Water-Mediated Interactions Influence the Binding of Thapsigargin to

Apr 11, 2013 - cavity of thapsigargin in sarco/endoplasmic reticulum calcium ATPase (SERCA) ... homeostasis by inhibition of the intracellular sarco/e...
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Water-Mediated Interactions Influence the Binding of Thapsigargin to Sarco/Endoplasmic Reticulum Calcium Adenosinetriphosphatase Eleonora S. Paulsen,*,† Jesper Villadsen,†,▼ Eleonora Tenori,†,△ Huizhen Liu,† Ditte F. Bonde,‡,§ Mette A. Lie,‡ Maike Bublitz,§,⊥ Claus Olesen,§,∥ Henriette E. Autzen,§,⊥ Ingrid Dach,§,∥ Pankaj Sehgal,§,∥ Poul Nissen,§ Jesper V. Møller,§,∥ Birgit Schiøtt,‡ and S. Brøgger Christensen*,† †

Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen Ø, Denmark ‡ iNANO and inSPIN Centers, Department of Chemistry, Langelandsgade 140, ∥Department of Biomedicine−Physiology and Biophysics, Ole Worms Alle 6, 1180, and ⊥Department of Molecular Biology and Genetics, Gustav Wieds Vej 10, Aarhus University, DK-8000 Aarhus C, Denmark § Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, Denmark S Supporting Information *

ABSTRACT: A crystal structure suggests four water molecules are present in the binding cavity of thapsigargin in sarco/endoplasmic reticulum calcium ATPase (SERCA). Computational chemistry indicates that three of these water molecules mediate an extensive hydrogenbonding network between thapsigargin and the backbone of SERCA. The orientation of the thapsigargin molecule in SERCA is crucially dependent on these interactions. The hypothesis has been verified by measuring the affinity of newly synthesized model compounds, which are prevented from participating in such water-mediated interactions as hydrogen-bond donors.

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INTRODUCTION In 2008, about 12.7 million new cancer cases were diagnosed, and 7.6 million patients have been estimated to die from the diseases.1 Global population aging as well as increasing adoption of unhealthy western lifestyle in developing countries contributes to an increasing incidence and numbers of deaths. Slowly developing malignant forms like prostate cancer are treated with only limited success with standard antiproliferative chemotherapy, especially if the disease has developed into the androgen-independent metastatic stage.2 Thapsigargin (1, Tg) is effective against proliferative as well as quiescent cells. The cytotoxic effect of Tg is caused by disruption of Ca 2+ homeostasis by inhibition of the intracellular sarco/endoplasmic reticulum calcium ATPase (SERCA).3 The ubiquitous presence of SERCA in all types of mammalian cells makes Tg a general cytotoxin and therefore highly toxic in vivo.2 Conjugation of a Tg derivative to a peptide creates a prodrug, the hydrophilicity of which prevents it from penetrating the cell membrane and consequently from reaching the SERCA pump. By choosing a peptide that is a substrate for prostate-specific antigen (PSA), a prodrug (GenSpera G115) is obtained, which is only cleaved in the vicinity of prostate cancer cells or prostate tissues expressing PSA, since this enzyme is inactivated in the blood.2 This strategy enables targeting of Tg toward prostate cancer tissue and thereby selective killing of malignant tissue.4 © 2013 American Chemical Society

An alternative possibility is to target the prodrug (GenSpera G202) against prostate-specific membrane antigen (PSMA), a proteolytic enzyme that predominantly is present in neovascular tissue like blood vessels in cancer tumors.5 The absence of anchoring groups like amino moieties in Tg prevents conjugation with a peptide, and consequently, a derivative has to be used in the prodrug. A successful outcome of the strategy, however, demands that the derivatives of Tg possess high affinity for SERCA. All previous attempts to design such inhibitors have been based on the hypothesis that Tg possesses only lipophilic interactions with SERCA.6−9 A recent X-ray high-resolution structure of SERCA1a in an E2-Pi-like state (a calcium-free state of dephosphorylation, mimicked by bound AlF4−), has provided a more detailed description of the Tg binding cavity and allows for the identification of four water molecules in close proximity to Tg (PDB code 3N5K).10 The present study was undertaken to investigate the importance of water molecules for binding of Tg to SERCA (Figure 1), using computational techniques in concert with ligand analogues unable to engage as hydrogen donors in water-mediated interactions with the protein. Received: January 23, 2013 Published: April 11, 2013 3609

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higher reactivity of OH-11 demands masking of this position prior to selective acylation of OH-7. Removal of the butanoyl group (Scheme 1) proceeds smoothly by use of triethylamine in methanol (2).11 Protection at O-8 and O-11 by reaction of 2 with acetone in the presence of 2,2-dimethoxypropane gives 3 in excellent yield.12 Even though a previous study had indicated that acylation of OH-7 of 3 by use of a carboxylic anhydride catalyzed with p-toluenesulfonic acid13 occurs to some extent, this reaction in this case did not afford the O-7 acylated derivatives 4. Acylation of OH-7 of 3 in good to fair yields to give 4a−d was achieved by treatment of 3 with the appropriate anhydride over a prolonged period of time in the presence of 4(N,N-dimethylamino)pyridine (DMAP). The target compounds 6a−d were obtained by acid-catalyzed hydrolysis of the ketal followed by selective acylation of the secondary alcohol at C-8. Compounds modified at C-12 were prepared from the acetal (7) by reduction of Tg with sodium borohydride (Scheme 2). Acetylation afforded the trisacetate (8). The relative configuration at C-12 was established by a correlation through space of H-12 and H-6 as verified in the rotating Overhauser effect (ROESY) spectrum. Unexpectedly, treatment of 8 with appropriate nitriles under acidic conditions affords the oxazolines (9a−c). In a Ritter reaction, carboxamides are formed from alcohols or alkenes by reaction under acidic conditions with nitriles. The mechanism is assumed to include formation of stabilized carbocation by reaction of the alcohol or alkene with the strong acid. The carbocation is attacked by the nitrogen of the nitrile to form a nitrilium ion, which after reaction with water is converted into an amide. The isopropylidene derivative of fructose reacts with some nitriles under acidic conditions to give a nitrilium ion. Subsequent cyclization by an intramolecular attack of a hydroxyl group yields an oxazoline.14,15 A similar mechanism of action might be operating in this case (Scheme 3).

Figure 1. Binding pocket of SERCA with thapsigargin (tube structure with cyan carbons and red oxygens) and four water molecules (tube structures with red oxygens and white hydrogens); PDB code 3N5K10. Color code for SERCA: TM 1 and 2 (purple), TM 3 and 4 (green), TM 5 and 6 (orange), TM 7−10 (gray), residue E255 (green tube), residues L828 and I829 (gray tubes).

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RESULTS Synthesis. On the basis of the assumption that OH-7 is crucial for anchoring of the water bridges, protocols were developed for derivatization at this position (Scheme 1). The Scheme 1. Synthesis of Thapsigargin Derivatives 6a−da

a

Reagents and conditions: (i) Et3N, rt, 3−4 h. (ii) AcOH, 2,2-dimethoxypropane, acetone, rt, 12 h (83%, over two steps). (iii) RCOOH, DMAP, DCC in dry DCM, rt, 48−60 h (23−85%). (iv) H2O, H+, rt, 3−12 h (84−89%). (v) (C3H7CO)2O, DMAP, dry DCM, rt (73−96%). 3610

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Scheme 2. Synthesis of Thapsigargin Derivatives 9a−ca

consistently was rotated 180° around an axis defined as the longest dimension of the molecule from the OH-7 to the octanoyl group, when compared to the cocrystallized ligand. As a result of this rotation, the lactone ring after docking points inward and to the left as opposed to pointing outward and to the right in the crystal structure when oriented as in Figure 1. A change in orientation is also observed for the butanoyl group, which in the crystal structure points into the binding pocket. In docking calculations where water molecules are not included, the group points directly out of the pocket and toward the interface between the protein and membrane lipid. A newly developed tool that incorporates displaceable water molecules during ligand docking (also known as the attached water model, AWM) was tested19 to verify the influence of water molecules on the orientation of Tg in SERCA. The method indicated that at least two water molecules are needed in the cavity to mediate the contact of Tg with SERCA in the right binding mode. One of the water molecules predicted by AWM overlays nicely with HOH782 (oxygen−oxygen distance of 1.08 Å), while the other reproduces the interaction of HOH783 with Tg in the PDB structure (Figure 1). Moreover, a newly developed protocol based on Glide16−18,20 and including water molecules inside the binding pocket has been applied to the series of thapsigargin derivatives (see detailed description in Experimental Section). The results of the calculations show a good correlation between the predicted and experimentally measured affinities of the ligands within the accuracy of the method (Table 2 and Figure 5). These data also support the hypothesis that water-mediated hydrogen bonds direct the ligand binding for optimal orientation in the cavity. Furthermore, the structure of compound 6a bound to SERCA was solved by X-ray crystallography (PDB code 4J2T; see Table S1 in Supporting Information for structural details) and shows that the compound indeed binds to the cavity in the way suggested by the theoretical calculations. Compound 6a is mainly stabilized by hydrophobic interactions with a single water-mediated hydrogen bond formed between the carbonyl oxygen at O-8 and the backbone of I829 (Figure 2 B). The orientation of 6a results in butanoyl moiety at O-7 pointing toward the lipid bilayer phase, most likely in a random orientation, as indicated by the lack of electron density for this part (Figure S1 in Supporting Information,).

a Reagents and conditions: (i) NaBH4, dry THF, rt (55%). (ii) Ac2O, DMAP, DCM, rt, 16 h (93%). (iii) SnCl4, dry RCN, rt, 15 h (33− 64%).

Scheme 3. Suggested Mechanism for Converting 8 into Oxazolines 9a−c

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DISCUSSION A previous pharmacophore model of Tg emphasizes hydrophobic interactions between the 2-methylbutenoyl (angeloyl) group at O-3 of Tg and residues N768, V769, and V263 of SERCA; between the butanoyl group at O-8 and residues P827, L828, and I829; between the acetyl group at O-10 and residues Y837 and F834; and between Me-15 and Q259 and the lactone ring and F256.8,13,21 None of these studies included hydrogen bonds for stabilizing the interaction between SERCA and Tg. The pharmacophore model has been confirmed by measuring the affinity of a series of model compounds to the protein21 and by point mutation studies.22,23 Furthermore, the results of a computational docking study using GOLD with the ChemScore function encompass only hydrophobic interactions.24 In contrast, the present study suggests water-mediated hydrogen bonds between the butanoyl carbonyl group and the backbone amide of L828 or the side chain of E255 and between OH-7 and the backbone of I829.

