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Tetrahedron 69 (2013) 3061e3066

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Tetrahedron

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Synthesis and biological activity of analogues of batzelladine F

Elliot L. Bennett a, Gregory P. Black a, Patrick Browne b, Amnon Hizi c,Mohammed Jaffar b, John P. Leyland a, Claire Martin a, Iris Oz-Gleenberg c,Patrick J. Murphy a,*, Terence D. Roberts a, Andrew J. Thornhill a, Steven A. Vale a

a School of Chemistry, Bangor University, Bangor, Gwynedd LL57 2UW, UKbMorvus Technology Ltd., NBGW, Llanarthne, Carmarthen SA31 1NZ, UKcDepartment of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

a r t i c l e i n f o

Article history:Received 23 October 2012Received in revised form 10 January 2013Accepted 28 January 2013Available online 6 February 2013

Keywords:Batzelladine F analoguesHIV-1 reverse transcriptase (RT)Anti-cancer activityGuanidine alkaloids

* Corresponding author. E-mail address: chs027@b

0040-4020/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.tet.2013.01.083

a b s t r a c t

Several analogues of the left hand portion of batzelladine F were prepared using a rapid and convergentmethodology and evaluated for activity against HIV-1 reverse transcriptase (RT) and several cancer celllines.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

In 1989 Kashman et al. isolated a unique polycyclic guanidinealkaloid ptilomycalin A (Fig. 1), from the sponge Ptilocaulis spicu-lifer.1 This molecule was shown to exhibit anti-tumour, anti-viraland anti-fungal properties and rekindled interest in polycyclicguanidium marine natural products. Similar structures were alsoisolated from a range of sponges and also species of starfish in-cluding the crambescidins2e4 and the batzelladine alkaloids.5e8

The batzelladine alkaloids are a class of these natural productsextracted from the sponge Batzella spiculifer, which was later foundto be taxonomically the same as P. spiculifer. Batzelladines AeE,were isolated in 1995 by Patil et al.5 and were shown to inhibit thebinding of gp-120, the surface protein of human immunodeficiencyvirus (HIV) to its cellular receptor, CD4. Batzelladines FeI wereisolated in 1997 by Patil et al.8 and these were shown to haveactivity in a p56lck-CD4 dissociation assay, which would suggestpotential applications in the treatment of auto-immune diseases.9

In 2007, Hamann et al.10 then isolated batzelladines KeM fromthe Jamaican sponge Monanchora unguifera and these were shownto have biological activity against cancer, protozoa, HIV-1, andAIDS-associated opportunistic infectious pathogens, includingMycobacterium tuberculosis.

angor.ac.uk (P.J. Murphy).

All rights reserved.

Work in our research group, has focused on synthetic protocolstowards ptilomycalin A11 and the batzelladines.12 Previously wehave prepared synthetic analogues of ptilomycalin A and havereported their anti-cancer activity, HIV-1 RT inhibiting ability andanti-malarial activity.13We now report the preparation of a range ofsimplified batzelladine analogues using related synthetic method-ology. Of direct relevance to this work is a report by the Overmangroup, who in 200414 detailed the activities of 28 syntheticanalogues of the batzelladine alkaloids and studied their ability toinhibit envelopemediated cellecell fusion of the HIV-1 virus. In thisstudy, the best inhibitors were found to contain two tricyclicguanidine motifs with an alkyl ester linkage and a methyl sub-stituent on the C1 position, such as 1, which closely resembles thenatural systems and displayed fusion inhibition of >90% at 5 mMconcentrations. Inhibitors containing one tricyclic guanidine motifand a methyl group on the C1 position, such as 2 displayed fusioninhibition of ca. 60% at 5 mM (Fig. 2).

Our strategy was to employ a convergent methodology based onour biomimetic approach12 to the batzelladine alkaloids to prepareanalogues and to investigate the effect of modification of the sidechain on HIV-1 RT activity and activity towards cancer cell lines.

2. Results and discussion

We have previously reported11 the preparation of phosphorane4a from the reaction of valerolactone 3 with methylenetri-

Fig. 1. The batzelladine alkaloids.

Fig. 2. Batzelladine analogues from Overman et al.14

E.L. Bennett et al. / Tetrahedron 69 (2013) 3061e30663062

phenylphosphorane and subsequent protection with tBDMSCl.Treatment of this ylide with excess succinaldehyde 7 gave aldehyde8a in 47% yield over three steps as well as the symmetrical ketone 9in 36% yield. Reaction of 8a with acetyl methylene-triphenylphosphorane gave the bis-enone 10a in 73% yield. Reactionof 8a with guanidine in DMF followed by reduction with sodium

Scheme 1. Synthesis of the batzelladine alkaloid analogues: (i) 2 equiv Ph3PCH3Br, n-BuLi, T�5 �Cert, 12 h. (iii) 6, THF, �60 �Cert, 16 h. (iv) 7, DCM, 16 h. (v) Acetyl methylenetriphenylp0 �Cert, 16 h. (vii) HCl (1 M), MeOH 48 h. (viii) Pd/C, EtOAc/MeOH, H2, 16 h (ix) Py, DMAP, Ac2MeOH, 0 �Cert, 16 h, (iii) NaBF4, DCM, 2 h.

