WO2022129041A1 - Prodrugs of 3-benzoamido-2-aminopropionic acid derivatives as modulators of the nmda receptor - Google Patents

Prodrugs of 3-benzoamido-2-aminopropionic acid derivatives as modulators of the nmda receptor Download PDF

Info

Publication number
WO2022129041A1
WO2022129041A1 PCT/EP2021/085681 EP2021085681W WO2022129041A1 WO 2022129041 A1 WO2022129041 A1 WO 2022129041A1 EP 2021085681 W EP2021085681 W EP 2021085681W WO 2022129041 A1 WO2022129041 A1 WO 2022129041A1
Authority
WO
WIPO (PCT)
Prior art keywords
amino
propanoate
ethyl
methyl
fluorobenzamido
Prior art date
Application number
PCT/EP2021/085681
Other languages
French (fr)
Inventor
John Paul Kilburn
Erhad ASCIC
Mauro Marigo
Laurent David
Original Assignee
H. Lundbeck A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by H. Lundbeck A/S filed Critical H. Lundbeck A/S
Publication of WO2022129041A1 publication Critical patent/WO2022129041A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/64—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings
    • C07C233/81—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups
    • C07C233/82—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
    • C07C233/83—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by carboxyl groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/06—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom containing only hydrogen and carbon atoms in addition to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/20—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/20—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D239/22—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with hetero atoms directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/10—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D241/12—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
    • C07D249/06—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles with aryl radicals directly attached to ring atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
    • C07D261/06—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members
    • C07D261/08—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D275/00—Heterocyclic compounds containing 1,2-thiazole or hydrogenated 1,2-thiazole rings
    • C07D275/02—Heterocyclic compounds containing 1,2-thiazole or hydrogenated 1,2-thiazole rings not condensed with other rings
    • C07D275/03—Heterocyclic compounds containing 1,2-thiazole or hydrogenated 1,2-thiazole rings not condensed with other rings with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/04—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D307/06—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/36—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms or radicals containing only hydrogen and carbon atoms, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2601/00—Systems containing only non-condensed rings
    • C07C2601/02—Systems containing only non-condensed rings with a three-membered ring

Definitions

  • the present invention is directed to compounds that are prodrugs of modulators of the NMDA receptor, pharmaceutical compositions comprising said compounds, and their use in the treatment of neurological disorders or neuropsychiatric disorders such as depression, in particular major depressive disorder (MDD) and treatment-resistant depression (TRD).
  • MDD major depressive disorder
  • TRD treatment-resistant depression
  • the North- American Center for Disease Control has reported that from 2005-2008, 8.9% of the US population was prescribed an antidepressant during any given month, antidepressants being also prescribed for anxiety, pain, and other non-mood disorders [Global Burden of Disease Study. Lancet. May 17, 1997; 349(9063): 1436-1442],
  • Antidepressants are marketed and thus known to the skilled person. Some examples of different types of antidepressant are selective serotonin reuptake inhibitors (SSRIs), Serotonin-norepinephrine reuptake inhibitors (SNRIs), Monoamine oxidase inhibitors (MAOIs), and Tricyclic antidepressants. Typical limitations of known antidepressants are delayed onset of efficacy and low remission rates after multiple courses of pharmacotherapy, and for some antidepressants severe side-effects [Antidepressants and the risk of suicidal behaviours. Jama. Jul. 21 2004; 292(3):338-343],
  • NMDA N-Methyl-D-Aspartate
  • TRD treatment-resistant depression
  • ketamine a pore-blocking antagonist of the NMDA receptor
  • MDD treatment with ketamine has the drawback of sedation, psychotomimetic side effects and requirement of intravenous or intransal administration.
  • NMDA receptors are tetrameric ligand-gated ion channels which are also involved in essential physiological processes such as synaptic plasticity and development.
  • NMDA receptors are heterotetramers comprising two GluNl subunits and two GluN2/GluN3 subunits. This means that they assemble as either diheteromeric or triheteromeric receptors.
  • the majority of native NMDA receptors consist of two GluNl subunits and two GluN2 subunits.
  • Activation of the NMDA receptors requires simultaneous binding at two different binding sites. Glutamate, the major excitatory neurotransmitter in the central nervous system, binds to the GluN2 subunits and glycine binds to the GluNl and GluN3 subunits.
  • D-cycloserine Another known modulator of the NMDA receptor is D-cycloserine, which is a partial glycine site agonist.
  • D-cycloserine has been intensively studied due to its neuroactive properties and potential utility in treatment of depression and depression disorders such as MDD [Heresco- Levy, U., Javitt, D.C., Gelfin, Y., Gorelik, E., Bar, M., Blanaru, M., Kremer, I., 2006. Controlled trial of d-cycloserine adjuvant therapy for treatment-resistant major depressive disorder. J. Affect. Disord.
  • Urwyler et al., J. Med. Chem. 2009, 52, 5093-5107 discloses 3-acylamino-2-amonipropionic acid derivatives with affinity for the glycine site of the NMDA receptor.
  • Maolanon et. al., ACS Chem. Neurosci. 2017, 8, 1681-1687 discloses Subtype-Specific Agonists for NMDA Receptor Glycine Binding Sites.
  • a prodrug of a compound being a modulator of the NMDA receptor with improved permeability and brain exposure compared to the parent compound may fulfil such unmet needs.
  • Summary of the invention 5 With this background, it is an object of the invention to provide a prodrug of modulators of the NMDA receptor.
  • R 1 is selected from the group consisting of hydrogen and halogen
  • R 2 and R 4 each independently are selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C 3 - C 6 )cycloalkyl and (C 1 -C 4 )alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C 1 - C 6 )alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy are optionally 15 substituted with one or more substituents independently selected from the group consisting of halogen, (C 1 -C 4 )alkyl, (C 1 -C 4 )alk
  • a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or diluents.
  • a method for the treatment of depression comprising the administration of a therapeutically effective amount of a compound of formula I, or acceptable salt thereof, or a pharmaceutical composition to a patient in need thereof.
  • a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of depression.
  • Figure 2 Effects of compound 2ba in the MEST model.
  • Y-axis Estimated seizure threshold (CC 50 ) current (mA) ;
  • X-axis bar furthest to the left: vehicle 10% HP ⁇ CD; bar second to the left: 3 mg/kg of compound 2ba; bar third to the left: 10 mg/kg of compound 2ba; bar furthest to the right: 30 mg/kg of compound 2ba.
  • Significance levels for post-hoc comparisons are indicated: * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001.
  • Figure 3 Effects of compound 2al and ketamine in the forced swim test model.
  • Y-axis Immobility time (s);
  • X-axis bar furthest to the left: Vehicle;
  • Significance levels for post-hoc comparisons are indicated: * ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001.
  • Figure 4 Effects of compound 2al in Resting state Electroencephalography (rsEEG) obtained in the Nucleus accumbens according to Example 5.
  • Y-axis Baseline-normalized power (dB); X-axis: bar furthest to the left: vehicle 10% HP ⁇ CD; bar second to the left: 3 mg/kg of compound 2al; bar third to the left: 10 mg/kg of compound 2al; bar furthest to the right: 30 mg/kg of compound 2al. Significance level for post-hoc comparison (relative to the vehicle group) is indicated: *** ⁇ 0.001.
  • Figure 5 Effects of compound 2ba in Resting state Electroencephalography (rsEEG) obtained in the Nucleus accumbens according to Example 5.
  • Y-axis Baseline-normalized power (dB); X-axis: bar furthest to the left: vehicle 10% HP ⁇ CD; bar second to the left: 3 mg/kg of compound 2ba; bar third to the left: 10 mg/kg of compound 2ba; bar furthest to the right: 30 mg/kg of compound 2ba. Significance level for post-hoc comparison (relative to the vehicle group) is indicated: *** ⁇ 0.001.
  • the present invention relates to ester prodrugs (Formula I) of the parent compounds (Formula V) or a pharmaceutically acceptable salt thereof, wherein: R 1 is selected from the group consisting of hydrogen and halogen; R 2 and R 4 each independently are selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C3- C6)cycloalkyl and (C 1 -C 4 )alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1- C6)alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C 1 -C 4 )alky
  • (Cx-Cy) alkyl refers to a hydrocarbyl radical obtained when one hydrogen atom is removed from a linear (i.e. unbranched) or branched saturated hydrocarbon.
  • the number of carbon atoms in the hydrocarbyl substituent (i.e. alkyl) is indicated by the prefix “(Cx-Cy)”, wherein x is the minimum and y is the maximum number of carbon atoms in the substituent.
  • Said ”(Cx-Cy) alkyl” comprises 1-6, preferably 1-4, more preferably 1-3, such as 1-2 or such as 2-3 carbon atoms.
  • (C 1 -C 6 ) alkyl refers to an alkyl substituent containing from 1 to 6 carbon atoms.
  • alkyl groups include the subclasses normal alkyl (n-alkyl), secondary and tertiary alkyl.
  • “(C 1 -C 6 ) alkyl” includes groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec.-butyl, tert. - butyl, n-pentyl, isopentyl, neopentyl, n-hexyl and isohexyl.
  • alkylene refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 6 carbon atoms, for example, of 1 to 4 carbon atoms.
  • Representative examples of alkylene include, but are not limited to, -CH 2 -, -CH 2 CH 2 -, - CH 2 CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH(CH 3 )CH 2 CH 2 -, and - CH 2 CH 2 CH 2 CH 2 CH 2 -.
  • (Cx-Cy) alkoxy refers to a moiety of the formula –OR’, wherein R’ indicates (Cx-Cy)alkyl as defined above.
  • R’ indicates (Cx-Cy)alkyl as defined above.
  • (C 1 -C 4 )alkoxy refers to such moiety wherein the alkyl contains from one to four carbon atoms.
  • (C1- C4)alkoxy include methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy and tert- butoxy.
  • (Cz-Cw)alkoxy(Cx-Cy)alkyl refers to an (Cz-Cw)alkoxy group, as defined herein, appended to the parent molecular moiety through an (C x -C y )alkyl group, as defined herein. Examples include, but are not limited to, methoxymethyl and ethoxymethyl.
  • halogen indicates a substituent selected from the 7 th main group of the periodic table, such as fluoro, chloro, bromo and iodo (F, Cl, Br and I).
  • halo(C x -C y )alkyl means a (C x -C y )alkyl group as defined herein, which is substituted with one or more halogen as defined herein, e.g. fluoro, chloro or bromo. Examples include, but are not limited to, trifluormethyl and difluoromethyl.
  • cyano refers to a CN group appended to the parent molecule through the carbon atom of the CN group.
  • hydroxyl or “hydroxy,” as used herein, means an -OH group.
  • phenyl is intended to mean a benzene radical, with one H removed at the attachment point.
  • hydroxy(Cx-Cy)alkyl or “hydroxyl(Cx-Cy)alkyl” as used herein, means a (Cx-Cy)alkyl group as defined herein, which is substituted with one or more -OH group(s). Examples include, but are not limited to hydroxymethyl, hydroxyethyl, hydroxypropyl.
  • heteroaryl refers to a radical of a heteroaromatic ring.
  • the term “5- or 6-membered heteroaryl” means a radical of a five or six membered heteroaromatic monocyclic ring containing at least one heteroatom independently selected from the group consisting of N, O and S, such as containing 1 to 4 heteroatoms independently selected from O, S, and N, such as 1 to 3 heteroatoms independently selected from O, S, and N, such as containing 1 to 2 heteroatoms independently selected from O, S, and N, such as containing 1 to 2 heteroatoms independently selected from O and N.
  • the five membered aromatic monocyclic rings have two double bonds and the six membered aromatic monocyclic rings have three double bonds.
  • the heteroaryl radical may be connected to the parent moiety through a carbon atom or a nitrogen atom contained anywhere within the heteroaryl group.
  • Representative examples of 5- or 6-membered heteroaryl include, but are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl, pyrrolyl, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl.
  • heterocyclyl as used herein, means a radical of an aliphatic heterocycle.
  • 4 - 6 membered heterocyclyl means a radical of a four-, five- or six- membered monocyclic ring containing at least one heteroatom independently selected from the group consisting of O, N, and S.
  • the four-membered ring contains zero or one double bond, and one heteroatom selected from the group consisting of O, N, and S.
  • the five- membered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S.
  • the six-membered ring contains zero, one or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S.
  • monocyclic heterocycles include, but are not limited to, azetidinyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, piperidinyl, tetrahydrofuranyl, tetrahydropyranyl.
  • the term ‘5- or 6-membered heterocyclic ring comprising 1 or 2 nitrogen’ comprises, but is not limited to, pyrrolidine, pyrazoline, piperidine, tetrahydropyrimidine dihydropyridine, imidazoline, imidazolidine.
  • (Cx-Cy)cycloalkyl refers to a saturated cycloalkane hydrocarbon radical comprising from x to y carbon atoms, such as from 3-6 carbon atoms, such as 3-5 or such as 3-4 carbon atoms.
  • the cycloalkyl may be monocyclic or bicyclic, wherein the two rings are bridged, fused, or spirocyclic.
  • cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. If substituents are described as being independently selected from a group, each substituent is selected independent of the other. Each substituent may therefore be identical or different from the other substituent(s).
  • optionally substituted means “unsubstituted or substituted”, and therefore the general formulas described herein encompasses compounds containing the specified optional substituent(s) as well as compounds that do not contain the optional substituent(s).
  • groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • the use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
  • the phrase “the compound” is to be understood as referring to various "compounds" of the invention or particular described aspect, unless otherwise indicated.
  • a modulator of the NMDA receptor with affinity to the glycine site is a compound which modulates through binding the orthosteric glycine binding site on the NMDA receptor so as to increase or decrease the flux of Ca + + through the ligand- gated channel.
  • therapeutically effective amount of a compound is intended to indicate an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease (e.g. depression) and its complications in a therapeutic intervention comprising the administration of said compound. An amount adequate to accomplish this is defined as “therapeutically effective amount”. Effective amounts for each purpose will depend on the severity of the disease (e.g. depression) or injury as well as the weight and general state of the subject.
  • determining an appropriate dosage may be achieved using routine experimentation, e.g. by constructing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician.
  • treatment and “treating” means the management and care of a patient for the purpose of combating a disease.
  • the term is intended to include the full spectrum of treatments for a given disease (e.g. depression) from which the patient is suffering, such as administration of the active compound to alleviate the symptoms or complications, to delay the progression of the disease (e.g. depression), to alleviate or relief the symptoms and complications, and/or to cure or eliminate the depression disease.
  • the patient to be treated is preferably a mammal, in particular a human being.
  • prodrug or “prodrug derivative” indicates a compound that, after administration to a living subject, such as a mammal, preferably a human is converted within the body into a pharmacologically active moiety. The conversion preferably takes place within a mammal, such as in a mouse, rat, dog, minipig, rabbit, monkey and/or human.
  • parent compound and “parent molecule” indicate the pharmacologically active moiety obtained upon conversion of a corresponding prodrug or administered as the active moiety to the patient.
  • the “parent compound” is to be understood as a compound of formula V, exemplified with compounds 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 1i, 1j, 1k, 1l, 1m, 1n, 1o, 1p, 1q, 1r, 1s, 1t, 1u, 1v, 1w, 1x, 1y, 1z, 1aa, 1ab, 1ac, 1ad, 1ae, 1af and 1ag.
  • the invention provides the compound according to Formula I above, or a pharmaceutically acceptable salt thereof, wherein R 1 is hydrogen.
  • the invention provides the compound according to Formula I above, or a pharmaceutically acceptable salt thereof, wherein R 3 is selected from the group consisting of hydrogen, fluoro, methoxy and (C1-C3)alkyl.
  • R 3 is selected from the group consisting of hydrogen, fluoro, methoxy and (C1-C3)alkyl.
  • the invention provides the compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R 1 and R 3 are hydrogen.
  • the invention provides the compound a according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R 2 is selected from the group consisting of hydrogen and halogen and wherein R 4 is selected from the group consisting of hydrogen, halogen, 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl and (C 3 -C 6 )cycloalkyl, wherein said 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4-6 membered heterocyclyl and(C 3 -C 6 )cycloalkyl are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy and halo(C 1 -C 4 )alkyl.
  • the invention provides the compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R 2 is fluoro.
  • the invention provides the compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R 4 is selected from the group consisting of 5- or 6-membered heteroaryl, wherein said 5- or 6-membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy and halo(C 1 -C 4 )alkyl.
  • the invention provides the compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R 4 is pyrazolyl, wherein said pyrazolyl is optionally substituted with one or more substituents independently selected from the group consisting of fluor, chloro, (C 1 -C 4 )alkyl and halo(C 1 - C 2 )alkyl.
  • the invention provides the compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R 4 is phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 -C 4 )alkyl and halo(C 1 -C 2 )alkyl.
  • the invention provides the compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R 5 is selected from the group consisting of (C 1 -C 6 )alkyl and (C 3 -C 6 )cycloalkyl, wherein said (C 1 -C 6 )alkyl and (C 3 -C 6 )cycloalkyl are optionally substituted with one or more substituents independently selected from the group consisting of -NH2, (C 1 -C 4 )alkoxy, (C 3 -C 6 )cycloalkyl, phenyl and 4-6 membered heterocyclyl.
  • the invention provides the compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R 2 and R 4 are not hydrogen.
  • the invention provides the compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 1 and R 3 are hydrogen;
  • R 2 is selected from the group consisting of halogen and (C 1 -C 6 )alkyl;
  • R 4 is selected from the group consisting of -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy, wherein said 5- or 6- membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alk
  • the invention provides the compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 2 is selected from the group consisting of hydrogen and halogen, and R4 is selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4 - 6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C 1 -C 4 )alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C 1 -C 6 )alkyl, 4-6 membered heterocyclyl, (C 3 -C 6 )cycloalkyl and (C1- C 4 )alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 -C 4 )alkyl, (C 1 -C 4 )
  • the invention provides the compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R 4 the 5- or 6- membered heteroaryl is selected from the group consisting of pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, furanyl and thienyl, wherein said 5- or 6- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy halo(C 1 -C 2 )alkyl, (C 1 -C 2 )alkoxy(C 1 - C 2 )alkyl, hydroxy(C 1 -C 2 )
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R 4 the 5- or 6- membered heteroaryl is selected from the group consisting of pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, furanyl and thienyl, wherein said 5- or 6- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1- C 4 )alkyl, (C 1 -C 4 )alkoxy, halo(C 1 -C 2 )alkyl, phenyl, phenoxy and -C(O)NH 2 .
  • the 5- or 6- membered heteroaryl is selected from the group consisting of pyrazolyl, pyridyl, pyrimidinyl, pyra
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R 4 the 5- membered heteroaryl is selected from the group consisting of pyrazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, furanyl and thienyl, wherein said 5- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy and halo(C 1 -C 2 )alkyl.
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 4 is selected from the group consisting of pyrazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, furanyl and thienyl, wherein said 5- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1- C4)alkyl, (C 1 -C 4 )alkoxy and halo(C 1 -C 2 )alkyl; and wherein R 2 is selected from halogen; and wherein R 1 and R 3 both represent hydrogen.
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 4 is selected from the group consisting of pyrazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, furanyl and thienyl, wherein said 5- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1- C 4 )alkyl, (C 1 -C 4 )alkoxy and halo(C 1 -C 2 )alkyl; and wherein R 2 is selected from halogen; and wherein R 1 and R 3 both represent hydrogen; and wherein R 5 is selected from methyl and ethyl.
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 4 represents phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy and halo(C 1 - C 2 )alkyl; and wherein R 2 is selected from halogen; and wherein R 1 and R 3 both represent hydrogen.
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 4 represents phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C 1 -C 4 )alkyl, (C 1 -C 4 )alkoxy and halo(C1- C2)alkyl; and wherein R 2 is selected from halogen; and wherein R 1 and R 3 both represent hydrogen; and wherein R 5 is selected from methyl and ethyl.
  • the invention provides a compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R 3 is selected from the group consisting of hydrogen, fluoro, methyl and ethyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 3 is hydrogen.
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 5 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl and isopentyl, wherein said methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl and isopentyl are optionally substituted with one or more substituents selected from (C 1 -C 4 )alkoxy and (C 3 - C 6 )cycloalkyl.
  • R 5 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl and isopentyl, wherein said methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl and isopenty
  • the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R 5 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl and isopentyl.
  • R 5 is selected from the group consisting of methyl and ethyl.
  • the invention provides the compound according to any one of the embodiments above, selected from the list consisting of (R)-methyl 2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoate, (+)(2R)-methyl 2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoate, (-)(2R)-methyl 2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(2-ethylfuran-3-yl)-5-fluorobenzamido)propanoate, (-)cis-(2R)-methyl 2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate
  • the invention provides a compound according to any one of the embodiments above as a salt of hydrochloric acid. In a further aspect the invention provides a compound according to any one of the embodiments above as a salt of hydrobromic acid. In a further aspect the invention provides a compound according to any one of the embodiments wherein the compound is selected from (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate with the formula 2al below Formula 2al, and (R)-ethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate with the formula 2ba below Formula 2ba, or a pharmaceutically acceptable salt thereof.
  • the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate, or a pharmaceutically acceptable salt thereof.
  • the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate hydrochloride.
  • the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido) hydrobromide.
  • the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-ethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoate or a pharmaceutically acceptable salt thereof.
  • the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-ethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoate hydrochloride.
  • the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-ethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoate hydrobromide.
  • the invention provides a compound according to formula I above, or a pharmaceutically acceptable salt thereof, wherein R 1 and R 2 together with the carbon atoms to which they are attached form a 5- or 6 membered heteroaromatic ring comprising 1 or 2 nitrogen(s), or a 5- or 6-membered heterocyclic ring comprising 1 or 2 nitrogen(s), and wherein said 5- or 6 membered heteroaryl is optionally substituted with one or more substituents independently selected from (C 1 -C 4 )alkyl, and wherein said 5- or 6-membered heterocycle is optionally substituted with one or more substituents independently selected from (C 1 -C 4 )alkyl and oxo; and wherein R 3 and R 4 each independently are selected from the group consisting of hydrogen, halogen, (C 1 -C 6 )alkyl and (C 1 -C 4 )alkoxy; or R 2 and R 3 , together with the carbon atoms to which they are attached form a 5- or 6 member
  • the compounds of the invention may possess tautomeric forms, stereoisomers, geometric isomers, and that these also constitute embodiments of the invention. Racemic forms may be resolved into the optical antipodes by known methods, for example, by separation of diastereomeric salts thereof with an optically active acid, and liberating the optically active amine compound by treatment with a base. Separation of such diastereomeric salts can be achieved, e.g. by fractional crystallization.
  • the optically active acids suitable for this purpose may include, but are not limited to d- or l- tartaric, mandelic or camphorsulfonic acids. Another method for resolving racemates into the optical antipodes is based upon chromatography on an optically active matrix.
  • the compounds of the present invention may also be resolved by the formation and chromatographic separation of diastereomeric derivatives from chiral derivatizing reagents, such as, chiral alkylating or acylating reagents, followed by cleavage of the chiral auxiliary.
  • chiral derivatizing reagents such as, chiral alkylating or acylating reagents
  • Any of the above methods may be applied either to resolve the optical antipodes of the compounds of the invention per se or to resolve the optical antipodes of synthetic intermediates, which can then be converted by methods described herein into the optically resolved final products which are the compounds of the invention. Additional methods for the resolution of optical isomers, known to those skilled in the art, may be used. Such methods include those discussed by J. Jaques, A. Collet and S.
  • Optically active compounds can also be prepared from optically active starting materials. Included in this invention are also isotopically labelled compounds, which are similar to those claimed in formula I, wherein one or more atoms are represented by an atom of the same element having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (e.g., 2 H, 3 H, 11 C, 13 C, 15 N, 18 F and the like). Particular mention is made of 2 H substituted compounds i.e. compounds wherein one or more H atoms are represented by deuterium.
  • one or more of the hydrogen atoms of the compound of formula I are represented by deuterium. It is recognized that elements are present in natural isotopic abundances in most synthetic compounds, and result in inherent incorporation of deuterium. However, the natural isotopic abundance of hydrogen isotopes such as deuterium is immaterial (about 0.015%) relative to the degree of stable isotopic substitution of compounds indicated herein. Thus, as used herein, designation of an atom as deuterium at a position indicates that the abundance of deuterium is significantly greater than the natural abundance of deuterium. Any atom not designated as a particular isotope is intended to rep-resent any stable isotope of that atom, as will be apparent to the ordinarily skilled artisan.
  • designation of a position as “D” in a compound has a minimum deuterium incorporation of greater than about 60% at that position such as greater than about 70% at that position such as greater than about 80% at that position such as greater than about 85% at that position.
  • designation of a position as “D” in a com-pound has a minimum deuterium incorporation of greater than about 90% at that position such as greater than about 95% at that position such as greater than about 97% at that position such as greater than about 99% at that position.
  • Pharmaceutically acceptable salts The compounds of this invention (parent compounds and their respective prodrugs) are generally utilized as the free substance or as a pharmaceutically acceptable salt thereof.
  • salts may be prepared in a conventional manner by treating a solution or suspension of a free base of a compound of the invention with a molar equivalent of a pharmaceutically acceptable acid.
  • suitable organic and inorganic acids are described below.
  • Pharmaceutically acceptable salts in the present context is intended to indicate non-toxic, i.e. physiologically acceptable salts.
  • pharmaceutically acceptable salts include salts formed with inorganic and/or organic acids on the nitrogen atoms in the parent molecule.
  • Said acids may be selected from for example hydrochloric acid, hydrobromic acid, phosphoric acid, nitrous acid, sulphuric acid, benzoic acid, citric acid, gluconic acid, lactic acid, maleic acid, succinic acid, tartaric acid, acetic acid, propionic acid, oxalic acid, maleic acid, fumaric acid, glutamic acid, pyroglutamic acid, salicylic acid, salicylic acid, saccharin, and sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid and benzenesulfonic acid.
  • the pharmaceutically acceptable salt is a hydrogen chloride salt.
  • the pharmaceutically acceptable salt is a hydrogen bromide salt.
  • pharmaceutically acceptable salts also include salts formed with inorganic and/or organic bases on the acidic groups of compounds of the invention.
  • Said bases may be selected from for example alkali metal bases, such as sodium hydroxide, lithium hydroxide, potassium hydroxide, alkaline earth bases, such as calcium hydroxide and magnesium hydroxide, and organic bases, such as trimethylamine and trimethylamine. Additional examples of useful acids and bases to form pharmaceutically acceptable salts can be found e.g. in Stahl and Wermuth (Eds) “Handbook of Pharmaceutical salts. Properties, selection, and use”, Wiley-VCH, 2008.
  • the invention encompasses compounds of the invention for use as a medicament, as well as for use in treatment of all diseases and disorders listed below.
  • compounds of the present invention may be useful in the treatment of depression and depressive disorders.
  • a compound of formula I or a pharmaceutically acceptable salt thereof is for use in in the treatment of depression.
  • the diagnosis of depression usually follows a clinical evaluation by a psychiatrist or other mental health professionals.
  • DSM IV Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV) published by the American psychiatric association and the ICD ( ICD-10: International Statistical Classification of Diseases and Related Health Problems – 10th Revision, published periodically by the World Health Organization) or any other psychiatric classification system.
  • Signs and symptoms of depression are for example depressed mood, loss of interest (anhendonia), weight or appetite changes, sleep problems, psychomotor activity (objective or subjective), fatigability, worthlessness, concentration difficulty, suicidal ideation, loss of confidence, sexual dysfunction and self-reproach.
  • compounds of the invention are for use to prevent, alter, reduce or alleviate one or more signs or symptoms of depression selected from the group consisting of depressed mood, loss of interest (anhendonia), weight or appetite changes, sleep problems, psychomotor activity (objective or subjective), fatigability, worthlessness, concentration difficulty, suicidal ideation, loss of confidence, sexual dysfunction and self-reproach.
  • the skilled person is familiar with various test for measuring the improvement of depressive symptoms. Examples of test for measuring the improvements are but not limited to the HAM-D or MADRS scale.
  • the depression is major depressive disorder.
  • the depression is treatment-resistant depression.
  • the depression is selected from major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, , bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
  • compound of the invention is used in the treatment of pain [Expert Rev Clin Pharmacol.2011 May 1; 4(3): 379–388].
  • the pain is neuropathic pain.
  • Preclinical animal models have demonstrated pro-cognitive and antidepressant-like effects with the use of NDMA glycine site modulators [Peyrovian et al., Progress in Neuropsychopharmacology & Biological Psychiatry.
  • a compound of formula I or a pharmaceutically acceptable salt thereof is for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus
  • a compound of formula I or a pharmaceutically acceptable salt thereof is for use in the treatment of suicidal ideation.
  • compound of the invention is for use in the treatment of a neurological disorder or neuropsychiatric disorder.
  • the invention provides a method for the treatment of depression, such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression, comprising the administration of a therapeutically effective amount of the compound of Formula I, or a pharmaceutically acceptable salt thereof to a patient (e.g. a human patient) in need thereof.
  • depression such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression
  • the invention provides a method for the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus, comprising the administration of a therapeutically effective amount of the compound of Formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of Formula I or a pharmaceutically acceptable salt thereof, to a patient (e.g. a human patient) in need thereof.
  • a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus comprising the administration of a therapeutically effective amount of the compound of Formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of Formula I or a pharmaceutically acceptable salt thereof, to a patient (e.g. a human patient) in need thereof.
  • the invention provides a method for the treatment of depression, such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression, comprising the administration of a pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, to a patient (e.g. a human patient) in need thereof.
  • depression such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression
  • the invention provides the use of the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of depression, such as for example major depressive disorder, treatment- resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
  • depression such as for example major depressive disorder, treatment- resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
  • the invention provides the use of the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus.
  • the invention provides the use of a pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of depression, such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
  • depression such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
  • the invention provides the use of a pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus.
  • a pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus.
  • Combination treatment In one embodiment of the invention, the compounds of the invention are for use as stand- alone treatment as the sole active compound. In another embodiment of the invention, the compounds of the invention may be used in combination with other agents useful in the treatment of disorders such as depression.
  • combined use in combination with” and “a combination of” and the like as used herein in the context of the method of the invention comprising the combined administration of therapeutically effective amounts of a compound of the invention, and another compound, which compound is useful in the treatment a neurodegenerative disease or disorder, is intended to mean the administration of a compound of the invention simultaneously or sequentially, in any order, together with said other compound.
  • the two compounds may be administered simultaneously or sequentially with a time gap between the administrations of the two compounds.
  • the two compounds may be administered either as part of the same pharmaceutical formulation or composition, or in separate pharmaceutical formulations or compositions.
  • the two compounds may be administered on the same day or on different days.
  • the patient to be treated may already be in treatment with one or more other compounds useful in the treatment of depression when treatment with a compound of the invention initiated.
  • the patient may already be in treatment with a compound of the invention when treatment with one or more other compounds useful in the treatment of a depression or psychosis is initiated.
  • therapeutically active compounds which may advantageously be combined with compounds of the invention include sedatives or hypnotics, such as benzodiazepines; anticonvulsants, such as lamotrigine, valproic acid, topiramate, gabapentin, carbamazepine; mood stabilizers such as lithium; dopaminergic drugs, such as dopamine agonists and L- Dopa; drugs to treat ADHD, such as atomoxetine; psychostimulants, such as modafinil, ketamine, methylphenidate and amphetamine; other antidepressants, such as mirtazapine, mianserin, vortioxetine, cipralex, and buproprion; hormones, such as T3, estrogen, DHEA and testosterone; atypical antipsychotics, such as olanzapine,
  • compositions comprising a compound of the invention, either as the sole active compound or in combination with another active compound, may be specifically formulated for administration by any suitable route such as the oral, rectal, nasal, buccal, sublingual, pulmonal, transdermal and parenteral (e.g. subcutaneous, intramuscular, and intravenous) route. It will be appreciated that the route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient. Doses In one embodiment, the compound of the present invention is administered in an amount from about 0.5 mg/kg body weight to about 50 mg/kg body weight per day. In particular, daily dosages may be in the range of 1 mg/kg body weight to about 30 mg/kg body weight per day.
  • the exact dosages will depend upon the frequency and mode of administration, the sex, the age, the weight, and the general condition of the subject to be treated, the nature and the severity of the condition to be treated, any concomitant diseases to be treated, the desired effect of the treatment and other factors known to those skilled in the art.
  • the frequency of administration is 1, 2, 3, 4, or 5 times per day
  • the frequency of administration is once weekly.
  • the frequency of administration is twice weekly.
  • a typical oral dosage for adults will be in the range of 1-3000 mg/day of a compound of the present invention, such as 700-2800 mg/day, such as 1000-2000 mg/day or 1200- 1700mg/day.
  • the compounds of the invention are administered in a unit dosage form containing said compounds in an amount of about 100 to 1000 mg, such as 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 750 mg or up to 1000 mg of a compound of the present invention.
  • the frequency of administration is 1, 2, 3, 4, or 5 times per day.
  • the frequency of administration is once weekly.
  • the frequency of administration is twice weekly.
  • a typical IV dosage for adults will be in the range of 20-300 mg/day of a compound of the present invention, such as 50-200 mg/day, such as 70-150 mg/day or 75-125 mg/day.
  • the compounds of the invention are administered in a unit dosage form containing said compounds in an amount of about 10 to 300 mg, such as 10 mg, 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg of a compound of the present invention.
  • the frequency of administration is once weekly. In an embodiment the frequency of administration is twice weekly.
  • excipient or “pharmaceutically acceptable excipient” refers to pharmaceutical excipients including, but not limited to, fillers, antiadherents, binders, coatings, colours, disintegrants, flavours, glidants, lubricants, preservatives, sorbents, sweeteners, solvents, vehicles and adjuvants.
  • the present invention also provides a pharmaceutical composition comprising a compound of the invention, such as one of the compounds disclosed in the Experimental Section herein.
  • the present invention also provides a process for making a pharmaceutical composition comprising a compound of the invention.
  • compositions according to the invention may be formulated with pharmaceutically acceptable excipients in accordance with conventional techniques such as those disclosed in Remington, “The Science and Practice of Pharmacy”, 22 th edition (2013), Edited by Allen, Loyd V., Jr.
  • Pharmaceutical compositions for oral administration include solid oral dosage forms such as tablets, capsules, powders and granules; and liquid oral dosage forms such as solutions, emulsions, suspensions and syrups as well as powders and granules to be dissolved or suspended in an appropriate liquid.
  • Solid oral dosage forms may be presented as discrete units (e.g. tablets or hard or soft capsules), each containing a predetermined amount of the active ingredient, and preferably one or more suitable excipients.
  • the solid dosage forms may be prepared with coatings such as enteric coatings or they may be formulated so as to provide modified release of the active ingredient such as delayed or extended release according to methods well known in the art.
  • the solid dosage form may be a dosage form disintegrating in the saliva, such as for example an oral-dispersible tablet.
  • excipients suitable for solid oral formulation include, but are not limited to, microcrystalline cellulose, corn starch, lactose, mannitol, povidone, croscarmellose sodium, sucrose, cyclodextrin, talcum, gelatin, pectin, magnesium stearate, stearic acid and lower alkyl ethers of cellulose.
  • the solid formulation may include excipients for delayed or extended release formulations known in the art, such as glyceryl monostearate or hypromellose.
  • the formulation may for example be prepared by mixing the active ingredient with solid excipients and subsequently compressing the mixture in a conventional tableting machine; or the formulation may for example be placed in a hard capsule e.g. in powder, pellet or mini tablet form.
  • the amount of solid excipient will vary widely but will typically range from about 25 mg to about 1 g per dosage unit.
  • Liquid oral dosage forms may be presented as for example elixirs, syrups, oral drops or a liquid filled capsule.
  • Liquid oral dosage forms may also be presented as powders for a solution or suspension in an aqueous or non-aqueous liquid.
  • excipients suitable for liquid oral formulation include, but are not limited to, ethanol, propylene glycol, glycerol, polyethylenglycols, poloxamers, sorbitol, poly-sorbate, mono and di-glycerides, cyclodextrins, coconut oil, palm oil, and water.
  • Liquid oral dosage forms may for example be prepared by dissolving or suspending the active ingredient in an aqueous or non-aqueous liquid, or by incorporating the active ingredient into an oil-in-water or water-in-oil liquid emulsion.
  • compositions for parenteral administration include sterile aqueous and nonaqueous solutions, dispersions, suspensions or emulsions for injection or infusion, concentrates for injection or infusion as well as sterile powders to be reconstituted in sterile solutions or dispersions for injection or infusion prior to use.
  • excipients suitable for parenteral formulation include, but are not limited to water, coconut oil, palm oil and solutions of cyclodextrins.
  • Aqueous formulations should be suitably buffered if necessary and rendered isotonic with sufficient saline or glucose.
  • compositions include suppositories, inhalants, creams, gels, dermal patches, implants and formulations for buccal or sublingual administration. It is requisite that the excipients used for any pharmaceutical formulation comply with the intended route of administration and are compatible with the active ingredients.
  • EXPERIMENTAL SECTION Preparation of the compounds of the invention
  • the compounds of the present invention of the general formula I, wherein R 1 , R 2 , R 3 , R 4 and R 5 are as defined above can be prepared by the methods outlined in the following reaction Schemes 1-11 and in the examples.
  • Compounds of general formula I may be prepared from compounds with general formula IV by standard de-protection procedures.
  • compounds of general formula I may be prepared from compounds with general formula IV where Pg 1 is a N-Carbobenzyloxy group (Cbz) and R 5 is defined as in general formula I.
  • Compounds with general formula IV may be prepared by compounds of general formula III with carboxylic acids (or salt thereof) of general formula II by standard peptide coupling such as using O-(Benzotriazol-1-yl)-N,N,N ⁇ ,N ⁇ -tetramethyluronium tetrafluoroborate in the presence of a base such as N,N-diisopropylethylamine in a solvent such as N,N- dimethylformamide.
  • Compounds of general formula I (Scheme 2) may be prepared from compounds with general formula V by standard esterification procedures.
  • Compounds of general formula V may be prepared from compounds with general formula IVa by standard de-protection procedures.
  • compounds of general formula V (Scheme 2) may be prepared from compounds with general formula IVa where Pg 1 is Cbz and Pg 2 is benzyl using conditions such as HBr in acetic acid.
  • Compounds with general formula IVa may be prepared by compounds of general formula IIIa with carboxylic acids (or salt thereof) of general formula II by standard peptide coupling such as using O-(Benzotriazol-1-yl)-N,N,N ⁇ ,N ⁇ -tetramethyluronium tetrafluoroborate in the presence of a base such as N,N-diisopropylethylamine in a solvent such as N,N- dimethylformamide.
  • Scheme 3 Benzoic acids of general formula II (or salt thereof), (Scheme 2) where R 2 can be hetaryl, aryl or alkenyl as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VII by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art.
  • Compounds of general formula VII can be obtained from compounds of general formula VI, under reaction conditions such as in the presence of a catalyst such as [1,1 ⁇ - Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane or [1,1 ⁇ -Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) and a base such as potassium triphosphate.
  • a catalyst such as [1,1 ⁇ - Bis(diphenylphosphino)ferrocene]dichloropalladium(II)
  • dichloromethane or [1,1 ⁇ -Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II)
  • a base such as potassium triphosphate.
  • similar reaction conditions may be applied for introducing R 4 substituents.
  • Benzoic acids of general formula II (or salt thereof), (Scheme 2) where R 2 is hetaryl or heterocycle as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VIII by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art.
  • Compounds of general formula VIII can be obtained from compounds of general formula VI, under reaction conditions such as in the presence of a catalyst such as copper (II) acetate and a base such as pyridine.
  • reaction conditions may be applied for introducing R 4 substituents.
  • Compounds of general formula VII can be obtained by reacting compounds of general formula X together with trialkylboranes, under reaction conditions such as in the presence of a catalyst such as [1,1 ⁇ - Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane and a base such as caesium acetate, or alkyl Zinc bromides, under reaction conditions such as in the presence of a catalyst such as palladium(II) acetate, and a ligand such as 2- dicyclohexylphosphino-2 ⁇ ,6 ⁇ -bis(N,N-dimethylamino)biphenyl (Cphos).
  • a catalyst such as [1,1 ⁇ - Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane and a base such as caesium acetate, or alkyl Zinc bromides
  • a catalyst such
  • Compounds of general formula VII can be obtained by reacting compounds of general formula XI together with trialkylboranes, under reaction conditions such as in the presence of a catalyst such as [1,1 ⁇ - Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane and a base such as caesium acetate, or alkyl Zinc bromides, under reaction conditions such as in the presence of a catalyst such as palladium(II) acetate, and a ligand such as 2- dicyclohexylphosphino-2 ⁇ ,6 ⁇ -bis(N,N-dimethylamino)biphenyl (Cphos).
  • a catalyst such as [1,1 ⁇ - Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane and a base such as caesium acetate, or alkyl Zinc bromides
  • a catalyst
  • reaction conditions may be applied for introducing R 1 substituents.
  • Scheme 8 Benzoic acids of general formula II (or salt thereof), (Scheme 2) where R 2 can be alkyloxy or cycloalkyoxy as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VII by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art.
  • Compounds of general formula VII can be obtained by reacting compounds of general formula XII together with aldehydes or ketones, under reaction conditions such as in the presence of a base such as isopropylmagnesium chloride complex with lithium chloride.
  • similar reaction conditions may be applied for introducing R 4 substituents.
  • Compounds of general formula VII can be obtained from compounds of general formula XIII, under reaction conditions such as in the presence of a catalyst such as [1,1 ⁇ - Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane or [1,1 ⁇ -Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) and a base such as cesium carbonate.
  • a catalyst such as [1,1 ⁇ - Bis(diphenylphosphino)ferrocene]dichloropalladium(II)
  • dichloromethane or [1,1 ⁇ -Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II)
  • a base such as cesium carbonate.
  • similar reaction conditions may be applied for introducing R 4 substituents.
  • Scheme 10 Compounds of general formula III where R 5 is as defined herein (Scheme 10) may be prepared by reacting carboxylic acids of formula XIV with alcohols of formula XV using standard esterification procedures.
  • Scheme 11 Compounds of general formula III where R 5 is as defined herein (Scheme 11) may be prepared by deprotection of compounds of general formula XVIII where Pg 3 could be a tert- butyloxycarbonyl protecting group (Boc) and Pg 1 could be a benzyloxy carbamate (Cbz).
  • Compounds of general formula XVIII where R 5 is as in general formula I may be prepared by reacting carboxylic acids of formula XVII with alcohols of formula XV using standard esterification procedures.
  • Preparative HPLC Preparative-HPLC
  • Method AA Instrument: Gilson GX-281 Liquid Handler, SHIMADZU LC-8A LCMS2010; Column: YMC-Actus Triart C18150*305 ⁇ m; Mobile Phase A: water (0.05% HCl v/v); Mobile phase B: MeCN; Gradient: B from 5% to 35% in 10 min then hold at 100% for 3 min; FlowRate(ml/min): 25; Column temperature: 35°C and Wavelength: 220nm 254nm
  • Method BB Instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector; Column: Xtimate C18 150*25mm*5 ⁇ m; Mobile Phase A: water (0.05% ammonia hydroxide v/v); Mobile phase B: MeCN; Gradient: from 42% to 72% in 10 min then hold at 100% for 2.5 min; Flow Rate (ml/min
  • NMR 1 H NMR spectra were recorded at 300, 400, 500 or 600 MHz on Bruker Avance instruments. TMS was used as internal reference standard. Chemical shift values are expressed in ppm.
  • Abbreviations are in accordance with to the ACS Style Guide: "The ACS Style guide – A manual for authors and editors" Janet S.
  • Step 2 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoic acid
  • the mixture was diluted with H 2 O (20 mL).
  • Step 4 (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate 60 mg, 0.11 mmol
  • HBr in AcOH 2 mL, 30% in AcOH
  • Step 2 A mixture of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate (0.35 g, 0.75 mmol) and HBr in AcOH (7 mL, 30% in AcOH) were stirred at 25°C for 15 hours. The residue was concentrated. The residue was purified by Preparative-HPLC (HCl). The crude product was dissolved with MeOH (3 mL) and methyl tert- butyl ether (12 mL) was added dropwise.
  • Step 2 (S)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate and (R)-ethyl 3-fluoro-5-(1- hydroxyethyl)benzoate
  • ethyl 3-fluoro-5-iodobenzoate 5.2 g, 17.68 mmol
  • i-PrMgCl-LiCl 1.3 M in THF, 19.04 mL
  • Step 3 (S)-ethyl 3-(1-ethoxyethyl)-5-fluorobenzoate To a solution of (S)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate (1.7 g, 8.01 mmol) in DMF (30 mL) was added NaH (481 mg, 12.02 mmol, 60% in mineral oil). The mixture was stirred at 0°C for 30 minutes. Then iodoethane (3.75 g, 24.03 mmol) was added. The mixture was stirred at 60°C for 15 hours. The reaction mixture was quenched by addition H 2 O (10 mL), and then concentrated under reduced pressure to give a residue.
  • Step 5 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((S)-1-ethoxyethyl)-5- fluorobenzamido)propanoate
  • (S)-3-(1-ethoxyethyl)-5-fluorobenzoic acid 200 mg, 0.942 mmol
  • DMF 10 mL
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (378 mg, 1.04 mmol, HCl), TBTU (454 mg, 1.41 mmol) and DIPEA (365 mg, 2.83 mmol).
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((R)-1-ethoxyethyl)-5- fluorobenzamido)propanoate
  • (R)-3-(1-ethoxyethyl)-5-fluorobenzoic acid 200 mg, 0.942 mmol
  • DMF 10 mL
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (378 mg, 1.04 mmol, HCl), TBTU (454 mg, 1.41 mmol) and DIPEA (365 mg, 2.83 mmol).
  • the mixture was stirred at 30°C for 4 hours.
  • the reaction mixture was diluted with H 2 O (30 mL) and extracted with ethyl acetate (30 mL ⁇ 3). The combined organic layers were washed with brine (30 mL ⁇ 2), dried over Na 2 SO 4 , filtered and concentrated.
  • Step 2 Methyl 3-(2-ethylfuran-3-yl)-5-fluorobenzoate To a solution of 3-bromo-2-ethylfuran (1.50 g, 8.57 mmol) and (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (1.70 g, 8.57 mmol) in a mixture of DME (40 mL) and H 2 O (4 mL) was added Pd(PPh3)4 (990 mg, 0.86 mmol) and Na2CO3 (3.55 g, 25.7 mmol) under N2 atmosphere. The mixture was stirred at 85 °C for 16 hours.
  • Pd(PPh3)4 990 mg, 0.86 mmol
  • Na2CO3 3.55 g, 25.7 mmol
  • Step 3 3-(2-ethylfuran-3-yl)-5-fluorobenzoic acid
  • a solution of methyl 3-(2-ethylfuran-3-yl)-5-fluorobenzoate (80 mg, 0.32 mmol) in a mixture of MeOH (4 mL) and H 2 O (1 mL) was added LiOH.H 2 O (41 mg, 0.97 mmol).
  • the mixture was stirred at 20 °C for 14 hours.
  • the mixture was concentrated.
  • the residue was dissolved in water (5 mL) and acidified with 2N HCl to adjust pH to 5 and extracted with ethyl acetate (15 mL ⁇ 3).
  • Step 5 (R)-2-amino-3-(3-(2-ethylfuran-3-yl)-5-fluorobenzamido)propanoic acid
  • ethyl acetate 2 mL
  • Pd/C 40 mg, 10% Pd, 50% water
  • Step 2 cis-3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzoic acid
  • a solution of cis-methyl 3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzoate (350 mg, 1.39 mmol) in a mixture of MeOH (5 mL) and H 2 O (2 mL) was added LiOH.H 2 O (175 mg, 4.16 mmol). The mixture was stirred at 20 °C for 16 hours. The mixture was concentrated. The residue was dissolved in water (5 mL) and acidified with 2N HCl to adjust pH to 5 and extracted with ethyl acetate (15 mL ⁇ 3).
  • Step 3 cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate
  • TBTU 303 mg, 0.94 mmol
  • DIPEA 244 mg, 1.89 mmol
  • R benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (230 mg, 0.63 mmol, HCl salt).
  • the mixture was stirred at 20 °C for 16 hours.
  • the mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL ⁇ 3). The combined organic layers were washed with brine (20 mL ⁇ 2), dried over Na 2 SO 4 , filtered and concentrated.
  • Step 5 (-)cis-(2R)-2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid
  • cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate (30 mg, 0.05 mmol) in MeOH (5 mL) was added Pd/C (20 mg, 10% Pd, 50% water) under N 2 .
  • Pd/C 20 mg, 10% Pd, 50% water
  • Step 2 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoic acid To a solution of methyl 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoate (290 mg, 1.11 mmol) in the mixture of MeOH (6 mL) and H 2 O (6 mL) was added LiOH.H 2 O (94 mg, 2.23 mmol). The mixture was stirred at 25°C for 1 hour. The mixture was concentrated.
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyrazin-2-yl)-5- fluorobenzamido)propanoate
  • 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoic acid 150 mg, 0.61 mmol
  • (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (267 mg, 0.73 mmol, HCl salt)
  • TBTU (293 mg, 0.91 mmol
  • DIPEA 236 mg, 1.83 mmol
  • Step 4 (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyrazin-2-yl)-5- fluorobenzamido)propanoate 240 mg, 0.43 mmol
  • 30% HBr/HOAc 5 mL
  • Step 2 3-(4-Ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
  • methyl 3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate 230 mg, 0.88 mmol
  • THF 2 mL
  • H 2 O 2 mL
  • LiOH.H 2 O 110 mg, 2.63 mmol
  • the mixture was stirred at 20 °C for 16 hours.
  • Step 3 (R)-Benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate
  • 3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid 200 mg, 0.81 mmol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (353 mg, 0.97 mmol, HCl salt)
  • TBTU 388 mg, 1.21 mmol
  • DIPEA 312 mg, 2.42 mmol
  • Step 4 (R)-2-Amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-methyl-1H-pyrazol- 5-yl)-5-fluorobenzamido)propanoate 270 mg, 0.48 mmol
  • 30% HBr in AcOH 5 mL
  • Step 2 Methyl 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoate and methyl 3-(3-ethyl- 1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoate
  • 3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid 300 mg, 1.52 mmol
  • dioxane 10 mL
  • 4-bromo-5-ethyl-1-methyl-1H-pyrazole and 4- bromo-3-ethyl-1-methyl-1H-pyrazole 300 mg, crude.
  • the reaction mixture was stirred at 100°C for 16 hours.
  • reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL ⁇ 3). The combined organic layers were washed with brine (10 mL ⁇ 3), dried over Na 2 SO 4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 30% to 40%) to give the mixture of two isomers (200 mg), which were separated by SFC separation to give methyl 3- (3-ethyl-1-methyl-pyrazol-4-yl)-5-fluoro-benzoate (70 mg), and methyl 3-(5-ethyl-1-methyl- pyrazol-4-yl)-5-fluoro-benzoate (90 mg).
  • Step 3 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoic acid
  • methyl 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoate 90 mg, 0.34 mmol
  • MeOH 3 mL
  • H 2 O 2 mL
  • LiOH.H 2 O 43 mg, 1.03 mmol
  • the reaction mixture was stirred at 25°C for 1 hour.
  • the MeOH was removed.
  • Step 2 3-(2-ethylpyridin-3-yl)-5-fluorobenzoic acid
  • a solution of methyl 3-(2-ethylpyridin-3-yl)-5-fluorobenzoate (200 mg, 0.77 mmol) in a mixture of H 2 O (2.5 mL) and MeOH (2.5 mL) was added LiOH.H 2 O (97 mg, 2.31 mmol).
  • the mixture was stirred at 25°C for 3 hours.
  • the mixture was concentrated, diluted with water (5 mL) then acidified with aq.
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylpyridin-3-yl)-5- fluorobenzamido)propanoate
  • 3-(2-ethylpyridin-3-yl)-5-fluorobenzoic acid 280 mg, crude
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (262 mg, 0.72 mmol, HCl)
  • HATU 410 mg, 1.08 mmol
  • DIPEA 279 mg, 2.16 mmol
  • Step 4 (R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylpyridin-3-yl)-5- fluorobenzamido)propanoate 100 mg, 0.18 mmol
  • 30% HBr/AcOH 3 mL
  • Step 1 methyl 3-fluoro-5-vinylbenzoate
  • methyl 3-bromo-5-fluorobenzoate 400 mg, 1.72 mmol
  • 4,4,5,5- tetramethyl-2-vinyl-1,3,2-dioxaborolane 318 mg, 2.06 mmol
  • dioxane 10 mL
  • H 2 O 1 mL
  • Pd(dppf)Cl2 126 mg, 0.17 mmol
  • K 2 CO 3 713 mg, 5.16 mmol
  • Step 3 3-ethyl-5-fluorobenzoic acid To a solution of methyl 3-ethyl-5-fluorobenzoate (130 mg, 0.71 mmol) in MeOH (10 mL) was added LiOH.H 2 O (90 mg, 2.14 mmol) in H 2 O (3 mL). The mixture was stirred at 15°C for 16 hours. The mixture was concentrated under reduced pressure.
  • Step 4 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate
  • TBTU (286 mg, 0.89 mmol
  • DIPEA 231 mg, 1.78 mmol
  • R -benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (217 mg, 0.59 mmol, HCl salt). The mixture was stirred at 20°C for 16 hours.
  • Step 5 (R)-2-amino-3-(3-ethyl-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate 100 mg, 0.21 mmol
  • 33% HBr in AcOH 5 mL
  • the mixture was concentrated under reduced pressure.
  • the residue was purified by Preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-ethyl-5- fluorobenzamido)propanoic acid (30 mg) as HCl salt.
  • Step 2 A solution of (R)-isobutyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (1 g, 2.25 mmol) in 30% HBr in AcOH (15 mL) was stirred at 20°C for 16 hours. The solvent was removed. The residue was added a mixture of AcOH (3 mL) and methyl tert-butyl ether (30 ml).
  • Step 2 Preparation of (R)-(S)-1-azidopropan-2-yl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl- 5-fluorobenzamido)propanoate
  • DCM a solution of (R)-2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluorobenzamido)propanoic acid (1.7 g, 4.38 mmol) in DCM (30 mL) was added DMAP (160 mg, 1.31 mmol), EDCI (1.68 g, 8.75 mmol) and DIPEA (1.13 g, 8.75 mmol) at 0 °C and stirred at for 30 min.
  • Step 3 To a solution of (R)-(S)-1-azidopropan-2-yl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (430 mg, 0.91 mmol) in MeOH (10 mL) was added Pd/C (150 mg, 10% Pd, 50% water) and HCl/MeOH (4 M, 1 mL) under N 2 . The suspension was degassed under vacuum and purged with H 2 several times. The mixture was stirred under H 2 (15 psi) at 25 °C for 1.5 hour. The mixture was filtered and the filtrate was concentrated.
  • Step 2 A solution of (R)-isopentyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (400 mg, 0.872 mmol) in 30% HBr in AcOH (4 mL) was stirred at 20°C for 2 hours. The mixture was concentrated, then ethyl acetate (5 mL) was added and the ethyl acetate phase was extracted with water (10 mL ⁇ 2), and the water phase was lyophilized to give (R)-isopentyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate (270 mg) as HBr salt.
  • Step 2 A mixture of (R)-2-methoxyethyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (550 mg, 1.23 mmol) and Pd/C (100 mg, 10% Pd, 50% water) in ethyl acetate (10 mL) was degassed and purged with H23 times, and then the mixture was stirred at 20 °C for 16 hours under H2 (15 PSI) atmosphere. The reaction mixture was filtered through celite and the filtrate was concentrated.
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-ethyl-3- fluorobenzamido)propanoate
  • TBTU (344 mg, 1.07 mmol)
  • DIPEA 277 mg, 2.14 mmol
  • Step 4 (R)-2-amino-3-(4-ethyl-3-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-ethyl-3- fluorobenzamido)propanoate 120 mg, 0.25 mmol
  • HOAc 4 mL, 30%
  • the mixture was concentrated.
  • the residue was purified by preparative- HPLC (Method AA) to give (R)-2-amino-3-(4-ethyl-3-fluorobenzamido)propanoic acid (50 mg) as HCl salt.
  • Step 2 A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-ethyl-3- fluorobenzamido)propanoate (150 mg, 0.37 mmol) in HBr in HOAc (3 mL, 30%) was stirred at 15°C for 2 hours. The mixture was concentrated. The residue was added water (15 mL) and then lyophilized to give (R)-methyl 2-amino-3-(4-ethyl-3-fluorobenzamido)propanoate (92 mg) as HBr salt.
  • Step 2 3-ethyl-5-fluoro-4-methylbenzoic acid To a solution of methyl 3-ethyl-5-fluoro-4-methylbenzoate (220 mg, 1.12 mmol) in a mixture of THF (4 mL) and H 2 O (2 mL) was added LiOH.H 2 O (141 mg, 3.36 mmol) and the mixture was stirred at 20°C for 16 hours, then the reaction mixture was stirred at 35°C for another 3 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoate
  • TBTU 264 mg, 0.82 mmol
  • DIPEA 213 mg, 1.65 mmol
  • Step 4 (R)-2-amino-3-(3-ethyl-5-fluoro-4-methylbenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoate 230 mg, 0.47 mmol
  • 30% HBr in AcOH 10 mL
  • Step 2 A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoate (120 mg, 0.29 mmol) in the solution of 30% HBr in AcOH (5 mL) was heated at 30°C for 2 hours. The mixture was concentrated. The residue was diluted with MeOH (0.5 mL) and methyl tert-butyl ether (5 mL). The mixture was stirred at 15°C for 0.5 hours.
  • Step 1 methyl 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoate
  • 3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid 877 mg, 4.43 mmol
  • 4- bromo-5-ethyl-isoxazole 0.6 g, 3.41 mmol
  • Na 2 CO 3 723 mg, 6.82 mmol
  • Pd(dppf)Cl 2 .CH 2 Cl 2 (557 mg, 0.68 mmol) under N 2 .
  • the mixture was purged with N 2 , sealed and heated at 120°C for 40 minutes in the microwave.
  • Step 2 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoic acid
  • the mixture was diluted with H 2 O (20 mL).
  • Step 4 (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate 60 mg, 0.11 mmol
  • HBr in AcOH 2 mL, 30% in AcOH
  • Step 2 A mixture of (R)-isobutyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate (0.8 g, 1.56 mmol) and 30% HBr/AcOH (10 mL) were stirred at 25°C for 16 hours. The mixture was concentrated. The residue was washed with methyl tert- butyl ether (5 mL ⁇ 2).
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-4- fluorobenzamido)propanoate
  • TBTU (458 mg, 1.43 mmol)
  • DIPEA 369 mg, 2.85 mmol
  • Step 4 (R)-2-amino-3-(3-ethyl-4-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-4- fluorobenzamido)propanoate 350 mg, 0.73 mmol
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-4- fluorobenzamido)propanoate 350 mg, 0.73 mmol
  • the mixture was concentrated.
  • the residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-ethyl-4- fluorobenzamido)propanoic acid (110 mg) as HCl salt.
  • Step 2 A solution of ((R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-4- fluorobenzamido)propanoate (300 mg, 0.75 mmol) in the solution of 30% HBr in AcOH (10 mL) was stirred at 15°C for 2 hours. The mixture was concentrated. The residue was diluted with MeOH (2 mL) and methyl tert-butyl ether (10 mL). The mixture was stirred at 15°C for 0.5 hours. The precipitate was filtered and washed with methyl tert-butyl ether (2 mL ⁇ 2).
  • Step 2 5-bromo-1-ethyl-4-methyl-1H-pyrazole and 3-bromo-1-ethyl-4-methyl-1H-pyrazole To a solution of 1-ethyl-4-methyl-1H-pyrazole (2.0 g, 18.16 mmol) in CHCl3 (10 mL) was added NBS (3.55 g, 19.97 mmol) and the reaction mixture was stirred at 30°C for 3 hours.
  • reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL ⁇ 3). The combined organic layers were washed with brine (20 mL ⁇ 3), dried over Na 2 SO 4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 5%) to give a mixture of 5-bromo-1-ethyl- 4-methyl-1H-pyrazole and 3-bromo-1-ethyl-4-methyl-1H-pyrazole (1.9 g).
  • Step 4 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoic acid To a solution of methyl 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoate (240 mg, 0.98 mmol) in a mixture of THF (4 mL) and H 2 O (2 mL) was added LiOH.H 2 O (82 mg, 1.96 mmol) and the mixture was stirred at 20°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 5 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5- yl)benzamido)propanoate
  • 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoic acid (220 mg, 0.96 mmol)
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (383 mg, 1.05 mmol, HCl salt)
  • TBTU 460 mg, 1.43 mmol
  • DIPEA 370 mg, 2.87 mmol
  • Step 6 (R)-2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H-pyrazol- 5-yl)benzamido)propanoate (380 mg, 0.70 mmol) in the solution of 30 % HBr in AcOH (10 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated.
  • Step 2 A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H-pyrazol- 5-yl)benzamido)propanoate (100 mg, 0.22 mmol) in the solution of 30 % HBr in AcOH (5 mL) was stirred at 10°C for 1 hour. The reaction mixture was concentrated. The sample was lyophilized to give (R)-methyl 2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5- yl)benzamido)propanoate (55mg) as HBr salt.
  • Step 2 methyl 3-bromo-5-fluoro-4-methoxybenzoate To a solution of methyl 3-bromo-5-fluoro-4-hydroxybenzoate (700 mg, 2.81 mmol) in DMF (10 mL) was added K 2 CO 3 (971 mg, 7.03 mmol) and iodomethane (1.20 g, 8.43 mmol), then the mixture was stirred at 60°C for 3 hours.
  • Step 3 methyl 3-ethyl-5-fluoro-4-methoxybenzoate
  • methyl 3-bromo-5-fluoro-4-methoxybenzoate 700 mg, 2.66 mmol
  • triethylborane 1 M in THF, 8 mL
  • Pd(dppf)Cl 2 195 mg, 0.27 mmol
  • CsOAc 1.53 g, 7.98 mmol
  • THF 5 mL
  • the mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL ⁇ 3).
  • Step 6 (R)-2-amino-3-(3-ethyl-5-fluoro-4-methoxybenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methoxybenzamido)propanoate 100 mg, 0.20 mmol
  • Pd/C 50 mg, 10% Pd, 50% water
  • the sealed tube was heated at 120 °C for 60 min under microwave.
  • the mixture was diluted with water (10 mL) and extracted with ethyl acetate (15 mL ⁇ 3).
  • the combined organic layers were washed with brine (20 mL), dried over Na 2 SO 4 , filtered and concentrated.
  • the residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 20%) to give methyl 3-(5-ethylisothiazol-4- yl)-5-fluorobenzoate (160 mg, crude).
  • Step 2 3-(5-ethylisothiazol-4-yl)-5-fluorobenzoic acid
  • methyl 3-(5-ethylisothiazol-4-yl)-5-fluorobenzoate 160 mg, crude
  • MeOH MeOH
  • H 2 O 2 mL
  • LiOH.H 2 O 37 mg, 0.88 mmol
  • the mixture was stirred at 15 °C for 15 hours.
  • the mixture was concentrated.
  • the residue was dissolved in water (15 mL) and extracted with ethyl acetate (20 mL ⁇ 2). The organic layer was discarded.
  • Step 4 (R)-2-amino-3-(3-(5-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate 40 mg, 0.07 mmol
  • 30% HBr in AcOH (4 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated.
  • Step 2 A mixture of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate (90 mg, 0.19 mmol) in 30% HBr in AcOH (4 mL) was stirred at 25 °C for 1 hour. The mixture was concentrated.
  • reaction mixture was stirred at 0°C for 3 hours.
  • the reaction mixture was poured into 100 mL of sat. Na2SO3, extracted with DCM (80 mL ⁇ 3), the organic layers were washed with brine (50 mL ⁇ 3), dried over Na 2 SO 4 and concentrated.
  • the residue was purified by Combi Flash on silica gel (MeOH/DCM with MeOH from 0% to 10%) to give 5- bromo-1-ethyl-1H-imidazole (1.55 g).
  • 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoic acid 70 mg, crude
  • (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate 131 mg, 358.6 ⁇ mol, HCl salt
  • TBTU 144 mg, 448.3 ⁇ mol
  • DIPEA 116 mg, 896.6 ⁇ mol
  • Step 2 methyl 3-(3-bromoisothiazol-4-yl)-5-fluorobenzoate A mixture of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.0 g, 5.05 mmol), 3-bromo- 4-iodoisothiazole (1.46 g, 5.05 mmol), Pd(dppf)Cl2 (370 mg, 0.51 mmol) and K2CO3 (2.09 g, 15.15 mmol) in a mixture of dioxane (20 mL) and H 2 O (2 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85°C for 16 hours under N2 atmosphere.
  • Step 3 methyl 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoate
  • Step 4 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoic acid
  • a solution of methyl 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoate (110 mg, 0.41 mmol) in a mixture of THF (4 mL) and H 2 O (2 mL) was added LiOH.H 2 O (52 mg, 1.24 mmol) and the mixture was stirred at 10°C for 16 hours.
  • the reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 5 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate
  • TBTU 86 mg, 0.27 mmol
  • DIPEA 69 mg, 0.54 mmol
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4- yl)benzamido)propanoate
  • 3-(5-ethylisoxazol-4-yl)benzoic acid (135 mg, 621.5 ⁇ mol)
  • (R)-benzyl 3- amino-2-(((benzyloxy)carbonyl)amino)propanoate (227 mg, 621.5 ⁇ mol, HCl salt) was added TBTU (299 mg, 932.2 ⁇ mol) and DIPEA (24.0 mg, 1.86 mmol).
  • Step 4 (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4- yl)benzamido)propanoate 130 mg, 246.4 ⁇ mol
  • 30% HBr in AcOH 5 mL
  • the reaction mixture was concentrated, the residue was dissolved in water (5 mL), washed with methyl tert-butyl ether(5 mL).
  • Step 2 A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4- yl)benzamido)propanoate (140 mg, 0.310 mmol) in 30% HBr/AcOH (5 mL) was stirred at 20°C for 1 hour. The reaction mixture was concentrated. The residue was dissolved in water (5 mL), washed with methyl tert-butyl ether (5 mL).
  • Step 3 3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzoic acid
  • MeOH MeOH
  • LiOH.H 2 O LiOH.H 2 O
  • H 2 O H 2 O
  • the mixture was stirred at 20 °C for 16 hours.
  • the mixture was concentrated.
  • the residue was added water (5 mL) and acidified with 2N HCl to adjust pH to 6 ⁇ 7 and extracted with ethyl acetate (20 mL ⁇ 3).
  • Step 4 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5- fluorobenzamido)propanoate
  • TBTU 246 mg, 0.76 mmol
  • DIPEA 198 mg, 1.53 mmol
  • R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (186 mg, 0.51 mmol, HCl salt).
  • Step 5 (R)-2-amino-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5- fluorobenzamido)propanoate 70 mg, 0.13 mmol
  • 30% HBr in AcOH 5 mL
  • Step 2 A mixture of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)- 5-fluorobenzamido)propanoate (100 mg, 0.21 mmol) in 30% HBr in AcOH ( 5 mL) was stirred at 20 °C for 1 hour. The mixture was concentrated. The residue was washed with a mixture of MeOH (1 mL) and methyl tert-butyl ether (5 mL).
  • Step 1 (E)-1-(dimethylamino)hex-1-en-3-one A mixture of pentan-2-one (10 g, 116.10 mmol), DMF-DMA (16.6 g, 139.32 mmol) were stirred at 110°C for 16 hours. The reaction mixture was concentrated to give (E)-1- (dimethylamino)hex-1-en-3-one (2.5 g).
  • Step 3 4-bromo-5-propylisoxazole To a solution of 5-propylisoxazole (1.6 g, crude) in DMF (15 mL) was added NBS (3.33 g, 18.7 mmol) and the reaction mixture was stirred at 15°C for 16 hours. The reaction mixture was quenched with water (10 mL), and extracted with ethyl acetate (20 mL ⁇ 3). The combined organic layers were washed with brine (20 mL ⁇ 3), dried over Na 2 SO 4 and concentrated.
  • Step 4 Methyl 3-fluoro-5-(5-propylisoxazol-4-yl)benzoate (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.0 g, 5.05 mmol), 4-bromo-5- propylisoxazole (960 mg, 5.05 mmol), KF (880 mg, 15.15 mmol) and Pd(t-Bu3P)2 (258 mg, 0.51 mmol) were taken up into a microwave tube in a mixture of dioxane (10 mL) and H 2 O (1 mL) under N2 atmosphere. The sealed tube was heated at 100°C for 60 minutes under microwave.
  • Step 5 3-fluoro-5-(5-propylisoxazol-4-yl)benzoic acid
  • a mixture of methyl 3-fluoro-5-(5-propylisoxazol-4-yl)benzoate (310 mg, 1.18 mmol) in conc.HCl (10 mL) was stirred at 80°C for 14 hours.
  • the mixture was extracted with ethyl acetate (20 mL ⁇ 3).
  • the combined organic layers were washed with brine (20 mL), dried over Na 2 SO 4 , filtered and concentrated to give 3-fluoro-5-(5-propylisoxazol-4-yl)benzoic acid (180 mg).
  • Step 6 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-propylisoxazol-4- yl)benzamido)propanoate
  • 3-fluoro-5-(5-propylisoxazol-4-yl)benzoic acid 90 mg, 0.36 mmol
  • (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate 145 mg, 0.40 mmol, HCl salt
  • TBTU 174 mg, 0.54 mmol
  • DIPEA 140 mg, 1.08 mmol
  • Step 7 (R)-2-amino-3-(3-fluoro-5-(5-propylisoxazol-4-yl)benzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-propylisoxazol-4- yl)benzamido)propanoat (90 mg, 0.16 mmol) in 30 % HBr in AcOH (5 mL) was heated at 50°C for 16 hours. The reaction mixture was concentrated. The residue was washed with a mixture of MeCN (1 mL) and methyl tert-butyl ether (5 mL).
  • Step 2 A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-propylisoxazol-4- yl)benzamido)propanoate (80 mg, 0.17 mmol) in the solution of 30% HBr in AcOH (5 mL) was heated at 20°C for 1 hour. The mixture was concentrated. The residue was washed with a mixture of MeOH (1 mL) and methyl tert-butyl ether (5 mL).
  • Step 2 methyl 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoate
  • 3-(2-chloro-4-(trifluoromethyl)pyridin-3-yl)-5-fluorobenzoate 550 mg, 1.97 mmol
  • triethylborane 1 M in THF, 5.9 mL
  • Pd(dppf)Cl 2 144 mg, 196.64 ⁇ mol
  • CsOAc (1.13 g, 5.90 mmol) in THF (7 mL) was degassed and purged with N 2 3 times, and then the mixture was stirred at 65 °C for 16 hours under N 2 atmosphere. The mixture was concentrated.
  • Step 3 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoic acid
  • methyl 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoate 430 mg, 1.57 mmol
  • MeOH 3 mL
  • LiOH.H 2 O 198 mg, 4.72 mmol
  • the mixture was stirred at 10 °C for 16 hours.
  • the mixture was concentrated to remove MeOH.
  • Step 5 (R)-2-amino-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyl-4-methylpyridin-3-yl)- 5-fluorobenzamido)propanoate 140 mg, 245.78 ⁇ mol
  • 30% HBr in AcOH (3 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated.
  • Step 2 A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyl-4-methylpyridin-3-yl)- 5-fluorobenzamido)propanoate (110 mg, 0.223 mmol) in 30% HBr in AcOH (3 mL) was stirred at 15 °C for 3 hours. The mixture was concentrated.
  • Step 3 methyl 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzoate (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (400 mg, 2.02 mmol), 5-iodo-1-propyl- 1H-1,2,3-triazole (479 mg, 2.02 mmol), K2CO3 (838 mg, 6.06 mmol) and Pd(dppf)Cl2.CH 2 Cl2 (165 mg, 0.20 mmol) were taken up into a microwave tube in a mixture of dioxane (8 mL) and H 2 O (0.8 mL) under N2 atmosphere. The sealed tube was heated at 100°C for 60 minutes under microwave.
  • Step 4 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzoic acid
  • a solution of methyl 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzoate (470 mg, 1.79 mmol) in a mixture of THF (8 mL) and H 2 O (4 mL) was added LiOH.H 2 O (187 mg, 4.46 mmol) and the mixture was stirred at 30°C for 16 hours.
  • the reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 6 (R)-2-amino-3-(3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-1H-1,2,3- triazol-5-yl)benzamido)propanoate 400 mg, 0.71 mmol
  • the solution of 30% HBr in AcOH 15 mL
  • Step 2 A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-1H-1,2,3- triazol-5-yl)benzamido)propanoate (350 mg, 0.72 mmol) in the solution of 30% HBr in AcOH (15 mL) was stirred at 30°C for 1 hour. The mixture was concentrated. The residue was added water (5 mL) and washed with methyl tert-butyl ether (5 mL ⁇ 2).
  • Step 2 methyl 3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoate To a solution of methyl 3-fluoro-5-(1-propyl-1H-pyrazol-5-yl)benzoate (0.89 g, 3.39 mmol) in THF (20 mL) was added NCS (680 mg, 5.09 mmol). The mixture was stirred at 70 °C for 2 hours. The mixture was concentrated.
  • Step 3 3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
  • Step 5 (R)-2-amino-3-(3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-propyl-1H-pyrazol- 5-yl)-5-fluorobenzamido)propanoate (0.18 g, 0.3 mmol) and HBr in AcOH (8 mL, 30% in AcOH) were stirred at 30°C for 15 hours. The mixture was concentrated. The residue was diluted with AcOH (3 mL).
  • Step 2 mixture of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-propyl-1H-pyrazol- 5-yl)-5-fluorobenzamido)propanoate (0.2 g, 0.39 mmol) and HBr in AcOH (8 mL, 30% in AcOH) were stirred at 30°C for 15 hours. The mixture was concentrated. The residue was diluted with AcOH (3 mL) and methyl tert-butyl ether (50 mL) was added to the mixture. The precipitate was filtered under N2. The filter cake was lyophilized.
  • Step 2 1-ethyl-5-iodo-3-methyl-1H-pyrazole
  • n-BuLi 20 mL, 2.5 M in hexane
  • I2 (12.1 g, 47.66 mmol) was added and the reaction was stirred at -78°C for 1 hour.
  • H 2 O 50 ml was added to quench the reaction and extracted with DCM (100 mL ⁇ 3).
  • Step 4 methyl 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate
  • a mixture of methyl 3-(1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (900 mg, 3.43 mmol) and NCS (500 mg, 3.74 mmol) in THF (2 mL) was stirred at 70°C for 2 hours. The mixture was concentrated.
  • Step 5 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
  • H 2 O (150 mg, 3.57 mmol) in a mixture of MeOH (8 mL) and H 2 O (2 mL) was stirred at 30°C for 4 hours. The mixture was concentrated. H 2 O (2 mL) was added, acidified with satd.
  • Step 7 (R)-2-amino-3-(3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-3-methyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate 270 mg, 0.46 mmol
  • 30% HBr in AcOH 5 mL
  • Step 2 A mixture of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-3-methyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (40 mg, 0.078 mmol) in 30% HBr in AcOH (5 mL) was stirred at 30°C for 2 hours. The mixture was concentrated.
  • Step 1 1-ethyl-5-iodo-1H-pyrazole
  • THF 60 mL
  • n- BuLi 25.0 mL, 2.5M in hexane
  • I2 15.8 g, 62.4 mmol, dissolving in 40 mL THF
  • the reaction mixture was allowed to warm to 30°C for 2 hours.
  • Step 3 methyl 3-(1-ethyl-4-iodo-1H-pyrazol-5-yl)-5-fluorobenzoate
  • THF methyl 3-(1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate
  • NIS 1049 mg, 4.65 mmol
  • the reaction mixture was stirred at 70°C for 4 hours.
  • the reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL ⁇ 3). The combined organic layers were washed with brine (20 mL ⁇ 3), dried over Na 2 SO 4 and concentrated.
  • Step 4 methyl 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoate
  • a mixture of methyl 3-(1-ethyl-4-iodo-1H-pyrazol-5-yl)-5-fluorobenzoate (500 mg, 1.34 mmol), methyl 2,2-difluoro-2-fluorosulfonyl-acetate (385 mg, 2.00 mmol), CuI (51 mg,0.27 mmol) in DMF (8 mL) was heated at 120°C for 4 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL ⁇ 3).
  • Step 5 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoic acid
  • methyl 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoate 240 mg, 0.76 mmol
  • LiOH.H 2 O 48 mg, 1.14 mmol
  • the reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL ⁇ 3).
  • Step 6 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-(trifluoromethyl)-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate
  • a mixture of 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoic acid 113 mg, 0.37 mmol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate 150 mg, 0.41 mmol, HCl salt
  • TBTU 180 mg, 0.56 mmol
  • DIPEA 145 mg, 1.12 mmol
  • reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL ⁇ 3). The combined organic layers were washed with brine (20 mL ⁇ 3), dried over Na 2 SO 4 and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 30%).
  • Step 7 (R)-2-amino-3-(3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-(trifluoromethyl)- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate 160 mg, 0.26 mmol
  • the solution of 30% HBr in AcOH 5 mL
  • reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL ⁇ 3). The combined organic layers were washed with brine (20 mL ⁇ 3), dried over Na 2 SO 4 and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 30%).
  • Step 2 A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-(trifluoromethyl)- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (93 mg, 0.17 mmol) in the solution of 30% HBr in AcOH (5 mL) was stirred at 20°C for 1 hour. The mixture was concentrated.
  • Step 3 3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
  • MeOH methyl 3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoate (235 mg, 0.75 mmol) in MeOH (4 mL)
  • LiOH.H 2 O 95 mg, 2.26 mmol
  • H 2 O 1 mL
  • Step 5 (R)-2-amino-3-(3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-(difluoromethyl)-1-propyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate 140 mg, 0.23 mmol
  • AcOH 3 HBr
  • the mixture was concentrated.
  • Step 2 A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-(difluoromethyl)-1-propyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (60 mg, 0.11 mmol) in 30% HBr in AcOH (2 mL) was stirred at 25°C for 2 hours. The solvent was removed under reduced pressure.
  • Step 2 methyl 3-fluoro-5-(4-iodo-1-propyl-1H-pyrazol-5-yl)benzoate
  • NIS 1.03 g, 4.58 mmol
  • the reaction mixture was stirred at 70°C for 2 hours.
  • the reaction mixture was concentrated.
  • the residue was added water (15 mL), extracted with ethyl acetate (15 mL ⁇ 3).
  • the organic layers was washed with brine (10 mL ⁇ 3), dried over Na 2 SO 4 , filtered and concentrated.
  • Step 3 methyl 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzoate
  • methyl 3-fluoro-5-(4-iodo-1-propyl-1H-pyrazol-5-yl)benzoate 590 mg, 1.52 mmol
  • methyl 2,2-difluoro-2-(fluorosulfonyl)acetate 1.46 g, 7.60 mmol
  • the mixture was stirred at 120°C for 15 hours.
  • the reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (15 mL ⁇ 3).
  • Step 4 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzoic acid
  • a solution of methyl 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzoate (280 mg, 0.85 mmol) in a mixture of MeOH (10 mL) and H 2 O (2 mL) was added LiOH.H 2 O (90 mg, 2.14 mmol). The mixture was stirred at 30°C for 1 hour. The reaction mixture was concentrated. The residue was added water (10 mL), adjusted pH to 5-6 with 1N HCl and extracted with ethyl acetate (15 mL ⁇ 3).
  • Step 5 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoate
  • Step 6 (R)-2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoate 110 mg, 0.18 mmol
  • 30% HBr in AcOH 6 mL
  • Step 2 A mixture of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoate (70 mg, 0.13 mmol) in 30% HBr in AcOH (6 mL) was stirred at 20°C for 1 hour. The reaction mixture was concentrated. The residue was added water and extracted methyl tert-butyl ether (10 ml ⁇ 3).
  • Step 1 methyl 3-(1-ethyl-4-formyl-1H-pyrazol-5-yl)-5-fluorobenzoate
  • POCl 3 7.43 g, 48.42 mmol
  • DMF 7.43 g, 19.50 mmol
  • the mixture was heated to 100°C for 16 hours.
  • the mixture was poured into H 2 O (10 mL) at 0°C, neutralize with sat. aqueous Na 2 CO 3 to pH 7 ⁇ 8 and extracted with ethyl acetate (20 mL ⁇ 2).
  • Step 2 methyl 3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate
  • DCM methyl 3-(1-ethyl-4-formyl-1H-pyrazol-5-yl)-5-fluorobenzoate
  • DAST 8.40 g, 52.12 mmol
  • the resulting mixture was stirred at 25°C for 2 hours.
  • the mixture was poured into sat. aqueous Na 2 CO 3 to pH 7 ⁇ 8 at 0°C and extracted with DCM (30 mL ⁇ 2).
  • Step 3 3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
  • MeOH methyl 3-[4-(difluoromethyl)-2-ethyl-pyrazol-3-yl]-5-fluoro-benzoate (410 mg, 1.37 mmol) in MeOH (8 mL)
  • LiOH LiOH .
  • H 2 O (173 mg, 4.12 mmol) in H 2 O (4 mL)
  • the mixture was concentrated to move MeOH, then added sat.
  • Step 4 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-(difluoromethyl)-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate
  • a mixture of 3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid 180 mg, 0.63 mmol
  • (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (277 mg, 0.76 mmol, HCl salt)
  • TBTU 305 mg, 0.95 mmol
  • DIPEA 164 mg, 1.27 mmol
  • Step 2 methyl 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate To a solution of methyl 3-(1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (180 mg, 0.73 mmol) in THF (10 mL) was added NCS (97 mg, 0.73 mmol), the mixture was stirred at 70°C for 1 hour. The mixture was concentrated.
  • Step 3 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
  • a solution of methyl 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (240 mg, 0.85 mmol) in the mixture of MeOH (5 mL) and H 2 O (5 mL) was added LiOH.H 2 O (53 mg, 1.27 mmol), the mixture was stirred at 20°C for 1 hour. The mixture was concentrated.
  • Step 2 A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol- 5-yl)-5-fluorobenzamido)propanoate (200 mg, 0.40 mmol) in HBr in HOAc (3 mL, 30%) was stirred at 15°C for 2 hours. The mixture was concentrated. The residue was added water (20 mL) and then lyophilized to give (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)- 5-fluorobenzamido)propanoate (158 mg) as HBr salt.
  • Step 2 A mixture of (R)-isopropyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (300 mg, 0.57 mmol) in HBr/AcOH (30 mL) was stirred at 25°C for 3 hours, The residue was diluted with methyl tert-butyl ether (40 mL) and propan-2-ol (5 mL). The precipitate was filtered under N2.
  • Step 2 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (200 mg, 0.3 mmol) was added to AcOH (4 mL) and 30% HBr in AcOH (0.4 mL). Then mixture was heated at 20°C for 1.5 hours. To the mixture was added methyl tert-butyl ether (5 mL).
  • Step 2 A solution of (R)-phenyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5- yl)-5-fluorobenzamido)propanoate (190 mg, 0.34 mmol) in the solution of 30% HBr in AcOH (6 mL) was stirred at 20°C for 0.5 hours. To the mixture was added methyl tert-butyl ether (5 mL).
  • Step 2 A solution of (R)-cyclopropyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (100 mg, 0.19 mmol) in a mixture of AcOH (3 mL) and 30% HBr in AcOH (0.3 mL) was heated and stirred at 20°C for 2 hours. The mixture was washed with methyl tert-butyl ether (5 mL).
  • Step 2 To a solution of (R)-2-methoxyethyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (200 mg, 0.366 mmol) in AcOH (5 mL) was added 30% HBr in AcOH (0.5 mL) and the mixture was stirred at 15 °C for 3 hours. The mixture was concentrated and methyl tert-butyl ether (30 mL) was added and the mixture was stirred at 15°C for 30 minutes.
  • Step 2 A solution of (R)-cyclohexyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (120 mg, 0.21 mmol) in 30% HBr in AcOH (2 mL) was stirred at 20°C for 30 min. The mixture was concentrated. The residue was washed with TBME (2 mL ⁇ 3) and the organic layer was decanted.
  • Step 2 To a mixture of (R)-2-oxo-2-(pyrrolidin-1-yl)ethyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4- chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (180 mg, 0.30 mmol) in AcOH (4 mL) was added 30% HBr in AcOH (0.6 mL). The mixture was stirred at 15°C for 2 hours. The reaction mixture was added methyl tert-butyl ether (15 mL).
  • Step 2 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylic acid
  • methyl 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylate 915 mg, 3.54 mmol
  • THF 5 mL
  • H 2 O 10 mL
  • LiOH.H 2 O 446 mg, 10.63 mmol
  • the mixture was stirred at 25°C for 16 hours.
  • Step 3 (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate
  • TBTU 7.4 mmol
  • DIPEA 7.9 mmol
  • R 4-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (657 mg, 1.80 mmol, HCl salt).
  • Step 4 (R)-2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid
  • (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]- 3-ylcarboxamido)propanoate 620 mg, 1.12 mmol
  • AcOH AcOH
  • Step 2 A mixture of (R)-isopropyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoate (3.6 g, 7.11 mmol) in 30% HBr in AcOH (20 mL) was stirred at 25°C for 1 hour. The mixture was concentrated.
  • the reaction mixture was stirred at 20°C for 1 hour.
  • the mixture was added water (80 mL) and extracted with ethyl acetate (100 mL ⁇ 3), the organic layers were washed with brine (80 mL ⁇ 3), dried over Na 2 SO 4 , filtered and concentrated.
  • Step 2 (R)-cyclopropyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate (4.90 g, 9.71 mmol) in a mixture of AcOH (30 mL) and 30% HBr in AcOH (3 mL) was stirred at 20°C for 2 hours. The mixture was washed with methyl tert-butyl ether (30 mL). The organic layer was decanted, then the residue was submitted to lyophilization.
  • Step 2 (R)-ethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate
  • (R)-ethyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate (670 mg, 1.36 mmol) in 30% HBr/AcOH (10 mL) was stirred at 20°C for 1 hour. The reaction mixture was concentrated.
  • Example 1a Affinity data for the glycine binding site of the NMDA receptor and Permeability Data of parent compounds of prodrugs of formula (I) Scintillation proximity assay (SPA): To determine the affinity of the compounds of the present invention for the glycine binding site of the NMDA receptor a SPA was used.
  • NMDA N-methyl-D-aspartate
  • the assay buffer contained 100 mM HEPES-NaOH, 150 mM NaCl, 1 mM EDTA, 10% glycerol at pH 7.4 in ultra-pure water.
  • Non-specific binding wass defined by inclusion of 10 ⁇ M L-689,560 (highly potent NMDA antagonist) and total binding by 1% DMSO.
  • the SPA beads were allowed to settle for 3 hours after which the signal was read on a Viewlux instrument (Perkin Elmer). Normalized data were used to calculate IC50 and Ki values.
  • MDR1-MDCKII permeability assay Papp(AB) A MDR1-MDCKII permeability assay can be used to measure the ability of compounds to passively permeate a cell membrane.
  • MDR1-MDCKII cells obtained from Piet Borst at the Netherlands Cancer Institute
  • PET polyethylene membranes
  • Test compounds were diluted with the transport buffer (HBSS with 10mM HEPES, added 1% BSA, pH 7.4) from DMSO stock solution to a concentration of 0.5 ⁇ M (DMSO: 0.4%) and applied to the apical or basolateral side of the cell monolayer. Permeation of the test compounds from A to B direction or B to A direction was determined in triplicate over a 60- minute incubation at 37°C and 5% CO2 with a relative humidity of 95%. In addition, the efflux ratio of each compound was also determined. Test and reference compounds were quantified by LC/MS/MS analysis based on the peak area ratio of analyte/IS.
  • Reference compounds Fenoterol (conc: 2 ⁇ M) was used as low permeability marker and Propranolol (conc: 2 ⁇ M) was used as high permeability marker in A to B Permeability, furthermore was bi-directional permeability of a P-glycoprotein substrate (digoxin) included.
  • the mass balance is defined as: the sum of the compound recovered from the acceptor chamber and the compound remaining in the donor chamber at the end of the experiment, divided by the initial donor amount. The mass balance should be as high as possible. Criterion: Recovery ⁇ 50% is insufficient.
  • Lucifer Yellow permeability was measured in one direction (A to B). The percentage of Lucifer Yellow was calculated in control wells as an estimation of the overall cell membrane integrity. The wells were considered fully acceptable if %Lucifer Yellow was less than 2%.
  • Table 3a Permeability data and Ki data of parent compounds of the invention
  • Example 1b Permeability Data of prodrug compounds of the invention
  • the experiments for the prodrugs of the invention were the same as for the parent compounds exemplified in Table 3a. The results are listed below in Table 3b. Results shows that prodrugs of the parent compounds have an improved permeability compared to the respective parent compounds.
  • Table 3b Permeability of prodrug compounds of the invention.
  • Example 2 In vivo exposure data
  • Plasma samples were dissected once the animal had been sacrificed, slightly “dipped” on filter paper to remove blood overflow on the outside, and transferred into Covaris AFA tubes. Plasma and brain samples were stored at -80°C until analysis. Sample preparation: Seven calibration standards and three QC samples were prepared in plasma and brain homogenate, respectively, in the concentration range 10-10000 ng/mL. Blank samples (control matrix with internal standard) were prepared and treated in the same way as calibration standards. Prior to analysis, the brain samples were homogenized with milliQ water 1:4 (w/v) using a Covaris focused-ultrasonicator. Study samples with expected concentration above upper limit of quantification were diluted with blank matrix.
  • LC-MS/MS Samples were analyzed using an AB Sciex API4000 triple quadrupole (TQ) mass spectrometer operated in positive and negative electrospray ionization and MS/MS mode (multiple reaction monitoring, MRM).
  • the mass spectrometer was coupled to a Waters Acquity UPLC equipped with a Waters Acquity UPLC HSS C18 SB (1.7 ⁇ m, 30 mm x 2.1 mm) analytical column. Chromatographic separation was achieved by a 3-minute gradient starting with 98% mobile phase A (0.1% Formic Acid in water) and 2% mobile phase B (0.1% Formic Acid in Acetonitrile) increasing to 95% mobile phase B. Flow rate was 0.6 mL/min and the column temperature was 40 o C.
  • Multiple reaction monitoring (MRM) transitions (m/z) were as follows: 380 ⁇ 248, 350 ⁇ 263, Tolbutamide: 269 ⁇ 106 (neg) and 271 ⁇ 155 (pos).
  • the individual treatment groups were dosed with either 10% hydroxypropyl- ⁇ -cyclodextrin (vehicle), compound 2al (3, 10, or 30 mg/g) or compound 2ba (3, 10, or 30 mg/g),
  • the dosing of the animals were performed by subcutaneous injection 30 minutes before test according to treatment groups.
  • Rats were individually assessed for the production of a tonic hind limb extensor seizure using a Hugo Sachs Electronik stimulator, which delivered an adjustable constant current (1–300 mA) of 0.3 seconds duration via corneal electrodes.
  • the stimulus intensity was varied, from a typical baseline of 25mA, by an ‘up and down’ method of shock titration.
  • the first rat within a treatment group was given a shock at the expected or estimated seizure threshold (CC50) current, that is, the current producing tonic hind limb extensor seizure in 50% of animals.
  • CC50 seizure threshold
  • the stimulus intensity was lowered or raised in log 0.06 :10 ⁇ (1+x*0.06) mA intervals if the preceding rat did or did not show tonic hind limb extension, respectively.
  • This procedure continued for all rats within a treatment group.
  • NMDA receptor antagonists are known to demonstrate anticonvulsive properties in various rodent seizure/epilepsy models (Parsons CG et.al., Neuropharmacology, (1995), 34, 1239; P. Wlaz et. al., Epilepsia, (1996), 37, 610)
  • Example 4 Porsolt swim test Adult male Wistar Kyoto (WKY) rats from Envigo (former Harlan; Blackthorn, UK). Animals were maintained under controlled conditions (21 ⁇ 1 °C, 37 ⁇ 1 %, 12 / 12 h light / dark cycles, lights on at 8 a.m.) with food and water available ad libitum.
  • saline vehicle
  • ketamine 5 mg/kg
  • compound 2al 3, 10, or 30 mg/kg
  • the Fisher least significant difference (LSD) test was used for post hoc analyses. Probability values of P ⁇ 0.05 were considered as statistically significant.
  • Statistical analyses were performed using SPSS. As shown in Figure 3, administration of compound 2al dosed at 1, 3, 10 and 20 mg/kg subcutaneously showed significant effects in time of immobility at the 3, 10 and 20 mg/kg doses.
  • the Porsolt swim test is a well-established model used in basic research and the pharmaceutical screening of potential antidepressant treatments. Ketamine (5mg/kg) induces decreased immobility in this test 24 hours following a single dose. Decreased immobility in the Porsolt swim test was the main endpoint to measure prolonged efficacy (Bogdanova OV et. al. Physiol.
  • Rats were habituated for 2 hours followed by 45 minutes of baseline recording, where after rats were injected subcutaneously with 10% hydroxypropyl- ⁇ -cyclodextrin (HP ⁇ CD, vehicle), 10 mg/kg ketamine in saline, or 20 mg/kg of compound 2al or 2ba in 10 % HP ⁇ CD and left in the box for two more hours. Rats only went through recording sessions once a week with at least six days between recordings to allow for wash-out of compounds.
  • HP ⁇ CD hydroxypropyl- ⁇ -cyclodextrin
  • the analog local field potential and electrocorticogram (LFP and ECoG) signals were amplified and band-pass filtered at 0.01-300 Hz (Precision Model 440; Brownlee, Palo Alto, CA, USA) and converted to a digital signal at a sampling rate of 1 kHz (CED Power 1401, Power 1 (625 kHz, 16 bit) and CED Expansion ADC16; CED, Cambridge, England).
  • An analog 50 Hz notch filter (Precision Model 440, Brownlee) was applied to the LFP/ECoG signals of the first dataset but was not applied in the following pharmaco-EEG experiments.
  • Video recordings were processed in EthoVision producing the mobility signal, which was subsequently collected in Spike2 along with the LFP/ECoG signals with a delay used subsequently to synchronize the signals.
  • Data analysis The development of the locomotive state-detection algorithm and the state-specific pharmaco-EEG analyses were carried out in MATLAB R2017a (The MathWorks, Inc., Natick, MA, USA) using functions from the sigTOOL toolbox. Significant differences between drug- treated animals and vehicle were assessed according to Turkey’s honest significant difference.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Pain & Pain Management (AREA)
  • Psychiatry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention is directed to novel prodrugs of modulators of the NMDA receptor. Separate aspects of the inventions are directed to pharmaceutical compositions comprising said compounds and uses of the compounds to treat neurological disorders or neuropsychiatric disorders such as depression.

