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RECENT ADVANCES IN ANTITUMOUR BERBERINE1
Gaetano Fiorillo, Tanjia Monir Syeda, and Paolo Lombardi
1EuroTransBio transnational project BERTA (BERberine as antiTumour Agents)
Naxospharma srl, via G. Di Vittorio 70, 20026 Novate Milanese, Milano, Italy
Berberine: Background history
Extracted from plants of the genus Berberis, Coptis and others.
In use in the Ayurvedic and Chinese medicines1.
Anti-microbial,anti-parasitic Anti-diarrheal, anti-inflammatory Anti-arryhthmic Cholesterol-lowering Anticancer2
N
O
O
OCH3
OCH3
Cl
Berberine Chloride
1L. M. Guamán Ortiz, P.Lombardi, M. Tillhon, A. I. Scovassi, Biochemical Pharmacology, 2012, 84, 1260–12672L. M. Guamán Ortiz, P. Lombardi, M. Tillhon, A. I. Scovassi, Molecules, 2014, 19, 12349-12367
2011: 7 clinical trials 2016: 25 clinical trials
S. Mazzini, M. C. Bellucci, R. Mondelli,Bioorg Med Chem, 2003, 505–514 (NMR Studies)
M. Ferraroni, C. Bazzicalupi, A. R. Bilia, P. Gratteri, Chem.Comm. 2011, 4917-4919 (X-rays studies)
minor groove binding“ Interaction between nucleic acids and berberine sulfate “
Journal of Cellular Biology, 15, 1962, 589
…has been reported since decades…Minor groove binding or Intercalation ????
Berberine: Background history
Berberine represents an interesting and attractive natural lead compound
Chemical modifications might select more specific medical indications resulting in derivatives with better (or
different) biological effects compared to the parent berberine
Performing rational chemical modifications of berberine structure led to
a new class of derivatives with antitumour properties
Chemical Programme
Aromatic interactions are ubiquitous in nature,their geometry is relevant for the molecular recognition
in biological systems1
L = Linker with different functionalities
NOO
OCH3
OCH3
X
L
Ar(Ar)
13
1 Waters, M.L., Curr Opin Chem Biol. 2002, 6, 736
Chemical Programme
from very low to low yields - better with activated halides or iodides - berberine back from loss of acetone major by-product
Alkylation of enamine (7,8-dihydroberberine)
1)
from low to moderate yields - berberine and tetrahydroberberine from disproportionation of enamine as major by-products
2)
Chemical Programme Prior Art
generally from good to very good yields
Uncommon aldehyde-enamine condensation1,2
1 Cook, AG, Enamines Synthesis, Structure and reaction, 1988, 2002 Iwasa, K, et al., Planta Medica, 1997, 196
Chemical Programme Instant Art
N
O
O
OMe
OMeCOOH
X
1) CHO-COOH, EtOH 80%, AcOH, 90°C reflux
2) HX
Esters or Amides
1) Commercially available aldehydes2) Commercially available alcohols followed by oxidation
(e.g. : PCC or TEMPO)3) Homologation starting from the above
Commercially available
OH
TEMPO
or PCC
O
Ph3P
1) H2, Pd/C
2) TFA 60%
O
3-4
O
O
1-2O
O
1-2
3
OMe
2MeO
OMe
TsOH
MeOH
Chemical Programme Aldehydes Synthesis
N
O
Ph3P=CH-COOEtheat,
no solvent N
O
Et1) H2, Pd/C
2) LiAlH4N
OH
TEMPO
N
O
N
TEMPO
N
OH O
Commercially available
Commercially available
Commercially availables
Cl Cl
O
Br
OEtOtBuOK
THF dry Cl Cl
COOEtO1) NaOH, EtOH2) HCl 1M Cl Cl
O+
Chemical Programme Aldehydes Synthesis
N
O
O
OCH3
OCH3
Cl
(H2C)n
n = 1 NAX 044n = 2 NAX 043n = 3 NAX 039n = 4 NAX 042n = 5 NAX 056n = 6 NAX 057
Berberine
NAX 044
NAX 043
NAX 039
NAX 042
NAX 056
NAX 057
0
2
4
6
8
10
12
1.770.35
2.11
11.01
7.6 7.586.8
Interaction constants of NAX s 1
Ki x
10-
5 (M
-1)
Berberine
NAX 045
NAX 046
NAX 035
NAX 053
NAX 080
NAX 081
0
2
4
6
8
10
12
1.770.48 0.51
7.07
10.048.9
7.48
Interaction constants of NAXs2
Ki x
10-
5 (M
-1)
N
O
O
OCH3
OCH3
Cl
(H2C)n
n = 0 NAX 045n = 1 NAX 046n = 2 NAX 035 n = 3 NAX 053n = 4 NAX 080n = 5 NAX 081
1D. Bhowmik, M. Hossain, F. Buzzetti, R. D’Auria, P. Lombardi, G.S.Kumar, J. Phys. Chem. B, 2012, 116, 2314−24.2D. Bhowmik, F. Buzzetti, G. Fiorillo, F. Orzi, T. Syeda Monir, P. Lombardi, G.S. Kumar, Med. Chem. Comm., 2014, 5, 226-31.
