WO2015048306A1 - Novel agents targeting cyp51 - Google Patents

Abstract

The invention provides inhibitors of a sterol C14-demethylase, a new series of 4- aminopyridyl-based lead inhibitors targeting Trypanosoma cruzi CYP51 (TcCYP51) developed using structure-based drug design as well as structure -property relationship (SPR) analyses. The screening hit starting point, LP 10 (K_D < 42 nM; EC₅₀ of 0.65 μΜ), has been optimized to give the potential leads that have low nanomolar binding affinity to TcCYP51 and significant activity against T. cruzi amastigotes cultured in human myoblasts. Many of the optimized compounds have improved microsome stability, and most are selective against the T. cruzi CYP51 relative to human CYPs 1A2, 2D6 and 3A4 (<50% inhibition at 1 μΜ). A rationale for the improvement of microsome stability and selectivity of inhibitors against human metabolic CYP enzymes is presented. In addition, the binding mode of several compounds of the invention with the T. brucei CYP51 (TbCYP51) ortholog has been characterized by x-ray structure analysis. Orally active compounds and their cyclodextrin complexes have been shown to be effective against Chagas-infected mice.

Description

NOVEL AGENTS TARGETING CYP51

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority of U.S. Ser. No. 61/882,819, filed September 26, 2013, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant number R01

AI095437, awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.

BACKGROUND

Chagas disease, or American trypanosomiasis is a chronic tropical infection caused by the protozoan parasite Trypanosoma cruzi. The infection can be lethal if untreated. Chagas disease is the leading cause of heart failure in Latin America.¹ Although first described a century ago,- it is still a major public health challenge in South America. Furthermore, many cases have been reported in North America, Europe, and Asia due to human population movements, migration of the triatomine insect vectors, HIV-coinfections, and contaminated blood transfusion.¹⁵

Currently nifurtimox and benznidazole are the only drugs approved for treatment of Chagas disease.- Although these drugs, which date from the late 1960s, show considerable efficacy in the acute stage of Chagas disease, their efficacy is debated in the chronic stage, which involves chronic Chagas cardiomyopathy, leading to congestive heart failure, thromboembolic phenomena, severe arrhythmias, and sudden unexpected death. - Moreover, these old drugs are associated with frequent side effects such as dermatitis, gastrointestinal, and neurologic toxicities, and even a rare case of bone marrow suppression.-^ Therefore, the need exists to develop new therapeutics bearing better safety profiles and improved efficacy to treat T. cruzi infections and prevent cardiovascular Chagas disease.

Sterol biosynthesis is a recognized target for the development of new therapeutic agents to treat T. cruzi infections - Sterol 14-demethylase (CYP51) has been successfully targeted for combating pathogenic fungal infections with azole drugs such as fluconazole, ketoconazole, and posaconazole, among others.- CYP51 catalyzes the oxidative removal of the 14-methyl group of lanosterol and produces A^{14 15}-unsaturated intermediates in ergosterol biosynthesis. - Due to the similarity of sterols and their biosynthesis pathways in fungi and T. cruzi, the anti-parasitic effects of these azole drugs against T. cruzi in infected mammalian cells have been observed Therefore, clinical trials of posaconazole and other antifungal agents in combination with benznidazole are underway for treatment of chronic Chagas disease. - Recently, tipifarnib, a class of farnesyl transferase inhibitors, has been repurposed as an anti-parasitic agent in the laboratory setting.² Hit-to-lead optimization of a new "NEU" series, identified via a HTS campaign at Northeastern University, has been achieved (Fig \) ^Si In addition, x-ray co-crystal structures of T. cruzi CYP51 (TcCYP51) with bound

posaconazole, fluconazole, VNF, and NEU321 have been determined for use in structure- based design approaches to the development of anti-Chagas agents. ---^]-^] However, all of these lead compounds are azoles, and there is an emerging issue of the rapid appearance of laboratory-induced resistance to azoles in T. cruzi cell culture.-- Thus, the development of therapeutics with different scaffolds targeting 7cCYP51 is an important undertaking. For all of these antifungal agents, including the new T. cruzi CYP51 inhibitors tipifarnib, NEU321, and VNI analogs, are azole-type compounds, resistance is an issue, and resistance to azoles in cell culture and in T. cruzi infected mice has been reported. Therefore, an important drug development objective for T. cruzi infection is to develop CYP51 inhibitors with improved properties and new, non-azole chemotypes.

Recently, non-azole hits of the LP 10 series were identified from a HTS campaign at UC San Francisco.^ The binding modes of these non-azole hits were characterized, and their co-crystal structures with M. tuberculosis CYP51 (M YP51) were determined.-³ A 60% cure rate was attained in a mouse model of T. cruzi infection using the non-azole CYP51 inhibitor LP 10.^

SUMMARY

In an effort to develop more potent non-azole CPY51 inhibitor leads as anti-Chagas disease and antifungal agents, we embarked on the optimization of LP 10 by using structure- based drug design considerations in conjunction with in vitro DMPK analysis (microsome stability and CYP inhibition) to drive rounds of inhibitor optimization. In particular, we strived to increase compound stability in human, rat, and mouse liver microsome

preparations, while retaining or increasing inhibition potency toward T. cruzi in infected mammalian cells, in order to identify candidates with properties appropriate to advance into animal models of T. cruzi infection. In addition, selectivity against human cytochrome P450 enzymes CYP1A2, CYP2C9, CYP2D6, and CYP3A4, the most important CYPs involved in drug metabolism and drug-drug interactions,^ has also been monitored and used for compound prioritization in our structure optimization efforts.

In various embodiments, the invention provides a compound of formula (I) Het

(I)

wherein Het is a 5- or 6-membered heteroaryl comprising a nitrogen atom, R is independently at each occurrence H or (Ci-Ce)alkyl, X and Y are each independently C(=0) or SO2, R¹ is an aryl(Co-C6)alkyl or a heteroaryl(Co-C6)alkyl wherein the aryl or the heteroaryl is substituted with 0-3 J, and R² is a group of formula

wherein Ar¹ and Ar² are independently selected aryl or heteroaryl, wherein each aryl or heteroaryl is substituted with 0-3 J, and Z is a 5- to 7-membered heterocyclyl comprising one or more nitrogen atoms, n = 0 or 1; J is halo, halo(Ci-C6)alkyl, (Ci-Ce)alkyl, cyano, (Ci-C6)alkoxyl, (Ci-C6)alkoxycarbonyl, C(=0)NR2, or NRC(=0)(Ci-C6)alkyl; wherein χ signifies a chiral center;

or a pharmaceutically acceptably salt thereof.

a

CYP-II-277 CYP-II-276

or a pharmaceutically acceptable salt thereof.

In various embodiments, the invention provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable excipient. For instance, the excipient can comprise a cyclodextrin or solutol.

The invention further provides, in various embodiments, a method of inhibiting a sterol C14-demethylase comprising contacting the demethylase with an effective amount or concentration of a compound of the invention, such as a compound of formula (I), i.e., of any of the various specific structural formulas disclosed and claimed herein. For instance, the sterol C14 demethylase can be CYP51.

The invention further provides, in various embodiments, a method of treatment of Chagas disease, comprising administering to a patient afflicted therewith an effective dose of a compound of the invention. For example, the compound can be orally administered to the patient as a cyclodextrin inclusion complex.

The invention further provides, in various embodiments, a method of treatment of a fungal disease, comprising administering to a patient afflicted therewith an effective dose of a compound of the invention.

Accordingly, in various embodiments, the invention provides CYP51 inhibitors of high potency and selectivity, having demonstrated bioactivity versus Chagas disease in a mouse model. Various embodiments of the present invention also provide methods of inhibition of CYP51 in vitro and in vivo. Furthermore, various embodiments provide a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable excipient. A pharmaceutical composition of the invention can comprise a compound of the invention and a cyclodextrin; this composition can be highly suitable for oral administration for treatment of Chagas disease, the cyclodextrin complex of the inventive compound providing for an unexpectedly high degree of oral bioavailability.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Inhibitors of 7cCYP51. (A) Azole type CYP51 inhibitors. (B) Pyridinyl type CYP51 inhibitors.

Figure 2. X-ray co-crystal structure of the 7¾CYP51-14t complex. (A) Electron density map (blue mesh) contoured at 1.0 σ delineates the positions of 14t (CYP-I-181)(yellow sticks) in the active site. In purple are amino acid residues providing hydrophobic contacts within 5 A to the indol moiety of 14t plus F105. Heme is displayed as grey sticks. (B) View of the 14t-bound CYP51 clipped by a plane through the binding site compares the binding modes of 14t (yellow) and posaconazole (cyan). The structure of 7¾CYP51 complexed with posaconazole (PDB code: 2X2N) is superimposed on that of with 14t. The active site surface is colored by hydrophobicity from orange (lipophilic) to blue (hydrophylic). (C) View of bound inhibitors from the entrance to the active site. The enzyme is represented by a gray surface. The hydrophobic units of posaconazole and 14t occupy different hydrophobic tunnels in corresponding co-crystal structures. The images here and otherwise were generated using PYMOU2 ₀r CHIMERA^.

Figure 3. Comparison of the 14t binding mode with NEE and VNF. View of the 14t- bound 7¾CYP51 clipped by a plane through the binding site. A hydrophobic cavity accommodating the indole ring of 14t (CYP-I- 181) (yellow) extends toward Fl 10 (A). This extension accommodates a substituted benzyl ring in NEE (purple) (PDB ID 4H60) (B) or biaryl moiety of VNF (pink) (PDB ID 3KSW) in the corresponding 7cCYP51 co-crystal structures. (C). Both structures were superimposed on the 14t-bound 7¾CYP51.

Figure 4. Predicted binding modes of inhibitors in 7icCYP51. Binding modes of 271 (A), 27s (B), 27k (C), and 27r (D) resulting from molecular docking using Glide XP. Inhibitors are in stick mode colored by atom type: carbon in yellow, oxygen in red, nitrogen in blue, fluorine in cyan, hydrogen on the tertiary amino group of 27s is in gray. The protein is shown as a semi-transparent gray surface; heme is displayed as orange spheres.

Figure 5. Comparison of the binding modes of 1 (blue) and posaconazole (red) in the active site of CYP51. Protein is shown as solvent accessible surface with M460 side chain present (A) or omitted (B) from the coordinates.

Figure 6. (A) Comparision of the binding poses of (S)-l (blue) and (R)-2 (red). Heme is shown in semi-transparent spheres. (B) Dose-response curves for 1 (blue) and 2 (red) show >500-fold difference in hit potency. (C) (R)-3 (red) protruding through the hydrophobic tunnel to the solvent accessible area (PDB ID 4BMM). Protein is represented by grey semi- transparent surface.

Figure 7. Rationale for (R)-2 optimization. (A) (R)-2 in T. cruzi CYP51 binding site (PDB ID 4BY0). Predicted binding modes for (R)-6 (C) and (R)-7 (B). Compounds are in stick mode, protein is represented by grey opaque surface.

Figure 8. Solutol vs. cyclodextrin compound administration efficacies. Data are averaged between five mice.

Figure 9. Dose-response on compound administration in solutol. Data are averaged between five mice.

Figure 10: Binding modes of S- and R-enantiomers. A. An overlay between all the available 7cCYP51 co-structures with bound S- (purple) or R- (yellow) enantiomers of the N- indolyl-oxopyridinyl-4-aminopropanyl scaffold is shown. Inhibitors are in stick

representation; protein Cot traces are shown in black lines. The coordinates PDB ID used for superimposition are as following: S-configuration: 4BJK; R-configuration: 4BMM, 4BYO; 4C0C and 2YMC. B. The overlapped 259- and 71-7cCYP51 complexes emphasize fit induced differences in positions of the 214-227 fragment in the FG-loop. Protein is in ribbon representation color-coded yellow for compound 11-71 and blue for compound 11-259. The β- sheet 1, the largest in the P450 molecule, is in pink. C. Superimposed binding poses for 11-71 (yellow) and 11-259 (blue). Piperazine ring flexes and twists in opposite directions to accommodate each inhibitor molecule in the binding site. Heme is shown as spheres colored according to the chemical elements: carbon grey, oxygen red, nitrogen blue, iron ochre. Images here and otherwise were generated using PyMOL (46) or CHIMERA (47).

Figure 11. Compound 11-259 in the active site of 7icCYP51. A. Piperazine group separating two aryl rings in the 11-259 structure (yellow sticks) allows smooth bending of the long substituent at the chiral center along the b-sheet 1 saddle. Electron density map (blue mesh) is contoured at 1.2 s. B. Residues within 5 A from 11-259 are highlighted in dark blue, heme is in gray. C. Slice through the binding site shows nearly perfect fit of 11-259 (yellow spheres) and the protein surface colored by hydrophobicity, hydrophobic areas are in orange and hydrophilic areas are in blue. Heme is shown in dark red spheres.

Figure 12. Compound 11-71 in the active site of 7icCYP51. A. Electron density map (blue mesh) contoured at 1.2 s delineates the 11-71 position in chain A. B. Indole ring of 11-71 is flipping in the active site in chain B. C. Slice through the binding site shows 11-71 (yellow spheres) and the protein surface colored by hydrophobicity. Color scheme is same as in Figure 1 1.

Figure 13. Compounds 11-250, 11-251, 11-255, 11-257, 11-258, and 11-259 are effective versus T. cruzi in mice. Luminescence assay shows the reduction in trypanosome populations in treated mice versus controls.

Figure 14. A 4-day mouse model oral delivery of selected compounds of the invention in 20% solutol. Compounds of the invention 11-250, 11-251, 11-258, and 11-259 in 20% solutol were administered orally to mice. High light emission intensity shows high trypanosome populations in the mice.

Figure 15. A comparison of orally administered compound 11-250 in hydroxypropyl-γ- cyclodextrin (HPpCD) versus in solutol. Luminescence assay shows comparative effects of orally delivered compound of the invention 11-250. High light emission intensity shows high trypanosome populations in the mice.

DETAILED DESCRIPTION

Definitions

As used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

The term "about" as used herein, when referring to a numerical value or range, allows for a degree of variability in the value or range, for example, within 10%, or within 5% of a stated value or of a stated limit of a range.

All percent compositions are given as weight-percentages, unless otherwise stated. All average molecular weights of polymers are weight- average molecular weights, unless otherwise specified.

As used herein, "individual" (as in the subject of the treatment) or "patient" means both mammals and non-mammals. Mammals include, for example, humans; non-human primates, e.g. apes and monkeys; and non-primates, e.g. dogs, cats, cattle, horses, sheep, and goats. Non-mammals include, for example, fish and birds.

The term "disease" or "disorder" or "malcondition" are used interchangeably, and are used to refer to diseases or conditions wherein CYP51 plays a role in the biochemical mechanisms involved in the disease or malcondition or symptom(s) thereof such that a therapeutically beneficial effect can be achieved by acting on CYP51. "Acting on" CYP51 , or "modulating" CYP51, can include binding to CYP51 and/or inhibiting the bioactivity of CYP51 and/or allosterically regulating the bioactivity of CYP51 in vivo.

The expression "effective amount", when used to describe therapy to an individual suffering from a disorder, refers to the amount of a compound of the invention that is effective to inhibit or otherwise act on CYP51 in the individual's tissues wherein CYP51 involved in the disorder is active, wherein such inhibition or other action occurs to an extent sufficient to produce a beneficial therapeutic effect.

"Substantially" as the term is used herein means completely or almost completely; for example, a composition that is "substantially free" of a component either has none of the component or contains such a trace amount that any relevant functional property of the composition is unaffected by the presence of the trace amount, or a compound is

"substantially pure" is there are only minimal traces of impurities present.

"Treating" or "treatment" within the meaning herein refers to an alleviation of symptoms associated with a disorder or disease, or inhibition of further progression or worsening of those symptoms, or prevention or prophylaxis of the disease or disorder, or curing the disease or disorder. Similarly, as used herein, an "effective amount" or a "therapeutically effective amount" of a compound of the invention refers to an amount of the compound that alleviates, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms, or prevents or provides prophylaxis for the disorder or condition. In particular, a "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of compounds of the invention are outweighed by the therapeutically beneficial effects. Phrases such as "under conditions suitable to provide" or "under conditions sufficient to yield" or the like, in the context of methods of synthesis, as used herein refers to reaction conditions, such as time, temperature, solvent, reactant concentrations, and the like, that are within ordinary skill for an experimenter to vary, that provide a useful quantity or yield of a reaction product. It is not necessary that the desired reaction product be the only reaction product or that the starting materials be entirely consumed, provided the desired reaction product can be isolated or otherwise further used.

By "chemically feasible" is meant a bonding arrangement or a compound where the generally understood rules of organic structure are not violated; for example a structure within a definition of a claim that would contain in certain situations a pentavalent carbon atom that would not exist in nature would be understood to not be within the claim. The structures disclosed herein, in all of their embodiments are intended to include only

"chemically feasible" structures, and any recited structures that are not chemically feasible, for example in a structure shown with variable atoms or groups, are not intended to be disclosed or claimed herein.

An "analog" of a chemical structure, as the term is used herein, refers to a chemical structure that preserves substantial similarity with the parent structure, although it may not be readily derived synthetically from the parent structure. A related chemical structure that is readily derived synthetically from a parent chemical structure is referred to as a "derivative." When a substituent is specified to be an atom or atoms of specified identity, "or a bond", a configuration is referred to when the substituent is "a bond" that the groups that are immediately adjacent to the specified substituent are directly connected to each other in a chemically feasible bonding configuration.

All single enantiomer, diastereomeric, racemic forms of a structure are intended, unless a particular stereochemistry or isomeric form is specifically indicated. In several instances though an individual stereoisomer is described among specifically claimed compounds, the stereochemical designation does not imply that alternate isomeric forms are less preferred, undesired, or not claimed. Compounds used in the present invention can include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions, at any degree of enrichment. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these are all within the scope of the invention. As used herein, the terms "stable compound" and "stable structure" are meant to indicate a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent. Only stable compounds are contemplated herein.

In general, "substituted" refers to an organic group as defined herein in which one or more bonds to a hydrogen atom contained therein are replaced by one or more bonds to a non-hydrogen atom such as, but not limited to, a halogen (i.e., F, CI, Br, and I); an oxygen atom in groups such as hydroxyl groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxylamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents J that can be bonded to a substituted carbon (or other) atom include F, CI, Br, I, OR', OC(0)N(R')₂, CN, NO, N0₂, ONO2, azido, CF3, OCF3, R', O (oxo), S (thiono), methylenedioxy, ethylenedioxy, N(R , SR, SOR, SO2R, S02N(R , SO3R, C(0)R, C(0)C(0)R, C(0)CH₂C(0)R, C(S)R, C(0)OR, OC(0)R, C(0)N(R)₂,

OC(0)N(R)₂, C(S)N(R)₂, (CH₂)o-2N(R)C(0)R, (CH₂)o-2N(R)N(R)₂, N(R)N(R)C(0)R, N(R)N(R)C(0)OR, N(R)N(R)CON(R)₂, N(R)S0₂R, N(R)S0₂N(R)2, N(R)C(0)OR, N(R)C(0)R, N(R)C(S)R, N(R)C(0)N(R)₂, N(R)C(S)N(R)₂, N(COR)COR, N(OR)R, C(=NH)N(R)₂, C(0)N(OR)R, or C(=NOR)R wherein R' can be hydrogen or a carbon- based moiety, and wherein the carbon-based moiety can itself be further substituted; for example, wherein R' can be hydrogen, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl, wherein any alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl or R' can be independently mono- or multi- substituted with J; or wherein two R' groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl, which can be mono- or independently multi-substituted with J.

In various embodiments, J is any of halo, (Cl-C6)alkyl, (Cl-C6)alkoxy, (Cl- C6)haloalkyl, hydroxy(Cl-C6)alkyl, alkoxy(Cl-C6)alkyl, (Cl-C6)alkanoyl, (Cl-

C6)alkanoyloxy, cyano, nitro, azido, R2N, RzNQO), R2NC(0)0, R2NC(0)NR, (Cl- C6)alkenyl, (Cl-C6)alkynyl, (C6-C10)aryl, (C6-C10)aryloxy, (C6-C10)aroyl, (C6- C10)aryl(Cl-C6)alkyl, (C6-C10)aryl(Cl-C6)alkoxy, (C6-C10)aryloxy(Cl-C6)alkyl, (C6- C10)aryloxy(Cl-C6)alkoxy, (3- to 9-membered)heterocyclyl, (3- to 9- membered)heterocyclyl(Cl-C6)alkyl, (3- to 9-membered)heterocyclyl(Cl-C6)alkoxy, (5- to 10-membered)heteroaryl, (5- to 10-membered)heteroaryl(Cl-C6)alkyl, (5- to 10- membered)heteroaryl(Cl-C6)alkoxy, or (5- to 10-membered)heteroaroyl. For example, R independently at each occurrence can be H, (C 1 -C6)alkyl, or (C6-C10)aryl, wherein any alkyl or aryl group is substituted with 0-3 J.

When a number of carbon atoms in a group, e.g., an alkyl, alkenyl, alkynyl, cycloalkyl, aryl, etc., is specified as a range, each individual integral number representing the number of carbon atoms is intended. For example, recitation of a (Ci-C4)alkyl group indicates that the alkyl group can be any of methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, or tert-butyl. It is understood that a specification of a number of carbon atoms must be an integer.

When a number of atoms in a ring is specified, e.g., a 3- to 9-membered heterocyclyl ring, the heterocyclyl ring can include any of 3, 4, 5, 6, 7, 8, or 9 atoms, which can be atoms of any element capable of forming two or more bonds, e.g., carbon, nitrogen, oxygen, sulfur, and the like. The number of atoms in a ring is understood to necessarily be an integer.

Alkyl groups include straight chain and branched alkyl groups and cycloalkyl groups having from 1 to about 20 carbon atoms, and typically from 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n- hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2- dimethylpropyl groups. As used herein, the term "alkyl" encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed above, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. Exemplary alkyl groups include, but are not limited to, straight or branched hydrocarbons of 1-6, 1-4, or 1-3 carbon atoms, referred to herein as Ci ealkyl, Ci-4alkyl, and Ci salkyl, respectively. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2 -methyl- 1 -butyl, 3-methyl-2-butyl, 2 -methyl- 1 -pentyl, 3 -methyl- 1-pentyl, 4- methyl- 1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl- 1 - butyl, 3, 3- dimethyl- 1 -butyl, 2-ethyl- l -butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, etc.

Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, pyridinyl, pyrimidinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed above. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed above.

Heteroarylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined above.

The term "alkoxy" or "alkoxyl" refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined above. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy, tert- butoxy, isopentyloxy, isohexyloxy, and the like. Exemplary alkoxy groups include, but are not limited to, alkoxy groups of 1-6 or 2-6 carbon atoms, referred to herein as Ci ealkoxy, and C2-6alkoxy, respectively. Exemplary alkoxy groups include, but are not limited to methoxy, ethoxy, isopropoxy, etc.

An alkoxy group can include one to about 12-20 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group is an alkoxy group within the meaning herein. A methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structures are substituted therewith.

The terms "halo" or "halogen" or "halide" by themselves or as part of another substituent mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine. A "haloalkyl" group includes mono-halo alkyl groups, poly-halo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1-dichloroethyl, 1,2-dichloroethyl, l,3-dibromo-3,3-difluoropropyl, perfluorobutyl, and the like.

Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined above. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6- substituted phenyl or 2-8 substituted naphthyl groups, which can be substituted with carbon or non-carbon groups such as those listed above.

Aralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl group are alkenyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above.

It will be understood that when compounds of the present invention contain one or more chiral centers, the compounds may exist in, and may be isolated as single and substantially pure enantiomeric or diastereomeric forms or as racemic mixtures. The present invention therefore includes any possible enantiomers, diastereomers, racemates or mixtures thereof of the compounds of the invention.

The compounds of the invention, or compounds used in practicing methods of the invention, may contain one or more chiral centers and, therefore, exist as stereoisomers. The term "stereoisomers" when used herein consist of all enantiomers or diastereomers. These compounds may be designated by the symbols "(+)," "(-)," "R" or "S," depending on the configuration of substituents around the stereogenic carbon atom, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. The present invention encompasses various stereoisomers of these compounds and mixtures thereof. Mixtures of enantiomers or diastereomers may be designated "(±)" in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. The compounds of the disclosure may contain one or more double bonds and, therefore, exist as geometric isomers resulting from the arrangement of substituents around a carbon-carbon double bond. The symbol =r. denotes a bond that may be a single, double or triple bond as described herein. Substituents around a carbon-carbon double bond are designated as being in the "Z" or "£"' configuration wherein the terms "Z" and are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the "E" and "Z" isomers. Substituents around a carbon-carbon double bond alternatively can be referred to as "cis" or "trans," where "cis" represents substituents on the same side of the double bond and "trans" represents substituents on opposite sides of the double bond.

Compounds of the invention, or compounds used in practicing methods of the invention, may contain a carbocyclic or heterocyclic ring and therefore, exist as geometric isomers resulting from the arrangement of substituents around the ring. The arrangement of substituents around a carbocyclic or heterocyclic ring are designated as being in the "Z" or "E" configuration wherein the terms "Z" and "E" are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting carbocyclic or heterocyclic rings encompass both "Z" and "E" isomers. Substituents around a carbocyclic or heterocyclic rings may also be referred to as "cis" or "trans", where the term "cis" represents substituents on the same side of the plane of the ring and the term "trans" represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated "cis/trans."

Individual enantiomers and diastereomers of contemplated compounds can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, (3) direct separation of the mixture of optical enantiomers on chiral liquid chromatographic columns or (4) kinetic resolution using stereoselective chemical or enzymatic reagents. Racemic mixtures can also be resolved into their component enantiomers by well known methods, such as chiral-phase liquid chromatography or crystallizing the compound in a chiral solvent. Stereoselective syntheses, a chemical or enzymatic reaction in which a single reactant forms an unequal mixture of stereoisomers during the creation of a new stereocenter or during the transformation of a pre-existing one, are well known in the art. Stereoselective syntheses encompass both enantio- and diastereoselective transformations, and may involve the use of chiral auxiliaries. For examples, see Carreira and Kvaerno, Classics in Stereoselective Synthesis, Wiley- VCH: Weinheim, 2009.

The isomers resulting from the presence of a chiral center comprise a pair of non-superimposable isomers that are called "enantiomers." Single enantiomers of a pure compound are optically active, i.e., they are capable of rotating the plane of plane polarized light. Single enantiomers are designated according to the Cahn-Ingold-Prelog system. The priority of substituents is ranked based on atomic weights, a higher atomic weight, as determined by the systematic procedure, having a higher priority ranking. Once the priority ranking of the four groups is determined, the molecule is oriented so that the lowest ranking group is pointed away from the viewer. Then, if the descending rank order of the other groups proceeds clockwise, the molecule is designated as having an (R) absolute

configuration, and if the descending rank of the other groups proceeds counterclockwise, the molecule is designated as having an (S) absolute configuration. In the example in the Scheme below, the Cahn-Ingold-Prelog ranking is A > B > C > D. The lowest ranking atom, D is oriented away from the viewer.

(R) configuration (S) configuration

A carbon atom bearing the A-D atoms as shown above is known as a "chiral" carbon atom, and the position of such a carbon atom in a molecule is termed a "chiral center." Compounds of the invention may contain more than one chiral center, and the configuration at each chiral center is described in the same fashion.

The present invention is meant to encompass diastereomers as well as their racemic and resolved, diastereomerically and enantiomerically pure forms and salts thereof.

Diastereomeric pairs may be resolved by known separation techniques including normal and reverse phase chromatography, and crystallization.

"Isolated optical isomer" or "isolated enantiomer" means a compound which has been substantially purified from the corresponding optical isomer(s) of the same formula.

Preferably, the isolated isomer is at least about 80%, more preferably at least 90% enantiomerically pure, even more preferably at least 98% enantiomerically pure, most preferably at least about 99% enantiomerically pure, by weight. By "enantiomeric purity" is meant the percent of the predominant enantiomer in an enantiomeric mixture of optical isomers of a compound. A pure single enantiomer has an enantiomeric purity of 100%.

Isolated optical isomers may be purified from racemic mixtures by well-known chiral separation techniques. According to one such method, a racemic mixture of a compound of the invention, or a chiral intermediate thereof, is separated into 99% wt.% pure optical isomers by HPLC using a suitable chiral column, such as a member of the series of

DAICEL^® CHIRALPAK^® family of columns (Daicel Chemical Industries, Ltd., Tokyo, Japan). The column is operated according to the manufacturer's instructions.

Another well-known method of obtaining separate and substantially pure optical isomers is classic resolution, whereby a chiral racemic compound containing an ionized functional group, such as a protonated amine or carboxylate group, forms diastereomeric salts with an oppositely ionized chiral nonracemic additive. The resultant diastereomeric salt forms can then be separated by standard physical means, such as differential solubility, and then the chiral nonracemic additive may be either removed or exchanged with an alternate counter ion by standard chemical means, or alternatively the diastereomeric salt form may retained as a salt to be used as a therapeutic agent or as a precursor to a therapeutic agent.

Description

The known compound (LP10) is a mixture of two racemic diastereomers: S- and R- isomers at the tryptophan unit and cis/trans-isomers within the methylcyclohexane ring. We began by identifying the most active enantiomer of the LP 10 scaffold as a starting point for structure optimization. To eliminate the impact of cis/trans isomerization in the

methylcyclohexane ring, we evaluated two enantiomerically pure LP 10 analogs with a simple cyclohexyl unit (Table 1). The S-isomer 1 has ~30-fold better binding affinity (KD) toward T. cruzi CYP51 than the R-isomer 2. This result is consistent with the co-crystal structures of LP9 (the methionine analog of LP 10) and of LP 1 1 (the valine analog of LP 10) with

AftCYP51 , which have S-enantiomers in the bound structures.^13a In addition, the S-isomer 1 had two-fold higher potency (EC50) against T. cruzi in infected cells than the R-isomer. Both isomers had similar microsome stability and both were potent inhibitors of human CYP enzymes 2C9, 2D6 and 3A4 (>90% inhibition at 10 uM). Accordingly, we first pursued S- enantiomers of LP 10 analogs in the development of non-azole CYP51 leads.

Table 1. Biochemical and cell-based activities, microsome stability and CYP inhibition properties of LP 10 and analogs 1 and 2

Stability of compounds in human (h) and mouse (m) liver microsomes, using sunitinib

Reference compounds for microsome stability and human CYP inhibition

^CKD of <5 nM (a hundredth of the target concentration) was estimated from the titration curves at 0.5 μΜ 7cCYP51 for the tightest binding inhibitors, if a plateau was reached at the stoichiometric enzyme-inhibitor ratio.

dValues in parentheses are % inhibition of the indicated human CYPs at 1 μΜ.

At the outset of these studies, the x-ray structure of 7cCYP51 with bound LP 10 was not available. Thus, design of initial analog sets was guided by our previous studies on AftCYP51 demonstrating that the hydrophobic contacts experienced by the indole ring accounts for ca. 100-fold increased binding affinity of LP 10 toward 7cCYP51 (KD < 42 nM), compared to LP9 and LP 1 1 (KD = 6,900 and 9,200 nM, respectively) which have smaller (methylthio) ethyl and isopropyl groups (methionine and valine side chains), respectively.^13a First, we explored the role of the aromatic nitrogen atom in drug-target interactions. Introduction of a methoxy substituent at the 2-position of the 4-acylaminopyridine unit (3a in Table 2) resulted in loss of binding affinity toward 7cCYP51 ; 3a was also inactive against T. cruzi in infected cells even at 10 μΜ, the highest concentration tested. Similarly, 3b with a 3,5-dimethylisoxazole unit as a pyridine replacement did not bind to CYP51 and was not active against the parasite in cell-based assays. These results indirectly confirm the binding mode of LP 10 that requires coordination of the pyridine nitrogen to the heme iron. Thus, a functional group that hinders the pyridine nitrogen will destabilize the interaction with the heme iron, resulting in a loss of biochemical and cell-based activity. Consistent with this analysis, the ability of 3a and 3b to inhibit human CYP enzymes was also significantly decreased compared to LP 10.

