Synthesis and biological activity of analogues of batzelladine F

Article abstract of DOI:10.1016/j.tet.2013.01.083

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

Full text of DOI:10.1016/j.tet.2013.01.083

Tetrahedron 69 (2013) 3061e3066
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
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,
,
a
a
a
Patrick J. Murphy a , Terence D. Roberts , Andrew J. Thornhill , Steven A. Vale
*
a School of Chemistry, Bangor University, Bangor, Gwynedd LL57 2UW, UK
b Morvus Technology Ltd., NBGW, Llanarthne, Carmarthen SA31 1NZ, UK
c Department 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
a b s t r a c t
Article history:
Several analogues of the left hand portion of batzelladine F were prepared using a rapid and convergent
Received 23 October 2012
Received in revised form 10 January 2013
Accepted 28 January 2013
Available online 6 February 2013
methodology and evaluated for activity against HIV-1 reverse transcriptase (RT) and several cancer cell
lines.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Batzelladine F analogues
HIV-1 reverse transcriptase (RT)
Anti-cancer activity
Guanidine alkaloids
1. Introduction
Work in our research group, has focused on synthetic protocols
towards ptilomycalin A11 and the batzelladines.12 Previously we
In 1989 Kashman et al. isolated a unique polycyclic guanidine
alkaloid ptilomycalin A (Fig. 1), from the sponge Ptilocaulis spicu-
lifer.1 This molecule was shown to exhibit anti-tumour, anti-viral
and anti-fungal properties and rekindled interest in polycyclic
guanidium marine natural products. Similar structures were also
isolated 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 products
extracted from the sponge Batzella spiculifer, which was later found
to be taxonomically the same as P. spiculifer. Batzelladines AeE,
were isolated in 1995 by Patil et al.5 and were shown to inhibit the
binding of gp-120, the surface protein of human immunodeficiency
virus (HIV) to its cellular receptor, CD4. Batzelladines FeI were
isolated in 1997 by Patil et al.8 and these were shown to have
activity in a p56lck-CD4 dissociation assay, which would suggest
potential applications in the treatment of auto-immune diseases.9
In 2007, Hamann et al.10 then isolated batzelladines KeM from
the Jamaican sponge Monanchora unguifera and these were shown
to have biological activity against cancer, protozoa, HIV-1, and
AIDS-associated opportunistic infectious pathogens, including
Mycobacterium tuberculosis.
have prepared synthetic analogues of ptilomycalin A and have
reported their anti-cancer activity, HIV-1 RT inhibiting ability and
anti-malarial activity.13 We now report the preparation of a range of
simplified batzelladine analogues using related synthetic method-
ology. Of direct relevance to this work is a report by the Overman
group, who in 200414 detailed the activities of 28 synthetic
analogues of the batzelladine alkaloids and studied their ability to
inhibit envelope mediated cellecell fusion of the HIV-1 virus. In this
study, the best inhibitors were found to contain two tricyclic
guanidine motifs with an alkyl ester linkage and a methyl sub-
stituent on the C1 position, such as 1, which closely resembles the
natural systems and displayed fusion inhibition of >90% at 5 mM
concentrations. Inhibitors containing one tricyclic guanidine motif
and a methyl group on the C1 position, such as 2 displayed fusion
inhibition of ca. 60% at 5 mM (Fig. 2).
Our strategy was to employ a convergent methodology based on
our biomimetic approach12 to the batzelladine alkaloids to prepare
analogues and to investigate the effect of modification of the side
chain on HIV-1 RT activity and activity towards cancer cell lines.
2. Results and discussion
We have previously reported11 the preparation of phosphorane
* Corresponding author. E-mail address: chs027@bangor.ac.uk (P.J. Murphy).
4a from the reaction of valerolactone 3 with methylenetri-
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.01.083

3062
E.L. Bennett et al. / Tetrahedron 69 (2013) 3061e3066
Fig. 1. The batzelladine alkaloids.
borohydride gave the tricyclic guanidine 11a in 42% yield. Depro-
tection of 9a was attempted using a range of methods and was found
to be problematic. The use of TBAF was problematic due to problems
encountered separating alcohol 12 from the reaction by-products.
