Poster presented at "8th International Conference Inhibitors of Protein Kinases" - Warsaw - September 21 - 25, 2014 (IPK 2014). Preliminary report on new compounds with potential activity against PDE5 (phosphodiesterase-5), by chemical modification of isoflavones extracted from Maclura pomifera (Osajin and Pomiferin). The full paper was published in Fitoterapia - Volume 105, September 2015, Pages 132–138: Semi-synthetic derivatives of natural isoflavones from Maclura pomifera as a novel class of PDE-5A inhibitors http://dx.doi.org/10.1016/j.fitote.2015.06.020

1. 17
ISOFLAVONES and ERECTILE DYSFUNCTION:
is PDE5 the link?
Ribaudo G.1
, Pavan V.1
, Vendrame T.2
, Redaelli M.3
, Mucignat-Caretta C.3
, Zagotto G.1
1
Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Italy
2
-----Treviso, Italy
3
Department of Molecular Medicine, University of Padova, Italy
[1] Goldstein I. Male sexual circuitry. Sci Am. 2000 Aug; 283(2) :70-5.
[2] Mesquita JF, Clinics (Sao Paulo) 2012; 67: 181–3.
[3] Aversa A. International Journal of Urology 2010; 17(1): 38–47.
[4] Gareri P. International Journal of Endocrynology 2014; 2014: 1-15.
[5] Decaluwe K. Biochemistry and Behavior 2014; 121: 146–157.
[6] Ojewole JAO. Phytochemistry 2006; 67: 610-7.
Pictures: PDB-ID 2H42, 1UHO, 2I42; ispot.org.za; ; afrigetics.co.za; wikipedia.org
ERECTILE DYSFUNCTION: PATOPHYSIOLOGY
Erectile dysfunction (ED) is “a consistent inability to get or keep
an erection that is satisfactory for sexual performance” [1]. Its
prevalence reaches the 50% of men in the 40-70 age range
and it is related to arterial, neurogenic, hormonal, cavernosal,
iatrogenic and psychogenic alterations [2] leading to an
abnormal penile circulation that is the result of a progressive
worsening of some conditions influencing erectile biology:
- Reduced responsivity to contraction-relaxation stimuli (α1
receptors) in the prostatic, bladder and erectile tissue.
- Compromised nitric oxide (NO) production by penile
endothelium, often coupled with the presence of reactive
oxygen species (ROS) promoting an inflammatory state.
- Reduction of smooth muscle in corpus cavernosum and of
androgens levels [3].
TREATMENT STRATEGIES
NON-PHARMACOLOGICAL: counseling, life style changes
(weight loss, increased physical exercise, quitting smoking),
medication changes. Proved risk factors comprehend
hypertension, diabetes mellitus, hyperlipidemia, smoking and
aging in general [3].
PHARMACOLOGICAL: phosphodiesterase-5 inhibitors (PDE5-
I), yohimbine, prostaglandin E1 (PGE1) and papaverine.
Current pharmacological treatments do not “cure” ED but
provide a general improvement of the erectile function [4].
MECHANISM OF PDE INHIBITION EXPERIMENTAL SECTION
I. Extraction and characterization
Fruits from Maclura pomifera were cut, desiccated and treated
with petroleum and diethyl ether in a Soxhlet apparatus. Osajin
and pomiferin were separated through precipitation with a Pb(II)
solution and subsequent recrystallizations. The two natural
isoflavones were fully characterized by NMR and high
resolution mass spectrometry. Purity profile was assessed by
HPLC analysis.
RESULTS, DISCUSSION AND FUTURE DIRECTIONS
N
N
S
O
O
O
N
HN
N
N
O
N
N
O
O
H
O
O
N
H
N
N
S
O
O
O
N
N
HN
N
O
1
2
3
The crystal structures of sildenafil (1), tadalafil (2) and
vardenafil (3) complexed with PDE5 are reported above. 2013
has been the year of the 15th anniversary of the introduction of
the first commercially available highly selective PDE5-I for the
treatment of ED and recently novel enhanced formulations
(orodispersible tablets, controlled release systems) are
reaching the market. Sildenafil, vardenafil, tadalafil and avanafil
are approved worldwide while udenafil and mirodenafil are
approved only in Korea [4]. The activity of these non-
hydrolysable analogues of cGMP consists in slowing the
degradation of cGMP by PDE5 promoting the relaxation of
penile smooth muscle [2-4].
