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