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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, Italy2 -----Treviso, Italy3 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

NS

OO

O

N

HNN

N

O

N

N

O

O

H

OO

NH

N

NS

OO

O

NN

HNN

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

HOO

HO

O OH

OH

12 4 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

OOHO

OH

13

HO O

OOHO

OH

14 6 7

OO

OOHOH

OO

OOHOH

OH

OO

OOHOH

OO

OOHOH

OH

II. Semi-synthetic derivatives

Several different modifications were carried out on the convenient scaffolds available.

OO

OOH

OHO

OOH

NO2

OO

OOHOH

NO2

OO

OOHO

NO2

OH

OH

OO

OOH

OO

OOH

OO

OOH

OH

OH

O

O

O

O

OHO

OOH

NH2

OH

OHO

OOH

NO2

OH

OO

OOHOH

OO

OOHOH

O

O

OO

OOO

O

O

O

O

O

O

SO

O

Cl

O

O

S

O

O

N

O

O

S

O

O

NN

O

N

NHN

N

O

SO

O

NN

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, TBABDCM/Et2O

reflux

NaNO2, CH3COOHEtOH, rt

HNO3, TBABDCM/Et2O, rt

CH3II, acetonereflux

DibromoethaneKI, EtOAc, reflux

mCPBADCM, reflux

Fe, NH4ClEtOH/H2O, rt

Acetylchloridepyridine, rt

chlorosulphonic acidDCM, rt

amineDCM

OO

OOO

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

OOHOH

OO

OOHOH

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.