Isolation and synthesis of N-acyladenine and adenosine alkaloids from a southern Australian marine sponge, Phoriospongia sp.

Article abstract of DOI:10.1016/j.tetlet.2014.08.116

Chemical fractionation of the southern Australian marine sponge Phoriospongia sp. (CMB-03107) yielded phorioadenine A (1) as a nematocidal agent and the first reported example of a 6-N-acyladenine natural product. The structure of 1 was confirmed by spectroscopic analysis and the chemical synthesis of racemic (1a) and enantiomeric (1b) analogues. HPLC-ESIMS analysis of the crude sponge extract with comparisons to the synthetic 6-N-acyladenosine 2a provided evidence that the biosynthetically related adenosine, phorioadenosine A (2), was present as a trace co-metabolite. The rare starfish metabolite asterubine (3) was also isolated as a co-metabolite, and its structure confirmed by spectroscopic analysis and chemical synthesis. Biological investigations confirmed that natural products 1-3 and synthetic analogues 1a-e and 2a were not cytotoxic to multiple mammalian cancer cell lines, or Gram-positive or -negative bacteria. Nematocidal activity (inhibition of larval development of Haemonchus contortus) detected in the Phoriospongia sp. extract was attributed to 1 (LD₉₉31 μg/mL), with preliminary structure-activity relationship investigations confirming the importance of the N-acyl side chain.

Full text of DOI:10.1016/j.tetlet.2014.08.116

Tetrahedron Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Isolation and synthesis of N-acyladenine and adenosine alkaloids
from a southern Australian marine sponge, Phoriospongia sp.
Michelle Farrugia a, Nicholas Trotter a, Soumini Vijayasarathy a, Angela A. Salim a, Zeinab G. Khalil a,
Ernest Lacey b, Robert J. Capon a,
⇑
a Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia
b Microbial Screening Technology Pty Ltd, Building C, 28-54 Percival Rd., Smithfield, NSW 2164, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Chemical fractionation of the southern Australian marine sponge Phoriospongia sp. (CMB-03107) yielded
phorioadenine A (1) as a nematocidal agent and the first reported example of a 6-N-acyladenine natural
product. The structure of 1 was confirmed by spectroscopic analysis and the chemical synthesis of
racemic (1a) and enantiomeric (1b) analogues. HPLC–ESIMS analysis of the crude sponge extract with
comparisons to the synthetic 6-N-acyladenosine 2a provided evidence that the biosynthetically related
adenosine, phorioadenosine A (2), was present as a trace co-metabolite. The rare starfish metabolite
asterubine (3) was also isolated as a co-metabolite, and its structure confirmed by spectroscopic analysis
and chemical synthesis. Biological investigations confirmed that natural products 1–3 and synthetic
analogues 1a–e and 2a were not cytotoxic to multiple mammalian cancer cell lines, or Gram-positive
or -negative bacteria. Nematocidal activity (inhibition of larval development of Haemonchus contortus)
Received 27 May 2014
Revised 19 August 2014
Accepted 28 August 2014
Available online xxxx
Keywords:
Phoriospongia sp.
6-N-Acyladenine
6-N-Acyladenosine
Haemonchus contortus
Nematocidal agent
detected in the Phoriospongia sp. extract was attributed to 1 (LD99 31 lg/mL), with preliminary
structure–activity relationship investigations confirming the importance of the N-acyl side chain.
Ó 2014 Elsevier Ltd. All rights reserved.
The ruminant livestock industry is heavily dependent on the use
of anthelmintic agents (drenches) to combat gastrointestinal
nematode parasites, with the cost of treating infected livestock
(i.e., sheep, goats, cattle), combined with parasite-related reduc-
tions in productivity, representing a significant economic burden.1
For much of the last three decades, commercial anthelmintics have
been largely limited to the benzimidazoles, levamisoles and
avermectins, with widespread multidrug resistance to all three
classes demanding the use of double and even triple drenches.2 In
response to the ever growing threat of anthelmintic resistance, in
2009 Novartis introduced ZolvixÒ (monepantel)3 as a new synthetic
class of amino-acetonitrile anthelmintics, while in 2010 Pfizer
launched StartectÒ (derquantel–abamectin), a formulation of the
new fungal anthelmintics 2-deoxyparaherquamide (derquantel)
and avermectins (abamectin).4 While ZolvixÒ and StartectÒ were
welcome contributions, resistance to these new drugs is inevitable.