The use of HF/pyridine complex gave reasonable yields (40e60%),
but was capricious, and yields were found to be much lower on scale
up. Methanolic HCl gave reproducible yields of 30e40%, however
close monitoring of the reaction was required as 12 was found to
undergo ring opening on prolonged exposure to these conditions.
We finally tried aqueous HCl in propan-2-ol and gratifyingly
obtained excellent reproducible yields of 70e85% (Scheme 1). We
also attempted to, improve on this final deprotection step by pre-
paring the benzylated analogue 11b. Thus the lithium enolate of
acetyl methylenetriphenylphosphorane 6 was reacted with iodide
515 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.
Reactionof8bwithacetyl methylenetriphenylphosphoranegavethe
bis-enone 10b in 65% yield. Treatment of bis-enone 10b with gua-
nidine in DMF followed by borohydride reduction gave the
Fig. 2. Batzelladine analogues from Overman et al.14
phenylphosphorane and subsequent protection with tBDMSCl.
Treatment of this ylide with excess succinaldehyde 7 gave aldehyde
8a in 47% yield over three steps as well as the symmetrical ketone 9
in 36% yield. Reaction of 8a with acetyl methylene-
triphenylphosphorane gave the bis-enone 10a in 73% yield. Reaction
of 8a with guanidine in DMF followed by reduction with sodium
Scheme 1. Synthesis of the batzelladine alkaloid analogues: (i) 2 equiv Ph3PCH3Br, n-BuLi, THF, 0 ꢀC, 1 h, then rt, 3 h, then 3, ꢁ78 ꢀCert, 12 h (ii) Imidazole, DMAP, tBDMSCl, DMF,
ꢁ5 ꢀCert, 12 h. (iii) 6, THF, ꢁ60 ꢀCert, 16 h. (iv) 7, DCM, 16 h. (v) Acetyl methylenetriphenylphosphorane DCM, rt, 48 h (vi) (i) Guanidine, DMF, 0 ꢀCert, 5 h, (ii) NaBH4, H2O, MeOH,
0 ꢀCert, 16 h. (vii) HCl (1 M), MeOH 48 h. (viii) Pd/C, EtOAc/MeOH, H2, 16 h (ix) Py, DMAP, Ac2O or CH3(CH2)nCOCl, 0 ꢀCert, 24 h. (x) (i) Guanidine, DMF, 0 ꢀCert, 5 h, (ii) NaBH4, H2O,
MeOH, 0 ꢀCert, 16 h, (iii) NaBF4, DCM, 2 h.

E.L. Bennett et al. / Tetrahedron 69 (2013) 3061e3066
3063
guanidine 11b in 31% yield. Unfortunately on attempted hydroge-
nation,11b was found to give only a low yield of the required alcohol
and considerable decomposition. With alcohol 12 in hand, a series of
six acylated derivatives 13e18 were easily prepared in 38e69% yield,
by reaction of 12 with either acetic anhydride or the corresponding
acid chloride. The guanidine 19 was also prepared from enone 9 in
18% yield by reaction under our standard guanylating conditions
(Scheme 1).
a panel of human cancer cell lines (Table 2). Compounds 11b,
12e18, 21 and 22 showed little toxicity (IC50 values 33.1e100 M)
indicating that there was little effect on the cancer cells. However,
compounds 11a, 19 and 20 showed increased toxicity (in the
range) with a range of the cell lines (IC50 range: 69.1 Me1.14
This may in part be due to these compounds having a greater
lipophilic 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 further
m
m
mM).
M
m
evaluation as anti-cancer agents (IC50 range 2.7
the whole panel of cells).
mMe4.0 mM across
3. Biological evaluation
Compounds 11a, 11b, 12e19 and the previously prepared
20e2212 (Fig. 3) were tested for percentage inhibition16 of the en-
zyme reverse transcriptase (RT) of HIV-1 that serves as key enzyme
in the life cycle of all retroviruses. Such an inhibition can signify the
potential to also inhibit the infectivity of the virus.17 The results of
these experiments are shown in Table 1.
