Patent Abstract:
disclosed are pyrimidyl guanidine derivatives having anticancer activity . the pyimidyl guanidine derivatives have the structure : r 1 is a cyclic or acyclic aliphatic , aromatic , heterocyclic groups optionally substituted , r 2 is a cyclic aliphatic , aromatic , heterocyclic groups optionally substituted by functional groups and r 3 is a cyclic aliphatic , aromatic , heterocyclic groups .

Detailed Description:
in a continuous effort to optimize the physicochemical properties of sc161 , we generated a unique type of pharmacophore model , that we refer to as the conformationally - biased common feature model to generate possible feature combinations . the model development was based on dynamic behavior recorded form the same 10 - ns md simulations recently employed . subsequently , the top - ten ranking pharmacophore models were applied to screen a subset of our small molecule database . finally , we selected structurally diverse and novel compounds which were frequently retrieved by multiple - queries and tested them against a panel of cancer cell lines to validate their cytotoxicity . md studies of sc161 were carried out by gromacs &# 39 ;, a widely used software to simulate various biomolecular systems in aqueous or lipid bilayer environment . 15 - 19 the partial charges of sc161 with polar hydrogens were obtained by ab initio calculation using the unrestricted hartree - fock method in gaussian program , at the high performance computer center , university of southern california . the basis set , the set of one - electron wave functions used to build molecular orbital wave functions , was set to sto - 3g with spin multiplicity of 2 . the topology file of sc161 consistent with gromacs force field was generated by the prodrg server , and was modified with the partial atomic charges by using the values calculated from the above ab initio procedure . next , sc161 was centered in a box of 414 flexible spc , or simple point charge water molecules with a size of 2 . 82 × 2 . 49 × 1 . 86 nm 3 . gromacs force field was used to describe bonding and nonbonding interactions . the whole system was gradually equilibrated for 50 ps at 50k , 100k , 150k , 200k , 250k , and 300k . finally , the production phase was simulated for 10 ns at 300k in a canonical ensemble ( nvt , the number of particles n , the volume v and the temperature t were set to constant values ). the chemical bond lengths involving hydrogen atoms were fixed using shake algorithm . 7 a 2 fs time - step was used and both the energy and trajectory output were collected at 2 ps interval . van der waats interactions and short - range electrostatic interactions were truncated at 10 . 0 with the particle mesh ewald method 21 setting used for long - range electrostatic interactions . trajectory analysis including cluster analysis was carried out using the vega software package 22 , 23 and recently reported elsewhere . the following examples are intended to illustrate , but not to limit , the scope of the invention . while such examples are typical of those that might be used , other procedures known to those skilled in the art may alternatively be utilized . indeed , those of ordinary skill in the art can readily envision and produce further embodiments , based on the teachings herein , without undue experimentation . the pharmacophore models were built on the selected snapshots of sc161 conformation , which were taken from the md trajectory . initially , catalyst software ( accelrys , inc .) package was used to import the sc161 conformation and map the functional features ( h - bond donor , h - bond acceptor , hydrophobic feature , or aromatic ring ) onto the frame . to develop the feature model , geometrical constraints were assigned to each feature . for example , coordinate and size of the feature , centered at the mapped atom or motif was assigned a radius of 1 . 3 å for h - bond donor to avoid feature overlapping and 1 . 