During my experimental thesis I synthesized and characterized 5 ligands in which the main scaffold is similar to curcumin setting up the procedure to introduce various groups and atoms in position 9 of these curcumin-like ligands. Obtaining three new ligands, two of which reacted with 123I in order to give a radiolabeled curcumin. This radiolabeled curcumin was then administered in mice during in-vivo tests and its bioaccumulation was followed with PET device to check its binding with β-amyloid plaques.
Moreover I synthesized and characterized a Ruthenium p-cymene complex containing the iodo-curcumin. On this complex were then realized in-vitro cytotoxicity and antioxidant activity tests that showed how the presence of these functionalization on the aromatic moiety lead to a lower activity of the complex itself. It could anyway interesting to evaluate through the use of a PET device its in-vivo biodistribution and accumulation at a systemic level in order to confirm or not the loss of this activity.
Circulatory Shock, types and stages, compensatory mechanisms
Synthesis and biological evaluation of radiolabeled curcumins and their ruthenium-arene complexes
1. UNIVERSITY OF CAMERINO
SCHOOL OF PHARMACY
MASTER’S DEGREE
PHARMACEUTICAL CHEMISTRY AND TECHNOLOGY
SYNTHESIS AND BIOLOGICAL EVALUATION OF
RADIOLABELED CURCUMINS AND THEIR RUTHENIUMARENE COMPLEXES
STUDENT:
LUCA PALMIERI
TUTOR:
PROF. CLAUDIO PETTINARI
DR. DOMENICO MARTINI
3. Curcumin
Molecular targets and biological activity
Anti-inflammatory
(pancreatitis, arthritis, IB
D and gastritis)
Well-known
contraindications
(GERD, stomach upset, slow
blood clotting)
Antioxidant
Antiproliferative
Tumoricidal
4. Curcumin
and its role in Alzheimer Disease (AD)
Neurofibrillary tangles
Cognitive deterioration
Amyloid-β aggregation
Progressive memory loss
Senile plaques
Behavioral disorders
5. Curcumin
Structure-Activity relationship
R2 studies
The aromatic end groups
Linker length
require one or more polar
hydrogen bonding
Compounds with an approximate
substitutions for an Å are better
linker length of 8-16 optimal
Aβ aggregation inhibition
inhibitors
R3 studies
R1 studies
Compounds
Linker flexibility lacking
a 2° phenyl group
have than
Compounds with more a less one or
inhibitory activity
two sp3-hybridized carbons don’t
properly react with Aβ plaques
(Reinke et al., Chem. Biol. Drug Des., 2007)
6. Curcumin and radioisotopes
what if we could use it for imaging ?
Half-life
13.22 hours
Within 6h and 4 weeks
123I
Energy emitted
Suitable energy rays emitter
• Pure gamma for medical use
• Positrons emitter
7. 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.
8. Synthesis of the 123I-Curcumin
HCl
n-Butylamine
1)
2)
2)
3)
2)
3)
4)
4)
3)
5)
5)
6)
6)
Pd(dppf)Cl2 + bis(neopentylglicolato)diboron
B2O3 + Precursor + B2O stirred in ethyl
2,4 pentanedionestirred3in ethyl acetate at 80 °C
dissolved 80 °C for 30’.
Iodo-vanillin + (n-BuO) B left to stir at in
acetate atin potassium3acetate, flushed80 °C for
argon and added with first solution
30’, then added to the dry DMSO. Solution
Vanillin + (n-BuO)3B piperidinethe mixture and
left to stir for 15’
Mixture added with added to and stirred at
left to stir of Ligand 1 and continue to stir at
Addiction at 80 °C
80°C for more 30’ for further 30’
Addictionmore almost at 50 at 100further130’.
80 °C for of with HCl 4h
Acidification n-Butylamine °C for °C for h
Acidification withunder reduced pressure
Solvent removed HCl at 50 °C for more and
Extraction with ethyl acetate and water 30’
Extraction with with Na SO and water and
anhydrification ethyl acetate
2
4
anhydrification with Na2SO4
Recrystallization with ethanol
Purification through flash chromatography
and recrystallization with ethanol
+
Pd(dppf)Cl2
bis(neopentylglicolato)diboron
KHF2
9. 1)
2)
2)
3)
3)
Na123I Ligand 3 in methanol
KHF2 ++ BF3K-Curcumin + Chloramine-T
stirred at 60 °C
Hand-agitation for 30’.
