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

CHEMOKINE RECEPTORSCHEMOKINE RECEPTORS

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)

8

Blocking receptor functions

Cell

DrugCXCL12/HIV

Over expression of CXCR4 receptors

Normal cell Cancer cell

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

Restrict to one configuration

N N

N N

N N

N N

H

H

H

H

Only cis V

trans-II

NN X

N

N

R

R

X

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.

Copper(II) coordination

Cu

N

N N

Cl

N

Cu

N

N N

N

Cl

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

MODELING THE INTERACTIONMODELING THE INTERACTION

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

MACROCYCLE SYNTHESISMACROCYCLE SYNTHESIS

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%).

Bisaminal precursorsN N

NN

H

H

N N

NN

Br- N N

NNBr-

N N

NN

N N

NN

NaBH4

H

H

H

H

N N

NN

Br- N N

NNBr-

H

H

H

H

MeI

COMPLEX CHARACTERIZATIONCOMPLEX CHARACTERIZATION

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

Cu

N

N N

Cl

N

Cu

N

N N

N

Cl

Cu

N

N N

Asp

N

Cu

N

N N

N

Asp

shorter /stronger

longer /weaker

BINDING TO THE PROTEINBINDING TO THE PROTEIN

N N

HNN

NH N

NN

SB

EQUATORIAL

N N

NN

N N

NN

CB

AXIAL

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)

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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

Summary of mAb 12G5 binding to CXCR4 in the presence of bound antagonists.

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.

BLOCKING SIGNALING PROCESSESBLOCKING SIGNALING PROCESSES

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

ANTI-CANCER ACTIVITYANTI-CANCER ACTIVITY

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

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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

Control

Drug/ no CXCL12

CXCL12

Drug + CXCL12

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.

CONCLUSIONSCONCLUSIONS

• 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)

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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

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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

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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

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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

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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)