borohydride gave the tricyclic guanidine 11a in 42% yield. Depro-tection of 9awas attempted using a range ofmethods andwas foundto be problematic. The use of TBAFwas problematic due to problemsencountered separating alcohol 12 from the reaction by-products.The use of HF/pyridine complex gave reasonable yields (40e60%),butwas capricious, and yieldswere found to bemuch lower on scaleup. Methanolic HCl gave reproducible yields of 30e40%, howeverclose monitoring of the reaction was required as 12 was found toundergo ring opening on prolonged exposure to these conditions.We finally tried aqueous HCl in propan-2-ol and gratifyinglyobtained excellent reproducible yields of 70e85% (Scheme 1). Wealso attempted to, improve on this final deprotection step by pre-paring the benzylated analogue 11b. Thus the lithium enolate ofacetyl methylenetriphenylphosphorane 6 was reacted with iodide515 to give the ylide 4b. This in turn was treated with excess succi-naldehyde 7 to give the aldehyde 8b in 65% yield over two steps.Reactionof8bwithacetylmethylenetriphenylphosphoranegave thebis-enone 10b in 65% yield. Treatment of bis-enone 10b with gua-nidine in DMF followed by borohydride reduction gave the

HF, 0 �C, 1 h, then rt, 3 h, then 3, �78 �Cert, 12 h (ii) Imidazole, DMAP, tBDMSCl, DMF,hosphorane DCM, rt, 48 h (vi) (i) Guanidine, DMF, 0 �Cert, 5 h, (ii) NaBH4, H2O, MeOH,O or CH3(CH2)nCOCl, 0 �Cert, 24 h. (x) (i) Guanidine, DMF, 0 �Cert, 5 h, (ii) NaBH4, H2O,

E.L. Bennett et al. / Tetrahedron 69 (2013) 3061e3066 3063

guanidine 11b in 31% yield. Unfortunately on attempted hydroge-nation,11bwas found to give only a lowyield of the required alcoholand considerable decomposition.With alcohol12 in hand, a series ofsix acylatedderivatives13e18wereeasily prepared in 38e69%yield,by reaction of 12with either acetic anhydride or the correspondingacid chloride. The guanidine 19 was also prepared from enone 9 in18% yield by reaction under our standard guanylating conditions(Scheme 1).

3. Biological evaluation

Compounds 11a, 11b, 12e19 and the previously prepared20e2212 (Fig. 3) were tested for percentage inhibition16 of the en-zyme reverse transcriptase (RT) of HIV-1 that serves as key enzymein the life cycle of all retroviruses. Such an inhibition can signify thepotential to also inhibit the infectivity of the virus.17 The results ofthese experiments are shown in Table 1.

Fig. 3. Batzelladine analogues assayed.

Table 1HIV-1 RTa inhibition results for compounds 11e22

Compound n¼ 10 mM 40 mM

11a d 15.5 69.611b d 13.4 29.312 d 0.0 14.613 0 4.3 0.314 2 0.0 8.615 4 2.2 12.516 6 16.5 12.117 8 7.7 30.718 10 3.5 36.119 d 0.0 5.820 d 47.9 77.621 d 0.0 0.022 d 5.9 0.6

a The RNA-dependant DNA polymerase activity of HIV-1 RT was determined afterpre-incubation of the enzyme with each compound dissolved in DMSO for 15 min at4 �C prior to assaying the activity as performed previously.16

Table 2Cancer cell growth inhibition (IC50/mM)18 results for compounds 11e22

Compound n¼ A2780a MCF7b MIA PaCa-2c HT-29d PC3e LoVof BEg CaSkih

11a d 2.9 2.7 2.8 3.2 3.2 3.3 ND ND11b d 41.0 33.1 37.7 35.0 42.5 41.0 57.7 35.012 d >100 >100 >100 >100 >100 >100 ND ND13 0 >100 >100 >100 >100 >100 >100 ND ND14 2 >100 >100 >100 >100 >100 >100 ND ND15 4 >100 >100 >100 >100 >100 >100 ND ND16 6 >100 >100 >100 >100 >100 >100 ND ND17 8 42.5 49.9 46.8 75.9 44.6 48.3 ND ND18 10 45.3 42.5 45.4 46.8 41.8 45.1 ND ND19 d 3.8 3.6 3.4 3.4 3.5 4.0 3.8 4.020 d 9.4 9.7 1.1 5.2 6.5 2.1 12.0 9.021 d 52.8 43.4 51.0 69.2 43.4 51.3 >100 52.622 d >100 >100 >100 >100 >100 >100 >100 >100

ND; not determined.a A2780cis ovarian cancer.b MCF7 breast cancer.c MIA PaCa-2, pancreatic cancer.d HT-29 colon cancer.e PC3 prostate cancer.f LoVo colon cancer.g BE colon cancer.h CaSki human cervical cancer.

