AMYLOIDOGENIC IMMUNOGLOBULIN LIGHT CHAINS

RELATED APPLICATIONS

[0001] The present application claims the benefit of priority to U.S. Provisional Patent

Application No. 62/826,476, filed on March 29, 2019, and which application is incorporated by reference as if fully set forth herein.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers DK046335 and TR002550 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0003] Light chain (LC) amyloidosis is a progressive and often fatal degenerative disease caused by conformational changes within immunoglobulin light chains after secretion by clonal plasma cells that result in organ toxicity, e.g. cardiomyopathy, nephrotic syndrome and end-stage renal failure. The light chain conformational changes also often lead to light chain aggregation, which may also drive proteotoxicity in some post-mitotic tissues. The pathologic mechanisms of disease leading to organ toxicity include both toxicity of amyloidogenic LC and mass effects of deposits, both modulated by misfolded LC concentration. Light chain amyloidosis patients are treated today by targeting the cancer component of this disease (proliferating clonal plasma cells) employing chemotherapy cocktails typically involving proteasome inhibitors (and when possible, stem cell transplants), which ideally eliminate the clonal plasma cells secreting full-length light chains. However, complete clonal plasma cell eradication is achieved in only 30-40% of the patients and most eventually relapse. Organ response, as a measure of improvement in organ function, is often limited (< 50%). Organ damage remains the major source of mortality and morbidity. Moreover, light chain amyloidosis patients exhibiting cardiac involvement are often too sick to tolerate chemotherapy and die within a year of diagnosis. SUMMARY

[0004] Light chain amyloidosis is caused by conformational changes within immunoglobulin light chains that generally lead to aggregation. Current chemotherapy treatments aim to eliminate the clonal plasma cells that secrete full-length light chains; however, complete eradication is achieved in only 30-40% of the patients. The kinetic stabilizer strategy introduced herein does not require an understanding of the non-native light chain structure-proteotoxicity relationships driving organ degeneration in light chain amyloidosis (AL) to be successful at stopping disease progression because it stops light chain conformational excursions at the beginning of the aggregation cascade. The small molecule kinetic stabilizers identified herein bind to conserved residues at the variable domain-variable domain interface in the native dimer, stabilizing this putative non-toxic structure. Without being bound by any theoiy, we hypothesize that small molecule kinetic stabilizers of immunoglobulin light chains will be successful at stopping disease progression because they stop light chain conformational excursions at the beginning of the aggregation cascade. These function strictly analogously to tafamidis, which has proven to be efficacious for the transthyretin amyloidoses. The small molecule kinetic stabilizers disclosed herein bind to conserved residues at the variable domain-variable domain interface in the native light chain dimer, stabilizing this putative non-toxic structure against the conformational changes and aggregation that cause light chain amyloidosis.

[0005] The present disclosure provides in various embodiments an LC kinetic stabilizer, i.e., a compound of Formula la, Formula lb, Formula P or a pharmaceutically acceptable salt thereof as defined herein:

wherein

A is H or

X ¹ is O or NR°; X ² is selected from the group consisting of a bond, NR ¹² , 0, C(O), C(0)NR ¹¹ , and CR ¹² R ¹¹ ; X ³ is O or NR ¹³ ;

W is selected from the group consisting of a bond, OC(O), C(0)0, C(0)NR ¹⁴ , NR ¹⁴ C(0)0, 0C(0)NR ¹⁴ , NR ¹⁴ C(0), NR ¹⁴ C(0)NR ¹⁵ , NR ¹⁴ ,

, and

m is an integer selected from 1, 2, 3, 4, 5, and 6;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

p is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

r is an integer selected from 0, 1, 2, and 3;

Z ¹ is selected from the group consisting of H, C ₁ -C ₆ -alkyl, C ₃ -Cs-cycloalkyl, 3- to 6- membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₂ -C ₆ -alkenyl, C ₃ -C ₈ -cycloalkenyl, 3- to 6-membered heterocycloalkenyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₆ -C ₁₀ -aryl, 5- to 10-member ed heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), -(C ₁ -C ₆ -alkyl)(C ₆ -C ₁₀ -aryl), -(C ₁ -C ₆ -alkyl)(5- to 10-membered heteroaryl) (wherein 1-4 heteroaryl members are independently selected from N, O, and S), halogen, NR ¹⁶ R ¹⁷ , COOR ¹⁸ ; OR ¹⁸ , NR ¹⁶ S0 ₂ R ¹⁸ , NR ¹⁶ COR ¹⁸ ,

X ⁴ (CR ²¹ R ²² )aCONR ¹⁶ R ¹⁷ , X ⁴ (CR ²¹ R ²² ) _a COOR ¹⁸ , X ⁴ (CR ²¹ R ²² ) _a COR ¹⁸ ,

X ⁴ (CR ²¹ R ²² ) _a NR ¹⁶ R ¹⁷ , X ⁴ (CR ²¹ R ²² ) _a OR ¹⁸ , S02NR ¹⁶ R ¹⁷ , X ⁴ (CR ²¹ R ²² ) _a NR ¹⁶ COR ¹⁸ , C(N=R ²³ )NR ²⁴ 0H, and X

wherein

X ⁴ is a bond, O, or NR°; and a is an integer selected from 0, 1, 2, 3, 4, 5, and 6; Z ^z is selected from the group consisting of H, C1-C6-alkyl, C3-C8-cycloalkyl, 3- to 6- membered heterocycloalkyl (wherein 1 -4 ring members are independently selected from N, Q, and S), C ₂ -C ₆ -alkenyl, C ₂ -C ₆ -alkynyl, C ₃ -C ₈ cycloalkenyl, 3- to 6-membered heterocycloalkenyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₆ -C ₁₀ -aryl, 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, Q, and S), -(C ₁ -C ₆ -alkyl)(C ₆ -C ₁₀ -aryl), -(C ₁ -C ₆ -alkyl)(5- to 10-membered heteroaryl) (wherein 1 -4 heteroaryl members are independently selected from N, O, and S), CN, OR ¹⁸ and NR ¹⁹ R ²⁰ ;

Ar ¹ is a divalent moiety selected from C ₆ -C ₁₀ -arylene and 5- to 10-membered heteroarylene (wherein 1-4 heteroaryl members are independently selected from N, O, and S);

R ¹ is selected from the group consisting of H, C ₁ -C ₆ -alkyl, C ₃ -C ₈ -cycloalkyl, 3- to 6- membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₂ -C ₆ -alkenyl, C ₃ -C ₈ -cycloalkenyl, 3- to 6-membered heterocycloalkenyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₆ -C ₁₀ -aryl, 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), -(C ₁ -C ₆ -alkyl)(C ₆ -C ₁₀ -aryl), -(C ₁ -C ₆ -alkyl)(5- to 10-membered heteroaryl) (wherein 1-4 heteroaryl members are independently selected from N, Q, and S), OH, (CR ^2l R ²² ) _b OR ¹⁸ (b is an integer selected from 0, 1, 2, 3, 4, 5, and 6), halogen, and (C ₁ -C ₆ )haloalkyl;

R ² , R ³ , and R ⁴ are independently selected from the group consisting of H, C ₁ -C ₆ -alkyl, -(C ₁ .- C ₆ -aikyl)(C ₆ -C ₁₀ -aryl), OH, (CR ² R ²² )' _b QR ¹⁸ , halogen, C3-C8-cycloalkyl, 3- to 6- membered heterocycloalkyl (wherein 1 -4 ring members are independently selected from N, O, and S), C ₂ -C ₆ -alkenyl, C ₃ -C ₈ -cycloalkenyl, 3- to 6-membered heterocycloalkenyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₆ C ₁₀ -aryl, 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), -(C ₁ -C ₆ -alkyl)(C ₆ -C ₁₀ -aryl), -(C ₁ -C ₆ -alkyl)(5- to 10-membered heteroaryl) (wherein 1-4 heteroaryl members are independently selected from N, O, and

S); R° and R ⁵ - R ²² are independently selected from the group consisting of H, C ₁ -C ₆ -alkyl, Ci- C6-haloalkyl, C ₃ -C ₈ -cycloalkyl, 3- to 6-membered heterocycloalkyl (wherein 1 -4 ring members are independently selected from N, O, and S), C ₂ -C ₆ -alkenyl, C ₂ -C ₆ -alkynyl, C ₃ -C ₈ -cycloalkenyl, 3- to 6-membered heterocycloalkenyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₆ -C ₁₀ -aryl, 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), -(C ₁ -C ₆ - alkyl)(C ₆ -C ₁₀ -aryl), -(C ₁ -C ₆ -alkyl)(5- to 10-membered heteroaryl) (wherein 1-4 heteroaryl members are independently selected from N, O, and S);

R ¹⁶ and R ¹⁷ are independently selected from the group consisting of H, C ₁ -C ₆ -alkyl, C ₃ -C ₈ - cycloalkyl, 3- to 6-membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₂ -C ₆ -alkenyl, C ₃ -C ₈ -cycloalkenyl, 3- to 6- membered heterocycloalkenyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₆ -C ₁₀ -aryl, 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), -( C ₁ -C ₆ -alkyl)(C ₆ -C ₁₀ -aryl), -(C ₁ - C ₆ -alkyl)(5- to 10-membered heteroaryl) (wherein 1-4 heteroaryl members are independently selected from N, O, and S), C(0)0R ¹⁸ , C(0)R ¹⁸ , and SO2R ¹⁸ ;

R ¹⁴ and R ⁹ , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring;

R ¹⁵ and R ⁹ , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring;

R ¹⁶ and R ⁹ , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring;

R ¹⁶ and R ¹⁴ , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring;

R ¹⁶ and R ¹⁵ , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring;

R ¹⁶ and R ¹⁷ , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring wherein ring members are selected from C, O and N; R ¹⁹ and R ²⁰ , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring;

R ²¹ and R ²² , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring;

OR a compound according to Formula II, or a pharmaceutically acceptable salt thereof:

(II) wherein

R ²³ - R ²⁸ are independently selected from the group consisting of H, halo, C ₁ -C ₆ -alkyl, C ₃ - C ₈ -cycloalkyl, 3- to 6-member ed heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₂ -C ₆ -alkenyl, C ₃ -C ₈ -cycloalkeny 1, 3- to 6- membered heterocycloalkenyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₆ -C ₁₀ -aryl, 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), -(C ₁ -C ₆ -alkyl)(C ₆ -C ₁₀ -aryl), -(C ₁ - C ₆ -alkyl)(5- to 10-membered heteroaryl) (wherein 1-4 heteroaryl members are independently selected from N, O, and S);

Ar ² is C ₆ -C ₁₀ -aryl or 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S; q is an integer selected from 0, 1, 2, 3, and 4; t is an integer selected from 0, 1, 2, 3, and 4; wherein any alkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, and ring moiety in Formula la, Formula lb, or Formula II is optionally substituted with one to six substituents selected from the group consisting of hydroxy, halo, C ₁ -C ₆ -haloalkyl, -NR’ ₂ (wherein each R’ is independently selected from the group consisting of H, C ₁ -C ₆ -alkyl, C2-C6-alkenyl, C ₂ -C ₆ -alkynyl, and C ₆ -C ₁₀ -aryl), - NHC(0)(0C ₁ -C ₆ -alkyl), -NO2, -CN, oxo, -C(0)0H, -C(0)0(C ₁ -C ₆ -alkyl), -C ₁ -C ₆ - alkyl(C ₁ -C ₆ -alkoxy), -C(0)NH ₂ , -C(0)C ₁ -C ₆ -alkyl, -OC ₁ -C ₆ -alkyl, -Si(C ₁ -C ₆ - alkyl) ₃ , -S(0) _0-2 -(C ₁ -C ₆ -alkyl), C<i-Cio-aryl, -(C ₁ -C ₆ -alkyl)(C ₆ -C ₁₀ -aryl), 3- to 14- membered heterocycloalkyl and -(C ₁ -C ₆ -alkyl)-(3- to 14-membered heterocycle) (wherein 1-4 heterocycle members are independently selected from N, O, and S), and - 0(C ₆ -C ₁₄ -aryl).

[0006] In some embodiments, the compound of Formula la, Formula lb, and Formula P does not include any of the following compounds:

[0007] In various embodiments, provided is a method of stabilizing an immunoglobulin light chain dimer in a native conformation thereof, comprising contacting the dimer and an effective amount of any compound described herein, including all enumerated compounds, such as any one of the formulae 1-21 (see Table 1, below).

[0008] Further, in various embodiments, provided is a method of treatment of light chain amyloidosis in a patient, comprising administering to the patient an effective amount of any compound described herein, including all enumerated compounds, such as any one of the formulae 1-21 (see Table 1, below). [0009] The present disclosure also provides in an embodiment a pharmaceutical composition comprising a compound of Formula la, Formula lb, Formula P, or any enumerated compound described herein, in combination with a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

[0010] Figure 1: High-throughput screen for LC kinetic stabilizers a) LCs consist of variable (V, yellow) and constant (C, grey) immunoglobulin domains, which form homodimers linked by a disulfide bond between C214 residues (black line). Cleavage of dye-labeled protein by protease leads to a decrease in FP in the resulting peptides. The rate of proteolysis depends on a LC’s kinetic stability, which can be enhanced by the binding of a stabilizing small molecule b) Proteolysis of fluorescein-labeled and unlabeled LCs, co- incubated with 100 nM PK and visualized by SDS-PAGE. Symbols show normalized band intensities and lines show fits to a single exponential decay model, n=3. Black, Coomassie stained total LC (10 mM); green, fluorescence of labeled LC (20 nM). c) Kinetics of fluorescein-labeled LC proteolysis measured by FP at 37 °C in a fluorimeter cuvette. Lines show fits to a single exponential decay model d) Kinetics of proteolysis measured by FP in a 384-well plate at 22 °C. Points show data for individual wells and lines show the mean, n=16. e) The extent of proteolysis after 24 h at 22 °C depends on the concentration of the LC-stabilizing kosmotrope Na ₂ SO ₄ , but not on the concentration of protease f) Post-proteolysis compound addition to LCs identifies false-positive screen hits. Each point represents the mean measurement (n=3) for a single compound. Green shaded areas indicate compounds considered to be hits g) Compounds that provide protection from both serine protease PK and metalloprotease thermolysin (green area) are likely to be stabilizers of LCs, rather than protease inhibitors.

[0011] Figure 2: 7-Diethylamino-4-methylcoumarin (1) is an environment-sensitive fluorophore that stabilizes LCs. a) SDS-PAGE gel showing proteolysis of unlabeled WIL-FL (10 mM) in the presence of 1% DMSO vehicle and 100 mM small molecule; t = 2 h. Modifications to the core coumarin structure (21) are shown in red for each small molecule b) Fluorescence emission (lex = 373 nm) of 1 (1 mM), but not 19 (1 mM), increases in the presence of unlabeled WIL-FL (20 mM). c) Titrations of LC constructs into 1 (1 mM), n=3. Lines indicate fits to a one-site binding model d) Affinities of 1 for recombinant LC variants, n=3, measured as for (c). e) SV-AUC of WIL-FL C214S (20 mM) and WIL-V (20 mM) in the absence (red) or presence (blue) of 1 (100 mM). Data for dimeric WIL-FL (20 mM) are shown in grey f) Titration of k I 018/08-FL (blue) and AL12-FL (red) into 1 (1 mM). Data for WIL-FL (grey) are shown for comparison. Inset: fluorescence spectra of 1 (1 mM) in PBS (dashed grey line) or the presence of 20 mM k I

018/08-FL (blue) or AL12-FL (red) g) Competition of 1 (1 mM) for the WIL-FL binding site indicates that the presence of other hit molecules (10 mM) reduces binding of 1 (1 mM). The unrelated molecule diflunisal (Dif) does not compete with 1 for LC binding h) Binding of 1 to human serum albumin (HSA) in the presence or absence of diflunisal determined by

fluorescence of 1, measured as for (c). i) Fluorescence of 1 in the presence of the LC homodimer (black) or LC:HC heterodimer (Fab, red) of the human IgG antibody 5J8.

[0012] Figure 3: Kinetic stabilizer binding to the V-domain-V -domain dimer interface. Crystal structures of JTO-FL with bound 1 (in orange) (LC blue, cyan) and without 1 (LC grey) a) Overview of structures aligned by one chain of the dimer b) Kinetic stabilizer 1 binds in a pocket formed by the side chains of residues 44, 87 and 98 from one protomer and 36', 44', and 46' from the other protomer. Residues are numbered according to the Kabat system c) Surface representation of the binding site region. 1 binds in a pocket that is not present in the unliganded LC. d) Conservation of binding site residues in human germline LC genes mapped onto the JTO- FL·1 structure. Red, highly conserved residues; blue, weakly conserved residues.

[0013] Figure 4: Kinetic stabilizer 1 binds to LCs in solution a) Superposition of selected region of ¹ H- ¹⁵ N HSQC NMR spectra of WIL-FL (0.05 mM monomeric concentration) in the absence (blue contours) or presence of 1 at LC:1 ratios (LC monomer: ligand) of 1:0.8 (red) and 1:2.2 (green) b) Residues with combined chemical shift changes Dv _HN larger than 0.1 ppm upon binding of 1 are mapped as red spheres onto the structure of the JTO-FL· 1 complex; the ligand is shown as yellow or green sticks in the two possible but mutually exclusive binding sites c) Intensity of peaks associated with free LC dimer (LC ₂ , blue) and complex (LC ₂ •1, green) as a function of 1 binding to WIL-FL. d) HSQC spectral region highlighting correlations for residue Q38 of JTO-V. Colors are as for (a). Note the presence of exchange peaks connecting free and ligand bound correlations when approximately equal amounts ofLC ₂ and 1 are added e) ¹⁵ N transverse relaxation rates (R ₂ ) for WIL-V (averaged over 29 residues) and JTO-V (32 residues) with (green) or without (blue) addition of 1. f) Spectra of WIL-V residue Q38, in the absence (blue) or presence of 1, at LC:l ratios of 1:0.9 (red) and 1:2.2 (green). Note that the cross peaks from the bound form at LC:1 of 1:0.9 are broadened. [0014] Figure 5: Small molecule kinetic stabilizer binding stabilizes LCs against proteolysis, unfolding and aggregation a) Proteolysis of recombinant WIL-FL C214S (10 mM) in the presence of vehicle (red) or 1 (100 mM; blue). Symbols show LC band quantitation and lines show fits to a single exponential decay model b) Unfolding rates of WIL-FL (5 mM) in the presence of 5 (Table 1; 50 mM; orange) or vehicle (red) as a function of urea concentration. Symbols represent rates calculated for individual kinetic transients and lines are fits to a 2-state unfolding model c) Equilibrium urea titrations of WIL-FL (5 mM) in the presence of 1 (100 mM; blue symbols), 5 (Table 1; 50 mM; orange symbols) or vehicle (red symbols) d) NMR hydrogen- deuterium exchange measurements of JTO-V (100 mM, left panel) and WIL-V (200 mM, right panel) in the presence (blue) or absence (red) of 1 (400 mM). Exchange rates were converted to free energies. Symbols show residues for which rates could be measured. Dashed lines indicate the average stability for each V-domain. e) Proteolysis of the plasma cell-secreted LC, ALMC2 (5 mM), in the presence of vehicle (red) or 1 (100 mM; blue). The line represents a fit to a single exponential decay model f) Residual soluble WIL-FL C214S dimer (10 uM) measured by SEC at pH 5, 37 °C in the presence (blue) or absence (red) of 1 (200 mM), as a function of time to assess aggregation kinetics by starting material disappearance. Individual reactions are shown by dashed lines; curves represent fits of all samples in a category (± 1) to a single exponential decay model.

DETAILED DESCRIPTION

[0015] In AL, the current treatments aim to eradicate the clonal plasma cells that secrete FL LCs (2, 46). However, complete suppression of the production of amyloidogenic light chain (a complete hematologic response) is achieved in only 30-40% of the patients and most eventually relapse. Kinetic stabilization of LCs is unlikely to contribute to plasma cell death but could reduce organ proteotoxicity and the progression of AL. Patients with prominent cardiac involvement currently have few available options for treatment and represent an urgent unmet medical need, as they are often too sick to tolerate chemotherapy. Kinetic stabilizer pretreatment is useful by allowing these patients to tolerate chemotherapy. In this context, FL LC stabilization also is useful in a method for maintenance therapy upon recurrence of AL after treatment Because reemergence of the clonal plasma cells is generally slow, organ toxicity caused by conformationally unstable circulating LC can be minimized by kinetic stabilizer treatment (47). An advantage of the small molecule LC kinetic stabilizers disclosed herein is their ability to bind to and stabilize the FL LCs of most patients because conserved residues comprise the kinetic stabilizer binding site. Thus, in accordance with an embodiment, the present disclosure provides a method for identifying patients whose LCs are amenable to kinetic stabilization by measuring susceptibility of their LC in plasma to PK endoproteolysis in the presence and absence of the kinetic stabilizer, determining the relative difference in susceptibilities, and identifying the patients most likely to benefit from kinetic stabilization of LCs.

[0016] Definitions

[0017] "Alkyl" refers to straight or branched chain hydrocarbyl including from 1 to about 20 carbon atoms. For instance, an alkyl can have from 1 to 10 carbon atoms or 1 to 6 carbon atoms. Exemplary alkyl includes straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, and the like, and also includes branched chain isomers of straight chain alkyl groups, for example without

limitation, -CH(CH ₃ ) ₂ , -CH(CH3)(CH ₂ CH ₃ ), -CH(CH ₂ CH3) ₂ , -C(CH ₃ ) ₃ , -C(CH ₂ CH ₃ ) ₃ , -CH ₂ CH( CH ₃ ) ₂ , -CH ₂ CH(CH ₃ )(CH ₂ CH ₃ ), -CH ₂ CH(CH ₂ CH ₃ ) ₂ , -CH ₂ C(CH ₃ ) ₃ , -CH ₂ C(CH ₂ CH ₃ ) ₃ , -CH(CH 3)CH(CH ₃ )(CH2CH ₃ ), -CH2CH ₂ CH(CH3) ₂ , -CH ₂ CH ₂ CH(CH ₃ )(CH ₂ CH ₃ ), -CH ₂ CH ₂ CH(CH ₂ CH ₃ ) ₂ , -CH ₂ CH ₂ C(CH ₃ ) ₃ , -CH ₂ CH ₂ C(CH ₂ CH ₃ ) ₃ , -CH(CH ₃ )CH ₂ CH(CH ₃ ) ₂ , -CH(CH ₃ )CH(CH ₃ )CH(C H ₃ ) ₂ , and the like. Thus, alkyl groups include primary alkyl groups, secondary alkyl groups, and tertiary alkyl groups. An alkyl group can be unsubstituted or optionally substituted at 1, 2, 3, 4,

5, or even 6 positions, which substituents are attached at any available atom to produce a stable compound, with substitution as described herein.“Optionally substituted alkyl” refers to alkyl or substituted alkyl.

[0018] Each of the terms“halogen,”“halide,” and“halo” refers to -F or fluoro, -Cl or chloro, -Br or bromo, or -I or iodo.

[0019] The term“alkenyl” refers to straight or branched drain hydrocarbyl groups including from 2 to about 20 carbon atoms having 1-3, 1-2, or at least one carbon to carbon double bond. An alkenyl group can be unsubstituted or optionally substituted with one or more substituents at 1 or more, e.g., 1, 2, 3, 4, 5, or even 6 positions, which substituents are attached at any available atom to produce a stable compound, with substitution as described herein. “Optionally substituted alkenyl” refers to alkenyl or substituted alkenyl.

[0020]“Alkyne or“alkynyl” refers to a straight or branched chain unsaturated hydrocarbon having the indicated number of carbon atoms, such as 2 to 6, and at least one triple bond

Examples of a (C ₂ -C ₈ )alkynyl group include, but are not limited to, acetylene, propyne, 1-butyne, 2-butyne, 1 -pentyne, 2-pentyne, 1-hexyne, 2-hexyne, 3-hexyne, 1-heptyne, 2-heptyne, 3-heptyne, 1-octyne, 2-octyne, 3-octyne and 4-octyne. An alkynyl group can be unsubstituted or optionally substituted with one or more substituents at 1 or more, e.g., 1 , 2, 3, 4, 5, or even 6 positions, which substituents are attached at any available atom to produce a stable compound, with substitution as described herein. “Optionally substituted alkynyl" refers to alkynyl or substituted alkynyl.

[0021] The term“cycloalkyl” refers to a saturated monocyclic, bicyclic, tricyclic, or polycyclic, 3- to 14-membered ring system, such as a C ₃ -C ₈ -cycloalkyl. The cycloalkyl may be attached via any atom. Representative examples of car cycloalkyl bocyclyl include, but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl. A cycloalkyl group can be unsubstituted or optionally substituted with one or more substituents at 1 or more _; , e.g., 1, 2, 3, 4, 5, or even 6 positions, which substituents are attached at any available atom to produce a stable compound, with substitution as described herein.

[0022]“Aryl” when used alone or as part of another term means a carbocyclic aromatic group whether or not fused having the number of carbon atoms designated or if no number is designated, up to 14 carbon atoms, such as a C ₆ -C ₁₄ -aryl. Particular aryl groups are phenyl, naphthyl, biphenyl, phenanthrenyl, naphthacenyl, and the like (see e.g. Lang’s Handbook of Chemistry (Dean, J. A, ed) 13 ^th ed. Table 7-2 [1985]). A particular aryl is phenyl. “Aryl” can be optionally fused with a carbocyclyl ring, as herein defined. An aryl group can be

unsubstituted or optionally substituted with one or more substituents attached at any available atom to produce a stable compound, wherein the substituents are as described herein.

“Optionally substituted aryl” refers to aryl or substituted aryl. [0023] The term“heteroatom” refers to N, O, and S. Compounds of the present disclosure that contain N or S atoms can be optionally oxidized to the corresponding N-oxide, sulfoxide, or sulfone compounds.

[0024]“Heteroaryl,” alone or in combination with any other moiety described herein, refers to a monocyclic aromatic ring structure containing 5 to 10, such as 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10 atoms, containing one or more, such as 1-4, 1-3, or 1-2, heteroatoms independently selected from the group consisting of O, S, and N. Heteroaryl is also intended to include oxidized S or N, such as sulfmyl, sulfonyl and N-oxide of a tertiary ring nitrogen. A carbon or heteroatom is the point of attachment of the heteroaryl ring structure such that a stable compound is produced. Examples of heteroaryl groups include, but are not limited to, pyridinyl, pyridazinyl, pyrazinyl, quinaoxalyl, indolizinyl, benzo[b]thienyl, quinazolinyl, purinyl, indolyl, quinolinyl, pyrimidinyl, pyrrolyl, pyrazolyl, oxazolyl, thiazolyl, thienyl, isoxazolyl, oxathiadiazolyl, isothiazolyl, tetrazolyl, imidazolyl, triazolyl, furanyl, benzofuryl, 1- pyrazolopyrimidinyl, imidazopyrazinyl, triazolopyrazinyl,_and indolyl. A heteroaryl group can be unsubstituted or optionally substituted with one or more substituents, e.g., 1, 2, 3, 4 or 5, also 1, 2, or 3 substituents, also 1 substituent, attached at any available atom to produce a stable compound, wherein the substituents are as described herein.“Optionally substituted heteroaryl” refers to heteroaryl or substituted heteroaryl.

[0025]“Heterocycloalkyl” means a saturated or partially unsaturated non-aromatic monocyclic, bicyclic, tricyclic or polycyclic ring system that has from 3 to 14, such as 3 to 6, atoms in which from 1 to 3 carbon atoms in the ring are replaced by heteroatoms of O, S or N. A

heterocycloalkyl is optionally fused with aryl or heteroaryl of 5-6 ring members, and includes oxidized S or N, such as sulfmyl, sulfonyl and N-oxide of a tertiary ring nitrogen. The point of attachment of the heterocycloalkyl ring is at a carbon or heteroatom such that a stable ring is retained. Examples of heterocycloalkyl groups include without limitation morpholino, tetrahydrofuranyl, dihydropyridinyl, piperidinyl, pyrrolidinyl, piperazinyl, dihydrobenzofuryl, and dihydroindolyl. A heterocycloalkyl group can be unsubstituted or optionally substituted with one or more substituents, such asl to 3 substituents, e.g., 1, 2 or 3 substituents, attached at any available atom to produce a stable compound, wherein the substituents are as described herein. [0026] The term“nitrile” or“cyano” can be used interchangeably and refer to a -CN group which is bound to a carbon atom of a heteroaryl ring, aryl ring and a heterocycloalkyl ring.

[0027] The term“oxo” refers to a =0 atom bound to an atom that is part of a saturated or unsaturated moiety. Thus, the =0 atom can be bound to a carbon, sulfur, or nitrogen atom that is part of a cyclic or acyclic moiety.

[0028] A“hydroxyl” or“hydroxy” refers to an -OH group.

[0029] The substituent -CO2H may be replaced with bioisosteric replacements such as:

and the like, wherein R has the same definition as R ^A as defined herein. See, e.g., THE PRACTICE OF MEDICINAL CHEMISTRY (Academic Press: New York, 1996), at page 203.

[0030] Compounds described herein can exist in various isomeric forms, including

configurational, geometric, and conformational isomers, including, for example, cis- or trans- conformations. The compounds may also exist in one or more tautomeric forms, including both single tautomers and mixtures of tautomers. The term“isomer” is intended to encompass all isomeric forms of a compound of this disclosure, including tautomeric forms of the compound. The compounds of the present disclosure may also exist in open-chain or cyclized forms. In some cases, one or more of the cyclized forms may result from the loss of water. The specific composition of the open-chain and cyclized forms may be dependent on how the compound is isolated, stored or administered. For example, the compound may exist primarily in an open- chained form under acidic conditions but cyclize under neutral conditions. All forms are included in the disclosure.

[0031] Some compounds described herein can have asymmetric centers and therefore exist in different enantiomeric and diaster eomeric forms. A compound as described herein can be in the form of an optical isomer or a diaster eomer. Accordingly, the disclosure encompasses compounds and their uses as described herein in the form of their optical isomers,

diaster eoisomers and mixtures thereof, including a racemic mixture. Optical isomers of the compounds of the disclosure can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, simulated moving bed technology or via chemical separation of stereoisomers through the employment of optically active resolving agents.

[0032] Unless otherwise indicated, the term“stereoisomer” means one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. Thus, a

stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound having two chiral centers will be substantially free of other diaster eomers of the compound. A typical stereomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound, or greater than about 99% by weight of one stereoisomer of the compound and less than about 1% by weight of the other stereoisomers of the compound. The stereoisomer as described above can be viewed as composition comprising two stereoisomers that are present in their respective weight percentages described herein. [0033] If there is a discrepancy between a depicted structure and a name given to that structure, then the depicted structure controls. Additionally, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it. In some cases, however, where more than one chiral center exists, the structures and names may be represented as single enantiomers to help describe the relative stereochemistry. Those skilled in the art of organic synthesis will know if the compounds are prepared as single enantiomers from the methods used to prepare them.

[0034] As used herein, and unless otherwise specified to the contrary, the term“compound” is inclusive in that it encompasses a compound or a pharmaceutically acceptable salt, stereoisomer, isotopologue, and/or tautomer thereof. Thus, for instance, a compound of Formula IA or Formula IB includes a pharmaceutically acceptable salt of a tautomer of the compound.

[0035] In this disclosure, a“pharmaceutically acceptable salt” is a pharmaceutically acceptable, organic or inorganic acid or base salt of a compound described herein. Representative pharmaceutically acceptable salts include, e.g., alkali metal salts, alkali earth salts, ammonium salts, water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene- 2, 2-disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium, calcium edetate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate, oxalate, palmitate, pamoate (l,l-methene-bis-2-hydroxy-3-naphthoate, einbonate), pantothenate,

phosphate/diphosphate, picrate, polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate, subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate salts. A pharmaceutically acceptable salt can have more than one charged atom in its structure. In this instance the pharmaceutically acceptable salt can have multiple counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterions. [0036] The terms“treat”,“treating” and“treatment” refer to the amelioration or eradication of a disease or symptoms associated with a disease. In various embodiments, the terms refer to minimizing the spread or worsening of the disease resulting from the administration of one or more prophylactic or therapeutic compounds described herein to a patient with such a disease.

[0037] The terms“prevent,”“preventing,” and“prevention” refer to the prevention of the onset recurrence, or spread of the disease in a patient resulting from the administration of a compound described herein.

[0038] The term“effective amount” refers to an amount of a compound as described herein or other active ingredient sufficient to provide a therapeutic or prophylactic benefit in the treatment or prevention of a disease or to delay or minimize symptoms associated with a disease. Further, a therapeutically effective amount with respect to a compound as described herein means that amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or prevention of a disease. Used in connection with a compound as described herein, the term can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease, or enhances the therapeutic efficacy of or is synergistic with another therapeutic agent

[0039] A“patient” or subject” includes an animal, such as a human, cow, horse, sheep, lamb, pig, chicken, turkey, quail, cat, dog, mouse, rat rabbit or guinea pig. In accordance with some embodiments, the animal is a mammal such as a non-primate and a primate (e.g., monkey and human). In one embodiment, a patient is a human, such as a human infant child, adolescent or adult. In the present disclosure, the terms“patient” and“subject” are used interchangeably.

COMPOUNDS

[0040] The present disclosure provides in various embodiments an LC kinetic stabilizer, i.e., a compound of Formula la or lb, or a pharmaceutically acceptable salt thereof:

wherein

A is H or

X ¹ is O or NR°;

X ² is selected from the group consisting of a bond, NR ¹² , 0, C(O), C(0)NR ¹¹ , and CR ¹² R ¹¹ ; X ³ is O or NR ¹³ ;

W is selected from the group consisting of a bond, OC(O), C(0)0, , C(0)NR ¹⁴ , NR ¹⁴ C(0)0, 0C(0)NR ¹⁴ , NR ¹⁴ C(0), NR ¹⁴ C(0)NR ¹⁵ , NR ¹⁴ ,

m is an integer selected from 1, 2, 3, 4, 5, and 6;

n is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

p is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

r is an integer selected from 0, 1, 2, and 3;

Z ¹ is selected from the group consisting of H, C ₁ -C ₆ -alkyl, C ₃ -C ₈ -cycloalkyl, 3- to 6- membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₂ -C ₆ -alkenyl, C ₃ -C ₈ -cycloalkenyl, 3- to 6-membered heterocycloalkenyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₆ -C ₁₀ -aryl, 5- to 10-member ed heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), -(C ₁ -C ₆ -alkylXC ₆ -C 10-aryl), -(C ₁ -C ₆ -alkyl)(5- to 10-membered heteroaryl) (wherein 1-4 heteroaryl members are independently selected from N, O, and S), halogen, NR ^,6 R ¹⁷ , COOR ¹⁸ ; OR ¹⁸ , NR ¹⁶ S0 ₂ R ¹⁸ , NR ¹⁶ COR ¹⁸ ,

wherein

X ⁴ is a bond, O, or NR°; and a is an integer selected from 0, 1, 2, 3, 4, 5, and 6;

Z ² is selected from the group consisting of H, C ₁ -C ₆ -alkyl, C ₃ -C ₈ -cycloalkyl, 3- to 6- membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₁ -C ₆ -alkenyl, C ₁ -C ₆ -alkynyl, C ₃ -C ₈ -cycloalkenyl, 3- to 6-member ed heterocycloalkenyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₆ -C ₁₀ -aryl, 5- to 10-membered heteroaryl (wherein 1 -4 heteroaryl members are independently selected from N, O, and S), -(C ₁ -C ₆ -alkylXC ₆ -C ₁₀ -aryl), -(C ₁ -C ₆ -alkyl)(5- to 10-membered heteroaryl) (wherein 1-4 heteroaryl members are independently selected from N, O, and S), CN, OR ¹⁸ andNR ¹⁹ R ²⁰ ;

Ar ¹ is a divalent moiety selected from C ₆ -C ₁₀ -arylene and 5- to 10-membered heteroarylene (wherein 1-4 heteroaryl members are independently selected from N, O, and S);

R ¹ is selected from the group consisting of H, C ₁ -C ₆ -alkyl, C ₃ -C ₈ -cycloalkyl, 3- to 6- membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₂ -C ₆ -alkenyl, C ₃ -C ₈ -cycloalkenyl, 3- to 6-membered heterocycloalkenyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₆ -C ₁₀ -aryl, 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), -(C ₁ -C ₆ -alkyl)(C ₆ -C ₁₀ -aryl), -(C ₁ -C ₆ -alkyl)(5- to 10-membered heteroaryl) (wherein 1-4 heteroaryl members are independently selected from N, O, and S), OH, (CR ²¹ R ²² ) _b OR ¹⁸ (b is an integer selected from 0, 1, 2, 3, 4, 5, and 6), halogen, and (C ₁ -C ₆ )haloalkyl; R ² , R ³ , and R ⁴ are independently selected from the group consisting of H, C ₁ -C ₆ -alkyl, -(Ci- C ₆ -alkyl)(C ₆ -C ₁₀ -aryl), OH, (CR ²¹ R ²² ) _b OR ¹⁸ , halogen, C ₃ -C ₈ -cycloalkyl, 3- to 6- membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), C^-C ₆ -alkenyl, C ₃ -C ₈ -cycloalkenyl, 3- to 6-membered heterocycloalkenyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₆ -C ₁₀ -aryl, 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), -(C ₁ -C ₆ -alkyl)(C ₆ -C ₁₀ -aryl), -(C ₁ -C ₆ -alkyl)(5- to 10-membered heteroaryl) (wherein 1-4 heteroaryl members are independently selected from N, O, and

S);

R ⁰ and R ⁵ - R ²² are independently selected from the group consisting of H, C ₁ -C ₆ -alkyl, Ci- C ₆ -haloalkyl, C ₃ -C ₈ -cycloalkyl, 3- to 6-membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₂ -C ₆ -alkenyl, C ₂ -C ₆ -alkynyl, C ₃ -C ₈ -cycloalkenyl, 3- to 6-membered heterocycloalkenyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₆ -C ₁₀ -aryl, 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), -(C ₁ -C ₆ - alkyl)(C ₆ -C ₁₀ -aryl), -(C ₁ -C ₆ -alkyl)(5- to 10-membered heteroaryl) (wherein 1-4 heteroaryl members are independently selected from N, O, and S);

R ¹⁶ and R ¹⁷ are independently selected from the group consisting of H, C ₁ -C ₆ -alkyl, C ₃ -C ₈ - cycloalkyl, 3- to 6-membered heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₂ -C ₆ -alkenyl, C ₃ -C ₈ -cycloalkeny 1, 3- to 6- membered heterocycloalkenyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₆ -C ₁₀ -aryl, 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), -(C ₁ -C ₆ -alkyl)(C ₆ -C ₁₀ -aryl), -(Ci- C ₆ -alkyl)(5- to 10-membered heteroaryl) (wherein 1-4 heteroaryl members are independently selected from N, O, and S), C(0)0R ¹⁸ , C(0)R ¹⁸ , and SO2R ¹⁸ ;

R ¹⁴ and R ⁹ , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring;

R ¹⁵ and R ⁹ , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring; R ¹⁶ and R ⁹ , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring;

R ¹⁶ and R ¹⁴ , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring;

R ¹⁶ and R ¹⁵ , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring;

R ¹⁶ and R ¹⁷ , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring wherein ring members are selected from C, O and N;

R ¹⁹ and R ²⁰ , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring;

R ²¹ and R ²² , together with the atoms to which they are bound, optionally are a 3- to 8- membered ring;

OR a compound according to Formula P, or a pharmaceutically acceptable salt thereof:

wherein

R ²³ - R ²⁸ are independently selected from the group consisting of H, halo, C ₁ -C ₆ -alkyl, C ₃ - Cg-cycloalkyl, 3- to 6-member ed heterocycloalkyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₂ -C ₆ -alkenyl, C ₃ -C ₈ -cycloalkenyl, 3- to 6- membered heterocycloalkenyl (wherein 1-4 ring members are independently selected from N, O, and S), C ₆ -C ₁₀ -aryl, 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S), -(C ₁ -C ₆ -alkyl)(C ₆ -C ₁₀ -aryl), -(Ci- C ₆ -alkyl)(5- to 10-membered heteroaryl) (wherein 1-4 heteroaryl members are independently selected from N, O, and S);

Ar ² is C ₆ -C ₁₀ -aryl or 5- to 10-membered heteroaryl (wherein 1-4 heteroaryl members are independently selected from N, O, and S; q is an integer selected from 0, 1, 2, 3, and 4; t is an integer selected from 0, 1, 2, 3, and 4; wherein any alkyl, cycloalkyl, heterocycloalkyl, alkenyl, cycloalkenyl, heterocycloalkenyl, aryl, heteroaryl, and ring moiety in Formula la, Formula lb, or Formula P is optionally substituted with one to six substituents selected from the group consisting of hydroxy, halo, C ₁ -C ₆ -haloalkyl, -NR’2 (wherein each R’ is independently selected from the group consisting of H, C ₁ -C ₆ -alkyl, C ₂ -C ₆ -alkenyl, C2-C6-alkynyl, and C ₆ -C ₁₀ -aryl), - NHC(0)(0Ci -C ₆ -alkyl), -NO2, -CN, oxo, -C(0)0H, -C(0)0(C ₁ -C ₆ -alkyl), -C ₁ -C ₆ - alkyl(C ₁ -C ₆ -alkoxy), -C(0)NH ₂ , -C(0)C ₁ -C ₆ -alkyl, -OC ₁ -C ₆ -alkyl, -Si(C ₁ -C ₆ - alkyl) ₃ , -S(0) _0-2 -(C ₁ -C ₆ -alkyl), C ₆ -C ₁₀ -aryl, -(C ₁ -C ₆ -alkyl)(C ₆ -C ₁₀ -aryl), 3- to 14- membered heterocycloalkyl and -(C ₁ -C ₆ -alkyl)-(3- to 14-membered heterocycle) (wherein 1-4 heterocycle members are independently selected from N, O, and S), and - 0(C ₆ -Ci ₄ -aryl).

