SYNTHESIS AND BIOLOGICAL EVALUATION OF RADIOLABELED CURCUMINS AND THEIR

RUTHENIUM-ARENE COMPLEXES

STUDENT: LUCA PALMIERI

TUTOR:

PROF. CLAUDIO PETTINARIDR. DOMENICO MARTINI

UNIVERSITY OF CAMERINOSCHOOL OF PHARMACY

MASTER’S DEGREEPHARMACEUTICAL CHEMISTRY AND TECHNOLOGY

Yellow-gold pigment

Obtained through extraction usually from Curcuma-Longa plant

Used as a spice and food colorant

HO OH

OO

O O

HO OH

O

O O

OH

HO OH

O

O

HO OH

HO

O

OH

Curcumin

Demethoxycurcumin

Bisdemethoxycurcumin

O O

O

OH

OH

OH

Chemically

Polyphenols family

Hydrophobic compound

Curcumin

CurcuminMolecular targets and biological activity

Anti-inflammatory(pancreatitis, arthritis,

IBD and gastritis)

Antioxidant

Antiproliferative

Tumoricidal

Well-known contraindications

(GERD, stomach upset, slow blood clotting)

Curcuminand its role in Alzheimer Disease (AD)

Neurofibrillary tangles

Amyloid-β aggregation

Senile plaquesBehavioral disorders

Cognitive deterioration

Progressive memory loss

CurcuminStructure-Activity relationship

R1 studies

Compounds lacking a 2° phenyl group

have a less inhibitory activity

R2 studies

The aromatic end groups require one or more polar

hydrogen bonding substitutions for an optimal

Aβ aggregation inhibition

R3 studies

Linker length

Compounds with an approximate linker length of 8-

16 Å are better inhibitors

Linker flexibility

Compounds with more than one or two sp3-hybridized carbons don’t properly react with Aβ plaques

(Reinke et al., Chem. Biol. Drug Des., 2007)

Curcumin and radioisotopeswhat if we could use it for imaging ?

Half-life

Within 6h and 4 weeks

Energy emitted

Suitable energy for medical use123I

Half-life

13.22 hours

Energy emitted

• Pure gamma rays emitter

• Positrons emitter

Aim of the work

Synthesis of Curcumin as a tin or borate precursor for the 123I labeling of Curcumin and the binding of the compound with β-amyloid plaques in

Alzheimer disease (AD) through in-vivo tests.

Synthesis of the 123I-Curcumin

+n-Butylamine

HCl

1) 2,4 pentanedione + B2O3 stirred in ethyl acetate at 80 °C for 30’.

2) Vanillin + (n-BuO)3B added to the mixture and left to stir at 80 °C for further 30’

3) Addiction of n-Butylamine at 100 °C for 1 h4) Acidification with HCl at 50 °C for more 30’5) Extraction with ethyl acetate and water and

anhydrification with Na2SO4

6) Purification through flash chromatography and recrystallization with ethanol

1) B2O3 + Precursor stirred in ethyl acetate at 80 °C2) Iodo-vanillin + (n-BuO)3B left to stir at 80 °C for

30’, then added to the first solution3) Mixture added with piperidine and stirred at

80°C for more 30’4) Acidification with HCl at 50 °C for further 30’.5) Extraction with ethyl acetate and water and

anhydrification with Na2SO4

6) Recrystallization with ethanol

1) Pd(dppf)Cl2 + bis(neopentylglicolato)diboron dissolved in potassium acetate, flushed in argon and added with dry DMSO. Solution left to stir for 15’

2) Addiction of Ligand 1 and continue to stir at 80 °C for more almost 4h

3) Solvent removed under reduced pressure

Pd(dppf)Cl2

bis(neopentylglicolato)diboron

KHF2

1) KHF2 + Ligand 3 in methanol2) Hand-agitation3) Wash with hexane and filtration through

gooch

1) Na123I + BF3K-Curcumin + Chloramine-T stirred at 60 °C for 30’.

2) Addiction of sodium thiosulfite3) Isolation of product via silica Sep-Pak® C18

cartridge

Na123I 123I-Curcumin

Na123I

Synthesis of the 123I-Curcumin

+n-Butylamine

HCl

1) B2O3 + Precursor stirred in ethyl acetate at 80 °C2) Bromo-vanillin + (n-BuO)3B left to stir at 80 °C

for 30’, then added to the first solution3) Mixture added with piperidine and stirred at

80°C for more 30’4) Acidification with HCl at 50 °C for further 30’.5) Extraction with ethyl acetate and water and

anhydrification with Na2SO4

6) Recrystallization with ethanol

Pd(Ph3P)4

Me3Sn-SnMe3

1) Na123I + Me3Sn-Curcumin+ HCl stirred at 60 °C for 30’.

