1) The document describes the synthesis and antiproliferative effects of novel berberine derivatives in HER2+ breast cancer cells. Berberine shows anticancer activity but the authors developed derivatives to improve properties. 2) Compounds NAX012, NAX013, NAX014, and NAX035 showed stronger antiproliferative effects than berberine against HER2+ breast cancer cells. NAX014 in particular had the most potent effects. 3) In a mouse model of HER2+ breast cancer, oral and injected administration of NAX014 delayed tumor onset and progression at well-tolerated doses, and also reduced lung metastases. NAX014 shows promise as an anticancer agent

1. SYNTHESIS AND ANTIPROLIFERATIVE EFFECT OF
NOVEL 13-HALOPHENYLALKYL BERBERINES IN
HER-2+
BREAST CANCER CELLS
Paolo Lombardi,a,c
Franco Buzzetti,c
Gaetano Fiorillo,c
Cristina Geroni,a
Elisa Pierpaoli,a,b
Carmen Plasencia,a,d
Mauro Provinciali,b
Carmela Salvatore,a
Tanjia Monir Syedac
a
Aesis Therapeutics, Incubatore di Impresa JCube, via della Barchetta 1, 60035 Jesi, Ancona
b
Centro Tecnologie Avanzate dell'Invecchiamento, INRCA-IRCCS, Via Birarelli 8,
60121 Ancona c
Naxospharma srl, via G. Di Vittorio 70, 20026 Novate Milanese, Milano
d
Aromics SL, Edif Hèlix,c/Baldiri Reixac, 15-21, 08028 Barcelona, Spain
Email: p.lombardi@naxospharma.eu

2. BerberineBerberine
Berberine
A definite medical potential has been established in a wide spectrum of clinical
applications in therapeutic areas such as hyperlipidemia, metabolic
syndrome, polycistic ovary syndrome, obesity, fatty liver disease,
coronary artery disease, where berberine has drawn most extensive
attention as come out of scientific and patent literature and ongoing
clinical trials (9, 2011  18, 2014)
Isoquinoline quaternary plant alkaloid used in the
Ayurvedic and Chinese medicines since hundreds of years
shows diverse pharmacological properties and activities:
anti-microbial/parasitic, anti-diarrheal, anti-inflammatory,
anti-arrythmic, cholesterol-lowering, and anti-tumour
The precise molecular basis of its many biological activities are
still debated.

3. BerberineBerberine
Modulation of protein expression by interaction with nucleic acids is postulated
Interactions between berberine and nucleic acids have been reported since 19621
However, is the alkaloid a minor groove binder.....
...or an intercalator?
1
Yamagishi H, Interaction between nuclei acid and berberine sulfate, 1962, J Cell Biol, 15,589

4. BerberineBerberine
Berberine represents an interesting and
attractive natural lead compound.
Rational chemical modifications might lead to more
specific and selective medical indications
Berberine

5. Chemistry programme
(Hetero)aromatic groups pending from a suitable position of the parent
alkaloid skeleton through linkers of variable length and functionality
possibly creating a geometric propensity for additional stacking-type,
non-covalent aromatic interactions (intramolecular and/or molecule-
cellular target).

6. X= Cl, I, Br
Since aromatic interactions are ubiquitous in nature, and their geometry
is relevant for the molecular recognition in biological systems1
that could result in better (or different) biological effects with respect to
the parent berberine
1
Waters ML, Curr Opin Chem Biol. 2002, 6, 736,
Chemistry programme
Patent US8188109B2

7. 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)
Chemistry programme
Prior art
from low to moderate yields -
berberine and tetrahydroberberine
from disproportionation of enamine
as major by-products

8. generally from good to very good yields
Uncommon aldehyde-enamine condensation1,2
Chemistry programme
Instant art
2
Iwasa, K, et al., Planta Medica, 1997, 1961
Cook, AG, Enamines Synthesis, Structure and reaction, 1988, pag 200-201

9. N
O
O
OCH3
OCH3
dihydroberberine
N
O
O
OCH3
OCH3
N
O
O
OCH3
OCH3
tetrahydroberberine berberine
Berberine and tetrahydroberberine are the major by-
products from disproportionation reaction of
dihydroberberine
Chemistry programme

10. Chemistry programme
Even with glyoxylic acid 1
1
Fiorillo, G et al, An uncommon aldehyde-enamine condensation. Synthesis and antiproliferative activity of new berberine-derived
(hetero)aryl amides. XXXIV Convegno Nazionale SCI, Divisione di Chimica Organica. Pavia (I), 10 September 2012.

