{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,25]],"date-time":"2026-02-25T06:13:47Z","timestamp":1772000027644,"version":"3.50.1"},"reference-count":47,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,2,17]],"date-time":"2026-02-17T00:00:00Z","timestamp":1771286400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pharmaceuticals"],"abstract":"<jats:p>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\u201380 \u00b5M). 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 (\u221211.2 kcal\/mol) and DHFR (\u221211.5 kcal\/mol), which was validated by Western blot and enzymatic inhibition (IC50 \u2248 20 nM). Correlation analyses showed significant relationships between hybrid steroidal antimetabolites\u2019 cytostatic efficacy and DBS for TS (r = \u22120.75) and DHFR (r = \u22120.76), and combined DBS values predicted growth inhibition (r = \u22120.81, p &lt; 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\u2013in vitro concordance provides a compelling preclinical rationale for further evaluation of steroidal antimetabolites as next-generation therapeutics for resistant gastrointestinal malignancies.<\/jats:p>","DOI":"10.3390\/ph19020331","type":"journal-article","created":{"date-parts":[[2026,2,18]],"date-time":"2026-02-18T07:50:30Z","timestamp":1771401030000},"page":"331","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Novel Active Homo-Aza (Lactam) Steroidal Antimetabolites for the Treatment of Human Pancreatic and Colorectal Cancer"],"prefix":"10.3390","volume":"19","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4347-413X","authenticated-orcid":false,"given":"Konstantinos E.","family":"Alifieris","sequence":"first","affiliation":[{"name":"Laboratory of Pharmacology, Faculty of Medicine, National and Kapodistrian University of Athens, Mikras Asias 75, Goudi, 11527 Athens, Greece"},{"name":"Recanati-Miller Transplantation Institute, Icahn School of Medicine at Mount Sinai, Manhattan, New York, NY 10029, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7680-7685","authenticated-orcid":false,"given":"Panagiotis","family":"Dalezis","sequence":"additional","affiliation":[{"name":"Laboratory of Pharmacology, Faculty of Medicine, National and Kapodistrian University of Athens, Mikras Asias 75, Goudi, 11527 Athens, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9281-7947","authenticated-orcid":false,"given":"Sofia","family":"Sagredou","sequence":"additional","affiliation":[{"name":"Laboratory of Pharmacology, Faculty of Medicine, National and Kapodistrian University of Athens, Mikras Asias 75, Goudi, 11527 Athens, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ioanna A.","family":"Anastasiou","sequence":"additional","affiliation":[{"name":"Laboratory of Pharmacology, Faculty of Medicine, National and Kapodistrian University of Athens, Mikras Asias 75, Goudi, 11527 Athens, Greece"},{"name":"Diabetes Center, First Department of Propaedeutic Internal Medicine, Medical School, National and Kapodistrian University of Athens, Laiko General Hospital, Goudi, 11527 Athens, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maria","family":"Deligiorgi","sequence":"additional","affiliation":[{"name":"Laboratory of Pharmacology, Faculty of Medicine, National and Kapodistrian University of Athens, Mikras Asias 75, Goudi, 11527 Athens, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4102-9638","authenticated-orcid":false,"given":"Christos","family":"Siokatas","sequence":"additional","affiliation":[{"name":"Laboratory of Organic Chemistry, Department of Chemistry, Faculty of Science, Aristotle University of Thessaloniki, University Campus, 54124 Thessaloniki, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nikolaos","family":"Spanakis","sequence":"additional","affiliation":[{"name":"Department of Microbiology, School of Medicine, National and Kapodistrian University of Athens (NKUA), Goudi, 15772 Athens, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Konstantinos","family":"Almpanakis","sequence":"additional","affiliation":[{"name":"Laboratory of Organic Chemistry, Department of Chemistry, Faculty of Science, Aristotle University of Thessaloniki, University Campus, 54124 Thessaloniki, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maria","family":"Voura","sequence":"additional","affiliation":[{"name":"Laboratory of Organic Chemistry, Department of Chemistry, Faculty of Science, Aristotle University of Thessaloniki, University Campus, 54124 Thessaloniki, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-5124-6727","authenticated-orcid":false,"given":"Kyriakos","family":"Orfanakos","sequence":"additional","affiliation":[{"name":"Laboratory of Pharmacology, Faculty of Medicine, National and Kapodistrian University of Athens, Mikras Asias 75, Goudi, 11527 Athens, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1450-3552","authenticated-orcid":false,"given":"Mihalis","family":"Panayiotidis","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Mississippi State University, Starkville, MS 39762, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6128-8277","authenticated-orcid":false,"given":"Vasiliki","family":"Sarli","sequence":"additional","affiliation":[{"name":"Laboratory of Organic Chemistry, Department of 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