4. Conclusions
A range of batzelladine F analogues were prepared in a rapid
convergent manner and evaluated for HIV-1 reverse transcriptase
and anti-cancer activity. Analogues with highly lipophilic silyl
Fig. 3. Batzelladine analogues assayed.
Table 1
Table 2
HIV-1 RTa inhibition results for compounds 11e22
Cancer cell growth inhibition (IC50/m
M)18 results for compounds 11e22
Compound
n¼
10
mM
40
m
M
Compound n¼ A2780a MCF7b MIA PaCa-2c HT-29d PC3e LoVof BEg CaSkih
11a
11b
12
13
14
15
16
17
18
19
20
21
22
d
d
d
0
2
4
6
8
10
d
d
d
d
15.5
13.4
0.0
4.3
0.0
2.2
16.5
7.7
3.5
0.0
69.6
29.3
14.6
0.3
11a
11b
12
13
14
15
16
17
18
19
20
21
22
d
d
d
0
2
4
2.9
41.0
2.7
33.1
2.8
37.7
3.2
35.0
3.2
3.3
ND ND
42.5 41.0 57.7 35.0
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 >100 >100
>100 >100 >100 ND ND
>100 >100 >100 ND ND
>100 >100 >100 ND ND
>100 >100 >100 ND ND
>100 >100 >100 ND ND
75.9
46.8
3.4
8.6
12.5
12.1
30.7
36.1
5.8
77.6
0.0
0.6
6
8
42.5
49.9
42.5
3.6
9.7
43.4
46.8
45.4
3.4
1.1
51.0
44.6 48.3 ND ND
41.8 45.1 ND ND
3.5
6.5
10 45.3
d
d
d
d
3.8
9.4
52.8
4.0
2.1
3.8
4.0
47.9
0.0
5.9
5.2
69.2
12.0 9.0
43.4 51.3 >100 52.6
>100 >100 >100
>100 >100 >100 >100 >100
a
The RNA-dependant DNA polymerase activity of HIV-1 RT was determined after
pre-incubation of the enzyme with each compound dissolved in DMSO for 15 min at
4 ꢀC prior to assaying the activity as performed previously.16
ND; not determined.
a
A2780cis ovarian cancer.
MCF7 breast cancer.
MIA PaCa-2, pancreatic cancer.
HT-29 colon cancer.
PC3 prostate cancer.
LoVo colon cancer.
BE colon cancer.
b
c
d
e
f
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 best
g
h
CaSki human cervical cancer.
inhibitors at 40 mM concentration, although it is worth noting that
containing side chains 11a and 19 gave the best percentage in-
hibition16 of HIV-1 reverse transcriptase and some correlation was
also observed with analogues containing varying length ester de-
rived side chains. A range of anti-cancer activity has been observed
and further novel analogues of compound 19 would provide
a greater understanding of the underlying mechanism of activity
(including structureeactivity relationship) of these derivatives as
potential anti-cancer therapeutics. In addition the simplicity and
the ester analogues containing nonyl-17 and undecyl-18 chains
show appreciably higher inhibition than for the shorter chain
members of the series 13e16 with some correlation between chain
length and activity. The best inhibitor in the lower 10
mM con-
centration appeared to be the ester analogue 14 with a heptyl side
chain. However, here there appears to be less correlation between
percentage inhibition and alkyl chain length at this concentration.
The compounds (11a, 11b, 12e22) were also evaluated against

3064
E.L. Bennett et al. / Tetrahedron 69 (2013) 3061e3066
bio-activity of these analogues suggests that further alterations can
be made to enhance inhibition properties.
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, 2ꢂ 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,
2ꢂ 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 cmꢁ1 m/z: (CI) [MþNH4þ] (C19H38NO3Siþ)
expected 356.2615, found 356.2614.
