Anti-Tuberculosis natural products: Synthesis and biological evaluation of pyridoacridine alkaloids related to ascididemin

Article abstract of DOI:10.1016/j.tet.2010.05.033

There is an urgent need for novel therapeutics possessing new modes of action to treat tuberculosis (TB) infections. In this study we report on the synthesis and biological evaluation of a series of pyrido[2,3,4-kl]acridin-6-one alkaloids related to the a

Full text of DOI:10.1016/j.tet.2010.05.033

Tetrahedron 66 (2010) 4977e4986
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
anti-Tuberculosis natural products: synthesis and biological evaluation
of pyridoacridine alkaloids related to ascididemin
,
b
b
David R. Appleton a , A. Norrie Pearce , Brent R. Copp
*
a Centre for Natural Products Research and Drug Discovery, Department of Pharmacology, Faculty of Medicine, Universiti Malaya, 50603 Kuala Lumpur, Malaysia
b Department of Chemistry, The University of Auckland, Private Bag 92019, Auckland, New Zealand
a r t i c l e i n f o
a b s t r a c t
Article history:
There is an urgent need for novel therapeutics possessing new modes of action to treat tuberculosis (TB)
infections. In this study we report on the synthesis and biological evaluation of a series of pyrido[2,3,4-kl]
Received 8 February 2010
Received in revised form 22 April 2010
Accepted 10 May 2010
acridin-6-one alkaloids related to the anti-TB (MIC 0.35
product ascididemin (1). The most interesting compounds identified were 21 and 24, which were found
to inhibit the growth of Mycobacterium tuberculosis (Mtb) H37Rv with MIC 2.0 M, but with negligible
cytotoxicity towards Vero and P388 cells (IC50>25 M). Another analogue (10) was evaluated against
mM) but cytotoxic (IC50 <0.14 mM) marine natural
Available online 1 June 2010
m
m
Keywords:
a range of singly-drug-resistant strains of Mtb and was found to exhibit no cross-resistance. These results
suggest that the pyrido[2,3,4-kl]acridin-6-one skeleton may provide a useful scaffold for future studies
directed towards possible anti-TB drugs.
Tuberculosis
Natural Products
Ascididemin
pyridoacridine
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
O
N
It has been estimated that two billion people or one-third of the
world’s population are infected with Mycobacterium tuberculosis
(Mtb), the microbe that causes tuberculosis (TB). Of those infected,
10% will become sick in their lifetime, making TB one of the leading
causes of death in the world.1 New drugs that provide shorter
treatment regimes than those currently available with existing
drugs and that can overcome the increasing emergence of drug
resistant strains are urgently needed. Screening programmes are in
place, e.g., Tuberculosis Antimicrobial Acquisition and Coordinating
Facility (TAACF),2 to discover new chemical entities that can act as
scaffolds for future drug development. Whilst much recent atten-
tion has focused on purely synthetic leads, such as R2079103 and
PA-824,4 there is continuing interest in the ability of natural
products to act as new drug leads.5
N
N
1
Figure 1. Chemical structure of ascididemin.
regulation in Mtb.7 Ascididemin, like other marine-derived pyr-
idoacridine alkaloids is known to exert cytotoxicity via inhibition of
DNA topoisomerase II, leading to cleavage of DNA and cell death, with
IC50s against mammalian cell lines as low as 0.3 m
M.8 While these
cytotoxic effects clearly limit the potential antibiotic utility of the
natural product, we were interested in exploring modifications to the
pentacyclic-core of ascididemin in an effort to decouple mammalian
cell toxicity from anti-tuberculosis activity. Herein, we describe the
synthesis, biological evaluation and structure/activity relationships
(SAR) of a series of natural product and natural product-related pyr-
idoacridine and pyridoacridone alkaloids.
As part of our search for new antituberculosis lead compounds,
we have screened a purified compound library of marine natural
products for activity against Mtb through The TAACF Program.2
Among a number of hits identified as exhibiting a minimum in-
hibitory concentration (MIC) of <6.25
mg/mL was the pyr-
2. Chemistry
idoacridone alkaloid ascididemin (1)6 (Fig. 1).
Preliminary investigation of themechanism ofactionof1, utilizing
differential gene expression technology, identified aneffect uponiron
Our ongoing interest in the secondary metabolites of New
Zealand marine organisms from the Class Ascidiacea (ascidians) has
afforded us a purified compound library comprised of a number of
examples of pyridoacridine and pyridoacridone alkaloids. Previous
investigation of extracts from a Northland, New Zealand collection
of the ascidian Lissoclinum notti yielded the pyridoacridone alkaloid
* Corresponding author. Tel.: þ60 3 79673199; fax: þ60 3 79674791; e-mail
address: david@um.edu.my (D.R. Appleton).
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.05.033

4978
D.R. Appleton et al. / Tetrahedron 66 (2010) 4977e4986
14
9
diplamine (2), which was first reported from a Fijian collection of
the ascidian Diplosoma sp.,9 along with previously unreported an-
alogues isodiplamine (3) and lissoclinidine (4).10 Specimens of the
same organism species collected in the south of New Zealand
provided samples of the previously reported alkaloids kuanoni-
amine D (5)11 and shermilamine B (6).12 The addition of two other
structurally related marine natural products to our study was of
interest: 11-hydroxyascididemin (7)13 and kuanoniamine A (8),11
were both prepared by literature methods14,15 (Fig. 2).
O
O
H
O
1
N
a
7
5
11
RS
RS
O
O
13a R = C6H5
14a
13b
R = C6H5
R = CH3(CH2)7
14b R = CH3(CH2)7
15b R = CH3CH2O2CCH2CH2
15a R = CH3CH2O2CCH2CH2
b
O
O
10
N
5
8
N
c
3
O
O
O
1
RS
N
RS
NH
NH
NH
O
11
H
R1
O
N
H
13 R = C6H5
13c R = C6H5
N
S
N
S
N
14 R = CH3(CH2)7
14c R = CH3(CH2)7
O
15
15c
R = CH3CH2O2CCH2CH2
R = CH3CH2O2CCH2CH2
N
R2
N
N
2 R1=SCH3, R2=H
O
O
N
4
5
N
3 R1=H, R2=SCH3
d
O
S
N
S
N
O
NH
10
16
OH O
O
H
S
N
N
N
S
Scheme 1. Reagents and conditions: (a) CeCl3$7H2O, 20-aminoacetophenone, air,
45e61% yield; (b) AcOH, H2SO4, 79e92% yield; (c) NH4Cl, (CH2O)n, AcOH, reflux,
50e83% yield; (d) m-CPBA, NaOAc, CH2Cl2, 73%.
