted bicyclic moiety (e.g. , indole, iso-indole, pyrrolo-pyridine, or napthylene).
  • particular compounds include those wherein Ri is hydrogen or optionally substituted alkyl.
  • R 2 is hydrogen or optionally substituted alkyl.
  • n is 1 or 2.
  • X is a bond or S.
  • X is -O-, -C(R 3 R 4 )O-, or -OC(R 3 R 4 )-, and, for example, R 3 is hydrogen or optionally substituted alkyl, and R 4 is hydrogen or optionally substituted alkyl.
  • R 3 is hydrogen and R 4 is trifluromethyl.
  • X is -S(O 2 )-, -S(O 2 )N(R 5 )-, -N(R 5 )S(O 2 )-, -C(R 3 R 4 )S(O 2 )-, or -S(O 2 )C(R 3 R 4 )-, and, for example, R 3 is hydrogen or optionally substituted alkyl, R 4 is hydrogen or optionally substituted alkyl, and R 5 is hydrogen or optionally substituted alkyl.
  • X is -N(R 5 )-, -N(R 5 )C(O)N(R 5 )-, -C(R 3 R 4 )N(R 5 )-, or -N(R 5 )C(R 3 R 4 )-, and, for example, R 3 is hydrogen or optionally substituted alkyl, R 4 is hydrogen or optionally substituted alkyl, and each R 5 is independently hydrogen or optionally substituted alkyl.
  • R 3 is trifluoromethyl. Others are encompassed by the formula:
  • R 3 is hydrogen
  • each of Zi, Z 2 , Z 3 , and Z 4 is independently N or CR 6 ; each R 6 is independently hydrogen, cyano, halogen, OR 7 , NRgR ⁇ , amino, hydroxyl, or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each R 7 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Rg is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each R 9 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; and m is 1-4. Certain such compounds are of the formula:
  • R 3 is trifluoromethyl. Others are of the formula:
  • R 3 is hydrogen
  • some compounds are such that all of Zi, Z 2 , Z3, and Z 4 are N. In others, only three of Zi, Z 2 , Z3, and Z 4 are N. In others, only two of Zi, Z 2 , Z 3 , and Z 4 are N. In others, only one of Zi, Z 2 , Z 3 , and Z 4 is N. In others, none of Zi, Z 2 , Z 3 , and Z 4 are N.
  • each of Z'i, Z' 2 , and Z' 3 is independently N, NH, S, O or CR 6 ; each R 6 is independently amino, cyano, halogen, hydrogen, OR 7 , SR 7 , NRgR 9 , or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each R 7 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Rg is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each R 9 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; and p is 1-3. Certain such compounds are of the formula:
  • R 3 is trifluoromethyl. Others are of the formula: wherein, for example, R 3 is hydrogen.
  • some compounds are such that all of Z'i, Z' 2 , and Z' 3 are N or NH. In others, only two of Z' I , Z' 2 , and Z' 3 are N or NH. In others, only one of Z'i, Z' 2 , and Z' 3 is N or NH. In others, none of Z' h Z' 2 , and Z' 3 are N or NH.
  • each of Z"i, Z" 2 , Z" 3 , and Z" 4 is independently N or CR10; each Ri 0 is independently amino, cyano, halogen, hydrogen, ORn, SRn, NR12R13, or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Rn is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Ri 2 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; and each Ri 3 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle. Certain such compounds are of the formula:
  • R 3 is hydrogen
  • some compounds are such that all of Z"i, Z" 2 , Z"3, and Z" 4 are N. In others, only three of Z" ls Z" 2 , Z' f 3, and Z" 4 are N. In others, only two of Z"i, Z" 2 , Z" 3 , and Z" 4 are N. In others, only one of Z"i, Z" 2 , Z" 3 , and Z" 4 is N. In others, none of Z" ls Z" 2 , Z" 3 , and Z" 4 are N.
  • R 3 is trifluoromethyl. Others are of the formula:
  • R 3 is hydrogen
  • some compounds are such that all of Z" ls Z" 2 , Z" 3 , and Z" 4 are N. In others, only three of Z" ls Z" 2 , Z" 3 , and Z" 4 are N. In others, only two of Z"i, Z" 2 , Z" 3 , and Z" 4 are N. In others, only one of Z" ls Z" 2 , Z" 3 , and Z" 4 is N. In others, none of Z" ls Z" 2 , Z" 3 , and Z" 4 are N.
  • each Ri 4 is independently amino, halogen, hydrogen, C(O)R A , OR A , NR B R C , S(O 2 )R A , or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each R A is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each R B is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Rc is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle; and m is 1-4.
  • particular compounds include those wherein both A and E are optionally substituted phenyl and, for example, X is -O-, -C(RsR 4 )O-, or -OC(RsR 4 )- and, for example, R3 is hydrogen and R 4 is trifluoromethyl and, for example, n is 1.
  • Stereoisomers may be asymmetrically synthesized or resolved using standard techniques such as chiral columns, chiral resolving agents, or enzymatic resolution. See, e.g., Jacques, J., et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S. H., et al, Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistry of Carbon Compounds (McGraw Hill, NY, 1962); and Wilen, S. H., Tables of Resolving Agents and Optical Resolutions, p. 268 (EX. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).
  • Particular compounds of the invention are potent TPHl inhibitors.
  • Specific compounds have a TPHl IC 50 of less than about 10, 5, 2.5, 1, 0.75, 0.5, 0.4, 0.3, 0.2, 0.1, or 0.05 ⁇ M.
  • Particular compounds are selective TPHl inhibitors.
  • Specific compounds have a TPHl IC 50 that is about 10, 25, 50, 100, 250, 500, or 1000 times less than their TPH2_IC 50 .
  • Particular compounds do not significantly inhibit human tyrosine hydroxylase (TH).
  • specific compounds have an IC 50 for TH of greater than about 100, 250, 500 or 1000 ⁇ M.
  • Particular compounds do not significantly inhibit human phenylalanine hydroxylase (PAH).
  • PAH human phenylalanine hydroxylase
  • specific compounds have an IC50 for PAH of greater than about 100, 250, 500 or 1000 ⁇ M.
  • Particular compounds of the invention do not significantly bind (e.g., inhibit with an IC 50 of greater than about 10, 25, 50, 100, 250, 500, 750, or 1000 ⁇ M) to one or more of the following: angiotensin converting enzyme, erythropoietin (EPO) receptor, factor IX, factor XI, integrin (e.g., ⁇ 4), isoxazoline or isoxazole fibrinogen receptor, metalloprotease, neutral endopeptidase (NEP), phosphatase (e.g., tyrosine phosphatase), phosphodiesterase (e.g., PDE-4), polymerase, PPAR ⁇ , TNF- ⁇ , vascular cell adhesion molecule- 1 (VCAM-I), or the vitronectin receptor.
  • angiotensin converting enzyme EPO
  • factor IX factor IX
  • factor XI factor XI
  • integrin e.g., ⁇ 4
  • certain compounds of the invention do not readily cross the blood/brain barrier (e.g., less than about 5, 2.5, 2, 1.5, 1, 0.5, or 0.01 percent of compound in the blood passes into the brain).
  • the ability or inability of a compound to cross the blood/brain barrier can be determined by methods known in the art. See, e.g. , Riant, P. et al. , Journal of Neurochemistry 51 :421 -425 (1988); Kastin, A.J., Akerstrom, V., J. Pharmacol. Exp. Therapeutics 294:633-636 (2000); W. A. Banks, W. A., et al., J. Pharmacol. Exp. Therapeutics 302:1062-1069 (2002).
  • A is optionally substituted phenyl, biphenyl or napthyl.
  • Pi is Ri or a protecting group
  • P 2 is a protecting group
  • P3 is OR 2 or a protecting group
  • X' is, for example, O or N
  • Yi and Y 3 are halogen (e.g., Br, Cl) or an appropriate pseudohalide (e.g., triflate); and each R' is independently hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle, or are taken together with the oxygen atoms to which they are attached to provide a cyclic dioxaborolane (e.g., 4,4,5,5-tetramethyl- 1,3,2-dioxaborolane).
  • a cyclic dioxaborolane e.g., 4,4,5,5-tetramethyl- 1,3,2-dioxaborolane.
  • the groups A, R 1 , R 2 , R 3 , R 6 and m are defined elsewhere herein.
  • the moieties Z"i, Z M 2, Z M 3, and Z M 4 are also defined herein, although it is to be understood that with regard to the scheme shown above, one of them is attached to the phenyl ring.
  • Z"i and Z M 4 may be independently CRio (which is defined herein), while Z M 2 is N and Z" 3 is a carbon atom bound to the adjacent phenyl ring.
  • the individual reactions shown above can be performed using conditions known in the art. For example, palladium catalysts and conditions suitable for the Suzuki coupling of the boron and halogen-containing moieties are well known, and examples are provided below.
  • types and appropriate uses of protecting groups are well known, as are methods of their removal and replacement with moieties such as, but not limited to, hydrogen (e.g., hydrolysis under acidic or basic conditions).
  • the A moiety can be bicyclic (e.g., optionally substituted biphenyl).
  • the starting material containing A can be prepared as shown below:
  • Y 2 is halogen or pseudohalogen, and each R is independently hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle, or are taken together with the oxygen atoms to which they are attached to provide a cyclic dioxaborolane (e.g., 4,4,5,5- tetramethyl-l,3,2-dioxaborolane).
  • a cyclic dioxaborolane e.g., 4,4,5,5- tetramethyl-l,3,2-dioxaborolane.
  • X is N, O or S
  • cyclic moiety D can be any of a variety of structures, which are readily incorporated into compounds of the invention.
  • D is oxazole
  • Scheme 7 compounds wherein D is oxazole can be prepared as shown below in Scheme 7:
  • This invention encompasses methods of affecting ⁇ e.g., slowing) gastrointestinal transit and gastric emptying, which comprise inhibiting peripheral tryptophan hydroxylase ⁇ e.g., TPHl) in patients in need thereof.
  • Patients in need thereof include patients with diarrhea and patients susceptible to diarrhea ⁇ e.g., patients taking medications or undergoing therapies, such as chemotherapy, that can cause diarrhea).
  • Preferred methods avoid measurably affecting serotonin levels in the central nervous system (CNS).
  • CNS central nervous system
  • One embodiment encompasses a method of slowing gastrointestinal transit in a patient, which comprises administering to the patient a sufficient amount of a potent TPHl inhibitor.
  • Another embodiment encompasses a method of slowing gastric emptying in a patient, which comprises administering to the patient a sufficient amount of a potent TPHl inhibitor.
  • the amount of active pharmaceutical ingredient e.g., a potent TPHl inhibitor
  • amount of active pharmaceutical ingredient sufficient to achieve the desired pharmacological effect can be readily determined by those skilled in the art. For example, a patient can be administered a low dose of a compound, and then increasingly larger doses over time until the desired effect is achieved.
  • Particular methods of the invention avoid adverse effects associated with alteration of CNS serotonin levels.
  • adverse effects include agitation, anxiety disorders, depression, and sleep disorders (e.g., insomnia and sleep disturbance).
  • compositions comprising one or more compounds of the invention.
  • Certain pharmaceutical compositions are single unit dosage forms suitable for oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, bolus injection, intramuscular, or intraarterial), or transdermal administration to a patient.
  • dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g.
  • crystalline or amorphous solids that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.
  • the formulation should suit the mode of administration.
  • the oral administration of a compound susceptible to degradation in the stomach may be achieved using an enteric coating.
  • a formulation may contain ingredients that facilitate delivery of the active ingredient(s) to the site of action.
  • compounds may be administered in liposomal formulations in order to protect them from degradative enzymes, facilitate transport in circulatory system, and effect their delivery across cell membranes.
  • poorly soluble compounds may be incorporated into liquid dosage forms (and dosage forms suitable for reconstitution) with the aid of solubilizing agents, emulsif ⁇ ers and surfactants such as, but not limited to, cyclodextrins (e.g., ⁇ -cyclodextrin, ⁇ -cyclodextrin, Captisol ® , and EncapsinTM (see, e.g., Davis and Brewster, Nat. Rev. Drug Disc.
  • solubilizing agents emulsif ⁇ ers and surfactants
  • cyclodextrins e.g., ⁇ -cyclodextrin, ⁇ -cyclodextrin, Captisol ®
  • EncapsinTM see, e.g., Davis and Brewster, Nat. Rev. Drug Disc.
  • Labrasol ® Labrafil ® , Labrafac ® , cremafor, and non-aqueous solvents, such as, but not limited to, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, dimethyl sulfoxide (DMSO), biocompatible oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, and mixtures thereof (e.g., DMSOxornoil).
  • DMSO dimethyl formamide
  • biocompatible oils e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils
  • glycerol tetrahydr
  • Nanoparticles of a compound may be suspended in a liquid to provide a nanosuspension (see, e.g., Rabinow, Nature Rev. Drug Disc. 3:785-796 (2004)).
  • Nanoparticle forms of compounds described herein may be prepared by the methods described in U.S. Patent Publication Nos. 2004-0164194, 2004-0195413, 2004-0251332, 2005-0042177 Al, 2005-0031691 Al, and U.S. Patent Nos.
  • the nanoparticle form comprises particles having an average particle size of less than about 2000 nm, less than about 1000 nm, or less than about 500 nm.
  • the composition, shape, and type of a dosage form will typically vary depending with use. For example, a dosage form used in the acute treatment of a disease may contain larger amounts of one or more of the active ingredients it comprises than a dosage form used in the chronic treatment of the same disease.
  • a parenteral dosage form may contain smaller amounts of one or more of the active ingredients it comprises than an oral dosage form used to treat the same disease. How to account for such differences will be apparent to those skilled in the art. See, e.g. , Remington 's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).
  • compositions of the invention suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups).
  • dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington 's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).
  • Typical oral dosage forms are prepared by combining the active ingredient(s) in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration.
  • tablets and capsules represent the most advantageous oral dosage unit forms.
  • tablets can be coated by standard aqueous or non-aqueous techniques.
  • Such dosage forms can be prepared by conventional methods of pharmacy.
  • pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.
  • Disintegrants may be incorporated in solid dosage forms to facility rapid dissolution. Lubricants may also be incorporated to facilitate the manufacture of dosage forms (e.g. , tablets).
  • Parenteral dosage forms can be administered to patients by various routes including subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses patients' natural defenses against contaminants, parenteral dosage forms are specifically sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
  • Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to those skilled in the art. Examples include: Water for Injection USP; aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as ethyl alcohol, polyethylene glycol, and polypropylene glycol; and nonaqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. 6.
  • Water for Injection USP Water for Injection USP
  • aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection
  • water-miscible vehicles such as ethyl alcohol
  • HPLC high performance liquid chromatography
  • the organic layer was separated and washed with H 2 O (2x100ml), dried over Na 2 SO 4 , and concentrated in vacuo to give crude intermediate.
  • the crude compound was dissolved in 5ml of MeCN and 5ml of H 2 O in a 20ml microwave reaction vial. To this solution were added L-/?-borono-phenylalanine (253mg, 1.21mmol), sodium carbonate (256mg, 2.42mmol) and catalytic amount of dichlorobis(triphenylphosphine)-palladium(II) (42.1mg, O.O ⁇ mmol). The mixture was sealed and stirred in the microwave reactor at 150 0 C for 5 minutes, followed by the filtration through celite.
  • (R)-l-(l-(Napthalen-2-yl) ethyl) cyanoguanidine was prepared by forming a mixture of naphthalene amine (1 equivalent), sodium dicyanide (0.95 eq.) and followed by 5N HCl (1 eq.) in n-BuOH: H 2 O (1 : 1). The mixture was refluxed for 1 day in a sealed tube at 160 0 C, and progress of reaction was monitored by LCMS. After completion of reaction, solvent (n- BuOH) was removed under reduced pressure and IN HCl was added to adjust pH to 3-5 range. The aqueous solution was extracted with EtOAc (2x100) and combined organic phase was dried over Na 2 SO 4 .
  • the crude intermediate was then dissolved in 1.5ml of MeCN and 1.5ml of H 2 O in a 5ml microwave vial. To this solution were added L-/?-borono- phenylalanine (126mg, 0.606mmol), sodium carbonate (128mg, 1.21mmol) and catalytic amount of dichlorobis(triphenylphosphine)-palladium(II) (21. lmg, 0.03mmol). The mixture was sealed and stirred in the microwave reactor at 150 0 C for 5 minutes followed by the filtration through celite. The filtrate was concentrated and dissolved in MeOH and H 2 O (1 :1) and purified by preparative HPLC using MeOH/H 2 O/TFA solvent system.
  • Tetrabutylammonium fluoride (0.1 ml; 1.0 M solution in tetrahydrofuran) was added to a solution of 2-trifluoromethyl-benzaldehyde (1.74g, lOmmol) and trifluoromethyltrimethylsilane (TMSCF 3 ) (1.8ml, 12 mmol) in 10 ml THF at 0 0 C.
  • TMSCF 3 trifluoromethyltrimethylsilane
  • the formed mixture was warmed up to room temperature and stirred for 4 hours.
  • the reaction mixture was then treated with 12 ml of IN HCl and stirred overnight.
  • the product was extracted with ethyl acetate (3x20ml).
  • the organic layer was separated and dried over sodium sulfate.
  • the organic solvent was evaporated to give 2.2g of l-(2- trifluoromethylphenyl)-2,2,2-trifluoro-ethanol, yield 90%.
  • Tetrabutylammonium fluoride (0.1 ml; 1.0 M solution in tetrahydrofuran) was added to a solution of 4-methyl-benzaldehyde (1.2g, lOmmol) and TMSCF3 (1.8ml, 12 mmol) in 10 ml THF at 0 0 C. The formed mixture was warmed up to room temperature and stirred for 4 hours. The reaction mixture was then treated with 12 ml of IN HCl and stirred overnight. The product was extracted with ethyl acetate (3x20ml). The organic layer was separated and dried over sodium sulfate. The organic solvent was evaporated to give 1.6g of l-(4- methylphenyl)-2,2,2-trifluoro-ethanol, yield 86%.
  • a microwave vial was charged with 4-chloro-2-amino-6-[l-(4-methylphenyl)-2,2,2- trifluoro-ethoxy]-pyrimidine (33mg, 0. lmmol), 4-borono-L-phenylalanine (31mg, 0.15mmol) and 1 ml of acetonitrile, 0.7ml of water.
  • Aqueous sodium carbonate (0.3 ml, IN) was added to above solution followed by 5 mol percent of dichlorobis(triphenylphosphine)- palladium(II).
  • the reaction vessel was sealed and heated to 150 0 C for 5 minutes with microwave. After cooling, the reaction mixture was evaporated to dryness.
  • Cyclohexanecarbaldehyde (0.9 g, 5mmol) was dissolved in 10ml aqueous 1,4- dioxane, to which 200mg (10 mmol) sodium borohydride was added. The reaction was run overnight at room temperature. After completion of the reaction, 5ml 10% HCl solution was added and the product was extracted with ethyl acetate. The organic layer was separated and dried over sodium sulfate. The organic solvent was evaporated to give 0.8g of 1-cyclohexyl- 2,2,2-trifluoro-ethanol, yield 88%.
  • a microwave vial (2ml) was charged with 4-chloro-6-[2-fluorophenoxy]-pyrimidine, (33mg, 0. lmmol), 4-borono-L-phenylalanine(31mg, 0.15mmol) and 1 ml of actonitrile, 0.7ml of water, 0.3 ml of aqueous sodium carbonate (IM) was added to above solution followed by 5 mol % of dichlorobis(triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 150 0 C for 5 minutes by microwave.
  • 3-(4-Chlorophenyl)piperidine (232mg, lmmol) was added to a solution of 2,4- dichlorotriazine (149.97mg, lmmol), and 300mg diisopropylethyl amine in 10ml THF at 0 0 C.
  • the formed mixture was warmed up to room temperature and stirred for 1 hour.
  • the product was extracted with ethyl acetate (3x20ml).
  • the organic layer was separated and dried over sodium sulfate.
  • the organic solvent was evaporated to give 328mg of 2-chloro-4-[3-(4- chlorophenyl)-piperidin-l-yl]-[l, 3, 5] triazine.
  • a microwave vial was charged with 2-chloro-4-[3-(4-chlorophenyl)-piperidin-l-yl]- [1, 3, 5]triazine (62mg, 0.2mmol), 4-borono-L-phenylalanine(60mg, 0.3mmol), 1 ml of acetonitrile, and 0.7ml of water.
  • Aqueous sodium carbonate (0.6 ml; IM) was added to the solution, followed by 5 mol percent dichlorobis(triphenylphosphine)-palladium(II).
  • the reaction vessel was sealed and heated to 150 0 C for 5 minutes with microwave. After cooling, the reaction mixture was evaporated to dryness.
  • a microwave vial was charged with 4-chloro-6-[2,2,2-trifluoro-l-phenyl-ethoxy]- [l,3,5]triazine-2-ylamine (33mg, 0. lmmol), 4-borono-L-phenylalanine(31mg, 0.15mmol), 1ml of actonitrile, and 0.7ml of water.
  • Aqueous sodium carbonate (0.3 ml, IM) was added to above solution followed by 5 mol percent dichlorobis(triphenylphosphine)-palladium(II).
  • the reaction vessel was sealed and heated to 150 0 C for 5 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness.
  • a microwave vial was charged with 6-chloro-N-[l-naphthalen-2yl-ethyl]- [l,3,5]triazine-2,4-diamine (30mg, O.lmmol), 2-boc protected-amino-3- ⁇ 5-[4,4,5,5,- tetramethyl-[l,3,2]dioxaborolan-2-yl)-pyridin2-yl-]-propionic acid (50mg, 0.15mmol) 1 ml of acetonitrile, and 0.7ml of water.
  • Aqueous sodium carbonate (0.3 ml; IN) was added to the solution, followed by 5 mol percent dichlorobis(triphenylphosphine)-palladium(II).
  • the reaction vessel was sealed and heated to 150 0 C for 5 mintues by microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol, and was then purified by Prep-LC to give 7 mg of boc protected 2-amino-3- ⁇ 5-[4- amino-6-( 1 -naphthalen-2-yl-ethylamino)- [ 1 ,3 ,5 ]triazin-2-yl] -pyridin-2-yl ⁇ proionic acid.
  • reaction vessel was sealed and heated to 150 0 C for 5 minutes with microwave. After cooling, the reaction mixture was evaporated to dryness, the residue was dissolved in 2.5 ml of methanol, and then was purified with Prep- LC to give 6.8 mg of boc protected 2-amino-3- ⁇ 3-[4-amino-6-(l-naphthalen-2-yl- ethylamino)[ 1 ,3,5]triazin-2-yl]-pyrazol- 1 -yl ⁇ proionic acid.
  • Aqueous sodium carbonate (2 ml, IM) was added to above solution followed by 5 mol percent of dichlorobis- (triphenylphosphine)-palladium(II).
  • the reaction vessel was sealed and heated to 15O 0 C for 5 minutes with microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol and purified with Prep-LC to give 5.3 mg of 2-amino-3- ⁇ 4-[6-(3-cyclopentyloxy-4-methoxy-benzylamino)-pyrimidin-4-yl]-phenyl ⁇ - propionic acid, yield 6%.
  • Emrys process vial (2-5ml) for microwave was charged with (6-chloro-pyrazin-2- yl)-(3-cyclopentyloxy-4-methoxy-benzyl)-amine (50mg, 0.15mmol), 4-borono-L- phenylalanine (31mg, 0.15mmol) and 2 ml of acetonitrile.
  • Aqueous sodium carbonate (2 ml, IM) was added to the solution followed by 5 mol percent of dichlorobis(triphenylphosphine)- palladium(II).
  • the reaction vessel was sealed and heated to 15O 0 C for 5 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness.
  • An Emrys process vial (2-5ml) for microwave was charged with (5-bromo-pyrazin-2- yl)-(4'-methyl-biphenyl-2-ylmethyl)-amine (25mg, 0.071mmol), 4-borono-L-phenylalanine (22mg, 0.1 lmmol) and 1 ml of acetonitrile.
  • Aqueous sodium carbonate (1 ml, IM) was added to the solution followed by 5 mol percent dichlorobis(triphenylphosphine)- palladium(II).
  • the reaction vessel was sealed and heated to 15O 0 C for 5 mintues by microwave. After cooling, the reaction mixture was evaporated to dryness.
  • An Emrys process vial (2-5ml) for microwave was charged with 4-chloro-6-(2,2,2- trifluoro-l-phenyl-ethoxy)-pyrimidine (30mg, O.l lmmol), 4-borono-L-phenylalanine (32mg, O.l ⁇ mmol), 1 ml of acetonitrile and 0.6 ml of water.
  • Aqueous sodium carbonate (0.42 ml, IM) was added to above solution followed by 10 mol percent of POPd 2 (dihydrogen di- ⁇ - chlorodichlorobis(di-tert-butylphosphinito- ⁇ P) dipalladate.
  • reaction vessel was sealed and heated to 12O 0 C for 30 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol, and the product was purified with Prep-LC to give 4.8mg of 2-amino-3- ⁇ 4-[6-(2,2,2-trifluoro-lphenyl-ethoxy)- pyrimidin-4-yl]-phenyl ⁇ -propionic acid, yield 11%.
  • Tetrabutylammonium fluoride (TBAF: 0.1 ml, IM) in THF was added to a solution of 3,4-difluro-benzaldehyde (1.42g, lOmmol) and (trifluromethyl)trimethylsilane (1.7Og, 12mmol) in 10 ml THF at O 0 C.
  • the mixture was warmed up to room temperature and stirred for 4 hours.
  • the reaction mixture was treated with 12 ml of IM HCl and stirred overnight.
  • the product was extracted with dicloromethane (3x20ml), the organic layer was combined and passed through a pad of silica gel. The organic solvent was evaporated to give 1.9g of 1- (3,4-difiuoro-phenyl)-2,2,2-trifluoro-ethanol, yield 90%.
  • Aqueous sodium carbonate (0.3 ml, IM) was added to above solution followed by 5 mol % of dichlorobis(triphenylphosphine)-palladium(II).
  • the reaction vessel was sealed and heated to 15O 0 C for 5 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol, then purified with Prep-LC to give 10 mg of 2-amino-3-(4- ⁇ 6-[l-(3,4-difluoro-phenyl)-2,2,2-trifluoro-ethoxy]-pyridin-4-yl ⁇ - phenyl)-propionic acid, yield 21%.
  • reaction mixture was cooled, filtered through a syringe filter and then separated by a reverse phase preparative-HPLC using YMC-Pack ODS 100x30 mm ID column (MeOH/H 2 O/TFA solvent system). The pure fractions were concentrated in vacuum. The product was then suspended in 5 ml of water, frozen and lyophilized to give the title compound as a trifluoro salt (12 mg, 20 %).
  • the above alcohol (290 mg, 1.151 mmol) was dissolved in anhydrous THF (10 ml).
  • Sodium hydride 55 mg, 1.375 mmol was added all at once, and the mixture was stirred at room temperature for 30 minutes.
  • the solution was then transferred into a flask that contained a suspension of 2-amino-4,6-dichloro-triazine (190 mg, 1.152 mmol) in THF (20 ml). The mixture was stirred at room temperature overnight.
  • the reaction mixture was stirred at about -40 0 C for 0.5 hours, then the cold bath was removed and the temperature was allowed to rise slowly to room temperature.
  • the solvent was evaporated and the residue was extracted with hexane (4x20 ml). The collected extractions were washed with cold 10% aqueous NaHCO 3 and dried over Na 2 SO 4 .
  • the solvent was evaporated at reduced pressure to afford 3,5-difluorophenyl-l- trimethylsilyloxyalkene (2.03g, 7.929 mmol, 57% crude yield), which was used in the successive reaction without further purification.
  • Powered calcium carbonate (3.806g, 38.06 mmol) and ethyl vinyl ether (2.184g, 30.329 mmol) were added to a solution of eerie ammonium nitrate (10.43Og, 19.033 mmol) in methanol (40 ml) under nitrogen atmosphere.
  • the water layer was basified to pH « 10 with aqueous sodium hydroxide (IM), and was extracted with dichloromethane and the organic layers were combined, dried over magnesium sulfate and concentrated to afford 290 mg of l-(5,7-difluoro-naphthalen-2-yl)- ethylamine (38% yield).
  • IM aqueous sodium hydroxide
  • the fresh made amine (290mg, 1.401mmol) was added directly to a suspension of 2- amino-4,6-dichloro triazine (277mg, 1.678 mmol) in anhydrous 1,4-dioxane (60 ml), and followed by addition of N,N-diisopropylethylamine (1 ml, 5.732 mmol).
  • the mixture was heated to mild reflux for about 3 hours.
  • the reaction mixture was then cooled, and the solvent was removed under reduced pressure. To the residue was added water and the mixture was sonicated for 2-3 minutes.
  • 5-Chloro-pyrazine-2 carboxylic acid (3,4-dimethoxy-phenyl)-amide (0.18 g, 0.61 mmol), L-p-borono phenylalanine (0.146 g, 0.70 mmol), CH 3 CN (2.5 ml), H 2 O (2.5 ml), Na 2 CO 3 (0.129 g, 1.22 mmol) were combined in a microwave vial. The mixture was sealed and kept at 150 0 C for 5 minutes. The mixture was filtered and concentrated.
  • 2-Amino 4,6-dichloro pyrimidine 0.327 g, 2 mmol
  • methyl-(l-naphthalen-2yl- ethyl)-amine (0.360 g, 2 mmol)
  • cesium carbonate 0.717 g, 2.2 mmol
  • the vial was sealed and stirred at 210 0 C for 20 minutes in a microwave reactor.
  • N-(biphenyl-4-ylmethyl)-5-bromopyrazin-2-amine 60 mg, 0.176 mmol
  • L-p- boronophenylalanine 37 mg, 0.176 mmol
  • palladiumtriphenylphosphine dichloride 3.6 mg, 0.0052 mmol
  • Na 2 CO 3 37 mg, 0.353 mmol
  • acetonitrile 1.25 mis
  • water (1.25 mis
  • Benzylmercaptan (0.14g, 1.11 mmol) was treated with NaH (60% in mineral oil, 67 mg, 1.66 mmol) in dry THF (15 ml) for 30 minutes.
  • 2-Amino-4,6-dichloropyrimidine (0.2 g, 1.22 mmol) was added and the mixture was stirred overnight.
  • the mixture was diluted with methylenechloride, washed with water, then brine, dried over MgSO4, and concentrated to give 0.11 g of 4-(benzylthio)-6-chloropyrimidin-2-amine.
  • 2-Mercaptonapthalene (0.2 g, 1.148) was treated with NaH (60% in Mineral oil, 92 mg, 2.30 mmol) in dry THF (10 ml) for 30 minutes.
  • 2-Amino-4,6-dichloropyrimidine (0.21 g, 1.26 mmol) was added and the mixture was stirred overnight.
  • the mixture was diluted with methylenechloride, washed with water, then brine, dried over MgSO4, and concentratred to give 0.18 g 4-chloro-6-(naphthalen-2-ylmethylthio)pyrimidin-2-amine.
  • 3,5-Difluorophenyl-trifluoromethyl ketone was treated with NaBH 4 (0.18 g, 4.76 mmol) in THF (5 ml) for 2 hours. The mixture was quenched with water, extracted with methylene chloride (2x). The organics were combined, filtered through silica gel and concentrated to give 0.46g of l-(3,4-difluorophenyl)-2,2,2-trifluoroethanol. l-(3,4-Difluorophenyl)-2,2,2-trifluoroethanol (0.1 g, 0.471 mmol) was treated with NaH (60% in mineral oil, 38 mg, 0.943 mmol) in dry THF (3 ml) for 30 minutes.
  • tetrabutylammoniumfluoride (TBAF 1.0 N in THF 13 uL, 3.3 mg, 0.013 mmol) was added to a mixture of 3-methyl-biphenyl-2-carboxaldehyde (0.25g, 1.27 mmol) and trifluoromethytrimethyl silane (0.25 g, 1.53 mmol), in THF (1.5 ml) at 0 0 C.
  • the reaction was warmed to room temperature and stirred for 4 hours.
  • HCl (3.0 N, 2.0 ml) was added, and the mixture was stirred for 3 hours.
  • the mixture was concentrated, dissolved in methylene chloride, filtered through silica gel, and concentrated to give 0.15 g of 2,2,2- trifluoro-l-(3'-methylbiphenyl-2-yl)ethanol.
  • reaction vessel was sealed and heated to 19O 0 C for 10 minutes with microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 10 ml of THF, to which was added 5N.HC1 (5ml). The mixture was refluxed for 2 hours in order to deprotect the benzophone and tert-butyl groups. The resulting reaction mixture was concentrated and dissolved in methanol (8ml) and purified with Prep-LC to afford 15mg of 2-amino-3-(4(4-amino-6-((R)-l-(naphthalene-2-yl)ethylamino)-l,3,5-trizin-2- yl)phenyl)propanoic acid.
  • Emrys process vial (20ml) for microwave was charged with tert-butyl 3-(4- bromo-2-fluorophenyl)-2-(diphenylmethylene-amino)propanoate (600mg, 1.24mmol), Pd(dba)2 (71mg, 0.124mmol), PCy3 (35mg, 0.124mmol), 4,4,4 > ,4 > ,5,5,5',5'-octamethyl-2,2 > - bi(l,3,2-dioxaborolane (346mg, l.leq. 1.36mmol) and KOAc (182mg, 1.5eq., 1.86mmol) 20ml of DMF.
  • reaction vessel was sealed and heated to 16O 0 C for 20 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in H 2 O (30ml), extracted with EtOAc (2x40ml), and purified with Prep-LC to give 220mg of tert-butyl 2-(diphenylmethyleneamino)-3-(2-fluoro- 4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenyl)propanoate.
  • Aqueous sodium carbonate (2 ml, IM) was added to above solution followed by 10 mol percent dichlorobis(triphenylphosphine)-palladium(II).
  • the reaction vessel was sealed and heated to 19O 0 C for 10 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 10 ml of THF, to which 5N.HC1 (2ml) was then added. The mixture was refluxed for 2 hours (deprotection of benzophone and tert-butyl groups).
  • the crude intermediate (250mg, 0.83mmol) was then dissolved in 6.0ml of MeCN and 6ml of H 2 O in a 20ml microwave vial. To this solution were added L-p-borono- phenylalanine (173.6mg, 0.83mmol), sodium carbonate (173.6mg, 1.66mmol) and catalytic amount of dichlorobis(triphenylphosphine)-palladium(II) (11.6mg, 0.0166mmol). The reaction vial was then sealed and stirred in the microwave reactor at 150 0 C for 7 minutes.
  • the crude intermediate (150mg, 0.497mmol) was then dissolved in 3.0ml of MeCN and 3ml of H 2 O in a 10 ml microwave vial.
  • L-p-borono- phenylalanine (104mg, 0.497mmol)
  • sodium carbonate 150mg, 0.994mmol
  • catalytic amount of dichlorobis(triphenylphosphine)-palladium(II) (6.9mg, 0.00994mmol).
  • the reaction vial was then sealed and stirred in the microwave reactor at 150 0 C for 5 minutes.
  • Reaction mixture was cooled, filtered through a syringe filter and then separated by a reverse phase preparative-HPLC using YMC-Pack ODS 100x30 mm ID column (MeOH/H 2 O/TFA solvent system). The pure fractions were concentrated in vacuum. The product was then suspended in 5 ml of water, frozen and lyophilized to give 5 mg of pure product, 2-amino-3-[5-(5-phenyl-thiophen-2-yl)- lH-indol-3-yl]-propionic acid.
  • Residue was purified by preparative HPLC using MeOH/H 2 O/TFA as solvent system to obtain (S)-2-amino-3-[4-(2- amino-6-phenylethynyl-pyrimidin-4-yl(-phenyl]-propionic acid as a TFA salt.
  • 1 H-NMR 400 MHz, CD 3 OD: ⁇ (ppm) 3.20-3.42 (m, 2H), 4.31 (m, IH), 7.40-7.51 (m, 6H), 7.62 (d, 2H), 8.18 (d, 2H). 6.52.
  • Step 1 Synthesis of l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanone.
  • thionyl chloride 29.2 ml, 400 mmol
  • the ice water bath was removed, and 2-bromo-4-chloro-benzoic acid (25 g, 106 mmol) was added.
  • the mixture was heated to mild reflux for 12h. Progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, the reaction mixture was concentrated.
  • reaction mixture was cooled to -78°C (dry ice/acetone bath), and l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanone (40 g, 139 mmol) in THF (400 ml) was added dropwise over 2h.
  • the reaction mixture was allowed to warm to -36°C, and was stirred at that temperature for 24 h, and further stirred at -32°C for another 24h.
  • 3N NaOH 250 ml was added, and the cooling bath was replaced by ice-water bath. Then 30 % hydrogen peroxide in water (250 ml) was added dropwise over 30 minutes.
  • Step 3 Synthesis of R-l-[4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro- ethanol.
  • R-l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanol (15.65g, 54.06 mmol)
  • 3- methylpyrazole (5.33 g, 65 mmol)
  • K 2 CO 3 (15.7 g, 113.5 mmol)
  • (lR,2R)-N,N'-dimethyl-cyclohexane-l,2-diamine (1.54 g, 10.8 mmol) and toluene (80 ml) were combined in a 250 ml pressure tube and heated to 130 0 C (oil bath temperature) for 12 h.
  • reaction mixture was diluted with ethyl acetate and washed with H 2 O (4 x 100 ml), brine, and dried over sodium sulfate. Removal of solvent gave a crude product, which was purified by ISCO column chromatography using 5-10 % ethyl acetate in hexane as solvent to get R-I- [4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro-ethanol (13.5 g; 86 %).
  • H 3 PO 4 40 ml, 85 % in water
  • water 300 ml
  • 50 % NaOH in water 50 % NaOH in water to adjust pH to 6.15.
  • the temperature was raised to 58°C and the above acidic aqueous solution was added dropwise into the buffer with simultaneous addition of 50 % NaOH solution in water so that the pH was maintained between 6.1 to 6.3.
  • H 3 PO 4 40 ml, 85 % in water
  • water 300 ml
  • 50 % NaOH in water 50 % NaOH in water to adjust the pH to 6.15.
  • the temperature was raised to 58°C, and the aqueous Li-salt of the compound was added dropwise into the buffer with simultaneous addition of 3N HCl so that the pH was maintained at 6.1 to 6.2.
  • tetra-n-butyl ammonium fluoride (0.05 eq.) was added to a mixture of substituted benzaldehyde (1 eq.) and trifluoromethyl trimethylsilane (1.2 eq.) in THF at 0 0 C. The temperature was then allowed to warm to room temperature. The mixture was stirred at room temperature for 5h, then diluted with ethyl acetate, washed with water, brine and dried by MgSO 4 . The solvent was removed under reduced pressure to give the trifluoro- alcohol as crude product, which was used in next step without further purification.
  • the above crude product (1 eq.) was added to a 5 ml microwave vial containing 4- borono-L-phenylalanine (1 eq.), Na 2 CO 3 (2 eq.), acetonitrile (2 ml), water (2 ml) and dichlorobis(triphenylphosphine)-palladium (0.05 eq.).
  • the vial was capped, and the mixture was heated at 150 0 C for 5 min under microwave radiation.
  • the mixture was cooled, filtered through a syringe filter, and then separated by a reverse phase preparative-HPLC using YMC-Pack ODS 100x30 mm ID column (MeOH/H 2 O/TFA solvent system). The pure fractions were combined and concentrated in vacuum.
  • the product was then suspended in 5 ml of water, frozen and lyophilized to give the product as a trifluoro acetic acid (TFA) salt.
  • TFA trifluoro acetic acid
  • the monochloride (0.460 g, 1.104 mmol) made above was added to a 20 ml microwave vial, which contained 4-borono-L-phenylalanine (0.277 g, 1.325 mmol), Na 2 CO 3 (0.234 g, 2.208 mmol), acetonitrile (8 ml) / water (8 ml) and dichlorobis(triphenylphosphine)- palladium (0.039 g, 0.055 mmol).
  • the vial was capped and the mixture stirred at 150 0 C for 10 minutes under microwave radiation.
  • Emrys process vial (20 ml) for microwave was charged with l-(4-(2-amino-6- chloro-pyrimidin-4-yloxy)-2,2,2-trifluoro-ethyl)-phenyl)-pyrrolidine-2-one (200 mg, 0.51 mmol), 4-borono-L-phenylalanine (108 mg, 0.51 mmol) and 5 ml of acetonitrile. 5 ml of aqueous sodium carbonate (IM) was added to above solution followed by 5 mol % of dichlorobis(triphenylphosphine)-palladium (II). The reaction vessel was sealed and heated to 16O 0 C for 7min with microwave irradiation.
  • IM aqueous sodium carbonate
  • R-l-(2-Bromo-5-fluoro-phenyl)-2,2,2-trifluoro-ethanol (4.Og, 14.65 mmol), 3-methyl pyrazole (1.56 g, 19.04 mmol), CuI (0.557g, 2.93 mmol), K 2 CO 3 (4.25 g, 30.76 mmol), (lR,2R)-N,N'-dimethyl-cyclohexane-l,2-diamine (0.416 g, 2.93 mmol) and toluene (15 ml) were taken in 50 ml of sealed tube and the resulting mixture was heated at 130 0 C (oil bath temperature) for 2 days.
  • the reaction vessel was sealed, and the mixture was heated at 16O 0 C for 10 minutes with microwave irradiation. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in H 2 O (10 ml) and extracted with ether. The ethereal layer was discarded. Then most of the water in the aqueous phase was removed in vacuo followed by addition of 10 ml of methanol. The crude product was purified with Prep-HPLC to give 1.163 g (yield 75%) of product.
  • Tetrabutylammonium fluoride (0.1 ml of IM in THF) was added to a solution of 4-(6-methoxy-pyridine-2-yl)-benzaldehyde (213 mg, 1 mmol) and trifluoromethyl trimethylsilane (0.2 ml, 1.2 mmol) in 10 ml THF at 0 0 C. The mixture was warmed up to room temperature and stirred for 4 hours. The reaction mixture was then treated with 12 ml of IM HCl and stirred overnight. The product was extracted with ethyl acetate (3x20ml). The organic layer was separated and dried over sodium sulfate. The organic solvent was evaporated to give 0.25g of l-[4-(6-methoxy-pyridine-2-yl)-phenyl]-2,2,2-trifluoro-ethanol which was directly used in next step without purification, yield: 90%.
  • TBAF Tetrabutylammonium fluoride
  • Tetrabutylammonium fluoride (TBAF, 0.1 ml of IM in THF) was added to a solution of 2-pyrimidin-5-yl-benzaldehyde (184 mg, 1 mmol) and trifluoromethyl trimethylsilane (TMSCF 3 , 0.2 ml, 1.2 mmol) in 10 ml THF at 0 0 C.
  • TMSCF 3 trifluoromethyl trimethylsilane
  • the product was extracted with ethyl acetate (3x20ml).
  • the organic layer was separated and dried over sodium sulfate.
  • the organic solvent was evaporated to give 0.21 g of 2,2,2-trifluoro-l-(2-pyrimidin-5-yl-phenyl)-ethanol (yield: 84%), which was directly used in next step without purification.
  • Human TPHl, TPH2, tyrosine hydroxylase (TH) and phenylalanine hydroxylase (PH) were all generated using genes having the following accession numbers, respectively: X52836, AY098914, X05290, and U49897.
  • the full-length coding sequence of human TPHl was cloned into the bacterial expression vector pET24 (Novagen, Madison, WI, USA).
  • a single colony of BL21(DE3) cells harboring the expression vector was inoculated into 50 ml of L broth (LB)- kanamycin media and grown up at 37°C overnight with shaking.
  • Expression of TPHl was induced with 15% D-lactose over a period of 10 hours at 25°C. The cells were spun down and washed once with phosphate buffered saline (PBS). TPHl was purified by affinity chromatography based on its binding to pterin.
  • PBS phosphate buffered saline
  • the cell pellet was resuspended in a lysis buffer (100 ml/20 g) containing 50 mM Tris-Cl, pH 7.6, 0.5 M NaCl, 0.1% Tween-20, 2 mM EDTA, 5 mM DTT, protease inhibitor mixture (Roche Applied Science, Indianapolis, IN, USA) and 1 mM phenylmethanesulfonyl fluoride (PMSF), and the cells were lyzed with a microfluidizer.
  • a lysis buffer 100 ml/20 g
  • PMSF phenylmethanesulfonyl fluoride
  • the lysate was centrifuged and the supernatant was loaded onto a pterin-coupled sepharose 4B column that was equilibrated with a buffer containing 50 mM Tris, pH 8.0, 2 M NaCl, 0.1% Tween-20, 0.5 mM EDTA, and 2 mM DTT.
  • the column was washed with 50 ml of this buffer and TPHl was eluded with a buffer containing 30 mM NaHCO 3 , pH 10.5, 0.5 M NaCl, 0.1% Tween-20, 0.5 mM EDTA, 2 mM DTT, and 10% glycerol.
  • Eluted enzyme was immediately neutralized with 200 mM KH 2 PO 4 , pH 7.0, 0.5 M NaCl, 20 mM DTT, 0.5mM EDTA, and 10% glycerol, and stored at -80 0 C.
  • Human tryptophan hydroxylase type II (TPH2), tyrosine hydroxylase (TH) and phenylalanine hydroxylase (PAH) were expressed and purified essentially in the same way, except the cells were supplemented with tyrosine for TH and phenylalanine for PAH during growth.
  • TPHl and TPH2 activities were measured in a reaction mixture containing 50 mM 4- morpholinepropanesulfonic acid (MOPS), pH 7.0, 60 ⁇ M tryptophan, 100 mM ammonium sulfate, 100 ⁇ M ferrous ammonium sulfate, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), 0.3 mM 6-methyl tetrahydropterin, 0.05 mg/ml catalase, and 0.9 mM DTT.
  • v is the initial velocity at a given compound concentration C
  • b is the background signal
  • D is the Hill slope which is approximately equal to 1
  • I c so is the concentration of the compound that inhibits half of the maximum enzyme activity.
  • Human TH and PAH activities were determined by measuring the amount of 3 H 2 O generated using L- [3, 4- H] -tyrosine and L- [4- H] -phenylalanine, respectively.
  • the enzyme 100 nM was first incubated with its substrate at 0.1 mM for about 10 minutes, and added to a reaction mixture containing 50 mM MOPS, pH 7.2, 100 mM ammonium sulfate, 0.05% Tween-20, 1.5 mM TCEP, 100 ⁇ M ferrous ammonium sulfate, 0.1 mM tyrosine or phenylalanine, 0.2 mM 6-methyl tetrahydropterin, 0.05 mg/ml of catalase, and 2 mM DTT.
  • RBL2H3 is a rat mastocytoma cell line, which contains TPHl and makes 5-hydroxytrypotamine (5HT) spontaneously
  • BON is a human carcinoid cell line, which contains TPHl and makes 5-hydroxytryptophan (5HTP).
  • the CBAs were performed in 96-well plate format.
  • the mobile phase used in HPLC contained 97% of 100 mM sodium acetate, pH 3.5 and 3% acetonitrile.
  • a Waters Cl 8 column (4.6 x 50 mm) was used with Waters HPLC (model 2795).
  • a multi-channel fluorometer (model 2475) was used to monitor the flow through by setting at 280 nm as the excitation wavelength and 360 nm as the emission wavelength.
  • RBL CBA Cells were grown in complete media (containing 5 % bovine serum) for 3-4 hours to allow cells to attach to plate wells (7K cell/well). Compounds were then added to each well in the concentration range of 0.016 ⁇ M to 11.36 ⁇ M. The controls were cells in complete media without any compound present. Cells were harvested after 3 days of incubation at 37°C. Cells were >95% confluent without compound present. Media were removed from plate and cells were lysed with equal volume of 0.1 N NaOH. A large portion of the cell lysate was treated by mixing with equal volume of IM TCA and then filtered through glass fiber. The filtrates were loaded on reverse phase HPLC for analyzing 5HT concentrations. A small portion of the cell lysate was also taken to measure protein concentration of the cells that reflects the cytotoxicity of the compounds at the concentration used. The protein concentration was measured by using BCA method.
  • the average of 5HT level in cells without compound treated was used as the maximum value in the IC50 derivation according to the equation provided above.
  • the minimum value of 5HT is either set at 0 or from cells that treated with the highest concentration of compound if a compound is not cytotoxic at that concentration.
  • BON CBA Cells were grown in equal volume of DMEM and F12K with 5 % bovine serum for 3-4 hours (2OK cell/well) and compound was added at a concentration range of 0.07 ⁇ M to 50 ⁇ M. The cells were incubated at 37°C overnight. Fifty ⁇ M of the culture supernatant was then taken for 5HTP measurement. The supernatant was mixed with equal volume of IM TCA, then filtered through glass fiber. The filtrate was loaded on reverse phase HPLC for 5HTP concentration measurement. The cell viability was measured by treating the remaining cells with Promega Celltiter-Glo Luminescent Cell Viability Assay. The compound potency was then calculated in the same way as in the RBL CBA.
  • GI gastrointestinal
  • the effect of a potent TPHl inhibitor of the invention on gastrointestinal (GI) transit time and gastric emptying was determined in Sprague-Dawley rats.
  • the compound was administred at doses of 50, 125 and 250 mpk, po, qd, for 14 days.
  • Each dosing group utilized nine rats.
  • Nine rats were also used as a negative control group (vehicle administration only), and another six were used as a positive control (Atropine).
  • the rats were dosed compound or vehicle at 10 ml/kg.
  • Atropine was given to the positive control group on day 14 only, whereas vehicle was given on days 1-14.
  • Body weights and observations were taken through out study, and the rats were fasted overnight on day 13 prior to the charcoal meal.
  • the potent TPHl inhibitor, Atropine or vehicle wereorally dosed 30 minutes prior to the charcoal meal.
  • the charcoal meal (5% charcoal in vehicle) was orally dosed at 15 ml/kg.
  • Necropsy was performed 25 minutes after the charcoal meal dose.
  • GI transit times were determined by measuring the distance the charcoal meal traveled down the small intestine, and dividing that distance by the total length of the small intestine. Gastric emptying times were determined by weighing the stomachs of the rats.
  • Brain 5 -HT levels were unaffected by the compound.

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Abstract

Methods and compounds are disclosed for affecting gastrointestinal motility and gastric emptying, which comprise inhibiting tryptophan hydroxylase (TPH) in patients in need thereof.

Description

METHODS OF AFFECTING GASTROINTESTINAL TRANSIT AND GASTRIC EMPTYING, AND COMPOUNDS USEFUL THEREIN
This application claims priority to U.S. provisional application no. 60/952,071, filed July 26, 2007, the entirety of which is incorporated herein by reference.
1. FIELD OF THE INVENTION
This invention relates to methods of affecting gastric transit and gastric emptying, and to compounds and compositions useful therein.
2. BACKGROUND
The neurotransmitter serotonin [5-hydroxytryptamine (5-HT)] is involved in multiple central nervous facets of mood control and in regulating sleep, anxiety, alcoholism, drug abuse, food intake, and sexual behavior. It has also been implicated in the regulation of vascular tone, gut motility and cell-mediated immune responses. Walther, D. J., et al., Science 299:76 (2003). 5-HT also plays a role in clotting and hemostasis: platelets — which cannot themselves make 5-HT — take up large amounts of peripheral 5-HT. Goodman & Gilman's The Pharmacological Basis of Therapeutics, 10th ed., p. 274-5 (McGraw-Hill, 2001).
Serotonin is synthesized in two steps from the amino acid tryptophan. Goodman & Gilman's, p. 270. The first step is rate-limiting, and is catalyzed by the enzyme tryptophan hydroxylase (TPH), which has two known isoforms: TPHl, which is expressed in the periphery, and TPH2, which is expressed primarily in the brain. Walther, D. J., et al., Science 299:76 (2003). The principle route by which serotonin is removed from the body involves the enzyme monoamine oxidase (MAO), which converts the compound to 5-hydroxyindole acetaldehyde, which is then converted to 5-hydroxyindole acetic acid (5 -HIAA) by the enzyme aldehyde dehydrogenase. Goodman & Gilman's, p. 270-2. Mice genetically deficient for the tphl gene ("knockout mice") have been reported.
In one case, the mice reportedly expressed normal amounts of serotonin in classical serotonergic brain regions, but largely lacked serotonin in the periphery. Id. In another, the knockout mice exhibited abnormal cardiac activity, which was attributed to a lack of peripheral serotonin. Cote, F., et al, PNAS 100(23): 13525-13530 (2003). Because serotonin is involved in so many biochemical processes, drugs that affect serotonin levels or affect serotonin receptors are often attended by adverse effects. For example, parenteral injection of the TPH inhibitor p-chlorophenylalanine (p-CPA) to rats reportedly decreased their gastrointestinal motility. Sailer, C. F., Strieker, E. M., Communications, J. Pharm. Pharmac. 30:646 (1978). And at high doses (3000 mg/day), oral administration of the compound reportedly causes constipation in humans. Cremata, V.Y., and Koe, B.K., Clin. Pharmacol. Therapeutics 7(6):768-776, 773 (1966). But p-CPA readily gets into the central nervous system, and is associated with a number of adverse psychological effects, such as dizziness, nausea and uneasiness. Id.
3. SUMMARY OF THE INVENTION
This invention is directed, in part, to methods of affecting gastrointestinal transit and gastric emptying, which comprise inhibiting peripheral tryptophan hydroxylase (TPH) in patients in need thereof, without substantially affecting their brain 5 -HT levels.
In particular methods, the TPH is inhibited by administering to the patient an effective amount of a compound of formula I:
Figure imgf000003_0001
I and pharmaceutically acceptable salts and solvates thereof, wherein: A is optionally substituted cycloalkyl, aryl, or heterocycle; X is a bond {i.e., A is directly bound to D), -O-, -S-, -C(O)-, -C(R4)=, =C(PM)-, -C(R3R4)-, -C(PM)=C(R4)-, -C=C-, -N(R5)-, -N(R5)C(O)N(R5)-, -C(R3R4)N(R5)-, -N(R5)C(R3R4)-, -ONC(R3)-, -C(R3)NO-, -C(R3R4)O-, -OC(R3R4)-, -S(O2)-, -S(O2)N(R5)-, -N(R5)S(O2)-, -C(R3R4)S(O2)-, or -S(O2)C(R3R4)-; D is optionally substituted aryl or heterocycle; Ri is hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle; R2 is hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle; R3 is hydrogen, alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted alkyl; R4 is hydrogen, alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted alkyl or aryl; each R5 is independently hydrogen or optionally substituted alkyl or aryl; and n is 0-3.
4. BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the effect of oral administration of a potent TPHl inhibitor on the gastrointestinal (GI) motility of rats. The asterisk identifies data wherein p < 0.01 when compared with vehicle control using the t test or one-way ANOVA test. Figure 2 shows the effect of oral administration of a potent TPHl inhibitor on the gastric emptying of rats. The asterisk identifies data wherein p < 0.01 when compared with vehicle control using the t test or one-way ANOVA test.
Figure 3 shows the effect of oral administration of a potent TPHl inhibitor on the blood and proximal colon levels of 5-HT of the rats for which data is presented in figures 1 and 2. In both cases, p < 0.0001 using one-way ANOVA.
5. DETAILED DESCRIPTION
This invention is based, in part, on the discovery of compounds that are potent inhibitors of TPH (e.g., TPHl). When administered to mammals, preferred compounds of the invention reduce peripheral serotonin levels.
5.1. Definitions
Unless otherwise indicated, the term "alkenyl" means a straight chain, branched and/or cyclic hydrocarbon having from 2 to 20 (e.g., 2 to 10 or 2 to 6) carbon atoms, and including at least one carbon-carbon double bond. Representative alkenyl moieties include vinyl, allyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-l-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 1-heptenyl, 2- heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1- decenyl, 2-decenyl and 3-decenyl.
Unless otherwise indicated, the term "alkyl" means a straight chain, branched and/or cyclic ("cycloalkyl") hydrocarbon having from 1 to 20 (e.g., 1 to 10 or 1 to 4) carbon atoms. Alkyl moieties having from 1 to 4 carbons are referred to as "lower alkyl." Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl and dodecyl. Cycloalkyl moieties may be monocyclic or multicyclic, and examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl. Additional examples of alkyl moieties have linear, branched and/or cyclic portions (e.g., l-ethyl-4-methyl- cyclohexyl). The term "alkyl" includes saturated hydrocarbons as well as alkenyl and alkynyl moieties.
Unless otherwise indicated, the term "alkoxy" means an -O-alkyl group. Examples of alkoxy groups include -OCH3, -OCH2CH3, -O(CH2)2CH3, -O(CH2)3CH3, -O(CH2)4CH3, and -O(CH2)5CH3. Unless otherwise indicated, the term "alkylaryl" or "alkyl-aryl" means an alkyl moiety bound to an aryl moiety.
Unless otherwise indicated, the term "alkylheteroaryl" or "alkyl-heteroaryl" means an alkyl moiety bound to a heteroaryl moiety. Unless otherwise indicated, the term "alkylheterocycle" or "alkyl-heterocycle" means an alkyl moiety bound to a heterocycle moiety.
Unless otherwise indicated, the term "alkynyl" means a straight chain, branched or cyclic hydrocarbon having from 2 to 20 (e.g., 2 to 20 or 2 to 6) carbon atoms, and including at least one carbon-carbon triple bond. Representative alkynyl moieties include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-l-butynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl, 6-heptynyl, 1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl, 8-nonynyl, 1-decynyl, 2-decynyl and 9-decynyl.
Unless otherwise indicated, the term "aryl" means an aromatic ring or an aromatic or partially aromatic ring system composed of carbon and hydrogen atoms. An aryl moiety may comprise multiple rings bound or fused together. Examples of aryl moieties include anthracenyl, azulenyl, biphenyl, fluorenyl, indan, indenyl, naphthyl, phenanthrenyl, phenyl, 1,2,3,4-tetrahydro-naphthalene, and to IyI.
Unless otherwise indicated, the term "arylalkyl" or "aryl-alkyl" means an aryl moiety bound to an alkyl moiety. Unless otherwise indicated, the terms "biohydrolyzable amide," "biohydrolyzable ester," "biohydrolyzable carbamate," "biohydrolyzable carbonate," "biohydrolyzable ureido" and "biohydrolyzable phosphate" mean an amide, ester, carbamate, carbonate, ureido, or phosphate, respectively, of a compound that either: 1) does not interfere with the biological activity of the compound but can confer upon that compound advantageous properties in vivo, such as uptake, duration of action, or onset of action; or 2) is biologically inactive but is converted in vivo to the biologically active compound. Examples of biohydrolyzable esters include lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters, and choline esters. Examples of biohydrolyzable amides include lower alkyl amides, α-amino acid amides, alkoxyacyl amides, and alkylaminoalkyl-carbonyl amides. Examples of biohydrolyzable carbamates include lower alkylamines, substituted ethylenediamines, aminoacids, hydroxy alkylamines, heterocyclic and heteroaromatic amines, and polyether amines.
Unless otherwise indicated, the phrases "disease or disorder mediated by peripheral serotonin" and "disease and disorder mediated by peripheral serotonin" mean a disease and/or disorder having one or more symptoms, the severity of which are affected by peripheral serotonin levels.
Unless otherwise indicated, the terms "halogen" and "halo" encompass fluorine, chlorine, bromine, and iodine. Unless otherwise indicated, the term "heteroalkyl" refers to an alkyl moiety (e.g., linear, branched or cyclic) in which at least one of its carbon atoms has been replaced with a heteroatom (e.g., N, O or S).
Unless otherwise indicated, the term "heteroaryl" means an aryl moiety wherein at least one of its carbon atoms has been replaced with a heteroatom (e.g., N, O or S). Examples include acridinyl, benzimidazolyl, benzofuranyl, benzoisothiazolyl, benzoisoxazolyl, benzoquinazolinyl, benzothiazolyl, benzoxazolyl, furyl, imidazolyl, indolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, phthalazinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidyl, pyrrolyl, quinazolinyl, quinolinyl, tetrazolyl, thiazolyl, and triazinyl. Unless otherwise indicated, the term "heteroarylalkyl" or "heteroaryl-alkyl" means a heteroaryl moiety bound to an alkyl moiety.
Unless otherwise indicated, the term "heterocycle" refers to an aromatic, partially aromatic or non-aromatic monocyclic or polycyclic ring or ring system comprised of carbon, hydrogen and at least one heteroatom (e.g., N, O or S). A heterocycle may comprise multiple (i.e., two or more) rings fused or bound together. Heterocycles include heteroaryls.
Examples include benzo[l,3]dioxolyl, 2,3-dihydro-benzo[l,4]dioxinyl, cinnolinyl, furanyl, hydantoinyl, morpholinyl, oxetanyl, oxiranyl, piperazinyl, piperidinyl, pyrrolidinonyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl and valerolactamyl. Unless otherwise indicated, the term "heterocyclealkyl" or "heterocycle-alkyl" refers to a heterocycle moiety bound to an alkyl moiety.
Unless otherwise indicated, the term "heterocycloalkyl" refers to a non-aromatic heterocycle.
Unless otherwise indicated, the term "heterocycloalkylalkyl" or "heterocycloalkyl- alkyl" refers to a heterocycloalkyl moiety bound to an alkyl moiety.
Unless otherwise indicated, the terms "manage," "managing" and "management" encompass preventing the recurrence of the specified disease or disorder, or of one or more of its symptoms, in a patient who has already suffered from the disease or disorder, and/or lengthening the time that a patient who has suffered from the disease or disorder remains in remission. The terms encompass modulating the threshold, development and/or duration of the disease or disorder, or changing the way that a patient responds to the disease or disorder.
Unless otherwise indicated, the term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases. Suitable pharmaceutically acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, N5N'- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Suitable non-toxic acids include inorganic and organic acids such as acetic, alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethenesulfonic, formic, fumaric, furoic, galacturonic, gluconic, glucuronic, glutamic, glycolic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phenylacetic, phosphoric, propionic, salicylic, stearic, succinic, sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonic acid. Specific non-toxic acids include hydrochloric, hydrobromic, phosphoric, sulfuric, and methanesulfonic acids. Examples of specific salts thus include hydrochloride and mesylate salts. Others are well-known in the art. See, e.g., Remington' s Pharmaceutical Sciences, 18 ed. (Mack Publishing, Easton PA: 1990) and Remington: The Science and Practice of Pharmacy, 19th ed. (Mack Publishing, Easton PA: 1995). Unless otherwise indicated, the term "potent TPHl inhibitor" is a compound that has a TPH 1 IC50 of less than about 10 μM.
Unless otherwise indicated, the terms "prevent," "preventing" and "prevention" contemplate an action that occurs before a patient begins to suffer from the specified disease or disorder, which inhibits or reduces the severity of the disease or disorder, or of one or more of its symptoms. The terms encompass prophylaxis.
Unless otherwise indicated, the term "prodrug" encompasses pharmaceutically acceptable esters, carbonates, thiocarbonates, N-acyl derivatives, N-acyloxyalkyl derivatives, quaternary derivatives of tertiary amines, N-Mannich bases, Schiff bases, amino acid conjugates, phosphate esters, metal salts and sulfonate esters of compounds disclosed herein. Examples of prodrugs include compounds that comprise a biohydrolyzable moiety {e.g., a biohydrolyzable amide, biohydrolyzable carbamate, biohydrolyzable carbonate, biohydrolyzable ester, biohydrolyzable phosphate, or biohydrolyzable ureide analog). Prodrugs of compounds disclosed herein are readily envisioned and prepared by those of ordinary skill in the art. See, e.g., Design of Prodrugs, Bundgaard, A. Ed., Elseview, 1985; Bundgaard, H., "Design and Application of Prodrugs," A Textbook of Drug Design and Development, Krosgaard-Larsen and H. Bundgaard, Ed., 1991, Chapter 5, p. 113-191; and Bundgaard, H., Advanced Drug Delivery Review, 1992, 8, 1-38.
Unless otherwise indicated, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or condition, or one or more symptoms associated with the disease or condition, or prevent its recurrence. A prophylactically effective amount of a compound is an amount of therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the disease. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.
Unless otherwise indicated, the term "protecting group" or "protective group," when used to refer to part of a molecule subjected to a chemical reaction, means a chemical moiety that is not reactive under the conditions of that chemical reaction, and which may be removed to provide a moiety that is reactive under those conditions. Protecting groups are well known in the art. See, e.g., Greene, T.W. and Wuts, P.G.M., Protective Groups in Organic Synthesis (3rd ed., John Wiley & Sons: 1999); Larock, R.C., Comprehensive Organic Transformations (2nd ed., John Wiley & Sons: 1999). Some examples include benzyl, diphenylmethyl, trityl, Cbz, Boc, Fmoc, methoxycarbonyl, ethoxycarbonyl, and pthalimido.
Unless otherwise indicated, the term "pseudohalogen" refers to a polyatomic anion that resembles a halide ion in its acid-base, substitution, and redox chemistry, generally has low basicity, and forms a free radical under atom transfer radical polymerization conditions. Examples of pseudohalogens include azide ions, cyanide, cyanate, thiocyanate, thiosulfate, sulfonates, and sulfonyl halides.
Unless otherwise indicated, the term "selective TPHl inhibitor" is a compound that has a TPH2_IC50 that is at least about 10 times greater than its TPH 1 IC50.
Unless otherwise indicated, the terms "serotonin-mediated disease," "serotonin- mediated disorder" and "serotonin-mediated disease or disorder" refer to a disease or disorder having one or more symptoms that are attributable to increased levels of peripheral 5- hydroxytryptamine (5-HT). Unless otherwise indicated, the term "stereomerically enriched composition of a compound refers to a mixture of the named compound and its stereoisomer(s) that contains more of the named compound than its stereoisomer(s). For example, a stereoisomerically enriched composition of (S)-butan-2-ol encompasses mixtures of (S)-butan-2-ol and (R)- butan-2-ol in ratios of, e.g., about 60/40, 70/30, 80/20, 90/10, 95/5, and 98/2. Unless otherwise indicated, the term "stereoisomer^ mixture" encompasses racemic mixtures as well as stereomerically enriched mixtures (e.g., R/S = 30/70, 35/65, 40/60, 45/55, 55/45, 60/40, 65/35 and 70/30).
Unless otherwise indicated, the term "stereomerically pure" means a composition that comprises one stereoisomer of a compound and is substantially free of other stereoisomers of that compound. For example, a stereomerically pure composition of a compound having one stereocenter will be substantially free of the opposite stereoisomer of the compound. A stereomerically pure composition of a compound having two stereocenters will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound, or greater than about 99% by weight of one stereoisomer of the compound and less than about 1% by weight of the other stereoisomers of the compound.
Unless otherwise indicated, the term "substituted," when used to describe a chemical structure or moiety, refers to a derivative of that structure or moiety wherein one or more of its hydrogen atoms is substituted with an atom, chemical moiety or functional group such as, but not limited to, alcohol, aldehylde, alkoxy, alkanoyloxy, alkoxycarbonyl, alkenyl, alkyl (e.g. , methyl, ethyl, propyl, t-butyl), alkynyl, alkylcarbonyloxy (-OC(O)alkyl), amide (-C(O)NH-alkyl- or -alkylNHC(O)alkyl), amidinyl (-C(NH)NH-alkyl or -C(NR)NH2), amine (primary, secondary and tertiary such as alkylamino, arylamino, arylalkylamino), aroyl, aryl, aryloxy, azo, carbamoyl (-NHC(O)O-alkyl- or -OC(O)NH-alkyl), carbamyl (e.g., CONH2, as well as CONH-alkyl, CONH-aryl, and CONH-arylalkyl), carbonyl, carboxyl, carboxylic acid, carboxylic acid anhydride, carboxylic acid chloride, cyano, ester, epoxide, ether (e.g., methoxy, ethoxy), guanidino, halo, haloalkyl (e.g., -CCI3, -CF3, -C(CF3)3), heteroalkyl, hemiacetal, imine (primary and secondary), isocyanate, isothiocyanate, ketone, nitrile, nitro, oxygen (i.e., to provide an oxo group), phosphodiester, sulfide, sulfonamido (e.g., SO2NH2), sulfone, sulfonyl (including alkylsulfonyl, arylsulfonyl and arylalkylsulfonyl), sulfoxide, thiol (e.g., sulfhydryl, thioether) and urea (-NHCONH-alkyl-). Unless otherwise indicated, a "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease or condition, or to delay or minimize one or more symptoms associated with the disease or condition. A therapeutically effective amount of a compound is an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment or management of the disease or condition. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of a disease or condition, or enhances the therapeutic efficacy of another therapeutic agent. Unless otherwise indicated, the term "TPH1_IC5O" is the IC50 of a compound for
TPHl as determined using the in vitro inhibition assay described in the Examples, below.
Unless otherwise indicated, the term "TPH2_IC50" is the IC50 of a compound for TPH2 as determined using the in vitro inhibition assay described in the Examples, below.
Unless otherwise indicated, the terms "treat," "treating" and "treatment" contemplate an action that occurs while a patient is suffering from the specified disease or disorder, which reduces the severity of the disease or disorder, or one or more of its symptoms, or retards or slows the progression of the disease or disorder.
Unless otherwise indicated, the term "include" has the same meaning as "include" and the term "includes" has the same meaning as "includes, but is not limited to." Similarly, the term "such as" has the same meaning as the term "such as, but not limited to."
Unless otherwise indicated, one or more adjectives immediately preceding a series of nouns is to be construed as applying to each of the nouns. For example, the phrase "optionally substituted alky, aryl, or heteroaryl" has the same meaning as "optionally substituted alky, optionally substituted aryl, or optionally substituted heteroaryl." It should be noted that a chemical moiety that forms part of a larger compound may be described herein using a name commonly accorded it when it exists as a single molecule or a name commonly accorded its radical. For example, the terms "pyridine" and "pyridyl" are accorded the same meaning when used to describe a moiety attached to other chemical moieties. Thus, the two phrases "XOH, wherein X is pyridyl" and "XOH, wherein X is pyridine" are accorded the same meaning, and encompass the compounds pyridin-2-ol, pyridin-3-ol and pyridin-4-ol.
It should also be noted that if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or the portion of the structure is to be interpreted as encompassing all stereoisomers of it. Similarly, names of compounds having one or more chiral centers that do not specify the stereochemistry of those centers encompass pure stereoisomers and mixtures thereof. Moreover, any atom shown in a drawing with unsatisfied valences is assumed to be attached to enough hydrogen atoms to satisfy the valences. In addition, chemical bonds depicted with one solid line parallel to one dashed line encompass both single and double (e.g. , aromatic) bonds, if valences permit.
5.2. Compounds
Particular methods of this invention comprise the use of potent TPHl inhibitors. Examples of potent TPHl inhibitors are disclosed herein and in U.S. patent application nos. 11/638,677 and 60/874,596, both filed December 12, 2006. These compounds are significantly more potent than p-chlorophenylalanine, which has a TPHl IC5O of about 93 μM.
Particular methods of the invention utilize compounds of formula I:
Figure imgf000011_0001
I and pharmaceutically acceptable salts and solvates thereof, wherein: A is optionally substituted cycloalkyl, aryl, or heterocycle; X is a bond, -O-, -S-, -C(O)-, -C(R4)=, =C(Pv4)-, -C(R3R4)-, -C(PM)=C(R4)-, -C=C-, -N(R5)-, -N(R5)C(O)N(R5)-, -C(R3R4)N(R5)-, -N(R5)C(R3R4)-, -ONC(R3)-, -C(R3)NO-, -C(R3R4)O-, -OC(R3R4)-, -S(O2)-, -S(O2)N(R5)-, -N(R5)S(O2)-, -C(R3R4)S(O2)-, or -S(O2)C(R3R4)-; D is optionally substituted aryl or heterocycle; Ri is hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle; R2 is hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle; R3 is hydrogen, alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted alkyl; R4 is hydrogen, alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted alkyl or aryl; each R5 is independently hydrogen or optionally substituted alkyl or aryl; and n is 0-3.
Particular compounds are of formula 1(A):
Figure imgf000012_0001
I(A)
Others are of formula II:
Figure imgf000012_0002
II and pharmaceutically acceptable salts and solvates thereof, wherein: A is optionally substituted cycloalkyl, aryl, or heterocycle; X is a bond, -O-, -S-, -C(O)-, -C(R4)=, =C(R4)-, -C(R3R4)-, -C(JLO=C(R4)-, -C=C-, -N(R5)-, -N(R5)C(O)N(R5)-, -C(R3R4)N(R5)-, -N(R5)C(R3R4)-, -ONC(R3)-, -C(R3)NO-, -C(R3R4)O-, -OC(R3R4)-, -S(O2)-, -S(O2)N(R5)-, -N(R5)S(O2)-, -C(R3R4)S(O2)-, or -S(O2)C(R3R4)-; D is optionally substituted aryl or heterocycle; E is optionally substituted aryl or heterocycle; Ri is hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle; R2 is hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle; R3 is hydrogen, alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted alkyl; R4 is hydrogen, alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted alkyl or aryl; R5 is hydrogen or optionally substituted alkyl or aryl; and n is 0-3. Particular compounds are of formula H(A):
Figure imgf000012_0003
ii(A)
With regard to the formulae disclosed herein (e.g., I, I(A), II and H(A)), particular compounds include those wherein A is optionally substituted cycloalkyl (e.g. , 6-membered and 5-membered). In some, A is optionally substituted aryl (e.g., phenyl or naphthyl). In others, A is optionally substituted heterocycle (e.g., 6-membered and 5-membered). Examples of 6-membered heterocycles include pyridine, pyridazine, pyrimidine, pyrazine, and triazine. Examples of 5-membered heterocycles include pyrrole, imidazole, triazole, thiazole, thiophene, and furan. In some compounds, A is aromatic. In others, A is not aromatic. In some, A is an optionally substituted bicyclic moiety (e.g., indole, iso-indole, pyrrolo-pyridine, or napthylene).
Particular compounds are of the formula:
Figure imgf000013_0001
wherein: each of Ai and A2 is independently a monocyclic optionally substituted cycloalkyl, aryl, or heterocycle. Compounds encompassed by this formula include those wherein Ai and/or A2 is optionally substituted cycloalkyl (e.g., 6-membered and 5-membered). In some, Ai and/or A2 is optionally substituted aryl (e.g., phenyl or naphthyl). In others, Ai and/or A2 is optionally substituted heterocycle (e.g., 6-membered and 5-membered). Examples of 6- membered heterocycles include pyridine, pyridazine, pyrimidine, pyrazine, and triazine. Examples of 5-membered heterocycles include pyrrole, imidazole, triazole, thiazole, thiophene, and furan. In some compounds, Ai and/or A2 is aromatic. In others, Ai and/or A2 is not aromatic.
With regard to the formulae disclosed herein, particular compounds include those wherein D is optionally substituted aryl (e.g., phenyl or naphthyl). In others, D is optionally substituted heterocycle (e.g., 6-membered and 5-membered). Examples of 6-membered heterocycles include pyridine, pyridazine, pyrimidine, pyrazine, and triazine. Examples of 5- membered heterocycles include pyrrole, imidazole, triazole, thiazole, thiophene, and furan. In some compounds, D is aromatic. In others, D is not aromatic. In some, D is an optionally substituted bicyclic moiety (e.g. , indole, iso-indole, pyrrolo-pyridine, or napthylene).
With regard to the various formulae disclosed herein, particular compounds include those wherein E is optionally substituted aryl (e.g., phenyl or naphthyl). In others, E is optionally substituted heterocycle (e.g., 6-membered and 5-membered). Examples of 6- membered heterocycles include pyridine, pyridazine, pyrimidine, pyrazine, and triazine. Examples of 5-membered heterocycles include pyrrole, imidazole, triazole, thiazole, thiophene, and furan. In some compounds, E is aromatic. In others, E is not aromatic. In some, E is an optionally substituted bicyclic moiety (e.g. , indole, iso-indole, pyrrolo-pyridine, or napthylene).
With regard to the various formulae disclosed herein, particular compounds include those wherein Ri is hydrogen or optionally substituted alkyl. In some, R2 is hydrogen or optionally substituted alkyl. In some, n is 1 or 2.
In some, X is a bond or S. In others, X is -C(R4)=, =C(R4)-, -C(R3R4)-, -C(R^=C(R4)-, or -C≡C-, and, for example, R4 is independently hydrogen or optionally substituted alkyl. In others, X is -O-, -C(R3R4)O-, or -OC(R3R4)-, and, for example, R3 is hydrogen or optionally substituted alkyl, and R4 is hydrogen or optionally substituted alkyl. In some, R3 is hydrogen and R4 is trifluromethyl. In some compounds, X is -S(O2)-, -S(O2)N(R5)-, -N(R5)S(O2)-, -C(R3R4)S(O2)-, or -S(O2)C(R3R4)-, and, for example, R3 is hydrogen or optionally substituted alkyl, R4 is hydrogen or optionally substituted alkyl, and R5 is hydrogen or optionally substituted alkyl. In others, X is -N(R5)-, -N(R5)C(O)N(R5)-, -C(R3R4)N(R5)-, or -N(R5)C(R3R4)-, and, for example, R3 is hydrogen or optionally substituted alkyl, R4 is hydrogen or optionally substituted alkyl, and each R5 is independently hydrogen or optionally substituted alkyl.
Other compounds are of the formula:
Figure imgf000014_0001
wherein, for example, R3 is trifluoromethyl. Others are encompassed by the formula:
Figure imgf000014_0002
wherein, for example, R3 is hydrogen.
Some compounds are encompassed by the formula:
Figure imgf000015_0001
wherein: each of Zi, Z2, Z3, and Z4 is independently N or CR6; each R6 is independently hydrogen, cyano, halogen, OR7, NRgRθ, amino, hydroxyl, or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each R7 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Rg is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each R9 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; and m is 1-4. Certain such compounds are of the formula:
Figure imgf000015_0002
Others are of the formula:
Figure imgf000015_0003
wherein, for example, R3 is trifluoromethyl. Others are of the formula:
Figure imgf000015_0004
wherein, for example, R3 is hydrogen.
Referring to the various formulae above, some compounds are such that all of Zi, Z2, Z3, and Z4 are N. In others, only three of Zi, Z2, Z3, and Z4 are N. In others, only two of Zi, Z2, Z3, and Z4 are N. In others, only one of Zi, Z2, Z3, and Z4 is N. In others, none of Zi, Z2, Z3, and Z4 are N.
Some compounds are of the formula:
Figure imgf000016_0001
wherein: each of Z'i, Z'2, and Z'3 is independently N, NH, S, O or CR6; each R6 is independently amino, cyano, halogen, hydrogen, OR7, SR7, NRgR9, or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each R7 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Rg is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each R9 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; and p is 1-3. Certain such compounds are of the formula:
Figure imgf000016_0002
Others are of the formula:
Figure imgf000016_0003
wherein, for example, R3 is trifluoromethyl. Others are of the formula:
Figure imgf000017_0001
wherein, for example, R3 is hydrogen.
Referring to the various formulae above, some compounds are such that all of Z'i, Z'2, and Z'3 are N or NH. In others, only two of Z'I, Z'2, and Z'3 are N or NH. In others, only one of Z'i, Z'2, and Z'3 is N or NH. In others, none of Z'h Z'2, and Z'3 are N or NH.
Some compounds are encompassed by the formula:
Figure imgf000017_0002
wherein: each of Z"i, Z"2, Z"3, and Z"4 is independently N or CR10; each Ri0 is independently amino, cyano, halogen, hydrogen, ORn, SRn, NR12R13, or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Rn is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Ri2 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; and each Ri3 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle. Certain such compounds are of the formula:
Figure imgf000017_0003
Others are of the formula:
Figure imgf000018_0001
wherein, for example, R3 is trifluoromethyl. Others are of the formula:
Figure imgf000018_0002
wherein, for example, R3 is hydrogen.
Referring to the various formulae above, some compounds are such that all of Z"i, Z"2, Z"3, and Z"4 are N. In others, only three of Z"ls Z"2, Z'f3, and Z"4 are N. In others, only two of Z"i, Z"2, Z"3, and Z"4 are N. In others, only one of Z"i, Z"2, Z"3, and Z"4 is N. In others, none of Z"ls Z"2, Z"3, and Z"4 are N.
Some compounds are of the formula:
Figure imgf000018_0003
wherein: each of Z"i, Z"2, Z"3, and Z"4 is independently N or CR10; each Ri0 is independently amino, cyano, halogen, hydrogen, ORn, SRn, NR12R13, or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Rn is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Ri2 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; and each R13 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle. Certain such compounds are of the formula:
Figure imgf000019_0001
Others are of the formula:
Figure imgf000019_0002
wherein, for example, R3 is trifluoromethyl. Others are of the formula:
Figure imgf000019_0003
wherein, for example, R3 is hydrogen.
Referring to the various formulae above, some compounds are such that all of Z"ls Z"2, Z"3, and Z"4 are N. In others, only three of Z"ls Z"2, Z"3, and Z"4 are N. In others, only two of Z"i, Z"2, Z"3, and Z"4 are N. In others, only one of Z"ls Z"2, Z"3, and Z"4 is N. In others, none of Z"ls Z"2, Z"3, and Z"4 are N.
Some are of the formula:
Figure imgf000020_0001
the substituents of which are defined herein. Others are of the formula:
Figure imgf000020_0002
the substituents of which are defined herein. Others are of the formula:
Figure imgf000020_0003
the substituents of which are defined herein. Others are of the formula:
Figure imgf000020_0004
the substituents of which are defined herein.
Some compounds of the invention are of the formula:
Figure imgf000020_0005
wherein: each Ri4 is independently amino, halogen, hydrogen, C(O)RA, ORA, NRBRC, S(O2)RA, or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each RA is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each RB is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Rc is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle; and m is 1-4.
Referring to the various formulae disclosed herein, particular compounds include those wherein both A and E are optionally substituted phenyl and, for example, X is -O-, -C(RsR4)O-, or -OC(RsR4)- and, for example, R3 is hydrogen and R4 is trifluoromethyl and, for example, n is 1.
This invention encompasses stereomerically pure compounds and stereomerically enriched compositions of them. Stereoisomers may be asymmetrically synthesized or resolved using standard techniques such as chiral columns, chiral resolving agents, or enzymatic resolution. See, e.g., Jacques, J., et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S. H., et al, Tetrahedron 33:2725 (1977); Eliel, E. L., Stereochemistry of Carbon Compounds (McGraw Hill, NY, 1962); and Wilen, S. H., Tables of Resolving Agents and Optical Resolutions, p. 268 (EX. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972).
Particular compounds of the invention are potent TPHl inhibitors. Specific compounds have a TPHl IC50 of less than about 10, 5, 2.5, 1, 0.75, 0.5, 0.4, 0.3, 0.2, 0.1, or 0.05 μM.
Particular compounds are selective TPHl inhibitors. Specific compounds have a TPHl IC50 that is about 10, 25, 50, 100, 250, 500, or 1000 times less than their TPH2_IC50.
Particular compounds do not significantly inhibit human tyrosine hydroxylase (TH). For example, specific compounds have an IC50 for TH of greater than about 100, 250, 500 or 1000 μM.
Particular compounds do not significantly inhibit human phenylalanine hydroxylase (PAH). For example, specific compounds have an IC50 for PAH of greater than about 100, 250, 500 or 1000 μM.
Particular compounds of the invention do not significantly bind (e.g., inhibit with an IC50 of greater than about 10, 25, 50, 100, 250, 500, 750, or 1000 μM) to one or more of the following: angiotensin converting enzyme, erythropoietin (EPO) receptor, factor IX, factor XI, integrin (e.g., α4), isoxazoline or isoxazole fibrinogen receptor, metalloprotease, neutral endopeptidase (NEP), phosphatase (e.g., tyrosine phosphatase), phosphodiesterase (e.g., PDE-4), polymerase, PPARγ, TNF-α, vascular cell adhesion molecule- 1 (VCAM-I), or the vitronectin receptor. The ability of a compound to bind to (e.g., inhibit) any of these targets can be readily determined using methods known in the art, as described in references cited above. Specific compounds of the invention do not inhibit cell adhesion.
When administered to mammals (e.g., mice, rats, dogs, monkeys or humans), certain compounds of the invention do not readily cross the blood/brain barrier (e.g., less than about 5, 2.5, 2, 1.5, 1, 0.5, or 0.01 percent of compound in the blood passes into the brain). The ability or inability of a compound to cross the blood/brain barrier can be determined by methods known in the art. See, e.g. , Riant, P. et al. , Journal of Neurochemistry 51 :421 -425 (1988); Kastin, A.J., Akerstrom, V., J. Pharmacol. Exp. Therapeutics 294:633-636 (2000); W. A. Banks, W. A., et al., J. Pharmacol. Exp. Therapeutics 302:1062-1069 (2002).
5.3. Synthesis of Compounds
Compounds of the invention can be prepared by methods known in the art, and by methods described herein.
For example, with reference to formula I, compounds in which E is phenyl and D is optionally substituted pyrazine, pyridiazine, pyridine or phenyl can generally be prepared by the method shown in Scheme 1 :
( A VcHO + H2N-h D 4-Br
Figure imgf000022_0001
jN- -Br
Figure imgf000022_0002
SOCI2, Ethanol
Figure imgf000022_0003
Figure imgf000022_0004
Scheme 1
wherein, for example:
Figure imgf000023_0001
Compounds wherein X is -OCR3- can generally be prepared using the method shown in Scheme 2, wherein R3 is CF3 and D is pyrimidine:
Figure imgf000023_0002
Scheme 2
wherein, for example, A is optionally substituted phenyl, biphenyl or napthyl.
Compounds of the invention can also be prepared using the approach shown below in Scheme 3:
Figure imgf000024_0001
Figure imgf000024_0002
Scheme 3
wherein Pi is Ri or a protecting group; P2 is a protecting group; P3 is OR2 or a protecting group; X' is, for example, O or N; Yi and Y3 are halogen (e.g., Br, Cl) or an appropriate pseudohalide (e.g., triflate); and each R' is independently hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle, or are taken together with the oxygen atoms to which they are attached to provide a cyclic dioxaborolane (e.g., 4,4,5,5-tetramethyl- 1,3,2-dioxaborolane). The groups A, R1, R2, R3, R6 and m are defined elsewhere herein. The moieties Z"i, ZM2, ZM3, and ZM4 are also defined herein, although it is to be understood that with regard to the scheme shown above, one of them is attached to the phenyl ring. For example, Z"i and ZM4 may be independently CRio (which is defined herein), while ZM2 is N and Z"3 is a carbon atom bound to the adjacent phenyl ring. The individual reactions shown above can be performed using conditions known in the art. For example, palladium catalysts and conditions suitable for the Suzuki coupling of the boron and halogen-containing moieties are well known, and examples are provided below. In addition, types and appropriate uses of protecting groups are well known, as are methods of their removal and replacement with moieties such as, but not limited to, hydrogen (e.g., hydrolysis under acidic or basic conditions).
The A moiety can be bicyclic (e.g., optionally substituted biphenyl). In such cases, the starting material containing A can be prepared as shown below:
Figure imgf000025_0001
wherein Y2 is halogen or pseudohalogen, and each R is independently hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle, or are taken together with the oxygen atoms to which they are attached to provide a cyclic dioxaborolane (e.g., 4,4,5,5- tetramethyl-l,3,2-dioxaborolane).
Another approach to the preparation of compounds wherein D is optionally substituted pyrimidine or triazine is shown below in Scheme 4:
Figure imgf000026_0001
Scheme 4
wherein, for example, X is N, O or S, and FG is defined below: FG = B(OH)2 when E is optionally substituted Phenyl
Figure imgf000026_0002
Ester derivatives of these and other compounds of the invention can be readily prepared using methods such as that shown below in Scheme 5, wherein E is optionally substituted phenyl:
Figure imgf000027_0001
Scheme 5
An alternate approach to the preparation of triazine-based compounds is shown below in Scheme 6:
>C*NH2 +
Figure imgf000027_0002
dry n-BuOH/ tBuOK 3.5 eq. 16O0C, sealed tube, 2 days
Figure imgf000027_0003
Figure imgf000027_0004
Scheme 6 The cyclic moiety D can be any of a variety of structures, which are readily incorporated into compounds of the invention. For example, compounds wherein D is oxazole can be prepared as shown below in Scheme 7:
Figure imgf000028_0001
Scheme 7
Using methods known in the art, the synthetic approaches shown above are readily modified to obtain a wide range of compounds. For example, chiral chromatography and other techniques known in the art may be used to separate stereoisomers of the final product. See, e.g., Jacques, J., et ah, Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S. H., et al. Tetrahedron 33:2125 (1977); Eliel, E. L.,
Stereochemistry of Carbon Compounds (McGraw Hill, NY, 1962); and Wilen, S. H., Tables of Resolving Agents and Optical Resolutions, p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN, 1972). In addition, as shown in some of the schemes above, syntheses may utilize chiral starting materials to yield stereomerically enriched or pure products.
5.4. Methods of Use
This invention encompasses methods of affecting {e.g., slowing) gastrointestinal transit and gastric emptying, which comprise inhibiting peripheral tryptophan hydroxylase {e.g., TPHl) in patients in need thereof. Patients in need thereof include patients with diarrhea and patients susceptible to diarrhea {e.g., patients taking medications or undergoing therapies, such as chemotherapy, that can cause diarrhea). Preferred methods avoid measurably affecting serotonin levels in the central nervous system (CNS).
One embodiment encompasses a method of slowing gastrointestinal transit in a patient, which comprises administering to the patient a sufficient amount of a potent TPHl inhibitor. Another embodiment encompasses a method of slowing gastric emptying in a patient, which comprises administering to the patient a sufficient amount of a potent TPHl inhibitor.
The amount of active pharmaceutical ingredient (e.g., a potent TPHl inhibitor) sufficient to achieve the desired pharmacological effect can be readily determined by those skilled in the art. For example, a patient can be administered a low dose of a compound, and then increasingly larger doses over time until the desired effect is achieved.
Particular methods of the invention avoid adverse effects associated with alteration of CNS serotonin levels. Examples of such adverse effects include agitation, anxiety disorders, depression, and sleep disorders (e.g., insomnia and sleep disturbance).
5.5. Pharmaceutical Compositions
This invention encompasses pharmaceutical compositions comprising one or more compounds of the invention. Certain pharmaceutical compositions are single unit dosage forms suitable for oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, or rectal), parenteral (e.g., subcutaneous, intravenous, bolus injection, intramuscular, or intraarterial), or transdermal administration to a patient. Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g. , crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient. The formulation should suit the mode of administration. For example, the oral administration of a compound susceptible to degradation in the stomach may be achieved using an enteric coating. Similarly, a formulation may contain ingredients that facilitate delivery of the active ingredient(s) to the site of action. For example, compounds may be administered in liposomal formulations in order to protect them from degradative enzymes, facilitate transport in circulatory system, and effect their delivery across cell membranes.
Similarly, poorly soluble compounds may be incorporated into liquid dosage forms (and dosage forms suitable for reconstitution) with the aid of solubilizing agents, emulsifϊers and surfactants such as, but not limited to, cyclodextrins (e.g., α-cyclodextrin, β-cyclodextrin, Captisol®, and Encapsin™ (see, e.g., Davis and Brewster, Nat. Rev. Drug Disc. 3:1023-1034 (2004)), Labrasol®, Labrafil®, Labrafac®, cremafor, and non-aqueous solvents, such as, but not limited to, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, dimethyl sulfoxide (DMSO), biocompatible oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acid esters of sorbitan, and mixtures thereof (e.g., DMSOxornoil).
Poorly soluble compounds may also be incorporated into suspensions using other techniques known in the art. For example, nanoparticles of a compound may be suspended in a liquid to provide a nanosuspension (see, e.g., Rabinow, Nature Rev. Drug Disc. 3:785-796 (2004)). Nanoparticle forms of compounds described herein may be prepared by the methods described in U.S. Patent Publication Nos. 2004-0164194, 2004-0195413, 2004-0251332, 2005-0042177 Al, 2005-0031691 Al, and U.S. Patent Nos. 5,145,684, 5,510,118, 5,518,187, 5,534,270, 5,543,133, 5,662,883, 5,665,331, 5,718,388, 5,718,919, 5,834,025, 5,862,999, 6,431,478, 6,742,734, 6,745,962, the entireties of each of which are incorporated herein by reference. In one embodiment, the nanoparticle form comprises particles having an average particle size of less than about 2000 nm, less than about 1000 nm, or less than about 500 nm. The composition, shape, and type of a dosage form will typically vary depending with use. For example, a dosage form used in the acute treatment of a disease may contain larger amounts of one or more of the active ingredients it comprises than a dosage form used in the chronic treatment of the same disease. Similarly, a parenteral dosage form may contain smaller amounts of one or more of the active ingredients it comprises than an oral dosage form used to treat the same disease. How to account for such differences will be apparent to those skilled in the art. See, e.g. , Remington 's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990).
5.5.1. Oral Dosage Forms
Pharmaceutical compositions of the invention suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington 's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton PA (1990). Typical oral dosage forms are prepared by combining the active ingredient(s) in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms. If desired, tablets can be coated by standard aqueous or non-aqueous techniques. Such dosage forms can be prepared by conventional methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary. Disintegrants may be incorporated in solid dosage forms to facility rapid dissolution. Lubricants may also be incorporated to facilitate the manufacture of dosage forms (e.g. , tablets).
5.5.2. Parenteral Dosage Forms Parenteral dosage forms can be administered to patients by various routes including subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses patients' natural defenses against contaminants, parenteral dosage forms are specifically sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to those skilled in the art. Examples include: Water for Injection USP; aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as ethyl alcohol, polyethylene glycol, and polypropylene glycol; and nonaqueous vehicles such as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. 6. EXAMPLES
6.1. HPLC Characterization
In some of the following synthetic examples, high performance liquid chromatography (HPLC) retention times are provided. Unless otherwise noted, the various conditions used to obtain those retention times are described below:
Method A: YMC-PACK ODS-A 3.0x50mm; Solvent A = 90% water, 10% MeOH, 0.1% TFA; Solvent B = 90% MeOH, 10% water, 0.1% TFA; B% from 0 to 100% over 4 min.; flow rate = 2 ml/min; observation wavelength = 220 nm.
Method B: YMC-PACK ODS-A 3.0x50mm; Solvent A = 90% water, 10% MeOH, 0.1% TFA; Solvent B = 90% MeOH, 10% water, 0.1% TFA; %B from 10 to 100% over 4 min.; flow rate = 3 ml/min; observation wavelength = 220 nm.
Method C: YMC-PACK ODS-A 3.0x50mm; Solvent A = 90% water, 10% MeOH, 0.1% TFA; Solvent B = 90% MeOH, 10% water, 0.1% TFA; B% from 0 to 100% over 5 min.; flow rate = 2 ml/min. ; observation wavelength = 220 nm. Method D: Shim VP ODS 4.6x50 mm; Solvent A = 90% water, 10% MeOH, 0.1%
TFA; Solvent B = 90% MeOH, 10% water, 0.1% TFA; B% from 0 to 100% over 4 min.; flow rate = 3 ml/min.; observation wavelength = 220 nm.
Method E: Shim VP ODS 4.6x50 mm; Solvent A = 90% water, 10% MeOH, 0.1% TFA; Solvent B = 90% MeOH, 10% water, 0.1% TFA; B% from 0 to 100% over 4 min.; flow rate = 3 ml/min; observation wavelength = 254 nm.
Method F: YMC-PACK ODS-A 4.6x33mm; Solvent A = 90% water, 10% MeOH, 0.1% TFA; Solvent B = 90% MeOH, 10% water, 0.1% TFA; B% from 0 to 100% over 4 min.; flow rate = 3 ml/min.; observation wavelength = 220 nm.
Method G: YMC-PACK ODS-A 4.6x50mm; Solvent A = 90% water, 10% MeOH, 0.1 % TFA; Solvent B = 90% MeOH, 10% water, 0.1% TFA; B% from 0 to 100% over 2 min.; flow rate = 2.5 ml/min.; observation wavelength = 220 nm.
Method H: Cl 8 4.6x20mm; Solvent A = 90% water, 10% MeOH, 0.1% TFA; Solvent B = 90% MeOH, 10% water, 0.1% TFA; B% from 0 to 100% over 2 min. flow rate = 2ml/min.; observation wavelength = 220 nm. Method I: YMC PACK ODS-A 3.0 x 50 mm; Solvent A = 90% water, 10% MeOH,
0.1% TFA; Solvent B = 90% MeOH, 10% water, 0.1% TFA; B% from 10 to 100% over 4 min.; flow rate = 2ml/min.; observation wavelength = 220 nm. Method J: YMC Pack ODS-A 3.0x50mm; Solvent A = H2O, 0.1% TFA; Solvent B = MeOH, 0.1% TFA; %B from about 10 to about 90% over 4 min.; flow rate = 2ml/min.; observation wavelength = 220 nm.
Method K: Sunfire Cl 8 50 mm x 4.6 mm x 3.5 μm; Solvent A = IO mM NH4OAc in water; Solvent B = MeCN; B% from 10 to 95% over 2 min.; flow rate = 4.5 ml/min.; observation wavelength = 220 nm.
Method L: Sunfire C18 50 mm x 4.6 mm x 3.5 μm; Solvent A = IO mM NH4OAc; Solvent B = MeCN; B% from 2 to 20% over 0.8 min, then to 95% B over 2 min; flow rate = 4.5 ml/min.; observation wavelength = 220 nm. Method M: YMC-PACK ODS-A 4.6x33mm; Solvent A = 90% water, 10% MeOH,
0.1% TFA; Solvent B = 90% MeOH, 10% water, 0.1% TFA; B% from 0 to 100% over 5 min.; flow rate = 2.5 ml/min.; observation wavelength = 254 nm.
Method N: YMC-PACK ODS-A 3.0x50mm; Solvent A = H2O, 0.1% TFA; Solvent B = MeOH, 0.1% TFA; B% from 10 to 90% over 4 min.; flow rate = 2 ml/min.; observation wavelength = 220 and 254 nm.
Method O: YMC-PACK ODS-A 3.0x50mm; Solvent A = 90% water, 10% MeOH with 0.1% TFA; Solvent B = 90% MeOH, 10% water with 0.1% TFA; B% from 0 to 100% over 4 min.; flow , rate = 2 ml/min.; observation wavelength = 220 and 254 nm.
Method P: ShimPack VP ODS 4.6x50mm; Solvent A = 90% H2O, 10% MeOH, 1%TFA; Solvent B = 10% H2O, 90% MeOH, 1%TFA; B% from 0 to 100% over 2 min.; flow rate = 3.5 ml/min.; observation wavelength = 220 and 254 nm.
Method Q: Shim VP ODS 4.6x50 mm; Solvent A = H2O with 0.1 % TFA; Solvent B = MeOH with 0.1 % TFA; B% from 0 to 100% over 4 min.; flow rate = 3 ml/min.; observation wavelength = 254 nm. Method R: YMC Pack ODS-A 4.6 x 33 mm; Solvent A = H2O, 0.1% TFA; Solvent B
= MeOH with 0.1% TFA; B% from 10 to 90% over 3 min.; flow rate 2 ml/min.; observation wavelength 220 and 254 nm.
Method S: YMC-Pack ODS-A 3.0x50 mm; Solvent A = 90% H2O, 10% MeOH, 1% TFA; Solvent B = 10% H2O, 90% MeOH, 1%TFA; B% from 10 to 90% over 4 min.; flow rate = 2 ml/min. observation wavelength = 220 and 254 nm. 6.2. Synthesis of (S)-2-Amino-3-(4-(4-amino-6-((R)-l-(naphthalen-2- yl)ethylamino)-l,3i5-triazin-2-yl)phenyl)propanoic acid
Figure imgf000034_0001
A mixture of 2-amino-4,6-dichloro-[l,3,5]triazine (200mg, 1.21mmol), (R)-(+)-l-(2- naphthyl)ethylamine (207mg, 1.21mmol) and diisopropyl-ethylamine (3.63mmol) was dissolved in 150 ml of 1,4-dioxane. The solution was refluxed at 900C for 3 hours. After the completion of reaction (monitored by LCMS), solvent was removed and the reaction mixture was extracted with CH2Cl2 (100ml) and H2O (100ml). The organic layer was separated and washed with H2O (2x100ml), dried over Na2SO4, and concentrated in vacuo to give crude intermediate. The crude compound was dissolved in 5ml of MeCN and 5ml of H2O in a 20ml microwave reaction vial. To this solution were added L-/?-borono-phenylalanine (253mg, 1.21mmol), sodium carbonate (256mg, 2.42mmol) and catalytic amount of dichlorobis(triphenylphosphine)-palladium(II) (42.1mg, O.Oβmmol). The mixture was sealed and stirred in the microwave reactor at 1500C for 5 minutes, followed by the filtration through celite. The filtrate was concentrated and dissolved in MeOH and H2O (1 : 1) and purified by preparative HPLC using MeOH/H2O/TFA solvent system. The combined pure fractions were evaporated in vacuo and further dried on a lyophilizer to give 238mg of 2- amino-3-{4-[4-amino-6-(l-naphthalen-2-yl)-ethylamino)-[l,3,5]triazin-2-yl]-phenyl}- propionic acid (yield: 46%, LC: Column: YMC Pack ODS-A 3.0x50mm, %B=0~100%, Gradient time = 4min, Flow Rate = 2ml/min, wavelength=220, Solvent A= 90 : 10 water:MeOH w/ 0.1%TFA, Solvent B=90:10 MeOH:water w/0.1%TFA , RT = 2.785 min, MS: M+l = 429). NMR: 1H-NMR (400 MHz, CD3OD): δ 1.65 (d, 3H), 3.22-3.42 (m, 2H), 4.3 (m, IH), 5.45 (m, IH), 7.4(m, IH), 7.6(m 4H), 7.8(m, 4H), 8.2(m, 2H).
6.3. Alternative Synthesis of (S)-2-Amino-3-(4-(4-amino-6-((R)-l-(naphthalen- 2-yl)ethylamino)-l,3i5-triazin-2-yl)phenyl)propanoic acid
(R)-l-(l-(Napthalen-2-yl) ethyl) cyanoguanidine was prepared by forming a mixture of naphthalene amine (1 equivalent), sodium dicyanide (0.95 eq.) and followed by 5N HCl (1 eq.) in n-BuOH: H2O (1 : 1). The mixture was refluxed for 1 day in a sealed tube at 1600C, and progress of reaction was monitored by LCMS. After completion of reaction, solvent (n- BuOH) was removed under reduced pressure and IN HCl was added to adjust pH to 3-5 range. The aqueous solution was extracted with EtOAc (2x100) and combined organic phase was dried over Na2SO4. Solvent was removed in vacuo to give crude product. The compound was purified by ISCO column chromatography using as the solvent system EtOAc:hexane (7:3 and 1 : 1), to obtain white solid 48-71% yield for Ig to 22.5 gram scale. NMR: 1H-NMR (400 MHz, CD3OD): δ 1.5(d, 3H), 5.1(m, IH), 7.5 (m, 4H), 7.8(s, IH), 7.9 (m, 2H); LCMS: RT 1.69, M+l : 239, Yield: 71%.
The title compound was prepared from (R)-I -(I -(napthalen-2-yl) ethyl) cyanoguanidine according to the method shown in Scheme 6.