Given the ongoing nature of the resistance challenge, there remains
an urgent and compelling case to continue research aimed at
discovering new classes of anthelmintics, in particular those
with modes-of-action not compromised by existing resistance
pathways. In noting the contribution made by microbial natural
product anthelmintics, with the avermectins reigning supreme as
the gold standard anthelmintic for many decades, we hypothesised
that marine biodiversity may offer an alternative source of natural
product anthelmintics.
To test this hypothesis we screened a library of >2500 southern
Australian and Antarctic marine invertebrates and algae in a
Haemonchus contortus larval development assay.5 This approach
has proved very successful in the past, facilitating our discovery
of many new classes of nematocides spanning such biosynthetic
classes as lipids (thiocyanatins,6 notheians7), alkaloids (esmodil,8
echinobetaines,9 bromoindoles10), peptides (phoriospongins11
)
and polyketides (amphilactams,12 geodin A Mg salt,13 onna-
mides14). In a continuation of these studies, this report describes
our investigation of the nematocidal extract obtained from a south-
ern Australian marine sponge, Phoriospongia sp. (CMB-03107), col-
lected during scientific SCUBA operations off St Leonards Pier, Port
Phillip Bay, Victoria. On subjecting the crude EtOH extract to bioas-
say guided solvent partitions and triturations, followed by C18 SPE
and HPLC chromatography, we recovered phorioadenine A (1) as
the first example of a 6-N-acyladenine natural product, and a new
nematocidal agent, together with the rare marine metabolite
asterubine (3) (Fig. 1), first reported in 1935 from a North Sea
starfish15 and patented in 2013 for the treatment of metabolic
⇑
Corresponding author. Tel.: +61 7 3346 2979; fax: +61 7 3346 2090.
E-mail address: r.capon@uq.edu.au (R.J. Capon).
http://dx.doi.org/10.1016/j.tetlet.2014.08.116
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Farrugia, M.; et al. Tetrahedron Lett. (2014), http://dx.doi.org/10.1016/j.tetlet.2014.08.116

2
M. Farrugia et al. / Tetrahedron Letters xxx (2014) xxx–xxx
O
N
2''
NH
6
O
N
N
N
8
2'
4'
NH
2
N
O
6
H
1
N
3
2
N
H2N
HO
8
SO3H
1'
2
N
H
NMe2
N
OH OH
phorioadenosine A (2)
Figure 2. Synthetic phorioadenine analogues 1a,b.
asterubine (3)
phorioadenine A (1)
Figure 1. Metabolites from Phoriospongia sp. (CMB-03107).
syndrome.16 The structure elucidation of 1 and 3 were supported by
detailed spectroscopic analysis and chemical synthesis, as detailed
below. During the course of these investigations we prepared the
6-N-acylated adenosine 2a, and used HPLC–ESIMS with single ion
extraction to detect and propose phorioadenosine A (2) as a trace
co-metabolite.
Scheme 1. Synthesis of achiral phorioadenine analogues 1c–e. Conditions: K2CO3,
MeCN, reflux, overnight.
HRESI(+)MS analysis of 1 returned a pseudo-molecular ion
(M+Na)+ consistent with a molecular formula (C10H13N5O,
D
mmu
+0.4) requiring seven double bond equivalents (DBE). The UV–vis
spectra for
together with 1H NMR (DMSO-d6) resonances
1
three were inactive in the nematode larval development assay.
While limited, these structure–activity relationship considerations
suggest that nematocidal activity is correlated with and is
dependent on the nature and chirality of the N-acyl side chain.
Having identified the natural product 1, we speculated that the
corresponding adenosine analogue may exist as a co-metabolite.
