Preparation of an ABC tricyclic model of the cylindrospermopsin alkaloids via a biomimetically inspired pathway

Abstract

Two tricyclic guanidine model compounds 28 and 29 have been prepared in 12 steps from 1,5-pentanediol using a biomimetic synthetic approach. The pivotal reaction in the sequence is a tethered Biginelli condensation between 21/22 and β-keto esters 23 and 24.

---

Introduction

The cylindrospermopsin alkaloids are a family of polyketide derived marine natural products currently known to be produced by 12 different species of cyanobacteria.¹ Cylindrospermopsin 1 was first isolated from the cyanobacteria cylindrospermopsis raciborskii in 1992 (ref. 1a) and retrospectively identified as the causative agent in an outbreak of hepatoenteritis which affected 148 residents of Great Palm Island Australia.² Subsequent to the discovery of cylindrospermopsin, two further metabolites were isolated and characterised, namely the related diastereomer 7-epi-cylindrospemropsin³2 and the C-7 deoxygenated biosynthetic intermediate 7-deoxy-cylindrospermopsin⁴3. Cylindrospermopsin has been shown to display hepatotoxic,⁵ cytotoxic,⁶ neurotoxic⁷ and carcinogenic effects,⁸ which are shared with its equally toxic diastereoisomer 7-epicylindrospemropsin⁹2, however the deoxygenated analogue 7-deoxycylindrospermopsin 3 has been shown to be devoid of toxicity in vivo¹⁰ (Fig. 1). These alkaloids have also been the subject of considerable synthetic interest, resulting in four total syntheses¹¹ and several general methodological approaches.¹² These approaches generally rely upon the initial construction of either the A or B rings of the toxin, with the tricyclic core being completed by the installation of the C ring at a later stage.

Fig. 1 The cylindrospermopsin alkaloids.

Recent advances have led to the characterisation of the gene cluster responsible for the biosynthesis of cylindrospermopsin in C. raciborskii strain AWT205.¹³ As previously indicated the starter unit for the polyketide chain is guanidinoacetate 4 which has been shown to be derived from a novel L-arginine–glycine amidinotransferase enzyme.¹⁴ It is believed that the tricyclic guanidinium core of this family of natural products is constructed in a modular fashion from 4, with sequential polyketide extensions and Michael type ring closures generating in turn the C ring (5 → 6), the A ring (7 → 8) and the B ring (9 → 10) (Scheme 1).

Scheme 1 Proposed biosynthesis of the cylindrospermopsin alkaloids.

Using this biosynthesis as a guide for our synthetic investigations we envisioned a strategy in which cylindrospermopsin could be separated into two distinct fragments consisting of the uracil D ring and the tricyclic ABC guanidinium core. Initially we concentrated on a preparing a simplified version of the tricyclic guanidinium moiety anticipating that the stereochemistry generated during the formation of the initial C ring juncture would dictate the stereochemistry generated during the closure of the A and B rings (Fig. 2).^12d,15

Fig. 2 Retrosynthetic analysis of cylindrospermopsin.

Results and discussion

To test this approach we began with the simple diol, 1,5-pentanediol 11 which was monosilylated in 61% yield by treatment with TBSCl¹⁶ followed by oxidation of the remaining alcohol function under Swern conditions to furnish aldehyde 12 in 89% yield. The β-nitro alcohol 13 was then prepared in 81% yield via the nitro-aldol (Henry) reaction between 12 and nitromethane. The nitro functionality of 13 was then reduced with the NiCl₂–NaBH₄ system followed by the guanylation of the primary amine formed with 14 which gave the protected guanidine 15 in 78% yield.¹⁷ Subsequently a Mitsunobu type cyclodehydration gave the required C-ring heterocycle 16 in 96% yield.¹⁸ The alkyl chain of 16 was then elaborated by removing the TBS protecting group with TBAF followed by oxidation of alcohol 17 with Dess–Martin periodinane to generate aldehyde 18. Aldehyde 18 then underwent a Horner–Wadsworth–Emmons reaction with the simple phosphonate 19 to give the model substrate, enone 20 in 86% yield. It was attempted to cyclise enone 20 under a variety of conditions; however the only isolated material was representative of bicyclic guanidine 21 which displays an anti-configuration between the two bridging methine groups as confirmed by X-ray crystallography (Fig. 3). Efforts were made to epimerise this material under both basic and acidic conditions via retro-Michael addition of the guanidine, however all attempts proved unsuccessful (Scheme 2).¹⁹

Fig. 3 X-ray crystal structure of bicyclic guanidine 21.

Scheme 2 Reagents and conditions: (a) TBSCl, NaH, THF, 2 h, 61%; (b) oxalyl chloride, DMSO, NEt₃, CH₂Cl₂, −78 °C, 3 h, 89%; (c) MeNO₂, DIPEA, CH₂Cl₂, 36 h, 81%; (d) NiCl₂·6H₂O, NaBH₄, MeOH, NEt₃, 0 °C, 3 h; (e) 14, 48 h, 78% (2 steps from 13); (f) PPh₃, I₂, imidazole, CH₂Cl₂, −20 °C, 1 h, 96%; (g) TBAF, THF, 0 °C to rt, 24 h, 99%; (h) Dess–Martin periodinane, pyridine, CH₂Cl₂, 24 h, 96%; (i) LiCl, DIPEA, 19, CH₃CN, 48 h, 86%; (j) TFA–CH₂Cl₂ (1 : 1), 2 h; (k) NEt₃, CH₂Cl₂, 48 h, 70% (2 steps from 20).

Being unable to elaborate enone 20 into a suitable tricycle guanidine, the initial synthetic approach was revised utilising the previously prepared aldehyde 18. Thus the Boc-protecting groups of 18 were removed to give a mixture of hemiaminal 22 and N-acyliminium species 23. The resulting mixture was then immediately condensed with allyl acetoacetate 24 in the presence of morpholinium acetate under a variety of conditions based on the work of Aaron and Overman.²⁰ This tethered Biginelli protocol successfully generated tricyclic guanidine 26 the relative stereochemistry of which was determined by 2D NOESY NMR where a strong nOe correlation was observed between the two piperidine methine protons confirming the desired all syn relationship (Scheme 3 and Table 1).

Scheme 3 Reagents and conditions: (a) see Table 1.