Description

PRODRUGS OF 3-BENZOAMIDO-2-AMINOPROPIONIC ACID DERIVATIVES AS MODULATORS OF THE NMDA RECEPTOR
Field of the invention
The present invention is directed to compounds that are prodrugs of modulators of the NMDA receptor, pharmaceutical compositions comprising said compounds, and their use in the treatment of neurological disorders or neuropsychiatric disorders such as depression, in particular major depressive disorder (MDD) and treatment-resistant depression (TRD).
Background of the invention
The World Health Organization estimates 350 million people will be affected with MDD and has projected that depression will constitute the largest health burden on society worldwide by 2030. A rough working estimate of prevalence is that depression affects l/5th of the population at some point, affecting women in a higher proportion than men (5-9% and 2-3% incidence respectively in the US, representing an overall incidence of 6.6%). The North- American Center for Disease Control has reported that from 2005-2008, 8.9% of the US population was prescribed an antidepressant during any given month, antidepressants being also prescribed for anxiety, pain, and other non-mood disorders [Global Burden of Disease Study. Lancet. May 17, 1997; 349(9063): 1436-1442],
Antidepressants are marketed and thus known to the skilled person. Some examples of different types of antidepressant are selective serotonin reuptake inhibitors (SSRIs), Serotonin-norepinephrine reuptake inhibitors (SNRIs), Monoamine oxidase inhibitors (MAOIs), and Tricyclic antidepressants. Typical limitations of known antidepressants are delayed onset of efficacy and low remission rates after multiple courses of pharmacotherapy, and for some antidepressants severe side-effects [Antidepressants and the risk of suicidal behaviours. Jama. Jul. 21 2004; 292(3):338-343],
In recent years, modulators of the N-Methyl-D-Aspartate (NMDA) receptors have received more attraction in treatment of MDD, in particular for treatment-resistant depression (TRD). Especially, ketamine, a pore-blocking antagonist of the NMDA receptor, is used for treating MMD due to its antidepressant effect and fast onset. However, MDD treatment with ketamine has the drawback of sedation, psychotomimetic side effects and requirement of intravenous or intransal administration.
NMDA receptors are tetrameric ligand-gated ion channels which are also involved in essential physiological processes such as synaptic plasticity and development. NMDA receptors are heterotetramers comprising two GluNl subunits and two GluN2/GluN3 subunits. This means that they assemble as either diheteromeric or triheteromeric receptors. The majority of native NMDA receptors consist of two GluNl subunits and two GluN2 subunits. Activation of the NMDA receptors requires simultaneous binding at two different binding sites. Glutamate, the major excitatory neurotransmitter in the central nervous system, binds to the GluN2 subunits and glycine binds to the GluNl and GluN3 subunits.
Another known modulator of the NMDA receptor is D-cycloserine, which is a partial glycine site agonist. D-cycloserine has been intensively studied due to its neuroactive properties and potential utility in treatment of depression and depression disorders such as MDD [Heresco- Levy, U., Javitt, D.C., Gelfin, Y., Gorelik, E., Bar, M., Blanaru, M., Kremer, I., 2006. Controlled trial of d-cycloserine adjuvant therapy for treatment-resistant major depressive disorder. J. Affect. Disord. 93, 239-243] and PTSD [Olden, M., Wyka, K., Cukor, J., Peskin, M., Altemus, M., Lee, F.S., Finkelstein-Fox, L., Rabinowitz, T., Difede, J., 2017. Pilot study of a telehealth- delivered medication augmented exposure therapy protocol for PTSD. J. Nerv. Ment. Dis. 205, 154-160], However, treatment of D-cycloserine suffers from frequent complaints of psychopathological stimulation such as anxiety, euphoria, agitation, feeling stimulated, dizziness/drowsiness, fatigue, headache, and gastrointestinal disturbance [Schade, S., Paulus, W., 2016. D-Cycloserine in neuropsychiatric diseases: a systematic review. Int. J. Neuropsychopharmacol]
Urwyler et al., J. Med. Chem. 2009, 52, 5093-5107 discloses 3-acylamino-2-amonipropionic acid derivatives with affinity for the glycine site of the NMDA receptor. Maolanon et. al., ACS Chem. Neurosci. 2017, 8, 1681-1687 discloses Subtype-Specific Agonists for NMDA Receptor Glycine Binding Sites.
Despite the longstanding interest in the field, there is evidently still an unmet need as regards developing efficient, well tolerated and active drugs for the treatment of depression in particular MDD and TRD. A prodrug of a compound being a modulator of the NMDA receptor, with improved permeability and brain exposure compared to the parent compound may fulfil such unmet needs. Summary of the invention 5 With this background, it is an object of the invention to provide a prodrug of modulators of the NMDA receptor. Accordingly, the present invention relates to compounds of formula I, or a pharmaceutically acceptable salt thereof:
Figure imgf000005_0001
wherein 10 R1 is selected from the group consisting of hydrogen and halogen; R2 and R4 each independently are selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3- C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1- C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy are optionally 15 substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkyl, hydroxy(C1-C4)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl; R3 is selected from the group consisting of hydrogen, halogen, (C1-C6)alkyl and (C1-C4)alkoxy; 20 R5 is selected from the group consisting of (C1-C6)alkyl, (C3-C6)cycloalkyl and phenyl, wherein said (C1-C6)alkyl, (C3-C6)cycloalkyl and phenyl are optionally substituted with one or more substituents independently selected from the group consisting of -NH2, (C1-C4)alkoxy, (C3- C6)cycloalkyl, phenyl, 4-6 membered heterocyclyl and -L-R6, wherein -L- represents -C(O)- and R6 is selected from the group consisting of 4-6 membered heterocyclyl; or pharmaceutically acceptable salts thereof, with the proviso that at least one of R1, R2, R3 and R4 is not hydrogen, and with the proviso that when R2, R3 and R4 each represents hydrogen, R1 does not represent fluoro. In a further aspect is provided a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or diluents. In a further aspect is provided a method for the treatment of depression comprising the administration of a therapeutically effective amount of a compound of formula I, or acceptable salt thereof, or a pharmaceutical composition to a patient in need thereof. In a further aspect is provided a compound of formula I, or a pharmaceutically acceptable salt thereof for use as a medicament. In a further aspect is provide a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof, for use in the treatment of depression. In a further aspect is provided a use of a compound of formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising a compound of formula I, or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for use in the treatment of depression. These and other aspects of the invention will become apparent upon reference to the following detailed description. It should be understood that the various aspects, embodiments, implementations and features of the invention mentioned herein may be claimed separately, or in any combination. All references, including publications, patent applications and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety. Headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way. Brief Description of Drawings Figure 1: Effects of compound 2al in the MEST model. Y-axis: Estimated seizure threshold (CC50) current (mA) ; X-axis: bar furthest to the left: vehicle 10% HPβCD; bar second to the left: 3 mg/kg of compound 2al; bar third to the left: 10 mg/kg of compound 2al; bar furthest to the right: 30 mg/kg of compound 2al. Significance levels for post-hoc comparisons (relative to the vehicle group) are indicated: *<0.05, **<0.01, ***<0.001. Figure 2: Effects of compound 2ba in the MEST model. Y-axis: Estimated seizure threshold (CC50) current (mA) ; X-axis: bar furthest to the left: vehicle 10% HPβCD; bar second to the left: 3 mg/kg of compound 2ba; bar third to the left: 10 mg/kg of compound 2ba; bar furthest to the right: 30 mg/kg of compound 2ba. Significance levels for post-hoc comparisons (relative to the vehicle group) are indicated: *<0.05, **<0.01, ***<0.001. Figure 3: Effects of compound 2al and ketamine in the forced swim test model. Y-axis: Immobility time (s); X-axis: bar furthest to the left: Vehicle; bar second to the left: ketamine (5 mg/kg); bar third to the left: compound 2al (1mg/kg); bar third to the right: compound 2al (3 mg/kg); bar second to the right: compound 2al (10 mg/kg); bar furthest to the right: compound 2al (20 mg/kg) Significance levels for post-hoc comparisons (relative to the vehicle group) are indicated: *<0.05, **<0.01, ***<0.001. Figure 4: Effects of compound 2al in Resting state Electroencephalography (rsEEG) obtained in the Nucleus accumbens according to Example 5. Y-axis: Baseline-normalized power (dB); X-axis: bar furthest to the left: vehicle 10% HPβCD; bar second to the left: 3 mg/kg of compound 2al; bar third to the left: 10 mg/kg of compound 2al; bar furthest to the right: 30 mg/kg of compound 2al. Significance level for post-hoc comparison (relative to the vehicle group) is indicated: ***<0.001. Figure 5: Effects of compound 2ba in Resting state Electroencephalography (rsEEG) obtained in the Nucleus accumbens according to Example 5. Y-axis: Baseline-normalized power (dB); X-axis: bar furthest to the left: vehicle 10% HPβCD; bar second to the left: 3 mg/kg of compound 2ba; bar third to the left: 10 mg/kg of compound 2ba; bar furthest to the right: 30 mg/kg of compound 2ba. Significance level for post-hoc comparison (relative to the vehicle group) is indicated: ***<0.001. Detailed Description of the Invention The present invention relates to ester prodrugs (Formula I) of the parent compounds (Formula V) or a pharmaceutically acceptable salt thereof, wherein:
Figure imgf000008_0001
R1 is selected from the group consisting of hydrogen and halogen; R2 and R4 each independently are selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3- C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1- C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkyl, hydroxy(C1-C4)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl; R3 is selected from the group consisting of hydrogen, halogen, (C1-C6)alkyl and (C1-C4)alkoxy; or pharmaceutically acceptable salts thereof, with the proviso that at least one of R1, R2, R3 and R4 is not hydrogen, and with the proviso that when R2, R3 and R4 each represents hydrogen, R1 does not represent fluoro. The inventors observed that the prodrugs of the invention have an improved permeability compared to the parent compounds of the invention as shown in Tables 3a and 3b. The inventors observed that dosing of compound 2al and 2ba gave a significant response in the Maximal Electro Shock Threshold test compared to vehicle. The inventors observed that dosing of compound 2al gave a significant response in the forced swim test compared to vehicle and a similar response to that observed with ketamine. The inventors observed that the administration of compounds 2al and 2ba showed significant effects in resting state Electroencephalography as shown in Figure 5 and Figure 3b. Definitions As used herein, the term ”(Cx-Cy) alkyl” refers to a hydrocarbyl radical obtained when one hydrogen atom is removed from a linear (i.e. unbranched) or branched saturated hydrocarbon. The number of carbon atoms in the hydrocarbyl substituent (i.e. alkyl) is indicated by the prefix “(Cx-Cy)”, wherein x is the minimum and y is the maximum number of carbon atoms in the substituent. Said ”(Cx-Cy) alkyl” comprises 1-6, preferably 1-4, more preferably 1-3, such as 1-2 or such as 2-3 carbon atoms. Thus, for example, “(C1-C6) alkyl” refers to an alkyl substituent containing from 1 to 6 carbon atoms. Examples of alkyl groups include the subclasses normal alkyl (n-alkyl), secondary and tertiary alkyl. Thus “(C1-C6) alkyl” includes groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec.-butyl, tert. - butyl, n-pentyl, isopentyl, neopentyl, n-hexyl and isohexyl. The term “alkylene,” as used herein, refers to a divalent group derived from a straight or branched chain hydrocarbon of 1 to 6 carbon atoms, for example, of 1 to 4 carbon atoms. Representative examples of alkylene include, but are not limited to, -CH2-, -CH2CH2-, - CH2CH2CH2-, -CH2CH(CH3)CH2-, -CH2CH2CH2CH2-, -CH2CH(CH3)CH2CH2-, and - CH2CH2CH2CH2CH2-. As used herein, the term “(Cx-Cy) alkoxy” refers to a moiety of the formula –OR’, wherein R’ indicates (Cx-Cy)alkyl as defined above. Thus, for example “(C1-C4)alkoxy” refers to such moiety wherein the alkyl contains from one to four carbon atoms. Examples of “(C1- C4)alkoxy” include methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy and tert- butoxy. The term “(Cz-Cw)alkoxy(Cx-Cy)alkyl”, as used herein refers to an (Cz-Cw)alkoxy group, as defined herein, appended to the parent molecular moiety through an (Cx-Cy)alkyl group, as defined herein. Examples include, but are not limited to, methoxymethyl and ethoxymethyl. As used in the context of the present invention, the term “halogen” indicates a substituent selected from the 7th main group of the periodic table, such as fluoro, chloro, bromo and iodo (F, Cl, Br and I). As used herein, the term “halo(Cx-Cy)alkyl” means a (Cx-Cy)alkyl group as defined herein, which is substituted with one or more halogen as defined herein, e.g. fluoro, chloro or bromo. Examples include, but are not limited to, trifluormethyl and difluoromethyl. As used herein, the term “cyano”, refers to a CN group appended to the parent molecule through the carbon atom of the CN group. The term “hydroxyl” or “hydroxy,” as used herein, means an -OH group. The term “phenyl” is intended to mean a benzene radical, with one H removed at the attachment point. The term “hydroxy(Cx-Cy)alkyl” or “hydroxyl(Cx-Cy)alkyl” as used herein, means a (Cx-Cy)alkyl group as defined herein, which is substituted with one or more -OH group(s). Examples include, but are not limited to hydroxymethyl, hydroxyethyl, hydroxypropyl. The term “oxo” as used herein means an oxygen atom which is connected to the parent moiety via a double bond (=O). The group C(O) is intended to represent a carbonyl group (C=O). The term “heteroaryl,” as used herein, refers to a radical of a heteroaromatic ring. As used herein the term “5- or 6-membered heteroaryl” means a radical of a five or six membered heteroaromatic monocyclic ring containing at least one heteroatom independently selected from the group consisting of N, O and S, such as containing 1 to 4 heteroatoms independently selected from O, S, and N, such as 1 to 3 heteroatoms independently selected from O, S, and N, such as containing 1 to 2 heteroatoms independently selected from O, S, and N, such as containing 1 to 2 heteroatoms independently selected from O and N. The five membered aromatic monocyclic rings have two double bonds and the six membered aromatic monocyclic rings have three double bonds. The heteroaryl radical may be connected to the parent moiety through a carbon atom or a nitrogen atom contained anywhere within the heteroaryl group. Representative examples of 5- or 6-membered heteroaryl include, but are not limited to, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, pyrazolyl, pyrrolyl, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl. As used herein, the term ‘5- or 6-membered heteroaromatic rings comprising 1 or 2 nitrogen’ comprises, but is not limited to, pyrrol, pyrazol, imidazole, pyridine, pyrimidin, pyridazine and pyrazine. The term “heterocyclyl” as used herein, means a radical of an aliphatic heterocycle. The term ‘4 - 6 membered heterocyclyl’ as used herein, means a radical of a four-, five- or six- membered monocyclic ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. The four-membered ring contains zero or one double bond, and one heteroatom selected from the group consisting of O, N, and S. The five- membered ring contains zero or one double bond and one, two or three heteroatoms selected from the group consisting of O, N and S. The six-membered ring contains zero, one or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, piperidinyl, tetrahydrofuranyl, tetrahydropyranyl. As used herein, the term ‘5- or 6-membered heterocyclic ring comprising 1 or 2 nitrogen’ comprises, but is not limited to, pyrrolidine, pyrazoline, piperidine, tetrahydropyrimidine dihydropyridine, imidazoline, imidazolidine. The term “(Cx-Cy)cycloalkyl,” as used herein, refers to a saturated cycloalkane hydrocarbon radical comprising from x to y carbon atoms, such as from 3-6 carbon atoms, such as 3-5 or such as 3-4 carbon atoms. The cycloalkyl may be monocyclic or bicyclic, wherein the two rings are bridged, fused, or spirocyclic. Representative examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl. If substituents are described as being independently selected from a group, each substituent is selected independent of the other. Each substituent may therefore be identical or different from the other substituent(s). The term “optionally substituted” means “unsubstituted or substituted”, and therefore the general formulas described herein encompasses compounds containing the specified optional substituent(s) as well as compounds that do not contain the optional substituent(s). For compounds described herein, groups and substituents thereof may be selected in accordance with permitted valence of the atoms and the substituents, such that the selections and substitutions result in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. For example, the phrase "the compound" is to be understood as referring to various "compounds" of the invention or particular described aspect, unless otherwise indicated. The description herein of any aspect or aspect of the invention using terms such as “comprising”, “having,” “including,” or “containing” with reference to an element or elements is intended to provide support for a similar aspect or aspect of the invention that “consists of”, “consists essentially of”, or “substantially comprises” that particular element or elements, unless otherwise stated or clearly contradicted by context (e.g., a composition described herein as comprising a particular element should be understood as also describing a composition consisting of that element, unless otherwise stated or clearly contradicted by context). In the present context, the term “a modulator of the NMDA receptor with affinity to the glycine site” is a compound which modulates through binding the orthosteric glycine binding site on the NMDA receptor so as to increase or decrease the flux of Ca + + through the ligand- gated channel. In the present context, the term "therapeutically effective amount" of a compound is intended to indicate an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease (e.g. depression) and its complications in a therapeutic intervention comprising the administration of said compound. An amount adequate to accomplish this is defined as "therapeutically effective amount". Effective amounts for each purpose will depend on the severity of the disease (e.g. depression) or injury as well as the weight and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, e.g. by constructing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician. In the present context, the term "treatment" and "treating" means the management and care of a patient for the purpose of combating a disease. The term is intended to include the full spectrum of treatments for a given disease (e.g. depression) from which the patient is suffering, such as administration of the active compound to alleviate the symptoms or complications, to delay the progression of the disease (e.g. depression), to alleviate or relief the symptoms and complications, and/or to cure or eliminate the depression disease. The patient to be treated is preferably a mammal, in particular a human being. In the present context, “disease” can be used synonymous with disorder, condition, malfunction, dysfunction and the like. In the present context, the terms “prodrug” or “prodrug derivative” indicates a compound that, after administration to a living subject, such as a mammal, preferably a human is converted within the body into a pharmacologically active moiety. The conversion preferably takes place within a mammal, such as in a mouse, rat, dog, minipig, rabbit, monkey and/or human. In the present context, the terms “parent compound” and “parent molecule” indicate the pharmacologically active moiety obtained upon conversion of a corresponding prodrug or administered as the active moiety to the patient. For example, the “parent compound” is to be understood as a compound of formula V, exemplified with compounds 1a, 1b, 1c, 1d, 1e, 1f, 1g, 1h, 1i, 1j, 1k, 1l, 1m, 1n, 1o, 1p, 1q, 1r, 1s, 1t, 1u, 1v, 1w, 1x, 1y, 1z, 1aa, 1ab, 1ac, 1ad, 1ae, 1af and 1ag. Embodiments of the invention In an embodiment the invention provides the compound according to Formula I above, or a pharmaceutically acceptable salt thereof, wherein R1 is hydrogen. In a further embodiment the invention provides the compound according to Formula I above, or a pharmaceutically acceptable salt thereof, wherein R3 is selected from the group consisting of hydrogen, fluoro, methoxy and (C1-C3)alkyl. In a further aspect the invention provides the compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R1 and R3 are hydrogen. In yet another aspect the invention provides the compound a according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R2 is selected from the group consisting of hydrogen and halogen and wherein R4 is selected from the group consisting of hydrogen, halogen, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl and (C3-C6)cycloalkyl, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl and(C3-C6)cycloalkyl are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy and halo(C1-C4)alkyl. In a further aspect the invention provides the compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R2 is fluoro. In a further aspect the invention provides the compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from the group consisting of 5- or 6-membered heteroaryl, wherein said 5- or 6-membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy and halo(C1-C4)alkyl. In a further aspect the invention provides the compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R4 is pyrazolyl, wherein said pyrazolyl is optionally substituted with one or more substituents independently selected from the group consisting of fluor, chloro, (C1-C4)alkyl and halo(C1- C2)alkyl. In a further aspect the invention provides the compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R4 is phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1-C4)alkyl and halo(C1-C2)alkyl. In a further aspect the invention provides the compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R5 is selected from the group consisting of (C1-C6)alkyl and (C3-C6)cycloalkyl, wherein said (C1-C6)alkyl and (C3-C6)cycloalkyl are optionally substituted with one or more substituents independently selected from the group consisting of -NH2, (C1-C4)alkoxy, (C3-C6)cycloalkyl, phenyl and 4-6 membered heterocyclyl. In a further aspect the invention provides the compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R2 and R4 are not hydrogen. In a further aspect the invention provides the compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R1 and R3 are hydrogen; R2 is selected from the group consisting of halogen and (C1-C6)alkyl; R4 is selected from the group consisting of -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6- membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkyl, hydroxy(C1-C4)alkyl, phenyl, phenoxy and -C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl. In a further aspect the invention provides the compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R2 is selected from the group consisting of hydrogen and halogen, and R4 is selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1- C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1-C4)alkyl, (C1-C4)alkoxy halo(C1-C2)alkyl, (C1- C2)alkoxy(C1-C2)alkyl, hydroxy(C1-C2)alkyl, phenyl, phenoxy and -C(O)NH2. In a further aspect the invention provides the compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R4 the 5- or 6- membered heteroaryl is selected from the group consisting of pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, imidazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, furanyl and thienyl, wherein said 5- or 6- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1-C4)alkyl, (C1-C4)alkoxy halo(C1-C2)alkyl, (C1-C2)alkoxy(C1- C2)alkyl, hydroxy(C1-C2)alkyl, phenyl, phenoxy and -C(O)NH2. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R4 the 5- or 6- membered heteroaryl is selected from the group consisting of pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, furanyl and thienyl, wherein said 5- or 6- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1- C4)alkyl, (C1-C4)alkoxy, halo(C1-C2)alkyl, phenyl, phenoxy and -C(O)NH2. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein in R4 the 5- membered heteroaryl is selected from the group consisting of pyrazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, furanyl and thienyl, wherein said 5- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1-C4)alkyl, (C1-C4)alkoxy and halo(C1-C2)alkyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from the group consisting of pyrazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, furanyl and thienyl, wherein said 5- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1- C4)alkyl, (C1-C4)alkoxy and halo(C1-C2)alkyl; and wherein R2 is selected from halogen; and wherein R1 and R3 both represent hydrogen. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from the group consisting of pyrazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, furanyl and thienyl, wherein said 5- membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1- C4)alkyl, (C1-C4)alkoxy and halo(C1-C2)alkyl; and wherein R2 is selected from halogen; and wherein R1 and R3 both represent hydrogen; and wherein R5 is selected from methyl and ethyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R4 represents phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1-C4)alkyl, (C1-C4)alkoxy and halo(C1- C2)alkyl; and wherein R2 is selected from halogen; and wherein R1 and R3 both represent hydrogen. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R4 represents phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1-C4)alkyl, (C1-C4)alkoxy and halo(C1- C2)alkyl; and wherein R2 is selected from halogen; and wherein R1 and R3 both represent hydrogen; and wherein R5 is selected from methyl and ethyl. In a further aspect the invention provides a compound according to any one of the embodiments above, or a pharmaceutically acceptable salt thereof, wherein R3 is selected from the group consisting of hydrogen, fluoro, methyl and ethyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R3 is hydrogen. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R5 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl and isopentyl, wherein said methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl and isopentyl are optionally substituted with one or more substituents selected from (C1-C4)alkoxy and (C3- C6)cycloalkyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R5 is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl and isopentyl. In a further aspect the invention provides a compound according to any one of the above embodiments, or a pharmaceutically acceptable salt thereof, wherein R5 is selected from the group consisting of methyl and ethyl. In a further aspect the invention provides the compound according to any one of the embodiments above, selected from the list consisting of (R)-methyl 2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoate, (+)(2R)-methyl 2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoate, (-)(2R)-methyl 2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(2-ethylfuran-3-yl)-5-fluorobenzamido)propanoate, (-)cis-(2R)-methyl 2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate, (+)cis-(2R)-methyl 2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate, methyl (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5-fluorobenzamido)propanoate, methyl (R)-2-amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, methyl (R)-2-amino-3-(3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5- fluorobenzamido)propanoate, methyl (R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3- (3-ethyl-5-fluorobenzamido)propanoate, (R)-isobutyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate, (R)-isopropyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate, (R)-(S)-1-aminopropan-2-yl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate, (R)-isopentyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate, (R)-2-methoxyethyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate, (R)-2-morpholinoethyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(4-ethyl-3-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-ethyl-5-fluoro-4-methylbenzamido)propanoate, (R)-isobutyl 2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-ethyl-4-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzamido)propanoate, (R)-methyl 2-amino-3-(3-ethyl-5-fluoro-4-methoxybenzamido)propanoate, (R)-methyl 2-amino-3-(3-(5-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(3-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoate, (R)-methyl 2-amino-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-fluoro-5-(5-propylisoxazol-4-yl)benzamido)propanoate, (R)-methyl 2-amino-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5- yl)benzamido)propanoate, (R)-methyl 2-amino-3-(3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoate, (R)-methyl 2-amino-3-(3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-isobutyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-isopropyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-benzyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-phenyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-cyclopropyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-cyclopropylmethyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-2-methoxyethyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-cyclohexyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-2-oxo-2-(pyrrolidin-1-yl)ethyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate, (R)-isopropyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate, (R)-benzyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate, (R)-cyclopropylmethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate, (R)-cyclopropyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate and (R)-ethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate or a pharmaceutically acceptable salt thereof. In a further aspect the invention provides a compound according to any one of the embodiments above as a salt of hydrochloric acid. In a further aspect the invention provides a compound according to any one of the embodiments above as a salt of hydrobromic acid. In a further aspect the invention provides a compound according to any one of the embodiments wherein the compound is selected from (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate with the formula 2al below
Figure imgf000023_0001
Formula 2al, and (R)-ethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate with the formula 2ba below
Figure imgf000023_0002
Formula 2ba, or a pharmaceutically acceptable salt thereof. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate, or a pharmaceutically acceptable salt thereof. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate hydrochloride. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido) hydrobromide. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-ethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoate or a pharmaceutically acceptable salt thereof. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-ethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoate hydrochloride. In a further aspect the invention provides a compound according to any one of the embodiments above, wherein the compound is (R)-ethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoate hydrobromide. In a further aspect the invention provides a compound according to formula I above, or a pharmaceutically acceptable salt thereof, wherein R1 and R2 together with the carbon atoms to which they are attached form a 5- or 6 membered heteroaromatic ring comprising 1 or 2 nitrogen(s), or a 5- or 6-membered heterocyclic ring comprising 1 or 2 nitrogen(s), and wherein said 5- or 6 membered heteroaryl is optionally substituted with one or more substituents independently selected from (C1-C4)alkyl, and wherein said 5- or 6-membered heterocycle is optionally substituted with one or more substituents independently selected from (C1-C4)alkyl and oxo; and wherein R3 and R4 each independently are selected from the group consisting of hydrogen, halogen, (C1-C6)alkyl and (C1-C4)alkoxy; or R2 and R3, together with the carbon atoms to which they are attached form a 5- or 6 membered heteroaromatic ring comprising 1 or 2 nitrogen(s), or a 5- or 6-membered heterocyclic ring comprising 1 or 2 nitrogen(s), and wherein said 5- or 6 membered heteroaryl is optionally substituted with one or more substituents independently selected from (C1-C4)alkyl, and wherein said 5- or 6-membered heterocycle is optionally substituted with one or more substituents independently selected from (C1-C4)alkyl and oxo; and wherein R1 is selected from the group consisting of hydrogen and halogen and R4 is selected from the group consisting of hydrogen, halogen, (C1-C6)alkyl and (C1-C4)alkoxy, and R5 is selected from the group consisting of (C1-C6)alkyl, (C3-C6)cycloalkyl and phenyl, wherein said (C1-C6)alkyl, (C3-C6)cycloalkyl and phenyl are optionally substituted with one or more substituents independently selected from the group consisting of -NH2, (C1-C4)alkoxy, (C3-C6)cycloalkyl, phenyl, 4-6 membered heterocyclyl and -L-R6, wherein -L- represents -C(O)- and R6 is selected from the group consisting of 4-6 membered heterocyclyl. Reference to compounds encompassed by the invention includes the free substance of compounds of the invention, pharmaceutically acceptable salts of compounds of the invention, such as acid addition salts or base addition salts, and polymorphic and amorphic forms of compounds of the invention and of pharmaceutically acceptable salts thereof. Furthermore, the compounds of the invention and pharmaceutically acceptable salts thereof may potentially exist in unsolvated as well as in solvated forms with pharmaceutically acceptable solvents such as water, ethanol and the like. Both solvated and unsolvated forms are encompassed by the present invention. Compound names can be assigned by using the Struct=Name naming algorithm as part of CHEMDRAW®. It should be understood that the compounds of the invention may possess tautomeric forms, stereoisomers, geometric isomers, and that these also constitute embodiments of the invention. Racemic forms may be resolved into the optical antipodes by known methods, for example, by separation of diastereomeric salts thereof with an optically active acid, and liberating the optically active amine compound by treatment with a base. Separation of such diastereomeric salts can be achieved, e.g. by fractional crystallization. The optically active acids suitable for this purpose may include, but are not limited to d- or l- tartaric, mandelic or camphorsulfonic acids. Another method for resolving racemates into the optical antipodes is based upon chromatography on an optically active matrix. The compounds of the present invention may also be resolved by the formation and chromatographic separation of diastereomeric derivatives from chiral derivatizing reagents, such as, chiral alkylating or acylating reagents, followed by cleavage of the chiral auxiliary. Any of the above methods may be applied either to resolve the optical antipodes of the compounds of the invention per se or to resolve the optical antipodes of synthetic intermediates, which can then be converted by methods described herein into the optically resolved final products which are the compounds of the invention. Additional methods for the resolution of optical isomers, known to those skilled in the art, may be used. Such methods include those discussed by J. Jaques, A. Collet and S. Wilen in Enantiomers, Racemates, and Resolutions, John Wiley and Sons, New York, 1981. Optically active compounds can also be prepared from optically active starting materials. Included in this invention are also isotopically labelled compounds, which are similar to those claimed in formula I, wherein one or more atoms are represented by an atom of the same element having an atomic mass or mass number different from the atomic mass or mass number usually found in nature (e.g., 2H, 3H, 11C, 13C, 15N, 18F and the like). Particular mention is made of 2H substituted compounds i.e. compounds wherein one or more H atoms are represented by deuterium. In one embodiment of the invention one or more of the hydrogen atoms of the compound of formula I are represented by deuterium. It is recognized that elements are present in natural isotopic abundances in most synthetic compounds, and result in inherent incorporation of deuterium. However, the natural isotopic abundance of hydrogen isotopes such as deuterium is immaterial (about 0.015%) relative to the degree of stable isotopic substitution of compounds indicated herein. Thus, as used herein, designation of an atom as deuterium at a position indicates that the abundance of deuterium is significantly greater than the natural abundance of deuterium. Any atom not designated as a particular isotope is intended to rep-resent any stable isotope of that atom, as will be apparent to the ordinarily skilled artisan. In one embodiment, designation of a position as “D” in a compound has a minimum deuterium incorporation of greater than about 60% at that position such as greater than about 70% at that position such as greater than about 80% at that position such as greater than about 85% at that position. In a further embodiment, designation of a position as “D” in a com-pound has a minimum deuterium incorporation of greater than about 90% at that position such as greater than about 95% at that position such as greater than about 97% at that position such as greater than about 99% at that position. Pharmaceutically acceptable salts The compounds of this invention (parent compounds and their respective prodrugs) are generally utilized as the free substance or as a pharmaceutically acceptable salt thereof. When a compound of the invention contains a free base, such salts may be prepared in a conventional manner by treating a solution or suspension of a free base of a compound of the invention with a molar equivalent of a pharmaceutically acceptable acid. Representative examples of suitable organic and inorganic acids are described below. Pharmaceutically acceptable salts in the present context is intended to indicate non-toxic, i.e. physiologically acceptable salts. The term “pharmaceutically acceptable salts” include salts formed with inorganic and/or organic acids on the nitrogen atoms in the parent molecule. Said acids may be selected from for example hydrochloric acid, hydrobromic acid, phosphoric acid, nitrous acid, sulphuric acid, benzoic acid, citric acid, gluconic acid, lactic acid, maleic acid, succinic acid, tartaric acid, acetic acid, propionic acid, oxalic acid, maleic acid, fumaric acid, glutamic acid, pyroglutamic acid, salicylic acid, salicylic acid, saccharin, and sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid and benzenesulfonic acid. In an embodiment of the invention, the pharmaceutically acceptable salt is a hydrogen chloride salt. In an embodiment of the invention, the pharmaceutically acceptable salt is a hydrogen bromide salt. The term pharmaceutically acceptable salts also include salts formed with inorganic and/or organic bases on the acidic groups of compounds of the invention. Said bases may be selected from for example alkali metal bases, such as sodium hydroxide, lithium hydroxide, potassium hydroxide, alkaline earth bases, such as calcium hydroxide and magnesium hydroxide, and organic bases, such as trimethylamine and trimethylamine. Additional examples of useful acids and bases to form pharmaceutically acceptable salts can be found e.g. in Stahl and Wermuth (Eds) “Handbook of Pharmaceutical salts. Properties, selection, and use”, Wiley-VCH, 2008. Conditions for treatment The invention encompasses compounds of the invention for use as a medicament, as well as for use in treatment of all diseases and disorders listed below. As described above compounds of the present invention may be useful in the treatment of depression and depressive disorders. Hence in one embodiment, a compound of formula I or a pharmaceutically acceptable salt thereof is for use in in the treatment of depression. The diagnosis of depression usually follows a clinical evaluation by a psychiatrist or other mental health professionals. The two most recognized sets of diagnostic criteria for major depressive disorder and other depressive, or mood disorders, are outlined in the DSM, Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, (DSM IV) published by the American psychiatric association and the ICD ( ICD-10: International Statistical Classification of Diseases and Related Health Problems – 10th Revision, published periodically by the World Health Organization) or any other psychiatric classification system. Signs and symptoms of depression are for example depressed mood, loss of interest (anhendonia), weight or appetite changes, sleep problems, psychomotor activity (objective or subjective), fatigability, worthlessness, concentration difficulty, suicidal ideation, loss of confidence, sexual dysfunction and self-reproach. Thus in an embodiment of the invention, compounds of the invention are for use to prevent, alter, reduce or alleviate one or more signs or symptoms of depression selected from the group consisting of depressed mood, loss of interest (anhendonia), weight or appetite changes, sleep problems, psychomotor activity (objective or subjective), fatigability, worthlessness, concentration difficulty, suicidal ideation, loss of confidence, sexual dysfunction and self-reproach. The skilled person is familiar with various test for measuring the improvement of depressive symptoms. Examples of test for measuring the improvements are but not limited to the HAM-D or MADRS scale. In an embodiment the depression is major depressive disorder. In a further embodiment the depression is treatment-resistant depression. In a further embodiment the depression is selected from major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, , bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression. In an embodiment of the invention, compound of the invention is used in the treatment of pain [Expert Rev Clin Pharmacol.2011 May 1; 4(3): 379–388]. In a further embodiment the pain is neuropathic pain. Preclinical animal models have demonstrated pro-cognitive and antidepressant-like effects with the use of NDMA glycine site modulators [Peyrovian et al., Progress in Neuropsychopharmacology & Biological Psychiatry. 92 (2019) 387-404]. Hence, In an embodiment of the invention, a compound of formula I or a pharmaceutically acceptable salt thereof is for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus In a further embodiment a compound of formula I or a pharmaceutically acceptable salt thereof is for use in the treatment of suicidal ideation. In an embodiment of the invention, compound of the invention is for use in the treatment of a neurological disorder or neuropsychiatric disorder. In an embodiment the invention provides a method for the treatment of depression, such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression, comprising the administration of a therapeutically effective amount of the compound of Formula I, or a pharmaceutically acceptable salt thereof to a patient (e.g. a human patient) in need thereof. In an embodiment the invention provides a method for the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus, comprising the administration of a therapeutically effective amount of the compound of Formula I, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising the compound of Formula I or a pharmaceutically acceptable salt thereof, to a patient (e.g. a human patient) in need thereof. In an embodiment the invention provides a method for the treatment of depression, such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression, comprising the administration of a pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, to a patient (e.g. a human patient) in need thereof. In an embodiment the invention provides the use of the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of depression, such as for example major depressive disorder, treatment- resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression. In an embodiment the invention provides the use of the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus. In an embodiment the invention provides the use of a pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of depression, such as for example major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression. In an embodiment the invention provides the use of a pharmaceutical composition comprising the compound of Formula I, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for use in the treatment of a condition selected from suicidal ideation, bipolar disorder (including bipolar depression), obsessive compulsive disorder and status epilepticus. Combination treatment In one embodiment of the invention, the compounds of the invention are for use as stand- alone treatment as the sole active compound. In another embodiment of the invention, the compounds of the invention may be used in combination with other agents useful in the treatment of disorders such as depression. The terms “combined use”, “in combination with” and “a combination of” and the like as used herein in the context of the method of the invention comprising the combined administration of therapeutically effective amounts of a compound of the invention, and another compound, which compound is useful in the treatment a neurodegenerative disease or disorder, is intended to mean the administration of a compound of the invention simultaneously or sequentially, in any order, together with said other compound. The two compounds may be administered simultaneously or sequentially with a time gap between the administrations of the two compounds. The two compounds may be administered either as part of the same pharmaceutical formulation or composition, or in separate pharmaceutical formulations or compositions. The two compounds may be administered on the same day or on different days. They may be administered by the same route, such for example by oral administration, by depot, by intramuscular injection or intravenous injection; or by different routes wherein one compound is for example administered orally or placed by depot and the other compound is for example injected. The two compounds may be administered by the same dosage regime or interval, such as once or twice daily, weekly, or monthly; or by different dosage regimes for example wherein one is administered once daily and the other is administered twice daily or weekly or monthly. In some instances, the patient to be treated may already be in treatment with one or more other compounds useful in the treatment of depression when treatment with a compound of the invention initiated. In other instances, the patient may already be in treatment with a compound of the invention when treatment with one or more other compounds useful in the treatment of a depression or psychosis is initiated. In other instances, the treatment with a compound of the invention and treatment with one or more other compounds useful in the treatment of psychosis initiated at the same time. Compounds for combination treatment Examples of therapeutically active compounds which may advantageously be combined with compounds of the invention include sedatives or hypnotics, such as benzodiazepines; anticonvulsants, such as lamotrigine, valproic acid, topiramate, gabapentin, carbamazepine; mood stabilizers such as lithium; dopaminergic drugs, such as dopamine agonists and L- Dopa; drugs to treat ADHD, such as atomoxetine; psychostimulants, such as modafinil, ketamine, methylphenidate and amphetamine; other antidepressants, such as mirtazapine, mianserin, vortioxetine, cipralex, and buproprion; hormones, such as T3, estrogen, DHEA and testosterone; atypical antipsychotics, such as olanzapine, brexpiprazole and aripiprazole; typical antipsychotics, such as haloperidol; drugs to treat Alzheimer's diseases, such as cholinesterase inhibitors and memantine, folate; S-Adenosyl-Methionine; immunmodulators, such as interferons; opiates, such as buprenorphins; angiotensin II receptor 1 antagonists (AT1 antagonists); ACE inhibitors; statins; and alpha1 adrenergic antagonist , such as prazosin. Administration routes The pharmaceutical compositions comprising a compound of the invention, either as the sole active compound or in combination with another active compound, may be specifically formulated for administration by any suitable route such as the oral, rectal, nasal, buccal, sublingual, pulmonal, transdermal and parenteral (e.g. subcutaneous, intramuscular, and intravenous) route. It will be appreciated that the route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient. Doses In one embodiment, the compound of the present invention is administered in an amount from about 0.5 mg/kg body weight to about 50 mg/kg body weight per day. In particular, daily dosages may be in the range of 1 mg/kg body weight to about 30 mg/kg body weight per day. The exact dosages will depend upon the frequency and mode of administration, the sex, the age, the weight, and the general condition of the subject to be treated, the nature and the severity of the condition to be treated, any concomitant diseases to be treated, the desired effect of the treatment and other factors known to those skilled in the art. In an embodiment the frequency of administration is 1, 2, 3, 4, or 5 times per day In an embodiment the frequency of administration is once weekly. In an embodiment the frequency of administration is twice weekly. A typical oral dosage for adults will be in the range of 1-3000 mg/day of a compound of the present invention, such as 700-2800 mg/day, such as 1000-2000 mg/day or 1200- 1700mg/day. Conveniently, the compounds of the invention are administered in a unit dosage form containing said compounds in an amount of about 100 to 1000 mg, such as 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 750 mg or up to 1000 mg of a compound of the present invention. In an embodiment the frequency of administration is 1, 2, 3, 4, or 5 times per day. In one embodiment the frequency of administration is once weekly. In an embodiment the frequency of administration is twice weekly. A typical IV dosage for adults will be in the range of 20-300 mg/day of a compound of the present invention, such as 50-200 mg/day, such as 70-150 mg/day or 75-125 mg/day. Conveniently, the compounds of the invention are administered in a unit dosage form containing said compounds in an amount of about 10 to 300 mg, such as 10 mg, 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg of a compound of the present invention. In an embodiment the frequency of administration is once weekly. In an embodiment the frequency of administration is twice weekly. Pharmaceutical formulations and excipients In the following, the term, “excipient” or “pharmaceutically acceptable excipient” refers to pharmaceutical excipients including, but not limited to, fillers, antiadherents, binders, coatings, colours, disintegrants, flavours, glidants, lubricants, preservatives, sorbents, sweeteners, solvents, vehicles and adjuvants. The present invention also provides a pharmaceutical composition comprising a compound of the invention, such as one of the compounds disclosed in the Experimental Section herein. The present invention also provides a process for making a pharmaceutical composition comprising a compound of the invention. The pharmaceutical compositions according to the invention may be formulated with pharmaceutically acceptable excipients in accordance with conventional techniques such as those disclosed in Remington, “The Science and Practice of Pharmacy”, 22th edition (2013), Edited by Allen, Loyd V., Jr. Pharmaceutical compositions for oral administration include solid oral dosage forms such as tablets, capsules, powders and granules; and liquid oral dosage forms such as solutions, emulsions, suspensions and syrups as well as powders and granules to be dissolved or suspended in an appropriate liquid. Solid oral dosage forms may be presented as discrete units (e.g. tablets or hard or soft capsules), each containing a predetermined amount of the active ingredient, and preferably one or more suitable excipients. Where appropriate, the solid dosage forms may be prepared with coatings such as enteric coatings or they may be formulated so as to provide modified release of the active ingredient such as delayed or extended release according to methods well known in the art. Where appropriate, the solid dosage form may be a dosage form disintegrating in the saliva, such as for example an oral-dispersible tablet. Examples of excipients suitable for solid oral formulation include, but are not limited to, microcrystalline cellulose, corn starch, lactose, mannitol, povidone, croscarmellose sodium, sucrose, cyclodextrin, talcum, gelatin, pectin, magnesium stearate, stearic acid and lower alkyl ethers of cellulose. Similarly, the solid formulation may include excipients for delayed or extended release formulations known in the art, such as glyceryl monostearate or hypromellose. If solid material is used for oral administration, the formulation may for example be prepared by mixing the active ingredient with solid excipients and subsequently compressing the mixture in a conventional tableting machine; or the formulation may for example be placed in a hard capsule e.g. in powder, pellet or mini tablet form. The amount of solid excipient will vary widely but will typically range from about 25 mg to about 1 g per dosage unit. Liquid oral dosage forms may be presented as for example elixirs, syrups, oral drops or a liquid filled capsule. Liquid oral dosage forms may also be presented as powders for a solution or suspension in an aqueous or non-aqueous liquid. Examples of excipients suitable for liquid oral formulation include, but are not limited to, ethanol, propylene glycol, glycerol, polyethylenglycols, poloxamers, sorbitol, poly-sorbate, mono and di-glycerides, cyclodextrins, coconut oil, palm oil, and water. Liquid oral dosage forms may for example be prepared by dissolving or suspending the active ingredient in an aqueous or non-aqueous liquid, or by incorporating the active ingredient into an oil-in-water or water-in-oil liquid emulsion. Further excipients may be used in solid and liquid oral formulations, such as colourings, flavourings and preservatives etc. Pharmaceutical compositions for parenteral administration include sterile aqueous and nonaqueous solutions, dispersions, suspensions or emulsions for injection or infusion, concentrates for injection or infusion as well as sterile powders to be reconstituted in sterile solutions or dispersions for injection or infusion prior to use. Examples of excipients suitable for parenteral formulation include, but are not limited to water, coconut oil, palm oil and solutions of cyclodextrins. Aqueous formulations should be suitably buffered if necessary and rendered isotonic with sufficient saline or glucose. Other types of pharmaceutical compositions include suppositories, inhalants, creams, gels, dermal patches, implants and formulations for buccal or sublingual administration. It is requisite that the excipients used for any pharmaceutical formulation comply with the intended route of administration and are compatible with the active ingredients. Compounds of the invention Table 1: Exemplified parent compounds of the invention
