Patent Application
20220064128
This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 24, 2021, is named P36352-US sequence listing_ST25.txt and is 13,863 bytes in size.
The invention relates to peptidomimetic (or peptide-like) compounds, specifically viral protease inhibitors, for the treatment of viral infections, and methods of preparing and using such compounds.
In December 2019, a new coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), caused an outbreak of the novel coronavirus disease COVID-19, which has spread to more than 200 countries with over 9 million confirmed cases and over 479,133 confirmed deaths worldwide as of Jun. 26, 2020 (WHO COVID-19 situation report -157). The WHO declared the coronavirus outbreak a public health emergency of international concern. Currently, there are no clinically effective vaccine or specific antiviral drug available for the prevention and treatment of SARS-CoV-2 infections.
Coronaviruses (CoVs) are enveloped, positive-sense, single-stranded RNA viruses. Seven human coronaviruses (HCoVs) have been so far identified, namely HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and the novel coronavirus (SARS-CoV-2). While SARS-CoV, MERS-CoV, and SARS-CoV-2 are highly pathogenic, the others generally cause mild to moderate upper-respiratory tract illness and contribute to 15%-30% cases of common colds in human adults.
The RNA genome of SARS-CoV-2 is about 30 kilobases in length shares approximately 80% sequence identity with SARS-CoV (Zhou P. et al. “A pneumonia outbreak associated with a new coronavirus of probable bat origin.” Nature 579(7798): 270-273, 2020). It consists six major open-reading frames (ORFs). ORF 1a/b, which is about two thirds of the whole genome length, directly translates two polyproteins, pp1a and pp1ab, which encodes 16 nonstructural proteins (nsps) to form the replication transcription complex. Nsp3, which encodes papain-like protease (PLpro), and nsp5, which encodes 3-chymotrypsin-like cysteine protease (3CLpro, also known as main protease, Mpro), are essential for processing these polyproteins. 3CLpro cleaves the polyprotein at 11 distinct sites to generate various nsps that are important for viral replication. Accordingly, inhibitors that block the cleavage function of 3CLpro could inhibit virus replication. In addition, 3CLpro is highly conserved between SARS-CoV and SARS-CoV-2 (96% sequence identity), as well as the other human coronaviruses. Furthermore, no human proteases with a similar cleavage specificity is known. These desired properties make 3CLpro one of the most attractive targets against coronavirus infections.
The invention relates to novel compounds of formula (II), as well as to pharmaceutically acceptable salts thereof,
wherein X, L, R1, R2, R3, R4, and R5 are as defined herein.
Another object of the present invention is related to novel compounds of formula (IIa), as well as to pharmaceutically acceptable salts thereof,
wherein X, L, R1, R2, R3, R4, and R5 are as defined herein.
The invention is also concerned with processes for the manufacture of compounds of formula (II) or (IIa).
The invention also relates to pharmaceutical compositions comprising a compound of formula (II) or (IIa) as described above and a pharmaceutically acceptable carrier and/or adjuvant.
A further aspect of the invention is the use of compounds of formula (II) or (IIa) as therapeutic active substances for the treatment of diseases which are associated with the inhibition of 3CLpro. The invention thus relates to a method for the treatment of a disease associated with the inhibition of 3CLpro activity such as for example coronavirus infections. The compounds of formula (II) or (IIa) show superior 3Clpro inhibition activity. In addition, the compounds of formula (II) or (IIa) also show good cytotoxicity, phototoxicity, solubility, hPBMC, human microsome stability and SDPK profiles, as well as low Cytochrome P450 (CYP) inhibition.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Furthermore, the following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention.
The nomenclature used in this application is based on IUPAC systematic nomenclature, unless indicated otherwise.
The term “chiral” denotes the ability of non-superimposability with the mirror image, while the term “achiral” refers to embodiments which are superimposable with their mirror image. Chiral molecules are optically active, i.e., they have the ability to rotate the plane of plane-polarized light. Whenever a chiral center is present in a chemical structure, it is intended that all stereoisomers associated with that chiral center are encompassed by the present invention.
The term “compound(s) of this invention” and “compound(s) of the present invention” refers to compounds of formula (II) or (IIa) and stereoisomers, solvates or salts thereof (e.g., pharmaceutically acceptable salts).
The term “substituent” denotes an atom or a group of atoms replacing a hydrogen atom on the parent molecule.
The term “C1-6alkyl” denotes a saturated, linear or branched chain alkyl group containing 1 to 6, particularly 1 to 4 carbon atoms, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl and the like. Particular “C1-6alkyl” groups are methyl, ethyl and n-propyl.
The term “C3-7cycloalkyl” denotes a saturated moncyclic hydrocarbon group containing from 3 to 7 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl, more particularly cyclopropyl. In addition, the term “cycloalkyl” also embraces bicyclic hydrocarbon groups containing from 3 to 10 carbon atoms. Bicyclic means consisting of two saturated carbocycles having one or more carbon atoms in common. Examples for bicyclic cycloalkyl are bicyclo[2.2.1]heptanyl or bicyclo[2.2.2]octanyl.
The term “C2-6alkenyl” denotes an unsaturated, linear or branched chain alkenyl group containing 2 to 6, particularly 2 to 4 carbon atoms, for example vinyl, propenyl, allyl, butenyl and the like. Particular “C2-6alkenyl” groups are allyl and vinyl.
The term “C1-6alkoxy” denotes C1-6alkyl-O—.
The term “CxH2x” alone or in combination signifies a saturated, linear or branched chain alkyl group containing x carbon atoms, wherein x is 1, 2, 3, 4, 5, or 6.
The term “oxy” denotes —O—. Example such as benzyloxy refers to benzyl-O—.
The term “oxo” denotes ═O.
The term “halogen” and “halo” are used interchangeably herein and denote fluoro, chloro, bromo, or iodo.
The term “pharmaceutically acceptable salts” denotes salts which are not biologically or otherwise undesirable. Pharmaceutically acceptable salts include both acid and base addition salts.
The term “pharmaceutically acceptable acid addition salt” denotes those pharmaceutically acceptable salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, carbonic acid, phosphoric acid, and organic acids selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, gluconic acid, lactic acid, pyruvic acid, oxalic acid, malic acid, maleic acid, maloneic acid, succinic acid, fumaric acid, tartaric acid, citric acid, aspartic acid, ascorbic acid, glutamic acid, anthranilic acid, benzoic acid, cinnamic acid, mandelic acid, embonic acid, phenylacetic acid, methane sulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicyclic acid.
The term “pharmaceutically acceptable base addition salt” denotes those pharmaceutically acceptable salts formed with an organic or inorganic base. Examples of acceptable inorganic bases include sodium, potassium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Salts derived from pharmaceutically acceptable organic nontoxic bases includes salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperizine, piperidine, N-ethylpiperidine, and polyamine resins.
The term “therapeutically effective amount” denotes an amount of a compound or molecule of the present invention that, when administered to a subject, (i) treats or prevents the particular disease, condition or disorder, (ii) attenuates, ameliorates or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition or disorder described herein. The therapeutically effective amount will vary depending on the compound, the disease state being treated, the severity of the disease treated, the age and relative health of the subject, the route and form of administration, the judgement of the attending medical or veterinary practitioner, and other factors.
The term “pharmaceutical composition” denotes a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with pharmaceutically acceptable excipients to be administered to a mammal, e.g., a human in need thereof.
Inhibitors of 3CLpro
In a first aspect, the present invention provides a compound of formula (II),
wherein
X is carbonyl or SO2;
R1 is H, C1-6alkyl, C3-7cycloalkylC1-6alkyl, or (C1-6alkyl)3Si—C1-6alkyl, wherein said C3-7cycloalkyl is optionally substituted with one to two substituents independently selected from halogen and C1-6alkyl;
R2 is C1-6alkoxy, phenylC2-6alkenyl, benzyloxy, oxazolyl, isoxazolyl, 1H-pyrrolo[2,3-b]pyridinyl, 1H-pyrrolo[3,2-c]pyridinyl, 6H-pyrrolo[2,3-c]pyridinyl, 1H-indolyl, 1H-benzimidazolyl, benzyl, imidazolyl, pyrazinyl, thiazolyl, imidazo[1,2-a]pyridine, pyrazolo[1,5-a]pyridine, phenyl-NH—C1-6alkyl; wherein each of said phenylC2-6alkenyl, phenyl-NH—C1-6alkyl, oxazolyl, isoxazolyl, thiazolyl, benzimidazolyl, 6H-pyrrolo[2,3-c]pyridinyl, and 1H-indolyl is unsubstituted or substituted with one to two substituents independently selected from halogen, C1-6alkyl, halo-C1-6alkyl, and C1-6alkoxy;
R3 is H or C1-6alkyl;
R4 is H, carbamoyl, oxopyrrolidinyl, oxopiperidinyl, 1H-pyrazolyl, 1H-imidazolyl, or (C1-6alkylamino)carbonyl;
R5 is C1-6alkyl substituted with one to two substituents independently selected from halogen and phenyl;
L is —CxH2x—, wherein x is 1, 2, 3, 4, 5, or 6;
or a pharmaceutically acceptable salt thereof.
In one embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (II) is a compound of formula (IIa),
wherein X, L, R1, R2, R3, R4, and R5 are as defined in claim 1.
In one embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (II) is a compound of formula (I),
wherein L, R1, R2, R3, R4, and R5 are as defined herein.
In a preferred embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein R1 is C1-6alkyl, (C1-6alkyl)3Si—C1-6alkyl, or C3-7cycloalkylC1-6alkyl, wherein said C3-7cycloalkyl is optionally substituted with one C1-6alkyl substituent.
In a particularly preferred embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein R1 is isobutyl, isopentyl, 2,2,dimethylpropyl, trimethylsilylmethyl, cyclopropylmethyl, (1-methylcyclopropyl)methyl, 1-bicyclo[1.1.1]pentanylmethyl, or cyclohexylmethyl.
In a preferred embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein R2 is benzyloxy, 1H-indolyl, benzimidazolyl, or isoxazolyl, wherein each of said 1H-indolyl, benzimidazolyl, and isoxazolyl is unsubstituted or substituted with one substituent selected from halogen, C1-6alkyl, halo-C1-6alkyl, and C1-6alkoxy.
In a particularly preferred embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein R2 is benzyloxy, 1H-indolyl, benzimidazolyl, 5-methylisoxazolyl, 5-(difluoromethyl)isoxazolyl, 5-(trifluoromethyl)isoxazolyl, 5-chloro-1H-benzimidazolyl, 5-chloro-1H-indolyl, or 4-methoxy-1H-indolyl.
In a particularly preferred embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein R3 is H.
In a particularly preferred embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein R4 is carbamoyl or oxopyrrolidinyl.
In a particularly preferred embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein L is —CH2— or —CH2—CH2—.
In a preferred embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein R5 is C1-6alkyl substituted twice with halogen.
In a particularly preferred embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein R5 is chlorofluoromethyl.
In a preferred embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein
In a particularly preferred embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein
In one embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein said compound of formula (II) is selected from:
In a preferred embodiment, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, wherein said compound of formula (II) is selected from:
The present invention also relates to the following enumerated embodiments. Thus, in one aspect, the present invention relates to (i) a compound of formula (I),
wherein
R1 is H, C1-6alkyl, or C3-7cycloalkylC1-6alkyl;
R2 is C1-6alkoxy, phenylC2-6alkenyl, benzyloxy, oxazolyl, isoxazolyl, 1H-pyrrolo[2,3-b]pyridinyl, 1H-pyrrolo[3,2-c]pyridinyl, 6H-pyrrolo[2,3-c]pyridinyl, 1H-indolyl, or 1H-benzimidazolyl; wherein each of said phenylC2-6alkenyl, oxazolyl, isoxazolyl, 6H-pyrrolo[2,3-c]pyridinyl, and 1H-indolyl is unsubstituted or substituted with one to two substituents independently selected from halogen, C1-6alkyl, and C1-6alkoxy;
R3 is H or C1-6alkyl;
R4 is H, carbamoyl, oxopyrrolidinyl, oxopiperidinyl, 1H-pyrazolyl, 1H-imidazolyl, or (C1-6alkylamino)carbonyl;
R5 is C1-6alkyl substituted with one to two substituents independently selected from halogen and phenyl;
L is —CxH2x—, wherein x is 1, 2, 3, 4, 5, or 6;
or a pharmaceutically acceptable salt thereof.
Another embodiment of present invention is (ii) a compound of formula (Ia),
wherein
R1 is H, C1-6alkyl, or C3-7cycloalkylC1-6alkyl;
R2 is C1-6alkoxy, phenylC2-6alkenyl, benzyloxy, oxazolyl, isoxazolyl, 1H-pyrrolo[2,3-b]pyridinyl, 1H-pyrrolo[3,2-c]pyridinyl, 6H-pyrrolo[2,3-c]pyridinyl, 1H-indolyl, or 1H-benzimidazolyl; wherein each of said phenylC2-6alkenyl, oxazolyl, isoxazolyl, 6H-pyrrolo[2,3-c]pyridinyl, and 1H-indolyl is unsubstituted or substituted with one to two substituents independently selected from halogen, C1-6alkyl, and C1-6alkoxy;
R3 is H or C1-6alkyl;
R4 is H, carbamoyl, oxopyrrolidinyl, oxopiperidinyl, 1H-pyrazolyl, 1H-imidazolyl, or (C1-6alkylamino)carbonyl;
R5 is C1-6alkyl substituted with one to two substituents independently selected from halogen and phenyl;
L is —CxH2x—, wherein x is 1, 2, 3, 4, 5, or 6;
or a pharmaceutically acceptable salt thereof.
A further embodiment of present invention is (iii) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to (i) or (ii), wherein R1 is C1-6alkyl or C3-7cycloalkylC1-6alkyl.
A further embodiment of present invention is (iv) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to (iii), wherein R1 is isobutyl or cyclohexylmethyl.
A further embodiment of present invention is (v) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (iv), wherein R2 is benzyloxy, 1H-indolyl, or C1-6alkoxy-1H-indolyl.
A further embodiment of present invention is (vi) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to (v), wherein R2 is benzyloxy, 1H-indolyl, or 4-methoxy-1H-indolyl.
A further embodiment of present invention is (vii) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (vi), wherein R3 is H.
A further embodiment of present invention is (viii) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (vii), wherein R4 is carbamoyl.
A further embodiment of present invention is (ix) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (viii), wherein L is —CxH2x—, and wherein x is 2.
A further embodiment of present invention is (x) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to (ix), wherein L is —CH2—CH2—.
A further embodiment of present invention is (xi) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (x), R5 is C1-6alkyl substituted twice with halogen.
A further embodiment of present invention is (xii) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to (xi), wherein R5 is chlorofluoromethyl.
A further embodiment of present invention is (xiii) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to any one of (i) to (xii), wherein
R1 is C1-6alkyl or C3-7cycloalkylC1-6alkyl;
R2 is benzyloxy, 1H-indolyl, or C1-6alkoxy-1H-indolyl;
R3 is H;
R4 is carbamoyl;
R5 is C1-6alkyl substituted twice with halogen;
L is —CxH2x—, and wherein x is 2.
A further embodiment of present invention is (xiv) a compound of formula (I) or (Ia), or a pharmaceutically acceptable salt thereof, according to (xiii), wherein
R1 is isobutyl or cyclohexylmethyl;
R2 is benzyloxy, 1H-indolyl, or 4-methoxy-1H-indolyl;
R3 is H;
R4 is carbamoyl;
R5 is chlorofluoromethyl;
L is —CH2—CH2—.
A further embodiment of present invention is (xv) a compound selected from:
A further embodiment of present invention is (xvi) a compound selected from:
A further embodiment of present invention is (xvii) a compound selected from:
Synthesis
The compounds of the present invention can be prepared by any conventional means. Suitable processes for synthesizing these compounds as well as their starting materials are provided in the schemes below and in the examples. All substituents, in particular, R1 to R5 and L are as defined above unless otherwise indicated. Furthermore, and unless explicitly otherwise stated, all reactions, reaction conditions, abbreviations and symbols have the meanings well known to a person of ordinary skill in organic chemistry.
General synthetic routes for preparing the compound of formula (I) or (Ia) are shown below.
Wherein R4a is R4—CyH2y—, y is 0, 1, 2, 3, or 4, and wherein R4b is R4—CzH2z—, z is 0, 1, 2, 3, 4, or 5.
Compound of formula (IIIa) can be prepared by a coupling reaction of (IIa), tert-butyl N-aminocarbamate and coupling reagent(s) in the presence of an organic base. Deprotection of compound of formula (IIIa) in the presence of a strong acid can afford compound of formula (IVa). Compound of formula (VIa) can be prepared by Michael addition of compound of formula (IVa) with compound for formula (Va) in the presence of an organic base in alcoholic solvent. Alternatively a reduction amination of compound of formula (IVa) with compound of formula (Vb) using NaBH4, NaBH3CN in the presence of TsOH affords compound of formula (VIa). Compound of formula (I-1) can be obtained by a coupling reaction using acid (VIIa), compound of formula (VIa), and coupling reagent(s), such as T3P, HATU, PyBOP or EDCI/HOBt, in the presence of an organic base, such TEA, DIEPA or DMAP. Alternatively, compound of formula (I-1) can be obtained by a reaction of compound of formula (VIa) and acylchloride (VIIb) in the presence of an organic base, such as TEA, DIEPA or DMAP.
Wherein R4a and R4b are as defined in Scheme 1.
Compound of formula (IIIb) can be prepared by a coupling reaction of compound of formula (IIb) with benzyl N-aminocarbamate and coupling reagent(s) in the presence of an organic base. Hydrogenolysis of compound of formula (IIIb) can afford compound of formula (IVb). Compound of formula (VIb) can be prepared by Michael addition of compound of formula (IVb) with compound of formula (Va) in the presence of an organic base in alcoholic solvent. Alternatively a reduction amination of compound of formula (IVb) with compound of formula (Vb) using NaBH4, NaBH3CN in the presence of TsOH affords compound of formula (VIb). Compound of formula (I-2) can be obtained by a coupling reaction using acid (VIIa), compound of formula (VIb), and coupling reagent(s), such as T3P, HATU, PyBOP or EDCI/HOBt, in the presence of an organic base, such TEA, DIEPA or DMAP. Alternatively, compound of formula (I-2) can be obtained by a reaction of compound of formula (VIb) and acylchloride (VIIb) in the presence of an organic base, such as TEA, DIEPA or DMAP. Deprotection of compound of formula (I-2) can afford compound of formula (VIII) in the presence of a strong acid, such as TFA. Compound of formula (I) can be prepared by a coupling reaction of compound of formula (IX) with compound of formula (VIII) and coupling reagent(s), such as T3P, HATU, PyBOP or EDCI/HOBt in the presence of an organic base, such as TEA, DIEPA or DMAP.
Wherein R4a and R4b are as defined in Scheme 1.
Deprotection of compound of formula (IIIb) from Scheme 2 can afford compound of formula (IIIc) in the presence of a strong acid. Compound of formula (IIId) can be prepared by a coupling reaction of compound (IIIc) with compound of formula (IX) and coupling reagent(s) in the presence of an organic base. Hydrogenolysis of compound of formula (IIId) in the presence of Pd/C in organic solvent can afford compound of formula (IVc). Compound of formula (VIc) can be prepared by Michael addition of compound of formula (IVc) and compound of formula (Va) in the presence of an organic base in alcoholic solvent. Alternatively a reduction amination of compound of formula (IVc) with aldehyde (Vb) using NaBH4, NaBH3CN in the presence of TsOH affords compound of formula (VIc). Compound of formula (I) can be obtained by a coupling reaction using acid (VIIa), compound of formula (VIc), and coupling reagent(s), such as T3P, HATU, PyBOP or EDCI/HOBt, in the presence of an organic base, such TEA, DIEPA or DMAP. Alternatively, compound of formula (I) can be obtained by a reaction of compound of formula (VIc) and acylchloride (VIIb) in the presence of an organic base, such as TEA, DIEPA or DMAP.
Compound of formula (IVc) can be prepared by a coupling reaction of compound (IIIc) with carbonyl chloride compound of formula (IX) in the presence of base, such as DIEPA. Compound of formula (IIIf) can be prepared by a coupling reaction of compound (IVc) with (S)-tert-butyl 1-((2-oxopyrrolidin-3-yl)methyl)hydrazinecarboxylate in the presence of coupling reagent(s) such as T3P, HATU, PyBOP or EDCI/HOBt, in the presence of an organic base, such TEA, DIEPA or DMAP.
Deprotection of compound of formula (IIIf) can afford compound of formula (IIIc) in the presence of a strong acid, such as HCl, TFA. Compound of formula (I) can be obtained by a coupling reaction using acid (VIIa), and coupling reagent(s), such as T3P, HATU, PyBOP or EDCI/HOBt, in the presence of an organic base, such TEA, DIEPA or DMAP, or using carbonyl chloride (VIIb) in the presence of organic base, such as DIEPA.
This invention also relates to a process for the preparation of a compound of formula (I) or (Ia) comprising one of the following steps:
a) coupling reaction using compound of formula (VIII),
compound of formula (IX),
and a coupling reagent in the presence of an organic base;
b) a reaction of compound of formula (VIc), and
acid (VIIa)
and a coupling reagent in the presence of an organic base; or
c) a reaction of compound of formula (VIc),
with acylchloride (VIIb), and
in the presence of an organic base;
wherein the coupling agent is selected from T3P, HATU, PyBOP or EDCI/HOBt, the organic base is selected from TEA, DIEPA, and DMAP.
Compounds of this invention can be obtained as mixtures of diastereomers or enantiomers, which can be separated by methods well known in the art, e.g. (chiral) HPLC or SFC.
Chiral compounds, such as compound of formula (Ia), can be obtained according to the schemes above with chiral starting materials.
Pharmaceutical Compositions and Administration
Another embodiment provides pharmaceutical compositions or medicaments containing the compounds of the invention and a therapeutically inert carrier, diluent or excipient, as well as methods of using the compounds of the invention to prepare such compositions and medicaments. In one example, compounds of formula (II) or (IIa) may be formulated by mixing at ambient temperature at the appropriate pH, and at the desired degree of purity, with physiologically acceptable carriers, i.e., carriers that are non-toxic to recipients at the dosages and concentrations employed into a galenical administration form. The pH of the formulation depends mainly on the particular use and the concentration of compound, but preferably ranges anywhere from about 3 to about 8. In one example, a compound of formula (II) or (IIa) is formulated in an acetate buffer, at pH 5. In another embodiment, the compounds of formula (II) or (IIa) are sterile. The compound may be stored, for example, as a solid or amorphous composition, as a lyophilized formulation or as an aqueous or nonaqueous solution.
Compositions are formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The “effective amount” of the compound to be administered will be governed by such considerations, and is the minimum amount necessary to inhibit 3CL protease. For example, such amount may be below the amount that is toxic to normal cells, or the mammal as a whole.
In one example, the pharmaceutically effective amount of the compound of the invention administered parenterally per dose will be in the range of about 0.01 to 100 mg/kg, alternatively about 0.1 to 100 mg/kg of patient body weight per day, with the typical initial range of compound used being 0.3 to 15 mg/kg/day. In another embodiment, oral unit dosage forms, such as tablets and capsules, preferably contain from about 1 to about 500 mg of the compound of the invention.
The compounds of the invention may be administered by any suitable means, including oral, topical (including buccal and sublingual), rectal, vaginal, transdermal, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intradermal, intrathecal and epidural and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
The compounds of the present invention may be administered in any convenient administrative form, e.g., tablets, powders, capsules, solutions, dispersions, suspensions, syrups, sprays, suppositories, gels, emulsions, patches, etc. Such compositions may contain components conventional in pharmaceutical preparations, e.g., diluents, carriers, pH modifiers, sweeteners, bulking agents, and further active agents.
A typical formulation is prepared by mixing a compound of the present invention and a carrier or excipient. Suitable carriers and excipients are well known to those skilled in the art and are described in detail in, e.g., Ansel, Howard C., et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems. Philadelphia: Lippincott, Williams & Wilkins, 2004; Gennaro, Alfonso R., et al. Remington: The Science and Practice of Pharmacy. Philadelphia: Lippincott, Williams & Wilkins, 2000; and Rowe, Raymond C. Handbook of Pharmaceutical Excipients. Chicago, Pharmaceutical Press, 2005. The formulations may also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents, diluents and other known additives to provide an elegant presentation of the drug (i.e., a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e., medicament).
An example of a suitable oral dosage form is a tablet containing about 0.01 to 500 mg of the compound of the invention compounded with about 50 to 400 mg anhydrous lactose, about 5 to 40 mg sodium croscarmellose, about 5 to 30 mg polyvinylpyrrolidone (PVP) K30, and about 0.1 to 10 mg magnesium stearate. The powdered ingredients are first mixed together and then mixed with a solution of the PVP. The resulting composition can be dried, granulated, mixed with the magnesium stearate and compressed to tablet form using conventional equipment. An example of an aerosol formulation can be prepared by dissolving the compound, for example 0.01 to 400 mg, of the invention in a suitable buffer solution, e.g. a phosphate buffer, adding a tonicifier, e.g. a salt such sodium chloride, if desired. The solution may be filtered, e.g., using a 0.2 micron filter, to remove impurities and contaminants.
An embodiment, therefore, includes a pharmaceutical composition comprising a compound of Formula (II) or (IIa), or a stereoisomer or pharmaceutically acceptable salt thereof. In a further embodiment includes a pharmaceutical composition comprising a compound of Formula (II) or (IIa), or a stereoisomer or pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier or excipient.
Another embodiment includes a pharmaceutical composition comprising a compound of Formula (II) or (IIa) for use in the treatment of coronavirus infections.
The following embodiments illustrate typical compositions of the present invention, but serve merely as representative thereof.
Composition A
A compound of the present invention can be used in a manner known per se as the active ingredient for the production of tablets of the following composition:
Composition B
A compound of the present invention can be used in a manner known per se as the active ingredient for the production of capsules of the following composition:
Indications and Methods of Treatment
The present invention provides peptidomimetic (or peptide-like) compounds that inhibit the enzymatic activity of 3C-like proteases from coronaviruses including, but not limited to, severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Accordingly, the compounds of the invention are useful for blocking coronavirus replication in infected cells. As such, the compounds can be used as a therapeutic or prophylactic agent for treatments of coronavirus infections.
In one aspect, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, for use as therapeutically active substance.
The present invention also provides methods for treatment or prophylaxis of viral infections caused by coronaviruses including, but not limited to, SARS-CoV, SARS-CoV-2 and MERS-CoV in a patient in need thereof.
Another embodiment includes a method of treating or preventing viral infections caused by coronaviruses including, but not limited to, SARS-CoV, SARS-CoV-2 and MERS-CoV in a mammal in need of such treatment, wherein the method comprises administering to said mammal a therapeutically effective amount of a compound of formula (II), a stereoisomer, tautomer, prodrug or pharmaceutically acceptable salt thereof.
In one aspect, the present invention provides the use of a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, for the treatment or prophylaxis of coronavirus infections, e.g. for the treatment or prophylaxis of severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or Middle East Respiratory Syndrome Coronavirus (MERS-CoV).
In one aspect, the present invention provides the use of a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for the treatment or prophylaxis of coronavirus infections, e.g. for the preparation of a medicament for the treatment or prophylaxis of SARS-CoV, SARS-CoV-2 or MERS-CoV infections.
In one aspect, the present invention provides the use of a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for inhibiting enzymatic activity of 3C-like proteases.
In one aspect, the present invention provides a compound of formula (II) as described herein, or a pharmaceutically acceptable salt thereof, for use in the treatment or prophylaxis of coronavirus infections, e.g. for use in the treatment or prophylaxis of SARS-CoV, SARS-CoV-2 or MERS-CoV infections.
The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention.
Abbreviations used herein are as follows:
General Experimental Conditions
Intermediates and final compounds were purified by flash chromatography using one of the following instruments: i) Biotage SP1 system and the Quad 12/25 Cartridge module, ii) ISCO combi-flash chromatography instrument. Silica gel Brand and pore size: i) KP-SIL 60 Å, particle size: 40-60 μm; ii) CAS registry NO: Silica Gel: 63231-67-4, particle size: 47-60 micron silica gel; iii) ZCX from Qingdao Haiyang Chemical Co., Ltd, pore: 200-300 or 300-400.
Alternatively, intermediates and final compounds were purified by preparative HPLC on reversed phase column using X Bridge™ Perp C18 (5 μm, OBD™ 30×100 mm) column, SunFire™ Perp C18 (5 μm, OBD™ 30×100 mm) column, Phenomenex Luna C18 75*30 mm*3 μm column or Phenomenex Synergi C18 150*25 mm*10 μm column.
For SFC chiral separation, intermediates were separated by chiral column (Daicel chiralpak IC, 5 μm, 30×250 mm), AS (10 μm, 30×250 mm) or AD (10 μm, 30×250 mm) using Mettler Toledo Multigram III system SFC, Waters 80Q preparative SFC or Thar 80 preparative SFC, solvent system: CO2 and IPA (0.5% TEA in IP A) or CO2 and MeOH (0.1% NH3—H2O in MeOH), back pressure 100 bar, detection UV@ 254 or 220 nm.
LC/MS spectra were obtained using a Waters UPLC-SQD Mass. Standard LC/MS conditions were as follows (running time 3 mins):
Acidic condition: A: 0.1% formic acid and 1% acetonitrile in H2O; B: 0.1% formic acid in acetonitrile;
Basic condition: A: 0.05% NH3H2O in H2O; B: acetonitrile.
Mass spectra (MS): generally only ions which indicate the parent mass are reported, and unless otherwise stated the mass ion quoted is the positive mass ion (M+H)+.
NMR Spectra were obtained using Bruker Avance 400 MHz.
All reactions involving air-sensitive reagents were performed under an argon atmosphere. Reagents were used as received from commercial suppliers without further purification unless otherwise noted.
