COMPOUNDS FOR THE TREATMENT OF CANCER AND INFLAMMATORY DISEASE
20220332725 · 2022-10-20
Inventors
- Yaron R. Hadari (Harrison, NY)
- Luca Carta (Scarsdale, NY, US)
- Michael Schmertzler (St. Petersburg, FL)
- Theresa M. Williams (Harleysville, PA)
- Charles H. Reynolds (Austin, TX)
Cpc classification
A61P1/04
HUMAN NECESSITIES
A61P29/00
HUMAN NECESSITIES
A61P1/02
HUMAN NECESSITIES
A61P19/08
HUMAN NECESSITIES
C07D271/06
CHEMISTRY; METALLURGY
A61P1/18
HUMAN NECESSITIES
A61P1/16
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
C07D401/12
CHEMISTRY; METALLURGY
C07D401/04
CHEMISTRY; METALLURGY
A61P15/00
HUMAN NECESSITIES
International classification
C07D271/06
CHEMISTRY; METALLURGY
C07D401/04
CHEMISTRY; METALLURGY
C07D401/12
CHEMISTRY; METALLURGY
Abstract
Provided herein are compounds that inhibit the phosphorylation of MAPK and thus are useful in compositions and methods for treating cancer and inflammatory disease.
Claims
1-5. (canceled)
6. A compound of Formula VIf: ##STR01150## or a pharmaceutically acceptable salt thereof, wherein R.sup.1f is 2-pyridyl; and R.sup.2f is —C(O)R, wherein R is aryl, heteroaryl or alkyl.
7. (canceled)
8. A method of treating, preventing or ameliorating one or more symptoms of cancer, comprising administering to a subject the compound or pharmaceutically acceptable salt thereof of claim 6.
9. The method of claim 8, wherein the cancer is a solid tumor.
10. The method of claim 9, wherein the solid tumor is hepatocellular carcinoma, prostate cancer, pancreatic cancer, ovarian cancer, or glioblastoma.
11. The method of claim 10, wherein the solid tumor is pancreatic cancer.
12. A method of treating, preventing or ameliorating one or more symptoms of an inflammatory disease, comprising administering to a subject the compound or pharmaceutically acceptable salt thereof of claim 6.
13. The method of claim 12, wherein the inflammatory disease is gastritis, schistosomiasis, cholangitis, chronic cholecystitis, pelvic inflammatory disease, chronic cervicitis, osteomyelitis, inflammatory bowel disease, reflux esophagitis, Barrett's esophagus, bladder inflammation (cystitis), asbestosis, silicosis, gingivitis, lichen planus, pancreatitis, protease mutation, lichen sclerosis, slaladenitis, bronchitis, Sjogren syndrome or Hashimoto's thyroiditis.
14. A pharmaceutical composition, comprising the compound or pharmaceutically acceptable salt thereof of claim 6 and a pharmaceutically acceptable carrier.
15. The compound or pharmaceutically acceptable salt thereof of claim 6, wherein the compound is selected from the group consisting of: ##STR01151## ##STR01152## ##STR01153## ##STR01154##
16. A method of treating, preventing or ameliorating one or more symptoms of cancer, comprising administering to a subject the compound or pharmaceutically acceptable salt thereof of claim 15.
17. The method of claim 16, wherein the cancer is a solid tumor.
18. The method of claim 17, wherein the solid tumor is hepatocellular carcinoma, prostate cancer, pancreatic cancer, ovarian cancer, or glioblastoma.
19. The method of claim 18, wherein the solid tumor is pancreatic cancer.
20. A method of treating, preventing or ameliorating one or more symptoms of an inflammatory disease, comprising administering to a subject the compound or pharmaceutically acceptable salt thereof of claim 15.
21. The method of claim 20, wherein the inflammatory disease is gastritis, schistosomiasis, cholangitis, chronic cholecystitis, pelvic inflammatory disease, chronic cervicitis, osteomyelitis, inflammatory bowel disease, reflux esophagitis, Barrett's esophagus, bladder inflammation (cystitis), asbestosis, silicosis, gingivitis, lichen planus, pancreatitis, protease mutation, lichen sclerosis, slaladenitis, bronchitis, Sjogren syndrome or Hashimoto's thyroiditis.
22. A pharmaceutical composition, comprising the compound or pharmaceutically acceptable salt thereof of claim 15 and a pharmaceutically acceptable carrier.
Description
8. EXAMPLES
[1735] The following examples are offered to illustrate but not to limit the disclosure.
Example 1
Protocol for MAPK Cell-Based Phosphorylation Assay
[1736] Cell lines: Tumor-derived pancreatic cancer cell lines PANC-1 were purchased from ATCC and were maintained according to ATCC recommendation.
