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

Send Orders for Reprints to [email protected] Current Pharmaceutical Design, 2015, 21, 5501-5517 5501

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 antigen (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 configuration 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]

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. Treatment Trilobolide (16): R = 2-MeBut Nortrilobolide (17): R = But of 22 with strong base affords 23 and traces of 24 (Scheme 3) [20, Thapsivillosin F (18): R = Sen 21]. Fig. (3). Structures of naturally occurring pentaoxygenated guaianolides [trilobolides] (16-18) found T. garganica or L.trilobum. Thapsigargin, Origin, Chemistry, Structure-Activity Relationships Current Pharmaceutical Design, 2015, Vol. 21, No. 38 5503

O O O O O MeO OMe O O MeO OMe O H O H O H H O H O O O H O O OH O O Acetone O OH OH OH OH OH Acetone OH O OH O O OH OH O O 20 O 22 O 29 O 32 O OH OH HO H HO H KOH O O O O O HO O H H O O O MeOH OH OH MeO O O O O O O O OH OH O O 23 O 24 O O O 30 O 31 O Scheme 3. Masking OH-8 and OH-11. Minor Major

As expected the ,-unsaturated angeloyl group at C-3 is more Scheme 5. Formation of isopropylidene derivatives 30 and 31. stable towards basic hydrolysis than the other acyl groups in the molecule. Secondary alcohols are known to react faster with acyl 2.2.4. Selective Substitution of the Angloyl Group at O-3 groups than tertiary alcohols. Consequently acylation of OH-2 pro- Whereas the double bond between C-2 and C-3 in the angeloyl ceeds faster than acylation of OH-10 enabling selective acylation to moiety protects the ester bond from saponification the same group give 25, which subsequently can be acylated at O-10 to give 26 also enable selective hydrolysis of this group. Oxidation of the (Scheme 4). Removal of the isopropylidene group with acid in an group with reagents like osmium tetraoxide or permanganate fol- aqueous medium affords 27 in which O-8 selectively can be acylat- lowed by treatment with periodate enables formation of the pyruvic ed affording 28 (Scheme 4). In total, the procedure enables selec- ester 33, which can be solvolyzed under mild conditions like pyri- tive replacement of all the acyl groups in the starting material ex- dine in methanol to give the 3-hydroxy compound 34 (Scheme 6) cept for the angeloyl group [20, 21]. [20]. Surprisingly attempts to convert the debutanoyl nortrilobolide 29 into the corresponding isopropylidene derivative 30 only afford- O O O O ed the target compound in trace amounts whereas the major product O O H O H O O - O O MnO4 or OsO4 O was the 3-methylether 31 (Scheme 5). The most likely explanation O O O O for the different behavior of the hexa- and the pentaoxygenated OH OH OH HO HO OH guaianolides is that the voluminous octanoyloxy group in the 2- O O O position prevents an attack from the methanol on the intermediary 1 O

- formed carbocation 32, whereas the absence of the 2-subsituent in IO4 29 favors the competing reaction. O O O O O H O O H O O O Pyridine O O HO O O O HO OH R O OH OH MeOH O OH H (RCO) O, H OH OH O 2 O O O 2 10 DMAP O 8 O O O 34 O 33 O OH OH Scheme 6. Selective angelate cleavage at O-3. O O O O

23 O 25 O Again a significant difference between the penta- and the hexaoxygenated guaianolide has been observed in the selectivity of oxidation of the angeloyl double bond. In the case of the pentaoxy- (R'CO)2O, DMAP genated guaianolide, nortrilobolide (17), the C-4-C-5 double bond is sensitive to oxidation, e.g. ozonolysis, affording a cleavage of the O O five-membered ring to form 35 and 36 (Scheme 7). Again it is as- R' R' R O O O sumed that the sterical effect of the 2-octanoyl substituent prevents H R O H O O O the attack of the C-4-C-5 double bond in the hexaoxygenated guai- H O O OH O O anolides. OH MeOH OH OH O O O O O 27 O 26 O O H O O H O O O 1) O3 then PPh3 O O O OH 2) Pyridine, MeOH OH OH (R''CO) O, O 2 OH OH DMAP O O 17 O 35 O O R' R O O H O O O O O R" O H O OH O O OH O O O OH O HO OH 28 O

