Design and synthesis of new bicyclic diketopiperazines as scaffolds for receptor probes of structurally diverse functionality

Abstract

Diketopiperazines (DKPs) are a common motif in various biologically active natural products, and hence they may be useful scaffolds for the rational design of receptor probes and therapeutic agents. We constructed a new bicyclic scaffold that combines a DKP bridged with a 10-membered ring. In this way we obtained a three-dimensional molecular skeleton, with several amendable sites that provide a starting point to design a new combinatorial library having diverse substituent groups. Structural variation is based upon the flexibility of alkylation of the nitrogen atoms of the DKP and on the side-chain olefin. We obtained a 10-membered secondary ring through a ring-closure metathesis reaction using the second generation Grubbs catalyst. Rings containing both O-ethers and S-ethers were compared. N-Alkyl or arylalkyl groups were introduced optionally at the two Nα-atoms. This is a general scheme that will allow us to test rings of varying sizes, linkages, and stereochemical parameters. The DKP derivatives were tested for activity in astrocytoma cells expressing receptors coupled to phospholipase C. Inhibitory effects were observed for signaling elicited by activation of human nucleotide P2Y receptors but not m3 muscarinic receptors. Compound 20 selectively inhibited calcium mobilization (IC₅₀ value of 486 ± 16 nM) and phosphoinositide turnover elicited by a selective P2Y₁ receptor agonist, but this compound did not compete for binding of a radiolabeled nucleotide-competitive receptor antagonist. Therefore, the new class of DKP derivatives shows utility as pharmacological tools for P2Y receptors.

Introduction

The diketopiperazine (DKP) ring has been noted as a motif in various biologically active natural products. Examples include the commercial antibiotic bicyclomycin,¹ the fungal metabolites TAN-14 A, C and E,² and the thaxtomin A and B.³ DKPs may be useful scaffolds for the rational design of receptor probes and drugs. Owing to the presence of multiple H-bond acceptors and donors and the side chains of the component amino acids, DKPs have multiple sites for structural elaboration of defined stereochemistry. Others have explored the synthesis of DKPs of varied structure,^4–8 and combinatorial/polymer-supported syntheses of DKPs have been reported.^9–12

One of the limitations of conventional medicinal chemical agents, as applied to the new domain of chemical genetics,^13,14 is the typical planarity found in most known pharmaceutical substances discovered through organic synthesis. We desired to explore a three-dimensional molecular skeleton, which could be regarded as a starting point to design a new combinatorial library having diverse substituent groups. In the present study we have built a new bicyclic scaffold that combines a DKP additionally bridged with a 10-membered ring. A similar approach to synthesize a series of bridged DKPs, which were desired as mimics of a peptide β-turn found in peptide hormones, was introduced by Reitz and co-workers.¹⁵

Construction of the DKP skeleton was achieved with amino acid derivatives N-Boc-L-serine and L-allylglycine, thus providing great flexibility for structural variation, including substitution at the nitrogen atoms of the DKP and on the side-chain olefin. The 10-membered ring was obtained through a ring-closure metathesis (RCM) reaction using the second generation Grubbs catalyst.^16,17 This methodology provides a bicyclic DKP template with ether and olefinic groups present. This is a general scheme that will allow us to test larger and smaller rings, vary the ether linkage and manipulate stereochemical parameters. The ether and olefinic functionality is desired for both chemical advantages (i.e. the ability to derivatize at these positions) and biological advantages (i.e. to provide additional recognition elements for interaction with receptors and other biopolymers).

Selected bicyclic DKP analogues were subjected to biological assays in a variety of receptor-based systems. Several of the derivatives were found to inhibit the activity of extracellular nucleotides through G protein-coupled P2Y receptors and subsequent biochemical steps. Eight mammalian subtypes of ^18–21 such receptors (P2Y_{1,2,4,6,11–14}) are currently sequence-defined. The P2Y receptors are present in the cardiovascular, immune, and nervous systems, as well as in other systems. They are currently the focus of intense drug discovery efforts to design selective agonists and antagonists, which are lacking for most of the subtypes. There are numerous therapeutic possibilities under exploration for P2Y receptor modulators. For example, antagonists of the P2Y₁ and P2Y₁₂ receptors are of interest as antithrombotic agents.^22–32 Agonists of the P2Y₂ receptor are of interest in the treatment of pulmonary diseases, including cystic fibrosis. The P2Y₆ receptor has recently been implicated in protection against apoptosis induced by TNFα.³³

Results

Chemical synthesis

A new bicyclic scaffold that combines a DKP bridged with a 10-membered ring was prepared. This 10-membered secondary ring was obtained through a RCM reaction using the second generation Grubbs catalyst. The starting materials in this synthesis (Schemes 1–3) were two amino acid derivatives of defined stereochemistry, N-Boc-L-serine and L-allylglycine, which provided the bicyclic DKP template with both ether and olefinic groups present. In order to substitute the O-ether moiety with an S-ether, L-cysteine 6 was used as the starting material instead of N-Boc-L-serine 1.

Scheme 1.

Preparation of protected Ser and Cys derivatives. Reagents and conditions: (i) NaH, allylbromide, DMF, rt, 94%; (ii) TMSCHN₂, Et₂ O–MeOH 1: 1, rt, 78%; (iii) TFA, CH₂ Cl₂, rt, 93% (5); (iv) EtONa, allylbromide, EtOH, rt, 76%; (v) TMSCHN₂, Et₂ O–CH₂ Cl₂ –MeOH 1: 5: 5, rt, 33% (13), 32% (14), 55% (15), 22% (16); (vi) 2,4-dimethoxybenzaldehyde or 2,4,6-trimethoxybenzaldehyde, NaBH₃ CN, MeOH, rt, 53% (9), 53% (10), 41% (11), 41% (12); (vii) 2,4-dimethoxybenzaldehyde or 2,4,6-trimethoxybenzaldehyde, NaBH(OAc)₃, CH₂ Cl₂, rt, 85% (13), 82% (14), 74% (15), 58% (16), *partial racemization was obtained in this reaction.

Scheme 3.

Synthesis of bicyclic DKP derivatives. The closure of the DKP ring preceded the RCM reaction. DMB = 2,4-(MeO)₂ PhCH₂. Reagents and conditions: (i) piperidine–CH₂ Cl₂ 1: 4, rt, 42%; (ii) 37 (0.2 mM), second generation Grubbs catalyst, CH₂ Cl₂, rt, 21% (20), 43% (38); 37 (40 mM), 79% (38).

The first step of the synthesis was the introduction of the allyl group in the N-Boc-L-serine and L-cysteine using the methods reported in the literature.^34,35 Esterification of compound 2 with TMSCHN₂ followed by removal of the Boc group with TFA afforded the amino ester 5. The intermediate 8 was obtained after esterification of the amino acid 7 (Scheme 1).

The amino methyl ester 5 was subjected to reductive amination in the presence of 2,4-dimethoxybenzaldehyde or 2,4,6-trimethoxybenzaldehyde to give compounds 13 and 14, respectively. Following the same procedure, the thio derivatives 15 and 16 were obtained from 8 (Scheme 1). Our initial procedure, in order to minimize racemization, involved mixing the amine, NaBH(OAc)₃ and the aldehyde simultaneously in CH₂Cl₂. ^15,36,37 The reactions carried at room temperature for 2 h afforded the desired compounds but with racemization that was partial (70: 30) for the dimethoxybenzyl derivatives 13 and 15 and total (50: 50) for the trimethoxybenzyl derivatives 14 and 16. An alternative route consisted of the reductive amination followed by esterification. The amino acid derivative 4 was subjected to reductive amination using NaBH₃CN in methanol at room temperature for 24 h to obtain 9, which after esterification with TMSCHN₂ provided 13 with less than 5% of racemization. Following the same procedure, 14–16 were obtained from 4 or 7 with a similar degree of racemization.

Fmoc-L-allylglycine (17) was coupled with the secondary amine 13 using O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate (HATU), 1-hydroxy-7-azabenzo-triazole (HOAt) and N-ethylmorpholine (NEM) in a mixture of CH₂Cl₂–DMF (9: 1) to obtain the dipeptide 18 (Scheme 2). RCM of 18 (1 mM) using the second generation Grubbs catalyst in dichloromethane at room temperature provided 19. When the first generation Grubbs catalyst was used with 18 no reaction was observed. Finally, removal of the Fmoc group of 19 using a mixture of piperidine–CH₂Cl₂ (1: 4) was accompanied by in situ formation of the DKP, and compound 20 was obtained in high yield (93%).

Scheme 2.

Synthesis of bicyclic DKP derivatives. The RCM reaction preceded the closure of the DKP ring. Reagents and conditions: (i) HATU, HOAt, NEM, CH₂ Cl₂ –DMF 9: 1, rt, 53% (18), 40% (23), 37% (31), 41% (34); (ii) second generation Grubbs catalyst, CH₂ Cl₂, rt, 60% (19), 57% (24), 57% (32), 71% (35); (iii) piperidine–CH₂ Cl₂ 1: 4, rt, 93% (20), 73% (25), 73% (33), 60% (36); (iv) CH₂ Cl₂ –TFA 2: 1, rt, 96%; (v) MeI or BnBr, NaH, THF, rt, 63% (21), 60% (22), 96% (27), 85% (28); (vi) CH₂ Cl₂ –TFA 2: 1, rt, 63% (29), 89% (30).

Similar reaction sequences starting from compounds 14, 15 and 16, instead of 13, provided the bicyclic DKP templates 25, 33 and 36.