Second, analogs with secondary or tertiary amine units, such as 3c, 3d, 3e, and 3f, were also found to have substantially reduced biochemical and cell-based potency, presumably because they are unable to bind in the 7cCYP51 active site which accommodates the highly lipophilic natural substrate, lanosterol. However, the microsome stability of 3c, 3d, 3e, and 3f was significantly increased (30- 120 min half-life), and inhibition of human CYP enzymes by these compounds was greatly decreased relative to LP 10. Therefore, balancing the charge or polarity distribution of inhibitor analogs was viewed as an important factor to address in the development of more active analogs.

Third, the indole ring of LP 10 was replaced with several isosteres. While 3g and 3i possessing 3-benzothiophene and N-methyl indole have similar binding affinity to CYP51 as compared to 1 (KD <5 nM), analogs 3h and 3j possessing 1 -naphthyl and 5-hydroxy indole units have substantially decreased binding affinity (KD = 91 and 220 nM, respectively). In contrast, introduction of larger hydrophobic substituents at the 5-position of the indole (as in analog 3k) did not reduce binding affinity to 7cCYP51, suggesting that room in the binding site is available to accommodate the bulky substituents at the 5-position of the indole ring (discussed subsequently in a context of the x-ray structure) without the loss of binding affinity and inhibition potency.

Table 2. Biochemical and cell-based activities, microsome stability and CYP inhibition properties of inhibitors 3.^a

aSee notes to Table 1

bn/b: no binding at 10 μΜ

cn/e: not effective at 10 μΜ

Finally, in order to explore the site of cyclohexane ring binding in 7cCYP51 , the cyclohexanecarboxamide unit was replaced with other aliphatic carboxylamides containing a terminal phenyl ring. We found that analogs 3m - 3r have similar binding affinity to 7cCYP51 and comparable inhibition potency against T. cruzi in infected cells, compared to those of 1. However, all of these non-azole inhibitors were rapidly degraded by liver microsome preparations in our standard stability assay using 1 mg/mL hepatic microsomal protein (ti/₂ = < 6 min).

Based on the results of this early stage SAR effort (Table 2, above), plans to increase inhibitor potency of analogs of 1 were formulated and included strategic decisions to: (1) retain the 4-acylamimopyridine moiety as a heme binding unit; (2) retain the indole ring or extend the 5-position of the indole; and, most importantly, (3) to identify replacements for the methylcyclohexane ring to increase microsome stability and decrease inhibition of drug- metabolizing CYP enzymes.

Enantiomeric pure 1 was synthesized by the sequence summarized in Scheme 1. Briefly, the S-enantiomer of N-Boc tryptophan (L-tryptophan) was coupled with 4- aminopyridine to produce 5. Treatment of 5 with 4N HCl in dioxane followed by treatment of the deprotected amine with cyclohexanecarbonyl chloride produced enantiomerically pure 1. A similar sequence, starting from R-N-Boc tryptophan, was used to synthesize 2 (not shown). Analogs 3a and 3b were synthesized by replacing 4-aminopyridine with 4-amino-2- methoxypyridine and 4-amino-3,5-dimethylisoxazole, respectively. Acylation of 6 with N- Boc-isonipecotic acid followed by treatment with TFA to effect deprotection of the Boc unit provided 3c, isolated as the HC1 salt. Amine salt 3f was obtained by the alkylation of 6 and benzyl bromide. Various carboxylic acids were coupled with 6 using pentafluorophenyl trifluoroacetate as the dehyrdrating agent¹² to generate 3d, 3e, and 3m - 3r. Replacements for the indole moiety of 1 were explored by using commercially available 5-hydroxytryptophan 10 as a starting material. The carboxyl and amine groups were blocked as the methyl ester and t-butyl carbamate (Boc), respectively, and 11 was treated with benzyl bromide and CS2CO3 in acetone to produce 12. Hydrolysis of the methyl ester unit of 12 produced 13, from which 3k was obtained by following the procedure for the synthesis of 1.

Cyclohexyl ring replacement.

Based on the initial SAR/SPR analysis, a series of inhibitors were synthesized by using various carboxylic acids to replace the cyclohexylcarboxamide unit of LP 10 (Scheme 2 and Table 3). The objective was to increase microsome stability and selectivity against human CYP enzymes, while retaining or increasing inhibition potency against T. cruzi in infected cells. First, a benzamide was used to replace the cyclohexylcarboxamide moiety of 1. The binding affinity (KD = <5 nM), T. cruzi inhibition potency (EC50 = 0.39 nM), microsome stability and inhibition of human CYPs of 14a were similar to those of 1 (Table 3, below). The successful replacement of the cyclohexylcarboxamide encouraged us to explore additional substituted benzamide derivatives. Because the phenyl ring of the benzamide is a potential site of metabolism by human CYP enzymes, we anticipated that substitution at the appropriate "soft sites" of the aryl ring could block metabolic oxidation reactions and lead to inhibitors with enhanced microsome stability, while at the same time exploring available space in the active site and hopefully enhancing inhibitor potency against CYP51.

Table 3. Biochemical and cell-based activities, microsome stability and CYP inhibition roperties of inhibitors 14.^a

aSee notes to Table 1

CYP inhibition for this compound was performed at 10 μΜ Fluoro, chloro, bromo and other substituents were added to the benzamide unit in attempts to block the potential soft site(s) on the phenyl ring of 14a. Of the set of inhibitors 14b - 14k that were synthesized in this effort, several had increased activity against T. cruzi in cell culture (14b, 14h, 14j, 14k) and while retaining microsome stability. Acyl groups with larger naphthyl and biaryl units were used in attempts to further improve the microsome stability (141 - 14t). Gratifyingly, many of these inhibitors had increased microsome stability, while retaining inhibitor potency. For instance, substitution of a fluorine or a bromine at the 6-position of the naphthyl ring led to 14n and 14o with enhanced microsome stability compared to 1. Analogs with biaryl units also had enhanced microsome stability (14p, 14r, 14s, and 14t) compared to 1 and 14a. Of these, analog 14t (CYP-I-181) was of particular interest as it displayed good inhibition potency (EC50 = 0.19 μΜ) against T. cruzi in infected cells and moderate (but improved) microsome stability (ti/2 = 17/25/36 min) against human/rat/mouse liver microsomes.

X-ray structure of 14t complexed with TbCYP51.

Although inhibitor design ultimately targeted 7cCYP51, the best co-crystals with 14t (CYP-I- 181) that diffracted to 2.67 A were obtained for the 7¾CYP51 ortholog (85% sequence identity). The majority of the first tier active site residues are identical between these two parasite CYP51 enzymes, with the exception of substrate-specific F105, which is isoleucine in the T. cruzi counterpart. With this difference, the structural information obtained for the 7¾CYP51- 14t complex facilitated further rounds of structure-based design of 7cCYP51 inhibitors.

Inhibitor 14t bound in the active site of CYP51 has several interesting features. First, the pyridine nitrogen of the 4-acylaminopyridine unit is coordinated to the heme iron (Fig 2), as expected from the series of the co-structures for the LP 10 analogs bound to M YP51--. Second, the indole ring of 14t (PDB small molecule code 181) occupies the hydrophobic area enclosed largely by the heme macrocycle and the p-electron rich residues Y103, Ml 06, Fl 10, Yl 16, F290 plus A287 (Fig. 2A); this is the same area where the 2,4-difluorophenyl unit of posaconazole binds (Fig. 2B). Variable F105 is >5 A away from the indole ring and within 4 A of the carbonyl group adjacent to the biaryl moiety of 14t, suggesting additional hydrophobic contacts with the inhibitor in 7¾CYP51 which are missing from T. cruzi ortholog. Last, the biaryl ring of 14t projects towards the solvent exposed area, as does the tail part of the posaconazole,™ but via a different hydrophobic tunnel between the FG-loop (residues 205-210) and the hairpin of the two-stranded b-sheet at the protein C-terminus (residues 459-461) (Fig. 2C).

A hydrophobic cavity accommodating the indole ring of 14t extends further toward Fl 10 (Fig. 3 A) to provide space sufficient to bind a substituted benzyl ring of NEE (PDB ID 4H60) (Fig. 3B ) or a rigid biaryl moiety of VNF (PDB ID 3KSW) (Fig. 3C). In addition to Fl 10, the cavity is enclosed by Yl 16, providing stacking interactions with the 4-chloro-3,5- dimethylbenzyl unit of NEE, as well as by the aliphatic hydrophobic residues Al 15, M123, L127, L130, A287, and hydrophilic neutral Q126. Presumably, the bulky substituents at the 5-position of the indole ring in analog 3k bind in this cavity.

Synthesis of aryl carboxylic acids.

The biaryl carboxylic acid intermediates were prepared by palladium-mediated coupling reactions of commercially available 4-bromo-2-fluorobenzoic acid 15 with various aryl boronic acids (Scheme 3, below). This reaction was performed under microwave irradiation (100 °C for 1 h) and provided products 16 in >90% yields. Intermediate 17 was obtained by the Heck reaction of 15 and 1 -chloro-4-vinylbenzene in the presence of

Pd(OAc)2. Nucleophilic aromatic substitution of 15 with aniline yielded biarylamine 19. Phenylacetylene was coupled with ester 18 to provide the biaryl acetylene 21a, which was hydrolyzed under basic conditions to afford 21b. Treatment of methyl 4-bromo-2- fluorobenzoate (18) with morpholine or N-Boc-piperazine at 50° C in toluene gave 22 and 23; by-products were obtained when this reaction was performed at higher temperature (ca. 100 °C). The N-Boc protecting group of 23 was removed by treatment with TFA. Aryl sulfonylamide 25b and N-benzylpiperazine 26b were obtained by the reactions of amine 24 with benzenesulfonyl chloride and benzyl bromide followed by ester hydrolysis under basic conditions, respectively. All benzoic acid derivatives were coupled with indole derivative 6 to generate the inhibitors 27 (Table 4) by using the reaction conditions in Scheme 2. Table 4. Biochemical and cell-based activities, microsome stability and CYP inhibition properties of inhibitors 27.^a

Values in parentheses are % inhibition of the indicated human CYPs at 10 μΜ.

Structure and property guided optimization of 14t.

The terminal phenyl ring of 14t (CYP-I- 181) was extensively modified since it is oriented toward the solvent accessible area and opportunities existed to enhance microsome stability and minimize inhibition of human CYP enzymes through such modifications.

Indeed, the substituted biaryl derivatives 27a - 27n exhibited enhanced microsome stability, while retaining their inhibition of T. cruzi in cell-based assays. It should be noted that all these compounds had low nM affinity for 7cCYP51 (Table 4, above). Of particular interest is that the potency of 27i in the cell-based T. cruzi assay was increased 50-fold (EC50 = 0.014 μΜ) compared to that of LP 10. Furthermore, 27i exhibited substantially diminished inhibition of human CYP enzymes (10/91/38/69% inhibition of human CYP1A2/2C9/2D6/3A4 at 1 μΜ). The microsome stability of analog 27k significantly increased (ti/2 = 34/125/83 min, for human, rat and mouse liver microsomes, respectively), and its inhibition of human CYP enzymes was also significantly decreased (% inhibition = 0/85/16/21% at 1 μΜ), while retaining sub-micromolar potency against T. cruzi in infected cells (EC50 = 0.23 μΜ).

Lastly, the potency, microsome stability, and CYP selectivity of several aminoaryl- containing analogs was assessed. The morpholinoaryl and sulfonylpiperazine derivatives 27q (CYP-II-5) and 27r (CYP-II-23) showed excellent anti-Γ. cruzi potency (EC50 = 0.057 and 0.018 μΜ, respectively), but were moderately stable only in rat liver microsomes (ti/2 = 19 min). Interestingly, the inhibition potency of amine salt 27s was slightly improved compared to that of LP 10. As discussed previously, the amine salts 3c - 3f lost binding affinity and inhibition potency due to the conflict between the polar ammonium ion and the hydrophobic active site of 7cCYP51. However, based on the co-crystal structure of 14t and 7¾CYP51 (Fig. 3), the polar ammonium ion of 27s can be oriented toward the solvent accessible area (Fig. 4). The microsome stability of 27s was also significantly increased, particularly in rat and mouse liver microsomes (ti/2 = 36 and 41 min respectively), and inhibition of human CYPs was notably decreased. Finally, the inhibition potency of 27t (CYP-II- 168), which also possesses a basic amine, was ca. 20-fold increased (EC50 = 0.039 μΜ) compared to LP 10; 27t was also fairly stable in rat and mouse liver microsomes (ti/2 = 26 and 20 min, respectively) and exhibited good selectivity toward human CYP enzymes (0/87/51/61% inhibition at 1 μΜ).

Binding poses of inhibitors 27.

Conditions under which X-ray crystal structures were obtained are shown in Table 5 and described in greater detail in the Examples section, below.

Table 5. Data collection and refinement statistics

Protein T. brucei CYP51

Inhibitor 14t (Small molecule code 181)

PDB ID 4BJK

Data collection

Space group C2

Cell dimensions

a, b, c (A) 199.3, 114.7, 136.22

a, β, r(°) 90.0, 135.5, 90.0

Molecules in AU 4

Wavelength 1.11587

Resolution (A) 2.67

Or Emerge (%) 8.6 (88.8)^d

II al 7.7 (1.4)

Completeness (%) 99.9 (99.9)

Redundancy 4.1 (4.1)

Crystallization 12% pentaerythritol propoxylate (5/4 PO/OH)

conditions 50 niM HEPES, pH 7.5

50 mM KC1

10% Jeffamine 600

Refinement

No. reflections 60761

iiwork / i&ree (%) 19.4/27.4

No. atoms

Protein 14083

Heme 172

Ligand 100

Solvent 136

Mean B value 66.1

5-factors

Protein 67.1

Heme 54.8

Ligand 84.9 Solvent 54.8

R.m.s deviations

Bond lengths (A) 0.011

Bond angles (°) 1.515

dValues in parentheses are for highest-resolution.

All potent inhibitors 27 in Table 4 were docked in the 3D structure of 7cCYP51, generated from the x-ray co-crystal structure of 7¾CYP51 complexed with 14t, by using

Glide XP mode.²² In the results of docking studies, the terminal 3-fluoro-4-hydroxylphenyl ring of 271 (Fig 4A) and the protonated secondary amine of the piperazine unit of 27s (Fig

4B) are oriented towards the solvent accessible area. This analysis suggested that unfilled space near the phenolic hydroxyl group of 271, could be filled by other substituents, as is the case with O-methyl (in 27i) and O-benzyl (in 27k) derivatives. This speculation is consistent with the excellent potency of benzenesulfonamide (27r) and benzylamine (27t) substituted inhibitors against T. cruzi, both of which have relatively large groups that may project into the unfilled region identified above (Fig. 4C, D).

The non-azole, indolylpyridinecarboxamide-based CYP51 inhibitor LP 10, identified by HTS, was shown in prior studies to have moderate potency (EC50 = 0.65 μΜ) against T. cruzi in cell culture and to be effective in an acute mouse model of T. cruzi infection (60% cure rate).¹⁴ Accordingly, LP 10 was selected as the starting point for hit- to-lead optimization, with the objective of improving activity against T. cruzi in cell culture, as well as improving microsome stability and enhancing selectivity against the human CYPs 1A2, 2C9, 2D6 and 3A4. A series of first-generation biaryl inhibitors (e.g., 14t) were synthesized and shown to have improved microsome stability and enhanced in vitro inhibitor potency (Table 2). The x-ray co-crystal structure of 7¾CYP51 with bound 14t (CYP-I-181) was determined and employed in structure-based design of the next round of CYP51 inhibitors. Several potent inhibitors such as 27i (CYP-II-9), 27q (CYP-II-5), 27r (CYP-II-23), and 27t (CYP-II-168) (EC50 = 14, 57, 18 and 39 nM against T. cruzi) were developed and had the same or better microsome stability compared to LP 10, as well as enhanced selectivity against human CYPs.

The microsome stability of many other inhibitors containing biaryl units, particularly 14t, 27k, 271, 27p, and 27s, was improved, as was the selectivity of 27k and 27s when tested against the battery of human CYPs. However, 14t, 27k, 271, and 27p were only moderately more potent against T. cruzi in cell culture than LP 10 (EC50 = 190 to 470 nM).

Especially noteworthy is that the binding mode of 14t in the co-crystal structure with

7¾CYP51 is similar to that of posaconazole with the exception that the biaryl unit of 14t extends towards the solvent accessible area though a different hydrophobic tunnel than used by posaconazole (Fig. 2B,C). The indole ring of 14t occupies the same hydrophobic cavity as the 2,4-difluorophenyl moiety of posaconazole. The cavity extends beyond these groups along the heme macrocycle and has sufficient space to accommodate an alkoxy group attached to C5 of the indole nucleus (inhibitor 3k).

X-ray crystallographic data were used to guide the development of a series of potent, selective N-indolyl-oxopyridinyl-4-aminopropanyl D-tryptophan-derived inhibitors of T. cruzi CYP51 with in vivo activity. Initially, the S-isomers of LP 10 analogs were explored because of better binding affinity and potency compared to those with R-configuration at the tryptophan center. From an in-depth comparison of the binding modes of the (S)-enantiomer

- 10, CYP-I- 181 :

ole in the active site of CYP51 , we identified critical features that were used to design the next generation of improved CYP51 inhibitors. Specifically, although the biaryl unit of CYP-I- 181 and the tail portion of posaconazole are oriented toward a solvent-accessible area, they protrude through different hydrophobic tunnels (Fig. 5). An ample void space surrounding the biaryl unit of the (S)-enantiomer CYP-I- 181 or the "tail" of posaconazole suggested the possibility of a different binding

(R)-enantiomer CYP-II-34:

CYP-II-34 was synthesized starting from D-tryptophan, and its biological properties were assessed. The potency of CYP-II-34 as an inhibitor of T. cruzi in infected cells was observed to increase remarkably relative to CYP-I-181, from EC50 of 1.3e-6 M to 2.2e-9 M. Compound CYP-II-34 also retained microsome stability and had an acceptable profile for inhibition of human CYPs (14/91/30/60% inhibition of CYP1A2/2C9/2D6/3A4 at 1 μΜ).

The x-ray co-crystal structure of the (R)-enantiomer CYP-II-34 with T. cruzi CYP51 was determined to a resolution of 3.1 A (PDB ID 4BY0), indicating that binding of the 4- acylaminopyridine and indole moieties of the R-enantiomer are same as for the (S)- enantiomer CYP-I-181, but the biaryl unit of the (R)-enantiomer is oriented toward the hydrophobic tunnel accommodating the tail of posaconazole (Fig. 6). CYP-II-34 is distinguished by an L-shape in the active site of T. cruzi CYP51, while the (S)-enantiomer CYP-I-181 shape is more linear (Fig. 6A). The different binding modes of the (S)-enantiomer CYP-I- 181 and the (R)-enantiomer CYP-II-34 affect the conformations of interacting amino acid residues, thus changing the landscape of the CYP51 binding site. The entrance from the solvent-accessible area remains open when the (S)-enantiomer is bound, partially due to flexibility of the M460 side chain (which is not resolved in the x-ray structure of T. brucei CYP51 complexed with the (S)-enantiomer (Fig. 5B). When the (R)-enantiomer is bound, the interactions between M460 and the central phenyl ring of the inhibitor are established and partially block the entrance to the active site. Improved stability of the drug-target complex is consistent with the increased potency of the (R)-enantiomer in inhibiting T. cruzi growth in mammalian cell culture (Fig. 6B).

The resolution of the T. cruzi CYP51 co-crystal structures was improved to 2.84 A by ed CYP-II-34, trifluorinated CYP-II-64:

(PDB ID 4BMM), which showed binding features similar to that of the (R)-enantiomer CYP- II-34. Based on this higher resolution structure, hydrophobic amino acids V213, L357, M358, and M360 are seen to surround the terminal phenyl ring of difluoro (R)-enantiomer CYP-II-34 or trifluoro (R)-enantiomer CYP-II-64, allowing enough space to introduce additional binding-enhancing substituents (Fig 6C). In addition, the terminal phenyl ring faces the solvent accessible area through the hydrophobic tunnel where posaconazole also binds.

Table 6 provides a summary of X-ray crystallographic data obtained in this crystal structure determination.

Table 6. Data collection and refinement statistics

Protein T. cruzi CYP51 T. cruzi CYP51

Inhibitor (R)-2 (Small molecule code 5PS) (R)-3 (Small molecule code TU1)

PDB ID 4BY0 4BMM

Data collection

Space group P3₂21 C2

Cell dimensions

a, b, c (A) 124.2, 124.2, 119.8 272.5, 66.5, 122.2

α, β, Π 90.0, 120, 90.0 90.0, 110.7, 90.0 Molecules in AU

Wavelength

Resolution (A)

Ι/ σΙ

Completeness (%)

Redundancy

Crystallization M ammonium sulfate M ammonium acetate

conditions M Bis-Tris, pH M Bis-Tris, pH

PEG PEG

Refinement

No. reflections

No. atoms

Protein

Heme

Ligand

Solvent

Mean B value

-factors

Protein

Heme

Ligand

Solvent

R.m.s deviations

Bond lengths (A)

Bond angles (°)

Values in parentheses are for highest-resolution.

Inhibitor (R)-enantiomer CYP-II-34 was further optimized to increase potency against T. cruzi in infected cells. Thus, a flexible piperazine ring was introduced at the position of the terminal phenyl ring of CYP-II-34 to probe binding interactions in the solvent accessible area. Finally, we decided to introduce additional substituents on the terminal phenyl ring of CYP-II-34 to fill the hydrophobic pocket elucidated by the crystal structures shown in Figure 7, below.

Therefore, the (R)-enantiomers CYP numbers 11-64, II- 1 11, 11-123, 11- 142, 11- 154, and 11-158 were synthesized (Scheme 4, below), and their biological properties were evaluated (Table 7).

The new inhibitors exhibit similar inhibition potency against T. cruzi in infected cells, compared to (R)-enantiomer CYP-II-34. In addition, CYP-II-1 1 1, CYP-II-123, and CYP-II- 154 have substantially improved microsome stability, and are weaker inhibitors of human CYP enzymes except for CYP 2C9.

Table 7: Stability and Activity versus Human CYP for Selected (RVenantiomers Compound Stability Stability Stability % Inhib. % Inhib. % Inhib. % Inhib.

CYP # Ti/2(min) Ti/2(min) Ti/2(min) @ 1 μΜ @ 1 μΜ @ 1 μΜ @ 1 μΜ human rat mouse CYP CYP CYP CYP

1A2 2C9 2D6 3A4

11-64 17 17 22 3 92 47 74

11-1 1 1 18 22 39 -21 90 48 48

11-123 33 31 51 -16 92 14 2

11-142 6 18 20 5 95 52 84

11-154 15 39 67 -14 89 19 48

11-158 64 18 13 -13 76 14 67

Stability of compounds in human (h), rat (r) and mouse (m) liver microsomes as evaluated compared to the Sunitinib reference.

The efficacy of the five of these new highly potent, metabolically stable and selective inhibitors derived from 11-64 was assessed in a 4-day dosing mouse model of infection with the transgenic T. cruzi luc strain expressing firefly luciferase. Briefly, mice were infected with T. cruzi for three days, and starting on day 4 the infected mice were treated with 40 mg/kg of test compounds via intraperitoneal injection for four consecutive days b.i.d. At day 7 post- infection, T. cruzi luminescent signal in the mice was read. It was found that the parasite load in the untreated animals significantly increased. Posaconazole used as a positive control produced >99% inhibition of parasitemia. The parental hit, LP 10, showed little efficacy under these treatment conditions, while the new, rationally designed analogs 11- 123, 11-142, and 11-154 suppressed parasite load by >97% over the 4-day treatment period. The compounds II-l 1 1 (84%) and 11-158 reduced the infection by 84% and 52% respectively over the 4-day treatment period..

Table 8, below provides a summary of the EC50 values, % inhibition in vivo, stability, and % inhibition of human CYPs at 1 μΜ concentration for the (R)-enantiomer II- 34 and the seven related compounds 11-64, II-l 1 1, 11-123, 11- 142, 11- 154, and 11-158 discussed above, compared to LP 10. The stability of compounds in human (h), rat (r), and mouse (m) liver microsomes, was determined using Sunitinib as a reference control.

The oral bioavailability of selected hits was assessed in the 4-day dosing mouse model of T. cruzi infection. 25 mg/kg of test compounds re-suspended in 20% HP CD were administered b.i.d. by oral gavage to reach a cumulative daily dose of 50 mg/kg. The compounds potency was evaluated following oral (o.p.) administration. Compounds were administered as suspension in 20% 2-hydroxypropyl- -cyclodextrin (HP CD) (VWR International) or as solution in Kolliphor HS 15 (Sigma #42966), also known as solutol. Only one compound, CYP-II-258, was fully soluble in ΗΡβΟϋ, while all dissolved in 20% solutol upon overnight incubation. Treatment with compounds started same day, in groups of five mice, and specified doses were administered twice a day (b.i.d.) for four consecutive days. Benznidazole at 50 mg/kg administered b.i.d. was used as a positive control and produced >99% inhibition of parasitemia under these treatment conditions. The luciferase activity associated with the transgenic T. cruzi parasites was monitored in live animals by direct count of photons upon injection of D-luciferine. Compounds 11-123, 11-142, and 11-154, showed >95% inhibition by i.p. administration. When administered orally, compound II- 154 was inactive, whereas 11-123 and 11-142 inhibited T. cruzi proliferation by 95.6% and 77.3%, respectively (Table 8, below). Thus, compound 11- 123 was equally potent by oral and intraperitoneal administration. The two most active hits belonged to distinct sub-scaffolds, referred as sub-scaffold A and sub-scaffold B. Both sub-scaffolds retained the invariant N- indolyl-oxopyridinyl-4-aminopropanyl portion of the skeleton while carrying a variable substituent at the chiral carbon center. The sub-scaffold A carried a biaryl (biphenyl) moiety, while a piperazine ring separated two aromatic rings of the biaryl structure in sub-scaffold B.

Table 8: In vitro properties and in vivo efficacy of hits in the 4-day mouse model upon intraperitoneal (i.p.) or oral (p.p.) administrations

Stability of compounds in human (h), rat (r), and mouse (m) liver microsomes, using Sunitinib as a reference control.

N/D not determined

N/A not applicable

Six analogs derived from the two most active hits by further halogenation of the terminal phenyl ring were orally bioavailable and more potent than the immediate precursors (Table 9). Between these six analogs, sub-scaffold A had a notably longer half-life and less inhibition of human CYPs. With the exception of CYP2C9, which was inhibited 90-96% by all the compounds, inhibition of three other human CYPs by sub-scaffold A dropped largely below 30%.

The plasma concentration-time curves following oral administration of a single 50 mg/kg dose orally in 20% HP CD were obtained for the five hits which suppressed parasite load in mice >98%. These compounds had notably different pharmacokinetic behavior. Compound 11-259 had the longest half-life (6.9 h) followed by 11-258 (5.2 h). The highest maximum plasma concentration was achieved by compounds 11-251 and 11-258 followed by 11-259. These three compounds had the highest AUC values and the lowest clearance. The shortest half-life, lowest Cmax and highest clearance were observed for 11-257.

Each measurement is an average of 5 mice; p.o. = oral administration

"Each measurement is an average of 3 mice

"Stability of compounds in human (h), rat (r), and mouse (m) liver microsomes, using Sunitinib as a reference control. N/D not determ ined

Tissue distribution was assessed for all five hits after 2 h and 8 h of exposure following oral administration. Compounds 11-251 , 11-257 and 11-259 accumulated in intestines at high concentrations, particularly 11-259, whose intestine concentration remained high even after 8 h exposure (Table 10).

Table 10. Tissue distribution

Formulation: 50mg/kg in 10 mg/ml suspension in 20% HP CD Given that no non-dissolved materials were observed in the intestines, compounds likely permeated the mucosa of the gut from the apical to the basolateral side. Compounds II- 250, 11-251 , 11-258, and 11-259 penetrated lever, lung and heart tissues. Compounds 11-251 , II- 258, and 11-259 were also detected in skeletal muscles after 8 h exposure. Compound 11-257 efficiently crossed the blood-brain barrier where it was detected after 2 h exposure.

The T. cruzi parasites in chronic infection reside intracellularly, largely in heart, gut and skeletal muscles making it important for anti-Chagas drugs to be lipophilic enough to penetrate cell membrane and deep tissues. On the other hand, poor water solubility of a drug leads to ineffective absorption. To identify the optimal formulation for long-term dosing in mice, both HP CD and solutol were tested side by side for oral administration of 250 and 259. The difference between two vehicles was profound for 11-250, which was more active at all concentrations if administered in solutol (Fig. 8). The potency of 11-259 was comparable in both vehicles. Dose-response curves were obtained for the four most potent inhibitors: Compounds 11-250, 11-251, 11-258 and 11-259 (Fig. 9). The highest dose of 50 mg/kg b.i.d. was the most efficient for all four compounds. Table 1 1 : Compounds containing sub-scaffold with sulfonyl group

control. NE = not effective.

The x-ray structures at resolution of 2.0 A determined in this work provided atomic details of drug-target interactions for two N-indolyl-oxopyridinyl-4-aminopropanyl compounds containing piperazine ring in the structure of the longest substituent. One is a low nanomolar orally bioavailable hit, compound 11-259, (R)-N-(3-(lH-indol-3-yl)- l -oxo- l - (pyridin-4-ylamino)propan-2-yl)-4-(4-(2,4-difluorophenyl)piperazin- l -yl)-2- fluorobenzamide. The other is a hit belonging to a sulfonyl-containing subset of the R- stereoisomers synthesized and tested over the course of hit- to-lead optimization (Table 1 1, above).

While many of the compounds in the sulfonyl subset were inactive in the cell-based assay, compound 11-71, 2-fluoranyl-N-[(2R)-3-(lH-indol-3-yl)- l-oxidanylidene- l -(pyridin-4- ylamino)propan-2-yl]-4-(4-thiophen-2-ylsulfonylpiperazin- 1 -yl)benzamide, had impressive low nanomolar EC50. Being shorter than posaconazole (43), both inhibitors fit entirely within the protein interior. In both complexes, the pyridinyl moiety of inhibitor coordinated to the heme iron and indole ring pointed at the heme macrocycle. In contrast to the S-stereisoomer, whose longest substituent binds within the a-domain, in the R-configuration of both compound 11-259 and 11-71 it protruded deeply into the β-domain establishing multiple contacts with the hydrophobic residues (Fig. 10). Flexibility of the piperazine ring allowed each inhibitor to fit the CYP51 binding site while the fit-induced protein conformations enhanced the drug-target interactions.

