Med Chem Res DOI 10.

1007/s00044-010-9523-y

MEDICINAL CHEMISTRY RESEARCH

ORIGINAL RESEARCH

3D-QSAR of amino-substituted pyrido[3,2B]pyrazinones as PDE-5 inhibitors

Omprakash Tanwar Rikta Saha M. Mumtaz Alam Mymoona Akhtar

Received: 16 August 2010 / Accepted: 19 November 2010 Springer Science+Business Media, LLC 2010

Abstract A 3D-QSAR study on amino-substituted pyrido[3,2b]pyrazinones as PDE-5 inhibitors was successfully performed by means of pharmacophore mapping using PHASE module of Schrodinger-9. The 3D-QSAR obtained from AADHRR-183 hypothesis was found to be statistically good with r2 = 0.95 and q2 = 0.81 taking PLS factor 4. The statistical signicance of the model was also conrmed by a high value of Fisher ratio of 85.1 and a very low value of RMSE 0.29. One of the other parameters which signify the model predictivity is Pearson R. Its value of 0.91 shows that the correlation between predicted and observed activities for the test set compounds is excellent. Hydrophobic groups are important for PDE-5 inhibition while H-bond donor groups are less favorable for the same. Electron withdrawing groups are favorable if include at ring A in the structures while unfavorable at other sites. Thus, it can be assumed that the present QSAR analysis is enough to demonstrate PDE-5 inhibition with the help of AADHRR-183 hypothesis and will help in designing novel and potent PDE-5 inhibitors. Keywords 3D-QSAR Pharmacophore PHASE Phosphodiesterases Pyrido[3,2b]pyrazinones Schrodinger Abbreviations 3D-QSAR Three-dimensional quantitative structure activity relationship

2D-QSAR PDEs RMSE cAMP cGMP PLS LOO r2 q2

Two-dimensional quantitative structure activity relationship Phosphodiesterases Root mean squared error Cyclic monophosphate Cyclic guanosine monophosphate Partial least square Leave one out Cross-validated correlation coefcient Internal predictivity

Introduction The Three-dimensional quantitative structureactivity relationship (3D-QSAR) involves analysis of the quantitative relationship between the biological activity of a set of compounds and their three-dimensional structural properties, using statistical correlation methods. Threedimensional QSAR approach is one of the most powerful techniques which come in the class of indirect drug design. Lead optimization without receptor 3D structure is one of the most important applications of 3D-QSAR. It allows 3D visual analysis for spatial arrangement of structural features with biological activity thus is advantageous over 2D-QSAR where model data has to be taken into consideration. PDEs are the enzymes which are responsible for the degradation of cyclic monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) that are imperative intracellular second messengers (Francis and Corbin, 1999). Erectile dysfunction is the most commonly encountered form of sexual dysfunction in men and can cause signicant

O. Tanwar R. Saha M. M. Alam M. Akhtar (&) Drug Design and Medicinal Chemistry Lab, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi 110062, India e-mail: mymoonaakhter@gmail.com

Med Chem Res

distress, impaired quality of life, and has a negative effect on overall self-esteem and intimate relationships (Eardley et al., 2010). Phosphodiesterase-5 (PDE-5) inhibitors have been proven to be effective for erectile dysfunction (Wallace, 2005). Cyclic guanosine monophosphate (cGMP) is a second messenger signaling molecule that is central to the regulation of many physiological processes, including smooth muscle tone (relaxes smooth muscles), visual transduction, platelet aggregation, bone growth, and electrolyte and uid homeostasis (Wall et al., 2003). Phosphodiesterase 5 (PDE-5) converts enzymatically the intracellular second messenger molecule cyclic guanosine monophosphate (cGMP) to its inactive form. By its preservation, cGMP activates cGMPdependent protein kinase I, which pivotally drives a biochemical cascade resulting in corporal smooth muscle relaxation and, hence, penile erection. This system mechanistically requires the synthesis of cGMP, secondary to the production and release of NO during sexual arousal. Accordingly, PDE-5 inhibitors augment the erectile response (Burnett, 2005). It was also found that selective increase in tumor capillary permeability appears to be mediated by a selective increase in tumor cGMP levels (Black et al., 2008). Sildenal citrate is the most popular PDE-5 selective inhibitor used in the treatment of erectile dysfunction (ED) (Terrett et al., 1996; Moreland et al., 1999). Some other PDE-5 inhibitors are also known like Vardenal-hydrochloride (Haning et al., 2002) and FR226807 (Hosogai et al., 2001) given in Fig. 1. The objective of this study is to develop the 3D pharmacophore for PDE5 inhibition and to provide the basis to design the novel and potent PDE-5 inhibitors.