In order to introduce more diversity in the scaffold, the amide in the DKP was alkylated with methyl and benzyl groups.³⁸ Compounds 20 and 25 were treated with NaH and the resulting anions reacted with methyl iodide and benzyl bromide to give 21, 22, 27 and 28.

The trimethoxybenzyl group in 25, 27 and 28 was removed easily under acidic conditions; however, the normally acid-labile dimethoxybenzyl group in 20 was stable under these conditions.³⁶

By this route the new bicyclic scaffold was obtained through RCM followed by the formation of the DKP ring (Scheme 2). The possibility was explored to obtain these derivatives through a new route, by which the DKP ring was built first and then the 10-membered ring was formed (Scheme 3). In this case, compound 18 reacted with the mixture of piperidine–CH₂Cl₂ (1: 4) to obtain the DKP 37. The RCM reaction of 37 using first generation Grubbs catalyst was not satisfactory. When the second generation catalyst was used and the reaction was performed in conditions of high dilution (0.2 mM), 20 (21%) was obtained with the dimer 38 (43%). However, with higher concentrations (40 mM) only the dimer 38 was obtained (79%).

Pharmacological activity

Compounds 20–22, 25–30, 33, and 36 were screened at a 10 lM concentration in an assay of phospholipase C in 1321N1 human astrocytoma cells stably expressing the human P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptors. The biological characterization of the dimeric 38 will be reported elsewhere. In P2Y₁ receptor-expressing cells, inhibition of agonist (30 nM 2-MeSADP)-induced phospholipase C (PLC) was observed only for 20 (45 ± 10%), 33 (33 ± 4%), and 36 (18 ± 4%). At the other P2Y receptor subtypes, substantial inhibition of PLC activity (ca. 50% at 10 μM) was observed only for compounds 21 and 36 at the P2Y₄ receptor and compound 20 at the P2Y₆ receptors. Compound 20 and other DKPs (Fig. 1) inhibited the mobilization of intracellular [Ca²⁺] in astrocytoma cells stably expressing the human P2Y₁ receptor, with concentration-dependent reduction of the maximal effect. Compound 20 was the most potent with complete inhibition of [Ca²⁺] mobilization at 10 μM. Increasing concentrations of compound 20 progressively attenuated the 2MeSADP effect (Fig. 2). Compound 20 was found to inhibit [Ca²⁺]i transients elicited by 2MeSADP with an IC₅₀ value of 486 ± 16 nM. Control experiments in which PLC was stimulated via an endogenous m3 muscarinic receptor (using carbachol) in the control astrocytoma cells or via heterologously expressed P2Y_2,4,6 receptors (using the appropriate nucleotide) demonstrated insignificant or weak inhibition by compound 20. Thus, the inhibitory effects of 20, albeit insurmountable, were selective for signaling induced by P2Y₁ receptor activation. However, in a radioreceptor binding assay compound 20 at 10 μM failed to compete for the binding of a specific P2Y₁ antagonist radioligand, [³H]MRS2279.³⁹ Therefore, the binding of the DKP was not occurring at the principal nucleotide binding site of this receptor.

Fig. 1.

Effects of DKP derivatives (10 μM, structures in Scheme 2) on concentration–response curves for intracellular Ca²⁺ changes induced by 2-MeSADP acting at P2Y₁ receptors expressed in 1321N1 astrocytoma cells. The cells were pre-treated for 20 min at room temperature with antagonist before application of the agonist 2-MeSADP.

Fig. 2.

Effect of compound 20 on the concentration–response curve of the [Ca²⁺]i transient induced by 2MeSADP in P2Y₁ receptors expressed in 1321N1 astrocytoma cells. The cells were pre-treated for 20 min at room temperature with different concentrations of compound 20 before application of the agonist 2-MeSADP.

As an indication of the selectivity of these derivatives for the P2Y receptor pathway, interactions with a limited number of other receptors were examined (data not shown). Binding experiments at the human A₁ AR expressed in CHO cell membranes⁴⁰ indicated that compound 20 at 10 μM displayed no significant inhibition of radioligand binding. Similarly, this compound failed to displace binding at the mouse TRH receptors, or to either activate or antagonize functional effects of the m3 muscarinic receptor or the human calcium sensing receptor.

By means of a molecular dynamics simulation we carried out a conformational analysis of compound 20. The DKP ring-puckering coordinates (Θ≅ −80°; P₂ ≅ −30°; Q ≅ 40°) indicated a boat–screw/boat conformation.

Two different stable conformations were found for the 10-membered ring: in the first conformation the ring bends toward the dimethoxybenzyl group, while in the second one it bends in the opposite direction. Two diametrically opposite stable conformations were also found for the dimethoxybenzyl group. The coordinates of two representative conformations, in PDB format, are supplied as ESI.^†

Consistent with the non-competitive nature of these ligands, the stable conformations of compound 20 do not resemble the bound conformation of the competitive P2Y₁ antagonist MRS2279, neither by steric nor electronic criteria.⁴¹

Discussion

These DKP scaffold molecules were intended for broad screening with no a priori bias for any particular protein target. Among the targets thus far examined, inhibition was associated exclusively with the P2Y receptors. These receptors are diverse in the nature of their endogenous nucleotide ligands. Adenine nucleotides are required for activation of the P2Y₁, P2Y₁₁, P2Y₁₂, and P2Y₁₃ subtypes, and uracil nucleotides activate P2Y₂, P2Y₄, and P2Y₆ subtypes. The P2Y₂ receptor is equipotently activated by UTP and ATP. The most recently identified subtypes are the P2Y₁₃ receptor, which is present in human monocytes and lymphocytes,⁴² and the P2Y₁₄ receptor, which is activated by UDP-glucose.⁴³ P2Y receptor ligands are being investigated for therapeutic applications in the cardiovascular, endocrine, and other systems. Exploration of selective agonists and antagonists modulators is most advanced at the P2Y₁ and P2Y₁₂ receptors, but at most of the P2Y receptors selective pharmacological probes are lacking.^22–32 Compound 20 was shown to antagonize signaling of the P2Y₁ receptor selectively and with moderate potency. At the P2Y₁ receptor, the previously described high affinity antagonists are nucleotide derivatives and therefore highly charged, which is highly limiting in pharmacological studies due to low bioavailability and stability.

Therefore, owing to the ability to modify the chemical functionality of this new class of DKP derivatives and the already demonstrated biological activity, it shows promise as an approach to designing new pharmacological tools. Further study will be required to explore the mechanism of inhibition of P2Y receptor-induced effects and to determine the overall pharmacological selectivity of this class of compounds. It must still be determined if manipulation of functional groups on the DKP derivatives is able to vary the spectrum of interaction of these scaffold molecules with signaling of specific P2Y receptor subtypes, and among varied biochemical target proteins in general. If the inhibition by the DKP is found to occur in direct association with P2Y receptors, e.g. at an allosteric site, several of the compounds already prepared, such as compound 20, will provide insurmountable antagonists with receptor selectivity.

Experimental

Chemical synthesis

Reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO). Compounds 2 and 7 were synthesized as reported.^34,35

¹H NMR spectra were obtained with a Varian Gemini 300 spectrometer using CDCl₃, CD₃OD or D₂O as solvents. The chemical shifts are expressed as ppm downfield from TMS. All melting points were determined with a Thomas-Hoover apparatus (A.H. Thomas Co.) and are uncorrected.

Purity of the compounds was checked using a Hewlett-Packard 1100 HPLC equipped with a Luna 5 μm RP-C18(2) analytical column (250 × 4.6 mm; Phenomenex, Torrance, CA). System A: linear gradient solvent system: H₂O–CH₃CN from 80: 20 to 0: 100 in 20 min, then isocratic for 5 min; the flow rate was 1 mL min⁻¹. System B: linear gradient solvent system: 5 mM TBAP–CH₃CN from 80: 20 to 20: 80 in 20 min, then isocratic for 2 min; the flow rate was 1 mL min⁻¹. Peaks were detected by UV absorption with a diode array detector. All derivatives tested for biological activity showed >98% purity in the HPLC systems.

TLC analysis was carried out on aluminium sheets precoated with silica gel F₂₅₄ (0.2 mm) from Aldrich. Low-resolution CI–NH₃ (chemical ionization) mass spectra were measured with a Finnigan 4600 mass spectrometer, and low-resolution FAB (fast atom bombardment) mass spectrometry was performed with a JEOL SX102 spectrometer with 6 kV Xe atoms following desorption from a glycerol matrix or on LC/MS 1100 Agilent, 1100 MSD, with a Waters Atlantis C18 column. High-resolution mass measurements were performed on a Micromass/Waters LCT Premier Electrospray Time of Flight (TOF) mass spectometer coupled with a Waters HPLC system.

(2S)-3-Allyloxy-2-tert-butoxycarbonylamino-propionic acid methyl ester (3)

To a solution of compound 2 (4.48 g, 18.3 mmol) in 130 mL of Et₂O–MeOH (1: 1) was added TMSCHN₂ (2 M in Et₂O) dropwise until a yellow tint persisted. The reaction mixture was stirred at room temperature for 30 min. The solvent was removed in vacuo, and the residue was purified by silica gel column chromatography (petroleum ether–ethyl acetate, 15: 1) to afford 3 (3.71 g, 78%). ¹H NMR (CDCl₃) δ 5.83 (m, 1H, CH =CH₂), 5.38 (br d, J = 8.8 Hz, 1H, NH), 5.21 (m, 2H, CH=CH ₂), 4.43 (m, 1H, H-2), 3.98 (m, 2H, CH ₂CH=CH₂), 3.85 (m, 1H, 1H-3), 3.77 (s, 3H, OCH₃), 3.65 (m, 1H, 1H-3), 1.46 [s, 9H, C(CH₃)₃]; MS (FAB) m/z 260 (M + H)⁺.

