Bioorganic & Medicinal Chemistry Letters 25 (2015) 2421–2424

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Synthesis of new ent-labdane diterpene derivatives from andrographolide and evaluation on cytotoxic activities Yan Luo a, Ke Wang b, Meng-han Zhang b, Da-yong Zhang b,⇑, Yang-chang Wu c,⇑, Xiao-ming Wu b, Wei-yi Hua b a b c

High Magnetic Field Laboratory, Chinese Academy of Sciences, Shu Shan Hu Rd No. 350, Hefei 230031, China Center of Drug Discovery & State Key Laboratory of Natural Medicines, China Pharmaceutical University, No. 24, Tongjia Xiang, Nanjing 210009, China Graduate Institute of Pharmaceutical Chemistry, College of Pharmacy, China Medical University, No. 91, Hsueh-Shih Road, Taichung 40402, Taiwan

a r t i c l e

i n f o

a b s t r a c t

Article history: Received 12 November 2014 Revised 22 March 2015 Accepted 31 March 2015 Available online 4 April 2015

There are many reports for andrographolide modification regarding antitumor effects. Transformation of the five-membered lactone ring to furan aromatic ring still results in compounds with good cytotoxicity. To determine further the importance of the five-membered lactone ring and to obtain better lead compounds, we transformed the five-membered lactone ring in andrographolide. New types of ent-labdane diterpene derivatives were made, whose cytotoxic activities were measured in vitro. Preliminary SAR was summarized and two compounds, 7 and 26, with good cytotoxic activity were obtained, which have the potential to be developed into new antitumor drugs. Ó 2015 Published by Elsevier Ltd.

Keywords: Andrographolide ent-Labdane diterpene SAR Cytotoxic activity

Andrographolide(1) is a labdane diterpenoid isolated from the leaves or whole plant of Andrographis paniculata. Previous research shows that andrographolide exhibits a wide spectrum of biological activity, including antibacterial,1 anti-inflammatory,2 anti-HIV,3 cardiovascular effects,4 anti-malarial,5 a-glucosidase inhibition,6 and antioxidant.7 14

HO

O O 12

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Additionally, andrographolide and its various semi-synthetic analogues have been reported for antitumor activity.8,9 There could be great value in discovering a potential antitumor, semi-synthetic drug from these compounds. Nanduri et al.10 studied the structure– activity relationship (SAR) of cytotoxic andrographolide derivatives. In their report, the a-alkylidene-c-butyrolactone moiety of andrographolide was opened to yield andrographolic acid, and the ⇑ Corresponding authors. Tel./fax: +86 25 83271307 (D.-y.Z.). E-mail address: [email protected] (D.-y. Zhang). http://dx.doi.org/10.1016/j.bmcl.2015.03.086 0960-894X/Ó 2015 Published by Elsevier Ltd.

exocyclic double bond (412(13)) was selectively reduced. From comparing the cytotoxic activity between the compounds, the intact a-alkylidene-c-butyrolactone moiety of andrographolide was considered the key part for the cytotoxic activity. However, no other structural modification had been reported to verify the significance of the a-alkylidene-c-butyrolactone moiety of andrographolide. Other intriguing studies11–14 revealed labdane-related diterpenes possessed good antitumor activity. These compounds have an aromatic ring, including a furan ring, and have a similar structure to andrographolide. To further confirm the role of a-alkylidene-c-butyrolactone moiety in cytotoxic activity and to discover potential antitumor, semi-synthetic analogues of some value, we designed a synthetic procedure to obtain the semi-synthetic analogues of furan ring-containing andrographolide. The first series of this synthetic process for andrographolide derivatives are represented in Scheme 1 by the use of excess acetic anhydride in a solvent of DCM (dichloromethane) at reflux. Compound 2 can be obtained from andrographolide in very high yield. Compound 3 was prepared from 2 using potassium borohydride. In this process, acetyl groups at the C-14 position in 2 were removed, and the double bond between C-12 and C-13 was shifted to C-13 and C-14. Compound 4 was obtained from 3 using 5% potassium bicarbonate in methanol at reflux. Reduction of 4 with DIBAl-H at 80 °C yielded the target product 5, this being the andrographolide derivative containing the desired furan ring. Compounds 6 and 7 were obtained by esterification of the two