6 å , for the rest of the features as default . finally , all the selected features were merged into one pharmacophore model . the generated model was used as an independent search query to screen a subset of our 5 , 000 , 000 small - molecule databases . on the basis of intuitive structural classification , we selected compounds representing the diverse chemical and structural space for their cytotoxic properties . in this study , we focused on two classes of compounds representing oxadiazolopyrazines and quinolins . human breast cancer cells mda - mb - 435 were purchased from the american type culture collection ( manassas , va .). the hct116 p53 +/+ and hct116 p53 −/− cells were kindly provided by dr . bert vogelstein ( johns hopkins medical institutions , baltimore , md .). the human ovarian carcinoma cell line ( hey ) which is naturally resistant to cisplatin , was kindly provided by dr . louis dubeau , university of southern california ( usc ) norris cancer center , and nih3t3 normal mouse fibroblast cells were kindly provided by dr . michael press from usc . hey and nih3t3 cells were maintained as monolayer cultures in rpmi , hct 116 cells in mccoy1s5a media and mda - mb 435 in dmem . media were purchased from ( mediatech , virginia ) and supplemented with 10 % fetal bovine serum ( gemini - bioproducts , woodland , calif . ), 2 mm l - glutamine and 5 % penicillin / streptomycin from bio whittaker were purchased from vwr . cells were incubated at 37 ° c . in a humidified atmosphere of 5 % co 2 . to remove the adherent cells from the flask for passaging and counting , cells were washed with pbs without calcium or magnesium , incubated with a small volume of 0 . 25 % trypsin - edta solution ( sigma - aldrich , st . louis , mo .) for 5 - 10 min , and washed with culture medium and centrifuged . all experiments were performed using cells at exponential growth stage . cells were routinely checked for mycoplasma contamination using a pcr - based assay ( stratagene , tex ., usa ). stock solutions ( 10 mm ) of all compounds were prepared in dmso and stored at − 20 ° c . further dilutions were made fresh in pbs or cell - culture media right before cell treatment . cytotoxicity was assessed by a 3 -( 4 , s - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide ( mtt ) assay as previously described . 24 briefly , cells were seeded in 96 - well microtiter plates and allowed to attach overnight . cells were subsequently treated with continuous exposure to the corresponding drug for 72 h . a mtt solution ( at a final concentration of 0 . 5 mglml ) was added to each well , and cells were incubated for 4 h at 37 ° c . after removal of the medium , dmso was added and the absorbance was read at 570 nm . all assays were done in triplicate . the ic 50 was then determined for each drug from a plot of log ( drug concentration ) versus percentage of cells killed . colony formation assays were also performed to confirm the activity of these compounds as described . 1 , 25 briefly , cells were plated in 6 - well plates at a density of 500 cells / well and allowed to attach . the next day , serial dilutions of the corresponding compounds were added and allowed to incubate for 24 h . after exposure , cells were washed in pbs and cultured in drug - free media until colonies were formed ( 8 - 10 days ). cells were subsequently washed , fixed with a 1 % glutaraldehyde solution for 30 min , and stained with a solution of crystal violet ( 2 %) for 30 min . after staining , cells were thoroughly washed with water . colonies were imaged on the chemidoc imaging system ( bio - rad ) and counted using the quantity one quantitation software package ( bio - rad ). the data reported represent means of at least three independent experiments . md is a well - accepted molecular mechanics approach to sample the time dependent conformational change by solving newton &# 39 ; s equation of motion . previously , we applied md simulations to develop dynamic pharmacophore models bearing features complementary to hiv - 1 integrase . the models were used as searching queries to screen small molecule databases and identified novel integrase inhibitor . 8 , 26 , 27 in this study , we collected data from sc161 simulation in its explicit water environment at the canonical ensemble . energy profile over all the 10 ns showed that the whole system had an average potential energy of − 16576 . 1 kj / mol with & lt ; 0 . 59 % fluctuation , and an average kinetic energy of 3189 kj / mol with & lt ; 2 . 0 % fluctuation . therefore , the total energy of the whole equilibrated system was − 13386 kjmol with 0 . 