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1
Design of Macrocyclic Chelators for Biomedical Applications
Dr. Tim Hubin
Department of Chemistry and Physics
Sept. 16, 2010 Oklahoma State University
Metal containing drugs
• Therapeutic and diagnostic– Cisplatin– Magnevist/ Dotarem– Zevalin (Indium, Yttrium)
N
N
NM
O
OOO
OOO
O
O
O
AntibodyDTPA
ZEVALIN
PtCl
ClH3N
H3N
N
NN
N
Gd
O
O
O
O
O
O
O
O
OH2
-
DOTA
Generally used chelators
N
N N
CO2H
HO2CHO2C
CO2H CO2H
NN
CO2H
CO2H
HO2C
HO2C
N N
N N
CO2HHO2C
HO2C CO2H
N
NN
N
HO2C CO2H
CO2HHO2C
N N
N N
H H
HH
N
NN
N
H H
HH
DTPA
EDTA
TETA
DOTA
cyclam
cyclen
CXCR4 chemokine receptor
• Important role in embryonic development:– Organogenesis (liver, heart)– Stem cell movement– Cerebellar neuron migration
(formation of brain)
• Seven transmembrane G-protein-coupled receptor
• 27% of amino acids are Asp, His or Tyr.
• Expressed on : » Leukocytes» T-lymphocytes» Endothelial cells» Neuronal cells
Khan, A.; Greenman, J.; Archibald, S. J. Curr. Med. Chem. 2007, 14, 2257.
CXCL12
• 67 residue highly basic protein
• Only known natural ligand (chemokine) for CXCR4
• Secreted by stromal, lung and liver cells, and lymph nodes
• Attracts leukocytes to sites of inflammation and lymphoid organs
A
B C
D E
Disease states
• Role in disease
– Tumor growth and metastasis– Human Immunodeficiency Virus– Stem cell mobilization– Autoimmune disorders (rheumatoid arthritis)
CXCR4 antagonists
• Peptide based
• Side chains protonated atphysiological pH
O
N
NH
NH2NH
NH2NH
NH
N
O
NH
NH2
O
N
NH
NH2NH
O
N
O
N
H
NH2
N
O
H
NH2
O
N
H
NH
NH2NH
N
OO NH2
CGP64222
N
NH
NH
O
NH
NH2 NH
NH
O CH3
KRH-1636
ArgArgCysTyrArgLys
Lys
ProTyr Arg Cys Arg COOH
NH2Nal
Cit
T140
Arg Arg ArgArg
ArgArgArg
ArgArg
Ac
COOH
RRWCYRKCYK
GY C Y R K C R CONH2
NH2
ALX40-4C
T22
11
Plerixafor/ AMD3100
N N
N N
NN
NN
H
H H
H
HH
AMD3100
The first bicyclams were discovered as impurities in a sample of cyclam. Amongst the most active anti-HIV agents in vitro.
Likely a prodrug; complexation of Zn2+ will occur in plasma
Anti-HIV clinical testing discontinued.
Stem cell mobilization
For example:Mol. Pharm., 1999, 55, 67.J. Med. Chem., 1995, 38, 366.Biochemistry, 2003, 42, 715.
Molecular shape
Bosnich, B.; Poon, C. K.; Tobe, M. L. Inorg. Chem.,1965, 4,1102
N N
N N
H
H
H
H
trans-I trans-II trans-III trans-IV trans-Vcis-V
N N
N N
NN
NN
H
H H
H
HH
AMD3100
N N
N N
NN
NNH H
N
NN
NN
NN
N
Me Me
Lewis, E. A.; Hubin, T. J.; Archibald, S. J. Patent WO2005121109, 2005.
CB chelator
N N
N N
N N
N N
H
H H H H
H
AMD3100
N N
N N
N N
N N
CuN
N
N
N
LCu
NN
N
N L
2+
SB chelator
N N
N N
N N
N NH
copper(II) complexes
H
Molecular modeling studies
• Docking studies?– No known X-ray structure of CXCR4– Develop a homology model to allow evaluation of
the full set of interactions in the binding pocket
• DFT calculations– Mono-macrocycle compounds at BP86/ TZP level– Bis-macrocycle compounds with mixed treatment
at QM/ MM level.G. McRobbie, G. C. Valks, C. J. Empson, A. Khan, J. D. Silversides, C. Pannecouque, E. De Clercq, S. G. Fiddy, A. J. Bridgeman, N. A. Young and S. J. Archibald, Dalton Trans., 2007, 5008.
