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3D-QSAR STUDIES ON 2- ARYLCARBONYL -3-TRIFLUOROMETHYLQUINOXALINE 1, 4-DI-NOXIDE DERIVATIVES AND THEIR REDUCED ANALOGUES USING K-NN MFA APPROACH Joohee Pradhan1*, Dr. Rajesh Sharma 2, Dr. Anju Goyal3 Geetanjali Institute of Pharmacy,Dabok, Udaipur, Rajasthan, India-313022 2 School of Pharmacy, Devi Ahilya Vishwavidyalaya, Takshshila Campus, Khandwa Road, Indore, M.P., India-452 017 3 B.N Girls College of Pharmacy, Sewashram Road, Udaipur, Rajasthan, India-313001 Email: juhipradhan123@gmail.com
1

Joohee Pradhan

ABSTRACT A set of thirty three Quinoxaline derivatives with cytotoxic activities against nine types of tumoral subgroups was subjected to three dimensional quantitative structure activity relationship studies through recently introduced k- nearest neighbor molecular field analysis with step wise, simulated annealing and genetic algorithm as variable selection methods resulting in eight statistically significant models for leukemia, nonsmall lung, colon, CNS, ovarian, prostate and breast cancers. These models gave a value of q2 as high as 0.9122 for model 1 and value of pred_r2 as high as 0.6738 for model 3.The k-NN MFA contour plots provided further understanding of the relationship between the structural features of quinoxaline derivatives and their activities, which should be applicable to design new, potential cytotoxic agents. Key Words: 3D-QSAR, k-NN MFA, Quinoxaline derivatives, Anticancer.

INTRODUCTION The search of anticancer compounds has always been on the desk top of molecular modeling and drug design specialists. In

spite of this intensive search, the discovery of selective anti cancer compounds has remained a largely illusive goal of cancer research. Subsequently, new approaches

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are needed in order to make an efficient search for candidates to be assayed as anti cancer drugs [1-6]. Quinoxaline derivatives are a class of substances possessing a broad spectrum of pharmacological activities including [7]. A series of thirty three anticancer quinoxaline derivatives (Fig. 1) having good activities against leukemia, nonsmall lung, colon, CNS, melanoma, ovarian, renal, prostate and breast cancers were subjected to three dimensional quantitative structure activity relationship (3D-QSAR) studies in order to find new, potent anticancer agents. Resent trends in 2D/3D-QSAR have focused on the development of procedure that allows selection of optimal variables from the pool of descriptors of chemical structures i.e. ones that are most meaningful and statistically significant in terms of correlation with biological activity. This is accomplished by combining one of the stochastic search methods such as SA, GAs, or evolutionary algorithms with the correlation methods such as MLR, PLSR, or artificial neural networks [8-13]. The k- nearest neighbor molecular field analysis (k-NN MFA), used for 3D-QSAR analysis of the present data set adopts a k-nearest neighbor principle for generating relationships of molecular fields with the experimentally reported activity. The variables and optimal k values were chosen using three variable selection methods viz. step wise (SW), simulated annealing (SA), and genetic algorithm (GA). Like many 3DQSAR methods, k-NN MFA requires suitable alignment of given set of molecules. This is followed by generation of a common rectangular grid around the molecules. The steric and electrostatic interaction energies are computed at the lattice points of the grid using a methyl probe of charge +1. These interaction energy values are considered for relationship generation and utilized as

descriptors to decide nearness between molecules. The optimal training and test sets are generated using the sphere exclusion algorithm. This algorithm allows the construction of training sets covering descriptor space occupied by representative points. Once the training and test sets are generated, k-NN methodology is applied to the descriptors generated over the grid [14]. EXPERIMENTAL WORK The growth inhibitory data of quinoxaline derivatives (Table 1) against nine types of tumor cells were collected from reported work of Beatriz Solano et.al. [15]. All the biological activity data (Gi50 in M) were converted to log Gi50 (Table 2A and 2B) to reduce skew ness of data set. The molecular modeling was carried out on Compaq PC having Pentium IV processor and windows XP operating system, using the software namely: Molecular Design Suite supplied by the VLife Sciences, Pune (VLife MDS) [16]. The structures were constructed using the 2D draw application and converted to 3D structures. Energy minimization and geometry optimization was conducted using Merck Molecular Force Field (MMFF) method with Root Mean Square (RMS) gradient set to 0.01 Kcal/mol and iteration limit to 10000. Alignment of all the thirty-three compounds was done using template based alignment in MDS; the aligned structures were used for the study. In the template based alignment method, a template structure was defined and used as a basis for alignment of a set of molecules. In the present case, benzene nucleus was the template used for template-based alignment, as it was common to all structures. The alignment of molecules is shown in Fig.2. For calculation of field descriptor values, using Tripos force field, both electrostatic and steric field type with cut offs 10.0 and 30.0 Kcal/mol respectively 2