Whereas the formation of intermediates I and II is analogous to the mechanism of the Ritter reaction, the transformation of II to the oxazoline 9 is not straightforward. The use of analogue 7 not acetylated at O-11 did not increase the yield. The strain of the five-membered oxazoline ring and the tetrahydrofuran (THF) ring forces the two five-membered rings to be cisannelated. Consequently, α-disposal of the 11-oxygen group enables only the relative configuration shown in 9. Molecular Modeling. To further explore the role of water molecules mediating the interaction of Tg and SERCA, we undertook molecular modeling simulations. Standard docking procedures in Glide16−18 were unable to predict the correct orientation of Tg inside the binding pocket, as the molecule 3611

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carbonyl oxygen of butanoyl to HOH782 further on to NH of L828. The latter hydrogen-bond network appears to be stabilized additionally by an interaction between HOH782 and HOH808 and the side chain of E255. The absence of hydrogen bonds from HOH809 to Tg suggests that this molecule has a steric function. Furthermore, exclusion of HOH809 from the model does not affect calculated binding modes of the ligands. Models in which HOH782 and HOH783 have been removed from the binding cavity result in a less stable binding mode, with the Tg molecule rotated 180° around an axis through C-2 and C-7. This orientation enables a hydrogen bond to be formed from the carbonyl oxygen of I829 to the carbonyl oxygen of the lactone ring. However, such a binding mode corresponds to a sufficiently lower predicted affinity than the experimentally measured value. To verify experimentally the importance of the interaction between the water molecules and OH-7, a series of analogues, in which the hydroxyl group has been transformed, has been designed, synthesized and tested (Tables 1 and 2). In addition to elucidating the importance of OH-7, this study also challenged the prediction of the binding mode by the increased volumes of some of the Tg side chains (Table 1). As appears from Table 2, removal of the butanoate group and thereby removal of the carbonyl oxygen as a hydrogen-bond acceptor decreases the affinity 50 times (entry 2). This observation has previously been interpreted as the result of decreased lipophilic interactions between the butanoyl group and the SERCA pump.21 On the basis of the present observation, the diminished affinity might be assigned to the missing lipophilic group at O-8 as well as the missing hydrogen-bond acceptor at the carbonyl group of the butanoate. The good affinity of 6e leads to the conclusion that OH-7 acts as a hydrogen-bond acceptor and that a small substituent at O-7 only to a limited extent introduces steric clash with the backbone. The increasing size of the O-7 side chain from 6a to 6d gradually increases the experimentally found KD values. Computational experiments suggest that all the analogues are located in the binding site in the same way (Figure 3). Moreover, the binding affinities predicted by the modeling calculations correspond well with the experimentally measured values to an accuracy as reported for the XP-docking protocol to be a few kilocalories per mole16−18,20 (Figure 5). Comparison of the calculated binding

Figure 2. Structural characterization of 6a binding to SERCA. (A) Overview of the binding pocket relative to the membrane position of SERCA. Compound 6a (yellow space-filling model) binds to the same pocket as Tg, which is located in the TM domain of SERCA (TM 1− 10). (B) Close-up of the inhibitor binding pocket (alignment of PDB structures 4J2T and 3N5K). Structural alignment of Tg (tube structure with cyan carbons and red oxygens) and compound 6a (tube structure with yellow carbons and red oxygens) including water molecules (red spheres). The interaction between 6a and SERCA is hydrophobic in nature, with a single water-mediated hydrogen bond between the carbonyl oxygen at O-8 and the backbone of SERCA as for Tg.

Inspection of the structure 3N5K (Figure 1) reveals water molecules HOH782 and HOH783 in the proximity of OH-7 and the carbonyl group of the butanoyl side chain. Two additional water molecules, HOH808 and HOH809, have been observed in the binding site close to the O-11 position at the lactone ring of Tg (Figure 1). Redocking of Tg into the binding groove of SERCA, taking advantage of the mentioned hydrogen bonds mediating a contact to the protein through water molecules, results in an energetically favorable binding mode. This calculated location of Tg is in agreement with the crystallographic data within root-mean-square deviation (RMSD) of 0.7 Å. The position of Tg is stabilized by an interaction between OH-7 further on to HOH783 and a carbonyl oxygen of I829 and by a hydrogen bond from the

Table 1. Thapsigargin Analogues Modified at OH-7 and OH-11 and with Replaced Acyl Groups

compd

R1

R2

R3

R4

R5

1, Tg 2 6a 6b 6c 6d 6e 10 11a 11b

COC7H15 COC7H15 COC7H15 COC7H15 COC7H15 COC7H15 COC7H15 COC7H15 COC7H15 COC6H5

COC3H7 H COC3H7 COC3H7 COC3H7 COC3H7 COC3H7 COC3H7 COC3H7 COC3H7

COCH3 COCH3 COCH3 COCH3 COCH3 COCH3 COCH3 COCH3 COC3H7 COCH3

OH OH OCOC3H7 OCOC5H11 OCOCH(CH3)2 OCOC7H15 OCOCH3

α-OH α-OH α-OH α-OH α-OH α-OH α-OH

3612

−O− OH OH

α-OH α-OH

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Table 2. Summary of Results of Docking Calculations and Experimentally Measured Binding Affinities of Thapsigargin and Its Derivatives compd 1 2 6a 6b 6c 6d 6e 8 9a 9b 9c 10 11a 11b

binding affinitya (KD, nM) 0.2 10.0 11 8.5 13 55 2.6 9.4 1.7 20 79 13 2.5 1.5

± ± ± ± ± ± ± ± ± ± ± ± ±

0.5 1 1.9 1 5 0.3 0.6 0.4 3.8 13 1 0.3 0.7

affinitya conv to free energy of binding (kcal/mol) −13.1 −10.8 −10.7 −10.9 −10.6 −9.8 −11.6 −10.8 −11.8 −10.4 −9.6 −10.6 −11.6 −11.9

GScoreb (kcal/mol)

RMSDc (Å)

−14.6 −11.3 −13.4 −13.0 −12.8 −11.9 −12.4 −13.4 −13.4 −12.7

0.26 0.36 0.37 0.22 0.33 0.14 0.60 0.55 0.40

−12.7 −13.1 −13.3

0.43 0.50 0.23

Figure 4. Predicted binding modes of thapsigargin (black tube) and derivatives modified at O-11 and O-12 (8, 9a, 9b) in SERCA binding pocket. Superimposition of six docking poses of ligands with the best GScore values (violet tubes) is shown. Water molecules are shown as tube structures with red oxygens and white hydrogens, residue E255 as a green tube, and residues L828 and I829 as gray tubes.

a

Experimentally measured. bCalculated in GlideXP . cRMSD measured between atoms of the sesquiterpene lactone skeletons of the docked compound (best docking pose) and thapsigargin.

Figure 5. Comparison of binding affinities of thapsigargin and its derivatives predicted by molecular modeling calculations and measured experimentally (based on data from Table 2).

Figure 3. Predicted binding modes of thapsigargin derivatives modified at O-2, O-7, O-8, and O-10 positions. Superimposition of the docking poses of ligands with the best GScore values (violet tubes) is shown. Water molecules are shown as tube structures with red oxygens and white hydrogens, residue E255 as a green tube, and residues L828 and I829 as gray tubes.

suggest a different position of the ligand or the nearby residues of the SERCA. In contrast to Tg, the Tg epoxide 10 (Table 1) has previously been found neither to release histamine from rat mast cells nor to introduce skin irritation of mouse ears. The compound therefore was concluded not to have affinity for the biological target molecule SERCA.25,26 The epoxide structure increases the distance from the epoxy oxygen atom to HOH783, indicating a poor interaction. Surprisingly, however, in spite of the missing effect in the whole-cell assay, the epoxide possesses significant inhibition of the isolated SERCA pump (Table 2). According to our modeling calculations, the binding between 10 and SERCA takes advantage of the water molecules placed in proximity of the backbone of SERCA. The low-energy binding mode of the epoxide analogue overlays nicely with that of thapsigargin with an RMSD of 0.43 Å calculated for the sesquiterpene lactone scaffold. When an increased distance between ligand and protein is kept in mind, the role of the

affinities to those measured in bioassays shows a general trend of modest overestimation of the predicted values. The model compounds 8, 9a, and 9b (Scheme 2) illustrate that even a large substituent is allowed at the α-surface below O-11 and the carbonyl group at C-12. To some extent the steric clash may be removed by exclusion of one or two water molecules. Meanwhile, all attempts to dock 9c resulted in a different binding mode from that of Tg and rotation of the ligand around the C-2−C-7 axis. Exclusion of HOH808 and HOH809 from the cavity was not sufficient to accommodate the extensive side chain of 9c. The relatively lower experimental affinity of the compound indicates some weaker interactions between the binding pocket and the ligand and may therefore 3613

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water molecules may be described as filling void space as well as engaging in a hydrogen-bonding network between the ligand and SERCA.

