Insights into the structural features of anticancer 1,6-naphthyridines and pyridopyrimidines as VEGFR-2 inhibitors: 3D-QSAR study

INTRODUCTION

The process of the development of arterioles from pre-existing vessels is known as angiogenesis, which plays a climacteric role in proliferation, migration, and survival of endothelial cells (Carmeliet and Jain, 2011). Stimulation of angiogenesis is amidst the hallmarks of tumor growth and cancer (Fouad and Aanei, 2017; Zhao and Adjei, 2015). Vascular endothelial growth factors (VEGFs) and vascular endothelial growth factor receptors (VEGFRs) have a remarkable impact on angiogenesis. Five VEGFs, namely VEGFA, VEGFB, VEGFC, VEGFD, and placental growth factor (PLGF), are involved in the activation of VEGFRs signaling cascade. VEGFRs consist of three receptors, namely VEGFR-1 (Flt-1), VEGFR-2 [Flk-1/kinase domain receptor (KDR)], and VEGFR-3 (Flt-4) (Takahashi and Shibuya, 2005). Among the VEGFR family, KDR is a well-established receptor for the discovery of novel antineoplastic agents (Modi and Kulkarni, 2019). Additionally, the disproportional elevation of VEGFs has been observed due to the activation of oncogenes, loss of tumor suppressor function, and alterations in glucose or oxygen levels. In contrast, autophosphorylation of VEGFR-2 in cancer has been observed due to the overexpression of VEGFs. VEGFR-2 signaling cascade and its role in cancer are shown in Figure 1. Furthermore, several small molecule VEGFR-2 modulators have been developed successfully, some of which are in the clinical trials (Fig. 2) (Frampton, 2012; Harris et al., 2008; Ho and Jonasch, 2011; Roskoski, 2007; Woo and Heo, 2012; Yakes et al., 2011). However, the failure of VEGFR-2 inhibitors in the clinic can be due to both acquired and intrinsic resistance. The occurrence of resistance is due to redundant signaling of receptors, development of hypoxia tolerant tumor cells, hypoxia-resistant malignant clones’ selection, elevation in circulating nontumor proangiogenic factors, and mutations of endothelial cells (Abdullah and Perez-Soler, 2012; Ellis and Hicklin, 2008; Jayson et al., 2016). Furthermore, toxicity associated with VEGFR-2 inhibitors includes thromboembolic complications, proteinuria, hemorrhage, anal fistula, gastrointestinal (GI) perforation, posterior reversible encephalopathy syndrome, hand–foot skin reaction, oral mucositis, diarrhea, thyroid disease, and bone marrow suppression (Kumar et al., 2009; Shepard and Garcia, 2009). These indicate that the discovery of selective VEGFR-2 inhibitors that can overcome the problem of resistance and toxicity remains a considerable task.

Figure 1. VEGFR-2 signaling cascades and its role in cancer. FAK = Focal Adhesion Kinase; HPC = Hematopoietic Progenitor Cell; DAG = Diacylglycerol; HSP-27 = Heat-Shock Protein-27; eNOS = Endothelial Nitric Oxide Synthase; MAPK = Mitogen-Activated Protein Kinase; PI3K = Phosphatidylinositol 3′ Kinase; PKC = Protein Kinase C; TSAd = T-cell-specific Adaptor molecule. PLCγ, Phospholipase C-γ; SHB, SH2, and β-cells. *Figure prepared using online tool BioRender (https://biorender.com). [Click here to view]

3D-QSAR and pharmacophore modeling is a type of ligand-based drug design approach and a widely used method for designing novel analogues with potent inhibitory activity against the biological target. 3D-QSAR enables the identification of pharmacophoric descriptors like hydrophobic, steric, electrostatic, H-bond donor, and acceptor, responsible for the interaction of the molecules with the active site of the receptor. In comparative molecular field analysis (CoMFA), steric and electrostatic descriptors (independent variables) are correlated with the inhibitory activity of ligands (Cramer et al., 1988; Zhu et al., 2005). In 1994, comparative molecular similarity indices analysis (CoMSIA) method was instigated by Klebe et al. (1994) It is an amended technique of CoMFA in which H-bond donor and acceptor, hydrophobic, electrostatic and steric descriptors are correlated with the inhibitory activity of ligands. The most prominent obstacle during drug discovery is the insufficient ADME (Absorption, Distribution, Metabolism, Excretion) parameters of drug molecules. Therefore, a computer-aided drug design approach with the prediction of ADME can rectify the problem of developing new hits during the drug discovery process.

