•
`
`Pergamon
`
`Bioorganic & Medicinal Cumistry Leners, Vol. 6, No. 15, pp. 1819-1824, 1996
`Copyright Q 1996 Elsevier Science Ltd
`Printed in Great Britain. All rights teserVed
`0960-894X/96 $15.00 + 0.00
`
`PII: S0960-894X(96)00323-X
`
`SILDENAFIL (VIAGRA ™), A POTENT AND SELECTIVE INHffiiTOR OF TYPE 5
`CGMP PHOSPHODIESTERASE WITH UTILITY FOR THE TREATMENT OF
`MALE ERECTILE DYSFUNCTION
`
`Nicholas K. Terrett,* AndrewS. Bell, David Brown1 and Peter Ellis,*
`
`Departments of Discovery Chemistry and Discovery Biology, t
`Pfizer Central Research, Sandwich, Kent, CT13 9NJ, UK
`
`Abstract: 5-(2'-Alkoxyphenyl)pyrazolo[4,3-d]pyrimidin-7-ones, and in particular our preferred compound,
`sildenaf!l (VIAGRA™), discovered through a rational drug design programme, are potent and selective
`inhibitors of the type 5 cGMP phosphodiesterase from both rabbit platelets and human corpus cavemosum.
`Sildenafil is currently in the clinic for the oral treatment of male erectile dysfunction.
`Copyright © 1996 Elsevier Science Ltd
`
`Introduction: Cyclic guanosine monophosphate (cGMP) is the ubiquitous second messenger for those G(cid:173)
`protein coupled receptors activated by endogenous substances such as nitric oxide (NO or EDRF) and atrial
`natriuretic peptide (ANP).
`Intracellular levels of cGMP are controlled by activation of cyclic nucleotide
`cyclases and breakdown by phosphodiesterases (PDE). Specifically, there are a number of PDE isozymes that
`will hydrolyse cGMP to the inactive GMP and thus cGMP levels may be raised by the use of a selective cGMP
`PDE inhibitor. 2 There are at least seven families of PDE3
`, of which three (types 1, 5 and 6) selectively
`5
`to cAMP.4
`hydrolyse cGMP relative
`PDE
`type 5,
`the calcium/calmodulin
`insensitive cGMP
`'
`phosphodiesterase, occurs in lung, platelets and various forms of smooth muscle. 6
`
`We considered that selective inhibitors of PDE type 5 would be attractive targets for the therapy of a range of
`cardiovascular disorders. As a consequence of our work on ANP/ we anticipated that a potent inhibitor would
`have utility in the therapy of hypertension and angina. However, we found that type 5 cGMP PDE is also the
`predominant cGMP hydrolysing activity in the cytosolic fraction from human corpus cavemosum.8 As penile
`erection is mediated by N0,9 and thus cGMP, 10 inhibitors of type 5 PDE improve erection by enhancing
`relaxation of the corpus cavemosal smooth muscle, and thereby have utility for the treatment of male erectile
`dysfunction (impotence). We here report the discovery of sildenaf!l (VIAGRA ™) (X), a potent and highly
`selective inhibitor for the type 5 PDE, that is an orally active treatment for male erectile dysfunction.
`
`Results and Discussion: Prior to our work, very little had been reported on the design, synthesis and
`screening of selective cGMP PDE inhibitors. Zaprinast (I, M&B 22,948), u developed as an anti-allergy agent,
`was one of the first type 5 PDE inhibitors to be reported, albeit only weakly active and a poorly selective
`
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`1820
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`N. K. TERRETI et al.
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`compound. Possibly as a consequence of its PDE inhibitory activity, zaprinast is a vasodilator in vitro 12 and
`lowers mean arterial blood pressure in anaesthetised dogs. 13 Zaprinast was screened against cGMP PDE type 5
`isolated from rabbit platelets, and demonstrated only modest affmity for this cGMP-hydrolysing enzyme and
`little selectivity over PDE 1. 14
`
`In order to find novel compounds with improved potency and selectivity over zaprinast, we explored a range of
`novel 2-alkoxyphenyl-substituted heterocyclic systems. Of the many series investigated, we found that
`derivatives of pyrazolo[4,3-d]pyrimidin-7-one (e.g. ll, see Figure 1) gave potent cGMP PDE type 5 inhibition.
`As we sought compounds with type 5 selectivity, all compounds were also screened against the other
`widespread cGMP degrading PDE, type 1, isolated from rat liver, and cAMP PDE type 3 isolated from rabbit
`platelets. 15 Type 5 PDE from human corpus cavemosal tissue was obtained and we demonstrated that it was
`essentially identical to the rabbit platelet enzyme and that standard inhibitors such as zaprinast have similar
`affmities for both enzymes (corpus cavemosum PDE type 5 ICso = 800nM).