Dataset and method Materials and methods A successful 3D-QSAR study was performed to establish relationship between the spatial three-dimensional pharmacophoric features and PDE-5 activity of a class of amino

substituted pyrido[3,2b] pyrazinone derivatives, synthesized by Owen et al., (2009). Present 3D-QSAR study was performed with the dataset of two different series containing total 30 compounds with well-dened PDE-5 inhibitory activity given as IC50 values in nM concentration. For the correlation purpose IC50 values then converted to their molar values and subsequently calculated to free energy-related terms, i.e., -log (1/IC50). The compounds of selected series with their inhibition data are summarized in Table 1. This dataset was then chosen for generating common pharmacophore hypotheses and then performing QSAR analysis. PHASE-3.1 module of Maestro-9 molecular modeling software was used to generate 3D pharmacophore models for selected series of PDE-5 inhibitors (Phase 3.1, Schrodinger, LLC, 2009). A pharmacophore conveys characteristics of the three-dimensional arrangement of the pharmacophoric elements which are purported to be critical for binding. A given hypothesis may be combined with known activity data to create a 3D-QSAR model that identies overall aspects of molecular structure that govern activity. The structures were sketched using maestro builder toolbar and were imported to develop pharmacophore model panel of the PHASE with their respective activity values. The ligands were assigned as actives and inactives by giving an appropriate activity threshold value ([7.87). The activity threshold value was selected in the basis of dataset activity distribution and active ligands are chosen to derive a set of suitable pharmacophores. Sketched structures were energy minimized/cleaned up by Ligprep module using OPLS_2005 force eld and proper protonation states were assigned with the ionizer subprogram at pH 7.2 0.2 (LigPrep, Schrodinger, LLC, 2009). The conformations were generated with the help of MacroModel torsional sampling using OPLS_2005 force eld (MacroModel, Schrodinger, LLC, 2009). Prepared ligands were then used for generating common pharmacophore hypothesis (CHP) and QSAR model generation. PHASE utilizes ne-grained conformational sampling and some

Fig. 1 Some phosphodiesterase inhibitors

O HN

O N N N

O
HO

HN N N

N
O

HN

NO2 NH

S N

S N

SILDENAFIL

VERDENAFIL

FR226807

Med Chem Res

scoring methods to identify CHPs for a series of molecules which have particular target specicity. Each hypothesis conveys a particular 3D conformation of a set of ligands in which the ligands are going to bind to the receptor. The best hypothesis is then correlated with known biological activity values to generate a 3D-QSAR model which identies the whole structural features of molecules that govern activity (Samantha et al., 2008; Almerico et al., 2010).

Active analog approach was used to identify a CPH which has been applied in generating signicant 3D-QSAR models (Narkhede and Degani, 2007; Lather et al., 2008; Mahipal et al., 2010). The common pharmacophores were culled from the conformations of the set of active ligands using a tree-based partitioning technique which groups together similar pharmacophores according to their intersite distances. A tree depth of ve with initial box size of 25.6 A and nal box size of 0.8 A was used (Samantha

Table 1 Structures of the compounds of the selected series with their inhibition data