[0041] It should be understood that, notwithstanding the definitions of Formula la, Formula lb, and Formula P, in some embodiments the compound does not include any of the following compounds:

[0042] In various embodiments, the compound according to claim 1, wherein the compound is of Formula la. In other embodiments, the compound is of Formula lb.

[0043] In an embodiment, each of X ¹ and X ³ is O.

[0044] A further embodiment is wherein each of R ² , R ³ , and R ⁴ is independently selected from H and C ₁ -C ₆ -alkyl. For example, each of R ² , R ³ , and R ⁴ is H

[0045] In an embodiment, R ¹ is H or C ₁ -C ₆ -alkyl. For example, R ¹ is C ₁ -C ₆ -alkyl, such as methyl.

[0046] In various embodiments, X ² is NR ¹² and p is 0, 1, or 2.

[0047] A further embodiment provides a compound wherein each of Z ² and R ¹² is independently selected from the group consisting of H, C ₁ -C ₆ -alkyl, C ₂ -C ₆ -alkenyl, C ₂ -C ₆ -alkynyl, and C ₃ -C ₈ - cycloalkyl. For example, per an embodiment, each of Z ² and R ¹² is C ₁ -C ₆ -alkyl. In additional embodiments, p is 0 and each of Z ² and R ¹² is ethyl.

[0048] In other embodiments, X ² is O; Z ² is C ₆ -C ₁₀ -aryl or -(C ₁ -C ₆ -alkyl)(C ₆ -C ₁₀ -aryl); p is 1, 2, or 3; and R ⁷ and R ⁸ are independently selected from H, C ₁ -C ₆ -alkyl. For instance, Z ² is phenyl or benzyl, and R ⁷ and R ⁸ are independently selected from H, methyl, trifluoromethyl, and ethyl. ^' R ⁵ R ^{8 '}

w-

R ⁶ 10 0.1

Jm

[0049] In various embodiments, A is . Thus, in an embodiment, A is

[0050] In still another embodiment, m is 2 and n is 1.

[0051] Additional embodiments provide a compound wherein W is selected from the group

consisting

[0052] Examples of Ar ¹ , per various embodiments, include phenyl, pyrazolopyrimidinyl, imidazopyrazinyl, pyridinyl, and triazolopyrazinyl.

[0053] In some embodiments, Z ¹ is selected from H, C ₁ -C ₆ -alkyl, and 5-membered

heterocycloalkyl.

[0054] Exemplary embodiments are compounds in which the compound is of formula la or lb; each of X ¹ and X ³ is O; X ² is NR ¹² , wherein R ¹² is C ₁ -C ₆ -alkyl; p is 0; Z ² is C ₁ -C ₆ -alkyl; A is

Z ¹

0.1

; m is 2 and n is 0 or 1 ;W is selected from the group consisting of are independently selected from H and C ₁ -C ₆ -alkyl; Ar ¹ is selected from the group consisting of phenyl, pyrazolopyrimidinyl, imidazopyrazinyl, pyridinyl, and triazolopyrazinyl; and Z ¹ is selected from the group consisting of H, C ₁ -C ₆ -alkyl, and a 5-membered heterocycloalkyl.

[0055] In exemplary embodiments, the compounds of the present disclosure, including for use in the methods described herein, include those in the table below: 1 9

Ό ccc k

,0,

2 10

Ό'

k J00C

3

Ό- <4- 11

Ό

NH ₂

'T'

4 12

o

k*

5 ,-JOL 13

Ό

0 ^™ L= _N

" /=\ G

6 14

^' q— N*

¾

o H

N

HI N

7 F 15 Ό' Ό'

F

19 28

Hi ^• O' Ό

20 29

H _? N

21 ccx 30

,o

K

^• N'

22 ,F 31

F

Enantiomer 1 o

H.

^• N'

23 F 32

^• F

Enantiomer 2

99 CX-kUXXT 100 r

O

M

103 104 k

105 Ί

Ί 106

k

107 108 k. k k

109 110 k k

O

:0

,0,

111 112

U y

k k N^S,

»

113 114 k k

N— W

X

115 U 116 k k

,o_ "1 «

117 Ί 118

k

r

119 r 120 r r

,0M ,w¼

121 r 122 r

n X Ix

1 3 r r

2 r 124 r 'Wi - t> . _G

125 126

K/

0,

127 Ί 128 Ί

LLG

o

129 130

O.

O

131 Ί 132 Ί

H H

133 134

Nr F,

.O. _* . ^

135 1 136

I

0- 0 - ]

141 n~ΐ 142 eon uoxr Ί

o. 1

o o

143

HK· Ί 144

r - jo. ^

145 146 Ί

1

161 Ί 162 Ί

r WI

169 F, 170 r

F·

171 172 173 174 N.

N·^

o,

.NH H

IH

175 176

k

k k

O

177 178 k k

s

.NH

NH

179 180 V)

k k

k k

0

181 'k 182 Y

k

k k

185 186

k k

187 188 V) k X/'

k

.W

189 190

k k

191 192 V k k

F

,F

195 196 k k

197 198 k k

199 Y)

k 200 I

k

k k

N.

O.

X

201 202 k k

0

/ y w\ ^k

203 204 k k

205 V 206 V) k k 207 Yn V 208 k k

209 210 Y k k

211 U 212 k k

o.

,NH O

•N'

213 214 k k

215 Y U>

216

V

k k 7 U CH

21 218 Ί

U

k F

OH!HO

0„ .0. _^ ,c. 0„ A ^

219 Ί 220 Ί

0.

Y '"L

221 222 k k

223 224

o

k

225 H 226 H

O, N

I

227 a 228 H

OCr N

HO,

H

a, N

229 230 H

N

Hi

il o.

244 243

246 245 ^• N'

o.

248 247

o.

250 249

N

H

F, ,F

252 251

F' N

H

O. O.

260 259

o.

262 ^• N' 261

O. o.

264 263

F

F

266 265 F.

·N·

F ^* N

H 268 267

^• N'

270 269

N

H

II

272 271

'hr ’hr

F,

274 273

'hr

O.

O

276 Ί 275

'hr

278 1 277 k

280 279 Ί

Ί

282 JUXXr Ί 281 v ccr

"1

284 Ί 283

Ί

286 Ί 285

"1

(V .0. ^ ^

288 Ί 287 Ί

H H

Ί o. o „

290 Ί "I

289 292 Ί 291 /Ί

a ^ ^

294 293

a Ί

96 Ί Ί

2 ocr· 295

r

298 r 297 r

Ί

300 Ί f o

299 r V*

r Ί

302 r 301 Ί Ί r

304 Ί 3 r 0

30 v

X

r

306 r 305 r

r

308 r 307 r

310 r

r 309 r

r

r

312 r 311 r cL \

[0056] In other embodiments, the compound is of Formula II.

[0057] In Formula P compounds, per an embodiment, each of q and t is 0 or 1 and AT ² is C ₆ -C ₁₀ - aryl. For example, q is 1 and t is 0. In an embodiment, Ar ² is phenyl optionally substituted with -NR’ 2 (wherein each R’ is independently selected from the group consisting of H, C ₁ -C ₆ -alkyl) and C ₁ -C ₆ -haloalkyl. [0058] In Formula P compounds, still further embodiments provide each of R ²⁵ , R ²⁶ , R ²⁷ , and R ²⁸ as independently H or C ₁ -C ₆ -alkyl. For example, each of R ²⁵ , R ²⁶ , R ²⁷ , and R ²⁸ is H.

[0059] Specific examples of Formula P compounds are chosen from the following table:

O O

H H

N N

N N

HI HI

7 F 8 F

F F

O O

a. .H,

^• N' ‘N'

22 F 23 ,F

^• F ^• F

Enantiomer 1 Enantiomer 2

PHARMACEUTICAL COMPOSITION

[0060] The disclosure also provides a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds described herein, or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof in admixture with a pharmaceutically acceptable carrier. In some embodiments, the composition further contains, in accordance with accepted practices of pharmaceutical compounding, one or more additional therapeutic agents, pharmaceutically acceptable excipients, diluents, adjuvants, stabilizers, emulsifiers,

preservatives, colorants, buffers, flavor imparting agents.

[0061] In one embodiment, the pharmaceutical composition comprises any enumerated compound described herein or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof, and a pharmaceutically acceptable carrier.

[0062] The pharmaceutical composition of the present disclosure is formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular subject being treated, the clinical condition of the subject, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[0063] The“therapeutically effective amount” of a compound or a pharmaceutically acceptable salt, stereoisomer, and/or tautomer thereof that is administered is governed by such

considerations, and is the minimum amount necessary to exert a therapeutic effect. Such amount may be below the amount that is toxic to normal cells, or the subject as a whole. Generally, the initial therapeutically effective amount of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure that is administered is in the range of about 0.01 to about 200 mg/kg or about 0.1 to about 20 mg/kg of patient body weight per day, with the typical initial range being about 0.3 to about 15 mg/kg/day. Oral unit dosage forms, such as tablets and capsules, may contain from about 0.1 mg to about 1000 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In another embodiment, such dosage forms contain from about 50 mg to about 500 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In yet another embodiment, such dosage forms contain from about 25 mg to about 200 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In still another embodiment, such dosage forms contain from about 10 mg to about 100 mg of a compound (or a pharmaceutically acceptable salt,

stereoisomer, or tautomer thereof) of the present disclosure. In a further embodiment, such dosage forms contain from about 5 mg to about 50 mg of a compound (or a pharmaceutically acceptable salt, stereoisomer, or tautomer thereof) of the present disclosure. In any of the foregoing embodiments the dosage form can be administered once a day or twice per day.

[0064] The compositions of the present disclosure can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrastemal injection or infusion techniques. [0065] Suitable oral compositions as described herein include without limitation tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, syrups or elixirs.

[0066] In another aspect, also encompassed are pharmaceutical compositions suitable for single unit dosages that comprise a compound of the disclosure or its pharmaceutically acceptable stereoisomer, salt, or tautomer and a pharmaceutically acceptable carrier.

[0067] The compositions of the present disclosure that are suitable for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions. For instance, liquid formulations of the compounds of the present disclosure contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically palatable preparations of a compound of the present disclosure.

[0068] For tablet compositions, a compound of the present disclosure in admixture with nontoxic pharmaceutically acceptable excipients is used for the manufacture of tablets. Examples of such excipients include without limitation inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, com starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known coating techniques to delay disintegration and absorption in the gastrointestinal tract and thereby to provide a sustained therapeutic action over a desired time period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

[0069] Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

[0070] For aqueous suspensions, a compound of the present disclosure is admixed with excipients suitable for maintaining a stable suspension. Examples of such excipients include without limitation are sodium carboxymethylcellulose, methylcellulose,

hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia.

[0071] Oral suspensions can also contain dispersing or wetting agents, such as naturally- occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0072] Oily suspensions may be formulated by suspending a compound of the present disclosure in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.

[0073] Sweetening agents such as those set forth above, and flavoring agents may be added to provide palatable oral preparations. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

[0074] Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide a compound of the present disclosure in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

[0075] Pharmaceutical compositions of the present disclosure may also be in the form of oil-inwater emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monoleate, and condensation reaction products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monoleate. The emulsions may also contain sweetening and flavoring agents.

[0076] Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable, an aqueous suspension or an oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

[0077] The compounds of Formula LA or Formula IB may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

[0078] Compositions for parenteral administrations are administered in a sterile medium.

Depending on the vehicle used and concentration the concentration of the drug in the

formulation, the parenteral formulation can either be a suspension or a solution containing dissolved drug. Adjuvants such as local anesthetics, preservatives and buffering agents can also be added to parenteral compositions. METHODS OF USE

[0079] Secretion of an immunoglobulin light chain (LC) by a clonally expanded plasma cell population can lead to the disease light chain amyloidosis (AL)— both a cancer and a proteinopathy (1, 2).“Free” LCs secreted without an associated antibody heavy chain initially adopt a well-defined homodimeric structure, wherein the monomers may be covalently linked by an inter-chain disulfide bond (Fig. la) (3). LC monomers comprise an N-terminal variable (V) domain attached to a C-terminal constant (C) domain. Each patient’s clonal plasma cells secrete a single, unique LC sequence. Most LCs are rapidly removed by the kidney.

[0080] However, since amyloidogenic full-length (FL) LCs are generally less stable than non- amyloidogenic FL LCs, they can misfold, or misfold and misassemble, into non-native species including cross-P-sheet amyloid fibrils, which are a hallmark of AL (4-8). Sequence also seems to play a role, as not all destabilized FL LCs aggregate in patients (4-8). How aggregation occurs in patients is not known, but several processes have been described in vitro, including destabilization-dependent endoproteolysis that releases amyloidogenic LC fragments (4, 9, 10). LC fragments including V-domains are observed in patient deposits alongside FL LCs (11-13).

[0081] Since the structure-proteotoxicity relationships driving AL are not well understood, a conservative strategy is to block FL LC misfolding at its origin by stabilizing the FL LC native state. Such a strategy has been effective at ameliorating the transthyretin amyloidoses (14-19). A small molecule that stabilizes FL LC dimers prevents any misfolding and/or endoproteolysis required for LC aggregation and organ toxicity. Such molecules are referred to herein as kinetic stabilizers, since they reduce the rate at which LCs transiently visit non-native, aggregation- prone and protease-sensitive conformations (20). The interfaces between the domains of the LC dimer are an important determinant of stability and aggregation propensity that have been identified as potential targets for stabilization (21, 22). Thus, it is an object herein to provide treatments that prevent newly synthesized and secreted FL LCs from misfolding and

aggregating, thus reducing organ proteotoxicity, such that patients can eventually tolerate effective chemotherapy regimens.

[0082] Using a protease-coupled fluorescence polarization assay that assesses LC kinetic stability, 650,000 small molecules were screened, which identified FL LC kinetic stabilizers in four structural classes that protect recombinant and plasma cell-secreted X6a LCs from endoproteolysis. Nuclear magnetic resonance (NMR) and x-ray crystallography revealed that these small molecules bind to the V-domain-V-domain interface in the FL LC dimer and in isolated V-domains, utilizing conserved residues found in most patient-derived LCs.

[0083] In light of these discoveries and other data disclosed herein, the present disclosure provides in various embodiments a method of stabilizing an immunoglobulin light chain dimer in a native conformation thereof. The method comprises contacting the dimer and an effective amount of any compound disclosed herein, include a compound of Formula la, Formula lb, Formula P, and all enumerated compounds disclosed herein. The contacting can occur in vivo, ex vivo, and in vitro.

[0084] In another embodiment, the present disclosure provides a method of treating light chain amyloidosis in a patient. The method comprises administering to the patient an effective amount of any compound disclosed herein, include a compound of Formula la, Formula lb, Formula P, and all enumerated compounds disclosed herein.

[0085] The present disclosure also provides a method of prophylactic treatment, comprising administering a therapeutically effective amount of a compound described herein to an AL patient who has undergone chemotherapy treatment, post-autologous stem cell transplantation, or has relapsed from prior chemotherapy treatment.

[0086] Any of the methods described herein are suitable for use in combination with

conventional chemotherapy (e.g., alkylating agents and steroid -based regimen: melphalan, dexamethasone and cyclophosphamide). The combination therapy method can include, per one embodiment, the administration of an immunomodulatory agent such as thalidomide, lenalidomide or pomalidomide, a proteasome inhibitor such as bortezomib, or both

[0087] In additional embodiments, any of the methods described herein are carried out in combination with anti-CD38 Ab and/or other chemotherapy treatments that target an underlying plasma cell clone.

[0088] Further combination therapies include any of the methods described herein with administration of UPR activators, such as ATF-6 activators and/or chemotherapy, or antiamyloid therapies, such as therapeutic antibodies directly targeting amyloid deposits. All of these combinations are contemplated. EXAMPLES

[0089] Compound Syntheses and Characterization

[0090] General Synthetic Procedures. All reagents and solvents were obtained from commercial suppliers and used without further purification. ¾ ¹³ C, and ¹⁹ F NMR spectra were recorded on either a Varian Mercury-400, a Bruker DRX-500, or a Bruker DRX-600 equipped with a DCH cryoprobe. Normal-phase chromatography was performed on a Teledyne Isco CombiFlash NextGen 3004- using Luknova SuperSep S1O2 columns. Preparative-scale reverse phase high performance liquid chromatography (RP-HPLC) was performed on an Agilent 1260 Infinity LC system using a Gemini NX-C18 column (110 A pore size, 5 pm particle size, 150 x 21.2 mm dimensions, mobile phase A = 0.1% TFA in H2O, mobile phase B = 0.1% TFA in MeCN). Final compound purities were determined by analytical RP-HPLC and were > 95% in purity. Mass spectrometry data were collected at The Scripps Research Institute C ₆ nter for Mass Spectrometry (ESI-MS; Agilent Technologies, LC/MSD TOF G1969A and GC-MS; Agilent Technologies, 6850 Network GC System, 5973 Mass Selective Detector).

[0091] Abbreviations

Boc = tert-butyloxy carbonyl

CataCXium® A = Di(l -adamantyl)-n-buty lphosphine

CDI = carbonyldiimidazole

DCM = dichloromethane

DIAD = diisopropyl azodicarbonate

DIPEA = diisopropyl ethylamine

DMF = N,N-dimethylformamide

DMP = Dess-Martin periodinane

DMPU = N,N’ -dimethy lpropyleneurea

HATU = l-[Bis(dimethylamino)methylene]-lH-l,2,3-triazolo[4,5-b]pyri dinium 3-oxid hexafluorophosphate

HMPT = jV-[bis(dimethylamino)phosphoryl]-/V-methyl-methanamine HPLC = high-performance liquid chromatography

LDA = lithium diisopropylamide

NBS = N-bromosuccinimide

NMR = nuclear magnetic resonance

MOM = methoxymethyl

RuPhos = 2-Dicyclohexylphosphino-2',6'-diisopropoxybiphenyl

TBAF = tetrabutylammonium fluoride

TBS = tert-butyldimethylsilyl

TEA = trifluoroacetic acid

THF = tetrahydrofuran

THP = tetrahydropyran

Xantphos = 4,5-Bis(diphenylphosphino)-9,9-dimethylxanthene

[0092] Compounds (22) and (23). Compounds 22 and 23 are enantiomers of racemate 8, and they were separated on a Waters UPC2 SFC with a Daicel LA column (3 pm, 4.6x250 mm) under isocratic conditions (4 mL/min, 30% MeOH / C02, 1600 psi backpressure) at 30 °C. The enantiomers were detected by UV absorbance (265 nm) and manually fractionated. After separation, the individual enantiomers were analyzed on the same instrument and method but were detected by UV absorbance (245 nm).

[0093] 7-(diethylamino)-4-methylquinolin-2(lH)-one (25). To a stirred suspension of 7- amino-4-methylquinolin-2(lH)-one (174 mg, 1 eq), acetic acid (240 mg, 4 eq), and NaBH(OAc)3 (848 mg, 4 eq) in 1 ,2-dichloroethane (10 mL) was added acetaldehyde (154 mg, 3.5 eq) on an ice- water bath. The yellow suspension was stirred at 0 °C for 3 hr, during which the crystalline starting material gradually dissolved. The mixture was partitioned between DCM (20 mL) and water (20 mL), then extracted with DCM (3 x 20 mL). The combined organics were washed with brine, dried over MgSO*, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (S1O2, 100% EtOAc) to afford 25 as a light yellow solid (177 mg, 77%). ESI-MS [M+l]: 231.0. ¹ H NMR (400 MHz, Chloroform-d) d 9.76 (s, 1H), 7.48 (d, J = 9.1 Hz, 1H), 6.63 (dd, J = 9.1, 2.5 Hz, 1H), 6.30 (d, J = 2.5 Hz, 1H), 6.23 (s, 1H), 3.45 (q, J = 7.1 Hz, 4H), 2.42 (s, 3H), 1.24 (t, J = 7.1 Hz, 6H).

[0094] 7-(diethyIamino)-2-imino-2H-chromene-3-carbonitrile (26). The title compound was prepared according to literature procedures, see e.g. : Zhou, J., et al. RSC Adv. 2014, 93(4), 51589-51592.

[0095] 7-(diethylammo)-4-hydroxy-2H-chn>men-2-one (27). The title compound was prepared according to literature procedures, see e.g. : Pan, S., et al. Chem. Comm. 2018, 39(54), 4955-4958.

[0096] Scheme 1.

[0097] 7-(benzylamino)-4-methyl-2H-chromen-2-one (28). To a stirred suspension of 7- amino-4-methyl-2H-chromen-2-one (174 mg, 1 eq) and benzaldehyde (159 mg, 1.5 eq) in MeCN (5 mL) was added NaBH(OAc) ₃ (414 mg, 2 eq)and the yellow suspension stirred at room temperature for 16 hours. The mixture was partitioned between DCM (20 mL) and water (20 mL), then extracted with DCM (3 x 20 mL). The combined organics were washed with brine, dried over MgSO«, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiCh, EtO Ac/hexanes) to afford 28 as a yellow solid (80 mg, 30%). ESI-MS [M+l]: 266.0. ^l U NMR (500 MHz, Chloroform-d) d 7.41 - 7.33 (m, 5H), 7.36 - 7.27 (m, 1H), 6.57 (dd, J = 8.7, 2.4 Hz, 1H), 6.49 (d, J = 2.3 Hz; 1H), 5.98 (q, J = 1.2 Hz, 1H), 4.76 (s, 1H), 4.41 (s, 2H), 2.34 (d, J = 1.2 Hz, 3H).

[0098] 7-((furan-3-ylmethyl)amino)-4-methyl-2H-chromen-2-one (29). The title compound was prepared analogously to 28, using furan-3-carbaldehyde. ESI-MS [M+l]: 256.0. ^! H NMR (500 MHz, Chloroform-d) d 7.43 (dt, J = 7.3, 1.6 Hz, 2H), 7.38 (d, J = 8.6 Hz, 1H), 6.57 (dd, J = 8.7, 2.3 Hz, 1H), 6.52 (d, J = 2.3 Hz, 1H), 6.42 (dd, J = 1.9, 0.9 Hz, 1H), 5.99 (d, J = 1.3 Hz, 1H), 4.56 (t, J = 5.5 Hz, 1H), 4.24 (d, J = 4.5 Hz, 2H), 2.35 (d, J = 1.2 Hz, 3H). [0099] 4-methyI-7-((3-phenylpropyl)amino)-2H-chromen-2-one (30). The title compound was prepared analogously to 28, using 3-phenylpropanal. ESI-MS [M+l]: 294.0. ¹ H NMR (500 MHz, Chloroform-d) d 7.40 - 7.29 (m, 3H), 7.28 - 7.19 (m, 3H), 6.47 (dd, J = 8.6, 2.4 Hz, 1H), 6.43 (d, J = 2.3 Hz, 1H), 6.00 (q, J = 1.2 Hz, 1H), 4.18 (s, 1H), 3.23 (t, J = 7.0 Hz, 2H), 2.77 (t, J = 7.5 Hz, 2H), 2.36 (d, J = 1.1 Hz, 3H), 2.06 - 1.96 (m, 2H).

[00100] 7-(ethyl(3-phenylpropyl)amino)-4-methyl-2H-chromen-2-one (31). To a stirred suspension of compound 30 (1 eq) and NaBH(OAc) ₃ (3 eq) in MeCN (2 mL) was added acetaldehyde (5 eq) on an ice-water bath, and the yellow suspension warmed to room

temperature over 16 hours. The mixture was partitioned between DCM (20 mL) and water (20 mL), then extracted with DCM (3 x 20 mL). The combined organics were washed with brine, dried over MgSO«, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (S1O2, EtO Ac/hexanes) to afford 31 as a viscous yellow oil. ESI-MS [M+l]: 322.1. ^X H NMR (500 MHz, Chloroform-d) d 7.40 - 7.30 (m, 3H), 7.28 - 7.20 (m, 3H), 6.56 - 6.47 (m, 2H), 5.99 - 5.94 (m, 1H), 3.47 - 3.33 (m, 4H), 2.70 (t, J = 7.7 Hz, 2H), 2.35 (d, J = 1.2 Hz, 3H), 2.03 - 1.93 (m, 2H), 1.20 (t, J = 7.1 Hz, 3H).

[00101] 7-(sec-butoxy)-4-methyl-2H-chromen-2-one (32). A stirred mixture of 7- hydroxy-4-methylcoumarin (176 mg, 1 eq), 2-bromobutane (274 mg, 2 eq), K ₂ CO ₃ (276 mg, 2 mmol), and KI (83 mg, 0.5 mmol) in DMF (5 mL) was heated at 80 °C overnight under argon. The mixture was diluted with 45 mL water, then extracted with diethyl ether (3 x 20 mL), dried over MgSO*, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (S1O2, 25% EtO Ac in hexanes) to afford 32 as a viscous light- yellow oil. ESI-MS [M+l]: 233.0.

[00102] 4-methyl-7-propoxy-2H-chromen-2-one (33). The title compound was prepared analogously to 32, using 1-bromopropane. ESI-MS [M+l]: 219.0.

[00103] 7-(2-aminoethoxy)-4-methyl-2H-chromen-2-one (34). A stirred mixture of 7- hydroxy-4- methyl-2H-chromen-2-one (132 mg), tert-butyl (2-bromoethyl)carbamate (203 mg), K ₂ CO ₃ (276 mg), and KI (83 mg) in DMF (5 mL) was heated at 70 °C overnight under argon. The mixture was diluted with 45 mL water, then extracted with DCM (3 x 20 mL), dried over MgS0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography. The intermediate was dissolved in 1.5 mL DCM and thereto was added 500 pL TFA, and the solution stirred for 2 hrs at room temperature. The residue was concentrated in vacuo and purified by reverse-phase preparative HPLC to afford the title compound as a viscous light-yellow oil. ESI-MS [M+l]: 220.0.

[00104] 7-(benzyl(ethyI)amino)-4-methyl-2H-chromen-2-one (35). The title compound was prepared analogously to 31, using 28 as starting material. ESI-MS [M+l ]: 294.0. 1H NMR (500 MHz, Chloroform-d) d 7.42 - 7.31 (m, 3H), 7.31 - 7.24 (m, 1H), 7.24 - 7.19 (m, 2H), 6.63 (dd, J = 8.9, 2.6 Hz, 1H), 6.57 (d, J = 2.6 Hz, 1H), 5.98 (q, J = 1.2 Hz, 1H), 4.62 (s, 2H), 3.58 (q, J = 7.1 Hz, 2H), 2.34 (d, J = 1.1 Hz, 3H), 1.34 - 1.25 (m, 4H).

[00105] 4-methyl-7-(pyrrolidin-l-yl)-2H-chromen-2-one (36). A suspension of 7- amino-4-methyl-2H-chromen-2-one (175 mg, 1 mmol), 1,4-dibromobutane (259 mg, 1.2 eq), K2CO3 (414 mg), and KI (83 mg, 0.5 mmol) in DMF (5 mL) was heated at 80 °C for 16 hours.. The mixture was partitioned between DCM (20 mL) and water (20 mL), then extracted with DCM (3 x 20 mL). The combined organics were washed with brine, dried over MgSO*, filtered, and concentrated under reduced pressure. The residue was purified by flash column

chromatography (S1O2, EtO Ac/hexanes) to afford 36 as a yellow solid. ESI-MS [M+l]: 230.0.

[00106] 7-(ethyl(phenethyl)amino)-4-methyl-2H-chromen-2-one (37). The title compound was prepared analogously to 31, using 40 as starting material. ESI-MS [M+l]: 308.0. Ή NMR (500 MHz, Chloroform-d) d 7.43 (d, J = 8.9 Hz, 1H), 7.39 - 7.31 (m, 2H), 7.30 - 7.20 (m, 3H), 6.64 (dd, J = 8.9, 2.6 Hz, 1H), 6.58 (d, J = 2.6 Hz, 1H), 5.99 (q, J = 1.2 Hz, 1H), 3.63 - 3.56 (m, 2H), 3.36 (q, J = 7.1 Hz, 2H), 2.96 - 2.89 (m, 2H), 2.38 (d, J = 1.2 Hz, 3H), 1.18 (t, J = 7.1 Hz, 3H).

[00107] 7-(benzyloxy)- 4-methyl- 2H-chromen-2- one (38). The title compound was prepared analogously to 34, using benzyl bromide. ¹ H NMR (500 MHz, Chloroform-d) d 7.53

(d, J = 8.8 Hz, 1H), 7.49 - 7.34 (m, 4H), 6.96 (dd, J = 8.8, 2.5 Hz, 1H), 6.92 (d, J = 2.5 Hz, 1H), 6.16 (q, J = 1.2 Hz, 1H), 5.16 (s, 1H), 2.42 (d, J = 1.2 Hz, 2H).

[00108] 4-methyl-7-((l-phenylethyl)amino)-2H-chromen-2-one (39). The title compound was prepared analogously to 34, using (l-bromoethyl)benzene and 7-amino-4-methyl- 2H-chromen-2-one as starting materials. ESI-MS [M+l]: 280.0. [00109] 4-methyl-7-(phenethylamino)-2H-chromen-2-one (40). The title compound was prepared analogously to 28, using 2-phenylacetaldehyde. ESI-MS [M+l ]: 280.0.

[00110] 4-methyl-7-(l-phenylpropoxy)-2H-chromen-2-one (41). The title compound was prepared analogously to 32, using (1 -bromopropyl)benzene, to afford 41 as a white solid (158.4 mg, 54%). ^l U NMR (400 MHz, Chloroform-d) d 7.44 (d, J = 8.8 Hz, 1H), 7.40 - 7.24 (m, 3H), 6.88 (dd, J = 8.8, 2.5 Hz, 1H), 6.74 (d, J = 2.5 Hz, 1H), 6.12 - 6.07 (m, 1H), 5.09 (dd, J = 7.3, 5.6 Hz, 1H), 2.36 (d, J = 1.3 Hz, 3H), 2.07 (dq, J = 14.7, 7.3 Hz, 1H), 1.93 (ddd, J = 13.5,

7.3, 5.7 Hz, 1H), 1.03 (t, J = 7.4 Hz, 3H).

[00111] 7-(benzhydryloxy)-4-methyl-2H-chromen-2-one (42). The title compound was prepared analogously to 32, using (bromomethylene)dibenzene. ¹ H NMR (500 MHz,

Chloroform-*/) d 7.51 - 7.25 (m, 10H), 6.99 (dd, 7= 8.8, 2.5 Hz, 1H), 6.87 (d, J= 2.5 Hz, 1H), 6.29 (s, 1H), 6.13 (q, J= 1.2 Hz, 1H), 2.38 (d, J= 1.2 Hz, 2H).

[00112] 4-methyl-7-(2-phenylpropoxy)-2H-chromen-2-one (43). The title compound was prepared analogously to 32, using 2-phenylpropyl 4-methylbenzenesulfonate, to afford 43 as a white solid (60.3 mg, 71%). ¹ H NMR (500 MHz, Chloroform-d) d 7.53 (d, J = 8.8 Hz, 1H), 7.49 - 7.34 (m, 5H), 6.96 (dd, J = 8.8, 2.5 Hz, 1H), 6.92 (d, J = 2.5 Hz, 1H), 6.16 (q, J = 1.2 Hz, 1H), 5.16 (s, 2H), 2.42 (d, J = 1.2 Hz, 3H), 1.29 (s, 3H).

[00113] 7-(l-(4-ffluoropheiyl)ethoxy)-4-mefliyl-2H-chromeii-2-one (44) The title compound was prepared analogously to 32, using l-(l-bromoethyl)-4-fluorobenzene. ¹ H NMR (500 MHz, Chloroform-d) d 7.46 (d, J = 8.8 Hz, 1H), 7.39 - 7.31 (m, 2H), 7.10 - 7.01 (m, 2H), 6.86 (dd, J = 8.8, 2.5 Hz, 1H), 6.74 (d, J = 2.5 Hz, 1H), 6.12 (q, J = 1.2 Hz, 1H), 5.37 (q, J = 6.4 Hz, 1H), 2.38 (d, J = 1.3 Hz, 3H), 1.68 (d, J = 6.4 Hz, 3H).

[00114] 4-methyl-7-(l-(o-tolyl)ethoxy)-2H-chromen-2-one (45). The title compound was prepared analogously to 32, using 1 -(1 -bromoethyl)-2-methylbenzene, to afford 58 as a white solid (172.7 mg, 78%). ‘H NMR (500 MHz, Chloroform-d) d 7.45 (d, J = 8.8 Hz, 1H), 7.39 - 7.33 (m, 1H), 7.23 - 7.13 (m, 3H), 6.84 (dd, J = 8.8, 2.5 Ήz, 1H), 6.61 (d, J = 2.5 Hz, 1H), 6.10 (q, J = 1.3 Hz, 1H), 5.53 (q, J = 6.4 Hz, 1H), 2.46 (s, 3H), 2.39 - 2.35 (m, 3H), 1.67 (d, J = 6.4 Hz, 3H). [00115] 4-methyl-7-(3-phenylbutoxy)-2H-chromeii-2-one (46). The title compound was prepared analogously to 32, using 3-phenylbutyl 4-methylbenzenesulfonate. ¹ H NMR (500

MHz, Chloroform-d) d 7.48 (d, J = 8.8 Hz, 1H), 7.37 - 7.29 (m, 2H), 7.23 (dtd, J = 7.2, 2.7, 1.0 Hz, 3H), 6.82 (dd, J = 8.8, 2.5 Hz, 1H), 6.75 (d, J = 2.5 Hz, 1H), 6.14 (q, J = 1.2 Hz, 1H), 3.94 (ddd, J = 9.4, 6.6, 5.6 Hz, 1H), 3.88 (ddd, J = 9.4, 7.4, 6.3 Hz, 1H), 3.09 - 2.97 (m, 1H), 2.41 (d, J = 1.2 Hz, 3H), 2.22 - 2.02 (m, 2H), 1.37 (d, J = 7.0 Hz, 3H).

[00116] 4-methyl-7-((l-phenylpropan-2-yl)oxy)-2H-chrOmen-2-one (47). The title compound was prepared analogously to 32, using l-phenylpropan-2-yl 4- methylbenzenesulfonate, to afford 56 as a white solid (154.5 mg, 70%). NMR (500 MHz, Chloroform-d) d 7.49 (d, J = 8.5 Hz, 1H), 7.38 - 7.21 (m, 5H), 6.88 - 6.81 (m, 2H), 6.14 (q, J = 1.2 Hz, 1H), 4.67 (q, J = 6.1 Hz, 1H), 3.13 (dd, J = 13.8, 6.1 Hz, 1H), 2.89 (dd, J = 13.8, 6.5 Hz, 1H), 2.41 (d, J = 1.2 Hz, 3H), 1.38 (d, J = 6.1 Hz, 3H).

[00117] 4-methyl-7-(piperidin-l-yl)-2H-chroinen-2-one (48). The title compound was prepared analogously to 36, using 1,5-dibromopropane. ESI-MS [M+l]: 244.0. 'H NMR (400

MHz, Chloroform-d) d 7.42 (d, J = 8.9 Hz, 1H), 6.84 (dd, J = 8.9, 2.6 Hz, 1H), 6.73 (d, J = 2.5 Hz, 1H), 6.03 (t, J = 1.2 Hz, 1H), 3.36 (t, J = 5.1 Hz, 5H), 2.38 (d, J = 1.2 Hz, 4H), 1.77 - 1.65 (m, 3H).

