Patent Application
20230014907
This disclosure provides compounds that inhibit apolipoprotein L1 (APOL1) and methods of using those compounds to treat APOL1 mediated kidney disease, including focal segmental glomerulosclerosis (FSGS) and/or non-diabetic kidney disease (NDKD). In some embodiments, the FSGS and/or NDKD is associated with common APOL1 genetic variants (G1: S342G:I384M and G2: N388del:Y389del).
FSGS is a disease of the podocyte (glomerular visceral epithelial cells) responsible for proteinuria and progressive decline in kidney function. NDKD is a disease characterized by hypertension and progressive decline in kidney function. Human genetics support a causal role for the G1 and G2 APOL1 variants in inducing kidney disease. Individuals with two APOL1 risk alleles are at increased risk of developing end-stage kidney disease (ESKD), including FSGS, human immunodeficiency virus (HIV)-associated nephropathy, NDKD, arterionephrosclerosis, lupus nephritis, microalbuminuria, and chronic kidney disease. See, P. Dummer et al., Semin Nephrol. 35(3): 222-236 (2015).
APOL1 is a 44 kDa protein that is only expressed in humans, gorillas, and baboons. APOL1 is produced mainly by the liver and contains a signal peptide that allows for secretion into the bloodstream, where it circulates bound to a subset of high-density lipoproteins. APOL1 is responsible for protection against the invasive parasite, Trypanosoma brucei brucei (T. b. brucei). APOL1 G1 and G2 variants confer additional protection against trypanosoma species that cause sleeping sickness. Although normal plasma concentrations of APOL1 are relatively high and can vary at least 20-fold in humans, circulating APOL1 is not causally associated with kidney disease.
However, APOL1 in the kidney is thought to be responsible for the development of kidney diseases, including FSGS and NDKD. Under certain circumstances, APOL1 protein synthesis can be increased by approximately 200-fold by pro-inflammatory cytokines such as interferons or tumor necrosis factor-α. In addition, several studies have shown that APOL1 protein can form pH-gated Na+/K+ pores in the cell membrane, resulting in a net efflux of intracellular K+, ultimately resulting in activation of local and systemic inflammatory responses, cell swelling, and death.
The risk of ESKD is substantially higher in people of recent sub-Saharan African ancestry as compared to those of European ancestry. In the United States, ESKD is responsible for nearly as many lost years of life in women as from breast cancer and more lost years of life in men than from colorectal cancer. Currently, FSGS and NDKD are managed with symptomatic treatment (including blood pressure control using blockers of the renin angiotensin system), and patients with FSGS and heavy proteinuria may be offered high dose steroids. Corticosteroids induce remission in a minority of patients and are associated with numerous and, at times, severe side effects, and are often poorly tolerated. These patients, and particularly individuals of recent sub-Saharan African ancestry with two APOL1 risk alleles, experience faster disease progression leading to ESKD.
Thus, there is an unmet medical need for treatment for APOL1 mediated kidney diseases, including FSGS, NDKD, and ESKD. In view of evidence that APOL1 plays a causative role in inducing and accelerating the progression of kidney disease, inhibition of APOL1 should have a positive impact on patients with APOL1 mediated kidney disease, particularly those who carry two APOL1 risk alleles (i.e., are homozygous or compound heterozygous for the G1 or G2 alleles).
One aspect of the disclosure provides at least one entity (e.g., at least one compound, deuterated derivative, or pharmaceutically acceptable salt) chosen from compounds of Formula I:
deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing, which can be employed in the treatment of diseases mediated by APOL1, such as, e.g., FSGS and NDKD, wherein:
(i) R is selected from —C(O)NR3R4, —NR5C(O)R3, —NR5C(O)NR3R4, —NR3R4, —OR3, —NR5—SO2R3, —OC(O)NR3R4, —C(O)OR3,
(ii) L is selected from divalent C1-C6 linear and branched alkyl (e.g., divalent C1-C6 linear and C3-C6 branched alkyl), divalent C2-C6 linear and branched alkenyl (e.g., divalent C2-C6 linear and C3-C6 branched alkenyl), divalent C2-C6 linear and branched alkynyl (e.g., divalent C2-C6 linear and C3-C6 branched alkynyl), and divalent 1- to 7-membered heteroalkyl, wherein the divalent alkyl and divalent heteroalkyl are optionally substituted with 1-4 groups independently selected from:
and
(vii) R5 is selected from hydrogen and C1-C6 linear or branched alkyl (e.g., C1-C6 linear or C3-C6 branched alkyl). In certain embodiments, the following compounds are excluded from Formula I:
and compounds where -L-R in Formula I is
and R3 and R4 are
In some embodiments, when L is a divalent C2 linear alkyl optionally substituted with 1-2 groups independently selected from methyl, halogen, and hydroxy and R is —NR3R4, then R3 and R4 are not
In some embodiments, when L is a divalent C2 linear alkyl optionally substituted with 1-2 groups independently selected from methyl, halogen, and hydroxy and R is —NR3R4, then R3 and R4 are not
In some embodiments, L is selected from divalent C1-C6 linear and branched alkyl, divalent C2-C6 linear and branched alkenyl, divalent C2-C6 linear and branched alkynyl, and divalent 1- to 6-membered heteroalkyl, wherein the divalent alkyl and divalent heteroalkyl are optionally substituted with 1-4 groups independently selected from:
each R2 is independently selected from:
R3 and R4 are independently selected from:
In one aspect of the disclosure, the at least one entity (e.g., the at least one compound, deuterated derivative, or pharmaceutically acceptable salt) is chosen from Compounds 1 to 527, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of the foregoing.
In some embodiments, the disclosure provides pharmaceutical compositions comprising a compound of Formula I, deuterated derivatives thereof, or a pharmaceutically acceptable salt of any of the foregoing. In some embodiments, the pharmaceutical compositions may comprise at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from Compounds 1 to 527, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of the foregoing. These compositions may further include at least one additional active pharmaceutical ingredient and/or at least one carrier.
Another aspect of the disclosure provides methods of treating FSGS and/or NDKD comprising administering to a subject in need thereof, at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from compounds of Formula I, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of the foregoing or a pharmaceutical composition comprising the compound, deuterated derivative, or pharmaceutically acceptable salt. In some embodiments, the methods comprise administering at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from Compounds 1 to 527, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of the foregoing or a pharmaceutical composition comprising the compound, deuterated derivative, or pharmaceutically acceptable salt.
In some embodiments, the methods of treatment include administration of at least one additional active agent to the subject in need thereof, either in the same pharmaceutical composition as the at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from compounds of Formula I, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of the foregoing. Alternatively, the additional active agent and the compound, deuterated derivative, or pharmaceutically acceptable salt may be administered as separate pharmaceutical compositions. In some embodiments, the methods comprise administering at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from Compounds 1 to 527, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of the foregoing with at least one additional active agent either in the same pharmaceutical composition or in separate pharmaceutical compositions.
Also provided herein are methods of inhibiting APOL1, comprising administering to a subject in need thereof, at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from compounds of Formula I, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing or a pharmaceutical composition comprising the compound, deuterated derivative, or pharmaceutically acceptable salt. In some embodiments, the methods of inhibiting APOL1 comprise administering at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from Compounds 1 to 527, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing or a pharmaceutical composition comprising the compound, deuterated derivative, or pharmaceutically acceptable salt.
The term “APOL1,” as used herein, means apolipoprotein L1 protein, and the term “APOL1” means apolipoprotein L1 gene.
As used herein, the term “APOL1 mediated kidney disease” refers to a disease or condition that impairs kidney function and can be attributed to APOL1. In some embodiments, APOL1 mediated kidney disease is associated with patients having two APOL1 risk alleles, e.g., patients who are homozygous or compound heterozygous for the G1 or G2 alleles. In some embodiments, the APOL1 mediated kidney disease is chosen from ESKD, NDKD, FSGS, HIV-associated nephropathy, arterionephrosclerosis, lupus nephritis, microalbuminuria, and chronic kidney disease.
The term “FSGS,” as used herein, means focal segmental glomerulosclerosis, which is a disease of the podocyte (glomerular visceral epithelial cells) responsible for proteinuria and progressive decline in kidney function. In some embodiments, FSGS is associated with two APOL1 risk alleles.
The term “NDKD,” as used herein, means non-diabetic kidney disease, which is characterized by severe hypertension and progressive decline in kidney function. In some embodiments, NDKD is associated with two APOL1 risk alleles.
The terms “ESKD” and “ESRD” are used interchangeably herein to refer to end stage kidney disease or end stage renal disease. ESKD/ESRD is the last stage of kidney disease, i.e., kidney failure, and means that the kidneys have stopped working well enough for the patient to survive without dialysis or a kidney transplant. In some embodiments, ESKD/ESRD is associated with two APOL1 risk alleles.
The term “compound,” when referring to a compound of this disclosure, refers to a collection of molecules having an identical chemical structure unless otherwise indicated as a collection of stereoisomers (for example, a collection of racemates, a collection of cis/trans stereoisomers, or a collection of (E) and (Z) stereoisomers), except that there may be isotopic variation among the constituent atoms of the molecules. Thus, it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms, will also contain lesser amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure. The relative amount of such isotopologues in a compound of this disclosure will depend upon a number of factors including the isotopic purity of reagents used to make the compound and the efficiency of incorporation of isotopes in the various synthesis steps used to prepare the compound. However, as set forth above, the relative amount of such isotopologues in toto will be less than 49.9% of the compound. In other embodiments, the relative amount of such isotopologues in toto will be less than 47.5%, less than 40%, less than 32.5%, less than 25%, less than 17.5%, less than 10%, less than 5%, less than 3%, less than 1%, or less than 0.5% of the compound.
As used herein, “optionally substituted” is interchangeable with the phrase “substituted or unsubstituted.” In general, the term “substituted,” whether preceded by the term “optionally” or not, refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. Unless otherwise indicated, an “optionally substituted” group may have a substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent chosen from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this disclosure are those that result in the formation of stable or chemically feasible compounds.
The term “isotopologue” refers to a species in which the chemical structure differs from only in the isotopic composition thereof. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C or 14C are within the scope of this disclosure.
Unless otherwise indicated, structures depicted herein are also meant to include all isomeric forms of the structure, e.g., racemic mixtures, cis/trans isomers, geometric (or conformational) isomers, such as (Z) and (F) double bond isomers, and (Z) and (F) conformational isomers. Therefore, geometric and conformational mixtures of the present compounds are within the scope of the disclosure. Unless otherwise stated, all tautomeric forms of the compounds of the disclosure are within the scope of the disclosure.
The term “tautomer,” as used herein, refers to one of two or more isomers of compound that exist together in equilibrium, and are readily interchanged by migration of an atom, e.g., a hydrogen atom, or group within the molecule.
“Stereoisomer,” as used herein, refers to enantiomers and diastereomers.
As used herein, “deuterated derivative” refers to a compound having the same chemical structure as a reference compound, but with one or more hydrogen atoms replaced by a deuterium atom (“D” or “2H”). It will be recognized that some variation of natural isotopic abundance occurs in a synthesized compound depending on the origin of chemical materials used in the synthesis. The concentration of naturally abundant stable hydrogen isotopes, notwithstanding this variation is small and immaterial as compared to the degree of stable isotopic substitution of deuterated derivatives described herein. Thus, unless otherwise stated, when a reference is made to a “deuterated derivative” of compound of the disclosure, at least one hydrogen is replaced with deuterium at well above its natural isotopic abundance (which is typically about 0.015%). In some embodiments, the deuterated derivatives of the disclosure have an isotopic enrichment factor for each deuterium atom, of at least 3500 (52.5% deuterium incorporation at each designated deuterium), at least 4500 (67.5% deuterium incorporation), at least 5000 (75% deuterium incorporation) at least 5500 (82.5% deuterium incorporation), at least 6000 (90% deuterium incorporation), at least 6333.3 (95% deuterium incorporation), at least 6466.7 (97% deuterium incorporation), or at least 6600 (99% deuterium incorporation).
The term “isotopic enrichment factor” as used herein means the ratio between the isotopic abundance and the natural abundance of a specified isotope.
The term “alkyl” or “aliphatic” as used herein, means a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic that has a single point of attachment to the rest of the molecule. Unless otherwise specified, alkyl groups contain 1 to 20 carbon atoms. In some embodiments, alkyl groups contain 1 to 10 carbon atoms. In some embodiments, alkyl groups contain 1 to 8 carbon atoms. In some embodiments, alkyl groups contain 1 to 6 carbon atoms, and in some embodiments, alkyl groups contain 1 to 4 carbon atoms, and in yet other embodiments alkyl groups contain 1 to 3 carbon atoms. Non-limiting examples of alkyl groups include, but are not limited to, linear or branched, and substituted or unsubstituted alkyl. In some embodiments, alkyl groups are substituted. In some embodiments, alkyl groups are unsubstituted. In some embodiments, alkyl groups are straight-chain. In some embodiments, alkyl groups are branched.
The terms “cycloalkyl,” “carbocycle,” or “cyclic alkyl” refer to a fused, spirocyclic, or monocyclic C3-8 hydrocarbon or a spirocyclic, bicyclic, bridged bicyclic, tricyclic, or bridged tricyclic C4-14 hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, wherein any individual ring in said bicyclic ring system has 3 to 7 members. Suitable cycloalkyl groups include, but are not limited to, cycloalkyl, bicyclic cycloalkyl (e.g., decalin), bridged bicycloalkyl such as norbornyl or [2.2.2]bicyclo-octyl, or bridged tricyclic such as adamantyl. In some embodiments, cycloalkyl groups are substituted. In some embodiments, cycloalkyl groups are unsubstituted.
The term “heteroalkyl,” as used herein, means aliphatic groups wherein one, two, or three carbon atoms are independently replaced by a non-carbon atom. In some embodiments, the heteroaryl contains one or more of oxygen, sulfur, and/or nitrogen. In some embodiments, one or more carbon atoms may be replaced by phosphorus, boron, and/or silicon. Heteroalkyl groups may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and include “heterocycle,” “heterocyclyl,” or “heterocyclic” groups. In some embodiments, the heteroalkyl is an aminoalkyl. In some embodiments, the heteroalkyl is a thioalkyl. In some embodiments, the heteroalkyl is an alkoxy. In some embodiments, the heteroalkyl has a combination of two or more heteroatoms independently selected from oxygen, nitrogen, phosphorus, and sulfur. In some embodiments, one, two, or three carbon atoms are replaced by nitrogen.
The term “alkenyl,” as used herein, means a straight-chain (i.e., unbranched), branched, substituted or unsubstituted hydrocarbon chain that contains one or more units of saturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that contains one or more units of unsaturation, but which is not aromatic (referred to herein as, “cyclic alkenyl”). In some embodiments, alkenyl groups are substituted. In some embodiments, alkenyl groups are unsubstituted. In some embodiments, alkenyl groups are straight-chain. In some embodiments, alkenyl groups are branched.
The term “heterocycle,” “heterocyclyl,” or “heterocyclic,” as used herein, means non-aromatic, monocyclic, bicyclic, or tricyclic ring systems in which one or more ring members is an independently chosen heteroatom. In some embodiments, the “heterocycle”, “heterocyclyl”, or “heterocyclic” group has 3 to 14 ring members in which one or more ring members is a heteroatom independently chosen from oxygen, sulfur, nitrogen, phosphorus, silicon, and boron. In some embodiments, each ring in a bicyclic or tricyclic ring system contains 3 to 7 ring members. In some embodiments, the heterocycle has at least one unsaturated carbon-carbon bond. In some embodiments, the heterocycle has at least one unsaturated carbon-nitrogen bond. In some embodiments, the heterocycle has one to three heteroatoms independently chosen from oxygen, sulfur, nitrogen, and phosphorus. In some embodiments, the heterocycle has one heteroatom that is a nitrogen atom. In some embodiments, the heterocycle has one heteroatom that is an oxygen atom. In some embodiments, the heterocycle has one heteroatom that is a sulfur atom. In some embodiments, the heterocycle has two heteroatoms that are each independently selected from nitrogen, sulfur, and oxygen. In some embodiments, the heterocycle has three heteroatoms that are each independently selected from nitrogen and oxygen. In some embodiments, heterocycles are substituted. In some embodiments, heterocycles are unsubstituted.
The term “heteroatom” refers to a non-carbon atom. In some embodiments, the heteroatom is selected from oxygen, sulfur, nitrogen, phosphorus, boron, and silicon (including any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or a substitutable nitrogen of a heterocyclic ring, for example, N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR+ (as in N-substituted pyrrolidinyl)).
The term “unsaturated,” as used herein, means that a moiety has one or more units or degrees of unsaturation. Unsaturation is the state in which not all of the available valance bonds in a compound are satisfied by substituents and thus the compound contains double or triple bonds.
The term “alkoxy” or “thioalkyl”, as used herein, refers to an alkyl group, as previously defined, wherein one carbon of the alkyl group is replaced by an oxygen (“alkoxy”) or sulfur (“thioalkyl”) atom, respectively. In some embodiments, one of the two carbon atoms that the oxygen or sulfur atom is linked between is not part of the alkoxy or thioalkyl groups, such as, e.g., when an “alkoxy” group is methoxy, ethoxy, or the like. A “cyclic alkoxy” refers to a monocyclic, spirocyclic, bicyclic, bridged bicyclic, tricyclic, or bridged tricyclic hydrocarbon that contains at least one alkoxy group, but is not aromatic. Non-limiting examples of cyclic alkoxy groups include tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, 8-oxabicyclo[3.2.1]octanyl, and oxepanyl. In some embodiments, “alkoxy” and/or “thioalkyl” groups are substituted. In some embodiments, “alkoxy” and/or “thioalkyl” groups are unsubstituted.
The terms “haloalkyl” and “haloalkoxy,” as used herein, means a linear or branched alkyl or alkoxy, as the case may be, which is substituted with one or more halogen atoms. Non-limiting examples of haloalkyl groups include —CHF2, —CH2F, —CF3, —CF2—, and perhaloalkyls, such as —CF2CF3. Non-limiting examples of haloalkoxy groups include —OCHF2, —OCH2F, —OCF3, and —OCF2—.
In some embodiments, the term “hydroxyalkyl” means a linear, branched, or cyclic alkyl which is substituted with one or more hydroxy groups.
The term “halogen” includes F, Cl, Br, and I, i.e., fluoro, chloro, bromo, and iodo, respectively.
The term “aminoalkyl” means an alkyl group which is substituted with or contains an amino group. An aminoalkyl group may be linear or branched.
As used herein, the term “alkylsulfonyl” refers to an alkyl group, as previously defined, wherein one carbon atom of the alkyl group, and the carbon atom's substituents, are replaced by a sulfur atom, and wherein the sulfur atom is further substituted with two oxo groups. An alkylsulfonyl group may be linear or branched. In some embodiments, alkylsulfonyl groups are substituted at the alkyl portion of the alkylsulfonyl group. In some embodiments, alkylsulfonyl groups are unsubstituted at the alkyl portion of the alkylsulfonyl group.
As used herein, an “amino” refers to a group which is a primary, secondary, or tertiary amine.
As used herein, a “carbonyl” group refers to C═O.
As used herein, a “cyano” or “nitrile” group refer to —C≡N.
As used herein, a “hydroxy” group refers to —OH.
As used herein, a “thiol” group refers to —SH.
As used herein, “tert” and “t-” each refer to tertiary.
As used herein, “Me” refers to methyl.
As used herein, an “amido” group refers to a carbonyl group, as previously defined, wherein the carbon of the carbonyl is bonded to an amino group, as previously defined. When a chemical group is said to be substituted by an amido group, that chemical group may be bonded to the carbonyl carbon or to the amino nitrogen of the amido group.
As used herein, a “carbamate” group refers to a carbonyl group, as previously defined, wherein the carbon of the carbonyl group is bonded to an amino group, as previously defined, and a divalent oxygen. When a chemical group is said to be substituted by a carbamate group, that chemical group may be bonded to the divalent oxygen or to the amino nitrogen of the carbamate group.
As used herein, “aromatic groups” or “aromatic rings” refer to chemical groups that contain conjugated, planar ring systems with delocalized pi electron orbitals comprised of [4n+2] p orbital electrons, wherein n is an integer ranging from 0 to 6. Non-limiting examples of aromatic groups include aryl and heteroaryl groups.
The term “aryl,” used alone or as part of a larger moiety as in “arylalkyl,” “arylalkoxy,” or “aryloxyalkyl,” refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in a bicyclic or tricyclic ring system contains 3 to 7 ring members. The term “aryl” also refers to heteroaryl ring systems as defined herein below. Non-limiting examples of aryl groups include phenyl rings. In some embodiments, aryl groups are substituted. In some embodiments, aryl groups are unsubstituted.
The term “heteroaryl,” used alone or as part of a larger moiety as in “heteroarylalkyl” or “heteroarylalkoxy,” refers to monocyclic, bicyclic, and tricyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic, at least one ring in the system contains one or more heteroatoms, and wherein each ring in a bicyclic or tricyclic ring system contains 3 to 7 ring members. In some embodiments, heteroaryl groups are substituted. In some embodiments, heteroaryl groups have one or more heteroatoms chosen from nitrogen, oxygen, and sulfur. In some embodiments, heteroaryl groups have one heteroatom. In some embodiments, heteroaryl groups have two heteroatoms. In some embodiments, heteroaryl groups are monocyclic ring systems having five ring members. In some embodiments, heteroaryl groups are monocyclic ring systems having six ring members. In some embodiments, heteroaryl groups are unsubstituted.
Non-limiting examples of useful protecting groups for nitrogen-containing groups, such as amine groups, include, for example, t-butyl carbamate (Boc), benzyl (Bn), tetrahydropyranyl (THP), 9-fluorenylmethyl carbamate (Fmoc), benzyl carbamate (Cbz), acetamide, trifluoroacetamide, triphenylmethylamine, benzylideneamine, and p-toluenesulfonamide. Methods of adding (a process generally referred to as “protecting”) and removing (process generally referred to as “deprotecting”) such amine protecting groups are well-known in the art and available, for example, in P. J. Kocienski, Protecting Groups, Thieme, 1994, which is hereby incorporated by reference in its entirety and in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Edition (John Wiley & Sons, New York, 1999) and 4th Edition (John Wiley & Sons, New Jersey, 2014).
Non-limiting examples of suitable solvents that may be used in this disclosure include, but are not limited to, water, methanol (MeOH), ethanol (EtOH), dichloromethane or “methylene chloride” (CH2Cl2), toluene, acetonitrile (MeCN), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), methyl acetate (MeOAc), ethyl acetate (EtOAc), heptanes, isopropyl acetate (IPAc), tert-butyl acetate (t-BuOAc), isopropyl alcohol (IPA), tetrahydrofuran (THF), 2-methyl tetrahydrofuran (2-Me THF), methyl ethyl ketone (MEK), tert-butanol, diethyl ether (Et2O), methyl-tert-butyl ether (MTBE), 1,4-dioxane, and N-methyl pyrrolidone (NMP).
Non-limiting examples of suitable bases that may be used in this disclosure include, but are not limited to, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), potassium tert-butoxide (KOtBu), potassium carbonate (K2CO3), N-methylmorpholine (NMM), triethylamine (Et3N; TEA), diisopropyl-ethyl amine (i-Pr2EtN; DIPEA), pyridine, potassium hydroxide (KOH), sodium hydroxide (NaOH), lithium hydroxide (LiOH) and sodium methoxide (NaOMe; NaOCH3).
The disclosure includes pharmaceutically acceptable salts of the disclosed compounds. A salt of a compound is formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the compound, such as a carboxyl functional group.
The term “pharmaceutically acceptable,” as used herein, refers to a component that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other mammals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. A “pharmaceutically acceptable salt” means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this disclosure. Suitable pharmaceutically acceptable salts are, for example, those disclosed in S. M. Berge, et al. J. Pharmaceutical Sciences, 1977, 66, 1 to 19.
Acids commonly employed to form pharmaceutically acceptable salts include inorganic acids such as hydrogen bisulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid, ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, as well as related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, butyne-1,4-dioate, hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylene sulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate and other salts. In some embodiments, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and those formed with organic acids such as maleic acid.
Pharmaceutically acceptable salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium, and N+(C1-4alkyl)4 salts. This disclosure also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Suitable non-limiting examples of alkali and alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium. Further non-limiting examples of pharmaceutically acceptable salts include ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate, and aryl sulfonate. Other suitable, non-limiting examples of pharmaceutically acceptable salts include besylate and glucosamine salts.
The terms “patient” and “subject” are used interchangeably and refer to an animal, including a human.
The terms “effective dose” and “effective amount” are used interchangeably herein and refer to that amount of compound that produces the desired effect for which it is administered (e.g., improvement in symptoms of FSGS and/or NDKD, lessening the severity of FSGS and/NDKD or a symptom of FSGS and/or NDKD, and/or reducing progression of FSGS and/or NDKD or a symptom of FSGS and/or NDKD). The exact amount of an effective dose will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).
As used herein, the term “treatment” and its cognates refer to slowing or stopping disease progression. “Treatment” and its cognates as used herein, include, but are not limited to the following: lessening the severity of a disease symptom, complete or partial remission, lower risk of kidney failure (e.g., ESRD), and disease-related complications (e.g., edema, susceptibility to infections, or thrombo-embolic events). Improvements in or lessening the severity of one or more symptoms can be readily assessed according to methods and techniques known in the art or subsequently developed. In some embodiments, the terms “treat,” “treating,” and “treatment,” refer to the lessening of severity of one or more symptoms of FSGS and/or NDKD. In some embodiments, “treatment” and its cognates refers to a reduction of the risk of ESRD.
The terms “about” and “approximately,” when used in connection with doses, amounts, or weight percent of ingredients of a composition or a dosage form, include the value of a specified dose, amount, or weight percent or a range of the dose, amount, or weight percent that is recognized by one of ordinary skill in the art to provide a pharmacological effect equivalent to that obtained from the specified dose, amount, or weight percent. In some embodiments, the term “about” refers to a value±10%, ±8%, ±6%, ±5%, ±4%, ±2%, or ±1% of a referenced value.
As used herein, the term “ambient conditions” means room temperature, open air, and uncontrolled humidity conditions.
The terms “selected from” and “chosen from” are used interchangeably herein.
The compound of Formula I, I′, II, or II′, a deuterated derivative thereof, or a pharmaceutically acceptable salt of any of the foregoing may be administered once daily, twice daily, or three times daily, for example, for the treatment of FSGS. In some embodiments, the compound of Formula I, I′, II, or II′, deuterated derivative thereof, or pharmaceutically acceptable salt of any of the foregoing is chosen from Compounds 1 to 527, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing. In some embodiments, at least one compound of Formula I, I′, II, or II′, deuterated derivative thereof, or pharmaceutically acceptable salt of any of the foregoing is administered once daily. In some embodiments, at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from Compounds 1 to 527, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing is administered once daily. In some embodiments, at least compound of Formula I, I′, II, or II′, deuterated derivative thereof, or pharmaceutically acceptable salt of any of the foregoing is administered twice daily. In some embodiments, at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from Compounds 1 to 527, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing is administered twice daily. In some embodiments, at least one compound of Formula I, I′, II, or II′, deuterated derivative thereof, or pharmaceutically acceptable salt of any of the foregoing is administered three times daily. In some embodiments, at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from Compounds 1 to 527, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing is administered three times daily.
In some embodiments, 2 mg to 1500 mg, 5 mg to 1000 mg, 10 mg to 500 mg, 20 mg to 300 mg, 20 mg to 200 mg, 30 mg to 150 mg, 50 mg to 150 mg, 60 mg to 125 mg, or 70 mg to 120 mg, 80 mg to 115 mg, 90 mg to 110 mg, 95 mg to 110 mg, or 100 mg to 105 mg of the at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from compounds of Formula I, I′, II, or II′, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing is administered once daily, twice daily, or three times daily. In some embodiments, 2 mg to 1500 mg, 5 mg to 1000 mg, 10 mg to 500 mg, 20 mg to 300 mg, 20 mg to 200 mg, 30 mg to 150 mg, 50 mg to 150 mg, 60 mg to 125 mg, or 70 mg to 120 mg, 80 mg to 115 mg, 90 mg to 110 mg, 95 mg to 110 mg, or 100 mg to 105 mg of the at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from Compounds 1 to 527, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing is administered once daily, twice daily, or three times daily.
One of ordinary skill in the art would recognize that, when an amount of compound is disclosed, the relevant amount of a pharmaceutically acceptable salt form of the compound is an amount equivalent to the concentration of the free base of the compound. The amounts of the compounds, pharmaceutically acceptable salts, solvates, and deuterated derivatives disclosed herein are based upon the free base form of the reference compound. For example, “10 mg of at least one compound chosen from compounds of Formula I, . . . and pharmaceutically acceptable salts thereof” includes 10 mg of a compound of Formula I, and a concentration of a pharmaceutically acceptable salt of that compound of Formula I that is equivalent to 10 mg of that compound of Formula I.
In some embodiments of the disclosure, the compound, deuterated derivative, or pharmaceutically acceptable salt for treating APOL1 mediated diseases, such as FSGS and/or NDKD, is selected from compounds of Formula I:
deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing, wherein:
(i) R is selected from —C(O)NR3R4, —NR5C(O)R3, —NR5C(O)NR3R4, —NR3R4, —OR3, —NR5—SO2R3, —OC(O)NR3R4, —C(O)OR3,
(ii) L is selected from divalent C1-C6 linear and branched alkyl (e.g., divalent C1-C6 linear and C3-C6 branched alkyl), divalent C2-C6 linear and branched alkenyl (e.g., divalent C2-C6 linear and C3-C6 branched alkenyl), divalent C2-C6 linear and branched alkynyl (e.g., divalent C2-C6 linear and C3-C6 branched alkynyl), and divalent 1- to 7-membered heteroalkyl, wherein the divalent alkyl and divalent heteroalkyl are optionally substituted with 1-4 groups independently selected from:
and
(vii) R5 is selected from hydrogen and C1-C6 linear or branched alkyl (e.g., C1-C6 linear or C3-C6 branched alkyl). In certain embodiments, the following compounds are excluded from Formula I:
and compounds where -L-R in Formula I is
and R3 and R4 are
In some embodiments, when L is a divalent C2 linear alkyl optionally substituted with 1-2 groups independently selected from methyl, halogen, and hydroxy and R is —NR3R4, then R3 and R4 are not
In some embodiments, when L is a divalent C2 linear alkyl optionally substituted with 1-2 groups independently selected from methyl, halogen, and hydroxy and R is —NR3R4, then R3 and R4 are not
In some embodiments, L is selected from divalent C1-C6 linear and branched alkyl, divalent C2-C6 linear and branched alkenyl, divalent C2-C6 linear and branched alkynyl, and divalent 1- to 6-membered heteroalkyl, wherein the divalent alkyl and divalent heteroalkyl are optionally substituted with 1-4 groups independently selected from:
each R2 is independently selected from:
R3 and R4 are independently selected from:
In some embodiments, R3 is hydrogen or methyl.
In some embodiments, R3 is hydrogen.
In some embodiments, each R1 is independently chosen from halogen groups.
In some embodiments, each R1 is fluoro.
In some embodiments, each R2 is independently chosen from halogen groups and methyl.
In some embodiments, each R2 is independently chosen from halogen groups.
In some embodiments, each R2 is fluoro.
In some embodiments, each n is 1 or 2.
In some embodiments, each n is 2.
In some embodiments, R5 is hydrogen.
In some embodiments, the compound of Formula I, deuterated derivative thereof, or pharmaceutically acceptable salt of any of the foregoing is selected from Compounds 1 to 527 depicted in Table 1, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing. A wavy line in a compound in Table 1 (i.e., ) depicts a bond between two atoms and indicates a position of mixed stereochemistry for a collection of molecules, such as a racemic mixture, cis/trans isomers, or (E)/(Z) isomers.
Another aspect of the disclosure provides methods for making compounds of Formula I, I′, II, or II′, Compounds 1 to 527, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of foregoing. The disclosure also provides intermediates for making any of compounds, deuterated derivatives, or pharmaceutically acceptable salts disclosed herein.
Another aspect of the disclosure provides pharmaceutical compositions comprising at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from compounds of Formula I, I′, II, or II′, Compounds 1 to 527, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of foregoing. In some embodiments, the pharmaceutical composition comprising at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from compounds of Formula I, I′, II, or II′, Compounds 1 to 527, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of foregoing is administered to a patient in need thereof.
A pharmaceutical composition may further comprise at least one pharmaceutically acceptable carrier. In some embodiments, the at least one pharmaceutically acceptable carrier is chosen from pharmaceutically acceptable vehicles and pharmaceutically acceptable adjuvants. In some embodiments, the at least one pharmaceutically acceptable is chosen from pharmaceutically acceptable fillers, disintegrants, surfactants, binders, and lubricants.
It will also be appreciated that a pharmaceutical composition of this disclosure can be employed in combination therapies; that is, the pharmaceutical compositions described herein can further include at least one additional active therapeutic agent. Alternatively, a pharmaceutical composition comprising at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from compounds of Formula I, I′, II, or II′, Compounds 1 to 527, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of foregoing can be administered as a separate pharmaceutical composition concurrently with, prior to, or subsequent to, a composition comprising at least one other active therapeutic agent. In some embodiments, a pharmaceutical composition comprising at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from compounds of Formula I, I′, II, or II′, Compounds 1 to 527, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of foregoing can be administered as a separate pharmaceutical composition concurrently with, prior to, or subsequent to, a composition comprising at least one other active therapeutic agent.
As described above, pharmaceutical compositions disclosed herein may optionally further comprise at least one pharmaceutically acceptable carrier. The at least one pharmaceutically acceptable carrier may be chosen from adjuvants and vehicles. The at least one pharmaceutically acceptable carrier, as used herein, includes any and all solvents, diluents, other liquid vehicles, dispersion aids, suspension aids, surface active agents, isotonic agents, thickening agents, emulsifying agents, preservatives, solid binders, and lubricants, as suited to the particular dosage form desired. Remington: The Science and Practice of Pharmacy, 21st edition, 2005, ed. D. B. Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988 to 1999, Marcel Dekker, New York discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier is incompatible with the compounds of this disclosure, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure. Non-limiting examples of suitable pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as, e.g., human serum albumin), buffer substances (such as, e.g., phosphates, glycine, sorbic acid, and potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts, and electrolytes (such as, e.g., protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugars (such as, e.g., lactose, glucose, and sucrose), starches (such as, e.g., corn starch and potato starch), cellulose and its derivatives (such as, e.g., sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate), powdered tragacanth, malt, gelatin, talc, excipients (such as, e.g., cocoa butter and suppository waxes), oils (such as, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil), glycols (such as, e.g., propylene glycol and polyethylene glycol), esters (such as, e.g., ethyl oleate and ethyl laurate), agar, buffering agents (such as, e.g., magnesium hydroxide and aluminum hydroxide), alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, phosphate buffer solutions, non-toxic compatible lubricants (such as, e.g., sodium lauryl sulfate and magnesium stearate), coloring agents, releasing agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, and antioxidants.
In some embodiments of the disclosure, the compounds and the pharmaceutical compositions described herein are used to treat APOL1 mediated kidney disease. In some embodiments, the APOL1 mediated kidney disease is chosen from ESKD, FSGS, HIV-associated nephropathy, NDKD, arterionephrosclerosis, lupus nephritis, microalbuminuria, and chronic kidney disease. In some embodiments, the APOL1 mediated kidney disease treated with the compound, deuterated derivative, pharmaceutically acceptable salt, and/or composition of the disclosure is FSGS. In some embodiments, the APOL1 mediated kidney disease treated with the compound, deuterated derivative, pharmaceutically acceptable salt, and/or composition of the disclosure is NDKD. In some embodiments, the APOL1 mediated kidney disease treated with the compound, deuterated derivative, and pharmaceutically acceptable salt and/or composition of the disclosure is ESKD. In some embodiments, the patient with APOL1 mediated kidney disease to be treated with the compound, deuterated derivative, pharmaceutically acceptable salt, and/or composition of the disclosure has two APOL1 risk alleles. In some embodiments, the patient with APOL1 mediated kidney disease is homozygous for APOL1 genetic risk alleles G1: S342G:I384M. In some embodiments, the patient with APOL1 mediated kidney disease is homozygous for APOL1 genetic risk alleles G2: N388del:Y389del. In some embodiments, the patient with APOL1 mediated kidney disease is heterozygous for APOL1 genetic risk alleles G1: S342G:I384M and G2: N388del:Y389del.
In some embodiments, the methods of the disclosure comprise administering to a patient in need thereof at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from compounds of Formula I, I′, II, or II′, Compounds 1 to 527, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of foregoing. In some embodiments, the compound, deuterated derivative, or pharmaceutically acceptable salt is chosen from Compounds 1 to 527, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of foregoing. In some embodiments, said patient in need thereof possesses APOL1 genetic variants, i.e., G1: S342G:I384M and G2: N388del:Y389del.
Another aspect of the disclosure provides methods of inhibiting APOL1 activity comprising contacting said APOL1 with at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from compounds of Formula I, I′, II, or II′, Compounds 1 to 527, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of foregoing. In some embodiments, the methods of inhibiting APOL1 activity comprise contacting said APOL1 with at least one compound, deuterated derivative, or pharmaceutically acceptable salt chosen from Compounds 1 to 527, deuterated derivatives of those compounds, and pharmaceutically acceptable salts of any of foregoing.
Without limitation, some example embodiments of this disclosure include:
1. A compound, deuterated derivative, or pharmaceutically acceptable salt selected from compounds of Formula I:
deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing, wherein:
(i) R is selected from —C(O)NR3R4, —NR5C(O)R3, —NR5C(O)NR3R4, —NR3R4, —OR3, —NR5—SO2R3, —OC(O)NR3R4, —C(O)OR3,
(ii) L is selected from divalent C1-C6 linear and branched alkyl (e.g., divalent C1-C6 linear and C3-C6 branched alkyl), divalent C2-C6 linear and branched alkenyl (e.g., divalent C2-C6 linear and C3-C6 branched alkenyl), divalent C2-C6 linear and branched alkynyl (e.g., divalent C2-C6 linear and C3-C6 branched alkynyl), and divalent 1- to 7-membered heteroalkyl, wherein the divalent alkyl and divalent heteroalkyl are optionally substituted with 1-4 groups independently selected from:
and
(vii) R5 is selected from hydrogen and C1-C6 linear or branched alkyl (e.g., C1-C6 linear or C3-C6 branched alkyl);
with the provisos that (1) the compound is not selected from
and
(2) when -L-R in Formula I is
then R3 and R4 are not
2. The compound, deuterated derivative, or pharmaceutically acceptable salt according to Embodiment 1, wherein L is selected from divalent C1-C6 linear and branched alkyl, divalent C2-C6 linear and branched alkenyl, divalent C2-C6 linear and branched alkynyl, and divalent 1- to 6-membered heteroalkyl, wherein the divalent alkyl and divalent heteroalkyl are optionally substituted with 1-4 groups independently selected from:
L is selected from divalent C1-C6 linear and branched alkyl, divalent C2-C6 linear and branched alkenyl, divalent C2-C6 linear and branched alkynyl, and divalent 1- to 6-membered heteroalkyl, wherein the divalent alkyl and divalent heteroalkyl are optionally substituted with 1-4 groups independently selected from:
each R2 is independently selected from:
R3 and R4 are independently selected from:
7. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-6, wherein, when L is a divalent C2 linear alkyl optionally substituted with 1-2 groups independently selected from methyl, halogen, and hydroxy and R is —NR3R4, then R3 and R4 are not
8. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-7, wherein each R1 is independently selected from halogen, hydroxy, amino, C1-C6 linear and branched alkyl (optionally substituted with 1-3 groups independently selected from hydroxy and halogen), C3-C6 cycloalkyl, and C1-C6 linear and branched alkoxy (optionally substituted with 1-3 groups independently selected from halogen).
9. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1, 2, 4, 6, and 7, wherein each R2 is independently selected from halogen, hydroxy, amino, cyano, C1-C6 linear and branched alkyl (optionally substituted with 1-3 groups independently selected from hydroxy and halogen), and C1-C6 linear and branched alkoxy (optionally substituted with 1-3 groups independently selected from halogen).
10. The compound, deuterated derivative, or pharmaceutically acceptable salt according to Embodiment 5, wherein each R2 is independently selected from halogen, hydroxy, amino, cyano, C1-C4 linear and branched alkyl (optionally substituted with 1-3 groups independently selected from hydroxy and halogen), and C1-C4 linear and branched alkoxy (optionally substituted with 1-3 groups independently selected from halogen).
11. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-10, wherein each R1 and/or R2 is fluorine.
12. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-11, wherein each n is independently selected from 0, 1, and 2.
13. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1, 3, 4, and 6-12, wherein L is selected from divalent C1-C6 linear and branched alkyl, and divalent C1-C6 linear and branched thioalkyl, wherein the divalent alkyl and divalent thioalkyl are optionally substituted with 1-2 groups independently selected from halogen.
14. The compound, deuterated derivative, or pharmaceutically acceptable salt according to Embodiment 13, wherein L is selected from divalent C1-C3 linear and branched alkyl, and divalent C1-C3 linear and branched thioalkyl, wherein the divalent alkyl and divalent thioalkyl are optionally substituted with 1-2 groups independently selected from halogen.
15. The compound, deuterated derivative, or pharmaceutically acceptable salt according to Embodiment 5, wherein L is selected from divalent C1-C6 linear and branched alkyl and divalent C1-C5 linear and branched thioalkyl, wherein the divalent alkyl and divalent thioalkyl are optionally substituted with 1-2 groups independently selected from halogen.
16. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-5 and 8-15, wherein R is —C(O)NR3R4, and wherein R3 and R4 are independently selected from:
R is
and
R3 is hydrogen.
30. A compound, deuterated derivative, or pharmaceutically acceptable salt selected from Compounds 1 to 527 (Table 1), deuterated derivatives thereof, or pharmaceutically acceptable salts of any of the foregoing.
31. A pharmaceutical composition comprising the compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-30 and a pharmaceutically acceptable carrier.
32. A method of treating APOL1 mediated kidney disease comprising administering to a patient in need thereof the compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-30 or the pharmaceutical composition according to
33. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-30, or the pharmaceutical composition according to Embodiment 31, for use in treating APOL1 mediated kidney disease.
34. Use of a compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Embodiments 1-30 in the manufacture of a medicament for treating APOL1 mediated kidney disease.
35. The method, compound, deuterated derivative, pharmaceutically acceptable salt, or pharmaceutical composition for use, or use according to any one of Embodiments 32-34, wherein the APOL1 mediated kidney disease is selected from ESKD, NDKD, FSGS, HIV-associated nephropathy, sickle cell nephropathy, diabetic neuropathy, arterionephrosclerosis, lupus nephritis, microalbuminuria, and chronic kidney disease.
36. The method, compound, deuterated derivative, pharmaceutically acceptable salt, or pharmaceutical composition for use, or use according to Embodiment 35, wherein the APOL1 mediated kidney disease is FSGS.
37. The method, compound, deuterated derivative, pharmaceutically acceptable salt, or pharmaceutical composition for use, or use according to Embodiment 35, wherein the APOL1 mediated kidney disease is NDKD.
38. The method, compound, deuterated derivative, pharmaceutically acceptable salt, or pharmaceutical composition for use, or use according to Embodiment 35, wherein the APOL1 mediated kidney disease is ESKD.
39. The method, compound, deuterated derivative, pharmaceutically acceptable salt, or pharmaceutical composition for use, or use according to any one of Embodiments 32-38, wherein the APOL1 is associated with APOL1 genetic alleles chosen from homozygous G1: S342G:I384M and homozygous G2: N388del:Y389del.
40. The method, compound, deuterated derivative, pharmaceutically acceptable salt, or pharmaceutical composition for use, or use according to any one of Embodiments 32-38, wherein the APOL1 is associated with compound heterozygous G1: S342G:I384M and G2: N388del:Y389del APOL1 alleles.
41. A method of treating APOL1 mediated kidney disease comprising administering to a patient in need thereof a compound, deuterated derivative, or pharmaceutically acceptable salt selected from compounds of Formula II:
deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing, wherein:
(i) R is selected from —C(O)NR3R4, —NR5C(O)R3, —NR5C(O)NR3R4, —NR3R4, —OR3, —NR5—SO2R3, —OC(O)NR3R4, —C(O)OR3,
(ii) L is selected from divalent C1-C6 linear and branched alkyl (e.g., divalent C1-C6 linear and C3-C6 branched alkyl), divalent C2-C6 linear and branched alkenyl (e.g., divalent C2-C6 linear and C3-C6 branched alkenyl), divalent C2-C6 linear and branched alkynyl (e.g., divalent C2-C6 linear and C3-C6 branched alkynyl), and divalent 1- to 7-membered heteroalkyl, wherein the divalent alkyl and divalent heteroalkyl are optionally substituted with 1-4 groups independently selected from:
and
(vii) R5 is selected from hydrogen and C1-C6 linear or branched alkyl (e.g., C1-C6 linear or C3-C6 branched alkyl);
with the provisos that
(1) the compound is not
and
(2) when L is a divalent C2 linear alkyl optionally substituted with 1-2 groups independently selected from methyl, halogen, and hydroxy and R is —NR3R4, then R3 and R4 are not
42. The method according to Embodiment 41, wherein the APOL1 mediated kidney disease is chosen from ESKD, NDKD, FSGS, HIV-associated nephropathy, arterionephrosclerosis, lupus nephritis, microalbuminuria, and chronic kidney disease.
43. The method according to Embodiment 41, wherein the APOL1 mediated kidney disease is FSGS.
44. The method according to Embodiment 41, wherein the APOL1 mediated kidney disease is NDKD.
45. The method according to Embodiment 41, wherein the APOL1 mediated kidney disease is ESKD.
46. The method according to any one of Embodiments 41-45, wherein the APOL1 mediated kidney disease is associated with APOL1 genetic alleles chosen from homozygous G1: S342G:I384M and homozygous G2: N388del:Y389del.
47. The method according to any one of Embodiments 41-45, wherein the APOL1 is associated with compound heterozygous G1: S342G:I384M and G2: N388del:Y389del APOL1 alleles.
48. A method of inhibiting APOL1 activity comprising contacting said APOL1 with a compound selected from Formula II, a deuterated derivative thereof, or a pharmaceutically acceptable salt of any of the foregoing.
49. The method according to Embodiment 48, wherein the APOL1 is associated with APOL1 genetic alleles chosen from homozygous G1: S342G:I384M and homozygous G2: N388del:Y389del.
50. The method according to Embodiment 48, wherein the APOL1 is associated with homozygous G1: S342G:I384M APOL1 alleles.
51. The method according to Embodiment 48, wherein the APOL1 is associated with compound heterozygous G1: S342G:I384M and G2: N388del:Y389del APOL1 alleles.
52. A compound, deuterated derivative, or pharmaceutically acceptable salt selected from compounds of Formula II, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing, for use in treating APOL1 mediated kidney disease.
53. The compound, deuterated derivative, or pharmaceutically acceptable salt for use according to Embodiment 52, wherein the APOL1 mediated kidney disease is chosen from ESKD, NDKD, FSGS, HIV-associated nephropathy, arterionephrosclerosis, lupus nephritis, microalbuminuria, and chronic kidney disease.
54. The method according to Embodiment 52, wherein the APOL1 mediated kidney disease is FSGS.
55. The method according to Embodiment 52, wherein the APOL1 mediated kidney disease is NDKD.
56. The method according to Embodiment 52, wherein the APOL1 mediated kidney disease is ESKD.
57. The compound, deuterated derivative, or pharmaceutically acceptable salt for use according to any one of Embodiments 52-56, wherein the APOL1 is associated with APOL1 genetic alleles chosen from homozygous G1: S342G:I384M and homozygous G2: N388del:Y389del.
58. The compound, deuterated derivative, or pharmaceutically acceptable salt for use according to Embodiments 52-56, wherein the APOL1 is associated with compound heterozygous G1: S342G:I384M and G2: N388del:Y389del APOL1 alleles.
59. Use of a compound, deuterated derivative, or pharmaceutically acceptable salt selected from compounds of Formula II, deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing, in the manufacture of a medicament for treating APOL1 mediated kidney disease.
60. The use according to Embodiment 59, wherein the APOL1 mediated kidney disease is selected from ESKD, NDKD, FSGS, HIV-associated nephropathy, arterionephrosclerosis, lupus nephritis, microalbuminuria, and chronic kidney disease.
61. The use according to Embodiment 59, wherein the APOL1 mediated kidney disease is FSGS.
62. The use according to Embodiment 59, wherein the APOL1 mediated kidney disease is NDKD.
63. The use according to Embodiment 59, wherein the APOL1 mediated kidney disease is ESKD.
64. The use according to any one of Embodiments 59-63, wherein the APOL1 is associated with APOL1 genetic alleles chosen from homozygous G1: S342G:I384M and homozygous G2: N388del:Y389del.
65. The use according to any one of Embodiments 59-63, wherein the APOL1 is associated with compound heterozygous G1: S342G:I384M and G2: N388del:Y389del APOL1 alleles.
66. The method, compound, deuterated derivative, or pharmaceutically acceptable salt for use, or use according to any one of Embodiments 41-65, wherein L is selected from divalent C1-C6 linear and branched alkyl, divalent C2-C6 linear and branched alkenyl, divalent C2-C6 linear and branched alkynyl, and divalent 1- to 6-membered heteroalkyl, wherein the divalent alkyl and divalent heteroalkyl are optionally substituted with 1-4 groups independently selected from:
L is selected from divalent C1-C6 linear and branched alkyl, divalent C2-C6 linear and branched alkenyl, divalent C2-C6 linear and branched alkynyl, and divalent 1- to 6-membered heteroalkyl, wherein the divalent alkyl and divalent heteroalkyl are optionally substituted with 1-4 groups independently selected from:
each R2 is independently selected from:
R3 and R4 are independently selected from:
and
and deuterated derivatives and pharmaceutically acceptable salts thereof, wherein:
(i) R is selected from —C(O)NR3R4, —NR5C(O)R3, —NR5C(O)NR3R4, —NRX—SO2R3, —OC(O)NR3R4, —C(O)OR3,
(ii) L is selected from divalent C1-C6 linear and branched alkyl, divalent C2-C6 linear and branched alkenyl, divalent C2-C6 linear and branched alkynyl, and divalent 1 to 6 membered heteroalkyl, wherein the divalent alkyl and divalent heteroalkyl are optionally substituted with 1-4 groups selected from:
and
(vii) R5 is selected from hydrogen and linear or branched C1-C6 alkyl;
with the provisos that (1) the compound is not selected from
and
(2) when -L-R in Formula I′ is
then R3 and R4 are not
2. The compound, deuterated derivative, or pharmaceutically acceptable salt according to Clause 1, wherein each R1 is independently selected from halogen, hydroxy, amino, C1-C6 linear, branched alkyl (optionally substituted with 1-3 groups independently selected from hydroxy and halogen), C3-C6 cycloalkyl, and C1-C6 linear and branched alkoxy (optionally substituted with 1-3 groups independently selected from halogen).
3. The compound, deuterated derivative, or pharmaceutically acceptable salt according to Clause 1 or Clause 2, wherein each R2 is independently selected from halogen, hydroxy, amino, cyano, C1-C6 linear and branched alkyl (optionally substituted with 1-3 groups independently selected from hydroxy and halogen), and C1-C6 linear and branched alkoxy (optionally substituted with 1-3 groups independently selected from halogen).
4. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Clauses 1-3, wherein each R3 and/or R2 are fluorine.
5. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Clauses 1-4, wherein each n is selected from 0, 1, and 2.
6. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Clauses 1-5, wherein L is selected from divalent C1-C6 linear and branched alkyl, and divalent C1-C6 linear and branched thioalkyl, wherein the divalent alkyl and divalent thioalkyl are optionally substituted with 1-2 groups independently selected from halogen.
7. The compound, deuterated derivative, or pharmaceutically acceptable salt according to Clause 6, wherein L is selected from divalent C1-C3 linear and branched alkyl, and divalent C1-C3 linear and branched thioalkyl, wherein the divalent alkyl and divalent thioalkyl are optionally substituted with 1-2 groups independently selected from halogen.
8. The compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Clauses 1-7, wherein R is —C(O)NR3R4, and wherein R3 and R4 are independently selected from:
and wherein R3 is hydrogen.
19. A compound selected from Compounds 1 to 527 (Table 1), deuterated derivatives thereof, or pharmaceutically acceptable salts of any of the foregoing.
20. A pharmaceutical composition comprising the compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Clauses 1-19 and a pharmaceutically acceptable carrier.
21. A method of treating APOL1 mediated kidney disease comprising administering to a patient in need thereof a compound selected from compounds of Formula II′:
deuterated derivatives thereof, and pharmaceutically acceptable salts of any of the foregoing, wherein:
(i) R is selected from —C(O)NR3R4, —NR5C(O)R3, —NR5C(O)NR3R4, —NR3R4, —OR3, —NRX—SO2R3, —OC(O)NR3R4, —C(O)OR3
(ii) L is selected from divalent C1-C6 linear and branched alkyl, divalent C2-C6 linear and branched alkenyl, divalent C2-C6 linear and branched alkynyl, and divalent 1 to 6 membered heteroalkyl, wherein the divalent alkyl and divalent heteroalkyl are optionally substituted with 1-4 groups selected from:
and wherein R3 is hydrogen.
63. A method of treating APOL1 mediated kidney disease comprising administering to a patient in need thereof the compound, deuterated derivative, or pharmaceutically acceptable salt according to any one of Clauses 1 to 19 or the composition according to claim 20.
64. The compound according to any one of Clauses 1 to 19, or the composition according to Clause 20 for use in treating APOL1 mediated kidney disease.
65. Use of a compound according to any one of Clauses 1 to 19 in the manufacture of a medicament for treating APOL1 mediated kidney disease.
66. The method, compound for use, or use according to any one of Clauses 63-65, wherein the APOL1 mediated kidney disease is selected from ESKD, NDKD, FSGS, HIV-associated nephropathy, sickle cell nephropathy, diabetic neuropathy, arterionephrosclerosis, lupus nephritis, microalbuminuria, and chronic kidney disease.
67. The method, compound for use, or use according to Clause 66, wherein the APOL1 mediated kidney disease is FSGS.
68. The method, compound for use, or use according to Clause 66, wherein the APOL1 mediated kidney disease is NDKD.
69. The method, compound for use, or use according to Clause 66, wherein the APOL1 mediated kidney disease is ESKD.
70. The method, compound for use, or use according to any one of Clauses 63-69, wherein the APOL1 is associated with APOL1 genetic alleles chosen from homozygous G1:
In order that the disclosure described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this disclosure in any manner.
Throughout the synthetic schemes and descriptions for preparing compounds of Formula I, I′, II, or II′, Compounds 1 to 527, deuterated derivatives of any of those compounds, and pharmaceutically acceptable salts of any of the foregoing, the following abbreviations are used:
Scheme 1 refers to processes for preparation of compounds of Formula 1-6 from compounds of Formula 1-1 or 1-4. X1 and X2 are halogens such as Cl, I, or Br. Any suitable conditions for coupling an alkyne to a can be used to convert aryl halides of Formula 1-1 and alkynes of Formula 1-4 to an alkyne of Formula 1-3. For example, the coupling may be performed in the presence of a CuI and Pd(PPh3)2Cl2 catalyst system. The reaction may be performed in the presence of a base (e.g. NEt3). Conversion of compounds of Formula 1-3 to indoles of Formula 1-6 may be accomplished by treatment with CuI or PdCl2 in a polar solvent (e.g. DMF or MeCN) in the presence of added heat (>100° C.). A compound of Formula 1-3 may also be prepared from a compound of Formula 1-4 and an aryl halide of Formula 1-5. Any suitable Sonagashira coupling condition may be used. For example, Pd(PPh3)2Cl2 and CuI in the presence of a base such as DIPEA or NEt3.
Scheme 2 refers to a process for preparation of compounds of Formula 1-6 from an indole such as that represented by Formula 2-1, and an alkyl halide of Formula 2-2, where X3 is a halogen (e.g., I or Br). R20 is an alkyl group such as Me or Et. The two R20 groups may be linked by a carbon carbon bond to form a cyclic boronate ester. In some embodiments, the reaction is performed in the presence of a catalyst such as PdCl2CN2, a ligand such as norbornylene, and a base (e.g., K2CO3). The reaction may be performed in a solvent such as dimethylacetamide at elevated temperature (e.g., 90° C.). Compounds of Formula 1-6 may also be prepared from indoles of Formula 2-1 and aryl boronic acids or esters of Formula 2-3. In some embodiments, the reaction is performed in the presence of a palladium catalyst (e.g., Pd(OAc)2 trimer) in a solvent such as AcOH. The reaction is performed in the presence of oxygen.
Scheme 3 refers to a process for the preparation of compounds of Formula 3-4 which may be used in the preparation of compounds of Formula 1. PG1 is any suitable group for the protection of a carboxylic acid as an ester. For example, PG1 may be methyl, ethyl, tert-Butyl or benzyl. Any suitable conditions for a performing a Fisher indole synthesis may be used in the reaction of a ketone of Formula 3-1 with a hydrazine of Formula 3-2. For example, ZnCl2 in a solvent such as AcOH and toluene at elevated temperature (110° C.). In an alternative embodiment, BF3.OEt2 in xylene solvent in the presence of added heat may be used. Any suitable conditions for the hydrolysis of an ester may be used in the preparation of compounds of Formula 3-4 from compounds of Formula 3-3.
Scheme 4 refers to processes for the preparation of compounds of Formula 4-4. PG2 refers to any suitable group for the protection of an amine. For example, PG2 may be Boc or CBz. A compound of Formula 4-4 may be prepared from a compound of Formula 4-1 and a hydrazine of Formula 4-2 using any suitable Fisher indole synthesis conditions. In some embodiments, ZnCl2 and AcOH may be used. The reaction may be performed in the presence of added heat. Hydrazines of Formula 4-2 may be used as free bases or as salts, such as the hydrochloride salt. A compound of Formula 4-4 may be prepared from compounds of Formula 4-3 using any suitable method for the removal of a nitrogen protecting group. For example, where PG2 is a CBz group, hydrogenation conditions such as hydrogen gas in the presence of a catalyst such as palladium on carbon and a solvent such as EtOH may be used.
Scheme 5 refers to processes for the preparation of Formula 5-5 from compounds of Formula 2-1 by reductive alkylation reactions with acetals of Formula 5-2. In some alternative embodiments, acetals of Formula 5-2 may be substituted with their corresponding aldehydes. PG1 is any suitable ester protecting group as defined above. L1 is any suitable linker group which is defined within formula I. Z1 is any suitable alkyl group (for example, ethyl or methyl). A compound of Formula 5-4 may be prepared from a compound of Formula 2-1 by reaction with an aldehyde of Formula 5-2. The reaction may be performed in the presence of an acid such as methanesulfonic acid or trifluoroacetic acid, and a reducing agent such as Et3SiH. Any suitable conditions, such as those for the hydrolysis of an ester, may be used for converting a compound of Formula 5-4 to a compound of Formula 5-5. For example, the reaction may be performed in the presence of a base (e.g., LiOH or NaOH) in an aqueous solvent mixture (e.g., THF and water).
Scheme 6 shows processes for the preparation of compounds of Formula 6-4 from compounds of formula 2-1 and acetals of Formula 6-2. Z1 is any suitable alkyl group (for example, ethyl or methyl). PG2 is any suitable nitrogen protecting group. L1 is any suitable linker group which is defined within Formula I. Any suitable conditions for reductive alkylation may be used to prepare compounds of Formula 6-3.
Scheme 7 shows processes for the preparation of compounds of Formula 7-2 from Formula 7-1. PG3 may be an alkyl group such as Et or Me. PG3 may also be hydrogen. L3 is any suitable linker group as described with Formula 1. Any suitable conditions for the reduction of an ester or carboxylic acid to an alcohol may be used. For example, a reducing agent such as LiAlH4 may be used. The reduction may be performed in a solvent such as THF. The reaction may be performed at reduced temperature, for example, at 0° C.
Compounds of Formula 8-1 may be prepared from compounds of Formula 2-1 using any suitable halogenating reagent (e.g., N-iodosuccinimide). Any suitable alkyne coupling reactions can be used for converting compounds of Formula 8-1 to such as those of Formula 8-3. For example, the reaction is performed in the presence of catalysts such as Pd(PPh3)2Cl2 and CuI, and a base (e.g., DIPEA or TEA).
Scheme 9 depicts processes for the preparation of compounds of Formula 9-2. Any suitable conditions, such as those for the formation of an amide from a carboxylic acid can be used for reacting a compound of Formula 5-5 with an amine of Formula 9-1 to provide compounds of Formula 9-2. In some embodiments, processes for preparing compounds of Formula 9-2 comprise reacting a compound of Formula 5-5 with an amine of Formula 9-1 in the presence of an amide coupling agent (e.g., HATU, CDMT, HDMC, or T3P) and a suitable base (e.g., DIPEA or TEA), as depicted in Scheme 9. In some embodiments, at least one solvent is DMF or dichloromethane.
Scheme 10 shows processes for the preparation of compounds of Formula 10-2 from an amine of Formula 4-4 and a carboxylic acid of Formula 10-1. Any suitable method for performing an amide coupling may be used.
Scheme 11 describes processes for the preparation of ureas of Formula 11-3. An intermediate of Formula 11-1 where LG′ is any suitable leaving group, may be prepared from an amine of Formula 4-4. For example, LG′ may be an activated phenol group such as p-nitro phenol. Compounds of Formula 11-1 may be prepared by treatment of an amine of Formula 4-4 with a carbonate reagent such as p-nitrophenol carbonate or (4-nitrophenyl) carbonochloridate. The reaction may be performed in a basic solvent such as pyridine. In alternative conditions, compounds of Formula 11-1 may be prepared by treatment with p-nitrophenol carbonate in the presence of a base such as DIPEA, in a solvent such as DMF. Addition of an amine of Formula 11-2 to a solution of an intermediate of Formula 11-1 afforded a compound of Formula 11-3. The reaction may be performed at room temperature or with added heat.
Scheme 12 shows processes for the preparation of sulfonamides of Formula 12-2. LG2 represents any suitable leaving group atom or group. For example, LG2 may be a chlorine atom. Reaction of an amine of Formula 4-4 with a sulfonyl reagent of Formula 12-1 in the presence of a base such as DIPEA and in a solvent such as DMF.
A process depicting methods for the preparation of carbamates of Formula 13-3 is shown in scheme 13. LG3 represents any suitable leaving group atom or group. For example, LG2 may be a p-nitrophenol group intermediate of Formula 13-1 is prepared from alcohols of Formula 7-2 and a reagent such as (4-nitrophenyl) carbonochloridate in a solvent such as pyridine. The reaction may be performed at room temperature. A compound of Formula 13-2 may be prepared from a p-nitrophenol group intermediate of Formula 13-1 by treatment with an amine of Formula 13-2. In some embodiments, the reaction may be performed in a solvent such as DMF. A base such as pyridine may be present. The reaction may be performed in the presence of added heat (e.g., 80° C.).
Unless otherwise stated, all final products were purified, as necessary, by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: 10-100% MeCN in H2O. Modifier: 0.2% formic acid or 0.1% Trifluoroacetic acid).
Products were analyzed by LCMS methods A, B, or C. LCMS m/z and retention times were collected.
LCMS Method A: HPLC Sunfire C18 column. Gradient: 2-98% MeCN/H2O over 3.8 minutes. TFA Modifier.
LCMS Method B: UPLC CSH C18 column. Gradient: 5-95% MeCN/H2O. TFA Modifier.
LCMS Method C: UPLC CSH C18 column. Gradient: 10-60% MeCN/H2O. TFA Modifier.
Potassium (2R,3R)-2,3,4-trihydroxybutanoate C1 (10 g, 57.1 mmol) was stirred with HBr in acetic acid (154 g, 103 mL of 30% w/w, 570.8 mmol) for 16 hours. Anhydrous MeOH (250 mL) was added and the mixture heated at reflux for 4 hours. The mixture was concentrated to dryness and the residue dissolved in EtOAc (100 mL). The solution was washed with water (50 mL) and brine (50 mL), then dried over Na2SO4, and concentrated in vacuo. Purification by silica gel chromatography (Gradient: 15-20% EtOAc in hexane) afforded the product as a colorless liquid. Methyl (2S,3R)-2,4-dibromo-3-hydroxy-butanoate (13 g, 83%). 1H NMR (400 MHz, Chloroform-d) δ 4.71 (d, J=3.4 Hz, 1H), 4.17-4.14 (m, 1H), 3.82 (s, 3H), 3.53-3.44 (m, 2H).
Potassium (2R,3R)-2,3,4-trihydroxybutanoate C1 (280 g) was stirred with a 33% solution of HBr in acetic acid (1 L) at room temperature for 24 hours. The reaction mixture was then poured into MeOH (5 L). The mixture was stirred at room temperature for 8 hours, then at 65° C. for 4 hours. The mixture was concentrated, the residue was dissolved in MeOH (1.2 L) and then concentrated sulfuric acid (30 mL) was slowly added. The mixture was heated under reflux for 6 hours, then concentrated. The residue was taken up with EtOAc (400 mL). The resulting solution was washed with water (250 mL), dried over Na2SO4, filtered and concentrated in vacuo to give the product as an oil which solidified upon storage at 4° C. (375 g, 74%).
Methyl (2R,3R)-2,4-dibromo-3-hydroxy-butanoate C2 (524.8 g, 1.9 mol) was dissolved in acetone (4.5 L) in a 12 L round-bottomed flask equipped with an overhead stirrer. The reaction was cooled to 0° C. in an ice-bath and Cs2CO3 (994 g, 3.1 mol) was added. The reaction was stirred for 30 minutes at 0° C. and then for 2 hours at room temperature. The mixture was filtered, washed with acetone, and then concentrated in vacuo to afford a dark gray oil residue. The product was dissolved in CH2Cl2 and filtered over a short plug of silica gel, eluting with CH2Cl2 (approximately 1 L). The filtrate was concentrated in vacuo to afford the product as a clear yellow oil (377.3 g, quantitative). 1H NMR (300 MHz, Chloroform-d) δ 3.83 (s, 3H), 3.71-3.61 (m, 2H), 3.61-3.53 (m, 1H), 3.46 (dd, J=9.9, 6.6 Hz, 1H). 13C NMR (75 MHz, Chloroform-d) δ 167.58, 55.89, 53.52, 52.77, 26.83.
To a solution of methyl (2R,3R)-2,4-dibromo-3-hydroxy-butanoate C2 (200 g, 0.73 mol) in acetone (2.0 L) was added anhydrous K2CO3 (151.1 g, 1.1 mol), while the reaction temperature was maintained at 0-5° C. The reaction was stirred at 0-5° C. for 2 hours, then gradually warmed to room temperature over 4 hours The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was distilled under vacuum 75-80° C./200-300 Pa to give the product as a colorless liquid (105 g, 74%).
Methyl (2R,3S)-3-(bromomethyl)oxirane-2-carboxylate C3 (52.6 g, 269.7 mmol) was dissolved in DMF (500 mL) in a 3 L round-bottomed flask equipped with a magnetic stir bar. NaN3 (25.3 g, 388.4 mmol) was added and the mixture was stirred at room temperature for 1 hour. The reaction was poured into water, and extracted with EtOAc. The extract was washed with water, dried over MgSO4, and concentrated in vacuo to afford a dark red oil. The oil residue was dissolved in CH2Cl2, and filtered over a plug of silica gel eluting with CH2Cl2. The filtrate was concentrated in vacuo to afford the product, C4, as a clear, light red oil (40.8 g, 96%). 1H NMR (300 MHz, Chloroform-d) δ 3.87-3.74 (m, 3H), 3.67-3.55 (m, 2H), 3.47 (dd, J=13.3, 5.1 Hz, 1H), 3.38 (ddd, J=6.3, 5.0, 4.4 Hz, 1H). 13C NMR (75 MHz, Chloroform-d) δ 167.76, 54.81, 52.67, 51.32, 48.74.
A 2 L 3-neck flask with overhead stirrer was charged with methyl (2R,3R)-3-(azidomethyl)oxirane-2-carboxylate C4 (67 g, 402.5 mmol) in toluene (500 mL), stirred for 10 minutes, and then warmed to 80° C. Bu3SnH (220 mL, 817.8 mmol) and AIBN (2 g, 12.2 mmol) were dissolved in toluene (500 mL) and then added to the reaction over 3 hours using an additional funnel. The resulting reaction mixture was stirred at 80-87° C. for 1 hour, then cooled to ambient temperature, and concentrated under reduced pressure. The residue was partitioned between acetonitrile (2 L) and pentane (1 L), stirred for 10 minutes and then the acetonitrile phase (bottom) was separated. The acetonitrile phase was washed with pentane (2×500 mL) and concentrated in vacuo to afford a light yellow solid. The solid residue was triturated with pentane (˜200 mL) to afford the product as a yellow solid which was used without further purification (52 g, 98%). 1H NMR (300 MHz, Chloroform-d) δ 5.89 (s, 1H), 4.00 (q, J=2.5 Hz, 1H), 3.74-3.50 (m, 2H), 3.44 (dd, J=12.4, 2.4 Hz, 1H). 13C NMR (75 MHz, Chloroform-d) δ 173.24, 53.28, 52.18, 44.00.
A Parr vessel containing (1R,5R)-6-oxa-3-azabicyclo[3.1.0]hexan-2-one C5 (60 g, 605.5 mmol) and NH3 (1.5 L, 58.6 mol) was pressurized to 200 psi and allowed to stir at 18° C. for 2 days. NH3 was released from the vessel to provide a grey solid. Heptane was added and the mixture stirred for 30 minutes. The solid was filtered, and then the filter cake was isolated, and then EtOAc and heptane to the solid. The mixture was concentrated in vacuo to afford the product (55 g, 78%). 1H NMR (300 MHz, Water-d2) δ 4.13 (q, J=7.2 Hz, 1H), 3.53 (dd, J=10.4, 7.4 Hz, 1H), 3.36 (d, J=7.5 Hz, 1H), 3.05 (dd, J=10.4, 6.8 Hz, 1H).
At −60° C., ammonia gas was condensed into an autoclave containing a frozen solution of methyl (2R,3S)-3-(bromomethyl)oxirane-2-carboxylate C3 (81 g, 0.42 mol) in 1,4-dioxane (160 mL) until approx. 400 mL of liquid was collected. The autoclave was closed, allowed to warm gradually to room temperature and then heated at 50-60° C. for 2 hours. The autoclave was then cooled back to −60° C. and depressurized. The reaction mixture was warmed gradually to allow the liquid ammonia to evaporate, leaving a viscous residue. The residue was taken up with MeOH (500 mL) and the suspension was treated with a 28% solution of sodium methoxide in MeOH (86 g, 0.42 mol). The mixture was stirred at room temperature for 30 minutes then concentrated. The residue was dissolved in water (500 mL), then Na2CO3 (89 g, 0.84 mol) and a solution of Boc2O (110 g, 0.5 mol) in THF (200 mL) was added. The mixture was stirred at room temperature for 10 hours. The aqueous phase was then saturated with NaCl solution and extracted with THF (3×200 mL). The combined organic phases were dried over Na2SO4 and concentrated in vacuo. The residue was triturated with warm MTBE (200 mL) and the precipitated solid was collected by filtration, washed with MTBE and dried under vacuum to afford the product as a white solid (28 g, 31%).
To solution of N-Boc-(3S,4R)-3-amino-4-hydroxypyrrolidin-2-one C7 (28 g, 129 mmol) in EtOH (300 mL) heated at 50-60° C. was added a solution of HCl in EtOH (5.0 M, 75 mL). The reaction mixture was kept at 50-60° C. for 2 hours. The suspension was cooled to room temperature and the solid was collected by filtration, washed with EtOH and dried in vacuo to afford the product as an off-white solid (18 g, 90%). 1H NMR (500 MHz, DMSO-d6) δ 8.73 (br, 3H), 8.28 (s, 1H), 6.03 (s, 1H), 4.42-4.37 (m, 1H), 3.74 (d, J=6.8 Hz, 1H), 3.48-3.39 (m, 1H), 3.03-3.00 (m, 1H).
To a solution of tert-butyl N-[(3R)-2-oxopyrrolidin-3-yl]carbamate C8 (458.6 mg, 2.290 mmol) in methanol (4 mL) was added HCl (2 mL of 4 M, 8.000 mmol) in 1,4-dioxane. Reaction was stirred at 50° C. for 30 minutes. Solvent was evaporated and crude product was washed with ether to afford crude (3R)-3-aminopyrrolidin-2-one (S2) (Hydrochloride salt) (350 mg, quantitative). LCMS m/z 130.05 [M+H]+.
To a suspension benzyl N-[(3R)-2,5-dioxopyrrolidin-3-yl]carbamate C9 (862 mg, 3.47 mmol) in MeOH (10 mL) and EtOAc (5 mL) was added 5% palladium on carbon catalyst (20 mg). The mixture was subjected to hydrogenation conditions of 50 psi H2 for 2 hours. Filtration through a pad of Celite®, washing with MeOH and CH2Cl2, then concentration of the filtrate in vacuo afforded the product (398 mg, 98%). 1H NMR (300 MHz, Methanol-d4) δ 3.86 (dd, J=8.8, 5.5 Hz, 1H), 2.98 (dd, J=17.9, 8.8 Hz, 1H), 2.43 (dd, J=17.9, 5.5 Hz, 1H). LCMS m/z 123.74 [M+H]+.
To a suspension of ethyl 1-aminocyclopropanecarboxylate C10 (Hydrochloride salt) (3.7 g, 22 mmol) in THF (100 mL) was added lithium boranuide (1 g, 45.91 mmol). Reaction bubbled immediately after addition. Reaction was stirred for 24 hours. MeOH (10 mL) was added. Solvent was removed in vacuo. MeOH (10 mL) was added and the solvent was again removed in in vacuo to give (1-aminocyclopropyl)methanol (S4) (720 mg, 37%). 1H NMR (300 MHz, Chloroform-d) δ 3.74 (s, 1H), 1.32-1.05 (m, 2H), 0.92-0.55 (m, 1H).
To a solution of 1H-pyrazole-4-carbonitrile C11 (10 g, 107.4 mmol) and pyridine (26 mL, 321.5 mmol) in DCM (350 mL) was added Tf2O (25 mL, 148.6 mmol) dropwise. The reaction was stirred at 30° C. Reaction turned orange. After complete addition the reaction was stirred for 3 hours. After 24 hours Tf2O (10 mL, 59.44 mmol) was added. Reaction was stirred for 72 hours. H2O (2 L) was added and aqueous layer was extracted with DCM (100 mL). Organic layer was washed with 1 M HCl (500 mL), extracted with DCM (50 mL) and combined with other organics. Combined organic extracts were dried over MgSO4, filtered, and concentrated to give 1-(trifluoromethylsulfonyl)pyrazole-4-carbonitrile C12 (20.54 g, 85%). 1H NMR (400 MHz, Chloroform-d) δ 8.54 (d, J=0.6 Hz, 1H), 8.20 (d, J=0.5 Hz, 1H).
To a solution of 3,3-difluorocyclobutanol (1.06 g, 9.81 mmol) in MeCN (10 mL) was added Cs2CO3 (3.5 g, 10.74 mmol). Reaction was cooled to 0° C. A solution of 1-(trifluoromethylsulfonyl)pyrazole-4-carbonitrile C12 (2 g, 8.883 mmol) in MeCN (10 mL) was slowly added to the reaction mixture keeping the temperature below 30° C. Reaction was warmed to room temperature and stirred for 30 minutes. Solids were filtered off and solvent was removed under reduced pressure. Water (30 mL) and DCM (30 mL) were added. The organic layer was separated and washed with brine, dried over Na2SO4, filtered, and the solvent was removed in vacuo. Silica gel chromatography (Gradient: 0-100% EtOAc in heptane) afforded the product 1-(3,3-difluorocyclobutyl)pyrazole-4-carbonitrile C13 (1.38 g, 81%). 1H NMR (400 MHz, Chloroform-d) δ 7.90 (d, J=4.3 Hz, 2H), 4.88-4.66 (m, 1H), 3.42-3.08 (m, 4H).
To a solution of 1-(3,3-difluorocyclobutyl)pyrazole-4-carbonitrile C13 (2.02 g, 11.03 mmol) in MeOH (80 mL) was added Raney nickel (approximately 187.1 mg, 21.02 μL, 3.187 mmol) and ammonia (100 mL of 7 M, 700.0 mmol). The mixture was subjected to hydrogenation conditions of 50 psi H2 for 2 hours. Filtration through a pad of Celite®, washing with MeOH, then concentration of the filtrate in vacuo afforded [1-(3,3-difluorocyclobutyl)pyrazol-4-yl]methanamine (S5) (2.0405 g, 99%). 1H NMR (400 MHz, Chloroform-d) δ 7.53 (d, J=7.2 Hz, 1H), 7.41 (s, 1H), 4.74-4.59 (m, 1H), 3.80 (s, 2H), 3.35-2.98 (m, 4H).
To a solution of (1S,2S)-1,2-bis[(4R)-2,2-dimethyl-1,3-dioxolan-4-yl]ethane-1,2-diol C14 (30 g, 114.4 mmol) in DCM (300 mL) was added NaHCO3 (12 mL) and the mixture was cooled to 10° C. before NaIO4 (49 g, 229.1 mmol) was added portion wise at such a rate that internal temperature stayed below 5° C. The reaction was stirred at room temperature for 2 hours, then was filtered on Celite®. The cake was washed with DCM and the filtrate was concentrated in vacuo, yielding (4R)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde (28.0 g, 94%) 1H NMR (400 MHz, DMSO-d6) δ 9.60 (s, 1H), 4.52 (ddd, J=7.3, 4.6, 1.6 Hz, 1H), 4.13-4.05 (m, 2H), 1.38 (s, 3H), 1.33 (s, 3H).
To a solution of (4R)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde C15 (9.9 g, 76 mmol) (prepared freshly) in DCM (90 mL) was added DAST (14.714 g, 12.061 mL, 91.285 mmol) at 0° C. After the addition, the solution was stirred at room temperature for 3 hours. The reaction was quenched by addition of 15% sodium bicarbonate aqueous solution. The organic layer was separated, and the aqueous layer was extracted with DCM (3×30 mL). The combined organic extracts were dried over magnesium sulfate, filtered, and the solvent carefully removed in vacuo to give crude product (4R)-4-(difluoromethyl)-2,2-dimethyl-1,3-dioxolane (12 g, quantitative). 1H NMR (400 MHz, Chloroform-d) δ 5.81-5.53 (m, 1H) 4.28-4.18 (m, 1H), 4.13-4.01 (m, 2H), 1.45 (s, 3H), 1.37 (s, 3H). The crude material was carried to the next step without further purification.
To a stirred solution of (4R)-4-(difluoromethyl)-2,2-dimethyl-1,3-dioxolane C16 (11.57 g, 76.049 mmol) in MeCN (95 mL) and H2O (5 mL), was added Pd(MeCN)2Cl2 (440.33 mg, 1.5210 mmol). Then the reaction mixture was heated at 60° C. for 5 hours. The reaction mixture was cooled to room temperature, filtered and the solvent was evaporated under reduced pressure. The crude product was dissolved in DCM (200 mL), followed by addition of imidazole (6.2127 g, 91.259 mmol). To the resulting solution was added dropwise a solution of TBSCl (12.608 g, 83.654 mmol) in DCM at 0° C. After addition the ice-bath was removed, and the solution was stirred at room temperature overnight. The resulting solution was diluted with DCM (60 mL) and water (60 mL). The organic layer was separated, and the water layer was extracted with DCM (2×30 mL). The combined organic layer was washed with water (2×30 mL) and brine (40 mL), dried over sodium sulfate, filtered and the solvent was removed in vacuo. Silica gel chromatography (Gradient: 5-10% EtOAc in heptane) afforded the product (2R)-3-[tert-butyl(dimethyl)silyl]oxy]-1,1-difluoro-propan-2-ol (8.6 g, 50%). 1H NMR (400 MHz, Chloroform-d) δ 5.29 (s, 1H), 3.83-3.70 (m, 2H), 2.58 (d, J=6.3 Hz, 1H), 1.25 (s, 1H), 0.90 (d, J=3.4 Hz, 10H), 0.09 (d, J=1.4 Hz, 6H).
To a stirred solution of (2R)-3-[tert-butyl(dimethyl)silyl]oxy-1,1-difluoro-propan-2-ol C17 (8.6 g, 37.997 mmol) in DCM (150 mL) was added pyridine (5.4100 g, 5.5317 mL, 68.395 mmol) and trifluoromethanesulfonic anhydride (13.937 g, 8.1982 mL, 49.396 mmol) in DCM (50 mL) dropwise at −20° C. The resulting mixture was stirred at −10° C., and then stirred at 0° C. for 2 hours. To the reaction mixture was added water (200 mL) and the aqueous layer was extracted with DCM (2×200 mL). The combined organic phase was washed with water and brine, then dried over Na2SO4 and evaporated under reduced pressure to get the crude product [(1R)-1-[[tert-butyl(dimethyl)silyl]oxymethyl]-2,2-difluoro-ethyl]trifluoromethanesulfonate (9 g, 66%). 1H NMR (400 MHz, Chloroform-d) δ 6.00 (td, J=4.32, 54.68 Hz, 2H), 4.88-4.82 (m, 1H), 4.12-4.10 (m, 1H), 3.98 (d, J=4.2 Hz), 0.85 (s, 9H), 0.13 (s, 6H).