36 O Scheme 4. Selective substitutions of acyl groups at O-2, O-10 and O-8. Scheme 7. Ozonolysis of nortrilobolide (17) to form 35 and 36. 5504 Current Pharmaceutical Design, 2015, Vol. 21, No. 38 Doan and Christensen

Selective removal of the angeloyl group in the pentaoxygentaed O O O O O O guaianolides, however, might be obtained taking advantage of the H O Ac2O H O O O DMAP O O possible nucleophilic removal of the angeloyloxy group by reacting O O O O OH OH nortrilobolide (17) with an acidic aqueous medium (Scheme 8). A OH O O similar reaction has been observed in the hexaoxygenated guaiano- O 38 OH 42 O O lide [14]. As expected a mixture of the two 3-alcohols 37 is ob- O O O tained. ScCl4 O H O RCN O O O O OH OH O O O O H O H O O + O 43 N O H (aq) R O O HO O OH MeCN OH OH OH O O Scheme 11. Formation of oxazoles 43 from hemiacetal 38. 17O 37 O 2.2.7. Synthesis of Thapsigargin (1) Scheme 8. Selective angelate cleavage of nortrilobolide (17) under acidic At the present the annual demand of paclitaxel is approximately aqueous conditions to yield 37. 1 ton per year. A successful outcome of the current clinical trials of mipsagargin is expected to create an annual demand of Tg (1) in the same order of magnitude. At the present Tg (1) is only available 2.2.5. Selective Reduction of the Lactone Group from the roots or fruits of the wild population of T. garganica. A Treatment of Tg (1) with sodium borohydride affords a selec- total synthesis of Tg (1) in 42 steps affording an overall yield of tive reduction of the lactone carbonyl group to form the hemiacetal 0.6% has been developed starting form (S)-carvone. (Scheme 12) 38, although only in modest yield (Scheme 9) [20, 22]. Sodium [24, 25]. bis(2-methoxyethoxy)ethoxyaluminium hydride (Red-Al) was found to be superior to sodium borohydride [22]. O

O O O O NaBH H H O O 4 O O O H O or O 42 steps O O H O O O Red-Al O O O O O O O OH OH OH OH O 12 OH OH O O O 1 O 38 OH (S)-carvone O O H 1 O Red-Al Na Al O O H Scheme 12. Total synthesis of thapsigargin (1) in 42 steps from (S)-carvone.

Scheme 9. Selective reduction of lactone carbonyl at C-12. In spite of the impressive academic achievement this synthesis, however, is not commercial feasible. An alternative procedure 2.2.6. Chemistry of the 12-Semiacetal (38) could be semi-synthesis from other available natural products. A few other studies towards synthesis of closely related compounds Attempts to O-12-alkylate 38 with ethyl orthoformate only to have been published [26, 27]. Trilobolide (16) might be a possibil- some extend afforded the acetal 39, instead the two orthoesteres 40 ity as an alternative starting material since this compound is easily and 41 were formed as the major products (Scheme 10) [22]. available and can be isolated from L. trilobum, which can be grown e.g. in The Czech Republic. O O A synthetic route has been developed in order to obtain Tg (1) O H O O O in four steps starting from nortrilobolide (17) (Scheme 13). O O OH OH Treatment of nortrilobolide (17) with chromium trioxide and O 39 aqueous hydrogen fluoride in an one pot reaction affords the ketone Minor O 44 in excellent yield. Oxidation of 44 with manganese(III) acetate O O OEt O O in the presence of excess amount of octanoic acid affords stereose- O H O O H O O O EtO OEt, H O O lective the -2-octanoyloxygentaed derivative 45. Reduction of 45 O O O O OH OH with zinc borohydride gives a mixture of the two epimeric alcohols OH O O O 38 40 46S and 46R, 46S being the major product. Finally, acylation of O OH Major O 46S with the mixed anhydride generated from angelic acid and O 2,4,6-trichlorobenzoyl chloride yields thapsigargin (1). O O H O O O O O 3. THE SARCO/ENDOPLASMIC RETICULUM CALCIUM OH ATPASE AS A DRUG TARGET O O 41 2+ Major O In the resting state any cells maintain a low Ca concentration O 2+ (100 nM) in the cytosol and a high Ca concentration (0.5 mM) in the sarco/endoplasmic reticulum. In the case of the endo/sarco- Scheme 10. Alkylation at O-12. plasmic reticulum the Sarco/Endoplasmic Reticulum Calcium ATPase (SERCA) maintain this gradient by transporting Ca2+ ions from the cytosol into the organelle. For each ATP consumed by the Another unexpected property of the 12-hemiacetal is the reac- 2+ tion of the diacetate 42, obtained from 38, with nitriles to form pump two Ca ions are transported across the membrane oxazoles 43 (Scheme 11) [23].