The figures highlighted in bold represent the highest percent-age inhibition of HIV-1 RT per final concentration. The silyl pro-tected batzelladine analogues 11a and 20 are by far the bestinhibitors at 40 mM concentration, although it is worth noting thatthe ester analogues containing nonyl-17 and undecyl-18 chainsshow appreciably higher inhibition than for the shorter chainmembers of the series 13e16with some correlation between chainlength and activity. The best inhibitor in the lower 10 mM con-centration appeared to be the ester analogue 14 with a heptyl sidechain. However, here there appears to be less correlation betweenpercentage inhibition and alkyl chain length at this concentration.The compounds (11a, 11b, 12e22) were also evaluated against

a panel of human cancer cell lines (Table 2). Compounds 11b,12e18, 21 and 22 showed little toxicity (IC50 values 33.1e100 mM)indicating that there was little effect on the cancer cells. However,compounds 11a, 19 and 20 showed increased toxicity (in the mMrange) with a range of the cell lines (IC50 range: 69.1 mMe1.14 mM).This may in part be due to these compounds having a greaterlipophilic character than the others and hence may enter the cell(via diffusion) more easily to exert their cytotoxic effect. Com-pounds 11a and 19 have emerged as potential leads for furtherevaluation as anti-cancer agents (IC50 range 2.7mMe4.0 mM acrossthe whole panel of cells).

4. Conclusions

A range of batzelladine F analogues were prepared in a rapidconvergent manner and evaluated for HIV-1 reverse transcriptaseand anti-cancer activity. Analogues with highly lipophilic silyl

containing side chains 11a and 19 gave the best percentage in-hibition16 of HIV-1 reverse transcriptase and some correlation wasalso observed with analogues containing varying length ester de-rived side chains. A range of anti-cancer activity has been observedand further novel analogues of compound 19 would providea greater understanding of the underlying mechanism of activity(including structureeactivity relationship) of these derivatives aspotential anti-cancer therapeutics. In addition the simplicity and

E.L. Bennett et al. / Tetrahedron 69 (2013) 3061e30663064

bio-activity of these analogues suggests that further alterations canbe made to enhance inhibition properties.

5. Experimental

5.1. General condition

Column chromatography was carried out on silica gel (particlesize 40e63 mm) and TLCswere conducted on precoated Kieselgel 60F254 (Art. 5554; Merck) with the eluent specified in each case. Allnon-aqueous reactions were conducted in oven-dried apparatusunder a static atmosphere of argon. Ether, THF and dichloro-methane were dried on a Pure Solv MD-3 solvent purificationsystem. Dry methanol and DMF was purchased from Aldrich.Chemical shifts are reported in d values relative to chloroform (7.27/77.0 ppm) as an internal standard. Proton and carbon were recor-ded in CDCl3 on a Bruker AC250/400/500 spectrometer unlessotherwise stated. Mass spectra data were obtained at the EPSRCMass Spectrometry Service Centre at the University of Wales,Swansea. Infrared spectra were recorded as thin films (oils) ona Bruker Tensor 27 series instrument.

5.1.1. 6-(tert-Butyl-dimethyl-silanyloxy)-1-(triphenyl-l5-phosphany-lidene)-hexan-2-one 4a.11e

5.1.1.1. Prepared using a modified literature method.11e n-Butyllithium (22.4 mL, 56.0 mmol, 2.5 M) was added to a stirred, cooled(0 �C) suspension of methyltriphenyl-phosphonium bromide(20.0 g, 55.9 mmol) in dry THF (180 mL) and warmed to rt over 4 h.The solution was then cooled (�78 �C) and freshly distilledd-valerolactone 3 (2.8 g, 28.0 mmol) was added dropwise and theresulting yellow solution warmed to rt overnight. After cooling(�5 �C), imidazole (2.40 g, 35.0 mmol) and DMAP (catalytic 0.1 g)were added and the mixture stirred for 30 min. tBDMSCl (4.70 g,31.0 mmol) dissolved in DMF (150 mL) was then slowly added andthe solution stirred for 5 days. The THF in the reaction was evapo-rated under reduced pressure, water (1 L) was added and themixture extracted with ethyl acetate (5�100 mL). After drying(MgSO4) and evaporation, crude 4a (20.8 g) was obtained as a vis-cous oil and was used in the next stage without further purification.Data were in accordance with the literature.11e dH (500 MHz, CDCl3)0.05 (6H, s, (CH3)2Si), 0.89 (9H, s, tBu), 1.59e1.64 (2H, m, CH2),1.68e1.74 (2H,m, CH2), 2.33 (2H, t, J 7.2 Hz, CH2), 3.64 (2H, t, J 6.7 Hz,CH2), 3.71 (1H, d, JPeH, 16.6 Hz, CH), 7.42e7.68 (15H, m, 3� Ph).