Remarkably, fit-induced conformational changes at the domain interface propagated -20 A through protein scaffolding to affect binding of the invariant portion of the inhibitor at the opposite end of the molecule. In the 11-259 complex, the indole ring adopted a single well- defined conformation, tightly enclosed by the π-electron rich BC-loop residues Y 103, M106, Fl 10 and Yl 16. In the 71 complex, repositioning of the bulky aromatic first tier side chains of Y103, Fl 10, and Yl 16, forced by the long-range propagation of the conformational changes caused ambiguity in the indole ring binding manifested in flipped alternative conformations (Fig. 12B). These allosteric modulations should be taken into account as a factor affecting SAR.

The piperazine ring of the variable substituent at the chiral carbon center faces the cleft between the a- and β-domains and is the only part of the inhibitor molecule loosely surrounded by protein amino acid residues. The latter allows piperazine to flex in an opposite direction in the complex of compound 11-71 to accommodate a -90° turn in the molecular skeleton enabled by the sulfonyl group pointing into the cleft between the a- and β-domains (Fig. 12). This positions thiophene moiety of compound 11-71 at -45° to the difluorophenyl ring of 259 (Fig. I OC), thus pushing the FG-loop away from the A-helix to accommodate the shape. In contrast, the 259 substituent extends flat along the β-sheet saddle, serving as a latch fastening the cleft and holding both domains together. At the end of the constricted channel, the 4-fluoro substituent of the terminal 2,4-difluorophenyl ring is at van der Waals distances of 170 and 172, and within 5 A of V77, 179 and F55. The 2-fluoro substituent is within 5 A of 145 and F48, all residues are part of the β-domain. Meantime, the 2-fluoro substituent of the benzamide ring in both inhibitors points toward a crevice formed by the a-domain residues Y103, 1105, Ml 06 and M480 residing at van der Waals distances of Y 103 and 1105. The 2- fluoro substituent was retained earlier in the hit-to-lead optimization for the increased half- life of hits in the macrosome stability assays.

Attenuated potency against pathogenic fungi.

Most of the amino acid residues concentrated around the invariant N-indolyl- oxopyridinyl-4-aminopropanyl part of the skeleton are either invariable, Y103 or Fl 10, or highly conserved across the phyla. This suggests that the N-indolyl-oxopyridinyl-4- aminopropanyl unit loaded with different substituents could be utilized in variety of CYP51 targets. On the other hand, the terminal difluorophenyl ring of 259 binds in the cavity formed by the residues least conserved across the CYP51 protein family compared to the rest of the 259 binding site constituted by the 24 exclusively hydrophobic amino acid residues (Fig. 12), aliphatic or aromatic: invariant, Y103, Fl 10; conserved across the phyla, F48, 172, Yl 16, A291, T295, L356, M460; Substrate-specific, 1105; Phylum-specific, F55, 179, M106, P210, V213, F290; more variable, 145, 170, V77, F214, A287, M358, M360, V461. This lack of conservation suggests that inhibitors tailored to fit the T. cruzi cavity would likely be T.cruzi specific, and that related inhibitors could be designed and/or selected from the broad genus to target organisms having differing cavity structures.

In concert with the lack of sequence conservation is attenuated potency of the selected 4-pyridinyl-based inhibitors, II- 181 , II- 142, II- 154 and 11-250, tested in vitro against a panel of pathogenic fungi including Candida parapsilosis, Candida krusei, Paecilomyces variotii, Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Cryptococcus neoformans, Rhizopus oryzae, and Scedosporium apiospermum. Being highly potent against T. cruzi, all hits inhibited fungi selectively and rather modestly, compared to the anti- fungal azoles used as positive controls. Two hits with different steric configurations at the chiral carbon, 11-181 (S) and 11-142 (R), were equipotent in inhibiting Candida parapsilosis and Candida krusei. Two R-configuration hits, 11-142 and 11-250, inhibited Candida albicans, while a single hit, II- 154, showed inhibition against Blastomyces dermatitidis. Compound II- 250 was the only hit tested against the Pneumocystis carinii and Pneumocystis murina strains, where it was found to inhibit both fungi > 97% after 48 h exposure at 100 μg/ml. In the drug discovery pipeline of The University of Texas Health Science Center, where the test was performed, 11-250 would be selected for IC50 determination and later toxicity testing should the compound produce an IC50 below 10μg/ml.

Using x-ray crystallography, we characterized the binding modes of two R-isomers from the scaffold family, one of them a low nanomolar and orally bioavailable hit, to a resolution of 2.0 A. X-ray data were obtained using the parameters shown in Table 12.

Table 12: Data collection and refinement statistics

Protein 7cCYP51 7cCYP51

PDB ID 2YMC 4C0C

Small molecule ID T9H WVH

Data collection

Space group P2i2i2i P6₃22

Cell dimensions

a, b, c (A) 79.2, 96.4, 137.1 128.5, 128.5, 116.7

α, β, Π 90, 90, 90 90, 120, 90

Molecules in AU 2 1

Wavelength 1.11587 1.11587

Resolution (A) 2.08 2.04 Or (%) 14.7 (88.7)¹ 9.2 (123.9)

I/ al 9.2 (1.8) 14.6 (1.5)

Completeness (%) 95.6 (76.0) 99.9 (99.2)

Redundancy 7.2 (4.9) 11.0 (7.1)

Crystallization 0.05 M ammonium citrate, pH 7.0 0.4 M ammonium sulfate

conditions 14% PEG 3350 0.1 M Bis-Tris, pH 5.0

19% PEG 3350

Refinement

No. reflections 57134 34827

17.4/24.3 19.9/25.4

No. atoms

Protein 7084 3540

Heme 86 43

Inhibitor 88 44

Solvent 676 125

Mean B value 25.1 43.5

S-factors

Protein 24.7 43.8

Heme 20.9 28.3

Inhibitor 17.4 37.7

Solvent 32.5 44.6

R.m.s deviations

Bond lengths 0.022 0.018

Bond angles (°) 2.012 1.963

'Values in parentheses are for highest-resolution

Induced fit demonstrated by the two low nanomolar CYP51 inhibitors in this work indicates that target flexibility should be taken into account as a factor affecting SAR.

Binding mode of the variable longest substituent created ambiguity in binding of the invariable indole ring in 11-71, which, as demonstrated previously (11-34), contributes an order of magnitude into hit binding affinity. This is in contrast to 11-259 which apparently has little space for additions to the skeleton (Fig. 12C). In addition to its near-perfect binding mode, compound 11-259 is characterized by oral bioavailability, relatively long terminal half- life, slow clearance and efficient distribution into the tissues. However, a clear drawback of the sub-scaffold B analogs, including compound 11-259, is attenuated stability, particularly in the human microsome fraction

Table 13, below, shows structures of specific compounds prepared and tested according to the methods described herein.

40

41

r- s

₀₀₆. o

S _{004 ±.}

s so

Q> Pi

OT

¾

G

m C

<= a

53

57

65

7|

73

74

76

79

80 Table 14: Biological Activity of Preferred Compounds of the Invention

% % Microsomal %inhibition of human stability⁶, CYPs^f

tm (min) at 1 μΜ

R^a=

b.i.d., b.i.d., h r m 1A2 2C9 2D6 3A p.o.^c p.o.^d

11-259 29±8 90.2±9.5 N/D^h 10 19 22 9 96 87 87

11-257 17±1 87.4±14.9 N/D 7 18 38 20 94 82 90

11-271 9.9 ± 6.0 93.7±4.0 N/D 7 24 26 10 90 88 86

11-269 92 ±8 96.8±1.8 N/D 6 29 46 6 86 84 82

11-275 N/D 5 13 17 1 93 50 74

III-118 8 37 7 89 77 76

III-119 16 39 -14 82 45 60

III-145 N/D 13 13 13 98 80 87

11-277 1 1 33 35 -1 1 69 44 65

11-276 9 30 28 -1 1 83 45 76

11-278 9 20 38 -3 84 63 69

11-279 6 22 26 8 90 77 88

11-270 0 17±9 80.3±9.6 N/D 7 22 14 -1 93 53 82 ^aTerminal N-phenyl rings of the compounds are depicted in orientations deduced based on the x-ray structure analysis and SAR. ^bEach measurement performed in triplicate (see SI); ^cEach measurement is an average of 5 mice treated 25 mg/kg (20% Kolliphor), p.o., b.i.d., for 4 days; ^dEach measurement is an average of 5 mice treated 10 mg/kg (20% Kolliphor), p.o., b.i.d., for 4 days; Stability of compounds in human (h), rat (r) and mouse (m) liver

microsomes as evaluated compared to the Sunitinib reference; inhibition of CYPs as

evaluated in human liver microsomes using selective marker substrates for each CYP; ^gThere was one outlier in this group with no effect of the compound (signal comparable to untreated controls); ^hN/D - not determined.

Table 15: PK parameters for compounds 11-277 and 11-276

aEach measurement is an average of three mice received a singe 25 mg/kg dose of test compound at 5 mg/ml suspension in 20% Kolliphor.

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30. Pettersen, E. F.; Goddard, T. D.; Huang, C. C; Couch, G. S.; Greenblatt, D. M.; Meng, E. C; Ferrin, T. E., UCSF Chimera— a visualization system for exploratory research and analysis. J Comput. Chem. 2004, 25, 1605-1612. Examples

Scheme 1

3f 3d, 3e, 31, 3m, 3n, 3o, 3p, 3q, and 3r

13 3k Reagents and conditions: (a) PyBOP, HOBt, NEt3, CH2CI2, 4-aminopyridine, 0 °C to room temp., lh, 94%. (b) 4N HC1 in dioxane, dioxane, room temp., 12h, >90% (crude) (c) cyclohexancarbonyl chloride, NEt3, CH2CI2, 0 °C to room temp., lh, 94%. (d) PyBOP, HOBt, NEt3, CH2CI2, 4-amino-2-methoxypyridine or 4-amino-3,5-dimethylisoxazole, 0 °C to room temp., lh, 84%. (e) pentafluorophenyl trifluoroacetate, 1 -Boc-isonipecotic acid, NEt3, CH2CI2, 0 °C to room temp., lh, 53%. (f) trifluoroacetic acid, CH2CI2, room temp., lh, 47%. (g) benzylbromide, NEt3, CH2CI2, room temp., 12h, 30%. (h) pentafluorophenyl trifluoroacetate, NEt3, CH2CI2, alkyl carboxylic acids, 0 °C to room temperature, lh, -80%. (i) SOCI2, CH3OH, 0 °C to room temp., 12h, then (Boc)₂0, NEfe, CH2CI2, 0 °C to room temp., 6h, 82%. (j) benzylbromide, CS2CO3, acetone, room temp., 12h, 77%. (k) 10% NaOH, CH3OH, 0 °C, 2h, 97%. (1) H2, Pd/C, CH3OH/THF, room temp., 24h, 29%.

Scheme 2

14

(a) PyBOP, HOBt, NEt3, CH2CI2, benzoic acids or naphthoic acids, 0 °C to room temp., lh, typically 80%.

cheme 3

(a) Aryl boronic acid, 5 mol% Pd2(dba , 10 mol% PCy3, 2M K3PO4, dioxane, 100 °C (microwave), lh, -90%. (b) 1 -chloro-4-vinylbenzene, 5 mol% Pd(OAc)2, 10 mol% P(o- tolyl)₃, EtaN, DMF, 100 °C (microwave), 2h, 73%. (c) SOCk, CH3OH, room temp. 12 h,

96%. (d) aniline, 5 mol% Pd(OAc)₂, 10 mol% BI AP, CS2CO3, toluene, 100 °C (microwave), 2h, 73%. (e) 10% NaOH (aq), CH3OH, 50 °C, lh, 91%. (f) ethynylbenzene, 5 mol%

Pd(OAc)2, 10 mol% BINAP, EtsN, Cul, toluene, 110 °C (microwave), 2h, 77%. (g) N-boc- piperazine or morpholine, Pd(OAc)2, P(o-tolyl)3, CS2CO3, toluene, 50 °C, 48h, 91%. (h) trifluoroacetic acid, CH2CI2, room temp. 1 h, >90% (crude), (i) phenylsulfonyl chloride,

Et3N, CH2CI2, 0 °C to room temp, lh, 81%. (j) 4-fluoro-benzyl bromide, Et3N, CH2CI2, room temp, lh, 88%. cheme 4

(a) Aryl boronic acid, 5 mol% Pd2(dba , 10 mol% PCy3, 2M K3PO4, dioxane, 100 °C (microwave), lh, >90% (b) FfeSC MeOH (2/1), 70 °C, 24 h, 92% (c) H₂ (balloon), Pd/C, MeOH/THF, room temp., 24 h, 92% (d) 4-fluorobenzyl bromide, K2CO3, acetone, 70 °C, 5 h, 95% (e) 10% NaOH (aq), MeOH/THF, 60 °C, 3 h, 95% (f) acetic anhydride, EfeN, CH2CI2, 0 °C to room temp., lh, 84%. (g) l-(3-fluorophenyl)piperazine, Pd(OAc)2, P(o-tolyl)3, CS2CO3, toluene, 60 °C, 48 h, 86% (h) PyBOP, HOBt, Et₃N, CH2CI2, room temp., lh, >90%. Chemistry, General Methods.

All reaction solvents were purified before use. Dichloromethane, tetrahydrofuran, dimethylformamide and toluene were purified by passing through a column of activated A- 1 alumina. All other reagents purchased from commercial suppliers were used as received. All reactions sensitive to moisture or oxygen were conducted under an argon atmosphere using flame-dried (under vacuum) or oven-dried (overnight) glassware. Removal of solvents was accomplished by using a rotary evaporator under reduced pressure in a water bath below 35 °C, followed by exposure to high vacuum using a vacuum pump. Microwave assisted reactions were performed using a Biotage® Initiator microwave reactor.

Proton nuclear magnetic resonance (¾ NMR) spectra and carbon (¹³C) NMR spectra were recorded on a commercially available NMR spectrometer at 400 MHz and 100 MHz, respectively. The proton signal for non-deuterated solvent (δ 7.26 for CHCb or δ 2.50 for DMSO) was used as an internal reference for ¾ NMR chemical shifts. Coupling constants (J) are reported in Hertz (Hz). ¹³C chemical shifts are reported relative to the δ 77.16 resonance of CDCb or the δ 39.52 resonance of DMSO-d6.

Analytical thin layer chromatography (TLC) was performed using glass plates precoated with a 0.25-mm thickness of silica gel. The TLC plates were visualized with UV light. Column chromatography was performed using a Biotage® Isolera flash purification system using Biotage® SNAP HP-SIL cartridge (30 μηι silica, 10 g to 100 g size). Unless noted otherwise, all compounds isolated by chromatography were sufficiently pure by ¾ NMR analysis for use in subsequent reactions. Polar compounds were purified using preparative high performance liquid chromatography (HPLC) using SunFire column (30 mm x 250 mm) with a linear gradient elution at 60 mL/min.

The purity of all final compounds (typically >96%) was assayed at 254 nm wavelength by using analytical HPLC (Varian 1 100 series) on a reverse phase ZORB AX

Eclipse XDB-C18 column (4.6 x 150 mm, 5 μηι). A linear gradient elution ranging from 2% to 98% CHsCN and H₂0 (containing 0.1% TFA and 1% CH3CN) at 1.5 mL/min was used. Compounds were lyophilized before dissolution in DMSO to give 10 mM stock solutions for use in biochemical and cell-based assays.

Synthesis of inhibitors 1. 2. 3. 14 and 27 by acylation of trytophan pyridinyl carboxamide (6). General Procedure A.

To a solution of a substituted benzoic or naphthoic acid (ca.1.2 eq), PyBOP (ca. 1.4 eq) and HOBt (ca. 10 mol%) in dry CH2CI2 (5 mL) was slowly added triethylamine (ca. 4 eq.) at ambient temperature over 15 min. After the reaction mixture became homogenous, 6 was added, and the reaction mixture was stirred at room temperature for 1 h. After completion of the reaction as determined by TLC analysis, the solvent was removed under reduced pressure. The crude mixture was dissolved in ethyl acetate (10 mL) and was washed with saturated aqueous NaHCCte (2 mL x 2) and brine (2 mL x 2). The organic layer was concentrated in vacuo and directly subjected to purification by flash chromatography to provide the amide products in ca. 80% yield.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)cyclohexanecarboxamide, (1). To a solution of 6 (0.1 12 g, 0.353 mmol) in CH2CI2 (10 mL) were added

cyclohexanecarbonyl chloride (0.06 mL) and (iPr)2EtN (0.1 mL) at 0 °C. After 10 min, the reaction mixture was warmed to ambient temperature and stirred for 1 h. After completion of the reaction monitored by TLC, ethyl acetate (40 mL) was added to the crude mixture, which was washed with saturated aqueous NaHCCb (10 mL x 2) and brine (10 mL x 2). The organic layer was dried over magnesium sulfate, filtered, and concentrated under reduced pressure. Purification of the crude product by flash chromatography provided 1 as a light yellow solid (0.103 g, 0.263 mmol, 75%). ¾ NMR (400 MHz, DMSO-de) δ 10.81 (d, J = 2.5 Hz, 1H), 10.56 (s, 1H), 8.50 - 8.38 (m, 2H), 8.06 (d, J = 7.6 Hz, 1H), 7.73 - 7.55 (m, 3H), 7.31 (d, J = 8.1 Hz, 1H), 7.15 (d, J = 2.3 Hz, 1H), 7.04 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 7.00 - 6.89 (m, 1H), 4.67 (td, J = 8.4, 5.7 Hz, 1H), 3.22 - 2.94 (m, 2H), 2.19 (ddd, J = 1 1.1, 7.7, 3.2 Hz, 1H), 1.61 (dt, J = 41.4, 10.3 Hz, 5H), 1.20 (dddd, J = 29.7, 15.4, 12.1, 5.8 Hz, 5H). ¹³C NMR (101 MHz, DMSO-de) δ 175.36, 172.31, 149.72, 146.03, 135.97, 127.19, 123.64, 120.88, 1 18.50, 1 18.16, 113.42, 1 1 1.24, 109.67, 54.21, 43.45, 29.09, 28.98, 27.58, 25.42, 25.21, 25.16. MS (ESI) 391 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-((2-methoxypyridin-4-yl)amino)-l-oxopropan-2- yl)cyclohexanecarboxamide (3a). Compound 3a was obtained by following the procedure for the synthesis of 1 (69%) as a yellow solid. ¾ NMR (400 MHz, CDCb) δ 8.78 (s, 1H), 8.20 (s, 1H), 7.95 (d, J = 5.7 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.23 - 7.15 (m, 1H), 7.14 - 7.05 (m, 1H), 7.02 (d, J = 2.3 Hz, 1H), 6.89 (d, J = 1.8 Hz, 1H), 6.78 (dd, J = 5.7, 1.9 Hz, 1H), 6.36 (d, J = 7.3 Hz, 1H), 4.93 (q, J = 7.0 Hz, 1H), 3.88 (s, 3H), 3.42 - 3.16 (m, 2H), 2.13 - 2.06 (m, 1H), 1.87 - 1.55 (m, 5H), 1.48 - 1.03 (m, 5H). ¹³C NMR (101 MHz, CDCb) δ 177.24, 170.87, 165.37, 147.42, 146.95, 136.35, 127.39, 123.32, 122.63, 120.07, 1 18.83, 1 1 1.49, 110.41, 108.43, 99.77, 54.54, 53.73, 45.26, 29.61, 29.53, 27.76, 25.74, 25.68, 25.63. MS (ESI) 421 m/z [M + H]⁺.

(S)-5-Bromo-N-(l-((3,5-dimethylisoxazol-4-yl)amino)-3-(lH-indol-3-yl)-l-oxopropan-2-yl)- 2-fluorobenzamide (3b). Compound 3b (75%) was obtained as a white solid by following the general procedure A with 5-bromo-2-fluorobenzoic acid. ¾ NMR (400 MHz, DMSO-d6) δ 10.90 (d, J = 2.5 Hz, 1H), 9.48 (s, 1H), 8.71 (dd, J = 7.3, 2.3 Hz, 1H), 7.82 - 7.67 (m, 2H), 7.64 (d, J = 7.9 Hz, 1H), 7.35 (dd, J = 8.1, 0.9 Hz, 1H), 7.29 (dd, J = 10.0, 8.6 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 7.07 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.98 (ddd, J = 7.9, 6.9, 1.0 Hz, 1H), 4.80 (td, J = 7.8, 6.5 Hz, 1H), 3.39 - 3.09 (m, 2H), 2.12 (s, 3H), 1.95 (s, 3H). ¹³C NMR (101 MHz, DMSO-de) δ 170.77, 162.42, 162.36, 159.74, 157.49, 157.25, 136.09, 135.13, 132.41, 132.38, 127.26, 125.69, 125.53, 123.89, 120.97, 1 18.77, 1 18.53, 1 18.44, 1 18.31, 1 15.91, 115.88, 1 13.94, 1 1 1.34, 109.48, 54.59, 27.43, 10.57, 9.22. MS (ESI) 499/501 m/z [M + H]⁺. (S)-N-(3-(lH-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)piperidine-4-carboxamide hydrochloride (3c). To a solution of 9 (0.12 g, 0.25 mmol) in CH2CI2 (10 mL) was added trifluoroacetic acid (0.5 mL), and the reaction mixture was stirred for 1 h at room

temperature. After removing solvent under reduced pressure, the reaction mixture was directly subjected to HPLC, and the product 3c (58 mg, 0.15 mmol, 47%) was obtained as a white solid. ¾ NMR (400 MHz, DMSO-de) δ 11.44 (s, 1H), 10.90 (d, J = 2.5 Hz, 1H), 8.80 (s, 1H), 8.68 - 8.61 (m, 2H), 8.49 (d, J = 7.3 Hz, 1H), 8.00 - 7.93 (m, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.36 - 7.23 (m, 2H), 7.21 (d, J = 1.9 Hz, 2H), 7.18 - 6.91 (m, 3H), 4.72 (ddd, J = 9.0, 7.3, 5.5 Hz, 1H), 3.30 - 3.15 (m, 2H), 3.09 (dd, J = 14.6, 9.0 Hz, 1H), 2.95 - 2.73 (m, 2H), 1.85 (dd, J = 14.4, 3.8 Hz, 1H), 1.79 - 1.51 (m, 3H). ¹³C NMR (101 MHz, DMSO-de) δ 173.41, 173.12, 158.33, 158.02, 151.00, 144.33, 136.02, 127.09, 123.92, 120.95, 1 18.24, 115.59, 1 14.26, 1 1 1.33, 109.20, 54.90, 42.28, 38.28, 27.28, 24.98. MS (ESI) 392.4 m/z [M + H]⁺.

(S)-N-(3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-l -methylpiperidine-4- carboxamide (3d). Compound 3d (47%) as a yellow solid was obtained by following the procedure for the synthesis of 9 with 1 -methylpiperidine-4-carboxylic acid. ¾ NMR (400 MHz, DMSO-de) δ 11.31 (s, 1H), 10.93 (d, J = 2.5 Hz, 1H), 8.67 - 8.44 (m, 3H), 7.84 (d, J = 6.0 Hz, 2H), 7.66 (d, J = 7.9 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 2.3 Hz, 1H), 7.08 - 6.99 (m, 1H), 6.94 (t, J = 7.3 Hz, 1H), 4.72 (q, J = 7.5 Hz, 1H), 3.44 - 3.27 (m, 2H), 3.27 - 3.02 (m, 2H), 2.99 - 2.76 (m, 2H), 2.66 (s, 3H), 2.04 - 1.57 (m, 5H). ¹³C NMR (101 MHz, DMSO-de) δ 173.03, 172.60, 148.52, 147.02, 139.68, 136.01, 127.21, 123.87, 120.91, 1 18.57, 118.22, 1 13.87, 1 1 1.32, 109.45, 108.75, 54.91, 52.45, 42.47, 38.41, 27.54, 25.67. MS (ESI) 406 m/z [M + H]⁺.

N-((S)-3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-l -methylpiperidine-3- carboxamide (3e). Compound 3e (light yellow, racemic mixture) was obtained by following the procedure for the synthesis of 9 with l-methylpiperidine-3-carboxylic acid (58%). ¾ NMR (400 MHz, DMSO-de) δ 11.57 (d, J = 6.3 Hz, 1H), 10.95 (dd, J = 14.9, 2.5 Hz, 1H), 8.75 (dd, J = 19.9, 7.4 Hz, 1H), 8.61 (dd, J = 6.1, 3.7 Hz, 2H), 7.96 (d, J = 5.9 Hz, 2H), 7.67 (d, J = 7.9 Hz, 1H), 7.45 (s, 1H), 7.37 - 7.28 (m, 2H), 7.27 - 7.16 (m, 2H), 7.1 1 - 6.99 (m, 1H), 6.95 (dd, J = 7.6, 4.8 Hz, 1H), 4.73 (t, J = 7.5 Hz, 1H), 3.40 - 2.72 (m, 6H), 2.67 (d, J = 4.2 Hz, 3H), 2.05 - 1.68 (m, 3H). ¹³C NMR (101 MHz, DMSO-de) δ 172.71, 158.41, 158.1 1, 150.31, 145.03, 136.03, 136.00, 127.15, 124.02, 120.91, 1 18.69, 1 18.57, 1 18.28, 1 15.71, 114.18, 1 14.12, 1 11.35, 109.26, 109.20, 55.10, 53.74, 52.86, 42.79, 27.24, 25.46, 22.52, 21.87. MS (ESI) 406 m/z [M + H]⁺.

(S)-2-((2-Fluorobenzyl)amino)-3-(lH-indol-3-yl)-N-(pyridin-4-yl)propanamide

hydrochloride (3f). To a solution of 6 (0.107 g, 0.338 mmol) in CH2CI2 were added Et3N (0.05 mL) and 2-fluorobenzylbromide (0.06 mL), and the reaction mixture was stirred at room temperature for 12 h. Solvent was removed by using rotary evaporator, and the product mixture was directly subjected to HPLC. The product 3f (38.7 mg, 0.0997 mmol, 30%) was obtained as a white solid. ¾ NMR (400 MHz, DMSO-de) δ 10.86 (d, J = 2.5 Hz, 1H), 8.94 (d, J = 8.3 Hz, 1H), 8.26 (dd, J = 7.5, 1.8 Hz, 1H), 8.12 (dd, J = 7.3, 1.9 Hz, 1H), 7.90 - 7.77 (m, 1H), 7.63 (d, J = 7.7 Hz, 1H), 7.51 - 7.42 (m, 1H), 7.42 - 7.21 (m, 6H), 7.18 (d, J = 2.4 Hz, 1H), 7.08 - 6.92 (m, 3H), 6.73 (dd, J = 7.6, 2.9 Hz, 1H), 5.39 (s, 2H), 4.51 (td, J = 8.6, 5.1 Hz, 1H), 3.44 - 2.98 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.66, 158.96, 156.65, 143.80, 141.67, 136.00, 131.30, 130.44, 130.41, 127.04, 125.12, 125.08, 124.07, 122.52, 120.95, 1 18.37, 1 15.92, 115.72, 1 1 1.32, 1 11.1 1, 109.13, 105.57, 56.65, 54.15, 28.01. MS (ESI) 389 m/z [M + H]⁺.

(S)-N-(3-(Benzo[b]thiophen-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2- yl)cyclohexanecarboxamide (3g). Compound 3g (68%) was obtained as a white solid by following the procedure for the synthesis of 1 with Boc-L-3-benzothienylalanine. ¾ NMR (400 MHz, DMSO-de) δ 10.58 (s, 1H), 8.54 - 8.37 (m, 2H), 8.23 (d, J = 7.9 Hz, 1H), 8.05 - 7.90 (m, 2H), 7.67 - 7.54 (m, 2H), 7.45 (s, 1H), 7.44 - 7.33 (m, 2H), 4.81 (ddd, J = 9.5, 7.9, 5.1 Hz, 1H), 3.46 - 3.05 (m, 2H), 2.17 (ddd, J = 10.9, 7.5, 3.5 Hz, 1H), 1.77 - 1.44 (m, 6H), 1.36 - 1.01 (m, 4H). ¹³C NMR (101 MHz, DMSO-de) δ 175.44, 171.62, 150.29, 145.48, 139.47, 138.62, 131.74, 124.27, 123.96, 123.89, 122.81, 121.99, 1 13.43, 52.91, 43.50, 30.44, 29.12, 28.89, 25.41, 25.22, 25.13. MS (ESI) 408.2 m/z [M + H]⁺.

(S)-N-(3-(Naphthalen-l-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2- yl)cyclohexanecarboxamide (3h). Compound 3h (67%) was obtained as a white solid by following the procedure for the synthesis of 1 with Boc-L-l-naphthylalanine. ¾ NMR (400 MHz, DMSO-de) δ 10.47 (s, 1H), 8.49 - 8.37 (m, 2H), 8.24 (t, J = 8.0 Hz, 2H), 7.91 (dd, J = 7.9, 1.4 Hz, 1H), 7.78 (dd, J = 7.5, 1.9 Hz, 1H), 7.64 - 7.46 (m, 4H), 7.46 - 7.32 (m, 2H), 4.82 (td, J = 8.5, 5.8 Hz, 1H), 3.61 - 3.26 (m, 2H), 2.16 (dtd, J = 15.7, 8.4, 7.6, 4.1 Hz, 1H), 1.73 - 1.41 (m, 5H), 1.36 - 0.95 (m, 5H). ¹³C NMR (101 MHz, DMSO-de) δ 175.39, 171.60, 150.31, 145.38, 133.33, 133.24, 131.67, 128.51, 127.29, 127.13, 126.07, 125.57, 125.22, 123.92, 1 13.48, 53.85, 43.47, 34.49, 29.14, 28.87, 25.41, 25.22, 25.12. MS (ESI) 402.3 m/z [M + H]⁺.

(S)-N-(3-(l -Methyl- lH-indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2- yl)cyclohexanecarboxamide (3i). Compound 3i (51%) was obtained as a yellow solid by following the procedures for the synthesis of 13 and 1 starting with 1 -methyl-L-tryptophan. Rf = 0.57 (10% MeOH in ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.46 (s, 1H), 8.51 - 8.33 (m, 2H), 8.04 (d, J = 7.8 Hz, 1H), 7.64 (d, J = 7.9 Hz, 1H), 7.60 - 7.51 (m, 2H), 7.35 (d, J = 8.2 Hz, 1H), 7.17 - 7.07 (m, 2H), 6.99 (t, J = 7.4 Hz, 1H), 4.66 (td, J = 8.2, 5.6 Hz, 1H), 3.70 (s, 3H), 3.21 - 2.95 (m, 2H), 2.29 - 2.14 (m, 1H), 1.77 - 1.49 (m, 5H), 1.40 - 1.01 (m, 5H). ¹³C NMR (101 MHz, DMSO-de) δ 175.37, 172.06, 150.30, 145.48, 136.45, 128.04, 127.53, 121.02, 118.80, 1 18.26, 1 13.35, 109.45, 109.13, 54.24, 43.45, 32.26, 29.05, 29.00, 27.50, 25.42, 25.20, 25.15. MS (ESI) 405 m/z [M + H]⁺.