11. Aldehyde sources
Chemistry programme
Commercially available aldehydes
Commercially available corresponding alcohols followed
by oxidation
Commercially available corresponding acids/esters
followed by reduction
Homologation procedures from the above and others
intermediates

12. DNA binding
Chemistry programme

13. Anticancer properties of berberine in several preclinical
studies have been published since many years
Derivatives possibly exhibiting ameliorated antitumour properties

14. Antiproliferative effects in human
malignant mesothelioma cell lines
N
O
O
O C H 3
O C H 3
C l
( H 2 C )
P h
P h
n
n = 0 N A X 4 5
n = 1 N A X 4 6
n = 2 N A X 3 5
n = 3 N A X 5 3

15. N
O
O
O C H 3
O C H 3
C l
C l
C l N A X 6 0
N
O
O
O C H 3
O C H 3
I
C l
N A X 1 4
N
O
O
O C H 3
O C H 3
I
O M e
N A X 3 8
N
O
O
OCH3
OCH3
Cl
OMe
OMeMeO
NAX 54
Antiproliferative effects in human
malignant mesothelioma cell lines

16. Antiproliferative effects in different
human cancer cell lines

17. Advanced Technology Center for Aging Research
Scientific Technological Area IRCCS - INRCA
Mauro ProvincialiElisa Pierpaoli

18. Project BackgroundProject Background
Breast CancerBreast Cancer
Berberine inhibits cellular growth of breast cancer cells and
promotes apoptosis by down-regulating the HER2/PI3K/Akt
signaling pathway1
Berberine inhibits cellular growth of breast cancer cells and
promotes apoptosis by down-regulating the HER2/PI3K/Akt
signaling pathway1

19. Breast Cancer (BC) is:
the second most common cancer worldwide,
the fifth most common cause of cancer death,
the leading cause of cancer death in women
(>1.4 million new cases and > 450,000 deaths
annually)
BC rates are rising around the world
The heterogeneity of BCs makes them both a fascinating and
challenging solid tumour to diagnose and to treat.
The heterogeneity of BCs makes them both a fascinating and
challenging solid tumour to diagnose and to treat.
Project BackgroundProject Background
Breast CancerBreast Cancer

20. BC characterized by overexpression of human epidermal growth
factor receptor 2 (HER2) has been associated with more aggressive
disease progression and a poorer prognosis.
Project BackgroundProject Background
Breast CancerBreast Cancer
HER2 is overexpressed in 20–30% of invasive BC thus, new
therapies are desperately needed.
HER2-targeting gold standard drugs show modest efficacy as single
agent and substantial toxicity in combination therapy.
HER2 + human BC
cells
(SK-BR-3)

21. TodayToday BC HER2BC HER2++
therapiestherapies
DRUGDRUG CompanyCompany MOAMOA Use - Cost x month &Use - Cost x month &
FDA Approval (yr)FDA Approval (yr)
TrastuzumabTrastuzumab
Herceptin®
Genentech (US)
Roche (EU)
Monoclonal Antibody
against HER2 receptor
Combo with
Chemio
$4,500
2006
PertuzumabPertuzumab
Perieta®
Genentech Monoclonal Antibody
against HER2 receptor
Combo with
Chemio &
Herceptin
$6,000
2012
TDM-1TDM-1
Kadcycla®
Genentech Antibody-drug conjugate
Ado-Trastuzumab
emtansine
Single agent
$9,800
2013
LapatinibLapatinib
Tykerb®
GSK EGFR & HER-2 tyrosine
kinases inhibitor
Combo with
Chemio
$3,625
2007

22. Competitive landscapeCompetitive landscape
HER2 pathway and targets
Phase III drugs MOA
BKM120, LEE011 (Novartis); Palbociclib
(Pfizer)
HER2 dowstream pathway
Me-too's of currently used antibodies HER2 transmembrane receptor

23. NAX compoundsNAX compounds
Chemical structures of Berberine chloride, NAX012, NAX013, NAX014, and NAX035.

24. Antiproliferative activity of NAX compoundsAntiproliferative activity of NAX compounds
against HER2+ breast cancer cellsagainst HER2+ breast cancer cells
SK-BR-3 cells
24h treatment
N202.1A cells
(murine)
24h treatment
Alamar Blue assay. The number of viable cells after treatment is expressed as a percentage of the vehicle treated control
SK-BR-3 cells
(human) 24h treatment
Cells
IC50 µM
NAX014 NAX012 NAX013 NAX035 Berberine (BRB)
SK-BR-3 52.3 94.2 >100 >100 91.8
N202.1A 20 40 >50 >50 NT
PirpaoliE.BioFactors,2013,39,672.