Table 1 Optimisation of reaction conditions^a

Entry	R	Deprotection	β-Keto ester (eqv.)	Morpholinium acetate (eqv.)	T	Reaction time	Yield
a All reactions were performed on an ∼0.5 mmol scale in 2,2,2-trifluoroethanol (1.5 mL) in the presence of sodium sulphate (1 g). b Over 12 reactions. c Reactions performed in a sealed tube. d N-Methylmorpholine (2.5 eqv.) and AcOH (2.5 eqv.) were added to the reaction as a solution in 2,2,2-trifluoroethanol.
1	Me	TFA	3	1.0	60 °C	48 h	4%
2	Me	TFA	2	1.5	60 °C	72 h	8%
3	Me	TFA	1	1.0	60 °C	96 h	10%
4	Me	TFA	3	1.0	60 °C	96 h	12%
5	Me	TFA	5	2.5	70 °C	12 d	21%
6	Me	AcOH	5	2.5	70 °C	12 d	36–43%^b
7^c	Me	AcOH	5	2.5	100 °C	6 d	26%
8^c	Me	AcOH	8	4.0	100 °C	12 d	37%
9	Me	AcOH	5	0^d	70 °C	12 d	0%
10	Ph	AcOH	5	2.5	70 °C	12 d	26%

---

Initial investigations were conducted using TFA for the deprotection of aldehyde 18 followed by condensation of the resultant material with varying equivalents of 24, in the presence of morpholinium acetate and Na₂SO₄ at 60 °C (entries 1–4). An incremental increase in the reaction time from 48 h to 96 h allowed for an appreciable increase in the yield from 4% to 10%, with an excess of β-keto ester 24 generating a further 2% increase in the isolated yield (entries 1–4). Because of the progressive increase in yield observed with increased reaction time the reaction was allowed to proceed over a 12 day period at a slightly higher temperature of 70 °C (entry 5). Using such a prolonged reaction time had the greatest observed effect on the isolated yield of guanidine 26 which increased to a more acceptable 21%. Further significant improvements to the yield were also observed when all the anionic species involved in the reaction were of the same nature. Under such condition the TFA used in the initial deprotection step was replaced with AcOH, which gave an increase in the isolated yield of between 36% and 43% (entry 6). The use of more forcing conditions were also investigated were the reaction was performed at 100 °C in a sealed tube, however, no improvement in the isolated yield was observed (entries 7–8). The condensation was also attempted using β-keto ester, 25 which under the best conditions gave the corresponding tricyclic guanidine 27 in an unoptimised 26% yield (entry 10). The relative stereochemistry of this tricyclic material was assigned by 2D NOESY NMR spectroscopy.

No other tricyclic products were apparent, however, with the hope of further increasing the reaction yield it was attempted to recycle bicyclic intermediates isolated during purification by re-subjecting them to the reaction conditions. Interestingly even though the reaction conditions employed are equilibrating²¹ no further tricyclic material could be generated suggesting that the addition of the β-keto ester to the N-acyliminium ion 23 occurs through an irreversible pathway. Another reaction product isolated in low yields was the β-keto ester 28 which is formed from the reaction of 24 with morpholine. No tricyclic material containing morpholine derived esters were observed during any of the reactions run. In an attempt to negate this side reaction which consumes the β-keto ester, N-methylmorpholine was used as the base in the reaction (entry 9). However under these conditions no tricyclic material was isolated.

With tricyclic guanidines 26 and 27 in hand deallylation/decarboxylation was undertaken by treatment with Pd(PPh₃)₄ followed by treating the resulting material with NaBH₃CN in a 1 : 1 mixture of AcOH–MeOH to give saturated tricyclic guanidines 29 and 30 in 57% and 53% yield respectively over two steps (Scheme 4).²² Again 2D NOESY NMR spectroscopy was utilised to confirm that tricyclic guanidines 29 and 30 displayed the correct relatively stereochemistry representative of the ABC core of the cylindrospermopsin alkaloids.

Scheme 4 Reagents and conditions: (a) Pd(PPh₃)₄, pyrrolidine, THF–MeOH, 1.5–2 h; (b) NaBH₃CN, AcOH–MeOH, 0 °C to rt, 16 h, 57% (29), 53% (30) (2 steps).

Conclusions

In conclusion we have demonstrated the use of a nitro-aldol reaction, intramolecular S_N2 cyclisation and tethered Biginelli condensation to produce two model tricyclic guanidinium species which possess the correct relative syn-geometry found in the cylindrospermopsin alkaloids. During our initial investigation of the key tethered Biginelli condensation the desired tricyclic dihydropyrimidine could only be generated in an initially low yield of 4%, this could be improved to between 36% and 43% employing optimised reaction conditions. The simple biologically inspired approach that we have demonstrated in combination with a key tethered Biginelli condensation hopefully demonstrates the potential of this methodology for the synthesis of 1–3 which we will report in due course.

Experimental section

General information

Column chromatography was carried out on silica gel (particle size 40–63 μm) and thin layer chromatography conducted on precoated Kieselgel 60 F254 (Art. 5554; Merck) with the eluent specified in each case. All anhydrous reactions were conducted in oven-dried apparatus under a static atmosphere of argon. THF and dichloromethane were dried on a Pure Solv MD-3 solvent purification system. Chemical shifts are reported in δ values relative to chloroform (7.27/77.0 ppm) or methanol (3.31/49.00 ppm) as an internal standard. NMR spectra were recorded on a Bruker Avance 400/500 spectrometer unless otherwise stated. Mass spectra data were obtained at the EPSRC Mass Spectrometry Service Centre at the University of Wales, Swansea.

5-(tert-Butyl-dimethyl-silanyloxy)-pentan-1-ol (16). To a stirred suspension of NaH (3.86 g, 100 mmol) in anhydrous THF (100 mL) was added pentane-1,5-diol (10.52 mL, 100 mmol), after 1 h TBSCl (15.07 g, 100 mol) was added and vigorous stirring continued for a further hour. The reaction mixture was then diluted with diethyl ether (600 mL), washed with potassium carbonate (aq. 10%, 200 mL), brine (200 mL) and the organic extracts dried over magnesium sulfate. After purification by flash column chromatography on silica gel using ethyl acetate–petroleum ether (10 : 90, 40 : 60), the title compound was obtained as a clear oil (14.7 g, 67.1 mmol, 67%). R_f = 0.36 (20% ethyl acetate in petrol); ν_max (chloroform)/cm⁻¹ 3417, 2932, 2858, 1471, 1389 and 1361; δ_H (500 MHz; CDCl₃) −0.01 (s, 6H), 0.84 (s, 9H), 1.30–1.36 (m, 2H), 1.46–1.54 (m, 4H), 2.74 (br s, 1H) and 3.53–3.57 (m, 4H); δ_C (125 MHz; CDCl₃) −5.4, 18.2, 21.9, 25.9, 32.3, 32.4, 62.4 and 63.1.