R1

N X

R2 N H N

Comp. code PDE009

log IC50 R1 R2 X PDE-5 8.89

OMe

PDE010
N

OMe

8.70

PDE011
N

8.60

PDE012
F

7.69

PDE013
F

8.57

PDE014

7.55

PDE015

8.52

PDE016

8.34

Med Chem Res Table 1 continued

PDE017

8.80

PDE018

7.87

PDE019

H
O

8.46

PDE020
O

7.28

PDE021

6.83

PDE022

7.65

PDE023

7.48

PDE024

6.65

Ar

HN R2

R2

Ar
N O

PDE025

9.00

Med Chem Res Table 1 continued

PDE026

9.05

PDE027

N
OMe

9.52

O
N

PDE028

OMe

9.52

PDE029

OMe

9.40

PDE030

O
N

OMe

9.52

PDE031

O
N
N

OH

8.54

PDE032

7.44

PDE033

7.47

PDE034

OMe
O
N

7.84

PDE035

OMe

8.64

PDE036

OMe

8.62

PDE037

OMe

7.57

PDE038

OH

6.27

PDE isoforms inhibition -logIC50 values (in molar units)

Med Chem Res Table 2 Score of different parameters of the hypothesis AADHRR183 S. No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Parameters Survival Survival inactive Post hoc Site Vector Volume Selectivity Must matches Energy Activity Inactive Score 8.4 5.831 3.65 0.91 0.983 0.755 2.297 14 0 8.52 2.569
O N N N

B
Hydrophobic Acceptor
N O

Acceptor

Donor

B
N

NH O

Survival weighted combination of the vector, site, volume, and survival scores, and a term for the number of matches, a large value of survival score indicates the better tness of the active ligands on the common pharmacophore and validates the model Survival inactive survival score for actives with a multiple of the survival score for inactive subtracted Post hoc this score is the result of rescoring Site score, this score measures how closely the site points are superimposed in an alignment to the pharmacophore of the structures that contribute to the hypothesis, based on the RMS deviation of the site points of a ligand from those of the reference ligand Vector alignment score Volume measures how much the volume of the contributing structures overlap when aligned on the pharmacophore Selectivity estimate of the rarity of the hypothesis, High selectivity means that the hypothesis is more likely to be unique to the actives Matches number of actives that match the hypothesis Energy relative energy of the reference ligand in kcal/mol Activity of the reference ligand Inactive survival score of inactives

Ring1

Ring1

Fig. 2 a Common pharmacophore for active ligands [two hydrogen bond acceptor (A) in pink color, one hydrogen bond donors (D) in sky blue color, one hydrophobic group (H) in green color and two aromatic ring (R) in yellow color] and b 2D representation of pharmacophore (Color gure online)

et al., 2008; Phase3.1, Schrodinger, LLC, 2009). The resulting pharmacophore was then scored and ranked. The scoring was done to identify the best candidate hypothesis, and which provided an overall ranking of all the hypotheses. The scoring algorithm included the contributions from the alignment of site points and vectors, volume overlap, selectivity, number of ligands matched, relative conformational energy, and activity. A detailed description about the scores can be found in the methodology research article where PHASE is described (Dixon et al., 2006). The selected AADHRR-183 hypothesis with various scores is summarized in Table 2. The best pharmacophore hypothesis AADHRR-183 (Fig. 2) was selected for further QSAR study. The abovementioned 3D pharmacophore hypothesis (Fig. 2a) encompass the following features: two hydrogen bond

acceptor (A) in pink color, one hydrogen bond donors (D) in sky blue color, one hydrophobic group (H) in green color and Two Aromatic ring (R) in yellow color. The 2D representation of the AADHRR-183 hypothesis is given in Fig. 2b. The 2D representation shows that the secondary amino group near to pyrazinones ring is hydrogen bond donor (D), N atom of pyridine and C=O group of pyrazinone ring are two hydrogen bond acceptors (A), substitution at ring A is hydrophobic groups (H), and rings A and B are two aromatic ring (R) are the pharmacophoric elements of AADHRR-183 hypothesis.

Building of QSAR model In this study, a signicant 3D-QSAR model was generated using AADHRR-183 hypothesis. For QSAR model generation, training and test partition was done by random selection method. Atom-based model selection criterion was chosen for model building (Shah et al., 2010). PLS factor was set as 04, the maximum number of PLS factors in each model can be 1/5 the total number of training set molecules. More the PLS factor value, more will be the reliability of models. Various models have been generated and the best model was selected on the basis of the statistical signicance given below.