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Table 2: Exemplified prodrugs of the invention
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
EXPERIMENTAL SECTION Preparation of the compounds of the invention The compounds of the present invention of the general formula I, wherein R1, R2, R3, R4 and R5 are as defined above can be prepared by the methods outlined in the following reaction Schemes 1-11 and in the examples. In the described methods, it is possible to make use of variants or modifications, which are themselves known to chemists skilled in the art or could be apparent to the person of ordinary skill in this art. Furthermore, other methods for preparing compounds of the invention will be readily apparent to the person skilled in the art in light of the following reaction schemes and examples. The schemes may involve the use of selective protecting groups during the synthesis of the compounds of the invention. One skilled in the art would be able to select the appropriate protecting group for a particular reaction. It may be necessary to incorporate protection and de-protection strategies for substituents such as amino, amido, carboxylic acid and hydroxyl groups in the synthetic methods described below to synthesize the compounds of Formula I. Methods for protection and de-protection of such groups are well known in the art, and may be found in T. Green, et al., Protective Groups in Organic Synthesis, 1991, 2nd Edition, John Wiley & Sons, New York. The schemes in this section are representative of methods useful in synthesizing the compounds of the present invention. They are not intended to constrain the scope of the invention in any way. Scheme 1
Figure imgf000051_0001
Compounds of general formula I (Scheme 1) may be prepared from compounds with general formula IV by standard de-protection procedures. As an example, compounds of general formula I (Scheme 1) may be prepared from compounds with general formula IV where Pg1 is a N-Carbobenzyloxy group (Cbz) and R5 is defined as in general formula I. Compounds with general formula IV may be prepared by compounds of general formula III with carboxylic acids (or salt thereof) of general formula II by standard peptide coupling such as using O-(Benzotriazol-1-yl)-N,N,Nʹ,Nʹ-tetramethyluronium tetrafluoroborate in the presence of a base such as N,N-diisopropylethylamine in a solvent such as N,N- dimethylformamide.
Figure imgf000051_0002
Compounds of general formula I (Scheme 2) may be prepared from compounds with general formula V by standard esterification procedures. As an example, compounds of general formula I (Scheme 2) may be prepared from compounds with general formula V by treatment with a reagent such as thionyl chloride in methanol (R5 = Me). Compounds of general formula V may be prepared from compounds with general formula IVa by standard de-protection procedures. As an example, compounds of general formula V (Scheme 2) may be prepared from compounds with general formula IVa where Pg1 is Cbz and Pg2 is benzyl using conditions such as HBr in acetic acid. Compounds with general formula IVa may be prepared by compounds of general formula IIIa with carboxylic acids (or salt thereof) of general formula II by standard peptide coupling such as using O-(Benzotriazol-1-yl)-N,N,Nʹ,Nʹ-tetramethyluronium tetrafluoroborate in the presence of a base such as N,N-diisopropylethylamine in a solvent such as N,N- dimethylformamide. Scheme 3
Figure imgf000052_0001
Benzoic acids of general formula II (or salt thereof), (Scheme 2) where R2 can be hetaryl, aryl or alkenyl as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VII by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art. Compounds of general formula VII can be obtained from compounds of general formula VI, under reaction conditions such as in the presence of a catalyst such as [1,1ʹ- Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane or [1,1ʹ-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) and a base such as potassium triphosphate. Likewise, similar reaction conditions may be applied for introducing R4 substituents.
Figure imgf000053_0001
Benzoic acids of general formula II (or salt thereof), (Scheme 2) where R2 is hetaryl or heterocycle as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VIII by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art. Compounds of general formula VIII can be obtained from compounds of general formula VI, under reaction conditions such as in the presence of a catalyst such as copper (II) acetate and a base such as pyridine. Likewise, similar reaction conditions may be applied for introducing R4 substituents. Scheme 5
Figure imgf000053_0002
Benzoic acids of general formula II (or salt thereof), (Scheme 2) where R2 is NRaRb as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VII by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art. Compounds of general formula VII can be obtained by reacting compounds of general formula IX together with amines of general formula RaRbNH. Likewise, similar reaction conditions may be applied for introducing R4 substituents. Scheme 6
Figure imgf000054_0001
Benzoic acids of general formula II (or salt thereof), (Scheme 2) where R2 can be alkyl as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VII by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art. Compounds of general formula VII can be obtained by reacting compounds of general formula X together with trialkylboranes, under reaction conditions such as in the presence of a catalyst such as [1,1ʹ- Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane and a base such as caesium acetate, or alkyl Zinc bromides, under reaction conditions such as in the presence of a catalyst such as palladium(II) acetate, and a ligand such as 2- dicyclohexylphosphino-2ʹ,6ʹ-bis(N,N-dimethylamino)biphenyl (Cphos). Likewise, similar reaction conditions may be applied for introducing R4 substituents. Scheme 7
Figure imgf000054_0002
Benzoic acids of general formula II (or salt thereof), (Scheme 2) where R3 can be alkyl as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VII by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art. Compounds of general formula VII can be obtained by reacting compounds of general formula XI together with trialkylboranes, under reaction conditions such as in the presence of a catalyst such as [1,1ʹ- Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane and a base such as caesium acetate, or alkyl Zinc bromides, under reaction conditions such as in the presence of a catalyst such as palladium(II) acetate, and a ligand such as 2- dicyclohexylphosphino-2ʹ,6ʹ-bis(N,N-dimethylamino)biphenyl (Cphos). Likewise, similar reaction conditions may be applied for introducing R1 substituents. Scheme 8
Figure imgf000055_0001
Benzoic acids of general formula II (or salt thereof), (Scheme 2) where R2 can be alkyloxy or cycloalkyoxy as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VII by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art. Compounds of general formula VII can be obtained by reacting compounds of general formula XII together with aldehydes or ketones, under reaction conditions such as in the presence of a base such as isopropylmagnesium chloride complex with lithium chloride. Likewise, similar reaction conditions may be applied for introducing R4 substituents. Scheme 9
Figure imgf000055_0002
Benzoic acids of general formula II (or salt thereof), (Scheme 2) where R2 can be hetaryl, aryl or alkenyly as described in general Formula I can be prepared from the corresponding ester where Pg can be methyl as in compounds of general formula VII by hydrolysis under aqueous conditions in a variety of conditions known to chemists skilled in the art. Compounds of general formula VII can be obtained from compounds of general formula XIII, under reaction conditions such as in the presence of a catalyst such as [1,1ʹ- Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane or [1,1ʹ-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) and a base such as cesium carbonate. Likewise, similar reaction conditions may be applied for introducing R4 substituents. Scheme 10
Figure imgf000056_0001
Compounds of general formula III where R5 is as defined herein (Scheme 10) may be prepared by reacting carboxylic acids of formula XIV with alcohols of formula XV using standard esterification procedures. Scheme 11
Figure imgf000056_0002
Compounds of general formula III where R5 is as defined herein (Scheme 11) may be prepared by deprotection of compounds of general formula XVIII where Pg3 could be a tert- butyloxycarbonyl protecting group (Boc) and Pg1 could be a benzyloxy carbamate (Cbz). Compounds of general formula XVIII where R5 is as in general formula I may be prepared by reacting carboxylic acids of formula XVII with alcohols of formula XV using standard esterification procedures. Compounds of general formula XVII where Pg3 could be a tert- butyloxycarbonyl protecting group (Boc) may be prepared by protection of compounds of general formula XVI using standard procedures. General Methods LC-MS methods Analytical LC-MS Data were obtained using one of the methods identified below. Method A: A Waters Acquity UPLC-MS was used. Column: Acquity UPLC BEH C181.7µm; 2.1x50mm; Column temperature: 60 °C; Solvent system: A = water/trifluoroacetic acid (99.965:0.035) and B = acetonitrile /water/trifluoroacetic acid (94.965:5:0.035); Method: Linear gradient elution with A:B = 90:10 to 0:100 in 1.0 minutes and with a flow rate of 1.2 mL/min. Method B: A Waters Acquity UPLC-MS was used. Column: Acquity UPLC BEH C181.7µm; 2.1x50mm; Column temperature: 60 °C; Solvent system: A = water/trifluoroacetic acid (99.5:0.5) and B = acetonitrile /water/trifluoroacetic acid (94.965:5:0.035); Method: Linear gradient elution with A:B = 98:2 to 0:100 in 1.15 minutes and with a flow rate of 1.2 mL/min. Method C: An Agilent 1200 LCMS system with ELS detector was used. Column: Waters XBridge Sheild RP18 5 µm; 2.1x50mm; Column temperature: 40 °C; Solvent system: A = water/ammonia (99.95:0.05) and B = acetonitrile; Method: Linear gradient elution with A:B = 95:5 to 0:100 in 4.0 minutes and with a flow rate of 0.8 mL/min. Method D: An Agilent 1200 LCMS system with ELS detector was used. Column: Agilent TC- C185 µm; 2.1x50mm; Column temperature: 50 °C; Solvent system: A = water/trifluoroacetic acid (99.9:0.1) and B = acetonitrile /trifluoroacetic acid (99.95:0.05); Method: Linear gradient elution with A:B = 99:1 to 0:100 in 4.0 minutes and with a flow rate of 0.8 mL/min. Method E: An Agilent 1200 LCMS system with ELS detector was used. Column: Agilent TC- C185 µm; 2.1x50mm; Column temperature: 50 °C; Solvent system: A = water/trifluoroacetic acid (99.9:0.1) and B = acetonitrile /trifluoroacetic acid (99.95:0.05); Method: Linear gradient elution with A:B = 90:10 to 0:100 in 4.0 minutes and with a flow rate of 0.8 mL/min. Method F: An Agilent 1200 LCMS system with ELS detector was used. Column: Phenomenex Luna-C18, 50×2mm, 5μm; Column temperature: 50 °C; Solvent system: A = water/trifluoroacetic acid (99.9:0.1) and B = acetonitrile /trifluoroacetic acid (99.95:0.05); Method: Linear gradient elution with A:B = 99:1 to 0:100 in 4.0 minutes and with a flow rate of 0.8 mL/min. Method G: An Agilent 1200 LCMS system with ELS detector was used. Column: Merck RP18e 5 µm; 2x25mm; Column temperature: 50 °C; Solvent system: A = water/trifluoroacetic acid (99.95:0.05) and B = acetonitrile /trifluoroacetic acid (99.95:0.05); Method: Linear gradient elution with A:B = 95:5 to 5:95 in 0.7 minutes and with a flow rate of 1.5 mL/min. Preparative HPLC Preparative-HPLC (Method AA): Instrument: Gilson GX-281 Liquid Handler, SHIMADZU LC-8A LCMS2010; Column: YMC-Actus Triart C18150*305μm; Mobile Phase A: water (0.05% HCl v/v); Mobile phase B: MeCN; Gradient: B from 5% to 35% in 10 min then hold at 100% for 3 min; FlowRate(ml/min): 25; Column temperature: 35°C and Wavelength: 220nm 254nm Preparative HPLC (Method BB): Instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector; Column: Xtimate C18 150*25mm*5μm; Mobile Phase A: water (0.05% ammonia hydroxide v/v); Mobile phase B: MeCN; Gradient: from 42% to 72% in 10 min then hold at 100% for 2.5 min; Flow Rate (ml/min): 25; Column temperature: 25°C and Wavelength: 220nm 254nm Preparative HPLC (Method CC): Instrument: Gilson GX-281 Liquid Handler, Gilson 322 Pump, Gilson 156 UV Detector; Column: Agela Durashell C18150 mm × 25mm × 5µm; Mobile Phase A: water (0.225%TFA, v/v); Mobile phase B: MeCN; Gradient: B from 32% to 62% in 10 min, hold 100% B for 2 min; Flow Rate (ml/min): 25; Column temperature: 40°C and Wavelength: 220nm 254nm Preparative HPLC (Method DD): Instrument: Gilson GX-281, Gilson 322 Pump, Gilson 156 UV Detector; Column: Waters Xbridge 150*25*5μm; Mobile Phase A: water (10mM NH4HCO3); Mobile phase B: MeCN; Gradient: B from 40% to 66% in 8.4 min then hold at 100% for 2 min; FlowRate(ml/min): 25; Column temperature: 30°C and Wavelength: 220nm 254nm. NMR 1H NMR spectra were recorded at 300, 400, 500 or 600 MHz on Bruker Avance instruments. TMS was used as internal reference standard. Chemical shift values are expressed in ppm. The following abbreviations are used for multiplicity of NMR signals: s = singlet, d = doublet, t = triplet, q = quartet, qui = quintet, h = heptet, dd = double doublet, dt = double triplet, dq = double quartet, tt = triplet of triplets, m = multiplet, br s = broad singlet and br = broad signal. Abbreviations are in accordance with to the ACS Style Guide: "The ACS Style guide – A manual for authors and editors" Janet S. Dodd, Ed.1997, ISBN: 0841234620 List of chemical abbreviations AcOH: Acetic Acid AcOK: Potassium acetate CuI: Copper(I) iodide Cu(OAc)2: Copper(II) acetate Cs2CO3: Cesium carbonate Cphos: 2-Dicyclohexylphosphino-2',6'-bis(N,N-dimethylamino)biphenyl CsOAc: Cesium acetate CPhos-Pd-G3: [(2-Dicyclohexylphosphino-2ʹ,6ʹ-bis(N,N-dimethylamino) -1,1ʹ-biphenyl)-2-(2ʹ- amino-1,1ʹ-biphenyl)] palladium(II) methanesulfonate DCM: Dichloromethane DEA: Diethylamine DIBAL-H: diisobutylaluminum hydride DIPEA: Diisopropylethylamine DMA: N,N-dimethylacetamide DME: Dimethoxyethane DMF: N,N-dimethylformamide ee: Enantiomeric excess EDC: N-(3-Dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride EtOAc: Ethyl acetate EtOH: Ethanol HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate HBr/HOAc: HBr in acetic acid HOAc: Acetic acid HOBt: 1-Hydroxybenzotriazole HPLC: High performance liquid chromatography IPA: Isopropanol iPrOAc: Isopropyl acetate i-PrMgCl-LiCl: Isopropylmagnesium chloride-lithium chloride complex iPrOH: Isopropanol IR[DF(CF3)PPY]2(DTBPY))PF6: [4,4ʹ-Bis(1,1-dimethylethyl)-2,2ʹ-bipyridine-N1,N1ʹ]bis[3,5- difluoro-2-[5-(trifluoromethyl)-2-pyridinyl-N]phenyl-C]Iridium(III) hexafluorophosphate MeCN: Acetonitrile MeI: Iodomethane MeOH: Methanol Me-THF: 2-Methyltetrahydrofuran MTBE: 2-Methoxy-2-methylpropane n-BuLi: n-Butyllithium NBS: N-Bromosuccinimide NCS: N-Chlorosuccinimide NaHMDS: Sodium bis(trimethylsilyl)amide NiCl2.glyme: Nickel(II) chloride ethylene glycol dimethyl ether complex NIS: N-Iodosuccinimide P2S5: Phosphorus(V) sulfide Pd/C: Palladium on carbon PEMB: 5-Ethyl-2-methylpyridine borane Pd(dtbpf)Cl2: [1,1ʹ-Bis(di-tert-butylphosphino)ferrocene]dichloropalladium(II) Pd(dppf)Cl2.CH2Cl2 : (1,1-Bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane complex) Pd(dppf)Cl2: [1,1ʹ-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) Pd(PPh3)4: Tetrakis(triphenylphosphine)palladium Pd(PPh3)2Cl2: dichloropalladium;triphenylphosphanium Pd(OAc)2: Palladium(II) acetate Pd(t-Bu3P)2: bis(tri-t-butylphosphine)palladium Py: SEM-Cl: 2-(Trimethylsilyl)ethoxymethyl chloride STAB: Sodium triacetoxyborohydride TBME: Tert-butyl methyl ether TBTU: 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate TEA: Triethylamine Tf2O: Trifluoromethanesulfonic anhydride TFA: Trifluoroacetic acid THF: Tetrahydrofuran TMSCHN2: Trimethylsilyldiazomethane TMSCl: Chlorotrimethylsilane T3P: Propylphosphonic anhydride Me-THF: 2-Methyltetrahydrofuran TsOH: p-Toluenesulfonic acid w/w: Weight per weight ZnEt2: Diethylzinc Preparation of the intermediates Intermediate 1 (R)-3-(Benzyloxy)-2-(((benzyloxy)carbonyl)amino)-3-oxopropan-1-aminium chloride
Figure imgf000062_0001
To phenylmethanol (56.0 mL) was added thionyl chloride (2.1 g, 17.6 mmol) dropwise at 28°C. After completion of the addition, (R)-3-amino-2- (((benzyloxy)carbonyl)amino)propanoic acid (3.8 g, 15.9 mmol) was added in several portions and the reaction was stirred for 24 h at 28°C. LCMS showed the reaction was completed. The excess benzyl alcohol was removed at 80°C/0.02 bar and the residue was stirred in cyclohexane (35 mL) for 16 h. Filtration and trituration of the filter cake with methyl tert-butyl ether (50 mL) afforded the title compound (3.0 g, yield: 51.6%). LCMS (m/z) 329.2 [M+H+], tR = (min, Method AA) = 0.52. Intermediate 2 (R)-2-(((benzyloxy)carbonyl)amino)-3-((tert-butoxycarbonyl)amino)propanoic acid
Figure imgf000062_0002
To a solution of (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoic acid (4.5 g, 18.9 mmol) in 10% sodium carbonate aqueous solution (50 mL) were added dioxane (30 mL) and di-tert-butyl dicarbonate (6.18 g, 28.32 mmol) at 0°C. The reaction mixture was stirred at 20°C for 16 h. The mixture was diluted with water (100 mL) and washed with methyl tert- butyl ether (50 mL × 3). The aqueous layer was acidified with 2N HCl to pH to 5~6 and extracted with ethyl acetate (80 mL × 3). The combined organic layers were dried over sodium sulfate, filtered and concentrated. The residue was slurried in diisopropyl ether (40 mL) and collected to give compound (R)-2-(((benzyloxy)carbonyl)amino)-3-((tert- butoxycarbonyl)amino)propanoic acid (5.5 g). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.43 - 7.28 (m, 5H), 5.20 - 5.09 (m, 2H), 4.52 - 4.28 (m, 1H), 3.69 - 3.40 (m, 2H), 1.43 (s, 9H). Intermediate 3 Methyl (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoate
Figure imgf000063_0001
To a solution of (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoic acid (200 g, 839 mmol) in methanol (1 L) was added dropwise thionyl chloride (656 g, 5.5 mol) at 0 °C. The mixture was aloowed to warm to 25 °C and stirred for 18 h. The reaction was concentrated and the residue washed with methyl tert-butyl ether (500 mL X 2) to give methyl (R)-3-amino- 2-(((benzyloxy)carbonyl)amino)propanoate (240 g) as HCl salt. 1H NMR (400 MHz, MeOD) δ 7.31-7.39 (m, 5H), 5.13 (s, 2H), 4.50-4.54 (m, 1H), 3.77 (s, 3H), 3.43-3.48 (m, 1H), 3.24-3.27 (m,1H) Intermediate 4 Ethyl (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoate
Figure imgf000064_0001
To a solution of (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoic acid (5 g, 21 mmol) in ethanol (60 mL) was added dropwise thionyl chloride (4.99 g, 42 mmol). The mixture was stirred at 30°C for 16 h. The reaction was concentrated to give ethyl (R)-3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (6.0 g) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 8.38 (br s, 3H), 7.94 (d, 1H), 7.41 - 7.30 (m, 5H), 5.07 (s, 2H), 4.46-4.37 (m, 1H), 4.12 (q, 2H), 3.25 - 3.15 (m, 1H), 3.11 - 2.99 (m, 1H), 1.18 (t, 3H). Intermediate 5 Isopropyl (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoate hydrochloride
Figure imgf000064_0002
To a solution of (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoic acid (5 g, 21 mmol) in i-PrOH (60 mL) was added drop wise thionyl chloride (5.0 g, 42 mmol). The mixture was stirred at 30°C for 16 h. Then additional thionyl chloride (3 mL) was added, the reaction was then stirred at 30°C for 24 h. The mixture was concentrated to give 10 g crude product. The crude product was treated with cyclohexane (30 mL) and stirred for 3 h. The solid was collected and dried to give isopropyl (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (6.3 g) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 8.38 (br, 3H), 7.92 (d, J = 8.0 Hz, 1H), 7.39 - 7.29 (m, 5H), 5.06 (s, 2H), 4.95 - 4.86 (m, 1H), 4.39 - 4.31 (m, 1H), 3.22 - 3.12 (m, 1H), 3.10 - 2.99 (m, 1H), 1.17 (dd, J = 9.2, 6.4 Hz, 6H). Intermediate 6 Cyclopropyl (R)-2-(((benzyloxy)carbonyl)amino)-3-((tert-butoxycarbonyl)amino)propanoate
Figure imgf000065_0001
To a mixture of (R)-2-(((benzyloxy)carbonyl)amino)-3-((tert- butoxycarbonyl)amino)propanoic acid (1.1 g, 3.25 mmol) and cyclopropanol (208 mg, 3.58 mmol) in DMF (25 mL) was added N,N-diisopropylethylamine (1.26 g, 9.75 mmol) and HATU (1.85 g, 4.88 mmol). The reaction mixture was stirred at 50°C for 16 h. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3) and concentrated. The residue was purified by Combi Flash on silica gel to give cyclopropyl (R)-2-(((benzyloxy)carbonyl)amino)- 3-((tert-butoxycarbonyl)amino)propanoate (1.1 g,). 1H NMR (400 MHz, CDCl3) δ 7.40 - 7.30 (m, 5H), 5.75 (br s, 1H), 5.12 (s, 2H), 4.80 (br s, 1H), 4.40 - 4.34 (m, 1H), 4.20 - 4.15 (m, 1H), 3.57 - 3.50 (m, 2H), 1.43 (s, 9H), 0.78 - 0.70 (m, 4H). Intermediate 7 Cyclopropyl (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoate
Figure imgf000065_0002
A mixture of cyclopropyl (R)-2-(((benzyloxy)carbonyl)amino)-3-((tert- butoxycarbonyl)amino)propanoate (1.0 g, 2.64 mmol) in HCl/Ethyl acetate (15 mL) was stirred at 15°C for 1 hour. The mixture was concentrated below 40°C to give (R)-cyclopropyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (810 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.00 (br s, 3H), 7.86 (br d, J = 8.4 Hz, 1H), 7.42 - 7.31 (m, 5H), 5.07 (s, 2H), 4.37 - 4.30 (m, 1H), 4.15 - 4.09 (m, 1H), 3.25 - 3.00 (m, 2H), 0.74 - 0.57 (m, 4H). Intermediate 8 Cyclohexyl (R)-2-(((benzyloxy)carbonyl)amino)-3-((tert-butoxycarbonyl)amino)propanoate
Figure imgf000066_0001
A mixture of (R)-2-(((benzyloxy)carbonyl)amino)-3-((tert-butoxycarbonyl)amino)propanoic acid (1 g, 2.96 mmol), HATU (1.69 g, 4.43 mmol) and N,N-diisopropylethylamine (1.15 g, 8.9 mmol) in DMF (25 mL) was stirred at 20°C for 30 min, cyclohexanol (326 mg, 3.25 mmol) was added and the resulting mixture was stirred at 50°C for 16 h. The mixture was diluted with water (25 mL) and extracted with ethyl acetate (50 mL × 2). The combined organic phases were washed with brine (50 mL × 2), dried over anhydrous sodium sulfate and concentrated. The residue was purified by Combi Flash (silica gel, from 0 to 30%, Ethyl acetate in petroleum ether) to give cyclohexyl (R)-2-(((benzyloxy)carbonyl)amino)-3-((tert- butoxycarbonyl)amino)propanoate (520 mg). 1H NMR (400MHz, CDCl3) δ 7.41 - 7.28 (m, 5H), 5.89-5.65 (m, 1H), 4.92 - 4.70 (m, 2H), 4.49- 4.33 (m, 1H), 3.68 - 3.47 (m, 2H), 1.91 - 1.73 (m, 4H), 1.61 - 1.34 (m, 15H). Intermediate 9 Cyclohexyl (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoate hydrochloride
Figure imgf000066_0002
A solution of cyclohexyl (R)-2-(((benzyloxy)carbonyl)amino)-3-((tert- butoxycarbonyl)amino)propanoate (520 mg, 1.24 mmol) in HCl/ Ethyl acetate (4 mL, 4 M) was stirred at 20°C for 1 hour. The mixture was concentrated to give cyclohexyl (R)-3-amino- 2-(((benzyloxy)carbonyl)amino)propanoate (440 mg) as HCl salt. 1H NMR (400MHz, CDCl3) δ 8.44 - 8.06 (m, 3H), 7.34 - 7.13 (m, 5H), 6.58 (br d, J = 6.8Hz, 1H), 5.03 (s, 2H), 4.78 - 4.33 (m, 2H), 3.59 - 3.23 (m, 2H), 1.89 - 1.19 (m, 10H). Intermediate 10 Phenyl (R)-2-(((benzyloxy)carbonyl)amino)-3-((tert-butoxycarbonyl)amino)propanoate
Figure imgf000067_0001
To a solution of (R)-2-(((benzyloxy)carbonyl)amino)-3-((tert- butoxycarbonyl)amino)propanoic acid (1 g, 2.9 mmol) in DMF (25 mL) was added HATU (1.69 g, 4.4 mmol), N,N-diisopropylethylamine (1.15 g, 8.9 mmol) and phenol (306 mg, 3.25 mmol). The mixture was stirred at 50°C for 16 h. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 25%) to give compound phenyl (R)-2-(((benzyloxy)carbonyl)amino)-3-((tert-butoxycarbonyl)amino)propanoate (890 mg). 1H NMR (400 MHz, CDCl3) δ 7.42 - 7.32 (m, 7H), 7.27 - 7.21 (m, 1H), 7.19 - 7.09 (m, 2H), 5.91 (br s, 1H), 5.15 (s, 2H), 4.91 (br s, 1H), 4.72 - 4.62 (m, 1H), 3.88 - 3.76 (m, 1H), 3.74 - 3.63 (m, 1H), 1.44 (s, 9H). Intermediate 11 Phenyl (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoate
Figure imgf000067_0002
)carbonyl)amino)-3-((tert- butoxycarbonyl)amino)propanoate (890 mg, 2.15 mmol) in HCl/ Ethyl acetate (4 M, 15 mL) was stirred at 15°C for 1 hour. The mixture was concentrated below 40°C to give compound phenyl (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (870 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 8.35 (br s, 3H), 8.19 (br d, J = 8.0 Hz, 1H), 7.46 - 7.27 (m, 8H), 7.11 (d, J = 7.6 Hz, 2H), 5.22 - 5.03 (m, 2H), 4.74 - 4.62 (m, 1H), 3.30 - 3.14 (m, 1H). Intermediate 12 2-Oxo-2-(pyrrolidin-1-yl)ethyl (R)-2-(((benzyloxy)carbonyl)amino)-3-((tert- butoxycarbonyl)amino)propanoate
Figure imgf000068_0001
To a solution of (R)-2-(((benzyloxy)carbonyl)amino)-3-((tert- butoxycarbonyl)amino)propanoic acid (1 g, 2.96 mmol), HATU (1.69 g, 4.43 mmol) and N,N- diisopropylethylamine (1.15 g, 8.87 mmol) in DMF (15 mL) was added 2-hydroxy-1- (pyrrolidin-1-yl)ethanone (382 mg, 2.96 mmol). The mixture was stirred at 50°C for 16 h. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 54%) to give 2-oxo-2-(pyrrolidin-1-yl)ethyl (R)-2-(((benzyloxy)carbonyl)amino)-3- ((tert-butoxycarbonyl)amino)propanoate (700 mg). 1H NMR (400MHz, CDCl3) δ 7.41 - 7.29 (m, 5H), 6.20 (br, 1H), 6.11 (d, J = 6.8 Hz, 1H), 5.18 - 5.07 (m, 2H), 5.01 (d, J = 14.4 Hz, 1H), 4.55 - 4.39 (m, 2H), 3.90 - 3.70 (m, 1H), 3.65 - 3.31 (m, 5H), 2.05 - 1.97 (m, 2H), 1.92 - 1.85 (m, 2H), 1.43 (s, 9H). Intermediate 13 2-Oxo-2-(pyrrolidin-1-yl)ethyl (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoate
Figure imgf000068_0002
To a solution of (R)-2-oxo-2-(pyrrolidin-1-yl)ethyl 2-(((benzyloxy)carbonyl)amino)-3-((tert- butoxycarbonyl)amino)propanoate (700 mg, 1.56 mmol) in ethyl acetate (4 mL) was added HCl/ Ethyl acetate (4 M, 10 mL). The mixture was stirred at 15°C for 1 hour. The mixture was concentrated in vacuo to give 2-oxo-2-(pyrrolidin-1-yl)ethyl (R)-3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (600 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.39 (br s, 3H), 8.03 (d, J = 8.0 Hz, 1H), 7.39 - 7.32 (m, 5H), 5.07 (s, 2H), 4.98 (d, J = 15.2 Hz, 1H), 4.76 (d, J = 15.2 Hz, 1H), 4.60 - 4.51 (m, 1H), 3.39 (t, J = 6.4 Hz, 2H), 3.30 (t, J = 7.2 Hz, 2H), 3.26 - 3.14 (m, 2H), 1.92 - 1.86 (m, 2H), 1.81 - 1.72 (m, 2H). Intermediate 14 2-Methoxyethyl (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoate
Figure imgf000069_0001
To a solution of (R)-3-amino-2-(((benzyloxy)carbonyl)amino)propanoic acid (5 g, 21 mmol) in 2-methoxyethanol (50 mL) was added thionyl chloride (5 g, 42 mmol). The mixture was stirred at 20°C for 16 h. The mixture was concentrated to give 2-methoxyethyl (R)-3-amino- 2-(((benzyloxy)carbonyl)amino)propanoate (7 g) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.35 (br, 3H), 7.95 (d, 1H), 7.38 - 7.31 (m, 5H), 5.11 - 5.00 (m, 2H), 4.49 - 4.40 (m, 1H), 4.25-4.15 (m, 2H), 3.50 (m, 2H), 3.25 (m, 4H), 3.05 (m, 1H). Preparation of the exemplified compounds of the invention Compound 1a (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)- 5-fluorobenzamido)propanoic acid is shown below
Figure imgf000070_0001
To a mixture of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (877 mg, 4.43 mmol), 4- bromo-5-ethyl-isoxazole (0.6 g, 3.41 mmol) and Na2CO3 (723 mg, 6.82 mmol) in a mixture of H2O (4 mL) and dioxane (16 mL) was added Pd(dppf)Cl2.CH2Cl2 (557 mg, 0.68 mmol) under N2. The mixture was purged with N2, sealed and heated at 120°C for 40 minutes in the microwave. The mixture was concentrated. The residue was diluted with H2O (20 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 5%) to give methyl 3-(5- ethylisoxazol-4-yl)-5-fluorobenzoate (100 mg). 1H NMR (400MHz, CDCl3) δ 8.36 (s, 1H), 7.85-7.81 (m, 1H), 7.71-7.63 (m, 1H), 7.28-7.20 (m, 1H), 3.94 (s, 3H), 2.95 (q, J = 7.6 Hz, 2H), 1.35 (t, J = 7.6 Hz, 3H). Step 2: 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoic acid
Figure imgf000071_0001
A mixture of methyl 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoate (0.125 g, 0.5 mmol) and LiOH.H2O (42 mg, 1.0 mmol) in a mixture of THF (2 mL) and H2O (2 mL) was stirred at 25°C for 0.5 hour. The mixture was diluted with H2O (20 mL). The aqueous phase was adjusted to pH=3~4 with saturated aqueous KHSO4 at 0°C and extracted with ethyl acetate (10 mL × 2). The organic phase was dried and concentrated to give 3-(5-ethylisoxazol-4-yl)-5- fluorobenzoic acid (0.11 g, crude). LC-MS (MH+): m/z = 236.0 tR (min, Method G) = 0.685 Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000071_0002
A mixture of 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoic acid (0.11 g, crude), (R)-benzyl 3-amino- 2-(((benzyloxy)carbonyl)amino)propanoate (171 mg, 0.47 mmol, HCl salt), DIPEA (302 mg, 2.34 mmol) and TBTU (225 mg, 0.7 mmol) in DMF (5 mL) was stirred at 25°C for 16 hours. The mixture was poured into H2O (50 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)- 3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoate (0.12 g). 1H NMR (400MHz, CDCl3) δ 8.35 (s, 1H), 7.53 (s, 1H), 7.42-7.27 (m, 11H), 7.23-7.15 (m, 1H), 7.05 (br s, 1H), 5.97 (br s, 1H), 5.21 (s, 2H), 5.10 (s, 2H), 4.67-4.52 (m, 1H), 3.99-3.87 (m, 1H), 3.86-3.76 (m, 1H), 2.95 (q, J = 7.6 Hz, 2H), 1.35 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000072_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate (60 mg, 0.11 mmol) and HBr in AcOH (2 mL, 30% in AcOH) were stirred at 50°C for 16 hours. The mixture was concentrated. The residue was purified by Preparative-HPLC (HCl) to give (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoic acid (20 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.14 (br s, 1H), 8.98 (s, 1H), 8.52 (br s, 3H), 7.91 (s, 1H), 7.71- 7.64 (m, 1H), 7.60-7.54 (m, 1H), 4.13-4.06 (m, 1H), 3.84-3.72 (m, 2H), 3.02 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 322.1 tR (min, Method c) = 0.995 [α]20,D = 6.0 (c = 0.5 mg/mL, CH3OH). Compound 2a (R)-methyl 2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)- 5-fluorobenzamido)propanoate
Figure imgf000072_0002
A mixture of 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoic acid (0.49 g, crude), (R)-methyl 3- amino-2-(((benzyloxy)carbonyl)amino)propanoate (526 mg, 2.08 mmol, HCl salt), DIPEA (1.35 g, 10.42 mmol) and TBTU (1.00 g, 3.12 mmol) in DMF (20 mL) was stirred at 25°C for 16 hours. The mixture was poured into H2O (50 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-methyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoate (0.35 g). 1H NMR (400MHz, CDCl3) δ 8.36 (s, 1H), 7.58 (s, 1H), 7.47 - 7.38 (m, 1H), 7.33-7.28 (m, 5H), 7.25 - 7.16 (m, 2H), 5.94 (br d, J=6.4 Hz, 1H), 5.11 (s, 2H), 4.62-4.50 (m, 1H), 3.99 - 3.88 (m, 1H), 3.84-3.71 (m, 4H), 2.95 (q, J=7.6 Hz, 2H), 1.35 (t, J=7.6 Hz, 3H). Step 2:
Figure imgf000073_0001
A mixture of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate (0.35 g, 0.75 mmol) and HBr in AcOH (7 mL, 30% in AcOH) were stirred at 25°C for 15 hours. The residue was concentrated. The residue was purified by Preparative-HPLC (HCl). The crude product was dissolved with MeOH (3 mL) and methyl tert- butyl ether (12 mL) was added dropwise. The precipitate was filtered and dried to give (R)- methyl 2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoate (0.137 g) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.14 (br s, 1H), 9.00 (s, 1H), 8.64 (br s, 3H), 7.89 (s, 1H), 7.67 (d, J = 9.6 Hz, 1H), 7.60 (d, J = 9.6 Hz, 1H), 4.26-4.20 (m, 1H), 3.81-3.77 (m, 2H), 3.74 (s, 3H), 3.04 (q, J = 7.6 Hz, 2H), 1.27 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 336.0, tR (min, Method C) = 1.665 min [α]20,D = -16 (c = 0.25 mg/mL, CH3OH). Compound 1b (+) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (+) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5- fluorobenzamido)propanoic acid is shown below
Figure imgf000074_0001
To a solution of 3-fluoro-5-iodobenzoic acid (5 g, 18.80 mmol) in EtOH (40 mL) was added sulfurous dichloride (6.71 g, 56.39 mmol). The mixture was stirred at 50°C for 14 hours. The reaction mixture was concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate=100:0 to 98:2) to give ethyl 3-fluoro- 5-iodobenzoate (5.2 g). 1H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 7.72-7.69 (m, 1H), 7.64-7.61 (m, 1H), 4.40 (q, J = 7.2 Hz, 2H), 1.40 (t, J = 7.2 Hz, 3H). Step 2: (S)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate and (R)-ethyl 3-fluoro-5-(1- hydroxyethyl)benzoate
Figure imgf000075_0001
To a solution of ethyl 3-fluoro-5-iodobenzoate (5.2 g, 17.68 mmol) in THF (60 mL) was added i-PrMgCl-LiCl (1.3 M in THF, 19.04 mL) at -70°C. The mixture was stirred at -70°C for 30 minutes. Then acetaldehyde (935 mg, 21.22 mmol) was added. The mixture was stirred at 20°C for 1 hour. The reaction mixture was quenched with saturated solution NH4Cl (20 mL) and concentrated. The residue was diluted with water (50 mL) and extracted with ethyl acetate (50 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether : Ethyl acetate=100:0 to 90:10). The residue was further separated by SFC (Instrument: Thar200; Column: DAICEL CHIRALPAK AD 250 × 50 mm I.D.5 μm; Mobile phase: supercritical CO2/MeOH (0.1% NH3`H2O, 15%) = 85/15; Flow Rate: 180 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give (S)-ethyl 3-fluoro-5-(1- hydroxyethyl)benzoate (1.7 g) and (R)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate (1.6 g). (S)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate: 1H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.63- 7.59 (m, 1H), 7.34-7.31 (m, 1H), 4.97 (q, J = 6.4 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 2.04 (s, 1H), 1.52 (d, J = 6.4 Hz, 3H), 1.40 (t, J = 7.2 Hz, 3H). (R)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate: 1H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.63- 7.59 (m, 1H), 7.34-7.31 (m, 1H), 4.97 (q, J = 6.4 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 2.05 (d, J = 4.0 Hz, 1H), 1.52 (d, J = 6.4 Hz, 3H), 1.40 (t, J = 7.2 Hz, 3H). Note: The (S) and (R) configuration was assigned arbitrarily after SFC separation. Step 3: (S)-ethyl 3-(1-ethoxyethyl)-5-fluorobenzoate
Figure imgf000076_0001
To a solution of (S)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate (1.7 g, 8.01 mmol) in DMF (30 mL) was added NaH (481 mg, 12.02 mmol, 60% in mineral oil). The mixture was stirred at 0°C for 30 minutes. Then iodoethane (3.75 g, 24.03 mmol) was added. The mixture was stirred at 60°C for 15 hours. The reaction mixture was quenched by addition H2O (10 mL), and then concentrated under reduced pressure to give a residue. The residue was diluted with ethyl acetate (60 mL) and washed with brine (30 mL × 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, Petroleum ether: Ethyl acetate=100:0 to 95:5) to give (S)-ethyl 3-(1- ethoxyethyl)-5-fluorobenzoate (1.45 g). 1H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.63-7.59 (m, 1H), 7.29-7.25 (m, 1H), 4.46 (q, J = 6.4 Hz, 1H), 4.40 (q, J = 7.2 Hz, 2H), 3.43-3.33 (m, 2H), 1.45-1.39 (m, 6H), 1.21 (t, J = 7.2 Hz, 3H). Step 4: (S)-3-(1-ethoxyethyl)-5-fluorobenzoic acid
Figure imgf000076_0002
To a solution of (S)-ethyl 3-(1-ethoxyethyl)-5-fluorobenzoate (600 mg, 2.50 mmol) in a mixture of THF (10 mL) and H2O (5 mL) was added LiOH.H2O (315 mg, 7.50 mmol). The mixture was stirred at 30°C for 16 hours. The reaction mixture was added 2 M HCl to pH=2~3 and concentrated. The residue was diluted with ethyl acetate (40 mL) and washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated to give (S)-3-(1-ethoxyethyl)-5- fluorobenzoic acid (480 mg). 1H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.72-7.68 (m, 1H), 7.36-7.33 (m, 1H), 4.50 (q, J = 6.4 Hz, 1H), 3.44-3.37 (m, 2H), 1.46 (d, J = 6.4 Hz, 3H), 1.23 (t, J = 7.2 Hz, 3H). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((S)-1-ethoxyethyl)-5- fluorobenzamido)propanoate
Figure imgf000077_0001
To a solution of (S)-3-(1-ethoxyethyl)-5-fluorobenzoic acid (200 mg, 0.942 mmol) in DMF (10 mL) was added (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (378 mg, 1.04 mmol, HCl), TBTU (454 mg, 1.41 mmol) and DIPEA (365 mg, 2.83 mmol). The mixture was stirred at 30°C for 16 hours. The reaction mixture was diluted with H2O (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether : Ethyl acetate=100:0 to 60:40) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-((S)-1-ethoxyethyl)-5-fluorobenzamido)propanoate (400 mg). LC-MS (MH+): m/z = 523.2 tR (min, Method G) = 0.831 Step 6: (+) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoic acid
Figure imgf000077_0002
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((S)-1-ethoxyethyl)-5- fluorobenzamido)propanoate (200 mg, 0.383 mmol) in AcOH (8 mL) was added Pd/C (50 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 20°C for 1 hour. The reaction mixture was filtered and concentrated. The residue was purified by preparation HPLC (Method AA) to give (+) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoic acid (30 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.07-8.99 (m, 1H), 8.56 (br s, 3H), 7.73 (s, 1H), 7.66-7.63 (m, 1H), 7.35-7.31 (m, 1H), 4.51 (q, J = 6.4 Hz, 1H), 4.12-4.09 (m, 1H), 3.78-3.76 (m, 2H), 3.48- 3.24 (m, 2H), 1.35 (d, J = 6.4 Hz, 3H), 1.10 (t, J = 6.8 Hz, 3H). LC-MS (MH+): m/z = 299.1 tR (min, Method D) = 1.534 [α]20,D = +56.0 (c = 1.0 mg/mL, CH3OH). Compound 2b (+)(2R)-methyl 2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((S)-1-ethoxyethyl)-5- fluorobenzamido)propanoate
Figure imgf000078_0001
To a solution of (S)-3-(1-ethoxyethyl)-5-fluorobenzoic acid (200 mg, 0.942 mmol) in DMF (10 mL) was added (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (327 mg, 1.13 mmol, HCl), TBTU (454 mg, 1.41 mmol) and DIPEA (365 mg, 2.83 mmol). The mixture was stirred at 30°C for 4 hours. The reaction mixture was diluted with H2O (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate=100:0 to 60:40) to give (R)- methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((S)-1-ethoxyethyl)-5- fluorobenzamido)propanoate (380 mg). LC-MS (MH+): m/z = 447.2 tR (min, Method G) = 0.757 Step 2:
Figure imgf000079_0001
To a solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((S)-1-ethoxyethyl)-5- fluorobenzamido)propanoate (200 mg, 0.448 mmol) in AcOH (10 mL) was added Pd/C (80 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 20°C for 1 hour. The reaction mixture was filtered through celite and concentrated. The residue was purified by preparation HPLC (neutral condition) to give (+)(2R)-methyl 2-amino-3-(3-(1-ethoxyethyl)-5- fluorobenzamido)propanoate (45 mg). 1H NMR (400MHz, CDCl3) δ 7.48 (s, 1H), 7.39-7.36 (m, 1H), 7.22-7.19 (m, 1H), 6.80 (br s, 1H), 4.45 (q, J=6.4 Hz, 1H), 3.90-3.84 (m, 1H), 3.79 (s, 3H), 3.74-3.70 (m, 1H), 3.54-3.48 (m, 1H), 3.44-3.34 (m, 2H), 1.43 (d, J=6.4 Hz, 3H), 1.20 (t, J=6.4 Hz, 3H). LCMS (MH+): m/z = 313.2, tR (min, Method C) = 1.753 min [α]20,D = +14.0 (c = 1.0 mg/mL, CH3OH). Compound 1c The overall synthesis scheme for the preparation of (-) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5- fluorobenzamido)propanoic acid is shown below
Figure imgf000079_0002
Step 1: (R)-ethyl 3-(1-ethoxyethyl)-5-fluorobenzoate
Figure imgf000080_0001
To a solution of (R)-ethyl 3-fluoro-5-(1-hydroxyethyl)benzoate (1.6 g, 7.54 mmol) in DMF (30 mL) was added NaH (452 mg, 11.31 mmol, 60% in mineral oil). The mixture was stirred at 0°C for 30 minutes. Then iodoethane (3.53 g, 22.62 mmol) was added. The mixture was stirred at 60°C for 15 hours. The reaction mixture was quenched by addition H2O (10 mL), and then concentrated. The residue was diluted with ethyl acetate (60 mL) and washed with brine (30 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate=100:0 to 95:5) to give (R)-ethyl 3-(1- ethoxyethyl)-5-fluorobenzoate (1.3 g). 1H NMR (400 MHz, CDCl3) δ 7.77 (s, 1H), 7.64-7.59 (m, 1H), 7.29-7.25 (m, 1H), 4.46 (q, J = 6.4 Hz, 1H), 4.40 (q, J = 7.2 Hz, 2H), 3.43-3.33 (m, 2H), 1.60-1.39 (m, 6H), 1.21 (t, J = 7.2 Hz, 3H). Step 2: (R)-3-(1-ethoxyethyl)-5-fluorobenzoic acid
Figure imgf000080_0002
To a solution of (R)-ethyl 3-(1-ethoxyethyl)-5-fluorobenzoate (600 mg, 2.50 mmol) in a mixture of THF (10 mL) and H2O (5 mL) was added LiOH.H2O (315 mg, 7.50 mmol). The mixture was stirred at 30°C for 16 hours. The reaction mixture was added 2 M HCl to pH=2~3 and concentrated. The residue was diluted with ethyl acetate (40 mL) and washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated to give (R)-3-(1-ethoxyethyl)-5- fluorobenzoic acid (480 mg). 1H NMR (400 MHz, CDCl3) δ 7.85 (s, 1H), 7.72-7.68 (m, 1H), 7.36-7.33 (m, 1H), 4.50 (q, J = 6.4 Hz, 1H), 3.44-3.37 (m, 2H), 1.46 (d, J = 6.4 Hz, 3H), 1.23 (t, J = 7.2 Hz, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((R)-1-ethoxyethyl)-5- fluorobenzamido)propanoate
Figure imgf000081_0001
To a solution of (R)-3-(1-ethoxyethyl)-5-fluorobenzoic acid (200 mg, 0.942 mmol) in DMF (10 mL) was added (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (378 mg, 1.04 mmol, HCl), TBTU (454 mg, 1.41 mmol) and DIPEA (365 mg, 2.83 mmol). The mixture was stirred at 30°C for 16 hours. The reaction mixture was diluted with H2O (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate=100:0 to 60:40) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-((R)-1-ethoxyethyl)-5-fluorobenzamido)propanoate (400 mg). LC-MS (MH+): m/z = 523.2 tR (min, Method G) = 0.831 Step 4: of (-) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoic acid
Figure imgf000081_0002
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((R)-1-ethoxyethyl)-5- fluorobenzamido)propanoate (200 mg, 0.383 mmol) in AcOH (8 mL) was added Pd/C (50 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 20°C for 0.5 hour. The reaction mixture was filtered and concentrated. The residue was purified by preparation HPLC (Method AA) to give (-) (2R)-2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoic acid (30 mg) as HCl 1H NMR (400MHz, DMSO-d6) δ 9.10-9.01 (m, 1H), 8.50 (br s, 3H), 7.73 (s, 1H), 7.66-7.63 (m, 1H), 7.35-7.30 (m, 1H), 4.51 (q, J = 6.4 Hz, 1H), 4.01-3.98 (m, 1H), 3.84-3.67 (m, 2H), 3.38- 3.23 (m, 2H), 1.35 (d, J = 6.4 Hz, 3H), 1.10 (t, J = 6.8 Hz, 3H). LC-MS (MH+): m/z = 299.1 tR (min, Method D) = 1.524 [α]20,D = -34.0 (c = 1.0 mg/mL, CH3OH). Compound 2c (-) (2R)-methyl 2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((R)-1-ethoxyethyl)-5- fluorobenzamido)propanoate
Figure imgf000082_0001
To a solution of (R)-3-(1-ethoxyethyl)-5-fluorobenzoic acid (200 mg, 0.942 mmol) in DMF (10 mL) was added (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((R)-1-ethoxyethyl)-5- fluorobenzamido)propanoate (327 mg, 1.13 mmol, HCl), TBTU (454 mg, 1.41 mmol) and DIPEA (365 mg, 2.83 mmol). The mixture was stirred at 30°C for 4 hours. The reaction mixture was diluted with H2O (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, Petroleum ether:Ethyl acetate=100:0 to 60:40) to give (-)(2R)-methyl 2-(((benzyloxy)carbonyl)amino)-3- (3-((R)-1-ethoxyethyl)-5-fluorobenzamido)propanoate (380 mg) LC-MS (MH+): m/z = 447.2 tR (min, Method G) = 0.757 Step 2:
Figure imgf000083_0001
To a solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-((R)-1-ethoxyethyl)-5- fluorobenzamido)propanoate (200 mg, 0.448 mmol) in AcOH (10 mL) was added Pd/C (80 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15psi) at 20°C for 1 hour. The reaction mixture was filtered through celite and concentrated. The residue was purified by preparation HPLC (neutral condition) to give (-)(2R)-methyl 2-amino-3-(3-(1-ethoxyethyl)-5- fluorobenzamido)propanoate (30 mg). 1H NMR (400MHz, CDCl3) δ 7.48 (s, 1H), 7.40-7.36 (m, 1H), 7.22-7.19 (m, 1H), 6.79 (br s, 1H), 4.45 (q, J=6.4 Hz, 1H), 3.92-3.83 (m, 1H), 3.79 (s, 3H), 3.74-3.70 (m, 1H), 3.54-3.48 (m, 1H), 3.44-3.34 (m, 2H), 1.43 (d, J=6.4 Hz, 3H), 1.20 (t, J=6.4 Hz, 3H). LCMS (MH+): m/z = 313.2, tR (min, Method C) = 1.752 min [α]20,D = -46.0 (c = 1.0 mg/mL, CH3OH). Compound 1d (R)-2-amino-3-(3-(2-ethylfuran-3-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(2-ethylfuran-3-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000084_0001
To a solution of 2,3-dibromofuran (2.3 g, 10.2 mmol) in THF (50 mL) was added Pd(dppf)Cl2 (745 mg, 1.02 mmol), cesium acetate (4.89 g, 25.46 mmol) and triethylborane (1 M in THF, 12.2 mL). The mixture was stirred at 65 °C for 7 hours. The mixture was added water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were concentrated below 30 °C. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 1%) and concentrated below 30 °C to give compound 3- bromo-2-ethylfuran (1.5 g). 1H NMR (400MHz, CDCl3) δ 7.27 (d, J = 1.2 Hz, 1H), 6.35 (d, J = 2.0 Hz, 1H), 2.67 (q, J = 7.6 Hz, 2H), 1.24 - 1.20 (m, 3H). Step 2: Methyl 3-(2-ethylfuran-3-yl)-5-fluorobenzoate
Figure imgf000084_0002
To a solution of 3-bromo-2-ethylfuran (1.50 g, 8.57 mmol) and (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (1.70 g, 8.57 mmol) in a mixture of DME (40 mL) and H2O (4 mL) was added Pd(PPh3)4 (990 mg, 0.86 mmol) and Na2CO3 (3.55 g, 25.7 mmol) under N2 atmosphere. The mixture was stirred at 85 °C for 16 hours. The mixture was diluted with water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 20%) to give 1.1 g of the crude product. The crude product was further purified by preparative-HPLC (Method AA) to give compound methyl 3- (2-ethylfuran-3-yl)-5-fluorobenzoate (630 mg). 1H NMR (400MHz, CDCl3) δ 7.87-7.84 (m, 1H), 7.65-7.58 (m, 1H), 7.38 (d, J = 1.6 Hz, 1H), 7.28- 7.24 (m, 1H), 6.52 (d, J = 1.6 Hz, 1H), 3.95 (s, 3H), 2.83 (q, J = 7.6 Hz, 2H), 1.30 (t, J = 7.6 Hz, 3H). Step 3: 3-(2-ethylfuran-3-yl)-5-fluorobenzoic acid
Figure imgf000085_0001
To a solution of methyl 3-(2-ethylfuran-3-yl)-5-fluorobenzoate (80 mg, 0.32 mmol) in a mixture of MeOH (4 mL) and H2O (1 mL) was added LiOH.H2O (41 mg, 0.97 mmol). The mixture was stirred at 20 °C for 14 hours. The mixture was concentrated. The residue was dissolved in water (5 mL) and acidified with 2N HCl to adjust pH to 5 and extracted with ethyl acetate (15 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated to give compound 3-(2-ethylfuran-3-yl)-5-fluorobenzoic acid (68 mg). 1H NMR (400MHz, DMSO-d6) δ 13.39 (br s, 1H), 7.79 (s, 1H), 7.65 (d, J = 1.6 Hz, 1H), 7.59-7.48 (m, 2H), 6.80 (d, J = 1.6 Hz, 1H), 2.82 (q, J = 7.6 Hz, 2H), 1.22 (t, J = 7.6 Hz, 3H). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylfuran-3-yl)-5- fluorobenzamido)propanoate
Figure imgf000086_0001
To a solution of 3-(2-ethylfuran-3-yl)-5-fluorobenzoic acid (80 mg, 0.21 mmol, ~60% purity) in DMF (5 mL) was added TBTU (99 mg, 0.31 mmol), DIPEA (80 mg, 0.62 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (125 mg, 0.34 mmol, HCl salt). The mixture was stirred at 15 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give 150 mg of the crude product. The crude product was further purified by preparative- HPLC (Method AA) to give compound (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethylfuran-3-yl)-5-fluorobenzamido)propanoate (65 mg). 1H NMR (400MHz, CDCl3) δ 7.51 (s, 1H), 7.39-7.29 (m, 11H), 7.26-7.17 (m, 2H), 6.79 (br s, 1H), 6.51 (d, J = 1.6 Hz, 1H), 5.93 (br d, J = 6.8 Hz, 1H), 5.27-5.17 (m, 2H), 5.16-5.08 (m, 2H), 4.65- 4.57 (m, 1H), 3.99-3.89 (m, 1H), 3.86-3.77 (m, 1H), 2.82 (q, J = 7.6 Hz, 2H), 1.29 (t, J = 7.6 Hz, 3H). Step 5: (R)-2-amino-3-(3-(2-ethylfuran-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000086_0002
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylfuran-3-yl)-5- fluorobenzamido)propanoate (40 mg, 0.07 mmol) in ethyl acetate (2 mL) was added Pd/C (40 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 1 hour. The mixture was filtered through Celite and the filtrate was concentrated. The residue was purified by preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-(2-ethylfuran-3-yl)-5- fluorobenzamido)propanoic acid (15 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.95-8.93 (m, 1H), 8.34 (br s, 2H), 7.75 (s, 1H), 7.66 (d, J=2.0 Hz, 1H), 7.60-7.54 (m, 1H), 7.47-7.41 (m, 1H), 6.78 (d, J = 2.0 Hz, 1H), 4.10-4.04 (m, 1H), 3.83- 3.75 (m, 1H), 3.74-3.68 (m, 1H), 2.83 (t, J = 7.6 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 320.9 tR (min, Method D) = 1.758 [α]20 ,D = 3.1 (c = 1.3 mg/mL, CH3OH). Compound 2d (R)-methyl 2-amino-3-(3-(2-ethylfuran-3-yl)-5-fluorobenzamido)propanoate
Figure imgf000087_0001
To a solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylfuran-3-yl)-5- fluorobenzamido)propanoate (90 mg, 0.19 mmol) in AcOH (4 mL) was added 30% HBr in AcOH (1 mL). The mixture was stirred at 15 °C for 1 hour. The mixture was added MeOH (3 mL) and concentrated. The residue was purified by prep-HPLC (HCl) to give (R)-methyl 2- amino-3-(3-(2-ethylfuran-3-yl)-5-fluorobenzamido)propanoate (15 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.99-8.94 (m, 1H), 8.53 (br s, 3H), 7.75 (s, 1H), 7.66 (d, J = 2.0 Hz, 1H), 7.60 - 7.54 (m, 1H), 7.48 - 7.42 (m, 1H), 6.80 (d, J = 2.0 Hz, 1H), 4.26 - 4.19 (m, 1H), 3.80 - 3.69 (m, 5H), 2.84 (q, J = 7.6 Hz, 2H), 1.22 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 335.1, tR (min, Method C) = 2.153 min [α]20,D = 1.5 (c =1.3 mg/mL, CH3OH). Compound 1e (-)cis-(2R)-2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (-)cis-(2R)-2-amino-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000088_0001
Step 1: cis-methyl 3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzoate
Figure imgf000088_0002
To a solution of methyl 3-(2-ethylfuran-3-yl)-5-fluorobenzoate (500 mg, 2.01 mmol) in AcOH (5 mL) was added Pd/C (200 mg, 10% Pd, <1% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 18 hours. The mixture was filtered through Celite and the filtrate was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 20%) to give cis-methyl 3-(2-ethyltetrahydrofuran-3-yl)- 5-fluorobenzoate (400 mg). 1H NMR (400MHz, CDCl3) δ 7.65 (s, 1H), 7.60-7.55 (m, 1H), 7.14-7.08 (m, 1H), 4.23-4.15 (m, 1H), 3.93 (s, 3H), 3.90-3.79 (m, 2H), 3.42-3.35 (m, 1H), 2.53-2.42 (m, 1H), 2.14-2.05 (m, 1H), 1.31-1.21 (m, 1H), 1.15-1.05 (m, 1H), 0.84 (t, J = 7.6 Hz, 3H). Step 2: cis-3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzoic acid
Figure imgf000089_0001
