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Design and Anticancer Evaluation of Novel Norcantharidin Derivatives with 3D-QSAR studies.

Byline: Chunqi Hu, Guofang Wang, Chunlei Wu and Liping Deng

Summary: Norcantharidin (NCTD) is a demethylated form of cantharidin (CAN) which was discovered from traditional Chinese medicine and is a treatment for cancers. In this study, two series of modification of CAN are designed and proposed based on the x-ray crystal structures of protein phosphatase 1 (PP1) and 2A (PP2A) which have been identified as molecular targets of CAN and NCTD for the anticancer activities. The synthesized compounds were tested by in vitro assays to verify the anticancer efficacy. Furthermore, the structure-activity relationship was discussed of the new efficacious compounds and their 3D-quantitative structure-activity relationship (3D-QSAR) was employed for further investigation on potent target molecules.

Keywords: Anticancer Evaluation, Norcantharidin Derivatives, Aromatic Amines, CoMFA


Cantharidin (Fig. 1), the principle active ingredient of Mylabris, a compound that has been used in China as a medicinal agent for 2000 years and for the treatment of cancer, particularly hepatoma [1].

Cantharidin is potentially attractive for the treatment of leukemia because it does not cause myelosuppression [2, 3] and is effective against cells exerting the multidrug resistance phenotype [4]. Despite such qualities, the nephrotoxicity of cantharidin has prevented it from entering mainstream oncology. Norcantharidin (NCTD Fig. 1), the demethylated analogue of cantharidin, also possesses anticancer activity and stimulates the bone marrow, but without the urinary toxicity. Both agents are known protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) inhibitors [5]. PP1 and PP2A, known as protein serine/threonine phosphatases (PPs), are a family of metalloproteins comprising seven subtypes PP1, PP2A, PP2B, PP4, PP5, PP6 and PP7. The x-ray structures of catalytic domain of PP1 and PP2A have been published, which renders the possibility to design CAN or NCTD analogues based on PP1 and PP2A structures.

Using the structures of PP1 ( pdb code: 3E7A) and PP5c-canthardinc acid complex (pdb code: 3H61) as references, we can get a very clear picture on how CAN binds to the active site of PP1, as shown in Fig. 2(A) ; one of designed CAN analogues docked to PP1 active site, as shown in Fig. 2(B).The bridged O atom in bicycle [2, 2, 1] skeleton is a key group to coordinate to metal atom in the enzyme and anchors the whole CAN to the active site.

There are enough spaces in the active site pocket to accommodate extended groups at the two ends of the CAN scaffold. In our previous study, we have investigated all known cantharidin structure-activity relationship (SAR) data, which can be summarized briefly as following: no modification of the bicycle [2.2.1] skeleton is permissible, the 7-oxa bridge is required to maintain activity, the presence of a double bond (5, 6-ene) has little effect on activity. Replacement of the O-atom (anhydride) with N (as N-H and N-R, where R= aryl or thiazolyl) allow the development of a new series of anhydride modified. Also, pyrazoles and isozazoles [6] have been the subject of chemical and biological studies due to their interesting pharmacology including antipyretic, analgesic, antiinflammatory potential herbicidal, fungicidal and leishmanicidal [7-10] properties.

Stimulated by these findings, we combine pyrazoles and isozazoles with norcantharidin derivatives and designed several series of novel norcantharidin derivatives (Fig. 3).All targted molecules were successfully synthesized [11-13] and tested for their in vitro activities(Table-1), furthermore, based on obtained data, 3D-QSAR study was carried out for a specific SAR discussion for the norcantharidin derivatives[14].


The tumor cell line A549 cultured was centrifuged after it had been digested by pancreatin. As re-suspended with a new medium, the cells were plated in triplicate in 50ul mediums at a density of 300 cells/well in 384-well plates.

All cells were cultured at 37 degC in 5% CO2 for 24 hours. To split charging compounds 1-19 with Bravo. Every compound was in concentrations: 16uM, 8uM, 4uM, 2uM, 1uM, 0.5uM, 0.25uM, 0.125uM. Repeat four times for each treatment.