N
N
N
O
N
H
N
N
6
7
8
Figure 2. Chemical structures of other natural pyridoacridines investigated for anti-TB
activity.
taken advantage of in the preparation of compounds 11,12,17, 21 and
23. Thus the reaction of 9 with tert-butyl N-(2-aminoethyl)carbamate
in the presence of a catalytic amount of sodium methoxide in MeOH
afforded 17 in 53% yield (Scheme 2). Deprotection using TFA in CH2Cl2
provided the unstable ammonium salt 18, which upon reaction with
tosylchloride in MeCN afforded sulfonamide 19 in 61% yield, or re-
action with phenylisothiocyanate also in MeCN provided thiourea 20
in 45% yield. Amide 21 was prepared by direct coupling of the mono-
substituted pyrazine ethylene diamine 22 with sulfide 9.
Preparation of alkaloids with variation in size of the pentacyclic-
core of ascididemin was targeted as a priority, with the express de-
sire of reducing DNA binding properties and inherent cytotoxicity.
We have previously reported the syntheses of tetracyclic pyrido
[2,3,4-kl]acridin-6-one analogues 9, 10, 11 and 12 as part of a struc-
ture/activity relationship study of 11-hydroxyascididemin14 (Fig. 3).
O
O
O
O
N
N
N
N
a
4
H
N
tBuO
12
N
H
RS
RS
H
S
11
tBuO
N
14
N
1
O
N
9
NH2
O
N
17
2
O
N
N
9
R=CH3C6H4
10 R=CH3CH2
9c R=CH3C6H4
H
N
NH2
e
b
O
O
N
O
N
22
H3CO
NH2
-
O
O
CF3CO2
N
N
N
N
N
H
12
11
N
H3N
N
N
N
H
H
O
N
21
N
18
Figure 3. Chemical structures of tetracyclic pyrido[2,3,4-kl]acridin-6-ones initially
investigated for anti-TB activity.
c
d
This series was initially extended by preparation of the thio-
phenyl(13), thio-n-octyl(14) and thioethylpropanate (15) analogues
via a similar route (Scheme 1). Thus, the thio-substituted 1,4-ben-
zoquinones 13a,16 14a and 15a were prepared in good yield by re-
action of the appropriate thiol (1.0 equiv) with benzoquinone
(2.0 equiv) in ethanol at room temperature. Subsequent oxidative
coupling of these quinones with 20-aminoacetophenone yielded the
desired 5-(2-acetylanilino)-2-thio substituted 1,4-benzoquinones
13b, 14b and 15b (45e61%), which upon acid catalysed cyclisation
led to acridinediones 13c, 14c and 15c in yields of 79e92%. Appli-
cation of a one pot annulation using paraformaldehyde and am-
monium chloride in glacial acetic acid,15 led to pyrido[2,3,4-kl]
acridin-6-one analogues 13,14 and 15. Oxidation of sulfide 1014 with
m-chloroperoxybenzoic acid yielded the sulfoxide 16 in 73% yield.
The propensity of 4-(4-methylphenylthio)pyrido[2,3,4-kl]acridin-
6-one (9) to undergo substitutionwith avarietyof nucleophiles14 was
O
N
O
N
N
H
H
N
H
N
N
O
N
S
N
H
H
O
N
S
19
20
Scheme 2. Reagents and conditions: (a) tert-butyl N-(2-aminoethyl)carbamate,
NaOMe, Et3N, N2, 53%; (b) TFA, 100%; (c) Tosylchloride, Et3N, 61%; (d) Phenyl-
isothiocyanate, Et3N, 45%; (e) NaOMe, Et3N, N2, 36%.
Aminoalcohol 23 was synthesised by nucleophilic substitution
of sulfide 9 with ethanolamine in the presence of sodium meth-
oxide in MeOH in low yield (25%) (Scheme 3). Subsequent prepa-
ration of ester 24 was achieved by reaction of 23 with
pyrazinecarboxylic acid in the presence of BOP-Cl and Et3N.

D.R. Appleton et al. / Tetrahedron 66 (2010) 4977e4986
4979
O
3. Results and discussion
O
N
N
N
a
HO
Pyrido[2,3,4-kl]acridin-6-one analogues were prepared and
evaluated for the ability to inhibit the in vitro growth of M. tuber-
culosis H37Rv at Southern Research Institute under the auspices of
the TAACF program.2 All of the active, and soluble, compounds were
also assessed for in vitro cytotoxicity towards the Vero and murine
leukaemia P388 cell-lines. Comparison of the in vitro anti-TB ac-
tivity determined for ascididemin (1) to a series of structurally re-
lated natural pyridoacridines 2e8 revealed that 1 was by far the
most potent and that the complete iminoquinone moiety, as seen in
compounds 1, 7 and 8, was required for biological activity (Table 1).
Interestingly, 11-hydroxyascididemin (7) exhibited weak activity
N
H
S
N
23
9
b
O
N
N
N
N
O
O
N
H
24
against Mtb H37Rv with an MIC >42
intact iminoquinone functionality. However, despite ascididemin
(1) exhibiting extremely good potency (MIC 0.35 M) against Mtb
in vitro, the selectivity index was low, with similar cytotoxicity
values being observed against both Vero (<0.14 M) and P388
(0.4 M) cell-lines.
mM despite still having the
Scheme 3. Reagents and conditions: (a) Ethanolamine, NaOMe, N2, 25%; (b) Pyrazine
carboxylic acid, BOP-Cl, Et3N, 38%.
m
The synthesis of the ring A modified analogues of ascididemin
(1) possessing either methyl carboxylate thiophene (25) or furan
(26) functionality was achieved using previously reported meth-
odology starting from the appropriately functionalised quinones,
2717 and 28, respectively. Reaction of thiophene quinone 27 with 20-
aminoacetophenone in the presence of cerium chloride and in situ
oxidation using air yielded adduct 29 in highyield (92%) (Scheme 4).
The purple adduct 29 was then subjected to dehydrative cyclisation
in the presence of 10:1 glacial acetic acid/H2SO4 at 100 ꢀC toyield the
yellow acridinedione 30 in 94% yield. Final one pot annulation, by
reaction with paraformaldehyde and ammonium chloride in the
presence of acid15 afforded the desired thiopheno-acridinone 25 in
83% yield.
m
m
Table 1
In vitro anti-TB activity and cytotoxicity of pyridoacridines and pyridoacridone
natural products
Compound
H37Rv MIC (
m
M)
Vero cells IC50
(m
M)
P388 cells IC50 (mM)
1
2
3
4
5
6
7
8
0.35
>17
>17
>17
>34
>32
>42
10.7
0.152
<0.14
ND
ND
ND
ND
ND
NDa
Ib
0.4
1.9
2.1
4.6
ND
ND
2.3
0.3
ND
RMPc
154
17
O
a
O
O
O
O
ND: not determined.