Med Chem Res Table 3 3D-QSAR statistical parameters PLS factors 1 2 3 4 SD 0.584 r2 F P RMSE q2 Pearson R

0.6248 35

7.195e- 0.4672 0.5024 0.7301 06

0.4171 0.8177 44.9 4.048e- 0.4459 0.5467 0.7645 08 0.3196 0.8984 56 1.271e- 0.3323 0.7482 0.8778 09 0.2887 0.81 0.9127

0.2309 0.9498 85.1 1.95e11

SD standard deviation of the regression, r2 for the regression, F variance ratio. Large values of F indicate a more statistically signicant regression, P signicance level of variance ratio. Smaller values indicate a greater degree of condence, RMSE root-mean-square error, q2 for the predicted activities, Pearson R value for the correlation between the predicted and observed activity for the test set

Fig. 3 QSAR visualization of various substituents affect: a electron withdrawing feature, b hydrogen-bond donor, and positive (c) and negative (d) hydrophobic effects (Color gure online)

Result and discussion A 3D-QSAR study has been performed successfully on the series of substituted pyrido[3,2b]pyrazinone derivatives to

understand the effect spatial arrangement of structural features on PDE-5 inhibition. Result of the 3D-QSAR can be visualized from Fig. 3. The blue cubes in 3D plots of the 3D pharmacophore regions refer to ligand regions in which the specic feature is important for better activity, whereas the red cubes demonstrates that particular structural feature or functional group is not essential for the activity or likely to reason for decreased binding potential. The statistical results of 3D-QSAR study are summarized in Table 3. The reliability of the present 3D-QSAR analysis can be justied by the fact that all statistical measures are signicant to any level. The model express 99% variance exhibited by pyrido[3,2b]pyrazinones, which is near to one and signifying a very close agreement of tting points on the regression line for the observed and PHASE predicted activity. The tness graph can be visualized from Fig. 4 and the observed and PHASE predicted activity data are summarized in Table 4. Validity of the model can be expressed by internal predictivity (q2 = 0.81) which is obtained by leave-one-out (LOO) or leave n out method. The q2 by leave-one-out method is more reliable and robust statistical parameter than r2 because it is obtained by external validation method of dividing the dataset into training and test set. The large value of F (85.1) indicates a statistically signicant regression model, which is supported by the small value of the variance ratio (P), an indication of a high degree of condence. Further small values of standard deviation of the regression (0.23) and RMSE (0.29) make an obvious implication that the data used for model generation are best for the QSAR analysis. Apart from the above-mentioned features, PLS factor also conrms the reliability of the model. In this study, number of PLS factor was taken as 4 and for each increment it gives one equation and there should be stepwise improvement each time the model generated. In addition to

Med Chem Res Fig. 4 Fitness graph between observed activity versus PHASE predicted activity for training and test set compounds

the above parameters it is interesting to note that active ligands are closely tted to the regression line and inactive ligands are scattered (Fig. 5). Figure 3 shows 3D-pharmacophore regions around compounds. For the selected pharmacophore blue and red cubes represent favorable and unfavorable regions, respectively. Molecular substitutions which increase the number of blue cubes will denitely lead to increases binding afnity of the molecules towards PDE-5 inhibition, while molecular substitutions which increase the number of red cubes will lead to decreased activity. Figure 3a represents electron withdrawing characteristic for the selected hypothesis. Visual analysis of Fig. 3a demonstrates that the throng of the blue cubes at the ring A site is pointing out the positive potential of electron withdrawing characteristic of the molecules and is requisite for the activity. It can be suggested that addition of appropriate electron withdrawing groups at the ring A and at the side of methoxy group will append the PDE-5 inhibition, whereas the addition of electron withdrawing groups at ring B site and at pyran ring site will lead to decreased receptor binding which in turn will result in lower potency of compounds. The potency of the compounds can be increased by addition of small electron withdrawing groups like uoro, chloro, bromo, etc., at para and meta positions on ring A. Figure 3b illustrates H-donor characteristic for the selected hypothesis. The crowd of red cube suggests that addition of hydrogen bond donor groups at ring A site will