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O

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Scheme 1. Reagents and conditions: (a) acetic anhydride, DCM, ZnCl2, reflux, 20 min, 74%; (b) KBH4, MeOH, rt, 3 h, 89%; (c) 5% KHCO3, MeOH, reflux, 10 h, 81%; (d) DIBAl-H, THF, 80 °C, 3 h, 81%; (e) DMAP, DCM, rt, 8 h, 97–98%, 6–7; EDCI. DMAP. DCM, rt, 20–60%, 8–11.

hydroxyl groups at C-3 and C-19 with suitable anhydrides. Additionally, compounds 8–11 were gained from appropriate aromatic acids. In order to identify the importance of the 48(17)double bond in activity, compound 15 was designed and synthesized according to Scheme 2. Compound 12 was formed after C-3 and C-19 position of 5 was protected by acetyl. Compound 13 without the 48(17)double bond was obtained after 12 reacting with excess potassium permanganate. At the same time, by-product 14 was formed. Product 15 was gained by deprotecting the acetyl of 13 under basic conditions. The synthetic pathway of new ent-labdane diterpene derivatives containing hydroxyl at C-7 is represented in Scheme 3. Compound 16 was prepared by combining 6 with selenium dioxide and t-butyl hydroperoxide. The synthesis of 16 could determine the activity of the hydroxyl at C-7. The synthetic pathway of andrographolide derivatives containing a carbonyl group at C-3 is represented in Scheme 4. With excess triphenylchloromethane catalyzed by N,N-diisopropylethylamine, compound 17 was yielded from 5. Under the trityl protecting group at C-19 oxygen, compound 18 was obtained from 17 with excess

pyridinium dichromate in N,N-dimethylformamide at 50 °C. After hydrolysis by toluenesulfonic acid in methanol, compound 19 was prepared from 18. As a result, new ent-labdane diterpene derivatives containing a carbonyl group at C-3 were obtained. Scheme 5 shows the synthetic route of furan-andrographolide with an additional hydroxyl at C-12. The hydroxyl groups at C-3 and C-19 on andrographolide were first protected with acetonide to produce compound 20. With a 1.2 times equivalent amount of pyridinium dichromate, allylic hydroxyl at C-14 in compound 20 was relocated to C-12. As a result, the key intermediate, compound 21 was formed. Compound 22 is the de-protect product of compound 21. The lactone group in 22 was reduced to furanolabdane 23, with DIBAl-H. So far, furanoandrographolide containing hydroxyl groups at C-12 was produced. Propionylation of C-3, C-12, and C-19 hydroxyl groups produced compound 24. In addition, compound 22 was reported to have good antitumor activity in vitro, but derivative effects have rarely carried out, so compound 26 was synthesized. New ent-labdane diterpene derivatives and andrographolide were tested for antitumor activity in vitro using the method of

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Scheme 3. Reagents and conditions: (a) SeO2, t-BuOOH, DCM, rt, 12 h, 73%.

targeting the five-membered lactone is an unfavorable transformation. The new ent-labdane diterpene, compound 5, which contains furan in the C-ring, still possessed some cytotoxic activity compared to andrographolide. However, cytotoxic activity of the new ent-labdane diterpene compound 6, which contained C-12 hydroxyl group, was lost, emphasizing that the C-12 hydroxyl group was unfavorable for activity in this new ent-labdane diterpene containing furan in the C-ring. Activity of compound 15 was lost in comparison to compound 5, which proved the importance of the 48(17)double bond in cytotoxic activity. Compound 16 had better activity than compound 6, which illustrated that introducing a hydroxyl group at C-7 was beneficial to its activity. Compounds 6, 7, 8, 9, 10, and 11 were esterified compounds at C-3, C-19 of compounds 5. Compounds 6 and 7 contained an aliphatic chain compared with 5. The cytotoxic activity of 6 was the same as 5, but 7 had stronger activity, which significantly exceeded that of andrographolide. These suggested that the introduction of an electron-withdrawing group at C-3 and C-19 could greatly enhance activity. Compounds 8, 9, 10, and 11 possessed aromatic rings at C-3 and C-19 in comparision with 5. The order of activity is as follows: 11 > 8 > 9, which shows that the activity of compounds containing an electron-donating group at the para position of the benzene ring were better than compounds without groups or an electron-withdrawing group. The activity of 10 was lost compared with 9, which indicates compounds possessing aromatic esters both at C-3 and C-19 could lose activity. Compound 22 (14-deoxy-12-hydroxyandrographolide) had good cytotoxicity, and compound 26, which contained an acetyl group at C-3, C-12 and trityl at C-19, showed good cytotoxic activity. This proved that the structure of 14-deoxy-12-hydroxyandrographolide was an excellent nucleus. Therefore, the preliminary SAR of this new ent-labdane diterpene can be summarized as follows:

