(+)-Agelasine D: Improved synthesis and evaluation of antibacterial and cytotoxic activities

Article abstract of DOI:10.1021/np050424c

An improved synthesis of (+)-agelasine D (10) from (+)-manool is reported together with cytotoxic and antibacterial data for agelasine D and structurally close synthetic analogues. These compounds display a broad spectrum of antibacterial activities including effects on M. tuberculosis and Gram-positive and Gram-negative bacteria (both aerobes and anaerobes). They exhibit profound cytotoxic activity against several cancer cells, including a multidrug-resistant cell line. (+)-Agelasine D (10) has been isolated earlier from a marine sponge (Agelas sp.).

Full text of DOI:10.1021/np050424c

J. Nat. Prod. 2006, 69, 381-386
381
(+)-Agelasine D: Improved Synthesis and Evaluation of Antibacterial and Cytotoxic Activities#
Anders Vik,† Erik Hedner,‡ Colin Charnock,§ Ørjan Samuelsen, Rolf Larsson,| Lise-Lotte Gundersen,*,† and Lars Bohlin‡
Department of Chemistry, UniVersity of Oslo, PO Box 1033 Blindern, N-0315 Oslo, Norway, DiVision of Pharmacognosy, Department of
Medicinal Chemistry, Biomedical Centre, Uppsala UniVersity, PO Box 574, S-751 23, Sweden, Faculty of Health Sciences, Oslo UniVersity
College, PO Box 4 St. OlaVs plass, N-0130 Oslo, Norway, Department of Medical Microbiology, UniVersity Hospital of North Norway,
Tromsø, Norway, and DiVision of Clinical Pharmacology, Department of Medicinal Sciences, Uppsala Academic Hospital, S-751 85 Uppsala,
Sweden
ReceiVed October 23, 2005
An improved synthesis of (+)-agelasine D (10) from (+)-manool is reported together with cytotoxic and antibacterial
data for agelasine D and structurally close synthetic analogues. These compounds display a broad spectrum of antibacterial
activities including effects on M. tuberculosis and Gram-positive and Gram-negative bacteria (both aerobes and anaerobes).
They exhibit profound cytotoxic activity against several cancer cells, including a multidrug-resistant cell line. (+)-
Agelasine D (10) has been isolated earlier from a marine sponge (Agelas sp.).
Marine sponges are rich sources of natural products with a range
of biological activities.1 Agelasines are isolated from marine sponges
(Agelas spp.), and they are associated with bioactivities such as
antimicrobial and cytotoxic effects, as well as contractive responses
of smooth muscles and inhibition of Na- and K-ATPase.2 More
than 10 agelasines are currently known, but only agelasines A,3
B,4 D,5 E,6 and (()-agelasine F7 (Figure 1) have been synthesized.
We recently published the first synthesis of (+)-agelasine D (10).5
Even though the reaction sequence constitutes only a few steps
from commercially available (+)-manool, the total yield of the target
compound is less than desired due to moderate regio- or stereo-
selectivity in some steps. We herein report an improved conversion
of (+)-manool to (+)-agelasine D (10) and also the first antibacterial
and cytotoxic data for this natural product and its synthetic
intermediates.
and (Z)-2 was obtained at 0 °C. Decreasing the reaction temperature
below 0 °C did not improve selectivity further. The acetate was
hydrolyzed to give, after chromatography, pure (E)-3, which was
converted to the alkylating agent 4 by treatment of PBr3. Compound
3 is found in nature and referred to as (+)-copalol or anti-copalol.
To the best of our knowledge, the transformation of (+)-manool
into (+)-copalol (3), described herein, is the most convenient
synthesis of this natural product published to date.9
A second drawback with our first (+)-agelasine D synthesis is
the low regioselectivity in the N-7 alkylation step (conversion of
6a to 9a, Scheme 3).5 Introduction of an alkyl group in the
7-position of an N-9-alkylated adenine derivative requires a
directing group at N6. A methoxy group is commonly used, but
substantial amounts of N6-alkylated products are generally formed
together with the desired 7,9-dialkylpurinium salt.4-7,10 Also acyl
groups have been suggested as directing groups.11 We introduced
the acetyl, benzoyl, and tert-butanoyl functionality at N6 of
9-methyladenine. All compounds reacted with excess benzyl
bromide in a highly regioselective manner to give 7,9-dialkyl-
purinium salts, but the isolated yields were often quite modest due
to decomposition during workup and purification. Instead we chose
to use alkoxy directing groups, and we assumed that increased
sterical hindrance at N6 would favor N-7 alkylation. The 6-chlo-
ropurine 5 was reacted with different N-alkoxyamines to give
compounds 6 (Scheme 2). In DMSO-d6 solution compounds 6 exist
mainly as the tautomer shown in Scheme 2. Compounds 6a-c were
isolated in acceptable yields. Syntheses of compounds 6 with R )
-Ph, -CPh3, or -TBDMS were not successful. In these latter
cases, the RONH2 reagents used were of limited stability at the
temperatures generally required for nucleophilic substitution on
compound 5. When compounds 6a-c reacted with benzyl bromide,
it was confirmed that the regioselectivity in the alkylation was
highly dependent on the sterical shielding of N6 (Table 1). The
tert-butoxy group was found to be an excellent directing group and
used in the agelasine D synthesis below. Compound 6b was reacted
with copalyl bromide 4 to give the desired product 9b in high yield
(Scheme 3). The alkylation occurred with complete regioselectivity,
as judged by 1H NMR of the crude product. Agelasine D (10) was
isolated after reductive cleavage of the directing group.