for 3−20 h. The conversion was monitored by thin-layer chromatography (TLC). After the completed reaction, the crude mixture was concentrated in vacuo. The crude residue was dissolved in ethyl acetate, extracted three times with saturated aqueous NaHCO3, washed with brine, dried with MgSO4, filtered, and concentrated in vacuo. General Procedure C: Acylation at O-8 Position. To a solution of diol 5 (1 equiv) in dry DCM (1−2 mL) were added butyric anhydride (1.5 equiv) and DMAP (cat.), and the mixture was stirred for 20 min at room temperature. After full conversion (followed by TLC), the mixture was diluted with DCM and extracted three times with saturated aqueous NaHCO3. The organic phase was washed with brine, dried with MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography with petroleum ether−ethyl acetate (3:1) as an eluent. (1S,2S,3bS,3b1R,5aS,8aS,10S,10aR)-10-Acetoxy-3b1-(butyryloxy)3,5a,7,7,10-pentamethyl-2-{[(Z)-2-methylbut-2-enoyl]oxy}-5-oxo1,2,3b,3b1,5,5a,8a,9,10,10a-decahydro-4,6,8-trioxabenzo[cd]cyclopenta[h]azulen-1-yl Octanoate (4a). The title compound was prepared from 3 (0.11 g, 0.180 mmol, 1 equiv), butyric anhydride (74 μL, 0.5 mmol, 2.5 equiv), DCC (55 mg, 0.270 mmol, 1.5 equiv), and DMAP (cat.) according to general procedure A. The mixture was stirred for 20 h before the precipitate was filtered out and the mixture was washed with NaHCO3. Purification by flash column chromatography resulted in 4a (105 mg, 85%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 6.07 (qq, J = 1.5, 7.0 Hz, 1H, angeloyl H-3), 5.92 (br s, 1H, H-6), 5.75 (br s, 1H, H-3), 5.42 (t, J = 4.5 Hz, 1H, H-2), 5.06 (br s, 1H, H-8), 3.70 (br s, 1H, H-1), 2.78−2.67 (dd, J = 3.5, 15.5 Hz, 1H, H-9a), 2.66−2.57 (dd, J = 2.9, 14.9 Hz, 1H, H-9b), 2.47−2.34 (m, 2H, butanoyl H-2), 2.34−2.24 (m, 2H, octanoyl H-2), 1.97 (dq, J = 1.5, 6.5 Hz, 3H, angeloyl CH3), 1.90−1.88 (m, 3H, angeloyl H-4), 18.9 (s, 3H, H15), 1.86 (s, 3H, H15, acetyl CH3), 1.72−1.58 (m, 4H, butanoyl H-3 and octanoyl H-3), 1.55 (s, 3H, acetal CH3), 1.53 (s, 3H, H-13), 1.46 (s, 3H, acetal CH3), 1.40 (s, 3H, H-14), 1.35−1.19 (m, 8H, octanoyl H-4−H-7,), 0.95 (t, J = 7.0 Hz, 3H, butanoyl H-4), 0.85 (t, J = 7.0 Hz, 3H, octanoyl H-8). 13C NMR (101 MHz, CDCl3) δ 172.4 (C-12), 171.9 (butanoyl C-1)*, 171.6 (octanoyl C-1)*, 170.1 (acetyl C-1), 167.2 (angeloyl C-1), 138.5 (angeloyl C-3), 137.9 (C-4), 127.4 (C-5), 127.2 (angeloyl C-2), 101.2 [acetal C(CH3)2], 83.9 (C10), 83.7 (C-3), 78.3 (C-7), 78.00 (C-2), 77.0 (C-11), 76.4 (C-6), 63.6 (C-8), 56.6 (C-1), 38.8 (C-9), 36.4 (octanoyl C-2), 34.3 (butanoyl C-2), 31.6 octanoyl C-6)*, 30.3 (acetal CH3), 29.1 (octanoyl C-5)*, 28.95 (octanoyl C-4), 24.7 (octanoyl C-3), 23.3 (acetal CH3), 22.6 (octanoyl C-7), 22.6 (acetyl CH3), 21.6 (C-14), 20.6 (angeloyl CH3), 18.1 (octanoyl C-5), 17.2 (C-13), 15.8 (angeloyl C-2), 14.1 (octanoyl C-8), 13.9, 13.8 (butanoyl C-4), 13.6, 13.4, 12.4 (C-15). (1S,2S,3bS,3b1R,5aS,8aS,10S,10aR)-10-Acetoxy-3b1-(hexanoyloxy)-3,5a,7,7,10-pentamethyl-2-{[(Z)-2-methylbut-2-enoyl]oxy}-5oxo-1,2,3b,3b1,5,5a,8a,9,10,10a-decahydro-4,6,8-trioxabenzo[cd]cyclopenta[h]azulen-1-yl Octanoate (4b). The title compound was prepared from 3 (0.32 g, 0.48 mmol, 1 equiv), hexanoic acid (0.20 mL, 1.4 mmol, 3 equiv), DCC (0.2 g, 1.0 mmol, 2.0 equiv), and DMAP (cat.) according to general procedure A. The reaction mixture was stirred for 60 h before it was diluted with DCM and washed with NaHCO3. Purification by flash column chromatography gave 4b (0.24 g, 64%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) δ 6.09 (qq, J = 1.5, 7.3 Hz, 1H, angeloyl H-3), 5.98−5.91 (m, 1H, H-6), 5.82−5.73 (m, 1H, H-3), 5.45 (dd, J = 4.5, 5.3 Hz, 1H, H-2), 5.07 (br s, 1H, H8), 3.72 (br s, 1H, H-1), 2.69 (dd, J = 4.5, 14.8 Hz, 1H, H-9a), 2.63 (dd, J = 2.3, 14.8 Hz, 1H, H-9b), 2.39−2.35 (m, 2H, hexanoyl H-2), 2.33−2.21 (m, 2H, octanoyl H-2), 1.99 (dq, J = 1.5, 7.3 Hz, 3H, angeloyl CH3), 1.92 (quin, J = 1.6 Hz, 3H, angeloyl H-3), 1.89 (br s, 3H, H-15), 1.88 (s, 3H, acetyl H-2), 1.68−1.59 (m, 4H, octanoyl H-3 and hexanoyl H-3), 1.57 (s, acetal CH3), 1.55 (s, 3H, H-13), 1.48 (s, 3H, acetal CH3), 1.43 (s, 3H, H-14), 1.38−1.23 (m, 8H, octanoyl H4−H-7, hexanoyl H-4, and H-5), 0.86, (t overlaid, 3H, octanoyl H-8)*, 0.85 (t overlaid, 3H, hexanoyl H-6)*. 13C NMR (101 MHz, CDCl3) δ 172.7 (C-12), 172.3 (hexanoyl C-1)*, 171.9 (octanoyl C-1)*, 170.3 (acetyl C-1), 167.5 (angeloyl C-1), 138.7 (angeloyl C-3), 138.2 (C-4),

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CONCLUSIONS This study suggests that an important factor for the binding of thapsigargin and analogues to SERCA is water-mediated hydrogen bonds between O-7 and the carbonyl group of butanoyl and the backbone of residues I829 and L828 of SERCA. The conclusion that water molecules are an essential part of the binding system is evidenced by docking calculations and by the use of an attached water model.19 This hypothesis was further verified by testing the binding affinities of two new series of thapsigargin derivatives. Docking of these series of derivatives to the model of SERCA with bound water molecules shows a correlation between predicted and experimentally measured affinities within the accuracy of the method.

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EXPERIMENTAL SECTION

Chemistry. Thapsigargin (1) was obtained from the seeds of Thapsia garganica and used as a starting material for the syntheses.27 Syntheses of the epoxide (10) and debutanoylthapsigargin (2) were performed according to previously described procedures.28,29 NMR spectra were recorded on a Varian Mercury (300 MHz), a Varian Gemini 2000, and a Bruker Avance (400 MHz) spectrometer. The chemical shifts (δ) are given in parts per million (ppm) relative to residual signals of the solvents (CDCl3 and CD3OD). Abbreviations used for multiplicities: br, broad signal; s, singlet; d, doublet; t, triplet; q, quartet; quin, quintet; sept, septet. Asterisk-marked shifts may be interchanged. Two-dimensional (2D) NMR spectra were recorded using standard pulse sequences. Assignments of the NMR signals were performed by correlation (COSY), nuclear Overhauser effect (NOESY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlation (HMBC) spectroscopy. In case of overlaid or weak signals, interpretation of HSQC or HMBC has been used for assignment. High-resolution mass spectrometry (HRMS) was recorded on a QTof1 (Micromass). Column chromatography was performed on Silica Gel 60 (Merck 1.07734). Reversed-phase chromatography was performed on LiChroprep RP-18 (Merck 1.13900). Purities of the compounds tested in the biological assay (>95% with the exception of 9c, the purity of which was determined to be 90.0%) was confirmed on a Shimadzu HPLC system with a reverse-phase C18 column [Phenomenex Luna C18(2) 3 μm, 4.6 mm × 150 mm] eluted at rate of 0.8 mL/min. The compounds were detected at 220 nm. Injection volumes were 3−5 μL of a 1 mg/mL solution, and analysis was performed at 40 °C. A linear gradient system started from A/B (30%/70% for 9a, 20%/80% for 9b and 9c, and 15%/85% for 6a−d) at time 0 to A/B (0%/100%) at 30 min. Preparative HPLC purification was carried out on an XBridge Prep C18, 5 μm, OBD 50 × 250 mm column at a rate of 20 mL/min with detection at 220 nm. Solvent mixtures A (5% CH3CN in H2O) and B (5% H2O in CH3CN) were applied for both analytical and preparative purposes. General Procedure A: Acylation of Thapsigargin Derivatives at O-7 Position. The mixture of isopropylidene (3) (1 equiv), carboxylic acid or its anhydride (2−3 equiv), dicyclocarbodiimide (DCC, 2 equiv) and 4-(N,N-dimethylamino)pyridine (DMAP, catalytic amount) was dissolved in dry dichloromethane (DCM) and stirred at room temperature for 20−60 h. The crude mixture was filtered and the precipitate was washed with DCM. The filtrate was diluted and washed with saturated aqueous NaHCO3 solution, washed with brine, dried with MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash column chromatography with petroleum ether (PE)−ethyl acetate (4:1 → 3:1) as eluents. General Procedure B: Cleavage of Acetal Group. To the solution of acetal-protected Tg derivative (4a−d) in MeOH (5−10 mL) was added a few drops of 6 M HCl, and the mixture was stirred 3614

