Accepted Manuscript Design, synthesis, and SAR of embelin analogues as the inhibitors of PAI-1 (Plasminogen Activator Inhibitor-1) Fanglei Chen, Guiping Zhang, Zebin Hong, Zhonghui Lin, Min Lei, Mingdong Huang, Lihong Hu PII: DOI: Reference:
S0960-894X(14)00272-8 http://dx.doi.org/10.1016/j.bmcl.2014.03.045 BMCL 21438
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
20 January 2014 12 March 2014 15 March 2014
Please cite this article as: Chen, F., Zhang, G., Hong, Z., Lin, Z., Lei, M., Huang, M., Hu, L., Design, synthesis, and SAR of embelin analogues as the inhibitors of PAI-1 (Plasminogen Activator Inhibitor-1), Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.03.045
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Graphical Abstract
Design, synthesis, and SAR of embelin analogues as the inhibitors of PAI-1 (Plasminogen Activator Inhibitor-1) Fanglei Chen, Guiping Zhang, Zhonghui Lin, Zebin Hong, Min Lei, Mingdong Huang, Lihong Hu
Leave this area blank for abstract info.
1
Bioorganic & Medicinal Chemistry Letters j o ur n al h om e p a g e : w w w . e l s e v i er . c o m
Design, synthesis, and SAR of embelin analogues as the inhibitors of PAI-1 (Plasminogen Activator Inhibitor-1) Fanglei Chena, Guiping Zhanga, Zebin Hongb, Zhonghui Linb, Min Leia, Mingdong Huangb,∗ and Lihong Hu a,∗ a
Shanghai Research Center for the Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P.R. China. b State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002, P.R. China.
A R T IC LE IN F O
A B S TR A C T
Article history: Received Revised Accepted Available online
The natural product embelin was found to have PAI-1 inhibitory activity with the IC50 value of 4.94 µM. Based on the structure of embelin, a series of analogues were designed, synthesized, and evaluated for their ability to inhibit PAI-1. The SAR study on these compounds disclosed that the inhibitory potency largely depended on the hydroxyl groups at C2 and C5, and the length of the alkyl chains at C3 and C6. Compound 11 displayed the best PAI-1 inhibitory potency with the IC50 value of 0.18 µM.
Keywords: Embelin Natural product PAI-1 Inhibitory activity SAR study
As the primary physiological inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) has been participated in a wide range of metabolic responses, such as blood coagulation, wound healing, angiogenesis, cell attachment and detachment, cell migration, and tumor-cell invasion.1–7 In human malignant tumors, the levels of uPA and PAI-1 are significantly higher than those in the corresponding normal tissues. Over-expressed uPA and PAI-1 can promote angiogenesis in tumor, facilitating the progression, dissemination and metastasis of tumors.8,9 In breast cancer, uPA and PAI-1 have been identified as the most extensively validated biological prognostic factors.10–12 Furthermore, increased levels of PAI-1 have been associated with increased risk of cardiovascular diseases.13–20 Therefore, PAI-1 is a potential target for treatment of cancer and cardiovascular diseases. Although many PAI-1 inhibitors have been reported,21–28 most of them have a certain degree of limitations, such as low affinity to bind with PAI-1, poor physicochemical properties, or lack of potency to inhibit PAI-1 in the presence of its cofactor. Therefore, searching for novel PAI-1 inhibitors with improved potencies is quite necessary and meaningful.
2009 Elsevier Ltd. All rights reserved.
Embelin (Figure 1) comes from the fruit of Embelia ribes Burm plant (Myrsinaceae), which has been used for the treatment of fever, inflammatory and gastrointestinal diseases for thousands of years. 29 As the active component of this plant, embelin has been shown to have analgesic, 30 anti-inflammatory,30 antidiabetes,31 and antitumor properties.30,32–35 In our previous drug screening test, we found that embelin was able to inhibit PAI-1 activity with the IC50 value of 4.94 µM. 36 Comparing with other small molecule inhibitors of PAI-1, embelin is distinctive. Specifically, the source is natural and rich, the structure is simple and stable, the potency is prominent, and the binding site is clear. To explore its structure and PAI-1 inhibitory activity relationship, we focused on our crystal structure of PAI-1 in complex with embelin (Figure 2).36
Figure 1. The structure of embelin.