(2S)-3-Allyloxy-2-amino-propionic acid (4)

To a solution of compound 2 (242 mg, 0.99 mmol) in CH₂Cl₂ (15 mL) was added TFA (1 mL). The reaction mixture was stirred at room temperature for 2 h. The solvent was removed in vacuo to obtain compound 4 as a crude solid (520 mg), which then was used directly in the next step without further purification. ¹H NMR (D₂O) δ 5.96 (m, 1H, CH =CH₂), 5.33 (m, 2H, CH=CH ₂), 4.11 (m, 2H, CH ₂CH=CH₂), 3.93 (m, 3H, H-2, H-3).

(2S)-3-Allyloxy-2-amino-propionic acid methyl ester (5)

To a solution of compound 3 (1.02 g, 3.93 mmol) in CH₂Cl₂ (60 mL) was added TFA (10 mL). The reaction mixture was stirred at room temperature for 30 min, and then was neutralized with the addition of saturated NaHCO₃ (225 mL). The product was extracted with CH₂Cl₂, dried over Na₂SO₄, and evaporated to obtain compound 5 (585 mg, 93%). ¹H NMR (CDCl₃) δ 5.87 (m, 1H, CH=CH₂), 5.22 (m, 2H, CH=CH ₂), 4.01 (m, 2H, CH ₂CH=CH₂), 3.75 (s, 3H, OCH₃), 3.68 (m, 3H, H-2, H-3).

(2R)-3-Allylsulfanyl-2-amino-propionic acid methyl ester (8)

To a suspension of compound 7 (1.21 g, 7.49 mmol) in 55 mL of Et₂O–MeOH–CH₂Cl₂ (1:5:5), was added TMSCHN₂ (2 M in Et₂O) dropwise until a yellow tint persisted. The reaction mixture was stirred at room temperature for 2 h. The solvent was removed in vacuo to obtain compound 8 as a crude solid (1.46 g), which then was used directly in the next step without further purification. ¹H NMR (CDCl₃) δ 5.78 (m, 1H, CH =CH₂), 5.12 (m, 2H, CH=CH ₂), 3.75 (s, 3H, OCH₃), 3.64 (m, 1H, H-2), 3.15 (m, 2H, CH ₂CH=CH₂), 2.88 (m, 1H, 1H-3), 2.71 (m, 1H, 1H-3); MS (CI) m/z 176 (M + H)⁺.

(2S)-3-Allyloxy-2-(2,4-dimethoxybenzylamino)-propionic acid (9)

(Procedure A) To a solution containing the crude solid 4 (260 mg) in MeOH (20 mL) was added NaBH₃CN (38 mg, 0.58 mmol) and 2,4-dimethoxybenzaldehyde (75 mg, 0.45 mmol). The reaction mixture was stirred at room temperature for 24 h. Water (3 mL) was added, and the product was extracted with CH₂Cl₂ and dried over Na₂SO₄. After removal of the solvent, the residue was purified by column chromatography on silica gel (CH₂Cl₂–MeOH, 92: 8) to give 9 (77 mg, 53% from 2) as a white solid. ¹H NMR (CDCl₃) δ 7.25 (d, J = 8.7 Hz, 1H, Ar–H), 6.47 (m, 2H, Ar–H), 5.83 (m, 1H, CH=CH₂), 5.19 (m, 2H, CH=CH ₂), 4.76 (br s, 1H, NH), 4.39 (d, J = 13.0 Hz, 1H, HCHAr), 4.24 (d, J = 13.0 Hz, 1H, HCHAr), 4.01–3.69 (m, 4H, CH ₂CH=CH₂, H-3), 3.85 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 3.56 (m, 1H, H-2). HRMS m/z found 296.1501 (M + H)⁺. C₁₅H₂₂NO₅ requires 296.1498. Mp 135–136 °C.

(2S)-3-Allyloxy-2-(2,4,6-trimethoxybenzylamino)-propionic acid (10)

(Procedure A) Compound 10 (84 mg, 53% from 2) was obtained from a solution of crude solid 4 (260 mg), NaBH₃CN (38 mg, 0.58 mmol) and 2,4,6-trimethoxybenz-aldehyde (89 mg, 0.45 mmol) in MeOH (20 mL). ¹H NMR (CDCl₃) δ 6.11 (s, 2H, Ar–H), 5.79 (m, 1H, CH =CH₂), 5.54 (br s, 1H, NH), 5.17 (m, 2H, CH=CH ₂), 4.49 (d, J = 13.2 Hz, 1H, HCHAr), 4.40 (d, J = 13.2 Hz, 1H, HCHAr), 3.98–3.74 (m, 3H, CH ₂CH=CH₂, 1H-3), 3.82 (s, 9H, OCH₃), 3.68 (m, 1H, 1H-3), 3.54 (m, 1H, H-2). HRMS m/z found 326.1588 (M +H)⁺. C₁₆H₂₄NO₆ requires 326.1604.

(2R)-3-Allylsulfanyl-2-(2,4-dimethoxybenzylamino)-propionic acid (11)

(Procedure A) Compound 11 (150 mg, 41%) as a white solid was obtained from 7 (206 mg, 1.28 mmol), NaBH₃CN (100 mg, 1.51 mmol) and 2,4-dimethoxybenzaldehyde (197 mg, 1.16 mmol) in MeOH (40 mL). ¹H NMR (CDCl₃) δ 7.30 (d, J = 8.7 Hz, 1H, Ar–H), 6.81 (br s, 1H, NH), 6.48 (m, 2H, Ar–H), 5.64 (m, 1H, CH=CH₂), 5.05 (m, 2H, CH=CH ₂), 4.45 (d, J = 13.2 Hz, 1H, HCHAr), 4.18 (d, J = 13.2 Hz, 1H, HCHAr), 3.87 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃), 3.41 (m, 1H, H-2), 3.16 (m, 1H, HCHCH=CH₂), 2.92 (m, 3H, HCH CH=CH₂, H-3). HRMS m/z found 312.1265 (M + H)⁺. C₁₅H₂₂NO₄S requires 312.1270. Mp 150–151 °C.

(2R)-3-Allylsulfanyl-2-(2,4,6-trimethoxybenzylamino)-propionic acid (12)

(Procedure A) Compound 12 (168 mg, 41%) was obtained from 7 (214 mg, 1.33 mmol), NaBH₃CN (104 mg, 1.57 mmol) and 2,4,6-trimethoxybenzaldehyde (242 mg, 1.21 mmol) in MeOH (40 mL). ¹H NMR (CDCl₃) δ 6.13 (s, 2H, Ar–H), 6.06 (br s, 1H, NH), 5.61 (m, 1H, CH=CH₂), 5.06 (m, 2H, CH=CH ₂), 4.49 (d, J = 13.3 Hz, 1H, H CHAr), 4.41 (d, J = 13.3 Hz, 1H, HCHAr), 3.85 (s, 6H, OCH₃), 3.82 (s, 3H, OCH₃), 3.34 (m, 1H, H-2), 3.17 (m, 1H, HCHCH=CH₂), 2.84 (m, 3H, HCHCH=CH₂, H-3). MS (API-ES) m/z 342 (M + H)⁺.

(2S)-3-Allyloxy-2-(2,4-dimethoxybenzylamino)-propionic acid methyl ester (13)

(Procedure B) To a solution of compound 5 (169 mg, 1.06 mmol) in CH₂Cl₂ (30 mL) was added NaBH(OAc)₃ (280 mg, 1.26 mmol) and 2,4-dimethoxybenzaldehyde (164 mg, 0.97 mmol). The reaction mixture was stirred at room temperature for 2 h. Water (30 mL) was added, the product was extracted with CH₂Cl₂ and dried over Na₂SO₄. After removal of the solvent, the residue was purified by column chromatography on silica gel (petroleum ether–ethyl acetate, 2: 1) to give 13 (253 mg, 85%). (Procedure C) To a suspension of compound 9 (23 mg, 0.08 mmol) in 5.5 mL of Et₂O–MeOH–CH₂Cl₂ (1: 5: 5) was added TMSCHN₂ (2 M in Et₂O) until a yellow tint persisted. The reaction mixture was stirred at room temperature for 30 min. After removal of the solvent, the product was purified by preparative thin-layer chromatography (petroleum ether–ethyl acetate, 2: 1) to give 13 (8 mg, 33%). ¹H NMR (CDCl₃) δ 7.14 (d, J = 8.7 Hz, 1H, Ar–H), 6.42 (m, 2H, Ar–H), 5.84 (m, 1H, CH=CH₂), 5.19 (m, 2H, CH=CH ₂), 3.95 (m, 2H, CH ₂CH=CH₂), 3.80 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃), 3.75 (d, J = 8.4 Hz, 2H, CH ₂Ar), 3.69 (s, 3H, OCH₃), 3.63 (m, 2H, H-3), 3.49 (m, 1H, H-2), 2.12 (br s, 1H, NH); MS (FAB) m/z 310 (M + H)⁺.