To a solution of cis-methyl 3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzoate (350 mg, 1.39 mmol) in a mixture of MeOH (5 mL) and H2O (2 mL) was added LiOH.H2O (175 mg, 4.16 mmol). The mixture was stirred at 20 °C for 16 hours. The mixture was concentrated. The residue was dissolved in water (5 mL) and acidified with 2N HCl to adjust pH to 5 and extracted with ethyl acetate (15 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated to give cis-3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzoic acid (310 mg). 1H NMR (400MHz, CDCl3) δ 13.26 (br s, 1H), 7.60 (s, 1H), 7.52-7.47 (m, 1H), 7.30-7.25 (m, 1H), 4.10-4.02 (m, 1H), 3.78-3.70 (m, 2H), 3.53-3.44 (m, 1H), 2.45-2.34 (m, 1H), 2.05-1.95 (m, 1H), 1.08-0.90 (m, 2H), 0.79-0.71 (m, 3H). Step 3: cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate
Figure imgf000089_0002
To a solution of cis-3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzoic acid (150 mg, 0.63 mmol) in DMF (5 mL) was added TBTU (303 mg, 0.94 mmol), DIPEA (244 mg, 1.89 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (230 mg, 0.63 mmol, HCl salt). The mixture was stirred at 20 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 40%) to give compound cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate (100 mg). LC-MS (MH+): m/z = 549.3 tR (min, Method G) = 0.924 Step 4: cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate and cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate
Figure imgf000090_0001
The start material cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate (100 mg, 0.18 mmol) was separated by SFC (Instrument: Waters; Column: DAICEL CHIRALCEL OJ- H(250mm*30mm,5μm); Mobile phase: supercritical CO2/EtOH (0.1% NH3`H2O, v%) = 80/20; Flow Rate: 50 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate (30 mg, peak 1, tR = 2.7 min) and the other diastereoisomer of cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate (32 mg, peak 2, tR = 2.87 min). Step 5: (-)cis-(2R)-2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000090_0002
To a solution of cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate (30 mg, 0.05 mmol) in MeOH (5 mL) was added Pd/C (20 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 1 hour. The mixture was filtered through Celite and the filtrate was concentrated. The residue was purified by prep-HPLC (HCl) to give compound (-)cis-(2R)-2- amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid (10 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.90-8.84 (m, 1H), 8.39 (br s, 3H), 7.59-7.50 (m, 2H), 7.24-7.17 (m, 1H), 4.15-4.03 (m, 2H), 3.81-3.66 (m, 4H), 3.50-3.40 (m, 1H), 2.43-2.32 (m, 1H), 2.08-1.96 (m, 1H), 1.09-0.89 (m, 2H), 0.80-0.72 (m, 3H). LC-MS (MH+): m/z = 325.2 tR (min, Method D) = 1.580
Figure imgf000091_0001
1.5 mg/mL, CH3OH). Preparation of cis-(2R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyltetrahydrofuran- 3-yl)-5-fluorobenzamido)propanoate
Figure imgf000091_0002
To a solution of cis-3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzoic acid (160 mg, 0.67 mmol) in DMF (3 mL) was added TBTU (323 mg, 1.01 mmol), DIPEA (260 mg, 2.01 mmol) and (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (194 mg, 0.67 mmol, HCl salt).The mixture was stirred at 20 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetatefrom 0 to 40%) to give 300 mg of the crude product. The crude product was further purified by prep-HPLC (basic) to give cis-(2R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyltetrahydrofuran- 3-yl)-5-fluorobenzamido)propanoate (170 mg). LC-MS (MH+): m/z = 473.2 tR (min, Method G) = 0.760 Preparation of cis-(2R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyltetrahydrofuran- 3-yl)-5-fluorobenzamido)propanoate and cis-(2R)-methyl 2-(((benzyloxy)carbonyl)amino)-3- (3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate
Figure imgf000092_0001
The start material cis-(2R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate (170 mg, 359.78 µmol) was separated by SFC (Instrument: Berger, MULTIGR AM-II; Column: Chiralpak AD 250 × 30 mm I.D.20 μm; Mobile phase: supercritical CO2/i-PrOH (0.1% NH3`H2O, v%) = 55/45; Flow Rate: 60 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give cis-(2R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate (70 mg, peak 1, tR = 6.011 min) and cis-(2R)-methyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate (75 mg, peak 2, tR = 6.350 min). 1H NMR (400MHz, CDCl3) δ 7.36 - 7.30 (m, 5H), 7.08 - 6.94 (m, 2H), 5.91 (br s, 1H), 5.12 (s, 2H), 4.61 - 4.52 (m, 1H), 4.24 - 4.17 (m, 1H), 3.92 - 3.79 (m, 7H), 3.40 - 3.32 (m, 1H), 2.52 - 2.40 (m, 1H), 2.12 - 2.00 (m, 1H), 1.25 - 1.18 (m, 1H), 1.15 - 1.05 (m, 1H), 0.87 - 0.81 (m, 3H). Compound 2e (-)cis-(2R)-methyl 2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate
Figure imgf000092_0002
To a solution of cis-(2R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate (70 mg, 0.15 mmol) in MeOH (5 mL) was added Pd/C (30 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 1 hour. The mixture filtered through Celite and the filtrate was concentrated to give (-)cis-(2R)-methyl 2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate (20 mg). 1H NMR (400MHz, CDCl3) δ 7.38 - 7.31 (m, 2H), 7.07 - 7.02 (m, 1H), 6.92 (br s, 1H), 4.23 - 4.15 (m, 1H), 3.93 - 3.78 (m, 6H), 3.75 - 3.70 (m, 1H), 3.56 - 3.48 (m, 1H), 3.41 - 3.34 (m, 1H), 2.51 - 2.40 (m, 1H), 2.14 - 2.03 (m, 1H), 1.30 - 1.22 (m, 1H), 1.14 - 1.05 (m, 1H), 0.85 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 339.1, tR (min, Method C) = 1.636 min [α]20,D = -66.7 (c = 1.6 mg/mL, CH3OH). Compound 1f (+)cis-(2R)-2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid The synthesis scheme for the preparation of (+)cis-(2R)-2-amino-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000093_0001
To a solution of cis-(2R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate (32 mg, 0.03 mmol, 48.6% purity) in MeOH (5 mL) was added Pd/C (20 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 0.5 hour. The mixture was filtered through Celite and the filtrate was concentrated. The residue was purified by prep-HPLC (HCl) to give (+)cis- (2R)-2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid (7 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.90-8.84 (m, 1H), 8.39 (br s, 3H), 7.57-7.51 (m, 2H), 7.24-7.18 (m, 1H), 4.12-4.03 (m, 2H), 3.82-3.68 (m, 4H), 3.50-3.40 (m, 1H), 2.44-2.39 (m, 1H), 2.07-1.96 (m, 1H), 1.09-0.90 (m, 2H), 0.81-0.71 (m, 3H). LC-MS (MH+): m/z = 325.0 tR (min, Method D) = 1.429 for (+)cis-(2R)-2-amino-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoic acid [α]20,D = 34.5 (c = 1.1 mg/mL, CH3OH). Compound 2f (+)cis-(2R)-methyl 2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate
Figure imgf000094_0001
To a solution of cis-(2R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2- ethyltetrahydrofuran-3-yl)-5-fluorobenzamido)propanoate (75 mg, 0.16 mmol) in MeOH (5 mL) was added Pd/C (20 mg, 10% Pd, 50% water) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 20 °C for 0.5 hour. The mixture filtered through celite and the filtrate was concentrated to give (+)cis-(2R)-methyl 2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate (35 mg). 1H NMR (400MHz, CDCl3) δ 7.38 - 7.30 (m, 2H), 7.08 - 7.02 (m, 1H), 6.83 (br s, 1H), 4.22- 4.14 (m, 1H), 3.93 - 3.77 (m, 6H), 3.75 - 3.69 (m, 1H), 3.54 - 3.46 (m, 1H), 3.41 - 3.32 (m, 1H), 2.52 - 2.41 (m, 1H), 2.12 - 2.01 (m, 1H), 1.26 - 1.20 (m, 1H), 1.14 - 1.04 (m, 1H), 0.85 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 339.1, tR (min, Method D) = 1.692 min [α]20,D = 21.0 (c = 2.0 mg/mL, CH3OH). Compound 1g (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5- fluorobenzamido)propanoic acid is shown below
Figure imgf000095_0001
Step 1: methyl 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoate
Figure imgf000095_0002
To a mixture of 2-chloro-3-ethylpyrazine (200 mg, 1.40 mmol) and (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (278 mg, 1.40 mmol) in the mixture of dioxane (5 mL) and H2O (0.5 mL) was added K2CO3 (582 mg, 4.21 mmol) and Pd(dppf)Cl2 (103 mg, 0.14 mmol), the mixture was stirred at 90°C for 14 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 10%) to give methyl 3-(3- ethylpyrazin-2-yl)-5-fluorobenzoate (290 mg). 1H NMR (400MHz, CDCl3) δ 8.56 (d, J=2.0 Hz, 1H), 8.50 (d, J=2.0 Hz, 1H), 8.04 (d, J=1.6 Hz, 1H), 7.82 (dd, J=1.2 Hz, 8.4 Hz,1H), 7.49 (dd, J=2.4 Hz, 7.2 Hz,1H), 3.96 (s, 3H), 2.91 (q, J=7.6 Hz, 2H), 1.30 (t, J=7.6 Hz, 2H). Step 2: 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoic acid
Figure imgf000096_0001
To a solution of methyl 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoate (290 mg, 1.11 mmol) in the mixture of MeOH (6 mL) and H2O (6 mL) was added LiOH.H2O (94 mg, 2.23 mmol). The mixture was stirred at 25°C for 1 hour. The mixture was concentrated. The residue was added water (20 mL), then dropwise added 2N aqueous HCl to pH=3~4 and extracted with ethyl acetate (30 mL × 3), the organic layers were dried over Na2SO4, filtered and concentrated to give 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoic acid (270 mg). LC-MS (MH+): m/z = 247.1 tR (min, method c) = 0.595. Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyrazin-2-yl)-5- fluorobenzamido)propanoate
Figure imgf000096_0002
To a mixture of 3-(3-ethylpyrazin-2-yl)-5-fluorobenzoic acid (150 mg, 0.61 mmol) and (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (267 mg, 0.73 mmol, HCl salt) in DMF (6 mL) was added TBTU (293 mg, 0.91 mmol) and DIPEA (236 mg, 1.83 mmol). The mixture was stirred at 20°C for 14 hours. The mixture was added water (60 mL) and extracted with ethyl acetate (40 mL × 3), the organic layers were washed with brine (40 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 40%) to give (R)- benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyrazin-2-yl)-5- fluorobenzamido)propanoate (240 mg). 1H NMR (400MHz, CDCl3) δ 8.57 (d, J=2.0 Hz, 1H), 8.49 (d, J=2.0 Hz, 1H), 7.69 (s, 1H), 7.47 (d, J=8.4 Hz, 1H), 7.41 (d, J=8.8 Hz, 1H), 7.37 - 7.28 (m, 10H), 6.85 (br s, 1H), 5.92 (br d, J=6.4 Hz, 1H), 5.22 (s, 2H), 5.11 (s, 2H), 4.64 - 4.56 (m, 1H), 3.98 - 3.79 (m, 2H), 2.91 (q, J=7.2 Hz, 2H), 1.29 (t, J=7.2 Hz, 3H). Step 4: (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000097_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylpyrazin-2-yl)-5- fluorobenzamido)propanoate (240 mg, 0.43 mmol) in 30% HBr/HOAc (5 mL) was stirred at 50°C for 14 hours. The mixture was concentrated. The residue was purified by preparative- HPLC (Method AA) to give (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5- fluorobenzamido)propanoic acid (116 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.18-9.11 (m, 1H), 8.65 (s, 1H), 8.64 - 8.57 (m, 4H), 7.95 (s, 1H), 7.89 (d, J=8.8 Hz, 1H), 7.65 (d, J=8.8 Hz, 1H), 4.15-4.08 (m, 1H), 3.84-3.76 (m, 2H), 2.86 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 333.0 tR (min, Method D) = 1.446 [α]20 ,D = +3.5 (c = 4.0 mg/mL, CH3OH). Compound 2g Methyl (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5-fluorobenzamido)propanoate
Figure imgf000097_0002
To a stirred solution of (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5-fluorobenzamido)propanoic acid (30 mg, 0.09 mmol) in methanol (0.5 mL), at 0 °C, was added, thionyl chloride (33.0 μl, 0.45 mmol) dropwise. The reaction mixture was warmed to room temperature and stirred for 20 hours. The mixture was concentrated and then diluted with sat. aq. NaHCO3 (2 mL) and extracted with ethyl acetate (3x2mL). The combined organic phase was concentrated and then diluted with a methanolic HCl solution (200µL, 1.25M) and stirred for 10 minutes. After concentration methyl (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5- fluorobenzamido)propanoate (14 mg) was afforded as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.05-8.98 (m, 1H), 8.67 (d, J = 2.5 Hz, 1H), 8.60 (d, J = 2.5 Hz, 1H), 8.58 (br s, 3H), 7.92-7.89 (m, 1H), 7.83-7.79 (m, 1H), 7.69-7.65 (m, 1H), 4.24-4.19 (m, 1H), 3.81-3.71 (m, 5H), 2.86 (q, J = 7.5 Hz, 2H), 1.18 (t, J = 7.5 Hz, 3 H). LCMS (MH+): m/z = 347.2, tR (min, Method c) = 0.36 min Compound 1h (R)-2-amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-ethyl-1-methyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoic acid is shown below.
Figure imgf000098_0001
A mixture of 5-bromo-4-ethyl-1-methyl-1H-pyrazole (200 mg, 1.06 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (251 mg, 1.27 mmol), Pd(dtbpf)Cl2 (69 mg, 0.11 mmol), K3PO4 (674 mg, 3.17 mmol) in the mixture of dioxane (3 mL) and H2O (0.5 mL) was degassed and purged with N2 (3 times), and then the mixture was stirred at 110 °C for 16 hours under N2 atmosphere. The mixture was concentrated, then added water (5 mL) and extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with saturated brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 20%) to give methyl 3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (230 mg). 1H NMR (400MHz, CDCl3) δ 7.82-7.79 (m, 2H), 7.44 (s, 1H), 7.24-7.21 (m, 1H), 3.96 (s, 3H), 3.77 (s, 3H), 2.41 (q, J = 7.6 Hz, 2H), 1.13 (t, J = 7.6 Hz, 3H). Step 2: 3-(4-Ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000099_0001
To a solution of methyl 3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (230 mg, 0.88 mmol) in the mixture of MeOH (2 mL), THF (2 mL) and H2O (2 mL) was added LiOH.H2O (110 mg, 2.63 mmol). The mixture was stirred at 20 °C for 16 hours. The mixture was concentrated to remove MeOH and THF, then added HCl (2 M) to adjust PH=3~4, and then extracted with ethyl acetate (10 mL × 3). The combined organic layer was washed with saturated brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(4-ethyl-1-methyl-1H-pyrazol-5- yl)-5-fluorobenzoic acid (200 mg). 1H NMR (400MHz, CDCl3) δ 8.52 (br, 1H), 7.92-7.85 (m, 2H), 7.53 (s, 1H), 7.30-7.25 (m, 1H), 3.84 (s, 3H), 2.43 (q, J = 7.6 Hz, 2H), 1.15 (t, J = 7.6 Hz, 3H). Step 3: (R)-Benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000099_0002
To a solution of 3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (200 mg, 0.81 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (353 mg, 0.97 mmol, HCl salt) in DMF (5 mL) was added TBTU (388 mg, 1.21 mmol) and DIPEA (312 mg, 2.42 mmol). The mixture was stirred at 20 °C for 16 hours. The mixture was added water (5 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with saturated brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 40%) twice to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-methyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (270 mg). 1H NMR (400MHz, CDCl3) δ 7.49-7.42 (m, 3H), 7.38-7.28 (m, 10H), 7.17-7.12 (m, 1H), 7.11- 7.06 (m, 1H), 5.99-5.97 (m, 1H), 5.21 (s, 2H), 5.10 (s, 2H), 4.63-4.56 (m, 1H), 3.99-3.78 (m, 2H), 3.73 (s, 3H), 2.40 (q, J = 7.6 Hz, 2H), 1.12 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-Amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000100_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-ethyl-1-methyl-1H-pyrazol- 5-yl)-5-fluorobenzamido)propanoate (270 mg, 0.48 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50°C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)- 5-fluorobenzamido)propanoic acid (60 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.23-9.19 (m, 1H), 8.65 (br, 3H), 7.86-7.83 (m, 1H), 7.80 (s, 1H), 7.54-7.50 (m, 1H), 7.46 (s, 1H), 4.17-4.07 (m, 1H), 3.84-3.78 (m, 2H), 3.73 (s, 3H), 2.36 (q, J = 7.6 Hz, 2H), 1.05 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 335.0, tR (min, Method c) = 0.99. [α]20 ,D = 14 (c = 1 mg/mL,CH3OH). Compound 2h Methyl (R)-2-amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000101_0001
To a stirred solution of (R)-2-amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid (20 mg, 0.06 mmol) in methanol (0.5 mL), at 0 °C, was added, thionyl chloride (22.0 μl, 0.30 mmol) dropwise. The reaction mixture was warmed to room temperature and stirred for 40 hours. The mixture was concentrated to methyl (R)-2- amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (11 mg) as HCl salt. 1H NMR (600 MHz, DMSO) δ 9.08-9.02 (m, 1H), 8.60 (br s, 3H), 7.82-7.75 (m, 1H), 7.74-7.72 (m, 1H), 7.57-7.51 (m, 1H), 7.43 (s, 1H), 4.26-4.18 (m, 1H), 3.80 – 3.75 (m, 2H), 3.73 (s, 3H), 3.72 (s, 3H), 2.37 (q, J = 7.6 Hz, 2H), 1.06 (t, J = 7.5 Hz, 3H). LCMS (MH+): m/z = 349.3, tR (min, Method c) = 0.40 min. Compound 1i (R)-2-amino-3-(3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-(5-ethyl-1-methyl-1H- pyrazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000101_0002
Step 1: 4-Bromo-5-ethyl-1-methyl-1H-pyrazole and 4-bromo-3-ethyl-1-methyl-1H-pyrazole
Figure imgf000102_0001
To a solution of 4-bromo-5-ethyl-1H-pyrazole (300 mg, 1.71 mmol) in DMF (5 mL) were added NaH (82 mg, 2.06 mmol, 60% in mineral oil) at 0°C. The reaction mixture was stirred at 0°C for 30 minutes. CH3I (268 mg, 1.89 mmol) was added to the reaction mixture and stirred at 20°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give a mixture of 4-bromo-5-ethyl- 1-methyl-1H-pyrazole and 4-bromo-3-ethyl-1-methyl-1H-pyrazole (300 mg, crude). Step 2: Methyl 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoate and methyl 3-(3-ethyl- 1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoate
Figure imgf000102_0002
To a solution of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (300 mg, 1.52 mmol) in dioxane (10 mL) were added a mixture of 4-bromo-5-ethyl-1-methyl-1H-pyrazole and 4- bromo-3-ethyl-1-methyl-1H-pyrazole (300 mg, crude). The reaction mixture was stirred at 100°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 30% to 40%) to give the mixture of two isomers (200 mg), which were separated by SFC separation to give methyl 3- (3-ethyl-1-methyl-pyrazol-4-yl)-5-fluoro-benzoate (70 mg), and methyl 3-(5-ethyl-1-methyl- pyrazol-4-yl)-5-fluoro-benzoate (90 mg). Methyl 3-(3-ethyl-1-methyl-pyrazol-4-yl)-5-fluoro-benzoate: 1H NMR (400 MHz, CDCl3) δ 7.84 (t, J=1.2 Hz, 1H), 7.61 - 7.56 (m, 1H), 7.47 (s, 1H), 7.27 - 7.24 (m, 1H), 3.94 (s, 3H), 3.90 (s, 3H), 2.80 (q, J=7.6 Hz, 2H), 1.26 (t, J=7.6 Hz, 3H) Methyl 3-(5-ethyl-1-methyl-pyrazol-4-yl)-5-fluoro-benzoate: 1H NMR (400 MHz, CDCl3) δ 7.84 (t, J=1.2 Hz, 1H), 7.62-7.59 (m, 1H), 7.58 (s, 1H), 7.26 - 7.22 (m, 1H), 3.94 (s, 3H), 3.89 (s, 3H), 2.80 (q, J=7.6 Hz, 2H), 1.26 (t, J=7.6 Hz, 3H). Step 3: 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoic acid
Figure imgf000103_0001
To a solution of methyl 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoate (90 mg, 0.34 mmol) in a mixture of MeOH (3 mL) and H2O (2 mL) were added LiOH.H2O (43 mg, 1.03 mmol). The reaction mixture was stirred at 25°C for 1 hour. The MeOH was removed. The residue was added 1M HCl to adjust pH =3. The reaction mixture was extracted with ethyl acetate (10 mL × 3), and the combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5- fluorobenzoic acid (70 mg). LC-MS (MH+): m/z = 249.1 tR (min, Method G) = 0.706 Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000103_0002
To a solution of 3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzoic acid (70 mg, 0.28 mmol) in DMF (5 mL) were added (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (123 mg, 0.34 mmol, HCl salt), TBTU (136 mg, 0.44 mmol) and DIPEA (109 mg, 0.85 mmol). The reaction mixture was stirred at 20°C for 3 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 60% to 70%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5- fluorobenzamido)propanoate (100 mg). LC-MS (MH+): m/z = 559.3 tR (min, Method G) = 0.882 Step 5: (R)-2-amino-3-(3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000104_0001
To a solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethyl-1-methyl-1H- pyrazol-4-yl)-5-fluorobenzamido)propanoate (100 mg, 0.18 mmol) in 30% HBr in AcOH (5 mL) were stirred at 50°C for 16 hours. The solvent was removed. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)- 5-fluorobenzamido)propanoic acid (65 mg) as 2HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.04 (br s, 1H), 8.51 (br, 2H), 7.77 (s, 1H), 7.70 (s, 1H), 7.60 - 7.54 (m, 1H), 7.39 - 7.33 (m, 1H), 4.11 - 4.04 (m, 1H), 3.81 (s, 3H), 3.79 - 3.69 (m, 2H), 2.81 (q, J = 7.6 Hz, 2H), 1.15 (t, J=7.6 Hz, 3H). LC-MS (MH+): m/z = 335.0 tR (min, Method c) = 0.967 [α]20 ,D = +4.0 (c = 1.0 mg/mL, CH3OH). Compound 2i Methyl (R)-2-amino-3-(3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000105_0001
(R)-2-amino-3-(3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5-fluorobenzamido)propanoic acid (30 mg, 0.090 mmol) was dissolved in 1.25 M hydrogen chloride-methanol solution (3.0 mL) and the reaction was stirred at room temperature for 17 h. The reaction mixture heated to 80 °C and stirred for additional 24 hours. The mixture was concentrated and then diluted with sat. aq. NaHCO3 (2 mL) and extracted with ethyl acetate (3x2mL). The combined organic phase was concentrated and then diluted with a methanolic HCl solution (1.0 mL, 1.25M) and stirred for 5 minutes. After concentration the resulting product was washed with THF to afford methyl (R)-2-amino-3-(3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5- fluorobenzamido)propanoate (23 mg) as HCl salt. 1H NMR (600 MHz, DMSO) δ 9.15-9.09 (m, 1H), 8.83-8.67 (m, 3H), 7.79-7.76 (m, 1H), 7.74 (s, 1H), 7.61 – 7.55 (m, 1H), 7.39 – 7.35 (m, 1H), 4.24-4.17 (m, 1H), 3.84 – 3.74 (m, 5H), 3.72 (s, 3H), 2.83 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.5 Hz, 3H). LCMS (MH+): m/z = 349.3, tR (min, Method c) = 0.38 min. Compound 1j (R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5- fluorobenzamido)propanoic acid is shown below
Figure imgf000105_0002
Step 1: methyl 3-(2-ethylpyridin-3-yl)-5-fluorobenzoate
Figure imgf000106_0001
To a solution of 3-bromo-2-ethyl-pyridine (200 mg, 1.07 mmol) and (3-fluoro-5- methoxycarbonyl-phenyl)boronic acid (213 mg, 1.07 mmol) in a mixture of dioxane (5 mL) and H2O (0.5 mL) was added Pd(dppf)Cl2 (79 mg, 0.11 mmol) and K2CO3 (446 mg, 3.22 mmol). The mixture was heated to 90°C and stirred for 16 hours. The reaction mixture was diluted with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layer was dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 20%) to give methyl 3-(2- ethylpyridin-3-yl)-5-fluorobenzoate (0.2 g). 1H NMR (400MHz, CDCl3) δ 8.61 (dd, J=1.2, 4.8 Hz, 1H), 7.81 (dd, J=1.2, 1.2 Hz, 1H), 7.78 - 7.73 (m, 1H), 7.50 (dd, J=1.6, 7.6 Hz, 1H), 7.26 - 7.18 (m, 2H), 3.95 (s, 3H), 2.76 (q, J=7.6 Hz, 2H), 1.21 (t, J=7.6 Hz, 3H). Step 2: 3-(2-ethylpyridin-3-yl)-5-fluorobenzoic acid
Figure imgf000106_0002
To a solution of methyl 3-(2-ethylpyridin-3-yl)-5-fluorobenzoate (200 mg, 0.77 mmol) in a mixture of H2O (2.5 mL) and MeOH (2.5 mL) was added LiOH.H2O (97 mg, 2.31 mmol). The mixture was stirred at 25°C for 3 hours. The mixture was concentrated, diluted with water (5 mL) then acidified with aq. HCl (2M) to pH=4-5, the mixture was concentrated, the residue was washed with ethyl acetate/MeOH(10 mL, 5:1), filtered, the filtrate was concentrated to give 3-(2-ethylpyridin-3-yl)-5-fluorobenzoic acid (300 mg, crude). LC-MS (MH+): m/z = 246.1 tR (min, method c) = 0.505. Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylpyridin-3-yl)-5- fluorobenzamido)propanoate
Figure imgf000107_0001
To a solution of 3-(2-ethylpyridin-3-yl)-5-fluorobenzoic acid (280 mg, crude) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (262 mg, 0.72 mmol, HCl) in DMF (5 mL) was added HATU (410 mg, 1.08 mmol) and DIPEA (279 mg, 2.16 mmol). The mixture was stirred at 25°C for 16 hours. The reaction mixture was diluted with water (10 mL), extracted with ethyl acetate (10 mL × 3), the organic layers were washed with brine, dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (Petroleum ether /ethyl acetate with ethyl acetate from 10 to 50%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(2-ethylpyridin-3-yl)-5-fluorobenzamido)propanoate (100 mg). 1H NMR (400MHz, CDCl3) δ 8.62 (dd, J=1.6, 4.8 Hz, 1H), 7.50-7.30 (m, 13H), 7.28-7.18 (m, 2H), 6.83 (br, 1H), 5.92 (br d, J=5.6 Hz, 1H), 5.22 (s, 2H), 5.10 (s, 2H), 4.64 - 4.56 (m, 1H), 3.98 - 3.92 (m, 1H), 3.88 - 3.79 (m, 1H), 2.76 (q, J=7.2 Hz, 2H), 1.22 (t, J=7.2 Hz, 3H). Step 4: (R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000107_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethylpyridin-3-yl)-5- fluorobenzamido)propanoate (100 mg, 0.18 mmol) in 30% HBr/AcOH (3 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated. The residue was purified by Preparative HPLC (HCl condiiton) to give (R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5- fluorobenzamido)propanoic acid (40 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.32-9.25 (m, 1H), 8.85-8.80 (m, 1H), 8.66 (br s, 3H), 8.43-8.37 (m, 1H), 7.98-7.88 (m, 3H), 7.68-7.61 (m, 1H), 4.15-4.13 (m, 1H), 3.84-3.79 (m, 2H), 3.00 (q, J=7.6 Hz, 2H), 1.16 (t, J=7.6 Hz, 3H). LC-MS (MH+): m/z = 332.0 tR (min, Method D) = 1.076. [α]20,D = +3.3 (c = 1.8 mg/mL, CH3OH). Compound 2j Methyl (R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5-fluorobenzamido)propanoate
Figure imgf000108_0001
To a stirred solution of (R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5-fluorobenzamido)propanoic acid (20 mg, 0.060 mmol) in methanol (0.5 mL), at 0 °C, was added, thionyl chloride (44 μl, 0.604 mmol) dropwise. The reaction mixture was warmed to room temperature and stirred over the weekend (72 hours). Concentration and wash with THF afforded methyl (R)-2- amino-3-(3-(2-ethylpyridin-3-yl)-5-fluorobenzamido)propanoate (17 mg) as HCl salt. 1H NMR (600 MHz, DMSO) δ 9.29-9.22 (m, 1H), 8.83 – 8.70 (m, 4H), 8.30 (br s, 1H), 7.91 – 7.82 (m, 3H), 7.65-7.60 (m, 1H), 4.25-4.18 (m, 1H), 3.85 – 3.76 (m, 2H), 3.71 (s, 3H), 2.96 (q, J = 7.6 Hz, 2H), 1.16 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 346.3, tR (min, Method D) = 0.32 min. Compound 1k (R)-2-amino-3-(3-ethyl-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-ethyl-5- fluorobenzamido)propanoic acid
Figure imgf000109_0001
Step 1: methyl 3-fluoro-5-vinylbenzoate
Figure imgf000109_0002
To a solution of methyl 3-bromo-5-fluorobenzoate (400 mg, 1.72 mmol) and 4,4,5,5- tetramethyl-2-vinyl-1,3,2-dioxaborolane (318 mg, 2.06 mmol) in dioxane (10 mL) and H2O (1 mL) was added Pd(dppf)Cl2 (126 mg, 0.17 mmol) and K2CO3 (713 mg, 5.16 mmol) under N2 atmosphere. The mixture was stirred at 100°C for 16 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (15 mL × 3), the combined organic layers were concentrated under reduced pressure. The residue was purified by Combi Flash (petroleum ether: ethyl acetate with ethyl acetate from 0 to 15%) to give methyl 3-fluoro-5- vinylbenzoate (150 mg). 1H NMR (400 MHz, CDCl3) δ 7.87 (s, 1H), 7.64-7.56 (m, 1H), 7.33-7.27 (m, 1H), 6.71 (dd, J = 17.6, 11.2 Hz, 1H), 5.84 (d, J = 17.6 Hz, 1H), 5.39 (d, J = 11.2 Hz, 1H), 3.94 (s, 3H). Step 2: methyl 3-ethyl-5-fluorobenzoate
Figure imgf000109_0003
To a solution of methyl 3-fluoro-5-vinylbenzoate (140 mg, 0.78 mmol) in MeOH (8 mL) was added Pd-C (10% w.t, 50% water, 80 mg) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (balloon) at 20°C for 3 hours. The mixture was filtered and the filtrate was concentrated under reduced pressure to give methyl 3-ethyl-5-fluorobenzoate (130 mg). The product was used next step directly. Step 3: 3-ethyl-5-fluorobenzoic acid
Figure imgf000110_0001
To a solution of methyl 3-ethyl-5-fluorobenzoate (130 mg, 0.71 mmol) in MeOH (10 mL) was added LiOH.H2O (90 mg, 2.14 mmol) in H2O (3 mL). The mixture was stirred at 15°C for 16 hours. The mixture was concentrated under reduced pressure. The residue was dissolved in water (5 mL) and washed with ethyl acetate (6 mL), and the organic layer was discarded. The aqueous layers was acidified with 2N HCl to adjust pH to 2 and extracted with DCM (10 mL × 3). The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to give 3-ethyl-5-fluoro-benzoic acid (100 mg). 1H NMR (400 MHz, CD3OD) δ 7.70 (s, 1H), 7.52-7.47 (m, 1H), 7.23-7.18 (m, 1H), 2.72 (q, J = 7.6 Hz, 2H), 1.26 (t, J=7.6 Hz, 3H). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate
Figure imgf000110_0002
To a solution of 3-ethyl-5-fluoro-benzoic acid (100 mg, 0.59 mmol) in DMF (6 mL) was added TBTU (286 mg, 0.89 mmol), DIPEA (231 mg, 1.78 mmol) and (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (217 mg, 0.59 mmol, HCl salt). The mixture was stirred at 20°C for 16 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with water (20 mL) and brine (20 mL) and concentrated under reduced pressure. The residue was purified by Combi Flash (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino) -3-(3-ethyl-5-fluorobenzamido)propanoate (220 mg).1H NMR (400 MHz, CDCl3) δ 7.38-7.30 (m, 11H), 7.15 (d, J = 9.2 Hz, 1H), 7.05 (d, J = 9.2 Hz, 1H), 6.63 (br s, 1H), 5.94 (d, J = 6.8 Hz, 1H), 5.22 (s, 2H), 5.13 (s, 2H), 4.63-4.57 (m, 1H), 3.96-3.78 (m, 2H), 2.68 (q, J=7.6 Hz, 2H), 1.25 (t, J=7.6 Hz, 3H). Step 5: (R)-2-amino-3-(3-ethyl-5-fluorobenzamido)propanoic acid
Figure imgf000111_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (100 mg, 0.21 mmol) in 33% HBr in AcOH (5 mL) was stirred at 50°C for 16 hours. The mixture was concentrated under reduced pressure. The residue was purified by Preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-ethyl-5- fluorobenzamido)propanoic acid (30 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 8.96-8.90 (m, 1H), 8.49 (br s, 3H), 7.63 (s, 1H), 7.51 (d, J = 9.6 Hz, 1H), 7.28 (d, J = 9.6 Hz, 1H), 4.12-4.06 (m, 1H), 3.90-3.64 (m, 2H), 2.67 (q, J = 7.6 Hz, 2H), 1.20 (t, J=7.6 Hz, 3H). LC-MS (MH+): m/z = 255.0 tR (min, Method D) = 1.641 [α]20,D = 6.0, (c = 1 mg/mL, MeOH). Compound 2k (R)-methyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido) propanoate
Figure imgf000112_0001
Figure imgf000112_0003
To a solution of 3-ethyl-5-fluoro-benzoic acid (850 mg, 5.05 mmol) in DMF (10 mL) were added (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (1.61 g, 5.56 mmol, HCl salt), TBTU (2.43 g, 7.58 mmol) and DIPEA (1.96 g, 15.16 mmol). The reaction mixture was stirred at 15 °C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with brine (3 × 20 mL), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 40% to 50%) to give (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluorobenzamido) propanoate (1.5 g). LC-MS (MH+): m/z = 403.1 tR (min, Method G) = 0.930
Figure imgf000112_0002
To a solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluorobenzamido) propanoate (1.5 g, 3.73 mmol) in HBr in AcOH (15 mL, 30% purity) was stirred at 10°C for 16 hours. The solvent was removed. The residue was washed with MeOH/methyl tert-butyl ether=1/10 (30 mL). The reaction mixture was filtered, the filtration cake was washed with methyl tert-butyl ether (2 ml) and the filtration cake was collected to give (R)-methyl 2- amino-3- (3-ethyl-5-fluorobenzamido)propanoate (1.0 g) as HBr salt. 1H NMR (400 MHz, DMSO-d6) δ 8.78 (s, 1H), 8.42 (br s, 3H), 7.54 (s, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.27 (d, J = 9.6 Hz, 1H), 4.20-4.15 (m, 1H), 3.77-3.63 (m, 5H), 2.65 (q, J=7.6 Hz, 2H), 1.18 (t, J=7.6 Hz, 3H). LCMS (MH+): m/z = 269.1, tR (min, Method C) = 1.756 min [α]20,D = +2.0 (c = 1.0 mg/mL, CH3OH). Compound 2l (R)-isobutyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate Step 1: Preparation of (R)-isobutyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate
Figure imgf000113_0001
To a solution of 3-ethyl-5-fluoro-benzoic acid (500 mg, 2.97 mmol) in DMF (10 mL) were added (R)-isobutyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (1.08 g, 3.27 mmol, HCl salt), TBTU (1.43 g, 4.46 mmol) and DIPEA (1.15 g, 8.92 mmol). The reaction mixture was stirred at 20°C for 16 hours. The reaction mixture was quenched with water (30 mL), extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 10% to 20%) to give (R)-isobutyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (1 g). 1HNMR (400MHz, CDCl3) δ 7.37 (s, 1H), 7.33-7.29 (m, 5H), 7.27-7.23 (m, 1H), 7.02 (d, J = 9.2 Hz, 1H), 6.95-6.90 (m, 1H), 5.93 (d, J = 6.8 Hz, 1H), 5.10 (s, 2H), 4.60-4.50 (m, 1H), 3.99-3.91 (m, 2H), 3.90-3.75 (m, 2H), 2.66 (q, J = 7.6 Hz, 2H), 2.00-1.90 (m, 1H), 0.91 (d, J = 6.4 Hz, 6H). Step 2:
Figure imgf000113_0002
A solution of (R)-isobutyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (1 g, 2.25 mmol) in 30% HBr in AcOH (15 mL) was stirred at 20°C for 16 hours. The solvent was removed. The residue was added a mixture of AcOH (3 mL) and methyl tert-butyl ether (30 ml). The reaction mixture was filtered, the filtration cake was washed with methyl tert-butyl ether (2 ml) and collected to give (R)-isobutyl 2-amino-3- (3-ethyl-5-fluorobenzamido)propanoate (750 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.81-8.77 (m, 1H), 8.44 (br s, 3H), 7.56 (s, 1H), 7.48-7.39 (m, 1H), 7.30-7.23 (m, 1H), 4.22-4.17 (m, 1H), 3.96-3.86 (m, 2H), 3.72-3.68 (m, 2H), 2.65 (q, J = 7.6 Hz, 2H), 1.91-1.75 (m, 1H), 1.18 (t, J = 7.6 Hz, 3H), 0.84-0.82 (m, 6H). LCMS (MH+): m/z = 311.0, tR (min, Method D) = 1.895 min [α]20,D = +2.0(c =5 mg/mL, CH3OH). Compound 2m (R)-isopropyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate Step 1: Preparation of (R)-isopropyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate
Figure imgf000114_0001
A mixture of 3-ethyl-5-fluoro-benzoic acid (500 mg, 2.97 mmol), (R)-isopropyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate(1 g, 3.16 mmol, HCl salt), TBTU (1.43 g, 4.46 mmol) and DIPEA (1.92 g, 14.87 mmol) in DMF (10 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 25°C for 10 hours under N2 atmosphere. The reaction mixture was diluted with H2O (15 mL) and extracted ethyl acetate (30 mL). The organic layer was concentrated under reduced pressure to give a residue. The residue was purified by Preparative HPLC (HCl condition) to give (R)-isopropyl 2-(((benzyloxy)carbonyl)amino)-3-(3- ethyl-5-fluorobenzamido)propanoate (640 mg). LCMS (MH+): m/z = 431.1, tR (min, Method G) = 0.987 min Step 2:
Figure imgf000115_0001
A mixture of (R)-isopropyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (500 mg, 1.16 mmol) and 30% HBr in AcOH (10 mL) was stirred at 20°C for 10 hours under N2 atmosphere. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was washed by DCM (5 mL) to give (R)- isopropyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate (360 mg) as HBr salt. 1H NMR (400 MHz, DMSO-d6) δ 8.76 (br s, 1H), 8.36 (br s, 3H), 7.55 (s, 1H), 7.45 (d, J = 9.6 Hz, 1H), 7.30 (d, J = 9.6 Hz, 1H), 5.02-4.93 (m, 1H), 4.16-4.13 (m, 1H), 3.74-3.65 (m, 2H), 2.71- 2.63 (m, 2H), 1.25-1.15 (m, 9H); LCMS (MH+): m/z = 297.0, tR (min, Method D) = 1.815 min [α]20,D = 4 (c = 1 mg/mL, CH3OH). Compound 2n (R)-(S)-1-aminopropan-2-yl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate The overall synthesis scheme for the preparation (R)-(S)-1-aminopropan-2-yl 2-amino-3-(3- ethyl-5-fluorobenzamido)propanoate
Figure imgf000115_0002
Step 1: Preparation of (S)-1-azidopropan-2-ol
Figure imgf000115_0003
To a solution of (S)-2-methyloxirane (3 g, 51.65 mmol) in a mixture of EtOH (30 mL) and H2O (7 mL) was added NaN3 (4.37 g, 67.15 mmol) and NH4Cl (3.59 g, 67.15 mmol). The mixture was stirred at 80 °C for 16 hours. The mixture was concentrated. The residue was dissolved in water (30 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, filtered and concentrated to give (S)-1- azidopropan-2-ol (3.5 g). 1H NMR (400 MHz, CDCl3) δ 3.99-3.90 (m, 1H), 3.36 - 3.28 (m, 1H), 3.25 - 3.17 (m, 1H), 2.55 (br s, 1H), 1.20 (d, J = 6.4 Hz, 3H), [α]20,D = 12.0, (c = 2.0 mg/mL, MeOH). Step 2: Preparation of (R)-(S)-1-azidopropan-2-yl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl- 5-fluorobenzamido)propanoate
Figure imgf000116_0001
To a solution of (R)-2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluorobenzamido)propanoic acid (1.7 g, 4.38 mmol) in DCM (30 mL) was added DMAP (160 mg, 1.31 mmol), EDCI (1.68 g, 8.75 mmol) and DIPEA (1.13 g, 8.75 mmol) at 0 °C and stirred at for 30 min. Then (S)-1- azidopropan-2-ol (531 mg, 5.25 mmol) was added and the mixture was stirred at 25 °C for 16 hours. The mixture was concentrated. The residue was added water (30 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 50%) to give 1.2 g of crude product.1 g of the crude product was further purified by preprative-HPLC (free base) to give 590 mg product.590 mg of the product was further purified by SFC (Instrument: Berger, MULTIGR AM-II; Column: Chiralpak AD 250 × 30 mm I.D. 5 μm; Mobile phase: supercritical CO2/EtOH (0.1% NH3`H2O, v%) = 70/30; Flow Rate: 80 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give (R)-(S)-1-azidopropan-2-yl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (430 mg).1H NMR (400 MHz, CDCl3) δ 7.33 - 7.27 (m, 5H), 7.25 - 7.17 (m, 2H), 6.99 (d, J = 9.6 Hz, 1H), 6.80 (br s, 1H), 6.06 - 5.82 (m, 1H), 5.87 (br d, J = 6.4 Hz, 1H), 5.12 - 5.01 (m, 3H), 4.53-4.45 (m, 1H), 3.96 - 3.74 (m, 2H), 3.40 - 3.21 (m, 2H), 2.63 (q, J = 7.6 Hz, 2H), 1.28 - 1.23 (m, 3H), 1.22 - 1.17 (m, 3H). Step 3:
Figure imgf000117_0001
To a solution of (R)-(S)-1-azidopropan-2-yl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (430 mg, 0.91 mmol) in MeOH (10 mL) was added Pd/C (150 mg, 10% Pd, 50% water) and HCl/MeOH (4 M, 1 mL) under N2. The suspension was degassed under vacuum and purged with H2 several times. The mixture was stirred under H2 (15 psi) at 25 °C for 1.5 hour. The mixture was filtered and the filtrate was concentrated. The residue was purified by prep-HPLC (HCl) to give (R)-(S)-1-aminopropan-2-yl 2-amino-3-(3-ethyl-5- fluorobenzamido)propanoate (150 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.09-0.04 (m, 1H), 8.88-8.85 (m, 3H), 8.38 (br s, 3H), 7.66 (s, 1H), 7.54 (d, J = 9.2 Hz, 1H), 7.27 (d, J = 9.2 Hz, 1H), 5.14 - 5.04 (m, 1H), 4.30 - 4.20 (m, 1H), 3.90 - 3.82 (m, 1H), 3.81 - 3.73 (m, 1H), 3.14-3.05 (m, 1H), 3.02 - 2.91 (m, 1H), 2.69-2.50 (m, 2H), 1.23 - 1.18 (m, 6H). LCMS (MH+): m/z = 312.0, tR (min, Method D) = 1.367 min [α]20,D = 12.2, (c = 1.8 mg/mL, MeOH). Compound 2o (R)-isopentyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate Step 1:
Figure imgf000118_0001
To a solution of 3-ethyl-5-fluorobenzoic acid (200 mg, 1.19 mmol) and (R)-isopentyl 3-amino- 2-(((benzyloxy)carbonyl)amino)propanoate (492 mg, 1.43 mmol, HCl salt) in DMF (10 mL) was added TBTU (573 mg, 1.78 mmol) and DIPEA (461 mg, 3.57 mmol). The mixture was stirred at 20 °C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 20%) to give (R)-isopentyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluorobenzamido)propanoate (400 mg). 1H NMR (400MHz, CDCl3) δ 7.38 (s, 1H), 7.36 - 7.22 (m, 6H), 7.04 (d, J = 9.2 Hz, 1H), 6.94 (br, 1H), 5.93 (d, J = 6.4 Hz, 1H), 5.17 - 5.05 (m, 2H), 4.60 - 4.48 (m, 1H), 4.28 - 4.16 (m, 2H), 3.95 - 3.76 (m, 2H), 2.68 (q, J = 7.6 Hz, 2H), 1.69 - 1.60 (m, 1H), 1.58 - 1.50 (m, 2H), 1.25 (t, J = 7.6 Hz, 3H), 0.91 (d, J = 6.8 Hz, 6H). Step 2:
Figure imgf000118_0002
A solution of (R)-isopentyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (400 mg, 0.872 mmol) in 30% HBr in AcOH (4 mL) was stirred at 20°C for 2 hours. The mixture was concentrated, then ethyl acetate (5 mL) was added and the ethyl acetate phase was extracted with water (10 mL × 2), and the water phase was lyophilized to give (R)-isopentyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate (270 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.83-8.80 (m, 1H), 8.46 (br s, 3H), 7.58 (s, 1H), 7.47 (d, J = 9.6 Hz, 1H), 7.29 (d, J = 9.6 Hz, 1H), 4.24 - 4.07 (m, 3H), 3.77 - 3.66 (m, 2H), 2.67 (q, J = 7.6 Hz, 2H), 1.65 - 1.52 (m, 1H), 1.41 (q, J = 7.2 Hz, 2H), 1.20 (t, J = 7.6 Hz, 3H), 0.79 (t, J = 7.2 Hz, 6H). LCMS (MH+): m/z = 325.1, tR (min, Method D) = 1.955 min [α]20,D = -8 (c = 3 mg/mL, CHCl3). Compound 2p (R)-2-methoxyethyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate Step 1:
Figure imgf000119_0001
To a solution of 3-ethyl-5-fluorobenzoic acid (400 mg, 2.38 mmol) and (R)-2-methoxyethyl 3- amino-2-(((benzyloxy)carbonyl)amino)propanoate (1.19 g, 3.57 mmol, HCl salt) in DMF (13 mL) was added TBTU (1.15 g, 3.57 mmol) and DIPEA (922 mg, 7.14 mmol). The mixture was stirred at 20 °C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 40%) twice to give (R)-2-methoxyethyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (600 mg). 1H NMR (400MHz, CDCl3) δ 7.40 (s, 1H), 7.37 - 7.24 (m, 6H), 7.13 - 7.00 (m, 2H), 6.11 (d, J = 6.8 Hz, 1H), 5.18 - 5.07 (m, 2H), 4.65 - 4.45 (m, 2H), 4.27-4.18 (m, 1H), 4.04 - 3.94 (m, 1H), 3.85 - 3.74 (m, 1H), 3.69 - 3.56 (m, 2H), 3.33 (s, 3H), 2.68 (q, J = 7.6 Hz, 2H), 1.225 (t, J = 7.6 Hz, 3H). Step 2:
Figure imgf000120_0001
A mixture of (R)-2-methoxyethyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (550 mg, 1.23 mmol) and Pd/C (100 mg, 10% Pd, 50% water) in ethyl acetate (10 mL) was degassed and purged with H23 times, and then the mixture was stirred at 20 °C for 16 hours under H2 (15 PSI) atmosphere. The reaction mixture was filtered through celite and the filtrate was concentrated. The residue was purified by SFC (Instrument: Berger, MULTIGR AM-II; Column: DAICEL CHIRALPAK AD- H(250mm*30mm,5um),Mobile phase: supercritical CO2/IPE (0.1% NH3`H2O, v%) = 80/20; Flow Rate: 50 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm). The obtained material was dissolved in ethyl acetate (5 mL), washed with NaHCO3 (10 mL × 3), then extracted with HCl (0.5 M, 10 mL × 3) and the water phase was lyophilized to give (R)-2-methoxyethyl 2-amino-3-(3-ethyl-5- fluorobenzamido)propanoate (150 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.01 (br s, 1H), 8.73 (br s, 3H), 7.64 (s, 1H), 7.53 (d, J = 9.6 Hz, 1H), 7.27 (d, J = 9.6 Hz, 1H), 4.31 - 4.15 (m, 3H), 3.86 - 3.68 (m, 2H), 3.56 - 3.43 (m, 2H), 3.17 (s, 3H), 2.67 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 313.0, tR (min, Method D) = 1.714 min [α]20,D = 5.6 (c = 5 mg/mL, CH3OH). Compound 2q (R)-2-morpholinoethyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate Step 1: Preparation of (R)-2-morpholinoethyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate
Figure imgf000121_0001
To a solution of (R)-2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluorobenzamido)propanoic acid (1 g, 2.57 mmol) and 2-morpholinoethanol (507 mg, 3.86 mmol) in DMF (15 mL) was added HATU (1.47 g, 3.86 mmol) and DIPEA (998 mg, 7.72 mmol). The mixture was stirred at 20 °C for 3 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel ( ethyl acetate/Petroleum ether with ethyl acetate from 0% to 100%) twice to give 800 mg product, which was further separated by SFC (Instrument: MG-II; Column: DAICEL CHIRALCEL OD-H(250mm*30mm,5um)Mobile phase: supercritical CO2/IPE (0.1% NH3`H2O, v%) =75/25; Flow Rate: 50 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm). The obtained material was dissolved in ethyl acetate (5 mL), washed with NaHCO3 (10 mL × 3) and concentrated to give (R)-2-morpholinoethyl 2- (((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluorobenzamido)propanoate (400 mg). 1H NMR (400MHz, CDCl3) δ 7.38 (s, 1H), 7.36 - 7.28 (m, 5H), 7.23 (d, J = 8.8 Hz, 1H), 7.10 - 6.99 (m, 2H), 5.99 (d, J = 6.4 Hz, 1H), 5.17 - 5.08 (m, 2H), 4.62 - 4.50 (m, 1H), 4.44 - 4.23 (m, 2H), 3.98 - 3.75 (m, 2H), 3.67 - 3.55 (m, 4H), 2.74 - 2.55 (m, 4H), , 2.50 - 2.39 (m, 4H), 1.25 (t, J = 7.6 Hz, 3H). Step 2:
Figure imgf000121_0002
A solution of (R)-2-morpholinoethyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5- fluorobenzamido)propanoate (400 mg, 0.798 mmol) in 30% HBr in AcOH (6 mL) was stirred at 20°C for 1 hour. The mixture was concentrated. Then AcOH:methyl tert-butyl ether = 1:2 (15 mL) was added and stirred at 20°C for 30 minutes. The sticky solid was collected and lyophilized to give (R)-2-morpholinoethyl 2-amino-3-(3-ethyl-5- fluorobenzamido)propanoate (280 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 10.20 (br s, 1H), 8.91-8.84 (m, 1H), 8.56 (br s, 3H), 7.62 (s, 1H), 7.51 (d, J = 8.8 Hz, 1H), 7.29 (d, J = 8.8 Hz, 1H), 4.63 - 4.45 (m, 2H), 4.36 - 4.25 (m, 1H), 4.02 - 3.69 (m, 6H), 3.60 - 3.51 (m, 4H), 3.29 - 3.11 (m, 2H), 2.68 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 368.1, tR (min, Method C) = 1.735 min [α]20,D = -2 (c = 6 mg/mL, DMSO). Compound 1l (R)-2-amino-3-(4-ethyl-3-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(4-ethyl-3- fluorobenzamido)propanoic acid is shown below
Figure imgf000122_0001
Step 1: methyl 4-ethyl-3-fluorobenzoate
Figure imgf000122_0002
To a mixture of methyl 4-bromo-3-fluorobenzoate (500 mg, 2.15 mmol) and triethylborane (6.44 mL, 1M in hexane) in THF (10 mL) was added caesium acetate (1.24 g, 6.44 mmol) and Pd(dppf)Cl2 (157 mg, 0.21 mmol), the mixture was stirred at 65 °C for 14 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 5%) to give methyl 4-ethyl-3-fluorobenzoate (350 mg). 1H NMR (400MHz, CDCl3) δ 7.75 (dd, J = 1.6, 8.0 Hz, 1H), 7.66 (dd, J = 1.6, 11.2 Hz, 1H), 7.29 - 7.24 (m, 1H), 3.91 (s, 3H), 2.72 (q, J=7.6 Hz, 2H), 1.25 (t, J=7.6 Hz, 3H). Step 2: 4-ethyl-3-fluorobenzoic acid
Figure imgf000123_0001
To a solution of methyl 4-ethyl-3-fluorobenzoate (350 mg, 1.92 mmol) in the mixture of MeOH (5 mL) and H2O (5 mL) was added LiOH.H2O (161 mg, 3.84 mmol), the mixture was stirred at 20°C for 3 hours. The mixture was concentrated. The residue was added water (30 mL), then drop-wise added 2N aqueous HCl to pH=3~4 and extracted with ethyl acetate (30 mL × 3), the organic layers were dried over Na2SO4, filtered and concentrated to give 4-ethyl- 3-fluorobenzoic acid (300 mg). 1H NMR (400MHz, CDCl3) δ 7.84 (dd, J = 1.6, 8.0 Hz, 1H), 7.66 (dd, J = 1.6, 10.4 Hz, 1H), 7.32 (dd, J = 7.2, 8.0 Hz, 1H), 2.76 (q, J = 7.6 Hz, 2H), 1.27 (t, J = 7.6 Hz, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-ethyl-3- fluorobenzamido)propanoate
Figure imgf000123_0002
To a mixture of 4-ethyl-3-fluorobenzoic acid (120 mg, 0.71 mmol) and (R)-benzyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (312 mg, 0.86 mmol, HCl salt) in DMF (5 mL) was added TBTU (344 mg, 1.07 mmol) and DIPEA (277 mg, 2.14 mmol), the mixture was stirred at 20°C for 14 hours. The mixture was added water (50 mL) and extracted with ethyl acetate(40 mL × 3), the organic layers were washed with brine (40 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 20%) to give (R)- benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-ethyl-3-fluorobenzamido)propanoate (120 mg). 1H NMR (400MHz, CDCl3) δ 7.39 - 7.31 (m, 12H), 7.22 (t, J = 7.6 Hz, 1H), 6.65 (br s, 1H), 5.97 (br d, J = 6.8 Hz, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.64 - 4.54 (m, 1H), 3.95 - 3.77 (m, 2H), 2.71 (q, J = 7.6 Hz, 2H), 1.25 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(4-ethyl-3-fluorobenzamido)propanoic acid
Figure imgf000124_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(4-ethyl-3- fluorobenzamido)propanoate (120 mg, 0.25 mmol) in HBr in HOAc (4 mL, 30%) was stirred at 50 °C for 14 hours. The mixture was concentrated. The residue was purified by preparative- HPLC (Method AA) to give (R)-2-amino-3-(4-ethyl-3-fluorobenzamido)propanoic acid (50 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.98-8.90 (m, 1H), 8.49 (br s, 2H), 7.72-7.66 (m, 2H), 7.46-7.37 (m, 1H), 4.04-3.99 (m, 1H), 3.82-3.67 (m, 2H), 2.66 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 255.0 tR (min, Method D) = 1.531 [α]20,D = +6.0 (c = 2.0 mg/mL, CH3OH). Compound 2r (R)-methyl 2-amino-3-(4-ethyl-3-fluorobenzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-ethyl-3- fluorobenzamido)propanoate
Figure imgf000124_0002
To a mixture of 4-ethyl-3-fluorobenzoic acid (180 mg, 1.07 mmol) and (R)-methyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (324 mg, 1.28 mmol, HCl salt) in DMF (4 mL) was added TBTU (516 mg, 1.61 mmol) and DIPEA (415 mg, 3.21 mmol). The mixture was stirred at 15°C for 14 hours. The mixture was added water (50 mL) and extracted with ethyl acetate (40 mL × 3), the organic layers were washed with brine (40 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 30%) to give (R)-methyl 2- (((benzyloxy)carbonyl)amino)-3-(4-ethyl-3-fluorobenzamido)propanoate (350 mg). 1H NMR (400MHz, CDCl3) δ 7.46 - 7.40 (m, 2H), 7.37 - 7.31 (m, 5H), 7.26 - 7.22 (m, 1H), 6.81 (br s, 1H), 5.88 (br s, 1H), 5.17 - 5.08 (m, 2H), 4.60 - 4.50 (m, 1H), 3.95 - 3.75 (m, 5H), 2.71 (q, J = 7.6 Hz, 2H), 1.25 (t, J = 7.6 Hz, 3H). Step 2:
Figure imgf000125_0001
A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(4-ethyl-3- fluorobenzamido)propanoate (150 mg, 0.37 mmol) in HBr in HOAc (3 mL, 30%) was stirred at 15°C for 2 hours. The mixture was concentrated. The residue was added water (15 mL) and then lyophilized to give (R)-methyl 2-amino-3-(4-ethyl-3-fluorobenzamido)propanoate (92 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.77-8.72 (m, 1H), 8.41 (br s, 3H), 7.66 - 7.57 (m, 2H), 7.45- 7.41 (m, 1H), 4.22-4.16 (m, 1H), 3.78 - 3.65 (m, 5H), 2.67 (q, J = 7.6 Hz, 2H), 1.18 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 269.0, tR (min, Method D) = 1.644 min [α]20,D = +2.0 (c = 2.0 mg/mL, CH3OH). Compound 1m (R)-2-amino-3-(3-ethyl-5-fluoro-4-methylbenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoic acid is shown below
Figure imgf000126_0001
Step 1: methyl 3-ethyl-5-fluoro-4-methylbenzoate
Figure imgf000126_0002
A mixture of methyl 3-bromo-5-fluoro-4-methylbenzoate (300 mg, 1.21 mmol), triethylborane (1 M in hexane, 3.64 mL), Pd(dppf)Cl2 (89 mg, 0.12 mmol) and CsOAc (699 mg, 3.64 mmol) in THF (5 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 65 °C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 3-ethyl-5-fluoro-4-methylbenzoate (220 mg). 1H NMR (400MHz, CDCl3) δ 7.65 (s, 1H), 7.51 (d, J = 10.0 Hz, 1H), 3.91 (s, 3H), 2.68 (q, J = 7.6 Hz, 2H), 2.26 (s, 3H), 1.23 (t, J = 7.6 Hz, 3H). Step 2: 3-ethyl-5-fluoro-4-methylbenzoic acid
Figure imgf000127_0001
To a solution of methyl 3-ethyl-5-fluoro-4-methylbenzoate (220 mg, 1.12 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (141 mg, 3.36 mmol) and the mixture was stirred at 20°C for 16 hours, then the reaction mixture was stirred at 35°C for another 3 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-ethyl-5-fluoro-4-methylbenzoic acid (200 mg). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoate
Figure imgf000127_0002
To a solution of a mixture of 3-ethyl-5-fluoro-4-methylbenzoic acid (100 mg, 0.55 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (220 mg, 0.60 mmol, HCl salt) in DMF (5 mL) was added TBTU (264 mg, 0.82 mmol) and DIPEA (213 mg, 1.65 mmol). The reaction mixture was stirred at 20°C for 2 hours. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoate (230 mg). 1H NMR (400MHz, CDCl3) δ 7.40 - 7.30 (m, 11H), 7.14 (d, J = 9.6 Hz, 1H), 6.64 (br, 1H), 5.99 (br d, J = 7.2 Hz, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.65 - 4.55 (m, 1H), 3.95 - 3.65 (m, 2H), 2.65 (q, J = 7.6 Hz, 2H), 2.25 (s, 3H), 1.21 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-ethyl-5-fluoro-4-methylbenzamido)propanoic acid
Figure imgf000128_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoate (230 mg, 0.47 mmol) in the solution of 30% HBr in AcOH (10 mL) was heated at 50°C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoic acid (80 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.90-8.82 (m, 1H), 8.46 (br s, 2H), 7.59 (s, 1H), 7.55-7.49 (m, 1H), 4.04-4.01 (m, 1H), 3.81-3.70 (m, 2H), 2.66 (q, J = 7.6 Hz, 2H), 2.21 (s, 3H), 1.17 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 269.0 tR (min, Method c) = 1.096 [α]20,D = +6.0 (c = 2 mg/mL,CH3OH). Compound 2s (R)-methyl 2-amino-3-(3-ethyl-5-fluoro-4-methylbenzamido)propanoate Step 1: (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoate
Figure imgf000128_0002
To a solution of a mixture of 3-ethyl-5-fluoro-4-methylbenzoic acid (100 mg, 0.55 mmol) and (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (152 mg, 0.60 mmol, HCl salt) in DMF (5 mL) was added TBTU (264 mg, 0.82 mmol) and DIPEA (213 mg, 1.65 mmol). The reaction mixture was stirred at 20°C for 2 hours. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoate (120 mg). 1H NMR (400MHz, CDCl3) δ 7.39 - 7.29 (m, 6H), 7.24 (s, 1H), 6.78 (br, 1H), 5.91 (br d, J = 5.6 Hz, 1H), 5.18 - 5.05 (m, 2H), 4.60 - 4.52 (m, 1H), 3.93 - 3.75 (m, 5H), 2.66 (q, J = 7.6 Hz, 2H), 2.25 (s, 3H),), 1.21 (t, J = 7.6 Hz, 3H). Step 2:
Figure imgf000129_0001
A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methylbenzamido)propanoate (120 mg, 0.29 mmol) in the solution of 30% HBr in AcOH (5 mL) was heated at 30°C for 2 hours. The mixture was concentrated. The residue was diluted with MeOH (0.5 mL) and methyl tert-butyl ether (5 mL). The mixture was stirred at 15°C for 0.5 hours. The precipitate was filtered and washed with methyl tert-butyl ether (1 mL × 2) to give (R)-methyl 2-amino-3-(3-ethyl-5-fluoro-4-methylbenzamido)propanoate (70 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.73 (br s, 1H), 8.40 (br s, 3H), 7.52 (s, 1H), 7.45 (d, J = 10.4 Hz, 1H), 4.22 - 4.16 (m, 1H), 3.78 - 3.64 (m, 5H), 2.66 (q, J = 7.6 Hz, 2H), 2.21 (d, J = 1.6 Hz, 3H), 1.15 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 283.0, tR (min, Method D) = 1.757 min [α]20,D = +1.0 (c = 2 mg/mL,CH3OH). Compound 1n (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)- 5-fluorobenzamido)propanoic acid is shown below
Figure imgf000130_0001
Step 1: methyl 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoate
Figure imgf000130_0002
To a mixture of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (877 mg, 4.43 mmol), 4- bromo-5-ethyl-isoxazole (0.6 g, 3.41 mmol) and Na2CO3 (723 mg, 6.82 mmol) in a mixture of H2O (4 mL) and dioxane (16 mL) was added Pd(dppf)Cl2.CH2Cl2 (557 mg, 0.68 mmol) under N2. The mixture was purged with N2, sealed and heated at 120°C for 40 minutes in the microwave. The mixture was concentrated. The residue was diluted with H2O (20 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 5%) to give methyl 3-(5- ethylisoxazol-4-yl)-5-fluorobenzoate (100 mg). 1H NMR (400MHz, CDCl3) δ 8.36 (s, 1H), 7.85-7.81 (m, 1H), 7.71-7.63 (m, 1H), 7.28-7.20 (m, 1H), 3.94 (s, 3H), 2.95 (q, J = 7.6 Hz, 2H), 1.35 (t, J = 7.6 Hz, 3H). Step 2: 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoic acid
