Amino Acids (20 different ones) Glycine Aspartate Metal Ions Fe, Mn, Cu, Zn Can bind to N and O atoms (Ligands) Often bind to 6 atoms at once CH3 N N O M N C CH3 O N CH3 Can bind to Amino Acids in Proteins Proteins = long chains of amino acids Structure: collagen, keratin Enzymes: perform reactions Receptors: signal changes to occur The CXCR4 Chemokine Receptor › 7-helix, trans-membrane receptor › Multiple roles in both normal and disease functions › Known co-receptor for HIV entry into immune cells › Expressed in multiple cancers and may play a role in metastasis AMD-3100 CXCR4 Receptor › Synthetic bis-macrocycle › Binds CXCR4 at Aspartate Amino Acids #171 and #262 › Has been in clinical trials as a “fusion inhibitor” of HIV › Currently in clinical trials against cancer and for stem cell mobilization as “Mozobil” or “Plerixafor” › Genzyme recently purchased AnorMED for $584 million NH N NH HN N HN NH HN AMD3100 (Ligand 1) InteractIon… Binding between AMD3100 & a CXCR4 Receptor Background • Importance of Metal Binding – – – – AMD-3100 is protonated at physiological pH, and can bind through H-bonding AMD-3100 strongly binds metal ions; Zinc(II) is 20 mM in blood plasma Zn(II) and Cu(II) AMD-3100 complexes are more effective at binding to CXCR4 NMR studies of AMD-3100 suggest that complex configuration is important • Modification of the macrocycle can select certain configurations NH HN N HN N NH HN N HN NH Cyclam All HN N Side-Bridged Cross-Bridged Trans-II Cis-V L3 N N N N N N N N CH3 H3C L1 NH N NH HN N HN NH HN “AMD3100” Research Plan • Diversity of Potential Complexes • Collaborators and Division of Labor – Tim Hubin (Weatherford, USA) • Synthesis of cross-bridged ligands and complexes • Development of cyclen and homocyclen chemistry – Steve Archibald (University of Hull, UK) • Synthesis of side-bridged ligands and complexes • X-ray crystallography • CXCR4 binding studies – Eric De Clercq (University of Leuven, Belgium) has agreed to test antiviral activity of compounds – Tony Ng (King’s College, London) is carrying out anti-cancer experiments What We knoW… • The bis-linked complexes are highly efficient antagonists, while single-macrocycle analogues are much less effective. My assIgned Project… The Objectives • Synthesize C3-symmetric tris-linked analogues of our most effective bis-tetraazamacrocycle metal complexes • Characterize their chemical and physical properties in preparation for determining if the added macrocycle enhances their antagonism of CXCR4 Reaction Step 1: Linking Macrocycles Characterization 13C NMR Tris-Cyclen Tetracycle Salt Elemental Analysis Calculated as C39H63N12Br3 ∙ 10.1 H2O: Calc: C 41.90, H 7.46, N 15.03; Found: C 42.27, H 7.21, N 14.69 Reaction Plan Diverges: Methylation and Forming the Side-Bridged Variations Methylated Tris-Cyclen Tetracycle Salt Characterization Methylated Tris-Cyclen Tetracycle Salt Elemental Analysis Calculated as C42H72N12I6 ∙ 3 H2O: Calc: C 32.33, H 5.04, N 10.77; Found: C 32.47, H 5.39, N 10.38 1H NMR Characterization Tris-Side-Bridged Cyclams Elemental Analysis Calculated as C45H84N12 ∙ 7.8 H2O: Calc: C 57.88, H 10.75, N 18.00; Found: C 10.35, H 5.39, N 17.60 M2+ Electrospray Mass Spectrum M+ Synthesis of Tris Cross-Bridged Cyclam & Cyclen 1. 15 eq NaBH, 95% EtOH sol 2. 5 days, N2 room temp 3. H20, HCl, KOH, CH2Cl2extractions Tris Cross-Bridged Cyclam and Cyclen Characterization Tris-Cross-Bridged Cyclams Electrospray M2+ M3+ Mass Spectrum M+ Final Reaction: Metal Complexations [M3(trisligand)(Oac)3](PF6)3 M= Co2+, Ni2+, Cu2+, Zn2+ Characterization Typical Metal Complex: [Ni3(tris-CB-Cyclens)(OAc)3](PF6)3 ∙ 6H2O Elemental Analysis Calculated as Ni3C48H87N12O6P3F18 ∙ 6 H2O: Calc: C 35.00, H 6.06, N 10.20; Found: C 34.66, H 5.61, N 10.20 Results: o The ligand syntheses of the side-bridged and cross-bridged C3-symmetric ligands proceeded similarly to the previously developed bis-ligand routes. o Complexation with the desired metal ions proceeded as expected. Characterization of the metal complexes is ongoing. Conclusions: o C3-symmetric tris-linked bridged tetraazamacrocycles are easily produced, using an appropriate linker and following synthetic methods adapted from the bis-linked analogues. o Metal ion complexation proceeds smoothly following known procedures. o The resulting complexes will inform our understanding of the requirements for producing even more efficient CXCR4 antagonists of this class.