5. Experimental
5.1. General condition
Column chromatography was carried out on silica gel (particle
size 40e63 mm) and TLCs were conducted on precoated Kieselgel 60
F254 (Art. 5554; Merck) with the eluent specified in each case. All
non-aqueous reactions were conducted in oven-dried apparatus
under a static atmosphere of argon. Ether, THF and dichloro-
methane were dried on a Pure Solv MD-3 solvent purification
system. Dry methanol and DMF was purchased from Aldrich.
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 in
DMF (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-
tion was slowly warmed to rt for 4 h, then cooled (0 ꢀC) and diluted
with 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 the
resulting solution warmed to rt overnight. Dichloromethane
(75 mL) and water (75 mL) were added and HCl (aq,1 M) was added
dropwise until effervescence ceased. The organic layer was sepa-
rated and the aqueous phase extracted with dichloromethane
(3ꢂ20 mL). The combined organic extracts were washed with
water (3ꢂ500 mL) and brine (3ꢂ50 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. Rf
0.23 (10% MeOH/CHCl3) dH (500 MHz, CDCl3) 0.01 (6H, s, 2ꢂ CH3),
0.85 (9H, s, 3ꢂ 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 cmꢁ1 m/z: (ESI) [M]þ
(C20H40N3OSiþ) expected 366.2935, found 366.2934.
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 unless
otherwise stated. Mass spectra data were obtained at the EPSRC
Mass Spectrometry Service Centre at the University of Wales,
Swansea. Infrared spectra were recorded as thin films (oils) on
a Bruker Tensor 27 series instrument.
5.1.1. 6-(tert-Butyl-dimethyl-silanyloxy)-1-(triphenyl-l
5-phosphany-
lidene)-hexan-2-one 4a.11e
5.1.1.1. Prepared using a modified literature method.11e n-Butyl
lithium (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 distilled
d-valerolactone 3 (2.8 g, 28.0 mmol) was added dropwise and the
resulting yellow solution warmed to rt overnight. After cooling
(ꢁ5 ꢀC), imidazole (2.40 g, 35.0 mmol) and DMAP (catalytic 0.1 g)
t
were added and the mixture stirred for 30 min. BDMSCl (4.70 g,
31.0 mmol) dissolved in DMF (150 mL) was then slowly added and
the solution stirred for 5 days. The THF in the reaction was evapo-
rated under reduced pressure, water (1 L) was added and the
mixture extracted with ethyl acetate (5ꢂ100 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, Bu), 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, 3ꢂ Ph).
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. Silyl
ether 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 h
TLC indicated the completion of the reaction, which was diluted with
dichloromethane (50 mL) and brine (50 mL), the organic layer was
separated and the aqueous layer extracted with dichloromethane
(50 mL). The aqueous phase was then further extracted with
dichloromethane/propan-2-ol (9:1, 3ꢂ20 mL) and the combined ex-
tracts dried (MgSO4), filtered and evaporated to give an oil, which was
purified by column chromatography (gradient elution with 0e16%
MeOH/CHCl3) gave alcohol 12 (238 mg, 0.828 mmol, 84%) as a pale
yellow 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 cmꢁ1 m/
z: (ESI) [M]þ (C14H26N3Oþ) expected 252.2070, found 252.2069.
t
5.1.2. 10-(tert-Butyl-dimethyl-silanyloxy)-6-oxo-dec-4-enal 8a and
1,16-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 in
dichloromethane (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 was
then washed with water (5ꢂ100 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) as
a clear oil, an impure fraction of 8a (1.62 g, ca. 80e90% pure) and
bis-enone 9 (2.60 g, 36% from 3) also as a clear oil. Data were in
accordance with the literature.11e
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 added
at this temperature). A solution of n-BuLi (9.50 mL, 2.5 M, 23.8 mmol)
was then added dropwise over 5 min and the reaction warmed to
between ꢁ50 ꢀC and ꢁ60 ꢀC for 1 h to generate the lithium enolate 6.