n = 3
n = 4
Monophenyl derivatives
Diphenyl derivatives
DNA interaction of berberine derivatives
In collaboration with
N
O
O
OCH3
OCH3
Cl
(H2C)n
Nn = 1 NAX 071n = 2 NAX 120n = 3 NAX 075n = 4 NAX 077n = 5 NAX 079
S. Chatterjee, S. Mallick, F. Buzzetti, G. Fiorillo, T. M. Syeda, P. Lombardi, K. Das Saha, G. S. Kumar, RCS Adv., 2015, 5, 90632
n = 5
Berberine
NAX 071
NAX 120
NAX 075
NAX 077
NAX 0790
2
4
6
8
10
12
Interaction constants of NAXs
Ki x
10-
5 (M
-1)
Pyridylalkyl derivatives
DNA interaction of berberine derivatives
In collaboration with
Binding to human telomeric G quadruplex
1P. Gratteri, M. Ferraroni, C. Bazzicalupi, F. Papi, G. Fiorillo, L. M. Guamán Ortiz, A. Nocentini, A. I. Scovassi, P. LombardiChemistry: An Asian Journal, 2016, 11, 1107-15
2D. Bhowmik, G. Fiorillo, P. Lombardi, G. S. Kumar, Journal of Molecular Recognition, 2015, 28, 722–730.
NAX 053 – d[TAG3(T2AG3)T]adduct crystal structure
NO
O
OCH3
OCH3
Cl
(H2C)3
NAX 053
DNA interaction of berberine derivatives
Table 1. Binding parameters for the association of berberine analogues with the quadruplex DNA dAGGG(TTAGGG)3
Adsorbance Fluorescence
10-5 Ki/ (M-1) n 10-5 Ki /(M-1) n
Berberine 8,51 0,97 8,66 1,02
NAX 012 10,9 1,10 12,6 1,01
NO
O
OCH3
OCH3
Cl
NAX 012
In collaboration with1
In collaboration with2
In vitro StudiesAntiproliferative effect in pleural mesothelioma MSTO-211H cells
R=4-OMe, NAX38R=3-,4-,5-(OMe)3, NAX54
In collaboration with
N
O
O
OCH3
OCH3
I
NAX 012
N
O
O
OCH3
OCH3
I
NAX 013H3CO
N
O
O
OCH3
OCH3
I
NAX 014Cl
N
O
O
OCH3
OCH3
Cl
NAX 035
NAX 012 NAX 013 NAX 014 NAX 035 Berberine
24 h 94.2±1.2 >100 52.3±3.2 >100 91.8±2.8
48 h 46.6±2.5 >100 30.7±2.1 >100 58.4±1.9
72 h 31.9±2.9 >100 26.5±6.7 48.6±6.7 36.0±1.8
Antiproliferative effect (IC50 mM)
Pierpaoli, E.; Arcamone, A.G.; Buzzetti, F.; Lombardi, P.; Salvatore, C.; Provinciali, M.Biofactors, 39, 2013, 672-679
Antiproliferative effects on HER2 + human Breast Cancer (BC) cells (SK-BR-3)
(SK-BR-3)
In vitro StudiesIn collaboration with
Time-course in mesothelioma MSTO-211H cells at the IC50 dose at 72 h
NAX038
Berberine NAX014 NAX012
NAX035
Effects of NAX compounds on TS protein expression levels
HER2
p-HER2
β-actin
Ctrl
Lap
atin
ib +
Tras
tuzu
mab
NO
O
OCH3
OCH3
I
Cl
NO
O
OCH3
OCH3
Cl
NAX 035 NAX 014
Effects of NAX compounds on HER2/neu expression and phosphorylation
Thymidylate Synthase (TS) is present as an enzymatically active dimeric form in equilibrium with a catalytically inactive monomeric form.
TS monomer is in equilibrium with a TS-mRNA complex, by binding to its own mRNA.
The cellular levels (and activity) of TS depends on an autoregulatory loop which allows TS to control its own translation from its own mRNA.
NAX035
Conclusions
NAX035 suppresses nascent TS protein synthesis instead of targeting TS catalytic activity as currently used TS inhibitor drugs do
Conclusions
Our technogical approach targets post-transcriptional control mechanisms. The selective targeting of transcript will be achieved by new low
molecular weight ligands structurally related to berberine.
Unique ability to reduce cellular HER2 expression via a postulated novel mechanism, in contrast to currently used drugs for the treatment of
HER2+ BC which target either the protein itself or its signalling pathway