To a stirred solution of [(1R)-1-[[tert-butyl(dimethyl)silyl]oxymethyl]-2,2-difluoro-ethyl] trifluoromethanesulfonate; methane C18 (9 g, 24.036 mmol) in DMF (100 mL) was added NaN3 (4.6877 g, 14.107 mL, 72.108 mmol) and stirred at room temperature for 2 hours. The reaction mixture was then diluted with water (500 mL), and extracted with DCM (2×200 mL). Combined organic layer was washed with water (3×500 mL), and brine and dried over Na2SO4 to provide crude desired compound [(2S)-2-azido-3,3-difluoro-propoxy]-cert-butyl-dimethyl-silane (5 g, 83%). 1H NMR (400 MHz, Chloroform-d) δ 5.82 (td, J=4.4, 55.28 Hz, 1H), 3.86-3.84 (m, 2H), 3.62-3.60 (m, 1H), 0.89 (s, 9H), 0.05 (s, 6H).
To a stirred solution of [(2S)-2-azido-3,3-difluoro-propoxy]-cert-butyl-dimethyl-silane C19 (5 g, 19.893 mmol) in methanol (100 mL) under argon atmosphere, was added Pd(OH)2/C (3.4 g, 20% w/w, 4.8421 mmol). The reaction was then hydrogenated under a H2 balloon atmosphere for 2 hours. The mixture was filtered through Celite® and washed with methanol. The combined organic layer was evaporated to dryness to afford the crude compound. Purification by silica gel chromatography (Gradient: 0-30% EtOAc in heptane) yielded the product (2S)-3-[tert-butyl(dimethyl)silyl]oxy-1,1-difluoro-propan-2-amine (2.6 g, 58%). 1H NMR (400 MHz, Methanol-d4) δ 5.73 (td, J=4.4, 55.28 Hz, 1H), 3.76-3.65 (m, 2H), 3.04-2.99 (m, 1H), 0.88 (s, 9H), 0.06 (s, 6H).
To a stirred solution of (2S)-3-[tert-butyl(dimethyl)silyl]oxy-1,1-difluoro-propan-2-amine C20 (400 mg, 1.7750 mmol) in MeOH (2 mL), was added HCl in dioxane (2.2188 mL of 4 M, 8.8750 mmol) at 0° C. and stirred at room temperature for 5 hours. The reaction mixture was evaporated under reduced pressure and the crude product obtained was triturated with diethyl ether (2×5 mL) and then dried properly to get the desired compound (2S)-2-amino-3,3-difluoro-propan-1-ol (hydrochloride salt) (230 mg, 88%). 1H NMR (400 MHz, DMSO-d6) δ 8.56 (s, 3H), 6.28 (td, J=3.48, 51.08 Hz, 1H), 5.56 (bs, 1H), 3.70-3.58 (m, 3H).
3-[5-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid (S7) was obtained from commercial sources. S7 may be prepared using analogous method to that described for S8.
To a flask containing 2,4-difluoro-6-iodo-aniline C23 (134 g, 525.5 mmol) was added NEt3 (1.3 L), followed by DMF (250 mL), 1-ethynyl-4-fluoro-benzene (83.5 g, 695.1 mmol), CuI (20.5 g, 107.6 mmol), and PdCl2(PPh3)2 (25 g, 35.6 mmol). The mixture was allowed to stir at room temperature for 2 hours. Solvent was removed under reduced pressure and water (500 mL) was added. The mixture was extracted with Ethyl acetate, filtered and concentrated in vacuo. The product mixture was filtered through a silica gel plug (Eluent: CH2Cl2), followed by a second silica plug filtration (Eluent: 30-40% EtOAc in heptane). Silica gel chromatography (Gradient: 0-20% EtOAc in heptane) afforded the product as a pale yellow solid. (87 g, 60%). 1H NMR (300 MHz, Chloroform-d) δ 7.58-7.45 (m, 2H), 7.14-7.02 (m, 2H), 6.92 (ddd, J=8.8, 2.8, 1.7 Hz, 1H), 6.87-6.71 (m, 1H), 4.15 (s, 2H). LCMS m/z 248.0 [M+H]+.
To a solution of 2,4-difluoro-6-[2-(4-fluorophenyl)ethynyl]aniline C24 (46 g, 167.5 mmol) in DMF (600 mL) was added CuI (1.9 g, 10.0 mmol) and the reaction was heated at reflux. Water (800 mL) was added and the mixture extracted with MTBE. The mixture was then washed with sat. NaCl solution, dried over Na2SO4 and then concentrated in vacuo to afford the product, which was used in subsequent steps without further purification (41 g, 87%). 1H NMR (300 MHz, Chloroform-d) δ 8.43 (s, 1H), 7.72-7.58 (m, 2H), 7.27-7.15 (m, 2H), 7.09 (dd, J=9.0, 2.1 Hz, 1H), 6.85-6.63 (m, 2H). LCMS m/z 248.0 [M+H]+.
A 12 L flask with overhead stirrer was charged with 5,7-difluoro-2-(4-fluorophenyl)-1H-indole C25 (300 g, 1.2 mol), CH2Cl2 (3 L), methyl 3,3-dimethoxypropanoate (195 mL, 1.4 mol) and TFA (300 mL, 3.9 mol). The reaction was heated to reflux for 4 hours. Additional CH2Cl2 was added to facilitate stirring. Upon cooling to room temperature, the solid product was filtered, washed with minimal CH2Cl2 and dried to afford the product (388 g, 96%). 1H NMR (400 MHz, DMSO-d6) δ 12.66 (s, 1H), 7.77-7.57 (m, 4H), 7.56-7.37 (m, 2H), 7.19 (ddd, J=11.0, 9.7, 2.1 Hz, 1H), 6.47 (d, J=16.1 Hz, 1H), 3.69 (s, 3H). LCMS m/z 332.4 [M+H]+.
To a suspension of methyl (E)-3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]prop-2-enoate C26 (80 g, 236.5 mmol) in EtOH (1.5 L) under a nitrogen atmosphere was added Pd(OH)2 (6 g of 20% w/w 8.5 mmol) and ammonium formate (160 g, 2.5 mol). The mixture was heated at reflux for ˜3 hours, then filtered to remove catalyst. The filtrate was concentrated in vacuo to afford the product as an off-white solid which was used without further purification (82 g, 100%). 1H NMR (300 MHz, Chloroform-d) δ 8.18 (s, 1H), 7.65-7.47 (m, 2H), 7.27-7.14 (m, 2H), 7.14-7.00 (m, 1H), 6.76 (ddd, J=10.8, 9.4, 2.2 Hz, 1H), 3.65 (s, 3H), 3.27-3.04 (m, 2H), 2.75-2.49 (m, 2H). LCMS m/z 334.3 [M+H]+.
LiOH (67 g, 2.8 mol) was added to a solution of methyl 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoate C27 (217 g, 651.1 mmol) in THF (1 L) and water (100 mL). The mixture was heated at reflux for 2 hours, and then allowed to cool overnight. THF was removed by concentration under reduced pressure, and water was added (approx. 1 L). The mixture was cooled on an ice bath and HCl (250 mL of 11.7 M, 2.9 mol) was added to adjust pH to ˜4. EtOAc (300 mL) was added, and the aqueous layer extracted with further EtOAc (100 mL). Combined organic extracts were dried over sodium sulfate (Na2SO4), filtered through a plug of silica gel rinsing with EtOAc. The filtrate was concentrated in vacuo to afford an orange oil (50-75 mL). Heptanes (˜50 mL) were added and the mixture chilled on dry ice. Upon agitation, a crystalline solid formed. The mixture was allowed to stir on an ice-bath until to allow completion of the crystallization process. The solid was filtered, washed with heptane and air dried to afford the product (S8) (208 g, 96%). 1H NMR (300 MHz, Chloroform-d) δ 8.15 (s, 1H), 7.60-7.46 (m, 2H), 7.27-7.15 (m, 2H), 7.09 (dd, J=9.1, 2.2 Hz, 1H), 6.77 (ddd, J=10.8, 9.4, 2.2 Hz, 1H), 3.26-3.05 (m, 2H), 2.78-2.57 (m, 2H). LCMS m/z 320.0 [M+H]+.
A reactor was charged with 5,7-difluoro-2-(4-fluorophenyl)-1H-indole C25 (4.0 kg, 16.5 mol), CH2Cl2 (37 L) and methyl 3,3-dimethoxypropanoate (2.6 L, 18.1 mol) followed by TFA (3.9 L, 51.0 mol) at ambient temperature. The resulting mixture was heated to reflux for 6 hours. The batch was then cooled to 20° C., charged with n-heptane (2 vol) and filtered. The filter cake was dried under vacuum at 45° C. to afford the product in ˜90% yield. 1H NMR (300 MHz, DMSO-d6) δ 12.63 (s, 1H), 7.76-7.54 (m, 4H), 7.55-7.39 (m, 2H), 7.18 (ddd, J=11.1, 9.7, 2.2 Hz, 1H), 6.46 (d, J=16.1 Hz, 1H), 3.69 (s, 3H). LCMS m/z 332.1 [M+H]+.
Methyl (E)-3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]prop-2-enoate C26 (1.5 kg, 9.06 mol) was slurried with THF (7 L) in a vessel. Pd(OH)2 (10 g of 20% w/w, ˜50% water, 0.014 mol) was charged. The mixture was purged with N2 three times, then once with H2 and the vessel pressurized to 50 psi with H2. The mixture was agitated at 20° C. until H2 uptake ceased. After 1.5 hours, the mixture was purged with N2 (3 times) and filtered through Solka-Floc using a THF (2 vol) rinse. The resulting filtrate was concentrated in vacuo at 45° C. (to 1.5 vol), charged with cyclohexane (1 vol), and concentrated again (to 1.5 vol) at 45° C. The slurry was cooled to 15-20° C. and filtered. The filter cake was then washed with cold cyclohexane (1 vol) and dried under vacuum at 45° C. to afford the product in 95% yield.
A mixture of methyl 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoate C27 (9 kg, 27 mmol) in 2-MeTHF (54 L, 6 vol) and MeOH (8.1 L, 0.9 vol) was charged with 20% KOH (2 equiv, 54 mol). The mixture was stirred at 35° C. for 6 hours. The mixture was then distilled under vacuum to 27 L (3 vol) and cooled to 10-15° C. Water (7.5 L) and 2-MeTHF (16 L) were charged and the resulting biphasic mixture was pH adjusted with 6 M HCl to a pH ˜2. The temperature was adjusted to 20° C. and the phases separated. The organic phase was washed with water (15 L), filtered through Celite® with 2-MeTHF rinse (18 L, 2 vol), and concentrated under vacuum to 18 L (2 vol). 18 L (2 vol) of n-heptane was charged and the batch again concentrated under vacuum to 18 L (3 vol). This cycle was repeated once more, and the batch was seeded. 16 L (1.8 vol) n-heptane was charged and the temperature adjusted to 20° C. The slurry was stirred for 2 hours, filtered and the cake washed with 2×18 L (2×2 vol) n-heptane. The filter cake was dried under vacuum at 45° C. to afford the desired product in 90% yield. 1H NMR (300 MHz, Chloroform-d) δ 8.28 (s, 1H), 7.53 (ddd, J=8.7, 5.4, 2.8 Hz, 2H), 7.27-7.13 (m, 2H), 7.08 (dd, J=9.1, 2.1 Hz, 1H), 6.76 (ddd, J=11.3, 9.4, 2.2 Hz, 1H), 3.91-3.69 (m, 4H), 3.28-3.07 (m, 2H), 2.79-2.53 (m, 2H), 2.00-1.74 (m, 3H). LCMS m/z 320.4 [M+H]+.
4-Chloro-2-fluoro-6-iodo-aniline (5 g, 17.498 mmol) was added to a solution of 1-ethynyl-4-fluoro-benzene C30 (2.7601 g, 2.6287 mL, 22.747 mmol) and triethylamine (4.0929 g, 5.6376 mL, 40.245 mmol) in DMF (100 mL) at room temperature and stirred for 1 hour. Water (150 mL) was added, and the mixture was stirred for 0.5 hours. The precipitate was filtrated and washed with water (100 mL). The crude material was purified by silica gel chromatography (Gradient: 0-20% EtOAc in hexane) to give 4-chloro-2-fluoro-6-[2-(4-fluorophenyl)ethynyl]aniline C31 (5.1 g, 99%) as a brown solid. 1H NMR (300 MHz, DMSO-d6) δ 7.77-7.69 (m, 2H), 7.31-7.22 (m, 3H), 7.16 (s, 1H), 5.75 (s, 2H). LCMS m/z 264.1 [M+H]+.
4-chloro-2-fluoro-6-[2-(4-fluorophenyl)ethynyl]aniline C31 (4.85 g, 16.555 mmol) and palladium(II) chloride (593.06 mg, 3.3110 mmol) were suspended in acetonitrile (485.00 mL) and stirred at 80° C. under argon atmosphere for 5 hours. Then acetonitrile was evaporated under reduced pressure (2 mbar, 40° C.). The residue was dissolved in DCM (200 mL) and washed with water (100 mL) and brine (100 mL). Organic layer was dried over MgSO4 and concentrated to dryness. The crude material (6.1 g) was purified by column chromatography (Gradient: 0-4% EtOAc in hexane) to give 5-chloro-7-fluoro-2-(4-fluorophenyl)-1H-indole C32 (3.7 g, 81%), as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 12.08 (s, 1H), 7.98 (dd, J=8.7, 5.4 Hz, 2H), 7.44 (s, 1H), 7.32 (t, J=8.8 Hz, 2H), 7.07 (d, J=10.8 Hz, 1H), 6.96 (d, J=3.3 Hz, 1H). LCMS m/z 264.2 [M+H]+.
To 5-chloro-7-fluoro-2-(4-fluorophenyl)-1H-indole C32 (654 mg, 2.480 mmol) in toluene (10 mL) was added methanesulfonic acid (251 μL, 3.868 mmol) and triethylsilane (1.3 mL, 8.139 mmol) followed by methyl 3,3-dimethoxypropanoate (440 μL, 3.103 mmol). The reaction was heated at 70° C. for 2 hours. Water (50 mL) was added, and the aqueous layer was extracted with EtOAc (3×30 mL). The combined organic layer was dried over MgSO4 and concentrated to dryness. The crude material was purified by silica gel chromatography (Gradient: 0-100% EtOAc in hexane) to provide methyl 3-[5-chloro-7-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoate C33 (528 mg, 59%). 1H NMR (300 MHz, Methanol-d4) δ 7.76-7.50 (m, 2H), 7.37 (d, J=1.7 Hz, 1H), 7.30-7.10 (m, 2H), 6.90 (dd, J=10.8, 1.7 Hz, 1H), 3.57 (s, 3H), 3.25-3.00 (m, 2H), 2.60 (dd, J=8.4, 7.0 Hz, 2H). LCMS m/z 350.16 [M+H]+.
A mixture of methyl 3-[5-chloro-7-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoate C33 (526 mg, 1.470 mmol) and LiOH (353 mg, 14.74 mmol) in methanol (4 mL), THF (4 mL), and water (4 mL) was stirred at 60° C. for 3 hours. The reaction mixture was concentrated, and added water (50 mL), then acidified with concentrated HCl to pH=1. The aqueous layer was extracted with DCM (3×50 mL). The combined organic layer was washed with brine, dried over Na2SO4, and evaporated to afford product S9 (545 mg, quantitative). LCMS m/z 335.99 [M+H]+.
To a mixture of 1-ethynyl-4-fluoro-benzene (1.74 g, 1.66 mL, 14.3 mmol), Et3N (2.58 g, 3.56 mL, 25.4 mmol), CuI (343 mg, 1.77 mmol) and PdCl2(PPh3)2 (395 mg, 0.55 mmol) in DMF (55 mL) was added 2-fluoro-6-iodo-4-methyl-aniline C34 (2.77 g, 11.0 mmol). The resulting suspension was stirred at ambient temperature for 2 hours. The reaction was quenched with H2O (100 mL) and extracted with DCM (3×100 mL). The combined organic extracts were dried over MgSO4, filtered and concentrated to afford the title compound as a brown solid (3.2 g, 95%). The crude was used in the subsequent step without further purification. 1H NMR (300 MHz, DMSO-d6) δ 7.73-7.65 (m, 2H), 7.26 (t, J=8.9 Hz, 2H), 6.92 (t, J=5.8 Hz, 2H), 5.28 (s, 2H), 2.15 (s, 3H). LCMS m/z 244.1 [M+H]+.
2-Fluoro-6-[2-(4-fluorophenyl)ethynyl]-4-methyl-aniline C35 (3.2 g, 10.5 mmol) and PdCl2 (189 mg, 1.05 mmol) were suspended in acetonitrile (320 mL) under an argon atmosphere. The reaction was stirred at 80° C. for 5 hours. The volatile was then removed in vacuo. The crude was dissolved in DCM (100 mL) and washed with H2O (50 mL) and brine (50 mL). The organic layer was separated and dried over MgSO4, filtered and concentrated. The crude was purified using silica gel chromatography (0-2% EtOAc in hexanes) to afford C36 (1.82 g, 69%). 1H NMR (300 MHz, DMSO-d6) δ 11.69 (s, 1H), 7.96 (dd, J=8.7, 5.4 Hz, 2H), 7.29 (t, J=8.8 Hz, 2H), 7.13 (s, 1H), 6.86 (t, J=2.8 Hz, 1H), 6.76 (d, J=12.4 Hz, 1H), 2.36 (s, 3H). LCMS m/z 244.2 [M+H]+.
To a solution of 7-fluoro-2-(4-fluorophenyl)-5-methyl-1H-indole C36 (518 mg, 2.13 mmol) in toluene (10 mL) was added sequentially methanesulfonic acid (220 μL, 3.39 mmol), triethylsilane (1.2 mL, 7.51 mmol) and methyl 3,3-dimethoxypropanoate (380 μL, 2.68 mmol). The resulting mixture was stirred at 80° C. overnight. The reaction was then cooled down to ambient temperature and quenched with H2O (50 mL), extracted with EtOAc (3×30 mL). The combined organic extracts were concentrated. The crude was purified using silica gel chromatography (0-100% EtOAc in hexanes) to afford the title compound as an off-white solid C37 (481 mg, 69%). 1H NMR (400 MHz, Chloroform-d) δ 8.05 (s, 1H), 7.59-7.47 (m, 2H), 7.26-7.11 (m, 3H), 6.79 (dd, J=12.0, 1.2 Hz, 1H), 3.67 (s, 3H), 3.28-3.11 (m, 2H), 2.76-2.61 (m, 2H), 2.50 (s, 3H). LCMS m/z 330.1 [M+H]+.
To a solution of methyl 3-[7-fluoro-2-(4-fluorophenyl)-5-methyl-1H-indol-3-yl]propanoate C37 (480 mg, 1.46 mmol) in MeOH (4 mL), THF (4 mL) and H2O (4 mL) was added LiOH (480 mg, 1.46 mmol). The resulting mixture was stirred at 60° C. for 3 hours. The volatile was removed and H2O (50 mL) was added. The pH of the reaction mixture was adjusted to 1 using concentrated HCl. The reaction was extracted with DCM (3×50 mL) and the combined organic extracts were washed with brine, dried over Na2SO4, filtered and concentrated to afford S10 (489 mg, 100%). LCMS m/z 316.1 [M+H]+.
To a solution of 4-bromo-2-fluoro-6-iodo-aniline C38 (40 g, 126.6 mmol) in DMF (80 mL) was added TEA (400 mL), 1-ethynyl-4-fluoro-benzene (20 g, 166.5 mmol), iodocopper (4 g, 21.00 mmol), and PdCl2(PPh3)2 (4.6 g, 6.6 mmol). Reaction was stirred at room temperature for 5 hours. Solvent was removed under reduced pressure and water (500 mL) was added. The mixture was extracted with MTBE, filtered and concentrated in vacuo. The product mixture was filtered through a silica gel plug (Eluent: CH2Cl2), followed by a second silica plug filtration (Eluent 20% EtOAc in heptane). Silica gel chromatography (Gradient: 0-15% EtOAc in heptane) afforded the product 4-bromo-2-fluoro-6-[2-(4-fluorophenyl) ethynyl] aniline (C39) (32 g, 66%). 1H NMR (300 MHz, Chloroform-d) δ 7.63-7.42 (m, 2H), 7.34-7.25 (m, 1H), 7.22-6.97 (m, 3H), 4.31 (s, 2H). LCMS m/z 308.21 [M+H]+.
A solution of 4-bromo-2-fluoro-6-[2-(4-fluorophenyl) ethynyl] aniline C39 (32 g, 103.9 mmol) in DMF (400 mL) was heated to 150° C. for 4 hours. The reaction was then stirred at room temperature overnight. Iodocopper (2 g, 10.50 mmol) was added and the reaction was heated to 150° C. for 3 hours. Reaction was cooled and water (800 mL) was added. The mixture was extracted with MTBE, washed with sat. NaCl and water, dried over sodium sulfate, filtered and concentrated in vacuo. The product mixture was filtered through a silica gel plug (Eluent 20% EtOAc in heptane). Silica gel chromatography (Gradient: 0-20% in heptane) afforded the product 5-bromo-7-fluoro-2-(4-fluorophenyl)-1H-indole (C40) (14.4 g, 45%). 1H NMR (300 MHz, Chloroform-d) δ 8.47 (s, 1H), 7.73-7.59 (m, 2H), 7.55 (dd, J=1.5, 0.7 Hz, 1H), 7.26-7.14 (m, 2H), 7.08 (dd, J=10.2, 1.6 Hz, 1H), 6.73 (dd, J=3.4, 2.3 Hz, 1H). LCMS m/z 307.01 [M+H]+.
To a solution of 5-bromo-7-fluoro-2-(4-fluorophenyl)-1H-indole C40 (966 mg, 3.131 mmol) in toluene (10 mL) was added methane sulfonic acid (320 μL, 4.931 mmol) and triethylsilane (1.55 mL, 9.704 mmol) followed by methyl 3,3-dimethoxypropanoate (536 μL, 3.781 mmol). Reaction was heated at 70° C. for 2 hours. Water was added (50 mL), and the organic layer was extracted with EtOAc (3×30 mL), dried over sodium sulfate, filtered and concentrated in vacuo. Silica gel chromatography (Gradient: EtOAc in heptane) afforded the product methyl 3-[5-bromo-7-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoate (C41) (890 mg, 72%). 1H NMR (300 MHz, Chloroform-d) δ 8.19 (s, 1H), 7.61-7.46 (m, 3H), 7.22 (dd, J=8.9, 8.4 Hz, 2H), 7.10 (dd, J=10.2, 1.5 Hz, 1H), 3.66 (s, 3H), 3.25-3.05 (m, 2H), 2.75-2.51 (m, 2H). LCMS m/z 394.04 [M+H]+.
A solution of methyl 3-[5-bromo-7-fluoro-2-(4-fluorophenyl)-1˜{H}-indol-3-yl]propanoate C41 (520 mg, 1.319 mmol) and LiOH (500 mg, 20.88 mmol) in MeOH (5 mL), THF (8 mL) and water (10 mL) was stirred and heated at 50° C. overnight. Organic solvent was evaporated and water (100 mL) was added. The solution was acidified to pH 1 by adding concentrated HCl. The aqueous layer was extracted with DCM. The combined organic layers were washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give 3-[5-bromo-7-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid (S11) (599 mg, quantitative). 1H NMR (300 MHz, Chloroform-d) δ 8.10 (s, 1H), 7.52-7.35 (m, 3H), 7.17-7.07 (m, 2H), 7.01 (dd, J=10.2, 1.5 Hz, 1H), 3.14-2.95 (m, 2H), 2.68-2.47 (m, 2H). LCMS m/z 380.09 [M+H]+.
To a solution of 4,7-difluoro-1H-indole (500 mg, 3.265 mmol) C45 and 1-fluoro-4-iodo-benzene (C46) (490 μL, 4.249 mmol) in DMA (4 mL) and water (1 mL) was added K2CO3 (1.2 g, 8.683 mmol), bicyclo[2.2.1]hept-2-ene (923 mg, 9.803 mmol), and Bis(acetonitrile)dichloropalladium(II) (85 mg, 0.3276 mmol). The reaction was warmed to 90° C. The reaction was heated for 14 hours before being allowed to cool to room temperature. The reaction was diluted with water and extracted with EtOAc. The organic extract was dried with MgSO4, filtered and concentrated in vacuo. Purification by silica gel chromatography (Eluent: 10% EtOAc in heptane) afforded the product 4,7-difluoro-2-(4-fluorophenyl)-1H-indole (C47) (689 mg, 84%). 1H NMR (300 MHz, Chloroform-d) δ 8.48 (s, 1H), 7.75-7.56 (m, 2H), 7.25-7.06 (m, 2H), 6.93-6.77 (m, 2H), 6.74-6.50 (m, 1H). LCMS m/z 248.13 [M+H]+.
To 4,7-difluoro-2-(4-fluorophenyl)-1H-indole C47 (383 mg, 1.532 mmol) in toluene (10 mL) was added methanesulfonic acid (155 μL, 2.389 mmol) and triethylsilane (750 μL, 4.696 mmol) followed by methyl 3,3-dimethoxypropanoate (262 μL, 1.848 mmol). The reaction was heated at 70° C. for 12 hours. Water (50 mL) was added, and the aqueous layer was extracted with EtOAc (3×30 mL). The combined organic layer was dried over MgSO4 and concentrated to dryness. The crude material was purified by silica gel chromatography (Gradient: 0-100% EtOAc in hexane) to provide methyl 3-[4,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoate (C48) (48 mg, 9%). 1H NMR (300 MHz, Chloroform-d) δ 8.18 (s, 1H), 7.63-7.51 (m, 2H), 7.27-7.16 (m, 2H), 6.82 (ddd, J=10.0, 8.6, 3.5 Hz, 1H), 6.69 (ddd, J=10.3, 8.6, 3.3 Hz, 1H), 3.64 (s, 3H), 3.35-3.16 (m, 2H), 2.82-2.62 (m, 2H). LCMS m/z 334.01 [M+H]+.
A mixture of methyl 3-[4,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoate C48 (47 mg, 0.1402 mmol) and LiOH (68 mg, 2.839 mmol) in MeOH (5 mL), THF (4 mL) and water (4 mL) was stirred at 60° C. for 20 hours. The reaction mixture was concentrated, and water (50 mL) was added. The solution was acidified with concentrated HCl to pH=1. The aqueous layer was extracted with DCM (3×30 mL). The combined organic layer was washed with brine, dried over Na2SO4, and evaporated to afford product 3-[4,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid (S12) (52 mg, 100%). 1H NMR (300 MHz, Chloroform-d) δ 8.12 (s, 1H), 7.51-7.35 (m, 2H), 7.11 (t, J=8.5 Hz, 2H), 6.73 (td, J=9.3, 3.4 Hz, 1H), 6.60 (ddd, J=11.8, 8.6, 3.3 Hz, 1H), 3.12 (t, J=8.0 Hz, 2H), 2.67 (t, J=8.0 Hz, 2H). LCMS m/z 320.1 [M+H]+.
A solution of 5,7-difluoro-1H-indole C49 (3.0 g, 19.59 mmol), 4-iodobenzonitrile C50 (4.9 g, 21.40 mmol), DMA (35 mL), water (4 mL), K2CO3 (5.64 g, 40.81 mmol), bicyclo[2.2.1]hept-2-ene (3.1 g, 32.92 mmol), and acetonitrile; dichloropalladium (421 mg, 1.623 mmol) was warmed to 90° C. and stirred overnight. The reaction was cooled to room temperature. The reaction was diluted with water (75 mL) and was extracted with ethyl acetate (2×50 mL). The combined organics were washed with brine, dried with MgSO4, filtered, and concentrated in vacuo. Purification by silica gel chromatography (Eluent: 10% EtOAc in heptane) afforded the product 4-(5,7-difluoro-1H-indol-2-yl)benzonitrile (C51) (2.79 g, 52%). LCMS m/z 254.97 [M+H]+.
4-(5,7-difluoro-1H-indol-2-yl)benzonitrile C51 (101 mg, 0.371 mmol), methyl 3,3-dimethoxypropanoate (79 μL, 0.5572 mmol), methanesulfonic acid (49 μL, 0.7551 mmol) and triethylsilane (180 μL, 1.127 mmol) were added to diethyl carbonate (2 mL) in a microwave tube. The reaction mixture was subjected to the microwave at 180° C. for 1 hour. Additional methyl 3,3-dimethoxypropanoate (79 μL, 0.5572 mmol), methanesulfonic acid (49 μL, 0.7551 mmol), triethylsilane (180 μL, 1.127 mmol) and TFA (58 μL, 0.7528 mmol) was added and the reaction was reheated to 180° C. for 2 hours in the microwave. The reaction was cooled to room temperature and water (100 mL) was added. Aqueous layer was extracted with EtOAc (3×50 mL). The combined organic layer was dried over MgSO4 and concentrated to dryness. Purification by silica gel chromatography (Gradient: 0-100% EtOAc in hexane) afforded methyl 3-[2-(4-cyanophenyl)-5,7-difluoro-1H-indol-3-yl]propanoate (C52) (16 mg, 12%). LCMS m/z 341.07 [M+H]+.
To a solution of methyl 3-[2-(4-cyanophenyl)-5,7-difluoro-1H-indol-3-yl]propanoate C52 (16 mg, 0.04436 mmol) in MeOH (1 mL) and THF (2 mL) was added aqueous LiOH solution (1 mL). The reaction mixture was stirred at 50° C. for 3 h. The reaction mixture was cooled to room temperature, concentrated, and water (40 mL) was added. The reaction was acidified with concentrated HCl to pH=1. The aqueous layer was extracted with DCM (3×25 mL). The combined organic layer was washed with brine, dried over Na2SO4, and evaporated to afford product 3-[2-(4-cyanophenyl)-5,7-difluoro-1H-indol-3-yl]propanoic acid (S13) (14 mg, 97%). LCMS m/z 327.03 [M+H]+.
3-(5-fluoro-2-phenyl-1H-indol-3-yl)propanoic acid (S14) was obtained from commercial sources.
To a solution of methyl 4-oxobutanoate (1 g, 8.612 mmol) and 5,7-difluoro-2-(4-fluorophenyl)-1H-indole C25 (1.5 g, 6.068 mmol) in DCM (40 mL) was added Et3SiH (4.8 mL, 30.05 mmol) and TFA (2.3 mL, 29.85 mmol). The mixture was stirred at room temperature overnight. Solvent was removed in vacuo. EtOAc and saturated NaHCO3 aqueous solution were added. The aqueous layer was extracted with EtOAc (2×50 mL). The combined organic fractions were washed with NaHCO3 and brine, dried over sodium sulfate, filtered, and the solution was concentrated to dryness. Purification by silica gel chromatography (Eluent: 50% EtOAc in heptane) afforded (C53) methyl 4-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]butanoate (1.9 g, 76%). 1H NMR (300 MHz, Chloroform-d) δ 8.34 (s, 1H), 7.51 (ddq, J=10.2, 5.0, 2.5, 2.0 Hz, 2H), 7.25-7.12 (m, 2H), 7.09 (dd, J=9.1, 2.2 Hz, 1H), 6.75 (ddd, J=10.8, 9.5, 2.2 Hz, 1H), 3.64 (s, 3H), 2.98-2.65 (m, 2H), 2.35 (t, J=7.2 Hz, 2H), 2.13-1.80 (m, 2H). LCMS m/z 348.2 [M+H]+.
To a solution of methyl 4-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]butanoate C53 (1.9 g, 5.5 mmol) in THF (30 mL) was added NaOH (30 mL of 2 M, 60 mmol). The reaction mixture was stirred at room temperature overnight. Organic layer was removed in vacuo, the pH was adjusted to 6. The aqueous layer was extracted with EtOAc. Combined organic fractions were washed with H2O (20 mL), brine (20 mL), dried over sodium sulfate, filtered, and the solvent was removed in vacuo to obtain (S15) 4-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]butanoic acid (1.7 g, 81%). 1H NMR (300 MHz, Chloroform-d) δ 8.10 (s, 1H), 7.63-7.41 (m, 2H), 7.25-7.13 (m, 2H), 7.10 (dd, J=9.1, 2.2 Hz, 1H), 6.76 (ddd, J=10.8, 9.4, 2.1 Hz, 1H), 3.01-2.66 (m, 2H), 2.41 (t, J=7.1 Hz, 2H), 2.11-1.87 (m, 2H). LCMS m/z 334.25 [M+H]+.
To a solution of tert-butyl N-[(3S)-2-oxopyrrolidin-3-yl]carbamate C54 (426 mg, 2.128 mmol) in methanol (4 mL) was added HCl (2 mL of 4 M, 8 mmol) in 1,4-dioxane. Reaction was stirred at 50° C. for 30 minutes. Solvent was evaporated and crude product was washed with ether to afford crude (S)-3-aminopyrrolidin-2-one (S16) (Hydrochloride salt) (302 mg, 99%). LCMS m/z 100.0 [M+H]+.
To a suspension benzyl N-[(3S)-2,5-dioxopyrrolidin-3-yl]carbamate C55 (649 mg, 2.614 mmol) in MeOH (10 mL) and EtOAc (5 mL) was added 5% palladium on carbon catalyst (20 mg). The mixture was subjected to hydrogenation conditions of 50 psi H2 for 2 hours. Filtration through a pad of Celite®, washing with MeOH and CH2Cl2, then concentration of the filtrate in vacuo afforded (3S)-3-aminopyrrolidine-2,5-dione (S17) (398 mg, 98%). 1H NMR (300 MHz, Chloroform-d) δ 4.00 (dd, J=8.7, 5.7 Hz, 1H), 3.12 (dd, J=18.2, 8.8 Hz, 1H), 2.69-2.37 (m, 1H), 1.73 (s, 2H). LCMS m/z 124.01 [M+H]+.
To a stirred solution of (2S)-2-aminopentanedioic acid C56 (6 g, 40.781 mmol) in MeOH (30 mL) was added thionyl chloride (29.111 g, 17.849 mL, 244.69 mmol) dropwise under cooling conditions. The reaction mixture was stirred at room temperature for 20 hours. After completion reaction was concentrated in vacuo to remove excess thionyl chloride to afford dimethyl (2S)-2-aminopentanedioate (C57) (7 g, 98%) as colorless oily liquid. 1H NMR (400 MHz, DMSO-d6) δ 8.70 (s, 3H), 4.03 (t, J=6.6 Hz, 1H), 3.72 (s, 3H), 3.60 (s, 3H), 2.54 (dd, J=15.9, 8.3 Hz, 1H), 2.06 (q, J=7.4 Hz, 2H). One proton is obscured by the DMSO solvent peak.
A solution of dimethyl (2S)-2-aminopentanedioate C57 (16 g, 91.334 mmol) in toluene (150 mL) was stirred at reflux at 110° C. for 5 hours. Solvent was evaporated and purified by column-chromatography (eluted at 5% MeOH in DCM) to afford the desire product methyl (2S)-5-oxopyrrolidine-2-carboxylate (C58) (4.2 g, 32%). 1H NMR (400 MHz, Chloroform-d) δ 6.14 (s, 1H), 4.32 (s, 1H), 3.78 (s, 3H), 2.52 (s, 1H), 2.40 (s, 2H), 2.29 (s, 1H).
To a stirred solution of methyl (2S)-5-oxopyrrolidine-2-carboxylate C58 (4.2 g, 29.342 mmol) in IPA (40 mL) was added NaBH4 (6.6604 g, 7.0480 mL, 176.05 mmol). The reaction was stirred at room temperature for 20 hours. Reaction was quenched by methanol and evaporated through reduced pressure. Purification by column chromatography (eluted at 5% MeOH in DCM) afforded (5S)-5-(hydroxymethyl)pyrrolidin-2-one (C59) (3.3 g, 98%). 1H NMR (400 MHz, Chloroform-d) δ: 7.29 (s, 1H), 4.25 (s, 1H), 3.79-3.74 (m, 1H), 3.64 (d, J=11.8 Hz, 1H), 3.43 (t, J=10.2 Hz, 1H), 2.35-2.30 (m, 2H), 2.27-2.20 (m, 1H), 1.81-1.73 (m, 1H).
To a stirred solution of (5S)-5-(hydroxymethyl)pyrrolidin-2-one C59 (3.3 g, 28.663 mmol) in toluene (45 mL) was added benzaldehyde (4.8669 g, 4.6797 mL, 45.861 mmol) and PTSA (272.62 mg, 1.4332 mmol) at room temperature. The reaction was stirred at room temperature for 17 hours. Reaction mixture was refluxed under Dean-Stark water separator for 6 hours. Solvent was evaporated. Purification by silica gel chromatography (Eluent: 30% EtOAc in heptane) afforded the desired product (C60) (3R,7aS)-3-phenyl-3,6,7,7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-one (4.8 g, 48%). 1H NMR (400 MHz, Chloroform-d) δ: 7.43 (d, J=7 Hz, 2H), 7.37-7.29 (m, 3H), 6.32 (s, 1H), 4.24-4.17 (m, 1H), 4.15-4.11 (m, 1H), 3.48 (t, J=8.04 Hz, 1H), 2.85-2.76 (m, 1H), 2.59-2.51 (m, 1H), 2.42-2.33 (m, 1H), 1.98-1.91 (m, 1H). LCMS m/z 204.0 [M+H]+.
LDA (2.9522 mL of 2 M, 5.9045 mmol) was cooled to −78° C. then (3R,7aS)-3-phenyl-3,6,7,7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-5-one (C60) (1 g, 4.9204 mmol) in THF (5 mL) was added. Reaction was stirred at −78° C. for 30 minutes. DPPA (2.7082 g, 2.1158 mL, 9.8408 mmol) was added and the reaction mixture was stirred for 10 minutes, then Boc anhydride (2.1477 g, 2.2607 mL, 9.8408 mmol) was added and the reaction was stirred for 17 hours. Reaction mixture was partitioned between ethyl acetate (2×125 mL) and brine (2×200 mL). Organic layer was dried over anhydrous magnesium sulfate and evaporated through reduced pressure. Purification by silica gel chromatography (Eluent: 20% EtOAc in heptane) afforded the desired product (C61) tert-butyl N-[(3R,6S,7aS)-5-oxo-3-phenyl-3,6,7,7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-6-yl]-carbamate (522 mg, 33%)1H NMR (400 MHz, Chloroform-d) δ 7.46-7.28 (m, 5H), 5.20 (d, J=6.5 Hz, 1H), 4.62 (s, 1H), 4.25 (dd, J=8.4, 6.3 Hz, 1H), 4.11-4.00 (m, 1H), 3.62 (dd, J=8.4, 6.9 Hz, 1H), 3.00 (d, J=13.0 Hz, 1H), 1.75 (q, J=11.6 Hz, 1H), 1.45 (s, 9H). LCMS m/z 319.0 [M+H]+.
A solution of tert-butyl N-[(3R,6S,7aS)-5-oxo-3-phenyl-3,6,7,7a-tetrahydro-1H-pyrrolo[1,2-c]oxazol-6-yl]carbamate (C61) (1.5 g, 4.7115 mmol) in DCM (15 mL) was cooled to 0° C., then TFA (11.100 g, 7.5 mL, 97.349 mmol) was added. The reaction mixture was stirred at room temperature for 2 hours. Solvent was removed in vacuo. 1,4-Dioxane-HCl (4 M) was added and reaction was stirred for 30 minutes. Solvent was removed to give (S18) (3S,5S)-3-amino-5-(hydroxymethyl)pyrrolidin-2-one (Hydrochloric Acid) (750 mg, 96%)1H NMR (400 MHz, DMSO-d6) δ 8.58 (m, 4H), 4.40 (m, 1H), 3.93 (m, 1H), 3.56 (m, 1H), 3.42 (m, 2H), 2.38 (m, 1H), 1.68 (m, 1H). LCMS m/z 131.0 [M+H]+.
To a flask containing 2,4-difluoro-6-iodo-aniline C23 (134 g, 525.5 mmol) was added NEt3 (1.3 L), followed by DMF (250 mL), 1-ethynyl-4-fluoro-benzene (83.5 g, 695.1 mmol), CuI (20.5 g, 107.6 mmol), and PdCl2(PPh3)2 (25 g, 35.6 mmol). The mixture was allowed to stir at room temperature for 2 hours. Solvent was removed under reduced pressure and water (500 mL) was added. The mixture was extracted with Ethyl acetate, filtered and concentrated in vacuo. The product mixture was filtered through a silica gel plug (Eluent: CH2Cl2), followed by a second silica plug filtration (Eluent: 30-40% EtOAc in Heptane). The resulting crude was purified via silica gel chromatography (Gradient: 0-20% EtOAc in heptane) to afford the product 2,4-difluoro-6-[2-(4-fluorophenyl)ethynyl]aniline (C24) (87 g, 60%) as a pale yellow solid. 1H NMR (300 MHz, Chloroform-d) δ 7.58-7.45 (m, 2H), 7.14-7.02 (m, 2H), 6.92 (ddd, J=8.8, 2.8, 1.7 Hz, 1H), 6.87-6.71 (m, 1H), 4.15 (s, 2H). LCMS m/z 248.0 [M+H]+.