(S)-N-(3-(5-Hydroxy-lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2- yl)cyclohexanecarboxamide (3j). To a solution of 3k (0.328 g, 0.66 mmol) in methanol (10 mL) was added 10% Pd/C (ca. 30 mg) at room temperature. After air was removed from the flask using a vacuum pump, hydrogen gas was introduced using a balloon. The reaction mixture was stirred for lh, and the flask was evacuated under vacuum and refilled with hydrogen gas. This procedure was repeated three times, and the reaction mixture was stirred overnight at ambient temperature. Palladium on carbon was removed by filtration through Celite pad. The filtrate was collected and evaporated to give the crude product, which was purified by flash chromatography to afford the product 3j as a brown solid (0.077 g, 0.19 mmol, 29%). ¾ NMR (400 MHz, DMSO-de) δ 10.47 (d, J = 3.6 Hz, 1H), 8.55 (s, 1H), 8.48 - 8.33 (m, 2H), 8.01 (d, J = 7.7 Hz, 1H), 7.66 - 7.49 (m, 2H), 7.09 (d, J = 8.5 Hz, 1H), 7.04 (d, J = 2.4 Hz, 1H), 6.93 (d, J = 2.2 Hz, 1H), 6.58 (dd, J = 8.6, 2.3 Hz, 1H), 4.63 (td, J = 8.2, 6.0 Hz, 1H), 3.15 - 2.83 (m, 2H), 2.19 (ddd, J = 14.4, 9.5, 3.5 Hz, 1H), 1.91 (s, 1H), 1.63 (dd, J = 32.3, 1 1.3 Hz, 5H), 1.38 - 1.00 (m, 5H). ¹³C NMR (101 MHz, DMSO-de) δ 175.33, 172.27, 150.30, 150.20, 145.50, 130.54, 127.96, 123.98, 1 13.36, 11 1.46, 1 1 1.24, 108.72, 102.55, 54.02, 43.46, 29.13, 28.99, 27.67, 25.43, 25.23, 25.17, 21.07. MS (ESI) 407 m/z [M + H]⁺. (S)-N-(3-(5-(Benzyloxy)-lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2- yl)cyclohexanecarboxamide (3k). The procedure for the synthesis of 1 was followed using 13 to provide 3k as a light yellow solid (52%, over 3 steps). Rf = 0.21 (100% ethyl acetate), Rf = 0.63 (10% MeOH in ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.67 (d, J = 2.6 Hz, 1H), 10.52 (s, 1H), 8.41 (d, J = 5.5 Hz, 2H), 8.08 (d, J = 7.8 Hz, 1H), 7.58 (d, J = 5.5 Hz, 2H), 7.46 (d, J = 7.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.32 (t, J = 7.2 Hz, 1H), 7.24 (d, J = 2.2 Hz, 1H), 7.21 (d, J = 8.7 Hz, 1H), 7.12 (d, J = 2.3 Hz, 1H), 6.77 (dd, J = 8.6, 2.3 Hz, 1H), 5.16 - 4.90 (m, 2H), 4.66 (td, J = 8.3, 5.4 Hz, 1H), 3.17 - 2.90 (m, 2H), 2.27 - 2.13 (m, 1H), 1.61 (dt, J = 32.1, 1 1.7 Hz, 5H), 1.36 - 1.00 (m, 5H). ¹³C NMR (101 MHz, DMSO-de) δ 175.32, 172.24, 152.06, 150.32, 145.51, 137.78, 131.34, 128.33, 127.65, 127.60, 124.45, 1 13.34,

11 1.80, 1 1 1.40, 109.58, 102.22, 69.90, 54.28, 43.51, 29.08, 29.00, 27.73, 25.43, 25.22, 25.18. MS (ESI) 497 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-4,4- difluorocyclohexanecarboxamide (31). The procedure for the synthesis of 9 was followed using 4,4-difluorocyclohexanecarboxylic acid to provide 31 as a light yellow solid (41%). Rf = 0.39 (100% ethyl acetate), ¾ NMR (400 MHz, DMSO-de) δ 10.81 (d, J = 2.4 Hz, 1H), 10.51 (s, 1H), 8.52 - 8.37 (m, 2H), 8.23 (d, J = 7.8 Hz, 1H), 7.63 (d, J = 7.8 Hz, 1H), 7.60 - 7.52 (m, 2H), 7.31 (dt, J = 8.1, 0.9 Hz, 1H), 7.15 (d, J = 2.3 Hz, 1H), 7.05 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 6.96 (ddd, J = 8.0, 6.9, 1.1 Hz, 1H), 4.71 (td, J = 8.4, 5.5 Hz, 1H), 3.24 - 2.94 (m, 2H), 2.42 - 2.29 (m, 1H), 2.07 - 1.37 (m, 8H). ¹³C NMR (101 MHz, DMSO-de) δ 173.93, 172.03, 150.33, 145.46, 135.99, 127.19, 123.65, 120.91, 1 18.52, 1 18.17, 1 13.36, 1 1 1.25, 109.63, 54.19, 40.44, 30.67, 27.65, 25.52, 25.43, 25.38, 25.29. MS (ESI) 427.1 m/z [M + H]⁺. (S)-3-( lH-Indol-3-yl)-2-(2-phenylacetamido)-N-(pyridin-4-yl)propanamide (3m). The procedure for the synthesis of 1 was followed using 2-phenylacetyl chloride to provide 3m as a yellow solid (61%). Rf = 0.30 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ

10.94 - 10.73 (m, 1H), 10.54 (s, 1H), 8.50 (d, J = 7.6 Hz, 1H), 8.46 - 8.31 (m, 2H), 7.63 (d, J = 7.9 Hz, 1H), 7.59 - 7.49 (m, 2H), 7.32 (d, J = 8.1 Hz, 1H), 7.29 - 7.09 (m, 6H), 7.05 (t, J = 7.5 Hz, 1H), 6.95 (t, J = 7.4 Hz, 1H), 4.72 (td, J = 8.3, 5.6 Hz, 1H), 3.46 (d, J = 2.2 Hz, 2H), 3.25 - 2.97 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.93, 170.16, 150.33, 145.42, 136.20, 136.02, 128.96, 128.08, 127.17, 126.21, 123.74, 120.90, 1 18.51, 1 18.22, 1 13.37, 11 1.26, 109.47, 68.24, 54.48, 41.85. MS (ESI) 399 m/z [M + H]⁺.

(S)-3-(lH-Indol-3-yl)-2-(3-phenylpropanamido)-N-(pyridin-4-yl)propanamide (3n). The procedure for the synthesis of 9 was followed using 3-phenylpropanoic acid to provide 3n as a yellow solid (73%). Rf = 0.33 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.81 (d, J = 2.5 Hz, 1H), 10.50 (s, 1H), 8.50 - 8.37 (m, 2H), 8.30 (d, J = 7.7 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.60 - 7.54 (m, 2H), 7.35 - 7.28 (m, 1H), 7.25 - 7.09 (m, 6H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.97 (ddd, J = 8.0, 6.9, 1.1 Hz, 1H), 4.72 (td, J = 8.2, 5.9 Hz, 1H), 3.25 - 2.92 (m, 2H), 2.74 (dd, J = 8.7, 6.8 Hz, 2H), 2.42 (dd, J = 8.8, 6.7 Hz, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 172.01, 171.53, 150.31, 145.46, 141.20, 136.02, 128.19, 128.14, 127.19, 125.79, 123.64, 120.91, 1 18.50, 1 18.21, 1 13.37, 1 1 1.27, 109.62, 54.38, 36.68, 30.96, 27.66. MS (ESI) 413 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-4-phenylbutanamide (3o). The procedure for the synthesis of 9 was followed using 4-phenylbutanoic acid to provide 3o as a yellow solid (67%). Rf = 0.33 (100% ethyl acetate). ¾ NMR (400 MHz, CDCb) δ 9.28 (s, 1H), 8.37 - 8.26 (m, 2H), 7.57 (dd, J = 8.0, 1.0 Hz, 1H), 7.38 - 7.34 (m, 2H), 7.32 (dt, J = 8.1, 1.0 Hz, 1H), 7.26 (s, 3H), 7.20 - 7.14 (m, 2H), 7.09 - 7.04 (m, 2H), 7.04 - 7.00 (m, 2H), 6.42 (d, J = 7.3 Hz, 1H), 4.96 (q, J = 7.1 Hz, 1H), 3.29 (dd, J = 7.0, 1.9 Hz, 2H), 2.54 (t, J = 7.6 Hz, 2H), 2.18 (td, J = 7.4, 2.0 Hz, 2H), 1.87 (p, J = 7.9 Hz, 2H). ¹³C NMR (101 MHz, CDCb) δ 174.04, 171.16, 149.27, 145.96, 141.20, 136.36, 128.60, 128.53, 127.32, 126.26, 123.40, 122.64, 120.09, 118.58, 1 14.05, 1 1 1.62, 1 10.09, 54.97, 35.73, 35.09, 27.78, 27.01. MS (ESI) 427 m/z [M + H]⁺.

(S)-3-(lH-Indol-3-yl)-2-(3-(4-methoxyphenyl)propanamido)-N-(pyridin-4-yl)propanamide (3p). The procedure for the synthesis of 9 was followed using 3-(4-methoxyphenyl)propanoic acid to provide 3p as a yellow solid (34%). ¾ NMR (400 MHz, CDCb) δ 8.98 (s, 1H), 8.42

- 8.33 (m, 2H), 8.33 - 8.24 (m, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.38 - 7.32 (m, 1H), 7.32 - 7.27 (m, 2H), 7.18 (ddd, J = 8.2, 7.0, 1.1 Hz, 1H), 7.07 (ddd, J = 8.1, 7.0, 1.0 Hz, 1H), 7.02 - 6.94 (m, 2H), 6.90 (d, J = 2.4 Hz, 1H), 6.75 - 6.70 (m, 2H), 6.40 (d, J = 7.4 Hz, 1H), 4.90 (q, J = 7.0 Hz, 1H), 3.71 (s, 3H), 3.34 - 3.12 (m, 2H), 2.88 - 2.74 (m, 2H), 2.46 (t, J = 7.7 Hz, 2H). ¹³C NMR (101 MHz, CDCb) δ 173.49, 170.91, 158.26, 149.75, 145.45, 136.33, 132.29, 129.35, 127.36, 123.40, 122.62, 120.08, 118.57, 1 14.14, 1 14.01, 11 1.59, 1 10.04, 55.38, 54.84, 38.40, 30.65, 27.40. MS (ESI) 443 m/z [M + H]⁺.

(S)-3-( lH-Indol-3-yl)-N-(pyridin-4-yl)-2-(3-(4-( trifluoromethyl)phenyl)propanamido)- propanamide (3q). The procedure for the synthesis of 9 was followed using 3-(4- (trifluoromethyl)phenyl)propanoic acid to provide 3q as a yellow solid (17%). ¾ NMR (400 MHz, CDCb) δ 8.71 (s, 1H), 8.41 - 8.34 (m, 2H), 8.29 (s, 1H), 7.59 (d, J = 7.8 Hz, 1H), 7.44 (d, J = 8.1 Hz, 2H), 7.36 (dt, J = 8.2, 0.9 Hz, 1H), 7.29 - 7.16 (m, 5H), 7.09 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H), 6.44 (d, J = 7.3 Hz, 1H), 4.90 (q, J = 7.2 Hz, 1H), 3.42

- 3.13 (m, 2H), 2.94 (t, J = 7.5 Hz, 2H), 2.51 (td, J = 7.5, 2.7 Hz, 2H). ¹³C NMR (101 MHz, CDCb) δ 172.70, 170.71, 149.97, 145.17, 144.48, 136.38, 128.74, 127.26, 125.66, 125.62, 125.58, 123.41, 122.77, 120.22, 1 18.55, 1 13.93, 1 1 1.68, 1 10.03, 54.90, 37.53, 31.13, 27.62. MS (ESI) 481 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-4-(4- fluorophenyl) butanamide (3r). The procedure for the synthesis of 9 was followed using 4-(4- fluorophenyl)butanoic acid to provide 3r as a solid (86%). Rf = 0.24 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.91 - 10.76 (m, 1H), 10.54 (s, 1H), 8.53 - 8.37 (m, 2H), 8.23 (d, J = 7.7 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.61 - 7.56 (m, 2H), 7.31 (d, J = 8.0 Hz, 1H), 7.17 (d, J = 2.3 Hz, 1H), 7.15 - 7.00 (m, 5H), 6.96 (t, J = 7.4 Hz, 1H), 4.73 (td, J = 8.5, 5.7 Hz, 1H), 3.23 - 2.93 (m, 2H), 2.44 (t, J = 7.6 Hz, 2H), 2.23 - 2.01 (m, 2H), 1.70 (p, J = 7.4 Hz, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 172.22, 172.05, 161.75, 159.35, 150.07, 145.72, 137.85, 137.82, 136.02, 130.01, 129.93, 127.17, 123.66, 120.91, 1 18.53, 1 18.19, 114.93, 1 14.72, 1 13.39, 11 1.27, 109.67, 54.36, 34.30, 33.54, 27.60, 26.99. MS (ESI) 445 m/z [M + H]⁺.

(S)-tert-Butyl (3-(lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)carbamate, (5). To a solution of N-Boc-L-tryptophan (1.0 g, 3.3 mmol), PyBOP (2.0 g, 3.9 mmol), and HOBt (0.29 g) in dry CH2CI2 (20 mL) was slowly added triethylamine (1.5 mL, ca. 4 eq.) at 0 °C, and the reaction mixture was stirred and warmed to ambient temperature for 15 min. After the mixture was cooled to 0 °C, 4-aminopyridine (0.39 g, 4.1 mmol) was added, and the reaction mixture was stirred at room temperature for 1 h. After completion of the reaction monitored by TLC, ethyl acetate (80 mL) was added to the crude mixture, which was washed with saturated aqueous NaHCCte (20 mL x 2) and brine (20 mL x 2). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification of the crude product by flash chromatography provided 0.98 g (94%) of 5 as a light yellow solid. Rf = 0.45 (100% ethyl acetate). ¾ NMR (400 MHz, CDCb) δ 9.07 (s, 1H), 8.86 (dd, J= 23.3, 1 1.0 Hz, 1H), 8.36 - 8.23 (m, 2H), 7.58 (d, J= 8.0 Hz, 1H), 7.31 (d, J= 8.3 Hz, 1H), 7.23 (d, J= 5.7 Hz, 2H), 7.14 (t, J= 7.7 Hz, 1H), 7.03 (t, J= 7.6 Hz, 1H), 6.98 (s, 1H), 5.57 (q, J= 7.5, 6.2 Hz, 1H), 4.67 (s, 1H), 3.40 - 3.15 (m, 2H), 1.39 (s, 9H). ¹³C NMR (101 MHz, CDCb) δ 171.75, 156.25, 150.26, 145.03, 136.47, 127.27, 123.51, 122.34, 1 19.79, 1 18.62, 1 13.90, 1 1 1.52, 109.94, 80.82, 56.04, 28.37, 22.61. MS (ESI) 381 m/z [M + H]⁺.

(S)-N-(3-( lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)benzamide (14a). The procedure for the synthesis of 1 was followed using benzoyl chloride to provide 14a as a solid (99%). Rf = 0.39 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.82 (d, J = 2.5 Hz, 1H), 10.64 (s, 1H), 8.74 (d, J = 7.5 Hz, 1H), 8.56 - 8.37 (m, 2H), 7.92 - 7.80 (m, 2H), 7.75 (d, J = 7.7 Hz, 1H), 7.67 - 7.61 (m, 2H), 7.58 - 7.39 (m, 3H), 7.32 (d, J = 8.1 Hz, 1H),

7.28 (d, J = 2.3 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.99 (ddd, J = 8.1, 6.9, 1.1 Hz, 1H), 4.89 (ddd, J = 9.1, 7.5, 5.6 Hz, 1H), 3.41 - 3.15 (m, 2H). ¹³C NMR (101 MHz, DMSO- de) δ 172.19, 166.50, 150.33, 145.55, 136.03, 133.75, 131.42, 128.19, 127.50, 127.15, 123.87, 120.95, 1 18.60, 118.22, 1 13.41, 1 11.32, 109.95, 55.28, 27.24. MS (ESI) 385 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-2-fluorobenzamide (14b). The procedure for the synthesis of 1 was followed using 2-fluorobenzoyl chloride to provide 14b as a light yellow solid (49%). Rf = 0.18 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.87 (d, J = 2.5 Hz, 1H), 10.63 (s, 1H), 8.50 (dd, J = 7.3, 3.6 Hz, 1H), 8.47 -

8.29 (m, 2H), 7.66 (d, J = 7.8 Hz, 1H), 7.60 (qd, J = 5.3, 1.8 Hz, 3H), 7.57 - 7.49 (m, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.31 - 7.21 (m, 3H), 7.06 (ddd, J = 8.0, 6.9, 1.2 Hz, 1H), 6.96 (ddd, J = 7.9, 6.9, 1.1 Hz, 1H), 4.89 (td, J = 8.1, 5.5 Hz, 1H), 3.40 - 3.1 1 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.52, 163.60, 160.61, 158.13, 150.37, 145.46, 136.06, 132.77, 130.33, 130.30, 127.17, 124.45, 124.41, 123.93, 123.09, 122.95, 120.97, 1 18.48, 1 18.25, 1 16.24, 116.02, 1 13.43, 1 1 1.33, 109.35, 55.08, 27.41. MS (ESI) 403 m/z [M + H]⁺.

(S)-N-(3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-3-fluorobenzamide (14c). The procedure for the synthesis of 1 was followed using 3-fluorobenzoyl chloride to provide 14c as a yellow solid (51%). Rf = 0.21 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.82 (d, J = 2.5 Hz, 1H), 10.66 (s, 1H), 8.88 (d, J = 7.5 Hz, 1H), 8.44 (d, J = 5.6 Hz, 2H), 7.74 (d, J = 7.8 Hz, 1H), 7.71 (dt, J = 7.7, 1.2 Hz, 1H), 7.69 - 7.63 (m, 1H), 7.63 - 7.59 (m, 2H), 7.51 (td, J = 8.0, 5.8 Hz, 1H), 7.38 (td, J = 8.3, 2.7 Hz, 1H), 7.31 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.02 - 6.94 (m, 1H), 4.89 (ddd, J = 9.4, 7.5, 5.4 Hz, 1H), 3.45 - 3.16 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.99, 165.18, 163.07, 150.26, 145.60, 136.03, 130.45, 127.13, 123.86, 123.72, 120.97, 1 18.59, 118.24, 1 13.45, 1 1 1.33, 109.86, 55.38, 27.22. MS (ESI) 403.2 m/z [M + H]⁺.

(S)-N-(3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-3-phenoxybenzamide (14d). The procedure for the synthesis of 9 was followed using 3-phenoxybenzoic acid to provide 14d as a white solid (84%). R_f = 0.60 (10% MeOH in ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.80 (d, J = 2.5 Hz, 1H), 10.63 (s, 1H), 8.82 (d, J = 7.6 Hz, 1H), 8.55 - 8.40 (m, 2H), 7.73 (d, J = 7.8 Hz, 1H), 7.65 (dt, J = 7.8, 1.2 Hz, 1H), 7.63 - 7.57 (m, 2H), 7.53 - 7.36 (m, 4H), 7.31 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 7.21 - 7.13 (m, 2H), 7.09 - 7.00 (m, 3H), 7.00 - 6.92 (m, 1H), 4.86 (ddd, J = 9.3, 7.5, 5.5 Hz, 1H), 3.32 - 3.1 1 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 172.06, 165.66, 156.62, 156.39, 150.31, 145.54, 136.01, 135.63, 130.15, 129.98, 127.12, 123.85, 123.73, 122.54, 121.68, 120.94, 1 18.73, 1 18.59, 1 18.22, 1 17.57, 113.42, 1 1 1.31, 109.91, 55.34, 27.18. MS (ESI) 477 m/z [M + H]⁺. (S)-N-(3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-4-

(trifluoromethyl)benzamide (14e). The procedure for the synthesis of 1 was followed using 4- trifluoromethylbenzoyl chloride to provide 14e as a white solid (50%). Rf = 0.27 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.81 (d, J = 2.5 Hz, 1H), 10.66 (s, 1H), 9.04 (d, J = 7.5 Hz, 1H), 8.58 - 8.27 (m, 2H), 8.05 (d, J = 8.1 Hz, 2H), 7.84 (d, J = 8.3 Hz, 2H), 7.74 (d, J = 7.6 Hz, 1H), 7.66 - 7.56 (m, 2H), 7.38 - 7.28 (m, 1H), 7.26 (d, J = 2.3 Hz, 1H), 7.06 (ddd, J = 8.2, 7.0, 1.3 Hz, 1H), 6.99 (ddd, J = 7.9, 6.8, 1.1 Hz, 1H), 4.92 (ddd, J = 9.3, 7.5, 5.4 Hz, 1H), 3.39 - 3.15 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.87, 165.37, 150.36, 145.48, 137.51, 136.03, 128.43, 127.14, 125.26, 123.82, 120.97, 1 18.58, 1 18.24, 1 13.44, 1 1 1.32, 109.84, 55.39, 27.25. MS (ESI) 453.3 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-4-methoxybenzamide (14f). The procedure for the synthesis of 1 was followed using 4-methoxybenzoyl chloride to provide 14f as a yellow solid (90%). ¾ NMR (400 MHz, DMSO-de) δ 10.81 (d, J = 2.5 Hz, 1H), 10.62 (s, 1H), 8.58 (d, J = 7.6 Hz, 1H), 8.47 - 8.37 (m, 2H), 7.90 - 7.81 (m, 2H), 7.74 (d, J = 7.8 Hz, 1H), 7.66 - 7.58 (m, 2H), 7.31 (dt, J = 8.1, 1.0 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.14 - 6.88 (m, 4H), 4.86 (ddd, J = 9.1, 7.5, 5.7 Hz, 1H), 3.80 (d, J = 2.8 Hz, 3H), 3.39 - 3.18 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 172.39, 165.96, 161.71, 150.34, 145.57, 136.02, 129.38, 127.16, 125.94, 123.85, 120.93, 1 18.61, 1 18.22, 1 13.39, 1 1 1.31, 1 10.02, 55.34, 55.25, 27.24. MS (ESI) 415 m/z [M + H]⁺.

(S)-N-(3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-2-chlorobenzamide (14g). The procedure for the synthesis of 1 was followed using 2-chlorobenzoyl chloride to provide 14g as a yellow solid (83%). Rf = 0.54 (10% MeOH in ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.95 - 10.79 (m, 1H), 10.59 (s, 1H), 8.85 (d, J = 7.6 Hz, 1H), 8.50 - 8.35 (m, 2H), 7.68 (d, J = 7.9 Hz, 1H), 7.62 - 7.56 (m, 2H), 7.50 - 7.40 (m, 2H), 7.40 - 7.28 (m, 3H), 7.24 (d, J = 2.3 Hz, 1H), 7.11 - 7.03 (m, 1H), 6.98 (t, J = 7.5 Hz, 1H), 4.90 (td, J = 8.5, 5.9 Hz, 1H), 3.42 - 3.08 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.53, 166.33, 150.35, 145.49, 136.16, 136.04, 130.90, 130.06, 129.59, 129.09, 127.19, 126.89, 123.84, 120.93, 1 18.53, 1 18.21, 1 13.40, 1 1 1.28, 109.57, 54.85, 27.39. MS (ESI) 419 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-2,5-difluorobenzamide (14h). The procedure for the synthesis of 1 was followed using 2,5-difluorobenzoyl chloride to provide 14h as a light yellow solid (50%). Rf = 0.39 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 12.31 (s, 1H), 1 1.00 (d, J = 2.5 Hz, 1H), 8.83 (dd, J = 7.0, 2.8 Hz, 1H), 8.72 (d, J = 7.0 Hz, 2H), 8.31 - 8.15 (m, 2H), 7.72 (d, J = 7.8 Hz, 1H), 7.47 - 7.27 (m, 5H), 7.10 - 6.98 (m, 1H), 6.93 (t, J = 7.4 Hz, 1H), 4.96 (ddd, J = 8.9, 7.0, 5.4 Hz, 1H), 3.50 - 3.18 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 172.85, 162.66, 158.86, 156.79, 156.46, 154.35, 152.93, 142.15, 136.07, 127.15, 124.34, 124.27, 124.18, 124.1 1, 120.98, 1 19.60, 1 19.51, 119.36, 1 19.27, 1 18.58, 118.30, 1 18.23, 1 18.06, 1 17.98, 1 16.53, 1 16.50, 1 16.28, 1 16.25, 114.62, 1 14.56, 1 1 1.38, 108.94, 55.95, 26.99. MS (ESI) 421 m/z [M + H]⁺.

(S)-N-(3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-2,4-difluorobenzamide (14i). The procedure for the synthesis of 1 was followed using 2,4-difluorobenzoyl chloride to provide 14i as a light yellow solid (68%). Rf = 0.39 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.93 - 10.83 (m, 1H), 10.65 (s, 1H), 8.54 (dd, J = 7.4, 3.2 Hz, 1H), 8.49 - 8.39 (m, 2H), 7.66 (dd, J = 8.3, 6.9 Hz, 2H), 7.63 - 7.55 (m, 2H), 7.34 (t, J = 8.6 Hz, 2H), 7.24 (d, J = 2.3 Hz, 1H), 7.17 (td, J = 8.5, 2.4 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 6.97 (t, J = 7.4 Hz, 1H), 4.88 (td, J = 8.2, 5.7 Hz, 1H), 3.53 - 3.08 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.48, 162.83, 162.22, 150.37, 145.45, 136.05, 131.99, 127.15, 123.92, 120.97, 1 19.77, 1 19.73, 1 18.47, 118.25, 1 13.43, 1 1 1.65, 1 1 1.33, 109.33, 104.59, 55.13, 27.40. MS (ESI) 421 m/z [M + H]⁺.

(S)-N-(3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-5-bromo-2- fluorobenzamide (14j). The general procedure A was followed using 5-bromo-2- fluorobenzoic acid to provide 14j as a light yellow solid (67%). ¾ NMR (400 MHz, DMSO- de^ 10.87 (d, J = 2.4 Hz, 1H), 10.63 (s, 1H), 8.74 (dd, J = 7.5, 2.3 Hz, 1H), 8.51 - 8.37 (m, 2H), 7.72 (ddd, J = 8.7, 4.4, 2.6 Hz, 1H), 7.69 - 7.63 (m, 2H), 7.62 - 7.56 (m, 2H), 7.33 (d, J = 8.1 Hz, 1H), 7.28 (dd, J = 10.0, 8.8 Hz, 1H), 7.22 (d, J = 2.4 Hz, 1H), 7.10 - 7.02 (m, 1H), 6.97 (t, J = 7.5 Hz, 1H), 4.88 (td, J = 8.3, 5.6 Hz, 1H), 3.39 - 3.07 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.33, 162.38, 150.37, 145.41, 136.03, 132.41, 127.17, 123.85, 120.98, 118.55, 1 18.48, 1 18.26, 115.94, 1 13.44, 1 11.33, 109.37, 55.16, 27.40. MS (ESI) 481/483 m/z [M + H]⁺.

(S)-N-(3-(lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-4-bromo-2- fluorobenzamide (14k). The general procedure A was followed using 4-bromo-2- fluorobenzoic acid to provide 14k as a yellow solid (70%). Rf = 0.30 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.86 (d, J = 2.3 Hz, 1H), 10.67 (s, 1H), 8.62 (dd, J = 7.4, 2.8 Hz, 1H), 8.51 - 8.36 (m, 2H), 7.70 - 7.63 (m, 2H), 7.63 - 7.58 (m, 2H), 7.51 (d, J = 6.4 Hz, 2H), 7.33 (d, J = 8.0 Hz, 1H), 7.22 (d, J = 2.3 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 6.96 (t, J = 7.4 Hz, 1H), 4.88 (td, J = 8.2, 5.6 Hz, 1H), 3.50 - 3.04 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.44, 162.92, 157.93, 150.14, 145.64, 136.04, 131.77, 127.70, 127.14, 123.90, 120.97, 1 19.41, 1 18.47, 118.25, 1 13.46, 1 11.33, 109.29, 55.14, 27.39. MS (ESI) 481/483 m/z [M + H]⁺.

(S)-N-(3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-2-naphthamide (141). The general procedure A was followed using 2-naphthoic acid to provide 14k as a yellow solid (24%). R_f = 0.30 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.83 (s, 1H), 10.67 (s, 1H), 8.91 (d, J = 7.5 Hz, 1H), 8.48 (s, 1H), 8.44 (d, J = 5.4 Hz, 2H), 8.12 - 7.87 (m, 4H), 7.78 (d, J = 7.8 Hz, 1H), 7.70 - 7.52 (m, 4H), 7.32 (d, J = 7.9 Hz, 2H), 7.03 (dt, J = 23.4, 7.2 Hz, 2H), 4.96 (q, J = 7.5 Hz, 1H), 3.44 - 3.21 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 172.15, 166.52, 150.35, 145.54, 136.04, 134.20, 132.03, 131.08, 128.82, 127.88, 127.77, 127.69, 127.61, 127.22, 126.74, 124.31, 123.86, 120.96, 1 18.63, 118.25, 1 13.43, 1 1 1.33,

109.98, 55.42, 27.33. MS (ESI) 435 m/z [M + H]⁺.

(S)-N-(3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-l -fluoro-2-naphthamide (14m). The procedure for the synthesis of 1 was followed using 1 -fluoro-2-naphthoic acid to provide 14m as a yellow solid (61%). ¾ NMR (400 MHz, DMSO-de) δ 10.97 - 10.83 (m, 1H), 10.67 (s, 1H), 8.67 (dd, J = 7.5, 3.5 Hz, 1H), 8.51 - 8.42 (m, 2H), 8.23 - 8.08 (m, 1H), 8.08 - 7.98 (m, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.75 - 7.56 (m, 6H), 7.34 (d, J = 8.0 Hz, 1H), 7.27 (d, J = 2.3 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 6.96 (t, J = 7.4 Hz, 1H), 4.97 (td, J = 8.0, 5.6 Hz, 1H), 3.48 - 3.15 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.51, 163.76, 150.31, 145.52, 136.06, 135.32, 128.53, 127.68, 127.45, 127.22, 125.52, 123.89, 123.76, 122.60, 122.43, 120.97, 1 18.49, 118.26, 1 13.45, 1 11.33, 109.39, 55.21, 27.49. MS (ESI) 453 m/z [M + H]⁺.

(S)-N-(3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-6-fluoro-2-naphthamide (14n). The general procedure A was followed using 6-fluoro-2-naphthoic acid to provide 14n as a light yellow solid (42%). Rf = 0.27 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO- de^ 10.83 (d, J = 2.5 Hz, 1H), 10.67 (s, 1H), 8.91 (d, J = 7.6 Hz, 1H), 8.52 (d, J = 1.2 Hz,

1H), 8.48 - 8.38 (m, 2H), 8.10 (dd, J = 9.1, 5.8 Hz, 1H), 7.97 (d, J = 1.2 Hz, 2H), 7.84 - 7.73 (m, 2H), 7.69 - 7.60 (m, 2H), 7.51 (td, J = 8.9, 2.6 Hz, 1H), 7.40 - 7.26 (m, 2H), 7.06 (ddd, J = 8.2, 7.0, 1.3 Hz, 1H), 7.03 - 6.95 (m, 1H), 4.96 (ddd, J = 9.1, 7.5, 5.6 Hz, 1H), 3.44 - 3.18 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 172.12, 166.33, 159.74, 150.31, 145.58, 136.04, 135.22, 135.13, 131.96, 131.86, 130.65, 129.23, 128.01, 127.31, 127.21, 125.40, 123.85,

120.96, 1 18.62, 1 18.26, 116.89, 1 13.44, 1 11.34, 109.95, 55.41, 27.35. MS (ESI) 453 m/z [M + H]⁺.