5.1.2. 10-(tert-Butyl-dimethyl-silanyloxy)-6-oxo-dec-4-enal 8a and1,16-bis-(tert-butyl-dimethyl-silanyloxy)-hexadeca-6,10-diene-5,12-dione 9.11e

5.1.2.1. Prepared using a modified literature method..11e Phos-phorane 4a from the previous stage (20.8 g) was dissolved indichloromethane (100 mL) and added dropwise to a stirred solu-tion of succinaldehyde 7 (9.30 g, 108.0 mmol) in dichloromethane(100 mL) and the mixture stirred at rt for 3 days. The reaction wasthen washed with water (5�100 mL), dried (MgSO4), and evapo-rated. Column chromatography under gradient elution (0e40%Et2O/petroleum ether) gave pure enal 8a (3.96 g, 47% from 3) asa clear oil, an impure fraction of 8a (1.62 g, ca. 80e90% pure) andbis-enone 9 (2.60 g, 36% from 3) also as a clear oil. Data were inaccordance with the literature.11e

5.1.3. 13-(tert-Butyl-dimethyl-silanyloxy)-trideca-3,7-diene-2,9-dione 10a. Acetylmethylenetriphenylphosphorane (5.67 g,17.8 mmol) was added to a cooled (0 �C) solution of 8a (3.54 g,11.86mmol) in dichloromethane (45 mL). After stirring at rt for twodays silica gel (12 g) was added and the solvent evaporated. The freeflowing powder was loaded onto a silica gel column and gradient

eluted (20e50% Et2O/petroleum ether) to give bis-enone 10a(2.93 g, 73%) as a clear oil. Rf 0.16 (40% Et2O/petroleum ether) dH(500 MHz, CDCl3) 0.01 (6H, s, (CH3)2Si), 0.83 (9H, s, tBu), 1.46e1.51(2H, m, CH2), 1.58e1.66 (2H, m, CH2), 2.19 (3H, s, CH3), 2.34e2.38(4H, m, 2� CH2), 2.51 (2H, t, J 7.5, CH2), 3.56 (2H, t, J 6.5, CH2), 6.05(1H, d, J 15.6 Hz, CH), 6.08 (1H, d, J 15.6 Hz, CH), 6.69e6.77 (2H, m,2� CH) dC (62.5 MHz, CDCl3) �5.6, 18.0, 20.3, 25.6, 26.7, 30.3, 30.4,31.9, 39.7, 62.5, 130.6, 131.6, 144.2, 145.5, 197.7, 199.8 nmax 2930,1674, 1628, 1472, 1361 cm�1 m/z: (CI) [MþNH4

þ] (C19H38NO3Siþ)expected 356.2615, found 356.2614.

5.1.4. (2aR,4S,7R,8aS)-4-(4-((tert-Butyldimethylsilyl)oxy)butyl)-7-methyl-1,2,2a,3,4,5,6,7,8,8a-decahydro-2a1,5,6-triazaacenaphthylen-2a1-ium chloride 11a. Guanidine (256 mg, 4.34 mmol) dissolved inDMF (4 mL) was added to a stirred, cooled (0 �C) solution of bis-enone 10a (0.98 g, 2.90 mmol) in DMF (6 mL). After 1 h the solu-tionwas slowly warmed to rt for 4 h, then cooled (0 �C) and dilutedwith water (1.5 mL) and methanol (5 mL). Sodium borohydride(0.60 g, 15.7 mmol) was then added in portions over 3 min and theresulting solution warmed to rt overnight. Dichloromethane(75mL) andwater (75mL) were added and HCl (aq,1M) was addeddropwise until effervescence ceased. The organic layer was sepa-rated and the aqueous phase extracted with dichloromethane(3�20 mL). The combined organic extracts were washed withwater (3�500 mL) and brine (3�50 mL), dried (MgSO4) and evap-orated. Column chromatography (gradient elution from 0 to 5%MeOH/CHCl3) gave guanidine 11a (480 mg, 41%) as a yellow oil. Rf0.23 (10% MeOH/CHCl3) dH (500 MHz, CDCl3) 0.01 (6H, s, 2� CH3),0.85 (9H, s, 3� CH3), 1.13e1.25 (2H, m, CH2), 1.33 (3H, d, J 6.5 Hz,CH3), 1.40e1.77 (8H, m), 2.12e2.23 (4H, m), 3.26e3.64 (6H, m), 8.56(1H, s, NH), 8.68 (1H, s, NH) dC (125 MHz, CDCl3) �5.3, 18.2, 20.4,21.6, 25.9, 30.3, 30.3, 32.3, 33.9, 34.5, 36.0, 45.6, 50.1, 55.7, 55.8, 62.6,149.9 nmax (CH2Cl2) 3271, 2931, 2858, 1628 cm�1 m/z: (ESI) [M]þ

(C20H40N3OSiþ) expected 366.2935, found 366.2934.