Figure 2. Structures of Food and Drug Administration (FDA)-approved VEGFR-2 inhibitors. [Click here to view]

Naphthyridine and pyridopyrimidine analogous are well-known heterocyclic compounds for cancer chemotherapy. Wang et al. (2020) reported pyridine derivatives bearing 1,6-naphthyridine scaffold as MET and VEGFR-2 inhibitors using scaffold hopping approach (MET, IC₅₀ = 9.8; VEGFR-2 IC₅₀ = 8.8 nM). Kiselev et al. (2010) reported Dibenzo[c,h][1,6]naphthyridines as anticancer agents targeting topoisomerase I enzyme. Salerno et al. (2019) reported Pyrido [3′, 2′: 5, 6] thiopyrano [4, 3-d]pyrimidine analogues as multitargeted tyrosine kinase inhibitors with potent VEGFR-2 inhibitory activity, IC₅₀ = 16 nM. The US20160096832 invention pertains to the synthesis of 7,8-dihydropyrido[4,3-d]pyrimidine derivatives as TKIs for the treatment of cancer (Allergan, Inc., US20160096832, 2016). The US20160096837 invention discloses the synthesis of pyrazolo[1,5-a]pyrido[3,4-e]pyrimidine analogues as tyrosine kinase inhibitors (TKIs) (Allergan, Inc., US20160096837, 2016). With this fact in the current research, CoMFA and CoMSIA models were established for a series of 1,6-naphthyridine and pyridopyrimidine analogues to specify the regions surrounding the molecule where modification might be possible to enhance their activity against VEGFR-2. The developed models were validated using the external validation method and were used further to study contour maps and the structure–activity relationship (SAR). Lastly, the CoMSIA model was used for designing novel analogues and predictions of their inhibitory activity.

MATERIAL AND METHODS

CoMFA and CoMSIA field generation

For the calculation of CoMFA and CoMSIA fields, a 3D cubic lattice (defined with a grid spacing of 2 Å extended to 4 Å units) beyond the aligned molecules in X, Y, Z directions was used. At the lattice intersection, electrostatic and steric interaction descriptors were evaluated by Lennard-Jones and Columbic potentials. In CoMFA, electrostatic and steric descriptors were derived using Sp³ carbon atom (van der Waals radius of 1.52 Å and +1.0 charge) as a probe atom. However, the cutoff value of steric and electrostatic descriptors was shortened to its original value (±30 kcal/mol), and the scale was set to the CoMFA standard.

Table 1. Structure of 1,6-naphthyridine and pyridopyrimidine analogues and their inhibitory activity against VEGFR-2. [Click here to view]

In CoMSIA, the binding capacities of the molecule were related to the alterations in the molecular feature exposed by the field, which is predominantly an extension of CoMFA. At all grid points, the distance of the probe atom and molecular atom was calculated using the Gaussian function. The calculation for CoMSIA is as follow:

${\mathrm{A}}_{\mathrm{F}\mathrm{,}\mathrm{k}\left(\mathit{j}\right)}^{\mathrm{q}}=\sum {\mathrm{W}}_{\mathrm{probe}\mathrm{,}\mathrm{k}}{\mathrm{W}}_{\mathrm{ik}}{\mathit{e}}^{-\alpha {\mathrm{r}}_{\mathrm{iq}}^2}$

Where A represents the similarity index at grid point q, summed over all atoms i of the molecule j under investigation. W_probe,k is the probe atom with radius 1 Å, charge +1, hydrophobicity +1, hydrogen bond donating +1, hydrogen bond accepting +1. w_ik is the actual value of the physicochemical property k of atom i. r_iq is the mutual distance between the probe atom at grid point q and atom i of the test molecule. α is the attenuation factor, with a default value of 0.3, and an optimal value normally between 0.2 and 0.4 (Bhansali and Kulkarni, 2014).

Figure 3. Common fragment for the alignment of the data set. [Click here to view]

Figure 4. The aligned data set for CoMFA and CoMSIA) model generation. [Click here to view]

RESULTS AND DISCUSSION

CoMFA and CoMSIA model generation

Electrostatic and steric descriptors were used for the generation of the CoMFA model. Initially, the PLS analysis was performed by “LOO” cross-validation correlation (q² = 0.659; ONC = 6). Non-cross-validation correlation was done by column filtering 2.0 and the same ONC (r²_ncv = 0.987; F value = 439.868; SEE = 0.123). The steric and electrostatic field contributions obtained 5.921 and 0.000, respectively (Table 2). The bootstrap analysis r²_bs (0.994) and cross-validation coefficient r²_cv (0.621) supported the consistency of the developed QSAR model. The pIC₅₀ values (experimental and predicted) of the data set obtained from the CoMFA model are given in Table 3. and 4. The correlation between actual and predicted activities of training and test set analogues based on the CoMFA model is shown in Figure 5.

