Carmefur Analogs – Dual-Activity Drug Repurposing, Scalable Synthesis & Membrane-Disruptive SAR

Introduction & Session Roadmap

• Topics addressed in the talk
  • Introduction & historical background of Carmefur
  • Analog design strategy & optimized synthesis workflow
  • GUV (Giant Unilamellar Vesicle) rupturing kinetics as a biophysical assay
  • Lipophilicity, micelle‐formation and critical micelle concentration (CMC)
  • Future experimental plans and formal acknowledgments

Background on Carmefur

• Definition
  • Carmefur (also written Carmifer/Carmofur in some older literature) is a pro-drug of the clinical chemotherapeutic agent 5-fluorouracil (5-FU).
• Dual therapeutic profile
  • Early indication: anticancer (esp. colorectal) activity discovered in $(1988)$.
  • Re-discovered during the COVID-$19$ pandemic as a potent antiviral against SARS-CoV-2 main protease (M tsubscript{pro}).
• Timeline of mechanistic discoveries
  • $(2013)$ – Covalent inhibition of acid ceramidase (AC) elucidated.
  • $(2020)$ – High-throughput repurposing screens identify M$_{pro}$ inhibition.

Mechanism of Action in Cancer

• Biological context
  • Ceramide = pro-apoptotic sphingolipid; accumulation triggers programmed cell death.
  • Acid ceramidase degrades ceramide → lowers intracellular ceramide levels → apoptosis avoided → tumor growth.
  • Overexpression stats: > $(40\%)$ of prostate tumors up-regulate AC.
• Carmefur’s molecular action
  • Covalent attachment to catalytic Cys$_{143}$ in AC.
  • Reaction releases active 5-FU moiety after thiocarbamate deactivation.
  • Restores ceramide signaling → re-initiates apoptosis → inhibits cell proliferation & tumor growth.

Mechanism of Action in SARS-CoV-2

• Target enzyme: SARS-CoV-2 main protease (M$_{pro}$) – essential for cleavage/maturation of viral polyproteins.
• Carmefur reacts analogously:
  • Covalent modification of catalytic Cys$_{145}$.
  • Same thiocarbamate release of 5-FU.
• Advantage: M$_{pro}$ sequence highly conserved across viral variants → broad antiviral potential.

Drug Repurposing Perspective

• General advantages
  • Shorter development timeline (see left chart): regulatory approval path compressed vs. de-novo NCEs.
  • Higher clinical success probability (see right chart).
• Historical success stories
  • Zidovudine: cancer → HIV.
  • Fingolimod: transplant rejection → multiple sclerosis.
  • Topiramate: epilepsy → obesity.
• COVID-$19$ urgency amplified repurposing strategies – large high-throughput screens led to Carmefur identification.

Synthesis Challenges & Optimization

• Literature route issues
  • Used toxic solvent pyridine.
  • Required $90\,^{\circ}\text{C}$, $72\,\text{h}$ reaction time.
  • Yield < $35\%$ – not scalable.
• Laboratory optimization (published Canadian J. Chem. $2023$)
  • Real-time monitoring with benchtop $^{19}\text{F}$ NMR.
  • Greener solvent, $90\,\text{min}$ total time, yield > $70\%$.
  • Strategy enabled rapid analog library construction.

First-Generation Analog Library Design

• Guiding rationale: vary urethane (NH–CO–O), carbamate (O–CO–NH) or amide (CO–NH) at C4 of 5-FU.
• Sub-libraries
  • Alkyl-chain length scans (C$<em>{5}$ → C$</em>{18}$).
  • Single-atom substitutions to modulate electrophilicity & metabolic stability.
  • Lipid-mimetic motifs (e.g.
  • Dodecylurethane (C$_{12}$)
  • Octadecylurethane (C$_{18}$))
  • Sterol & unsaturated tails: sterylamide vs. oleylamide.
  • Exotic constructs: bis-cyclohexyl, “two-headed” analog, cholesterol carbamate.