To test this hypothesis we synthesised the 6-N-acyladenosine 2a
(Scheme 2), and used it as an authentic standard to probe the
HPLC–ESIMS data of the Phoriospongia extract, successfully employ-
ing single ion extraction technology to detect phorioadenosine A (2)
as a very minor co-metabolite (Fig. 3).
diagnostic for two deshielded heteroaromatic protons (dH 8.41, s;
8.63, s), was suggestive of an adeninyl moiety (C5H4N5). Further
analysis of the 1D and 2D NMR data (Table 1) revealed resonances
consistent with a 2-methylbutyryl amide residue (C5H9ON) (dH
1.25, d, 20-Me; 0.99, dd, 40; 1.55 and 1.81, ddd, H2-30; 2.68, ddq,
H-20), accounting for the remaining DBE and satisfying the molec-
ular formula. Given the very low yields of 1 from the Phoriospongia
sp., the structure assignment and absolute configuration were con-
firmed by chemical synthesis. Treatment of adenine with ( )-2-
methylbutyryl chloride, followed by standard work-up and
stepwise gradient C18 SPE chromatography yielded the racemic
product rac-phorioadenine A (1a), which was spectroscopically
indistinguishable from 1. Significantly, key 2D 1H–15N HMBC
NMR (methanol-d4) correlations from H-20 (dH 2.68) to 6-N (dN
ꢀ239), from H-2 (dH 8.63) to N1 (dN ꢀ133) and N3 (dN ꢀ130),
and from H-8 (dH 8.41) to N7/N9 (dN ꢀ150/224) and N3 (dN
ꢀ130), confirmed 6-N-acyl substitution. Repeating the synthesis
HRESI(+)MS analysis of 3 returned a pseudo-molecular ion
(MꢀH) consistent with a molecular formula (C5H13N3O3S,
Dmmu
ꢀ0.8) suggestive of the known marine metabolite asterubine, first
reported in 1935 from two North Sea starfish of the genus Asterias,15
and synthesised in the same year.17 As a detailed spectroscopic
characterisation of 3 had not been reported in the scientific litera-
ture, to confirm our re-isolation we synthesised and independently
characterised this metabolite (Table 2). Optimisation of the conden-
sation of taurine with dimethylcyanamide delivered a simple
one-step (66%) synthesis of asterubine that was spectroscopically
identical to that isolated from Phoriospongia sp. (CMB-03107).
In addition to evaluation in a nematode larval development
assay, we determined that the crude extract, and the natural/syn-
thetic compounds 1a–e and 2a, were not cytotoxic towards the
Gram-negative bacteria Escherichia coli (ATCC 25922) and
Pseudomonas aeruginosa (ATCC 27853), or the Gram-positive bacte-
ria Staphylococcus aureus (ATCC 25923), Staphylococcus epidermidis
(ATCC 12228) and Bacillus subtilis (ATCC 6633), or human colon
(SW620) and lung (NCIH460) cancer cell lines. While of modest
potency, the selective cytotoxicity (i.e., nematocidal activity)
using (S)-2-methylbutyryl chloride yielded the enantiomer ent-
21
phorioadenine A (1b) ([
a]
+10.3), confirming the structure and
D
22
absolute configuration of phorioadenine A (1) ([
a
]
D
ꢀ14.1).
The nematocidal activity against H. contortus detected in the
crude Phoriospongia sp. (CMB-03107) extract was attributed to
phorioadenine A (1) (LD99 31
that of the commercial anthelmintics levamisole and closantel
(LD99 5–10 g/mL). Of note, the synthetic analogues rac-phorioad-
lg/mL), being slightly weaker than
l
enine A (1a) and ent-phorioadenine A (1b) (Fig. 2) were inactive
in the nematode larval development assay. To further explore the
importance of the structure–activity relationship (SAR) significance
of the N-acyl side chain, we synthesised and tested the three achiral
phorioadenine homologues 1c–e (Scheme 1), determining that all
Table 1
NMR (400 MHz, CD3OD) data of phorioadenine A (1)a
Position
dC
dH, m (J in Hz)
2
8
153.0
146.1
179b
43.6
8.63, s
8.41, s
—
2.68, ddq (6.8, 6.8, 6.8)
a 1.81, ddd (14, 7.4, 6.8)
b 1.55, ddd (14, 7.4, 6.8)
0.99, dd (7.4, 7.4)
1.25, d (6.8)
10
20
30
28.2
40
20-Me
12.0
17.6
Scheme 2. Synthesis of 6-N-acyladenosine (2a). Reagents and conditions: (a)
TBDMSCl, DMF, N2, rt, 21 h, 67%. (b) Pyridine, 2-methylbutanoyl chloride, CH2Cl2,
N2, rt, 21 h, 74%. (c) NH4F, anhyd MeOH, reflux, N2, 18 h, 40%.
a
Assignments supported by HMQC data.