dx.doi.org/10.1021/jm4001083 | J. Med. Chem. 2013, 56, 3609−3619

Journal of Medicinal Chemistry

Article

gave the product (87 mg, 89%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 6.11 (dd, J = 1.5, 7.2 Hz, 1H, angeloyl H-3), 6.01 (br s, 1H, H-6), 5.70 (br s, 1H, H-3), 5.42 (dd, J = 2.8, 3.5 Hz, 1H, H-2), 5.32 (br s, 1H, OH), 5.19 (br s, 1H, H-8), 4.19 (br s, 1H, H-1), 2.82 (d, J = 14.3 Hz, 1H, H-9a), 2.51−2.38 (m, 2H, butanoyl, H-2), 2.32−2.23 (m, 2H, octanoyl H-2), 2.01−1.96 (dq, J = 1.5, 6.5 Hz, 3H, angeloyl H-4), 1.92−1.89 (m, 6H, angeloyl CH3 and H-15), 1.88−1.85 (m, 3H, acetyl CH3), 1.69−1.59 (m, 4H, octanoyl H-3 and butanoyl H-3), 1.48 (s, 3H, H-13), 1.40 (s, 3H, H-14), 1.35−1.25 (m, 8H, octanoyl H-4−H7), 0.95 (t, J = 7.3 Hz, 3H, butanoyl H-4), 0.88 (t, J = 7.3 Hz, 3H, octanoyl H-8). 13C NMR (101 MHz, CDCl3) δ 174.2 (C-12), 172.5 (octanoyl C-1)*, 172.4 (butanoyl C-1)*, 170.6 (acetyl C-1)*, 167.1 (angeloyl C-1), 138.8 (angeloyl C-3), 129.0 (angeloyl C-2), 128.2, 127.4 (C-5), 84.2 (C-3), 78.8 (C-2), 76.1 (C-6), 36.7 (butanoyl C-2), 34.3 (octanoyl C-2), 31.2 (octanoyl C-6), 29.0, 24.8, 24.4 (C-14), 23.8, 22.6 (octanoyl C-7), 22.2 (C-13), 21.4 (angeloyl CH3), 20.6 (butanoyl C-3), 17.9 (octanoyl C-3), 15.8 (angeloyl C-2), 14.0 (acetyl CH3), 13.9 (butanoyl C-4), 13.6 (octanoyl C-8), 12.7 (C-15). (3S,3aR,4S,6S,6aR,7S,8S,9bS)-6-Acetoxy-3a-(hexanoyloxy)-3,4-dihydroxy-3,6,9-trimethyl-8-{[(Z)-2-methylbut-2-enoyl]oxy}-2-oxo2,3,3a,4,5,6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-7-yl Octanoate (5b). The title compound was prepared from 4b (238 mg, 0.331 mmol, 1 equiv) according to general procedure B. The reaction mixture was heated up to 37 °C for 4 h before it was concentrated in vacuo. Purification by flash column chromatography (PE−EtOAc 3:1) gave the product (160 mg, 84%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 6.11 (qd, J = 1.5, 7.2 Hz, 1H, angeloyl H-3), 5.99 (br s, 1H, H-6), 5.70 (d, J = 1.0 Hz, 1H, H-3), 5.44−5.41 (br s, 1H, H-2), 5.18 (br s, 1H, H-8), 4.74 (s, 1H, OH), 4.09 (br s, 1H, H-1), 4.02 (s, 1H, OH), 2.81 (d, J = 14.6 Hz, 1H, H-9a), 2.52−2.34 (m, 2H, hexanoyl H-2), 2.34−2.21 (m, 2H, octanoyl H-2), 2.01−1.96 (m, 3H, angeloyl H-4), 1.93−1.89 (m, 6H, angeloyl CH3 and H-15), 1.89−1.83 (m, 3H, acetyl CH3), 1.66−1.55 (m, 4H, octanoyl H-3 and hexanoyl H-3), 1.51−1.47 (s, 3H, H-13), 1.40 (s, 3H, H-14), 1.36−1.24 (m, 12H, octanoyl H-4−H-7, hexanoyl H-4, and H-5), 0.93−0.85 (m, 6H, octanoyl H-8 and hexanoyl H-6). 13C NMR (101 MHz, CDCl3) δ 174.4 (C-12), 172.6 (octanoyl C-1)*, 172.5 (hexanoyl C-1)*, 170.6 (acetyl C-1)*, 167.1 (angeloyl C-1), 138.8 (angeloyl C-3), 127.4 (C5), 84.2 (C-3), 79.1 (C-2), 77.8, 76.2 (C-6), 34.8 (hexanoyl C-2), 34.3 (octanoyl C-2), 31.7 (octanoyl C-6), 31.2, 29.0, 24.8, 24.3 (C-14), 23.8, 22.6 (octanoyl C-7), 22.3 (C-13), 20.5 (angeloyl CH3), 19.3, 15.8 (acetyl CH3), 14.0 (hexanoyl C-6), 13.9 (octanoyl C-8), 12.7 (C15). (3S,3aR,4S,6S,6aR,7S,8S,9bS)-6-Acetoxy-3,4-dihydroxy-3a-(isobutyryloxy)-3,6,9-trimethyl-8-{[(Z)-2-methylbut-2-enoyl]oxy}-2-oxo2,3,3a,4,5,6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-7-yl Octanoate (5c). The title compound was prepared from 4c (26 mg, 0.040 mmol, 1 equiv) according to general procedure B. The reaction mixture was stirred at room temperature for 16 h before it was concentrated in vacuo. The product was used in the next step without purification. (3S,3aR,4S,6S,6aR,7S,8S,9bS)-6-Acetoxy-3,4-dihydroxy-3,6,9-trimethyl-8-{[(Z)-2-methylbut-2-enoyl]oxy}-2-oxo2,3,3a,4,5,6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-3a,7-diyl Dioctanoate (5d). The title compound was prepared from 4d (32 mg, 0.043 mmol, 1 equiv) according to general procedure B. The reaction mixture was stirred at room temperature for 20 h before it was concentrated in vacuo. The product was used for further synthesis without purification. (3S,3aR,4S,6S,6aR,7S,8S,9bS)-6-Acetoxy-3-hydroxy-3,6,9-trimethyl-8-{[(Z)-2-methylbut-2-enoyl]oxy}-7-(octanoyloxy)-2-oxo2,3,3a,4,5,6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-3a,4-diyl Dibutyrate (6a). The title compound was prepared from 5a (87 mg, 0.134 mmol, 1 equiv) according to general procedure C. Purification by column chromatography gave the product (77 mg, 80%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 6.54 (br s, 1H, H-8), 6.12 (qq, J = 1.4, 7.3 Hz, 1H, angeloyl H-3), 5.78 (br s, 1H, H-6), 5.67 (br s, 1H, H-3), 5.44 (t, J = 2.5 Hz, 1H, H-2), 4.21 (br s, 1H, H-1), 2.89 (dd, J = 3.1, 15.4 Hz, 1H, H-9a), 2.69 (s, 1H, OH), 2.47 (m, 1H, butanoyl H-2), 2.39 (m, 1H, butanoyl H-2′), 2.28 (m, 4H, butanoyl H2 and octanoyl H-2), 2.10 (dd, J = 4.1, 15.4 Hz, 1H, H-9b), 2.00 (dq, J

