Advances in structural modifications of celastrol

Celastrol, a quinone methide triterpenoid from the Chinese medicinal root bark of Tripterygium wilfordii , possesses beneficial therapeutic properties and affects numerous cellular pathways. Recently, structure and activity relation studies based on celastrol semi-synthetic derivatives have contributed greatly to our understanding of its mechanism of action and cellular targets. These modifications have resulted in better activity and improvement of the pharmacokinetic properties of celastrol. This review will mainly cover advances in the structural modifications of celastrol at the C-20 carboxylic acid functionality, alterations of the A ring, and modifications at C-6.


Introduction
Over the past few decades the high activity of extracts from the medicinal herb Tripterygium wilfordii have attracted intensive attention.2][3][4][5][6] Celastrol shows a great variety of biological effects such as anti-oxidant, 7,8 anti-tumor, [9][10][11][12] anti-obesity, 13,14 cardiac protection, 15,16 anti-inflammatory [17][18][19] and antiangiogenic 20,21 activities, in both in vitro and in vivo models.It exhibits astonishing clinical potential as a therapeutic and chemopreventive agent toward various cancers.Celastrol has also been reported to inhibit growth, 22,23 induce apoptosis, [24][25][26][27] and suppress invasion and metastasis [28][29][30] of tumor cells.It is extremely effective towards many molecular targets, but there are some inherent deficiencies including high toxicity, poor stability and water solubility which impede its further application. 31he screening and assignment of active new derivatives from synthetic or semi-synthetic strategies has attracted intense interest from medicinal scientists.This review provides a summary of the recent advances in celastrol structural modifications of functionality of the C-20 carboxylic acid, alteration of the A ring at C-2, and modification of the B ring at C-6.

Modification of the C-20 Carboxylic Acid Group Functionality
In order to improve solubility, reduce toxicity, and develop new activities of celastrol analogues, simple modifications strategies are still the esterification or amidification of the 20-carboxylic acid group, 32 for example, the N-propylamide (2) and cetyl ester (3), which showed antiproliferation activity similar to or better than that of celastrol in various cancer cell line assays (Scheme 1). 33

Scheme 1
In 2010, Xu and coworkers prepareded a series of celastrol analogues (4-16) and investigated their neuroprotective effect against t-BHP-induced cell damage in neuronal PC12 cells (Scheme 2). 35Cells treated with celastrol and compound 15 were subjected to Western blot analysis to check Hsp70 protein expression.Celastrol enormously increased the Hsp70 expression at 0.4 and 1.6 μM.Compound 15 improved Hsp70 expression at a concentration as low as 0.1 μM, but its neuroprotective effect was not evident until its concentration reached 0.4 μM.Compared with celastrol, 15 is more effective in protecting cells from t-BHP-induced cell damage and against t-BHP-induced cytotoxicity.Compound 15 up-regulated Hsp70 protein expression dose-dependently and probably helped to strengthen the cellular capability to resist oxidative stress, and eventually promote cell survival.These results suggest that 15 may be used as a potential candidate for intervention in neurodegenerative diseases.

Scheme 2
To improve the aqueous solubility of celastrol, in 2012 San et al. designed and synthesized a series of celastrol derivatives of amino carbamic acid ester at C-3 of the A ring and esterification of the 20-carboxylic acid (Scheme 3). 36Although the aqueous solubility of derivatives was significantly improved, these compounds were less active than celastrol in inhibiting the proliferation activity of Bel and A-549 tumor cells.
Celastrol from the natural source possesses numerous beneficial therapeutic properties and influences diverse cellular pathways.In order to understand the mechanism of action and cellular target(s) of celastrol, Morimoto and coworkers designed and synthesized a family of celastrol derivatives 27 and 28 for the identification of protein targets and investigated them as inducers toward heat shock response (Scheme 5). 38In the celastrol structure, conversion of the carboxylic acid functionality into amides or long-chain analogues leads to improvement in its potency and yields bioactive compounds that induce the heat shock response (HSR) and antioxidant response, and inhibit Hsp90 activity.The biotinylated conjugates of celastrol 27 and 28 were used as affinity reagents in extracts of human Panc-1 cells to identify Annexin II, eEF1A, and β-tubulin.The plausible results confirmed a previous suggestion that β-tubulin is another potential target of celastrol and celastrol targets Hsp90 indirectly by invoking a redox imbalance.