25. Alamar Blue assay. The number of viable cells after treatment is expressed as a percentage of the vehicle treated control
Time-dependent activity of NAX compoundsTime-dependent activity of NAX compounds
against HER2+ breast cancer cellsagainst HER2+ breast cancer cells
50 µM
0
20
40
60
80
100
120
0 24 48 72
Time
%ofcontrol
NAX12B
NAX13B
NAX14C
NAX35B
BRB
10 µM
0
20
40
60
80
100
120
0 24 48 72
Time
%ofcontrol
NAX12B
NAX13B
NAX14C
NAX35B
BRB
SK-BR-3 cellsSK-BR-3 cells
Time
IC50 µM
NAX014 NAX012 NAX013 NAX035
Berberine
(BRB)
24 h 52.3 ±3.2 94.2 ±1.2 >100 >100 91.8±2.8
48h 30.7 ±2.1 46.6 ±2.5 >100 >100 58.4 ±1.9
72 h 26.5 ±6.7 31.9 ±2.9 >100 48.6 ±6.7 36.0 ±1.8
PirpaoliE.BioFactors,2013,39,672.

26. PI assay (SKBR3 + [50 uM] BRB analogs)
0
10
20
30
40
50
60
70
80
Apoptoticnuclei(%)
24h
48h
72h
24h 2,97 18,00 15,70 15,20 52,60
48h 7,88 27,70 54,20 44,40 65,10
72h 8,10 44,20 71,60 68,40 45,20
Ctrl BRB NAX12B NAX14C NAX35B
SK-BR-3 cellsSK-BR-3 cells
Induction of apoptosis of NAX compoundsInduction of apoptosis of NAX compounds
against HER2+ breast cancer cellsagainst HER2+ breast cancer cells
Time-dependent apoptotic effectTime-dependent apoptotic effect
Quantification of Apoptosis by Flow Cytometry: apoptosis was measured through sub-diploid DNA peak
analysis after staining with propidium iodide
Time 72hTime 72h
ControlControl BerberineBerberine
NAX014NAX014NAX012NAX012
8.7%
71.6%
68.4%
44.2
%
PirpaoliE.BioFactors,2013,39,672.

27. Breast whole mount1
Week 12
Breast whole mount1
Week 25
FVB-N 233 transgenic mouse model
expresses the HER2/neu oncogene
Female mice develop spontaneous malignant,
fatal, breast tumours into the mammary gland
and metastases (Muller et al. 1988).
BC is palpable starting on Week 25.
FVB-N 233 transgenic mouse model
expresses the HER2/neu oncogene
Female mice develop spontaneous malignant,
fatal, breast tumours into the mammary gland
and metastases (Muller et al. 1988).
BC is palpable starting on Week 25.
Tumour model: FVB mice Her2/neuTumour model: FVB mice Her2/neu
Mice bearing breast tumors (Week 25)
1. 3D technique.
TUM
TUM
Tumor expression

28. EXP 1: Antitumour efficacy inEXP 1: Antitumour efficacy in
HER-2/neu transgenic female miceHER-2/neu transgenic female mice
FVB-N 233FVB-N 233 Her2/neu miceHer2/neu mice treated IPtreated IP with 2.5mg/kg of compounds (2xweek)x12with 2.5mg/kg of compounds (2xweek)x12
Tumour Number Tumour Growth Inhibition
NAX014 is effectiveNAX014 is effective in delaying the onsetin delaying the onset and theand the
progression of HER2+ BC at well tolerated dosesprogression of HER2+ BC at well tolerated doses
NAX014 is effectiveNAX014 is effective in delaying the onsetin delaying the onset and theand the
progression of HER2+ BC at well tolerated dosesprogression of HER2+ BC at well tolerated doses

29. High % ofHigh % of tumour freetumour free
micemice after NAX014after NAX014
treatmenttreatment
High % ofHigh % of tumour freetumour free
micemice after NAX014after NAX014
treatmenttreatment
FVB-N 233FVB-N 233 Her2/neu miceHer2/neu mice treated IPtreated IP with 2.5mg/kg of compounds (2xweek)x12with 2.5mg/kg of compounds (2xweek)x12
EXP 1: Antitumour efficacy in HER-2/neuEXP 1: Antitumour efficacy in HER-2/neu
transgenic female mice (2)transgenic female mice (2)

30. EXP 2: Antitumour efficacy in HER-2/neuEXP 2: Antitumour efficacy in HER-2/neu
transgenic female micetransgenic female mice
FVB-N 233FVB-N 233 Her2/neu mice treatedHer2/neu mice treated per os with 20 mg/kgper os with 20 mg/kg of NAX014 (2xweek)x8of NAX014 (2xweek)x8
NAX014 is effective byNAX014 is effective by oral routeoral route
in delaying the onset and the progression of HER2+ BCin delaying the onset and the progression of HER2+ BC
NAX014 is effective byNAX014 is effective by oral routeoral route
in delaying the onset and the progression of HER2+ BCin delaying the onset and the progression of HER2+ BC
Age (Weeks)
Tumour Number
Age (Weeks)
Tumour Growth
Inhibition