MTT15 against four cancer cell lines (human bladder cancer cell line NTUB1, cis-platin-resistant human bladder cancer cell line NP14, human breast cancer MCF-7, human breast cancer (MDA-MB231). All tested samples were dissolved in DMSO (0.1%). cis-platin was used as positive control. The results expressed as IC50 values (drug concentration causing 50% growth inhibition) in lM are shown in Table 1. With 15 compounds under examination for possible cytotoxic activities, 7 and 26 displayed good cytotoxic effects more than andrographolide and cis-platin. Cytotoxicity of compound 11 was superior to andrographolide against MCF-7 and NTUB1 cells. Compounds 9, 16 and 22 showed better cytotoxic activities than andrographolide against NTUB1 cells, while compound 8 displayed better cytotoxic activity than andrographolide for NP14. Cytotoxic activity of compound 4 was lost compared to 1, which illustrated the removal of the C-14 hydroxyl group

1. Transformation of the hydroxyl group at C-3 to a carbonyl group could result in a decrease of cytotoxic activity. 2. Esterification at C-3 and C-19 could enhance activity. Introducing electron-withdrawing aliphatic esters at C-3 and C-19 could significantly enhance activity. When an aromatic ester is introduced at C-19, the order of activity is such: electron-donating group at the para position of aromatic ring>no group at the para position of aromatic ring>electron-withdrawing group at the para position of aromatic ring. Introducing aromatic esters both at C-3 and C-19 could lose activity. 3. For the compounds esterified at C-3 and C-19, introducing hydroxyl groups at C-7 could enhance the activity. 4. The 48(17)double bond had great influence on cytotoxic activity, while breakage of the 48(17)double bond could result in the loss of activity. 5. Introducing hydroxyl groups at C-12 decreases activity.

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Scheme 2. Reagents and conditions: (a) Ac2O, DMAP, CH2Cl2, rt, 5 h, 98%; (b) KMnO4, MgSO.47H2O, TBAB, benzene/water, 60 °C, 12 h, 21%; (c) 5% KHCO3, MeOH, rt, 2 h, 84%.

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Scheme 4. Reagents and conditions: (a) TrCl, DIPEA, DCM, rt, 18 h, 81%; (b) PDC, DMF, 50 °C, 18 h, 66%; (c) p-toluenesulfonic acid, MeOH, rt, 3 h, 94%.

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Scheme 5. Reagents and conditions: (a) 2,2-dimethoxypropane, p-toluenesulfonic acid, toluene-DMSO = 7:1, reflux, 3 h, 95%; (b) PDC, DCM, rt, 5 h, 80%; (c) AcOH-H2O = 3:1, rt, 2 h, 90%; (d) DIBAl-H, THF, 80 °C, 3 h, 30%; (e) propionic anhydride, DMAP, DCM, 77%; (f) TrCl, DIPEA, DCM, 18 h, rt, 80%; (g) acetic anhydride, DMAP, DCM, 8 h, rt, 98%.

Acknowledgments

Table 1 Cytotoxic activity against four human cancer cellsa Cytotoxic activity in 48 hb (lM)

Compds

4 5 6 7 8 9 10 11 15 16 19 22 23 24 26 Andrographolide cis-Platin

NTUB1

MCF-7

M231

NP14

>100 39.80 42.88 11.25 >100 23.82 >100 26.34 >100 29.59 63.08 14.93 >100 89.74 8.45 32.52 5.38

NAc 79.51 >100 22.92 36.76 62.11 >100 25.17 >100 44.01 87.28 78 >100 >100 6.41 30.82 63.37

NA >100 40.41 2.64 35.03 37.47 >100 28.55 56.26 40.92 71.24 27.60 >100 >100 6.37 16.55 >100

>100 53.31 94.17 4.43 18.64 80.91 >100 40.38 >100 27.72 43.24 49.43 >100 43.11 14.31 21.24 65.27

a

Inhibition of cell growth by the listed compounds was determined using MTT assay. b Data represent the mean value of three independent determinations. c NA means not active.