57 % fluctuation , indicating the system was in its well established stable state . the average simulation temperature was at 300 k with fluctuation less than 6 k . to monitor the conformational changes , six parameters were initially defined to analyze the dynamic behavior of sc161 . as shown in fig1 , two variables , d 1 and d 2 were defined to sample the distance fluctuation between atom pairs n24 to 018 , and 018 to n13 , respectively . in addition , three flexible torsional angles were monitored during the simulations . those were t 1 defined by atoms n13 - c14 - n15 - n16 t , 2 defined by c14 - n15 - n16 - cla7n d t 3 defined by atoms n16 - c17 - c19 - c20 . last , a planar angle was defined describing the orientation between the planes set by the two aromatic fragments of sc161 . plane i ( p 1 hereafter ) was defined by three atoms , c19 , n21 , and n24 of pyrazine motif , and plane ii ( p 2 hereafter ) was defined by n5 , n13 , and c14 of quinoxalin scaffold . apparently , the conformational behavior was strictly dependent on the three flexible dihedral angles , which could consistently be monitored by the above defined planar angle and the two distances d 1 and d 2 . fig2 describes the dynamic behavior of sc161 by means of the time - dependence of the defined variables . the snapshots were recorded at intervals of 2 ps , and the total of 5001 frames representing the 10 - ns simulation studies . among the three torsional angles ( fig2 a ), t 1 is highly populated at a value close to 0 ° or 360 ° which represents the same orientation of the molecule , while t 2 stabilizes at its average of 180 . 0 ° with very small fluctuation . this indicates that the two torsion angles , t 1 and t 2 are rather stable , and that no major conformational change occurred to the quinoxalinhydrazine motif of sc161 which is consistently depicted by the d 2 index averaged at 4 . 8 å ± 0 . 1 å ( fig2 b ). this observation indicates that the conformational change is independent of t 1 , t 2 and d 2 , which are therefore not considered for further conformational analysis . however , torsion angle , t 3 , apparently samples a higher angle space than t 1 and t 2 and its value covers an entire range of 0 °- 360 °. this consequently leads to the wide range fluctuation of both planar angle value ranging from 0 °- 180 °, and d 1 value ranging from 2 . 7 a ( cis position ) to 3 . 8 å ( trans position ) around its average of 3 . 5 å . therefore , the hydrazine motif actively flips at different angles leading to the various conformational clusters of sc161 . we performed further regression analysis for better understanding the correlation of torsion angle t 3 , distance d 1 , and planar angle . fig3 a shows a nearly symmetric profile of distance d 1 versus torsion angle t 3 with respect to the t 3 value of 180 °, and a strong correlation between d 1 and t 3 with the correlation coefficient of 0 . 92 ( r 2 = 0 . 85 ). likewise , the planar angle was selected and further correlated with the torsion angle , t 3 , as described by the scatter plot in fig3 b . this shows a strong linear correlation between the two variables as aligned well by the linear equation functions ( symmetric with respect to the t 3 value of 180 ) and with a correlation coefficient of 0 . 95 ( r 2 = 0 . 895 , 0 . 897 , respectively ). the regression analysis therefore suggests that the conformational change of sc161 solely depends on the behavior of torsion t 3 . as discussed above , we clustered the conformation of sc161 simply based on the distributions of t 3 value , which were calculated from 5001 snapshots . fig4 shows the population of the twelve clusters with an incremental step of 30 ° in t 3 value . due to the overlap between the conformations at t 3 values of 0 ° and 360 °, the first cluster ( cluster a ) contained the frames with t 3 ranging from 0 - 15 ° and 345 °- 360 °. subsequently , cluster b was formed by frames with t 3 values of 15 - 45 °, c with t 3 values of 45 - 75 ° and so on till the twelfth cluster l formed at last . the population of cluster frames shows a gaussian distribution ( fig4 ) with the peak value of 1961 snapshots reached in the middle cluster ( g ) of t 3 range between 180 °± 15 °. on the basis of the above cluster analysis , we focused on the conformations collected in the top - ranked cluster g to build the pharmacophore model . the average structure of the snapshots was first generated , which essentially characterized the dynamic behavior of the frames in the cluster . then , seven features were mapped onto the average structure . they are three h - bond acceptors defined by nitrogens of pyrazine and oxygen atom of the ketone , two h - bond donors defined by the hydrazine linker , and the two hydrophobic features were defined by the three - ring fragment of sc161 ( fig5 ). the pharmacophore model was applied to a subset of our database containing 350 , 000 small molecules where each compound