(2S)-3-Allyloxy-2-(2,4,6-trimethoxybenzylamino)-propionic acid methyl ester (14)

(Procedure B) Compound 14 (934 mg, 82%) was obtained from 5 (585 mg, 3.68 mmol), NaBH(OAc)₃ (970 mg, 4.35 mmol) and 2,4,6-trimethoxybenzaldehyde (670 mg, 3.34 mmol) in CH₂Cl₂ (70 mL). (Procedure C) Compound 14 (12 mg, 32%) was obtained from 10 (36 mg, 0.11 mmol) and TMSCHN₂ (2 M in Et₂O) in Et₂O–MeOH–CH₂Cl₂ (1: 5: 5) (5.5 mL). ¹H NMR (CDCl₃) δ 6.09 (s, 2H, Ar–H), 5.83 (m, 1H, CH=CH₂), 5.17 (m, 2H, CH=CH ₂), 3.92 (m, 2H, CH ₂CH=CH₂), 3.83 (d, J = 2.1 Hz, 2H, CH ₂Ar), 3.80 (s, 3H, OCH₃), 3.79 (s, 6H, OCH₃), 3.63 (s, 3H, OCH₃), 3.59 (m, 2H, H-3), 3.50 (m, 1H, H-2), 2.71 (br s, 1H, NH); MS (FAB) m/z 340 (M + H)⁺.

(2R)-3-Allylsulfanyl-2-(2,4-dimethoxybenzylamino)-propionic acid methyl ester (15)

(Procedure B) Compound 15 (797 mg, 74% from 7) was obtained from a solution containing crude solid 8 (731 mg), NaBH(OAc)₃ (988 mg, 4.43 mmol) and 2,4-dimethoxybenzaldehyde (578 mg, 3.41 mmol) in CH₂Cl₂ (65 mL). The product was purified by column chromatography on silica gel (petroleum ether–ethyl acetate, 3: 1). (Procedure C) Compound 15 (76 mg, 55%) was obtained from 11 (132 mg, 0.42 mmol) and TMSCHN₂ (2 M in Et₂O) in Et₂O–MeOH (1: 1) (10 mL). The product was purified by column chromatography on silica gel (petroleum ether–ethyl acetate, 4: 1). ¹H NMR (CDCl₃) δ 7.12 (d, J = 8.7 Hz, 1H, Ar–H), 6.42 (m, 2H, Ar–H), 5.73 (m, 1H, CH=CH₂), 5.06 (m, 2H, CH=CH ₂), 3.81 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃), 3.76 (m, 2H, CH ₂Ar), 3.69 (s, 3H, OCH₃), 3.41 (m, 1H, H-2), 3.05 (m, 2H, CH ₂CH=CH₂), 2.74 (m, 2H, H-3); MS (CI) m/z 326 (M + H)⁺.

(2R)-3-Allylsulfanyl-2-(2,4,6-trimethoxybenzylamino)-propionic acid methyl ester (16)

(Procedure B) Compound 16 (697 mg, 58% from 7) was obtained from a solution containing crude solid 8 (731 mg), NaBH(OAc)₃ (988 mg, 4.43 mmol) and 2,4,6-trimethoxybenzaldehyde (682 mg, 3.41 mmol) in CH₂Cl₂ (65 mL). The product was purified by column chromatography on silica gel (petroleum ether–ethyl acetate, 2: 1). (Procedure C) Compound 16 (35 mg, 22%) was obtained from 12 (154 mg, 0.45 mmol) and TMSCHN₂ (2M in Et₂O) in Et₂O–MeOH (1: 1) (12 mL). The product was purified by column chromatography on silica gel (petroleum ether–ethyl acetate, 4: 1). ¹H NMR (CDCl₃) δ 6.10 (s, 2H, Ar–H), 5.72 (m, 1H, CH =CH₂), 5.05 (m, 2H, CH=CH ₂), 3.81 (m, 2H, CH ₂Ar), 3.80 (s, 3H, OCH₃), 3.79 (s, 6H, OCH₃), 3.65 (s, 3H, OCH₃), 3.37 (m, 1H, H-2), 3.03 (m, 2H, CH ₂CH=CH₂), 2.72 (m, 2H, H-3), 2.42 (br s, 1H, NH); MS (CI) m/z 356 (M + H)⁺.

(2S)-(9H -Fluoren-9-ylmethoxycarbonylamino)-pent-4-enoic acid (17)

A solution of Fmoc-Cl (900 mg, 3.38 mmol) in dioxane (5 mL) was added, at 0 °C, to a suspension of L-allylglycine (299 mg, 2.60 mmol) and NaHCO₃ (655 mg, 7.79 mmol) in 16.8 mL H₂O–dioxane (1.4: 1). The mixture was stirred for 15 min at 0 °C and then at room temperature for 4.5 h. The pH was adjusted to approximately 9 with the addition of solid NaHCO₃ and the mixture was diluted with H₂O (120 mL) and washed with Et₂O. The aqueous layer was acidified to pH = 3 with an aqueous HCl solution (6 M). The product was extracted with EtOAc, dried over Na₂SO₄ and the solvent was removed under reduced pressure. The resulting residue was purified by column chromatography on silica gel (CH₂Cl₂–MeOH, 97: 3) to afford 17 (615 mg, 70%). ¹H NMR (CDCl₃) δ 7.75 (d, J = 7.5 Hz, 2H, Ar–H), 7.57 (d, J = 6.9 Hz, 2H, Ar–H), 7.39 (t, J = 7.4 Hz, 2H, Ar–H), 7.29 (t, J = 7.4 Hz, 2H, Ar–H), 5.71 (m, 1H, H-4), 5.34 (br d, J = 7.8 Hz, 1H, NH), 5.17 (m, 2H, H-5), 4.47 (m, 1H, H-2), 4.39 (d, J = 6.9 Hz, 2H, CHCH ₂), 4.21 (t, J = 6.9 Hz, 1H, CH CH₂), 2.58 (m, 2H, H-3); MS (FAB) m/z 338 (M + H)⁺.

(2S,2′S)-3-Allyloxy-2-{(2,4-dimethoxybenzyl)-[2′-(9H-fluoren-9-ylmethoxycarbonylamino)-pent-4′-enoyl]-amino}-propionic acid methyl ester (18)

(Procedure D) A solution containing compound 17 (638 mg, 1.89 mmol), HATU (742 mg, 1.89 mmol), HOAt (3.79 mL, 1.89 mmol, solution 0.5–0.7 M in DMF) and NEM (0.49 mL, 3.79 mmol) in CH₂Cl₂ (34.2 mL) was added to a solution of compound 13 (117 mg, 0.38 mmol) in 15.5 mL CH₂Cl₂–DMF (9: 1). The reaction mixture was stirred at room temperature overnight. The solvent was removed in vacuo, and the residue was purified by silica gel column chromatography (petroleum ether–ethyl acetate, 2: 1) to afford 18 (126 mg, 53%). ¹H NMR (CDCl₃) δ 7.77 (d, J = 7.5 Hz, 2H, Ar–H), 7.61 (d, J = 7.2 Hz, 2H, Ar–H), 7.40 (t, J = 7.4 Hz, 2H, Ar–H), 7.31 (t, J = 7.4 Hz, 2H, Ar–H), 7.17 (d, J = 7.8 Hz, 1H, Ar–H), 6.43 (m, 2H, Ar–H), 5.74 (m, 2H, CH=CH₂), 5.14 (m, 4H, CH=CH ₂), 4.80 (m, 1H, H CHAr), 4.53–4.02 (m, 6H, HCHAr, CHCH ₂OCO, CH CH₂OCO, CH CH₂CH=CH₂, H-2), 3.88 (m, 2H, OCH ₂CH=CH₂), 3.84–3.55 (m, 2H, H-3), 3.79 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 3.59 (s, 3H, OCH₃), 2.48 (m, 2H, CH ₂CH=CH₂); MS (FAB) m/z 629 (M + H)⁺.

(3S,6S)-4-(2,4-Dimethoxybenzyl)-6-(9H -fluoren-9-ylmethoxy-carbonylamino)-5-oxo-3,4,5,6,7,10-hexahydro-2H-[1,4]oxazecine-3-carboxylic acid methyl ester (19)

(Procedure E) To a solution of compound 18 (70 mg, 0.11 mmol) in CH₂Cl₂ (500 mL) was added second generation Grubbs catalyst (35 mg, 0.04 mmol). The reaction mixture was stirred at room temperature for 48 h. The solvent was removed in vacuo, and the residue was purified by column chromatography on silica gel (petroleum ether–ethyl acetate, 2: 1) to afford 19 (40 mg, 60%). ¹H NMR (CDCl₃) δ 7.78 (d, J = 7.2 Hz, 2H, Ar–H), 7.63 (d, J = 7.2 Hz, 2H, Ar–H), 7.41 (t, J = 7.2 Hz, 2H, Ar–H), 7.32 (t, J = 7.1 Hz, 2H, Ar–H), 7.08 (d, J = 8.1 Hz, 1H, Ar–H), 6.40 (m, 2H, Ar–H), 6.07 (d, J = 7.5 Hz, 1H, NH), 5.83 (m, 2H, H-8, H-9), 5.25 (m, 1H, H-6), 4.94 (d, J = 15.0 Hz, 1H, HCHAr), 4.51–4.34 (m, 3H, CHCH ₂OCO, 1H-10), 4.29–4.08 (m, 3H, CH CH₂OCO, H-2), 3.90 (m, 2H, HCHAr, 1H-10), 3.79 (s, 3H, OCH₃), 3.75 (m, 1H, H-3), 3.69 (s, 3H, OCH₃), 3.43 (s, 3H, OCH₃), 2.68 (m, 1H, 1H-7), 2.53 (m, 1H, 1H-7); MS (FAB) m/z 601 (M + H)⁺.

