Drug Discovery And Technology Of Sildenafil Biology Essay

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Sildenafil, a drug which is used for treatment of erectile dysfunction (impotence) in men, was discovered back in 1986. It's discovery can be called as an example of serendipity. A cardiovascular research programme was carried by the Pfizer to obtain a novel drug for the pulmonary hypertension or angina. This search had resulted in discovery of sildenafil, the first oral treatment for the erectile dysfunction. It is a phosphodiestrase (PDE) inhibitor type 5, which increases blood flow to spongy tissues of penis and maintains erection which is necessary for sexual intercourse. This discovery had brought solution to impotence which was suffered by millions of men. It was patented in 1996 and was approved by the USFDA at march 27, 1998. Sildenafil was released in the market by the brand name VIAGRA for erectile dysfunction and also available as REVATIO as treatment of pulmonary hypertension(Hossein A.Ghofrani et al. 2006). Sildenafil is the first oral treatment which is available in the market as oral treatment for erectile dysfunction in men(Jim Kling 1998).


Sildenafil is used to treat erectile dysfunction. Erectile dysfunction is a disease in which erection of penis is not attained or maintained which is required for sexual intercourse. Erectile dysfunction can be caused by diseases such as diabetes, hypertension or psychological disorders such as stress, anxiety etc. It is a common disease between men aged between 40 and seventy years. Before the discovery of Sildenafil, treatments such as vacuum therapy by penile pump or penile prosthesis surgery are used to treat erectile dysfunction. Vacuum pumps were available on doctor's prescription but penile prosthesis surgery was done as a last resort. It involves insertion of artificial rods in penis which was not much favorable(Anon 2008a;Chris Steidle 2009)


Sildenafil was discovered by a group of pharmaceutical scientists of Pfizer Company situated at sandwich, Kent. In 1984, a cardiovascular research programme was carried out by these scientists to find a new drug which has significant effect in case of angina or hypertension. Nitrates are the diffusible gases which starts conversion of guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) which is responsible for a cascade or reactions causing vascular muscle relaxation. Thus, nitrate show anti-anginal effect which is required but it's action is limited by prolonged exposure to nitrates(Parker and Parker 1998). PDE enzymes act on cGMP and cause it's degradation by converting it again to guanosine mono- or triphosphate. So, a biological hypothesis was put forward by the scientists that to block the PDE enzyme and potentiate the action of nitric oxide (NO). At that time, 5 types of PDE enzymes were identified from which PDE 5 type of enzyme was acting only on cGMP and not on any other cyclic nucleotide. This PDE5 enzyme is present in spongy tissues of penis known as corpus cavernosum. There were no compounds available at that time showing potent PDE5 inhibitory activity. A search of documentation of various compounds showing vasodilator effects lead to a compound, Zaprinast which shows weak and non-selective inhibitor of PDE5 enzyme. It showed it's vasodilatory activity in anaesthetized dog. Later on, it was developed as an anti-allergy agent. But, zaprinast act as a lead compound for Sildenafil and further modifications were carried out on structure of zaprinast to inhibit PDE5 enzyme(Simon F .Campbell 2000).

Zaprinast (figure 1) and guanine ring analogues shares a structural similarity in their basic structure. The triazolopyrimidinone structure of zaprinast and the guanine ring of GTP have similar dipole moment in magnitude and direction (9.5 and 7.4 respectively).


Figure 1: structure of Zaprinast(Illarion V.Turko et al. 1999).

From the structure of zaprinast and cGMP, it was suggested that this heterocyclic rings are recognized by the active site of PDE enzyme. So, modifications were carrying out on triazolopyrimidinone ring to obtain a potent drug with high enyme affinity. Moreover, computational studies showed that cGMP attains a syn conformation and zaprinast allows isosteric replacements which favours binding with phosphate moiety. Lastly, it was supported by X-ray structure that heterocyclic ring is essential for the inhibitory activity so it was remained unchanged(J.A.Beavo 1995).