Synthetic Workflow (General)

• Starting material: $1\,\text{eq}$ 5-fluorouracil.
• Apparatus: two-neck RBF + Teflon stir bar under N$_{2}$ atmosphere.
• Solvent: N-methyl-2-pyrrolidone (NMP).
• Electrophile: chloroformate / isocyanate / acyl chloride depending on target.
• Base: triethylamine to scavenge HCl.
• Temperature: $60\,^{\circ}\text{C}$ in sand bath.
• Progress monitoring: $^{19}\text{F}$ NMR ppm shift.
• Purity confirmation: $^{1}\text{H}$ NMR; example peaks
  • Most deshielded NH (adjacent to two EWG) appears downfield.
  • Terminal alkyl triple peak at $0.8\,\text{ppm}$.

Stability Issues with Amide Series

• Observation: Oleylamide sample returned from high-field showed peak at $12\,\text{ppm}$ ⇒ decomposition to 5-FU + oleic acid.
• Controlled study with lauroylamide (C$_{12}$)
  • Solvent = CDCl$<em>{3}$: stable $\ge$ $99\,\text{h}$ even after H$</em>{2}$O spike.
  • Solvent = DMSO-d$_{6}$: $\approx$ $50\%$ decomposed at $t=0$; fully decomposed within $2\,\text{h}$ after water.
• Conclusion
  • DMSO + trace water triggers rapid hydrolysis.
  • Decision: amide sub-library excluded from bio-assays until stability can be engineered.

GUV (Giant Unilamellar Vesicle) Rupture Assay

• Why GUVs?
  • Cell-sized synthetic membranes replicate biophysical properties without biological complexity.
  • Cost-effective vs. live cells; enables direct visualization of membrane disruption.
• Experimental setup (Posi Lab)
  • Incubate GUVs with analogs at graded concentrations & time points.
  • Microscopy images before/after; quantify % ruptured.
  • Visual marker: patchy, non-circular morphology denotes lysis.
• Key findings (15-min incubation data)
  • Lipid-tail analogs vastly outperform parent Carmefur.
  • Drop-off in activity starts near $5\,\mu\text{M}$ yet persists down to pM/fM.
  • Hit compounds
  • Dodecylurethane: > $90\%$ rupture into \textit{µM} range; active to $100\,\text{aM}$.
  • Octadecylurethane: $60!–!90\%$ rupture even at $\text{fM}$ (≈ one ant / $10^{3}$ Olympic pools analogy).
  • Tail–oxygen swap (carbamate) lowers but retains high potency (≥ $90\%$ rupture down to pM).
• Hypotheses generated
  1. Optimal hydrophobic tail length for maximal rupture?
  2. Effect of single-atom (NH → O) replacement on membrane activity & dual-enzyme inhibition.

Critical Micelle Concentration (CMC) Determination

• Concept
  • Amphiphilic molecules self-assemble into micelles above a threshold (CMC);
    micelle formation may correlate with membrane-disruptive ability.
• Method
  • Measure GUV contact angle on slide vs. compound concentration.
  • Sharp inflection in angle plot ≡ CMC.
• Ongoing analysis aims to link CMC values with GUV rupture %.

Future Directions

• Complete SAR matrix
  • Systematically evaluate chain length (C$<em>{5}$–C$</em>{18}$) vs. rupture efficacy.
  • Compare urethane vs. carbamate vs. (stabilized) amide where possible.
• Correlate self-assembly (CMC) with potency.
• Transition to in-vitro cell models
  • MTT (metabolic viability) and LDH release (membrane leakage) assays in cancer cell lines.
  • Examine concordance between GUV lysis and cellular cytotoxicity.
• Expand dual-target profiling
  • Enzymatic IC$<em>{50}$ vs. AC and M$</em>{pro}$ to ascertain selectivity.

Acknowledgments & Resources

• Project members (current & alumni) – Carmefur Analog Team.
• Collaborators
  • Posi Lab (GUV & micelle studies).
  • Dr. Edward New (principal investigator).
  • Steve Lynch – high-field NMR support at Stanford.
• Funding & publication
  • Optimization work: Canadian J. Chem. $2023$.
  • Analog series: Springer Nature (QR codes in slides).
• Note: unpublished data not publicly shareable; see peer-reviewed papers for details.

Q&A Highlights

• Concern: Amide hydrolytic instability vs. biological (aqueous) milieu.
  • Response: Amides currently excluded from biological testing; urethane & carbamate forms exhibit stability.
• Why GUVs before live cells?
  • Rapid, inexpensive, mechanistic clarity; cells introduce confounding toxicities unrelated to membrane rupture.
• Availability of slides/data
  • Published pieces via two cited papers; in-progress data kept internal until publication.