4- aminoquinoline inhibitors of the autophagy-lysosomal pathway (ALP) as a therapeutic approach for pancreatic cancer

Abstract

Described herein are compounds of Formula (I), pharmaceutical compositions comprising the compounds, and methods of using the compounds for treating cancer and for inhibiting the autophagy-lysosomal pathway (ALP).

Description

Atty. Dkt. No.: 115872-33254- AMINOQUINOLINE INHIBITORS OF THE AUTOPHAGY- LYSOSOMAL PATHWAY (ALP) AS A THERAPEUTIC APPROACH FOR PANCREATIC CANCERCROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application 63 / 688,792, filed August 29, 2024, and U.S. Provisional Application 63 / 690,535, filed September 4, 2024, the entirety of each of which is incorporated herein by reference.BACKGROUND

[0002] Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy and often deemed a fatal disease. The current 5-year survival rate is a devastating 11% marred by lack of early detection methods and ineffective treatment options. Large efforts have been made to develop targeted therapeutics for use in PDAC, with little success. The uncontrolled tumor growth driven by activating KRAS mutations coupled with the highly desmoplastic, nutrient void, stroma force PDAC to rely upon metabolic reprogramming to keep up with energetic needs. Nutrient scavenging pathways, namely autophagy and macropinocytosis, have been shown to be essential for PDAC tumor growth. These pathways converge on degradative lysosomes to break down cargo into biological building blocks to be used by the cell, making the autophagy-lysosomal pathway (ALP) an attractive point of therapeutic intervention.

[0003] There remains a need developing small molecule-based pharmaceuticals that inhibit the ALP and that act as a therapy for pancreatic cancer.SUMMARYJ0004] In an aspect, this disclosure provides a compound according to Formula (I):-1-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325wherein:Ring A is a 5 to 14-membered cycloalkyl, a 6 to 14-membered aryl, a 5 to 16- membered heterocycloalkyl, or a 5 to 16-membered heteroaryl;Ring B is absent, a 5 to 14-membered cycloalkyl, a 6 to 14-membered aryl, a 5 to 16-membered heterocycloalkyl, or a 5 to 16-membered heteroarylR1is H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -CN, -ORla, -COORla, or -NRlaRlb, wherein Rlaand Rlbare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R2is H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -CN, -OR2a, -COOR2a, or -NR2aR2b, wherein R2aand R2bare each independently H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl; or R1and R2together form a 5 to 10-membered cycloalkyl, a 6 to 10-membered aryl, a 5 to 10-membered heterocycloalkyl, or a 5 to 10-membered heteroaryl;R3is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -OR3a, -SR3a, -SO2R3a, -COOR3a, -NR3aR3b, -NO2, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, wherein R3aand R3bare each independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heteroaryl, or optionally substituted heterocycloalkyl, and n is an integer from 0 to 5;X1is a bond, an optionally substituted alkylenyl, an optionally substituted alkenylenyl, an optionally substituted heteroalkylenyl, or an optionally substituted heteroalkenylenyl;-2-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325X2 is a bond, an optionally substituted alkylenyl, an optionally substituted alkenylenyl, an optionally substituted heteroalkylenyl, or an optionally substituted heteroalkenyl enyl.

[0005] In another aspect, this disclosure provides a composition comprising a compound of any embodiment disclosed herein, and a pharmaceutically acceptable carrier.

[0006] In yet another aspect, provided is a pharmaceutical composition for treating a disease or disorder, wherein the pharmaceutical composition comprises a therapeutically effective amount of a compound described herein, and a pharmaceutically acceptable carrier.

[0007] In another aspect, this disclosure is directed to a pharmaceutical composition for inhibiting the autophagy lysosomal pathway (ALP), wherein the pharmaceutical composition comprises a therapeutically effective amount of a compound described herein, and a pharmaceutically acceptable carrier

[0008] In another aspect, provided is a method of treating a disease or disorder in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound described herein or a pharmaceutical composition described herein

[0009] In yet another aspect, provided is a method of inhibiting the ALP in a patient, the method comprising administering to the patient a therapeutically effective amount of a compound described herein or a pharmaceutical composition described herein.

[0010] In another aspect, this disclosure is directed to the use of a therapeutically effective amount of a compound described herein for treating a disease or disorder in a patient in need thereof.

[0011] In any embodiment herein, the disease or disorder is a cancer (e.g., pancreatic ductal adenocarcinoma).

[0012] In yet another aspect, this disclosure is directed to the use of a therapeutically effective amount of a compound described herein for inhibiting the ALP.-3-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1. JTC-801 induces nuclear translocation of TFEB without inhibition of mTOR. (A) JTC-801, selective opioid receptor-like 1 (ORL1) receptor antagonist, was a hit compound from the TFEB phenotypic screen. (B) U2-OS mCh-TFEB cells were treated with varying doses of JTC-801 and nuclear translocation of TFEB was quantified as nuclear / cytoplasmic ratio. Nuclear translocation was induces at concentrations >1 pM in U2-OS mCh-TFEB cells. (C) U2-OS cells were treated with vehicle (DMSO), Torin-1 [1 pM] or JTC-801 [5 pM] for 3h. pS6 levels and S6 expression was detect by Western blot. Representative Western blot is shown. (D) pS6 levels were quantified and normalized to Tubulin levels. Torin-1 treatment induced a significant (ordinary one-way ANOVA, p<0.0001) reduction in pS6 levels. JTC-801 had no significant effect on pS6 levels. All data from n=3 biological replicates and is represented as mean ± SD.

[0014] FIG. 2. JTC-801 inhibits lysosomal degradation in PDAC cells. PaTu 8988T cells were pre-incubated with DQ-BSA and then treated with JTC-801 [2.5, 1.25 pM] or vehicle (DMSO) for 12h. DQ-BSA fluorescence intensity was quantified and normalized to DMSO treated cells and displayed as percent inhibition. JTC-801 causes significant (ordinary one-way ANOVA, p=0.0113) inhibition of lysosomal degradation at [2.5 pM], All data from n=3 biological replicates and is represented as mean ± SD.

[0015] FIG. 3. Development of GN Series from hit small molecule, JTC-801. Based on the hit, JTC-801, modifications were made to generate a library of derivatives known of the GN Series. Modifications explored included alterations to the quinoline (green), various linker groups (blue), and conversion of the phenolic ether (orange) to a phenyl piperazine with various aromatic substitutions (brown). The free amine of the quinoline (red) was deemed essential for activity (data not shown)

[0016] FIG. 4. PDAC Cytotoxicity profiling of GN Series reveals trifluoromethyl and nitro functional groups confer cytotoxicity in PDAC cells. PDAC cells (MIA PaCa-2, PaTu 8988T) were treated with GN Series small molecules [5 pM], JTC-801 [5 pM], or vehicle (DMSO) for 72h. Cell growth was quantified using the SRB assay and normalized to DMSO treated cells and displayed as percentage of cell growth. GN Series small-4-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 molecules show a range of cytotoxicity profiles with those containing a phenyl piperazine with a trifluoromethyl and or nitro substitution (*) showing high levels of cytotoxicity. All data from n=3 biological replicates and is represented as the mean.

[0017] FIG. 5. Various linkers used in the GN Series. The GN Series is comprised of analogs with chemical modifications to the quinoline (Ri) and para (R2) or ortho (R3) substitutions of the phenyl piperazine. Modifications to the linker group of GN Series included (A) furan, (B) PEG, or (C) phenyl-based linkers.

[0018] FIG. 6. Structure of GN17 and GN64. Cytotoxicity and lysosomal inhibition profiling identified (A) GN17 and (B) GN64 as lead small molecules.

[0019] FIG. 7. GN17 and GN64 confer cytotoxicity in PDAC cell lines with greater potency than HCQ. PDAC cells (BxPC-3, MIA PaCa-2, PANC-1, PaTu 8988T) were treated with (A) GN17 and (B) GN64 [10-0.078 pM, half-fold dilutions], (C) HCQ [250- 1.95 pM] or vehicle (DMSO) for 72h. Cell viability was quantified using the CellTiter-Glo 2.0 Cell Viability Assay and normalized to DMSO treated cells and displayed as percentage of cytotoxicity. All data from n=3 biological replicates and is represented as the mean ± SEM. (D) IC50 values were calculated from each dose response curve. GN17 and GN64 gave similar cytotoxicity profiles with >10-fold potency compared to HCQ. Data is represented as the mean.

[0020] FIG. 8. GN64 inhibits lysosomal degradation with higher potency than GN17. PaTu 8988T cells were treated with GN17 and GN64 [10-0.078 pM, half-fold dilutions], BafAl [100 nM], Torin-1 [1 pM], or vehicle (DMSO) for 12h. (A) Representative fluorescence images of DQ-BSA (red) and nuclear stain, Hoechst (blue) (B) DQ-BSA fluorescence intensity was quantified and normalized to DMSO (0% inhibition) and BafAl (100% inhibition) treated cells and displayed as percent inhibition. GN64 shows greater potency than GN17. All data from n=3 biological replicates and is represented as the mean ± SEM.

[0021] FIG. 9. GN64 induces nuclear translocation of TFEB. U2-OS mCh-TFEB cells were treated with GN64 [2.5, 1.25, 0.5 pM], Torin-1 [1 pM], or vehicle (DMSO) control for 5h. Nuclei were labeled with Hoechst and cells were fixed in 4% PF A. (A) Representative -5-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 fluorescence images of mCh-TFEB (red) and Hoechst (blue) (B) Nuclear translocation of TFEB was quantified as nuclear / cytoplasmic intensity ratio and compared to levels induced by Torin-1 and DMSO (dotted lines). All data from at least n=2 biological replicates and is represented as mean ± SD.

[0022] FIG. 10. Cytotoxicity profiling of GNU in HPOs. A panel of PDAC HPO lines were treated with GNU [30-1 nM, 10 dilution range], cytotoxic positive control, SN38 [10 pM] or vehicle (DMSO) for 6d. Cell viability was quantified using the CellTiter-Glo 3D Cell Viability Assay and normalized to DMSO (0% cytotoxicity) and SN38 (100% cytotoxicity). IC50 was calculated for each HPO line. Data is represented as the IC50 for each replicate (black dots) and the median (red cross). All data from n=2 biological replicates.

[0023] FIG. 11. GN64 induces accumulation of LC3-II in PDAC cells. PDAC cells (MIA PaCa-2, PaTu 8988T) were treated with GN64 [2.5, 1, 0.5 pM], HCQ [20 pM], or vehicle (DMSO) for 24h. LC3 (LC3-I and LC3-II) levels were detect by Western blot. (A) Representative Western blot is shown. (B, C) LC3-II levels were quantified and normalized to Tubulin levels and DMSO treatment. GN64 [2.5 pM] induced accumulation of LC3-II in both MIA PaCa-2 and PaTu 8988T cells (ordinary one-way ANOVA, p=0.0353 and p=0.0334, respectively). All data from n>3 biological replicates and is represented as mean ± SD.

[0024] FIG. 12. GN64 induces the accumulation of p62 in PDAC cells. PDAC cells (MIA PaCa-2, PaTu 8988T) were treated with GN64 [2.5, 1, 0.5 pM], HCQ [20 pM], or vehicle (DMSO) for 24h. p62 levels were detect by Western blot. (A) Representative Western blot is shown. (B, C) p62 levels were quantified and normalized to Tubulin levels and DMSO treatment. GN64 [2.5 pM] induced accumulation of p62 in both MIA PaCa-2 and PaTu 8988T cells (ordinary one-way ANOVA, both p<0.0001). In PaTu 8988T cells, GN64 [1 pM] and [0.5 pM] also induced accumulation of p62 (ordinary one-way ANOVA, both p<0.0001). All data from n>3 biological replicates and is represented as mean ± SD.

[0025] FIG. 13. HCQ, but not GN64, reduces levels of mature-CTSD in PDAC cells.PDAC cells (MIA PaCa-2, PaTu 8988T) were treated with GN64 [2.5, 1, 0.5 pM], HCQ [20-6-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 pM], or vehicle (DMSO) for 24h. CTSD levels (Pro- and mature-CTSD) were detect by Western blot. (A) Representative Western blot is shown. Non-specific bands are denoted with an (*) (B, C) Mature CTSD levels were quantified and normalized to Tubulin levels and DMSO treatment. HCQ [20 pM] reduced levels of mature-CTSD in PDAC cells (ordinary one-way ANOVA, p=0.0043 and p=0.0134, respectively). GN64 did not alter levels of mature-CTSD. All data from n>3 biological replicates and is represented as mean ± SD

[0026] FIG. 14. GN64 increases protein levels of LAMP1 in PDAC cells. PDAC cells (MIA PaCa-2, PaTu 8988T) were treated with GN64 [2.5, 1, 0.5 pM], HCQ [20 pM], or vehicle (DMSO) for 24h. LAMP1 levels were detect by Western blot. (A) Representative Western blot is shown. (B, C) LAMP1 levels were quantified and normalized to Tubulin levels and DMSO treatment. GN64 [2.5 pM] induced accumulation of LAMP1 in both MIA PaCa-2 and PaTu 8988T cells (ordinary one-way ANOVA, p= 0.0090 and p= 0.0222, respectively). In PaTu 8988T cells, GN64 [1 pM] also induced accumulation of LAMP2 (ordinary one-way ANOVA, both p=0.0150). All data from n>3 biological replicates and is represented as mean ± SD.

[0027] FIG. 15. GN64 induces enlargement of lysosomes. PaTu 8988T cells were treated with GN64 [2.5, 1.25 pM] or vehicle (DMSO) control for 24h. Cells were fixed in ice cold methanol. Immunofluorescence was performed with antibody against LAMP2 and nuclear stain, DAPI. (A) Representative immunofluorescence images (scale bar: 10 pM). ROI of LAMP2 shown in bottom row for each treatment. (B) Lysosomal size was calculated using quantitative image analysis. GN64 [2.5, 1.25 pM] induces an enlargement in lysosome (one-way ANOVA, p<0.0001 for both concentrations). Data is representative from one biological replicate and is represented as mean ± SD. Experiment has been repeated three times with similar results.

[0028] FIG. 16. GN64 enhances LysoTracker labeling in PDAC cells. PDAC cells (MIA PaCa-2, PaTu 8988T) were treated with GN64 [2.5, 1.25, 0.625 pM], HCQ [20 pM], BafAl [100 nM] or vehicle (DMSO) for 24h. Labeling with LysoTracker Red was performed as well as nuclear stain, Hoechst. Cells were fixed with 4% PFA and fluorescence microscopy was performed. Representative fluorescent images showing an increase in LysoTracker Red -7-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325(red) labeling induced by GN64 and a decrease by both HCQ and BafAl. Experiment has been repeated three times with similar results.

[0029] FIG. 17. GN64 treatment induces increased LysoSensor fluorescence, indicative of increased acidification of lysosomes. MIA PaCa-2 cells were treated with GN64 [2.5, 1.25, 0.625, 0.3215 pM], BafAl [100 nM] or vehicle (DMSO) control for 24h. Labeling with LysoSensor Green was performed as well as nuclear stain, Hoechst. Fluorescence microscopy was performed. (A) Representative fluorescence images (scale bar: 10 pM) (B) An upper threshold for positive LysoTracker Green signal was established and the area occupied by positive LysoTracker Green labeling per cell (%) was calculated using quantitative image analysis. GN64 induces increases LysoSensor fluorescence (one-way ANOVA, p<0.0001 for all concentrations). Data is representative from one biological replicate and is represented as mean ± SD. Experiment has been repeated three times with similar results.

[0030] FIG. 18. GN64 induces hyperacidification of lysosomes in PDAC cells. PaTu 8988T cells were treated with GN64 [2.5, 1.25, 0.625, 0.3215 pM], BafAl [100 nM] or vehicle (DMSO) control for 24h with simultaneous labeling with ApHID-Alexa647. Fluorescence microscopy was performed. (A) Representative fluorescence images showing pH sensitive ApHID (green) and pH insensitive Alexa647 (red). Using untreated, ApHID labeled cells and pH calibration buffers, a standard curve was generated. pH was calculated based on the ratio of ApHID / Alexa647. (B) Using this standard curve, the lysosomal pH of treated cells was calculated. GN64 [2.5, 1.25 pM] induces hyperacidification of lysosomes (one-way ANOVA, p= 0.0073, p=0.0357, respectively). (C) As a control, BafAl showed an increase in lysosomal pH (unpaired t-test, p=0.0047). All data from n>3 biological replicates and is represented as mean ± SD.

[0031] FIG. 19. GN64 induces transcriptional upregulation of lysosomal genes and specifically, those related to ganglioside catabolism. PaTu 8988T cells were treated with GN64 [2.5 pM] or vehicle (DMSO) control for 24h. Bulk RNA sequencing was performed on the samples. Pathway analysis was performed on statistically significantly upregulated genes using (A) KEGG (FDR < 0.05) and (B) GO Biological Process (FDR < 0.05) pathway databases. (C) A heatmap of statistically significant upregulated genes from the -8-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325KEGG Lysosome pathway. (D) A heatmap showing all analyzed genes from the GO Biological Processes Ganglioside Catabolic Process pathway. For (B, C) data is displayed as the mean log2(Fold Change) from n=3 biological replicates. Statistical significance was set at adjusted p<0.05 and expressed as *<0.05, **<0.01, ***<0.001, and ****<0.0001.

[0032] FIG. 20. GN64 does not affect Rab5 protein levels. PDAC cells (MIA PaCa-2, PaTu 8988T) were treated with GN64 [2.5, 1, 0.5 pM], HCQ [20 pM], or vehicle (DMSO) for 24h. Rab5 levels were detect by Western blot. (A) Representative Western blot is shown. (B, C) Rab5 levels were quantified and normalized to Tubulin levels and DMSO treatment. All data from n>3 biological replicates and is represented as mean ± SD.

[0033] FIG. 21. GN64 induces accumulation of Rab7. PDAC cells (MIA PaCa-2, PaTu 8988T) were treated with GN64 [2.5, 1, 0.5 pM], HCQ [20 pM], or vehicle (DMSO) for 24h. Rab7 levels were detect by Western blot. (A) Representative Western blot is shown. (B, C) Rab7 levels were quantified and normalized to Tubulin levels and DMSO treatment. All data from n>3 biological replicates and is represented as mean ± SD.

[0034] FIG. 22. GNP2 2 is a photoprobe based on the pharmacophore of GN64. TheGN Pharmacophore of GN64 (orange) was modified to add a photoreactive benzophenone (purple) and click-chemistry amendable alkyne handle (green) for use in target identification studies.

[0035] FIG. 23. GNP2 2 retains ALP inhibitory properties and PDAC cytotoxicity.PaTu 8988T cells were treated with GNP2_2 and GN64 [10-0.078 pM, half-fold dilutions], BafAl [100 nM], Torin-1 [1 pM], or vehicle (DMSO) for 12h. (A) Representative fluorescence images of DQ-BSA (red) and nuclear stain, Hoechst (blue) (B) DQ-BSA fluorescence intensity was quantified and normalized to DMSO (0% inhibition) and BafAl (100% inhibition) treated cells and displayed as percent inhibition. GNP2 2 shows similar lysosomal degradation inhibition profile to GN64. (C) PDAC cells (BxPC-3, MIA PaCa-2, PANC-1, PaTu 8988T) were treated with GNP2_2 [10-0.078 pM, half-fold dilutions] or vehicle (DMSO) for 72h. Cell viability was quantified using the CellTiter-Glo 2.0 Cell Viability Assay and normalized to DMSO treated cells and displayed as percentage of cytotoxicity. All data from n=3 biological replicates and is represented as the mean ± SEM.-9-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0036] FIG. 24. Experimental scheme for in situ photolabeling for subcellular visualization of GNP2_2. Combining in situ photolabeling with immunofluorescence can be used to visualize the subcellular localization of GNP2 2. PDAC cells are treated with GNP2 2 (Ih) and subjected to UV-crosslinking. Cells are fixed with ice cold methanol. Click-chemistry is performed on fixed cells using a fluorescent-azide for attachment to the alkyne handle of GNP2 2. Following click-chemistry, traditional immunofluorescence techniques are used and combined with fluorescent microscopy to visualize specific proteins markers and GNP2_2.

[0037] FIG. 25. GNP2 2 is localized to lysosomes. In situ photolabeling was performed in PaTu 8988T cells. Cells were treated with DMSO or GNP2_2 [1 pM] followed by UV irradiation and ice-cold methanol fixation. Click-chemistry with AlexaFluor(AF)647-N3 (magenta) was performed on fixed cells. Immunofluorescence was performed with antibodies against LAMP 1 (green), CTSD (red), and nuclear stain, DAPI (blue). Representative fluorescence images are shown. GNP2_2 labels lysosomes (LAMP1+, CTSD+) as shown by colocalization (Column A and ROI). Click-chemistry and secondary fluorescent antibodies without the presence of primary antibodies show no background signal (Column B). Click-chemistry without presence of GNP2 2 shows minimal background signal (Column C). Experiment has been repeated three times with similar results.

[0038] FIG. 26. Experimental scheme for live cell photolabeling for target identification. Combining live cell photolabeling with affinity enrichment can be used to determine protein targets of GN64. Cells are first treated with and without GN64 parent blocking to be able to identify specific protein interactions. Cells are next treated with GNP2 2 and subjected to UV-crosslinking. Cells are lysed using tip sonication and ultracentrifugation-based cell fractionation (membrane versus soluble fraction) is performed. Click-chemistry is performed using TAMRA-Biotin-Azide with a cleavable Dde linker. Affinity enrichment is performed with streptavidin-agarose beads. Proteins are eluted from the beads using hydrazine. Eluent can be subjected to proteomic or immunoblotting analyses.-10-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0039] FIG. 27. Live cell photolabeling and in-gel fluorescence reveal specifically labeled protein bands of interest. Live cell photolabeling was performed in PaTu 8988T cells. Parent blocking was performed with either GN64 [25 pM] or vehicle (DMSO). Cells were then incubated with DMSO or GNP2_2 [100 nM] followed by UV irradiation, lysis, and cellular fractionation. Click-chemistry with TAMRA-Biotin-Azide was performed on membrane fraction followed by subsequent streptavidin affinity enrichment. Eluents were analyzed by SDS PAGE. In-gel fluorescence was used to visualize labeled protein bands. Specifically labeled protein bands of interest were identified at 200, 120, 80, 50 kDa.

[0040] FIG. 28. Volcano plot of proteins labeled by GNP2 2 and identified by quantitative proteomics. Live cell photolabeling was performed in PaTu 8988T cells. Parent blocking was performed with either GN64 [10 pM] or vehicle (DMSO). Cells were then incubated with DMSO or GNP2_2 [100 nM] followed by UV irradiation, lysis, and cellular fractionation. Click-chemistry with TAMRA-Biotin-Azide was performed on membrane fraction followed by subsequent streptavidin affinity enrichment. Eluents were analyzed by LC-MS / MS. Proteins enriched in the GNP2 2 photolabeling without parent blocking are on the left side of the volcano plot. Proteins >1.5 fold enrichment are highlighted in orange and those also reaching statistical significance are shown in green.

[0041] FIG. 29. GNP2 2 specifically photolabels NPC1. Live cell photolabeling was performed in PaTu 8988T cells. Parent blocking was performed with either GN64 [10 pM] or vehicle (DMSO). Cells were then incubated with DMSO or GNP2_2 [50, 10 nM] followed by UV irradiation, lysis, and cellular fractionation. Click-chemistry with TAMRA- Biotin-Azide was performed on membrane fraction followed by subsequent streptavidin affinity enrichment. Input and streptavidin enrichment eluents were analyzed by Western blot. Representative Western blots are shown. Input levels of NPC1 across treatments were equivalent (upper blot). GNP2_2 [50, 10 nM] specifically labels NPC1 (Lower blot, lanes 2, 4, respectively) and labeling is blocked by GN64 [10 pM] (Lower blot, lanes 3, 4). A control treatment for non-specific binding to the streptavidin-agarose beads, without GNP2 2, shows no enrichment of NPC1 (Lower blot, lane 1). Experiment has been repeated three times with similar results.-11-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0042] Fig. 30. HCQ fails to block GNP2 2 specific photolabeling of NPC1. In vitro photoaffinity labeling was performed using membrane fraction from PDAC (PaTu 8988T) cells. Affinity labeling of NPC1 with GNP2_2 is specifically blocked by both GN64 and GN79 but not with HCQ.|0043] FIG. 31. Structure of NPC1 inhibitor, U18666A and its photoprobe, U-X. (A)U18666A is a cationic amphiphilic drug discovered to inhibit NPC1. U18666A was discovered to bind the sterol sensing domain of NPC1 through studies utilizing (B) U-X, a photoprobe based on the core structure of U18666 A.

[0044] FIG. 32. GN64 does not induce cholesterol accumulation. PaTu 8988T cells were treated with GN64 [2.5, 1.25, 0.0625 pM], NPCi, U18666A [5 pM] or vehicle (DMSO) control for 24h. Cells were fixed in 1.5% PF A. Free cholesterol fluorescence labeling was performed with fllipin. (A) Representative fluorescence images (B) Lysosomal storage organelle (LSO) compartment ratio was calculated using quantitative image analysis. U18666A [5 pM] induces accumulation of free cholesterol (one-way ANOVA, p<0.0001). GN64 did not affect levels of free cholesterol. All data from n=3 biological replicates and is represented as mean ± SD.

[0045] FIG. 33. GN64 synergizes with U18666A to induce cholesterol accumulation.PaTu 8988T cells were pre-treated with U18666A [500 nM] or vehicle (DMSO) control for 3h. GN64 [2.5, 1.25, 0.0625 pM] or DMSO was added for an additional 21h. Cells were fixed in 1.5% PF A. Free cholesterol fluorescence labeling was performed with fllipin. (A) Representative fluorescence images (B) Lysosomal storage organelle (LSO) compartment ratio was calculated using quantitative image analysis and normalized to DMSO. GN64 [2.5, 1.25 pM] synergizes with U18666A [500 nM] to induce accumulation of free cholesterol. All data from n=5 biological replicates and is represented as mean ± SD.

[0046] FIG. 34. NPCli are weakly cytotoxic to PDAC cells. PDAC cells ((A) MIA PaCa- 2, (B) PaTu 8988T) were treated with GN64, Itraconazole (ITZ), U18666A [10-0.156 pM, half-fold dilutions] or vehicle (DMSO) for 72h. Cell viability was quantified using the CellTiter-Glo 2.0 Cell Viability Assay and normalized to DMSO treated cells and displayed as percentage of cytotoxicity. GN64 shows low micro-molar cytotoxicity while NPCli-12-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 show minimal cytotoxicity. All data from n=3 biological replicates and is represented as the mean ± SEM.

[0047] FIG. 35. Combination treatment with U18666A does not enhance cytotoxicity of GN64. PDAC cells ((A) MIA PaCa-2, (B) PaTu 8988T) were pre-treated with U18666A [500 nM] for 3h. GN64 [10-0.156 pM, half-fold dilutions] or vehicle (DMSO) was added for 72h. Cell viability was quantified using the CellTiter-Glo 2.0 Cell Viability Assay and normalized to DMSO treated cells and displayed as percentage of cytotoxicity. Pretreatment with U18666A does not alter the cytotoxicity profile of GN64. All data from n=3 biological replicates and is represented as the mean ± SEM.

[0048] FIG. 36. GN64 and U18666A cross compete photolabeling of photoprobesGNP2_2 and U-X. Live cell photolabeling was performed in PaTu 8988T cells. Parent blocking was performed with either GN64 [10 pM], U18666A [10 pM], or vehicle (DMSO). Cells were then incubated with DMSO, U-X [lOOnM] or GNP2_2 [100 nM] followed by UV irradiation, lysis, and cellular fractionation. Click-chemistry with TAMRA- Biotin-Azide was performed on membrane fraction followed by subsequent streptavidin affinity enrichment. Input and streptavidin enrichment eluents were analyzed by Western blot. Representative Western blots are shown. Input levels of NPC1 across treatments were equivalent (upper blot). U-X specifically labels NPC1 (Lower blot, lane 1) and labeling is blocked by U18666A (Lower blot, lane 2) and GN64 (Lower blot, lane 3). GNP2_2 specifically labels NPC1 (Lower blot, lane 3) and labeling is blocked by U18666A (Lower blot, lane 5) and GN64 (Lower blot, lane 6). Experiment has been repeated three times with similar results.

[0049] FIG. 37. GNP2 2 fails to label NPC1-P691S. Live cell photolabeling was performed in PaTu 8988T cells expression endogenous NPC1 as well as NPC1-WT-FLAG as well as with the following mutations: P202F203A, F503 / 4A, P691S, or D786N. Parent blocking was performed with GN64 [5 pM] or vehicle (DMSO). Cells were then incubated with GNP2_2 [50 nM] followed by UV irradiation, lysis, and cellular fractionation. Clickchemistry with TAMRA-Biotin-Azide was performed on membrane fraction followed by subsequent streptavidin affinity enrichment. Input and streptavidin enrichment eluents were analyzed by Western blot. Representative Western blots are shown. Input levels of NPC1- -13-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325FLAG across treatments were equivalent with and without parent blocking (upper blot). GNP2 2 specifically photolabeled NPC1-WT-FLAG and NPC1-P202 / F203A-FLAG (Lower blots, lanes 1-4). Expression of NPC1-F503 / 4A-FLAG was not detected. Although NPC1-P691 S-FLAG expression was greater than NPC1-WT-FLAG (Upper blot, lanes 1,2 vs. 7, 8), GNP2_2 failed to photolabel NPC1-P691 S-FLAG (Lower blots, lanes 7, 8). GNP2_2 specific photolabeling of NPC1-D786N-FLAG was enhanced (Lower blots, lanes 9, 10) compared to NPC1-WT-FLAG (Lower blots, lanes 1, 2). Experiment has been repeated three times with similar results.

[0050] FIG. 38. CRISPR / Cas9 generation of NPC1 KO in PDAC cells. PDAC cells (MIA PaCa-2, PaTu 8988T) were transfected with the PX459 plasmid containing Cas9 and sgRNA against NPC 1. Single clones were generated and assessed for NPC 1 expression. Representative Western blots are shown for (A) MIA PaCa-2 and (B) PaTu 8988T cells. Western blot confirmed NPC1 KO in two clones for MIA PaCa-2 (#1, #2) and three clones for PaTu 8988T (#1, #2, #3).

[0051] FIG. 39. PDAC NPC1 KO cells show resistance to GN64 cytotoxicity. (A) MIA PaCa-2 (WT and NPC1 KO #1, 2) and (D) PaTu 8988T (WT and NPC1 KO #1, 2, 3) were treated with GN64 [10-0.3125 pM, half-fold dilutions] or vehicle (DMSO) for 24h. Cell viability was quantified using the CellTiter-Glo 2.0 Cell Viability Assay and normalized to DMSO treated cells and displayed as percent cell viability. Data displayed as the mean ± SEM. (B, E) Area under the curve was calculated for each dose response curve. NPC1 KO cells show increased AUC, suggesting resistance to GN64. Data displayed as the mean ± SD (C) Cell viability of MIA PaCa-2 cells treated with GN64 [2.5 pM]. NPC1 KO cells show enhanced viability. Data displayed as the mean ± SD. (F) Cell viability of PaTu 8988T cells treated with GN64 [5 pM], NPC1 KO cells show enhanced viability. Data displayed as the mean ± SD. All data from n=3 biological replicates.

[0052] FIG. 40. The pH of PDAC NPC1 KO lysosomes trends lower compared to WT cells. PaTu 8988T WT and NPC1 KO cells labeled with ApHID-Alexa647 for 24h. Fluorescence microscopy was performed. Using untreated, ApHID labeled cells and pH calibration buffers, a standard curve was generated. pH was calculated based on the ratio of ApHID / Al exa647. Using this standard curve, the lysosomal pH of WT vs. NPC1 KO cells -14-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 was calculated. The pH of NPC1 KO cells trended lower compared to WT (pH 4.103 vs pH 4.386, one-way ANOVA). All data from n=3 biological replicates and is represented as mean ± SD.

[0053] FIG. 41. Effect of U18666A on lysosomal pH of PDAC WT and NPC1 KO cells.PaTu 8988T (WT and NPC1 KO) were treated with U18666A [5 pM] or vehicle (DMSO) control for 24h with simultaneous labeling with ApHID-Alexa647. Fluorescence microscopy was performed. Using untreated, ApHID labeled cells and pH calibration buffers, a standard curve was generated. pH was calculated based on the ratio of ApHID / Alexa647. Using this standard curve, the lysosomal pH of treated cells was calculated. The pH of U18666A treated WT trended lower compared to untreated WT cells (pH 3.4636 vs pH 4.166, one-way ANOVA). U18666A had no effect of lysosomal pH of NPC1 KO cells (pH 3.800 vs 3.875). All data from n>3 biological replicates and is represented as mean ± SD.

[0054] FIG. 42. GN64 induces hyperacidification of lysosomes in PDAC WT cells but not NPC1 KO. PaTu 8988T (WT and NPC1 KO) were treated with Gn64 [2.5, 1.25, 0.625, 0.3125 pM] or vehicle (DMSO) control for 24h with simultaneous labeling with ApHID- Alexa647. Fluorescence microscopy was performed. Using untreated, ApH ID labeled cells and pH calibration buffers, a standard curve was generated. pH was calculated based on the ratio of ApHID / Alexa647. Using this standard curve, the lysosomal pH of treated cells was calculated. GN64 [2.5, 1.25 pM] induces hyperacidification of lysosomes (one-way ANOVA, p=0.0001, p=0.0024, respectively). GN64 failed to induce hyperacidification in NPC1 KO cells. All data from n>3 biological replicates and is represented as mean ± SD.

[0055] FIG. 43. GNP2 2 specifically photolabels mature TPP1 form. Live cell photolabeling was performed in PaTu 8988T cells. Parent blocking was performed with either GN64 [10 pM] or vehicle (DMSO). Cells were then incubated with DMSO or GNP2_2 [50, 10 nM] followed by UV irradiation, lysis, and cellular fractionation. Clickchemistry with TAMRA-Biotin-Azide was performed on membrane fraction followed by subsequent streptavidin affinity enrichment. Input and streptavidin enrichment eluents were analyzed by Western blot. Representative Western blots are shown. Input levels of NPC1 and TPP1 (mature) across treatments were equivalent (upper blot). GNP2 2 [50, 10 nM]-15-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 specifically labels NPC1 (Lower blot, lanes 2, 4, respectively) and labeling is blocked by GN64 [10 pM] (Lower blot, lanes 3, 4). GNP2_2 [50 nM] specifically labels TPP1 (mature) (Lower blot, lane 2) and labeling is blocked by GN64 [10 pM] (Lower blot, lane 3). A control treatment for non-specific binding to the streptavidin-agarose beads, without GNP2 2, shows no enrichment of NPC1 or TPP1 (Lower blot, lane 1). Experiment has been repeated three times with similar results.

[0056] FIG. 44. TPP1 KO in PDAC cell lines does not alter sensitivity profile to GN64.PDAC cells (MIA PaCa-2, PaTu 8988T) were transfected with the PX459 plasmid containing Cas9 and sgRNA against Tppl. Single clones were generated and assessed for TPP1 expression. Representative Western blots are shown for (A) MIA PaCa-2 and (C) PaTu 8988T cells. Western blot confirmed TPP1 KO in three clones for MIA PaCa-2 (#1, #2, #3) and three clones for PaTu 8988T (#1, #2, #3). Cytotoxicity profiling (72h) was performed in (B) MIA PaCa-2 and (D) PaTu 8988T WT and TPP1 KO cells. Cell viability was quantified using the CellTiter-Glo 2.0 Cell Viability Assay and normalized to DMSO treated cells and displayed as percent cell viability. Data displayed as the mean ± SEM.

[0057] FIG. 45. GN64 fails to alter TPP1 activity in vitro. PDAC cells lysates were generated in Acetate buffer (0.1M sodium acetate, pH 4.0) containing E64-d [10 pM] and Pepstatin A [10 pM] Lysates were treated with GN64 [10, 5, 2.5, 1.25 pM], TPPli, AAF- CMK [20 pM] or vehicle (DMSO) control. TPP1 substrate, AAF-AMC [62.5 pM] was added to lysates as well. Cells were incubated at 37°C, shaking for 24h. Reaction was halted by adding 0.5M EDTA, pH 12.0. AMC fluorescence was measured using a fluorescence plate reader. Background subtraction was performed using fluorescence from AAF-AMC in Acetate buffer alone (without cells) and normalized to DMSO (100% activity). Data displayed as the mean ± SD. All data from n>2 biological replicates.

[0058] FIG. 46. GN64 fails to photolabel SLC19A1. Live cell photolabeling was performed in PaTu 8988T cells. Parent blocking was performed with either GN64 [10 pM] or vehicle (DMSO). Cells were then incubated with DMSO or GNP2_2 [50, 10 nM] followed by UV irradiation, lysis, and cellular fractionation. Click-chemistry with TAMRA- Biotin-Azide was performed on membrane fraction followed by subsequent streptavidin affinity enrichment. Input and streptavidin enrichment eluents were analyzed by Western -16-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 blot. Representative Western blots are shown. Input levels of NPC1 and SLC19A1 across treatments were equivalent (upper blot). GNP2_2 [50, 10 nM] specifically labels NPC1 (Lower blot, lanes 2, 4, respectively) and labeling is blocked by GN64 [10 pM] (Lower blot, lanes 3, 4). GNP2 2 fails to photolabel SLC19A1. Experiment has been repeated three times with similar results.

[0059] FIG. 47. GNP2 2 specifically photolabels IFITM3. Live cell photolabeling was performed in PaTu 8988T cells. Parent blocking was performed with either GN64 [10 pM] or vehicle (DMSO). Cells were then incubated with DMSO or GNP2_2 [50, 10 nM] followed by UV irradiation, lysis, and cellular fractionation. Click-chemistry with TAMRA- Biotin-Azide was performed on membrane fraction followed by subsequent streptavidin affinity enrichment. Input and streptavidin enrichment eluents were analyzed by Western blot. Representative Western blots are shown. Input levels of NPC1 and IFITM3 across treatments were equivalent (upper blot). GNP2_2 [50, 10 nM] specifically labels NPC1 (Lower blot, lanes 2, 4, respectively) and labeling is blocked by GN64 [10 pM] (Lower blot, lanes 3, 4). GNP2_2 [50 nM] specifically labels IFITM3 (Lower blot, lane 2) and labeling is blocked by GN64 [10 pM] (Lower blot, lane 3). A control treatment for nonspecific binding to the streptavidin-agarose beads, without GNP2 2, shows no enrichment of NPC1 or IFITM3 (Lower blot, lane 1). Experiment has been repeated three times with similar results.

[0060] FIG. 48. IFITM3 KO in PDAC cell lines does not alter sensitivity profile to GN64. PDAC cells (MIA PaCa-2, PaTu 8988T) were transfected with the PX459 plasmid containing Cas9 and sgRNA against IFITM3. Single clones were generated and assessed for IFITM3 expression. Representative Western blots are shown for (A) MIA PaCa-2 and (C) PaTu 8988T cells. Western blot confirmed IFITM3 KO in two clones for MIA PaCa-2 (#1, #2) and one clone for PaTu 8988T (#1). Cytotoxicity profiling (72h) was performed in (B) MIA PaCa-2 and (D) PaTu 8988T WT and IFITM3 KO cells. Cell viability was quantified using the CellTiter-Glo 2.0 Cell Viability Assay and normalized to DMSO treated cells and displayed as percent cell viability. Data displayed as the mean ± SEM.