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were selected and charge type was selected as Gasteiger Marsili. Dielectric constant was set to 1.0 considering the distance dependent dielectric function. Probe setting was carbon atom with charge 1.0 and grid setting as follows: From X: - 6.730200 Y: - 7.636500 Z: - 12.37640 To 14.603500 10.074900 8.074400 Interval 2.0000 2.0000 2.0000

This resulted in calculation of 2080 field descriptors (1040 for each electrostatic and steric) for all the compounds in separate columns. For performing QSAR analysis, all the invariable columns were removed from the work sheet, as they do not contribute to QSAR. The optimal test and training data set were generated using sphere exclusion method. The dissimilarity level was set to 5, as the higher the dissimilarity level, the lesser the predictive ability of QSAR model. The method resulted in selection of eight compounds as test set and remaining others as training set. Building k-NN MFA Models Since there was a large pool of descriptors available to build model, various variable selection methods viz. stepwise variable selection, simulated annealing, and genetic algorithm were used along with k-nearest neighborhood (k-NN) to find optimal sub-set of descriptors for k-NN MFA model. Step-wise variable selection method The k-NN MFA models were developed using step-wise forward-backward method with cross correlation limit set to 0.5 and term selection criteria as q2. F-test in was set to 4.0 and F-test out to 3.99. As some additional parameters, variance cut-off was set as 2 Kcal/mol and scaling and auto scaling, additionally the k-nearest neighbor parameter setting was done within the range

of 2-5 and prediction method was selected as distance based weighted average. Simulated annealing variable selection method The cross correlation limit was set as 0.5, terms in model as 4, maximum temperature as 100 0C, min temperature as 0.01, iteration at given temperature as 5, decrease temperature by as 10, seed as 0, perturbation limit as 1, and term selection criteria as q2. Rests of the settings were as described in stepwise variable selection method. Genetic algorithm variable selection method Cross correlation limit as 0.5, crossover probability as 0.95, Mutation probability as 0.05, term selection criteria as q2, population as 10, number of generations 10000, print after iterations: 100, seed as 0. Convergence criteria 0.01, convergence ending criteria: 5 and chromosome length as 3. Additional parameter settings were as described in step-wise variable selection method. Among the various models generated for the selected members of training and test sets, 8 statistically significant models were identified. The software VLife MDS allows user to choose probe, grid size, and grid interval for the generation of descriptors. The variable selection methods along with the corresponding parameters are allowed to be chosen and optimum models are generated by maximizing q2. The fitness plots for the entire statistically significant models viz. model 1 to 8 are given in Fig.3 and corresponding observed and predicted activities are depicted in Tables 3A and 3B. The 3D plots included in Fig.4 shows the electrostatic and steric fields important for interaction of molecules with space, and thus modification of these fields according to the ranges obtained may result into more potent compounds. 3

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RESULTS AND DISCUSSION The k-NN MFA of quinoxaline derivatives with reported cytotoxic activities against all the nine types of tumor cells using SW, SA, and GA variable selection methods resulted in several statistically significant models, of which corresponding best models are reported herein. The model selection criteria being the value of q2, the internal predictive ability of the model, and that of pred_r2, the ability of model to predict the activity of external test set. For activity against leukemia cell lines, model 1 generated with SW variable selection method, found to be statistically most significant especially with respect to internal Predictive ability (q2=0.9122) of the model. The model showed an external predictive ability of about 50 %( pred_r2=0.4826). Model 1: Model for leukemia S_424 (-0.7613 to 30.0000); E_350(0.4939 to 10.0000); E_732 (-1.6457 to -6.1262); S_774 (-0.1099 to -0.0814) q2 = 0.9122; q2_se = 0.3034; Pred_r2 = 0.4826 ;pred_r2se = 0.7773 Another statistically significant model model 2 was obtained for cytotoxic activity against nonsmall lung through SW k-NN MFA justified by internal and external Predictive ability of the model as 87% (q2=0.8696) and 43% (pred_r2=0.4357) respectively. Model 2: Model for Non small lung cancer S_425 (1.6809 to 14.9994); E_566 (-4.1910 to 4.4985)

