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Thapsigargin, Origin, Chemistry, Structure-Activity Relationships and Prodrug Development

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Thapsigargin, Origin, Chemistry, Structure-Activity Relationships and Prodrug Development Send Orders for Reprints to [email protected] Current Pharmaceutical Design, 2015, 21, 5501-5517 5501 Thapsigargin, Origin, Chemistry, Structure-Activity Relationships and Prodrug Development Nhu Thi Quynh Doan and Søren Brøgger Christensen* Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenha- gen, Copenhagen, Denmark Abstract: Thapsigargin was originally isolated from the roots of the Mediterranean umbelliferous plant Thapsia garganica in order to characterize the skin irritant principle. Characteristic chemical properties and semi-syntheses are reviewed. The biological activity was related to the subnanomolar affinity for the sarco/endoplasmic reticulum calcium ATPase. Prolonged inhibition of the pump afforded collapse of the calcium homeostasis and eventually apoptosis. Structure-activity relationships enabled design of an equipotent analogue containing a linker. Conjuga- tion of the analogue containing the linker with peptides, which only are substrates for either prostate specific anti- gen (PSA) or prostate specific membrane antigen (PSMA) enabled design of prodrugs targeting a number of cancer diseases including prostate cancer (G115) and hepatocellular carcinoma (G202). Prodrug G202 has under the name Søren B. Christensen of mipsagargin in phase II clinical trials shown promising properties against hepatocellular carcinoma. Keywords: Thapsigargin, mipsagargin, drug development, clinical trials, structure activity relationships, prostate specific antigen, prostate specific membrane antigen, prodrug, anti-angiogenesis. 1. INTRODUCTION prodrugs selectively activated in tumors. Mipsagargin a prodrug Already Theophrastos (372 – 287 B.C.) described the skin irri- designed only to be cleaved in neovascular tumors has in phase II tant properties of the resin of Mediterranean plant Thapsia gargani- clinical trials showed an encouraging effect against sorafenib re- ca L. (Apiaceae, previous Umbelliferae). Later scholars like Dios- sistant patients. corides (approximately 50 A.D.) and Plinius (24 – 79 A.D.) also mentioned the use of preparations from the plant for treatment of 2. CHEMISTRY e.g. pulmonary diseases, catarrh and as a counterirritant for relief of rheumatic pain [1, 2]. Radix thapsiae and resina thapsiae have been 2.1. The Structures and Diversity of Polyoxygenated Guaiano- included in several pharmacopeias latest in the French pharmacope- lides ia from 1937. In spite of this extended traditional use of the plant Thapsigargin (1) was isolated together with the related the first publication describing the constitution was not published thapsigargicin (2, Tc, Fig. 1). Besides Thapsigargin (1) several before 1978 [3]. The relative configuration of the major skin irritat- hexaoxygenated guaianolides only differing from Tg (1) by the acyl ing principle of thapsigargin (1, Tg, Fig. 1) was published in 1980 groups at O-2 and O-8 have been found in the genus Thapsia (2-13, [4] and 1982 [5] and the absolute configuration in 1985 [6]. A Fig. 1) [14, 15]. Two additional thapsigargins have been found in number of cellular assays revealed that the compound was a very Laser trilobum Borkh. (14, 15, Fig. 2) [16]. As well the hexa- as the potent secretagogue for histamine release from peritoneal mast cells pentaoxygenated guaianolides are only present in either Thapsia [7, 8] and activated a number of cells belonging to the humane in- species or in L. trilobum (16-18, Fig. 3). Comparison of the 1H and flammatory response [9]. In addition to being a skin irritant Tg (1) 13C NMR spectra of Tg (1) and the related Tc (2) revealed that both also showed to be a cocarcinogen facilitating skin cancer develop- compounds possessed an acetoxy, a butanoyloxy and an an- ment in mice [10]. Intensive interest for Tg (1), however, first ap- geoyloxy group present on the hydrogen poor guaianolide skeleton. peared when the potent biological activities were related to the In addition the presence of an octanoyloxy residue was present in ability of inhibiting the Sarco/Endoplasmic Reticulum Calcium Tg (1) whereas a hexanoyloxy group was present in Tc (2). Com- ATPase (SERCA) [11]. Today Tg (1) has become a positive stand- parison with the spectra of trilobolide (16, Fig. 3) [17], which just ard in all experiments for calcium homeostasis in cells [12]. Pro- had been published at this time, revealed the absence of the two longed inhibition of the SERCA pump affords a persistent high protons at C-2, whereas an additional acyloxy group was present. concentration of calcium ions in the cytosol, which after 12 – 24 hours induces