[0061] FIG. 49. Mitochondrial proteins are specifically labeled by GNP2 2 only after preincubation with GN64. Live cell photolabeling was performed in PaTu 8988T cells.-17-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325Parent blocking was performed with either GN64 [10 pM] or vehicle (DMSO). Cells were then incubated with DMSO or GNP2_2 [100 nM] followed by UV irradiation, lysis, and cellular fractionation. Click-chemistry with TAMRA-Biotin-Azide was performed on membrane fraction followed by subsequent streptavidin affinity enrichment. Eluents were analyzed by LC-MS / MS. Proteins enriched in the GNP2 2 photolabeling without parent blocking are on the left side of the volcano plot. Proteins enriched in the parent (GN64) blocking samples are on the right side of the volcano plot. Mitochondrial proteins are highlighted in red and are specifically enriched in the parent blocking samples.

[0062] FIG. 50. GN64 effect on NPC1 protein levels in PDAC cells. PDAC cells (MIA PaCa-2, PaTu 8988T) were treated with GN64 [2.5, 1, 0.5 pM], HCQ [20 pM], or vehicle (DMSO) for 24h. NPC1 levels were detect by Western blot. (A) Representative Western blot is shown. NPC1 levels were quantified and normalized to Tubulin levels and DMSO treatment. (A) GN64 [0.5 pM] decreased NPC1 expression in MIA PaCa-2 cells (one-way ANOVA, p=0.0355) (B) GN64 [2.5, 1 pM] increased NPC1 expression in PaTu 8988T cells (ordinary one-way ANOVA, p=0.0370 and p=0.0054, respectively). All data from n>3 biological replicates and is represented as mean ± SD.

[0063] FIG. 51. GN64 increases NPC2 expression. PDAC cells (MIA PaCa-2, PaTu 8988T) were treated with GN64 [2.5, 1, 0.5 pM], HCQ [20 pM], or vehicle (DMSO) for 24h. NPC2 levels were detect by Western blot. (A) Representative Western blot is shown. NPC2 levels were quantified and normalized to Tubulin levels and DMSO treatment. (A) GN64 [2.5 pM] increased NPC2 expression in MIA PaCa-2 cells (one-way ANOVA, p=0.0176) (B) GN64 [1, 0.5 pM] increased NPC2 expression in PaTu 8988T cells (ordinary one-way ANOVA, p=0.0149 and p=0.0398, respectively). All data from n>3 biological replicates and is represented as mean ± SD.

[0064] FIG. 52. GN79*HC1 (“GN79HC1”) retains cytotoxicity and ALP inhibitory properties in PDAC cells. PaTu 8988T cells were treated with GN79HC1 and GN64 [10- 0.078 pM, half-fold dilutions], BafAl [100 nM], Torin-1 [1 pM], or vehicle (DMSO) for 12h. (A) Representative fluorescence images of DQ-BSA (red) and nuclear stain, Hoechst (blue) (B) DQ-BSA fluorescence intensity was quantified and normalized to DMSO (0% inhibition) and BafAl (100% inhibition) treated cells and displayed as percent inhibition.-18-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325GN79HC1 shows lysosomal degradation inhibition. (C) PDAC cells (BxPC-3, MIA PaCa-2, PANC-1, PaTu 8988T) were treated with GN79HC1 [10-0.078 pM, half-fold dilutions] or vehicle (DMSO) for 72h. Cell viability was quantified using the CellTiter-Glo 2.0 Cell Viability Assay and normalized to DMSO treated cells and displayed as percentage of cytotoxicity. All data from n=3 biological replicates and is represented as the mean ± SEM.

[0065] FIG. 53. GN79HC1 confers cytotoxicity to HPO lines. A panel of PDAC HPO lines were treated with GN79 [30-1 nM, 10 dilution range], cytotoxic positive control, SN38 [10 pM] or vehicle (DMSO) for 6d. Cell viability was quantified using the CellTiter-Glo 3D Cell Viability Assay and normalized to DMSO (0% cytotoxicity) and SN38 (100% cytotoxicity). IC50 was calculated for each HPO line. Data is represented as the IC50 for each replicate (black dots) and the median (red cross). All data from n=2 biological replicates.

[0066] FIG. 54. GN79HC1 blocks photolabeling with GNP2 2. Live cell photolabeling was performed in PaTu 8988T cells. Parent blocking was performed with either GN64 [5 pM], GN79HC1 [5 pM], or vehicle (DMSO). Cells were then incubated withGNP2_2 [25 nM] followed by UV irradiation, lysis, and cellular fractionation. Click-chemistry with TAMRA-Biotin-Azide was performed on membrane fraction followed by subsequent streptavidin affinity enrichment. Input and streptavidin enrichment eluents were analyzed by Western blot. Representative Western blots are shown. Input levels of NPC1 across treatments were equivalent (upper blot). GNP2_2 specifically labels NPC1 (Lower blot, lane 1) and labeling is blocked by GN64 [10 pM] (Lower blot, lane 2) and GN79HC1 (Lower blow, lane 3). Experiment has been repeated three times with similar results.

[0067] FIG. 55. GN79HC1 in cyclodextrin based formulations retains PDAC cytotoxicity. PDAC cells ((A)MIA PaCa-2, (B) PANC-1, (C) PaTu 8988T) were treated with GN79HC1 in DMSO, 10% HP-[3-CD, or 12% Captisol [10-0.078 pM, half-fold dilutions] or vehicle for 72h. Cell viability was quantified using the CellTiter-Glo 2.0 Cell Viability Assay and normalized to respective vehicle control treated cells and displayed as percentage of cytotoxicity. All data from n=3 biological replicates and is represented as the mean ± SEM.-19-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0068] FIG. 56. Acute pharmacokinetics of GN79HC1 (10% HP-|J-CD). B6 female mice (6-8 weeks, n=12) were dosed with 30 mg / kg of GN79HC1 (10% HP-P-CD) or vehicle control via intraperitoneal (IP) injection. Following single administration, 3 mice from each group were bled at the following timepoints: 30 min, 4hr, 8hr, 24hr. (A) Plasma GN79 concentration was measured via LC / MS analysis. (B) At time of mouse sacrifice, pancreases were removed, and flash frozen. GN79 concentration was measured via LC / MS analysis.

[0069] FIG. 57. GN79HC1 is well-tolerated at lOmg / kg. NSG female mice (6-8 weeks) were dosed with 100, 75, 50, 25, or 10 mg / kg of GN79HC1 (10% HP-P-CD) or vehicle control via IP injection Q.D. x 5d for 2 weeks. Mice weight was monitored for (A) High dose regimines (100, 75, 50 mg / kg) and showed substantial weight loss and death. (B) For 25 mg / kg dosing, mice experienced weight loss but a switch to every other day (QOD) reverted cachexia. (C) GN79HC1 (10% HP-P-CD) was well-tolerated without any observable weight loss or toxicities.

[0070] FIG. 58. GN79HC1 reduces tumor burden in vivo. NSG female mice (6-8 weeks) were subcutaneously injected with five million MIA PaCa-2 cells and once tumors were established, were treated with 10 mg / kg of GN79HC1 (10% HP-P-CD) or vehicle control via IP injection Q.D. x 5d for 2 weeks. Tumor volume was measured twice a week. (A, B) GN79HC1 significantly reduces tumor volume after seven (16% reduction; p = 0.0256), ten (16% reduction; p = 0.0019), and 14 days (23% reduction; p < 0.0001) of treatment compared to vehicle control (two-way ANOVA). All data from n>5 mice per group and is represented as mean ± SD.

[0071] FIG. 59. GN79HC1 treatment induces increased expression of lysosomal and ganglioside catabolism genes. MIA PaCa-2 cells were treated with GN79HC1 [5, 2.5, 1.25 pM] or vehicle (DMSO) control for 24h. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed. (A) GN79HC1 did not cause in increase in expression of GM2A. GN79HC1 induced an increase in expression of ganglioside catabolism genes: (B) HEXA, (C) HEXB, (D) NEU1. GN79HC1 induced an increase in expression of lysosomal gene, (E) NPC2. Data is displayed as the relative expression n=3-20-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 biological replicates. Statistical significance was set at adjusted p<0.05 and expressed as *<0.05, **<0.01, ***<0.001, and ****<0.0001 (ordinary one-way ANOVA),

[0072] FIG. 60. Pharmacokinetic (PK) data obtained for GN64 in plasma and selected organs of B6 female mice (6-8 weeks old). Mice received a single intraperitoneal (i.p.) dose of GN64 monohydrochloride salt at 10 mg / kg, formulated in 10% hydroxypropyl-P- cyclodextrin (HPBCD).DETAILED DESCRIPTION

[0073] Before the present disclosure is further described, it is to be understood that the disclosure is not limited to the particular embodiments set forth herein, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.Definitions]0074] The following terms are used throughout as defined below.

[0075] As used herein and in the appended claims, singular articles such as “a” and “an” and “the” and similar referents in the context of describing the elements (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the embodiments and does not pose a limitation on the scope of the claims unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential.

[0076] As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent depending upon the context in which it is used. If there are uses of-21-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term - for example, “about 10 wt.%” would be understood to mean “9 wt.% to 11 wt.%.” It is to be understood that when “about” precedes a term, the term is to be construed as disclosing “about” the term as well as the term without modification by “about” - for example, “about 10 wt.%” discloses “9 wt.% to 11 wt.%” as well as disclosing “10 wt.%.”

[0077] The phrase “and / or” as used in the present disclosure will be understood to mean any one of the recited members individually or a combination of any two or more thereof - for example, “A, B, and / or C” would mean “A, B, C, A and B, A and C, B and C, or the combination of A, B, and C.”|0078] Generally, reference to a certain element such as hydrogen or H is meant to include all isotopes of that element. For example, if an R group is defined to include hydrogen or H, it also includes deuterium and tritium. Compounds comprising radioisotopes such as tritium, C14, P32and S35are thus within the scope of the present technology. Procedures for inserting such labels into the compounds of the present technology will be readily apparent to those skilled in the art based on the disclosure herein.

[0079] In general, “substituted” refers to an organic group as defined below (e.g., an alkyl group) in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non-hydrogen or non-carbon atoms. Substituted groups also include groups in which one or more bonds to a carbon(s) or hydrogen(s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom. Thus, a substituted group is substituted with one or more substituents, unless otherwise specified. In some embodiments, a substituted group is substituted with 1, 2, 3, 4, 5, or 6 substituents.Examples of substituent groups include: halogens (z.e., F, Cl, Br, and I); hydroxyls; alkoxy, alkenoxy, aryloxy, aralkyloxy, heterocyclyl, heterocyclylalkyl, heterocyclyloxy, and heterocyclylalkoxy groups; carbonyls (oxo); carboxylates; esters; urethanes; oximes; hydroxylamines; alkoxyamines; aralkoxyamines; thiols; sulfides; sulfoxides; sulfones; sulfonyls; pentafluorosulfanyl (z.e., SFs), sulfonamides; amines; N-oxides; hydrazines; hydrazides; hydrazones; azides; amides; ureas; amidines; guanidines; enamines; imides;-22-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 isocyanates; isothiocyanates; cyanates; thiocyanates; imines; nitro groups; and nitriles (z.e., CN).

[0080] Substituted ring groups such as substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups also include rings and ring systems in which a bond to a hydrogen atom is replaced with a bond to a carbon atom. Therefore, substituted cycloalkyl, aryl, heterocyclyl and heteroaryl groups may also be substituted with substituted or unsubstituted alkyl, alkenyl, and alkynyl groups as defined below.

[0081] Alkyl groups include straight chain and branched chain alkyl groups having from 1 to 12 carbon atoms, and typically from 1 to 10 carbons or, in some embodiments, from 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Alkyl groups may be substituted or unsubstituted. Examples of straight chain alkyl groups include groups such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, tert-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. Representative substituted alkyl groups may be substituted one or more times with substituents such as those listed above, and include without limitation haloalkyl (e.g., trifluoromethyl), hydroxyalkyl, thioalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, alkoxyalkyl, carboxyalkyl, and the like.

[0082] Cycloalkyl groups include mono-, bi- or tricyclic alkyl groups having from 3 to 12 carbon atoms in the ring(s), or, in some embodiments, 3 to 10, 3 to 8, or 3 to 4, 5, or 6 carbon atoms. Cycloalkyl groups may be substituted or unsubstituted. Exemplary monocyclic cycloalkyl groups include, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group has 3 to 8 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 5, 3 to 6, or 3 to 7. Bi- and tricyclic ring systems include both bridged cycloalkyl groups and fused rings, such as, but not limited to, bicyclo[2.1.1]hexane, adamantyl, decalinyl, and the like. Substituted cycloalkyl groups may be substituted one or more times with non-hydrogen and non-carbon groups as defined above. However, substituted cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined above. Representative substituted cycloalkyl groups may be mono-substituted or substituted more than once, such as, but not -23- 4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 limited to, 2,2-, 2,3-, 2,4- 2,5- or 2, 6-di substituted cyclohexyl groups, which may be substituted with substituents such as those listed above.

[0083] Cycloalkylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a cycloalkyl group as defined above. Cycloalkylalkyl groups may be substituted or unsubstituted. In some embodiments, cycloalkylalkyl groups have from 4 to 16 carbon atoms, 4 to 12 carbon atoms, and typically 4 to 10 carbon atoms. Substituted cycloalkylalkyl groups may be substituted at the alkyl, the cycloalkyl or both the alkyl and cycloalkyl portions of the group. Representative substituted cycloalkylalkyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri -substituted with substituents such as those listed above.

[0084] Alkenyl groups include straight and branched chain alkyl groups as defined above, except that at least one double bond exists between two carbon atoms. Alkenyl groups may be substituted or unsubstituted. Alkenyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkenyl group has one, two, or three carboncarbon double bonds. Examples include, but are not limited to vinyl, allyl, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, among others. Representative substituted alkenyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri -substituted with substituents such as those listed above.

[0085] Cycloalkenyl groups include cycloalkyl groups as defined above, having at least one double bond between two carbon atoms. Cycloalkenyl groups may be substituted or unsubstituted. In some embodiments the cycloalkenyl group may have one, two or three double bonds but does not include aromatic compounds. Cycloalkenyl groups have from 4 to 14 carbon atoms, or, in some embodiments, 5 to 14 carbon atoms, 5 to 10 carbon atoms, or even 5, 6, 7, or 8 carbon atoms. Examples of cycloalkenyl groups include cyclohexenyl, cyclopentenyl, cyclohexadienyl, cyclobutadienyl, and cyclopentadienyl.-24-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0086] Cycloalkenylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of the alkyl group is replaced with a bond to a cycloalkenyl group as defined above. Cycloalkenylalkyl groups may be substituted or unsubstituted. Substituted cycloalkenylalkyl groups may be substituted at the alkyl, the cycloalkenyl or both the alkyl and cycloalkenyl portions of the group. Representative substituted cycloalkenylalkyl groups may be substituted one or more times with substituents such as those listed above.

[0087] Alkynyl groups include straight and branched chain alkyl groups as defined above, except that at least one triple bond exists between two carbon atoms. Alkynyl groups may be substituted or unsubstituted. Alkynyl groups have from 2 to 12 carbon atoms, and typically from 2 to 10 carbons or, in some embodiments, from 2 to 8, 2 to 6, or 2 to 4 carbon atoms. In some embodiments, the alkynyl group has one, two, or three carboncarbon triple bonds. Examples include, but are not limited to -C=CH, -C=CCH3, -CH2C=CCH3, and -C=CCH2CH(CH2CH3)2, among others. Representative substituted alkynyl groups may be mono-substituted or substituted more than once, such as, but not limited to, mono-, di- or tri -substituted with substituents such as those listed above.

[0088] Aryl groups are cyclic aromatic hydrocarbons that do not contain heteroatoms. Aryl groups herein include monocyclic, bicyclic and tricyclic ring systems. Aryl groups may be substituted or unsubstituted. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, fluorenyl, phenanthrenyl, anthracenyl, indenyl, indanyl, pentalenyl, and naphthyl groups. In some embodiments, aryl groups contain 6-14 carbons, and in others from 6 to 12 or even 6-10 carbon atoms in the ring portions of the groups. In some embodiments, the aryl groups are phenyl or naphthyl. The phrase “aryl groups” includes groups containing fused rings, such as fused aromatic-aliphatic ring systems (e.g., indanyl, tetrahydronaphthyl, and the like). Representative substituted aryl groups may be mono-substituted (e.g., tolyl) or substituted more than once. For example, monosubstituted aryl groups include, but are not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or naphthyl groups, which may be substituted with substituents such as those listed above.

[0089] Aralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined above. Aralkyl -25-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 groups may be substituted or unsubstituted. In some embodiments, aralkyl groups contain 7 to 16 carbon atoms, 7 to 14 carbon atoms, or 7 to 10 carbon atoms. Substituted aralkyl groups may be substituted at the alkyl, the aryl or both the alkyl and aryl portions of the group. Representative aralkyl groups include but are not limited to benzyl and phenethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-indanylethyl. Representative substituted aralkyl groups may be substituted one or more times with substituents such as those listed above.

[0090] Heterocyclyl groups include aromatic (also referred to as heteroaryl) and nonaromatic (also referred to as heterocycloalkyl) ring compounds containing 3 or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Heterocyclyl groups may be substituted or unsubstituted. In some embodiments, the heterocyclyl group contains 1, 2, 3 or 4 heteroatoms. In some embodiments, heterocyclyl groups include mono-, bi- and tricyclic rings having 3 to 16 ring members, whereas other such groups have 3 to 6, 3 to 10, 3 to 12, or 3 to 14 ring members. Heterocyclyl groups encompass aromatic, partially unsaturated and saturated ring systems, such as, for example, imidazolyl, imidazolinyl and imidazolidinyl groups. The phrase “heterocyclyl group” includes fused ring species including those comprising fused aromatic and non-aromatic groups, such as, for example, benzotri azolyl, 2,3-dihydrobenzo[l,4]dioxinyl, and benzofl, 3]dioxolyl. The phrase also includes bridged polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. The phrase includes heterocyclyl groups that have other groups, such as alkyl, oxo or halo groups, bonded to one of the ring members, referred to as “substituted heterocyclyl groups”. Heterocyclyl groups include, but are not limited to, aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, thiazolidinyl, tetrahydrothiophenyl, tetrahydrofuranyl, dioxolyl, furanyl, thiophenyl, pyrrolyl, pyrrolinyl, imidazolyl, imidazolinyl, pyrazolyl, pyrazolinyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, thiazolinyl, isothiazolyl, thiadiazolyl, oxadiazolyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydropyranyl, tetrahydrothiopyranyl, oxathiane, dioxyl, dithianyl, pyranyl, pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, triazinyl, dihydropyridyl, dihydrodithiinyl, dihydrodithionyl, homopiperazinyl, quinuclidyl, indolyl, indolinyl, isoindolyl,azaindolyl (pyrrolopyridyl), indazolyl, indolizinyl, benzotri azolyl, benzimidazolyl, benzofuranyl, benzothiophenyl, benzthiazolyl, benzoxadi azolyl,-26-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 benzoxazinyl, benzodithiinyl, benzoxathiinyl, benzothiazinyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[l,3]dioxolyl, pyrazolopyridyl, imidazopyridyl (azabenzimidazolyl), tri azol opyridyl, isoxazolopyridyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, quinolizinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, pteridinyl, thianaphthyl, dihydrobenzothiazinyl, dihydrobenzofuranyl, dihydroindolyl, dihydrobenzodioxinyl, tetrahydroindolyl, tetrahydroindazolyl, tetrahydrobenzimidazolyl, tetrahydrobenzotriazolyl, tetrahydropyrrolopyridyl, tetrahydropyrazolopyridyl, tetrahydroimidazopyridyl, tetrahydrotriazolopyridyl, and tetrahydroquinolinyl groups. Representative substituted heterocyclyl groups may be mono-substituted or substituted more than once, such as, but not limited to, pyridyl or morpholinyl groups, which are 2-, 3-, 4-, 5-, or 6-substituted, or disubstituted with various substituents such as those listed above.

[0091] Heteroaryl groups are aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S. Heteroaryl groups may be substituted or unsubstituted. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, thiophenyl, benzothiophenyl, furanyl, benzofuranyl, indolyl, azaindolyl (pyrrolopyridinyl), indazolyl, benzimidazolyl, imidazopyridinyl (azabenzimidazolyl), pyrazolopyridinyl, triazolopyridinyl, benzotri azolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups include fused ring compounds in which all rings are aromatic such as indolyl groups and include fused ring compounds in which only one of the rings is aromatic, such as 2,3-dihydro indolyl groups. Representative substituted heteroaryl groups may be substituted one or more times with various substituents such as those listed above.

[0092] Heterocyclylalkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heterocyclyl group as defined above. Heterocyclylalkyl groups may be substituted or unsubstituted. Substituted heterocyclylalkyl groups may be substituted at the alkyl, the heterocyclyl or both the alkyl-27-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 and heterocyclyl portions of the group. Representative heterocyclyl alkyl groups include, but are not limited to, morpholin-4-yl-ethyl, furan-2-yl-methyl, imidazol-4-yl-methyl, pyri din-3 -yl-m ethyl, tetrahydrofuran-2-yl-ethyl, and indol-2-yl-propyl. Representative substituted heterocyclylalkyl groups may be substituted one or more times with substituents such as those listed above.

[0093] Heteroaralkyl groups are alkyl groups as defined above in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined above. Heteroaralkyl groups may be substituted or unsubstituted. Substituted heteroaralkyl groups may be substituted at the alkyl, the heteroaryl or both the alkyl and heteroaryl portions of the group. Representative substituted heteroaralkyl groups may be substituted one or more times with substituents such as those listed above.

[0094] Groups described herein having two or more points of attachment (i.e., divalent, trivalent, or polyvalent) within the compound of the present technology are designated by use of the suffix, “ene.” For example, divalent alkyl groups are alkylene groups, divalent aryl groups are arylene groups, divalent heteroaryl groups are divalent heteroarylene groups, and so forth. Substituted groups having a single point of attachment to the compound of the present technology are not referred to using the “ene” designation. Thus, e.g., chloroethyl is not referred to herein as chloroethylene.

[0095] Alkoxy groups are hydroxyl groups (-OH) in which the bond to the hydrogen atom is replaced by a bond to a carbon atom of a substituted or unsubstituted alkyl group as defined above. Alkoxy groups may be substituted or unsubstituted. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, and the like. Examples of branched alkoxy groups include but are not limited to isopropoxy, sec-butoxy, tert-butoxy, isopentoxy, isohexoxy, and the like. Examples of cycloalkoxy groups include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. Representative substituted alkoxy groups may be substituted one or more times with substituents such as those listed above.

[0096] The terms “alkanoyl” and “alkanoyloxy” as used herein can refer, respectively, to - C(O)-alkyl groups and -O-C(O)-alkyl groups, each containing 2-5 carbon atoms.-28-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325Similarly, “aryloyl” and “aryloyloxy” refer to -C(O)-aryl groups and -O-C(O)-aryl groups.

[0097] The terms "aryloxy" and “arylalkoxy” refer to, respectively, a substituted or unsubstituted aryl group bonded to an oxygen atom and a substituted or unsubstituted aralkyl group bonded to the oxygen atom at the alkyl. Examples include but are not limited to phenoxy, naphthyloxy, and benzyloxy. Representative substituted aryloxy and arylalkoxy groups may be substituted one or more times with substituents such as those listed above.[00981 The term “carboxylate” as used herein refers to a -COOH group.

[0099] The term “ester” as used herein refers to -COOR70and -C(O)O-G groups. R70is a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. G is a carboxylate protecting group. Carboxylate protecting groups are well known to one of ordinary skill in the art. An extensive list of protecting groups for the carboxylate group functionality may be found in Protective Groups in Organic Synthesis, Greene, T.W.; Wuts, P. G. M., John Wiley & Sons, New York, NY, (3rd Edition, 1999) which can be added or removed using the procedures set forth therein and which is hereby incorporated by reference in its entirety and for any and all purposes as if fully set forth herein.

[0100] The term “amide” (or “amido”) includes C- and N-amide groups, i.e., -C(O)NR71R72, and -NR71C(O)R72groups, respectively. R71and R72are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. Amido groups therefore include but are not limited to carbamoyl groups (-C(O)NH2) and formamide groups (-NHC(O)H). In some embodiments, the amide is -NR71C(O)-(CI-5 alkyl) and the group is termed "carbonylamino," and in others the amide is -NHC(O)-alkyl and the group is termed "alkanoylamino."

[0101] The term “nitrile” or “cyano” as used herein refers to the -CN group.-29-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0102] Urethane groups include N- and O-urethane groups, i.e., -NR73C(O)OR74and -OC(O)NR73R74groups, respectively. R73and R74are independently a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. R73may also be H.|0103] The term “amine” (or “amino”) as used herein refers to -NR75R76groups, wherein R75and R76are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl or heterocyclyl group as defined herein. In some embodiments, the amine is alkylamino, dialkylamino, arylamino, or alkylarylamino. In other embodiments, the amine is NH2, methylamino, dimethylamino, ethylamino, diethylamino, propylamino, isopropylamino, phenylamino, or benzylamino.|0104] The term “sulfonamido” includes S- and N-sulfonamide groups, i.e., -SO2NR78R79and -NR78SO2R79groups, respectively. R78and R79are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclylalkyl, or heterocyclyl group as defined herein. Sulfonamido groups therefore include but are not limited to sulfamoyl groups (-SO2NH2). In some embodiments herein, the sulfonamido is -NHSCh-alkyl and is referred to as the "alkylsulfonylamino" group.

[0105] The term “thiol” refers to -SH groups, while “sulfides” include -SR80groups, “sulfoxides” include -S(O)R81groups, “sulfones” include -SO2R82groups, and “sulfonyls” include -SO2OR83. R80, R81, R82, and R83are each independently a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein. In some embodiments the sulfide is an alkylthio group, -S-alkyl.

[0106] The term “urea” refers to -NR84-C(O)-NR85R86groups. R84, R85, and R86groups are independently hydrogen, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl, or heterocyclylalkyl group as defined herein.

[0107] The term “amidine” refers to -C(NR87)NR88R89and -NR87C(NR88)R89, wherein R87, R88, and R89are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.-30-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0108] The term “guanidine” refers to -NR90C(NR91)NR92R93, wherein R90, R91, R92and R93are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0109] The term “enamine” refers to -C(R94)=C(R95)NR96R97and -NR94C(R95)=C(R96)R97, wherein R94, R95, R96and R97are each independently hydrogen, a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.

[0110] The term “halogen” or “halo” as used herein refers to bromine, chlorine, fluorine, or iodine. In some embodiments, the halogen is fluorine. In other embodiments, the halogen is chlorine or bromine.

[0111] The term “hydroxyl” as used herein can refer to -OH or its ionized form, -O . A “hydroxyalkyl” group is a hydroxyl-substituted alkyl group, such as HO-CH2-.

[0112] The term “imide” refers to -C(O)NR98C(O)R", wherein R98and R" are each independently hydrogen, or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein.[0113 J The term “imine” refers to -CR100(NR101) and -N(CR100R101) groups, wherein R100and R101are each independently hydrogen or a substituted or unsubstituted alkyl, cycloalkyl, alkenyl, alkynyl, aryl aralkyl, heterocyclyl or heterocyclylalkyl group as defined herein, with the proviso that R100and R101are not both simultaneously hydrogen.

[0114] The term “nitro” as used herein refers to an -NO2 group.]0115] The term “trifluorom ethyl” as used herein refers to -CF3.

[0116] The term “trifluorom ethoxy” as used herein refers to -OCF3.

[0117] The term “azido” refers to -N3.

[0118] The term “trialkyl ammonium” refers to a -N(alkyl)s group. A trialkylammonium group is positively charged and thus typically has an associated anion, such as halogen anion.-31-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0119] The term “isocyano” refers to -NC.

[0120] The term “isothiocyano” refers to -NCS.

[0121] The term “pentafluorosulfanyl” refers to -SFs.

[0122] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 atoms refers to groups having 1, 2, or 3 atoms. Similarly, a group having 1-5 atoms refers to groups having 1, 2, 3, 4, or 5 atoms, and so forth.

[0123] As understood by one of ordinary skill in the art, “molecular weight” (also known as “relative molar mass”) is a dimensionless quantity but is converted to molar mass by multiplying by 1 gram / mole or by multiplying by 1 Da - for example, a compound with a weight-average molecular weight of 5,000 has a weight-average molar mass of 5,000 g / mol and a weight-average molar mass of 5,000 Da.

[0124] Pharmaceutically acceptable salts of compounds described herein are within the scope of the present technology and include acid or base addition salts which retain the desired pharmacological activity and is not biologically undesirable (e.g., the salt is not unduly toxic, allergenic, or irritating, and is bioavailable). When the compound of the present technology has a basic group, such as, for example, an amino group, pharmaceutically acceptable salts can be formed with inorganic acids (such as hydrochloric acid, hydroboric acid, nitric acid, sulfuric acid, and phosphoric acid), organic acids (e.g., alginate, formic acid, acetic acid, benzoic acid, gluconic acid, fumaric acid, oxalic acid, -32-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 tartaric acid, lactic acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid, naphthalene sulfonic acid, and p-toluenesulfonic acid) or acidic amino acids (such as aspartic acid and glutamic acid). When the compound of the present technology has an acidic group, such as for example, a carboxylic acid group, it can form salts with metals, such as alkali and earth alkali metals (e.g., Na+, Li+, K+, Ca2+, Mg2+, Zn2+), ammonia or organic amines (e.g., dicyclohexylamine, trimethylamine, tri ethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine) or basic amino acids (e.g., arginine, lysine and ornithine). Such salts can be prepared in situ during isolation and purification of the compounds or by separately reacting the purified compound in its free base or free acid form with a suitable acid or base, respectively, and isolating the salt thus formed.

[0125] Those of skill in the art will appreciate that compounds of the present technology may exhibit the phenomena of tautomerism, conformational isomerism, geometric isomerism, and / or stereoisomerism. As the formula drawings within the specification and claims can represent only one of the possible tautomeric, conformational isomeric, stereochemical or geometric isomeric forms, it should be understood that the present technology encompasses any tautomeric, conformational isomeric, stereochemical and / or geometric isomeric forms of the compounds having one or more of the utilities described herein, as well as mixtures of these various different forms.|0126] “ Tautomers” refers to isomeric forms of a compound that are in equilibrium with each other. The presence and concentrations of the isomeric forms will depend on the environment the compound is found in and may be different depending upon, for example, whether the compound is a solid or is in an organic or aqueous solution. For example, in aqueous solution, quinazolinones may exhibit the following isomeric forms, which are referred to as tautomers of each other:4921 -4618-5569.4Atty. Dkt. No.: 115872-3325As another example, guanidines may exhibit the following isomeric forms in protic organic solution, also referred to as tautomers of each other:Because of the limits of representing compounds by structural formulas, it is to be understood that all chemical formulas of the compounds described herein represent all tautomeric forms of compounds and are within the scope of the present technology.

[0127] Stereoisomers of compounds (also known as optical isomers) include all chiral, diastereomeric, and racemic forms of a structure, unless the specific stereochemistry is expressly indicated. Thus, compounds used in the present technology include enriched or resolved optical isomers at any or all asymmetric atoms as are apparent from the depictions. Both racemic and diastereomeric mixtures, as well as the individual optical isomers can be isolated or synthesized so as to be substantially free of their enantiomeric or diastereomeric partners, and these stereoisomers are all within the scope of the present technology.

[0128] The compounds of the present technology may exist as solvates, especially hydrates. Hydrates may form during manufacture of the compounds or compositions comprising the compounds, or hydrates may form over time due to the hygroscopic nature of the compounds. Compounds of the present technology may exist as organic solvates as well, including DMF, ether, and alcohol solvates among others. The identification and preparation of any particular solvate is within the skill of the ordinary artisan of synthetic organic or medicinal chemistry.

[0129] The terms “patient” or “subject” are used interchangeably to refer to a human or a non-human animal (e.g., a mammal).

[0130] The terms “treat”, “treating”, treatment” and the like refer to a course of action that eliminates, reduces, suppresses, mitigates, ameliorates, or prevents the worsening of, either temporarily or permanently, a disease, disorder or condition to which the term applies, or at-34-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 least one of the symptoms associated therewith. Treatment includes alleviation of symptoms, diminishment of extent of disease, inhibiting (e.g., arresting the development or further development of the disease, disorder or condition or clinical symptoms association therewith) an active disease, delaying or slowing of disease progression, improving the quality of life, and / or prolonging survival of a subject as compared to expected survival if not receiving treatment or as compared to a published standard of care therapy for a particular disease.

[0131] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. Also within this disclosure are Arabic numerals referring to referenced citations, the full bibliographic details of which are provided immediately preceding the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the present technology.

[0132] Overview

[0133] Pancreatic cancer

[0134] Epidemiology

[0135] Pancreatic cancer (PC) is a highly aggressive malignancy often deemed a fatal disease. Although PC can manifest in either the endocrine or exocrine compartments, cancers of the latter are far more common; specifically, pancreatic ductal adenocarcinoma (PDAC) accounts for about 90% of all PC cases. Although early stages of localized disease usually carry a favorable prognosis, early diagnosis is extremely challenging due in part to the asymptomatic nature. However, about 85% of patients are diagnosed with locally advanced disease and / or with metastases, primarily due to the lack of symptoms and markers for this disease. For all patients, regardless of staging at the time of diagnoses, current five-year survival rate is a devastating 11%. Globally, PC diagnoses are on the rise, and it is projected to become the second leading cause of cancer-related deaths by 2030.

[0136] Genetic alterations of PDAC-35-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0137] Only about 10-15% of PDAC cases have a familial basis. Many genes that have been associated with a general risk of cancer have also been identified as PC susceptibility genes, including: BRCA1 / 2, PALB2, and CDKN2A. BRCA1 / 2 and PALB2 are tumor suppressor genes (TSGs), all involved in the BRCA complex, which plays a critical role in the DNA damage response through homologous recombination (HR). Additional genes with germ line mutations associated with PC include ATM, STK11, TP53, MLH1, MSH2, MSH6, PMS2, and PRSS1. A majority of these genes are also involved with the DNA damage response.

[0138] Over 90% of PDAC cases contain activating KRAS mutations. KRAS is a small GTPase with many roles in cell signaling via its interaction with cell membrane growth factor receptors. Specifically in PDAC, the most common KRAS mutations are G12D (40%) and G12V (33%). These activating mutations have been found to occur very early in the carcinogenesis of PDAC, triggering the initial development of initiating pancreatic intraepithelial neoplasias, which explains their high prevalence in PDAC patients. In vivo work with genetically engineered mouse (GEM) models harboring KRAS mutations has shown that, although these mice readily develop precursor pancreatic intraepithelial neoplasias, these mice fail to develop malignant progression. This suggests that the combined effect of subsequent acquired mutations is required for PDAC progression.

[0139] Current therapeutic strategies

[0140] Surgical resection is typically an option only for patients presenting with localized disease, which comprises approximately 15-20% of PDAC patients, however, recurrence rates are high. For the vast majority of patients — those who present with locally advanced or metastatic PDAC (80-85%), the disease is generally deemed non-curative, and they are often treated with palliative intent.

[0141] Almost all intent-to-treat patients receive a combination chemotherapeutic regimen. The current standard of care involves combination chemotherapy in the form of either FOLFIRINOX (folinic acid [leucovorin], fluorouracil [5-FU], irinotecan and oxaliplatin) or modified FOLFIRINOX (mFOLFIRINOX; without bolus Fluorouracil, which is typically responsible for the hematologic toxic effects and diarrhea). mFOLFIRINOX has-36-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 demonstrated an increased safety profile while maintaining efficacy. However, over 75% of patients receiving mFOLFIRINOX still experience Grade 3-4 adverse events. The high rate of adverse events restricts these aggressive chemotherapy regimens to well-performing individuals. For those patients with worse performance status (Eastern Cooperative Oncology Group [ECOG] of 2), the combination of gemcitabine plus nab-paclitaxel or gemcitabine and capecitabine is typically administered. In the metastatic setting, where most patients present, these treatments offer only a modest survival benefit alongside significant toxicities. The current standard of care for metastatic PDAC fails to prolong life expectancy by more than a year.

[0142] Clinical benefit from other forms of targeted cancer therapies for PDAC remains elusive. Immunotherapy has failed to provide clinical benefits to PDAC patients. Typically, immunotherapy is more effective in tumors with mismatch repair deficiency (dMMR), high microsatellite instability (MSI-H), and high tumor mutational burden (TMB-H), all of which contribute to the production of neoantigens that can be recognized by the body’s immune system. PDAC is characterized by an immunosuppressive tumor microenvironment (TME) and only about 2% of PDAC patients have dMMR and / or MSI-H tumors.

[0143] Most recently, personalized vaccine approaches have been utilized to harness the power of patients’ immune systems against PDAC. Although carrying a high lethality, T cell immunity has been associated with the reported long-term survivors of PDAC, providing the foundation for T cell activation as a therapeutic approach. Further studies are underway in larger cohorts to determine the clinically utility of personalized mRNA vaccines, but the initial data are very promising for the subset of patients who present with resectable disease.

[0144] With over 90% of PDAC patients having activating KRAS mutations, and considering the roles of KRAS in the tumorigenesis of various cancer subtypes, significant efforts have been directed toward the development of small molecule direct and indirect inhibitors of KRAS. However, KRAS is typically deemed “undruggable” due to the lack of exploitable binding pockets and its high affinity for GTP. The localization of KRAS to the plasma membrane, within proximity of growth factor receptors, is crucial for its-37-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 transformational activity. Attempts to block this localization by inhibiting post-translational lipid modifications of KRAS (prenylation) in PDAC have shown no clinical efficacy.

[0145] Taken together, the low overall survival rates and limited treatment options with adequate outcomes highlight the clinical challenges facing PDAC.

[0146] Autophagy lysosomal pathway (ALP)

[0147] Autophagy as a cellular recycling program

[0148] Autophagy is a conserved cellular recycling process that degrades unnecessary or damaged intracellular components via lysosomes. It maintains cellular health, recycles building blocks during stress (e.g., starvation), and removes misfolded proteins from endoplasmic reticulum (ER) stress.

[0149] Autophagy can be divided into three main categories based on how cargo is recognized and delivered to the lysosome. “Macroautophagy” involves the encapsulation of cytoplasm, including organelles, in an isolation membrane to form “autophagosomes,” which fuse with lysosomes to deliver the internal material for degradation.“Microautophagy” describes the direct invagination of lysosomal membrane to deliver cytoplasm to the lumen of degradative lysosomes. Finally, “chaperone mediated autophagy (CMA)” is a uniquely selective pathway in which individual cytosolic proteins with a pentapeptide motif are recognized by chaperones and transferred to the lumen of lysosomes via lysosome-associated membrane protein type 2A (LAMP-2A). Macroautophagy has been studied most extensively and is recognized as the major subtype of autophagy; it will be referred to simply as “autophagy” herein.