Figure imgf000131_0002
A mixture of methyl 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoate (0.125 g, 0.5 mmol) and LiOH.H2O (42 mg, 1.0 mmol) in a mixture of THF (2 mL) and H2O (2 mL) was stirred at 25°C for 0.5 hour. The mixture was diluted with H2O (20 mL). The aqueous phase was adjusted to pH=3~4 with sat. aq. KHSO4 at 0°C and extracted with ethyl acetate (10 mL × 2). The organic phase was dried and concentrated to give 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoic acid (0.11 g, crude). LC-MS (MH+): m/z = 236.0 tR (min, Method G) = 0.685 Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000131_0001
A mixture of 3-(5-ethylisoxazol-4-yl)-5-fluorobenzoic acid (0.11 g, crude), (R)-benzyl 3-amino- 2-(((benzyloxy)carbonyl)amino)propanoate (171 mg, 0.47 mmol, HCl salt), DIPEA (302 mg, 2.34 mmol) and TBTU (225 mg, 0.7 mmol) in DMF (5 mL) was stirred at 25°C for 16 hours. The mixture was poured into H2O (50 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)- 3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoate (0.12 g). 1H NMR (400MHz, CDCl3) δ 8.35 (s, 1H), 7.53 (s, 1H), 7.42-7.27 (m, 11H), 7.23-7.15 (m, 1H), 7.05 (br s, 1H), 5.97 (br s, 1H), 5.21 (s, 2H), 5.10 (s, 2H), 4.67-4.52 (m, 1H), 3.99-3.87 (m, 1H), 3.86-3.76 (m, 1H), 2.95 (q, J = 7.6 Hz, 2H), 1.35 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000132_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate (60 mg, 0.11 mmol) and HBr in AcOH (2 mL, 30% in AcOH) were stirred at 50°C for 16 hours. The mixture was concentrated. The residue was purified by Preparative-HPLC (HCl) to give (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoic acid (20 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.14 (br s, 1H), 8.98 (s, 1H), 8.52 (br s, 3H), 7.91 (s, 1H), 7.71- 7.64 (m, 1H), 7.60-7.54 (m, 1H), 4.13-4.06 (m, 1H), 3.84-3.72 (m, 2H), 3.02 (q, J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 322.1 tR (min, Method c) = 0.995 [α]20,D = 6.0 (c = 0.5 mg/mL, CH3OH). Compound 2t (R)-isobutyl 2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoate Step 1: (R)-isobutyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000132_0002
A mixture of 3-(5-ethylisoxazol-4-yl)-5-fluoro-benzoic acid (0.5 g, crude), (R)-isobutyl 3- amino-2-(((benzyloxy)carbonyl)amino)propanoate (703 mg, 2.13 mmol, HCl salt), TBTU (1.02 g, 3.19 mmol) and DIPEA (1.37 g, 10.63 mmol) in DMF (10 mL) was stirred at 30°C for 15 hours. The mixture was poured into H2O (50mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL), dried over anhydrous Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-isobutyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoate (0.8 g). 1H NMR (400MHz, CDCl3) δ 8.36 (s, 1H), 7.57 (s, 1H), 7.41 (d, J=8.6 Hz, 1H), 7.35 - 7.25 (m, 5H), 7.24 - 7.14 (m, 2H), 5.89 (d, J=6.0 Hz, 1H), 5.10 (s, 2H), 4.64-4.52 (m, 1H), 4.03 - 3.88 (m, 3H), 3.82 - 3.70 (m, 1H), 2.95 (q, J=7.6 Hz, 2H), 2.00-1.89 (m, 1H), 1.34 (t, J=7.6 Hz, 3H), 0.92 (d, J=6.8 Hz, 6H). Step 2:
Figure imgf000133_0001
A mixture of (R)-isobutyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)-5- fluorobenzamido)propanoate (0.8 g, 1.56 mmol) and 30% HBr/AcOH (10 mL) were stirred at 25°C for 16 hours. The mixture was concentrated. The residue was washed with methyl tert- butyl ether (5 mL × 2). The product was lyophilized to give (R)-isobutyl 2-amino-3-(3-(5- ethylisoxazol-4-yl)-5-fluorobenzamido)propanoate (0.35 g) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.99 - 8.91 (m, 2H), 8.47 (br s, 3H), 7.83 (s, 1H), 7.70 - 7.59 (m, 2H), 4.29-4.23 (m, 1H), 4.02 - 3.88 (m, 2H), 3.82 - 3.72 (m, 2H), 3.03 (q, J=7.6 Hz, 2H), 1.94- 1.82 (m, 1H), 1.27 (t, J=7.6 Hz, 3H), 0.89-0.83 (m, 6H). LCMS (MH+): m/z = 378.0, tR (min, Method D) = 2.045 min [α]20,D = 0.67 (c = 1.5 mg/mL, CH3OH). Compound 1o (R)-2-amino-3-(3-ethyl-4-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-ethyl-4- fluorobenzamido)propanoic acid is shown below
Figure imgf000134_0001
A mixture of methyl 3-bromo-4-fluorobenzoate (1 g, 4.29 mmol), triethylborane (12.9 mL, 1M in hexane), Pd(dppf)Cl2 (314 mg, 0.43 mmol) and CsOAc (2.47 g, 12.87 mmol) in THF (10 mL) was degassed and purged with N23 times, and then the mixture was stirred at 65 °C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 3-ethyl-4-fluorobenzoate (510 mg). 1H NMR (400MHz, CDCl3) δ 7.95 - 7.85 (m, 2H), 7.08 - 7.02 (m, 1H), 3.91 (s, 3H), 2.70 (q, J = 7.6 Hz, 2H), 1.26 (t, J = 7.6 Hz, 3H). Step 2: 3-ethyl-4-fluorobenzoic acid
Figure imgf000135_0001
To a solution of methyl 3-ethyl-4-fluorobenzoate (200 mg, 1.10 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (138 mg, 3.29 mmol) and the mixture was stirred at 20°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-ethyl-4-fluorobenzoic acid (160 mg). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-4- fluorobenzamido)propanoate
Figure imgf000135_0002
To a solution of a mixture of 3-ethyl-4-fluorobenzoic acid (160 mg, 0.95 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (382 mg, 1.05 mmol, HCl salt) in DMF (5 mL) was added TBTU (458 mg, 1.43 mmol) and DIPEA (369 mg, 2.85 mmol). The reaction mixture was stirred at 20°C for 16 hours. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-4-fluorobenzamido)propanoate (400 mg). 1H NMR (400MHz, DMSO-d6) δ 7.61 (d, J = 6.0 Hz, 1H), 7.44 - 7.29 (m, 11H), 7.03 - 6.96 (m, 1H), 6.65 (br s, 1H), 5.99 (br d, J = 6.8 Hz, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.63 - 4.56 (m, 1H), 3.94 - 3.88 (m, 2H), 2.66 (q, J = 7.6 Hz, 2H), 1.22 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-ethyl-4-fluorobenzamido)propanoic acid
Figure imgf000136_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-4- fluorobenzamido)propanoate (350 mg, 0.73 mmol) in the solution of 30% HBr in AcOH (10 mL) was heated at 50°C for 15 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-ethyl-4- fluorobenzamido)propanoic acid (110 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.84-8.74 (m, 1H), 7.89-7.80 (m, 1H), 7.79-7.74 (m, 1H), 7.28- 7.20 (m, 1H), 3.81-3.70 (m, 2H), 3.65-3.57 (m, 1H), 2.65 (q, J = 7.2 Hz, 2H), 1.19 (t, J = 7.2 Hz, 3H). LC-MS (MH+): m/z = 255.0 tR (min, Method c) = 0.945 [α]20 ,D = +6.0 (c = 2 mg/mL,CH3OH). Compound 2u (R)-methyl 2-amino-3-(3-ethyl-4-fluorobenzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-4- fluorobenzamido)propanoate
Figure imgf000136_0002
To a solution of a mixture of 3-ethyl-4-fluorobenzoic acid (170 mg, 1.01 mmol) and (R)- methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (321 mg, 1.11 mmol, HCl salt) in DMF (5 mL) was added TBTU (487 mg, 1.52 mmol) and DIPEA (392 mg, 3.03 mmol). The reaction mixture was stirred at 20°C for 2 hours. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 30% to 40%) to give (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-4- fluorobenzamido)propanoate (300 mg). 1H NMR (400MHz, CDCl3) δ 7.66 (d, J = 7.2 Hz, 1H), 7.55 - 7.52 (m, 1H), 7.34 - 7.30 (m, 5H), 7.05 - 7.00 (m, 1H), 6.83 (br, 1H), 5.95 (br, 1H), 5.12 (s, 2H), 4.53 - 4.50 (m, 1H), 3.94 - 3.76 (m, 5H), 2.67 (q, J = 7.6 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H). Step 2:
Figure imgf000137_0001
A solution of ((R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-4- fluorobenzamido)propanoate (300 mg, 0.75 mmol) in the solution of 30% HBr in AcOH (10 mL) was stirred at 15°C for 2 hours. The mixture was concentrated. The residue was diluted with MeOH (2 mL) and methyl tert-butyl ether (10 mL). The mixture was stirred at 15°C for 0.5 hours. The precipitate was filtered and washed with methyl tert-butyl ether (2 mL × 2). The residue was purified by preparative HPLC (Method AA) to give (R)-methyl 2-amino-3-(3- ethyl-4-fluorobenzamido)propanoate (100 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.87 (br s, 1H), 8.62 (br s, 3H), 7.89 - 7.83 (m, 1H), 7.81 - 7.75 (m, 1H), 7.29 - 7.22 (m, 1H), 4.20-4.15 (m, 1H), 3.78 - 3.70 (m, 5H), 2.66 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.2 Hz, 3H). LCMS (MH+): m/z = 269.0, tR (min, Method D) = 1.65 min [α]20,D = +2.0 (c = 2 mg/mL,CH3OH). Compound 1p (R)-2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(1-ethyl-4-methyl-1H- pyrazol-5-yl)benzamido)propanoic acid is shown below
Figure imgf000138_0001
Step 1: 1-ethyl-4-methyl-1H-pyrazole
Figure imgf000138_0002
To a solution of 4-methyl-1H-pyrazole (3.0 g, 36.5 mmol) in DMF (10 mL) was added NaH (1.90 g, 47.50 mmol, 60% in mineral oil) at 0°C and stirred at 0°C for 0.5 hour. Then iodoethane (6.84 g, 43.85 mmol) was added at 0°C and stirred at 20°C for 3 hours. The reaction mixture was quenched with water (20 mL), extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 5% to 10%) to give 1-ethyl-4-methyl-1H-pyrazole (4.0 g). 1H NMR (400 MHz, CD3Cl) δ 7.29 (s, 1H), 7.16 (s, 1H), 4.10 (q, J = 7.6 Hz, 2H), 2.07 (s, 3H), 1.45 (t, J = 7.6 Hz, 3H). Step 2: 5-bromo-1-ethyl-4-methyl-1H-pyrazole and 3-bromo-1-ethyl-4-methyl-1H-pyrazole
Figure imgf000139_0001
To a solution of 1-ethyl-4-methyl-1H-pyrazole (2.0 g, 18.16 mmol) in CHCl3 (10 mL) was added NBS (3.55 g, 19.97 mmol) and the reaction mixture was stirred at 30°C for 3 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 5%) to give a mixture of 5-bromo-1-ethyl- 4-methyl-1H-pyrazole and 3-bromo-1-ethyl-4-methyl-1H-pyrazole (1.9 g). LC-MS (MH+): m/z = 191.0 tR (min, Method G) = 0.718 Step 3: methyl 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoate
Figure imgf000139_0002
To a solution of (3-(methoxycarbonyl)phenyl)boronic acid (952 mg, 5.29 mmol) in DME (10 mL) were added a mixture of 5-bromo-1-ethyl-4-methyl-1H-pyrazole and 3-bromo-1-ethyl- 4-methyl-1H-pyrazole (1.0 g, 5.29 mmol), Pd(PPh3)4 (611 mg, 0.53 mmol) and Na2CO3 (1.68 g, 15.87 mmol). The reaction was degassed and purged with N23 times, and then the mixture was stirred at 85°C for 16 hours under N2 atmosphere. The reaction mixture was quenched with water (20 mL), extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 10% to 15%) to give methyl 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoate (240 mg). 1H NMR (400 MHz, CD3Cl) δ 8.10 (d, J = 7.6 Hz, 1H), 8.00 (s, 1H), 7.60 - 7.54 (m, 1H), 7.53 - 7.48 (m, 1H), 7.42 (s, 1H), 4.04 (q, J = 7.2 Hz, 2H), 3.95 (s, 3H), 1.99 (s, 3H), 1.34 (t, J = 7.2 Hz, 3H). Step 4: 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoic acid
Figure imgf000140_0001
To a solution of methyl 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoate (240 mg, 0.98 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (82 mg, 1.96 mmol) and the mixture was stirred at 20°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(1-ethyl-4- methyl-1H-pyrazol-5-yl)benzoic acid (220 mg). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5- yl)benzamido)propanoate
Figure imgf000140_0002
To a solution of 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoic acid (220 mg, 0.96 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (383 mg, 1.05 mmol, HCl salt) in DMF (5 mL) was added TBTU (460 mg, 1.43 mmol) and DIPEA (370 mg, 2.87 mmol). The reaction mixture was stirred at 20°C for 16 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 40% to 45%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1- ethyl-4-methyl-1H-pyrazol-5-yl)benzamido)propanoate (380 mg). 1H NMR (400 MHz, CDCl3) δ 7.69-7.65 (m, 2H), 7.53 - 7.48 (m, 1H), 7.45 - 7.40 (m, 2H), 7.38 - 7.28 (m, 10H), 6.88 (br, 1H), 5.99 (br, 1H), 5.21 (s, 2H), 5.10 (s, 2H), 4.63 - 4.56 (m, 1H), 4.01 (q, J = 7.2 Hz, 2H), 3.98 - 3.78 (m, 2H), 1.98 (s, 3H), 1.30 (t, J = 7.2 Hz, 3H). Step 6: (R)-2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzamido)propanoic acid
Figure imgf000141_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H-pyrazol- 5-yl)benzamido)propanoate (380 mg, 0.70 mmol) in the solution of 30 % HBr in AcOH (10 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated. The sample was purified by preparation HPLC (Method AA) to give (R)-2-amino-3-(3-(1-ethyl-4-methyl-1H- pyrazol-5-yl)benzamido)propanoic acid (140 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 8.94-8.84 (m, 1H), 7.96 (d, J = 7.6 Hz, 1H), 7.86 (s, 1H), 7.64 (dd, J = 7.6 Hz, 7.6 Hz, 1H ), 7.55 (d, J = 7.6 Hz, 1H), 7.38 (s, 1H), 4.02-3.96 (m, 3H), 3.81-3.66 (m, 2H), 1.94 (s, 3H), 1.20 (t, J = 7.2 Hz, 3H). LC-MS (MH+): m/z = 317.0 tR (min, Method c) = 1.027 [α]20 ,D = +9.0 (c = 2.0 mg/mL, CH3OH). Compound 2v (R)-methyl 2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzamido)propanoate Step 1: (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5- yl)benzamido)propanoate
Figure imgf000142_0001
To a solution of 3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzoic acid (100 mg, 0.43 mmol) and (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (121 mg, 0.48 mmol, HCl salt) in DMF (5 mL) was added TBTU (209 mg, 0.65 mmol) and DIPEA (168 mg, 1.30 mmol). The reaction mixture was stirred at 10°C for 2 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 55% to 60%) twice to give (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3- (3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzamido)propanoate (100 mg). 1H NMR (400 MHz, CDCl3) δ 7.81-7.71 (m, 2H), 7.58 - 7.51 (m, 1H), 7.47 - 7.40 (m, 2H), 7.32 (s, 5H), 7.02 (br, 1H), 5.91 (br d, J = 6.0 Hz, 1H), 5.10 (s, 2H), 4.60 - 4.54 (m, 1H), 4.07 - 3.90 (m, 3H), 3.87 - 3.76 (m, 4H), 1.99 (s, 3H), 1.32 (t, J = 7.2 Hz, 3H). Step 2:
Figure imgf000142_0002
A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-methyl-1H-pyrazol- 5-yl)benzamido)propanoate (100 mg, 0.22 mmol) in the solution of 30 % HBr in AcOH (5 mL) was stirred at 10°C for 1 hour. The reaction mixture was concentrated. The sample was lyophilized to give (R)-methyl 2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5- yl)benzamido)propanoate (55mg) as HBr salt. 1H NMR (400 MHz, DMSO-d6) δ 8.85-8.80 (m, 1H), 8.45 (br s, 3H), 7.97 - 7.91 (m, 1H), 7.83 (s, 1H), 7.68 - 7.62 (m, 1H), 7.60 - 7.56 (m, 1H), 7.41 (s, 1H), 4.27 - 4.19 (m, 1H), 4.00 (q, J = 7.2 Hz, 2H), 3.82 - 3.66 (m, 5H), 1.94 (s, 3H), 1.21 (t, J = 7.2 Hz, 3H). LCMS (MH+): m/z = 331.2, tR (min, Method D) = 1.554 min [α]20,D = +1.0 (c = 2.0 mg/mL, CH3OH). Compound 1q (R)-2-amino-3-(3-ethyl-5-fluoro-4-methoxybenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-ethyl-5-fluoro-4- methoxybenzamido)propanoic acid
Figure imgf000143_0001
Step 1: methyl 3-bromo-5-fluoro-4-hydroxybenzoate
Figure imgf000143_0002
To a solution of methyl 3-fluoro-4-hydroxybenzoate (500 mg, 2.94 mmol) in DMF (5 mL) was added NBS (628 mg, 3.53 mmol) and the reaction mixture was stirred at 30°C for 1 hour. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 5%) to give methyl 3-bromo-5-fluoro-4- hydroxybenzoate (700 mg). 1H NMR (400 MHz, CD3Cl) δ 8.02 (dd, J = 2.0 Hz, 1.6 Hz, 1H), 7.74 (dd, J = 10.4 Hz, 1.6 Hz, 1H), 6.17 (s, 1H), 3.91 (s, 3H). Step 2: methyl 3-bromo-5-fluoro-4-methoxybenzoate
Figure imgf000144_0001
To a solution of methyl 3-bromo-5-fluoro-4-hydroxybenzoate (700 mg, 2.81 mmol) in DMF (10 mL) was added K2CO3 (971 mg, 7.03 mmol) and iodomethane (1.20 g, 8.43 mmol), then the mixture was stirred at 60°C for 3 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 2%) to give methyl 3-bromo-5-fluoro-4-methoxybenzoate (700 mg). 1H NMR (400 MHz, CD3Cl) δ 8.02 (dd, J = 2.0 Hz, 2.0 Hz, 1H), 7.74 (dd, J = 11.6 Hz, 2.0 Hz, 1H), 4.05 (d, J = 2.8 Hz, 3H), 3.91 (s, 3H). Step 3: methyl 3-ethyl-5-fluoro-4-methoxybenzoate
Figure imgf000144_0002
A mixture of methyl 3-bromo-5-fluoro-4-methoxybenzoate (700 mg, 2.66 mmol), triethylborane (1 M in THF, 8 mL), Pd(dppf)Cl2 (195 mg, 0.27 mmol) and CsOAc (1.53 g, 7.98 mmol) in THF (5 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 65°C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 3-ethyl-5-fluoro-4-methoxybenzoate (360 mg). 1H NMR (400MHz, CDCl3) δ 7.66 (s, 1H), 7.59 (dd, J = 12.4 Hz, 2.0 Hz, 1H), 4.00 (d, J = 2.8 Hz, 3H), 3.89 (s, 3H), 2.67 (q, J = 7.6 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H). Step 4: 3-ethyl-5-fluoro-4-methoxybenzoic acid
Figure imgf000145_0001
To a solution of methyl 3-ethyl-5-fluoro-4-methoxybenzoate (360 mg, 1.70 mmol) in a mixture of THF (6 mL) and H2O (3 mL) was added LiOH.H2O (214 mg, 5.09 mmol) and the mixture was stirred at 10°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-ethyl-5-fluoro- 4-methoxybenzoic acid (330 mg). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methoxybenzamido)propanoate
Figure imgf000145_0002
To a solution of a mixture of 3-ethyl-5-fluoro-4-methoxybenzoic acid (100 mg, 0.50 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (202 mg, 0.56 mmol, HCl salt) in DMF (5 mL) was added TBTU (243 mg, 0.76 mmol) and DIPEA (196 mg, 1.51 mmol). The reaction mixture was stirred at 20°C for 2 hours. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methoxybenzamido)propanoate (250 mg). 1H NMR (400MHz, CDCl3) δ 7.35-7.29 (m, 11H), 7.27-7.21 (m, 1H), 6.61 (br s, 1H), 5.98 (br d, J = 6.8 Hz, 1H), 5.21 (s, 2H), 5.12 (s, 2H), 4.62-4.54 (m, 1H), 3.98 (d, J = 2.8 Hz, 3H), 3.94-3.76 (m, 2H), 2.65 (q, J = 7.6 Hz, 2H), 1.20 (t, J = 7.6 Hz, 3H). Step 6: (R)-2-amino-3-(3-ethyl-5-fluoro-4-methoxybenzamido)propanoic acid
Figure imgf000146_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methoxybenzamido)propanoate (100 mg, 0.20 mmol) and Pd/C (50 mg, 10% Pd, 50% water) in AcOH (5 mL) was degassed and purged with H2 for 3 times, and then the mixture was stirred at 10°C for 1 hour under H2 atmosphere (15 psi). The reaction mixture was filtered and concentrated. The sample was submitted to lyophilization to give (R)-2-amino-3-(3-ethyl- 5-fluoro-4-methoxybenzamido)propanoic acid (8 mg). 1H NMR (400MHz, DMSO-d6) δ 8.80 (br s, 1H), 7.60-7.52 (m, 2H), 3.88 (s, 3H), 3.69-3.59 (m, 1H), 3.50-3.40 (m, 2H), 2.62 (q, J = 7.6 Hz, 2H), 1.16 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 285.0 tR (min, Method E) = 1.443 [α]20,D = +17.0 (c = 2 mg/mL,CH3OH). Compound 2w (R)-methyl 2-amino-3-(3-ethyl-5-fluoro-4-methoxybenzamido)propanoate Step 1: (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methoxybenzamido)propanoate
Figure imgf000147_0001
To a solution of a mixture of 3-ethyl-5-fluoro-4-methoxybenzoic acid (100 mg, 0.51 mmol) and (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (160 mg, 0.56 mmol, HCl salt) in DMF (5 mL) was added TBTU (243 mg, 0.76 mmol) and DIPEA (196 mg, 1.51 mmol). The reaction mixture was stirred at 20°C for 2 hours. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methoxybenzamido)propanoate (200 mg, 91.7% yield). 1H NMR (400MHz, CDCl3) δ 7.40 - 7.29 (m, 7H), 6.78 (br, 1H), 5.92 (br d, J = 6.4 Hz, 1H), 5.13 (s, 2H), 4.60 - 4.51 (m, 1H), 3.98 (d, J = 2.4 Hz, 3H), 3.92 - 3.76 (m, 5H), 2.67 (q, J = 7.2 Hz, 2H), 1.21 (t, J = 7.6 Hz, 3H).
Figure imgf000147_0002
A mixture of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-ethyl-5-fluoro-4- methoxybenzamido)propanoate (100 mg, 0.23 mmol) and Pd/C (50 mg, 10% Pd, 50% water) in AcOH (5 mL) was degassed and purged with H2 for 3 times, and then the mixture was stirred at 10°C for 1 hour under H2 atmosphere (15 psi). The reaction mixture was filtered and concentrated. The sample was purified by preparation HPLC (neutral condition) to give (R)-methyl 2-amino-3-(3-ethyl-5-fluoro-4-methoxybenzamido)propanoate (18 mg). 1H NMR (400MHz, DMSO-d6) δ 8.52-8.48 (m, 1H), 7.57 - 7.52 (m, 2H), 3.89 (s, 3H), 3.60 (s, 3H), 3.56 - 3.51 (m, 1H), 3.43 - 3.38 (m, 2H), 2.63 (q, J = 7.6 Hz, 2H), 1.17 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 299.2, tR (min, Method D) = 1.715 min [α]20,D = -14.0 (c = 2 mg/mL,CH3OH). Compound 1r (R)-2-amino-3-(3-(5-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(5-ethylisothiazol-4-yl)- 5-fluorobenzamido)propanoic acid is shown below
Figure imgf000148_0001
Step 1: methyl 3-(5-ethylisothiazol-4-yl)-5-fluorobenzoate
Figure imgf000148_0002
4-bromo-5-ethylisothiazole (500 mg, 1.11 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (286 mg, 1.44 mmol), Pd(dppf)Cl2.CH2Cl2 (91 mg, 0.11 mmol) and Na2CO3 (353 mg, 3.33 mmol) were taken up into a microwave tube in a mixture of dioxane (6 mL) and H2O (0.7 mL). The sealed tube was heated at 120 °C for 60 min under microwave. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 20%) to give methyl 3-(5-ethylisothiazol-4- yl)-5-fluorobenzoate (160 mg, crude). Step 2: 3-(5-ethylisothiazol-4-yl)-5-fluorobenzoic acid
Figure imgf000149_0001
To a solution of methyl 3-(5-ethylisothiazol-4-yl)-5-fluorobenzoate (160 mg, crude) in a mixture of MeOH (5 mL) and H2O (2 mL) was added LiOH.H2O (37 mg, 0.88 mmol). The mixture was stirred at 15 °C for 15 hours. The mixture was concentrated. The residue was dissolved in water (15 mL) and extracted with ethyl acetate (20 mL × 2). The organic layer was discarded. The aqueous layer was acidified with 2N HCl to adjust pH to 4 and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3-(5-ethylisothiazol-4-yl)- 5-fluorobenzoic acid (60 mg). LC-MS (MH+): m/z = 252.1 tR (min, Method D) = 0.797 Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000149_0002
To a solution of 3-(5-ethylisothiazol-4-yl)-5-fluorobenzoic acid (60 mg, 0.12 mmol) in DMF (5 mL) was added TBTU (60 mg, 0.19 mmol), DIPEA (48 mg, 0.37 mmol) and (R)-benzyl 3-amino- 2-(((benzyloxy)carbonyl)amino)propanoate (55 mg, 0.15 mmol, HCl salt). The mixture was stirred at 15 °C for 16 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 40%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoate (45 mg. 1H NMR (400 MHz, CDCl3) δ 8.43 (s, 1H), 7.52 (s, 1H), 7.38-7.32 (m, 11H), 7.25-7.19 (m, 1H), 6.87 (br s, 1H), 5.91 (br d, J = 6.0 Hz, 1H), 5.23 (s, 2H), 5.12 (s, 2H), 4.65-4.56 (m, 1H), 4.01- 3.92 (m, 1H), 3.86-3.78 (m, 1H), 3.00 (q, J = 7.6 Hz, 2H), 1.36 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-(5-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000150_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate (40 mg, 0.07 mmol) in 30% HBr in AcOH (4 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(5-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoic acid (18 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.16-9.10 (m, 1H), 8.71 (s, 1H), 8.54 (br s, 3H), 7.89 (s, 1H), 7.76-7.69 (m, 1H), 7.63-7.56 (m, 1H), 4.16-4.08 (m, 1H), 3.82-3.76 (m, 2H), 3.07 (q, J = 7.6 Hz, 2H), 1.27 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 338.0 tR (min, Method D) = 1.543 [α]20,D = 1.0 (c = 2.5 mg/mL, CH3OH). Compound 2x (R)-methyl 2-amino-3-(3-(5-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisothiazol-4- yl)-5-fluorobenzamido)propanoate
Figure imgf000150_0002
To a solution of 3-(5-ethylisothiazol-4-yl)-5-fluorobenzoic acid (70 mg, 0.28 mmol) in DMF (5 mL) was added TBTU (134 mg, 0.42 mmol), DIPEA (108 mg, 0.84 mmol) and (R)-methyl 3- amino-2-(((benzyloxy)carbonyl)amino)propanoate (80 mg, 0.28 mmol, HCl salt). The mixture was stirred at 15 °C for 16 hours. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 40%) to give (R)- methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate (90 mg). 1H NMR (400MHz, CDCl3) δ 8.45 (s, 1H), 7.59 (s, 1H), 7.50 - 7.44 (m, 1H), 7.33 (s, 5H), 7.26 - 7.21 (m, 1H), 7.07 (br s, 1H), 5.87 (br d, J = 6.4 Hz, 1H), 5.12 (s, 2H), 4.62 - 4.54 (m, 1H), 4.01 - 3.91 (m, 1H), 3.87 - 3.76 (m, 4H), 3.02 (q, J = 7.6 Hz, 2H), 1.36 (t, J = 7.6 Hz, 3H). Step 2:
Figure imgf000151_0001
A mixture of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate (90 mg, 0.19 mmol) in 30% HBr in AcOH (4 mL) was stirred at 25 °C for 1 hour. The mixture was concentrated. The residue was washed with MeCN (2 mL) and methyl tert-butyl ether (5 mL) and dried to give (R)-methyl 2-amino-3-(3-(5- ethylisothiazol-4-yl)-5-fluorobenzamido)propanoate (72 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.92-8.87 (m, 1H), 8.67 (s, 1H), 8.43 (br s, 3H), 7.80 (s, 1H), 7.70 - 7.65 (m, 1H), 7.64 - 7.59 (m, 1H), 4.28 - 4.18 (m, 1H), 3.78 - 3.68 (m, 5H), 3.05 (q, J = 7.6 Hz, 2H), 1.27 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 352.0, tR (min, Method D) = 1.604 min [α]20,D = -0.9 (c =3.6mg/mL, CH3OH). Compound 1s (R)-2-amino-3-(3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(1-ethyl-1H-imidazol- 5-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000152_0001
Step 1: 5-bromo-1-ethyl-1H-imidazole
Figure imgf000152_0002
1-ethyl-1H-imidazole (5.0 g, 52.0 mmol) was dissolved in DCM (70 mL), and 1,3-dibromo-5,5- dimethylimidazolidine-2,4-dione (7.58 g, 26.5 mmol) dissolved in DCM (50 ml) was added dropwise at 0°C. The reaction mixture was stirred at 0°C for 3 hours. The reaction mixture was poured into 100 mL of sat. Na2SO3, extracted with DCM (80 mL × 3), the organic layers were washed with brine (50 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (MeOH/DCM with MeOH from 0% to 10%) to give 5- bromo-1-ethyl-1H-imidazole (1.55 g). 1H NMR (400MHz, CDCl3) δ 7.53 (s, 1H), 6.97 (s, 1H), 3.95 (q, J = 7.2 Hz, 2H), 1.39 (t, J = 7.2 Hz, 3H). Step 2: methyl 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoate
Figure imgf000153_0001
A mixture of 5-bromo-1-ethyl-1H-imidazole (800 mg, 4.57 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (1.09 g, 5.48 mmol), Pd(dppf)Cl2 (298 mg, 457.0 µmol), K3PO4 (2.91 g, 13.71 mmol) in the mixture of H2O (2 mL) and dioxane (10 mL) was degassed by purging with N2, and then the mixture was stirred at 100 °C for 14 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (MeOH/DCM with MeOH from 0% to 5%). The obtained material was further purified by preparative HPLC (Method BB) to give methyl 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoate (150 mg). 1H NMR (400MHz, CDCl3) δ 7.87 (s, 1H), 7.76-7.70 (m, 1H), 7.63 (s, 1H), 7.31-7.27 (m, 1H), 7.16 (s, 1H), 4.06 (q, J = 7.2 Hz, 2H), 3.96 (s, 3H), 1.36 (t, J = 7.2 Hz, 3H) Step 3: 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoic acid
Figure imgf000153_0002
To a solution of methyl 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoate (150 mg, 604.2 µmol) in MeOH (2 mL) and H2O (2 mL) was added LiOH.H2O (76.0 mg, 1.81 mmol). The mixture was stirred at 5 °C for 16 hours. The mixture was concentrated to remove MeOH, then HCl (2 M) was added to adjust PH=5~6. Following concentration on the rotavapor afforded 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoic acid (141 mg, crude). Step: 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-imidazol-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000154_0001
To a solution of 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoic acid (70 mg, crude) and (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (131 mg, 358.6 µmol, HCl salt) in DMF (3 mL) was added TBTU (144 mg, 448.3 µmol) and DIPEA (116 mg, 896.6 µmol). The mixture was stirred at 5 °C for 16 hours. To the reaction mixture was added water (5 mL), extracted with ethyl acetate (10 mL × 4), and the organic layers were washed with brine (10 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (MeOH/DCM with MeOH from 0% to 10%) and further purified by preparative HPLC (Method AA) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H- imidazol-5-yl)-5-fluorobenzamido)propanoate (60 mg). LC-MS (MH+): m/z = 545.3 tR (min, Method G) = 0.801. Step 5: (R)-2-amino-3-(3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000154_0002
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-imidazol-5-yl)-5- fluorobenzamido)propanoate (60.0 mg, 110.2 µmol) in 30% HBr in AcOH (2 mL) was stirred at 50°C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(1-ethyl-1H-imidazol-5-yl)-5- fluorobenzamido)propanoic acid (15 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.33 (br s, 1H), 9.18 (br s, 1H), 8.64 (br s, 3H), 8.03 (s, 1H), 7.96-7.87 (m, 2H), 7.72 (d, J = 8.8 Hz, 1H), 4.28 (q, J = 7.2 Hz, 2H), 4.19-4.09 (m, 1H), 3.86-3.76 (m, 2H), 1.30 (t, J = 7.2 Hz, 3H). LC-MS (MH+): m/z = 321.0 tR (min, Method D) = 1.107. [α]20,D = 4 (c = 1 mg/mL, MeOH). Compound 2y (R)-methyl 2-amino-3-(3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-imidazol- 5-yl)-5-fluorobenzamido)propanoate
Figure imgf000155_0001
To a solution of 3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzoic acid (70 mg, crude) and (R)- methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (90 mg, 358.63 µmol, HCl salt) in DMF (3 mL) was added TBTU (144 mg, 448.29 µmol) and DIPEA (116 mg, 896.58 µmol). The mixture was stirred at 5 °C for 16 hours. The reaction mixture was added water (5 mL), extracted with ethyl acetate (10 mL × 4), the organic layers were washed with brine (10 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (MeOH/DCM with MeOH from 0% to 5%) and further purified by preparative HPLC (Method DD) to give (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-imidazol-5- yl)-5-fluorobenzamido)propanoate (25 mg). LC-MS (MH+): m/z = 469.2 tR (min, Method G) = 0.701. Step 2:
Figure imgf000156_0001
A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-imidazol-5-yl)-5- fluorobenzamido)propanoate (25 mg, 53.4 µmol) in 30% HBr in AcOH (2 mL) was stirred at 15°C for 1 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-methyl 2-amino-3-(3-(1-ethyl-1H-imidazol-5-yl)-5- fluorobenzamido)propanoate (10 mg) as HCl salt. 1H NMR (400MHz, CD3OD-d4) δ 9.17 (s, 1H), 7.97 (s, 1H), 7.85 (d, J = 9.2 Hz, 1H), 7.79 (s, 1H), 7.64 (d, J = 8.4 Hz, 1H), 4.40 - 4.29 (m, 3H), 4.08 - 3.98 (m, 1H), 3.96 – 3.85 (m, 4H), 1.42 (t, J = 7.2 Hz, 3H). LCMS (MH+): m/z = 335.1, tR (min, Method D) = 0.938 min [α]20,D = -2 (c = 2 mg/mL, MeOH). Compound 1t (R)-2-amino-3-(3-(3-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(3-ethylisothiazol-4-yl)- 5-fluorobenzamido)propanoic acid
Figure imgf000156_0002
Step 1: 3-bromo-4-iodoisothiazole
Figure imgf000157_0001
To a solution of 3-bromoisothiazole (1.0 g, 6.10 mmol) in H2SO4 (20 mL) was added I2 (1.08 g, 4.27 mmol). After 30 minutes, NaIO4 (652 mg, 3.05 mmol) was added in small portions to the reaction mixture and the mixture was stirred at 30°C for 16 hours. The reaction mixture was poured onto ice (~100 g), decolorized with Na2SO3 (20 mL), and extracted with CHCl3 (20 mL × 3). The combined organic extracts were washed with water (10 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give 3-bromo-4- iodoisothiazole (1.46 g). 1H NMR (400MHz, CDCl3) δ 8.63 (s, 1H). Step 2: methyl 3-(3-bromoisothiazol-4-yl)-5-fluorobenzoate
Figure imgf000157_0002
A mixture of (3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.0 g, 5.05 mmol), 3-bromo- 4-iodoisothiazole (1.46 g, 5.05 mmol), Pd(dppf)Cl2 (370 mg, 0.51 mmol) and K2CO3 (2.09 g, 15.15 mmol) in a mixture of dioxane (20 mL) and H2O (2 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 85°C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 3-(3- bromoisothiazol-4-yl)-5-fluorobenzoate (1.2 g). 1H NMR (400MHz, CDCl3) δ 8.63 (s, 1H), 7.99-7.97 (m, 1H), 7.81-7.77 (m, 1H), 7.50-7.45 (m, 1H), 3.97 (s, 3H). Step 3: methyl 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoate
Figure imgf000158_0001
A mixture of methyl 3-(3-bromoisothiazol-4-yl)-5-fluorobenzoate (500 mg, 1.58 mmol), triethylborane (1 M in THF, 4.74 mL), Pd(dppf)Cl2 (116 mg, 0.16 mmol) and CsOAc (911 mg, 4.74 mmol) in THF (10 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 65°C for 16 hours under N2 atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 5%) to give methyl 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoate (110 mg). 1H NMR (400MHz, CDCl3) δ 8.56 (s, 1H), 7.87-7.86 (m, 1H), 7.76-7.73 (m, 1H), 7.31-7.28 (m, 1H), 3.96 (s, 3H), 2.86 (q, J = 7.6 Hz, 2H), 1.30 (t, J = 7.2 Hz, 3H). Step 4: 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoic acid
Figure imgf000158_0002
To a solution of methyl 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoate (110 mg, 0.41 mmol) in a mixture of THF (4 mL) and H2O (2 mL) was added LiOH.H2O (52 mg, 1.24 mmol) and the mixture was stirred at 10°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(3- ethylisothiazol-4-yl)-5-fluorobenzoic acid (90 mg). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate
Figure imgf000159_0001
To a solution of a mixture of 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoic acid (45 mg, 0.18 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (72 mg, 0.20 mmol, HCl salt) in DMF (3 mL) was added TBTU (86 mg, 0.27 mmol) and DIPEA (69 mg, 0.54 mmol). The reaction mixture was stirred at 10°C for 1 hour. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(7-(pentan-3- yl)benzo[d]thiazole-2-carboxamido)propanoate (45 mg). LC-MS (MH+): m/z = 562.3 tR (min, Method G) = 0.953 Step 6: (R)-2-amino-3-(3-(3-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000159_0002
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(7-(pentan-3-yl)benzo[d]thiazole- 2-carboxamido)propanoate (45 mg, 0.08 mmol) in 30% HBr in AcOH (3 mL) was heated at 50°C for 16 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(3-(3-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoic acid (10 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.13 (s, 1H), 9.10 (br, 1H), 8.52 (br s, 3H), 7.84 (s, 1H), 7.75 (d, J = 8.8 Hz, 1H), 7.58 (d, J = 8.8 Hz, 1H), 4.15-4.09 (m, 1H), 3.81-3.77 (m, 2H), 2.84 (q, J = 7.6 Hz, 2H), 1.19 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 338.1 tR (min, Method c) = 1.105 [α]20,D = +1.0 (c = 2 mg/mL,CH3OH). Compound 2z (R)-methyl 2-amino-3-(3-(3-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylisothiazol-4- yl)-5-fluorobenzamido)propanoate
Figure imgf000160_0001
To a solution of a mixture of 3-(3-ethylisothiazol-4-yl)-5-fluorobenzoic acid (45 mg, 0.18 mmol) and (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (50 mg, 0.20 mmol, HCl salt) in DMF (3 mL) was added TBTU (86 mg, 0.27 mmol) and DIPEA (69 mg, 0.54 mmol). The reaction mixture was stirred at 10°C for 1 hour. The mixture was added water (20 mL), extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with saturated brines (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 20% to 30%) to give (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylisothiazol-4-yl)- 5-fluorobenzamido)propanoate (45 mg). LC-MS (MH+): m/z = 486.2 tR (min, Method G) = 0.883 Step 2:
Figure imgf000160_0002
A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(3-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate (45 mg, 0.09 mmol) in the solution of 30% HBr in AcOH (3 mL) was stirred at 10°C for 1 hour. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-methyl 2-amino-3-(3-(3-ethylisothiazol-4-yl)-5- fluorobenzamido)propanoate (10 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.12 (s, 1H), 9.02 (br s, 1H), 8.56 (br s, 3H), 7.79 (s, 1H), 7.72 (d, J = 8.8 Hz, 1H), 7.57 (d, J = 10.0 Hz, 1H), 4.26 - 4.17 (m, 1H), 3.79 - 3.70 (m, 5H), 2.84 (q, J = 7.6 Hz, 2H), 1.18 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 352.1, tR (min, Method D) = 1.757 min [α]20,D = -6.0 (c = 1 mg/mL,CH3OH). Compound 1u (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(5-ethylisoxazol-4- yl)benzamido)propanoic acid
Figure imgf000161_0001
Step 1: ethyl 3-(5-ethylisoxazol-4-yl)benzoate
Figure imgf000161_0002
4-bromo-5-ethyl-isoxazole (500 mg, 2.84 mmol), (3-ethoxycarbonylphenyl)boronic acid (551 mg, 2.84 mmol), Pd(t-Bu3P)2 (145 mg, 284.1 μmol) and KF (413 mg, 7.10 mmol) were taken up into a microwave tube in a mixture of dioxane (6 mL) and H2O (0.6 mL) under N2 atmosphere. The sealed tube was heated at 100 °C for 60 min under microwave. The reaction mixture was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether /ethyl acetate with ethyl acetate from 0% to 10%) to give ethyl 3-(5-ethylisoxazol-4- yl)benzoate (340 mg).1H NMR (400MHz, CDCl3) δ 8.39 (s, 1H), 8.06-8.00 (m, 2H), 7.56-7.50 (m, 2H), 4.42 (q, J = 7.2Hz, 2H), 2.97 (q, J = 7.6Hz, 2H), 1.42 (t, J = 7.2Hz, 3H), 1.37 (t, J = 7.6Hz, 3H). Step 2: 3-(5-ethylisoxazol-4-yl)benzoic acid
Figure imgf000162_0001
A solution of ethyl 3-(5-ethylisoxazol-4-yl)benzoate (430 mg, 1.75 mmol) in conc. HCl (10 mL) was stirred at 80 °C for 16 hours. The reaction mixture was extracted with ethyl acetate (20 mL × 3). The organic layer was concentrated to give 3-(5-ethylisoxazol-4-yl)benzoic acid (270 mg).1H NMR (400MHz, DMSO-d6) δ 13.0 (br, 1H), 8.90 (s, 1H), 7.98 (s, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 7.6 Hz, 1H), 7.56 (dd, J = 8.0 Hz, 7.6Hz, 1H), 2.94 (q, J = 7.6Hz, 2H), 1.23 (t, J = 7.6Hz, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4- yl)benzamido)propanoate
Figure imgf000162_0002
To a solution of 3-(5-ethylisoxazol-4-yl)benzoic acid (135 mg, 621.5 μmol) and (R)-benzyl 3- amino-2-(((benzyloxy)carbonyl)amino)propanoate (227 mg, 621.5 μmol, HCl salt) in DMF (5 mL) was added TBTU (299 mg, 932.2 μmol) and DIPEA (24.0 mg, 1.86 mmol). The mixture was stirred at 15°C for 16 hours. The reaction mixture was diluted with water (10 mL), extracted with ethyl acetate (10 mL × 3), the organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 5% to 38%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoate (230 mg). 1H NMR (400MHz, CDCl3) δ 8.37 (s, 1H), 7.77 (s, 1H), 7.58-7.52 (m, 1H), 7.51-7.44 (m, 2H), 7.39-7.29 (m, 10H), 6.85 (br s, 1H), 5.95 (br d, J = 6.8 Hz, 1H), 5.23 (s, 2H), 5.12 (s, 2H), 4.62 (br d, J = 4.0 Hz, 1H), 4.02-3.90 (m, 1H), 3.88-3.76 (m, 1H), 2.96 (q, J = 7.6 Hz, 2H), 1.35 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoic acid
Figure imgf000163_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4- yl)benzamido)propanoate (130 mg, 246.4 μmol) in 30% HBr in AcOH (5 mL) was stirred at 50°C for 16 hours. The reaction mixture was concentrated, the residue was dissolved in water (5 mL), washed with methyl tert-butyl ether(5 mL). The water phase was lyophizised to give (R)-2-amino-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoic acid (85 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.89 (s, 1H), 8.85-8.78 (m, 1H), 8.31 (br s, 3H), 7.96 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.69 (d, J = 8.0 Hz, 1H), 7.63-7.56 (m, 1H), 4.20-4.10 (m, 1H), 3.83-3.66 (m, 2H), 3.00 (q, J=7.6 Hz, 2H), 1.26 (t, J=7.6 Hz, 3H). LC-MS (MH+): m/z = 304.1 tR (min, Method D) = 1.53 [α]20,D = +1.2 (c = 5 mg/mL,CH3OH). Compound 2aa (R)-methyl 2-amino-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoate Step 1: (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4- yl)benzamido)propanoate
Figure imgf000164_0001
To a solution of 3-(5-ethylisoxazol-4-yl)benzoic acid (135 mg, 621.49 μmol) and (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (179 mg, 621.49 μmol, HCl salt) in DMF (5 mL) was added TBTU (299 mg, 932.23 μmol) and DIPEA (24 mg, 1.86 mmol). The mixture was stirred at 15°C for 16 hours. The reaction mixture was diluted with water (10 mL), extracted with ethyl acetate (10 mL × 3), the organic layer was washed with brine (10 mL), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 5% to 38%) to give (R)-methyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoate (140 mg). 1H NMR (400MHz, CDCl3) δ18.38 (s, 1H), 7.82 (s, 1H), 7.67 (br s, 1H), 7.54 - 7.47 (m, 2H), 7.36 - 7.29 (m, 5H), 6.98 (br s, 1H), 5.89 (br d, J=6.4 Hz, 1H), 5.13 (s, 2H), 4.60-4.58 (m, 1H), 4.03 - 3.90 (m, 1H), 3.88 - 3.74 (m, 4H), 2.97 (q, J=7.6 Hz, 2H), 1.36 (t, J=7.7 Hz, 3H). Step 2:
Figure imgf000164_0002
A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(5-ethylisoxazol-4- yl)benzamido)propanoate (140 mg, 0.310 mmol) in 30% HBr/AcOH (5 mL) was stirred at 20°C for 1 hour. The reaction mixture was concentrated. The residue was dissolved in water (5 mL), washed with methyl tert-butyl ether (5 mL). The water phase was lyophilized to give (R)- methyl 2-amino-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoate (100 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.90 (s, 1H), 8.82 (t, J=5.6 Hz, 1H), 8.45 (br s, 3H), 7.95 (s, 1H), 7.83 (d, J=8.0 Hz, 1H), 7.69 (d, J=7.6 Hz, 1H), 7.59 (dd, J =8.0 Hz, 7.6 Hz, 1H), 4.23 (t, J=5.6 Hz, 1H), 3.80 - 3.69 (m, 5H), 3.00 (q, J=7.6 Hz, 2H), 1.26 (t, J=7.6 Hz, 3H). LCMS (MH+): m/z = 318.1, tR (min, Method D) = 1.594 min [α]20,D = +1.7 (c = 3.5 mg/mL,CH3OH). Compound 1v (R)-2-amino-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(1-ethyl-1H-1,2,3- triazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000165_0001
Step 1: 5-bromo-1-ethyl-1H-1,2,3-triazole
Figure imgf000165_0002
To a solution of 1-ethyl-1H-1,2,3-triazole (300 mg, 3.09 mmol) in THF (5 mL) was added n- BuLi (2.5 M in hexane, 1.50 mL) at -40 °C and stirred for 1 hour. Then Br2 (592 mg, 3.71 mmol) was added and the mixture was stirred at 20 °C for 2 hours. The mixture was quenched with water (10 mL) and extracted with ethyl acetate (15 mL × 2). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 5-bromo-1-ethyl-1H-1,2,3-triazole (530 mg). 1H NMR (400MHz, CDCl3) δ 7.66 (s, 1H), 4.43 (q, J = 7.2 Hz, 2H), 1.54 (t, J = 7.2 Hz, 3H). Step 2: Methyl 3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzoate
Figure imgf000166_0001
5-bromo-1-ethyl-1H-1,2,3-triazole (480 mg, 2.73 mmol), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (648 mg, 3.27 mmol), Pd(dppf)Cl2.CH2Cl2 (223 mg, 0.27 mmol) and K2CO3 (1.13 g, 8.18 mmol) were taken up into a microwave tube in a mixture of dioxane (10 mL) and H2O (1 mL). The sealed tube was heated at 100 °C for 60 min under microwave. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL) and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give methyl 3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzoate (540 mg). 1H NMR (400MHz, CDCl3) δ 7.91 - 7.87 (m, 1H), 7.87-7.81 (m, 1H), 7.76 (s, 1H), 7.35-7.29 (m, 1H), 4.44 (q, J = 7.2 Hz, 2H), 3.97 (s, 3H), 1.51 (t, J = 7.2 Hz, 3H). Step 3: 3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzoic acid
Figure imgf000166_0002
To a solution of methyl 3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzoate (540 mg, 2.17 mmol) in MeOH (10 mL) was added LiOH.H2O (273 mg, 6.50 mmol) in H2O (2 mL). The mixture was stirred at 20 °C for 16 hours. The mixture was concentrated. The residue was added water (5 mL) and acidified with 2N HCl to adjust pH to 6~7 and extracted with ethyl acetate (20 mL × 3). The combined organic layer was concentrated to give 3-(1-ethyl-1H- 1,2,3-triazol-5-yl)-5-fluorobenzoic acid (480 mg). 1H NMR (400MHz, DMSO-d6) δ 8.00 (s, 1H), 7.90-7.86 (m, 1H), 7.82-7.75 (m, 2H), 4.44 (q, J = 7.2 Hz, 2H), 1.36 (t, J = 7.2 Hz, 3H). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000167_0001
To a solution of 3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzoic acid (120 mg, 0.51 mmol) in DMF (5 mL) was added TBTU (246 mg, 0.76 mmol), DIPEA (198 mg, 1.53 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (186 mg, 0.51 mmol, HCl salt). The mixture was stirred at 15 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 50%) twice to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-1,2,3- triazol-5-yl)-5-fluorobenzamido)propanoate (70 mg). 1H NMR (400MHz, CDCl3) δ 7.70 (s, 1H), 7.58-7.49 (m, 2H), 7.39-7.28 (m, 11H), 7.24-7.19 (m, 1H), 6.00 (br d, J = 6.4 Hz, 1H), 5.22 (s, 2H), 5.11 (s, 2H), 4.65-4.57 (m, 1H), 4.39 (q, J = 7.2 Hz, 2H), 4.02-3.92 (m, 1H), 3.88-3.77 (m, 1H), 1.47 (t, J = 7.2 Hz, 3H). Step 5: (R)-2-amino-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000167_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5- fluorobenzamido)propanoate (70 mg, 0.13 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50 °C for 5 hours. The mixture was concentrated. The residue was added water (5 mL) and washed with methyl tert-butyl ether (5 mL × 2). The aqueous layer was lyophilized to give (R)- 2-amino-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoic acid (50 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.00-8.93 (m, 1H), 8.32 (br s, 3H), 7.98 (s, 1H), 7.85 (s, 1H), 7.82-7.77 (m, 1H), 7.76-7.70 (m, 1H), 4.47 (q, J = 7.2 Hz, 2H), 4.17-4.08 (m, 1H), 3.82-3.75 (m, 2H), 1.35 (t, J = 7.2 Hz, 3H). LC-MS (MH+): m/z = 322.1 tR (min, Method D) = 1.271 [α]20,D = 0.9 (c = 4.4 mg/mL, CH3OH). Compound 2ab (R)-methyl 2-amino-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-1,2,3- triazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000168_0001
To a solution of 3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzoic acid (120 mg, 0.51 mmol) in DMF (5 mL) was added TBTU (246 mg, 0.77 mmol) and DIPEA (198 mg, 1.53 mmol) and (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (147 mg, 0.51 mmol, HCl salt). The mixture was stirred at 15 °C for 16 hours. The mixture was diluted with water (15 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 60%) three times to give (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3- (1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoate (100 mg). 1H NMR (400MHz, CDCl3) δ 7.71 (s, 1H), 7.66 - 7.58 (m, 2H), 7.44 (br s, 1H), 7.32 (s, 5H), 7.26 - 7.21 (m, 1H), 5.97 (br d, J = 6.0 Hz, 1H), 5.17 - 5.07 (m, 2H), 4.63 - 4.54 (m, 1H), 4.41 (q, J = 7.2 Hz, 2H), 4.04 - 3.91 (m, 1H), 3.88 - 3.76 (m, 4H), 1.48 (t, J = 7.2 Hz, 3H). Step 2:
Figure imgf000169_0001
A mixture of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)- 5-fluorobenzamido)propanoate (100 mg, 0.21 mmol) in 30% HBr in AcOH ( 5 mL) was stirred at 20 °C for 1 hour. The mixture was concentrated. The residue was washed with a mixture of MeOH (1 mL) and methyl tert-butyl ether (5 mL). The solid was collected and dried to give (R)-methyl 2-amino-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5-fluorobenzamido)propanoate (85 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.01 - 8.95 (m, 1H), 8.47 (br s, 3H), 7.99 (s, 1H), 7.85 (s, 1H), 7.82 - 7.77 (m, 1H), 7.76 - 7.70 (m, 1H), 4.47 (q, J = 7.2 Hz, 2H), 4.29 - 4.19 (m, 1H), 3.79 - 3.72 (m, 5H), 1.35 (t, J = 7.2 Hz, 3H). LCMS (MH+): m/z = 336.2, tR (min, Method D) = 1.375 min [α]20,D = 0.7 (c =2.8 mg/mL, CH3OH). Compound 1w (R)-2-amino-3-(3-fluoro-5-(5-propylisoxazol-4-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(5- propylisoxazol-4-yl)benzamido)propanoic acid
Figure imgf000170_0001
Step 1: (E)-1-(dimethylamino)hex-1-en-3-one
Figure imgf000170_0002
A mixture of pentan-2-one (10 g, 116.10 mmol), DMF-DMA (16.6 g, 139.32 mmol) were stirred at 110°C for 16 hours. The reaction mixture was concentrated to give (E)-1- (dimethylamino)hex-1-en-3-one (2.5 g). 1H NMR (400 MHz, CD3Cl) δ 7.54 (d, J = 12.4 Hz, 1H), 5.02 (d, J = 12.4 Hz, 1H), 3.07 (s, 6H), 2.48-2.38 (m, 2H), 1.67-1.58 (m, 2H), 0.93 (t, J = 7.2 Hz, 3H). Step 2: 5-propylisoxazole
Figure imgf000170_0003
A mixture of (E)-1-(dimethylamino)hex-1-en-3-one (2.5 g, 17.7 mmol) and NH2OH.HCl (2.46 g, 35.4 mmol) in EtOH (20 mL) was stirred at 80°C for 16 hours. The mixture was diluted with H2O (20 mL) and extracted with pentane (30 mL × 3). The organic phase was distilled to give 5-propylisoxazole (1.6 g, crude). 1H NMR (400 MHz, CD3Cl) δ 8.14 (d, J = 1.2 Hz, 1H), 5.98 (d, J = 1.2 Hz, 1H), 2.78 (t, J = 7.6 Hz, 2H), 1.80-1.69 (m, 2H), 0.99 (t, J = 7.6 Hz, 3H). Step 3: 4-bromo-5-propylisoxazole
Figure imgf000171_0001
To a solution of 5-propylisoxazole (1.6 g, crude) in DMF (15 mL) was added NBS (3.33 g, 18.7 mmol) and the reaction mixture was stirred at 15°C for 16 hours. The reaction mixture was quenched with water (10 mL), and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 5%) to give 4-bromo-5-propylisoxazole (1.6 g). 1H NMR (400 MHz, CD3Cl) δ 8.17 (s, 1H), 2.78 (t, J = 7.2 Hz, 2H), 1.81-1.71 (m, 2H), 0.98 (t, J = 7.2 Hz, 3H). Step 4: Methyl 3-fluoro-5-(5-propylisoxazol-4-yl)benzoate
Figure imgf000171_0002
(3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.0 g, 5.05 mmol), 4-bromo-5- propylisoxazole (960 mg, 5.05 mmol), KF (880 mg, 15.15 mmol) and Pd(t-Bu3P)2 (258 mg, 0.51 mmol) were taken up into a microwave tube in a mixture of dioxane (10 mL) and H2O (1 mL) under N2 atmosphere. The sealed tube was heated at 100°C for 60 minutes under microwave. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 10%) to give methyl 3- fluoro-5-(5-propylisoxazol-4-yl)benzoate (180 mg). 1H NMR (400 MHz, CD3Cl) δ 8.38 (s, 1H), 7.86-7.84 (m, 1H), 7.72-7.68 (m, 1H), 7.27-7.24 (m, 1H), 3.97 (s, 3H), 2.92 (t, J = 7.6 Hz, 2H), 1.85-1.78 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H). Step 5: 3-fluoro-5-(5-propylisoxazol-4-yl)benzoic acid
Figure imgf000172_0001
A mixture of methyl 3-fluoro-5-(5-propylisoxazol-4-yl)benzoate (310 mg, 1.18 mmol) in conc.HCl (10 mL) was stirred at 80°C for 14 hours. The mixture was extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL), dried over Na2SO4, filtered and concentrated to give 3-fluoro-5-(5-propylisoxazol-4-yl)benzoic acid (180 mg). Step 6: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-propylisoxazol-4- yl)benzamido)propanoate
Figure imgf000172_0002
To a solution of 3-fluoro-5-(5-propylisoxazol-4-yl)benzoic acid (90 mg, 0.36 mmol) and (R)- benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (145 mg, 0.40 mmol, HCl salt) in DMF (5 mL) was added TBTU (174 mg, 0.54 mmol) and DIPEA (140 mg, 1.08 mmol). The reaction mixture was stirred at 10°C for 1 hour. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5- propylisoxazol-4-yl)benzamido)propanoate (90 mg). 1H NMR (400 MHz, CDCl3) δ 8.34 (s, 1H), 7.54 (s, 1H), 7.38-7.29 (m, 11H), 7.22-7.18 (m, 1H), 6.95 (br, 1H), 5.91 (br d, J = 6.4 Hz, 1H), 5.23 (s, 2H), 5.12 (s, 2H), 4.64-4.58 (m, 1H), 4.00-3.76 (m, 2H), 2.91 (t, J = 7.6 Hz, 2H), 1.84-1.77 (m, 2H), 0.99 (t, J = 7.2 Hz, 3H). Step 7: (R)-2-amino-3-(3-fluoro-5-(5-propylisoxazol-4-yl)benzamido)propanoic acid
Figure imgf000173_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-propylisoxazol-4- yl)benzamido)propanoat (90 mg, 0.16 mmol) in 30 % HBr in AcOH (5 mL) was heated at 50°C for 16 hours. The reaction mixture was concentrated. The residue was washed with a mixture of MeCN (1 mL) and methyl tert-butyl ether (5 mL). The organic layer was decanted, then the residue was submitted to lyophilization to give (R)-2-amino-3-(3-fluoro-5-(5- propylisoxazol-4-yl)benzamido)propanoic acid (45 mg) as HBr salt. 1H NMR (400 MHz, DMSO-d6) δ 8.93 (s, 1H), 8.93-8.87 (m, 1H), 8.25 (br s, 2H), 7.82 (s, 1H), 7.68-7.58 (m, 2H), 4.07-4.03 (m, 1H), 3.84-3.64 (m, 2H), 2.97 (t, J = 7.6 Hz, 2H), 1.76-1.66 (m, 2H), 0.91 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 336.1 tR (min, Method D) = 1.757 [α]20,D = +4.0 (c = 2.0 mg/mL, CH3OH). Compound 2ac (R)-methyl 2-amino-3-(3-fluoro-5-(5-propylisoxazol-4-yl)benzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5- propylisoxazol-4-yl)benzamido)propanoate
Figure imgf000174_0001
To a solution of a mixture of 3-fluoro-5-(5-propylisoxazol-4-yl)benzoic acid (90 mg, 0.36 mmol) and (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (100 mg, 0.40 mmol, HCl salt) in DMF (5 mL) was added TBTU (174 mg, 10.54 mmol) and DIPEA (140 mg, 1.08 mmol). The reaction mixture was stirred at 10°C for 1 hour. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 30%) twice to give (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3- fluoro-5-(5-propylisoxazol-4-yl)benzamido)propanoate (80 mg). 1H NMR (400MHz, CDCl3) δ 8.38 (s, 1H), 7.60 (s, 1H), 7.41 (d, J = 9.2 Hz, 1H), 7.35 - 7.30 (m, 5H), 7.20 (d, J = 9.2 Hz, 1H), 7.14 (br, 1H), 5.88 (br d, J = 5.6 Hz, 1H), 5.13 (s, 2H), 4.62 - 4.55 (m, 1H), 3.99 - 3.92 (m, 1H), 3.85 - 3.75 (m, 4H), 2.92 (t, J = 7.6 Hz, 2H), 1.86 - 1.77 (m, 2H), 0.98 (t, J = 7.6 Hz, 3H). Step 2:
Figure imgf000174_0002
A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(5-propylisoxazol-4- yl)benzamido)propanoate (80 mg, 0.17 mmol) in the solution of 30% HBr in AcOH (5 mL) was heated at 20°C for 1 hour. The mixture was concentrated. The residue was washed with a mixture of MeOH (1 mL) and methyl tert-butyl ether (5 mL). The organic layer was decanted, then the residue was submitted to lyophilization to give (R)-methyl 2-amino-3-(3-fluoro-5-(5- propylisoxazol-4-yl)benzamido)propanoate (15 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.93 (s, 1H), 8.92-8.87 (m, 1H), 8.45 (br s, 3H), 7.80 (s, 1H), 7.66 - 7.59 (m, 2H), 4.25 - 4.21 (m, 1H), 3.83 - 3.68 (m, 5H), 2.98 (t, J = 7.6 Hz, 2H), 1.75 - 1.65 (m, 2H), 0.91 (t, J = 7.2 Hz, 3H). LCMS (MH+): m/z = 350.0, tR (min, Method D) = 2.033 min [α]20,D = +8.0 (c = 1 mg/mL,CH3OH). Compound 1x (R)-2-amino-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(2-ethyl-4- methylpyridin-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000175_0001
Step 1: methyl 3-(2-chloro-4-(trifluoromethyl)pyridin-3-yl)-5-fluorobenzoate
Figure imgf000175_0002
A mixture of 3-bromo-2-chloro-4-(trifluoromethyl)pyridine (1.5 g, 7.27 mmol), (3-fluoro-5- methoxycarbonylphenyl)boronic acid (1.44 g, 7.27 mmol), Pd(dppf)Cl2 (532 mg, 726.5 µmol), Na2CO3 (2.31 g, 21.80 mmol) in the mixture of H2O (2 mL) and Dioxane (18 mL) was degassed and purged with N23 times, and then the mixture was stirred at 80 °C for 3 hours under N2 atmosphere. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 20%) to give 3-(2-chloro-4- (trifluoromethyl)pyridin-3-yl)-5-fluorobenzoate (1.6 g). 1H NMR (400MHz, CDCl3) δ 8.30 (d, J = 5.2 Hz, 1H), 7.84-7.77 (m, 1H), 7.73-7.69 (m, 1H), 7.22- 7.13 (m, 2H), 3.95 (s, 3H), 2.13 (s, 3H). Step 2: methyl 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoate
Figure imgf000176_0001
A mixture of 3-(2-chloro-4-(trifluoromethyl)pyridin-3-yl)-5-fluorobenzoate (550 mg, 1.97 mmol), triethylborane (1 M in THF, 5.9 mL), Pd(dppf)Cl2 (144 mg, 196.64 µmol), CsOAc (1.13 g, 5.90 mmol) in THF (7 mL) was degassed and purged with N23 times, and then the mixture was stirred at 65 °C for 16 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 40%) to give methyl 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoate (430 mg). 1H NMR (400MHz, CDCl3) δ 8.45 (d, J = 5.2 Hz, 1H), 7.79-7.73 (m, 1H), 7.67 (dd, J = 1.6, 1.6 Hz, 1H), 7.13-7.08 (m, 1H), 7.05 (d, J = 5.2 Hz, 1H), 3.94 (s, 3H), 2.52 (q, J = 7.6 Hz, 2H), 2.02 (s, 3H), 1.13 (t, J = 7.6 Hz, 3H). Step 3: 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoic acid