Table 2. Summary of CoMFA and CoMSIA models. [Click here to view]

Based on the CoMFA results, CoMSIA was carried out using a similar data set. The cross-validation correlation coefficient (q²) was found to be 0.689, and non-cross validation correlation coefficient (r²_ncv) was found to be 0.985 with six ONC. The contribution of CoMFA descriptors are as follows: steric (0.759), electrostatic (0.000), hydrophobic (0.349), H-bond donor (0.189), and H-bond acceptor (0.310). The internal reliability of the data set was evaluated using cross-validation method (r²_cv = 0.678). The robustness of the model was determined by bootstrap analysis r²_bs (0.992) and SEE_bs (0.096) (Table 2). The pIC₅₀ values (actual and predicted) of training and test set analogues based on the CoMSIA model are presented in Table 3 and 4. The correlation between actual and predicted activities of the training and the test set analogues based on the CoMSIA model is shown in Figure 6. The residual activities differences of analogues as histogram based on the CoMFA and CoMSIA model are shown in Figure 7.

3D-QSAR visualization

CoMFA contour maps

3D contour maps are the essential features of the CoMFA and CoMSIA. Contour maps were generated as a result of alternations in the molecular fields (standard deviations and the least squares coefficients; StDev*Coeff) allotted to every single grid intersection within the active site. Based on these contour maps, the chemical structures are modified to optimize the inhibitory activity. The most potent analogue 20 (IC₅₀ = 0.003 µM) and least active analogue 45 were chosen for the analysis of contour maps. Here, around the molecule, a transparent style was selected to visualize the generated contour maps. The steric field is characterized by green (favorable) and yellow contour (unfavorable). In green contour, the incorporation of bulky substituents lead to the enhancement of inhibitory activity, whereas in yellow contour, the incorporation of bulky substituent lead to the loss of biological activity. For better understanding, the potent analogue 20 alienated into three regions: “a,” “b,” and “c” (Fig. 8).

The green contour in Figure 9a at the “b” region suggests that the bulky substituent and substituted urea moieties may be favorable for inhibitory activity. This is evident from the experimental IC₅₀ values (µM) of analogues 14 (0.003), 17 (0.005), 19 (0.006), 20 (0.003), 23 (0.006), 26 (0.004), 102 (0.062), 103 (0.021), and 105 (0.071). In these analogues, -NHCONHtBu, the bulky substituent present at the second position of 1,6-naphthyridine ring and pyridopyrimidine ring, leads to potent inhibitory activity. Whereas in analogues 15 (IC₅₀ = 0.18 µM), 21 (IC₅₀ = 0.22 µM), 27 (IC₅₀ = 2.9 µM), 30 (IC₅₀ = 3.6 µM), and 35 (IC₅₀ = 1.2 µM), -NH₂, substituent present at the second position of 1,6-naphthyridine ring, leads to poor efficacy against VEGFR-2. Furthermore, as observed in Figure 9a, the yellow contour at “a” region at -NH group suggests that the bulky substituents are unfavorable for inhibitory activity, which is apparent from the actual IC₅₀ values (µM) of analogues 39 (0.67), 40 (0.65), 44 (1.9), 45 (13), and 46 (7.6), where -N(Ac)(CH2)_xOBn bulky substituent present at the seventh position of 1,6-naphthyridine ring leads to a remarkable loss of biological activity. Furthermore, the green contours were observed at the phenyl ring (third position of 1,6-naphthyridine and pyridopyrimidine ring), which indicates that phenyl ring substituted with bulky groups is favorable for inhibitory activity. Here, phenyl ring substituted with 3,5-diOMe and 2,6-diCl substituents exhibits potent activity in comparison to the unsubstituted phenyl ring. This is apparent from the experimental IC₅₀ values (µM) of analogues 27 (2.9), 29 (8.7), 30 (3.6), and 31 (0.18).