5.1.5. (2aR,4S,7R,8aS)-4-(4-Hydroxybutyl)-7-methyl-1,2,2a,3,4,5,6,7,8,8a-decahydro-2a1,5,6-triazaacenaphthylen-2a1-ium chloride 12. Silylether 11a (395 mg, 0.982 mmol) was dissolved in propan-2-ol (3 mL),following which HCl (aq, 1 M, 3 mL) was added dropwise. After 16 hTLC indicated the completion of the reaction, which was diluted withdichloromethane (50 mL) and brine (50 mL), the organic layer wasseparated and the aqueous layer extracted with dichloromethane(50 mL). The aqueous phase was then further extracted withdichloromethane/propan-2-ol (9:1, 3�20 mL) and the combined ex-tracts dried (MgSO4), filtered and evaporated to give an oil, whichwaspurified by column chromatography (gradient elution with 0e16%MeOH/CHCl3) gave alcohol 12 (238 mg, 0.828 mmol, 84%) as a paleyellow oil. Rf 0.12 (15% MeOH/CHCl3) dH (500 MHz, CDCl3) 1.15e1.25(3H, m, CH2, OH), 1.34 (3H, d, J 6.0 Hz, CH3), 1.50e1.70 (8H, m),2.10e2.30 (4H, m), 3.35e3.70 (6H, m), 8.28 (1H, s, NH), 8.45 (1H, s,NH) dC (125 MHz, CDCl3) 20.4, 21.1, 30.2, 30.3, 31.8, 33.9, 34.0, 35.9,45.7, 50.0, 55.8, 55.9, 61.1,149.9 nmax (CDCl3) 3270, 2933,1626 cm�1m/z: (ESI) [M]þ (C14H26N3Oþ) expected 252.2070, found 252.2069.

5.1.6. (E)-10-(Benzyloxy)-6-oxodec-4-enal 8b. Acetylmethylene-tri-phenylphosphorane (7.85 g, 24.7 mmol) was dissolved in dry THF(100 mL) with stirring and then cooled (�78 �C: if precipitation, oc-curs the reaction can be rewarmed to ca.�60 �C and the n-BuLi addedat this temperature). A solution of n-BuLi (9.50mL, 2.5 M, 23.8 mmol)was then added dropwise over 5 min and the reaction warmed tobetween�50 �C and�60 �C for 1 h to generate the lithium enolate 6.The reactionwas then cooled (�78 �C) and a solution of (3-iodoprop-1-oxymethyl)benzene 515 (5.68 g, 20.6 mmol) in THF (10 mL) wasadded over 10 min. After warming slowly to rt overnight, water(500 mL) was added and the mixture extracted with ethyl acetate(3�100mL). The combined organic extractswerewashedwithwater

E.L. Bennett et al. / Tetrahedron 69 (2013) 3061e3066 3065

(2�50 mL), dried (MgSO4) and the solvent removed to give the crudephosphorane 4b. This was dissolved in dichloromethane (50 mL) anda solution of succinaldehyde 7 (2.63 g, 30.6mmol) in dry DCM (15mL)was added. After 24 h at rt the reaction mixture was washed withwater (4 � 250 mL), dried (MgSO4) and evaporated. Silica gel chro-matography (gradient elution 10e50% EtOAc/petroleum ether) gave8b (3.64 g, 64%) as a clear unstable oil, whichwas used immediately inthe next step. Rf 0.22 (25% EtOAc/petroleum ether) dH (250 MHz,CDCl3) 1.54e1.68 (4H, m, 2� CH2), 2.44e2.48 (2H,m, CH2), 2.49 (2H, t,J 7.0 Hz, CH2), 2.57 (2H, tt, J 7.0, 1.0 Hz, CH2), 3.42 (2H, t, J 6.0 Hz, CH2),4.43 (2H, s, CH2), 6.05 (1H, dt, J 15.5, 2.0 Hz, CH), 6.73 (1H, dt, J 15.5,7.0 Hz, 1H), 7.18e7.30 (5H, m, Ar), 9.73 (1H, t, J 1.0 Hz, CHO) dC(125 MHz, CDCl3) 20.8, 24.5, 29.1, 39.9, 41.8, 69.9, 72.8, 127.4, 127.6,128.3, 130.9, 138.5, 144.1, 200.0, 200.3 nmax 3030, 2939, 2860, 1724,1696,1672,1630,1454,1103.3 cm�1m/z: (CI) [MþH]þ m/z: (C17H23O3)expected 275.1642, found 275.1643.

5.1.7. (3E,7E)-13-(Benzyloxy)trideca-3,7-diene-2,9-dione 10b. Acety-lmethylenetriphenylphosphorane (6.33 g, 19.9 mmol) dissolved indichloromethane (100 mL) was added over 5 min to a cooled (0 �C)solution of aldehyde 8b (3.46 g, 12.6 mmol) in dichloromethane(100 mL). After warming to rt overnight the reaction was evaporatedand purified using silica gel column chromatography (25% EtOAc/petroleum ether) to give 10b (2.73 g, 69%) as a clear oil. Rf 0.23 (25%EtOAc/petroleum ether) dH (500 MHz, CDCl3) 1.61e1.75 (4H, m, 2�CH2), 2.24 (3H, s, CH3), 2.36e2.43 (4H,m, 2� CH2), 2.56 (2H, t, J 6.0 Hz,CH2), 3.49 (2H, t, J 7.0 Hz, CH2), 4.50 (2H, s, CH2), 6.10 (1H, br d, J15.9 Hz, CH), 6.12 (1H, br d, J 15.2 Hz, CH), 6.73e6.81 (2H, m, 2� CH),7.25e7.37 (5H, m, Ph) dC (500 MHz, CDCl3) 20.9, 27.1, 29.2, 30.6, 30.7,40.0, 70.0, 72.9, 127.5, 127.6, 128.3, 130.9, 131.9, 138.5, 144.5, 145.7,198.2, 200.1 nmax 3031, 2937, 2860, 1697, 1674, 1628, 1454, 1362, 1255,1104, 981 cm�1 m/z: (CI) [MþNH4]þ (C20H30NO3

þ) expected 332.2220,found 332.2217.

