# Novel Active Homo-Aza (Lactam) Steroidal Antimetabolites for the Treatment of Human Pancreatic and Colorectal Cancer

## TL;DR

Researchers developed new steroidal drugs that show promise in treating pancreatic and colorectal cancers by targeting specific enzymes and inducing cell death.

## Contribution

The study introduces novel lactam steroidal antimetabolites with dual enzyme inhibition and strong preclinical efficacy.

## Key findings

- CS23 showed potent antiproliferative activity and induced apoptosis in cancer cells.
- CS23 exhibited high binding affinity for thymidylate synthase and dihydrofolate reductase.
- Strong correlations were found between docking scores and biological activity of the compounds.

## Abstract

Background: Colorectal and pancreatic cancers remain therapeutically challenging, with limitations in efficacy and limitations due to toxicity from conventional antimetabolites such as 5-fluorouracil (5-FU), methotrexate (MTX), and gemcitabine (GEM). Steroidal conjugation offers an approach to enhance selectivity and toxicokinetics. Methods: Five novel hybrid homo-aza (lactam) steroidal antimetabolites (GE23, CS18, CS23, KA44, MV16) were synthesized and tested against three pancreatic and four colorectal carcinoma cell lines with distinct molecular characteristics. Antiproliferative activity (MTT), apoptosis (Annexin V/PI), and cell cycle effects were assessed. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibition was examined via molecular docking, Western blot, and enzymatic assays. Correlations between docking binding scores (DBS) and biological data were analyzed, and effects were compared with reference drugs (5-FU, MTX, GEM). Results: CS23, CS18, and KA44 exhibited the most potent cytostatic activity (mean GI50 10–80 µM). CS23 also induced high cytocidal effects, strong apoptosis (40% at 72 h), and G1/S arrest. Moreover, docking predicted the high binding affinity of CS23 for both TS (−11.2 kcal/mol) and DHFR (−11.5 kcal/mol), which was validated by Western blot and enzymatic inhibition (IC50 ≈ 20 nM). Correlation analyses showed significant relationships between hybrid steroidal antimetabolites’ cytostatic efficacy and DBS for TS (r = −0.75) and DHFR (r = −0.76), and combined DBS values predicted growth inhibition (r = −0.81, p < 0.01). No simple, universal correlation with single mutations of KRAS, BRAF, PI3K, or TP53 was found. Conclusions: Lactam steroidal antimetabolite hybrids, particularly CS23, act as dual TS/DHFR inhibitors, inducing apoptosis and cell cycle arrest with improved selectivity. Their strong in silico–in vitro concordance provides a compelling preclinical rationale for further evaluation of steroidal antimetabolites as next-generation therapeutics for resistant gastrointestinal malignancies.