5-(tert-Butyl-dimethyl-silanyloxy)-pentanal (12)²³. To a cooled (−78 °C) and stirred solution of oxalyl chloride (3.98 mL, 46.4 mmol) in anhydrous CH₂Cl₂ (120 mL) was added anhydrous DMSO (5.96 mL, 84.0 mmol). After 20 min a solution of alcohol 11 (6.34 g, 29.0 mmol) in anhydrous CH₂Cl₂ (50 mL) was added to the reaction mixture. After a further 20 min NEt₃ (24.2 mL, 173.9 mmol) was also added. After 3 h TLC analysis indicated the complete consumption of 11 and the reaction was diluted with brine (100 mL) and warmed to rt. After separation the organic layer was washed with brine (2 × 100 mL), HCl (0.25 M, 3 × 100 mL) and water (3 × 100 mL). After drying and evaporation the resulting yellow oil was dissolved in petroleum ether (50 mL) and passed through a plug of silica (ca. 1 cm) eluting with further petroleum ether (200 mL). After evaporation 12 was obtained as a pale yellow oil (5.57 g, 25.7 mmol, 89%) which was used without further purification. R_f = 0.62 (20% ethyl acetate in petrol); ν_max (chloroform)/cm⁻¹ 2954, 2930, 2857, 1728, 1472, 1388 and 1361; δ_H (500 MHz; CDCl₃) 0.01 (s, 6H), 0.85 (s, 9H), 1.49–1.54 (m, 2H), 1.64–1.70 (m, 2H), 2.42 (td, J = 7.4, 1.7 Hz, 2H), 3.59 (t, J = 6.2 Hz, 2H) and 9.73 (t, J = 1.7 Hz, 1H); δ_C (125 MHz; CDCl₃) −5.4, 18.2, 18.6, 25.9, 32.0, 43.5, 62.5 and 202.4.

6-(tert-Butyl-dimethyl-silanyloxy)-1-nitro-pentan-2-ol (13). To a stirred solution of aldehyde 12 (4.93 g, 22.7 mmol) in CH₂Cl₂ (80 mL) at rt was added nitromethane (73.7 mL, 1.36 mol) and the mixture cooled (0 °C) and DIPEA (9.90 mL, 56.9 mmol) added. Reaction progress was monitored via TLC and after 5 days the reaction was quenched with an ammonium chloride solution (sat, 200 mL), separated and the aqueous layer extracted with chloroform (3 × 100 mL). The combined organic extracts were washed with brine (100 mL), dried over anhydrous magnesium sulfate and purified by flash column chromatography on silica gel using ethyl acetate–petroleum ether (10 : 90, 30 : 70). Fractions eluting in 10 : 90 ethyl acetate–petroleum ether gave 13 as a pale yellow oil (5.11 g, 18.4 mmol, 81%). R_f = 0.29 (20% ethyl acetate in petrol); ν_max (chloroform)/cm⁻¹ 3418, 2953, 2931, 2858, 1556, 1471, 1463, 1421, 1385 and 1361; δ_H (500 MHz; CDCl₃) 0.04 (s, 6H), 0.88 (s, 9H), 1.43–1.59 (m, 6H), 2.87 (br s, 1H), 3.62 (t, J = 5.9 Hz, 2H), 4.30–4.34 (m, 1H), 4.37 (dd, J = 12.6, 8.5 Hz, 1H) and 4.42 (dd, J = 12.6, 3.1 Hz, 1H); δ_C (125 MHz; CDCl₃) −5.4, 18.3, 21.6, 25.9, 32.1, 33.4, 62.8, 68.6 and 80.6; LRMS, CI, m/z 295 ([M + NH₃]⁺, 3%), 278 ([M + H]⁺, 9%), 235 (5), 234 (30), 219 (7), 218 (20), 217 (100), 176 (5), 159 (8), 132 (6), 102 (3), 91 (6) and 85 (6); HRMS, ESI, m/z C₁₂H₂₈NO₄Si, requires 287.1782, found 287.1784 [M + H]⁺.

N,N′-bis-(tert-Butyloxycarbonyl)-N′′-(6-(tert-butyldimethylsilyloxy)-2-hydroxyhexyl)-guanidine (15). To a vigorously stirred, cooled (0 °C) solution of nickel(II) chloride hexahydrate (8.67 g, 36.5 mmol) in methanol (50 mL) was added NaBH₄ (4.10 g, 109.4 mmol) and stirring continued for 30 min. To the resultant black suspension was added nitro-alcohol 13 (3.38 g, 12.2 mmol) in methanol (20 mL), followed by the portion-wise addition of further NaBH₄ (9.23 g, 246.8 mmol). The reaction mixture was stirred for a further 2 h at rt and then filtered through a pad of Celite® (ca. 5 cm) and washed with methanol (3 × 50 mL). NEt₃ (149 mL, 1.07 mol) was added and after stirring for 45 min, the guanylating agent 14 (4.14 g, 13.4 mmol) dissolved in methanol (15 mL) was also added. After 48 h the reaction was diluted with water (400 mL) and extracted with ethyl acetate (3 × 250 mL). The combined organic extracts were dried over magnesium sulfate, evaporated and the resultant reaction material purified by flash column chromatography on silica gel using ethyl acetate–petroleum ether (5 : 95, 25 : 75). Fractions eluting in 10 : 90 ethyl acetate–petroleum ether gave 15 as a clear oil (4.66 g, 9.5 mmol, 78%). R_f = 0.34 (40% ether in petrol); ν_max (chloroform)/cm⁻¹ 3330, 3291, 3156, 2982, 2933, 2859, 1724, 1618, 1601, 1577, 1472, 1460, 1411, 1394, 1368 and 1335; δ_H (500 MHz; CDCl₃) 0.04 (s, 6H), 0.88 (s, 9H), 1.34–1.62 (m, 6H), 1.47 (s, 9H, Boc), 1.49 (s, 9H, Boc), 3.33–3.39 (m, 1H), 3.54 (ddd, J = 14.2, 6.3, 2.2 Hz, 1H), 3.61 (t, J = 6.3 Hz, 2H), 3.71–3.80 (br m, 1H), 4.58 (br s, 1H), 8.64–8.69 (br m, 1H) and 11.47 (s, 1H); δ_C (125 MHz; CDCl₃) −5.3, 18.4, 21.8, 26.0, 28.0, 28.2, 32.7, 34.9, 47.6, 63.1, 71.8, 79.5, 83.4, 153.1, 157.4 and 162.9; LRMS, CI, m/z 491 ([M + H]⁺, 40%), 434 (4), 390 (3), 373 (4), 370 (1), 320 (2), 275 (3), 274 (9), 273 (14), 248 (6), 232 (11), 231 (44), 217 (100), 204 (11), 175 (4), 174 (5), 160 (15) 159 (21), 145 (20), 132 (16), 104 (18), 92 (18), 60 (30), 58 (49) and 45 (71); HRMS, ESI, m/z C₂₃H₄₈N₃O₆Si, requires 490.3307, found 490.3312 [M + H]⁺.