[00118] 7-(l-(2-ffluoropheiyl)ethoxy)-4-mefliyl-2H-chromeii-2-one (49). The title compound was prepared analogously to 32, using l-(l-bromoethyl)-2-fluorobenzene, to afford 49 as a white solid (185.6 mg, 83%). ^X H NMR (400 MHz, Chloroform-d) d 7.46 (d, J = 8.8 Hz, 1H), 7.40 (td, J = 7.6, 1.8 Hz, 1H), 7.33 - 7.23 (m, 1H), 7.22 - 7.05 (m, 2H), 6.88 (dd, J = 8.8,

2.5 Hz, 1H), 6.76 (d, J = 2.5 Hz, 1H), 6.12 (q, J = 1.3 Hz, 1H), 5.72 (q, J = 6.4 Hz, 1H), 2.38 (d, J = 1.2 Hz, 3H), 1.71 (d, J = 6.4 Hz, 3H).

[00119] 7-(diethylamino)-4-(hydroxymethyl)-2H-chrOmen-2-one (50). The title compound was prepared using literature procedures, see e.g. : Kawaguchi, M., et al. Chem. Comm. 2018, 73(54), 10371-10374.

[00120] 7-(l-(2,6-difluorophenyl)ethoxy)-4-mefliyl-2H-dirom«i-2-one (51). The title compound was prepared analogously to 36, using 2-(l-bromoethyl)-l,3-difluorobenzene. ^X H NMR (400 MHz, Chloroform-d) d 7.47 (d, J = 8.8 Hz, 1H), 7.25 (tt, J = 8.4, 6.3 Hz, 1H), 6.95 - 6.85 (m, 3H), 6.83 (d, J = 2.5 Hz; 1H), 6.12 (q, J = 1.2 Hz, 1H), 5.81 (q, J = 6.6 Hz, 1H), 2.38 (d, J = 1.2 Hz, 3H), 1.84 (d, J = 6.6 Hz, 3H).

[00121] 7-(diethylammo)-l,4-dimethyIquinolln-2(lH)-one (52). Under argon, a solution of 25 (40 mg, 1 eq) in DMF (1 mL) was added to NaH (8.2 mg, 60% in mineral oil, 1.2 eq) on ice, and the suspension stirred for 10 min on ice. Thereto was added a solution of methyl iodide (30 mg, 1.2 eq) in DMF (1 mL) and stirred for 16 hr at room temperature. The reaction was quenched with 1 mL saturated aqueous NEUCl. The mixture was diluted with 20 mL HzO and extracted with EtOAc (3 x 20 mL). The combined organics were washed once with brine, dried over MgSO*, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography to afford the title compound as a tan solid. ESI-MS [M+l]: 245.0. ^] H NMR (400 MHz, Chloroformed) d 7.51 (d, J= 9.0 Hz, 1H), 6.64 (dd, J= 9.0, 2.4 Hz, 1H), 6.42 (d, J= 2.4 Hz, 1H), 6.30 (s, 1H), 3.67 (s, 3H), 3.48 (q, J= 7.1 Hz, 4H), 2.39 (s, 3H), 1.26 (t, J= 7.1 Hz,

6H).

[00122] Scheme 2.

THPO^ ^BF * ^K

cataCXium A,

[00123] 3-bromo-7-(diethylammo)-4-methyl-2H-chromai-2-one (Int 2-1). The title intermediate was prepared by literature procedures, see e.g. : Kitamura, K., et al. Bioorg. Med. Chem. Lett 2014, 24(24), 5660-5662. [00124] 7-(diethylamino)-4-metiiyl-3-(2-((tetrahydro-2H-pyran-2-yI)o xy)ethyI)-2H- chromen-2-one (Int 2-2). A mixture of Int 2-1 (1.3 g, 4.19 mmol, 1 eq), potassium trifluoro(2- ((tetrahydro-2H-pyran-2- yl)oxy)ethyl)borate (1.09 g, 4.61 mmol, 1.1 eq), bis(l-adamantyl)- butyl-phosphane (300 mg, 838 mthoΐ, 0.2 eq), Pd(OAc)2 (282 mg, 1.26 mmol, 0.3 eq) and CS2CO3 (4.10 g, 12.5 mmol, 3 eq) in dioxane (30 mL) and H2O (13 mL) was degassed and purged with N2 for 3 times, and then the mixture was stirred at 100 °C for 12 hours under N2 atmosphere. The reaction mixture was quenched by addition of H2O (300 mL), and then extracted with EtOAc (100 mL * 3). The combined organic layers were dried ova- Na ₂ S0 ₄ , filtered and concentrated under reduced pressure to give a residue. The residue was purified by reverse-phase preparative HPLC to give Int 2-2 (1.2 g, 3.34 mmol, 79% yield) as a yellow solid. ¹ H NMR (400MHz, chloroform-d) d = 7.44 - 7.39 (m, 1H), 6.64 - 6.58 (m, 1H), 6.51 (d, J=2.8 Hz, 1H), 4.59 (t, J=4.0 Hz, 1H), 3.94 - 3.77 (m, 2H), 3.59 (td, J=6.9, 9.6 Hz, 1H), 3.53 - 3.45 (m, 1H), 3.41 (q, J=6.8 Hz, 4H), 2.98 - 2.89 (m, 2H), 2.42 - 2.36 (m, 3H), 1.81 - 1.72 (m, 1H), 1.72 - 1.62 (m, 1H), 1.59 - 1.43 (m, 4H), 1.20 (t, J=6.8 Hz, 6H).

[00125] 7-(diethylainino)-3-(hydroxymetfayI)-4-methyl-2H-chromen-2-o ne (Int 2-3).

A mixture of Int 2-1 (3 g, 9.67 mmol, 1 eq), tributylstannylmethanol (4.04 g, 12.6 mmol, 1.3 eq) and PdfPPhs)* (559 mg, 484 mihoΐ, 0.05 eq) in dioxane (30 mL) was bubbled with nitrogen and vial was sealed. The mixture was stirred at 100 °C for 16 hours and then 120 °C for 6 hours. The mixture was purified by column chromatography over silica gel (petroleum ether/ethyl acetate 10/1 to 1/1). The desired fractions were collected and concentrated under vacuum to give Int 2-3 (924 mg, 3.39 mmol, 35% yield, 96% purity) as a yellow solid. LC-MS [ESI, M+l]: 262. *H NMR (400 MHz, CDCh) d = 7.45 (d, J=9.2 Hz, 1H), 6.62 (dd, J=2.8, 9.2 Hz, 1H) 6.50 (d, J=2.8, 1H), 4.67 (d, J=6.8, 1H), 3.42 (q, 1=1.2, 4H), 2.97 (t, J=6.8, 1H), 2.42 (s, 1H), 1.22 (q, J=7.2,

4H).

[00126] 7-(diethylamino)-3-(2-hydroxyefliyl)-4-methyl-2H-chromen-2-o ne (53). A solution of Int 2-2 (1.9 g, 5.29 mmol, 1 eq) in HCl/dioxane (4 M, 50 mL) was stirred at 20 °C for 30 min. The mixture was concentrated under reduced pressure. The residue was purified by reverse-phase preparative HPLC. The desired fractions were collected and lyophilized to give 53 (734 mg, 45%) as a brown gum. ^X H NMR (400 MHz, DMSO-d6) d = 7.58 - 7.48 (m, 1H), 6.75 - 6.63 (m, 1H), 6.52 - 6.42 (m, 1H), 4.61 - 4.53 (m, 1H), 3.75 -3.59 (m, 2H), 3.46 - 3.37 (m, 6H), 2.84 - 2.73 (m, 2H), 2.37 - 2.27 (m, 3H), 1.73 - 1.52 (m, 2H), 1.49 - 1.34 (m, 4H), 1.16 - 1.07 (m, 6H). LC-MS [ESI, M+l]: 276.1.

[00127] Scheme 3.

o o

LLA

n ^■ 1: Int 3-1-1

n ^■ 2: Int 3-1-2

OP GDI, carboxylic

LIOH -CCCr acids

/<N n = 1: 54

n - 2: 62

[00128] Ethyl 2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)acetate (Lit 3-1-

1). To a stirred solution of 3-diethylaminophenol (412.5 mg, 1 eq) and diethyl 2-acetylsuccinate (703 mg, 1.3 eq) in toluene (5 mL) was added ClTi(OiPr)3 (717 mg) and the dark brown suspension refluxed overnight Thereto was added sat. aqueous Rochelle’s salt (10 mL) and the emulsion was filtered through C ₆ lite. The filtrate was extracted with EtOAc (3 x 20 mL). The combined organics were washed once with brine, dried over MgSCU, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography to afford a mixture of the title intermediate and the isopropyl ether as a brown oil.

[00129] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)acetic acid (54). To a solution of Int 3-1-1 in EhO (2 mL), MeOH (2 mL), and THF (2 mL) was added lithium hydroxide monohydrate (3 eq) and the light brown suspension stirred at room temperature for 16 hours. The mixture was acidified by addition of 1 M HC1 and extracted with EtOAc (3 x 20 mL). The combined organics were washed once with brine, dried over MgSO*, filtered, and concentrated in vacuo, to afford a brown solid A sample was purified by reverse-phase preparative HPLC to afford the title compound as a yellow solid. LC-MS [ESI, M+l]: 290.0. ¾ NMR (400 MHz, Chloroform-c/) d 7.59 (d, J= 9.0 Hz, 1H), 6.95 (dd, ./= 8.9, 2.5 Hz, 1H), 6.80 (d, ./= 2.5 Hz, 1H), 3.78 (s, 2H), 3.51 (q, ./= 7.1 Hz, 3H), 2.44 (s, 2H), 1.23 (t, J= 7.1 Hz, 6H).

[00130] ethyl 3-(7-(diethylammo)-4-methyl-2-oxo-2H-chromen-3-yl)propanoate (Int 3-1-2). The title intermediate was prepared analogously to Int 3-1-1, using diethyl 2- acetylpentanedioate.

[00131] 2-(7-(diethyIammo)-4-methyl-2-oxo-2H-chromen-3-yI)-N-pheneth ylacetamide (55). To a stirred solution of 54 (40 mg, 1 eq) in DMF (1 mL) was added CDI (33 mg, 1.5 eq) and stirred at room temperature for 1 hrs, during which the color changed from orange-red to yellow. Thereto was added 2-phenylethan-l -amine (49 mg, 3 eq) and stirred for an additional hour at room temperature. The mixture was diluted with 19 mL H2O and extracted with EtOAc (3 x 20 mL). The combined organics were washed once with brine, dried over MgSO*, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography (S1O2, 0 to 45% EtOAc in hexanes) to afford the title compound as a light yellow solid. In other examples, purification was achieved using reverse-phase preparative HPLC. E SI-MS [M+l]: 393.2. ^X H NMR (500 MHz, Chloroform-i/) d 7.45 (d, J= 9.1 Hz, 1H), 7.25 - 7.16 (m, 2H), 7.16 - 7.09 (m, 3H), 6.65 (dd, J = 9.1, 2.6 Hz, lH), 6.53 (d, J = 2.6 Hz, 1H), 6.46 (s, 1H), 3.53 - 3.40 (m, 8H), 2.78 (t, J= 7.1 Hz, 2H), 2.46 (s, 3H), 1.24 (t, J= 7.1 Hz, 6H).

[00132] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)-N-(3- phenylpropyl)acetamide (56). The title compound was prepared analogously to 55, using 3- phenylpropan-1 -amine. ESI-MS [M+l]: 407.2. ¹ H NMR (500 MHz, Chloroform-i/) d 7.46 (d, J = 9.1 Hz, 1H), 7.30 - 7.22 (m, 2H), 7.25 - 7.13 (m, 3H), 6.64 (dd, J = 9.1, 2.6 Hz, 1H), 6.57 - 6.49 (m, 2H), 3.54 (s, 2H), 3.44 (q, J= 7.1 Hz, 4H), 3.24 (td, J= 7.1, 5.9 Hz, 2H), 2.66 - 2.58 (m, 2H), 2.52 (s, 3H), 1.89 - 1.76 (m, 2H), 1.23 (t , J= 7.1 Hz, 6H).

[00133] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)-N-(4- phenylbutyl)acetamide (57). The title compound was prepared analogously to 55, using 4- phenylbutan-1 -amine. ESI-MS [M+l]: 421.2. ¹ H NMR (500 MHz, Chloroform·*/) d 7.45 (d, J= 9.1 Hz, 1H), 7.25 - 7.16 (m, 2H), 7.13 (d, J= 7.0 Hz, 3H), 6.65 (dd, J= 9.1, 2.6 Hz, 1H), 6.53 (d, J= 2.6 Hz, 1H), 6.46 (s, 1H), 3.53 - 3.40 (m, 9H), 2.78 (t, J= 7.1 Hz, 2H), 2.46 (s, 3H), 1.24 (t, J= 7.1 Hz, 6H). [00134] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)-N-(2- (dimethylamino)ethyl)acetamide (58). The title compound was prepared analogously to 55, using N ¹ ,N ¹ -dimethylpropane-, 2l -diamine. ESI-MS [M+l]: 360.2. ¹ H NMR (400 MHz,

Chloroform-d) d 7.45 (d, 7= 9.0 Hz, 1H), 6.72 (s, 1H), 6.62 (dd, 7= 9.1, 2.6 Hz, 1H), 6.51 (d, J = 2.6 Hz, 1H), 3.55 (s, 2H), 3.43 (q, J= 7.1 Hz, 4H), 3.30 (q, J= 6.1 Hz, 2H), 2.50 (s, 3H), 2.41 (q, J= 6.0 Hz, 2H), 2.24 (s, 6H), 1.22 (t, J= 7.1 Hz, 6H).

[00135] 2-(7-(diethylammo)-4-methyl-2-oxo-2H-chromen-3-yl)-N-(3- (dimethylamino)propyl)acetamide (59). The title compound was prepared analogously to 55, using N ¹ ,N ¹ -dimethylpropane-l, 3-diamine. ESI-MS [M+l]: 374.2. ¹ H NMR (400 MHz, Chloroform-d) d 7.44 (d, 7= 9.0 Hz, 1H), 6.62 (dd, 7= 9.0, 2.6 Hz, 1H), 6.50 (d, 7= 2.6 Hz,

1H), 3.55 (s, 3H), 3.43 (q, 7= 7.1 Hz, 4H), 3.28 (dt, 7= 10.0, 5.0 Hz, 3H), 2.45 (s, 4H), 2.32 (t, 7 = 6.6 Hz, 3H), 2.09 (s, 6H), 1.22 (t, 7 = 7.1 Hz, 6H).

[00136] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen -3-yl)-N-methylacetamide (60). The title compound was prepared analogously to 55, using methylamine. ESI-MS [M+l]: 303.1. ¹ H NMR (400 MHz, Chloroform d) d 7.45 (d, 7= 9.1 Hz, 1H), 6.62 (dd, 7= 9.1, 2.6 Hz, 1H), 6.53 - 6.43 (m, 2H), 3.54 (s, 2H), 3.43 (q, 7= 7.1 Hz, 4H), 2.76 (d, 7= 4.9 Hz, 3H), 2.50 (s, 3H), 1.22 (t, 7= 7.1 Hz, 6H).

[00137] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)-N,N- dimethylacetamide (61). The title compound was prepared analogously to 55, using dimethylamine. ESI-MS [M+l]: 317.1 ¹ H NMR (400 MHz, Chloroformed) d 7.43 (d, 7= 9.0 Hz, 1H), 6.60 (dd, 7= 9.0, 2.6 Hz, 1H), 6.51 (d, 7= 2.6 Hz, 1H), 3.70 (s, 2H), 3.42 (q, 7= 7.1 Hz, 4H), 2.98 (s, 3H), 2.39 (s, 3H), 1.21 (t, 7= 7.0 Hz, 6H).

[00138] 3-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)propanoic acid (62). The title compound was prepared analogously to 54, using Int 3-1-2. ESI-MS [M+l]: 304.0

[00139] N-benzyl-3-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl) propenamide (63). The title compound was prepared analogously to 55, using 62 and benzylamine. ESI-MS [M+l]: 393.2. ¹ H NMR (500 MHz, Chloroformed) d 7.41 (d, 7= 9.0 Hz, 1H), 7.35 - 7.14 (m, 6H), 6.62 (dd , 7 = 9.0, 2.6 Hz, 1H), 6.47 (d, 7 = 2.6 Hz, 1H), 5.24 (s, 1H), 4.38 (t, 7= 6.0 Hz, 3H), 3.42 (q, 7= 7.1 Hz, 4H), 2.95 (t, 7= 7.5 Hz, 2H), 2.51 (t, .J= 7.5 Hz, 2H), 2.37 (s, 3H), 1.22 (t, 7= 7.1 Hz, 6H). [00140] 3-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)-N- phenethylpropanamide (64). The title compound was prepared analogously to 55, using 62 and 2-phenylethan-l -amine. ESI-MS [M+l]: 407.2. 1+R (400 MHz, Chloroform-d ) d 7.43 (d, J = 9.0 Hz, 1H), 7.33 - 7.10 (m, 6H), 6.62 (dd, J= 9.1, 2.6 Hz, 1H), 6.51 (d, J= 2.6 Hz, 1H), 5.84 (s, 1H), 3.47 (dq, J = 29.3, 7.1 Hz, 7H), 2.94 (t, J= 7.5 Hz, 2H), 2.78 (t, J= 7.1 Hz, 2H), 2.44 (z J= 7.5 Hz, 2H), 1.22 (t, J = 7.1 Hz, 6H).

[00141] 3-(7-(diethylammo)-4-methyl-2-oxo-2H-chromen-3-yl)-N-(3- phenylpropyI)propenamide (65). The title compound was prepared analogously to 55, using 62 and 3-phenylpropan-l -amine. ESI-MS [M+l]: 421.2. NMR (400 MHz, Chloroform-*/) d 7.41 (d, J= 9.0 Hz, 1H), 7.33 - 7.21 (m, 4H), 7.25 - 7.08 (m, 6H), 6.60 (dd, J = 9.0, 2.6 Hz, 1H), 6.49 (d, J= 2.6 Hz, 1H), 3.42 (q, J= 7.1 Hz, 3H), 3.27 (q, J= 6.7 Hz, 1H), 2.95 (t, J= 7.4 Hz, 1H), 2.61 (dt, J= 16.8, 7.7 Hz, 4H), 2.48 (t, J= 7.4 Hz, 1H), 2.40 (s, 2H), 1.80 (h, J= 7.3 Hz, 4H), 1.22 (t, J= 7.1 Hz, 6H).

[00142] 3-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)-N- methylpropanamide (66). The title compound was prepared analogously to 55, using 62 and methylamine. ESI-MS [M+l]: 317.1. ¹ H NMR (500 MHz, Chloroform-d ) d 7.42 (d, J= 9.0 Hz, 1H), 6.61 (dd, J= 9.0, 2.6 Hz, 1H), 6.48 (d, J= 2.6 Hz, 1H), 6.00 (s, 1H), 3.41 (q, J= 7.1 Hz, 4H), 2.95 (q, J= 7.7 Hz, 2H), 2.78 (d, J= 4.9 Hz, 3H), 2.46 (t, J= 7.5 Hz, 2H), 2.40 (s, 3H),

1.21 (t, J= 7.1 Hz, 6H).

[00143] 3-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)-N-(2- (dimethylamino)ethyl)propenamide (67). The title compound was prepared analogously to 55, using 62 and N ¹ ,N ¹ -dimethylpropane- ,2l -diamine. ESI-MS [M+l]: 317.1. ¹ H NMR (500 MHz, Chloroform-*/) d 7.41 (d, J= 9.1 Hz, 1H), 6.60 (dd, J= 9.0, 2.6 Hz, 1H), 6.52 (s, 1H), 6.49 (d, J = 2.6 Hz, 1H), 3.63 (s, 1H), 3.41 (q, J= 7.1 Hz, 4H), 3.35 (q, J= 5.7 Hz, 2H), 2.94 (dd, J= 8.4, 6.8 Hz, 2H), 2.54 -2.39 (m, 4H), 2.40 (s, 3H), 2.28 (s, 5H), 1.21 (t, J= 7.1 Hz, 6H).

[00144] 3-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)-N-(3- (dimethylamino)propyl)propenamide (68). The title compound was prepared analogously to 55, using 62 and N ¹ ,N ¹ -dimethylpropane-l, 3-diamine. ESI-MS [M+l]: 388.1. ¹ H NMR (500 MHz, Chloroform-*/) d 7.42 (d, J= 9. \ Hz, 1H), 6.93 (d, J= 6.0 Hz, 1H), 6.60 (dd, J= 9.0, 2.6 Hz, 1H), 6.49 (d, J= 2.6 Hz, 1H), 3.41 (q, J= 7.1 Hz, 5H), 3.30 (td, J= 6.5, 5.4 Hz, 2H), 2.94 (dd, J= 8.4, 6.8 Hz, 2H), 2.43 (dd, J= 8.3, 6.9 Hz, 2H), 2.33 (t, J= 6.6 Hz, 2H), 2.20 (s, 6H), 1.62 (p, J= 6.6 Hz, 2H), 1.21 (t, J= 7.1 Hz, 6H).

[00145] Scheme 4.

[00146] Tert-butyl (2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3- yl)ethyl)carbamate (69). A mixture of Jut 2-1 (310 mg, 1 eq), potassium 2-(Boc- aminoethyl)trifluoroborate (301 mg, 1.2 eq), RuPhos (93 mg, 0.2 eq) and Cs ₂ CO ₃ (977 mg, 3 eq) in toluene (4.5 mL) and H2O (1.5 mL) in a microwave tube was sparged with argon for 5 minutes while stirring. Thereto was added Pd(dba)2 (58 mg, 0.1 eq) and further sparged for an additional minute. The vial was sealed and heated at 90 °C for 16 hr. The initially red biphasic mixture turned dark orange, and a black precipitate appeared. The reaction was then diluted with water (15 mL) and extracted with EtOAc (3 x 20 mL). The combined organics were washed once with brine, dried over MgSO*, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (S _i O ₂ , 0 to 25% EtOAc in hexanes) to afford 69 as a yellow solid (249.3 mg, 67%). ESI-MS [M+l]: 375.2. ¹ H NMR (400 MHz, CDCb) d = 7.42 (br d, J=9.2 Hz, 1H), 6.60 (dd, J=2.4, 9.2 Hz, 1H), 6.50 (d, J=2.4 Hz, 1H), 4.95 - 4.77 (m, 1H), 3.41 (q, J=7.2 Hz, 4H), 3.31 (q, J=6.4 Hz, 2H), 2.84 (br t, J=6.7 Hz, 2H), 2.39 (s, 3H), 1.43 (s, 9H), 1.21 (t, J=7.2 Hz, 6H).

[00147] 3-(2-aminoethyl)-7-(diethylamino)-4-inethyl-2H-chromen-2~one (Int 4-1). To a stirred solution of 69 (450 mg) in DCM (4 mL) was added neat TEA (1 mL) and stirred at room temperature for 16 hours. The mixture was concentrated in vacuo and then treated with saturated NaHC0 ₃ . The yellow precipitate that appeared was filtered and washed with H2O to afford the title intermediate as a yellow solid (321 mg, 98%). ESI-MS [M+l]: 275.2 ¹ H NMR (600 MHz, DMSO-d ₆ ) d 7.80 (s, 3H), 7.56 (d, J= 9.1 Hz, 1H), 6.72 (dd, J = 9.1, 2.6 Hz, 1H), 6.50 (d, J=

2.6 Hz, 1H), 3.43 (q, J= 7.0 Hz, 3H), 3.40 (s, 1H), 2.96 - 2.87 (m, 2H), 2.82 (dd, J= 8.6, 6.6 Hz, 2H), 2.37 (s, 3H), 1.12 (t, J= 7.0 Hz, 6H).

[00148] N-(2-(7-(diethylamino)-4-methyl- 2-oxo- 2H-chromen-3-yl)ethyI)-3- phenylpropanamide (70). The title compound was prepared analogously to 55, using 3- phenylpropanoic acid and Int 4-1. ESI-MS [M+l]: 407.2. ¹ H NMR (400 MHz, Chloroform-d ) d 7.43 (d, J = 9.0 Hz, 1H), 7.31 - 7.17 (m, 4H), 7.23 - 7.09 (m, 2H), 6.63 (dd, J= 9.0, 2.6 Hz, 1H), 6.50 (d, J= 2.6 Hz, 1H), 6.18 (t, J= 5.0 Hz, 1H), 3.49 - 3.34 (m, 6H), 2.95 (dd, J= 8.9, 6.9 Hz, 2H), 2.83 (t, J= 6.8 Hz, 2H), 2.47 (dd, J= 8.9, 6.9 Hz, 2H), 1.23 (t, J= 7.1 Hz, 6H).

[00149] 3-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)-N-(2- f1uorophenethyl)propenamide (71). The title compound was prepared analogously to 55, using 62 and 2-(2-fluorophenyl)ethan-l -amine. ESI-MS [M+l]: 425.2. ¹ H NMR (400 MHz,

Chloroform-d) d 7.42 (d, J= 9.1 Hz, 1H), 7.28 - 6.93 (m, 6H), 6.61 (dd, J= 9.0, 2.6 Hz, 1H), 6.49 (d, J= 2.6 Hz, 1H), 5.95 (t, J= 5.5 Hz, 1H), 3.57 - 3.33 (m, 7H), 2.97 - 2.78 (m, 6H), 2.43 (t, J= 7.5 Hz, 2H), 1.21 (t, J= 7.1 Hz, 6H).

[00150] 3-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)-N-(3- fluorophenethyl)propenamide (72). The title compound was prepared analogously to 55, using 62 and 2-(3-fluorophenyl)ethan-l-amine. ESI-MS [M+l]: 425.2. ¹ H NMR (400 MHz,

Chloroform-d) d 7.43 (d, J= 9.0 Hz, 1H), 7.30 - 7.16 (m, 1H), 7.02 - 6.82 (m, 3H), 6.62 (dd, J= 9.0, 2.6 Hz, 1H), 6.51 (d, J= 2.6 Hz, 1H), 5.91 (s, 1H), 3.55 - 3.34 (m, 6H), 2.93 (t, J= 7.5 Hz, 2H), 2.80 (dt, J= 14.1, 7.0 Hz, 2H), 2.51 - 2.38 (m, 4H), 1.35 - 1.15 (m, 6H).

[00151] 3-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)-N-(4- fluorophenethyl)propenamide (73). The title compound was prepared analogously to 55, using 62 and 2-(4-fluorophenyl)ethan-l -amine. ESI-MS [M+l]: 425.2. ¹ H NMR (400 MHz,

Chloroform-*/) d 7.43 (d, J= 9.0 Hz, 1H), 6.62 (dd, J= 9.0, 2.6 Hz, 1H), 6.50 (d, J= 2.6 Hz,

1H), 4.40 (t, J= 5.9 Hz, 3H), 2.91 (t, J= 7.4 Hz, 2H), 2.83 - 2.70 (m, 9H), 1.22 (t, J= 7.1 Hz,

6H). [00152] 3-(7-(diethylamino)-4-methiyl-2-oxo-2H-chromen-3-yl)-N-(2-(p yridin-2- yl)ethyl)propenamide (74). The title compound was prepared analogously to 55, using 62 and 2-(pyridin-2-yl)ethan-l -amine. ESI-MS [M+l]: 408.2.

[00153] 3-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)-N-(2-(py ridm-3- yl)ethyl)propenamide (75). The title compound was prepared analogously to 55, using 62 and 2-(pyridin-3-yl)ethan-l -amine. ESI-MS [M+l]: 408.2.

[00154] 3-(7-(diethylamino)-4-methyi-2-oxo-2H-chromen-3-yl)-N-(2-(py ridin-4- yl)ethyl)propenamide (76). The title compound was prepared analogously to 55, using 62 and 2-(pyridin-4-yl)ethan-l -amine. ESI-MS [M+l]: 408.2.

[00155] 3-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)-N-(3- phenylpropyl)propenamide (77). The title compound was prepared analogously to 55, using 62 and 3-phenylpropan-l -amine. ESI-MS [M+l]: 421.2. ¹ H NMR (500 MHz, Chloroform-*/) d 7.43 (d, J = 9.1 Hz, 1H), 7.26 (d, J= 7.7 Hz, 1H), 7.24 - 7.11 (m, 4H), 6.62 (dd, J= 9.0, 2.6 Hz, 1H), 6.49 (d, J = 2.6 Hz, 1H), 6.13 (s, 1H), 3.50 - 3.37 (m, 7H), 2.88 (t, J = 6.8 Hz, 2H), 2.63 (t, J= 7.6 Hz, 2H), 2.22 - 2.14 (m, 2H), 2.02 - 1.90 (m, 2H), 1.22 (t, J= 7.1 Hz, 6H).

[00156] Scheme 5.

[00157] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyI (4-nitrophenyl) carbonate (Int 5-1). To a stirred solution of 53 (500 mg, 1.82 mmol, 1 eq) and pyridine (440 uL, 5.46 mmol, 3 eq) in DCM (2 mL) under argon was added a solution of 4-nitrophenyl chloroformate (440 mg, 2.18 mmol, 1.2 eq) in DCM (2 mL), dropwise, at room temperature. The reaction was stirred for 3 hours at room temperature, during which the solution turned orange and a crystalline precipitate appeared. The mixture was partitioned between DCM (20 mL) and 0.1 M aqueous HC1 (20 mL), then extracted with DCM (3 x 20 mL). The combined organics were washed with brine, dried over MgSO*, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (S1O2, 0 to 25% EtOAc in hexanes) to afford Iht 5-1 as a yellow-orange solid (621.1 mg, 78%), which was stored at -20 °C. LC-MS [ESI, M+l]: 441.2. ¹ H NMR (400 MHz, CDC1 ₃ ) d = 8.27-8.23 (m, 2H), 7.43 (d, J = 8.8 Hz, 1H), 7.36 - 7.31 (m, 2H), 6.61 - 6.56 (m, 1H), 6.51 (d, J = 2.8 Hz, 1H), 4.48 (t, J = 6.8 Hz, 2H), 3.42 (q, J = 7.2 Hz, 4H), 3.11 (t, J = 6.8 Hz, 2H), 2.41 (s, 3H), 1.22 (t, J = 7.2 Hz, 6H).

[00158] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyI benzylcarbamate (78). To a stirred solution of Lit 5-1 (37 mg, 1 eq) and DIPEA (2 eq) in DMF (1 mL) was added benzylamine (26 mg, 3 eq). The solution immediately turned a bright yellow upon addition of amine. The reaction was stirred at room temperature for 2 hours, then diluted with 20 mL H2O and basified with addition of solid NazCOs. The suspension was extracted with EtOAc (3 x 10 mL). The combined organics were washed once with brine, dried over MgSO*, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (S1O2, EtO Ac/hexanes) to afford the title compound as a yellow solid (26.1 mg, 80%). ESI-MS [M+l]: 409.2. ¹ H NMR (400 MHz, Chloroform-d) d 7.41 (d, J= 9.0 Hz, 1H), 7.37 - 7.27 (m, 3H), 6.61 (dd, J= 9.0, 2.6 Hz, 1H), 6.51 (d, J= 2.6 Hz, 1H), 4.97 (s, 1H), 4.39 (d, J= 6.0 Hz, 2H), 4.28 (t, J= 7.0 Hz, 2H), 3.43 (q, J= 7.1 Hz, 5H), 3.01 (t, J= 7.0 Ήz, 2H), 2.39 (s, 3H), 1.23 (t, J= 7.1 Hz, 6H).

[00159] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chrOmen-3-yl)ethyl methyl carbamate (79). The title compound was prepared analogously to 78, using methylamine. ESI-MS [M+l]: 333.1. ^! H NMR (400 MHz, Chloroform-*/) d 7.42 (d, J= 9.0 Hz, 1H), 6.61 (dd, J= 9.0, 2.6 Hz, 1H), 6.50 (d, J= 2.6 Hz, 1H), 4.61 (s, 1H), 4.23 (t, J= 7.1 Hz, 2H), 3.42 (q, J= 7.1 Hz, 4H),

2.98 (t, J= 7.1 Hz, 2H), 2.80 (d, J= 4.8 Hz, 3H), 2.40 (s, 3H), 1.22 (t, J= 7.1 Hz, 6H).

[00160] 2-(7-(diethylamino)-4-inethyl-2-oxo-2H-chromen-3-yl)ethyl

phenethylcarbamate (80). The title compound was prepared analogously to 78, using 2- phenylethan-1 -amine. ESI-MS [M+l]: 423.2. ¹ H NMR (400 MHz, Chloroform-*/) d 7.41 (d, J= 9.0 Hz, 1H), 7.38 - 7.20 (m, 4H), 7.20 (d, J= 7.3 Hz, 2H), 6.61 (dd, J= 9.0, 2.6 Hz, 1H), 6.51 (d, J= 2.5 Hz, 1H), 4.69 (d, J= 6.8 Hz, 1H), 4.23 (t, J= 7.1 Hz, 2H), 3.43 (q, J= 7.1 Hz, 6H), 2.97 (z J= 7.1 Hz, 2H), 2.82 (t, J= 7.1 Hz, 2H), 2.37 (s, 3H), 1.22 (t, J = 7.1 Hz, 6H).

[00161] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyI (3- phenylpropyI)carbamate (81). The title compound was prepared analogously to 78, using 3- phenylpropan-1 -amine. ESI-MS [M+l ]: 437.2. H NMR (400 MHz, Chloroform-*/) d 7.42 (d, J = 9.0 Hz, 1H), 7.36 - 7.26 (m, 3H), 7.26 - 7.15 (m, 3H), 6.60 (dd, J= 9.0, 2.6 Hz, 1H), 6.51 (d, J = 2.6 Hz, 1H), 4.68 (s, 1H), 4.23 (t, J= 7.0 Hz, 2H), 3.43 (q, J= 7.1 Hz, 4H), 3.22 (q, J= 6.7 Hz, 2H), 2.99 (t, J= 7.0 Hz, 2H), 2.67 (q, J= 7.4 Hz, 2H), 2.40 (s, 3H), 1.85 (h, J = 6.8 Hz, 2H),

1.22 (t, J= 7.1 Hz, 6H).

[00162] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyI (2-(pyridin-2- yl)ethyl)carbamate (82). The title compoimd was prepared analogously to 78, using 2-(pyridin- 2-yl)ethan-l -amine. ESI-MS [M+l]: 424.1. ¹ H NMR (500 MHz, Chloroform-*/) d 8.57 - 8.50 (m, 1H), 7.62 (td, J = 7.6, 1.8 Hz, 1H), 7.39 (s, 1H), 7.20 - 7.11 (m, 2H), 6.60 (dd, J = 9.0, 2.6 Hz, 1H), 6.50 (d, J= 2.6 Hz, 1H), 5.37 (d, J= 6.0 Hz, 1H), 4.22 (t, J= 7.0 Hz, 2H), 3.61 (q, J= 6.3 Hz, 2H), 3.42 (q, J= 7.1 Hz, 4H), 2.98 (dt, J= 14.9, 6.7 Hz, 4H), 2.37 (s, 3H), 1.21 (t, J=

7.1 Hz, 6H).

[00163] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (2-(pyridin-3- yl)ethyl)carbamate (83). The title compound was prepared analogously to 78, using 2-(pyridin-

3-yl)ethan-l -amine, to afford 83 as a tan solid (20.7 mg, 78%). ESI-MS [M+l]: 424.1.

[00164] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (2-(pyridin-4- yl)ethyl)carbamate (84). The title compound was prepared analogously to 78, using 2-(pyridin-

4-yl)ethan-l -amine. ESI-MS [M+l]: 424.1. ^l H NMR (400 MHz, Chloroform-*/) d 8.53 - 8.43 (m, 2H), 7.53 (d, J= 7.9 Hz, 1H), 7.24 (dd, J= 7.8, 4.8 Hz, 1H), 6.60 (dd, J= 9.0, 2.6 Hz, 1H), 6.50 (d, J= 2.6 Hz, 1H), 4.84 (s, 1H), 4.23 (t, J= 7.0 Hz, 2H), 3.42 (q, J= 7.1 Hz, 6H), 2.97 (dd, J= 8.3, 5.7 Hz, 2H), 2.83 (t, J= 7.2 Hz, 2H), 2.37 (s, 3H), 1.21 (t, J= 7.1 Hz, 6H).

[00165] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)rthyl (pyridin-2- ylmethyl)carbamate (85). The title compound was prepared analogously to 78, using pyridin-2- ylmethanamine. ESI-MS [M+l]: 410.2. ¹ H NMR (400 MHz, Chloroforms/) d 8.54 (d , J= 4.9 Hz, 1H), 7.65 (t, J= 7.7 Hz, 1H), 7.40 (d, J= 9.0 Hz, 1H), 7.28 (d, J= 5.8 Hz, 1H), 7.19 (dd, J= 7.5, 5.0 Hz, 1H), 6.60 (dd, J= 9.1, 2.6 Hz, 1H), 6.50 (d, J= 2.6 Hz, 1H), 5.76 (t, J= 5.5 Hz, 1H), 4.49 (d, J= 5.5 Hz, 2H), 4.28 (t, J= 7.0 Hz, 2H), 3.42 (q, J= 7.1 Hz, 4H), 3.00 (t, J= 6.9 Hz, 2H), 2.38 (s, 3H), 1.21 (t, J= 7.0 Hz, 6H).

[00166] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyI (pyridin-4- ylmethyl)carbamate (86). The title compound was prepared analogously to 78, using pyridin-4- ylmethanamine. ESI-MS [M+l]: 410.2. ¹ H NMR (400 MHz, Chloroform-*/) 5 8.54 (d, J= 5.1 Hz, 2H), 7.41 (d, .7= 9.0 Hz, 1H), 7.20 (d, J= 5.0 Hz, 2H), 6.61 (dd, J= 9.1, 2.6 Hz, 1H), 6.50 (d, J = 2.5 Hz, 1H), 5.26 (d, J= 6.5 Hz, 1H), 4.39 (d, J= 6.3 Hz, 2H), 4.29 (t, J = 7.0 Hz, 2H), 3.42 (q, J= 7.1 Hz, 4H), 3.00 (t, J= 7.0 Hz, 2H), 2.38 (s, 3H), 1.22 (t, J = 7.0 Hz, 6H).

[00167] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyl (furan-2- ylmethyl)carbamate (87). The title compound was prepared analogously to 78, using furan-2- ylmethanamine. ESI-MS [M+l]: 399.2. ¹ H NMR (400 MHz, Chloroform-*/) d 7.41 (d, J = 9.0 Hz, 1H), 6.61 (dd, J= 9.0, 2.6 Hz, 1H), 6.50 (d, J = 2.6 Hz, 1H), 6.32 (s, 1H), 6.27 - 6.19 (m, 1H), 5.00 (s, 1H), 4.36 (d, J= 5.9 Hz, 2H), 4.26 (t, J= 7.0 Hz, 2H), 3.43 (q, J= 7.1 Hz, 4H), 2.99 (t, J= 7.0 Hz, 2H), 2.37 (s, 3H), 1.22 (t , J= 7.1 Hz, 6H).