Preparation of Intermediate
To a solution of (2S)-2-amino-3-cyclopropyl-propanoic acid (1.0 g, 7.74 mmol, Shanghai Titan Scientific Co., Ltd, CAS number: 102735-53-5), Na2CO3 (1.6 g, 15.48 mmol) and NaHCO3 (0.65 g, 7.74 mmol) in water (30 mL) and acetone (4 mL) was added CbzCl (1.65 g, 9.68 mmol) at 0° C. After being stirred at 20° C. for 3 hrs, the reaction mixture was extracted with EtOAc (20 mL) twice. The aqueous layer was acidized by HCl (1M) until pH=2 and then extracted with EtOAc (20 mL) three times. The combined organic layers were washed with brine (10 mL), dried over Na2SO4 and concentrated in vacuo. (2S)-2-(Benzyloxycarbonylamino)-3-cyclopropyl-propanoic acid (1.3 g, Intermediate AA) was obtained as a yellow oil, which was used in next step without further purification. MS obsd. (ESI+) [(M+Na)+]: 286.1.
Intermediate AB was prepared in analogy to Intermediate AA by using (2S)-2-amino-3-cyclopentyl-propanoic acid (1.0 g, 6.36 mmol, Bidepharm, CAS number: 99295-82-6) instead of (2S)-2-amino-3-cyclopropyl-propanoic acid. (S)-2-(((Benzyloxy)carbonyl)amino)-3-cyclopentylpropanoic acid (1.3 g, Intermediate AB) was obtained as a light yellow oil. 1H NMR (400 MHz, CDCl3) δ ppm: 7.33-7.36 (m, 5H), 5.22 (d, J=8.8 Hz, 1H), 5.16 (s, 2H), 4.37-4.40 (m, 1H), 1.80-1.89 (m, 5H), 1.61-1.63 (m, 2H), 1.52-1.56 (m, 2H), 1.11-1.15 (m, 2H).
To a solution of (S)-2-(((benzyloxy) carbonyl)amino)-4-methylpentanoic acid (10.0 g, 37.69 mmol, Wuxi Apptec (Wuhan) Co., Ltd.) and tert-butyl hydrazinecarboxylate (5.48 g, 41.46 mmol) in EtOAc (150 mL) was added T3P (33.6 mL, 56.54 mmol, 50% in EtOAc) and DIEPA (18.7 mL, 113.1 mmol) at 0° C. After being stirred at 25° C. for 3 hrs, the reaction mixture was poured into water (200 mL) and extracted with EtOAc (200 mL) three times. The combined organic phases were washed with HCl (1M, 200 mL) twice, saturated aqueous NaHCO3 (200 mL) twice, brine (200 mL) twice, dried over anhydrous Na2SO4 and concentrated in vacuo. (S)-tert-Butyl 2-(2-(((benzyloxy)carbonyl)amino)-4-methyl pentanoyl) hydrazinecarboxylate (14.0 g, Intermediate AC-1) was obtained as a light yellow oil and used in next step without further purification. MS obsd. (ESI+) [(M−tBu+H)+]: 323.9.
To a solution of (S)-tert-butyl 2-(2-(((benzyloxy)carbonyl)amino)-4-methyl pentanoyl) hydrazinecarboxylate (5.0 g, Intermediate AC-1) in methanol (50 mL) was added Pd/C (500.0 mg, 10% purity). The mixture was degassed in vacuo and purged with H2 three times. After being stirred under H2 balloon at 25° C. for 12 hrs, the resulting mixture was filtered through a pad of celite. The filtrate was concentrated in vacuo to give (S)-tert-butyl 2-(2-amino-4-methylpentanoyl) hydrazinecarboxylate (3.0 g, Intermediate AC) as a light yellow oil and used in next step without further purification. 1H NMR (400 MHz, CDCl3) δ ppm: 3.48-3.51 (m, 1H), 1.67-1.76 (m, 2H), 1.46 (s, 9H), 1.38-1.44 (m, 1H), 0.94 (dd, J=6.4 Hz, J=15.6 Hz, 6H).
To a solution of tert-butyl (2R,3S)-6-oxo-2,3-diphenylmorpholine-4-carboxylate (1.0 g, 2.83 mmol, GL Biochem, CAS 112741-49-8), 3-bromo-2-methylprop-1-ene (413 mg, 3.06 mmol, Shanghai Titan, CAS: 1458-98-6) and HMPA (1.0 mL) in THF (10 mL) was added NaHMDS (3.08 mL, 3.08 mmol, 1M) at −70° C. under N2. The reaction mixture was stirred at −70° C. for 1 hr, then quenched with sat. aq. solution of NH4Cl (30 mL), extracted with EtOAc (50 mL) 3 times. The combined organic layers were washed with brine (20 mL) and concentrated in vacuo. The residue was purified by column, eluted with EtOAc (0˜2%) in petroleum ether to afford tert-butyl (3S,5S,6R)-3-(2-methylallyl)-2-oxo-5,6-diphenylmorpholine-4-carboxylate (600 mg, Intermediate AD-1) as a yellow oil. MS obsd. (ESI+) [M−C4H8+H]+: 352.2.
To a solution of liquid NH3 (500.0 mL) in ethanol (40 mL) and THF (400 mL) was added tert-butyl (3S,5S,6R)-3-(2-methylallyl)-2-oxo-5,6-diphenylmorpholine-4-carboxylate (47.0 g, AD-1) at −70° C. Then Li (7.2 g) was added. After stirred at −70° C. for 1 hr, the reaction mixture was quenched with NH4Cl solution (41 mL, 20%) at 0° C. After addition, the mixture was warmed to 25° C. and diluted with water (300 mL). The aqueous phase was washed with MTBE (300 mL) 2 times. Then the aqueous layers was adjusted by HCl (1M) until pH=1 at 0° C. and extracted with EtOAc (500 mL) 3 times. The combined organic layers were concentrated in vacuo to afford (S)-2-((tert-butoxycarbonyl)amino)-4-methylpent-4-enoic acid (18.93 g, Intermediate AD-2) as a light yellow oil. 1H NMR (400 MHz, CDCl3) δ ppm: 5.00-4.76 (m, 3H), 4.41 (d, J=4.4 Hz, 1H), 2.67-2.50 (m, 1H), 2.43-2.38 (m, 1H), 1.77 (s, 3H), 1.45 (s, 9H)
To a mixture of (S)-2-((tert-butoxycarbonyl)amino)-4-methylpent-4-enoic acid (18.9 g, Intermediate AD-2) and K2CO3 (34.19 g) in DMF (300 mL) was added Mel (23.4 g) at 20° C. After stirred at 20° C. for 1 hr, the reaction mixture was filtered and the mother liquor was diluted with brine (400 mL) and extracted with EtOAc (300 mL) three times. The combined organic layers were washed with brine (300 mL) two times, dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by column, eluted with EtOAc (0-6%) in petroleum ether to afford methyl (S)-2-((tert-butoxycarbonyl)amino)-4-methylpent-4-enoate (17.2 g, Intermediate AD-3) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ ppm: 4.94 (d, J=6.8 Hz, 1H), 4.86 (s, 1H), 4.76 (s, 1H), 4.48-4.34 (m, 1H), 3.74 (s, 3H), 2.59-2.45 (m, 1H), 2.39-2.34 (m, 1H), 1.75 (s, 3H), 1.44 (s, 9H)
A mixture of methyl (S)-2-((tert-butoxycarbonyl)amino)-4-methylpent-4-enoate (17.2 g, Intermediate AD-3) in HCl/dioxane (200.0 mL, 4M) was stirred at 20° C. for 2 hrs. Then the reaction mixture was concentrated in vacuo to afford methyl (S)-2-amino-4-methylpent-4-enoate hydrochloride (12.7 g, Intermediate AD-4) and used directly without purification.
To a solution of methyl (S)-2-amino-4-methylpent-4-enoate hydrochloride (12.7 g, Intermediate AD-4) in THF (100 mL) was added a solution of Na2CO3 (29.97 g) in water (100 mL) at 0° C. CbzCl (18.09 g) was added to the mixture at 0° C. After stirred at 20° C. for 1 hr, the reaction mixture was diluted with water (200 mL) and extracted with EtOAc (200 mL) three times. The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by column, eluted with EtOAc (0%˜7%) in petroleum ether to afford methyl (S)-2-(((benzyloxy)carbonyl) amino)-4-methylpent-4-enoate (14.03 g, Intermediate AD-5) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ ppm: 7.43-7.29 (m, 5H), 5.20 (d, J=7.2 Hz, 1H), 5.12 (s, 2H), 4.89-4.83 (m, 1H), 4.76 (s, 1H), 4.55-4.45 (m, 1H), 3.75 (s, 3H), 2.60-2.50 (m, 1H), 2.43-2.38 (m, 1H), 1.75 (s, 3H). MS obsd. (ESI+) [(M+H)+]: 278.2.
ZnEt2 (196.2 mL, 1 Min hexane) was added to DCM (200 mL) at 0° C. Then TFA (15.0 mL) in DCM (75 mL) was added at 0° C. under N2 protection. After stirred at 0° C. for 20 mins, Diiodomethane (16.0 mL, Energy Chemical, CAS: 75-11-6) in DCM (35 mL) was added into above solution at 0° C. under N2 protection. The mixture was stirred at 0° C. for another 20 min. Then to the mixture, methyl (S)-2-(((benzyloxy)carbonyl)amino)-4-methylpent-4-enoate (5.0 g AD-5) in DCM (35 mL) was added at 0° C. The resulting mixture was stirred at 20° C. for 2 hrs. After reaction, the reaction mixture was quenched with 1 M aq. solution of HCl (80 mL) at 0° C. The aqueous layer was separated. The organic layer was washed with sat.aq. NaHCO3 (30 mL) and concentrated in vacuo. The residue was purified by column, eluted with EtOAc (0˜7.5%) in petroleum ether to afford methyl (S)-2-(((benzyloxy)carbonyl)amino)-3-(1-methylcyclopropyl)propanoate (4.16 g, Intermediate AD-6) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ ppm: 7.40-7.32 (m, 5H), 5.21 (d, J=7.6 Hz, 1H), 5.17-5.03 (m, 2H), 4.51 (q, J=8.0 Hz, 1H), 3.75 (s, 3H), 1.76-1.68 (m, 1H), 1.67-1.58 (m, 1H), 1.10 (s, 3H), 0.36-0.17 (m, 4H). MS obsd. (ESI+) [(M+H)+]: 292.1.
To a mixture of methyl (S)-2-(((benzyloxy)carbonyl)amino)-3-(1-methylcyclopropyl)propanoate (4.16 g, Intermediate AD-6) in THF (20 mL) was added a solution of LiOH H2O (1.2 g) in water (20 mL) at 0° C. After stirred at 0° C. for 2 hrs, the reaction mixture was extracted with EtOAc (100 mL) two times. The combined organic layers were dried over anhydrous Na2SO4 and concentrated in vacuo to afford (S)-2-(((benzyloxy)carbonyl)amino)-3-(1-methylcyclopropyl)propanoic acid (3.58 g, Intermediate AD) as a light yellow oil. 1H NMR (400 MHz, CDCl3) δ ppm: 8.18 (br. s, 1H), 7.40-7.29 (m, 5H), 5.27 (d, J=8.0 Hz, 1H), 5.20-5.10 (m, 2H), 4.60-4.48 (m, 1H), 1.87 (dd, J1=5.6 Hz, J2=14.0 Hz, 1H), 1.60 (dd, J1=8.4 Hz, J2=14.0 Hz, 1H), 1.11 (s, 3H), 0.38-0.21 (m, 4H). MS obsd. (ESI+) [(M+H)+]: 278.2.
To a solution of ethyl chlorofluoroacetate (1000 g) in ethanol (9 L) was added water (1 L) and NaOH (313 g). After stirred at 25° C. for 12 hrs, the reaction mixture was concentrated under reduced pressure to remove most of EtOH. Then the residue was diluted with water (1500 mL) and acidified with HCl (2M) until pH=4˜5. The mixture was extracted with MTBE (1 L) 4 times. The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to give crude compound without further purification. The compound 2-chloro-2-fluoro-acetic acid (718 g, AE-1) was obtained as a colorless oil which was used directly. 1H NMR (400 MHz, CDCl3) δ 6.16 (d, J=50.4 Hz, 1H)
To a solution of 2-chloro-2-fluoro-acetic acid (718.0 g, AE-1) in EtOAc (3000 mL) was added 0° C. solution of (S)-1-phenylethanamine (386.73 g) in EtOAc (3000 mL). The mixture was stirred at 0° C. for 2 hrs and then stood over night. The reaction mixture was filtered and the solid was dissolved with acetone (760 g, 7600 mL) under 80° C. The resulting solution was slowly cooled to 20° C. and stood overnight. The precipitate was filtrated, collected and dissolved with acetone (100 g/1 L) at 80°. The recrystallization operation was repeated for 2 times. The collected solid was triturated with acetone for 3 times (acetone, 100 g/0.5 L). The solid was collected and dissolved with water (1000 mL) and acidified with HCl (1M, 750 mL). The mixture was extracted with MTBE (1 L) three times. The combined organic layers were dried over Na2SO4, filtered and concentrated under reduced pressure to afford (R)-2-chloro-2-fluoroacetic acid (121.15 g, 1080 mmol, 61% purity, containing MTBE, Intermediate AE-2) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ 6.29 (d, J=50.8 Hz, 1H)
To (R)-2-chloro-2-fluoroacetic acid (12.0 g) was added PCl5 (24.46 g). The reaction mixture was stirred at 20° C. for 1 hr. Then the reaction mixture was stirred at 70° C. for 1 hr. 1H NMR showed the reaction was finished. (R)-2-chloro-2-fluoroacetyl chloride (22.84 g, 40% purity with POCl3 and MTBE) was collected by distillation (70° C., 20 mmHg) as a colorless liquid. 1H NMR (400 MHz, CDCl3) δ 6.40 (d, J=50.8 Hz, 1H).
To a solution of tert-butyl 2-oxopyrrolidine-1-carboxylate (100.0 g, Wuxi catalog: CAS number: 85909-08-6) in THF (1000 mL) was added to a stirring solution of LiHMDS (1133.79 mL) at 0° C. The reaction mixture was allowed to warm up to 20° C. over 30 min before 2,2,2-trifluoroethyl trifluoroacetate (211.69 g, 1080 mmol) was added. After stirred for additional 20 min at 20° C., the reaction mixture was extracted with EtOAc (600 mL) two times. The combined organic phases were dried over Na2SO4, filtered and concentrated in vacuo. The oil was dissolved in toluene (1000 mL) then formaldehyde (48.64 g) and K2CO3 (164.16 g) were added into the solution. The reaction mixture was heated at 110° C. for 2 hours. The mixture was diluted with ethyl acetate (800 mL), washed with brine (200 mL) three times, dried over anhydrous Na2SO4, filtered and concentrated in vacuo to afford the desired product (tert-butyl 3-methylene-2-oxo-pyrrolidine-1-carboxylate (80 g, AF-1) as a light yellow oil which was used directly. MS obsd. (ESI+) [M−Bu+H]+: 142.1.
To a solution of tert-butyl 3-methylene-2-oxo-pyrrolidine-1-carboxylate (80.0 g) in IP A (800 mL) was added benzyl carbazate (67.41 g). After stirred at 85° C. for 12 hrs under N2 protection, the reaction mixture was concentrated in vacuo to afford crude tert-butyl 3-[(2-benzyloxycarbonylhydrazino)methyl]-2-oxo-pyrrolidine-1-carboxylate (147 g, AF-2) as a yellow oil which was used directly. MS obsd. (ESI+) [2M+H]+: 727.2
To a solution of tert-butyl 3-[(2-benzyloxycarbonylhydrazino)methyl]-2-oxo-pyrrolidine-1-carboxylate (147.0 g, Intermediate AF-2) in DCM (700 mL) was added TFA (500.0 mL). After stirred at 20° C. for 2 hrs, the reaction mixture was concentrated in vacuo to afford benzyl 2-((2-oxopyrrolidin-3-yl)methyl)hydrazinecarboxylate (147.0 g, Intermediate AF-3) as a yellow oil which was used directly. MS obsd. (ESI+) [M+H]+: 264.0.
To a solution of benzyl N-[(2-oxopyrrolidin-3-yl)methylamino]carbamate (150.0 g, Intermediate AF-3) in ethanol (800 mL) was added DIEA (220.89 g) and Boc2O (149.21 g). After stirred at 50° C. for 12 hrs, the reaction mixture was concentrated in vacuo. The residue was diluted with ethyl acetate (1000 mL), washed with brine (200 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column, eluted with petroleum ether/ethyl acetate from 1:1 to 0:1 to afford an impure product which was conducted a further purification by reverse flash (0.1% TFA) to afford 2-benzyl 1-ten-butyl 1-((2-oxopyrrolidin-3-yl)methyl)hydrazine-1,2-dicarboxylate (50 g, Intermediate AF-4) as a light yellow oil. MS obsd. (ESI+) [M−Boc+H]+: 264.2
To a solution of tert-butyl N-(benzyloxycarbonylamino)-N-[(2-oxopyrrolidin-3-yl)methyl]carbamate (160.0 g, Intermediate AF-4) in methanol (1500 mL) was added Pd/C (15.0 g, 10% purity) and Pd(OH)2/C (15.0 g) under N2. The reaction mixture was degassed under vacuum and purged H2 for 3 times. After stirred at 25° C. for 12 hrs under H2 balloon, the resulting mixture was filtered through a pad of celite and the filtrate was concentrated in vacuo to afford tert-butyl 1-((2-oxopyrrolidin-3-yl)methyl)hydrazinecarboxylate (100 g) as a colorless oil.
The racemate was purified with SFC preparation: (Column: Phenomenex-Cellulose-2 (250 mm*50 mm, 10 um); Condition: Neu-MeOH; B %: 45-45%; FlowRate (ml/min): 220) to afford (S)-tert-butyl 1-((2-oxopyrrolidin-3-yl)methyl)hydrazinecarboxylate (faster eluting, 41.4 g, Intermediate AF) as a light yellow solid. 1H NMR (400 MHz, CDCl3) δ 5.77 (s, 1H), 3.55-3.75 (m, 2H), 3.30-3.45 (m, 2H), 2.75-2.85 (m, 1H), 2.20-2.30 (m, 1H), 1.89-1.99 (m, 1H), 1.42-1.51 (m, 9H). MS obsd. (ESI+) [M+H]+: 230.1. SFC: RT=1.615 min, ee %=98.05%.
The absolute configuration was determined by X-Ray.
To a suspension of 1,1-dibromo-2,2-bis(chloromethyl)cyclopropane (10 g, WuXi catalog, CAS number: 98577-44-7) in diethyl ether (11.49 mL) was added methyllithium QAM, 20.56 g) dropwise at −50 to −60° C. under N2 atmosphere. Then the reaction mixture was allowed to warm to 0° C. and stirred for 2 hrs. After reaction, a distillation apparatus is connected and the receiving flask is cooled to −78° C. Tricyclo[1.1.1.01,3]pentane (Intermediate AG-1) is obtained as a solution in Et2O (0.3 to 0.6M) in 70-95% yield.
A solution of (2R)-2-(tert-butoxycarbonylamino)-3-iodo-propionic acid methyl ester (658 mg) in diethyl ether (5 mL) was added 0.300 M tricyclo[1.1.1.01,3]pentane (6.66 mL, Intermediate AG-1) at room temperature. After stirred at this temperature for 15 min, triethylborane (199.92 uL, 1M) was added and stirred for another 2 hrs. The reaction mixture was concentrated and purified by silica gel chromatography (EtOAc/PE form 0% to 12%) to afford (2S)-2-(tert-butoxycarbonylamino)-3-(3-iodo-1-bicyclo[1.1.1]pentanyl)propionic acid methyl ester (788 mg, Intermediate AG-2) as a white solid. MS obsd. (ESI+) [M+H]+:396.
To a solution of (2S)-2-(tert-butoxycarbonylamino)-3-(3-iodo-1-bicyclo[1.1.1]pentanyl)propionic acid methyl ester (287 mg, Intermediate AG-2) and 2,6-lutidine (233.42 mg) in tetrahydrofuran (2 mL) was added bis(trimethylsilyl)silyl-trimethyl-silane (180.57 mg) and stirred at rt for 30 min. Then triethylborane (142.31 mg) was added and stirred for another 1 hr. The resulting mixture was stirred for 15 min at rt and concentrated in vacuo. The residue was purified by column chromatography (EtOAc/PE from 10% to 30%) to afford (2S)-3-(1-bicyclo 1.1.1 pentanyl)-2-(tert-butoxycarbonylamino)propionic acid methyl ester (85 mg intermediate AG-3) as a colorless oil.
A solution of (2S)-3-(1-bicyclo[1.1.1]pentanyl)-2-(tert-butoxycarbonylamino)propionic acid methyl ester (85 mg, Intermediate AG-3) in dichloromethane (1 mL) was added TFA (1.48 g, 1 mL) at 0° C. After stirred at rt for 1 hour, the resulting mixture was concentrated in vacuo to afford crude (2S)-2-amino-3-(1-bicyclo[1.1.1]pentanyl)propionic acid methyl ester TFA salt (80 mg, Intermediate AG) as a light yellow oil which can be used for next step without purification.
To a solution of 2-(benzyloxycarbonylamino)acetic acid (5.0 g, 23.9 mmol) and tert-butyl carbazate (3.16 g, 23.9 mmol, Wuxi Apptec (Wuhan) Co., Ltd.) in EtOAc (80 mL) was added T3P (21.3 mL, 35.9 mmol) and DIEPA (11.9 mL, 71.7 mmol) at 0° C. The mixture was stirred at 25° C. for 3 hrs, and then diluted with water (100 mL), extracted with EtOAc (100 mL) three times. The combined organic layer was washed with HCl (1M, 100 mL) twice, saturated aqueous NaHCO3 (100 mL) twice, and brine (100 mL) twice, dried over anhydrous Na2SO4 and concentrated in vacuo. tert-Butyl N-[[2-(benzyloxycarbonylamino)acetyl]amino]carbamate (8.0 g, compound 1a) was obtained as a light yellow oil and used in next step without further purification. MS obsd. (ESI+) [(M−Boc+H)+]: 223.9.
A solution of tert-butyl N-[[2-(benzyloxycarbonylamino)acetyl]amino]carbamate (2.0 g, compound 1a) in HCl/dioxane (4M, 20.0 mL) was stirred at 25° C. for 2 hrs. After reaction, the resulting reaction mixture was concentrated in vacuo to afford benzyl (2-hydrazinyl-2-oxo-ethyl)carbamate (1.7 g, compound 1b) as a yellow solid and used in next step without further purification. MS obsd. (ESI+) [(M+H)+]: 223.9.
To a solution of benzyl (2-hydrazinyl-2-oxo-ethyl)carbamate (300.0 mg, compound 1b) in 2-propanol (3 mL) was added acrylamide (49.3 mg). After stirred at 60° C. for 1.5 hrs and at 85° C. for another 2 hrs, the reaction mixture was concentrated in vacuo. The residue was purified by prep-HPLC to afford benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (80 mg, compound 1c) as a light yellow oil. MS obsd. (ESI+) [(M+H)+]: 295.0.
To a solution of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (80.0 mg, 0.270 mmol, compound 1c) and TEA (55.0 mg, 0.540 mmol) in THF (2 mL) was added chloroacetyl chloride (30.7 mg, 0.270 mmol) at 0° C. The mixture was warmed to 25° C. and stirred for 2 hrs. After reaction, the resulting mixture was diluted with H2O (10 mL) and extracted with EtOAc (10 mL) three times. The combined organic phases were dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by prep-HPLC) to afford benzyl N-[2-[2-(3-amino-3-oxo-propyl)-2-(2-chloroacetyl)hydrazino]-2-oxo-ethyl]carbamate (23.0 mg, Example 1)) as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.47 (s, 1H), 7.65 (t, J=5.6 Hz, 1H), 7.33-7.39 (m, 5H), 7.29-7.32 (m, 1H), 6.85 (s, 1H), 5.04 (s, 2H), 4.10-4.31 (m, 2H), 3.70 (s, 2H), 3.60-3.62 (m, 2H), 2.29-2.32 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 371.3.
Compound 2a was prepared in analogy to Example 1, Step 1 by using (2S)-2-(benzyloxycarbonylamino)-4-methyl-pentanoic acid (Shanghai Titan Scientific Co., Ltd. CAS number: 2018-66-8) instead of (2-(benzyloxycarbonylamino) acetic acid. tert-Butyl N-[[(2S)-2-(benzyloxycarbonylamino)-4-methyl-pentanoyl]amino]carbamate (3.8 g, compound 2a) was obtained as a white solid.
Compound 2b was prepared in analogy to Example 1, Step 2 by using tert-butyl N-[[(2S)-2-(benzyloxycarbonylamino)-4-methyl-pentanoyl]amino]carbamate (compound 2a) instead of tert-butyl N-[[2-(benzyloxycarbonylamino)acetyl]amino]carbamate (compound 1a). Benzyl N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]carbamate (730 mg, compound 2b) was obtained as a yellow oil. MS obsd. (ESC) [(M+H)+]: 280.
Compound 2c was prepared in analogy to Example 1, Step 3 by using benzyl N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]carbamate (compound 2b) instead of benzyl (2-hydrazinyl-2-oxo-ethyl)carbamate (compound 1b). Benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]carbamate (500 mg, compound 2c) was obtained as a viscous oil. MS obsd. (ESI+) [(M+H)+]: 351.
The title compound was prepared in analogy to Example 1, Step 4 by using benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]carbamate (compound 2c) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (80 mg, compound 1c). Benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (7.3 mg, Example 2) was obtained as a white solid. 1H NMR (400 MHz, CD3OD, 25° C.) δ ppm: 7.14-7.34 (m, 5H), 5.01 (br d, J=5.14 Hz, 2H), 3.40-4.32 (m, 5H), 2.15-2.53 (m, 2H), 1.38-1.77 (m, 3H), 0.87 (dd, J=12.72, 6.48 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 427.
The title compound was prepared in analogy to Example 1, Step 4 by using benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]carbamate (50.0 mg, compound 2c) and 2-chloro-2-phenylacetyl chloride (Sigma-Aldrich, CAS number: 2018-66-8) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c) and chloroacetyl chloride. Benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-phenyl-acetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (20.4 mg, Example 3) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.59-11.06 (m 1H), 7.52-7.81 (m, 1H), 7.47-7.59 (m, 1H), 7.23-7.46 (m, 10H), 6.84 (br. s, 1H), 5.85-5.88 (m, 1H), 4.99-5.19 (m, 2H), 3.95-4.03 (m, 1H), 3.59-3.86 (m, 1H), 3.29 (s, 1H), 2.24-2.37 (m, 2H), 1.62-1.75 (m, 1H), 1.49-1.60 (m, 1H), 1.32-1.47 (m, 1H), 0.84-0.95 (m, 6H). MS obsd. (ESI+) [(M+H)+]: 503.2.
To a solution of benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]carbamate (50.0 mg, compound 2c) and 2-chloropropanoic acid (18.6 mg) in DMF (0.500 mL) was added DIEPA (0.1 mL, 0.570 mmol) and T3P (0.13 mL, 50% in EtOAc) at 0° C. for 1 hr. The resulting mixture was diluted with water (15 mL) and extracted with EtOAc (10 mL) three times. The combined organic layers were washed with brine (15 mL), dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by prep-HPLC to afford benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloropropanoyl)amino]carbamoyl]-3-methyl-butyl]carbamate (22.3 mg, Example 4) as a light yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm: 9.37-9.71 (m, 1H), 7.29-7.40 (m, 5H), 6.10-6.31 (m, 1H), 5.75 (br. s, 1H), 5.36-5.57 (m, 1H), 5.13 (s, 2H), 4.50-4.55 (m, 1H), 4.20-4.27 (m, 1H), 3.83-4.16 (m, 1H), 3.52-3.63 (m, 1H), 2.40-2.60 (m, 2H), 1.67-1.89 (m, 3H), 1.51-1.61 (m, 3H), 0.95-1.00 (m, 6H). MS obsd. (ESI+) [(M+H)+]: 441.1.
The title compound was prepared in analogy to Example 4 by using 2-chlorobutanoic acid instead of 2-chloropropanoic acid (Shanghai Aladdin Bio-Chem Technology Co., Ltd, CAS number: 7623-11-2). Benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chlorobutanoyl)amino]carbamoyl]-3-methyl-butyl]carbamate (33.9 mg, Example 5) was obtained as a white yellow solid. 1H NMR (400 MHz, CDCl3) δ ppm: 9.28-9.65 (m, 1H), 7.29-7.43 (m, 5H), 6.03-6.19 (m, 1H), 5.67 (br. s, 1H), 5.45-5.29 (m, 1H), 5.12-5.14 (m, 2H), 4.18-4.42 (m, 2H), 4.00-4.16 (m, 1H), 3.43-3.66 (m, 1H), 2.38-2.76 (m, 2H), 1.95-2.04 (m, 1H), 1.80-1.87 (m, 1H), 1.51-1.71 (m, 3H), 0.90-1.05 (m, 9H). MS obsd. (ESI+) (M+H)+: 455.1.
The title compound was prepared in analogy to Example 1, Step 4 by using benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]carbamate (50.0 mg, compound 2c) and bromoacetyl bromide instead of benzyl (2-(2-(3-amino-3-oxo-propyl)hydrazinyl)-2-oxo-ethyl)carbamate (compound 1c) and chloroacetyl chloride. (S)-Benzyl (1-(2-(3-amino-3-oxo-propyl)-2-(2-bromoacetyl)hydrazinyl)-4-methyl-1-oxo-pentan-2-yl)carbamate (4 mg, Example 7) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.52-10.65 (m, 1H), 7.66-7.71 (m, 1H), 7.33-7.48 (m, 5H), 7.29-7.32 (m, 1H), 6.86 (s, 1H), 5.04 (s, 2H), 3.97-4.10 (m, 1H), 3.64-3.95 (m, 2H), 3.43-3.50 (m, 2H), 2.20-2.33 (m, 2H), 1.60-1.74 (m, 1H), 1.35-1.58 (m, 2H), 0.77-0.91 (m, 6H). MS obsd. (ESI+) [M+H]+]: 471.1.