Thapsigargin, Origin, Chemistry, Structure-Activity Relationships Current Pharmaceutical Design, 2015, Vol. 21, No. 38 5505

O O 3.1. Structure-Activity Relationships H O O H O O O CrO3, HF (aq) A requirement for use of the prodrug approach is an intimate O O O O MeCN OH OH knowledge of structure-activity relationships (SAR). Only such OH OH O O knowledge enables location of the linker to give an agent, which 17O 44 O still possesses activity. Furthermore SAR analysis might indicate O how structural changes of the molecule might make the compound O O more drugable. Below are presented Tables 1-10 illustrating the H O O Mn(OAc3) Zn(BH4)2 O O effects of changes of the structure of Tg (1). The data given in the Octanoic acid OH table are mainly taken from the references: [21, 30-35]. OH O 3.1.1. Changes at the 8-Position 45 O O O When interpreting the relative activities presented in Table 1 O O O H O O H O (analogues 20, 47-72) it is important to remember that the IC50 O O value of Tg (1) is subnanomolar [23, 35] meaning that even an ana- HO O HO O OH OH logue 100 times less active than Tg (1) is a potent compound. The OH OH O O IC50 value is the concentration, in which the SERCA pump is inhib- 46S O 42R O ited 50%. Considering this fact it can be concluded that even dramatic changes of the side chain at O-8 only to a minor extent reduc- 2,4,6-trichlorobenzoyl chloride es activity. Even hydrolysis to give the 8-hydroxy analogue 20 still Et3N, Angelic acid affords a potent compound. From the point of view of designing a prodrug this finding is encouraging since it means that a broad O O spectrum of linkers possessing a terminal amino group may be used O H O O O as linkers. O O OH The key then may be a peptide bond. Noticeable is the analogue OH O terminating with a tert-butyloxycarbonyl protecting group (Boc) 62. 1 O The significant lower activity of this compound might indicate that the presence of terminal hydrophilic groups could be of importance. Scheme 13. Semi-synthesis of Tg (1) starting from nortrilobolide Important for defining the pharmacophore of Tg (1) is the finding (17). that inversion of the stereochemistry at C-8 dramatically reduces the potency as seen for analogue 72 (Table 2). and 2-3 H+ ions released into the cytosol [28]. Blockage of the 3.1.2. Changes at the 3-Position pump results in a collapse of the Ca2+-gradient. As a consequence 2+ In contrast to changes at O-8 dramatic changes can be seen by the cytosolic Ca concentration increases to 500 nM for about 3 changes at O-3 (analogues 34, 73-82, Table 3). hours where after it again decreases to low nM concentration. A continued blockage of the pump affords a new increase after about No major reduction of activity is observed by replacing the 19 hours, but now the concentration reaches 1500 nM. The later angeloyl with a benzoyl 76 or a flexible octanoyl group 74 (Table burst induces a cascade reaction, which eventually results in apop- 3). However, introduction of a 4-methylbenzoyl 77 causes a severe tosis [13]. From a chemotherapeutic point of view the cytotoxicity drop in activity, whereas a 3-methylbenzoyl 78 is tolerated. is interesting since cells are killed in a proliferation independent A similar trend is seen by replacement with a 2-phenylbenzoate way [13]. Most presently used chemotherapeutics like paclitaxel, 79 and a 4-phenylbenzoate 80. Adding even a little flexibility into vincristine and doxorubicin only kill cells during the proliferation the side chain as is seen in biphenylacetoyl 81 again regains activi- and consequently slowly developing cancer diseases like prostate ty. cancer are not affected [13]. The drawback by targeting SERCA, As was the case for C-8, inversion of the stereochemistry at C-3 however, is that this pump is essential for survival of almost all provokes a dramatic decrease in activity as seen in analogue 82 kind of cells meaning that Tg (1) is a general cell toxin. This is (Table 4). supported by the low lethal dosis for mice (0.8 mg/kg) [29]. The use of Tg (1) per se as a drug thus is excluded. However, a prodrug 3.1.3 Changes at the 2-Position targeting the compound towards cancer tissue has been developed. The binding site is very tolerant toward substitution in the 2- A prodrug is a drug that by itself is inactive, but is cleaved near the position since no dramatic shifts in activity occurs by replacements pharmacological target to release the active agent. The moiety used in this position (analogues 83-86, Table 5 and 6). This conclusion is to inactivate the agent is named the promoiety. An optimum bond further confirmed by inspecting the naturally occurring hexao- for conjugating the active drug with the promoiety is a bond that xygentaed guaianloides (Fig. 1), all of which have very similar only is cleaved in the target tissue (Fig. 4). activities. 3.1.4. Changes in the 10-Position In contrast to the 2-position, the 10-position is located in an area in the binding cavity, which severely reacts towards introduction of voluminous groups by reducing the affinity of the ligand (analogues 87-92, Table 7). 3.1.5 Changes at the 7- and 11-Positions Important to notice is that replacement of the hydroxy groups with an acyl group and thereby preventing the group form being hydrogen donors only to a limiting extent effect the IC value (ana- Fig. (4). The prodrug principle. An agent (gray oval) is coupled to an inacti- 50 logues 19, 22, 93-101, Table 8 and 9). vating group (the promoiety, circle) via a linker (wavy line) and a bond sensitive to cleavage only in the target tissue (bold binding). At the pharma- Acylation of O-7 even with small acyl group only affords a cological target the sensitive bond is cleaved and the active agent released. minor decrease in activity but flexible large substituents decrease activity. The epoxide 19 also still possesses some affinity for the 5506 Current Pharmaceutical Design, 2015, Vol. 21, No. 38 Doan and Christensen