[00168] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chrOmen-3-yl)ethyl (2-(lH-imidazol- 4-yI)ethyl)carbamate (88). The title compound was prepared analogously to 78, using 2-(lH- imidazol-4-yl)ethan-l -amine. ESI-MS [M+l]: 413.2. ¹ H NMR (400 MHz, Chloroform-*/) d 7.67 (s, 1H), 7.43 (s, 1H), 6.85 (s, 1H), 6.61 (dd, J= 9.0, 2.6 Hz, 1H), 6.48 (d, J= 2.5 Hz, 1H), 5.30 (t, J= 6.2 Hz, 1H), 4.22 (t, J= 6.8 Hz, 2H), 3.42 (q, J= 7.1 Hz, 6H), 2.96 (t, J= 6.8 Hz, 2H), 2.82 (d , J= 6.6 Hz, 2H), 2.38 (s, 3H), 1.21 (t, J= 7.1 Hz, 6H).

[00169] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (pyridin-3- ylmethyl)carbamate (89). The title compound was prepared analogously to 78, using pyridin-3- ylmethanamine, to afford 89 as a tan solid (18.7 mg, 71%). ESI-MS [M+l]: 410.2. ¹ H NMR (400 MHz, Chloroform-*/) d 8.53 (dd, J= 9.1, 3.6 Hz, 2H), 7.65 (d, J= 7.9 Hz, 1H), 7.40 (d, J= 9.0 Hz, 1H), 7.25 (dd, J= 7.8, 5.0 Hz, 1H), 6.60 (dd, J= 9.0, 2.6 Hz, 1H), 6.49 (d, J= 2.6 Hz, 1H), 5.25 (s, 1H), 4.38 (d, J= 6.2 Ήz, 2H), 3.42 (q, J= 7.1 Hz, 4H), 2.98 (t, J= 7.0 Hz, 2H),

2.36 (s, 3H), 1.21 (t, J= 7.1 Hz, 6H).

[00170] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (pyrimidin-4- ylmethyl)carbamate (90). The title compound was prepared analogously to 78, using pyrimidin-4-ylmethanamine. ESI-MS [M+l]: 411.2. ^! H NMR (400 MHz, Chloroform-*/) d 8.72 (d, J= 4.9 Hz, 2H), 7.42 (d, J= 9.0 Hz, 1H), 7.21 (d, J= 9.9 Hz, 1H), 6.60 (dd, J= 9.0, 2.6 Hz, 1H), 6.50 (d, J = 2.6 Hz, 1H), 5.85 (s, 1H), 4.66 (d, J= 5.1 Hz, 2H), 4.29 (t, J = 7.1 Hz, 2H), 3.42 (q, y= 7.1 Hz, 4H), 3.07 - 2.94 (m, 2H), 2.41 (s, 3H), 1.22 (t, J = 7.1 Hz, 6H).

[00171] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (pyrazin-2- ylmetiiyl)carbamate (91). The title compound was prepared analogously to 78, using pyrazin- 2-ylmethanamine. ESI-MS [M+l]: 411.2. ^! H NMR (400 MHz, Chloroform-i/) d 8.63 (s, 1H), 8.59 - 8.47 (m, 2H), 7.40 (d , J= 9.0 Hz, 1H), 6.60 (dd, J= 9.0, 2.6 Hz, 1H), 6.50 (d, J = 2.6 Hz, 1H), 5.63 (s, 1H), 4.55 (d , J= 5.7 Hz, 2H), 4.28 (t, J= 7.0 Hz, 2H), 3.42 (q, 7= 7.1 Hz, 4H), 3.00 (t, J= 7.0 Hz, 2H), 2.38 (s, 3H), 1.22 (t, J= 7.1 Hz, 6H).

[00172] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyI ((lH-pyrazol-5- yl)methyl)carbamate (92). The title compound was prepared analogously to 78, using (1H- pyrazol-5-yl)methanamine. ESI-MS [M+l]: 399.2. ¹ H NMR (400 MHz, Chloroform-t/) d 7.51 (d, J = 2.1 Hz, 1H), 7.40 (d, J= 9.0 Hz, 1H), 6.60 (dd, J= 9.1, 2.6 Hz, 1H), 6.49 (d, J = 2.6 Hz, 1H), 6.26 - 6.18 (m, 1H), 5.55 (s, 1H), 4.41 (d, J= 5.8 Hz, 2H), 4.26 (t, J= 6.9 Hz, 2H), 3.42 (q, J= 7.1 Hz, 4H), 2.98 (q, J= 5.5, 4.4 Hz, 3H), 2.36 (s, 3H), 1.21 (t, J= 7.1 Hz, 6H).

[00173] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (pyrimidin-2- ylmethyl)carbamate (93). The title compound was prepared analogously to 78, using pyrimidin-2-ylmethanamine. ESI-MS [M+l]: 411.2. ’H NMR (500 MHz, Chloroform-i/) d 8.71 (d, J= 4.9 Hz, 2H), 7.41 (s, 1H), 7.21 (t, J= 5.0 Hz, 1H), 6.60 (dd, J= 9.0, 2.6 Hz, 1H), 6.50 (d, J= 2.6 Hz, 1H), 5.85 (t, J= 5.3 Hz, 1H), 4.66 (d, J= 5.1 Hz, 2H), 4.29 (t, J= 7.0 Hz, 2H), 3.42 (q, J= 7.1 Hz, 4H), 3.02 (t, J= 7.0 Hz, 2H), 2.41 (s, 3H), 1.21 (t, J= 7.1 Hz, 6H).

[00174] N-(2-(7-(diethylammo)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl)a cetamide (94). The title compound was prepared analogously to 55, using acetic acid and Lit 4-1. ESI- MS [M+l]: 317.1. ¹ H NMR (500 MHz, Chloroform-i/) d 7.42 (d, J= 9.0 Hz, 1H), 6.61 (dd, J= 9.1, 2.6 Hz, 1H), 6.49 (d, J= 2.6 Hz, 1H), 6.26 (d, ^= 5.1 Hz, 1H), 3.48 - 3.37 (m, 6H), 2.87 (t, J= 6.8 Hz, 2H), 2.40 (s, 3H), 1.95 (s, 3H), 1.21 (t, J= 7.1 Hz, 6H).

[00175] N-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl) -3-(pyridin-3- yl)propenamide (95). The title compound was prepared analogously to 55, using 3-(pyridin-3- yl)propanoic acid and Int 4-1. ESI-MS [M+l]: 408.1. ¹ H NMR (500 MHz, Chloroform-i/) d 8.46 (d, J= 2.2 Hz, 1H), 8.38 (dd, J= 4.8, 1.6 Hz, 1H), 7.52 (dt, J= 7.9, 2.0 Hz, 1H), 7.41 (d, J = 9.0 Hz, 1H), 7.16 (ddd, 7= 7.8, 4.8, 0.8 Hz, 1H), 6.61 (dd, 7= 9.1, 2.6 Hz, 1H), 6.46 (dd, 7 = 6.1, 3.5 Hz, 2H), 3.42 (q, 7= 7.0 Hz, 6H), 2.95 (dd, 7= 8.5, 6.9 Hz, 2H), 2.82 (t, 7= 6.8 Hz, 2H), 2.47 (dd, 7= 8.5, 7.0 Hz, 2H), 2.37 (s, 3H), 1.21 (t, 7= 7.1 Hz, 6H).

[00176] N-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyI) -3-(pyridin-4- yl)propanamide (96). The title compound was prepared analogously to 55, using 3-(pyridin-4- yl)propanoic acid and Int 4-1. ESI-MS [M+l]: 408.1. ¹ H NMR (500 MHz, Chloroform-7) d 8.49 - 8.42 (m, 2H), 7.42 (d , 7 = 9.0 Hz, 1H), 7.16 - 7.09 (m, 2H), 6.62 (dd, 7= 9.1, 2.6 Hz, 1H), 6.48 (d, 7= 2.6 Hz, 1H), 6.43 (t, 7= 5.1 Hz, 1H), 3.42 (qd, 7= 6.8, 2.9 Hz, 6H), 2.95 (dd, 7=

8.6, 6.9 Hz, 2H), 2.84 (t, 7 = 6.7 Hz, 2H), 2.48 (dd, 7= 8.6, 7.0 Hz, 2H), 2.37 (s, 3H), 1.21 (t, 7= 7.1 Hz, 6H).

[00177] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyl ((6- chloropyridin-3-yl)methyl)carbamate (97). The title compound was prepared analogously to 78, using (6-chloropyridin-3-yl)methanamine. ESI-MS [M+l]: 444.6. ¹ H NMR (500 MHz, Chloroform-7) d 8.31 (d, 7= 2.4 Hz, 1H), 7.66 - 7.58 (m, 1H), 7.39 (s, 1H), 7.26 (d, 7= 8.3 Hz, 1H), 6.60 (dd, 7= 9.0, 2.6 Hz, 1H), 6.49 (d, 7= 2.6 Hz, 1H), 4.35 (d, 7= 6.2 Hz, 2H), 4.32 (s, 1H), 4.27 (t, 7= 6.9 Hz, 2H), 3.42 (q, 7= 7.1 Hz, 4H), 2.98 (t, 7= 6.9 Hz, 2H), 2.36 (s, 3H), 1.22 (t, 7= 7.1 Hz, 6H).

[00178] l-(2-(7-(diethylamino)-4-mefliyI-2-oxo-2H-chromai-3-yl)efliy l)-3-(pyridin-3- ylmethyl)urea (98).

[00179] To a stirred solution of carbonyldiimidazole (16.5 mg, 1 eq) in 0.5 mL DMF on an ice-water bath was added a solution of Int 4-1 (28 mg, 1 eq) in 0.5 mL DMF dropwise. The yellow solution was stirred for 30 min at 0 °C and an additional 30 ^e min at room temperature. Thereto was added pyridin-3-ylmethanamine (16.2 mg, 1.5 eq) and stirred for 16 hrs at room temperature. The reaction was diluted with 20 mL H2O and extracted with EtOAc (3 x 10 mL). The combined organics were washed once with brine, dried over MgSO*, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (S1O2, EtOAc/hexanes) to afford the title compound as a yellow solid. ESI-MS [M+l]: 409.1. ¹ H NMR (500 MHz, Chloroform-i/) d 8.54 (d, J= 2.0 Hz, 1H), 8.45 (dd, J= 4.8, 1.7 Hz, 1H), 7.65 (dt, J= 7.9, 2.0 Hz, 1H), 7.40 (d, J= 9.1 Hz, 1H), 7.19 (dd, J= 7.8, 4.8 Hz, 1H), 6.60 (dd, J = 9.1, 2.6 Hz, 1H), 6.43 (d, J= 2.6 Hz, 1H), 5.77 (s, 1H), 5.51 (s, 1H), 4.39 (d, J= 5.9 Hz, 2H), 3.41 (q, J= 7.1 Hz, 4H), 3.32 (q, J= 6.7 Hz, 2H), 2.81 (t, J= 7.2 Hz, 2H), 2.37 (s, 3H), 1.21 (t, J = 7.1 Hz, 6H).

[00180] l-(2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)ethyl)-3-(2-(pyridin- 3-yl)ethyl)urea (99). The title compound was prepared analogously to 98, using 2-(pyridin-3- yl)ethan-l -amine. ESI-MS [M+l]: 423.2. ^X H NMR (400 MHz, Chloroform-d) d 8.43 (dd, J = 14.1, 3.4 Hz, 2H), 7.55 (d, J= 7.6 Hz, 1H), 7.41 (d, J= 9.0 Hz, 1H), 7.20 (dd, J= 7.8, 4.9 Hz, 1H), 6.61 (dd, J = 9.1, 2.6 Hz, lH), 6.46 (d, J = 2.5 Hz, 1H), 5.27 (d, J = 9.1 Hz, 2H), 3.44 (dq, J = 21.1, 6.9 Hz, 6H), 3.30 (q, J= 6.7 Hz, 2H), 2.82 (q, J= 7.4 Hz, 4H), 2.39 (s, 3H), 1.21 (t, J=

7.1 Hz, 6H).

[00181] Pyridin-3-ylmethyl (2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3- yl)ethyl)carbamate (100). The title compound was prepared analogously to 78, using Int 4-1 and 4-nitrophenyl (pyridin-3 -y lmethy 1) carbonate. ESI-MS [M+l]: 410.2. ^X H NMR (500 MHz, Chloroform-i/) d 8.62 (d, J= 2.1 Hz, 1H), 8.60 - 8.54 (m, 1H), 7.70 (dt, J= 7.9, 2.0 Hz, 1H), 7.41 (d, J= 9.0 Hz, 1H), 7.30 (d, J= 4.9 Hz, 1H), 7.28 (s, 1H), 6.61 (dd, J= 9.0, 2.6 Hz, 1H), 6.49 (d, J= 2.6 Hz, 1H), 5.12 (s, 2H), 3.41 (dq, J= 16.2, 6.8 Hz, 6H), 2.88 (t, J= 6.9 Hz, 2H), 2.36 (s, 3H), 1.22 (t, J= 7.1 Hz, 6H).

[00182] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl ((6- (trifluoromethyl)pyridin-3-yl)methyl)carbamate (101). The title compound was prepared analogously to 78, using (6-(trifluoromethyl)pyridin-3-yl)methanamine. ESI-MS [M+l]: 478.2. ¹ H NMR (400 MHz, Chloroform-i/) d 8.66 (s, 1H), 7.83 (d, J= 8.1 Hz, 1H), 7.62 (d, J= 8.1 Hz, 1H), 7.40 (d, J= 9.0 Hz, 1H), 6.61 (dd, J= 9.0, 2.5 Hz, 1H), 6.49 (d, J= 2.6 Hz, 1H), 5.38 (s, 1H), 4.46 (d, J= 6.3 Hz, 2H), 4.29 (t, J= 7.0 Hz, 2H), 3.43 (q, J= 7.1 Hz, 5H), 2.99 (t, J= 6.9 Hz, 2H), 1.22 (t, J= 7.1 Hz, 6H).

[00183] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl methyl(2- (pyridin-3-yl)ethyl)carbamate (102). The title compound was prepared analogously to 78, using N-methyl-2-(pyridin-3-yl)ethan-l-amine, to afford 102 as a viscous yellow oil (15.1 mg, 84%). ESI-MS [M+l]: 438.2. ^l U NMR (400 MHz, Chloroform-7) d 8.57 - 8.48 (m, 1H), 7.65 - 7.54 (m, 1H), 7.46 - 7.35 (m, 1H), 7.18 - 7.05 (m, 2H), 6.60 (dd, 7= 9.0, 2.6 Hz, 1H), 6.50 (d, 7 = 2.6 Hz, 1H), 4.19 (ddt, 7 = 20.8, 13.8, 7.0 Hz, 2H), 3.63 (dt, 7= 14.5, 7.4 Hz, 2H), 3.42 (q, 7= 7.1 Hz, 4H), 2.96 (q, 7= 7.3, 5.9 Hz, 5H), 2.87 (s, 2H), 2.80 (s, 2H), 1.21 (t, 7= 7.1 Hz, 6H).

[00184] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (5- acetamidopyridin-2-yl)carbamate (103). The title compound was prepared analogously to 78, using N-(6-aminopyridin-3-yl)acetamide. ESI-MS [M+l]: 453.2.

[00185] l-(2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)ethyl) 3-methyl azetidine- 1 ,3-dicar boxylate (104). The title compound was prepared analogously to 78, using methyl azetidine-3-carboxylate. ESI-MS [M+l]: 417.2. ¹ H NMR (400 MHz, Chloroform-7) d 7.43 (d, 7= 9.0 Hz, 1H), 6.61 (dd, 7= 9.0, 2.6 Hz, 1H), 6.51 (d, 7= 2.6 Hz, 1H), 4.23 (t, 7= 6.9 Hz, 2H), 4.15 (d, 7= 7.5 Hz, 4H), 3.81 - 3.68 (m, 5H), 3.49 - 3.35 (m, 5H), 2.98 (t, 7= 6.9 Hz, 2H), 2.40 (s, 3H), 1.22 (t, 7= 7.1 Hz, 6H).

[00186] Ethyl 4-(((2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3- yl)ethoxy)carbonyl)amino)piperidine-l-carboxylate (105). The title compound was prepared analogously to 78, using ethyl 4-aminopiperidine-l-carboxylate. ESI-MS [M+l]: 474.2. 'H NMR (400 MHz, Chloroform-7) d 7.42 (d, 7= 9.0 Hz, 1H), 6.61 (dd, 7= 9.1, 2.6 Hz, 1H), 6.50 (d, 7= 2.5 Hz, 1H), 4.65 (d, 7= 8.0 Hz, 1H), 4.14 (q, 7= 7.1 Hz, 3H), 3.42 (q, 7= 7.1 Hz, 6H), 2.39 (s, 4H), 1.24 (dt, 7= 20.8, 6.9 Hz, 17H).

[00187] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl 3- (hydroxymethyl)pyrrolidine-l-carboxylate (106). The title compound was prepared analogously to 78, using pyrrolidin-3-ylmethanol. ESI-MS [M+l]: 403.2. ¾ NMR (400 MHz, Chloroform-7) d 7.42 (d, 7= 9.0 Hz, 2H), 6.61 (dd, 7= 9.0, 2.5 Hz, 2H), 6.50 (d, 7= 2.6 Hz, 2H), 3.42 (q, 7= 7.1 Hz, 10H), 1.22 (t, 7= 7.1 Hz, 13H).

[00188] 2-(7-(diethylamino)-4-mefliyl-2-oxo-2H-chromen-3-yl)ethyl 4- (cyclopropylsulfonyl)piperazine-l-carboxylate (107). The title compound was prepared analogously to 78, using l-(cyclopropylsulfonyl)piperazine. ESI-MS [M+l]: 492.3. ^X H NMR (500 MHz, Chloroform-7) d 7.42 (d, 7= 9.0 Hz, 1H), 6.61 (dd, 7= 9.0, 2.6 Hz, 1H), 6.50 (d, 7= 2.6 Hz, 1H), 4.28 (t, 7= 7.0 Hz, 2H), 3.57 (s, 4H), 3.43 (q, 7= 7.1 Hz, 4H), 3.27 (d, 7= 6.5 Hz, 4H), 3.01 (t, J= 7.0 Hz, 2H), 2.39 (s, 3H), 2.25 (tt, J= 7.9, 4.9 Hz, 1H), 1.26 - 1.12 (m, 7H), 1.16 (s, 1H), 1.07 - 0.96 (m, 2H).

[00189] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyI 3,3- difluoroazetidine-l-carboxylate (108). The title compound was prepared analogously to 78, using 3,3-difluoroazetidine. ESI-MS [M+l]: 395.1. ¹ H NMR (500 MHz, Chloroform-*/) d 7.43 (d, J= 9.0 Hz, 1H), 6.62 (dd, J= 9.0, 2.6 Hz, 1H), 6.51 (d, J= 2.6 Hz, 1H), 4.36 - 4.23 (m, 6H), 3.43 (q, J = 7.1 Hz, 4H), 3.00 (t, J= 6.9 Hz, 2H), 2.39 (s, 3H), 1.22 (t, J= 7.1 Hz, 6H).

[00190] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl 4-(pyrazin-2- yl)piperazine-l-carboxylate (109). The title compound was prepared analogously to 78, using 2-(piperazin-l-yl)pyrazine. ESI-MS [M+l]: 466.3. ¹ H NMR (500 MHz, Chloroform-*/) d 8.15 (d, 7= 1.6 Hz, 1H), 8.08 (dd, 7= 2.6, 1.5 Hz, 1H), 7.90 (d, J = 2.6 Hz, 1H), 7.41 (d, 7= 9.0 Hz, 1H), 6.60 (dd, J = 9.0, 2.6 Hz, lH), 6.51 (d, J = 2.6 Hz, 1H), 4.30 (t, J= 7.0 Hz, 2H), 3.60 (s, 9H), 3.42 (q, J= 7.1 Hz, 4H), 3.03 (t, J= 7.0 Hz, 2H), 2.41 (s, 3H), 1.22 (t, J = 7.1 Hz, 6H).

[00191] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl 2-thia-6- azaspiro[3.3]heptane-6-carboxylate 23-dioxide (110). The title compound was prepared analogously to 78, using 2-thia-6-azaspiro[3.3]heptane 2,2-dioxide. ESI-MS [M+l]: 449.2. 'H NMR (500 MHz, Chloroform-*/) d 7.42 (d, J= 9.0 Hz, 1H), 6.62 (dd, J= 9.0, 2.6 Hz, 1H), 6.50 (d, J= 2.6 Hz, 1H), 4.33 (s, 4H), 4.24 (t, J= 6.7 Hz, 2H), 4.20 (s, 4H), 3.43 (q, J= 7.1 Hz, 4H), 3.02 - 2.93 (m, 2H), 2.38 (s, 3H), 1.22 (t, J= 7.1 Hz, 6H).

[00192] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl 3-hydroxy-3- methylpyrrolidine-l-carboxylate (111). The title compound was prepared analogously to 78, using 3 -methylpyrrolidin-3 -ol. ESI-MS [M+l]: 403.2. ¹ H NMR (500 MHz, Chloroform-*/) d 7.42 (d, J= 9.0 Hz, 1H), 7.28 (s, 1H), 6.60 (dd, J= 9.0, 2.6 Hz, 1H), 6.51 (d, J= 2.6 Hz, 1H), 4.35 - 4.14 (m, 2H), 3.42 (q, J= 7.1 Hz, 5H), 3.03 (td, J= 13.8, 13.3, 6.7 Hz, 1H), 3.01 - 2.91 (m, 1H), 2.40 (s, 3H), 1.99 - 1.89 (m, 1H), 1.94 - 1.79 (m, 1H), 1.43 (d, J= 9.4 Hz, 3H), 1.22 (t, J= 7.1 Hz, 6H).

[00193] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl thiomorpholine- 4-carboxylate 1,1-dioxide (112). The title compound was prepared analogously to 78, using thiomorpholine 1,1 -dioxide. ESI-MS [M+l]: 437.2. ¹ H NMR (500 MHz, Chloroform-*/) d 7.42 (d, J= 9.0 Hz, 1H), 6.62 (dd, J= 9.0, 2.6 Hz, 1H), 6.51 (d, J= 2.6 Hz, 1H), 4.31 (t, J= 6.7 Hz, 2H), 3.97 (s, 4H), 3.43 (q, .7= 7.1 Hz, 4H), 3.03 (q, J= 6.8, 5.3 Hz, 6H), 2.39 (s, 3H), 1.22 (t, J = 7.1 Hz, 6H).

[00194] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyI 4-(3- methylisoxazol-5-yl)piperazine-l-carboxylate (113). The title compound was prepared analogously to 78, using 3-methyl-5-(piperazin-l-yl)isoxazole. ESI-MS [M+l]: 469.3. NMR (500 MHz, Chloroform-*/) d 7.41 (d, J= 9.0 Hz, 1H), 6.61 (dd, J= 9.0, 2.6 Hz, 1H), 6.50 (d, J= 2.6 Hz, 1H), 4.29 (t, J= 6.9 Hz, 2H), 3.58 (s, 9H), 3.43 (q, J= 7.1 Hz, 4H), 3.02 (t, J= 7.0 Hz, 2H), 2.40 (s, 3H), 2.24 (s, 3H), 1.22 (t, J = 7.1 Hz, 6H).

[00195] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyl 4-(pyridin-2- yl)piperazine-l-carboxylate (114). The title compound was prepared analogously to 78, using 1 -(pyridin-2-y l)piperazine. ESI-MS [M+l]: 465.3. ¹ H NMR (500 MHz, Chloroform-*/) d 8.55 (dt, J = 4.5, 1.5 Hz, lH), 7.64 (td, J= 7.7, 1.9 Hz, 1H), 7.42 (d, J= 9.0 Hz, 1H), 7.15 (dd, J=

7.5, 5.0 Hz, 2H), 6.60 (dd, J= 9.1, 2.6 Hz, 1H), 6.51 (d, J= 2.6 Hz, 1H), 4.35 (s, 1H), 3.42 (q, J = 7.1 Hz, 4H), 3.02 (t, J= 7.1 Hz, 2H), 2.88 (ddt, J= 15.7, 8.4, 3.6 Hz, 4H), 2.42 (s, 3H), 1.22 (t, J= 7.1 Hz, 6H).

[00196] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl 4-(2H-tetrazol-5- yl)piperazine-l-carboxylate (115). The title compound was prepared analogously to 78, using l-(2H-tetrazol-5-yl)piperazine. ESI-MS [M+l]: 456.2.

[00197] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl 4-(pyrimidin-2- yl)piperazine-l-carboxyIate (116). The title compound was prepared analogously to 78, using 2-(piperazin-l-yl)pyrimidine. ESI-MS [M+l]: 466.2. ¹ H NMR (500 MHz, Chloroform-*/) d 8.33 (d, J= 4.7 Hz, 2H), 7.41 (d, J= 9.0 Hz, 1H), 6.60 (dd, J= 9.0, 2.6 Hz, 1H), 6.57 - 6.48 (m, 2H), 4.30 (t, J= 7.0 Hz, 2H), 3.82 (s, 4H), 3.54 (s, 4H), 3.42 (q, J= 7.1 Hz, 4H), 3.03 (t, J= 7.0 Hz, 2H), 2.41 (s, 3H), 1.22 (t, J= 7.1 Hz, 6H).

[00198] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (3- acetamidob«izyl)carbamate (117). The title compound was prepared analogously to 78, using N-(3-(aminomethyl)phenyl)acetamide, to afford 117 as a white solid (11.0 mg, 59%). ESI-MS [M+l]: 466.2. ^X H NMR (500 MHz, Chloroform-*/) d 7.91 (s, 1H), 7.66 (d, J= 7.6 Hz, 1H), 7.41 (d, J= 9.0 Hz, 1H), 7.31 (s, 1H), 7.28 (s, 1H), 7.25 (d, J= 7.9 Ήz, 1H), 7.04 (s, 1H), 6.97 (d, J= 7.6 Hz, 1H), 6.47 (d, J= 2.6 Hz, 1H), 4.35 (d, J= 6.2 Hz, 2H), 4.31 (s, 2H), 3.42 (q, J= 7.1 Hz, 4H), 3.01 (t, J= 6.9 Hz, 2H), 2.38 (s, 3H), 2.18 (s, 3H), 1.22 (t, .7= 7.1 Hz, 6H).

[00199] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyI 4-(pyridin-3- ylmethyl)piperidine-l-carboxylate (118). The tide compound was prepared analogously to 78, using 3-(piperidin-4-ylmethyl)pyridine. ESI-MS [M+l]: 478.3. ^X H NMR (500 MHz,

Chloroform-*/) d 8.47 (dd, J= 4.8, 1.6 Hz, 1H), 8.41 (d, 7 = 2.2 Hz, 1H), 7.49 - 7.37 (m, 2H), 7.23 (dd, J = 7.8, 4.8 Hz, 1H), 6.60 (dd, J = 9.1, 2.6 Hz, 1H), 6.50 (d, J= 2.6 Hz, 1H), 4.23 (t, J = 7.0 Hz, 2H), 3.42 (q, J= 7.1 Hz, 5H), 2.99 (t, J = 7.1 Hz, 3H), 2.68 (t, J= 12.9 Hz, 3H), 2.54 (d, J= 7.1 Hz, 2H), 2.38 (s, 3H), 1.68 (ddp, 7= 11.0, 7.3, 3.6 Hz, 1H), 1.64 (s, 1H), 1.21 (t , J= 7.1 Hz, 6H).

[00200] 4-((((2-(7-(diethylamino)-4-methyi-2-oxo-2H-chromen-3- yl)ethoxy)carbonyl)amino)methyl)benzoic acid (119). The title compound was prepared analogously to 78, using 4-(aminomethyl)benzoic acid. ESI-MS [M+l]: 453.2.

[00201] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (4- ((aminooxy)carbonyl)benzyl)carbamate (120). The title compound was prepared analogously to 55, using 119 and ammonium chloride. ESI-MS [M+l]: 468.2.

[00202] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (4- (((methylamino)oxy)carbonyl)benzyl)carbamate (121). The title compound was prepared analogously to 55, using 119 and methylamine. ESI-MS [M+l]: 482.2.

[00203] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (4- (((dimethylamino)oxy)carbonyl)benzyl)carbamate (122). The title compound was prepared analogously to 55, using 119 and dimethylamine. ESI-MS [M+l]: 496.3.

[00204] 3-((((2-(7-(diethylamino)-4-methyi-2-oxo-2H-chromen-3- yl)ethoxy)carbonyl)amino)methyl)benzoic acid (123). The title compound was prepared analogously to 78, using 3-(aminomethyl)benzoic acid. ESI-MS [M+l]: 453.2.

[00205] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromcn-3-yl)ethyl (3- ((aminooxy)carbonyl)benzyl)carbamate (124). The title compound was prepared analogously to 55, using 123 and ammonium chloride. ESI-MS [M+l]: 468.2. [00206] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (3- (((methylamino)oxy)carbonyl)benzyl)carbamate (125). The title compound was prepared analogously to 55, using 123 and methylamine. ESI-MS [M+l]: 482.2.

[00207] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (3- (((dimethylamino)oxy)carbonyI)benzyI)carbamate (126). The title compound was prepared analogously to 55, using 123 and dimethylamine. ESI-MS [M+l]: 496.3.

[00208] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl ((5- acetamidopyridin-2-yI)methyl)carbamate (127). The title compound was prepared analogously to 78, using N-(6-(aminomethyl)pyridin-3-yl)acetamide. ESI-MS [M+l]: 467.2.

[00209] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyI ((5- acetamidopyridin-3-yl)methyl)carbamate (128). The title compound was prepared analogously to 78, using N-(5-(aminomethyl)pyridin-3-yl)acetamide, to afford 24 as a light yellow solid (10.3 mg, 65%). ESI-MS [M+l]: 467.2.

[00210] Scheme 6.

BocNH BF.K

X»/ ³

129

[00211] 3-(aminomethyl)-7-(diethylamino)-4-inethyl-2H-chromen-2-one (Int 6-1). The title intermediate was prepared analogously to Int 4-1, using N-(5-(aminomethyl)pyridin-3- yl)acetamide, to afford Int 6-1 as a yellow solid (683 mg, 15% over 2 steps). ESI-MS [M+l]: 261. 1H NMR (400MHz, DMSO-d6) 5 = 8.29 (br s, 3H), 7.66 (d, J=9.2 Hz, 1H), 6.88 - 6.72 (m, 1H), 6.68 - 6.53 (m, 1H), 3.91 (q, J=5.6 Hz, 2H), 3.44 (q, J=7.2 Hz, 4H), 2.47 (s, 3H), 1.11 (t, J=7.2 Hz, 6H).

[00212] 3-((7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)methyl)imidazolidine- 2,4-dione (129). The title compound was prepared in 2 steps from Int 6-1. First, urea formation with glycine methyl ester hydrochloride was performed analogously to preparation of compound 98, and the urea intermediate was purified by flash column chromatography (SiCh,

EtO Ac/hexanes). Second, the urea intermediate (25 mg) was refluxed in 5M aqueous HC1 for 1 hr. The solution was neutralized with solid Na2COs and extracted with EtO Ac (3 x 20 mL). The combined organics were washed once with brine, dried over MgS0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (S1O2, 100% EtO Ac) to afford the title compound as a yellow solid. ESI-MS [M+l]: 344.1.

[00213] 3-(2-(7-(diethylamino)-4-mefliyl-2-oxo-2H-chromai-3-yl)efliy l)imidazolidine- 2,4-dione (130). The title compound was prepared analogously to 129, in two steps starting from Int 4-1. ESI-MS [M+l]: 358.1.

[00214] l-(2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)ethyl)-3-methylurea (131). The title compound was prepared analogously to 98, using methylamine. ESI-MS [M+l]: 332.1.

[00215] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (2-(pyridin-2- ylamino)ethyl)carbamate (132). The title compound was prepared analogously to 78, using N ¹ - (pyridin-2-yl)ethane- 1,2-diamine. ESI-MS [M+l]: 439.2.

[00216] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chrOmen-3-yl)ethyl (3- (dimethylamino)propyl)carbamate (133). The title compound was prepared analogously to 78, using N ¹ ,N ¹ -dimethy lpropane- 1 , 3 -diamine. ESI-MS [M+l]: 404.2.

[00217] N,N-diethyl-4-methylquinolin-7-amine (134). A stirred solution of compound 25 (30 mg) in POCI3 (2 mL) was heated at 100 °C for 1 hr. Afterwards, the solution was poured on ice and basified with solid Na ₂ CC> ₃ . The suspension was extracted with EtO Ac (3 x 20 mL). The combined organics were washed once with brine, dried over MgSCh, filtered, and concentrated under reduced pressure. The residue was dissolved in acetic acid (5 mL) and thereto was added 10% palladium on carbon (10 mg). The suspension was hydrogenated using a hydrogen balloon (1 atm) at room temperature for 1 hour, then filtered through C ₆ lite and concentrated under reduced pressure. The residue was purified by flash column chromatography (S1O2, EtO Ac/hexanes) to afford the title compound as a viscous, bright yellow oil. ESI-MS [M+l]: 215.0.

[00218] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyI (3- ((dimethylamino)methyl)benzyl)carbamate (135). The title compound was prepared analogously to 78, using l-(3-(aminomethyl)phenyl)-N,N-dimethylmethanamine. ESI-MS

[M+l]: 466.2.

[00219] 7-(diethylamino)-4-ethyl-2H-chromen-2-one (136). The title compound was prepared analogously to Iht 3-1-1, using methyl 3-oxopentanoate. ESI-MS [M+l]: 246.0.

[00220] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyl ((lH-indol-5- yl)methyl)carbamate (137). The title compound was prepared analogously to 78, using (1H- indol-5-yl)methanamine. ESI-MS [M+l]: 448.2.

[00221] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl ((6-oxo-l,6- dihydropyridin-3-yl)methyl)carbamate (138). The title compound was prepared analogously to 78, using 5-(aminomethyl)pyridin-2(lH)-one. ESI-MS [M+l]: 426.2.

[00222] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chrOmen-3-yl)ethyl (isoquinolin-1- ylmethyl)carbamate (139). The title compound was prepared analogously to 78, using isoquinolin-l-ylmethanamine. ESI-MS [M+l]: 460.3.

[00223] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (isoquinolin-4- ylmethyl)carbamate (140). The title compound was prepared analogously to 78, using isoquinolin-4-ylmethanamine. ESI-MS [M+l]: 460.3.

[00224] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chrOmen-3-yl)ethyl (3-(lH-imidazol- 4-yl)benzyl)carbamate (141). The title compound was prepared analogously to 78, using (3- (lH-imidazol-4-yl)phenyl)methanamine, to afford 141 as a yellow solid (13.1 mg, 61%). ESI- MS [M+l]: 475.3. [00225] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl ((1H- benzo[d]imidazol-6-yl)methyl)carbamate (142). The title compound was prepared analogously to 78, using (lH-benzo[d]imidazol-6-yl)methanamine. ESI-MS [M+l]: 449.2.

[00226] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl ((l-oxo-1,2- dihydroisoquinoIin-7-yl)methyI)carbamate (143). The title compound was prepared analogously to 78, using 7-(aminomethyl)isoquinolin-l(2H)-one. ESI-MS [M+l]: 476.3.

[00227] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyl (3-carbamoyl-4- fluorobenzyl)carbamate (144). The title compound was prepared analogously to 78, using 5- (aminomethyl)-2-fluorobenzamide. ESI-MS [M+l]: 470.2.

[00228] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (3-carbamoyl-5- fluorobenzyl)carbamate (145). The title compound was prepared analogously to 78, using 5- (aminomethyl)-3-fluorobenzamide. ESI-MS [M+l]: 470.2.

[00229] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyl (piperidin-3- ylmethyl)carbamate (146). The title compound was prepared in two steps. First, the Boc- protected amine was prepared analogously to 78, using tert-butyl 3-(aminomethyl)piperidine-l- carboxylate, and purified by flash column chromatography (S1O2, EtO Ac/hexanes). Second, to a stirred solution of the Boc-protected amine in DCM (2 mL) was added neat TFA (1 mL) and the mixture stirred at room temperature for 16 hrs. The mixture was concentrated under reduced pressure and purified by reverse-phase preparative HPLC to afford 146 as a yellow gum (10.2 mg, 71% over 2 steps). ESI-MS [M+l]: 416.2.

[00230] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chrOmen-3-yl)ethyl (piperidin-4- ylmethyl)carbamate (147). The title compound was prepared analogously to 146, using tert- butyl 4-(aminomethyl)piperidine- 1 -carboxylate. ESI-MS [M+l]: 416.2.

[00231] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl ((1- methylpiperidin-4-yl)methyl)carbamate (148). The title compound was prepared analogously to 78, using (l-methylpiperidin-4-yl)methanamine. ESI-MS [M+l]: 430.3.

[00232] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chrOmen-3-yl)ethyl (3- sulfamoylbenzyl)carbamate (149). The title compound was prepared analogously to 78, using 3-(aminomethyl)benzenesulfonamide. ESI-MS [M+l]: 488.3. [00233] N-(4-(diethylamino)phenyl)-2-(2,5-dioxoimidazolidm-l-yl)acet amide (150).

To a stirred solution of 2-(2,5-dioxoimidazolidin-l-yl)acetic acid (20 mg, 1 eq) and DIPEA (50 pL) in DMF (1 mL) was added HATU (53 mg, 1.1 eq) and stirred for 5 min at room temperature. Thereto was added N^N ¹ -diethylbenzene-1, 4-diamine (32 pL, 1.5 eq) and stirred for 16 hrs at room temperature. The mixture was concentrated under reduced pressure and purified by reverse-phase preparative HPLC. The desired fractions were lyophilized, dissolved in 20 mM aqueous HC1, and lyophilized to afford the title compound as a hygroscopic white solid ESI-MS [M+l]: 305.2.

[00234] 2-(2,5-dioxoimidazolidin-l-yl)-N-(4-(tiifluoromethyl)phenyl) acetamide (151). The title compound was prepared analogously to 150, using 4-(trifluoromethyl)aniline hydrochloride. ESI-MS [M-l]: 300.2.

[00235] Scheme 7.