Figure imgf000177_0001
To a solution of methyl 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoate (430 mg, 1.57 mmol) in the mixture of H2O (3 mL) and MeOH (3 mL) was added LiOH.H2O (198 mg, 4.72 mmol). The mixture was stirred at 10 °C for 16 hours. The mixture was concentrated to remove MeOH. Then HCl (2 M) was added to adjust PH=5~6 and concentrated to give 3-(2- ethyl-4-methylpyridin-3-yl)-5-fluorobenzoic acid (465 mg, crude) as HCl salt. LC-MS (MH+): m/z = 260.1 tR (min, Method D) = 0.584 Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5- fluorobenzamido)propanoate
Figure imgf000177_0002
To a solution of 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoic acid (116 mg, HCl salt, crude) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate hydrochloride (157 mg, 431.47 µmol, HCl salt) in DMF (3 mL) was added TBTU (189 mg, 588.4 µmol) and DIPEA (253 mg, 1.96 mmol). The mixture was stirred at 20 °C for 3 hours. The reaction mixture was quenched with H2O (5 mL), extracted with ethyl acetate (10 mL × 2). The combined organic layers were washed with brine (10 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 60%) twice to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5- fluorobenzamido)propanoate (140 mg). 1H NMR (400MHz, CDCl3) δ 8.45 (d, J = 5.2 Hz, 1H), 7.49-7.28 (m, 12H), 7.09-7.01 (m, 2H), 6.93 (m, 1H), 5.95 (d, J = 6.0 Hz, 1H), 5.29-5.15 (m, 2H), 5.08 (s, 2H), 4.64-4.52 (m, 1H), 4.02- 3.75 (m, 2H), 2.51 (q, J = 7.6 Hz, 2H), 2.02 (s, 3H), 1.13 (t, J = 7.6 Hz, 3H). Step 5: (R)-2-amino-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000178_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyl-4-methylpyridin-3-yl)- 5-fluorobenzamido)propanoate (140 mg, 245.78 µmol) in 30% HBr in AcOH (3 mL) was stirred at 50 °C for 16 hours. The mixture was concentrated. The residue was dissolved in MeCN (3 mL) and methyl tert-butyl ether (10 mL), and the solid was collected and lyophilizated. The residue was dissolved in AcOH (3 mL) and methyl tert-butyl ether (10 mL), and the solid was collected and lyophilizated to give (R)-2-amino-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5- fluorobenzamido)propanoic acid (70 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.99 (br s, 1H), 8.81 (br s, 1H), 8.37 (br s, 3H), 8.06-7.77 (m, 2H), 7.76-7.49 (m, 2H), 4.20-4.07 (m, 1H), 3.80-3.66 (m, 2H), 2.74-2.63 (m, 2H), 2.26 (s, 3H), 1.09 (m, 3H). LC-MS (MH+): m/z = 346.0 tR (min, Method D) = 0.983 [α]20,D = 1.5 (c = 4 mg/mL, MeOH). Compound 2ad (R)-methyl 2-amino-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzamido)propanoate Step 1:
Figure imgf000179_0001
To a solution of 3-(2-ethyl-4-methylpyridin-3-yl)-5-fluorobenzoic acid (116 mg, HCl salt, crude) and (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate hydrochloride (109 mg, 431.46 µmol, HCl salt) in DMF (3 mL) was added TBTU (189 mg, 588.36 µmol) and DIPEA (253 mg, 1.96 mmol). The mixture was stirred at 20 °C for 3 hours. The reaction mixture was quenched with H2O (5 mL), extracted with ethyl acetate (10 mL × 2). The combined organic layers were washed with brine (10 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel ( ethyl acetate/Petroleum ether with ethyl acetate from 0% to 80%) twice to give (R)-methyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5- fluorobenzamido)propanoate (110 mg). 1H NMR (400MHz, CDCl3) δ 8.44 (d, J = 4.8 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 7.42 - 7.28 (m, 6H), 7.21 - 7.01 (m, 3H), 6.01 - 5.85 (m, 1H), 5.09 (s, 2H), 4.67 - 4.48 (m, 1H), 3.99 - 3.73 (m, 5H), 2.52 (q, J = 7.6 Hz, 2H), 2.03 (s, 3H), 1.11 (t, J = 7.6 Hz, 3H). Step 2:
Figure imgf000179_0002
A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(2-ethyl-4-methylpyridin-3-yl)- 5-fluorobenzamido)propanoate (110 mg, 0.223 mmol) in 30% HBr in AcOH (3 mL) was stirred at 15 °C for 3 hours. The mixture was concentrated. The residue was dissolved in MeOH (3 mL) and methyl tert-butyl ether (10 mL) was added, the solid was collected and lyophilizated to give (R)-methyl 2-amino-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5- fluorobenzamido)propanoate (80 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.03-8.98 (m, 1H), 8.80 (d, J = 6.0 Hz, 1H), 8.50 (br s, 3H), 7.94 (d, J = 6.0 Hz, 1H), 7.86 (d, J = 9.2 Hz, 1H), 7.71 (s, 1H), 7.57 (d, J = 8.8 Hz, 1H), 4.28 - 4.19 (m, 1H), 3.85 - 3.65 (m, 5H), 2.68 (q, J = 7.6 Hz, 2H), 2.26 (s, 3H), 1.19 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 360.0, tR (min, Method D) = 1.033 min [α]20,D = -0.55 (c = 11 mg/mL, MeOH). Compound 1y (R)-2-amino-3-(3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-fluoro-5-(1-propyl-1H- 1,2,3-triazol-5-yl)benzamido)propanoic acid
Figure imgf000180_0001
Step 1: 1-propyl-1H-1,2,3-triazole
Figure imgf000180_0002
To a solution of 2-(trimethylsilyl)-2H-1,2,3-triazole (2 g, 14.16 mmol) and 1-iodopropane (2.65 g, 15.58 mmol) in dioxane (20 mL) was added KOH (794 mg, 14.16 mmol). The resulting solution was stirred at 100°C for 16 hours. The reaction mixture was quenched with water (10 mL), and extracted with ethyl acetate (20 mL × 3). The combined organic extracts were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 50%) to give 1-propyl-1H-1,2,3-triazole (1.1 g). 1H NMR (400MHz, DMSO-d6) δ 8.12 (s, 1H), 7.71 (s, 1H), 4.31 (t, J = 7.2 Hz, 2H), 1.87-1.77 (m, 2H), 0.81 (t, J = 7.2 Hz, 3H). Step 2: 5-iodo-1-propyl-1H-1,2,3-triazole
Figure imgf000181_0002
To a stirred solution of 1-propyl-1H-1,2,3-triazole (400 mg, 3.60 mmol) in THF (10 mL) was added n-BuLi (2.5 M in hexane, 1.73 mL) dropwise at -78oC under nitrogen and the mixture was stirred for 1 hour at the same temperature. Then I2 (1.10 g, 4.32 mmol) (dissolving in 5 mL THF) was added dropwise at -78oC over 15 minutes. The reaction mixture was allowed to warm to 20°C for 2 hours. The reaction mixture was quenched with ice water (20 mL), and extracted with ethyl acetate (30 mL × 3). The combined organic extracts were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 35%) to give 5-iodo-1-propyl-1H-1,2,3-triazole (740 mg). 1H NMR (400MHz, CDCl3) δ 7.72 (s, 1H), 4.37 (t, J = 7.6 Hz, 2H), 2.01-1.91 (m, 2H), 0.98 (t, J = 7.6 Hz, 3H). Step 3: methyl 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzoate
Figure imgf000181_0001
(3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (400 mg, 2.02 mmol), 5-iodo-1-propyl- 1H-1,2,3-triazole (479 mg, 2.02 mmol), K2CO3 (838 mg, 6.06 mmol) and Pd(dppf)Cl2.CH2Cl2 (165 mg, 0.20 mmol) were taken up into a microwave tube in a mixture of dioxane (8 mL) and H2O (0.8 mL) under N2 atmosphere. The sealed tube was heated at 100°C for 60 minutes under microwave. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3) and dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 35%) to give methyl 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzoate (470 mg). 1H NMR (400MHz, CDCl3) δ 7.88 (t, J = 1.2 Hz, 1H), 7.86-7.82 (m, 1H), 7.76 (s, 1H), 7.33-7.29 (m, 1H), 4.35 (t, J = 7.2 Hz, 2H), 3.98 (s, 3H), 1.94 - 1.84 (m, 2H), 0.88 (t, J = 7.2 Hz, 3H). Step 4: 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzoic acid
Figure imgf000182_0001
To a solution of methyl 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzoate (470 mg, 1.79 mmol) in a mixture of THF (8 mL) and H2O (4 mL) was added LiOH.H2O (187 mg, 4.46 mmol) and the mixture was stirred at 30°C for 16 hours. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-fluoro-5- (1-propyl-1H-1,2,3-triazol-5-yl)benzoic acid (420 mg). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5- yl)benzamido)propanoate
Figure imgf000183_0001
To a solution of a mixture of 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzoic acid (210 mg, 0.84 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (338 mg, 0.93 mmol, HCl salt) in DMF (8 mL) was added TBTU (406 mg, 1.25 mmol) and DIPEA (327 mg, 2.53 mmol). The reaction mixture was stirred at 30°C for 1 hour. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 50%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl- 1H-1,2,3-triazol-5-yl)benzamido)propanoate (400 mg). 1H NMR (400 MHz, CDCl3) δ 7.70 (s, 1H), 7.58 (s, 1H), 7.54-7.49 (m, 1H), 7.40-7.28 (m, 11H), 7.23-7.19 (m, 1H), 6.02 (br d, J = 6.4 Hz, 1H), 5.22 (s, 2H), 5.10 (s, 2H), 4.65-4.58 (m, 1H), 4.31 (t, J = 7.2 Hz, 2H), 4.00-3.80 (m, 2H), 1.89-1.79 (m, 2H), 0.84 (t, J = 7.2 Hz, 3H). Step 6: (R)-2-amino-3-(3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzamido)propanoic acid
Figure imgf000183_0002
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-1H-1,2,3- triazol-5-yl)benzamido)propanoate (400 mg, 0.71 mmol) in the solution of 30% HBr in AcOH (15 mL) was heated at 50°C for 15 hours. The mixture was concentrated. The residue was added water (5 mL) and washed with methyl tert-butyl ether (5 mL × 2). The aqueous layer was lyophilized to give (R)-2-amino-3-(3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5- yl)benzamido)propanoic acid (250 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.05-8.98 (m, 1H), 8.37 (br s, 3H), 8.00 (s, 1H), 7.88 (s, 1H), 7.84-7.79 (m, 1H), 7.75-7.71 (m, 1H), 4.46-4.39 (m, 2H), 4.18-4.10 (m, 1H), 3.84-3.67 (m, 2H), 1.75-1.64 (m, 2H), 0.77-0.71 (m, 3H). LC-MS (MH+): m/z = 336.0 tR (min, Method D) = 1.249 [α]20,D = +0.6 (c = 7 mg/mL,CH3OH). Compound 2ae (R)-methyl 2-amino-3-(3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl- 1H-1,2,3-triazol-5-yl)benzamido)propanoate
Figure imgf000184_0001
To a solution of a mixture of 3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzoic acid (210 mg, 0.84 mmol) and (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (268 mg, 0.93 mmol, HCl salt) in DMF (8 mL) was added TBTU (406 mg, 1.26 mmol) and DIPEA (327 mg, 2.56 mmol). The reaction mixture was stirred at 30°C for 1 hour. The mixture was added water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 55%) twice to give (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3- fluoro-5-(1-propyl-1H-1,2,3-triazol-5-yl)benzamido)propanoate (350 mg). 1H NMR (400 MHz, CDCl3) δ 7.71 (s, 1H), 7.64 - 7.59 (m, 2H), 7.46 (br s, 1H), 7.35 - 7.29 (m, 5 H), 7.25 - 7.21 (m, 1H), 5.97 (br d, J = 6.8 Hz, 1H), 5.11 (s, 2H), 4.62 - 4.55 (m, 1H), 4.32 (t, J = 7.2 Hz, 2H), 4.01 - 3.78 (m, 5H), 1.90 - 1.80 (m, 2H), 0.87 (t, J = 7.6 Hz, 3H). Step 2:
Figure imgf000185_0001
A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-1H-1,2,3- triazol-5-yl)benzamido)propanoate (350 mg, 0.72 mmol) in the solution of 30% HBr in AcOH (15 mL) was stirred at 30°C for 1 hour. The mixture was concentrated. The residue was added water (5 mL) and washed with methyl tert-butyl ether (5 mL × 2). The aqueous layer was lyophilized to give (R)-methyl 2-amino-3-(3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5- yl)benzamido)propanoate (200 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.03-8.96 (m, 1H), 8.48 (br s, 3H), 7.99 (s, 1H), 7.85 (s, 1H), 7.82 - 7.78 (m, 1H), 7.76 - 7.71 (m, 1H), 4.41 (t, J = 7.6 Hz, 2H), 4.27 - 4.20 (m, 1H), 3.77 - 3.74 (m, 5H), 1.75 - 1.65 (m, 2H), 0.75 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 350.1, tR (min, Method D) = 1.329 min [α]20,D = +4.3 (c = 1.4 mg/mL,CH3OH). Compound 1z (R)-2-amino-3-(3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-chloro-1-propyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000185_0002
Step 1: methyl 3-fluoro-5-(1-propyl-1H-pyrazol-5-yl)benzoate
Figure imgf000186_0001
A mixture of 5-bromo-1-propyl-pyrazole (0.885 g, 4.68 mmol), (3-fluoro-5-methoxycarbonyl- phenyl)boronic acid (772 mg, 3.90 mmol), Pd(dtbpf)Cl2 (254 mg, 0.39 mmol), K3PO4 (2.48 g, 11.70 mmol) in the mixture of dioxane (35 mL) and H2O (3.5 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 100 °C for 3 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give methyl 3- fluoro-5-(1-propyl-1H-pyrazol-5-yl)benzoate (0.9 g). 1H NMR (400MHz, CDCl3) δ 7.88 (t, J = 1.2 Hz, 1H), 7.79-7.74 (m, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.33-7.27 (m, 1H), 6.33 (d, J = 1.8 Hz, 1H), 4.12-4.08 (m, 2H), 3.95 (s, 3H), 1.88-1.79 (m, 2H), 0.82 (t, J = 7.6 Hz, 3H). Step 2: methyl 3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000186_0002
To a solution of methyl 3-fluoro-5-(1-propyl-1H-pyrazol-5-yl)benzoate (0.89 g, 3.39 mmol) in THF (20 mL) was added NCS (680 mg, 5.09 mmol). The mixture was stirred at 70 °C for 2 hours. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 10%) to give methyl 3-(4-chloro- 1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoate (0.9 g). 1H NMR (400MHz, CDCl3) δ 7.87 (t, J=1.6 Hz, 1H), 7.85-7.79 (m, 1H), 7.54 (s, 1H), 7.35-7.29 (m, 1H), 4.05-3.99 (m, 2H), 3.95 (s, 3H), 1.80-1.74 (m, 2H), 0.80 (t, J=7.6 Hz, 3H). Step 3: 3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000187_0001
To a solution of methyl 3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoate (0.90 g, 3.03 mmol) in a mixture of MeOH (10 mL) and H2O (10 mL) was added LiOH.H2O (255 mg, 6.07 mmol). The mixture was stirred at 25°C for 1 hour .The mixture was concentrated. The residue was adjust pH=4 with aq. KHSO4 (6 mL). The precipitate was filtered to give 3-(4- chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (0.80 g). LC-MS (MH+): m/z = 283.1 tR (min, Method G) = 0.804 Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-propyl-1H-pyrazol-5-yl)- 5-fluorobenzamido)propanoate
Figure imgf000187_0002
A mixture of 3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (0.20 g, 0.71 mmol), (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (258 mg, 0.71 mmol, HCl salt), TBTU (341 mg, 1.06 mmol) and DIPEA (457 mg, 3.54 mmol) in DMF (20 mL) was stirred at 30°C for 16 hours. The mixture was diluted with H2O (50 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 35%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (0.18 g). 1H NMR (400MHz, CDCl3) δ 7.55 (s, 1H), 7.51 (s, 1H), 7.46 (br d, J=8.4 Hz, 1H), 7.37-7.29 (m, 10H), 7.26-7.23 (m, 1H), 6.98 (br, 1H), 5.94 (br d, J=6.4 Hz, 1H), 5.26-5.16 (m, 2H), 5.10 (s, 2H), 4.65-4.54 (m, 1H), 4.00 (t, J=7.2 Hz, 2H), 3.97-3.90 (m, 1H), 3.85-3.76 (m, 1H), 1.82-1.72 (m, 2H), 0.79 (t, J=7.6 Hz, 3H). Step 5: (R)-2-amino-3-(3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000188_0001
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-propyl-1H-pyrazol- 5-yl)-5-fluorobenzamido)propanoate (0.18 g, 0.3 mmol) and HBr in AcOH (8 mL, 30% in AcOH) were stirred at 30°C for 15 hours. The mixture was concentrated. The residue was diluted with AcOH (3 mL). methyl tert-butyl ether (50 mL) was added to the mixture. The precipitate was filtered under N2. The filter cake was lyophilized to give (R)-2-amino-3-(3-(4-chloro-1- propyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid (50 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.99-8.93 (m, 1H), 8.34 (br s, 3H), 7.86 (d, J=8.8 Hz, 1H), 7.79 (s, 1H), 7.75 (s, 1H), 7.63 (d, J=8.8 Hz, 1H), 4.17-4.11 (m, 1H), 4.04 (t, J=6.8 Hz, 2H), 3.85-3.77 (m, 1H), 3.74-3.66 (m, 1H), 1.70-1.58 (m, 2H), 0.70 (t, J=7.8 Hz, 3H). LC-MS (MH+): m/z = 369.0 tR (min, Method D) = 1.663 [α]20,D = 2.0 (c = 2.0 mg/mL, CH3OH). Compound 2af (R)-methyl 2-amino-3-(3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-propyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000188_0002
A mixture of 3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (0.2 g, 0.71 mmol), (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (178 mg, 0.71 mmol, HCl salt), TBTU (341 mg, 1.06 mmol) and DIPEA (457 mg, 3.54 mmol) in DMF (20 mL) was stirred at 30°C for 16 hours. The mixture was diluted with H2O (50 mL) and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (20 mL), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 45%) to give (R)-methyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (0.2 g). 1H NMR (400MHz, CDCl3) δ 7.62 - 7.52 (m, 3H), 7.36-7.29 (m, 5H), 7.27 - 7.24 (m, 1H), 7.18 (br, 1H), 5.90 (br d, J=6.0 Hz, 1H), 5.10 (s, 2H), 4.62-4.52 (m, 1H), 4.02 (t, J=7.2 Hz, 2H), 3.97 - 3.89 (m, 1H), 3.85 - 3.75 (m, 4H), 1.83 - 1.73 (m, 2H), 0.80 (t, J=7.6 Hz, 3H). Step 2:
Figure imgf000189_0001
mixture of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-propyl-1H-pyrazol- 5-yl)-5-fluorobenzamido)propanoate (0.2 g, 0.39 mmol) and HBr in AcOH (8 mL, 30% in AcOH) were stirred at 30°C for 15 hours. The mixture was concentrated. The residue was diluted with AcOH (3 mL) and methyl tert-butyl ether (50 mL) was added to the mixture. The precipitate was filtered under N2. The filter cake was lyophilized. The residue was purified by preparative HPLC (Method AA) to give (R)-methyl 2-amino-3-(3-(4-chloro-1-propyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (14 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.18-9.12 (m, 1H), 8.68 (br s, 3H), 7.94 - 7.87 (m, 1H), 7.82- 7.80 (m, 1H), 7.75 (s, 1H), 7.64-7.62 (m, 1H), 4.26-4.20 (m, 1H), 4.05 (t, J=6.8 Hz, 2H), 3.81- 3.78 (m, 2H), 3.74 (s, 3H), 1.70-1.59 (m, 2H), 0.70 (t, J=6.8 Hz, 3H). LCMS (MH+): m/z = 383.2, tR (min, Method D) = 1.845 min [α]20,D = 3.0 (c = 2.0 mg/mL, CH3OH). Compound 1aa (R)-2-amino-3-(3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-chloro-1-ethyl-3- methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000190_0001
Step 1: 1-ethyl-3-methyl-1H-pyrazole
Figure imgf000190_0002
To a solution of 3-methyl-1H-pyrazole (4 g, 48.72 mmol) in THF (100 mL) was added NaH (2.92 g, 73.01 mmol, 60% in mineral oil) at 0°C and the reaction was stirred at 0°C for 1 hour. iodoethane (9.12 g, 58.46 mmol) was added at 0°C and the resulting mixture was stirred at 30°C for 16 hours. H2O (100 ml) was added to quench the reaction at 0°C and extracted with DCM (100 mL × 3). The combined organic layers were washed with brine (50 mL × 2), dried over Na2SO4, filtered and concentrated to give 1-ethyl-3-methyl-1H-pyrazole (5 g, crude). Step 2: 1-ethyl-5-iodo-3-methyl-1H-pyrazole
Figure imgf000191_0001
To a solution of 1-ethyl-3-methyl-1H-pyrazole (5 g, crude) in THF (100 mL) was added n-BuLi (20 mL, 2.5 M in hexane) at -78°C and the reaction was stirred at -78°C for 1 hour. I2 (12.1 g, 47.66 mmol) was added and the reaction was stirred at -78°C for 1 hour. H2O (50 ml) was added to quench the reaction and extracted with DCM (100 mL × 3). The combined organic layers were washed with brine (100 mL × 2), dried over Na2SO4, filtered and concentrated to give 1-ethyl-5-iodo-3-methyl-1H-pyrazole (10 g, crude). Step 3: methyl 3-(1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000191_0002
A mixture of 1-ethyl-5-iodo-3-methyl-1H-pyrazole (1.0 g, crude), (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (840 mg, 4.24 mmol), Pd(dppf)Cl2 (310 mg, 0.42 mmol) and K2CO3 (1.76 g, 12.73 mmol) in a mixture of dioxane (50 mL) and H2O (5 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 80°C for 2 hours under N2 atmosphere. The mixture was diluted with H2O (50 ml) and extracted with ethyl acetate (100 mL × 3). The combined organic layers were washed with brine (100 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi flash (silica gel, petroleum ether/ethyl acetate with ethyl acetate from 0~30%) to give methyl 3-(1-ethyl- 3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (900 mg). 1H NMR (400 MHz, CDCl3) δ 7.86 (s, 1H), 7.78-7.72 (m, 1H), 7.31-7.27 (m, 1H), 6.11 (s, 1H), 4.10 (q, J = 7.2 Hz, 2H), 3.95 (s, 3H), 2.31 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H). Step 4: methyl 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000192_0001
A mixture of methyl 3-(1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (900 mg, 3.43 mmol) and NCS (500 mg, 3.74 mmol) in THF (2 mL) was stirred at 70°C for 2 hours. The mixture was concentrated. The residue was purified by Combi flash (silica gel, petroleum ether/ethyl acetate with ethyl acetate from 0~20%) and further purified by preparative HPLC (Method CC) to give methyl 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (300 mg). 1H NMR (400 MHz, CDCl3) δ 7.88 (dd, J = 1.6 Hz, 1.2 Hz, 1H), 7.84-7.79 (m, 1H), 7.36-7.30 (m, 1H), 4.06 (q, J = 7.2 Hz, 2H), 3.97 (s, 3H), 2.30 (s, 3H), 1.35 (t, J = 7.2 Hz, 3H). Step 5: 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000192_0002
A mixture of methyl 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoate (350 mg, 1.18 mmol) and LiOH.H2O (150 mg, 3.57 mmol) in a mixture of MeOH (8 mL) and H2O (2 mL) was stirred at 30°C for 4 hours. The mixture was concentrated. H2O (2 mL) was added, acidified with satd. aqueous KHSO4 to pH 3~4 and extracted with ethyl acetate (10 mL × 5). The combined organic layers were washed with brine (10 mL), dried over Na2SO4, filtered and concentrated to give 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (300 mg). LC-MS (MH+): m/z = 283.0 tR (min, Method D) = 0.761 Step 6: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-3-methyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000193_0001
A mixture of 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (150 mg, 0.53 mmol), (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (233 mg, 0.64 mmol, HCl salt), TBTU (256 mg, 0.80 mmol) and DIPEA (148 mg, 1.15 mmol) in DMF (5 mL) was stirred at 30°C for 16 hours. The mixture was diluted with H2O (10 mL) and extracted with ethyl acetate (15 mL × 3). The organic phase was washed with brine (20 mL × 2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi flash (silica gel, petroleum ether/ethyl acetate with ethyl acetate from 0~40%) to give (R)-benzyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (270 mg). 1H NMR (400 MHz, CDCl3) δ 7.50 (s, 1H), 7.48-7.41 (m, 1H), 7.37-7.26 (m, 11H), 6.97-6.84 (m, 1H), 5.91 (d, J = 6.8 Hz, 1H), 5.27-5.17 (m, 2H), 5.15-5.04 (m, 2H), 4.65-4.55 (m, 1H), 4.04 (q, J = 7.2 Hz, 2H), 4.00-3.75 (m, 2H), 2.30 (s, 3H), 1.34 (t, J = 7.2 Hz, 3H). Step 7: (R)-2-amino-3-(3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000193_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-3-methyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (270 mg, 0.46 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50°C for 3 hours. The solvent was removed under reduced pressure. The solid was suspended in TBME (5 mL), filtered, washed with TBME (1 mL × 2) and lyophilized to give (R)-2-amino-3-(3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid (105 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.02-8.85 (m, 1H), 8.45-8.20 (m, 3H), 7.83 (d, J = 9.2 Hz, 1H), 7.77 (s, 1H), 7.59 (d, J = 9.2 Hz, 1H), 4.15-4.10 (m, 1H), 4.05-4.00 (m, 2H), 3.75-3.65 (m, , 2H), 2.20 (s, 3H), 1.22 (t, J=7.2 Hz, 3H). LC-MS (MH+): m/z = 369.1 tR (min, Method D) = 1.721
Figure imgf000194_0001
Compound 2ag (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-3- methyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000194_0002
A mixture of 3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (150 mg, 0.530 mmol), (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (184 mg, 0.64 mmol, as HCl salt), TBTU (256 mg, 0.80 mmol) and DIPEA (148 mg, 1.15 mmol) in DMF (5 mL) was stirred at 30°C for 16 hours. The mixture was diluted with H2O (10 ml) and extracted with ethyl acetate (15 mL×3). The combined organic layers were washed with brine (20 mL×2), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi flash (silica gel, petroleum ether/ethyl acetate with ethyl acetate from 0~40%) to give (R)-methyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (45 mg). 1H NMR (400 MHz, CDCl3) δ 7.65-7.55 (m, 2H), 7.42-7.40 (m, 1H), 7.37-7.26 (m, 5H), 7.25- 7.22 (m, 1H), 6.04 (d, J = 6.8 Hz, 1H), 5.15-5.04 (m, 2H), 4.62-4.49 (m, 1H), 4.55 (q, J = 7.2 Hz, 2H), 4.95-3.82 (m, 2H), 3.79 (s, 3H), 2.28 (s, 3H), 1.33 (t, J = 7.6 Hz, 3H). Step 2:
Figure imgf000195_0001
A mixture of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-3-methyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (40 mg, 0.078 mmol) in 30% HBr in AcOH (5 mL) was stirred at 30°C for 2 hours. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl-3-methyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (25 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.10-9.03 (m, 1H), 8.60 (br s, 3H), 7.85 (d, J = 9.6 Hz, 1H), 7.77 (s, 1H), 7.59 (d, J = 8.8 Hz, 1H), 4.30-4.15 (m, 1H), 4.02 (q, J = 7.6 Hz, 2H), 3.74-3.81 (m, 2H), 3.73 (s, 3H), 2.20 (s, 3H), 1.22 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 383.0, tR (min, Method D) = 1.720 min
Figure imgf000195_0002
-4.00 (c = 2 mg/mL, MeOH). Compound 1ab (R)-2-amino-3-(3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(1-ethyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000196_0001
Step 1: 1-ethyl-5-iodo-1H-pyrazole
Figure imgf000196_0002
To a stirred solution of 1-ethyl-1H-pyrazole (5.0 g, 52.01 mmol) in THF (60 mL) was added n- BuLi (25.0 mL, 2.5M in hexane) dropwise at -78oC under nitrogen and the mixture was stirred for 1 hour at the same temperature. Then I2 (15.8 g, 62.4 mmol, dissolving in 40 mL THF) was added dropwise at -78oC over 15 minutes. The reaction mixture was allowed to warm to 30°C for 2 hours. The reaction was quenched with ice water (20 mL) and extracted with ethyl acetate (30 mL × 3). The organic layer was washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 20%) to give 1-ethyl-5-iodo-1H- pyrazole (11 g).1H NMR (400MHz, CDCl3) δ 7.51 (d, J = 2.0 Hz, 1H), 6.42 (d, J = 2.0 Hz, 1H), 4.26 (q, J = 7.2 Hz, 2H), 1.44 (t, J = 7.2 Hz, 3H). Step 2: methyl 3-(1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000197_0001
(3-fluoro-5-(methoxycarbonyl)phenyl)boronic acid (1.0 g, 5.05 mmol), 1-ethyl-5-iodo-1H- pyrazole (1.12 g, 5.05 mmol), K2CO3 (2.09 g, 15.15 mmol) and Pd(dppf)Cl2.CH2Cl2 (413 mg, 0.51 mmol) were taken up into a microwave tube in a mixture of dioxane (10 mL) and H2O (1 mL) under N2 atmosphere. The sealed tube was heated at 100 °C for 60 minutes under microwave. The reaction was quenched with ice water (20 mL), and extracted with ethyl acetate (30 mL × 3). The organic layer was washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 20%) to give methyl 3-(1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzoate (1.0 g). 1H NMR (400MHz, CDCl3) δ 7.90 (s, 1H), 7.80-7.76 (m, 1H), 7.58 (s, 1H), 7.34-7.29 (m, 1H), 6.35 (s, 1H), 4.20 (q, J = 7.2 Hz, 2H), 3.97 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H). Step 3: methyl 3-(1-ethyl-4-iodo-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000197_0002
To a solution of methyl 3-(1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (350 mg, 1.41 mmol) in THF (8 mL) was sadded NIS (1049 mg, 4.65 mmol) and the reaction mixture was stirred at 70°C for 4 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 10%) to give methyl 3-(1-ethyl-4-iodo-1H-pyrazol-5-yl)-5-fluorobenzoate (500 mg). 1H NMR (400MHz, CDCl3) δ 7.88-7.83 (m, 2H), 7.62 (s, 1H), 7.32-7.29 (m, 1H), 4.13 (q, J = 7.2 Hz, 2H), 3.97 (s, 3H), 1.36 (t, J = 7.2 Hz, 3H). Step 4: methyl 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000198_0001
A mixture of methyl 3-(1-ethyl-4-iodo-1H-pyrazol-5-yl)-5-fluorobenzoate (500 mg, 1.34 mmol), methyl 2,2-difluoro-2-fluorosulfonyl-acetate (385 mg, 2.00 mmol), CuI (51 mg,0.27 mmol) in DMF (8 mL) was heated at 120°C for 4 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with saturated brines (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 15%) to give methyl 3-(1-ethyl-4-(trifluoromethyl)-1H- pyrazol-5-yl)-5-fluorobenzoate (240 mg). 1H NMR (400MHz, CDCl3) δ 7.91-7.87 (m, 1H), 7.87-7.85 (m, 1H), 7.80 (s, 1H), 7.32-7.28 (m, 1H), 4.04 (q, J = 6.8 Hz, 2H), 3.97 (s, 3H), 1.39 (t, J = 7.2 Hz, 3H). Step 5: 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000198_0002
To a solution of methyl 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoate (240 mg, 0.76 mmol) in the mixture of THF (4 mL) and H2O (1 mL) was added LiOH.H2O (48 mg, 1.14 mmol), the mixture was stirred at 20°C for 1 hour. The reaction mixture was quenched with water (5 mL), extracted with ethyl acetate (10 mL × 3). The aqueous phase was adjusted to pH=3~4 with HCl (6M), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated to give 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoic acid (226 mg). Step 6: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-(trifluoromethyl)-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000199_0001
To a mixture of 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoic acid (113 mg, 0.37 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (150 mg, 0.41 mmol, HCl salt) in DMF (6 mL) was added TBTU (180 mg, 0.56 mmol) and DIPEA (145 mg, 1.12 mmol), the mixture was stirred at 20°C for 1 hour. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 30%). The obtained material was further purified by Chiral SFC (Instrument: Berger, MULTIGR AM-II; Column: DAICEL CHIRALCEL OD- H(250mm*30mm,5μm); Mobile phase: supercritical CO2/IPA (0.1% NH3`H2O, v%) = 65/35; low Rate: 60 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (160 mg). 1H NMR (400MHz, CDCl3) δ 7.81 (s, 1H), 7.54-7.48 (m, 2H), 7.38-7.30 (m, 10H), 7.25-7.22 (m, 1H), 6.95 (br s, 1H), 5.89 (br d, J = 6.0 Hz, 1H), 5.26-5.19 (m, 2H), 5.11 (s, 2H), 4.64-4.57 (m, 1H), 4.09-3.93 (m, 3H), 3.83-3.74 (m, 1H), 1.38 (t, J = 7.6 Hz, 3H). Step 7: (R)-2-amino-3-(3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000200_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-(trifluoromethyl)- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (160 mg, 0.26 mmol) in the solution of 30% HBr in AcOH (5 mL) was stirred at 50°C for 2 hours. The mixture was concentrated. The residue was added HOAc (3 mL), then added methyl tert-butyl ether (5 mL). The formed solid was collected by filtration and added water (10 mL) for freeze-drying. The obtained solid was further purified by preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-(1-ethyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid (28 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.97-8.92 (m, 1H), 8.34-8.30 (m, 3H), 8.04 (s, 1H), 7.90 (d, J = 9.2 Hz, 1H), 7.78 (s, 1H), 7.67 (d, J = 9.2 Hz, 1H), 4.16-4.10 (m, 1H), 4.00 (q, J = 7.2 Hz, 2H), 3.84-3.65 (m, 2H), 1.27 (t, J = 7.2 Hz, 3H). LC-MS (MH+): m/z = 389.0 tR (min, Method D) = 1.756 [α]20,D = -2.7 (c = 4.5 mg/mL, CH3OH). Compound 2ah (R)-methyl 2-amino-3-(3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000200_0002
To a mixture of 3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzoic acid (113 mg, 0.37 mmol) and (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (104 mg, 0.41 mmol, HCl salt) in DMF (6 mL) was added TBTU (180 mg, 0.56 mmol) and DIPEA (145 mg, 1.12 mmol), the mixture was stirred at 20°C for 1 hour. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 30%). The obtained material was further purified by Chiral SFC (Instrument: Thar200; Column: DAICEL CHIRALCEL OJ-H(250mm*30mm,5μm); Mobile phase: supercritical CO2/MeOH (0.1% NH3`H2O, v%) = 80/20; low Rate: 50 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give (R)-methyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (93 mg). 1H NMR (400MHz, CDCl3) δ 7.80 (s, 1H), 7.65 - 7.55 (m, 2H), 7.37 - 7.31 (m, 5H), 7.26 - 7.22 (m, 1H), 7.14 (br, 1H), 5.87 (br, 1H), 5.11 (s, 2H), 4.61 - 4.54 (m, 1H), 4.04 (q, J = 7.6 Hz, 2H), 4.00 - 3.79 (m, 5H), 1.39 (t, J = 7.2 Hz, 3H). Step 2:
Figure imgf000201_0001
A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(1-ethyl-4-(trifluoromethyl)- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (93 mg, 0.17 mmol) in the solution of 30% HBr in AcOH (5 mL) was stirred at 20°C for 1 hour. The mixture was concentrated. The sample was added water (15 mL) and lyophilized to give (R)-methyl 2-amino-3-(3-(1-ethyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (70 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.98-8.91 (m, 1H), 8.44 (br s, 3H), 8.05 (s, 1H), 7.88 (d, J = 9.6 Hz, 1H), 7.77 (s, 1H), 7.68 (d, J = 8.8 Hz, 1H), 4.28 - 4.20 (m, 1H), 4.01 (q, J = 6.8 Hz, 2H), 3.82 - 3.66 (m, 5H), 1.27 (t, J = 6.8 Hz, 3H). LCMS (MH+): m/z = 403.0, tR (min, Method D) = 1.825 min [α]20,D = +2.0 (c = 1.0 mg/mL, CH3OH). Compound 1ac (R)-2-amino-3-(3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-(4-(difluoromethyl)-1- propyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000202_0001
Step 1: methyl 3-fluoro-5-(4-formyl-1-propyl-1H-pyrazol-5-yl)benzoate
Figure imgf000202_0002
To a stirred solution of methyl 3-fluoro-5-(1-propyl-1H-pyrazol-5-yl)benzoate (300 mg, 1.14 mmol) in POCl3 (4.95 g, 32.28 mmol) was added DMF (950 mg, 13.00 mmol) and the mixture was heated to 100°C for 16 hours. The mixture was poured into H2O (5 mL) at 0°C, neutralize with sat. aqueous Na2CO3 to pH 7~8 and extracted with ethyl acetate (5 mL × 2 ). The organic phase was washed with brine (5 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash (silica gel, from 0 to 30%, Ethyl acetate in petroleum ether) to give methyl 3-fluoro-5-(4-formyl-1-propyl-1H-pyrazol-5-yl)benzoate (220 mg). 1H NMR (400MHz, CDCl3) δ 9.63 (s, 1H), 8.08 (s, 1H), 7.93 - 7.87 (m, 2H), 7.36 - 7.31 (m, 1H), 4.01 (t, J = 7.6 Hz, 2H), 3.97 (s, 3H), 1.88 - 1.80 (m, 2H), 0.83 (t, J = 7.6 Hz, 3H). Step 2: methyl 3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000203_0001
To a solution of methyl 3-fluoro-5-(4-formyl-1-propyl-1H-pyrazol-5-yl)benzoate (250 mg, 0.86 mmol) in DCM (5 mL) was added DAST (1.22 g, 7.57 mmol) at 0°C and the resulting mixture was stirred at 25°C for 1 hour. Additional DAST (1.22 g, 7.57 mmol) was added and the resulting mixture was stirred at 25°C for 1 hour. Additional DAST (1.22 g, 7.57 mmol) was added and the resulting mixture was stirred at 25°C for 1 hour. The mixture was poured into sat. aqueous NaHCO3 to pH 7~8 at 0°C and extracted with DCM (30 mL × 2). The organic phase was washed with brine (10 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash (silica gel, from 0 to 30%, Ethyl acetate in petroleum ether) to give methyl 3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoate (210 mg). 1H NMR (400MHz, CDCl3) δ 7.90 - 7.82 (m, 2H), 7.77 (s, 1H), 7.32-7.26 (m, 1H), 6.45 (t, J = 55.6 Hz, 1H), 4.01 - 3.93 (m, 5H), 1.85 - 1.75 (m, 2H), 0.82 (t, J=7.6 Hz, 3H). Step 3: 3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000203_0002
To a solution of methyl 3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoate (235 mg, 0.75 mmol) in MeOH (4 mL) was added a solution of LiOH.H2O (95 mg, 2.26 mmol) in H2O (1 mL) and the resulting mixture was stirred at 25°C for 2 hours. The mixture was concentrated to move MeOH, then added sat. aqueous KHSO4 to adjust pH = 4~5 and extracted with EtOAc (10 mL × 2). The organic phase was washed with brine (10 mL × 2), dried over Na2SO4 and concentrated to give 3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzoic acid (210 mg, 93.6% yield). LC-MS (MH+): m/z = 299.1 tR (min, Method C) = 0.78 Step 4: 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000204_0001
A mixture of 3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (100 mg, 0.334 mmol), (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (147 mg, 0.40 mmol, HCl salt), TBTU (161 mg, 0.50 mmol) and DIPEA (87 mg, 0.67 mmol) in DMF (5 mL) was stirred at 25 °C for 16 hours. The mixture was diluted with H2O (5 mL) and extracted with ethyl acetate (5 mL × 2). The organic phase was washed with brine (5 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash (silica gel, from 0 to 40%, Ethyl acetate in petroleum ether) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4- (difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (150 mg). 1H NMR (400MHz, CDCl3) δ 7.77 (s, 1H), 7.58 - 7.49 (m, 2H), 7.39 - 7.27 (m, 10H), 7.25 - 7.16 (m, 2H), 6.44 (t, J = 55.6 Hz, 1H), 6.04 (d, J = 6.8 Hz, 1H), 5.20 (s, 2H), 5.08 (s, 2H), 4.65-4.51 (m, 1H), 4.00 - 3.87 (m, 3H), 3.85 - 3.75 (m, 1H), 1.84-1.73 (m, 2H), 0.79 (t, J=7.6 Hz, 3H). Step 5: (R)-2-amino-3-(3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000205_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-(difluoromethyl)-1-propyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (140 mg, 0.23 mmol) in 30% HBr in AcOH (3 mL) was at 25°C for 2 hours, and then stirred at 50°C for 1 hour. The mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2- amino-3-(3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid (50 mg, 50.1% yield) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.11 (t, J = 4.8 Hz, 1H), 8.60-8.35 (m, 3H), 7.89 (d, J = 9.2 Hz, 1H), 7.85 (s, 1H), 7.79 (s, 1H), 7.59 (d, J = 8.8 Hz, 1H), 7.89 (t, J = 55.2 Hz,, 1H), 4.19 - 4.07 (m, 1H), 3.99 (t, J = 6.8 Hz, 2H), 3.85 - 3.71 (m, 2H), 1.73-1.61 (m, 2H), 0.70 (t, J = 7.2 Hz, 3H), COOH not observed. LC-MS (MH+): m/z = 385.1 tR (min, Method D) = 1.14 [α]20,D = 1.8 (c = 30 mg/mL, CH3OH). Compound 2ai (R)-methyl 2-amino-3-(3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate Step 1: Preparation of methyl (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4- (difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000205_0002
A mixture of 3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (100 mg, 0.34 mmol), (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (116 mg, 0.40 mmol, HCl salt), TBTU (161 mg, 0.50 mmol) and DIPEA (87 mg, 0.67 mmol) in DMF (5 mL) was stirred at 25°C for 16 hours. The mixture was diluted with H2O (5 mL) and extracted with ethyl acetate (5 mL × 2). The organic phase was washed with brine (5 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash (silica gel, from 0 to 30%, Ethyl acetate in petroleum ether) to give (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4- (difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (135 mg). 1H NMR (400MHz, CDCl3) δ 7.80-7.75 (m, 1H), 7.61 (d, J = 8.8 Hz, 1H), 7.57 (s, 1H), 7.38 - 7.28 (m, 5H), 7.27 - 7.19 (m, 2H), 6.46 (t, J = 55.2 Hz, 1H), 5.92 (d, J = 6.4 Hz, 1H), 5.10 (s, 2H), 4.62 - 4.50 (m, 1H), 4.04 - 3.89 (m, 3H), 3.85 - 3.69 (m, 4H), 1.85 - 1.75 (m, 2H), 0.81 (t, J = 7.2 Hz, 3H). Step 2:
Figure imgf000206_0001
A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-(difluoromethyl)-1-propyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (60 mg, 0.11 mmol) in 30% HBr in AcOH (2 mL) was stirred at 25°C for 2 hours. The solvent was removed under reduced pressure. The residue was washed with TBME (2 mL × 3) and the organic layer was decanted, then the residue was lyophilized to give (R)-methyl 2-amino-3-(3-(4-(difluoromethyl)-1-propyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (41 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.98-8.92 (m, 1H), 8.45 (br s, 3H), 7.88 - 7.81 (m, 2H), 7.74 (s, 1H), 7.65 - 7.58 (m, 1H), 6.88 (t, J = 55.2 Hz, 1H), 4.28-4.20 (m, 1H), 3.99 (t, J = 7.2 Hz, 2H), 3.81-3.74 (m, 5H), 1.72-1.62 (m, 2H), 0.70 (t, J=7.2 Hz, 3H). LCMS (MH+): m/z = 399.0, tR (min, Method D) = 1.797 min [α]20,D = 1.20 (c = 5.0 mg/mL, CH3OH). Compound 1ad (R)-2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoic acid
Figure imgf000207_0001
Step 1: methyl 3-fluoro-5-(1-propyl-1H-pyrazol-5-yl)benzoate
Figure imgf000207_0002
A mixture of 5-bromo-1-propyl-pyrazole (0.885 g, 4.68 mmol), (3-fluoro-5-methoxycarbonyl- phenyl)boronic acid (772 mg, 3.90 mmol), Pd(dtbpf)Cl2 (254 mg, 0.39 mmol), K3PO4 (2.48 g, 11.70 mmol) in the mixture of dioxane (35 mL) and H2O (3.5 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 100 °C for 3 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give methyl 3- fluoro-5-(1-propyl-1H-pyrazol-5-yl)benzoate (0.90 g). 1H NMR (400MHz, CDCl3) δ 7.88 (t, J=1.2 Hz, 1H), 7.79-7.74 (m, 1H), 7.55 (d, J=2.0 Hz, 1H), 7.33-7.27 (m, 1H), 6.33 (d, J=1.8 Hz, 1H), 4.12 - 4.08 (m, 2H), 3.95 (s, 3H), 1.88-1.79 (m, 2H), 0.82 (t, J=7.6 Hz, 3H). Step 2: methyl 3-fluoro-5-(4-iodo-1-propyl-1H-pyrazol-5-yl)benzoate
Figure imgf000208_0001
To a solution of methyl 3-fluoro-5-(1-propyl-1H-pyrazol-5-yl)benzoate (600 mg, 2.29 mmol) in THF (20 mL) was added NIS (1.03 g, 4.58 mmol). The mixture was stirred at 70°C for 2 hours. The reaction mixture was concentrated. The residue was added water (15 mL), extracted with ethyl acetate (15 mL × 3). The organic layers was washed with brine (10 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 20 %) to give methyl 3-fluoro-5-(4-iodo-1-propyl-1H-pyrazol-5-yl)benzoate (660 mg). 1H NMR (400MHz, CDCl3) δ 7.87-7.81 (m, 2H), 7.61 (s, 1H), 7.32-7.27 (m, 1H), 4.04 (t, J = 7.2 Hz, 2H), 3.97 (s, 3H), 1.81-1.71 (m, 2H), 0.80 (t, J = 7.6 Hz, 3H). Step 3: methyl 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzoate
Figure imgf000208_0002
To a mixture of methyl 3-fluoro-5-(4-iodo-1-propyl-1H-pyrazol-5-yl)benzoate (590 mg, 1.52 mmol) and methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (1.46 g, 7.60 mmol) in DMF (15 mL) was added CuI (29 mg, 0.15 mmol). The mixture was stirred at 120°C for 15 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (15 mL × 3). The organic layers were washed with brine (15 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 20%) twice to give methyl 3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzoate (280 mg). 1H NMR (400MHz,CDCl3) δ 7.88-7.79 (m, 3H), 7.29-7.26 (m, 1H), 3.96 (s, 3H), 3.95-3.90 (m, 2H), 1.84-1.75 (m, 2H), 0.82 (t, J = 7.6 Hz, 3H). Step 4: 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzoic acid
Figure imgf000209_0001
To a solution of methyl 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzoate (280 mg, 0.85 mmol) in a mixture of MeOH (10 mL) and H2O (2 mL) was added LiOH.H2O (90 mg, 2.14 mmol). The mixture was stirred at 30°C for 1 hour. The reaction mixture was concentrated. The residue was added water (10 mL), adjusted pH to 5-6 with 1N HCl and extracted with ethyl acetate (15 mL × 3). The organic layers was dried over Na2SO4, filtered and concentrated to give 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzoic acid (255 mg). 1H NMR (400MHz, DMSO-d6) δ 7.99 (s, 1H), 7.80-7.71 (m, 2H), 7.38 (d, J = 8.8 Hz, 1H), 3.93 (t, J = 7.2 Hz, 2H), 1.72-1.63 (m, 2H), 0.70 (t, J = 7.2 Hz, 3H). Step 5: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoate
Figure imgf000210_0001
To a mixture of 3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzoic acid (100 mg, 0.32 mmol), TBTU (152 mg, 0.47 mmol) and DIPEA (123 mg, 0.95 mmol) in DMF (15 mL) was added (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (138 mg, 0.38 mmol, HCl salt). The mixture was stirred at 35°C for 16 hours. The reaction mixture was added water (10 mL), extracted with ethyl acetate (15 mL × 3). The organic layers was washed with brine (15 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 40%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl- 4-(trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoate (110 mg). 1H NMR (400MHz, CDCl3) δ 7.81 (s, 1H), 7.52-7.47 (m, 2H), 7.37-7.31 (m, 10H), 7.25-7.20 (m, 1H), 6.99-6.69 (m, 1H), 5.90 (br d, J = 6.0 Hz, 1H), 5.28-5.17 (m, 2H), 5.11 (s, 2H), 4.60-4.55 (m, 1H), 4.01-3.91 (m, 3H), 3.85-3.74 (m, 1H), 1.85-1.75 (m, 2H), 0.81 (t, J = 7.2 Hz, 3H). Step 6: (R)-2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoic acid
Figure imgf000210_0002
A mixture of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoate (110 mg, 0.18 mmol) in 30% HBr in AcOH (6 mL) was stirred at 20°C for 16 hours. The reaction mixture was concentrated. The residue was added water and extracted with methyl tert-butyl ether (10 ml × 3). The aqueous layer was lyophilized. The product was purified by preparative-HPLC (Method AA) to give (R)- 2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoic acid (14 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.09-9.04 (m, 1H), 8.44 (br s, 3H), 8.04 (s, 1H), 7.95-7.92 (m, 1H), 7.79 (s, 1H), 7.66-7.61 (m, 1H), 4.17-4.08 (m, 1H), 3.96-3.92 (m, 2H), 3.85-3.78 (m, 1H), 3.76-3.70 (m, 1H), 1.72-1.63 (m, 2H), 0.71 (t, J = 7.6 Hz, 3H). LC-MS (MH+): m/z = 403.2 tR (min, Method D) = 1.891 [α]20,D = 11.3 (c = 1.0 mg/mL, CH3OH). Compound 2aj (R)-methyl 2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoate
Figure imgf000211_0001
To a solution of 3-fluoro-5-[2-propyl-4-(trifluoromethyl)pyrazol-3-yl]benzoic acid (150 mg, 0.47 mmol), TBTU (228 mg, 0.71 mmol) and DIPEA (184 mg, 1.42 mmol) in DMF (15 mL) was added (R)-methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (164 mg, 0.57 mmol, HCl salt). The mixture was stirred at 20°C for 3 hours. The reaction mixture was added water (10 mL), extracted with ethyl acetate (15 mL × 3). The organic layers was washed with brine (15 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 58%) to give (R)- methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H- pyrazol-5-yl)benzamido)propanoate (140 mg). 1H NMR (400MHz, CDCl3) δ 7.80 (s, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.57 (s, 1H), 7.39 - 7.28 (m, 5H), 7.24 (d, J = 8.4 Hz, 1H), 7.17-7.09 (m, 1H), 5.86 (br d, J = 6.0 Hz, 1H), 5.11 (s, 2H), 4.65- 4.50 (m, 1H), 4.01 - 3.92 (m, 3H), 3.84 - 3.77 (m, 4H), 1.84-1.75 (m, 2H), 0.82 (t, J = 7.2 Hz, 3H). Step 2:
Figure imgf000212_0001
A mixture of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-fluoro-5-(1-propyl-4- (trifluoromethyl)-1H-pyrazol-5-yl)benzamido)propanoate (70 mg, 0.13 mmol) in 30% HBr in AcOH (6 mL) was stirred at 20°C for 1 hour. The reaction mixture was concentrated. The residue was added water and extracted methyl tert-butyl ether (10 ml × 3). The aqueous layer was lyophilized to give (R)-methyl 2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)- 1H-pyrazol-5-yl)benzamido)propanoate (28 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.98-8.91 (m, 1H), 8.44 (br s, 3H), 8.05 (s, 1H), 7.89 (d, J = 9.6 Hz, 1H), 7.75 (s, 1H), 7.66 (d, J = 8.8 Hz, 1H), 4.28 - 4.18 (m, 1H), 3.95 (t, J = 6.8 Hz, 2H), 3.83 - 3.63 (m, 5H), 1.72-1.63 (m, 2H), 0.72 (t, J = 6.8 Hz, 3H). LCMS (MH+): m/z = 417.0, tR (min, Method D) = 1.896 min [α]20,D = 0.95 (c = 2.1 mg/mL, CH3OH). Compound 1ae (R)-2-amino-3-(3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(3-(4-(difluoromethyl)-1- ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000213_0001
Step 1: methyl 3-(1-ethyl-4-formyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000213_0002
To a stirred solution of methyl 3-(1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (500 mg, 2.01 mmol) in POCl3 (7.43 g, 48.42 mmol) was added DMF (1.43 g, 19.50 mmol) and the mixture was heated to 100°C for 16 hours. The mixture was poured into H2O (10 mL) at 0°C, neutralize with sat. aqueous Na2CO3 to pH 7~8 and extracted with ethyl acetate (20 mL × 2). The organic phase was washed with brine (10 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash (silica gel, from 0 to 30%, Ethyl acetate in petroleum ether) to give methyl 3-(1-ethyl-4-formyl-1H-pyrazol-5-yl)-5-fluorobenzoate (480 mg). 1H NMR (400MHz, CDCl3) δ 9.65 (s, 1H), 8.09 (s, 1H), 7.94-7.88 (m, 2H), 7.38-7.32 (m, 1H), 4.11 (q, J = 7.2 Hz, 2H), 3.98 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H). Step 2: methyl 3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000214_0001
To a solution of methyl 3-(1-ethyl-4-formyl-1H-pyrazol-5-yl)-5-fluorobenzoate (480 mg, 1.74 mmol) in DCM (10 mL) was added DAST (8.40 g, 52.12 mmol) at 0°C and the resulting mixture was stirred at 25°C for 2 hours. The mixture was poured into sat. aqueous Na2CO3 to pH 7~8 at 0°C and extracted with DCM (30 mL × 2). The organic phase was washed with brine (10 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash (silica gel, from 0 to 20%, Ethyl acetate in petroleum ether) to give methyl 3-(4-(difluoromethyl)-1- ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (410 mg).1H NMR (400MHz, CDCl3) δ 7.90-7.83 (m, 2H), 7.78 (s, 1H), 7.33-7.27 (m, 1H), 6.65 (t, J = 55.6 Hz, 1H), 4.07 (q, J = 7.2 Hz, 2H), 3.97 (s, 3H), 1.40 (t, J = 7.2 Hz, 3H). Step 3: 3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000214_0002
To a solution of methyl 3-[4-(difluoromethyl)-2-ethyl-pyrazol-3-yl]-5-fluoro-benzoate (410 mg, 1.37 mmol) in MeOH (8 mL) was added a solution of LiOH.H2O (173 mg, 4.12 mmol) in H2O (4 mL) and the resulting mixture was stirred at 25°C for 2 hours. The mixture was concentrated to move MeOH, then added sat. aqueous KHSO4 to adjust pH = 4~5 and extracted with ethyl acetate (10 mL × 2). The organic phase was washed with brine (10 mL × 2), dried over Na2SO4 and concentrated to give 3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5- yl)-5-fluorobenzoic acid (365 mg). 1H NMR (400MHz, DMSO-d6) δ 7.87-7.79 (m, 2H), 7.77 (t, J = 1.6 Hz, 1H), 7.70-7.63 (m, 1H), 7.85 (t, J = 55.2 Hz, 1H), 4.03 (t, J = 7.2 Hz, 2H), 1.27 (t, J = 7.2 Hz, 3H). Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-(difluoromethyl)-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000215_0001
A mixture of 3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (180 mg, 0.63 mmol), (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (277 mg, 0.76 mmol, HCl salt), TBTU (305 mg, 0.95 mmol) and DIPEA (164 mg, 1.27 mmol) in DMF (5 mL) was stirred at 25°C for 16 hours. The mixture was diluted with H2O (5 mL) and extracted with ethyl acetate (5 mL × 2). The organic phase was washed with brine (5 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash (silica gel, from 0 to 40%, Ethyl acetate in petroleum ether) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4- (difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (240 mg). 1H NMR (400MHz, CDCl3) δ 7.76 (s, 1H), 7.56-7.48 (m, 2H), 7.39-7.16 (m, 12H), 6.43 (t, J = 55.6 Hz, 1H), 6.15-6.01 (m, 1H), 5.19 (s, 2H), 5.07 (s, 2H), 4.62-4.52 (m, 1H), 4.02 (q, J = 7.2 Hz, 2H), 3.96-3.74 (m, 2H), 1.35 (t, J = 7.2 Hz, 3H). Step 5: (R)-2-amino-3-(3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid
Figure imgf000216_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-(difluoromethyl)-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (240 mg, 0.40 mmol) in 30% HBr in AcOH (5 mL) was stirred at 50°C for 5 hours. The reaction was concentrated. The residue was washed with TBME (2 mL × 3) and the organic layer was decanted, then the residue was submitted to lyophilize to give (R)-2-amino-3-(3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid (150 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.99-8.93 (m, 1H), 8.45-8.25 (m, 3H), 7.89-7.81 (m, 2H), 7.76 (s, 1H), 7.63-7.56 (m, 1H), 6.87 (d, J = 55.2 Hz, 1H), 4.13-4.17 (m, 1H), 4.05-3.99 (m, 2H), 3.85- 3.65 (m, 2H), 1.25 (t, J = 7.2 Hz, 3H). LC-MS (MH+): m/z = 471.1 tR (min, Method E) = 1.316 [α]20,D = 2.71 (c = 14 mg/mL, CH3OH). Compound 2ak (R)-methyl 2-amino-3-(3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000216_0002
A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-(difluoromethyl)-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (200 mg, 0.39 mmol) in 30% HBr in AcOH (5 mL) was stirred at 30°C for 2 hours. The reaction was concentrated. The residue was washed with TBME (2 mL × 3) and the organic layer was decanted, then the residue was submitted to lyophilize to give (R)-methyl 2-amino-3-(3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (98 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.01-8.94 (m, 1H), 8.47 (br s, 3H), 7.91 - 7.80 (m, 2H), 7.75 (s, 1H), 7.62 (d, J = 9.2 Hz, 1H), 6.88 (t, J = 55.2 Hz, 1H), 4.32 - 4.16 (m, 1H), 4.04 (d, J = 7.2 Hz, 2H), 3.76 - 3.68 (m, 5H), 1.26 (t, J = 7.2 Hz, 3H). LCMS (MH+): m/z = 385.1, tR (min, Method D) = 1.706 min [α]20,D = 1.07 (c = 15 mg/mL, CH3OH). Compound 1af (R)-2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid The overall synthesis scheme for the preparation of (R)-2-amino-3-(3-(4-chloro-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoic acid is shown below
Figure imgf000217_0001
Step 1: methyl 3-(1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000217_0002
To a mixture of 5-bromo-1-ethyl-1H-pyrazole (200 mg, 1.14 mmol) and (3-fluoro-5- (methoxycarbonyl)phenyl)boronic acid (271 mg, 1.37 mmol) in the mixture of dioxane (5 mL) and H2O (0.5 mL) was added K3PO4 (728 mg, 3.43 mmol) and Pd(dtbpf)Cl2 (74 mg, 0.11 mmol). The mixture was stirred at 100°C for 14 hours under N2 atmosphere. The mixture was concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 20%) to give methyl 3-(1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzoate (230 mg). 1H NMR (400MHz, CDCl3) δ 7.89 (d, J = 1.6 Hz, 1H), 7.80 - 7.75 (m, 1H), 7.57 (d, J = 1.6 Hz, 1H), 7.33 - 7.28 (m, 1H), 6.34 (d, J = 2.0 Hz, 1H), 4.19 (q, J = 7.2 Hz, 2H), 3.96 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H). Step 2: methyl 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate
Figure imgf000218_0001
To a solution of methyl 3-(1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (180 mg, 0.73 mmol) in THF (10 mL) was added NCS (97 mg, 0.73 mmol), the mixture was stirred at 70°C for 1 hour. The mixture was concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 10%) to give methyl 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (190 mg). 1H NMR (400MHz, CDCl3) δ 7.87 (s, 1H), 7.86 - 7.82 (m, 1H), 7.56 (s, 1H), 7.36 - 7.32 (m, 1H), 4.11 (q, J = 7.2 Hz, 2H), 3.97 (s, 3H), 1.38 (t, J = 7.2 Hz, 3H). Step 3: 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid
Figure imgf000218_0002
To a solution of methyl 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoate (240 mg, 0.85 mmol) in the mixture of MeOH (5 mL) and H2O (5 mL) was added LiOH.H2O (53 mg, 1.27 mmol), the mixture was stirred at 20°C for 1 hour. The mixture was concentrated. The residue was added water (30 mL), then drop-wise added 2N aqueous HCl to pH=3~4 and extracted with ethyl acetate (30 mL × 3), the organic layers were dried over Na2SO4, filtered and concentrated to give 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (210 mg). LC-MS (MH+): m/z = 269.0 tR (min, Method G) = 0.659 Step 4: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000219_0001