`
`Figure 1 Enzyme inhibitory data. !Cso values are in nanomolar unless otherwise stated, and are the mean
`values of at least 2 determinations.
`
`ICso (nM)
`PDE 1
`PDE3
`PDE5
`
`I (zaprinast)
`9400
`>100JJ.M
`2000
`
`IT
`3300
`>100JJ.M
`330
`
`the
`substituents around
`selectivity with
`increasing potency and
`for
`scope
`the
`We explored
`pyrazolopyrimidinone structure. Modelling studies suggested that the nucleus may mimic the guanosine base of
`cGMP, as both are·of similar size, shape and have a similar dipole moment (see Figure 2). 16 We considered that
`
`Figure 2 Modelling studies on cGMP and a pyrazolo£4,3-d]pyrimidin-7-one
`
`1~:6
`H,No~
`
`\ _..o OH
`~P,
`0
`0
`
`cGMP
`
`0
`
`5'-position
`
`pyrawlo[4,3-d]pyrimidin-7-one
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`Sildenafil (VIA ORA"")
`
`1821
`
`extending the 3-substituent might fill a space in the enzyme active site occupied by ribose, and substituents on
`the 5' -position of the phenyl ring could, depending on the conformation of cGMP in the enzyme active site,
`reproduce the role of the phosphate in binding. Replacement of the 3-methyl group in II by n-propyl gave a
`much more potent compound (ill see Figure 3) with increased selectivity over type 1 POE. Removal of the !(cid:173)
`methyl group (IV) from the pyrazole reduced type 5 activity.
`
`Figure 3 Enzyme inhibitory data. !Cso values are in nanomolar unless otherwise stated, and are the mean
`values of at least 2 determinations. ~0
`Me
`;l;lN
`V
`
`"Pr
`
`ICso (nM)
`POE 1
`POE3
`POE5
`
`m
`790
`>lOOilM
`27
`
`IV
`860
`>lOOilM
`82
`
`Exploration around the 2' -substituent in this series suggested that ethoxy was preferred over many other
`groups (see Figure 4). Replacement of ethoxy with hydrogen (V) reduced type 5 POE affmity some 200-fold,
`and hydroxy, nitro or sulphonamide derivatives (VI, vm and IX respectively) are all weaker in activity. The
`SAR suggested that key features in the 2' -alkoxy series were a hydrogen bond between the pyrimidinone NH
`and oxygen lone pair of the alkoxy group maintaining coplanarity between the phenyl and heterocyclic systems
`(confirmed by an X-ray crystal structure17
`) and a requirement for a small lipophilic substituent.
`
`Figure 4 The effect of the 2 '-substituent on PDE 5 inhibitory activity. IC50 values are in nanomolar unless
`otherwise stated, and are the mean values of at least 2 determinations. ND = not determined.
`0
`Me
`
`, ~x,
`
`Compound
`
`Structure
`R=
`
`ICso(nM)
`POE 1
`
`v
`VI
`m
`VII
`
`vm
`IX
`
`H
`HO
`EtO
`~0
`N02
`NHS02Me
`
`NO
`NO
`790
`NO
`
`NO
`NO
`
`"p r
`
`POE3
`
`63,000
`>lOOuM
`>lOOilM
`47,000
`
`>lOOilM
`83,000
`
`POE5
`(platelet)
`4,500
`1,000
`27
`960
`
`4,400
`780
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`1822
`
`N. K. TERRETT et al.
`
`As mentioned above, a 5'-substituent on the 2-ethoxyphenyl ring has the potential to fill a space occupied by
`the phosphate of cGMP in the PDE active site. Access to 5' -substituted analogues is synthetically
`straightforward as electrophilic attack occurs selectively at this position and can be effected at a late stage in
`the synthesis, permitting rapid production of many analogues. Additionally, in order to improve the low
`solubility of compound ill (log D = 4.0), we wanted to make analogues with lower lipophilicity. The
`introduction of polar or charged substituents in 5' -sulphonamides (see Figure 5) gave derivatives with lower
`values of log D. These were demonstrated to possess greater solubility, as compared with m and furthermore
`we found clear increases in enzyme affinity (see compounds X (sildenafiJ.), XI, XD, and Xill). Intriguingly, 5'(cid:173)
`substitution of zaprinast with sulphones or sulphonamides enhanced the antiallergic activity of this series, 18
`although it was not clear that this activity was mediated through inhibition of cGMP PDE.
`
`Figure 5 The lipophilicity of 5-(2 '-alkoxyphenyl)pyrazolo[4,3-d]pyrimidin-7-ones could be varied by the use
`of polar or charged 5'-sulphonamide substituents. /C50 values are in nanomolar unless otherwise stated, and
`are the mean values of at least 2 determinations.