NB3 BF,K

Pd(dba)2. RuPhos

CsgCO _g , toluene/HgO

Int 7-1 "ho 162

[00236] 3-bromo-4-methyI-7-(l-phenylethoxy)-2H-chromen-2-one (Int 7-1). To a stirred solution of 4-methyl-7-(l-phenylethoxy)-2H-chr omen-2 -one (50 mg, 1 eq) in MeCN (2 mL) was added NBS (35.6 mg, 0.2 mmol, 1.1 eq) and the reaction stirred at room temperature for 16 hours. The colorless solution was concentrated under reduced pressure and purified by flash column chromatography (S1O2, 0 to 20% EtOAc in hexanes) to afford Int 7-1 as a white solid (58.2 mg, 90%).

[00237] 4-methyl- 3-(morpholinomethyl)-7-(l-phenylethoxy)-2H-chromen-2-one (152).

A mixture of Int 7-1 (20 mg, 0.056 mmol, 1 eq), potassium (morpholin-4- yl)methyltrifluoroborate (18 mg, 0.084 mol, 1.5 eq), RuPhos (5 mg, 0.011 mmol, 0.2 eq) and CS2CO3 (55 mg, 0.17 mmol, 3 eq) in toluene (0.375 mL) and H2O (0.125 mL) in a microwave vial was sparged with argon for 2 minutes while stirring. To the mixture was added Pd(dba)2 (3.2 mg, 0.056 mmol, 0.1 eq) and further sparged for an additional minute. The vial was sealed and heated at 90 °C for 16 hr. The initially red biphasic mixture turned dark yellow-orange, and a black precipitate appeared. The reaction was then diluted with water (15 mL) and extracted with EtOAc (3 x 10 mL). The combined organics were washed once with brine, dried over Na2S04, filtered, and concentrated under reduced pressure. The residue was purified by reverse-phase preparative HPLC to afford 152 as a viscous, colorless oil (3.5 mg, 17%). ESI-MS [M+l]: 380.2.

[00238] Scheme 8.

aryl chlorid· or bromide,

Pd(PPhi) ₄ . l¾CO,·

dioxin·, H,O

/''N cticV^

J Int 8-1

[00239] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (3-(4,4,5- trimethyl-l,3,2~dioxaborolan-2-yl)benzyl)carbamate (Int 8-1). The title intermediate was prepared analogously to 78, using (3-(4,4,5-trimethyl-l,3,2-dioxaborolan-2- yl)phenyl)methanamine, to afford Int 8-1 as a yellow solid (204.4 mg, 86%). LC-MS [ESI, M+l]: 535.2

[00240] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (3-(lH-imidazol- 2-yI)benzyl)carbamate (153). A mixture of Int 8-1 (15 mg, 1 eq), tert-butyl 2-iodo-lH- imidazole-l-carboxylate (12 mg, 1.5 eq), and K2CO3 (15.5 mg, 4 eq) in 1,4-dioxane (0.9 mL) and H2O (0.3 mL) in a microwave vial was sparged with argon for 2 minutes while stirring. To the mixture was added Pd(PPli3)4 (5 mg, 0.15 eq) and further sparged for an additional minute. The vial was sealed and heated at 90 °C for 8 hr. The reaction was then diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organics were washed once with brine, dried over Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (S1O2, 10 to 100% EtO Ac/hexanes) to afford 153 as a yellow solid. LC-MS [ESI, M+l]: 475.3.

[00241] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyl (3-(pyridin-2- yl)benzyl)carbamate (154). The title compound was prepared analogously to 153, using 2- chloropyridine. LC-MS [ESI, M+l]: 486.3. [00242] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (3-(pyrimidin-2- yl)benzyl)carbamate (155). The title compound was prepared analogously to 153, using 2- chloropyrimidine. LC-MS [ESI, M+l]: 487.3.

[00243] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (3-(pyrimidin-5- yl)benzyl)carbamate (156). The title compound was prepared analogously to 153, using 5- bromopyrimidine. LC-MS [ESI, M+l]: 487.3.

[00244] 2-(7-(diethylamino)-4-methyi-2-oxo-2H-chromeii-3-yl)ethyl (3-(pyrazin-2- yl)benzyl)carbamate (157). The title compound was prepared analogously to 153, using chloropyrazine. LC-MS [ESI, M+l]: 487.3.

[00245] Scheme 9.

[00246] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)acetaldeh yde (Lit 9-1)

[00247] To a mixture of 53 (200 mg, 726 pmol, 1 eq.) in DCM (5 mL) was added DMP (462 mg, 1.09 mmol, 1.5 eq.) at 0 °C. The mixture was stirred at 20 °C for 12 hours. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by reversed phase flash chromatography. The desired fractions were collected and neutralized with saturated NaHCCb solution, concentrated under vacuum to remove MeCN and extracted with EtOAc (2 x 50 mL). The organic layers were dried over Na2S04 and concentrated under vacuum to give Int 9-1 (160 mg, 574 pmol, 79% yield, 98% purity) as a viscous, bright yellow-orange oil, which was used immediately in the next step. LC-MS [ESI, M+l]: 274.2.

[00248] Tert-butyl 4- amino-4-(((2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3- yl)ethyl)amino)methyI)piperidme-l-carboxylate (Int 9-2). To a solution of Int 9-1 (380 mg, 1.39 mmol, 1 eq.) in MeOH (5 mL) was added tert-butyl 4-amino-4-(aminomethyl)piperidine-l- carboxylate (351 mg, 1.53 mmol, 1.1 eq.) and AcOH (83.5 mg, 1.39 mmol, 79.5 uL, 1 eq.). The mixture was stirred at 20 °C for 1 hr. Then NaBfbCN (262 mg, 4.17 mmol, 3 eq) was added at 0 °C, and the mixture was stirred at 20 °C for 12 hrs. After completion, the reaction mixture was quenched with water (1 mL) at 0 °C, and then concentrated under reduced pressure. The residue was purified by reverse-phase preparative HPLC. The desired fractions were collected and neutralized with saturated NaHCCh solution, concentrated under vacuum to remove MeCN and extracted with EtOAc (6 x 30 mL). The organic layers were dried over Na ₂ SC> ₄ and concentrated under vacuum to give compound Int 9-2 (275 mg, 554 pmol, 40% yield, 98% purity) as a viscous yellow oil. LC-MS [ESI, M+l]: 487.5.

[00249] Tert-butyl 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl) -2- oxo-l,3,8-triazaspiro[4.5] decane- 8- carboxylate (Int 9-3). To a mixture of Int 9-2 (260 mg, 534 pmol, 1 eq.) in DCM (10 mL) was added CDI (260 mg, 1.60 mmol, 3 eq.) at 0 °C under N2. The mixture was stirred at 20 °C for 12 hrs. The reaction mixture was quenched with water (1 mL) and then dried over Na ₂ SC> ₄ , filtered and concentrated under reduced pressure. The residue was purified by reverse-phase preparative HPLC. The desired fractions were collected and neutralized with saturated NaHCCb solution, concentrated under vacuum to remove MeCN and extracted with EtOAc (3 x 30 mL). The organic layers were dried over Na ₂ $0 ₄ and concentrated under vacuum to give Int 9-3 (186 mg, 359 pmol, 67% yield, 99% purity) as a viscous yellow oil which solidified under vacuum. LC-MS [ESI, M+l]: 513.4. ‘H NMR (400 MHz, CDCh-d) d = 7.40 (d, J = 9.2 Hz, 1 H), 6.60 (dd, J = 2.4 Hz, 8.8 HZ, 1 H), 6.50 (d, J = 2.4 Hz, 1 H), 4.66 (s, 1 H), 3.47 - 3.35 (m, 12 H), 2.92 - 2.80 (m, 2 H), 2.42 (s, 3 H), 1.67 - 1.61 (m, 4 H), 1.48 (s, 9 H), 1.21 (t, J = 7.2 Hz, 6 H).

[00250] 3-(2-(7-(diethylamino)- 4-methyl- 2-oxo- 2H-chromen-3-yl)ethyl)-l, 3,8- triazaspiro[4.5]decan-2-one (Int 9-4). To a mixture of Int 9-3 (160 mg, 312 pmol, 1 eq.) in MeCN (1 mL) was added HCl/dioxane (4 M, 5 mL, 64.1 eq.). The mixture was stirred at 15 °C for 30 min. The reaction mixture was concentrated under reduced pressure. Then the residue was dissolved in DCM (15 mL) and adjusted pH to 8 with saturated NaHCCb solution. The separated water layer was extracted with DCM (10 mL x 3). The combined organic layers were washed with brine (5 mL ^c 1), dried over sodium sulfate, filtered and concentrated under reduced pressure to give Int 9-4 (90 mg, 216 mhioΐ, 69% yield, 99% purity) as a yellow solid. LC-MS [ESI, M+l]: 413.3.

[00251] 3-(2-(7-(diethylamino)- 4-methyl· 2-oxo-2H-chromen-3-yl)ethyl)-8-phenyl- l,3,8-triazaspiro[4.5]decan-2-one (158). To a mixture of Int 9-4 (70.0 mg, 170 mihoΐ, 1 eq.) and iodobenzene (138 mg, 678 mihoΐ, 75.7 pL, 4 eq.) in toluene (6 mL) was added Xantphos (19.6 mg, 33.9 pmol, 0.2 eq.), Xantphos Pd G3 (16.1 mg, 17.0 mihoΐ, 0.1 eq) and CS2CO3 (166 mg, 509 mpioΐ, 3 eq.) in one portion in the atmosphere of N2. The mixture was stirred at 110 °C for 12 hours. To the reaction mixture was added water (10 mL) and then extracted with ethyl acetate (20 mL x 3). The combined organic layers were washed with brine (10 mL x 1), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (S1O2, DCMXMeOH = 100/1 to 5/1) and triturated with MeOH to give the title compound (46.6 mg, 88.5 mihoΐ, 52% yield, 92.8% purity) as an off-white solid. LCMS [ESI, M+l]: 489.4. ¹ H NMR (400 MHz, CDCb-d) d = 7.42 (d, J = 8.8 Hz, 1 H), 7.29 (s,

1 H), 7.25 (s, 1 H), 6.93 (d, J = 7.6 Hz, 2 H), 6.87 (t, J = 7.2 Hz, 1 H), 6.60 (dd, J = 2.4, 8.8 Hz, 1 H), 6.50 (d, J = 2.4 Hz, 1 H), 4.54 (s, 1 H), 3.41 - 3.31 (m, 8 H), 3.25 - 3.12 (m, 4 H), 2.88 (t, J = 7.6 Hz, 2 H), 2.43 (s, 3 H), 1.86 - 1.82 (m, 4 H), 1.21 (t, J = 7.2 Ήz, 6 H).

[00252] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (3-(lH-pyrazol-5- yl)benzyl)carbamate (159). The title compound was prepared analogously to 153, using tert- butyl 5-bromo-lH-pyrazole-l-carboxylate. LC-MS [ESI, M+l]: 475.3.

[00253] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (3-(5-methyl-lH- imidazol-4-yl)benzyl)carbamate (160). The title compound was prepared analogously to 153, using tert-butyl 4-bromo-5 -methyl- 1 H-imidazole- 1 -carboxylate. LC-MS [ESI, M+l]: 489.3.

[00254] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chroinen-3-yl)ethyl (3-(pyridin-3- yl)benzyl)carbamate (161). The title compound was prepared analogously to 153, using 3- bromopyridine. LC-MS [ESI, M+l]: 486.3. [00255] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (3-(pyridin-4- yl)benzyl)carbamate (162). The title compound was prepared analogously to 153, using 4- bromopyridine hydrochloride. LC-MS [ESI, M+l]: 486.3.

[00256] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (3-(2-oxo-l,2- dihydropyridin-3-yl)benzyl)carbamate (163). The title compound was prepared analogously to 153, using 3-bromopyridin-2(lH)-one, to afford 163 as a white solid (4.4 mg, 40%). LC-MS [ESI, M+l]: 502.3.

[00257] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (3-(lH-pyrazol-4- yl)benzyl)carbamate (164). The title compound was prepared analogously to 153, using tert- butyl 4-bromo-lH-pyrazole-l-carboxylate. LC-MS [ESI, M+l]: 475.3.

[00258] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyI (3-(1H- 1,2,4- triazoI-5-yl)benzyl)carbamate (165). The title compound was prepared analogously to 153, using tert-butyl 5-bromo- 1 H- 1 ,2,4-triazole- 1 -car boxy late. LC-MS [ESI, M+l]: 476.3.

[00259] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyI (3-(pyridazin-3- yl)benzyl)carbamate (166). The title compound was prepared analogously to 153, using 3- chloropyridazine. LC-MS [ESI, M+l]: 487.3.

[00260] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyl (3-(oxazol-2- yl)benzyl)carbamate (167). The title compound was prepared analogously to 153, using 2- chlorooxazole. LC-MS [ESI, M+l]: 476.3.

[00261] 4-methyl-7-(pentan-3-yi)-2H-chromen-2-one (168). A mixture of 7-iodo-4- methyl-2H-chromen-2-one (40 mg, 1 eq), l,2-bis(diphenylphosphaneyl)benzene (6.3 mg, 0.1 eq), 4,4'-di-tert-butyl-2,2 ^, -bipyridine (3.8 mg, 0.1 eq), and NIL· (9.4 mg, 0.214 eq) in DMPU (1 mL) was sparged with argon for 3 min while stirring. Under a flow of argon, pyridine (2.3 pL, 0.2 eq), 3-bromopentane (25.4 mg, 1.2 eq), and manganese metal (15.4 mg, 2 eq) were sequentially added. The vial was sealed and the dark red/gray suspension was heated at 80 °C for 16 hours, turning dark orange/brown and finally dark yellow/brown. The suspension was filtered through C ₆ lite and diluted with EtOAc (20 mL). The organic layer was washed with H2O (2 x 20 mL), once with brine, dried over MgSO*, and concentrated. The residue was purified by reverse- phase preparative HPLC to afford the title compound as a viscous, colorless oil. NMR (400 MHz, Chloroform-i/) d 7.54 (d, J= 8.0 Hz, 1H), 7.17 - 7.08 (m, 2H), 6.26 (q, ./= 1.3 Hz, 1H), 2.51 - 2.39 (m, 4H), 1.83 - 1.68 (m, 2H), 1.67 - 1.51 (m, 2H), 0.80 (t, ./= 7.4 Hz, 6H).

[00262] 4-methyl-7-(trifluoromethyl)-2H-chromen-2-one (169). A mixture of 7-iodo-4- methyl-2H-chromen-2-one (30 mg, 1 eq), Cul (4 mg, 0.2 eq), 1,10-phenanthroline (3.8 mg, 0.2 eq), and KF (18.3 mg, 3 eq) was placed under argon in a sealed microwave vial. Afterwards, DMSO (0.5 mL), B(OMe)3 (55 mg, 5 eq), and TMSCF3 (75 mg, 5 eq) were added sequentially. The dark orange solution was stirred at 60 °C for 16 hours and turned dark green. The reaction was then diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organics were washed once with brine _; , dried over MgSCL, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (S1O2, 0 to 15%

EtO Ac/hexanes) to afford the title compound as a yellow solid. NMR (400 MHz,

Chloroform-*/) d 7.75 (d, J= 8.3 Hz, 1H), 7.64 - 7.54 (m, 2H), 6.43 (q, J= 1.4 Hz, 1H), 2.51 (d, J= 1.3 Hz, 3H). ¹⁹ F NMR (376 MHz, Chloroform·*/) d -63.15 ppm.

[00263] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl (3-(N- hydroxycarbamimidoyl)benzyl)carbamate (170). The title compound was prepared in two steps. In the first step, Lit 5-1 was reacted with 3-(aminomethyl)benzonitrile analogous to the preparation of 78. The intermediate compound was suspended in EtOH (4 mL) and to the stirred suspension was added H2NOH (150 pL, 50% aqueous solution) and stirred for 16 hrs at room temperature. The reaction was then diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organics were washed once with brine, dried over MgSCL, filtered, and concentrated under reduced pressure. The residue was purified by flash column chromatography (S1O2, 0 to 100% EtOAc in hexanes) to afford the title compound as a yellow solid. LC-MS [ESI, M+l]: 467.2.

[00264] 4-methyl- 7-(l-(pyridin-3-yl)ethoxy)-2H-chromen-2-one (171). The title compound was prepared analogously to 32, using 3-(l-bromoethyl)pyridine. LC-MS [ESI, M+l]: 282.0.

[00265] 4-methyl-7-(l-(pyridin-4-yl)ethoxy)-2H-chromen-2-one (172). The title compound was prepared analogously to 32, using 4-(l-bromoethyl)pyridine. LC-MS [ESI, M+l]: 282.0. [00266] 4-methyl-7-(l-(pyrazin-2-yl)ethoxy)-2H-chromen-2-one (173). The title compound was prepared analogously to 32, using 2-(l -bromoethyl)pyrazine. LC-MS [ESI, M+l]: 283.0.

[00267] 4-methyl-7-(l-(pyridin-2-yl)ethoxy)-2H-chromen-2-one (174). The title compound was prepared analogously to 32, using 2-(l-bromoethyl)pyridine. LC-MS [ESI, M+l]: 282.0.

[00268] 3-(2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)ethyl)- 1,3,7- triazaspiro[4.4]nonane-2,4-dione (175). A suspension of 53 (200 mg, 0.73 mmol, 1 eq), tert- butyl 2,4-dioxo-l,3,8-triazaspiro[4.5]decane-8-carboxylate (215 mg, 0.80 mmol, 1.1 eq), and PPh ₃ (382 mg, 1.46 mmol, 2 eq) in anhydrous THF (5 mL) was placed under argon and cooled on an ice bath. To the stirred suspension was added diisopropyl azodicarboxylate (288 pL, 1.46 mmol, 2 eq) dropwise over 2 minutes. The orange suspension was stirred on ice for 10 minutes, followed by at room temperature for an additional 4 hr, during which the hydantoin starting material gradually dissolved. The mixture was concentrated under reduced pressure and purified by flash column chromatography (S1O2, 0 to 50% EtOAc/hexanes) to afford a mixture of the Boc-protected spiro-hydantoin coumarin and Ph ₃ PO as a viscous yellow oil. To a stirred solution of this residue in MeOH (3 mL) was then added concentrated HC1 (1 mL) dropwise and stirred overnight at room temperature, during which a white precipitate appeared. The reaction was diluted with water (30 mL) and washed once with EtOAc (15 mL) to remove Ph ₃ PO. Afterwards, the aqueous layer was basified with addition of solid NazC0 ₃ to give a bright yellow solution, which was extracted with DCM (6 x 20 mL). The combined organics were washed once with brine, dried over Na ₂ SC> ₄ , filtered, and concentrated to afford the title compound as a white solid (257.5 mg, 71% over 2 steps). A sample was further purified by reverse-phase preparative HPLC. LC-MS [ESI, M+l]: 427.4.

[00269] 3-(2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)ethyl)-8-(lH- imidazole-4-carbonyl)-l,3,8-triazaspiro[4.5]decane-2,4-dione (176). The title compound was prepared analogously to 150, using 175 and lH-imidazole-4-carboxylic acid as starting materials, to afford 176 as a yellow solid (6.6 mg, 37%, 2HC1 salt). LC-MS [ESI, M+l]: 521.3.

[00270] 3-(2-(7-(diethylamino}- 4-methyl- 2-oxo-2H-chromen-3-yl)ethyl)-8-(lH- imidazole-2-carbonyl)-l,3,8-triazaspiro[4.5]decane-2,4-dione (177). The title compound was prepared analogously to 150, using 175 and lH-imidazole-2-carboxylic acid as starting materials. LC-MS [ESI, M+l]: 521.3.

[00271] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl) -8-(2-oxo-l,2- dihydropyridine-3-carbonyl)-l,3,8-triazaspiro[4.5] decan e-2,4-dione (178). The title compound was prepared analogously to 150, using 175 and 2-oxo-l,2-dihydropyridine-3- carboxylic acid as starting materials, to afford 176 as a yellow solid (8.1 mg, 46%, HC1 salt). LC-MS [ESI, M+l]: 548.3.

[00272] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yI)etiiyl )-l,3,7- triazaspiro[4.4]nonane-2,4-dione (179). The title compoimd was prepared analogously to 175, using tert-butyl 2,4-dioxo-l,3,7-triazaspiro[4.4]nonane-7-carboxylate, to afford 179 as a yellow solid (274.3 mg, 91% over 2 steps). LC-MS [ESI, M+l]: 413.2.

[00273] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chroinen-3-yl)ethyl )-7-(lH- imidazole-4-carbonyl)-l,3,7-triazaspiro[4.4]nonane-2,4-dione (180). The title compound was prepared analogously to 150, using 179 and 1 H-imidazole-4-carboxy lie acid as starting materials, to afford 180 as a yellow solid (8.6 mg, 50%, 2HC1 salt). LC-MS [ESI, M+l]: 507.3.

[00274] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chroinen-3-yl)ethyl )-7-(lH- imidazole-2-carbonyl)-l,3,7-triazaspiro[4.4]nonane-2,4-dione (181). The title compound was prepared analogously to 150, using 179 and 1 H-imidazole-2-carboxylic acid as starting materials. LC-MS [ESI, M+l]: 507.3.

[00275] 3-(2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)ethyl)-7-(2-oxo-l,2- dihydropyridine-3-carbonyl)-l,3,7-triazaspiro[4.4]nonane-2,4 -dione (182). The title compound was prepared analogously to 150, using 179 and 2-oxo-l,2-dihydropyridine-3- carboxylic acid as starting materials, to afford 182 as a yellow solid (10.6 mg, 62%, HC1 salt). LC-MS [ESI, M+l]: 534.3.

[00276] 4-methyl-3-(morpholinomethyl)-7-(2-phenylpropoxy)-2H-chromei -2-one (183). The title compound was prepared in two steps, analogously to 152, using 43 as starting material. LC-MS [ESI, M+l]: 394.2. [00277] 4-methyl-3-(morpholinomethyl)-7-((l-phenylprOpan-2-yl)oxy)-2 H-chromen- 2-one (184). The title compound was prepared in two steps, analogously to 152, using 47 as starting material. LC-MS [ESI, M+l]: 394.2.

[00278] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yI)etiiyl )-8-(pyrimidin- 2-yl)-l _y 3,8-triazaspiro[4.5]decane-2,4-dione (185). A stirred solution of 175 (10 mg, 1 eq), 2- chloropyrimidine (5.3 mg, 2 eq), DIPEA (12 pL, 3 eq) in DMF (1 mL) was heated at 80 °C for 16 hrs. The mixture was concentrated under reduced pressure and purified by flash column chromatography to afford the title compound as a yellow solid (8.7 mg, 75%). In other examples, purification was achieved through reverse-phase preparative HPLC. LC-MS [ESI, M+l]: 505.3.

[00279] 3-(2-(7-(diethylamino)-4-methyI-2-oxo-2H-chromai-3-yl)etiiyl )-8-(5- (trifluoromethyl)pyrimidin-2-yl)-l,3,8-triazaspiro[4.5]decan e-2,4-dione (186). The title compound was prepared analogously to 185, using 2-chloro-5-(trifluoromethyl)pyrimidine. LC- MS [ESI, M+l]: 573.3.

[00280] 3-(2-(7-(diethylaimno)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyl )-8-(5- (trifluoromethyl)pyridin-2-yl)-l,3,8-triazaspiro[4.5] decan e-2,4-dione (187). The title compound was prepared analogously to 185, using 2-chloro-5-(trifluoromethyl)pyridine. LC- MS [ESI, M+l]: 572.3.

[00281] 3-(2-(7-(diethylamino)-4-methyI-2-oxo-2H-chromeii-3-yl)ethyl )-8-(9H-purin- 6-yl)-l,3,8-triazaspiro [4.5] decan e-2,4-dione (188). The title compound was prepared analogously to 185, using 6-chloropurine, to afford 188 as a yellow solid (9.2 mg, 69%, HC1 salt). LC-MS [ESI, M+l]: 545.3.

[00282] 2-(3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)eth yl)-2,4-dioxo- l,3,8-triazaspiro[4.5]decan-8-yl)pyrimidine-5-carbonitrile (189). The title compound was prepared analogously to 185, using 2-chloropyrimidine-5-carbonitrile. LC-MS [ESI, M+l]: 530.3.

[00283] 2-(3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)eth yl)-2,4-dioxo- l,3,8-triazaspiro[4.5]decan-8-yl)pyrimidine-4-carbonitrile (190). The title compound was prepared analogously to 185, using 2-chloropyrimidine-4-carbonitrile. LC-MS [ESI, M+l]: 530.3. [00284] 6-(3-(2-(7-(diediylamino)-4-methyl-2-oxo-2H-chromen-3-yl)eth yl)-2,4-dioxo- l,3,8-triazaspiro[4.5]decan-8-yl)nicotinonitrile (191). The title compound was prepared analogously to 185, using 6-chloronicotinonitrile.. LC-MS [ESI, M+l]: 529.3.

[00285] 3-(2-(7-(diethylammo)-4-methyl-2-oxo-2H-chromen-3-yI)ethyl)- 8-(lH- pyrazolo[3,4-d]pyrimidin-4-yl)-l,3,8-triazaspiro[4.5]decane- 2,4-dione (192). The title compound was prepared analogously to 185, using 4-chloro-lH-pyrazolo[3,4-d]pyrimidine, to afford 192 as a yellow solid (8.1 mg, 61%, HC1 salt).. LC-MS [ESI, M+l]: 545.6.

[00286] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yI)etiiyl )-8-(9H-purin-

2-yI)-l,3,8-triazaspiro[4.5] decane-2, 4-dione (193). The title compound was prepared analogously to 185, using 2-chloro-9H-purine. LC-MS [ESI, M+l]: 545.6.

[00287] 3-(2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)eth yl)-7-(pyrimidin- 2-yl)-l,3,7-triazaspiro [4.4] nonane-2, 4-dione (194). The title compound was prepared analogously to 185, using 179, to afford 194 as a yellow solid (8.6 mg, 68%). LC-MS [ESI, M+l]: 491.3.

[00288] 3-(2-(7-(diethylammo)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl)- 7-(5- (trifluoromethyl)pyrimidin-2-yl)-l _y 3,7-triazaspiro[4.4]nonane-2, 4-dione (195). The title compound was prepared analogously to 185, using 179 and 2-chloro-5- (trifluoromethyl)pyrimidine as starting materials. LC-MS [ESI, M+l]: 559.3.

[00289] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl) -7-(5- (trifluoromethyl)pyridin-2-yI)-l,3,7-triazaspiro[4.4]nonane- 2, 4-dione (196). The title compound was prepared analogously to 185, using 179 and 2-chloro-5-(trifluoromethyl)pyridine as starting materials. LC-MS [ESI, M+l]: 558.3.

[00290] 3-(2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)ethyl)-7-(9H-purin-

6-yl)-l,3,7-triazaspiro [4.4] nonane-2, 4-dione (197). The title compound was prepared analogously to 185, using 179 and 6-chloropurine as starting materials, to afford 197 as a yellow solid (10.1 mg, 74%, HC1 salt). LC-MS [ESI, M+l]: 531.3.

[00291] 4-(3-(2-(7-(diefliylamino)-4-methyl-2-oxo-2H-chiOmen-3-yl)et hyl)-2,4-dioxo- l,3,8-triazaspiro[4.5]decane-8-carbonyl)benzamide (198). The title compound was prepared analogously to 150, using 175 and 4-carbamoylbenzoic acid as starting materials. LC-MS [ESI, M+l]: 574.4.

[00292] 4-(3-(2-(7-(diethylammo)-4-methyl-2-oxo-2H-chromen-3-yl)ethy l)-2,4-dioxo- l,3,7-triazaspiro[4.4]nonane-7-carbonyl)benzamide (199). The title compound was prepared analogously to 150, using 179 and 4-carbamoylbenzoic acid as starting materials. LC-MS [ESI, M+l]: 560.3.

[00293] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl) -8- (phenylsulfony 1)-13,8-triazaspiro [4.5] decan e-2/4-dione (200). To a stirred solution of 175 (10 mg, 1 eq) and DIPEA (12 pL) in DMF (1 mL) was added benzenesulfonyl chloride (3.8 pL, 1.3 eq) on an ice-water bath. The reaction was stirred at 0 °C for 3 hr, then concentrated under reduced pressure. The residue was purified by flash column chromatography (S1O2,

EtO Ac/hexanes) to afford the title compound as a yellow solid. LC-MS [ESI, M+l]: 567.4.

[00294] 3-(2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)eth yl)-7- (phenylsulfonyI)-l,3,7-triazaspiro[4.4]nonane-2,4-dione (201). The title compound was prepared analogously to 200, using 179, to afford 201 as a yellow solid (12.7 mg, quant). LC- MS [ESI, M+l]: 553.4.

[00295] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl) -7-(pyridin-3- ylsulfonyl)-l,3,7-triazaspiro[4.4]nonane-2,4-dione (202). The title compound was prepared analogously to 200, using 179 and pyridine-3-sulfonyl chloride as starting materials, to afford 202 as a yellow solid (11.8 mg, 93%). LC-MS [ESI, M+l]: 554.3.

[00296] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chroinen-3-yl)ethyl )-7- (ethylsulfonyl)-l,3,7-triazaspiro[4.4]nonane-2,4-dione (203). The title compound was prepared analogously to 200, using 179 and ethanesulfonyl chloride as starting materials. LC- MS [ESI, M+l]: 505.3.

[00297] 3-(2-(7-(diethylammo)-4-methyl-2-oxo-2H-chroinen-3-yl)ethyl) -N-methyl-2,4- dioxo-l _y 3,7-triazaspiro[4.4]nonane-7-sulfonamide (204). The title compound was prepared analogously to 200, using 179 and methylsulfamoyl chloride as starting materials. LC-MS [ESI, M+l]: 506.3. [00298] 3-(2-(7-(dietiiylamino)-4-metiiyl-2-oxo-2H-chromen-3-yl)ethy l)-7- (pyrimidine-2-carbonyl)-l,3,7-triazaspiro[4.4]nonane-2,4-dio ne (205). The title compound was prepared analogously to 150, using 179 and pyrimidine-2-carboxylic acid as starting materials. LC-MS [ESI, M+l]: 519.3.

[00299] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-din>men-3-yl)eth yl)-8- (pyrimidine-2-carbonyI)-l,3,8-triazaspiro[4.5]decane-2,4-dio ne (206). The title compound was prepared analogously to 150, using 175 and pyrimidine-2-carboxylic acid as starting materials. LC-MS [ESI, M+l]: 533.3.

[00300] 3-(2-(7-(diethylammo)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl)- 7- (pyrimidine-4-carbonyl)-l,3,7-triazaspiro[4.4]nonane-2,4-dio ne (207). The title compound was prepared analogously to 150, using 179 and pyrimidine-4-carboxylic acid as starting materials. LC-MS [ESI, M+l]: 519.3.

[00301] 3-(2-(7-(diethylammo)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl)- 8- (pyrimidine-4-carbonyl)-l,3,8-triazaspiro[4.5]decane-2,4-dio ne (208). The title compound was prepared analogously to 150, using 175 and pyrimidine-4-carboxylic acid as starting materials. LC-MS [ESI, M+l]: 533.3.

[00302] 3-(2-(7-(diethylammo)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl)- 7- (pyrimidine-5-carbonyl)-l,3,7-triazaspiro[4.4]nonane-2,4-dio ne (209). The title compound was prepared analogously to 150, using 179 and pyrimidine-5-carboxylic acid as starting materials. LC-MS [ESI, M+l]: 519.3.

[00303] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chroinen-3-yl)ethyl )-8- (pyrimidine-5-carbonyl)-l,3,8-triazaspiro[4.5]decane-2,4-dio ne (210). The title compound was prepared analogously to 150, using 175 and pyrimidine-5-carboxylic acid as starting materials. LC-MS [ESI, M+l]: 533.3.

[00304] 3-(2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)ethyl)-7-(pyraz in

2-carbonyl)-l,3,7-triazaspiro[4.4]nonane-2,4-dione (211). The title compound was prepared analogously to 150, using 179 and pyrazine-2-carboxylic acid as starting materials. LC-MS [ESI, M+l]: 519.3. [00305] 3-(2-(7-(dietiiylamino)-4-metiiyl-2-oxo-2H-chromen-3-yl)ethy l)-8-(pyrazine- 2-carbonyl)-l,3,8-triazaspiro[4.5] decan e-2,4-dione (212). The title compound was prepared analogously to 150, using 175 and pyrazine-2-carboxylic acid as starting materials. LC-MS [ESI, M+l]: 533.3.

[00306] 3-((7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)methyl)- 5- isobutylimidazolkline-2,4-dione (213). The title compound was prepared analogously to the first step for preparation of 175, using Int 2-3 and 5-isobutylimidazolidine-2,4-dione as starting materials. LC-MS [ESI, M+l]: 400.2.

[00307] 3-(2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)ethyl)- 5- isobutylimidazolkline-2,4-dione (214). The title compound was prepared analogously to the first step for preparation of 175, using 5-isobutylimidazolidine-2,4-dione. LC-MS [ESI, M+l]: 414.2.

[00308] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl) -8- (imidazo[l,2-a] pyrazin-8-yl)-l,3,8-triazaspiro[4.5] decan e-2,4-dione (215). The title compound was prepared analogously to 185, using 8-chloroimidazo[l,2-a]pyrazine, to afford 215 as a yellow solid (6.4 mg, 48%, HC1 salt). LC-MS [ESI, M+l]: 544.3.

[00309] 3-(2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)ethyl)-8-(l-methyl- lH-pyrazolo[3,4-d]pyrimidin-4-yl)-l,3,8-triazaspiro[4.5]deca ne-2,4-dione (216). The title compound was prepared analogously to 185, using 8-chloroimidazo[l,2-a]pyrazine. LC-MS [ESI, M+l]: 559.3.

[00310] 8-([l, 2, 4]triazolo[4,3-a]pyrazin-8-yl)-3-(2-(7-(diethylainino)- 4-methyl- 2-oxo-

2H-chromen-3-yl)ethyl)-l,3,8-triazaspin)[4.5]decane-2,4-d ione (217). The title compound was prepared analogously to 185, using 8-chloro-[l,2,4]triazolo[l,5-a]pyrazine, to afford 217 as a light yellow solid (10.9 mg, 76%, HC1 salt). LC-MS [ESI, M+l]: 545.3.

[00311] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chrOmen-3-yl)ethyl ((2'-hydroxy- [l,l'-biphenyl]-3-yl)methyl)carbamate (218). The title compound was prepared in two steps. The first step was analogous to preparation of 153, using 1 -iodo-2-(methoxymethoxy)benzene. Next, a solution of the MOM-protected intermediate (~15 mg) in 0.5M HC1 in MeOH (3 mL) was stirred at room temperature for 16 hrs. The reaction was quenched by addition of saturated NaHCCb and extracted with EtO Ac (3 x 10 mL). The combined organics were washed once with brine, dried over MgSO*, filtered, and concentrated under reduced pressure to afford the title compound as a brown solid. LC-MS [ESI, M+l]: 501.3.

[00312] 3'-((((2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3- yl)ethoxy)carbonyl)amino)methyl)-[l,V-biphenyl]-2-yl sulfurofluoridate (219). The atmosphere above a solution of 218 (12.2 mg, 1 eq) and DIPEA (3 eq) in MeCN (2 mL) was evacuated and refilled with SO2F2 gas (1 atm) using a balloon. The solution was stirred for 2 hr at room temperature and concentrated under reduced pressure. The residue was purified by flash column chromatography (S1O2, EtO Ac/hexanes) to afford the title compound as a yellow gum. LC-MS [ESI, M+l]: 583.3.

[00313] 2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3-yl)ethyl ((2'-oxo-l',2'- dihydro-[3,3'-bipyridin]-5-yi)methyl)carbamate (220). The title compound was prepared analogously to 78, using 5'-(aminomethyl)-[3,3'-bipyridin]-2(lH)-one. LC-MS [ESI, M+l]: 503.3.

[00314] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chroinen-3-yl)ethyl )-8-(pyrimidin- 2-yl)-l,3,8-triazaspiro[4.5]decan-2-one (221). The title compound was prepared analogously to 185, using Int 9-4, to afford 221 as a yellow solid (8.1 mg, 40%, HC1 salt). LC-MS [ESI, M+l]: 491.3.

[00315] 3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chroinen-3-yl)ethyl )-8-(lH- pyrazolo[3,4-d]pyrimidin-4-yl)-l,3,8-triazaspiro[4.5]decan-2 -one (222). The title compound was prepared analogously to 185, using Int 9-4 and 4-chloro-lH-pyrazolo[3,4-d]pyrimidine as starting materials, to afford 222 as a light yellow solid (8.4 mg, 38%, HC1 salt). LC-MS [ESI, M+l]: 531.3.

[00316] 3-(2-(7-(diethylamino)-4-mefliyl-2-oxo-2H-chromai-3-yl)efliy l)-8- (imidazo[l,2-a]pyrazin-8-yl)-l,3,8-triazaspiro[4.5]decan-2-o ne (223). The title compound was prepared analogously to 185, using Int 9-4 and 8-chloroimidazo[l,2-a]pyrazine as starting materials, to afford 223 as a brown solid (4.3 mg, 20%, HC1 salt). LC-MS [ESI, M+l]: 530.4.

[00317] 4-methyl-2-oxo-2H-chromene-7-carbonitrile (224). The title compound was prepared using literature procedures. Characterization data matched the literature. [00318] 4-methyl-2-oxo-2H-chromene-7-carboxylic acid (225). The title compound was prepared using literature procedures. Characterization data matched the literature.

[00319] 4-methyl-2-oxo-N-(pyridin-2-ylmethyl)-2H-chrOmene-7-carboxam ide (226). The title compound was prepared analogously to 150, using 225 and pyridin-2-ylmethanamine as starting materials. LC-MS [ESI, M+l]: 295.0. ‘HNMR (600 MHz, DMSO-d6) d 9.38 (t, J = 5.9 Hz, 1H), 8.56 (ddd, J = 4.9, 1.8, 0.9 Hz, 1H), 7.93 - 7.88 (m, 2H), 7.83 (td, J = 7.7, 1.8 Hz, 1H), 7.41 (d, J = 7.9 Hz, lH), 7.36 - 7.31 (m, 1H), 5.76 (s, 1H), 4.63 (d, J = 5.9 Hz, 1H), 2.48 (d, J = 1.3 Hz, 2H).

[00320] 4-methyl-N-(naphthalen-2-yl)-2-oxo-2H-chromene-7-carboxamide (227). The title compound was prepared analogously to 150, using 225 and naphthalen-2-amine as starting materials. LC-MS [ESI, M-l]: 328.0.

[00321] 4-methyl- 2-oxo-N-(pyridin-4-ylmethyl)-2H-chromene-7-carboxamide (228).

The title compound was prepared analogously to 150, using 225 and pyridin-4-ylmethanamine as starting materials. LC-MS [ESI, M+l]: 295.0.