Compound 8a was prepared in analogy to Example 1, Step 1 by using (2S)-2-(benzyloxycarbonylamino)-3-cyclohexyl-propanoic acid (10 g, Bidepharm, CAS number: 25341-42-8) instead of (2-(benzyloxycarbonylamino)acetic acid. tert-Butyl N-[[(2S)-2-(benzyloxycarbonylamino)-3-cyclohexyl-propanoyl]amino]carbamate (13.0 g, compound 8a) was obtained as a white solid. MS obsd. (ESI+) [(M−Boc+H)+]: 319.9.
Compound 8b was prepared in analogy to Example 1, Step 2 by using tert-butyl N-[[(2S)-2-(benzyloxycarbonylamino)-3-cyclohexyl-propanoyl]amino]carbamate (3.0 g, compound 8a) instead of tert-butyl N-[[2-(benzyloxycarbonylamino)acetyl]amino]carbamate (compound 1a). Benzyl N-[(1S)-1-(cyclohexylmethyl)-2-hydrazino-2-oxo-ethyl]carbamate (2.2 g, compound 8b) was obtained as a white solid. MS obsd. (ESI+) [(M+H)+]: 320.
Compound 8c was prepared in analogy to Example 1, Step 3 by using benzyl N-[(1S)-1-(cyclohexylmethyl)-2-hydrazino-2-oxo-ethyl]carbamate (2.2 g, compound 8b) instead of benzyl (2-hydrazinyl-2-oxo-ethyl)carbamate (compound 1b). Benzyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (650 mg, compound 8c) was obtained as a white solid. MS obsd. (ESI+) [(M+H)+]: 391.0.
The title compound was prepared in analogy to Example 1, Step 4 by using benzyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (50.0 mg, compound 8c) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c). Benzyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)-2-(2-chloroacetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (12.2 mg, Example 8) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.51-10.70 (m, 1H), 7.72 (br. s, 1H), 7.32-7.38 (m, 6H), 6.87 (s, 1H), 5.01 (s, 2H), 4.21-4.39 (m, 1H), 3.95-4.03 (m, 2H), 3.75-3.81 (m, 1H), 2.22-2.33 (m, 2H), 1.48-1.67 (m, 8H), 1.24-1.33 (m, 1H), 1.09-1.16 (m, 3H), 0.84-0.95 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 467.2.
Compound 10a was prepared in analogy to Example 1, Step 1 by using (2S)-2-(benzyloxycarbonylamino)-3-cyclopentyl-propanoic acid (0.9 g, Intermediate AB) instead of (2-(benzyloxycarbonylamino)acetic acid, tert-Butyl N-[[(2S)-2-(benzyloxycarbonylamino)-3-cyclopentyl-propanoyl]amino]carbamate (1.0 g, compound 10a) was obtained as a white solid. MS obsd. (ESI+) [(M+H)+]: 406.2.
Compound 10b was prepared in analogy to Example 1, Step 2 by using tert-butyl N-[[(2S)-2-(benzyloxycarbonylamino)-3-cyclopentyl-propanoyl]amino]carbamate (700 mg, compound 10a) instead of tert-butyl N-[[2-(benzyloxycarbonylamino)acetyl]amino]carbamate (compound 1a). Benzyl N-[(1S)-1-(cyclopentylmethyl)-2-hydrazino-2-oxo-ethyl]carbamate (300 mg, compound 10b) was obtained as a light yellow solid. (ESI+) [(M+H)+]: 306.
Compound 10c was prepared in analogy to Example 1, Step 3 by using benzyl N-[(1S)-1-(cyclopentylmethyl)-2-hydrazino-2-oxo-ethyl]carbamate (500 mg, compound 10b) instead of benzyl (2-hydrazinyl-2-oxo-ethyl)carbamate (compound 1b). Benzyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)hydrazino]-1-(cyclopentylmethyl)-2-oxo-ethyl]carbamate (300 mg, compound 10c) was obtained as a light yellow solid. (ESI+) [(M+H)+]: 377.
The title compound was prepared in analogy to Example 1, Step 4 by using benzyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)hydrazino]-1-(cyclopentylmethyl)-2-oxo-ethyl]carbamate (80 mg, compound 10c) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c). Benzyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)-2-(2-chloroacetyl)hydrazino]-1-(cyclopentylmethyl)-2-oxo-ethyl]carbamate (48 mg, Example 10) was obtained as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.49-10.89 (m, 1H), 7.71 (br. s, 1H), 7.33-7.39 (m, 6H), 6.85 (s, 1H), 5.04 (s, 2H), 4.19-4.35 (m, 1H), 3.96-4.12 (m, 2H), 3.77-3.84 (m, 1H), 3.36-3.45 (m, 1H), 2.29-2.33 (m, 2H), 1.58-1.85 (m, 7H), 1.45-1.51 (m, 2H), 1.11-1.18 (m, 2H), MS obsd. (ESI+) [(M+H)+]: 453.1.
Compound 11a was prepared in analogy to Example 1, Step 1 by using (2S)-2-(benzyloxycarbonylamino)-3-cyclopropyl-propanoic acid (1.6 g, Intermediate AA) instead of (2-(benzyloxycarbonylamino)acetic acid, tert-Butyl N-[[(2S)-2-(benzyloxycarbonylamino)-3-cyclopropyl-propanoyl]amino]carbamate (2 g, compound 11a) was obtained as a yellow oil.
Compound 11b was prepared in analogy to Example 1, Step 2 by using tert-butyl N-[[(2S)-2-(benzyloxycarbonylamino)-3-cyclopropyl-propanoyl]amino]carbamate (2 g, compound 11a) instead of tert-butyl N-[[2-(benzyloxycarbonylamino)acetyl]amino]carbamate (compound 1a). Benzyl N-[(1S)-1-(cyclopropylmethyl)-2-hydrazino-2-oxo-ethyl]carbamate hydrochloride (1.6 g, compound 11b) was obtained as a yellow solid. MS obsd. (ESI+) [(M+H)+]: 278.
Compound 11c was prepared in analogy to Example 1, Step 3 by using benzyl N-[(1S)-1-(cyclopropylmethyl)-2-hydrazino-2-oxo-ethyl]carbamate hydrochloride (1.6 g, compound 11b) instead of benzyl (2-hydrazinyl-2-oxo-ethyl)carbamate (compound 1b). Benzyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)hydrazino]-1-(cyclopropylmethyl)-2-oxo-ethyl]carbamate (400 mg, compound 11c) was obtained as a yellow oil. MS obsd. (ESI+) [(M+H)+]: 349.0.
The title compound was prepared in analogy to Example 1, Step 4 by using benzyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)hydrazino]-1-(cyclopropylmethyl)-2-oxo-ethyl]carbamate (50.0 mg, compound 11c) instead of benzyl (2-(2-(3-amino-3-oxo-propyl)hydrazinyl)-2-oxo-ethyl)carbamate (compound 1c). Benzyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)-2-(2-chloroacetyl)hydrazino]-1-(cyclopropylmethyl)-2-oxo-ethyl]carbamate (19.4 mg, Example 11) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.45-10.73 (m, 1H), 7.74-7.76 (m, 1H), 7.29-7.37 (m, 6H), 6.85 (s, 1H), 5.02 (s, 2H), 4.21-4.41 (m, 1H), 4.06-4.21 (m, 2H), 3.70-3.92 (m, 1H), 2.52-2.58 (m, 1H), 2.22-2.34 (m, 2H), 1.60-1.72 (m, 1H), 1.33-1.45 (m, 1H), 0.72-0.83 (m 1H), 0.37-0.47 (m, 2H), 0.14-0.20 (m, 1H), 0.04-0.11 (m, 1H). MS obsd. (ESI+) [(M+H)+]: 425.1.
The title compound was prepared in analogy to Example 1, Step 4 by using benzyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (compound 8c) and 2-fluoroacetyl chloride instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c) and chloroacetyl chloride. Benzyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)-2-(2-fluoroacetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (32.5 mg, compound 15) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.19-10.52 (m, 1H), 7.70 (br s, 1H), 7.28-7.35 (m, 7H), 6.86 (br.s, 1H), 5.03 (br.s, 2H), 4.59-4.75 (m, 1H), 3.95-4.08 (m, 1H), 3.74-3.86 (m, 1H), 2.20-2.27 (m, 2H), 1.54-1.82 (m, 6H), 1.31-1.48 (m, 3H), 1.10-1.18 (m, 3H), 0.86-0.90 (m, 2H). MS obsd. (ESI+)[(M+H)+]: 451.
To a solution of ethyl chlorofluoroacetate (8.0 g) in ethanol (114 mL) and water (7 mL) was added NaOH (2.73 g). After stirred at 20° C. for 12 hr, the solvent was removed in vacuo. The residue was dissolved in HCl (3 M 80 mL). The aqueous solution was extracted with MTBE (100 mL) four times. The combined organic phase was dried over anhydrous Na2SO4 and concentrated to afford 2-chloro-2-fluoro-acetic acid (4.2 g, compound 16a) as a yellow liquid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 6.88 (s, 0.5H), 6.76 (s, 0.5H).
Example 16 was prepared in analogy to Example 4 by using benzyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (50.0 mg, compound 8c) and 2-chloro-2-fluoro-acetyl chloride instead of benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]carbamate (compound 2c) and 2-chloropropanoic acid. Benzyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)-2-(2-chloro-2-fluoro-acetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (Example 16) obtained as a white solid. MS obsd. (ESI+) [(M+H)+]: 485.4.
Separation of Example 16 by chiral HPLC afforded Example 16-A (faster eluting, 6.3 mg) and Example 16-B (slower eluting, 6.2 mg) as a white solid with 5%-40% MeOH (0.05% DEA)/CO2 on CHIRALCEL OJ-3 column.
Example 16-A: 1H NMR (400 MHz, CDCl3) δ ppm: 9.54 (br. s, 1H), 7.23-7.33 (m, 5H), 6.07-6.58 (m, 2H), 5.63 (br. s, 1H), 5.44 (br. s, 1H), 5.02-5.10 (m, 2H), 4.15-4.21 (m, 1H), 3.83-4.02 (m, 1H), 3.25-3.60 (m, 1H), 2.31-2.63 (m, 2H), 1.44-1.70 (m, 4H), 0.99-1.37 (m, 6H), 0.89-0.99 (m, 3H). MS obsd. (ESI+) [(M+H)+]: 485.4.
Example 16-B: 1H NMR (400 MHz, CDCl3) δ ppm: 9.48 (br. s, 1H), 7.28-7.39 (m, 5H), 6.28-6.47 (m, 1H), 5.94-6.13 (m, 1H), 5.63 (br. s, 1H), 5.27 (br. s, 1H), 5.05 (s, 2H), 4.13-4.19 (m, 1H), 3.70-4.00 (m, 1H), 3.20-3.60 (m, 1H), 2.35-2.68 (m, 2H), 1.49-1.60 (m, 4H), 1.08-1.41 (m, 6H), 0.85-0.92 (m, 3H). 485.4.
Example 17 was prepared in analogy to Example 4 by using 2-chloro-2-fluoro-acetic acid instead of 2-chloropropanoic acid. Benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (60 mg, Example 17) was obtained as a white solid. MS obsd. (ESI+) [(M+H)+]: 445.4.
Separation of example of 17 by chiral SFC afford Example 17-A (faster eluting, 28 mg) and Example 17-B (Slower eluting, 40 mg) with isopropanol 5%-40% (0.05% DEA)/CO2 on Chiralcel OD-3 column.
Example 17-A: 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.64-10.70 (m, 1H), 7.72-7.76 (m, 1H), 7.29-7.41 (m, 6H), 6.50-7.02 (m, 2H), 5.02-5.06 (m, 2H), 3.98-4.02 (m, 1H), 3.66-3.92 (m, 1H), 3.37-3.43 (m, 1H), 2.32-2.36 (m, 2H), 1.46-1.66 (m, 3H), 0.88 (dd, J=6.4 Hz, 12.8 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 445.4.
Example 17-B: 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.44-10.97 (m, 1H), 7.65-7.82 (m, 1H), 7.34-7.43 (m, 5H), 7.29-7.33 (m, 1H), 6.60-7.20 (m, 2H), 5.03-5.06 (m, 2H), 3.92-4.10 (m, 1H), 3.66-3.90 (m, 1H), 3.42-3.57 (m, 1H), 2.28-2.36 (m, 2H), 1.66-1.69 (m, 1H), 1.52-1.56 (m, 1H), 1.32-1.49 (m, 1H), 0.88 (dd, J=6.4, 10.4 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 445.3.
To a solution of tert-butyl 2-oxo-pyrrolidine-1-carboxylate (10.0 g, 53.99 mmol, Wuxi Apptec (Wuhan) Co., Ltd) in THF (100 mL) was added a solution of LiHMDS (113.4 mL) at 0° C. The reaction mixture was allowed to warm up to rt over 30 mins before 2,2,2-trifluoroethyl trifluoroacetate (21.2 g) was added. After additional 20 mins at rt, the reaction mixture was quenched with saturated NH4Cl (500 mL) and extracted with EtOAc (500 mL) twice. The combined organic phases were dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was dissolved in toluene (100 mL), then formaldehyde (40.53 g) and K2CO3 (16.42 g) were added to the solution. After stirred at 108° C. for 2 hrs, the reaction mixture was diluted with EtOAc (500 mL). The organic layer was washed with brine (500 mL) twice, dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by flash chromatography on silica gel to afford tert-butyl 3-methylene-2-oxo-pyrrolidine-1-carboxylate (3.5 g, compound 18a) as a yellow oil. MS obsd. (ESI+) [(M−C4H8+H)+]: 142.1.
To a solution of benzyl N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]carbamate (700.0 mg, compound 2a) in 2-propanol (10 mL) was added tert-butyl 3-methylene-2-oxo-pyrrolidine-1-carboxylate (494.3 mg, compound 18a). After stirred at 90° C. for 48 hrs, the reaction mixture was purified by reversed flash chromatography to afford tert-butyl 3-[[2-[(2S)-2-(benzyloxycarbonylamino)-4-methyl-pentanoyl]hydrazino]methyl]-2-oxo-pyrrolidine-1-carboxylate (450 mg, compound 18b) as a yellow oil. MS obsd. (ESI+) [(M−Boc+Na)+]: 399.4.
To a solution of tert-butyl 3-[[2-[(2S)-2-(benzyloxycarbonylamino)-4-methyl-pentanoyl]hydrazino]methyl]-2-oxo-pyrrolidine-1-carboxylate (450.0 mg, compound 18b) in 1,4-dioxane (5 mL) was added HCl/dioxane (2.0 mL). After stirred at 20° C. for 2 hrs, the reaction mixture was concentrated in vacuo. The residue was purified by reversed flash chromatography to afford benzyl N-[(1S)-3-methyl-1-[[(2-oxo-pyrrolidin-3-yl)methylamino]carbamoyl]butyl]carbamate (150 mg, compound 18c) as a yellow oil. MS obsd. (ESI+) [(M+H)+]: 377.2.
The title compound was prepared in analogy to Example 1, Step 4 by using benzyl N-[(1S)-3-methyl-1-[[(2-oxo-pyrrolidin-3-yl)methylamino]carbamoyl]butyl]carbamate (150 mg, compound 18c) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c). Benzyl N-[(1S)-1-[[(2-chloroacetyl)-[(2-oxo-pyrrolidin-3-yl)methyl]amino]carbamoyl]-3-methyl-butyl]carbamate (15.1 mg, Example 18) was obtained as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm: 9.68-10.23 (m, 1H), 7.34-7.38 (m, 5H), 5.60-5.78 (m, 1H), 5.16-5.42 (m, 1H), 5.10 (s, 2H), 4.24-4.56 (m, 3H), 3.74-4.24 (m, 1H), 3.24-3.38 (m, 2H), 2.67-2.87 (m, 1H), 2.17-2.44 (m, 1H), 1.64-1.78 (m, 2H), 1.47-1.51 (m, 3H), 0.89-1.04 (m, 6H). MS obsd. (ESI+) (M+H)+: 453.2.
Compound 20a was prepared in analogy to Example 18, Step 1 by using tert-butyl 2-oxo-piperidine-1-carboxylate instead of tert-butyl 2-oxo-pyrrolidine-1-carboxylate (Wuxi Apptec (Wuhan) Co., Ltd). tert-Butyl 3-methylene-2-oxo-piperidine-1-carboxylate (1.0 g, compound 20a) was obtained as a yellow oil. MS obsd. (ESI+) [2M+Na]+]: 445.1
Compound 20b was prepared in analogy to Example 18, Step 2 by using tert-butyl 3-methylene 2-oxo-piperidine-1-carboxylate (1.0 g, compound 20a) instead of tert-butyl 3-methylene-2-oxo-pyrrolidine-1-carboxylate (compound 18a). tert-Butyl 3-((2-((S)-2-(((benzyloxy)carbonyl)amino)-4-methylpentanoyl)hydrazinyl)methyl)-2-oxo-piperidine-1-carboxylate (170 mg, compound 20b) was obtained as a colorless oil. MS obsd. (ESI+) [2M+H]+]: 981.7.
Compound 20c was prepared in analogy to Example 18, Step 3 by using tert-butyl 3-((2-((S)-2-(((benzyloxy)carbonyl)amino)-4-methylpentanoyl)hydrazinyl)methyl)-2-oxo-piperidine-1-carboxylate (190 mg, compound 20b) instead of tert-butyl 3-[[2-[(2S)-2-(benzyloxycarbonylamino)-4-methyl-pentanoyl]hydrazino]methyl]-2-oxo-pyrrolidine-1-carboxylate (compound 18b). Benzyl N-[(1S)-3-methyl-1-[[(2-oxo-3-piperidyl)methylamino]carbamoyl]butyl]carbamate (100 mg, compound 20c) was obtained as a yellow oil. MS obsd. (ESI+) [(M+H)+]: 391.4.
The title compound was prepared in analogy to Example 1, Step 4 by using benzyl ((2S)-4-methyl-1-oxo-1-(2-((2-oxo-piperidin-3-yl)methyl)hydrazinyl)pentan-2-yl)carbamate (50 mg, compound 20c) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c). Benzyl N-[(1S)-1-[[(2-chloroacetyl)-[(2-oxo-3-piperidyl)methyl]amino]carbamoyl]-3-methyl-butyl]carbamate (7.2 mg, Example 20) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.44-10.79 (m, 1H), 7.62-7.77 (m, 1H), 7.43-7.57 (m, 1H), 7.24-7.42 (m, 5H), 4.97-5.11 (m, 2H), 4.20-4.40 (m, 1H), 3.92-4.10 (m, 2H), 3.46-3.57 (m, 3H), 3.10 (s, 2H), 1.62-1.93 (m, 3H), 1.30-1.58 (m, 4H), 0.86-0.91 (m, 6H). MS obsd. (ESI+) [(M+H)+]: 467.2.
To a solution of benzyl N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]carbamate (450.0 mg, compound 2b) in toluene (2 mL) was added propionaldehyde (102.92 mg). After being stirred at 60° C. for 2 hrs and at 20° C. for another 12 hrs, the reaction mixture was concentrated in vacuo. The residue was dissolve in THF (2 mL), then TsOH (831.27 mg, 4.83 mmol) and NaBH3CN (10.12 mg, 0.160 mmol) were added. After being stirred at 40° C. for 2 hrs, the reaction mixture was concentrated in vacuo. The residue was purified by prep-HPLC to afford benzyl N-[(1S)-3-methyl-1-(propylaminocarbamoyl)butyl]carbamate (160 mg, compound 21a) as a colorless oil. MS obsd. (ESI+) [(M+H)+]: 322.0.
The title compound was prepared in analogy to Example 1, Step 4 by using benzyl N-[(1S)-3-methyl-1-(propylaminocarbamoyl)butyl]carbamate (160 mg, compound 21a) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c). Benzyl N-[(1S)-1-[[(2-chloroacetyl)-propyl-amino]carbamoyl]-3-methyl-butyl]carbamate (8.5 mg, Example 21) was obtained as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.49-10.74 (m, 1H), 7.70-7.71 (br. s, 1H), 7.31-7.36 (m, 5H), 4.99-5.08 (m, 2H), 4.21-4.37 (m, 1H), 3.96-4.10 (m, 2H), 3.41-3.43 (m, 1H), 3.01-3.17 (m, 1H), 1.61-1.68 (m, 1H), 1.50-1.57 (m, 1H), 1.41-1.46 (m, 2H), 1.23 (s, 1H), 0.80-0.91 (m, 9H). MS obsd. (ESI+) [(M+H)+]: 398.
Compound 23a was prepared in analogy to Example 21, Step 1 by using 1H-pyrazole-3-carbaldehyde (276.0 mg, Wuxi Apptec (Wuhan) Co., Ltd) instead of propionaldehyde. Benzyl N-[(1S)-3-methyl-1-[(1H-pyrazol-3-ylmethylamino) carbamoyl]butyl]carbamate (500 mg, compound 23a) was obtained as a yellow oil. MS obsd. (ESI+) [(M+H)+]: 360.4.
The title compound was prepared in analogy to Example 1, Step 4 by using benzyl N-[(1S)-3-methyl-1-[(1H-pyrazol-3-ylmethylamino) carbamoyl]butyl]carbamate (500 mg, compound 23a) instead of benzyl (2-(2-(3-amino-3-oxo-propyl)hydrazinyl)-2-oxo-ethyl)carbamate (compound 1c). Benzyl N-[(1S)-1-[[(2-chloroacetyl)-(1H-pyrazol-3-ylmethyl)amino]carbamoyl]-3-methyl-butyl]carbamate (20 mg, Example 23) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 12.70 (br. s, 1H), 10.51-10.58 (m, 1H), 7.62-7.63 (m, 2H), 7.30-7.34 (m, 5H), 6.14-6.15 (m, 1H), 5.01-5.03 (m, 3H), 4.25-4.40 (m, 1H), 3.91-4.16 (m, 3H), 1.23-1.55 (m, 3H), 0.82 (s, 6H). MS obsd. (ESI+) [(M+H)+]: 436.1.
Compound 24a was prepared in analogy to Example 21, Step 1 by using 1H-imidazole-4-carbaldehyde (34.4 mg, Wuxi Apptec (Wuhan) Co., Ltd) instead of propionaldehyde. Benzyl N-[(1S)-1-(1H-imidazol-4-ylmethylamino)carbamoyl]-3-methyl-butyl carbamate (40 mg, compound 24a) was obtained as a yellow oil. MS obsd. (ESI+) [(M+H)+]: 360.0.
The title compound was prepared in analogy to Example 1, Step 4 by using (S)-benzyl (1-(2-((1H-imidazol-4-yl)methyl) hydrazinyl)-4-methyl-1-oxo-pentan-2-yl)carbamate (40 mg, compound 24a) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c). Benzyl N-[(1S)-1-[[(2-chloroacetyl)-(1H-imidazol-4-ylmethyl)amino]carbamoyl]-3-methyl-butyl]carbamate (20.2 mg, Example 24) was obtained as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 14.24 (br. S, 1H), 10.55-10.73 (m, 1H), 8.98 (s, 1H), 7.71 (s, 1H), 7.53 (s, 1H), 7.30-7.36 (m, 5H), 5.03 (s, 2H), 4.88 (d, J=14.4 Hz, 1H), 4.30-4.50 (m, 2H), 4.13 (d, J=15.6 Hz, 1H), 3.92 (m, 1H), 1.30-1.53 (m, 3H), 0.83 (d, J=6.0 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 436.1.
To a solution of benzyl hydrazinecarboxylate (8 g, 48.14 mmol), (S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanoic acid (10 g, 43.24 mmol, Wuxi Apptec (Wuhan) Co., Ltd) and DIEPA (22.6 mL, 129.71 mmol) in EtOAc (100 mL) was added T3P (38.57 mL, 64.85 mmol, 50% solution in EtOAc) at 0° C. The reaction mixture was stirred for 1 hr at 0° C., then diluted with EtOAc (300 mL), washed with HCl (1N, 100 mL), saturated aqueous NaHCO3 (200 mL) and brine (300 mL). The organic layer was dried over anhydrous Na2SO4, and concentrated in vacuo to afford (S)-benzyl 2-(2-((tert-butoxycarbonyl)amino)-4-methylpentanoyl)hydrazinecarboxylate (16 g, compound 26a) as a yellow oil and used in next step without further purification. MS obsd. (ESI+) [(M+H)+]: 380.0.
To a solution of tert-butyl N-[(1S)-1-(benzyloxycarbonylaminocarbamoyl)-3-methyl-butyl]carbamate (16.0 g, compound 26a) in methanol (150 mL) was added Pd/C (1.0 g, 10% purity) under N2. The mixture was degassed in vacuo and purged with H2 three times. The resulting mixture was stirred at 25° C. for 4 hrs under H2 balloon. The reaction mixture was filtered and concentrated in vacuo to afford tert-butyl N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]carbamate (10 g, compound 26b) as a yellow gum and used in next step without further purification. MS obsd. (ESI+) [M−C4H8+H]+]: 189.9.
Compound 26c was prepared in analogy to Example 1, Step 3 by using tert-butyl N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]carbamate (10 g, compound 26b) instead of benzyl (2-hydrazinyl-2-oxo-ethyl)carbamate (compound 1b). tert-Butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]carbamate (350 mg, compound 26c) was obtained as a yellow oil and used in next step without further purification. MS obsd. (ESI+) [(M+H)+]: 317.1
The title compound was prepared in analogy to Example 1, Step 4 by using tert-butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]carbamate (50.0 mg, compound 26c) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c). tert-Butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (18.4 mg, Example 26) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.35-10.63 (m, 1H), 7.29-7.38 (m, 2H), 6.83 (s, 1H), 4.15-4.34 (m, 1H), 3.97-4.14 (m, 1H), 3.82-3.89 (m, 1H), 3.67-3.80 (m, 1H), 3.33-3.41 (m, 1H), 2.28-2.33 (m, 2H), 1.58-1.69 (m, 1H), 1.49-1.53 (m, 1H), 1.40-1.44 (m, 1H), 1.38 (s, 9H), 0.88 (dd, J=6.4, 13.2 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 393.2.
To a solution of tert-butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (350.0 mg, Example 26) in 1,4-dioxane (5 mL) was added HCl/dioxane (4M, 5.0 mL). After being stirred at 25° C. for 3 hrs, the reaction mixture was concentrated in vacuo to afford 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloroacetyl)amino]propanamide (290 mg, compound 27a) as a yellow solid and used in next step without further purification.
To a solution of 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloroacetyl)amino]propanamide (80.0 mg, compound 27a) and 4-methoxy-1H-indole-2-carboxylic acid (69.7 mg, Shanghai Haohong Pharmaceutical Co., Ltd, CAS number: 103260-65-7) in DMF (1.5 mL) at 0° C. was added DIEPA (0.17 mL) and T3P (0.29 mL, 50% in EtOAc). After stirred at 0° C. for 1 hr, the reaction mixture was diluted with H2O (50 mL) and extracted with EtOAc (20 mL) twice. The combined organic layers were washed with brine (50 mL), dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by prep-HPLC to afford N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]-4-methoxy-1H-indole-2-carboxamide (16 mg, Example 27) as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.61 (s, 1H), 10.43-10.95 (br. s, 1H), 8.59 (d, J=6.8 Hz, 1H), 7.39 (d, J=2.0 Hz, 1H), 7.38 (br. s, 1H), 7.08-7.14 (m, 1H), 6.98-7.05 (m, 1H), 6.86 (s, 1H), 6.51 (d, J=7.6 Hz, 1H), 4.13-4.41 (m, 2H), 3.98-4.17 (m, 1H), 3.89 (s, 3H), 3.72-3.84 (m, 1H), 3.40-3.53 (m, 1H), 2.32-2.33 (m, 2H), 1.69-1.87 (m, 2H), 1.45-1.63 (m, 1H), 0.86-0.98 (m, 6H). MS obsd. (ESI+) [(M+H)+]: 466.4.
The title compound was prepared in analogy to Example 27, Step 2 by using 1H-indole-2-carboxylic acid instead of 4-methoxy-1H-indole-2-carboxylic acid. N-[(1S)-1-[[(3-Amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]-1H-indole-2-carboxamide (36.2 mg, Example 28) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.61 (s, 1H), 10.51-10.85 (m, 1H), 8.66 (d, J=7.2 Hz, 1H), 7.63 (d, J=8.0 Hz, 1H), 7.42-7.44 (m, 1H), 7.37 (br. s, 1H), 7.29 (d, J=1.6 Hz, 1H), 7.17-7.21 (m, 1H), 7.00-7.07 (m, 1H), 6.86 (br. s, 1H), 4.12-4.43 (m, 2H), 3.95-4.19 (m, 1H), 3.69-3.94 (m, 1H), 3.39-3.54 (m, 1H), 2.26-2.37 (m, 2H), 1.71-1.89 (m, 2H), 1.48-1.65 (m, 1H), 0.89-0.99 (m, 6H). (ESI+) [(M+Na)+]: 458.4.
The title compound was prepared in analogy to Example 27, Step 2 by using 5-methylisoxazole-3-carboxylic acid instead of 4-methoxy-1H-indole-2-carboxylic acid (Bidepharm, CAS number: 3405-77-4). N-[(1S)-1-[[(3-Amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]-2-methyl-oxazole-4-carboxamide (31.9 mg, Example 29) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.50-10.69 (br. s, 1H), 8.85-9.04 (m, 1H), 7.36 (br. s, 1H), 6.85 (br. s, 1H), 6.58 (s, 1H), 4.36-4.50 (m, 1H), 4.18-4.30 (m, 1H), 3.95-4.17 (m, 1H), 3.72-3.86 (m, 1H), 3.37-3.46 (m, 1H), 2.47 (d, J=0.4 Hz, 3H), 2.24-2.34 (m, 2H), 1.74-1.85 (m, 1H), 1.61-1.71 (m, 1H), 1.51-1.60 (m, 1H), 0.91 (dd, J=6.4, 14.8 Hz, 6H). MS obsd. (ESI+) [(M+Na)+]: 424.4.
Compound 30a was prepared in analogy to Example 4 by using (S)-tert-butyl (1-(2-(3-amino-3-oxo-propyl)hydrazinyl)-3-cyclohexyl-1-oxo-propan-2-yl)carbamate (compound 26c) and fluoroacetic acid instead of benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]carbamate (compound 2c) and 2-chloropropanoic acid. tert-Butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-fluoroacetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (400 mg, compound 30a) was obtained as a white solid. MS obsd. (ESI+) [M+H]+]: 377.2.