q2 = 0.8696 ;q2_se = 0.2707 Predr2 = 0.4357 ;pred_r2se = 0.6130 For colon cancer cells, Model 3 had a value of q2=0.8008 and that of pred_r2=0.6738 that explained 80% of total variance (internal Predictive ability) and 67% predictive power for the external test set. Model 3: Model for colon cancer S_647 (-0.7403 to -0.5645); S_481 (-0.6315 to -0.5299) q2 = 0.8008; q2_se = 0.3954 Pred_r2 = 0.6738; pred_r2se = 0.4552 Another statistically significant model, model 4 was generated for activity against CNS cancer cells having a value of q2=0.8480 and that of pred_r2=0.4054. Model 4: Model for CNS cancer S_425 (1.6809 to 1.9651); E_262 (-10.000 to -3.4808) q2 = 0.8480; q2_se = 0.2711 Pred_r2 = 0.4054; pred_r2se = 0.5683 Model 5 and model 6 with values of q2 0.8969 and 0.8622 also proved their statistical significance supported by fairly well values of pred_r2 (0.3740 and 0.3371 respectively). Model 5 and 6: Models for ovarian cancer Model 5 S_424 (-0.5352 to 30.0000); E_262 (10.0000 to 3.7342) q2 = 0.8969; q2_se = 0.2395 Pred_r2 = 0.3470; pred_r2se = 0.4966 Model 6 S_481 (-0.6315 to 30.0000); E_251 (10.0000 to -6.4378) q2 = 0.8622 ;q2_se = 0.2769 Predr_2 = 0.3371; pred_r2se = 0.5003

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The measure of goodness of internal Predictive ability q2 also allowed mentioning here model 7 and 8. Model 8C developed through SA k-NN MFA for cytotoxic activity against prostate cells was having internal Predictive ability of 77% (q2=0.7721) and 59% (pred_r2=0.5950) respectively. Model 7: Model for prostate cancer S_424 (-0.5352 to -0.5197); E_163 (-3.3142 to 0.2325) q2 = 0.7712; q2_se = 0.4124 Predr2 = 0.5950; pred_r2se = 0.3999 The last significant model, model 8 was selected as model for activity against breast cancer as justified by value of q2=0.7882 and that of pred_r2=0.5728. Model 8 : Model for breast cancer S_647 (-0.7403 to -0.5645) ; S_481 (0.6315 to -0.5299) q2 = 0.7882; q2_se = 0.3607 Predr2 = 0.5728; pred_r2se = 0.4250 The k-NN MFA contour plots which showed the relative position and ranges of the corresponding important electrostatic/steric fields in the model provided guidelines for new molecule design. A negative range indicated that negative electrostatic potential was favorable for increase in the activity and hence a more electro negative substituent group was preferred in that region. Conversely, a Positive range indicated that positive electrostatic potential was favorable for increase in the activity and hence a less electronegative substituent group was preferred in that region. As far as steric field is concerned, negative range indicated that negative steric potential was favorable for increase in the activity and hence less bulky substituent group was preferred in that region. A positive range indicated that positive steric potential was favorable for increase in the activity and hence more bulky substituent group was preferred in that region. A deep insight into the resulted models revealed that in almost

all the models, both electrostatic and steric descriptors are equally important (contribution 50:50) in describing the biological activity of the quinoxaline derivatives, except for activity against colon and breast cancer cells, where only steric descriptors play the role. For activity against leukemia, electronegative sustituents at Ra/Rb along with furyl or thienyl group at position A may result into more active compounds. A less activity of compounds 5, 10, 11, 15, and 24 further proves the fact. In order to find potent and selective agent against non small lung cancer, a deep insight into model 2 revealed that sterically bulky group is favored at position A with a more flexible range of substitution at position 3. The thienyl group without substitution at position Ra and Rb would be favorable for activity as is evident from compound 17, having maximum activity against non small lung cancer cells. In addition, a methyl group at position Ra/Rb would also be beneficial. For activity against colon cancer, model 3 having electrostatic and steric contribution as 66.6 and 33.3 percent respectively, allows less bulky substituents to be preferred at position A and less electro negativity is required at position 4. Consequently, naphthyl ring at position A and reduced compounds at position 1 and 4 would be detrimental for the activity. Further, a more electro negative substituent at Ra and Rb and a more bulky group at A would be required for activity against CNS cancer cells; methoxy group at Rb is discouraged. Models 5 and 6 for ovarian cancer incorporate both steric and electrostatic descriptors equally where more flexibility is allowed sterically, while more electro negative substitution at Ra and/or Rb would be beneficial for the activity supported by 5