To a solution of 2,4-difluoro-6-[2-(4-fluorophenyl)ethynyl]aniline C24 (46 g, 167.5 mmol) in DMF (600 mL) was added CuI (1.9 g, 10.0 mmol) and the reaction was heated at reflux. Water (800 mL) was added and the mixture extracted with MTBE. The mixture was then washed with sat. NaCl solution, dried over Na2SO4 and then concentrated in vacuo to afford the product 5,7-difluoro-2-(4-fluorophenyl)-1H-indole C25, which was used in subsequent steps without further purification (41 g, 87%). 1H NMR (300 MHz, Chloroform-d) δ 8.43 (s, 1H), 7.72-7.58 (m, 2H), 7.27-7.15 (m, 2H), 7.09 (dd, J=9.0, 2.1 Hz, 1H), 6.85-6.63 (m, 2H). LCMS m/z 248.0 [M+H]+.
To a solution of 5,7-difluoro-2-(4-fluorophenyl)-1H-indole C25 (5.43 g, 22.0 mmol) in DCM (50 mL) was added methanesulfonic acid (4.5 mL, 69.4 mmol). After stirring for 20 minutes, benzyl N-(2,2-dimethoxyethyl)carbamate (5.5 g, 23.0 mmol) and triethylsilane (10.5 mL, 65.7 mmol) were added and the reaction was stirred overnight. The reaction was concentrated, and the crude was dissolved in DMSO and purified by reversed-phase chromatography (C18 column; Gradient: 0-100% MeCN in H2O with 0.1% TFA) to afford the product benzyl N-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]carbamate (C62) as a solid (9 g, 77%). LCMS m/z 425.2 [M+H]+.
To a solution of benzyl N-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]carbamate C62 (5.3 g, 12.5 mmol) in EtOH (50 mL) was added Pd/C (200 g, 1.88 mol). The reaction was stirred under a H2 atmosphere using a balloon overnight. The reaction was then filtered and concentrated. The crude was dissolved in DMSO and purified by reversed-phase chromatography (C18 column; Gradient: 0-100% MeCN in H2O with 0.1% TFA) to afford the title compound as a TFA salt (S19) (2.5 g, 49%). 1H NMR (300 MHz, Acetone-d6) δ 10.90 (s, 1H), 7.95-7.64 (m, 2H), 7.44 (dd, J=9.4, 2.2 Hz, 1H), 7.39-7.25 (m, 2H), 6.86 (ddd, J=11.1, 9.6, 2.2 Hz, 1H), 4.32-3.91 (m, 2H), 3.56-3.36 (m, 2H), 2.85 (s, 2H). LCMS m/z 291.1 [M+H]+.
To a solution of (2S)-2-amino-3,3,3-trifluoro-propan-1-ol hydrochloride (16 mg, 0.097 mmol) in DMSO (1 mL) was added 3-[5-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid S7 (20 mg, 0.066 mmol), HATU (40 mg, 0.11 mmol), and NEt3 (50 μL, 0.36 mmol). The mixture was allowed to stir at room temperature for 12 hours. The mixture was then purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient:
MeCN in H2O with 0.1% trifluoroacetic acid) to afford the product (1) (21 mg, 74%). 1H NMR (300 MHz, Methanol-d4) δ 7.71-7.55 (m, 2H), 7.42-7.04 (m, 4H), 6.97-6.68 (m, 1H), 4.70-4.56 (m, 1H), 3.75 (dd, J=11.8, 4.8 Hz, 1H), 3.64 (dd, J=11.8, 6.7 Hz, 1H), 3.21-3.08 (m, 2H), 2.72-2.51 (m, 2H). LCMS m/z 413.1 [M+H]+.
To a solution of tert-butyl (3S,4S)-3-amino-4-hydroxy-pyrrolidine-1-carboxylate C21 (20 mg, 0.099 mmol) in DMSO (1 mL) was added 3-[5-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid S7 (20 mg, 0.066 mmol), HATU (40 mg, 0.11 mmol), and NEt3 (50 μL, 0.36 mmol). The mixture was allowed to stir at room temperature for 12 hours. The mixture was then purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford the product (25 mg, 75%). 1H NMR (400 MHz, Methanol-d4) δ 7.64 (ddd, J=8.8, 5.2, 2.2 Hz, 2H), 7.30 (td, J=6.2, 5.7, 2.9 Hz, 2H), 7.25-7.18 (m, 2H), 6.87 (tt, J=8.8, 2.2 Hz, 1H), 4.02 (ddt, J=11.1, 5.5, 2.3 Hz, 1H), 3.89 (ddt, J=9.0, 4.4, 2.4 Hz, 1H), 3.55 (ddd, J=11.5, 5.9, 4.2 Hz, 1H), 3.45 (m, 1H), 3.25-3.08 (m, 4H), 2.60-2.43 (m, 2H), 1.43 (d, J=1.0 Hz, 9H). LCMS m/z 486.2 [M+H]+.
To a solution of tert-butyl (3S,4S)-3-[3-[5-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoylamino]-4-hydroxy-pyrrolidine-1-carboxylate C22 (25.3 mg, 0.0501 mmol) in methanol (1 mL) was added HCl in 1,4-dioxane (0.5 mL of 4 M, 2 mmol). The mixture was stirred at 50° C. for 20 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was triturated with diethyl ether and n-heptane. Lyophilization afforded the product (2) (22 mg, 94%). 1H NMR (400 MHz, Methanol-d4) δ 7.65 (dd, J=8.5, 5.2 Hz, 2H), 7.36-7.16 (m, 4H), 6.88 (td, J=9.1, 2.3 Hz, 1H), 4.17 (s, 1H), 4.15-4.06 (m, 1H), 3.63-3.55 (m, 1H), 3.20-3.13 (m, 4H), 2.64-2.47 (m, 2H). LCMS m/z 486.2 [M+H]+.
Compounds 3-90 (see Table 2) were prepared from intermediate S7 using the appropriate reagent and using the amide formation methods as described for compounds 1-2. Amines were prepared by methods described above or obtained from commercial sources. Any modifications to methods are noted in Table 2 and accompanying footnotes.
1H NMR; LCMS m/z
1H NMR (300 MHz, Methanol-d4) δ 7.78-7.56 (m, 2H), 7.36-7.09 (m, 4H), 6.87 (ddd, J = 9.4, 8.8, 2.5 Hz, 1H), 5.87 (td, J = 55.6, 3.3 Hz, 1H), 4.24 (tdq, J = 12.1, 5.9, 3.4, 3.0 Hz, 1H), 3.68-3.53 (m, 2H), 3.22- 3.03 (m, 2H), 2.72-2.52 (m, 2H); LCMS m/z 395.12 [M + H]+.
1H NMR (300 MHz, Chloroform-d) δ 8.14 (s, 1H), 7.60-7.47 (m, 2H), 7.41- 7.30 (m, 2H), 7.24-7.13 (m, 2H), 6.99 (td, J = 9.0, 2.5 Hz, 1H), 5.60 (s, 1H), 3.93 (s, 1H), 3.66 (s, 2H), 3.20 (dd, J = 8.1, 7.0 Hz, 2H), 2.54 (dd, J = 8.2, 6.8 Hz, 2H), 2.14 (tq, J = 6.2, 3.2, 2.7 Hz, 2H), 1.91- 1.68 (m, 4H); LCMS m/z 385.09 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.69-7.55 (m, 2H), 7.37-7.16 (m, 4H), 6.88 (ddd, J = 9.4, 8.8, 2.5 1H), 3.74 (ddd, J = 5.9, 4.9, 1.3 Hz, 1H), 3.17 (dd, J = 8.2, 7.3 Hz, 2H), 2.96 (dd, J = 12.9, 4.4 Hz, 1H), 2.82 (dd, J = 12.9, 8.9 Hz, 1H), 2.63- 2.51 (m, 2H), 1.05 (d, J = 6.9 Hz, 3H); LCMS m/z 358.21 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.74-7.59 (m, 2H), 7.39-7.26 (m, 2H), 7.26-7.14 (m, 2H), 6.87 (ddd, J = 9.4, 8.8, 2.5 Hz, 1H), 4.23 (q, J = 7.2 Hz, 1H), 3.20-3.06 (m, 2H), 2.66 (s, 3H), 2.58 (td, J = 7.5, 3.0 Hz, 2H), 1.19 (d, J = 7.2 Hz, 3H); LCMS m/z 386.1 [M + H]+.
1H NMR (400 MHz, Methanol-d4) δ 7.69-7.58 (m, 2H), 7.37-7.26 (m, 2H), 7.25-7.14 (m, 2H), 6.87 (ddd, J = 9.4, 8.6, 2.5 Hz, 1H), 4.23 (q, J = 7.2 Hz, 1H), 3.15 (dddd, J = 8.5, 7.2, 5.1, 1.4 Hz, 4H), 2.57 (td, J = 7.6, 3.9 Hz, 2H), 1.19 (d, J = 7.2 Hz, 3H), 1.06 (t, J = 7.3 Hz, 3H); LCMS m/z 400.18 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 10.68 (s, 1H), 8.06 (d, J = 7.7 Hz, 1H), 7.72- 7.54 (m, 2H), 7.42-7.03 (m, 4H), 6.87 (ddd, J = 9.4, 8.8, 2.5 Hz, 1H), 4.36-4.20 (m, 1H), 3.25-3.01 (m, 2H), 2.57 (td, J = 7.4, 2.0 Hz, 2H), 1.22 (d, J = 7.2 Hz, 3H); LCMS m/z 372.13 [M + H]+.
1H NMR (400 MHz, Methanol-d4) δ 7.72-7.59 (m, 2H), 7.36-7.28 (m, 2H), 7.28-7.10 (m, 2H), 6.87 (ddd, J = 9.4, 8.7, 2.5 Hz, 1H), 4.38-4.30 (m, 1H), 4.24 (td, J = 7.7, 4.0 Hz, 1H), 3.24- 3.06 (m, 2H), 2.62-2.53 (m, 2H), 2.17-1.96 (m, 2H), 1.91- 1.75 (m, 1H); LCMS m/z 371.16 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.73-7.52 (m, 2H), 7.41-7.12 (m, 4H), 6.87 (ddd, J = 9.5, 8.7, 2.5 Hz, 1H), 3.19-3.04 (m, 2H), 2.63-2.49 (m, 2H), 1.37 (s, 6H); LCMS m/z 386.07 [M + H]+.
1H NMR (400 MHz, Methanol-d4) δ 7.73-7.51 (m, 2H), 7.39-7.28 (m, 2H), 7.26-7.13 (m, 2H), 6.87 (ddd, J = 9.5, 8.7, 2.5 Hz, 1H), 3.70-3.45 (m, 4H), 3.20- 3.07 (m, 2H), 2.58-2.48 (m, 2H), 1.13 (s, 3H); LCMS m/z 389.17 [M + H]+.
1H NMR (300 MHz, Chloroform-d) δ 8.17 (s, 1H), 7.64-7.45 (m, 2H), 7.38- 7.28 (m, 2H), 7.24-7.09 (m, 2H), 6.98 (td, J = 9.0, 2.5 Hz, 1H), 5.29 (s, 1H), 3.45 (s, 2H), 3.19 (dd, J = 8.2, 6.9 Hz, 2H), 2.53 (dd, J = 8.1, 6.9 Hz, 2H), 1.09 (s, 6H); LCMS m/z 373.11 [M + H]+.
1H NMR (400 MHz, Methanol-d4) δ 7.72-7.60 (m, 2H), 7.36-7.27 (m, 2H), 7.27-7.14 (m, 2H), 6.88 (ddd, J = 9.4, 8.6, 2.5 Hz, 1H), 3.19-3.08 (m, 2H), 2.62- 2.52 (m, 2H), 1.39-1.25 (m, 2H), 0.83-0.67 (m, 2H); LCMS m/z 384.16 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.73-7.60 (m, 2H), 7.42-7.13 (m, 4H), 6.87 (ddd, J = 9.4, 8.7, 2.5 Hz, 1H), 3.56 (qd, J = 11.1, 5.2 Hz, 2H), 3.28-3.03 (m, 3H), 2.56 (dd, J = 8.7, 7.0 Hz, 2H), 0.95-0.73 (m, 1H), 0.46 (tdd, J = 8.2, 5.1, 3.6 Hz, 1H), 0.32 (dddd, J = 9.5, 8.3, 4.8, 3.4 Hz, 1H), 0.26-0.05 (m, 2H); LCMS m/z 385.16 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.69-7.55 (m, 2H), 7.38-7.12 (m, 4H), 6.87 (td, J = 9.1, 2.5 Hz, 1H), 3.88 (dq, J = 8.3, 5.5 Hz, 1H), 3.31 (p, J = 1.6 Hz, 4H), 3.23- 3.03 (m, 2H), 2.63-2.46 (m, 2H); LCMS m/z 375.09 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.68-7.54 (m, 2H), 7.39-7.11 (m, 4H), 6.87 (ddd, J = 9.4, 8.7, 2.5 Hz, 1H), 4.28 (dd, J = 10.7, 6.0 Hz, 1H), 3.26 (dd, J = 7.1, 5.0 Hz, 2H), 3.21-3.04 (m, 2H), 2.62-2.50 (m, 2H), 2.03- 1.90 (m, 1H), 1.83 (tq, J = 7.1, 3.7 Hz, 1H), 1.68-1.59 (m, 1H). One proton obscured by solvent peak. LCMS m/z 398.09 [M + H]+.
1H NMR (300 MHz, Chloroform-d) δ 8.15 (2s,, 1H), 7.56 (ddt, J = 6.8, 5.3, 1.7 Hz, 2H), 7.29 (d, J = 2.3 Hz, 2H), 7.23-7.12 (m, 2H), 7.06-6.85 (m, 1H), 4.45 (s, 1H), 3.72-3.44 (m, 2H), 3.30 (d, J = 12.4 Hz, 1H), 3.19 (td, J = 7.2, 2.1 Hz, 3H), 2.57 (td, J = 9.9, 6.5 Hz, 2H), 2.18- 2.01 (m, 1H), 1.93-1.62 (m, 1H), 1.56-1.20 (m, 12H); LCMS m/z 484.03 [M + H]+.
1H NMR (400 MHz, Methanol-d4) δ 7.76-7.55 (m, 2H), 7.40-7.27 (m, 2H), 7.27-7.13 (m, 2H), 6.87 (ddd, J = 9.5, 8.8, 2.5 Hz, 1H), 3.93 (qd, J = 6.4, 3.1 Hz, 1H), 3.78 (td, J = 6.3, 3.1 Hz, 1H), 3.56 (dd, J = 11.0, 6.3 Hz, 1H), 3.47 (dd, J = 11.0, 6.2 Hz, 1H), 3.16 (td, J = 7.3, 1.6 Hz, 2H), 2.72-2.52 (m, 2H), 0.99 (d, J = 6.5 Hz, 3H); LCMS m/z 389.17 [M + H]+.
1H NMR (400 MHz, Methanol-d4) δ 7.69-7.58 (m, 2H), 7.36-7.26 (m, 2H), 7.26-7.12 (m, 2H), 6.94- 6.81 (m, 1H), 4.03 (dt, J = 9.7, 8.0 Hz, 1H), 3.87 (q, J = 7.9 Hz, 1H), 3.16-3.05 (m, 2H), 2.50 (ddd, J = 8.1, 6.6, 2.3 Hz, 2H), 1.94 (dddd, J = 10.7, 9.4, 8.0, 1.4 Hz, 1H), 1.49 (tt, J = 11.0, 9.0 Hz, 1H), 1.23-1.12 (m, 1H); LCMS m/z 371.16 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.71-7.57 (m, 2H), 7.37-7.11 (m, 4H), 6.87 (ddd, J = 9.4, 8.8, 2.5 Hz, 1H), 4.00 (dtd, J = 12.3, 6.8, 5.6 Hz, 1H), 3.25 (s, 3H), 3.25-3.19 (m, 1H), 3.17- 3.06 (m, 3H), 2.58-2.46 (m, 2H), 1.00 (d, J = 6.8 Hz, 3H); LCMS m/z 373.11 [M + H]+.
1H NMR (300 MHz, Chloroform-d) δ 8.03 (s, 1H), 7.61-7.46 (m, 2H), 7.37- 7.30 (m, 1H), 7.25-7.09 (m, 2H), 6.99 (td, J = 9.0, 2.4 Hz, 1H), 5.37 (s, 1H), 3.98 (dp, J = 10.4, 3.4 Hz, 1H), 3.54 (dd, J = 11.0, 3.5 Hz, 1H), 3.40 (dd, J = 11.0, 6.0 Hz, 1H), 3.21 (dd, J = 8.3, 6.8 Hz, 2H), 2.55 (t, J = 7.6 Hz, 2H), 1.02 (d, J = 6.9 Hz, 3H); LCMS m/z 359.1 [M + H]+.
1H NMR (300 MHz, Chloroform-d) δ 7.52 (s, 1H), 7.01 (m, 3H), 6.66 (m, 3H), 6.45 (s, 1H), 5.15 (s, 1H), 3.92 (d, J = 6.5 Hz, 2H), 3.82 (d, J = 7.0 Hz, 2H), 3.35 (s, 2H), 2.68 (s, 2H), 2.05 (d, J = 7.2 Hz, 2H); LCMS m/z 387.08 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.72-7.52 (m, 2H), 7.36-7.14 (m, 4H), 6.87 (td, J = 9.2, 2.5 Hz, 1H), 3.78 (d, J = 11.0 Hz, 1H), 3.56 (d, J = 11.0 Hz, 1H), 3.21-3.06 (m, 2H), 2.61- 2.48 (m, 2H), 1.52 (s, 3H); LCMS m/z 384.15 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.73-7.59 (m, 2H), 7.41-7.13 (m, 4H), 6.87 (td, J = 9.1, 2.5 Hz, 1H), 3.85-3.68 (m, 1H), 3.67- 3.51 (m, 1H), 3.14 (ddd, J = 8.7, 6.7, 1.7 Hz, 2H), 2.61- 2.41 (m, 2H), 0.99 (ddd, J = 11.1, 10.1, 6.4 Hz, 6H); LCMS m/z 373.17 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.72-7.54 (m, 2H), 7.35-7.27 (m, 2H), 7.27-7.08 (m, 2H), 6.87 (ddd, J = 9.4, 8.7, 2.5 Hz, 1H), 4.38 (t, J = 5.2 Hz, 1H), 3.73 (dd, J =11.1, 5.3 Hz, 1H), 3.65 (dd, J = 11.1, 5.1 Hz, 1H), 3.24-3.00 (m, 2H), 2.70-2.50 (m, 2H); LCMS m/z 388.05 [M + H]+.
1H NMR (400 MHz, Methanol-d4) δ 7.72-7.54 (m, 2H), 7.40-7.16 (m, 4H), 6.87 (ddd, J = 9.4, 8.8, 2.5 Hz, 1H), 3.80-3.69 (m, 2H), 3.62 (d, J = 4.9 Hz, 2H), 3.22- 3.11 (m, 2H), 2.58 (dd, J = 8.6, 7.3 Hz, 2H), 1.03 (d, J = 6.1 Hz, 3H); LCMS m/z 389.17 [M + H]+.
1H NMR (300 MHz, Chloroform-d) δ 8.11 (s, 1H), 7.63-7.50 (m, 2H), 7.32 (dd, J = 8.8, 4.4 Hz, 1H), 7.29- 7.11 (m, 4H), 6.99 (td, J = 9.0, 2.5 Hz 1H) 5.76 (s 1H) 3.72 (s, 1H), 3.47 (s, 2H), 3.19 (dd, J = 8.2, 6.9 Hz, 2H), 2.52 (dd, J = 8.2, 6.9 Hz, 2H), 0.85-0.72 (m, 2H), 0.66- 0.45 (m, 2H); LCMS m/z 371.09 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 10.69 (s, 1H), 7.75-7.55 (m, 2H), 7.41- 7.13 (m, 5H), 6.95-6.67 (m, 1H), 3.43 (s, 1H), 3.16 (t, J = 7.5 Hz, 2H), 2.62-2.52 (m, 5H); LCMS m/z 399.17 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 6.22-6.07 (m, 2H), 5.87-5.75 (m, 2H), 5.75-5.62 (m, 2H), 5.34 (ddd, J = 9.4, 8.8, 2.5 Hz, 1H), 3.08 (ddt, J = 8.1, 6.7, 4.1 Hz, 0H), 2.22 (dd, J = 11.8, 4.8 Hz, 1H), 2.11 (dd, J = 11.8, 6.7 Hz, 1H), 1.70- 1.56 (m, 2H), 1.09 (ddd, J = 9.0, 6.3, 1.2 Hz, 2H); LCMS m/z 413.13 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.75-7.53 (m, 2H), 7.40-7.12 (m, 4H), 6.88 (dddd, J = 9.6, 8.8, 2.5, 1.0 Hz, 1H), 4.72-3.74 (m, 1H), 3.55-3.33 (m, 2H), 3.16 (t, J = 7.9 Hz, 2H), 2.96- 2.80 (m, 1H), 2.72 (d, J = 1.7 Hz, 3H), 2.69-2.57 (m, 1H), 0.87 (dd, J = 53.0, 6.8 Hz, 3H); LCMS m/z 373.17 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.73-7.58 (m, 2H), 7.38-7.16 (m, 4H), 6.87 (td, J = 9.1, 2.5 Hz, 1H), 4.40 (td, J = 8.3, 4.4 Hz, 1H), 3.86-3.68 (m, 2H), 3.61- 3.53 (m, 1H), 3.14 (t, J = 7.5 Hz, 2H), 2.51 (t, J = 7.5 Hz, 2H), 2.40 (tm, 2H), 2.27 (m, 2H); LCMS m/z 370.23 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.74 (d, J = 8.1 Hz, 1H), 7.69-7.57 (m, 2H), 7.36-7.25 (m, 2H), 7.24 (d, J = 8.8 Hz, 1H), 6.87 (ddd, J = 9.5, 8.8, 2.5 Hz, 1H), 6.45 (s, 1H), 3.90 (p, J = 6.7 Hz, 1H), 3.13 (t, J = 7.9 Hz, 2H), 3.00 (q, J = 7.1, 6.5 Hz, 2H), 2.50 (td, J = 7.5, 3.1 Hz, 2H), 1.39 (s, 9H), 0.98 (d, J = 6.8 Hz, 3H); LCMS m/z 458.04 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.76-7.51 (m, 2H), 7.43-7.05 (m, 4H), 6.87 (td, J = 9.1, 2.5 Hz, 1H), 4.28 (qt, J = 7.2, 3.5 Hz, 1H), 3.14 (dd, J = 8.7, 7.1 Hz, 2H), 2.57 (td, J = 7.4, 2.0 Hz, 2H), 1.23 (d, J = 7.2 Hz, 3H); LCMS m/z 372.19 [M + H]+;
1H NMR (400 MHz, Methanol-d4) δ 7.71-7.51 (m, 2H), 7.42-7.09 (m, 4H), 6.87 (ddd, J = 9.4, 8.7, 2.5 Hz, 1H), 3.59 (d, J = 11.2 Hz, 1H), 3.47 (d, J = 11.2 Hz, 1H), 3.20-3.06 (m, 2H), 2.61- 2.42 (m, 2H), 1.76 (dq, J = 13.7, 7.5 Hz, 1H), 1.58-1.44 (m, 1H), 1.10 (s, 3H), 0.71 (t, J = 7.5 Hz, 3H); LCMS m/z 387.19 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.73-7.58 (m, 2H), 7.37-7.28 (m, 2H), 7.24-7.12 (m, 4H), 7.12- 7.05 (m, 1H) 7.02-6.93 (m, 2H), 6.92-6.79 (m, 1H), 3.17 (t, J = 7.5 Hz, 2H), 2.59 (dd, J = 8.1, 7.0 Hz, 2H), 1.20- 0.95 (m, 4H); LCMS m/z 417.17 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.77-7.57 (m, 2H), 7.41-7.09 (m, 4H), 6.87 (ddd, J = 9.4, 8.7, 2.5 Hz, 1H), 3.65 (h, J = 5.7, 5.3 Hz, 1H), 3.55-3.40 (m, 2H), 3.15 (td, J = 7.4, 1.9 Hz, 2H), 2.59 (ddd, J = 9.4, 6.4, 1.3 Hz, 2H), 1.78 (h, J = 6.8 Hz, 1H), 0.82 (d, J = 6.8 Hz, 3H), 0.75 (d, J = 6.8 Hz, 3H); LCMS m/z 387.11 [M + H]+.
1H NMR (400 MHz, Methanol-d4) δ 7.64 (ddd, J = 8.8, 5.2, 2.2 Hz, 2H), 7.30 (td, J = 6.2, 5.7, 2.9 Hz, 2H), 7.25- 7.18 (m, 2H), 6.87 (tt, J = 8.8, 2.2 Hz, 1H), 4.02 (ddt, J = 11.1, 5.5, 2.3 Hz, 1H), 3.89 (ddt, J = 9.0, 4.4, 2.4 Hz, 1H), 3.55 (ddd, J = 11.5, 5.9, 4.2 Hz, 1H), 3.25-3.08 (m, 4H), 2.60-2.43 (m, 2H), 1.43 (d, J = 1.0 Hz, 9H); LCMS m/z 486.17 [M + H]+.
1H NMR (400 MHz, Methanol-d4) δ 7.66-7.58 (m, 2H), 7.40-7.26 (m, 2H), 7.27-7.16 (m, 2H), 6.87 (ddd, J = 9.5, 8.8, 2.5 Hz, 1H), 3.71 (pd, J = 6.3, 5.3 Hz, 1H), 3.22-2.94 (m, 4H), 2.59- 2.47 (m, 2H), 1.02 (d, J = 6.3 Hz, 3H); LCMS m/z 359.17 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.73-7.56 (m, 2H), 7.37-7.15 (m, 4H), 6.97-6.78 (m, 1H), 3.71 (td, J = 6.4, 5.4 Hz, 1H), 3.19- 3.02 (m, 4H), 2.54 (dd, J = 8.9, 6.8 Hz, 2H), 1.02 (d, J = 6.3 Hz, 3H); LCMS m/z 359.1 [M + H]+.
1H NMR (300 MHz, Chloroform-d) δ 8.06 (s, 1H), 7.63-7.50 (m, 2H), 7.35- 7.28 (m, 2H), 7.27-7.12 (m, 2H), 6.98 (td, J = 9.0, 2.5 Hz, 1H), 5.39 (d, J = 7.7 Hz, 1H), 3.81-3.68 (m, 1H), 3.55 (dd, J = 11.1, 3.4 Hz, 1H), 3.45 (dd, J = 11.1, 5.7 Hz, 1H), 3.22 (dd, J = 8.2, 6.9 Hz, 2H), 2.58 (t, J = 7.5 Hz, 2H), 1.59- 1.39 (m, 1H), 1.32 (dq, J = 14.2, 7.4 Hz, 1H), 0.79 (t, J = 7.5 Hz, 3H); LCMS m/z 373.11 [M + H]+.
1H NMR (300 MHz, Chloroform-d) δ 8.06 (d, J = 23.2 Hz, 1H), 7.63-7.49 (m, 2H), 7.37-7.25 (m, 3H), 7.26- 7.12 (m, 2H), 6.98 (td, J = 9.0, 2.5 Hz, 1H), 5.42 (d, J = 7.8 Hz, 1H), 3.86-3.66 (m, 0H), 3.55 (dd, J = 11.1, 3.4 Hz, 1H), 3.44 (dd, J = 11.1, 5.7 Hz, 1H), 3.21 (dd, J = 8.2, 6.9 Hz, 2H), 2.58 (t, J = 7.5 Hz, 2H), 1.59-1.14 (m, 2H), 0.79 (t, J = 7.4 Hz, 3H); LCMS m/z 373.11 [M + H]+.
1H NMR (300 MHz, Methanol-d4) δ 7.75-7.58 (m, 2H), 7.40-7.11 (m, 4H), 6.96-6.75 (m, 1H), 4.61- 4.18 (m, 2H), 4.15-4.01 (m, 1H), 3.57-3.44 (m, 2H), 3.26- 3.06 (m, 2H), 2.61-2.46 (m, 2H); LCMS m/z 377.15 [M + H]+.
1C8 BEH Column was used for purification.
To a solution (1-aminocyclobutyl)methanol (13 mg, 0.1285 mmol) in DMSO (1 mL) was added 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid S8 (27 mg, 0.08457 mmol), HATU (45 mg, 0.1183 mmol) and NEt3 (40 μL, 0.2870 mmol). The mixture was allowed to stir at room temperature for 5 hours. Purification by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) afforded the product 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[1-(hydroxymethyl)-cyclobutyl]propanamide (91) (20 mg, 59%). 1H NMR (300 MHz, Methanol-d4) δ 7.76-7.55 (m, 2H), 7.32-7.04 (m, 3H), 6.72 (ddd, J=11.1, 9.6, 2.2 Hz, 1H), 3.66 (s, 2H), 3.21-3.01 (m, 2H), 2.56-2.38 (m, 2H), 2.18-1.92 (m, 4H), 1.84-1.65 (m, 2H). LCMS m/z 403.17 [M+H]+.
A microwave tube was charged with a solution of 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid S8 (18 mg, 0.056 mmol), 5-methyl-1,2,4-oxadiazol-3-amine (11.2 mg, 0.1130 mmol), and 1-methylsulfonylbenzotriazole (23 mg, 0.1122 mmol) in THF (1 mL). TEA (35 μL, 0.2511 mmol) was added and the reaction was heated at 130° C. for 30 minutes. Purification by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.2% formic acid) afforded 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-(5-methyl-1,2,4-oxadiazol-3-yl)propanamide (92) (2.1 mg, 9%). 1H NMR (300 MHz, Methanol-d4) δ 7.74-7.57 (m, 2H), 7.34-7.20 (m, 2H), 7.11 (dd, J=9.4, 2.2 Hz, 1H), 6.73 (ddd, J=11.1, 9.6, 2.2 Hz, 1H), 3.30 (s, 3H), 3.20-3.01 (m, 2H), 2.68-2.45 (m, 2H). LCMS m/z 401.12 [M+H]+.
To 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid S8 (29 mg, 0.089 mmol), methanesulfonamide (17 mg, 0.1787 mmol) and DMAP (22 mg, 0.1801 mmol) in DMF (1 mL) was added 3-(ethyliminomethyleneamino)-N,N-dimethyl-propan-1-amine (Hydrochloride salt) (26 mg, 0.1356 mmol). Reaction was stirred for 1 hour. After 1 hour a further eq. of DMAP was added. Reaction was stirred at room temperature for 48 hours. Purification by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) afforded the product 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-methylsulfonyl-propanamide (93) (18 mg, 50%). 1H NMR (300 MHz, Methanol-d4) δ 7.79-7.54 (m, 2H), 7.34-7.19 (m, 2H), 7.17 (dd, J=9.5, 2.2 Hz, 1H), 6.76 (ddd, J=11.1, 9.6, 2.2 Hz, 1H), 3.24-3.02 (m, 2H), 3.15 (s, 3H), 2.73-2.50 (m, 2H). LCMS m/z 396.88 [M+H]+.
To a solution of 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[(3S,4S)-4-hydroxy-2-oxo-pyrrolidin-3-yl]propanamide S1 in DMSO (1 mL) was added 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid S8 (25 mg, 0.08 mmol), HATU (33 mg, 0.09 mmol) and NEt3 (30 μL, 0.22 mmol). The mixture was allowed to stir at room temperature for 2 hours. The mixture was then purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.2% formic acid) to afford the product C28 (6 mg, 40%). 1H NMR (300 MHz, Methanol-d4) δ 7.68 (ddd, J=9.2, 5.1, 2.3 Hz, 2H), 7.37-7.19 (m, 3H), 6.75 (ddt, J=11.4, 9.6, 1.9 Hz, 1H), 4.68 (d, J=5.1 Hz, 1H), 4.40 (dd, J=5.1, 3.9 Hz, 1H), 3.65-3.57 (m, 1H), 3.26 (d, J=11.3 Hz, 1H), 3.20-3.08 (m, 2H), 2.75-2.64 (m, 2H). LCMS m/z 418.1 [M+H]+.
To a solution of 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[(3S,4R)-4-hydroxy-2-oxo-pyrrolidin-3-yl]propanamide C28 (105 mg, 0.2516 mmol) in DCM (2 mL) was added Et3N (53 μL, 0.3803 mmol). After cooling to 0° C. MSCl (24 μL, 0.3101 mmol) was added and, after stirring for 5 minutes at 0° C., the mixture was allowed to warm up to room temperature and stirred for a further 2.5 hours. The mixture was evaporated in vacuo. Purification by silica gel chromatography (Eluent: 50% EtOAc in heptane) afforded the product [(3R,4S)-4-[3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoylamino]-5-oxo-pyrrolidin-3-yl]methanesulfonate (C29) (45 mg, 31%). 1H NMR (300 MHz, Methanol-d4) δ 7.61 (ddt, J=8.3, 5.2, 2.6 Hz, 2H), 7.32-7.16 (m, 2H), 7.12 (dd, J=9.4, 2.2 Hz, 1H), 6.72 (ddd, J=11.1, 9.6, 2.2 Hz, 1H), 5.46 (dd, J=5.6, 4.1 Hz, 1H), 4.45 (d, J=5.6 Hz, 1H), 3.61 (dd, J=12.0, 4.2 Hz, 1H), 3.23-3.08 (m, 3H), 3.04 (s, 3H), 2.84-2.59 (m, 2H). LCMS m/z 496.35 [M+H]+.
A mixture of [(3R,4S)-4-[3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoylamino]-5-oxo-pyrrolidin-3-yl] methanesulfonate C29 (15 mg, 0.03027 mmol) and KOAc (9 mg, 0.09170 mmol) in DMF (2 mL) was heated to 80° C. overnight. Reaction was cooled to room temperature, diluted with water followed by extraction with EtOAc (3×5 mL). Combined organic fractions were washed with H2O (2 mL), and brine (2 mL). The organic layer was dried over sodium sulfate and filtered. Purification by silica gel chromatography (Eluent: EtOAc) afforded the product 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-(5-oxo-1,2-dihydropyrrol-4-yl)propanamide (94) (5.4 mg, 40%). 1H NMR (300 MHz, Methanol-d4) δ 7.72-7.54 (m, 2H), 7.29-7.04 (m, 4H), 6.71 (ddd, J=11.1, 9.6, 2.2 Hz, 1H), 3.95 (d, J=2.2 Hz, 2H), 3.18 (dd, J=8.5, 6.8 Hz, 2H), 2.71 (dd, J=8.5, 6.8 Hz, 2H). LCMS m/z 400.14 [M+H]+.
Compounds 95-193 (see Table 3) were prepared in a single step from intermediate S8 using the appropriate reagent and using the amide formation methods as described for compounds 91-94 (using coupling reagents such as HATU, or 1-methylsulfonylbenzotriazole). Amines were prepared by methods described above or obtained from commercial sources. Any modifications to methods are noted in Table 3 and accompanying footnotes.
1H NMR; LCMS m/z
1H NMR (400 MHz, Methanol-d4) δ 7.70-7.60 (m, 2H), 7.28-7.21 (m, 2H), 7.19 (dd, J = 9.5, 2.2 Hz, 1H), 6.73 (ddd, J = 11.0, 9.6, 2.2 Hz, 1H), 3.62 (s, 6H), 3.17-3.05 (m, 2H), 2.62-2.51 (m, 2H); LCMS m/z 423.45 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 8.60 (d, J = 8.0 Hz, 1H), 7.73-7.59 (m, 2H), 7.32-7.18 (m, 2H), 7.15 (dd, J = 9.4, 2.2 Hz, 1H), 6.72 (ddd, J = 11.1, 9.6, 2.2 Hz 1H) 4.40-4.25 (m, 1H), 3.53-3.39 (m, 1H), 3.22-3.04 (m, 3H), 2.60-2.44 (m, 2H), 1.30 (d, J = 6.2 Hz, 3H); LCMS m/z 402.3 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 7.78-7.59 (m, 2H), 7.35-7.11 (m, 3H), 6.75 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 4.62-4.17 (m, 2H), 4.05 (dq, J = 22.0, 5.3 Hz, 1H), 3.59-3.51 (m, 2H), 3.23-3.08 (m, 2H), 2.62-2.49 (m, 2H); LCMS m/z 395.27 [M + H]+
1H NMR (300 MHz, Chloroform-d) δ 8.28 (s, 1H), 7.63-7.50 (m, 2H), 7.25-7.14 (m, 2H), 7.10 (dd, J = 9.1, 2.2 Hz, 1H), 6.86-6.68 (m, 1H), 5.54 (d, J = 7.3 Hz, 1H), 3.99 (dtt, J = 10.6, 7.2, 3.7 Hz, 1H), 3.57 (dd, J = 11.1, 3.5 Hz, 1H), 3.43 (dd, J = 11.0, 6.0 Hz, 1H), 3.18 (dd, J = 8.5, 6.9 Hz, 2H), 2.53 (dd, J = 8.4, 6.9 Hz, 2H), 1.04 (d, J = 6.9 Hz, 3H); LCMS m/z 377.2 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 7.71-7.55 (m, 2H), 7.34-7.08 (m, 3H), 6.72 (ddd, J = 11.4, 9.7, 2.2 Hz, 1H), 3.77 (s, 2H), 3.22-3.02 (m, 2H), 2.68-2.39 (m, 2H); LCMS m/z 376.1 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 8.46-8.30 (m, 2H), 7.78-7.52 (m, 4H), 7.30-7.07 (m, 3H), 6.74 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 3.25 (dd, J = 8.4, 6.8 Hz, 2H), 2.73 (dd, J = 8.4, 6.8 Hz, 2H); LCMS m/z 396.14 [M + H]+
1H NMR (300 MHz, Chloroform-d) δ 8.14 (s, 1H), 7.63-7.43 (m, 2H), 7.27-7.17 (m, 2H), 7.11 (dd, J = 9.1, 2.2 Hz, 1H), 6.85-6.70 (m, 1H), 5.43 (s, 1H), 4.00 (ddt, J = 10.2, 6.8, 3.5 Hz, 1H), 3.58 (dd, J = 11.0, 3.5 Hz, 1H), 3.44 (dd, J = 11.0, 5.9 Hz, 1H), 3.27- 3.09 (m, 2H), 2.59-2.41 (m, 2H), 1.05 (d, J = 6.9 Hz, 3H); LCMS m/z 377.2 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 7.71-7.54 (m, 2H), 7.31-7.13 (m, 3H), 6.72 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 4.64-4.54 (m, 1H), 3.80-3.69 (m, 1H), 3.64 (dd, J = 11.8, 6.6 Hz, 1H), 3.20-3.09 (m, 2H), 2.71-2.50 (m, 2H); LCMS m/z 431.09 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 7.74-7.48 (m, 2H), 7.33-7.05 (m, 3H), 6.72 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 4.61 (tdd, J = 9.6, 5.5, 2.4 Hz, 1H), 3.84-3.70 (m, 1H), 3.64 (dd, J = 11.8, 6.6 Hz, 1H), 3.22- 3.03 (m, 2H), 2.75-2.53 (m, 2H); LCMS m/z 431.12 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ7.80-7.48 (m, 2H), 7.39-7.11 (m, 3H), 6.72 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 5.88 (td, J = 55.6, 3.4 Hz, 1H), 4.24 (tdq, J = 12.0, 5.9, 3.0 Hz, 1H), 3.62 (q, J = 1.0 Hz, 1H), 3.23-3.07 (m, 2H), 2.68-2.49 (m, 2H); LCMS m/z 413.04 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 8.20 (s, 2H), 7.77-7.58 (m, 2H), 7.31- 7.17 (m, 4H), 7.14 (dd, J = 9.5, 2.2 Hz, 1H), 6.73 (ddd, J = 11.0, 9.6, 2.2 Hz, 1H), 4.45 (s, 2H), 3.21-2.96 (m, 2H), 2.61-2.37 (m, 2H); LCMS m/z 399.17 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 7.68-7.62 (m, 2H), 7.26 (t, J = 8.8 Hz, 2H), 7.16 (dt, J = 9.3, 1.8 Hz, 1H), 6.87-6.71 (m, 1H), 4.76-4.56 (m, 1H), 3.98-3.84 (m, 1H), 3.58- 3.40 (m, 2H), 3.15 (t, J = 7.9 Hz, 3H), 2.75 (d, J = 4.1 Hz, 4H), 1.05-0.65 (m, 3H); LCMS m/z 391.15 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 7.71-7.51 (m, 2H), 7.31-7.17 (m, 2H), 7.12 (ddd, J = 13.0, 9.4, 2.2 Hz, 1H), 6.73 (dddd, J = 12.8, 9.6, 3.1, 2.2 Hz, 1H), 3.94 (2s, 2H), 3.12 (ddd, J = 9.2, 7.4, 3.3 Hz, 2H), 2.92 (2s, 3H), 2.83- 2.53 (m, 2H); LCMS m/z 390.14 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 7.71-7.56 (m, 2H), 7.41 (d, J = 2.3 Hz, 1H), 7.29-7.11 (m, 3H), 6.72 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 6.45 (d, J = 2.3 Hz, 1H), 3.76 (s, 3H), 3.26- 3.04 (m, 2H), 2.81-2.57 (m, 2H); LCMS m/z 399.17 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 7.74-7.53 (m, 2H), 7.37 (d, J = 3.6 Hz, 1H), 7.27-7.11 (m, 3H), 7.07 (d, J = 3.6 Hz, 1H), 6.71 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 3.23 (dd, J = 8.4, 6.8 Hz, 2H), 2.77 (dd, J = 8.4, 6.8 Hz, 2H); LCMS m/z 402.23 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 7.72-7.51 (m, 2H), 7.29-7.07 (m, 3H), 6.73 (ddd, J = 11.0, 9.6, 2.2 Hz, 1H), 4.27 (ddd, J = 9.7, 7.5, 3.8 Hz, 1H), 3.84-3.61 (m, 3H), 3.41 (dd, J = 9.1, 3.6 Hz, 1H), 3.11 (dd, J = 8.3, 6.9 Hz, 2H), 2.49 (dd, J = 8.2, 7.0 Hz, 2H), 2.10 (dq, J = 13.0, 7.6 Hz, 1H), 1.65 (dddd, J = 13.1, 7.4, 5.7, 3.8 Hz, 1H); LCMS m/z 389.18 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 8.65 (d, J = 2.5 Hz, 1H), 8.25 (dd, J = 4.9, 1.5 Hz, 1H), 8.03 (ddd, J = 8.3, 2.5, 1.4 Hz, 1H), 7.76-7.57 (m, 2H), 7.40 (ddd, J = 8.4, 4.9, 0.8 Hz, 1H), 7.31-7.10 (m, 3H), 6.74 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 3.26 (t, J = 7.6 Hz, 2H), 2.85-2.45 (m, 2H); LCMS m/z 396.11 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 7.70-7.56 (m, 2H), 7.31-7.06 (m, 3H), 6.72 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 5.95 (d, J = 3.1 Hz, 1H), 5.86 (dq, J = 3.2, 1.1 Hz, 1H), 4.20 (s, 2H), 3.11 (dd, J = 8.7, 6.8 Hz, 2H), 2.52 (dd, J = 8.7, 6.8 Hz, 2H), 2.19 (dd, J = 1.0, 0.5 Hz, 3H); LCMS m/z 413.14 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 7.61 (dd, J = 8.5, 5.3 Hz, 2H), 7.17 (q, J = 8.6 Hz, 3H), 7.03 (s, 1H), 6.72 (t, J = 10.6 Hz, 1H), 3.22 (t, J = 7.7 Hz, 2H), 2.75 (t, J = 7.6 Hz, 2H), 2.37 (s, 3H); LCMS m/z 416.11 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 7.74-7.55 (m, 3H), 7.30-7.08 (m, 4H), 6.83-6.76 (m, 1H), 6.77-6.67 (m, 1H), 4.37 (s, 2H), 3.19 (t, J = 7.5 Hz, 2H), 2.75-2.55 (m, 2H), 2.51 (s, 3H); LCMS m/z 424.18 [M + H]+
1H NMR (300 MHz, Methanol-d4) δ 8.47 (ddd, J = 5.0, 1.8, 0.9 Hz, 1H), 7.73 (td, J = 7.7, 1.8 Hz, 1H), 7.67-7.57 (m, 2H), 7.40- 7.08 (m, 6H), 6.73 (ddd, J = 11.5, 9.6, 2.2 Hz, 1H), 5.01 (t, J = 5.8 Hz, 1H), 3.76 (dd, J = 5.8, 2.5 Hz, 2H), 3.22- 3.05 (m, 2H), 2.75-2.58 (m, 2H); LCMS m/z 440.15 [M + H]+
1DIPEA was used as a base
2DMF was used as solvent
3Stirred for 12 hours
4Stirred at 40° C. for 10 minutes
5Diluted with EtOAc, washed with water and brine, dried with magnesium sulfate and filtered.