(S)-N-(3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-6-bromo-2-naphthamide (14o). The general procedure A was followed using 6-bromo-2-naphthoic acid to provide 14o as a light yellow solid (76%). Rf = 0.36 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO- de) δ 10.92 - 10.74 (m, 2H), 8.98 (d, J = 7.5 Hz, 1H), 8.57 - 8.41 (m, 3H), 8.28 (d, J = 2.0 Hz, 1H), 7.98 (d, J = 6.1 Hz, 3H), 7.85 - 7.63 (m, 4H), 7.32 (d, J = 7.8 Hz, 2H), 7.03 (dt, J = 24.7, 7.2 Hz, 2H), 4.96 (td, J = 8.4, 5.7 Hz, 1H), 3.32 (qd, J = 14.6, 7.3 Hz, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 172.23, 166.27, 149.45, 146.35, 136.03, 135.29, 131.63, 131.03, 130.58, 129.80, 129.59, 127.95, 127.19, 127.07, 125.49, 123.88, 121.07, 120.96, 1 18.60, 118.25, 1 13.56, 1 1 1.34, 109.88, 55.54, 27.29. MS (ESI) 515 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-[l,l '-biphenyl]-4- carboxamide (14p). The general procedure A was followed using [l,l '-biphenyl]-4- carboxylic acid to provide 14p as a light yellow solid (28%). Rf = 0.36 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.83 (d, J = 2.5 Hz, 1H), 10.66 (s, 1H), 8.81 (d, J = 7.6 Hz, 1H), 8.51 - 8.39 (m, 2H), 7.96 (d, J = 8.4 Hz, 2H), 7.81 - 7.70 (m, 5H), 7.67 - 7.60 (m, 2H), 7.56 - 7.45 (m, 2H), 7.44 - 7.37 (m, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.29 (d, J = 2.3 Hz, 1H), 7.1 1 - 7.03 (m, 1H), 7.00 (t, J = 7.4 Hz, 1H), 4.92 (td, J = 8.1, 7.7, 5.8 Hz, 1H), 3.45 - 3.21 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 172.21, 166.15, 150.36, 145.56, 142.92, 139.10, 136.04, 132.54, 129.02, 128.21, 128.07, 127.17, 126.86, 126.40, 123.86, 120.95, 1 18.61, 1 18.24, 1 13.42, 1 1 1.32, 109.98, 55.32, 27.25. MS (ESI) 461 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-[l,l '-biphenyl]-3- carboxamide (14q). The general procedure A was followed using [l,l '-biphenyl]-3- carboxylic acid to provide 14q as a yellow solid (42%). Rf = 0.30 (100%) ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.90 - 10.80 (m, 1H), 10.66 (s, 1H), 8.94 (d, J = 7.7 Hz, 1H), 8.50 - 8.37 (m, 2H), 8.14 (d, J = 1.8 Hz, 1H), 7.83 (dd, J = 7.8, 1.8 Hz, 2H), 7.78 (d, J = 7.8 Hz, 1H), 7.76 - 7.71 (m, 2H), 7.66 - 7.61 (m, 2H), 7.53 (dt, J = 1 1.7, 7.7 Hz, 3H), 7.41 (t, J = 7.3 Hz, 1H), 7.35 - 7.26 (m, 2H), 7.05 (t, J = 7.5 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 5.01 - 4.88 (m, 1H), 3.42 - 3.17 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 172.15, 166.41,

150.36, 145.53, 140.07, 139.47, 136.01, 134.42, 129.59, 128.98, 128.95, 127.78, 127.22, 126.84, 126.73, 125.65, 123.81, 120.94, 118.62, 1 18.25, 1 13.42, 1 1 1.32, 1 10.03, 55.44, 27.30. MS (ESI) 461 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-2-fluoro-[l,l '-biphenyl]- 4-carboxamide (14r). The general procedure A was followed using 3-fluoro-[l, l'-biphenyl]- 4-carboxylic acid to provide 14r as a light yellow solid (78%). Rf = 0.36 (100% ethyl acetate). 'H NMR (400 MHz, DMSO-de) δ 10.83 (d, J = 2.5 Hz, 1H), 10.66 (s, 1H), 8.93 (d, J = 7.6 Hz, 1H), 8.60 - 8.35 (m, 2H), 7.91 - 7.71 (m, 3H), 7.70 - 7.56 (m, 5H), 7.55 - 7.40 (m, 3H), 7.32 (d, J = 7.9 Hz, 1H), 7.28 (d, J = 2.3 Hz, 1H), 7.10 - 7.03 (m, 1H), 7.00 (t, J = 7.5 Hz, 1H), 4.92 (ddd, J = 9.3, 7.4, 5.4 Hz, 1H), 3.33 (s, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.99, 164.88, 159.88, 157.44, 150.36, 145.53, 136.04, 134.24, 131.02, 130.71, 128.85, 128.83, 128.71, 128.40, 127.15, 124.04, 123.86, 120.98, 1 18.60, 1 18.26, 1 13.46, 1 1 1.35, 109.89, 55.38, 27.25. MS (ESI) 479 m/z [M + H]⁺.

(S)-N-(3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-3-fluoro-fl ,1 '-biphenyl) '- 4-carboxamide (14s). The general procedure A was followed using 16s to provide 14s as a yellow solid (37%). ¾ NMR (400 MHz, DMSO-de) δ 10.88 (d, J = 2.7 Hz, 1H), 10.64 (s, 1H), 8.58 - 8.37 (m, 3H), 7.79 - 7.57 (m, 8H), 7.54 - 7.46 (m, 2H), 7.47 - 7.40 (m, 1H), 7.36 - 7.31 (m, 1H), 7.25 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.96 (ddd, J = 8.1, 6.9, 1.0 Hz, 1H), 4.91 (td, J = 7.9, 5.5 Hz, 1H), 3.32 (s, 2H). ¹³C NMR (101 MHz,

DMSO-de) δ 171.49, 150.35, 145.44, 137.75, 136.05, 129.07, 127.16, 126.90, 123.91, 121.49, 121.35, 120.95, 1 18.46, 118.25, 1 13.92, 1 13.42, 109.31, 55.08, 27.42. MS (ESI) 479 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-3,3'-difluoro-[ 1,1 '- biphenyl] -4-carboxamide (14t). The general procedure A was followed using 16t to provide 14t as a light yellow solid (54%). Rf = 0.45 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.88 (d, J = 2.4 Hz, 1H), 10.65 (s, 1H), 8.53 (dd, J = 7.4, 3.8 Hz, 1H), 8.45 (d, J = 5.6 Hz, 2H), 7.88 - 7.58 (m, 8H), 7.58 - 7.47 (m, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.31 - 7.19 (m, 2H), 7.06 (t, J = 7.5 Hz, 1H), 6.97 (t, J = 7.5 Hz, 1H), 4.97 - 4.84 (m, 1H), 3.46 - 3.17 (m, 2H). ¹³C NMR (101 MHz, DMSO) δ 171.49, 163.89, 163.22, 161.47, 161.08,

158.60, 150.37, 145.46, 136.07, 131.09, 131.00, 127.18, 123.92, 123.03, 122.61, 122.17, 122.03, 120.98, 1 18.48, 1 18.26, 1 13.87, 113.65, 1 13.43, 1 1 1.34, 109.34, 55.1 1, 27.43. MS (ESI) 497 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-3,4'-difluoro-[ 1,1 '- biphenyl] -4-carboxamide (27a). The general procedure A was followed using 16a to provide 27a as a light yellow solid (64%). R_f = 0.42 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.88 (d, J = 2.6 Hz, 1H), 10.67 (s, 1H), 8.54 - 8.39 (m, 3H), 7.88 - 7.76 (m, 2H), 7.74 - 7.55 (m, 6H), 7.33 (ddd, J = 8.8, 6.7, 2.2 Hz, 3H), 7.25 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.01 - 6.88 (m, 1H), 4.91 (td, J = 8.1, 5.6 Hz, 1H), 3.45 - 3.17 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.53, 163.25, 161.28, 161.12, 158.64, 150.21,

145.61, 143.65, 143.57, 136.07, 130.98, 129.15, 129.06, 127.18, 123.92, 122.42, 120.98, 1 18.47, 1 18.26, 1 16.03, 115.82, 1 13.94, 1 13.46, 1 1 1.34, 109.32, 55.12, 27.42. MS (ESI) 497 m/z [M + H]⁺. (S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-2',3-difluoro-[l,r- biphenyl] -4-carboxamide (27b). The general procedure A was followed using 16b to provide 27b as a light yellow solid (74%). ¾ NMR (400 MHz, DMSO-de) δ 10.88 (d, J = 2.5 Hz, 1H), 10.67 (s, 1H), 8.58 (dd, J = 7.3, 3.5 Hz, 1H), 8.53 - 8.36 (m, 2H), 7.73 - 7.64 (m, 2H), 7.64 - 7.57 (m, 3H), 7.48 (ddt, J = 9.7, 6.4, 1.4 Hz, 3H), 7.40 - 7.30 (m, 3H), 7.25 (d, J = 2.3 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.97 (ddd, J = 7.9, 6.9, 1.0 Hz, 1H), 4.91 (td, J = 8.2, 5.6 Hz, 1H), 3.44 - 3.15 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.53, 163.31, 160.49, 160.24, 157.79, 150.14, 145.68, 139.51, 136.07, 130.75, 130.49, 127.17, 125.14, 125.10, 123.94, 122.30, 122.16, 120.98, 1 18.48, 1 18.26, 1 16.40, 1 16.18, 1 13.49, 1 1 1.34, 109.33, 55.14, 27.41. MS (ESI) 497 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-3,3',5'-trifluoro-fl, - biphenyl] -4-carboxamide (27c). The general procedure A was followed using 16c to provide 27c as a light yellow solid (73%). Rf = 0.30 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.88 (d, J = 2.5 Hz, 1H), 10.65 (s, 1H), 8.57 (dd, J = 7.4, 3.6 Hz, 1H), 8.51 - 8.34 (m, 2H), 7.83 - 7.72 (m, 1H), 7.73 - 7.64 (m, 3H), 7.63 - 7.55 (m, 4H), 7.37 - 7.27 (m, 2H), 7.25 (d, J = 2.3 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.01 - 6.93 (m, 1H), 4.91 (td, J = 8.2, 5.6 Hz, 1H), 3.40 - 3.16 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.48, 164.17, 164.03, 163.18, 161.72, 161.59, 161.01, 150.37, 145.46, 136.07, 130.93, 127.18, 123.92, 122.81, 122.72, 122.68, 120.99, 1 18.49, 1 18.27, 1 16.00, 1 14.71, 1 14.48, 1 13.45, 11 1.34, 1 10.37, 1 10.1 1, 109.34, 55.13, 27.43. MS (ESI) 515 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-2',3,5'-trifluoro-[l, - biphenyl] -4-carboxamide (27 d). The general procedure A was followed using 16d to provide 27d as a light yellow solid (70%). Rf = 0.42 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.88 (d, J = 2.5 Hz, 1H), 10.64 (s, 1H), 8.61 (dd, J = 7.3, 3.3 Hz, 1H), 8.45 (d, J = 5.5 Hz, 2H), 7.73 - 7.65 (m, 2H), 7.63 - 7.58 (m, 2H), 7.57 - 7.47 (m, 3H), 7.42 (td, J = 9.5, 4.7 Hz, 1H), 7.37 - 7.29 (m, 2H), 7.25 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.97 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 4.91 (td, J = 7.9, 5.5 Hz, 1H), 3.46 - 3.14 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.47, 163.26, 162.71, 160.45, 159.54, 157.97, 157.14, 156.42, 152.87, 150.62, 150.36, 145.48, 136.06, 130.54, 127.18, 123.93, 120.98, 1 18.48, 118.27, 1 17.12, 1 13.45, 1 1 1.34, 109.34, 55.13, 27.42. MS (ESI) 515 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-4'-chloro-3-fluoro-[l,l '- biphenyl] -4-carboxamide (27 e). The general procedure A was followed using 16e to provide 27e as a light yellow solid (60%). Rf = 0.32 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.88 (d, J = 2.6 Hz, 1H), 10.65 (s, 1H), 8.51 (dd, J = 7.3, 3.9 Hz, 1H), 8.47 - 8.36 (m, 2H), 7.84 - 7.76 (m, 2H), 7.75 - 7.65 (m, 3H), 7.62 (ddd, J = 10.6, 5.3, 1.8 Hz, 3H), 7.58 - 7.52 (m, 2H), 7.33 (d, J = 8.1 Hz, 1H), 7.25 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.96 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 4.91 (td, J = 8.0, 5.4 Hz, 1H), 3.45 - 3.15 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.50, 163.25, 161.12, 158.64, 154.72, 153.81, 150.35, 145.49, 136.58, 136.07, 133.57, 131.04, 129.04, 128.75, 127.19, 123.93, 122.46, 121.74, 120.99, 1 18.48, 118.27, 1 14.25, 1 14.01, 1 13.45, 1 1 1.35, 109.33, 55.12, 27.43. MS (ESI) 513 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-3'-chloro-3-fluoro-[l,l '- biphenyl] -4-carboxamide (27f). The general procedure A was followed using 16f to provide 27f, which was further purified by HPLC (36%, a white solid). ¾ NMR (400 MHz, DMSO- de) δ 10.88 (d, J = 2.5 Hz, 1H), 10.66 (s, 1H), 8.54 (dd, J = 7.3, 3.8 Hz, 1H), 8.49 - 8.39 (m, 2H), 7.85 (t, J = 1.9 Hz, 1H), 7.78 - 7.63 (m, 5H), 7.63 - 7.59 (m, 2H), 7.56 - 7.46 (m, 2H), 7.37 - 7.31 (m, 1H), 7.25 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.96 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 4.92 (td, J = 7.9, 5.5 Hz, 1H), 3.44 - 3.15 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.50, 163.24, 161.07, 158.59, 150.28, 145.55, 143.03, 142.95, 139.89,

136.06, 133.93, 130.97, 130.89, 128.45, 127.18, 126.71, 125.67, 123.92, 122.69, 122.21,

122.07, 120.99, 1 18.48, 118.27, 1 14.55, 1 14.31, 1 13.45, 1 1 1.34, 109.34, 55.13, 27.43. MS (ESI) 513 m/z [M + H]⁺.

(S)-N-(3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-2'-chloro-3-fluoro-fl , 1 '- biphenyl] -4-carboxamide (27 g). The general procedure A was followed using 16g to provide 27g as a white solid (80%). Rf = 0.51 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.88 (d, J = 2.4 Hz, 1H), 10.65 (s, 1H), 8.62 (dd, J = 7.3, 3.2 Hz, 1H), 8.51 - 8.41 (m, 2H), 7.75 - 7.64 (m, 2H), 7.61 (dd, J = 4.8, 1.6 Hz, 4H), 7.49 - 7.43 (m, 3H), 7.38 (dd, J = 11.4, 1.6 Hz, 1H), 7.34 (dd, J = 7.9, 1.8 Hz, 2H), 7.26 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.97 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 4.91 (td, J = 8.3, 5.6 Hz, 1H), 3.43 - 3.17 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 172.59, 171.53, 163.42, 160.13, 157.65, 155.83, 150.29, 145.55, 143.07, 137.78, 136.07, 131.62, 131.39, 131.1 1, 130.16, 130.03, 129.98, 127.67, 127.17, 125.44, 123.95, 122.42, 122.28, 120.99, 1 18.49, 1 18.26, 1 17.14, 1 16.90, 113.45, 1 1 1.34, 109.37, 55.15, 27.40. MS (ESI) 513.3 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-3,3',4'-trifluoro-[l, l '- biphenyl] -4-carboxamide (27h). The general procedure A was followed using 16h to provide 27h as a white solid (78%). Rf = 0.45 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.88 (d, J = 2.6 Hz, 1H), 10.65 (s, 1H), 8.53 (dd, J = 7.3, 3.8 Hz, 1H), 8.49 - 8.38 (m, 2H), 7.92 (ddd, J = 12.1, 7.7, 2.3 Hz, 1H), 7.75 - 7.49 (m, 8H), 7.33 (d, J = 8.0 Hz, 1H), 7.25 (d, J = 2.3 Hz, 1H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.01 - 6.90 (m, 1H), 4.91 (td, J = 8.1, 5.6 Hz, 1H), 3.46 - 3.16 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.50, 163.22, 161.07, 158.59, 150.35, 145.49, 136.07, 130.97, 127.18, 123.93, 122.55, 122.08, 121.95, 120.99, 1 18.48, 1 18.27, 1 18.18, 118.00, 1 16.29, 1 16.1 1, 1 14.45, 1 14.21, 113.45, 1 1 1.35, 109.34, 55.14, 27.43. MS (ESI) 515.3 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-3,3'-difluoro-4'-methoxy- [1, 1 '-biphenyl] -4-carboxamide (27 i). The general procedure A was followed using 16i to provide 27i, which was further purified by HPLC (45%, a white solid). ¾ NMR (400 MHz, DMSO-de) δ 10.89 (d, J = 2.5 Hz, 1H), 10.81 (s, 1H), 8.48 (dd, J = 7.0, 4.2 Hz, 3H), 7.74 - 7.64 (m, 5H), 7.64 - 7.58 (m, 3H), 7.37 - 7.31 (m, 1H), 7.31 - 7.23 (m, 2H), 7.06 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 6.96 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 4.90 (td, J = 7.8, 5.6 Hz, 1H), 3.89 (s, 3H), 3.53 - 3.16 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.77, 163.31, 158.73, 152.96, 150.54, 149.03, 147.66, 147.56, 146.71, 136.07, 130.89, 127.16, 123.96, 123.22, 121.00, 1 18.45, 1 18.28, 1 14.27, 1 14.22, 113.74, 1 13.65, 1 13.51, 1 1 1.36, 109.25, 56.14, 55.21, 27.34. MS (ESI) 527.2 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-3'-cyano-3-fluoro-[l,l '- biphenyl] -4-carboxamide (27 j). The general procedure A was followed using 16j to provide 27j as a white solid (47%). ¾ NMR (400 MHz, DMSO-de) δ 10.89 (d, J = 2.5 Hz, 1H), 10.80 (s, 1H), 8.60 (dd, J = 7.4, 3.6 Hz, 1H), 8.48 (d, J = 5.6 Hz, 2H), 8.29 (t, J = 1.8 Hz, 1H), 8.12 (dt, J = 8.1, 1.3 Hz, 1H), 7.90 (dt, J = 7.8, 1.4 Hz, 1H), 7.80 - 7.74 (m, 1H), 7.74 - 7.64 (m, 6H), 7.33 (d, J = 8.1 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.11 - 7.02 (m, 1H), 6.96 (t, J = 7.3 Hz, lH), 4.92 (td, J = 8.1, 5.6 Hz, 1H), 3.29 (dqt, J = 23.3, 14.6, 9.3, 8.5 Hz, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.68, 163.27, 161.08, 158.60, 149.34, 142.31, 138.83, 136.06, 132.14, 131.71, 131.03, 130.63, 130.27, 127.17, 123.95, 122.75, 120.99, 1 18.59, 118.47, 1 18.28, 1 14.70, 114.46, 1 13.60, 1 12.28, 1 1 1.35, 109.30, 55.23, 27.36. MS (ESI) 504 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-4'-(benzyloxy)-3,3'- difluoro-fl, 1 '-biphenyl] -4-carboxamide (27k). The general procedure A was followed using 16k to provide 27k as a light yellow solid (88%). Rf = 0.39 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.90 (d, J = 2.5 Hz, 1H), 10.69 (s, 1H), 8.48 (dd, J = 7.4, 4.2 Hz, 1H), 7.87 - 7.52 (m, 8H), 7.51 - 7.38 (m, 5H), 7.38 - 7.30 (m, 4H), 7.25 (d, J = 2.4 Hz, 1H), 7.1 1 - 7.02 (m, 1H), 6.96 (t, J = 7.4 Hz, 1H), 5.26 (s, 2H), 4.91 (td, J = 8.0, 5.6 Hz, 1H), 3.40 - 3.16 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.55, 163.25, 161.21, 158.73, 153.26, 150.84, 150.22, 146.62, 146.52, 145.55, 143.14, 143.06, 136.39, 136.07, 130.93, 130.90, 130.83, 130.78, 128.53, 128.11, 127.80, 127.19, 123.94, 123.17, 123.14, 122.00, 121.97, 121.19, 121.06, 120.99, 118.49, 1 18.27, 1 15.72, 1 14.67, 1 14.47, 1 13.78, 1 13.54, 1 1 1.35, 109.33, 70.24, 55.13, 29.60, 27.43. MS (ESI) 603 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-3,3'-difluoro-4

[1, 1 '-biphenyl] -4-carboxamide (271). The procedure for the synthesis of 3j was followed using 27k to provide 271 as a brown solid (81%). ¾ NMR (400 MHz, DMSO-de) δ 10.88 (d, J = 2.5 Hz, 1H), 10.75 (s, 1H), 10.20 (s, 1H), 8.60 - 8.36 (m, 3H), 7.71 - 7.53 (m, 7H), 7.45 (dd, J = 8.4, 2.2 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.25 (d, J = 2.4 Hz, 1H), 7.10 - 7.00 (m, 2H), 6.96 (t, J = 7.4 Hz, 1H), 4.90 (td, J = 8.0, 5.6 Hz, 1H), 3.47 - 3.1 1 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.70, 158.76, 149.42, 136.07, 127.16, 126.73, 123.95, 123.20, 121.74, 120.99, 1 18.46, 118.27, 1 1 1.36, 109.27, 55.17, 27.37. MS (ESI) 513 m/z [M + H]⁺. (S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-4'-amino-3-fluoro-[l, l '- biphenyl] -4-carboxamide (27m). The general procedure A was followed using 161 to provide 27m as a yellow solid (59%). ¾ NMR (400 MHz, DMSO-de) δ 10.88 (d, J = 2.5 Hz, 1H), 10.64 (s, 1H), 8.52 - 8.38 (m, 2H), 8.27 (dd, J = 7.2, 5.3 Hz, 1H), 7.64 (dd, J = 8.1, 6.2 Hz, 2H), 7.62 - 7.57 (m, 2H), 7.51 - 7.41 (m, 4H), 7.33 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.2, 6.9, 1.2 Hz, 1H), 6.99 - 6.92 (m, 1H), 6.64 (d, J = 8.6 Hz, 2H), 5.47 (s, 2H), 4.90 (td, J = 7.8, 5.5 Hz, 1H), 3.49 - 3.16 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.58, 163.30, 161.48, 150.32, 149.68, 145.52, 136.08, 127.59, 127.19, 124.41, 123.93, 120.99, 120.66, 1 18.92, 118.79, 1 18.47, 1 18.27, 1 14.04, 1 13.44, 1 12.05, 1 1 1.81, 1 1 1.35, 109.29, 55.06, 27.45. MS (ESI) 494 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-2-fluoro-4- (phenylethynyl)benzamide (27n). The general procedure A was followed using 21b to provide 27n as a light yellow solid (86%). ¾ NMR (400 MHz, DMSO-de) δ 10.89 (d, J = 2.5 Hz, 1H), 10.68 (s, 1H), 8.66 (dd, J = 7.4, 3.1 Hz, 1H), 8.52 - 8.36 (m, 2H), 7.68 (d, J = 7.9 Hz, 1H), 7.65 - 7.56 (m, 5H), 7.52 (dd, J = 1 1.1, 1.5 Hz, 1H), 7.45 (dd, J = 7.0, 2.8 Hz, 4H), 7.34 (d, J = 8.1 Hz, 1H), 7.25 (d, J = 2.4 Hz, 1H), 7.07 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.01 - 6.93 (m, 1H), 4.97 - 4.84 (m, 1H), 3.34 - 3.14 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.45, 163.05, 160.32, 157.83, 150.39, 145.45, 136.07, 131.59, 130.76, 130.72, 129.43, 128.86, 127.54, 127.51, 127.17, 126.43, 126.33, 123.93, 123.38, 123.24, 121.50, 121.00, 1 18.90,

1 18.65, 1 18.50, 1 18.27, 113.44, 1 1 1.35, 109.34, 91.92, 87.54, 87.51, 55.15, 27.42. MS (ESI) 503.3 m/z [M + H]⁺.

(S,E)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-4-(4-chlorostyryl)-2- fluorobenzamide (27o). The general procedure A was followed using 17 to provide 27o as a light yellow solid (78%). Rf = 0.51 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.88 (d, J = 2.5 Hz, 1H), 10.67 (s, 1H), 8.55 - 8.33 (m, 3H), 7.72 - 7.57 (m, 6H), 7.54 (dd, J = 12.4, 1.5 Hz, 1H), 7.51 - 7.41 (m, 4H), 7.37 - 7.28 (m, 2H), 7.24 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 6.96 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 4.90 (td, J = 8.0, 5.6 Hz, 1H), 3.44 - 3.12 (m, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.56, 163.23, 158.65, 150.17, 145.66, 141.97, 141.89, 136.07, 135.40, 132.67, 130.79, 130.31, 128.82, 128.50, 127.18, 123.94, 122.66, 121.32, 120.99, 1 18.48, 118.26, 1 13.46, 1 13.27, 1 1 1.35, 109.31, 55.13, 27.43. MS (ESI) 539.3 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-2-fluoro-4- (phenylamino)benzamide (27p). The general procedure A was followed using 20 to provide 27p as a yellow solid (88%). Rf = 0.33 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 10.89 (d, J = 2.5 Hz, 1H), 10.66 (s, 1H), 8.83 (s, 1H), 8.53 - 8.38 (m, 2H), 8.18 (s, 1H), 7.81 (t, J = 7.4 Hz, 1H), 7.70 - 7.54 (m, 3H), 7.42 - 7.27 (m, 3H), 7.22 (d, J = 2.3 Hz, 1H), 7.17 (d, J = 7.8 Hz, 2H), 7.03 (dt, J = 19.1, 7.4 Hz, 2H), 6.94 (t, J = 7.4 Hz, 1H), 6.85 (dd, J = 8.7, 2.2 Hz, 1H), 6.74 (dd, J = 14.4, 2.2 Hz, 1H), 4.96 - 4.80 (m, 1H), 3.27 (qd, J = 14.7, 6.8 Hz, 2H). ¹³C NMR (101 MHz, DMSO-de) δ 171.74, 162.93, 162.90, 162.54, 160.09, 150.20, 148.88, 148.76, 145.65, 140.97, 136.1 1, 132.1 1, 132.07, 129.42, 127.19, 123.94, 122.23, 121.01, 1 19.48, 1 18.46, 118.28, 1 13.45, 1 11.37, 1 1 1.04, 1 10.92, 1 10.71, 109.20, 100.72, 100.44, 54.93, 27.56. MS (ESI) 494.2 m/z [M + H]⁺.

(S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-2-fluoro-4- morpholinobenzamide (27 q). The general procedure A was followed using 16m to provide 27q as a white solid (49%). Rf = 0.48 (10% MeOH in ethyl acetate), Rf = 0.15 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-de) δ 1 1.29 (s, 1H), 10.91 (d, J = 2.5 Hz, 1H), 8.68 - 8.49 (m, 2H), 7.96 - 7.78 (m, 3H), 7.70 - 7.53 (m, 2H), 7.32 (d, J = 8.1 Hz, 1H), 7.24 (d, J = 2.4 Hz, 1H), 7.04 (ddd, J = 8.1, 6.9, 1.1 Hz, 1H), 6.93 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 6.86 - 6.69 (m, 2H), 4.88 (q, J = 6.8 Hz, 1H), 3.71 (dd, J = 5.9, 3.9 Hz, 4H), 3.44 - 3.18 (m, 6H). ¹³C NMR (101 MHz, DMSO-de) δ 172.48, 136.08, 127.12, 124.03, 1 18.40, 1 18.29, 1 14.00, 11 1.37, 1 10.19, 109.53, 109.01, 65.71, 46.89, 45.87, 25.93. MS (ESI) 488.3 m/z [M + H]⁺. (S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-2-fluoro-4-(4- (phenylsulfonyl)piperazin-l-yl)benzamide (27r). The general procedure A was followed using 25b to provide 27r as a white solid (56%). ¾ NMR (400 MHz, DMSO-de) δ 10.85 (d, J = 2.5 Hz, 1H), 10.61 (s, 1H), 8.43 (d, J = 5.5 Hz, 2H), 7.85 - 7.70 (m, 4H), 7.70 - 7.62 (m, 2H), 7.62 - 7.51 (m, 4H), 7.31 (d, J = 8.1 Hz, 1H), 7.18 (d, J = 2.4 Hz, 1H), 7.04 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 6.92 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 6.79 - 6.67 (m, 2H), 4.92 - 4.79 (m, 1H), 3.39 (t, J = 5.1 Hz, 4H), 3.35 - 3.14 (m, 2H), 2.97 (t, J = 5.0 Hz, 4H). ¹³C NMR (101 MHz, DMSO-de) δ 171.65, 162.87, 162.49, 160.03, 153.45, 150.22, 145.57, 136.06, 134.56, 133.45, 131.65, 131.60, 129.51, 127.58, 127.17, 123.89, 120.97, 1 18.41, 1 18.25, 1 13.42, 1 1 1.33, 110.86, 1 10.23, 109.18, 101.42, 54.91, 46.30, 45.34, 27.50. MS (ESI) 627 m/z [M + H]⁺. (S)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-2-fluoro-4-(piperazin-l- yl)benzamide hydrochloride (27s). The general procedure A was followed using 4-(4-(tert- butoxycarbonyl)piperazin- 1 -yl)-2-fluorobenzoic acid obtained by hydrolysis of 23 (79%). ^!H NMR (400 MHz, CDCb) δ 9.37 (s, 1H), 8.60 (d, J = 2.5 Hz, 1H), 8.31 - 8.21 (m, 2H), 7.81 (t, J = 9.1 Hz, 1H), 7.56 (d, J = 7.9 Hz, 1H), 7.44 - 7.28 (m, 4H), 7.14 - 7.02 (m, 2H), 6.91 (t, J = 7.5 Hz, 1H), 6.61 (dd, J = 9.1, 2.4 Hz, 1H), 6.41 (dd, J = 16.0, 2.4 Hz, 1H), 5.07 (q, J =

6.6, 6.1 Hz, 1H), 3.54 (dd, J = 6.8, 3.9 Hz, 4H), 3.41 - 3.33 (m, 2H), 3.25 (dd, J = 6.6, 4.1 Hz, 4H), 1.47 (s, 9H). The procedure for the synthesis of 3c was followed to provide 27s, which was further purified by HPLC (83%, a brown solid). ¾ NMR (400 MHz, DMSO-de) δ 12.43 (s, 1H), 11.03 (d, J = 2.5 Hz, 1H), 9.63 (s, 2H), 8.72 (d, J = 6.9 Hz, 2H), 8.34 - 8.18 (m, 2H), 8.02 (t, J = 6.8 Hz, 1H), 7.69 (d, J = 7.9 Hz, 1H), 7.61 (t, J = 8.9 Hz, 1H), 7.37 - 7.28 (m, 2H), 7.03 (t, J = 7.5 Hz, 1H), 6.90 (t, J = 7.6 Hz, 1H), 6.87 - 6.75 (m, 2H), 4.95 (dt, J = 8.2, 6.2 Hz, 1H), 3.56 (t, J = 5.1 Hz, 4H), 3.37 (qd, J = 14.6, 6.9 Hz, 2H), 3.15 (p, J = 4.3 Hz, 4H). ¹³C NMR (101 MHz, DMSO-de) δ 173.23, 163.08, 163.06, 162.64, 160.19, 153.49, 153.37, 153.11, 142.02, 136.12, 131.78, 131.74, 127.16, 124.23, 121.01, 1 18.58, 1 18.33, 1 14.57, 11 1.41, 1 1 1.01, 1 10.88, 110.27, 108.91, 101.64, 101.36, 55.80, 43.91, 42.01, 27.01. MS (ESI) 487.3 m/z [M + H]⁺.