I: insoluble in Vero cell line assay media.
RMP: rifampin.
H
N
9
3
b
c
12
a
CH3O2C
CH3O2C
6
X
X
In order to investigate if it was possible to synthesise analogues
27 X=S
28 X=O
29 X=S
31
X=O
that maintain or improve anti-TB activity while reducing cytotox-
icity, we investigated a series of synthetic pyridoacridines possess-
ing substitution at the 4-position of a pyrido[2,3,4-kl]acridin-6-one
core. Previous work by our group had shown that the cytotoxicity of
the pyridoacridine pharmacophore could be attenuated by altering
the substitution at this position and synthetic routes were well
established and high-yielding.14 A variety of aryl- and alkyl-sulfides
was prepared and assessed for in vitro anti-TB activity and cyto-
toxicity. The most potent analogue of this series was identified as 4-
b
O
6
O
N
9
6
3
N
13
12
c
CH3O2C
CH3O2C
X
N
X
10
O
1
25 X=S
26 X=O
30 X=S
32 X=O
ethylthiopyrido[2,3,4-kl]acridin-6-one (10) with an MIC of 0.34 mM,
Scheme 4. Reagents and conditions: (a) CeCl3$7H2O, 20-aminoacetophenone, air, 92%
comparable to ascididemin (1) (Table 2). Encouragingly, the level of
cytotoxicity observed for 10 was 1e2 orders of magnitude less,
yielding a selectivity index of 20. Interestingly, oxidation of the
sulfide to sulfoxide 16 resulted in a significant reduction in the anti-
TB activity. In general, for the sulfide series, the alkyl sulfides showed
greater potency than the aryl sulfides and had comparable cyto-
toxicity values. Some activity was also observed for this series
against the Gram-positive bacterium Bacillus subtilis and the fungus
Trichophyton mentagrophytes in disc diffusion assays. The trend of
antibacterial activity for the pyridoacridines correlated reasonably
well with anti-TB activity while the antifungal activity seemed to
correlate with cytotoxicity, with possibly some solubility effects for
the relatively non-polar examples 9 and 14 explaining their low
activity in the disc diffusion assays. Replacement of the thio-sub-
stituent with a methoxyl group (11) resulted in a slight reduction of
anti-TB activity while increasing the cytotoxicity. The thiophene (25)
and furan (26) analogues also exhibited similar potencies against
Mtb H37Rv and cytotoxicity as 10 but were not investigated further
due their poor solubilities and more complicated synthetic routes.
The 2,3,4-trisubstituted pyridine ring present in 9, 14 and 15 is
for X¼S, 22% for X¼O
*
; (b) AcOH, H2SO4, 81e94%; (c) NH4Cl, (CH2O)n, AcOH, reflux,
76e83%. *X¼O also yields the spontaneously cyclised compound (61%).
Furan quinone 28 was prepared from commercially available 33
after esterification (34) and either methyl ether cleavage with bo-
ron tribromide in CH2Cl2 to yield 35 followed by CAN oxidation to
28, or direct formation of 28 from the methyl ether 34 using cerium
ammonium sulfate in quantitative yield (Scheme 5). A subsequent
reaction sequence of substitution, cyclisation and annulation using
quinone 28, carried out in similar fashion to that performed on
quinone 27, yielded the target furano-acridinone 26 (Scheme 4).
a
b
HO2C
CH3O2C
CH3O2C
c
O
O
O
OCH3
33
OCH3
34
OH
35
d
28
Scheme 5. Reagents and conditions: (a) MeOH, H2SO4, reflux, 100%; (b) BBr3, 86%; (c)
CAN, 83%; (d) Ce(NH4)4(SO4)4, H2SO4, 100%.

4980
D.R. Appleton et al. / Tetrahedron 66 (2010) 4977e4986
Table 2
for analogues 17,19 and 20, it was considered desirable to incorporate
a bulky but basic group. Pyrazinecarboxylic acid was chosen as it is
known to be the active pharmacophore of the anti-TB drug pyr-
azinecarboxamide18 after intracellular amide-bond cleavage, and
should provide higher aqueous solubility along with the possibility of
a hybrid drug interaction with Mtb. Amide (21) and ester (24) ana-
logues were found to exhibit moderate anti-TB activity with MIC’s of
In vitro biological activities of substituted thio- and oxo- acridinediones and pyrido
[2,3,4-kl]acridin-6-ones
Compound H37Rv MIC B.subb T.mentc Vero cells Selectivity P388 cells
(
m
M)a
IC50
(
m
M) indexd
2.6
IC50 (mM)
9c
9
10
11
13
14c
14
15c
15
16
9.0
1
0
3
8
1
1
0
6
0
0
1
6
1
0
0
3
23.8
Ie
6.8
10.7
I
4.9
14.1
15.2
5.8
ND
I
28.0
19.5
6.8
3.3
21.1
3.3
27.1
1.0
17.9
ND
8.5
3.0
2.20
0.34
1.5
20
7.1
2.0
mM against M. tuberculosis H37Rv while having no measurable
cytotoxicity of doses up to 25
mM against both Vero and P388 cells.
18.4
17.0
1.0
17.6
2.1
0.3
14
0.9
2.7
The amide 21 also exhibited the highest antibacterial activity of the
series in the disc diffusion assay against B. subtilis with a radius of
10 mm at 120 mg/disc.
In a preliminary effort directed towards determination of the
mechanism of action, analogue 10 was profiled against a panel of
singly-drug-resistant strains of M. tuberculosis and found to be
6
3
ND
>20
0.58
0.61
18.5
19.5
0.152
NDf
25
26
30
1 (20) 0 (20)
3 (20) 7 (20)
ND
ND
I
I
I
ND
ND
ND
ND
extremely potent with MIC’s 0.34 mM or less against all strains
32
RMPg
154
(Table 4). This suggests the mechanism of action of pyrido[2,3,4-kl]
acridin-6-one alkaloids is different from those of current antitu-
berculosis agents.
a
MIC (
m
M) against M. tuberculosis H37Rv.
B. subtilis inhibition zone (mm) at 120
T. mentagrophytes inhibition zone (mm) at 120
b
c
m
g/disc unless indicated in brackets.
g/disc unless indicated in
m
brackets.
Table 4
d
Selectivity index: Vero cell line cytotoxicity/Mtb MIC.
I: insoluble in Vero cell line media.
ND: not determined.
In vitro anti-TB profiling of 4-ethylthiopyrido[2,3,4-kl]acridin-6-one (10) against
singly-drug-resistant strains of Mtb
e
f
g
MIC (
mM)
RMP: rifampin.