Table 1. Changes at O-8.

O O O H O O R * Thapsigargin analogues O 8 O Relative IC50 OH O OH

20 R = H 5.4/50 [30] [21]

O H N 47 R = 327 [21] O

NH2

O O 48 R = 81 [21] N NH2 H

O O 49 R = 4.4 [21] N NH2 H

50 R = 1.7 [21]

NH2

51 R = O 1.9 [21] N O H

52 R = 1.5 [21]

NH2

53 R = O 1.8 [21] N O H

H N O 54 R = O 1.3 [21] O

NH O 2 55 R = 1.7 [21]

O NH 56 R = N 2 35 [31] H

57 R = NH2 99 [31]

Thapsigargin, Origin, Chemistry, Structure-Activity Relationships Current Pharmaceutical Design, 2015, Vol. 21, No. 38 5507

(Table 1) Contd....

O 58 R = 90 [31] NH2

59 R = NH2 17 [31]

O 60 R = 3 [31] NH2

61 R = NH2 2.6 [31]

O H 62 R = N O 240 [31] O

O H 63 R = N 287 [31] NH2 O

O H 64 R = N 3.4 [31] NH2 O

O H 65 R = N 1.2 [31] NH2 O

OH O H 66 R = N 0.8 [31] NH2 O

O O

67 R = N O 30 [35] H OH

O H N O 68 R = O 8.5 [35] O

O O H 69 R = N 11.5 [35] O N O H O

O O 70 R = O O 44 [35] O N O H

O O H O O N O O N 71 R = H 70 [35] O

*The relative IC50 value is the ratio between IC50 value of the analogue and the IC50 value of thapsigargin (1) [IC50(analogue)/IC50(thapsigargin)].