——— ∗ Corresponding author. Tel.: +86-591-8370-4996; fax: +86-591-8370-4996; e-mail: [email protected] (M. Huang) ∗ Corresponding author. Tel.: +86-21-2023-1965; fax: +86-21-2023-1965; e-mail: [email protected] (L. Hu)
2
Bioorganic & Medicinal Chemistry
In this crystal structure, embelin binds to the groove constituted by α helixes D and F and β strand 2A. The polar, sixmember ring of embelin sits in the middle of the groove forming the direct strong contacts with Tyr-79 (α helix D), Asp-95 and Thr-93 (β strand 2A), and His-143 (α helix F). The aliphatic carbon chain of embelin stretches out of the cavity involving in the interactions with Ser-119 (hE-s1A connecting loop) and Trp139 (α helix F). 36 This interaction mode was proposed to fix β strand 2A to the neighboring α helixes D and F, delaying the insertion of the reaction center loop into β strand 2A that is the perquisite for translocation of the protease to the opposite pole of PAI-1 and subsequent covalent complex formation.36–38 From the inhibitory mechanism of embelin, it is clear that both the nucleus and the alkyl chain played an important role in the process of fixing β strand 2A to the neighboring α helixes D and F. Therefore, to explore the importance of the 2,5-dihydroxyl-pbenzoquinone nucleus and the optimal length of the alkyl chain, we designed and synthesized series of embelin analogues (Figure 3).
intermediates 1a, 1b and 1c were prepared successively. After the reaction of 1c with cerium (IV) ammonium nitrate (CAN), compounds 2 and 3 could be obtained. 39,40 In order to evaluate the PAI-1 inhibitory activity of embelin analogues, a chromogenic assay was adopted, in which the activity of PAI-1 was evaluated by its inhibition towards uPAmediated hydrolysis of chromogenic substrate.36 The PAI-1 inhibitory activities of compounds 2 and 3 were tested and exhibited in Table 1. From this table, we found that methylation of 5-hydroxyl (2) resulted in a sharp decrease of potency, while methylation of both 2-hydroxyl and 5-hydroxyl (3) led to a complete loss of potency even at 100 µM. Hence, retaining hydroxyl groups at C2 and C5 were crucial for the compounds to keep PAI-1 inhibitory activity. O
OH OH
OMe OMe
a, b
HO
OMe
c
MeO
MeO
O 1
OH 1a
OMe
O OMe
d, e MeO
OMe 1b
OR2
f
C11H23
2: R1 = Me, R2 = H R1O
OMe
C11H23
3: R1 = Me, R2 = Me
O
1c
Scheme 1. Reagents and conditions: (a) con. HCl, MeOH; (b) Na2S2O4, H2O; (c) KOH, MeI, DMSO; (d) n-BuLi, THF, –10 o C; (e) 1-Bromo-undecane, –10 oC~rt; (f) CAN, CH3CN, –10 o C~rt. Table 1. IC50 Values of compounds 2 and 3 against PAI-1a Figure 2. Close-up view of the embelin binding site with direct contacts shown as yellow dashed lines. Selected residues of PAI-1 binding to embelin are given in stick. Tyr-79, located on top of embelin, is not shown for clarity (PDB code: 3UT3).
Compd.
IC50 value (µM)
embelin
4.94 ± 0.41
2
90.19 ± 4.04
3
–b
a b
IC50 values are based on at least three independent experiments. No activity at 100 µM.
O
O
O
O O
O
a-2 R3 =
O
O O a, b
R3
OH
c
4a-8a n
R3
HO O 4-8
4: n = 3; 5: n = 4; 6: n = 5; 7: n = 6; 8: n = 7.
Scheme 2. Reagents and conditions: (a) n-BuLi, THF, –10 oC; (b) R3Br, –10 o C~rt; (c) 1,4-dioxane, 6 mol/L HCl, air, reflux.
Figure 3. Design of embelin analogues.
Firstly, we prepared two etherification derivatives (2, 3) (Scheme 1). Using 2,5-dihydroxycyclohexa-2,5-diene-1,4-dione (1) as the starting material, via four steps of reactions,
Then, to explore the relationship between the length of the side chain at C3 and PAI-1 inhibitory activity, compounds 4–8 containing different length of alkyl chains at C3 were synthesized. As shown in Scheme 2, compound a-2 reacted with n-BuLi, and alkyl bromide in THF to generate intermediates 4a– 8a which deprotected with 6 mol/L HCl in 1,4-dioxane to obtain compounds 4–8.
3
As shown in Table 2, 4 and 5 kept the activity, and 6–8 showed slightly increased activity. Among them, compound 8 with the n-nonyl group substituted at C3 exhibited the best potency.