6Stirred with DCM for 20 minutes while precipitation occurred. Solid filtered and rinsed with ether.
7Stirred overnight.
8Amine was Boc protected. Stirred in TFA (1 mL) for 30 minutes. Evaporated to dryness and used in the amidation step without further purification.
To 3-[5-chloro-7-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid S9 (12 mg, 0.03288 mmol), HATU (20 mg, 0.05260 mmol) and (1-aminocyclobutyl)methanol (7 mg, 0.06921 mmol) in DMSO (0.5 mL) was added TEA (30 μL, 0.2152 mmol). The reaction was stirred at room temperature for 12 hours. The crude material was purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford 3-[5-chloro-7-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[1-(hydroxymethyl)cyclobutyl]propanamide 194 (11 mg, 80%). LCMS m/z 418.97 [M+H]+.
Compounds 195-212 (see Table 4) were prepared in a single step from intermediate S9 using the method described for the preparation of compound 194. Amines were prepared by methods described above or obtained from commercial sources. Any modifications to methods are noted in Table 4.
1H NMR; LCMS
To a solution of 3-[7-fluoro-2-(4-fluorophenyl)-5-methyl-1H-indol-3-yl]propanoic acid S10 (12 mg, 0.036 mmol) and (2R)-2-aminopropan-1-ol (5 mg, 0.067 mmol) in DMSO (0.5 mL) was added HATU (25 mg, 0.066 mmol) and Et3N (30 μL, 0.22 mmol). The reaction was stirred at ambient temperature for 12 hours. The mixture was then purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford the (6.6 mg, 49%). LCMS m/z 373.2 [M+H]+.
Compounds 214-226 (see Table 5) were prepared in a single step from intermediate S10 using the appropriate reagents and the amide coupling method as described for compound 213. Amines were prepared by methods described above or obtained from commercial sources. Any modifications to methods are noted in Table 5 and accompanying footnotes.
1H NMR; LCMS m/z
To a solution of 3-[5-bromo-7-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid S11 (20.5 mg, 0.05367 mmol), (2R)-2-aminopropan-1-ol (6 mg, 0.07988 mmol) and HATU (41 mg, 0.1078 mmol) in DMSO (1 mL) was added TEA (50 μL, 0.3587 mmol). The reaction mixture was stirred at room temperature for 12 hours. Purification by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) afforded 3-[5-bromo-7-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[(1R)-2-hydroxy-1-methyl-ethyl]propanamide (229) (16 mg, 68%). 1H NMR (300 MHz, Methanol-d4) δ 11.26 (s, 1H), 7.70-7.54 (m, 3H), 7.34-7.14 (m, 2H), 7.02 (dd, J=10.5, 1.6 Hz, 1H), 3.89 (dt, J=7.2, 5.9 Hz, 1H), 3.50-3.34 (m, 2H), 3.11 (dd, J=8.7, 6.8 Hz, 2H), 2.53-2.35 (m, 2H), 1.02 (d, J=6.8 Hz, 3H). LCMS m/z 437.05 [M+H]+.
Compounds 228-229 (see Table 6) were prepared in a single step from intermediate S11 using the method described for the preparation of compound 227. Amines were obtained from commercial sources.
1H NMR; LCMS m/z [M + H]+
1H NMR (300 MHz, Methanol- d4) δ 11.27 (s, 1H), 8.46 (d, J = 9.3 Hz, 1H), 7.73-7.54 (m, 3H), 7.35-7.12 (m, 2H), 7.02 (dd, J = 10.5, 1.6 Hz, 1H), 4.62 (dddd, J = 9.3, 8.0, 6.4, 4.8 Hz, 1H), 3.75 (dd, J = 11.8, 4.8 Hz, 1H), 3.65 (dd, J = 11.8, 6.6 Hz, 1H), 3.22-2.97 (m, 2H), 2.77- 2.46 (m, 2H); LCMS m/z 491.02 [M + H]+
1H NMR (300 MHz, Methanol- d4) δ 7.70-7.50 (m, 3H), 7.33- 7.10 (m, 2H), 7.02 (dd, J = 10.5, 1.6 Hz, 1H), 4.71-4.47 (m, 1H), 3.76 (dd, J = 11.8, 4.8 Hz, 1H), 3.65 (dd, J = 11.8, 6.7 Hz, 1H), 3.22-3.02 (m, 2H), 2.77-2.52 (m, 2H); LCMS m/z 491.06 [M + H]+
To a solution of 3-(7-bromo-2-phenyl-1H-indol-3-yl)propanoic acid C42 (413 mg, 1.160 mmol), (3S,4R)-3-amino-4-hydroxy-pyrrolidin-2-one S1 (138 mg, 1.188 mmol), and HATU (542 mg, 1.425 mmol) in DMSO (5 mL) was added TEA (500 μL, 3.587 mmol). The reaction was stirred at room temperature for an hour. Purification by reversed-phase chromatography (Column: C18. Gradient: 0-100% MeCN in water with 0.1% TFA) afforded the product. The desired peak fractions were evaporated in vacuo. Ethyl acetate (180 mL) was added. The organic layer was washed with brine, and NaHCO3, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to afford 3-(7-bromo-2-phenyl-1H-indol-3-yl)-N-[(3S,4R)-4-hydroxy-2-oxo-pyrrolidin-3-yl] propanamide (C43) (421 mg, 81%). 1H NMR (300 MHz, Chloroform-d) δ 8.23 (s, 1H), 7.65-7.51 (m, 5H), 7.45 (t, J=7.2 Hz, 1H), 7.38 (dd, J=7.6, 0.9 Hz, 1H), 7.06 (t, J=7.8 Hz, 1H), 6.21 (s, 1H), 5.55 (s, 1H), 5.52 (s, 1H), 4.22-4.16 (m, 1H), 4.00 (dd, J=8.1, 2.0 Hz, 1H), 3.67-3.55 (m, 1H), 3.37-3.15 (m, 3H), 2.70-2.57 (m, 2H). LCMS m/z 442.21 [M+H]+
To a solution of 3-(7-bromo-2-phenyl-1H-indol-3-yl)-N-[(3S,4R)-4-hydroxy-2-oxo-pyrrolidin-3-yl]propenamide C43 (26 mg, 0.05827 mmol) in 1,4-dioxane (2 mL) and water (0.5 mL) in a microwave tube was added 2-cyclopropyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (20 mg, 0.1190 mmol), [1,1′-Bis(diphenylphosphino)ferrocene]dichloro-palladium(II) (8 mg, 0.009796 mmol), Cs2CO3 (60 mg, 0.1842 mmol), and 2-cyclopropyl-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (20 mg, 0.1190 mmol). Reaction mixture was heated at 140° C. in the microwave for an hour. After cooling to room temperature, water (10 mL) was added. The aqueous layer was extracted with DCM. The organic layer was collected, and the solvent was removed in vacuo. Purification by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) afforded the product (230) 3-(7-cyclopropyl-2-phenyl-1H-indol-3-yl)-N-(5-oxo-1,2-dihydropyrrol-4-yl)propanamide (2 mg, 9%). LCMS m/z 386.16 [M+H]+.
To HATU (30 mg, 0.07890 mmol), (2S)-2-amino-3,3-difluoro-propan-1-ol (hydrochloride salt) S6 (8 mg, 0.04934 mmol) and 3-[7-chloro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid C44 (16 mg, 0.05036 mmol) in DMSO (0.5 mL) was added TEA (30 μL, 0.2152 mmol). The reaction was stirred at room temperature for 12 hours. The crude material was purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford 3-[7-chloro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[(1S)-2,2-difluoro-1-(hydroxymethyl)ethyl]propanamide (14.5 mg, 71%). LCMS m/z 411.08 [M+H]+
To 3-[4,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid S12 (12 mg, 0.03232 mmol), (2S)-2-amino-3,3,3-trifluoro-propan-1-ol (hydrochloride salt) (8 mg, 0.04833 mmol) and HATU (25 mg, 0.06575 mmol) in DMSO (1 mL) was added TEA (30 μL, 0.2152 mmol). The reaction was stirred at room temperature for 12 hours. Purification by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) afforded 3-[4,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[(1S)-2,2,2-trifluoro-1-(hydroxymethyl)ethyl]propanamide (8.5 mg, 61%). LCMS m/z 431.09 [M+H]+.
3-[4,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[(3 S)-2-oxotetrahydrofuran-3-yl]propenamide 233 was prepared in a single step from intermediate S12 using HATU as described for compound 232. Final product was isolated as 3-[4,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[(3S)-2-oxotetrahydrofuran-3-yl]propanamide (8.3 mg, 63%). LCMS m/z 402.97 [M+H]+.
To a solution of 3-[2-(4-cyanophenyl)-5,7-difluoro-1H-indol-3-yl]propanoic acid S13 (14 mg, 0.04288 mmol), (2S)-2-amino-3,3,3-trifluoro-propan-1-ol (hydrochloride salt) (10.65 mg, 0.06433 mmol) and HATU (33 mg, 0.08679 mmol) in DMSO (1 mL) was added TEA (30 μL, 0.2152 mmol). The reaction was stirred at room temperature for 12 hours. The crude material was purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford 3-[2-(4-cyanophenyl)-5,7-difluoro-1H-indol-3-yl]-N-[(1S)-2,2,2-trifluoro-1-(hydroxymethyl)ethyl]propanamide (5.1 mg, 26%). 1H NMR (300 MHz, Methanol-d4) δ 7.92-7.82 (m, 4H), 7.19 (ddd, J=20.2, 9.3, 2.2 Hz, 1H), 6.79 (ddd, J=11.5, 9.6, 2.2 Hz, 1H), 4.60 (td, J=7.1, 4.9 Hz, 1H), 3.75 (dd, J=11.8, 4.8 Hz, 1H), 3.63 (dd, J=11.8, 6.6 Hz, 1H), 3.19 (td, J=8.0, 2.6 Hz, 2H), 2.74-2.49 (m, 2H). LCMS m/z 438.19 [M+H]+.
To a solution of (2S)-2-amino-3,3-difluoro-propan-1-ol hydrochloride S14 (8 mg, 0.05 mmol) in DMSO (0.5 mL) was added 3-(5-fluoro-2-phenyl-1H-indol-3-yl)propanoic acid S6 (16 mg, 0.056 mmol), HATU (16 mg, 0.056 mmol), and NEt3 (30 μL, 0.22 mmol). The mixture was allowed to stir at room temperature for 12 hours. The mixture was then purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford the product (21 mg, 74%). LCMS m/z 377.1 [M+H]+.
Compound 236 (see Table 7) was prepared from intermediate S14 using the appropriate reagent and using the amide formation method as described for compound 235.
1H NMR; LCMS m/z
1H NMR (400 MHz, Chloroform-d) δ 8.13 (s, 1H), 7.64-7.56 (m, 2H), 7.56-7.49 (m, 2H), 7.48- 7.36 (m, 1H), 7.38-7.29 (m, 2H), 6.98 (td, J = 9.0, 2.5 Hz, 1H), 5.75 (s, 1H), 3.58 (d, J = 5.2 Hz, 2H), 3.32 (td, J = 5.6, 4.4 Hz, 2H), 3.28-3.21 (m, 2H), 2.62-2.51 (m, 2H); LCMS m/z 327.21 [M + H]+
Compounds 237-238 (see Table 8) were obtained from commercial sources and may be prepared using analogous procedures to those used in the preparation of compounds 1-236.
1H NMR; LCMS m/z
To a solution of 4-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]butanoic acid S15 (16 mg, 0.097 mmol) in DMF (1 mL) was added methanamine (2 mg, 0.65 mmol), HATU (30 mg, 0.07 mmol), and NEt3 (18 μL, 0.13 mmol). The mixture was allowed to stir at room temperature for 2 hours. The mixture was then purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford the product (21 mg, 74%). 1H NMR (300 MHz, Chloroform-d) δ 7.73-7.46 (m, 2H), 7.31-7.15 (m, 2H), 7.10 (dd, J=9.4, 2.2 Hz, 1H), 6.72 (ddd, J=11.0, 9.6, 2.2 Hz, 1H), 2.93-2.73 (m, 2H), 2.67 (s, 3H), 2.20 (t, J=7.3 Hz, 2H), 1.94 (p, J=7.5 Hz, 2H).
Compounds 240-256 (see Table 9) were prepared from intermediate S15 using the appropriate reagent and using the amide formation method as described for compound 239. Amines were prepared by methods described above or obtained from commercial sources.
1H NMR; LCMS m/z
1H NMR (300 MHz, Chloroform-d) δ 8.23 (s, 1H), 7.64-7.44 (m, 2H), 7.24-7.12 (m, 2H), 7.09 (dd, J = 9.1, 2.2 Hz, 1H), 6.76 (ddd, J = 10.8, 9.4, 2.2 Hz, 1H), 5.51 (d, J = 7.1 Hz, 1H), 4.06 (ddt, J = 10.3, 6.8, 3.4 Hz, 1H), 3.66 (dd, J = 10.9, 3.5 Hz, 1H), 3.51 (dd, J = 11.0, 6.2 Hz, 1H), 2.83 (dd, J = 8.6, 6.7 Hz, 2H), 2.21 (t,
1H NMR (300 MHz, Chloroform-d) δ 8.21 (s, 1H), 7.66-7.43 (m, 2H), 7.25-7.13 (m, 2H), 7.10 (dd, J = 9.1, 2.2 Hz, 1H), 6.76 (ddd, J = 11.4, 9.5, 2.2 Hz, 1H), 5.50 (d, J = 7.2 Hz, 1H), 4.06 (dtq, J = 10.3, 6.8, 3.3 Hz, 1H), 3.66 (dd, J = 10.9, 3.4 Hz, 1H), 3.51 (dd, J = 11.0, 6.2 Hz, 1H), 2.92-2.82 (m, 2H), 2.22 (t, J = 7.3
1H NMR (300 MHz, Chloroform-d) δ 7.63 (dd, J = 8.7, 5.3 Hz, 2H), 7.22 (d, J = 8.8 Hz, 2H), 7.12 (dd, J = 9.3, 2.2 Hz, 1H), 6.80-6.66 (m, 1H), 2.96- 2.65 (m, 2H), 2.25 (t, J = 7.3 Hz, 2H), 1.95 (p, J = 7.5 Hz, 2H); LCMS m/z 333.1 [M + H]+
1H NMR (300 MHz, Chloroform-d) δ 8.84- 8.62 (m, 1H), 7.72-7.52 (m, 2H), 7.39 (d, J = 1.5 Hz, 1H), 7.32-7.15 (m, 2H), 7.07 (dd, J = 9.4, 2.2 Hz, 1H), 6.73 (ddd, J = 11.0, 9.6, 2.2 Hz, 1H), 4.35 (d, J = 0.9 Hz, 2H), 3.87 (s, 3H), 2.98-2.69 (m, 2H), 2.27 (t, J = 7.4 Hz, 2H), 1.96 (p, J = 7.5 Hz, 2H); LCMS m/z 427.14 [M + H]+
1H NMR (300 MHz, Chloroform-d) δ 8.29 (s, 1H), 7.61-7.41 (m, 2H), 7.24-7.12 (m, 2H), 7.09 (dd, J = 9.1, 2.2 Hz, 1H), 6.74 (ddd, J = 10.8, 9.4, 2.2 Hz, 1H), 6.19-5.86 (m, 2H), 4.30 (ddd, J = 10.9, 8.2, 5.3 Hz, 1H), 3.37 (ddd, J = 9.8, 3.5, 1.3 Hz, 2H), 2.90-2.68
To a solution of 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethanamine (75 mg, 0.2282 mmol) S19 and DIPEA (100 μL, 0.575 mmol) in DMF (3 mL) was added (2S)-2-(tert-butoxycarbonylamino)propanoic acid (55 mg, 0.2907 mmol and HATU (100 mg, 0.2630 mmol). The mixture was stirred overnight and purified by reversed-phase chromatography (Column: C18. Gradient: 0-100% MeCN in water with 0.1% trifluoroacetic acid) to yield tert-butyl N-[(1S)-2-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethylamino]-1-methyl-2-oxo-ethyl]carbamate (55 mg, 46%). LCMS m/z 462.18 [M+H]+.
To solution of tert-butyl N-[(1S)-2-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethylamino]-1-methyl-2-oxo-ethyl]carbamate (25 mg, 0.05417 mmol) C63 in DCM (10 mL) was added TFA (100 μL, 1.298 mmol) the reaction was stirred overnight. The reaction was then concentrated and re-dissolved in DMSO (5 mL). The crude residue was then purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to yield (2S)-2-amino-N-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]propanamide (5.5 mg, 25%). 1H NMR (300 MHz, Acetone-d6) δ 7.84-7.73 (m, 2H), 7.35-7.23 (m, 3H), 6.85 (ddd, J=11.3, 9.6, 2.2 Hz, 1H), 4.90 (q, J=7.0 Hz, 1H), 3.58-3.47 (m, 2H), 3.30 (d, J=0.8 Hz, 1H), 3.11-3.00 (m, 2H), 2.75-2.47 (m, 1H), 1.57 (dd, J=10.9, 7.0 Hz, 3H). LCMS m/z 362.14 [M+H]+.
To a solution of (2S)-2-amino-N-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]propenamide C64 (25 mg, 0.06581 mmol) dissolved in DCM (5 mL) was added triphosgene (20 mg, 0.0674 mmol) and DIPEA (50 μL, 0.287 mmol). The reaction was stirred for 3 hours at room temperature at which point it was concentrated and re-dissolved in DMSO (5 mL). This crude solution was purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to yield (5 S)-3-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]-5-methyl-imidazolidine-2,4-dione (17.5 mg, 59%). 1H NMR (300 MHz, Acetone-d6) δ 7.99-7.67 (m, 3H), 7.44-7.21 (m, 3H), 7.14 (s, 1H), 6.84 (ddd, J=11.0, 9.7, 2.2 Hz, 1H), 4.01 (qd, J=7.0, 1.2 Hz, 1H), 3.91 (ddt, J=13.3, 6.7, 3.4 Hz, 1H), 3.86-3.60 (m, 2H), 3.39 (qd, J=7.4, 4.4 Hz, 2H), 3.21-2.93 (m, 2H), 1.25 (d, J=7.0 Hz, 3H). LCMS m/z 388.1 [M+H]+.
To a solution of 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethanamine (75 mg, 0.2282 mmol) S19 and DIPEA (100 μL, 0.575 mmol) in DMF (3 mL) was added 2-(tert-butoxycarbonylamino)acetic acid (50 mg, 0.2854 mmol) and HATU (100 mg, 0.2630 mmol). The mixture was stirred overnight and purified by reversed-phase chromatography (Column: C18. Gradient: 0-100% MeCN in water with 0.1% trifluoroacetic acid) to yield tert-butyl N-[2-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethylamino]-2-oxo-ethyl]carbamate (65 mg, 35%). LCMS m/z 448.23 [M+H]+.
To a solution of tert-butyl N-[(1S)-2-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethylamino]-1-methyl-2-oxo-ethyl]carbamate C65 (25 mg, 0.04930 mmol) in DCM (10 mL) was added TFA (100 μL, 1.298 mmol) and reaction was stirred overnight. The reaction was then concentrated, dissolved in DMSO (1 mL), and purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to yield 2-amino-N-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]acetamide (Trifluoroacetate salt) (5.3 mg, 21%). 1H NMR (300 MHz, Acetone-d6) δ 7.83-7.72 (m, 2H), 7.35-7.23 (m, 3H), 6.84 (ddd, J=11.1, 9.7, 2.2 Hz, 1H), 4.58 (s, 2H), 3.57 (dt, J=15.4, 7.7 Hz, 2H), 3.06 (dd, J=9.1, 6.4 Hz, 2H). LCMS m/z 348.14 [M+H]+.
To a solution of 2-amino-N-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]acetamide C66 (25 mg, 0.0654 mmol) dissolved in DCM (5 mL) was added triphosgene (20 mg, 0.0674 mmol) and DIPEA (50 μL, 0.287 mmol) and the reaction was stirred overnight. The reaction was then concentrated, re-dissolved in DMSO (5 mL), and purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: 0-100% MeCN in H2O with 0.1% trifluoroacetic acid) to yield 3-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]imidazolidine-2,4-dione (15.9 mg, 58%). 1H NMR (300 MHz, Acetone-d6) δ 8.03-7.63 (m, 2H), 7.49-7.23 (m, 3H), 7.05 (s, 1H), 6.84 (ddd, J=11.1, 9.7, 2.2 Hz, 1H), 3.85 (d, J=1.0 Hz, 2H), 3.79-3.68 (m, 2H), 3.20-2.92 (m, 2H). LCMS m/z 374.02 [M+H]+.
To a solution of 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethanamine as a TFA salt S19 (297 mg, 0.73 mmol) in DMF (5 mL) was added a solution of bis(4-nitrophenyl) carbonate (268 mg, 0.88 mmol) in DMF (5 mL) and DIPEA (300 μL, 1.72 mmol). The reaction was stirred for 1 hour and then 2-amino-3-fluoro-propan-1-ol (HCl salt) (120 mg, 0.93 mmol) was added and the reaction was stirred overnight. The reaction was purified by reverse-phase chromatography (C18 column; Gradient: 0-100% MeCN in H2O with 0.1% TFA) to afford the title compound (280 mg, 85%). 1H NMR (400 MHz, Acetone-d6) δ 7.97-7.73 (m, 2H), 7.36-7.25 (m, 2H), 6.92-6.72 (m, 1H), 4.58 (dd, J=8.9, 4.5 Hz, 1H), 4.47 (ddd, J=8.9, 7.6, 5.0 Hz, 1H), 4.36 (dd, J=8.9, 5.5 Hz, 1H), 3.71-3.64 (m, 1H), 3.64-3.54 (m, 2H), 3.54-3.40 (m, 3H), 3.19-2.85 (m, 2H). LCMS m/z 410.2 [M+H]+.
Compounds 260-303 (see Table 10) were prepared in a single step from intermediate S19 using the appropriate reagents and the amide coupling method as described for the preparation of compound 259. Amines were prepared by methods described above or obtained from commercial sources. Any modifications to methods are noted in Table 10 and accompanying footnotes.
1H NMR; LCMS m/z [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.70 (s, 1H), 7.99-7.74 (m, 2H), 7.53-7.05 (m, 3H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 6.10 (s, 1H), 5.90 (s, 1H), 3.74 (d, J = 9.5 Hz, 2H), 3.45 (d, J = 8.0 Hz, 2H), 3.19- 2.88 (m, 4H); LCMS m/z 406.89 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.70 (s, 1H), 7.93-7.66 (m, 2H), 7.44-7.11 (m, 3H), 6.83 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 6.08 (d, J = 9.7 Hz, 1H), 4.58 (ddp, J = 13.1, 8.6, 4.3 Hz, 1H), 4.03-3.69 (m, 3H), 3.55-3.45 (m, 3H), 3.18- 2.88 (m, 2H); LCMS m/z 446.16 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.70 (s, 1H), 7.91-7.72 (m, 2H), 7.47-7.19 (m, 3H), 6.83 (ddd, J = 11.2, 9.7, 2.2 Hz, 1H), 6.09 (d, J = 9.3 Hz, 2H), 4.76-4.45 (m, 1H), 3.99-3.69 (m, 3H), 3.35 (s, 1H), 3.17- 2.91 (m, 3H); LCMS m/z 446.2 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 7.77 (dd, J = 8.5, 5.4 Hz, 2H), 7.29 (t, J = 10.4 Hz, 2H), 6.82 4, J = 10.1 Hz, 1H), 3.65 (t, J = 5.5 Hz, 2H), 3.52 (t, J = 7.7 Hz, 2H), 3.15-2.99 (m, 3H), 3.01-2.71 (m, 2H); LCMS m/z 441.81 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 8.00-7.54 (m, 2H), 7.34 (dd, J =9.5, 2.2 Hz, 1H), 7.29- 7.09 (m, 3H), 7.00-6.54 (m, 4H), 5.82 (d, J = 28.3 Hz, 2H), 4.29 (d, J = 5.2 Hz, 2H), 3.48 (d, J = 8.5 Hz, 2H), 3.15-2.87 (m, 4H); LCMS m/z 440.27 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.70 (s, 1H), 8.09-7.66 (m, 2H), 7.51-7.06 (m, 3H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 5.60 (s, 1H), 4.30 (s, 1H), 4.10 (s, 1H), 3.73 (dd, J = 10.5, 3.2 Hz, 1H), 3.50 (ddd, J = 9.9, 4.9, 1.9 Hz, 4H), 3.02 (d, J = 6.7 Hz, 2H), 0.93 (s, 9H); LCMS m/z 434.33 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 7.94-7.68 (m, 2H), 7.44- 7.12 (m, 3H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.48 (dd, J = 8.7, 6.4 Hz, 4H), 3.31 (s, 2H), 3.12-2.84 (m, 2H), 1.93-1.67 (m, 2H), 1.67-1.34 (m, 6H); LCMS m/z 432.28 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 8.08-7.44 (m, 2H), 7.41- 7.15 (m, 3H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 5.76 (td, J = 55.8, 4.5 Hz, 2H), 3.89- 3.69 (m, 1H), 3.45 (d, J = 14.9 Hz, 3H), 3.36-3.22 (m, 1H), 3.21-2.91 (m, 3H); LCMS m/z 427.8 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.75 (s, 1H), 8.00-7.61 (m, 2H), 7.29 (dtd, J = 8.9, 6.7, 2.2 Hz, 3H), 6.82 (ddd, J = 11.0, 9.6, 2.2 Hz, 1H), 5.50 (s, 1H), 4.50 (s, 2H), 3.68 (q, J = 6.7 Hz, 1H), 3.48 (dd, J = 8.5, 6.6 Hz, 2H), 3.22-2.84 (m, 2H), 1.10 (dd, J = 12.3, 5.8 Hz, 9H); LCMS m/z 419.82 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.80 (s, 1H), 7.98-7.68 (m, 2H), 7.47-7.11 (m, 3H), 7.05-6.64 (m, 1H), 5.50 (s, 1H), 3.85 (s, 2H), 3.66-3.40 (m, 5H), 3.25-2.82 (m, 2H), 1.90 (dq, J = 13.5, 6.9 Hz, 1H), 0.90 (dd, J = 11.1, 6.9 Hz, 6H); LCMS m/z 420.29 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 7.99-7.62 (m, 2H), 7.44- 7.14 (m, 3H), 6.82 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 3.45 (dd, J = 8.8, 6.4 Hz, 3H), 3.36- 3.09 (m, 4H), 3.14-2.90 (m, 3H), 1.82-1.51 (m, 2H), 1.18 (s, 6H); LCMS m/z 419.82 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.72 (s, 1H), 7.87-7.74 (m, 2H), 7.36-7.20 (m, 3H), 7.01 (s, 1H), 6.81 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 6.30 (s, 1H), 4.11 (h, J = 6.4 Hz, 1H), 3.50-3.39 (m, 2H), 3.06- 2.95 (m, 2H), 2.43 (dd, J = 14.8, 5.9 Hz, 1H), 2.28 (dd, J = 14.8, 6.2 Hz, 1H), 1.15 (d, J = 6.6 Hz, 3H); LCMS m/z 419 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 8.54-8.08 (m, 1H), 7.94- 7.70 (m, 2H), 7.55-7.40 (m, 1H), 7.37-7.14 (m, 2H), 6.96- 6.68 (m, 1H), 4.35-4.12 (m, 1H), 4.06 (s, 1H), 3.54-3.38 (m, 2H), 3.10-2.95 (m, 3H), 1.99-1.80 (m, 2H), 1.67 (d, J = 3.2 Hz, 2H), 1.54-1.35 (m, 1H); LCMS m/z 417.8 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 8.02-7.72 (m, 2H), 7.41- 7.09 (m, 2H), 6.82 (ddd, J = 11.2, 9.7, 2.2 Hz, 1H), 4.27 (dd, J = 7.8, 4.9 Hz, 2H), 3.44 (t, J = 7.5 Hz, 2H), 3.00 (dd, J = 8.7, 6.4 Hz, 2H), 2.61-2.48 (m, 1H), 2.01-1.77 (m, 4H), 1.69-1.44 (m, 2H), 1.33 (ddd, J = 12.6, 9.0, 6.5 Hz, 2H); LCMS m/z 417.7 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.75 (s, 1H), 7.79 (dd, J = 8.8, 5.4 Hz, 2H), 7.29 (t, J = 8.4 Hz, 3H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 6.50 (s, 1H), 4.19 (s, 2H), 3.95 (s, 1H), 3.65 (s, 1H), 3.42 (q, J = 6.5 Hz, 2H), 3.05 (dd, J = 9.1, 6.3 Hz, 3H), 1.20 (q, J = 4.0 Hz, 2H), 0.98 (q, J = 4.0 Hz, 3H); LCMS m/z 417.8 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.75 (s, 1H), 7.81 (ddd, J = 8.1, 5.1, 2.4 Hz, 2H), 7.46- 7.10 (m, 3H), 6.82 (ddd, J = 11.6, 9.7, 2.2 Hz, 1H), 5.90 (s, 1H), 3.63-3.36 (m, 2H), 3.35 (s, 2H), 3.17-2.82 (m, 2H), 2.00-1.84 (m, 5H), 1.77-1.56 (m, 2H), 1.56-1.15 (m, 1H); LCMS m/z 417.83 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 7.98-7.66 (m, 2H), 7.40- 7.21 (m, 3H), 6.86-6.76 (m, 1H), 3.48 (dd, J = 8.7, 6.4 Hz, 2H), 3.16-2.96 (m, 5H), 0.64- 0.20 (m, 5H); LCMS m/z 417.8 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.72 (s, 1H), 7.89-7.66 (m, 2H), 7.44-7.21 (m, 3H), 7.05 (s, 1H), 6.81 (ddd, J = 11.0, 9.6, 2.2 Hz, 1H), 4.24 (t, J = 9.4 Hz, 1H), 3.58-3.42 (m, 2H), 3.42-3.31 (m, 2H), 3.18-2.90 (m, 2H), 2.56 (ddt, J = 12.1, 8.0, 3.9 Hz, 1H), 1.97- 1.70 (m, 1H); LCMS m/z 417.13 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.70 (s, 1H), 7.96-7.70 (m, 2H), 7.46-7.18 (m, 3H), 6.82 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 5.80 (s, 1H), 3.72 (ddd, J = 9.6, 6.3, 3.4 Hz, 1H), 3.51-3.31 (m, 3H), 3.14-2.86 (m, 5H), 1.67-1.44 (m, 1H), 1.36 (ddt, J = 13.7, 9.5, 4.8 Hz, 1H), 1.09 (d, J = 6.2 Hz, 3H); LCMS m/z 405.81 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 7.92-7.64 (m, 2H), 7.46- 7.15 (m, 3H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.61- 3.41 (m, 2H), 3.12 (s, 2H), 3.09-2.80 (m, 2H), 1.11 (s, 6H); LCMS m/z 405.85 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 7.99-7.70 (m, 2H), 7.47- 7.08 (m, 3H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.61- 3.35 (m, 3H), 3.34-3.18 (m, 2H), 3.18-2.84 (m, 3H), 1.64 (qt, J = 6.8, 4.7 Hz, 1H), 0.82 (d, J = 6.9 Hz, 3H); LCMS m/z 406.13 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 7.93-7.62 (m, 2H), 7.40- 7.08 (m, 3H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 6.02 (s, 1H), 4.33 (p, J = 7.0 Hz, 1H), 3.50 (s, 2H), 3.14-2.91 (m, 3H), 1.33 (d, J = 7.2 Hz, 3H); LCMS m/z 405.91 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 8.00-7.72 (m, 2H), 7.40- 7.08 (m, 3H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.79 (d, J = 7.3 Hz, 1H), 3.65-3.25 (m, 5H), 3.14-2.86 (m, 3H), 1.08 (d, J = 6.7 Hz, 3H); LCMS m/z 391.84 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.70 (s, 1H), 7.93-7.64 (m, 2H), 7.40-7.14 (m, 3H), 6.93-6.60 (m, 1H), 5.68 (s, 1H), 5.36 (s, 1H), 4.13 (t, J = 5.3 Hz, 1H), 3.79 (dt, J = 12.1, 6.0 Hz, 1H), 3.44 (td, J = 5.2, 1.7 Hz, 2H), 3.30 (s, 2H), 3.11- 2.89 (m, 2H), 1.08 (d, J = 6.8 Hz, 3H); LCMS m/z 392.24 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 7.99-7.62 (m, 2H), 7.42- 7.14 (m, 3H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 5.81 (d, J = 43.8 Hz, 2H), 3.96-3.66 (m, 1H), 3.57-3.35 (m, 2H), 3.20 (dt, J = 13.8, 4.7 Hz, 1H), 3.09-2.89 (m, 4H), 1.07 (d, J = 6.3 Hz, 3H); LCMS m/z 391.94 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.75 (s, 1H), 7.94-7.72 (m, 2H), 7.55-7.35 (m, 3H), 7.36-7.08 (m, 1H), 6.85-6.75 (m, 1H), 6.10 (s, 1H), 5.90 (s, 1H), 3.99-3.78 (m, 2H), 3.64- 3.38 (m, 2H), 3.16-2.90 (m, 2H); LCMS m/z 391.77 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 8.31-8.05 (m, 1H), 7.89- 7.68 (m, 2H), 7.44-7.21 (m, 3H), 7.15-6.98 (m, 1H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.71-3.54 (m, 2H), 3.54- 3.41 (m, 2H), 3.25 (dd, J = 5.7, 5.0 Hz, 2H), 3.10-2.84 (m, 2H); LCMS m/z 377.83 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 7.93-7.66 (m, 2H), 7.42- 7.15 (m, 3H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 5.95 (s, 1H), 5.76 (s, 1H), 3.70-3.55 (m, 2H), 3.24 (t, J = 6.4 Hz, 2H), 3.08-2.83 (m, 6H); LCMS m/z 439.85 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 8.92-8.63 (m, 2H), 7.80 (dt, J = 5.3, 3.8 Hz, 4H), 7.29 (dtd, J = 8.9, 7.1, 6.6, 2.2 Hz, 3H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.54 (t, J = 6.8 Hz, 2H), 3.44 (dd, J = 8.7, 6.4 Hz, 2H), 3.14-2.83 (m, 4H); LCMS m/z 439.26 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 7.93-7.68 (m, 2H), 7.46- 7.12 (m, 3H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.44 (t, J = 7.6 Hz, 2H), 3.26 (d, J = 5.4 Hz, 2H), 3.08-2.97 (m, 2H), 2.56 (ddd, J = 13.5, 7.5, 5.5 Hz, 2H), 2.45-2.13 (m, 3H); LCMS m/z 437.77 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.71 (s, 1H), 7.89-7.76 (m, 2H), 7.39-7.21 (m, 3H), 7.06 (s, 1H), 6.81 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 6.21 (d, J = 22.5 Hz, 2H), 4.74-4.59 (m, 1H), 3.48-3.39 (m, 2H), 3.07-2.96 (m, 2H), 2.69 (s, 3H), 2.56-2.21 (m, 2H), 1.12 (d, J = 6.8 Hz, 3H); LCMS m/z 433.14 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 7.81 (ddt, J = 8.6, 5.4, 2.7 Hz, 2H), 7.42-7.19 (m, 3H), 6.82 (ddd, J = 11.6, 9.6, 2.2 Hz, 1H), 3.95-3.73 (m, 2H), 3.45 (dd, J = 8.6, 6.3 Hz, 2H), 3.27 (td, J = 11.7, 2.1 Hz, 3H), 3.16-2.90 (m, 5H), 1.71-1.48 (m, 3H), 1.33-1.06 (m, 2H); LCMS m/z 431.81 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.75 (s, 1H), 8.03-7.60 (m, 2H), 7.42-7.19 (m, 3H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.55-3.36 (m, 4H), 3.30- 3.10 (m, 7H), 3.03-2.88 (m, 2H), 0.74-0.25 (m, 4H); LCMS m/z 431.81 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 8.00-7.68 (m, 2H), 7.41- 7.14 (m, 3H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 4.02- 3.65 (m, 2H), 3.63-3.42 (m, 2H), 3.31 (ddd, J = 11.2, 10.1, 3.1 Hz, 2H), 3.26-2.80 (m, 6H), 1.91-1.02 (m, 5H); LCMS m/z 432.28 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 7.98-7.63 (m, 2H), 7.44- 7.21 (m, 3H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 4.49- 4.23 (m, 1H), 3.69 (dd, J = 10.2, 6.9 Hz, 1H), 3.50-3.28 (m, 2H), 3.20 (dd, J = 10.2, 4.0 Hz, 1H), 3.14-2.95 (m, 2H), 2.77 (d, J = 1.6 Hz, 3H), 2.63 (ddq, J = 16.9, 8.3, 0.8 Hz, 1H), 2.23-2.10 (m, 1H); LCMS m/z 430.76 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 7.96-7.68 (m, 2H), 7.47- 7.09 (m, 4H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 6.31 (d, J = 1.9 Hz, 1H), 4.09 (s, 2H), 3.46 (t, J = 7.7 Hz, 2H), 3.17- 2.83 (m, 4H), 2.24 (s, 3H); LCMS m/z 428.28 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.71 (s, 1H), 7.81 (dd, J = 8.8, 5.4 Hz, 2H), 7.34-7.22 (m, 3H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.94 (s, 1H), 3.52-3.39 (m, 4H), 3.34-3.20 (m, 2H), 3.05 (dd, J = 8.4, 6.3 Hz, 2H), 2.04-1.74 (m, 3H), 1.63 (dt, J = 9.8, 5.3 Hz, 1H); LCMS m/z 417.96 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 8.12-7.57 (m, 2H), 7.46- 7.09 (m, 3H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.90- 3.73 (m, 2H), 3.73-3.54 (m, 1H), 3.46 (t, J = 7.6 Hz, 2H), 3.30-2.80 (m, 6H), 1.97-1.63 (m, 3H), 1.64-1.45 (m, 1H); LCMS m/z 417.8 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 11.35 (s, 1H), 7.93-7.60 (m, 2H), 7.42-7.12 (m, 3H), 6.84 (ddd, J = 11.6, 9.7, 2.2 Hz, 1H), 4.30 (d, J = 10.7 Hz, 2H), 4.07 (d, J = 10.8 Hz, 2H), 3.55 (d, J = 5.2 Hz, 3H), 3.30 (d, J = 12.4 Hz, 1H), 3.23- 2.95 (m, 4H), 0.95 (s, 3H); LCMS m/z 418.27 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 7.93-7.68 (m, 2H), 7.54- 7.42 (m, 1H), 7.41-7.14 (m, 3H), 6.82 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 6.42 (dd, J = 1.8, 0.8 Hz, 1H), 4.18 (s, 2H), 3.73- 3.35 (m, 2H), 3.21-2.83 (m, 3H); LCMS m/z 414.28 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 8.11-7.65 (m, 2H), 7.44- 7.15 (m, 3H), 6.98-6.60 (m, 1H), 4.31 (dp, J = 13.7, 7.1 Hz, 1H), 3.70-3.36 (m, 4H), 3.25- 2.95 (m, 2H), 2.73 (s, 3H), 1.07 (d, J = 6.9 Hz, 3H); LCMS m/z 406.16 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 8.05-7.68 (m, 2H), 7.29 (dtd, J = 8.8, 7.0, 2.2 Hz, 3H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 5.95-5.48 (m, 2H), 4.04 (p, J = 6.2 Hz, 1H), 3.46 (p, J = 7.2 Hz, 2H), 3.01 (dd, J = 7.8, 6.8 Hz, 2H), 2.88-2.54 (m, 2H), 1.24 (d, J = 6.8 Hz, 3H); LCMS m/z 401.28 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.71 (s, 1H), 7.87-7.75 (m, 2H), 7.38-7.20 (m, 3H), 6.82 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 6.09 (s, 1H), 3.74- 3.59 (m, 2H), 3.52-3.41 (m, 2H), 3.36 (t, J = 5.4 Hz, 2H), 3.13-2.97 (m, 2H), 2.89 (s, 3H); LCMS m/z 392.16 [M + H]+
1H NMR (300 MHz, Acetone- d6) δ 10.72 (s, 1H), 7.86-7.74 (m, 2H), 7.38-7.23 (m, 3H), 6.82 (ddd, J = 11.6, 9.7, 2.1 Hz, 1H), 3.84 (s, 1H), 3.64- 3.53 (m, 2H), 3.43 (d, J = 5.4 Hz, 2H), 3.06 (t, J = 7.5 Hz, 2H), 2.83 (s, 3H), 1.05 (d, J = 6.7 Hz, 3H); LCMS m/z 406.24 [M + H]+
To a solution of 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethanamine (Trifluoroacetate salt) S19 (50 mg, 0.1092 mmol) in DMF (2 mL) was added 4-amino-2-(tert-butoxycarbonylamino)-4-oxo-butanoic acid (25 mg, 0.108 mmol), HATU (40 mg, 0.105 mmol), and DIPEA (50 μL, 0.2871 mmol). The mixture was allowed to stir at room temperature overnight. The mixture was then purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford the product tert-butyl N-[3-amino-1-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethylcarbamoyl]-3-oxo-propyl]carbamate (2.6 mg, 8%). 1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.80 (ddt, J=8.7, 5.5, 3.8 Hz, 2H), 7.41-7.22 (m, 4H), 6.83 (ddt, J=11.2, 9.6, 2.4 Hz, 1H), 4.36 (s, 1H), 3.60-3.47 (m, 3H), 3.13-2.96 (m, 2H), 2.82-2.52 (m, 1H), 2.10-2.07 (m, 1H), 1.39 (s, 9H). LCMS m/z 505.2 [M+H]+.