tert-Butyl-2-((tert-butoxycarbonyl)imino)-5-(4-((tert-butyldimethylsilyl)oxy)butyl)imidazolidine-1-carboxylate (16). A stirred solution of alcohol 15 (7.79 g, 15.9 mmol) in CH₂Cl₂ (150 mL) was cooled (−20 °C) and PPh₃ (9.59 g, 36.6 mmol), imidazole (4.22 g, 62.0 mmol, 3.9 eqv.) and I₂ (7.48 g, 31.8 mmol, 2 eqv.) were added. Reaction progress was monitored by TLC and after stirring for 2 h 30 min the reaction mixture was diluted with chloroform (150 mL), washed with an ammonium chloride solution (sat, 150 mL) and then brine (150 mL). After drying over magnesium sulfate and evaporation, the reaction material was purified by flash column chromatography on silica gel using ethyl acetate–petroleum ether (10 : 90, 40 : 60). Fractions eluting in 30 : 70 ethyl acetate–petroleum ether gave 16 as a pale yellow oil (7.31 g, 15.5 mmol, 98%). R_f = 0.18 (40% ethyl acetate in petrol); ν_max (chloroform)/cm⁻¹ 3313, 2954, 2930, 2858, 1760, 1703, 1651, 1604, 1532, 1473, 1460, 1438 and 1368; δ_H (500 MHz; CDCl₃) 0.02 (s, 6H), 0.87 (s, 9H), 1.28–1.39 (m, 2H), 1.44–1.67 (m, 4H), 1.47 (s, 9H), 1.50 (s, 9H), 3.35–3.50 (br m, 1H), 3.58 (t, J = 6.5 Hz, 2H), 3.73–3.86 (br m, 1H) and 4.03–4.15 (br m, 1H); δ_C (125 MHz; CDCl₃) −5.3, 18.3, 20.7, 25.9, 28.1, 28.1, 32.6, 33.6, 56.4, 56.4, 62.7 and 82.8, four quaternary carbon signals were not detected; LRMS, CI, m/z 473 ([M + H]⁺, 100%), 430 (2), 416 (20), 372 (76), 357 (35), 330 (2), 316 (16), 302 (12), 272 (18), 257 (31), 243 (7), 214 (8), 184 (15), 159 (6), 148 (13), 132 (29), 116 (11), 84 (57), 69 (37), 58 (96) and 45 (44); HRMS, ESI, m/z C₂₃H₄₆N₃O₅Si, requires 472.3201, found 472.3201 [M + H]⁺.

2-tert-Butoxycarbonylimido-4-(4-hydroxy-butyl)-imidazolidine-1-carboxylic acid tert-butyl ester (17). To a stirred solution of guanidine heterocycle 16 (72.4 mg, 0.154 mmol) in THF (2 mL) at 0 °C was added a solution of TBAF in THF (1 M, 0.16 mL, 0.16 mmol). Reaction progress was monitored by TLC and after 24 h the reaction was quenched with an ammonium chloride solution (sat, 2 mL) and extracted with chloroform (3 × 5 mL). The combined organic extracts were then washed with brine (5 mL) and dried over magnesium sulfate. After evaporation, purification was achieved by flash column chromatography on silica gel using ethyl acetate–petroleum ether (80 : 20 to 100 : 0) and ethyl acetate–methanol (90 : 10). Fractions eluting in ethyl acetate gave 17 as a clear oil (54.4 mg, 0.152 mmol, 99%). R_f = 0.03 (100% ethyl acetate); ν_max (chloroform)/cm⁻¹ 3318, 2981, 2932, 2865, 1745, 1701, 1649, 1602, 1532, 1476, 1457 and 1370; δ_H (500 MHz; CDCl₃) 1.20–1.27 (m, 2H), 1.36 (s, 9H), 1.40 (s, 9H), 1.42–1.58 (m, 4H), 3.24–3.43 (br m, 1H), 3.50 (t, J = 6.3 Hz, 2H), 3.62–3.77 (br m, 1H) and 3.93–4.08 (br m, 1H); δ_C (125 MHz; CDCl₃) 20.2, 27.8, 27.8, 32.0, 33.2, 56.0, 56.0, 61.8, 80.2 and 82.6, three quaternary carbon signals were not detected; LRMS, CI, m/z 358 ([M + H]⁺, 55%), 319 (5), 302 (36), 279 (7), 258 (29), 243 (18), 211 (5), 202 (30), 186 (13), 156 (8), 133 (40), 116 (29), 98 (20), 79 (48), 69 (53), 58 (100) and 45 (54); HRMS, ESI, m/z C₁₇H₃₁N₃O₅, requires 358.2336, found 358.2335 [M + H]⁺.