127.8 (angeloyl C-2), 127.4 (C-5), 101.5 [acetal C(CH3)2], 84.2 (C7), 84.0 (C-3), 78.6 (C-7), 78.4 (C-2), 77.0 (C-11), 76.3 (C-4), 63.9 (C-8), 56.9 (C-1), 39.1 (C-9), 34.8 (octanoyl C-2)*, 34.6 (hexanoyl C-2)*, 31.9 (octanoyl C-6), 31.4 (hexanoyl C-4), 30.6 (acetal CH3), 29.4 (octanoyl C-5), 25.0 (hexanoyl C-4), 24.7 (octanoyl C-3), 24.5 (hexanoyl C-3), 23.6 (acetal CH3), 22.8 (hexanoyl C-5), 22.6 (octanoyl C-7), 21.8 (acetyl CH3), 21.6 (C-14), 20.8 (angeloyl CH3), 17.5 (octanoyl C-3), 16.0 (C-13), 14.3 (angeloyl C-2), 14.2 (hexanoyl C-6), 14.1 (octanoyl C-8), 12.8 (C-15). (1S,2S,3bS,3b1R,5aS,8aS,10S,10aR)-10-Acetoxy-3b1-(isobutyryloxy)-3,5a,7,7,10-pentamethyl-2-{[(Z)-2-methylbut-2-enoyl]oxy}-5oxo-1,2,3b,3b1,5,5a,8a,9,10,10a-decahydro-4,6,8-trioxabenzo[cd]cyclopenta[h]azulen-1-yl Octanoate (4c). The title compound was prepared according to general procedure A. The mixture of 3 (82 mg, 0.132 mmol, 1 equiv), isobutyric anhydride (66 μL, 0.396 mmol, 3 equiv), DCC (82 mg, 0.396 mmol, 3 equiv), and DMAP (cat.) was stirred for 36 h before it was washed with NaHCO3. Purification by flash column chromatography gave 4c (60 mg, 70%) as a pale yellow oil. 1H NMR (300 MHz, CDCl3) δ 6.09 (qq, J = 1.5, 7.3 Hz, 1H, angeloyl H-3), 5.95 (br s, 1H, H-6), 5.79−5.70 (br s, 1H, H-3), 5.46 (dd, J = 4.2, 5.4 Hz, 1H, H-2), 5.18−5.03 (br s, 1H, H-8), 3.75 (br s, 1H, H-1), 2.84−2.71 (m, 1H, H-9a), 2.71−2.52 (m, 1 H, H-9b), 2.28 (sept, J = 7.2 Hz, 1H, isobutanoyl H-2), 2.02−1.87 (m, 6H, angeloyl CH3 and acetyl CH3), 1.86−1.75 (m, 3H, H-15), 1.59 (s, 3H, H-13), 1.53 (s, 3H, acetal CH3), 1.47 (s, 3H, acetal CH3), 1.43 (s, 3H, H-14), 1.34−1.13 (m, 14H, octanoyl H-4−H-7 and isobutanoyl CH3), 0.88 (t, J = 6.5 Hz, 3H, octanoyl H-8). 13C NMR (75 MHz, CDCl3) δ 172.5 (C-12), 171.8 (octanoyl C-1)*, 170.2 (isobutanoyl C-1)*, 170.5 (acetyl C-1)*, 167.3 (angeloyl C-1), 138.7 (angeloyl C-3), 127.6 (C5), 101.4 [acetal C(CH3)2], 84.2 (C-7), 84.1 (C-10, C-3), 78.8 (C-7), 78.1 (C-2), 76.6 (C-4), 66.2 (C-8), 56.9 (C-1), 50.2, 35.0 (octanoyl C2)*, 34.6 (isobutanoyl C-2)*, 34.1, 33.1, 32.0 (octanoyl C-6), 31.4, 30.7 (acetal CH3), 29.3 (octanoyl C-5), 26.7, 25.7, 25.1 (C-14), 23.7 (acetal CH3), 23.0 (octanoyl C-7), 22.1 (acetyl CH3), 20.9 (C-13), 20.5 (angeloyl CH3), 19.2, 17.4 (C-13), 16.2, 15.7 (angeloyl C-2), 14.5 (octanoyl C-8), 12.8 (C-15). (1S,2S,3bS,3b1R,5aS,8aS,10S,10aR)-10-Acetoxy-3,5a,7,7,10-pentamethyl-2-{[(Z)-2-methylbut-2-enoyl]oxy}-5-oxo1,2,3b,3b1,5,5a,8a,9,10,10a-decahydro-4,6,8-trioxabenzo[cd]cyclopenta[h]azulene-1,3b1-diyl Dioctanoate (4d). The title compound was prepared from 3 (130 mg, 0,209 mmol, 1 equiv), octanoic acid (67 μL, 0,419 mmol, 2 equiv), DCC (86 mg, 0,419 mmol, 2 equiv), and DMAP (cat.) according to general procedure A. The reaction mixture was stirred at room temperature for 60 h. Purification by flash column chromatography gave 4d (32 mg, 23%) as a pale yellow oil. 1H NMR (300 MHz, CDCl3) δ 6.09 (qq, J = 1.5, 7.3 Hz, 1H, angeloyl H-3), 5.76 (br s, 1H, H-6), 5.70 (br s, 1H, H-3), 5.46 (dd, J = 4.5, 5.3 Hz, 1H, H-2), 4.26 (br s, 1H, H-1), 3.99 (br s, 1H, H1), 2.81−2.72 (dd, J = 4.0, 15.2 Hz, 1H, H-9a), 2.64−2.56 (dd, J = 2.9, 15.2 Hz, 1H, H-9b), 2.39−2.20 (m, 4H, 2 × octanoyl H-2), 1.97 (dq, J = 1.5, 7.2 Hz, 3H, angeloyl CH3), 1.90−1.88 (m, 3H, angeloyl H-4), 1.86 (m, 6H, acetyl H-2 and H-15), 1.68−1.56 (m, 4H, 2 × octanoyl H-3), 1.54 (s, 3H, H-13), 1.51 (s, 3H, acetal CH3), 1.44 (s, 3H, acetal CH3), 1.40 (s, 3H, H-14), 1.37−1.19 (m, 16H, octanoyl H-4−H-7), 0.91−0.81 (m, 6H, 2 × octanoyl H-8). 13C NMR (75 MHz, CDCl3) δ 173.0 (C-12)*, 172.5 (octanoyl C-1)*, 171.9 (octanoyl C-1), 170.0 (acetyl C-1)*, 167.3 (angeloyl C-1), 137.6 (C-4), 127.2 (C-5), 101.2 [acetal C(CH3)2], 83.9 (C-10), 83.8 (C-3), 78.5 (C-7), 78.0 (C-2), 76.3 (C-4), 63.6 (C-8), 56.4 (C-1), 38.7 (C-9), 36.4 (octanoyl C-2), 36.1 (octanoyl C-2), 34.3 (butanoyl C-2), 31.7 (octanoyl C-6), 30.3 (octanoyl C-6), 29.1 (octanoyl C-4), 29.0 (octanoyl C-4), 24.7 (octanoyl C-3), 23.3 (octanoyl C-7), 22.6 (octanoyl C-7), 18.4 (octanoyl C-5), 18.1 (octanoyl C-5), 17.2 (C-13), 16.2 (angeloyl C-2), 14.1 (octanoyl C-8), 13.6 (butanoyl C-4), 12.4 (C-15). (3S,3aR,4S,6S,6aR,7S,8S,9bS)-6-Acetoxy-3a-(butyryloxy)-3,4-dihydroxy-3,6,9-trimethyl-8-{[(Z)-2-methylbut-2-enoyl]oxy}-2-oxo2,3,3a,4,5,6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-7-yl Octanoate (5a). The title compound was prepared from 4a (105 mg, 0.152 mmol, 1 equiv) according to general procedure B. The reaction mixture was heated up to 37 °C for 3 h before it was concentrated in vacuo. Purification by flash column chromatography (PE−EtOAc 3:1) 3615

dx.doi.org/10.1021/jm4001083 | J. Med. Chem. 2013, 56, 3609−3619

Journal of Medicinal Chemistry

Article

= 1.5, 7.3 Hz, 3H, angeloyl CH3), 1.92 (quin, 3H, angeloyl H-3), 1.89 (s, 6H, H-15, acetyl H-2), 1.68−1.59 (m, 4H, butanoyl H-3 and octanoyl H-3), 1.49 (s, 3H, H-13), 1.34 (s, 3H, H-14), 1.29 (m, 8H, octanoyl H-4−H-7), 0.96, 0.95 (t, J = 7.4 Hz, 2 × 3H, butanoyl H-4), 0.88 (t, J = 7.3 Hz, 3H, octanoyl H-8). 13C NMR (101 MHz, CDCl3) δ 173.9 (C-12), 172.3, 172.0, 170.4 (2 × butanoyl C-1 and octanoyl C1), 170.3 (acetyl C-1), 166.9 (angeloyl C-1), 141.5 (C-4), 138.9 (angeloyl C-3), 130.1 (C-5), 127.4 (angeloyl C-2), 87.7 (C-7), 84.2 (C-4), 83.4 (C-10), 78.4 (C-11), 77.2 (C-2), 76.2 (C-6), 64.4 (C-8), 56.8 (C-1), 39.1 (C-9), 37.0, 36.7, 34.3 (octanoyl C-2 and 2 × butanoyl C-2), 31.6, 31.2 (octanoyl C-6 and C-5), 24.7 (C-14), 24.0, 22.5 (octanoyl C-4 and C-3), 22.5 (acetyl CH3), 22.3 (octanoyl C-7), 20.6 (angeloyl CH3), 19.7 (C-13), 18.0, 17.9 (2 × butanoyl C-3) 15.8 (angeloyl C-4), 14.0 (octanoyl C-8), 13.9, 13.5 (2 × butanoyl C-4), 13.0 (C-15). HRMS calcd for C38H56NaO13 (M + Na+) 743.3613, found 743.3584. (3S,3aR,4S,6S,6aR,7S,8S,9bS)-6-Acetoxy-4-(butyryloxy)-3a-(hexanoyloxy)-3-hydroxy-3,6,9-trimethyl-8-{[(Z)-2-methylbut-2-enoyl]oxy}-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-7yl Octanoate (6b). The title compound was prepared from 5b (95 mg, 0.140 mmol, 1 equiv) according to general procedure C. Purification by column chromatography gave the product (89 mg, 85%) as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 6.60 (br s, 1H, H-8), 6.12 (qq, J = 1.5, 7.3 Hz, 1H, angeloyl H-3), 5.78 (br s, 1H, H-6), 5.69 (br s, 1H, H-3), 5.44 (t, J = 2.5 Hz, 1H, H-2), 4.21 (br s, 1H, H-1), 2.89 (dd, J = 3.3, 15.4 Hz, 1H, H-9a), 2.72 (br s, 1H, OH), 2.55−2.38 (m, 2H, hexanoyl H-2), 2.29 (t, J = 8.0 Hz, 2H, octanoyl H-2), 2.25 (t, J = 8.0 Hz, 2H, butanoyl H-2), 2.12 (dd, J = 4.4, 15.4 Hz, 1H, H-9a), 2.01 (dq, J = 1.5, 7.2 Hz, 3H, angeloyl H-4), 1.92 (dq, J = 1.5, 1.6 Hz, 3H, angeloyl CH3), 1.89 (s, 6H, H-15, acetyl CH3), 1.63 (m, 6H, octanoyl H-3, hexanoyl H-3, and butanoyl H-3), 1.49 (s, 3H, H-13), 1.35 (s, 3H, H-14), 1.33−1.26 (m, 12H, hexanoyl H-4−H-5 and octanoyl H4−H-7), 0.95 (t, J = 7.3 Hz, 3H, butanoyl H-4), 0.89 (t overlaid, J = 7.3 Hz, 6H, octanoyl H-8 and hexanoyl H-6). 13C NMR (101 MHz, CDCl3) δ 174.0 (C-12), 172.4 (hexanoyl C-1)*, 172.3 (butanoyl C1)*, 172.0 (octanoyl C-1)*, 170.4 (acetyl C-1), 167.0 (angeloyl C-1), 141.5 (C-4), 138.9 (angeloyl C-3), 130.2 (C-5), 127.4 (angeloyl C-2), 87.7 (C-7), 84.2 (C-3), 83.4 (C-10), 78.4 (C-2), 77.3 (C-2), 76.2 (C6), 64.4 (C-8), 56.8 (C-1), 39.0 (C-9), 36.5 (butanoyl C-2)*, 34.9 (hexanoyl C-2)*, 34.3 (octanoyl C-2)*, 31.7 (octanoyl C-5)*, 31.2 (hexanoyl C-4)*, 29.1 (octanoyl C-4)*, 29.0 (octanoyl C-6)*, 24.8 (hexanoyl C-3)*, 24.5, 24.1 (C-14), 22.6 (acetyl C-1), 22.5 (octanoyl C-7), 22.3, 20.6 (angeloyl CH3), 19.7 (C-13), 18.0 (butanoyl C-3), 15.8 (angeloyl C-4), 14.0 (octanoyl C-8)*, 13.9 (hexanoyl C-6)*, 13.7 (butanoyl C-4)*, 13.0 (C-15). HRMS calcd for C40H60NaO13 (M + Na+) 771.3926, found 771.3905. (3S,3aR,4S,6S,6aR,7S,8S,9bS)-6-Acetoxy-4-(butyryloxy)-3-hydroxy-3a-(isobutyryloxy)-3,6,9-trimethyl-8-{[(Z)-2-methylbut-2enoyl]oxy}-2-oxo-2,3,3a,4,5,6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-7-yl Octanoate (6c). The title compound was prepared from 5c (25 mg, 0.038 mmol, 1 equiv) according to general procedure C. Purification by flash column chromatography gave the product (20 mg, 73%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 6.62 (br s, 1H, H-8), 6.13 (qq, J = 1.5, 7.3 Hz, 1H, angeloyl H-3), 5.79 (br s, 1H, H6), 5.67 (br s, 1H, H-3), 5.46 (t, J = 2.6 Hz, 1H, H-2), 4.19 (br s, 1H, H-1), 2.89 (dd, J = 2.9, 15.5 Hz, 1H, H-9a), 2.67 (sept, J = 7.5 Hz, 1H, isobutanoyl H-2), 2.44 (br s, 1H, OH), 2.27 (t, J = 7.5 Hz, 2H, octanoyl H-2), 2.11 (dd, J = 4.7, 15.5 Hz, 1H, H-9b), 2.05 (dd, J = 1.5, 7.2 Hz, 3H, angeloyl H-4), 1.92 (m, 3H, angeloyl H-4), 1.89 (s, 6H, H15 and acetyl CH3), 1.64 (m, 6H, butanoyl H-3 and octanoyl H-3), 1.47 (s, 3H, H-13), 1.34 (s, 3H, H-14), 1.27 (m, 8H, octanoyl H-4−H7), 1.21 (d, J = 6.9 Hz, 3H, isobutanoyl H-3), 0.95 (t, J = 7.5 Hz, 3H, butanoyl H-4), 0.87 (t, J = 7.3 Hz, 3H, octanoyl H-8). 13C NMR (75 MHz, CDCl3) δ 175.5 (isobutanoyl C-1), 173.3 (C-12), 172.4 (octanoyl C-1)*, 172.2 (butanoyl C-1)*, 170.5 (acetyl C-1), 166.3 (angeloyl C-1), 141.8 (C-4), 139.3 (angeloyl C-3), 130.2 (C-5), 127.5 (angeloyl C-2), 88.0 (C-7), 84.5 (C-3), 83.5 (C-10), 78.7 (C-11), 77.5 (C-2), 76.7 (C-6), 64.1 (C-8), 57.3 (C-1), 38.1 (C-9), 36.9 (butanoyl C-2), 35.4 (isobutanoyl C-2), 34.6 (octanoyl C-2), 32.0, 30.1, 29.4 (octanoyl C-6−C-4), 24.4 (C-14), 23.0 (octanoyl C-7), 21.1 (acetyl