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Table 2. Inversion at C-8.

* Thapsigargin analogue Relative IC50

O O

O O H O O 72 O 8 O 3124 [32] OH O OH

*The relative IC50 value is the ratio between IC50 value of the analogue and the IC50 value of thapsigargin (1) [IC50(analogue)/IC50(thapsigargin)].

Table 3. Changes at O-3.

O O O H O O R O 3 * Thapsigargin analogues O Relative IC50 OH O OH

34 R = H 565 [21]

O [32] O O H O O 73 R = O 3 O 66.5 OH OH O O

O 74 R = 11.2 [32]

O 75 R = 1.53 [32]

O 76 R = 4.0 [21]

O 77 R = 220 [21]

78 R = 45 [21]

O 79 R = 15 [21]

80 R = 350 [21]

81 R = O 90 [21]

*The relative IC50 value is the ratio between IC50 value of the analogue and the IC50 value of thapsigargin (1) [IC50(analogue)/IC50(thapsigargin)].

Thapsigargin, Origin, Chemistry, Structure-Activity Relationships Current Pharmaceutical Design, 2015, Vol. 21, No. 38 5509

Table 4. Inversion at C-3.

* Thapsigargin analogue Relative IC50

O O O O H O O 82 O 3 O 438 [32] OH O OH

*The relative IC50 value is the ratio between IC50 value of the analogue and the IC50 value of thapsigargin (1) [IC50(analogue)/IC50(thapsigargin)].

Table 5. Changes at O-2.

* Thapsigargin analogue Relative IC50

O O O H O 83 O O 8 [21] O O OH O OH

*The relative IC50 value is the ratio between IC50 value of the analogue and the IC50 value of thapsigargin (1) [IC50(analogue)/IC50(thapsigargin)].

Table 6. Changes at O-2.

* Thapsigargin analogue Relative IC50

O H O O O O O 84 40 [35] OH O OH

O H O O O O O 85 0.1/17.5 [33] [35] OH H OH O O

O H O O O O 86 0.3 [33] OH H OH O O

* The relative IC50 value is the ratio between IC50 value of the analogue and the IC50 value of thapsigargin (1) [IC50(analogue)/IC50(thapsigargin)].

5510 Current Pharmaceutical Design, 2015, Vol. 21, No. 38 Doan and Christensen

Table 7. Changes at O-10.

O R O O H O O 10 Relative Thapsigargin analogues O O IC * OH 50 O OH

87 R = 42/135 [32] [21] H

O 88 R = 12.5 [21]

O 89 R = 80 [21]

78 [21] 90 R =

91 R = 350 [21]

92 R = O 135 [21]

*The relative IC50 value is the ratio between IC50 value of the analogue and the IC50 value of thapsigargin (1) [IC50(analogue)/IC50(thapsigargin)].

Table 8. Changes at O-7 and O-11.

O O O H O O O Relative Thapsigargin analogues O O * 7 IC O R1 50 2 O 11 O R

O 93 R1 = R2 = H 2.8 [32]

O 94 R1 = R2 = H 55 [23]

O 95 R1 = R2 = H 65 [23]

O 96 R1 = R2 = H 42.5 [23]

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(Table 8) Contd....

O 97 R1 = R2 = H 275 [23]

O 98 R1 = H R2 = 2.5 [32]

O 99 R1 = H R2 = 55 [21]

O O 100 R1 = R2 = 15 [32]

*The relative IC50 value is the ratio between IC50 value of the analogue and the IC50 value o thapsigargin (1) [IC50(analogue)/IC50(thapsigargin)].

Table 9. Changes at O-7 and O-11.