Compound 30b was prepared in analogy to Example 27, step 1 by using (S)-tert-butyl (1-(2-(3-amino-3-oxo-propyl)-2-(2-fluoroacetyl)hydrazinyl)-4-methyl-1-oxo-pentan-2-yl)carbamate (400 mg, compound 30a) instead of N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (Example 26). 3-[[[(2S)-2-Amino-4-methyl-pentanoyl]amino]-(2-fluoroacetyl)amino]propanamide (400 mg, compound 30b) was obtained as a yellow oil.
The title compound was prepared in analogy to Example 27, Step 2 by using (S)-3-(2-(2-amino-4-methylpentanoyl)-1-(2-fluoroacetyl)hydrazinyl)propanamide (100 mg, compound 30b) instead of (S)-3-(2-(2-amino-4-methylpentanoyl)-1-(2-chloroacetyl)hydrazinyl)propanamide (compound 27a). N-[(1S)-1-[[(3-Amino-3-oxo-propyl)-(2-fluoroacetyl)amino]carbamoyl]-3-methyl-butyl]-4-methoxy-1H-indole-2-carboxamide (32.1 mg, Example 30) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.60 (s, 1H), 10.46-10.62 (m, 1H), 8.61 (d, J=6.8 Hz, 1H), 7.39 (s, 2H), 7.05-7.16 (m, 1H), 7.01 (d, J=8.0 Hz, 1H), 6.86 (s, 1H), 6.51 (d, J=7.6 Hz, 1H), 4.91-5.24 (m, 1H), 4.51-4.87 (m, 1H), 4.29-4.49 (m, 1H), 3.88 (s, 3H), 3.72-3.84 (m, 1H), 3.41-3.46 (m, 1H), 2.20-2.39 (m, 2H), 1.73-1.81 (m, 2H), 1.52-1.60 (m, 1H), 0.88-0.98 (m, 6H). MS obsd. (ESI+) [M+H]+]: 450.1.
The title compound was prepared in analogy to Example 27, Step 2 by using (S)-3-(2-(2-amino-4-methylpentanoyl)-1-(2-fluoroacetyl)hydrazinyl)propanamide (100 mg, compound 30b) and 1H-indole-2-carboxylic acid instead of (S)-3-(2-(2-amino-4-methylpentanoyl)-1-(2-chloroacetyl)hydrazinyl)propanamide (compound 27a) and 4-methoxy-1H-indole-2-carboxylic acid. N-[(1S)-1-[[(3-Amino-3-oxo-propyl)-(2-fluoroacetyl)amino]carbamoyl]-3-methyl-butyl]-1H-indole-2-carboxamide (30.8 mg, Example 31) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.60 (s, 1H), 10.47-10.63 (m, 1H), 8.67 (d, J=7.2 Hz, 1H), 7.63 (d, J=8.0 Hz, 1H), 7.43 (d, J=8.8 Hz, 1H), 7.38 (s, 1H), 7.29 (s, 1H), 7.17-7.21 (m, 1H), 7.01-7.07 (m, 1H), 6.86 (s, 1H), 4.72-5.13 (m, 2H), 4.33-4.39 (m, 1H), 3.69-3.91 (m, 1H), 3.37-3.45 (m, 1H), 2.31-2.33 (m, 2H), 1.72-1.85 (m, 2H), 1.51-1.62 (m, 1H), 1.02-0.87 (m, 6H). MS obsd. (ESI+) [M+H]+]: 420.1.
Compound 32a was prepared in analogy to Example 26, Step 1 by using (S)-2-((tert-butoxycarbonyl)amino)-3-cyclohexylpropanoic acid (8.6 g, 31.69 mmol, Thbiochem, CAS number: 37736-82-6) instead of (S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanoic acid. tert-Butyl N-[(1S)-2-(2-benzyloxycarbonylhydrazino)-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (12 g, compound 32a) was obtained as a white solid. MS obsd. (ESI+) [(M+H)+]: 420.5.
Compound 32b was prepared in analogy to Example 26, Step 2 by using (S)-benzyl 2-(2-((tert-butoxycarbonyl)amino)-3-cyclohexylpropanoyl)hydrazinecarboxylate (compound 32a) instead of (Si-benzyl 2-(2-((tert-butoxycarbonyl)amino)-4-methylpentanoyl)hydrazinecarboxylate (compound 26a). tert-Butyl N-[(1S)-1-(cyclohexylmethyl)-2-hydrazino-2-oxo-ethyl]carbamate (6.5 g, compound 32b) was obtained as a white solid. MS obsd. (ESI+) [(M−Bu+H)+]: 230.4.
Compound 32c was prepared in analogy to Example 26, Step 3 by using (S)-tert-butyl (3-cyclohexyl-1-hydrazinyl-1-oxo-propan-2-yl)carbamate (compound 32b) instead of tert-butyl N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]carbamate (compound 26b). tert-Butyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (2.2 g, compound 32c) was obtained as a white solid. MS obsd. (ESI+) [(M+H)+]: 357.0.
Compound 32d was prepared in analogy to Example 1, Step 4 by using tert-butyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (2.2 g, compound 32c) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c). tert-Butyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)-2-(2-chloroacetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (140 mg, compound 32d) was obtained as a light yellow oil.
Compound 32e was prepared in analogy to Example 27, Step 1 by using tert-butyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)-2-(2-chloroacetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (140 mg, compound 32d) instead of ten-butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (350.0 mg, Example 26). 3-[[[(2S)-2-Amino-3-cyclohexyl-propanoyl]amino]-(2-chloroacetyl)amino]propanamide (100 mg, compound 32e) was obtained as a colorless oil. MS obsd. (ESI+) [(M+H]+]: 333.4.
The title compound was prepared in analogy to Example 27, Step 2 by using 3-[[[(2S)-2-amino-3-cyclohexyl-propanoyl]amino]-(2-chloroacetyl)amino]propanamide (50.0 mg, compound 32e) instead of 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloroacetyl)amino]propanamide (compound 27a). N-[(1S)-2-[2-(3-Amino-3-oxo-propyl)-2-(2-chloroacetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]-4-methoxy-1H-indole-2-carboxamide (14.1 mg, Example 32) was obtained as a light yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 11.60 (s, 1H), 10.60-10.75 (m, 1H), 8.57 (d, J=6.8 Hz, 1H), 7.39 (d, J=2.0 Hz, 1H), 7.37 (br. s, 1H), 7.10 (t, J=8.0 Hz, 1H), 7.01 (d, J=8.0 Hz, 1H), 6.86 (br. s, 1H), 6.51 (d, J=8.0 Hz, 1H), 4.39-4.42 (m, 3H), 4.08-4.14 (m, 1H), 3.89 (s, 3H), 2.27-2.32 (m, 2H), 1.72-1.79 (m, 3H), 1.60-1.70 (m, 4H), 1.39-1.41 (m, 1H), 1.21-1.25 (m, 1H), 1.11-1.19 (m, 3H), 0.89-0.99 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 506.3.
The title compound was prepared in analogy to Example 27, Step 2 by using 3-[[[(2S)-2-amino-3-cyclohexyl-propanoyl]amino]-(2-chloroacetyl)amino]propanamide (compound 32e) and (E)-3-(4-chloro-2-fluoro-phenyl)prop-2-enoic acid instead of 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloroacetyl)amino]propanamide (compound 27a) and 4-methoxy-1H-indole-2-carboxylic acid. 3-[(2-Chloroacetyl)-[[(2S)-2-[[(E)-3-(4-chloro-2-fluoro-phenyl)prop-2-enoyl]amino]-3-cyclohexyl-propanoyl]amino]amino]propanamide (33 mg, Example 33) was obtained as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm: 9.74 (s, 1H), 7.66 (d, J=7.6 Hz, 1H), 7.42 (t, J=8.4 Hz, 1H), 7.14 (t, J=8.4 Hz, 2H), 6.59-6.63 (m, 2H), 6.27 (s, 1H), 5.81 (s, 1H), 4.65 (q, J=7.4 Hz, 1H), 4.11-3.70 (m, 4H), 2.57 (s, 2H), 1.75-1.81 (m, 5H), 1.35-1.48 (m, 2H), 0.93-1.26 (m, 6H). MS obsd. (ESI+) [M+H]+: 515.2.
Compound 34a was prepared in analogy to Example 27, Step 2 by using (E)-3-(4-chloro-2-fluorophenyl)acrylic acid (Yantai ChengKaiLun Chemical technology Co., Ltd, CAS number: 312693-55-3) and tert-butyl N-[[(2S)-2-amino-4-methyl-pentanoyl]amino]carbamate (Intermediate AC) instead of 4-methoxy-1H-indole-2-carboxylic acid and 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloroacetyl)amino]propanamide (compound 27a). tert-Butyl N-[[(2S)-2-[[(E)-3-(4-chloro-2-fluoro-phenyl)prop-2-enoyl]amino]-4-methyl-pentanoyl]amino]carbamate (1.29 g, compound 34a) was obtained as a yellow solid.
Compound 34b was prepared in analogy to Example 1, Step 2 by using tert-butyl N-[[(2S)-2-[[(E)-3-(4-chloro-2-fluoro-phenyl)prop-2-enoyl]amino]-4-methyl-pentanoyl]amino]carbamate (1.29 g, compound 34a) instead of tert-butyl N-[[2-(benzyloxycarbonylamino)acetyl]amino]carbamate (compound 1a). (E)-3-(4-chloro-2-fluoro-phenyl)-N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]prop-2-enamide (420 mg, compound 34b) was obtained as a yellow solid. MS obsd. (ESI+) [(M+H)+]: 328.1.
Compound 34c was prepared in analogy to Example 1, Step 3 by using (E)-3-(4-chloro-2-fluoro-phenyl)-N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]prop-2-enamide (compound 34b) instead of benzyl (2-hydrazinyl-2-oxo-ethyl)carbamate (compound 1b). 3-[2-[(2S)-2-[[(E)-3-(4-Chloro-2-fluoro-phenyl)prop-2-enoyl]amino]-4-methyl-pentanoyl]hydrazino]propanamide (110 mg, compound 34c) was obtained as a yellow solid. MS obsd. (ESI+) [(M+H)+]: 399.3
The title compound was prepared in analogy to Example 1, Step 4 by using 3-[2-[(2S)-2-[[(E)-3-(4-chloro-2-fluoro-phenyl)prop-2-enoyl]amino]-4-methyl-pentanoyl]hydrazino]propanamide (110 mg, compound 34c) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c). 3-[(2-Chloroacetyl)-[[(2S)-2-[[(E)-3-(4-chloro-2-fluoro-phenyl)prop-2-enoyl]amino]-4-methyl-pentanoyl]amino]amino]propanamide (9 mg, Example 34) was obtained as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm: 9.72 (br. s, 1H), 7.65 (d, J=16.0 Hz, 1H), 7.42 (t, J=8.4 Hz, 1H), 7.14 (t, J=8.0 Hz, 2H), 6.59 (d, J=16.0 Hz, 1H), 6.53 (br. s 1H), 6.21 (br. s, 1H), 5.82 (br. s, 1H), 4.62-4.65 (m, 1H), 3.40-4.12 (m, 4H), 2.52-2.60 (m, 2H), 1.71-1.78 (m, 3H), 1.00 (dd, J=5.6, 10.4 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 475.1.
The title compound was prepared in analogy to Example 27, Step 2 by using 3-[[[(2S)-2-amino-3-cyclohexyl-propanoyl]amino]-(2-chloroacetyl)amino]propanamide (compound 32e) and indole-2-carboxylic acid instead of 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloroacetyl)amino]propanamide (compound 27a) and 4-methoxy-1H-indole-2-carboxylic acid. N-[(1S)-2-[2-(3-Amino-3-oxo-propyl)-2-(2-chloroacetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]-1H-indole-2-carboxamide (31.6 mg, Example 35) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.60 (s, 1H), 10.59-10.78 (m, 1H), 8.65 (d, J=6.4 Hz, 1H), 7.63 (d, J=8.4 Hz, 1H), 7.43 (d, J=8.4 Hz, 1H), 7.37 (s, 1H), 7.29 (d, J=2.0 Hz, 1H), 7.19 (t, J=8.0 Hz, 1H), 7.04 (d, J=8.0 Hz, 1H), 6.86 (s, 1H), 4.01-4.49 (m, 3H), 3.71-3.85 (m, 1H), 2.27-2.32 (m, 2H), 1.72-1.79 (m, 3H), 1.60-1.70 (m, 4H), 1.39-1.49 (m, 1H), 1.21-1.24 (m, 1H), 1.03-1.17 (m, 3H), 0.89-0.99 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 476.2.
Compound 36a was prepared in analogy to Example 4 by using (S)-tert-butyl (1-(2-(3-amino-3-oxo-propyl)hydrazinyl)-3-cyclohexyl-1-oxo-propan-2-yl)carbamate (compound 32c) and fluoroacetic acid instead of benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]carbamate (compound 2c) and 2-chloropropanoic acid. (S)-tert-Butyl (1-(2-(3-amino-3-oxo-propyl)-2-(2-fluoroacetyl)hydrazinyl)-3-cyclohexyl-1-oxo-propan-2-yl)carbamate (320 mg, compound 36a) was obtained as a light yellow solid. MS obsd. (ESI+) [(M+H)+]: 417.1.
Compound 36b was prepared in analogy to Example 27, Step 1 by using (S)-tert-butyl (1-(2-(3-amino-3-oxo-propyl)-2-(2-fluoroacetyl)hydrazinyl)-3-cyclohexyl-1-oxo-propan-2-yl)carbamate (200.0 mg, compound 36a) instead of tert-butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (Example 26).
(S)-3-(2-(2-Amino-3-cyclohexylpropanoyl)-1-(2-fluoroacetyl)hydrazinyl)propanamide (150 mg, compound 36b) was obtained as a light yellow oil.
The title compound was prepared in analogy to Example 27, Step 2 by using (S)-3-(2-(2-amino-3-cyclohexylpropanoyl)-1-(2-fluoroacetyl)hydrazinyl)propanamide (150 mg, compound 36b) and 1H-indole-2-carboxylic acid instead of 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloroacetyl)amino]propanamide (compound 27a) and 4-methoxy-1H-indole-2-carboxylic acid. N-[(1S)-2-[2-(3-Amino-3-oxo-propyl)-2-(2-fluoroacetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]-1H-indole-2-carboxamide (12.8 mg, Example 36) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.60 (s, 1H), 10.40-10.62 (m, 1H), 8.65 (d, J=6.0 Hz, 1H), 7.63 (d, J=8.0 Hz, 1H), 8.65 (dd, J=0.8, 8.4 Hz, 1H), 7.37 (s, 1H), 7.29 (d, J=1.2 Hz, 1H), 7.17-7.21 (m, 1H), 7.02-7.07 (m, 1H), 6.87 (s, 1H), 4.46-5.26 (m, 2H), 4.38-4.49 (m, 1H), 3.79-3.88 (m, 1H), 3.32-3.34 (m, 1H), 2.27-2.34 (m, 2H), 1.71-1.82 (m, 3H), 1.68-1.78 (m, 3H), 1.40-1.47 (m, 1H), 1.23-1.29 (m, 2H), 1.06-1.19 (m, 2H), 0.89-1.03 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 460.3.
The title compound was prepared in analogy to Example 27, Step 2 by using (S)-3-(2-(2-amino-3-cyclohexylpropanoyl)-1-(2-fluoroacetyl)hydrazinyl)propanamide (150 mg, compound 36b) instead of 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloroacetyl)amino]propanamide (compound 27a). (S)—N-(1-(2-(3-Amino-3-oxo-propyl)-2-(2-fluoroacetyl)hydrazinyl)-3-cyclohexyl-1-oxo-propan-2-yl)-4-methoxy-1H-indole-2-carboxamide (35.2 mg, Example 37) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.60 (s, 1H), 10.40-10.60 (m, 1H), 8.57 (d, J=6.4 Hz, 1H), 7.34-7.43 (m, 2H), 7.06-7.15 (m, 1H), 7.00-7.02 (m, 1H), 6.86 (s, 1H), 6.51 (d, J=8.0 Hz, 1H), 455-5.33 (m, 2H), 4.30-4.50 (m, 1H), 3.89 (s, 3H), 3.49-3.80 (m, 2H), 2.31-2.32 (m, 2H), 1.63-1.81 (m, 5H), 1.55-1.62 (m, 2H), 1.42-1.44 (m, 1H), 1.14-1.26 (m, 3H), 0.87-1.05 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 490.2.
Compound 38a was prepared in analogy to Example 27, Step 2 by using 1H-indole-2-carboxylic acid and tert-butyl N-[[(2S)-2-amino-4-methyl-pentanoyl]amino]carbamate (Intermediate AC) instead of 4-methoxy-1H-indole-2-carboxylic acid and 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloroacetyl)amino]propanamide (compound 27a). tert-Butyl N-[[(2S)-2-(1H-indole-2-carbonylamino)-4-methyl-pentanoyl]amino]carbamate (3.0 g, compound 38a) was obtained as a yellow solid. MS obsd. (ESI+) [(M−tBu+H)+]: 333.0.
Compound 38b was prepared in analogy to Example 1, Step 2 by using tert-butyl N-[[(2S)-2-(1H-indole-2-carbonylamino)-4-methyl-pentanoyl]amino]carbamate (2.5 g, compound 38a) instead of tert-butyl N-[[2-(benzyloxycarbonylamino)acetyl]amino]carbamate (compound 1a). N-[(1S)-1-(Hydrazinecarbonyl)-3-methyl-butyl]-1H-indole-2-carboxamide (1.4 g, compound 38b) was obtained as a yellow solid. MS obsd. (ESI+) [(M+H)+]: 289.0.
Compound 38c was prepared in analogy to Example 18, Step 2 by using N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]-1H-indole-2-carboxamide (1.4 g, compound 38b) instead of benzyl N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]carbamate (compound 2a). tert-Butyl 3-[[2-[(2S)-2-(1H-indole-2-carbonylamino)-4-methyl-pentanoyl]hydrazino]methyl]-2-oxo-pyrrolidine-1-carboxylate (350 mg, compound 38c) was obtained as a yellow solid. MS obsd. (ESI+) [(M−Boc+H)+]: 386.2.
Compound 38d was prepared in analogy to Example 18, Step 3 by using tert-Butyl 3-[[2-[(2S)-2-(1H-indole-2-carbonylamino)-4-methyl-pentanoyl]hydrazino]methyl]-2-oxo-pyrrolidine-1-carboxylate (320 mg, compound 38c) instead of (compound 18c). N-((2S)-4-Methyl-1-oxo-1-(2-((2-oxo-pyrrolidin-3-yl)methyl)hydrazinyl) pentan-2-yl)-1H-indole-2-carboxamide (228.0 mg, compound 38d) was obtained as a yellow solid.
The title compound was prepared in analogy to Example 4 by using N-((2S)-4-methyl-1-oxo-1-(2-((2-oxo-pyrrolidin-3-yl)methyl)hydrazinyl) pentan-2-yl)-1H-indole-2-carboxamide (80 mg, compound 38d) and fluoroacetic acid instead of benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]carbamate (compound 2c) and 2-chloropropanoic acid. N—((S)-1-(2-(2-Fluoroacetyl)-2-((2-oxo-pyrrolidin-3-yl)methyl) hydrazinyl)-4-methyl-1-oxo-pentan-2-yl)-1H-indole-2-carboxamide (65.6 mg, Example 38) was obtained as a white solid. MS obsd. (ESI+) [(M+H)+]: 446.4.
Separation of compound 38 by chiral SFC separation afforded Example 38-A (faster eluting, 18.3 mg) and Example 38-B (slower eluting, 15.9 mg) as a white solid with 40% MeOH+ACN (0.05% DEA) (0.05% DEA)/CO2 on Chiralpak IC-3 column.
Example 38-A: 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.60 (s, 1H), 10.38-10.69 (m, 1H), 8.63-8.69 (m, 1H), 7.69-7.74 (m, 1H), 7.63 (d, J=8.0 Hz, 1H), 7.43 (d, J=8.0 Hz, 1H), 7.29 (d, J=1.2 Hz, 1H), 7.16-7.20 (m, 1H), 7.02-7.06 (m, 1H), 4.62-5.22 (m, 2H), 4.37-4.44 (m, 1H), 3.77-3.88 (m, 1H), 3.11-3.26 (m, 3H), 2.45 (br s, 1H), 2.06-2.17 (m, 1H), 1.73-1.78 (m, 3H), 1.54-1.58 (m, 1H), 0.94 (dd, J=5.6 Hz, 21.2 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 446.4.
Example 38-A: 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.61 (s, 1H), 10.44-10.58 (m, 1H), 8.66 (d, J=6.8 Hz, 1H), 7.70-7.73 (m, 1H), 7.62 (d, J=8.0 Hz, 1H), 7.42 (d, J=8.0 Hz, 1H), 7.29 (d, J=1.6 Hz, 1H), 7.16-7.20 (m, 1H), 7.02-7.06 (m, 1H), 4.68-5.20 (m, 2H), 4.37-4.46 (m, 1H), 3.76-3.89 (m, 1H), 3.26-3.30 (m, 1H), 3.08-3.19 (m, 2H), 2.40 (br s, 1H), 2.06-2.20 (br. s, 1H), 1.76-1.78 (m, 3H), 1.54-1.58 (m, 1H), 0.94 (dd, J=6.4, 20.8 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 446.4.
Compound 39a was prepared in analogy to Example 26, Step 1 by using (2S)-2-[tert-butoxycarbonyl(methyl)amino]-3-cyclohexyl-propanoic acid (270 mg, GL Biochem, CAS number: 97269-22-2) instead of (S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanoic acid. (S)-Benzyl 2-(2-((tert-butoxycarbonyl)(methyl)amino)-3-cyclohexylpropanoyl)hydrazinecarboxylate (360 mg, compound 39a) was obtained as a colorless oil. MS obsd. (ESI+) [M−Boc+H]+: 334.1.
Compound 39b was prepared in analogy to Example 26, Step 2 by using (S)-benzyl 2-(2-((tert-butoxycarbonyl)(methyl)amino)-3-cyclohexylpropanoyl)hydrazinecarboxylate (360 mg, compound 39a) instead of (S)-benzyl 2-(2-((tert-butoxycarbonyl)amino)-4-methylpentanoyl)hydrazinecarboxylate (compound 26a). (S)-tert-Butyl (3-cyclohexyl-1-hydrazinyl-1-oxo-propan-2-yl)(methyl)carbamate (240 mg, compound 38b) was obtained as a light yellow oil. MS obsd. (ESI+) [(M−Boc)+]: 200.1.
Compound 39c was prepared in analogy to Example 26, Step 3 by using tert-butyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]-N-methyl-carbamate (compound 39b) instead of tert-butyl N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]carbamate (compound 26b). tert-Butyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]-N-methyl-carbamate (300 mg, compound 39c) was obtained as a colorless oil. MS obsd. (ESI+) [(M+H)+]: 371.1.
Compound 39d was prepared in analogy to Example 1, Step 4 by using tert-butyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]-N-methyl-carbamate (300 mg, compound 39c) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c). tert-Butyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)-2-(2-chloroacetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]-N-methyl-carbamate (100 mg, compound 39d) was obtained as a colorless oil. MS obsd. (ESI+) [(M+H)+]: 447.1
Compound 39e was prepared in analogy to Example 27, Step 1 by using tert-butyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)-2-(2-chloroacetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]-N-methyl-carbamate (100 mg, compound 39d) instead of tert-butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (350.0 mg, Example 26). (S)-3-(1-(2-Chloroacetyl)-2-(3-cyclohexyl-2-(methylamino)propanoyl)hydrazinyl)propanamide (70 mg, compound 39e) was obtained as a light yellow oil. MS obsd. (ESI+) [(M+H)+]: 347.0.
The title compound was prepared in analogy to Example 27, Step 2 by using 3 (S)-3-(1-(2-chloroacetyl)-2-(3-cyclohexyl-2-(methylamino)propanoyl)hydrazinyl)propanamide (70 mg, compound 39e) instead of 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloroacetyl)amino]propanamide (compound 27a). N-[(1S)-2-[2-(3-Amino-3-oxo-propyl)-2-(2-chloroacetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]-N-methyl-1H-indole-2-carboxamide (13.2 mg, Example 39) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.61 (s, 1H), 10.54 (br. s, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.45 (d, J=8.0 Hz, 1H), 7.38 (s, 1H), 7.18-7.22 (m, 1H), 6.92-7.08 (m, 2H), 6.88 (s, 1H), 5.13 (br. s, 1H), 4.16-4.30 (m, 2H), 3.80 (br. s, 1H), 3.38 (m, 1H), 3.32 (s, 3H), 2.32-2.33 (m, 2H), 1.59-1.86 (m, 7H), 0.98-1.23 (m, 6H). MS obsd. (ESI+) [(M+H)+]: 490.4.
Compound 40a was prepared in analogy to Example 26, Step 1 by using (S)-2-((tert-butoxycarbonyl)(methyl)amino)-4-methylpentanoic acid (2.0 g, Shanghai Haohong Pharmaceutical Co., Ltd, CAS number: 53363-89-6) instead of (S)-2-((tert-butoxycarbonyl)amino)-4-methylpentanoic acid. tert-Butyl N-[(1S)-1-(benzyloxycarbonylaminocarbamoyl)-3-methyl-butyl]-N-methyl-carbamate (2.9 g, compound 40a) was obtained as a light yellow oil. MS obsd. (ESI+) [M−Boc+H]+: 294.2.
Compound 40b was prepared in analogy to Example 26, Step 2 by using tert-butyl N-[(1S)-1-(benzyloxycarbonylaminocarbamoyl)-3-methyl-butyl]-N-methyl-carbamate (2.9 g, compound 40a) instead of (S)-benzyl 2-(2-((tert-butoxycarbonyl)amino)-4-methylpentanoyl)hydrazinecarboxylate (compound 26a). tert-Butyl N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]-N-methyl-carbamate (900 mg, compound 40b) was obtained as a colorless oil. MS obsd. (ESI+) [M−Boc+H]+: 160.4.
Compound 40c was prepared in analogy to Example 26, Step 3 by using tert-butyl N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]-N-methyl-carbamate (compound 40b) instead of tert-butyl N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]carbamate (compound 26b). tert-Butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]-N-methyl-carbamate (480 mg, compound 40c) was obtained as a colorless oil. MS obsd. (ESI+) [(M−Boc+H)+]: 230.9.
Compound 40d was prepared in analogy to Example 1, Step 4 by using tert-butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]-N-methyl-carbamate (240 mg, compound 40c) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c). tert-Butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]-N-methyl-carbamate (270 mg, compound 40d) was obtained as a colorless oil. MS obsd. (ESI+) [M−Boc+H]+: 307.2.
Compound 40e was prepared in analogy to Example 27, Step 1 by using 3-[(2-chloroacetyl)-[[(2S)-4-methyl-2-(methylamino)pentanoyl]amino]amino]propanamide (compound 40d) instead of tert-butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (350.0 mg, Example 26). 3-[(2-Chloroacetyl)-[[(2S)-4-methyl-2-(methylamino)pentanoyl]amino]amino]propanamide (60 mg, compound 40e) was obtained as a light yellow oil.
The title compound was prepared in analogy to Example 27, Step 2 by using 3-[(2-chloroacetyl)-[[(2S)-4-methyl-2-(methylamino)pentanoyl]amino]amino]propanamide (60 mg, compound 40e) (70 mg, compound 39e) instead of 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloroacetyl)amino]propanamide (compound 27a). N-[(1S)-1-[[(3-Amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]-N-methyl-1H-indole-2-carboxamide (29.2 Example 40) was obtained as a white solid. 1H NMR (400 MHz, CDCl3) 4 ppm: 9.83 (br. s, 1H), 9.51 (br. s, 1H), 7.67 (d, J=7.6 Hz, 1H), 7.44 (d, J=8.4 Hz, 1H), 7.32 (t, J=8.0 Hz, 1H), 7.16 (t, J=7.2 Hz, 1H), 6.93 (s, 1H), 5.80-6.05 (m, 2H), 5.15-5.25 (m, 1H), 3.95-4.15 (m, 2H), 3.39 (s, 3H), 2.45-2.65 (m, 2H), 1.84-1.91 (m, 2H), 1.60-1.65 (m, 1H), 1.21-1.29 (m, 2H), 1.02 (d, J=6.8 Hz, 3H), 0.96 (d, J=6.0 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 450.4.
Compound 41a was prepared in analogy to Example 1, Step 3 by using N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]-1H-indole-2-carboxamide (200 mg, compound 38b) instead of benzyl (2-hydrazinyl-2-oxo-ethyl)carbamate (compound 1b). N-[(1S)-1-[[(3-Amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]-1H-indole-2-carboxamide (100 mg, compound 41a) was obtained as a light yellow solid. MS obsd. (ESI+) [(M+H)+]: 360.4.
The title compound was prepared in analogy to Example 4 by using N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]-1H-indole-2-carboxamide (50.0 mg, compound 41a) and 2-chloro-2-fluoro-acetic acid instead of benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]carbamate (compound 2c) and 2-chloropropanoic acid. N-[(1S)-1-[[(3-Amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]-1H-indole-2-carboxamide was obtained as a white solid (11.0 mg, Example 41, MS obsd. (ESI+) [(M+H)+]: 454.1).
Separation of Example 41 by chiral SFC separation afforded Example 41-A (faster eluting, 2.1 mg) and Example 41-B (slower eluting, 1.9 mg) as a white solid with 5%-40% MeOH (0.05% DEA)/CO2 on DAICEL CHIRALCEL OJ column.
Example 41-A: 1H NMR (400 MHz, CD3OD) δ ppm: 7.61 (d, J=8.0 Hz, 1H), 7.43 (d, J=8.0 Hz, 1H), 7.21-7.24 (m, 2H), 7.06 (t, J=8.0 Hz, 1H), 6.81 (d, J=50.4 Hz, 1H), 4.45-4.51 (m, 1H), 3.68-3.94 (m, 2H), 2.46-2.55 (m, 2H), 1.84-1.89 (m, 2H), 1.68-1.72 (m, 1H), 1.04 (dd, J=6.4, 16.8 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 454.1.