CH3), 19.8 (C-13), 19.3 (butanoyl C-3), 18.6 (isobutanoyl C-3), 18.4 (isobutanoyl C-3′), 16.3 (angeloyl C-4), 14.3 (octanoyl C-8, butanoyl C-4), 14.2 (angeloyl CH 3 ), 13.4 (C-15). HRMS calcd for C38H56NaO13 (M + Na+) 743.3613, found 743.3590. (3S,3aR,4S,6S,6aR,7S,8S,9bS)-6-Acetoxy-4-(butyryloxy)-3-hydroxy-3,6,9-trimethyl-8-{[(Z)-2-methylbut-2-enoyl]oxy}-2-oxo2,3,3a,4,5,6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-3a,7-diyl Dioctanoate (6d). The title compound was prepared from 5d (22 mg, 0.031 mmol, 1 equiv) according to general procedure C. Purification by column chromatography gave the product (23 mg, 95%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 6.61 (br s, 1H, H-8), 6.11 (qq, J = 1.5, 7.3 Hz, 1H, angeloyl H-3), 5.77 (br s, 1H, H-6), 5.68 (br s, 1H, H-3), 5.37 (t, J = 2.8 Hz, 1H, H-2), 4.19 (br s, 1H, H-1), 2.89 (dd, J = 2.9, 15.2 Hz, 1H, H-9a), 2.55−2.37 (m, 2H, octanoyl H-2), 2.35−2.25 (m, 4H, butanoyl H-2 and octanoyl H-2′), 2.1 (dd, J = 4.2, 15.2 Hz, 1H, H-9b), 2.01 (dq, J = 1.5, 7.2 Hz, 3H, angeloyl H-4), 1.92 (dq, J = 1.5, 1.6 Hz, 3H, angeloyl CH3) 1.89 (s, 6H, acetyl CH3 and H15), 1.62 (m, 6H, octanoyl H-3 and butanoyl H-3), 1.49 (s, 3H, H-13), 1.34 (s, 3H, H-14), 1.32−1.22 (m, 16H, octanoyl H-4−H-7), 0.95 (t, J = 7.3 Hz, butanoyl H-4), 0.86 (m, 6H, 2 × octanoyl H-8). 13C NMR (75 MHz, CDCl3) δ 172.5 (C-12), 172.4 (octanoyl C-1)*, 172.4 (octanoyl C-1′)*, 172.2, (butanoyl C-1)*, 170.5 (acetyl C-1), 167.4 (angeloyl C-1), 141.6 (C-4), 139.0 (angeloyl C-3), 130.0 (C-5), 127.4 (angeloyl C-2), 87.8 (C-7), 84.3 (C-3), 83.5 (C-10), 78.6 (C-11), 77.6 (C-2), 76.4 (C-6), 64.5 (C-8), 56.9 (C-1), 39.2 (C-9), 38.0 (angeloyl C-4), 36.7 (octanoyl C-2)*, 35.2 (octanoyl C-2′)*, 34.5 (butanoyl C2)*, 3.94 (octanoyl C-3), 29.3 (octanoyl C-4), 29.1 (octanoyl C-4′), 25.0 (octanoyl C-5), 24.2 (octanoyl C-5′), 24.6 (C-14), 24.2 (acetyl C2), 22.8 (octanoyl C-7 and C-3), 20.8 (angeloyl CH3), 20.0 (C-13), 18.2 (butanoyl C-3), 16.1 (angeloyl C-4), 14.3 (octanoyl C-4), 13.9 (butanoyl C-4), 13.4 (octanoyl C-8), 13.2 (C-15). HRMS calcd for C42H64NaO13 (M + Na+) 799.4239, found 799.4215. (3S,3aR,4S,6S,6aR,7S,8S,9bS)-6-Acetoxy-4-(butyryloxy)-2,3,3a-trihydroxy-3,6,9-trimethyl-8-{[(Z)-2-methylbut-2-enoyl]oxy}2,3,3a,4,5,6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-7-yl Octanoate (7). To a solution of 1 (1000 mg, 1.537 mmol, 1 equiv) in dry THF (11 mL) was added NaBH4 (1000 mg, 13.2 mmol, 17.2 equiv). The mixture was stirred for 65 h at room temperature before 1 M HCl (20 mL) was added, and the mixture was extracted with DCM (2 × 30 mL). The combined organic phase was washed with brine, dried with MgSO4, filtered, and concentrated in vacuo. The crude product was purified by column chromatography (toluene−EtOAc 2:1) to give a colorless oil (547 mg, 55%). 1H NMR (300 MHz, CDCl3) δ 6.08 (qd, J = 1.5, 7.2 Hz, 1H, angeloyl H-3), 5.70 and 5.65 (br s, 1H, H-3), 5.56 (dd, J = 3.3, 4.3 Hz, 1H, H-8), 5.52 and 5.46 (dd, J = 2.8, 3.3 Hz, 1H, H-2), 5.17 and 5.12 (br s, 1H, H-6), 5.06 (d, J = 3.0 Hz, 1H, H-12), 4.22−4.08 (m, 1H, H-1), 2.90 (dd, J = 3.1, 14.7 Hz, 1H, H-9a), 2.81 (dd, J = 3.4, 14.7 Hz, 1H, H-9b), 2.64 (s, 1H), 2.48 (dd, J = 4.3, 14.7 Hz, 1H), 2.44−2.35 (m, 2H), 2.35−2.21 (m, 6H), 2.02−1.95 (m, 3H, angeloyl CH3), 1.94−1.87 (m, 6H, O-10acetyl and H-2), 1.87−1.79 (m, 3H, H-15), 1.69−1.55 (m, 4H, butanoyl H-3 and octanoyl H-3), 1.59 and 1.47 (s, 3H, H-13), 1.39 (s, 3H, H-14), 1.35−1.23 (m, 8H, octanoyl H-4−H-7), 0.96 (dt, J = 4.5, 7.4 Hz, 3H, butanoyl H-4), 0.90−0.84 (m, 3H, octanoyl H-8). 13C NMR (101 MHz, CDCl3) δ 173.1 (butanoyl C-1)*, 173.0 (octanoyl C-1)*, 172.5 (acetyl C-1)*, 170.3 (angeloyl C-1), 138.9 (angeloyl C3), 138.5, 138.2 (C-5)*, 134.0 (C-4)*, 132.6, 127.7, 103.5 (C-12)*, 100.3 (C-12)*, 84.7 (C-10)*, 84.4 (C-3)*, 80.7 (C-7), 79.8 (C-2), 78.1 (C-6), 77.9, 74.6 (C-11), 67.2 (C-8), 58.1 (C-1), 36.7 (C-9), 34.4 (octanoyl C-2), 31.7 (octanoyl C-6), 29.0 (octanoyl C-5), 24.9 (octanoyl C-7), 22.6 (C-14)*, 22.2 (acetyl CH3), 20.6 (angeloyl CH3), 18.1 (C-13), 16.5, 15.8, 14.1 (angeloyl C-2), 13.7 (octanoyl C-8), 12.8 (C-15). (3S,3aR,4S,6S,6aR,7S,8S,9bS)-4-(Butyryloxy)-3a-hydroxy-3,6,9-trimethyl-8-{[(Z)-2-methylbut-2-enoyl]oxy}-7-(octanoyloxy)2,3,3a,4,5,6,6a,7,8,9b-decahydroazuleno[4,5-b]furan-2,3,6-triyl Triacetate (8). To a solution of 7 (60 mg, 0.100 mmol, 1 equiv) in DCM (2 mL) were added DMAP (cat.) and an acetic anhydride (30 mg, 0.3 mmol, 3 equiv). The reaction mixture was stirred at room temperature overnight before saturated aqueous NH4Cl solution was added. The mixture was extracted with DCM. The combined organic phase was 3616