(S)-N-(3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-2-fluoro-4-(4-(4- fluorobenzyl)piperazin-l-yl)benzamide hydrochloride (27t). The general procedure A was followed using 26b to provide 27t, which was further purified by HPLC (26%, a white solid). ¾ NMR (400 MHz, DMSO-de) δ 1 1.59 (s, 1H), 10.96 (d, J = 2.5 Hz, 1H), 8.69 (d, J = 6.8 Hz, 2H), 8.12 - 7.93 (m, 3H), 7.72 - 7.53 (m, 5H), 7.33 (ddd, J = 8.7, 6.3, 2.8 Hz, 3H), 7.26 (d, J = 2.4 Hz, 1H), 7.13 - 7.00 (m, 1H), 6.93 (t, J = 7.4 Hz, 1H), 6.90 - 6.77 (m, 2H), 4.87 (dt, J = 8.2, 6.0 Hz, 1H), 4.36 (s, 2H), 3.86 - 3.02 (m, 7H). ¹³C NMR (101 MHz, DMSO-de) δ 172.94, 163.99, 163.1 1, 161.54, 160.12, 158.53, 158.21, 153.03, 152.92, 151.54, 143.88, 136.11, 133.64, 133.55, 131.73, 127.08, 126.05, 124.13, 121.05, 1 18.34, 1 15.94, 1 15.72,

115.50, 1 14.39, 1 1 1.43, 11 1.26, 1 1 1.13, 1 10.33, 108.90, 101.72, 101.44, 57.87, 55.52, 49.87, 44.16, 27.04. MS (ESI) 595.4 m/z [M + H]⁺, 298.3 m/z [M + 2H]²⁺. NHBoc

(R)-tert-Butyl (3-(lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)carbamate, (2). To a solution of N-Boc-D-tryptophan (1.0 g, 3.3 mmol), PyBOP (2.0 g, 3.9 mmol), and HOBt (0.29 g) in dry CH2CI2 (20 mL) was slowly added triethylamine (1.5 mL, 4 eq.) at 0 °C, and the reaction mixture was stirred and warmed to ambient temperature for 15 min. After the mixture was cooled to 0 °C, 4-aminopyridine (0.39 g, 4.1 mmol) was added, and the reaction mixture was stirred at room temperature for 1 h. After completion of the reaction monitored by TLC, ethyl acetate (80 mL) was added to the crude mixture, which was washed with saturated aqueous NaHCCte (20 mL x 2) and brine (20 mL x 2). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. Purification of the crude product by flash chromatography provided 0.98 g (94%) of 1 as a light yellow solid. Rf = 0.45 (100% ethyl acetate). ¾ NMR (400 MHz, CDCb) δ 9.07 (s, 1H), 8.86 (dd, J= 23.3, 1 1.0 Hz, 1H), 8.36 - 8.23 (m, 2H), 7.58 (d, J= 8.0 Hz, 1H), 7.31 (d, J= 8.3 Hz, 1H), 7.23 (d, J= 5.7 Hz, 2H), 7.14 (t, J= 7.7 Hz, 1H), 7.03 (t, J= 7.6 Hz, 1H), 6.98 (s, 1H), 5.57 (q, J= 7.8 Hz, 1H), 4.67 (s, 1H), 3.40 - 3.15 (m, 2H), 1.39 (s, 9H). ¹³C NMR (101 MHz, CDCb) δ 171.75, 156.25, 150.26, 145.03, 136.47, 127.27, 123.51, 122.34, 1 19.79, 118.62, 1 13.90, 1 1 1.52, 109.94, 80.82, 56.04, 28.37, 22.61. MS (ESI) 381 m/z [M + H]⁺.

(R)-2-Amino-3-(lH-indol-3-yl)-N-(pyridin-4-yl)propanamide hydrochloric acid (3). To a solution of 2 (0.48 g, 1.2 mmol) in dioxane (10 ml) was added 4N HCI in dixoane (5 ml), and the reaction mixture was stirred at room temperature for 12 hours. The solvent was removed with a rotary evaporator, and water (20 mL) and ether (20 mL) were added to dissolve the reaction mixture. By-products and impurities were extracted with ether layer, and removed. The aqueous layer was dried by using lyophilizer to obtain the crude product 3 as a brown solid (0.35 g, 1.09 mmol, 88%). ¾ NMR (400 MHz, DMSO-d6) δ 1 1.97 (s, 1H), 1 1.09 (d, J = 2.5 Hz, 1H), 8.82 - 8.63 (m, 2H), 8.44 (s, 3H), 8.06 - 7.89 (m, 2H), 7.57 (d, J = 7.9 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.25 (d, J = 2.5 Hz, 1H), 7.05 (ddd, J = 8.1, 6.9, 1.1 Hz, 1H), 6.91 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 4.35 (t, J = 6.8 Hz, 1H), 3.47 - 3.20 (m, 2H); ¹³C NMR (101 MHz, DMSO-d6) 8 169.83, 158.72, 158.40, 150.35, 144.69, 136.25, 126.96, 125.17, 121.21, 1 18.52, 1 18.30, 118.22, 1 14.67, 1 11.54, 106.08, 54.01, 26.94; [α]ο²⁵ = - 69.8 (c = 1.0, MeOH); MS (ESI) m/z 281.1 [M+H]⁺; HRMS (ESI) m/z for C16H17N4O [M+H]⁺ calcd 281.1402, found 281.1400.

2',3-Difluoro-5'-(trifluoromethyl)-[l , l '-biphenyl]-4-carboxylic acid (5). A reaction mixture of 4-bromo-2-fluorobenzoic acid (0.318 g, 1.4 mmol), (2-fluoro-5-

(trifluoromethyl)phenyl)boronic acid (0.335 g, 1.6 mmol), Pd2(dba)3 (62.1 mg, 0.069 mmol), PCy3 (32.3 mg, 0.12 mol), and K3PO4 (2 M, 5 mL) in dioxane (12 mL) was stirred under microwave heating (120 °C) for 1 h. The palladium catalyst was removed by filtration through Celite pad, which was washed with ethyl acetate. The filtrate was acidified with 2N HC1 (aq). The product mixture was diluted with ethyl acetate (50 mL) and washed with water (10 mL x 2) and brine (10 mL x 2). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. The resulting product was purified by flash

chromatography to afford the title product 5 as a white solid (0.382 g, 1.3 mmol, 93%); ¾ NMR (400 MHz, DMSO-d6) δ 13.40 (s, 1H), 8.06 - 7.93 (m, 2H), 7.89 (ddd, J = 8.6, 4.4, 2.4 Hz, 1H), 7.68 - 7.59 (m, 2H), 7.56 (dt, J = 8.1, 1.7 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d6) δ 164.67, 164.64, 162.29, 162.21, 159.77, 159.65, 139.51, 139.42, 132.15, 128.27, 128.23, 128.20, 128.16, 128.10, 128.05, 127.19, 127.05, 126.21, 126.18, 125.89, 125.85, 125.1 1, 125.07, 125.03, 122.32, 119.26, 1 19.16, 1 17.79, 1 17.67, 1 17.64, 1 17.55, 1 17.43, 1 17.40. 3, 4 '-Difluoro-3 '-( trifluoromethyl)-[l , 1 '-biphenyl] -4-carboxylic acid (6). The procedure for the synthesis of 5 was followed using (4-fluoro-3-(trifluoromethyl)phenyl)boronic acid at 100 °C to provide 6 as a white solid (90%); ¾ NMR (400 MHz, DMSO-d6) δ 13.33 (s, 1H), 8.20 - 8.06 (m, 2H), 7.94 (t, J = 8.0 Hz, 1H), 7.76 (dd, J = 12.3, 1.8 Hz, 1H), 7.68 (dd, J = 8.2, 1.8 Hz, 1H), 7.63 (dd, J = 10.6, 8.7 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d6) δ 164.73, 164.70, 162.79, 160.41, 160.24, 157.87, 143.71, 143.62, 134.73, 134.70, 133.95, 133.86, 132.56, 125.95, 125.90, 125.86, 123.85, 122.82, 122.78, 121.14, 1 18.60, 1 18.49, 1 18.07, 1 17.86, 117.50, 1 17.37, 1 17.17, 1 17.05, 1 15.52, 115.28. 3'-Chloro-3,4'-difluoro-fl ,1 '-biphenylJ-4-carboxylic acid (7). The procedure for the synthesis of 5 was followed using (3-chloro-4-fluorophenyl)boronic acid at 100 °C to provide 7 as a white solid (83%), which includes a by-product, 3"-chloro-3,4',4"-trifluoro-[l,l':3',l"- terphenyl]-4-carboxylic acid; ¾ NMR (400 MHz, DMSO-d6) δ 13.28 (s, 1H), 8.03 (dd, J = 7.1, 2.4 Hz, 1H), 7.93 (t, J = 8.0 Hz, 1H), 7.80 (ddd, J = 8.7, 4.6, 2.4 Hz, 1H), 7.71 (dd, J = 12.4, 1.8 Hz, 1H), 7.65 (dd, J = 8.2, 1.8 Hz, 1H), 7.53 (t, J = 8.9 Hz, 1H). "C NMR (101 MHz, DMSO-d6) 8 164.72, 164.69, 162.78, 160.23, 158.82, 156.35, 143.86, 143.77, 135.43, 135.39, 132.51, 132.43, 129.22, 127.86, 127.79, 122.60, 122.57, 120.44, 120.26, 1 18.38, 118.27, 1 17.56, 1 17.35, 1 15.26, 1 15.02.

4 8

Methyl 4-bromo-2-fluorobenzoate (8). To a solution of 4 (13.1 g, 60.0 mmol) in methanol (60 mL) was slowly added SOCk (12 mL) at 0 °C. After stirring overnight (ca. 12 h) at ambient temperature, the solvent was removed under reduced pressure, and the reaction mixture was basified with saturated NaHCCte at 0 °C. Ether (200 mL) was added to the crude mixture, which was washed with saturated NaHCCte (100 mL x 2) and brine (50 mL x 2). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. The product mixture was purified by flash chromatography to provide the title product 8 as a white solid (13.4 g, 58 mmol, 96%). ¾ NMR (400 MHz, CDCb) δ 7.86 - 7.78 (m, 1H), 7.39 - 7.30 (m, 2H), 3.92 (s, 2H). ¹³C NMR (101 MHz, CDCb) δ 164.35, 164.31, 163.06, 160.43, 133.30, 128.10, 128.00, 127.71, 127.67, 120.96, 120.71, 1 17.86, 1 17.76, 52.64. MS (ESI) m/z 233/235 [M+H]⁺.

Methyl 4-(4-(2,4-dtfluorophenyl)piperazin-l-yl)-2-fluorobenzoate (9). A reaction mixture of 8 (0.235 g, 1.0 mmol), l-(2,4-difluorophenyl)piperazine (0.208 g, 1.1 mmol), Pd(OAc)₂ (28.8 mg, 0.13 mmol), BINAP (0.135 g, 0.22 mmol), and CS2CO3 (0.427 g, 1.3 mol) in dry toluene (5 mL) was stirred at 50 °C for 48 h. After cooling, the palladium catalyst was removed by filtration through Celite pad. The product mixture was diluted with ethyl acetate (50 mL) and washed with aqueous NaHCCte (10 mL x 2) and brine (10 mL x 2). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. The resulting product was purified by flash chromatography to yield 9 as a white solid (0.252 g, 0.72 mmol, 72%); Rf = 0.36 (20% ethyl acetate in hexane). ¾ NMR (400 MHz, CDCb) δ 7.83 (t, J = 8.8 Hz, 1H), 6.95 - 6.87 (m, 1H), 6.86 - 6.76 (m, 2H), 6.65 (dd, J = 9.0, 2.5 Hz, 1H), 6.54 (dd, J = 14.6, 2.5 Hz, 1H), 3.86 (s, 3H), 3.49 - 3.41 (m, 4H), 3.13 (dd, J = 6.2, 4.0 Hz, 4H). ¹³C NMR (101 MHz, CDCb) δ 165.09, 164.93, 164.89, 162.53, 159.53, 159.42, 157.1 1, 157.06, 157.00, 156.95, 155.53, 155.42, 154.59, 154.47, 136.30, 136.26, 136.21, 136.17, 133.44, 133.41, 119.80, 1 19.76, 1 19.71, 119.67, 1 1 1.02, 1 10.99, 1 10.81, 1 10.78, 109.50, 109.48, 107.70, 107.60, 105.16, 104.92, 104.90, 104.66, 101.75, 101.48, 77.16, 51.84, 50.60, 50.57, 47.43. MS (ESI) m/z 351 [M+H]⁺.

4-(4-(2,4-Difluorophenyl)piperazin-l-yl)-2-fluorobenzoic acid (10). To a solution of 9 (0.814 g, 2.3 mmol) in methanol/THF (20/5 mL) was added 10% KOH (10 mL), and the reaction mixture was stirred for 2 h at 70 °C. After completion of the reaction, the reaction mixture was cooled to ambient temperature, and 2N HC1 was added to the reaction mixture until white solid precipitated. The white solid was diluted with ethyl acetate (100 mL) and THF (20 mL) and washed with brine (20 mL x 2). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. The resulting product was purified by flash chromatography to yield 9 (0724 g, 2.2 mmol, 94%) as a white solid; Rf = 0.30 (50% ethyl acetate in hexane). ¾ NMR (400 MHz, THF-d8) δ 10.89 (s, 1H), 7.81 (t, J = 8.8 Hz, 1H), 7.07 (td, J = 9.3, 5.8 Hz, 1H), 6.96 (ddd, J = 12.0, 8.8, 2.9 Hz, 1H), 6.92 - 6.83 (m, 1H), 6.77 (dd, J = 8.9, 2.5 Hz, 1H), 6.69 (dd, J = 14.6, 2.4 Hz, 1H), 3.54 - 3.44 (m, 4H), 3.20 - 3.09 (m, 4H). ¹³C NMR (101 MHz, THF-d8) δ 166.27, 165.43, 165.39, 163.72, 160.47, 160.36, 158.18, 158.07, 157.95, 156.78, 156.67, 155.71, 155.60, 137.97, 137.93, 137.88, 137.84, 134.56, 134.53, 121.08, 121.03, 120.98, 120.94, 1 1 1.78, 1 1 1.74, 1 1 1.56, 1 1 1.53, 1 10.21, 109.10, 108.99, 105.67, 105.42, 105.16, 102.48, 102.21, 51.69, 51.66, 48.40.

CYP-II-250 (R)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-2 ', 3- difluoro-5'-(trifluoromethyl)-[l, -biphenyl]-4-carboxamide (11-250). To a solution of 5 (0.419 g, 1.4 mmol), PyBOP (0.804 g, 1.5 mmol) and HOBt (87.6 mg) in dry CH2CI2 (20 mL) was slowly added triethylamine (0.8 mL, 4 eq.) at ambient temperature, and the reaction mixture was stirred for 15 min. After the reaction mixture became homogenous, 3 (0.434 g, 1.4 mmol) was added, and the reaction mixture was stirred at room temperature for 1 h. After completion of the reaction monitored by TLC, the solvent was removed under reduced pressure. Ethyl acetate (80 mL) was added to the crude mixture, which was washed with saturated aqueous NaHCCb (20 mL x 2) and brine (20 mL x 2). The organic layer was concentrated in vacuo and directly subjected to purification by flash chromatography to provide the title product as a light yellow solid (0.604 g, 1.1 mmol, 85%); Rf = 0.30 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-d6) δ 10.90 (d, J = 2.5 Hz, 1H), 10.68 (s, 1H), 8.67 (dd, J = 7.4, 3.2 Hz, 1H), 8.51 - 8.40 (m, 2H), 7.98 (dd, J = 7.1, 2.4 Hz, 1H), 7.89 (ddd, J = 8.5, 4.4, 2.4 Hz, 1H), 7.76 - 7.66 (m, 2H), 7.66 - 7.57 (m, 4H), 7.54 (dt, J = 8.0, 1.7 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.07 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.02 - 6.92 (m, 1H), 4.99 - 4.83 (m, 1H), 3.39 - 3.16 (m, 2H); ¹³C NMR (101 MHz, DMSO- d6) δ 171.48, 163.26, 162.31, 160.47, 159.78, 157.98, 150.38, 145.48, 137.75, 137.66, 136.07, 130.59, 130.56, 128.21, 128.18, 128.14, 127.77, 127.38, 127.25, 127.19, 126.19, 126.16, 125.87, 125.83, 125.14, 125.1 1, 125.06, 123.94, 123.08, 122.93, 122.35, 120.99, 118.50, 1 18.28, 1 17.78, 117.54, 1 16.93, 1 16.70, 1 16.67, 1 13.45, 1 1 1.35, 109.36, 55.16, 27.44. MS (ESI) m/z 565.2 [M+H]⁺.

CYP-II-251 (R)-N-(3-(lH-Indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-3,4'- difluoro-3'-(trifluoromethyl)-[l, -biphenyl]-4-carboxamide (11-251). The procedure for the synthesis of 11-250 was followed using 6 to provide 11-251 as a light yellow solid (68%); Rf = 0.30 (100% ethyl acetate); ¾ NMR (400 MHz, DMSO-d6) δ 10.90 (d, J = 2.5 Hz, 1H), 10.73 (s, 1H), 8.59 (dd, J = 7.3, 3.7 Hz, 1H), 8.50 - 8.42 (m, 2H), 8.22 - 8.06 (m, 2H), 7.82 - 7.75 (m, 1H), 7.74 - 7.66 (m, 4H), 7.66 - 7.60 (m, 3H), 7.34 (d, J = 8.1 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.06 (ddd, J = 8.0, 6.9, 1.2 Hz, 1H), 7.01 - 6.93 (m, 1H), 4.92 (td, J = 7.9, 5.5 Hz, 1H), 3.39 - 3.17 (m, 2H). ¹³C NMR (101 MHz, DMSO-d6) δ 171.57, 163.23, 161.09,

160.30, 158.60, 150.05, 145.79, 142.06, 141.98, 136.09, 134.87, 133.88, 133.79, 131.04, 131.01, 127.20, 125.82, 125.77, 123.95, 123.89, 122.87, 122.84, 122.29, 122.15, 121.17,

121.00, 1 18.49, 1 18.29, 118.08, 1 17.87, 1 14.79, 1 14.55, 1 13.50, 1 1 1.36, 109.34, 55.19, 27.43. MS (ESI) m/z 565.2 [M+H]⁺.

CYP-II-258 (R)-N-( 3-(l H-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl)-3 '-chloro- 3,4'-difluoro-[l ,1 '-biphenyl] -4-carboxamide hydrochloride (11-258). The procedure for the synthesis of 11-250 was followed using 7 to provide 11-258, which was further purified by HPLC to yield a white solid (58%); Rf = 0.30 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-d6) δ 12.29 (s, 1H), 1 1.00 (d, J = 2.5 Hz, 1H), 8.79 - 8.67 (m, 3H), 8.27 - 8.17 (m, 2H), 8.03 (dd, J = 7.1, 2.4 Hz, 1H), 7.79 (ddd, J = 8.7, 4.7, 2.4 Hz, 1H), 7.76 - 7.67 (m, 3H), 7.64 (dd, J = 8.1, 1.7 Hz, 1H), 7.53 (t, J = 9.0 Hz, 1H), 7.47 (t, J = 100.0 Hz, 1H), 7.39 - 7.29 (m, 2H), 7.04 (ddd, J = 8.1, 6.9, 1.1 Hz, 1H), 6.99 - 6.88 (m, 1H), 4.98 (ddd, J = 8.8, 6.8, 5.4 Hz, 1H), 3.51 - 3.23 (m, 2H); ¹³C NMR (101 MHz, DMSO-d6) δ 173.02, 163.40, 161.12, 158.72, 158.64, 156.25, 152.96, 142.32, 142.24, 142.12, 136.07, 135.54, 130.99, 130.96, 129.10, 127.79, 127.71, 127.14, 124.22, 122.66, 122.63, 121.82, 121.69, 120.98, 120.42, 120.24, 1 18.56, 1 18.31, 117.57, 1 17.36, 1 14.59, 1 14.33, 1 1 1.37, 108.98, 55.93, 26.97. MS (ESI) m/z 531.2 [M+H]⁺.

CYP-II-259 (R)-N-(3-(lH-Indol-3-yl)-l -oxo-1 -(pyridin-4-ylamino)propan-2-yl^

difluorophenyl)piperazin-l-yl)-2-fluorobenzamide (11-259). The procedure for the synthesis of 11-250 was followed using 10 to provide 11-259 as a light yellow solid (74%); Rf = 0.18 (100% ethyl acetate). ¾ NMR (400 MHz, DMSO-d6) δ 10.89 (d, J = 2.5 Hz, 1H), 10.64 (s, 1H), 8.50 - 8.38 (m, 2H), 7.82 (t, J = 7.5 Hz, 1H), 7.69 - 7.56 (m, 4H), 7.33 (dt, J = 8.1, 0.9 Hz, 1H), 7.28 - 7.18 (m, 2H), 7.16 - 6.97 (m, 3H), 6.94 (ddd, J = 8.0, 7.0, 1.0 Hz, 1H), 6.89 - 6.77 (m, 2H), 4.96 - 4.81 (m, 1H), 3.44 (dd, J = 6.7, 3.5 Hz, 4H), 3.34 - 3.19 (m, 2H), 3.16 - 3.01 (m, 4H); ¹³C NMR (101 MHz, DMSO-d6) δ 171.69, 162.95, 162.92, 162.68, 160.24, 158.48, 156.15, 156.09, 156.03, 155.96, 154.30, 154.19, 153.69, 153.56, 150.36, 145.50, 136.48, 136.44, 136.39, 136.35, 136.10, 131.72, 131.67, 127.21, 123.92, 121.01, 120.31, 120.27, 120.22, 120.18, 118.46, 1 18.29, 1 13.43, 1 1 1.37, 1 1 1.21, 1 1 1.17, 1 1 1.00, 1 10.96,

110.31, 1 10.18, 109.92, 109.22, 104.95, 104.69, 104.44, 100.95, 100.67, 54.91, 50.06, 46.92, 27.57. MS (ESI) m/z 599.3 [M+H]⁺. Synthesis and Chemical Characterization of Exemplary Compounds of the Invention General procedure B— synthesis of substituted biphenyl carboxlic acids.

A mixture of 2-fluoro-4-bromobenzoic acid (ca. 0.10 g, 0.46 mmol), phenyl boronic acid (ca. 1.1 eq), Pd2(dba)₃ (3 mol%), PCys (6 mol %), and K3PO4 (2 M, 1 mL) in dioxane (4 mL) was stirred under microwave heating (100 °C) for 1 h. The palladium catalyst was removed by filtration. The filtrate was acidified with 2N HCI (aq), and a white solid precipitated. The product mixture was diluted with ethyl acetate (30 mL) and washed with water (10 mL x 2) and brine (10 mL x 2). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. The resulting product was purified by flash chromatography to afford 2-fluorobiphenylcarboxylic acid in ca. 90% yield.

General procedure for the synthesis of preferred inhibitors on the invention. To a solution of the appropriate benzoic acid (ca.1.2 eq), PyBOP (ca. 1.4 eq) and HOBt (ca. 10 mol%) in dry CH2CI2 (5 mL) was slowly added triethylamine (ca. 4 eq.) at ambient temperature. The reaction mixture was stirred for 15 min until it became homogenous. D-tryptophan-(4- aminopyridyl)amide 26 was added, and the reaction mixture was stirred at room temperature for 1 h.

26

After confirming that the reaction was complete by using TLC analysis, the solvent was removed under reduced pressure. Ethyl acetate (10 mL) was added to the crude product mixture, and this solution was washed then with saturated aqueous NaHCCte (2 mL x 2) and brine (2 mL x 2). The organic layer was concentrated in vacuo and the crude product was directly subjected flash chromatographic purification to provide the titled products.

Scheme 5: Synthesis of Precursor Carboxyiic Acids for Preferred Compounds of the Invention

CYP-II-271, (R)-N-(3-(lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-4-(4-(4- mide TFA

The general procedure was followed using carboxylic acid 15b as the acylating agent. The crude product was further purified by HPLC to afford product 11-271 as a white solid (71%): ¾ NMR (400 MHz, DMSO-de) δ 1 1.48 (s, 1H), 10.93 (d, J = 2.5 Hz, 1H), 8.78 - 8.58 (m, 2H), 8.1 1 - 7.89 (m, 3H), 7.60 (dd, J = 8.5, 7.0 Hz, 2H), 7.33 (d, J = 8.1 Hz, 1H), 7.30 - 7.21 (m, 3H), 7.14 - 6.97 (m, 3H), 6.94 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 6.89 - 6.77 (m, 2H), 4.86 (q, J = 6.7 Hz, 1H), 3.45 (dd, J = 6.7, 3.7 Hz, 4H), 3.39 - 3.16 (m, 6H). ¹³C NMR (176 MHz, DMSO-de) δ 173.01, 163.29, 162.20, 160.79, 158.06, 157.87, 154.22, 154.15, 151.64, 149.51, 143.80, 136.1 1, 131.66, 131.64, 128.69, 127.06, 124.12, 122.71, 121.07, 1 18.34, 1 17.10, 1 14.38, 1 1 1.43, 1 10.00, 109.93, 109.84, 108.89, 100.84, 100.68, 55.48, 47.63, 46.49, 27.05; MS (ESI) m/z 597.2 [M+H]⁺; HRMS (ESI) m/z for C33H32N6O2FCI [M+H]⁺ calcd 597.2181, found 597.2180.

CYP-II-269, (R)-N-(3-(lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-2-fluoro-4- benzamide TFA

The general procedure was followed using carboxylic acid 16b as the acylating agent. The crude product was further purified by HPLC to afford compound 11-269 as a white solid (42%): ¾ NMR (400 MHz, DMSO-de) δ 1 1.53 (s, 1H), 10.94 (d, J = 2.5 Hz, 1H), 8.81 - 8.60 (m, 2H), 8.1 1 - 8.00 (m, 2H), 7.97 (t, J = 6.8 Hz, 1H), 7.61 (dt, J = 9.0, 5.0 Hz, 2H), 7.53 (d, J = 8.7 Hz, 2H), 7.33 (d, J = 8.1 Hz, 1H), 7.27 (d, J = 2.4 Hz, 1H), 7.11 (d, J = 8.7 Hz, 2H), 7.05 (ddd, J = 8.0, 6.9, 1.2 Hz, 1H), 6.94 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 6.88 - 6.77 (m, 2H), 4.86 (q, J = 6.7 Hz, 1H), 3.45 (ddt, J = 10.6, 8.0, 4.2 Hz, 8H), 3.39 - 3.19 (m, 2H). ¹³C NMR (176 MHz, DMSO-de) δ 173.06, 163.30, 163.28, 162.21, 160.81, 158.18, 157.99, 154.08, 154.02, 152.86, 151.88, 143.52, 136.12, 131.69, 131.66, 127.06, 126.26, 126.24, 126.21, 126.19, 125.75, 124.21, 124.13, 121.07, 1 18.34, 1 18.1 1, 1 17.93, 1 16.00, 1 14.42, 114.20, 1 1 1.43, 109.92, 109.85, 109.69, 108.88, 100.68, 100.52, 55.51, 46.36, 46.21, 27.03; MS (ESI) m/z 631.2 [M+H]⁺; HRMS (ESI) m/z for C34H32N6O2F4 [M+H]⁺ calcd 631.2445, found 631.2450. CYP-II-275, (R)-N-(3-(lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propa

orobenzamide TFA

The general procedure was followed using carboxylic acid 17b as the acylating agent. The crude product was further purified by HPLC to afford compound 11-275 as a white solid (23%): ¾ NMR (400 MHz, DMSO-de) δ 1 1.40 (s, 1H), 10.93 (d, J = 2.5 Hz, 1H), 8.69 -

8.61 (m, 2H), 8.01 - 7.91 (m, 3H), 7.65 - 7.56 (m, 2H), 7.44 (dd, J = 7.9, 1.5 Hz, 1H), 7.37 - 7.29 (m, 2H), 7.26 (d, J = 2.4 Hz, 1H), 7.20 (dd, J = 8.1, 1.6 Hz, 1H), 7.12 - 7.01 (m, 2H), 6.94 (ddd, J = 8.0, 6.8, 1.0 Hz, 1H), 6.91 - 6.78 (m, 2H), 4.87 (q, J = 6.7 Hz, 1H), 3.46 (dd, J = 6.5, 3.6 Hz, 4H), 3.39 - 3.19 (m, 2H), 3.10 (t, J = 5.0 Hz, 4H). ¹³C NMR (176 MHz, DMSO-de) δ 172.87, 163.26, 162.17, 160.77, 157.92, 157.74, 154.43, 154.36, 148.61, 144.48, 136.11, 131.64, 131.61, 130.38, 128.17, 127.64, 127.07, 124.24, 124.10, 121.06, 120.96, 1 18.34, 1 14.27, 1 1 1.42, 110.10, 1 10.03, 109.86, 108.93, 100.87, 100.71, 55.42, 50.47, 47.05, 27.10; MS (ESI) m/z 597.2 [M+H]⁺; HRMS (ESI) m/z for C33H32N6O2FCI [M+H]⁺ calcd 597.2181, found 597.2172.

CYP-III-118, (R)-N-(3-(lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-4-(4-(3,4- dichlorophenyl)piperazin-l-yl)-2-fluorobenzamide TFA

The general procedure was followed using carboxylic acid 18b as the acylating agent. The crude product was further purified by HPLC to afford compound III-118 as a white solid (29%): ¾ NMR (400 MHz, DMSO-de) δ 1 1.46 (s, 1H), 10.93 (d, J = 2.5 Hz, 1H), 8.74 - 8.60 (m, 2H), 8.03 - 7.87 (m, 3H), 7.60 (t, J = 7.9 Hz, 2H), 7.42 (d, J = 8.9 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.19 (d, J = 2.8 Hz, 1H), 7.05 (ddd, J = 8.1, 6.9, 1.1 Hz, 1H), 6.99 (dd, J = 9.1, 2.9 Hz, 1H), 6.94 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 6.89 - 6.78 (m, 2H), 4.86 (q, J = 6.8 Hz, 1H), 3.44 (dd, J = 6.9, 3.6 Hz, 4H), 3.39 - 3.20 (m, 6H). ¹³C NMR (176 MHz, DMSO-de) δ 172.97, 163.27, 162.19, 160.79, 158.03, 157.85, 154.12, 154.05, 151.48, 150.39, 143.95, 136.11, 131.67, 131.65, 131.54, 130.52, 127.07, 124.1 1, 121.07, 119.85, 1 18.34, 1 16.39, 115.45, 1 14.36, 1 11.43, 1 10.02, 109.94, 109.81, 108.90, 100.82, 100.66, 55.47, 47.02, 46.30, 27.06; MS (ESI) m/z 631.2 [M+H]⁺; HRMS (ESI) m/z for C33H31N6O2FCI2 [M+H]⁺ calcd 631.1791, found 631.1784.