2) Reaction initiated adding H2O2 and stirring for 10’

3) Addiction of NaHSO3 4) Extraction using ethyl acetate and passage

through an anhydrous Na2SO4 plug5) Removal of the solvent with a gentle N2 flow6) Purification by HPLC using a semipreparative

column

1) Hexamethylditin + Ligand 2 + 1,4-dioxane stirred at 100 °C for about 1h

2) Removal of the solvent under reduced pressure

3) Purification by column chromatography

Na123I 123I-Curcumin

Na123I

Starting product: Yield:

20%

75%

Summarizing

In-vivo test

Mouse 1 Mouse 2 Mouse 3 Mouse 415 min post injection 30 min post injection 60 min post injection 120 min post injection

(no CT shown) (no CT shown)

Ventral view of co-registered SPECT/CT images of 123I-Curcumin

The tracer was cleared within 15 min by the liver showing hepatobilliary clearance from the circulation and no sign of accumulation in deposit sites.

Metal complexes and coordination with curcumin

Side effects in normal tissues• Nephrotoxicity• Neurotoxicity• Ototoxicity• Nausea and vomiting• Acquired resistance during therapy

Ruthenium has:

Lower toxicity

Higher selectivity

Platinum-based complexes Ruthenium-based complexes

Aim of the work

The role of a complex between the iodo-curcumin and the Ruthenium p-cymene dimer will be researched as a possible antioxidant and antitumoral compound. Evaluating his chemical synthesis, characterization, and in-vitro

studies.

Synthesis of the Ru-complex 1

KOHMeOH

O OH

O

HO

O

OH

I

+

Ligand 1

RuCl

ClClRu

Cl

O O

O

HO

O

OH

I

Ru Cl

Ru-complex 1

Temp. Time Other conditions

1st attempt RT 24 h

2nd attempt 50 °C 4 h RT 24 h

3rd attempt RT 3 d

4th attempt 40-50 °C 5 h reflux

5th attempt 40-50 °C 24 h reflux+ KOH

6th attempt 40-50 °C 2 d reflux

RT 3 d

Purification of the Ru-complex 1

O O

O

HO

O

OH

I

Ru Cl

Ru-complex 1

RuCl

ClClRu

Cl

Synthesis of the Ru-complex 2

+ RuCl

ClClRu

Cl

O OH

O

HO

O

OH

BF3KLigand 2

O O

O

HO

O

OH

BF3K

Ru Cl

Ru-complex 2

KOHMeOH Temp. Time Other conditions

1st attempt RT 24 h

2nd attempt 50 °C 4 h

RT 24 h

3rd attempt RT 3 d

4th attempt 40-50 °C 5 h reflux

5th attempt 40-50 °C 24 h reflux + KOH

6th attempt 40-50 °C 2 d reflux

RT 3 d

7th attempt RT 24 h + AgCF3SO3

Bio-evaluation of Ru-complex 1MTT cytotoxic assay

(in-vitro test)

Vehicle 800 µg/mL 400 µg/mL 200 µg/mL 100 µg/mL 50 µg/mL 25 µg/mL 12,5 µg/mL 6,25 µg/mL

0

20

40

60

80

100

120

72 h incubation

MDA-MBHCT116

Concentration

Abs 5

40 n

m

The compound wasn’t significantly active, especially compared with the cytotoxic activity of the sole curcumin which is cytotoxic just at concentrations of 10 µg/mL.

DPPH ABTS IC50

M

IC50

M

Ru-complex 1 373.3(9.5) 22.5(1.2)

Curcumin 32.6 (±5) 15.4(1.4)

Trolox 5.1(0.2) 69.0(0.5)

IC50 = The concentration of compound that affords a 50% reduction in the assay

The complex showed an important decrease of activity, compared to the natural curcumin.

Bio-evaluation of Ru-complex 1Antioxidant activity assay

(in-vitro test)

DPPH method

Based upon the discoloration of DPPH

radical in presence of an antioxidant compound,

and measuring it spectrophotometrically

ABTS method

Based upon the discoloration of the ABTS radical in presence of an antioxidant compound,

and measuring it spectrophotometrically

Conclusions• Five curcumin-like ligands were synthesized and chemically characterized

• Two of these ligands reacted with a radioisotope to give a new radio-ligand

• Unfortunately, the radio-ligand showed no sign of bio-accumulation in β-amyloid

deposit sites in mice

• A new Ru-complex was synthesized, chemically characterized, and tested in-vitro for its

antioxidant and cytotoxic activities

• The complex showed to possess a decreased cytotoxic and antioxidant effects

compared to curcumin

Future perspectives

It should be interesting to synthesize a complex containing radiolabeled curcumin and test

it, not just in-vitro but also, in-vivo in order to follow its biodistribution and bio-

accumulation.

Prof. Giulio Lupidi