(1S,8S)-11-(2,4-Dimethoxybenzyl)-3-oxa-9,11-diazabicyclo-[6.2.2]dodec-5-ene-10,12-dione (20)

(Procedure F) Compound 19 (24 mg, 0.04 mmol) was stirred at room temperature in 5 mL of CH₂Cl₂–piperidine (4: 1) for 2 h. After the solvent was removed, the resulting residue was purified by silica gel column chromatography (CH₂Cl₂–MeOH, 96: 4) to obtain 20 (13 mg, 93%) as a white solid. ¹H NMR (CDCl₃) δ 7.24 (m, 1H, Ar–H), 6.45 (m, 2H, Ar–H), 5.93 (br s, 1H, NH), 5.62 (m, 2H, H-5, H-6), 4.98 (d, J = 14.3 Hz, 1H, HCHAr), 4.25 (m, 3H, H-4, H-8), 4.14 (d, J = 14.3 Hz, 1H, HCHAr), 3.89 (m, 3H, H-1, H-2), 3.79 (s, 6H, OCH₃), 3.11 (m, 1H, 1H-7), 2.83 (m, 1H, 1H-7); ¹³C NMR (CDCl₃) δ 169.5, 168.1, 161.0, 158.9, 132.0, 131.4, 125.9, 116.4, 104.9, 98.6, 73.2, 71.5, 61.5, 55.6, 53.8, 41.6, 34.0; HRMS m/z found 347.1591 (M + H)⁺. C₁₈H₂₃N₂O₅ requires 347.1607; HPLC (System A) 8.1 min (99%), (System B) 9.1 min (99%). Mp 155–156 °C.

(1S,8S)-11-(2,4-Dimethoxybenzyl)-9-methyl-3-oxa-9,11-diaza-bicyclo[6.2.2]dodec-5-ene-10,12-dione (21)

(Procedure G) A solution of compound 20 (10 mg, 0.03 mmol) in THF (1 mL) was added, at 0 °C, to a suspension of NaH (3.5 mg, 0.09 mmol, 60% dispersion in mineral oil) in THF (1 mL). After stirring at room temperature for 30 min, the reaction mixture was cooled to 0 °C and CH₃I (18 μL, 0.29 mmol) was added. The reaction mixture was stirred at room temperature for 5 h, followed by quenching with H₂O. The product was extracted with CH₂Cl₂, dried over Na₂SO₄, and the solvent was removed under reduced pressure. The resulting residue was purified by preparative thin-layer chromatography (CH₂Cl₂–MeOH, 96: 4) to obtain 21 (6.6 mg, 63%). ¹H NMR (CDCl₃) δ 7.25 (m, 1H, Ar–H), 6.44 (m, 2H, Ar–H), 5.67 (m, 1H, H-6), 5.55 (m, 1H, H-5), 4.95 (d, J = 14.4 Hz, 1H, HCHAr), 4.21 (m, 2H, H-4), 4.16 (d, J = 14.4 Hz, 1H, HCHAr), 4.15 (m, 1H, H-8), 3.90 (m, 3H, H-1, H-2), 3.79 (s, 6H, OCH₃), 3.07 (m, 1H, 1H-7), 2.91 (s, 3H, NCH₃), 2.88 (m, 1H, 1H-7); ¹³C NMR (CDCl₃) δ 168.1, 167.3, 161.0, 158.9, 132.1, 130.8, 125.7, 116.5, 104.9, 98.6, 72.8, 71.4, 61.7, 61.1, 55.6, 41.3, 33.9, 32.9; HRMS m/z found 377.1777 (M+ H)⁺. C₁₉H₂₅N₂O₅ requires 361.1763; HPLC (System A) 9.1 min (98%), (System B) 10.4 min (98%).

(1S,8S)-9-Benzyl-11-(2,4-dimethoxybenzyl)-3-oxa-9,11-diaza-bicyclo[6.2.2]dodec-5-ene-10,12-dione (22)

(Procedure G) Compound 22 (7.5 mg, 60%) was obtained as a white solid from 20 (10 mg, 0.03 mmol), NaH (4.2 mg, 0.10 mmol, 60% dispersion in mineral oil) and BnBr (54 μL, 0.45 mmol) in THF (2 mL) after for 8 h stirring at room temperature. The product was purified by preparative thin-layer chromatography (CH₂Cl₂–MeOH, 97: 3). ¹H NMR (CDCl₃) δ 7.29 (m, 3H, Ar–H), 7.19 (m, 3H, Ar–H), 6.44 (m, 2H, Ar–H), 5.68 (m, 1H, H-6), 5.55 (m, 1H, H-5), 5.29 (d, J = 14.7 Hz, 1H, HCHPh), 4.96 (d, J = 14.7 Hz, 1H, HCHAr), 4.20 (m, 2H, H-4), 4.16 (m, 2H, H-8, HCHAr), 3.99 (m, 2H, H-1, 1H-2), 3.80 (s, 3H, OCH₃), 3.79 (m, 2H, 1H-2, HCH Ph), 3.77 (s, 3H, OCH₃), 2.89 (m, 2H, H-7); ¹³C NMR (CDCl₃) δ 167.9, 167.4, 161.1, 158.9, 135.7, 131.8, 130.5, 129.1, 128.4, 128.1, 126.3, 116.4, 104.9, 98.7, 72.8, 71.5, 61.7, 57.8, 55.6 (2C), 48.7, 41.7, 32.9; HRMS m/z found 437.2095 (M + H)⁺. C₂₅H₂₉N₂O₅ requires 437.2076; HPLC (System A) 13.4 min (98%), (System B) 16.2 min (98%). Mp 129–130 °C.

(2S,2′S)-3-Allyloxy-2-[[2′-(9H -fluoren-9-ylmethoxycarbonyl-amino)-pent-4′-enoyl]-(2,4,6-trimethoxybenzyl)-amino]-propionic acid methyl ester (23)

(Procedure D) Compound 23 (278 mg, 40%) was obtained from 14 (357 mg, 1.05 mmol) in 45 mL CH₂Cl₂–DMF (9: 1), and 17 (1.77 g, 5.26 mmol), HATU (2.06 g, 5.26 mmol), HOAt (10.5 mL, 5.26 mmol, solution 0.5–0.7 M in DMF) and NEM (1.35 mL, 10.5 mmol), in CH₂Cl₂ (99 mL) and DMF (0.47 mL). ¹H NMR (CDCl₃) δ 7.76 (d, J = 7.5 Hz, 2H, Ar–H), 7.61 (d, J = 7.5 Hz, 2H, Ar–H), 7.40 (t, J = 7.2 Hz, 2H, Ar–H), 7.30 (t, J = 7.2 Hz, 2H, Ar–H), 6.10 (s, 2H, Ar–H), 5.84 (m, 2H, CH=CH₂), 5.15 (m, 4H, CH=CH ₂), 4.61 (m, 2H, CH ₂Ar), 4.49–4.07 (m, 5H, CHCH ₂OCO, CH CH₂OCO, CH CH₂CH=CH₂, H-2), 3.98 (m, 2H, OCH ₂CH=CH₂), 3.81 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 3.70 (m, 2H, H-3), 3.50 (s, 3H, OCH₃), 2.58 (m, 1H, HCHCH=CH₂), 2.44 (m, 1H, HCH CH=CH₂); MS (API-ES) m/z 681 (M + Na)⁺.

(3S,6S)-6-(9H -Fluoren-9-ylmethoxycarbonylamino)-5-oxo-4-(2,4,6-trimethoxybenzyl)-3,4,5,6,7,10-hexahydro-2H -[1,4]oxa-zecine-3-carboxylic acid methyl ester (24)

(Procedure E) Compound 24 (5.5 mg, 57%) was obtained from 23 (10 mg, 0.015 mmol) and second generation Grubbs catalyst (4 mg, 0.005 mmol) in CH₂Cl₂ (60 mL). The product was purified by preparative thin-layer chromatography (petroleum ether–ethyl acetate, 2: 1). ¹H NMR (CDCl₃) δ 7.78 (d, J = 7.5 Hz, 2H, Ar–H), 7.63 (d, J = 7.5 Hz, 2H, Ar–H), 7.41 (t, J = 7.4 Hz, 2H, Ar–H), 7.32 (t, J = 7.4 Hz, 2H, Ar–H), 6.08 (m, 3H, Ar–H, NH), 5.83 (m, 2H, H-8, H-9), 5.33 (m, 1H, H-6), 4.78–4.37 (m, 5H, CH ₂Ar, CHCH ₂OCO, 1H-10), 4.29–4.08 (m, 3H, CHCH₂OCO, H-2), 3.92 (m, 2H, H-3, 1H-10), 3.80 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃), 3.74 (s, 3H, OCH₃), 3.40 (s, 3H, OCH₃), 2.70 (m, 1H, 1H-7), 2.49 (m, 1H, 1H-7); MS (API-ES) m/z 653 (M + Na)⁺.

