asthma and chronic obstructive pulmonary disease ( copd ) are the two most prevalent chronic airway diseases . copd is a treatable and preventable disease but current predictions are that it will continue to increase as an important cause of mortality and morbidity worldwide [ 12 ] . phosphodiesterases ( pdes ) have been classified into at least 11 families ( pde 111 ) according to their substrate sensitivity , inhibitor selectivity , ca / calmodulin requirement and amino acid sequences [ 34 ] . phosphodiesterase-4 ( pde4 ) is a key enzyme in the hydrolysis of camp in mast cells , basophils , eosinophils , monocytes and lymphocytes , as well as areas in the brain and airway smooth muscle [ 56 ] . pde4 plays a significant role in modulating the activity of camp , an important second messenger that mediates the relaxation of airway smooth muscle and suppresses inflammatory cell function , thereby attenuating the inflammatory response . increasing the intracellular concentration of camp in the airway tissues and cells suppresses inflammatory cell function and thus should be beneficial for treatment of asthma and copd . over the last two decades only a small number of these drugs have the potential to be approved for market [ 910 ] . comparative molecular field analysis ( comfa ) is one of the well known 3d - qsar descriptors which has been used regularly to produce the three dimensional models to indicate the regions that affect biological activity with a change in the chemical substitution . the advantages of comfa are the ability to predict the biological activities of the molecules and to represent the relationships between steric / electrostatic property and biological activity in the form of contour maps gives key features on not only the ligand - receptor interaction but also the topology of the receptor . we present here our 3d - qsar studies using comfa method on a training set of 5,6-dihydro-(9h)-pyrazolo-[4,3-c]-1,2,4-triazolo-[4,3-]-pyridine derivatives as pde4 inhibitors by considering the steric and electrostatic influences . the model deduced from this investigation provides underlying structural requirements and good predictive ability , which could aid new pde4 inhibitors prior to their synthesis . the structures of the 5,6-dihydro-(9h)-pyrazolo-[4,3-c]-1,2,4-triazolo-[4,3-]-pyridine derivatives and the biological activities data were obtained from the reference . the negative logarithm of ic50 ( pic50 ) was used as the biological activity in the 3d - qsar study ( table 1 ) . three - dimensional structure building and all modeling were performed using the sybyl 7.0 program package   on a personal computer equipped with a pentium iv processor . geometry optimization was carried out using maximin molecular mechanics and tripos force field , gasteiger hckle charge supplied within sybyl7.0 , with the convergence criterion set at 0.05 kcal/( mol ) . an external test set consisting of four molecules was used to validate the comfa models . a common substructure - based alignment was adopted in the present study , which attempted to align molecules to the template molecule on a common backbone . the alignment of the training set molecules was derived by sybyl 7.0 ( figure 2 ) . comfa of these molecules was carried out on the steric and electrostatic fields using the default values . the steric and electrostatic comfa potential fields were calculated at each lattice intersection of a regularly spaced grid of 2.0  . the grid box dimensions were determined automatically in such a way that the region boundaries were extended beyond 4  in each direction from the coordinates of each molecule . the steric and electrostatic fields were calculated separately for each molecule using sp carbon atom probe with a charge of 1 ( default probe atom in sybyl ) and energy cut - off values of 30 kcal / mol for both steric and electrostatic fields . the relationship between the structural parameters ( comfa interaction energies ) and the biological activities has been quantified by the pls algorithm . pls regression technique is especially useful in quite common case where the number of descriptors ( independent variables ) is comparable to or greater than the number of compounds ( data points ) and/or there exist other factors leading to correlations between variables . the cross - validation analysis was carried out using leave - one - out ( loo ) method where one compound is removed from the dataset and its activity is predicted using the model derived from the rest of the dataset . the cross - validated q and the optimum number of components were obtained . to speed up the analysis and reduce noise , a minimum column filtering value (  ) of 2.00 kcal / mol was used for the cross - validation . finally , non - cross - validated analysis was performed to calculate non - cross - validated r using the optimal number of previously identified components and was employed to analyze the result of the comfa . the structures of the 5,6-dihydro-(9h)-pyrazolo-[4,3-c]-1,2,4-triazolo-[4,3-]-pyridine derivatives and the biological activities data were obtained from the reference . the negative logarithm of ic50 ( pic50 ) was used as the biological activity in the 3d - qsar study ( table 1 ) . three - dimensional structure building and all modeling were performed using the sybyl 7.0 program package   on a personal computer equipped with a pentium iv processor . geometry optimization was carried out using maximin molecular mechanics and tripos force field , gasteiger hckle charge supplied within sybyl7.0 , with the convergence criterion set at 0.05 kcal/( mol ) . an external test set consisting of four molecules was used to validate the comfa models . a common substructure - based alignment was adopted in the present study , which attempted to align molecules to the template molecule on a common backbone . the alignment of the training set molecules was derived by sybyl 7.0 ( figure 2 ) . comfa of these molecules was carried out on the steric and electrostatic fields using the default values . the steric and electrostatic comfa potential fields were calculated at each lattice intersection of a regularly spaced grid of 2.0  . the grid box dimensions were determined automatically in such a way that the region boundaries were extended beyond 4  in each direction from the coordinates of each molecule . the steric and electrostatic fields were calculated separately for each molecule using sp carbon atom probe with a charge of 1 ( default probe atom in sybyl ) and energy cut - off values of 30 kcal / mol for both steric and electrostatic fields . the relationship between the structural parameters ( comfa interaction energies ) and the biological activities has been quantified by the pls algorithm . pls regression technique is especially useful in quite common case where the number of descriptors ( independent variables ) is comparable to or greater than the number of compounds ( data points ) and/or there