NOVEL PYRAZOLES AS INHIBITORS OF THE
RECEPTOR OF THE TGF-β FACTOR FOR
APPLICATION IN NANOMEDICINE.
IN VITRO ANTI CANCER ACTIVITY EVALUATION
AGAINST MALIGNANT GLIOMAS
Mauro Comes-Franchini
Department of Organic Chemistry “A. Mangini”
Faculty of Industrial Chemistry
University of Bologna (Italy)
Camerino 23 ottobre 2008
OUTLINE
Glioblastoma (GBM), Trasforming Growth Factor TGF-β
Heterocycles against TGF-β
Synthesis of novel pyrazoles ad In Vitro Test
Towards In vivo test: Nanomedicine applications
Incidence of primitive brain tumors increased in the last decades and
tumors of the astrocytic series are the most frequent. Glioblastoma (GBM),
the astrocytic tumor of the highest grade, remains almost universally fatal.
Of the approximately 12,000 people who are diagnosed with GBM annually
in the U.S., half will die within a year, and the rest within 3 years. Currently,
the only treatments that stretch survival limits are exceptionally invasive
surgeries to remove the tumor.
Gliomas are often characterized by resistance to the conventional chemoor radio-therapy. It appears important to search innovative and powerful
therapeutic tools, as the molecular targeting, to inhibit specific pathways
that play a role in malignant gliomas.
Transforming growth factor-β (TGF-β) is a multifunctional cytochine that plays a
critical role in the regulation of cell growth, differentation and development in a wide
range of biological systems.
Malignant gliomas overexpress membrane TGF-β receptors. Therefore, specific
Inhibition of this growth factor could offer a novel therapy for the treatment of GBM
OUTLINE
Glioblastoma (GBM), Trasforming Growth Factor TGF-β
Heterocycles against TGF-β
Synthesis of novel pyrazoles and In Vitro Test
Towards In vivo test: Nanomedicine applications
Heterocycles as inhibitors of the transforming growth factor-β
O
SB-431542
O
O
N
O
N
H
N
N
OMe
O
NH2
H
N
N S
NH2
HN
H
N
O
N
F
N
Johnson & Johnson/Scios
Pfizer
Glaxo-Smith-Kline
N
OMe
F
N
N
N
N
H
N
N
GW6604
N
N
N
NH
N
N
N
N
Biogen Idec
Glaxo-Smith-Kline
Lilly Research Laboratories
Nature Review (Drug Discovery), 2004, 3, 1011.
Co-crystallization data for TGF-β inhibitors
….. suggested that the minimum requirements for tight binding at the active site consist
of the presence of a 2-pyridyl group on the 3-position of the pyrazole ring and an aryl
or heteroaryl substituent at the 4-position featuring a hydrogen bond acceptor.
J. Scott Sawyer et al. Bioorg. Med. Chem. Lett. 2004, 14, 3581–3584.
To characterize the binding affinity of putative lead candidates the binding constant
has been determined (Autodock 4, Docking Calculation on PDB code 1PY5)
and we planned the following…
http://www.rcsb.org/pdb/home/home.do
Amide: Restricted CN bond rotation,
coplanarity of the attached atom, H-bond
acceptor.
2-Pyridil group or simply an aryl group?
(just flat to enter into the pocket???)
4 3
N
N
Of course, we wanted to have a simple multi-gram chemistry…
OUTLINE
Glioblastoma (GBM), Trasforming Growth Factor TGF-β
Heterocycles against TGF-β
Synthesis of novel pyrazoles and In Vitro Test
Towards In vivo test: Nanomedicine applications
Synthesis of the 4-amido-1,3-diphenyl-1H-pyrazoles
O
O
O
Br + KN
O
DMF
Yield=85%
N
2h, RT
O
O
O
O
O
N
(Me)2NCH(OMe)2
16h, 103°C
O
PhNHNH2
N
EtOH (90%)
2h, 80°C
O
Ph
Yield= 76%
N
N
Ph
O
N
O
Ph
NH2NH2
N
N
Ph
EtOH
1.5h, rfx
Yield= 45%
H2N
1
Ph
N
N
Ph
Up to 10 grams
Synthesis of the 4-amido-1,3-diphenyl-1H-pyrazoles
Amidation via acyl chlorides
O
NH
O
NH
Ph
H2N
N
Ph
N
4; Yield=81%
N
Ph
Ph
C11H23
N
Ph
2; Yield=64%
N
N
Ph
1
O
MeO
NH
Ph
3; Yield=70%
N
Ph
N
Reagent and conditions: RCOCl, EtN(iPr)2, CH2Cl2, 24h, rt.
Synthesis of the 4-amido-1,3-diphenyl-1H-pyrazoles
Amidation via carboxylic acids
O
NH
Ph
H2N ()11
Boc
N
N
Ph
O
N
H
9
OH
O
1
HO
NH
HO ()11
N
Ph
Ph
N
8; Yield=31%
O
9
OH
NH
H2N
OH
H2N
7; Yield=41%
O
NHBoc
O
N
Ph
Ph
N
N
Ph
Ph
N
5; Yield=49%
HS
O
O
9
OH
NH
HS ()11
N
Ph
Ph
N
6; Yield=70%%
Reagent and conditions: RCOOH, DCC-DMAP, CH2Cl2, rt, 48h.
Boc removal: HCl/AcOEt, 3h, rt.