* Thapsigargin analogue Relative IC50

O O O H O O O [Private communi- 19 O O 100 cation] O O

O O O H O O 22 O O 95 [21] OH O O

O OH O H O 101 O O 350 [21] O O O O O

* The relative IC50 value is the ratio between IC50 value of the analogue and the IC50 value of thapsigargin (1) [IC50(analogue)/IC50(thapsigargin)].

Table 10. Changes at the 12- and 11-positions.

* Thapsigargin analogue Relative IC50

O O O H O O O 102 O O 1.06 [34] OH O OH

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(Table 10) Contd....

* Thapsigargin analogue Relative IC50

O O O H O O O O O 38R OH 1.63 [22] O OH

O O O H O O O O O 38S OH 1.63 [22] O OH

O O O H O O O O O 39 OH 1.17 [22] O OH

O O O H O O O O O 103 OH 1.92 [22] O OH

O O

O O O H O O O O O OH 42 47 [23] O O O O

O O O H O O O O O 104 OH 5.39 [22] O O H

Thapsigargin, Origin, Chemistry, Structure-Activity Relationships Current Pharmaceutical Design, 2015, Vol. 21, No. 38 5513

(Table 10) Contd....

* Thapsigargin analogue Relative IC50

O O O H O O O O O 40 OH 2.51 [22] O O H O O

O O O H O O O O O 41 OH 40.4 [22] O O H O O

O O O H O O O O O 105 OH 16.4 [22] O O H O

O OH O H O O O O 106 OH 582 [22] O O H O

OH HO H O O O O 107 OH 6312 [22] O O H O

O O O H O O O O O 108 OH 8.5 [23] O O H N

5514 Current Pharmaceutical Design, 2015, Vol. 21, No. 38 Doan and Christensen

(Table 10) Contd....

* Thapsigargin analogue Relative IC50

O O O H O O O O 109 O 100 [23] OH O O H N

O O O H O O O O 110 O 395 [23] OH O O H N O

* The relative IC50 value is the ratio between IC50 value of the analogue and the IC50 value of thapsigargin (1) [IC50(analogue)/IC50(thapsigargin)]. binding site. However no effect was observed in a whole cell assays indicating that inhibition of the pump was not efficient [8]. 3.1.6. Changes at the 12- and 11-Positions The trend observed for position 10 is observed again in the 12 and 11 positions. Introduction of large non-flexible groups causes reduction in affinity whereas smaller groups are tolerated (analogues 38-42, 102-110, Table 10).

3.2. The Pharmacophore of Thapsigargin

The first X-ray structure of SERCA bound to Tg (1) was published by Toyoshina in 2002 [36]. Important for interpreting the Structure-Activity Relationships is proof of the same binding site for all analogues. X-ray structures have revealed that a number of analogues with high affinity all bind to SERCA at the same binding site [23, 35, 37]. The structures verify that the most important interactions between the pump and the analogues are hydrophobic interactions. Four hydrophobic interactions have been suggested: 1) the angeloyl group at O-3 and N768, V769 and V263, 2) the acyl group at O-8 and residues P827, L828 and I829, 3) the acetyl group at O- 10 and residues Y837 and F834, and 4) the methyl group at C-15 Fig. (5). Hydrophobic interactions between the backbone of the binding site and Q259. The octanoyl at O-2 extends into the lipophilic mem- of SERCA (helices) and angeloyl at O-3 (N768, V769, V263), butanoyl at brane with only limited interaction with the ATPase [35]. A hydro- O-8 (P827, L828, I829), acetyl at O-10 (Y837) and the lactone ring (residue phobic interaction between the lactone ring and F256 has also been F256) of Tg (1) (ball and sticks). The interacting residues are marked in suggested (Fig. 5). In addition, to verifying the same binding site gray. PDB 4J2T. calculations indicate that beside hydrophobic interactions some water molecules also are of significance for correct localization of the ligand in the binding site [23], in particular hydrogen bridge from the carbonyl of the acyl group at O-8 to the backbone amide of L828 or the side chain of E255. In addition a hydrogen bond between OH-7 and the backbone of I829 may stabilize the binding [23]. The importance of hydrophobic interactions from the angeloyl and the acetyl group to the pump explains the drastic drop in affinity if either C-3 or C-8 is inverted. After inversion the two acyl group would be prevented from forming hydrophobic interactions with the backbone.