Example 41-B: 1H NMR (400 MHz, CD3OD) δ ppm: 7.62 (d, J=8.0 Hz, 1H), 7.44 (d, J=8.0 Hz, 1H), 7.21-7.24 (m, 2H), 7.06 (t, J=12 Hz, 1H), 6.54 (d, J=50.4 Hz, 1H), 4.54-4.56 (m, 1H), 3.72-3.92 (m, 2H), 2.48-2.53 (m, 2H), 1.82-1.88 (m, 2H), 1.72-1.77 (m, 1H), 1.04 (dd, J=6.0, 18.0 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 454.1.
The title compound was prepared in analogy to Example 27, Step 2 by using 1H-benzimidazole-2-carboxylic acid instead of 4-methoxy-1H-indole-2-carboxylic acid. (S)—N-(1-(2-(3-Amino-3-oxo-propyl)-2-(2-chloroacetyl)hydrazinyl)-4-methyl-1-oxo-pentan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (10.1 mg, Example 42) was obtained as a white solid.
1H NMR (400 MHz, CD3OD) δ ppm: 7.52-7.79 (m, 2H), 7.35 (dd, J=2.4, 5.6 Hz, 2H), 3.59 (s, 1H), 4.01-4.49 (m, 2H), 3.52-3.99 (m, 2H), 2.52 (s, 2H), 1.86-1.93 (m, 2H), 1.75-1.80 (m, 1H), 1.04 (dd, J=6.4, 14.0 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 437.1.
The title compound was prepared in analogy to Example 27, Step 2 by using 3-isoxazolecarboxylic acid (Wuxi Apptec (Wuhan) Co., Ltd) instead of 4-methoxy-1H-indole-2-carboxylic acid. N-[(1S)-1-[[(3-Amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]isoxazole-3-carboxamide (6.7 mg, Example 43) was obtained as a colorless gum. 1H NMR (400 MHz, CD3OD) δ ppm: 8.96 (d, J=6.0 Hz, 1H), 8.81 (d, J=1.6 Hz, 1H), 6.85 (d, J=1.6 Hz, 1H), 4.59-4.52 (m, 1H), 4.09-4.45 (m, 1H), 3.55-4.05 (m, 3H), 2.46-2.55 (m, 2H), 1.68-1.91 (m, 3H), 1.02 (dd, J=6.4, 14.4 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 388.0.
The title compound was prepared in analogy to Example 27, Step 2 by using 1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid (Wuxi Apptec (Wuhan) Co., Ltd) instead of 4-methoxy-1H-indole-2-carboxylic acid. N-[(1S)-1-[[(3-Amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]-1H-pyrrolo[2,3-b]pyridine-2-carboxamide (7.9 mg, Example 44) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 12.16 (s, 1H), 10.65-10.68 (m, 1H), 8.69 (d, J=6.8 Hz, 1H), 8.34 (dd, J=1.2, 4.4 Hz, 1H), 8.07-8.12 (m, 1H), 7.36 (s, 1H), 7.26 (d, J=2.0 Hz, 1H), 7.13 (dd, J=4.4, 8.0 Hz, 1H), 6.86 (s, 1H), 4.42-4.53 (m, 1H), 3.99-4.32 (m, 2H), 3.73-3.93 (m, 2H), 2.23-2.33 (m, 2H), 1.73-1.79 (m, 2H), 1.58-1.64 (m, 1H), 0.95 (dd, J=6.4, 20.0 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 436.9.
The title compound was prepared in analogy to Example 27, Step 2 by using 2-methyloxazole-4-carboxylic acid (Bidepharm, CAS number 23012-17-1) instead of 4-methoxy-1H-indole-2-carboxylic acid. N-[(1S)-1-[[(3-Amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]-2-methyl-oxazole-4-carboxamide (4.9 mg, Example 45) was obtained as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm: 9.86 (s, 1H), 8.13 (s, 1H), 7.35 (d, J=6.0 Hz, 1H), 6.44 (s, 1H), 6.09 (br. s, 1H), 4.58-4.63 (m, 1H), 3.88-4.29 (m, 4H), 2.56 (br. s, 2H), 2.48 (s, 3H), 1.75-1.77 (m, 3H), 0.98 (dd, J=6.0, 17.6 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 402.3.
The title compound was prepared in analogy to Example 27, Step 2 by using 5-methylisoxazole-4-carboxylic acid (Bidepharm, CAS number: 42831-50-5) instead of 4-methoxy-1H-indole-2-carboxylic acid. (S)—N-(1-(2-(3-Amino-3-oxo-propyl)-2-(2-chloroacetyl)hydrazinyl)-4-methyl-1-oxo-pentan-2-yl)-5-methylisoxazole-4-carboxamide (6.7 mg, Example 46) was obtained as a colorless gum. 1H NMR (400 MHz, CD3OD) δ ppm: 8.74 (s, 1H), 4.34-4.51 (m, 2H), 3.94-4.20 (m, 2H), 3.55-3.80 (m, 1H), 2.66 (s, 3H), 2.45-2.60 (m, 2H), 1.75-1.85 (m, 2H), 1.64-1.71 (m, 1H), 1.02 (dd, J=6.4, 16.8 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 402.1.
The title compound was prepared in analogy to Example 27, Step 2 by using 5-methoxy-1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid (Bidepharm, CAS number: 17288-36-7) instead of 4-methoxy-1H-indole-2-carboxylic acid. N-[(1S)-1-[[(3-Amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]-5-methoxy-6H-pyrrolo[2,3-c]pyridine-2-carboxamide (5.5 mg, Example 47) was obtained as a white solid. 1H NMR (400 MHz, CD3OD) δ ppm: 8.42 (s, 1H), 7.13 (s, 1H), 7.00 (s, 1H), 4.61-4.64 (m, 2H), 4.56-4.58 (m, 1H), 4.07-4.25 (m, 1H), 3.91 (s, 3H), 3.60-3.90 (m, 1H), 2.49-2.57 (m, 2H), 1.79-1.92 (m, 2H), 1.71-1.76 (m, 1H), 1.03 (dd, J=6.4, 17.2 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 467.1.
The title compound was prepared in analogy to Example 27, Step 2 by using 3-[[[(2S)-2-amino-3-cyclohexyl-propanoyl]amino]-(2-chloroacetyl)amino]propanamide (70.0 mg, compound 32e) and 5-methylisoxazole-3-carboxylic acid instead of 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloroacetyl)amino]propanamide (compound 27a) and 4-methoxy-1H-indole-2-carboxylic acid. (S)—N-(1-(2-(3-Amino-3-oxo-propyl)-2-(2-chloroacetyl)hydrazinyl)-3-cyclohexyl-1-oxo-propan-2-yl)-5-methylisoxazole-3-carboxamide (48.9 mg, Example 36) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.30-10.73 (m, 1H), 8.87-8.94 (m, 1H), 7.36 (s, 1H), 6.85 (s, 1H), 6.58 (s, 1H), 4.20-4.50 (m, 2H), 3.98-4.12 (m, 1H), 3.72-3.82 (m, 1H), 2.46 (s, 3H), 2.25-2.35 (m, 2H), 1.71-1.79 (m, 2H), 1.57-1.70 (m, 5H), 1.27-1.37 (m, 1H), 1.05-1.45 (m, 4H), 0.87-0.99 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 442.4.
Compound 49a was prepared in analogy to Example 27, Step 2 by using tert-butyl N-[[(2S)-2-amino-4-methyl-pentanoyl]amino]carbamate (Intermediate AC) instead of 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloroacetyl)amino]propanamide (compound 27a). tert-Butyl N-[[(2S)-2-[(4-methoxy-1H-indole-2-carbonyl)amino]-4-methyl-pentanoyl]amino]carbamate (1.3 g, compound 49a) was obtained as a yellow solid. MS obsd. (ESI+) [(M−C4H8+H)+]: 363.2.
Compound 49b was prepared in analogy to Example 1, Step 2 by using tert-Butyl N-[[(2S)-2-[(4-methoxy-1H-indole-2-carbonyl)amino]-4-methyl-pentanoyl]amino]carbamate (1.3 g, compound 49a) instead of tert-butyl N-[[2-(benzyloxycarbonylamino)acetyl]amino]carbamate (compound 1a). 4-Methoxy-N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]-1H-indole-2-carboxamide (428 mg, Compound 49b) was obtained as a yellow solid. MS obsd. (ESI+) [(M+H)+]: 319.1.
Compound 49c was prepared in analogy to Example 1, Step 3 by using 4-methoxy-N-[(1S)-1-(hydrazinecarbonyl)-3-methyl-butyl]-1H-indole-2-carboxamide (100 mg, Compound 49b) instead of benzyl (2-hydrazinyl-2-oxo-ethyl)carbamate (compound 1b). N-[(1S)-1-[[(3-Amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]-4-methoxy-1H-indole-2-carboxamide (100 mg, compound 49c) was obtained as a yellow oil. MS obsd. (ESI+) [(M+H)+]: 390.4
The title compound was prepared in analogy to Example 4 by using (S)—N-(1-(2-(3-amino-3-oxo-propyl)hydrazinyl)-4-methyl-1-oxo-pentan-2-yl)-4-methoxy-1H-indole-2-carboxamide (50.0 mg, compound 49c) and 2-chloro-2-fluoro-acetic acid instead of benzyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)amino]carbamoyl]-3-methyl-butyl]carbamate (compound 2c) and 2-chloropropanoic acid. N-((2S)-1-(2-(3-Amino-3-oxo-propyl)-2-(2-chloro-2-fluoroacetyl)hydrazinyl)-4-methyl-1-oxo-pentan-2-yl)-4-methoxy-1H-indole-2-carboxamide (6.1 mg, Example 49) was obtained as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm: 10.09 (br. s, 1H), 7.16-7.20 (m, J=7.6 Hz, 2H), 7.05-6.94 (m, 1H), 6.64, (br. s, 1H), 6.46 (d, J=7.6 Hz, 1H), 6.35-6.15 (m, 1H), 4.63-4.68 (m, 1H), 3.29-4.25 (m, 5H), 2.12-2.66 (m, 2H), 1.62-1.87 (m, 3H), 0.64-1.11 (m, 6H). MS obsd. (ESI+) [(M+H)+]: 484.0.
Compound 50a was prepared in analogy to Example 1, Step 3 by using of (S)-tert-butyl (1-hydrazinyl-4-methyl-1-oxo-pentan-2-yl)(methyl)carbamate (900.0 mg, compound 40b) and N-methylprop-2-enamide (Bidepharm) instead of benzyl (2-hydrazinyl-2-oxo-ethyl)carbamate (compound 1b) and acrylamide. (S)-tert-Butyl methyl(4-methyl-1-(2-(3-(methylamino)-3-oxo-propyl)hydrazinyl)-1-oxo-pentan-2-yl)carbamate (160 mg, compound 50a) was obtained as a colorless oil. MS obsd. (ESI+) [(M+H)+]: 345.1.
Compound 50d was prepared in analogy to Example 1, Step 4 by using (S)-tert-butyl methyl(4-methyl-1-(2-(3-(methylamino)-3-oxo-propyl)hydrazinyl)-1-oxo-pentan-2-yl)carbamate (160 mg, compound 50a) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c). (S)-tert-Butyl (1-(2-(2-chloroacetyl)-2-(3-(methylamino)-3-oxo-propyl)hydrazinyl)-4-methyl-1-oxo-pentan-2-yl)(methyl)carbamate (180 mg, compound 50b) was obtained as a colorless oil. MS obsd. (ESI+) [(M+H)+]: 421.4.
Compound 50c was prepared in analogy to Example 27, step 1 by using ((S)-tert-butyl (1-(2-(2-chloroacetyl)-2-(3-(methylamino)-3-oxo-propyl)hydrazinyl)-4-methyl-1-oxo-pentan-2-yl)(methyl)carbamate (90 mg, compound 50b) instead of N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (Example 26). (S)-3-(1-(2-Chloroacetyl)-2-(4-methyl-2-(methylamino)pentanoyl)hydrazinyl)-N-methylpropanamide (60 mg, compound 50c) was obtained as a colorless oil.
The title compound was prepared in analogy to Example 27, Step 2 by using (S)-3-(1-(2-chloroacetyl)-2-(4-methyl-2-(methylamino)pentanoyl)hydrazinyl)-N-methylpropanamide (60 mg, compound 50c)) and 1H-indole-2-carboxylic acid instead of (S)-3-(2-(2-amino-4-methylpentanoyl)-1-(2-chloroacetyl)hydrazinyl)propanamide; hydrochloride (compound 27a) and 4-methoxy-1H-indole-2-carboxylic acid. (S)—N-(1-(2-(2-Chloroacetyl)-2-(3-(methylamino)-3-oxo-propyl)hydrazinyl)-4-methyl-1-oxo-pentan-2-yl)-N-methyl-1H-indole-2-carboxamide (9.7 mg, Example 50) was obtained as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm: 9.83 (br. s, 1H), 9.49 (br. s, 1H), 7.68 (d, J=8.4 Hz, 1H), 7.44 (d, J=8.8 Hz, 1H), 7.32 (t, J=7.2 Hz, 1H), 7.16 (t, J=7.6 Hz, 1H), 6.93 (s, 1H), 5.77 (br. s, 1H), 5.21 (t, J=IB Hz, 1H), 3.95-4.15 (m, 2H), 3.36 (s, 3H), 2.77 (d, J=4.8 Hz, 3H), 2.35-2.65 (m, 2H), 1.89 (t, J=7.6 Hz, 2H), 1.69-1.66 (m, 1H), 1.35-1.21 (m, 2H), 1.03 (d, J=6.4 Hz, 3H), 0.98 (d, J=6.4 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 464.1.
The title compound was prepared in analogy to Example 1, step 4 by using N-((2S)-4-methyl-1-oxo-1-(2-((2-oxo-pyrrolidin-3-yl)methyl)hydrazinyl) pentan-2-yl)-1H-indole-2-carboxamide (80 mg, compound 38d) instead of benzyl N-[2-[2-(3-amino-3-oxo-propyl)hydrazino]-2-oxo-ethyl]carbamate (compound 1c). N-[(1S)-1-[[(2-chloroacetyl)-[(2-oxo-pyrrolidin-3-yl)methyl]amino]carbamoyl]-3-methyl-butyl]-1H-indole-2-carboxamide (70.8 mg, Example 51) was obtained as a white solid.
Separation of compound of Example 51 by chiral SFC separation afford Example 51-A (faster eluting, 20.1 mg) and Example 51-B (slower eluting, 23.8 mg) as a white solid with 40% MeOH+ACN (0.05% DEA) (0.05% DEA)/CO2 on Chiralpak IC-3 column.
N-[(1S)-1-[[(2-chloroacetyl)-[[(3S)-2-oxo-pyrrolidin-3-yl]methyl]amino]carbamoyl]-3-methyl-butyl]-1H-indole-2-carboxamide and N-[(1S)-1-[[(2-chloroacetyl)-[[(3R)-2-oxo-pyrrolidin-3-yl]methyl]amino]carbamoyl]-3-methyl-butyl]-1H-indole-2-carboxamide (51-A and 51-B)
Example 51-A: 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.60 (m, 1H), 10.52-10.84 (m, 1H), 8.67 (br. s, 1H), 7.70-7.74 (m, 1H), 7.62 (d, J=7.6 Hz, 1H), 7.43 (d, J=8.4 Hz, 1H), 7.29 (d, J=1.6 Hz, 1H), 7.16-7.20 (m, 1H), 7.02-7.05 (m, 1H), 4.30-4.57 (m, 2H), 4.06-4.16 (m, 1H), 3.77-3.90 (m, 1H), 3.23-3.27 (m, 1H), 3.14 (br. s, 2H), 2.44-2.50 (m, 1H), 2.05-2.11 (m, 1H), 1.72-1.79 (m, 3H), 1.57-1.59 (m, 1H), 0.94 (dd, J=10.0, 20.0 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 462.3.
Example 51-B: 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.60 (m, 1H), 10.58-10.73 (m, 1H), 8.65 (d, J=6.8 Hz, 1H), 7.71 (br s, 1H), 7.62 (d, J=8.0 Hz, 1H), 7.43 (d, J=8.4 Hz, 1H), 7.29 (d, J=1.2 Hz, 1H), 7.16-7.20 (m, 1H), 7.02-7.06 (m, 1H), 4.31-4.46 (m, 2H), 4.08-4.15 (m, 1H), 3.78-3.90 (m, 1H), 3.28-3.30 (m, 1H), 3.08-3.19 (m, 2H), 2.38-2.43 (m, 1H), 2.16-2.23 (m, 1H), 1.73-1.82 (m, 3H), 1.51-1.59 (m, 1H), 0.94 (dd, J=6.4, 19.6 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 462.3.
The tide compound was prepared in analogy to Example 27, Step 2 by using 1H-pyrrolo[3,2-c]pyridine-2-carboxylic acid (38.4 mg) instead of 4-methoxy-1H-indole-2-carboxylic acid. N-[(1S)-1-[[(3-Amino-3-oxo-propyl)-(2-chloroacetyl)amino]carbamoyl]-3-methyl-butyl]-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (23.2 mg, Example 52) was obtained as a yellow solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 13.33 (s, 1H), 10.90-10.92 (m, 1H), 9.50 (s, 1H), 9.30 (d, J=6.8 Hz, 1H), 8.48 (s, 1H), 7.72-8.02 (m, 2H), 7.40 (s, 1H), 6.89 (s, 1H), 4.26-4.67 (m, 2H), 4.01-4.16 (m, 1H), 3.63-3.93 (m, 2H), 2.30-2.33 (m, 2H), 1.72-1.95 (m, 2H), 1.56-1.63 (m, 1H), 0.90-0.99 (m, 6H). MS obsd. (ESI+) [(M+H)+]: 436.9.
To a solution of(S)-tert-butyl (1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4-methyl-1-oxopentan-2-yl)carbamate (3.0 g, 26c), (2-chloro-2-fluoro-acetyl)oxysodium (6.38 g, 47.41 mmol) and DIEA (3.68 g, 28.45 mmol) in DMF (60 mL) was added T3P (9.05 g, 14.22 mmol, 50% in EtOAc) at 0° C. After stirred at 20° C. for 12 hrs, the reaction mixture was poured into ice-water (50 mL) and extracted with EtOAc (100 mL) three times. The combined organic phase was washed with brine (60 mL) and over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column (eluted with EtOAc:MeOH=1:0˜100:1) to afford tert-butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (1.3 g, compound 53a) as a white solid. MS obsd. (ESI+) [(M+H)+]: 411.1.
To a solution of tert-butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (200.0 mg, compound 53a) in DCM (2 mL) was added TFA (0.5 mL). After stirred at 20° C. for 1 hr, the reaction mixture was concentrated in vacuo to afford 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloro-2-fluoro-acetyl)amino]propanamide (151 mg, compound 53b) as a yellow oil.
To a solution of 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloro-2-fluoro-acetyl)amino]propanamide (74.61 mg, 53b), 4-chloro-1H-indole-2-carboxylic acid (46.97 mg, Bidepharm, CAS number: 24621-73-6) and DIEA (92.92 mg) in DMF (1 mL) was added T3P (198.6 mg) at 0° C. After stirred at 25° C. for 2 hrs, the reaction mixture was purified by prep-HPLC) to afford N-((2S)-1-(2-(3-amino-3-oxopropyl)-2-(2-chloro-2-fluoroacetyl)hydrazinyl)-4-methyl-1-oxopentan-2-yl)-4-chloro-1H-indole-2-carboxamide (30 mg, Example 53) as a white solid.
Separation of compound of compound 53 by chiral SFC separation afford Example 53-A (faster eluting, 6.3 mg) and Example 53-B (slower eluting, 4.5 mg) as a white solid with 40% MeOH+ACN (0.05% DEA) (0.05% DEA)/CO2 on Chiralpak IC-3 column.
Example 53-A: 1H NMR (400 MHz, CD3OD) δ ppm: 7.39 (d, J=8.4 Hz, 1H). 7.34 (s, 1H), 7.18 (t, J=8.0 Hz, 1H), 7.09 (d, J=7.6 Hz, 1H), 6.88 (d, J=50.0 Hz, 1H), 4.43-4.46 (m, 1H), 3.93-3.97 (m, 1H), 3.66-3.71 (m, 1H), 2.48-2.56 (m, 2H), 1.84-1.96 (m, 2H), 1.65-1.72 (m, 1H), 1.07 (d, J=6.4 Hz, 3H), 1.03 (d, J=6.4 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 488.1. SFC: RT=1.664 min, ee %=100%.
Example 53-B: 1H NMR (400 MHz, CD3OD) δ ppm: 7.40 (d, J=8.0 Hz, 1H). 7.36 (s, 1H), 7.19 (t, J=8.0 Hz, 1H), 7.09 (d, J=7.6 Hz, 1H), 6.48-6.98 (m, 1H), 4.54-5.58 (m, 1H), 3.92-3.96 (m, 1H), 3.70-3.76 (m, 1H), 2.47-2.63 (m, 2H), 1.73-1.93 (m, 3H), 1.06 (d, J=6.0 Hz, 3H), 1.00 (d, J=6.0 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 488.3 SFC: RT=1.862 min, ee %=97.7%.
The title compound was prepared in analogy to Example 53, Step 3 by using 4-methoxy-1H-indole-2-carboxylic acid instead of 4-chloro-1H-indole-2-carboxylic acid. N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]-4-methoxy-1H-indole-2-carboxamide (compound 54) was obtained as a yellow solid.
Separation of compound of compound 54 by chiral SFC separation afford Example 54-A (faster eluting, 14 mg) and Example 54-B (slower eluting, 10.7 mg) as a white solid with mobile phase: [0.1% NH3H2O MEOH]; 40%-40%, 70 min on DAICEL CHIRALCEL OJ column.
Example 54-A: 1H NMR (400 MHz, CDCl3) δ ppm: 10.30-10.11 (m, 2H), 7.37-7.27 (m, 1H), 7.16 (s, 2H), 7.05-6.97 (m, 1H), 6.70-6.52 (m, 1H), 6.51-6.12 (m, 2H), 4.73-4.67 (m, 1H), 4.05-3.96 (m, 1H), 3.95 (s, 3H), 3.68-3.49 (m, 1H), 2.53-2.31 (m, 2H), 1.79-1.72 (m, 2H), 1.27-1.23 (m, 1H), 0.99-0.90 (m, 6H). MS obsd. (ESI+) [(M+H)+]: 484.2 SFC: RT=0.972 min, ee %=100%
Example 54-B: 1H NMR (400 MHz, CD3Cl3) δ ppm: 10.50-9.86 (m, 1H), 7.27-7.20 (m, 2H), 7.03 (br. s, 1H), 6.88-5.87 (m, 3H), 4.69-4.67 (m, 1H), 4.32-3.03 (m, 5H), 2.58-2.22 (m, 2H), 1.83-1.69 (m, 3H), 1.00-0.87 (m, 6H). MS obsd. (ESI+) [(M+H)+]: 441.3. SFC: RT=2.503 min, ee %=97.734%
Compound 56a was prepared in analogy to Example 53, step 1 by using tert-butyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (2.0 g, compound 32c) instead of (S)-tert-butyl (1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4-methyl-1-oxopentan-2-yl)carbamate (26c). tert-Butyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)-2-(2-chloro-2-fluoro-acetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (470 mg, 56a) was obtained as a white solid MS obsd. (ESI+) [(M+H)+]: 451.42.
Compound 56b was prepared in analogy to Example 53, step 2 by using ten-butyl N-[(1S)-2-[2-(3-amino-3-oxo-propyl)-2-(2-chloro-2-fluoro-acetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]carbamate (470 mg, 56a) instead of tert-butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (53a). 3-(2-((S)-2-amino-3-cyclohexylpropanoyl)-1-(2-chloro-2-fluoroacetyl)hydrazinyl)propanamide (compound 56b, 370 mg) was obtained as a yellow oil. MS obsd. (ESI+) [(M+H)+]: 351.2.
The title compounds were prepared in analogy to Example 53, Step 3 by using 4-methoxy-1H-indole-2-carboxylic acid and 3-(2-((S)-2-amino-3-cyclohexylpropanoyl)-1-(2-chloro-2-fluoroacetyl)hydrazinyl)propanamide (compound 56b) instead of 4-chloro-1H-indole-2-carboxylic acid and 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloro-2-fluoro-acetyl)amino]propanamide (53b). N-[(1S)-2-[2-(3-Amino-3-oxo-propyl)-2-(2-chloro-2-fluoro-acetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]-4-methoxy-1H-indole-2-carboxamide (mg, compound 56) was obtained as a yellow solid.
Separation of compound 56 by chiral prep-SFC afforded Example 56-A (faster eluting, 20.3 mg) and Example 56-B (slower eluting, 8.4 mg) as a white solid with on column as a white solid with mobile phase: [0.1% NH3H2O MEOH]; 40%-40%, 70 min on DAICEL CHIRALCEL OJ column.
Example 56-A: 1H NMR (400 MHz, DMSO-d6) δ ppm 11.62-11.64 (m, 1H), 10.76 (d, J=27.2 Hz, 1H), 8.59-8.65 (m, 1H), 7.40-7.48 (m, 2H), 7.11 (t, J=8.0 Hz, 1H), 6.91-7.03 (m, 1.7H), 6.66 (s, 0.3H), 6.50-6.55 (m, 1H), 4.39-4.50 (m, 1H), 3.70-3.88 (m, 4H), 3.45-3.51 (m, 1H), 2.50-2.57 (m, 2H), 1.52-1.84 (m, 6H), 1.06-1.50 (m, 5H), 0.79-1.01 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 524.4. SFC: RT=1.661 min, ee %=100%.
1H NMR (400 MHz, CD3OD) δ ppm: 7.31 (s, 1H), 7.15 (t, J=8.0 Hz, 1H), 7.04 (d, J=8.0 Hz, 1H), 6.87 (d, J=50.0 Hz, 1H), 6.52 (d, J=8.0 Hz, 1H), 4.44-4.52 (m, 1H), 3.80-4.20 (m, 4H), 3.63-3.76 (m, 1H), 2.48-2.59 (m, 2H), 1.67-1.90 (m, 6H), 1.18-1.58 (m, 5H), 0.97-1.12 (m, 2H) MS obsd. (ESI+) [(M+H)+]: 524.4. SFC: RT=1.922 min, ee %=100%.
The title compound was prepared in analog to Example 53, step 3 by using 5-fluoro-1H-indole-2-carboxylic acid (Wuxi catalog, CAS Number: 399-76-8) instead of 4-chloro-1H-indole-2-carboxylic acid. 5-Fluoro-N-[rac-(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]-1H-indole-2-carboxamide was obtained (50 mg, compound 57) was obtained as a white solid.
Separation of compound 57 by chiral prep-SFC afforded Example 57-A (faster eluting, 19.1 mg) and Example 56-B (slower eluting, 20.7 mg) as a white solid with Neu-MeOH B:30 Gradient Time (min): 4.2; 25 FlowRate (ml/min): 60 on DAICEL CHIRALCEL OJ-H column.
Example 57-A: 1H NMR (400 MHz, CD3OD) δ ppm: 7.42 (q, J=4.4 Hz, 1H), 7.29 (dd, J=3.2 Hz, 9.6 Hz, 1H), 7.18 (s, 1H), 7.01 (td, 2.4 Hz, 9.2 Hz, 1H), 6.76-6.89 (d, J=50.4 Hz, 1H), 4.43-4.45 (m, 1H), 3.94-4.10 (m, 1H), 3.61-3.69 (m, 1H), 2.44-2.55 (m, 2H), 1.81-1.92 (m, 2H), 1.69-1.72 (m, 1H), 1.06 (d, J=6.4 Hz, 3H), 1.02 (d, J=6.4 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 472.3.
Example 57-B: 1H NMR (400 MHz, CD3OD) δ ppm. 7.43 (q, J=4.4 Hz, 1H), 77.29 (dd, J=2.4 Hz, 9.2 Hz, 1H), 7.19 (s, 1H), 7.02 (td, J=2.4 Hz, 9.2 Hz, 1H), 6.48-6.90 (m, 1H), 4.54-4.57 (m, 1H), 3.91-3.96 (m, 1H), 3.65-3.72 (m, 1H), 2.46-2.66 (m, 2H), 1.69-1.91 (m, 3H), 1.06 (d, J=6.4 Hz, 3H), 1.02 (d, J=6.4 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 472.4.
The title compound was prepared in analogy to Example 53, Step 3 by using 5-chloro-1H-indole-2-carboxylic acid instead of 4-chloro-1H-indole-2-carboxylic acid. 5-Chloro-N-[rac-(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]-1H-indole-2-carboxamide (10 mg) was obtained as a white solid.
Separation of compound 58 by chiral prep-SFC afforded Example 58-A (faster eluting, 17.8 mg) and Example 58-B (slower eluting, 10 mg) as a white solid with Neu—MeOH; MeOH %: 30%˜30%; Flow Rate (mL/min): 60 on DAICEL CHIRALCEL OJ-H column.
Example 58-A: 1H NMR (400 MHz, CD3OD) δ ppm: 7.62 (d, J=1.6 Hz, 1H), 7.41 (d, J=8.8 Hz, 1H), 7.17-7.22 (m, 2H), 6.80 (d, J=50.0 Hz, 1H), 4.44-4.52 (m, 1H), 3.79-4.04 (m, 1H), 3.60-3.73 (m, 1H), 2.49-2.59 (m, 2H), 1.65-1.90 (m, 3H), 1.05 (d, J=6.4 Hz, 3H), 1.02 (d, J=6.4 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 488.4.
Example 58-B: 1H NMR (400 MHz, CD3OD) δ ppm: 7.62 (d, J=2.8 Hz, 1H), 7.42 (d, J=8.8 Hz, 1H), 7.17-7.21 (m, 2H), 6.47-6.95 (m, 1H), 4.54-4.57 (m, 1H), 3.77-4.05 (m, 1H), 3.62-3.76 (m, 1H), 2.47-2.63 (m, 2H), 1.71-1.89 (m, 3H), 1.05 (d, J=6.4 Hz, 3H), 1.02 (d, J=6.0 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 488.3.