Alteration of the A ring and modification at C6
In 2014, Pan and coworkers synthesized a series of new C-6-modified celastrol derivatives and evaluated them for their cytotoxic activity toward seven human cancer cell lines (BGC-823, H4, Bel7402, H522, Colo 205, HepG2 and MDA-MB-468) in vitro (Scheme 6). 31Most of the compounds exhibited higher potent inhibition against BGC823 (IC 50 = 0.42-0.77μM), H4 (IC 50 = 0.35-1.37μM), and Bel7402 (IC 50 = 0.45-1.55μM) than that of celastrol (IC 50 = 3.73 μM, IC 50 = 2.09 μM and IC 50 = 1.55 μM).It was clear that the C-6 functionalized celastrol derivatives possessed increased cytotoxicity potency in vitro against the human cancer cell BGC823, H4, Bel7401.In particular, 2,3-acetylation was more effective than propionylation, but the C(6)carbonated derivatives dramatically dropped in cytotoxicity potency.The compound 29-A displayed excellent inhibition of tumor growth in vivo antitumor assay on nude mice bearing Colo 205 xenografts with a 0.06 μM IC 50 value.The results indicate that C-6 functionalized celastrol derivatives may be regarded as potential drug candidates for treating cancer.

Scheme 7
In a later work, Pan and coworkers have reported a series of C-6-indole-substituted celastrol derivatives and evaluated their cytotoxic activity against human hepatocellular carcinoma Bel7402 and human glioblastoma cell line H4 (Scheme 7). 39The active compounds exhibited inhibition activities against Bel7402 (IC 50 = 0.01-23.91μM) and H4 (IC 50 = 2.03-14.56μM), and the IC 50 of celastrol as positive control against Bel7402 and H4 were 2.09 μM, 1.55 μΜ, respectively.The results from their structure-activity relationships indicated that the analogues 33f and 33h showed significant antiproliferative activities against Bel7402 cancer cells in vitro (IC 50 0.02 μM and 0.01 μM, respectively).Celastrol derivatives at C-6 position bearing a C-C bond still displayed potent in vitro anticancer activities.In fact, the addtion of a C-C bond at C-6 inevitably influences the Michael acceptor action and formation of a protein complex.  At t same time, the study strongly suggested that the Michael-acceptor action of celastrol is not necessary for its antitumor activity.

Summary and Outlook
Celastrol, as a representative of the natural quinone methide triterpenoids, displays wide antiinflammatory and anticancer activity and produces multiple biological effects at the molecular level.In particular, it has excellent prospects as an anti-cancer therapeutic drug in view of its perceived beneficial effects on a variety of cancers in vitro and in vivo.At present, modifications of celastrol have chiefly been focused on the C-20 carboxylic acid functionality, and alteration at the A ring and C-6-modification at the B ring.Some authors have stated that the quinone methide moiety is required for its antitumor activity; 32,38 however, while the A ring is transformed into a benzene ring with an alkoxy group, the corresponding derivatives still exhibit significant activities against certain tumor cell such as, BGC823, H4 and Bel7402.Some more recent studies have indicated that the C(6)-position of celastrol was suitable for modification and have shown that the Michael acceptor property is not necessary for its anti-tumor activity. 31,39herefore, the modifications of celastrol should focus on developing new biological activities, improving its pharmacokinetic properties, and exploring novel modifications, strategies and location points.With better understanding of the mechanism of action of celastrol, we believe that more and better celastrol derivatives will be brought about in the future.

2 .
Modification of the C-20 Carboxylic Acid Group Functionality 3. Alteration of the A-ring and Modification at C-

Financial
support was provided by the National Natural Science Foundation of China (21562016), the Natural Science Foundation of the Guizhou Science and Technology Department (JZ [2014]2010) and The Science and Technology Planning Project of Guiyang City [(2012204)23].