is represented by an ensemble of up to 250 conformations . all together , 938 compounds were mapped by the derived model . with considerations for structural diversity and calculated pharmacokinetic properties , we initially tested 20 compounds and realized that those with oxadiazolopyrazine or quinolin motif showed significant cytotoxicity . we thus extended our search to include more compounds bearing the same scaffolds . therefore , a total of 35 novel compounds were tested for their cytotoxic properties in a panel of cancer cell lines . table 1 lists the compounds identified from this work along with their predicted physiochemical properties and model fitting values . cytotoxicity of these compounds against a panel of cancer cell lines are summarized in table 2 . seventeen compounds showed ic 50 values & lt ; 10 μm . representatives of the oxadiazolopyrazine containing compounds are 15 , 2 , and 6 with icso values & lt ; 3 μm in mda - mb - 435 , hct116 p53 +/+, hct116 p53 −/−, and hey cells . quinolin analogues such as 30 and 32 were , in general , less potent than oxadiazolopyrazines . the best compound , 2 , displayed ic 50 values & lt ; 2 μm in hct116 p53 +/+, hct116 p53 −/−, and hey cells . additionally , 2 also exhibited mild toxicity against nih3t3 ( ic 50 = 8 μm ) compound 15 exhibited ic 50 value of 2 . 8 μm and 6 . 1 μm against mda - mb - 435 cell and hey cell , respectively . 19 was toxic to hct116 p53 +/+ and p53 −/− cells , while not toxic to other selected cells at dose up to 10 . 0 μm . cytotoxicity of 2 was further confirmed by colony formation assay . fig6 shows a representative result of a colony formation assay in hct p53 +/+, and hey cells treated with 2 at various doses . at a dose of 1 μm of 2 , & gt ; 95 % colonies were killed in hct p53 +/+ cells . fig7 shows the representative compounds , 2 and 17 , mapped against the pharmacophore model derived from the structural conformations of cluster g . because of the multiple conformations of each compound , various mapping orientations against the model were observed . in oxadiazolopyrazines , the three h - bond acceptors could favorably be mapped by the nitrogen atoms , while the n — h could map either of the h - bond donors . on the other hand , only one of the hydrophobic features could be mapped by the aromatic motif . we applied the “ hiphop ” module in the catalyst software package ( accelrys , inc .) to generate a “ qualitative model ” based on multiple snapshot inputs without taking the biological data into consideration . this model represents the essential 3d arrangement of functional groups common to the selected set of molecules . in the traditional application of the module , the multiple molecules are the exact input set , however in this work , we concentrated on single active molecules but regarded the multiple conformations of the inputs as described below . we first created ten ensembles to represent the dynamic conformations of sc161 . the 1001 frames of sc161 were collected at every lops interval of the 10 - ns md study , and were represented by 10 ensembles referred to as r 1 , r 2 through r 10 . each ensemble has 100 conformations or frames except rep 1 which has 101 frames with 0 - ps as the beginning snapshot . therefore , r 1 containing 101 conformations of sc161 covers the trajectory : 0 ps - 1000 ps , 1 - 2 : 1010 ps - 2000 ps , r 3 : 2010 ps - 3000 ps , r 4 : 3010 ps - 4000 ps , and so on , through r 10 , which ranges from 9010 ps to 1011 s . the purpose of creating ten compounds representing the conformational diversity of sc161 is to generate a set of inputs for the common feature model development using the catalyst software package . it is not necessary to use ten representations ; we could divide the trajectory into any desired number of subsets as long as it is more than two , the minimum requirement of the common feature model approach . next , we mapped the compound with the chemical functions ( h - bond donor , h - bond acceptor , hydrophobic , negative ionizable charge feature , and positive ionizable feature , etc ) available from the feature dictionary in catalyst . a total of ten top ranking hypotheses were collected and then used as 3d queries for database mining . on the basis of frequency of mapping by the various queries and the intuitive structural classification , we selected compounds representing diverse chemical and structural space for their cytotoxic properties . for this study , we initially tested 93 compounds in a panel of cancer cell lines . human breast cancer cell lines mda - mb - 435 and skbr - 3 were purchased from the american type culture collection ( manassas , va .). the hct116 p53 +/+ and hct116 p53 −/− cell lines were kindly provided by dr . bert vogelstein ( johns hopkins medical institutions , baltimore , md .). the hey human ovarian carcinoma cell line was kindly provided by dr . louis dubeau ( university of southern california norris cancer center ). cells were maintained as monolayer cultures in media supplemented with 10 % fetal bovine serum ( gemini - bioproducts , woodland , calif .) and 2 mmo / ll - glutamine at 37 ° c . in a humidified atmosphere of 5 % c02 . to remove the adherent cells from the flask for passaging and counting , cells were washed with pbs without calcium or magnesium , incubated with a small volume of 0 . 25 % trypsin - edta solution ( sigma - aldrich , st . louis , mo .) for 5 - 10 min , and washed with culture medium and centrifuged . all experiments were performed using cells in exponential growth stage . cells were routinely checked for mycoplasma contamination using a pcr - based assay ( stratagene , cambridge , uk ). stock solutions ( 10 mm ) of all compounds were prepared in dmso and stored at − 20 ° c . further dilutions were made fresh in cell - culture media just prior to cell treatment . cytotoxicity was assessed by a 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazolium bromide ( mtt ) assay as previously described . 23 cells were seeded in 96 - well plates and allowed to attach overnight . cells were subsequently treated with continuous exposure to the corresponding drug for 72 h . an mtt solution ( at a final concentration of 0 . 5 mg / ml ) was added to each well , and cells were incubated for 4 h at 37 ° c . after removal of the medium , dmso was added and the absorbance was read at 570 nm . all assays were performed in triplicate . the ic 50 was determined for each drug from a plot of log ( drug concentration ) versus percentage of cells killed . the overall strategies for deriving the novel pharmacophore model on the basis of sc161 and applying the pharmacophore models in retrieving novel compounds is summarized in fig8 . the molecule sc161 is represented by a set of ten representative ensembles coving the entire trajectory of the 1001 frames taken from the 10 ns md simulation . the top ten ranking common feature pharmacophore models , labeled as m1 - m10 ( fig9 ) were generated on the basis of various conformational behaviors represented by the ten input ensembles . each of the models has five features as listed in fig9 , which show the most favorable mapping of sc161 against each of the ten models . the features derived in the models are an h - bond acceptor , a hydrophobic feature or a positive ionizable feature . table 3 summarizes the feature mapping of the exported hypotheses . according to the ranking score , these ten models have very close scores values . each of the ten input ensembles could map all five - feature in the model as indicated by the value of “ 1 ” in the table about “ direct hit mask ”, while the last part of “ partial hit mask ” with value of “ 0 ” indicates that none of the molecules mapped all but one feature in the model . these hit masks provide a brief way together with the score as well to compare the common feature hypothesis . in table 3 , the pharmacophore models m1 - m8 have similar types of features , i . e . two hydrophobic , one positive ionizable and two h - bond acceptor features . models m9 and m10 , however , have no positive ionizable features , but instead bear three h - bond acceptor features . the positive ionizable feature defined by catalyst not only contains a single positively charged center , but it also includes those having the potential to be positively charged , such as primary , secondary or tertiary amide , amidine , and amidine with substituted hydrogen atom . in comparison with our recently published seven - feature model , which was derived from the single frame of the most probable conformation calculated from the same trajectory ; four of the features from both models are conserved . these are the two h - bond acceptors mapped by the ketone oxygen or either nitrogen of the pyrazine , and the two hydrophobic features mapped by the pyrrolo - quinoxaline motif . superimposition of the original seven - feature model and mi ( the top - ranked model as a representative of the common feature models from this work ), is shown in