[0150] In nutrient-rich conditions, autophagy is suppressed by mTORCl via inhibition of the ULK1 complex (ULK1, ATG13, FIP200, ATG101). Upon induction, the ULK1 complex activates the class III PI3K complex to initiate phagophore formation at the ER.

[0151] The ubiquitin-like, microtubule-associated protein 1 light chain 3 (LC3) conjugation system drives phagophore growth. LC3 is processed by ATG4 to LC3-I, then conjugated to phosphatidylethanolamine (PE) via ATG7, ATG3, and the ATG12-ATG5-ATG16L1-38-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 complex to form LC3-II, which anchors to membranes and recruits proteins with LC3 Interaction Regions (LIRs). Cargo adaptors such as p62 / SQSTMl and NBR1 bind ubiquitinated proteins and deliver them to autophagosomes via LIR-LC3 interactions.

[0152] Phagophore closure into a sealed autophagosome involves ESCRT machinery, with ATG proteins recycled afterward. Autophagosomes are transported to lysosomes along microtubules, tether via SNARE protein interactions, and fuse to form autolysosomes. Inside autolysosomes, hydrolases break down cargo into reusable building blocks for cytoplasmic recycling.

[0153] Hydrolytic capabilities of the lysosomes

[0154] Once considered merely the cell’s “garbage can,” lysosomes are now recognized as central hubs for degradation, secretion, repair, signaling, and energy metabolism — critical for cellular homeostasis. They are single-membrane organelles lined internally with a protective glycocalyx, embedded with transmembrane proteins that serve as nutrient sensors, transporters, ion channels, structural supports, and trafficking machinery.

[0155] The lysosomal lumen contains over 60 acid hydrolases (e.g., proteases, lipases, phosphatases, nucleases) capable of digesting diverse biomolecules. These enzymes are synthesized in the ER, glycosylated, and tagged with mannose-6-phosphate (M6P) in the Golgi, ensuring their delivery via M6P receptors to endosomes, which mature into lysosomes.

[0156] Lysosomal acidity (pH 4.2-5.3) is essential for releasing hydrolases from M6P receptors, converting them to their active forms, and optimizing their activity. pH is maintained by V-type ATPases that pump protons into the lumen, creating a voltage gradient that must be balanced by counterion movement (cation efflux or anion influx). Potential pH-regulatory channels include C1C-7 (a 2C171H+antiporter) and TRPML1 (mediates Ca2+release), though their exact contributions to lysosomal acidification remain unclear.

[0157] Transcriptional regulation of the ALP-39-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0158] Autophagy-lysosomal pathway activity is primarily regulated at the transcriptional level by transcription factor EB (TFEB) and related microphthalmia (MiT) family transcription factors. Lysosomal genes feature a conserved 10-base CLEAR (Coordinated Lysosomal Expression and Regulation) motif, to which TFEB binds, promoting transcription of autophagy-related and lysosomal biogenesis / function genes. In nutrientrich conditions, TFEB is phosphorylated by mTOR and ERK2 / MAPK1, causing its retention in the cytoplasm and thus inactivation. Under stress or starvation, mTOR is inhibited, TFEB becomes dephosphorylated, and translocates into the nucleus to activate the CLEAR gene network. This nuclear translocation serves as a key marker of ALP activation or inhibition.

[0159] Metabolic reprogramming and essentiality of the ALP in PDAC

[0160] Stromal component

[0161] PDAC is characterized by a fibrous, desmoplastic stroma and significant deposition of extracellular matrix, creating a nutrient-deprived microenvironment and making it one of the most hypoxic human cancers. The tumor microenvironment (TME) of PDAC is rich in collagen and hyaluronic acid, which constitute the majority of the tumor mass, while PDAC tumor cells represent only a small fraction. Additionally, PDAC tumors exhibit dysfunctional vasculature, partly due to increased interstitial pressure that leads to collapsed blood vessels. In most normal tissues, efficient nutrient exchange from circulation maintains near-equilibrium nutrient levels; however this process is severely impaired in PDAC due to compromised blood flow. The desmoplastic TME also hinders the removal of cellular waste, further exacerbating the stresses faced by tumor cells.

[0162] Metabolic reprogramming and “autophagy ” addiction

[0163] To survive in this harsh TME, PDAC cells undergo metabolic reprogramming, driven largely by constitutively active KRAS. This includes enhanced glucose uptake, altered glutamine metabolism for redox balance, and control of reactive oxygen species (ROS) levels. KRAS also upregulates nutrient scavenging pathways — chiefly autophagy and macropinocytosis — to supply essential metabolites.-40-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0164] Autophagy is hyperactivated and indispensable in PDAC (“autophagy addiction”), serving to reduce ROS, prevent DNA damage, and support mitochondrial function and oxidative phosphorylation. Genetic inhibition of core autophagy genes (e.g., ATG5 or ATG7) blocks KRAS-driven PDAC development in GEM models, highlighting autophagy’s necessity. This heightened ALP activity is sustained by constitutive expression of TFEB and other MiT transcription factors.

[0165] PDAC cells also rely on macropinocytosis to internalize extracellular proteins and other substrates, which are then degraded in lysosomes to release amino acids and nutrients, further fueling cell growth in nutrient-limited conditions.

[0166] ALP therapeutic strategies

[0167] Because PDAC cells depend heavily on autophagy for survival, the ALP is an attractive therapeutic target. Chloroquine (CQ) and hydroxychloroquine (HCQ) — lysosomotropic drugs that block autophagic cargo degradation and disrupt endocytic trafficking — are FDA-approved for other uses, enabling repurposing. However, HCQ monotherapy has not improved PDAC survival.

[0168] Autophagy upregulation contributes to resistance to chemotherapy, radiotherapy, and targeted therapies, supporting combination ALP inhibition strategies. HCQ plus gemcitabine / nab-paclitaxel has shown improved response rates in localized and metastatic PDAC. Combining HCQ with MEK or ERK inhibitors exploits the increased autophagy dependence seen after KRAS inhibition, producing synergistic antitumor activity in PDAC models — including patient-derived xenografts (PDX) — and reduced tumor burden in at least one clinical case (HCQ + Trametinib).

[0169] PDAC’s poor immunotherapy response is partly due to nutrient scavenging- mediated suppression of surface MHC-I expression, with MHC-I retained in autophagosomes / lysosomes. Autophagy inhibition restores MHC-I surface levels, boosts T- cell response, and reduces tumor growth in GEM models, providing rationale for combining CQ / HCQ with immune checkpoint blockade (ICB) therapy.

[0170] Niemann-Pick Type C (NPC1)-41-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0171] Role in cholesterol homeostasis

[0172] After lysosomal degradation of cargo, the released molecules — including free cholesterol — must be exported to the cytoplasm or other organelles. LDL-derived cholesterol is delivered to lysosomes via LDL receptor-mediated endocytosis; LDL is released at low pH, and cholesterol esters are hydrolyzed by lysosomal acid lipases.

[0173] The lysosomal membrane protein NPC1 (with a sterol-sensing domain, SSD) and the luminal protein NPC2 jointly mediate cholesterol export. NPC2 binds cholesterol in the lysosomal lumen and transfers it to the N-terminal domain (NTD) of NPC1. NPC1 then shuttles cholesterol — likely via its middle-luminal domain (MLD) — to the luminal membrane leaflet, bypassing the glycocalyx. Cholesterol transport is proton gradientdependent, suggesting a proton-driven export mechanism from lumen to cytosolic membrane face, potentially handed off to unknown carrier proteins.

[0174] Structural analyses and yeast models (NCR1 / NPC2) reveal a hydrophobic tunnel for cholesterol transfer. Mutations in NPCl’s NTD (e.g., P202A / F203A) disrupt cholesterol binding, while Loop 2 mutations (F503 / 4A) impair NPC2 interaction — both causing lysosomal cholesterol accumulation. SSD mutations (e.g., P692S) inactivate transport while preserving NPC1 localization.

[0175] Small-molecule NPC1 inhibitors, such as U18666A, block cholesterol export and mimic NPC1 loss-of-function. U18666A also inhibits cholesterol biosynthesis via oxidosqualene cyclase and desmosterol reductase. Binding studies reveal U18666A targets NPCl’s SSD, confirmed using photoreactive and clickable derivatives.

[0176] Niemann-Pick Disease (NPD) and biological consequences

[0177] Niemann-Pick Type C disease (NPCD) is a rare lysosomal storage disorder (~1 in 150,000 births) affecting the liver, spleen, brain, and motor control, often leading to early death. It is caused by mutations in either NPC1 (-95% of cases) or NPC2, which impair cholesterol export from late endosomes / lysosomes.-42-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0178] Loss of NPC1 / NPC2 function results in lysosomal accumulation of cholesterol, altered sphingomyelin metabolism, and secondary buildup of glycolipids (e.g., glucosylceramide, lactosylceramide, GM2, GM3). Lysosomes become enlarged, with defective trafficking and reduced proteolysis of autophagic / endocytic cargo, alongside impaired autophagosome-lysosome fusion.

[0179] Deficient cholesterol export decreases ER and Golgi cholesterol levels. Since the Golgi monitors cellular cholesterol, this defect mimics cholesterol starvation, activating SREBPs and upregulating enzymes for cholesterol synthesis and uptake — despite lysosomal overload.

[0180] In neurons, sphingomyelin buildup can erode the protective glycocalyx, compromising lysosomal integrity. Lysosomal leakage releases cathepsin proteases into the cytoplasm, damaging mitochondria and inducing mitochondrial outer membrane permeabilization (MOMP) and apoptosis. NPCD also features elevated reactive oxygen species (ROS), which activate c-Abl kinase, further promoting MOMP and cell death.

[0181] The Present Technology

[0182] Using phenotypic, high-throughput screening (HTS) and subsequent hit-to-lead optimization, the inventors generated a class of 4-aminoquinoline inhibitors of the autophagy lysosomal pathway (ALP). The inventors showed that the compounds described herein inhibit lysosomal degradation and lead to hyperacidification of lysosomes. Given the reliance on the ALP exhibited by pancreatic ductal adenocarcinoma (PDAC), the ALP inhibition induced by the compounds described herein also prove to be cytotoxic in PDAC cell lines and human patient derived-organoids (HPOs).

[0183] The present disclosure generally relates to compounds that inhibit the ALP.

[0184] In one aspect, the disclosure is directed to a compound according to Formula (I):-43-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325wherein:Ring A is a 5 to 14-membered cycloalkyl, a 6 to 14-membered aryl, a 5 to 16- membered heterocycloalkyl, or a 5 to 16-membered heteroaryl;Ring B is absent, a 5 to 14-membered cycloalkyl, a 6 to 14-membered aryl, a 5 to 16-membered heterocycloalkyl, or a 5 to 16-membered heteroarylR1is H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -CN, -ORla, -COORla, or -NRlaRlb, wherein Rlaand Rlbare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R2is H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -CN, -OR2a, -COOR2a, or -NR2aR2b, wherein R2aand R2bare each independently H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl; or R1and R2together form a 5 to 10-membered cycloalkyl, a 6 to 10-membered aryl, a 5 to 10-membered heterocycloalkyl, or a 5 to 10-membered heteroaryl;R3is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -OR3a, -SR3a, -SO2R3a, -COOR3a, -NR3aR3b, -NO2, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, wherein R3aand R3bare each independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heteroaryl, or optionally substituted heterocycloalkyl, and n is an integer from 0 to 5;X1is a bond, an optionally substituted alkylenyl, an optionally substituted alkenylenyl, an optionally substituted heteroalkylenyl, or an optionally substituted heteroalkenylenyl;-44-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325X2 is a bond, an optionally substituted alkylenyl, an optionally substituted alkenylenyl, an optionally substituted heteroalkylenyl, or an optionally substituted heteroalkenyl enyl.

[0185] In any embodiment herein, it may be that the compound of Formula (I) is a compound of Formula (IA), (IB), (IC), or (ID):|0186] In any embodiment herein, it may be that Ring A is a phenyl, piperazinyl, or furanyl.|0187] In any embodiment herein, it may be that Ring A is a phenyl.-45-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0188] In any embodiment herein, it may be that Ring B is piperidinyl, piperazinyl, morpholinyl, phenothiazinyl, or iminodibenzyl.

[0189] In any embodiment herein, it may be that Ring B is piperazinyl.

[0190] In any embodiment herein, it may be that R3is alkyl, -OR3a, -SO2R3a, -COOR3a, optionally substituted aryl, or optionally substituted heteroaryl, wherein R3ais H, alkyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0191] In any embodiment herein, it may be that R3is methyl, ethyl, propyl, butyl, or t- butyl.

[0192] In any embodiment herein, it may be that R3is -OR3a, wherein R3ais optionally substituted aryl.

[0193] In any embodiment herein, it may be that R3is optionally substituted phenyl.

[0194] In any embodiment herein, it may be that R3is -COOR3a, wherein R3ais alkyl.

[0195] In any embodiment herein, it may be thatis:4921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0196] In any embodiment herein, it may be that R1is H, alkyl, or -COORla, wherein Rlais alkyl.

[0197] In any embodiment herein, it may be that R1is H.

[0198] In any embodiment herein, it may be that R1is methyl.

[0199] In any embodiment herein, it may be that R2is H, alkyl, or -COOR2a, wherein R2ais alkyl.

[0200] In any embodiment herein, it may be that R2is H.

[0201] In any embodiment herein, it may be that R2is methyl.

[0202] In any embodiment herein, it may be that R2is -COOR2a, wherein R2ais ethyl.

[0203] In any embodiment herein, it may be that R1and R2taken together are a 6- membered cycloalkyl.

[0204] In any embodiment herein, it may be that X1is a bond, an alkylenyl, an optionally substituted alkenylenyl, or a heteroalkylenyl.-49-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0205] In any embodiment herein, it may be that X1is a bond.

[0206] In any embodiment herein, it may be that X1is -CH2-.

[0207] In any embodiment herein, it may be that X1is -CH2-O-(CH2)2-.

[0208] In any embodiment herein, it may be that X1is a C2 alkenylenyl substituted with -CN.

[0209] In any embodiment herein, it may be that X2is a bond or an optionally substituted alkylenyl.

[0210] In any embodiment herein, it may be that X2is a bond.

[0211] In any embodiment herein, it may be that X2is -CH2-.

[0212] In any embodiment herein, it may be that the compound is selected from:4921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-3325-53-4921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0213] Therapeutic Uses

[0214] The present disclosure relates to methods of treating a disease or disorder in a patient in need thereof, the method comprising administering to the patient a therapeutically effective amount of a compound described herein or a pharmaceutical composition described herein.

[0215] The present disclosure relates to the use of a therapeutically effective amount of a compound described herein or a pharmaceutical composition described herein for treating a disease or disorder in a patient in need thereof.

[0216] In any embodiment herein, it may be that the disease or disorder is cancer. A nonlimiting example of the cancer includes pancreatic ductal adenocarcinoma.-59-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0217] The present disclosure relates to methods of inhibiting ALP in a patient, the method comprising administering to the patient a therapeutically effective amount of a compound described herein or a pharmaceutical composition described herein.

[0218] The present disclosure relates to the use of a therapeutically effective amount of a compound described herein for inhibiting ALP.

[0219] Routes of Administration

[0220] In any embodiment herein, it may be that pharmaceutical compositions containing a compound according to this disclosure may be in a form suitable for oral administration. Oral administration may involve swallowing the formulation thereby allowing the compound to be absorbed into the bloodstream in the gastrointestinal tract. Alternatively, oral administration may involve buccal, lingual or sublingual administration, thereby allowing the compound to be absorbed into the blood stream through oral mucosa.{0221] In another embodiment, the pharmaceutical compositions containing a compound according to this disclosure may be in a form suitable for parenteral administration. Forms of parenteral administration include, but are not limited to, intravenous, intraarterial, intramuscular, intradermal, intraperitoneal, intrathecal, intracisternal, intracerebral, intracerebroventricular, intraventricular, and subcutaneous. Pharmaceutical compositions suitable for parenteral administration may be formulated using suitable aqueous or nonaqueous carriers. Depot injections, which are generally administered subcutaneously or intramuscularly, may also be utilized to release the compounds disclosed herein over a defined period of time.

[0222] Other routes of administration are also contemplated by this disclosure, including, but not limited to, nasal, vaginal, intraocular, rectal, topical (e.g., transdermal), and inhalation.Pharmaceutical Compositions[0223J The compounds of the present disclosure may be in the form of compositions suitable for administration to a subject. In general, such compositions are pharmaceutical compositions comprising a compound according to this disclosure or a pharmaceutically-60-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 acceptable salt thereof and one or more pharmaceutically acceptable excipients. In certain embodiments, the compound may be present in an effective amount. The pharmaceutical compositions may be used in the methods of the present disclosure; thus, for example, the pharmaceutical compositions comprising a compound according to this disclosure can be administered to a subject in order to practice the therapeutic methods and uses described herein.

[0224] The pharmaceutical compositions of the present disclosure can be formulated to be compatible with the intended method or route of administration. Routes of administration may include those known in the art. Exemplary routes of administration are oral and parenteral. Furthermore, the pharmaceutical compositions may be used in combination with one or more other therapies described herein in order to treat or prevent the diseases, disorders and conditions as contemplated by the present disclosure. In one embodiment, one or more other therapeutic agents contemplated by this disclosure are included in the same pharmaceutical composition that comprises the compound according to this disclosure. In another embodiment, the one or more other therapeutical agents are in a composition that is separate from the pharmaceutical composition comprising the compound according to this disclosure.

[0225] In one aspect, the compounds described herein may be administered orally. Oral administration may be via, for example, capsule or tablets. In making the pharmaceutical compositions that include the compounds of the present disclosure (e.g., a compound of Formula I), or a pharmaceutically acceptable salt thereof, the tablet or capsule includes at least one pharmaceutically acceptable excipient. Non-limiting examples of pharmaceutically acceptable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, polyethylene glycol, cellulose, sterile water, syrup, and methyl cellulose. Additional pharmaceutically acceptable excipients include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl and propylhydroxy -benzoates.-61-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0226] In another aspect, the compounds of the present disclosure, or a pharmaceutically acceptable salt thereof, may be administered parenterally, for example by intravenous injection. A pharmaceutical composition appropriate for parenteral administration may be formulated in solution for injection or may be reconstituted for injection in an appropriate system such as a physiological solution. Such solutions may include sterile water for injection, salts, buffers, and tonicity excipients in amounts appropriate to achieve isotonicity with the appropriate physiology.

[0227] The pharmaceutical compositions described herein may be stored in an appropriate sterile container or containers. In any embodiment herein, it may be that the container is designed to maintain stability for the pharmaceutical composition over a given period of time.Administering

[0228] In general, the disclosed methods comprise administering a compound described herein, or a composition thereof, in an effective amount to a subject in need thereof. An “effective amount” with reference to an ALP inhibitor of the present disclosure means an amount of the compound that is sufficient to engage the target (e.g., by inhibiting the target) at a level that is indicative of the potency of the compound. For ALP, target engagement can be determined by one or more biochemical or cellular assays resulting in an EC50, ED50, EC90, IC50, or similar value which can be used as one assessment of the potency of the compound. Assays for determining target engagement include, but are not limited to, those described in the Examples. The effective amount may be administered as a single quantity or as multiple, smaller quantities (e.g., as one tablet with “x” amount, as two tablets each with “x / 2” amount, etc.).

[0229] In any embodiment herein, it may be that the disclosed methods include administering a therapeutically effective amount of a compound described herein to a subject in need thereof. As used herein, the phrase “therapeutically effective amount” with reference to compound disclosed herein means a dose regimen (z.e., amount and interval) of the compound that provides the specific pharmacological effect for which the compound is administered to a subject in need of such treatment. For prophylactic use, a therapeutically effective amount may be effective to eliminate or reduce the risk, lessen the severity, or -62-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 delay the onset of the disease, including biochemical, histological and / or behavioral signs or symptoms of the disease. For treatment, a therapeutically effective amount may be effective to reduce, ameliorate, or eliminate one or more signs or symptoms associated with a disease, delay disease progression, prolong survival, decrease the dose of other medication(s) required to treat the disease, or a combination thereof. With respect to cancer specifically, a therapeutically effective amount may, for example, result in the killing of cancer cells, reduce cancer cell counts, reduce tumor burden, eliminate tumors or metastasis, or reduce metastatic spread. A therapeutically effective amount may vary based on, for example, one or more of the following: the age and weight of the subject, the subject’s overall health, the stage of the subject’s disease, the route of administration, and prior or concomitant treatments.

[0230] Administration may include one or more (e.g., one, two, or three or more) dosing cycles.EXAMPLES

[0231] Example 1: High-throughput screening identifies JTC-801 as an inducer of TFEB nuclear translocation

[0232] First, using a phenotypic high-throughput screen (HTS), inventors identified small molecules that modulated the ALP. Secondary screening was conducted to identify specific inhibitors of the ALP, including hit small molecule, the 4-aminoquinoline, JTC-801, an opioid receptor-like 1 (ORL-1) receptor antagonist. Hit-to-lead optimization was performed to enhance ALP inhibitory properties and cytotoxicity to PDAC cell lines in vitro, leading to the generation of a library of 4-aminoquinoline derivatives referred to as the GN Series. The lead small molecule is GN64, based on the 4-aminoquinoline pharmacophore of JTC- 801.

[0233] Assessing small molecule modulation of the ALP can be performed by analyzing the localization of TFEB following treatment. A fluorescent reporter tag can be used to visualize the subcellular localization of TFEB. Using this approach, inventors previously conducted high-throughput screening to identify small molecule modulators of the ALP. 9,794 biologically active and structurally diverse compounds were screened using a well- -63-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 characterized osteosarcoma cell line, U2-OS, stably expressing N-terminally mCherry(mCh)-tagged TFEB (U2-OS mCh-TFEB). Nuclear stain, Hoechst, was used to segment cellular nuclei, and the nuclear to cytoplasmic ratio of mCh-TFEB fluorescence was measured. A total of 27 compounds were identified as hits (0.28%). Toxic and fluorescent compounds were filtered out.

[0234] Among these hits, many compounds target the TKR-PI3K-mTOR axis, such as Torinl, Torin2, AZD8055, PKI-587, INK128, BEZ235, Wortmannin and Foretinib, for which the mechanism has well been established, further validating this high-content assay. One hit was the selective opioid receptor-like 1 (ORL1) receptor antagonist, JTC-801 (FIG. 1A). Confirmation studies revealed that JTC-801 induces the nuclear translocation of TFEB with a half maximal effective concentration (EC50) of 2.82 pM (5h) (FIG. IB).

[0235] The goal of the study was to identify small molecules with novel mechanisms for inducing TFEB translocation. As the nuclear translocation of TFEB can be regulated by phosphorylation status, specifically through mTOR, JTC-801 was investigated for mTOR inhibitory activity. S6 is a target for phosphorylation by active mTOR, whereas inhibition of mTOR leads to a decrease in phospho-S6. Following acute treatment, JTC-801 does not inhibit the phosphorylation of S6, supporting the conclusion that the observed nuclear translocation of TFEB is not due to direct mTOR inhibition (FIGs. 1C-1D).

[0236] JTC-801 inhibits lysosomal degradation in PDAC cells

[0237] Nuclear translocation of TFEB can serve as a readout of ALP modulation, either via starvation induction or ALP inhibition. Once in the nucleus, TFEB activates the expression of genes involved in lysosomal biogenesis and function, either as a starvation response or as a compensatory mechanism to overcome the ALP inhibition.

[0238] Inventors first aimed to identify if the observed TFEB nuclear translocation induced by JTC-801 resulted in functional ALP. To assess lysosomal activity, the degradation of endocytic or autophagic cargo can be evaluated. Endocytosis involves the uptake of extracellular material and trafficking to the lysosomes for degradation. Dye-quenched (DQ)- bovine serum albumin (BSA) is heavily fluorescently labeled BSA, that self-quenches its fluorescence in its intact form. Cells supplemented with DQ-BSA will uptake it through -64-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 endocytosis, traffic the cargo to the lysosomes, leading to eventual degradation. Cleavage of DQ-BSA in the lysosome by active proteases leads to the release of fluorescent fragments, which can be observed via microscopy or flow cytometry.

[0239] The effect of JTC-801 on lysosomal activity was assessed in the PDAC cell line, PaTu 8988T. JTC-801 inhibits lysosomal degradation at low micromolar concentrations (FIG. 2). These data support the conclusion that the nuclear translocation of TFEB is due to ALP dysfunction

[0240] Hit-to-lead optimization: development of 4-aminoquinoline library, the GN Series

[0241] Hit-to-lead optimization studies involve chemical modifications around the validated hit to enhance its affinity for the target and to increase potency. Based on the core 4- aminoquinoline structure of JTC-801, 51 derivatives were initially generated through alterations to the linker group and conversion of the phenolic ether to a phenyl piperazine with various substitutions. This small library of analogs is referred to as the GN Series (FIG. 3).

[0242] The goal of this project was to identify novel inhibitors of the ALP that confer cytotoxicity to PDAC. The GN Series was initially screened for cytotoxicity in PDAC cells due to the suitability of this assay for larger screening endeavors. Cytotoxicity (72h) was assessed in two PDAC cell lines: MIA PaCa-2 and PaTu 8988T. GN Series analogs containing trifluoromethyl and / or nitro group substitutions on the phenyl piperazine (8 / 50 GN Series analogs) yielded the highest cytotoxicity (FIG. 4).

[0243] Based on this observation, additional analogs were generated to expand the library of trifluoromethyl and / or nitro group phenyl piperazine substitutions to 21 small molecules. These GN Series analogs encompassed alterations to the pharmacophore, including (1) furan, phenyl, or PEG linker groups and (2) monomethyl, dimethyl, or cyclohexyl modifications to the quinoline along with para and ortho trifluoromethyl and or nitro group substitutions on the phenyl piperazine (FIG. 5A-5C).

[0244] A second round of cytotoxicity screening was performed in three PDAC cell lines: MIA PaCa-2, PANC-1, and PaTu 8988T at concentrations of 2.5 and 1 pM. Cytotoxic GN-65-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325Series analogs were distinguished as those exhibiting >50% cytotoxicity at 2.5 pM and >15% cytotoxicity at 1 pM on average across all three cell lines tested. Based on these criteria, 12 out of 21 GN Series analogs were identified with PDAC cytotoxicity (Table 1).

[0245] Table 1. PDAC Cytotoxicity profiling of GN Series trifluoromethyl and or nitro containing small molecules. PDAC cells (MIA PaCa-2, PANC-1, PaTu 8988T) were treated with GN Series small molecules [2.5, 1 pM] or vehicle (DMSO) for 72h. Cell growth was quantified using the CellTiter-Glo 2.0 Cell Viability Assay and normalized to DMSO treated cells and displayed as percentage of cytotoxicity. The average cytotoxicity across all three cell lines was calculated. All data from n=3 biological replicates and is represented as the mean.

[0246] The lysosomal inhibitory activity of the 21 small molecules of the GN Series with trifluoromethyl and / or nitro group substitutions was then tested. Using the DQ-BSA assay, the lysosomal inhibitory effects were assessed in the PDAC cell line, PaTu 8988T.-66-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325Lysosomal inhibitory GN Series analogs was distinguished as those exhibiting >50% DQ- BSA inhibition at 1.25 pM. Based on these criteria, eight lysosomal inhibitory GN Series analogs were identified (Table 2).

[0247] Table 1.1 PDAC lysosomal degradation inhibition profiling of GN Series trifluoromethyl and or nitro containing small molecules. PaTu 8988T cells were treated with GN Series small molecules [1.25 pM], V-ATPase inhibitor, BafAl [100 nM] or vehicle (DMSO) for 12h. DQ-BSA fluorescence intensity was quantified and normalized to DMSO (0% inhibition) and BafAl (100% inhibition) treated cells and displayed as percent inhibition. All data from n=3 biological replicates and is represented as the mean.-67-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0248] Taking these two screens together, five GN Series analogs were identified that were both cytotoxic and lysosomal inhibitory. Three of the five compounds utilized a cyclohexyl substitution on the quinoline; these compounds were not pursued further due to the potential hydrophobic effects of the cyclohexyl group. The focus was then directed to the two analogs with cytotoxic and lysosomal inhibitory properties, GN17 and GN64 (FIG. 6).

[0249] GN64 as the lead small molecule-68-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0250] Confirmatory studies were then performed to assess the cytotoxicity and lysosomal inhibition of GN17 and GN64 more robustly. Using four PDAC cell lines, BxPC-3, MIA PaCa-2, PANC-1, and PaTu 8988T, the cytotoxicity IC50 was determined for both GN17 and GN64 at 72h (FIGs. 7A-7B). Both leads exhibited low micromolar IC50 values in all four cell lines tested, confirming the results from the screen (FIG. 7D). The cytotoxicity was compared to the known 4-aminoquinoline ALP inhibitor HCQ (FIG. 7C). GN I 7 and GN64 demonstrated > 10-fold potency when compared to HCQ in all four PDAC cell lines tested (FIG. 7D).

[0251] Next, the lysosomal inhibitory properties of GN I 7 and GN64 was further investigated. Using the DQ-BSA assay and PaTu 8988T cells, GN64 inhibited lysosomal degradation at sub-micromolar levels, showing increased potency compared to GN I 7 (FIGs. 8A-8B). Based on the confirmed cytotoxicity and enhanced potency for ALP inhibition, GN64 was selected as the lead GN Series small molecule.

[0252] Discussion

[0253] JTC-801 was previously discovered as an inducer of TFEB nuclear translocation without evidence of acute mTOR inhibition. It was investigated whether the nuclear translocation of TFEB was due to activation of functional autophagy or as a compensatory mechanism in the face of ALP inhibition. It was demonstrated that JTC-801 inhibits lysosomal degradative capabilities in PDAC cells, supporting the idea that the nuclear translocation of TFEB occurs through a compensatory mechanism in response to ALP inhibition. The hit-to-lead optimization strategies led to the development of the GN Series of compound and the identification of GN64, based on PDAC cytotoxicity and ALP inhibitory properties. GN64, inhibits lysosomal degradation at sub-micromolar levels and confers cytotoxicity to PDAC cell lines.

[0254] TFEB modulation and the ALP

[0255] The initial HTS was centered around the nuclear translocation of TFEB. The nuclear translocation and subsequent activation of the CLEAR gene network, which induces the expression of genes related to lysosomal biogenesis and function, is triggered by a variety of-69-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 stress stimuli, including nutrient starvation, infection, as well as mitochondrial, ER, or lysosomal dysfunction.

[0256] Small molecules can induce the cellular effects of starvation or the stresses that lead to TFEB nuclear translocation. Given that the subcellular localization of TFEB is primarily controlled by its phosphorylation status, small molecules that inhibit phosphorylation or induce dephosphorylation can lead to its nuclear translocation. Most notably, mTOR inhibitors, such as Rapamycin, Torinl, CCI-779, and Curcumin, prevent the phosphorylation of TFEB. Trehalose, which causes lysosomal calcium release and activates the phosphatase PPP3CB / calcineurin, leads to the dephosphorylation of TFEB and subsequent nuclear translocation. In these instances, the activation of TFEB triggers the expression of the CLEAR gene network, leading to increased lysosomal biogenesis and function.

[0257] Nuclear translocation can also occur as a compensatory mechanism when the ALP is inhibited. Examples include treatment with CQ and BafAl both of which lead to the alkalinization of the lumen of lysosomes through distinct mechanisms. Chronic ALP inhibition with CQ or BafAl, leads to the accumulation of cargo within the lysosomes due to the altered pH and inhibition of acid hydrolases, which will eventually trigger mTOR inhibition.

[0258] Although small molecules that promote the nuclear translocation of TFEB and result in upregulated, functional ALP are of clinical interest in various pathologies, the focus was on the discovery of novel small molecules that lead to ALP inhibition. As such, the inventors directed the attention to small molecules that caused ALP dysfunction and subsequent TFEB nuclear translocation. The secondary screening of hits utilized DQ-BSA to assess lysosomal degradative capabilities. This assay demonstrated that JTC-801 inhibits lysosomal degradation and induces the nuclear translocation of TFEB.

[0259] Furthermore, JTC-801 was investigated for its potential as an mTOR inhibitor. Based on Western blot (WB) analysis of mTOR phosphorylation substrates, including S6, JTC-801 did not show mTOR inhibition during acute treatment, supporting the idea that the-70-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 nuclear translocation of TFEB is mTOR-independent. However, the debate regarding mTOR-independent versus dependent regulation of TFEB is hotly contested.

[0260] 4-aminoquinoline structure of GN Series

[0261] The inventors sought to modify the core structure of JTC-801 to increase potency as an ALP inhibitor and inducer of cytotoxicity in ALP dependent PDAC cells. Initial modifications began with substitutions of the phenolic ether of JTC-801 to a phenyl piperazine. Initial SAR studies showed the importance of the aromatic substitution of the piperazine ring over the simple aliphatic substitutions. Furthermore, these substitutions also indicated that the scaffold could tolerate the transition from phenolic ethers to amines.

[0262] SAR studies investigation modifications to the quinoline demonstrated that the free amine of JTC-801 was essential for TFEB nuclear translocation. Primary amines can undergo N-acetylation and oxidation by monoamine oxidase (MAO) in first pass metabolism. The introduction of steric hindrance can prevent this conversion. Additionally, it was found that increasing hydrophobicity via carbon content on the quinoline typically increased the scaffold's efficacy. However, it was chosen to avoid the cyclohexyl derivatives for fear of the effect being too dependent on the increased hydrophobic interactions.

[0263] Preliminary screening of the GN Series revealed that GN Series analogs containing a trifluoromethyl and or nitro group substitutions of the phenyl piperazine yielded highest cytotoxicity. Based on this information the focus was directed to expanding the GN Series to include additional trifluoromethyl and or nitro group containing analogs. It was discovered that dimethyl quinoline analogs with a phenyl linker and either a trifluoromethyl (GN17) or a nitro group (GN64) in the ortho position were the most potent for both ALP inhibition and cytotoxicity. Upon further review, GN64 showed more promising ALP inhibition in PDAC cells compared to GN17.

[0264] Interestingly, a collection of small molecules was identified that showed either cytotoxicity without ALP inhibition or ALP inhibition without cytotoxicity. Most notably, GN54 and GN57, two analogs both with a furan linker, para nitro phenyl piperazine, and either a dimethyl or cyclohexyl quinoline, respectively, showed strong lysosomal inhibition -71-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 but failed to induce cytotoxicity at the concentrations and timepoints tested. Conversely, GN18, which contains a phenyl linker, ortho-nitro and para-trifluoromethyl phenyl piperazine, with a dimethyl quinoline, showed strong cytotoxicity but surprisingly displayed ALP activation.

[0265] In the field of ALP inhibition, quinolines are well known. CQ is a well-studied 4- aminoquinoline, like that of JTC-801 and GN64. The exact mechanism by which CQ and HCQ inhibit the ALP is contested. However, it is widely accepted that CQ / HCQ accumulates in lysosomes via lysosomotropism leading to the inhibition of their hydrolytic functions. CQ / HCQ can also accumulate in the Golgi and endosomes which can inhibit post-translational processing in the ER and Golgi.

[0266] Structurally, GN64 contains a unique free amine, shown to be essential for activity, that is not found on CQ / HCQ. Furthermore, cytotoxicity profiling shows a >10-fold potency when compared to HCQ.

[0267] GN64 induced cytotoxicity and ALP inhibition

[0268] As previously mentioned, PDAC undergoes metabolic reprogramming and increased reliance on nutrient scavenging pathway, namely autophagy and macropinocytosis. Both pathways utilize degradative lysosomes to break down cargo into biological building blocks to be used by the cell. Autophagy has been shown to be both upregulated and essential for tumorigenesis in PDAC. Pharmacological inhibition of autophagy in PDAC models leads to impaired mitochondrial function and decreased proliferation in vitro, and reduced tumor burden in vivo. Pancreatic cancer cells also utilize macropinocytosis as a nutrient scavenging pathway to take up extracellular material for lysosomal degradation. Macropinocytosis is essentially for supporting PDAC cell growth. Inhibition of lysosomal function impairs both essential pathways in PDAC.

[0269] GN64 both inhibits lysosomal degradation and confers cytotoxicity in PDAC cell lines. Given the “ALP addiction” of PDAC, it is plausible the inhibition of lysosomal degradation, which ultimately impairs both autophagy and macropinocytosis, results in the PDAC cell death observed. This GN64 mediated inhibition of lysosomal degradation can be of therapeutic relevance to PDAC.-72-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0270] The use of DQ-BSA to study effects on lysosomal degradation requires careful experimental design to accurately test the posed hypothesis. DQ-BSA enters cells via endocytosis and is ultimately cleaved and visualized once endosomes fuse with lysosomes. DQ-BSA analysis can be affected by mechanisms of endocytosis and endolysosomal fusion events. Inhibition of uptake or delivery to lysosomes would also be indicated by a lack of DQ-BSA signal. In the experiments, pre-loaded cells were first with DQ-BSA, washed away excess, and then drug treatments began. By pre-incubating with the lysosomal substrate before initiating drug treatment, the analysis would omit any effects on endocytosis and would focus on lysosomal mediate degradation events. This design allows for proper hypothesis testing if our small molecules inhibit lysosomal activity.

[0271] To further test the cytotoxicity in a more clinically relevant model, cytotoxicity profiling was performed in a panel of HPOs. HPOs represent three-dimensional (3D) constructs of target organs, which recapitulate the architecture and function of native tissue better than 2D cell culture. HPOs have been successfully derived from pancreatic tissue and accurately represent phenotypic and genotypic distinctions found in original patient tumors4. HPOs have successfully predicted therapeutic responses in gastric cancer, validating their use in pharmacological testing. A panel of >60 HPO lines were employed, spanning transcriptional subtype (classical, basal-like, hybrid), tissue of origin (primary, metastatic), patient treatment status (naive, treated), and mutational statuses for cytotoxicity profiling.

[0272] Early development of the GN Series initially utilized GNU; GNU is identical to GN64 except for a monomethyl 4-aminoqunioline versus the dimethyl of GN64. Cytotoxicity profiling of HPOs was performed using GNU. Using this similar analog to GN64, it was found that GNU conferred low-micromolar cytotoxicity in a majority of the HPO lines tested, recapitulating what was observed in PDAC cell lines (FIG. 10).

[0273] Of note, certain HPO lines displayed intrinsic resistance, with IC50 values nearly 20-fold higher than sensitive HPO lines. All the HPO lines tested have been comprehensively characterized by both exome and transcriptome sequencing. These data can be used to identify associations between mutational status and expression levels to define biomarkers of sensitivity or resistance in the future.-73-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0274] Methods

[0275] Cell line culture

[0276] The following cells were cultured in RPMI with penicillin, streptomycin and 10% FBS: U2-OS (American Type Culture Collection (ATCC), wild-type (WT) and mCh- TFEB) and BxPC-3 (ATCC). The following cells were cultured in DMEM medium with high glucose, penicillin, streptomycin and 10% FBS: MIA PaCa-2 (ATCC), PANC-1 (ATCC), PaTu 8988T (a kind gift from Dr. Nancy Du, Weill Cornell Medical College). All cells were cultured at 37 °C with 5% CO2 / 95% air.