H
0.34
37Rv
EMB-Ra
<0.17
INH-Rb
0.34
RMP-Rc
<0.17
KM-Rd
<0.17
CIP-Re
<0.17
necessary for good selectivity indices: in general, the respective
acridinediones 9c, 14c, 15c, 30 and 32 were less potent against
M. tuberculosis and were more cytotoxic that the corresponding
pyridoacridines and therefore, were also not investigated further. It
was evident from this initial study that separation of anti-TB activity
and cytotoxicity could be achieved through the size-reduced ana-
logues of ascididemin (1).
A series of 4-aminopyrido[2,3,4-kl]acridin-6-one analogues were
then prepared and evaluated for anti-TB activity and cytotoxicity.
While the 4-amino derivative 12 exhibited less potent anti-TB activity
a
Ethambutol resistant.
Isoniazid resistant.
Rifampicin resistant.
Kinamycin resistant.
Ciprofloxacin resistant.
b
c
d
e
4. Conclusion
The marine natural product ascididemin (1) exhibits potent in
vitro growth inhibition of M. tuberculosis H37Rv (MIC 0.35 M) but
also significant levels of cytotoxicity (IC50<0.14 M). This in-
m
(MIC 3.2 mM)and alowerselectivityindexof7.3(Table 3), itwasfound
m
that analogues bearing a 2-carbon spacer and a terminal bulky group
were significantly less cytotoxic while maintaining respectable anti-
TB activity. Initial compounds prepared included the tert-butyloxy-
carbonyl derivative 17, tosylate 19 and phenylthiourea 20, all of which
exhibited reasonable anti-TB activity and much reduced cytotoxicity
against both Vero and P388 cells. The free terminal amine 18 or al-
cohol 23 was relatively more cytotoxic. Good antibacterial activity
was also observed for these compounds against B. subtilis, and along
with the low cytotoxicity, no antifungal activity was observed for any
of the amino derivatives. Due to the observation of limited solubility
vestigation of a series of 4-substituted pyrido[2,3,4-kl]acridin-6-
ones led to the identification of several compounds found to
possess M. tuberculosis MICs in the low micromolar range while
exhibiting significantly reduced cytotoxicity, and exemplifies the
value of screening natural products libraries as a starting point for
further lead development programmes. In addition, the observa-
tion of growth inhibition by thioethyl analogue 10 towards a range
of single drug resistant strains of Mtb is particularly encouraging,
and suggests that size-reduced analogues of ascididemin may
provide a useful scaffold for future studies.
Table 3
5. Experimental section
In vitro Biological Activities of Substituted 4-aminopyrido[2,3,4-kl]acridin-6-ones
12, 17e21, 23 and 24
5.1. Chemistry: general methods
Compound H37Rv MIC B.subb T.mentc Vero cells Selectivity P388 cells
(m
M)a
IC50
(
m
M) indexd
IC50 (mM)
All solvents used were analytical grade or better and/or purified
according to standard procedures. Chemical reagents used were
either purchased from standard chemical suppliers and used as
purchased or prepared according to literature procedures and pu-
rified to match the reported physical and spectral data. Analytical
thin layer chromatography was carried out on Merck DC-plas-
tikfolien Kieselgel 60 F254 plates and products were visualised by
UV fluorescence. Flash column chromatography was performed
using Merck 40e63 mm silica gel. Mass spectra were recorded on
a VG-7070 mass spectrometer operating at a nominal accelerating
voltage of 70 eV. All spectra were obtained using EI, FAB or CI
12
17
18
19
20
21
23
3.2
2.0
5.5
3.5
3.7
2.0
1.3
2.0
0.152
NDe
5
8
5
5
10
1
3
ND
0
0
0
0
0
0
0
23.5
>25
2.1
If
7.3
>12.5
0.4
ND
ND
>12.5
9.8
>12.5
ND
41.0
8.6
51.6
>59
>63
50.2
>63
I
>25
12.7
>25
154
24
RMPg
a
MIC (
m
M) against M. tuberculosis H37Rv.
b
c
d
e
f
B. subtilis inhibition zone (mm) at 120
T. mentagrophytes inhibition zone (mm) at 120
Selectivity index: Vero cell line cytotoxicity/Mtb MIC.
ND: not determined.
I: insoluble in Vero cell line media.
RMP: rifampin.
m
g/disc.
m
g/disc.
ionisation
techniques
using
perfluorokerosene,
3-nitro-
benzylalcohol or polyethylene glycol as the internal standards. In-
frared spectra were recorded as dry films on sodium chloride with
g

D.R. Appleton et al. / Tetrahedron 66 (2010) 4977e4986
4981
a Spectrum One Fourier Transform infrared spectrometer, with the
1603 cmꢁ1 absorption band of polystyrene being used as a refer-
ence. Melting points were recorded on a Reichert/Kofler block and
are uncorrected. NMR spectra were recorded at 298 K at either
300.13 or 400.13 MHz for 1H and 75.47 or 100.62 MHz for 13C on
Bruker Avance 300 or 400 spectrometers. Chemical shifts are given
yield) as yellow/brown crystals. Mp 210e211 ꢀC; Rf (100% CH2Cl2)
0.23; IR nmax (film) 1650, 1644, 1599, 1552, 1469, 1433, 1358, 1242,
1168, 974, 858, 759 cmꢁ1; 1H NMR (400 MHz, CDCl3)
d 9.05 (1H, d,
J¼5.7 Hz, H-2), 8.61 (1H, dd, J¼8.4, 1.4 Hz, H-11), 8.57 (1H, dd, J¼8.4,
1.7 Hz, H-8), 8.49 (1H, d, J¼5.7 Hz, H-1), 7.97 (1H, ddd, J¼8.4, 7.0,
1.4 Hz, H-9), 7.90 (1H, ddd, J¼8.4, 7.1, 1.4 Hz, H-10), 7.67 (2H, m, H-
13/17), 7.53 (3H, m, H-14/15/16), 6.29 (1H, s, H-5); 13C NMR
in parts per million on the d scale referenced to the residual solvent
peaks (CHCl3: 1H 7.25, 13C 77.0 ppm; DMSO-d6: 1H 2.50, 13C
39.4 ppm; MeOH-d4: 1H 3.30, 13C 49.0 ppm). Assignments were
aided by DEPT135, HSQC and HMBC experiments. Microanalyses
were carried out by the Campbell Laboratory, University of Otago,
Dunedin, New Zealand. Target compounds ascididemin (1), 11-
hydroxyascididemin (7), kuanoniamine A (8) and pyridoacridines 9,
10, 11 and 12 were prepared as previously described,14,15 as were
the quinone starting materials 13a16 and 27.17
(100 MHz, CDCl3) d 180.3 (C-6), 160.5 (C-4), 149.5 (C-3a), 148.2 (C-
2),146.6 (C-6a),145.9 (C-7a),137.0 (C-11b),136.1 (C-13/17),133.1 (C-
8), 131.8 (C-9), 130.6 (C-15), 130.6 (C-10), 130.3 (C-14/16), 127.9
(C-12), 124.9 (C-5), 122.9 (C-11), 122.8 (C-11a), 117.4 (C-1), 116.3 (C-
3b); MS m/z (%) 340 (Mþ, 100), 311 (MþꢁCHO, 79); HREIMS calcd
for C21H12N2OS 340.0670, found 340.0668.
5.1.4. 2-(Octylthio)benzo[l,4]quinone (14a). A solution of octane-
thiol (2.98 g, 20.4 mmol) in MeOH (5 mL) was added to a suspen-
sion of 1,4-benzoquinone (4.4 g, 40.7 mmol) in MeOH (30 mL). The
dark orange solution was swirled for 5 min, poured into ice-cold
water (60 mL), then cooled to yield the bright orange crude prod-
uct, which was filtered and dried to give 14a (3.42 g, 67%). Mp
96e97 ꢀC; Rf (100% CH2Cl2) 0.8; IR nmax (KBr) 1656, 1637,
5.1.1. 2-(Phenylthio)-5-(2-acetylanilino)-1,4-benzoquinone
(13b). 20-Aminoacetophenone (0.44 g, 3.3 mmol) in methanol
(10 mL) was added to a solution of 2-phenythio-l,4-benzoquinone
(13a)16 (0.8 g, 3.7 mmol) and cerium trichloride heptahydrate
(0.56 g, 1.5 mmol) in ethanol (60 mL). The solution was stirred vig-
orously for 24 h, yielding a dark red precipitate that was filtered and
recrystallised from methanol to afford 13b (0.8 g, 61% yield) as a dark
1622 cmꢁ1
;
1H NMR (CDCl3, 300 MHz)
d
6.78 (1H, d, J¼10.1 Hz, H-
6), 6.69 (1H, dd, J¼10.1, 2.3 Hz, H-5), 6.35 (1H, d, J¼2.3 Hz, H-3), 2.74
red crystalline solid. Mp 164e168 ꢀC; Rf (100% CH2Cl2) 0.71; IR nmax
(film) 1658, 1651, 1614, 1558, 1620, 1452, 1300, 1250, 1210, 997,
752 cmꢁ1; 1H NMR (CDCl3, 300 MHz)
11.02 (1H, s, NH), 7.92 (1H br
,
(2H, t, J¼7.3 Hz, H2-7), 1.71 (2H, m), 1.43 (2H, m), 1.26 (8H, m), 0.86
(3H, t, J¼7.0 Hz, H3-14); 13C NMR (CDCl3, 75 MHz)
d 183.9 (C-1 or C-
d
4), 183.8 (C-4 or C-1), 153.2 (C-2), 137.4 (C-5), 136.1 (C-6), 124.7 (C-
3), 31.6 (C-12), 30.4 (C-7), 29.0 (C-9, C-10 and C-11), 27.2 (C-8), 22.5
(C-13), 14.0 (C-14); EIMS m/z (%) 254 (Mþþ2H, 22), 252 (Mþ, 28),
142 (100); HREIMS calcd for C14H20O2S 252.1184, found 252.1180.
Anal. Calcd for C14H20O2S: C, 66.63; H, 7.99. Found: C, 66.42; H, 8.28.
d, J¼7.8 Hz, ArH), 7.55 (2H, brd, J¼5.0 Hz, ArH), 7.50(5H, m, ArH), 7.17
(1H, m, ArH), 6.47 (1H, s), 5.84 (1H, s), 2.63 (3H, s, COCH3); 13C NMR
(CDC13, 75 MHz) d 201.2,183.2,179.7,158.6,143.2,139.8,135.5,134.1,
132.3,130.4,130.2,127.5,125.6,123.2,122.0,120.7,102.4, 28.4; MS m/
z (%) 349, 331 (MþꢁH2O, 48), 307 (MþꢁCHCO, 100), 186 (Mþꢁ163,
60); HREIMS calcd for C20H15NO3S 349.0773, found 349.0773.
5.1.5. 2-(2-Acetylanilino)-5-(octylthio)benzo[l,4]quinone
(14b). A
mixture of 20-aminoacetophenone (1.95 g, 14.4 mmol), quinone 14a
(3.32 g, 13.1 mmol), cerium trichloride heptahydrate (2.34 g,
6.57 mmol) and EtOH (100 mL) was stirred at room temperature
with air bubbling through it for 4 days. The dark red crude product
was removed by filtration every day and dried to afford 14b (2.56 g,
51%). Mp 115e117 ꢀC; Rf (100% CH2Cl2) 0.72; IR nmax (KBr) 2926,1644,
5.1.2. 2-(Phenylthio)-9-methyl-l,4-acridinedione (13c). 2-(Phenyl-
thio)-5-(2-acetylanilino)-1,4-benzoquinone (13b) (1.1 g, 3.3 mmol)
was dissolved in a premixed solution of glacial acetic acid (36 mL)
and concd H2SO4 (4 mL) and heated at 80 ꢀC for 10 min. Addition of
water (100 mL) afforded a precipitate, which was filtered, washed
with water (5ꢂ20 mL) and cold methanol (2ꢂ30 mL). Recrystalli-
sation from chloroform/methanol yielded 13c (0.78 g (79% yield))
as yellow needle-like crystals. Mp 207e210 ꢀC; Rf (100% CH2Cl2)
0.34; IR nmax (film) 1660sh, 1576, 1560, 1376, 1337, 1250, 1184,
1614, 1581, 1557, 1532, 1454 cmꢁl; 1H NMR (CDCl3, 300 MHz)
d 11.13
(1H, s, NH-7), 7.94 (1H, d, J¼8.4 Hz, H-10), 7.56 (2H, m, H-12,13), 7.18
(1H, m, H-11), 6.47 (1H, s, H-3), 6.36 (1H, s, H-6), 2.78 (2H, t, J¼7.2 Hz,
H2-16), 2.67 (3H, s, H3-15), 1.75 (2H, m, H2-17), 1.48 (2H, m, H2-18),
1.29 (8H, m), 0.89 (3H, t, J¼6.9 Hz, H3-23); 13C NMR (CDCl3, 75 MHz)
758 cmꢁ1
;
1H NMR (CDCl3, 400 MHz)
d
8.40 (1H, dm, J¼9.0 Hz,
H-5), 8.33 (1H, dm, J¼8.6 Hz, H-8), 7.89 (1H, ddd, J¼8.4, 6.8, 1.8 Hz,
H-6), 7.75 (1H, ddd, J¼8.4, 6.9, 1.8 Hz, H-7), 7.52 (5H, m, H-
13,14,15,16,17), 6.36 (1H, s, H-3), 3.21 (3H, s, H3-11); 13C NMR
d 201.2 (C-14), 183.1 (C-4), 179.3 (C-1), 157.4 (C-5), 143.1 (C-2), 139.9
(C-8),134.1 (C-12),132.3 (C-10),125.7 (C-9),123.1 (C-11),120.9 (C-13
or C-6),120.6 (C-6 or C-13),102.9 (C-3), 31.7, 30.6 (C-16), 29.0 (3ꢂC),
28.4 (C-15), 27.3 (C-17), 22.6,14.0 (C-23); EIMS m/z (%) 387 (Mþþ2H,
100), 385 (Mþ, 10), 352 (70), 274 (35), 256 (48); HREIMS calcd for
C22H27NO3S 385.1712, found 385.1712. Anal. Calcd for C22H27NO3S:
C, 68.54; H, 7.06; N, 3.63. Found: C, 68.44; H, 6.95; N, 3.51.
(CDCl3, 100 MHz)
d 183.6 (C-1), 180.2 (C-4), 160.2 (C-2), 151.8 (C-9),
148.4 (C-10a), 147.2 (C-4a), 135.7 (C-14,16), 132.6 (C-6), 132.2 (C-5),
130.7 (C-15), 130.5 (C-13,17), 129.5 (C-7), 129.2 (C-8a), 128.4 (C-3),
127.2 (C-12), 125.5 (C-8), 123.2 (C-9a), 16.2 (C-11); MS m/z (%) 331
(Mþ, 100), 302 (MþꢁCHO, 20), 275 (23), 274 (50), 254 (41); HREIMS
calcd for C20H13NO2S 331.0667, found 331.0667.
5.1.6. 2-(Octylthio)-9-methyl-acridine[l,4]dione (14c). A solution of
14b (209 mg, 0.54 mmol) in glacial acetic acid-H2SO4 (10:1, 5 mL)
was heated at 100 ꢀC for 5 min. The dark red reaction mixture was
cooled, diluted with water (30 mL) and neutralised with concd
ammonia. The mixture was filtered, the residue was washed with
water then cold methanol and recrystallised from MeOH to afford
a dark yellow product 14c (170 mg, 85%). Mp 136e137 ꢀC; Rf (100%
5.1.3. 4-Phenylthiopyrido[2,3,4-kl]acridin-6-one (13). 2-Phenylthio-
9-methyl-1,4-acridione (13c, 33 mg, 0.10 mmol) and ammonium
chloride (380 mg, 7.10 mmol) in glacial acetic acid (1.0 mL) were
stirred at 100 ꢀC. Paraformaldehyde (24 mg, 0.8 mmol) in glacial
acetic acid (10 mL) was added and heating continued at 94 ꢀC for
5 min under nitrogen. The dark purple reaction mixture was
cooled, poured onto ice, made basic with concd ammonia and
extracted with chloroform (3ꢂ100 mL). The combined organic ex-
tracts were washed with water/concd ammonia (4:1, three times),
then brine (100 mL), dried over magnesium sulfate, and the solvent
removed in vacuo. The yellow/orange residue was purified by flash
column chromatography (silica gel, chloroform), with recrystalli-
sation from dichloromethane/methanol yielding 13 (28 mg, 83%
CH2Cl2) 0.31; IR nmax (KBr) 2925, 1664, 1574, 1562 cmꢁ1
;
1H NMR
(CDCl3, 300 MHz)
d
8.33 (1H, d, J¼8.4 Hz, H-5), 8.25 (1H, d, J¼7.5 Hz,
H-8), 7.83 (1H, t, J¼7.5 Hz, H-6), 7.68 (1H, t, J¼7.7 Hz, H-7), 6.80 (1H,
s, H-3), 3.11 (3H, s, H-11), 2.81 (2H, t, J¼7.4 Hz, H-12),1.73 (2H, m, H-
13), 1.45 (2H, m, H-14), 1.25 (8H, m), 0.85 (3H, t, J¼6.8 Hz, H-19); 13C
NMR (CDCl3, 75 MHz)
d 183.5 (C-1), 179.6 (C-4), 158.8 (C-2), 151.6
(C-9), 148.3 (C-10a), 147.1 (C- 4a), 132.5 (C-6), 132.1 (C-5), 129.4 (C-
7), 129.1 (C-8a), 127.1 (C-3), 125.4 (C-8), 123.4 (C-9a), 31.7, 30.9 (C-

4982
D.R. Appleton et al. / Tetrahedron 66 (2010) 4977e4986
12), 29.0 (3ꢂC), 27.3 (C-13), 22.5, 16.1 (C-11), 14.0 (C-19); EIMS m/z
(%) 369 (Mþþ2H, 35), 367 (Mþ, 40), 256 (100); HREIMS calcd for
C22H25NO2S 367.1606, found 367.1606. Anal. Calcd for C22H25NO2S:
C, 71.90; H, 6.86; N, 3.81. Found: C, 71.88; H, 6.82; N, 4.06.
(3ꢂ100 mL), washed with brine and the solvent removed in vacuo.
The residue was subjected to silica flash chromatography eluting
with CH2Cl2 to afford 15c (175 mg, 92%) as yellow needles from
CH2Cl2. Mp 174e176 ꢀC; Rf (100% CH2Cl2) 0.21; IR nmax (KBr) 1729,
1662, 1573, 1561, 1377, 1341, 1252, 1200 cmꢁ1 1H NMR (CDCl3,
;
5.1.7. 4-Octylthiopyrido[2,3,4-kl]acridin-6-one (14). A mixture of
14c (170 mg, 0.464 mmol), ammonium chloride (750 mg, 14 mmol)
and paraformaldehyde (70 mg, 2.32 mmol) were heated in glacial
acetic acid (40 mL) at 100 ꢀC for 10 min. The purple solution was then
made alkalinewith2 MNaOHand extracted withCH2Cl2 (3ꢂ100 mL).
The extract was washed with brine, dried over MgSO4 and concen-
trated in vacuo. The residue was chromatographed (silica), eluting
with CH2C12/MeOH (100:1) to afford 14 as a yellow, amorphous solid
(88 mg, 50%). Mp 119e120 ꢀC; Rf (100% CH2Cl2) 0.21; IR nmax (KBr)
300 MHz)
d
8.31 (1H, d, J¼8.4, H-5), 8.23 (1H, d, J¼8.5 Hz, H-8), 7.83
(1H, t, J¼7.6 Hz, H-6), 7.68 (1H, t, J¼7.7 Hz, H-7), 6.82 (1H, s, H-3),
4.16 (2H, q, J¼7.1 Hz, H2-15), 3.12 (2H, t, J¼7.1 Hz, H2-12), 3.09 (3H,
S, H3-11), 2.75 (2H, t, J¼7.3 Hz, H2-13), 1.23 (3H, t, J¼7.1 Hz, H3-16);
13C NMR (CDCl3, 75 MHz)
d 183.3 (C-1), 179.5 (C-4), 170.6 (C-14),
157.5 (C-2),151.7 (C-9), 148.2 (C-10a),146.9 (C-4a),132.6 (C-6), 132.1
(C-5), 129.4 (C-7), 129.0 (C-8a), 127.1 (C-3), 125.4 (C-8), 123.2 (C-9a),
61.1 (C-15), 32.2 (C-13), 25.4 (C-12), 16.0 (C-11), 14.1 (C-16); EIMS
m/z (%) 355 (Mþ, 75), 256 (100), 254 (60), 55 (75); HREIMS calcd for
C19H17NO4S 355.0878, found 355.0881.
1651, 1644,1597, 1551 cmꢁ1; 1H NMR (CDCl3, 300 MHz)
d 8.91 (1H, d,
J¼5.8 Hz, H-2), 8.50 (1H, dd, J¼8.1, 1.2 Hz, H-11), 8.46 (1H, dd, J¼8.1,
1.2 Hz, H-8), 8.35(1H,d, J¼5.8 Hz, H-1), 7.89(1H, ddd,J¼8.1, 8.1, 1.2 Hz,
H-9), 7.81 (1H, ddd, J¼8.1, 8.1,1.2 Hz, H-10), 6.72 (1H, s, H-5), 2.96 (2H,
t, J¼7.4 Hz, H2-12), 1.83 (2H, m, H2-13), 1.52 (2H, m, H2-14), 1.32e1.26
5.1.11. Ethyl
3-(4-thiopyrido[2,3,4-kl]acridin-6-one)propanoate
(15). A mixture of dione 15c (89 mg, 0.251 mmol), ammonium
chloride (402 mg, 7.52 mmol) and paraformaldehyde (38 mg,
1.25 mmol) in glacial acetic acid (25 mL) was heated at 100 ꢀC for
10 min. Using2 MNaOH, thecooledpurplereactionmixturewasthen
neutralised. The mixture was extracted with CH2Cl2 (3ꢂ100 mL),
washed with brine (2ꢂ100 mL) and evaporated. The residue was
subjected to silica flash chromatography eluting with CH2Cl2/MeOH
(95:5) to yield 15 (60 mg, 66%). Mp 173e174 ꢀC; Rf (100% CH2Cl2) 0.10;
(8H, m), 0.86(3H, t, J¼7.0 Hz, H3-19);13C NMR(CDCl3, 75 MHz)
d179.6
(C-6), 159.0 (C-4), 149.5 (C-3a), 148.1 (C-2), 146.3 (C-6a), 145.7 (C-7a),
136.7 (C-11b), 132.9 (C-8), 131.6 (C-9), 130.4 (C-10), 123.5 (C-5), 122.7
(C-11), 122.6 (C-11a), 117.0 (C-1), 116.2 (C-11c), 31.7, 31.1 (C-12), 29.1
(3ꢂC), 27.5 (C-13), 22.5, 14.0 (C-19); FABMS m/z (%) 379 (Mþþ2HþH,
34), 307 (27), 289 (14), 154 (100), 136 (66); HRFABMS calcd for
C23H27N2OS 379.1844 [Mþ2HþH]þ, found 379.1841.
IR nmax (KBr)2923,1730,1645,1598,1551,1429,1357,1242,1183 cmꢁ1
1H NMR (CDCl3, 300 MHz)
;
d
8.97 (1H, d, J¼5.5 Hz, H-2), 8.58 (1H, dd,
5.1.8. 2-(3-Ethylpropanoatethio)benzo[l,4]quinone (15a). Ethyl-3-
mercaptopropionate (2.48 g, 18.5 mmol) in MeOH (5 mL) was added
dropwise to a solution of 1,4-benzoquinone (4.00 g, 37.0 mmol) in
MeOH (40 mL) and the dark orange solution was stirred for 10 min.
Pouring into water (40 mL) and cooling produced crude product,
which was filtered and recrystallised from MeOH to yield 15a (3.86 g,
87%) as orange-red prisms. Mp 98e99 ꢀC; Rf (100% CH2Cl2) 0.63; IR
nmax (KBr) 1732, 1655, 1632, 1373, 1212, 1196 cmꢁ1; 1H NMR (CDCl3,
J¼8.3, 1.1 Hz, H-11), 8.55 (1H, dd, J¼8.4, 1.0 Hz, H-8), 8.43 (1H, d,
J¼5.5 Hz, H-1), 7.96 (1H, ddd, J¼7.0, 7.0, 1.5 Hz, H-9), 7.88 (1H, ddd,
J¼7.0, 7.0,1.5 Hz, H-10), 6.80 (1H, s, H-5), 4.21 (2H, q, J¼7.2 Hz, H2-15),
3.29 (2H, t, J¼7.4 Hz, H2-12), 2.85 (2H, t, J¼7.3 Hz, H2-13), 1.29 (3H, t,
J¼7.2 Hz, H3-16); 13C NMR (CDCl3, 75 MHz)
d 179.8 (C-6),170.8 (C-14),
158.0 (C-4), 149.3 (C-3a), 148.2 (C-2), 146.3 (C-6a), 145.9 (C-7a), 136.9
(C-11b), 133.1 (C-8), 131.8 (C-9), 130.6 (C-10), 123.5 (C-5), 122.8 (C-
11a), 122.8 (C-11), 117.3 (C-1), 116.3 (C-11c), 61.2 (C-15), 32.5 (C-13),
25.7 (C-12), 14.2 (C-16); EIMS m/z (%) 367 (Mþþ2HþH, 15), 307 (30),
289 (15), 154 (100), 136 (65), 120 (10), 107 (20), 89 (20), 77 (20);
HREIMS calcd for C20H19N2O3S 367.1111 [Mþ2HþH]þ, found 367.1112.
300 MHz)
d
6.76(1H, d,J¼10.1 Hz, H-6), 6.68(1H, dd, J¼10.1, 2.2 Hz, H-
5), 6.38 (1H, d, J¼2.2 Hz, H-3), 4.13 (2H, q, J¼7.2 Hz, H2-10), 3.02 (2H, t,
J¼7.3 Hz, H2-7), 2.67 (2H, t, J¼7.3 Hz, H2-8), 1.22 (3H, t, J¼7.2 Hz, H3-
11); 13C NMR (CDCl3, 75 MHz)
d 183.7 (C-1 or C-4), 183.5 (C-4 or C-1),
170.5 (C-9),151.8(C-2),137.3(C-5),136.0(C-6),124.8(C-3), 61.0(C-10),
32.1 (C-8), 24.9 (C-7),14.0 (C-11); EIMS m/z (%) 240 (Mþ, 75),153 (70),
55 (100); HREIMS calcd for C11H12O4S 240.0456, found 240.0453.