Synthesis of the 4-amido-1-methyl-3-(pyridin-2-yl)1H-pyrazoles
O
O
(Me)2NCH(OMe)2
N
N
Yield=58%
N
EtOH, rfx, 6h
O
N
2-methoxyethanol
N
+ MeNHNH2
+
3h,rfx
N
N
N
N
Me
1) HNO3/H2SO4
48h RT, 1h 100°C
2) H2, Pd/C
EtOH/DMF
2h,RT
Yield= 31%
N
N
N
N
H2N
ratio= 1:1
N
N
9
Yield=65%
N
N
Me
Up to 5 grams
Synthesis of the 4-amido-1-methyl-3-(pyridin-2-yl)1H-pyrazoles
Amidation via acyl chlorides
O
NH
O
H2N
NH
Ph
N
C11H23
N
Me
N
Me
N
12; Yield=65%
N
N
10; Yield=73%
N
N
Ph
9
O
MeO
NH
N
Me
N
N
11; Yield=69%%
Reagent and conditions: RCOCl, EtN(iPr)2, CH2Cl2, 24h, rt.
Synthesis of the 4-amido-1-methyl-3-(pyridin-2-yl)1H-pyrazoles
Amidation via carboxylic acids
O
NH
N
H2N ()11
N
Me
N
Boc
N
H
O
9
OH
1
N
HO
NH
N
HO ()11
N
Me
N
16; Yield=30%
O
N
OH
N
13; Yield=35%
HS
O
O
9
OH
9
N
N
Me
Ph
Ph
O
NH
H2N
OH
H2N
15 Yield=37%
O
NHBoc
O
NH
N
HS ()11
N
Me
N
14 Yield=53%%
Reagent and conditions: RCOOH, DCC-DMAP, CH2Cl2, rt, 48h.
Boc removal: HCl/AcOEt, 3h, rt.
Cytotoxicity test: MTT assay
In Vitro screening on U87 MG
(Human, Caucasian, Tissue: brain; Tumor: glioblastoma-astrocytoma).
96-well microtiter
plates
MTT
N
NH
Treated cells
3-(4,5-Dimethylthiazol-2-yl)-2,5
-diphenyltetrazolium bromide
+ MTT
N
+
Br-
N
N
N
Incubation 3h
+ Solubilization
Solution
MTT Assay
Measurement
Elisa-reader 540nm
is a colorimetric assay used to test cell
proliferation and cytotoxicity: metabolic activity
of viable cells reduces the tetrazolium salt MTT
in water insoluble formazan violet crystals.
Screening was done by
Dipartimento Clinico Scienze Radiologiche ed Istocitopatologiche (Ospedale Bellaria, Bologna)
pyr7 molecular docking
All the heterocyclic scaffold enter into the
pocket
Simple Ph group on C3 have similar effect
with respect to 2-Pyr group
It is important to have amides on C4 with
long alkylic chain ending with a NH2 (Hbond with Asp 290)
O
H2N ()11
7
NH
N
Ph
Ph
N
Pyr7
Binding Energy: -10.22 kcal/mol
KI : 32.47 nM
Additional H-bonding interation between the
Asp 351 carboxyl group and the NH of the CO
Synthesis of the 2-amino-N-(1,3-diphenyl1H-pyrazol-4-yl)ethanethioamide
S
H-bond acceptor strength similar
to that of an amide O-atom
R
Stronger Acid (pKa 11-13)
Stronger H-bond donor
H
N
D. Seebach. Helvetica Chimica Acta 1999, 82, 2067.
O
NH
BocHN ( )11
N
Ph
Ph
Lawesson Reagent
N
NH
BocHN ( )11
Toluene, 55°C, 18h
Ph HCl/AcOEt
N
rt, 3h
NH
N
N
Lawesson Reagent
Toluene, 55°C, 18h
H2N ( )11
Ph
N
7S, Yield= 97%
S
S
BocHN ( )11
NH
N
Ph
N
Ph
Yield= 92%
O
N
Me
S
S
NH
N HCl/AcOEt
BocHN ( )11
N
N
Me
Yield= 88%
rt, 3h
NH
N
H2N ( )11
N
Me
N
15S, Yield= 95%
pyr7 and pyr7S molecular docking
As you can see in this picture from the binding energy the two
molecules similarly docked into the ATP pocket of the TGFβ-I
binding site. So, a different ligand interaction with the receptor
due to different strenght of atoms/groups cannot explain the
different in vitro citotoxicity.
pyr7 and pyr7S : geometry and chemical group
involved into binding interaction
O acceptor
Aromatic
N acceptor
N donor
Aromatic
N donor
Aromatic
S acceptor
N acceptor
Aromatic
Chemical group involved into binding interaction:
•set of atoms
•depends on the local environment
Position:
•physical position
•functional position
Proper geometry
BINDING INTERACTIONS ARE
SIMILAR FOR THE 2
COMPOUNDS
logP
(logarithm of partition
coefficient between n-octanol and
water) is a well established measure of
the compound's hydrophobicity.
OUTLINE
Glioblastoma (GBM), Trasforming Growth Factor TGF-β
Heterocycles against TGF-β
Synthesis of novel pyrazoles and In Vitro Test
Towards In vivo test: Nanomedicine applications
Future developments
Synthesis: Improving of the molecular docking and alternative synthetic
methods (Rational combinatorial library design).
Drug Delivery: Conjugation on the ending COOH group of the nanocarrier
with chlorotoxin, a glioma tumor-targeting peptide.
Diagnostic and Therapeutic: Encapsulation of metallic nanoparticles
into the nanocarrier for magnetic resonance imaging (MRI) and thermal
ablation.
Acknowledgment
Dipartimento di Chimica Organica “A. Mangini” (University of Bologna):
Denis Gentili,
Molecular Docking: Carlo Maurizio Camaggi, Elena Strocchi
Dipartimento Clinico Scienze Radiologiche ed Istocitopatologiche,
Ospedale Bellaria, (University of Bologna):
Annalisa Pession, Monica Rani.