lead to decreased PDE-5 inhibition. Similar diminished potential of the compounds can be found if hydrogen bond donor groups are added at NH at ring B site. While proper orientation of hydrogen bond donor groups may slightly lead to increased inhibition toward PDE-5 as indicated by fewer blue cubes. Figure 3c and d demonstrating the effect of positive and negative hydrophobic potential, respectively. It can be deduced from gure that hydrophobic groups are well tolerated at cyclohexyl moiety on ring B (blue cubes), while the substitution of hydrophobic groups at ring A site and pyridine ring are unacceptable (red cubes) or may hinder the binding of the molecules to the receptor active site and will result in decreased PDE-5 inhibition. An interesting nding of the present analysis is that H atom (on NH group) of pyrazinone ring should substituted only with hydrophobic groups as there is dense crowd of blue cube is present. At the same time no hydrophobic substituents should be present on pyridine ring for better PDE-5 inhibition. It is clear that cyclohexyl ring can be substituted with more hydrophobic rings like substituted cyclohexyl rings, saturated naphthalene rings, etc., along with the some hydrophobic groups at ring A. Although PDE-6 and PDE-11 activities were also reported for some ligands, but no signicant statistics have been developed so they are not reported in the present. It may also be due to the fact that the molecules are more selective towards PDE-5 as compared to PDE-6 and PDE11. Thus, it can be assumed that this study shows the

Med Chem Res Table 4 Fitness and PHASE predicted activity data for all compounds

Ligand name PDE009 PDE010 PDE011 PDE012 PDE013 PDE014 PDE015 PDE016 PDE017 PDE018 PDE019 PDE020 PDE021 PDE022 PDE023 PDE024 PDE025 PDE026 PDE027 PDE028 PDE029 PDE030 PDE031 PDE032 PDE033 PDE034 PDE035 PDE036 PDE037 PDE038

QSAR set Training Training Test Training Training Training Test Training Training Training Training Test Training Test Training Training Training Training Training Training Test Training Test Training Training Test Training Training Training Training

PLS factors 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234 1234

PHASE predicted activity 9.06 8.97 8.21 7.93 8.39 7.53 8.54 8.54 8.65 8.03 8.62 7.23 6.92 7.41 7.12 6.57 8.86 8.83 9.53 9.43 9.4 9.46 8.77 7.36 7.22 8.4 8.68 8.54 7.39 6.83

Pharm set Active Active Active Inactive Active Inactive Active Active Active Inactive Active Inactive Inactive Inactive Inactive Inactive Active Active Active Active Active Active Active Inactive Inactive Inactive Active Active Inactive Inactive

Fitness 2.67 2.62 2.83 2.83 2.78 2.76 3 2.84 2.82 2.92 2.89 2.66 2.58 2.57 2.64 2.63 2.24 2.32 2.39 2.51 2.52 2.71 2.35 2.2 2.34 2.5 2.44 2.45 2.5 2.27

selectivity towards and will help in designing more selective PDE-5 inhibitors.

Summary and conclusion A meaningful 3D-QSAR study was derived for the series of amino-substituted pyrido[3,2b]pyrazinones as PDE-5 inhibitors, to identify how three-dimensional arrangements of various substituents will affect the PDE-5 inhibition. Selected model is very much signicant to draw unambiguous inferences. The statistical parameter values are well above the acceptance limits. So it is easy to draw clear inference to design novel compounds for better PDE-5 inhibition. The 3D-QSAR model discussed above explains how and at what extent electron withdrawing, hydrophobic, and H-donor properties should be modied

to achieve better PDE-5 inhibition. The model shows that PDE-5 inhibition can be increased, if the hydrophobic character at ring B is supplemented by appropriate hydrophobic functional groups. Hydrophobic groupsubstituted cyclohexyl rings or naphthalene rings at B ring can provide potent PDE-5 inhibitors. The present nding suggests that H-bond donor substituents are mostly unfavorable in the structures. Thus, it is clear that inclusion of such groups will lead to deceased PDE-5 inhibition. Electron withdrawing characteristic is benecial at ring A, whereas electron withdrawing groups at pyrane ring and at pyrazinone are unfavorable. This suggests that inclusion of ouro, chloro, bromo, etc., at ring A will provide better compounds. Thus, it is clear from this study that it is possible to develop novel and potent PDE5 inhibitors if suitable substituents are added to the parent structures.