O O
Compd.
IC50 value (µM)
embelin
4.94 ± 0.41
4
4.56 ± 0.12
5
4.18 ± 0.25
6
3.87 ± 0.33
7
1.87 ± 0.15
8 a
O
a, b O
O
Table 2. IC50 values of compounds 4–8 against PAI-1a
a-2 R5 =
c
R5
O
O
O
O
R5
OH R5
HO O
15a-20a
m
R5
15-20
15: m = 1; 16: m = 2; 17: m = 3; 18: m = 4; 19: m = 5; 20: m = 6.
Scheme 4. Reagents and conditions: (a) n-BuLi, THF, –10 oC; (b) R5Br, –10 o C~r.t; (c) 1,4-dioxane, 6 mol/L HCl, air, reflux.
1.78 ± 0.18 IC50 values are based on at least three independent experiments.
On the basis of 8, we further introduced carbon chains at C6 (Scheme 3). The PAI-1 inhibitory activities of compounds 9–14 revealed that introducing carbon chains at C6 could significantly improve the potencies of compounds. Specifically, 9, 12 and 13 showed 13-fold, 8-fold and 5-fold improved potency respectively, 10 and 14 exhibited 6.5-fold improved potency. Compound 11 afforded the best potency in this series of compounds with the IC50 value of 0.18 µM, which was 27-fold more potent than embelin (Table 3).
Table 4. IC50 values of compounds 15–20 against PAI-1a Compd.
IC50 value (µM)
embelin
4.94 ± 0.41
15
5.68 ± 0.15
16
7.11 ± 0.19
17
0.92 ± 0.13
18
0.58 ± 0.14
19
0.51 ± 0.05
20
0.89 ± 0.08
a
IC50 values are based on at least three independent experiments.
To further explore the optimal length of carbon chains at C3 and C6, we designed and synthesized compounds 15–20 (Scheme 4). The synthetic methods of these compounds were similar to that of 4–8. As shown in Table 4, 15 and 16 were slightly less potent than embelin. 17 and 20 showed 5-fold improved potency relative to embelin. 18 and 19 exhibited 9-fold and 10-fold improved potency, respectively. According to IC50 values of compounds 9–20, it could be concluded that the lengths of alkyl chains at C3 and C6 have significant effect on the potencies of compounds. Introducing long carbon chain at C3 and short carbon chain at C6 is optimal, such as compounds 9 and 11.
Scheme 3. Reagents and conditions: (a) n-BuLi, THF, –10 o C; R4Br, –10 oC~rt; (b) 1,4-dioxane, 6 mol/L HCl, reflux. Table 3. IC50 values of compounds 9–14 against PAI-1a Compd.
IC50 value (µM)
embelin
4.94 ± 0.41
9
0.38 ± 0.19
10
0.75 ± 0.06
11
0.18 ± 0.05
12
0.63 ± 0.09
13
0.95 ± 0.05
14
0.77 ± 0.16
a
IC50 values are based on at least three independent experiments.
In conclusion, we have found embelin which was able to inhibit PAI-1 with the IC50 value of 4.94 µM. Based on the structure of PAI-1/embelin complex, a series of embelin analogs were designed and synthesized for the study of SAR. From the IC50 values of these compounds, the SAR could be summarized as follows: (1) the hydroxyl groups at C2 and C5 were indispensable for the inhibitory potency, because both of them have been participated in the hydrogen-bonding interaction with the binding pocket; (2) suitably shortening the length of the alkyl chain at C3 could slightly increase activity. Introducing alkyl chains at C6 could significantly improve potency. In sum, several compounds have exhibited much better potencies than that of embelin. Among them, Compound 11 showed the best activity with the IC50 value of 0.18 µM. In addition, as our initial study, these analogues have retained the structural character of embelin. For the purpose to obtain more potent PAI-1 inhibitors, we are exploring embelin analogues with more diverse and complex substituents at C3 and C6, and the corresponding work will be disclosed in due course.
Acknowledgments
4
Bioorganic & Medicinal Chemistry
This work was supported by the National Natural Science Foundation of China (grants 30925040, 81102329, 81273397), the Chinese National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (grants 2013ZX09508104), and the Science Foundation of Shanghai (grant 12XD1405700). Supplementary Material Supplementary data (synthesis of 2−20, copies of 1H and 13C NMR for final products, and biological assay methods) can be found in the online version, at References and notes 1.
2. 3. 4. 5. 6.
7. 8.
9.
10.
11.
12.
13. 14. 15. 16.