tert-Butyl 2-(tert-butoxycarbonylimino)-5-(4-oxobutyl)imidazolidine-1-carboxylate (18). To a solution of alcohol 17 (260 mg, 0.73 mmol) in CH₂Cl₂ (2 mL) at rt was added pyridine (0.18 mL, 2.19 mmol) and Dess–Martin periodinane (310 mg, 0.73 mmol) and the mixture stirred for 24 h. The reaction was then filtered through a tight plug of cotton wool, diluted with chloroform (20 mL) and washed with NaHCO₃ (sat, 3 × 10 mL). Residual pyridine was removed by azeotropic evaporation with toluene (3 × 10 mL) to give 18 as a clear oil (249 mg, 0.70 mmol, 96%) which was used immediately without further purification. R_f = 0.30 (1% methanol in ethyl acetate); ν_max (chloroform)/cm⁻¹ 3336, 2987, 2942, 2882, 1758, 1710, 1656, 1609, 1536, 1482, 1443, 1374 and 1320; δ_H (500 MHz; CDCl₃) 1.42 (s, 9H), 1.47 (s, 9H), 1.51–1.63 (m, 4H), 2.44 (td, J = 6.7, 1.0 Hz, 2H), 3.44 (dd, J = 12.6, 3.2 Hz, 1H), 3.80 (dd, J = 12.6, 9.3 Hz, 1H), 4.05–4.09 (m, 1H) and 9.71 (s, 1H); δ_C (125 MHz; CDCl₃) 16.6, 27.9, 28.0, 33.0, 43.2, 55.9, 55.9, 80.6, 83.1 and 201.2, three quaternary carbon signals were not detected; LRMS, ESI, m/z 414 (15), 388 ([M + MeOH + H]⁺, 100%), 356 ([M + H]⁺, 15%), 332 (6), 316 (12), 300 (10), 260 (14), 214 (12), 182 (10) and 164 (4); HRMS, ESI, m/z C₁₇H₃₀N₃O₅, requires 356.2180, found 356.2186 [M + H]⁺.

tert-Butyl-2-(tert-butoxycarbonylimino)-5-((E)-6-oxohept-4-enyl)imidazolidine-1-carboxylate (20). To a stirred suspension of lithium chloride (28.2 mg, 0.65 mmol) in anhydrous acetonitrile (3 mL) was added phosphonate 19 (75 μL, 0.54 mmol). After 10 min, DIPEA (0.11 mL, 0.65 mmol) was then added and the mixture stirred for a further 10 min. At this point aldehyde 18 (192 mg, 0.54 mmol) dissolved in acetonitrile (2 mL) was added and the mixture stirred for 48 h. The reaction was diluted with water (15 mL) and extracted with diethyl ether (3 × 15 mL) and the combined organic extracts washed with brine (10 mL) and dried over magnesium sulfate. After evaporation, enone 20 (183 mg, 0.46 mmol, 86%) was obtained as a pale yellow oil and used without further purification. R_f = 0.22 (100% ethyl acetate); ν_max (chloroform)/cm⁻¹ 3320, 2980, 2933, 2860, 1764, 1707, 1678, 1650, 1605, 1532, 1374 and 1327; δ_H (500 MHz; CDCl₃) 1.34–1.45 (m, 2H), 1.47 (s, 9H), 1.51 (s, 9H), 1.59–1.71 (m, 4H), 2.23 (s, 3H), 3.40–3.54 (br m, 1H), 3.72–3.88 (m, 1H), 4.09–4.20 (br m, 1H), 6.07 (d, J = 15.8 Hz, 1H) and 6.74 (dt, J = 15.8, 7.0 Hz, 1H); δ_C (125 MHz; CDCl₃) 22.7, 27.0, 28.1, 28.1, 32.1, 33.3, 56.1, 56.1, 80.8, 83.1, 131.7, 146.9 and 198.3, three quaternary carbon signals were not detected; LRMS, ESI, m/z 791 ([2M + H]⁺, 79%), 762 (7), 708 (3), 692 (6), 593 (7), 448 (12), 430 (17), 412 (49), 396 ([M + H]⁺, 100%), 374 (2), 356 (13), 340 (27), 296 (10), 279 (19), 256 (6), 240 (23), 212 (3) and 196 (17); HRMS, ESI, m/z C₂₀H₃₄N₃O₅, requires 396.2493, found 396.2497 [M + H]⁺.

(5S,8aS)-5-(2-Oxopropyl)hexahydroimidazo[1,5-a]pyridin-3(2H)-iminium trifluoroacetate (21). To a stirred solution of enone 20 (162 mg, 0.41 mmol) in CH₂Cl₂ (1.5 mL) was added trifluoroacetic acid (1.5 mL) and the mixture stirred at rt for 24 h. After evaporation under reduced pressure, residual trifluoroacetic acid was removed by azeotropic evaporation with chloroform (3 × 15 mL) and the residue dried under high vacuum for 4 h. The resultant guanidinium salt was dissolved in CH₂Cl₂ (4 mL) and triethylamine (70 μL, 0.45 mmol) was added and the mixture stirred at rt for 48 h. After evaporation, purification by flash column chromatography on silica gel using methanol–chloroform (0 : 100 to 14 : 86 in 1% increments) gave 198 (89 mg, 0.28 mmol, 70%) as a viscous oil. An analytical sample was obtained by slow crystallisation from ethyl acetate. R_f = 0.08 (10% methanol in chloroform); m.p. 149–153 °C (dec.); ν_max (chloroform)/cm⁻¹ 3400, 2953, 2875, 1714, 1682, 1579, 1530, 1482, 1430, 1367 and 1320; δ_H (500 MHz; CDCl₃) 1.43 (qd, J = 13.6, 3.2 Hz, 1H), 1.50–1.59 (m, 1H), 1.63 (d, J = 13.6 Hz, 1H), 1.71–1.75 (m, 1H) 1.80 (dt, J = 13.6, 3.2 Hz, 1H), 1.92 (dd, J = 12.8, 3.0 Hz, 1H), 2.20 (s, 3H), 2.54 (dd, J = 18.3, 3.2 Hz, 1H), 3.05 (dd, J = 18.3, 9.3 Hz, 1H), 3.23 (dd, J = 9.5, 7.3 Hz, 1H), 3.77 (t, J = 9.5 Hz, 1H), 3.86 (app td, J = 11.5, 3.6 Hz, 1H), 4.36–4.43 (m, 1H) and 9.75 (s, 1H, NH); δ_C (125 MHz; CDCl₃) 18.2, 28.9, 30.1, 31.0, 44.3, 45.4, 47.6, 53.7, 157.9 and 206.7; LRMS, positive ESI, m/z 391 ([2M + H]⁺, 22%), 196 ([M + H]⁺, 100%), 138 (5) and 130 (8); LRMS, negative ESI m/z 227 ([2M − H]⁻, 44%), and 113 ([M − H]⁻, 100%); HRMS, ESI, m/z C₁₀H₁₈N₃O, requires 196.1444, found 196.1441 [M + H]⁺.