Homology modeling
• Predict the structure using X-ray data from a related protein
• Align the sequences using conserved regions
• Five CXCR4 sequences were used
• Disulfide bridges of key importance
N N
NN
N N
NN
Br- N N
NNBr-
Br
Br
N N
HNN
NH N
NN
NaBH4
H
H
H
H(a)
(b)
H
H
Reagents: (a) acetonitrile, RT, 24 h (89%); (b) NaBH4, EtOH reflux, 1 h (65%).
Cu-O1 2.28(1) Å
Cu-O2 2.90(1) Å
Side bridged (SB) Cross bridged (CB)
Cu-O1 1.95(1) Å
Cu-O2 2.66(1) Å
Selecting the cell line• Use anti-CXCR4
antibodies to screen cell lines
• Two identified Jurkat and Molt-4 (T-cell leukemia)
• Four anti-CXCR4 antibodies used (variation in binding epitopes)
33
Binding by flow cytometry
CXCR4
Drug molecule
Receptor specific antibody
Fluorescent antibody
Key Name Parameter- control.001 FL1-H
+ Control 717.019 FL1-H
L2 717.010 FL1-H
L1 717.009 FL1-H
Competitive Binding Studies
35
IC50 and EC50 concentrations for CXCR4 antagonists in competition with mAb 44717 in Jurkat cells.
Residence time
G. McRobbie, A. Khan, G. Nicholson, L. Madden, J. Greenman C. Pannecouque, E. De Clercq, T. J. Hubin and S. J. Archibald, J. Am. Chem. Soc, 2009, 3416.
Ca2+ signaling
• Signal transduction by chemokine receptors leads to elevation of cytosolic free calcium.
• Signaling induced by CXCL12 was monitored in CXCR4 transfected U87 cells.
• IC50 values were in the range of ng/ml with no signal blocking observed for other chemokine receptors (CCR5)
Ca2+ Ion Signaling Assays
40
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
0 50 100 150
Control
1000 ng/ml
200 ng/ml
40 ng/ml
8 ng/ml
Time (sec)
Flu
ore
sc
en
ce
Ch
an
ge
(c
ou
nts
)
Ca-signaling data for AMD3100 CXCR4 experiment by collaborator Schols.
CALCIUM SIGNALING RESULTS
Compound Calcium signalingAv IC50(nM)
Cu2(ClO4)4(SB) 47.48Cu2Cl2(PF6)4(CB) 4.64
AMD3100 18.67
Cu2AMD3100 56.08
CXCR4 and Cancer Cell Metastasiso CXCL12 is normally responsible for trafficking of lymphocytes
o CXCL12 is secreted by stromal, lung and liver cells, and lymph nodes
o The interaction at the cell membrane is through CXCR4, which is over-expressed in some cancers
o Potential mechanism of metastasis
Normal cell Cancer cell
44
Invasion assays• Cell invasion assays in response to a chemokine gradient.
• Initially used SJSA cells (osteosarcoma).
• Experiments run in presence and absence of antagonist.
ANTI-CANCER ACTIVITYANTI-CANCER ACTIVITY
Invasive CXCR4 mutants
0
50
100
150
200
250
300
350
0 12.5 37.5 75 100 5% FBS
CXCL12 (nM)
Cel
l n
um
ber
/fie
ld Delta34
Delta23
4C
6A
AA
Cancer Cell Invasion Assay
47
Invasion of SJSA cells in matrigel with CXCL12 (12.5 nM) and CXCR4 antagonists (20-200 nM). Cells were counted in five different fields (x40 obj) in duplicates. Mean of the values plotted. Asterisk represents significance (p < 0.01) from B. A = no CXCL12 and no antagonist; B = CXCL12 only; C = 20 nM Cu-Cross Bridged antagonist; D = 200 nM Cu-Cross Bridged antagonist; E = 20 nM AMD3100; F = 200 nM AMD3100.
• Strong and specific CXCR4 antagonism from a cross-bridged bicylam analogue
• Axial vs. equatorial coordination makes all the difference in copper(II) containing protein binding drugs.
• Promising early anti-metastatic properties in vitro. In vivo testing to follow.