Detected from the HMBC spectrum.
b
Please cite this article in press as: Farrugia, M.; et al. Tetrahedron Lett. (2014), http://dx.doi.org/10.1016/j.tetlet.2014.08.116

M. Farrugia et al. / Tetrahedron Letters xxx (2014) xxx–xxx
3
column, with a 2 mL/min gradient from 100% H2O to 100% MeOH
over 30 min) to yield phorioadenine A (1) (0.9 mg, 0.33%) and
asterubine (3) (1.1 mg, 0.41%). [Note–% yields are estimates in
the crude EtOH extract].
Phorioadenine A (1): a colourless amorphous solid. [
0.035, MeOH); UV (MeOH) kmax (log
22
a]
ꢀ14.1 (c
D
e
) 209 (3.96), 227 (sh), 256
(sh), 273 (3.63), 281 (3.63), 2.91 (3.48) nm; NMR (MeOH-d4) data
see Table 1; HRESI(+)MS m/z 242.1016 [M+Na]+ (calculated for C10-
H13N5ONa, 242.1012,
Dmmu +0.4).
Phorioadenosine A (2): detected by HPLC–ESIMS (SIE) as a trace
metabolite. HRESI(+)MS m/z 352.1617 [M+H]+ (calculated for
C15H22N5O5, 352.1615, Dmmu +0.2).
Asterubine (3): a colourless solid. NMR (400 MHz, D2O) data see
Table 2; ESI(+)MS m/z 234 [M+K]+, 218 [M+Na]+, 196 [M+H]+;
HRESI(ꢀ)MS m/z 194.0597 [MꢀH]ꢀ (calculated C5H12N3O3S,
194.0605,
D
mmu ꢀ0.8).
Acknowledgments
We acknowledge the assistance of L. Goudie in identifying
sponge material, and D. Howse for database support. This research
was partially funded by the Institute for Molecular Bioscience, The
University of Queensland, and the Australian Research Council
(LP120100088).
Supplementary data
Supplementary data (general experimental, sponge collection
and taxonomy, bioassays, synthetic procedures and 1H and 13C
NMR spectra for 1b) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.
08.116.
Figure 3. (a) HPLC chromatogram (UV 298 nm) of Phoriospongia extract and HPLC-
ESI(+)MS chromatograms of Phoriospongia extract with single ion monitoring on
m/z (M+H) ions for the natural products (b) 1 and (d) 2, compared to ESI(+)MS
chromatograms for the synthetic authentic standards (c) 1a and (e) 2a.
References and notes
Table 2
1. (a) Barger, I. A.; Cox, H. W. Vet. Parasitol. 1984, 15, 169–175; (b) Besier, R. B.;
Love, S. C. J. Aust. J. Exp. Agric. 2003, 43, 1383–1391.
NMR (400 MHz, D2O) data of asterubine (3)a
2. Sangster, N. C.; Gill, J. Parasitol. Today 1999, 15, 141–146.
3. (a) Kaminsky, R.; Ducray, P.; Jung, M.; Clover, R.; Rufener, L.; Bouvier, J.; Weber,
S. S.; Wenger, A.; Wieland-Berghausen, S.; Goebel, T.; Gauvry, N.; Pautrat, F.;
Skripsky, T.; Froelich, O.; Komoin-Oka, C.; Westlund, B.; Sluder, A.; Maeser, P.
Nature 2008, 452, 176–180; (b) Kaminsky, R.; Gauvry, N.; Schorderet Weber, S.;
Skripsky, T.; Bouvier, J.; Wenger, A.; Schroeder, F.; Desaules, Y.; Hotz, R.;
Goebel, T.; Hosking, B. C.; Pautrat, F.; Wieland-Berghausen, S.; Ducray, P.
Parasitol. Res. 2008, 103, 931-939.; (c) Kaminsky, R.; Mosimann, D.; Sager, H.;
Stein, P.; Hosking, B. Int. J. Parasitol 2009, 39, 443–446.
Position
dC
dH, m (J in Hz)
1
2
3
50.2
38.8
157b
38.5
3.00, t (6.4)
3.49, t (6.4)
—
NMe2
2.85, s
a
Assignments supported by HMQC data.