As revealed from Fig. 6 a long flexible substituent at O-8 al- lows affinity for the pump since it is able to adjust between the fifth, third and fourth transmembrane segments. The Boc-group is placed in a cavity between the first, second, third and fourth helices towards the surface of the pump [37]. This binding site explains the Fig. (6). The location of the N-Boc-12-aimnododecanoyl residue of com- poor activity of Ile-N-6-aminohexanoic acid since the nonflexible pound 62 in the binding site of SERCA. PDB 2BY4. amid group would be located between the transmembrane residues. Thapsigargin, Origin, Chemistry, Structure-Activity Relationships Current Pharmaceutical Design, 2015, Vol. 21, No. 38 5515

Whereas a large diversity of substituents at O-2 and O-8 are O accepted only a limited number of nonflexible groups are allowed O at O-3. Very pronounced is the significant loss of activity by mov- O H O O O H N ing the methyl group from the meta to the para position of benzoic O O R acid [21]. A little more diversity is allowed in the cavity near the O- OH OH 10 [21]. O Based on the structure activity-relationships the pharmacophore O model shown in Fig. 7 has been developed for Tg (1). HO O O O O H H H N N N 111: R = N N N O O H H H H O O O O HO NH O H O N O H2N O O 10 2 NH O 3 8 O 2 7 O H PSA clevage 11 OH O H 15 O 12 H HO O O O O O H H H 112: R = N N N N N N N H H H O O O O Fig. (7). Pharmacophore model of thapsigargin (1). The ovals indicate resi- HO NH dues that interact with the backbone of SERCA via hydrophobic interactions N H2N O and Bu-C=O and O-7 are assumed to be involved in hydrogen bridges from NH2 Tg (1) to to the backbone of SERCA. 4. PRODRUG DESIGN 113: R = NH2 The potency of analogues with a long but flexible acyl residue O at O-8 and the possibility for formation of a peptide bond if a - Fig. (8). Prodrug developed for cleavage by PSA. Prodrug only cleaved by amino group was introduced was used as a starting point for pro- PSA and thereby selective toxic in the prostate and in prostate cancer tissue. drug development. Targeting of the analogue towards cancer tissue was obtained taking advantage of proteolytic enzymes expressed to a larger extent in tumors. The obtained prodrug 112 was tested in mice. No toxicity was observed but inoculated prostate cancer tumors were found to 4.1. Prostate Specific Antigen shrink by treatment with the drug. In comparison, tumors on not Prostate specific antigen (PSA) is a proteolytic enzyme secreted treated control mice grew exponentially [47]. The compound was into the semen of by the epithelial cells of the prostate, with the under the name of G115 taken into the pipeline of GenSpera in physiologic function to regulate the viscosity of the ejaculate [38, order to develop it into a drug. 39]. A healthy man typically has a serum concentration of less than 4 ng/mL. An steady increase beyond this level might indicate the 4.2. Prostate Specific Membrane Antigen presence of malign prostate cells [38]. PSA entrance into the blood Like PSA, prostate specific membrane antigen (PSMA) is a causes inactivation either by complexation to or proteolysis by ma- proteolytic enzyme but in contrast to PSA PSMA is not secreted jor serum protease inhibitors like 1-antichymotrypsin or 2- into the lumen but is bound to the membrane of the epithelial cells. macroglobulin [40-42].