fig1 c while fig1 a and 10 b describe the individual mapping respectively . the two h - bond donors mapped by the hydrazine in the seven - feature model were not defined in this work . however , the positive ionizable feature was derived ( m1 - m8 ) based on the amidine scaffold of sc161 , while an extra h - bond acceptor was defined by the quinoxaline nitrogen in m9 and m10 . table 4 lists the cluster analysis of the ten models by hierarchical average linkage method available in catalyst . if only two clusters formed , models m1 - m8 are in the same cluster , while m9 and m10 are in the other cluster . apparently , in most situations , the two models m9 and m10 are always clustered in the same group . not surprisingly , in all circumstances , m1 and m2 are always in the same cluster because of the identical feature locations mapped against the same conformation of sc161 ; however , the orientation defined by both the small sphere and the big sphere pair of one h - bond acceptor mapped by a ketone motif is slightly different . in total , 93 compounds were selected from the database mapping against the pharmacophore models . three compounds , smd6 , smd18 and smd58 , displayed potency showing an ic 50 & lt ; 10 μm in skbr - 3 , hct116 p53 +/+, and hey cell lines . smd58 was the most potent with an ic 50 & lt ; 3 . 5 μm in five cancer cell lines . we then carried out structure activity relationship ( sar ) studies to test various analogues of smd6 , smd18 and smd58 . the structures of these compounds , and their tested cytotoxic data are listed in table 5 - 7 . in addition , some inactive compounds are provided in the supporting information . eleven substituents of smd6 bearing n -( 4 , 6 - dimethylpyrimidin - 2 - yl )- acetamidin were tested . three of these smd6 analogues showed cytotoxicity at doses & lt ; 10 um . smd6 - 4 and smd6 - 8 had compatible toxicity profiles with an ic 50 & lt ; 3 . 0 um in certain cancer cell lines . furthermore , 16 additional smd18 substituents were tested by mtt assay . interestingly , only smd18 - 7 and smd18 - 14 showed slightly more potency than their parent smd8 , exhibiting activity at doses & lt ; 10 um finally , we tested 27 additional compounds bearing the core of smd58 ( table 6 ), and 16 of them showed potency against various cancer cell lines . both smd58 and smd58 - 1 had an ic 50 & lt ; 1 um in p53 +/+ cell line indicating much more potency than sc161 . none of the ten models could map all three active compounds ; however , smd6 could be mapped by 4 models , m3 , m7 , m8 , and m10 . smd18 could fit all ten models , while smd58 could fit models m1 - m8 . the possible model mapping of the three potent compounds against certain pharmacophore model is depicted in fig1 . our 10 ns md studies illustrate the dynamic behavior of sc161 and its preference for a planar conformation . as summarized in fig8 , the pharmacophore model using a unique approach derived from the ions - md trajectory , was successfully applied to identify novel cytotoxic compounds . this method comprehensively takes into account all possible conformations of the small molecule . in this study , we show that conformational sampling of a single lead molecule is an efficient approach to build pharmacophore hypotheses and identify compounds with different physicochemical and drug like properties . many modifications and variation of the invention as hereinbefore set forth can be made without departing from the spirit and scope thereof and therefore only such limitations should be imposed as are indicated by the appended claims . all patent and literature references cited in the present specification are hereby incorporated by reference in their entirety . 1 . grande , f . ; aiello , f . ; grazia , 0 . d . ; brizzi , a . ; garofalo , a ; neamati , n . synthesis and antitumor activities of a series of novel quinoxalinhydrazides . bioorg med chem 2007 , 15 , 288 - 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1 integrase . j med chem 2000 , 43 , 2100 - 21 14 . values with standard deviation are from at least three independent experiments and others are as explicitly stated . each experiment was generated from an average of four independent wells . a the column indicates the total number of desired clusters , and the row indicates the total ten - model generated . the entries in the table indicate which cluster the certain model belongs to . the model with the same value as shown in table entry indicates belongs to the same cluster .