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the fact that compound 17 without any substitution at these positions shows no activity against prostate cancer. Thienyl group at position A is again preferred for activity. As far as activity against breast cancer is concerned, only steric factor decides the specificity where a less bulky substitution at position 4 would be beneficial with phenyl or naphthyl ring discouraged at position A. CONCLUSIONS 3D-QSAR studies were performed using kNN MFA on a series of quinoxaline derivatives with growth inhibitory activity against nine type of tumoral subgroup of cells. Eight statistically significant models were obtained with LOO cross validated q2 values 0.9122, 0.8696, 0.8008, 0.8480, 0.8969, 0.8622, 0.7721, 0.7882 for models 1to 9 respectively. The developed models also possess promising predictive ability as discerned by testing on the external test set and should be useful to elucidate the relationship between compound structure and biological activities and to facilitate design of more potent and selective cytotoxic agents. For example thienyl or furyl ring at position A may result in more potent inhibitors than phenyl, naphthyl, since the increase in bulk due to ring at this place would not fit the ranges so obtained. Similarly, a methoxy substitution at Rb may always be unfavorable for cytotoxic activity. ACKNOWLEDGEMENT The authors are thankful to Head, School of Pharmacy to provide facilities and Vlife Science Technologies Pvt. Ltd, 1, Akshay 50, Anand Park, Aundh, Pune, India to provide the software. REFERENCES:

1. Estrada E, Uriarte E, Montero A, Teijeria M, Santana L, and Clercq E De, A Novel Approach for the Virtual Screening and Rational Design of Anticancer Compounds, J. Med. Chem., 43, 2000,19751985. 2. Denny WA, in The search for anticancer drugs, M J Warring, B A J Ponder (Eds), Kluwer, Dordrecht, 1992, 320-338. 3. Kubinyi H, J. Recept. Signal Transduct. Res, 19, 1999,15-18. 4. Walters WP, Stahl MT and Murcko MA, Virtual Screening an overview, Drug Discov. Today, 3, 1998, 160-178. 5. Ferrante K, Winograd B and Canetta R, Cancer Chemother. Pharmacol., 43, 1999, S61. 6. Menta E and Palumbo M, Antineoplastic agents, Expert Opin. Ther Pat., 8, 1998, 1627-1672. 7. Porter A E A, In Comprehensive Heterocyclic Chemistry, Pergarmon, New York, , 1984, 157-197. 8. Sutter J M, Dixon S L and Jurs P C, Automated Descriptor Selection for Quantitative Structure-Activity Relationships Using Generalized Simulated Annealing, Chem Inf. Comput.Sci.,35,1995,77-84. 9. Rogers D and Hopfinger A J, Application of Genetic Function Approximation to Quantitative Structure-Activity Relationships and Quantitative Structure-Property Relationships, J. Chem. Inf. Comput. Sci., 34, 1994, 854-866. 10. Kubinyi H, Variable Selection in QSAR Studies. I. An Evolutionary Algorithm, Quant.Struct.-Act.Relat.,13,1994,285-294. 11. Kubinyi H, Variable Selection in QSAR Studies. II. A Highly Efficient Combination of Systematic Search and Evolution, Quant. Struct.-Act. Relat., 13, 1994, 393-401. 12. Luke B T, Evolutionary Programming Applied to the Development of Quantitative Structure-Activity Relationships and Quantitative StructureProperty 6

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Relationships, J. Chem. Inf. Comput. Sci., 34, 1994, 1279-1287. 13. So S S and Karplus M, Evolutionary Optimization in Quantitative StructureActivity Relationship: An Application of Genetic Neural Networks, J. Med. Chem., 39, 1994, 1521-1530. 14. Ajmani S, Jadhav K and Kulkarni S A, Three Dimensional QSAR using K-Nearest Neighbor, Method & Its interpretation, J. Chem. Inf. Model, 46, 2006, 24-31. 15. Solano B, Junnotula V R, Marn A, Villar R, Burguete A, Vicente E, PerezTables and Figures:

Silanes S, Aldana I, Monge A, Dutta S, Sarkar U, and Gates KS, Synthesis and Biological Evaluation of New 2-Arylcarbonyl3-trifluoromethylquinoxaline1, 4-Di-N-oxide Derivatives and Their Reduced Analogues, J. Med. Chem., 2007, accessed 10 oct 2007. 16. Vlife MDS software package, version 3.0, supplied by Vlife science technologies Pvt. Ltd, 1, Akshay 50, Anand park, Aundh, Pune,India 411007.