After carrying out modifications on the parent ring, a pyrazolopyrimidinone structure (UK-122764, figure 2) was found to have 10 fold increase in potency against PDE5 and decreased affinity towards other PDE enzymes. But, still modifications were required to be done as to replace cyclic phosphate ring of original substrate in the spatial region. Thus 3- methyl substitution was carried out on the parent ring and a tetrahedreal group of sulphonamide was attached to the ring to replace the cyclic phosphate ring(Hossein A.Ghofrani, Ian H.Osterloh, & Friedrich Grimminger 2006).

Figure 2: Structure of pyrazolopyrimidinone derivative(Illarion V.Turko, Stephen A.Ballard, Sharron H.Francis, & Jackie D.Corbin 1999).

A significant breakthrough came in 1989, when a compound was synthesized named UK-92480 (Sildenafil, figure 3) which was having 100 fold increase in PDE5 enzyme selectivity and very less selectivity against the rest of the enzymes of PDE family. The piperazinosulphonamide moiety imparts lipophilicity to the structure of Sildenafil and and makes it more soluble. Also, it potentiates the action of nitric oxide effectively and increase cGMP levels intracellularly with lesser side effects. It resembles the structure of cGMP which shows competitive inhibition of PDE5 enzyme and it was confirmed as it gave a dose related increase of 2-4 fold of cGMP in canine coronary arteries(Robert B.Moreland et al. 1998).


Sildenafil cGMP

Figure 3: structures of Sildenafil and cyclic Guanosine monophosphate (cGMP)(Illarion V.Turko, Stephen A.Ballard, Sharron H.Francis, & Jackie D.Corbin 1999).

Clinical Development:

In 1991, Healthy volunteers were given a single dose of 200mg of Sildenafil as a part of phase-1 clinical trials. These doses were well tolerated among the volunteers but when dose exceed from 150mg, it shows disturbances in color vision. When Sildenafil was given intravenously with glycerin trinitrate, it increases the vasodilatory effect of nitrates among the subjects. So, Sildenafil should be contraindicated or a safe way needs to be developed to administer both drugs simultaneously. In 1992, studies were carried out on healthy volunteers who were given 75mg of Sildenafil three times a day for 10 days. Healthy volunteers reported flushing, headaches, indigestion and some of them also reported penile erections as side effects. But this was not taken seriously at that time and further trials were carried out as a cardiovascular study till 1993(Hossein A.Ghofrani, Ian H.Osterloh, & Friedrich Grimminger 2006). But, results show that Sildenafil was not a promising drug for the treatment of hypertension. So in late 1993, new clinical trials were carried out in healthy individuals suffering from erectile dysfunction were carried out. A Rigiscan device having two loops was fitted, one loop at the base and another at the tip of penis to determine the girth and hardness during the sexual intercourse. After carrying out two different clinical studies for almost one year, results showed that single dose of Sildenafil showed erection with a linear dose-response relationship(M Bollel et al. 1996).

Further, clinical trials were carried out in more than 3000 patients suffering from erectile dysfunction of mixed type, with a mean duration of five years and they were given 25mg, 50mg or 100mg of Sildenafil as dosage. These patients were aged between 18-87 years and 21 trials were carried out in randomized, placebo controlled, double blind for 6 months with different study designs such as parallel, cross over, fixed dose and titration(Pfizer Labs 2007).

Effectiveness of Sildenafil was assessed with instruments such as Rigiscan but primarily it was assessed on the basis of International Index of Erectile Function (IIEF) which consist of 15 questions mainly which serves two main end points(Rosen R.C. et al. 1997). They are:

Ability to achieve erection required for sexual inter course

Maintainance of erection after penetration

The answers were recorded as categorical responses after 4 weeks period of time. They are:

(0) No attempted intercourse

(1) Never or almost never

(2) A few times

(3) Some times

(4) Most times

(5) Almost or always

Also, a diary was kept by the patients and their sexual data was recorded by themselves daily and an optional questionnaire was also administered for their partners.

Results of patients showing improvement of erections assessed by questionnaire in any of the four different study designs for 12 to 24 weeks of time is shown below which was obtained on the 1797 patients that were characterized by median scores of 2 (few times) on IIEF question(Pfizer Labs 2007).


Treatment group

Figure 4: Graph showing improvement in patients with different groups(Pfizer Labs 2007).