Detected from the HMBC spectrum.
b
4. Little, P. R.; Hodge, A.; Watson, T. G.; Seed, J. A.; Maeder, S. J. N. Z. Vet. J. 2010, 58,
121–129.
exhibited by the 6-N-acyladenine 1 is noteworthy, and may be
indicative of a mode of action that deserves closer investigation.
In conclusion, we have isolated the first example of a 6-N-acy-
ladenine (1) from a marine sponge, and synthesised its enantiomer
to confirm its structure and absolute configuration. Compound 1
5. Gill, J. H.; Redwin, J. M.; Van Wyk, J. A.; Lacey, E. Int. J. Parasitol. 1995, 25, 463–
470.
6. (a) Capon, R. J.; Skene, C.; Liu, E. H.-T.; Lacey, E.; Gill, J. H.; Heiland, K.; Friedel, T.
J. Org. Chem. 2001, 66, 7765–7769; (b) Capon, R. J.; Skene, C.; Liu, E. H.-T.; Lacey,
E.; Gill, J. H.; Heiland, K.; Friedel, T. J. Nat. Prod. 2004, 67, 1277–1282.
7. Capon, R. J.; Barrow, R. A.; Rochfort, S.; Jobling, M.; Skene, C. Tetrahedron 1998,
54, 2227–2242.
8. Capon, R. J.; Skene, C.; Liu, E. H.; Lacey, E.; Gill, J. H.; Heiland, K.; Friedel, T. Nat.
Prod. Res. 2004, 18, 305–309.
9. (a) Capon, R. J.; Vuong, D.; Lacey, E.; Gill, J. H. J. Nat. Prod. 2005, 68, 179–182; (b)
Capon, R. J.; Vuong, D.; McNally, M.; Peterle, T.; Trotter, N.; Lacey, E.; Gill, J. H.
Org. Biomol. Chem. 2005, 3, 118–122.
inhibits larval development of H. contortus (LD99 31 lg/mL), and
was not cytotoxic to multiple mammalian cancer cell lines,
Gram-positive and Gram-negative bacteria. SAR investigations
revealed that the chirality at position 20 is essential for its anthel-
minthic activity.
10. Capon, R. J.; Skene, C.; Vuong, D.; Lacey, E.; Gill, J. H.; Heiland, K.; Friedel, T. J.
Nat. Prod. 2002, 65, 368–370.
11. Capon, R. J.; Ford, J.; Lacey, E.; Gill, J. H.; Heiland, K.; Friedel, T. J. Nat. Prod. 2002,
65, 358–363.
Fractionation and characterisation
12. Ovenden, S. P. B.; Capon, R. J.; Lacey, E.; Gill, J. H.; Friedel, T.; Wadsworth, D. J.
Org. Chem. 1999, 64, 1140–1144.
13. Capon, R. J.; Skene, C.; Lacey, E.; Gill, J. H.; Wadsworth, D.; Friedel, T. J. Nat. Prod.
1999, 62, 1256–1259.
14. Vuong, D.; Capon, R. J.; Lacey, E.; Gill, J. H.; Heiland, K.; Friedel, T. J. Nat. Prod.
2001, 64, 640–642.
15. Ackermann, D. Z. Physiol. Chem. 1935, 232, 206–212.
16. Jang, H. U.; Na, M. G.; Jung, Y. T.; Kim, C. H.; Hwang, S. R. KR 2013127607,
Korea, Nov 25, 2013.
A portion of the crude EtOH extract of Phoriospongia sp.
(CMB-03107) was concentrated in vacuo and the residue
(270 mg) triturated with CH2Cl2, leaving an insoluble residue that
was partitioned between n-BuOH and H2O. All three fractions were
concentrated in vacuo, and based on bioassay data the n-BuOH
fraction was selected for chromatographic fractionation (C18 SPE
with a 20% stepwise gradient elution from 60% H2O/MeOH to
17. (a) Ackermann, D. Z. Physiol. Chem. 1935, 234, 208–211; (b) Ackermann, D.;
Muller, E. Z. Physiol. Chem. 1935, 235, 233–234.
100% MeOH, followed by a Phenomenex C18, 250 ꢁ 9.6 mm, 5
lm
Please cite this article in press as: Farrugia, M.; et al. Tetrahedron Lett. (2014), http://dx.doi.org/10.1016/j.tetlet.2014.08.116