[00322] N-(3,5-dichloro-4-hydroxyphenyl)-4-methyl-2-oxo-2H-chromene- 7- carboxamide (229). The title compound was prepared analogously to ISO, using 225 and 4- amino-2,6-dichlorophenol as starting materials. LC-MS [ESI, M+l]: 365.0.

[00323] N-(4-hydroxybenzyl)-4-methyl-2-oxo-2H-chrom«ie-7-carboxamid e (230). The title compound was prepared analogously to 150, using 225 and 4-(aminomethyl)phenol as starting materials. LC-MS [ESI, M+l]: 310.0.

[00324] 7-(diallylamino)-4-methyl-2H-chromen-2-one (231). The title compound was prepared analogously to 36, using 225 and allyl bromide as starting materials, to afford 231 and 240 as a mixture of products. LC-MS [ESI, M+l]: 256.0.

[00325] N-(4-hydroxyphenethyI)-4-methyi-2-oxo-2H-chromene-7-carboxam ide (232). The title compound was prepared analogously to 150, using 225 and 4-(2-aminoethyl)phenol as starting materials. LC-MS [ESI, M+l]: 324.0.

[00326] 4-methyl- 2-oxo-N-(quinolin-2-yl)-2H-chromene-7-carboxamide (233). The title compound was prepared analogously to 150, using 225 and quinolin-2-amine as starting materials. LC-MS [ESI, M+l]: 331.0. [00327] 4-methyl-2-oxo-N-(qiimolin-4-yl)-2H-chromene-7-carboxamide (234). The title compound was prepared analogously to 150, using 225 and quinolin-4-amine as starting materials. LC-MS [ESI, M+l]: 331.0.

[00328] 4-methyl- 2-oxo-N-(quinolin-6-yI)-2H-diromene-7-carboxamide (235). The title compound was prepared analogously to 150, using 225 and quinolin-6-amine as starting materials. LC-MS [ESI, M+l]: 331.0.

[00329] N-((lH-indol-3-yi)methyl)-4-methyl-2-oxo-2H-chromene-7-carbo xamide (236). The title compound was prepared analogously to 150, using 225 and (lH-indol-3- yl)methanamine as starting materials. LC-MS [ESI, M+l]: 333.1.

[00330] N-((lH-indol-5-yl)methyl)-4-methyl-2-oxo-2H-chromene-7-carbo xamide (237). The title compound was prepared analogously to 150, using 225 and (lH-indol-5- yl)methanamine as starting materials. LC-MS [ESI, M+l]: 333.1.

[00331] N-(lH-benzo [d] imidazol-6-yl)-4-methyl-2-oxo-2H-chromene-7-carboxamide

(238). The title compound was prepared analogously to 150, using 225 and 1H- benzo[d]imidazol-6-amine as starting materials. LC-MS [ESI, M+l]: 320.0.

[00332] N-(benzo[d]thiazol-5-yl)-4-methyl- 2-oxo- 2H-chromene-7-carboxamide (239).

The title compound was prepared analogously to 150, using 225 and benzo[d]thiazol-5-amine as starting materials. LC-MS [ESI, M+l]: 337.1.

[00333] 7-(allyiamino)-4-methyl-2H-chromen-2-one (240). The title compound was prepared analogously to 36, using 225 and allyl bromide as starting materials, to afford 231 and 240 as a mixture of products that was separated by flash column chromatography (S1O2, EtOAc/hexanes). LC-MS [ESI, M+l]: 216.0.

[00334] 7-(dipropylamino)-4-methyl-2H-chromen-2-one (241). The title compound was prepared analogously to 36, using 225 and 1-iodopropane as starting materials, to afford 241 and 242 as a mixture of products that was separated by flash column chromatography (S1O2, EtOAc/hexanes). LC-MS [ESI, M+l]: 260.0. ^X H NMR (600 MHz, Chloroform-*/) d 7.39 (d, J= 9.0 Hz, 1H), 6.59 (dd, J= 9.0, 2.6 Hz, 1H), 6.50 (d, J= 2.6 Hz, 1H), 5.97 (q, J= 1.2 Hz, 1H), 3.34 - 3.28 (m, 4H), 2.36 (d, J= 1.2 Hz, 3H), 1.72 - 1.61 (m, 3H), 0.97 (t, J= 7.4 Hz, 5H). [00335] 4-methyl-7-(propylamino)-2H-diromen-2-one (242). The title compound was prepared analogously to 36, using 225 and 1 -iodopropane as starting materials, to afford 241 and 242 as a mixture of products that was separated by flash column chromatography (S1O2, EtOAc/hexanes). LC-MS [ESI, M+l ]: 218.0. ^l U NMR (600 MHz, Chloroform-7) d 7.37 (d, 7 = 8.7 Hz, 1H), 6.54 (dd, 7= 8.6, 2.3 Hz, 1H), 6.48 (d, 7= 2.3 Hz, 1H), 6.01 (d, 7= 1.3 Hz, 1H), 3.83 (s, 3H), 3.17 (t, 7= 7.1 Hz, 2H), 2.37 (d, 7= 1.2 Hz, 3H), 1.70 (h, 7= 7.3 Hz, 2H), 1.04 (t, 7 = 7.4 Hz, 3H).

[00336] 7-(bis(2-methylallyl)amino)-4-methyE2H-chromen-2-one (243). The title compound was prepared analogously to 36, using 225 and 3 -bromo-2-methylprop- 1 -ene as starting materials, to afford 243 and 244 as a mixture of products that was separated by flash column chromatography (SiCh, EtOAc/hexanes). LC-MS [ESI, M+l]: 284.1. ^X H NMR (600 MHz, Chloroform-7) d 7.38 (d, 7= 8.9 Hz, 1H), 6.56 (dd, 7= 8.9, 2.6 Hz, 1H), 6.49 (d, 7= 2.6 Hz, 1H), 5.97 (t, 7= 1.3 Hz, 1H), 4.75 (t, 7= 1.5 Hz, 2H), 3.88 (s, 4H), 2.34 (d, 7= 1.2 Hz, 3H), 1.77 (s, 6H).

[00337] 4-methyl-7-((2-methylallyl)amino)-2H-chrOmen-2-one (244). The title compound was prepared analogously to 36, using 225 and 3 -bromo-2-methylprop- 1 -ene as starting materials, to afford 243 and 244 as a mixture of products that was separated by flash column chromatography (S1O2, EtO Ac/hexanes) . LC-MS [ESI, M+l]: 230.0. ¹ H NMR (600 MHz, Chloroform-60 d 7.36 (d, 7= 8.7 Hz, 1H), 6.54 (dd, 7= 8.7, 2.4 Hz, 1H), 6.46 (d, 7= 2.4 Hz, 1H), 5.98 (q, 7= 1.2 Hz, 1H), 4.96 (tt, 7= 1.6, 0.9 Hz, 1H), 4.92 (p, 7= 1.4 Hz, 1H), 3.75 (d, 7= 1.5 Hz, 2H), 2.34 (d, 7 = 1.2 Hz, 3H), 1.79 (dd, 7= 1.6, 0.8 Hz, 3H).

[00338] 7-(bis(cyclopropylmethyl)amino)-4-methyl-2H-chromen-2-one (245). The title compound was prepared analogously to 36, using 225 and (bromomethyl)cyclopropane as starting materials, to afford 245 and 246 as a mixture of products that was separated by flash column chromatography (S1O2, EtO Ac/hexanes). LC-MS [ESI, M+l]: 284.1. ¹ H NMR (600 MHz, Chloroform-7) d 7.46 (d, 7= 8.9 Hz, 1H), 6.88 (dd, 7= 8.9, 2.6 Hz, 1H), 6.77 (d, 7= 2.6 Hz, 1H), 6.04 (d, 7= 1.3 Hz, 1H), 2.76 (s, 3H), 2.39 (d, 7 = 1.2 Hz, 2H), 1.14 - 1.02 (m, 2H), 0.66 - 0.54 (m, 3H), 0.29 (h, 7= 4.8, 4.1 Hz, 3H).

[00339] 7-((cyclopropylmethyl)amino)-4-methyl-2H-chroinen-2-one (246). The title compound was prepared analogously to 36, using 225 and (bromomethyl)cyclopropane as starting materials, to afford 245 and 246 as a mixture of products that was separated by flash column chromatography (S1O2, EtOAc/hexanes). LC-MS [ESI, M+l]: 230.0. ¹ H NMR (600 MHz, Chloroform-i/) d 7.37 (d, J= 8.7 Hz, 1H), 6.54 (dd, J= 8.7, 2.3 Hz, 1H), 6.45 (d, J= 2.3 Hz, 1H), 6.00 (t, J= 1.2 Hz, 1H), 3.61 (s, 2H), 3.03 (d , J= 7.0 Hz, 2H), 2.36 (d, J= 1.2 Hz, 3H), 1.18 - 1.07 (m, 1H), 0.63 - 0.53 (m, 1H), 0.30 (dt, J= 6.1, 4.6 Hz, 2H).

[00340] 7-(but-3-en-l-ylammo)-4-methyl-2H-chrOmen-2-one (247). The title compound was prepared analogously to 36, using 225 and 4-iodobut-l-ene as starting materials. LC-MS [ESI, M+l]: 230.0. ‘H NMR (600 MHz, Chloroform-t/) d 7.36 (d, J= 8.7 Hz, 1H), 6.52 (dd, J = 8.6, 2.3 Hz, 1H), 6.46 (d, J= 2.4 Hz, 1H), 5.99 (q , J= 1.1 Hz, 1H), 5.84 (ddt, J= 17.0, 10.2, 6.8 Hz, 1H), 5.22 - 5.15 (m, 1H), 5.19 - 5.13 (m, 1H), 3.26 (t, J = 6.7 Hz, 2H), 2.43 (qt, J = 6.8, 1.3 Hz, 2H), 2.35 (d, J= 1.2 Hz, 4H).

[00341] 7-(di(prop-2-yn-l-yl)amino)-4-methyl-2H-chromen-2-one (248). The title compound was prepared analogously to 36, using 225 and propargyl bromide as starting materials, to afford 248 and 249 as a mixture of products that was separated by flash column chromatography (SiCh, EtO Ac/hexanes). LC-MS [ESI, M+l]: 252.0. ^X H NMR (600 MHz, Chloroform-i/) d 7.51 (d, J= 8.8 Hz, 1H), 6.87 (dd, J= 8.8, 2.6 Hz, 1H), 6.84 (d, J= 2.5 Hz, 1H), 6.10 (q, J= 1.2 Hz, 1H), 4.21 (d, J= 2.4 Hz, 4H), 2.40 (d, J= 1.2 Hz, 3H), 2.32 (t, J= 2.4 Hz, 2H).

[00342] 4-methyl-7-(prop-2-yn-l-ylamino)-2H-chn)men-2-one (249). The title compound was prepared analogously to 36, using 225 and propargyl bromide as starting materials, to afford 248 and 249 as a mixture of products that was separated by flash column chromatography (S1O2, EtOAc/hexanes). Characterization data matched that of the literature.

[00343] 7-(bis(3-fluoropropyl)amino)-4-mefliyl-2H-chrom«i-2-one (250). The title compound was prepared analogously to 36, using 225 and l-fluoro-3-iodopropane as starting materials, to afford 250 and 251 as a mixture of products that was separated by flash column chromatography (SiCh, EtO Ac/hexanes). LC-MS [ESI, M+l]: 296.0.

[00344] 7-((3-fluoropropyl)amino)-4-methyl-2H-chromen-2-one (251). The title compound was prepared analogously to 36, using 225 and l-fluoro-3-iodopropane as starting materials, to afford 250 and 251 as a mixture of products that was separated by flash column chromatography (S1O2, EtOAc/hexanes). LC-MS [ESI, M+l]: 236.0. [00345] 7-(aIlyl(metiiyl)ammo)-4-methyl-2H-chromen-2-one (252). The title compound was prepared analogously to 36, using 240 and methyl iodide as starting materials. LC-MS [ESI, M+l]: 230.0. ¹ H NMR (600 MHz, DMSO^fc) d 7.52 (d , J= 8.9 Hz, 1H), 6.73 (dd, J= 8.9, 2.6 Hz, 1H), 6.55 (d, .7= 2.6 Hz, 1H), 5.97 (q, J= 1.2 Hz, 1H), 5.84 (ddt , J = 17.3, 10.1, 4.8 Hz, 1H), 5.15 (dq, J= 10.3, 1.7 Hz, 1H), 4.07 (dt, .7 = 4.9, 1.8 Hz, 2H), 3.02 (s, 3H), 2.34 (d, J= 1.2 Hz, 2H).

[00346] 4-methyl-7-(methyl(propyl)amino)-2H-chromen-2-one (253). The title compound was prepared analogously to 36, using 242 and methyl iodide as starting materials. LC-MS [ESI, M+l]: 232.0. ¹ H NMR (600 MHz, DMSO^fc) d 7.50 (d , J= 8.9 Hz, 1H), 6.73 (dd,

J= 9.0, 2.6 Hz, 1H), 6.52 (d, J = 2.6 Hz, 1H), 5.95 (d, J = 1.3 Hz, 1H), 3.41 - 3.36 (m, 2H), 2.99 (s, 3H), 2.33 (d, J= 1.2 Hz, 3H), 1.56 (hept, J= 7.4, 7.0 Hz, 2H), 0.89 (t, J= 7.4 Hz, 3H).

[00347] 4-methyl-7-(methyl(2-methylallyl)amino)-2H-chromen-2-one (254). The title compound was prepared analogously to 36, using 244 and methyl iodide as starting materials. LC-MS [ESI, M+l]: 244.0. ¹ H NMR (600 MHz, DMSCWe) d 7.51 (d, J= 9.0 Hz, 1H), 6.70 (dd, J= 9.0, 2.6 Hz, 1H), 6.52 (d, J= 2.6 Hz, 1H), 5.96 (t, J = 1.2 Hz, 1H), 4.83 (p, J = 1.5 Hz, 1H), 3.98 (s, 2H), 3.04 (s, 3H), 2.34 (d, J= 1.2 Hz, 3H), 1.69 (t, J= 1.1 Hz, 3H).

[00348] 7-((cyclopropylmethyl)(methyl)amino)-4-methyl-2H-chrOmen-2-o ne (255).

The title compound was prepared analogously to 36, using 246 and methyl iodide as starting materials. LC-MS [ESI, M+l]: 244.0. ^l H NMR (600 MHz, DMSO^fc) d 7.52 (d, J= 9.0 Hz,

1H), 6.79 (dd, J= 9.0, 2.6 Hz, 1H), 6.58 (d, J = 2.6 Hz, 1H), 5.96 (t, J= 1.2 Hz, 1H), 3.36 (s, 1H), 3.03 (s, 3H), 2.34 (d, J= 1.2 Hz, 4H), 1.08 - 0.97 (m, 1H), 0.52 - 0.42 (m, 2H), 0.34 - 0.24 (m, 2H).

[00349] 7-(but-3-en-l-yl(methyl)amino)-4-methyl-2H-chromen-2-one (256). The title compound was prepared analogously to 36, using 247 and methyl iodide as starting materials. LC-MS [ESI, M+l]: 244.0. ^l H NMR (600 MHz, DMSO^/b) d 7.52 (d, J= 9.0 Hz, 1H), 6.74 (dd, J= 8.9, 2.6 Hz, 1H), 6.54 (d, J= 2.6 Hz, 1H), 5.96 (q, J= 1.1 Hz, 1H), 5.86 (ddt, J= 17.2, 10.2, 6.9 Hz, 1H), 5.03 (ddd, J= 10.2, 2.1, 1.1 Hz, 1H), 3.54 - 3.48 (m, 2H), 3.00 (s, 3H), 2.36 - 2.26 (m, 5H).

[00350] 4-methyl-7-(methyl(prop-2-yn-l-yl)ammo)-2H-chromen-2-one (257). The title compound was prepared analogously to 36, using 249 and methyl iodide as starting materials. LC-MS [ESI, M+l]: 228.0. ¹ H NMR (600 MHz, DMSO-de) d 7.58 (d, J= 8.9 Hz, 1H), 6.86 (dd, J= 8.9, 2.5 Hz, 1H), 6.76 - 6.69 (m, 1H), 6.04 (q, J= 1.1 Hz, 1H), 4.27 (d, J= 2.4 Hz, 1H), 3.16 (t, J= 2.4 Hz, 1H), 3.05 - 3.00 (m, 4H), 2.35 (dt, J= 11.3, 0.9 Hz, 3H).

[00351] 7-((3-fluoropropyI)(methyl)amino)-4-methyI-2H-chromen-2-one (258). The title compound was prepared analogously to 36, using 251 and methyl iodide as starting materials. LC-MS [ESI, M+l]: 250.0. ¹ H NMR (600 MHz, DMSCWe) d 7.52 (d, J = 8.9 Hz, 1H), 6.74 (dd, J= 9.0, 2.6 Hz, 1H), 6.56 (d, J= 2.5 Hz, 1H), 5.97 (t, J = 1.2 Hz, 1H), 4.51 (dt, J = 47.4, 5.8 Hz, 2H), 3.62 - 3.43 (m, 2H), 3.00 (s, 3H), 2.34 (d, J = 1.2 Hz, 3H), 2.03 - 1.81 (m,

2H).

[00352] 7-(allyl(prop-2-yn-l-yl)amino)-4-methyl-2H-chrom«i-2-one (259). The title compound was prepared analogously to 36, using 249 and allyl bromide as starting materials. LC-MS [ESI, M+l]: 254.0. ‘H NMR (600 MHz, DMSCWe) d 7.57 (d, J= 9.0 Hz, 1H), 6.81 (dd, J= 8.9, 2.6 Hz, 1H), 6.66 (d, J= 2.5 Hz, 1H), 6.03 (z J= 1.3 Hz, 1H), 5.88 (ddt, J = 17.2, 10.2, 5.0 Hz, 1H), 5.22 (dq, J= 17.2, 1.8 Hz, 1H), 5.18 (dq, J= 10.4, 1.6 Hz, 1H), 4.25 (d, J= 2.4 Hz, 2H), 4.11 (dt, J= 5.\, 1.8 Hz, 2H), 3.23 (t, J= 2.4 Hz, 1H), 2.36 (d, J= 1.2 Hz, 3H).

[00353] 4-methyl-7-(prop-2-yn-l-yl(propyl)amino)-2H-chromen-2-one (260). The title compound was prepared analogously to 36, using 249 and 1-iodopropane as starting materials. LC-MS [ESI, M+l]: 256.0. ¹ H NMR (600 MHz, DMSCWe) d 7.55 (d, J= 8.9 Hz, 1H), 6.81 (dd, J= 9.0, 2.6 Hz, 1H), 6.65 (d, J= 2.6 Hz, 1H), 6.01 (d, J= 1.3 Hz, 1H), 4.23 (d, J= 2.5 Hz, 2H), 3.42 (s, 1H), 3.20 (t, J= 2.4 Hz, 1H), 2.35 (d, J= 1.2 Hz, 4H), 1.62 (h, J= 7.4 Hz, 2H), 0.91 (t, J = 7.4 Hz, 4H).

[00354] 4-methyl-7-((2-methylallylXprop-2-yn-l-yl)amino)-2H-chromen- 2-one (261). The title compound was prepared analogously to 36, using 249 and 3-bromo-2-methylprop-l-ene as starting materials. LC-MS [ESI, M+l]: 268.0. ¹ H NMR (600 MHz, DMSO-e/e) d 7.56 (d, J= 8.9 Hz, 1H), 6.78 (dd, J= 8.9, 2.6 Hz, 1H), 6.62 (d, J= 2.5 Hz, 1H), 6.02 (t, J= 1.2 Hz, 1H),

4.81 - 4.77 (m, 1H), 4.23 (d, J= 2.4 Hz, 2H), 3.99 (s, 2H), 3.24 (t, J= 2.4 Hz, 1H), 2.35 (d, J= 1.2 Hz, 3H), 1.72 (s, 3H).

[00355] 7-((cyclopropylmethyl)(prop-2-yn-l-yl)amino)-4-methyl-2H-chr omen-2-one (262). The title compound was prepared analogously to 36, using 249 and

(bromomethyl)cyclopropane as starting materials. LC-MS [ESI, M+l]: 268.0. ^! H NMR (600 MHz, DMSO-7 ₆ ) d 7.57 (d, J= 8.9 Hz; 1H), 6.88 (dd, J= 9.0, 2.6 Hz, 1H), 6.71 (d, J= 2.6 Hz, 1H), 6.03 (d, J= 1.3 Hz, 1H), 4.29 (d, J= 2.4 Hz, 2H), 3.36 (d, J= 6.6 Hz, 2H), 3.18 (t, J= 2.3 Hz, 1H), 2.36 (d, J= 1.2 Hz, 3H), 1.08 (dtdd, J= 11.4, 7.8, 6.4, 4.9 Hz, 1H), 0.53 - 0.45 (m, 2H), 0.43 - 0.30 (m, 2H).

[00356] 7-(but-3-en-l-yI(prop-2-yn-l-yl)amino)-4-methyI-2H-chromen-2 -one (263). The title compound was prepared analogously to 36, using 249 and 4-iodobut-l-ene as starting materials. LC-MS [ESI, M+l]: 268.0. 'H NMR (600 MHz, DMSO^fc) d 7.57 (d, J= 8.9 Hz, 1H), 6.83 (dd, J = 9.0, 2.6 Hz, lH), 6.67 (d, J = 2.6 Hz, 1H), 6.02 (d, J = 1.4 Hz, 1H), 5.88 (ddt, 7= 17.1, 10.2, 6.8 Hz, 1H), 5.14 (dt, J = 17.2, 1.8 Hz, 1H), 5.05 (dd, J= 10.2, 2.0 Hz, 1H), 4.25 (d, J= 2.4 Hz, 2H), 3.53 (dd, J= 8.6, 6.4 Hz, 2H), 3.21 (t, J= 2.3 Hz, 1H), 2.43 - 2.32 (m, 5H).

[00357] 7-((3-fluoroprOpyl)(prop-2-yn-l-yl)amino)- 4-methyl- 2H-chromen-2-one (264). The title compound was prepared analogously to 36, using 249 and l-fluoro-3- iodopropane as starting materials. LC-MS [ESI, M+l]: 274.0. ¹ H NMR (600 MHz, DMSO-ifc) d 7.58 (d, J= 8.9 Hz, 1H), 6.83 (dd, J= 8.9, 2.6 Hz, 1H), 6.69 (d, J= 2.6 Hz, 1H), 6.03 (d, J= 1.4 Hz, 1H), 4.55 (dt, J= 47.3, 5.8 Hz, 2H), 4.25 (d, J= 2.4 Hz, 2H), 3.69 - 3.48 (m, 2H), 3.22 (t , J= 2.4 Hz, 1H), 2.36 (d, J= 1.2 Hz, 3H), 2.14 - 1.84 (m, 2H).

[00358] 4-methyl-7-((3,3,3-trifluoropropyl)amino)-2H-chromen-2-one (265). The title compound was prepared analogously to 36, using l,l,l-trifluoro-3-iodopropane. LC-MS [ESI, M+l]: 271.9.

[00359] 7-(dimethylamino)-4-methyl-2H-chromeii-2-one (266). The title compound was prepared analogously to 36, using methyl iodide. Characterization data matched that of the literature.

[00360] 4-methyl-2-oxo-N-(prop-2-yn-l-yl)-2H-chrOmene-7-carboxamide (267). The title compound was prepared analogously to 150, using 225 and propargylamine as starting materials. LC-MS [ESI, M+l]: 242.0.

[00361] 7-(ethyl(methyl)amino)-4-methyl-2H-chromen-2-one (268). The title compound was prepared analogously to 36, using 270 and methyl iodide as starting materials. LC-MS [ESI, M+l]: 218.0. [00362] 2-((4-methyl-2-oxo-2H-chromen-7-yl)amino)acetonitrile (269). The title compound was prepared analogously to 36, using bromoacetonitrile. LC-MS [ESI, M+l]: 214.9. Ή NMR (600 MHz, Acetone-de) d 7.60 (d, J= 8.7 Hz, 1H), 6.83 (ddd, J = 8.6, 2.3, 0.8 Hz, 1H), 6.70 (dd, J= 2.4, 1.4 Hz, 1H), 6.42 (s, 1H), 6.03 (s, 1H), 4.47 (d, J= 6.4 Hz, 1H), 2.41 (d, ./=

1.2 Hz, 2H).

[00363] 7-(ethylamino)-4-methyl-2H-chn)men-2-one (270). The title compound was prepared analogously to 36, using iodoethane. LC-MS [ESI, M+l]: 203.9. 'H NMR (600 MHz,

Acetone-de) d 7.45 (d, J= 8.7 Hz, 1H), 6.64 (dd, J= 8.7, 2.3 Hz, 1H), 6.42 (d, J= 2.3 Hz, 1H), 5.89 (q, J= 1.2 Hz, 1H), 5.83 (s, 1H), 3.25 (qd, J= 7.1, 5.1 Hz, 2H), 2.36 (d, J = 1.2 Hz, 2H), 1.27 (td, J= 7.2, 1.2 Hz, 4H).

[00364] 7-(ethyl(prop-2-yn-l-yl)amino)-4-methyl-2H-chromen-2-one (271). The title compound was prepared analogously to 36, using 270 and propargyl bromide as starting materials. LC-MS [ESI, M+l]: 242.0.

[00365] 2-(ethyl(4-metiiyi-2-oxo-2H-chrom«i-7-yl)ammo)acetonitrile (272). The title compound was prepared analogously to 36, using 270 and bromoacetonitrile as starting materials. LC-MS [ESI, M+l]: 243.0.

[00366] 7-(ethyl(2-fluoroethyl)amino)-4-methyl-2H-chromen-2-one (273). The title compound was prepared analogously to 36, using 270 and l-fluoro-3-iodopropane as starting materials. LC-MS [ESI, M+l]: 250.0.

[00367] 7-(ethyl(isopropyl)amino)-4-methyl-2H-chromen-2-one (274). The title compound was prepared analogously to 36, using 270 and 2-iodopropane as starting materials. LC-MS [ESI, M+l]: 246.0. ¹ H NMR (600 MHz, Acetone-de) d 7.52 (d, J= 9.0 Hz, 1H), 6.82 (dd, J= 9.0, 2.6 Hz, 1H), 6.58 (d, J= 2.7 Hz, 1H), 5.90 (q, J= 1.2 Hz, 1H), 4.27 (hept, J= 6.6 Hz, 1H), 3.44 (q, J= 7.1 Hz, 2H), 2.38 (d, J= 1.2 Hz, 2H), 1.28 (d, J= 6.6 Hz, 5H), 1.23 (t , J= 7.1 Hz, 3H).

[00368] 7-(ethyl(propyl)amino)-4-methyl-2H-chromen-2-one (275). The title compound was prepared analogously to 36, using 270 and 1-iodopropane as starting materials. LC-MS [ESI, M+l]: 246.0. ‘H NMR (600 MHz, Acetone-de) d 7.50 (d, J= 9.0 Hz, 1H), 6.72

(dd, J= 9.0, 2.6 Hz, 1H), 6.49 (d, J= 2.6 Hz, 1H), 5.89 (q, J= 1.2 Hz, 1H), 3.53 (q, J= 7.1 Hz, 2H), 3.43 - 3.37 (m, 2H), 2.37 (d, J= 1.2 Hz, 3H), 1.74 - 1.64 (m, 2H), 1.21 (t, J= 7.1 Hz, 3H), 0.99 (t, J= 7.4 Hz, 3H).

[00369] l-(2-(7-(diethylamino)- 4-methyl· 2-oxo-2H-chromen-3-yl)ethyl)-3-(pyridin-2- ylmethyl)urea (276). The title compound was prepared analogously to 98, using pyridin-2- ylmethanamine. LC-MS [ESI, M+l]: 409.2.

[00370] l-(2-(7-(diethylamino)- 4-methyl· 2-oxo-2H-chromen-3-yl)ethyl)- 3- (pyridin -4- ylmethyl)urea (277). The title compound was prepared analogously to 98, using pyridin-4- ylmethanamine. LC-MS [ESI, M+l]: 409.2.

[00371] l-benzyl-3-(2-(7-(dietiiylamino)-4-methyl-2-oxo-2H-chromen-3 -yl)ethyl)urea (278). The title compound was prepared analogously to 98, using benzylamine. LC-MS [ESI, M+l]: 408.2.

[00372] l-((lH-indoI-5-yl)mediyl)-3-(2-(7-(diethylammo)-4-methyl-2-o xo-2H- chromen-3-yl)ethyl)urea (279). The title compound was prepared analogously to 98, using (lH-indol-5-yl)methanamine. LC-MS [ESI, M+l]: 447.3.

[00373] l-(2-(7-(diethylamino)- 4-methyl· 2-oxo-2H-chromen-3-yl)ethyl)-3-(4- hydroxyph«iethyi)urea (280). The title compound was prepared analogously to 98, using 4-(2- aminoethyl)phenol. LC-MS [ESI, M+l]: 438.2. ¹ H NMR (600 MHz, DMSO-de) d 9.15 (s, 1H), 7.51 (d, J= 9.0 Hz, 1H), 6.99 - 6.91 (m, 2H), 6.72 - 6.63 (m, 3H), 6.49 (d , J = 2.6 Hz, 1H), 5.94 (s, 1H), 5.76 (s, 1H), 3.42 (q, J= 7.0 Ήz, 4H), 3.15 - 3.07 (m, 4H), 2.62 (t , J= 6.9 Hz, 2H), 2.49 (d, 7.2 Hz, 1H), 2.31 (s, 3H), 1.12 (t, J= 7.0 Hz, 6H).

[00374] l-(2-(lH-imidazol-5-yl)ethyl)-3-(2-(7-(diethylamino)-4-methy I-2-oxo-2H- chromoi-3-yl)ethyl)urea (281). The title compound was prepared analogously to 98, using 2- ( 1 H-imidazol- 5 -y 1) ethan- 1 -amine. LC-MS [ESI, M+l]: 412.2.

[00375] l-((6-chloropyridin-2-yl)mediyl)-3-(2-(7-(dietiiylamino)-4-m ethyl-2-oxo-2H- chromen-3-yl)ethyl)urea (282). The title compound was prepared analogously to 98, using (6- chloropyridin-2-yl)methanamine. LC-MS [ESI, M+l]: 443.6. ¹ H NMR (600 MHz, DMSO-dls) d 7.75 (t, J= 7.7 Hz, 1H), 7.52 (d, J= 9.1 Hz, 1H), 7.37 - 7.31 (m, 1H), 7.22 (dd, J= 7.6, 0.8 Hz, 1H), 6.70 (dd, J= 9.1, 2.6 Hz, 1H), 6.50 (d, J = 2.6 Hz, 1H), 6.49 (s, 1H), 6.23 (s, 2H), 4.24 (s, 2H), 3.42 (q, ./= 7.0 Hz, 4H), 3.15 (t, J= 6.9 Hz, 2H), 2.64 (t, J= 6.9 Hz; 2H), 2.32 (s, 3H), 1.12 (t, J= 7.0 Hz, 6H).

[00376] l-([l,V-biphenyl]-4-ylmethyl)-3-(2-(7-(diethylammo)-4-methyl -2-oxo-2H- chromen-3-yl)ethyl)urea (283). The title compound was prepared analogously to 98, using [1,1 '-biphenyl]-4-ylmethanamine. LC-MS [ESI, M+l]: 484.3. NMR (600 MHz, DMSO-de) d 7.66 - 7.60 (m, 2H), 7.58 - 7.50 (m, 3H), 7.50 - 7.42 (m, 2H), 7.39 - 7.32 (m, 1H), 7.31 - 7.24 (m, 2H), 6.69 (dd, J= 9.1, 2.6 Hz, 1H), 6.49 (d, J = 2.6 Hz, 1H), 6.42 (s, 1H), 6.05 (s, 1H), 4.22 (s, 2H), 3.41 (q, J= 7.0 Hz, 4H), 3.18 (t, J= 6.9 Hz, 2H), 2.66 (t , J = 6.8 Hz, 2H), 2.34 (s, 3H), 1.10 (t, J= 7.0 Hz, 6H).

[00377] l-([l,l'-biphenyl]-3-ylmethyl)-3-(2-(7-(diethylamino)-4-meth yl-2-oxo-2H- chromen-3-yl)ethyl)urea (284). The title compound was prepared analogously to 98, using [1,1 '-biphenyl]-3-ylmethanamine. LC-MS [ESI, M+l]: 484.3. ¹ H NMR (600 MHz, DMSO-de) d 7.67 - 7.60 (m, 2H), 7.57 - 7.42 (m, 5H), 7.42 - 7.34 (m, 2H), 7.22 (dt, J= 7.6, 1.4 Hz, 1H), 6.48 (d, J= 2.5 Hz, 1H), 6.40 (s, 1H), 6.06 (s, 1H), 4.26 (s, 2H), 3.42 (q, J= 7.0 Hz, 4H), 3.16 (t, J= 6.9 Hz, 2H), 2.65 (t, J= 6.9 Hz, 2H), 2.54 (s, 1H), 2.28 (s, 3H), 1.12 (t, J= 7.0 Hz, 6H).

[00378] l-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl) -3- (naphthalen- l-ylmethyl)urea (285). The title compound was prepared analogously to 98, using naphthalen-1 -ylmethanamine. LC-MS [ESI, M+l]: 458.3. ^X H NMR (600 MHz, DMSO-tfc) d 8.12 - 8.04 (m, 1H), 7.98 - 7.90 (m, 1H), 7.82 (d, J= 8.1 Hz, 1H), 7.58 - 7.50 (m, 2H), 7.54 - 7.47 (m, 1H), 7.47 - 7.36 (m, 2H), 6.49 (d, J= 2.6 Hz, 1H), 6.35 (s, 1H), 6.01 (s, 1H), 4.65 (s, 2H), 3.42 (q, J= 7.0 Hz, 4H), 3.18 (t, J= 6.9 Hz, 2H), 2.66 (t, J= 6.9 H z, 2H), 2.53 (s, 1H), 2.31 (s, 3H), 1.12 (t, J= 7.0 Hz, 6H).

[00379] l-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)ethyl) -3-((6-oxo-l,6- dihydropyridin-3-yl)methyl)urea (286). The title compound was prepared analogously to 98, using 5-(aminomethyl)pyridin-2(lH)-one. LC-MS [ESI, M+l]: 425.2. ¹ H NMR (600 MHz, DMSOnifc) d 7.51 (d, J= 9.0 Hz, 1H), 7.36 (dd, J= 9.4, 2.6 Hz, 1H), 7.21 (d, J= 2.6 Hz, 1H), 6.69 (dd, J= 9.1, 2.6 Hz, 1H), 6.49 (d, J= 2.6 Hz, 1H), 6.31 (d, J= 9.4 Hz, 1H), 6.21 (s, 1H), 6.02 (s, 1H), 3.90 (s, 2H), 3.42 (q, J= 7.0 Hz, 4H), 3.13 (dt, J= 13.8, 5.0 Hz, 2H), 2.63 (t, J= 6.9 Hz, 2H), 2.30 (s, 3H), 1.27 (dd, J= 9.2, 6.7 Hz, 2H), 1.12 (t, J= 7.0 Hz, 6H). [00380] 1-(2-(7-(ΰίbί1^ΐ8ΐhΐho)-4-ihbί1^2-oco-2H-<LGqpibh- 3^1)bί1^1)-3-^hίho1ίh-4- ylmethyl)urea (287). The title compound was prepared analogously to 98, using quinolin-4- ylmethanamine. LC-MS [ESI, M+l]: 459.3. ¹ H NMR (600 MHz, DMSO-ifc) d 11.07 (s, 1H), 8.50 (s, 1H), 8.32 (s, 1H), 8.12 (dd, J= 10.3, 4.8 Hz, 1H), 8.01 (d, ./= 8.7 Hz, 1H), 7.85 (tt, J = 16.9, 7.3 Hz, 1H), 7.76 - 7.69 (m, 1H), 7.60 - 7.49 (m, 1H), 6.69 (s, 1H), 6.54 - 6.47 (m, lH), 3.62 (ddt, J= 11.1, 7.4, 4.2 Hz, 1H), 3.49 - 3.38 (m, 4H), 3.15 (qd, J= 7.5, 4.3 Hz, 1H), 2.83 (d, J= 6.7 Hz, lH), 2.44 - 2.37 (m, 2H), 1.12 (p, 7= 6.3, 5.7 Hz, 5H).

[00381] l-(2-(7-(diethylamino)- 4-methyl· 2-oxo-2H-chromen-3-yl)ethyl)-3-(quinolin-3- ylmethyl)urea (288). The title compound was prepared analogously to 98, using quinolin-3- ylmethanamine. LC-MS [ESI, M+l]: 459.3.

[00382] l-(2-(7-(diethylamino)- 4-methyl· 2-oxo-2H-chromen-3-yl)ethyl)- 3- (isoquinolin-4-ylmethyl)urea (289). The title compound was prepared analogously to 98, using isoquinolin-4-ylmethanamine. LC-MS [ESI, M+l]: 459.3. ¹ H NMR (600 MHz, DMSO-t/e) d 8.48 (d, J= 8.2 Hz, 1H), 8.45 (s, 1H), 8.40 (d, J= 8.5 Hz, 1H), 8.15 (ddd, J= 8.4, 6.9, 1.3 Hz, 1H), 7.98 (ddd, J= 8.1, 6.9, 1.0 Hz, 1H), 7.46 (d, J= 9.1 Hz, 1H), 6.68 (dd, J= 9.1, 2.6 Hz, 1H), 6.64 (s, 1H), 6.47 (d, J= 2.6 Hz, 1H), 6.25 (s, 1H), 4.76 (d, J= 5.3 Hz, 2H), 3.42 (q, J= 7.0 Hz, 4H), 2.65 (t , J= 6.9 Hz, 2H), 2.27 (s, 3H), 1.12 (t, J= 7.0 Hz, 6H).

[00383] 1-(2-(7-(ΰίbί1^ΐ8ΐhΐho)-4-iheί1^2-oco-2H-ί1ii·oihbh -3^1)bί1^1)-3-

(isoquinolin-l-ylmethyl)urea (290). The title compound was prepared analogously to 98, using isoquinolin-l-ylmethanamine. LC-MS [ESI, M+l]: 459.3.