Results and Discussion
Synthesis. In our previously reported synthesis of (+)-agelasine
D,5 (+)-manool was converted directly into the allylic bromide 4
(Scheme 1). However, an inseparable mixture of the E- and
Z-isomers of the bromide had to be used in the alkylation step
(conversion of 6a to 9a, Scheme 3). Pure (E)-9a was isolated in
modest yield after crystallization. Therefore, we sought a method
for converting (+)-manool to pure (E)-4. Even though iodination
of tertiary allylic acetates with Ph3PI2 occurs with complete
E-selectivity, this reaction could not be used for our purpose since
isomerization of exocyclic double bonds readily takes place under
these conditions.8 Geometrically pure primary allylic bromides can
be formed by Pd-catalyzed rearrangement of tertiary allylic esters,
such as the acetates of linalool and geranyllinalool, followed by
ester hydrolysis and bromination.6 When manool acetate (1) was
subjected to rearrangement catalyzed by PdCl2(MeCN)2 at ambient
temperature, full conversion of the starting material took place in
less than an hour (Scheme 1), whereas the same rearrangement of
geranyllinalyl acetate required more than 1 day.6 At ambient
temperature the E:Z ratio of 2 was 87:13. The ratio was somewhat
improved by lowering the temperature, and a 92:8 mixture of (E)-
# Dedicated to Dr. Norman R. Farnsworth of the University of Illinois
at Chicago for his pioneering work on bioactive natural products.
* Corresponding author. Tel: +47 22 857019. Fax: +47 22 855507.
E-mail: l.l.gundersen@kjemi.uio.no.
Antibacterial Activity. Many natural products from marine
sponges are associated with potent antibacterial activities.1c Age-
lasines B and F (Figure 1) exhibit activity against several types of
bacteria.2c,g Agelasine F is even reported to inhibit Mycobacterium
tuberculosis.12 On the other hand, epi-agelasine C is found to be
inactive against several microorganisms including Staphylococcus
aureus and Escherichia coli.2e The same is true for the agelasine
† University of Oslo.
‡ Uppsala University.
§ Oslo University College.
University Hospital of North Norway.
Uppsala Academic Hospital.
|
10.1021/np050424c CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/05/2006

382 Journal of Natural Products, 2006, Vol. 69, No. 3
Vik et al.
Figure 1. Structures of agelasines discussed in the text.
Scheme 1a
a (i) Ac2O, DMAP, Et3N, THF, ∆; (ii) PdCl2(MeCN)2, THF, 0 °C; (iii) K2CO3, MeOH; (iv) PBr3, pyridine, Et2O, 0 °C.
Scheme 2a
Scheme 3a
a (i) Comp. 4, DMA, 50 °C; (ii) Zn, AcOH, MeOH, H2O, 75 °C.
shown an increase in resistance to most of the antimicrobial agents
traditionally used for treating anaerobic infections.13 The MIC values
for compounds 9 and 10 were comparable with those obtained using
metronidazole, which has been the drug of choice for preventing
and treating infections by the B. fragilis group species for 40 years.14
Agelasine analogues of low cytotoxicity might thus have potential
as antibiotics for treatment of infections by these organisms. All
compounds were profound inhibitors of M. tuberculosis at 6.25 µg/
mL. Only minor differences in activity between agelasine D and
the alkoxyoxy derivatives 9 were found, even though the alkoxy
group appears to be very important for antimycobacterial activity
in many agelasine analogues.6 The tert-butoxy derivative of
agelasine D, 9b, was somewhat less active against S. aureus and
E. coli, compared to compounds 9a and 10, but activities against
M. tuberculosis, B. fragilis, and B. thetaiotaomicron were compa-
rable to those found for compound 9a.
Cytotoxicity. Agelasine G is cytotoxic against murine lymphoma
cells,15 but cytotoxicity data for other agelasines are, to the best of
our knowledge, not available in the literature. We chose to screen
agelasine D (10) and the analogues 9a,b against four human tumor
cell lines: lymphoma (U-937 GTB), myeloma (RPMI 8226/s),
leukemia (CEM/s), and renal cells (ACHN). The latter is a
multidrug-resistant cell line, whereas the others are regarded as
a (i) RONH3Cl or RONH2 Et3N, n-BuOH, ∆; (ii) BnBr, DMA, 50 °C.
Table 1. Regioselectivity in Alkylation of Compounds 6
R
ratio 7:8a
yield (%) 7b
yield (%) 8b
Me
t-Bu
Bn
3:2
9:1
4:1
55, 7a
86, 7b
72, 7c
30, 8a
9, 8b
13, 8c
1
a From H NMR of the crude product. b Isolated yields.