6. Actually, introduction of furan ring by replacing a-alkylene-cbutyrolactone moiety might affect the antitumor activity more or less, since a Michael receptor is lost, that is why compounds 22 and 26 display better antitumor activities than compounds 23 and 24. In conclusion, a new kind of ent-labdane diterpenoid was formed after transformation of the C-ring to a furan-ring in andrographolide, which was the main structure in the synthesis of many derivatives. Those structures still possessed certain cytotoxic activity, and it is suggested that the antitumor mechanism of andrographolide is determined by multiple parts of the structure, not just part of a-alkylene-c-butyrolactone. At the same time, the structure of 14-deoxy-12-hydroxyandrographolide was an excellent nucleus with great potential for modification. Two compounds, 7 and 26 displayed good cytotoxic activity, which may serve as potential lead compounds in the development of new antitumor drugs.

This work was supported by the grants from the National Natural Science Foundation of China – China (No. 30973607 and No. 81172934), National Science Council – Taiwan (NSC 1032911-I-002-303), National Health Research Institutes – Taiwan (NHRI-EX102-10241BI), and in part from the grant from Chinese Medicine Research Center, China Medical University (the Ministry of Education, the Aim for the Top University Plan). Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.03. 086. References and notes 1. Arifullah, M.; Namsa, N. D.; Mandal, M.; Chiruvella, K. K.; Vikrama, P.; Gopal, G. R. Asian Pac. J. Trop. Biomed. 2013, 3, 604. 2. Shen, T.; Yang, W. S.; Yi, Y. S.; Sung, G. H.; Rhee, M. H.; Poo, H.; Kim, M. Y.; Kim, K. W.; Kim, J. H.; Cho, J. Y. Evid. Based Complement. Altern. Med. 2013, 2013, 210736. 3. Tang, C.; Liu, Y.; Wang, B.; Gu, G.; Yang, L.; Zheng, Y.; Qian, H.; Huang, W. Archiv der Pharmazie 2012, 345, 647. 4. Awang, K.; Abdullah, N. H.; Hadi, A. H.; Fong, Y. S. J. Biomed. Biotechnol. 2012, 2012, 876458. 5. Dua, V. K.; Ojha, V. P.; Roy, R.; Joshi, B. C.; Valecha, N.; Devi, C. U.; Bhatnagar, M. C.; Sharma, V. P.; Subbarao, S. K. J. Ethnopharmacol. 2004, 95, 247. 6. Xu, H. W.; Dai, G. F.; Liu, G. Z.; Wang, J. F.; Liu, H. M. Bioorg. Med. Chem. 2007, 15, 4247. 7. Pandeti, S.; Sonkar, R.; Shukla, A.; Bhatia, G.; Tadigoppula, N. Eur. J. Med. Chem. 2013, 69, 439. 8. Chen, D.; Song, Y.; Lu, Y.; Xue, X. Bioorg. Med. Chem. Lett. 2013, 23, 3166. 9. Sirion, U.; Kasemsook, S.; Suksen, K.; Piyachaturawat, P.; Suksamrarn, A.; Saeeng, R. Bioorg. Med. Chem. Lett. 2012, 22, 49. 10. Nanduri, S.; Nyavanandi, V. K.; Thunuguntla, S. S.; Kasu, S.; Pallerla, M. K.; Ram, P. S.; Rajagopal, S.; Kumar, R. A.; Ramanujam, R.; Babu, J. M.; Vyas, K.; Devi, A. S.; Reddy, G. O.; Akella, V. Bioorg. Med. Chem. Lett. 2004, 14, 4711. 11. Jung, M.; Ko, I.; Lee, S.; Choi, S. J.; Youn, B. H.; Kim, S. K. A. Bioorg. Med. Chem. Lett. 1998, 8, 3295. 12. Oh, S.; Jeong, I. H.; Shin, W. S.; Lee, S. Bioorg. Med. Chem. Lett. 2009, 2003, 13. 13. Reddy, P. P.; Rao, R. R.; Shashidhar, J.; Sastry, B. S.; Rao, J. M.; Babu, K. S. Bioorg. Med. Chem. Lett. 2009, 19, 6078. 14. Nanduri, S.; Nyavanandi, V. K.; Thunuguntla, S. S. R.; Velisoju, M.; Kasu, S.; Rajagopal, S.; Kumar, R. A.; Rajagopalan, R.; Iqbal, J. Tetrahedron Lett. 2004, 45, 4883. 15. Zhang, D. Y.; Zhu, C. Z.; Wang, K.; Zhang, M. H.; Wu, Y. C.; Wu, X. M.; Hua, W. Y. Synthesis 2014, 46, 2574.