CYP-III-119, (R)-N-(3-(lH ndol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)^ 4- benzamide TFA

The general procedure was followed using carboxylic acid 19b as the acylating agent. The crude product was further purified by HPLC to afford compound III-119 as a white solid

(67%): ¾ NMR (400 MHz, DMSO-de) δ 1 1.66 (s, 1H), 10.94 (d, J = 2.5 Hz, 1H), 8.80 - 8.63 (m, 2H), 8.14 - 8.03 (m, 2H), 7.98 (t, J = 6.8 Hz, 1H), 7.60 (dd, J = 10.3, 8.3 Hz, 2H), 7.33 (d, J = 8.1 Hz, 1H), 7.27 (d, J = 2.4 Hz, 1H), 7.05 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 7.00 (d, J = 1.8 Hz, 2H), 6.93 (td, J = 7.5, 6.9, 1.1 Hz, 1H), 6.89 (t, J = 1.7 Hz, 1H), 6.88 - 6.77 (m, 2H), 4.95 - 4.80 (m, 1H), 3.43 (dd, J = 7.1, 3.5 Hz, 4H), 3.40 - 3.22 (m, 6H). ¹³C NMR (176 MHz, DMSO-de) δ 173.15, 163.29, 162.19, 160.79, 158.07, 157.88, 154.06, 154.00, 152.28, 143.00, 136.10, 134.67, 131.67, 131.64, 127.05, 124.13, 121.06, 1 18.34, 1 17.20, 1 15.63, 114.47, 1 13.09, 1 1 1.42, 109.95, 109.88, 109.74, 108.86, 100.75, 100.59, 55.56, 46.60, 46.18, 26.99; MS (ESI) m/z 631.2 [M+H]⁺; HRMS (ESI) m/z for C33H31N6O2FCI2 [M+H]⁺ calcd 631.1791, found 631.1783.

CYP-III-145, (R)-N-(3-(lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-2-fluoro-4- -l-yl)benzamide TFA

The general procedure was followed using carboxylic acid 20b as the acylating agent. The crude product was further purified by HPLC to afford compound III-145 as a white solid (77%): ¾ NMR (400 MHz, DMSO-de) δ 1 1.65 (s, 1H), 10.94 (d, J = 2.4 Hz, 1H), 8.77 - 8.65 (m, 2H), 8.36 (d, J = 5.2 Hz, 1H), 8.13 - 8.01 (m, 2H), 7.97 (t, J = 6.8 Hz, 1H), 7.60 (t, J = 8.6 Hz, 2H), 7.33 (d, J = 8.1 Hz, 1H), 7.27 (d, J = 2.4 Hz, 1H), 7.15 (s, 1H), 7.05 (ddd, J = 8.1, 6.9, 1.1 Hz, 1H), 6.99 - 6.89 (m, 2H), 6.89 - 6.75 (m, 2H), 4.87 (q, J = 6.6 Hz, 1H), 3.79 - 3.69 (m, 4H), 3.50 - 3.41 (m, 4H), 3.40 - 3.21 (m, 2H). ¹³C NMR (176 MHz, DMSO-de) δ 173.15, 163.29, 162.22, 160.82, 158.82, 158.31, 158.12, 157.93, 157.74, 154.15, 154.09, 152.32, 149.48, 143.02, 138.57, 138.39, 138.20, 138.02, 136.1 1, 131.67, 131.65, 127.06, 124.14, 124.09, 122.53, 121.06, 120.98, 1 18.34, 1 17.37, 1 15.69, 114.47, 1 1 1.43, 109.81,

109.74, 109.66, 108.86, 107.57, 102.55, 102.53, 100.62, 100.46, 55.56, 46.14, 43.76, 27.00; MS (ESI) m/z 632.2 [M+H]⁺; HRMS (ESI) m/z for C33H30N7O2F4 [M+H]⁺ calcd 632.2397, found 632.2398. CYP-II-277, (R)-N-(3-(lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl^

dichlorophenyl)piperazin-l-yl)-2-fluorobenzamide TFA

The general procedure was followed using carboxylic acid 21b as the acylating agent. The crude product was further purified by HPLC to afford compound 11-277 as a white solid

(54%): ¾ NMR (400 MHz, DMSO-de) δ 1 1.47 (s, 1H), 10.93 (d, J = 2.5 Hz, 1H), 8.75 - 8.62 (m, 2H), 8.10 - 7.90 (m, 3H), 7.60 (dd, J = 8.5, 6.7 Hz, 2H), 7.33 (d, J = 8.1 Hz, 1H), 7.26 (d, J = 2.4 Hz, 1H), 7.05 (ddd, J = 8.1, 7.0, 1.2 Hz, 1H), 7.00 (d, J = 1.8 Hz, 2H), 6.94 (ddd, J = 8.0, 6.9, 1.0 Hz, 1H), 6.89 (t, J = 1.7 Hz, 1H), 6.87 - 6.76 (m, 2H), 4.86 (q, J = 6.7 Hz, 1H), 3.57 - 3.18 (m, 10H). ¹³C NMR (176 MHz, DMSO-de) δ 172.98, 163.26, 162.19, 160.79, 158.06, 157.88, 154.06, 153.99, 152.28, 151.53, 143.88, 136.1 1, 134.67, 131.67, 131.65, 127.06, 124.10, 121.06, 1 18.33, 1 17.79, 1 17.21, 1 16.09, 1 14.36, 1 13.09, 1 1 1.42, 109.97, 109.90, 109.74, 108.89, 100.74, 100.58, 79.16, 78.97, 78.78, 55.47, 46.60, 46.18, 27.06; MS (ESI) m/z 631.2 [M+H]⁺; HRMS (ESI) m/z for C33H31N6O2FCI2 [M+H]⁺ calcd 631.1791, found 631.1782. CYP-II-276, (R)-N-(3-(lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-4-(4-(5- l)-2-fluorobenzamide TFA

The general procedure was followed using carboxylic acid 22b as the acylating agent. The crude product was further purified by HPLC to afford compound 11-276 as a white solid

(60%): ¾ NMR (400 MHz, DMSO-de) δ 1 1.55 (s, 1H), 10.94 (d, J = 2.5 Hz, 1H), 8.77 - 8.59 (m, 2H), 8.06 - 8.02 (m, 2H), 7.99 (t, J = 6.8 Hz, 1H), 7.61 (dt, J = 9.1, 5.1 Hz, 2H), 7.33 (d, J = 8.1 Hz, 1H), 7.27 (d, J = 2.4 Hz, 1H), 7.25 - 7.17 (m, 1H), 7.09 - 7.01 (m, 3H), 6.98 - 6.90 (m, 1H), 6.90 - 6.78 (m, 2H), 4.87 (q, J = 6.7 Hz, 1H), 3.43 (dd, J = 6.5, 3.5 Hz, 4H), 3.39 - 3.18 (m, 2H), 2.97 (t, J = 4.9 Hz, 4H), 2.26 (s, 3H). ¹³C NMR (176 MHz, DMSO- de) 8 173.04, 163.30, 162.17, 160.76, 158.09, 157.90, 154.47, 154.40, 152.26, 151.80, 143.61 , 136.1 1 , 132.29, 131.64, 131.61 , 130.76, 130.66, 127.06, 124.12, 122.77, 121.07, 1 19.07, 1 18.33, 1 14.41 , 1 1 1.43, 1 10.07, 109.99, 109.85, 108.89, 100.86, 100.70, 55.50, 50.68, 47.17, 27.03, 17.20; MS (ESI) m/z 61 1.2 [M+H]⁺; HRMS (ESI) m/z for

C34H34N6O2FCI [M+H]⁺ calcd 61 1.2338, found 61 1.2336.

CYP-II-278, (R)-N-(3-(lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-4-(4-(2,3- dichlorophenyl)piperazin-l-yl)-2-fluorobenzamide TFA

The general procedure was followed using carboxylic acid 23b as the acylating agent. The crude product was further purified by HPLC to afford compound 11-278 as a white solid

(58%): ¾ NMR (400 MHz, DMSO-de) δ 1 1.48 (s, 1H), 10.94 (d, J = 2.5 Hz, 1H), 8.76 - 8.60 (m, 2H), 8.05 - 7.92 (m, 3H), 7.68 - 7.54 (m, 2H), 7.40 - 7.29 (m, 3H), 7.27 (d, J = 2.4 Hz, 1H), 7.20 (q, J = 4.8, 4.4 Hz, 1H), 7.05 (ddd, J = 8.2, 6.9, 1.1 Hz, 1H), 7.00 - 6.90 (m, 1H), 6.90 - 6.75 (m, 2H), 4.95 - 4.80 (m, 1H), 3.46 (dd, J = 6.4, 3.5 Hz, 4H), 3.40 - 3.21 (m, 2H), 3.1 1 (t, J = 4.9 Hz, 4H). ¹³C NMR (176 MHz, DMSO-de) δ 173.00, 163.29, 162.16, 160.76, 158.30, 158.1 1 , 157.93, 157.75, 154.39, 154.32, 151.57, 150.77, 143.86, 136.12, 132.67, 131.64, 131.61 , 128.56, 127.07, 126.10, 124.75, 124.12, 121.06, 1 19.73, 1 18.34, 1 14.38, 1 1 1.43, 1 10.16, 1 10.09, 109.89, 108.90, 100.92, 100.76, 55.48, 50.53, 47.02, 27.05; MS (ESI) m/z 631.2 [M+H]⁺; HRMS (ESI) m/z for C33H31N6O2FCI2 [M+H]⁺ calcd 631.1791 , found 631.1793.

CYP-II-279, (R)-N-(3-(lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan-2-yl)-4-(4-(3- chloro-4-fluorophenyl)piperazin-l-yl)-2-fluorobenzamide TFA

The general procedure was followed using carboxylic acid 24b as the acylating agent. The crude product was further purified by HPLC to afford compound 11-279 as a white solid

(69%): ¾ NMR (400 MHz, DMSO-de) δ 1 1.47 (s, 1H), 10.93 (d, J = 2.5 Hz, 1H), 8.73 - 8.62 (m, 2H), 8.04 - 7.91 (m, 3H), 7.60 (t, J = 8.0 Hz, 2H), 7.38 - 7.22 (m, 3H), 7.15 (dd, J = 6.4, 3.0 Hz, 1H), 7.05 (ddd, J = 8.2, 7.0, 1.2 Hz, 1H), 6.99 (dt, J = 9.2, 3.6 Hz, 1H), 6.94 (ddd, J = 7.9, 6.9, 1.0 Hz, 1H), 6.89 - 6.76 (m, 2H), 4.86 (q, J = 6.9, 6.5 Hz, 1H), 3.44 (dd, J = 6.7, 3.7 Hz, 4H), 3.39 - 3.17 (m, 6H). ¹³C NMR (176 MHz, DMSO-de) δ 172.97, 163.26, 162.19, 160.79, 158.09, 157.91, 154.19, 154.13, 151.63, 151.47, 150.28, 148.22, 143.96, 136.1 1, 131.66, 131.64, 127.07, 124.11, 121.06, 1 19.64, 1 19.53, 1 18.34, 1 16.95, 1 16.92, 1 16.80, 115.88, 1 15.84, 1 14.35, 11 1.43, 1 10.06, 109.98, 109.88, 108.90, 100.89, 100.73, 55.47, 47.99, 46.50, 27.06; MS (ESI) m/z 615.2 [M+H]⁺; HRMS (ESI) m/z for C33H31N6O2F2CI [M+H]⁺ calcd 615.2087, found 615.2090. CYP-II-270, (R)-N-(3-(lH-indol-3-yl)-l-oxo-l-(pyridin-4-ylamino)propan^

chloro-2-methoxyphenyl)piperazin-l-yl)-2-fluorobenzamide TFA

The general procedure was followed using carboxylic acid 25b as the acylating agent. The crude product was further purified by HPLC to afford compound 11-270 as a white solid

(69%): ¾ NMR (400 MHz, DMSO-de) δ 1 1.62 (s, 1H), 10.94 (d, J = 2.4 Hz, 1H), 8.75 - 8.65 (m, 2H), 8.13 - 8.02 (m, 2H), 7.97 (t, J = 6.8 Hz, 1H), 7.72 - 7.51 (m, 2H), 7.41 - 7.30 (m, 1H), 7.27 (d, J = 2.3 Hz, 1H), 7.09 - 6.91 (m, 4H), 6.89 (d, J = 2.4 Hz, 1H), 6.88 - 6.77 (m, 2H), 4.87 (q, J = 6.6 Hz, 1H), 3.80 (s, 3H), 3.42 (dd, J = 6.7, 3.6 Hz, 4H), 3.39 - 3.22 (m, 2H), 3.09 (dd, J = 6.2, 3.8 Hz, 4H). ¹³C NMR (176 MHz, DMSO-de) δ 173.17, 163.29, 162.19, 160.79, 158.14, 157.95, 154.43, 154.37, 152.46, 150.82, 142.85, 142.02, 136.1 1, 131.62, 131.60, 127.06, 124.49, 124.14, 121.90, 121.06, 1 18.36, 1 18.34, 1 18.05, 1 17.27, 115.60, 1 14.49, 1 13.15, 11 1.42, 109.97, 109.90, 109.81, 108.86, 100.80, 100.64, 55.76, 55.57, 49.37, 46.90, 26.99; MS (ESI) m/z 627.2 [M+H]⁺; HRMS (ESI) m/z for

C34H34N6O3FCI [M+H]⁺ calcd 627.2287, found 627.2282.

General procedure for the synthesis of 4-(4-(aryl)piperazin-l-yl)benzoic acids 15b, 16b, 17b, 18b. 19b. 20b. 21b. 22b. 23b. 24b. and 25b. To a solution of methyl 2-fluoro-4-(4- aryl)piperazin- l-yl)benzoates (15a - 25a) (ca. 0.25 g) in methanol/THF (10/10 mL) was added 10% NaOH (10 mL), and the reaction mixture was stirred for 3 h at 60 °C. After the reaction was complete as indicated by TLC analysis, the mixture was cooled to ambient temperature, and 2N HC1 was added until a white solid precipitated. The mixture was diluted with ethyl acetate (60 mL) and washed with brine (10 mL x 2). The organic layer was dried over magnesium sulfate, filtered, and concentrated in vacuo. The resulting product yielded the appropriate 4-(4-(aryl)piperazin- 1 -yl)benzoic acids as a white solid in quantitative yield (>98%), and the crude product was used for the next amide coupling reaction with 26 without further purification.

11-263, Methyl 4-(4-(4-chlorophenyl)piperazin-l-yl)-2-fluorobenzoate (15a). The general N- arylation procedure was followed using 1 -(4-chlorophenyl)piperazine to provide 15a as a white solid (47%): R_f= 0.30 (20% ethyl acetate in hexane); ¾ NMR (400 MHz, CDCb) δ 7.85 (t, J = 8.8 Hz, 1H), 7.26 (s, 2H), 6.93 - 6.82 (m, 2H), 6.67 (dd, J = 9.0, 2.5 Hz, 1H), 6.55 (dd, J = 14.5, 2.5 Hz, 1H), 3.88 (s, 3H), 3.55 - 3.42 (m, 4H), 3.32 - 3.23 (m, 4H). ¹³C NMR (101 MHz, CDCb) δ 165.17, 165.01, 164.97, 162.61, 155.40, 155.29, 149.57, 133.56, 133.53, 129.25, 125.38, 1 17.67, 109.54, 109.52, 107.89, 107.78, 101.80, 101.54, 51.93, 49.04, 47.23; MS (ESI) m/z 349.3 [M+H]⁺.

11-261, Methyl 2-fluoro-4-(4-(4-(trifluoromethyl)phenyl)piperazin-l-yl)benzoate (16a). The general N-arylation procedure was followed using 1 -(4-trifluoromethylphenyl)piperazine to provide 16a as a white solid (68%): Rf= 0.30 (20% ethyl acetate in hexane); ¾ NMR (400 MHz, CDCb) δ 7.86 (t, J = 8.8 Hz, 1H), 7.56 - 7.48 (m, 2H), 6.95 (d, J = 8.6 Hz, 2H), 6.66 (dd, J = 9.0, 2.5 Hz, 1H), 6.55 (dd, J = 14.5, 2.4 Hz, 1H), 3.88 (s, 3H), 3.58 - 3.47 (m, 4H), 3.44 (dt, J = 7.6, 2.6 Hz, 4H). ¹³C NMR (101 MHz, CDCb) δ 165.19, 165.01, 164.97, 162.63, 155.24, 155.13, 152.85, 133.62, 133.59, 126.74, 126.70, 126.66, 126.63, 126.12, 123.43, 121.39, 121.06, 120.74, 1 14.76, 109.42, 109.39, 107.96, 107.86, 101.70, 101.43, 51.95, 47.66, 46.96; MS (ESI) m/z 383.2 [M+H]⁺. II- 264, Methyl 4-(4-(2-chlorophenyl)piperazin-l-yl)-2-fluorobenzoate (17a). The general N- arylation procedure was followed using 1 -(2-chlorophenyl)piperazine to provide 17a as a white solid (41%): R_f= 0.39 (20% ethyl acetate in hexane); ¾ NMR (400 MHz, CDCb) δ 7.85 (t, J = 8.8 Hz, 1H), 7.39 (dd, J = 7.9, 1.5 Hz, 1H), 7.29 - 7.19 (m, 1H), 7.09 - 6.97 (m, 2H), 6.68 (dd, J = 8.9, 2.5 Hz, 1H), 6.56 (dd, J = 14.7, 2.4 Hz, 1H), 3.88 (s, 3H), 3.55 - 3.43 (m, 4H), 3.24 - 3.1 1 (m, 4H). ¹³C NMR (101 MHz, CDCb) δ 165.15, 165.02, 164.98, 162.59, 155.74, 155.63, 148.79, 133.45, 133.42, 130.85, 128.99, 127.80, 124.32, 120.45, 109.48, 109.46, 107.62, 107.52, 101.72, 101.44, 51.86, 50.95, 47.56; MS (ESI) m/z 349.2 [M+H]⁺.

III- 102, Methyl 4-(4-(3,4-dichlorophenyl)piperazin-l-yl)-2-fluorobenzoate (18a). The general N-arylation procedure was followed using 1 -(3,4-dichlorophenyl)piperazine to provide 18a as a white solid (37%): Rf= 0.30 (20% ethyl acetate in hexane); ¾ NMR (400 MHz, CDCb) δ 7.86 (t, J = 8.8 Hz, 1H), 7.33 (d, J = 8.9 Hz, 1H), 7.26 (s, OH), 7.05 (d, J = 2.9 Hz, 1H), 6.85 (dd, J = 8.9, 2.9 Hz, 1H), 6.67 (dd, J = 9.0, 2.5 Hz, 1H), 6.56 (dd, J = 14.4, 2.5 Hz, 1H), 3.88 (s, 3H), 3.57 - 3.47 (m, 4H), 3.37 - 3.29 (m, 4H). ¹³C NMR (101 MHz, CDCb) δ 165.14, 164.94, 162.58, 155.12, 155.01, 149.63, 133.63, 133.61, 133.23, 130.86, 1 18.06, 1 16.08, 109.67, 109.65, 108.23, 108.12, 102.01, 101.74, 52.01, 48.93, 46.97; MS (ESI) m/z 383.1 [M+H]⁺.

III-103, Methyl 4-(4-(2,4-dichlorophenyl)piperazin-l-yl)-2-fluorobenzoate (19a). The general N-arylation procedure was followed using 1 -(2,4-dichlorophenyl)piperazine to provide 19a as a white solid (73%): Rf= 0.30 (20% ethyl acetate in hexane); ¾ NMR (400 MHz, CDCb) δ 7.86 (t, J = 8.7 Hz, 1H), 6.88 (t, J = 1.7 Hz, 1H), 6.83 (d, J = 1.7 Hz, 2H), 6.67 (dd, J = 8.9, 2.5 Hz, 1H), 6.56 (dd, J = 14.2, 2.5 Hz, 1H), 3.89 (s, 3H), 3.58 - 3.45 (m, 4H), 3.38 (dd, J = 6.5, 3.9 Hz, 4H). ¹³C NMR (101 MHz, CDCb) δ 165.13, 164.98, 164.94, 162.57, 157.56, 155.02, 151.82, 135.81, 133.65, 133.62, 120.04, 1 14.34, 109.62, 101.98, 101.71, 52.02, 48.23, 46.95; MS (ESI) m/z 383.1 [M+H]⁺.

III-143, Methyl 2-fluoro-4-(4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-l-yl)benzoate (20a). The general N-arylation procedure was followed using l-(5-(trifluoromethyl)pyridin-2- yl)piperazine to provide 29a as a light yellow solid (53%): Rf= 0.24 (20% ethyl acetate in hexane); ¾ NMR (400 MHz, CDCb) δ 8.37 - 8.26 (m, 1H), 7.84 (t, J = 8.8 Hz, 1H), 6.82 (d, J = 3.6 Hz, 2H), 6.63 (dd, J = 9.0, 2.5 Hz, 1H), 6.52 (dd, J = 14.4, 2.5 Hz, 1H), 3.86 (s, 3H), 3.83 - 3.74 (m, 4H), 3.52 - 3.44 (m, 4H). ¹³C NMR (101 MHz, CDCb) δ 165.13, 164.95, 164.91, 162.57, 158.80, 155.15, 155.03, 149.12, 140.60, 140.27, 139.94, 139.61, 133.55, 133.52, 127.24, 124.52, 121.80, 1 19.09, 109.23, 109.20, 108.77, 108.73, 108.70, 108.67, 107.71, 107.61, 107.60, 102.69, 102.64, 101.47, 101.20, 51.88, 46.62, 44.39; MS (ESI) m/z 384.3 [M+H]⁺.

11-266, Methyl 4-(4-(3,5-dichlorophenyl)piperazin-l-yl)-2-fluorobenzoate (21a). The general N-arylation procedure was followed using l-(3,5-dichlorophenyl)piperazine to provide 21a as a light yellow solid (61%): R/= 0.39 (20% ethyl acetate in hexane); ¾ NMR (400 MHz, CDCb) δ 7.85 (t, J = 8.7 Hz, 1H), 6.84 (t, J = 1.7 Hz, 1H), 6.76 (d, J = 1.7 Hz, 2H), 6.65 (dd, J = 8.9, 2.4 Hz, 1H), 6.53 (dd, J = 14.4, 2.4 Hz, 1H), 3.88 (s, 3H), 3.47 (dd, J = 7.0, 3.6 Hz, 4H), 3.34 (dd, J = 6.4, 4.0 Hz, 4H). ¹³C NMR (101 MHz, CDCb) δ 165.15, 164.97, 164.93,

162.59, 155.17, 155.06, 152.18, 135.74, 133.60, 133.57, 1 19.51, 1 14.01, 109.49, 109.47, 108.05, 107.95, 101.79, 101.52, 51.95, 47.92, 46.93; MS (ESI) m/z 383.3 [M+H]⁺.

11-265, Methyl 4-(4-(5-chloro-2-methylphenyl)piperazin-l-yl)-2-fluorobenzoate (22a). The general N-arylation procedure was followed using l-(5-chloro-2-methylphenyl)piperazine to provide 22a as a light yellow solid (50%): Rf= 0.42 (20% ethyl acetate in hexane); ¾ NMR (400 MHz, CDCb) δ 7.85 (t, J = 8.8 Hz, 1H), 7.18 - 7.06 (m, 1H), 7.05 - 6.93 (m, 2H), 6.68 (dd, J = 9.0, 2.5 Hz, 1H), 6.57 (dd, J = 14.6, 2.5 Hz, 1H), 3.88 (s, 3H), 3.51 - 3.40 (m, 4H), 3.06 - 2.98 (m, 4H), 2.29 (s, 3H). ¹³C NMR (101 MHz, CDCb) δ 165.16, 165.05, 165.01,

162.60, 155.74, 155.63, 152.08, 133.49, 133.46, 132.23, 131.98, 131.06, 123.69, 1 19.66, 109.54, 109.52, 107.77, 107.67, 101.79, 101.52, 51.91, 51.39, 47.74, 17.58; MS (ESI) m/z 363.3 [M+H]⁺.

11-267, Methyl 4-(4-(2,3-dichlorophenyl)piperazin-l-yl)-2-fluorobenzoate (23a). The general N-arylation procedure was followed using l-(2,3-dichlorophenyl)piperazine to provide 23a as a white solid (68%): R_f= 0.33 (20% ethyl acetate in hexane); ¾ NMR (400 MHz, CDCb) δ 7.84 (t, J = 8.8 Hz, 1H), 7.23 - 7.1 1 (m, 2H), 6.96 (dd, J = 7.4, 2.2 Hz, 1H), 6.67 (dd, J = 9.0, 2.5 Hz, 1H), 6.56 (dd, J = 14.6, 2.5 Hz, 1H), 3.87 (s, 3H), 3.52 - 3.44 (m, 4H), 3.15 (dd, J = 6.0, 3.9 Hz, 4H). ¹³C NMR (101 MHz, CDCb) δ 165.12, 164.97, 164.93, 162.56, 155.65, 155.54, 150.73, 134.28, 133.45, 133.43, 127.74, 127.68, 125.22, 1 18.68, 109.51, 109.49, 107.73, 107.63, 101.77, 101.51, 51.86, 51.02, 47.53; MS (ESI) m/z 383.3 [M+H]⁺.

11-272, Methyl 4-(4-(3-chloro-4-fluorophenyl)piperazin-l-yl)-2-fluorobenzoate (24a). The general N-arylation procedure was followed using 1 -(3-chloro-4-fluorophenyl)piperazine to provide 24a as a white solid (67%): Rf= 0.24 (20% ethyl acetate in hexane); ¾ NMR (400 MHz, CDCb) δ 7.85 (t, J = 8.8 Hz, 1H), 7.05 (t, J = 8.8 Hz, 1H), 6.95 (dd, J = 6.2, 2.9 Hz, 1H), 6.80 (ddd, J = 9.0, 3.8, 2.9 Hz, 1H), 6.67 (dd, J = 9.0, 2.5 Hz, 1H), 6.55 (dd, J = 14.5, 2.5 Hz, 1H), 3.88 (s, 3H), 3.52 - 3.42 (m, 4H), 3.28 - 3.19 (m, 4H). ¹³C NMR (101 MHz, CDCb) δ 165.14, 165.00, 164.96, 162.58, 155.35, 155.24, 153.95, 151.54, 148.09, 148.07, 133.56, 133.53, 121.36, 121.18, 118.54, 1 17.01, 1 16.79, 1 16.39, 1 16.33, 109.61, 109.59, 107.96,

107.86, 101.89, 101.62, 51.97, 49.55, 47.25; MS (ESI) m/z 367.3 [M+H]⁺.

11-262, Methyl 4-(4-(5-chloro-2-methoxyphenyl)piperazin-l-yl)-2-fluorobenzoate (25a). The general N-arylation procedure was followed using l-(5-chloro-2-methoxyphenyl)piperazine to provide 25a as a white solid (31%): R/= 0.24 (20% ethyl acetate in hexane); ¾ NMR (400 MHz, CDCb) δ 7.85 (t, J = 8.8 Hz, 1H), 6.99 (dd, J = 8.7, 2.5 Hz, 1H), 6.89 (d, J = 2.5 Hz, 1H), 6.80 (d, J = 8.7 Hz, 1H), 6.68 (dd, J = 9.0, 2.5 Hz, 1H), 6.56 (dd, J = 14.6, 2.4 Hz, 1H), 3.88 (s, 3H), 3.87 (s, 3H), 3.54 - 3.44 (m, 4H), 3.23 - 3.13 (m, 4H). ¹³C NMR (101 MHz, CDCb) δ 165.20, 165.09, 165.05, 162.64, 155.72, 155.61, 151.09, 141.91, 133.52, 133.49, 126.09, 122.91, 1 18.88, 112.42, 109.54, 109.52, 107.76, 107.66, 101.79, 101.52, 55.94, 51.93, 50.22, 47.53; MS (ESI) m/z 379.3 [M+H]⁺.

II-263-2, 4-(4-(4-Chlorophenyl)piperazin-l-yl)-2-fluorobenzoic acid (15b). The general procedure of hydrolysis was followed using 15a to provide 15b as a white solid: Rf= 0.75 (100% ethyl acetate); ¾ NMR (400 MHz, DMSO-de) δ 12.51 (s, 1H), 7.72 (t, J = 8.9 Hz, 1H), 7.35 - 7.17 (m, 2H), 7.10 - 6.93 (m, 2H), 6.91 - 6.70 (m, 2H), 3.57 - 3.41 (m, 4H), 3.30 - 3.19 (m, 4H). ¹³C NMR (176 MHz, DMSO-de) δ 164.87, 164.85, 163.95, 162.51, 154.98, 154.92, 149.48, 133.17, 133.15, 128.68, 122.67, 1 17.05, 109.30, 106.95, 106.90, 101.06, 100.91, 47.60, 46.21 ; MS (ESI) m/z 333.1 [M-H]^".

II-261-2, 2-Fluoro-4-(4-(4-(trifluoromethyl)phenyl)piperazin-l-yl)benzoic acid (16b). The general procedure of hydrolysis was followed using 16a to provide 16b as a white solid: Rf= 0.30 (50% ethyl acetate); ¾ NMR (400 MHz, DMSO-de) δ 12.51 (s, 1H), 7.73 (t, J = 8.9 Hz, 1H), 7.53 (d, J = 8.7 Hz, 2H), 7.10 (d, J = 8.7 Hz, 2H), 6.90 - 6.72 (m, 2H), 3.52 (dd, J = 6.8, 3.5 Hz, 4H), 3.44 (dd, J = 6.6, 3.5 Hz, 4H). ¹³C NMR (176 MHz, DMSO-de) δ 164.89,

164.87, 163.97, 162.53, 154.84, 154.78, 152.81, 133.19, 133.17, 127.29, 126.25, 126.23, 126.21, 126.19, 125.76, 124.22, 122.69, 1 18.23, 1 18.05, 1 17.87, 1 17.69, 1 14.1 1, 109.13,

106.87, 106.81, 100.89, 100.73, 46.31, 45.91 ; MS (ESI) m/z 367.2 [M-H]^".

II-264-2, 4-(4-(2-Chlorophenyl)piperazin-l-yl)-2-fluorobenzoic acid (17b). The general procedure of hydrolysis was followed using 17a to provide 17b as a white solid: Rf= 0.30 (50% ethyl acetate); ¾ NMR (400 MHz, DMSO-de) δ 12.51 (s, 1H), 7.72 (t, J = 9.0 Hz, 1H), 7.44 (dd, J = 7.9, 1.5 Hz, 1H), 7.36 - 7.29 (m, 1H), 7.20 (dd, J = 8.1, 1.6 Hz, 1H), 7.08 (td, J = 7.6, 1.5 Hz, 1H), 6.89 - 6.75 (m, 2H), 3.58 - 3.44 (m, 4H), 3.15 - 3.03 (m, 4H). ¹³C NMR (176 MHz, DMSO-de) δ 164.87, 164.85, 163.94, 162.50, 155.21, 155.14, 148.60, 133.15, 133.14, 130.38, 128.16, 127.65, 124.24, 120.97, 109.35, 107.03, 106.97, 101.12, 100.96, 50.48, 46.79; MS (ESI) m/z 335.3 [M+H]⁺. III-102-2, 4-(4-(3,4-Dichlorophenyl)piperazin-l-yl)-2-fluorobenzoic acid (18b). The general procedure of hydrolysis was followed using 18a to provide 18b as a white solid: Rf= 0.60 (100% ethyl acetate); ¾ NMR (400 MHz, DMSO-de) δ 12.52 (s, 1H), 7.72 (t, J = 8.9 Hz, 1H), 7.42 (d, J = 9.0 Hz, 1H), 7.19 (d, J = 2.9 Hz, 1H), 6.99 (dd, J = 9.0, 2.9 Hz, 1H), 6.87 - 6.74 (m, 2H), 3.56 - 3.43 (m, 4H), 3.33 (dd, J = 6.5, 3.9 Hz, 4H). ¹³C NMR (176 MHz,

DMSO-de) δ 164.88, 164.85, 163.95, 162.50, 154.88, 154.82, 150.36, 133.17, 133.15, 131.53, 130.51, 1 19.80, 1 16.34, 115.39, 109.27, 106.99, 106.93, 101.04, 100.89, 46.98, 46.01 ; MS (ESI) m/z 369.1 [M+H]⁺.