To a mixture of 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (210 mg, 0.78 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (342 mg, 0.94 mmol, HCl salt) in DMF (6 mL) was added TBTU (376 mg, 1.17 mmol) and DIPEA (303 mg, 2.34 mmol), the mixture was stirred at 20°C for 14 hours. The mixture was added water (50 mL) and extracted with ethyl acetate (40 mL × 3), the organic layers were washed with brine (40 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 40%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5- yl)-5-fluorobenzamido)propanoate (350 mg). 1H NMR (400MHz, CDCl3) δ 7.56 (s, 1H), 7.52 - 7.44 (m, 2H), 7.37 - 7.25 (m, 11H), 6.92 (br s, 1H), 5.91 (br d, J = 6.4 Hz, 1H), 5.22 (d, J = 3.6 Hz, 2H), 5.11 (s, 2H), 4.65 - 4.55 (m, 1H), 4.09 (q, J = 7.2 Hz, 2H), 4.03 - 3.75 (m, 2H), 1.37 (t, J = 7.2 Hz, 3H). Step 5: (R)-2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoic acid
Figure imgf000220_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5- yl)-5-fluorobenzamido)propanoate (150 mg, 0.26 mmol) in HBr/HOAc (4 mL, 30%) was stirred at 50°C for 14 hours. The mixture was concentrated. The residue was added HOAc (3 mL), then added methyl tert-butyl ether (5 mL). The formed solid was collected by filtration and added water (10 mL) for freeze-drying. The obtained solid was further purified by preparative-HPLC (Method AA) to give (R)-2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)- 5-fluorobenzamido)propanoic acid (28 mg) as HCl salt. NMR (400MHz, DMSO-d6) δ 8.96 (br t, J = 5.6 Hz, 1H), 8.34 (br d, J = 3.6 Hz, 3H), 7.84 (d, J = 9.2 Hz, 1H), 7.78 (s, 1H), 7.74 (s, 1H), 7.63 (d, J = 8.8 Hz, 1H), 4.17 - 4.03 (m, 3H), 3.84 - 3.66 (m, 2H), 1.23 (t, J = 7.2 Hz, 3H) LC-MS (MH+): m/z = 355.0 tR (min, Method D) = 1.674
Figure imgf000220_0002
+3.0 (c = 2.0 mg/mL, CH3OH). Compound 2al (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000220_0003
To a mixture of 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid [cf. synthesis of compound 1af] (190 mg, 0.71 mmol) and (R)-methyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (245 mg, 0.85 mmol, HCl salt) in DMF (6 mL) was added TBTU (341 mg, 1.06 mmol) and DIPEA (274 mg, 2.12 mmol), the mixture was stirred at 15°C for 14 hours. The mixture was added water (40 mL) and extracted with ethyl acetate (40 mL × 3), the organic layers were washed with brine (40 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 30%) to give (R)-methyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (200 mg). 1H NMR (400MHz, CDCl3) δ 7.61 - 7.54 (m, 3H), 7.36 - 7.28 (m, 6H), 7.11 (br s, 1H), 5.86 (br d, J = 6.8 Hz, 1H), 5.12 (s, 2H), 4.62 - 4.53 (m, 1H), 4.12 (q, J = 7.2 Hz, 2H), 4.00 - 3.75 (m, 5H), 1.38 (t, J = 7.2 Hz, 3H). Step 2:
Figure imgf000221_0001
A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol- 5-yl)-5-fluorobenzamido)propanoate (200 mg, 0.40 mmol) in HBr in HOAc (3 mL, 30%) was stirred at 15°C for 2 hours. The mixture was concentrated. The residue was added water (20 mL) and then lyophilized to give (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)- 5-fluorobenzamido)propanoate (158 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.95 (t, J = 6.0 Hz, 1H), 8.44 (br s, 3H), 7.83 (d, J = 9.2 Hz, 1H), 7.77-7.74 (m, 2H), 7.65 (d, J = 9.2 Hz, 1H), 4.27 - 4.21 (m, 1H), 4.08 (q, J = 7.2 Hz, 2H), 3.82 - 3.67 (m, 5H), 1.24 (t, J = 7.2 Hz, 3H). LCMS (MH+): m/z = 369.0, tR (min, Method C) = 1.902 min [α]20,D = +2.0 (c = 2.0 mg/mL, CH3OH). Compound 2am (R)-isobutyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate Step 1: Preparation of (R)-isobutyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000222_0001
To a solution of 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (150 mg, 0.56 mmol) in DMF (10 mL) were added (R)-isobutyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (203 mg, 0.61 mmol, HCl salt), TBTU (269 mg, 0.84 mmol) and DIPEA (216 mg, 1.67 mmol). The reaction mixture was stirred at 20°C for 2 hours. The reaction mixture was added water (10 mL), extracted with ethyl acetate (10 mL × 3). The combined organic layers were washed with brine (10 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by column chromatography on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 20% to 30%) to give (R)-isobutyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (250 mg). 1H NMR (400MHz, CDCl3) δ 7.62 - 7.56 (m, 2H), 7.55 (s, 1H), 7.35 - 7.27 (m, 6H), 7.25-7.15 (m, 1H), 5.88 (d, J = 6.0 Hz, 1H), 5.11 (s, 2H), 4.62-4.52 (m, 1H), 4.14 - 4.10 (m, 2H), 4.03 - 3.91 (m, 3H), 3.84 - 3.74 (m, 1H), 2.0-1.94 (m, 1H), 1.37 (t, J = 7.2 Hz, 3H), 0.94 (d, J = 6.8 Hz, 6H) Step 2:
Figure imgf000222_0002
A solution of (R)-isobutyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol- 5-yl)-5-fluorobenzamido)propanoate (250 mg, 0.46 mmol) in 30% HBr in AcOH (2 mL) were stirred at 10°C for 1 hours. The solvent was removed. The residue was washed with a mixture of MeCN (1 mL) and methyl tert-butyl ether (5 mL). The organic layer was decanted. Then the residue was lyophilized to give (R)-isobutyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5- yl)-5-fluorobenzamido)propanoate (220 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.02-8.95 (m, 1H), 8.48 (br s, 3H), 7.86 (d, J = 9.2 Hz, 1H), 7.79 (s, 1H), 7.75 (s, 1H), 7.65 (d, J = 9.2 Hz, 1H), 4.33 - 4.20 (m, 1H), 4.09 (q, J = 7.2 Hz, 2H), 4.00 - 3.87 (m, 2H), 3.80 - 3.72 (m, 2H), 1.90-1.80 (m, 1H), 1.24 (t, J = 7.2 Hz, 3H), 0.85 (d, J = 6.4 Hz, 6H). LCMS (MH+): m/z = 411.0, tR (min, Method D) = 1.978 min [α]20,D = 2 (c = 1 mg/mL, MeOH). Compound 2an (R)-isopropyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate Step 1: Preparation of (R)-isopropyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000223_0001
To a solution of (R)-isopropyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate hydrochloride (236 mg, 0.74 mmol，)， 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzoic acid (200 mg, 0.74 mmol) in DMF (25 mL) was added TBTU (359 mg, 1.12 mmol), DIPEA (481 mg, 3.72 mmol). The mixture was stirred at 30°C for 2 hours. The mixture was diluted with H2O (100 mL) and extracted with EtOAC (30 mL × 2). The organic phase was washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give (R)-isopropyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)- 5-fluorobenzamido)propanoate(300 mg, 0.57 mmol). 1H NMR (400MHz, CDCl3) δ7.61 - 7.57 (m, 2H), 7.55 (s, 1H), 7.32 – 7.25 (m, 7H), 5.89 (d, J = 6.0 Hz, 1H), 5.14 - 5.06 (m, 3H), 4.56 - 4.50 (m, 1H), 4.11 (q, J = 7.2 Hz, 2H), 3.95 - 3.92 (m, 1H), 3.81 - 3.74 (m, 1H), 1.37 (t, J = 7.2 Hz, 3H), 1.27 (d, J = 6.0 Hz, 6H). Step 2:
Figure imgf000224_0001
A mixture of (R)-isopropyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (300 mg, 0.57 mmol) in HBr/AcOH (30 mL) was stirred at 25°C for 3 hours, The residue was diluted with methyl tert-butyl ether (40 mL) and propan-2-ol (5 mL). The precipitate was filtered under N2. The filter cake was diluted with methyl tert-butyl ether (40 mL) and propan-2-ol (5 mL) once again then filtered. The mother liquid was concentrated and lyophilized to give (R)-isopropyl 2-amino-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (100 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.00-8.94 (m, 1H), 8.46 (br s, 3H), 7.84 (d, J = 9.2 Hz, 1H), 7.78 (s, 1H), 7.74 (s, 1H), 7.64 (d, J = 8.8 Hz, 1H), 5.01-4.92 (m, 1H), 4.20 - 4.16 (m, 1H), 4.08 (q, J = 7.2 Hz, 2H), 3.78 - 3.70 (m, 2H), 1.25 - 1.20 (m, 6H), 1.20 - 1.14 (m, 3H). LCMS (MH+): m/z = 397.2, tR (min, Method D) = 1.933 min [α]20,D =4.54 (c = 4.4 mg/mL, CH3OH). Compound 2ao (R)-benzyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate Step 1: Preparation of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000225_0001
To a mixture of 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (210 mg, 0.78 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (342 mg, 0.94 mmol, HCl salt) in DMF (6 mL) was added TBTU (376 mg, 1.17 mmol) and DIPEA (303 mg, 2.34 mmol), the mixture was stirred at 20°C for 14 hours. The mixture was added water (50 mL) and extracted with ethyl acetate (40 mL × 3), the organic layers were washed with brine (40 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 40%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5- yl)-5-fluorobenzamido)propanoate (350 mg). 1H NMR (400MHz, CDCl3) δ 7.56 (s, 1H), 7.52 - 7.44 (m, 2H), 7.37 - 7.25 (m, 11H), 6.92 (br s, 1H), 5.91 (br d, J = 6.4 Hz, 1H), 5.22 (d, J = 3.6 Hz, 2H), 5.11 (s, 2H), 4.65 - 4.55 (m, 1H), 4.09 (q, J = 7.2 Hz, 2H), 4.03 - 3.75 (m, 2H), 1.37 (t, J = 7.2 Hz, 3H). Step 2:
Figure imgf000225_0002
(R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (200 mg, 0.3 mmol) was added to AcOH (4 mL) and 30% HBr in AcOH (0.4 mL). Then mixture was heated at 20°C for 1.5 hours. To the mixture was added methyl tert-butyl ether (5 mL). The organic layer was decanted, Then the residue was submitted to lyophilization to give (R)-benzyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5- yl)-5-fluorobenzamido)propanoate (105 mg) as HBr salt. 1H NMR (400MHz, CD3OD) δ 7.73 - 7.67 (m, 2H), 7.62 (s, 1H), 7.49 - 7.28 (m, 6H), 5.37 - 5.23 (m, 2H), 4.40 - 4.34 (m, 1H), 4.12 (q, J = 7.2 Hz, 2H), 4.03 - 3.85 (m, 2H), 1.31 (t, J = 7.2 Hz, 3H). LCMS (MH+): m/z = 445.0, tR (min, Method D) = 2.213 min [α]20,D = +1.5 (c = 4.0 mg/mL, CH3OH). Compound 2ap (R)-phenyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate Step 1: Preparation of (R)-phenyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000226_0001
To a mixture of 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (150 mg, 0.56 mmol) and (R)-phenyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (193 mg, crude, HCl salt) in DMF (5 mL) was added TBTU (269 mg, 0.84 mmol) and DIPEA (216 mg, 1.67 mmol), the mixture was stirred at 20°C for 16 hours. The mixture was added water (10 mL) and extracted with ethyl acetate (20 mL × 3), the organic layers were washed with brine (20 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 30%) to give (R)-phenyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (200 mg). 1H NMR (400MHz, CDCl3) δ 7.62 - 7.54 (m, 3H), 7.43 - 7.28 (m, 9H), 7.16 - 7.10 (m, 2H), 7.05 (br, 1H), 5.94 (br d, J = 6.8 Hz, 1H), 5.15 (s, 2H), 4.85 – 4.75 (m, 1H), 4.16 - 4.09 (m, 4H), 1.27 (t, J = 7.2 Hz, 3H). Step 2:
Figure imgf000227_0001
A solution of (R)-phenyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5- yl)-5-fluorobenzamido)propanoate (190 mg, 0.34 mmol) in the solution of 30% HBr in AcOH (6 mL) was stirred at 20°C for 0.5 hours. To the mixture was added methyl tert-butyl ether (5 mL). The organic layer was decanted, then the residue was submitted to lyophilize to give (R)-phenyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (105 mg) as HBr salt.1H NMR (400MHz, DMSO-d6) δ 9.15-9.09 (m, 1H), 8.63 (br s, 3H), 7.89 - 7.84 (m, 1H), 7.81 (s, 1H), 7.74 (s, 1H), 7.67 - 7.62 (m, 1H), 7.49 - 7.43 (m, 2H), 7.34 - 7.28 (m, 1H), 7.25 - 7.21 (m, 2H), 4.57 - 4.50 (m, 1H), 4.04 (q, J = 7.2 Hz, 2H), 4.00 - 3.87 (m, 2H), 1.21 (t, J = 7.2 Hz, 3H). LCMS (MH+): m/z = 431.0, tR (min, Method D) = 2.456 min [α]20,D = -1.3 (c = 3.0 mg/mL, CH3OH). Compound 2aq (R)-cyclopropyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate Step 1: Preparation of (R)-cyclopropyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1- ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000227_0002
To a mixture of 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (150 mg, 0.56 mmol) and (R)-cyclopropyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (171 mg, crude, 0.61 mmol, HCl salt) in DMF (5 mL) was added TBTU (269 mg, 0.84 mmol) and DIPEA (216 mg, 1.67 mmol), the mixture was stirred at 15°C for 16 hours. The mixture was added water (10 mL) and extracted with ethyl acetate (20 mL × 3), the organic layers were washed with brine (20 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 30%) to give (R)-cyclopropyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1- ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (260 mg). 1H NMR (400MHz, CDCl3) δ 7.61 - 7.55 (m, 3H), 7.35 - 7.28 (m, 6H), 7.13 (br s, 1H), 5.85 (br d, J = 5.6 Hz, 1H), 5.11 (s, 2H), 4.55 - 4.48 (m, 1H), 4.26 - 4.20 (m, 1H), 4.11 (q, J = 6.8 Hz, 2H), 3.95 - 3.73 (m, 2H), 1.37 (t, J = 7.2 Hz, 3H), 0.76 (d, J = 4.4 Hz, 4H). Step 2:
Figure imgf000228_0001
A solution of (R)-cyclopropyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (100 mg, 0.19 mmol) in a mixture of AcOH (3 mL) and 30% HBr in AcOH (0.3 mL) was heated and stirred at 20°C for 2 hours. The mixture was washed with methyl tert-butyl ether (5 mL). The organic layer was decanted, then the residue was submitted to lyophilization to give (R)-cyclopropyl 2-amino-3-(3-(4-chloro-1- ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (75 mg) HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.96-8.91 (m, 1H), 8.44 (br s, 3H), 7.84 - 7.80 (m, 1H), 7.76 (s, 1H), 7.75 (s, 1H), 7.67 - 7.63 (m, 1H), 4.24 - 4.04 (m, 4H), 3.78 - 3.65 (m, 2H), 1.24 (t, J = 7.2 Hz, 3H), 0.76 - 0.58 (m, 4H). LCMS (MH+): m/z = 395.2, tR (min, Method D) = 1.837 min [α]20,D = +1.2 (c = 1.7 mg/mL, CH3OH). Compound 2ar (R)-cyclopropylmethyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate
Figure imgf000229_0001
To a solution of (R)-2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoic acid (380 mg, 0.74 mmol, HBr salt) in cyclopropylmethanol (4.56 g, 63.2 mmol) was added SOCl2 (175 mg, 1.47 mmol). The mixture was stirred at 40°C for 16 hours. The mixture was concentrated. The residue was added methyl tert-butyl ether (5 mL) and then hexane (10 mL). The solid was collected and dried. The residue was purified by preparative HPLC (Method AA) to give (R)-cyclopropylmethyl 2-amino-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (180 mg) as HCl salt. 1H NMR (400 MHz, DMSO-d6) δ 9.20-9.14 (m, 1H), 8.67 (br s, 3H), 7.90 (d, J = 9.2 Hz, 1H), 7.82 (s, 1H), 7.74 (s, 1H), 7.63 (d, J = 9.2 Hz, 1H), 4.29 - 4.18 (m, 1H), 4.09 (q, J = 7.2 Hz, 2H), 4.03 - 3.97 (m, 1H), 3.96 - 3.89 (m, 1H), 3.86 - 3.72 (m, 2H), 1.23 (t, J = 7.2 Hz, 3H), 1.10 - 1.00 (m, 1H), 0.49 - 0.37 (m, 2H), 0.30 - 0.19 (m, 2H). LCMS (MH+): m/z = 409.0, tR (min, Method D) = 1.990 min [α]20,D = 4.4 (c = 3.4 mg/mL, CH3OH). Compound 2as (R)-2-methoxyethyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate Step 1: Preparation of (R)-2-methoxyethyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1- ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000230_0001
To a solution of 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (200 mg, 744.40 µmol) and (R)-2-methoxyethyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (272 mg, 818.85 µmol, HCl salt) in DMF (6 mL) was added TBTU (359 mg, 1.12 mmol) and DIPEA (289 mg, 2.23 mmol). The mixture was stirred at 15 °C for 2 hours. The reaction mixture was quenched with water (10 mL), extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (20 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 60%) twice to give (R)-2-methoxyethyl 2-(((benzyloxy)carbonyl)amino)- 3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (200 mg). 1H NMR (400MHz, CDCl3) δ 7.63 - 7.56 (m, 2H), 7.55 (s, 1H), 7.38 - 7.28 (m, 6H), 7.26 - 7.19 (m, 1H), 6.01 (d, J = 6.8 Hz, 1H), 5.17 - 5.08 (m, 2H), 4.66 - 4.51 (m, 2H), 4.28 - 4.20 (m, 1H), 4.10 (q, J = 7.2, 2H), 4.07 - 4.00 (m, 1H), 3.88 - 3.73 (m, 1H), 3.70 - 3.56 (m, 2H), 3.31 (s, 3H), 1.37 (t, J = 7.2 Hz, 3H). Step 2:
Figure imgf000230_0002
To a solution of (R)-2-methoxyethyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (200 mg, 0.366 mmol) in AcOH (5 mL) was added 30% HBr in AcOH (0.5 mL) and the mixture was stirred at 15 °C for 3 hours. The mixture was concentrated and methyl tert-butyl ether (30 mL) was added and the mixture was stirred at 15°C for 30 minutes. The solid was collected and dried and the resulting residue was purified by preparation HPLC (HCl condition) to give (R)-2-methoxyethyl 2-amino-3-(3- (4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (80 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.22-9.17 (m, 1H), 8.73 (br s, 3H), 7.91 (d, J = 9.2 Hz, e1H), 7.82 (s, 1H), 7.74 (s, 1H), 7.62 (d, J = 9.2 Hz, 1H), 4.33 - 4.18 (m, 3H), 4.09 (q, J = 7.2 Hz, 2H), 3.88 - 3.71 (m, 2H), 3.54 - 3.46 (m, 2H), 3.16 (s, 3H), 1.24 (t, J = 7.2 Hz, 3H). LCMS (MH+): m/z = 413.2, tR (min, Method C) = 1.944 min [α]20,D = -6 (c = 1 mg/mL, MeOH). Compound 2at (R)-cyclohexyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate Step 1: Preparation of (R)-cyclohexyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl- 1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000231_0001
A mixture of 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (280 mg, 1.04 mmol), (R)-cyclohexyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (420 mg, 1.18 mmol, as HCl salt), TBTU (502 mg, 1.56 mmol) and DIPEA (404 mg, 3.13 mmol) in DMF (5 mL) was stirred at 20°C for 16 hours. The mixture was diluted with H2O (5 mL) and extracted with ethyl acetate (5 mL × 2). The organic phase was washed with brine (5 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash (silica gel, from 0 to 30%, Ethyl acetate in petroleum ether) to give 200 mg of crude . The crude was further purified by SFC separation to give (R)-cyclohexyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1- ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (125 mg). 1H NMR (400MHz, CDCl3) δ 7.61 - 7.53 (m, 2H), 7.52 (s, 1H), 7.33 - 7.23 (m, 7H), 5.89 (d, J = 6.4 Hz, 1H), 5.08 (s, 2H), 4.89-4.76 (m, 1H), 4.58 - 4.46 (m, 1H), 4.08 (d, J = 7.2 Hz, 2H), 3.96 - 3.87 (m, 1H), 3.81 - 3.66 (m, 1H), 1.88 - 1.76 (m, 2H), 1.75 - 1.62 (m, 3H), 1.57 - 1.29 (m, 8H). Step 2:
Figure imgf000232_0001
A solution of (R)-cyclohexyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H- pyrazol-5-yl)-5-fluorobenzamido)propanoate (120 mg, 0.21 mmol) in 30% HBr in AcOH (2 mL) was stirred at 20°C for 30 min. The mixture was concentrated. The residue was washed with TBME (2 mL × 3) and the organic layer was decanted. Then the residue was submitted to lyophilize to give (R)-cyclohexyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (96 mg)) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 9.02-8.94 (m, 1H), 8.61-8.33 (m, 3H), 7.85 (d, J = 9.2 Hz, 1H), 7.78 (s, 1H), 7.74 (s, 1H), 7.64 (d, J = 8.8 Hz, 1H), 4.84 - 4.70 (m, 1H), 4.28 - 4.15 (m, 1H), 4.08 (q, J = 7.2 Hz, 2H), 3.75 (t, J = 6.0 Hz, 2H), 1.86 - 1.12 (m, 13H). LCMS (MH+): m/z = 437.2, tR (min, Method E) = 1.852 min [α]20,D = 2.50 (c = 8.0 mg/mL, CH3OH). Compound 2au (R)-2-oxo-2-(pyrrolidin-1-yl)ethyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate Step 1: Preparation of (R)-2-oxo-2-(pyrrolidin-1-yl)ethyl 2-(((benzyloxy)carbonyl)amino)-3- (3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate
Figure imgf000232_0002
To a solution of 3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzoic acid (300 mg, 1.12 mmol), TBTU (538 mg, 1.67 mmol) and DIPEA (433 mg, 3.35 mmol) in DMF (10 mL) was added (R)-2-oxo-2-(pyrrolidin-1-yl)ethyl 3-amino-2- (((benzyloxy)carbonyl)amino)propanoate (600 mg, crude, HCl salt). The mixture was stirred at 40°C for 16 hours. The reaction mixture was added water (15 mL), extracted with ethyl acetate (15 mL × 3). The combined organic layers were washed with brine (15 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether/ethyl acetate with ethyl acetate from 0% to 80%) twice to give 300 mg of product, which was further purified by SFC(Instrument: Berger, MULTIGR AM-II; Column: DAICEL CHIRALPAK AD(250mm×30mm,10μm); Mobile phase: supercritical CO2/ IPA = 60/40; Flow Rate: 70 mL/min; Column Temperature: 38oC; Nozzle Pressure: 100 bar; Nozzle Temperature: 60oC; Evaporator Temperature: 20oC; Trimmer Temperature: 25oC; Wavelength: 220 nm) to give (R)-2-oxo-2-(pyrrolidin-1-yl)ethyl 2- (((benzyloxy)carbonyl)amino)-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate (180 mg). 1H NMR (400MHz, CDCl3) δ 9.00 - 8.86 (m, 1H), 7.93 - 7.82 (m, 2H), 7.51 (s, 1H), 7.40 - 7.29 (m, 5H), 7.24 (d, J = 8.4 Hz, 1H), 6.12 (d, J = 7.2 Hz, 1H), 5.21 - 5.02 (m, 3H), 4.69 - 4.58 (m, 1H), 4.51 - 4.36 (m, 2H), 4.15 - 4.05 (m, 2H), 3.82 - 3.70 (m, 1H), 3.45 - 3.24 (m, 3H), 3.20 - 3.05 (m, 1H), 2.07 - 1.97 (m, 2H), 1.92 - 1.80 (m, 2H), 1.35 (t, J = 7.2 Hz, 3H). Step 2:
Figure imgf000233_0001
To a mixture of (R)-2-oxo-2-(pyrrolidin-1-yl)ethyl 2-(((benzyloxy)carbonyl)amino)-3-(3-(4- chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (180 mg, 0.30 mmol) in AcOH (4 mL) was added 30% HBr in AcOH (0.6 mL). The mixture was stirred at 15°C for 2 hours. The reaction mixture was added methyl tert-butyl ether (15 mL). The precipitate formed was washed with methyl tert-butyl ether (15 mL × 3) and the organic solvent was discarded. The solid was collected by filtration and dried. The product was further purified by preparation HPLC (HCl condition) to give (R)-2-oxo-2-(pyrrolidin-1-yl)ethyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5-fluorobenzamido)propanoate (52 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.05 (br s, 1H), 8.79 - 8.44 (m, 3H), 7.93 - 7.78 (m, 2H), 7.74 (s, 1H), 7.64 (d, J = 8.4 Hz, 1H), 5.15 - 5.02 (m, 1H), 4.89-4.83 (m, 1H), 4.55 - 4.45 (m, 1H), 4.18 - 4.02 (m, 3H), 3.85 - 3.69 (m, 1H), 3.34 - 3.26 (m, 2H), 3.22 - 2.98 (m, 2H), 1.96 - 1.83 (m, 2H), 1.79 - 1.73 (m, 2H), 1.24 (t, J = 7.2 Hz, 3H). LCMS (MH+): m/z = 466.1, tR (min, Method D) = 1.890 min [α]20,D = 28.57 (c = 7.0 mg/mL, CH3OH). Compound 1ag (R)-2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid The overall synthesis scheme for the preparation (R)-2-amino-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoic acid
Figure imgf000234_0001
Step 1: methyl 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylate
Figure imgf000234_0002
To a solution of methyl 3-bromo-5-fluoro-benzoate (1 g, 4.29 mmol) and (2- ethylphenyl)boronic acid (644 mg, 4.29 mmol) in a mixture of dioxane (15 mL) and H2O (1.5 mL) was added Pd(dppf)Cl2 (314 mg, 0.43 mmol) and K2CO3 (1.78 g, 12.87 mmol). The mixture was heated to 90°C for 16 hours. The reaction mixture was diluted with water (20 mL), extracted with ethyl acetate (20 mL × 3), the organic layer was washed with brine (20 mL), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (eluent : petroleum) to give methyl 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylate (915 mg). 1H NMR (400MHz, CDCl3) δ 7.82 (m, 1H), 7.75-7.70 (m, 1H), 7.38-7.34 (m, 2H), 7.27-7.15 (m, 3H), 3.95 (s, 3H), 2.60 (q, J=7.6 Hz, 2H), 1.12 (t, J=7.6 Hz, 3H). Step 2: 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylic acid
Figure imgf000235_0001
To a solution of methyl 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylate (915 mg, 3.54 mmol) in a mixture of MeOH (10 mL), THF (5 mL) and H2O (10 mL) was added LiOH.H2O (446 mg, 10.63 mmol). The mixture was stirred at 25°C for 16 hours. The reaction mixture was concentrated, diluted with water (10 mL), adjusted pH = 4~5 with aq HCl (2M). The mixture was extracted with ethyl acetate (20 mL × 3), the organic layer was dried over Na2SO4 and concentrated to give 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylic acid (800 mg). 1H NMR (400MHz, DMSO-d6) δ 7.69-7.65 (m, 2H), 7.50-7.45 (m, 1H), 7.38-7.36 (m, 2H), 7.29- 7.25 (m, 1H), 7.20 (d, J=7.6 Hz, 1H), 2.53 (q, J=7.6Hz, 2H), 1.03 (t, J=7.6Hz, 3H). Step 3: (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate
Figure imgf000235_0002
To a solution of 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylic acid (400 mg, 1.64 mmol) in DMF (10 mL) was added TBTU (789 mg, 2.46 mmol), DIPEA (635 mg, 4.91 mmol) and (R)-benzyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (657 mg, 1.80 mmol, HCl salt). The mixture was stirred at 10 °C for 1 hour. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (30 mL × 3). The combined organic layers were washed with brine (30 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 20% to 30%) to give (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoate (620 mg). 1H NMR (400MHz, CDCl3) δ 7.43-7.26 (m, 15H), 7.20-7.14 (m, 2H), 6.71 (br s, 1H), 5.94-5.91 (m, 1H), 5.26-5.16 (m, 2H), 5.09 (s, 2H), 4.63-4.54 (m, 1H), 3.99- 3.76 (m, 2H), 2.59 (q, J = 7.6 Hz, 2H), 1.11 (t, J = 7.6 Hz, 3H). Step 4: (R)-2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid
Figure imgf000236_0001
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]- 3-ylcarboxamido)propanoate (620 mg, 1.12 mmol) in the solution of 30% HBr in AcOH (10 mL) was heated at 50°C for 1 hour. The reaction mixture was concentrated. The residue was purified by preparative HPLC (Method AA) to give (R)-2-amino-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoic acid (310 mg) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 8.94 (br s, 1H), 7.72-7.64 (m, 2H), 7.41-7.34 (m, 3H), 7.31-7.25 (m, 1H), 7.19 (d, J = 7.6 Hz, 1H), 3.80-3.75 (m, 1H), 3.65-3.55 (m, 2H), 2.55 (q, J = 7.6 Hz, 2H), 1.03 (t, J=7.6 Hz, 3H). LC-MS (MH+): m/z = 331.1 tR (min, Method D) = 1.832 [α]20D = +10.0 (c = 1.0 mg/mL, CH3OH). Compound 2av (R)-methyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate Step 1: Preparation of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoate
Figure imgf000237_0001
To a solution of 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylic acid (2.1 g, 8.60 mmol) and (R)- methyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (2.73 g, 9.46 mmol, HCl salt) in DMF (40 mL) was added TBTU (4.14 g, 12.90 mmol) and DIPEA (3.33 g, 25.79 mmol). The reaction mixture was stirred at 25°C for 2 hours. The mixture was added water (50 mL), extracted with ethyl acetate (60 mL × 3). The combined organic layers were washed with saturated brines (50 mL × 3), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash on silica gel (ethyl acetate/Petroleum ether with ethyl acetate from 0% to 30%) twice to give (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro- [1,1'-biphenyl]-3-ylcarboxamido)propanoate (4 g). 1H NMR (400MHz, CDCl3) δ 7.49 - 7.44 (m, 2H), 7.39 - 7.29 (m, 7H), 7.26 - 7.23 (m, 1H), 7.20 - 7.16 (m, 2H), 6.91 (br, 1H), 5.88 (br d, J = 6.8 Hz, 1H), 5.10 (s, 2H), 4.59 - 4.53 (m, 1H), 3.95 - 3.77 (m, 5H), 2.59 (q, J = 7.6 Hz, 2H), 1.11 (t, J = 8.0 Hz, 3H). Step 2:
Figure imgf000237_0002
A solution of (R)-methyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]- 3-ylcarboxamido)propanoate (4.0 g, 8.36 mmol) in the solution of 30% HBr in AcOH (40 mL) was stirred at 20°C for 1 hour. The mixture was concentrated. The residue was washed with MeOH/methyl tert-butyl ether=1/5 (100 mL). The reaction mixture was filtered, and the filtration cake was washed with methyl tert-butyl ether (20 mL), collected and lyophilized to give (R)-methyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate (3.0 g) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.89-8.83 (m, 1H), 8.41 (br s, 3H), 7.69 - 7.63 (m, 2H), 7.45 - 7.36 (m, 3H), 7.33 - 7.27 (m, 1H), 7.20 (d, J = 7.6 Hz, 1H), 4.24 - 4.20 (m, 1H), 3.81 - 3.66 (m, 5H), 2.56 (q, J = 7.6 Hz, 2H), 1.04 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 345.1, tR (min, Method D) = 2.0 min [α]20,D = +3.6 (c = 0.28 g/100mL, MeOH). Compound 2aw (R)-isopropyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate Step 1: Preparation of (R)-isopropyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro- [1,1'-biphenyl]-3-ylcarboxamido)propanoate
Figure imgf000238_0001
A mixture of 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylic acid (2 g, 8.19 mmol), (R)-isopropyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (3.11 g, 9.83 mmol, HCl salt), TBTU (3.94 g, 12.28 mmol) and DIPEA (2.12 g, 16.38 mmol) in DMF (100 mL) was stirred at 25°C for 16 hours. The mixture was diluted with H2O (100 mL) and extracted with ethyl acetate (100 mL × 2). The organic phase was washed with brine (100 mL × 2), dried over Na2SO4 and concentrated. The residue was purified by Combi Flash (silica gel, from 0 to 40%, Ethyl acetate in petroleum ether) to give (R)-isopropyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl- 5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate (3.6 g). 1H NMR (400MHz, CDCl3) δ 7.49 - 7.40 (m, 2H), 7.36 - 7.27 (m, 7H), 7.24 - 7.19 (m, 1H), 7.17 - 7.12 (m, 2H), 7.07 - 6.95 (m, 1H), 5.85 (br d, J = 6.0 Hz, 1H), 5.12 - 5.00 (m, 3H), 4.54 - 4.41 (m, 1H), 3.93 - 3.74 (m, 2H), 2.56 (q, J = 7.6 Hz, 2H), 1.30 - 1.23 (m, 6H), 1.08 (t, J = 7.6 Hz, 3H). Step 2:
Figure imgf000239_0001
A mixture of (R)-isopropyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoate (3.6 g, 7.11 mmol) in 30% HBr in AcOH (20 mL) was stirred at 25°C for 1 hour. The mixture was concentrated. The residue was washed with n- hexane (10 mL × 3) and the organic layer was decanted, then the residue was submitted to lyophilization to give (R)-isopropyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate (2.6 g) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.95-8.88 (m,, 1H), 8.48 (br s, 3H), 7.75 - 7.62 (m, 2H), 7.45 - 7.34 (m, 3H), 7.32 - 7.24 (m, 1H), 7.20 (d, J = 7.2 Hz, 1H), 5.03 - 4.86 (m, 1H), 4.17 (t, J = 6.0 Hz, 1H), 3.83 - 3.66 (m, 2H), 2.55 (q, J = 7.6 Hz, 2H), 1.20 (d, J = 6.4 Hz, 3H), 1.14 (d, J = 6.4 Hz, 3H), 1.01 (t, J=7.6 Hz, 3H). LCMS (MH+): m/z = 373.2, tR (min, Method E) = 2.153 min
Figure imgf000239_0002
4.0 (c = 11 mg/mL, CH3OH). Compound 2ax (R)-benzyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate
Figure imgf000239_0003
A solution of (R)-benzyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]- 3-ylcarboxamido)propanoate (4.0 g, 7.21 mmol) and 30% HBr in AcOH (7.21 mmol, 8 mL) in AcOH (80 mL) was stirred at 20°C for 1.5 hours. The solvent was removed. The residue was washed with a mixture of MeCN (30 mL) and methyl tert-butyl ether (150 mL). The organic layer was decanted. Then the residue was lyophilized to give (R)-benzyl 2-amino-3-(2'-ethyl- 5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate (3 g) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.92-8.85 (m, 1H), 8.46 (br s, 3H), 7.68 - 7.61 (m, 2H), 7.47 - 7.41 (m, 1H), 7.40 - 7.35 (m, 4H), 7.34 - 7.28 (m, 4H), 7.20 (d, J = 7.2 Hz, 1H), 5.30 - 5.23 (m, 1H), 5.18 - 5.12 (m, 1H), 4.32-4.25 (m, 1H), 3.81 - 3.73 (m, 2H), 2.56 (q, J = 7.6 Hz, 2H), 1.04 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 421.1, tR (min, Method E) = 2.081 min [α]20,D = +8.0 (c = 1 mg/mL,CH3OH). Compound 2ay (R)-cyclopropylmethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate
Figure imgf000240_0001
To a solution of (R)-2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoic acid (3.0 g, crude) in cyclopropylmethanol (60 mL) was added SOCl2 (2.16 g, 18.16 mmol). The mixture was stirred at 40°C for 32 hours. The solvent was removed. The residue was purified by Preparative HPLC (HCl condition) to give (R)-cyclopropylmethyl 2-amino-3-(2'- ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate (2.7 g) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.07 (br s, 1H), 8.66 (br s, 3H), 7.74 (d, J = 9.6 Hz, 1H), 7.69 (s, 1H), 7.44 - 7.36 (m, 3H), 7.31-7.28 (m, 1H), 7.21 (d, J = 7.6 Hz, 1H), 4.25-4.18 (m, 1H), 4.05 - 3.88 (m, 2H), 3.86 - 3.69 (m, 2H), 2.56 (q, J = 7.6 Hz, 2H), 1.07 - 1.00 (m, 4H), 0.47 - 0.39 (m, 2H), 0.27-0.22 (m, 2H). LCMS (MH+): m/z = 385.1, tR (min, Method E) = 1.986 min [α]20,D = 4.0 (c = 0.1 g/100 mL, CH3OH). Compound 2az (R)-cyclopropyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate Step 1: Preparation of (R)-cyclopropyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro- [1,1'-biphenyl]-3-ylcarboxamido)propanoate
Figure imgf000241_0001
To a solution of a mixture of 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylic acid (3 g, 12.28 mmol) and (R)-cyclopropyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (3.87 g, 12.28 mmol, HCl salt) in DMF (50 mL) was added TBTU (5.92 g, 18.42 mmol) and DIPEA (4.76 g, 36.85 mmol). The reaction mixture was stirred at 20°C for 1 hour. The mixture was added water (80 mL) and extracted with ethyl acetate (100 mL × 3), the organic layers were washed with brine (80 mL × 3), dried over Na2SO4, filtered and concentrated. The residue was purified by Combi Flash on silica gel chromatography (Petroleum ether: Ethyl acetate, Ethyl acetate from 0% to 20%) twice to give (R)-cyclopropyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5- fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate (5 g). LCMS (MH+): m/z = 502.2, tR (min, Method G) = 1.020 min Analytic Chiral SFC method: ee% = 94.83%, tR = 4.998 min (Instrument: Agilent 1260 with DAD detector, Column: ChiralPak AD-3150×4.6mm I.D., 3µm, Mobile phase: A: CO2 B:IPA (0.05% DEA), Gradient: from 5% to 40% of B in 5.5min and hold 40% for 3 min, then 5% of B for 1.5 min, Column Temp: 40°C, Flow rate: 2.5 mL/min, Back pressure: 100 bar). Step 2:
Figure imgf000242_0001
(R)-cyclopropyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate (4.90 g, 9.71 mmol) in a mixture of AcOH (30 mL) and 30% HBr in AcOH (3 mL) was stirred at 20°C for 2 hours. The mixture was washed with methyl tert-butyl ether (30 mL). The organic layer was decanted, then the residue was submitted to lyophilization. The residue was purified by preparative HPLC (Method AA) to give (R)- cyclopropyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate (1.45 g) as HCl salt. 1H NMR (400MHz, DMSO-d6) δ 9.06-8.99 (m, 1H), 8.66 (br s, 3H), 7.74 - 7.69 (m, 1H), 7.67 (s, 1H), 7.43 - 7.35 (m, 3H), 7.32 - 7.26 (m, 1H), 7.21 (d, J = 7.6 Hz, 1H), 4.20 - 4.10 (m, 2H), 3.82 - 3.68 (m, 2H), 2.56 (q, J = 7.6 Hz, 2H), 1.03 (t, J = 7.6 Hz, 3H), 0.73 - 0.58 (m, 4H). LCMS (MH+): m/z = 371.0, tR (min, Method E) = 1.895 min [α]20,D = +4.0 (c = 0.1 g/100mL, MeOH). Compound 2ba (R)-ethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate Step 1: (R)-ethyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate
Figure imgf000242_0002
To a solution of 2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-carboxylic acid [cf. synthesis of compound 1ag] (500 mg, 2.05 mmol) in DMF (10 mL) was added TBTU (986 mg, 3.07 mmol), DIPEA (794 mg, 6.14 mmol) and (R)-ethyl 3-amino-2-(((benzyloxy)carbonyl)amino)propanoate (620 mg, 2.05 mmol, HCl salt). The mixture was stirred at 20 °C for 16 hours. The mixture was diluted with water (30 mL) and extracted with ethyl acetate (20 mL × 3). The combined organic layers were washed with brine (30 mL × 2) and concentrated. The residue was purified by Combi Flash on silica gel (petroleum ether: ethyl acetate with ethyl acetate from 0 to 30%) to give 800 mg of the crude product. The crude product was further purified by preparative HPLC (Method DD) to give (R)-ethyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'- biphenyl]-3-ylcarboxamido)propanoate (670 mg). 1H NMR (400MHz, CDCl3) δ 7.51 - 7.44 (m, 2H), 7.38 - 7.27 (m, 7H), 7.27 - 7.22 (m, 1H), 7.21 - 7.15 (m, 2H), 6.97 (br s, 1H), 5.86 (br d, J=6.4 Hz, 1H), 5.09 (s, 2H), 4.59 - 4.50 (m, 1H), 4.31 - 4.19 (m, 2H), 3.97 - 3.79 (m, 2H), 2.59 (q, J=7.6 Hz, 2H), 1.29 - 1.24 (m, 3H), 1.11 (t, J=7.6 Hz, 3H). Step 2: (R)-ethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate
Figure imgf000243_0001
A solution of (R)-ethyl 2-(((benzyloxy)carbonyl)amino)-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate (670 mg, 1.36 mmol) in 30% HBr/AcOH (10 mL) was stirred at 20°C for 1 hour. The reaction mixture was concentrated. The residue was diluted with tBuOMe (4 mL), and the solvent was treated with n-hexane (15 mL), in which a solid was formed. The solvent was removed. The residue was lyophilized to give (R)-ethyl 2-amino-3- (2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate (430 mg) as HBr salt. 1H NMR (400MHz, DMSO-d6) δ 8.87 (t, J = 5.6 Hz, 1H), 8.40 (br s, 3H), 7.68-7.64 (m, 2H), 7.45 - 7.37 (m, 3H), 7.32-7.27 (m, 1H), 7.20 (d, J = 7.6 Hz, 1H), 4.21-4.14 (m, 3H), 3.76 - 3.70 (m, 2H), 2.56 (q, J = 7.6 Hz, 2H), 1.18 (t, J = 7.2 Hz, 3H), 1.03 (t, J = 7.6 Hz, 3H). LCMS (MH+): m/z = 359.0, tR (min, Method E) = 1.91 min [α]20,D = 2.7 (c = 5.2 mg/mL, CH3OH). In vitro and in vivo characterization of compounds of the invention Example 1a: Affinity data for the glycine binding site of the NMDA receptor and Permeability Data of parent compounds of prodrugs of formula (I) Scintillation proximity assay (SPA): To determine the affinity of the compounds of the present invention for the glycine binding site of the NMDA receptor a SPA was used. The assay was run in a 384-plate format (OptiPlate-384) where each well contained a mix of 5 µL of test compound, 5 µL NR1s1s2 (ligand binding domains of the NMDA receptor, MW 35.6 kDa, 0.075 ug/well final), 5 µL [3H]- MDL-105,519 (radiolabelled, high affinity N-methyl-D-aspartate (NMDA) glutamate receptor antagonist which binds selectively to the glycine site obtained from Sigma Aldrich, final concentration 5 nM, Kd =1.3 nM), 5 µL streptavidin coated imaging beads (Perkin Elmer cat. No.: RPNQ0273, 8 ug/well). The assay buffer contained 100 mM HEPES-NaOH, 150 mM NaCl, 1 mM EDTA, 10% glycerol at pH 7.4 in ultra-pure water. Non-specific binding wass defined by inclusion of 10 µM L-689,560 (highly potent NMDA antagonist) and total binding by 1% DMSO. Following 30 minutes incubation in the dark (shaker, Multi-microplate Genie), the SPA beads were allowed to settle for 3 hours after which the signal was read on a Viewlux instrument (Perkin Elmer). Normalized data were used to calculate IC50 and Ki values. MDR1-MDCKII permeability assay Papp(AB): A MDR1-MDCKII permeability assay can be used to measure the ability of compounds to passively permeate a cell membrane. MDR1-MDCKII cells (obtained from Piet Borst at the Netherlands Cancer Institute) were seeded onto polyethylene membranes (PET) in 96-well BD insert systems at 2.5 x 105 cells/ mL until to 4-7 days for confluent cell monolayer formation. Experimental Procedure: Test compounds were diluted with the transport buffer (HBSS with 10mM HEPES, added 1% BSA, pH 7.4) from DMSO stock solution to a concentration of 0.5 µM (DMSO: 0.4%) and applied to the apical or basolateral side of the cell monolayer. Permeation of the test compounds from A to B direction or B to A direction was determined in triplicate over a 60- minute incubation at 37°C and 5% CO2 with a relative humidity of 95%. In addition, the efflux ratio of each compound was also determined. Test and reference compounds were quantified by LC/MS/MS analysis based on the peak area ratio of analyte/IS. Reference compounds: Fenoterol (conc: 2 µM) was used as low permeability marker and Propranolol (conc: 2 µM) was used as high permeability marker in A to B Permeability, furthermore was bi-directional permeability of a P-glycoprotein substrate (digoxin) included. Data Analysis: The apparent permeability coefficient Papp (cm/s) was calculated using the equation: Papp = (dCr/dt) x Vr / (A x C0) (1) Where dCr/dt is the cumulative concentration of compound in the receiver chamber as a function of time (µM/s); Vr is the solution volume in the receiver chamber (0.05 mL on the apical side; 0.25 mL on the basolateral side); A is the surface area for the transport, i.e. 0.0804 cm2 for the area of the monolayer; C0 is the initial concentration in the donor chamber (µM). The efflux was calculated using the equation: Efflux ratio = Papp(BA)/Papp(AB) (2) The mass balance (Recovery) was calculated. The mass balance is defined as: the sum of the compound recovered from the acceptor chamber and the compound remaining in the donor chamber at the end of the experiment, divided by the initial donor amount. The mass balance should be as high as possible. Criterion: Recovery < 50% is insufficient. To evaluate the integrity of the cell monolayer, Lucifer Yellow permeability was measured in one direction (A to B). The percentage of Lucifer Yellow was calculated in control wells as an estimation of the overall cell membrane integrity. The wells were considered fully acceptable if %Lucifer Yellow was less than 2%. Table 3a: Permeability data and Ki data of parent compounds of the invention
Figure imgf000246_0001
Figure imgf000247_0001
BLOQ = Below Level of Quantification NT =Not tested Table 3a shows that compounds of formula V have affinity to the glycine site of the NMDA receptor. Example 1b: Permeability Data of prodrug compounds of the invention The experiments for the prodrugs of the invention were the same as for the parent compounds exemplified in Table 3a. The results are listed below in Table 3b. Results shows that prodrugs of the parent compounds have an improved permeability compared to the respective parent compounds. Table 3b: Permeability of prodrug compounds of the invention.
Figure imgf000248_0001
Figure imgf000249_0001
Figure imgf000250_0001
BLOQ = Below Level of Quantification NT =Not tested Tables 3a and 3b disclose that prodrugs of the invention have an improved permeability compared to the parent compounds. Example 2: In vivo exposure data In vivo procedure: Brain disposition of test compounds was evaluated in male Sprague Dawley rats (standard body weight range). Briefly, discrete (nominal dose: 2 mg/kg, 2 ml/kg) or cassetted (nominal dose: 1 mg/kg/compound, 2 ml/kg ) test compounds were administered by intravenous bolus injection (formulated in 10% hydroxypropyl-β-cyclodextrin or 10-20% Captisol, pH=3). Sample collection: Serial blood samples were collected from a lateral tail vein at designated time points (n=3 per time point) then rats were put under deep isofluorane induced anaesthesia prior to removal of brains (n=3 per time point). Blood samples were stabilized against further metabolism ex vivo by addition of an esterase inhibitor (100 µM diisopropyl fluorophosphate). Similarly, esterase inhibitor (100-125 µM) was included in the brain homogenate buffer. Blood was collected into K3-EDTA-coated tubes and the samples are gently turned upside- down to ensure a homogenous sample. The tubes were centrifuged at 3300 x g for 10 minutes at max 4 °C and plasma samples were transferred to Micronic tubes. Brain samples were dissected once the animal had been sacrificed, slightly “dipped” on filter paper to remove blood overflow on the outside, and transferred into Covaris AFA tubes. Plasma and brain samples were stored at -80°C until analysis. Sample preparation: Seven calibration standards and three QC samples were prepared in plasma and brain homogenate, respectively, in the concentration range 10-10000 ng/mL. Blank samples (control matrix with internal standard) were prepared and treated in the same way as calibration standards. Prior to analysis, the brain samples were homogenized with milliQ water 1:4 (w/v) using a Covaris focused-ultrasonicator. Study samples with expected concentration above upper limit of quantification were diluted with blank matrix. Brain homogenate and plasma from study samples, calibration standards, quality controls and blank samples were subsequently treated with the same extraction procedure, i.e. protein precipitation by adding 150 µL acetonitrile with internal standard (Tolbutamide) to 25 μL of sample. Samples were centrifuged and the supernatant from each sample was diluted 1:1 with water to lower the content of organic solvent. LC-MS/MS: Samples were analyzed using an AB Sciex API4000 triple quadrupole (TQ) mass spectrometer operated in positive and negative electrospray ionization and MS/MS mode (multiple reaction monitoring, MRM). The mass spectrometer was coupled to a Waters Acquity UPLC equipped with a Waters Acquity UPLC HSS C18 SB (1.7 µm, 30 mm x 2.1 mm) analytical column. Chromatographic separation was achieved by a 3-minute gradient starting with 98% mobile phase A (0.1% Formic Acid in water) and 2% mobile phase B (0.1% Formic Acid in Acetonitrile) increasing to 95% mobile phase B. Flow rate was 0.6 mL/min and the column temperature was 40oC. Multiple reaction monitoring (MRM) transitions (m/z) were as follows: 380→248, 350→263, Tolbutamide: 269→106 (neg) and 271→155 (pos). Quantification was performed by linear regression, 1/x2 weighting. Concentrations of prodrug and drug in plasma and brain were quantified against matrix matched calibration standards. The blood brain deposition data is shown in the Table 4 below. Table 4: in vivo exposure data of corresponding parent compound after administration a prodrug of the invention in male Sprague Dawley rats
Figure imgf000252_0001
Figure imgf000253_0001
Thus, after administration of compounds of formula I (prodrugs) the corresponding parent compound is observed in rat brain homogenate. Example 3- Maximal Electro Shock Threshold Naïve rats were acclimatized to the procedure room in their home cages, with food and water available ad libitum. All rats were weighed at the beginning of the study and randomly assigned to treatment groups. The individual treatment groups were dosed with either 10% hydroxypropyl-β-cyclodextrin (vehicle), compound 2al (3, 10, or 30 mg/g) or compound 2ba (3, 10, or 30 mg/g), The dosing of the animals were performed by subcutaneous injection 30 minutes before test according to treatment groups. Rats were individually assessed for the production of a tonic hind limb extensor seizure using a Hugo Sachs Electronik stimulator, which delivered an adjustable constant current (1–300 mA) of 0.3 seconds duration via corneal electrodes. The stimulus intensity was varied, from a typical baseline of 25mA, by an ‘up and down’ method of shock titration. Thus, the first rat within a treatment group was given a shock at the expected or estimated seizure threshold (CC50) current, that is, the current producing tonic hind limb extensor seizure in 50% of animals. For subsequent animals, the stimulus intensity was lowered or raised in log 0.06 :10^(1+x*0.06) mA intervals if the preceding rat did or did not show tonic hind limb extension, respectively. This procedure continued for all rats within a treatment group. Data generated from treatment groups of n=12-16 were used to calculate the CC50 values according to the method of Kimball et al. (Kimball A, Burnett W, Doherty D. Chemical protection against ionizing radiation. I. Sampling methods for screening compounds in radiation protection studies with mice. Radiat Res.1957;7(1):1–12). Significant differences between drug-treated animals and vehicle were assessed by a one-way ANOVA with Dunnett’s post hoc test. As shown in Figures 1 and 2, administration of compound 2al dosed at 1, 3 and 10 mg/kg subcutaneously and compound 2ba dosed at 3, 10 and 30 mg/kg subcutaneously showed dose dependent effects on the maximal electro shock threshold. Thus, the administration of the prodrugs 2al and 2ba resulted in a significant change in electrophysiological properties of the brain in rat. NMDA receptor antagonists are known to demonstrate anticonvulsive properties in various rodent seizure/epilepsy models (Parsons CG et.al., Neuropharmacology, (1995), 34, 1239; P. Wlaz et. al., Epilepsia, (1996), 37, 610) Example 4 Porsolt swim test Adult male Wistar Kyoto (WKY) rats from Envigo (former Harlan; Blackthorn, UK). Animals were maintained under controlled conditions (21 ± 1 °C, 37 ± 1 %, 12 / 12 h light / dark cycles, lights on at 8 a.m.) with food and water available ad libitum. In all studies WKY rats were randomly allocated to a maximum of 5 treatment groups (n=20 per group). WKY rats received either a single administration of either saline (vehicle), ketamine (5 mg/kg) or compound 2al (3, 10, or 30 mg/kg) according to treatment group. The dosing of the animals was performed by subcutaneous injection 24 hours before test. The WKY rats were individually placed into a glass cylinder (50 cm height, 20 cm diameter) containing 30 cm of water at 25 ± 1 °C for a 5 minutes test phase. The test session recorded (using a video camera placed above the cylinder for subsequent behavioural analysis) the time of immobility (s). One-way analysis of variance (ANOVA) was used to detect statistical significance in the FST data. The Fisher least significant difference (LSD) test was used for post hoc analyses. Probability values of P<0.05 were considered as statistically significant. Statistical analyses were performed using SPSS. As shown in Figure 3, administration of compound 2al dosed at 1, 3, 10 and 20 mg/kg subcutaneously showed significant effects in time of immobility at the 3, 10 and 20 mg/kg doses. The Porsolt swim test is a well-established model used in basic research and the pharmaceutical screening of potential antidepressant treatments. Ketamine (5mg/kg) induces decreased immobility in this test 24 hours following a single dose. Decreased immobility in the Porsolt swim test was the main endpoint to measure prolonged efficacy (Bogdanova OV et. al. Physiol. Behav.2013; 118: 227-239). Example 5– Resting state Electroencephalography (rsEEG) in rats Surgical procedure On the day of surgery, rats (270-300g) were anesthetized with a 0.25 ml/100g subcutaneous (SC) injection of 1:1 hypnorm/Dormicum and mounted in a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA) with blunt ear bars. Marcain (0.2 ml SC) was injected under the scalp, and gel (Neutral Opthta Eye Gel) put on the eyes to prevent the mucous membrane drying out. Holes were burred in the scull to allow for placement of two depth electrodes (E363-series; PlasticsOne, Roanoke, VA, USA) in left and right pre/infralimbic PFC (AP: 3.0 mm from the bregma suture, Medial-Lateral (ML): +/- 0.7 mm from the sagittal suture and DV: 3.0 mm from the dura) and thalamus (AP: -2.8 mm from the bregma suture, ML: +0.7 mm from the sagittal suture and DV: 4.4 mm from the dura) and three screw electrodes at vertex (AP: -2 mm from the bregma suture, ML: +2.0 mm from the sagittal suture), a reference electrode (AP: +8.0 mm and ML: −2.0 mm), and a ground electrode (AP: -5 mm, ML: +5 mm). During surgeries, nails were cut to prevent rats from scratching wounds following surgery. After completion of surgeries, rats were placed under warming lamps until recovery of consciousness (maximum 4 hours). Water soaked food pellets were placed in the home cage, so the rat easily and quickly could start feeding. Extra muesli was supplied to aid the recovery. Rats were treated with Norodyl and Noromox for 5 days in total and closely observed during a 10-14-day post-surgery recovery period. Animal bodyweights were recorded daily. No rats lost more than 10 % of their pre-surgery bodyweight. Sutures were removed after 7-10 days. At the end of experiments electrical lesions were performed in all recording electrodes and brains were cut for visual microscopy inspection of electrode placement. The differences between depth- and screw-electrode impedances were handled by investigating relative power changes and common-mode noise sources were reduced from recording in shielded boxes and excluding power estimates around 50, 100, and 150 Hz from analyses. Electrophysiological recordings Rats were handled daily and habituated to recording box the week before recording sessions. Recordings were performed during the dark phase of the light/dark cycle. At 8 AM, rats (400- 500 g) were individually transferred to an acrylic chamber (30 cm wide 45 cm deep 55 cm high) placed within an electrically shielded sound-proof box (90 cm wide 55 cm deep 65 cm high) and were tethered to a six-pin wire suspended from a rotating swivel, allowing free movement within the recording box. Rats were habituated for 2 hours followed by 45 minutes of baseline recording, where after rats were injected subcutaneously with 10% hydroxypropyl-β-cyclodextrin (HPβCD, vehicle), 10 mg/kg ketamine in saline, or 20 mg/kg of compound 2al or 2ba in 10 % HPβCD and left in the box for two more hours. Rats only went through recording sessions once a week with at least six days between recordings to allow for wash-out of compounds. The analog local field potential and electrocorticogram (LFP and ECoG) signals were amplified and band-pass filtered at 0.01-300 Hz (Precision Model 440; Brownlee, Palo Alto, CA, USA) and converted to a digital signal at a sampling rate of 1 kHz (CED Power 1401, Power 1 (625 kHz, 16 bit) and CED Expansion ADC16; CED, Cambridge, England). An analog 50 Hz notch filter (Precision Model 440, Brownlee) was applied to the LFP/ECoG signals of the first dataset but was not applied in the following pharmaco-EEG experiments. Video recordings were processed in EthoVision producing the mobility signal, which was subsequently collected in Spike2 along with the LFP/ECoG signals with a delay used subsequently to synchronize the signals. Data analysis The development of the locomotive state-detection algorithm and the state-specific pharmaco-EEG analyses were carried out in MATLAB R2017a (The MathWorks, Inc., Natick, MA, USA) using functions from the sigTOOL toolbox. Significant differences between drug- treated animals and vehicle were assessed according to Turkey’s honest significant difference. As shown in Figures 4 and Figure 5 respectively, administration of compound 2al dosed at 3, 10 and 30 mg/kg subcutaneously and administration of compound 2ba dosed at 3, 10 and 30 mg/kg subcutaneously shows a clear effect on High Frequency Oscillations in the range of 130-160 Hz (HFO) in the nucleus accumbens compared to vehicle (10% captisolHPbetaCD). Thus, after a subcutaneous injection of 10 and 30 mg/kg of compounds 2al and 2ba a significant effect can be observed on the neuronal activity in the nucleus accumbens. Ketamine and d-cycloserine have been shown to exhibit similar effects (I.H. Hansen et. al., Eur J Neurosci., 2019, 50, 1948–1971).