General procedure for tethered Biginelli condensations. A solution of aldehyde 18 (∼0.5 mmol) in glacial acetic acid (3 mL) was stirred at rt for 24 h. After evaporation under reduced pressure, residual acetic acid was removed by co-evaporation with chloroform (3 × 15 mL) and the residue dried under high vacuum for 4 h. The resultant salts where then dissolved in trifluoroethanol (1.5 mL) and morpholine acetate (2.5 eqv.), anhydrous sodium sulfate (1 g) and the relevant β-keto ester (5 eqv.) added and the mixture stirred at 70 °C for 12 days. After cooling to rt and evaporation, purification was achieved by flash column chromatography on silica gel using methanol–chloroform (stepwise gradient of 0.5% increments 0 : 100 to 4 : 96) containing 1% acetic acid.

(5aR,8aS)-5-((Allyloxy)carbonyl)-4-methyl-3,5a,6,7,8,8a-hexahydro-1H-2,2a¹,3-triazaacenaphthylen-2-ium acetate (26). Following the general procedure outlined above tricyclic guanidine 26 was obtained as a tan oil in yields of 36–43%. Upon standing or in a methanol solution the acetate counterion was slowly converted to the corresponding carbonate by atmospheric CO₂. R_f = 0.26 (10% methanol in chloroform); ν_max (chloroform)/cm⁻¹ 3400, 2949, 2932, 2905, 2862, 1696, 1679, 1637, 1605, 1449, 1407, 1391 and 1337; δ_H (500 MHz; CDCl₃) 1.32–1.40 (m, 1H), 1.51–1.66 (m, 2H), 1.70 (dd, J = 12.9, 3.2 Hz, 1H) 1.87 (dt, J = 13.3, 2.9 Hz, 1H), 1.96 (dd, J = 13.1, 3.0 Hz, 1H) 2.00 (s, 3H), 2.36 (s, 3H), 3.39 (dd, J = 10.4, 1.9 Hz, 1H), 3.75 (t, J = 9.5 Hz, 1H), 3.89–3.93 (m, 1H), 4.41 (dd, J = 2.8, 11.4 Hz, 1H), 4.63–4.64 (m, 2H), 5.23–5.32 (m, 2H) and 5.89–5.97 (m, 1H); δ_C (125 MHz; CDCl₃) 18.6, 21.3, 24.0, 29.5, 31.3, 48.0, 52.6, 57.9, 65.0, 101.5, 118.2, 132.2, 146.7, 154.6, 165.1 and 179.5; LRMS, positive ESI, m/z 523 ([2M + H]⁺, 5%), 402 (4), 314 (15), 304 (6), 262 ([M + H]⁺, 100) and 222 (2); LRMS, negative ESI m/z 165 (6) 113 (20) 77 (100), 75 (62), 60 ([CO₃]²⁻, 37%) and 59 ([M − H]⁻, 68%); HRMS, ESI, m/z C₁₄H₂₀N₃O₂, requires 262.1550, found 262.1548 [M + H]⁺.

(5aR,8aS)-5-((Allyloxy)carbonyl)-4-phenyl-3,5a,6,7,8,8a-hexahydro-1H-2,2a¹,3-triazaacenaphthylen-2-ium acetate (27). (5aR,8aS)-5-((Allyloxy)carbonyl)-4-phenyl-3,5a,6,7,8,8a-hexahydro-1H-2,2a¹,3-triazaacenaphthylen-2-ium acetate (27) was prepared following the general procedure outlined above giving tricyclic guanidine 27 as a tan oil in 26% yield after chromatography. R_f = 0.24 (10% methanol in chloroform); ν_max (chloroform)/cm⁻¹ 3338, 2926, 2859, 1693, 1668, 1648, 1606, 1493, 1448 and 1375; δ_H (500 MHz; CDCl₃) 1.50–1.71 (m, 4H) 1.81–1.88 (m, 1H), 1.98–2.05 (m, 1H), 3.15–3.23 (m, 1H), 3.62 (t, J = 7.2 Hz, 1H), 3.85–3.93 (m, 1H), 4.37 (d, J = 5.4 Hz, 2H), 4.53 (dd, J = 11.1, 2.7 Hz, 1H), 4.93–5.03 (m, 2H), 5.50–5.57 (m, 1H) and 7.30–7.36 (m, 5H); δ_C (125 MHz; CDCl₃) 21.3, 29.6 31.3, 48.2, 53.4, 58.0, 65.3, 103.5, 118.3, 128.1, 128.4, 130.2, 131.2, 133.2, 144.5, 153.5 and 164.8; LRMS, positive ESI, m/z 422 ([M + 2AcOH]⁺, 5%), 390 (6), 382 ([M + AcOH]⁺, 9%), 342 (13), 324 ([M + H]⁺, 100%), 316 (69), 290 (12) and 276 (8); LRMS, negative ESI m/z 121 (4) 113 (3) 77 (18), 59 ([M − H]⁻, 100%), 46 (3) and 45 (12); HRMS, ESI, m/z C₁₉H₂₂N₃O₂, requires 324.1707, found 324.1705 [M + H]⁺.

1-Morpholinobutane-1,3-dione (28). Following the general procedure outlined above amide 28 was isolated as a pale yellow oil (13% (based on 23)). R_f = 0.39 (10% methanol in chloroform); δ_H (500 MHz; CDCl₃) 2.24 (s, 3H), 3.38 (t, J = 4.8 Hz, 2H), 3.53 (s, 2H) 3.58–3.60 (m, 2H) and 3.62–3.65 (m, 4H); δ_C (125 MHz; CDCl₃) 30.2, 42.1, 46.7, 49.7, 66.5, 66.6, 165.0 and 202.0; LRMS, positive EI, m/z 172 ([M + H]⁺, 34%), 171 (M⁺, 54%), 128 (77), 87 (30), 86 (96), 85 (30), 57 (68), 56 (52) and 43 (100).