11.2g We have demonstrated profound antimycobacterial activity
for several synthetic analogues of agelasines, but only moderate
activity for agelasine E.6 It is claimed that other agelasines,
including agelasine D, are antimicrobial, but no details regarding
microorganisms nor quantitative activity were reported.2a Syntheti-
cally prepared (+)-agelasine D (10) and the N6-alkoxy derivatives
9a,b were screened against Gram-positive and Gram-negative
bacteria, as well as M. tuberculosis, and the results are summarized
in Table 2. The compounds examined were found to exhibit
relatively broad antibacterial spectra. Highest activities were found
against the Gram-positive bacteria S. aureus and S. pyogenes. The
Bacteroides fragilis group of organisms are the most commonly
isolated anaerobes in clinical settings. Species of this group have

(+)-Agelasine D
Journal of Natural Products, 2006, Vol. 69, No. 3 383
Figure 2. Survival index (SI) curves of agelasine 10 (left) and analogue 9a (right) at 10 concentrations on the human cancer cell lines
U-937 GTB, RPMI 8226/s, CEM/s, and ACHN. Data points are mean ( SEM of triplicates values, calculated using nonlinear regression
in GraphPad Prism 4 (GraphPad Software, San Diego, CA).
Table 2. Antibacterial Activity of Compounds 9a, 9b, and Agelasine D (10) and Clinically Used Drugs against Mycobacterium
tuberculosis, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa,
Bacteroides fragilis, and Bacteroides thetaiotaomicron
% inhibition
M. tuberculosis
H37Rv
MIC (µg mL-1
)
M.
S.
S.
E.
E.
P.
B.
B.
compound
at 6.25 mg/mL
tuberculosis
aureus
pyogenes
faecalis
coli
aeruginosa
fragilis
thetaiotaomicron
9a
9b
96
100
92
>90
n.d.
n.d.
>6.25
3.13
>6.25
0.25
2
6
1
n.d.
0.03
n.d.
2
8
n.d.
8
n.d.
>4
n.d.
8
32
8
n.d.
0.13
n.d.
32
n.d.
16
n.d.
0.13
n.d.
4-8
4
16
n.d.
n.d.
0.5
4-8
4-8
8-16
n.d.
n.d.
1
n.d.a
2
agelasine D (10)
rifampicin
gentamycin
n.d.
1-2
n.d.
metronidazole
a No data obtained.
Table 3. Cytotoxic Activity of Compounds 9a, 9b, and
Agelasine D (10), as Well as Three Clinically Used Drugs on
the Cell Lines U-937 GTB, RPMI 8226/s, CEM/s, and ACHN
involved in the host defense of sponges from Agelas spp. Further
investigations with relevant assays are needed to clarify the role of
agelasine D in the sponge. Our results demonstrate that agelasines
are highly interesting antineoplastic agents, and the less cytotoxic
analogues may have a potential as antibiotics. There is an urgent
need for new antibacterial agents with novel mechanisms and
chemical structures since resistance has been reported for virtually
all existing antibacterial drugs.18
IC50 (µM)
U-937 GTB RPMI 8226/s
CEM/s
ACHN
compound
(lymphoma)
(myeloma)
(leukemia) (renal)
9a
9b
1.03
>80
0.2
>80
2.33 8.01
>80 >80
agelasine D (10)
doxorubicin
cisplatin
5.59
3.7
4.68
0.18
2.48
0.007
24.97
14.2
17.8
31.5
0.11
2.56
0.0059
0.13
14.83
0.007
Experimental Section
palitaxel
General Experimental Procedures. Melting points are uncorrected.
1
The H NMR spectra were acquired on a Bruker Avance DPX 300
spectrometer at 300 MHz, and the 1H decoupled 13C NMR spectra were
recorded at 75 MHz using the above-mentioned spectrometer. Mass
spectra under electron impact conditions were recorded with a VG
Prospec instrument at 70 eV ionizing voltage and are presented as m/z
(% rel int). Electrospray MS were recorded with a Bruker Apex 47e
FT-ICR mass spectrometer. Elemental analyses were performed by Ilse
Beetz Mikroanalytisches Laboratorium, Kronach, Germany. DMA and
n-butanol were distilled from BaO and stored over molecular sieves (3
Å), THF and diethyl ether were distilled from Na/benzophenone, and
triethylamine was distilled from CaH2. Acetic acid anhydride was
refluxed over CaCl2 for 5 days prior to distillation and kept over
molecular sieves (3 Å). O-tert-Butylhydroxylamine hydrochloride was
synthesized according to the literature19 from N-(tert-butoxy)phthal-
imide.20 Compounds 5,6 6a,6 and 9a5 were prepared as described before,
and antimycobacterial activities against M. tuberculosis H37Rv
(ATCC27294) were determined as reported before.6
drug-sensitive cell lines. The results are illustrated in Figure 2 and
summarized in Table 3. Agelasine D (10) and the analogue 9a
exhibited potent activity against the myeloma cell line examined,
as well as the other three cell lines. Both compounds showed a
dose-dependent cytotoxicity and were cytotoxic in concentrations
in the range of clinical drugs used today (Table 3). The activities
were comparable with that reported for agelasine G against another
lymphoma cell line (L1210; IC50 3.1 µg/mL ≈ 4.8 µM).10 Renal
cell carcinomas (RCC) are quite resistant to chemotherapy;16 hence
it was especially intriguing to find that compound 9a was more
effective against the primary multidrug-resistant (MDR) cell line
ACHN (renal adenocarcinoma cells)17 than any of the anticancer
drugs used as positive controls. The tert-butoxy derivative 9b
showed no activity at 80 µM against any of the cell lines examined.