[0277] Plasmids

[0278] The following plasmids were used: pcDNA3.1-mCh-TFEB.

[0279] DQ-BSA assay for lysosomal degradation activity

[0280] Cells were plated in 96-well plates and cultured for 24h. Cells were labeled with Calcein AM [1 pM] and nuclear stain, Hoechst [1 pg / mL] in Live Cell Imaging Solution (Invitrogen) for 45min to label live cell bodies for imaging quantification and nuclei. Cells were then incubated with DQ-BSA Red (Invitrogen) at 25 pg / mL in appropriate media for Ih. Cells were washed lx with PBS and then 100 pL of fresh media was added. Cells were imaged using a Cytation 5 Cell Imaging Multi-Mode Reader (Agilent) every 2h for up to 12h. After initial read at timepoint Oh, treatment was added as 100 pL at 2X final concentration. mTOR (mTORCl and mTORC2) inhibitor, Torin-l[l pM] was used as a positive control and the specific vacuolar H+ ATPase (V-ATPase) inhibitor, Bafilomycin- A1 (BafAl) [100 nM] was used as a negative control. Quantitative image analysis was performed using Gen5 (Agilent). Briefly, cell masks were generated using Calcein AM labeling and DQ-BSA Red average intensity was calculated within cell masks at each 2h interval timepoints up to 12h. Data was normalized using DMSO (0% inhibition) and BafAl (100% inhibition). Following 12h of treatment, cells were fixed in 4% PF A, with retention of labeling.

[0281] Cytotoxicity profding (PDAC cell lines)-74-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0282] Cytotoxicity profiling was performed PDAC cell lines. 3000-4000 cells were plated in 100 pL of media in 96-well plates and cultured for 24h. Treatment and vehicle (DMSO) control were added to cells as 25 pL in media at 5X final concentration. Cells were cultured for indicated time interval.|0283] For cytotoxicity screening of the GN Series, cell growth was measured using the Sulforhodamine B (SRB) colorimetric as previously described. Cell growth was normalized to DMSO control (100% cell growth).

[0284] For confirmatory cytotoxicity profiling, including screening GN series small molecules with trifluoromethyl and or nitro phenyl piperazine substitutions and cytotoxicity profiling of GN Series leads, GN17, GN64 and comparisons to HCQ, cell viability was determined using CellTiter-Glo® 2.0 Cell Viability Assay. Cell viability was normalized to DMSO control (100% viability).

[0285] Nuclear translocation of mCh-TFEB

[0286] Using U2-OS mCh-TFEB cells, 6000 cells were plated in 100 pL of media in 96- well plates and cultured for 24h. Treatment was added as 25 pL at 5X final concentration for 5h. mTOR (mTORCl and mT0RC2) inhibitor, Torin-l[l pM] was used as a positive control. Nuclei labeling (Hoechst) and fixation with 4% PFA were performed simultaneously. Cells were imaged using a Zeiss Axio Observer 3. Nuclear translocation of TFEB was quantified as nuclear / cytoplasmic intensity ratio and compared to levels induced by Torin-1 and DMSO (FIG. 9).

[0287] Cytotoxicity profiling (HPO lines)

[0288] HPO studies were performed in collaboration with the Center for Pancreatic Cancer Research at MSKCC. Briefly 2,000 single organoid cells were plated into matrigel-coated wells in a 384-well plate format and cultured for 4-5 days in complete organoid medium to allow the formation of multicellular 3D organoids. Organoids were then exposed to drugs over a 10-concentration range (from 30pM to InM) for 6 days. Vehicle (DMSO) and SN38 at 10 pM as negative and positive controls were used for normalization. At the endpoint, cell viability was assessed using the CellTiter-Glo 3D Cell Viability Assay kit (Promega)-75-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 and acquired on a Cytation 3 Imaging reader (Biotek) equipped with Gen5 Image+ 2.09 software. Response parameters (IC50 and AUC) were calculated using a built-in 4 parameter sigmoidal dose-response curve-fit model (GraphPad Prism).

[0289] Example 2: GN64 induces hyperacidification of lysosomes leading to ALP inhibition

[0290] Mechanistic studies were performed on the inhibition of the ALP by GN64 in PDAC cells. GN64 inhibits lysosomal degradation, leading to the accumulation of autophagy cargo adaptor proteins, without impacting the maturation of cathepsin D (CTSD). GN64 treatment in PDAC resulted in increased lysosomal mass, seemingly without affecting lysosomal integrity. Additionally, GN64 treatment resulted in hyperacidification of PDAC lysosomes. Furthermore, the transcriptional alterations induced by GN64 in PDAC cells were investigated. GN64 upregulates the expression of genes related to the ALP, specifically those involved in lysosomal acidification and ganglioside catabolism.

[0291] Results

[0292] GN64 induces accumulation of autophagic markers without affecting CTSD maturation

[0293] It was shown that GN64 inhibited lysosomal degradation in PDAC cells at submicromolar levels. Functional autophagy involves the fusion of autophagosomes with lysosomes, giving rise to autolysosomes. LC3-II facilitates the maturation of phagophore formation and functions as a docking site for autophagic adaptor proteins and their cargo. As autophagosomes fuse with lysosomes, LC3-II itself is degraded. The accumulation of LC3-II in cells can be interpreted as either (1) increased autophagosome formation or (2) inhibition of lysosomal degradation.

[0294] To further investigate the ALP inhibitory effects of GN64, its effect on LC3-II protein levels in PDAC cells was tested. GN64 treatment led to the accumulation of autophagic marker LC3-II, in both MIA PaCa-2 (FIGs. 11 A-l IB) and PaTu 8988T (FIGs. 11 A, 11C) PDAC cells. As a control, HCQ, which inhibits acidification of lysosomes and disrupts autophagosome-lysosome fusion, also showed an accumulation of LC3-II in treated-76-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325PDAC cells (FIGs. 11A-11C). Although GN64 mediated inhibition of lysosomal degradation was shown previously, the accumulation of LC3-II may still be due to increased autophagosome formation.

[0295] To better assess autophagy blockade, interrogating the levels of other autophagy substrates can help further elucidate the consequences of LC3-II accumulation. The autophagic adaptor protein, p62, is able to bind ubiquitinated, misfolded proteins and also to LC3-II, bringing its cargo to autophagosomes for lysosomal degradation. p62 is also a substrate of autophagy and its accumulation has been identified as a marker of reduced autophagic flux. GN64 treatment led to the accumulation of p62 in both MIA PaCa-2 (FIGs. 12A-12B) and PaTu 8988T (FIGs. 12A, 12C) PDAC cells. As a control, HCQ also showed an accumulation of p62 in treated PDAC cells (FIGs. 12A-12C). Taken together, GN64 inhibits lysosomal degradation and causes accumulation of LC3-II and p62 demonstrating that GN64 inhibits the ability of lysosomes to degrade cargo.

[0296] Many small molecules have been described as inhibiting the ALP based on their ability to alkalinize the lysosomal lumen. Alkalization of lysosomes can be triggered by lysosomotropic agents, which can freely diffuse across the membrane but become trapped in acidic lumens following protonation, such as CQ / HCQ. Others can inhibit the V-ATPase, such as BafAl, or counterion channels ultimately leading to reduced proton content in the lysosomal lumen.

[0297] The maturation and activity of cathepsins depend on the acidic environment of the lysosome, where alkalization would result in inhibition of both these processes. To this point, it was next sought to investigate the effect of GN64 treatment on the maturation of the most abundant lysosomal cathepsin, CTSD. CTSD undergoes a multistep maturation process where the final events occur in the lysosome mediated by cysteine cathepsins, CTSB and CTSL. GN64 treatment did not affect the levels of mature CTSD in PDAC cells (FIGs. 13A-13C). On the contrary, HCQ significantly reduced the levels of mature CTSD in PDAC cells. This observation supports a distinct mechanism of action for GN64 compared to HCQ.

[0298] GN64 increases lysosomal mass and LysoTr acker labeling-n-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0299] The effect of GN64 on lysosomal mass was examined next. First, immunoblotting was used to assess the levels of LAMP 1, a highly glycosylated transmembrane protein that is one of the most abundant proteins in lysosomes. LAMP1 accumulation has been shown upon treatment with CQ and has been explained as a consequence of impaired lysosomal turnover, or activated lysosomal biogenesis following compensatory TFEB nuclear translocation. GN64 treatment led to the accumulation of LAMP 1 in both MIA PaCa-2 (FIGs. 14A-14B) and PaTu 8988T PDAC cells (FIG. 14A, FIG. 14C). HCQ also showed an accumulation of LAMP1 in treated PDAC cells (FIG.s 14A-14C). These data further support GN64 mediated modulation of the ALP.

[0300] Lysosomotropic agents cause enlargement of lysosomes via a “proton sponge” effect, where, as they absorb protons, additional protons are pumped into the lysosome along with chloride ions and water molecules. It has been shown that alterations to lysosomal morphology can be indicative of inhibitory consequences. Using immunofluorescence, it was further examined the size and morphology of lysosomes in GN64 treated PDAC cells. GN64 treatment increased the size of lysosomes in PDAC cells (FIGs. 15A-15B).

[0301] To assess potential LMP, lysosomotropic dyes can be used. LysoTracker is a hydrophobic fluorophore linked to a weak base, which can diffuse across cell membranes in its neutral state but becomes protonated, and subsequently accumulated, in acidic compartments, such as lysosomes. Decreased LysoTracker labeling can indicate alkalinization of lysosomes or induction of LMP, as the dye will freely diffuse without accumulation. Upon treatment with GN64, PDAC cells show a marked increase in LysoTracker labeling (FIGs. 16). HCQ, on the other hand, leads to reduced LysoTracker labeling, demonstrating another example of divergent phenotypes between these two small molecules. These data support the known consequences of HCQ as a lysosomotropic agent, capable of inducing both lysosomal alkalinization and LMP. However, the increased LysoTracker labeling observed with GN64 supports the opposite; GN64 does not appear to induce LMP or alkalinization of lysosomes.

[0302] GN64 causes hyperacidification of lysosomes-78-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0303] Without any evidence of GN64 induced alkalinization of lysosomes, it was hypothesized that GN64 could be inducing “hyperacidification” of lysosomes, which could also lead to the observed ALP inhibition. Given the importance of lysosomal pH regarding their function, significant efforts have been made to develop methodologies to perform direct assessment of lysosomal pH. pH-sensitive dyes can be delivered to lysosomes using labeled dextrans or proteins and then a qualitative assessment of lysosomal pH can be made using a ratiometric calculation with a non-pH sensitive dye or by including a ratio-to-pH calibration curve. However, the sensitivity of these probes is significantly affected by their pKa, bleaching properties, and fluorescent spectra. Also, many lysosomal pH sensors are lysosomotropic agents that can alter lysosomal pH.

[0304] Commercially available LysoSensor dye is an acidotropic probe, which is fluorescent in a pH-dependent manner. In more acidic compartments, LysoSensor displays increased fluorescence. Therefore, an increase in intensity following treatment supports hyperacidification. LysoTracker labeling was utilized following GN64 treatment in PDAC cells to further assess effects on acidification of lysosomes. GN64 treatment led to an increase in LysoSensor labeling (FIGs. 17A-17B); as fluorescence increases with decreasing pH, this data indicates that GN64 treatment leads to hyperacidification of lysosomes in PDAC cells. It is worth noting that LysoSensor is primarily semiquantitative and qualitative, without allowing for estimation of intracellular pH.]0305] It was next sought to more quantitatively assess the pH of untreated versus GN64 treated lysosomes. Newly developed Acid pH Indicator Dye (ApHID) shows superior dynamic range and brightness increases with acidity as well as low photobleaching and resistance to oxidation. ApHID was employed to determine the pH of PDAC lysosomes following GN64 treatment. Using a prepared calibration curve, the pH of lysosomes can be quantified. In PaTu 8988T cells, GN64 significantly reduced the pH of lysosomes in a dosedependent manner (FIGs. 18A-18B). Untreated PaTu 8998T cells showed a lysosomal pH of 4.17, in line with the reported pH of homeostatic lysosomes. As a positive control, BafAl was used and a significant increase in pH was observed as expected (pH 7.44) (FIG. 18C). GN64 treatment induced a dose dependent decrease to less than pH 2.5.-79-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0306] GN64 treatment leads to upregulation of lysosomal genes, specifically those related to lipid catabolism

[0307] Next, transcriptional changes induced by GN64 in PDAC cell lines was investigated. Changes to the transcriptional profile of PDAC cells can reveal relevant pathways to further study the mechanism of GN64. RNA sequencing was performed in PaTu 8988T cells following 24h treatment with GN64 [2.5 pM] (n=3). I l l genes were identified as being significantly transcriptionally upregulated in response to treatment and 24 genes as downregulated. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene ontology (GO) pathway analysis was performed for the significantly upregulated genes (ShinyGO 0.80). KEGG analyses revealed significantly upregulated genes were associated with the lysosomes (FIG. 19A) further supporting GN64-induced alterations to the ALP.Significantly upregulated genes associated with the lysosomes include cathepsins (CTSD, CTSA, and CTSL), M6PR, and lysosomal membrane proteins (CD63 and LAMP1) (FIG. 19C). GO Biological Process analyses revealed many gene members of the ganglioside catabolic process to be significantly upregulated (FIGs. 19B, 19D). Ganglioside accumulation has been shown to lead to inhibition of lysophagy and accumulation of autophagic markers in lysosomes. Together, these data support GN64-induced ALP modulation and may reveal GN64 induced alteration to gangliosides degradation.

[0308] Discussion

[0309] It was previously shown that GN64 inhibits lysosomal degradation using the DQ- BSA assay and confers cytotoxicity to a panel of PDAC cell lines. Additional orthogonal assays were employed to further confirm inhibition of lysosomal degradative capabilities. It was demonstrated that GN64 induced accumulation of autophagic markers, LC3-II and p62, both typically degraded during functional autophagy. It was also shown that GN64 does not affect the maturation of CTSD. Furthermore, lysosomal mass and morphology was investigated. GN64 caused increased levels of lysosomal marker, LAMP1 and caused an enlargement of lysosomal size. Utilizing lysosomotropic LysoTracker dye, it was shown that GN64 induces increased labeling, supporting that GN64 inhibits lysosomal degradation in a distinct mechanism that does not involve alkalization or LMP. The divergent ALP inhibitory phenotypes induced by HCQ and GN64 were highlighted, both of which are 4--80-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 aminoquinoline small molecules. The pH of GN64 treated PDAC lysosomes was directly assessed and it was shown that GN64 induced hyperacidification of lysosomes, which is the proposed mechanism by which lysosomal function is impaired. Transcriptomic analysis using bulk RNA sequencing further support the ALP inhibitory properties of GN64, revealing upregulation of lysosomal genes and specifically, those related to ganglioside catabolic processes.

[0310] GN64 as an inhibitor of the ALP

[0311] The fusion of autophagosomes with lysosomes delivers cargo to the acid hydrolases of the lysosomal lumen. LC3-II also enters the autolysosomal lumen and is degraded during functional autophagy. However, as a standalone assay, the levels of LC3-II can be indicative of either increased autophagosome number or autophagic flux blockade. It is more common to simultaneously study other autophagic substrates. Autophagic adaptor protein p62 is also a substrate of autophagy and its accumulation can be a sign of autophagy dysfunction.

[0312] The levels of LC3-II and p62 were measured using immunoblotting with GN64- treated lysates from PDAC cells. As the DQ-BSA assay already showed evidence of lysosomal degradation inhibition, it was sought to use these assays as orthogonal methods for rigor and robustness of the results. GN64 treatment led to increased levels of both LC3- II and p62, further supporting lysosomal inhibition.

[0313] It was shown that GN64 induces an increase in lysosomal mass as detected by the lysosomal marker LAMP1. It is unclear if the effect is due to inhibited turnover through lysophagy or as a consequence of the nuclear translocation of TFEB or a combination effect. Investigations into CQ-induced accumulation of LAMP 1 support a potential block of lysophagy over new protein synthesis. Most of what is known about lysophagy is based on studies involving the induction of lysosomal damage, and not basal lysosomal turnover.

[0314] It was also shown that GN64 induces enlargement of lysosomes. Swelling of lysosomes can be induced by a variety of mechanisms including osmotic imbalance and accumulation of undegraded cargo. These triggers can also occur simultaneously, making it difficult to identify the exact mechanisms. Nevertheless, lysosomal dysfunction is a-81-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 hallmark of enlarged lysosomes, with degradation and trafficking being negatively impacted. This deleterious change in lysosomal morphology further shows GN64 inhibits the ALP.

[0315] The effects of GN64 on markers of the endosomal pathway, another route mediating cargo delivery to lysosomes, was also explored. The proper trafficking and recruitment of accessory proteins for endosomal function are mediated by the family of Rab GTPases. As endosomes mature from early endosomes (EE) to late endosomes (LE) and ultimately lysosomal fusion, they utilize a Rab cascade where one Rab is replaced with another along the pathway. Rab5 is associated with EE. Rab5 is the main RabGTPase associated with EE; it mediates the recruitment of early endosome antigen 1 (EEA1), which is necessary for proper tethering between endocytic vesicles and early endosomes. Rab5 also recruits and activates the PI-3-kinase, Vps34, which promotes the synthesis of specific lipids on endosomes. As EE mature to LE, Rab5 is exchanged for Rab7, which ultimately mediates fusion with lysosomes. Interestingly, Rab7 is also found on lysosomes and autophagosomes and is required for the fusion of autophagosomes and lysosomes.

[0316] Inventors monitored the levels of Rab5 and Rab7 following GN64 treatment. GN64 treatment did not affect protein levels of EE marker, Rab5 (FIGs. 20A-20C). These data support that GN64 does not cause early endocytic disturbance. However, both HCQ and GN64 caused an accumulation of LE marker, Rab7 (FIGs. 21 A-21C). It is difficult to decipher if this is a direct effect on the late endosomal pathway or a further consequence of the autophagy inhibition previously shown as Rab7 is found on both late endosomes and autophagosomes. But, Rab7 turnover has been shown to be lysosomal dependent, whereas inhibitors of lysosomal activity (BafAl) induce an accumulation of Rab7. The accumulation of Rab 7 can be further evidence of ALP inhibition.

[0317] Differential ALP inhibitory properties of GN64 and other 4-aminoquinoline, HCQ

[0318] GN64 shares 4-aminoquinoline structure with known ALP inhibitor, HCQ. HCQ is a lysosomotropic agent, known to accumulate in acidic organelles. As a weak base, HCQ directly neutralizes the pH of the lysosomal lumen. It was previously demonstrated that GN64 has over 10-fold greater cytotoxic potency compared to HCQ. This effect alone does-82-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 not prove that GN64 and HCQ have distinct mechanisms of action. Investigations into CTSD maturation have shown that HCQ inhibits the maturation of CTSD; this effect can be a consequence of the known lysosomal alkalization. Deviation from the optimal acidic pH inhibits the activity of CTSB and CTSL, which mediate the final processing of CTSD.

[0319] However, treatment with GN64 results in no alteration to the levels of mature CTSD. The inability of GN64 to affect CTSD maturation does not indicate luminal alkalinization, as seen with HCQ. This supports unique mechanisms for these two 4- aminoquinolines. GN64’s divergent mechanism of action from lysosomal alkalization is further supported by the marked increase in LysoTracker labeling observed after treatment. LysoTracker requires an intact, acidic lumen to become protonated and accumulate within. In contrast, lower levels of LysoTracker labeling were observed with HCQ, supporting its known mechanism of alkalization. Lower levels of LysoTracker labeling can also indicate LMP, as compromised membrane integrity prevents accumulation of the dye. GN64 also did not display this phenotype, suggesting a mechanism of ALP inhibition that is not mediated by alkalization or LMP.

[0320] GN64 as an inducer of hyperacidification of lysosomes

[0321] Without evidence of alkalization of pH, it was investigated if GN64 induced hyperacidification of lysosomes. The maturation of CTSD and increased LysoTracker labeling are not indicative of lysosomal alkalization, as we observed with HCQ. Using LysoSensor green, we observed evidence of hyperacidification, as demonstrated by the increased fluorescence of this pH-sensitive dye. However, this allows for qualitative assessment without being able to quantify the pH directly. Using the novel pH-sensitive dye, ApHID, we demonstrate that GN64 induces hyperacidification of lysosomes in PDAC cells with GN64 [1.25 pM] results in a pH of 2.21 compared to a pH of 4.17 in untreated cells. The function of lysosomes is strongly dependent on a narrow window of optimal pH. Deviation from pH range 4-5 will have deleterious consequences on ALP function, as we show with GN64. The GN64 induced hyperacidification can explain the increased LysoTracker labeling that was observed. A lysosomal lumen with more protons can accommodate and protonate more LysoTracker dye, leading to higher signal observed.-83-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0322] It was also shown that GN64 did not affect levels of CTSD maturation. A likely explanation could be maturation occurring in endosomes before reaching the hyperacidified lysosomes. Endosomes, like lysosomes, also contain V-ATPase proton pumps, and show decreasing pH as they mature and eventually fuse with lysosomes. Late endosomes can exhibit a pH of 5.0-5.5. CTSL has been shown to be activated at slightly acidic pH 5.5-6.02 With this information, it is plausible that CTSD can be activated before reaching the hyperacidified lysosomes. HCQ has been shown to alkalinize various cellular compartments, including the Golgi and endosomes. This would prevent any acid-dependent maturation of CTSD. If the hyperacidification induced by GN64 is lysosomal specific, maturation can occur prior to fusion with lysosomes. Even though matured, the cathepsins and other pH sensitive acid hydrolases may have limited activity in the hyperacidified lysosomes.

[0323] It is unlikely that maturation can occur in the hyperacidified lysosomes of GN64 treated PDAC cells. It has been reported using in vitro assays that CTSL is irreversibly inactivated at acidic pH (pH < 4.0) and that its pH dependent stability relies on a delicate balance of hydrophobic and ionic interactions. Small synthetic substrates and competitive inhibitors have been shown to substantially stabilize, and even protect, CTSL against pH- induced denaturation, which may allow for CTSL to remain active in hyperacidified lysosomes. However, the binding of CTSL to substrates is also pH dependent with much lower binding observed at acidic pH.

[0324] Overall, the data presented demonstrates that GN64 inhibits the ALP via mechanisms resulting in hyperacidification of lysosomes. Concurrently, GN64 is cytotoxicity to ALP-dependent PDAC cells. It is difficult to say directly if the impaired ALP leads to the cytotoxicity observed. However, the multifaceted support of the ALP in PDAC, supports the therapeutic strategy of ALP inhibition.

[0325] GN64 transcriptional alterations

[0326] GN64 treatment upregulates expression of many genes related to lysosomal function. Taken together with our data showing GN64 inhibits the ALP, these transcriptional responses may represent a compensatory response. In fact, many genes found-84-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 to be upregulated are known TFEB target genes, whose expression is induced following nuclear translocation of TFEB in response to lysosomal dysfunction.

[0327] Previously, we showed that CTSD maturation was not affected by GN64 treatment. Transcriptional analysis reveals GN64 induces expression of cathepsin genes, including CTSD and CTSL, which is responsible for the maturation of CTSD. We also observed increased LAMP1 protein expression, following GN64 treatment, indicative of an increase in lysosomal mass. GN64 treatment also caused an increase in LAMP1 gene expression, matching the observed increases at the protein level.

[0328] Interestingly, an increased expression of genes related to glycolipid catabolic processes was seen, specifically, ganglioside catabolism. GN64 induces the expression of lysosomal enzymes that mediate the breakdown of these glycolipids. It’s plausible that GN64 may induce the accumulation of glycosphingolipids within lysosomes and lead to the induced expression of glycosphingolipid catabolism as a compensatory mechanism. It’s been shown that accumulation of glycolipids within lysosomes leads to swelling of lysosomes, which we observe following GN64 treatment. In fact, the optimal pH of HexA was shown to be pH 4.0-4.4; deviation from this range, including the hyperacidification induced by GN64, would reasonably impair activity and glycosphingolipid degradation. Further work is necessary to better interrogate potential glycolipid accumulation in lysosomes following GN64 treatment. Lipidomics can be used to assess lysosomal content after treatment, which may reveal aberrant accumulation of lipids in PDAC lysosomes.]0329] Methods]0330] Cell line culture

[0331] Cell line culture performed as previously described in Example 1.

[0332] Cell lysis and immunoblotting

[0333] PDAC cells were seeded in 6-well plates (MIA PaCa-2: 300,000, PaTu 8988T: 180,000) and cultured for 24h. Treatments including hydroxychloroquine [20 pM] (C6628- 25G, Sigma) and GN64 [2.5-0.5 pM] were added to cells for 24h. Cells were washed IX-85-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 with PBS and lysed with IX RIP A Buffer (9806, Cell Signaling Technologies) supplemented with PMSF (36978, Thermo Scientific) and cOmplete Protease Inhibitor Cocktail (11697498001, Millipore Sigma) for 30 min. Samples were collected in 1.5 mL Eppendorff tubes and subjected to centrifugation at 21,000 x g for 15 min. Cleared lysates were collected and protein quantification was performed using DC protein assay (Bio-rad). Lysates were combined with 4X Laemmli Sample Buffer (1610747, Bio-red) supplemented with 2-Mercaptoethanol (M3148, Millipore Sigma).

[0334] Protein samples were subjected to SDS-PAGE gels (Bio-Rad), electrophoresed, transferred to Immobilon-FL PVDF membranes (Millipore) and WB. For quantification, Image Studio Lite (LI-COR) was used.

[0335] The following primary antibodies were used for immunoblotting: p62 (SQSTM1, PM045, MBL International Corporation (MBLI); 1 : 1000), LC3B (NB 100-2220, Novus Biologicals; 1 : 1000), Tubulin (ab56676, Abeam; 1 :2000), Cathepsin D (ab6313, Abeam; 1 : 1000), LAMP1 (9091, Cell Signaling Technology; 1 : 1000), Rab5 (3547, Cell Signaling Technology, 1 : 1000), Rab7 (sc-376362, Santa Cruz Biotechnology; 1 :500). Horseradish peroxidase (HRP)-conjugated anti-rabbit and mouse secondary antibodies (NA9340V, NXA931V, GE Healthcare) for enhanced chemiluminescence (ECL) substrate (Millipore) and IRDye 800CW goat anti-rabbit IgG (H+L) or anti-mouse IgG (H+L) secondary antibodies (925-32211, 925-32210, LLCOR) for Odyssey CLx Imaging (LLCOR) were used.

[0336] Immunofluorescence and lysosomal size quantification

[0337] PaTu 8988T cells were seeded in 8w chamber slides (100,000 per well) (EMD Millipore) coated with Poly-D-Lysine (A-003-E, Millipore) and cultured for 24h.Treatments including GN64 [2.5-0.5 pM] were added to cells for 24h. Cells were washed IX with ice cold PBS. Cells were then fixed in ice cold methanol for 5 mins. Cells were washed 3X with PBS and subsequently blocked with 5% Normal Donkey Serum (017-000- 121, Jackson ImmunoResearch) in PBS for Ih. Immunofluorescence was performed using antibodies against LAMP2 conjugated to Alexa Fluor 488 (sc-18822, Santa Cruz; 1 : 50). Cells were stained with 4',6-Diamidino-2-Phenylindole, Dihydrochloride (DAPI, D1306,-86-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325ThermoFisher) for 5 min and mounted using Fluoromount-G (17984-25, Electron Microscopy Sciences). Cells were imaged using a Zeiss Axio Observer 3.

[0338] For quantification of lysosomal size, following background subtraction, individual cell masks were generated by thresholding and dilation of the DAPI fluorescence signal. LAMP2 fluorescence was thresholded within cell masks and a watershed transformation was applied to separate individual lysosomes. The area (pm2) was calculated for each lysosome.

[0339] Lysotracker Red labeling

[0340] PDAC cells were seeded in 96-well plates (MIA PaCa-2: 25,000, PaTu 8988T: 8,000) in 100 pL of media and cultured for 24h. Treatment was prepared at 5X desired final concentration and 25 pL was added to the cells in respective wells for 24h. BafAl treatments were performed for only the final 2h. Cells were washed with PBS and incubated with LysoTracker Red (L7528, ThermoFisher) [50 nM] and Hoechst (in complete media) for 30min. Cells were washed with PBS and then fixed in 4% PF A. Cells were washed 3X with PBS and then imaged on a Cytation 5 Cell Imaging Multi-Mode Reader (Agilent).

[0341] Ly sosensor Green labeling

[0342] MIA PaCa-2 cells were seeded in 35 mm dishes with a No. 1.5 PDL coated coverslip (P35GC-1.5-10-C, Mattek) (500,000 per dish) and cultured for 24h. Media was aspirated, and 2 mL of treatment added at final concentration for 24h. Cells were washed with PBS and incubated with LysoSensor Green (L7535, ThermoFisher) [1 pM] and Hoechst (in complete media) for Ih. Cells were washed with PBS and imaged using a Zeiss Axio Observer 7.

[0343] For quantification of LysoSensor Green, following background subtraction, individual cell masks were generated by thresholding and dilation of the DAPI fluorescence signal. LysoSensor Green fluorescence was thresholded within cell masks and a watershed transformation was applied to separate individual lysosomes. The area (pm2) of each cell and corresponding area occupied by positive LysoSensor Green labeling was calculated for each cell and displayed as a percentage.-87-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0344] Quantification of lysosomal pH using Acid pH Indicator Dye (ApHID)

[0345] PaTu 8988T cells were seeded in 35 mm dishes with a No. 1.5 PDL coated coverslip (P35GC-1.5-10-C, Mattek) (400,000 per dish) and cultured for 24h. Dextran (70kDa ApHID- pH dependent and Alexa647- pH independent) was solubilized in PBS to final concentration of 15-25mg / mL and diluted in media at 1 mg / mL. Media-dextran solution was added to cells at 1 mg / mL to each well along with appropriate treatment. Cells were incubated overnight for uptake. Dextran was washed off with PBS and chased with media for 2 hours.

[0346] For calibration dishes, cells were washed 3X with PBS and fixed with 0.5% PFA for 5 minutes followed by 3X washes with PBS. Fixed calibration wells were incubated in pH calibration buffers. Calibration buffers: pH 2, 3 (50 mM Citric Acid, 50 mM Sodium Citrate + 20mM Methylamine + 20mM Sodium Acetate) pH 4, 5 (50mM Tris Maleate + 20mM Methylamine + 20mM Sodium Acetate) pH 6, 7 (50mM Sodium Phosphate Monobasic Anhydrous + 20mM Methylamine + 20mM Sodium Acetate). 2.5 pM Nigericin + 2.5 pM Monensin was added to buffers right before incubating with fixed cells to ensure buffer equilibration across membranes.

[0347] For sample dishes, cells were washed 3X with IX Medium-2 (170 mM NaCl, 20 mM 4-(2-Hydroxyethyl)piperazine-l -ethane-sulfonic acid (HEPES), 1.3 mM CaC12, 5 mM KC1, 1 mM MgC12 + 0.2% Glucose) and imaged in IX Medium-2.

[0348] Calibration and sample dishes were imaged at 40x using a Zeiss Axio Observer 7, with a minimum of 16 fields per well. Lysosomes were masked using the pH-insensitive Alexa647 signal. Both Alexa647 intensity and pH-sensitive 70kDa ApHID intensity was measured within each lysosome and the resultant ratio was calculated. A sigmoidal calibration curve of buffer pH vs signal ratio was built, and sample pH values were interpolated using GraphPad.

[0349] Bulk RNA sequencing and pathway analysis

[0350] PDAC cells were seeded in pl 00 dishes (PaTu 8988T: 2,000,000) and cultured for 24h. GN64 treatment [2.5 pM] was added to cells for 24h. Cells were washed IX with PBS,-88-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 trypsinized, and pelleted. Cells were resuspended in media before counting. One million cells were pelleted and washed with PBS and then stored at -80C as a dry pellet. Once three biological replicates were obtained, all samples were transferred to the Integrated Genomic Operation Core (MSKCC) for processing including RNA extraction, PolyA sequencing, and analysis. Pathway analysis was performed on statistically significantly upregulated genes using (1) Kyoto Encyclopedia of Genes and Genomes (KEGG) (False discovery rate (FDR) < 0.05) and (2) Gene Ontology (GO) Biological Process (FDR < 0.05) pathway databases.

[0351] Example 3: Photoaffinity labeling identifies NPC Intracellular Cholesterol Transporter 1 (NPC1) as a molecular target of GN64

[0352] Chemical biology approaches were utilized to determine the molecular target of GN64. GNP2 2, a photoactivatable probe with a click chemistry-amenable alkyne handle, was developed based on the pharmacophore of GN64. GNP2 2 retained ALP inhibitory properties and cytotoxicity in PDAC cells, supporting its use in target identification studies utilizing photoaffinity labeling (PAL) approaches in PDAC cells. In situ photolabeling revealed that GNP2 2 localized to lysosomes in PDAC cells. Using quantitative proteomics, it was determined that GNP2 2 specifically binds to NPC1, and this interaction could be blocked by GN64. NPC1 KO cells were generated to further examine the role of NPC1 in the ALP inhibitory and cytotoxic mechanisms of GN64. PDAC NPC1 KO cells exhibited resistance to GN64 cytotoxicity. Furthermore, GN64 failed to induce hyperacidification of lysosomes in NPC1 KO cells, supporting NPC1 as the protein target of GN64.

[0353] The development of the photoprobe GNP2 2 and its use in identifying the mechanistic target of GN64 are described. The addition of a benzophenone and alkyne handle was confirmed not to affect ALP inhibitory or cytotoxic properties. Using GNP2 2, the small molecule was shown to localize to lysosomes, consistent with previous data. Combining PAL with quantitative proteomics identified NPC1 as a putative target protein of GN64. Given the canonical role of NPC1 in mediating the egress of cholesterol from lysosomes, it was investigated whether GN64 could induce cholesterol accumulation; however, this phenotype was not observed. Interestingly, although GN64 alone did not induce cholesterol accumulation in lysosomes, it synergized with the NPC1 inhibitor -89-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325U18666A. This synergistic effect did not result in increased cytotoxicity, supporting the conclusion that cholesterol accumulation is unlikely to drive cell death. To further interrogate the binding of GN64 to NPC1, cross-competition PAL studies were performed with U18666A and its photoprobe, U-X. Cross-competition was observed, in which labeling of NPC1 by U-X was blocked by GN64, and labeling of NPC1 by GNP2_2 was blocked by U18666A, supporting a shared binding site. Mutation studies demonstrated that a P691S mutation in the sterol-sensing domain (SSD) of NPC1 abolished labeling of NPC1 by GN64; this same residue is critical for U18666A binding, further supporting a shared binding site. Validation studies using PDAC NPC1 KO cells showed that these cells exhibited enhanced resistance to GN64 treatment. Furthermore, NPC1 KO cells were immune to GN64-induced hyperacidification, supporting NPC1 as the mechanistic target of GN64.

[0354] Results

[0355] GNP2 2 is a photoprobe based on the pharmacophore of GN64

[0356] GN64 was previously been shown to inhibit the ALP, inducing hyperacidification of lysosomes and transcriptomic upregulation of lysosomal genes, particularly those related to ganglioside catabolic processes. However, the direct biological target responsible for these effects remains elusive.

[0357] Small molecule protein target identification can be achieved through modifying the candidate pharmacophore to include a moiety capable of UV-inducible covalent linkage to the target protein and an affinity handle for target isolation, known as photoaffinity labeling (PAL). Benzophenone is a photoreactive moiety, which can react with adjacent C-H bonds. Alkyne moieties allow for click chemistry to be performed post-labeling, to attach a fluorescent dye or affinity handle for in-gel fluorescence or target enrichment, respectively.

[0358] GNP2 2 (FIG. 22) is based on the 4-aminoquinoline, aromatic linker, and piperazine pharmacophore of GN64, modified to include a photoreactive benzophenone and alkyne click-chemistry handle for affinity handle and / or fluorescence attachment.-90-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0359] For photoprobes to be useful in target identification endeavors, they must retain the ability to bind the same target protein. Chemical modification to the original small molecule may alter binding properties. To ensure relevance in target identification studies, photoprobes can be assessed for retained activity. In this case, it was investigated if GNP2 2 retained (1) PDAC cytotoxicity and (2) ALP inhibitory properties. Using the DQ- BSA assay, it was demonstrated that GNP2 2 retains ALP inhibition (FIGs. 23 A-23B). GNP2 2 also conferred cytotoxicity in PDAC cells (FIG. 23C), demonstrating that these modifications do not alter activity.

[0360] GNP2 2 labels lysosomes

[0361] The alkyne handle of GNP2 2 allows for attachment of a variety of chemical moieties via click chemistry. To visualize the subcellular localization of GNP2 2, following covalent attachment of GNP2 2 via UV crosslinking, an azide fluorophore, can be added in fixed cells to perform in situ click chemistry (FIG. 24). This developed methodology is amenable to downstream use with immunofluorescence as well, allowing for visualization of GNP2_2 along with various structural markers.

[0362] This strategy was applied using immunofluorescence antibodies against LAMP1 and CTSD, where positive co-staining indicates lysosomes. This method revealed that GNP2 2 localizes to lysosomes (FIG. 25, Column A), supporting a lysosomal subcellular site of action. Negative controls were incorporated to determine background fluorescence, nonspecific labeling, and potential fluorescence bleed-through (FIG. 25, Columns B, C). In Column B, primary antibodies were omitted during immunofluorescence processing. The absence of fluorescent signal for CTSD or LAMP1 confirmed the lack of non-specific secondary labeling and the absence of bleed-through from the AF647 channel into those used for CTSD or LAMP1 visualization. In Column C, GNP2 2 treatment was omitted, but click chemistry with AF647-N3 was still performed. The absence of signal in the AF647 channel demonstrated that fluorescence was entirely dependent on GNP2 2 and that no background signal was generated from the click-chemistry reaction. Furthermore, the retention of primary and secondary antibodies in Column C confirmed that there was no fluorescent bleed-through into the AF647 channel. These controls further validated the-91-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 visualization of GNP2 2 and supported its lysosomal subcellular localization, as highlighted in the region of interest (FIG. 25, ROI).

[0363] GNP2 2 specifically labels NPC1

[0364] Beyond visualization, PDAC cells treated with GNP2 2 can be subjected to UV crosslinking and lysis followed by biotin-azide click-chemistry and streptavidin enrichment to isolate putative protein targets, which can then be identified by quantitative proteomics (FIG. 26). PaTu 8988T cells were treated with GNP2 2, with and without parent, GN64, pre-blocking to determine specific targets.

[0365] Click chemistry mediated attachment of TAMRA-Biotin- Azide with a cleavable [N- l-(4,4-dimethyl-2,6-dioxocyclohex-l-ylidene)-3-ethyl] (Dde) linker to the alkyne handle of GNP2 2 was performed. Using streptavidin-agarose beads for affinity enrichment and hydrazine elution, we initially performed in-gel TAMRA fluorescence to identify protein bands of interest that are enriched in the GNP2 2 alone samples compared to the GN64 parent blocking samples. Live cell photolabeling with subsequent TAMRA-Biotin-Azide click-chemistry and affinity enrichment and downstream SDS-PAGE reveal multiple specifically labeled protein bands (FIG. 27).