(1S,8S)-11-(2,4,6-Trimethoxybenzyl)-3-oxa-9,11-diazabicyclo-[6.2.2]dodec-5-ene-10,12-dione (25)

(Procedure F) Compound 25 (35 mg, 73%) was obtained as a white solid from 24 (80 mg, 0.13 mmol) and 15 mL of CH₂Cl₂–piperidine (4: 1). ¹H NMR (CDCl₃) δ 6.17 (br s, 1H, NH), 6.09 (s, 2H, Ar–H), 5.63 (m, 2H, H-5, H-6), 5.26 (d, J = 14.1 Hz, 1H, HCHAr), 4.29 (m, 2H, H-4), 4.24 (m, 1H, H-8), 4.10 (d, J = 14.1 Hz, 1H, HCHAr), 4.04 (m, 1H, 1H-2), 3.81 (s, 3H, OCH₃), 3.77 (s, 6H, OCH₃), 3.74 (m, 1H, 1H-2), 3.64 (m, 1H, H-1), 3.11 (m, 1H, 1H-7), 2.81 (m, 1H, 1H-7); ¹³C NMR (CDCl₃) δ 169.9, 167.7, 161.8, 160.3, 131.5, 126.0, 103.2, 90.7, 73.0, 71.4, 59.7, 55.9, 55.6, 53.8, 35.5, 34.1; HRMS m/z found 377.1705 (M + H)⁺. C₁₉H₂₅N₂O₆ requires 377.1713; HPLC (System A) 8.3 min (99%), (System B) 9.3 min (99%). Mp 123–124 °C.

(1S,8S)-3-Oxa-9,11-diazabicyclo[6.2.2]dodec-5-ene-10,12-dione (26)

(Procedure H) To a solution of compound 25 (10 mg, 0.03 mmol) in CH₂Cl₂ (1.5 mL) was added TFA (0.75 mL). After stirring at room temperature for 7 h, the solvent was removed under reduced pressure. The residue was purified by preparative thin-layer chromatography (CH₂Cl₂–MeOH, 94: 6) to afford 26 (5 mg, 96%) as a white solid. ¹H NMR (CD₃OD) δ 5.59 (m, 2H, H-5, H-6), 4.33 (m, 2H, H-4), 4.06 (m, 1H, H-8), 3.95 (m, 1H, 1H-2), 3.77 (m, 2H, H-1, 1H-2), 3.04 (m, 1H, 1H-7), 2.77 (m, 1H, 1H-7); ¹³C NMR (CD₃OD) δ 173.0, 171.1, 132.5, 126.2, 76.0, 74.9, 59.1, 54.4, 35.0; HRMS m/z found 197.0932 (M + H)⁺. C₉H₁₃N₂O₃ requires 197.0926.

(1S,8S)-9-Methyl-11-(2,4,6-trimethoxybenzyl)-3-oxa-9,11-diazabicyclo[6.2.2]dodec-5-ene-10,12-dione (27)

(Procedure G) Compound 27 (10 mg, 96%) was obtained from 25 (10 mg, 0.03 mmol), NaH (3.5 mg, 0.09 mmol, 60% dispersion in mineral oil) and CH₃I (18 μL, 0.29 mmol) in THF (2 mL) after 7 h of stirring at room temperature. The product was purified by preparative thin-layer chromatography (CH₂Cl₂–MeOH, 96: 4). ¹H NMR (CDCl₃) δ 6.08 (s, 2H, Ar–H), 5.68 (m, 1H, H-6), 5.57 (m, 1H, H-5), 5.23 (d, J = 13.8 Hz, 1H, HCHAr), 4.24 (m, 2H, H-4), 4.15 (m, 1H, H-8), 4.10 (d, J = 13.8 Hz, 1H, HCHAr), 3.96 (m, 1H, 1H-2), 3.80 (s, 3H, OCH₃), 3.78 (m, 1H, 1H-2), 3.77 (s, 6H, OCH₃), 3.69 (m, 1H, H-1), 3.08 (m, 1H, 1H-7), 2.89 (s, 3H, NCH₃), 2.86 (m, 1H, 1H-7); ¹³C NMR (CDCl₃) δ 167.7, 167.6, 161.8, 160.3, 130.7, 126.0, 103.2, 90.7, 72.5, 71.2, 61.1, 60.0, 55.9, 55.6, 35.2, 33.8, 32.9; HRMS m/z found 391.1867 (M + H)⁺. C₂₀H₂₇N₂O₆ requires 391.1869; HPLC (System A) 9.1 min (98%), (System B) 10.6 min (98%). Mp 158–159 °C.

(1S,8S)-9-Benzyl-11-(2,4,6-trimethoxybenzyl)-3-oxa-9,11-diazabicyclo[6.2.2]dodec-5-ene-10,12-dione (28)

(Procedure G) Compound 28 (10.6 mg, 85%) was obtained from 25 (10 mg, 0.03 mmol), NaH (4 mg, 0.1 mmol, 60% dispersion in mineral oil) and BnBr (64 μL, 0.53 mmol) in THF (2 mL) after 24 h of stirring at room temperature. The product was purified by preparative thin-layer chromatography (CH₂Cl₂–MeOH, 97: 3). ¹H NMR (CDCl₃) δ 7.28 (m, 3H, Ar–H), 7.16 (m, 2H, Ar–H), 6.10 (s, 2H, Ar–H), 5.70 (m, 1H, H-6), 5.60 (m, 1H, H-5), 5.33 (d, J = 15.3 Hz, 1H, HCHPh), 5.20 (d, J = 14.0 Hz, 1H, HCHAr), 4.27 (m, 2H, H-4), 4.15 (m, 1H, H-8), 4.13 (d, J =14.0 Hz, 1H, HCHAr), 3.89 (m, 2H, H-2), 3.82 (s, 3H, OCH₃), 3.75 (s, 6H, OCH₃), 3.73 (m, 1H, H-1), 3.70 (d, J = 15.3 Hz, 1H, HCHPh), 2.98 (m, 1H, 1H-7), 2.86 (m, 1H, 1H-7); ¹³C NMR (CDCl₃) δ 167.9, 167.7, 161.8, 160.3, 135.8, 130.6, 128.9, 128.1, 128.0, 126.5, 103.1, 90.7, 72.4, 71.1, 59.9, 57.7, 55.9, 55.6, 48.4, 35.3, 32.8; HRMS m/z found 467.2208 (M + H)⁺. C₂₆H₃₁N₂O₆ requires 467.2182; HPLC (System A) 13.3 min (98%), (System B) 16.3 min (98%).

(1S,8S)-9-Methyl-3-oxa-9,11-diazabicyclo[6.2.2]dodec-5-ene-10,12-dione (29)

(Procedure H) Compound 29 (1.7 mg, 63%) was obtained from 27 (5 mg, 0.01 mmol) and TFA (0.35 mL) in CH₂Cl₂ (0.7 mL). ¹H NMR (CDCl₃) δ 6.12 (br s, 1H, NH), 5.62 (m, 2H, H-5, H-6), 4.32 (m, 2H, H-4), 4.07 (m, 2H, 1H-2, H-8), 3.96 (m, 1H, H-1), 3.73 (m, 1H, 1H-2), 3.14 (m, 1H, 1H-7), 2.95 (s, 3H, NCH₃), 2.88 (m, 1H, 1H-7); HRMS m/z found 211.1086 (M+ H)⁺. C₁₀H₁₅N₂O₃ requires 211.1083.

(1S,8S)-9-Benzyl-3-oxa-9,11-diazabicyclo[6.2.2]dodec-5-ene-10,12-dione (30)

(Procedure H) Compound 30 (2.7 mg, 89%) was obtained from 28 (5 mg, 0.01 mmol) and TFA (0.35 mL) in CH₂Cl₂ (0.7 mL). The product was purified by preparative thin-layer chromatography (CH₂Cl₂–MeOH, 96: 4). ¹H NMR (CDCl₃) δ 7.31 (m, 3H, Ar–H), 7.25 (m, 2H, Ar–H), 6.22 (br s, 1H, NH), 5.64 (m, 2H, H-5, H-6), 5.38 (d, J = 15.0 Hz, 1H, HCHPh), 4.35 (m, 2H, H-4), 4.11 (m, 2H, 1H-2, H-8), 4.04 (m, 1H, H-1), 3.73 (d, J = 15.0 Hz, 1H, HCHPh), 3.70 (m, 1H, 1H-2), 3.01 (m, 1H, 1H-7), 2.86 (m, 1H, 1H-7); HRMS m/z found 287.1391 (M + H)⁺. C₁₆H₁₉N₂O₃ requires 287.1396.

(2R,2′S)-3-Allylsulfanyl-2-{(2,4-dimethoxybenzyl)-[2′-(9H -fluoren-9 - ylmethoxycarbonylamino) - pent - 4′ - enoyl] - amino } -propionic acid methyl ester (31)

(Procedure D) Compound 31 (107 mg, 37%) was obtained from 15 (146 mg, 0.45 mmol) in 20 mL CH₂Cl₂–DMF (9: 1), and 17 (755 mg, 2.24 mmol), HATU (878 mg, 2.24 mmol), HOAt (4.48 mL, 2.24 mmol, solution 0.5–0.7 M in DMF) and NEM (0.58 mL, 4.48 mmol) in CH₂Cl₂ (40.3 mL). The product was purified by column chromatography on silica gel (petroleum ether–ethyl acetate, 3: 1). ¹H NMR (CDCl₃) δ 7.77 (d, J = 7.2 Hz, 2H, Ar–H), 7.61 (d, J = 7.2 Hz, 2H, Ar–H), 7.40 (t, J = 7.4 Hz, 2H, Ar–H), 7.31 (t, J = 7.4 Hz, 2H, Ar–H), 7.16 (d, J = 8.7 Hz, 1H, Ar–H), 6.43 (m, 2H, Ar–H), 5.75 (m, 2H, CH=CH₂), 5.11 (m, 4H, CH=CH ₂), 4.78 (d, J = 15.6 Hz, 1H, H CHAr), 4.49–4.18 (m, 5H, HCHAr, CHCH ₂OCO, CH CH₂OCO, CH CH₂CH=CH₂), 4.07 (m, 1H, H-2), 3.78 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.55 (s, 3H, OCH₃), 3.10 (m, 3H, SCH ₂CH=CH₂, 1H-3), 2.94 (m, 1H, 1H-3), 2.61 (m, 1H, HCHCH=CH₂), 2.46 (m, 1H, HCHCH=CH₂); MS (API-ES) m/z 667 (M + Na)⁺.