31. Antimetastatic efficacy inAntimetastatic efficacy in
HER-2/neu transgenic female miceHER-2/neu transgenic female mice
NAX014 oral administration (20 mg/kg)
Lung metastases NAX014 Control
Mice with metastases (%) 12.5 55.5
Cumulative no. of metastases 1 7
Mean size of metaststases (mm) 6 ±0 5.7 ±1.8
Maximum size of metastases
(mm)
6 8
NAX014 shows antimetastatic efficacy by oral routeNAX014 shows antimetastatic efficacy by oral routeNAX014 shows antimetastatic efficacy by oral routeNAX014 shows antimetastatic efficacy by oral route
FVB-N 233FVB-N 233 Her2/neu mice treated per os with 20 mg/kg of NAX014 (2xweek)x8Her2/neu mice treated per os with 20 mg/kg of NAX014 (2xweek)x8

32. Tolerability in miceTolerability in mice
(repeated IP administration)(repeated IP administration)
NAX012
0
25
50
75
100
125
0 5 10 15 20 25 30
Day
BodyWeight(%initialweight)
0 mg/kg
2.5 mg/kg
5 mg/kg
10 mg/kg
20 mg/kg
NAX014
0
25
50
75
100
125
0 5 10 15 20 25 30
Day
BodyWeight(%initialweight)
0 mg/kg
2.5 mg/kg
5 mg/kg
10 mg/kg
20 mg/kg
NAX012
(Body weight reduction) BBR
0
25
50
75
100
125
0 5 10 15 20 25 30
Day
BodyWeight(%initial
weight)
0 mg/kg
2.5 mg/kg
5 mg/kg
10 mg/kg
Berberine
(Body weight reduction)
NAX014
(Body weight reduction)Dose
(total
dose)
(mg/kg)
% Letality
NAX014 NAX012 Berberine
2.5 (20) 0 0 0
5 (40) 0 0 0
10 (80) 0 0 85.7
20 (160) 25 50 100
FVB mice; intraperitoneal (IP)treatment
(2xweek)x4

33. NAX014NAX014
Recovery of the antiproliferative activityRecovery of the antiproliferative activity
SK-BR-3 human breast carcinoma cells treated with
NAX014 (20µM)for 48h and then in drug-free medium
for 96 and 120h
IrreversibleIrreversible
antiproliferatiantiproliferati
ve effectve effect
IrreversibleIrreversible
antiproliferatiantiproliferati
ve effectve effect

34. HER2
p-HER2
β-actin
NAX
014
Effect of NAX compounds on HER2/neuEffect of NAX compounds on HER2/neu
expression and phosphorylationexpression and phosphorylation
in HER2+ SK-BR-3 cellsin HER2+ SK-BR-3 cells
Graphs B and C represent quantification of the blots by the GS-670 imaging densitometer after HER2 normalization respect to bactin
(B) and pospho-HER2/b-actin normalization respect to HER2/b-actin (C). Results shown are representative of 2 independent
experiments.
Treatment: NAX012 and NAX014
and berberine
50µM for 24h
Unique ability of NAX014Unique ability of NAX014
to block total HER2to block total HER2
expression, most probablyexpression, most probably
by forming complex(es)by forming complex(es)
with DNA and/or mRNA).with DNA and/or mRNA).
Unique ability of NAX014Unique ability of NAX014
to block total HER2to block total HER2
expression, most probablyexpression, most probably
by forming complex(es)by forming complex(es)
with DNA and/or mRNA).with DNA and/or mRNA).
PirpaoliE.BioFactors,2013,39,672.

35. Effect of NAX compounds on expression ofEffect of NAX compounds on expression of
thymidylate synthase (TS) in MSTO cell linethymidylate synthase (TS) in MSTO cell line

36. Postulated mechanismPostulated mechanism
HER2 pathway and targets
NAX014NAX014
is a novel anti-HER2is a novel anti-HER2
agent, targeting HER2agent, targeting HER2
expressionexpression

37. Dual Hispano-Italian spin outDual Hispano-Italian spin out
newconewco
Cristina Geroni
Carmela Salvatore
Carmen Plasencia Narcis Clavell

38. ConclusionsConclusions
Unique ability to reduce cellular HER2 expression
NAX014
Aknowledgements: Financial supports were provided by Ministero dello Sviluppo
Economico (Grant. 01705 to Naxospharma) and by Agència per a la competitivitat de
l'empresa ACC1O (Grant RDNET11-1-0001 to Aromics) under the 6th call of the
EuroTransBio initiative, transnational project BERTA (BERberine as antiTumour Agents).
In vitro activity at µM concentrations
Antitumour and anti-metastatic efficacy on HER2+
tumours and tolerability at the effective doses in
vivo by oral administration
Compound leading to a new series of 13-
halophenylalkyl berberine derivatives with activity on
HER2 overexpressing cancers
First candidate for further development into Phase I
clinical studies