Analogues Prepared and (Tested)
50
NN
N N
N N
NN
H3C CH3
1
NN
N HN
N N
NNH
7
NN
N N
N N
NN
H3C CH3
2
NN
N HN
N N
NNH
8
NN
N N
N N
NN
H3C CH3
3
NN
N HN
N N
NNH
9
NN
N N
N N
NN
H3C CH3
4
NN
N HN
N N
NNH
10
NN
N N
N N
NN
H3C CH3
5
NN
N HN
N N
NNH
11
NN
N N
N N
NN
H3C CH3
6
NN
N HN
N N
NNH
12
Current Leads:Cu(Ligand 1)Zn(Ligand 7)
Recent Developments
51
1. Synthesis of Unsubstituted Cross-Bridged Linked Ligands
NN
N N
N N
NN
H3C CH3
All current cross-bridged analogues have fourtertiary nitrogens, due to synthetic method.
NN
NH N
N N
HNN
The presence of a secondary nitrogen may enhance CXCR4 binding through H-bonding.
NN
N N
NN
N N
1. p-dibromoxylene2. allyl bromide
NN
N N
3 eq tBuOKMW 45 min100 oCnormal workup
NaBH3(CN), CH3CNMW, closed vessel110 oC, 45 min
NN
N N
NN
N N
NN
NH N
NN
N HN
52
2. If two is good, three is better? C3 Symmetric Compounds
NN
N N
N N
NN
H3C CH3
All current linked analogues two macrocycles.
The presence of a third macrocycle may enhance CXCR4 binding through another AA.
NN
N N
Br
Br
Br
NN
N N
NN
N N
N
N
N
N3Br-
1. 15 eq. NaBH4 added to 0 oC MeOH solution2. 4h room temp stirring3. H2O, HCl, KOH, CH2Cl2 extraction
NN
N HN
NN
NH N
N
NH
N
N
dry CH3CN, N2, 5 days
60% yield
15 eq. CH3I, CH3CN, N2, 5 d
NN
N N
NN
N N
N
N
N
N
CH3
CH3
H3C
6I-
NN
N N
NN
N N
N
N
N
N
CH3
CH3
H3C
1. 15 eq. NaBH, 95% EtOH sol.2. 5 days, N2, room temp.3. H2O, HCl, KOH, CH2Cl2 extraction
53
3. Targeting CXCR4 is good, is targeting CXCR4/CCR5 better?
NN
N N
N N
NN
H3C CH3
None of our current compounds works on CCR5.
NNH
NH HN
N
Cl
Cl
A Dual CXCR4/CCR5 Antagonist incorporates macrocycles and a 2,6-dichloropyridine pharmacophore.
NN
N N
NN
N N
Br
THF
N
Br
N
Cl
Cl
1 eq., 5 days
Cl
Cl
EtOH
NaBH4
NNH
N N
N
Cl
Cl
NN
N N
NN
N N
Br
THF
N
Br
N
Cl
Cl
1 eq., 5 days
Cl
Cl
EtOH
NaBH4
NNH
N N
N
Cl
Cl
NN
N N
NN
N N
Br
THF
N
Br
N
Cl
Cl
1 eq., 5 days
Cl
Cl
CH3I, CH3CNNN
N N
N
Cl
Cl
EtOH, NaBH4
I
CH3
I
NN
N N
N
Cl
Cl
CH3
54
AcknowledgementsFunding
– OK-INBRE (NIH)– Research Corporation– SWOSU
COLLABORTORS
Dr. Steve Archibald (Hull)Abid Khan
Prof. Erik De Clercq (Leuven)Dr. Christophe Pannecouque(Leuven)Dr. Dominique Schols (Leuven)
Prof Tony Ng (KCL)Dr. Gilbert Fruhwirth (KCL)
Dr. Jana Barlic (OMRF)
Current research group: Courtney Garcia (Pre-Med)Desiray Cannon (Chemistry)Kevin Wilson (Chemistry)Past members:Robert Ullom—University of Kansas (Medicine)TauLyn Snell—Wichita State University (PA)Joe Blas—Creighton (Medicine)Danny Maples—OSU (Chemistry)Randall Maples—OSU (Chemistry)Dallas Matz—Arizona State University (Chemistry)Mike McClain—OU (Chemistry)Amy Cain—U. British Columbia (Chemistry)Neil Funwie—OU (Petroleum Engineering)Orry Birdsong—UT Galveston (Medicine)Kimberly Roewe—OSU (Chemistry)Kiet Ngyuen—SWOSU (Pharmacy)Katherine Coats (Chemistry)Josh Priddle—OSU (Medicine)