Table1 Structures of 2 Arylcarbonyl 3-trifluromethylquinoxaline 1, 4 DiN oxide derivatives and their reduced analogs
Compound 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Ra H H CF3 F Cl H H H CF3 H CH3 F Cl H H H H H CF3 H F Cl H H H H H Rb F CF3 H F Cl F Cl CF3 H H CH3 F Cl CH3 OCH3 F Cl CF3 H H F Cl CH3 OCH3 F Cl CF3 A phenyl phenyl phenyl naphthyl naphthyl naphthyl naphthyl naphthyl naphthyl thienyl thienyl thienyl thienyl thienyl thienyl thienyl thienyl thienyl thienyl furyl furyl furyl furyl furyl furyl furyl furyl

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28
O F3 C N O
N

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H furyl
OO O N+

CF3
O O

30

31

F3C

29
N -O
+

CF3

F3 C

N+ OOO

CF3
N CF3

32

33
N+ O

F3 C

F3C

CF3

CF3

Table 2A Cytotoxic activities (mean Gi50 M Inhibition Cell Growth and pGi50) against tumoral subgroup cell lines of Quinoxaline derivatives
Com p. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Leukemia Gi50 0.23 0.24 0.77 0.77 1.58 0.44 0.65 0.26 1.20 13.9 3 2.71 0.05 0.02 0.49 4.43 0.27 0.07 0.20 0.03 0.07 0.43 0.27 0.26 3.96 0.30 0.19 0.21 0.13 51.0 9 48.4 pGi50 -0.638 -0.619 -0.113 -0.113 0.198 -0.356 -0.187 -0.585 0.079 1.144 0.433 -1.301 -1.699 -0.309 0.646 -0.568 -1.155 -0.699 -1.523 -1.155 -0.366 -0.568 -0.585 0.597 -0.522 -0.721 -0.677 -0.886 1.708 1.685 Nonsmall lung Gi50 pGi50 0.80 -0.097 0.64 -0.194 1.66 0.220 3.47 0.540 1.70 0.100 1.54 0.187 1.51 0.178 1.42 0.152 1.78 0.250 0.35 2.81 0.83 2.04 0.56 3.93 1.68 0.07 0.45 0.30 0.35 0.73 0.28 0.47 3.49 3.13 0.36 0.76 0.48 48.1 4 41.9 -0.456 0.448 -0.081 0.309 -0.252 0.594 0.225 -1.154 -0.346 -0.522 -0.456 -0.136 -0.553 -0.328 0.542 0.495 -0.443 0.1191 -0.318 1.682 1.622 Colon Gi50 0.54 0.46 1.04 1.71 1.55 0.74 1.29 0.66 1.73 0.83 5.31 0.27 0.59 0.88 7.08 0.92 0.04 0.38 0.15 0.51 0.19 0.50 1.17 2.51 1.06 0.21 0.57 0.28 41.4 1 75.1 pGi50 -0.267 -0.337 0.017 0.233 0.190 -0.130 0.110 -0.180 0.238 -0.081 0.725 -0.568 -0.229 -0.055 0.850 -0.036 -1.397 -0.420 -0.824 -0.292 -0.721 -0.301 0.068 0.399 0.025 -0.677 -0.244 -0.552 1.617 1.875 CNS Gi50 1.45 0.69 1.71 2.51 1.63 1.61 1.47 0.95 1.72 1.45 3.05 0.44 0.67 0.75 6.24 1.63 0.09 0.48 0.18 0.40 0.37 0.79 0.74 3.20 2.35 0.77 0.91 0.81 45.5 3 35.4 pGi50 0.161 -0.161 0.233 0.399 0.212 0.206 0.167 -0.022 0.235 0.161 0.484 -0.356 -0.174 -0.125 0.795 0.212 -1.045 -0.318 -0.744 -0.398 -0.431 -0.102 -0.130 0.505 0.371 -0.113 -0.040 -0.091 1.658 1.549 Melanoma Gi50 0.95 1.13 1.23 2.50 1.57 1.12 1.23 0.97 1.48 1.39 3.99 0.39 0.94 0.90 5.89 1.51 0.12 0.90 0.33 0.67 0.56 1.57 0.88 3.03 1.56 0.49 1.11 0.86 42.9 0 46.1 pGi50 -0.022 0.053 0.089 0.397 0.195 0.049 0.089 -0.013 0.170 0.143 0.600 -0.408 -0.026 -0.045 0.770 0.178 -0.920 -0.045 -0.481 -0.173 -0.251 60.195 -0.055 0.481 0.193 -0.309 0.045 -0.065 1.632 1.663

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2 88.1 0 43.6 5 53.9 5 3 39.4 1 47.7 4 81.5 2 1 49.7 9 85.3 9 100. 0 8 25.3 1 24.4 5 75.8 6 1 31.5 2 80.5 8 100. 0

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31 32 33

1.945 1.639 1.732

1.595 1.679 1.911

1.697 1.931 2.000

1.403 1.388 1.880

1.498 1.906 2.000

Table 2B Cytotoxic activities (mean Gi50 M Inhibition Cell Growth and pGi50) against tumoral subgroup cell lines of Quinoxaline derivatives
Comp. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 Ovarian Gi50 pGi50 1.34 0.127 0.58 -0.236 1.18 0.071 3.80 0.579 1.51 0.178 1.20 0.079 1.20 0.079 0.47 -0.327 1.69 0.227 0.75 -0.124 2.34 0.369 0.53 -0.275 1.16 0.064 0.27 -0.568 4.99 0.698 1.17 0.068 0.16 -0.795 0.43 -0.366 0.20 -0.698 1.45 0.161 0.39 -0.408 1.65 0.217 0.85 -0.070 2.51 0.399 2.03 0.307 0.56 -0.251 0.72 -0.142 0.65 -0.187 42.49 1.628 42.99 1.633 29.29 1.466 40.43 1.606 90.85 1.958 Renal Gi50 pGi50 1.14 0.056 0.33 -0.481 1.47 0.167 2.18 0.338 1.64 0.214 1.48 0.170 1.38 0.139 0.58 -0.236 1.55 0.190 0.48 -0.318 4.62 0.664 0.50 -0.301 1.60 0.204 0.25 -0.602 5.11 0.708 1.65 0.206 0.03 -1.522 0.31 -0.508 0.24 -0.619 0.33 -0.481 0.36 -0.443 0.31 -0.508 0.58 -0.236 1.74 0.240 2.41 0.382 0.20 -0.698 0.43 -0.366 0.49 -0.309 60.60 1.782 49.69 1.696 28.59 1.456 34.57 1.538 100.00 2.000 Prostate Gi50 pGi50 0.53 -0.275 0.27 -0.568 1.32 0.120 1.46 0.164 1.55 0.190 1.62 0.209 1.48 0.170 0.57 -0.244 1.51 0.178 0.40 -0.397 3.89 0.589 0.51 -0.292 0.79 -0.102 0.89 -0.050 2.48 0.394 1.51 0.178 0.20 -0.698 0.15 -0.823 0.20 -0.698 0.42 -0.376 0.33 -0.481 0.24 -0.619 3.16 0.499 2.60 0.414 0.11 -0.958 0.42 -0.376 0.19 -0.721 51.88 1.715 35.08 1.545 38.46 1.585 44.67 1.650 100.00 2.000 Breast Gi50 pGi50 0.73 -0.136 0.54 -0.267 1.41 0.149 2.36 0.372 1.62 0.209 1.70 0.230 1.15 0.060 0.99 -0.004 1.67 0.222 0.85 -0.070 3.08 0.488 0.41 -0.387 0.79 -0.102 0.56 -0.251 4.92 0.691 1.19 0.075 0.08 -1.096 0.50 -0.301 0.18 -0.744 0.76 -0.119 0.20 -0.698 1.38 0.139 0.66 -0.180 2.88 0.459 1.33 0.123 0.28 -0.552 0.57 -0.244 0.48 -0.318 32.04 1.505 59.11 1.771 34.33 1.535 38.52 1.585 84.00 1.924