dx.doi.org/10.1021/jm4001083 | J. Med. Chem. 2013, 56, 3609−3619

Journal of Medicinal Chemistry

Article

washed with brine, dried with MgSO4, filtered, and concentrated in vacuo. Purification by flash column chromatography with toluene− EtOAc (9:1) plus added 1% acetic acid as eluent gave 63 mg (93%) of 8. 1H NMR (400 MHz, CDCl3) δ 6.56 (s, 1H, H-12), 6.07 (qq, J = 1.5, 7.3 Hz, 1H, angeloyl H-3), 5.68 (s, 1H, H-3), 5.60 (t, J = 3.6 Hz, 1H, H-8), 5.50 (t, J = 3.0 Hz, 1H, H-2), 5.06 (br s, 1H, H-6), 4.22 (br s, 1H, H-1), 2.92 (dd, J = 3.3, 14.6 Hz, 1H, H-9a), 2.86 (br s, 1H, OH), 2.37 (dd, J = 4.3, 14.6 Hz, 1H, H-9b), 2.35−2.25 (m, 4H, octanoyl H-2 and butanoyl H-2), 2.15 (s, 3H, acetyl O-12), 1.99 (dq, J = 1.5, 7.3 Hz, 3H, angeloyl H-4), 1.96 (s, 3H, acetyl O-11), 1.91 (dq, 3H, angeloyl CH3), 1.89 (s, 3H, acetyl O-10), 1.80 (br s, 3H, H-15), 1.72 (m, 2H, butanoyl H-3), 1.65 (s, 3H, H-13), 1.62 (m, 2H, octanoyl H-3), 1.40 (s, 3H, H-14), 1.36−1.21 (m, 8H, octanoyl H-4−H-7), 0.98 (t, J = 7.4 Hz, 3H, butanoyl H-4), 0.87 (t, J = 6.5 Hz, 3H, octanoyl H8). 13C NMR (101 MHz, CDCl3) δ 172.4 (butanoyl C-1)*, 171.3 (octanoyl C-1)*, 170.2 (O-12-acetyl C-1)*, 169.0 (O-11-acetyl C-1)*, 167.9 (O-10-acetyl C-1)*, 167.1 (angeloyl C-1), 140.8 (C-4), 138.4 (angeloyl C-3), 132.3 (C-5)*, 127.6 (angeloyl C-2), 96.6 (C-12), 90.3 (C-11), 84.4 (C-10)*, 84.3 (C-3)*, 79.3 (C-7), 77.9 (C-2), 77.2 (C6), 65.8 (C-8), 58.0 (C-1), 38.0 (C-9), 36.6 (butanoyl C-2), 34.3 (octanoyl C-2), 31.7 (octanoyl C-7), 29.1 (octanoyl C-5), 29.0 (octanoyl C-4), 24.9 (acetyl O-11 C-2), 22.6 (C-14), 21.9 (O-11acetyl C-2), 21.2 (O-10-acetyl C-2), 20.6 (angeloyl C-4), 18.1 (butanoyl C-3), 15.8 (angeloyl C-4), 14.1 (octanoyl C-8), 13.7 (butanoyl C-4), 12.8 (C-13), 12.7 (C-15). HRMS calcd for C38H56NaO14 (M + Na+) 759.3562, found 759.3540. (2S,3S,3aR,4S,6S,6aR,6bS,9aR,10aS)-4-Acetoxy-6-(butyryloxy)6a-hydroxy-1,4,6b,8-tetramethyl-2-{[(Z)-2-methylbut-2-enoyl]oxy}2,3,3a,4,5,6,6a,6b,9a,10a-decahydroazuleno[5′,4′:4,5]furo[2,3-d]oxazol-3-yl Octanoate (9a). To a solution of SnCl4 (70 μL, 0.070 mmol) in dry acetonitrile (1 mL) was added a solution of 8 (59 mg, 0.080 mmol, 1.0 equiv) in dry acetonitrile (5 mL), and the mixture was stirred at room temperature for 16 h under nitrogen. The mixture was quenched with water (15 mL) and extracted with DCM (2 × 30 mL). The combined organic phase was washed with brine, dried with MgSO4, filtered, and concentrated in vacuo. Purification by flash column chromatography with toluene−ethyl acetate (2:1) plus added 1% acetic acid as eluent gave the product (18 mg, 33%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 6.09 (dd, J = 1.5, 7.3 Hz, 1H, angeloyl H-3), 5.66 (t, J = 3.0 Hz, 1H, H-8), 5.64 (s, 3H, H-3), 5.46 (s, 1H, H-12), 5.45 (t, J = 2.0 Hz, 1H, H-2), 4.41 (br s, 1H, H-6), 4.10 (br s, 1H, H-1), 2.87 (dd, J = 3.1, 14.5 Hz, 1H, H-9a), 2.46 (dd, J = 4.0, 14.6 Hz, 1H, H-9b), 2.32 (dt, J = 13.2, 7.0 Hz, 2H, butanoyl H-2), 2.26 (m, 2H, octanoyl H-2), 2.01 (s, 3H, H-2′), 1.98 (dq, J = 7.0, 1.5 Hz, 3H, angeloyl H-4), 1.89 (br s, 3H, angeloyl CH3), 1.87 (s, 3H, O-10acetyl), 1.80 (br s, 3H, H-15), 1.68−1.60 (m, 4H, butanoyl H-3 and octanoyl H-3), 1.47 (s, 3H, H-13), 1.34 (s, 3H, H-14), 1.36−1.23 (m, 8H, octanoyl H-4−H-7), 0.98 (t, J = 7.0 Hz, 3H, butanoyl H-4), 0.87 (t, J = 7.0 Hz, 3H, octanoyl H-8). 13C NMR (75 MHz, CDCl3) δ 172.3 (butanoyl C-1), 171.3 (octanoyl C-1), 170.0 (acetyl C-1), 168.9 (C-1′), 167.0 (angeloyl C-1), 141.4 (C-5)*, 138.7 (angeloyl C-3), 131.3 (C-4)*, 127.4 (angeloyl C-2), 102.9 (C-12), 94.0 (C-11), 84.4 (C-10), 84.2 (C-3), 79.6 (C-7), 77.9 (C-2), 73.3 (C-6), 65.7 (C-8), 58.2 (C-1), 37.8 (C-9), 36.5 (octanoyl C-2), 34.3 (butanoyl C-2), 31.7 (octanoyl C-6), 29.1 (octanoyl C-4), 29.0 (octanoyl C-5), 29.0 (octanoyl C-3), 22.7 (C-2′), 22.7 (C-14), 22.6 (acetyl C-2), 21.5 (octanoyl C-7), 20.7 (angeloyl C-4), 18.1 (butanoyl C-3), 16.0 (angeloyl C-4), 15.9 (C-13), 14.1 (butanoyl C-4), 13.9 (octanoyl C-8), 13.1 (C-15). HRMS calcd for C36H54NNaO11 (M + Na+) 676.3691, found 676.3659. (2S,3S,3aR,4S,6S,6aR,6bS,9aR,10aS)-4-Acetoxy-6-(butyryloxy)6a-hydroxy-1,4,6b-trimethyl-2-{[(Z)-2-methylbut-2-enoyl]oxy}-8propyl-2,3,3a,4,5,6,6a,6b,9a,10a-decahydroazuleno[5′,4′:4,5]furo[2,3-d]oxazol-3-yl Octanoate (9b). A solution of SnCl4 (50 μL, 0.050 mmol) in dry butyronitrile (1 mL) was added to a solution of 8 (33 mg, 0.043 mmol, 1.0 equiv) in dry butyronitrile (4 mL), and the mixture was stirred at room temperature for 20 min. The reaction was monitored by TLC, showing full conversion of the starting material. DCM (15 mL) was added and the mixture was washed with brine. The aqueous phase was extracted with DCM (15 mL). The combined