`0
`Me
`
`E~ HN~:N
`MN~
`y
`
`"Pr
`
`Compound
`
`m
`X
`Sildenafil
`(VIAGRATM)
`XI
`
`XII
`
`xm
`
`R
`
`Structure
`R=
`
`H
`
`S02N l
`~NMe
`1 \
`S02N N \_
`\_ /
`OH
`
`S02NJ--CONH2
`
`1 \
`NH
`S02N
`\_/
`
`ICso (nM)
`PDE 1
`
`PDE3
`
`790
`260
`
`>100~M
`65,000
`
`460
`
`62,000
`
`PDE5
`. (platelet)
`27
`3.6 (platelet)
`3.0 (corpus
`cavernosum)
`1.9
`
`110
`
`34,000
`
`390
`
`>1001J.M
`
`2.1
`
`5.7
`
`LogO
`
`4.0
`2.7
`
`2.0
`
`2.3
`
`1.5
`
`Overall, our results demonstrated that a range of different 5' -substituents are tolerated by PDE type 5, and
`amongst these, sildenafl.l (X) gave an excellent combination of enzyme inhibitory potency, selectivity, solubility
`and in vivo characteristics.
`
`The synthesis of pyrazolo[4,3-d]pyrimidin-7-ones commenced with the preparation of the pyrazole ring from
`the diketoester (1) and hydrazine (see Figure 6). Following the regioselective N-methylation of the pyrazole,
`hydrolysis gave the carboxylic acid (3). Nitration followed by carboxamide formation and nitro group
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`Sildenafll (VIAGRA"')
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`reduction gave the key substituted pyrazole intermediate (4). Acylating the amine with a 2-substituted benzoyl
`chloride and cyclisation under basic conditions produced the pyrazolopyrimidinone (6). For the preferred 2'(cid:173)
`ethoxy series, chlorosulphonylation proceeds selectively on the 5'-position of the phenyl ring, allowing ready
`coupling with a range of amines to afford the sulphonamide products (7).
`
`Figure 6. The synthetic route to pyrazolo[4,3-d]pyrimidin-7-ones
`0
`0
`
`
`
`Et0~0 ~'
`
`R~OEt
`
`(1)
`
`0
`
`(2)
`
`I N
`"
`R
`
`0
`
`Me
`
`R
`
`0
`
`Me
`
`~~,
`n /NH
`\:~ (5)
`M~'
`y
`
`!orR= EtO
`1.CIS03H
`2.aminas, EtOH
`
`R
`
`o,s,N,R
`I
`R
`
`(7)
`
`In summary, 5-(2'-al.koxyphenyl)pyrazolo[4,3-d]pyrimidin-7-ones, and sildenaftl in particular, are potent and
`selective inhibitors of the type 5 cGMP phosphodiesterase from both rabbit platelets and human corpus
`cavemosum. We have demonstrated that structural modification has achieved a 500-fold increase in cGMP
`PDE affinity over our early leads. The efficacy of sildenaftl (VIAGRA TM) for the oral therapy of male erectile
`dysfunction is currently being assessed through clinical trials, and results from these studies will be published in
`the near future.
`
`Acknowledgements: The authors are grateful to S.F. Campbell and D.A. Roberts for initial suggestions and
`modelling work with zaprinast and cGMP, to M.F. Burslem for enzyme isolation and screening, and also to J.
`Bordner (Pfizer Central Research, Groton, CT, USA) for X-ray crystallographic studies.
`
`References and Notes
`I. Current address, Medicinal Sciences, Glaxo Wellcome Research and Development. Medicines Research Centre,
`Gunnel Wood Road, Stevenage, Hertfordshire, SG I 2NY, UK
`2. Goy, M.F. cGMP: theWaywardChildoftheCyclicNucleotideFamily. TrendsNeurosci.1991,14, 293-299
`3. Beavo, J.A.; Conti, M.; Heaslip, R.I. Multiple Cyclic Nucleotide Phosphodiesterases. Mol. Pharmacal. 1994, 46,
`399-400
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`1824
`
`N. K. TERREIT et al.
`
`4. Murray, K.J.; England, P.J. Inhibitors of Cyclic Nucleotide Phosphodiesterases as Therapeutic Agents. Biochem.
`Soc. Trans. 1992, 20, 460-464.
`5. Beavo, J.A.; Reifsnyder, D.H. Primary Sequences of Cyclic Nucleotide Phosphodiesterase Isozymes and the Design
`of Selective Inhibitors. Trends Pharmacal. Sci. 1990, 11, 150-155.
`6. Murray, K.J. Phosphodiesterase VA Inhibitors. Drug News Perspectives, 1993, 6, 150-156.