Claims

CLAIMS 1. A compound according to general formula I
Figure imgf000258_0001
wherein R1 is selected from the group consisting of hydrogen and halogen; R2 and R4 each independently are selected from the group consisting of hydrogen, halogen, -NRaRb, 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl, (C3-C6)cycloalkyl and (C1-C4)alkoxy, wherein said 5- or 6- membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl, (C3- C6)cycloalkyl and (C1-C4)alkoxy are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1- C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkyl, hydroxy(C1-C4)alkyl, phenyl, phenoxy and - C(O)NH2, and wherein Ra and Rb each independently are selected from the group consisting of hydrogen and (C1-C4)alkyl; R3 is selected from the group consisting of hydrogen, halogen, (C1-C6)alkyl and (C1- C4)alkoxy; R5 is selected from the group consisting of (C1-C6)alkyl, (C3-C6)cycloalkyl and phenyl, wherein said (C1-C6)alkyl, (C3-C6)cycloalkyl and phenyl are optionally substituted with one or more substituents independently selected from the group consisting of -NH2, (C1-C4)alkoxy, (C3-C6)cycloalkyl, phenyl, 4-6 membered heterocyclyl and -L-R6, wherein -L- represents -C(O)- and R6 is selected from the group consisting of 4-6 membered heterocyclyl; or pharmaceutically acceptable salts thereof, with the proviso that at least one of R1, R2, R3 and R4 is not hydrogen, and with the proviso that when R2, R3 and R4 each represents hydrogen, R1 does not represent fluoro.
2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein R1 is hydrogen.
3. The compound according to any one of claims 1 - 2, or a pharmaceutically acceptable salt thereof, wherein R3 is selected from the group consisting of hydrogen, fluoro, methoxy and (C1-C3)alkyl.
4. The compound according to any one of claims 1 - 3, or a pharmaceutically acceptable salt thereof, wherein R1 and R3 are hydrogen. 5. The compound according to any one of claims 1 - 4, or a pharmaceutically acceptable salt thereof, wherein R2 is selected from the group consisting of hydrogen and halogen and wherein R4 is selected from the group consisting of hydrogen, halogen,
5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4 - 6 membered heterocyclyl and (C3-C6)cycloalkyl, wherein said 5- or 6-membered heteroaryl, phenyl, (C1-C6)alkyl, 4-6 membered heterocyclyl and(C3-C6)cycloalkyl are optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1- C4)alkyl, (C1-C4)alkoxy and halo(C1-C4)alkyl.
6. The compound according to any one of claims 1 - 5, or a pharmaceutically acceptable salt thereof, wherein R2 is fluoro.
7. The compound according to any one of claims 1 - 6, or a pharmaceutically acceptable salt thereof, wherein R4 is selected from the group consisting of 5- or 6- membered heteroaryl, wherein said 5- or 6-membered heteroaryl is optionally substituted with one or more substituents independently selected from the group consisting of halogen, (C1-C4)alkyl, (C1-C4)alkoxy and halo(C1-C4)alkyl.
8. The compound according to any one of claims 1 - 7, or a pharmaceutically acceptable salt thereof, wherein R4 is pyrazolyl, wherein said pyrazolyl is optionally substituted with one or more substituents independently selected from the group consisting of fluor, chloro, (C1-C4)alkyl and halo(C1-C2)alkyl.
9. The compound according to any one of claims 1 - 7, or a pharmaceutically acceptable salt thereof, wherein R4 is phenyl, wherein said phenyl is optionally substituted with one or more substituents independently selected from the group consisting of fluoro, chloro, (C1-C4)alkyl and halo(C1-C2)alkyl.
10. The compound according to any one of claims 1 – 9, wherein R5 is selected from the group consisting of (C1-C6)alkyl and (C3-C6)cycloalkyl, wherein said (C1-C6)alkyl and (C3-C6)cycloalkyl are optionally substituted with one or more substituents independently selected from the group consisting of -NH2, (C1-C4)alkoxy, (C3- C6)cycloalkyl, phenyl and 4-6 membered heterocyclyl.
11. The compound according to any one of claims 1 - 10, selected from the list consisting of (R)-methyl 2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoate, (+)(2R)-methyl 2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoate, (-)(2R)-methyl 2-amino-3-(3-(1-ethoxyethyl)-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(2-ethylfuran-3-yl)-5-fluorobenzamido)propanoate, (-)cis-(2R)-methyl 2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate, (+)cis-(2R)-methyl 2-amino-3-(3-(2-ethyltetrahydrofuran-3-yl)-5- fluorobenzamido)propanoate, methyl (R)-2-amino-3-(3-(3-ethylpyrazin-2-yl)-5-fluorobenzamido)propanoate, methyl (R)-2-amino-3-(3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, methyl (R)-2-amino-3-(3-(5-ethyl-1-methyl-1H-pyrazol-4-yl)-5- fluorobenzamido)propanoate, methyl (R)-2-amino-3-(3-(2-ethylpyridin-3-yl)-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3- (3-ethyl-5-fluorobenzamido)propanoate, (R)-isobutyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate, (R)-isopropyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate, (R)-(S)-1-aminopropan-2-yl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate, (R)-isopentyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate, (R)-2-methoxyethyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate, (R)-2-morpholinoethyl 2-amino-3-(3-ethyl-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(4-ethyl-3-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-ethyl-5-fluoro-4-methylbenzamido)propanoate, (R)-isobutyl 2-amino-3-(3-(5-ethylisoxazol-4-yl)-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-ethyl-4-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(1-ethyl-4-methyl-1H-pyrazol-5-yl)benzamido)propanoate, (R)-methyl 2-amino-3-(3-ethyl-5-fluoro-4-methoxybenzamido)propanoate, (R)-methyl 2-amino-3-(3-(5-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(1-ethyl-1H-imidazol-5-yl)-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(3-ethylisothiazol-4-yl)-5-fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(5-ethylisoxazol-4-yl)benzamido)propanoate, (R)-methyl 2-amino-3-(3-(1-ethyl-1H-1,2,3-triazol-5-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-fluoro-5-(5-propylisoxazol-4-yl)benzamido)propanoate, (R)-methyl 2-amino-3-(3-(2-ethyl-4-methylpyridin-3-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-fluoro-5-(1-propyl-1H-1,2,3-triazol-5- yl)benzamido)propanoate, (R)-methyl 2-amino-3-(3-(4-chloro-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl-3-methyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(1-ethyl-4-(trifluoromethyl)-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(4-(difluoromethyl)-1-propyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-fluoro-5-(1-propyl-4-(trifluoromethyl)-1H-pyrazol-5- yl)benzamido)propanoate, (R)-methyl 2-amino-3-(3-(4-(difluoromethyl)-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-isobutyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-isopropyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-benzyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-phenyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-cyclopropyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-cyclopropylmethyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-2-methoxyethyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-cyclohexyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-2-oxo-2-(pyrrolidin-1-yl)ethyl 2-amino-3-(3-(4-chloro-1-ethyl-1H-pyrazol-5-yl)-5- fluorobenzamido)propanoate, (R)-methyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate, (R)-isopropyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate, (R)-benzyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate, (R)-cyclopropylmethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate, (R)-cyclopropyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3- ylcarboxamido)propanoate and (R)-ethyl 2-amino-3-(2'-ethyl-5-fluoro-[1,1'-biphenyl]-3-ylcarboxamido)propanoate or a pharmaceutically acceptable salt thereof.
12. A pharmaceutical composition comprising a compound, or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 11, and one or more pharmaceutically acceptable carriers or diluents.
13. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11 for use as a medicament.
14. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, or the pharmaceutical composition according to claim 12 for use in the treatment of depression.
15. The compound or pharmaceutical composition according to claim 14, wherein the depression is selected from major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
16. A method for the treatment of depression comprising the administration of a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, or the pharmaceutical composition according to claim 12 to a patient (e.g. a human patient) in need thereof.
17. A method for the treatment of depression comprising the administration of a therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, or the pharmaceutical composition according to claim 12 to a patient (e.g. a human patient) in need thereof.
18. The method for the treatment of depression according to claim 17, wherein depression is selected from major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
19. Use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, or the pharmaceutical composition according to claim 12 for the manufacture of a medicament for use in the treatment of depression.
20. The use of a compound or pharmaceutical composition according to claim 19, wherein the depression is selected from the group consisting of major depressive disorder, treatment-resistant depression, catatonic depression, melancholic depression, atypical depression, psychotic depression, perinatal depression, postpartum depression, bipolar depression, including bipolar I depression and bipolar II depression, and mild, moderate or severe depression.
PCT/EP2021/085681 2020-12-16 2021-12-14 Prodrugs of 3-benzoamido-2-aminopropionic acid derivatives as modulators of the nmda receptor WO2022129041A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP20214467.1 2020-12-16
EP20214467 2020-12-16

Publications (1)

Publication Number Publication Date
WO2022129041A1 true WO2022129041A1 (en) 2022-06-23

Family

ID=73854751

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2021/085681 WO2022129041A1 (en) 2020-12-16 2021-12-14 Prodrugs of 3-benzoamido-2-aminopropionic acid derivatives as modulators of the nmda receptor

Country Status (1)

Country Link
WO (1) WO2022129041A1 (en)

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
"ACS Style Guide", 1997, article "he ACS Style guide - A manual for authors and editors"
"Antidepressants and the risk of suicidal behaviours", JAMA, vol. 292, no. 3, 21 July 2004 (2004-07-21), pages 338 - 343
"Global Burden of Disease", STUDY. LANCET., vol. 349, no. 9063, 17 May 1997 (1997-05-17), pages 1436 - 1442
"Handbook of Pharmaceutical salts. Properties, selection, and use", 2008, WILEY-VCH
BOGDANOVA OV, PHYSIOL. BEHAV., vol. 118, 2013, pages 227 - 239
EXPERT REV CLIN PHARMACOL., vol. 4, no. 3, 1 May 2011 (2011-05-01), pages 379 - 388
GREENFIELD A ET AL: "Synthesis and biological activities of aryl-ether-, biaryl-, and fluorene-aspartic acid and diaminopropionic acid analogs as potent inhibitors of the high-affinity glutamate transporter EAAT-2", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 15, no. 22, 15 November 2005 (2005-11-15), pages 4985 - 4988, XP027801275, ISSN: 0960-894X, [retrieved on 20051115] *
HERESCO-LEVY, U.JAVITT, D.C.GELFIN, Y.GORELIK, E.BAR, M.BLANARU, M.KREMER, I.: "Controlled trial of d-cycloserine adjuvant therapy for treatment-resistant major depressive disorder", J. AFFECT. DISORD., vol. 93, 2006, pages 239 - 243, XP024997304, DOI: 10.1016/j.jad.2006.03.004
I.H. HANSEN, EUR J NEUROSCI., vol. 50, 2019, pages 1948 - 1971
J. JAQUESA. COLLETS. WILEN: "Enantiomers, Racemates, and Resolutions", 1981, JOHN WILEY AND SONS
KIMBALL ABURNETT WDOHERTY D: "Chemical protection against ionizing radiation. I. Sampling methods for screening compounds in radiation protection studies with mice", RADIAT RES., vol. 7, no. 1, 1957, pages 1 - 12
MAOLANON ALEX R. ET AL: "Subtype-Specific Agonists for NMDA Receptor Glycine Binding Sites", ACS CHEMICAL NEUROSCIENCE, vol. 8, no. 8, 16 August 2017 (2017-08-16), US, pages 1681 - 1687, XP055890912, ISSN: 1948-7193, Retrieved from the Internet <URL:https://pubs.acs.org/doi/pdf/10.1021/acschemneuro.7b00117> DOI: 10.1021/acschemneuro.7b00117 *
MAOLANON, ACS CHEM. NEUROSCI., vol. 8, 2017, pages 1681 - 1687
NAKADA Y ET AL: "Novel acyl coenzyme A (CoA): Diacylglycerol acyltransferase-1 inhibitors: Synthesis and biological activities of diacylethylenediamine derivatives", BIOORGANIC, ELSEVIER, AMSTERDAM, NL, vol. 18, no. 7, April 2010 (2010-04-01), pages 2785 - 2795, XP027027287, ISSN: 0968-0896, [retrieved on 20100204] *
OLDEN, M.WYKA, K.CUKOR, J.PESKIN, M.ALTEMUS, M.LEE, F.S.FINKELSTEIN-FOX, L.RABINOWITZ, T.DIFEDE, J.: "Pilot study of a telehealth-delivered medication augmented exposure therapy protocol for PTSD.", J. NERV. MENT. DIS., vol. 205, 2017, pages 154 - 160
PEYROVIAN ET AL., PROGRESS IN NEUROPSYCHOPHARMACOLOGY & BIOLOGICAL PSYCHIATRY, vol. 92, 2019, pages 387 - 404
SCHADE, S.PAULUS, W.: "D-Cycloserine in neuropsychiatric diseases: a systematic review", INT. J., 2016
STEPHAN URWYLER ET AL: "Drug Design, in Vitro Pharmacology, and Structure-Activity Relationships of 3-Acylamino-2-aminopropionic Acid Derivatives, a Novel Class of Partial Agonists at the Glycine Site on the N-Methyl-D-aspartate (NMDA) Receptor Complex", JOURNAL OF MEDICINAL CHEMISTRY, vol. 52, no. 16, 27 August 2009 (2009-08-27), US, pages 5093 - 5107, XP055721101, ISSN: 0022-2623, DOI: 10.1021/jm900363q *
T. GREEN ET AL.: "Protective Groups in Organic Synthesis", 1991, JOHN WILEY & SONS
URWYLER ET AL., J. MED. CHEM., vol. 52, 2009, pages 5093 - 5107

Similar Documents

Publication Publication Date Title
US11419869B2 (en) Dosage forms and regimens for amino acid compounds
CN103097340B (en) Therapeutic activity composition and its application method
JP6065052B2 (en) Tetrahydrocarboline derivative
DE60008372T2 (en) PHARMACEUTICAL ACTIVE COMPOUNDS
EP3316890B1 (en) Novel breathing control modulating compounds, and methods of making and using same
KR102659213B1 (en) Jak1 selective inhibitors
CA2924705A1 (en) Quinazoline derivative and preparation method therefor
EP3344613B1 (en) Heteroaryl compounds and their use as therapeutic drugs
CN107922400B (en) Muscarinic M1 receptor positive allosteric modulators
AU2013344653A1 (en) Novel orally bioavailable breathing control modulating compounds, and methods of using same
KR102276072B1 (en) Cyclic amine derivative and pharmaceutical use thereof
WO2022129047A2 (en) Novel modulators of the nmda receptor
US20220213117A1 (en) Prodrugs of modulators of the nmda receptor
WO2022129041A1 (en) Prodrugs of 3-benzoamido-2-aminopropionic acid derivatives as modulators of the nmda receptor
US11466027B2 (en) Modulators of the NMDA receptor
US20230028658A1 (en) Expanded dosage regimens for integrin inhibitors
TW201130811A (en) Aryl benzylamine compounds
TW202341983A (en) Compounds for mutant kras protein degradation and uses thereof
EA045414B1 (en) SUBSTITUTED ALKYNYLENE COMPOUNDS AS ANTI-CANCER AGENTS
JP2001058991A (en) Sulfonamide derivative
EA040603B1 (en) TRIAZOL-FUSED PIPERAZINE DERIVATIVES AND THEIR APPLICATION AS MODULATORS OF mGLU5 RECEPTORS
WO2019018890A1 (en) Novel spirocyclic compounds

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21824599

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21824599

Country of ref document: EP

Kind code of ref document: A1