(3R,6S)-4-(2,4-Dimethoxybenzyl)-6-(9H -fluoren-9-ylmethoxy-carbonylamino)-5-oxo-3,4,5,6,7,10-hexahydro-2H-[1,4]thiazecine-3-carboxylic acid methyl ester (32)

(Procedure E) Compound 32 (52 mg, 57%) was obtained from 31 (95 mg, 0.15 mmol) and second generation Grubbs catalyst (46 mg, 0.05 mmol) in CH₂Cl₂ (148 mL). The product was purified by column chromatography on silica gel (petroleum ether–ethyl acetate, 2.5: 1). ¹H NMR (CDCl₃) δ 7.77 (d, J = 7.5 Hz, 2H, Ar–H), 7.63 (d, J = 7.5 Hz, 2H, Ar–H), 7.41 (t, J = 7.4 Hz, 2H, Ar–H), 7.32 (t, J = 7.5 Hz, 2H, Ar–H), 7.12 (d, J = 7.5 Hz, 1H, Ar–H), 6.41 (m, 2H, Ar–H), 6.15 (br d, J = 6.3 Hz, 1H, NH), 5.67 (m, 2H, H-8, H-9), 5.30 (m, 1H, H-6), 5.05 (d, J = 14.4 Hz, 1H, HCHAr), 4.43 (m, 2H, CHCH ₂OCO), 4.29–4.07 (m, 2H, CHCH₂OCO, HCH Ar), 3.79 (s, 3H, OCH₃), 3.76–3.63 (m, 1H, H-3), 3.71 (s, 3H, OCH₃), 3.52–3.28 (m, 2H, H-10), 3.37 (s, 3H, OCH₃), 3.02–2.72 (m, 3H, H-2, 1H-7), 2.45 (m, 1H, 1H-7); MS (API-ES) m/z 639 (M + Na)⁺.

(1R,8S)-11-(2,4-Dimethoxybenzyl)-3-thia-9,11-diazabicyclo-[6.2.2]dodec-5-ene-10,12-dione (33)

(Procedure F) Compound 33 (19 mg, 73%) was obtained from 32 (45 mg, 0.07 mmol) and 5 mL of CH₂Cl₂–piperidine (4: 1). The product was purified by preparative thin-layer chromatography (CH₂Cl₂–MeOH, 96: 4). ¹H NMR (CDCl₃) δ 7.26 (d, J = 8.4 Hz, 1H, Ar–H), 6.45 (m, 3H, Ar–H, NH), 5.71 (m, 2H, H-5, H-6), 5.21 (d, J = 14.6 Hz, 1H, HCHAr), 4.33 (m, 1H, H-1), 4.28 (d, J = 14.6 Hz, 1H, HCHAr), 4.24 (m, 1H, H-2), 3.79 (s, 6H, OCH₃), 3.45 (m, 1H, 1H-4), 3.25–3.02 (m, 4H, H-2, 1H-4, 1H-7), 2.55 (m, 1H, 1H-7); ¹³C NMR (CDCl₃) δ 169.6, 167.0, 161.1, 159.1, 132.5, 130.7, 127.4, 116.2, 104.7, 98.7, 60.1, 56.9, 55.6, 55.2, 45.4, 32.4, 30.4, 29.3; HRMS m/z found 363.1371 (M + H)⁺. C₁₈H₂₃N₂O₄S requires 363.1379; HPLC (System A) 9.8 min (99%), (System B) 11.4 min (99%).

(2R,2′S)-3-Allylsulfanyl-2-[[2′ -(9H -fluoren-9-ylmethoxycar-bonylamino)-pent-4′-enoyl]-(2,4,6-trimethoxybenzyl)-amino]-propionic acid methyl ester (34)

(Procedure D) Compound 34 (124 mg, 41%) was obtained from 16 (159 mg, 0.45 mmol) in 20 mL CH₂Cl₂–DMF (9: 1), and 17 (755 mg, 2.24 mmol), HATU (878 mg, 2.24 mmol), HOAt (4.48 mL, 2.24 mmol, solution 0.5–0.7 M in DMF) and NEM (0.58 mL, 4.48 mmol) in CH₂Cl₂ (40.3 mL). The product was purified by column chromatography on silica gel (petroleum ether–ethyl acetate, 2.5: 1). ¹H NMR (CDCl₃) δ 7.76 (d, J = 7.5 Hz, 2H, Ar–H), 7.62 (m, 2H, Ar–H), 7.40 (t, J = 7.2 Hz, 2H, Ar–H), 7.30 (m, 2H, Ar–H), 6.10 (s, 2H, Ar–H), 5.82 (m, 2H, CH=CH₂), 5.13 (m, 4H, CH=CH ₂), 4.72–4.18 (m, 5H, CH ₂Ar, CHCH ₂OCO, CHCH₂OCO), 3.89–3.66 (m, 2H, CH CH₂CH=CH₂, H-2), 3.81 (s, 3H, OCH₃), 3.78 (s, 6H, OCH₃), 3.47 (s, 3H, OCH₃), 3.15 (m, 3H, SCH ₂CH=CH₂, 1H-3), 2.98 (m, 1H, 1H-3), 2.65 (m, 1H, HCHCH=CH₂), 2.48 (m, 1H, HCH CH=CH₂); MS (API-ES) m/z 697 (M + Na)⁺.

(3R,6S)-6-(9H -Fluoren-9-ylmethoxycarbonylamino)-5-oxo-4-(2,4,6-trimethoxybenzyl)-3,4,5,6,7,10-hexahydro-2H -[1,4]thiaze-cine-3-carboxylic acid methyl ester (35)

(Procedure E) Compound 35 (30 mg, 71%) was obtained from 34 (44 mg, 0.07 mmol) and second generation Grubbs catalyst (17 mg, 0.02 mmol) in CH₂Cl₂ (65 mL). The product was purified by preparative thin-layer chromatography (petroleum ether–ethyl acetate, 2: 1). ¹H NMR (CDCl₃) δ 7.77 (d, J = 7.2 Hz, 2H, Ar–H), 7.63 (m, 2H, Ar–H), 7.40 (t, J = 7.4 Hz, 2H, Ar–H), 7.31 (t, J = 7.2 Hz, 2H, Ar–H), 6.16 (d, J = 6.9 Hz, 1H, NH), 6.09 (m, 2H, Ar–H), 5.65 (m, 2H, H-8, H-9), 5.36 (m, 1H, H-6), 4.70 (m, 2H, CH ₂Ar), 4.43 (m, 2H, CHCH ₂OCO), 4.24 (t, J = 7.2 Hz, 1H, CHCH₂OCO), 3.84–3.62 (m, 1H, H-3), 3.80 (s, 3H, OCH₃), 3.77 (s, 6H, OCH₃), 3.52–3.27 (m, 2H, H-10), 3.36 (s, 3H, OCH₃), 3.05–2.65 (m, 3H, H-2, 1H-7), 2.43 (m, 1H, 1H-7); MS (API-ES) m/z 669 (M + Na)⁺.

(1R,8S)-11-(2,4,6-Trimethoxybenzyl)-3-thia-9,11-diazabicyclo-[6.2.2]dodec-5-ene-10,12-dione (36)

(Procedure F) Compound 36 (9 mg, 60%) was obtained from 35 (25 mg, 0.04 mmol) and 3.75 mL of CH₂Cl₂–piperidine (4: 1). The product was purified by preparative thin-layer chromatography (CH₂Cl₂–MeOH, 96: 4). ¹H NMR (CDCl₃) δ 6.09 (s, 3H, Ar–H, NH), 5.71 (m, 2H, H-5, H-6), 5.39 (d, J = 13.8 Hz, 1H, HCHAr), 4.41 (d, J = 13.8 Hz, 1H, HCHAr), 4.25 (m, 1H, H-8), 4.07 (m, 1H, H-1), 3.80 (s, 3H, OCH₃), 3.78 (s, 6H, OCH₃), 3.45 (m, 1H, 1H-4), 3.26–3.01 (m, 4H, H-2, 1H-4, 1H-7), 2.54 (m, 1H, 1H-7); ¹³C NMR (CDCl₃) δ 169.6, 166.4, 161.8, 160.6, 130.7, 127.5, 103.2, 90.7, 60.1, 56.0, 55.6, 55.3, 55.2, 38.2, 32.9, 30.6, 29.5; HRMS m/z found 393.1495 (M + H)⁺. C₁₉H₂₅N₂O₅S requires 393.1484; HPLC (System A) 9.7 min (99%), (System B) 11.3 min (99%).