`7. Danilewicz, J.C.; Barclay, P.L.; Brown, D.; Campbell, S.F.; James, K.; Samuels, G.M.R.; Terrett, N.K.; Wythes,
`M.J. UK-69,578, a Novel Inhibitor of EC 3.4.24.11 which Increases Endogenous ANF Levels and is Natriuretic and
`Diuretic. Biochem. Biophys. Res. Commlin. 1989, 164, 58-65.
`8. Ballard, S.A.; Burslem, M.F.; Gingell, J.C.; Price, M.E.; Tang, K.; Turner, L.A.; Naylor, A.M. In Vitro Cyclic GMP
`Hydrolysing Activity in the Cytosolic Fraction from Corpus Cavemosum. J. Urology, In press.
`9. Kim, N.; Azadzoi, K.M.; Goldstein, I.; De Tejeda, I.S. A Nitric Oxide-like Factor Mediates Non-adrenergic Non(cid:173)
`cholinergic Neurogenic Relaxation of Penile Corpus Cavemosum Smooth Muscle. J. Clin. Invest. 1991, 88, 112-118.
`Raifer, J.; Aronson, W.J.; Bush, P.A.; Dorey, F.J.; Ignarro, L.J. Nitric Oxide as a Mediator of Relaxation of the Corpus
`Cavemosum in Response to Nonadrenergic Noncholinergic Neurotransrnission. N. Engl. J. Med. 1992, 326, 90-94.
`Burnett, A.L.; Lowenstein, C.J.; Bredt, D.S.; Chang, T.C.K.; Snyder, S.H. Nitric Oxide: a Physiologic Mediator of
`Penile Erection. Science 1992, 257,401-403.
`10. Mirone, V.; Palmieri, A.; Nistico, G. Valida Risposta Erettile Indotta dal Ciclico GMP in un Gruppo di Pazienti
`Affetti da Impotenza e suoi Effetti sui Sistema Ossido Nitrico-guanilato Ciclasi. Acta. Urol. ltal. 1992, Suppl.4, 11-12.
`11. Holland, A.; Jackson, D.; Chaplen, P.; Lunt, E.; Marshall, S.; Pain, D.; Wooldridge, K.; Antiallergic Activity of 8-
`Azapurin-6-ones with Heterocyclic 2-Substituents. Eur. J. Med. Chern. 1975, 10,447-449.
`12. Komas, N.; Lugnier, C.; Stoclet, J.C. Endothelium-dependent and Independent Relaxation of the Rat Aorta by Cyclic
`Nucleotide Phosphodiesterase Inhibitors. Brit. J. Pharmacal. 1991, 104,495-503.
`13. Lee, K.C.; Canniff, P.C.; Hamel, D.W.; Pagani, E.D.; Gorcyzca, W.P.; Ezrin, A.M.; Silver, P.J. Comparative
`Hemodynamic and Renal Effects of the Low Km cGMP Phosphodiesterase Inhibitors Cicletanine and Zaprinast in
`Anesthetized Dogs. Drug Develop. Res. 1991,23,127-144.
`14. Meanwell, N.A.; Drug News Perspectives 1991, 4, 400-413.
`15. Compound affinities for the PDEs were assessed by determination of their ICso values. The PDE enzymes were
`isolated from rat kidney (type 1), rabbit platelets (types 3 and 5) and human corpus cavernosum (type 5) by the method of
`Thompson, W.J. et al. Biochem., 1971, 10, 311, and assays performed by the 'batch' method of Thompson, W.J.;
`Appleman, M.M.; Biochem., 1971, 18,5228. Inhibition studies were performed using a substrate concentration of
`500nM. Inhibitors were dissolved in DMSO and concentration-response curves were constructed over the range 3xl0-10
`to lxl04 in halflog increments. IC50 values were calculated using a sigmoidal curve fitting algorithm.
`16. Dipole moments were calculated using SPARTAN, Wavefunction, Inc., Irvine, CA, USA.
`17. X-ray crystallographic studies of sildenafil (see below) and related compounds demonstrated that in the solid state at
`least, this hydrogen bond was important.
`
`18. Wilson, S.R.; Wilson, R.B.; Shoemaker, A.L.; Wooldridge, K.R.H.; Hodgson, D.J. Antiallergic 8-Aza-purines 3.
`Structural Characterization of 2-(2-Propoxyphenyl)-8-azahypoxanthine,-2-(2-Propoxy-5-(propylsulfonyl)phenyl)-8-
`azahypoxanthine, and 2-(2-Propoxy-5-(N-methyl-N-isopropylsulfamoyl)phenyl-8-azahypoxanthine. J. Am. Chern. Soc.
`1982,104,259-264.
`
`(Received in Belgium 15 May 1996; accepted 4 July 1996)
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