17.
18. 19. 20. 21. 22.
Schmitt, M.; Harbeck, N.; Thomssen, C.; Wilhelm, O.; Magdolen, V.; Reuning, U.; Ulm, K.; Hofler, H.; Janicke, F.; Graeff, H. Thromb. Haemost. 1997, 78, 285. Angenete, E.; Langenskioeld, M.; Palmgren, I.; Falk, P.; Oeresland, T.; Ivarsson, M.-L. J. Surg. Res. 2009, 153, 46. Takahashi, T.; Suzuki, K.; Ihara, H.; Mogami, H.; Kazui, T.; Urano, T. Thromb. Hemost. 2005, 31, 356. Chazaud, B.; Ricoux, R.; Christov, C.; Plonquet, A.; Gherardi, R. K.; Barlovatz-Meimon, G. Am. J. Pathol. 2002, 160, 237. Lijnen, H. R. J. Thromb. Haemost. 2005, 3, 35. Macfelda, K.; Weiss, T. W.; Kaun, C.; Breuss, J. M.; Zorn, G.; Oberndorfer, U.; Voegele-Kadletz, M.; Huber-Beckmann, R.; Ullrich, R.; Binder, B. R.; Losert, U. M.; Maurer, G.; Pacher, R.; Huber, K.; Wojta, J. J. Mol. Cell. Cardiol. 2002, 34, 1681. Czekay, R.-P.; Loskutoff, D. J. Exp. Biol. Med. 2004, 229, 1090. (a) Duffy, M. J. Curr. Pharm. Des. 2004, 10, 39; (b) Blasi, F.; Carmeliet, P. Nat. Rev. Mol. Cell Biol. 2002, 3, 932; (c) Danø, K.; Behrendt, N.; Høyer-Hansen, G.; Johnsen, M.; Lund, L. R.; Ploug, M.; Rømer, J. Thromb. Haemost. 2005, 93, 676. Durand, M. K. V.; Bodker, J. S.; Christensen, A.; Dupont, D. M.; Hansen, M.; Jensen, J. K.; Kjelgaard, S.; Mathiasen, L.; Pedersen, K. E.; Skeldal, S.; Wind, T.; Andreasen, P. A. Thromb. Haemost. 2004, 91, 438. Janicke, F.; Prechtl, A.; Thomssen, C.; Harbeck, N.; Meisner, C.; Untch, M.; Sweep, C. G. J.; Selbmann, H.-K.; Graeff, H.; Schmitt, M. J. Natl. Cancer Inst. 2001, 93, 913. Harris, L.; Fritsche, H.; Mennel, R.; Norton, L.; Ravdin, P.; Taube, S.; Somerfield, M. R.; Hayes, D. F.; Bast, R. C. Jr. J. Clin. Oncol. 2007, 25, 5287. Look, M. P.; van Putten, W. L. J.; Duffy, M. J.; Harbeck, N.; Christensen, I. J.; Thomssen, C.; Kates, R.; Spyratos, F.; Ferno, M.; Eppenberger-Castori, S.; Fred Sweep, C. G. J.; Ulm, K.; Peyrat, J.-P.; Martin, P.-M.; Magdelenat, H.; Brünner, N.; Duggan, C.; Lisboa, B. W.; Bendahl, P.-O.; Quillien, V.; Daver, A.; Ricolleau, G.; Meijer-van Gelder, M. E.; Manders, P.; Fiets, W. E.; Blankenstein, M. A.; Broët, P.; Romain, S.; Daxenbichler, G.; Windbichler, G.; Cufer, T.; Borstnar, S.; Kueng, W.; Beex, L. V. A. M.; Klijn, J. G. M.; O’Higgins, N.; Eppenberger, U.; Jänicke, F.; Schmitt, M.; Foekens, J. A. J. Natl. Cancer Inst. 2002, 94, 116. Van De Craen, B.; Declerck, P. J.; Gils, A. Thromb. Res. 2012, 130, 576. Hamsten, A.; Wiman, B.; Defaire, U.; Blomback, M. New Engl. J. Med. 1985, 313, 1557. Hamsten, A.; Defaire, U.; Walldius, G.; Dahlen, G.; Szamosi, A.; Landou, C.; Blomback, M.; Wiman, B. Lancet 1987, 2, 3. Juhan-Vague, I.; Valadier, J.; Alessi, M. C.; Aillaud, M. F.; Ansaldi, J.; Philip-Joet, C.; Holvoet, P.; Serradimigni, A.; Collen, D. Thromb. Haemost. 1987, 57, 67. Meltzer, M. E.; Lisman, T.; de Groot, P. G.; Meijers, J. C. M.; le Cessie, S.; Doggen, C. J. M.; Rosendaal, F. R. Blood 2010, 116, 113. Garg, N.; Fay, W. P. Current Drug Targets 2007, 8, 1003. Konstantinides, S.; Schafer, K.; Loskutoff, D. J. Arterioscler. Thromb. Vasc. Biol. 2002, 22, 1943. Binder B. R.; Mihaly, J. Immunol. Lett. 2008, 118, 116. Folkes, A.; Roe, M. B.; Sohal, S.; Golec, J.; Faint, R.; Brooks, T.; Charlton, P. Bioorg. Med. Chem. Lett. 2001, 11, 2589. Ye, B.; Bauer, S.; Buckman, B. O.; Ghannam, A.; Griedel, B. D.; Khim, S.-K.; Lee, W.; Sacchi, K. L.; Shaw, K. J.; Liang, A.; Wu, Q.; Zhao, Z. Bioorg. Med. Chem. Lett. 2003, 13, 3361.