The title compound was prepared in analogy to Example 53, Step 3 by using 1H-indole-2-carboxylic acid and 3-(2-((S)-2-amino-3-cyclohexylpropanoyl)-1-(2-chloro-2-fluoroacetyl)hydrazinyl)propanamide (compound 56b) instead of 4-chloro-1H-indole-2-carboxylic acid and 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloro-2-fluoro-acetyl)amino]propanamide (53b). N-[(1S)-2-[2-(3-Amino-3-oxo-propyl)-2-(2-chloro-2-fluoro-acetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]-1H-indole-2-carboxamide (20 mg, compound 59) was obtained as a yellow solid.
Separation of compound 59 by chiral SFC separation afforded Example 59-A (faster eluting, 20.3 mg) and Example 59-B (slower eluting, 8.4 mg) by prep-SFC with Neu-MeOH; MeOH %: 50%-50%; FlowRate (mL/min): 70 on DAICEL CHIRALCEL OJ column.
Example 59-A: 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.60-11.62 (m, 1H), 10.71-10.79 (m, 1H), 8.68-8.71 (m, 1H), 7.63 (d, J=8.0 Hz, 1H), 7.45 (d, J=8.0 Hz, 1H), 7.45 (s, 1H), 7.30 (s, 1H), 7.19 (t, J=8.0 Hz, 1H), 7.04 (t, J=8.0 Hz, 1H), 6.51-6.98 (m, 2H), 4.47-4.48 (m, 1H), 3.72-3.89 (m, 1H), 3.45-3.52 (m, 1H), 2.32-2.41 (m, 2H), 1.55-1.85 (m, 7H), 1.35-1.48 (m, 1H), 1.11-1.25 (m, 3H), 0.79-1.02 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 494.1. ee %=100%.
Example 59-B: 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.59-11.62 (m, 1H), 11.05 (s, 1H), 8.69 (d, J=4.8 Hz, 1H), 7.63 (d, J=8.0 Hz, 1H), 7.43 (t, J=7.6 Hz, 2H), 7.31 (s, 1H), 7.19 (t, J=6.8 Hz, 1H), 7.04 (t, J=7.6 Hz, 1H), 6.79-6.98 (m, 2H), 4.31-4.52 (m, 1H), 3.75-3.89 (m, 1H), 3.39-3.49 (m, 1H), 2.27-2.41 (m, 2H), 1.55-1.92 (m, 7H), 1.42-1.54 (m, 1H), 1.11-1.28 (m, 3H), 0.88-1.02 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 494.1. SFC: RT=2.002 min, ee %=100%
The title compound was prepared in analogy to Example 53, Step 3 by using 5-chloro-1H-benzimidazole-2-carboxylic acid instead of 4-chloro-1H-indole-2-carboxylic acid. 5-Chloro-N-[rac-(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]-1H-benzimidazole-2-carboxamide (20 mg, compound 60) was obtained as a yellow solid.
Separation of compound 60 by chiral SFC separation afforded Example 60-A (faster eluting, 3.1 mg) and Example 60-B (slower eluting, 5 mg) with Neu-ETOH; B %: 30-30%; FlowRate (ml/min): 60) on DAICEL CHIRALPAK AD-H column.
Example 60-A: 1H NMR (400 MHz, CD3OD) δ ppm: 7.83-7.71 (m, 1H), 7.68-7.53 (m, 1H), 7.39-7.30 (m, 1H), 7.05-6.48 (m, 1H), 4.59-4.54 (m, 1H), 4.05-3.85 (m, 1H), 3.73-3.62 (m, 1H), 2.58-2.45 (m, 2H), 1.89-1.75 (m, 3H), 1.06 (d, J=6.0 Hz, 3H), 1.02 (d, J=6.0 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 489.0. SFC: RT=6.523 min, ee %=100%
Example 60-B: 1H NMR (400 MHz, CD3OD) δ ppm: 7.75-7.52 (m, 2H), 7.34-7.31 (m, 1H), 6.92-6.75 (m, 1H), 4.55-4.45 (m, 1H), 4.02-3.82 (m, 1H), 3.71-3.60 (m, 1H), 2.57-2.47 (m, 2H), 1.92-1.73 (m, 3H), 1.06 (d, J=6.0 Hz, 3H), 1.02 (d, J=6.0 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 489.0. SFC: RT=6.199 min, ee %=100%
To a solution of (S)-2-((tert-butoxycarbonyl)amino)-3-cyclopropylpropanoic acid (10.0 g) and benzyl hydrazinecarboxylate (7.97 g, 48.0 mmol) in DMF (150 mL) was added DIEA (31.14 mL). The reaction mixture was cooled down to 0° C. and T3P (38.91 mL, 65.43 mmol, 50% purity in EtOAc) was added. After stirred at 0° C. for 2 hrs, the reaction mixture was added into water (500 mL) and extracted with EtOAc (200 mL) for three times. The combined organic layers were washed with sat. aq. solution of sodium bicarbonate (200 mL), sat. aq. solution of citric acid (200 mL) and brine (200 mL), dried over anhydrous Na2SO4, concentrated in vacuo to afford tert-butyl N-[rac-(1S)-1-(cyclopropylmethyl)-2-oxo-2-10 [2-(2-phenoxyacetyl)hydrazino]ethyl]carbamate (16 g, compound 62a) as a yellow oil. MS obsd. (ESI+) [M+Na]+: 400.2.
A solution of tert-butyl N-[rac-(1S)-1-(cyclopropylmethyl)-2-oxo-2-[2-(2-phenoxyacetyl)hydrazino]ethyl]carbamate (16.0 g, compound 62a) in methanol (30 mL) was purged with nitrogen three times. Then Pd/C (1.5 g, 10% purity) was added to the solution under nitrogen. The mixture was degassed under vacuum and purged H2 for hydrogen three times. After stirred at 20° C. for 16 hrs under H2 balloon, the reaction mixture was filtered, concentrated in vacuo to afford tert-butyl (S)-(3-cyclopropyl-1-hydrazinyl-1-oxopropan-2-yl)carbamate (11 g, compound 62b) as a yellow oil. MS obsd. (ESI+) [M−C4H8+H]+: 187.8.
To a solution of acrylamide (876.45 mg) in 2-propanol (10 mL) was added tert-butyl (S)-(3-cyclopropyl-1-hydrazinyl-1-oxopropan-2-yl)carbamate (11 g, compound 62b) (HCl salt). The mixture was purged and degassed with nitrogen for three times. After stirred at 50° C. for 2 hrs, the reaction mixture was added water (200 mL) and basified with sodium bicarbonate until pH=7, extracted with EtOAc (50 mL) three times. The combined organic layers were washed with brine (200 mL), dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column (eluted with ethyl acetate in petroleum ether:EtOAc=30-100% to methanol in ethyl acetate=0˜10%) to afford tert-butyl (S)-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-3-cyclopropyl-1-oxopropan-2-yl)carbamate (4.5 g, compound 62c) as a white solid. MS obsd. (ESI+) [M−C4H8+H]+: 258.8.
Compound 62d was prepared in analogy to Example 53, step 1 by using tert-butyl (S)-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-3-cyclopropyl-1-oxopropan-2-yl)carbamate (4.5 g, compound 62c) instead of (S)-tert-butyl (1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4-methyl-1-oxopentan-2-yl)carbamate (26c). tert-Butyl((2S)-1-(2-(3-amino-3-oxopropyl)-2-(2-chloro-2-fluoroacetyl) hydrazinyl)-3-cyclopropyl-1-oxopropan-2-yl)carbamate (200 mg, compound 62d) was obtained as a white solid. MS obsd. (ESI+) [M−C4H8+H]+: 352.8.
Compound 62e was prepared in analogy to Example 53, step 2 by using tert-butyl((2S)-1-(2-(3-amino-3-oxopropyl)-2-(2-chloro-2-fluoroacetyl) hydrazinyl)-3-cyclopropyl-1-oxopropan-2-yl)carbamate (200 mg, compound 62d) instead of tert-butyl N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]carbamate (200.0 mg, compound 53a). 3-(2-((S)-2-Amino-3-cyclopropylpropanoyl)-1-(2-chloro-2-fluoroacetyl)hydrazinyl)propanamide (120 mg, compound 62e) was obtained as a yellow oil.
The title compound was prepared in analogy to Example 53, Step 3 by using 1H-indole-2-carboxylic acid (22.87 mg) and 3-(2-((S)-2-amino-3-cyclopropylpropanoyl)-1-(2-chloro-2-fluoroacetyl)hydrazinyl)propanamide (TFA salt) (40.0 mg, compound 62e) instead of 4-chloro-1H-indole-2-carboxylic acid and 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloro-2-fluoro-acetyl)amino]propanamide (74.61 mg, compound 53b). N-((2S)-1-(2-(3-Amino-3-oxopropyl)-2-(2-chloro-2-fluoroacetyl)hydrazinyl)-3-cyclopropyl-1-oxopropan-2-yl)-1H-indole-2-carboxamide (4.89 mg, Example 62) was obtained as a white solid.
1H NMR (400 MHz, CD3OD) δ ppm: 7.63 (d, J=8.4 Hz, 1H), 7.50-7.43 (m, 1H), 7.27-7.17 (m, 2H), 7.13-6.90 (m, 1H), 7.02-6.81 (m, 1H), 4.02-3.50 (m, 1H), 3.26-3.17 (m, 2H), 2.77-2.37 (m, 2H), 2.10-1.54 (m, 2H), 1.05-0.76 (m, 1H), 0.66-0.45 (m, 2H), 0.32-0.11 (m, 2H) MS obsd. (ESI+) [(M+H)+]: 452.3.
The title compound was prepared in analogy to Example 53, Step 3 by using 1H-benzimidazole-2-carboxylic acid instead of 4-chloro-1H-indole-2-carboxylic acid. N-[(1S)-1-[[(3-Amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]-1H-benzimidazole-2-carboxamide (40 mg, compound 63) was obtained as a yellow solid.
Separation of compound 63 by chiral SFC afforded Example 63-A (faster eluting, 11.8 mg) and Example 63-B (slower eluting, 12.9 mg) with Neu-ETOH; B %: 30-30%; FlowRate (ml/min): 60) on DAICEL CHIRALCEL OJ-H column.
Example 63-A: 1H NMR (400 MHz, CD3OD) δ ppm: 7.68-7.72 (m, 2H), 7.33-7.42 (m, 2H), 6.67-6.95 (m, 1H), 4.42-4.53 (m, 1H), 3.94-4.02 (m, 1H), 3.67-3.72 (m, 1H), 2.49-2.59 (m, 2H), 1.82-1.99 (m, 2H), 1.66-1.80 (m, 1H), 1.05 (dd, J=6.0 Hz, 15.2 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 455.1. SFC: RT=1.030 min, ee %=100%
Example 63-B: 1H NMR (400 MHz, CD3OD) δ ppm: 7.59-7.76 (m, 2H), 7.29-7.42 (m, 2H), 6.38-6.85 (m, 1H), 4.42-4.53 (m, 1H), 3.84-4.02 (m, 1H), 3.57-3.83 (m, 1H), 2.49-2.59 (m, 2H), 1.78-1.95 (m, 3H), 1.05 (dd, J=6.4 Hz, 17.2 Hz, 6H). MS obsd. (ESI+) [(M+H)+]: 455.2. SFC: RT=1.177 min, ee %=96.564%
The title compound was prepared in analogy to Example 53, Step 3 by using pyrazolo[1,5-a]pyridine-2-carboxylic acid (Bidepharm, CAS Number: 63237-88-7) instead of 4-chloro-1H-indole-2-carboxylic acid. N-[(1S)-2-[2-(3-Amino-3-oxo-propyl)-2-(2-chloro-2-fluoro-acetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]-1H-indole-2-carboxamide (compound 64) was obtained as a yellow solid.
Separation of compound 64 by chiral SFC afforded Example 64-A (faster eluting, 4.0 mg) and Example 64-B (slower eluting, 6.5 mg) with Neu-ETOH; B %: 30-30%; FlowRate (ml/min): 60) on DAICEL CHIRALPAK AD-H column
Example 64-A: 1H NMR (400 MHz, CD3OD) δ ppm: 8.61 (d, J=6.4 Hz, 1H), 7.72 (d, J=9.2 Hz. 1H), 7.25-7.29 (m, 1H), 7.08 (s, 1H), 6.99-7.03 (m, 1H), 6.46-6.60 (m, 1H), 4.63-4.70 (m, 1H), 3.92-4.01 (m, 1H), 3.65-3.75 (m, 1H), 2.45-2.59 (m, 2H), 1.74-1.90 (m, 3H), 1.05 (d, J=6.4 Hz, 3H), 1.02 (d, J=6.4 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 455.3. SFC: RT=1.909 min, ee %=96.9%
Example 64-B: 1H NMR (400 MHz, CD3OD) δ ppm: 8.61 (d, J=6.4 Hz, 1H), 7.72 (d, J=8.8 Hz, 1H), 7.25-7.29 (m, 1H), 7.08 (s, 1H), 7.01-7.07 (m, 1H), 6.82 (d, J=50.0 Hz, 1H), 4.54-4.58 (m, 1H), 3.93-3.97 (m, 1H), 3.66-3.70 (m, 1H), 2.46-2.61 (m, 2H), 1.70-1.90 (m, 3H), 1.06 (d, J=6.0 Hz, 3H), 1.03 (d, J=6.4 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 455.3. SFC: RT=2.104 min, ee %=96.5%
To a solution of (S)-2-amino-4,4-dimethylpentanoic acid (TFA salt) (230.0 mg, compound 65a, GL Biochem, CAS number: 57224-50-7) in DCM (10 mL) was added DIEA (1023.6 mg) and 1H-benzimidazole-2-carbonyl chloride (310.0 mg). After stirred at 25° C. for 2 hrs, the reaction mixture was concentrated in vacuo. The residue was dissolved with EtOAc (60 mL), washed with HCl (1 M 30 mL), brine (20 mL), dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column, eluted with MeOH in DCM: MeOH=0˜10% to afford (S)-2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoic acid (120 mg, compound 65b) as a yellow solid. MS obsd. (ESI+) [(M+H)+]: 290.1
To a solution of (S)-2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoic acid (120.0 mg, compound 65b), tert-butyl 1-(3-Amino-3-oxopropyl)hydrazine-1-carboxylate (84.29 mg) and DIEA (160.81 mg) in DCM (3 mL) was added T3P (395.67 mg, 0.620 mmol, 50% purity in EtOAc). After stirred at 25° C. for 12 hrs, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column, eluted with MeOH in DCM: =0˜10% to afford tert-butyl (S)-2-(2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoyl)-1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (100 mg, compound 65c) as a yellow solid. MS obsd. (ESI+) [(M+H)+]: 475.2.
To a solution of tert-butyl (S)-2-(2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoyl)-1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (100.0 mg, compound 65c) in DCM (2 mL) was added TFA (2.0 mL). After stirred at 25° C. for 1 hr, the reaction mixture was concentrated in vacuo to afford (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (TFA salt) (75 mg, compound 65d) as a light yellow oil.
To a solution of (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (TFA salt) (75.0 mg, compound 65d), 2-chloro-2-fluoroacetic acid (66.5 mg) and DIEA (129.43 mg) in DMF (2 mL) was added T3P (191.09 mg, 0.300 mmol, 50% purity in EtOAc) at 0° C. After stirred at 25° C. for 12 hrs, the reaction mixture was purified by prep-HPLC (Phenomenex Synergi C18 150*25 mm*10 um; ACN in water (0.1% TFA): 30%-60%) to afford N-((2S)-1-(2-(3-amino-3-oxopropyl)-2-(2-chloro-2-fluoroacetyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (1.82 mg, Example 65) as a yellow solid. 1H NMR (400 MHz, CD3OD) δ ppm: 7.69 (dd, J1=3.2 Hz, J2=6.0 Hz, 2H), 7.38 (dd, J1=3.2 Hz, J2=6.0 Hz, 2H), 6.82-6.50 (m, 1H), 4.71-4.48 (m, 1H), 4.13-3.80 (m, 1H), 3.76-3.61 (m, 1H), 2.61-2.46 (m, 2H), 2.02-1.82 (m, 2H), 1.07-1.00 (m, 9H). MS obsd. (ESI+) [(M+H)+]: 469.4.
Compound 66a was prepared in analogy to Example 65, step 1 by using (S)-2-amino-3-cyclobutylpropanoic acid (150.56 mg, GL Biochem) instead of (S)-2-amino-4,4-dimethylpentanoic acid. (S)-2-(1H-benzo[d]imidazole-2-carboxamido)-3-cyclobutylpropanoic acid (140 mg, compound 66a) was obtained as a yellow oil. MS obsd. (ESI+) [(M+Na)+]: 309.8.
Compound 66b was prepared in analogy to Example 65, step 2 by using (S)-2-(1H-benzo[d]imidazole-2-carboxamido)-3-cyclobutylpropanoic acid (140 mg, compound 66a) instead of (2S)-2-(1H-benzimidazole-2-carbonylamino)-4,4-dimethyl-pentanoic acid (compound 65b). tert-Butyl (S)-2-(2-(1H-benzo[d]imidazole-2-carboxamido)-3-cyclobutylpropanoyl)-1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (100 mg, compound 66b) was obtained as a light yellow solid. MS obsd. (ESI+) [(M+H)+]:473.2.
Compound 66c was prepared in analogy to Example 65, step 3 by using tert-butyl (S)-2-(2-(1H-benzo[d]imidazole-2-carboxamido)-3-cyclobutylpropanoyl)-1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (100 mg, compound 66b) instead of tert-butyl (S)-2-(2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoyl)-1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (100.0 mg, compound 65c). (S)—N-(1-(2-(3-Amino-3-oxopropyl)hydrazinyl)-3-cyclobutyl-1-oxopropan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (75 mg, compound 66c) was obtained as a light yellow oil.
The title compound was prepared in analogy to Example 65, step 4 by using (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-3-cyclobutyl-1-oxopropan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (75 mg, compound 66c) instead of (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (TFA salt) (75.0 mg, compound 65d). N-((2S)-1-(2-(3-Amino-3-oxopropyl)-2-(2-chloro-2-fluoroacetyl)hydrazinyl)-3-cyclobutyl-1-oxopropan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (1.77 mg, Example 66) was obtained as a white solid. 1H NMR (400 MHz, CD3OD) δ ppm: 7.68-7.66 (m, 2H), 7.37-7.35 (m, 2H), 6.92-6.50 (m, 1H), 4.51-4.46 (m, 1H), 4.15-3.88 (m, 1H), 3.76-3.61 (m, 1H), 2.64-2.46 (m, 2H), 2.21-2.13 (m, 2H), 2.12-1.99 (m, 2H), 1.99-1.76 (m, 4H), 1.28-1.25 (m, 1H). MS obsd. (ESI+) [(M+H)+]: 467.4.
To a solution of tert-butyl hydrazinecarboxylate (10.0 g) in IPA (100 mL) was added acrylamide (5.38 g). After stirred at 70° C. for 3 hrs, the reaction mixture was concentrated in vacuo. The residue was dissolved with ethyl acetate (60 mL), washed with HCl (1 M 30 mL), brine (20 mL) and dried over anhydrous Na2SO4 and concentrated in vacuo. tert-Butyl 2-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (8 g, compound 67a) was obtained as a colorless oil.
To a solution of tert-butyl 2-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (1.2 g) was added benzyl (2,5-dioxopyrrolidin-1-yl) carbonate (1.47 g) in DMF (30 mL) at 25° C. After stirred at 25° C. for 12 hrs, the reaction mixture was diluted with H2O (100 mL) and extracted with EtOAc (100 mL) three times. The combined organic phase was washed with brine (80 mL) three times, dried over anhydrous Na2SO4 and filtrated. The filtrate was concentrated in vacuo. The residue was purified by silica gel column, eluted with MeOH in DCM=0˜10% to afford 1-benzyl 2-(tert-butyl) 1-(3-amino-3-oxopropyl)hydrazine-1,2-dicarboxylate (580 mg, compound 67b) as a white solid. MS obsd. (ESI+) [(M+Na)+]: 360.1
To a solution of 1-benzyl 2-(tert-butyl) 1-(3-amino-3-oxopropyl)hydrazine-1,2-dicarboxylate (1.0 g) in DCM (10 mL) was added TFA (10 mL). After stirred at 25° C. for 2 hrs, the reaction mixture was concentrated in vacuo to afford benzyl 1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (TFA salt) (145 mg, 67c) as a colorless oil.
To a solution of (S)-3-(bicyclo[1.1.1]pentan-1-yl)-2-((tert-butoxycarbonyl)amino)propanoic acid (550.0 mg, compound 67c) and benzyl 1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (511.12 mg) in DCM (10 mL) was added DIEA (1.11 g) and T3P (1.37 g, 50% purity in EtOAc) at 0° C. After stirred at 20° C. for 12 hrs, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column, eluted with MeOH in DCM=0˜1% to afford benzyl (S)-1-(3-amino-3-oxopropyl)-2-(3-(bicyclo[1.1.1]pentan-1-yl)-2-((tert-butoxycarbonyl) amino)propanoyl)hydrazine-1-carboxylate (500 mg, compound 67d) as a yellow oil.
MS obsd. (ESC) [M+H]+: 475.3.
To a solution of benzyl (S)-1-(3-amino-3-oxopropyl)-2-(3-(bicyclo[1.1.1]pentan-1-yl)-2-((tert-butoxycarbonyl)amino)propanoyl)hydrazine-1-carboxylate (500.0 mg) in methanol (10 mL) was added Pd/C (50.0 mg, 10% purity). The mixture was degassed under vacuum and purged H2 three times. The mixture was stirred at 20° C. for 2 hrs under H2 balloon. LCMS showed the reaction was completed. The reaction mixture was filtered. The filtrate was concentrated in vacuo to afford tert-butyl (S)-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-3-(bicyclo[1.1.1]pentan-1-yl)-1-oxopropan-2-yl)carbamate (300 mg, compound 67e) as a yellow solid. MS obsd. (ESI+) [M+H]+: 341.2.
To a solution of tert-butyl (S)-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-3-(bicyclo[1.1.1]pentan-1-yl)-1-oxopropan-2-yl)carbamate (300.0 mg, compound 67e) in DCM (6 mL) was added DIEA (569.47 mg) and 2-chloro-2-fluoro-acetyl chloride (346.15 mg) at 0° C. The mixture was stirred at 0° C. for 1 hr. LCMS showed the reaction was completed. It was concentrated in vacuo. The residue was purified by silica gel column, eluted with MeOH in DCM=0˜2% to afford tert-butyl ((2S)-1-(2-(3-amino-3-oxopropyl)-2-(2-chloro-2-fluoroacetyl)hydrazinyl)-3-(bicyclo[1.1.1]pentan-1-yl)-1-oxopropan-2-yl)carbamate (260 mg, compound 67f) as a yellow oil. MS obsd. (ESI+) [M+H]+: 435.2.
To a solution of tert-butyl ((2S)-1-(2-(3-amino-3-oxopropyl)-2-(2-chloro-2-fluoroacetyl) hydrazinyl)-3-(bicyclo[1.1.1]pentan-1-yl)-1-oxopropan-2-yl)carbamate (260.0 mg, compound 67f) in DCM (3 mL) was added TFA (3.0 mL). After stirred at 20° C. for 1 hr, the reaction mixture was concentrated in vacuo to afford 3-(2-((S)-2-amino-3-(bicyclo[1.1.1]pentan-1-yl) propanoyl)-1-(2-chloro-2-fluoroacetyl)hydrazinyl)propanamide (200 mg, compound 67g) as a yellow oil.
The title compound was prepared in analogy to Example 53, Step 3 by using 1H-benzimidazole-2-carboxylic acid and 3-(2-((S)-2-amino-3-(bicyclo[1.1.1]pentan-1-yl) propanoyl)-1-(2-chloro-2-fluoroacetyl)hydrazinyl)propanamide (compound 67g) instead of 4-chloro-1H-indole-2-carboxylic acid and 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloro-2-fluoro-acetyl)amino]propanamide (74.61 mg, 53b). N-((2S)-1-(2-(3-Amino-3-oxopropyl)-2-(2-chloro-2-fluoroacetyl) hydrazinyl)-3-(bicyclo[1.1.1]pentan-1-yl)-1-oxopropan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (145 mg, Example 67) was obtained as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm: 9.97 (br. s, 1H), 7.72 (m, 2H), 7.53 (m, 2H), 6.68-6.42 (m, 2H), 4.66 (m, 1H), 4.15-3.73 (m, 4H), 2.52 (m, 1H), 2.44-2.42 (m, 1H), 2.11 (m, 2H), 1.73-1.69 (m, 7H). MS obsd. (ESI+) [M+H]+: 479.2.
Compound 68a was prepared in analogy to Example 65, step 1 by using 5-methylisoxazole-3-carbonyl chloride instead of 1H-benzimidazole-2-carbonyl chloride. (2S)-4,4-dimethyl-2-[(5-methylisoxazole-3-carbonyl)amino]pentanoic acid (3 g, compound 68a) was obtained as a white solid. MS obsd. (ESI+) [M+H]+: 255.2
Compound 68b was prepared in analogy to Example 65, step 2 by using (2S)-4,4-dimethyl-2-[(5-methylisoxazole-3-carbonyl)amino]pentanoic acid (3 g, compound 68a) and tert-butyl (S)-1-((2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (2.71 g, intermediate AF) instead of (S)-2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoic acid (120.0 mg, compound 65b) and tert-butyl 1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate. tert-Butyl N-[[(2S)-4,4-dimethyl-2-[(5-methylisoxazole-3-carbonyl)amino]pentanoyl]amino]-N-[[(3S)-2-oxopyrrolidin-3-yl]methyl]carbamate (3.3 g, compound 68c) was obtained as a white solid.
MS obsd. (ESI+) [(M+H)+]: 466.2
Compound 68c was prepared in analogy to Example 65, step 3 by using tert-butyl N-[[(2S)-4,4-dimethyl-2-[(5-methylisoxazole-3-carbonyl)amino]pentanoyl]amino]-N-[[(3S)-2-oxopyrrolidin-3-yl]methyl]carbamate (compound 68c) instead of tert-butyl (S)-2-(2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoyl)-1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (compound 65c). N—((S)-4,4-dimethyl-1-oxo-1-(2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)pentan-2-yl)-5-methylisoxazole-3-carboxamide, hydrochloride (2.2 g, 68d) was obtained as a white solid. MS obsd. (ESI+) [(M+H)+]: 366.2
The title compound was prepared in analogy to Example 65, step 4 by using N—((S)-4,4-dimethyl-1-oxo-1-(2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)pentan-2-yl)-5-methylisoxazole-3-carboxamide, hydrochloride (2.2 g, 68d) and (R)-2-chloro-2-fluoroacetyl chloride instead of (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (TFA salt) (75.0 mg, compound 65d) and 2-chloro-2-fluoroacetic acid. N—((S)-1-(2-((R)-2-Chloro-2-fluoroacetyl)-2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)-5-methylisoxazole-3-carboxamide (1637 mg, compound 68) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 11.12-10.30 (m, 1H), 9.16-8.83 (m, 1H), 7.78-7.70 (m, 1H), 7.04-6.59 (m, 2H), 4.58-4.32 (m, 1H), 3.98-3.81 (m, 1H), 3.30-3.10 (m, 3H), 2.52-2.50 (m, 1H), 2.49 (s, 3H), 2.28-2.09 (m, 1H), 1.90-1.64 (m, 3H), 0.93 (s, 9H). MS obsd. (ESI+) [M+H]+: 460.1.
Compound 70a was prepared in analogy to Example 65, step 2 by using (S)-2-(((benzyloxy)carbonyl)amino)-3-(1-methylcyclopropyl)propanoic acid and (Intermediate AD) and tert-butyl (S)-1-((2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (2.96 g, Intermediate AF) instead of (S)-2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoic acid (120.0 mg, compound 65b) and tert-butyl 1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate. tert-Butyl 2-((S)-2-(((benzyloxy)carbonyl)amino)-3-(1-methylcyclopropyl)propanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (5.76 g, compound 70a) was obtained as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm: 9.20-8.95 (m, 1H), 7.43-7.29 (m, 5H), 6.29 (br. s, 1H), 5.42 (d, J=5.6 Hz, 1H), 5.21-5.07 (m, 2H), 4.38 (br. s, 1H), 3.96-3.29 (m, 5H), 2.81-2.68 (m, 1H), 2.34-2.20 (m, 1H), 2.00-1.82 (m, 2H), 1.41 (s, 9H), 1.08 (s, 3H), 0.41-0.23 (m, 4H). MS obsd. (ESI+) [(M+H)+]: 489.3
To a solution of tert-butyl 2-((S)-2-(((benzyloxy)carbonyl)amino)-3-(1-methylcyclopropyl)propanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (5.76 g, compound 70a) in ethanol (100 mL) was added Pd/C (1.0 g, 10% purity on charcoal, wet). The mixture was degassed under vacuum and purged three times. After stirred at 20° C. for 1.5 hrs under H2 balloon, the mixture was filtered and the filtrate was concentrated in vacuo to afford tert-butyl 2-((S)-2-amino-3-(1-methylcyclopropyl)propanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (4.0 g, compound 70b) as an off-white solid. 1H NMR (400 MHz, CDCl3) δ ppm: 9.60-9.32 (m, 1H), 5.92 (br. s, 1H), 3.98-3.33 (m, 5H), 2.75-2.67 (m, 1H), 2.43-1.97 (m, 6H), 1.52-1.37 (m, 9H), 1.11 (s, 3H), 0.37-0.25 (m, 4H).
To a solution of tert-butyl 2-((S)-2-amino-3-(1-methylcyclopropyl)propanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (3.5 g, compound 70b) and DIEA (5.1 g) in THF (70 mL) was added 5-methylisoxazole-3-carbonyl chloride (2.16 g) at 0° C. After stirred at 0° C. for 30 minutes, the reaction mixture was quenched by sat.aq. solution of NaHCO3 (20 mL) and acidified with HCl (1M) until pH=2˜3, and extracted with EtOAc (100 mL) three times. The combined organic layers were dried over anhydrous Na2SO4, filtered and the filtrate was concentrated in vacuo. The residue was purified by reversed-phase flash (MeCN in water (0.1% TFA)=0˜ 70%) to afford tert-butyl 2-((S)-3-(1-methylcyclopropyl)-2-(5-methylisoxazole-3-carboxamido)propanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (3.47 g, compound 70c) as a light yellow oil. MS obsd. (ESI+) [(M+H)+]: 464.2.