apoptosis [13]. The ubiquitous presence of SERCA A combination of these observations led to the suggestion of the in all living cells and the subnanomolar affinity for the SERCA constitution of Tg (1) [4]. The poor number of protons at the guaia- pump therefore makes Tg (1) a potent universal cell toxin. Howev- nolide skeleton prevented establishment of the relative configura- er, the overexpression of proteolytic enzyme in neovascular tissue tion. Fortunately, treatment of Tg (1) with thionyl chloride afforded in tumors and the presence of prostate specific antigen only in the a crystalline epoxide 19 (Scheme 1) the structure of which was prostate gland or in prostate cancer tumors have enabled design of solved through an X-ray crystallographic analysis [18]. The X-ray analysis did neither enable establishment of the absolute configura- tion nor of the relative configuration at C-7 or C-11. Resistance towards periodic acid of the debutanoyl thapsigargin *Address correspondence to this author at the Department of Drug Design 20 (Scheme 2), however, revealed that the three hydroxyl groups and Pharmacology, Faculty of Health and Medical Sciences University of had to be trans disposed. Copenhagen, Universitetsparken 2, DK-2100 Copehagen Ø, Denmark; Tel/Fax: +45-3533-6253, +45-3533-6041; E-mail: [email protected] 1381-6128/15 $58.00+.00 © 2015 Bentham Science Publishers 5502 Current Pharmaceutical Design, 2015, Vol. 21, No. 38 Doan and Christensen 1 14 The 1-disposed H-1 is typically found in guaianolides isolated R O O H O from species belonging to Apiaceae (umbelliferous plants) but a O 10 9 2 2 1 R few examples have been found in plants belonging to Asteraceae O 3 8 O 6 7 (composites) [19]. 4 5 OH 11 OH 15 O 2.2. Chemistry of Polyoxygenated Guaianolides 12 13 O From a chemical point of view the polyoxygentaed guaianolides possess three functional groups: ester groups, tertiary alcohols and a 1 2 Thapsigargin (1): R = Oct, R = But double bond between C-4 and C-5. In spite of this, methods have Thapsigargicin (2): R1 = Hex, R2 = But been developed for selective modifications of the structures. 1 2 Thapsitranstagin (3): R = iVal, R = 2-MeBut 2.2.1. Epoxide Formation 1 2 Thapsivillosin A (4): R = Ang, R = Sen 1 2 Treatment of as well pentaoxygenated as hexaoxygenated guai- Thapsivillosin B (5): R = Ang, R = 2-MeBut 1 2 anolides converts the 7,11-dihydroxy diol into an epoxide with Thapsivillosin C (6): R = Oct, R = 2-Mebut 1 2 conversion of the stereochemistry at C-11 (Scheme 1). Epoxide Thapsivillosin D (7): R = 6-MeOct, R = Sen formation from a diol is a very seldom reaction and probably locked Thapsivillosin E (8): R1 = 6-MeOct, R2 = 2-Mebut 1 2 optimal conformation of the 7- and 11-hydroxy groups enables the Thapsivillosin G (9): R = 6-MeHep, R = 2-Mebut 1 2 reaction [17, 18]. Thapsivillosin H (10): R or R = Ang or Sen 1 2 Thapsivillosin I (11): R = Ang, R = But 1 2 O O Thapsivillosin J (12): R = iVal, R = But 1 2 O O O O Thapsivillosin K (13): R = Sen, R = 2-MeBut H O O H O O O SOCl2 O O O O O O O O OH O OH O O Ang But Hex 1 19 O O O O Scheme 1. Conversion of thapsigargin (1) into epoxide (19). 2-MeBut 6-MeHep 2.2.2. Selective Hydrolysis of the Ester Group at O-8 O O Treatment of as well penta- as hexaoxygenated guaianolides 6-MeOct Non with triethylamine in a protolytic solvent like methanol affords a selective hydrolysis of the O-8 ester group (Scheme 2) [20]. The O O O hydrolysis of the labile O-8 ester group might be facilitated by the Oct Sen iVal juxtaposed OH-11 since the hydrolysis of the ester group in the epoxide 19 demands harsher reaction conditions. The use of a stronger base like sodium carbonate results in opening of the 6,12- Fig. (1). Structures of naturally occurring hexaoxygenated guaianolides lactone. As a consequence, a mixture of the 6,12- (20) and 8,12- found in Thapsia [thapsigargins] (1-13) with traditional numbering. (21) guaianolides are obtained after workup under acidic conditions (Scheme 2) [18]. R1 14 O R2 O H O O O O 10 9 2 1 O O O 3 8 O O O O H O H O 5 6 7 O Et3N O 4 OH O O O OH 11 MeOH 15 OH OH OH O OH OH 12 13 O O O 120O O 1 2 2-Hydroxy-10-desacetoxytrilobolide (14): R = R = H Na2CO3 (aq) 1 2 MeOH 2-Acetoxytrilobolide (15): R = R = Ac O O O O Fig. (2). Structures of the naturally occurring hexaoxygenated guaianolides O H O H O O O O H found in L. trilobum [thapsigargins] (14-15). O OH 20 O O OH OH HO O HO HO HO CH -O 3 14 O O 19 21 H O O 10 9 Scheme 2. Formation of 6,12- (20) and 8,12- (21) guaianolides. 2 1 O 3 8 OR 6 7 4 5 OH 11 OH 2.2.3. Selective Substitutions of the Ester Group at O-2, O-10 and 15 O O-8 12 13 O Masking of the OH-8 and OH-11 by reaction with 2,2-dime- thoxypropane yields an isopropylidende derivative (22, Scheme 3) in which the polycyclic nature prevents relactonization.
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