23. Gorlatova, N. V.; Cale, J. M.; Elokdah, H.; Li, D. H.; Fan, K.; Warnock, M.; Crandall, D. L.; Lawrence, D. A. J. Biol. Chem. 2007, 282, 9288. 24. Miyazaki, H.; Ogiku, T.; Sai, H.; Ohmizu, H.; Murakami, J.; Ohtani, A. Bioorg. Med. Chem. Lett. 2008, 18, 6419. 25. Miyazaki, H.; Miyake, T.; Terakawa, Y.; Ohmizu, H.; Ogiku, T.; Ohtani, A. Bioorg. Med. Chem. Lett. 2010, 20, 546. 26. Cale, J. M.; Li, S. H.; Warnock, M.; Su, E. J.; North, P. R.; Sanders, K. L.; Puscau, M. M.; Emal, C. D.; Lawrence, D. A. J. Biol. Chem. 2010, 285, 7892. 27. Yamaoka, N.; Kawano, Y.; Izuhara, Y.; Miyata, T.; Meguro, K. Chem. Pharm. Bull. 2010, 58, 615. 28. Pandya, V.; Jain, M.; Chakrabarti, G.; Soni, H.; Parmar, B.; Chaugule, B.; Patel, J.; Joshi, J.; Joshi, N.; Rath, A.; Raviya, M.; Shaikh, M.; Sairam, K. V. V. M.; Patel, H.; Patel, P. Bioorg. Med. Chem. Lett. 2011, 21, 5701. 29. Gupta, O. P.; Ali, M. M.; Ghatak, B. J. Ray; Atal, C. K. Indian J. Physiol. Pharmacol. 1977, 21, 31. 30. Chitra, M.; Sukumar, E.; Suja, V.; Devi, C. S. S. Chemotherapy 1994, 40, 109. 31. Mahendran, S.; Badami, S.; Maithili, V. Biomed. Prev. Nutr. 2011, 1, 25. 32. Reuter, S.; Prasad, S.; Phromnoi, K.; Kannappan, R.; Yadav, V. R.; Aggarwal, B. B. Mol. Cancer Res. 2010, 8, 1425. 33. Taghiyev, A.; Sun, D. G.; Gao, Z. M.; Liang, R.; Wang, L. M. Exp. Ther. Med. 2012, 4, 649. 34. Hu, R.; Zhu, K.; Li, Y. C.; Yao, K.; Zhang, R.; Wang, H. H.; Yang, W.; Liu, Z. G. Med. Oncol. 2011, 28, 1584. 35. Mori, T.; Doi, R.; Kida, A.; Nagai, K.; Kami, K.; Ito, D.; Toyoda, E.; Kawaguchi, Y.; Uemoto, S. J. Surg. Res. 2007, 142, 281. 36. Lin, Z. H.; Jensen, J. K.; Hong, Z. B.; Shi, X. L.; Hu, L. H.; Andreasen, P. A.; Huang, M. D. Chem. Biol. 2013, 20, 253. 37. Huntington, J. A. Trends Biochem. Sci. 2006, 31, 427. 38. Dupont, D. M.; Madsen, J. B.; Kristensen, T.; Bodker, J. S.; Blouse, G. E.; Wind, T.; Andreasen, P. A. Front. Biosci. 2009, 14, 1337. 39. Poigny, S.; Guyot, M.; Samadi, M. Tetrahedron 1998, 54, 14791. 40. Mizuno, C. S.; Rimando, A. M.; Duke, S. O. J. Agric. Food Chem. 2010, 58, 4353.