After 48 hours, it was the time to detect the activity of cells with CCK-8 kit: CCK-8 solution was added (5.0 ul/well), and then, the plates were read on a Microplate Reader at 450 nm; Cells were incubated for 100 minutes in the cell incubator, after that, the plates were read again on a Microplate Reader at 450 nm. Using this variation of the two absorption values divided the variance of the two absorption values which were untreated by norcantharidin analogues. Well then, the average of each of four repeated was the cellular activity in well (Table-2). The inhibition efficiency of the compounds on cells was 1 minus the cell activity in well. At last, the IC50 values were gotten by calculation.

Results and Discussion

Biological Activities

All synthesized norcantharidin derivatives were screened for anti-proliferation activities by MTT assay in vitro against human lung adenocarcinoma eptithelial cells A549: cantharidin (CAN for short) and norcantharidin (NCAD for short) were included as internal standards. The results are presented in Table-1. The cytotoxicity of norcantharidin derivatives was dose-and time-dependent. Table-1, norcantharidin did not exhibit any potency in this test. Thus nineteen compounds displayed improved activities than that of norcantharidin. In this nineteen compounds, four compounds (3d, 4a, 4c and 8d) displayed moderate to potent activities (0.5) with 5 components. The non-cross validated partial least squares analysis results in a conventional r2 of 0.995, F = 264.70, and a standard error of estimated (SEE) of 0.045. The steric eld descriptors explain 51.3% of the variance, while the electrostatic descriptors explain 48.7 %. The predictive correlation coefficient (r2pred) value based on molecules of the test set was 0.605 for the CoMFA model. The CoMFA predicted activities are listed in Table 2. The correlations between the predicted activities and the experimental activities are depicted in Fig. 5.

Table-3: Cross-validated analyses of the CoMFA and CoMSIA models.


Statistical###q2###N###r2###SEE###F###Steric Electrostatic




For the CoMSIA model, LOO analysis gives a result that the cross-validated q2 is 0.633 (>0.5) with 5 components. The non-cross validated PLS analysis results in a conventional r2 of 0.993, F = 166.19, and a standard error of estimated (SEE) of 0.053. The corresponding eld contributions are 17.4, 17.8, 17.2, 35.2 and 12.4%, respectively. The predictive correlation coefficient (r2pred) value based on molecules of the test set was 0.597 for the CoMSIA model. Fig. 6 shows the relationship between the predicted and the experimental pI50 values for the CoMSIA model.

Based on the results of the 3D-QSAR analyses undertaken in the present study, the CoMFA/CoMSIA contour maps offer enough information for us to understand the 3D-QSAR between the compound structures and their inhibitory activities.

3D-QSAR Contour Maps

CoMFA steric and electrostatic contour maps were shown in Fig. 7 The green/yellow contour (Fig. 7A) at R2 group indicated that bulky groups in certain region on R2 could increase the potency, and more positively charged group in this region (blue contour, Fig.7B) enhanced activity. For R1 substituent, a big green contour at the pyrazole or isoxazole, indicating that bulky groups introduced in this region enhanced activity, but for substituent on 7-oxa-bicyclo ring, the yellow contour indicated that introduction a bulky group would reduce the activity. This phenomenon would explain why compound 8d presented as the most potent molecule of all the compounds tested.

CoMSIA contour maps were shown in Fig. 8 Steric and electrostatic contour maps(Fig. 8A-B) also indicated the conclusion made by CoMFA model; In hydrophobic contour map (Fig. 8C), yellow/white contours indicate regions where hydrophobic groups increase/decrease activity, which consistent with the docking result that the molecule was surrounded with hydrophobic amino-acid residue. In hydrogen donor and acceptor fields, the purple (Fig.8D) and red (Fig.8E) contours indicated the introduction of both hydrogen donor or acceptor on core structure were disfavor for the potencies. As for R2 group, the introduction of hydrogen donor moieties was beneficial for the potency. One bulk red contours around the benzyl indicated that a hydrogen bond acceptor substituent at these sites would increase the activity. The inference obtained by Fig. 8E satisfactorily matched the hydrogen bond donor contour map.


In this study, two series of modification of CAN were tested for their anticancer activities. Some of the targeted molecules exhibited improved potency than CAN. To study the structure-relationship of those molecules for better modification on this series of compounds, the 3D-QSAR study (including CoMFA and CoMSIA) was applied and two predicted models with good quality were built. The structural information of active site can be realized by the crystal structure of ligand-protein complex. The in silico modeling methods described here can be used to facilitate, expedite and streamline further more potent norcantharidin derivatives discovery and development.