Table 3. Actual and predicted pIC50 values and residual values of training set analogues by CoMFA and CoMSIA. [Click here to view]

Table 4. Actual and predicted pIC50 values and residual values of test set analogues by CoMFA and CoMSIA. [Click here to view]

Figure 5. The plot of actual and predicted activities of the training and the test set analogues based on the CoMFA model. [Click here to view]

Figure 6. The plot of actual and predicted activities of the training and the test set analogues based on the CoMSIA model. [Click here to view]

Comparative Molecular Similarity Indices Analysis contour maps

CoMSIA steric field contour maps are almost homogenous to CoMFA. Additionally, in CoMSIA steric map, the green contour at the “c” region in Figure 10a indicates that the bulky substituents favored the potent activity. In most of the analogues, the 1,3-dimethoxyphenyl ring present at the third position of 1,6-naphthyridines and pyridopyrimidines suggests that the replacement with more bulky groups may enhance the VEGFR-2 inhibitory activity. Furthermore, similar to CoMFA steric maps, the yellow contour was observed at the “a” region specifically at -NH group, which suggests that the tertiary amine group is unfavorable for the activity. This is apparent from the IC₅₀ values of analogues 39, 40, 44, 45, and 46, where -N(Ac)(CH2)_xOBn substituent present which results in a remarkable loss of activity. Additionally, at “a” region green contour near aliphatic linker indicates that bulky substituents are favored in this region. The generated contour maps from CoMSIA for the most active analogue 20 and the least active analogue 45 represented in Figures 10 and 11.

The hydrophobic field are represented by yellow and white contour maps (Fig. 10b). To enhance the inhibitory activity, hydrophobic groups are favorable in the yellow contour, while hydrophilic groups are favorable in white contour. In Figure 10b at “c’ region near phenyl ring, a large yellow contour suggests that hydrophobic substituent can be favorable for inhibitory activity. While in “a” and “b” region, white contour indicates that the hydrophilic group can enhance the activity.

Figure 7. (a, b) Histogram of CoMFA and CoMSIA residual values for the training set analogues; (c, d) Histogram of CoMFA and CoMSIA residual values for the test set analogues. [Click here to view]

Figure 8. The most potent analogue, 20 alienated into (a), (b), and (c) regions. [Click here to view]

The CoMSIA donor field is indicated by cyan and purple contour maps. H-bond donor groups are favorable in cyan contour maps. In contrast, purple contour indicates that the H-bond donor groups are unfavorable. Here, in Figure 10c at the “b” region, H-bond donor groups are unfavorable for the biological activity. The acceptor field is characterized by magenta and red contour maps. Magenta contour represents a H-bond acceptor substituent that is favorable, while red contour represents a H-bond acceptor substituent that is not favorable. In Figure 10d, at “b” region magenta contour present at -C=O functional group, which suggests that urea or amide moiety is necessary for the significant biological activity. Replacement of urea functional group with amine leads to loss of activity i.e., analogues 15 (IC₅₀ = 0.18 µM), 21 (IC₅₀ = 0.22 µM), 27 (IC₅₀ = 2.9 µM), and 35 (IC₅₀ = 1.2 µM). While red contour observed at the “b” region indicates that acceptor substituents are unfavoured for inhibitory activity. This is evident from the experimental IC₅₀ values of analogues 39, 40, 44, 45, and 46.

ADME Studies

The in silico prediction of ADME properties identifies whether the new molecules could be toxic or nontoxic, able or unable to cross membranes, and can be metabolized by the body into an active or inactive form. ADME predictions using QikProp involves the use of molecular descriptors. The pharmaceutically relevant descriptors are shown in Table 6. Log P represents lipophilicity, which is a crucial factor governing passive membrane partitioning. Increased log P value increases permeability while reducing solubility. The octanol–water partition coefficient (QPlogPo/w) and aqueous solubility (QPlogS) were found in the range of 3.140–5.056 and –6.227 to –3.923, respectively. The blood-brain barrier (BBB) partition coefficient (QPlogBB) was found ranging from –0.019 to 0.269. QPlogHERG (predicted IC₅₀ value for the blockage of human Ether-à-go-go-Related Gene (HERG) K+ channels) ranged from –8.144 to –6.479. QPPCaco (Caco-2 cell permeability; mm/second) and QPlogKhsa (Prediction of binding to human serum albumin) varied from 12.733 to 170.97 and 1.020 to 0.328, respectively. Moreover, the percentage of human oral absorption was found in the range of 39.190%–88.895% for all the analogues. The pharmacokinetic parameters for the 16 designed analogues fell under the acceptable range that can be suitable for human use, which also reveals the potential of designed analogues as possible drug-like candidates.