S0960-894X(14)00272-8 http://dx.doi.org/10.1016/j.bmcl.2014.03.045 BMCL 21438
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
20 January 2014 12 March 2014 15 March 2014
Please cite this article as: Chen, F., Zhang, G., Hong, Z., Lin, Z., Lei, M., Huang, M., Hu, L., Design, synthesis, and SAR of embelin analogues as the inhibitors of PAI-1 (Plasminogen Activator Inhibitor-1), Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.03.045
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Graphical Abstract
Design, synthesis, and SAR of embelin analogues as the inhibitors of PAI-1 (Plasminogen Activator Inhibitor-1) Fanglei Chen, Guiping Zhang, Zhonghui Lin, Zebin Hong, Min Lei, Mingdong Huang, Lihong Hu
Leave this area blank for abstract info.
1
Bioorganic & Medicinal Chemistry Letters j o ur n al h om e p a g e : w w w . e l s e v i er . c o m
Design, synthesis, and SAR of embelin analogues as the inhibitors of PAI-1 (Plasminogen Activator Inhibitor-1) Fanglei Chena, Guiping Zhanga, Zebin Hongb, Zhonghui Linb, Min Leia, Mingdong Huangb,∗ and Lihong Hu a,∗ a
Shanghai Research Center for the Modernization of Traditional Chinese Medicine, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P.R. China. b State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350002, P.R. China.
A R T IC LE IN F O
A B S TR A C T
Article history: Received Revised Accepted Available online
The natural product embelin was found to have PAI-1 inhibitory activity with the IC50 value of 4.94 µM. Based on the structure of embelin, a series of analogues were designed, synthesized, and evaluated for their ability to inhibit PAI-1. The SAR study on these compounds disclosed that the inhibitory potency largely depended on the hydroxyl groups at C2 and C5, and the length of the alkyl chains at C3 and C6. Compound 11 displayed the best PAI-1 inhibitory potency with the IC50 value of 0.18 µM.
Keywords: Embelin Natural product PAI-1 Inhibitory activity SAR study
As the primary physiological inhibitor of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) has been participated in a wide range of metabolic responses, such as blood coagulation, wound healing, angiogenesis, cell attachment and detachment, cell migration, and tumor-cell invasion.1–7 In human malignant tumors, the levels of uPA and PAI-1 are significantly higher than those in the corresponding normal tissues. Over-expressed uPA and PAI-1 can promote angiogenesis in tumor, facilitating the progression, dissemination and metastasis of tumors.8,9 In breast cancer, uPA and PAI-1 have been identified as the most extensively validated biological prognostic factors.10–12 Furthermore, increased levels of PAI-1 have been associated with increased risk of cardiovascular diseases.13–20 Therefore, PAI-1 is a potential target for treatment of cancer and cardiovascular diseases. Although many PAI-1 inhibitors have been reported,21–28 most of them have a certain degree of limitations, such as low affinity to bind with PAI-1, poor physicochemical properties, or lack of potency to inhibit PAI-1 in the presence of its cofactor. Therefore, searching for novel PAI-1 inhibitors with improved potencies is quite necessary and meaningful.
2009 Elsevier Ltd. All rights reserved.
Embelin (Figure 1) comes from the fruit of Embelia ribes Burm plant (Myrsinaceae), which has been used for the treatment of fever, inflammatory and gastrointestinal diseases for thousands of years. 29 As the active component of this plant, embelin has been shown to have analgesic, 30 anti-inflammatory,30 antidiabetes,31 and antitumor properties.30,32–35 In our previous drug screening test, we found that embelin was able to inhibit PAI-1 activity with the IC50 value of 4.94 µM. 36 Comparing with other small molecule inhibitors of PAI-1, embelin is distinctive. Specifically, the source is natural and rich, the structure is simple and stable, the potency is prominent, and the binding site is clear. To explore its structure and PAI-1 inhibitory activity relationship, we focused on our crystal structure of PAI-1 in complex with embelin (Figure 2).36
Figure 1. The structure of embelin.