Addiction of sodium thiosulfite through
Wash with hexane and filtration
Isolation
gooch of product via silica Sep-Pak® C18
cartridge
Na123I
Na123I
123I-Curcumin
10. Synthesis of the 123I-Curcumin
HCl
n-Butylamine
1)
2)
3)
4)
5)
6)
B2O3 + Precursor stirred in ethyl acetate at 80 °C
Bromo-vanillin + (n-BuO)3B left to stir at 80 °C
for 30’, then added to the first solution
Mixture added with piperidine and stirred at
80°C for more 30’
Acidification with HCl at 50 °C for further 30’.
Extraction with ethyl acetate and water and
anhydrification with Na2SO4
Recrystallization with ethanol
+
Pd(Ph3P)4
Me3Sn-SnMe3
11. 1)
2)
3)
4)
5)
6)
Hexamethylditin + Ligand 2 + 1,4-dioxane
Na123I + Me3Sn-Curcumin+ HCl stirred at 60
stirred at
°C for 30’.100 °C for about 1h
Removal initiated adding H2O and stirring
Reaction of the solvent under 2reduced
pressure
for 10’
Purification NaHSO3
Addiction ofby column chromatography
Extraction using ethyl acetate and passage
through an anhydrous Na2SO4 plug
Removal of the solvent with a gentle N2 flow
Purification by HPLC using a semipreparative
column
Na123I
Na123I
123I-Curcumin
Starting product:
Yield:
20%
75%
13. In-vivo test
Ventral view of co-registered SPECT/CT images of 123I-Curcumin
Mouse 1
Mouse 2
Mouse 3
Mouse 4
15 min post injection
30 min post injection
60 min post injection
(no CT shown)
120 min post injection
(no CT shown)
The tracer was cleared within 15 min by the liver showing hepatobilliary clearance from the
circulation and no sign of accumulation in deposit sites.
14. Metal complexes and coordination with
curcumin
Platinum-based complexes
•
•
•
•
•
Side effects in normal tissues
Nephrotoxicity
Neurotoxicity
Ototoxicity
Nausea and vomiting
Acquired resistance during therapy
Ruthenium-based complexes
Ruthenium has:
Lower toxicity
Higher selectivity
15. Aim of the work
The role of a complex between the iodo-curcumin and the Ruthenium pcymene dimer will be researched as a possible antioxidant and antitumoral
compound. Evaluating his chemical synthesis, characterization, and in-vitro
studies.
16. Synthesis of the Ru-complex 1
Temp.
Time
st attempt
1MeOH
RT
24 h
2nd attempt
50 °C
RT
4h
24 h
3rd attempt
RT
Other conditions
3d
KOH
4th attempt 40-50 °C
5h
reflux
5th attempt 40-50 °C
24 h
6th attempt 40-50 °C
2d
reflux
+ KOH
reflux
RT
3d
18. Synthesis of the Ru-complex 2
KOH
MeOH
Temp.
Time
1st attempt
RT
24 h
2nd attempt
50 °C
4h
RT
24 h
RT
3d
3rd attempt
Other conditions
4th attempt 40-50 °C
5h
reflux
5th attempt 40-50 °C
24 h
reflux + KOH
6th attempt 40-50 °C
2d
reflux
RT
7th attempt
3d
RT
24 h
+ AgCF3SO3
19. Bio-evaluation of Ru-complex 1
MTT cytotoxic assay
(in-vitro test)
72 h incubation
120
Abs 540 nm
100
80
60
40
20
MDA-MB
0
HCT116
Concentration
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.
20. Bio-evaluation of Ru-complex 1
Antioxidant activity assay
(in-vitro test)
DPPH method
DPPH
ABTS
ABTS method
Based upon the
discoloration of DPPH
radical in presence of an
antioxidant compound,
and measuring it
spectrophotometrically
IC50
IC50
M
M
Ru-complex 1
373.3( 9.5)
22.5( 1.2)
Based upon the
discoloration of the ABTS
radical in presence of an
antioxidant compound,
and measuring it
spectrophotometrically
Curcumin
32.6 (±5)
5.1( 0.2)
15.4( 1.4)
69.0( 0.5)
Trolox
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.
21. 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 bioaccumulation.