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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
Email: [email protected]
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
13-halophenyl alkyl berberines:Synthetic Methods
generally from good to very good yields
Uncommon aldehyde-enamine condensation1,2
2 Iwasa, K, et al., Planta Medica, 1997, 196
1 Cook, AG, Enamines Synthesis, Structure and reaction, 1988, pag 200-201
13-halophenyl alkyl berberines:Synthetic Methods
Commercially available aldehydes
Commercially available corresponding alcohols followed by oxidation
Commercially available corresponding acids/esters followed by reduction to corresponding alcohols
Homologation procedures from the above and others intermediates
Aldehyde Reagents
Synthesis of NAX 014
1. Pyridinium Chloro Cromate (PCC), CH2Cl2
2. Onodera Oxidation: DMSO, TEA, P2O5 in CH2Cl2
3. TEMPO/NaOCl oxidation: 1% TEMPO, NaOCl, NaHCO3, KBr, H2O and CH2Cl
Aldehyde Synthesis
Cl
O
Cl
OH
OX
Chromium is not recommended
It smells!!!
Few and non-toxic waste
Cl
O N
O
O
OCH3
OCH3
dihydroberberine
1) EtOH 80%, AcOH, 90°C
2) HI 1M
N
O
O
OCH3
OCH3
Cl
ClNAX 014
Enamine-Aldehyde condensation
TEMPO
Antiproliferative effectLinker with n = 2
NAX 014
NAX 043
NAX 048
NAX 051
NAX 103
NAX 1140.0
20.0
40.0
60.0
80.0
100.0
120.0
48h
0,4 uM2 uM10 uM50 uM
cell
viab
ility
(% o
f ctr
l)
NAX 014
NAX 043
NAX 048
NAX 051
NAX 103
NAX 1140.0
20.0
40.0
60.0
80.0
100.0
120.0
24h
0,4 uM2 uM10 uM50 uM
cell
viab
ility
(% o
f ctr
l)
N
O
O
OCH3
OCH3
I
(H2C)2
Cl
NAX 014
N
O
O
OCH3
OCH3
Cl
(H2C)2
NAX 043
N
O
O
OCH3
OCH3
Cl
(H2C)2
F
NAX 048
H
N
O
O
OCH3
OCH3
Cl
(H2C)2
Cl
NAX 051
N
O
O
OCH3
OCH3
Cl
(H2C)2
Cl
NAX 103
Cl
N
O
O
OCH3
OCH3
Cl
(H2C)2
Cl
NAX 114Cl
Breast Cancer (SKBR3) cells
Antiproliferative effectLinker with n = 3
N
O
O
OCH3
OCH3
Cl
(H2C)3
Br
NAX 072
N
O
O
OCH3
OCH3
Cl
(H2C)3
Cl
NAX 060
Cl
N
O
O
OCH3
OCH3
Cl
(H2C)3
Cl
NAX 105
N
O
O
OCH3
OCH3
Cl
(H2C)3
F
NAX 107
N
O
O
OCH3
OCH3
Cl
(H2C)3
Cl
NAX 111Cl
N
O
O
OCH3
OCH3
I
(H2C)2
Cl
NAX 014
NAX 014
NAX 072
NAX 060
NAX 105
NAX 107
NAX 1110.0
20.0
40.0
60.0
80.0
100.0
120.0
24h
0,4 uM2 uM10 uM50 uM
cell
viab
ility
(% o
f ctr
l)
NAX 014
NAX 072
NAX 060
NAX 105
NAX 107
NAX 1110.0
20.0
40.0
60.0
80.0
100.0
120.0
48h
0,4 uM2 uM10 uM50 uM
cell
viab
ility
(% o
f ctr
l)
Breast Cancer (SKBR3) cells
XO
OEtLiAlH4
THF X
OH
XO
PCC
DCMX=Cl,F
Cl Cl
O
Br
OEtOtBuOK
THF dry Cl Cl
COOEtO1) NaOH, EtOH
2) HCl 1M Cl Cl
O
Cl
O
Cl
+
Ph3PCHOMe Br
tBuOK, THF dry ClCl
OMe
HCl 2M, THF
65° C ClCl
O
X
OPh3P= CH2COOEt
no solvent X
COOEt
1) H2 ,Pd/C
X X2) LiAlH4,THF
PCC
DCM
O
OH
X=Cl,F
Aldehyde Intermadiates
Conclusions & Future goals
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.