[0366] Specifically labeled protein bands identified with in-gel fluorescence supported combining live cell photolabeling with mass spectrometry to identify these putative targets. Quantitative proteomics on post-affinity eluents revealed 27 putative targets with >1.5 fold enrichment (-log2(GN64 / DMSO) < -0.0585) in the unblocked vs. GN64 blocked conditions, of which nine reached statistical significance (p<0.05; n=3 biological replicates) (FIG. 28, Table 1.3).Table 1.2 Proteins specifically photolabeled by GNP2 2 and identified by quantitative proteomics.-92-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0367] One of the 27 putative protein targets was Niemann-Pick disease, type Cl (NPC1). NPC1 is a membrane protein that mediates intracellular cholesterol and lipids, including sphingosine (a ganglioside precursor) trafficking, specifically from lysosomes to post- lysosomal destination. Mutations to NPC1 result in Niemann-Pick disease, type C, a lysosomal storage disorder which leads to accumulation of cholesterol in lysosomes.-93-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0368] PAL studies were repeated for downstream SDS-PAGE and immunoblotting with antibodies specific for NPC1. In PDAC cells, GNP2_2 specifically labels NPC1 as shown with blocked photolabeling with pre-treatment of GN64 (FIG. 29).

[0369] PAL studies were repeated using purified membrane fraction from PDAC (PaTu 8988T) cells, referred to as in vitro photoaffinity labeling. Membrane fraction was preincubated with GN64, GN79, and HCQ at 10 pM followed by incubation with GNP2_2 [100 nM], In vitro photoaffinity labeling reveals GNP2_2 specific labeling of NPC1, as shown by blocking with both GN64 and GN79 whereas HCQ blocking fails to block GNP2 2 specific labeling of NPC1, further highlighting a unique mechanistic target of GN64 and GN79 compared to HCQ (FIG. 30).] 03701 GN64 fails to induce cholesterol accumulation yet synergizes with U18666AJ0371] The canonical function of NPC1 is mediating cholesterol egress from lysosomes. Low density lipoprotein (LDL) internalized via clatherin-mediated endocytosis and trafficking to lysosomes to be broken down into free cholesterol by lysosomal lipases. NPC1, in coordination with lysosomal luminal protein NPC2, facilitates the egress of cholesterol from the lysosome to other parts of the cell or for storage in the ER.

[0372] A hallmark of NPC1 loss of function is accumulation of free cholesterol within lysosomes. NPC1 loss of function is characterized by the accumulation of cholesterol in endocytic organelles that have characteristics of late endosomes and / or lysosomes; these abnormal organelles can be referred to as lysosome-like storage organelles (LSOs). The accumulation of cholesterol can be detected using fl lipin, a naturally fluorescent antifungal antibiotic detergent that binds specifically to free cholesterol. Use of fllipin and automated microscopy techniques have been used to identify compounds that both inhibit NPC1 and revert cholesterol accumulation in NPC1 loss of function cells.

[0373] Small molecule inhibitors of NPC1 (NPCli) already exist. The cationic amphiphilic drug, U18666A (FIG. 31 A) was initially developed as a cholesterol synthesis inhibitor.U18666A was found to mimic loss of function of NPC1 and shown to prevent the egress of cholesterol from the late endosomes and lysosomes. More recently, the binding site of U18666A was mapped to the SSD of NPC1. This binding site was discovered through -94-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 studies using U-X (FIG. 3 IB), a U18666A derivative with a benzophenone and alkyne handle that permits ultraviolet-induced crosslinking and click-chemistry affinity handle attachment.

[0374] PDAC cells were treated with GN64 and the NPC1 inhibitor U18666A, and fllipin accumulation in lysosomal storage organelles (LSOs) was assessed using fllipin staining. U18666A caused robust accumulation of cholesterol, indicated by a large increase in fllipin staining (FIGs. 32A-32B). In contrast, GN64 treatment did not result in cholesterol accumulation (FIGs. 32A-32B). Inhibition of the canonical role of NPC1 would be expected to prevent cholesterol egress from lysosomes; however, this effect was not observed with GN64 treatment.

[0375] Although specific binding of GN64 to NPC1 was observed based on PAL studies, inhibition of canonical NPC1 function was not detected. It was then investigated whether GN64 might act as an activator of NPC1, in which binding would enhance the canonical function of cholesterol egress. To test this hypothesis, PDAC cells were pre-treated with a low dose of U18666A followed by GN64 treatment. This approach was intended to determine whether GN64 could reduce the cholesterol accumulation induced by U18666A, a method previously used to identify small molecules capable of reversing cholesterol accumulation in Niemann-Pick C cells. Unexpectedly, GN64 synergized with low-dose U18666A to produce massive increases in cholesterol accumulation in LSOs (FIGs. 33 A- 33B).

[0376] Given the synergistic accumulation of cholesterol observed with U18666A and GN64, it was next investigated whether these effects resulted in synergy in cytotoxicity. The cytotoxicity of the NPC1 inhibitors U18666A and itraconazole (ITZ) was first assessed. Both U18666A and ITZ exhibited minimal toxicity at concentrations below 10 pM, in contrast to GN64, which was cytotoxic at low-micromolar concentrations in PDAC cells (FIGs. 34A-34B).

[0377] Combination approaches were then employed, with NPC1 inhibitor pre-treatment followed by GN64 treatment, to determine whether the synergistic effects on cholesterol accumulation would result in cytotoxic synergy. No enhancement in GN64 toxicity was-95-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 observed with U18666A pre-treatment (FIGs. 35A-35B). Based on these findings, cholesterol accumulation induced by U18666A alone, or the synergistic cholesterol accumulation resulting from combination treatment with GN64, does not appear to be responsible for cytotoxicity.|0378] GNP2 2 labeling is blocked by NPC1 inhibitor U18666A

[0379] Cross-competition studies were employed to investigate whether (1) the NPC1 inhibitor U18666A would block GNP2 2 photolabeling, and (2) GN64 would block photolabeling of NPC1 by the U18666A photoprobe, U-X. These studies were designed to further validate NPC1 as a binding partner of GN64 and to potentially reveal shared binding sites. U-X was confirmed to photolabel NPC1, and this labeling was blocked by U18666A (FIG. 36, Lanes 1-2). Pretreatment with GN64 also inhibited U-X photolabeling of NPC1 (FIG. 36, Lanes 1-3). Additionally, pretreatment with U18666A inhibited GNP2 2 photolabeling of NPC1 (FIG. 36, Lanes 4-5). These cross-competition results support the conclusion that binding during pretreatment with either GN64 or U18666A prevents the binding of U-X or GNP2_2.

[0380] GNP2 2 fails to label P691S mutant NPC1

[0381] An amino acid substitution in the membrane region of NPC1 (P691S) does not affect NPC1 trafficking to lysosomes but prevents the transport of cholesterol out of the organelle. This sterol-sensing domain (SSD) mutation also abolished labeling with U-X, suggesting the presence of a U18666A binding site within the SSD. PDAC cells were generated to express endogenous NPC1 as well as FL AG-tagged NPC1 containing the following mutations: P202A / F203A, F503 / 4A, P691S, D786N, or wild-type (WT). Antibodies against NPC1 (detecting both endogenous and NPC1-FLAG) and against FLAG (detecting only exogenous NPC1) were used. NPC1-P691S-FLAG showed abolished GNP2_2 labeling (FIG. 37, Lanes 7-8), while NPC1-D786N-FLAG exhibited enhanced GNP2 2 labeling (FIG. 37, Lanes 9-10). NPC1-P202A / F203A-FLAG showed no change in photolabeling compared with NPC1-WT-FLAG (FIG. 37, Lanes 5-6). Expression of NPC1-F503 / 4A was very low, preventing conclusions regarding the impact of this mutation on GNP2_2 labeling. Specific GNP2_2 labeling of NPC1 was detected in all samples using-96-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 the anti-NPCl antibody, allowing visualization of both endogenous and exogenous NPC1. These findings further support the localization of a GN64 / GNP2 2 binding site within the SSD ofNPCl.

[0382] NPC1 KO cells show resistance to GN64 induced cytotoxicity

[0383] Validation is a critical step in target identification, as it is necessary to distinguish mechanistic targets from simple binding partners. To validate NPC1 as the mechanistic target of GN64, CRISPR / Cas9 and specific single guide RNAs (sgRNAs) targeting NPC1 were used. In theory, PDAC cells lacking the GN64 target would display increased resistance to GN64 treatment, as the compound would be unable to exert its effects. For NPC1 knockout (KO) generation, a plasmid enabling temporal expression of Cas9 and an sgRNA targeting NPC1 was transiently introduced into both MIA PaCa-2 and PaTu 8988T cell lines. Single-cell clones were analyzed for complete NPC1 KO by immunoblotting (FIG. 38). NPC1 KO clones were successfully generated in both MIA PaCa-2 and PaTu 8988T cells.

[0384] Cells lacking the mechanistic protein target of GN64 would, in theory, be resistant to its cytotoxic effects. To assess this, the cytotoxicity profiles ofNPCl KO cells were compared with those of wild-type (WT) cells. PDAC cell lines lacking NPC1 exhibited increased resistance to GN64 treatment (FIGs. 39A-39F). This finding supports NPC1 as the mechanistic target underlying GN64 cytotoxicity.

[0385] NPC1 KO lysosomes do not become hyperacidified following GN64 treatment

[0386] To further validate NPC1 as the target of GN64-mediated ALP inhibition, lysosomal pH was examined under conditions of both genetic and pharmacological inhibition of NPC1. Lysosomal pH in PDAC wild-type (WT) cells was compared with that in NPC1 knockout (KO) cells to determine whether genetic deletion ofNPCl could phenocopy the effects of GN64 treatment. Using the ApHID lysosomal pH sensor as previously described, NPC1 KO cells exhibited a non-significant trend toward lower lysosomal pH compared to WT cells (FIG. 40), with mean values of pH 4.10 for NPC1 KO and pH 4.39 for WT cells.-97-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0387] To further investigate the role of NPC1 in lysosomal acidification, PDAC cells were treated with the known NPC1 inhibitor U18666A. Although not statistically significant, U18666A treatment resulted in a reduction in lysosomal pH in PDAC wild-type (WT) cells, with no measurable effect in PDAC NPC1 knockout (KO) cells (FIG. 41). In PDAC WT cells, lysosomes from DMSO-treated controls had a pH of 4.166 (SD = 0.48), compared with a pH of 3.46 (SD = 0.72) following U18666A treatment. In NPC1 KO cells, lysosomal pH was 3.87 (SD = 0.46) for DMSO-treated cells and 3.80 (SD = 0.29) for U18666A-treated cells.

[0388] GN64 was previously shown to induce hyperacidification of lysosomes in PDAC wild-type (WT) cells expressing endogenous NPC1. To determine whether this effect is mediated by NPC1, the ability of GN64 to alter lysosomal pH was assessed in NPC1 knockout (KO) cells. In PDAC WT cells, GN64 treatment resulted in hyperacidification of lysosomes; however, in NPC1 KO cells, GN64 failed to induce hyperacidification (FIG. 42). These findings further support NPC1 as the mechanistic target of GN64, leading to lysosomal hyperacidification and ALP inhibition.

[0389] Discussion

[0390] GN64 has previously been shown to inhibit the autophagy-lysosomal pathway (ALP) and confer cytotoxicity to PDAC cell lines. Mechanistic studies demonstrated that GN64 inhibits the ALP through multiple orthogonal assays, while also inducing accumulation of lysosomal mass and hyperacidification of lysosomes. Transcriptional profiling of GN64-treated cells revealed upregulation of lysosomal genes, including those involved in lipid catabolism.

[0391] Biochemical target identification approaches were employed to determine the molecular target of GN64. A photoprobe, GNP2_2, was developed based on the pharmacophore of GN64, incorporating a photoreactive benzophenone and a versatile clickchemistry handle for downstream assays. GNP2 2 was shown to retain both cytotoxic and ALP inhibitory properties, supporting its utility in target identification studies.J0392] Exploiting the click-chemistry handle for fluorophore attachment enabled visualization of GNP2 2 subcellular localization, which was found to be lysosomal in -98-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325PDAC cells, consistent with its ALP inhibitory mechanism and site of action. When the click-chemistry handle was used to attach a biotin-based affinity tag, quantitative proteomics identified specific, putative binding targets of GNP2 2. Proteomic analysis revealed the lysosomal membrane protein NPC1 as one such putative target. Crosscompetition studies using the known NPC1 inhibitor U18666A and its photoprobe U-X demonstrated mutual competition between U18666A / U-X and GN64 / GNP2_2, further supporting a GN64-NPC1 interaction. Additionally, studies in PDAC cells expressing NPC1 mutant proteins showed that a P691S mutation abolished GNP2 2 photolabeling, providing evidence for a GN64 binding site within the sterol-sensing domain (SSD) of NPC1.

[0393] Validation studies showed that CRISPR / Cas9 KO of NPC1 in PDAC cells conferred resistance to GN64 cytotoxicity. Furthermore, NPC1 KO cells showed increased lysosomal pH and GN64 failed to hyperacidify lysosomes of NPC1 KO cells. Taken together, this data supports the mechanism of action by which an inhibitory interaction of GN64 with NPC1 leads to hyperacidification of lysosomes, ultimately leading to ALP inhibition and cytotoxicity.

[0394] Development of GNP2 2

[0395] Using photoaffinity labeling in drug target identification requires modifications to the pharmacophore to include photoreactive moieties and either a direct affinity handle or a click-chemistry handle for post photolabeling attachment of an affinity handle. The latter is preferred due to the smaller size of an alkyne handle and lower likelihood of altering the properties of the photoprobe. Nevertheless, significant SAR knowledge of the biologically active molecule is necessary to understand where these modifications can be implemented without altering activity. Preliminary studies determined the 4-aminoquinoline and free amine were essential to activity. Furthermore, alterations to the linker group also greatly affected activity regarding ALP inhibition and cytotoxicity.

[0396] Early SAR studies did reveal that various substitutions to the phenyl piperazine affected activity. GNP2 2 lacks the ability to test these modifications and thus precludes the ability to differentiate how various phenyl piperazine modifications would affect-99-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 photolabeling. However, with limited ability to modify the core pharmacophore of the GN Series, this photoprobe is the closest design possible without affecting activity.Furthermore, the implementation of parent blocking with GN64 during photoaffinity labeling studies ensures specific interactions, that could be blocked by preincubation with GN64, were investigated.

[0397] Putative targets of GN64 identified by quantitative proteomics

[0398] PAL and quantitative proteomics revealed 27 putative targets with >1.5-fold enrichment in the unblocked vs. GN64 blocked conditions, with nine reaching statistical significance. Other putative targets were investigated in addition to NPC1 but failed to confirm any as mechanistic targets.

[0399] The most statistically significantly enriched putative protein, tripeptidyl peptidase 1 (TPP1), was initially investigated. TPP1 is a lysosomal serine protease that has N-terminal tripeptidyl exopeptidase activity. Mutations to TPP1 lead to neuronal ceroid lipofuscinosis, marked by the accumulation of storage material containing auto-fluorescent lipopigments in lysosomes. The function and biological consequences of observed patient mutations made this a very exciting putative target given the ALP inhibitory effects observed with GN64.

[0400] Using PAL and immunoblotting, GNP2 2 was confirmed to specifically photolabel the mature form of TPP1 (FIG. 43). CRISPR / Cas9 was used to generate TPP1 knockout (KO) PDAC cells with three different sgRNAs targeting TPP1 (#1, #2, #3) (FIGs. 44A, 44C). GN64 cytotoxicity profiling in PDAC wild-type (WT) and TPP1 KO cells produced inconclusive results, with no significant differences in sensitivity observed (FIGs. 44B, 44D).

[0401] The ability of GN64 to impair TPP1 activity in vitro was also assessed. Lysates were generated from PDAC wild-type (WT) and TPP1 knockout (KO) cells and incubated with GN64 or, as a control, the TPP1 inhibitor AAF-CMK. Using the TPP1 substrate AAF- AMC, which releases fluorescence upon cleavage, TPP1 activity was quantified following treatment. GN64 did not alter TPP1 activity in PDAC cell lysates (FIGs. 45 A, 45B). Based on these findings, TPP1 was not pursued further as a putative mechanistic target of GN64.-100-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0402] The most enriched protein in the GNP2 2 labeling sample set compared to the GN64 blocked set was reduced folate carrier (RFC), SLC19A1. SLC19A1 is ubiquitously expressed throughout the body and is an anionic exchanger mediating the major route of delivery of folates. However, immunoblotting confirmation failed to show enrichment of SLC19A1 following PAL (FIG. 46). No further explorations into SLC19A1 as a mechanistic target of GN64 were performed.

[0403] The second most enriched protein in the GNP2 2 labeling sample set compared to the GN64 blocked set was interferon induced transmembrane protein 3 (IFITM3). IFITM3 is most commonly known as an antiviral effector protein, interfering with the virus- endosomal membrane fusion process. IFITM3 was of specific interest because IFITM3 was identified as a y-secretase modulatory protein.

[0404] GNP2 2 photolabeling of IFITM3 in PDAC cells was confirmed (FIG. 47). CRISPR / Cas9 was used to generate IFITM3 knockout (KO) PDAC cells, resulting in two MIA PaCa-2 clones (#1, #2) (FIG. 48A) and one PaTu 8988T clone (#1) (FIG. 48C). GN64 cytotoxicity profiling of wild-type (WT) and IFITM3 KO cells revealed no significant differences in sensitivity (FIGs. 48B, 48D). Based on these results, IFITM3 was not pursued further as a putative mechanistic target of GN64.

[0405] Several additional proteins were identified as being specifically labeled by GNP2 2. Putative targets were investigated based on the extent to which they have been studied and their potential involvement in ALP modulation. Although the evidence supporting NPC1 as the mechanistic target of GN64 is compelling, investigation of other labeled proteins may yield additional insights. It is possible that some of these proteins are part of complexes with NPC1, with GNP2_2 interaction with NPC1 mediating proximity to these other proteins. Further studies into the NPC1 interactome, both in the presence and absence of GN64 treatment, may provide a means to explore these associations.

[0406] Segmentation of proteins detected in the proteomic analysis by organelle marker revealed that mitochondrial proteins were detected exclusively in the parent-blocked samples, in which cells were pretreated with GN64 prior to incubation with the photoprobe GNP2 2 (FIG. 49). Although these are not considered specific interactions — given that-101-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325GN64 occupancy of the binding site could preclude GNP2 2 recognition — the exclusive presence of mitochondrial proteins in the parent-blocked samples is notable. One possible explanation is that GN64 treatment may lead to mitochondrial membrane permeabilization, thereby increasing accessibility for GNP2 2 to non-specifically label mitochondrial targets. As previously described, autophagy is a key regulator of mitochondrial health in PDAC. It is therefore plausible that GN64-mediated ALP inhibition could have consequences for mitochondrial health, which may contribute to the cytotoxic effects observed.

[0407] GN64 does not induce cholesterol accumulation as a single agent but synergizes with U18666A

[0408] The effect of GN64 on NPC1 protein levels in PDAC cells was investigated. Results were inconclusive across the two PDAC cell lines examined (FIGs. 50A-50C). In PaTu 8988T cells, an increase in NPC1 protein levels was observed following GN64 treatment (FIGs. 50A, 50C). As an increase in LAMP1 protein levels was also detected, this effect may be a consequence of increased lysosomal mass, given that NPC1 is primarily localized to lysosomal membranes.

[0409] Given the role of NPC1 in mediating cholesterol egress from lysosomes and the demonstrated binding of GN64 to NPC1, the ability of GN64 to phenocopy NPC1 loss of function was investigated. A hallmark of NPC1 loss of function is the accumulation of free cholesterol within lysosomes. As expected, treatment with the known NPC1 inhibitor U18666A led to cholesterol accumulation in PDAC cells. U18666A inhibits cholesterol egress through interaction with the sterol-sensing domain (SSD) of NPC1. In contrast, GN64 treatment did not result in robust cholesterol accumulation. Cross-competition photolabeling studies demonstrated that both U18666A and GN64 bind NPC1 and can mutually block photoprobe labeling with U-X and GNP2 2, supporting the presence of a shared binding site. Furthermore, the P691S mutation — known to abolish U-X photolabeling and U18666A binding to NPC1 — also prevented photolabeling by GNP2 2, further supporting a shared binding mechanism. However, despite this overlap in binding, GN64 and U18666A diverge in their induced phenotypes with respect to cholesterol accumulation.-102-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0410] More interestingly, GN64 was shown to synergize with U18666A to induce cholesterol accumulation. The exact mechanism for this effect remains elusive. One plausible explanation is that GN64 may mediate enhanced U18666A-NPC1 binding; however, in our cross-competition PAL studies, GN64 did not show enhanced NPC1 photolabeling with U-X. Another explanation may be in the hyperacidification of lysosomes induced by GN64 concomitantly affects the ability of NPC1 to transport cholesterol. The ability of the NTD of NPC1 has also been shown to bind cholesterol at acidic pH (4.5), and loss of binding was observed at neutral pH. Variation from homeostatic pH would likely perturb the ability of NPC1 to properly traffic cholesterol. The hyperacidification of lysosomes by GN64 may disrupt cholesterol -NTD interaction and the binding of U18666A to the SSD may prevent the egress of cholesterol from the lysosome. The synergistic cholesterol accumulation may be a product of these two distinct effects, which may prevent the egress at two distinct points.

[0411] The observed synergy for cholesterol accumulation, did not translate to synergistic cytotoxicity. Initial cytotoxicity profiling studies showed U18666A, although able to induce greater cholesterol accumulation, was less cytotoxic in PDAC cells compared to GN64. Taken together, it is unlikely that cholesterol accumulation leads to cytotoxicity in PDAC cells given this robust cholesterol accumulation and muted cytotoxicity that was observed with U18666A.|0412] Differential labeling of NPC 1 mutants|0413] Explorations into PAL of mutant forms of NPC1 revealed unique patterns of GNP2 2 labeling. Mutational analysis can identify key residues involved in small molecule binding. Overexpression of NPC 1-WT-FL AG resulted in GNP2 2 specific photolabeling, with robust blocking by GN64. The adjacent P202 / F203A mutations in the NTD of NPC1 are known to abolish cholesterol binding; the NTD is thought to receive cholesterol from NPC2 before hand-off event to the SSD. These mutations failed to affect GNP2 2 specific labeling, which remained blockable by pretreatment with GN64. Mutations to Loop 2 of NPC1, were found to inhibit the interaction with NPC2; specifically mutations to F503 / 4A reduced binding affinity to NPC2. Unfortunately, NPC-F503 / 4A-FLAG expression was very low, and we could not assess impact of these residues in GNP2_2 labeling.-103-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0414] Mutations to the SSD of NPC1 were found to inactivate the protein and lead to cholesterol accumulation in late endosomal / lysosomal compartments without affect proper subcellular localization. Furthermore, using murine NPC1, a P692S (P691S in human) inhibits labeling with photoactivable cholesterol. This mutation completely abolished GNP2_2 labeling. Interestingly, this mutant was expressed at very high levels. Even with the greater abundance of this protein, no labeling was detected.

[0415] A variety of known NPCli, namely U18666A and itraconazole, block lysosomal cholesterol egress by binding the SSD, and the P691S mutation has been shown to prevent the binding of these small molecules; the P691S mutation has been shown to phenocopy loss of NPC1 function and induce cholesterol accumulation. Using U18666A photoprobe, U-X, we confirm that U-X photolabels NPC1 and is blockable by U18666A pre-treatment. To further interrogate the possibility of a shared binding site, GN64 was used to see if it can also block U-X photolabeling of NPC1. It was demonstrated that GN64 pre-treatment can also block U-X labeling of NPC1. In further cross-competition studies, U18666A could also block GNP2 2 photolabeling of NPC1. These cross-competition studies and reciprocal blocking and the ablation of GNP2 2 photolabeling of the P691S mutation highlight a potential shared binding site. Another possibly explanation is the binding of U18666A or the P691S mutation inflicts a conformational change in NPC1, that disrupts a different binding pocket targeted by GN64, precluding its interaction.J0416] Conversely, photolabeling of NPC1-D786N-FLAG was enhanced compared to NPC1-WT-FLAG. The D786N mutation is predicted to be a gain-of-function mutation, leading to enhanced cholesterol transport activity based on homology to the SSD of both cholesterol sensor, SCAP. The equivalent mutation in SCAP (D443N) renders it immune to sterol suppression, whereas the protein does not undergo a conformational change in response to cholesterol and does not bind suppressor protein, Insig. In NPC1, studies have shown that this mutation enhances the rate of cholesterol egress to the PM and ER, supporting a regulatory role for the SSD of NPC1. In our experiments, NPC1-D786N- FLAG photolabeling was greatly enhanced compared to NPC1-WT-FLAG. Assuming a similar regulatory role in NPC1 as in SCAP, a D786N mutation may result in increased activity. GN64 and GNP2_2 binding may be enhanced in relation to NPC1 activity.-104-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0417] Transcriptomic alterations and implications with NPC1

[0418] Transcriptional alterations induced by GN64 further implicate NPC1 as the mechanistic target. Among the significantly upregulated genes identified was NPC2, a cooperative protein partner of NPC1. NPC2 binds cholesterol in the lysosomal lumen and transfers it to the transmembrane protein NPC1 for cholesterol egress. The effect of GN64 on NPC2 protein levels was assessed, and treatment of PDAC cell lines with GN64 led to increased NPC2 protein expression (FIG. 51). These findings are consistent with RNA sequencing data demonstrating GN64-induced transcriptional upregulation of NPC2 in PDAC cells. It is plausible that impaired cholesterol trafficking triggers a feedback mechanism resulting in increased NPC2 expression to compensate for the dysregulation. Interestingly, treatment with hydroxychloroquine (HCQ) did not induce an increase in NPC2 protein expression, further highlighting a mechanistic divergence between these 4-aminoquinoline ALP inhibitors.

[0419] Mutations in NPC1 result not only in the accumulation of cholesterol within endolysosomal compartments but also in the accumulation of glycolipids. Altered sphingomyelin metabolism and secondary accumulation of glycolipids — including glucosylceramide, lactosylceramide, and the gangliosides GM2 and GM3 — have been reported. GN64 treatment resulted in significant enrichment of genes related to glycolipid catabolic processes, including ceramide, sphingolipid, glycosphingolipid, and ganglioside catabolism. Although direct assessment of glycolipid accumulation in GN64-treated lysosomes has not been performed, the observed transcriptional upregulation of these genes may represent a compensatory response to glycolipid accumulation caused by dysfunctional catabolic enzyme activity.

[0420] Role ofNPCl in lysosomal Homeostasis and Acidification

[0421] GN64 induces lysosomal hyperacidification in PDAC lysosomes. Based on our PAL studies, NPC1 may have an indirect role in lysosomal acidification. NPC1 has been implicated in regulating membrane contact sites (MCS), a mechanism by which it is able to hand off cholesterol to other cellular compartments including the ER and mitochondria. Loss of function ofNPCl results in remodeling to ER-Lysosome, ER-Golgi, and ER--105-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325Mitochondrial MCSs. Specific ER-plasma membrane (PM) MCSs help regulate the transfer of cholesterol to the PM, where over 40-90% of it is found in cells. In neurons, the voltagegated potassium channel Kv2.1, is a key regulator of ER-PM MCSs through a physical interaction with ER localized proteins. NPC1 loss of function was shown to remodel voltage-gated ion channels at the PM and mitochondria, leading to enhanced calcium signaling and elevated calcium levels in mitochondria causing neural toxicity. Given the importance of proton and counter ion channels are in proper lysosomal acidification, investigations into how NPCl inhibition, specifically with GN64, alters ion channel composition in lysosomes is warranted. Alterations in membrane composition, including reduced cholesterol, would greatly affect cell signaling processes and potentially necessary ion channels related to lysosomal acidification.

[0422] NPC1 relies on an acidic environment for function. Studies involving the yeast NCR1 identified a “proton-relay network” of charged residues in the transmembrane region of yeast NCR1, supporting that cholesterol transport is proton-driven. In both yeast and mammalian systems, sterol transport strictly depends on an actively maintained proton gradient across the lysosomal and vacuolar membrane, supporting the idea that cholesterol transport is pH dependent. Many proteins bind ligands in a pH specific manner; one example, as we discussed earlier, are cathepsins namely CTSL where lower binding is observed at acidic pH. The ability of the NTD of NPC1 has also been shown to bind cholesterol at acidic pH, and loss of binding was observed at neutral pH. Variation from homeostatic pH would likely perturb the ability of NPC1 to properly traffic cholesterol.

[0423] Methods

[0424] Cell line culture

[0425] Cell line culture performed as previously described in Example 1. The following cells were cultured in DMEM medium with high glucose, penicillin, streptomycin and 10% FBS: Lenti-X 293T (ATCC). All cells were cultured at 37 °C with 5% CO2 / 95% air.

[0426] Plasmids-106-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0427] The following plasmids were used: pSpCas9(BB)-2A-Puro (PX459) containing sgRNA against NPC1, IFITM3, and TPP1 (Designed by the Gene Editing & Screening Core at Memorial Sloan Kettering Cancer Center), pLVX-NPCl(WT)-FLAG (164972, Addgene), pLVX-NPCl(P202A / F203A)-FL AG (164976, Addgene), pLVX- NPC1(F5O3 / 4A)-FLAG (164974, Addgene), pLVX-NPCl(P691S)-FLAG (164973, Addgene), pLVX-NPCl(D786N)-FLAG (164975, Addgene).

[0428] The following plasmids were used for lentiviral production: psPAX2 (12260, Addgene) and pMD2.G (12259, Addgene).

[0429] DQ-BSA assay for lysosomal degradation activity

[0430] DQ-BSA assay for lysosomal degradation activity was performed as previously described in Example 1.

[0431] Cytotoxicity profding (PDAC cell lines)

[0432] Cytotoxicity profiling of PDAC cell lines was performed as previously described in Example 1. Briefly, PDAC cells were plated in 100 pL of media in 96-well (3,000-4000 for 72h treatment schemes, 10,000 for 24h treatment schemes) plates and cultured for 24h. Treatment and vehicle (DMSO) control were added to cells as 25 pL in media at 5X final concentration. Cells were cultured for indicated time interval, cell viability was determined using CellTiter-Glo® 2.0 Cell Viability Assay. Cell viability was normalized to DMSO control (100% viability).]0433] In situ photolabeling and immunofluorescenceJ0434] PaTu 8988T cells were seeded in 8w chamber slides (100,000 per well) (EMD Millipore) coated with Poly-D-Lysine (A-003-E, Millipore) and cultured for 24h. Cells were treated with photoprobe (GNP2_1 or GNP2_2) for Ih. Cells were washed IX with ice cold PBS. UV-crosslinking was performed with 350A light for 15 min on ice. Cells were then fixed in ice cold methanol for 5 mins. Click-chemistry was performed with the addition of BTTES [2 mM] (CCT-1237, Vector Laboratories), CuSCh [1 mM], and (+)-Sodium L- ascorbate [20 mM] (A7631, Sigma) in PBS with AZDye 647 Azide [50 pM] (CCT-1299,-107-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325Vector Laboratories) for Ih. Cells were washed 3X with PBS and subsequently blocked with 5% Normal Donkey Serum (017-000-121, Jackson ImmunoResearch) for Ih. Immunofluorescence was performed using antibodies against Cathepsin D (ab6313, Abeam; 1 :200) and LAMP1 (9091, Cell Signaling Technology; 1 :500). Cells were stained with 4', 6- Diamidino-2-Phenylindole, Dihydrochloride (DAPI, D1306, ThermoFisher) for 5 min and mounted using Fluoromount-G (17984-25, Electron Microscopy Sciences). Cells were imaged using a Zeiss Axio Observer 3.

[0435] Live cell photolabeling and affinity enrichment

[0436] PDAC cells were grown to -80% confluency in plOO dishes. Cells were initially treated with parent blocking in DMEM medium with high glucose, penicillin, and streptomycin (without serum) for 30 min. Photoaffinity probe was then added for an additional 30 min. Cells were washed IX with ice cold PBS and fresh ice cold PBS was added to the dishes. UV-crosslinking was performed with 350A light for 15 min on ice. Cells were scrapped and collected into 15 mL centrifuge tubes and centrifuged at 500 x g for 5 min. Cell pellets were resuspended in HEPES [50 mM], NaCl [150 mM], pH 7.2, supplemented with PMSF and protease inhibitor without EDTA (A32955, ThermoFisher) and TCEP [2.5 mM], Cells were tip-sonicated and centrifuged at 1000 x g for 5 min to pellet debris.

[0437] The supernatant was subjected to ultracentrifugation at 100,000 x g for 45 min at 4°C to pellet the membrane fraction. The supernatant was discarded, and the pellet was resuspended in HEPES [50 mM], NaCl [150 mM], pH 7.2, supplemented with PMSF and protease inhibitor without EDTA (A32955, ThermoFisher) and TCEP [2.5 mM] using tipsonication. Protein quantification was performed using DC protein assay (Bio-rad) and all samples were prepared at 1 pg / pL.

[0438] Click-chemistry was performed with the addition of 5% tert-butanol, 1% DMSO, TBTA [0.1 mM], CuSCU [1 mM], and TCEP [1 mM] in dH2O with Dde TAMRA Biotin Azide [50 pM] (BP-24299, BroadPharm) and incubated for Ih at room temperature. Samples were subjected to ultracentrifugation at 100,000 x g for 45 min at 4°C to pellet the membrane fraction. The supernatant was discarded and the pellet was resuspended in IX-108-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325RIPA Buffer (9806, Cell Signaling Technologies) using tip-sonication. Samples were rotated at 4°C for 30 min and centrifuged at 21,000 x g for 10 min at 4°C. The supernatant was collected.

[0439] For input samples, a portion was saved and combined with 4X Laemmli Sample Buffer (1610747, Bio-red) supplemented with 2-Mercaptoethanol (M3148, Millipore Sigma). The remaining supernatant was incubated with high-capacity streptavidin agarose beads (20357, ThermoFisher) and allowed to rotate overnight at 4°C.

[0440] Bead-sample mixtures were centrifuged at 300 x g and the supernatant was removed. Beads were subsequently washed twice with IX RIPA, twice with TBS-T, and once with dFbO. Bead elution was performed with 2% Hydrazine and 0.05% SDS for 30 min, shaking at RT.

[0441] Eluent was combined with 4X Laemmli Sample Buffer (1610747, Bio-red) for immunoblotting analysis or transferred as is for downstream proteomic analysis.

[0442] Quantitative proteomics

[0443] Quantitative proteomics was performed by the laboratory of Dr. Alban Ordureau.

[0444] Immunoblotting

[0445] Immunoblotting was performed as previously described in Example 2.

[0446] The following antibodies were used for immunoblotting: H3 (07-690, Millipore; 1 :2000), Tubulin (ab 56676, Abeam; 1 :2000), Niemann Pick Cl (abl34113, Abeam;1 : 1000), Niemann Pick C2 (HPA000835, Millipore Sigma; 1 :750), TPP1 (ab54685, Abeam; 1 : 1000), IFITM3 (anti-Fragilis, ab 109429, Abeam; 1 :5000), SLC19A1 (PB9504, Boster Bio; 1 :500). HRP-conjugated anti-rabbit and mouse secondary antibodies (NA9340V, NXA93 IV, GE Healthcare) for ECL substrate (Millipore) and IRDye 800CW goat antirabbit IgG (H+L) or anti-mouse IgG (H+L) secondary antibodies (925-32211, 925-32210, LI-COR) for Odyssey CLx Imaging (LLCOR) were used. For quantification, Image Studio Lite (LLCOR) was used.-109-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0447] Filipin labeling

[0448] PaTu 8988T cells (8,000) were seeded in 96-well plates in 100 pL of media and cultured for 24h. Treatment was prepared at 5X desired final concentration and 25 pL was added to the cells in respective wells for 24h. Cells were fixed in 1.5% PF A. Free cholesterol labeling was performed with filipin (F9765, MilliporeSigma) [50 pg / mL] in PBS for 45min. Cells were imaged on a Cytation 5 Cell Imaging Multi-Mode Reader (Agilent).

[0449] Lysosomal storage organelle (LSO) compartment ratio was calculated as previously described. Briefly, two different filipin intensity thresholds were applied. A lower threshold was used to generate cell masks. The number of pixels within cell masks was calculated. A second, higher threshold was used to identify LSOs. The total intensity within the pixels defined by the higher threshold was also calculated. To calculate the LSO compartment ratio, the total intensity within the pixels defined by the higher threshold was divided by the number of pixels in the cell mask.

[0450] Viral production and transduction ofNPCl-WT-FLAG andNPCl mutants

[0451] Lenti-X 293T cells were grown to -80% confluency in plOO dishes. 5 pg each of psPAX2 and pMD2.G were prepared in 450 pL of CaCh [0.25 M] along with 10 pg of target lentiviral plasmid. The DNA-CaCh solution was dropwise added to 450 pL of 2X BES Buffered Saline (14280, Millipore Sigma) and allowed to incubate for 30 min at room temperature. The solution was dropwise added to the Lenti-X 293T cells and cultured for 24h. Media was aspirated and changed to DMEM medium with high glucose, penicillin, streptomycin and 30% FBS and cultured for 48h. The media containing lentiviral particles was passed through a 0.45 pm filter. The filtered virus was immediately used for transduction or aliquoted and frozen at -80C.

[0452] Filtered virus containing media at a 1 :3 ratio with fresh media was added to cells at 50% confluency. Cells were cultured for 48h. Puromycin selection was performed for 4 days at [2 pg / mL],

[0453] CRJSPR / Cas9 gene editing-110-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0454] PDAC cells were seeded in 12-well plates (MIA PaCa-2: 125,000, PaTu 8988T: 70,000) and cultured for 24h. Media was changed to DMEM medium with high glucose, and 10% FBS (without antibiotics). Transfection was performed using Lipofectamine™ 3000 Transfection Reagent (L3000008, ThermoFisher) and appropriate plasmid containing targeting sgRNA overnight. Media was changed to DMEM medium with high glucose, penicillin, streptomycin and 10% FBS. Cells were cultured for an additional 24h. Puromycin selection was performed for 4 days at [2 pg / mL], Cells were expanded and then seeded as single cells in 96w plates for single cell clone generation. Individual clones were checked for successful KO using immunoblotting.10455] Quantification of lysosomal pH using Acid pH Indicator Dye (ApHID)J0456] Quantification of lysosomal pH using Acid pH Indicator Dye (ApHID) was performed as previously described in Example 2.

[0457] TPP1 activity assay

[0458] PDAC cells were resuspended in Acetate buffer (0.1M sodium acetate, pH 4.0) containing E64-d [10 pM] (E8640, Sigma) and Pepstatin A [10 pM] (P4265, Sigma). Cells were passed through a 25-gauge needle, ten times. Protein quantification was performed using DC protein assay (Bio-rad). Samples were prepared at 0.133 pg / pL. A total of 120 pg of protein (150 pL) was added to a 96w plate in technical triplicate for each treatment. Treatment and vehicle (DMSO) control were added to lysates as 1 pL to achieve desired final concentration. TPP1 inhibitor, AAF-CMK [20 pM] was used as a positive control. TPP1 substrate, AAF-AMC [62.5 pM] was added to each well. Cells were incubated at 37°C, shaking for 24h. Reaction was halted by adding 0.5M EDTA, pH 12.0. AMC fluorescence was measured using a fluorescence plate reader (Envision 2104 Multi-label reader). Background subtraction was performed using fluorescence from AAF-AMC in Acetate buffer alone (without cells) and normalized to DMSO (100% activity).