[00384] l-(3-(lH-imidazol-4-yl)benzyl)- 3-(2-(7-(diethyi amino)- 4-methyi-2-oxo-2H- chromen-3-yl)ethyl)urea (291). The title compound was prepared analogously to 98, using (3- (lH-imidazol-4-yl)phenyl)methanamine. LC-MS [ESI, M+l]: 474.3. ^! H NMR (600 MHz, DMSO-dis) d 7.90 - 7.80 (m, 3H), 7.70 (d, J= 1.0 Hz, 1H), 7.53 - 7.43 (m, 3H), 7.38 (dd, J= 8.5, 1.8 Hz, 1H), 6.69 (dd, J= 9.1, 2.6 Hz, 1H), 6.49 (d, J= 2.6 Hz, 1H), 6.44 (s, 1H), 6.08 (s, 1H), 5.76 (s, 2H), 4.36 (s, 2H), 3.42 (q, J= 7.0 Hz, 4H), 3.18 (t, J= 6.8 Hz, 2H), 2.67 (t, J= 6.9 Hz, 2H), 2.33 (s, 3H), 1.12 (t, J= 7.0 Hz, 6H).

[00385] l-(2-(7-(diethylamino)-4-mefllyl·2-oxo-2H-chromal-3-yl)efll yl)-3- (naphthalen-2-ylmethyl)urea (292). The title compound was prepared analogously to 98, using naphthalen-2-ylmethanamine. LC-MS [ESI, M+l]: 474.3. ^! H NMR (600 MHz, DMSO-tfc) d 7.90 - 7.80 (m, 3H), 7.70 (s, 1H), 7.53 - 7.43 (m, 3H), 7.38 (dd, ./= 8.4, 1.8 Hz, 1H), 6.69 (dd, J = 9.1, 2.6 Hz, 1H), 6.49 (d, J= 2.6 Hz, 1H), 6.44 (s, 1H), 6.08 (s, 1H), 4.36 (s, 2H), 3.42 (q, J = 7.0 Hz, 4H), 3.18 (t, J= 6.9 Hz, 2H), 2.67 (t, J = 6.9 Hz, 2H), 2.33 (s, 3H), 1.12 (t, J = 7.0 Hz,

6H).

[00386] l-(2-(7-(dietiiylamino)-4-metiiyl-2-oxo-2H-cliromen-3-yl)eth yl)-3-(quinolm-2- ylmethyl)urea (293). The title compound was prepared analogously to 98, using quinolin-2- ylmethanamine. LC-MS [ESI, M+l]: 459.3.

[00387] l-(2-(7-(diethylammo)-4-methyl-2-oxo-2H-chromen-3-yI)ethyl)- 3-(pyrimidin- 4-ylmetfayl)urea (294). The title compound was prepared analogously to 98, using pyrimidin-4- ylmethanamine. LC-MS [ESI, M+l]: 410.2.

[00388] l-(2-(7-(diethylamino)- 4-methyl- 2-oxo-2H-chromen-3-yl)ethyl)- 3- (isoquinolin-3-ylmethyl)urea (295). The title compound was prepared analogously to 98, using isoquinolin-3-ylmethanamine. LC-MS [ESI, M+l]: 459.3.

[00389] l-(2-(7-(diethylamino)-4-methyI-2-oxo-2H-chromai-3-yl)etiiyl )-3-((l-oxo-l,2- dihydroisoquinolin-6-yl)methyl)urea (296). The title compound was prepared analogously to 98, using 6-(aminomethyl)isoquinolin-l(2H)-one. LC-MS [ESI, M+l]: 475.3.

[00390] 3-((3-(2-(7-(diethylamlno)-4-methyl-2-oxo-2H-dirom«i-3- yl)ethyl)ureido)methyl)-5-fluorobenzoic acid (297). The title compound was prepared analogously to 98, using 3-(aminomethyl)-5-fluorobenzoic acid. LC-MS [ESI, M+l]: 470.2.

[00391] 3-((3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3- yl)ethyl)ureido)methyl)benzamide (298). The title compound was prepared analogously to 98, using 3-(aminomethyl)benzamide. LC-MS [ESI, M+l]: 451.2.

[00392] 3-((3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-dirom«i-3- yl)ethyl)ureido)methyl)benzenesulfonamide (299). The title compound was prepared analogously to 98, using 3-(aminomethyl)benzenesulfonamide. LC-MS [ESI, M+l]: 487.3. [00393] Scheme 10.

OTBS .OTBS

HMPT, LDA,

THF

/Ni

/I J

1 Int 10-1

[00394] 4-(3-((tert-butyldimethylsilyl)oxy)propyl)-7-(diethylamino)- 2H-chromen-2- one (Int 10-1). To solution of LDA (2 M in hexanes, 32.4 mL, 1.50 eq.) in THF (50 mL) was added N-[bis(dimethylamino)phosphoryl]-N-methyl-methanamine (11.6 g, 64.9 mmol, 11.4 mL, 1.50 eq.) at 0 °C. After stirring at 0 °C for 5 mins, a solution of 7-(diethylamino)-4-methyl- chromen-2-one (10.0 g, 43.2 mmol, 1.00 eq.) in THF (50 mL) was added slowly over 10 mins at 0 °C. The resultant mixture was allowed to stir at 0 °C for 45 mins, and thereto was added a solution of tert-butyl-(2-iodoethoxy)-dimethyl-silane (12.4 g, 43.3 mmol, 1.00 eq.) in THF (50 mL) slowly at 0 °C. The temperature was maintained for another 30 mins, then the mixture was warmed up to 30 °C slowly. After completion the mixture was quenched with saturated sodium bicarbonate (20 mL) and diluted with ethyl acetate (50 mL). The separated organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to give a residue.

The mixture was purified by column chromatography over silica gel (petroleum ether/ethyl acetate = 50/1 to 3/1). The title intermediate Int 10-1 (5.00 g, 12.8 mmol, 30% yield) was obtained as a yellow gum. ^X H NMR (400 MHz, chloroform-d) 5= 7.46 (d, J = 8.8 Hz, 1H), 6.58 (dd, J = 2.4, 8.8 Hz, 1H), 6.50 (d, J = 2.4 Hz, 1H), 5.94 (s, 1H), 3.71 (br t, J = 5.6 Hz, 2H), 3.43 (q, J = 7.2 Hz, 4H), 2.80 - 2.70 (m, 2H), 1.90 - 1.82 (m, 2H), 1.20 (t, J = 7.2 Hz, 6H), 0.92 (s, 9H), 0.07 (s, 6H).

[00395] 7-(diethylamino)-4-(3-hydroxypropyl)-2H-chromen-2-one (Int 10-2). To a solution of 4-[3-[tert-butyl(dimethyl)silyl]oxypropyl] -7-(diethylamino)chromen-2-one (5.00 g, 12.8 mmol, 4.00 eq.) in THF (100 mL) was added TBAF (1 M, 19.3 mL, 6.00 eq.). The reaction mixture was stirred at 30 °C for 2 hrs. The reaction mixture was diluted with water (200 mL) and extracted with ethyl acetate (200 mL x 3). The combined organic layers were washed with brine (100 mL x 2), dried ova- anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography over silica gel (petroleum ether/ethyl acetate = 10/1 to 1/1). Compound 7-(diethylamino)-4-(3- hydroxypropyl)chromen-2-one (0.27 g, 976 mihoΐ, 30% yield, 99% purity) was obtained as a yellow oil. LC-MS [ESI, M+l]: 276.1. ¹ H NMR (400 MHz, chloroform-d) d = 7.46 (d, J = 9.2 Hz, 1H), 6.60 (dd, J = 2.4, 9.2 Hz, 1H), 6.50 (d, J = 2.8 Hz, 1H), 6.0 (s, 1H), 3.78 (br t, J = 5.6 Hz, 2H), 3.40 (q, J = 7.2 Hz, 4H), 2.84 - 2.78 (m, 2H), 1.98 - 1.88 (m, 2H), 1.63 - 1.57 (m, 1H), 1.22 (t, J = 7.2 Hz, 6H).

[00396] 3-(7-(diethylammo)-2-oxo-2H-chiOmen-4-yl)propyl (4-nitrophenyl) carbonate (Int 10-3). The title intermediate was prepared analogously to Int 5-1, using Int 10- 2, to afford an orange solid. LC-MS [ESI, M+l]: 441.2. ¹ H NMR (600 MHz, DMSO^e) d 8.36 - 8.29 (m, 2H), 7.60 - 7.52 (m, 3H), 6.69 (dd, J= 9.0, 2.6 Hz, 1H), 6.52 (d, J= 2.6 Hz, 1H), 5.97 (d, J= 0.9 Hz, 1H), 4.35 (t, J= 6.4 Hz, 2H), 3.43 (q, J= 7.0 Hz, 4H), 2.86 - 2.79 (m, 2H), 2.11 - 2.00 (m, 2H), 1.12 (t, J= 7.0 Hz, 6H).

[00397] 3-(7-(diethylamino)-2-oxo-2H-chromen-4-yl)propyl benzyl carbamate (300). The title compound was prepared analogously to 78, using Int 10-3 and benzylamine as starting materials. LC-MS [ESI, M+l]: 409.3. ¹ H NMR (600 MHz, DMSO-ifc) d 7.74 (t, J= 6.2 Hz, 1H), 7.53 (d, J= 9.0 Hz, 1H), 7.32 (t, J= 7.6 Hz, 2H), 7.29 - 7.20 (m, 3H), 6.67 (dd, J= 9.1, 2.6 Hz, 1H), 6.52 (d, J= 2.5 Hz, 1H), 5.92 (s, 1H), 4.20 (d, J= 6.2 Hz, 2H), 4.05 (t, J= 6.4 Hz, 2H), 3.43 (q, J= 7.0 Hz, 4H), 2.76 (t, J= 7.7 Hz, 2H), 1.90 (t, J= 7.6 Hz, 2H), 1.13 (t, J= 7.0 Hz,

6H).

[00398] 3-(7-(diethylammo)-2-oxo-2H-chromen-4-yl)pn>pyl (pyridin-4- ylmethyl)carbamate (301). The title compound was prepared analogously to 78, using Int 10-3 and pyridin-4-ylmethanamine as starting materials. LC-MS [ESI, M+l]: 410.2. ¹ H NMR (600 MHz, DMSO-Je) 6 8.76 (s, 2H), 8.27 (d, J= 8.1 Hz, 1H), 7.92 (t, ./= 6.0 Hz, 1H), 7.53 (d, J= 9.0 Hz, 1H), 6.68 (dd, J= 9.0, 2.6 Hz, 1H), 6.52 (d, J= 2.5 Hz, 1H), 5.91 (s, 1H), 4.37 (d, .7= 6.1 Hz, 2H), 4.06 (t, J= 6.5 Hz, 2H), 3.43 (q, J= 7.0 Hz, 4H), 2.75 (dd, J= 14.1, 6.5 H z, 2H), 1.91 (p, .7= 6.7 Hz, 2H), 1.13 (t, J= 7.0 Hz, 6H). [00399] 3-(7-(diethylamino)-2-oxo-2H-chromen-4-yl)propyl (3- carbamoylbenzyl)carbamate (302). The title compound was prepared analogously to 78, using Int 10-3 and 3-(aminomethyl)benzamide as starting materials. LC-MS [ESI, M+l]: 452.2. ^X H NMR (600 MHz, DMSO-7 ₆ ) 6 7.95 (s, 1H), 7.82 - 7.71 (m, 3H), 7.53 (d, J= 9.0 Hz, 1H), 7.46 - 7.36 (m, 2H), 7.34 (s, 1H), 6.67 (dd, J= 9.1, 2.6 Hz, 1H), 6.52 (d, J= 2.6 Hz, 1H), 5.92 (s, 1H), 4.24 (d, J= 6.2 Hz, 2H), 4.06 (t, J= 6.4 Hz, 2H), 3.43 (q , J= 7.0 Hz, 4H), 2.75 (q, J= 6.9, 6.1 Hz, 2H), 1.90 (p, J= 6.7 Hz, 2H), 1.12 (t, J= 7.0 Hz, 6H).

[00400] 3-(7-(diethylammo)-2-oxo-2H-chromen-4-yl)propyl (3- sulfamoylbenzyl)carbamate (303). The title compound was prepared analogously to 78, using Iht 10-3 and 3-(aminomethyl)benzenesulfonamide as starting materials. LC-MS [ESI, M+l]: 488.3. ’H NMR (600 MHz, DMSO^fc) d 7.86 (t, J= 6.2 Hz, 1H), 7.77 - 7.68 (m, 2H), 7.58 - 7.47 (m, 2H), 7.48 (dd, J = 7.8, 1.5 Hz, 1H), 7.36 (s, 2H), 6.69 (dd, 7 = 9.0, 2.5 Hz, 1H), 6.52 (d, J= 2.5 Hz, 1H), 5.92 (s, 1H), 4.27 (d, J= 6.2 Hz, 2H), 4.06 (t, J= 6.5 Hz, 2H), 3.43 (q, J= 7.0 Hz, 4H), 2.76 (q, J= 7.5 Hz, 2H), 1.91 (p, J= 6.7 Hz, 2H), 1.13 (t, J= 7.0 Hz, 6H).

[00401] 3-(7-(diethylamino)-2-oxo-2H-chiOmen-4-yl)propyl (3-(lH-imidazol-4- yl)benzyl)carbamate (304). The title compound was prepared analogously to 78, using Int 10-3 and (3-(lH-imidazol-4-yl)phenyl)methanamine as starting materials. LC-MS [ESI, M+l]: 475.3. ¹ H NMR (600 MHz, DMSO-ί/b) d 9.20 (s, 1H), 8.14 (s, 1H), 7.80 (t, J= 6.1 Hz, 1H), 7.74 - 7.66 (m, 2H), 7.57 - 7.44 (m, 2H), 7.39 - 7.32 (m, 1H), 6.66 (dd, J= 9.1, 2.6 Hz, 1H), 6.52 (d, J= 2.6 Hz, 1H), 5.92 (s, 1H), 4.76 (s, 1H), 4.27 (d, J= 6.1 Hz, 2H), 4.06 (t, J= 6.5 Hz, 2H), 3.42 (q, J= 7.1 Hz, 4H), 2.76 (dd, J= 16.2, 8.5 Hz, 2H), 1.91 (p, J= 6.6 Hz, 2H), 1.12 (t, J= 7.0 Hz, 6H).

[00402] 3-(3-((((3-(7-(diethyIamino)-2-oxo-2H-chromen-4- yl)propoxy)carbonyl)amino)methyl)phenyl)propanoic acid (305). The title compound was prepared analogously to 78, using Int 10-3 and 3-(3-(aminomethyl)phenyl)propanoic acid as starting materials. LC-MS [ESI, M+l]: 481.3. ‘H NMR (600 MHz, DMSO-t/e) d 7.70 (t, J= 5.8 Hz, 1H), 7.54 (d, J= 9.0 Hz, 1H), 7.23 (t, J= 7.6 Hz, 1H), 7.15 - 7.06 (m, 3H), 6.67 (dd, J= 9.0, 2.6 Hz, 1H), 6.52 (d, J= 2.5 Hz, 1H), 5.92 (s, 1H), 4.17 (d, J= 6.2 Hz, 2H), 4.05 (t, J= 6.4 Hz, 2H), 3.43 (q, J= 7.0 Hz, 5H), 2.78 (dt, J= 24.4, 7.8 Hz, 4H), 1.90 (p, J= 6.7 Hz, 2H), 1.33 - 1.22 (m, 1H), 1.13 (t, J= 7.0 Hz, 6H). [00403] Methyl 3-(3-((((3-(7-(dietiiylamino)-2-oxo-2H-dirOmen- 4yl)propoxy)carbonyl)amino)methyl)phenyl)propanoate (306). The title compound was prepared analogously to 78, using Int 10-3 and methyl 3-(3-(aminomethyl)phenyl)propanoate as starting materials. LC-MS [ESI, M+l]: 495.3.

[00404] 3-(3-((3-(2-(7-(diethylamino)-4-methyI-2-oxo-2H-chrOmen-3- yl)ethyl)ureido)methyl)phenyl)propanoic acid (307). The title compound was prepared analogously to 98, using 3-(3-(aminomethyl)phenyl)propanoic acid. LC-MS [ESI, M+l]: 480.3.

[00405] Methyl 3-(3-((3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3- yl)ethyl)ureido)methyl)phenyl)propanoate (308). The title compound was prepared analogously to 98, using methyl 3-(3-(aminomethyl)phenyl)propanoate. LC-MS [ESI, M+l]: 494.3.

[00406] 3-(3-((((2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3- yl)ethoxy)carbonyl)amino)methyl)phenyl)propanoic acid (309). The title compound was prepared analogously to 78, using 3-(3-(aminomethyl)phenyl)propanoic acid. LC-MS [ESI, M+l]: 481.3. ¾ NMR (600 MHz, DMSO^fc) d 7.73 (t, J= 6.2 Hz, 1H), 7.54 (d, J= 9.1 Hz, 1H), 7.23 (t, J= 7.9 Hz, 1H), 6.82 (t, J= 2.0 Hz, 1H), 6.77 (dd, J= 8.2, 2.6 Hz, 1H), 6.68 (dd, J = 9.1, 2.6 Hz, 1H), 6.52 (d, J= 2.6 Hz, 1H), 4.64 (s, 2H), 4.17 (d, J= 6.2 Hz, 2H), 3.43 (q, J= 7.0 Hz, 5H), 2.80 -2.71 (m, 2H), 1.13 (t, J= 7.0 Hz, 6H).

[00407] Methyl 3-(3-((((2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeii-3- yl)ethoxy)carbonyl)amino)methyl)phenyl)propanoate (310). The title compound was prepared analogously to 78, using methyl 3-(3-(aminomethyl)phenyl)propanoate. LC-MS [ESI, M+l]: 495.3.

[00408] 2-(3-((3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3- yl)ethyl)ureido)methyl)phenoxy)acetic acid (311). The title compound was prepared analogously to 98, using 2-(3-(aminomethyl)phenoxy)acetic acid. LC-MS [ESI, M+l]: 482.3. ¹ H NMR (600 MHz, DMSCMi) d 7.52 (d, J= 9.1 Hz, 1H), 7.19 (t, J= 7.8 Hz, 1H), 6.84 - 6.72 (m, 3H), 6.69 (dd, J= 9.1, 2.6 Hz, 1H), 6.49 (d, J= 2.6 Ήz, 1H), 6.32 (s, 1H), 6.04 (s, 1H), 4.64 (s, 2H), 3.42 (q, J= 7.0 Hz, 4H), 3.15 (t, J= 6.8 Hz, 2H), 2.65 (t , J= 6.9 Hz, 2H), 2.32 (s, 3H), 1.12 (t, J= 7.0 Hz, 6H). [00409] Methyl 2-(3-((3-(2-(7-(diethylamino)-4-methyl-2-oxo-2H-chromeni-3- yl)ethyl)ureido)methyl)phenoxy)acetate (312). The title compound was prepared analogously to 98, using methyl 2-(3-(aminomethyl)phenoxy)acetate. LC-MS [ESI, M+l]: 496.3.

[00410] General Biological Methods and Materials

[00411] Recombinant proteins were expressed in E. coli and purified as previously described (4). WIL-FL K83C and JTO-FL K83C were conjugated with fluorescein maleimide (Vector Laboratories) and the labeled proteins, WIL-FL* and JTO-FL*, respectively, purified by ion exchange chromatography. From 400 mL of bacterial culture, we obtained approximately 40 mg (1.7 pmol monomer equivalent) of pure, natively folded WIL-FL K79C. 500 nmol of this LC was reacted with 2 equivalents of fluorescein maleimide, yielding 420 nmol of WIL-FL* after repurification. 75 nmol of JTO-FL* was produced at similar overall yield. The screening campaign required 223 nmol of WIL-FL* and 40 nmol of JTO-FL*. Proteolysis assays were generally carried out by incubating LC (10 mM for unlabeled LCs, 20 nM for fluorescein-labeled LCs) with proteinase K (Thermo Fisher, 200 nM), in PBS at 37 °C or ambient temperature (22 °C) for screening. FP was measured using either a Jasco 8600 fluorimeter (Xex = 488 nm, Xe _m = 520 nm) or Perkin Elmer EnVision plate reader equipped with polarizing excitation and emission filters (lbc = 485 ± 20 nm, Xe _m = 535 ± 20 nm) and a 505 nm dichroic mirror. FP values were calculated using the formula:

FS - FP

P = G

FS + FP

[00412] P is the polarization, FS and FP are the emission intensities of parallel and perpendicular-polarized light, respectively, and G is a dye- and instrument-specific correction factor. Values are reported as mP. For the high-throughput screen, 653,085 small molecules from the Scripps Florida screening library were assayed in 1536-well plates. Final LC concentration was 10 nM in 5 mΐ. Small molecules were added by pintool from DMSO stock plates to a final concentration of 6.8 mM compound and 0.68% DMSO. Fluorogenic binding of 1 to LCs was measured using a Molecular Devices Gemini platereader (Xex = 373 nm, Xem = 480 nm). Crystals of JTO-FL were grown via sitting-drop vapor diffusion using a crystallization buffer consisting of 20% PEG 3350 and 0.2 M NH4H2PO4 at 23 °C. To generate JTO-FL·! complexes, crystals of apo JTO-FL were soaked with a tenfold molar excess of 1 for 10 days. Diffraction data were collected at 80 K and a wavelength of 1.0332 A at beamline 23-ID-D (for apo JTO-FL) or 23- ID-B (for JTO-FL·!) at the Advanced Photon Source (Argonne, IL). The refined models were deposited in the Protein Data Bank under accession codes 6MG4 for apo JTO-FL and 6MG5 for JTO-FL·!. All samples for NMR were buffered in 50 mM Bis Tris, pH 6.4, 1 mM EDTA, 10% D2O. 'H, ¹⁵ N experiments were recorded at 37 °C on a 14.1 T Broker AVANCE III HD spectrometer equipped with a cryogenically cooled x,y,z gradient probe. SV-AUC, unfolding and aggregation experiments were performed as previously described (4).

[00413] Protein expression and purification. Full-length LCs were expressed as inclusion bodies in E. coli as previously described (1). Briefly, inclusion bodies were isolated from cells by five rounds of sonication and centrifugation; dissolved in 4 M GuHCl containing 5 mM DTT; refolded by dropwise dilution into Tris-Cl at pH 8.5 containing 5 mM reduced glutathione and 0.5 mM oxidized glutathione; and purified by ammonium sulfate precipitation followed by ion exchange and size exclusion chromatography. LC V-domains were expressed in the periplasm of E. coli using a pelB leader sequence; isolated by periplasmic shock; and purified by ammonium sulfate precipitation followed by ion exchange and size exclusion

chromatography.

[00414] ALMC2 LC was purified after secretion from the ALMC2 plasma cell line (2). ALMC2 cells were grown in Iscove's modified Dulbecco's media (IMDM) containing 5% fetal bovine serum (FBS), 292 pg/ml glutamine, 100 U/ml penicillin, 100 gg/ml streptomycin and 2 ng/ml nM interleukin-6 (Thermo). Approximately 450,000,000 cells (300 ml of culture) were pelleted and resuspended in 300 ml of IMDM lacking FBS and phenol red indicator, but containing glutamine, penicillin, streptomycin and interleukin-6. C ₆ lls were allowed to secrete protein for 24 h, after which cells were removed by centrifugation and filtratioa Media containing ALMC2 LC was dialyzed overnight against 25 mM Tris-Cl, pH 8. LC was concentrated by ion exchange chromatography using a FastflowQ column (GE). ALMC IgG was removed by passing the eluate through a protein A column (GE), and the LC was dialyzed and then purified by ion exchange chromatography using a MonoQ column (GE).

[00415] 5 J8 Fab was expressed in HEK293F suspension cells and purified by Ni-NTA and ion exchange chromatography as previously described (3). [00416] Protein labeling. Surface-exposed cysteine residues were incorporated into LCs by mutation using the Novagen QuikChange or NEB SDM mutagenesis protocols according to the manufacturers’ instructions. LCs (20-50 mM) were expressed as described above and conjugated with fluorescein by reaction with equimolar fluorescein maleimide (Vector

Laboratories) for 1 h at room temperature (22 °C). Unreacted fluorescein was removed by ion exchange chromatography. Labeling efficiency was determined to be 50% by absorbance spectroscopy, corresponding to a single fluorescein molecule per LC dimer. LC misfolding (4) or modification of K83C LCs by glutathione upon refolding may account for some of the heterogeneity in the conjugated protein prodcuct. Experiments (Fig. 1) showed that proteolysis of the LCs resulted in a sufficiently large change in fluorescence polarization, so we did not attempt to optimize the labeling and purification further.

[00417] Proteolysis assays. Unless otherwise described, LCs were incubated with proteinase K (100 nM; Thermo) in phosphate buffered saline (PBS, 10 mM Na ₂ HPC> ₄ , 1.8 mM KH2PO4, 137 mM NaCl, and 2.7 mM KC1, pH 7.4), after which the reaction was quenched with phenylmethyl sulfonyl fluoride (100 mM) and remaining LC quantified by SDS-PAGE or fluorescence polarization. The experiment in Fig. lb measured simultaneous proteolysis of labeled and unlabeled LC by adding 20 nM WTL-FL* or JTO-FL* to 5 mM WIL-FL or JTO-FL, respectively. Fluorescein fluorescence images of the SDS-PAGE gels were recorded, after which the gels were Coomassie stained and imaged. Gel images shown have had their contrast increased after quantification for clarity.

[00418] Fluorescence polarization measurements. FP in cuvettes was measured in a Jasco 8600 fluorimeter fitted with polarizing filters and excitation and emission

monochromators, using an excitation wavelength of 488 ran and emission wavelength of 520 ran. FP on plates was measured using a Perkin Elmer EnVision plate reader equipped with polarizing excitation and emission filters (l» = 485±20 nm, ¾e _m = 535±20 run) and a 505 nm dichroic mirror. Fluorescein-labeled LC (20 nM) in PBS containing 0.02% (v/v) Pluronic F-127 detergent (Thermo) was used for all measurements. Fluorescence polarization was calculated using the formula: [00419] P is the polarization, FS and FP are the emission intensities of parallel and perpendicular-polarized light, respectively, and G is a dye- and instrument-specific correction factor. Total fluorescence was calculated using the formula:

TF = FS + 2FP

[00420] Addition of detergent appears to prevent denaturation of the LC upon binding to the polystyrene surface of the plate, since it did not affect the kinetics of proteolysis in quartz cuvettes.

[00421] Pilot screen. For screening in a plate format, each compound’s effect on the PK proteolysis of WIL-FL* was measured by FP after 24 h incubation at room temperature

(typically 22 °C). The measured FP was normalized plate wise by comparing the compound wells to high and low control wells on each plate (n=32). The high FP control wells contained JTO- FL* and DMSO vehicle without proteinase K. Low FP control wells contained WIL-FL*,

DMSO vehicle and proteinase K, to determine the rate of proteolysis of unliganded WIL-FL*. Percent activity was calculated for each plate using the formula:

Test well— median (data wells)

Percent activity = 100 x

Median(high control)— median(data wells)

[00422] To screen the commercial library, WIL-FL* and JTO-FL* in PBS, pH 7.4, containing 0.02% Pluronic F-127 detergent were dispensed into black polystyrene microplates (Greiner catalog # 788076) using a Beckman Coulter BioRAPTR. Final LC concentration was 20 nM in 10 mΐ, equivalent to 10 nM fluorescein. Compounds (n=l) were added by 50 nl pintool from 10 mM DMSO stock solutions using a Beckman Coulter BioMek robot for a final concentration of 10 mM compound and 0.5% DMSO. Plates were incubated at room temperature (22 °C) for 10 min to allow compounds to dissolve, after which proteinase K was dispensed to a final concentration of 500 nM Plates were centrifuged at 1000 rpm for 1 minute, stacked, wrapped to minimize evaporation, and incubated for 24 h at 22 °C. FP was measured as described above. We used hits from the commercial library screen (compounds 17 and 18,) to validate the assay for use on 1,536-well plates.

[00423] Primary screen. 653,085 small molecules from the Scripps Florida screening library were screened using the PCFP assay in 1,536-well plates. Final LC concentration was 10 nM in 5 mΐ. Small molecules were added by pintool from DMSO stock plates to a final concentration of 6.75 mM compound and 0.675% DMSO. Activity relative to the high control (JTO-FL* with no protease) and low control (WIL-FL* with DMSO vehicle) was calculated using equation 3, above. 2,779 small molecules were selected as hits based on an activity greater than three standard deviations above the mean activity of the plate. This result corresponds to an initial hit rate of 0.4%. Assay performance was consistent with a platewise average Z’ of 0.75 ± 0.03 and an average signal-to-background ratio of 1.36 ± 0.02 (mean ± sd, n=526 plates). Two compounds were unavailable for further study, so 2,777 small molecules were replated at 10 mM in DMSO to create a stock plate for secondary screens.

[00424] Secondary screens. We used three rounds of secondary screening to remove false positive hits from the primary screen. The 2,777 small molecules identified in the primary screen were used for each secondary screen.

[00425] Firstly, the PCFP assay was repeated in triplicate with each of the 2,777 primary screening hits, with an average Z’ of 0.71 ± 0.03. We identified 1,422 molecules with >20.6% activity (the mean plus three standard deviations of this dataset). Note that many compounds show apparent protection greater than the JTO-FL* positive control, which we attribute to autofluorescence of the small molecule affecting the FP measurement.

[00426] Secondly, to remove compounds that interfere with the FP measurement, the primary screen was repeated in triplicate but compounds were added after 24 h of proteinase K treatment (Fig. If). This assay had an average Z’ of 0.73 ± 0.01. Bona fide hits should retain no FP beyond the low control. Six hundred sixty two small molecules that showed 1.25-fold lower mean activity in this assay compared to the secondary screen described in the previous paragraph were retained.

[00427] Thirdly, to remove molecules that inhibit proteinase K, the assay was repeated in triplicate using 100 nM thermolysin (Thermo). The assay was carried out in 50 mM Tris-Cl, pH 7.5, containing 150 mM NaCl, 100 mM ZnCh, 100 mM CaCh and 0.02% v/v Pluronic F-127 detergent The additional metal ions were added to bind to ion-chelating small molecules and prevent them from inhibiting the metalloproteinase. This assay yielded an average Z’ of 0.64 ± 0.02. We identified 1,578 compounds that were active in this assay, which we define as >20 % activity (mean plus three standard deviations of this dataset). Of these, 1,243 molecules showed similar activity against PK and thermolysin. [00428] Two hundred sixty nine molecules that passed all three secondary screens were retained for further analysis. We also retained 147 additional molecules that passed the FP artefact secondary screen but failed one of the two other secondary screens assessing protease inhibition (PCFP with PK or with thermolysin).

[00429] The concentration-dependence assay employed the same reagents, protocols, and detection systems as the secondary assays, but tested each of the selected compounds as 10-point dose-response titrations (3-fold dilutions) in triplicate, in each of the three formats described above. A four-parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc.). The reported ECso values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. Small molecules which showed similar concentration dependent FP retention in the PK and thermolysin screens and contrasting activity in the FP artefact counterscreen were retained.

[00430] All compounds used for titration assays were analyzed by LC-MS to confirm purity and identify mass. Of the 416 samples submitted for LC-MS analysis, 386 samples confirmed mass, i.e., the molecular weight of the structure in Scripps’ database matched that identified by LC-MS analysis of the screening sample. As determined by nominal methods (UV- vis spectroscopy, MS and ELSD), 379 samples demonstrated purity of >80%.

[00431] Protease inhibition assay. PK (5 mΐ of 200 nM), or buffer without protease, were dispensed into wells of a microplate. Compounds were added by pintool (n=2) and incubated for

5 min at room temperature (22 °C). Five mΐ of EnzCheck Red fluorogenic protease substrate (Thermo) were dispensed into the wells. Fluorescence (lbc = 580 nm, Xem = 620 ran) was read at 30 sec intervals for 5 min. An increase in fluorescence indicated protease activity. Some compounds exhibit an apparently higher protease activity than vehicle, which we attribute to autofluorescence.

[00432] Equilibrium dialysis. Eight compounds were dialyzed against 20 mM WIL-FL in PBS containing 0.02% Pluronic F-127 detergent in 8 kDa-cutoff Rapid Equilibrium Dialysis cartridges (Thermo) for 4 h according to the manufacturers’ instructions. Small molecule concentrations inside and outside the dialysis cartridge were measured by reverse phase HPLC and dissociation constants calculated using the equation: [LC] x [Ligand _out ]

K _D =

[Ligand _ln ] - [Ligand _out ]

[00433] [LC] is the LC concentration inside the dialysis cartridge, [Ligand _m ] and

[Ligandou _t ] are the measured small molecule concentrations inside and outside the dialysis cartridge, respectively.

[00434] Isothermal Titration Calorimetry (ITC). WIL-FL was dialyzed into PBS, and the dialysate was filtered and used to dilute the LC and small molecules for ITC analysis. DMSO and Pluronic F-127 concentrations were matched as closely as possible between the protein and ligand solutions. Small molecule (100 mM) was titrated into 20 mM LC (monomer equivalent) with a Microcal Auto ITC 200. Heats were analyzed using Origin software (OriginLab

Corporation).

[00435] Fluorogenic coumarin titration. LCs were concentrated to 200 mM in centrifugal concentrators with a 10 kDa membrane, unless the available material was limiting. 1.5-fold serial dilutions of LCs in PBS containing 1 mM 1 and 0.02% (v/v) Pluronic F-127 were prepared in 384-well plates. Fluorescence of 1 was measured on a Gemini plate reader

(Molecular Devices) using an excitation wavelength of 373 nm and emission wavelength of 445 nm. Data were normalized platewise to the plateau fluorescence value for WIL-FL to calculate the fraction of 1 bound. These data were initially fit to a 2-state, 1-site binding equation:

[LC]

fraction bound =

ligand ^ [LC]

[00436] KD _l igan _d is the LC*1 dissociation constant For LCs lacking an inter-chain disulfide bond, 1 binds to the dimer and shifts the monomer-dimer equilibrium towards dimer (Fig. 2e).

To estimate the affinity of the e fit the fluorescence data to a 3-state, 1 -site binding equation:

[LC] ² /KD dimerization

fraction bound =

K D ligand X [LC]2/K _D dimerization

[00437] The dimerization dissociation constant, Kouga _nd , is constrained to 1-10 mM based on NMR affinity measurements (5).

[00438] Analytical Ultracentrifugation (AUC). WIL-FL and WIL-V (20 mM) in PBS containing either 0.5% DMSO vehicle or 100 mM 1 were run in a Beckman XL-1 analytical ultracentrifuge at 50,000 rpm at 20 °C. Sedimentation was measured by absorbance at 280 nm. Data were processed using SedFit (6).

[00439] Crystal structure determination. Purified JTO-FL was concentrated to 200 mM and subjected to crystallization trials using the high-throughput robotic Rigaku CrystalMation platform at The Scripps Research Institute. Crystals of JTO-FL were grown via sitting-drop vapor diffusion using a crystallization buffer consisting of 20% PEG 3350 and 0.2 M NH ₄ H ₂ PO ₄ at 23 °C. Both diamond-shaped and plate-shaped crystals were generated in the same drop using these conditions, but only the plate-shaped crystals produced usable diffraction data. To generate JTO-FL·! complexes, crystals of apo JTO-FL were soaked with a tenfold molar excess of 1 for 10 days. Crystals were harvested and immediately flash cooled in liquid nitrogen.

[00440] Diffraction data were collected from crystals at 80 K and a wavelength of 1.0332 A at beamline 23-ID-D (for apo JTO-FL) or 23-ID-B (for JTO-FL·!) at the Advanced Photon Source (Argonne, IL). Frames were indexed and integrated using HKL-2000 (7) (for apo JTO- FL) or XDS (8) (for JTO-FL·!), the space group was assigned as P2i2i2i using Pointless, and data were scaled in Scala (9). Five percent of reflections (randomly distributed) were flagged for model cross-validation using Rf _h * (10).

[00441] The apo JTO-FL structure was solved to 1.75 A resolution by molecular replacement (MR) with Phaser (11), using the existing full-length CLE light chain structure (12) (PDB: 1LIL) as the search model. The JTO-FL·! structure was similarly solved to 1.8 A

resolution with MR using apo JTO-FL as the search model. Both models were refined with iterative cycles of manual adjustment in Coot (13), and refinement in RefmacS (14) using isotropic thermal parameters and hydrogen atoms at calculated positions. Final adjustments were made after analysis with MolProbity (15). The refined models were deposited in the Protein Data Bank under accession codes 6MG4 for apo JTO-FL and 6MG5 for JTO-FL·!.

[00442] NMR A stock solution of 1 (40 mM) was made by dissolving the ligand into ethanol. All samples were buffered in 50 mM Bis Tris, pH 6.4, 1 mM EDTA, 10% D2O. All in- phase (for ¹⁵ N WIL-V and ¹⁵ N JTO-V) and TROSY (for ¹⁵ N WIL-FL) based ¾ ¹⁵ N experiments were recorded at 37 °C on a 14.1 T Bruker AVANCE IP HD spectrometer equipped with a cryogenically cooled x,y,z gradient probe. [00443] For the titrations, samples with different protein:! ratios were generated starting from two mother solutions, the first without the ligand, the second with a ratio of 1 :2.2 between concentration of monomeric protein and ligand. The concentration of ethanol was identical in the two mother solutions. The concentration of monomeric protein was 0.05, 0.4 and 0.14 mM respectively for WIL-FL, WIL-V and JTO-V. Intensities for 8 LCz peaks (Asn 30, Tyr 32, Trp 35, Gin 38, Gly 41, Phe 50, Gly 101) and 4 LCz*L peaks (Asn 30, Gin 38, Gly 41, Asp 52) were fitted to get an estimate of the KD for WIL-FL.

[00444] ¹⁵ N transverse relaxation rates were calculated from Ri and Rip experiments acquired for JTO-V and WIL-V at 0.4 mM protein concentration with or without 1 mM 1. A ¹⁵ N gBi field of 1.7 kHz was applied during the relaxation delay for Ri _p experiments.

[00445] Chemical exchange saturation transfer (CEST) experiments were acquired on WIL-V samples at 0.4 mM in the presence of 0.2 or 0.3 mM 1 using a multi-site excitation scheme (16). 4.25 ps DANTE pulses with gBi = 6.4 kHz were applied every 9.1 ms during a saturation delay of 0.35 s, giving an effective ¹⁵ N Bi field of 3 Hz and a spacing on 110 Hz between consecutive excitation frequencies. Longitudinal two-spin order ZZ-exchange experiments were acquired on JTO-V samples at 0.14 mM in the presence of 0.06 mM 1, with the 1-50 ms ZZ-mixing delay occurring after ti evolution (17). Further details of the analysis of these experiments are given in the Supplemental NMR Analysis described below.