5.1.8. (2aR,4S,7R,8aS)-4-(4-(Benzyloxy)butyl)-7-methyl-1,2,2a,3,4,5,6,7,8,8a-decahydro-2a1,5,6-triazaacenaphthylen-2a1-ium chloride11b. Guanidine (101 mg, 1.71 mmol) in dry DMF (3 mL) was addeddropwise to a cooled (0 �C) solution of 10b (860 mg, 2.74 mmol) indry DMF (5mL). Themixture waswarmed slowly to rt and stirred for4 h. Methanol (5 mL) and water (2 mL) were added and the mixturecooled (0 �C). Sodium borohydride (500 mg, 13.2 mmol) was thenadded in small portions over 5 min and the mixture stirred to rtovernight. Dichloromethane (25 mL) was added and the reactioncautiously acidified with HCl (aq 2 M) until effervescence ceased.After separation of the organic phase the reaction was furtherextracted with dichloromethane (4 � 50 mL) and the combined ex-tracts were washed with water (4 � 250 mL) and brine (200 mL),then dried (MgSO4). Column chromatography on silica gel (gradientelution 0e15% MeOH/CHCl3) to gave 11b (320 mg, 31%) as a paleyellow gum. Rf 0.16 (5% MeOH/CHCl3) dH (500 MHz, CDCl3) 1.00e1.25(2H, m, CH2), 1.32 (3H, d, J 6.5 Hz, CH3) 1.33e1.80 (8H, m), 2.09e2.21(m, 4H), 3.31e3.66 (6H, m), 4.49 (2H, s, CH2), 6.72 (1H, br s, NH), 6.81(1H, br s, NH), 7.24e7.38 (5H, s, Ph) dC (125 MHz, CDCl3) 20.1, 21.6,29.1, 29.9, 30.0, 33.3, 34.0, 35.4, 45.9, 50.1, 55.7, 55.8, 69.7, 72.7, 127.3,127.5,128.1,138.4,149.1 nmax 3300e3600 (br), 2939, 2866,1625,1496,1217 cm�1 m/z (ESI) [M]þ (C21H32N3Oþ) expected 342.2540, found342.2541.

5.1.9. (2aR,4S,7R,8aS)-4-(4-Hydroxybutyl)-7-methyl-1,2,2a,3,4,5,6,7,8,8a-decahydro-2a1,5,6-triazaacenaphthylen-2a1-ium chloride12. Benzyl ether 11b (320 mg, 0.88 mmol) was dissolved in a 1:1mixture of ethyl acetate andmethanol (10mL) and 10% Pd/C (150mg)was added. The mixture was stirred under a H2 atmosphere for 4 hthen filtered through Celite�, which was washed with further ethylacetate (3 � 15 mL) and 10% methanol in ethyl acetate (15 mL). Afterevaporation of the filtrate chromatography of the residue on silica gel

(gradient elution, 0e20% MeOH/CHCl3) gave 12 (50.0 mg, 20%) asa pale yellow oil with data as reported above.

5.1.10. Preparation of esters 13e18

5.1.10.1. General method. Alcohol 12 (ca. 0.05 mmol) and DMAP(5 mg) were dissolved in pyridine (2 mL) and cooled (0 �C). Aceticanhydride or the acid chloride (3 equiv) was then added and thereaction stirred at rt for 24 h. Chloroform (50 mL) was added andthe reaction mixture was poured into HCl (aq, 0.5 M, 250 mL), theorganic layer separated, dried (MgSO4) and evaporated. Silica gelcolumn chromatography (gradient elution 0e10% MeOH/CHCl3)gave the desired compounds as oils.

5.1.11. (2aR,4S,7R,8aS)-4-(4-Acetoxybutyl)-7-methyl-1,2,2a,3,4,5,6,7,8,8a-decahydro-2a1,5,6-triazaacenaphthylen-2a1-ium chloride13. Alcohol 12 (16.8mg, 0.0496mmol) gave 13 (11.3mg, 69%) Rf 0.04(10% MeOH/CHCl3) dH (500 MHz, CDCl3) 1.17e1.27 (2H, m), 1.34 (3H,d, J 6.2 Hz, CH3), 1.45e1.77 (8H, m), 2.05 (3H, s, CH3), 2.13e2.25 (4H,m), 3.34e3.42 (1H, m, CH), 3.48e3.55 (1H, m, CH), 3.60e3.68 (2H,m), 4.02e4.11 (2H, m, CH2), 8.66 (1H, br s, NH), 8.68 (1H, br s, NH) dC(125 MHz) 20.6, 21.1, 21.9, 28.4, 30.4, 30.5, 34.2, 34.5, 36.1, 45.8, 50.0,55.8, 55.9, 64.0, 150.1, 171.3 m/z: (CI) [M]þ (C16H28N3O2

þ) expected294.2176, found 294.2176.