Compound 70d was prepared in analogy to Example 65, step 3 by using tert-butyl 2-((S)-3-(1-methylcyclopropyl)-2-(5-methylisoxazole-3-carboxamido)propanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (3.47 g, compound 70c) instead of tert-butyl (S)-2-(2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoyl)-1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (compound 65c). 5-Methyl-N—((S)-3-(1-methylcyclopropyl)-1-oxo-1-(2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)propan-2-yl)isoxazole-3-carboxamide (2.21 g, compound 70d) was obtained as a white solid. 1H NMR (400 MHz, CD3OD) δ ppm: 6.48 (s, 1H), 4.81-4.69 (m, 1H), 3.54-3.35 (m, 4H), 2.94-2.92 (m, 1H), 2.49 (d, J=0.8 Hz, 3H), 2.45-2.33 (m, 1H), 2.02-1.88 (m, 2H), 1.79-1.73 (m, 1H), 1.13 (s, 3H), 0.53-0.44 (m, 1H), 0.37-0.26 (m, 3H). MS obsd. (ESI+) [M+H]+: 364.3.
The title compound was prepared in analogy to Example 65, step 4 by using 5-Methyl-N—((S)-3-(1-methylcyclopropyl)-1-oxo-1-(2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)propan-2-yl)isoxazole-3-carboxamide (2.21 g, compound 70d) and (R)-2-chloro-2-fluoroacetyl chloride instead of (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (TFA salt) (compound 65d) and 2-chloro-2-fluoroacetic acid. N—((S)-1-(2-((R)-2-chloro-2-fluoroacetyl)-2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)-3-(1-methylcyclopropyl)-1-oxopropan-2-yl)-5-methylisoxazole-3-carboxamide (905.1 mg, Example 70) was obtained as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm: 10.03 (br. s, 1H), 7.33-7.29 (m, 1H), 6.49 (d, J=50.8 Hz, 1H), 6.45 (d, J=0.4 Hz, 1H), 5.65 (br. s, 1H), 4.62-4.59 (m, 1H), 4.50-4.36 (m, 1H), 3.43-3.39 (m, 2H), 2.92-2.79 (m, 2H), 2.50 (s, 3H), 2.45-2.32 (m, 1H), 2.14-2.00 (m, 1H), 1.90-1.75 (m, 1H), 1.59-1.54 (m, 1H), 1.13 (s, 3H), 0.49-0.41 (m, 2H), 0.38-0.29 (m, 2H). MS obsd. (ESI+) [M+H]+: 458.2
To a solution of methyl (S)-2-amino-3-(bicyclo[1.1.1]pentan-1-yl)propanoate (HCl salt) (1.6 g, Intermediate AG) and Na2CO3 (2.47 g) in THF (20 mL) and water (15 mL) was added 5-methylisoxazole-3-carbonyl chloride (1.13 g) at 0° C. After stirred at 20° C. for 12 hrs, the reaction mixture was poured into HCl (1M, 60 mL) and extracted with EtOAc (30 mL) three times. The combined organic layer was concentrated in vacuo. The residue was purified by silica gel column (eluted with ethyl acetate in petroleum ether=0˜30%) to afford methyl (S)-3-(bicyclo 1.1.1 pentan-1-yl)-2-(5-methylisoxazole-3-carboxamido)propanoate (1.6 g, 71a) as a yellow oil. MS obsd. (ESI+) [M+H]+: 279.1.
To a solution of methyl (S)-3-(bicyclo[1.1.1]pentan-1-yl)-2-(5-methylisoxazole-3-carboxamido)propanoate (1.6 g, compound 71a) in THF (15 mL) and water (15 mL) was added LiOH H2O (482.47 mg). After stirred at 20° C. for 1 hour, the mixture was poured into HCl (1 M, 30 mL) and extracted with EtOAc (40 mL) three times. The combined organic phase was dried over anhydrous Na2SO4, concentrated in vacuo to afford (S)-3-(bicyclo[1.1.1]pentan-1-yl)-2-(5-methylisoxazole-3-carboxamido)propanoic acid (400 mg, compound 71b) as a white solid. MS obsd. (ESI+) [M+H]+: 265.2.
Compound 71c was prepared in analogy to Example 65, step 2 by using (S)-3-(bicyclo[1.1.1]pentan-1-yl)-2-(5-methylisoxazole-3-carboxamido)propanoic acid (400 mg, compound 71b) and tert-butyl (S)-1-((2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (500 mg, Intermediate AF) instead of (S)-2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoic acid (compound 65b) and tert-butyl 1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate. tert-Butyl 2-((S)-3-(bicyclo[1.1.1]pentan-1-yl)-2-(5-methylisoxazole-3-carboxamido)propanoyl)-1-((2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (1.7 g, compound 71c) was obtained as a white solid. MS obsd. (ESI+) [Ms+H]+: 476.2.
Compound 71 d was prepared in analogy to Example 65, Step 3 by using rm-butyl 2-((S)-3-(bicyclo[1.1.1]pentan-1-yl)-2-(5-methylisoxazole-3-carboxamido)propanoyl)-1-((2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (1.7 g, compound 71c) instead of tert-butyl (S)-2-(2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoyl)-1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (compound 65c). N-[(1S)-1-(1-Bicyclo[1.1.1]pentanylmethyl)-2-oxo-2-[2-[[(3S)-2-oxopyrrolidin-3-yl]methyl]hydrazino]ethyl]-5-methyl-isoxazole-3-carboxamide (1.3 g, compound 71d) was obtained as a white solid. MS obsd. (ESI+) [M+H]+: 376.2.
The title compound was prepared in analogy to Example 65, step 4 by using N-((2S)-3-(bicyclo[1.1.1]pentan-1-yl)-1-oxo-1-(2-((2-oxopyrrolidin-3-yl) methyl)hydrazinyl)propan-2-yl)-5-methylisoxazole-3-carboxamide (1.3 g, compound 71d) and (R)-2-chloro-2-fluoroacetyl chloride instead of (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (TFA salt) (75.0 mg, compound 65d) and 2-chloro-2-fluoroacetic acid. N-[(1S)-1-(1-bicyclo[1.1.1]pentanylmethyl)-2-[2-[(2R)-2-chloro-2-fluoro-acetyl]-2-[[(3S)-2-oxopyrrolidin-3-yl]methyl]hydrazino]-2-oxo-ethyl]-5-methyl-isoxazole-3-carboxamide (139.66 mg, Example 71) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.99-10.41 (m, 1H), 8.99-8.88 (m, 1H), 7.83-7.72 (m, 1H), 6.95-6.58 (m, 2H), 4.38-4.30 (m, 1H), 3.95-3.79 (m, 1H), 3.31-3.21 (m, 1H), 3.19-3.05 (m, 2H), 2.55-2.53 (m, 1H), 2.49-2.46 (m, 3H), 2.38-2.35 (m, 1H), 2.15-2.05 (m, 1H), 2.04-1.90 (m, 2H), 1.73-1.61 (m, 7H). MS obsd. (ESI+) [(M+H)+]: 470.1. SFC: RT=1.843 min, ee %=100%.
The title compound was prepared in analogy to Example 53, Step 3 by using 5-methylisoxazole-3-carboxylic acid and 3-(2-((S)-2-amino-3-cyclohexylpropanoyl)-1-(2-chloro-2-fluoroacetyl)hydrazinyl)propanamide (compound 56b) instead of 4-chloro-1H-indole-2-carboxylic acid and 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloro-2-fluoro-acetyl)amino]propanamide (53b). 5-Methyl-N-[rac-(1S)-2-[2-(3-amino-3-oxo-propyl)-2-(2-chloro-2-fluoro-acetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]isoxazole-3-carboxamide (21.3 mg, Example 74) was obtained as a white solid. 1H NMR (400 MHz, CD3OD) δ ppm: 6.76-6.43 (m, 1H), 6.49 (s, 1H), 4.58-4.54 (m, 1H), 4.02-3.86 (m, 1H), 3.75-3.55 (m, 1H), 2.56-2.51 (m, 2H), 2.49 (s, 3H), 1.86-1.69 (m, 7H), 1.50-1.49 (m, 1H), 1.38-1.20 (m, 3H), 1.10-0.95 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 460.3
The title compound was prepared in analogy to Example 53, Step 3 by using 5-methylisoxazole-3-carboxylic acid and 3-(2-((S)-2-amino-3-cyclopropylpropanoyl)-1-(2-chloro-2-fluoroacetyl)hydrazinyl)propanamide (TFA salt) (40.0 mg, compound 62e) instead of 4-chloro-1H-indole-2-carboxylic acid and 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloro-2-fluoro-acetyl)amino]propanamide (74.61 mg, 53b). 5-Methyl-N-[rac-(1S)-2-[2-(3-amino-3-oxo-propyl)-2-(2-chloro-2-fluoro-acetyl)hydrazino]-1-(cyclopropylmethyl)-2-oxo-ethyl]isoxazole-3-carboxamide (3.2 mg, Example 77) was obtained as a white solid. 1H NMR (400 MHz, CD3OD) δ ppm: 6.84-6.38 (m, 2H), 4.57-4.42 (m, 1H), 4.03-3.59 (m, 2H), 2.59-2.49 (m, 5H), 1.95-1.68 (m, 2H), 0.92-0.82 (m, 1H), 0.59-0.51 (m, 2H), 0.25-0.18 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 418.3.
Compound 78a was prepared in analogy to Example 65, step 2 by using (S)-4,4-dimethyl-2-(pyrazine-2-carboxamido)pentanoic acid (1.3 g, compound 77c) instead of (S)-2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoic acid (120.0 mg, compound 65b). tert-butyl (S)-1-(3-amino-3-oxopropyl)-2-(4,4-dimethyl-2-(pyrazine-2-carboxamido)pentanoyl)hydrazine-1-carboxylate (700 mg, compound 78a) was obtained as a yellow solid. MS obsd. (ESI+) [(M+H)+]: 437.2.
Compound 78b was prepared in analogy to Example 65, step 3 by using tert-butyl (S)-1-(3-amino-3-oxopropyl)-2-(4,4-dimethyl-2-(pyrazine-2-carboxamido)pentanoyl)hydrazine-1-carboxylate (700 mg, compound 78a) instead of tert-butyl (S)-2-(2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoyl)-1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (100.0 mg, compound 65c). (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)pyrazine-2-carboxamide (400 mg, compound 78b) was obtained as a yellow solid.
MS obsd. (ESI+) [(M+H)+]: 337.1.
The title compound was prepared in analogy to Example 65, step 4 by using (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)pyrazine-2-carboxamide (400 mg, compound 78b) instead of (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (TFA salt) (75.0 mg, compound 65d). N-((2S)-1-(2-(3-amino-3-oxopropyl)-2-(2-chloro-2-fluoroacetyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)pyrazine-2-carboxamide (114.6 mg, compound 78) was obtained as a white solid. 1H NMR (400 MHz, CDCl3) 3 ppm: 9.38 (d, J=6.0 Hz, 1H), 8.81 (d, J=2.0 Hz, 1H), 8.60 (s, 1H), 8.15 (d, J=5.6 Hz, 1H), 6.56-6.05 (m, 3H), 4.67-4.56 (m, 1H), 3.82-3.60 (m, 3H), 2.71-2.55 (m, 2H), 2.05-1.95 (m, 1H), 1.83-1.71 (m, 1H), 1.03 (s, 9H). MS obsd. (ESI+) [(M+H)+]: 431.1.
To a solution of methyl (S)-2-((tert-butoxycarbonyl)amino)-3-cyclobutylpropanoate (350.0 mg) in DCM (4.0 mL) was added TFA (4.0 mL). After stirred at 25° C. for 1 hr, the mixture was concentrated in vacuo to afford methyl (S)-2-amino-3-cyclobutylpropanoate (TFA salt) (210 mg, compound 79a) as a colorless oil.
To a solution of methyl (S)-2-amino-3-cyclobutylpropanoate (TFA salt) (210.0 mg, compound 79a), pyrazine-2-carboxylic acid (165.77 mg) and DIEA (1.0 g) in DCM (5 mL) was added and T3P (1.3 g, 2 mmol, 50% in EtOAc). After stirred at 25° C. for 12 hrs, the reaction mixture was diluted with ethyl acetate (100 mL) and washed with brine (30 mL). The organic phase was dried over Na2SO4. The residue was purified by silica gel column, eluted with MeOH in DCM=0˜10% to afford methyl (S)-3-cyclobutyl-2-(pyrazine-2-carboxamido)propanoate (330 mg, compound 79b) as a colorless oil. MS obsd. (ESI+) [(M+H)+]: 264.2.
To a solution of methyl (S)-3-cyclobutyl-2-(pyrazine-2-carboxamido)propanoate (330.0 mg, compound 79b) in THF (4 mL) was added LiOH H2O (110.57 mg) in water (4 mL) at 0° C. After stirred at 0° C. for 2 hrs, the reaction mixture was diluted with EtOAc (100 mL), washed with HCl solution (1M 30 mL) and brine (30 mL) in turns. The organic phase was dried over anhydrous Na2SO4. After filtration and concentration, (S)-3-cyclobutyl-2-(pyrazine-2-carboxamido)propanoic acid (300 mg, compound 79c) was obtained as a white solid. MS obsd. (ESI+) [(M+H)+]: 250.2
Compound 79b was prepared in analogy to Example 65, step 2 by using (S)-3-cyclobutyl-2-(pyrazine-2-carboxamido)propanoic acid (300 mg, compound 79c) instead of (2S)-2-(1H-benzimidazole-2-carbonylamino)-4,4-dimethyl-pentanoic acid (compound 65b). tert-Butyl (S)-1-(3-amino-3-oxopropyl)-2-(3-cyclobutyl-2-(pyrazine-2-carboxamido)propanoyl)hydrazine-1-carboxylate (400 mg, compound 79d) was obtained as a white solid. MS obsd. (ESI+) [(M−Boc+H)+]: 355.3.
Compound 79e was prepared in analogy to Example 65, step 3 by using tert-butyl (S)-1-(3-amino-3-oxopropyl)-2-(3-cyclobutyl-2-(pyrazine-2-carboxamido)propanoyl)hydrazine-1-carboxylate (400 mg, compound 79d) instead of tert-butyl (S)-2-(2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoyl)-1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (100.0 mg, compound 65c). (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-3-cyclobutyl-1-oxopropan-2-yl)pyrazine-2-carboxamide (TFA salt) (160 mg, compound 79e) was obtained as a yellow oil. MS obsd. (ESI+) [(M+H)+]: 355.3.
The title compound was prepared in analogy to Example 65, step 4 by using (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-3-cyclobutyl-1-oxopropan-2-yl)pyrazine-2-carboxamide (TFA salt) (160 mg, compound 79e) instead of (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (TFA salt) (75.0 mg, compound 65d). N-((2S)-1-(2-(3-Amino-3-oxopropyl)-2-(2-chloro-2-fluoroacetyl)hydrazinyl)-3-cyclobutyl-1-oxopropan-2-yl)pyrazine-2-carboxamide (56.9 mg, Example 79) was obtained as a white solid. 1H NMR (400 MHz, CD3OD) δ ppm: 9.26-9.24 (m, 1H), 8.82 (t, J=2.4 Hz, 1H), 8.72 (d, J=1.2 Hz, 1H), 6.84-6.49 (m, 1H), 4.53-4.33 (m, 1H), 4.13-3.85 (m, 1H), 3.72-3.61 (m, 1H), 2.58-2.46 (m, 3H), 2.16-2.02 (m, 4H), 1.94-1.75 (m, 4H). MS obsd. (ESI+) [(M+H)+]:429.3.
The title compound was prepared in analogy to Example 53, Step 3 by using imidazo[1,2-a]pyridine-2-carboxylic acid and 3-(2-((S)-2-amino-3-cyclohexylpropanoyl)-1-(2-chloro-2-fluoroacetyl)hydrazinyl)propanamide (compound 56b) instead of 4-chloro-1H-indole-2-carboxylic acid and 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloro-2-fluoro-acetyl)amino]propanamide (compound 53b). N-((2S)-1-(2-(3-amino-3-oxopropyl)-2-(2-chloro-2-fluoroacetyl)hydrazinyl)-3-cyclohexyl-1-oxopropan-2-yl)imidazo[1,2-a]pyridine-2-carboxamide (2.02 mg, Example) was obtained as a white solid 1H NMR (400 MHz, CD3OD) δ ppm: 8.60-8.63 (m, 1H), 8.49 (d, J=8.4 Hz, 1H), 7.71-7.73 (m, 1H), 7.60-7.66 (m, 1H), 7.14-25 (m, 1H), 6.72a-6.85 (m, 1H), 4.41-4.43 (m, 0.5H), 3.88-4.05 (m, 1H), 3.61-3.71 (m, 1H), 2.46-2.64 (m, 2H), 1.69-1.86 (m, 7H), 1.49-1.59 (m, 1H), 1.23-1.34 (m, 3H), 0.98-1.09 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 495.0.
To a solution of m-aminoanisole (1.0 g) and ethyl bromoacetate (1.36 g) in ethanol (20 mL) was added CH3COONa (667.0 mg). After stirred at 90° C. for 12 hrs, the reaction mixture was concentrated in vacuo. The residue was purified by silica gel column, eluted with petroleum ether:EtOAc=10:1˜3:1 to afford ethyl 2-((3-methoxyphenyl)amino)acetate (1.5 g, compound 83a) as a yellow oil. MS obsd. (ESI+) [(M+H)+]: 210.0
To a solution of ethyl 2-(3-methoxyanilino)acetate (1.5 g, 83a) in THF (10 mL) was added NaOH (574.0 mg) in water (10 mL) at 0° C. After stirred at 20° C. for 2 hrs, the mixture was poured into HCl (1M, 15 mL) and extracted with EtOAc (30 mL) three times. The combined organic layer was washed with brine (40 mL), dried over anhydrous Na2SO4 and concentrated in vacuo to afford 2-((3-methoxyphenyl)amino)acetic acid (1.1 g, compound 83b) as a yellow oil. MS obsd. (ESI+) [(M+H)+]: 181.8.
The title compound was prepared in analogy to Example 53, Step 3 by using 2-((3-methoxyphenyl)amino)acetic acid instead of 4-chloro-1H-indole-2-carboxylic acid. 3-(1-(2-chloro-2-fluoroacetyl)-2-((S)-2-(2-((3-methoxyphenyl)amino)acetamido)-4-methylpentanoyl)hydrazinyl)propanamide (20 mg, Example 83) was obtained as a white solid.
Separation of compound 83 by chiral SFC afforded Example 83-A (faster eluting, 2.18 mg) and Example 83-B (slower eluting, 3.65 mg) with Neu-ETOH; B %: 30-30%; FlowRate (ml/min): 60) on DAICEL CHIRALPAK AD-H column
Example 83-A: 1H NMR (400 MHz, CD3OD) δ 8.09-8.12 (m, 1H), 7.03 (t, J=8.0 Hz, 1H), 6.47-6.80 (m, 1H), 6.15-6.33 (m, 3H), 4.20-4.46 (m, 1H), 3.82-4.02 (m, 2H), 3.49-3.77 (m, 5H), 2.35-2.49 (m, 2H), 1.48-1.75 (m, 3H), 0.91 (d, J=6.0 Hz, 3H), 0.87 (d, J=5.6 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 474.3. SFC: RT=4.058 min, ee %=100%.
Example 83-B: 1H NMR (400 MHz, CD3OD) δ 7.03 (t, J=8.0 Hz, 1H), 6.34-6.90 (m, 1H), 6.20-6.32 (m, 3H), 4.37-4.41 (m, 1H), 3.83-3.90 (m, 2H), 3.58-3.79 (m, 5H), 2.41-2.60 (m, 2H), 1.47-1.69 (m, 3H), 0.87-0.96 (m, 6H). MS obsd. (ESI+) [(M+H)+]: 474.3. SFC: RT=4.420 min, ee %=91.9%.
The title compound was prepared in analogy to Example 53, Step 3 by using alpha-toluenesulfonyl chloride and 3-(2-((S)-2-amino-3-cyclohexylpropanoyl)-1-(2-chloro-2-fluoroacetyl)hydrazinyl)propanamide (compound 56b) instead of 4-chloro-1H-indole-2-carboxylic acid and 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloro-2-fluoro-acetyl)amino]propanamide (compound 53b). 3-(1-(2-Chloro-2-fluoroacetyl)-2-((S)-3-cyclohexyl-2-(phenylmethylsulfonamido)propanoyl)hydrazinyl)propanamide (33.18 mg, Example 84) was obtained as a white solid. 1H NMR (400 MHz, CD3OD) δ ppm: 7.45-7.47 (m, 2H), 7.38-7.22 (m, 3H), 6.39-6.74 (m, 1H), 4.33-4.40 (m, 2H), 3.55-4.03 (m, 3H), 2.39-2.64 (m, 2H), 1.44-1.79 (m, 7H), 1.15-1.48 (m, 4H), 0.84-0.98 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 505.2.
The title compound was prepared in analogy to Example 53, Step 3 by using alpha-toluenesulfonyl chloride and 3-(2-((S)-2-amino-3-cyclopropylpropanoyl)-1-(2-chloro-2-fluoroacetyl)hydrazinyl)propanamide (TFA salt) (40.0 mg, compound 62e) instead of 4-chloro-1H-indole-2-carboxylic acid and 3-[[[(2S)-2-amino-4-methyl-pentanoyl]amino]-(2-chloro-2-fluoro-acetyl)amino]propanamide (74.61 mg, compound 53b). 3-[[[(2S)-2-(Benzylsulfonylamino)-3-cyclopropyl-propanoyl]amino]-(2-chloro-2-fluoro-acetyl)amino]propanamide (2.71 mg, Example 85) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm: 9.91 (br. s, 1H), 7.38-7.49 (m, 5H), 6.27-6.34 (m, 3H), 4.33 (s, 1H), 3.55-4.11 (m, 3H), 2.40-2.58 (m, 2H), 1.57-1.64 (m, 2H), 0.69-0.75 (m, 1H), 0.32-0.50 (m, 2H), 0.02-0.21 (m, 2H). MS obsd. (ESI+) [M+H]+: 463.1.
The title compound was prepared in analogy to Example 53, Step 3 by using (E)-3-phenylprop-2-enoic acid instead of 4-chloro-1H-indole-2-carboxylic acid 3-[(2-chloro-2-fluoro-acetyl)-[[(2S)-4-methyl-2-[[(E)-3-phenylprop-2-enoyl]amino]pentanoyl]amino]amino]propanamide (compound 86) was obtained as a yellow solid.
Separation of compound 86 by chiral SFC afforded Example 86-A (faster eluting, 8.13 mg) and Example 86-B (slower eluting, 11.9 mg) with Neu-ETOH; B %: 30-30%; flow rate 60 ml/min) on DAICEL CHIRALPAK AD-H column
Example 86-A: 1H NMR (400 MHz, CD3OD) δ ppm: 7.55-7.64 (m, 3H), 7.36-7.44 (m, 3H), 6.68-6.82 (m, 2H), 4.31-4.50 (m, 1H), 3.87-4.02 (m, 1H), 3.53-3.77 (m, 1H), 2.39-2.68 (m, 2H), 1.71-1.97 (m, 2H), 1.55-1.68 (m, 1H), 1.04 (d, J=6.8 Hz, 3H), 1.00 (d, J=6.8 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 441.3. SFC: RT=2.225 min, ee %=99.34%
Example 86-B: 1H NMR (400 MHz, CD3OD) δ ppm: 7.56-7.62 (m, 3H), 7.36-7.42 (m, 3H), 6.45-6.95 (m, 2H), 4.44-4.47 (m, 1H), 3.85-4.10 (m, 1H), 3.52-3.76 (m, 1H), 2.45-2.68 (m, 2H), 1.63-1.86 (m, 3H), 1.04 (d, J=6.0 Hz, 3H), 1.00 (d, J=6.0 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 441.3. SFC: RT=2.503 min, ee %=97.734%
The title compound was prepared in analogy to Example 53, Step 3 by using 1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid instead of 4-chloro-1H-indole-2-carboxylic acid. N-[(1S)-2-[2-(3-amino-3-oxo-propyl)-2-(2-chloro-2-fluoro-acetyl)hydrazino]-1-(cyclohexylmethyl)-2-oxo-ethyl]-1H-indole-2-carboxamide (compound 88) was obtained as a yellow solid.
Separation of compound 88 by chiral SFC afforded Example 88-A (faster eluting, 5.0 mg) and Example 88-B (slower eluting, 6.6 mg) with Neu-ETOH; B %: 30-30%; flow rate 60 ml/min on DAICEL CHIRALPAK AD-H column
Example 88-A: 1H NMR (400 MHz, CD3OD) δ ppm: 8.86 (s, 1H), 8.16 (d, J=5.6 Hz, 1H), 7.83 (d, J=5.6 Hz, 1H), 7.36 (s, 1H), 6.89 (d, J=50 Hz, 1H), 4.47-4.65 (m, 1H), 3.93-4.19 (m, 1H), 3.65-3.73 (m, 1H), 2.44-2.60 (m, 2H), 1.80-1.93 (m, 2H), 1.65-1.72 (m, 1H), 1.07 (d, J=6.4 Hz, 3H), 1.02 (d, J=6.4 Hz, 3H). MS obsd. (ESI+), (M+H)+: 455.1. SFC: RT=0.715 min, ee %=81.2%
Example 88-B: 1H NMR (400 MHz, CD3OD) δ ppm: 9.01 (s, 1H), 8.23-8.27 (m, 1H), 8.80-8.94 (m, 1H), 7.52 (s, 1H), 6.47-6.89 (m, 1H), 4.59-4.62 (m, 1H), 3.95-4.22 (m, 1H), 3.65-3.75 (m, 1H), 2.44-2.60 (m, 2H), 1.76-1.93 (m, 3H), 1.07 (d, J=6.4 Hz, 3H), 1.02 (d, J=6.4 Hz, 3H). MS obsd. (ESI+). (M+H)+: 455.1. SFC: RT=1.413 min, ee %=63%
The title compound was prepared in analogy to Example 53, Step 3 by using 5-methoxy-1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid instead of 4-chloro-1H-indole-2-carboxylic acid and. 5-Methoxy-N-[rac-(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]-1H-pyrrolo[2,3-c]pyridine-2-carboxamide (11.1 mg, Example 89) was obtained as a yellow solid. 51H NMR (400 MHz, CD3OD) δ ppm: 8.56 (d, J=2.8 Hz, 1H), 7.45 (s, 1H), 7.34 (s, 1H), 6.45-6.88 (m, 1H), 4.45-4.62 (m, 1H), 4.08 (s, 3H), 3.94-4.05 (m, 1H), 3.57-3.76 (m, 1H), 2.48-2.60 (m, 2H), 1.71-1.92 (m, 3H), 1.02-1.08 (m, 6H). MS obsd. (ESI+) [(M+H)+]: 485.1.
The title compound was prepared in analogy to Example 53, Step 3 by using pyrazolo[1,5-a]pyridine-2-carboxylic acid instead of 4-chloro-1H-indole-2-carboxylic acid. N,N-[rac-(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]pyrazolo[1,5-a]pyridine-2-carboxamide (mg, Example 90) was obtained as a yellow solid.
Separation of compound 90 by chiral SFC afforded Example 90-A (faster eluting, 5.0 mg) and Example 90-B (slower eluting, 6.6 mg) with Neu-IPA B: 50 Gradient Time (min): 4.2; 30 FlowRate (ml/min): 70 on DAICEL CHIRALPAK IC column
Example 90-A: 1H NMR (400 MHz, CD3OD) δ ppm: 8.61 (d, J=6.4 Hz, 1H), 7.72 (d, J=9.2 Hz, 1H), 7.25-7.29 (m, 1H), 7.08 (s, 1H), 6.99-7.03 (m, 1H), 6.46-6.60 (m, 1H), 4.63-4.70 (m, 1H), 3.92-4.01 (m, 1H), 3.65-3.75 (m, 1H), 2.45-2.59 (m, 2H), 1.74-1.90 (m, 3H), 1.05 (d, J=6.4 Hz, 3H), 1.02 (d, J=6.4 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 455.3. SFC: RT=1.909 min, ee %=96.9%.
Example 90-B: 1H NMR (400 MHz, CD3OD) δ ppm: 8.61 (d, J=6.4 Hz, 1H), 7.72 (d, J=8.8 Hz, 1H), 7.25-7.29 (m, 1H), 7.08 (s, 1H), 7.01-7.07 (m, 1H), 6.82 (d, J=50.0 Hz, 1H), 4.54-4.58 (m, 1H), 3.93-3.97 (m, 1H), 3.66-3.70 (m, 1H), 2.46-2.61 (m, 2H), 1.70-1.90 (m, 3H), 1.06 (d, J=6.0 Hz, 3H), 1.03 (d, J=6.4 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 455.3. SFC: RT=2.104 min, ee %=96.5%
The title compound was prepared in analogy to Example 53, Step 3 by using 1H-pyrrolo[3,2-b]pyridine-2-carboxylic acid instead of 4-chloro-1H-indole-2-carboxylic acid. N-[(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]-1H-pyrrolo[3,2-b]pyridine-2-carboxamide (mg, Example 91) was obtained as a yellow solid.