Due to its low toxicity, compound 9b is an interesting antibacterial
compound.
(+)-Manoyl Acetate (1). (+)-Manool (1.00 g, 3.44 mmol) and
DMAP (0.33 g, 2.7 mmol) were dissolved in dry THF (30 mL) under
N2. Triethylamine (4.8 mL, 34 mmol) and Ac2O (3.2 mL, 34 mmol)
were added, and the mixture was refluxed for 40 h. The reaction mixture
was cooled, diluted with diethyl ether (100 mL), washed with 2 M
aqueous HCl (3 × 30 mL), saturated aqueous NH4Cl (3 × 30 mL),
and saturated aqueous NaCl (30 mL), dried (MgSO4), and evaporated
in vacuo. The residue was purified by flash chromatography on silica
gel eluting with EtOAc-hexane (1:70); yield 1.04 g (91%) as a colorless
We have made significant improvements in our synthesis of (+)-
agelasine D (10) and found that this marine natural product exhibits
a variety of biological activities. Agelasine D and two close
analogues display a broad spectrum of antibacterial activities
including an effect on M. tuberculosis. The same compounds show
profound inhibitory activity against several cancer cells including
a multidrug-resistant cell line. It is plausible considering the
cytotoxic and antibacterial activity of agelsine D that it can be
oil: [R]20 +24.8 (c 2.52, CHCl3) (lit.21 [R]D +24.5; c 2.52, CHCl3);
D

384 Journal of Natural Products, 2006, Vol. 69, No. 3
Vik et al.
1H NMR (CDCl3, 300 MHz) δ 5.97 (1H, dd, J ) 17.5, 11.0 Hz), 5.16
(1H, dd, J ) 17.5, 0.9 Hz), 5.13 (1H, dd, J ) 11.0, 0.9 Hz), 4.92 (1H,
d, J ) 1.4 Hz), 4.50 (1H, d, J ) 1.4 Hz), 2.39 (1H, ddd, J ) 12.7, 4.2,
2.4 Hz), 2.05 (3H, s, CH3), 2.00-0.95 (18H, m), 0.81 (3H, s, CH3),
0.68 (3H, s, CH3); 13C NMR (CDCl3, 75 MHz) δ 170.3 (CO), 149.1,
142.4, 113.4, 106.8, 83.8, 57.6, 56.0, 42.6, 40.2, 39.7, 39.4, 38.7, 34.00,
33.97, 24.8, 23.9, 22.6, 22.1, 19.8, 17.8, 14.6.
233-235 °C (lit.23 232-234 °C); 1H NMR (DMSO-d6, 300 MHz, data
of major tautomer, as shown in Scheme 2) δ 11.22 (1H, br s, NH),
7.78 (1H, s, H-8), 7.60 (1H, d, J ) 3.3 Hz, H-2), 7.54 (2H, m, Ph),
7.45-7.22 (3H, m, Ph), 5.02 (2H, s, CH2), 3.63 (3H, s, CH3); EIMS
m/z 255 [M]+ (36), 238 (19), 149 (19), 91 (100).
7-Benzyl-6-methoxyamino-9-methyl-7H-purinium (7a) and N6-
Benzyl-N6-methoxy-9-methyl-9H-purin-6-amine (8a). N6-Methoxy-
9-methyl-9H-purin-6-amine (6a) (115 mg, 0.64 mmol) was dissolved
in DMA (10 mL) at 50 °C under N2 before benzyl bromide (0.12 mL,
0.96 mmol) was added. The mixture was stirred at 50 °C overnight
and evaporated in vacuo. The residue was purified by flash chroma-
tography on silica gel eluting with CH2Cl2-MeOH saturated with NH3
(9:1); yield 7a 95 mg (55%). The fractions containing isomer 8a were
combined, evaporated, and purified by flash chromatography eluting
with EtOH-EtOAc (1:18); yield 8a 52 mg (30%).
(+)-Copalyl Acetate (2).22 (+)-Manoyl acetate (1) (0.70 g, 2.1
mmol) and PdCl2(CH3CN)2 (0.1 mmol, 28 mg) were dissolved in dry
THF (20 mL) at 0 °C and stirred for 2 h. The mixture was evaporated
and filtered through a small pad of silica gel eluting with EtOAc-
hexane (1:20); yield 0.95 g (90%, E/Z: 92:8) as a colorless oil: [R]20
D
+30.1 (c 1.6, CHCl3) (lit.22 [R]D +29.5; c 1.6, CHCl3 for the E-isomer
1
and [R]D +8; c 9.2, CHCl3 for the Z-isomer); H NMR (CDCl3, 300
MHz) δ 5.32 (1H, m), 4.84 (1H, d, J ) 1.3 Hz), 4.60 (2H, d, J ) 7.1
Hz), 4.51 (1H, d, J ) 1.3 Hz), 2.40 (1H, ddd, J ) 12.6, 4.2, 2.4 Hz),
2.30-0.95 (21H, m), 0.88 (3H, s, CH3), 0.81 (3H, s, CH3), 0.69 (3H,
s, CH3); 13C NMR (CDCl3, 75 MHz) δ 171.5 (CO), 149.0, 143.5, 118.3,
106.6, 61.8, 56.6, 56.0, 42.6, 40.0, 39.5, 38.8, 38.7, 34.01, 33.97, 24.8,
22.1, 22.0, 21.5, 19.8, 16.9, 14.9; anal. C 79.42%, H 10.78%, calcd
for C22H36O2, C 79.46% H, 10.91%.
7-Benzyl-6-methoxyamino-9-methyl-7H-purinium (7a): colorless
crystals; mp 215-216 °C; 1H NMR (CDCl3, 300 MHz) δ 8.08 (1H, s,
H-8), 7.80 (1H, s, H-2), 7.46-7.43 (2H, m, Ph), 7.36-7.32 (3H, m,
Ph), 5.64 (2H, s, CH2), 3.83 (3H, s, OCH3), 3.69 (3H, s, NCH3); 13C
NMR (CDCl3, 75 MHz) δ 157.1 (CH, C-2), 147.8 (C, C-6), 145.0 (C,
C-4), 134.1 (C, Ph), 129.3 (CH, C-8), 129.3, 129.2, 129.1 (each CH,
Ph), 109.4 (C, C-5), 61.5 (CH3, OCH3), 53.0 (CH2), 31.0 (CH3, NCH3);
HRESIMS m/z 270.1345 (calcd for C14H15N5O+H, 270.1349).
N6-Benzyl-N6-methoxy-9-methyl-9H-purin-6-amine (8a):24 color-
less oil; 1H NMR (CDCl3, 300 MHz) δ 8.50 (1H, s, H-2), 7.81 (1H, s,
H-8), 7.45-7.42 (2H, m, Ph), 7.32-7.24 (3H, m, Ph), 5.34 (2H, s,
CH2O), 3.83, 3.82 (each 3H, s, CH3); 13C NMR (CDCl3, 75 MHz) δ
155.6 (C, C-6), 152.3 (CH, C-2), 151.7 (C, C-4), 141.0 (CH, C-8),
136.7 (C, Ph), 128.6, 128.3, 127.4 (each CH, Ph), 118.9 (C, C-5), 62.5
(CH3, OCH3), 53.8 (CH2), 29.7 (CH3, NCH3); EIMS m/z 269 [M]+
(12), 238 (100), 133 (12), 106 (23), 91 (52); HREIMS m/z 269.1275
(calcd for C14H15N5O, 269.1276).
(+)-Copalol (3). A mixture of (+)-copalyl acetate (2) (814 mg, 2.25
mmol) and K2CO3 in methanol (3%) (1.2 g of K2CO3 in 38 g of MeOH)
was stirred at ambient temperature overnight. The mixture was
evaporated, diluted with water (50 mL), and extracted with diethyl ether
(4 × 50 mL). The combined organic extracts were washed with
saturated aqueous NaCl (25 mL), dried (MgSO4), and evaporated in
vacuo. The residue was purified by flash chromatography on silica gel
eluting with EtOH-EtOAc-hexane (1:5:50); yield 537 mg (76%, pure
E-isomer) as a colorless oil: [R]20 +36.8 (c 1.14, CHCl3) (lit.9a [R]D
D
+31.2; c 1.14, CHCl3); 1H NMR (CDCl3, 300 MHz) δ 5.40 (1H, tq, J
) 6.9, 1.2 Hz), 4.82 (1H, d, J ) 1.4 Hz), 4.52 (1H, d, J ) 1.1 Hz),
4.14 (2H, d, J ) 6.9 Hz), 2.40 (1H, ddd, J ) 12.7, 4.2, 2.4 Hz), 2.16
(1H, m), 1.99 (1H, dt, J ) 12.9, 5.0 Hz), 1.90-0.95 (17H, m), 0.88
(3H, s, CH3), 0.81 (3H, s, CH3), 0.69 (3H, s, CH3); 13C NMR (CDCl3,
75 MHz) δ 149.0, 141.0, 123.4, 106.6, 59.8, 56.7, 55.9, 42.6, 40.1,
39.5, 38.83, 38.76, 34.01, 33.98, 24.9, 22.2, 22.1, 19.8, 16.8, 14.9; EIMS
m/z 290 [M]+ (17), 275 (70), 272 (22), 257 (34), 205 (16), 137 (100);
HREIMS m/z 290.2603 (calcd for C20H34NO, 290.2609).
7-Benzyl-6-tert-butoxyamino-9-methyl-7H-purinium (7b) and N6-
Benzyl-N6-tert-butoxy-9-methyl-9H-purin-6-amine (8b). The title
compounds were prepared from N6-tert-butoxy-9-methyl-9H-purin-6-
amine (6b) (221 mg, 1.00 mmol) and benzyl bromide (0.18 mL, 1.5
mmol) as described for compounds 7a and 8a above. The products
were isolated by flash chromatography on silica gel eluting with CH2-
Cl2-MeOH saturated with NH3 (11:1); yield 7b 268 mg (86%). The
fractions containing isomer 8b were combined, evaporated, and purified
by flash chromatography eluting with EtOAc-hexane (1:1); yield 8b
30 mg (9%).
Anticopalyl Bromide (4). (+)-Copalol (3) (870 mg, 3.00 mmol)
was dissolved in dry diethyl ether (10 mL) under N2 at 0 °C. PBr3
(813 mg, 3.00 mmol) was added, and the mixture was stirred at 0 °C
for 3 h. The mixture was diluted with diethyl ether (60 mL) and washed
with 10% aqueous NaHCO3 (20 mL). The aqueous phase was extracted
with diethyl ether (20 mL), and the combined organic extracts were
dried (MgSO4) and evaporated in vacuo; yield 841 mg (76%) of the
crude compound as a pale yellow oil. Spectroscopic data were in
agreement with those reported before.5
7-Benzyl-6-tert-butoxyamino-9-methyl-7H-purinium (7b): pale
1
yellow crystals; mp 210-211 °C (dec); H NMR (CDCl3, 300 MHz)
δ 9.61 (1H, s), 7.81 (1H, s), 7.52-7.49 (2H, m, Ph), 7.32-7.29 (3H,
m, Ph), 5.75 (2H, s, CH2), 3.80 (3H, s, CH3), 1.21 (9H, s, t-Bu); 13C
NMR (CDCl3, 75 MHz) δ 154.1 (CH, C-2), 143.5 (C, C-4), 142.1 (C,
C-6), 134.5 (C, Ph), 133.7 (CH, C-8), 129.1, 129.0, 128.6 (each CH,
Ph), 110.6 (C, C-5), 77.8 (C, t-Bu), 52.6 (CH2), 31.4 (CH3, NCH3),
27.6 (CH3, t-Bu); HRESIMS m/z 312.1831 (calcd for C17H21N5O+H,
312.1818).
N6-tert-Butoxy-9-methyl-9H-purin-6-amine (6b). 6-Chloro-9-meth-
yl-9H-purine (5) (0.60 g, 3.6 mmol) and O-tert-butylhydroxylamine
hydrochloride (1.33 g, 10.6 mmol) were dissolved in dry n-butanol
(18 mL) under N2. Triethylamine (4.9 mL, 35 mmol) was added, and
the mixture was refluxed overnight and evaporated in vacuo. The
residue was purified by flash chromatography on silica gel eluting with
EtOH-CH2Cl2 (1:14); yield 480 mg (61%) as colorless crystals, mp
235 °C. The compound existed as a 3:1 mixture of imino and amino
tautomers in DMSO-d6. 1H NMR (DMSO-d6, 300 MHz, data of major
tautomer, as shown in Scheme 2) δ 10.80 (1H, br s, NH), 7.76 (1H, s,
H-8), 7.56 (1H, d, J ) 3.7 Hz, H-2), 3.63 (3H, s, NCH3), 1.27 (9H, s,
t-Bu); 13C NMR (DMSO-d6, 75 MHz data of major tautomer, as shown
in Scheme 2) δ 145.0 (CH, C-2), 142.2 (C, C-4), 139.3 (CH, C-8),
141.6 (C, C-6), 119.4 (C, C-5), 77.6 (C, t-Bu), 30.3 (CH3, NCH3), 28.4
(CH3, t-Bu); EIMS m/z 221 [M]+ (2), 166 (9), 165 (100), 148 (8), 134
(7), 135 (69), 107 (6); HREIMS m/z 221.1271 (calcd for C10H15N5O,
221.1276); anal. C 54.14%, H 6.67%, N 31.52%, calcd for C10H15N5O,
C 54.28%, H 6.83%, N 31.65%.
N-Benzyl-N-tert-butoxy-9-methyl-9H-purin-6-amine (8b): color-
less oil; 1H NMR (CDCl3, 300 MHz) δ 8.53 (1H, s, H-2), 7.85 (1H, s,
H-8), 7.35-7.28 (2H, m, Ph), 7.26-7.16 (3H, m, Ph), 6.07 (1H, br s,
Ha in CH2), 4.92 (1H, br s, Hb in CH2), 3.85 (3H, s, NCH3), 1.44 (9H,
s, t-Bu); 13C NMR (CDCl3, 75 MHz) δ 158.9 (C, C-6), 152.1 (CH,
C-2), 151.6 (C, C-4), 140.6 (CH, C-8), 137.3 (C, Ph), 128.4, 128.1,
127.0 (each CH, Ph), 119.8 (C, C-5), 82.8 (C, t-Bu), 58.6 (CH2), 29.7
(CH3, NCH3), 27.3 (CH3, t-Bu); HRESIMS m/z 312.1806 (calcd for
C17H21N5O+H, 312.1818).
7-Benzyl-6-benzyloxyamino-9-methyl-7H-purinium (7c) and N6-
Benzyl-N6-benzyloxy-9-methyl-9H-purin-6-amine (8c). The title
compounds were prepared from N6-benzyloxy-9-methyl-9H-purin-6-
amine (6c) (255 mg, 1.00 mmol) and benzyl bromide (0.18 mL, 1.5
mmol) as described for compounds 7a and 8a above. The products
were isolated by flash chromatography on silica gel eluting with CH2-
Cl2-MeOH saturated with NH3 (12:1); yield 7c 249 mg (72%). The
fractions containing isomer 8c were combined, evaporated, and purified
by flash chromatography eluting with EtOAc-hexane (2:1); yield 8c
41 mg (13%).
7-Benzyl-6-benzyloxyamino-9-methyl-7H-purinium (7c): pale
yellow crystals; mp 183 °C (dec); 1H NMR (CDCl3, 300 MHz) δ 8.69
(1H, br s, H-8), 7.84 (1H, s, H-2), 7.45-7.15 (10H, m, 2×Ph), 5.57
(2H, s, NCH2), 5.07 (2H, s, OCH2), 3.64 (3H, s, CH3); 13C NMR
(CDCl3, 75 MHz,) δ 156.9 (CH, C-2), 147.6 (C, C-6), 145.1 (C, C-4),
N6-Benzyloxy-9-methyl-9H-purin-6-amine (6c). A mixture of
6-chloro-9-methyl-9H-purine (5) (1.06 g, 6.29 mmol), O-benzylhy-
droxylamine hydrochloride (2.70 g, 16.9 mmol), and triethylamine (7.55
mL, 74.8 mmol) in dry n-butanol (50 mL) was stirred at reflux under
N2 for 16 h. The solvent was removed in vacuo, and the residue was
stirred vigorously in MeOH (60 mL) at 50-60 °C until most of the
residue was dissolved. The mixture was cooled to ca -50 °C [MeCN/
CO2(s)] for 10 min. The solid was filtered off, washed with diethyl
ether (10 mL), and dried; yield 1.27 g (79%) as colorless crystals: mp

(+)-Agelasine D
Journal of Natural Products, 2006, Vol. 69, No. 3 385
139.7, 135.0 (each C, Ph), 131.9 (CH, C-8), 129.6, 129.5, 129.4, 128.0,
128.4, 127.7 (each CH, Ph), 109.7 (C, C-5), 76.1 (CH2, OCH2), 53.1
(CH2, NCH2), 31.4 (CH3); HRESIMS m/z 346.1659 (calcd for
C20H19N5O+H, 346.1662).
Standards26 using dimethyl sulfoxide and sterile deionized water as the
solvent and dilutant, respectively. Stock solutions were stored in
polyethylene vials at -80 °C until the day of use. After the antimicro-
bials were thawed, serial 2-fold dilutions were prepared in reduced
Wilkins-Chalgren broth (Oxoid). Working concentrations ranging from
1.25 to 320 µg/mL were prepared to obtain final antibiotic concentra-
tions of 0.125 to 32 µg/mL after dilution in broth. A direct suspension
of colonies was prepared in 10 mL of Wilkins-Chalgren broth. The
turbidity of the suspension was adjusted spectrophotometrically to match
a 0.5 McFarland standard. This suspension was further diluted 1:10
with broth to obtain the inoculum having a concentration of about 1.5
× 107 CFU/mL. Susceptibility testing was performed using the broth
microdilution technique.26 Dilutions (100 µL) were added in triplicate
to the wells of a microtiter plate. To each well was added 10 µL of the
inoculum. Broth alone, broth amended with cells, and broth amended
with antimicrobial without cells were used as controls. After inoculation,
plates were lidded and incubated anaerobically at 35 °C for 48 h. The
MIC value was read as the lowest concentration of antimicrobial agent
showing no visible growth. Tests were repeated at least once with fresh
samples and a fresh inoculum.
Determination of Cytotoxic Activities. A fluorometric microculture
cytotoxity assay (FMCA) was used to determine cytotoxicity of 9a,
9b, and 10.27 Compounds were dissolved in 10% DMSO and added to
96-well microtiter plates in the following 10 concentrations: 80, 40,
20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0.15625 µM. Each concentration
was applied in triplicate. Solvent controls (10% DMSO), blank controls
(cell-growth medium), positive controls (Triton X-100), and negative
controls (phosphate-buffered saline solution, PBS) were prepared for
each microtiter plate. The tumor cells, suspended in cell-growth media,
were dispensed in the prepared microtiter plates. After 72 h of
incubation, the cells were washed with PBS, and FDA was added. The
plates were reincubated for 40 min, and the generated fluorescence
was measured. The fluorescence is proportional to the number of living
cells, which reflects cell survival, and provides a quantifiable survival
index (SI).
N6-Benzyl-N6-benzyloxy-9-methyl-9H-purin-6-amine (8c): color-
1
less oil; H NMR (CDCl3, 200 MHz,) δ 8.57 (1H, s, H-2), 7.88 (1H,
s, H-8), 7.55-7.25 (10H, m, 2×Ph), 5.28, 5.05 (each 2H, s, CH2), 3.89
(3H, s, CH3); 13C NMR (CDCl3, 75 MHz) δ 156.7 (C, C-6), 152.8
(CH, C-2), 152.3 (C, C-4), 141.5 (CH, C-8), 137.1, 136.1 (each C,
Ph), 130.2, 129.4, 128.9, 128.8, 128.7, 127.9 (each CH, Ph), 119.7 (C,
C-5), 77.7 (CH2), 55.5 (CH2), 30.2 (CH3); EIMS m/z 345 [M]+ (43),
329 (18), 328 (77), 223 (49), 91 (100); HREIMS m/z 345.1581 (calcd
for C20H19N5O, 345.1589).
(+)-N6-tert-Butoxyagelasine D (9b). N6-tert-Butoxy-9-methyl-9H-
purin-6-amine (6b) (221 mg, 1.00 mmol) was dissolved in dry DMA
(4 mL) under N2 at 50 °C. Anticopalyl bromide (4) (493 mg, 1.40
mmol) in DMA (2 mL) was added. The mixture was stirred overnight
at 50 °C and evaporated in vacuo, and the residue was purified by
flash chromatography on silica gel eluting with CH2Cl2-MeOH
saturated with NH3 (10:1); yield 445 mg (90%) as pale yellow
1
crystals: mp 181 °C; [R]20 +25.1 (c 5.0, CHCl3); H NMR (CDCl3,
D
300 MHz) δ 8.97 (1H, s, H-8), 7.84 (1H, s, H-2), 5.53 (1H, t, J ) 7.2
Hz, CH), 5.10 (2H, d, J ) 7.2 Hz, CH2), 4.81 (1H, s), 4.69 (1H, s),
3.86 (3H, s, NCH3), 2.38 (1H, ddd, J ) 12.7, 3.5, 2.4 Hz), 2.22 (1H,
m), 1.82 (3H, s, CH3), 1.29 (9H, s, t-Bu), 2.00-0.90 (14H, m), 0.86
(3H, s, CH3), 0.79 (3H, s, CH3), 0.66 (3H, s, CH3); 13C NMR (CDCl3,
75 MHz) δ 155.7 (CH, C-2), 148.8, 145.9 (each C, Cd), 144.6 (C,
C-4), 143.8 (C, C-6), 131.7 (CH, C-8), 116.6 (CH, CHd), 111.1 (C,
C-5), 106.7 (CH2, dCH2), 79.8 (C, t-Bu), 56.8, 56.0, 48.1 (CH2), 42.1
(CH2), 40.1, 39.5 (CH2), 38.9 (CH2), 38.7 (CH2), 33.99, 33.96, 31.7
(CH3, NCH3), 28.1 (CH3, t-Bu), 24.8 (CH2), 22.1, 22.0 (CH2), 19.7
(CH2), 17.6, 14.9; HRESIMS m/z 494.3858 (calcd for C30H47N5O+H,
494.3853).
(+)-Agelasine D (10). A mixture of (+)-N6-tert-butoxyagelasine D
(9b) (197 mg, 0.400 mmol), Zn (0.33 g, 5.0 mmol), and AcOH (0.4
mL) in MeOH (20 mL) and water (2 mL) was stirred vigorously at 75
°C for 40 h. The mixture was filtered and the solid washed with MeOH
(20 mL). Saturated aqueous NaCl (10 mL) and water (10 mL) were
added to the MeOH solution, and the mixture was stirred for 1 h at
ambient temperature and evaporated in vacuo. The residue was
transferred to a separatory funnel using 50% saturated aqueous NaCl
(40 mL) and CHCl3 (50 mL). The phases were separated, and the
aqueous phase was extracted with CHCl3 (4 × 50 mL). The combined
organic layers were dried (MgSO4) and evaporated in vacuo. The
residue was purified by flash chromatography on silica gel eluting with
MeOH-CH2Cl2 (1:6); yield 90 mg (49%) as colorless crystals: mp
Acknowledgment. The Norwegian Research Council is greatly
acknowledged for a scholarship to A.V. (KOSK program, grant 149339/
431) as well as for partial financing of the Bruker Avance instrument
used in this study. The work has also been supported by the Swedish
Research for Environment, Agricultural Science and Spatial Planning.
Antimycobacterial data were provided by the Tuberculosis Antimicro-
bial Acquisition and Coordinating Facility (TAACF) through a research
and development contract with the U.S. National Institute of Allergy
and Infectious Diseases; we are grateful for all help provided by Dr.
C. Kwong and co-workers. M. C. Olsen at University Hospital of North
Norway is acknowledged for technical assistance with the antibacterial
susceptibility assays.
132-136 °C (lit.5 128-130 °C); [R]20 +11.0 (c 1.0, MeOH), (lit.2a
D
[R]D +10.4; c 1.1, MeOH). Spectroscopic data are as reported before.5
Determination of Antibacterial Activity against S. aureus (ATCC
25923), S. pyogenes (ATCC 19615), E. coli (ATCC 25922), E.
faecalis, and P. aeruginosa (ATCC27853). The minimum inhibitory
concentrations (MIC) of the agelasines were determined using a standard
broth microdilution technique as previously described.25 Overnight
cultures of bacteria were grown to exponential growth phase and diluted
in growth medium to give a final inoculum of 1 × 106 CFU/mL.
Bacteria (60 µL) and serial dilutions of agelasines in doubley distilled
water (60 µL) were added to a 96-well microtiter plate (Nunc, Roskilde,
Denmark) and incubated at 37 °C overnight. The MIC was determined
as the lowest concentration where no visible growth occurred. Bac-
topeptone water (2%) (Difco, Detroit) was used as growth medium for
S. aureus, E. coli, and P. aeruginosa and brain heart infusion broth
(Oxoid Ltd, Basingstroke, Hampshire, UK) for E. faecalis and S.
pyogenes. Gentamycin (Invitrogen Ltd, Paisley, UK) was used as
internal control in all experiments.
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NP050424C