5.1.12. (2aR,4S,7R,8aS)-4-(4-(Butyryloxy)butyl)-7-methyl-1,2,2a,3,4,5,6,7,8,8a-decahydro-2a1,5,6-triazaacenaphthylen-2a1-ium chloride14. Alcohol 12 (12.1 mg, 0.0357 mmol) gave 14 (6.5 mg, 51%) Rf 0.05(10% MeOH/CHCl3) dH (500 MHz, CDCl3) 0.95 (3H, t, J 6.6 Hz, CH3),1.18e1.30 (2H, m), 1.36 (3H, d, J 6.3 Hz, CH3), 1.45e1.80 (10H, m),2.13e2.25 (4H, m), 2.29 (2H, t, J 7.5 Hz, CH2), 3.34e3.31 (1H, m, CH),3.48e3.56 (1H, m, CH), 3.60e3.68 (2H, m), 4.05e4.12 (2H, m, CH2),8.73 (1H, br s, NH), 8.75 (1H, br s, NH) dC (125 MHz) 13.7, 18.5, 21.9,28.4, 29.7, 30.4, 30.5, 34.1, 34.4, 36.1, 36.2, 45.7, 50.0, 55.7, 55.9, 63.7,150.1, 173.8 m/z: (CI) [M]þ (C18H32N3O2

þ) expected 322.2489, found322.2492.

5.1.13. (2aR,4S,7R,8aS)-4-(4-(Hexanoyloxy)butyl)-7-methyl-1,2,2a,3,4,5,6,7,8,8a-decahydro-2a1,5,6-triazaacenaphthylen-2a1-ium chloride15. Alcohol 12 (17.2 mg, 0.0507mmol) gave 15 (10.1 mg, 51%) Rf 0.05(10% MeOH/CHCl3) dH (500 MHz, CDCl3) 0.90 (3H, t, J 6.6 Hz, CH3),1.18e1.34 (6H, m), 1.35 (3H, d, J 6.3 Hz, CH3), 1.45e1.80 (10H, m),2.13e2.24 (4H, m), 2.30 (2H, t, J 7.5 Hz, CH2), 3.34e3.41 (1H, m, CH),3.49e3.56 (1H, m, CH), 3.61e3.68 (2H, m), 4.05e4.12 (2H, m, CH2),8.76 (2H, br s, 2� NH) dC (125 MHz) 13.9, 20.5, 21.8, 22.3, 24.7, 28.4,30.3, 30.4, 31.3, 34.1, 34.3, 34.4, 36.1, 45.7, 50.0, 55.7, 55.9, 63.8, 150.1,174.0 m/z: (CI) [M]þ (C20H36N3O2

þ) expected 350.2802, found350.2803.

5.1.14. (2aR,4S,7R,8aS)-7-Methyl-4-(4-(octanoyloxy)butyl)-1,2,2a,3,4,5,6,7,8,8a-decahydro-2a1,5,6-triazaacenaphthylen-2a1-ium chloride16. Alcohol 12 (21.9mg, 0.0646mmol) gave 16 (15.6mg, 58%) Rf 0.05(10% MeOH/CHCl3) dH (500 MHz, CDCl3) 0.88 (3H, t, J 6.6 Hz, CH3),1.17e1.31 (10H, m), 1.35 (3H, d, J 6.3 Hz, CH3), 1.45e1.78 (10H, m),2.13e2.25 (4H, m), 2.29 (2H, t, J 7.5 Hz, CH2), 3.35e3.40 (1H, m, CH),3.48e3.55 (1H, m, CH), 3.61e3.68 (2H, m), 4.11e4.30 (2H, m, CH2),8.67 (1H, br s, NH), 8.69 (1H, br s, NH) dC (125 MHz) 14.0, 20.5, 21.8,22.6, 25.0, 28.4, 28.9, 29.1, 30.3, 30.4, 31.6, 34.1, 34.3, 34.4, 36.0, 45.7,50.0, 55.7, 55.9, 63.7, 150.0, 173.9 m/z (CI) [M]þ (C22H40N3O2

þ) ex-pected 378.3115, found 378.3118.

5.1.15. (2aR,4S,7R,8aS)-4-(4-(Decanoyloxy)butyl)-7-methyl-1,2,2a,3,4,5,6,7,8,8a-decahydro-2a1,5,6-triazaacenaphthylen-2a1-ium chloride17. Alcohol 12 (17.9 mg, 0.0528 mmol) gave 17 (8.9 mg, 38%) Rf 0.05(10% MeOH/CHCl3) dH (500 MHz, CDCl3) 0.88 (3H, t, J 6.6 Hz, CH3),1.18e1.33 (14H, m), 1.35 (3H, d, J 6.3 Hz, CH3), 1.44e1.79 (10H, m),2.14e2.25 (4H, m), 2.29 (2H, t, J 7.5 Hz, CH2), 3.34e3.41 (1H, m, CH),

E.L. Bennett et al. / Tetrahedron 69 (2013) 3061e30663066

3.49e3.56 (1H, m, CH), 3.61e3.68 (2H, m), 4.04e4.12 (2H, m, CH2),8.77 (2H, br s, 2� NH) dC (125 MHz) 14.1, 20.5, 21.8, 22.6, 25.0, 28.4,29.1, 29.2, 29.3, 29.4, 29.7, 30.3, 30.4, 31.9, 34.1, 34.3, 36.1, 45.7, 50.0,55.7, 55.9, 63.8, 150.1, 174.0 m/z (CI) [M]þ (C24H44N3O2

þ) expected406.3428, found 406.3433.