organic phase was dried with MgSO4, filtered, and concentrated in vacuo. The residue was purified by flash column chromatography with toluene−ethyl acetate (2:1) plus added 1% acetic acid as an eluent to give the product (20 mg, 64%) as a colorless oil. 1H NMR (300 MHz, CDCl3) δ 6.09 (dd, J = 1.5, 7.3 Hz, 1H, angeloyl H-3), 5.67 (t, J = 3.2 Hz, 1H, H-3), 5.64 (t, J = 4.4 Hz, 1H, H-8), 5.48 (br s, 1H, H-12), 5.46 (t, J = 2.6 Hz, 1H, H-2), 4.40 (br s, 1H, H-6), 4.07 (br s, 1H, H1), 2.87 (dd, J = 3.2, 14.5, Hz, 1H, H-9a), 2.46 (dd, J = 4.2, 14.6 Hz, 1H, H-9b), 2.32−2.26 (m, 2H, butanoyl H-2), 2.26 (m, 2H, H-2′), 2.22 (m, 2H, octanoyl H-2), 1.98 (d, J = 7.0 Hz, 3H, angeloyl H-4), 1.89 (s, 3H, acetyl H-2), 1.88 (br s, 3H, angeloyl CH3) 1.79 (br s, 3H, H-15), 1.68−1.60 (m, 6H, butanoyl H-3, octanoyl H-3, and H-3′) 1.47 (s, 3H, H-13), 1.34 (s, 3H, H-14), 1.36−1.23 (m, 8H, octanoyl H-4− H-7), 0.98 (t, J = 7.5 Hz, 6H, H-4′ and butanoyl H-4), 0.86 (t, J = 6.2 Hz, 3H, octanoyl H-8). 13C NMR (75 MHz, CDCl3) δ 172.5 (butanoyl C-1), 171.5 (octanoyl C-1), 170.2 (acetyl C-1), 167.2 (angeloyl C-1), 167.9 (C-1′), 141.9 (C-5)*, 138.9 (angeloyl C-3), 131.4 (C-4)*, 127.4 (angeloyl C-2), 102.9 (C-12), 93.8 (C-11), 84.5 (C-10), 84.4 (C-3), 79.9 (C-7), 78.0 (C-2), 73.5 (C-6), 65.9 (C-8), 58.4 (C-1), 37.9 (C-9), 36.7 (octanoyl C-21), 34.5 (butanoyl C-2), 31.9 (octanoyl C-6), 30.3 (C-2′), 29.3 (octanoyl C-5), 25.1 (octanoyl C-3), 22.7 (C-14), 22.7 (octanoyl C-7), 22.8 (acetyl C-2), 20.9 (angeloyl CH3-2), 19.6 (C-3′), 18.3 (butanoyl C-3), 16.3 (C-13), 16.1 (angeloyl C-4), 14.3 (octanoyl C-8), 14.0 (butanoyl C-4), 13.9 (C-4′), 13.1 (C-15). HRMS calcd for C38H58NO11 (M + H+) 704.4004, found 704.3993. (2S,3S,3aR,4S,6S,6aR,6bS,9aR,10aS)-4-Acetoxy-6-(butyryloxy)6a-hydroxy-1,4,6b-trimethyl-2-{[(Z)-2-methylbut-2-enoyl]oxy}-8ethoxyethyl-2,3,3a,4,5,6,6a,6b,9a,10a-decahydroazuleno[5′,4′:4,5]furo[2,3-d]oxazol-3-yl Octanoate (9c). To a solution of 8 (50 mg, 67.9 μmol) in dry DCM (2.0 mL) were added 3-ethoxypropionitrile (0.5 mL, 4.5 mmol) and SnCl4 (100 μL, 100 μmol) under a nitrogen atmosphere. The mixture was stirred for 1 h at room temperature and then DCM (20 mL) was added. The mixture was washed with brine (12 mL) and the aqueous phase was extracted with DCM (20 mL). The combined organic phases were washed with water (20 mL) and concentrated under vacuum. Purification by flash column chromatography with toluene−ethyl acetate (5:1) plus added 1% acetic acid as an eluent afforded 9c (31 mg, 62%). 1H NMR (300 MHz, CDCl3) δ 6.03 (qq, J = 1.5, 7.3 Hz, 1H, angeloyl H-4), 5.60 (t, J = 4.1 Hz, 1H, H-8), 5.62 (br s, 1H, H-3), 5.56 (br s, 1H, H-12), 5.40 (t, J = 2.6 Hz, 1H, H2), 4.42 (br s, 1H, H-6), 4.02 (br s, 1H, H-1), 3.64 (m, 2H, H-3′), 3.44 (q, J = 7.0 Hz, H-5′), 2.82 (dd, J = 14.7, 2.3 Hz, 1H, H-9a), 2.44 (t, J = 6.6 Hz, 2H, H-2′), 2.38 (dd, J = 14.7, 4.1 Hz, 1H, H-9b), 2.23 (m, 4H, octanoyl H-2 and butanoyl H-2), 1.91 (dq, J = 7.3, 1.2 Hz, 3H, angeloyl H-4) 1.82 (br s, 6H, angeloyl H-5 and acetyl H-2), 1.73 (br s, 3H, H-15), 1.58 (m, 4H, octanoyl H-3 and butanoyl H-3), 1.40 (s, 3H, H-13), 1.28 (s, 3H, H-14), 1.21 (m, 8H, octanoyl H-4−H-7), 1.08 (t, J = 7.03 Hz, 3H, H-6′), 0.90 (t, J = 7.3 Hz, 3H, butanoyl H-4), 0.80 (t, J = 7.3 Hz, 3H, octanoyl H-8). 13C NMR (75 MHz, CDCl3) δ 172.2 (octanoyl C-1), 171.5 (butanoyl C-1), 170.1 (acetyl C-1), 170.0 (angeloyl C-1), 166.9 (C-1′), 141.5 (C-5), 138.7 (angeloyl C-3), 131.4 (C-4), 127.3 (angeloyl C-2), 102.8 (C-12), 93.6 (C-11), 84.3 (C-10), 84.2 (C-3), 79.7 (C-7), 77.7 (C-2), 73.2 (C-6), 66.9 (C-3′), 66.5 (C4′), 65.5 (C-8), 58.4 (C-1), 37.8 (C-9), 36.3 (butanoyl C-2), 34.3 (octanoyl C-2), 31.7 (octanoyl C-6), 29.2 (C-2′), 29.0 (octanoyl C-5), 24.9 (octanoyl C-3), 22.7 (acetyl C-2), 22.6 (C-13), 22.4 (octanoyl C4), 20.7 (angeloyl C-5), 18.1 (butanoyl C-3), 15.9 (angeloyl C-4), 15.6 (C-14), 15.2 (C-6′), 14.2 (butanoyl C-4), 13.9 (octanoyl C-8), 13.0 (C-15). HRMS calcd for C39H60NO12 (M + H+) 734.4110, found 734.4135. The purity of the compound was determined to be 90.0% by HPLC. SERCA Inhibition Assay. The activity of SERCA in the presence of Tg and analogues was measured spectrophotometrically with a NADH coupled and ATP regenerating assay medium as previously described.13 Computational Methods. Molecular modeling calculations were based on crystallographic data of SERCA in complex with Tg (PDB code 3N5K).10 Molecular modeling calculations were performed in Schrödinger Suite 2010 (Windows version)30 and in the attached 3617

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water model19 as implemented in the Molegro Virtual Docker19 (MVD) for assessing the importance of water molecules mediating the interaction between Tg analogues and SERCA. The target protein was prepared in Protein Preparation Wizard according to the standard procedure including addition of hydrogen, assignment of bond orders, and protonation state. Optimization was performed with OPLS 2005 force field and minimized with converge cutoff of 0.01 kcal·mol−1·Å−1 root-mean-square force. Methodology for ligand preparation was inspired by the congeneric series approach.31 All analyzed compounds are derivatives of Tg with a rigid guaianolide skeleton in common. On the basis of the assumption that structure similarity corresponds to a similar binding mode, sesquiterpene lactone cores of prepared ligands were superimposed on the crystallographic data of the bound Tg. Ligand preparation procedure included conformational search performed by torsional sampling−Monte Carlo multiple minimum (MCMM) with maximum 10 000 steps and 1000 steps per rotatable bond followed by minimization of the lowest energy conformation (calculations were performed in water, termination criterion of 0.001 kcal·mol−1·Å−1 root-mean-square force). Docking experiments were carried out in Glide16−18,20 with extra precision mode applied. The grid was centered on the cocrystallized ligand. Different docking protocols were first tested with the focus on optimal predicting capacity. The presence of at least one cocrystallized water molecule inside the binding cavity of SERCA was validated as a required minimum for proper prediction of the Tg binding mode. The choice of water molecules was governed by the results of the AWM calculations (HOH782 and HOH783).19 The series of docking experiments with varied amount of cocrystallized water molecules was performed for each tested compound (see Table S2 in Supporting Information for details). Explicit water model and desolvation penalties were applied in calculations to correct for the different amount of water molecules included in the model. The best results were achieved by docking calculations carried out without scaling of van der Waals radii of the nonpolar ligand atoms. Epic32,33 state penalties were added to docking scores and ligand input partial charges were used. Additional constraints were applied by definition of hydrophobic cells in the grid placed at the sesquiterpene lactone cores. In order to ensure satisfying minimization of the docking simulations, the number of minimization steps was increased to 5000. During the docking calculations, 10 poses were saved per ligand. In MVD’s attached water model, Tg was solvated with 26 attached water molecules. The docking runs were performed with default settings in MVD version 4.0 and an entropy penalty value of 3. An attached water molecule was included during the docking calculation, if the interaction energy between the attached water and the surroundings was favorable.19 Crystallization and Structure Determination. For Ca2+ATPase, cocrystallization with the inhibitor was achieved by solubilization in 35 mM octa(ethylene glycol) dodecyl ether (C12E8) in 100 mM 3-(N-morpholino)propanesulfonic acid (MOPS; pH 6.8), 20% glycerol (v/v), 80 mM KCl, 3 mM MgCl2, and 2 mM ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) in the presence of 125 μM Tg analogue. The solubilization was followed by ultracentrifugation for 35 min at 50 000 rpm in a Beckman TLA 110 rotor to remove insoluble residues. The supernatant, with a protein concentration around 12 mg/mL, was stored overnight at 4 °C and subjected to another ultracentrifugation for 15 min at 70 000 rpm prior to the crystallization setup. The vapor diffusion method with hanging drops was used for crystallization at 12 °C with 2 + 2 μL volumes of protein solution and reservoir buffer. Single squared crystals appeared after a few days and grew to a maximum size of 250 × 250 × 50 μm within a couple of weeks. The crystals were mounted with a bent litho loop (Molecular Dimensions) and cryoprotected by transferring them shortly to a solution consisting of 1 μL of reservoir buffer and 1 μL of 50% (v/v) glycerol before flash-cooling in liquid nitrogen. Data was collected at the I911-3 beamline at MAX-lab in Lund, Sweden. Diffraction data were processed and scaled by use of the Xds package.34 Phases were obtained by molecular replacement

with the program PHASER from the PHENIX program package35 with a previously published SERCA structure (PDB ID 3NAN) as a search model. Model building was performed with Coot,36,37 and model refinement was performed with phenix.refine35 for all models. All structural figures in this study were prepared with PyMOL (The PyMOL Molecular Graphics System, version 1.5.0.4, Schrödinger LLC).

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR and 13C NMR spectra of new model compounds; one figure showing a close-up of the binding pocket of SERCA with 6a (difference in electron density of the ligand); and two tables listing data collection and model refinement of the X-ray structure (PDB code 4J2T) and water molecules included in docking calculations. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*. Present Addresses ▼

(J.V.) Department of Chemistry, Technical University of Denmark, Kemitorvet, Building 207, DK-2800 Kgs. Lyngby, Denmark. △ (E.T.) Dipartimento di Chimica, Universita di Firenze, Via della Lastruccia 3, I-50019 Sesto Fiorentino, Italy. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge The Danish Cancer Society and The Danish Strategic Research Council (SPOTLight) for financial support for this research.

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ABBREVIATIONS USED SERCA, sarco/endoplasmic reticulum calcium ATPase; Tg, thapsigargin; E2-P transition state, a calcium-free phosphorylated state of sarco/endoplasmic reticulum calcium ATPase; PSA, prostate-specific antigen; PSMA, prostate-specific membrane antigen; TM, transmembrane helix; AWM, attached water model; MCMM, Monte Carlo multiple minimum; rt, room temperature

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REFERENCES

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dx.doi.org/10.1021/jm4001083 | J. Med. Chem. 2013, 56, 3609−3619