2. Amino Acids (20 different ones) Proteins = long chains of amino acids Structure: collagen, keratin Enzymes: perform reactions Receptors: signal changes to occur Glycine Aspartate Metal Ions Fe, Mn, Cu, Zn Can bind to N and O atoms (Ligands) Often bind to 6 atoms at once Can bind to Amino Acids in Proteins

3.  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 › 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 AMD3100 (Ligand 1) CXCR4 Receptor

4. Interaction… Binding between AMD3100 & a CXCR4 Receptor

5. 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  M 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 Cyclam All Side-Bridged Trans-II Cross-Bridged Cis-V

6. L1L1 “AMD3100” L3L3

7. 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 Research Plan

8. The bis-linked complexes are highly efficient antagonists, while single-macrocycle analogues are much less effective. Synthesize C 3 -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 What we know… My Assigned Project… The Objectives

9. Reaction Step 1: Linking Macrocycles

10.  Tris-Cyclen Tetracycle Salt  Elemental Analysis Calculated as C 39 H 63 N 12 Br 3 ∙ 10.1 H 2 O:  Calc: C 41.90, H 7.46, N 15.03; Found: C 42.27, H 7.21, N 14.69 13 C NMR Characterization

11. Reaction Plan Diverges: Methylation and Forming the Side-Bridged Variations Methylated Tris-Cyclen Tetracycle Salt

12. 1 H NMR Methylated Tris-Cyclen Tetracycle Salt Elemental Analysis Calculated as C 42 H 72 N 12 I 6 ∙ 3 H 2 O: Calc: C 32.33, H 5.04, N 10.77; Found: C 32.47, H 5.39, N 10.38 Characterization

13. M 2+ M+M+ Tris-Side-Bridged Cyclams Elemental Analysis Calculated as C 45 H 84 N 12 ∙ 7.8 H 2 O: Calc: C 57.88, H 10.75, N 18.00; Found: C 10.35, H 5.39, N 17.60 Electrospray Mass Spectrum Characterization

14. Synthesis of Tris Cross-Bridged Cyclam & Cyclen 1. 15 eq NaBH, 95% EtOH sol 2. 5 days, N 2 room temp 3. H 2 0, HCl, KOH, CH 2 Cl 2 extractions Tris Cross-Bridged Cyclam and Cyclen

15. M 3+ M 2+ M+M+ Tris-Cross-Bridged Cyclams Electrospray Mass Spectrum Characterization

16. Typical Metal Complex: [Ni 3 (tris-CB-Cyclens)(OAc) 3 ](PF 6 ) 3 ∙ 6H 2 O Elemental Analysis Calculated as Ni 3 C 48 H 87 N 12 O 6 P 3 F 18 ∙ 6 H 2 O: Calc: C 35.00, H 6.06, N 10.20; Found: C 34.66, H 5.61, N 10.20 Final Reaction: Metal Complexations [M 3 (trisligand)(Oac) 3 ](PF 6 ) 3 M= Co 2+, Ni 2+, Cu 2+, Zn 2+ Characterization

17. o The ligand syntheses of the side-bridged and cross-bridged C 3 -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. o C 3 -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. Conclusions: Results: