Structural studies of cytotoxic marine alkaloids: Synthesis of novel ring-E analogues of ascididemin and their in vitro and in vivo biological evaluation

Article abstract of DOI:10.1016/S0040-4020(99)01038-8

The cytotoxic marine alkaloid ascididemin and various pyridine ring-E analogues have been synthesised in an attempt to determine the pharmaceutical utility and structure-activity requirements for the parent alkaloid. All compounds synthesised were evaluated in a wide range of biological screens for selective cytotoxicity, antiviral, antifungal and antimicrobial properties. Many analogues exhibited selective cytotoxicity to human solid tumour cell-lines in vitro, with one also exhibiting moderate antitumour activity in in vivo xenograft assays. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(99)01038-8

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 497–505
Structural Studies of Cytotoxic Marine Alkaloids: Synthesis of
Novel Ring-E Analogues of Ascididemin and their in vitro and in
vivo Biological Evaluation
*
Brent S. Lindsay, Holly C. Christiansen and Brent R. Copp
Department of Chemistry, University of Auckland, Private Bag 92019, Auckland, New Zealand
Received 13 September 1999; revised 10 November 1999; accepted 25 November 1999
Abstract—The cytotoxic marine alkaloid ascididemin and various pyridine ring-E analogues have been synthesised in an attempt to
determine the pharmaceutical utility and structure-activity requirements for the parent alkaloid. All compounds synthesised were evaluated
in a wide range of biological screens for selective cytotoxicity, antiviral, antifungal and antimicrobial properties. Many analogues exhibited
selective cytotoxicity to human solid tumour cell-lines in vitro, with one also exhibiting moderate antitumour activity in in vivo xenograft
assays. ᭧ 2000 Elsevier Science Ltd. All rights reserved.
Introduction
the chemistry of ring-E of the alkaloid ascididemin,
whereby investigations of the chemistry of enamine (and
related alkylamine) synthetic precursors have led to the
synthesis of novel 5- and 6-substituted analogues.
Over the last 15 years, more than 35 alkaloids based upon
the pyrido[2,3,4-kl]acridine skeleton have been reported
1
from the marine environment. Invariably these polycyclic
aromatic alkaloids have been isolated due to the range of
potentially useful biological activities they exhibit, which
include antifungal, antiviral and antitumour properties.
Some of the first examples of these compounds included
the ascidian-derived pyridoacridone alkaloids 2-bromo-
To explore the structural features responsible for the bio-
logical activities of ascididemin and related compounds, all
the derivatives in this study were tested for antiproliferative
properties against a range of targets, including leukaemia
cells, viruses and microorganisms. All compounds have also
been tested in the US National Cancer Institute (NCI) in
vitro primary screen, with several, including the natural
product parent compound ascididemin, also being studied
in Hollow Fibre and xenograft in vivo assays. The compre-
hensive testing by the NCI has shown that most of the
compounds synthesised in the current study exhibited selec-
tive cytotoxicity against certain cancer types in vitro, and
that a limited number of analogues also exhibited activity in
vivo.
2
3
leptoclinidinone (1) and ascididemin (2), with more recent
4
examples including 11-hydroxyascididemin (3), 9,10-dihy-
5
dro-11-hydroxyascididemin (4) and kuanoniamine A (5)
6
(
Fig. 1). Numerous approaches to the synthesis of the
7
pyridoacridone class of alkaloid have been reported while
only a limited number of biological studies of these
compounds have been presented.
8
Our interest in the ascididemin class of alkaloid was sparked
by the 1990 report by Schmitz et al. that ascididemin inhib-
ited the catalytic function of the DNA processing enzyme
9
topoisomerase II. As this enzyme is the molecular target of
many current clinical anticancer drugs we became interested
in assessing the human antitumour potential of ascididemin
and related compounds. Our initial studies established the
importance of N-8 in ring A, and a completed ring-E for the
observed topoisomerase II enzyme inhibition, human
tumour cytotoxicity and antifungal/antibacterial properties
8
b
of ascididemin. We now report more complete studies on
Keywords: marine metabolites; structure-activity; antitumour compounds;
alkaloids.
*
Corresponding author. Tel.: ϩ64-9-373-7599; fax: ϩ64-9-373-7422;
e-mail: b.copp@auckland.ac.nz
Figure 1.
0
040–4020/00/$ - see front matter ᭧ 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(99)01038-8

4
98
B. S. Lindsay et al. / Tetrahedron 56 (2000) 497–505
7
b
Scheme 1. Bracher’s synthesis of ascididemin (2) from dione 6 via enamine 7.
Chemistry
fashion (see Experimental section) and evaluated in the
same range of biological assays (see Biological Activities
section).
One of the more generally applicable procedures reported to
date for the synthesis of ascididemin is Bracher’s method-
ology which achieves a final ring-E annulation (i.e. 6 to 2 in
Scheme 1) using dimethylformamide-diethylacetal
DMFDEA) in DMF under nitrogen to form an enamine
7), which is then cyclised to the pentacyclic product with
We next investigated b-alkylation of enamine 7 as a method
with which to synthesise previously unrealised ring-E
derivatives of ascididemin. It was presumed that reaction
of 7 with Eschenmoser’s salt (or the more classical Mannich
reaction itself) would yield a difunctionalised side-chain
product (i.e. 13, which is a masked enal (14)). Both of
these side-chain functional groups should be capable of
(
(
7
b
ammonium chloride in acetic acid. As we, and others,
8
b,c
have previously noted that dione 6 is essentially devoid
of biological activity, we speculated that the more func-
tionalised enamine 7 could exhibit interesting biological
properties.
undergoing further reaction with NH to form pyridine
3
ring-E, leaving the other non-reacting functional group as
a substituent at the 5-position of ascididemin. Indeed,
reaction of enamine 7 with Eschenmoser’s salt yielded the
desired but unstable enal 14 in low yield. The observation of
an aldehydic (d 10.01) and two olefinic (d 6.77 and 6.37)
Reaction of quinone 6 with DMFDEA in DMF under nitro-
gen and subsequent removal of all volatiles in vacuo
afforded the previously uncharacterised enamine 7 as a
dark red solid. Introduction of an enamine functional
1
protons in the H NMR spectrum as well as carbon
1
group was confirmed by the observation of H NMR signals
resonances at d 190.8 (CH) and d 133.1 (CH ) supported
2
at d 7.50 and 7.15 (both 13 Hz doublets) and 3.08 (6H, s)
the formation of 14. In order to circumvent the instability of
14, cyclisation chemistry was investigated using a three
step-one pot reaction sequence. Thus dione 6 was reacted
with DMFDEA to afford enamine 7, that was in-turn reacted
with Eschenmoser’s salt in DMF to afford a product that was
1
3
0
0
and C NMR signals at d 94.4 (C-1 ), 155.2 (C-2 ) and
1.1 (NMe ). As discussed in the Biological Activities
4
2
section, 7 exhibited a wide range of potent biological
activities including selective human tumour cytotoxicity,
antiviral and antifungal properties. In order to extend the
structure activity relationship of this finding, analogues 8, 9,
finally cyclised with NH Cl in HOAc. Subsequent work up
4
and chromatography yielded 5-acetoxymethylascididemin
(15) (27%). (Fig. 3)
1
0
10
10
1
0, 11, and 12 (Fig. 2) were synthesised in similar
Figure 2.
Figure 3.

B. S. Lindsay et al. / Tetrahedron 56 (2000) 497–505
499
Figure 4.
That the putative enal intermediate had undergone a
Michael Reaction with acetate before final cyclisation was
confirmed by extensive spectroscopic characterisation of
the presence of an essentially intact fragment of ascidide-
min, as well as an 11-substituted pyrido[2,3-b]acridine-
5,12-dione fragment. The observation of benzenoid-type
1
1
5. Substitution at the 5-position was confirmed by the
JCH coupling constants for C-5 (d, Jˆ163 Hz) and particu-
observation of H-6 (d 9.35) as a singlet, while the obser-
vation of new H (at d 5.92 (2H, s) and 2.20 (3H, s)) and C
NMR resonances (d 64.2, CH ; 20.9, CH ) confirmed the
larly C-6 (d, Jˆ163 Hz), as well as HMBC correlations
from both H-5 and H-6 to a new aromatic carbon resonance
(C-7, d 137.7) supported the unusual non-pyridinoid nature
of ring-E of the ascididemin fragment. Finally, interfrag-
1
13
2
3
existence of an acetoxymethyl functional group. Further
0
confirmation of its placement at C-5 was made by the obser-
ment connectivity between C-7 and C-11 was established
1
13
vation of long range H– C correlations from the methyl-
ene protons at 5.92 ppm to carbons C-4b, C-5, C-6 and the
acetate carbonyl (d 170.5). Trace quantities (Ͻ5%) of a
product attributable to 5-formylascididemin (16) (dH
by the observation of an HMBC correlation between H-6 (d
0
7.69) and C-11 (d 157.8) thus securing the structure of 19.
Ethylamine analogues of enamine 7 were of interest as an
extension of the structure-activity relationship of the bio-
logically active enamines, and also because of their similarity
to heterocyclic analogues of the human antitumor agent
1
0.94 (CHO, s), 9.54 (H-6, s)) were also detected in the
product mixture, however, due to insufficient quantities,
this product was not isolated nor characterised.
12
mitoxantrone. Dimethylaminoethyl derivative 20 was
prepared by reaction of dione 6 with Eschenmoser’s salt
Whilst exploring optimisation of the synthesis of 15, we
found that the relative quantity of acetic acid used in the
final step was absolutely crucial in determining what
reaction products were obtained. Thus using the established
three step-one pot reaction but adding only half as much
acetic acid solvent in the final step yielded the novel ter-
pyridyl analogue 17 as the major product. HRFABMS indi-
cated a molecular formula of C H N O , while the
1
in DMF at 120ЊC (95%). The observation of new H
NMR resonances at d 4.09 and 3.06 (both br t, Jˆ8 Hz)
13
and d 2.72 (6H, s) and new C resonances at d 56.8 and
26.0 (both CH ) and d 43.4 (CH ) confirmed conversion of
2
3
the methyl group in 6 to a dimethylaminoethyl group in 20.
The diethylamino derivative 21 was prepared by Mannich
reaction with dione 6 utilising diethylamine as the base
(Fig. 5).
3
7
17
5
4
1
13
observation of 9 H and 19 C NMR resonances suggested
the product was symmetrical. The H NMR spectrum
1
showed signals typical for the 11-substituted pyrido[2,3-b]-
acridine-5,12-dione chromophore, while the observation
It was found that 20 could be readily converted into asci-
7
b
didemin in 69% by heating in acetic acid with NH Cl.
4
of an additional aromatic three-proton spin system, at d
Presumably the initial reaction product is 5,6-dihydroasci-
didemin but this is oxidised in situ to the more stable fully
aromatic ascididemin. A natural extension of this reaction
7h
7
.73 (1H, t, Jˆ2 Hz) and 8.69 (2H, d, Jˆ2 Hz), was
attributable to a 3,5-disubstituted pyridine ring. The deaza
analogue 18 was synthesised in similar fashion, starting with
1
1
11-methyl-benzo[b]acridine-5,12-dione (Fig. 4).
Further use of the three step-one pot reaction with an
additional doubling of the final step concentration yielded
no terpyridyl analogue 17 and only minor amounts of
5-acetoxymethyl- (15) and 5-formyl-ascididemin (16). The
major product of the reaction was, however, deduced to be
the unprecedented nonacycle alkaloid 19. The structural
elucidation of 19 relied on NMR spectroscopy and was
supported by HRFAB mass spectrometry. HRFABMS
determined the molecular formula to be C H N O ,
3
5
16
4
3
1
13
while inspection of the H and C NMR data suggested
Figure 5.

5
00
B. S. Lindsay et al. / Tetrahedron 56 (2000) 497–505
Table 1. In vitro antiviral and antimicrobial activities of 2 and its analogues
viruses. The saturated side-chain derivatives 20 and 21 both
showed diminished antimicrobial and antiviral properties
compared to enamine 7. As demonstrated by results for
a
b
Compound
Antiviral
HSV-1
?
Antimicrobial
13
PV1
?
Cyto (dose)
Ec
10
Bs
14
Ca
11
enamine 10, the quinoid functional group is not required for
the observation of antifungal or antiviral properties, but any
reduction in chromophore size below three fused rings (i.e.
11 and 12) resulted in less potent antiviral and antimicrobial
activities. This chromophore size dependence of enamine
biological activity is consistent with a mechanism action
2
1ϩ(10)
2
0
20
20
1
5
0
0
0
0
0
10
0
2
2
2
2
0
20
20
1
2
2
2
6
7
8
9
1
1
1
2
2
1
1
1
5
2
3
?
?
–
–
?
?
–
–
4ϩ(80)
4ϩ(20)
3ϩ(20)
Ϫ(20)
Ϫ(10)
1ϩ(10)
Ϫ(10)
2ϩ(10)
2ϩ(10)
2ϩ(80)
1ϩ(80)
Ϫ(10)
7
5
4
5
2
0
20
20
20
20
3
0
0
0
20
20
14
4
0
involving DNA intercalation.
c
–
–
10
12
4
12
9
1
0
9
10
4
1
4ϩ
2ϩ
4ϩ
4ϩ
?
?
2ϩ
–
4ϩ
2ϩ
?
4ϩ
?
?
–
–
–
12
0
2
4
0
0
5
8
0
0
0
10
10
21
11
4
0
2
1
0
The biological properties of all compounds were further
evaluated for cytotoxicity against the murine leukaemia
cell line P388 and by the NCI in their in vitro disease-
oriented primary antitumour screen (Table 2). Similar
P388 activity was observed for ascididemin (2) and
analogues 15, 19, 22, 23 and 24 irrespective of the nature
of the substituent, while all ring-E opened derivatives, with
the exception of enamine 7, were less cytotoxic than the
parent compound. The NCI testing provides observations
0
1
2
0
1
7
8
9
Ϫ(10)
Ϫ(80)
1ϩ(80)
1ϩ(80)
–
3ϩ
?
?
?
3
5
8
0
a
15a
on the mean response parameters (GI , TGI, LC ),
Assay methods have been described elsewhere. Zones of cytotoxicity
and antiviral activity were measured microscopically as excess radii
from the disc and indicated by–, none detectable; 1ϩ, 1–2 mm; 2ϩ,
–3.5 mm; 3ϩ, 3.5–4.5 mm; 4ϩ, greater than 4.5 mm; ‘?’ indicates
that cell death due to viral infection and compound toxicity has masked
any antiviral activity.
Zone of microbial inhibition against E. coli, B. subtilis and C. albicans
for 120 mg of test compound (except where indicated by superscripted
number, which is the loading in mg used) on a 6 mm paper disc. Incu-
bation was for 18 h at 35ЊC. Zones measured as excess radii in mm.
Data from Ref. 8b.
50 50
differential cellular sensitivity and subpanel-specific
15b
patterns of sensitivity. In these primary assays ascidide-
min (2) was found to be selective for the melanoma and
colo-rectal subpanels and to exhibit a panel average LC
2
50
16
b
c
value of 6.5 mM.
While the 5-acetoxymethyl (15) and 6-methyl (22)
derivatives exhibited similar potency and subpanel selec-
tivity to that observed for ascididemin, the more lipophilic
derivatives 23 and 24 were less cytotoxic towards human
tumor cell lines in vitro. Disruption of the pentacyclic struc-
ture of ascididemin generally resulted in derivatives that
exhibited less potent cytotoxicity in the NCI assays,
however enamines 7 and 8 and amine 20 exhibited sufficient
potency and selectivity to warrant further in vivo evaluation.
result was to then attempt a Mannich reaction of dione 6
with paraformaldehyde and NH Cl in acetic acid, which
conveniently afforded ascididemin in 80% yield. As we
have communicated, this reaction was found to be quite
general for a range of analogues of dione 6.
4
7
j,m
We have
now also found that the reaction is quite general for a range
of aldehydes, allowing for simple insertion of carbon-based
substituents at the 6-position of ascididemin. Thus reaction
COMPARE analysis of the NCI in vitro cytotoxicity profile
observed for ascididemin indicated high similarity with
those profiles observed for amine analogue 20 (Pairwise
Correlation Coefficientˆ0.79) and enamine 7 (0.69),
suggestive of similar mechanisms of cytotoxic action for
of dione 6 with NH Cl and acetaldehyde, benzaldehyde or
4
cinnamaldehyde in acetic acid afforded the increasingly
lipophilic ascididemin analogues 22, 23 and 24 in yields
of 90%, 81% and 72% respectively.
17
the three compounds. Given the propensity of para-
quinone containing compounds to exhibit cellular toxicity
via a mechanism involving redox cycling and the formation
18
Biological Activities
of reactive oxygen species (ROS) we have undertaken
studies to ascertain whether ascididemin is also capable of
exhibiting toxicity via this mechanism. Our preliminary
results suggest that ascididemin can indeed act to oxi-
datively damage DNA via a thiol-dependent conversion of
All compounds were initially assayed for cytotoxicity
against the BSC monkey kidney cell line, antiviral activity
towards DNA (HSV-1) and RNA (PV-1) viruses and for
antimicrobial activity against Gram positive and Gram
negative bacteria and a fungus (Table 1). Ascididemin (2)
exhibited more potent activity towards Gram positive and
Gram negative bacteria than towards fungi and had no
detectable antiviral activity due to cytotoxicity of the
compound towards the viral host cells. Substitution at the
19
oxygen to DNA-cleaving oxygen radicals.
The potency and tumour cell line selectivity observed in
vitro for ascididemin (2) and analogues 7, 9, 20, 22, 23
and 24 warranted further study in vivo. Preliminary in
vivo evaluation was made at the NCI using the Hollow
15f
5
or 6 positions of ascididemin resulted in reduced antibac-
Fibre protocol, but of the seven compounds tested only
three (2, 20 and 22) exhibited significant reductions in cell
viability. Ascididemin exhibited localised activity towards
breast (MDA-MB-231 and -435) and CNS (SF-295 and
U251) tumour cells, and more promisingly, towards the
remote subcutaneously located melanoma (LOX
IMVI) and non-small cell lung (NCI-H23) cell lines.
terial activities, while 15 and 22 exhibited a modest degree
of antifungal activity towards the human pathogenic fungi
C. albicans. Enamine functionalised diones exhibited
substantially increased antimicrobial activities compared
to the dione 6, and also resulted in the observation of anti-
viral activities towards both DNA (HSV-1) and RNA (PV1)

B. S. Lindsay et al. / Tetrahedron 56 (2000) 497–505
Table 2. In vitro antitumour activities (mM) of 2 and its analogues
501
a
b
c
Compound (NSC)
P388 IC50
GI50
TGI
LC50
2
1
2
2
2
6
7
8
9
1
1
1
2
2
1
1
1
(675670)
0.4
0.5
1.4
1.4
2.8
56
0.4
14
12
24
69
71
4
0.18 (2.6)
0.05 (1.9)
0.3 (2.3)
4.1 (2)
2.6 (1.7)
11
0.9 (2.7)
0.6 (3.7)
1.6 (3.8)
16 (1.7)
9 (1.8)
6.5 (2.5)
8.0 (2.8)
6.9 (2.4)
47 (1.3)
36 (1.3)
79
24 (2.2)
23 (1.9)
66 (1.2)
5 (694488)
2 (683787)
3 (686553)
4 (686555)
d
(659780)
56
(680733)
(680732)
(686556)
0
0.6 (2.6)
1.1 (2.1)
3.3 (2.7)
4.3 (2.3)
29 (1.6)
n.t.
n.t.
100 (0)
1.7 (3.5)
6.9 (2)
1.4 (2.6)
30 (1.6)
6.8 (2.4)
7.7 (1.9)
e
n.t.
n.t.
n.t.
1
n.t.
2 (690415)
0 (680734)
1 (683790)
7 (694492)
8 (694490)
9 (694491)
98 (0.5)
0.3 (3)
1.4 (2)
0.2 (2.3)
4.3 (2.8)
1.1 (2.8)
100 (0)
14 (2)
42 (1.4)
15.4 (2.2)
75 (1.2)
38 (1.3)
7.6
4
8
3.0
a
16
NSC number is the NCI reference number for each compound. Search for this number at the NCI website to view complete information of all in vitro
assay profiles.
IC50 against the P388 D1 murine leukaemia cell line.
GI50 (50% growth inhibition), TGI (total growth inhibition) and LC50 (50% cell kill) data are averaged calculated mean micro-molar values obtained from
b
c
two experiments at the NCI. Value in parenthesis is the observed range of data, being the number of log10 units between the most and least sensitive cell
line(s) in the panel.
P388 IC50 value from Ref. 8b, NCI data from Ref. 8c.
n.t. Not tested.
d
e
6
-Methylascididemin (22) exhibited a similar profile of
Experimental
activity, but also showed activity towards the colo-rectal
tumour cell lines COLO 205 and SW-620, while amine 20
only showed moderate activity towards the CNS tumour cell
line SF-295.
General methods
Details of instrumental methods and general experimental
7
m
15
procedures and biological assays have been reported
previously. NMR spectral assignments were made with
the aid of the 2-dimensional NMR experiments, and
Based upon the observed Hollow Fibre assay results, more
extensive subcutaneous xenograft in vivo assays were
undertaken on ascididemin and amine 20.
8
c,15f
1
13
Ascididemin
H– C coupling constants were determined by gated
decoupled C NMR experiments. The preparations of
11-methylpyrido[2,3-b]acridine-5,12-dione (dione 6),
8
c
13
was found to exhibit no significant ꢀT=C Ͻ 40%† anti-
7
b
tumour activity using non-toxic doses and standard NCI
2
0
11 21
delivery schedules (data not shown). The best result for
the compound was observed against the colo-rectal tumour
line HCT-116, with a T=C of 58% at 8 mg/kg/IP dose. A
similar result has been reported for xenograft evaluation of
11-methylbenzo[b]acridine-5,12-dione,
cleistopholine,
10
enamines 10, 11 and 12 have been reported previously.
0
11-[2 -(Dimethylamino)vinyl]pyrido[2,3-b]acridine-5,12-
dione (7). Dione 6 (200 mg, 0.73 mmol), dimethylforma-
mide diethyl acetal (DMFDEA) (0.37 mL, 2.16 mmol) and
8
c
2
-bromoleptoclinidinone (1, 2-bromoascididemin). Amine
20, however, exhibited moderate antitumour activity
ꢀ
T=C 31% at 36 mg/kg/IP dose) towards the SF-295 (CNS)
tumour cell line at non-toxic doses.
DMF (4 mL), was heated to 120ЊC under N for 1 h. The
2
reaction mixture was allowed to cool and the solvent and
unreacted DMFDEA was removed in vacuo to yield 7 as a
blood red solid (228 mg, 95%). mp 230–231ЊC. IR (film) n
Conclusion
1677, 1649, 1598, 1556, 1463, 1376, 1276, 1254,
Ϫ1
1
106 cm . UV (CH OH): l
(log e) 476 nm (4.0), 278
3
max
1
Although isolated as in vitro cytotoxic agents, it would
(4.4), 237 (4.4). H NMR (400 MHz, CDCl ) d 9.00 (1H,
3
8
c
appear from our results and those of others, that ascidide-
min and its 2-bromo analogue have limited in vivo anti-
tumour activity. Increasing lipophilicity at the 6-position
of ascididemin also results in further degradation of in
vivo activity. A limiting factor in attempting more detailed
in vivo evaluation (including dose and delivery route
optimisation) of examples of the pyridoacridone family of
alkaloids is the poor water solubility of the compounds, an
issue we are currently attempting to address. The discovery
however, of selective cytotoxicity for some ascididemin-
related compounds against certain human-tumour cell
lines is particularly encouraging and adds focus to the
considerable synthetic interest in this class of alkaloid.
dd, Jˆ4.6, 1.7 Hz, H-2), 8.53 (1H, dd, Jˆ7.9, 1.7 Hz, H-4),
8.13 (1H, d, Jˆ8.7 Hz, H-10), 8.11 (1H, d, Jˆ8.7 Hz, H-7),
7.62 (1H, t, Jˆ7.6 Hz, H-8), 7.59 (1H, dd, Jˆ7.9, 4.6 Hz,
0
H-3), 7.50 (1H, d, Jˆ12.9 Hz, H-1 ), 7.38 (1H, t, Jˆ7.6 Hz,
0
H-9), 7.15 (1H, d, Jˆ12.9 Hz, H-2 ), 3.08 (6H, s, NMe ).
2
13
C NMR (100 MHz, CDCl ) d 182.7 (d, Jˆ3 Hz, C-5),
3
0
181.7 (C-5), 155.2 (d, Jˆ163 Hz, C-2 ), 155.1 (ddd,
Jˆ182, 7, 3 Hz, C-2), 153.7 (s, C-11), 150.4 (dd, Jˆ11,
5 Hz, C-12a), 148.7 (t, Jˆ8 Hz, C-6a), 148.1 (s, C-5a),
135.0 (dd, Jˆ169, 6 Hz, C-4), 131.6 (dd, Jˆ162, 9 Hz,
C-8), 130.9 (dd, Jˆ165, 8 Hz, C-7), 129.0 (d, Jˆ7 Hz,
C-4a), 128.8 (dd, Jˆ163, 8 Hz, C-10), 127.2 (dd, Jˆ163,
8 Hz, C-9), 127.2 (m, C-10a), 126.9 (dd, Jˆ167, 9 Hz, C-3),

5
02
B. S. Lindsay et al. / Tetrahedron 56 (2000) 497–505
0
1
18.3 (s, C-11a), 94.4 (d, Jˆ159 Hz, C-1 ), 41.1 (br, NMe ).
which time the blood red coloration dissipated to orange/
2
ϩ
ϩ
EIMS m/z (%) 329 (M , 40). HREIMS (M ) found
brown. The mixture was then allowed to cool and NH Cl
4
3
29.1157, C H N O requires 329.1164.
(250 mg, 4.67 mmol) and acetic acid (25 mL volume
crucial) were added, followed by continued heating at
115ЊC for 30 min. After cooling, the dark reaction mixture
was poured onto ice, made basic with aqueous KOH (10%)
2
0
15
3
2
0
1-[2 -(Dimethylamino)vinyl]benzo[b]acridine-5,12-dione
11
1
(
8). 11-methylbenzo[b]acridine-5,12-dione (50 mg, 0.183
mmol), DMFDEA (63 mL, 0.37 mmol) in DMF (2 mL)
and extracted exhaustively with CHCl . The combined
3
were heated at 125ЊC under N for 0.5 h. Solvent was
organic extract was washed with brine, dried (MgSO4)
and the solvent removed in vacuo. The product was purified
by prep-TLC (SiO , CH Cl /MeOH/TEA 100:1:0.01) yield-
2
removed in vacuo to yield 8 as a red solid (85 mg, 94%).
mp 217–219ЊC (CHCl , cyclohexane); IR (film) n 1678,
3
2
2
2
Ϫ1
1
(
634, 1596, 1557 cm . UV (MeOH) l
(log e) 241 nm
ing 15 (16 mg, 27%) as a yellow powder. mp 257–259ЊC.
IR (film) n 2919, 1720, 1674, 1641, 1582, 1540, 1520, 1457,
max
1
4.2), 277 (4.6), 464 (4.3). H NMR (400 MHz, CDCl ) d
3
Ϫ1
1
8
1
1
3
.25 (2H, m), 8.18 (2H, d, Jˆ8.6 Hz), 7.73 (1H, td, Jˆ7.5,
.4 Hz), 7.67 (2H, td, Jˆ7.4, 1.2 Hz), 7.41 (1H, td, Jˆ7.6,
.2 Hz), 7.39 (1H, d, Jˆ13.0 Hz), 7.06 (1H, d, Jˆ13.0 Hz),
1415, 1382, 1365, 1340, 1311 cm . H NMR (400 MHz,
CDCl ) d 9.35 (1H, s, H-6), 9.19 (1H, dd, Jˆ4.6, 1.8 Hz,
3
H-9), 8.78 (1H, dd, Jˆ7.9, 1.9 Hz, H-11), 8.68 (1H, dd,
Jˆ8.1, 1.4 Hz, H-1), 8.67 (1H, dd, Jˆ8.1, 1.4 Hz, H-4),
8.04 (1H, ddd, Jˆ8.1, 7.1, 1.2 Hz, H-2), 7.96 (1H, ddd,
Jˆ8.5, 7.1, 1.4 Hz, H-3), 7.68 (1H, dd, Jˆ7.9, 4.6 Hz,
1
3
.10 (6H, s, NMe ). C NMR (100 MHz, CDCl ) d 183.2
2
3
(
d, Jˆ4 Hz), 183.0 (d, Jˆ3 Hz), 154.4 (d, Jˆ165 Hz), 153.4
(
m), 149.0 (m), 148.9 (m), 135.7 (d, Jˆ7 Hz), 134.2 (dd,
13
Jˆ162, 8 Hz), 132.9 (dd, Jˆ163, 8 Hz), 132.7 (t, Jˆ6 Hz),
H-10), 5.92 (2H, s, CH O), 2.20 (3H, s, CH ). C NMR
2 3
1
31.4 (ddd, Jˆ160, 9, 2 Hz), 131.0 (dd, Jˆ164, 8 Hz), 128.9
(100 MHz, CDCl ) d 181.7 (C-12), 170.5 (OC(O)Me),
3
(
dd, Jˆ161, 8 Hz), 127.6 (m), 127.1 (dm, Jˆ164 Hz), 127.0
155.8 (C-9), 152.4 (C-6), 152.3 (C-7b), 150.0 (C-7a),
146.6 (C-13a), 145.9 (C-12a), 137.5 (C-4b), 136.4 (C-11),
133.9 (C-1), 131.7 (C-2), 131.0 (C-3), 128.5 (C-11a), 127.0
(C-5), 126.9 (C-4), 125.8 (C-10), 123.6 (C-4a), 118.7 (C-
(
dm, Jˆ164 Hz), 126.9 (dm, Jˆ164 Hz), 118.6 (m), 94.2 (d,
ϩ
ϩ
Jˆ161 Hz). EIMS m/z (%) 278 (M , 70). HREIMS (M )
found 328.1213, C H N O requires 328.1212. Anal. calcd
2
1
16
2
2
ϩ
for C H N O ·H O: C, 72.8; H, 5.2; N, 8.1%. Found: C,
12b), 64.2 (CH O), 20.9 (CH ). EIMS m/z (%) 355 (M ,
2
1
16
2
2
2
2
3
7
3.1; H, 4.8; N, 7.9.
95). HREIMS found 355.0955, C H N O requires
21 13 3 3
3
55.0957.
0
4
-[2 -(Dimethylamino)vinyl]benzo[b]quinoline-5,10-qui-
2
1
0
0
none (9). Cleistopholine (25 mg, 0.11 mmol), DMFDEA
3 ,5 -Di(11-pyrido[2,3-b]acridine-5,12-dione)pyridine (17).
Dione 6 (200 mg, 0.73 mmol), DMFDEA (0.40 mL,
2.34 mmol) and DMF (4 mL) were heated to 120ЊC under
N2 for 1 h. To the reaction mixture was then added
Eschenmoser’s salt (290 mg, 1.57 mmol) and DMF
(4.0 mL) and heating continued at 120ЊC for 15 min under
N . The mixture was then allowed to cool and NH Cl
(
77 mL, 0.45 mmol) in DMF (2 mL) were heated at 125ЊC
under N for 1.3 h. The red mixture was poured onto cold
2
water (150 mL) and extracted with CH Cl ꢀ3×50 mL†: The
2
2
red organic layer was washed with water ꢀ4×100 mL†; then
brine ꢀ2×150 mL† and dried (MgSO ) and the solvent
4
removed in vacuo. Recrystallisation of the red solid
2
4
(
CHCl , cyclohexane) afforded 9 (28 mg, 93%). mp 241–
(1.000 g, 18.70 mmol) and acetic acid (50 mL volume
crucial) were added followed by continued heating at reflux
for 30 min. After cooling the dark reaction mixture was
poured onto ice, made basic with aqueous KOH (10%)
3
2
1
4
43ЊC. IR (smear) 1681, 1648, 1611, 1568, 1302, 1248,
Ϫ1
196, 1121 cm . UV (MeOH) l
(log e) 270 nm (5.6),
max
1
05 (4.4), 518 (3.4). H NMR (400 MHz, CDCl ) d 8.48
3
(
8
1H, d, Jˆ5.7 Hz, H-2), 8.28 (1H, dd, Jˆ7.8, 1.2 Hz, H-6),
and extracted exhaustively with CHCl . The combined
3
.23 (1H, dd, Jˆ7.5, 1.3 Hz, H-9), 7.77 (1H, td, Jˆ7.5,
organic extract was washed with brine, dried (MgSO4)
and the solvent removed in vacuo. Repeated silica gel
chromatography afforded 17 (36 mg, 17%). mp Ͼ310ЊC.
IR (film) n 1688, 1638, 1582, 1496, 1460, 1374, 1329,
1
.5 Hz, H-8), 7.72 (1H, td, Jˆ7.5, 1.5 Hz, H-7), 7.46 (1H,
0
d, Jˆ5.7 Hz, H-3), 7.42 (1H, d, Jˆ13.4 Hz, H-2 ), 7.12 (1H,
0
13
d, Jˆ13.4 Hz, H-1 ), 3.07 (6H, s, NMe ). C NMR
2
Ϫ1
1
(
100 MHz, CDCl ) d 185.3 (d, Jˆ4 Hz, C-10), 182.9 (d,
1263, 1223, 1101, 1066, 995, 752 cm . H NMR
3
Jˆ3 Hz, C-5), 151.5 (d, Jˆ12 Hz, C-10a), 150.8 (m, C-4),
(400 MHz, CDCl ) d 9.16 (2H, dd, Jˆ4.5, 1.7 Hz, H-2),
3
0
1
50.7 (d, Jˆ181 Hz, C-2), 148.3 (dm, Jˆ160 Hz, C-2 ),
34.8 (m, C-9a), 134.2 (dd, Jˆ163, 9 Hz, C-8), 133.2 (dd,
8.80 (2H, dd, Jˆ7.9, 1.7 Hz, H-4), 8.69 (2H, d, Jˆ2.1 Hz,
0
1
H-2 ), 8.54 (2H, d, Jˆ8.3 Hz, H-7), 8.17 (2H, d, Jˆ8.3 Hz,
Jˆ164, 7 Hz, C-7), 132.4 (m, C-5a), 127.0 (dd, Jˆ165,
H-10), 8.00 (2H, ddd, Jˆ8.2, 6.9, 1.2 Hz, H-8), 7.89 (2H,
ddd, Jˆ8.2, 6.9, 1.2 Hz, H-9), 7.81 (2H, dd, Jˆ8.0, 4.6 Hz,
7
Hz, C-9), 126.9 (dd, Jˆ166, 8 Hz, C-6), 122.4 (m,
0
13
C-4a), 119.7 (ddd, Jˆ160, 8, 6 Hz, C-3), 93.2 (d,
H-3), 7.73 (1H, t, Jˆ2.1 Hz, H-4 ). C NMR (100 MHz,
0
Jˆ160 Hz, C-1 ), 41.1 (br m, NMe ). EIMS m/z (%) 278
CDCl ) d 181.6 (s, C-12), 181.3 (d, Jˆ4 Hz, C-5), 155.8
2
3
ϩ
ϩ
(
M , 70). HREIMS (M ) found 278.1054, C H N O
(ddd, Jˆ184, 7, 4 Hz, C-2), 149.6 (m, C-12a), 149.6 (m,
1
7
14
2
2
requires 278.1055. Anal. calcd. for C H N O ·0.25H O:
C-6a), 149.2 (bs, C-11), 147.7 (ddd, Jˆ183, 12, 6 Hz,
1
7
14
2
2
2
0
C, 72.2; H, 5.2; N, 9.9%. Found: C, 72.3; H, 5.0; N, 10.0.
C-2 ), 147.2 (s, C-5a), 136.1 (dd, Jˆ170, 6 Hz, C-4),
0
1
35.6 (dt, Jˆ165, 6 Hz, C-4 ), 133.7 (dd, Jˆ163, 9 Hz,
0
5
-Acetoxymethylascididemin (15). Dione 6 (46 mg,
.17 mmol), DMFDEA (0.09 mL, 0.53 mmol) and DMF
C-8), 132.5 (obsc, C-3 ), 131.7 (dd, Jˆ163, 6 Hz, C-7),
131.4 (dd, Jˆ163, 9 Hz, C-9), 130.7 (d, Jˆ8 Hz, C-4a),
129.8 (t, Jˆ6 Hz, C-10a), 128.4 (dd, Jˆ165, 7 Hz, C-10),
128.3 (dd, Jˆ165, 9 Hz, C-3), 124.5 (s, C-11a). FABMS m/z
0
(1 mL) were heated to 120ЊC under N for 1 h. The reaction
2
mixture was allowed to cool and the solvent and unreacted
DMFDEA removed under reduced pressure. The blood red
residue, Eschenmoser’s salt (54 mg, 0.29 mmol) and DMF
ϩ
ϩ
(%) 620 ([(MϩNa) ϩ2H], 45), 619 ([(MϩNa) ϩ2H -H],
ϩ
ϩ
95), 618 ((MϩNa) , 100), 598 ([(MϩH) ϩ2H], 30),
597 ([(MϩH) ϩ2H -H], 65), 596 ((MϩH) , 85).
ϩ
ϩ
(
5.5 mL) were heated under N at 115ЊC for 30 min, over
2

B. S. Lindsay et al. / Tetrahedron 56 (2000) 497–505
503
0
0
HRFABMS found 596.1356, C H N O requires
(dd, Jˆ162, 8 Hz, C-7 ), 128.9 (obsc, C-10a ), 128.0 (dd,
3
7
18
5
4
0
0
5
96.1359.
Jˆ167, 9 Hz, C-3 ), 127.9 (d, Jˆ7 Hz, 4a ), 127.5 (dd,
0
Jˆ163, 7 Hz, C-10 ), 127.2 (obsc, C-11a), 125.2 (m,
0
0
3
,5 -Di(11-benzo[b]acridine-5,12-dione)pyridine (18). 11-
C-4a), 125.0 (d, Jˆ163 Hz, C-5), 123.3 (dd, Jˆ167, 8 Hz,
11
0
Methylbenzo[b]acridine-5,12-dione (200 mg, 0.73 mmol),
C-10), 123.0 (d, Jˆ7 Hz, C-12b), 122.9 (s, 11a ), 122.3 (dd,
ϩ
DMFDEA (0.40 mL, 2.34 mmol) and DMF (4 mL) were
Jˆ161, 8 Hz, C-4). FABMS m/z (%) 543 ([(MϩH) ϩ2H],
ϩ
ϩ
heated to 120ЊC under N for 30 min. To the reaction
15), 542 ([(MϩH) ϩ2H -H], 30) 541 ((MϩH) , 45).
2
ϩ
mixture were then added Eschenmoser’s salt (277 mg,
HRFABMS found 541.1301 (MH ), C H N O requires
35
17
4
3
1
.50 mmol) and DMF (4.0 mL) and heating continued at
541.1301.
1
20ЊC for 15 min under N . The mixture was then allowed
2
0
to cool and NH Cl (1.000 g, 18.70 mmol) and acetic acid
11-[2 -(Dimethylamino)ethyl]pyrido[2,3-b]acridine-5,12-
dione (20). Dione 6 (249 mg, 0.91 mmol), Eschenmoser’s
salt (246 mg, 1.33 mmol) and DMF (6 mL), were heated to
4
(
50 mL volume crucial) were added followed by continued
heating at reflux for 30 min. After cooling the dark reaction
mixture was poured onto ice, made basic with aqueous KOH
120ЊC under N for 30 min. The orange/brown reaction
2
(
10%) and extracted exhaustively with CHCl . The
mixture was cooled, poured onto ice-water (100 mL) and
the resulting mixture made basic with aqueous KOH (10%).
The product was extracted with CH Cl ꢀ3×50 mL†; washed
3
combined organic extract was washed with brine, dried
(
MgSO ) and the solvent removed in vacuo. Repeated silica
4
2
2
gel chromatography afforded 18 as a poorly soluble tan solid
with brine which had been made basic with a few drops of
1
(
20 mg, 9%). mp Ͼ310ЊC. H NMR (400 MHz, CDCl )
aqueous KOH (10%), dried (MgSO ) and the solvent
3
4
0
dˆ8.69 (2H, d, Jˆ2.0 Hz, H-2 ), 8.55 (2H, d, Jˆ8.4 Hz,
removed in vacuo to yield 20 as an orange brown residue
(285 mg, 95%), mp 158–159ЊC. IR (film) n 3060, 2955,
2919, 2861, 1690, 1643, 1614, 1584, 1502, 1467, 1414,
1373, 1331, 1267, 1220, 1102, 1067, 997, 967, 767, 732,
H-7), 8.46 (2H, m, H-2 or H-3), 8.24 (2H, m, H-2 or H-3),
8
.10 (2H, d, Jˆ8.5 Hz, H-10), 7.99 (2H, ddd, Jˆ8.4, 6.9,
1
.2 Hz, H-8), 7.86 (2H, m, H-1 and H-4) 7.83 (2H, ddd,
0
Ϫ1
1
Jˆ8.4, 6.9, 1.2 Hz, H-9), 7.64 (1H, t, Jˆ2.0 Hz, H-4 ).
697 cm . H NMR (400 MHz, CDCl ) d 9.12 (1H, dd,
3
ϩ
FABMS m/z (%) 594 ((MϩH) , 10). HRFABMS found
Jˆ4.6, 1.8 Hz, H-2), 8.68 (1H, dd, Jˆ8.0, 1.8 Hz, H-4),
ϩ
594.1453 (MH ), C H N O requires 594.1454.
8.57 (1H, dd, Jˆ8.4, 0.9 Hz, H-10), 8.38 (1H, dd, Jˆ8.4,
3
9
20
3
4
0
.9 Hz, H-7), 7.91 (1H, ddd, Jˆ8.3, 6.8 Hz, 1.4, H-8), 7.83
0
0
0
0
0
7
-(11 -Pyrido[2 ,3 -b]acridine-5 ,12 -dione)-7-carbaasci-
(1H, ddd, Jˆ8.3, 6.8, 1.4 Hz, H-9), 7.75 (1H, dd, Jˆ7.8,
0
didemin (19). Enamine 7 (65 mg, 0.20 mmol), Eschenmoser’s
salt (70 mg, 0.38 mmol) and DMF (6 mL) were heated at
4.6 Hz, H-3), 4.09 (2H, bt, Jˆ7.5 Hz, H-1 ), 3.06 (2H, bt,
0
13
Jˆ8.4 Hz, H-2 ), 2.72 (6H, s, NMe ). C NMR (100 MHz,
2
1
05ЊC for 10 min under N . The DMF was then removed
CDCl ) d 183.2 (s, C-12), 181.2 (d, Jˆ4 Hz, C-5), 155.7
2
3
under reduced pressure and the residue was dissolved in
(ddd, Jˆ182, 7, 3 Hz, C-2), 152.0 (m, C-11), 149.7 (dd,
Jˆ11, 5 Hz, C-12a), 149.0 (dd, Jˆ9, 7 Hz, C-6a), 147.3
(s, C-5a), 135.9 (dd, Jˆ169, 6 Hz, C-4), 133.3 (dd,
Jˆ162, 9 Hz, C-8), 132.3 (dd, Jˆ165, 8 Hz, C-7), 130.9
(dd, Jˆ163, 8 Hz, C-9), 130.1 (obsc, C-4a), 129.2 (m,
C-10a), 128.2 (dd, Jˆ167, 9 Hz, C-3), 125.7 (dd, Jˆ163,
CH Cl /MeOH (8 mL, 4:1) and poured onto glacial acetic
2
2
acid (5 mL), water (1.5 mL) and NH Cl (732 mg,
4
1
3.7 mmol). The mixture was stirred briskly at ambient
temperature for 24 h, diluted with water, made basic with
aqueous KOH (10%) and extracted exhaustively
0
with CHCl . The combined organic extract was washed
7 Hz, C-10), 124.9 (obsc, C-11a), 56.8 (t, Jˆ139 Hz, C-2 ),
3
0
with brine, dried (MgSO ) and the solvent removed in
43.4 (qd, Jˆ139, 4 Hz, NMe ) 26.0 (t, Jˆ131 Hz, C-1 ).
4
2
ϩ
vacuo. Repeated silica gel chromatography afforded 19 as
a tan solid (17 mg, 32%). mp (decomp.) Ͼ310ЊC. IR (film)
n 2924, 1689, 1667, 1650, 1581, 1461, 1375, 1328, 1259,
EIMS m/z (%) 331 (M , 15). HREIMS found 331.1317
ϩ
(M ), C H N O requires 331.1321. Anal. calcd for
20
17
3
2
C H N O ·0.75CH Cl : C, 63.1; H, 4.7; N, 10.6%.
20
17
3
2
2
2
Ϫ1
1
1
065, 1035, 992, 764 cm . H NMR (400 MHz, CDCl ) d
Found: C, 63.4; H, 4.7; N, 10.8.
3
8
.98 (1H, d, Jˆ8.8 Hz, H-5), 8.97 (1H, dd, Jˆ4.5, 1.7 Hz,
0
0
H-2 ), 8.79 (2H, dd, Jˆ7.9, 1.7 Hz, H-4 and H-4 ), 8.71 (1H,
Conversion of 20 to ascididemin (2). Dione 20 (926 mg,
dd, Jˆ8.1, 1.3 Hz, H-1), 8.64 (1H, dd, Jˆ7.8, 1.9 Hz, H-11),
2.8 mmol) and NH Cl (2.9 g, 53.2 mmol) were heated at
4
0
8
1
.58 (1H, d, Jˆ8.5 Hz, H-9 ), 7.98 (1H, ddd, Jˆ8.5, 7.0,
reflux in acetic acid (41 mL) for 30 min to afford, after
workup and chromatography ascididemin (541 mg, 69%)
.5 Hz, H-2), 7.97 (1H, ddd, Jˆ8.5, 7.0, 1.5 Hz, H-3), 7.91
0
3
(
1H, m, H-8 ), 7.88 (1H, dd, Jˆ4.6, 1.8 Hz, H-9), 7.72 (1H,
identical in all respects with the literature data.
0
dd, Jˆ7.9, 4.5 Hz, H-3 ), 7.69 (1H, d, Jˆ8.8 Hz, H-6), 7.49
0
0
0
(
2H, m, H-7 and H-10 ), 7.13 (1H, dd, Jˆ7.8, 4.6 Hz,
11-[2 -(Diethylamino)ethyl]pyrido[2,3-b]acridine-5,12-
1
3
H-10). C NMR (100 MHz, CDCl ) d 182.5 (d, Jˆ4 Hz,
dione (21). Diethylamine hydrochloride (108 mg,
0.99 mmol), paraformaldehyde (48 mg, 1.71 mmol) and
glacial acetic acid (2 mL), were heated to 70ЊC for 5 min.
Dione 6 (98 mg, 0.36 mmol) was then added to the reaction
mixture and the resulting mixture was heated for 20 min at
110ЊC. The orange/brown reaction mixture was cooled,
poured onto ice-water (50 mL) and the resulting mixture
3
0
0
C-12), 182.1 (d, Jˆ4 Hz, C-5 ), 181.2 (s, C-12 ), 157.8 (t,
0
0
Jˆ5 Hz, C-11 ), 155.6 (ddd, Jˆ183, 8, 3 Hz, C-2 ), 152.4
(
1
ddd, Jˆ181, 8, 3 Hz, C-9), 152.4 (dd, Jˆ11, 6 Hz, C-7b),
0
49.4 (dd, Jˆ10, 5 Hz, C-6a ), 149.3 (dd, Jˆ12, 6 Hz,
0
0
C-12a ), 147.9 (s, C-5a or C-12a), 145.8 (s, C-12a or
0
C-5a ), 144.7 (t, Jˆ8 Hz, C-13a), 137.7 (d, Jˆ8 Hz, C-7),
1
36.3 (dd, Jˆ167, 6 Hz, C-11), 135.9 (dd, Jˆ170, 6 Hz,
made basic with aq NH3 and extracted with CH Cl2
2
0
0
C-4 ), 133.6 (obsc, C-4b), 133.0 (dd, Jˆ171, 6 Hz, C-8 ),
ꢀ3×30 mL†: The orange brown CH Cl extract was washed
2
2
1
32.9 (dd, Jˆ171, 6 Hz, C-1), 132.7 (d, Jˆ163 Hz, C-6),
with aq NH ꢀ3×100mL†; then brine, dried (MgSO ) and the
3
4
0
1
31.8 (dd, Jˆ165, 5 Hz, C-9 ), 130.6 (obsc, C-7a), 130.5
solvent removed in vacuo to yield 21 as an orange brown
residue (127 mg, 99%). IR (film) n 3065, 2984, 1690, 1639,
(
dd, Jˆ162, 9 Hz, C-3), 130.1 (dd, Jˆ162, 8 Hz, C-2), 129.9

5
04
B. S. Lindsay et al. / Tetrahedron 56 (2000) 497–505
1
3
1
1
608, 1580, 1562, 1503, 1462, 1402, 1375, 1334, 1270,
211, 1102, 1065, 1024, 1001, 965, 769, 732, 714 cm .
C NMR (100 MHz, CDCl ) d 182.0 (d, Jˆ3 Hz, C-12),
3
Ϫ1
157.8 (t, Jˆ4 Hz, C-6), 155.5 (ddd, Jˆ181, 8, 4 Hz, C-9),
152.4 (dd, Jˆ11, 5 Hz, C-7b), 149.5 (s, C-7a), 145.9 (s,
C-12a), 145.8 (m, 13a), 138.7 (d, Jˆ3 Hz, C-4b), 138.5
(m, C-14), 136.5 (dd, Jˆ169, 6 Hz, C-11), 133.1 (dd,
Jˆ165, 7 Hz, C-1), 131.7 (dd, Jˆ162, 8 Hz, C-2), 130.6
(dd, Jˆ162, 9 Hz, C-3), 130.0 (dt, Jˆ160, 7 Hz, C-17),
129.1 (d, Jˆ7 Hz, C-11a), 129.0 (dd, Jˆ161, 7 Hz, C-16),
127.9 (dt, Jˆ161, 7 Hz, C-15), 125.4 (dd, Jˆ167, 9 Hz,
C-10), 123.7 (obsc, C-4a), 122.8 (dd, Jˆ160, 7 Hz, C-4),
116.9 (d, Jˆ6 Hz, C-12b), 112.7 (d, Jˆ163 Hz, C-5). EIMS
UV (CH OH) l
(log e) 478 nm (3.4), 293 (4.2), 272
3
max
1
(4.2), 231 (4.3). H NMR (400 MHz, CDCl ) d 9.09 (1H,
3
dd, Jˆ4.6, 1.7 Hz, H-2), 8.64 (1H, dd, Jˆ7.9, 1.7 Hz, H-4),
8
.35 (1H, dd, Jˆ8.3, 1.0 Hz, H-10), 8.32 (1H, dd, Jˆ8.3,
1
.0 Hz, H-7), 7.85 (1H, ddd, Jˆ8.2, 7.1, 1.2 Hz, H-8), 7.73
(
1H, ddd, Jˆ8.2, 7.1, 1.2 Hz, H-9), 7.71 (1H, dd, Jˆ7.9,
0
4
.6 Hz, H-3), 3.90 (2H, bt, Jˆ7.5 Hz, H-1 ), 2.88 (2H, bt,
0
Jˆ8.4 Hz, H-2 ), 2.76 (4H, q, Jˆ7.2 Hz, N(CH CH ) ), 1.09
2
3 2
1
3
(
6H, t, Jˆ7.2 Hz, N(CH CH ) ). C NMR (100 MHz,
2
3 2
ϩ
ϩ
CDCl ) d 182.8 (s, C-12), 181.5 (d, Jˆ3 Hz, C-5), 155.6
m/z (%) 359 (M , 100). HREIMS found 359.1069 (M ),
3
(
ddd, Jˆ183, 7, 4 Hz, C-2), 154.7 (obsc, C-11), 149.9 (dd,
C H N O requires 359.1059. Anal. calcd. for
C H N O: C, 80.2; H, 3.6; N, 11.7%. Found: C, 80.0; H,
3.5; N, 11.5.
2
4
13
3
Jˆ11, 5 Hz, C-12a), 148.7 (dd, Jˆ10, 6 Hz, C-6a), 147.5 (s,
C-5a), 135.6 (dd, Jˆ169, 6 Hz, C-4), 132.7 (dd, Jˆ163,
24 13 3
9
Hz, C-8), 132.4 (dd, Jˆ166, 8 Hz, C-7), 130.0 (dd,
Jˆ162, 8 Hz, C-9), 129.9 (d, Jˆ6 Hz, C-4a), 129.3 (m,
6-Styrylascididemin (24). Prepared following the general
procedure in 72% yield. mp 281–283ЊC. IR (film) n 1682,
1636, 1595, 1577, 1499, 1449, 1431, 1399, 1349, 1271,
1184, 1102 cm . UV (MeOH) l
7j
C-10a), 127.8 (dd, Jˆ168, 9 Hz, C-3), 125.2 (dd, Jˆ161,
7
Hz, C-10), 125.0 (t, Jˆ2 Hz, C-11a), 52.9 (tm, Jˆ135 Hz,
0
Ϫ1
C-2 ), 46.7 (tq, Jˆ132, 4 Hz, N(CH CH ) ), 26.7 (t,
(log e) 203 nm
max
1
2
3 2
0
Jˆ132 Hz, C-1 ), 12.4 (q, Jˆ125 Hz, N(CH CH ) ). EIMS
(4.74), 253 (4.65), 279 (4.61), 332 (4.79), 430 (3.98). H
2
3 2
ϩ
ϩ
m/z (%) 359 (M , 20). HREIMS found 359.1625 (M ),
NMR (400 MHz, CDCl ) d 9.17 (1H, dd, Jˆ4.7, 1.7 Hz,
3
C H N O requires 359.1634.
H-9), 8.72 (1H, dd, Jˆ7.9, 1.7 Hz, H-11), 8.61 (1H, dd,
Jˆ8.2, 1.4 Hz, H-4), 8.52 (1H, dd, Jˆ8.1, 1.4 Hz, H-1),
8.46 (1H, s, H-5), 7.96 (1H, d, Jˆ16.1 Hz, H-15), 7.92
(1H, ddd, Jˆ8.3, 6.9, 1.5 Hz, H-2), 7.85 (1H, ddd, Jˆ8.1,
7.1, 1.3 Hz, H-3), 7.66 (2H, d, Jˆ7.2 Hz, H-17), 7.61 (1H,
dd, Jˆ7.9, 4.6 Hz, H-10), 7.53 (1H, d, Jˆ16.1 Hz, H-14),
7.41 (2H, t, Jˆ7.6 Hz, H-18), 7.34 (1H, t, Jˆ7.3 Hz, H-19).
22
21
3
2
6
-Methylascididemin (22). Prepared following the general
7j
procedure that we have previously communicated in 90%
yield. mp (decomp.) 266–268ЊC. IR (film) n 2922, 2857,
1
1
676, 1643, 1606, 1578, 1503, 1428, 1395, 1349, 1264,
101, 1068, 1035, 947, 764, 736 cm . UV (MeOH) lmax
Ϫ1
13
(log e) 198 nm (4.48), 221 (4.60), 248 (4.56), 273 (sh 4.35),
C NMR (100 MHz, CDCl ) d 181.9 (d, Jˆ4 Hz, C-12),
3
1
300 (4.14), 338 (3.85), 391 (3.91). H NMR (400 MHz,
156.1 (d, Jˆ5 Hz, C-6), 155.5 (ddd, Jˆ181, 8, 4 Hz, C-9),
152.2 (dd, Jˆ11, 5 Hz, C-7b), 149.4 (s, C-7a), 145.8 (s,
C-12a), 145.8 (m, 13a), 138.5 (d, Jˆ3 Hz, C-4b), 136.5
(dd, Jˆ169, 7 Hz, C-11), 136.1 (dt, Jˆ154, 5 Hz, C-15),
136.1 (m, C-16), 133.0 (dd, Jˆ165, 7 Hz, C-1), 131.7 (dd,
Jˆ162, 9 Hz, C-2), 130.5 (dd, Jˆ162, 9 Hz, C-3), 129.1 (d,
Jˆ6 Hz, C-11a), 129.0 (dt, Jˆ161, 7 Hz, C-19), 128.8 (dd,
Jˆ160, 8 Hz, C-18), 127.6 (obsc, C-14), 127.6 (obsc, C-17),
125.4 (dd, Jˆ167, 8 Hz, C-10), 123.5 (obsc, C-4a), 122.8
(dd, Jˆ160, 8 Hz, C-4), 117.0 (d, Jˆ6 Hz, C-12b), 113.4
CDCl ) dˆ9.18 (1H, dd, Jˆ4.8, 1.8 Hz, H-9), 8.77 (1H,
3
dd, Jˆ7.9, 1.7 Hz, H-11), 8.63 (1H, bd, Jˆ8.2 Hz, H-4),
8
7
1
.56 (1H, dd, Jˆ8.2, 1.0 Hz, H-1), 8.37 (1H, bs, H-5),
.95 (1H, td, Jˆ8.3, 1.4 Hz, H-2), 7.88 (1H, td, Jˆ8.2,
.3 Hz, H-3), 7.64 (1H, dd, Jˆ7.9, 4.7 Hz, H-10), 3.05
1
3
(
3H, bs, CH ). C NMR (100 MHz, CDCl ) d 182.0 (d,
3
3
Jˆ4 Hz, C-12), 159.8 (qd, Jˆ5, 1 Hz, C-6), 155.6 (ddd,
Jˆ182, 7, 3 Hz, C-9), 152.4 (dd, Jˆ12, 5 Hz, C-7b),
1
49.2 (s, C-7a), 146.0 (s, C-12a), 145.7 (t, Jˆ8 Hz,
ϩ
C-13a), 138.5 (d, Jˆ3 Hz, C-4b), 136.6 (dd, Jˆ167, 6 Hz,
(dd, Jˆ163, 4 Hz, C-5). EIMS m/z (%) 385 (M , 85), 384
ϩ
C-11), 133.0 (dd, Jˆ165, 7 Hz, C-1), 131.6 (dd, Jˆ163,
(100). HRFABMS found 386.1310 (MH ), C H N O
26
16
3
8
Hz, C-2), 130.5 (dd, Jˆ163, 8 Hz, C-3), 129.0 (d,
requires 386.1293. Anal. calcd for C H N O·0.75H O:
C, 78.3; H, 4.2; N, 10.5%. Found: C, 78.1; H, 4.1; N, 10.4%
26 15 3 2
Jˆ8 Hz, C-11a), 125.4 (dd, Jˆ167, 8 Hz, C-10), 123.3
(
m, C-4a), 122.8 (dd, Jˆ160, 8 Hz, C-4), 116.4 (obsc,
C-12b), 115.7 (dq, Jˆ162, 4 Hz, C-5), 25.9 (qd, Jˆ128,
ϩ
3
Hz, CH ). EIMS m/z (%) 297 (M , 100). HREIMS
Acknowledgements
3
ϩ
found 297.0904 (M ), C H N O requires 297.0902.
1
9
11
3
Anal. calcd for C H N O·CH Cl : C, 63.0; H, 3.4; N,
We thank S. McGivern and B. Knowles for help with pre-
liminary aspects of this work, M. Walker for mass spectral
and NMR data acquisition, and Mrs G. Ellis (Canterbury
University) and staff at the National Cancer Institute (Dr
Ven Narayanan and Dr Melinda Hollingshead), for arranging
testing through the NCI’s preclinical antitumour drug
discovery screen. Financial assistance from the Auckland
Medical Research Foundation and the University of Auck-
land Research Committee is also gratefully acknowledged.
1
9
11
3
2
2
11.0%. Found: C, 63.1; H, 3.2; N, 11.1.
6
-Phenylascididemin (23). Prepared following the general
7
j
procedure in 81% yield. mp Ͼ310ЊC. IR (film) n 1680,
1
1
601, 1578, 1509, 1459, 1426, 1394, 1348, 1270, 1104,
071, 1035, 947, 873, 813, 740, 694 cm . UV (MeOH)
Ϫ1
lmax (log e) 202 nm (4.67), 248 (4.63), 272 (4.57), 303
1
(4.56), 407 (3.88). H NMR (400 MHz, CDCl ) d 9.18
3
(
1H, dd, Jˆ4.6, 1.8 Hz, H-9), 8.80 (1H, s, H-5), 8.75 (1H,
dd, Jˆ7.9, 1.8 Hz, H-11), 8.69 (1H, dd, Jˆ8.1, 1.5 Hz, H-4),
8
.55 (1H, dd, Jˆ8.2, 1.4 Hz, H-1), 8.31 (2H, dm, Jˆ8.5 Hz,
References
H-15), 7.94 (1H, td, Jˆ8.3, 1.3 Hz, H-2), 7.87 (1H, td,
Jˆ7.2, 1.2 Hz, H-3), 7.63 (1H, dd, Jˆ7.9, 4.7 Hz, H-10),
1. (a) Molinski, T. F. Chem. Rev. 1993, 93, 1825–1838. (b) Salomon,
C. E.; Faulkner, D. J. Tetrahedron Lett. 1996, 37, 9147–9148. (c)
7
.56 (2H, t, Jˆ7.6 Hz, H-16), 7.51 (1H, t, Jˆ7.1 Hz, H-17).

B. S. Lindsay et al. / Tetrahedron 56 (2000) 497–505
505
Plubrukarn, A.; Davidson, B. S. J. Org. Chem. 1998, 63, 1657–
659. (d) Copp, B. R.; Jompa, J.; Tahir, A.; Ireland, C. M. J. Org.
11. Peterson, J. R.; Zjawiony, J. K.; Liu, S.; Hufford, C. D.; Clark,
A. M.; Rogers, R. D. J. Med. Chem. 1992, 35, 4069–4077.
12. Krapcho, A. P.; Petry, M. E.; Getahun, Z.; Landi, J. J.;
Stallman, J.; Polsenberg, J. F.; Gallagher, C. E.; Maresch, M. J.;
Hacker, M. P. J. Med. Chem. 1994, 37, 828–837.
13. Enamine 7 and amine 20 had no detectable activity towards
HIV-1 in testing at the NCI (data not shown).
14. Albert, A. Selective Toxicity, 7; Chapman & Hall: New York,
1985.
15. (a) Copp, B. R.; Fulton, K. F.; Perry, N. B.; Blunt, J. W.;
Munro, M. H. G. J. Org. Chem. 1994, 59, 8233–8238. (b) Boyd,
M. R. Status of the NCI preclinical antitumor discovery screen:
implications for section of new agents for clinical trial. In Cancer.
Principles & Practise of Oncology; DeVita Jr., V. T., Hellman, S.,
Rosenberg, S. A., Eds.; Lippincott: Philadelphia, 1989; Vol. 3(10),
pp 1–12. (c) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.;
Paull, K. D.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-
Wolff, A.; Gray-Goodrich, M.; Campbell, H.; Boyd, M. R. J. Natl.
Cancer Inst. 1991, 83, 757–766. (d) Boyd, M. R. In Current
Therapy in Oncology; Neiderhuber, J. E., Ed.; Decker: Philadelphia,
1992; pp 11–22. (e) Boyd, M. R.; Paul, K. D.; Rubenstein, L. R. In
Antitumor Drug Discovery and Development; Valeriote, F. A.,
Corbett, T., Baker, L., Eds.; Kluwer Academic: Amsterdam,
1991; pp 11–34. (f) Plowman, J.; Dykes, D. J.; Hollingshead,
M.; Simpson-Herren, L.; Alley, M. C. In Anticancer Drug
Development Guide: Preclinical Screening, Clinical trials and
Approval; Teicher, B. Ed.; Humana Press: Totowa, NJ, 1997;
pp 101–125.
1
Chem. 1998, 63, 8024–8026. (e) Eder, C.; Schupp, P.; Proksch, P.;
Wray, V.; Steube, K.; Muller, C. E.; Frobenius, W.; Herderich, M.;
van Soest, R. W. M. J. Nat. Prod. 1998, 61, 301–305. (f) de
Guzman, F. S.; Carte, B.; Troupe, N.; Faulkner, D. J.; Harper,
M. K.; Concepcion, G. P.; Mangalindan, G. C.; Matsumoto, S. S.;
Barrows, L. R.; Ireland, C. M. J. Org. Chem. 1999, 64, 1400–1402.
2. (a) Bloor, S. J.; Schmitz, F. J. J. Am. Chem. Soc. 1987, 109,
6134–6136. (b) de Guzman, F. S.; Schmitz, F. J. Tetrahedron Lett.
1989, 30, 1069–1070. (c) Lindsay, B. S.; Oliver, A. G.; Rickard, C.
E. F.; Copp, B. R. J. Chem. Crystallogr. 1998, 28, 645–648.
. Kobayashi, J.; Cheng, J.-F.; Nakamura, H.; Ohizumi, Y.; Hirata,
Y.; Sasaki, T.; Ohta, T.; Nozoe, S. Tetrahedron Lett. 1988, 29,
3
1
4
177–1180.
. Schmitz, F. J.; De Guzman, F. S.; Hossain, M. B.; van der Helm,
D. J. Org. Chem. 1991, 56, 804–808.
. Zeng, C. M.; Ishibashi, M.; Matsumoto, K.; Nakaike, S.;
Kobayashi, J. Tetrahedron 1993, 49, 8337–8342.
5
6
4
7
. Carroll, A. R.; Scheuer, P. J. J. Org. Chem. 1990, 55,
426–4431.
. (a) Bracher, F. Liebigs Ann. Chem. 1990, 205–206. (b) Bracher,
F. Heterocycles 1989, 29, 2093–2095. (c) Moody, C. J.; Rees,
C. W.; Thomas, R. Tetrahedron Lett. 1990, 31, 4375–4376. (d)
Gomez-Bengoa, E.; Eschavarren, A. M. J. Org. Chem. 1991, 56,
3
497–3501. (e) Moody, C. J.; Rees, C. W.; Thomas, R. Tetra-
hedron 1992, 48, 3589–3602. (f) Bracher, F. Liebigs Ann. Chem.
992, 1205–1207. (g) Kitahara, Y.; Nakahara, S.; Yonezawa, T.;
1
Nagatsu, M.; Kubo, A. Heterocycles 1993, 36, 943–946. (h)
Gellerman, G.; Rudi, A.; Kashman, Y. Synthesis 1994, 3, 239–
16. Mean graphs depicting the dose-response data for compounds
submitted to the NCI can be viewed online, by searching for
the NSC number of the compound at the NCI web site
http://dtp.nci.nih.gov/docs/cancer/searches/cancer_open_compounds.
html
17. Paull, K. D.; Shoemaker, R. H.; Hodes, L.; Monks, A.;
Scudiero, D. A.; Rubinstein, L.; Plowman, J.; Boyd, M. R.
J. Natl. Cancer Inst. 1989, 81, 1088–1092.
2
1
41. (i) Kitahara, Y.; Tamura, F.; Kubo, A. Chem. Pharm. Bull.
994, 42, 1363–1364. (j) Lindsay, B. S.; Pearce, A. N.; Copp, B. R.
Synth. Commun. 1997, 27, 2587–2592. (k) Kitahara, Y.; Nakahara,
S.; Yonezawa, T.; Nagatsu, M.; Shibano, Y.; Kubo, A. Tetrahedron
1
997, 53, 17029–17038. (l) Bontemps, N.; Delfourne, E.; Bastide,
J.; Francisco, C.; Bracher, F. Tetrahedron 1997, 53, 1743–1750.
m) Copp, B. R.; Hansen, R. P.; Appleton, D. R.; Lindsay, B. S.;
Squire, C. J.; Clark, G. R.; Rickard, C. E. F. Synth. Commun. 1999,
(
18. Bachur, N.; Gordon, S. L.; Gee, M. V. Cancer Res. 1978, 38,
1745–1750.
2
8
9, 2665–2676.
19. Matsumoto, S. S.; Copp, B. R.; Holden, J. A.; Barrows, L.R.
Unique DNA cleaving ability of novel ascididemin analogs
through redox cycling and the production of reactive oxygen
species. Poster presentation (P1942) at the 37th Annual meeting
of the Society of Toxicology, Seattle, Washington, 1–5 March
1998.
. (a) Bonnard, I.; Bontemps, N.; Lahmy, S.; Banaigs, B.;
Combaut, G.; Francisco, C.; Colson, P.; Houssier, C.; Waring,
M. J.; Bailly, C. Anti-Cancer Drug Des. 1995, 10, 333–346. (b)
Lindsay, B. S.; Barrows, L. R.; Copp, B. R. Bioorg. Med. Chem.
Lett. 1995, 5, 739–742. (c) Bracher, F. Pharmazie 1997, 52, 57–
6
0. (d) Blanco, M. D.; de la Fuente, J. A.; Avendano, C.;
Menendez, J. C. Tetrahedron Lett. 1999, 40, 4097–4098.
. Schmitz, F. J.; de Guzman, F. S.; Choi, Y.-H.; Hossain, M. B.;
Rizvi, S. K.; van der Helm, D. Pure Appl. Chem. 1990, 62,
20. Xenograft assay treatment schedules: MDA-MB-435 and -231
Q4D×3, 13 (i.e. every four days for a total of three treatments with
the first treatment given on day 13 after tumour implantation).
LOX IMVI QD×5, 8. OVCAR-3 Q4D×3, 6. U251 Q4D×3, 14.
HCT-116 QD×5, 9. NCI-H460 QD×5, 2. NCI-H522 QD×5, 7.
SF-295 QD×5, 2. UACC Q4D×3, 15.
9
1
1
4
393–1396.
0. Brederick, H.; Simchen, G.; Wahl, R. Chem. Ber. 1968, 101,
048–4056.
21. Bracher, F. Liebigs Ann. Chem. 1989, 87.