exist other factors leading to correlations between variables . the cross - validation analysis was carried out using leave - one - out ( loo ) method where one compound is removed from the dataset and its activity is predicted using the model derived from the rest of the dataset . the cross - validated q and the optimum number of components were obtained . to speed up the analysis and reduce noise , a minimum column filtering value (  ) of 2.00 kcal / mol was used for the cross - validation . finally , non - cross - validated analysis was performed to calculate non - cross - validated r using the optimal number of previously identified components and was employed to analyze the result of the comfa . thirty - one molecules were randomly partitioned into a training set of 27 and a test set of four compounds with bias given to both chemical and biological diversity in both the training set and the test set molecules . despite the ambiguity of drug - receptor interactions in general , a statistically significant model was obtained from the comfa study . a  cross - validated q  may then be defined , completely analogously to the definition of the conventional q , as where press is the standard errors of the cross - validated predictions and sd is the sum of squared deviations of each biological property value from their mean and press , or predictive sum of squares , is the sum , over all compounds , of the squared differences between the actual and  predicted  biological property values . often for qsars developed with comfa a shift in the q values is observed as the grid spacing is altered . to examine this possibility with these data , the different grid boxes with 1.0 , 1.5 , 2.0 , 2.5 , and 3.0  grid spacing , the influence of the different grid spacing to comfa model is obvious . only from the q after leave - one - out cross - validation , the model with the grid spacing of 2.0  was selected as the best model ( see table 2 ) . the leave - one - out cross - validated pls analysis of the best model gave rise to a cross - validated value ( q ) of 0.741 , suggesting that the model is a useful tool for predicting pde4 inhibitory activity . the correlation coefficient between the calculated and experimental activities , non - cross - validated value ( r ) of 0.954 with standard error estimate 0.211 . the respective relative contributions of steric and electrostatic fields were 0.915 and 0.085 , indicating that steric field is more predominant . then the condition without electrostatic were studied and the new q and r values were 0.739 and 0.953 respectively . the actual and predicted values of the training set by the best model are given in table 1 , and the graph of observed activity versus predicted activities of training set molecules from comfa analysis is illustrated in figure 3 . the 3d - qsar model was validated using a test set ( in table 1 ) of 4 compounds , which were not included in the development of the model . on the basis of the pls statistics of comfa model , this is further validated by the residual values of the test set ( table 1 ) . figure 4 represents the graph of the actual versus predicted pic50 values of the test set molecules for comfa model . the contour plot representations of the comfa results for pde4 inhibitors are presented in figures 5 and 6 using compound 24 as reference structure . the green - colored regions indicate areas where steric bulk enhances pde4 inhibitory activity , while the yellow contours indicate regions where steric bulk is detrimental for the biological activity . blue - colored regions show areas where electropositive charged groups enhance pde4 inhibitory activity , while red regions represent where electronegative charged groups improve the activity . the electrostatic contour map displayed in figure 5 shows a region of red polyhedral space , indicating that the electron - rich groups are beneficial to the activity . compounds 4 and 7 have higher inhibition activity than compound 10 because the chlorine atom is placed within the electron - rich charge red contour . additionally , a blue polyhedron in the top left corner of figure 5 , indicates that electron - rich substituents will reduce the biological activity . compound 11 , with a strong electron withdrawing substituent , 4-trifluoromethylphenyl , can not fit into the electropositively charged blue contour . the plot shows the cyclobutyl group of molecule 24 placed between two big green polyhedrons . if a substituent , such as 3-pentyl group , is attached on molecule 28 , it occupies the green contour and enhances the activity . in compounds 25 and 29 , where the substituent group r is cyclopentyl and 1-methylcyclohex-1-yl , respectively , these fit into the green contour , and the activity of these compounds compounds 20 , 21 and 22 , with methyl , ethyl and propyl substituents , show less activity than compound 28 . additionally , the contour plot shows a yellow polyhedron in the top left corner of figure 6 . if a bulky substituent occupies the yellow contour this will depress the biological activity . compounds 8 , 9 and 11 , with 4-methoxyphenyl , 4-chlorophenyl and 4-trifluoromethylphenyl substituents , respectively , are very close to the yellow contour which represents the unfavored steric region . 3d qsar studies were performed using comfa on a series of derivatives with inhibitory activity on phosphodiesterase-4 . a satisfactory comfa model was obtained with loo cross - validation q and non - cross - validated r values of 0.741 and 0.954 respectively . the developed model also possesses promising predictive ability as discerned by the testing on the external test set , and should be useful to elucidate the relationship between compound structures and biological activities and to facilitate design of more potent phosphodiesterase-4 inhibitors . for example , use of a p - trifluoromethyl substituent in a cyclobutyl maybe increase the biological activity , since a fluorine atom at that position participates in the electron - rich charge red contour and the trifluoromethyl will fit into the green contour . similarly , other p - substituents on the cycylobutyl moiety might show even greater inhibitory power than a simple cyclobutyl .
phosphodiesterase-4 ( pde4 ) plays an important role in treatment of asthma and chronic obstructive pulmonary disease . 
 thirty - one analogs displaying variable inhibition of pde4 were selected to develop models for establishing three - dimensional quantitative structure - activity relationships ( 3d - qsar ) . comparative molecular field analysis ( comfa ) 
 was conducted on the group of analogs to determine the structural requirements for potency in inhibiting pde4 . 
 the resulting model exhibited good q2 and r2 values up to 0.741 and 0.954 for comfa . 
 the contributions from the steric and electrostatic fields were 0.915 and 0.085 respectively . 
 the 3d - qsar model should be very useful for design of novel pde 4 inhibitors .