6.4. Synthesis of (S)-2-Amino-3-(4-(4-amino-6-((4'-methylbiphenyl-4- yl)methylamino)-l,3i5-triazin-2-yl)phenyl)propanoic acid
Figure imgf000035_0001
A mixture of 2-amino-4,6-dichloro-[l,3,5]triazine (lOOmg, 0.606mmol), 4'-methyl- biphenyl-4-yl-methylamine (142mg, 0.606mmol), and cesium carbonate (394mg, 1.21mmol) was dissolved in 1,4-dioxane (1.5ml) and H2O (1.5ml) in a 5ml microwave vial. The mixture was stirred in microwave reactor at 1000C for 15 minutes. Solvent was removed and the residue was dissolved in CH2Cl2 (20ml) and washed with H2O (2x20ml), dried over Na2SO4 and then removed in vacuo. The crude intermediate was then dissolved in 1.5ml of MeCN and 1.5ml of H2O in a 5ml microwave vial. To this solution were added L-/?-borono- phenylalanine (126mg, 0.606mmol), sodium carbonate (128mg, 1.21mmol) and catalytic amount of dichlorobis(triphenylphosphine)-palladium(II) (21. lmg, 0.03mmol). The mixture was sealed and stirred in the microwave reactor at 1500C for 5 minutes followed by the filtration through celite. The filtrate was concentrated and dissolved in MeOH and H2O (1 :1) and purified by preparative HPLC using MeOH/H2O/TFA solvent system. The combined pure fractions were evaporated in vacuo and further dried on a lyophilizer to give 21.6 mg of 2-amino-3-(4-{4-amino-6-[(4'-methyl-biphenyl-4-ylmethyl)-amino]-[l,3,5]triazin-2-yl}- phenyl)-propionic acid (LC: Column: YMC Pack ODS-A 3.0x50mm, %B=0~100%, Gradient time = 4min, Flow Rate = 2ml/min, wavelength=220, Solvent A= 90:10 water :MeOH w/ 0.1%TFA, Solvent B=90:10 MeOH:water w/0.1%TFA , RT = 3.096 min, MS: M+l = 455). 1H NMR(400 MHz, CD3OD) δ 2.33 (s, 3H), 3.24-3.44 (m, 2H), 4.38 (m, IH), 7.02 (d, 2H), 7.42 (m, 2H), 7.50-7.60 (m, 6H), 8.22 (m, 2H).
6.5. Synthesis of ( S)-2-Amino-3-(4-(4-morpholino-6-(naphthalen-2- ylmethylamino)-l,3i5-triazin-2-yl)phenyl)propanoic acid
Figure imgf000036_0001
A mixture of 2,4-dichloro-6-morpholin-4-yl-[l,3,5]triazine (121mg, 0.516mmol), C- naphthalen-2-yl-methylamine hydrochloride (lOOmg, 0.516mmol), cesium carbonate (336mg, 1.03mmol) was dissolved in 1,4-Dioxane (1.5ml) and H2O (1.5ml) in a 5ml microwave vial. The mixture was stirred in microwave reactor at 1800C for 600 seconds. Solvent was removed, and the residue was dissolved in CH2Cl2 (10ml) and washed with H2O (2xl0ml), dried over Na2SO4 and then in vacuo. The residue was purified by preparative HPLC to give 20mg intermediate (yield 11%, M+l=356). The intermediate was then dissolved in 0.5ml of MeCN and 0.5ml of H2O in a 2ml microwave vial. To this solution were added L-/?-borono- phenylalanine (11.7mg, 0.0562mmol), sodium carbonate (11.9mg, 0.112mmol) and a catalytic amount of dichlorobis(triphenylphosphine)-palladium(II) (2.0mg, 5%). The mixture was sealed and stirred in the microwave reactor at 1500C for 5 minutes followed by the filtration through celite. The filtrate was concentrated and dissolved in MeOH and H2O (1 :1) and purified by preparative HPLC using MeOH/H2O/TFA solvent system. The combined pure fractions were evaporated in vacuo and further dried on lyophilizer to give 17mg of 2- amino-3-(4-{4-morpholin-4-yl-6-[(naphthalene-2-ylmethyl)-amino]-[l,3,5]triazin-2-yl}- phenyl)-propionic acid (yield: 63%, LC: Method B, RT = 3.108 min, MS: M+l = 486). 6.6. Synthesis of (2S)-2-Amino-3-(4-(2-amino-6-(2,2,2-trifluoro-l-(2- ftrifluoromethyl)phenyl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
Figure imgf000037_0001
Tetrabutylammonium fluoride (0.1 ml; 1.0 M solution in tetrahydrofuran) was added to a solution of 2-trifluoromethyl-benzaldehyde (1.74g, lOmmol) and trifluoromethyltrimethylsilane (TMSCF3) (1.8ml, 12 mmol) in 10 ml THF at 00C. The formed mixture was warmed up to room temperature and stirred for 4 hours. The reaction mixture was then treated with 12 ml of IN HCl and stirred overnight. The product was extracted with ethyl acetate (3x20ml). The organic layer was separated and dried over sodium sulfate. The organic solvent was evaporated to give 2.2g of l-(2- trifluoromethylphenyl)-2,2,2-trifluoro-ethanol, yield 90%.
NaH (80mg, 60%, 3.0mmol) was added to a solution of l-(2-trifluoromethylphenyl)- 2,2,2-trifluoro-ethanol (244mg, lmmol) in 10 ml of anhydrous THF. The mixture was stirred for 20 minutes, 2-amino-4, 6-dichloro-pyrimidine (164mg, lmmol) was added and then the reaction mixture was heated at 700C for 1 hour. After cooling, 5ml water was added and ethyl acetate (20ml) was used to extract the product. The organic layer was dried over sodium sulfate. The solvent was removed by rotovap to give 267 mg of 4-chloro-6-[2, 2, 2- trifluoro- 1 -(2-trifluoromethylphenyl)-ethoxy]-pyrimidin-2-ylamine, yield 71%.
In a microwave vial, 4-chloro-2-amino-6-[l-(2-trifluoromethylphenyl)-2, 2, 2- trifluoro-ethoxy]-pyrimidine (33mg, 0. lmmol), 4-borono-L-phenylalanine(31mg, 0.15mmol) and 1 ml of acetonitrile, 0.7ml of water. 0.3 ml of IN aqueous sodium carbonate was added to above solution followed by 5 mole percent of dichlorobis(triphenylphosphine)- palladium(II). The reaction vessel was sealed and heated at 1500C for 5 minutes with microwave irradiation. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol, and then was purified by Prep- LC to give 5.6 mg of 2-amino-3-(4- {2-amino-6-[2,2,2-trifluoro- 1 -(2-triifluoromethylphenyl)- ethoxy]- pyrimidin-4-yl}-phenyl)-propionic acid. 1H NMR (400MHz, CD3OD) δ 7.96 (m, 3H), 7.80 (d, J=8.06 Hz, IH), 7.74 (t, J=7.91 Hz IH), 7.63(t, J=8.06 Hz, IH), 7.41 (d, J=8.3Hz, 2 H), 7.21 (m, IH), 6.69 (s, IH), 3.87 (m, 1 H), 3.34 (m, 1 H), 3.08 (m, IH). 6.7. Synthesis of (2S)-2-Amino-3-(4-(2-amino-6-(2,2,2-trifluoro-l-p- tolylethoxy)pyrimidin-4-yl)phenyl)propanoic acid
Figure imgf000038_0001
Tetrabutylammonium fluoride (0.1 ml; 1.0 M solution in tetrahydrofuran) was added to a solution of 4-methyl-benzaldehyde (1.2g, lOmmol) and TMSCF3 (1.8ml, 12 mmol) in 10 ml THF at 00C. The formed mixture was warmed up to room temperature and stirred for 4 hours. The reaction mixture was then treated with 12 ml of IN HCl and stirred overnight. The product was extracted with ethyl acetate (3x20ml). The organic layer was separated and dried over sodium sulfate. The organic solvent was evaporated to give 1.6g of l-(4- methylphenyl)-2,2,2-trifluoro-ethanol, yield 86%.
NaH (80mg, 60%, 3.0mmol) was added to a solution of l-(4-methylphenyl)-2,2,2- trifluoro-ethanol (190mg, lmmol) in 10 ml of anhydrous THF. The mixture was stirred for 20 minutes, 2-amino-4,6-dichloro-pyrimidine (164mg, lmmol) was added and then the reaction mixture was heated at 700C for 1 hour. After cooling, 5ml water was added and ethyl acetate (20ml) was used to extract the product. The organic layer was dried over sodium sulfate. The solvent was removed by rotovap to give 209 mg of 4-chloro-6-[l-(4- methylphenyl)-2,2,2-trifluoro-ethoxy]-pyrimidin-2-ylamine, yield 66%.
A microwave vial was charged with 4-chloro-2-amino-6-[l-(4-methylphenyl)-2,2,2- trifluoro-ethoxy]-pyrimidine (33mg, 0. lmmol), 4-borono-L-phenylalanine (31mg, 0.15mmol) and 1 ml of acetonitrile, 0.7ml of water. Aqueous sodium carbonate (0.3 ml, IN) was added to above solution followed by 5 mol percent of dichlorobis(triphenylphosphine)- palladium(II). The reaction vessel was sealed and heated to 1500C for 5 minutes with microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol, was then purified by Prep-LC to give 14.6mg of 2-amino-3- (4- {2-amino-6-[2,2,2-trifluoro- 1 -(4-methylphenyl)- ethoxy]-pyrimidin-4-yl} -phenyl- propionic acid. 1H NMR (300MHz, CD3OD) δ 7.94 (d, J=8.20 Hz, 2H), 7.47 (d, J=7.24 Hz, 4 H), 7.27 (d, J=8.01 Hz, 2H) 6.80 (s, IH), 6.75 (m, IH), 4.30 (t, 1 H), 3.21-3.44 (m, 2 H), 2.37 (s, 3H). 6.8. Synthesis of αS)-2-Amino-3-(4-(2-amino-6-q-cvclohexyl-2,2,2- trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
Figure imgf000039_0001
Cyclohexanecarbaldehyde (0.9 g, 5mmol) was dissolved in 10ml aqueous 1,4- dioxane, to which 200mg (10 mmol) sodium borohydride was added. The reaction was run overnight at room temperature. After completion of the reaction, 5ml 10% HCl solution was added and the product was extracted with ethyl acetate. The organic layer was separated and dried over sodium sulfate. The organic solvent was evaporated to give 0.8g of 1-cyclohexyl- 2,2,2-trifluoro-ethanol, yield 88%. NaH (80mg, 60%, 3.Ommol) was added to the solution of 1 -cyclohexyl-2,2,2- trifluoro-ethanol (182mg, lmmol) in 10 ml of anhydrous THF, the mixture was stirred for 20 minutes, 2-amino-4,6-dichloro-pyrimidine (164mg, lmmol) was added and then the reaction mixture was heated at 700C for 1 hour. After cooling, 5ml water was added and ethyl acetate (20ml) was used to extract the product. The organic layer was dried over sodium sulfate. The solvent was removed by rotovap to give 202 mg of 4-chloro-6-[l-cyclohexyl-2,2,2- trifluoro-ethoxy]-pyrimidin-2-ylamine, yield 65%.
In a microwave vial, 4-chloro-2-amino-6-[l-cyclohexane-2,2,2-trifluoro-ethoxy]- pyrimidine (33mg, 0. lmmol), 4-borono-L-phenylalanine (31mg, 0.15mmol) and 1 ml of acetonitrile, 0.7ml of water, 0.3 ml of aqueous sodium carbonate (IM) was added to above solution followed by 5 mol percent of dichlorobis(triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 1500C for 5 minutes with a microwave. After cooling, the reaction mixture was evaporated to dryness, the residue was dissolved in 2.5 ml of methanol, and the product was purified by Prep-LC to give 4.9 mg 2-amino-3-{4-[2- amino-6-(l-cyclohexyl-2, 2, 2-trifluoro-ethoxy]-pyrimidin-4-yl}-phenyl)-propionic acid. 1H NMR (300MHz, CD3Cl) δ 7.95 (d, J=8.39Hz, 2 H), 7.49 (d, J=8.39Hz, 2 H), 6.72 (s, IH), 5.90(m, IH), 4.33 (t, 1 H), 3.21-3.44 (m, 2 H), 1.73-2.00 (m, 6H), 1.23-1.39 (m, 5H). 6.9. Synthesis of ( S)-2-Amino-3-(4-( 6-(2-fluorophenoxy)pyrimidin-4- vDphenvDpropanoic acid
Figure imgf000040_0001
NaH (80mg, 60%, 3.0mmol) was added to a solution of 2-fluorophenol (112 mg, lmmol) in 10 ml of anhydrous THF, the mixture was stirred for 20 minutes, 4,6-dichloro- pyrimidine (149mg, lmmol) was added and then the reaction mixture was heated at 700C for 1 hour. After cooling, 5ml water was added and ethyl acetate (20ml) was used to extract the product. The organic layer was dried over sodium sulfate. The solvent was removed by rotovap to give 146 mg of 4-chloro-6-(2-fluorophenoxy)-pyrimidine, yield 65%.
A microwave vial (2ml) was charged with 4-chloro-6-[2-fluorophenoxy]-pyrimidine, (33mg, 0. lmmol), 4-borono-L-phenylalanine(31mg, 0.15mmol) and 1 ml of actonitrile, 0.7ml of water, 0.3 ml of aqueous sodium carbonate (IM) was added to above solution followed by 5 mol % of dichlorobis(triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 1500C for 5 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness, the residue was dissolved in 2.5 ml of methanol, and the product was purified with Prep-LC to give 4.9 mg 2-amino-3-{4-[2-amino-6-(l-2-fluorophenyl-2,2,2- trifluoro-ethoxy]-pyrimidin-4-yl}-phenyl)-propionic acid. 1H NMR (400MHz, CD3OD) δ 8.74 (s, IH), 8.17 (d, J=8.06 Hz, 2H), 7.63 (s, IH), 7.50(d, J=8.06 Hz, 2H), 7.30 (m, 5H), 4.33 (m, 1 H), 3.34 (m, 1 H).
6.10. Synthesis of (2S)-2-Amino-3-(4-(4-(3-(4-chlorophenyl)piperidin-l-yl)- l,3i5-triazin-2-yl)phenyl)propanoic acid
Figure imgf000040_0002
3-(4-Chlorophenyl)piperidine (232mg, lmmol) was added to a solution of 2,4- dichlorotriazine (149.97mg, lmmol), and 300mg diisopropylethyl amine in 10ml THF at 00C. The formed mixture was warmed up to room temperature and stirred for 1 hour. The product was extracted with ethyl acetate (3x20ml). The organic layer was separated and dried over sodium sulfate. The organic solvent was evaporated to give 328mg of 2-chloro-4-[3-(4- chlorophenyl)-piperidin-l-yl]-[l, 3, 5] triazine.
A microwave vial was charged with 2-chloro-4-[3-(4-chlorophenyl)-piperidin-l-yl]- [1, 3, 5]triazine (62mg, 0.2mmol), 4-borono-L-phenylalanine(60mg, 0.3mmol), 1 ml of acetonitrile, and 0.7ml of water. Aqueous sodium carbonate (0.6 ml; IM) was added to the solution, followed by 5 mol percent dichlorobis(triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 1500C for 5 minutes with microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol, was then purified by Prep-LC to give 5.1mg of 2-amino-3-(4-{4-[3-(4- chlorophenyl)-piperidin-l-yl]-[l,3,5]triazin-2-yl}-phenyl)-propionic acid. 1H NMR (400MHz, CD3Cl) δ 8.58 (d, 2H), 8.05 (d, 2H), 7.47 (m, 5 H), 4.96 (m, 1 H), 4.23(m, 2H), 3.21-3.44 (m, 4 H), 2.37 (m, 5H).
6.11. Synthesis of (2S)-2-Amino-3-(4-(4-amino-6-(2,2,2-trifluoro-l- phenylethoxy)-l,3i5-triazin-2-yl)phenyl)propanoic acid
Figure imgf000041_0001
NaH (80mg, 60%, 3.0mmol) was added to a solution of 2,2,2-trifluoro-l-phenyl- ethanol (176mg, lmmol) in 10 ml of anhydrous 1,4- dioxane. The mixture was stirred for 20 minutes, then added to a solution of 2-amino-4,6-dichloro-triazine (164mg, lmmol) in 30ml of 1 ,4-dioxane at 00C for 1 hour. The reaction mixture was then warmed to room temperature. After completion of the reaction, 5ml of water was added and ethyl acetate (20ml) was used to extract the product. The organic layer was dried over sodium sulfate. The solvent was removed by rotovap to give 198 mg of 4-chloro-6-[2,2,2-trifluoro-l-phenyl- ethoxy]-[l,3,5]triazine-2-ylamine, yield 65%.
A microwave vial was charged with 4-chloro-6-[2,2,2-trifluoro-l-phenyl-ethoxy]- [l,3,5]triazine-2-ylamine (33mg, 0. lmmol), 4-borono-L-phenylalanine(31mg, 0.15mmol), 1ml of actonitrile, and 0.7ml of water. Aqueous sodium carbonate (0.3 ml, IM) was added to above solution followed by 5 mol percent dichlorobis(triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 1500C for 5 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol, was then purified with Prep-LC to give 3.2mg 2-amino-3-{4-[4-amino-6-(l- phenyl-2,2,2-trifluoro-ethoxy]-[l,3,5]triazin-2yl]-phenyl)-propionic acid. 1H NMR (300MHz, CD3OD) δ 8.22 (d, J=8.20 Hz, 2H), 7.52 (m, 2 H), 7.33 (m, 5H) 6.62 (m, IH), 4.19 (t, I H), 3.1-3.33 (m, 2 H).
6.12. Synthesis of (S)-2-Amino-3-(5-(4-amino-6-((R)-l-(naphthalen-2- yl)ethylamino)-l,3i5-triazin-2-yl)pyridin-2-yl)propanoic acid
Figure imgf000042_0001
A microwave vial was charged with 6-chloro-N-[l-naphthalen-2yl-ethyl]- [l,3,5]triazine-2,4-diamine (30mg, O.lmmol), 2-boc protected-amino-3-{5-[4,4,5,5,- tetramethyl-[l,3,2]dioxaborolan-2-yl)-pyridin2-yl-]-propionic acid (50mg, 0.15mmol) 1 ml of acetonitrile, and 0.7ml of water. Aqueous sodium carbonate (0.3 ml; IN) was added to the solution, followed by 5 mol percent dichlorobis(triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 1500C for 5 mintues by microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol, and was then purified by Prep-LC to give 7 mg of boc protected 2-amino-3-{5-[4- amino-6-( 1 -naphthalen-2-yl-ethylamino)- [ 1 ,3 ,5 ]triazin-2-yl] -pyridin-2-yl} proionic acid.
The above product (7.0 mg) was dissolved in 0.1ml of 10%TFA/DCM solution for 2 hours to provide 1.1 mg of 2-amino-3-{3-[4-amino-6-(l-naphthalen-2-yl-ethylamino)- [l,3,5]triazin-2-yl]-pyridin-2-yl}proionic acid. 1H NMR (300MHz, CD3Cl) δ 9.35 (d, 1 H), 8.57 (m, 1 H), 7.85 (m, 4H), 7.45 (m, 4 H), 6.94 (s, IH), 5.58(m, IH), 4.72 (m, 2H), 4.44 (m, I H), 1.42 (d, 3H). 6.13. Synthesis of (S)-2-Amino-3-(3-(4-amino-6-((R)-l-(naphthalen-2- yl)ethylamino)-l,3i5-triazin-2-yl)-lH-pyrazol-l-yl)propanoic acid
Figure imgf000043_0001
6-Chloro-N-[l-naphthalen-2yl-ethyl]-[l,3,5]triazine-2,4-diamine (30mg, O.lmmol), 2- boc-protected amino-3-{3-[4,4,5,5,-tetramethyl-[l,3,2]dioxaborolan-2-yl)-pyrazol-l-yl]- propionic acid (50mg, 0.15mmol), 1 ml of acetonitrile, and 0.7ml of water. Aqueous sodium carbonate (0.3 ml and IN) was added to a microwave vial, followed by 5 mol percent of dichlorobis(triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 1500C for 5 minutes with microwave. After cooling, the reaction mixture was evaporated to dryness, the residue was dissolved in 2.5 ml of methanol, and then was purified with Prep- LC to give 6.8 mg of boc protected 2-amino-3-{3-[4-amino-6-(l-naphthalen-2-yl- ethylamino)[ 1 ,3,5]triazin-2-yl]-pyrazol- 1 -yl}proionic acid.
The above product (6.8mg) was stirred in 0.1ml 10%TFA/DCM solution for 2 hours to provide 3mg of 2-amino-3-{3-[4-amino-6-(l-naphthalen-2-yl-ethylamino)-[l,3,5]triazin-2- yl]-pyrazol-l-yl}proionic acid. 1H NMR (300MHz, CD3Cl) δ 8.52 (s, 1 H), 8.21 (s, 1 H), 7.74 (m, 4 H), 7.36 (m, 3H), 5.35(m, IH), 4.72 (m, 2H), 4.44 (m, 1 H), 1.55 (d, 3H).
6.14. Synthesis of (S)-2-Amino-3-(4'-(3-(cvclopentyloxy)-4- methoxybenzylamino)biphenyl-4-yl)propanoic acid
Figure imgf000043_0002
Sodium triacetoxyl-borohydride (470mg, 2.21mmol) was added to a solution of 4- bromo-phenylamine (252mg, 1.47mmol) and 3-cyclopentyloxy-4-methoxy-benzaldehyde (324mg, 1.47mmol) in 10 ml of 1 ,2-dicloroethtane (DCE), 0.5 ml of HOAc was added. The mixture was stirred overnight at room temperature, followed by addition of 15 ml of DCE. The organic phase was washed with water and dried over sodium sulfate. The solvent was removed by rotovap to give 656 mg of crude (4-bromo-phenyl)-(3-cyclopentyloxy-4- methoxy-benzyl)-amine. It was used for next step without further purification.
An Emrys process vial (2-5ml) for microwave was charged with (4-bromo-phenyl)- (3-cyclopentyloxy-4-methoxy-benzyl)-amine (84mg, 0.22mmol), 4-borono-L- phenylalanine(46mg, 0.22mmol) and 2 ml of acetonitrile. Aqueous sodium carbonate (2 ml, IM) was added to above solution, followed by 5 mol percent of dichlorobis- (triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 15O0C for 5 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol and purified with Prep-LC to give 5 mg of 2- amino-3-[4'-(3-cyclophentyloxy-4-methoxy-benzylamino)-biphenyl-4-yl]-propionic acid, yield 5%. 1H-NMR (400 MHz, DMSO-d6): δ 1.46 (m, 2H), 1.62 (m, 4H), 3.01(m, 2H), 3.64 (s, 3H), 4.14 (s, 3H), 4.66(m, IH), 6.61(d, 2H), 6.81(s, 2H), 6.88(s, IH), 7.18(d, 2H), 7.31(d, 2H), 7.44(d, 2H), 7.60(m, IH), 8.19(s, 3H).
6.15. Synthesis of ( S)-2-Amino-3-(4-( 6-(3-(cyclopentyloxy)-4- methoxybenzylamino)pyrimidin-4-yl)phenyl)propanoic acid
Figure imgf000044_0001
Sodium tiracetoxyl-borohydride (985mg, 4.65mmol) was added to a solution of 6- chloro-pyrimidin-4-ylamine (200mg, 1.55mmol) and 3-cyclopentyloxy-4-methoxy- benzaldehyde (682mg, 3.1mmol) in 25 ml of DCE. 1 ml of HOAc was added, and the mixture was stirred overnight at 5O0C, followed by addition of 25 ml of DCE. The organic phase was washed with water, and the product was purified with column (silica gel, hexane:EtOAc 5:1) to give 64 mg of (6-chloro-pyrimidin-4-yl)-(3-cyclopentyloxy-4- methoxy-benzyl)-amine, yield 12%. An Emrys process vial (2-5ml) for microwave was charged with (6-chloro-pyrimidin- 4-yl)-(3-cyclopentyloxy-4-methoxy-benzyl)-amine (64mg, 0.19mmol), 4-borono-L- phenylalanine (40mg, 0.19mmol) and 2 ml of acetonitrile. Aqueous sodium carbonate (2 ml, IM) was added to above solution followed by 5 mol percent of dichlorobis- (triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 15O0C for 5 minutes with microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol and purified with Prep-LC to give 5.3 mg of 2-amino-3-{4-[6-(3-cyclopentyloxy-4-methoxy-benzylamino)-pyrimidin-4-yl]-phenyl}- propionic acid, yield 6%. 1H-NMR (400 MHz, DMSO-d6): δ 1.46 (m, 2H), 1.62 (m, 4H), 3.01(m, 2H), 3.08(m, 2H), 3.65(s, 3H), 4.20(m, IH), 4.46(d, 2H), 4.68(m, IH), 6.82(t, 2H), 6.87(d, 2H), 7.40(d, 2H), 7.90(s, 2H), 8.25(s, 2H), 8.6(s, IH).
6.16. Synthesis of (S)-2-Amino-3-(4-(6-(3-(cvclopentyloxy)-4- methoxybenzylamino)pyrazin-2-yl)phenyl)propanoic acid
Figure imgf000045_0001
Sodium triacetoxyl-borohydride (1315mg, 6.2mmol) was added to a solution of 6- chloro-pyrazin-2-yl-amine (400mg, 3.10mmol) and 3-cyclopentyloxy-4-methoxy- benzaldehyde (818mg, 3.7mmol) in 50 ml of DCE, 1 ml of HOAc was added and the mixture was stirred overnight at 5O0C, followed by addition of another 50 ml of DCE. The organic phase was washed with water, and the product was purified with column (silica gel, hexane:EtOAc 6:1) to give 50 mg of (6-chloro-pyrazin-2-yl)-(3-cyclopentyloxy-4-methoxy- benzyl)-amine, yield 10%.
An Emrys process vial (2-5ml) for microwave was charged with (6-chloro-pyrazin-2- yl)-(3-cyclopentyloxy-4-methoxy-benzyl)-amine (50mg, 0.15mmol), 4-borono-L- phenylalanine (31mg, 0.15mmol) and 2 ml of acetonitrile. Aqueous sodium carbonate (2 ml, IM) was added to the solution followed by 5 mol percent of dichlorobis(triphenylphosphine)- palladium(II). The reaction vessel was sealed and heated to 15O0C for 5 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol, and the product was purified with Prep- LC to give 5.5 mg of 2-amino-3- {4-[6-(3-cyclopentyloxy-4-methoxy-benzylamino)-pyrazin-2-yl]-phenyl} - propionic acid, yield 6%. 1H-NMR (400 MHz, DMSO-d6): δ 1.46 (m, 2H), 1.62 (m, 4H), 3.01(m, 2H), 3.08(m, 2H), 3.65(s, 3H), 4.0(m, IH), 4.45(d, 2H), 4.65(m, IH), 6.90(s, 2H), 6.95(s, IH), 7.32(d, 2H), 7.60(t, IH), 7.90(s, IH), 7.95(d, 2H), 8.25(s, IH).
6.17. Synthesis of (S)-2-Amino-3-(4-(5-((4'-methylbiphenyl-2- yl)methylamino)pyrazin-2-yl)phenyl)propanoic acid
Figure imgf000046_0001
Sodium tiracetoxyl borohydride (215mg, 1.02mmol) was added to the solution of 4'- methyl-biphenyl-2-carbaldehyde and 5-bromo-pyrazin-2-ylamine in 5 ml of DCE, 0.1 ml of HOAc was added and the mixture was stirred overnight at room temperature, followed by addition of 5 ml of DCE. The organic phase was washed with water, and purified with column (silica gel, hexane:EtOAc 6:1) to give 100 mg of (5-bromo-pyrazin-2-yl)-(4'-methyl- biphenyl-2-ylmethyl)-amine, yield 55%. An Emrys process vial (2-5ml) for microwave was charged with (5-bromo-pyrazin-2- yl)-(4'-methyl-biphenyl-2-ylmethyl)-amine (25mg, 0.071mmol), 4-borono-L-phenylalanine (22mg, 0.1 lmmol) and 1 ml of acetonitrile. Aqueous sodium carbonate (1 ml, IM) was added to the solution followed by 5 mol percent dichlorobis(triphenylphosphine)- palladium(II). The reaction vessel was sealed and heated to 15O0C for 5 mintues by microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol, and the product was purified with Prep-LC to give 19 mg of 2-amino-3-{4-[6-(3-cyclopentyloxy-4-methoxy-benzylamino)-pyrazin-2-yl]-phenyl}- propionic acid, yield 63%. 1H-NMR (400 MHz, CD3OD): δ 2.22(s, 3H), 3.09(m, IH), 3.25(m, IH), 4.18(t, IH), 4.40(s, 2H), 7.07(d, 2H), 7.14(m, 3H), 7.24(m, 4H), 7.36(m,lH), 7.72(d, 2H), 7.84(s, IH), 8.20(d, IH). 6.18. Synthesis of αS)-2-Amino-3-(4-(6-(2,2,2-trifluoro-l-phenylethoxy)- Pyrimidin-4-yl)phenyl)propanoic acid
Figure imgf000047_0001
NaH (60%, 120mg, 3.0mmol) was added to a solution of 2,2,2-trifluoro-l-phenyl- ethanol (350mg, 2.03mmol) in 5 ml of THF. The mixture was stirred for 20 minutes at room temperature. 4,6-Dichloro-pyrimidine (300mg, 2.03mmol) was added and then the reaction mixture was heated at 7O0C for 1 hour. After cooling, the THF was evaporated to provide a residue, which was dissolved in 15 ml of EtOAc, and then washed with water, and dried over sodium sulfate. The solvent was removed by rotovap to give 550 mg of 4-chloro-6-(2,2,2- trifluoro- 1 -phenyl-ethoxy)-pyrimidine, yield 95 % .
An Emrys process vial (2-5ml) for microwave was charged with 4-chloro-6-(2,2,2- trifluoro-l-phenyl-ethoxy)-pyrimidine (30mg, O.l lmmol), 4-borono-L-phenylalanine (32mg, O.lβmmol), 1 ml of acetonitrile and 0.6 ml of water. Aqueous sodium carbonate (0.42 ml, IM) was added to above solution followed by 10 mol percent of POPd2 (dihydrogen di-μ- chlorodichlorobis(di-tert-butylphosphinito-κP) dipalladate. The reaction vessel was sealed and heated to 12O0C for 30 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol, and the product was purified with Prep-LC to give 4.8mg of 2-amino-3-{4-[6-(2,2,2-trifluoro-lphenyl-ethoxy)- pyrimidin-4-yl]-phenyl}-propionic acid, yield 11%. 1H-NMR (400 MHz, CD3OD): δ 3.20(m, IH), 3.40(m, IH), 4.25(t, IH), 6.82(dd, IH), 7.43(m, 5H), 7.57(s, IH), 7.60(m, 2H),8.10(d, 2H),8.75(s, IH).
6.19. Synthesis of αS)-2-Amino-3-(4-(6-q-(3,4-difluorophenyl)-2,2,2- trifluoroethoxy)pyrimidin-4-yl)phenvl)propanoic acid
Figure imgf000047_0002
Tetrabutylammonium fluoride (TBAF: 0.1 ml, IM) in THF was added to a solution of 3,4-difluro-benzaldehyde (1.42g, lOmmol) and (trifluromethyl)trimethylsilane (1.7Og, 12mmol) in 10 ml THF at O0C. The mixture was warmed up to room temperature and stirred for 4 hours. The reaction mixture was treated with 12 ml of IM HCl and stirred overnight. The product was extracted with dicloromethane (3x20ml), the organic layer was combined and passed through a pad of silica gel. The organic solvent was evaporated to give 1.9g of 1- (3,4-difiuoro-phenyl)-2,2,2-trifluoro-ethanol, yield 90%.
NaH (80mg, 60%, 3.0mmol) was added to a solution of l-(3,4-Difluoro-phenyl)- 2,2,2-trifluoro-ethanol (212mg, lmmol) in 5 ml of THF, the mixture was stirred for 20 minutes at room temperature. 4,6-Dichloro-pyrimidine (149mg, lmmol) was added and then the reaction mixture was heated at 7O0C for 1 hour. After cooling, THF was evaporated. The residue was dissolved in 15 ml of EtOAc, and then washed with water, dried over sodium sulfate. The solvent was removed by rotovap to give 230 mg of 4-chloro-6-[l-(3,4-difluoro- phenyl)-2,2,2-trifluoro-ethoxy]-pyrimidine, yield 70%. An Emrys process vial (2-5ml) for microwave was charged with 4-chloro-6-[l-(3,4- difluoro-phenyl)-2,2,2-trifluoro-ethoxy]-pyrimidine (33mg, 0. lmmol), 4-borono-L- phenylalanine (31mg, 0.15mmol), 1 ml of acetonitrile and 0.7ml of water. Aqueous sodium carbonate (0.3 ml, IM) was added to above solution followed by 5 mol % of dichlorobis(triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 15O0C for 5 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol, then purified with Prep-LC to give 10 mg of 2-amino-3-(4-{6-[l-(3,4-difluoro-phenyl)-2,2,2-trifluoro-ethoxy]-pyridin-4-yl}- phenyl)-propionic acid, yield 21%. 1H-NMR (400 MHz, CD3OD): δ 3.1 l(m, IH), 3.27(m, IH), 4.19(dd, IH), 6.78(q, IH), 7.26(m, 2H), 7.35(d, 3H),7.49(m, 2H), 8.02(d, 2H),8.66(s, IH).
6.20. Synthesis of (S)-2-Amino-3-(4-(5-(3-(cvclopentyloxy)-4- methoxybenzylamino)-pyrazin-2-yl)phenyl)propanoic acid
Figure imgf000049_0001
A mixture of 3-cyclopentyloxy-4-methoxy-benzaldehyde (417 mg, 1.895 mmol), 2- amino-5-bromopyrazine (300 mg, 1.724 mmol), sodium triacetoxyborohydride (1.5 eq) and glacial acetic acid (3 eq) in dichloromethane (10 ml) was stirred at room temperature overnight. Then the reaction mixture was diluted with ethyl acetate, and washed with water. The oraganic layer was dried over MgSO4 and filtered. The filtrate was concentrated to give the crude product, which was purified by ISCO (SiO2 flash column chromatography) (Hexane/ethyl acetate = 100/0 to 3/2) to give about 400 mg of 6-bromo-pyrazin-2-yl)-(3- cyclopentyloxy-4-methoxy-benzyl)-amine. Yield: 61%.