O 4.1.1. Substrate Specificity of PSA O O From the point of view of creating prodrugs the substrate speci- O H O O H N ficity of PSA calls attention. PSA cleaves on the C-side of most Tyr O O R residues and certain Leu residues, but not on the C-site of Phe and OH Trp site [43, 44]. In addition during screening of a number of sub- OH O strates PSA was shown for cleave on the C-site of Gln [45]. O PSMA cleavage O OH O OH 4.1.2. PSA Specific Substrates O NH O O 2 H H A few attempts to develop prodrug based on the substrate speci- N N OH 114 R = N N H H ficity of PSA have been made [46]. In this case advantage of the O O O ability of PSA to cleave on the C-site of Gln was taken to construct O OH O OH a number of peptides terminating with Gln. Screening of a number O NH O 2 H of peptides terminating in Gln afforded His-Ser-Ser-Lys-Leu-Gln N (HSSKLQ) as a possible choice for a promoiety group. The peptide 115 R = OH O was conjugated to 7-amino-4-metylcoumarin and the product was O OH cleaved fast in the prostate by PSA but was stable in serum [47]. O NH Conjugation to the peptide, morpholino-Pro-Ser-Ser-Lys-Leu- 2 116 R = OH Gln afforded a prodrug 111 (Fig. 8), which was found not to be a O substrate for PSA. However, insertion of an additional Leu residue afforded a prodrug 112 with the wanted cleavage profile to produce 113 after PSA cleavage [47]. The morpholino group was added in Fig. (9). Prodrug developed for cleavage by PSMA. The arrows indicate the order to increase solubility. cleavage sites.

5516 Current Pharmaceutical Design, 2015, Vol. 21, No. 38 Doan and Christensen

4.2.1. Substrate Specificity of PSMA CONCLUSION PSMA is a carboxypeptidase, which cleaves poly--glutamoyl Thapsigargin (1) was originally isolated of curiosity in order to peptide bonds in glutamated folates and thereby enables uptake of understand the skin irritating properties of the resin of T. garganica folate [46, 48]. PSMA is expressed in the prostate but in spite of the [51]. Observed tumor promoting properties and general cytotoxicity name PSMA is overexpressed in neovasculature in a number of of the agent made it unlikely that the compound might be a drug tumors including hepatocellular carcinoma, mesothelioma, ovarian candidate [10]. However, after in depth studies to understand the cancer, renal cancer, bladder cancer, renal cancer and breast cancer mechanism of action [11] and use of prodrug design, a compound [48]. has successfully been developed, which currently is under phase II clinical trials showing promising results. Ingenol mebutate market- 4.2.2. PSMA Specific Substrates ed by Leo Pharma is another example of a drug developed from a The substrate specificity of PSMA also have inspired to design cocarcinogen [52]. prodrugs [46]. The thapsigargin prodrug 114 is shown in Figure 8. Incubation of the prodrug 114 with PSMA afforded a fast cleavage LIST OF ABBREVIATIONS of the three terminal Glu residues and a slower cleavage of the 12ADT = 8-(12-amindodecanoy)-8-debutanoyl)- fourth Glu to give 12-ADTAsp 116. Incubation of 12-ADTAsp 116 thapsigargin and 12-ADTAspGlu 115 showed that 12-ADTAsp 116 was almost PSA = Prostate specific antigen half as potent a cytotoxin towards TSU and LNCaP cells as Tg (1), whereas 12-ADTAspGlu 115 was 10 times less potent towards PSMA = prostate specific membrane antigen LNCaP cells and to a 100 times less potent towards TSU cells [48]. SERCA = sarco/endoplamsic reticulum calcium ATPase An X-ray study of the complex of 12-ADTAsp 116 bound to SER- Tc = thapsigargicin CA revealed that 12-ADTAsp 116 was bound very similar as Boc- Tg = thapsigargin 12ADT 62. The compound was under the name of G202 taken into the pipeline of GenSpera in order to develop it into a drug. CONFLICT OF INTEREST 4.3. Development of G202 into a Drug SBC is a member of the Scientific Board of GenSpera. NTQD has no conflict of interest. The project has been supported by the 4.3.1. In Vivo Studies Danish Cancer Society, GenSpera and the Danish Strategic Re- The in vitro results encouraged to perform in vivo studies. Mice search Council. inoculated with PSMA producing human prostate cancer cells (LNCaP and MDA-PCa2b cell lines) were treated with G202 (56 ACKNOWLEDGEMENTS mg/kg) for three consecutive days. 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Received: June 16, 2015 Accepted: August 17, 2015