The reaction was then cooled (ꢁ78 ꢀC) and a solution of (3-iodoprop-
1-oxymethyl)benzene 515 (5.68 g, 20.6 mmol) in THF (10 mL) was
added over 10 min. After warming slowly to rt overnight, water
(500 mL) was added and the mixture extracted with ethyl acetate
(3 ꢂ 100 mL). The combined organic extracts were washed with water
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,
11.86 mmol) in dichloromethane (45 mL). After stirring at rt for two
days silica gel (12 g) was added and the solvent evaporated. The free
flowing powder was loaded onto a silica gel column and gradient

E.L. Bennett et al. / Tetrahedron 69 (2013) 3061e3066
3065
(2ꢂ50 mL), dried (MgSO4) and the solvent removed to give the crude
phosphorane 4b. This was dissolved in dichloromethane (50 mL) and
a solution of succinaldehyde 7 (2.63 g, 30.6 mmol) in dry DCM (15 mL)
was added. After 24 h at rt the reaction mixture was washed with
water (4 ꢂ 250 mL), dried (MgSO4) and evaporated. Silica gel chro-
matography (gradient elution 10e50% EtOAc/petroleum ether) gave
8b (3.64 g, 64%) as a clear unstable oil, which was used immediately in
the next step. Rf 0.22 (25% EtOAc/petroleum ether) dH (250 MHz,
CDCl3) 1.54e1.68 (4H, m, 2ꢂ 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 cmꢁ1 m/z: (CI) [MþH]þ m/z: (C17H23O3)
expected 275.1642, found 275.1643.
(gradient elution, 0e20% MeOH/CHCl3) gave 12 (50.0 mg, 20%) as
a 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). Acetic
anhydride or the acid chloride (3 equiv) was then added and the
reaction stirred at rt for 24 h. Chloroform (50 mL) was added and
the reaction mixture was poured into HCl (aq, 0.5 M, 250 mL), the
organic layer separated, dried (MgSO4) and evaporated. Silica gel
column 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 chloride
13. Alcohol 12 (16.8 mg, 0.0496 mmol) gave 13 (11.3 mg, 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 (C16H28N3O2þ) expected
294.2176, found 294.2176.
5.1.7. (3E,7E)-13-(Benzyloxy)trideca-3,7-diene-2,9-dione 10b. Acety-
lmethylenetriphenylphosphorane (6.33 g, 19.9 mmol) dissolved in
dichloromethane (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 evaporated
and 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, 2ꢂ
CH2), 2.24 (3H, s, CH3), 2.36e2.43 (4H, m, 2ꢂ 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, J
15.9 Hz, CH), 6.12 (1H, br d, J 15.2 Hz, CH), 6.73e6.81 (2H, m, 2ꢂ 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 cmꢁ1 m/z: (CI) [MþNH4]þ (C20H30NOþ3 ) expected 332.2220,
found 332.2217.
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 chloride
14. 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 (C18H32N3Oþ2 ) expected 322.2489, found
322.2492.
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 chloride
11b. Guanidine (101 mg, 1.71 mmol) in dry DMF (3 mL) was added
dropwise to a cooled (0 ꢀC) solution of 10b (860 mg, 2.74 mmol) in
dry DMF (5 mL). The mixture was warmed slowly to rt and stirred for
4 h. Methanol (5 mL) and water (2 mL) were added and the mixture
cooled (0 ꢀC). Sodium borohydride (500 mg, 13.2 mmol) was then
added in small portions over 5 min and the mixture stirred to rt
overnight. Dichloromethane (25 mL) was added and the reaction
cautiously acidified with HCl (aq 2 M) until effervescence ceased.
After separation of the organic phase the reaction was further
extracted with dichloromethane (4 ꢂ 50 mL) and the combined ex-
tracts were washed with water (4 ꢂ 250 mL) and brine (200 mL),
then dried (MgSO4). Column chromatography on silica gel (gradient
elution 0e15% MeOH/CHCl3) to gave 11b (320 mg, 31%) as a pale
yellow 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 cmꢁ1 m/z (ESI) [M]þ (C21H32N3Oþ) expected 342.2540, found
342.2541.