To a 5 ml microwave vial, the above 6-bromo-pyrazin-2-yl)-(3-cyclopentyloxy-4- methoxy-benzyl)-amine (50 mg, 0.132 mmol), 4-borono-L-phenylalanine (30 mg , 0.144 mmol), Na2CO3 (31 mg, 0.288 mmol), acetonitrile (2 ml) and water (2 ml). Dichlorobis (triphenylphosphine)-palladium (5 mg, 0.007 mmol) was added. The vial was capped and stirred at 1500C for 5 minutes under microwave radiation. The reaction mixture was cooled, filtered through a syringe filter and then separated by a reverse phase preparative-HPLC using YMC-Pack ODS 100x30 mm ID column (MeOH/H2O/TFA solvent system). The pure fractions were concentrated in vacuum. The product was then suspended in 5 ml of water, frozen and lyophilized to give the title compound as a trifluoro salt (12 mg, 20 %). 1H NMR (CD3OD) δ 8.41 (s, IH), 7.99 (s, IH), 7.83 (d, J = 9.0 Hz, 2H), 7.37 (d, J = 6.0 Hz, 2H), 6.90- 6.95 (m, 3H), 4.78 (m, IH), 4.50 (s, 2H), 4.22-4.26 (m, IH), 3.79 (s, 3H), 3.12-3.39 (m, 2H), 1.80-1.81 (m, 6H), 1.60 (m, 2H). M+l = 463. 6.21. Synthesis of (S)-2-Amino-3-(4-(5-((3-(cyclopentyloxy)-4-methoxybenzyr)- (methyl)amino)pyrazin-2-yl)phenyl)propanoic acid
Figure imgf000050_0001
To a solution of (6-bromo-pyrazin-2-yl)-(3-cyclopentyloxy-4-methoxy-benzyl)-amine (70 mg, 0.185 mmol) in acetonitrile (10 ml) was added formaldehyde (18.5 mmol) and sodium cyanoborohydride (17 mg, 0.278 mmol). Then, concentrated aqueous HCl was added dropwise until the pH « 2. The mixture was stirred for about 6 hours at room temperature. It was then diluted with ethyl acetate, washed with water (3 X 5 ml), dried over MgSO4. The solvent was removed by vacuum to give 70 mg of crude product 5-(bromo-pyrazin-2-yl)-(3- cyclopentyloxy-4-methoxy-benzyl)-methyl-amine (95 % crude yield), which was used in the next step without further purification.
The 5 -(bromo-pyrazin-2-yl)-(3 -cy clopentyloxy-4-methoxy-benzyl)-methyl-amine (37 mg, 0.094 mmol) was subjected to a Suzuki coupling reaction as described above to afford 6 mg of the title compound. Yield: 13%. 1H NMR (CD3OD) δ 8.59 (s, IH), 8.12 (s, IH), 7.85 (d, 2H), 7.39 (d, 2H), 6.81-6.91 (m, 3H), 4.72 (m, IH), 4.30 (m, IH), 3.79 (s, 3H), 3.20-3.40 (m, 2H), 3.18 (s, 3H), 3.79 (s, 3H), 1.80 (m, 6H), 1.58 (m, 2H). M+l = 477.
6.22. Synthesis of (S)-2-Amino-3-(4-(5-(q,3-dimethyl-lH-pyrazol-4- yl)methylamino)pyrazin-2-yl)phenyl)propanoic acid
Figure imgf000050_0002
A mixture of 1,3 -dimethyl- lH-pyrazole-4-carbaldehyde (142 mg, 1.145 mmol), 2- amino-5-bromopyrazine (200 mg, 1.149mmol), borane trimethylamine complex (126 mg, 1.73mmol) and glacial acetic acid (137 mg, 2.29 mmol) in anhydrous methonol (3 ml) was stirred at room temperature overnight. The reaction mixture was then diluted with ethyl acetate, washed with water, dried over MgSO4 and filtered. The filtrate was concentrated to give 300 mg of (5-bromo-pyrazin-2-yl)-(l,3-dimethyl-lH-pyrazol-4-ylmethyl)amine as crude product, which was used for next step reaction without further purification. Crude yield: 93%.
The (5 -bromo-pyrazin-2-yl)-( 1,3 -dimethyl- lH-pyrazol-4-ylmethyl)amine (40 mg, 0.142 mmol) was used in the Suzuki coupling reaction described above to afford 19 mg of of the title compound. Yield: 36.5%. 1H NMR (CD3OD) δ 8.48 (s, IH), 8.05 (s, IH), 7.87 (d, 2H), 7.39 (d, 2H), 6.10 (s, IH), 4.81 (s, 2H), 4.30 (m, IH), 3.83 (s, 3H), 3.11-3.38 (m, 2H), 2.10 (s, 3H). M+l = 367.
6.23. Synthesis of (S)-2-Amino-3-(4-(4-amino-6-((S)-l-(naphthalen-2- yl)ethylamino)-l,3i5-triazin-2-yloxy)phenyl)propanoic acid
Figure imgf000051_0001
To a 250 ml flask, R-(+)-l-(2-naphthyl)ethylamine (400 mg, 2.424 mmol), 2-amino- 4,6-dichloro triazine (373mg, 2.181 mmol), anhydrous 1,4-dioxane (40 ml), and N,N- diisopropylethylamine (1 ml, 5.732 mmol) were added and heated to mild reflux for about 4 hours. The reaction was monitored carefully in order to avoid the formation of the disubstituted product. (It was observed that the longer the reaction, the more disubstituted product is formed). After 4 hours, the reaction mixture was cooled and the solvent was removed under reduced pressure. Water was added to the residue, and the solution was sonicated for 2-3 minutes. The solvent was then filtered, washed with water and dried to give 540 mg (83 % crude yield) of the mono-chloride, 6-chloro-N-(l-naphthalen-2yl-ethyl)- [l,3,5]triazine-2,2-diamine, which was used for the next step reaction without further purification. A mixture of 6-chloro-N-(l-naphthalen-2yl-ethyl)-[l,3,5]triazine-2,2-diamine (90 mg, 0.300 mmol), 2-tert-butoxycarbonylamino-3-(4-hydroxy-phenyl)-propionic acid tert-butyl ester (102 mg, 0.303 mmol) and potassium carbonate (82 mg, 0.594 mmol) in isopropanol (8 ml) was refluxed over night. The solvent was removed under reduced pressure and the residue was suspended in ethyl acetate. The solid was filtered and washed with ethyl acetate. The filtrate was concentrated and then redissolved in a mixture of methanol/water(90:10) and purified by a preparative-LC using a Sunfϊre C18 OBD 100x30mm ID column (MeOH/H2θ/TFA solvent system). The pure fractions were combined and concentrated to give 50 mg of pure product, 3-{4-[4-amino-6-(l-naphthalen-2-yl-ethylamino)-[l,3,5]triazin- 2yloxy] -phenyl} 2-tert-butoxycarbonvlamino-propionic acid tert-butyi ester, (28% yield).
The above product (50 mg, 0.083mmol) was dissolved in trifluoro acetic acid/dichloromethane (8ml/2ml) and stirred at room temperature over night. The solvent was removed under reduced pressure. The residue was then redissolved in a mixture of methanol/water(90:10) and purified by a preparative-LC using a Sunfϊre Cl 8 OBD 100x30mm ID column (MeOH/H2O/TFA solvent system). The pure fractions were combined and concentrated under reduced pressure to afford about 4 ml, which was frozen and lyophilized to give 4 mg of the title compound as a TFA salt (11 % yield). 1H NMR (CD3OD) δ 7.37-7.81 (m, 8H), 7.19 (m, 2H), 6.98 (m, IH), 5.37 (m, IH), 4.19 (m, IH), 3.17-3.38 (m, 2H), 1.56 (m, 3H). M+l = 445.
6.24. Synthesis of (S)-2-Amino-3-(4-(4-amino-6-((RM-(biphenyl-2-yl)-2,2,2- trifluoroethoxv)-l,3,5-triazin-2-vl)phenvl)propanoic acid
Figure imgf000052_0001
A mixture of l-biphenyl-2-yl-2,2,2-trifluoro-ethanone (300 mg, 1.2 mmol), borane tetrahydrofuran complexes (1.2 ml, IM in THF, 1.2 mmol) and S-2-methyl-CBS- oxazaborolidine (0.24 ml, IM in toluene, 0.24 mmol) in THF (8ml) was stirred at room temperature over night. Several drops of concentrated HCl were added and the mixture was stirred for 30 minutes. The product was purified by SiO2 chromatography (hexane/ethyl acetate = 100/0 to 3/1) to give 290 mg of l-biphenyl-2-yl-2,2,2-trifluoro-ethanol (96% yield). The above alcohol (290 mg, 1.151 mmol) was dissolved in anhydrous THF (10 ml). Sodium hydride (55 mg, 1.375 mmol) was added all at once, and the mixture was stirred at room temperature for 30 minutes. The solution was then transferred into a flask that contained a suspension of 2-amino-4,6-dichloro-triazine (190 mg, 1.152 mmol) in THF (20 ml). The mixture was stirred at room temperature overnight. Water was added and the mixture was then diluted with ethyl acetate. The organic layer was washed with water, dried over MgSO4 and then concentrated to give 400 mg of crude product 2-amino-4-(l-biphenyl- 2-yl-2,2,2-trifluoro-ethoxy-6-chloro-triazine.
The 2-amino-4-(l-biphenyl-2-yl-2,2,2-trifluoro-ethoxy-6-chloro-triazine (40 mg, 0.105 mmol) was subjected to the same Suzuki coupling reaction as described above to afford 5 mg of the title compound. Yield: 9.4%. 1H NMR (CD3OD) 5 8.18 (d, 2H), 7.86 (m, IH), 7.40-7.52 (m, 9H), 7.32 (m, IH), 7.07 (m, IH), 4.32 (m, IH), 3.22-3.41 (m, 2H). M+l = 510.
6.25. Synthesis of (2SV2- Amino-3-(4-(4-amino-6-(l-(6,8-difluoronaphthalen-2- yl)ethylamino)-l,3i5-triazin-2-yl)phenyl)propanoic acid
Figure imgf000053_0001
In a three-neck flask, copper iodine (CuI) (299 mg, 1.515 mmol) and lithium chloride (LiCl) (145 mg, 3.452 mmol) were added under nitrogen to anhydrous THF (60 ml). The mixture was stirred at room temperature until a pale yellow solution was obtained. After cooling to 00C, methyl vinyl ketone and chlorotrimethylsilane were added, and the mixture was stirred until an orange color was observed (~20 min). After cooling to about -400C, a solution of 3,5-difluorophenylmagnesium bromide (27.65 ml, 13.8mmol) in THF (0.5M) was slowly added. The reaction mixture was stirred at about -400C for 0.5 hours, then the cold bath was removed and the temperature was allowed to rise slowly to room temperature. The solvent was evaporated and the residue was extracted with hexane (4x20 ml). The collected extractions were washed with cold 10% aqueous NaHCO3 and dried over Na2SO4. The solvent was evaporated at reduced pressure to afford 3,5-difluorophenyl-l- trimethylsilyloxyalkene (2.03g, 7.929 mmol, 57% crude yield), which was used in the successive reaction without further purification. Powered calcium carbonate (3.806g, 38.06 mmol) and ethyl vinyl ether (2.184g, 30.329 mmol) were added to a solution of eerie ammonium nitrate (10.43Og, 19.033 mmol) in methanol (40 ml) under nitrogen atmosphere. To the resulting suspension was added a solution of above made 3,5-difluorophenyl-l-trimethylsilyloxyalkene (2.03g, 7.929 mmol) in ethyl vinyl (6 ml, 4.518g, 62.75 mmol) dropwise under vigorous stirring, and the mixture was stirred at room temperature overnight. The solid was filtered through a celite layer, and the filtrate was concentrated to one-fourth of its initial volume. The resulting thick mixture was slowly poured, under vigorous stirring, into 1 : lv/v diethyl ether- 10% aqueous NaHCO3. The precipitate was filtered off, the ethereal solution was separated, and the solvent was evaporated at reduced pressure to give clear liquid. The solution of resulting liquid (a mixture of acyclic and cyclic acetates) in methanol (4ml) was added dropwise to a suspension of dichlorodicyanobenzoquinone (1.77g, 7.797mmol) in 80% aqueous sulfuric acid at 00C. After the addition was complete, the ice bath was removed and stirring was continued for 30 minutes. The mixture was poured into ice water; and the resulting brown precipitate was filtered and dissolved in acetone. Silica gel was added to make a plug, and the crude product was purified by chromatography (hexane/ethyl acetate = 100/0 to 3/1) to give 760 mg of 1- (5,7-difluoro-naphthalen-2-yl)-ethanone (48% in two-step yield) as a light yellow solid.
The above ketone (760mg, 3.689mmol) was dissolved in methanol (40 ml). Then, ammonium acetate (2.841g, 36.896 mmol), sodium cyanoborohydride (232 mg, 3.389mmol) and molecular sieves (3A, 7.6 g) were added. The mixture was stirred at room temperature for two days. The solid was filtered and the filtrate was concentrated. The residue was dissolved in water and concentrated aqueous HCl was added dropwise until the pH « 2. The mixture was then extracted with ethyl acetate to remove the unfinished ketone and other byproducts. The water layer was basified to pH « 10 with aqueous sodium hydroxide (IM), and was extracted with dichloromethane and the organic layers were combined, dried over magnesium sulfate and concentrated to afford 290 mg of l-(5,7-difluoro-naphthalen-2-yl)- ethylamine (38% yield).
The fresh made amine (290mg, 1.401mmol) was added directly to a suspension of 2- amino-4,6-dichloro triazine (277mg, 1.678 mmol) in anhydrous 1,4-dioxane (60 ml), and followed by addition of N,N-diisopropylethylamine (1 ml, 5.732 mmol). The mixture was heated to mild reflux for about 3 hours. The reaction mixture was then cooled, and the solvent was removed under reduced pressure. To the residue was added water and the mixture was sonicated for 2-3 minutes. The resulting solid was filtered and washed with water and dried to give 395 mg (60 % crude yield) of 6-chloro-N-[l-(6,8-difluoro- naphthalen-2-yl-ethyl]-[l,3,5]triazine-2,4-diamine, which was used for the next step reaction directly without further purification.
The above made mono-chloride (48 mg, 0.144 mmol) was subjected to the same Suzuki coupling reaction as described above to afford 12 mg of the title product. Yield: 17.9%. 1H NMR (CD3OD) δ 8.14-8.22 (m, 2H), 8.05 (m, IH), 7.92 (m, IH), 7.63 (m, IH), 7.32-7.51 (m, 3H), 7.11 (m, IH), 5.48 (m, IH), 4.13 (m, IH), 3.13-3.41 (m, 2H), 1.66 (d, 3H). M+l = 465.
6.26. Synthesis of (2S)-2-Amino-3-(4-(4-amino-6-(2,2,2-trifluoro-l-(3'- methylbiphenyl-2-yl)ethoxy)-l,3i5-triazin-2-yl)phenyl)propanoic acid
Figure imgf000055_0001
To a mixture of 3 '-methyl- l-biphenyl-2-carbaldehyde (500mg, 2.551mmol) and trifluoromethyl trimethylsilane (435mg, 3.061mmol) in THF (3ml) was added tetrabutyl ammonium fluoride (13mg, 0.05 mmol) at 00C. The temperature was allowed to warm to room temperature. The mixture was stirred for 5 hours at room temperature, then diluted with ethyl acetate, washed with water and brine and dried by MgSO4. The solvent was removed under reduced pressure to give 660 mg (97% crude yield) of 2,2,2-trifluoro-l-(3'- methyl-biphenyl-2-yl)-ethanol as crude product, which was used for next step without further purification. The above-made alcohol (660 mg, 2.481 mmol) was dissolved in anhydrous 1,4- dioxane (10 ml). Sodium hydride (119 mg, 60% in mineral oil, 2.975 mmol) was added all at once and the mixture was stirred at room temperature for 30 minutes. The solution was transferred into a flask containing a suspension of 2-amino-4,6-dichloro-triazine (491 mg, 2.976 mmol) in 1,4-dioxane (70 ml). The mixture was stirred at room temperature for 6 hours. The solvent was removed, and the residue was suspended in ethyl acetate, which was washed with water, dried over MgSO4 and then concentrated to give 790 mg of crude product, which contained about 57% of the desired product 2-amino-4-( l-(3'-methyl- biphenyl-2-yl-2,2,2-trifluoro-ethoxy-6-chloro-triazine and about 43% byproduct (the bisubstituted product). The crude product was used without further purification.
The 2-amino-4-( 1 -(3 '-methyl-biphenyl-2-yl-2,2,2-trifluoro-ethoxy-6-chloro-triazine (98 mg, 57% purity, 0.142 mmol) was used to run the same Suzuki coupling reaction as described above to afford 9 mg of the title compound. Yield: 12.0%. 1H NMR (CD3OD) δ 8.09 (m, 2H), 7.85 (m, IH), 7.50 (m, 2H), 7.28-7.43 (m, 5H), 7.17-7.26 (m, 2H), 7.18 (m, IH), 3.85 (m, IH), 3.08-3.44 (m, 2H), 2.33 (s, 3H). M+l = 524.
6.27. Synthesis of fS)-2-Amino-3-f4-f5-f3,4-dimethoxyphenylcarbamoyl)- Pyrazin-2-yl)phenyl)propanoic acid
Figure imgf000056_0001
To a mixture of 3,4-dimethoxy phenylamine (0.306 g, 2 mmol) and triethylamine (0.557 ml, 4 mmol) in dichloromethane (20 ml) was added 5-chloro-pyrazine-2-carbonyl chloride (0.354 g, 2 mmol) at 0-50C. The mixture was allowed to stir at room temperature for 3 hours. The mixture was diluted with methylene chloride (20 ml), washed with saturated NaHCO3 (20 ml), brine (20 ml), dried (anhyd. Na2SO4) and concentrated to get 0.42 g of crude 5-chloro-pyrazine-2 carboxylic acid (3,4-dimethoxy-phenyl)-amide, which was directly used in the next reaction.
5-Chloro-pyrazine-2 carboxylic acid (3,4-dimethoxy-phenyl)-amide (0.18 g, 0.61 mmol), L-p-borono phenylalanine (0.146 g, 0.70 mmol), CH3CN (2.5 ml), H2O (2.5 ml), Na2CO3 (0.129 g, 1.22 mmol) were combined in a microwave vial. The mixture was sealed and kept at 1500C for 5 minutes. The mixture was filtered and concentrated. The residue was dissolved in methano I/water (1 :1) and purified by preparative HPLC, using MeOH/H2O/TFA as solvent system to afford 2-amino-3- {4-[5-(3,4-dimethoxy- phenylcarbomyl)-pyrazin-2yl]-phenyl} -propionic acid as a TFA salt (HPLC: Method A, Retention time = 2.846 min, LCMS M+l 423). 1H NMR (400 MHz, DMSO-d6) δ 3.10-3.30 (m, 2H), 3.72 (d, 6H), 4.05 (m, IH), 7.42-7.62 (m, 4H), 8.22 (m, 3H), 9.30 (m, 2H) . 6.28. Synthesis of (S)-2- Amino-3-(4-(2-amino-6-(4-q-(trifluoromethyl)phenyl)- piperidin-l-yl)pyrimidin-4-yl)phenyl)propanoic acid
Figure imgf000057_0001
2-Amino 4,6-dichloro pyrimidine (0.164 g, 1 mmol), 4-(2- trifluoromethyl-phenyl)- piperidine hydrochloride (0.266 g, 1 mmol), and cesium carbonate (0.684 g, 2.1mmol) were dissolved in a mixture of 1 ,4-dioxane (5 ml) and H2O (5 ml) in a 20 ml microwave vial. The mixture was stirred at 2100C for 20 minutes in a microwave reactor. Solvent was removed and the residue was dissolved in 5 % methanol in CH2Cl2 (20 ml), dried over Na2SO4 and concentrated to get the crude intermediate, 4-chloro-6-[4-(2-trifluoromethyl-phenyl)- piperidin-l-yl]-pyrimidin-2-ylamine (0.42 g) which was directly used in the following step.
The crude intermediate (0.42 g), L-p-borono-phenylalanine (0.209 g, 1 mmol), sodium carbonate (0.210 g, 2 mmol), and dichlorobis (triphenylphosphine)-palladium(II) (35 mg, 0.05 mmol) were dissolved in a mixture of MeCN (2.5 ml) and H2O (2.5 ml) in a 10 ml microwave vial. The vial was sealed and stirred in a microwave reactor at 1500C for 6 minutes. The mixture was filtered, and the filtrate was concentrated. The residue was dissolved in MeOH and H2O (1 : 1) and purified by preparative HPLC using MeOH/H2O/TFA as the solvent system to afford 2-amino-3-(4-{4-(2-trifluoromethyl-phenyl)-piperidine-l-yl]- pyrimidin-4yl}-phenyl)-propionic acid as a TFA salt. HPLC: Method A, Retention time = 3.203 min. LCMS M+l 486. 1H NMR (400 MHz, CD3OD) δ 1.80-2.20 (m, 5H), 3.0-3.16 (m,2H), 3.22-3.42 (m, 2H), 4.22(t, IH), 4.42-4.54 (m, IH), 5.22-5.34 (m, IH), 6.80(s, IH), 7.40(t, IH), 7.50-7.60(m, 4H), 7.68(d, IH), 7.82(d, 2H). 6.29. Synthesis of (S)-2-Amino-3-(4-(2-amino-6-((RM-(naphthalen-2- yl)ethylamino)pyrimidin-4-yl)phenyl)propanoic acid
Figure imgf000058_0001
2-Amino 4,6-dichloro pyrimidine (0.164 g, 1 mmol), (R)-(+)-l-(2-naphthyl)- ethylamine (0.171 g, 1 mmol), and cesium carbonate (0.358 g, 1.1 mmol) were dissolved in a mixture of 1 ,4-dioxane (4 ml) and H2O (4 ml) in a 20 ml microwave vial. The vial was sealed and stirred at 2100C for 20 minutes in a microwave reactor. Solvent was removed and the residue was dissolved in CH2Cl2 (50 ml), washed with water (20 ml), brine (20 ml), dried (Na2SO4) and concentrated to afford the crude intermediate, 6-chloro-N-4-(naphthalene-2yl- ethyl)-pyrimidine-2,4-diamine (0.270 g) which was directly used in the following step. The crude intermediate (0.27 g), L-p-borono-phenylalanine (0.210 g, 1 mmol), sodium carbonate (0.210 g, 2 mmol), and dichlorobis(triphenylphosphine)-palladium(II) (25 mg, 0.036 mmol) were dissolved in a mixture of MeCN (2.5 ml) and H2O (2.5 ml) in a microwave vial. The vial was sealed and stirred in the microwave reactor at 1500C for 6 minutes. The mixture was filtered and the filtrate was concentrated. The residue was dissolved in MeOH and H2O (1 : 1) and purified by preparative HPLC using MeOH/H2O/TFA as the solvent system to afford 2 amino-3-{4-[2-amino-6-(l-naphthalen-2yl-ethylamino)- pyrimidin-4-yl]-phenyl}-propionic acid as a TFA salt. HPLC: Method A, Retention time = 3.276 min. LCMS M+l 428. 1U NMR (400 MHz, CD3OD) δ 1.68 (d, 3H), 3.22-3.40 (m, 2H), 4.30(t, IH), 5.60 (q, IH), 6.42(s, IH), 7.42-7.54(m, 5H), 7.72(m, 2H), 7.82-7.84(m, 4H).
6.30. Synthesis of (S)-2-Amino-3-(4-(2-amino-6-(methyl((R)-l-(naphthalen-2- yl)ethyl)amino)pyrimidin-4-yl)phenyl)propanoic acid
Figure imgf000059_0001
2-Amino 4,6-dichloro pyrimidine (0.327 g, 2 mmol), methyl-(l-naphthalen-2yl- ethyl)-amine (0.360 g, 2 mmol), and cesium carbonate (0.717 g, 2.2 mmol) were dissolved in a mixture of 1 ,4-dioxane (7.5 ml) and H2O (7.5 ml) in a 20 ml microwave vial. The vial was sealed and stirred at 2100C for 20 minutes in a microwave reactor. Solvent was removed and the residue was dissolved in CH2Cl2 (50 ml), washed with water (20 ml), brine (20 ml) dried (Na2SO4) and concentrated to get the crude intermediate, 6-chloro-N-4-methyl-N-4-(l- napthalen-2-yl-ethyl)-pyrimidine-2,4-diamine (0.600 g), which was directly used in the following step.
The crude intermediate (0.30 g), L-p-borono-phenylalanine (0.210 g, 1 mmol), sodium carbonate (0.210 g, 2 mmol), and dichlorobis(triphenylphosphine)-palladium(II) (25 mg, 0.036 mmol) were dissolved in a mixture of MeCN (2.5 ml) and H2O (2.5 ml) in a microwave vial. The vial was sealed and stirred in the microwave reactor at 1500C for 6 minutes. The mixture was filtered and the filtrate was concentrated. The residue was dissolved in MeOH and H2O (1 : 1) and purified by preparative HPLC using MeOH/H2O/TFA as the solvent system to afford 2-amino-3-(4-{2-amino-6-[methyl-(l-naphthalen-2yl- ethyl)amino]-pyrimidin-4yl}-phenyl)-propionic acid as a TFA salt (HPLC: Method C, Retention time = 2.945 min, LCMS M+l 442) 1H NMR (400 MHz, CD3OD) δ 1.70 (m, 3H), 2.92(s, 3H), 3.22-3.42(m, 2H), 4.28(m, IH), 6.60(s, IH), 6.72(m, IH), 7.40-7.92 (m, HH). 6.31. Synthesis of (S)-2-Amino-3-(4-(2-amino-6-((S)-2,2,2-trifluoro-l-(6- methoxynaphthalen-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
Figure imgf000060_0001
2-Amino 4,6-dichloro pyrimidine (0.096 g, 0.6 mmol), 2,2,2-trifluoro-l-(6-methoxy- naphthalen-2-yl)-ethanol (0.140 g, 0.55 mmol), and NaH (96 mg, 0.60 mmol) were added to anhydrous dioxane (20 ml) under a nitrogen atmosphere. The reaction was stirred at 800C for 12 hours, cooled to room temperature, and quenched with water (0.2 ml). The reaction mixture was concentrated, and the residue dissolved in CH2Cl2 (50 ml), washed with water (20 ml), brine (20 ml) dried (Na2SO4) and concentrated to afford the crude intermediate, 4- chloro-6-[2,2,2-trifluoro- 1 -(6-methoxy-naphthalene-2-yl)-ethoxy]-pyrimidin-2-ylamine (0.22g) which was directly used in the following step.
The crude intermediate (0.22 g), L-p-borono-phenylalanine (0.126 g, 0.6 mmol), sodium carbonate (0.126 g, 1.2 mmol), and dichlorobis(triphenylphosphine)-palladium(II) (15 mg, 0.021 mmol) were dissolved in a mixture of MeCN (2.0 ml) and H2O (2.0 ml) in a microwave vial. The vial was sealed and stirred in the microwave reactor at 1500C for 6 minutes. The mixture was filtered and the filtrate was concentrated. The residue was dissolved in MeOH and H2O (1 : 1) and purified by preparative HPLC using MeOH/H2O/TFA as the solvent system to afford 2-amino-3-(4-{2-amino-6-[2,2,2-trifluoro-l-(6-methoxy- naphthalen-2-yl)-ethoxy]-pyrimidin-4-yl]-phenyl)-propionic acid as a TFA salt (HPLC: Method C, Retention time = 3.190 min. LCMS M+l 513. 1H NMR (400 MHz, CD3OD) δ 3.22-3.42(m, 2H), 3.86(s, 3H), 4.32(1H), 6.88 (m, IH), 6.92(1H), 7.20(dd, IH), 7.26(s, IH), 7.50(d, 2H), 7.63(d, IH), 7.80-7.90(m, 4H), 8.05(s, IH). 6.32. Synthesis of (^)-2-Amino-3-(4-(5-(biphenyl-4-ylmethylamino)pyrazin-2- vDphenvDpropanoic acid
Figure imgf000061_0001
4-Phenylbenzaldehyde (0.3 g, 1.65 mmol) and 2-amino-5-bromopyrazine (0.24 g, 1.37 mmol) were treated with Na(OAc)3BH (0.44 g, 2.06 mmol) in dichloroethane (7.0 mis) and acetic acid (0.25 mis) for 18 hours at room temperature. The mixture was diluted with dichloromethane, washed with 1.0 N NaOH, washed with brine, dried over MgSO4, and concentrated. Chromatography (SiO2, EtOAc : Hex, 1 :1) gave 0.18 g of N-(biphenyl-4- ylmethyl)-5-bromopyrazin-2-amine.
N-(biphenyl-4-ylmethyl)-5-bromopyrazin-2-amine (60 mg, 0.176 mmol), L-p- boronophenylalanine (37 mg, 0.176 mmol), palladiumtriphenylphosphine dichloride (3.6 mg, 0.0052 mmol), Na2CO3 (37 mg, 0.353 mmol), acetonitrile (1.25 mis) and water (1.25 mis) were heated in a microwave reactor at 1500C for 5 minutes. The mixture was concentrated, dissolved in 1.0 N HCl, washed twice with ether, concentrated and purified by preprative HPLC to give 41 mgs of the title compound. M+l = 425; 1U NMR (CD3OD) δ 8.42 (s, IH), 8.05 (s, IH), 7.92 (d, 2H), 7.58 (d, 4H), 7.40 (m, 7H), 4.60 (s, 2H), 4.25 (m, IH), 3.40 (m, IH), 3.20 (m ,1H).
6.33. Synthesis of (S)-2-Amino-3-(4-(5-(naphthalen-2-ylmethylamino)pyrazin-2- yl)phenyl)propanoic acid
Figure imgf000061_0002
2-Napthaldehyde (0.6 g, 3.84 mmol) and 2-amino-5-bromopyrazine (0.56 g, 3.201 mmol) were treated with Na(OAc)3BH (1.02 g, 4.802 mmol) in dichloroethane (15.0 mis) and acetic acid (0.5 mis) for 18 hours at room temperature. The mixture was diluted with dichloromethane, washed with 1.0 N NaOH, washed with brine, dried over MgSO4, and concentrated. Chromatography (SiO2, EtOAc : Hex, 1 :1) gave 0.49 g 5-bromo-N- (naphthalen-2-ylmethyl)pyrazin-2-amine.
5-Bromo-N-(naphthalen-2-ylmethyl)pyrazin-2-amine (0.2 g, 0.637 mmol), L-p- boronophenylalanine (0.13 g, 0.637 mmol), palladiumtriphenylphosphine dichloride (13 mg, 0.019 mmol), Na2CO3 (0.13 g, 1.27 mmol), acetonitrile (5 mis) and water (5 mis) were heated in a microwave reactor at 1500C for 5 minutes. The mixture was concentrated, dissolved in 1.0 N HCl, washed twice with ether, concentrated, dissolved in methanol, filtered and concentrated to yield 0.12 g of the captioned compound. M+l = 399; 1H NMR (CD3OD) δ 8.51 (s, IH), 8.37 (s, IH), 7.90 (m, 6H), 7.50 (m, 5H), 4.85 (s, 2H), 4.30 (t, IH), 3.38 (m, IH), 3.22 (m, IH).
6.34. Synthesis of (S)-2-(Tert-butoxycarbonylamino)-3-(4-(5-(naphthalen-2- ylmethylamino)pyrazin-2-yl)phenyl)propanoic acid
Figure imgf000062_0001
(S)-2-Amino-3-(4-(5-(naphthalen-2-ylmethylamino)pyrazin-2-yl)phenyl)propanoic acid (0.15 g, 0.345 mmol) was treated with triethylamine (87 mg, 0.862 mmol), and boc- anhydride (84 mg, 0.379) in dioxane (3 ml) and H2O (3 ml) at 00C. The mixture was warmed to room temperature and stirred overnight. The mixture was concentrated, and partitioned between EtOAc and H2O. The aqueous phase was acidified to pH = 1 with 1.0 N HCl and extracted with EtOAc. The organics were combined, washed with brine, dried over MgSO4, and concentrated to yield 48 mg of the captioned compound.
6.35. Synthesis of ( S)-2-Morpholinoethyl 2-amino-3-(4-( 5-(naphthalen-2- ylmethylamino)pyrazin-2-yl)phenvl)propanoate
Figure imgf000062_0002
(S)-2-(Tert-butoxycarbonylamino)-3-(4-(5-(naphthalen-2-ylmethylamino)pyrazin-2- yl)phenyl)propanoic acid (48 mg, 0.090 mmol), 4-(2-hydroxyethyl)morpholine (12 mg, 0.090 mmol), triethylamine (18 mg, 0.180 mmol), and benzotriazole-l-yloxytris(dimethylamino)- phosphonium hexaflurophosphate (BOP, 18 mg, 0.090 mmol), in dichloromethane (3.0 ml) were stirred at room temperature for 5 hours. Additional triethylamine (18 mg, 0.180 mmol) and BOP (18 mg, 0.090 mmol) were added, and the mixture was stirred overnight. The mixture was concentrated and purified via prep HPLC to give 2 mg of the captioned compound.
6.36. Synthesis of (2S)-2-Amino-3-(4-(2-amino-6-(2,2,2-trifluoro-l-(3'- fluorobiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
Figure imgf000063_0001
To 4'-bromo-2,2,2-trifiuoroacetophenone (5.0 g, 19.76 mmol) in THF (50 mis) at O0C was added NaBH4 (1.5 g, 39.52 mmol). The mixture was warmed to room temperature and stirred for 1 hour. The reaction was complete by TLC (CH2Cl2). The mixture was quenched with H2O, rotary evaporated to remove most of the THF, and extracted 2 times with CH2Cl2. The organics were combined, washed with brine, concentrated to a small volume and filtered through a plug of silica gel. The silica was washed with CH2Cl2 to elute the product, and the resulting solution was concentrated to give 4.65 g of l-(4-bromophenyl)-2,2,2- trifluoroethanol. Yield 92 %.
To Pd(PPli3)4 (2.1 g, 1.823 mmol) was added 3-fluorophenylmagnesium bromide (55 mis, 1.0 M in THF, 55 mmol) at O0C over 15 minutes. The ice bath was removed and the mixture was stirred for 30 minutes. l-(4-Bromophenyl)-2,2,2-trifluoroethanol (4.65 g, 18.23 mmol) in THF (50 mis) was added over 10 minutes. The mixture was heated to reflux for 3 hours and was shown complete by LC (Sunfire column, TFA). The mixture was cooled, quenched with H2O, rotary evaporated to remove most of the THF, and extracted 3 times with CH2Cl2. The organics were combined washed with brine, dried over MgSO4, and concentrated. Chromatography (SiO2, CH2Cl2) gave 4.64 g of 2,2,2-trifluoro-l-(3'- fluorobiphenyl-4-yl)ethanol. Yield 94 %.