General procedure for deallylation/decarboxylation of dihydropyrimidines. To a stirred solution of tricyclic guanidine (∼0.1 mmol) in a mixture of anhydrous methanol (1 mL) and anhydrous THF (1 mL) was added tetrakis(triphenylphosphine)-palladium(0) (0.02 eqv.) and pyrrolidine (1.2 eqv.). The reaction was stirred at rt and progress monitored by TLC. After 1.5–2 h the solvent was removed under reduced pressure and the residue dissolved in methanol (1 mL) and acetic acid (1 mL) and cooled (0 °C), whereupon NaBH₃CN (5 eqv.) was added and the mixture stirred to rt over 16 h. After evaporation, the reaction material was purified by flash column chromatography on silica gel using methanol–chloroform (stepwise gradient of 1% increments 0 : 100 to 10 : 90) containing 1% acetic acid.

(4S,5aR,8aS)-4-Methyl-3,4,5,5a,6,7,8,8a-octahydro-1H-2,2a¹,3-triazaacenaphthylen-2-ium acetate (29). Following the general procedure outlined above guanidine 29 was obtained as a pale yellow oil in a 57% yield after chromatography. Upon standing or in methanol solution the acetate counterion is slowly converted to the carbonate by atmospheric CO₂. R_f = 0.15 (10% methanol in chloroform); ν_max (chloroform)/cm⁻¹ 3145, 2924, 2853, 1666, 1445, 1338 and 1332; δ_H (500 MHz; CDCl₃) 1.11–1.19 (m, 1H), 1.27–1.49 (m, 4H), 1.33 (d, J = 6.3 Hz, 3H), 1.90–2.05 (m, 2H), 1.98 (s, 3H), 2.11 (app dt, J = 13.4, 3.6 Hz, 1H), 3.18–3.27 (m, 2H), 3.50–3.58 (m, 1H), 3.62–3.69 (m, 1H) and 3.77–3.84 (m, 1H); δ_C (125 MHz; CDCl₃) 20.6, 22.2, 24.5, 29.7, 31.1, 37.1, 45.5, 48.5, 50.1, 56.8 and 155.4; LRMS, positive ESI, m/z 395 (5), 239 (2) and 180 ([M + H]⁺, 100%); LRMS, negative ESI m/z 113 (7), 77 (100), 75 (7), 60 ([CO₃]²⁻, 30%), 59 ([M − H]⁻, 12%), 45 (14), 35 (6) and 33 (3); HRMS, ESI, m/z C₁₀H₁₈N₃, requires 180.1495, found 180.1492 [M + H]⁺.

(4R,5aR,8aS)-4-Phenyl-3,4,5,5a,6,7,8,8a-octahydro-1H-2,2a¹,3-triazaacenaphthylen-2-iumacetate (30). Following the general procedure outlined above guanidine 30 was obtained as a pale yellow oil in 53% yield after chromatography. Upon standing or in methanol solution the acetate counter-ion is slowly converted to the carbonate by atmospheric CO₂. R_f = 0.08 (10% methanol in chloroform); ν_max (chloroform)/cm⁻¹ 3156, 2942, 2928, 2863, 1671, 1607, 1556, 1455 and 1379; δ_H (500 MHz; CD₃OD) 1.22–1.29 (m, 1H), 1.35–1.45 (m, 1H), 1.50–1.59 (m, 1H), 1.80 (app dt, J = 13.5, 11.5, 11.0 Hz, 1H), 1.96–2.02 (m, 2H), 2.09–2.14 (m, 1H), 2.36 (app dt, J = 13.5, 3.3, 3.3 Hz, 1H), 3.25–3.29 (m, 1H), 3.52 (app tt, J = 11.3, 11.0, 4.0, 3.3 Hz, 1H), 3.82–3.89 (m, 2H), 4.62 (dd, J = 11.5, 3.3 Hz, 1H) and 7.31–7.45 (m, 5H); δ_C (125 MHz; CDCl₃) 22.2, 23.0, 29.5, 30.9, 38.7, 48.6, 50.4, 54.0, 57.1, 126.1, 128.5, 129.1, 139.1, 155.9 and 174.0; LRMS, positive ESI, m/z 483 ([2M + H]⁺, 6%), 284 (4) and 242 ([M + H]⁺, 100%); LRMS, negative ESI m/z 120 (10), 92 (12), 76 (42), 75 (100), 59 ([M − H]⁻, 18%), 45 (16) and 32 (5); HRMS, ESI, m/z C₁₅H₂₀N₃, requires 242.1648, found 242.1652 [M + H]⁺.

Acknowledgements

We gratefully acknowledge the EPSRC (EP/J01821X/1) and Bangor University for their financial support. We also thank the EPSRC National Mass Spectrometry Facility at Swansea, the EPSRC X-Ray crystallography service at Southampton, Professor Ōmura of Kitasato University for useful information and Jackie Hollinshead for NMR studies. The contributions of previous co-workers in the group, Andrew Thornhill, Alan Molyneux, Steven P Williams and Louise Jones (née Hall) are also acknowledged.