——— ∗ Corresponding author. Tel.: +86-591-8370-4996; fax: +86-591-8370-4996; e-mail: [email protected] (M. Huang) ∗ Corresponding author. Tel.: +86-21-2023-1965; fax: +86-21-2023-1965; e-mail: [email protected] (L. Hu)
2
Bioorganic & Medicinal Chemistry
In this crystal structure, embelin binds to the groove constituted by α helixes D and F and β strand 2A. The polar, sixmember ring of embelin sits in the middle of the groove forming the direct strong contacts with Tyr-79 (α helix D), Asp-95 and Thr-93 (β strand 2A), and His-143 (α helix F). The aliphatic carbon chain of embelin stretches out of the cavity involving in the interactions with Ser-119 (hE-s1A connecting loop) and Trp139 (α helix F). 36 This interaction mode was proposed to fix β strand 2A to the neighboring α helixes D and F, delaying the insertion of the reaction center loop into β strand 2A that is the perquisite for translocation of the protease to the opposite pole of PAI-1 and subsequent covalent complex formation.36–38 From the inhibitory mechanism of embelin, it is clear that both the nucleus and the alkyl chain played an important role in the process of fixing β strand 2A to the neighboring α helixes D and F. Therefore, to explore the importance of the 2,5-dihydroxyl-pbenzoquinone nucleus and the optimal length of the alkyl chain, we designed and synthesized series of embelin analogues (Figure 3).
intermediates 1a, 1b and 1c were prepared successively. After the reaction of 1c with cerium (IV) ammonium nitrate (CAN), compounds 2 and 3 could be obtained. 39,40 In order to evaluate the PAI-1 inhibitory activity of embelin analogues, a chromogenic assay was adopted, in which the activity of PAI-1 was evaluated by its inhibition towards uPAmediated hydrolysis of chromogenic substrate.36 The PAI-1 inhibitory activities of compounds 2 and 3 were tested and exhibited in Table 1. From this table, we found that methylation of 5-hydroxyl (2) resulted in a sharp decrease of potency, while methylation of both 2-hydroxyl and 5-hydroxyl (3) led to a complete loss of potency even at 100 µM. Hence, retaining hydroxyl groups at C2 and C5 were crucial for the compounds to keep PAI-1 inhibitory activity. O
OH OH
OMe OMe
a, b
HO
OMe
c
MeO
MeO
O 1
OH 1a
OMe
O OMe
d, e MeO
OMe 1b
OR2
f
C11H23
2: R1 = Me, R2 = H R1O
OMe
C11H23
3: R1 = Me, R2 = Me
O
1c
Scheme 1. Reagents and conditions: (a) con. HCl, MeOH; (b) Na2S2O4, H2O; (c) KOH, MeI, DMSO; (d) n-BuLi, THF, –10 o C; (e) 1-Bromo-undecane, –10 oC~rt; (f) CAN, CH3CN, –10 o C~rt. Table 1. IC50 Values of compounds 2 and 3 against PAI-1a Figure 2. Close-up view of the embelin binding site with direct contacts shown as yellow dashed lines. Selected residues of PAI-1 binding to embelin are given in stick. Tyr-79, located on top of embelin, is not shown for clarity (PDB code: 3UT3).
Compd.
IC50 value (µM)
embelin
4.94 ± 0.41
2
90.19 ± 4.04
3
–b
a b
IC50 values are based on at least three independent experiments. No activity at 100 µM.
O
O
O
O O
O
a-2 R3 =
O
O O a, b
R3
OH
c
4a-8a n
R3
HO O 4-8
4: n = 3; 5: n = 4; 6: n = 5; 7: n = 6; 8: n = 7.
Scheme 2. Reagents and conditions: (a) n-BuLi, THF, –10 oC; (b) R3Br, –10 o C~rt; (c) 1,4-dioxane, 6 mol/L HCl, air, reflux.
Figure 3. Design of embelin analogues.
Firstly, we prepared two etherification derivatives (2, 3) (Scheme 1). Using 2,5-dihydroxycyclohexa-2,5-diene-1,4-dione (1) as the starting material, via four steps of reactions,
Then, to explore the relationship between the length of the side chain at C3 and PAI-1 inhibitory activity, compounds 4–8 containing different length of alkyl chains at C3 were synthesized. As shown in Scheme 2, compound a-2 reacted with n-BuLi, and alkyl bromide in THF to generate intermediates 4a– 8a which deprotected with 6 mol/L HCl in 1,4-dioxane to obtain compounds 4–8.
3
As shown in Table 2, 4 and 5 kept the activity, and 6–8 showed slightly increased activity. Among them, compound 8 with the n-nonyl group substituted at C3 exhibited the best potency.
O O
Compd.
