Amino Acids (20 different ones) Proteins = long chains of amino acidsStructure: collagen, keratinEnzymes: perform reactionsReceptors: 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

M

N

N O

O

N

N

CH3

CH3

C CH3

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

NH

NH

HN

N

HN

HN

NH

N

AMD3100 (Ligand 1)

CXCR4 Receptor

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 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

NH

NH

HN

HN

N

N

HN

HN

NH

N

N

HN

CyclamAll

Side-BridgedTrans-II

Cross-BridgedCis-V

NH

NH

HN

N

HN

HN

NH

N

L1

“AMD3100”

N

N

N

N

N

N

N

N

H3CCH3

L3

• 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

• The bis-linked complexes are highly efficient antagonists, while single-macrocycle analogues are much less effective. 

• 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

What we know…

My Assigned Project…

The Objectives

Reaction Step 1: Linking Macrocycles

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

13C NMR

Characterization

Reaction Plan Diverges:Methylation and Forming the Side-Bridged Variations

Methylated Tris-Cyclen Tetracycle Salt

1H NMR

Methylated Tris-Cyclen Tetracycle SaltElemental 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

Characterization

M2+

M+

Tris-Side-Bridged CyclamsElemental 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

Characterization

Synthesis of Tris Cross-Bridged Cyclam & Cyclen

1. 15 eq NaBH, 95% EtOH sol2. 5 days, N2 room temp3. H20, HCl, KOH, CH2Cl2extractions

Tris Cross-Bridged Cyclam and Cyclen

M3+

M2+

M+

Tris-Cross-Bridged Cyclams

Electrospray Mass Spectrum

Characterization

Typical Metal Complex: [Ni3(tris-CB-Cyclens)(OAc)3](PF6)3 ∙ 6H2OElemental 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

Final Reaction: Metal Complexations[M3(trisligand)(Oac)3](PF6)3

M= Co2+, Ni2+, Cu2+, Zn2+

Characterization

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.

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.

Conclusions:

Results: