J . Nat. Prod. 1997, 60, 727-728
727
Isola tion a n d Ch a r a cter iza tion of 1,3-Dim eth ylisogu a n in e fr om th e Ber m u d ia n
Sp on ge Am ph im ed on vir id is
Scott S. Mitchell,† Andy B. Whitehill,† Henry G. Trapido-Rosenthal,‡ and Chris M. Ireland*,†
Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, and Bermuda Biological Station,
Ferry Reach GE-01, Bermuda
Received December 16, 1996X
The new compound 1,3-dimethylisoguanine has been isolated and characterized from the
Bermudian sponge Amphimedon viridis. Chemical conversion of the natural product to
theophylline and 2D NMR methods were used to determine the position of the methyl groups
on the purine ring. Analysis of the mass spectral fragmentation pattern allowed assignment
of the purine ring as isoguanine.
Marine sponges have proven to be an exceptionally
rich source of modified nucleosides. The isolation of
spongouridine and spongothymidine from Cryptotethia
crypta1 and subsequent development of antiviral ana-
logues demonstrated the potential medicinal importance
of these compounds. More recently, several groups have
reported the isolation of methylated guanine base
analogues from sponges, including 7,9-dimethylguanine
(herbipoline),2 1,7,9-trimethylguanine,3 1,3,7-trimeth-
ylguanine,4 and 3,7-dimethylisoguanine.5 No physi-
ological role for the myriad of methylated guanine
analogues isolated from sponges is apparent.
The crude MeOH extract of Amphimedon viridis
(Duchassaing and Michelotti, 1864, formerly known as
Haliclona viridis) was found to have potent activity in
an HCT 116 cytotoxicity assay. The majority of this
activity was traced to meta-substituted pyridinium
compounds similar to the halitoxins.6 Further exami-
nation of the extract led to the isolation of 1,3-dimeth-
ylisoguanine (1), theophylline (2), and thymine. To the
best of our knowledge, this is the first report of 1,3-
dimethylisoguanine as either a natural or synthetic
product.
ESIMS with deuterated electrospray solvent demon-
strated the presence of two exchangeable protons in the
neutral molecule. Both methyl proton signals showed
HMBC correlations to a quaternary carbon at δ 150.8
ppm, while the δ 3.60 ppm methyl proton and the δ 7.62
ppm proton both showed HMBC correlations to a carbon
at δ 152.7 ppm. This HMBC pattern was consistent
with several substitution patterns on the purine base.
Both guanines and isoguanines are known to deaminate
when heated with HCl, and the methylation positions
may be inferred by comparing the product with a
commercially available methylated xanthine analogue.
Conversion of the natural product to 2 by refluxing with
HCl demonstrated the positions of the methyl substit-
uents on the purine base. The reaction product was
1
found to be identical to commercial theophylline by H
NMR, 13C NMR, UV spectroscopy, MS/MS, and EIMS.
Fragmentation patterns for purines are relatively well
characterized,8,9 and EIMS has been used previously to
distinguish between guanines and isoguanines.4,9
A
characteristic mode of fragmentation for some guanines
via EIMS,4,8,9 but especially for xanthines and isogua-
nines,9 is the expulsion of neutral cyanamide fragments
consisting of N1, C2, and their substituents. Guanines
and isoguanines can be distinguished by MS due to a
one-mass-unit difference in this fragment; isoguanines
contain an oxygen substitutent on C2, while guanines
have an imino substituent in the same position. The
EIMS of 1 displays an abundant ion of m/z 122,
corresponding to the loss of CH3NCO, whereas no loss
of CH3NCNH is observed. HREIMS of the m/z 122 ion
gave an exact mass of 122.0589, corresponding to a
composition of C5H6N4 (+0.3 mmu error).10 This frag-
mentation is particularly clear in the negative ion
collision-induced dissociation mass spectrum of 1 (Fig-
ure 1). The loss of the CH3NCO moiety proves the
purine heterocycle is an isoguanine and unambiguously
assigns 1 as 1,3-dimethylisoguanine.
The freeze-dried sponge was extracted repeatedly
with MeOH and the resulting crude extract partitioned
according to a modified Kupchan fractionation protocol.7
The CHCl3-soluble material was subjected to counter-
current chromatography (CCC) using a CHCl3, MeOH,
and H2O solvent system to yield pure compound 1 (24
mg).
A molecular formula of C7H9N5O established by
HRFABMS+, and 13C-NMR chemical shifts suggested
compound 1 was a purine heterocycle with two N-
methyl substitutents. A very broad exchangeable peak
Compound 1 was tested in an assay of 26 human
cancer cell lines showing highest cytotoxicity to an
ovarian cancer cell line (IC50 2.1 µg/mL).
1
was observed centered about 7.60 ppm in the H-NMR
Exp er im en ta l Section
spectrum, and a deuterium exchange experiment using
Gen er a l Exp er im en ta l P r oced u r es. 1H- and 13C-
NMR experiments were performed on a Varian Unity
500 MHz spectrometer. Spectra were referenced to
residual undeuterated solvent peaks or solvent 13C
signals. HREIMS and LREIMS measurements were
* To whom correspondence should be addressed. Phone: (801) 581-
8305. FAX: (801) 581-6208. E-mail: cireland@deans.pharm.utah.edu.
† University of Utah.
‡ Bermuda Biological Station.
X Abstract published in Advance ACS Abstracts, J une 15, 1997.
S0163-3864(97)00015-3 CCC: $14.00
© 1997 American Chemical Society and American Society of Pharmacognosy