5.1.16. (2aR,4S,7R,8aS)-4-(4-(Dodecanoyloxy)butyl)-7-methyl-1,2,2a,3,4,5,6,7,8,8a-decahydro-2a1,5,6-triazaacenaphthylen-2a1-ium chlo-ride18. Alcohol 12 (15.4 mg, 0.0454mmol) gave 18 (13.0 mg, 61%) Rf0.05 (10% MeOH/CHCl3) dH (500 MHz, CDCl3) 0.88 (3H, t, J 6.5 Hz,CH3), 1.17e1.32 (18H, m), 1.34 (3H, d, J 6.2 Hz, CH3), 1.44e1.80 (10H,m), 2.13e2.25 (4H, m), 2.29 (2H, t, J 7.4 Hz, CH2), 3.33e3.41 (1H, m,CH), 3.47e3.56 (1H, m, CH), 3.60e3.69 (2H, m), 4.03e4.11 (2H, m,CH2), 8.72 (2H, br s, 2 NH) dC (125 MHz) 14.1, 20.5, 21.8, 22.6, 25.0,28.4, 29.1, 29.2, 29.3, 29.5, 29.6, 29.7, 30.3, 30.4, 31.9, 34.0, 34.3, 34.4,36.0, 45.7, 50.0, 55.7, 55.9, 63.8, 150.0, 174.0 m/z (CI) [M]þ

(C26H48N3O2þ) expected 434.3741, found 434.3740.

5.1.17. (2aR,4S,7R,8aS)-4,7-Bis(4-((tert-butyldimethylsilyl)oxy)butyl)-1,2,2a,3,4,5,6,7,8,8a-decahydro-2a1,5,6-triazaacenaphthylen-2a1-iumtetrafluoroborate 19. Guanidine (0.30 g, 5.08 mmol) in DMF (3.0 mL)was added dropwise to a cooled (0 �C) solution of enone 9 (2.60 g,5.09 mmol) in DMF (10 mL). The reaction was warmed slowly to rtand stirred for 5 h. After cooling (0 �C) water (4 mL) and methanol(8 mL) were added followed by the portion-wise addition of sodiumborohydride (1.16 g, 30.5 mmol) over 15 min. After 16 h dichloro-methane (100 mL) was added and the solution was acidified using1 M HCl (aq) until effervescence ceased. The organic phase wasseparated and the aqueous phase extracted with further dichloro-methane (2 � 50 mL). The combined extracts were washed withwater (3 � 250 mL), brine (50 mL) then dried (MgSO4). After evap-oration the crude productwas dissolved in dichloromethane (20mL)and stirred with sodium tetrafluoroborate solution (20 mL, satd aq)for 2 h. After separation, drying (MgSO4) and evaporation, chroma-tography on silica gel (gradient elution 0e10%MeOH/CHCl3) gave 19(0.57 g, 18%) as an oil. Rf 0.11 (5% MeOH/CHCl3) dH (500 MHz, CDCl3)0.05 (12H, s, 2� (CH3)2Si), 0.89 (18H, s, 2� tBu), 1.17e1.32 (2H, m, 2�CH), 1.40e1.79 (16H, m), 2.19e2.30 (2H, m, 2� CH), 3.30e3.46 (2H,m, 2� CH), 3.62 (4H, t, J 6.2 Hz, 2� CH2), 3.63e3.70 (2H, m, 2� CH),6.70 (2H, br s, 2� NH) dC (125 MHz) �5.3, 18.3, 21.2, 26.0, 30.3, 32.3,33.5, 34.1, 50.5, 56.1, 62.7,149.3 nmax 3367, 2930, 2858,1626 cm�1m/z(ESI) 538.4 (100% Mþ) �ve mode 86.8 (100%, BF4�) m/z (ESI) [M]þ

(C29H60N3O2Si2þ) expected 538.4219, found 538.4213.

5.1.18. SRB cytotoxicity inhibition assay. Growth inhibition wasmeasured by the sulpho-rhodamine B (SRB) method as describedpreviously.18 In essence, cells were seeded into 96-well microplatesin Eagle’s minimum essential medium supplemented with non-essential amino acids, glutamine (2.0 mM) and 10% (v/v) foetalbovine serum and incubated overnight. The test compounds weredissolved in DMSO, serially diluted in culture medium and addedto the cells. After 6 days exposure the cells were stained with SRBand absorbance measured at 570 nm. The cytotoxicity of eachcompound was expressed as that concentration producing 50%

inhibition of cell growth (IC50) compared with cells incubated withmedium alone.

Acknowledgements

Thanks are given to the KESS scheme for funding (C.M. andE.L.B.) and to the EPSRC Mass Spectrometry center at Swansea.

Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.tet.2013.01.083.

References and notes

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