(M1230-04-23) Design, Synthesis, and Evaluation of Claudin-1 Inhibitors for the Treatment of Metastatic Colorectal Cancer

Purpose: Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality worldwide.¹ While early-stage CRC can be treated by surgical interventions, late-stage CRC remains lethal due to therapy resistance and metastasis. Claudins comprise a family of tight junction membrane proteins often dysregulated in cancer cells, making them a protein of interest for therapeutic approaches. Several studies report that claudin-1 (CLDN1) expression is upregulated in CRC cells and promotes epithelial-mesenchymal transition, metastasis, and resistance to anoikis.² CLDN1 directly interacts with ephrin type-A receptor 2 (EPHA2) tyrosine kinase to inhibit its degradation and promote cancer stemness and chemoresistance in CRC.³ Therefore, CLDN1 can be a viable target against therapy-resistant CRC as depicted by its role in Figure 1a. In our previously published work, we reported the discovery of novel first-generation CLDN1 inhibitors, I-6 and PDS-0330, which selectively binds to CLDN1 with micromolar affinity and inhibits CLDN1-dependent CRC progression both in in vitro and in vivo models of CRC without exhibiting toxicity.²  We hypothesized that our synthesized inhibitors were binding to CLDN1 receptors in the tumor cells and interfering with cancer progression and metastasis. However, for concretely proving the binding of small-molecules to their target receptors, it is often necessary to use affinity-based pull down assays that require a fluorescent or biotin tag.⁴ In this context, the main objectives of our research were (i) to synthesize CLDN1 inhibitors with higher potency than our current leads using SAR-based approaches; (ii) to investigate the in vitro efficacy of our synthesized compounds on CLDN1 expressing metastatic colorectal cancer cells; (iii) to synthesize a fluorescent-labelled CLDN1 inhibitor and investigate whether our inhibitors are selectively binding to CLDN1 receptors.

Methods: Synthesis scheme of the CLDN1 inhibitors are shown in Figure 1b. The anilines used in the final step were either commercially purchased or synthesized using a method described by Qiao et al.⁵ Aromatic trifluoromethyl group (1) was reacted with amino nucleophiles containing a second NH, OH, or SH nucleophile (2) to form C-C linked aryl-heterocycles 3a-3l.  Additionally, anilines 7a-7c were synthesized by reacting 4-nitrobenzoyl chloride (4) with different acid hydrazides, followed by thiation and subsequent reduction. The final compounds were produced from conversion of 2-methoxy napthoic acid (8) to acid chloride (9), followed by reaction with ammonium thiocyanate and an appropriate aniline to yield the desired final targets 10-33. For synthesizing a fluorescent-tagged CLDN1 inhibitor, we utilized click chemistry (Figure 1c) to couple the alkyne handle of 34 with the azide derivative of a fluorescent tag 5-carboxyflourescein to produce 35. The compounds were tested in cell-based assay for cell survival against the CLDN1 overexpressing SW620 and SW480^cldn1 colorectal cancer cells. CLDN1 lacking HCT-116 cancer cells and normal IEC-6 cells were used as control. For the fluorescent-tagged affinity based pull down assay, 25 µM of compound 35 was administered to SW620, SW480^cldn1, and HCT-116 cells. Four hours after administration, the cells were visualized under fluorescence microscope to observe illumination at 490 nm. In vitro DMPK assays for selected compounds were performed to assess human and mouse liver microsomal intrinsic clearance, plasma protein binding, and half-life.

