Synthesis and antiproliferative effect of novel 13-halophenyl alkyl berberines in HER-2 + breast cancer cells Gaetano Fiorillo c , Franco Buzzetti, c Cristina Geroni, a Paolo Lombardi, a,c Elisa Pierpaoli, b Carmen Plasencia, a,d Mauro Provinciali, b Carmela Salvatore, a Tanji Monir Syeda c a Aesis Therapeutics, Incubatore di Impresa JCube, via della Barchetta 1, 60035 Jesi, Ancona, Italy b Centro Tecnologie Avanzate dell'Invecchiamento, INRCA-IRCCS, Via Birarelli 8, 60121 Ancona, Italy c Naxospharma srl, via G. Di Vittorio 70, 20026 Novate Milanese, Milano, Italy d Aromics SL, Edif Hèlix,c/Baldiri Reixac, 15-21, 08028 Barcelona, Spain Email: staff@naxospharma.eu
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Synthesis and antiproliferative effect of novel 13-halophenyl alkyl berberines in HER-2+
breast cancer cellsGaetano Fiorilloc, Franco Buzzetti,c Cristina Geroni,a Paolo Lombardi,a,c Elisa
Pierpaoli,b Carmen Plasencia,a,d Mauro Provinciali,b Carmela Salvatore,a Tanjia Monir Syedac aAesis Therapeutics, Incubatore di Impresa JCube, via della Barchetta 1, 60035 Jesi,
Ancona, ItalybCentro Tecnologie Avanzate dell'Invecchiamento, INRCA-IRCCS, Via Birarelli 8, 60121 Ancona, Italy cNaxospharma srl, via G. Di Vittorio 70, 20026 Novate Milanese, Milano, ItalydAromics SL, Edif Hèlix,c/Baldiri Reixac, 15-21, 08028 Barcelona, Spain
HER2+ Breast Cancer (Human Epidermal growth factor Receptor 2 positive)
HER2-targeting gold standard drugs show modest efficacy as single agent and substantial toxicity in
combination therapy
Represents 20–30% of invasive BC associated with more aggressive disease progression and a poorer prognosis
New agents exhibiting a mechanism of action different in respect to current therapies might offer a new
option for treating HERB2+ BC patients
BerberineBackground History
Bitter-tasting isoquinoline quaternary alkaloid extracted from plants of the genus Berberis, Coptis
and others.
In use in the Ayurvedic and Chinese medicines since hundreds of years.
It shows diverse pharmacological activities: Anti-microbial/parasitic, Anti-diarrheal, anti-inflammatory, Anti-arryhthmic, Cholesterol-lowering Anticancer
N
O
O
OCH3
OCH3
Cl
Berberine chloride
BerberineAnticancer Properties
The precise molecular basis of its many biological activities are still debated
Modulation of protein expression by interaction with nucleic acids is postulated
The interactions between berberine and nucleic acids, reported since 1962, could lead to its anticancer effect
Mazzini, S. et al, Bioorg Med Chem, 2003, 505–514(NMR Studies)
Ferraroni, M. et al, Chem. Commun. 2011, 4917-4919
(RX studies)
intercalation
minor groove binding
BerberineDNA Interaction Mechanism
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
1 Waters ML, Curr Opin Chem Biol. 2002, 6, 736
Aromatic interactions are ubiquitous in nature, and their geometry is relevant for the molecular recognition in
biological systems1
(Hetero)aromatic groups pending from a suitable position of the parent alkaloid skeleton
linkers of variable length and functionalitygeometric propensity for additional stacking-type, non-covalent aromatic interactions
Chemical Programme
Berberine could be a useful new therapeutic agent in the treatment of HER2-overexpressing BC
Berberine suppresses the growth of HER2+ BC cells and promotes apoptosis by down-regulating the HER2/PI3K/Akt signaling pathway
Berberine and Breast Cancer
(BC)
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)
Method - Alamar Blue assay. The number of viable cells after treatment is expressed as a % of the vehicle treated control
E. Pierpaoli, P. Lombardi, et al, Biofactors, 39, 2013, 672-679
Previous Findings
HER2 + human BC cells (SK-BR-3)
(SK-BR-3)
Lapa
tinib
+Tr
astu
zum
ab
Treatment: NAX012 and NAX014 and berberine (BBR) 50µM for 24h
In a transgenic mouse model which spontaneously develops HER2-positive mammary tumors, repeated i.p. or oral injections of a safety dose (2.5 mg/kg) of NAX014 delayed the development of tumors, reducing both the number and size of tumor masses1.
1E. Pierpaoli, E. Damiani, F. Orlando, G. Lucarini, B. Bartozzi, P. Lombardi, C. Salvatore, C. Geroni, A. Donati, M. Provinciali, Carcinogenesis, 2015, manuscript in press
NAX 014: First Lead Compound
Unique ability to reduce cellular HER2 expression via a postulated novel mechanism
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)
from low to moderate yields - berberine and tetrahydroberberine from disproportionation of enamine as major by-products
An extra chlorine substitution and a longer linker seems to improve the antiproliferative effect in cancer cells.
Presently, NAX060 is a second lead compound under further investigation. Results will be disclosed due course.
N
O
O
OCH3
OCH3
Cl
(H2C)3
Cl
NAX 060
Cl
Know
n c
ompo
und
Kim
et a
l. US
6,00
8,35
6
All the analogues bound DNA noncooperatively in Contrast to the cooperative binding of berberine.
Binding was dominated by nonelectrostatic forces (at least 75% contribution to binding free energy).
Intercalative binding with strong stabilization of the DNA helix, and weakening of the base stacking with moderate conformational changes within the B-form
Unfavourable nature of the effect of the phenyl group in proximity (in n =1), more favourable as the alkyl chain length increased, driven largely by entropy contributions in the case of n = 3−6.