Also, clinical studies were carried out in patients suffering from erectile dysfunction caused by conditions such as diabetes or spinal cord injury. In all cases, sildenafil showed it's effect and improved erection. On all trials, sildenafil improved conditions of erectile dysfunction in 43% of patients against 15% of placebo(Pfizer Labs 2007).

Target identification and Validation:

During the clinical development of sildenafil, 6 more families of PDE inhibitors were identified. So, there are total 11 types of PDE enzymes from which sildenafil showed high selectivity towards PDE5 than any other else except PDE6. It showed only 10 fold selectivity towards PDE6. Visual defects associated with sildenafil were explained by weak inhibition of PDE6 enzyme which was situated in photo receptors. It shows high selectivity towards PDE5 against PDE7-11 enzymes(Hossein A.Ghofrani, Ian H.Osterloh, & Friedrich Grimminger 2006).


Figure 5: Mechanism of action of sildenafil(Simon F .Campbell 2000).

Sildenafil shares a similar structure with cGMP. The 3' substituent of heterocyclic ring fill the space in the enzyme filled by ribose and the 5' end of sildenafil mimic the role of cyclic phosphate in substrate binding. Sildenafil blocks the PDE5 catalytic site which is acquired by cGMP. So cGMP binds to the allosteric site of the enzyme and cause enzyme inhibition. This will result in increase levels of cGMP as it is not catalyzed by the enzyme and will cause vasodilation which will result in increased blood flow to the tissue. Figure 5 illustrates the mechanism of action of sildenafil. As sildenafil has similar structure with cGMP and has high affinity towards PDE5 enzyme, question arises that sildenafil interacts with the cGMP for binding at allosteric site. A site directed mutagenesis study was carried out to assess the amino acids involved in catalysis of substrate. The main objective is to understand the inhibition of the enzyme at the molecular level and evaluating the inhibitory effects of drugs on the catalytic site. Analysis of binding of sildenafil to allosteric site was carried out and results showed that there is no interaction between sildenafil and cGMP for binding at allosteric site(Illarion V.Turko, Stephen A.Ballard, Sharron H.Francis, & Jackie D.Corbin 1999).working model.bmp

Figure 6: Working model of PDE5 enzyme(Hossein A.Ghofrani, Ian H.Osterloh, & Friedrich Grimminger 2006).

The first PDE5 enzyme was identified in laboratory. It was recognized when a search was carried out for cyclic nucleotide-binding proteins other than cyclic nucleotide dependant protein kinases as a cGMP binding protein in lung tissue. Figure 6 shows working model of PDE5 enzyme in which there is phosphorylation site at the amino terminal portions. There are two allosteric binding sites for cGMP near it. At catalytic sites consists of two Zn+2 binding motifs A and B near the carboxy terminal portion. The phosphorylation site is at serine residue at 92 position. The binding motifs are aspartic acid at positions 714 and 754. They facilitate the catalysis by acting as a catalytic base or as a coordinated ligand for required metal(Jackie D.Corbin and Sharron H.Francis 1999).

Lead Regeneration:

Reaction scheme for sildenafil was first reported in 1996 in Bioorganic & Medicinal chemistry Letters. Below is the reaction scheme (Figure 7) which was explained in the journal. It is a linear reaction which involves reaction of diketo ester (1) to hydrazine. It will result in cyclization of the product giving pyrazole ring (2). This ring upon hydrolysis with a base, gives a carboxylic acid derivative on which regioselcetive N-methylation has occurred (3). This compound on nitration with nitric acid in presence of sulphuric acid, and on reduction with selenium chloride and ammonium hydroxide, nitro group gets reduced to amino groups (4). Now this compound, upon acylation under

synthesis 1.bmp


Figure 7: Reaction scheme for synthesis of Sildenafil(Anon 2008b).

basic conditions, undergoes ring closure and forms a pyrazolopyrimidinone derivative (6).This upon chlorosulphonylation at 5' position of phenyl ring, result in sildenafil(David J.Dale et al. 1999;Nicholas K.Terrett et al. 1996). Different routes are available for synthesis of sildenafil but there are minor changes in the steps. Sildenafil is commercially produced by another route which was published later on.