This study was nancially supported by National Science Foundation for Young Scholars of China under Grant (No. 81502926 and No. 81202411). The authors also thank Prof. Yongzhou Hu of Zhejiang University for helping providing experiment facilities.


1. G. S. Wang, The Clinical Studies of Canthairidin, Acta. Pharm. Sinica., 15, 119 (1980).

2. X. H. Liu, I. Blazsek, M. Comisso, S. Legras, S. Marion and P. Quittet, Effects of Norcantharidin, a Protein Phosphatase Type-2A Inhibitor on the Growth of Normal and Malignant Haemopoietic Cells, Eur. J. Cancer, 31, 953 (1995).

3. S. N. Yi, J. Wass, P. Vincent and H. Iland, Inhibitory Effect of Norcantharidin on K562 Human Myeloid Leukemia Cells in Vitro, Leuk. Res., 15, 883 (1991).

4. T. Efferth, M. Davey, A. Olbrich, G. Rucher, E. Gebhart and R. Davery, Activity of Drugs from Traditional Chinese Medical Toward Sensitive and MDR1- or MRP1-Overexpressing Multidrug-Resistant Human CCRF-CEM Leukemia Cells, Blood Cells, Molecules and Diseases., 28, 160 (2002).

5. A. McCluskey, S. P. Ackland, M. C. Bowyer, M. L. Baldwin, J. Garner and C. C. Walkom. Cantharidin Analogues: Synthesis and Evaluation of Growth Inhibition in a Panel of Selected Tumor Cell Lines, Bioorg. Chem., 31, 68 (2003).

6. J. P. McCormick, J. E. Carrel, J. P. Doom, Origin of Oxygen Atoms in Cantharidin Biosynthesized by Beetles, J. Am. Chem. Soc., 108, 8071 (1986).

7. T. W. Waldrep, J. R. Beck, M. P. Lynch and F. L. Wright, Synthesis and Herbicidal Activity of 1-aryl-5-halo and 1-aryl-5-(trifluoromethyl)-1H-pyrazole-4-carbox amides, J. Agr. Food. Chem, 38, 541 (1990).

8. J. L. Hupartz, Systemic Fungicides the synthesis of pyrazolo [1, 5-a] pyrimidine analogues of carboxin, Aust. J. Chem., 38, 221 (1985).

9. J. L. Hupartz, Systemic Fungicides. The Synthesis of Certain Parazole Analogues of Carboxin, Aust. J. Chem., 36, 135 (1983).

10. V. J. Ram, U. K. Singha and P. Y. Guru, Chemotherapeutic agents XI: Synthesis of Pyrimidines and Azolopyrimidines as Leishmanicides, Eur. J. Med. Chem., 25, 533 (1990).

11. L. P. Deng, F. M. Liu and H. Y. Wang, 1,3-dipolar Cycloaddition Reaction: Synthesis of Novel 5,6-Dehydronorcantharidin Devirativies of Substituated Aromatic Amines with Potential Antitumor Activities, J. Heterocyclic. Chem., 42, 13 (2005).

12. L. P. Deng, Y. Zhang, W. F. Tao, J. F. Shen, and W. Wang. Synthesis of Norcantharidin-Dimer Derivatives, J. Heterocyclic. Chem., 48, 158 (2011).

13. L. P. Deng, Y. Zhang, W. F. Tao, J. F. Shen, and W. Wang. Synthesis of Heterocyclic Substituted Norcantharidin Derivatives. J. Heterocyclic. Chem., 48, 808 (2011).

14. J. Gasteiger, M. Marsili, Iterative Partial Equalization of Orbital Electro Negativity-a Rapid Access to Atomic Charges, Tetrahedron., 36, 3219 (1980).

15. Y. Ai, S. T. Wang, P. H. Sun, F. J. Song, Molecular Modeling Studies of 4,5-dihydro-1H-pyralozo[4,3-h] Quinazoline Derivatives as Potent CDK2/cycli A Inhibitors using 3D-QSAR and Docking, Int. J. Mol .Sci., 11, 3705 (2010).

16. S. N. Yi, J. Wass, P. Vincent and H. Iland, Inhibitory Effect of Norcantharidin on K562 Human Myeloid Leukemia Cells in Vitro. Leuk. Res., 15, 883 (1991).

17. S. Wold, Cross-Validatory Estimation of the Number of Components in Factor and Principal Components Models, Technometrics., 20, 397 (1978).
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Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Date:Apr 30, 2017
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