References

1. (a) I. Ohtani, R. E. Moore and M. T. C. Runnegar, J. Am. Chem. Soc., 1992, 114, 7941; (b) K. Harada, I. Ohtani, K. Iwamoto, M. Suzuki, M. F. Watanabe and K. Terao, Toxicon, 1994, 32, 73; (c) R. Banker, S. Carmeli, O. Hadas, B. Teltsch, R. Porat and A. Sukenik, J. Phycol., 1997, 33, 613; (d) R. Li, W. W. Carmichael, S. Brittain, G. K. Eaglesham, G. R. Shaw, Y. Liu and M. W. Watanabe, J. Phycol., 2001, 37, 1121; (e) M. A. Schembri, B. A. Neilan and C. P. Saint, Environ. Toxicol., 2001, 16, 413; (f) L. Spoof, K. A. Berg, J. Rapala, K. Lahti, L. Lepisto, J. Metcalf, G. A. Codd and J. Meriluoto, Environ. Toxicol., 2006, 21, 552; (g) K. Preußel, A. Stüken, C. Wiedner, I. Chorus and J. Fastner, Toxicon, 2006, 47, 156; (h) M. Seifert, G. McGregor, G. Eaglesham, W. Wickramasinghe and G. Shaw, Harmful Algae, 2007, 6, 73; (i) L. Brient, M. Lengronne, M. Bormans and J. Fastner, Environ. Toxicol., 2009, 24, 415; (j) R. Mazmouz, F. Chapuis-Hugon, S. Mann, V. Pichon, A. Méjean and O. Ploux, Appl. Environ. Microbiol., 2010, 76, 4943; (k) G. B. McGregor, B. C. Sendall, L. T. Hunt and G. K. Eaglesham, Harmful Algae, 2011, 10, 402; (l) M. Kokociński, J. Mankiewicz-Boczek, T. Jurczak, L. Spoof, J. Meriluoto, E. Rejmonczyk, H. Hautala, M. Vehniäinen, J. Pawełczyk and J. Soininen, Environ. Sci. Pollut. Res. Int., 2013, 20, 5243.
2. S. Byth, Med. J. Aust., 1980, 2, 40.
3. R. Banker, B. Teltsch, A. Sukenik and S. Carmeli, J. Nat. Prod., 2000, 63, 387.
4. R. L. Norris, G. K. Eaglesham, G. Pierens, G. R. Shaw, M. J. Smith, R. K. Chiswell, A. A. Seawright and M. R. Moore, Environ. Toxicol., 1999, 14, 163.
5. (a) M. T. Runnegar, S. M. Kong, Y. Z. Zhong, J. L. Ge and S. C. Lu, Biochem. Biophys. Res. Commun., 1994, 201, 235; (b) M. T. Runnegar, S. M. Kong, Y. Z. Zhong and S. C. Lu, Biochem. Pharmacol., 1995, 49, 219.
6. A. R. Humpage, F. Fontaine, S. Froscio, P. Burcham and I. R. Falconer, J. Toxicol. Environ. Health, Part A, 2005, 68, 739.
7. T. Kiss, A. Vehovszky, L. Hiripi, A. Kovacs and L. Voros, Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol., 2002, 131, 167.
8. I. R. Falconer and A. R. Humpage, Environ. Toxicol., 2001, 16, 192.
9. (a) R. Banker, B. Teltsch, A. Sukenik and S. Carmeli, J. Nat. Prod., 2000, 63, 387; (b) R. Banker, S. Carmeli, M. Werman, B. Teltsch, R. Porat and A. Sukenik, J. Toxicol. Environ. Health, Part A, 2001, 62, 281.
10. R. L. Norris, G. K. Eaglesham, G. Pierens, G. R. Shaw, M. J. Smith, R. K. Chiswell, A. A. Seawright and M. R. Moore, Environ. Toxicol., 1999, 14, 163.
11. (a) C. Xie, M. T. C. Runnegar and B. B. Snider, J. Am. Chem. Soc., 2000, 122, 5017; (b) G. R. Heintzelman, W. Fang, S. P. Keen, G. A. Wallace and S. M. Weinreb, J. Am. Chem. Soc., 2001, 123, 851; (c) G. R. Heintzelman, W. Fang, S. P. Keen, G. A. Wallace and S. M. Weinreb, J. Am. Chem. Soc., 2002, 124, 3939; (d) J. D. White and J. D. Hansen, J. Am. Chem. Soc., 2002, 124, 4950; (e) J. D. White and J. D. Hansen, J. Org. Chem., 2005, 70, 1963; (f) R. E. Looper and R. M. Williams, Angew. Chem., Int. Ed., 2004, 43, 2930; (g) R. E. Looper, M. T. C. Runnegar and R. M. Williams, Tetrahedron, 2006, 62, 4549.
12. (a) I. J. McAlpine and R. W. Armstrong, Tetrahedron Lett., 2000, 41, 1849; (b) J. F. Djung, D. J. Heart and E. R. R. Young, J. Org. Chem., 2000, 65, 5668; (c) H. Henon and Y. Troin, Synlett, 2007, 1446; (d) D. M. Evans and P. J. Murphy, J. Chem. Soc., Chem. Commun., 2011, 44, 3225.
13. T. K. Mihali, R. K. Kellmann, J. Muenchhoff, K. D. Barrow and B. A. Neilan, Appl. Environ. Microbiol., 2008, 74, 716.
14. (a) R. Kellmann, T. Mills and B. A. Neilan, J. Mol. Evol., 2006, 62, 267; (b) J. Muenchhoff, K. S. Siddiqui, A. Poljak, M. J. Raftery, K. D. Barrow and B. A. Neilan, FEBS J., 2010, 277, 3844; (c) J. Muenchhoff, K. S. Siddiqui and B. A. Neilan, FEBS J., 2012, 279, 805; (d) Á. Barón-Sola, M. A. Gutiérrez-Villanueva, F. F. del Campo and S. Sanz-Alférez, MicrobiologyOpen, 2013, 2, 447.
15. (a) M. Dennis, L. M. Hall, P. J. Murphy, A. J. Thornhill, R. Nash, A. L. Winters, M. B. Hursthouse, M. E. Light and P. Horton, Tetrahedron Lett., 2003, 44, 3075; (b) C. Albrecht, S. Barnes, H. Böckemeier, D. Davies, M. Dennis, D. M. Evans, M. D. Fletcher, I. Jones, V. Leitmann, P. J. Murphy, R. Rowles, R. Nash, R. A. Stephenson, P. N. Horton and M. B. Hursthouse, Tetrahedron Lett., 2008, 185.
16. P. G. McDougal, J. G. Rico, Y. Oh and B. D. Condon, J. Org. Chem., 1986, 51, 3388.
17. (a) T. Satoh and S. Suzuki, Tetrahedron Lett., 1969, 10, 4555; (b) M. B. Bernatowicz, Y. Wu and G. R. Matsueda, Tetrahedron Lett., 1993, 34, 3389.
18. T. Hirose, T. Sunazuka, S. Tsuchiya, T. Tanaka, Y. Kojima, R. Mori, M. Iwatsuki and S. Ōmura, Chem.–Eur. J., 2008, 14, 8220.
19. B. B. Snider and Z. Shi, J. Am. Chem. Soc., 1994, 116, 549.
20. Z. D. Aron and L. E. Overman, Chem. Commun., 2004, 253.
21. I. McDonald and L. E. Overman, J. Org. Chem., 1999, 64, 1520.
22. (a) F. Cohen and L. E. Overman, J. Am. Chem. Soc., 2001, 123, 10782; (b) B. B. Snider, J. Chen, A. D. Patil and A. J. Freyer, Tetrahedron Lett., 1996, 37, 6977.
23. B. M. Trost and K. D. Moeller, Heterocycles, 1989, 28, 321.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra03031a

---