To 2,2,2-trifluoro-l-(3'-fluorobiphenyl-4-yl)ethanol (1.4 g, 5.18 mmol) in THF (50 mis) at O0C was added NaH (60 % in mineral oil, 0.31 g, 7.77 mmol). The ice bath was removed and the mixture was stirred for 30 minutes. 2-Amino-4,6-dichloropyrimidine (1.0 g, 6.22 mmol) in THF (25 mis) was added at once. The mixture was heated to 5O0C for 5 hours. The reaction was complete by LCMS (Sunfϊre, TFA). The mixture was cooled, quenched with brine, and extracted 3 times with CH2Cl2. The organics were combined, washed with brine, dried over MgSO4, and concentrated. Chromatography (SiO2, CH2Cl2) afforded 1.48 g of 4-chloro-6-(2,2,2-trifluoro- 1 -(3'-fluorobiphenyl-4-yl)ethoxy)pyrimidin-2-amine. Yield 73%.
4-Chloro-6-(2,2,2-trifluoro- 1 -(3'-fluorobiphenyl-4-yl)ethoxy)pyrimidin-2-amine (0.75 g, 1.89 mmol), L-p-boronophenylalanine (0.47 g, 2.26 mmol), Pd(PPh3)2Cl2 (79 mgs, 0.113 mmol), Na2CO3 (0.44 g, 4.15 mmol), acetonitrile (10 mis), and H2O (10 mis) were combined in a 20 ml microwave reactor and heated in the microwave at 15O0C for 7 minutes. The reaction was complete by LCMS (Sunfϊre, neutral). The mixture was concentrated, dissolved in NaOH (20 mis 0.5 N), filtered, extracted with ether three times, and cooled to O0C. At 0 0C, 1.0 N HCl was added slowly until a pH of 6.5 was attained. The mixture was stirred at O0C for 30 minutes and the product was filtered, dried in air, treated with excess 2.0 N HCl in ether, concentrated, then triturated with CH2Cl2 to give 1.12 g, 99% (95.5 % purity). 385 mgs were purified via prep HPLC (Sunfire, TFA), concentrated, treated with excess 1.0 N HCl (aq.), concentrated to a small volume and lyophilized to afford 240 mgs of the captioned compound. M+l = 527; 1H NMR δ (CD3OD) 7.86 (d, 2H), 7.64 (s, 4H), 7.49 (d, 2H), 7.36 (m, 2H), 7.28 (m ,1H), 7.02 (m, IH), 6.95 (s, IH), 6.75 (q, IH), 4.26 (t, IH), 3.32 (m, IH), 3.21 (m, IH).
6.37. Synthesis of (S)-2-Amino-3-( 4-(2-amino-6-(benzylthio)pyrimidin-4- vDphenvDpropanoic acid
Figure imgf000064_0001
Benzylmercaptan (0.14g, 1.11 mmol) was treated with NaH (60% in mineral oil, 67 mg, 1.66 mmol) in dry THF (15 ml) for 30 minutes. 2-Amino-4,6-dichloropyrimidine (0.2 g, 1.22 mmol) was added and the mixture was stirred overnight. The mixture was diluted with methylenechloride, washed with water, then brine, dried over MgSO4, and concentrated to give 0.11 g of 4-(benzylthio)-6-chloropyrimidin-2-amine.
4-(Benzylthio)-6-chloropyrimidin-2-amine (0.1 g, 0.397 mmol), L-p- boronophenylalanine (0.1 g, 0.477 mmol), Pd(PPh3)2Cl2 (17 mg, 0.024 mmol), Na2CO3 (93 mg, 0.874 mmol), MeCN (2.5 ml) and water (2.5 ml) were heated at 1500C for 5 minutes in a microwave. The mixture was concentrated and purified via prep HPLC to give 0.42 g of the title compound. M+l = 381; 1H NMR (CD3OD) δ 7.8 (d, 2H), 7.37 (t, 4H), 7.23 (m, 2H), 7.16 (m, IH), 6.98 (s, IH), 4.43 (s, 2H), 4.20 (t, IH), 3.29 (m, IH), 3.13 (M, IH).
6.38. Synthesis of ( S)-2-Amino-3-(4-(2-amino-6-( naphthalen-2- ylmethylthio)pyrimidin-4-yl)phenyl)propanoic acid
Figure imgf000065_0001
2-Mercaptonapthalene (0.2 g, 1.148) was treated with NaH (60% in Mineral oil, 92 mg, 2.30 mmol) in dry THF (10 ml) for 30 minutes. 2-Amino-4,6-dichloropyrimidine (0.21 g, 1.26 mmol) was added and the mixture was stirred overnight. The mixture was diluted with methylenechloride, washed with water, then brine, dried over MgSO4, and concentratred to give 0.18 g 4-chloro-6-(naphthalen-2-ylmethylthio)pyrimidin-2-amine.
4-Chloro-6-(naphthalen-2-ylmethylthio)pyrimidin-2-amine (0.1 g, 0.331 mmol), L-p- boronophenylalanine (83 mg, 0.397 mmol), Pd(PPh3)2Cl2 (14 mg, 0.020 mmol), Na2CO3 (77 mg, 0.729 mmol), MeCN (2.5 ml) and water (2.5 ml) were heated at 1500C for 5 minutes in a microwave. The mixture was concentrated and purified via prep HPLC to give 57 mg of the title compound. M+l = 431; 1H NMR (CD3OD) δ 7.85 (s, IH), 7.79 (d, 2H), 7.72 (d, 3H), 7.46 (dd, IH), 7.35 (m, 4H), 6.95 (s, IH), 4.58 (s, 2H), 4.17 (m, IH), 3.26 (m, IH), 3.11 (m, IH). 6.39. Synthesis of αS)-2-Amino-3-(4-α-amino-6-q-(3,4-difluorophenyl)-2,2,2- trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid
Figure imgf000066_0001
3,5-Difluorophenyl-trifluoromethyl ketone was treated with NaBH4 (0.18 g, 4.76 mmol) in THF (5 ml) for 2 hours. The mixture was quenched with water, extracted with methylene chloride (2x). The organics were combined, filtered through silica gel and concentrated to give 0.46g of l-(3,4-difluorophenyl)-2,2,2-trifluoroethanol. l-(3,4-Difluorophenyl)-2,2,2-trifluoroethanol (0.1 g, 0.471 mmol) was treated with NaH (60% in mineral oil, 38 mg, 0.943 mmol) in dry THF (3 ml) for 30 minutes. 2-Amino- 4,6-dichloropyrimidine (77 mg, 0.471 mmol) was added and the mixture was stirred at 500C for 6 hours. The mixture was quenched with water and extracted with methylenechloride (2x). The organics were combined, washed with water, then brine, dried over MgSO4, and concentrated to give 0.14 g of 4-chloro-6-(l-(3,4-difluorophenyl)-2,2,2-trifluoroethoxy)- pyrimidin-2-amine. 4-Chloro-6-(l-(3,4-difluorophenyl)-2,2,2-trifluoroethoxy)pyrimidin-2-amine (0.14 g,
0.421 mmol), L-p-boronophenylalanine (110 mg, 0.505 mmol), Pd(PPh3)2Cl2 (18 mg, 0.025 mmol), Na2CO3 (98 mg, 0.926 mmol), MeCN (2.5 ml) and water (2.5 ml) were heated at 1500C for 5 minutes in a microwave. The mixture was concentrated and purified via prep HPLC to give 74 mg of the title compound. M+l = 469; 1H NMR (CD3OD) δ 7.83 (d, 2H), 7.47 (m, IH), 7.38 (m, 4H), 7.28 (m, IH), 4.21 (t, IH), 3.29 (m, IH), 3.15 (m, IH).
6.40. Synthesis of (2S)-2-Amino-3-(4-(2-amino-6-(2,2,2-trifluoro-l-(3'- methylbiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid
Figure imgf000066_0002
To 4'-bromo-2,2,2-trifluoroacetophenone (5.0 g, 19.76 mmol) in THF (50 mis) at O0C was added NaBH4 (1.5 g, 39.52 mmol). The mixture was warmed to room temperature and stirred for 1 hour. The reaction was complete by TLC (CH2Cl2). The mixture was quenched with H2O, rotary evaporated to remove most of the THF, and extracted 2 times with CH2Cl2. The organics were combined, washed with brine, concentrated to a small volume and filtered through a plug of silica gel. The silica was washed with CH2Cl2 to elute the product, and the resulting solution was concentrated to give 4.65 g of l-(4-bromophenyl)-2,2,2- trifluoroethanol. Yield: 92 %. l-(4-Bromophenyl)-2,2,2-trifluoroethanol (0.13 g, 0.525 mmol), m-tolylboronic acid (0.1 g, 0.736 mmol), Fibercat (4.28 % Pd, 47 mgs, 0.0157 mmol Pd), K2CO3 (0.22 g, 1.576 mmol), EtOH (3 mis), and H2O (0.5 mis) were combined and heated at 8O0C for 4 hours. The reaction was shown complete by TLC (CH2Cl2). The mixture was cooled, filtered, concentrated, slurried in CH2Cl2, and chromatographed over silica gel (CH2Cl2) to give 0.1 g of 2,2,2-trifluoro-l-(3'-methylbiphenyl-2-yl)ethanol. Yield: 72 %. Alternatively, l-(4-bromophenyl)-2,2,2-trifluoroethanol (0.98 g, 3.86 mmol), m- tolylboronic acid (0.63 g, 4.63 mmol), Pd(PPh3)2Cl2 (0.16 g, 0.232 mmol Pd), Na2CO3 (0.90 g, 8.49 mmol), AcCN (10 mis), and H2O (10 mis) were combined and heated in the microwave at 15O0C for 10 minutes. The reaction was shown complete by TLC (CH2Cl2). The mixture was cooled, concentrated, slurried in CH2Cl2, filtered, and chromatographed over silica gel (CH2Cl2) to give 0.80 g of 2,2,2-trifluoro-l-(3'-methylbiphenyl-2-yl)ethanol. Yield: 79 %.
Alternatively, tetrabutylammoniumfluoride (TBAF 1.0 N in THF 13 uL, 3.3 mg, 0.013 mmol) was added to a mixture of 3-methyl-biphenyl-2-carboxaldehyde (0.25g, 1.27 mmol) and trifluoromethytrimethyl silane (0.25 g, 1.53 mmol), in THF (1.5 ml) at 00C. The reaction was warmed to room temperature and stirred for 4 hours. HCl (3.0 N, 2.0 ml) was added, and the mixture was stirred for 3 hours. The mixture was concentrated, dissolved in methylene chloride, filtered through silica gel, and concentrated to give 0.15 g of 2,2,2- trifluoro-l-(3'-methylbiphenyl-2-yl)ethanol.
2,2,2-Trifluoro-l-(3'-methylbiphenyl-2-yl)ethanol (0.15 g, 0.563 mmol) was treated with NaH (60% in mineral oil, 45 mg, 1.12 mmol) in dry THF (5 ml) for 30 minutes. 2-
Amino-4,6-dichloropyrimidine (92 mg, 0.5633 mmol) was added and the mixture was stirred at 500C for 6 hours. The mixture was quenched with water and extracted wth methylenechloride (2x). The organics were combined, washed with water, then brine, dried over MgSO4, and concentrated to give 0.16 g of 4-chloro-6-(2,2,2-trifluoro-l-(3'- methylbiphenyl-2-yl)ethoxy)pyrimidin-2-amine.
4-Chloro-6-(2,2,2-trifluoro-l-(3'-methylbiphenyl-2-yl)ethoxy)pyrimidin-2-amine (0.16 g, 0.406 mmol), L-p-boronophenylalanine (10 mg, 0.487 mmol), Pd(PPh3)2Cl2 (17 mg, 0.024 mmol), Na2CO3 (95 mg, 0.894 mmol), MeCN (2.5 ml) and water (2.5 ml) were heated at 1500C for 5 minutes in a microwave. The mixture was concentrated and purified via prep HPLC to give 105 mg of the title compound. M+l = 523; 1H NMR (CD3OD) δ 7.85 (d, 2H), 7.70 (d, IH), 7.44 (m, 4H), 7.31 (t, IH), 7.21 (m, 2H), 7.10 (m, 2H), 6.87 (q, IH), 6.84 (s, IH), 4.25 (t, IH), 3.30 (m, IH), 3.18 (m, IH).
6.41. Synthesis of (S)-2-Amino-3-(4-(5-(3-(cvclopentyloxy)-4- methoxybenzylamino)pyridin-3-yl)phenyl)propanoic acid
Figure imgf000068_0001
Sodium triacetoxyl-borohydride (245mg, 1.16mmol) was added to the solution of 5- bromo-pyridine-3-amine(100mg, 0.57mmol) and 3-cyclopentyloxy-4-methoxy-benzaldehyde (127mg, 0.57mmol) in 10ml of 1 ,2-dicloroethtane (DCE), of HOAc (66μL, 2eq. 1.16mmol) was added, the mixture was stirred overnight at room temperature, followed by addition of 15 ml of DCE. The organic phase was washed with water, and dried over sodium sulfate. The solvent was removed by under reduced pressure to give 200 mg of crude 5-bromo-N-(3- (cyclopentyloxy)-4-methoxybenzyl) pyridin-3 -amine, which was used for the next step without further purification.
An Emrys process vial (2-5ml) for microwave was charged with 5-bromo-N-(3- (cyclopentyloxy)-4-methoxybenzyl)pyridin-3-amine (40mg, 0.106mmol), 4-borono-L- phenylalanine (22mg, 0.106mmol) and 2 ml of acetonitrile. Aqueous sodium carbonate (2 ml, IM) was added to above solution followed by 10 mol percent of dichlorobis (triphenylphosphine)-palladium (II). The reaction vessel was sealed and heated to 18O0C for 10 minutes with a microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 2.5 ml of methanol and purified with Prep-LC to give 20 mg of (S)-2-amino-3-(4-(5-3-(cyclophentyloxy-4-methoxy-benzylamino)pyridine-3-yl)phenyl)- propanoic acid. NMR: 1H-NMR (400 MHz, CD3OD): δ 1.59(m, 2H), 1.7 (m, 6H), 3.17(m, IH), 3.3 (m, IH), 3.75 (s, 3H), 4.2 (dd, IH) 4.39 (s, 2H), 4.7 (m, IH), 6.9(m, 3H), 7.4(d, 2H), 7.6(d, 2H), 7.7(s, IH), 7.9 (s, IH), 8.15(s, IH); Analytical HPLC: RT 2.69; M+l : 462(RT: 1.285).
6.42. Synthesis of 2-Amino-3-(3-(4-amino-6-((R)-l-(naphthalen-2- yl)ethylamino)-l,3i5-triazin-2-yl)phenyl)propanoic acid
Figure imgf000069_0001
To a solution of tert-butyl 2-(diphenylmethylene-amino) acetate (400 mg, 1.35mmol) in THF (25ml) was added a solution of LDA (1.8M in THF, 2eq, 2.7mmol, fresh bottle from Aldrich) over 5 minutes at -78°C, and the resulting mixture was stirred for 20 minutes. A solution of 2-(3-(bromomethyl) phenyl)-5,5-dimethyl-l, 3, 2-dioxaborinane (460mg, 1.2eq. 1.62mmol) in THF (10ml) was added drop-wise to the reaction mixture over 5 minutes. The reaction was continued at same (-780C) temperature for 30 minutes, and left for 3 hours at room temperature. The reaction was quenched with saturated NH4Cl, followed by the addition of water (30ml), and was extracted with EtOAc (2x40ml). The organic fractions were combined and dried over Na2SO4. The solvent was then concentrated at reduced pressure and crude te/t-Butyl-3-(3-(5, 5-dimethyl-l, 3, 2-dioxaborinan-2-yl)phenyl) 2(diphenylmethylene amino) propionate was purified by column chromatography to provide the product as a semi-solid.
An Emrys process vial (20ml) for microwave was charged with (R)-6-chloro-N2-(l- (naphthalene-2-yl)ethyl)-l,3,5-triazine-2,4-diamine (lOOmg, 0.33mmol), tert-butyl-3-(3-(5,5- dimethyl-l,3,2-dioxaborinan-2-yl)phenyl)-2-(diphenyl methyleneamino) propanoate (248mg, 0.5mmol, 1.5eq.) and 6ml of acetonitrile plus 6ml of aqueous sodium carbonate (IM) was added to above solution followed by 10 mol percent of dichlorobis(triphenylphosphine)- palladium(II). The reaction vessel was sealed and heated to 19O0C for 10 minutes with microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 10 ml of THF, to which was added 5N.HC1 (5ml). The mixture was refluxed for 2 hours in order to deprotect the benzophone and tert-butyl groups. The resulting reaction mixture was concentrated and dissolved in methanol (8ml) and purified with Prep-LC to afford 15mg of 2-amino-3-(4(4-amino-6-((R)-l-(naphthalene-2-yl)ethylamino)-l,3,5-trizin-2- yl)phenyl)propanoic acid. NMR: 1H-NMR (400 MHz, CD3OD): δ 1.85(d, 3H), 3.2-3.45 (m, 2H), 4.37(m, IH), 5.5 (m, IH), 7.4(m, IH), 7.6(m 4H), 7.9(m, 4H), 8.18(m, 2H), Analytical HPLC: RT 2.79 M+l : 429 (RT: 1.35).
6.43. Synthesis of 2-Amino-3-(4-(4-amino-6-((R)-l-(naphthalen-2- yl)ethylamino)-l,3i5-triazin-2-yl)-2-fluorophenyl)propanoic acid
Figure imgf000070_0001
To a solution of tert-butyl 2-(diphenylmethylene-amino) acetate (l.lg, 3.73mmol) in THF (30ml) was added a solution of LDA (1.8M in THF, leq, 3.73mmol, fresh bottle from Aldrich) over 5 minutes at -78°C, and the resulting mixture was stirred for 20 minutes. A solution of 4-bromo-l-(bromomethyl)-2-fluorobenezene (Ig, 3.74mmol) in THF (10ml) was added drop-wise to the reaction mixture over 5 minutes. The reaction was continued at -78°C for 30 minutes, after which it was left at room temperature for 3 hours. The reaction was quenched with saturated NH4Cl, after which water (30ml) was added. Product was extracted with EtOAc (2x40ml), and the organic fractions were combined and dried over Na2SO4. The solvent was concentrated at reduced pressure and crude tert-Butyl 3-(4-bromo-2- fluorophenyl)-2-(diphenylmethyleneamino)-propanoate was purified by column chromatography. The product was obtained as a solid.
An Emrys process vial (20ml) for microwave was charged with tert-butyl 3-(4- bromo-2-fluorophenyl)-2-(diphenylmethylene-amino)propanoate (600mg, 1.24mmol), Pd(dba)2 (71mg, 0.124mmol), PCy3 (35mg, 0.124mmol), 4,4,4>,4>,5,5,5',5'-octamethyl-2,2>- bi(l,3,2-dioxaborolane (346mg, l.leq. 1.36mmol) and KOAc (182mg, 1.5eq., 1.86mmol) 20ml of DMF. The reaction vessel was sealed and heated to 16O0C for 20 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in H2O (30ml), extracted with EtOAc (2x40ml), and purified with Prep-LC to give 220mg of tert-butyl 2-(diphenylmethyleneamino)-3-(2-fluoro- 4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)phenyl)propanoate.
An Emrys process vial (5ml) for microwave was charged with (R)-6-chloro-N2-(l- (naphthalene-2-yl)ethyl)-l,3,5-triazine-2,4-diamine (67mg, 0.22mmol), tert-butyl-2- (diphenylmethyleneamino)-3-(2-fluoro-4-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2- yl)phenyl)propanoate (120mg, 0.22mmol) and 2ml of acetonitrile. Aqueous sodium carbonate (2 ml, IM) was added to above solution followed by 10 mol percent dichlorobis(triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 19O0C for 10 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 10 ml of THF, to which 5N.HC1 (2ml) was then added. The mixture was refluxed for 2 hours (deprotection of benzophone and tert-butyl groups). After deprotection of two groups, the mixture was concentrated, dissolved in methanol (5ml), and purified with Prep-LC to afford lOmg of 2-amino-3-(4-(4-amino-6-((R)- l-(naphthalene-2-yl)ethylamino)-l,3,5-trizin-2-yl)-2-fluorophenyl)propanoic acid. NMR: 1H-NMR (400 MHz, CD3OD): δ 1.6 (d, 3H), 3.07 (m, IH), 3.45(m, IH), 3.8 (m, IH), 5.45 (m, IH), 7.4(m, 4H), 7.6(m IH), 7.8(m, 4H), 8.08(m, IH), Analytical HPLC: RT 2.88, M+l : 447 (RT: 1.44).
6.44. Synthesis of f2S)-2-Amino-3-f4-f4-amino-6-fl-fadamantyll)ethylamino)- l,3i5-triazin-2-yl)phenyl)propanoic acid
Figure imgf000071_0001
A solution of adamantine amine (1 equivalent), 2-amino-4,6-dichloro-[l,3,5] triazine (1 equivalent) and diisopropyl ethyl amine (5 equivalents, Aldrich) in anhydrous 1,4-dioxane was refluxed at 1300C for 3 hours. After completion of the reaction, the dioxane was removed under reduced pressure. The reaction was then cooled to room temperature, water was added, and product was extracted with dichloromethane (2x40ml). The combined organic solution was dried over Na2SO4 and concentrated to afford product, which was used in the next step without purification.
An Emrys process vial (20ml) for microwave was charged with adamantine trizine chloride (200mg, 0.65mmol), 4-borono-L-phenylalanine(135mg, 0.65mmol) and 5ml of acetonitrile. Aqueous sodium carbonate (5 ml, IM) was added to above solution followed by 5 mol percent dichlorobis(triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 19O0C for 20 minutes by microwave. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 4 ml of methanol and purified with Prep-LC to give 60 mg (yield 21%) of coupled product. NMR: 1H-NMR (400 MHz, CD3OD): δ 1.22 (m, 3H), 1.6-1-8 (m, 12H), 2.01(d, 3H), 3.25-3.42 (m, 2H), 4.0 (m, IH), 4.40(m, IH), 7.6(d, 2H), 8.2(d, 2H), Analytical HPLC: RT 3.11, M+l : 437 (RT: 1.76).
6.45. Alternative Synthesis of (2S)-2-Amino-3-(4-(4-amino-6-q- fadamantyll)ethylamino)-l,3i5-triazin-2-yl)phenyl)propanoic acid Adamantane (2-yl) ethyl cyanoguanidine was prepared by forming a solution of cyanoguanidine (1 equivalent), (S)-2-amino-3-(4-cyanophenylpropanoic acid (1 equivalent) and potassium tertiary butaoxide (3.5 equivalent, Aldrich) in dry n-BuOH, which was vigorously refluxed at 1600C in a sealed tube for 2 days. After completion of the reaction, the mixture was allowed to cool to room temperature, and the reaction was quenched with water. Solvent was removed under reduced pressure. Again, after allowing to cool to room temperature, the reaction mixture was brought to pH 12-14 by adding IN NaOH. Then, impurities were removed while extracting with Ether:EtOAc (9:1, 2x100 ml). The aqueous solution was cooled to 00C, IN HCl was then added to adjust pH to 7. The pale yellow product was slowly crashed out in H2O, the mixture was kept in a refrigerator for 30 minutes, and the solid was obtained by filtration with 92% purity. Compound was crystallized from MeOH to afford a white solid (>98% pure, 48-78% yield). 1H-NMR (400 MHz, CD3OD): δ 1.0(d, 3H), 1.45-1.6(m, 6H), 4.62-4.8(m, 4H) 2.0 (m, 2H), 3.3(m, IH), 3.5 (m, IH); Analytical HPLC: RT 2.69; M+l : 462(RT: 1.285).
The title compound was prepared from adamantane (2-yl) ethyl cyanoguanidine using the method shown in Scheme 6.
6.46. Synthesis of (S)-2-Amino-3-(4-(5-fluoro-4-((RM-(naphthalen-2- yl)ethylamino)pyrimidin-2-yl)phenvl)propanoic acid
Figure imgf000072_0001
A mixture of (R)-(+)-l-(2-napthyl)ethylamine (102.6mg, 0.599mmol), 2,4-dichloro-5- fluroro pyrimidine (lOOmg, 0.599mmol) and cesium carbonate (390mg, 1.2mmol) was dissolved in 1,4-dioxane (3ml) and H2O (3ml) in a 10 ml microwave vial. The mixture was stirred in the microwave reactor at 800C for 10 minutes. The residue was dissolved in CH2Cl2 (50 ml), washed with water (20 ml), brine (20 ml) dried (Na2SO4) and concentrated to get the crude intermediate 2-chloro-5-fluoro-pyrimidin-4-yl)-(l-naphthalen-2-yl-ethyl)- amine.
The crude intermediate (250mg, 0.83mmol) was then dissolved in 6.0ml of MeCN and 6ml of H2O in a 20ml microwave vial. To this solution were added L-p-borono- phenylalanine (173.6mg, 0.83mmol), sodium carbonate (173.6mg, 1.66mmol) and catalytic amount of dichlorobis(triphenylphosphine)-palladium(II) (11.6mg, 0.0166mmol). The reaction vial was then sealed and stirred in the microwave reactor at 1500C for 7 minutes. The contents were then filtered, and the filtrate was concentrated and dissolved in MeOH and H2O (1 :1) and purified by preparative HPLC using MeOH/H2O/TFA as the solvent system. The combined pure fraction were evaporated in vacuo and further dried on a lyophilizer to give 154mg of 2-amino-3- {4-[5-fluoro-4-(l -naphthalen-2-yl-ethylamino)-pryrimidin-2-yl]- phenyl} -propionic acid. NMR: 1H-NMR (400 MHz, CD3OD) δ 1.8(d, 3H) 3.2-3.4(m, 2H), 4.35(m, IH), 5.7(q, IH), 7.5(m, 4H), 7.6(d, IH), 7.8-7.9(m, 4H), 8.1(d, 2H), 8.3(d, IH). LCMS: M+l= 431.
6.47. Synthesis of (S)-2-Amino-3-(4-(2-amino-6-(4-(trifluor()methyl)- benzylamino)pyrimidin-4-yl)phenyl)propanoic acid
Figure imgf000073_0001
A mixture of trifluoromethyl benzylamine (106.8mg, 0.610mmol), 2-amino-4,6- dichloropyrimidine (lOOmg, 0.610mmol) and cesium carbonate (217mg, 1.2mmol) was dissolved in 1,4-dioxane (6ml) and H2O (6ml) in a 20 ml microwave vial. The mixture was stirred in the microwave reactor at 2100C for 25 minutes. The solvent was then removed. The residue was dissolved in CH2Cl2 (50 ml), washed with water (20 ml), brine (20 ml), dried (Na2SO4) and concentrated to get the crude intermediate 6-chloro-N-4'-(trifluoromethyl- benzy l)-pryrimidine-2-4-diamine .
The crude intermediate (150mg, 0.497mmol) was then dissolved in 3.0ml of MeCN and 3ml of H2O in a 10 ml microwave vial. To this solution were added L-p-borono- phenylalanine (104mg, 0.497mmol), sodium carbonate (150mg, 0.994mmol) and catalytic amount of dichlorobis(triphenylphosphine)-palladium(II) (6.9mg, 0.00994mmol). The reaction vial was then sealed and stirred in the microwave reactor at 1500C for 5 minutes. The contents were filtered, and the filtrate was concentrated and dissolved in MeOH and H2O (1 :1) and purified by preparative HPLC using a MeOH/H2O/TFA solvent system. The combined pure fractions were evaporated in vacuo and further dried on a lyophilizer to afford 2-amino-3-{4-[2-amino-6-(4-trifluoromethyl-benzylamino)-pyrimidin-4-yl]-phenyl}- propionic acid. NMR: 1H-NMR (300MHz, CD3OD) δ 3.1-3.3(m, 2H), 4.2(t, IH), 4.7(s, 2H), 6.3(s, IH), 7.4-7.5(m, 4H), 7.6(d, 2H), 7.7(d, 2H). LCMS: M+l=432.
6.48. Synthesis of 2-Amino-3-(5-(5-phenylthiophen-2-yl)-lH-indol-3- vDpropanoic acid
Figure imgf000074_0001
2-Amino-3-(5-bromo-lH-indol-3-yl)-propionic acid (0.020 g, 0.071 mmol) was added to a 5 ml microwave vial, which contained 5-phenyl-thiophen-2-boronic acid (0.016 g, 0.078mmol), Na2CO3 (0.015 g, 0.142 mmol), acetonitrile (1.5 ml) / water (1.5 ml) and dichlorobis(triphenylphosphine)-palladium (3 mg, 0.003 mmol). Microwave vial was capped and stirred at 1500C for 5 min under microwave radiation. Reaction mixture was cooled, filtered through a syringe filter and then separated by a reverse phase preparative-HPLC using YMC-Pack ODS 100x30 mm ID column (MeOH/H2O/TFA solvent system). The pure fractions were concentrated in vacuum. The product was then suspended in 5 ml of water, frozen and lyophilized to give 5 mg of pure product, 2-amino-3-[5-(5-phenyl-thiophen-2-yl)- lH-indol-3-yl]-propionic acid. IH-NMR (300 MHz, CD3OD): 3.21-3.26 (m, 2H), 4.25 (q, IH), 7.15-7.35 (m, 8H), 7.58 (d, 2H), 7.82 (d, IH). 6.49. Synthesis of (S)-2-Amino-3-(4-(4-(4-phenoxyphenyl)-lH-l,2,3-triazol-l- vDphenvDpropanoic acid
Figure imgf000075_0001
A mixture of l-ethynyl-4-phenoxy-benzene (126mg, 0.65mmol) and (S)-3-(4-azido- phenyl)-2-tert-butoxycarbonylamino-propionic acid (200mg, 0.65mg) in H2O:dioxane (5:1) was heated at 1000C in a sealed tube for overnight. After completion of reaction, 3N HCl (5 ml) was added and the mixture was stirred for 2hr at 500C. Removal of solvent gave crude product which was dissolved in MeOH and purified by preparative HPLC to give 45 mg of desired product (yield: 29%). 1H-NMR (400 MHz, CD3OD): δ (ppm) 3.2 (m, IH), 3.4 (m, IH), 4.3(m, IH), 6.9(d, 2H), 7.0(d, 2H), 7.2(m, IH), 7.3(d, 2H), 7.4-7.55 (m, 6H), 8.0(s, IH).
6.50. Synthesis of (S)-2-Amino-3-(4-(4-(4-(thiophene-2-carboxamido)phenyr)- lH-l,2,3-triazol-l-yl)phenyl)propanoic acid and (S)-2-Amino-3-(4-(5-(4- fthiophene-2-carboxamido)phenyl)-lH-l,2,3-triazol-l- yl)phenyl)propanoic acid
Figure imgf000075_0002
A mixture of thiophene-2-carboxylic acid (4-ethyl-phenyl) amide (117mg, 0.49mmol) and (S)-3-(4-azido-phenyl)-2-tert-butoxycarbonylamino-propionic acid (150mg, 0.49mg) in 5 ml of H2O:dioxane (5:1) was heated at 1000C in a sealed tube overnight. After completion of reaction, 3N HCl (5 ml) was added and the mixture was stirred for 2hr at 500C. Removal of solvent gave crude product which was dissolved in MeOH and purified by preparative HPLC. According to LCMS (retention time) and NMR, two regio-isomers were obtained (total yield: 70mg, 66%). The major product is (S)-2-amino-3-(4-(4-(4-(thiophene-2- carboxamido)phenyl)-lH-l,2,3-triazol-l-yl)phenyl)propanoic acid. NMR: 1H-NMR (400 MHz, CD3OD): δ 3.2 (m, IH), 3.4 (m, IH), 4.3(m, IH), 7.15(m, IH), 7.3(d, 2H), 7.6(m, 4H), 7.0(m, 3H), 7.95 (d, IH), 8.0(s, IH). The minor product is (S)-2-amino-3-(4-(5-(4- (thiophene-2-carboxamido)phenyl)- IH-1 ,2,3-triazol- 1 -yl)phenyl)propanoic acid. 1H-NMR (400 MHz, CD3OD): δ 3.2 (m, IH), 3.4 (m, IH), 4.35(m, IH), 7.2(m, IH), 7.3(d, 2H), 7.5- 7.6(m, 4H), 7.75(m, 3H), 7.95 (d, IH), 8.05(s, IH).
6.51. Synthesis of fS)-2-Amino-3-f4-f2-amino-6-fphenylethvnyl)pyrimidin-4- vDphenvDpropanoic acid
Figure imgf000076_0001
2-Amino 4,6-dichloro pyrimidine (0.180 g, 1.1 mmol), trimethyl-phenylethynyl- stannane (0.264 g, 1 mmol), were dissolved in THF (20 ml) and the mixture was stirred at 65°C for 12h. LCMS indicated the completion of reaction. Solvent was removed and the residue was directly used in the following step.
The crude intermediate (0.42 g), L-p-borono-phenylalanine (0.210 g, 1 mmol), sodium carbonate (0.210 g, 2 mmol), and dichlorobis (triphenylphosphine)-palladium(II) (25 mg, 0.036 mmol) were dissolved in a mixture of MeCN (3 ml) and H2O (3 ml) in a 10 ml microwave vial. The vial was sealed and stirred in the microwave reactor at 1500C for 6 min. The mixture was filtered and the filtrate was concentrated. Residue was purified by preparative HPLC using MeOH/H2O/TFA as solvent system to obtain (S)-2-amino-3-[4-(2- amino-6-phenylethynyl-pyrimidin-4-yl(-phenyl]-propionic acid as a TFA salt. 1H-NMR (400 MHz, CD3OD): δ (ppm) 3.20-3.42 (m, 2H), 4.31 (m, IH), 7.40-7.51 (m, 6H), 7.62 (d, 2H), 8.18 (d, 2H). 6.52. Synthesis of (S)-2-Amino-3-r4-q-amino-6-{R-l-r4-chloro-2-(3-methyl- Pyrazol-l-yl)-phenyll-2,2,2-trifluoro-ethoxy}-pyrimidin-4-yl)-phenyll- propionic acid ethyl ester
Figure imgf000077_0001
The title compound was prepared stepwise, as described below:
Step 1 : Synthesis of l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanone. To a 500 ml 2 necked RB flask containing anhydrous methanol (300 ml) was added thionyl chloride (29.2 ml, 400 mmol) dropwise at 0-50C (ice water bath) over 10 min. The ice water bath was removed, and 2-bromo-4-chloro-benzoic acid (25 g, 106 mmol) was added. The mixture was heated to mild reflux for 12h. Progress of the reaction was monitored by TLC and LCMS. After completion of the reaction, the reaction mixture was concentrated. Crude product was dissolved in dichloromethane (DCM, 250 ml), washed with water (50 ml), sat. aq. NaHCO3 (50 ml), brine (50 ml), dried over sodium sulfate, and concentrated to give the 2- bromo-4-chloro-benzoic acid methyl ester (26 g, 99 %), which was directly used in the following step.