Separation of compound 90 by chiral SFC afforded Example 90-A (faster eluting, 5.0 mg) and Example 90-B (slower eluting, 6.6 mg) with 45% MeOH (Neu) in Supercritical CO2 flow rate 70 g/min Cycle Time: 3.0 min, total time: 50 min Single injection volume: 2.0 ml Back Pressure: 100 bar to keep the CO2 in Supercritical flow on DAICEL CHIRALPAK IG column
Example 91-A: 1H NMR (400 MHz, CD3OD) δ ppm: 8.45-8.51 (m, 1H), 8.05 (d, J=8.0 Hz, 1H), 7.36-7.50 (m, 2H), 6.50-7.09 (m, 1H), 4.59-4.62 (m, 1H), 3.89-4.07 (m, 1H), 3.65-3.80 (m, 1H), 2.47-2.60 (m, 2H), 1.76-2.00 (m, 3H), 1.09 (d, J=6.4 Hz, 3H), 1.04 (d, J=6.4 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 455.1. SFC: RT=2.066 min, ee %=97.93%
Example 91-B: 1H NMR (400 MHz, CD3OD) δ ppm: 8.02-8.04 (m, 1H), 7.36-7.50 (m, 2H), 6.89 (d, J=50.4 Hz, 1H), 4.45-4.62 (m, 1H), 3.90-4.12 (m, 1H), 3.62-3.77 (m, 1H), 2.47-2.60 (m, 2H), 1.82-1.98 (m, 2H), 1.65-1.80 (m, 1H), 1.07 (d, J=6.0 Hz, 3H), 1.03 (d, J=6.0 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 455.1. SFC: RT=2.304 min, ee %=97.93%
The tide compound was prepared in analogy to Example 53, Step 3 by using imidazo[1,2-a]pyridine-2-carboxylic acid (Wuxi catalog, CAS number: 64951-08-2) instead of 4-chloro-1H-indole-2-carboxylic acid and N-[rac-(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]imidazo[1,2-a]pyridine-2-carboxamide (compound 95) was obtained as a yellow solid.
Separation of compound 95 by chiral SFC afforded Example 95-A (faster eluting, 5.0 mg) and Example 95-B (slower eluting, 6.6 mg) with Neu-IPA; B %: 50-50%; FlowRate (ml/min): 70) on DAICEL CHIRALPAK IC column.
Example 95-A: 1H NMR (400 MHz, CD3OD) δ ppm: 8.53 (d, J=6.8 Hz, 1H), 8.41 (s, 1H), 7.65 (d, J=9.2 Hz, 1H), 7.46-7.49 (m, 1H), 7.05 (t, J=6.8 Hz, 1H), 6.53 (d, J=50.4 Hz, 1H), 4.51-4.60 (m, 1H), 3.89-4.05 (m, 1H), 3.59-3.76 (m, 1H), 2.45-2.65 (m, 2H), 1.75-1.90 (m, 3H), 1.06 (d, J=6.0 Hz, 3H), 1.02 (d, J=5.6 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 455.3. SFC: RT=1.370 min, ee %=98.4%
Example 95-B: 1H NMR (400 MHz, CD3OD) δ ppm: 8.55 (d, J=6.0 Hz, 1H), 8.47 (s, 1H), 7.64-7.76 (m, 1H), 7.47-7.57 (m, 1H), 7.06-7.09 (m, 1H), 6.61-6.79 (m, 1H), 4.55-4.60 (m, 1H), 3.79-4.05 (m, 1H), 3.58-3.76 (m, 1H), 2.48-2.58 (m, 2H), 1.76-1.89 (m, 3H), 1.06 (d, J=6.4 Hz, 3H), 1.02 (d, J=6.0 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 455.1. SFC: RT=2.148 min, ee %=96.6%
The title compound was prepared in analogy to Example 53, Step 3 by using 1H-imidazole-2-carboxylic acid instead of 4-chloro-1H-indole-2-carboxylic acid. N-[rac-(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]-1H-imidazole-2-carboxamide (2 mg, compound 96) was obtained as a yellow solid. 1H NMR (400 MHz, CD3OD) δ ppm: 7.14-7.28 (m, 2H), 6.47-6.88 (m, 1H), 4.39-4.89 (m, 1H), 3.71-4.05 (m, 2H), 2.41-2.60 (m, 2H), 1.72-1.95 (m, 3H), 0.99-1.05 (m, 6H). MS obsd. (ESI+) [(M+H)+]: 405.1.
The title compound was prepared in analogy to Example 53, Step 3 by using 5-methylisoxazole-3-carboxylic acid instead of 4-chloro-1H-indole-2-carboxylic acid. 5-Methyl-N-[rac-(1S)-1-[[(3-amino-3-oxo-propyl)-(2-chloro-2-fluoro-acetyl)amino]carbamoyl]-3-methyl-butyl]isoxazole-3-carboxamide (20 mg, compound 97) was obtained as a yellow solid.
Separation of compound 97 by chiral SFC afforded Example 97-A (faster eluting, 5.0 mg) and Example 97-B (slower eluting, 6.6 mg) with Neu-IPA; B %: 50-50%; flow rate 70 ml/min on DAICEL CHIRALPAK IC column.
Example 97-A: 1H NMR (400 MHz, CD3OD) δ ppm: 6.50-7.02 (m, 2H), 4.51-4.56 (m, 1H), 3.50-4.09 (m, 2H), 2.50-2.60 (m, 2H), 2.49 (s, 3H), 1.64-1.92 (m, 3H), 1.03 (d, J=6.0 Hz, 3H), 0.99 (d, J=6.0 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 420.3. SFC: RT=1.032 min, ee %=97.82%.
Example 97-B: 1H NMR (400 MHz, CD3OD) δ ppm: 6.76 (d, J=50.4 Hz, 1H), 6.49 (s, 1H), 4.51-4.36 (m, 1H), 3.85-4.06 (m, 1H), 3.51-3.76 (m, 1H), 2.51-2.60 (m, 2H), 2.49 (s, 3H), 1.74-1.88 (m, 2H), 1.61-1.73 (m, 1H), 1.03 (d, J=6.4 Hz, 3H), 1.00 (d, J=6.4 Hz, 3H). MS obsd. (ESI+) [(M+H)+]: 420.3. SFC: RT=1.116 min, ee %=83.25%
To a solution of ethyl (Z)-2-chloro-2-(hydroxyimino)acetate (3.0 g) in ethyl acetate (50 mL) was added 2-bromo-3,3,3-trifluoroprop-1-ene (10.39 g, Shanghai Haohong, CAS Number: 1514-82-5) and NaHCO3 (5.49 g). After stirred at 20° C. for 12 hrs, the reaction mixture was concentrated in vacuo at 30° C. to afford ethyl 5-(trifluoromethyl)isoxazole-3-carboxylate (3.75 g, compound 98a) as a yellow oil. 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.93 (s, 1H), 4.44-4.39 (m, 2H), 1.34 (t, J=7.2 Hz, 3H)
To a solution of ethyl 5-(trifluoromethyl)isoxazole-3-carboxylate (3.75 g, compound 98a) in methanol (40 mL) was added the solution of NaOH (1.58 g) in water (40 mL) at 0° C. After stirred at 20° C. for 1 hr, the reaction mixture was concentrated in vacuo. The residue was dissolved with EtOAc (80 mL) and washed with HCl (1M 40 mL). The aqueous phase was extracted with EtOAc (80 mL) six times. The combined organic phase was dried with anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column, eluted with EtOAc in petroleum ether=30˜50% to afford 5-(trifluoromethyl)isoxazole-3-carboxylic acid (2.3 g, compound 98b) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm: 7.39 (br. s, 1H), 7.18 (s, 1H).
To a solution of 5-(trifluoromethyl)isoxazole-3-carboxylic acid (700.0 mg, compound 98b) in DCM (10 mL) was added DMF (0.05 mL) and oxalyl chloride (588.84 mg) dropwise at 0° C. After stirred at 20° C. for 1 hr, the reaction mixture was concentrated in vacuo at 20° C. to afford 5-(trifluoromethyl)isoxazole-3-carbonyl chloride (771 mg, compound 98c) as a yellow oil.
To a solution of tert-butyl 2-((S)-2-amino-3-(1-methylcyclopropyl)propanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (680.0 mg, compound 70b) and DIEA (1487.7 mg) in THF (25 mL) was added 5-(trifluoromethyl)isoxazole-3-carbonyl chloride (765.57 mg, compound 98c) at 0° C. After stirred at 0° C. for 1 hr, the reaction mixture was extracted with EtOAc (30 mL) three times. The combined organic layers were dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by reversed-phase flash, eluted with MeCN in water (0.1% TFA)=0˜70% to afford tert-butyl 2-((S)-3-(1-methylcyclopropyl)-2-(5-(trifluoromethyl)isoxazole-3-carboxamido)propanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (516 mg, compound 98d) as a white solid. MS obsd. (ESI+) [(M+H)+]: 518.2.
Compound 98e was prepared in analogy to Example 65, step 3 by using tert-butyl 2-((S)-3-(1-methylcyclopropyl)-2-(5-(trifluoromethyl)isoxazole-3-carboxamido)propanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (516 mg, compound 98d) instead of tert-butyl (S)-2-(2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoyl)-1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (100.0 mg, compound 65c). N—((S)-3-(1-Methylcyclopropyl)-1-oxo-1-(2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)propan-2-yl)-5-(trifluoromethyl)isoxazole-3-carboxamide (88 mg, compound 98e) was obtained as a white solid. MS obsd. (ESI+) [(M+H)+]: 418.0.
The title compound was prepared in analogy to Example 65, step 4 by using N—((S)-3-(1-methylcyclopropyl)-1-oxo-1-(2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)propan-2-yl)-5-(trifluoromethyl)isoxazole-3-carboxamide (88 mg, compound 98e) and (R)-2-chloro-2-fluoroacetyl chloride instead of (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (TFA salt) (75.0 mg, compound 65d) and 2-chloro-2-fluoroacetic acid. N—((S)-1-(2-((R)-2-Chloro-2-fluoroacetyl)-2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)-3-(1-methylcyclopropyl)-1-oxopropan-2-yl)-5-(trifluoromethyl)isoxazole-3-carboxamide (36.3 mg, Example 98) was obtained as a white solid.
1H NMR (400 MHz, CDCl3) δ ppm: 10.08 (br. s, 1H), 7.40-7.39 (m, 1H), 7.17 (s, 1H), 6.53 (d, J=50 Hz, 1H), 5.72 (d, J=3.2 Hz, 1H), 4.65-4.59 (m, 1H), 4.46-4.44 (m, 1H), 3.43 (dd, J1=4.4 Hz, J2=9.2 Hz, 2H), 2.88-2.84 (m, 2H), 2.42-2.38 (m, 1H), 2.18-2.04 (m, 1H), 1.89-1.73 (m, 1H), 1.62-1.52 (m, 1H), 1.13 (s, 3H), 0.53-0.43 (m, 2H), 0.41-0.34 (m, 2H).
MS obsd. (ESI+) [M+H]+: 512.0.
Compound 99a was prepared in analogy to Example 71, step 1 by using 5-(trifluoromethyl)isoxazole-3-carbonyl chloride instead of 5-methylisoxazole-3-carbonyl chloride. Methyl (S)-3-(bicyclo[1.1.1]pentan-1-yl)-2-(5-(trifluoromethyl)isoxazole-3-carboxamido) propanoate (940 mg, compound 99a) was obtained as a colorless oil. MS obsd. (ESI+) [(M+H)+]: 333.0.
Compound 99b was prepared in analogy to Example 71, step 2 by using methyl (S)-3-(bicyclo[1.1.1]pentan-1-yl)-2-(5-(trifluoromethyl)isoxazole-3-carboxamido) propanoate (890.0 mg, compound 99b) instead of methyl (S)-3-(bicyclo 1.1.1 pentan-1-yl)-2-(5-methylisoxazole-3-carboxamido)propanoate (compound 71a). (S)-3-(Bicyclo[1.1.1]pentan-1-yl)-2-(5-(trifluoromethyl)isoxazole-3-carboxamido)propanoic acid (850 mg, compound 99b) was obtained as a white solid. MS obsd. (ESI+) [(M+H)+]: 319.0
Compound 99c was prepared in analogy to Example 65, step 2 by using (S)-3-(bicyclo[1.1.1]pentan-1-yl)-2-(5-(trifluoromethyl)isoxazole-3-carboxamido)propanoic acid (850 mg, compound 99b) and tert-butyl (S)-1-((2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (1.0 g, Intermediate AF) instead of (S)-2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoic acid (120.0 mg, compound 65b) and tert-butyl 1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate. tert-Butyl 2-((S)-3-(bicyclo[1.1.1]pentan-1-yl)-2-(5-(trifluoromethyl)isoxazole-3-carboxamido)propanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (580 mg, compound 99c) was obtained as a yellow solid.
MS obsd. (ESI+) [(M+H)+]: 530.2.
Compound 99d was prepared in analogy to Example 65, step 3 by using tert-butyl 2-((S)-3-(bicyclo[1.1.1]pentan-1-yl)-2-(5-(trifluoromethyl)isoxazole-3-carboxamido)propanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (580 mg, compound 99c) instead of tert-butyl (S)-2-(2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoyl)-1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (100.0 mg, compound 65c). N—((S)-3-(bicyclo[1.1.1]pentan-1-yl)-1-oxo-1-(2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl) propan-2-yl)-5-(trifluoromethyl)isoxazole-3-carboxamide (400 mg, compound 99d) was obtained as a yellow solid. MS obsd. (ESI+) [(M+H)+]: 430.2.
The title compound was prepared in analogy to Example 65, step 4 by using afford N—((S)-3-(bicyclo[1.1.1]pentan-1-yl)-1-oxo-1-(2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl) propan-2-yl)-5-(trifluoromethyl)isoxazole-3-carboxamide (400 mg, compound 99d) and (R)-2-chloro-2-fluoroacetyl chloride instead of (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (TFA salt) (75.0 mg, compound 65d) and 2-chloro-2-fluoroacetic acid. N—((S)-3-(Bicyclo[1.1.1]pentan-1-yl)-1-(2-((R)-2-chloro-2-fluoroacetyl)-2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)-1-oxopropan-2-yl)-5-(trifluoromethyl) isoxazole-3-carboxamide (162.2 mg, Example 99) was obtained as a yellow solid. 1H NMR (400 MHz, CD3OD) δ ppm: 7.46 (s, 1H), 6.80-6.55 (m, 1H), 4.57-4.40 (m, 1H), 4.06 (dd, J1=8.4 Hz, J2=13.6 Hz, 1H), 3.44 (dd, J1=5.2 Hz, J2=13.6 Hz, 1H), 3.40-3.32 (m, 2H), 2.78-2.64 (m, 1H), 2.46 (s, 1H), 2.36-2.22 (m, 1H), 2.09-2.03 (m, 2H), 2.00-1.90 (m, 1H), 1.87-1.72 (m, 6H). MS obsd. (ESI+) [(M+H)+]: 524.1.
To a solution of ethyl 5-(hydroxymethyl)isoxazole-3-carboxylate (7.0 g, Bidepharm, CAS number: 123770-62-7), TEA (13.04 g) and DMSO (15.98 g) in DCM (110 mL) was added pyridine-SO3 complex (39.06 g, 245.4 mmol) at 0° C. under N2 protection. After stirred at 20° C. for 12 hrs, the reaction mixture was concentrated in vacuo. The residue was diluted with EtOAc (200 mL) and washed with H2O (100 mL). The aqueous phase was extracted with EtOAc (100 mL) two times. The combined organic phase was washed with brine (50 mL) three times, dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified by silica gel column, eluted with EtOAc in petroleum ether=0˜20% to afford ethyl 5-formylisoxazole-3-carboxylate (4 g, compound 100a) as a yellow oil. 1H NMR (400 MHz, CDCl3) δ ppm: 10.03 (s, 1H), 7.37 (s, 1H), 4.49 (q, J=6.8 Hz, 2H), 1.45 (t, J=6.8 Hz, 3H).
To a solution of ethyl 5-formylisoxazole-3-carboxylate (4.0 g, 23.65 mmol, compound 100a) in DCM (60 mL) was added DAST (11.44 g) at −40° C. under N2 protection. After stirred at 20° C. for 16 hrs, the reaction mixture was poured into ice water (60 mL) and basified with sat. aq. solution of NaHCO3 until pH=9. The resulting mixture was extracted with DCM (60 mL) three times. The combined organic phase was dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by column (eluted with EtOAc in petroleum ether=10˜20%) to afford ethyl 5-(difluoromethyl)isoxazole-3-carboxylate (2.5 g, compound 100b) as a yellow oil. 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.53-7.27 (m, 2H), 4.39 (q, J=12 Hz, 2H), 1.33 (t, J=12 Hz, 3H)
To a solution of ethyl 5-(difluoromethyl)isoxazole-3-carboxylate (2.5 g, 13.08 mmol, compound 100b) in methanol (30 mL) was added NaOH (1.15 g) in water (30 mL) at 0° C. After stirred at 0° C. for 1 hr, the reaction mixture was concentrated in vacuo to remove MeOH. The residue was dissolved with EtOAc (50 mL) and washed with HCl (1 M 30 mL). The aqueous phase was extracted with EtOAc (50 mL) six times. The combined organic phase was dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified by silica gel column, eluted with EtOAc in petroleum ether=10˜35% to afford 5-(difluoromethyl)isoxazole-3-carboxylic acid (1.6 g, 9.81 mmol, compound 100c) as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm: 7.05 (s, 1H), 6.83 (t, J=53.2 Hz, 1H).
To a solution of 5-(difluoromethyl)isoxazole-3-carboxylic acid (4.23 g, compound 100c) in DCM (50 mL) was added DMF (0.2 mL) and oxalyl chloride (3.98 g, 31.37 mmol) dropwise at 0° C. After stirred at 20° C. for 1 hr, the reaction mixture was concentrated in vacuo below 20° C. to afford 5-(difluoromethyl)isoxazole-3-carbonyl chloride (4.6 g, compound 100d) as a yellow oil.
Compound 100e was prepared in analogy to Example 65, step 2 by using 5-(difluoromethyl)isoxazole-3-carbonyl chloride (4.6 g, compound 100d) and tert-butyl (S)-1-((2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (2.71 g, Intermediate AF) instead of (S)-2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoic acid (120.0 mg, compound 65b) and tert-butyl 1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate. tert-Butyl 2-((S)-2-(5-(difluoromethyl)isoxazole-3-carboxamido)-3-(1-methylcyclopropyl)propanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (3.9 g, 7.81 mmol, compound 100e) was obtained as a yellow solid. MS obsd. (ESI+) [(M+H)+]: 500.2.
Compound 100e was prepared in analogy to Example 65, step 3 by using tert-butyl 2-((S)-2-(5-(difluoromethyl)isoxazole-3-carboxamido)-3-(1-methylcyclopropyl)propanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (3.9 g, compound 100d) instead of ten-butyl (S)-2-(2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoyl)-1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (100.0 mg, compound 65c). 5-(Difluoromethyl)-N—((S)-3-(1-methylcyclopropyl)-1-oxo-1-(2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)propan-2-yl)isoxazole-3-carboxamide (2.8 g, compound 100e) was obtained as a white solid. MS obsd. (ESI+) [(M+H)+]: 400.1.
The title compound was prepared in analogy to Example 65, step 4 by using 5-(difluoromethyl)-N—((S)-3-(1-methylcyclopropyl)-1-oxo-1-(2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)propan-2-yl)isoxazole-3-carboxamide (2.8 g, compound 100e) and (R)-2-chloro-2-fluoroacetyl chloride instead of (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (TFA salt) (75.0 mg, compound 65d) and 2-chloro-2-fluoroacetic acid. N—((S)-1-(2-((R)-2-Chloro-2-fluoroacetyl)-2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)-3-(1-methylcyclopropyl)-1-oxopropan-2-yl)-5-(difluoromethyl)isoxazole-3-carboxamide (1941 mg, Example 100) was obtained as a white solid. 1H NMR (400 MHz, CDCl3) δ ppm: 10.16 (br. s, 1H), 7.47 (d, J=3.6 Hz, 1H), 7.04 (s, 1H), 6.80 (t, J=53.6 Hz, 1H), 6.57-6.36 (m, 1H), 5.94 (s, 1H), 4.75-4.63 (m, 1H), 4.43 (d, J=9.6 Hz, 1H), 3.43-3.39 (m, 2H), 2.99-2.76 (m, 2H), 2.41-2.36 (m, 1H), 2.12-2.09 (m, 1H), 1.92-1.79 (m, 1H), 1.59-1.53 (m, 1H), 1.13 (s, 3H), 0.50-0.42 (m, 2H), 0.39-0.33 (m, 2H). MS obsd. (ESI+) [(M+H)+]: 494.1.
Compound 101a was prepared in analogy to Example 65, step 1 by using (2S)-2-amino-5-methyl-hexanoic acid (Energy chemical, CAS number: 31872-98-7) and 5-methylisoxazole-3-carbonyl chloride and instead of (S)-2-amino-4,4-dimethylpentanoic acid (TFA salt) (compound 65a) and 1H-benzimidazole-2-carbonyl chloride. (S)-5-methyl-2-(5-methylisoxazole-3-carboxamido)hexanoic acid (800 mg, compound 101a) was obtained as a white solid. MS obsd. (ESI+) [M+H]+: 254.8.
Compound 101b was prepared in analogy to Example 65, step 2 by using (S)-5-methyl-2-(5-methylisoxazole-3-carboxamido)hexanoic acid (800 mg, compound 101a) and tert-butyl (S)-1-((2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (2.71 g, Intermediate AF) instead of (S)-2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoic acid (120.0 mg, compound 65b) and tert-butyl 1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate. tert-Butyl 2-((S)-5-methyl-2-(5-methylisoxazole-3-carboxamido)hexanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (800 mg, compound 101b) was obtained as a yellow solid. MS obsd. (ESI+) [M+H]+: 466.2.
Compound 101c was prepared in analogy to Example 65, step 3 by using tert-butyl 2-((S)-5-methyl-2-(5-methylisoxazole-3-carboxamido)hexanoyl)-1-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazine-1-carboxylate (800 mg, compound 101b) instead of tert-butyl (S)-2-(2-(1H-benzo[d]imidazole-2-carboxamido)-4,4-dimethylpentanoyl)-1-(3-amino-3-oxopropyl)hydrazine-1-carboxylate (compound 65c). 5-Methyl-N—((S)-5-methyl-1-oxo-1-(2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)hexan-2-yl)isoxazole-3-carboxamide (620 mg, compound 101c) was obtained as a white solid. MS obsd. (ESI+) [M+H]+:366.3.
The title compound was prepared in analogy to Example 65, step 4 by using 5-methyl-N—((S)-5-methyl-1-oxo-1-(2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)hexan-2-yl)isoxazole-3-carboxamide (620 mg, compound 101c) and (R)-2-chloro-2-fluoroacetyl chloride instead of (S)—N-(1-(2-(3-amino-3-oxopropyl)hydrazinyl)-4,4-dimethyl-1-oxopentan-2-yl)-1H-benzo[d]imidazole-2-carboxamide (TFA salt) (75.0 mg, compound 65d) and 2-chloro-2-fluoroacetic acid. N—((S)-1-(2-((R)-2-Chloro-2-fluoroacetyl)-2-(((S)-2-oxopyrrolidin-3-yl)methyl)hydrazinyl)-5-methyl-1-oxohexan-2-yl)-5-methylisoxazole-3-carboxamide (479.2 mg, Example 101) was obtained as a white solid. 1H NMR (400 MHz, CD3OD) δ ppm: 6.80 (d, J=50.4 Hz, 1H), 6.50 (s, 1H), 4.45-4.24 (m, 1H), 4.15-4.09 (m, 1H), 3.43-3.33 (m, 3H), 2.83-2.64 (m, 1H), 2.49 (s, 3H), 2.40-2.24 (m, 1H), 2.08-1.80 (m, 3H), 1.65-1.59 (m, 1H), 1.43-1.38 (m, 1H), 1.34-1.23 (m, 1H), 0.95 (d, J=6.4 Hz, 6H). MS obsd. (ESI+) [M+H]+: 460.1.
The full-length gene encoding SARS-CoV-2 3CLpro was optimized and synthesized for Escherichia coli (E. coli) BL21 (DE3) expression, with the protein sequence of
The method of cloning and producing authentic SARS-CoV-2 3CLpro was followed by the protocol published for SARS-CoV 3CLpro previously (Grum-Tokars V. et al. “Evaluating the 3C-like protease activity of SARS-Coronavirus: Recommendations for standardized assays for drug discovery.” Virus Res. 2008 April; 133(1):63-73).
For the SARS-CoV-2 3CLpro assay, 2 μL of 0.02 μM recombinant SARS-CoV-2 3CL protease were mixed with serial dilutions of each compound in 4 μL assay buffer containing 40 mM HEPES, pH=8.0, 1 mM CHAPS, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP in a well of a 384-well plate and pre-incubated at rt for 1 hr. The custom-synthesized fluorogenic 3CLpro peptide substrate used in the assay is as follows: FAM-KTSAVLQSGFRKMEK-TAMRA (peptide sequence according to SEQ ID NO: 3). This FRET-based substrate contains a FAM fluorophore attached at the N-terminus of a canonical 3CLpro peptide substrate. The fluorophore is internally quenched by the TAMRA. The reaction was initiated by the addition of 10 μL of the substrate with a final concentration of 15 μM and each well was incubated at RT for 30 mins. The final concentration of the protease used at the assay was 25 nM and each compound was at a final concentration range of 100-0.0016 μM. The IC50 value which is the value causing 50% inhibition of the catalytic activity of the SARS-CoV 3CLpro was calculated by four parameters equation analysis.
SARS-CoV 3CLpro Inhibition Assay
The SARS-CoV 3CLpro was expressed in E. coli BL21 (DE3) with the protein sequence of
For the SARS-CoV 3CLpro assay, 2 μL of 0.02 μM recombinant SARS-CoV 3CL protease were mixed with serial dilutions of each compound in 4 μL assay buffer containing 40 mM HEPES, pH=8.0, 1 mM CHAPS, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP in a well of a 384-well plate and pre-incubated at rt for 1 hr. The custom-synthesized fluorogenic 3CLpro peptide substrate used in the assay is as follows: FAM-KTSAVLQSGFRKMEK-TAMRA (peptide sequence according to SEQ ID NO: 3). This FRET-based substrate contains a FAM fluorophore attached at the N-terminus of a canonical 3CLpro peptide substrate. The fluorophore is internally quenched by the TAMRA. The reaction was initiated by the addition of 10 μL of the substrate with a final concentration of 15 μM and each well was incubated at RT for 30 mins. The final concentration of the protease used at the assay was 25 nM and each compound was at a final concentration range of 100-0.0016 μM. The IC50 value which is the value causing 50% inhibition of the catalytic activity of the SARS-CoV 3CLpro was calculated by four parameters equation analysis.
MERS-CoV 3CLpro Inhibition Assay
The MERS-CoV 3CLpro was expressed in E. coli BL21 (DE3) with the protein sequence of
For the MERS-CoV 3CLpro assay, 2 μL of 0.02 μM recombinant MERS-CoV 3CL protease were mixed with serial dilutions of each compound in 4 μL assay buffer containing 40 mM HEPES, pH=8.0, 1 mM CHAPS, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP in a well of a 384-well plate and pre-incubated at rt for 1 hr. The custom-synthesized fluorogenic 3CLpro peptide substrate used in the assay is as follows: FAM-KTSAVLQSGFRKMEK-TAMRA (peptide sequence according to SEQ ID NO: 3). This FRET-based substrate contains a FAM fluorophore attached at the N-terminus of a canonical 3CLpro peptide substrate. The fluorophore is internally quenched by the TAMRA. The reaction was initiated by the addition of 10 μL of the substrate with a final concentration of 15 μM and each well was incubated at RT for 30 mins. The final concentration of the protease used at the assay was 25 nM and each compound was at a final concentration range of 100-0.0016 μM. The IC50 value which is the value causing 50% inhibition of the catalytic activity of the MERS-CoV 3CLpro was calculated by four parameters equation analysis.
HCoV-229E 3CLpro Inhibition Assay
The HCoV-229E 3CLpro was expressed in E. coli BL21 (DE3) with the protein sequence of
For the HCoV-229E 3CLpro assay, 2 μL of 0.02 μM recombinant HCoV-229E 3CL protease were mixed with serial dilutions of each compound in 4 μL assay buffer containing 40 mM HEPES, pH=8.0, 1 mM CHAPS, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP in a well of a 384-well plate and pre-incubated at rt for 1 hr. The custom-synthesized fluorogenic 3CLpro peptide substrate used in the assay is as follows: FAM-KTSAVLQSGFRKMEK-TAMRA (peptide sequence according to SEQ ID NO: 3). This FRET-based substrate contains a FAM fluorophore attached at the N-terminus of a canonical 3CLpro peptide substrate. The fluorophore is internally quenched by the TAMRA. The reaction was initiated by the addition of 10 μL of the substrate with a final concentration of 15 μM and each well was incubated at RT for 30 mins. The final concentration of the protease used at the assay was 25 nM and each compound was at a final concentration range of 100-0.0016 μM. The IC50 value which is the value causing 50% inhibition of the catalytic activity of the HCoV-229E 3CLpro was calculated by four parameters equation analysis.
HCoV-OC43 3CLpro Inhibition Assay
The HCoV-OC43 3CLpro was expressed in E. coli BL21 (DE3) with the protein sequence of
For the HCoV-OC43 3CLpro assay, 2 μL of 0.02 μM recombinant HCoV-OC43 3CL protease were mixed with serial dilutions of each compound in 4 μL assay buffer containing 40 mM HEPES, pH=8.0, 1 mM CHAPS, 150 mM NaCl, 1 mM EDTA, 1 mM TCEP in a well of a 384-well plate and pre-incubated at rt for 1 hr. The custom-synthesized fluorogenic 3CLpro peptide substrate used in the assay is as follows: FAM-KTSAVLQSGFRKMEK-TAMRA (peptide sequence according to SEQ ID NO: 3). This FRET-based substrate contains a FAM fluorophore attached at the N-terminus of a canonical 3CLpro peptide substrate. The fluorophore is internally quenched by the TAMRA. The reaction was initiated by the addition of 10 μL of the substrate with a final concentration of 15 μM and each well was incubated at RT for 30 mins. The final concentration of the protease used at the assay was 25 nM and each compound was at a final concentration range of 100-0.0016 μM. The IC50 value which is the value causing 50% inhibition of the catalytic activity of the HCoV-OC43 3CLpro was calculated by four parameters equation analysis.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/111852 | Aug 2020 | CN | national |
| PCT/CN2021/108676 | Jul 2021 | CN | national |
This application is entitled to the priority benefit of International Application No. PCT/CN2020/111852 filed on Aug. 27, 2020 and International Application No. PCT/CN2021/108676 filed on Jul. 27, 2021, the disclosures of which are incorporated herein by reference.