To a solution of 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethanamine (Trifluoroacetate salt) S19 (40 mg, 0.087 mmol) and DIPEA (33.3 μL, 0.191 mmol) dissolved in DMF (2 mL) was added (2R)-2-(tert-butoxycarbonylamino)-3-hydroxy-propanoic acid (60 mg, 0.2924 mmol) and HATU (40 mg, 0.105 mmol). The mixture was allowed to stir at room temperature overnight. The mixture was then purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford tert-butyl N-[(1R)-2-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethylamino]-1-(hydroxymethyl)-2-oxo-ethyl]carbamate (11.8 mg, 30%). 1H NMR (300 MHz, Acetone-d6) δ 7.88-7.60 (m, 3H), 7.43-7.06 (m, 3H), 6.83 (dddd, J=11.1, 9.7, 6.0, 2.2 Hz, 1H), 4.20-4.02 (m, 1H), 3.98-3.62 (m, 2H), 3.64-3.48 (m, 2H), 3.06 (q, J=7.0 Hz, 2H), 1.52-1.14 (m, 9H). LCMS m/z 478.2 [M+H]+.
To a solution of tert-butyl N-[(1R)-2-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethylamino]-1-(hydroxymethyl)-2-oxo-ethyl]carbamate 305 (15 mg, 0.030 mmol) in dichloromethane was added trifluoroacetic acid (20 μL, 0.2596 mmol) and the solution was stirred at room temperature for 3 hours. The reaction was concentrated and redissolved in dichloromethane (5 mL). To the solution was added triphosgene (15 mg, 0.05055 mmol) and diisopropylethylamine (20 μL, 0.1148 mmol) and stirred overnight. Reaction was concentrated and dissolved in DMSO (2 mL). The mixture was then purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford the product (4R)—N-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]-2-oxo-oxazolidine-4-carboxamide (4.7 mg, 33%). 1H NMR (300 MHz, Acetone-d6) δ 7.94-7.63 (m, 2H), 7.30 (dtd, J=8.9, 5.8, 2.9 Hz, 2H), 6.85 (t, J=10.5 Hz, 1H), 4.55 (dd, J=9.4, 8.4 Hz, 1H), 4.34 (ddd, J=9.4, 5.2, 1.7 Hz, 1H), 4.19 (d, J=5.2 Hz, 1H), 3.83-3.66 (m, 2H), 3.66-3.50 (m, 1H), 3.07 (td, J=8.7, 7.5, 4.1 Hz, 2H). LCMS m/z 404.22 [M+H]+.
Compounds 307-417 (see Table 11) were prepared from intermediate S19 using the appropriate reagents, using the amide formation methods as described for compounds 304, and 306 (using coupling reagents such as HATU, post amide formation modifications). Carboxylic acids were prepared by methods described above or obtained from commercial sources. Any modifications to methods are noted in Table 11 and accompanying footnotes.
1H NMR; LCMS m/z
1H NMR (300 MHz, Acetone-d6) δ, 10.8 (s, 1H), 7.80 (ddd, J = 8.5, 5.4, 2.6 Hz, 2H), 7.43-7.19 (m, 3H), 6.96-6.78 (m, 1H), 6.20 (d, J = 7.8 Hz, 1H), 4.37-4.09 (m, 1H), 3.72-3.44 (m, 4H), 3.05 (dd, J = 9.3, 6.4 Hz, 2H), 2.16-1.99 (m, 2H), 2.02-1.83 (m, 1H), 1.85- 1.57 (m, 1H), 1.40 (d, J = 1.9 Hz, 9H); LCMS m/z 492.24 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 10.8 (s, 1H), 7.93-7.73 (m, 2H), 7.41- 7.08 (m, 3H), 6.91-6.65 (m, 1H), 4.12 (s, 1H), 3.96-3.63 (m, 2H), 3.66-3.43 (m, 2H), 3.06 (q, J = 8.1 Hz, 2H), 1.42 (d, J = 7.8 Hz, 9H); LCMS m/z 478.29 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 10.75 (s, 1H), 7.84-7.74 (m, 2H), 7.55 (s, 1H), 7.36-7.22 (m, 3H), 6.89-6.75 (m, 1H), 3.76 (s, 1H), 3.57-3.48 (m, 2H), 3.24-3.13 (m, 1H), 3.09- 2.95 (m, 2H), 2.38 (s, 2H), 2.25-2.14 (m, 1H). LCMS m/z 431.2 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.81 (dddd, J = 17.3, 6.7, 5.3, 3.1 Hz, 3H), 7.42-7.17 (m, 3H), 6.98- 6.68 (m, 1H), 4.17-3.98 (m, 1H), 3.75 (dd, J = 11.0, 4.4 Hz, 1H), 3.65 (d, J = 6.0 Hz, 4H), 3.27-2.85 (m, 1H); LCMS m/z 379.1 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.95-7.72 (m, 2H), 7.40 (s,1H), 7.37-7.22 (m, 2H), 6.83 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 3.72-3.44 (m, 2H), 3.04 (s, 2H), 2.74- 2.50 (m, 2H), 2.15 (s, 1H), 1.92 (ddd, J = 16.1, 8.9, 4.2 Hz, 3H), 1.36 (s, 9H); LCMS m/z 488.24 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.98-7.65 (m, 2H), 7.38-7.19 (m, 3H), 6.83 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 3.49 (ddd, J = 9.6, 7.8, 6.0 Hz, 3H), 3.15-2.96 (m, 2H), 2.41 (s, 2H), 1.36 (s, 9H), 0.93-0.58 (m, 4H); LCMS m/z 488.3 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.94-7.70 (m, 2H), 7.44-7.16 (m, 3H), 6.98-6.67 (m, 1H), 4.27- 3.92 (m, 1H), 3.79-3.42 (m, 2H), 3.04 (q, J = 7.9 Hz, 2H), 1.40 (s, 3H), 1.26 (d, J = 7.1 Hz, 9H); LCMS m/z 462.3 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.99-7.65 (m, 2H), 7.35-7.12 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.69-3.36 (m, 2H), 3.23-2.88 (m, 2H), 2.84- 2.69 (m, 1H), 2.69-2.50 (m, 2H), 2.37 (dddd, J = 13.7, 8.1, 2.9, 1.4 Hz, 2H); LCMS m/z 457.2 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.89-7.61 (m, 2H), 7.32 (dd, J = 9.3, 2.2 Hz, 1H), 7.25-7.12 (m, 2H), 7.05 (s, 1H), 6.85 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 3.66 (td, J = 7.3, 6.1 Hz, 2H), 3.16 (dd, J = 8.1, 6.7 Hz, 2H); LCMS m/z 453.19 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 8.31 (dq, J = 42.6, 1.1 Hz, 1H), 7.92-7.73 (m, 2H), 7.37-7.08 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.66 (d, J = 6.1 Hz, 5H), 3.23-3.05 (m, 2H); LCMS m/z 453.09 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.86-7.71 (m, 2H), 7.36-7.18 (m, 3H), 6.95-6.76 (m, 1H), 3.66 (s, 2 H), 3.53 (q, J = 6.9 Hz, 2H), 3.15-2.95 (m, 2H), 1.41 (s, 9H); LCMS m/z 448.3 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 10.75 (s, 1H), 9.46 (s, 1H), 7.86-7.73 (m, 2H), 7.48 (s, 1H), 7.36-7.22 (m, 3H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.59-3.46 (m, 2H), 3.10-2.99 (m, 2H), 2.70-2.46 (m, 3H), 2.40- 2.28 (m, 2H), 2.25 (d, J = 6.6 Hz, 2H); LCMS m/z 444.2 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 7.91-7.73 (m, 3H), 7.55 (s, 1H), 7.41-7.14 (m, 3H), 6.96-6.60 (m, 1H), 3.92-3.40 (m, 3H), 3.34- 2.85 (m, 3H), 2.28 (d, J = 62.9 Hz, 3H), 1.83 (d, J = 11.5 Hz, 1H), 1.22 (s, 3H); LCMS m/z 430.3 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.98-7.72 (m, 2H), 7.53 (s, 1H), 7.42-7.16 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.69-3.32 (m, 2H), 3.25-2.80 (m, 3H), 2.73-2.41 (m, 3H); LCMS m/z 430.19 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.96-7.60 (m, 3H), 7.40-7.16 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 4.35 (ddd, J = 8.5, 5.1, 1.7 Hz, 1H), 3.73 (dd, J = 11.2, 8.5 Hz, 1H), 3.66- 3.53 (m, 2H), 3.53-3.40 (m, 1H), 3.21-2.83 (m, 2H); LCMS m/z 420.11 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 8.00-7.67 (m, 2H), 7.50-7.18 (m, 3H), 6.95-6.61 (m, 1H), 6.46 (s, 1H), 4.27-3.95 (m, 4H), 3.69-3.46 (m, 2H), 3.22- 2.92 (m, 2H), 1.57-1.09 (m, 2H); LCMS m/z 418.17 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.93-7.72 (m, 2H), 7.40-7.11 (m, 3H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.91 (dq, J = 4.9, 2.4 Hz, 1H), 3.85-3.41 (m, 2H), 3.30-2.91 (m, 2H), 2.20 (tt, J = 11.0, 3.4 Hz, 1H), 2.04-1.85 (m, 2H), 1.76 (dt, J = 13.1, 4.0 Hz, 2H), 1.50 (tdd, J = 12.2, 5.6, 3.1 Hz, 4H); LCMS m/z 417.23 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 10.74 (s, 1H), 7.85-7.74 (m, 2H), 7.39- 7.23 (m, 4H), 6.83 (ddd, J = 11.0, 9.7, 2.2 Hz, 1H), 3.54- 3.38 (m, 3H), 3.03 (dd, J = 8.5, 6.4 Hz, 2H), 2.28-1.06 (m, 8H); LCMS m/z 417.23 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 10.74 (s, 1H), 7.79 (ddt, J = 8.5, 5.3, 2.6 Hz, 2H), 7.54 (s, 1H), 7.36- 7.22 (m, 3H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.96 (s, 1H), 3.58-3.44 (m, 2H), 3.04 (t, J = 7.5 Hz, 2H), 2.28- 2.16 (m, 1H), 1.86 (td, J = 12.4, 3.5 Hz, 1H), 1.81- 1.56 (m, 3H), 1.52-1.14 (m, 4H); LCMS m/z 417.26 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.89-7.67 (m, 2H), 7.44-7.16 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 4.57 (q, J = 7.7 Hz, 1H), 3.76-3.46 (m, 2H), 3.35-2.89 (m, 2H); LCMS m/z 417.16 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.91-7.73 (m, 2H), 7.65 (s, 1H), 7.42-7.15 (m, 3H), 6.85 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.80-3.51 (m, 2H), 3.36-2.97 (m, 2H); LCMS m/z 417.1 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.89-7.68 (m, 3H), 7.42-7.12 (m, 4H), 6.84 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 4.00 (s, 1H), 3.56 (q, J = 7.4, 7.0 Hz, 2H), 3.06 (dd, J = 9.4, 6.3 Hz, 2H), 2.26 (t, J =6.4 Hz, 2H), 2.02- 1.58 (m, 4H); LCMS m/z 416.27 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.93-7.54 (m, 2H), 7.44-7.08 (m, 5H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.53 (q, J = 6.7 Hz, 2H), 3.30 (t, J = 5.9 Hz, 2H), 3.04 (t, J = 7.6 Hz, 2H), 2.14 (d, J = 8.7 Hz, 1H), 2.01- 1.47 (m, 3H); LCMS m/z 413.99 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.87-7.63 (m, 4H), 7.38-7.02 (m, 3H), 6.84 (ddd, J = 11.0, 9.7, 2.2 Hz, 1H), 3.70-3.36 (m, 3H), 3.05 (t, J =7.5 Hz, 2H), 2.95- 2.73 (m, 1H), 2.57 (qd, J = 18.0, 7.0 Hz, 2H), 2.05-1.64 (m, 3H); LCMS m/z 416.24 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.89-7.70 (m, 4H), 7.41-7.18 (m, 3H), 6.85 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.71-3.34 (m, 4H), 3.04 (dd, J = 16.2, 8.8 Hz, 2H), 2.81 (d, J = 13.5 Hz, 2H), 2.64 (s, 2H); LCMS m/z 416.2 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 10.12 (s, 1H), 7.87-7.74 (m, 2H), 7.77-7.65 (m, 1H), 7.38-7.22 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.76 (dd, J = 9.3, 4.6 Hz, 1H), 3.71-3.43 (m, 2H), 3.18-3.02 (m, 2H), 3.01- 2.70 (m, 1H); LCMS m/z 416.2 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 8.08 (d, J = 2.6 Hz, 1H), 7.95-7.85 (m, 2H), 7.77 (ddd, J = 8.5, 5.3, 2.7 Hz, 2H), 7.42-7.19 (m, 3H), 6.93-6.75 (m, 1H), 6.49 (d, J = 9.6 Hz, 1H), 3.63 (dt, J = 7.8, 6.1 Hz, 3H), 3.31-3.01 (m, 3H); LCMS m/z 412.14 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.98-7.59 (m, 2H), 7.43 (s, 1H), 7.28 (ddt, J = 12.0, 9.7, 2.6 Hz, 3H), 7.04 (s, 1H), 6.82 (ddd, J = 11.0, 9.7, 2.2 Hz, 1H), 6.39 (s, 1H), 3.70-3.40 (m, 2H), 3.01 (t, J = 7.7 Hz, 2H), 2.95- 2.72 (m, 1H), 2.50 (dd, J = 14.7, 7.7 Hz, 1H), 2.36-2.12 (m, 1H), 1.09 (d, J = 7.0 Hz, 3H); LCMS m/z 404.2 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 10.83 (s, 1H), 7.87-7.74 (m, 2H), 7.36- 7.23 (m, 3H), 6.94-6.77 (m, 2H), 4.54 (dd, J = 9.4, 8.4 Hz, 1H), 4.34 (ddd, J = 9.4, 5.3, 1.6 Hz, 1H), 4.17 (dd, J = 8.4, 5.2 Hz, 1H), 3.62- 3.49 (m, 2H), 3.08-3.00 (m, 1H); LCMS m/z 404.17 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 10.75 (s, 1H), 7.87-7.74 (m, 3H), 7.39- 7.21 (m, 3H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 6.67 (s, 1H), 4.90 (dd, J = 9.6, 5.9 Hz, 1H), 3.90-3.78 (m, 1H), 3.72-3.48 (m, 3H), 3.15-3.04 (m, 2H); LCMS m/z 404.12 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.96-7.72 (m, 2H), 7.44-7.15 (m, 4H), 6.84 (ddd, J =11.1, 9.7, 2.2 Hz, 1H), 4.14 (dd, J = 8.4, 4.6 Hz, 1H), 3.80-3.34 (m, 2H), 3.22-2.86 (m, 2H), 2.40 (t, J = 10.2 Hz, 1H), 2.36-2.20 (m, 2H), 1.99 (dd, J = 8.8, 4.8 Hz, 5H); LCMS m/z 402.21 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 8.02-7.63 (m, 2H), 7.44-7.08 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.74-3.41 (m, 2H), 3.32-2.95 (m, 2H), 1.60- 1.22 (m, 4H); LCMS m/z 402.2 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 8.04-7.68 (m, 2H), 7.37-7.21 (m, 3H), 6.84 (ddd, J = 11.0, 9.7, 2.2 Hz, 1H), 3.73-3.29 (m, 4H), 3.29-3.12 (m, 1H), 3.05 (dd, J = 8.5, 6.4 Hz, 2H), 2.58- 2.16 (m, 2H); LCMS m/z 402.1 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.94-7.69 (m, 2H), 7.40-7.19 (m, 3H), 6.97-6.63 (m, 1H), 3.63- 3.43 (m, 4H), 3.21-2.86 (m, 2H), 1.10 (s, 6H); LCMS m/z 391.23 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.96-7.67 (m, 2H), 7.53-7.37 (m, 1H), 7.28 (qt, J = 6.8, 2.1 Hz, 2H), 7.00-6.74 (m, 1H), 4.01 (dd, J = 8.4, 7.0 Hz, 1H), 3.60- 3.34 (m, 2H), 3.22-2.76 (m, 4H), 2.64-2.31 (m, 2H), 2.33-2.16 (m, 1H); LCMS m/z 390.13 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.92-7.55 (m, 3H), 7.46-7.14 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 6.29 (s, 2H), 3.79- 3.46 (m, 2H), 3.18 (s, 2H), 3.12-2.97 (m, 2H), 2.77 (d, J = 3.7 Hz, 3H); LCMS m/z 390.2 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 10.75 (s, 1H), 7.90-7.77 (m, 2H), 7.53 (s, 1H), 7.37 (dd, J = 9.4, 2.2 Hz, 1H), 7.33-7.23 (m, 2H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 4.69-4.43 (m, 1H), 3.68 (d, J = 6.1 Hz, 1H), 3.64-3.51 (m, 2H), 1.21- 1.00 (m, 1H), 0.57-0.24 (m, 4H); LCMS m/z 389.2 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.96-7.74 (m, 2H), 7.37 (dd, J = 9.4, 2.2 Hz, 1H), 7.34-7.11 (m, 2H), 6.92-6.70 (m, 1H), 4.55 (s, 1H), 3.74-3.44 (m, 3H), 3.27-2.90 (m, 2H), 1.37- 1.18 (m, 1H), 1.18-0.93 (m, 2H), 0.63 (q, J = 3.8 Hz, 1H); LCMS m/z 389.3 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 7.96-7.65 (m, 3H), 7.46-7.17 (m, 4H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 4.01 (dd, J = 5.8, 2.7 Hz, 1H), 3.70-3.44 (m, 2H), 3.25-3.01 (m, 5H); LCMS m/z 388.2 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.98-7.64 (m, 4H), 7.41 (dd, J = 9.5, 2.2 Hz, 1H), 7.36-7.05 (m, 2H), 6.96-6.64 (m, 3H), 3.89- 3.64 (m, 2H), 3.38-3.01 (m, 2H); LCMS m/z 385.2 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.74 (s, 1H), 8.20-7.65 (m, 5H), 7.38- 7.19 (m, 3H), 6.84 (ddd, J = 11.5, 9.6, 2.2 Hz, 1H), 3.70- 3.58 (m, 2H), 3.17-3.06 (m, 2H); LCMS m/z 385.16 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 10.79 (s, 1H), 9.18 (s, 1H), 8.55 (s, 1H), 8.14 (s, 1H), 7.75 (dd, J = 8.6, 5.3 Hz, 2H), 7.36-7.16 (m, 3H), 6.84 (ddd, J = 11.6, 9.7, 2.2 Hz, 1H), 3.70-3.62 (m, 2H), 3.17 (t, J = 7.4 Hz, 2H); LCMS m/z 385.13 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 10.68 (d, J = 30.3 Hz, 2H), 7.99 (s, 1H), 7.90-7.74 (m, 2H), 7.42- 7.32 (m, 1H), 7.31-7.18 (m, 2H), 6.96 (td, J = 2.7, 1.4 Hz, 1H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 6.71-6.63 (m, 1H), 6.17-6.08 (m, 1H), 3.67-3.57 (m, 2H), 3.15- 3.04 (m, 2H); LCMS m/z 384.15 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.89-7.79 (m, 2H), 7.38 (dd, J = 9.4, 2.2 Hz, 1H), 7.34-7.20 (m, 2H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.64-3.46 (m, 2H), 3.16-2.97 (m, 2H), 1.33 (s, 6H); LCMS m/z 377.2 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 7.99-7.70 (m, 2H), 7.54 (dd, J = 9.4, 2.2 Hz, 1H), 7.42-7.22 (m, 3H), 6.96-6.74 (m, 1H), 3.78- 3.41 (m, 3H), 3.23-2.93 (m, 2H), 2.46 (ddd, J = 12.1, 8.0, 6.1 Hz, 1H), 1.11 (dd, J = 33.0, 7.1 Hz, 3H); LCMS m/z 377.2 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 7.93-7.61 (m, 3H), 7.44-7.18 (m, 3H), 6.84 (ddd, J = 11.5, 9.7, 2.2 Hz, 3H), 3.59 (q, J = 7.4, 6.9 Hz, 2H), 3.30 (s, 2H), 3.08 (dd, J = 9.0, 6.4 Hz, 2H); LCMS m/z 376.17 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.91-7.35 (m, 2H), 7.54-7.29 (m, 1H), 7.35-7.12 (m, 2H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.76-3.45 (m, 2H), 3.32-3.03 (m, 2H), 2.81 (s, 3H); LCMS m/z 376.17 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 8.09-7.68 (m, 2H), 7.38 (dd, J = 9.4, 2.2 Hz, 1H), 7.35-7.11 (m, 2H), 6.83 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 3.73-3.41 (m, 2H), 3.21-2.99 (m, 2H), 1.28- 1.04 (m, 2H), 1.06-0.74 (m, 2H); LCMS m/z 375.16 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 10.75 (s, 1H), 7.89-7.74 (m, 2H), 7.60 (s, 1H), 7.35-7.23 (m, 3H), 6.83 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 3.76 (t, J = 5.9 Hz, 2H), 3.59-3.46 (m, 2H), 3.10-2.99 (m, 2H), 2.37 (t, J = 5.9 Hz, 2H); LCMS m/z 363.2 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 7.99-7.74 (m, 2H), 7.44-7.14 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 4.13 (q, J = 6.8 Hz, 1H), 3.74-3.38 (m, 2H), 3.23-3.03 (m, 2H), 1.29 (d, J = 6.8 Hz, 3H); LCMS m/z 363.2 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 7.84-7.73 (m, 2H), 7.35-7.23 (m, 3H), 6.85 (ddd, J = 11.3, 9.6, 2.2 Hz, 1H), 4.90 (q, J = 7.0 Hz, 1H), 3.58-3.47 (m, 2H), 3.30 (d, J = 0.8 Hz, 1H), 3.11-3.00 (m, 2H), 2.75- 2.47 (m, 1H), 1.57 (dd, J = 10.9, 7.0 Hz, 3H); LCMS m/z 362.1 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.77 (s, 1H), 8.36 (s, 1H), 7.87-7.74 (m, 2H), 7.66 (s, 1H), 7.43-7.24 (m, 3H), 7.01 (s, 1H), 6.84 (ddd, J = 11.0, 9.7, 2.2 Hz, 1H), 3.66-3.52 (m, 2H), 3.15-3.04 (m, 2H); LCMS m/z 362.1 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.88-7.75 (m, 2H), 7.40- 7.22 (m, 3H), 6.83 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 3.98 (s, 2H), 3.66-3.52 (m, 2H), 3.13-3.02 (m, 2H); LCMS m/z 349.1 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 7.83-7.72 (m, 2H), 7.35-7.23 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 4.58 (s, 2H), 3.57 (dt, J = 15.4, 7.7 Hz, 2H), 3.06 (dd, J = 9.1, 6.4 Hz, 2H); LCMS m/z 348.1 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 8.56 (s, 1H), 7.96-7.66 (m, 2H), 7.40 (dd, J = 9.4, 2.2 Hz, 1H), 7.29- 7.14 (m, 2H), 6.84 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 3.97- 3.57 (m, 2H), 3.34-3.06 (m, 2H); LCMS m/z 470.15 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 8.63-8.31 (m, 2H), 7.91-7.66 (m, 2H), 7.44-7.35 (m, 1H), 7.31- 7.12 (m, 2H), 6.95-6.68 (m, 1H), 3.93-3.64 (m, 2H), 3.40-3.06 (m, 2H); LCMS m/z 470.12 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 8.87 (dt, J = 5.0, 0.7 Hz, 1H), 8.08 (t, J = 1.1 Hz, 1H), 8.03-7.92 (m, 1H), 7.87-7.64 (m, 2H), 7.31 (dd, J = 9.4, 2.2 Hz, 1H), 7.27-6.99 (m, 2H), 6.85 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.88-3.57 (m, 2H), 3.21 (t, J = 7.2 Hz, 2H); LCMS m/z 464.18 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 8.01 (d, J = 3.6 Hz, 1H), 7.89 (s, 1H), 7.87-7.73 (m, 3H), 7.35-7.19 (m, 3H), 6.83 (ddd, J = 11.0, 9.7, 2.2 Hz, 1H), 4.25-4.02 (m, 2H), 3.63-3.47 (m, 2H), 3.13-3.00 (m, 2H); LCMS m/z 416.1 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 8.82 (t, J = 1.7 Hz, 1H), 8.62 (d, J = 2.8 Hz, 1H), 7.89 (ddd, J = 9.4, 2.8, 1.8 Hz, 1H), 7.86-7.73 (m, 2H), 7.33 (dd, J = 9.4, 2.2 Hz, 1H), 7.28-7.12 (m, 2H), 6.85 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 3.83-3.51 (m, 2H), 3.41-3.05 (m, 2H); LCMS m/z 414.12 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.95-7.66 (m, 2H), 7.44-7.15 (m, 3H), 6.84 (dddd, J = 11.1, 9.7, 2.2, 1.5 Hz, 1H), 4.38 (dd, J = 8.8, 8.2 Hz, 1H), 4.19 (dd, J = 8.8, 6.3 Hz, 1H), 3.66- 3.44 (m, 2H), 3.44-3.22 (m, 1H), 3.06 (td, J = 7.7, 7.2, 2.2 Hz, 2H), 2.89-2.44 (m, 2H); LCMS m/z 403.2 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 9.22 (d, J = 0.7 Hz, 1H), 8.34 (d, J = 0.7 Hz, 1H), 7.89-7.68 (m, 2H), 7.31 (dd, J = 9.4, 2.2 Hz, 1H), 7.27-7.11 (m, 2H), 6.85 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 3.80-3.50 (m, 2H), 3.28-2.92 (m, 2H); LCMS m/z 402.1 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 9.04 (d, J = 2.1 Hz, 1H), 8.24 (d, J = 2.1 Hz, 2H), 7.99-7.74 (m, 2H), 7.42 (dd, J = 9.4, 2.2 Hz, 1H), 7.34-7.16 (m, 2H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.92-3.57 (m, 2H), 3.40-2.97 (m, 2H); LCMS m/z 402.1 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 9.30 (d, J = 2.0 Hz, 1H), 8.85 (ddd, J = 8.1, 2.1, 1.4 Hz, 1H), 8.19 (ddd, J = 8.1, 5.6, 0.7 Hz, 1H), 8.00 (s, 1H), 7.89-7.67 (m, 2H), 7.32 (dd, J = 9.4, 2.2 Hz, 1H), 7.27-7.15 (m, 2H), 6.86 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.88-3.64 (m, 2H), 3.22 (dd, J = 8.0, 6.6 Hz, 2H); LCMS m/z 396.14 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.94-7.70 (m, 2H), 7.44-7.15 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.80-3.44 (m, 2H), 3.29-2.86 (m, 2H), 1.50 (s, 6H); LCMS m/z 386.2 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 10.74 (s, 1H), 7.86-7.61 (m, 2H), 7.40- 7.13 (m, 3H), 6.83 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 4.70- 4.52 (m, 4H), 3.78-3.64 (m, 1H), 3.57-3.44 (m, 2H), 3.09-2.98 (m, 2H); LCMS m/z 375.16 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.93-7.64 (m, 2H), 7.30 (ddd, J = 8.8, 5.4, 2.9 Hz, 3H), 6.85 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 6.06 (t, J = 54.1 Hz, 1H), 3.76- 3.48 (m, 2H), 3.28-2.88 (m, 2H); LCMS m/z 369.13 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.90-7.73 (m, 2H), 7.41-7.15 (m, 3H), 6.84 (ddd, J = 11.0, 9.6, 2.2 Hz, 1H), 3.67-3.32 (m, 4H), 3.23-2.91 (m, 2H); LCMS m/z 358.1 [M + H]+.
1H NMR (300 MHz, Acetone-d6) δ 7.94-7.68 (m, 2H), 7.41-7.19 (m, 3H), 6.84 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 6.35-6.06 (m, 2H), 5.56 (dd, J = 7.8, 4.5 Hz, 1H), 3.70-3.36 (m, 2H), 3.25-2.82 (m, 2H); LCMS m/z 345.1 [M + H]+.
1Boc deprotection was performed after amide formation.
2Purification by reversed-phase HPLC. Method: C18 Waters Sunfire column (30 × 150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid.
3Step 3. (triphosgene and DIPEA treatment) was omitted.
A solution of 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethanamine S19 (56 mg, 0.1929 mmol) in dichloromethane (2 mL) was treated with acetyl chloride (17 μL, 0.2391 mmol) followed by DIPEA (51 μL, 0.2928 mmol). The resulting solution was stirred at ambient temperature overnight then partitioned between dichloromethane and 1 M NaOH. The organic layer was concentrated in vacuo then purified by silica gel chromatography (Gradient: 0-20% EtOAc in heptane) to afford N-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]acetamide (32.9 mg, 49%). 1H NMR (300 MHz, Chloroform-d) δ 8.19 (s, 1H), 7.60-7.53 (m, 2H), 7.24-7.17 (m, 2H), 7.10 (dd, J=9.0, 2.2 Hz, 1H), 6.76 (ddd, J=10.7, 9.4, 2.1 Hz, 1H), 5.47 (s, 1H), 3.49 (q, J=6.9 Hz, 2H), 3.02 (t, J=7.1 Hz, 2H), 1.85 (s, 3H). LCMS m/z 333.15 [M+H]+
To a solution of 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethanamine (Trifluoroacetate salt) S19 (25 mg, 0.0546 mmol) was dissolved in dimethylformamide (2 mL). To the reaction was added 4-methyltetrahydrofuran-2-one (10.9 mg, 0.1092 mmol) and warmed to 110° C. overnight. The mixture was then purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford the product N-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]formamide (5.8 mg, 14%). 1H NMR (300 MHz, Acetone-d6) δ 8.14 (d, J=1.5 Hz, 1H), 7.96-7.66 (m, 2H), 7.41-7.15 (m, 3H), 6.92-6.73 (m, 1H), 3.72-3.46 (m, 2H), 3.25-2.95 (m, 2H). LCMS m/z 319.06 [M+H]+.
2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethanamine (160 mg, 0.4868 mmol) S19 was dissolved in DMF (16 mL). To this solution was added DIPEA (200 μL, 1.148 mmol) and the mixture was split into 8 equal portions. To one portion was added 2-(2,5-dioxoimidazolidin-4-yl)ethanesulfonyl chloride (50 mg, 0.2206 mmol). The reaction was stirred overnight. The crude mixture was purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford N-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]-2-(2,5-dioxoimidazolidin-4-yl)ethanesulfonamide (10.0 mg, 20%). 1H NMR (300 MHz, Acetone-d6) δ 7.94-7.67 (m, 2H), 7.59-7.19 (m, 3H), 7.07 (s, 1H), 6.84 (ddd, J=11.1, 9.7, 2.2 Hz, 1H), 6.42 (t, J=6.3 Hz, 1H), 4.28 (ddd, J=7.2, 5.4, 1.5 Hz, 1H), 3.55-3.35 (m, 2H), 3.35-3.03 (m, 4H), 2.38-2.14 (m, 1H). LCMS m/z 481.19 [M+H]+.
2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethanamine S19 (80 mg, 0.2434 mmol) was dissolved in DMF (8 mL), DIPEA (50 μL, 0.2871 mmol) was added, and the mixture was divided into 4 equal portions. To one portion was added, 2-[tert-butyl(dimethyl)silyl]oxypropane-1-sulfonyl chloride (70 mg, 0.2565 mmol). The reaction was stirred at room temperature overnight. The crude mixture was purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% TFA) to afford 2-[tert-butyl(dimethyl)silyl]oxy-N-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]propane-1-sulfonamide (15 mg, 17%). LCMS m/z 527.21 [M+H]+.
2-((tert-butyldimethylsilyl)oxy)-N-(2-(5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl)ethyl)propane-1-sulfonamide C67 (20 mg, 0.03797 mmol) was dissolved in THF (5 mL). To this solution was added TBAF (100 μL of 1M, 0.1000 mmol) and the reaction was stirred overnight. The mixture was concentrated and re-dissolved in DMSO (2 mL). The crude solution was purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford 1-((2-(5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl)ethyl)amino)propan-2-ol (3.2 mg, 17%). 1H NMR (300 MHz, Acetone-d6) δ 7.96-7.63 (m, 2H), 7.46-7.14 (m, 3H), 6.85 (ddd, J=11.1, 9.7, 2.2 Hz, 1H), 4.29-4.12 (m, 2H), 3.55-3.29 (m, 3H), 3.29-2.97 (m, 4H), 1.22 (d, J=6.3 Hz, 3H). LCMS m/z 413.08 [M+H]+.
Compounds 422-425 (see Table 12) were prepared from intermediate S19 using the appropriate reagents, using the sulfonamide formation method as described for compound 421. Sulfonyl chloride reagents were obtained from commercial sources. Any modifications to methods are noted in Table 12 and accompanying footnotes.
1H NMR; LCMS m/z
1H NMR (300 MHz, Acetone-d6) δ 7.79 (ddd, J = 8.8, 5.2, 2.6 Hz, 2H), 7.52- 7.14 (m, 2H), 6.99-6.72 (m, 1H), 3.94 (t, J = 6.3 Hz, 2H), 3.40 (d, J = 8.6 Hz, 1H), 3.33 (s, 5H), 3.20-2.95 (m, 2H); LCMS m/z 399.13 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 7.89-7.63 (m, 2H), 7.40-7.16 (m, 3H), 6.85 (ddd, J = 11.6, 9.7, 2.2 Hz, 1H), 4.12-3.82 (m, 2H), 3.53-3.38 (m, 2H), 3.28 (td, J = 11.9, 2.1 Hz, 2H), 3.22- 2.93 (m, 3H), 1.95-1.78 (m, 2H), 1.66 (qd, J = 12.3, 4.7 Hz, 2H); LCMS m/z 439.13 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.77 (s, 1H), 7.85-7.71 (m, 2H), 7.35- 7.19 (m, 4H), 6.99 (d, J = 1.1 Hz, 1H), 6.83 (ddd, J = 11.0, 9.7, 2.2 Hz, 1H), 3.90 (s, 3H), 3.58-3.42 (m, 3H), 3.20-2.99 (m, 2H); LCMS m/z 435.06 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 8.00-7.61 (m, 2H), 7.44-7.16 (m, 3H), 6.85 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 3.54-3.34 (m, 2H), 3.14 (dd, J = 9.5, 6.5 Hz, 2H), 2.83 (s, 6H); LCMS m/z 397.96 [M + H]+
To a solution of bis(4-nitrophenyl) carbonate (800 mg, 2.630 mmol) dissolved in DMF (20 mL) was added DIPEA (800 μL, 4.593 mmol) and 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethanamine S19 (750 mg, 2.282 mmol). The solution was stirred at room temperature for 30 min, and then used as is in subsequent steps without further purification (4-nitrophenyl) N-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]carbamate. LCMS m/z 456.1 [M+H]+.
To a solution of (4-nitrophenyl) N-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]carbamate C67 (20 mg, 0.044 mmol) dissolved in DMF (4 mL) was added 1-(hydroxymethyl)cyclopropanecarboxylic acid (20 mg, 0.1722 mmol) and the solution was stirred overnight at room temperature. The crude mixture was purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to yield 1-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethylcarba-moyloxymethyl]cyclopropanecarboxylic acid (4.2 mg, 23%). 1H NMR (300 MHz, Acetone-d6) δ 7.93-7.72 (m, 2H), 7.41-7.19 (m, 2H), 6.82 (ddd, J=11.5, 9.7, 2.2 Hz, 1H), 3.57 (t, J=5.8 Hz, 1H), 3.46 (t, J=7.4 Hz, 2H), 3.01 (dd, J=8.7, 6.3 Hz, 4H), 1.56 (t, J=5.8 Hz, 2H), 0.83-0.42 (m, 4H). LCMS m/z 432.85 [M+H]+.