[00446] Native-state hydrogen exchange experiments were measured for WIL-V at 0.2 mM and JTO-V at 0.1 mM with or without 0.5 mM 1. The proteins, previously desalted and lyophilized, were dissolved in a DzO based sodium citrate buffer, pD 5.0, containing 1.25% v/v of either pure ethanol or 40 mM 1 stock solution. Per-residue exchange rates kex were converted to free energies using the equation where krc are the residue-specific exchange rates of unprotected amides (18): kex

AG = -RT In (— )

KfC

[00447] Kinetic unfolding. WIL-FL (5 mM) in 50 mM sodium phosphate buffer, pH 7.0, containing 50 mM 5 was rapidly diluted into urea in 50 mM sodium phosphate buffer and 50 mM 5 using an APP SX-20 stopped flow fluorimeter at 37 °C. The change in fluorescence emission >335 nm was measured as a function of time (l« _c = 280 nm). Measurement of unfolding rates in the presence of 1 was not possible on this filter-based fluorimeter due to fluorescence of 1.

Kinetic transients were fitted to single exponential curves and rates extracted.

[00448] Equilibrium unfolding. WIL-FL (5 mM) was titrated against urea in 50 mM sodium phosphate buffer, pH 7.0, containing 50 mM 5 or 100 mM 1. After incubation overnight at 25 °C, intrinsic tryptophan fluorescence emission spectra were measured using a Jasco 8600 fluorimeter (Xcx = 280 tun, X«m = 300-420 nm). Average wavelength (also known as center of spectral mass) values were calculated using the equation:

Average wavelength

[00449] l, and ¾ are the are the wavelength and intensity at i, respectively. Average wavelength data were normalized for comparison. LCs do not refold fully reversibly from urea (1), so we cannot report free energies of unfolding. Midpoint urea concentrations were calculated by fitting the titration data to a 2-state unfolding equation:

G-mx

(a + bx)e RT + (c + dx)

Fraction folded

[00450] x is the urea concentration (M), G is the free energy of unfolding (kJ mol ^'1 ), m is the denaturant dependence of the unfolding reaction (U mol ^"1 M ^"1 ), R is the ideal gas constant, T is the temperature in K, a and c are the fluorescence of the folded and unfolded states, and b and d are the denaturant dependence of the fluorescence of the folded and unfolded states, respectively. Cm, the midpoint concentration, is:

G

Cm ^—

m

[00451] Aggregation. WIL C214S was dialyzed into ddHzO and filtered. Each 1 ml reaction contained 10 mM LC, ddHzO, 1% DMSO or 200 mM 1 in 1% DMSO, 150 mM NaCl, and ABC buffer (20 mM sodium acetate, 20 mM boric acid, 20 mM sodium citrate, pH 5 with HC1). Components are listed in the order they were added.

[00452] For analysis by SEC, reactions were set up in 2 ml tubes (Axygen #SCT-200-B-S) equipped with 4 mm stir bars. For each experiment, four reactions (two replicates each of vehicle and 1) were placed in a microfuge tube rack on top of the center of a stir plate. Reactions were stirred at approximately 2,000 rpm at 37 °C, and 60 mΐ aliquots were removed at the indicated timepoints. Aliquots were centrifuged at 16,000 g at 4 °C for 5 min. 10 mΐ of the supernatant was injected into a Waters Acquity H-Class Bio-UPLC (ultra performance liquid chromatography) instrument equipped with a BEH200 SEC column (Waters, Milford, MA) equilibrated with PBS with 1 mM EDTA At the end of an experiment, stir bars were washed for at least 3 h with 1 M NaOH, followed by at least 3 h with 6 M GuHCl at room temperature to remove residual aggregates.

[00453] Thioflavin T (ThT)-containing aggregation reactions were set up in 96- well plates with similar conditions used for the reactions described above, except each 100 mΐ reaction also contained 2 mM ThT. Aggregation was induced with shaking as previously described (4). In total, 21 replicates for both vehicle and 1, over four independent experiments (plates) were performed.

[00454] Electron micrographs were recorded by the Scripps Research microscopy core facility.

[00455] A protease-coupled fluorescence polarization assay of LC stability. To identify FL LC kinetic stabilizers, a protease-coupled fluorescence polarization (PCFP) assay was developed (Fig. la). This assay measures the protease resistance of FL LCs— a reflection of the kinetic stability imparted by small molecule binding (4, 5). Cleavage of the AL patient- derived l6-57 FL LC known as WIL (23) to smaller fragments requires conformational excursions (4) and leads to reduced fluorescence polarization (FP) of the LC-conjugated fluorescein fluorophore (Fig. la) (24). A surface-exposed K79C V-domain mutation (numbered according to the Rabat scheme (25)) was introduced into FL WIL and into the non-AL FL LC JTO (23), to which we conjugated fluorescein via an attached maleimide. The resulting conjugates, referred to as WIL-FL* and JTO-FL*, were purified by ion-exchange

chromatography. Both WIL-FL* and JTO-FL* are obligate dimers because of the disulfide bond between monomers. Proteinase K (PK), a broad-spectrum serine protease that efficiently cleaves transiently unfolded LCs under physiological conditions(4) was used for the PCFP assay in order to minimize any sequence-specific cleavage site differences between LCs. [00456] We measured PK proteolysis of WIL-FL* and JTO-FL* (20 nM) spiked into unlabeled WIL-FL and JTO-FL (10 mM), respectively, at 37 °C in phosphate buffered saline (PBS; pH 7.4). Both labeled LCs are degraded at increased rates compared to the parent sequences based on the disappearance of the LC band by SDS-PAGE (Fig. lb; ko _b s = 7.6 ± 0.3 * 10 ⁴ s ^'1 for WIL-FL* versus 2.4 ± 0.9 * 10 ⁴ s ^'1 for WIL-FL; 9.5 ± 3.5 * 10 ^"5 s ^'1 for JTO-FL* versus no measurable decrease for JTO-FL, mean ± sd, n=3). The K to C mutation and subsequent fluorescein conjugation reduces the kinetic stability of both LCs, possibly by decreasing the solvent entropy change upon folding in the region displaying the solvated dye, i.e., by attenuating the hydrophobic effect Importantly, endoproteolysis of AL-associated WIL- FL* is significantly faster than that of the more kinetically stable JTO-FL* (Fig. lb). Treatment of WIL-FL* (20 nM) with PK (500 nM) at 37 °C in PBS leads to reduction of FP (l» = 488 nm, Xcm = 520 nm) at a rate (kobs = 9.9* 10 ⁴ s ^'1 ) comparable to that measured by SDS-PAGE, whereas the FP of JTO-FL* is reduced more slowly (kobs = 1.8*10"* s ^'1 ), as expected (Fig. lc).

[00457] The PCFP assay was miniaturized to a 384- well microplate format and verified that we observed similar patterns of proteolysis under conditions suitable for high-throughput screening. In PBS at 22 °C, WIL-FL* (20 nM) is cleaved by PK (500 nM) over 24 h, whereas JTO-FL* is cleaved more slowly (Fig. Id). We previously observed correlations between rates of denaturant-induced unfolding and rates of endoproteolysis (4). Initial experiments showed that the FP of both WIL-FL* and JTO-FL* decreased at a similar rate in the presence of PK, and increased in the absence of PK This appears to be due to denaturation of the LCs on the plate surface. Addition of Pluronic F-127 detergent to untreated plates (Greiner #655076) prevented the increase in FP in the absence of PK and yielded proteolysis kinetics similar to those in cuvettes with PK. Employing low-binding plates with or without Pluronic F-127 detergent (0.02%) does not alter the rate of WIL-FL* endoproteolysis. Thus, we added Pluronic F-127 detergent to untreated plates for the high-throughput screen and subsequent experiments due to the increased cost of low-binding plates. This detergent may have the additional benefit of reducing colloidal aggregation of small molecules (26). Addition of the kosmotropic salt sodium sulfate reduces the extent of WIL-FL* proteolysis after 24 h, consistent with its stabilizing effect (Fig. le). The extent of WIL-FL* endoproteolysis is not influenced by the concentration of PK, consistent with FL LC conformational excursions from the native state being rate limiting for proteolysis (4), a condition referred to as EX1 kinetics (Fig. le). The assay exhibited a robust Z’ score of 0.8 (27).

[00458] Identification of small molecules that protect LCs from proteolysis. We screened a commercial library of 16,000 small molecules, employing the 384-well PCFP assay with WIL-FL*. The results guided further miniaturization of the assay to 1,536-well plates, optimizing the concentrations of WIL-FL* (10 nM), PK (250 nM) and kinetic stabilizer candidate (6.75 mM). We screened an additional library of 653,085 small molecules, identifying 2,777 primary hits exhibiting > 20.6 % (mean + 3 standard deviations) retention of WIL-FL* FP (relative to 0.68 % vehicle (DMSO) control assigned 0 %, with 100 % corresponding to the FP of JTO-FL* without PK). The PCFP assay was repeated in triplicate on the 2,777 primary screening hits, of which 1,422 molecules had > 20.6 % mean retention of WIL-FL* FP.

[00459] To identify and exclude molecules that interfered with FP measurements, the assay was repeated, but candidate stabilizers were not added until after 24 h of PK treatment (Fig. If). Compounds that artefactually increase FP would result in similar apparent retention of FP in both assay configurations. Six hundred sixty-two molecules had >1.25-fold greater mean activity (n=3) in the primary screen than in this counterscreen (2,115 molecules artefactually increased FP). To eliminate PK inhibitors, the PCFP screen was rerun in triplicate on the 2,777 hits using the protease thermolysin (200 nM; candidate stabilizer concentration 6.75 mM; Fig. lg). Thermolysin is a broad-spectrum metalloprotease that is likely not inhibited by compounds that inhibit PK, a serine protease. Plotting the thermolysin versus PK data identified 1,243 compounds on the diagonal that were not protease inhibitors (Fig. lg). The 269 small molecules that fulfilled all three assessment criteria (exhibited PCFP activity in triplicate, passed the FP artefact counterscreen (Fig. If), and exhibited > 20 % efficacy in the PCFP thermolysin counterscreen) were retained.

[00460] The potency of these surviving 269 LC kinetic stabilizers was measured by recording concentration-dependence data in the three PCFP configurations covered in the preceding paragraph, along with 147 candidates that appeared to inhibit one or both proteases. One hundred twenty-eight compounds exhibiting a dose-response curve were chosen for further evaluation. We further verified that these 128 compounds do not inhibit PK by measuring their ability to inhibit proteolysis of a fluorogenic peptide substrate. To exclude the possibility that differences in the initial FP amplitude could lead to misinterpretation of the endpoint FP measurement, we measured FP kinetics upon PK treatment. Ninety of the 128 compounds reduced the rate and amplitude of FP change relative to vehicle, consistent with LC kinetic stabilization. Finally, to verify that the FP measurements were reporting on FL LC proteolysis sensitivity, we measured protection of WIL-FL* from PK proteolysis imparted by the 128 compounds mentioned above using SDS-PAGE. Twenty-six molecules stabilize FL LCs by this assay. Sixteen molecules showed clear activity in both the kinetic FP and SDS-PAGE assays (Table 1).

[00461] The sixteen hit molecules fall into four classes (Table 1 -compounds 1-16):

coumarins (1-3, 9-15), an aryl cyanoacrylamide (4), biaryl hydrazones (5-6), and hydantoins (7- 8). Two sulfones (17-18) derived from the commercial library were also validated as hits.

[00462] Small molecules 1 to 16 were identified as hits from the Scripps Florida screen and showed clear activity in both the PCFP and SDS-PAGE assays. Small molecules 17 to 18 were identified as hits from the commercial library. Data for the related but inactive coumarins 19, 20 and 21 are included for reference. Activity is normalized to the activity of the high and low controls from either the pilot screen (n=l), primary screen (n=l), or counterscreen (mean ± sd, n=3). ECso values are calculated from dose response curves measured during the screening process. LC-MS validation was obtained for compounds 1-15, multiple peaks were observed for compound 16, not determined for compounds 17-21. ND = not determined.

[00463] We purchased eight representative small molecules and determined their midpoint effective concentration (ECso) values in the PCFP assay by titration. Dissociation constants (K _D ligand) measured for each compound using equilibrium dialysis are similar to the ECso values, indicating that the small molecule-bound FL LC is highly resistant to proteolysis.

[00464] Table 1 : Screening data for selected small molecules that stabilize LCs and selected inactive analogs.

PK

Thermolysin Counterscreen ECso

P) Structure activity

activity (%) activity (%) (mM)

(%)

1 47.8±0.7 36.4±2.0 -8.07±3.3 12.5

/<N Ό

k

p,

2 37.8±1.4 33.8±3.1 -4.13±5.2 6.92

o·

k

PK

Thermolysin Counterscreen

ID ECso

Structure activity

activity (%) activity (%) (mM)

(%)

o H

N

HI N

7 F 29.3±2.5 26.3±1.7 2.51±4.7 5.87

F o

H

N

HI

8 F 48.0±2.4 50.8±1.7 -2.05±2.9 7.36

F

9 XXX 37.9±1.1 27.U1.3 -3.45ti.89 7.9

PK

Thermolysin Counterscreen

ID ECso

Structure activity

activity (%) activity (%) (mM)

(%) o

14 41.9±3.0 34.8±3.1 1.67±4.7 9.04

Ό

[00465] 7-amino-coumarins are fluorogenic kinetic stabilizers of X6a LCs. Twenty four of 128 screening hits surviving the counterscreens are coumarins modified at the 7-position by either a diethylamino group (21 molecules, e.g., 1 and 2; Table 1) or a 1-phenylethoxy group (3 molecules, e.g. 3; Table 1). Coumarins are often regarded as promiscuous binders; however, 7,839 other coumarin-containing molecules in the library, of which 432 are modified at the 7 position, were not identified as hits after counterscreening. We initially focused on compound 1, 7-diethylamino-4-methylcoumarin, a commercially available dye known as“Coumarin 1”.

[00466] To verify the activity of 1 and investigate the structural requirements at the 7 position of coumarins, we measured protection from PK proteolysis of unlabeled WIL-FL.

Incubation of WIL-FL (10 mM) with 1 (100 mM; Table 1 and Fig. 2a), but not the related compounds 19, 20 and 21 (100 mM, Fig. 2a and Table 1), protects this FL LC against PK proteolysis (t = 2 h). Compound 9, which lacks a methyl group at the 4-position, also stabilizes WIL-FL, but is less efficacious than 1 (Fig. 2a and Table 1). Incubation of WIL-FL (20 mM) with 1 (1 mM) results in a large fluorescence emission increase of 1 relative to the weaker, red- shifted fluorescence of 1 in PBS (Fig. 2b, l« _c = 373 nm), suggesting that the altered environment of 1 associated with binding and the restricted mobility of the 7-diethylamino group contribute to its fluorogenicity (28). In contrast, 19 (1 mM) is fluorescent in PBS, but its emission intensity is reduced in the presence of WIL-FL (20 mM; Fig. 2b). The FP of 1 increases from 0.05 in buffer to 0.35 in the WIL-FL·! complex, consistent with an increase in rotational correlation time.

[00467] The fluorogenicity of 1 makes it a convenient probe to investigate whether LCs other than WIL-FL can be kinetically stabilized and whether other non-fluorescent candidate kinetic stabilizers bind LCs at the same site. Isothermal titration calorimetry measurements indicate that 1 binds to WIL-FL with a KD ligand of 3.08 ± 0.52 mM (mean ± sd, n=3). To measure binding we titrated different WIL constructs into 1 in PBS containing 0.02% Pluronic F-127 and measured the fluorescence emission of the LC*1 complex in a 384-well plate format (Fig. 2c, l _< * = 373 nm, Xe _m = 445 nm). We used a low concentration of 1 (1 mM) and varied LC concentration (serial dilution from 133 pMto 18 nM in 1.5-fold decrements) to minimize background fluorescence and simplify the analysis. Binding of compound 1 to WIL-FL is fit well by a 1-site model with an apparent KD ligand of 3.14 ± 0.3 mM (mean ± sd, n = 5), whereas binding to the WIL V-domain has a steeper dependence on LC concentration and is fit less well by a 1 : 1 binding model (apparent KD ligand of 79.3 ± 5.2 mM). No binding of 1 (1 mM) was detected to the isolated l3 C-domain (Fig. 2c). Recombinant FL X6-57 LCs form disulfide-linked dimers (4), but their isolated V-domains populate an equilibrium between monomers and dimers (4, 22, 29). The apparent positive cooperativity in the binding of 1 to WIL-V is consistent with induced dimerization of the LC V-domain.

[00468] Concentration-dependent NMR chemical shift data indicate that WIL-V has a dimerization equilibrium constant (KD dimer) of approximately 5 mM (22). Fitting the

fluorescence data to a model of binding-induced dimerization constrained by the NMR-derived KD dimer yields an apparent KD ligand of 3.0 ± 1.2 mM for WIL-VW indicating that the apparent low affinity for WIL-V compared to WIL-FL is due to a low concentration of WIL-V dimer at equilibrium. Since the affinities for WIL-FL and dimeric WIL-V are indistinguishable, we can assume that 1 binds only to dimeric WIL-V and, hence, estimate the apparent KD dimer from the fluorogenic binding data in the presence of 1 (1 mM) to be 1.45 mM, compared to 5 mM in the absence of 1.

[00469] A successful kinetic stabilizer should, but is not required to, bind to and stabilize patient LCs with a vast range of sequences (30, 31). To ask whether 1 can stabilize other FL LC sequences, we measured binding of 1 to three more recombinant l6-57 FL LC disulfide-linked dimers: JTO-FL; ALMC2-FL, an AL-associated LC sequence derived from the ALMC2 cell line (32); and the germline sequence 6aJL2-FL. Serial dilution of these LCs from 133 mM to 18 nM in 1.5-fold decrements into 1 (1 mM ) in PBS yields Ko ugand values of between 1 and 20 mM (Fig. 2d, left panel). The isolated V-domains from WIL, ALMC2 and 6aJL2 bind to 1 (1 mM) with reduced affinity (Fig. 2d, left panel), while JTO-V has Ko ugand of 16.5 ± 0.4 mM (mean ± sd, n=3), similar to that of JTO-FL (20.3 ± 1 mM), consistent with JTO-V being mainly dimeric at this concentration (22). Eliminating the inter-chain disulfide bond between the C-domains by the C214S mutation reduces the affinity of LCs for 1 (1 mM) (Fig. 2d), consistent with this mutation’s destabilization of the dimeric interface (4).

[00470] To further test the hypothesis that 1 binds at the interface between the V-domains, we measured binding of 1 to WIL-FL and JTO-FL variants with mutations designed to disrupt the dimer (22). The F98D mutation, which disrupts the V-domain-V-domain interface, rendered binding of 1 (1 mM) undetectable in the presence or absence of the C214 inter-chain disulfide bond (Fig. 2d, right panel). The FI 18D mutation, which destabilizes the C-domain-C-domain interface, or the LI 17G or PI 41 A mutations, which destabilize the hydrophobic core of the C- domain, resulted in reduced binding affinity of 1 (1 mM) to WIL-FL C214S and, to a lesser extent, JTO-FL C214S (Fig. 2d, right panel), with KD values similar to those exhibited by the isolated V-domains (-20-50 mM).

[00471] To further investigate the ability of small molecules to induce dimerization of LCs, we performed sedimentation velocity analytical ultracentrifugation (SV-AUC) analysis of WIL-V and WIL-FL C214S (20 mM) in the presence or absence of 1 (100 mM). Continuous sedimentation distributions (Fig. 2e) show the apparent size of the sedimenting species.

Deviations from the expected size indicate exchange between species, in this case monomer and dimer. The size distributions of both LCs (20 mM) increase upon binding 1 (100 mM). WIL-FL C214S has a KD dimer of approximately 16 mM, estimated by concentration-dependent SV-AUC experiments.

[00472] Of the two classes of human LCs, l and k, l LCs are more commonly observed in AL, but a fraction of patients secrete K1-33 LCS (12, 33). To ask whether k LCs possess a similar small molecule binding site as l LCs, we incubated 1 (1 mM) with two K1-33 LCS (serial dilution from 133 mM to 18 nM in 1.5-fold decrements), which represent the patient-derived sequence AL12-FL (34) and its germline precursor k 1 018/08-FL (7). Recombinant expression of these K1-33 LCS using a similar protocol to that used for X6-57 LCs yielded both monomeric and dimeric LCs, as shown by SV-AUC (SI Appendix Fig. ST). Consistent with this reduced propensity to dimerize, binding of 1 to K1-33 LCS is weaker than binding to X6-57 LCs (Fig. 2f). Binding of an excess of K1-33 LCS (20 mM) to 1 (1 mM) afforded FL LC ^e l fluorescence (Fig. 2f, inset). This result indicates that a binding site for 1 is present in dimers of both X6-57 and K1-33

LCs.

[00473] To ask whether the non-coumarin small molecules in Table 1 bind to FL LCs at the same or an overlapping site, we measured binding of 1 (1 mM) to WIL-FL (serial dilution from 150 mM to 20 nM in 1.5-fold decrements) in the presence of the other screening hits (10 mM). The apparent binding affinity of LCs for 1 was reduced by six non-fluorescent molecules identified in the screen, but not by the negative control diflunisal, an anti-inflammatory drug that stabilizes transthyretin (15, 35) (Fig. 2g). These data suggest that the hits have binding sites that overlap with that of 1, or exhibit negatively cooperative binding with 1 to WIL-FL.

[00474] The free monoclonal LC concentration in the plasma of AL patients is typically in the range of 1-20 mM (10-100 mg/dl) (36), whereas the concentration of antibodies and albumin is much higher. Therefore, kinetic stabilizer binding selectivity to FL LCs in preference to other plasma proteins is important to minimize the proportion of free FL LC. Albumin binds many small molecules in blood, typically with mM KD values. Titration of albumin (serial dilution from 400 mM to 53 nM in 1.5-fold decrements) with 1 (1 mM) results in increased coumarin fluorescence, with an apparent KD of 37 ± 3 mM (Fig. 2h). Affinity of 1 for albumin is reduced in the presence of 100 mM diflunisal (Fig. 2h), consistent with diflunisal binding competitively to albumin.

[00475] The heterodimeric interface between LCs and heavy chains (HCs) in an antibody is structurally distinct from that of the LC homodimer interface. To verify that our kinetic stabilizers bind FL LC dimers selectively over antibodies, we compared the binding of 1 (1 mM) to the LC and antigen binding fragment (Fab, the LC:HC dimer) of 5J8, a human anti-influenza hemagglutinin antibody (serial dilution from 120 mM to 16 nM in 1.5-fold decrements) (37). The 5J8 FL LC from the X3 class (not associated with AL) binds to 1 with an apparent KD of ~ 50 mM (Fig. 2i), an event that protects the 5J8 LC from proteolysis by PK In contrast, the 5J8 Fab shows no apparent binding to 1 as measured by fluorescence (Fig. 2i), and 1 does not protect the 5J8 Fab from PK proteolysis.

[00476] Wildtype (WT) full-length l6-57 LC constructs are obligate, disulfide-crosslinked dimers. Other species are in equilibrium between monomer and dimer. See Table 2. NMR measurements show that full-length, C214S LC constructs are dimeric at 200 mM The percentage of residues at the domain interfaces between protomers which have dimer-like chemical shifts is reported. NMR data are described further in Rennella et al. (5).

[00477] Table 2. Summary of monomer-dimer equilibria for different LC species.

LC NMR (200 mM) AUC (20 mM)

WT JTO-FL Obligate dimer Single peak 3.5 S

WT WIL-FL Obligate dimer Single peak 3.5 S

12.5 mM Single peak 2 S

5000 mM Single peak 1.5 S

dimer-like (V-domain interface)

Single peak 3.5 S

dimer-like (C-domain interface)

dimer-like (V-domain interface) Two peaks, 2 S and 3.2 S; KD dimer-like (C-domain interface) = 16 mM LC NMR (200 mM) AUC (20 mM) k ΐ 018/08-FL Not measured Two peaks, 2.4 S and 3.2 S

Major peak at 2.4 S with

AL12-FL Not measured

shoulder at 2.9 S

[00478] Structural insight into LC kinetic stabilizer binding. We determined the crystal structures of JTO-FL with and without bound 1. Our attempts to crystallize other LOkinetic stabilizer complexes (e.g., the WIL-FL·! complex) have so far been unsuccessful.

The unbound JTO-FL LC structure is very similar to previously published FL LC structures (3,

5, 38) (Fig. 3a). LC homodimers are covalently linked by an inter-chain disulfide bond between C214 residues in the C-domains of the monomers. In the apo JTO-FL structure, refined at 1.75 A resolution, the conformation of the V-domains is the same as that in the published JTO-V dimer structure (39).

[00479] The structure of the JTO-FL· 1 complex, refined at 1.8 A resolution,

unambiguously shows density for 1 at the interface formed by the two V-domains. This binding site is consistent with previous reports that the interface between V-domains in the dimer can accommodate various aromatic ligands (21, 40), although the exact binding site that we identify below has not been previously observed. The binding site for 1 comprises residues P44, Y87 and F98 from one protomer and Y36', P44' and T46' from the other protomer (Fig. 3b), which together form a cavity that is not present in the unbound structure (Fig. 3c; cf. right to left panels). The JTO-FL F98D variant (22) has no measurable affinity for 1 (Fig. 2d), consistent with disruption of the p- p stacking interaction observed in the structure. The 7-diethylamino substructure projects through the V-domain-V -domain interface, interacting with both P44 residues. These interactions may explain the apparent requirement for modification at the 7 position of the coumarin scaffold. The carbonyl oxygen of 1 hydrogen bonds with the sidechain hydroxyl and/or backbone amide of T46'. A CH-p interaction between 1 and Y87 may also contribute to the binding free energy. Binding of 1 causes a rotation of the V domains relative to each other, which creates the interface cavity (Fig. 3c). The distance between the Ca residues of F98 and P44' increases from 7.2 A to 9.7 A to accommodate 1. The LC dimer has two pairs of F98 and P44 residues, creating two potential binding sites, but only one molecule of 1 is observed per dimer. Aligning a second molecule of 1 utilizing the same intermolecular interactions results in a steric clash between the diethylamino substructures. [00480] The residues used by JTO to bind 1 are highly conserved (Fig. 3d). Residues Y36,

P44, Y87 and F98 are present in 88, 99, 95 and 100 % of all human germline LC genes, respectively, whereas T46 is only present in two functional germline genes. Mutation of residue 46 in WIL-FL and JTO-FL to leucine, which is the residue present in 75% of LC germline genes, slightly enhances the binding affinity of 1 for these FL LCs, indicating that the sidechain hydroxyl group of T46’ is not necessary for binding of 1.

[00481] To verify that 1 binds to different LCs in solution at the same site identified in the crystal structure, we recorded the NMR spectra of WIL-FL, W1L-V and JTO-V in the absence and presence of 1. We used high concentrations of LC (100-500 mM) and solubility-limited concentrations of 1 (up to 500 mM) to maximize the NMR signal from the bound state. 2- dimensional *H- ¹⁵ N heteronuclear single-quantum coherence (HSQC) experiments, wherein each amide in a protein is represented as a peak in the spectrum, allowed identification of residues perturbed by binding of 1. Chemical shift and intensity perturbations of similar residues in each spectrum occur upon addition of 1 (Fig. 4a). In each case, the perturbed residues are concentrated in the interface between the two V-domains, close to the site at which 1 binds according to the crystal structure (Fig. 4b). Upon titration of 1 into WIL-FL, which is an obligate dimer (4), new peaks of the bound state IC½·1 appear for the amides close to the binding site (Fig. 4a, b). The changes in intensity for LC ₂ and IC¼·1 peaks can be fit by a two-state binding model with an apparent KD of 19 ± 2 mM (Fig. 4c).

[00482] Concentration-dependent chemical shift measurements (22) indicate that JTO-V is dimeric at concentrations used for NMR (generally greater than 100 mM) with a dimerization KD of approximately 12.5 mM. Binding of 1 to JTO-V (140 mM) at approximately equimolar stoichiometry results in the appearance of exchange cross-peaks between peaks originating from LC ₂ and LC ₂ ^e l (Fig. 4d, red contours) in a regular HSQC experiment that is not designed to measure exchange rates. A ZZ-exchange experiment (41) measured on- and off-rates of 1.00 ±

0.10 mM ^'1 s ^'1 and 57 ± 2 s ^'1 , respectively, for the JTO-V2·! complex, yielding a KD of 57 ± 4 mM for a two-state model, in agreement with the value measured by fluorescence (Fig. 2d).

[00483] Binding of 1 to isolated WIL-V (400 mM) is more complex, since this V-domain is monomeric in the absence of ligand (KD ~ 5 mM) (22) and becomes dimeric upon addition of 1. ¹⁵ N transverse relaxation (R2) rates, which report on overall protein tumbling and hence on molecular size, double for WIL-V upon addition of excess 1 (500 mM), whereas they do not change for JTO-V (140 mM), which is dimeric without and with 1 bound (Fig. 4e). Notably, WIL-V bound to 1 exhibits multiple HSQC peaks for most interfacial residues and may represent alternative conformations of the dimer or of the bound ligand (Fig. 4f). The presence of multiple conformations for the LC ₂ ^e l complex of WIL-V prevents accurate quantification of KD.

Chemical exchange saturation transfer (CEST) experiments (42) indicate that the KD for WIL- V ₂ ·! is < 50 mM, in agreement with the effective KD of 3 ± 1.6 mM determined from fluorogenic binding titrations (Fig. 2c). Removal of 1 from WIL-V by dialysis yielded an HSQC spectrum indistinguishable from that of the starting material, indicating that binding is reversible and non- covalent. Thus, that the heterogeneity observed in the bound state spectra (Fig. 4f) is likely due to alternate binding modes.

[00484] We next asked how binding of kinetic stabilizers would affect the stability of different LCs. In these experiments, we generally used an excess of kinetic stabilizer (limited by small molecule solubility) to saturate LC binding and thus investigate the properties of ligand- stabilized LCs, as a proxy for the behavior of small molecules that bind to LCs with nanomolar affinity. Ablation of the inter-chain disulfide bond by the C214S mutation results in reduced LC kinetic stability (4) and reduced small molecule binding affinity (Fig. 2d). Nonetheless, binding of 1 (100 mM) reduces the rate constant of PK proteolysis of WIL-FL C214S (10 mM) from 6.9 x IQ ^"3 to 2.8 x IQ ^"3 s ^'1 (Fig. 5a).

[00485] To confirm that protease resistance is driven by stabilization of the LC native state ensemble by small molecule binding, we measured urea-mediated unfolding rates of LCs in solution by tryptophan emission fluorescence (4). The intrinsic fluorescence of 1 precluded its use in these experiments. WIL-FL (5 mM) in the presence of 5 (a non-fluorescent kinetic stabilizer used at a solubility-limited concentration of 50 mM, Table 1) unfolds with a rate constant (extrapolated to the absence of denaturant) of 2.43 s ^'1 compared to 3.57 s ^'1 for WIL-FL alone (Fig. 5b). Binding of 5 increases the kinetic stability of the FL LC, even though the > 5 M urea concentrations used in this experiment will lower the affinity of 5 for the FL LC. Titration of urea into WIL-FL (5 mM) in the absence or presence of 1 (100 mM) or 5 (50 mM) revealed an increase in the midpoint concentration of urea required to unfold the FL LC (Fig. 5c) from 1.4 to 2.0 M The magnitude of this stabilization imparted by 1 and 5 was indistinguishable (Fig. 5c), indicating that the ligand-bound states are similarly stable. [00486] To verify that binding of 1 reduces LC unfolding under non-denaturing conditions, we measured residue-specific hydrogen-deuterium exchange rates by NMR of WIL- V and JTO-V (100 mM) in the absence of or with 1 (500 mM). These rates (the decrease in intensity of the HSQC peak associated with each amide) are converted to free energies that report on each amide’s folding equilibrium by normalizing for the published exchange rates of amides in unstructured peptides. Both V-domains are stabilized by an average of 0.6 kcal/mol in the presence of 1 (500 mM, Fig. 5d), indicating that transient unfolding is reduced by binding of

1.

[00487] To verify that the kinetic stabilizers bind LCs secreted from cells analogously to recombinant FL LCs, we measured proteolysis of LCs secreted by ALMC2 cells, an AL patient- derived cell line (32). Purified ALMC2-derived LC (5 mM) is cleaved by PK (100 nM) with a rate constant of 3.83 x 10 ^"4 s ^'1 , whereas no proteolysis was detected after 1 h of incubation by PK in the presence of 1 (100 mM; Fig. 5e).

[00488] Inhibition of LC aggregation. Full-length LCs aggregate much less readily than their isolated V-domains in vitro (4, 7). Although aggregation can be measured in vitro, it is not clear to what extent these experiments recapitulate processes that occur in patients, where the mechanism of aggregation is unknown. Assessment of the effect of kinetic stabilizers on FL LC aggregation at the hit stage of drug discovery is complicated by the denaturing acidic pH used to destabilize the FL LCs to promote aggregation in vitro, which can also reduce the binding affinity of the kinetic stabilizers, sometimes dramatically.

[00489] To assess the influence of 1 on FL LC aggregation, we used WIL-FL C214S, which forms weak dimers (KD = 16 mM), increasing its propensity to aggregate compared to WIL-FL, while retaining affinity for 1 (Fig. 2d). WIL-FL C214S aggregates upon vigorous stirring at or below pH 5 to form heterogenous aggregate structures. WIL-FL C214S aggregation kinetics assessed by fluorescence of the amyloid-binding dye thioflavin T demonstrate that 1 decreases the rate of aggregation significantly versus vehicle control, but the variability in these experiments is greater than desired. This may be because a given LC can stochastically form different aggregate structures with different kinetics.

[00490] Thus, we also quantified aggregation by measuring the rate of disappearance of soluble dimeric LC using size exclusion chromatography (SEC) (4). Binding of 1 (200 mM) to WIL-FL C214S (10 mM), which stabilizes the dimer and enhances kinetic stability (Fig. 5a), results in a larger proportion of soluble, dimeric LC eluting from the SEC column after 64 h of shaking at pH 5, which promotes aggregation. While individual WIL-FL C214S aggregation reactions exhibit variation in kinetics from run-to-run, aggregation in the presence of 1 (200 mM; dashed blue lines, Fig. 5f, n = 10) is generally slower than in the absence of 1 (Fig. 5f, red dashed lines, n=10). Fitting all the aggregation data in Fig. 5f to a single exponential decay model reveals that WIL-FL C214S aggregates significantly more slowly in the presence of 1 (kagg = 0.011 hr ^'1 , Fig. 5f, bold blue curve) than in the absence of 1 (kagg = 0.066 hr ^"1 , Fig. 5f, bold red curve, P < 0.001, t-test on log-transformed rates).

[00491] Unlabeled LC Protease Sensitivity Assay. W1L-FL (5 mM) in phosphate- buffered saline (PBS, pH 7.4) + 0.02% Pluronic F-127 was incubated with proteinase K (50 nM) with small molecule (10 mM) in 1% DMSO vehicle, or vehicle alone. After 2 hours at 37 °C, reactions were quenched with 2 mM phenylmethyl sulfonyl fluoride in methanol and centrifuged at 14,000 rpm for 2 minutes. Remaining WIL-FL was quantified by injecting 10 pL of the supernatant into a Waters Acquity H-Class Bio-UPLC (ultra performance liquid

chromatography) instrument equipped with a BEH200 analytical size-exclusion column (1.7 pm, 4.6x150 mm) under isocratic conditions (0.2 mL/min, PBS + 1 mM EDTA, 2400 psi

backpressure, 15 min), monitoring absorbance at 280 run. Activity of each compound is given as fold protection from protease = (A compound - A vehicle) / (A_no_protease - A vehicle), where each“A” corresponds to peak integration of the respective treatment Results from the LC Protease Sensitivity Assay are shown in Table 3.

[00492] Table 3: Fold protection of LC from protease in the proteinase K sensitivity assay for WIL-FL at 10 mM Protection: A < 0.3); 3 < B < 0.6; and C ³ 0.6.

Compound Protection Compound Protection Compound Protection

22 A 62 A 102 A

23 A 63 A 103 B

24 A 64 A 104 A

25 A 65 A 105 B

26 A 66 A 106 A

27 A 67 A 107 A

28 A 68 A 108 A

29 A 69 A 109 A Compound Protection Compound Protection Compound Protection

30 A 70 A 110 B

31 A 71 A 111 A

32 A 72 A 112 A

33 A 73 A 113 A

34 A 74 A 114 A

35 A 75 C 115 A

36 A 76 A 116 A

37 A 77 A 117 C

38 A 78 A 118 B

39 A 79 A 119 B

40 A 80 A 120 B

41 A 81 A 121 B

42 A 82 A 122 B

43 B 83 A 123 B

44 A 84 A 124 C

45 A 85 B 125 C

46 A 86 B 126 C

47 A 87 B 127 B

48 A 88 B 128 B

49 A 89 C 129 B

50 A 90 A 130 B

51 A 91 B 131 B

52 A 92 B 132 C

53 A 93 B 133 B

54 A 94 A 134 A

55 A 95 B 135 C

56 A 96 A 136 A

57 A 97 A 137 B

58 A 98 C 138 A

59 A 99 B 139 A

60 A 100 A 140 A

61 A 101 A 141 C

142 C 182 B 222 C

143 B 183 B 223 C

144 C 184 A 224 A

145 C 185 C 225 A

146 B 186 A 226 A

147 B 187 A 227 A

148 B 188 C 228 A

149 C 189 A 229 A

150 A 190 C 230 A

151 A 191 A 231 A

152 B 192 C 232 A Compound Protection Compound Protection Compound Protection

153 C 193 C 233 A

154 C 194 C 234 A

155 C 195 A 235 A

156 B 196 A 236 A

157 C 197 C 237 A

158 B 198 B 238 A

159 C 199 B 239 A

160 C 200 A 240 A

161 C 201 B 241 A

162 C 202 C 242 A

163 C 203 C 243 A

164 C 204 B 244 A

165 C 205 B 245 A

166 C 206 A 246 A

167 C 207 B 247 A

168 A 208 B 248 B

169 A 209 B 249 A

170 C 210 B 250 A

171 A 211 B 251 A

172 A 212 B 252 A

173 A 213 C 253 A

174 A 214 C 254 A

175 C 215 C 255 A

176 B 216 C 256 A

177 C 217 C 257 A

178 A 218 C 258 A

179 B 219 A 259 A

180 C 220 A 260 A

181 C 221 C 261 A

262 A 302 A

263 A 303 A

264 A 304 A

265 A 305 A

266 A 306 A

267 A 307 B

268 A 308 B

269 A 309 A

270 A 310 A

271 A 311 B

272 A 312 C

273 A

274 A

275 A Compound Protection Compound Protection Compound Protection

276 A

277 B

278 C

279 A

280 B

281 B

282 B

283 A

284 B

285 A

286 A

287 A

288 B

289 C

290 C

291 C

292 C

293 B

294 A

295 B

296 B

297 A

298 B

299 B

300 A

301 A

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[00494] All patents and publications referred to herein, including numbered references appearing throughout the present disclosure, 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.