POLYPHENOLS AND (ISO)FLAVONES
Various micronutrients and polyphenols found in soy, green
tea, and many fruits and vegetables have been described to
impact ED. Polyphenols can represent an efficient approach to
the disease because they can act as antioxidants, improving
the NO-cGMP pathway and stimulating angiogenesis [5].
Resveratrol (contained in wine, 4) and quercetin (5) are two
examples [5].
OH
HO
OH
11
HO
HO
O
HO
O OH
OH
124 5
Kraussianones come from the roots of Eriosema kraussianum, a
South African plant already known as a traditional remedy for
curing or alleviating impotency. Ojewole et al. [6] assessed that
these compounds provide their effects following two steps: a
concentration-related, initial slight contractions, and then
relaxations of the isolated portal veins (the mechanisms still
remains unclear). This is peculiarly true for kraussianone 1 (6)
and 2 (7).
O O
O
OHO
OH
13
HO O
O
OHO
OH
146 7
OO
OOH
OH
OO
OOH
OH
OH
OO
OOH
OH
OO
OOH
OH
OH
II. Semi-synthetic derivatives
Several different modifications were carried out on the
convenient scaffolds available.
OO
OOH
OHO
OOH
NO2
OO
OOH
OH
NO2
OO
OOH
O
NO2
OH
OH
OO
OOH
OO
OOH
OO
OOH
OH
OH
O
O
O
O
OHO
OOH
NH2
OH
OHO
OOH
NO2
OH
OO
OOH
OH
OO
OOH
OH
O
O
OO
OO
O
O
O
O
O
O
O
S
O
O
Cl
O
O
S
O
O
N
O
O
S
O
O
N
N
O
N
NH
N
N
O
S
O
O
N
N
III. Synthetic hybrid analogues
A novel synthetic route was designed to obtain molecules
combining the structural features of known PDE5-I and
scaffolds of natural compounds.
8 910
11 12
13
14
8 1215
16
17
3D model of osajin (8)
3D model of pomiferin (9)
20
21
1
18 19
HNO3, TBAB
DCM/Et2O
reflux
NaNO2, CH3COOH
EtOH, rt
HNO3, TBAB
DCM/Et2O, rt
CH3II, acetone
reflux
Dibromoethane
KI, EtOAc, reflux
mCPBA
DCM, reflux
Fe, NH4Cl
EtOH/H2O, rt
Acetylchloride
pyridine, rt
chlorosulphonic acid
DCM, rt
amine
DCM
OO
OO
O
OH
NO2/NH2O
O
The toxicity and the potential safety of
the extracted and synthesized
molecules were assessed through an
MTT test, showing encouraging results.
The obtained molecules are now being
screened focusing on their inhibiting
capabilities towards PDE5 in an
enzymatic assay.
MACLURA POMIFERA
Focusing our attention on the structural features of the
compounds already described as potential PDE5-I or, in
general, ED traditional remedies we identified a family of
molecules of interest. Going back to natural products, we used
Maclura pomifera as a source of natural isoflavones. This wild
apple is readily available in our region (Veneto, northern Italy).
OO
OOH
OH
OO
OOH
OH
OH
8
9
sildenafil (1), osajin (8), hybrid derivative (21)
1 8
21
SYNTHETIC STRATEGIES
In our quest for novel compounds with a potential activity
against PDE5 we pursued three strategies: I. Extraction and full
characterization of osajin (8) and pomiferin (9); II. Modification
of the two isoflavones to obtain semi-synthetic derivatives; III.
Ex novo synthesis of hybrid derivatives combining structural
features of natural compounds and known PDE5-Is.
Extraction and synthetic
schemes were optimized
to investigate different
chemical features basing
on the available and
characterized scaffolds.