[0459] Example 4: Development of GN64 bioisostere, GN79 and preliminary in vivo studies-I l l-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0460] Example 4 describes the approach taken to enhance the drug-like properties of GN64. Bioisosteric replacement of the potentially mutagenic nitro group yielded GN79, containing a cyano moiety. GN79»HC1, the monohydrochloride salt of GN79 and also referred to herein as “GN79HC1,” was shown to (1) retain ALP inhibitory properties, (2) confer cytotoxicity to PDAC cell lines and PDAC HPO lines, and (3) interact with NPC1, as demonstrated by PAL studies with GNP2 2. Formulation optimization led to the development of the monohydrochloride salt GN79HC1 and a 10% HP-P-CD formulation to improve aqueous solubility. Preliminary pharmacokinetic studies indicated GN79HC1 accumulation in mouse tissues and confirmed 10 mg / kg as a well-tolerated dose for subsequent in vivo efficacy testing.

[0461] Results

[0462] GN79HC1 is a bioisostere of GN64

[0463] Nitro functional groups can present a metabolic liability, as their reduction may lead to DNA alkylation. Drug development strategies often aim to “de-risk” lead small molecules early by identifying suitable modifications that mitigate such liabilities without disrupting activity. Isoelectronicity studies have identified substitutions capable of retaining biological activity while reducing potential mutagenicity. In this context, bioisosteric substitution of the nitro group with a cyano functional group was performed to generate GN79. In parallel, the development of a salt form of GN79 was explored to enhance aqueous solubility for in vivo use. These efforts resulted in the synthesis of GN79»HC1 (also referred to herein as “GN79HC1”), the monohydrochloride salt of GN79.]0464] GN79HCI retains cytotoxic, ALP inhibitory, and NPC1 binding properties

[0465] Although previous studies showed cyano group substitutions of nitro groups have not affected the activity of other drug in development, it was imperative that these modifications to GN64 were assessed. Furthermore, salt generation has the potential to affect stability and activity in either direction. The PDAC cytotoxicity and ALP inhibitory properties of GN79HC1 was assessed and compared to GN64. Albeit less potent than GN64, GN79HC1 inhibits lysosomal degradation (FIGs. 52A, 52B) and confers cytotoxicity to PDAC cell lines (FIG. 52C)-112-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0466] To assess PDAC cytotoxicity in a more clinically relevant context, the effects of GN79HC1 were evaluated in a panel of human pancreatic organoid (HPO) lines. A panel of more than 60 HPO lines was utilized, encompassing variation in transcriptional subtype (classical, basal-like, hybrid), tissue of origin (primary, metastatic), patient treatment status (naive, treated), and mutational status (Table 4), to comprehensively profile sensitivity to GN79HC1.

[0467] Table 4. Characterisistics of HPO lines used in cytotoxicity profiling.NaiveTreated 27Mutant Deletion Wild-TypeKRAS _ 61 _ 0 _ 2 _TP53 49 0 14CDKN2A 16 35 12SMAD4 16 8 39

[0468] Low micromolar IC50 cytotoxicity values, that was observed in PDAC cell lines were recapitulated in a majority of HPO lines tested (FIG. 53). This data demonstrates that GN79HC1 exerts cytotoxicity in clinically relevant models of PDAC.

[0469] Given the implications of NPC1 as the mechanistic target of GN64, it was further sought to demonstrate that GN79HC1 also interacted with NPC1 by using photoaffinity labeling studies. Using photoprobe GNP2 2 and parent blocking with both GN64 and-113-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325GN79HC1, it was shown that GN79HC1 can block labeling of NPC1 (FIG. 54). The bioisosteric substitution of the nitro group to a cyano group proves to retain the properties of lead small molecule, GN64.

[0470] HP-fl-CD formulation of GN79HCI shows enhanced solubility and retains cytotoxicity

[0471] Formulation development studies were conducted to further increase the aqueous solubility of GN79HC1 for use in in vivo studies. Cyclodextrin (CD) excipients were explored, as these molecules can encapsulate an active pharmaceutical ingredient (API) within a hydrophobic cavity while presenting a hydrophilic exterior to enhance solubility and bioavailability. Formulations of GN79HC1 with the widely used CDs Captisol and hydroxypropyl-P-cyclodextrin (HP-P-CD) were evaluated. Both formulations achieved GN79HC1 solubility greater than 10 mg / mL at CD concentrations providing isotonicity with blood. Cytotoxicity assays against PDAC cell lines showed that both CD formulations exhibited similar cytotoxicity profiles to GN79HC1 in DMSO; however, greater variability and slightly reduced potency were observed with the 12% Captisol formulation (FIG. 55). Additionally, higher stability was observed with the HP-P-CD formulation (data not shown). Based on these results, GN79HC1 formulated in 10% HP-P-CD was selected for in vivo studies.

[0472] GN79HCI (10% HP-fi-CD) is well tolerated inMTD studies

[0473] Pharmacokinetic analysis was initiated by administering a single intraperitoneal (i.p.) dose of GN79HC1 (10% HP-P-CD formulation) at 30 mg / kg. In plasma, a Cmax of 3,433.5 ng / mL was observed (FIG. 56A). Accumulation of GN79HC1 within target pancreatic tissue was also detected (FIG. 56B), demonstrating that the compound reaches the intended site following a single dose. More comprehensive biodistribution studies will be required to further characterize tissue distribution and concentrations achieved.

[0474] The maximum tolerated dose (MTD) of GN79HC1 in healthy mice was investigated. High daily dosing regimens (100, 75, or 50 mg / kg, Q.D.) resulted in significant weight loss (FIG. 57A) and mortality. At 25 mg / kg Q.D., mice experienced weight loss by Day 20, which was reversed when the dosing frequency was reduced to every other day (Q.O.D.) -114-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325(FIG. 57B). However, mice in this group exhibited extensive gastrointestinal (GI) hemorrhages. In contrast, dosing at 10 mg / kg Q.D. was well tolerated, with no observable toxicities or weight loss (FIG. 57C). These findings support the use of GN79HC1 (10% HP-P-CD) at 10 mg / kg for future in vivo efficacy studies. GN79HC1 (10% HP-P-CD) reduces tumor volume in xenograft models of PDAC in vivo

[0475] The efficacy of GN79HC1 was evaluated using subcutaneous (SC) xenograft models of PDAC. Five million MIA PaCa-2 cells were injected SC into a single flank of NSG mice. Once tumors reached approximately 100 mm3, treatment with GN79HC1 (10% HP-P-CD) at 10 mg / kg via intraperitoneal (i.p.) injection was initiated for 14 days, and tumor size was monitored throughout the treatment period.

[0476] Following 14 days of treatment GN79HC1 led to a 23% reduction in tumor size compared to vehicle treated mice (FIGs. 58A, 58B). These results indicate GN79HC1 was effective in reducing tumor burden in vivo

[0477] Discussion

[0478] GN64 has previously been shown to be cytotoxic to PDAC cell lines and to inhibit the autophagy-lysosomal pathway (ALP) through multiple orthogonal assays, while also inducing accumulation of lysosomal mass and hyperacidification of lysosomes. Target identification studies utilizing PAL identified NPC1 as the molecular target of GN64, and genetic deletion of NPC1 conferred resistance to GN64-induced cytotoxicity while preventing GN64-mediated hyperacidification. In this chapter, efforts were undertaken to de-risk GN64 by performing bioisosteric replacement of the nitro group to mitigate potential mutagenic effects. This led to the development of GN79, a cyano-containing analog, and its monohydrochloride salt, GN79HC1, both of which retained PDAC cytotoxicity and ALP inhibitory activity. To enable in vivo evaluation, formulation studies were conducted to enhance the aqueous solubility of GN79HC1, with a 10% HP-P-CD formulation achieving solubility greater than 10 mg / mL at concentrations isotonic with blood. Using this formulation, preliminary pharmacokinetic studies were performed, identifying 10 mg / kg as the maximum tolerated dose (MTD). In subcutaneous xenograft models of PDAC, GN79HC1 treatment significantly reduced tumor burden in vivo.-115-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0479] GN79HCI as a bioisostere of GN64

[0480] Nitro functional groups are associated with specific toxicities through partial reduction to hydroxylamines and nitrosamines, which can lead to a wide range of adverse effects, including mutagenicity. Drug development strategies often aim to “de-risk” lead compounds by employing bioisosteric substitutions of nitro groups with alternative moieties that retain biological activity. To reduce the metabolic liabilities of GN64, a cyano group — previously identified as a nitro bioisostere — was incorporated, generating GN79. The monohydrochloride salt form, GN79HC1, also incorporates this cyano substitution. Both GN79 and GN79HC1 retain cytotoxic and ALP inhibitory properties, although GN79HC1 exhibits a modest reduction in potency in both assays compared to GN64. Despite this shift, the removal of the nitro group — particularly one on an aromatic system — significantly reduces potential translational risk. GN79HC1 maintains ALP inhibition and PDAC cytotoxicity in vitro and has been shown to reduce tumor burden in vivo, supporting its potential clinical utility.

[0481] Pharmacodynamic biomarker exploration

[0482] Preliminary pharmacokinetic investigations revealed pancreatic tissue accumulation of GN79 and that 10 mg / kg of GN79HC1 (10% HP-P-CD) was well tolerated in mice without observable toxicities. Furthermore, GN79HC1 was able to reduce tumor burden in vivo.

[0483] As a complement to efficacy studies, biomarker exploration can be used to assess on-tumor drug effects. Transcriptome analysis of GN64-treated cells identified significant upregulation of lysosome-associated genes (NPC2) and genes involved in ganglioside catabolism (GM2A, HEXA, HEXB, NEU1). Transcriptomic profiling of tumors following acute treatment may therefore reveal drug-induced effects. In MIA PaCa-2 cells treated in vitro with GN79HC1, four of these genes were validated, with increased expression observed (FIGs. 59A-59E). These genes were initially identified as upregulated in bulk RNA sequencing of GN64-treated PaTu 8988T cells. Validation using MIA PaCa-2 cells and GN79HC1 demonstrated that this compound also induces upregulation of these-116-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 biomarkers in a different PDAC cell line, supporting the earlier GN64 RNA sequencing findings.

[0484] Pharmacokinetic studies with GN64

[0485] A pharmacokinetic (PK) study was conducted to evaluate the distribution of GN64 in plasma and selected organs of B6 female mice. Mice received a single intraperitoneal (i.p.) dose of GN 64 monohydrochloride salt at 10 mg / kg, formulated in 10% hydroxypropyl-P-cyclodextrin (HPBCD).

[0486] In plasma, GN64 reached a peak concentration of approximately 150 ng / mL, indicating efficient absorption and systemic availability (FIG. 60). Tissue distribution revealed favorable penetration into key organs: both the pancreas and kidney achieved maximum concentrations of approximately 10 ng / mg, while the liver exhibited a peak of 0.1 ng / mg, suggesting a slower uptake or clearance profile.]0487] Methods

[0488] DQ-BSA assay for lysosomal degradation activity

[0489] DQ-BSA assay for lysosomal degradation activity was performed as previously described in Example 1.

[0490] Cytotoxicity profding (PDAC cell lines)

[0491] Cytotoxicity profiling of PDAC cell lines was performed as previously described in Example 1.

[0492] Cytotoxicity profding (HPO lines)

[0493] Cytotoxicity profiling of PDAC HPO lines was performed as previously described in Example 1.

[0494] Live cell photolabeling and affinity enrichment

[0495] Live cell photolabeling and affinity enrichment was performed as previously described in Example 3.-117-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0496] Formulation development

[0497] Formulation development was performed in collaboration with the Organic Synthesis Core Facility at MSKCC.

[0498] Pharmacokinetic profiling andMTD studies

[0499] In vivo studies were performed in collaboration with the Antitumor Assessment Core Facility at MSKCC. GN79HC1 was prepared in 10% (2-Hydroxypropyl)-beta-cyclodextrin (HP-P-CD). For acute dosing studies, B6 female mice (6-8 weeks, n=12) were dosed with 30 mg / kg of GN79 or vehicle control via intraperitoneal (IP) injection. Following single administration, 3 mice from each group were bled at the following timepoints: 30 min, 4hr, 8hr, 24hr. GN79 concentration was measured via LC / MS. At time of mouse sac, pancreases were removed, and flash frozen for downstream LC / MS analysis.

[0500] For MTD studies, NSG female mice (6-8 weeks) were dosed with 100, 75, 50, 25, or 10 mg / kg of GN79HC1 (10% HP-P-CD) or vehicle control via IP injection Q.D. x 5d for 2 weeks. Mice weight was monitored during the study and mice were observed for any evidence of toxicities.

[0501] In vivo PDAC xenograft studies

[0502] In vivo studies were performed in collaboration with the Antitumor Assessment Core Facility at MSKCC. NSG female mice (6-8 weeks) were injected with MIA PaCa-2 cells (five million) subcutaneously in a single flank. Tumors were allowed to establish, reaching 100 mm3. Once establish, treatment with GN79HC1 (10% HP-P-CD) at 10 mg / kg or vehicle control via i.p. injection was initiated. Treatment was administered Q.D. x 5 (every day for five days, followed by two days off) for two weeks. Tumor volumes and body weights were recorded twice a week.

[0503] Quantitative reverse transcription polymerase chain reaction (qRT-PCR)

[0504] MIA PaCa-2 cells were seeded in 6-well plates (MIA PaCa-2: 300,000) and cultured for 24h. GN79HC1 [5, 2.5 1.25 pM] and vehicle (DMSO) was added to cells for 24h. Cells were washed IX with PBS, trypsinized, and pelleted. Cells were frozen and stored at -80°C.-118-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325RNA was extracted using RNeasy Mini Kit (Qiagen Cat# 74104). 550ng RNA was used to synthesize cDNA using SuperScript III First-Strand Synthesis SuperMix (Invitrogen Cat# 11752-050, -250) as per manufacturer’s instructions. 100 ng cDNA was used for each qPCR reaction, and each sample was run in triplicates per probe. TaqMan Fast Advanced Master Mix (2x) (Thermo Fisher cat# 4444557) was used for the qPCR as per manufacturer’s instructions. TaqMan probes against genes of interest were used.

[0505] Pharmacokinetic Study

[0506] A pharmacokinetic (PK) study was conducted to evaluate the distribution of GN64 in plasma and selected organs of B6 female mice (6-8 weeks old). Mice received a single intraperitoneal (i.p.) dose of GN64 monohydrochloride salt at 10 mg / kg, formulated in 10% hydroxypropyl-P-cyclodextrin (HPBCD). Three mice sacrificed at each of the following time points post-administration: 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr, and 24 hr. Whole blood was collected into EDTA tubes, and plasma was separated and stored at -80°C for LC-MS analysis. Pancreas, liver, and kidney tissues were harvested and flash-frozen for subsequent LC-MS quantification.

[0507] Overall, these results showcase the development of a novel, NPC1 -targeting small molecule, GN79HC1 and examine the role of NPC1 in the ALP and as a potential therapeutic target in PDAC. The data presented herein support the clinical development of GN79HC1 as a potential therapy for the treatment of PDAC.

[0508] General Synthetic ProceduresCyclohexyl

[0509] In a flame-dried flask, the aniline (1 equiv) was suspended in toluene with various ketones (3 equiv). The vessel was assembled into a Dean-Stark for water collection and placed under a helium atmosphere. Then was cooled on ice, and TiCh (1 equiv) was added-119-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 dropwise. The reaction was refluxed overnight. The next day, the reaction vessel was cooled, and the solid was filtered off and washed with 2.5M NaOH, followed by water.eaR: Me, O2Et, Cyclohexyl

[0510] The amino quinoline was dissolved in pyridine and then cooled on ice. BOC2O (2 equiv) was added dropwise, and the reaction vessel was heated to reflux for 3 hours. The total conversion was confirmed via LC / MS. The pyridine was evaporated, and the resulting residue was purified by a silica gel column to give the desired product.

[0511] The Boc-protected amino quinoline was dissolved in MeOH, and catalytic amounts of Pd / C were added, followed by tri ethyl silane (5 equiv). The reaction vessel was fitted with a balloon filled with hydrogen, and the reaction was refluxed overnight. The next day, the Pd / C was filtered over celite and washed with MeOH. The organic layer was concentrated, followed by purification on a silica gel column.

[0512] The box-protected aminoquinoline was dissolved in DCM, and DIPEA (2 equiv) was added to the solution. The reaction vessel was cooled on ice, and the chlorobenzoic chloride (1 equiv) was added slowly. The reaction was then taken off ice and allowed to come to room temperature. The reaction was monitored via LC / MS. After complete-120-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 conversion, the reaction was worked up with 0. IM HCl / Brine solution and washed twice with 0. IM HC1. The organic layer was dried and concentrated to yield the desired product

[0513] The product was dissolved in DMF, and various piperidine / piperazine (2 equiv) derivatives were added. The reaction was left at room temperature for 30 minutes and then cooled on ice, followed by DIPEA (2 equiv) dropwise. The reaction was concentrated and then purified on a silica or alumina column.

[0514] Deprotection was achieved by dissolving the Boc-protected aniline in a mixture of TFA (20%) in DCM, which allowed it to react at room temperature. After complete deprotection, the product was washed twice with a concentrated bicarbonate solution. The organic layer was concentrated and the product was purified via preparatory TLC.4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0515] Various five-membered aromatic bromides (1 equiv) were dissolved in acetonitrile followed by the addition of piperazine or piperidine (1 equiv). After 10 minutes, potassium carbonate (2 equiv) was added. The reaction was refluxed overnight. The potassium carbonate was filtered off and washed with more acetonitrile. The organic was evaporated off, yielding the desired product.

[0516] Hydrolysis was achieved by dissolving the ester in methanol and adding an equal volume solution of NaOH (2.5M) in water. The reaction was refluxed overnight, and the formation of the acid was confirmed via LC / MS. The product was neutralized and purified via a neutralized silica gel column to give the desired product.

[0517] The acid (1.5 equiv) was added with amino quinoline (1 equiv) into DMF. Next, EDC (2 equiv) and catalytic amounts of DMAP were added. Finally, DIPEA was added to the reaction vessel, allowing the reaction to last overnight. The next day, the product was confirmed via LC / MS and dissolved in Ethyl acetate, followed by washing with 0.1M HCl / Brine solution. The crude product was purified via a silica gel column and deprotection with 20% TFA in DCM. The final desired product was obtained after separation on a preparatory TLC plate.-122-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0518] The acid chloride (1.5 equiv) and amino quinoline (1 equiv) were dissolved in DMF. The reaction was cooled on ice and then DIPEA was added to the reaction slowly.Completion was determined using LC / MS and the reaction was dissolved in ethyl acetate and washed with 0.1M HC1 twice. The residue was then purified using silica gel yielding the desired product.

[0519] Substituted piperazine or piperidine (1 equiv) were dissolved in acetonitrile with the aminoquinoline peg-Cl (1 equiv) derivative. Next (B NI (1 equiv) was slowly added and the reaction was slowly heated to 60C. The product was monitored via LC / MS. Upon completion the reaction was dissolved in ethyl acetate and washed with 0.1M HC1 and brine followed by silica gel purification to yield the desired product.

[0520] Compound CharacterizationGN-1

[0521] JHNMR (500 MHz, Chloroform-d): 5 (ppm) 11.72 (s, 1H), 8.60 (s, 1H), 7.95 (dd,J=7.5, 1.7 Hz, 1H), 7.84 (d, J=9.0 Hz, 1H), 7.29 - 7.43 (m, 3H), 7.13 (dd, J=7.3, 1.2 Hz,-123-4921 -4618-5569.4Atty. Dkt. No.: 115872-33251H), 6.40 (s, 1H), 5.06 (br. s., 2H), 3.58 (s, 2H), 3.44 (br. s., 4H), 2.40 - 2.52 (m, 7H), 1.39 (s, 9H).13C NMR (126MHz, Chloroform-d): 5 (ppm) 165.9, 157.0, 153.5, 149.3, 144.2,136.2, 133.5, 131.5, 131.4, 130.3, 129.6, 127.8, 122.3, 116.6, 110.2, 103.1, 79.2, 63.2, 61.3,51.2, 27.4, 24.3, 23.7. HRMS Expected: 476.2583, HRMS: 476.2673GN-2

[0522] 'HNMR (500 MHz, Methanol-d4): 5 (ppm) 8.51 (s, 1H), 8.02 (d, 7=9.1 Hz, 1H), 7.77 (d, 7=9.0 Hz, 1H), 7.60 (d, 7=7.1 Hz, 1H), 7.27 - 7.44 (m, 3H), 6.58 (s, 1H), 3.73 (s, 2H), 2.68 - 2.84 (m, 4H), 2.52 - 2.64 (m, 7H), 2.46 (s, 3H).13C NMR (126MHz, Methanoldi 8 (ppm) 170.9, 159.5, 153.5, 138.2, 137.9, 137.2, 136.3, 132.3, 131.8, 130.0, 129.4,129.2, 121.6, 117.2, 113.9, 103.4, 61.2, 55.3, 51.6, 44.9, 20.3. HRMS Expected: 390.2216, HRMS: 390.2293GN-3

[0523] 'HNMR (CHLOROFORM-d / DMSO-d ,500MHz): 5 (ppm) 12.03 (s, 1H), 8.64 (d, 7=2.0 Hz, 1H), 8.05 (d, 7=9.1 Hz, 1H), 7.88 (d, 7=9.0 Hz, 1H), 7.51 (d, 7=11.0 Hz, 1H),7.41 - 7.49 (m, 2H), 7.23 (d, 7=8.7 Hz, 1H), 7.08 (d, 7=8.4 Hz, 2H), 6.83 (d, 7=8.5 Hz, 2H),6.42 (s, 1H), 5.35 (br. s., 2H), 3.66 (s, 2H), 3.19 (br. s., 4H), 2.70 (br. s., 4H), 2.52 (s, 3H), 2.27 (s, 3H).13C NMR (CHLOROFORM-d ,126MHz): 5 (ppm) 176.6, 167.9, 156.5, 152.2,-124-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325148.5, 136.3, 136.1, 136.0, 133.3, 132.7, 131.4, 131.0, 129.8, 129.7, 128.7, 120.8, 116.5,115.8, 115.3, 102.7, 62.0, 52.2, 49.6, 20.4. HRMS Expected: 466.2529, HRMS: 466.2615GN-4

[0524] 'HNMR (500 MHz, Methanol-d4) 5 8.61 (d, J = 2.3 Hz, 1H), 7.99 (d, J = 9.1, 1H), 7.83 - 7.74 (m, 1H), 7.71 (d, J = 9.0 Hz, 1H), 7.53 (d, J = 7.2 Hz, 3H), 6.57 (s, 1H), 4.21 (s, 2H), 3.39 - 3.28 (m, 4H), 3.26 - 3.18 (m, 4H), 2.53 (s, 3H).13C NMR (126 MHz, Methanol-d4) 6 170.39, 159.62, 153.83, 138.02, 137.54, 137.21, 133.69, 132.69, 131.04, 130.08, 129.19, 121.30, 117.00, 114.13, 103.16, 61.07, 43.10, 19.88. Expected HRMS: 376.2059, HRMS: 376.2131.GN-5

[0525] XH NMR (500 MHz, Chloroform-d) 5 11.83 (s, 1H), 8.60 (s, 1H), 7.96 (d, J = 9.2 Hz, 1H), 7.84 (d, J = 8.9 Hz, 1H), 7.49 - 7.31 (m, 3H), 7.15 (s, 1H), 6.35 (s, 1H), 5.41 (s, 2H), 4.16 (s, 1H), 3.69 (t, J = 4.5 Hz, 4H), 3.53 (s, 2H), 2.52 - 2.37 (m, 7H).13C NMR (126 MHz, Chloroform-d) 5 171.24, 161.94, 155.48, 148.84, 140.79, 138.07, 137.96, 136.60, 136.41, 134.88, 134.66, 132.63, 131.89, 127.56, 127.44, 121.45, 115.56, 107.65, 70.54, 66.27, 56.65, 27.93, 27.90. Expected HRMS: 377.1899, HRMS: 377.19804921 -4618-5569.4Atty. Dkt. No.: 115872-3325GN-6

[0526] 'HNMR (500 MHz, Chloroform-d) 5 12.09 (s, 1H), 8.61 (d, J = 2.2 Hz, 1H), 7.95 (dd, J = 7.1, 2.2 Hz, 1H), 7.88 (d, J = 8.9 Hz, 1H), 7.45 - 7.33 (m, 3H), 7.15 (d, J = 6.8 Hz, 1H), 6.40 (s, 1H), 5.37 (s, 1H), 3.58 (s, 2H), 3.40 (s, 3H), 2.48 (s, 8H), 2.38 (q, J = 7.2 Hz, 2H), 1.02 (t, J = 7.2 Hz, 3H).13C NMR (126 MHz, Chloroform-d) 5 167.09, 157.35, 151.12, 144.15, 137.04, 134.91, 132.91, 132.59, 131.39, 130.67, 128.70, fz, 124.09, 117.38, 111.34, 103.85, 62.33, 50.33, 29.69, 25.59, 24.11, 11.99. Expected HRMS: 404.2372, HRMS: 404.2467GN-7

[0527] XH NMR (500 MHz, Chloroform-d) 5 11.91 (s, 1H), 8.53 (d, J = 2.2 Hz, 1H), 7.94 (d, J = 9.3 Hz, 1H), 7.79 (d, J = 8.9 Hz, 1H), 7.41 (d, J = 8.9 Hz, 1H), 7.37 - 7.31 (m, 2H), 7.12 (d, J = 9.0 Hz, 1H), 6.80 - 6.70 (m, 4H), 6.32 (s, 1H), 5.21 (s, 1H), 3.64 (s, 3H), 3.55 (s, 2H), 3.02 (t, J = 4.7 Hz, 4H), 2.59 (t, J = 4.8 Hz, 4H).13C NMR (126 MHz, Chloroform- d) 5 167.01, 157.78, 154.10, 150.61, 145.02, 137.23, 134.71, 132.80, 132.58, 131.40, 130.63, 128.75, 128.61, 123.89, 118.24, 117.56, 114.57, 111.41, 104.02, 62.21, 55.56, 52.50, 50.40, 24.55. Expected HRMS: 482.2478, HRMS: 482.2546GN-10-126-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0528] 'H NMR (500 MHz, Chloroform-d) 5 12.30 (s, 1H), 8.67 (s, 1H), 8.06 (d, J = 7.3 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.55 (d, J = 11.4 Hz, 1H), 7.52 - 7.40 (m, 2H), 7.24 (d, J = 7.3 Hz, 1H), 6.50 (s, 1H), 5.01 (s, 2H), 3.75 - 3.58 (m, 2H), 2.67 - 2.35 (m, 11H), 1.11 (s, 9H).13C NMR (126 MHz, Chloroform-d) 5 167.03, 158.00, 150.14, 137.26, 134.91, 132.97, 132.56, 131.36, 130.56, 128.66, 123.79, 117.59, 110.88, 104.07, 62.34, 53.79, 53.13, 45.45, 25.77, 25.37, 24.78. Expected HRMS: 482.2478, HRMS: 482.2546GN-11|0529]XH NMR (500 MHz, Chloroform-d) 5 11.38 (s, 1H), 8.48 (s, 1H), 8.03 (d, J = 9.3 Hz, 2H), 7.92 - 7.86 (m, 1H), 7.82 (d, J = 8.8 Hz, 1H), 7.54 (d, J = 9.2 Hz, 1H), 7.46 - 7.38 (m, 2H), 7.29 - 7.20 (m, 1H), 6.77 (d, J = 9.3 Hz, 2H), 6.46 (s, 1H), 5.01 (s, 2H), 3.71 (s, 2H), 3.46 - 3.37 (m, 4H), 2.65 (t, J = 5.0 Hz, 4H), 2.49 (s, 3H).13C NMR (126 MHz, Chloroform-d) 5 166.99, 161.46, 157.62, 154.45, 151.68, 149.73, 138.48, 137.31, 134.54, 132.91, 132.04, 130.65, 130.44, 128.58, 125.79, 123.61, 117.38, 112.85, 111.09, 103.62, 61.67, 51.82, 46.69, 24.36. Expected HRMS: 497.2223, HRMS: 497.2291GN-12

[0530] 'HNMR (500 MHz, Chloroform-d) 5 8.56 (s, 1H), 7.96 (d, J = 7.0 Hz, 1H), 7.79 (d, J = 8.9 Hz, 1H), 7.46 - 7.30 (m, 3H), 7.30 - 7.09 (m, 3H), 6.91 - 6.76 (m, 3H), 6.36 (s, 1H), 5.02 (s, 2H), 3.62 (s, 2H), 3.18 (s, 4H), 2.64 (s, 4H), 2.45 (s, 3H).13C NMR (126 MHz, Chloroform-d) 5 167.00, 157.99, 150.72, 150.36, 137.29, 134.63, 132.67, 132.61, 131.43, -127-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325130.66, 129.31, 128.87, 128.84, 123.61, 120.35, 117.58, 116.21, 111.15, 104.09, 62.31,52.45, 49.03, 24.69. Expected HRMS: 452.2372, HRMS: 452.2451GN-13

[0531] JH NMR (500 MHz, Chloroform-d) 5 11.45 (s, 1H), 8.58 (d, J = 2.4 Hz, 1H), 7.80 (d, J = 8.9 Hz, 1H), 7.34 (d, J = 9.0, 2.3 Hz, 1H), 7.28 (d, J = 1.9 Hz, 1H), 6.85 (d, J = 1.9 Hz, 1H), 6.42 (s, 1H), 4.71 (s, 2H), 3.73 (s, 2H), 2.49 (s, 10H), 2.38 - 1.99 (m, 4H), 1.56 - 1.29 (m, 2H), 0.83 (t, J = 7.4 Hz, 3H).13C NMR (126 MHz, Chloroform-d) 5 161.83, 158.22, 151.33, 149.62, 145.72, 141.50, 134.74, 129.41, 122.97, 121.45, 117.72, 112.43, 110.10, 104.19, 60.42, 55.22, 52.79, 52.71, 25.12, 19.99, 11.88. Expected HRMS: 408.2321, HRMS: 408.2416GN-14

[0532] JH NMR (600 MHz, Chloroform-; / ) 5 11.93 (s, 1H), 8.54 (d, J= 2.2 Hz, 1H), 7.99 (d, J= 7.4, 1.8 Hz, 1H), 7.82 (d, J= 8.9 Hz, 1H), 7.42 (m, 3H), 7.25 - 7.18 (m, 3H), 6.87 (d, J= 8.1 Hz, 2H), 6.83 (t, J= 13 Hz, 1H), 4.92 (br, 2H), 3.66 (s, 2H), 3.24 (br, 4H), 2.69 (m, 4H), 2.57 (s, 3H), 2.13 (s, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 167.03, 156.42,-128-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325150.73, 147.49, 137.33, 134.99, 132.64, 131.47, 130.68, 129.32, 128.87, 128.30, 122.99,120.36, 117.48, 116.24, 110.72, 109.34, 62.38, 52.49, 49.06, 23.53, 12.62.GN-15

[0533] 'HNMR (600 MHz, CDCh / MeOD) 5 8.66 (s, 1H), 8.04 (d, J= 7.3 Hz, 1H), 7.82 (d, J= 8.9 Hz, 1H), 7.54 - 7.46 (m, 1H), 7.38 (d, J= 8.9 Hz, 1H), 7.11 (d, J= 8.4 Hz, 2H), 6.88 (d, J= 8.5 Hz, 2H), 3.75 (s, 3H), 3.24 (t, J= 5.0 Hz, 5H), 2.76 (t, J= 5.0 Hz, 5H), 2.61 (s, 4H), 2.29 (s, 4H), 2.23 (s, 4H).13C NMR (151 MHz, CDCh / MeOD -d) 5 167.11, 157.20, 148.60, 147.55, 143.27, 137.08, 134.20, 132.81, 132.71, 131.37, 130.82, 130.17, 129.85, 128.87, 128.55, 122.39, 117.69, 116.70, 110.95, 109.30, 62.34, 52.52, 49.76, 23.50, 20.45, 12.59.GN-17

[0534] 'HNMR (600 MHz, CDCh / MeOD-tZ) 5 8.62 (s, 1H), 8.05 (d, J= 9.0 Hz, 1H), 7.97- 7.88 (m, 1H), 7.72 (d, J= 9.0 Hz, 1H), 7.54 - 7.49 (m, 2H), 7.47 (d, J= 8.6 Hz, 2H), 7.37- 7.33 (m, 2H), 6.93 (d, J= 8.6 Hz, 2H), 3.79 (s, 2H), 3.30 (t, J= 5.1 Hz, 4H), 2.73 (t, J= 5.0 Hz, 4H), 2.69 (s, 3H), 2.19 (s, 3H).13C NMR (151 MHz, CDCh / MeOD-tZ) 5 168.17, 153.69, 152.97, 151.50, 136.50, 136.16, 133.69, 132.34, 131.16, 130.72, 128.81, 126.52,-129-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325125.80, 123.86, 122.20, 121.21, 121.00, 116.07, 114.99, 111.86, 108.54, 61.88, 52.33, 48.04, 19.61, 11.86. Expected HRMS: 534.2481, HRMS: 534.2475GN-18

[0535] 'HNMR (600 MHz, CDCh / MeOD-tZ) 5 8.65 (s, 1H), 8.08 (s, 1H), 7.97 - 7.94 (m, 1H), 7.92 (d, J= 9.0 Hz, 1H), 7.74 (d, J= 8.7 Hz, 1H), 7.57 - 7.49 (m, 3H), 7.36 - 7.33 (m, 1H), 7.28 (d, J= 8.7 Hz, 1H), 3.82 (s, 2H), 3.22 (t, J= 4.8 Hz, 4H), 2.80 - 2.70 (m, 4H), 2.66 (s, 3H), 2.25 (s, 3H).13C NMR (151 MHz, CDCh / MeOD-tZ) 5 167.92, 147.93, 141.09, 136.85, 135.00, 133.30, 132.53, 131.11, 130.84, 130.62, 128.95, 124.25, 124.22, 123.83, 123.30, 122.45, 121.32, 117.20, 111.47, 109.14, 61.95, 52.26, 50.98, 21.84, 12.30.GN-19

[0536] JH NMR (600 MHz, CDCh / MeOD-tZ) 5 8.58 (d, J= 2.3 Hz, 1H), 7.81 (d, J= 8.9 Hz, 1H), 7.40 (d, J= 1.9 Hz, 1H), 7.32 (dd, J= 8.9, 2.3 Hz, 1H), 7.10 (d, J = 8.1 Hz, 2H), 6.94 (d, J= 2.0 Hz, 1H), 6.87 (d, J= 8.1 Hz, 2H), 3.89 (s, 2H), 2.81 (d, J= 8.3 Hz, 9H), -130-4921 -4618-5569.4Atty. Dkt. No.: 115872-33252.60 (s, 3H), 2.28 (s, 3H), 2.22 (s, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 161.99, 156.85, 151.59, 148.50, 147.68, 142.81, 141.85, 134.24, 130.29, 129.85, 128.32, 122.13,121.36, 117.65, 117.63, 116.80, 112.30, 112.28, 110.36, 109.29, 109.27, 55.09, 52.75,52.73, 23.32, 20.45, 12.55. Expected HRMS: 470.2551, HRMS: 470.2556GN-20

[0537] 'HNMR (600 MHz, Chloroform-; / ) 5 8.08 (s, 1H), 7.56 (d, J= 9.0 Hz, 1H), 7.37 (d, J= 7.6 Hz, 1H), 7.29 - 7.16 (m, 4H), 6.89 (d, J= 8.4 Hz, 2H), 6.60 (d, J= 8.5 Hz, 2H), 6.28 (s, 1H), 3.76 (s, 2H), 3.55 (s, 2H), 2.95 (t, J= 5.0 Hz, 4H), 2.60 (t, J= 5.0 Hz, 4H), 2.39 (s, 3H), 2.14 (s, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 170.73, 157.75, 151.23, 148.78, 144.59, 135.52, 135.05, 133.59, 131.62, 130.61, 129.92, 129.59, 128.50, 127.65, 127.21, 124.27, 117.22, 116.78, 111.72, 103.62, 61.73, 53.06, 41.41, 38.99, 23.91, 20.36. Expected HRMS: 480.2763, HRMS: 480.2765GN-21

[0538] 'HNMR (600 MHz, Chloroform-; / ) 5 8.00 (s, 1H), 7.66 (d, J= 8.9 Hz, 1H), 7.55 (d, J= 7.6 Hz, 1H), 7.42 - 7.31 (m, 2H), 7.27 (d, J= 4.4 Hz, 2H), 6.98 (d, J= 8.2 Hz, 2H), 6.68 (d, J= 8.5 Hz, 2H), 3.83 (s, 2H), 3.65 (s, 2H), 3.08 (t, J= 5.0 Hz, 4H), 2.73 (t, J= 5.0 Hz, 4H), 2.56 (s, 3H), 2.25 (s, 3H), 2.09 (s, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 170.08, 148.82, 135.36, 135.24, 134.07, 131.78, 130.55, 129.71, 129.66, 129.55, 128.69, 127.17,-131-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325123.56, 117.17, 116.57, 116.46, 111.21, 109.11, 62.05, 53.21, 49.22, 41.78, 20.44, 12.40.Expected HRMS: 494.2920, HRMS: 494.2922GN-22

[0539] 'HNMR (600 MHz, CDCh / MeOD-tZ) 5 8.58 (s, 1H), 7.96 (d, J= 5.6 Hz, 1H), 7.74 (d, J= 9.0 Hz, 1H), 7.42 (p, J= 7.4 Hz, 2H), 7.31 (d, J= 9.0 Hz, 1H), 7.22 (s, 1H), 7.06 (d, J= 7.4 Hz, 2H), 6.83 (d, J= 8.0 Hz, 2H), 5.21 (s, OH), 3.68 (s, 2H), 3.17 (s, 4H), 2.68 (s, 4H), 2.52 (d, J= 15.2 Hz, 5H), 2.15 (s, 3H), 1.14 (t, J= 8.6 Hz, 3H).13C NMR (151 MHz, CDCh / MeOD-tZ) 5 167.17, 156.97, 148.72, 147.86, 142.93, 136.99, 136.65, 134.21, 132.83, 132.69, 131.31, 130.84, 128.85, 128.63, 128.20, 122.51, 117.61, 116.67, 111.01, 109.25, 62.30, 52.54, 49.68, 27.94, 23.23, 15.75, 12.51.GN-23'HNMR (600 MHz, Chloroform-; / ) 5 12.17 (s, 1H), 8.07 (d, J= 7.7 Hz, 1H), 7.88 (d, J= 8.9 Hz, 1H), 7.51 - 7.40 (m, 3H), 7.24 (d, J= 7.5 Hz, 1H), 6.89 (d, J= 9.1 Hz, 2H), 6.84 (d, J= 9.1 Hz, 2H), 4.71 (s, 2H), 4.34 (s, 1H), 3.78 (s, 3H), 3.70 (s, 2H), 2.91 - 2.78 (m, 2H), 2.65 (s, 3H), 2.49 (s, 2H), 2.25 (s, 3H), 2.03 (s, 2H), 1.93 (s, 2H).13C NMR (151 MHz, Chloroform-; / ) 5 166.94, 154.28, 151.02, 137.40, 134.95, 133.08, 133.07, 132.56, 131.45,-132-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325131.43, 130.49, 129.47, 128.70, 122.33, 117.83, 117.77, 114.72, 110.27, 110.16, 109.45,62.41, 55.69, 30.53, 24.32, 14.14, 12.76. Expected HRMS: 552.2975, HRMS: 552.2971GN-24