[1737] Method: Cells were plated at 7500 cells/well density in 96-wells plate, starved ON, and the small molecules to be tested were added to the cells in the final concentration of 30 μM with 0.3% DMSO for 6 hours incubation at 37° C. Next, cells were stimulated with 1.5 ng/ml EGF for 15 min. followed by cell fixation with 4% Formaldehyde in PBS at RT for 20 min. Phosphorylation level of MAPK was determined by Cell-direct ELISA.
[1738] Cell-direct ELISA: For each well, cells were permeabilized with PBS-Triton 0.1%, quenched with H.sub.2O.sub.2 0.6% in PBS-Triton 0.1%, and probed with anti-phospho-MAPK antibodies (R&D Systems) followed by HRP-conjugated secondary antibody (Jackson Immunoresearch, West Grove, Pa.). Next, a solution 50 μM of the fluorescent substrate AmpliFlu Red (Sigma) was added and incubated at RT for 20 min. At the end of the incubation time, fluorescence was measured at 595 nm on a microplate reader (AF2200; Eppendorf, Inc., Hamburg, Germany).
[1739] Table 2 shows inhibition data for selected compounds tested in the cellular assay described above.
TABLE-US-00002 TABLE 2 % Inhibition of MAPK phosphorylation @ 30 μM in the PANC-1 pancreatic cancer cell line Compound PANC-1
Example 2
Protocol for Cell Proliferation Assay
[1740] Cell lines: Tumor-derived pancreatic cancer cell lines MIA-PACA2 were purchased from ATCC and grown in complete DMEM-High Glucose medium supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% heat-inactivated FBS at 37° C. in a humidified incubator with 5% CO2.
[1741] Method: Cells are plated at 1000 cells/well density in 96-wells plate, starved ON, and the next day tested small molecules are added to the cells in the final concentration of 30 μM with 0.3% DMSO 3 hours prior to 10% FBS addition. After serum addition cells are incubated for 6 days at 37° C. in a humidified incubator with 5% CO.sub.2.
[1742] Assay: At the end of the incubation period, cell cultures are tested using the CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega Corporation, Madison, Wis.) according to the manufacturer specifications. Briefly, assay is performed by adding 20 μl of the CellTiter 96 Aqueous One Solution Reagent directly to culture wells, followed by for 1-4 hours incubation at 37° C. in a humidified incubator with 5% CO.sub.2. At the end of incubation time, absorbance at 492 nm is recorded with the 96-well plate reader Eppendorf AF2200, and degree of small molecule-dependent proliferation inhibition is calculated from raw data assuming No Serum cells value as 100%.
[1743] Table 3 shows inhibition data for selected compounds tested in the cellular assay described above.
TABLE-US-00003 TABLE 3 % Inhibition of cell proliferation @ 30 μM in MIA-PACA2 pancreatic cancer cell lines MIA- Compound PACA2
Example 3
Protocol for Mouse TNF Alpha and IL6 Quantification Assay
[1744] Cell lines: Abelson murine leukemia virus transformed macrophage cell line RAW 264.7 was purchased from ATCC and grown in complete DMEM-High Glucose medium supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL), and 10% heat-inactivated FBS at 37° C. in a humidified incubator with 5% CO.sub.2.
[1745] Method: Cells were plated at 40000 cells/well density in a 96-wells plate. After a 3-hour incubation, macrophages were starved with DMEM plus 0.5% FBS o/n. The next day the small molecules to be tested were added to the cells in the final concentration of 30 μM (with 0.3% DMSO) 3 hours prior to LPS stimulation (100 ng/ml). After LPS stimulation cells were incubated at 37° C. for 16 h. At the end of the incubation period, culture media were collected and production of LPS-induced TNFα and IL6 cytokine was measured using ELISA detection kits.
[1746] Sandwich ELISA: The ELISA Immunoassays Quantikine Mouse TNF-alpha (catalog number MTA00B) and IL6 (catalog number M6000B) were purchased from R&D Systems Inc., Minneapolis, Minn. These 4.5 hours solid phase ELISAs were used to measure mouse TNFα or IL6 levels in macrophages culture supernatants. Assays were executed according to the manufacturer specifications.
[1747] Table 4 shows inhibition data for selected compounds tested in the cellular assay described above.
TABLE-US-00004 TABLE 4 IL-6 and TNFα % Inhibition @ 30 μM in RAW 264.7 murine leukemia virus transformed macrophage cell lines Compound IL-6 TNF-α
[1748] This disclosure is not to be limited in scope by the embodiments disclosed in the examples which are intended as single illustrations of individual aspects, and any methods which are functionally equivalent are within the scope of this disclosure. Indeed, various modifications in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
[1749] Various references such as patents, patent applications, and publications are cited herein, the disclosures of which are hereby incorporated by reference herein in their entireties.