A solution of (4S)-4-hydroxypyrrolidin-2-one (1.83 g, 18.1 mmol) C69 in DCM (50 mL) was cooled to −10° C. and N,N,N′,N′-tetramethylethane-1,2-diamine (11 mL, 72.89 mmol) was added. To this cooled reaction, chloro(trimethyl)silane (8 mL, 63.03 mmol) was then added dropwise as well as an additional portion of DCM (50 mL). The solution was then stirred at −10° C. for 30 minutes. at which point iodine (6 g, 23.64 mmol) was added and the slurry was stirred for 2 hours. The reaction was quenched with saturated aqueous Na2SO3 (80 mL) solution and stirred until the mixture forms 2 homogeneous layers. The layers were separated, and the aqueous layer was extracted with DCM. The combined organic layers were washed with 1 M HCl (80 mL), dried over Na2SO4, filtered, and concentrated to a yellow waxy solid. This solid was dissolved in EtOH (80 mL). To this solution was added K2CO3 (3.81 g, 27.57 mmol). The reaction was stirred at room temperature overnight. The reaction was filtered, and the precipitate was washed with DCM (40 mL) and the filtrate was concentrated. The resulting dark solid was rinsed with MeCN (40 mL) and filtered to obtain (1R,5R)-6-oxa-3-azabicyclo[3.1.0]hexan-2-one (2 g, 72% yield). LCMS m/z 128.78 [M+H]+.
(1R,5R)-6-oxa-3-azabicyclo[3.1.0]hexan-2-one S19 (50 mg, 0.5046 mmol) and 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethanamine C5 (140 mg, 0.4823 mmol) were combined in EtOH (5 mL). The reaction was heated to 130° C. for and stirred for 20 minutes. The reaction was then concentrated and re-dissolved in DMSO (5 mL). Purification by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) afforded the product (3S,4R)-3-((2-(5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl)ethyl)amino)-4-hydroxypyrrolidin-2-one (16.3 mg, 6.2%). 1H NMR (300 MHz, Acetone-d6) δ 10.92 (s, 1H), 7.99-7.76 (m, 2H), 7.50-7.37 (m, 1H), 7.37-7.19 (m, 2H), 6.86 (ddt, J=11.1, 9.7, 2.6 Hz, 1H), 4.93 (q, J=8.1 Hz, 1H), 4.14 (d, J=8.4 Hz, 1H), 3.98-3.79 (m, 1H), 3.74-3.41 (m, 4H), 3.28-3.01 (m, 1H). LCMS m/z 390.31 [M+H]+.
2,2-Dimethoxy-N-methyl-ethanamine (15 g, 125.9 mmol) was dissolved in DCM (100 mL) and to this solution was added DIPEA (22 mL, 126.3 mmol) and benzyl (2,5-dioxopyrrolidin-1-yl) carbonate C70 (32 g, 128.4 mmol). The reaction was stirred at room temperature for 3 hours and then washed with 1 M HCl (50 mL) followed by a wash with 1 M NaOH (50 mL). The resulting organic layer was washed with saturated NaCl (100 mL). The organics were concentrated to a yellow oil and used as it in the next step. Benzyl N-(2,2-dimethoxyethyl)-N-methyl-carbamate (28 g, 33%). LCMS m/z 254.11 [M+H]+.
Benzyl N-(2,2-dimethoxyethyl)-N-methyl-carbamate C71 (5.3 g, 20.92 mmol) was dissolved in DCM (50 mL). To this solution was added triethylsilane (10 mL, 62.61 mmol). In a separate flask, 5,7-difluoro-2-(4-fluorophenyl)-1H-indole C25 (4.925 g, 19.92 mmol), methanesulfonic acid (3.5 mL, 53.94 mmol), and DCM (50 mL) were combined into a solution which was added dropwise to the solution containing the Benzyl N-(2,2-dimethoxyethyl)-N-methyl-carbamate C71. The reaction was stirred overnight at room temperature at which point it was concentrated and re-dissolved in DMSO (25 mL). Purified by 275 g reversed-phase chromatography (Column: C18. Gradient: 0-100% MeCN in water with 0.1% formic acid) to afford benzyl N-[2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]-N-methyl-carbamate (4.5 g, 44%). LCMS m/z 437.25 [M+H]+.
Benzyl (2-(5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl)ethyl)(methyl)carbamate C72 (900 mg, 2.053 mmol) was dissolved in EtOH (20 mL) and to this solution was added Pd/C (50 mg, 0.4698 mmol). A balloon containing H2 was attached and the reaction was stirred overnight at room temperature. The reaction was then filtered, concentrated, and re-dissolved in DMSO (5 mL). The mixture was purified by reversed-phase chromatography (Column: C18. Gradient: 0-100% MeCN in water with 0.1% trifluoroacetic acid) to afford 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-methyl-ethanamine (Trifluoroacetate salt) (450 mg, 51%). 1H NMR (300 MHz, Acetone-d6) δ 10.84 (s, 1H), 7.84-7.73 (m, 2H), 7.48-7.34 (m, 1H), 7.35-7.22 (m, 2H), 6.84 (ddd, J=11.5, 9.7, 2.1 Hz, 1H), 3.49-3.32 (m, 4H), 2.82 (s, 3H). LCMS m/z 305.47 [M+H]+.
To a microwave vial was added 5,7-difluoro-2-(4-fluorophenyl)-1H-indole C25 (100 mg, 0.3882 mmol), 2-(dimethylamino)ethanol (117 μL, 1.164 mmol), Cs2CO3 (139 mg, 0.4266 mmol, and (pentamethylcyclopentadienyl)iridium(III) Chloride (4 mg, 0.01004 mmol). Heated the reaction mix at 150° C. for 48 hours. Cooled the reaction mix to room temperature. Purified by reversed-phase chromatography (Column: C18. Gradient: 5-95% MeCN in water with 0.1% TFA) to afford 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N,N-dimethyl-ethanamine (Trifluoroacetate salt) (68.6 mg, 39%). 1H NMR (300 MHz, DMSO-d6) δ 11.90 (s, 1H), 9.51 (s, 1H), 7.74-7.64 (m, 2H), 7.45-7.33 (m, 3H), 7.11-6.97 (m, 1H), 3.34-3.22 (m, 2H), 3.16-3.04 (m, 2H), 2.84 (s, 6H). LCMS m/z 319.19 [M+H]+.
A solution of tert-butyl N-[2-(5-methoxy-1H-indol-3-yl)ethyl]carbamate C73 (30 mg, 0.1033 mmol) in AcOH (2 mL) was treated with 4-fluorophenylboronic acid (22 mg, 0.1572 mmol) and palladium acetate (5 mg, 0.02227 mmol), and 02 balloon. The reaction was stirred at room temperature overnight. The reaction was concentrated and purified by silica gel chromatography (Gradient: 0-100% EtOAc in heptane) to afford tert-butyl N-[2-[2-(4-fluorophenyl)-5-methoxy-1H-indol-3-yl]ethyl]carbamate 430 (14.9 mg, 35%). 1H NMR (300 MHz, Methanol-d4) δ 7.66-7.57 (m, 2H), 7.27-7.08 (m, 4H), 6.77 (dd, J=8.7, 2.4 Hz, 1H), 3.85 (s, 3H), 3.37-3.32 (m, 2H), 2.99 (dd, J=8.7, 6.4 Hz, 2H), 1.41 (s, 9H). LCMS m/z 384.24 [M+H]+
A solution of N-[2-(2-bromo-1H-indol-3-yl)ethyl]acetamide C74 (65 mg, 0.2312 mmol), (4-fluorophenyl)boronic acid (50 mg, 0.3573 mmol), and K3PO4 (350 μL of 2 M, 0.7000 mmol) in dioxane (800 μL) was flushed with nitrogen, added ferrous; cyclopenta-1,4-dien-1-yl(diphenyl)phosphane; dichloromethane; dichloropalladium (40 mg, 0.04898 mmol) and stirred at 100° C. for 2 hours. The reaction mixture was then added to water, extracted with EtOAc (3 times), dried over sodium sulfate, filtered, and solvent was removed under reduced pressure. Purification by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) afforded N-[2-[2-(4-fluorophenyl)-1H-indol-3-yl]ethyl]acetamide (30 mg, 42%). LCMS m/z 296.8 [M+H]+.
Compound 432 (=S19) was prepared as described for S19.
To a mixture of methyl 4-(4-fluorophenyl)-4-oxo-butanoate C75 (1 g, 5 mmol) and (2,4-difluorophenyl)hydrazine hydrochloride (1.72 g, 9.53 mmol) in acetic acid (13 mL) and toluene (13 mL) was added zinc chloride (3.1 g, 23 mmol). The mixture was heated for 24 hours (at ˜115° C.) and concentrated in vacuo. The residue was partitioned between EtOAc and water. The aqueous layer was extracted with EtOAc, and the combined organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (Gradient: 0-20% EtOAc in heptane) to give the product (356.4 mg, 22%). 1H NMR (300 MHz, Chloroform-d) δ 8.23 (s, 1H), 7.68-7.56 (m, 2H), 7.24-7.15 (m, 2H), 7.13-7.05 (m, 1H), 6.75 (ddd, J=10.8, 9.5, 2.2 Hz, 1H), 3.74 (d, J=3.3 Hz, 5H). LCMS m/z 320.14 [M+H]+.
To a solution of methyl 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]acetate C76 (351.6 mg, 1.046 mmol) in THF (2 mL), MeOH (2 mL), and water (1 mL) was added LiOH (55 mg, 2.3 mmol). The mixture was stirred at room temperature for 2.5 hours. The mixture was then concentrated in vacuo, and the residue was acidified to pH 3 with 1 M HCl (aqueous). The aqueous layer was extracted with EtOAc, and the organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo to afford the product (317.5 mg, 94%), which was used in subsequent reactions without further purification. 1H NMR (300 MHz, DMSO-d6) δ 12.45 (s, 1H), 11.86 (s, 1H), 7.80-7.65 (m, 2H), 7.45-7.33 (m, 2H), 7.19 (dt, J=9.5, 2.6 Hz, 1H), 7.00 (ddd, J=11.2, 9.8, 2.2 Hz, 1H), 3.60 (s, 2H). LCMS m/z 306.14 [M+H]+.
To a solution of 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]acetic acid C77 (62.1 mg, 0.193 mmol) in THF (1.4 mL) at 0° C. under a nitrogen atmosphere was added lithium aluminum hydride (387 μL of 2 M in THF, 0.774 mmol). The ice bath was removed, and the mixture was allowed to warm to room temperature. The mixture was then refluxed for 2.5 hours. The mixture was cooled to room temperature, and the reaction was quenched at 0° C. by the addition of saturated aqueous solution of sodium potassium L(+)-tartrate tetrahydrate followed by ethyl acetate. The aqueous layer was extracted with ethyl acetate, and the combined organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo to afford the product (30.6 mg, 50%). 1H NMR (300 MHz, Methanol-d4) δ 7.73-7.64 (m, 2H), 7.27-7.18 (m, 2H), 7.10 (dd, J=9.4, 2.2 Hz, 1H), 6.72 (ddd, J=11.1, 9.6, 2.2 Hz, 1H), 3.79 (t, J=7.3 Hz, 2H), 3.01 (t, J=7.3 Hz, 2H). LCMS m/z 292.14 [M+H]+.
To a solution of 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethanol 433 (3000 mg, 6.90 mmol) in DCM (50 mL) was added (4-nitrophenyl) carbonochloridate (2 g, 9 mmol) followed by pyridine (1 g, 13 mmol). The reaction mixture was stirred for 3 hours. The mixture was concentrated in vacuo and partitioned between EtOAc and water. The organic layer separated and washed with 2 M NaOH) and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated in vacuo to give the product (2.7 g, 25%), which was used in the next step without additional purification. LCMS m/z 457.47 [M+H]+.
To a solution of 2-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]ethyl (4-nitrophenyl) carbonate S20 (50 mg, 0.1 mmol) in DMF (2 mL) was added 2-amino-2-methyl-propane-1,3-diol (13.8 mg, 0.13 mmol), followed by pyridine (17 mg, 0.22 mmol). The mixture was heated to 80° C. for 3 h. The mixture was then filtered and purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford the product (16.5 mg, 28%). LCMS m/z 423.14 [M+H]+.
Compounds 435-502 (see Table 13) were prepared from intermediate S20 using the appropriate amine and using the carbamate coupling method as described for compound 434. Amines were obtained from commercial sources. Any modifications to methods are noted in Table 13 and accompanying footnotes.
1H NMR; LCMS m/z
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 8.03-7.52 (m, 2H), 7.30 (td, J = 9.2, 2.5 Hz, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 5.74 (s, 1H), 4.25 (t, J = 7.3 Hz, 2H), 3.51 (s, 2H), 3.12 (t, J = 7.3 Hz, 2H), 1.25 (s, 6H); LCMS m/z 407.14 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.99-7.54 (m, 2H), 7.34-7.16 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 5.92 (s, 1H), 4.29 (td, J = 7.5, 1.9 Hz, 2H), 3.83-3.59 (m, 1H), 3.49 (qd, J = 10.6, 5.5 Hz, 2H), 3.13 (t, J = 7.3 Hz, 3H), 1.13 (d, J = 6.7 Hz, 3H); LCMS m/z 393.1 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 8.10-7.62 (m, 2H), 7.51-7.13 (m, 3H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 5.69 (d, J = 8.8 Hz, 1H), 4.48-4.19 (m, 2H), 4.08 (qd, J = 6.5, 2.6 Hz, 1H), 3.66 (h, J = 6.0, 5.5 Hz, 3H), 3.61-3.37 (m, 2H), 3.15 (t, J = 7.3 Hz, 2H), 1.13 (d, J = 6.4 Hz, 3H); LCMS m/z 423.23 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.90-7.61 (m, 2H), 7.42-7.15 (m, 3H), 6.84 (ddd, J = 11.2, 9.7, 2.2 Hz, 1H), 4.29 (t, J = 7.3 Hz, 2H), 3.79 (d, J = 6.5 Hz, 1H), 3.43-3.19 (m, 5H), 3.13 (t, J = 7.3 Hz, 2H), 1.11 (d, J = 6.8 Hz, 3H); LCMS m/z 407.01 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.92-7.63 (m, 2H), 7.30 (ddd, J = 8.9, 5.4, 2.9 Hz, 3H), 6.84 (ddd, J = 11.3, 9.7, 2.2 Hz, 1H), 5.87 (s, 1H), 4.30 (tq, J = 6.9, 3.5 Hz, 2H), 3.50 (d, J = 11.3 Hz, 4H), 3.13 (t, J = 7.3 Hz, 3H), 1.64 (s, 1H), 1.44 (dt, J = 13.8, 7.3 Hz, 1H), 0.91 (t, J = 7.5 Hz, 3H); LCMS m/z 407.14 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.78 (s, 1H), 7.80 (ddd, J = 8.6, 5.3, 2.4 Hz, 2H), 7.31 (ddt, J = 8.8, 5.2, 2.7 Hz, 3H), 6.84 (ddd, J = 11.5, 9.6, 2.2 Hz, 1H), 6.64 (s, 1H), 6.19-5.62 (m, 1H), 4.34 (t, J = 7.3 Hz, 2H), 3.50 (tt, J = 15.0, 5.4 Hz, 2H), 3.16 (t, J = 7.3 Hz, 2H); LCMS m/z 399.1 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.95-7.66 (m, 2H), 7.42-7.17 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 5.91 (s, 1H), 4.29 (td, J = 7.5, 1.9 Hz, 2H), 3.70 (s, 1H), 3.48 (qd, J = 10.6, 5.5 Hz, 2H), 3.13 (t, J = 7.3 Hz, 2H), 1.13 (d, J = 6.7 Hz, 3H); LCMS m/z 393.17 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.99-7.64 (m, 2H), 7.41-7.15 (m, 3H), 6.84 (ddd, J = 11.0, 9.6, 2.2 Hz, 1H), 5.81 (d, J = 51.0 Hz, 1H), 4.30 (t, J = 7.3 Hz, 2H), 3.91-3.34 (m, 3H), 3.14 (d, J = 14.6 Hz, 3H), 1.26- 0.72 (m, 6H); LCMS m/z 407.11 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.96-7.58 (m, 2H), 7.45-7.16 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 5.84 (d, J = 9.1 Hz, 1H), 4.55-4.13 (m, 2H), 3.68-3.34 (m, 4H), 3.13 (t, J = 7.3 Hz, 2H), 1.91 (h, J = 6.7 Hz, 1H), 0.91 (dd, J = 8.9, 6.8 Hz, 6H); LCMS m/z 421.35 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.79 (ddd, J = 8.6, 5.5, 2.5 Hz, 2H), 7.30 (td, J = 9.0, 2.6 Hz, 3H), 6.84 (ddd, J = 11.5, 9.7, 2.3 Hz, 1H), 6.51-6.27 (m, 1H), 4.47-3.97 (m, 3H), 3.96-3.59 (m, 3H), 3.53 (dd, J = 9.3, 3.9 Hz, 1H), 3.13 (t, J = 7.3 Hz, 2H), 2.13 (dt, J = 10.7, 5.2 Hz, 1H), 1.92-1.66 (m,
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.92-7.68 (m, 2H), 7.29 (td, J = 8.8, 2.1 Hz, 3H), 6.84 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 4.31 (t, J = 7.2 Hz, 2H), 4.02 (s, 1H), 3.15 (t, J = 7.2 Hz, 2H), 2.88 (h, J = 12.0, 11.3 Hz, 2H), 2.62 (d, J = 18.0 Hz, 2H); LCMS m/z 425.12 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.93-7.62 (m, 2H), 7.38-7.11 (m, 3H), 6.84 (dddd, J = 10.9, 9.7, 2.2, 0.9 Hz, 1H), 4.28 (q, J = 7.3 Hz, 3H), 4.00-3.78 (m, 1H), 3.63 (d, J = 9.8 Hz, 1H), 3.11 (d, J = 6.7 Hz, 2H), 2.99- 2.74 (m, 1H), 2.68- 2.40 (m, 2H), 2.25 (s, 1H), 1.84 (d, J = 10.8 Hz, 1H); LCMS m/z
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.80 (ddd, J = 8.3, 5.3, 2.4 Hz, 2H), 7.30 (ddt, J = 8.9, 6.4, 2.3 Hz, 3H), 6.84 (ddd, J = 10.2, 9.0, 2.5 Hz, 1H), 6.07 (s, 1H), 4.30 (t, J = 7.3 Hz, 2H), 3.64 (d, J = 64.4 Hz, 2H), 3.30-2.51 (m, 4H), 1.40 (ddt, J = 20.9, 13.7, 7.1 Hz, 2H), 0.93 (t, J = 6.9 Hz, 3H); LCMS m/z 407.04 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 8.01 (s, 1H), 7.87-7.70 (m, 2H), 7.40-7.21 (m, 3H), 7.00-6.68 (m, 3H), 4.57-4.19 (m, 4H), 3.15 (t, J = 7.3 Hz, 2H); LCMS m/z 416.13 [M + H]+
1H NMR (300 MHz, Acetic Acid-d4) δ 10.75 (s, 1H), 8.72 (s, 1H), 7.90-7.71 (m, 2H), 7.65 (s, 1H), 7.42 (s, 1H), 7.38-7.20 (m, 3H), 6.83 (ddd, J = 11.7, 9.8, 2.2 Hz, 1H), 4.51-4.22 (m, 4H), 4.02 (s, 3H), 3.11 (t, J = 7.2 Hz, 2H); LCMS m/z 428.97 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.93-7.66 (m, 2H), 7.39-7.14 (m, 3H), 6.84 (ddd, J = 11.6, 9.7, 2.2 Hz, 1H), 6.27 (s, 1H), 4.30 (t, J = 7.3 Hz, 2H), 3.35 (s, 3H), 3.25-2.95 (m, 3H), 2.50 (ddt, J = 8.0, 3.6, 1.7 Hz, 1H), 0.89 (ddd, J = 8.7, 6.6, 3.8 Hz, 1H), 0.73 (td, J = 6.8, 4.6 Hz, 1H); LCMS m/z 405.25 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.78 (s, 1H), 7.80 (ddd, J = 8.6, 5.3, 2.4 Hz, 2H), 7.31 (ddt, J = 8.8, 5.2, 2.7 Hz, 3H), 6.84 (ddd, J = 11.5, 9.6, 2.2 Hz, 1H), 6.64 (s, 1H), 6.19-5.62 (m, 1H), 4.34 (t, J = 7.3 Hz, 2H), 3.50 (tt, J = 15.0, 5.4 Hz, 2H), 3.16 (t, J = 7.3 Hz, 2H); LCMS m/z 414.96 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 8.03-7.54 (m, 2H), 7.41-7.16 (m, 3H), 6.84 (ddd, J = 11.5, 9.7, 2.2 Hz, 1H), 6.04 (s, 1H), 4.32 (t, J = 7.3 Hz, 2H), 3.74 (s, 1H), 3.39-2.94 (m, 4H), 2.00-1.77 (m, 4H), 1.84-1.15 (m, 2H); LCMS m/z 419.33 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.95-7.59 (m, 2H), 7.30 (tq, J = 9.7, 3.1, 2.6 Hz, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 6.78-6.50 (m, 2H), 4.49-4.25 (m, 4H), 3.16 (t, J = 7.3 Hz, 2H), 2.27 (d, J = 1.2 Hz, 3H); LCMS m/z 430.14 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.96-7.60 (m, 2H), 7.45-7.15 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 6.33 (s, 1H), 4.31 (t, J = 7.3 Hz, 2H), 3.86- 3.52 (m, 3H), 3.45 (dd, J = 8.6, 5.4 Hz, 1H), 3.13 (t, J = 7.1 Hz, 4H), 2.42 (dq, J = 13.6, 6.8 Hz, 1H), 1.95 (ddt, J = 10.8, 7.8, 4.0 Hz, 1H), 1.58 (dq, J = 13.2, 6.8 Hz, 1H);
1H NMR (300 MHz, Acetone-d6) δ 10.77 (s, 1H), 8.55 (s, 1H), 7.92-7.70 (m, 2H), 7.56 (s, 1H), 7.30 (ddd, J = 8.9, 5.3, 2.9 Hz, 4H), 6.83 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 5.30 (q, J = 8.7 Hz, 2H), 4.58-4.11 (m, 4H), 3.13 (t, J = 7.3 Hz, 2H); LCMS m/z 497.11 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 8.05-7.60 (m, 2H), 7.60-7.13 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 6.33 (d, J = 7.8 Hz, 1H), 4.28 (t, J = 7.3 Hz, 2H), 3.70 (p, J = 8.1 Hz, 3H), 3.12 (t, J = 7.3 Hz, 2H), 2.47- 2.16 (m, 2H), 2.05 (s, 1H), 1.31 (s, 3H); LCMS m/z 419.29 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.84 (s, 1H), 8.75 (s, 2H), 8.23 (d, J = 8.0 Hz, 1H), 7.81 (td, J = 8.6, 5.4 Hz, 3H), 7.41- 7.18 (m, 3H), 7.18- 6.64 (m, 2H), 4.64- 4.08 (m, 4H), 3.16 (t, J = 7.2 Hz, 2H); LCMS m/z 426.13 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.76 (s, 1H), 7.96-7.54 (m, 2H), 7.49-7.08 (m, 3H), 6.84 (ddd, J = 11.1, 9.7, 2.2 Hz, 1H), 6.27 (s, 1H), 4.51- 4.23 (m, 2H), 3.61 (s, 2H), 3.31 (d, J = 2.3 Hz, 2H), 3.20-2.95 (m, 4H), 1.08 (s, 3H); LCMS m/z 423.04 [M + H]+
1H NMR (300 MHz, Acetone-d6) δ 10.77 (s, 1H), 8.01-7.56 (m, 2H), 7.31 (td, J = 9.2, 2.4 Hz, 3H), 6.84 (ddd, J = 11.1, 9.6, 2.2 Hz, 1H), 5.77 (s, 1H), 4.29 (t, J = 7.3 Hz, 2H), 3.71 (s, 6H), 3.15 (t, J = 7.3 Hz, 3H), 2.08-2.05 (m, 2H); LCMS m/z 439.36 [M + H]+
To a mixture of 4-ethoxy-1-(4-fluorophenyl)butan-1-one C78 (255.3 mg, 1.214 mmol) and (2,4-difluorophenyl)hydrazine hydrochloride (440 mg, 2.4 mmol) in acetic acid (5 mL) and toluene (5 mL) was added zinc chloride (750 mg, 5.5 mmol). The mixture was heated at 115° C. overnight and concentrated in vacuo. The residue was partitioned between EtOAc and water. The aqueous layer was extracted with EtOAc, and the combined organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified by silica gel chromatography (Gradient: 0-20% EtOAc in heptane) to give the product (97.9 mg, 24%). 1H NMR (300 MHz, Chloroform-d) δ 8.10 (s, 1H), 7.66-7.58 (m, 2H), 7.22-7.15 (m, 2H), 7.10 (ddt, J=9.2, 2.2, 0.6 Hz, 1H), 6.74 (ddd, J=10.8, 9.5, 2.2 Hz, 1H), 3.68 (t, J=7.1 Hz, 2H), 3.48 (q, J=7.0 Hz, 2H), 3.05 (t, J=7.1 Hz, 2H), 1.19 (t, J=7.0 Hz, 3H). LCMS m/z 320.19 [M+H]+.
To a solution of 5-chloro-1-(4-fluorophenyl)pentan-1-one C79 (10 g, 46.58 mmol) and isoindoline-1,3-dione (6.90 g, 46.90 mmol) in DMF (100 mL) was added K2CO3 (6.90 g, 49.93 mmol). The reaction was heated to 70° C. for 16 hours. The reaction mixture was diluted with water and extracted with EtOAc (3×). The combined organic extracts were then washed with water and brine, dried over sodium sulfate, filtered, and concentrated in vacuo to afford crude product, 2-[5-(4-fluorophenyl)-5-oxo-pentyl]isoindoline-1,3-dione (S21) (15.2 g, 100%) as a residue which was used directly in the next reaction without further purification. Quantitative yield as assumed. LCMS m/z 326.15 [M+H]+.
A solution of 2-[5-(4-fluorophenyl)-5-oxo-pentyl]isoindoline-1,3-dione S21 (423 mg, 1.28 mmol), toluene (3 mL), AcOH (3 mL), dichlorozinc (900 mg, 6.6 mmol), and (2,4-difluorophenyl)hydrazine (737 mg, 5.11 mmol) was heated to 110° C. with stirring for 6 hours before cooling to room temperature. The reaction was quenched into aq. saturated sodium bicarbonate (150 mL) and extracted with ethyl acetate (2×100 mL). The combined organics were washed with brine, dried with MgSO4, filtered and concentrated in vacuo. Purification by silica gel chromatography (Gradient: 0-25% EtOAc/heptanes) afforded the product 2-[3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propyl]isoindoline-1,3-dione (C80) (510 mg, 40%) as a yellow solid. LCMS m/z 435.31 [M+H]+.
To a solution of 2-[3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propyl]iso-indoline-1,3-dione C80 (498 mg, 1.146 mmol) in ethanol (11 mL) was added hydrazine monohydrate (800 μL, 16.32 mmol). The reaction was heated to 80° C. with stirring for 8 hours before cooling to room temperature. The reaction was diluted with additional ethanol and filtered through a bed of Celite®. The filtrate was concentrated in vacuo to afford 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propan-1-amine (474 mg, 100%) as a light orange solid. LCMS m/z 305.24 [M+H]+.
To a solution of 5,7-difluoro-2-(4-fluorophenyl)-1H-indole C25 (100 mg, 0.39 mmol) and benzyl N-(3-oxopropyl)carbamate (93 mg, 0.45 mmol) in dichloromethane (6 mL) under nitrogen was added triethylsilane (188 μL, 1.177 mmol) and TFA (90 μL, 1.168 mmol). The reaction was heated to 40° C. overnight, at which time it was cooled to room temperature and partitioned between dichloromethane and aq. saturated sodium bicarbonate. The organics were collected through a phase separator and concentrated in vacuo. Purification by silica gel chromatography (Gradient: 0-100% EtOAc in Heptanes) afforded benzyl N-[3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propyl]carbamate (C81) (95.7 mg, 54%). 1H NMR (300 MHz, Chloroform-d) δ 8.12 (s, 1H), 7.53-7.42 (m, 2H), 7.34 (q, J=2.7, 1.6 Hz, 5H), 7.21-7.10 (m, 2H), 7.02 (dd, J=9.1, 2.1 Hz, 1H), 6.74 (ddd, J=10.8, 9.4, 2.2 Hz, 1H), 5.07 (s, 2H), 4.63 (s, 1H), 3.18 (q, J=6.7 Hz, 2H), 2.87-2.74 (m, 2H), 1.84 (p, J=7.3 Hz, 2H). LCMS m/z 439.33 [M+H]+.
A solution of benzyl N-[3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propyl]carbamate C81 (90 mg, 0.197 mmol) and palladium on carbon (97 mg of 2.2% w/w, 0.02 mmol) in ethanol (3 mL) was purged and evacuated with nitrogen and allowed to stir at room temperature for 3 hours under a hydrogen balloon. The reaction was filtered and concentrated in vacuo. Purification by silica gel chromatography (Gradient: 0-100% EtOAc in heptanes) afforded 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propan-1-amine (40.5 mg, 65%) 1H NMR (300 MHz, Chloroform-d) δ 8.19 (s, 1H), 7.51 (ddd, J=8.0, 5.1, 2.3 Hz, 2H), 7.22-7.12 (m, 2H), 7.06 (dd, J=9.2, 2.2 Hz, 1H), 6.73 (ddd, J=11.5, 9.5, 2.2 Hz, 1H), 2.86-2.78 (m, 2H), 2.72 (t, J=7.1 Hz, 2H), 1.84-1.73 (m, 2H). LCMS m/z 305.07 [M+H]+.
To a stirred solution of 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propan-1-amine 504 (780 mg, 0.0018 mol) in DMF (12 mL) were added DIPEA (1.4692 g, 2 mL, 0.0113 mol), AcOH (261.36 mg, 0.25 mL, 0.0043 mol) and HATU (1.1 g, 0.0028 mol) at room temperature. The reaction mixture was heated to 50° C. for 3 hours at which time the reaction mixture was poured into ice cold water (100 mL) and extracted with EtOAc (3×100 mL). The organic layer was washed with brine solution (80 mL), dried over sodium sulfate, filtered, concentrated in vacuo. Purification by reversed-phase HPLC (Method: C18 Luna column (25×150 mm, 10 micron). Gradient: MeOH in H2O with 10 mM ammonium bicarbonate) afforded an off-white solid which was washed with n-pentane (10 mL) and dried under vacuum to yield N-[3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propyl]acetamide (326 mg, 52%). 1H NMR (400 MHz, DMSO-d6) δ 11.64 (brs, 1H), 7.85-7.82 (m, 1H), 7.67-7.63 (m, 2H), 7.38-7.33 (m, 2H), 7.25-7.22 (m, 1H), 6.99-6.93 (m, 1H), 3.18-3.04 (m, 2H), 2.76-2.72 (m, 2H), 1.78 (s, 3H), 1.74-1.66 (m, 2H). 19F NMR (376.22 MHz, DMSO-d6) δ −113.76, −122.33, −129.31; LCMS m/z 347.1 [M+H]+.
To a solution of dichlorozinc (1.9 g, 14 mmol) and 2-[5-(4-fluorophenyl)-5-oxo-pentyl]isoindoline-1,3-dione S21 (2.2 g, 6.8 mmol) in AcOH (30 mL) was added (4-fluorophenyl)hydrazine (Hydrochloride salt) (1.5 g, 9.226 mmol). The reaction was heated to 70° C. for 4 hours. An extra portion of hydrazine (1 equiv.) was added to the mixture, and the mixture was heated at 70° C. for 2 more hours. The reaction mixture was filtered to remove ZnCl2 and the filtrate was concentrated in vacuo. The residue was dissolved in EtOAc, washed with water (2×), brine (2×) dried, and concentrated to afford 2-[3-[5-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propyl]isoindoline-1,3-dione (2.85 g, 100%) as an orange solid. The material was taken forward without further purification and quantitative yield was assumed. LCMS m/z 417.27 [M+H]+
To a solution of 2-[3-[5-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propyl]isoindoline-1,3-dione C82 (2.82 g, 6.77 mmol) in EtOH (30 mL) was added hydrazine (30 mL of 1 M, 30 mmol). The reaction was heated to 50° C. and stirred for 2 hours. Additional hydrazine (4 equiv.) was added and the mixture was heated for 3 more hours. The volatiles were removed under reduced pressure and the solid residue was taken up in EtOH and filtered. The filtrate was concentrated in vacuo to afford 3-[5-fluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propan-1-amine (S22) (1.82 g, 94%) which was directly used in the next step. LCMS m/z 287.2 [M+H]+.
Compounds 506 and 507 were prepared in one step according to the procedure as described for 505 using the appropriate amine. Acids were obtained from commercial sources.
1H NMR; LCMS m/z
A solution of 5-chloro-1-(4-fluorophenyl)pentan-1-one C83 (5.0 g, 23 mmol), tert-butyl N-acetylcarbamate (3.7 g, 23 mmol) and potassium carbonate (4.8 g, 35 mmol) in DMF (50 mL) was heated overnight at 80° C. The reaction mixture was filtered, and the filtrate partitioned with water and extracted with ethyl acetate. The organic layer was washed with brine, dried over sodium sulfate and concentrated in vacuo. Purification by silica gel chromatography (Gradient: 0-20% EtOAc in Heptanes) afforded tert-butyl N-acetyl-N-[5-(4-fluorophenyl)-5-oxo-pentyl]carbamate (5.5 g, 21%). LCMS m/z 337.16 [M+H]+.
A solution of (2-chlorophenyl)hydrazine (200 mg, 1.403 mmol), tert-butyl N-acetyl-N-[5-(4-fluorophenyl)-5-oxo-pentyl]carbamate C84 (473.4 mg, 1.403 mmol) and dichlorozinc (478.2 mg, 325.4 μL, 3.508 mmol) in toluene (5 mL) and AcOH (5 mL) were heated to 120° C. overnight. The reaction mixture was cooled to room temperature, filtered, and concentrated to dryness. The residue was brought up in water and extracted with ethyl acetate. The organic layer was concentrated in vacuo. Purification by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) afforded N-[3-[7-chloro-2-(4-fluorophenyl)-1H-indol-3-yl]propyl]acetamide (508) (16.9 mg) as the trifluoroacetate salt. 1H NMR (300 MHz, Methanol-d4) δ 7.72-7.44 (m, 3H), 7.27-6.91 (m, 4H), 3.15 (t, J=7.0 Hz, 2H), 2.84 (t, J=7.9 Hz, 2H), 1.86 (d, J=10.4 Hz, 5H). LCMS m/z 345.17 [M+H]+.
A solution of N-(2-sulfanylethyl)acetamide C85 (250 mg, 2.098 mmol) and 2-bromo-1-(4-fluorophenyl)ethanone C86 (456 mg, 2.10 mmol) in DMF (4 mL) was treated with DIPEA (406 μL, 2.331 mmol) at 22° C. The reaction was stirred for 2 hours. Water was added, and the mixture was extracted with EtOAc. The organic layer was washed with brine, dried over Na2SO4 and evaporated. Purification by silica gel chromatography (Gradient: 0-40% EtOAc in heptane) yielded the product: N-[2-[2-(4-fluorophenyl)-2-oxo-ethyl]sulfanylethyl]acetamide (305 mg, 54%). 1H NMR (300 MHz, Chloroform-d) δ 8.05-7.97 (m, 2H), 7.20-7.12 (m, 2H), 6.00 (s, 1H), 3.84 (s, 2H), 3.47 (q, J=6.0 Hz, 2H), 2.73 (dd, J=6.8, 5.7 Hz, 2H), 1.99 (s, 3H). LCMS m/z 256.18 [M+H]+.
A solution of N-[2-[2-(4-fluorophenyl)-2-oxo-ethyl]sulfanylethyl]acetamide C87 (305 mg, 1.141 mmol), (2,4-difluorophenyl)hydrazine (Hydrochloride salt) (413 mg, 2.287 mmol), acetic acid (3 mL) in toluene (3 mL) was treated with zinc chloride (702 mg, 5.150 mmol). The mixture was stirred at 115° C. overnight and then concentrated in vacuo. Purification by reversed-phase HPLC (Method: C18 column. Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) and further purification by silica gel chromatography (Gradient: 0-100% EtOAc in heptane) yielded the product. N-[2-[[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]sulfanyl]ethyl]acetamide (16.9 mg, 4%). 1H NMR (300 MHz, Chloroform-d) δ 8.67 (s, 1H), 7.85-7.78 (m, 2H), 7.26-7.18 (m, 3H), 6.81 (ddd, J=10.6, 9.3, 2.2 Hz, 1H), 5.32 (s, 1H), 3.05 (q, J=6.0 Hz, 2H), 2.69 (dd, J=6.7, 5.5 Hz, 2H), 1.72 (s, 3H). LCMS m/z 365.1 [M+H]+.
Phenylhydrazine (2.5 mL, 25.38 mmol) was added to a solution of zinc chloride (2.4 g, 17.61 mmol) and N-[2-[2-(4-fluorophenyl)-2-oxo-ethyl]sulfanylethyl]acetamide C87 (5.3 g, 20.76 mmol) in AcOH (80 mL). The reaction was heated to 70° C. for 4 hours and was concentrated in vacuo. The residue was dissolved in EtOAc and washed with water, brine and dried and concentrated under reduced pressure. The residue was then purified by silica gel chromatography (Gradient: 0-100% EtOAc in hexanes) to afford the title compound as a beige solid N-[2-[[2-(4-fluorophenyl)-1H-indol-3-yl]sulfanyl]ethyl]acetamide (3.7 g, 54%). 1H NMR (400 MHz, DMSO-d6) δ 11.81 (s, 1H), 8.06-7.97 (m, 2H), 7.82 (t, J=5.5 Hz, 1H), 7.67 (dt, J=7.5, 0.9 Hz, 1H), 7.43 (dt, J=8.0, 1.0 Hz, 1H), 7.38 (t, J=9.0 Hz, 2H), 7.19 (ddd, J=8.1, 7.0, 1.3 Hz, 1H), 7.13 (ddd, J=8.0, 7.0, 1.1 Hz, 1H), 3.01 (dt, J=8.0, 6.0 Hz, 2H), 2.70-2.63 (m, 2H), 1.69 (s, 3H). LCMS m/z 329.15 [M+H]+.
A solution of 6-chloro-1-(4-fluorophenyl)hexan-1-one C88 (1 g, 4.373 mmol) in DMF (45 mL) was treated with (1,3-dioxoisoindolin-2-yl)potassium (990 mg, 5.345 mmol) then heated/stirred at 80° C. overnight. The reaction was quenched with water causing precipitation of product as white solid. The product was collected by vacuum filtration, washed with water, dried under vacuum to afford 2-[6-(4-fluorophenyl)-6-oxo-hexyl]isoindoline-1,3-dione C90 (1.372 g, 88%)1H NMR (300 MHz, Chloroform-d) δ 8.03-7.95 (m, 2H), 7.86 (dd, J=5.4, 3.1 Hz, 2H), 7.78-7.69 (m, 2H), 7.18-7.09 (m, 2H), 3.73 (t, J=7.2 Hz, 2H), 3.00-2.92 (m, 2H), 1.80 (dq, J=14.2, 7.3 Hz, 4H), 1.47 (td, J=8.5, 7.7, 3.1 Hz, 2H). LCMS m/z 340.05 [M+H]+.
A mixture of 2-[6-(4-fluorophenyl)-6-oxo-hexyl]isoindoline-1,3-dione C90 (660 mg, 1.835 mmol), (2,4-difluorophenyl)hydrazine (Hydrochloride salt) (665 mg, 3.683 mmol), and zinc chloride (1.2 g, 8.803 mmol) in toluene (5 mL) and acetic acid (5 mL) was heated and stirred at 110° C. overnight. The reaction was concentrated in vacuo then partitioned between ethyl acetate/water. The combined organics were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo followed by purification by silica gel chromatography (Gradient: 0-20% EtOAc in heptane) yielded the product 2-[4-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]butyl]isoindoline-1,3-dione (C91) (450 mg, 48%). 1H NMR (300 MHz, Chloroform-d) δ 8.04 (s, 1H), 7.85-7.80 (m, 2H), 7.71 (dt, J=5.2, 3.5 Hz, 2H), 7.53-7.46 (m, 2H), 7.20-7.13 (m, 2H), 7.03 (dd, J=9.2, 2.2 Hz, 1H), 6.70 (ddd, J=10.8, 9.5, 2.2 Hz, 1H), 3.69-3.62 (m, 2H), 2.82 (t, J=7.1 Hz, 2H), 1.69 (d, J=7.1 Hz, 4H). LCMS m/z 449.27 [M+H]+.