[0540] 'HNMR (600 MHz, CDCh / MeOD-tZ) 5 8.71 (s, 1H), 7.96 (d, J= 8.0 Hz, 1H), 7.71 (d, J= 8.8 Hz, 1H), 7.45 - 7.41 (m, 2H), 7.36 (d, J= 8.9 Hz, 1H), 7.22 (t, J= 4.4 Hz, 1H), 7.06 (d, J= 8.5 Hz, 2H), 6.82 (d, J= 8.5 Hz, 2H), 4.35 (q, J= 7.1 Hz, 2H), 3.68 (s, 2H), 3.26 - 3.08 (m, 4H), 2.70 (d, J= 13.0 Hz, 7H), 2.51 (q, J= 7.6 Hz, 2H), 1.37 (t, J= 7.1 Hz, 3H), 1.14 (t, J= 7.6 Hz, 3H).13C NMR (151 MHz, CDCh / MeOD-tZ) 5 169.45, 167.17, 158.91, 154.24, 148.69, 144.27, 136.84, 136.69, 134.98, 132.74, 131.40, 130.95, 128.91, 128.77, 128.66, 124.63, 117.68, 116.65, 111.88, 102.48, 62.33, 61.03, 52.55, 27.95, 27.20, 15.76, 14.29.GN-25

[0541] XH NMR (600 MHz, Chloroforms / ) 5 12.17 (s, 1H), 8.07 (d, J = 7.7 Hz, 1H), 7.88 (d, J= 8.9 Hz, 1H), 7.51 - 7.40 (m, 3H), 7.24 (d, J= 7.5 Hz, 1H), 6.89 (d, J= 9.1 Hz, 2H), 6.84 (d, J= 9.1 Hz, 2H), 4.71 (s, 2H), 4.34 (s, 1H), 3.78 (s, 3H), 3.70 (s, 2H), 2.91 - 2.78 (m, 2H), 2.65 (s, 3H), 2.49 (s, 2H), 2.25 (s, 3H), 2.03 (s, 2H), 1.93 (s, 2H).13C NMR (151 MHz, Chloroforms / ) 5 166.94, 154.28, 151.02, 137.40, 134.95, 133.08, 133.07, 132.56,-133-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325131.45, 131.43, 130.49, 129.47, 128.70, 122.33, 117.83, 117.77, 114.72, 110.27, 110.16, 109.45, 62.41, 55.69, 30.53, 24.32, 14.14, 12.76. Expected HRMS: 511.2709, HRMS: 511.2706GN-26

[0542] 'HNMR (600 MHz, Chloroform-; / ) 5 8.56 (s, 1H), 7.98 (d, J= 7.5 Hz, 1H), 7.77 (d, J= 8.9 Hz, 1H), z7.45 - 7.34 (m, 2H), 7.19 (s, 1H), 7.04 (d, J= 8.5 Hz, 2H), 6.80 (d, J= 8.6 Hz, 2H), 4.60 (s, 2H), 3.65 (s, 3H), 3.19 (s, 5H), 2.68 (t, J= 4.6 Hz, 5H), 2.56 (s, 3H), 2.47 (t, J= 7.8 Hz, 3H), 1.50 (p, J= 7.6 Hz, 2H), 1.27 (h, J= 7.4 Hz, 2H), 0.85 (t, J= 13 Hz, H).13C NMR (151 MHZ, Chloroform-; / ) 5 166.96, 157.33, 148.74, 146.36, 143.73, 137.52, 135.03, 134.76, 132.62, 131.44, 130.56, 129.54, 129.19, 128.84, 122.32, 117.81, 116.36, 110.35, 109.49, 62.42, 52.58, 49.45, 34.72, 33.84, 24.37, 22.38, 14.02, 12.78. Expected HRMS: 522.3233, HRMS:522.3242-134-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325GN-27

[0543] 'HNMR (600 MHz, Chloroform-; / ) 5 8.69 (s, 1H), 7.94 (d, J= 7.2 Hz, 1H), 7.71 (d, J= 8.8 Hz, 1H), 7.43 - 7.34 (m, 3H), 7.17 (d, J= 7.2 Hz, 1H), 7.02 (s, 2H), 6.78 (d, J= 8.5 Hz, 2H), 4.33 (q, J= 7.1 Hz, 2H), 3.61 (s, 2H), 3.14 (s, 4H), 2.70 (s, 3H), 2.64 (d, J= 4.9 Hz, 4H), 2.49 - 2.36 (m, 2H), 1.47 (p, J= 7.6 Hz, 2H), 1.34 (t, J= 7.1 Hz, 3H), 1.25 (h, J= 7.4 Hz, 2H), 0.83 (t, J= 7.4 Hz, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 169.46, 169.44,167.15, 167.04, 158.80, 154.02, 148.69, 148.69, 144.38, 137.05, 136.99, 135.28, 135.18,135.17, 132.75, 132.72, 132.71, 132.70, 131.49, 131.47, 130.85, 129.23, 129.09, 128.89,124.79, 124.67, 117.68, 117.64, 116.46, 111.84, 111.83, 111.72, 102.52, 62.35, 60.99,52.59, 52.56, 49.55, 34.75, 33.85, 27.55, 22.39, 14.35, 14.04. Expected HRMS: 580.3288, HRMS: 580.3289GN-28

[0544] 'HNMR (600 MHz, Chloroform-; / ) 5 12.02 (s, 1H), 8.78 (s, 1H), 8.05 (d, J= 9.2 Hz, 1H), 7.80 (d, J= 8.8 Hz, 1H), 7.57 - 7.40 (m, 3H), 7.28 (d, J= 8.9 Hz, OH), 7.17 - 7.09 (m, 2H), 6.87 (d, J= 8.6 Hz, 2H), 4.43 (q, J= 7.1 Hz, 2H), 3.75 (s, 2H), 3.25 (s, 4H), 2.88 - 2.65 (m, 7H), 2.29 (s, 3H), 1.45 (t, J= 7.2 Hz, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 169.51, 167.03, 158.57, 153.45, 148.56, 144.60, 137.25, 135.57, 132.63, 132.52, 131.47, 130.70, 130.03, 129.84, 129.63, 128.90, 124.60, 117.42, 116.49, 111.00, 102.65, 62.41, 60.93, 52.57, 52.54, 49.59, 27.99, 20.45, 14.31. Expected HRMS: 538.2818, HRMS: 538.28044921 -4618-5569.4Atty. Dkt. No.: 115872-3325GN-29

[0545] 'H NMR (500 MHz, Chloroform-; / ) 5 8.71 (s, 1H), 7.77 (d, J= 8.9 Hz, 1H), 7.43 (d, J= 8.9 Hz, 1H), 7.38 (d, J= 1.9 Hz, 1H), 7.10 (d, J= 8.6 Hz, 2H), 6.94 (d, J= 1.9 Hz, 1H), 6.86 (d, J= 8.6 Hz, 2H), 4.42 (q, J= 7.1 Hz, 2H), 3.88 (s, 2H), 3.29 (s, 4H), 2.81 (d, J= 9.7 Hz, 7H), 2.28 (s, 3H), 1.44 (t, J= 7.1 Hz, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 169.53, 161.83, 158.43, 153.32, 151.13, 148.48, 144.49, 141.73, 135.50, 130.22, 129.85, 129.68, 124.12, 121.54, 117.43, 116.66, 112.44, 110.44, 102.68, 60.90, 55.16, 52.74, 49.62, 28.00, 20.45, 14.30. Expected HRMS: 528.2611, HRMS: 528.2608GN-30

[0546] XH NMR (600 MHz, Chloroform-; / ) 5 11.98 (s, 1H), 8.63 (s, 1H), 8.07 (d, J= 7.3 Hz, 1H), 7.87 (d, J= 8.9 Hz, 1H), 7.47 (q, J= 7.5, 6.7 Hz, 3H), 7.25 (d, J= 7.1 Hz, 1H), 7.11 (d, J= 8.2 Hz, 2H), 6.86 (d, J= 8.5 Hz, 2H), 4.88 (s, 2H), 3.67 (s, 2H), 3.23 (s, 4H), 3.02 (t, J= 5.9 Hz, 2H), 2.73 (t, J= 4.8 Hz, 4H), 2.66 - 2.52 (m, 2H), 2.30 (s, 3H), 1.92 (dt,4921 -4618-5569.4Atty. Dkt. No.: 115872-3325J= 7.6, 4.1 Hz, 4H).13C NMR (151 MHZ, Chloroforms / ) 5 167.01, 157.32, 148.59, 146.91, 143.41, 137.36, 134.37, 132.68, 132.51, 131.35, 130.49, 129.82, 129.76, 128.88, 128.70, 122.97, 117.26, 116.46, 110.87, 110.55, 62.20, 52.41, 49.51, 33.63, 23.68, 22.74, 22.68, 20.42. Expected HRMS: 506.2919, HRMS: 506.2920GN-31

[0547] JH NMR (600 MHz, Chloroforms / ) 5 11.76 (s, 1H), 8.51 (s, 1H), 7.96 (d, J= 8.9 Hz, 1H), 7.52 (d, J= 8.9 Hz, 1H), 7.35 (d, J= 1.9 Hz, 1H), 7.07 (d, J= 8.2 Hz, 2H), 6.93 (d, J= 1.9 Hz, 1H), 6.81 (d, J= 8.5 Hz, 2H), 5.38 (s, 2H), 4.40 (s, 1H), 3.85 (s, 2H), 2.88 (s, 2H), 2.64 (s, 3H), 2.57 (s, 2H), 2.28 (s, 3H), 2.18 (s, 3H), 2.03 (s, 2H), 1.94 (s, 2H).13C NMR (151 MHz, Chloroforms / ) 5 161.92, 155.22, 154.79, 151.92, 148.48, 141.48, 135.31, 130.56, 130.05, 126.97, 123.22, 121.19, 117.17, 116.18, 112.34, 110.40, 109.16, 55.11, 49.59, 30.31, 22.69, 20.49, 12.47. Expected HRMS: 485.2553, HRMS: 485.2531GN-32-137-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0548] 'H NMR (600 MHz, CDCL MeOD-; / ) 5 8.63 (s, 1H), 8.05 (d, J= 7.2 Hz, 1H), 7.85 (d, J= 9.0 Hz, 1H), 7.54 - 7.41 (m, 3H), 7.29 (d, J= 8.6 Hz, 1H), 7.17 - 7.08 (m, 4H), 3.73 (s, 2H), 3.08 (d, .7= 9.7 Hz, 2H), 2.61 (s, 4H), 2.31 (d, J= 21.1 Hz, 5H), 2.24 (s, 3H), 1.93 (d, J= 12.5 Hz, 2H), 1.86 - 1.74 (m, 2H).13C NMR (151 MHz, CDCL MeOD-; / ) 5 167.41, 157.29, 147.76, 142.41, 136.94, 136.12, 134.31, 132.75, 131.37, 130.86, 128.72, 128.41, 126.59, 117.79, 111.33, 109.35, 41.74, 33.26, 23.43, 21.00. Expected HRMS: 479.2811, HRMS: 479.2812GN-33

[0549] 'HNMR (600 MHz, Chloroform-; / ) 5 8.32 (s, 1H), 7.90 (d, J= 8.9 Hz, 1H), 7.44 (d, J= 8.9 Hz, 1H), 7.28 - 7.10 (m, 9H), 4.13 (s, 2H), 4.00 (s, 1H), 3.72 (t, J= 5.1 Hz, 2H), 2.67 (s, 3H), 2.64 (t, J= 5.1 Hz, 2H), 2.54 (s, 4H), 2.33 (s, 4H), 2.25 (s, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 168.76, 157.57, 146.17, 144.05, 142.01, 141.21, 133.09, 132.51, 129.68, 129.07, 128.64, 128.58, 127.71, 127.16, 122.95, 117.64, 110.43, 109.55, 75.53, 70.76, 69.32, 57.89, 53.79, 51.53, 24.44, 22.65, 12.77. Expected HRMS: 558.2636, HRMS: 558.2632GN-34-138-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0550] 'H NMR (600 MHz, Chloroform-; / ) 5 8.48 (s, 1H), 8.03 (s, 1H), 7.87 (d, J= 8.9 Hz, 1H), 7.59 (d, J= 7.4 Hz, 1H), 7.50 (d, J= 7.7 Hz, 1H), 7.36 (dq, J= 24.4, 8.2, 7.4 Hz, 3H), 7.01 (d, J= 7.8 Hz, 2H), 6.91 (d, J= 8.0 Hz, 1H), 6.84 (t, J = 8.1 Hz, 1H), 6.79 (d, J= 8.4 Hz, 1H), 6.72 (d, J= 8.0 Hz, 1H), 6.69 (s, 1H), 5.37 (s, 2H), 4.65 (s, 2H), 2.65 (s, 3H), 2.30 (s, 3H), 2.24 (s, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 167.49, 157.52, 146.37, 144.90, 144.39, 143.82, 137.32, 135.95, 135.07, 133.85, 130.74, 129.61, 129.14, 127.53, 127.23, 127.07, 127.05, 127.02, 124.03, 122.75, 121.86, 120.98, 120.97, 117.58, 115.80, 114.68, 109.56, 109.46, 49.59, 24.36, 16.30, 12.77. Expected HRMS: 549.1783, HRMS:549.1793GN-35

[0551] 'HNMR (600 MHz, Chloroform-; / ) 5 8.46 (s, 1H), 8.16 (s, 1H), 7.86 (d, J= 8.9 Hz, 1H), 7.63 (d, J= 7.8 Hz, 1H), 7.56 (d, J= 7.5 Hz, 1H), 7.35 (t, J= 6.9 Hz, 1H), 7.27 (t, J= 12.6 Hz, 4H), 7.20 (d, J= 8.0 Hz, 2H), 7.04 (d, J= 7.4 Hz, 4H), 6.88 (t, J= 6.8 Hz, 2H), 5.25 (s, 2H), 4.66 (s, 2H), 3.12 (s, 4H), 2.65 (s, 3H), 2.26 (s, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 167.75, 157.56, 148.09, 146.35, 143.86, 137.09, 135.70, 134.09, 133.87, 130.65, 130.49, 129.92, 129.72, 127.27, 127.26, 126.42, 122.91, 121.97, 120.31, 117.62, 109.63, 109.56, 52.19, 32.20, 24.37, 12.78. Expected HRMS: 499.2498, HRMS: 499.2511GN-36

[0552] 'HNMR (600 MHz, Chloroform-; / ) 5 12.13 (s, 1H), 8.59 (d, J= 2.2 Hz, 1H), 8.03(d, J= 9.2 Hz, 1H), 7.86 (d, J= 8.9 Hz, 1H), 7.77 - 7.64 (m, 1H), 7.45 (t, J= 7.6 Hz, 1H),-139-4921 -4618-5569.4Atty. Dkt. No.: 115872-33257.41 (t, J= 7.4 Hz, 1H), 7.20 (d, J= 7.5 Hz, 1H), 6.91 (d, J= 8.4 Hz, 2H), 6.67 (d, J= 8.5 Hz, 2H), 4.63 (s, 2H), 4.39 (br, 1H), 3.87 (d, J= 12.1 Hz, 1H), 3.50 (d, J= 12.1 Hz, 1H), 3.02 (t, J= 5.9 Hz, 2H), 2.76 (br, 1H), 2.59 (t, J= 6.0 Hz, 4H), 2.21 (s, 3H), 2.02 (br, 1H), 1.94 (m, 5H), 1.79 (br, 1H), 1.62 (br, 1H).13C NMR (151 MHz, Chloroform-; / ) 5 166.97, 157.63, 154.85, 146.29, 144.05, 137.57, 134.69, 132.70, 132.51, 131.39, 130.67, 130.39, 130.04, 129.51, 128.66, 123.06, 117.38, 116.06, 110.56, 110.36, 72.34, 62.67, 56.19, 53.26, 34.09, 28.80, 23.82, 22.94, 22.83, 21.84, 20.44. Expected HRMS: 521.2917, HRMS: 521.2896

[0553] 'HNMR (600 MHz, Chloroform-; / ) 5 12.17 (s, 1H), 8.60 (s, 1H), 8.06 (d, J= 7.6 Hz, 1H), 7.89 (d, J= 8.9 Hz, 1H), 7.55 - 7.35 (m, 3H), 7.21 (d, J= 7.5 Hz, 1H), 7.09 (d, J= 8.5 Hz, 2H), 6.83 (d, J= 8.5 Hz, 2H), 4.72 (s, 2H), 4.40 (br, 1H), 2.80 (br, 2H), 2.63 (s, 3H), 2.48 (br, 2H), 2.28 (s, 3H), 2.21 (s, 3H), 2.02 (br, 2H), 1.93 (br, 2H).13C NMR (151 MHz, Chloroform-; / ) 5 166.96, 157.29, 154.89, 146.40, 143.71, 137.34, 134.81, 133.11, 132.52, 131.42, 130.45, 130.05, 129.47, 128.62, 122.38, 117.83, 116.17, 110.49, 109.46, 107.78, 67.40, 62.32, 30.42, 24.28, 20.50, 12.72. Expected HRMS: 495.2760, HRMS: 495.2756GN-38|0554] 'HNMR (600 MHz, Chloroform-; / ) 5 12.16 (s, 1H), 8.59 (s, 1H), 8.04 (d, J= 6.1 Hz, 1H), 7.88 (d, J= 8.9 Hz, 1H), 7.71 (s, 1H), 7.50 - 7.37 (m, 2H), 7.21 (d, J= 7.4 Hz, 1H), 6.92 (d, J= 8.1 Hz, 2H), 6.67 (d, J= 8.5 Hz, 2H), 4.63 (s, 2H), 4.39 (p, J= 3.0 Hz,-140-4921 -4618-5569.4Atty. Dkt. No.: 115872-33251H), 3.88 (d, J= 12.2 Hz, 1H), 3.52 (d, J= 12.2 Hz, 1H), 2.65 (m, 5H), 2.22 (m, 6H), 2.02 (br, 2H), 1.81 (br, 1H), 1.63 (br, 1H).13C NMR (151 MHz, Chloroform-; / ) 5 166.97, 157.23, 154.85, 146.27, 143.83, 137.54, 134.98, 132.69, 132.52, 131.41, 130.69, 130.42, 130.05, 129.56, 128.68, 122.82, 117.80, 116.08, 110.46, 109.33, 72.35, 62.69, 56.14, 53.30, 28.77, 24.40, 21.81, 20.44, 12.74. Expected HRMS: 495.2760, HRMS: 495.2743GN-39

[0555] 'HNMR (600 MHz, Chloroform-^ 5 8.49 (s, 1H), 7.99 (s, 1H), 7.85 (d, J= 8.9 Hz, 1H), 7.60 (d, J= 8.9 Hz, 1H), 7.50 (d, J= 7.7 Hz, 1H), 7.42 - 7.29 (m, 3H), 7.01 (t, J= 7.2 Hz, 2H), 6.91 (d, J= 8.0 Hz, 1H), 6.84 (t, J= 6.9 Hz, 1H), 6.80 (d, J= 8.3 Hz, 1H), 6.72 (d, J= 8.0 Hz, 1H), 6.69 (s, 1H), 5.38 (s, 2H), 4.67 (s, 2H), 3.02 (t, J= 6.1 Hz, 2H), 2.62 (t, J= 6.2 Hz, 2H), 2.30 (s, 3H), 1.95 (dt, J= 10.6, 6.3 Hz, 4H).13C NMR (151 MHz, Chloroform- d) 5 167.46, 157.93, 146.41, 144.90, 144.39, 144.02, 137.32, 135.97, 135.08, 133.56, 130.74, 129.56, 129.14, 127.54, 127.23, 127.07, 127.02, 124.03, 122.74, 122.06, 121.96, 120.99, 117.14, 115.80, 114.67, 110.79, 109.28, 109.19, 49.60, 34.00, 23.84, 22.86, 22.76, 16.30. Expected HRMS: 575.1932, HRMS: 575.1939

[0556] 'HNMR (600 MHz, Chloroform-^ 5 8.60 (s, 1H), 7.85 (d, J= 8.9 Hz, 1H), 7.43 - 7.29 (m, 2H), 7.09 (d, J= 8.3 Hz, 2H), 6.94 (d, J= 1.9 Hz, 1H), 6.83 (d, J= 8.5 Hz, 2H), 4.43 (br, 1H), 3.83 (s, 2H), 3.01 (t, J= 6.0 Hz, 2H), 2.87 (br, 2H), 2.62 (t, J= 6.1 Hz, 2H), 2.56 (br, 2H), 2.29 (s, 3H), 2.06 (br, 2H), 2.02 - 1.88 (m, 6H).13C NMR (151 MHz,-141-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325Chloroform-; / ) 6 161.81, 157.56, 154.81, 151.50, 146.25, 143.78, 141.43, 134.58, 130.63, 130.07, 129.51, 122.07, 121.50, 117.39, 116.23, 112.45, 110.70, 109.51, 70.94, 55.19, 49.67, 34.03, 30.31, 23.84, 22.91, 22.80, 20.51. Expected HRMS: 511.2709, HRMS: 511.2694GN-41

[0557] 'HNMR (600 MHz, Chloroform-; / ) 5 8.66 (s, 1H), 8.04 (d, J= 7.1 Hz, 1H), 7.80 (d, J= 9.0 Hz, 1H), 7.55 - 7.44 (m, 2H), 7.39 (d, J= 8.9 Hz, 1H), 7.30 (d, J= 7.2 Hz, 2H), 6.94 (d, J= 9.0 Hz, 2H), 6.79 (d, J= 9.0 Hz, 2H), 3.75 (s, 2H), 3.17 (br, 4H), 2.98 (t, J= 6.1 Hz, 2H), 2.88 (s, 6H), 2.76 (br, 4H), 2.60 (t, J= 6.2 Hz, 2H), 1.98 - 1.87 (m, 4H).13C NMR (151 MHz, Chloroform-; / ) 5 167.17, 157.56, 147.74, 146.22, 143.43, 142.65, 137.05,133.91, 132.91, 132.73, 131.34, 130.83, 128.84, 128.34, 122.71, 118.57, 117.26, 114.82,110.92, 110.47, 62.33, 52.65, 52.62, 50.86, 41.62, 33.21, 23.69, 22.73, 22.72. Expected HRMS: 535.3185, HRMS: 535.3181GN-42

[0558] 'HNMR (600 MHz, Chloroform-; / ) 5 8.04 (d, J= 7.6 Hz, 1H), 7.86 (d, J= 8.9 Hz, 1H), 7.49 - 7.36 (m, 2H), 7.23 - 7.17 (m, 2H), 7.14 (d, J= 7.7 Hz, 2H), 3.66 (s, 2H), 3.54 (s, 2H), 2.65 (s, 13H), 2.34 (s, 3H), 2.24 (s, 3H), 2.10 (s, 2H).13C NMR (151 MHz, Chloroform-; / ) 5 166.95, 157.28, 146.39, 143.71, 137.33, 137.28, 136.90, 134.76, 134.38,-142-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325134.37, 132.85, 132.52, 131.35, 130.48, 129.34, 129.18, 128.99, 128.67, 122.42, 117.77,110.43, 109.40, 62.59, 62.35, 52.63, 52.45, 24.32, 21.12, 12.74. Expected HRMS:494.2903, HRMS:494.2920GN-43

[0559] 'HNMR (600 MHz, Chloroform-; / ) 5 8.33 (d, J= 2.3 Hz, 1H), 7.89 (d, J= 8.9 Hz, 1H), 7.44 (dd, J= 8.9, 2.3 Hz, 1H), 7.29 - 7.21 (m, 7H), 7.21 - 7.13 (m, 3H), 4.13 (s, 2H), 3.99 (s, 1H), 3.72 (t, J= 5.1 Hz, 2H), 3.04 (t, J= 6.0 Hz, 2H), 2.68 - 2.61 (br, 4H), 2.61 - 2.21 (br, 7H), 2.04 (d, J= 8.4 Hz, 1H), 2.00 - 1.89 (m, 4H).13C NMR (151 MHz, Chloroform-; / ) 5 168.75, 157.93, 146.27, 144.18, 142.03, 141.22, 132.84, 132.51, 129.56, 129.06, 128.64, 128.59, 127.70, 127.16, 123.24, 117.18, 110.78, 110.30, 75.56, 70.76, 69.32, 57.91, 53.79, 51.53, 34.05, 23.86, 22.90, 22.79. Expected HRMS: 484.2792, HRMS: 484.2769GN-44

[0560] 'HNMR (600 MHz, Chloroform-; / ) 5 12.05 (s, 1H), 8.66 (s, 1H), 8.04 (d, J= 9.2 Hz, 1H), 7.85 (d, J= 8.9 Hz, 1H), 7.52 - 7.37 (m, 4H), 7.24 - 7.18 (m, 4H), 7.14 (d, J= 7.7Hz, 3H), 4.70 (s, 3H), 3.66 (s, 3H), 3.54 (s, 3H), 3.02 (t, J= 6.0 Hz, 3H), 2.71 - 2.49 (m, 4H), 2.34 (s, 5H), 2.11 - 1.79 (m, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 166.95, 157.66, 146.34, 143.91, 137.40, 136.88, 134.59, 134.43, 132.84, 132.50, 131.35, 130.43, -143-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325129.32, 129.16, 128.98, 128.66, 122.67, 117.34, 110.66, 110.13, 62.59, 62.37, 52.64, 52.48, 34.04, 23.84, 22.91, 22.81, 21.12. Expected HRMS: 520.3076, HRMS: 520.3098GN-45

[0561] 'HNMR (600 MHz, Chloroform-; / ) 5 8.34 (s, 1H), 7.81 (d, J= 8.9 Hz, 1H), 7.47 (d, J= 8.9 Hz, 1H), 7.26 - 7.20 (m, 2H), 6.91 - 6.76 (m, 3H), 4.17 (s, 2H), 3.80 (t, J= 5.2 Hz, 2H), 3.28 - 3.12 (m, 4H), 2.99 (t, J= 6.0 Hz, 2H), 2.72 (q, J= 5.0 Hz, 6H), 2.56 (t, J= 6.1 Hz, 2H), 1.92 (q, J= 6.6, 6.0 Hz, 4H).13C NMR (151 MHz, Chloroform-; / ) 5 168.51, 157.80, 151.07, 146.28, 143.96, 132.88, 129.52, 129.13, 122.56, 119.97, 117.13, 116.15, 110.77, 109.62, 70.80, 69.24, 57.81, 53.60, 49.02, 33.94, 23.75, 22.83, 22.74. Expected HRMS: 460.2713, HRMS: 460.2717

[0562] 'HNMR (600 MHz, Chloroform-; / ) 5 8.30 (d, J= 2.3 Hz, 1H), 7.75 (d, J= 9.0 Hz, 1H), 7.41 (dd, J= 9.0, 2.2 Hz, 1H), 6.80 - 6.71 (m, 3H), 4.10 (s, 2H), 3.72 (t, J= 5.1 Hz, 2H), 3.70 (s, 2H), 3.05 - 2.99 (m, 3H), 2.93 (t, J= 6.0 Hz, 2H), 2.69 - 2.62 (m, 5H), 2.51 (t, J= 6.2 Hz, 2H), 1.86 (q, J= 6.6, 6.0 Hz, 4H).13C NMR (151 MHz, Chloroform-; / ) 5 168.55, 157.73, 154.01, 145.43, 133.00, 129.39, 122.70, 118.38, 117.13, 114.45, 110.74, 109.70, 70.86, 69.09, 57.84, 53.70, 50.51, 33.89, 23.78, 22.84, 22.76. Expected HRMS: 490.2818, HRMS: 490.2809-144-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0563] 'H NMR (600 MHz, Chloroform-; / ) 5 8.18 (s, 1H), 7.68 (d, J= 9.0 Hz, 1H), 7.56 (d, J= 9.0 Hz, 1H), 7.39 (d, J= 8.6 Hz, 2H), 6.84 (d, J= 8.6 Hz, 2H), 3.68 (t, J= 5.1 Hz, 2H), 3.25 (t, J = 5.1 Hz, 5H), 2.82 (t, J= 6.1 Hz, 2H), 2.68 (t, J= 5.1 Hz, 2H), 2.64 (t, J= 5.1 Hz, 4H), 2.38 (t, J= 6.2 Hz, 2H), 1.82 - 1.75 (m, 4H).13C NMR (151 MHz, Chloroform-; / ) 5 167.93, 152.14, 150.80, 136.65, 133.41, 125.39 (q, 3.7 Hz), 124.57, 123.98, 122.78,122.12, 119.93, 121.26 - 118.35 (m), 116.87, 110.01, 108.48, 69.79, 66.53, 56.68, 51.90, 46.49, 28.88, 21.70, 20.89, 20.51. Expected HRMS: 528.2586, HRMS: 528.2599

[0564] 'HNMR (600 MHz, Chloroform-; / ) 5 8.31 (s, 1H), 8.05 - 7.91 (s, 1H), 7.77 (d, J= 8.9 Hz, 1H), 7.55 (d, J= 8.8, Hz, 1H), 7.42 - 7.32 (d, J=8.8, Hz 1H), 6.95 (d, J= 8.7 Hz, 1H), 4.10 (s, 2H), 3.72 (t, J= 5.1 Hz, 2H), 3.13 - 3.00 (t, J=5.7 Hz, 4H), 2.94 (t, J= 6.0 Hz, 2H), 2.66 (m, 4.9 Hz, 6H), 2.53 (t, J= 6.2 Hz, 2H), 1.94 - 1.79 (m, 4H).13C NMR (151 MHz, Chloroform-; / ) 5 168.43, 158.00, 147.95, 146.36, 144.04, 141.03, 132.78, 130.26 (q, J = 3.3 Hz), 129.59, 127.10 - 119.50 (m), 117.22, 110.89, 109.88, 70.83, 69.08, 57.84, 53.10, 23.83, 22.84. Expected HRMS: 573.2437, HRMS: 573.24364921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0565] 'H NMR (600 MHz, Chloroform-; / ) 5 8.59 (s, 1H), 8.07 (d, J= 9.3 Hz, 2H), 7.94 (d, J= 9.2 Hz, 1H), 7.74 (d, J= 8.9 Hz, 1H), 7.48 - 7.40 (m, 1H), 7.26 (d, J= 8.9 Hz, 1H), 6.79 (d, J= 9.4 Hz, 2H), 3.70 (s, 3H), 3.44 (t, J= 5.1 Hz, 5H), 2.90 (t, J= 6.1 Hz, 3H), 2.67 (t, J = 5.0 Hz, 6H), 2.54 (t, J= 6.3 Hz, 3H), 1.90 - 1.83 (m, 4H).13C NMR (151 MHz, Chloroform-; / ) 5 167.13, 157.43, 154.51, 147.97, 143.11, 138.91, 137.09, 133.72, 132.52, 131.16, 130.86, 129.00, 128.16, 126.01, 122.40, 117.23, 113.11, 110.86, 110.51, 62.09, 52.03, 46.90, 33.05, 30.93, 23.63, 22.63. Expected HRMS: 537.2614, HRMS: 537.2595

[0566] 'HNMR (600 MHz, Chloroform-; / ) 5 8.56 (d, J= 2.2 Hz, 1H), 7.98 (dd, J= 7.5, 1.7 Hz, 1H), 7.76 (d, J= 8.9 Hz, 1H), 7.43 (dd, J= 8.0, 6.0 Hz, 3H), 7.40 (td, J= 7.4, 1.7 Hz, 1H), 7.31 (dd, J= 8.9, 2.3 Hz, 1H), 7.20 - 7.16 (m, 1H), 6.86 (d, J= 8.6 Hz, 2H), 3.65 (s, 2H), 3.30 (t, J= 4.8 Hz, 4H), 2.93 (t, J= 6.0 Hz, 2H), 2.67 (t, J= 5.1 Hz, 4H), 2.52 (t, J= 6.1 Hz, 2H), 1.91 - 1.81 (m, 4H).13C NMR (151 MHz, Chloroform-; / ) 5 166.93, 157.77, 152.76, 146.54, 143.83, 134.29, 132.57, 132.37, 131.42, 130.61, 129.40, 128.94, 126.58 (q, J= 3.7 Hz), 125.51, 123.72, 122.44, 121.21 (q, J= 32.8 Hz), 117.40, 114.81, 110.78, 110.40, 62.30, 52.20, 47.89, 33.94, 23.82, 22.86, 22.78. Expected HRMS: 560.2637, HRMS: 560.2634|0567] 'HNMR (600 MHz, Chloroform-; / ) 5 8.61 (s, 1H), 8.02 (s, 1H), 7.95 (d, J= 7.1 Hz, 1H), 7.77 (d, J= 8.9 Hz, 2H), 7.67 (d, J= 8.8 Hz, 1H), 7.46 - 7.39 (m, 2H), 7.28 - 7.21 (m, 1H), 7.17 (d, J= 8.7 Hz, 2H), 3.71 (s, 3H), 3.17 (t, J= 4.8 Hz, 6H), 2.92 (t, J= 6.1 Hz, 3H), -146-4921 -4618-5569.4Atty. Dkt. No.: 115872-33252.70 (t, J= 4.7 Hz, 6H), 2.54 (t, J= 6.2 Hz, 3H), 1.87 (dt, J= 11.6, 5.2 Hz, 5H).13C NMR (151 MHz, Chloroform-; / ) 6 167.02, 157.58, 147.74, 143.35, 141.15, 137.09, 133.81, 132.69, 132.46, 131.27, 130.88, 130.52, 129.00, 128.45, 124.19 (q, J= 4.0 Hz), 124.09, 123.33 (q, = 34.4 Hz), 122.32, 121.03, 117.31, 110.81, 110.57, 62.15, 52.13 (d, J= 3.6 Hz), 50.91, 33.28, 23.69, 22.70, 22.69. Expected HRMS: 605.2488, HRMS: 605.2501

[0568] 'HNMR (600 MHz, Chloroform-; / ) 5 8.32 (s, 1H), 8.31 (s, 1H), 7.85 (d, J= 7.9 Hz, 2H), 7.79 (d, J= 9.0 Hz, 1H), 7.57 (d, J= 11.3 Hz, 1H), 7.27 (d, J= 8.0 Hz, 2H), 2.55 (s, 3H), 2.39 (s, 3H), 2.16 (s, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 159.71, 153.69, 144.60, 133.41, 131.02, 130.10, 129.05, 127.31, 123.27, 117.09, 116.86, 111.17, 109.23, 103.08, 22.67, 21.81, 12.34. Expected HRMS: 357.1715, HRMS: 357.1713

[0569] 'HNMR (600 MHz, CDCh+MeOD) 5 8.20 (s, 1H), 7.70 (m, 2H), 7.38 (d, J= 8.6 Hz, 2H), 7.34 (d, J= 1.9 Hz, 1H), 6.91 (d, J= 2.0 Hz, 1H), 6.83 (d, J= 8.5 Hz, 2H), 3.90 (s, 2H), 3.24 (t, J= 5.1 Hz, 4H), 2.74 (t, J= 5.0 Hz, 4H), 2.46 (s, 4H), 1.99 (s, 3H).13C NMR (151 MHz, CDCh+MeOD) 5 161.83, 161.60, 161.37, 161.23, 161.13, 153.17, 151.75, 149.15, 140.98, 135.07, 133.11, 126.26, 125.32, 124.47, 122.67, 120.88, 120.47, 120.25, 120.03, 119.82, 119.72, 119.45, 119.39, 116.86, 114.92, 114.36, 113.86, 110.70, 110.64, 110.59, 110.56, 107.03, 53.50, 51.25, 46.88, 17.77, 10.57. Expected HRMS: 524.2195, HRMS: 524.2267-147-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0570] 'H NMR (600 MHz, Chloroform-; / ) 5 8.53 (s, 1H), 8.07 (d, J= 9.4 Hz, 2H), 7.79 (d, J= 9.0 Hz, 1H), 7.35 (d, J= 1.9 Hz, 1H), 7.24 (s, 1H), 6.88 (s, 1H), 6.81 (d, J= 9.5 Hz, 2H), 3.86 (s, 2H), 3.48 (t, J= 5.0 Hz, 4H), 2.74 (t, J= 5.0 Hz, 4H), 2.55 (s, 3H), 2.15 (s, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 155.19, 153.43, 138.10, 126.53, 121.29, 120.37, 116.42, 109.54, 108.21, 53.81, 45.90, 21.82, 11.42. Expected HRMS: 501.2172, HRMS: 501.2247

[0571] 'HNMR (600 MHz, CDCh+MeOD) 5 8.50 (s, 1H), 7.90 (s, 1H), 7.74 (d, J= 9.0 Hz, 1H), 7.60 (d, .7= 9.1 Hz, 1H), 7.55 (d, J= 8.8 Hz, 1H), 7.29 (s, 1H), 7.10 (d, J= 8.8 Hz, 1H), 6.83 (d, J= 2.0 Hz, 1H), 3.85 (s, 2H), 3.08 (t, J= 4.9 Hz, 4H), 2.66 (t, J= 4.9 Hz, 4H), 2.50 (s, 3H), 2.06 (s, 3H).13C NMR (151 MHz, CDCh+MeOD) 5 162.31, 154.84, 152.87, 150.15, 147.57, 142.13, 140.83, 136.22, 134.31, 130.36, 126.08, 122.98 (d, J = 34.3 Hz), 121.11, 120.72, 115.71, 111.43, 111.04, 108.23, 54.12, 52.14, 50.60, 18.69, 11.45. Expected HRMS: 569.2046, HRMS: 569.2122-148-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0572] 'H NMR (600 MHz, CDCh+MeOD) 5 8.19 (s, 1H), 7.71 (s, 1H), 7.58 (d, J = 9.0 Hz, 1H), 7.37 (d, J = 8.5 Hz, 2H), 7.34 (d, J = 1.5 Hz, 1H), 6.92 (d, J = 2.0 Hz, 1H), 6.81 (d, J = 8.4 Hz, 2H), 3.89 (s, 2H), 3.22 (t, J = 5.1 Hz, 4H), 2.84 (t, J = 4.5 Hz, 2H), 2.72 (t, J = 4.8, 3.0 Hz, 4H), 2.38 (t, J = 6.1 Hz, 2H), 1.78 (mz, J = 12.1, 5.7 Hz, 4H).13C NMR (151 MHz, CDCh+MeOD) 5 162.51 (m)f, 154.01, 152.71, 151.09, 141.94, 135.72, 134.50, 126.43, 125.47, 123.67, 121.88(m), 120.39, 119.86, 117.92, 115.97, 115.25, 111.86, 111.56, 109.18, 54.44, 52.25, 47.84, 28.11, 22.22, 21.50, 20.92. Expected HRMS: 524.2195, HRMS: 550.2424

[0573] 'HNMR (600 MHz, CDCh+MeOD) 5 8.00 (d, J= 9.2 Hz, 2H), 7.66 (d, J = 9.1 Hz, 1H), 7.37 (d, J= 1.9 Hz, 1H), 6.97 (d, J= 2.1 Hz, 1H), 6.75 (d, J= 9.4 Hz, 2H), 3.94 (s, 2H), 3.40 (d, J= 5.2 Hz, 4H), 2.86 (t, J= 6.1 Hz, 2H), 2.72 (t, J= 5.1 Hz, 4H), 2.42 (t, J= 6.2 Hz, 2H), 1.86 - 1.77 (m, 4H).13C NMR (151 MHz, CDCh+MeOD) 5 153.81, 153.56, 152.24, 149.65, 141.18, 137.64, 135.07, 133.05, 125.71, 124.94, 119.19, 114.27, 111.99, 110.78, 110.11, 108.19, 53.09, 51.09, 45.79, 26.87, 21.24, 20.48, 19.85.

