Development in Potential Anti-HIV & Antimetastatic Drug: C 3 -Symmetric Tris -Linked Bridged Tetraazamacrocycles

1. Development in Potential Anti-HIV & Antimetastatic Drug: C3-Symmetric Tris-Linked Bridged Tetraazamacrocycles as Potential CXCR4 Antagonists Courtney D. Garcia1, B. N. Shockey1, B. Gridley2, S. J. Archibald2, T. J. Hubin2 1. Department of Chemistry, Southwestern Oklahoma State University, 100 Campus Drive, Weatherford, OK 73096 USA 2. Department of Chemistry, University of Hull, Cottingham Road, Hull, HU6 7RX, UK 3. Medical Research Laboratory, University of Hull, Cottingham Road, Hull, HU6 7RX, UK

2. Basic Biochemistry--Proteins 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. Background • 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 CXCR4 Receptor AMD3100 (Ligand 1)

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 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 Cyclam All Side-Bridged Trans-II Cross-Bridged Cis-V

6. L1 “AMD3100” Figure 3. The inhibition  of anti-CXCR4 antibody binding over time after exposure to 32nM of the drug. A population of 100,000 cells was isolated for each data point and analyzed by flow cell cytometry using a secondary fluorescein tagged IgG antibody (negative values are not shown). L3

7. 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

8. 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

9. Reaction Step 1: Linking Macrocycles

10. 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

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

12. 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

13. 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 Electrospray Mass Spectrum M2+ M+

14. 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

15. Characterization Tris-Cross-Bridged Cyclams Electrospray Mass Spectrum M2+ M+ M3+

16. Final Reaction: Metal Complexations [M3(trisligand)(Oac)3](PF6)3 M= Co2+, Ni2+, Cu2+, Zn2+ 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 Characterization

17. What we found… Results: • The ligand syntheses of the side-bridged and cross-bridged C3-symmetric ligands proceeded similarly to the previously developed bis-ligand routes. • Complexation with the desired metal ions proceeded as expected. Characterization of the metal complexes is ongoing. Conclusions: • C3-symmetric tris-linked bridged tetraazamacrocycles are easily produced, using an appropriate linker and following synthetic methods adapted from the bis-linked analogues. • Metal ion complexation proceeds smoothly following known procedures. • The resulting complexes will inform our understanding of the requirements for producing even more efficient CXCR4 antagonists of this class.