Figure 9. Contour maps of CoMFA (StDev*Coeff) (a, b) steric field for analogue 20 and analogue 45. StDev*Coeff, standard deviations, and the least-squares coefficients allotted to each grid intersection were contoured within the binding pocket [Click here to view]

Figure 10. Contour maps of CoMSIA (stDev*Coeff) (a) steric (b) hydrophobic (c) H-bond donor and (d) H-bond acceptor fields for analogue 20. StDev*Coeff, standard deviations, and the least-squares coefficients assigned to each grid intersection were contoured within the binding pocket. [Click here to view]

Figure 11. Contour maps CoMSIA (stDev*Coeff) (a) steric (b) hydrophobic (c) H-bond donor and (d) H-bond acceptor fields for analogue 45. StDev*Coeff, standard deviations, and the least-squares coefficients assigned to each grid intersection were contoured within the binding pocket. [Click here to view]

Figure 12. SAR based on 3D-QSAR. [Click here to view]

Table 5. Predicted pIC50 values of designed analogues using the developed CoMSIA model. [Click here to view]

Table 6. ADME properties of designed analogues. [Click here to view]

CONCLUSION

VEGFR-2/KDR is a transmembrane receptor tyrosine kinase expressed on the endothelial cells and is activated by VEGFs, which is a promising target for angiogenesis inhibition and suppresses tumor growth. We performed 3D-QSAR using CoMFA and CoMSIA models to correlate the structural parameters of 1,6-naphthyridine and pyridopyrimidine analogues with their VEGFR-2 inhibitory activity. PLS analysis was carried out to evaluate the developed 3D-QSAR model, which indicates that all statistical parameters were obtained reasonably. The conventional correlation coefficient (r²_ncv) and cross-validation coefficient (q²) were found to be 0.985 and 0.987 and 0.659 and 0.689 for CoMFA and CoMSIA, respectively. The predictability of CoMSIA was found to be better than CoMFA. The results suggest that steric, hydrophobic, donor and acceptor descriptors have a remarkable impact on inhibitory activity; on the contrary, the electrostatic field has no contribution. In 1,6-naphthyridine analogues, the substituents present on the 3-phenyl ring are significant for inhibitory activity. The order of inhibitory activity was found as follows: (3-(3,5-dimethoxyphenyl) > 3-(2,6-dichlorophenyl) > 3-phenyl). The contour maps analysis suggests that bulky, hydrophobic and H-bond acceptor substituents are favored at the second position of 1,6-naphthyridine ring near the “b” region. The replacement of urea moieties in analogues with thiourea or amide may enhance the biological activity. Furthermore, at the third position of 1,6-naphthyridine ring at the “c” region, the bulky and hydrophobic substituent could enhance the biological activity. Moreover, majority of FDA approved VEGFR-2 inhibitors consist of (I) aromatic ring or nitrogen-containing heteroaromatic ring, (II) core aromatic or heteroaromatic ring either monocyclic or bicyclic, (III) amide/urea/thiourea functional group (H-bond acceptor and H-bond donor group), (IV) an aromatic ring substituted with halogens. On the same line, the reported 1,6-naphthyridine and pyridopyrimidine analogues also consist of an aromatic ring attached via an aliphatic linker, core nitrogen-containing aromatic bicyclic ring, urea functional group, and substituted aromatic ring. Based on the developed 3D-QSAR model, contour map analysis, and structure finding, novel inhibitors were designed with little modification on the most potent analogue 20, and their activities were predicted using the CoMSIA model. The ADME properties of designed analogues were also found to be satisfactory in order to become drug-like candidates. The synthesis and biological activity screening of designed compounds were undertaken. The 3D-QSAR analysis summarised in this research work can be useful for the designing of novel 1,6-naphthyridine and pyridopyrimidine analogues as VEGFR-2 inhibitors.

ACKNOWLEDGMENTS

The authors are grateful to Dr. Shivajirao Kadam, Chancellor, Bharati Vidyapeeth (Deemed to be University), Pune, and Dr. Kakasaheb R. Mahadik, Principal, Poona College of Pharmacy, Pune, Maharashtra, India, for their encouragement.

AUTHOR CONTRIBUTIONS

The authors have approved the final version of the manuscript and contributed equally.

CONFLICT OF INTEREST

The authors declare no conflict of interest pertaining to this manuscript.

FUNDING

There was no funding source pertaining to this research work.

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