[0574] 'HNMR (600 MHz, CDCh+MeOD) 5 8.57 (s, 1H), 8.03 (s, 1H), 7.75 (d, J= 8.9Hz, 1H), 7.66 (d, J= 8.7 Hz, 1H), 7.34 (d, J= 1.9 Hz, 1H), 7.18 (d, J = 8.7 Hz, 2H), 6.86 (d, -149-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325J= 1.9 Hz, 1H), 3.87 (s, 2H), 3.22 (t, J= 4.9 Hz, 4H), 2.91 (t, J= 6.2 Hz, 2H), 2.77 (t, J = 4.8 Hz, 4H), 2.55 (t, J= 6.1 Hz, 2H), 1.87 (m, J= 14.1, 7.1 Hz, 4H).13C NMR (151 MHz, CDCh+MeOD) 5 171.61, 161.80, 157.37, 151.19, 147.67, 142.04, 141.33, 133.71, 130.55, 130.53, 128.30, 124.15, 124.13, 124.04, 123.71, 123.48, 122.24, 122.00, 121.46, 121.18, 117.29, 112.18, 110.53, 110.31, 54.90, 52.29, 50.89, 33.13, 23.66, 22.66, 14.15. Expected HRMS: 595.2202, HRMS: 595.2272

[0575] 'HNMR (600 MHz, CDCh+MeOD) 5 9.23 (s, 1H), 8.27 (s, 1H), 7.72 (d, J= 8.9 Hz, 1H), 7.43 (d, J= 6.8 Hz, 1H), 6.97 (d, J= 8.4 Hz, 2H), 6.70 (d, J= 8.5 Hz, 2H), 4.08 (s, 2H), 3.69 (t, J= 5.2 Hz, 2H), 3.06 (t, J= 5.0 Hz, 4H), 2.90 (t, J= 5.9 Hz, 2H), 2.66 - 2.60 (m, 7H), 2.45 (t, J= 6.1 Hz, 2H), 2.19 (s, 3H), 1.86 - 1.78 (m, 3H).13C NMR (151 MHz, CDCh+MeOD) 5 168.52, 157.64, 148.97, 146.59, 133.01, 129.68, 129.58, 129.23, 122.70, 117.11, 116.54, 110.65, 109.84, 70.89, 68.84, 57.81, 53.53, 49.53, 33.82, 23.73, 22.82, 22.75, 20.47. Expected HRMS: 473.279, HRMS: 474.2866

[0576] 'HNMR (600 MHz, CDCh+MeOD) 5 8.28 (s, 1H), 7.99 (s, 1H), 7.74 (d, J = 9.0 Hz, 1H), 7.60 (d, J = 6.5 Hz, 1H), 7.50 (d, J = 11.3 Hz, 1H), 7.07 (d, J = 8.7 Hz, 1H), 4.10 (s, 2H), 3.69 (t, J = 5.0 Hz, 2H), 3.15 - 3.10 (m, 4H), 2.70 (t, J = 5.1 Hz, 2H), 2.66 (t, J = 4.9 Hz, 4H), 2.52 (s, 3H), 2.12 (s, 3H).13C NMR (151 MHz, Chloroform-d) 5 168.55, 156.57, 148.05, 147.90, 142.48, 141.01, 133.39, 130.41 - 130.29 (m), 127.83, 124.21 - 124.04 (m), 123.07, 123.10 - 122.08 (m), 120.87, 117.27, 110.47, 109.14, 70.96, 67.79, 57.72, 52.83, 50.50, 23.10, 12.45.-150-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0577] XH NMR (600 MHz, Chloroform-^ 5 11.86 (s, 1H), 8.58 (s, 1H), 7.98 (d, J= 7.5 Hz, 1H), 7.77 (d, J= 8.9 Hz, 1H), 7.49 - 7.29 (m, 3H), 7.18 (s, 1H), 7.06 (d, J= 8.5 Hz, 2H), 6.81 (d, J= 8.6 Hz, 2H), 4.70 (s, 2H), 3.64 (s, 2H), 3.18 (s, 4H), 2.94 (t, J= 6.0 Hz, 2H), 2.67 (d, .7= 5.2 Hz, 4H), 2.56 - 2.46 (m, 4H), 1.85 (td, J= 14.7, 4.4 Hz, 4H), 1.14 (t, J = 7.6 Hz, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 166.99, 157.48, 148.79, 146.73, 143.54, 137.49, 136.39, 134.53, 132.64, 132.60, 131.43, 130.56, 129.13, 128.82, 128.64, 122.74, 117.30, 116.49, 110.64, 110.38, 62.39, 52.56, 49.49, 27.95, 23.77, 22.82, 22.76, 15.78. Expected HRMS: 520.3076, HRMS: 520.3058

[0578] 'HNMR (600 MHz, Chloroform-; / ) 5 8.56 (s, 1H), 7.98 (d, J= 7.5 Hz, 1H), 7.77 (d, J= 8.9 Hz, 1H), 7.45 - 7.35 (m, 3H), 7.21 - 7.16 (m, 1H), 7.03 (d, J= 8.5 Hz, 2H), 6.79 (d, J= 8.6 Hz, 2H), 3.64 (s, 2H), 3.17 (s, 4H), 2.94 (t, J= 6.0 Hz, 2H), 2.67 (t, J= 4.9 Hz, 4H), 2.51 (t, J= 6.1 Hz, 2H), 2.47 (t, J= 7.4 Hz, 2H), 1.89 - 1.82 (m, 4H), 1.49 (p, J= 7.6 Hz, 2H), 1.27 (h, J= 7.4 Hz, 2H), 0.84 (t, J= 7.4 Hz, 3H).13C NMR (151 MHz, Chloroform-^ 5 167.02, 157.28, 148.72, 146.98, 137.45, 135.03, 134.58, 132.66, 132.60, 131.44, 130.58, 129.18, 128.82, 122.93, 117.24, 116.37, 110.60, 110.50, 62.38, 52.56, 49.46, 34.72, 33.83, 23.72, 22.75, 22.71, 22.37, 14.00.-151-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0579] 'HNMR (600 MHz, Chloroform-; / ) 5 9.08 (s, 1H), 8.29 (s, 1H), 7.76 (d, J= 8.9 Hz, 1H), 7.41 (d, J= 11.1 Hz, 1H), 7.21 - 7.14 (m, 2H), 6.84 - 6.77 (m, 3H), 4.50 (s, 2H), 4.11 (s, 2H), 3.73 (t, J= 5.2 Hz, 2H), 3.16 - 3.11 (m, 4H), 2.69 - 2.62 (m, 6H), 2.56 (s, 3H), 2.14 (s, 3H).13C NMR (151 MHZ, Chloroform-; / ) 5 168.51, 157.45, 151.08, 146.24, 143.82, 133.24, 129.67, 129.17, 122.33, 120.03, 117.61, 116.18, 109.73, 109.58, 70.86, 69.16, 57.84, 53.61, 49.04, 24.36, 12.72. Expected HRMS: 434.2556, HRMS: 434.2564

[0580] 'HNMR (600 MHz, CDCh+MeOD) 5 8.62 (s, 1H), 8.08 (d, J= 9.4 Hz, 2H), 7.97 (d, J= 7.5 Hz, 1H), 7.82 (d, J= 8.9 Hz, 1H), 7.49 - 7.40 (m, 2H), 7.26 (d, J= 8.9 Hz, 1H), 7.24 - 7.21 (m, 1H), 6.80 (d, J= 9.4 Hz, 2H), 3.70 (s, 2H), 3.45 (t, J= 5.2 Hz, 4H), 2.68 (t, J= 5.3 Hz, 4H), 2.57 (s, 3H), 2.17 (s, 3H).13C NMR (151 MHz, CDCh+MeOD) 5 166.97, 154.49, 139.09, 137.20, 134.27, 132.61, 132.37, 131.38, 131.36, 130.88, 129.08, 128.42, 126.04, 122.18, 117.65, 113.19, 110.78, 109.38, 62.23, 52.08, 46.97, 23.43, 12.60. Expected HRMS: 511.2458, HRMS: 511.2444-152-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0581] 'H NMR (600 MHz, Chloroform-; / ) 5 9.21 (s, 1H), 8.28 (s, 1H), 7.75 (d, J= 8.9 Hz, 1H), 7.44 (d, J= 8.9 Hz, 1H), 6.78 - 6.71 (m, 4H), 4.62 (s, 2H), 4.10 (s, 2H), 3.73 - 3.68 (m, 5H), 3.02 (t, 4H), 2.67 - 2.62 (m, 6H), 2.55 (s, 3H), 2.12 (s, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 168.55, 157.30, 154.00, 146.45, 145.42, 143.66, 133.30, 129.43, 122.50, 118.36, 117.57, 114.45, 109.94, 109.49, 70.88, 68.96, 57.83, 55.57, 53.65, 50.47, 24.23, 12.68.

[0582] 'HNMR (600 MHz, CDCh+MeOD) 5 8.30 (s, 1H), 8.04 (d, J= 9.4 Hz, 2H), 7.74 (d, J= 9.0 Hz, 1H), 7.46 (d, J= 9.0 Hz, 1H), 6.74 (d, J= 9.4 Hz, 2H), 4.11 (s, 2H), 3.71 (t, J = 5.1 Hz, 2H), 3.40 (t, J= 5.2 Hz, 4H), 2.76 - 2.68 (m, 6H), 2.53 (s, 3H), 2.13 (s, 3H).13C NMR (151 MHz, CDCh+MeOD) 5 168.50, 156.89, 154.67, 147.78, 142.84, 138.73, 133.33, 133.24, 128.14, 125.95, 122.80, 122.69, 117.35, 112.90, 110.39, 109.23, 70.98, 67.81, 57.75, 52.76, 46.56, 23.23, 12.47. Calc. HRMS: 479.2407, HRMS: 479.2397

[0583] 'HNMR (600 MHz, CDCh+MeOD) 5 8.64 (s, 1H), 7.97 (d, J= 7.1 Hz, 1H), 7.83 (d, J= 8.9 Hz, 1H), 7.73 (d, J= 8.2 Hz, 1H), 7.51 - 7.39 (m, 3H), 7.31 (d, J= 8.9 Hz, 1H), 7.25 - 7.21 (m, 1H), 7.15 (d, J = 8.3 Hz, 1H), 7.08 - 7.03 (m, 1H), 3.71 (s, 2H), 3.10 (t, J= 4.8 Hz, 4H), 2.70 (t, J= 4.7 Hz, 4H), 2.57 (s, 3H), 2.17 (s, 3H).13C NMR (151 MHz, CDCh+MeOD) 5 167.04, 156.97, 147.69, 145.55, 143.84, 142.96, 137.09, 137.04, 134.47, 134.36, 133.88, 132.78, 132.77, 132.66, 132.63, 131.39, 131.37, 130.88, 128.92, 128.41, 125.96, 122.83, 122.81, 122.35, 121.41, 117.72, 117.70, 110.91, 109.34, 109.32, 62.31, 52.55, 51.74, 23.50, 12.61. Calc. HRMS: 511.2458, HRMS: 511.2448-153-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0584] 'HNMR (600 MHz, Chloroform-; / ) 5 12.02 (s, 1H), 8.21 (s, 1H), 8.00 (d, J= 7.6 Hz, 1H), 7.89 (d, J= 9.0 Hz, 1H), 7.79 (d, J= 9.0 Hz, 1H), 7.72 (s, 1H), 7.61 (s, 1H), 7.45 - 7.38 (m, 2H), 7.34 (d, J= 8.6 Hz, 2H), 7.26 (d, J= 8.6 Hz, 2H), 7.20 - 7.16 (m, 1H), 4.90 (s, 2H), 4.21 - 4.15 (m, 1H), 3.68 (s, 2H), 3.05 (d, J= 11.0 Hz, 2H), 2.59 (s, 3H), 2.32 - 2.20 (m, 4H), 2.16 (s, 3H), 2.15 - 2.11 (m, 2H).13C NMR (151 MHz, Chloroform-; / ) 5 166.82, 137.44, 136.70, 136.69, 135.10, 132.92, 132.50, 132.11, 131.65, 131.62, 130.97, 130.67, 129.06, 128.90, 126.73, 124.56, 124.41, 123.11, 121.71, 117.64, 110.22, 109.33, 62.12, 51.61, 32.20, 24.00, 14.12, 12.71. Calc. HRMS: 565.2499, HRMS: 565.2483

[0585] 1H NMR (600 MHz, Chloroform-; / ) 5 11.56 (s, 1H), 8.04 - 7.93 (m, 4H), 7.47 - 7.40 (m, 2H), 7.22 (dd, J= 10.4, 7.8 Hz, 2H), 7.13 (d, J= 8.7 Hz, 1H), 3.74 (s, 2H), 3.20 (t, J= 4.8 Hz, 4H), 2.72 (t, J= 4.8 Hz, 4H), 2.68 (s, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 165.87, 157.07, 146.57, 143.86, 140.09, 136.25, 135.28, 134.90, 131.58, 131.17, 130.49, 129.74, 129.37 (q, J= 3.3 Hz), 128.12, 128.01, 126.10, 123.20 (q, J= 3.9 Hz), 123.01, 122.50, 122.26 (q, J= 34.4 Hz), 121.67, 121.21, 119.83, 116.22, 61.17, 51.07, 49.80, 24.08. Calc. HRMS: 550.2066, HRMS: 550.2075-154-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0586] 'HNMR (600 MHz, Chloroform-d) 5 12.54 (s, 1H), 8.34 (s, 1H), 7.96 - 7.84 (m, 1H), 7.34 (dd, J = 5.7, 3.3 Hz, 1H), 7.27 (dd, J = 17.1, 8.3 Hz, 4H), 7.19 (d, J = 1.6 Hz, 2H), 7.23 - 7.06 (m, 7H), 4.22 (d, J = 26.6 Hz, 1H), 3.57 (s, 2H), 3.11 (t, J = 5.1 Hz, 1H), 2.47 (s, 7H), 1.95 (s, 6H).13C NMR (151 MHZ, Chloroform-d) 5 154.17, 150.31, 136.48, 136.05, 133.21, 132.63, 131.18, 127.56, 127.11, 120.47, 117.91, 115.36, 108.06, 74.89, 74.69, 43.72, 11.68. Calc. HRMS: 590.2687, HRMS: 590.2679|0587] 'HNMR (600 MHz, Chloroform-; / ) 5 8.48 - 8.44 (m, 1H), 8.02 - 7.94 (m, 2H), 7.82 (d, J= 8.9 Hz, 1H), 7.72 (dd, J= 8.2, 1.8 Hz, 1H), 7.68 (d, J= 8.0 Hz, 1H), 7.53 (d, J= 8.9 Hz, 1H), 7.39 (m, J= 20.7, 7.4, 1.5 Hz, 2H), 7.15 (m, J= 7.4, 1.5 Hz, 1H), 3.84 (s, 2H), 3.59 (s, 2H), 2.54 (s, 3H), 2.50 - 2.47 (m, 9H), 2.11 (s, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 166.00, 148.73, 136.62, 135.95, 134.33, 131.78, 131.58, 130.78, 130.40, 129.88 (q, J= 34.1 Hz), 129.72, 127.92 - 127.70 (m), 122.81, 122.70, 120.89, 120.76 (q, J = 3.8 Hz), 119.09, 116.01, 110.00, 108.09, 61.17, 57.54, 51.70, 51.30. Calc. HRMS: 593.2488, HRMS: 593.2480-155-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0588] JH NMR (600 MHz, Chloroform-^ 5 11.59 (s, 1H), 8.65 (s, 1H), 7.98 (d, J= 7.4 Hz, 1H), 7.85 (t, J= 8.5 Hz, 1H), 7.76 (s, 1H), 7.67 (d, J= 8.8 Hz, 1H), 7.47 - 7.39 (m, 2H), 7.36 - 7.27 (m, 1H), 7.22 (d, J= 7.2 Hz, 1H), 7.02 (d, J= 8.7 Hz, 1H), 4.73 (s, 2H), 3.73 (s, 2H), 3.36 (t, J= 4.7 Hz, 4H), 2.82 - 2.75 (m, 4H), 2.59 (s, 3H), 2.18 (s, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 166.93, 157.09, 137.35, 136.16 - 133.11 (m), 132.71, 132.26, 131.98 - 131.76 (m), 131.39, 131.05 - 130.85 (m), 130.77, 129.03, 125.61 - 120.71 (m), 118.74, 117.98, 117.79, 117.22, 110.82, 110.50, 109.59, 105.24, 103.15, 62.22, 52.33 (d, J= 2.5 Hz), 50.84, 14.12, 12.73. Calc. HRMS: 559.2433, HRMS: 559.2437|0589] 'HNMR (600 MHz, Chloroform-; / ) 5 8.62 (dd, J= 12.6, 2.2 Hz, 1H), 7.99 (d, J= 7.3 Hz, 1H), 7.94 - 7.89 (m, 1H), 7.83 (s, 1H), 7.73 (d, J= 10.9 Hz, 1H), 7.47 - 7.36 (m, 5H), 7.22 (d, J= 7.1 Hz, 1H), 3.69 (s, 3H), 3.03 (t, J= 4.9 Hz, 4H), 2.69 (s, 4H), 2.60 (s, 3H), 2.15 (s, 3H).13C NMR (151 MHZ, Chloroform-^ 5 167.05, 154.69, 137.17, 135.09, 134.80, 132.69, 131.44, 131.43, 130.77, 130.22 - 129.89 (m), 128.89, 127.32 - 126.86 (m), 125.74 - 124.60 (m), 124.35, 124.24, 123.95, 123.85, 123.52, 122.55, 122.43, 117.85, 117.54, 111.08, 110.83, 109.46, 103.03, 62.32, 53.01, 52.66, 41.02, 14.04, 12.61. Calc. HRMS: 602.2355, HRMS: 602.2344-156-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325|0590] 'HNMR (600 MHz, Chloroform-d) 5 8.65 (s, 1H), 7.99 (d, J = 7.5 Hz, 2H), 7.48 - 7.40 (m, 2H), 7.36 (d, J = 6.1 Hz, 1H), 7.26 - 7.20 (m, 3H), 6.36 - 6.31 (m, 1H), 3.73 (s, 2H), 3.69 - 3.52 (m, 4H), 2.78 (t, J= 5.2 Hz, 4H), 2.61 (s, 3H), 2.17 (s, 3H).13C NMR (151 MHz, Chloroform-d) 5 167.33, 156.54, 150.55, 148.42, 145.58, 142.35, 139.38, 136.80, 134.07, 132.99, 132.72, 132.54, 130.97, 130.87, 128.82, 127.57, 122.57, 117.46, 111.19, 109.50, 109.16, 106.30, 62.06, 52.08, 22.78, 12.30.|0591] 'HNMR (600 MHz, Chloroform-d) 5 8.62 (s, 1H), 8.05 (d, J = 7.5 Hz, 1H), 7.86 (d, J = 8.9 Hz, 1H), 7.52 (d, J = 9.0 Hz, 2H), 7.50 - 7.46 (m, 1H), 7.42 (d, J = 9.3 Hz, 1H), 7.26 (d, J = 7.3 Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H), 3.74 (fs, 2H), 3.42 (t, J = 5.2 Hz, 4H), 2.74 (t, J = 5.1 Hz, 4H), 2.64 (s, 3H), 2.22 (s, 3H).13C NMR (151 MHz, Chloroform-d) 5 165.86, 151.86, 136.42, 133.57, 132.59, 131.51, 131.21, 130.36, 129.62, 127.95, 122.01, 121.20, 118.77, 116.90, 116.74, 113.48, 109.73, 109.48, 108.57, 102.10, 100.09, 61.20, 51.00, 46.01, 13.06, 11.69. Calc. HRMS: 491.2559, HRMS: 491.2553-157-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0592] 'HNMR (600 MHz, Chloroform-d) 5 8.58 (s, 1H), 7.91 (d, J = 9.1 Hz, 1H), 7.83 - 7.77 (m, 2H), 7.44 (t, J = 3.6 Hz, 2H), 7.35 (d, J = 8.9 Hz, 1H), 7.25 (s, OH), 6.32 (d, J = 8.1 Hz, 2H), 3.86 (t, J = 5.1 Hz, 6H), 3.74 (s, 3H), 2.81 (s, 10H), 2.74 (s, 1H), 2.55 (s, 4H).13C NMR (151 MHz, Chloroform-d) 5 155.02, 134.56, 132.66, 130.31, 125.86, 124.59, 121.97, 120.31, 120.02, 118.51, 118.50, 116.57, 110.27, 105.02, 99.58, 98.64, 96.80, 92.78, 49.53, 39.64, 36.09, 25.37, 10.31. Calc. HRMS: 615.2502, HRMS: 615.2513

[0593] 'HNMR (600 MHz, Chloroform-d) 5 8.62 (dd, J = 5.8, 2.1 Hz, 1H), 8.07 - 7.92 (m, 2H), 7.85 (t, J = 8.4 Hz, 1H), 7.61 (dd, J = 8.7, 2.3 Hz, 1H), 7.43 (pd, J = 7.4, 1.7 Hz, 2H), 7.34 (ddd, J = 22.2, 8.9, 2.3 Hz, 1H), 7.25 - 7.22 (m, 1H), 7.05 (dd, J = 8.6, 2.4 Hz, 1H), 3.86 (s, 3H), 3.71 (d, J = 2.2 Hz, 2H), 3.17 (t, J = 4.8 Hz, 4H), 2.79 - 2.66 (m, 4H), 2.58 (s, 3H), 2.17 (s, 3H).13C NMR (151 MHZ, Chloroform-d) 5 165.97, 165.77, 153.35, 136.05, 134.74 - 126.75 (m), 126.75 - 119.95 (m), 120.22, 117.95, 116.55, 110.11, 109.75, 108.24, 101.70, 61.27, 51.40 (d, J = 3.3 Hz), 50.73, 13.13, 11.53. Calc. HRMS: 592.2536, HRMS: 592.2528-158-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325

[0594] XH NMR (600 MHz, Chloroform-d) 5 11.14 (s, 1H), 8.51 - 8.46 (m, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.93 (d, J = 7.3 Hz, 1H), 7.77 (d, J = 8.9 Hz, 1H), 7.41 (dq, J = 17.4, 9.9, 8.9 Hz, 2H), 6.13 (d, J = 8.5 Hz, 1H), 4.71 (s, 3H), 4.06 (s, 5H), 3.69 (s, 3H), 2.77 (d, J = 5.2 Hz, 5H), 2.52 (s, 4H).13C NMR (151 MHz, Chloroform-d) 5 166.91, 157.47, 146.44,144.75, 144.63, 144.48, 143.65, 137.45, 134.84, 134.43, 132.44, 132.10, 131.15, 130.74, 129.64, 129.10, 124.00, 121.96, 117.86, 110.35, 109.71, 103.00, 61.82, 51.98, 48.88, 24.30,12.75. Calc. HRMS: 553.2312, HRMS: 553.2319

[0595] 'HNMR (600 MHz, Chloroform-d) 5 8.36 (s, 1H), 8.01 (d, J = 9.0 Hz, 1H), 7.91 (d, J = 6.7 Hz, 1H), 7.84 - 7.78 (m, 1H), 7.57 (d, J = 8.9 Hz, 1H), 7.47 (dd, J = 9.0, 6.7 Hz, 1H), 7.36 (dd, J = 5.7, 3.3 Hz, 2H), 7.14 (d, J = 9.0 Hz, 1H), 6.95 (d, J = 8.9 Hz, 1H), 3.60 (s, 2H), 3.33 (s, 4H), 2.55 (d, J = 13.4 Hz, 8H), 2.43 (s, 4H).13C NMR (151 MHz, Chloroform-d) 5 166.02, 156.09, 148.35, 146.34, 143.86, 141.94, 135.99, 133.38, 132.83, 131.55, 131.29, 129.77, 129.74, 129.70, 129.32, 127.84, 127.83, 127.41, 125.98, 120.83, 120.79, 116.56, 116.54, 109.68, 108.37, 60.57, 60.56, 50.94, 44.70, 22.57, 11.53. Calc. HRMS: 572.2080, HRMS: 572.2095-159-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325GN-85

[0596] JH NMR (600 MHz, Chloroform-; / ) 5 11.78 (s, 1H), 8.63 (d, J= 2.2 Hz, 1H), 8.06 (d, J= 7.6 Hz, 1H), 7.86 (d, J= 8.9 Hz, 1H), 7.49 (dt, J= 20.2, 7.4 Hz, 2H), 7.42 (d, J= 8.9 Hz, 1H), 7.28 (d, J= 7.3 Hz, 1H), 7.14 (d, J= 8.2 Hz, 2H), 6.91 (d, J= 9.1 Hz, 2H), 3.75 (s, 2H), 3.28 (t, J= 4.9 Hz, 4H), 2.76 (t, J= 5.1 Hz, 4H), 2.64 (s, 3H), 2.24 (s, 3H).13C NMR (151 MHz, Chloroform-; / ) 5 166.90, 149.46, 137.50, 134.72, 132.57, 132.45, 131.44, 128.91, 121.46, 119.76, 110.36, 109.53, 62.35, 24.69, 12.72.

[0597] While certain embodiments have been illustrated and described, a person with ordinary skill in the art, after reading the foregoing specification, can effect changes, substitutions of equivalents and other types of alterations to the compounds of the present technology or salts, pharmaceutical compositions, derivatives, prodrugs, metabolites, tautomers or racemic mixtures thereof as set forth herein. Each aspect and embodiment described above can also have included or incorporated therewith such variations or aspects as disclosed in regard to any or all of the other aspects and embodiments.

[0598] The present technology is also not to be limited in terms of the particular aspects described herein, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the appended claims. It is to be understood that this present technology is not limited to particular methods, reagents, compounds, compositions, labeled compounds or biological systems, which can, of course, vary. It is also to be understood that the terminology used-160-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 herein is for the purpose of describing particular aspects only, and is not intended to be limiting. Thus, it is intended that the specification be considered as exemplary only with the breadth, scope and spirit of the present technology indicated only by the appended claims, definitions therein and any equivalents thereof.

[0599] The embodiments, illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising,” “including,” “containing,” etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the claimed technology. Additionally, the phrase “consisting essentially of’ will be understood to include those elements specifically recited and those additional elements that do not materially affect the basic and novel characteristics of the claimed technology. The phrase “consisting of’ excludes any element not specified.

[0600] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0601] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and -161-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325 refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member.

[0602] All publications, patent applications, issued patents, and other documents (for examplejournals, articles and / or textbooks) referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

[0603] The present technology may include, but is not limited to, the features and combinations of features recited in the following lettered paragraphs, it being understood that the following paragraphs should not be interpreted as limiting the scope of the claims as appended hereto or mandating that all such features must necessarily be included in such claims:A. A compound according to Formula (I):wherein:Ring A is a 5 to 14-membered cycloalkyl, a 6 to 14-membered aryl, a 5 to 16- membered heterocycloalkyl, or a 5 to 16-membered heteroaryl;Ring B is absent, a 5 to 14-membered cycloalkyl, a 6 to 14-membered aryl, a 5 to 16-membered heterocycloalkyl, or a 5 to 16-membered heteroarylR1is H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -CN, -ORla, -COORla, or -NRlaRlb, wherein Rlaand Rlbare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;-162-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325R2is H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -CN, -OR2a, -COOR2a, or -NR2aR2b, wherein R2aand R2bare each independently H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl; or R1and R2together form a 5 to 10-membered cycloalkyl, a 6 to 10-membered aryl, a 5 to 10-membered heterocycloalkyl, or a 5 to 10-membered heteroaryl;R3is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -OR3a, -SR3a, -SO2R3a, -COOR3a, -NR3aR3b, -NO2, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, wherein R3aand R3bare each independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heteroaryl, or optionally substituted heterocycloalkyl, and n is an integer from 0 to 5;X1is a bond, an optionally substituted alkylenyl, an optionally substituted alkenylenyl, an optionally substituted heteroalkylenyl, or an optionally substituted heteroalkenylenyl;X2is a bond, an optionally substituted alkylenyl, an optionally substituted alkenylenyl, an optionally substituted heteroalkylenyl, or an optionally substituted heteroalkenyl enyl.B. The compound of Paragraph A, wherein Ring A is a phenyl, piperazinyl, or furanyl.C. The compound of Paragraph A or B, wherein Ring A is a phenyl.D. The compound of any one of Paragraphs A-C, wherein Ring B is piperidinyl, piperazinyl, morpholinyl, phenothiazinyl, or iminodibenzyl.E. The compound any one of Paragraphs A-D, wherein Ring B is piperazinyl.F. The compound any one of Paragraphs A-E, wherein R3is alkyl, -OR3a, -SO2R3a, -COOR3a, optionally substituted aryl, or optionally substituted heteroaryl, wherein R3ais H, alkyl, optionally substituted aryl, or optionally substituted heteroaryl.-163-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325G. The compound of any one of Paragraphs A-F, wherein R3is methyl, ethyl, propyl, butyl, or t-butyl.H. The compound of any one of Paragraphs A-F, wherein R3is -OR3a, wherein R3ais optionally substituted aryl.I. The compound of any one of Paragraphs A-F, wherein R3is optionally substituted phenyl.J. The compound of any one of Paragraphs A-F, wherein R3is -COOR3a, wherein R3ais alkyl.K. The compound of any one of Paragraphs A- J, wherein R1is H, alkyl, or -COORla, wherein Rlais alkyl.L. The compound of any one of Paragraphs A-K, wherein R1is H.M. The compound of any one of Paragraphs A-K, wherein R1is methyl.N. The compound of any one of Paragraphs A-M, wherein R2is H, alkyl, or -COOR2a, wherein R2ais alkyl.O. The compound of any one of Paragraphs A-N, wherein R2is H.P. The compound of any one of Paragraphs A-N, wherein R2is methyl.Q. The compound of any one of Paragraphs A-N, wherein R2is -COOR2a, wherein R2ais ethyl.R. The compound of any one of Paragraphs A- J, wherein R1and R2taken together are a 6- membered cycloalkyl.S. The compound of any one of Paragraphs A-R, wherein X1is a bond, an alkylenyl, an optionally substituted alkenylenyl, or a heteroalkylenyl.T. The compound of any one of Paragraphs A-R, wherein X1is a bond.-164-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325U. The compound of any one of Paragraphs A-R, wherein X1is -CH2-.V. The compound of any one of Paragraphs A-R, wherein X1is -CH2-O-(CH2)2-.W. The compound of any one of Paragraphs A-R, wherein X1is a C2 alkenylenyl substituted with -CN.X. The compound of any one of Paragraphs A-W, wherein X2is a bond or an optionally substituted alkylenyl.Y. The compound of any one of Paragraphs A-X, wherein X2is a bond.Z. The compound of any one of Paragraphs A-X, wherein X2is -CH2-.AA. The compound of any one of Paragraphs A-Z, wherein the compound is selected from:4921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-3325-167-4921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-3325-170-4921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-3325-172-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325AB. A pharmaceutical composition comprising a therapeutically effective amount of the compound of any one of Paragraphs A-AA and a pharmaceutically acceptable carrier.AC. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Paragraphs A-AA or the pharmaceutical composition of paragraph AB.AD. The method of Paragraph AC, wherein the disease is cancer.AE. The method of Paragraph AC or AD, wherein the disease is pancreatic ductal adenocarcinoma.AF. Use of a therapeutically effective amount of the compound of any one of ParagraphsA-AA or the pharmaceutical composition of paragraph AB for treating a disease or disorder in a subject in need thereof.-173-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325AG. The use of Paragraph AF, wherein the disease is cancer.AH. The use of Paragraph AF or AG, wherein the disease is pancreatic ductal adenocarcinoma.Al. Use of therapeutically effective amount of the compound of any one of Paragraphs A- AA or the pharmaceutical composition of paragraph AB for the manufacture of a medicament for the treatment of a disease or disorder in a subject in need thereof.AJ. The use of Paragraph Al, wherein the disease is cancer.AK. The use of Paragraph Al or AJ, wherein the disease is pancreatic ductal adenocarcinomaAL. A method of inhibiting the autophagy lysosomal pathway (ALP) in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Paragraphs A-AA or the pharmaceutical composition of paragraph AB.AM. Use of a therapeutically effective amount of the compound of any one of ParagraphsA-AA or the pharmaceutical composition of paragraph AB for inhibiting the autophagy lysosomal pathway (ALP).

[0604] Other embodiments are set forth in the following claims, along with the full scope of equivalents to which such claims are entitled.-174-4921 -4618-5569.4

Claims

Atty. Dkt. No.: 115872-3325WHAT IS CLAIMED IS:

1. A compound of Formula (I):wherein:Ring A is a 5 to 14-membered cycloalkyl, a 6 to 14-membered aryl, a 5 to 16- membered heterocycloalkyl, or a 5 to 16-membered heteroaryl;Ring B is absent, a 5 to 14-membered cycloalkyl, a 6 to 14-membered aryl, a 5 to 16- membered heterocycloalkyl, or a 5 to 16-membered heteroarylR1is H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -CN, -ORla, -COORla, or -NRlaRlb, wherein Rlaand Rlbare each independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;R2is H, halogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -CN, -OR2a, -COOR2a, or -NR2aR2b, wherein R2aand R2bare each independently H, optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl; or R1and R2together form a 5 to 10-membered cycloalkyl, a 6 to 10-membered aryl, a 5 to 10-membered heterocycloalkyl, or a 5 to 10-membered heteroaryl;R3is optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, -OR3a, -SR3a, -SO2R3a, -COOR3a, -NR3aR3b, -NO2, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, wherein R3aand R3bare each independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, optionally substituted heteroaryl, or optionally substituted heterocycloalkyl, and n is an integer from 0 to 5;-175-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325X1is a bond, an optionally substituted alkylenyl, an optionally substituted alkenylenyl, an optionally substituted heteroalkylenyl, or an optionally substituted heteroalkenylenyl;X2is a bond, an optionally substituted alkylenyl, an optionally substituted alkenylenyl, an optionally substituted heteroalkylenyl, or an optionally substituted heteroalkenylenyl,2. The compound of Claim 1, wherein Ring A is a phenyl, piperazinyl, or furanyl.

3. The compound of Claim 1, wherein Ring A is a phenyl.

4. The compound of Claim 1, wherein Ring B is piperidinyl, piperazinyl, morpholinyl, phenothiazinyl, or iminodibenzyl.

5. The compound of Claim 1, wherein Ring B is piperazinyl.

6. The compound of Claim 1, wherein R3is alkyl, -OR3a, -SO2R3a, -COOR3a, optionally substituted aryl, or optionally substituted heteroaryl, wherein R3ais H, alkyl, optionally substituted aryl, or optionally substituted heteroaryl.

7. The compound of Claim 1, wherein R3is methyl, ethyl, propyl, butyl, or t-butyl.

8. The compound of Claim 1, wherein R3is -OR3a, wherein R3ais optionally substituted aryl.

9. The compound of Claim 1, wherein R3is optionally substituted phenyl.

10. The compound of Claim 1, wherein R3is -COOR3a, wherein R3ais alkyl.

11. The compound of Claim 1, wherein R1is H, alkyl, or -COORla, wherein Rlais alkyl.

12. The compound of Claim 1, wherein R1is H.

13. The compound of Claim 1, wherein R1is methyl.

14. The compound of Claim 1, wherein R2is H, alkyl, or -COOR2a, wherein R2ais alkyl.

15. The compound of Claim 1, wherein R2is H.

16. The compound of Claim 1, wherein R2is methyl.

17. The compound of Claim 1, wherein R2is -COOR2a, wherein R2ais ethyl.-176-4921 -4618-5569.4Atty. Dkt. No.: 115872-332518. The compound of Claim 1, wherein R1and R2taken together are a 6-membered cycloalkyl.

19. The compound of Claim 1, wherein X1is a bond, an alkylenyl, an optionally substituted alkenylenyl, or a heteroalkylenyl.

20. The compound of Claim 1, wherein X1is a bond.

21. The compound of Claim 1, wherein X1is -CH2-.

22. The compound of Claim 1, wherein X1is -CH2-O-(CH2)2-.

23. The compound of Claim 1, wherein X1is a C2 alkenylenyl substituted with -CN.

24. The compound of Claim 1, wherein X2is a bond or an optionally substituted alkylenyl.

25. The compound of Claim 1, wherein X2is a bond.

26. The compound of Claim 1, wherein X2is -CH2-.-177-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325-178-4921 -4618-5569.4Atty. Dkt. No.: 115872-3325-179-4921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-3325-182-4921 -4618-5569.4Atty. Dkt. No.: 115872-33254921 -4618-5569.4Atty. Dkt. No.: 115872-3325-184-4921 -4618-5569.4Atty. Dkt. No.: 115872-332528. A pharmaceutical composition comprising a therapeutically effective amount of the compound of any one of Claims 1-27 and a pharmaceutically acceptable carrier.

29. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Claims 1-27 or the pharmaceutical composition of claim 28.

30. The method of Claim 29, wherein the disease is cancer.

31. The method of Claim 29, wherein the disease is pancreatic ductal adenocarcinoma.-185-4921 -4618-5569.4Atty. Dkt. No.: 115872-332532. Use of a therapeutically effective amount of the compound of any one of Claims 1-27 for treating a disease or disorder in a subject in need thereof.

33. The use of Claim 32, wherein the disease is cancer.

34. The use of Claim 32, wherein the disease is pancreatic ductal adenocarcinoma.

35. Use of therapeutically effective amount of the compound of any one of Claims 1-27 for the manufacture of a medicament for the treatment of a disease or disorder in a subject in need thereof.

36. The use of Claim 35, wherein the disease is cancer.

37. The use of Claim 35, wherein the disease is pancreatic ductal adenocarcinoma.

38. A method of inhibiting the autophagy lysosomal pathway (ALP) in a subject, the method comprising administering to the subject a therapeutically effective amount of the compound of any one of Claims 1-27.

39. Use of a therapeutically effective amount of the compound of any one of Claims 1-27 for inhibiting the autophagy lysosomal pathway (ALP).-186-4921 -4618-5569.4

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