(-) indicates absence of biological activity

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Table 3A Observed and predicted activities of statistically significant models obtained by k-NN MFA
Compd. 1 2 3 5 6 7 8 9 10 11 12 15 16 19 21 23 24 26 27 28 29 30 31 32 33 4* 13* 14* 17* 18* 20* 22* 25* Model 1 Obs Pred -0.6383 -0.5852 -0.6198 -0.6197 -0.1135 -0.5885 0.1978 -0.1871 -0.3565 -0.3437 -0.1871 -0.585 0.0792 1.144 0.433 -1.301 0.6464 -0.5686 -1.5229 -0.3665 -0.585 0.5977 -0.7212 -0.6778 -0.8661 1.7083 1.665 1.945 1.64 1.732 -0.1135 -1.699 -0.3098 -1.1549 -0.699 -1.1549 -0.5686 -0.5229 0.1091 -0.4112 0.3258 0.4035 0.8641 -1.1363 0.6771 -0.6338 -1.042 -0.8235 -0.4006 0.351 -0.6083 -0.6515 -0.4643 1.6901 1.7065 1.5557 1.7153 1.6879 -0.1354 -0.8263 0.367 0.3266 -0.6067 -0.2726 -0.657 -0.6642 Model 2 Obs Pred -0.0969 -0.2466 -0.1938 -0.0246 0.2201 0.3494 0.2304 -0.1699 0.1875 0.1939 0.179 0.1523 0.2504 -0.4559 0.4487 -0.0809 0.5944 0.2253 -0.5229 -0.1376 -0.3279 0.5428 -0.4437 -0.1192 -0.3188 1.6825 1.6225 1.5956 1.6789 1.9112 -1.1549 -0.5528 -0.4559 -0.3468 -0.2518 0.3096 -0.5528 0.4955 0.2001 0.2122 0.0677 -0.1541 0.0371 -0.302 1.6641 -0.2052 -0.1608 -0.2817 0.0676 0.2876 -0.1998 -0.3623 -0.2052 1.6508 1.6152 1.6619 1.6229 1.6508 0.5403 0.3096 -0.2518 -1.1549 -0.3468 -0.4559 -0.301 0.0253 Model 3 Obs Pred -0.2676 0.017 -0.3372 0.2142 0.017 -0.2676 0.1903 -0.0498 -0.1308 0.1741 0.1106 -0.1805 0.238 -0.0809 0.7251 -0.5686 0.85 -0.0362 -0.8239 -0.7212 0.0682 0.3997 -0.6778 -0.2441 -0.5528 1.6171 1.8757 1.6971 1.9314 2.000 0.233 -0.2291 -0.0555 -1.3979 -0.4202 -0.2924 -0.3265 -0.4617 0.0525 -0.0735 -0.0735 -0.1083 0.2341 -0.3567 -0.0098 -0.6231 -0.0887 -0.5528 0.5622 0.3965 0.078 -0.3805 -0.721 1.8798 1.6686 1.9637 1.8497 1.8221 0.2146 -0.3007 0.3964 -0.4304 -0.2079 -0.3561 -0.4617 0.2146 Model 4 Obs Pred 0.1614 0.196 -0.1612 0.0465 0.233 -0.1819 0.2122 -0.3902 0.2068 0.0397 0.1673 -0.0223 0.2355 0.1614 0.4843 -0.3565 0.7952 0.2122 -0.7447 -0.4318 -0.1308 0.5051 -0.1135 -0.041 -0.0915 1.6583 1.55 1.4033 1.3883 1.88 0.3997 -0.1739 -0.1249 -1.0458 -0.3188 -0.3979 -0.1024 0.3711 0.0397 -0.0968 0.1554 0.1957 0.4305 -0.7239 0.4941 -0.1387 -0.2918 0.0397 0.001 0.7569 -0.0248 -0.0834 -0.0737 1.6697 1.3883 1.7404 1.55 1.5774 -0.3058 -0.3043 -0.0281 0.0174 0.0045 0.186 -0.2974 0.0204