2-Bromo-4-chloro-benzoic acid methyl ester (12.4 g, 50 mmol) in toluene (200 ml) was cooled to -700C, and trifluoromethyl trimethyl silane (13 ml, 70 mmol) was added. Tetrabutylamonium fluoride (IM, 2.5 ml) was added dropwise, and the mixture was allowed to warm to room temperature over 4h, after which it was stirred for 1Oh at room temperature. The reaction mixture was concentrated to give the crude [l-(2-bromo-4-chloro-phenyl)-2,2,2- trifluoro-l-methoxy-ethoxy]-trimethyl-silane. The crude intermediate was dissolved in methanol (100 ml) and 6N HCl (100 ml) was added. The mixture was kept at 45-500C for 12h. Methanol was removed, and the crude was extracted with dichloromethane (200 ml). The combined DCM layer was washed with water (50 ml), NaHCO3 (50 ml), brine (50 ml), and dried over sodium sulfate. Removal of solvent gave a crude product, which was purified by ISCO column chromatography, using 1-2% ethyl acetate in hexane as solvent, to afford 1- (2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanone (10 g, 70%). 1H-NMR (300 MHz, CDCl3): δ (ppm) 7.50 (d,lH), 7.65(d,lH), 7.80(s,lH). Step 2: Synthesis of R-l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanol. To catechol borane (IM in THF 280 ml, 280 mmol) in a 2L 3-necked RB flask was added S-2- methyl-CBS oxazaborolidine (7.76 g, 28 mmol) under nitrogen, and the resulting mixture was stirred at room temperature for 20 min. The reaction mixture was cooled to -78°C (dry ice/acetone bath), and l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanone (40 g, 139 mmol) in THF (400 ml) was added dropwise over 2h. The reaction mixture was allowed to warm to -36°C, and was stirred at that temperature for 24 h, and further stirred at -32°C for another 24h. 3N NaOH (250 ml) was added, and the cooling bath was replaced by ice-water bath. Then 30 % hydrogen peroxide in water (250 ml) was added dropwise over 30 minutes. The ice water bath was removed, and the mixture was stirred at room temperature for 4h. The organic layer was separated, concentrated and re-dissolved in ether (200 ml). The aqueous layer was extracted with ether (2 x 200 ml). The combined organic layers were washed with IN aq. NaOH (4 x 100 ml), brine, and dried over sodium sulfate. Removal of solvent gave crude product which was purified by column chromatography using 2 to 5% ethyl acetate in hexane as solvent to give desired alcohol 36.2 g (90 %, e.e. >95%). The alcohol (36.2 g) was crystallized from hexane (80 ml) to obtain R-l-(2-bromo-4-chloro- phenyl)-2,2,2-trifiuoro-ethanol 28.2 g (70 %; 99-100 % e.e.). 1H-NMR (400 MHz, CDCl3) δ (ppm) 5.48 (m, IH), 7.40 (d, IH), 7.61 (d, 2H).
Step 3: Synthesis of R-l-[4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro- ethanol. R-l-(2-bromo-4-chloro-phenyl)-2,2,2-trifluoro-ethanol (15.65g, 54.06 mmol), 3- methylpyrazole (5.33 g, 65 mmol), CuI (2.06 g, 10.8 mmol), K2CO3 (15.7 g, 113.5 mmol), (lR,2R)-N,N'-dimethyl-cyclohexane-l,2-diamine (1.54 g, 10.8 mmol) and toluene (80 ml) were combined in a 250 ml pressure tube and heated to 1300C (oil bath temperature) for 12 h. The reaction mixture was diluted with ethyl acetate and washed with H2O (4 x 100 ml), brine, and dried over sodium sulfate. Removal of solvent gave a crude product, which was purified by ISCO column chromatography using 5-10 % ethyl acetate in hexane as solvent to get R-I- [4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro-ethanol (13.5 g; 86 %). 1H-NMR (400 MHz, CDCl3): δ (ppm) 2.30(s, 3H), 4.90(m, IH), 6.20(s, IH), 6.84(d, IH), 7.20(s, IH), 7.30(d, IH), 7.50(d, IH). Step 4: Synthesis of (S)-2-Amino-3- r4-(2-amino-6- (R-I- r4-chloro-2-(3-methyl- pyrazol- 1 -yl)-phenvH-2,2,2-trifluoro-ethoxy| -pyrimidin-4-vD-phenvU -propionic acid ethyl ester. R-l-[4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro-ethanol (17.78 g, 61.17 mmol), (S)-3-[4-(2-amino-6-chloro-pyrimidine-4-yl)-phenyl]-2-tert- butoxycarbonylamino-propionic acid (20.03 g, 51 mmol), 1,4-dioxane (250 ml), and Cs2CO3 (79.5 g, 244 mmol) were combined in a 3-necked 500 ml RB flask and heated to 1000C (oil bath temperature) for 12-24 h. The progress of reaction was monitored by LCMS. After the completion of the reaction, the mixture was cooled to 600C, and water (250 ml) and THF (400 ml) were added. The organic layer was separated and washed with brine (150 ml). The solvent was removed to give crude BOC protected product, which was taken in THF (400 ml), 3N HCl (200 ml). The mixture was heated at 35-400C for 12h. THF was removed in vacuo. The remaining aqueous layer was extracted with isopropyl acetate (2x 100 ml) and concentrated separately to recover the unreacted alcohol (3.5 g). Traces of remaining organic solvent were removed from the aqueous fraction under vacuum. To a IL beaker equipped with a temperature controller and pH meter, was added
H3PO4 (40 ml, 85 % in water) and water (300 ml) then 50 % NaOH in water to adjust pH to 6.15. The temperature was raised to 58°C and the above acidic aqueous solution was added dropwise into the buffer with simultaneous addition of 50 % NaOH solution in water so that the pH was maintained between 6.1 to 6.3. Upon completion of addition, precipitated solid was filtered and washed with hot water (50-600C) (2 x 200 ml) and dried to give crude (S)-2- amino-3-[4-(2-amino-6-{R-l-[4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro- ethoxy}-pyrimidin-4-yl)-phenyl} -propionic acid (26.8 g; 95 %). LCMS and HPLC analysis indicated the compound purity was about 96-97 %.
To anhydrous ethanol (400 ml) was added SOCl2 (22 ml, 306 mmol) dropwise at 0- 5°C. Crude acid (26.8 g ) from the above reaction was added. The ice water bath was removed, and the reaction mixture was heated at 40-450C for 6-12h. After the reaction was completed, ethanol was removed in vacuo. To the residue was added ice water (300 ml), and extracted with isopropyl acetate (2 x 100 ml). The aqueous solution was neutralized with saturated Na2CO3 to adjust the pH to 6.5. The solution was extracted with ethyl acetate (2 x 300 ml). The combined ethyl acetate layer was washed with brine and concentrated to give 24 g of crude ester (HPLC purity of 96-97 %). The crude ester was then purified by ISCO column chromatography using 5 % ethanol in DCM as solvent to give (S)-2-amino-3-[4-(2- amino-6- (R- 1 -[4-chloro-2-(3-methyl-pyrazol- 1 -yl)-phenyl]-2,2,2-trifluoro-ethoxy} - pyrimidin-4-yl)-phenyl} -propionic acid ethyl ester (20.5g; 70 %; HPLC purity of 98 %). LCMS M+l = 575. 1H-NMR (400 MHz, CD3OD): δ (ppm) 1.10 (t, 3H), 2.25 (s, 3H), 2.85 (m, 2H), 3.65 (m, IH), 4.00 (q, 2H), 6.35 (s, IH), 6.60 (s, IH), 6.90 (m, IH), 7.18 (d, 2H), 7.45 (m, 2H), 7.70 (d, IH), 7.85 (m, 3H). 6.53. Synthesis of (S)-2-amino-3-(4-q-amino-6-((R)-l-(4-chloro-2-(3-methyl- lH-pyrazol-l-yl)phenyl)-2,2,2-trifluoroethoxy)pyrimidin-4- vDphenvDpropanoic acid
Figure imgf000080_0001
(S)-2-Amino-3-[4-(2-amino-6- (R- 1 -[4-chloro-2-(3-methyl-pyrazol- 1 -yl)-phenyl]-
2,2,2-trifluoro-ethoxy}-pyrimidin-4-yl)-phenyl} -propionic acid ethyl ester (22.2 g, 38.6 mmol) was dissolved in THF (220 ml) and water (50 ml). Lithium hydroxide monohydrate (5.56 g, 132 mmol) was added. The reaction mixture was stirred at room temperature for 12 h. THF was removed, and water (100 ml) was added to the residue to get the clear solution. To a 1 L beaker equipped with a temperature controller and pH meter was added
H3PO4 (40 ml, 85 % in water), water (300 ml) and 50 % NaOH in water to adjust the pH to 6.15. The temperature was raised to 58°C, and the aqueous Li-salt of the compound was added dropwise into the buffer with simultaneous addition of 3N HCl so that the pH was maintained at 6.1 to 6.2. Upon completion of addition, precipitated solid was filtered and washed with hot water (50-600C) (2 x 200 ml) and dried to give (S)-2-amino-3-[4-(2-amino- 6- (R- 1 -[4-chloro-2-(3-methyl-pyrazol- 1 -yl)-phenyl]-2,2,2-trifluoro-ethoxy} -pyrimidin-4-yl)- phenyl} -propionic acid (19.39 g; 92 %). LCMS and the HPLC analysis indicated the compound purity was about 98-99%. LCMS M+l = 547. 1H-NMR (400 MHz, CD3OD): δ (ppm) 2.40 (s, 3H), 3.22-3.42 (m, 2H), 4.38 (t, IH), 6.42 (s, IH), 7.10 (s, IH), 7.21 (m, IH), 7.60 (m, 4H), 7.81 (d, IH), 7.92 (m, 3H).
6.54. Synthesis of (S)-2-Amino-3-(4-{2-amino-6-[2,2,2-trifluoro-l-α-thiazol-2- yl-phenvD-ethoxyl -pyrimidin-4-yl}-phenyl)-propionic acid
Figure imgf000080_0002
To a 40 ml microwave reactor, was added 1.04 g of 2-formyl phenylboronic acid (6.9 mmoles), 1.14 g of 2-bromo thiazole (6.9 mmoles), 240 mg of palladium bistriphenyl- phosphine dichloride (Pd(PPh3)2Cl2, 0.34 mmoles). Then, 13.8 ml of IM Na2CO3 (13.8 mmoles) and 10 ml of CH3CN were added to the mixture. The reactor was sealed, and the reaction was run under microwave at 1600C for 5 minutes. LCMS shows completion of the reaction with desired product. The reaction mixture was then poured into a separation funnel. Then 200 ml of methylene chloride and 100 ml of water were added for extraction. The methylene chloride layer was dried over MgSO4. Removal of solvent gave a crude product, which was purified by silica gel column chromatography eluting with hexanes/ethyl acetate mixture (5/1 to 2/1) to give pure 2-thiazol-2-yl-benzaldehyde (0.5 g, yield: 38%).
To a 50 ml round bottom flask, 184 mg of 2-thiazol-2-yl-benzaldehyde (0.97 mmole) and 10 ml of anhydrous tetrahydrofuran (THF) were added. Then, 145.4 mg of trifluoromethyltrimethylsilane (1.02 mmoles) and 20 μl of IM tert-butylammonium fluoride in THF (0.02 mmole) were added to solution. The mixture was stirred at room temperature overnight, after which 10 ml of 1 N HCl was added and the reaction mixture was stirred at r.t. for 15 minutes. THF was removed in vacuo, and the mixture was extracted with methylene chloride (3 x 50ml). The combined CH2Cl2 layer was dried over MgSO4. Removal of solvent gave 262 mg of crude product, which was about 95% pure, and was used in next step without further purification. 2,2,2-Trifiuoro-l-(2-thiazol-2-yl-phenyl)-ethanol (260 mg, 1 mmole), (S)-3-[4-(2- amino-6-chloro-pyrimidin-4-yl)-phenyl]-2-tert-butoxycarbonylamino-propionic acid (390 mg, 1 mmole), cesium carbonate (1.3 g, 4 mmoles) and 10 ml of 1 ,4-dioxane were mixed together in a 50 ml sealed tube. The reaction mixture was heated at 1000C for 3 days. Water (20 ml) was added, and then IN HCl aq. was added slowly to adjust the pH to 4, then the 1,4- dioxane was removed in vacuo and the resulting mixture was extracted with methylene chloride (3 x 50 ml). The combine methylene chloride layer was dried over MgSO4. Removal of solvent gave a crude product, which was taken to next step reaction without further purification.
The above crude product was dissolved in 5 ml of methylene chloride, and 0.4 ml of trifluoroacetic acid was added. The mixture was stirred at room temperature overnight. The trifluoroacetic acid was then removed in vacuo to give a crude product, which was purified by prep HPLC to give 63 mg of pure product. HPLC; YMC Pack ODS-A 3x50 mm, 7um; Solvent A = water with 0.1% TFA; Solvent B = methanol with 0.1% TFA. Solvent B from 10 to 90% over 4 minutes; Flow rate = 2 ml/min; RT = 3 min. HPLC purity = 100%. LCMS: M+l = 515.9. 1U NMR (400 MHz, CD3OD) δ 8.06 ppm (2H, m); 7.92 (2H, d, J=8 Hz); 7.84(1H, m); 7.81 (IH, m); 7.77 (IH, d, J = 4 Hz); 7.57 (2H, m); 7.45 (2H, d, J = 8 Hz); 6.84 (IH, s); 4.30 (2H, dd, J = 8 Hz); 3.38 (2H, dd, J = 12, 2 Hz); 3.23 (2H, dd, J = 12, 8 Hz).
6.55. Synthesis of (S)-2-Amino-3-r4-(2-amino-6-{2,2,2-trifluoro-l-r2-(pyridin-3- yloxy)-phenyll-ethoxy}-pyrimidin-4-yl)-phenyll-propionic acid; (S)-2- Amino-3-[4-f2-amino-6-{2,2,2-trifluoro-l-[4-fpyridin-3-yloxy)-phenyll- ethoxy}-pyrimidin-4-yl)-phenyll-propionic acid; (S)-2-Amino-3-r4-(6- {2,2,2-trifluoro-l-[4-fpyridin-3-yloxy)-phenyll-ethoxy}-pyrimidin-4-yl)- phenyll -propionic acid; (S)-2-Amino-3-(4-{2-amino-6-r2,2,2-trifluoro-l-(4- thiophen-2-yl-phenyl)-ethoxyl-pyrimidin-4-yl}-phenyl)-propionic acid; (S)-2-Amino-3-(4-{6-[2,2,2-trifluoro-l-(4-imidazol-l-yl-phenyl)-ethoxyl- pyrimidin-4-yl}-phenyl)-propionic acid; and (S)-2-Amino-3-(4-{2-amino- 6- [2,2,2-trifluoro- l-( 4- [ 1 ,2,41 triazol- l-yl-phenvD-ethoxyl -pyrimidin-4-yl}- phenyD-propionic acid
Figure imgf000082_0001
The title compounds were prepared using the general approach shown below: cιγ^/cι
Figure imgf000083_0001
Figure imgf000083_0002
In this approach, tetra-n-butyl ammonium fluoride (0.05 eq.) was added to a mixture of substituted benzaldehyde (1 eq.) and trifluoromethyl trimethylsilane (1.2 eq.) in THF at 00C. The temperature was then allowed to warm to room temperature. The mixture was stirred at room temperature for 5h, then diluted with ethyl acetate, washed with water, brine and dried by MgSO4. The solvent was removed under reduced pressure to give the trifluoro- alcohol as crude product, which was used in next step without further purification.
The above-made alcohol (1 eq.) was dissolved in anhydrous 1,4-dioxane. Sodium hydride ( 60% in mineral oil, 1.2 eq.) was added all at once, and the mixture was stirred at room temperature for 30 minutes. 2-Amino-4,6-dichloropyrimidine (1 eq.) was added, and the resulting mixture was stirred at 800C for 2 h. The solvent was removed, and the residue was suspended in ethyl acetate, which was washed with water, dried over MgSO4 and then concentrated to give the desired monochloride product, which was used in next step without further purification.
The above crude product (1 eq.) was added to a 5 ml microwave vial containing 4- borono-L-phenylalanine (1 eq.), Na2CO3 (2 eq.), acetonitrile (2 ml), water (2 ml) and dichlorobis(triphenylphosphine)-palladium (0.05 eq.). The vial was capped, and the mixture was heated at 1500C for 5 min under microwave radiation. The mixture was cooled, filtered through a syringe filter, and then separated by a reverse phase preparative-HPLC using YMC-Pack ODS 100x30 mm ID column (MeOH/H2O/TFA solvent system). The pure fractions were combined and concentrated in vacuum. The product was then suspended in 5 ml of water, frozen and lyophilized to give the product as a trifluoro acetic acid (TFA) salt.
(S)-2-Amino-3-[4-(2-amino-6-{2,2,2-trifluoro-l-[2-(pyridin-3-yloxy)-phenyl]- ethoxy}-pyrimidin-4-yl)-phenyl} -propionic acid. 1H-NMR (400 MHz, CD3OD) δ: 3.05-3.40 (m, 2H), 3.81 (m, IH), 6.64 (s, IH), 7.01(d, IH), 7.15-7.54 (m, 7H), 7.74 (d, IH), 7.94 (d, 2H), 8.35 (m, 2H).
(S)-2-Amino-3-[4-(2-amino-6-{2,2,2-trifluoro-l-[4-(pyridin-3-yloxy)-phenyl]- ethoxy}-pyrimidin-4-yl)-phenyl} -propionic acid. 1H-NMR (400 MHz, CD3OD) δ: 3.20-3.41 (m, 2H), 4.30 (m, IH), 6.81 (m, 2H), 7.17 (m, 2H), 7.46-7.69 (m, 6H), 7.93 (d, 2H), 8.41 (s, 2H).
(S)-2-Amino-3-[4-(6- {2,2,2-trifluoro- 1 -[4-(pyridin-3-yloxy)-phenyl]-ethoxy} - pyrimidin-4-yl)-phenyl}-propionic acid. 1H-NMR (300 MHz, CD3OD) δ: 3.15-3.35 (m, 2H), 4.25 (t, IH), 6.90 (q, IH), 7.25 (d, 2H), 7.45 (d, 2H), 7.71 (m, 3H), 7.99 (m, 3H), 8.14-8.18 (m, IH), 8.55 (d, IH), 8.63 (d, IH), 8.84 (d, IH).
(S)-2-Amino-3-[4-(2-amino-6-{2,2,2-trifluoro-l-(4-thiophen-2-yl-phenyl)-ethoxy]- pyrimidin-4-yl} -phenyl} -propionic acid. 1H-NMR (400 MHz, CD3OD) δ: 3.03-3.31 (m, 2H), 4.19 (m, IH), 6.68 (m, 2H), 7.00 (m, IH), 7.31-7.36 (m, 4H), 7.52 (m, 2H), 7.62 (d, 2H), 7.85 (d, 2H).
(S)-2-Amino-3-(4-{6-[2,2,2-trifluoro-l-(4-imidazol-l-yl-phenyl)-ethoxy]-pyrimidin- 4-yl}-phenyl)-propionic acid. 1H-NMR (400 MHz, CD3OD) δ: 3.03-3.31 (m, 2H), 4.19 (m, IH), 6.88 (m, IH), 7.32-8.63 (m, HH), 8.64 (s, IH), 9.25 (s, IH).
(S)-2-Amino-3-(4-{2-amino-6-[2,2,2-trifluoro-l-(4-[l,2,4]triazol-l-yl-phenyl)- ethoxy]-pyrimidin-4-yl}-phenyl)-propionic acid. 1H-NMR (400 MHz, CD3OD) δ: 3.07-3.36 (m, 2H), 4.16 (m, IH), 6.65 (s, IH), 6.75 (m, IH), 7.31 (d, 2H), 7.69 (d, 2H), 7.85 (m, 4H), 8.08 (s, IH), 9.03 (s, IH).
6.56. Synthesis of (S)-2-amino-3-[4-(2-amino-6-{2,2,2-trifluoro-l-r5-fluoro-2-(3- methyl-pyrazol-l-yl)-phenyll-ethoxy}-pyrimidin-4-yl)-phenyll-propionic acid
Figure imgf000084_0001
The mixture of 2-bromo-5-fluoro-benzoic acid methyl ester (1 g, 4.292 mmol), NaBH4 (0.423 g, 11.159 mmol) and LiCl (0.474 g, 11.159 mmol) in THF/EtOH (20 ml/10 ml) was stirred at room temperature overnight. Aqueous HCl (10 ml, 2N) was added and stirred for about 10 min. Then the organic solvent was removed under low vacuum. The residue was diluted with water and extracted by ethyl acetate. The organic layer was washed with aqueous NaHCO3 (10%), water and brine, and then dried (MgSO4) and concentrated to afford 852 mg (96.8% crude yield) crude product, (2- bromo-5-fluoro-phenyl)methanol, as a white solid, which was used without further purification. To the solution of (2-bromo-5-fluoro-phenyl)methanol (0.852 g, 4.156 mmol) in
DCM (15 ml) was added MnO2 (4.254 g, 85%, 41.56 mmol). The mixture was stirred at room temperature for two days, and then filtered and washed with DCM. The filtrate was concentrated to afford 777 mg 2-bromo-5-fluoro-benzaldehyde (92% yield). The newly made aldehyde (0.777 g, 3.828 mmol) was then dissolved in anhydrous THF (10 ml) and cooled to 00C. Trifluoromethyl trimethylsilane (1.13 ml, 7.656 mmol) was added, and followed by tetrabutyl ammonium fluoride (0.020 g, 0.076 mmol). The temperature was then allowed to warm to room temperature. The mixture was stirred for 5h at room temperature, then diluted with ethyl acetate, washed with water, brine and dried by MgSO4. The solvent was removed under reduced pressure to give 2-bromo-5-fluoro-phenyl)2,2,2-trifluoro- ethanol, 1.1 g (90% purity) as a crude product, which was used for the next step without further purification.
2-Bromo-5-fiuoro-phenyl)2,2,2-trifiuoro-ethanol (0.990 g, 3.263 mmol, 90%), 3- methyl pyrazole ( 0.476 g, 4.895 mmol), CuI (0.367 g, 1.632 mmol), K2CO3 (1.334 g, 8.158 mmol), (lR,2R)-N,N'-dimethyl-cyclohexane-l,2-diamine (0.110 g, 0.653 mmol) and toluene (10 ml) were combined in a 20 ml microwave vial, which was then sealed and heated at
1800C for 40 min. The mixture was filtered and washed with ethyl acetate. The filtrate was washed with water for 3 times and then silica gel was added to make a plug. The compound was purified by ISCO column chromatography using 5-10 % ethyl acetate in hexane as solvent to get l-(5-fluoro-2-(3-methyl-pyrazol-l-yl)-phenyl)-2,2,2-trifluoro-ethanol 75 mg. 1H-NMR (400 MHz, CDCl3) δ: 2.29(s, 3H), 4.90(m, IH), 6.21(d, IH), 7.07-7.1 l(m, IH), 7.19-7.22(m, IH), 7.29-7.32(m, IH), 7.5 l(d, IH).
The above -made alcohol (0.075 g, 0.273 mmol) was dissolved in anhydrous 1,4- dioxane (3 ml). Sodium hydride (0.013 g, 0.328 mmol, 60% in mineral oil) was added all at once, and the mixture was stirred at room temperature for 30 minutes. 2-Amino-4,6- dichloro-pyrimidine (0.045 g, 0.273 mmol) was added. The mixture was stirred at 800C for about 2 hours. The solvent was removed, and the residue was suspended in ethyl acetate, which was washed with water, dried over MgSO4 and then concentrated to give the desired monochloride product 100 mg (0.249 mmol), which was added to a 5 ml microwave vial containing 4-borono-L-phenylalanine (0.052 g, 0.249 mmol), Na2CO3 (0.053 g, 0.498 mmol), acetonitrile (2 ml) / water (2 ml) and dichlorobis(triphenylphosphine)-palladium (5 mg, 0.007 mmol). The vial was capped and stirred at 1500C for 5 min under microwave radiation. The reaction mixture was cooled, filtered through a syringe filter, and then separated by reverse phase preparative-HPLC using YMC-Pack ODS 100x30 mm ID column (MeOH/H2O/TFA solvent system). The pure fractions were concentrated in vacuum. The product was then suspended in 5 ml of water, frozen and lyophilized to give (S)-2-amino-3-[4-(2-amino-6- ((R)-I -[5-fluoro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro-ethoxy}-pyrimidin-4-yl)- phenyl} -propionic acid, 37 mg as a trifluoro salt. 1H-NMR (400 MHz, CD3OD): δ 2.29 (s, 3H), 3.08-3.30 (m, 2H), 4.19 (q, IH), 6.32 (d, IH), 6.82 (s, IH), 6.85 (m, IH), 7.26 (m, IH), 7.33 (d, 2H), 7.42 (m, 2H), 7.75 (d, IH), 7.87 (d, 2H).
6.57. Synthesis of (S)-2-amino-3-r4-q-amino-6{2,2,2-trifluoro-l-r5-chloro-2-(3- methyl-pyrazol-l-yl)-phenyll-ethoxy}-pyrimidin-4-yl)-phenyll-propionic acid
Figure imgf000086_0001
The title compounds was prepared from R-l-[5-chloro-2-(3-methyl-pyrazol-l-yl)- phenyl]-2,2,2-trifluoro-ethanol, which was prepared using the same approach as described above for R-l-[4-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro-ethanol. In particular, R-l-[5-chloro-2-(3-methyl-pyrazol-l-yl)-phenyl]-2,2,2-trifluoro-ethanol (0.959 g, 3.318 mmol) was dissolved in anhydrous 1,4-dioxane (8 ml). Sodium hydride (0.159 g, 3.982 mmol, 60% in mineral oil) was added all at once, and the mixture was stirred at room temperature for 30 minutes. 2-Amino-4,6-dichloro-pyrimidine (0.544 g, 3.318 mmol) was added. The mixture was stirred at 800C for about 2 hours. The solvent was removed, and the residue was suspended in ethyl acetate, which was washed with water, dried over MgSO4 and then concentrated to give the desired monochloride product 1.38 g, which was used directly without further purification.
The monochloride (0.460 g, 1.104 mmol) made above was added to a 20 ml microwave vial, which contained 4-borono-L-phenylalanine (0.277 g, 1.325 mmol), Na2CO3 (0.234 g, 2.208 mmol), acetonitrile (8 ml) / water (8 ml) and dichlorobis(triphenylphosphine)- palladium (0.039 g, 0.055 mmol). The vial was capped and the mixture stirred at 1500C for 10 minutes under microwave radiation. The mixture was cooled, filtered through a syringe filter and then separated by a reverse phase preparative-HPLC using YMC-Pack ODS 100x30 mm ID column (MeOHZH2OZTFA solvent system). The pure fractions were concentrated in vacuum. The product was then suspended in 5 ml of water, frozen and lyophilized to give 580 mg of (S)-2-amino-3-[4-(2-amino-6- (R- 1 -[5-chloro-2-(3-methyl-pyrazol- 1 -yl)-phenyl]- 2,2,2-trifluoro-ethoxy}-pyrimidin-4-yl)-phenyl} -propionic acid. 1H-NMR (400 MHz, CD3OD): δ 2.40 (s, 3H), 3.29-3.46 (m, 2H), 4.38 (q, IH), 6.45 (d, IH), 7.09 (s, IH), 7.24 (m, IH), 7.53-7.70 (m, 4H), 7.82 (s, IH), 7.90 (d, IH), 7.97 (d, 2H).
6.58. Synthesis of (S)-2-amino-3-[4-(2-amino-6-{2,2,2-trifluoro-l-r4-(2-oxo- Pyrrolidin-l-yl)-phenyll-ethoxy}-pyrimidin-4-yl)-phenyll-propionic acid
Figure imgf000087_0001
4-(2-Oxo-pyrrolidine-l-yl)-benzaldehyde (500 mg, 2.64 mmol) in THF (20 ml) was cooled to 00C and trifluoromethyl trimethyl silane (375mg, 2.64 mmol) was added. Tetrabutylammonium fluoride (IM, 0.1 ml) was added dropwise, and the mixture was allowed to warm to room temperature over Ih and stirred further for over-night at room termperature. After completion of the reaction, 3N HCl (5 ml) was added, and the reaction mixture was stirred for 2 hr. The mixture was concentrated. Water (20ml) was added and the mixture was extracted by EtOAc (2x20ml) and washed with NaHCO3 (20 ml), brine (20 ml), and dried over sodium sulfate and concentrated to give 590 mg of desired product, which was used in next step without further purification (yield of 86%).
A solution of 4,6-dichloro-pyrimidin-2-ylamine (700 mg, 2.69 mmol), NaH (194 mg, 8.07 mmol, 60%) and l-(4-(2,2,2-trifluoro-l-hydroxy-ethyl)-phenyl)-pyrrolidine-2-one (441 mg, 2.69 mmol) in dry THF (10 ml) was stirred at room temperature for overnight. After completion of the reaction, THF was removed under reduced pressure. Water (10 ml) was added while the mixture was cooled down to 00C. The mixture was then extracted with dichloromethane (2x40ml). The combined organic solution was dried over Na2SO4. Removal of solvent gave 498 mg of desired product with 92% purity, which was used in next step without further purification (yield of 498 mg, 48%).
An Emrys process vial (20 ml) for microwave was charged with l-(4-(2-amino-6- chloro-pyrimidin-4-yloxy)-2,2,2-trifluoro-ethyl)-phenyl)-pyrrolidine-2-one (200 mg, 0.51 mmol), 4-borono-L-phenylalanine (108 mg, 0.51 mmol) and 5 ml of acetonitrile. 5 ml of aqueous sodium carbonate (IM) was added to above solution followed by 5 mol % of dichlorobis(triphenylphosphine)-palladium (II). The reaction vessel was sealed and heated to 16O0C for 7min with microwave irradiation. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in 4 ml of methanol and purified with Prep-LC to give 153 mg of product (yield 58%). 1H-NMR (400 MHz, CD3OD): δ (ppm) 2.1 (m, 2H), 2.5 (t, 2H), 3.05-3.4(m, 2H), 3.85 (t, 2H), 4.2 (m, IH), 6.6(m, IH), 6.75(s, IH), 7.3(d, 2H), 7.5 (d, 2H), 7.6 (d, 2H), 7.9 (d, 2H).
6.59. Synthesis of (S)-2-Amino-3-r4-q-amino-6-{(R)-2,2,2-trifluoro-l-r5-fluoro-
2-f3-methyl-pyrazol-l-yl)-phenyll-ethoxy}-pyrimidin-4-yl)-phenyll- propionic acid
Figure imgf000088_0001
R-l-(2-Bromo-5-fluoro-phenyl)-2,2,2-trifluoro-ethanol (4.Og, 14.65 mmol), 3-methyl pyrazole (1.56 g, 19.04 mmol), CuI (0.557g, 2.93 mmol), K2CO3 (4.25 g, 30.76 mmol), (lR,2R)-N,N'-dimethyl-cyclohexane-l,2-diamine (0.416 g, 2.93 mmol) and toluene (15 ml) were taken in 50 ml of sealed tube and the resulting mixture was heated at 1300C (oil bath temperature) for 2 days. Mixture was diluted with ethyl acetate and washed with H2O (4 x 30 ml), brine, and dried over sodium sulfate. Removal of solvent gave a crude product, which was purified by ISCO column chromatography using 5-10 % ethyl acetate in hexane as solvent to give 1.75 g of R-2,2,2-trifluoro-l-[5-fluoro-2-(3-methyl-pyrazol-l-yl)-phenyl]- ethanol (Yield: 44 %). 1H-NMR (400 MHz, CDCl3): δ (ppm) 2.35(s, 3H), 5.0(m, IH), 6.3(s, IH), 7.1(m, IH), 7.20(s, IH), 7.35(d, IH), 7.50(s, IH).
A solution of 4, 6-dichloro-pyrimidin-2-ylamine (938 mg, 5.72 mmol), NaH (188 mg, 1.5 eq. 8.17 mmol, 60%) and R-2,2,2-trifluoro-l-[5-fluoro-2-(3-methyl-pyrazol-l-yl)- phenyl] -ethanol (1.5 g, 1 eq. 5.45 mmol) in dry THF (10 ml) was stirred at room temperature at 500C overnight. After completion of the reaction, THF was removed under reduced pressure. Water (10 ml) was added to quench the reaction. The mixture was then extracted with dichloromethane (2x40ml). The combined organic solution was dried over Na2SO4. Removal of solvent gave desired product with 92% purity, which was used in next step without purification (yield: 85%).
An Emrys process vial (20 ml) for microwave was charged with chloro-6-R-2,2,2- trifluoro- 1 -(5 -fluoro-2-(3 -methyl-pyrazol- 1 -yl)-phenyl)-ethoxy)-pyrimidin-2-ylamine (2.18 g, 5.45 mmol), 4-borono-L-phenylalanine (1.13 g, 5.45 mmol), sodium carbonate (1 M 10.90 ml, 2 eq.) was added to above solution followed by 5 mol % of dichlorobis (triphenylphosphine)-palladium(II) (191 mg, 0.27 mmol) and 5 ml of acetonitrile, and 5 ml H2O. The reaction vessel was sealed, and the mixture was heated at 16O0C for 10 minutes with microwave irradiation. After cooling, the reaction mixture was evaporated to dryness. The residue was dissolved in H2O (10 ml) and extracted with ether. The ethereal layer was discarded. Then most of the water in the aqueous phase was removed in vacuo followed by addition of 10 ml of methanol. The crude product was purified with Prep-HPLC to give 1.163 g (yield 75%) of product. 1H-NMR (400 MHz, CD3OD): δ (ppm) 2.4 (s, 3H), 3.35 (m, IH), 3.5 (m, IH), 4.36 (m, IH), 6.4 (s, IH), 7.0 (s, 1H),7.1 (m,lH), 7.4 (m, IH), 7.55 (m, 4H), 7.85 (s, IH), 8.0 (d, 2H).