III-103-2, 4-(4-(2,4-Dichlorophenyl)piperazin-l-yl)-2-fluorobenzoic acid (19b). The general procedure of hydrolysis was followed using 19a to provide 19b as a white solid: Rf= 0.60 (100% ethyl acetate); ¾ NMR (400 MHz, DMSO-de) δ 7.72 (t, J = 8.9 Hz, 1H), 6.98 (d, J =

1.8 Hz, 2H), 6.88 (t, J = 1.7 Hz, 1H), 6.85 - 6.72 (m, 2H), 3.46 (dd, J = 6.9, 3.5 Hz, 4H), 3.38 (dd, J = 6.5, 3.7 Hz, 4H). ¹³C NMR (176 MHz, DMSO-de) δ 164.99, 164.97, 163.95, 162.50, 154.79, 154.73, 152.29, 134.71, 133.20, 133.19, 1 17.19, 1 13.05, 109.23, 107.27, 107.21, 101.00, 100.85, 46.60, 45.96; MS (ESI) m/z 369.1 [M+H]⁺.

III-143-2, 2-Fluoro-4-(4-(5-(trifluoromethyl)pyridin-2-yl)piperazin-l-yl)benzoic acid (20b). The general procedure of hydrolysis was followed using 20a to provide 20b as a light yellow solid: Rf= 0.66 (100% ethyl acetate); ¾ NMR (400 MHz, DMSO-de) δ 12.49 (s, 1H), 8.36 (d, J = 5.1 Hz, 1H), 7.72 (t, J = 9.0 Hz, 1H), 7.14 (s, 1H), 6.92 (dd, J = 5.1, 1.3 Hz, 1H), 6.87 - 6.70 (m, 2H), 3.84 - 3.70 (m, 4H), 3.54 - 3.44 (m, 4H). ¹³C NMR (176 MHz, DMSO-de) δ 164.88, 164.87, 163.97, 162.53, 158.80, 154.90, 154.83, 149.48, 138.55, 138.37, 138.19, 138.00, 133.17, 133.16, 125.64, 124.09, 122.54, 120.98, 109.09, 107.55, 107.52, 106.78, 106.72, 102.52, 102.50, 102.47, 102.45, 100.82, 100.67, 45.85, 43.71 ; MS (ESI) m/z 370.1 [M+H]⁺.

II-266-2, 4-(4-(3,5-Dichlorophenyl)piperazin-l-yl)-2-fluorobenzoic acid (21b). The general procedure of hydrolysis was followed using 21a to provide 21b as a white solid: Rf= 0.33 (50% ethyl acetate in hexane); ¾ NMR (400 MHz, DMSO-de) δ 17.27 (s, 1H), 12.48 (t, J =

8.9 Hz, 1H), 1 1.75 (d, J = 1.8 Hz, 2H), 1 1.64 (t, J = 1.7 Hz, 1H), 1 1.62 - 11.48 (m, 2H), 8.23 (dd, J = 6.8, 3.6 Hz, 4H), 8.14 (dd, J = 6.8, 3.7 Hz, 4H). ¹³C NMR (176 MHz, DMSO-de) δ 164.88, 164.86, 163.95, 162.50, 154.83, 154.76, 152.24, 134.66, 133.17, 133.16, 1 17.15, 113.02, 109.19, 106.93, 106.87, 100.96, 100.81, 46.55, 45.89; MS (ESI) m/z 369.1 [M+H]⁺. II-265-2, 4-(4-(5-Chloro-2-methylphenyl)piperazin-l-yl)-2-fluorobenzoic acid (22b). The general procedure of hydrolysis was followed using 22a to provide 22b as a white solid: Rf= 0.36 (50% ethyl acetate in hexane); ¾ NMR (400 MHz, DMSO-de) δ 12.52 (s, 1H), 7.73 (t, J = 9.0 Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H), 7.04 (d, J = 7.1 Hz, 2H), 6.91 - 6.67 (m, 2H), 3.54 - 3.40 (m, 4H), 3.05 - 2.89 (m, 4H), 2.26 (s, 3H). ¹³C NMR (176 MHz, DMSO-de) δ 164.88, 164.86, 163.93, 162.49, 155.25, 155.18, 152.25, 133.15, 133.13, 132.28, 130.77, 130.67, 122.77, 1 19.09, 109.35, 107.05, 106.99, 101.12, 100.97, 50.68, 46.92, 17.21 ; MS (ESI) m/z 349.3 [M+H]⁺.

II-267-2, 4-(4-(2,3-Dichlorophenyl)piperazin-l-yl)-2-fluorobenzoic acid (23b). The general procedure of hydrolysis was followed using 23a to provide 23b as a white solid: Rf= 0.30 (50% ethyl acetate in hexane); ¾ NMR (400 MHz, DMSO-de) δ 12.52 (s, 1H), 7.72 (t, J = 8.9 Hz, 1H), 7.44 - 7.29 (m, 2H), 7.19 (q, J = 4.8, 4.4 Hz, 1H), 6.94 - 6.72 (m, 2H), 3.58 - 3.43 (m, 4H), 3.10 (dd, J = 6.1, 3.9 Hz, 4H). ¹³C NMR (176 MHz, DMSO-de) δ 164.87,

164.85, 163.93, 162.49, 155.16, 155.10, 150.75, 133.15, 133.14, 132.66, 128.54, 126.10, 124.74, 1 19.75, 109.37, 107.09, 107.04, 101.16, 101.01, 50.53, 46.76; MS (ESI) m/z 369.3 [M+H]⁺.

II-272-2, 4-(4-(3-Chloro-4-fluorophenyl)piperazin-l-yl)-2-fluorobenzoic acid (24b). The general procedure of hydrolysis was followed using 24a to provide 24b as a white solid: Rf= 0.18 (50% ethyl acetate in hexane); ¾ NMR (400 MHz, DMSO-de) δ 12.51 (s, 1H), 7.72 (t, J = 8.9 Hz, 1H), 7.27 (t, J = 9.1 Hz, 1H), 7.15 (dd, J = 6.3, 3.0 Hz, 1H), 6.99 (dt, J = 9.1, 3.5 Hz, 1H), 6.89 - 6.71 (m, 2H), 3.47 (dd, J = 6.6, 3.8 Hz, 4H), 3.25 (dd, J = 6.4, 3.9 Hz, 4H). ¹³C NMR (176 MHz, DMSO-de) δ 164.87, 164.85, 163.95, 162.50, 154.97, 154.91, 151.62, 150.26, 148.20, 133.16, 119.64, 1 19.53, 1 16.92, 1 16.79, 1 15.83, 1 15.80, 109.35, 107.00, 106.95, 101.12, 100.97, 47.97, 46.22; MS (ESI) m/z 353.3 [M+H]⁺.

II-262-2, 4-(4-(5-Chloro-2-methoxyphenyl)piperazin-l-yl)-2-fluorobenzoic acid (25b). The general procedure of hydrolysis was followed using 25a to provide 25b as a white solid: Rf= 0.21 (50% ethyl acetate in hexane); ¾ NMR (400 MHz, DMSO-de) δ 12.50 (s, 1H), 7.72 (t, J = 9.0 Hz, 1H), 7.05 - 6.94 (m, 2H), 6.89 (d, J = 2.4 Hz, 1H), 6.87 - 6.74 (m, 2H), 3.80 (s, 3H), 3.45 (dd, J = 6.5, 3.6 Hz, 4H), 3.16 - 3.03 (m, 4H). ¹³C NMR (176 MHz, DMSO-de) δ

164.86, 164.84, 163.94, 162.50, 155.19, 155.13, 150.82, 142.02, 133.1 1, 124.50, 121.88,

1 18.05, 1 13.13, 109.28, 106.96, 106.90, 101.04, 100.89, 55.75, 49.37, 46.64; MS (ESI) m/z 365.3 [M+H]⁺.

Hepatic microsomal stability.

Microsome stability was evaluated by incubating 1 μΜ compound with 1 mg/mL hepatic microsomes (human, rat, or mouse) in 100 mM potassium phosphate buffer, pH 7.4 at 37 °C with continuous shaking. The reaction was initiated by adding NADPH, 1 mM final concentration. The final incubation volume was 300 μΐ_^ and 40 μΐ_^ aliquots were removed at 0, 5, 10, 20, 40, and 60 minutes. The aliquots were added to 160 μΐ_^ acetonitrile to stop the reaction and precipitate the protein. NADPH dependence of the reaction is evaluated in parallel incubations without NADPH. At the end of the assay, the samples are centrifuged through a 0.45 micron filter plate (Millipore Solventer low binding hydrophilic plates, cat# MSRLN0450) and analyzed by LC-MS/MS. The data were log transformed and results are reported as half-life.

Binding affinity by UV-vis spectroscopy.

Binding affinity of compounds to CYP51 was approximated from the

spectrophotometry titration curves as previously described.- ' - Spectra were recorded using a Cary scanning spectrophotometer (Varian) in a 1-cm path length quartz cuvette at 23°C. Protein at 1 μΜ concentration in 1 mL of 100 mM potassium phosphate buffer (pH 7.5) containing 10% glycerol was titrated with l-μΐ aliquots of test compound (100 μΜ in DMSO) ranging from 0.1 μΜ to 4 μΜ. The organic solvent effect was compensated by adding the same volume of DMSO in the reference cuvette containing protein.

P450 inhibition.

Cytochrome P450 inhibition was evaluated in human liver microsomes using four selective marker substrates (CYP1A2, phenaceten demethylation to acetaminophen;

CYP2C9, tolbutamide hydroxylation to hydroxytolbutamide; CYP2D6, bufuralol hydroxylation to 4'-hydroxybufuralol; and CYP3A4, midazolam hydroxylation to - hydroxymidazolam) in the presence or absence of 10 or 1 μΜ test compound. The reaction is initiated by the addition of 1 mM NADPH and stopped after ten minutes by the addition of 2- times volume of acetonitrile containing dextrorphan as an internal standard. The

concentration of each marker substrate is approximately its Km. - Furafylline,

sulfaphenazole, quinidine, and ketoconazole were included in each run to validate that the assay could identify selective inhibitors of each isoform.

Molecular docking.

The homology model of 7cCYP51 was generated based on the x-ray co-crystal structure of 7¾CYP51 complexed with 14t (PDB ID code: 4BJK) by using the homology model module implemented in Molecular Operating Environment (MOE). The homology model was refined with Protein Preparation Wizard implemented in Maestro 9.3. A receptor grid was generated from the refined structure using default values except for positional constraint at the nitrogen of 4-acylaminopyridine (radius: 0.8). The structure of 14t was docked into the active site of 7cCYP51 by using Glide5.5 in extra precision (XP) mode with the predefined positional constraint (ligand feature: neutral acceptor). The binding pose of 14t was the same as that in the original co-crystal structure with 1.5 A RMSD of all atom pairs (maximum difference = 5.0 A of fluoro atoms). The structures of 27k, 271, 27r, and 27s were subsequently docked to the model structure of 7cCYP51 by applying the same parameters to predict their binding poses in the 7cCYP51 active site.

T. cruzi CYP51 expression, purification and UV-vis assay.

Recombinant 7cCYP51 was expressed and purified as described elsewhere.⁴--⁴-' 7cCYP51 was used to monitor compound binding in UV-vis spectral assay as previously described.- Binding affinity of hits was estimated from the titration curves using the quadratic tight-binding equation:^

{(S+Et+KD)-[(S+Et+KD)²-4SEtf -⁵} (1)

where Aobs is the absorption shift determined at any ligand concentration; A_max is the maximal absorption shift obtained at saturation; KD is dissociation constant for the inhibitor- enzyme complex; S is the ligand concentration; Et is the total enzyme concentration.

As differences in affinity between tight-binding ligands are reflected in the sharpness of the titration curve, KD values recovered from fits to experimental data approximated by equation (1) are disproportionally sensitive to error in data points near inflection point, setting a limit to the method's sensitivity. Thus, caution was exercised when comparing the tight binding constants. At 0.5 μΜ 7cCYP51 working concentration, only the upper limit of KD at <5 nM (a hundredth of the target concentration) could be estimated for the tightest binding inhibitors, if a plateau in the titration curve was reached at the stoichiometric enzyme- inhibitor ratio.

T. cruzi cell-based assay.

EC50 of compounds were determined in the automated cell-based assay adapted from Engel and co-authors-⁴ and modified as previously described.-⁴

X-ray Crystallography.

Recombinant 7¾CYP51 mutant V34M/D249A/D250A/D251 A modified by inserting a Hiss-tag at the C-terminus and replacing the first 31 residues upstream of P32 with the fragment MAKKTSSKGKL was used to obtain co-crystal structure with 14t. Concentrated purified protein stored at -80°C was diluted to 0.1 mM prior to crystallization by mixing with water supplemented with 14t to reach 1 : 1 protein:inhibitor ratio. Crystallization conditions were determined using commercial high-throughput screening kits available in deep-well format (Hampton Research), a nanoliter drop-setting Mosquito robot (TTP LabTech) operating with 96-well plates, and a hanging drop crystallization protocol. Crystals were further optimized in 96-well plates for diffraction data collection and harvested directly from the 200-nL drops. Prior to data collection, crystals were cryo-protected by plunging them into a drop of reservoir solution supplemented with 20% ethylene glycol, then flash frozen in liquid nitrogen.

Diffraction data were collected at 100- 1 10 K at Beamline 8.3.1, Advanced Light Source, Lawrence Berkeley National Laboratory, USA. Data indexing, integration, and scaling were conducted using MOSFLM- and the programs implemented in the ELVES software suite.-- The crystal structures were determined by molecular replacement using diffraction data processed in the C2 space group, with R_mer_ge of 8.6% and atomic coordinates of T. brucei CYP51 (PDB ID code: 2X2N) as a search model. The final model was built using COOT- and refinement was performed by using REFMAC5 software- until R and Rfiee converged to 19.4% and 27.4%, respectively. Data collection and refinement statistics are shown in Table 5, above.

Only one of the four protein chains (chain A) constituting an asymmetric unit contained electron density corresponding to the whole molecule of 14t; 14t was assigned PDB code 181. In three other chains, only the N-indolylpyridinyl portion of 14t could be unambiguously placed. Thus, coordinates for the disordered biaryl moiety in chains B, C and D were omitted from the PDB entry.

T. cruzi maintenance.

Trypanosoma cruzi, Y luc strain, episomally expressing the firefly luciferase gene, was developed as described elsewhere (36). Cultured trypomastigotes were obtained by weekly infection of C2C12 myoblasts, with trypomastigotes being released in the supernatant 4 to 7 days post infection, collected by centrifugation for 15 min at 3300 rpm. Without selective antibiotic pressure, the luciferase expression in the parasite is detectable for about as long as 7 passages in mammalian culture. To maintain high titer of luciferase marker in parasite population, the pressure of G418 antibiotic was applied to epimastigote form. For that, the epimastigotes were cultivated in LIT medium (Camargo et al., 1964), supplemented with 10% fetal bovine serum and 200 μg/ml of G418, at 28° C. Once a month, myoblast cultures were infected with 2-4 week old epimastigotes enriched with the metacyclic trypomastigotes. Forty-eight hours post-infection, the epimastigotes were removed from the medium by successive washing of cultures with phosphate buffer saline (PBS). Seven days post-infection, trypomastigote population enriched with the transgenic parasites expressing luciferase was released to the medium. 4-day dosing mouse model of T. cruzi infection.

Eight-week-old female Swiss Webster albino mice (average weight 20 g) were obtained from the Simonsen Labs. All animal procedures were approved and carried out in accordance with the guidelines established by the Institutional Animal Care and Use Committee from UCSF (Approval number AN087605-01). Mice were housed at a maximum of 5 per cage and kept in a specific -pathogen free (SPF) room at 20 to 24° C under a 12-h light/12-h dark cycle and provided with sterilized water and chow ad libitum. To infect the mice, trypomastigotes of T. cruzi Y luc strain were harvested from culture supernatant and injected intraperitonealy (i.p.), 10⁵ trypomastigotes per mouse. Three days post infection, mice were anesthetized by inhalation of isofluorane (controlled flow of 1.5% of isofluorane in air was administered through a nose cone via gas anesthesia system). Mice were injected i.p. with 150 mg/kg D-luciferin potassium salt (Gold Biotechnology) dissolved in PBS and imaged after 5 min using IVIS Spectrum Pre-clinical In Vivo Imaging System (Perkin Elmer, Waltham, MA) and the data acquisition and analysis software Livinglmage V4.1 (Perkin Elmer, Waltham, MA). Only mice with detectable luminescence were used for treatment. The compounds potency was evaluated following oral (o.p.) administration. Compounds were administered as suspension in 20% 2-hydroxypropyl-b-cyclodextrin (HPbCD) (VWR International) or as solution in Kolliphor HS 15 (Sigma #42966), also known as solutol. Only one compound, CYP-II-258, was fully soluble in HPbCD, while all dissolved in 20% solutol upon overnight incubation. Treatment with compounds started same day, in groups of five mice, and specified doses were administered twice a day (b.i.d.) for four consecutive days. Two control groups included untreated mice, which received 20% HPbCD or solutol, and the positive control group, which received benznidazole 50 mg/kg, both b.i.d. by oral gavage. After four days of treatment, mice were imaged again as described above. The absolute numbers of measured photons/s/cm² were averaged between all five mice in each group and compared directly between compound-treated mice and the control groups.

Anti-Γ. cruzi activity in cell-based assay.

EC50 values of compounds were determined in a cell-based assay performed in triplicate as described elsewhere. (Gunatilleke, S. S.; Calvet, C. M.; Johnston, J. B.; Chen, C. K.; Erenburg, G.; Gut, J.; Engel, J. C; Ang, K. K.; Mulvaney, J.; Chen, S.; Arkin, M. R.;

McKerrow, J. H.; Podust, L. M., Diverse inhibitor chemotypes targeting Trypanosoma cruzi CYP51. PLoS Negl. Trop. Dis. 2012, 6 (7), el 736.) Briefly, mouse C2C12 myoblasts (ATCC #CRL- 1772) used to harbor parasites were cultivated in Dulbecco's Modified Eagle's Medium H-21 containing 4.5 g/1 glucose (DMEM H-21), supplemented with 5% fetal bovine serum (FBS), 25 mM HEPES, 2 mM L-glutamine, 100 U/ml penicillin and 100 μ^ηιΐ streptomycin. T. cruzi CA-I/72 trypomastigotes were obtained from infected-culture supernatants after 4-7 days of infection. Cultures were maintained at 37°C with 5% CO2. Trypomastigotes and C2C 12 cells concentration was determined using a Neubauer hemocytometer. Sterile, black 384-well plates with clear-bottom wells (Greiner Bio-One) were seeded with 500 cells/well and then were infected with 2500 parasites/well in a final volume of 50 μΐ /well. Culture plates were incubated at 37°C with 5% CO2 for 24-hours. After that, culture medium was removed and test compounds were added in fresh medium. For this, an intermediate plate (384-well plate) was prepared by serial dilution (10 mM, 2 mM, 400 μΜ, 80 μΜ, 16 μΜ, 3 μΜ, 128 ηΜ, 25.6 ηΜ, 5.1 ηΜ) for all the compounds in 100 % DMSO. Then, 50 nl of each sample were diluted in 50 μΐ media (DMEM H-21) and added to the experimental plate followed by incubation at 37°C with 5% CO2 for 72h. Wells containing non-infected cells were used as a positive control (100% cell survival), while T. crwzz^'-infected but untreated cells (0% cell survival) were used as a negative control. Cells were then fixed for 2 h with 4% paraformaldehyde, and rinsed with a solution of 150 mM

NaCl, l OO mM NPMT, 0.1% Triton X- 100 and 0.1% NaNs. After that, they were treated for 4 h with 0.2 μg/ml of the DNA fluorescent dye, DAPI (4,6-diamidino-2-phenylindole), diluted in the same solution. Plates were kept at ambient temperature until image acquisition was performed. Images were acquired by an IN Cell Analyzer 2000 (GE Healthcare) and the procedure and analyses were performed according to previously described.

Inhibition of sterol biosynthesis in amastigotes.

Sterol profiling was performed on T. cruzi whole-cell lipid extracts. Posaconazole (100 nM) was used as a positive control while benznidazole (5 μΜ) and K777 (1.6 μΜ) served as a negative controls. Compounds 9 and 10 were tested at 100 nM. Briefly, C2C 12 mouse myoblasts were infected with T. cruzi (CAI/72 strain) and treated with compounds after 72h of infection. After 24 h of treatment, the cultures were detached, and the lipids from cell pellet were extracted with chloroform/methanol, chloroform and acetonitrile, each step followed by several rounds of washes with water to extract polar molecules. The organic layer was then dried under nitrogen gas and subsequently treated with 75 μΐ_^ Ν,Ν- bis(trimethylsilyl)-2,2,2-trifluoroacetamide (BSTFA) for 2 h at 37 °C to facilitate chemical derivatization with trimethylsilyl (TMS) groups (BSTFA, Sigma-Aldrich). The TMS- derivatized lipid mixture was analyzed by injecting 3 μΐ_^ directly into an Agilent HP5790 gas chromatography system outfitted with a DB5-MS analytical column (30 m, 0.25 mm i.d., 0.33 μηι film thickness, Agilent) coupled to a mass selective detector. The lipids were separated on the analytical column using a temperature profile that begins at 200 °C for 1 min, increases by 15 °C/min up to 300 °C and then holds at 300 °C for 20 minutes. The inlet temperatures of the GC and the MSD were held at 250 °C and 300 °C, respectively. The mass spectrometer scanned from m/z 50 - 750 during the course of analysis.

Animal model.

To assess in vivo efficacy of test compounds, a 4-day mouse model of infection by transgenic T.cruzi Y luc strain expressing firefly luciferase was used as previously described.(Andriani, G.; Chessler, A. D.; Courtemanche, G.; Burleigh, B. A.; Rodriguez, A., Activity in vivo of anti-Trypanosoma cruzi compounds selected from a high throughput screening. PLoSNegl. Trop. Dis. 2011, 5 (8), el298; Calvet, C. M.; Vieira, D. F.; Choi, J. Y.; Kellar, D.; Cameron, M. D.; Siqueira-Neto, J. L.; Gut, J.; Johnston, J. B.; Lin, L.; Khan, S.; McKerrow, J. H.; Roush, W. R.; Podust, L. M., 4-Aminopyridyl-based CYP51 inhibitors as anti-Trypanosoma cruzi drug leads with improved pharmacokinetic profile and in vivo potency. J. Med. Chem. 2014, 57 (16), 6989-7005.) Eight-week-old female Swiss Webster albino mice (average weight 20 g) were obtained from Simonsen Labs (Gilroy, CA). All animal protocols were approved and carried out in accordance with the guidelines established by the Institutional Animal Care and Use Committee from UCSF (Approval number

AN087605-01). Mice were housed at a maximum of 5 per cage and kept in a specific- pathogen free (SPF) room at 20 to 24° C under a 12-h light/ 12-h dark cycle and provided with sterilized water and chow ad libitum. To infect the mice, trypomastigotes of T. cruzi Y luc strain were harvested from culture supernatant and injected intraperitonealy, 10⁵ trypomastigotes per mouse. Three control groups included untreated mice, which received a vehicle, 20% Kolliphor HS 15 (also known as Solutol), and the positive control groups, which received 25 or 50 mg/kg benznidazole, all via oral gavage (p.o.), twice a day (b.i.d). Starting on day 4 the infected mice were treated with test compounds at 25 mg/kg administered in 20% Kolliphor, p.o., b.i.d., for four consecutive days. At day 7 post-infection, the luminescent signal in the mice was read upon injection of D-luciferin. The absolute numbers of measured photons/s/cm² were averaged between all five mice in each group and compared directly between compound-treated mice and the control groups. Two tailed paired Student t test was used to assess statistical significance between luminescence values from vehicle -treated and compound-treated groups at day 7 post-infection; values are statistically significant when p< 0.05. Single dose PK

Compounds were dose at 25 mg/kg in eight-week-old female Swiss Webster albino mice over oral gavage. All compounds were formulated to a concentration of 5 mg/ml in 20% Kolliphor/80% water. Three mice were dosed with each compound and n=3 plasma samples were collected at approximately 0.25, 0.5, 1, 2, 4, 6, 8, 12 and 24 h post exposure. Plasma samples were treated with 5 times v/v acetonitrile to precipitate protein and filtered through a 0.2 μηι filter prior to analysis by LC-MS/MS using ABSciex 5500.

CYP51 expression and purification.

Heterologous expression and purification of recombinant T. cruzi CYP51 modified by replacing the first 31 residues upstream of Pro32 with the fragment MAKKTSSKGKL (von Wachenfeldt, C; Richardson, T. H.; Cosme, J.; Johnson, E. F., Microsomal P450 2C3 is expressed as a soluble dimer in Escherichia coli following modification of its N-terminus. Arch. Biochem. Biophys. 1997, 339 (1), 107-1 14) and by inserting a His6-tag at the C- terminus were carried out according to the protocol described elsewhere. (See: Gunatilleke, S. S.; Calvet, C. M.; Johnston, J. B.; Chen, C. K.; Erenburg, G.; Gut, J.; Engel, J. C; Ang, K. K.; Mulvaney, J.; Chen, S.; Arkin, M. R.; McKerrow, J. H.; Podust, L. M., Diverse inhibitor chemotypes targeting Trypanosoma cruzi CYP51. PLoSNegl. Trop. Dis. 2012, 6 (7), el 736; Chen, C.-K.; Leung, S. S. F.; Guilbert, C; Jacobson, M. P.; McKerrow, J. H.; Podust, L. M., Structural characterization of CYP51 from Trypanosoma cruzi and Trypanosoma brucei bound to the antifungal drugs posaconazole and fluconazole. PLoSNegl. Trop. Dis. 2010, 4, e651.) Briefly, six liters of Terrific Broth medium supplemented with 100 μg/ml ampicillin, 1 mM thiamine and trace elements was inoculated with 60 ml of the overnight culture and was incubated at 28°C, 230 rpm until OD600nm reached 1.0. CYP51 expression was induced by adding 0.25 mM isopropyl- -thiogalactopyranoside (IPTG) and ImM δ-aminolevulinic acid, a precursor in heme biosynthesis, was added at that time. After induction, the growth was continued at 18°C at 180 rpm for 48 h. Cells were harvested, resuspended in 50 mM Tris, pH 8.5, 1 mM EDTA, 100 mM NaCl, 0.5 mM PMSF, 1 mM DTT and lysed using a microfluidizer. After centrifugation, the soluble fraction was purified by conventional Ni- NTA agarose chromatography using a linear gradient of imidazole (0 to 0.5 M) in 50 mM potassium phosphate, pH 8.0, 10% glycerol, 1 mM DTT, 0.5 mM EDTA, 500 mM NaCl. After dialysis overnight against 20 mM potassium phosphate pH 7.5, 10% glycerol, 1 mM DTT, 0.5 mM EDTA, the sample was applied on MonoQ column. The flow-through fractions were applied on Mono S column and the protein was eluted in the same buffer using linear NaCl gradient (0-0.5 M). Fractions containing CYP51 were combined and concentrated using Centriprep concentrating device (Millipore). These samples were stored at -80°C and used as needed for co-crystallization and binding assays.

All patents and publications referred to herein are incorporated by reference herein to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.

The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Claims

What is claimed is:

1. A compound of formul

wherein Het is a 5- or 6-membered heteroaryl comprising a nitrogen atom, R is independently at each occurrence H or (Ci-Ce)alkyl, X and Y are each independently C(=0) or SO2, R¹ is an aryl(Co-C6)alkyl or a heteroaryl(Co-C6)alkyl wherein the aryl or the heteroaryl is substituted with 0-3 J, and R² is a group of formula Ar'-Zn-Ar², wherein Ar¹ and Ar² are independently selected aryl or heteroaryl, wherein each aryl or heteroaryl is substituted with 0-3 J, and Z is a 5- to 7-membered heterocyclyl comprising one or more nitrogen atoms, n = 0 or 1 ; J is halo, halo(Ci-C6)alkyl, (Ci-C6)alkyl, cyano, (Ci-C6)alkoxyl, (Ci-C6)alkoxycarbonyl, C(=0)NR2, or NRC(=0)(Ci-C6)alkyl; wherein χ signifies a chiral center;

or a pharmaceutically acceptably salt thereof.

2. A compound of claim 1 of formula

3. A compound of claim 1 of formula

wherein J¹ is halo, halo(Ci-C6)alkyl, (Ci-C6)alkyl, cyano, (Ci-C6)alkoxyl, (Ci- Ce)alkoxycarbonyl, C(=0)NR2, or NRC(=0)(Ci-C₆)alkyl; and m = 0, 1, 2, or 3.

4. The compound of claim 3 of formula

5. The compound of claim 3 or 4 wherein m = 0.

6. The compound of any one of claims 1 -5 wherein the absolute configuration at the chiral center signified by χ is the (R) absolute configuration.

7. The compound of any one of claims 1 -5 wherein the absolute configuration at the chiral center signified by χ is the (S) absolute configuration.

8. The compound of claim 3 of formula

wherein ring Ar¹ and ring Ar² are each independently substituted with 0-3 fluoro, chloro, (Ci- C6)alkyl, (Ci-C6)alkoxyl, or trifluoromethyl groups, or any combination thereof.

9. The compound of claim 8 having the (R) absolute configuration at the chiral center signified by χ.

10. The compound of claim 9, wherein the compound is any one of

or a pharmaceutically acceptable salt thereof.

wherein ring Ar¹ and ring Ar² are each independently substituted with 0-3 fluoro, chloro, (Ci- C6)alkyl, (Ci-C6)alkoxyl, or trifluoromethyl groups, or any combination thereof. The compound of claim 1 1 having the (R) absolute configuration at the chiral center

or a pharmaceutically acceptable salt thereof.

CYP-II-277 CYP-II-276

or a pharmaceutically acceptable salt thereof.

15. A pharmaceutical composition comprising a compound of any one of claims 1-14 and a pharmaceutically acceptable excipient.

16. The pharmaceutical composition of claim 15 wherein the excipient comprises a cyclodextrin or solutol.

17. The composition of claim 16 wherein the cyclodextrin is a 2-hydroxypropyl-y- cyclodextrin.

18. An oral dosage form for treatment of Chagas disease or a fungal disease comprising the pharmaceutical composition of claim 17.

19. A method of inhibiting a sterol C14-demethylase comprising contacting the sterol C14-demethylase with an effective amount or concentration of a compound of any one of claims 1-14.

20. The method of claim 19 wherein the sterol C14-demethylase is CYP51.

21. The method of claim 19, wherein the sterol C14-demethylase is disposed within living tissue of a patient.

22. A method of treatment of Chagas disease, comprising administering to a patient afflicted therewith an effective dose of a compound of any one of claims 1-14.

23. The method of claim 22, wherein the compound is administered to the patient as a cyclodextrin inclusion complex.

24. The method of claim 22, wherein the compound is administered orally to the patient.

25. A method of treatment of a fungal disease, comprising administering to a patient afflicted therewith an effective dose of a compound of any one of claims 1-14.

26. The method of claim 25, wherein the compound is administered to the patient as a cyclodextrin inclusion complex.

27. The method of claim 25, wherein the compound is administered orally to the patient.