(3S,6S)-3-Allyl-6-allyloxymethyl-1-(2,4-dimethoxybenzyl)-piperazine-2,5-dione (37)

(Procedure F) Compound 37 (52 mg, 42%) was obtained from 18 (206 mg, 0.33 mmol) and 10 mL of CH₂Cl₂–piperidine (4: 1). The product was purified by column chromatography on silica gel (petroleum ether–ethyl acetate, 1: 2). ¹H NMR (CDCl₃) δ 7.24 (d, J = 8.2 Hz, 1H, Ar–H), 6.45 (m, 2H, Ar–H), 6.19 (br s, 1H, NH), 5.82 (m, 2H, CH=CH₂), 5.22 (m, 4H, CH=CH ₂), 5.10 (d, J = 14.7 Hz, 1H, H CHAr), 4.12 (d, J = 14.7 Hz, 1H, HCHAr), 4.01 (m, 1H, H-6), 3.98 (m, 2H, OCH ₂CH=CH₂), 3.93 (m, 1H, H-3), 3.82 (m, 2H, CHCH ₂O), 3.79 (2s, 6H, OCH₃), 2.90 (m, 1H, H CHCH=CH₂), 2.60 (m, 1H, HCHCH=CH₂); ¹³C NMR (CDCl₃) δ 166.9, 166.4, 161.0, 158.8, 134.1, 133.7, 131.9, 120.1, 118.2, 116.3, 104.8, 98.6, 72.6, 67.8, 60.1, 55.6, 55.4, 41.7, 40.6; MS (FAB) m/z 375 (M + H)⁺.

(1S,8S,11S,18S)-21,23-Bis-(2,4-dimethoxybenzyl)-3,13-dioxa-9,19,21,23-tetraazatricyclo[16.2.2.28,11]tetracosa-5,15-diene-10, 20,22,24-tetraone (38)

(Procedure E) To a solution of compound 37 (15 mg, 0.04 mmol) in CH₂Cl₂ (1 mL) was added second generation Grubbs catalyst (5 mg, 0.006 mmol). The reaction mixture was stirred at room temperature for 16 h. The solvent was removed in vacuo, and the residue was purified by preparative thin-layer chromatography (CHCl₃–MeOH, 97: 3) to afford 38 (11 mg, 79%). ¹H NMR (CDCl₃) δ 9.18 (m, 2H, NH), 7.24 (d, J = 8.3 Hz, 2H, Ar–H), 6.45 (m, 4H, Ar–H), 5.65 (m, 4H, H-5, H-6, H-15, H-16), 5.18 (d, J = 14.5 Hz, 2H, HCHAr), 4.33 (m, 2H, 1H-4, 1H-14), 4.25 (m, 2H, 1H-2, 1H-12), 4.11 (d, J = 14.5 Hz, 2H, HCHAr), 3.99 (m, 2H, H-8, H-18), 3.93 (m, 2H, H-1, H-11), 3.79 (s, 6H, OCH₃), 3.78 (s, 6H, OCH₃), 3.76 (m, 2H, 1H-2, 1H-12), 3.67 (m, 2H, 1H-4, 1H-14), 2.90 (m, 2H, 1H-7, 1H-17), 2.68 (m, 2H, 1H-7, 1H-17); ¹³C NMR (CDCl₃) δ 169.5, 166.3, 161.0, 158.8, 131.8, 130.6, 127.9, 116.2, 104.9, 98.6, 72.5, 69.8, 60.5, 55.6, 55.3, 40.8, 40.0; HRMS m/z found 693.3149 (M + H)⁺. C₃₆H₄₅N₄O₁₀ requires 693.3136.

Pharmacological analyses

Cell culture and membrane preparation

Human 1321N1 astrocytoma cells transfected individually with the hP2Y_1,2,4,6,11 receptors^26,33,40 were grown at 37 °C in a humidified incubator with 5% CO₂–95% air in Dulbecco’s modified Eagle’s medium (JRH Biosciences, Inc.) supplemented with 10% fetal bovine serum (FBS), 100 Units mL⁻¹ penicillin, 100 μg mL⁻¹ streptomycin and 2 mM L-glutamine. The cells were grown to ca. 60% confluence for the experiments.

For membrane preparation, human astrocytoma cells expressing human P2Y₁ receptors were grown to approximately 80% confluence and then harvested. The cells were homogenized and suspended and then centrifuged at 100 g for 5 min at room temperature. The pellet was resuspended in 50 mM tris(hydroxymethyl)aminomethane (Tris) hydrochloride buffer (pH 7.4). The suspension was homogenized with a Polytron homogenizer (Brinkmann) for 10 s and was then recentrifuged at 20 000 g for 20 min at 4 °C. The resultant pellets were resuspended in Tris buffer (pH 7.4), and the suspension was stored at −80 °C until the binding experiments. The protein concentration was measured with the Bradford assay.⁴⁴

Storage of test substances

Agonists were dissolved as stock solutions in Tris buffer (pH 7.4), and the DKP derivatives were stored as frozen stock solutions in DMSO (5 mM) at −20 °C. Prior to use the DMSO solutions were warmed briefly to 50–60 °C.

Determination of inositol phosphates

The quantity of inositol phosphates was measured essentially as reported.^25,26,33,41 The P2Y_1,2,4,6,11-1321N1 cells were grown to confluence in 6-well plates in the presence of myo-[³H]inositol (2 μCi mL⁻¹) for 24 h. Cells were then treated for 30 min at 37 °C with antagonists or buffer in the presence of 20 mM LiCl, followed by another 30 min of incubation at 37 °C with the appropriate agonist. Agonists used were: P2Y₁, 2-MeSADP; hP2Y₂, UTP; hP2Y₄, UTP; hP2Y₆, UDP; hP2Y₁₁, ATP. The reaction was terminated upon aspiration of the medium and addition of cold formic acid (20 mM). After 30 min, supernatants were neutralized with NH₄OH, and applied to Bio-Rad Dowex AG 1-X8 anion exchange columns. All of the columns were then washed with water followed by a 60 mM sodium formate solution containing 5 mM sodium tetraborate. Total inositol phosphates were eluted with 1 M ammonium formate containing 0.1 M formic acid, and radioactivity values were measured using a liquid scintillation counter.

Radioligand binding assay

P2Y₁ receptor binding experiments were performed as previously described.³⁹ Briefly, membranes (40 μg protein) from astrocytoma cells stably expressing human P2Y₁ receptors were incubated with [³H]MRS2279 (8 nM) for 30 min at 4 °C in a total assay volume of 200 μL. For adenosine A₁ receptor binding, an agonist radioligand [³H]R-PIA (2.0 nM) was incubated with membranes (40 lg protein per tube) from CHO cells stably expressing human adenosine A₁ receptors for 60 min at 25 °C.⁴⁰ Radiolabeled ligand concentrations used in all assays approximated the K_d values of the receptor. Binding reactions were terminated by filtration through Whatman GF/B glass-fiber filters under reduced pressure with an MT-24 cell harvester (Brandel), and radioactivity was determined with a 1414 liquid scintillation counter (Wallac, Win Spectral, Perkin Elmer Life Sciences).

Calcium mobilization assay

Human astrocytoma cells stably expressing human P2Y₁ receptors were cultured in Dulbecco’s modified Eagle’s medium (DMEM, JRH Biosciences, Inc., Lenexa, KS, USA) and F12 (1: 1) supplemented with 10% fetal bovine serum, 100 units penicillin mL⁻¹, 100 μg streptomycin mL⁻¹, 2 μmol glutamine mL⁻¹, and 500 μg geneticin mL⁻¹. For the assay, cells were grown overnight in 100 μL of media in 96-well flat-bottom plates at 37°C at 5% CO₂ or until approximately 60–80% confluency. The calcium assay kit (Molecular Devices, Sunnyvale, CA) was used as directed without washing of the cells, and with probenecid added to the loading dye at a final concentration of 2.5 mM to increase dye retention. Cells were loaded with 50 μL of dye with probenecid to each well and incubated for 45 min at room temperature. The compound plate was prepared using dilutions of various compounds in Hank’s buffer. For antagonist studies, both agonist and antagonist were added to the sample plate. Samples were run in duplicate using a Molecular Devices Flexstation I at room temperature. Cell fluorescence (excitation = 485 nm, emission = 525 nm) was monitored following exposure to the compound. Increases in intracellular calcium are reported as the maximum fluorescence value after exposure minus the basal fluorescence value before exposure.

Data analysis

IC₅₀ values obtained in radioligand binding assays and in assays of inhibition of agonist-stimulated inositol phosphate accumulation were calculated by a four-parameter logistic equation and the GraphPad software package (GraphPad, San Diego, CA). All concentration–effect curves were repeated in at least three separate experiments, carried out in duplicate or triplicate with different membrane preparations or different 1321N1 cell cultures.

Molecular modeling

Molecular modeling was carried out with the module Discover3 of InsightII (Accelrys, Inc.) on Silicon Graphics Origin 200 computer, using the CFF91 forcefield.⁴⁵

An NVT (constant-volume/constant-temperature) molecular dynamics simulation was carried out for 500 ns at 298 K, with a time step of 1 fs. Conformations were sampled at regular intervals of 100 ns and energy minimized employing the BFGS Newton method, until an RMS gradient lower than 0.00001 kcal mol⁻¹ Å ⁻¹ was reached.

For a description of the puckering of the DKP ring we used the coordinates Θ (phase angle of symmetrical interconversion), P₂ (phase angle of pseudorotation), and Q (total puckering amplitude) as defined by Haasnoot,⁴⁶ utilizing the final formulae that we recently reported.⁴⁰