6.60. Synthesis of (S)-2-Amino-3-[4-(2-amino-6-{2,2,2-trifluoro-l-r4-(6- methoxy-pyridin-2-yl)-phenyll-ethoxy}-pyrimidin-4-yl)-phenyll-propionic acid
Figure imgf000089_0001
Tetrabutylammonium fluoride (TBAF) (0.1 ml of IM in THF) was added to a solution of 4-(6-methoxy-pyridine-2-yl)-benzaldehyde (213 mg, 1 mmol) and trifluoromethyl trimethylsilane (0.2 ml, 1.2 mmol) in 10 ml THF at 00C. The mixture was warmed up to room temperature and stirred for 4 hours. The reaction mixture was then treated with 12 ml of IM HCl and stirred overnight. The product was extracted with ethyl acetate (3x20ml). The organic layer was separated and dried over sodium sulfate. The organic solvent was evaporated to give 0.25g of l-[4-(6-methoxy-pyridine-2-yl)-phenyl]-2,2,2-trifluoro-ethanol which was directly used in next step without purification, yield: 90%.
CS2CO3 (375 mg, 1 mmol) was added to a solution of l-[4-(6-methoxy-pyridine-2-yl)- phenyl]-2,2,2-trifluoro-ethanol (67mg, 0.2mmol) in 10 ml of anhydrous 1,4-dioxane. The mixture was stirred for 5 min, then was added (S)-3-[4-(2-amino-6-chloro-pyrimidin-4-yl)- phenyl]-2-tert-butoxycarbonylamino-propionic acid (78 mg, 0.2 mmol), and the mixture was heated at 1100C overnight. After cooling, 5 ml water was added and ethyl acetate (20 ml) was used to extract the product. The organic layer was dried over sodium sulfate. The solvent was removed by rotovap to give 112 mg (S)-3-[4-(2-Amino-6-{2,2,2-trifluoro-l-[4- (6-methoxy-pyridin-2-yl)-phenyl]-ethoxy}-pyrimidin-4-yl)-phenyl]-2-tert- butoxycarbonylamino-propionic acid (yield: 88%).
The above product (112 mg) was added into 5 ml of 30% TFA/DCM solution. Upon completion of the reaction, the solvent was evaporated to give a crude product, which was purified by preparative HPLC to give 5 mg of (S)-2-amino-3-[4-(2-amino-6-{2,2,2-trifluoro- 1 -[4-(6-methoxy-pyridin-2-yl)-phenyl]-ethoxy} -pyrimidin-4-yl)-phenyl]propionic acid. 1H NMR (300MHz, CD3OD) δ (ppm) 8.18 (d, J=8.4Hz, 2 H), 7.94 (d, J=8.4Hz, 2 H), 7.74 (m, 3 H), 7.60 (d, J=8.4Hz, 2 H), 7.52 (d, J=7.2Hz, 1 H), 7.08 (s, 1 H), 6.86(m, IH), 6.82 (d, J=8.1Hz IH), 4.37 (t, 1 H), 4.03(s, 3 H), 3.5 (m, 2 H).
6.61. Synthesis of (S)-2-Amino-3-[4-(2-amino-6-{2,2,2-trifluoro-l-r2-fluoro-4-(5- methoxy-pyridin-3-yl)-phenyll-ethoxy}-pyrimidin-4-yl)-phenyll-propionic acid
Figure imgf000090_0001
TBAF (0.1 ml) was added to a solution of 4-bromo-2-fluoro-benzaldehyde (2.03 g, 10 mmol) and TMSCF3 (20ml, 12 mmol) in 10 ml THF at 00C. The formed mixture was warmed up to room temperature and stirred for 4 hours. The reaction mixture was then treated with 12 ml of 3M HCl and stirred overnight. The product was extracted with ethyl acetate (3x20ml). The organic layer was separated and dried over sodium sulfate. The organic solvent was evaporated to give 2.4g of l-(4-bromo-2-fluoro-phenyl)-2,2,2-trifluoro- ethanol (yield: 90%). CS2CO3 (8.45g, 26mmol) was added to the solution of l-(4-bromo-2-fluoro-phenyl)-
2,2,2-trifluoro-ethanol (1.4g, 5.2mmol) in 10 ml of anhydrous 1,4-dioxane, the mixture was stirred for 5 minutes, then (S)-3-[4-(2-amino-6-chloro-pyrimidin-4-yl)-phenyl]-2-tert- butoxycarbonylamino-propionic acid (2.0 g, 5 mmol) was added, and the resulting mixture was heated at 1100C overnight. After cooling, 5 ml of water was added and ethyl acetate (20 ml) was used to extract the product. The organic layer was dried over sodium sulfate. The solvent was removed by rotovap to give 2.6 g of (S)-3-(4-{2-amino-6-[l-(4-bromo-2-fluoro- phenyl)-2,2,2-trifluoro-ethoxy]-pyrimidin-4-yl}phenyl)2tertbutoxycarbonylamino-propionic acid (yield: 82%).
A microwave vial (2 ml) was charged with (S)-3-(4-{2-amino-6-[l-(4-bromo-2- fluoro-phenyl)-2,2,2-trifluoro-ethoxy]-pyrimidin-4-yl} -phenyl)-2-tert-butoxycarbonylamino- propionic acid (130 mg, 0.2 mmol), 3-methoxy-5-(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2- yl)-pyridine (70 mg, 0.3 mmol) 1 ml of acetonitrile, and 0.7 ml of water. To this mixture was added 0.4 ml of aqueous sodium carbonate (IM), followed by 14 mg (5 mol %) of dichlorobis(triphenylphosphine) palladium(II). The reaction vessel was sealed and heated to 1500C for 5 minutes with microwave irradiation. After cooling, the reaction mixture was evaporated to dryness, the residue was dissolved in 2.5 ml of methanol and purified with Prep HPLC to give 51 mg of (S)-3-[4-(2-amino-6-{2,2,2-trifluoro-l-[2-fluoro-4-(5-methoxy- pyridin-3-yl)-phenyl]-ethoxy}-pyrimidin-4-yl)-phenyl]-2-tert-butoxycarbonylamino- propionic acid. The above-product (51 mg) was dissolved in 5 ml of 30% TFA/DCM solution. The mixture was stirred at room temperature overnight. Removal of solvent gave a crude product, which was purified by Prep HPLC to give 17 mg of (S)-2-amino-3-[4-(2-amino-6- {2,2,2- trifluoro- 1 -[2-fluoro-4-(5-methoxy-pyridin-3-yl)-phenyl]-ethoxy} -pyrimidin-4-yl)-phenyl]- propionic acid. 1H NMR (300MHz, CD3OD) δ (ppm): 8.73 (s, 1 H), 8.56 (s, 1 H), 8.25 (s, 1 H), 7.94 (d, J=8.2Hz, 2 H), 7.77(m, 3H), 7.55 (d, J=8.4Hz, 2 H), 7.16 (m, IH), 7.00(s, IH), 4.35 (t, 1 H), 4.09(s, 3 H), 3.4 (m, 2 H). 6.62. Synthesis of (S)-2-Amino-3- r4-(2-amino-6-{(S)-2,2,2-trifluoro-l- f4-(2- fluoro-pyridin-4-yl)-phenyll-ethoxy}-pyrimidin-4-yl)-phenyll-propionic acid
Figure imgf000092_0001
CS2CO3 (16.25g, 50 mmol) was added to the solution of (S)-I -(4-bromo-phenyl)-
2,2,2-trifluoro-ethanol (2.55 g, 11.0 mmol) in 10 ml of anhydrous 1,4-dioxane, and the mixture was stirred for 5 minutes, after which (S)-3-[4-(2-amino-6-chloro-pyrimidin-4-yl)- phenyl]-2-tert-butoxycarbonylamino-propionic acid (3.92 g, 10 mmol) was added. The resulting mixture was heated at 1100C overnight. After cooling, 5 ml of water was added and ethyl acetate (20 ml) was used to extract the product. The organic layer was dried over sodium sulfate. The solvent was removed by rotovap to give 5.2 g of (S)-3-(4-{2-amino-6- [(S)-I -(4-bromo-phenyl)-2,2,2-trifluoro-ethoxy]-pyrimidin-4-yl}phenyl)-2-tert-butoxy- carbonylamino-propionic acid (yield: 82%).
A microwave vial (2 ml) was charged with (S)-3-(4-{2-amino-6-[(S)-l-(4-bromo- phenyl)-2,2,2-trifluoro-ethoxy]-pyrimidin-4-yl} -phenyl)-2-tert-butoxycarbonylamino- propionic acid (139 mg, 0.23 mmol), 2-fluoropyridine-4-boronic acid (40 mg, 0.27 mmol) 1 ml of acetonitrile, and 0.7ml of water. To this mixture, 0.4 ml of aqueous sodium carbonate (IM) was added, followed by 14 mg (5 mol %) of dichlorobis(triphenylphosphine)- palladium(II). The reaction vessel was sealed and heated to 1500C for 5 minutes with microwave irradiation. After cooling, the reaction mixture was evaporated to dryness, and the residue was dissolved in 2.5 ml of methanol. The product was purified with Preparative HPLC to give 70 mg of (S)-3-[4-(2-amino-6-{(S)-2,2,2-trifluoro-l-[4-(2-fiuoro-pyridin-4-yl)- phenyl] -ethoxy } -pyrimidin-4-yl)-phenyl] -2-tert-butoxycarbonylamino-propionic acid.
The above product (70 mg) was dissolved in 5 ml 30% TFA in DCM. The reaction mixture was stirred at r.t. overnight. Removal of solvent gave crude product which was purified by preparative HPLC to give 52 mg of (S)-2-amino-3-[4-(2-amino-6-{(S)-2,2,2- trifluoro-l-[4-(2-fluoro-pyridin-4-yl)-phenyl]-ethoxy}-pyrimidin-4-yl)-phenyl]-propionic acid. 1H NMR (300MHz, CD3OD) δ (ppm) 8.17 (d, J=5.7Hz, 1 H), 7.85 (d, J=8.4Hz, 2 H), 7.77(d, J=6.9Hz ,2H), 7.67(d, J=8.2Hz ,2H), 7.53 (m, 1 H), 7.38 (d, J=8.4Hz,, 2H), 7.30(s, IH), 6.76 (m, 2H), 4.21 (t, 1 H), 3.2 (m, 2 H).
6.63. Synthesis of (S)-2-Amino-3- r4-(2-amino-6-{(S)-2,2,2-trifluoro-l- f4-(5- methoxy-pyridin-3-yl)-phenyll-ethoxy}-pyrimidin-4-yl)-phenyll-propionic acid
Figure imgf000093_0001
A microwave vial (2 ml) was charged with (S)-3-(4-{2-amino-6-[(S)-l-(4-bromo- phenyl)-2,2,2-trifluoro-ethoxy]-pyrimidin-4-yl}-phenyl)-2-tert-butoxycarbonylamino- propionic acid (139 mg, 0.23 mmol), 3-methoxy-5-(4,4,5,5-tetramethyl-[l,3,2]-dioxaborolan- 2-yl)-pyridine (69 mg, 0.27 mmol), 1 ml of acetonitrile, and 0.7ml of water. To this mixture was added 0.4 ml of aqueous sodium carbonate (IM), followed by 14 mg of dichlorobis- (triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 1500C for 5 minutes with microwave irradiation. After cooling, the reaction mixture was evaporated to dryness, the residue was dissolved in 2.5 ml of methanol and purified by preparative HPLC to give 60 mg of (S)-3-[4-(2-amino-6-{(S)-2,2,2-trifluoro-l-[4-(5-methoxy-pyridin-3-yl)- phenyl]-ethoxy} -pyrimidin-4-yl)-phenyl]-2-tert butoxycarbonylamino-propionic acid.
The above product (60 mg) was dissolved in 5 ml of 30% TFA in DCM. The reaction mixture was stirred at room temperature overnight. Removal of solvent gave a crude product which was purified by preparative HPLC to give 48 mg of (S)-2-amino-3-[4-(2-amino-6- {(S)-2,2,2-trifluoro- 1 -[4-(5-methoxy-pyridin-3-yl)-phenyl]-ethoxy} -pyrimidin-4-yl)-phenyl]- propionic acid. 1H NMR (300MHz, CD3OD) δ (ppm): 8.54 (d, J=I.5Hz, 1 H), 8.37 (d, J=2.7Hz, 1 H), 8.03 (dd, J=2.7Hz, 1.5Hz, IH), 7.84 (d, J=8.2Hz, 2 H), 7.78(d, J=8.4Hz ,2H), 7.70(d, J=8.4Hz ,2H), 7.41 (d, J=8.4Hz,, 2H), 6.81(s, IH), 6.75 (m, IH), 4.22 (t, 1 H), 3.95 (t, 3 H), 3.25 (m, 2 H). 6.64. Synthesis of (S)-2-Amino-3- r4-(2-amino-6-{(S)-2,2,2-trifluoro-l- f4-(4- trifluoromethyl-pyridin-3-yl)-phenyll-ethoxy}-pyrimidin-4-yl)-phenyll- propionic acid
Figure imgf000094_0001
A microwave vial (2 ml) was charged with (S)-3-(4-{2-amino-6-[(S)-l-(4-bromo- phenyl)-2,2,2-trifluoro-ethoxy]-pyrimidin-4-yl}-phenyl)-2-tert-butoxycarbonylamino- propionic acid (139 mg, 0.23 mmol), 4-trifluoromethylpyridine-3-boronic acid (61 mg, 0.3 mmol), 1 ml of acetonitrile, and 0.7 ml of water. To this mixture was added 0.4 ml of aqueous sodium carbonate (IM), followed by 14 mg of dichlorobis(triphenylphosphine)- palladium(II). The reaction vessel was sealed and heated to 1500C for 5 minutes with microwave irradiation. After cooling, the reaction mixture was evaporated to dryness, the residue was dissolved in 2.5 ml of methanol and was purified by preparative HPLC to give 20 mg of (S)-3-[4-(2-amino-6-{(S)-2,2,2-trifluoro-l-[4-(4-trifluoromethyl-pyridin-3-yl)-phenyl]- ethoxy } -pyrimidin-4-yl)-phenyl]-2-tert butoxycarbonylamino-propionic acid The above product (20 mg) was dissolved in 5 ml of 30% TFA in DCM. The reaction mixture was stirred at r.t. overnight. Removal of solvent gave crude product which purified by preparative HPLC to give 10 mg of (S)-2-amino-3-[4-(2-amino-6-{(S)-2,2,2-trifluoro-l- [4-(4-trifluoromethyl-pyridin-3-yl)-phenyl]-ethoxy}-pyrimidin-4-yl)-phenyl]-propionic acid. 1H NMR (300MHz, CD3OD) δ (ppm): 8.72 (d, J=5.1Hz, 1 H), 8.55 (s, 1 H), 7.87 (d, J=8.2, 2H), 7.72 (d, J=5.0Hz, 1 H), 7.63(d, J=8.2Hz ,2H), 7.36(m, 4H), 6.81(m, IH), 6.70 (s, IH), 4.20 (t, 1 H), 3.22 (m, 2 H). 6.65. Synthesis of (S)-2-Amino-3-(4-{2-amino-6-r(S)-2,2,2-trifluoro-l-(4- isoxazol-4-yl-phenyl)-ethoxyl-pyrimidin-4-yl}-phenyl)-propionic acid
Figure imgf000095_0001
A microwave vial (2 ml) was charged with (S)-3-(4-{2-amino-6-[(S)-l-(4-bromo- phenyl)-2,2,2-trifluoro-ethoxy]-pyrimidin-4-yl}-phenyl)-2-tert-butoxycarbonylamino- propionic acid (139 mg, 0.23 mmol), 4-(4,4,5,5-tetramethyl-[l,3,2]dioxaborolan-2-yl)- isoxazole (57.5 mg, 0.3 mmol), 1 ml of acetonitrile, and 0.7ml of water. To this mixture was added 0.4 ml of aqueous sodium carbonate (IM), followed by 14mg of dichlorobis- (triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 1500C for 5 minutes with microwave irradiation. After cooling, the reaction mixture was evaporated to dryness, the residue was dissolved in 2.5 ml of methanol and was purified by preparative HPLC to give 20 mg of (S)-3-(4-{2-amino-6-[(S)-2,2,2-trifiuoro-l-(4-isoxazol-4-yl-phenyl)- ethoxy]-pyrimidin-4-yl} -phenyl)-2-tert-butoxycarbonylamino propionic acid.
The above product (20 mg) was dissolved in 5 ml of 30% TFA in DCM. The mixture was stirred at r.t. overnight. Removal of solvent gave a crude product, which was purified by preparative HPLC to give 10 mg of (S)-2-amino-3-(4-{2-amino-6-[(S)-2,2,2-trifluoro-l-(4- isoxazol-4-yl-phenyl)-ethoxy]-pyrimidin-4-yl}-phenyl)-propionic acid. 1H NMR (300MHz, CD3OD) δ (ppm) 9.03 (s, IH), 8.77(s, IH), 7.84 (m, 2H), 7.63 (d, J=8.2, IH), 7.56 (d, J=8.4Hz, 1 H), 7.50(m, IH), 7.37(m, 3H), 6.70(m, 2H), 4.20 (t, 1 H), 3.22 (m, 2 H).
6.66. Synthesis of (S)-2-Amino-3-(4-{2-amino-6-[2,2,2-trifluoro-l-(2-pyrimidin-
5-yl-phenyl)-ethoxyl -pyrimidin-4-yl}-phenyl)-propionic acid
Figure imgf000095_0002
A microwave vial (20 ml) was charged with 2-formylphenylboronic acid (290 mg, 2.0 mmol), 5-bromo-pyrimidine (316 mg, 2.0 mmol) and 8 ml of acetonitrile. To this mixture was added 4 ml of aqueous sodium carbonate (IM), followed by 100 mg of dichlorobis- (triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated at 1500C for 5 minutes with microwave irradiation. After cooling, the reaction mixture was extracted with ethylacetate. The organic layer was evaporated to provide a crude material, which was purified by ISCO to give 220 mg of 2-pyrimidin-5-yl-benzaldehyde.
Tetrabutylammonium fluoride (TBAF, 0.1 ml of IM in THF) was added to a solution of 2-pyrimidin-5-yl-benzaldehyde (184 mg, 1 mmol) and trifluoromethyl trimethylsilane (TMSCF3, 0.2 ml, 1.2 mmol) in 10 ml THF at 00C. The mixture was warmed up to room temperature and stirred for 4 hours. The mixture was then treated with 3 ml of IM HCl and stirred overnight. The product was extracted with ethyl acetate (3x20ml). The organic layer was separated and dried over sodium sulfate. The organic solvent was evaporated to give 0.21 g of 2,2,2-trifluoro-l-(2-pyrimidin-5-yl-phenyl)-ethanol (yield: 84%), which was directly used in next step without purification.
Cs2CO3 (325 mg, 1.0 mmol) was added to a solution of 2,2,2-trifluoro-l-(2-pyrimidin- 5-yl-phenyl)-ethanol (72 mg, 0.28 mmol) in 10 ml of anhydrous THF. The mixture was stirred for 20 min, 2-amino-4,6-dichloro-pyrimidine (36.7 mg, 0.22 mmol) was added and then the reaction mixture was heated at 1100C until the reaction was completed. After cooling to room temperature, 5 ml of water was added and ethyl acetate (20 ml) was used to extract the product. The organic layer was dried over sodium sulfate. The solvent was removed by rotovap to give 76 mg of crude 4-chloro-6-[2,2,2-trifluoro-l-(2-pyrimidin-5-yl- phenyl)-ethoxy]-pyrimidin-2-ylamine (yield: 92%).
A microwave vial (2 ml) was charged with above crude intermediate (38 mg, 0.1 mmol), 4-borono-L-phenylalanine (31 mg, 0.15 mmol), 1 ml of acetonitrile, and 0.7ml of water. To this mixture was added 0.3 ml of aqueous sodium carbonate (IM), followed by 4 mg, 5 mol % of dichlorobis(triphenylphosphine)-palladium(II). The reaction vessel was sealed and heated to 1500C for 5 minutes with microwave irradiation. After cooling, the reaction mixture was evaporated to dryness, the residue was dissolved in 2.5 ml of methanol and then purified with preparative HPLC to give 10 mg of (S)-2-amino-3-(4-{2-amino-6-
[2,2,2-trifluoro- 1 -(2-pyrimidin-5-yl-phenyl)-ethoxy]-pyrimidin-4-yl} -phenyl)-propionic acid. 1H NMR (300MHz, CD3OD) δ (ppm) 9.21 (s, 1 H), 8.87 (s, 2 H), 7.86 (d, J=8.4, 2H), 7.75 (m, 1 H), 7.53(m, 2H), 7.37(d, J=8.2, IH), 7.33 (m, IH), 6.72(s, IH), 6.58 (m, IH), 4.20 (t, 1 H), 3.22 (m, 2 H). 6.67. Additional Compounds
Additional compounds prepared using methods known in the art and/or described herein are listed below:
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
Figure imgf000103_0001
Figure imgf000104_0001
Figure imgf000105_0001
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
6.68. In Vitro Inhibition Assays
Human TPHl, TPH2, tyrosine hydroxylase (TH) and phenylalanine hydroxylase (PH) were all generated using genes having the following accession numbers, respectively: X52836, AY098914, X05290, and U49897. The full-length coding sequence of human TPHl was cloned into the bacterial expression vector pET24 (Novagen, Madison, WI, USA). A single colony of BL21(DE3) cells harboring the expression vector was inoculated into 50 ml of L broth (LB)- kanamycin media and grown up at 37°C overnight with shaking. Half of the culture (25 ml) was then transferred into 3 L of media containing 1.5% yeast extract, 2% Bacto Peptone, 0.1 mM tryptophan, 0.1 mM ferrous ammonium sulfate, and 50 mM phosphate buffer (pH 7.0), and grown to ODβoo = 6 at 37°C with oxygen supplemented at 40%, pH maintained at 7.0, and glucose added. Expression of TPHl was induced with 15% D-lactose over a period of 10 hours at 25°C. The cells were spun down and washed once with phosphate buffered saline (PBS). TPHl was purified by affinity chromatography based on its binding to pterin. The cell pellet was resuspended in a lysis buffer (100 ml/20 g) containing 50 mM Tris-Cl, pH 7.6, 0.5 M NaCl, 0.1% Tween-20, 2 mM EDTA, 5 mM DTT, protease inhibitor mixture (Roche Applied Science, Indianapolis, IN, USA) and 1 mM phenylmethanesulfonyl fluoride (PMSF), and the cells were lyzed with a microfluidizer. The lysate was centrifuged and the supernatant was loaded onto a pterin-coupled sepharose 4B column that was equilibrated with a buffer containing 50 mM Tris, pH 8.0, 2 M NaCl, 0.1% Tween-20, 0.5 mM EDTA, and 2 mM DTT. The column was washed with 50 ml of this buffer and TPHl was eluded with a buffer containing 30 mM NaHCO3, pH 10.5, 0.5 M NaCl, 0.1% Tween-20, 0.5 mM EDTA, 2 mM DTT, and 10% glycerol. Eluted enzyme was immediately neutralized with 200 mM KH2PO4, pH 7.0, 0.5 M NaCl, 20 mM DTT, 0.5mM EDTA, and 10% glycerol, and stored at -800C. Human tryptophan hydroxylase type II (TPH2), tyrosine hydroxylase (TH) and phenylalanine hydroxylase (PAH) were expressed and purified essentially in the same way, except the cells were supplemented with tyrosine for TH and phenylalanine for PAH during growth. TPHl and TPH2 activities were measured in a reaction mixture containing 50 mM 4- morpholinepropanesulfonic acid (MOPS), pH 7.0, 60 μM tryptophan, 100 mM ammonium sulfate, 100 μM ferrous ammonium sulfate, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), 0.3 mM 6-methyl tetrahydropterin, 0.05 mg/ml catalase, and 0.9 mM DTT. The reactions were initiated by adding TPHl to a final concentration of 7.5 nM. Initial velocity of the reactions was determined by following the change of fluorescence at 360 nm (excitation wavelength = 300 nm). TPHl and TPH2 inhibition was determined by measuring their activities at various compound concentrations, and the potency of a given compound was calculated using the equation:
Figure imgf000109_0001
where v is the initial velocity at a given compound concentration C, Vo is the v when C = 0, b is the background signal, D is the Hill slope which is approximately equal to 1 , and Icso is the concentration of the compound that inhibits half of the maximum enzyme activity.
Human TH and PAH activities were determined by measuring the amount of 3H2O generated using L- [3, 4- H] -tyrosine and L- [4- H] -phenylalanine, respectively. The enzyme (100 nM) was first incubated with its substrate at 0.1 mM for about 10 minutes, and added to a reaction mixture containing 50 mM MOPS, pH 7.2, 100 mM ammonium sulfate, 0.05% Tween-20, 1.5 mM TCEP, 100 μM ferrous ammonium sulfate, 0.1 mM tyrosine or phenylalanine, 0.2 mM 6-methyl tetrahydropterin, 0.05 mg/ml of catalase, and 2 mM DTT. The reactions were allowed to proceed for 10-15 minutes and stopped by the addition of 2 M HCl. The mixtures were then filtered through activated charcoal and the radioactivity in the filtrate was determined by scintillation counting. Activities of of compounds on TH and PAH were determined using this assay and calculated in the same way as on TPHl and TPH2.
6.69. Cell-Based Inhibition Assays Two types of cell lines were used for screening: RBL2H3 is a rat mastocytoma cell line, which contains TPHl and makes 5-hydroxytrypotamine (5HT) spontaneously; BON is a human carcinoid cell line, which contains TPHl and makes 5-hydroxytryptophan (5HTP). The CBAs were performed in 96-well plate format. The mobile phase used in HPLC contained 97% of 100 mM sodium acetate, pH 3.5 and 3% acetonitrile. A Waters Cl 8 column (4.6 x 50 mm) was used with Waters HPLC (model 2795). A multi-channel fluorometer (model 2475) was used to monitor the flow through by setting at 280 nm as the excitation wavelength and 360 nm as the emission wavelength.
RBL CBA: Cells were grown in complete media (containing 5 % bovine serum) for 3-4 hours to allow cells to attach to plate wells (7K cell/well). Compounds were then added to each well in the concentration range of 0.016 μM to 11.36 μM. The controls were cells in complete media without any compound present. Cells were harvested after 3 days of incubation at 37°C. Cells were >95% confluent without compound present. Media were removed from plate and cells were lysed with equal volume of 0.1 N NaOH. A large portion of the cell lysate was treated by mixing with equal volume of IM TCA and then filtered through glass fiber. The filtrates were loaded on reverse phase HPLC for analyzing 5HT concentrations. A small portion of the cell lysate was also taken to measure protein concentration of the cells that reflects the cytotoxicity of the compounds at the concentration used. The protein concentration was measured by using BCA method.
The average of 5HT level in cells without compound treated was used as the maximum value in the IC50 derivation according to the equation provided above. The minimum value of 5HT is either set at 0 or from cells that treated with the highest concentration of compound if a compound is not cytotoxic at that concentration.
BON CBA: Cells were grown in equal volume of DMEM and F12K with 5 % bovine serum for 3-4 hours (2OK cell/well) and compound was added at a concentration range of 0.07 μM to 50 μM. The cells were incubated at 37°C overnight. Fifty μM of the culture supernatant was then taken for 5HTP measurement. The supernatant was mixed with equal volume of IM TCA, then filtered through glass fiber. The filtrate was loaded on reverse phase HPLC for 5HTP concentration measurement. The cell viability was measured by treating the remaining cells with Promega Celltiter-Glo Luminescent Cell Viability Assay. The compound potency was then calculated in the same way as in the RBL CBA.
6.70. Effects on Gastric Transit and Emptying
The effect of a potent TPHl inhibitor of the invention on gastrointestinal (GI) transit time and gastric emptying was determined in Sprague-Dawley rats. The compound was administred at doses of 50, 125 and 250 mpk, po, qd, for 14 days. Each dosing group utilized nine rats. Nine rats were also used as a negative control group (vehicle administration only), and another six were used as a positive control (Atropine).
The rats were dosed compound or vehicle at 10 ml/kg. Atropine was given to the positive control group on day 14 only, whereas vehicle was given on days 1-14. Body weights and observations were taken through out study, and the rats were fasted overnight on day 13 prior to the charcoal meal. On day 14, the potent TPHl inhibitor, Atropine or vehicle wereorally dosed 30 minutes prior to the charcoal meal. The charcoal meal (5% charcoal in vehicle) was orally dosed at 15 ml/kg. Necropsy was performed 25 minutes after the charcoal meal dose. GI transit times were determined by measuring the distance the charcoal meal traveled down the small intestine, and dividing that distance by the total length of the small intestine. Gastric emptying times were determined by weighing the stomachs of the rats.
As shown in Figure 1, administration of the potent TPHl inhibitor slowed GI motility in a dose-dependent manner. As shown in Figure 2, it also slowed gastric emptying in a dose-dependent manner. And as shown in Figure 3, the effects of the compound on GI transit and gastic emptying correlate with changes in 5 -HT levels in the blood and proximal colon.
Brain 5 -HT levels were unaffected by the compound.
All publications (e.g., patents and patent applications) cited above are incorporated herein by reference in their entireties.

Claims

CLAIMSWhat is claimed is:
1. The use of a compound of formula I:
Figure imgf000112_0001
I or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for slowing gastrointestinal motility in a patient, wherein:
A is optionally substituted cycloalkyl, aryl, or heterocycle; X is a bond, -O-, -S-, -C(O)-, -C(R4)=, KXR4)-, -C(R3R4)-, -C(R4)K(R4)-, -C≡C-,
-N(R5)-, -N(R5)C(O)N(R5)-, -C(R3R4)N(R5)-, -N(R5)C(R3R4)-, -ONC(R3)-, -C(R3)NO-, -C(R3R4)O-, -OC(R3R4)-, -S(O2)-, -S(O2)N(R5)-, -N(R5)S(O2)-, -C(R3R4)S(O2)-, or -S(O2)C(R3R4)-;
D is optionally substituted aryl or heterocycle; Ri is hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle;
R2 is hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle;
R3 is hydrogen, alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted alkyl;
R4 is hydrogen, alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted alkyl or aryl; each R5 is independently hydrogen or optionally substituted alkyl or aryl; and n is 0-3.
2. The use of claim 1 , wherein compound is of formula I(A):
Figure imgf000112_0002
3. The use of claim 1 , wherein the compound is of formula II :
Figure imgf000113_0001
II wherein: A is optionally substituted cycloalkyl, aryl, or heterocycle;
X is a bond, -O-, -S-, -C(O)-, -C(R4)=, ^(R4)-, -C(R3R4)-, -C(R^=C(R4)-, -C≡C-, -N(R5)-, -N(R5)C(O)N(R5)-, -C(R3R4)N(R5)-, -N(R5)C(R3R4)-, -ONC(R3)-, -C(R3)NO-, -C(R3R4)O-, -OC(R3R4)-, -S(O2)-, -S(O2)N(R5)-, -N(R5)S(O2)-, -C(R3R4)S(O2)-, or -S(O2)C(R3R4)-; D is optionally substituted aryl or heterocycle;
E is optionally substituted aryl or heterocycle;
Ri is hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle;
R2 is hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle;
R3 is hydrogen, alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted alkyl;
R4 is hydrogen, alkoxy, amino, cyano, halogen, hydroxyl, or optionally substituted alkyl or aryl; R5 is hydrogen or optionally substituted alkyl or aryl; and n is 0-3.
4. The use of claim 1 , wherein the compound is of formula H(A):
Figure imgf000113_0002
ii(A)
5. The use of claim 1, wherein the compound is of the formula:
Figure imgf000114_0001
wherein: each of Ai and A2 is independently a monocyclic optionally substituted cycloalkyl, aryl, or heterocycle.
6. The use of claim 1 , wherein the compound is of the formula:
Figure imgf000114_0002
7. The use of claim 1 , wherein the compound is of the formula:
Figure imgf000114_0003
8. The use of claim 1 , wherein the compound is:
Figure imgf000114_0004
wherein: each of Zi, Z2, Z3, and Z4 is independently N or CR6; each R6 is independently hydrogen, cyano, halogen, OR7, NRgRθ, amino, hydroxyl, or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each R7 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle; each Rg is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle; each R9 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle; and m is 1-4.
9. The use of claim 1 , wherein the compound is of the formula:
Figure imgf000115_0001
10. The use of claim 1, wherein the compound is nd of the formula:
Figure imgf000115_0002
11. The use of claim 1 , wherein the compound is of the formula:
Figure imgf000115_0003
wherein: each of Z'i, T2, and Z'3 is independently N, NH, S, O or CR6; each R6 is independently amino, cyano, halogen, hydrogen, OR7, SR7, NRSRΘ, or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each R7 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle; each R8 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle; each R9 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle; and p is 1-3.
12. The use of claim 1 , wherein the compound is of the formula:
Figure imgf000116_0001
13. The use of claim 1 , wherein the compound is of the formula:
Figure imgf000116_0002
14. The use of claim 1, wherein the compound is of the formula:
Figure imgf000117_0001
wherein: each of Z"i, Z"2, Z"3, and Z"4 is independently N or CRio; each Rio is independently amino, cyano, halogen, hydrogen, ORn, SRn, NR12R13, or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Rn is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle; each R12 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle; and each RB is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle.
15. The use of claim 1 , wherein the compound is of the formula:
Figure imgf000117_0002
16. The use of claim 1, wherein the compound is of the formula:
Figure imgf000118_0001
17. The use of claim 1, wherein the compound is of the formula:
Figure imgf000118_0002
wherein: each of Z"i, ZM2, ZM3, and ZM4 is independently N or CRio; each Rio is independently amino, cyano, halogen, hydrogen, ORn, SRn, NR12R13, or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Rn is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle; each R12 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle; and each Ri3 is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle.
18. The use of claim 1 , wherein the compound is of the formula:
Figure imgf000119_0001
19. The use of claim 1, wherein the compound is of the formula:
Figure imgf000119_0002
20. The use of a compound of the formula:
Figure imgf000119_0003
or a pharmaceutically acceptable salt or solvate thereof, in the manufacture of a medicament for slowing gastrointestinal motility in a patient, wherein:
A2 is a monocyclic optionally substituted cycloalkyl, aryl, or heterocycle;
Ri is hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle;
R2 is hydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle; each Rio is independently amino, cyano, halogen, hydrogen, ORn, SRn, NR12R13, or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Ri4 is independently amino, halogen, hydrogen, C(O)RA, ORA, NRBRC, S(O2)RA, or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each RA is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle; each RB is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each Rc is independently hydrogen or optionally substituted alkyl, alkyl-aryl or alkyl- heterocycle; and m is 1-4.
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