Med Chem Res Francis SH, Corbin JD (1999) Cyclic nucleotide-dependent protein kinases: intracellular receptors for cAMP and cGMP action. Crit Rev Clin Lab Sci 36:275328 Haning H, Niewohner U, Schenke T, Es-Sayed M, Schmidt G, Lampe T, Bischoff E (2002) Imidazo[5, 1-f][1, 2, 4]triazin-4(3H)-ones, a new class of potent PDE 5 inhibitors. Bioorg Med Chem Lett 12:865868 Hosogai NH, Hamada K, Tomita M, Nagashima A, Takahashi T, Sekizawa T, Mizutani T, Urano Y, Kuroda A, Sawada K, Ozaki T, Seki J, Goto T (2001) FR226807: a potent and selective phosphodiesterase type 5 inhibitor. Eur J Pharmacol 428: 295302 Lather V, Kristam R, Saini JS, Karthikeyan NA, Balaji VN (2008) QSAR models for prediction of glycogen synthase kinase-3b inhibitory activity of indirubin derivatives. QSAR Comb Sci 27:718728 LigPrep, version 2.3, Schrodinger, LLC, New York, NY, 2009 MacroModel, version 9.7, Schrodinger, LLC, New York, NY, 2009 Mahipal, Tanwar OP, Karthikeyan C, Moorthy NSHN, Trivedi P (2010) 3D-QSAR of aminophenyl benzamide derivatives as histone deacetylase inhibitors. Med Chem Res 6:277285 Moreland RB, Goldstein I, Kim NN, Traish A (1999) Sildenal citrate, a selective phosphodiesterase type 5 inhibitor: research and clinical implications in erectile dysfunction. TEM 10:97 104 Narkhede SS, Degani MS (2007) Pharmacophore renement and 3DQSAR studies of histamine H3 antagonists. QSAR Comb Sci 26:744753 Owen DR, Walker JK, Jon Jacobsen E, Freskos JN, Hughes RO, Brown DL, Bell AS, Brown DG, Phillips C, Mischke BV, Molyneaux JM, Fobian YM, Heasley SE, Moon JB, Stallings WC, Joseph Rogier D et al (2009) Identication, synthesis and SAR of amino substituted pyrido[3, 2b]pyrazinones as potent and selective PDE5 inhibitors. Bioorg Med Chem Lett 19: 40884091 PHASE-3.1, Schrodinger, LLC, New York, NY, 2009 Samantha L, Cesare M, Aleksey K, Mattia S, Stefano M, Elena C, Dorotea R, Alessandro C (2008) SAR and QSAR study on 2aminothiazole derivatives, modulators of transcriptional repression in Huntingtons disease. Bioorg Med Chem 16:56955703 Shah UA, Deokar HS, Kadam SS, Kulkarni VM (2010) Pharmacophore generation and atom-based 3D-QSAR of novel 2-(4methylsulfonylphenyl)pyrimidines as COX-2 inhibitors. Mol Divers 14:559568 Terrett NK, Bell AS, Brown D, Ellis P (1996) Sildenal (viagra), a potent and selective inhibitor of type 5 cgmp phosphodiesterase with utility for the treatment of male erectile dysfunction. Bioorg Med Chem Lett 6:18191824 Wall ME, Francis SH, Corbin JD, Grimes K, Jannetta RR, Kotera J, Macdonald BA, Gibson RR, Trewhella J (2003) Mechanisms associated with cGMP binding and activation of cGMP-dependent protein kinase. PNAS 100:23802385 Wallace WD (2005) Available and future treatments for erectile dysfunction. Clinical Cornerstone 1:3744

Fig. 5 Alignment of active (a) and inactive (b) ligands to the pharmacophore Acknowledgments The authors are thankful to University Grants Commission, New Delhi, India for providing nancial support. The authors also wish to acknowledge the team of Schrodinger for providing software facility.

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