*test set compounds

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Table 3B Observed and predicted activities of statistically significant models obtained by k-NN MFA
Compd. 1 2 3 5 6 7 8 9 10 11 12 15 16 19 21 23 24 26 27 28 29 30 31 32 33 4* 13* 14* 17* 18* 20* 22* 25* Model 5 Obs Pred 0.1271 -0.1023 -0.2366 -0.0305 0.0719 -0.3087 0.179 -0.0007 0.0792 -0.3102 0.0792 -0.3279 0.2279 -0.1249 0.3692 -0.2757 0.6981 0.0682 -0.699 -0.4089 -0.0706 0.3997 -0.2518 -0.1427 -0.1871 1.6283 1.6334 1.4667 1.6067 1.9583 0.5798 0.0645 -0.5686 -0.7959 -0.3665 0.1614 0.2175 0.3075 0.042 -0.2288 -0.0532 -0.0511 0.4733 -0.1546 0.3544 -0.0903 -0.1586 -0.2126 -0.0658 0.5069 -0.0269 -0.0478 -0.2569 1.9583 1.6067 1.7089 1.6334 1.6283 -0.1546 0.0206 -0.0286 -0.0286 -0.0618 -0.0505 0.0202 -0.0506 Model 6 Obs Pred 0.1271 0.0466 -0.2366 0.082 0.0719 -0.285 0.179 -0.1046 0.0792 0.0792 0.0792 -0.3279 0.2279 -0.1249 0.3692 -0.2757 0.6981 0.0682 -0.699 -0.4089 -0.0706 0.3997 -0.2518 -0.1427 -0.1871 1.6283 1.6334 1.4667 1.6067 1.9583 0.5798 0.0645 -0.5686 -0.7959 -0.3665 0.1614 0.2175 0.3075 0.0679 -0.2627 -0.3454 0.0895 -0.0831 -0.2733 0.3976 0.0734 -0.1944 -0.2463 0.1641 0.6935 0.0706 0.0357 -0.2912 1.6673 1.6543 1.6893 1.5854 1.5416 -0.1643 -0.1084 -0.0506 -0.699 -0.699 -0.4815 0.415 Model 7 Obs Pred -0.2757 -0.1951 -0.5686 -0.0462 0.1206 -0.5292 0.1903 0.1846 0.2095 -0.3328 0.1703 -0.2441 0.179 -0.3979 0.58 -0.2924 0.3845 0.179 -0.3239 -0.3768 -0.6198 0.4997 -0.9586 -0.3768 -0.7212 1.715 1.5451 1.585 1.65 2 -0.337 -0.1023 -0.0305 -0.5096 -0.1172 -0.3822 -0.0792 0.1836 -0.3504 -0.1194 -0.7596 -0.5068 -0.1466 0.5211 -0.4237 -0.0643 -0.1052 -0.6343 0.4446 -0.4207 -0.1946 -0.5388 1.713 1.6519 1.8396 1.5514 1.9862 -0.4304 -0.384 -0.3265 -0.3561 -0.3007 0.3964 -0.4617 0.2146 Model 8 Obs Pred -0.1367 0.1492 -0.2627 0.2162 0.1492 -0.1367 0.2095 -0.0226 0.2304 0.1414 0.0607 -0.0044 0.2227 -0.0706 0.4886 -0.3872 0.692 0.0755 -0.7447 -0.699 -0.1805 0.4594 -0.5528 -0.2441 -0.3188 1.5057 1.7717 1.5357 1.5857 1.9243 0.3729 -0.1024 -0.2518 -1.0969 -0.301 -0.1192 0.1399 0.1239 0.2266 -0.029 -0.029 0.0356 0.1397 -0.2383 0.1454 -0.4698 -0.2124 -0.3178 0.474 0.1539 0.0505 -0.4643 -0.6987 1.7767 1.562 1.745 1.7315 1.5623 0.2162 -0.3128 0.1538 -0.335 -0.1385 -0.2378 -0.23 -0.399

*test set compounds 11

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O Rb
7 6 3 8 1

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N
2

Ra

N
4

CF3

Fig 1 Parent structure of quinoxaline derivatives.

Fig.2 Template based alignment of Molecules.

Model 1

Model 2

Model 3

Model 4 12

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Model 5 Model 6 Model 7 Model 8 Fig 3 Graphsa of observed v/s predicted activities of Model 1 to 8 Obtained through kNN MFA.
a

Observed activity on X-axis; predicted activity on Y-axis; Red dots represent training set compounds; Blue dots represent test set compounds.

Model 1

Model 2

Model 3

Model 4

Model 5

Model 6 Model 7 Fig 4 3D Plots of Models 1to 8 th obtained

Model 8

13
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