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 chloride
15. Alcohol 12 (17.2 mg, 0.0507 mmol) 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, 2ꢂ 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 (C20H36N3Oþ2 ) expected 350.2802, found
350.2803.
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 chloride
16. Alcohol 12 (21.9 mg, 0.0646 mmol) gave 16 (15.6 mg, 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 (C22H40N3O2þ) ex-
pected 378.3115, found 378.3118.
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 chloride
12. Benzyl ether 11b (320 mg, 0.88 mmol) was dissolved in a 1:1
mixture of ethyl acetate and methanol (10 mL) and 10% Pd/C (150 mg)
was added. The mixture was stirred under a H2 atmosphere for 4 h
then filtered through CeliteÒ, which was washed with further ethyl
acetate (3 ꢂ 15 mL) and 10% methanol in ethyl acetate (15 mL). After
evaporation of the filtrate chromatography of the residue on silica gel
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 chloride
17. 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),

3066
E.L. Bennett et al. / Tetrahedron 69 (2013) 3061e3066
3.49e3.56 (1H, m, CH), 3.61e3.68 (2H, m), 4.04e4.12 (2H, m, CH2),
8.77 (2H, br s, 2ꢂ 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 (C24H44N3O2þ) expected
406.3428, found 406.3433.
inhibition of cell growth (IC50) compared with cells incubated with
medium alone.
Acknowledgements
Thanks are given to the KESS scheme for funding (C.M. and
E.L.B.) and to the EPSRC Mass Spectrometry center at Swansea.
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-
ride 18. Alcohol 12 (15.4 mg, 0.0454 mmol) gave 18 (13.0 mg, 61%) Rf
0.05 (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
(C26H48N3Oþ2 ) expected 434.3741, found 434.3740.
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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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-ium
tetrafluoroborate 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 rt
and stirred for 5 h. After cooling (0 ꢀC) water (4 mL) and methanol
(8 mL) were added followed by the portion-wise addition of sodium
borohydride (1.16 g, 30.5 mmol) over 15 min. After 16 h dichloro-
methane (100 mL) was added and the solution was acidified using
1 M HCl (aq) until effervescence ceased. The organic phase was
separated and the aqueous phase extracted with further dichloro-
methane (2 ꢂ 50 mL). The combined extracts were washed with
water (3 ꢂ 250 mL), brine (50 mL) then dried (MgSO4). After evap-
oration the crude product was dissolved in dichloromethane (20 mL)
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)
5. Patil, A. D.; Kumar, N. V.; Kokke, W. C.; Bean, M. F.; De Brosse, C.; Mai, S.;
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t
0.05 (12H, s, 2ꢂ (CH3)2Si), 0.89 (18H, s, 2ꢂ Bu), 1.17e1.32 (2H, m, 2ꢂ
CH), 1.40e1.79 (16H, m), 2.19e2.30 (2H, m, 2ꢂ CH), 3.30e3.46 (2H,
m, 2ꢂ CH), 3.62 (4H, t, J 6.2 Hz, 2ꢂ CH2), 3.63e3.70 (2H, m, 2ꢂ CH),
6.70 (2H, br s, 2ꢂ 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 cmꢁ1 m/z
(ESI) 538.4 (100% Mþ) ꢁve mode 86.8 (100%, BFꢁ4 ) m/z (ESI) [M]þ
(C29H60N3O2Siþ2 ) expected 538.4219, found 538.4213.
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Murphy, P. J.; Diquet, B. J. Antibiot. 2006, 59, 583e590.
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previously.18 In essence, cells were seeded into 96-well microplates
in Eagle’s minimum essential medium supplemented with non-
essential amino acids, glutamine (2.0 mM) and 10% (v/v) foetal
bovine serum and incubated overnight. The test compounds were
dissolved in DMSO, serially diluted in culture medium and added
to the cells. After 6 days exposure the cells were stained with SRB
and absorbance measured at 570 nm. The cytotoxicity of each
compound was expressed as that concentration producing 50%
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18. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.;
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1107e1112.