Results: Percent inhibition of live cells in the CLDN1 overexpressing cells and control cells are presented in Table 1. Among the synthesized CLDN1 inhibitors, compounds 21, 30¸ 32, and 33 had the highest inhibition of SW620 cells with 69.2%, 86.5%, 66%, and 77.5% inhibition. Compounds 32 and 33 demonstrated an impressive 90.3% and 94.1% inhibition of SW480^cldn1 cell line as well. Figure 2a illustrates the dose-dependent reduction of cell viability in the CLDN1 overexpressing cell lines by compounds 21, 30¸ 32, and 33. In vitro DMPK assays were conducted for a few selected compounds, which showed that 30 and 33 were moderately stable in human and mice liver microsomes (Table 2). However, all the tested compounds displayed poor reproducibility in the plasma protein binding assays, probably due to high plasma protein binding as the compounds were stable in plasma. Figure 2b exhibits the visualization of CLDN1 inhibitor 35 in selected cells under fluorescence microscope. The fluorescent-tagged inhibitor illuminated brightly when bound to the CLDN1 expressing SW620 and SW480^cldn1 cells but was not visible in the CLDN1 lacking HCT116 cells.

Conclusion: Expanding our previously discovered leads, we hereby described the synthesis and biological evaluation of a series of CLDN1 inhibitors. We identified four compounds (21, 30, 32, and 33) with increased activity against SW620 and SW480^cldn1 cells compared to our previous leads. Compound 33 had overall best potency and favorable pharmacokinetic parameters but was a bit cytotoxic towards control cells which we are working to address. We also synthesized a novel CLDN1 inhibitor with a fluorescent-tag 35, that provided evidence of the binding of our inhibitors with CLDN1 receptors. In future, we are working to take some of our new leads to in vivo studies and also a biotin-tagged CLDN1 inhibitor development is under progress.

References: 1. American Cancer Society. Cancer Facts & Figures 2024. Atlanta: ACS; 2024.
2. Fatima, I., Uppada, J. P., Chhonker, Y. S., Gowrikumar, S., Barman, S., Roy, S., ... & Dhawan, P. (2023). Identification and characterization of a first-generation inhibitor of claudin-1 in colon cancer progression and metastasis. Biomedicine & Pharmacotherapy, 159, 114255.
3. Primeaux, M., Liu, X., Gowrikumar, S., Fatima, I., Fisher, K. W., Bastola, D., ... & Dhawan, P. (2023). Claudin-1 interacts with EPHA2 to promote cancer stemness and chemoresistance in colorectal cancer. Cancer Letters, 579, 216479.
4. Tabana, Y., Babu, D., Fahlman, R., Siraki, A. G., & Barakat, K. (2023). Target identification of small molecules: an overview of the current applications in drug discovery. BMC biotechnology, 23(1), 44.
5. Qiao, J. X., Wang, T. C., Hu, C., Li, J., Wexler, R. R., & Lam, P. Y. (2011). Transformation of anionically activated trifluoromethyl groups to heterocycles under mild aqueous conditions. Organic Letters, 13(7), 1804-1807.
6. Mashinson, V., Webster, T. M., Vadukoot, A. K., Tolentino, K. T., Simeon, P., Fatima, I., ... & Hopkins, C. R. (2023). Discovery, synthesis and biological evaluation of a series of N-(phenylcarbamothioyl)-2-napthamides as inhibitors of Claudin-1. Bioorganic & Medicinal Chemistry, 92, 117416.
7. Bhat, A. A., Syed, N., Therachiyil, L., Nisar, S., Hashem, S., Macha, M. A., ... & Haris, M. (2020). Claudin-1, a double-edged sword in cancer. International journal of molecular sciences, 21(2), 569.


Figure 1. a) Schematic model of role and regulation of CLDN1 in CRC progression & metastasis [7]. b) Synthetic schemes for the production of CLDN1 inhibitors 10-33. c) Synthetic scheme and methodology for the affinity based pull-down assay of fluorescent-tagged CLDN1 inhibitor 35.


Table 1. % Inhibition of live cells in different cell lines and in vitro pharmacokinetic parameters of selected compounds.


Figure 2. a) Dose response assay of selected compounds 21, 30, 32 and 33 showed significantly decreased cell viability in the CLDN1 overexpressed SW620 and SW480cldn1 cells. b) Visualization of CLDN1 inhibitor in selected cells under fluorescence microscope. The fluorescent-tagged inhibitor illuminated brightly when bound to the CLDN1 expressing SW620 and SW480cldn1 cells but was absent in the CLDN1 lacking HCT116 cells.