IC50 value (µM)
embelin
4.94 ± 0.41
4
4.56 ± 0.12
5
4.18 ± 0.25
6
3.87 ± 0.33
7
1.87 ± 0.15
8 a
O
a, b O
O
Table 2. IC50 values of compounds 4–8 against PAI-1a
a-2 R5 =
c
R5
O
O
O
O
R5
OH R5
HO O
15a-20a
m
R5
15-20
15: m = 1; 16: m = 2; 17: m = 3; 18: m = 4; 19: m = 5; 20: m = 6.
Scheme 4. Reagents and conditions: (a) n-BuLi, THF, –10 oC; (b) R5Br, –10 o C~r.t; (c) 1,4-dioxane, 6 mol/L HCl, air, reflux.
1.78 ± 0.18 IC50 values are based on at least three independent experiments.
On the basis of 8, we further introduced carbon chains at C6 (Scheme 3). The PAI-1 inhibitory activities of compounds 9–14 revealed that introducing carbon chains at C6 could significantly improve the potencies of compounds. Specifically, 9, 12 and 13 showed 13-fold, 8-fold and 5-fold improved potency respectively, 10 and 14 exhibited 6.5-fold improved potency. Compound 11 afforded the best potency in this series of compounds with the IC50 value of 0.18 µM, which was 27-fold more potent than embelin (Table 3).
Table 4. IC50 values of compounds 15–20 against PAI-1a Compd.
IC50 value (µM)
embelin
4.94 ± 0.41
15
5.68 ± 0.15
16
7.11 ± 0.19
17
0.92 ± 0.13
18
0.58 ± 0.14
19
0.51 ± 0.05
20
0.89 ± 0.08
a
IC50 values are based on at least three independent experiments.
To further explore the optimal length of carbon chains at C3 and C6, we designed and synthesized compounds 15–20 (Scheme 4). The synthetic methods of these compounds were similar to that of 4–8. As shown in Table 4, 15 and 16 were slightly less potent than embelin. 17 and 20 showed 5-fold improved potency relative to embelin. 18 and 19 exhibited 9-fold and 10-fold improved potency, respectively. According to IC50 values of compounds 9–20, it could be concluded that the lengths of alkyl chains at C3 and C6 have significant effect on the potencies of compounds. Introducing long carbon chain at C3 and short carbon chain at C6 is optimal, such as compounds 9 and 11.
Scheme 3. Reagents and conditions: (a) n-BuLi, THF, –10 o C; R4Br, –10 oC~rt; (b) 1,4-dioxane, 6 mol/L HCl, reflux. Table 3. IC50 values of compounds 9–14 against PAI-1a Compd.
IC50 value (µM)
embelin
4.94 ± 0.41
9
0.38 ± 0.19
10
0.75 ± 0.06
11
0.18 ± 0.05
12
0.63 ± 0.09
13
0.95 ± 0.05
14
0.77 ± 0.16
a
IC50 values are based on at least three independent experiments.
In conclusion, we have found embelin which was able to inhibit PAI-1 with the IC50 value of 4.94 µM. Based on the structure of PAI-1/embelin complex, a series of embelin analogs were designed and synthesized for the study of SAR. From the IC50 values of these compounds, the SAR could be summarized as follows: (1) the hydroxyl groups at C2 and C5 were indispensable for the inhibitory potency, because both of them have been participated in the hydrogen-bonding interaction with the binding pocket; (2) suitably shortening the length of the alkyl chain at C3 could slightly increase activity. Introducing alkyl chains at C6 could significantly improve potency. In sum, several compounds have exhibited much better potencies than that of embelin. Among them, Compound 11 showed the best activity with the IC50 value of 0.18 µM. In addition, as our initial study, these analogues have retained the structural character of embelin. For the purpose to obtain more potent PAI-1 inhibitors, we are exploring embelin analogues with more diverse and complex substituents at C3 and C6, and the corresponding work will be disclosed in due course.
Acknowledgments
4
Bioorganic & Medicinal Chemistry
This work was supported by the National Natural Science Foundation of China (grants 30925040, 81102329, 81273397), the Chinese National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” (grants 2013ZX09508104), and the Science Foundation of Shanghai (grant 12XD1405700). Supplementary Material Supplementary data (synthesis of 2−20, copies of 1H and 13C NMR for final products, and biological assay methods) can be found in the online version, at References and notes 1.
2. 3. 4. 5. 6.
7. 8.
9.
10.
11.
12.
13. 14. 15. 16.
17.
18. 19. 20. 21. 22.
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