A solution of 2-[4-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]butyl]isoindoline-1,3-dione C91 (0.87 g, 1.843 mmol) in ethanol (18 mL) was treated with hydrazine (5.5 mL of 1 M, 5.500 mmol). The resulting solution was heated at reflux for 4 hours then cooled to ambient temperature and concentrated in vacuo. The residue was purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford 4-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]butan-1-amine (Trifluoroacetate salt) (228.4 mg, 27%). 1H NMR (300 MHz, DMSO-d6) δ 11.69 (s, 1H), 7.70-7.64 (m, 2H), 7.59 (s, 2H), 7.41-7.33 (m, 2H), 7.25 (dd, J=9.6, 2.2 Hz, 1H), 6.98 (ddd, J=11.2, 9.8, 2.2 Hz, 1H), 2.83-2.70 (m, 4H), 1.56 (td, J=15.5, 14.7, 7.7 Hz, 4H). LCMS m/z 319.07 [M+H]+.
A solution of 2-[4-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]butyl]isoindoline-1,3-dione C91 (450 mg, 0.8840 mmol) in ethanol (9 mL) was treated with hydrazine (2.7 mL of 1 M, 2.700 mmol). The resulting mixture was heated at 60° C. for approximately 3-4 hours. The reaction was concentrated in vacuo then suspended in additional ethanol and filtered. The filtrated was concentrated in vacuo then dried under high vacuum to afford 4-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]butan-1-amine which was used without further purification.
The product from the above reaction was dissolved in dichloromethane (9 mL) and treated with acetyl chloride (63 μL, 0.8860 mmol) followed by DIPEA (308 μL, 1.768 mmol). The resulting solution was stirred at room temperature overnight, followed by concentration in vacuo and purified by silica gel chromatography (Gradient: 10-100% EtOAc in heptane) afforded the product N-[4-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]butyl]acetamide (51.7 mg, 14% over 2 steps). 1H NMR (300 MHz, Chloroform-d) δ 8.11 (s, 1H), 7.53-7.46 (m, 2H), 7.19 (td, J=8.9, 2.4 Hz, 2H), 7.03 (dd, J=9.1, 2.2 Hz, 1H), 6.74 (ddd, J=10.7, 9.4, 2.1 Hz, 1H), 5.35 (s, 1H), 3.26-3.18 (m, 2H), 2.80 (t, J=7.5 Hz, 2H), 1.94 (s, 3H), 1.66 (t, J=8.0 Hz, 2H), 1.50 (dt, J=8.2, 6.7 Hz, 2H). LCMS m/z 361.24 [M+H]+.
To 5,7-difluoro-2-(4-fluorophenyl)-1H-indole (505 mg, 2.043 mmol) and methyl 3-oxobutanoate (353 μL, 3.271 mmol) in DCE (8 mL) at 70° C. was added, MsOH (265 μL, 4.084 mmol) and triethylsilane (980 μL, 6.136 mmol). The reaction was heated at 70° C. for 1 hour. Additional methyl 3-oxobutanoate (353 μL, 3.271 mmol), MsOH (265 μL, 4.084 mmol), triethylsilane (980 μL, 6.136 mmol) were added and the mixture allowed to stir for overnight at 70° C. Water (50 mL) was added and the mixture was extracted with dichloromethane (3×). The mixture was concentrated and purified by silica gel chromatography (Gradient: 0 to 100% EtOAc in hexanes) to afford the product. methyl 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]butanoate (144 mg, 20%). 1H NMR (300 MHz, Chloroform-d) δ 8.07 (s, 1H), 7.65-7.48 (m, 2H), 7.21 (dtd, J=9.3, 6.7, 2.2 Hz, 3H), 6.76 (ddd, J=10.8, 9.4, 2.2 Hz, 1H), 3.67 (p, J=7.3 Hz, 1H), 3.58 (s, 3H), 2.81 (dd, J=7.7, 1.5 Hz, 2H), 1.44 (d, J=7.1 Hz, 3H). LCMS m/z 348.23 [M+H]+.
A solution of methyl 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]butanoate (391.8 mg, 1.072 mmol) in THF (5 mL)/MeOH (5 mL)/Water (2 mL) was treated with LiOH (55 mg, 2.297 mmol), and the resulting reaction stirred for approximately 2 h at room temperature. The reaction was concentrated in vacuo then the aqueous layer was acidified to pH 3 with 1N HCl. The aqueous layer was then extracted with ethyl acetate 3×, the combined organics were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]butanoic acid (350.4 mg, 93%). 1H NMR (300 MHz, Chloroform-d) δ 8.03 (s, 1H), 7.49-7.40 (m, 2H), 7.20-7.11 (m, 3H), 6.74 (ddd, J=10.7, 9.4, 2.1 Hz, 1H), 3.58 (p, J=7.3 Hz, 1H), 2.88-2.72 (m, 2H), 1.44 (d, J=7.1 Hz, 3H). LCMS m/z 334.1 [M+1]+.
To 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-2-methyl-propanoic acid C94 (18 mg, 0.05400 mmol), (2S)-2-amino-3,3,3-trifluoro-propan-1-ol (Hydrochloride salt) (13 mg, 0.07853 mmol) and HATU (40 mg, 0.1052 mmol) in DMSO (1 mL) was added TEA (40 μL, 0.2870 mmol). The reaction was stirred at room temperature for 12 hours. Purification by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) yielded a pair of diastereomers. Compound 513 was the first eluting isomer and compound 514 was the second eluting isomer. 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-2-methyl-N-[(1S)-2,2,2-trifluoro-1-(hydroxymethyl)ethyl]propanamide 513 (5 mg, 20%)1H NMR (300 MHz, Methanol-d4) δ 8.29 (d, J=9.3 Hz, 1H), 7.72-7.59 (m, 2H), 7.34-7.13 (m, 3H), 6.72 (ddd, J=11.7, 9.6, 2.2 Hz, 1H), 4.61-4.42 (m, OH), 3.50 (t, J=6.0 Hz, 2H), 3.15 (dd, J=14.0, 6.9 Hz, 1H), 2.86 (ddd, J=26.7, 14.1, 7.3 Hz, 2H), 1.04 (d, J=6.7 Hz, 3H). LCMS m/z 445.13 [M+H]+.
3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[(1S)-2,2,2-trifluoro-1-(hydroxymethyl)ethyl]butanamide 514 (5 mg, 20%)1H NMR (300 MHz, Methanol-d4) δ 8.32 (d, J=9.3 Hz, 1H), 7.72-7.54 (m, 2H), 7.31 (dd, J=9.9, 2.2 Hz, 1H), 7.28-7.13 (m, 2H), 6.72 (ddd, J=11.1, 9.6, 2.1 Hz, 1H), 4.53 (dd, J=13.4, 5.1 Hz, 1H), 3.74 (dd, J=11.7, 4.8 Hz, 1H), 3.62 (dd, J=12.0, 6.8 Hz, 2H), 2.95-2.64 (m, 2H), 1.42 (d, J=7.1 Hz, 3H). LCMS m/z 445.13 [M+H]+.
To a 0° C. solution of (diisopropylamino)lithium (11 mL of 1 M, 11.00 mmol) in THF (9 mL) was added 1-(4-fluorophenyl)ethenone C95 (1.381 g, 10 mmol) followed by TMSCl (1.4 mL, 11.03 mmol). The resulting mixture was warmed to room temperature and stirred for 2 hours then quenched with saturated sodium bicarbonate solution. Extracted with ethyl acetate (3×), washed with sodium bicarbonate, and sodium chloride solutions 3×, then dried over sodium sulfate, filtered, and concentrated in vacuo to afford 1-(4-fluorophenyl)vinyloxy-trimethyl-silane (2.1 g, 100%) LCMS m/z 211.16 [M+H]+ which was used without further purification in the next reaction.
A solution of the 1-(4-fluorophenyl)vinyloxy-trimethyl-silane C96, diiodomethane (1.2 mL, 14.90 mmol), and dichloromethane (20 mL) was degassed with nitrogen and cooled to 0° C. To this solution was added diethylzinc (12.4 mL of 15% w/v, 15.06 mmol). The resulting mixture was warmed to room temperature then stirred overnight. The mixture was quenched with saturated ammonium chloride solution and then extracted DCM (3×). The combined organics were washed with sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford [1-(4-fluorophenyl)cyclopropoxy]-trimethyl-silane (2.2 g, 98%) 1H NMR (300 MHz, Chloroform-d) δ 7.26-7.13 (m, 2H), 6.98-6.87 (m, 2H), 1.17-1.09 (m, 2H), 0.93-0.87 (m, 2H), 0.08-0.14 (m, 9H). LCMS m/z 224.89 [M+H]+ which was used without further purification in the next reaction.
The crude [1-(4-fluorophenyl)cyclopropoxy]-trimethyl-silane C97 was dissolved in methanol (20 mL), degassed with nitrogen, and cooled to 0° C. A single drop of chlorotrimethyl silane was added to the reaction mixture, which was then warmed to room temperature and stirred overnight. The mixture was quenched with saturated ammonium chloride and extracted with ethyl acetate (3×). The combined organics were washed with brine, dried over sodium sulfate, filtered, and concentrated in vacuo. The residue was purified via silica gel chromatography (Gradient: 0-30% EtOAc in heptanes) to afford 1-(4-fluorophenyl)cyclopropanol (660 mg, 43%)1H NMR (300 MHz, Chloroform-d) δ 7.35-7.25 (m, 2H), 7.08-6.98 (m, 2H), 2.34 (s, 1H), 1.31-1.24 (m, 2H), 1.06-0.97 (m, 2H).
Under argon atmosphere, 1-(4-fluorophenyl)cyclopropanol C98 (660 mg, 4.337 mmol), ethyl 2-bromo-2,2-difluoro-acetate (3.5 g, 17.24 mmol), iodocopper (84 mg, 0.4411 mmol), 1,10-phenanthroline (158 mg, 0.8768 mmol), and K2CO3 (1.2 g, 8.683 mmol) were dissolved in acetonitrile (45 mL) and stirred at 80° C. for 5 hours. The reaction was quenched with water, then partitioned with ethyl acetate. The aqueous layer was separated and extracted with ethyl acetate (3×). The combined organics were washed with saturated NaCl (3×), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified via silica gel chromatography (Gradient: 0-20% EtOAc in heptanes) to afford ethyl 2,2-difluoro-5-(4-fluorophenyl)-5-oxo-pentanoate (389 mg, 29%)1H NMR (300 MHz, Chloroform-d) δ 8.05-7.95 (m, 2H), 7.21-7.07 (m, 2H), 4.33 (q, J=7.1 Hz, 2H), 3.26-3.16 (m, 2H), 2.65-2.46 (m, 2H), 1.35 (t, J=7.2 Hz, 3H). LCMS m/z 275.17 [M+H]+.
A solution of ethyl 2,2-difluoro-5-(4-fluorophenyl)-5-oxo-pentanoate C99 (389 mg, 1.418 mmol), (2,4-difluorophenyl)hydrazine (Hydrochloride salt) (510 mg, 2.824 mmol), and zinc chloride (915 mg, 6.712 mmol) in acetic acid (4 mL) and toluene (4 mL) was heated to 115° C. and stirred overnight. The reaction was concentrated in vacuo then partitioned between ethyl acetate and water. The aqueous layer washed with ethyl acetate (3×) and the combined organics were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified via silica gel chromatography (Gradient: 0-20% EtOAc in heptanes) to afford ethyl 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-2,2-difluoro-propanoate (228.4 mg, 40%)1H NMR (300 MHz, Chloroform-d) δ 8.27 (s, 1H), 7.56 (ddd, J=7.1, 5.3, 2.7 Hz, 2H), 7.25-7.09 (m, 3H), 6.77 (ddd, J=10.7, 9.4, 2.1 Hz, 1H), 4.19 (q, J=7.1 Hz, 2H), 3.53 (t, J=16.9 Hz, 2H), 1.24 (t, J=7.1 Hz, 3H). LCMS m/z 384.15 [M+H]+.
A solution of ethyl 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-2,2-difluoro-propanoate C100 (210 mg, 0.5205 mmol) in THF (2 mL), MeOH (2 mL), and Water (1 mL) was treated with LiOH (28 mg, 1.169 mmol) and allowed to stir at ambient temperature for 2 hours. The reaction was concentrated in vacuo, then the aqueous layer was acidified with 1 M HCl to pH 3, followed by extraction with ethyl acetate (3×). The combined organics were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-2,2-difluoro-propanoic acid (155.3 mg, 80%)1H NMR (300 MHz, Chloroform-d) δ 8.28 (s, 1H), 7.54 (ddd, J=8.8, 5.0, 2.3 Hz, 2H), 7.27-7.10 (m, 6H), 6.78 (ddd, J=10.7, 9.4, 2.2 Hz, 1H), 3.53 (d, J=17.1 Hz, 2H). LCMS m/z 356.02 [M+H]+.
A solution of 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-2,2-difluoro-propanoic acid C101 (102.1 mg, 0.2730 mmol), ammonium chloride (45 mg, 0.8413 mmol), HATU (157 mg, 0.4129 mmol), and DMF (2 mL) was treated with DIPEA (143 μL, 0.8210 mmol). The resulting solution was stirred at room temperature for 1.5 hours, then partitioned between ethyl acetate and water. The organics were concentrated in vacuo, combined with the crude from a smaller scale version of this reaction (50 mg of 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-2,2-difluoro-propanoic acid and purified via silica gel chromatography (Gradient: 0-100% EtOAc in heptanes) to afford ethyl 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-2,2-difluoro-propanamide (136.9 mg, 95%)1H NMR (300 MHz, DMSO-d6) δ 11.97 (s, 1H), 8.01 (d, J=64.0 Hz, 2H), 7.76-7.63 (m, 2H), 7.45-7.31 (m, 2H), 7.22 (ddd, J=9.4, 7.6, 2.2 Hz, 1H), 7.01 (ddd, J=11.2, 9.7, 2.2 Hz, 1H), 3.52 (t, J=17.9 Hz, 2H). LCMS m/z 355.07 [M+H]+.
A solution of 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-2,2-difluoro-propanamide C102 (103.9 mg, 0.2786 mmol) in THF (2 mL) under N2 was cooled to 0° C. in ice bath. Lithium aluminum hydride (560 μL of 2 M, 1.120 mmol) was added to the reaction. The ice bath was removed, and the reaction slowly warmed to room temperature then heated at reflux for 2.5 hours. The reaction was cooled to room temperature then 0° C. in an ice bath. The reaction was slowly quenched by addition of saturated Rochelle's salt followed by ethyl acetate. The organics were separated and the aqueous washed with ethyl acetate (3×), the combined organics were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-2,2-difluoro-propan-1-amine (70.9 mg, 69%) LCMS m/z 341.12 [M+H]+.
A solution of 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-2,2-difluoro-propan-1-amine C103 (70.9 mg, 0.1926 mmol) in dichloromethane (2 mL) was treated with acetyl chloride (15 μL, 0.2110 mmol) followed by DIPEA (51 μL, 0.2928 mmol). The resulting solution was stirred at room temperature overnight then partitioned between dichloromethane and 1N NaOH. The organic layer was concentrated in vacuo then purified via silica gel chromatography (Gradient: 0-20% EtOAc in heptanes) to afford ethyl N-[3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-2,2-difluoro-propyl]acetamide (22.5 mg, 30%). 1H NMR (300 MHz, DMSO-d6) δ 11.94 (s, 1H), 8.27 (t, J=6.1 Hz, 1H), 7.70-7.65 (m, 2H), 7.40-7.34 (m, 2H), 7.24-7.19 (m, 1H), 7.01 (ddd, J=11.2, 9.8, 2.3 Hz, 1H), 3.57 (td, J=14.7, 6.1 Hz, 2H), 3.36 (s, 2H), 1.85 (s, 3H). LCMS m/z 383.11 [M+H]+.
Anhydrous 4 Å molecular sieves were flame dried in flask under high vacuum. The flask was cooled under high vacuum then charged with methyl (E)-3-(2-aminophenyl)prop-2-enoate C104 (1 g, 5.643 mmol) and 4-fluorobenzaldehyde C105 (596 μL, 5.647 mmol), followed by anhydrous toluene (30 mL). The resulting solution was stirred at reflux overnight under nitrogen. The solution was concentrated in vacuo then dried overnight under high vacuum to afford product as a yellow oil methyl (E)-3-[2-[(E)-(4-fluorophenyl)methyleneamino]-phenyl]prop-2-enoate (1.01 g, 60%)1H NMR (300 MHz, Chloroform-d) δ 8.35 (s, 1H), 8.19 (d, J=16.1 Hz, 1H), 7.99-7.91 (m, 2H), 7.63 (dd, J=7.8, 1.5 Hz, 1H), 7.41 (td, J=7.7, 1.5 Hz, 1H), 7.26 (dd, J=7.6, 1.3 Hz, 1H), 7.23-7.15 (m, 2H), 7.00 (dd, J=7.9, 1.3 Hz, 1H), 6.47 (d, J=16.2 Hz, 1H), 3.79 (s, 3H). LCMS m/z 284.8 [M+H]+.
A solution of methyl (E)-3-[2-[(E)-(4-fluorophenyl)methyleneamino]phenyl]prop-2-enoate C106 (1.01 g, 3.387 mmol) in anhydrous THF (27 mL) was treated with DBU (608 μL, 4.066 mmol) and 1,4-dimethyl-1,2,4-triazol-1-ium iodide (230 mg, 1.022 mmol) under nitrogen. The resulting solution was heated/stirred at 80° C. for 4 hours. The reaction was quenched by partitioning between ethyl acetate and water, the combined organics were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The residue was purified via silica gel chromatography (Gradient: 10-100% EtOAc in heptanes) to afford methyl 2-[2-(4-fluorophenyl)-1H-indol-3-yl]acetate (716.1 mg, 70%)1H NMR (300 MHz, Chloroform-d) δ 8.13 (s, 1H), 7.71-7.63 (m, 3H), 7.40 (dt, J=8.0, 1.0 Hz, 1H), 7.27-7.17 (m, 4H), 3.83 (s, 2H), 3.74 (s, 3H). LCMS m/z 284.13 [M+H]+.
A solution of methyl 2-[2-(4-fluorophenyl)-1H-indol-3-yl]acetate C107 (4.5 g, 14.69 mmol) in THF (50 mL) and MeOH (50 mL) and Water (25 mL) was treated with LiOH (780 mg, 32.57 mmol). The resulting mixture was stirred at ambient temperature overnight. The reaction was quenched by concentration in vacuo of organics, then acidification of the aqueous layer with 1N HCl to pH 3. The resulting thick white precipitate was collected under vacuum filtration, washed with water, filtered, and dried to constant mass to afford 2-[2-(4-fluorophenyl)-1H-indol-3-yl]acetic acid (3.8902 g, 94%)1H NMR (300 MHz, DMSO-d6) δ 12.38 (s, 1H), 11.34 (s, 1H), 7.79-7.67 (m, 2H), 7.55 (d, J=7.8 Hz, 1H), 7.45-7.33 (m, 3H), 7.08 (dddd, J=29.7, 8.0, 7.0, 1.2 Hz, 2H). LCMS m/z 270.35 [M+H]+.
A solution of the 2-[2-(4-fluorophenyl)-1H-indol-3-yl]acetic acid C108 (25 mg) and HATU (42 mg) and DIPEA (33 μL) in DMF (1.0 mL) was added to a tube containing 3-aminopropane-1,2-diol. The reaction was agitated on an orbital shaker overnight. The reaction was then directly submitted to purification by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to afford N-(2,3-dihydroxypropyl)-2-(2-(4-fluorophenyl)-1H-indol-3-yl)acetamide. LCMS m/z 343.77 [M+H]+.
In a 250 mL round bottle flask, 3,4-dihydro-2H-pyran (3.8 mL, 41.65 mmol) and (2,4-difluorophenyl)hydrazine C110 (5 g, 34.69 mmol) were dissolved in DMA (100 mL) and H2SO4 (100 mL, 1.876 mol) was added. The mixture was heated to 100° C. for 2 hours. The reaction mixture was then cooled to room temperature, and water (250 mL) was added to the reaction mixture, followed by extraction with EtOAc (3×250 mL). Combined organic fractions were dried over sodium sulfate, after filtered off salt, the solution was concentrated in vacuo. Purification by silica gel chromatography (Gradient: 10-100% EtOAc in hexanes) to afford 3-(5,7-difluoro-1H-indol-3-yl)propan-1-ol (4.2 g, 47%). LCMS m/z 212.02 [M+H]+.
In a 20 mL microwave tube, (4-fluorophenyl)boronic acid (78 mg, 0.56 mmol) and 3-(5,7-difluoro-1H-indol-3-yl)propan-1-ol C111 (115 mg, 0.545 mmol) were dissolved in AcOH (3 mL). Then palladium acetate (15 mg, 0.06681 mmol) was added to the reaction mixture. The reaction mixture was degassed with 02 for 5 minutes, then the tube was sealed, and the mixture was stirred at room temperature for overnight. The solvent was then removed reduced pressure. Saturated NaHCO3 solution was added to the reaction mixture, followed by extraction with DCM (3×30 mL). Combined organic fractions were washed with NaHCO3 (20 mL), dried over MgSO4. Purification by reversed-phase HPLC (Method: C18 column. Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) provided 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propan-1-ol (2.5 mg, 3%). 1H NMR (300 MHz, Chloroform-d) δ 8.11 (s, 1H), 7.63-7.49 (m, 2H), 7.21 (t, J=8.6 Hz, 2H), 7.11 (dd, J=9.1, 2.2 Hz, 1H), 6.77 (ddd, J=10.8, 9.4, 2.2 Hz, 1H), 3.69 (t, J=6.3 Hz, 2H), 3.00-2.83 (m, 2H), 2.04-1.84 (m, 2H), 1.28 (s, 1H). LCMS m/z 306.19 [M+H]+.
A mixture of 5,7-difluoro-2-(4-fluorophenyl)-1H-indole (1.38 g, 5.582 mmol), DCM (15 mL), methyl 3,3-dimethoxypropanoate (900 μL, 6.348 mmol) and TFA (2.4 mL, 31.15 mmol) was heated to reflux for 14 hours. The reaction mixture was then cooled to room temperature and filtered, to collect the product as the solid: methyl (E)-3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]prop-2-enoate (1.6 g, 86%). LCMS m/z 332.21 [M+H]+.
A mixture of methyl (E)-3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]prop-2-enoate C26 (840 mg, 2.527 mmol) and KOH (1.6 g, 28.52 mmol) in MeOH (15 mL) and H2O (18 mL) was heated at 100° C. for 6 hours. The reaction mixture was then concentrated in vacuo. Concentrated HCl was added to adjust till pH=1. The mixture was then filtered, washed with water (3×), dried to yield product (E)-3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]prop-2-enoic acid (765 mg, 94%). LCMS m/z 318.12 [M+H]+.
To (E)-3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]prop-2-enoic acid C112 (65 mg, 0.2033 mmol), (3S,4R)-3-amino-4-hydroxy-pyrrolidin-2-one S1 (24 mg, 0.2067 mmol) and HATU (93 mg, 0.2446 mmol) in DMSO (1 mL) was added Et3N (115 μL, 0.8251 mmol). The reaction mixture was stirred at room temperature for 6 h. An additional HATU (46 mg, 0.12 mmol) and Et3N (58 μL, 0.42 mmol) were added. After another 6 h, the reaction mixture was directly purified by reversed-phase HPLC (Method: C18 Waters Sunfire column (30×150 mm, 5 micron). Gradient: MeCN in H2O with 0.1% trifluoroacetic acid) to give the product. (E)-3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[(3S,4R)-4-hydroxy-2-oxo-pyrrolidin-3-yl]prop-2-enamide (12.2 mg, 14%). 1H NMR (300 MHz, Methanol-d4) δ 12.01 (s, 1H), 7.79 (dd, J=15.8, 0.6 Hz, 1H), 7.71-7.56 (m, 2H), 7.52 (dd, J=9.8, 2.2 Hz, 1H), 7.39-7.20 (m, 2H), 6.73 (d, J=15.8 Hz, 1H), 4.49 (td, J=7.6, 6.9 Hz, 1H), 4.37 (d, J=7.8 Hz, 1H), 3.64 (dd, J=9.9, 7.6 Hz, 1H), 3.17 (dd, J=9.9, 7.0 Hz, 1H). LCMS m/z 416.0 [M+H]+.
To 3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]propanoic acid S8 (1280 mg, 3.809 mmol), (3S)-3-aminopyrrolidin-2-one (470 mg, 4.694 mmol), HATU (2.1 g, 5.523 mmol) in DMF (20 mL) was added Et3N (2 mL, 14.35 mmol). The reaction was stirred at room temperature for 5 hours. The reaction mixture was concentrated in vacuo. Then it was added EtOAc (300 mL), wash with 0.5 M HCl (100 mL), brine, and dried over Na2SO4. The organic layer was concentrated in vacuo. Purification by silica gel chromatography (Gradient: 0-10% MeOH in EtOAc). Second purification was performed using silica gel chromatography (Gradient: 0-20% MeOH in DCM). The title compound was isolated as a minor product: (E)-3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-N-[(3S)-2-oxopyrrolidin-3-yl]prop-2-enamide (16 mg, 1%). 1H NMR (300 MHz, Chloroform-d) δ 8.79 (s, 1H), 7.81 (d, J=15.6 Hz, 1H), 7.62-7.51 (m, 2H), 7.34 (dd, J=9.4, 2.1 Hz, 1H), 7.22 (t, J=8.6 Hz, 2H), 6.86 (ddd, J=11.1, 9.2, 2.1 Hz, 1H), 6.41 (d, J=15.6 Hz, 1H), 6.27 (d, J=5.4 Hz, 1H), 5.78 (s, 1H), 4.50 (ddd, J=11.0, 8.2, 5.3 Hz, 1H), 3.46 (dd, J=9.8, 4.3 Hz, 2H), 2.89 (dd, J=7.9, 4.7 Hz, 1H), 2.17-1.94 (m, 1H). LCMS m/z 400.17 [M+H]+.
Compound 520 was prepared in three steps from C47 and (3S)-3-aminopyrrolidin-2-one using the method described for the preparation of compound 519. LCMS m/z 400.11 [M+H]+.
Compound 521 was isolated as a minor product in the preparation of compound C51 described above. Methyl (E)-3-[2-(4-cyanophenyl)-5,7-difluoro-1H-indol-3-yl]prop-2-enoate (153 mg, 18%). 1H NMR (300 MHz, Chloroform-d) δ 8.67 (s, 1H), 7.93-7.80 (m, 3H), 7.78-7.66 (m, 2H), 7.45 (dd, J=9.3, 2.2 Hz, 1H), 6.92 (ddd, J=10.5, 9.2, 2.1 Hz, 1H), 6.52 (d, J=16.1 Hz, 1H), 3.83 (s, 3H). LCMS m/z 339.09 [M+H]+
To a mixture of 3-[2-(4-cyanophenyl)-5,7-difluoro-1H-indol-3-yl]propanoic acid S13 (45 mg, 0.1355 mmol) HATU (103 mg, 0.2709 mmol) and (3S)-3-aminopyrrolidin-2-one (27 mg, 0.2697 mmol) DMSO (2 mL) was added TEA (95 μL, 0.6816 mmol). The reaction was allowed to stir at room temperature for room temperature for 12 hours. Purification by reverse phase chromatography afforded the product as a minor component. E)-3-[2-(4-cyanophenyl)-5,7-difluoro-1H-indol-3-yl]-N-[(3S)-2-oxopyrrolidin-3-yl]prop-2-enamide (10 mg, 18%). 1H NMR (300 MHz, Methanol-d4) δ 7.58-7.50 (m, 2H), 7.44-7.38 (m, 2H), 7.27-7.20 (m, 1H), 7.13 (d, J=10.1 Hz, 1H), 6.78-6.64 (m, 1H), 5.50-5.36 (m, 1H), 4.33 (t, J=7.8 Hz, 1H), 3.25-3.17 (m, 1H), 2.25-2.09 (m, 1H), 1.65-1.52 (m, 1H). LCMS m/z 407.14 [M+H]+.
To a solution of ditert-butyl carbonate (5 g, 28.70 mmol) and 5,7-difluoro-2-(4-fluorophenyl)-1H-indole (6 g, 24.27 mmol) in THF (120 mL) was added Pyridine (4 mL, 49.46 mmol), followed by DMAP (295 mg, 2.415 mmol). The reaction mixture was stirred at room temperature overnight. The solvent was removed solvent under reduced pressure. Water was added to the reaction mixture followed by extraction with EtOAc (3×50 mL). Combined organic fractions were washed with H2O (1×20 mL), brine (1×20 mL), dried over sodium sulfate, after filtered off salt, the solution was concentrated to dryness. Purification Silica (Redi-Sep cartridge, 120 g), eluting with 0-60% EtOAc in Hexanes to afford tert-butyl 5,7-difluoro-2-(4-fluorophenyl)indole-1-carboxylate (8.3 g, 96%). LCMS m/z 345.64 [M+1]+.
was dissolved in CHCl3 (50 mL), the mixture was cooled to 0° C., to which, 1-iodopyrrolidine-2,5-dione (2.3 g, 10.22 mmol) was added. The mixture was stirred at 0° C. for 3 hours, the stirred at room temperature overnight. Additional 1-iodopyrrolidine-2,5-dione was added to the reaction mixture, which was allowed to stir until completion. The reaction mixture was quenched with sat. Na2SO3 (20 mL). Diluted with 30 mL of H2O, the organic layer was separated, the aqueous layer was extracted with DCM (3×50 mL), dried over Na2SO4, Filtered off Na2SO4 and removed solvent to afford the off white solid, which was washed with Heptane to afford clean compound. tert-butyl 5,7-difluoro-2-(4-fluorophenyl)-3-iodo-indole-1-carboxylate (4.1 g, 91%). 1H NMR (300 MHz, Chloroform-d) δ 7.51-7.36 (m, 2H), 7.26-7.15 (m, 2H), 7.02 (ddd, J=8.0, 2.4, 0.8 Hz, 1H), 6.93 (ddt, J=12.0, 9.3, 2.6 Hz, 1H), 1.37 (d, J=2.5 Hz, 9H). LCMS m/z 472.86 [M+H]+.
1 tert-Butyl 5,7-difluoro-2-(4-fluorophenyl)-3-iodo-indole-1-carboxylate (133 mg, 0.2810 mmol) and 1-prop-2-ynylcyclobutanol (115 mg, 1.044 mmol) were dissolved in N-ethylethanamine (6 mL), the mixture was degassed with Na for several minutes, then Pd(PPh3)2Cl2 (20 mg, 0.02849 mmol) and CuI (11 mg, 0.05776 mmol) were added. Sealed the tube and heated the reaction mixture to 60° C. for overnight. The mixture was concentrated. Water (10 mL) was added to the reaction mixture followed by extraction with EtOAc (3×10 mL). Combined organic fractions were washed with H2O (1×2 mL), brine (1×2 mL), dried over sodium sulfate, filtered and concentrated to dryness. Purification by silica gel (Gradient: 0-10% EtOAc in Hexanes) afforded tert-butyl 5,7-difluoro-2-(4-fluorophenyl)-3-[3-(1-hydroxycyclobutyl)prop-1-ynyl]indole-1-carboxylate (121 mg, 83%). 1H NMR (300 MHz, Chloroform-d) δ 7.61-7.42 (m, 2H), 7.25-7.08 (m, 3H), 6.88 (ddd, J=11.7, 9.3, 2.4 Hz, 1H), 2.72 (s, 2H), 2.14-2.02 (m, 4H), 1.89-1.68 (m, 2H), 1.65-1.47 (m, 1H), 1.41 (s, 9H).
tert-butyl 5,7-difluoro-2-(4-fluorophenyl)-3-[3-(1-hydroxycyclobutyl)prop-1-ynyl]in-dole-1-carboxylate (60 mg, 0.1161 mmol) was dissolved in DCM (2 mL) to which, TFA (500 μL, 6.490 mmol) was added. The mixture was stirred at RT for 1 hour, Removed the solvent, Purification by silica gel chromatography (Gradient: 0-100% EtOAc in Hexanes) afforded 4-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-2-methyl-but-3-yn-2-ol (5.6 mg, 5%) as the minor product. 1H NMR (300 MHz, Chloroform-d) δ 8.48 (s, 1H), 8.06-7.89 (m, 2H), 7.26-7.13 (m, 3H), 6.80 (ddd, J=10.7, 9.3, 2.2 Hz, 1H), 2.12 (s, 1H), 1.70 (s, 6H). LCMS m/z 330.49 [M+H]+. 1-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]-3-(1-hydroxycyclobutyl)propan-1-one (38 mg, 70%) was obtained as the major product. 1H NMR (300 MHz, Chloroform-d) δ 8.92 (s, 1H), 7.81 (dd, J=9.6, 2.2 Hz, 1H), 7.69-7.44 (m, 2H), 7.35-7.12 (m, 2H), 6.85 (ddd, J=11.1, 9.3, 2.2 Hz, 1H), 2.63 (t, J=6.6 Hz, 2H), 2.11-1.78 (m, 6H), 1.78-1.58 (m, 1H), 1.40 (dp, J=11.3, 8.9 Hz, 1H). LCMS m/z 374.24 [M+H]+.
Compound 524 was prepared from C114 and the appropriate alkyne using the method described for the preparation of compound 523.
3-[3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]prop-2-ynyl]oxetan-3-ol (26 mg, 64%). 1H NMR (300 MHz, Methanol-d4) δ 8.24-8.05 (m, 2H), 7.27-7.16 (m, 2H), 7.13 (dd, J=8.9, 2.3 Hz, 1H), 6.79 (ddd, J=11.5, 9.7, 2.3 Hz, 1H), 4.74 (d, J=6.6 Hz, 2H), 4.65 (d, J=6.6 Hz, 2H), 2.99 (s, 2H). LCMS m/z 358.02 [M+H]+;
Compound 525 was prepared from C114 and the appropriate alkyne using the method described for the preparation of compound 523.
1-[3-[5,7-difluoro-2-(4-fluorophenyl)-1H-indol-3-yl]prop-2-ynyl]cyclobutanol (9.2 mg, 19%). 1H NMR (300 MHz, Chloroform-d) δ 8.46 (s, 1H), 8.04-7.87 (m, 2H), 7.18 (dtd, J=8.7, 6.7, 2.2 Hz, 3H), 6.78 (ddd, J=10.7, 9.4, 2.2 Hz, 1H), 2.88 (s, 2H), 2.37 (s, 1H), 2.28-2.17 (m, 4H), 1.97-1.79 (m, 1H), 1.73-1.66 (m, 1H). LCMS m/z 356.24 [M+1]+.
Compound 526 was obtained from commercial sources, and may be prepared from S7 using analogous methods to that described for compound 1.
Compound 527 was obtained from commercial sources. Compound 525 may be prepared using methods described for compound 431. 1H NMR (400 MHz, DMSO-d6) δ 11.19 (s, 1H), 8.05 (t, J=5.9 Hz, 1H), 7.75-7.65 (m, 2H), 7.60 (d, J=7.9 Hz, 1H), 7.49 (d, J=7.9 Hz, 2H), 7.38 (td, J=8.3, 1.8 Hz, 2H), 7.11 (ddd, J=8.1, 7.0, 1.2 Hz, 1H), 7.02 (d, J=7.0 Hz, 1H), 3.33-3.27 (m, 2H), 3.02-2.89 (m, 2H), 1.79 (d, J=2.9 Hz, 3H). LCMS m/z 279.25 [M+H]
Acute APOL1 Thallium Assay with Inducible Stable Clones of HEK 293 Cells
Apolipoprotein L1 (APOL1) proteins form potassium-permeable cation pores in the plasma membrane. APOL1 risk variants (G1 and G2) induce greater potassium flux than G0 in HEK293 cells. This assay exploits the permeability of thallium (Tl+) through ligand-gated potassium channels. The dye produces a bright fluorescent signal upon binding to Tl+ conducted through potassium channels. The intensity of the Tl+ signal is proportional to the number of potassium channels in the open state. Therefore, it provides a functional indication of the potassium channel activities. During the initial dye-loading step, the Tl+ indicator dye as an acetoxymethyl (AM) ester enters the cells through passive diffusion. Cytoplasm esterases cleave the AM ester and relieve its active thallium-sensitive form. The cells are then stimulated with Tl+. The increase of fluorescence in the assay represents the influx of Tl+ into the cell specifically through the potassium channel (i.e. through APOL1 pores), providing a functional measurement of potassium channel/pore activity. The Thallium assay is conducted with cells expressing G1 APOL1.
APOL1 Cell Line (HEK T-Rex Stable Inducible Cell Line)
Tissue Culture Media
Reagents:
Materials
Instruments and Equipment
Cells Scaled Up from Frozen Vials
Cell Culture
T-Rex APOL1 HEK cells are split twice per week to keep the confluence state below 85% of the culture flask surface area. Cells can be kept until passage 25.
Day 1
Preparation of Cell Assay Plates
Preparation of Tetracycline
Day 2
Preparation of Thallium Loading Dye and Cells Loading
FLIPR@ Potassium Assay Kit R8223
Preparation of Drug Plates and Transfer of Compounds to Assay Plates
Preparation of the Thallium Sulfate Source Plate
Start Assay on FLIPR 384-Head
Parameters
Data Analysis
Trypanosoma brucei brucei is a blood stream parasite to which human, gorillas and baboon are immune due to the presence of the APOL1 protein in their HDL particles. The protein is uptaken by the parasite via the TbHpHb receptor located in its flagellar pocket and is bonded by the Hpr protein contained in the HDL particles which triggers the receptor endocytosis by the parasite.
Following endocytosis, the formed vesicle containing the HDL particle matures from early to late endosome, and subsequently to lysosome. The concomitant pH change in the lumen of the vesicle triggers the insertion of the APOL1 protein into the membrane of the late endosome/lysosome and hereby triggers lysosomal membrane permeabilization and as a further downstream event, trypanosome lysis. Trypanosoma brucei brucei is sensitive to lysis by all three APOL1 variants (G0, G1, and G2).
The Trypanosoma brucei brucei lysis assay is a lysis assay of the parasite using recombinant APOL1 protein variant followed by a fluorescent detection method of viability by the addition of AlamarBlue reagent to the assay well, a general metabolic redox indicator (AlamarBlue assay).
Briefly, the AlamarBlue active compound, the resazurin, a blue, water soluble, non-toxic and cell permeable molecule, which can be followed by absorbance, is reduced by various metabolic pathways into resorufin, a red compound which can be followed by either absorbance or fluorescence. The assay allows the calculation of the percent viability (percent of living Trypanosomes remaining in each well) at the end of a lysis relative to the untreated condition by interpolation of fluorescent values (FLU) on a standard curve with a known amount of seeded trypanosome/well.
Trypanosoma brucei brucei (ATCC, Cat. No. PRA-382)
Thaw/Expansion Media (ATCC Medium 2834 Modified HMI-9 Medium)
327.5 mL total
Assay Media (No Phenol Red/No FBS): Make on Day of Use
302.5 mL total
HMI-9 (10×)
Hypoxanthine Stock (100×)-9 (10×)
Media Reagents
Materials
Equipment
Assay Ready Plates (ARPs)
Step 1, Day 1
Step 2, Day 4
Step 3, Day 6
Step 1, Day 1
Step 2, Day 2
Step 3, Day 5
APOL1 G1 Protein
Multidrop
Bravo: Compound Transfer
Trypanosome Addition:
Trypanosomes as described in Step 3 of the Trypanosoma brucei brucei Culture section.
AlamarBlue Addition
The compounds of Formula I, deuterated derivatives thereof and pharmaceutically acceptable salts of any of the foregoing are useful as inhibitors of APOL1 activity. Table 16 below illustrates the IC50 of the compounds 1 to 527 using procedures described above (assays described above in Example 2A and 2B). In Table 16 below, the following meanings apply. For IC50: “+++” means<0.25 μM; “++” means 0.25 μM to 1.0 μM; “+” means greater than 1.0 μM. N.D.=Not determined.
This disclosure provides merely exemplary embodiments of the disclosed subject matter. One skilled in the art will readily recognize from the disclosure and embodiments, that various changes, modifications and variations can be made therein without departing from the spirit and scope of the disclosure as defined in the following claims.
This application claims the benefit of priority of U.S. Provisional Application No. 63/038,276, filed Jun. 12, 2020, which is incorporated by reference herein in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63038276 | Jun 2020 | US |