Anal. Calcd for C11H12O4S: C, 54.99; H, 5.03. Found: C, 54.86; H, 4.81.
5.1.12. 4-Ethylsulfoxopyrido[2,3,4-kl]acridin-6-one
(16). m-Chlor-
operoxybenzoic acid (85%) (31 mg, 0.15 mmol) was added to a solu-
tion of 4-ethylthiopyrido[2,3,4-kl]acridin-6-one (10) (40 mg,
0.137 mmol) and sodium acetate (50 mg) in CH2Cl2 (40 mL). The
solution was stirred at room temperature for 30 min, then washed
with water, dried over anhydrous magnesium sulfate and the solvent
removed in vacuo. Purification was achieved by flash chromatogra-
phy eluting with CH2Cl2/MeOH (200:1) yielding 16 (31 mg, 73%) and
starting material 10 (10%). IR nmax (film) 1654, 1577, 1430, 1359, 1232,
5.1.9. 2-(2-Acetylanilino)-5-(3-ethylpropanoatethio)benzo[l,4]qui-
none (15b). A mixture of 20-aminoacetophenone (2.35 g,
17.4 mmol), 15a (3.80 g, 15.8 mmol) and cerium trichloride hepta-
hydrate (2.95 g, 7.9 mmol) were dissolved in MeOH (100 mL) and
stirred for 7 days. The dark red precipitate was filtered and washed
to yield 15b (2.68 g, 45%). Mp 137e138 ꢀC; Rf (100% CH2Cl2) 0.45; IR
1062, 1038, 896, 846, 773, 762 cmꢁ1 1H NMR (CDCl3, 400 MHz)
;
d
8.96 (1H, d, J¼5.8 Hz, H-2), 8.65 (1H, dd, J¼8.4, 1.5 Hz, H-8), 8.62
nmax (KBr) 3434, 1732, 1651, 1614, 1557, 1529, 1451, 1303, 1251 cmꢁ1
1H NMR (CDCl3, 300 MHz)
;
(1H, dd, J¼8.3, 1.4 Hz, H-11), 8.49 (1H, d, J¼5.8 Hz, H-1), 8.04 (1H,
ddd, J¼8.4, 7.0, 1.4 Hz, H-9), 7.97 (1H, ddd, J¼8.3, 7.0, 1.5 Hz, H-10),
7.55 (1H, s, H-5), 3.52 (1H, m, H-13A), 3.24 (1H, m, H-13B),1.38 (3H, t,
d
11.14 (1H, s), 7.95 (1H, d, J¼8.1 Hz), 7.55
(2H, m), 7.17 (1H, q, J¼6.1 Hz), 6.46 (1H, s), 6.40 (1H, s), 4.19 (2H, q,
J¼7.1 Hz), 3.08 (2H, t, J¼7.1 Hz), 2.74 (2H, t, J¼7.3 Hz), 2.67 (3H, s),
J¼7.4 Hz, H2-14); 13C NMR (CDCl3, 100 MHz)
d 181.1 (C-6), 158.8 (C-
1.29 (3H, t, J¼4.8 Hz); 13C NMR (CDCl3, 75 MHz)
d
201.2, 182.7, 179.1,
4), 148.3 (C-2), 148.1 (C-3a), 146.3 (C-6a), 146.2 (C-7a), 137.4 (C-11b),
133.5 (C-8), 132.3 (C-5), 131.5 (C-9), 131.3 (C-10), 123.0 (C-11a), 122.9
(C-11), 117.4 (C-1), 116.8 (C-11c), 47.5 (C-13), 6.0 (C-14); EIMS m/z (%)
292 (MþꢁO, 20), 260 (50), 259 (41), 204 (100), 177 (22); HRFABMS
calcd for C17H15N2O2S 311.0849 [Mþ2HþH]þ, found 311.0850.
170.6, 156.1, 142.9, 139.7, 134.0, 132.2, 125.6, 123.1, 120.9, 120.5,
102.8, 61.0, 32.1, 28.3, 25.1, 14.0; EIMS m/z (%) 375 (Mþþ2H, 100),
373 (Mþ, 35), 274 (60), 256 (45), 55(40); HREIMS calcd for
C19H19NO5S 373.0984, found 373.0973. Anal. Calcd for C19H19NO5S:
C, 61.11; H, 5.13; N, 3.75. Found: C, 61.04; H, 4.90; N, 3.74.
5.1.13. tert-Butyl
2-(6-oxo-6H-pyrido[2,3,4-kl]acridin-4-ylamino)
5.1.10. 3-(9-Methyl-1,4-acridinedion-2-ylthio)propanoate (15c). Qu-
inone adduct 15b (200 mg, 0.54 mmol) was dissolved in glacial
acetic acid/H2SO4 (10:1, 5 mL) and heated to 100 ꢀC for 5 min. The
cooled reaction mixture was diluted with ice and then neutralised
with concd ammonia. The mixture was extracted with CH2Cl2
ethylcarbamate (17). To a solution of 4-(4-methylphenylthio)pyrido
[2,3,4-kl]acridin-6-one (9) (130 mg, 0.37 mmol), tert-butyl 2-ami-
noethylcarbamate (296 mg, 1.84 mmol), Et3N (619
in dry CH2Cl2/MeOH (1:1, 6 mL), solution of Na (17 mg,
0.74 mmol) in dry methanol (4 mL) was added. The reaction
mL, 4.44 mmol)
a

D.R. Appleton et al. / Tetrahedron 66 (2010) 4977e4986
4983
mixture was then heated to 65 ꢀC under N2 for 7 h. H2O (100 mL)
was then added and the resulting mixture was neutralised with 1 M
H2SO4, and extracted with CH2Cl2 (3ꢂ60 mL). The combined or-
ganic extracts were washed with brine (2ꢂ60 mL), dried over an-
hydrous magnesium sulfate and the solvents removed in vacuo.
Silica flash column chromatography eluting with CH2Cl2/MeOH
(98:2) yielded 17 as an orange solid (76 mg, 53%). Mp 187e190 ꢀC
(decomp.); Rf (10% MeOH/CH2Cl2) 0.61; IR nmax (film) 3358, 2928,
and Et3N (29
isothiocyanate (33
m
L, 0.21 mmol) in dry MeCN (20 mL), phenyl-
mL, 0.28 mmol) was added. The reaction mixture
was stirred under N2 for 1 h. H2O (100 mL) was added and extracted
with CH2Cl2 (3ꢂ60 mL) then washed with 0.2 M HCl (50 mL), H2O
(50 mL) and brine (50 mL) then dried over anhydrous magnesium
sulfate. Silica flash column chromatography eluting with CH2Cl2/
MeOH (100:0 to 97:3) yielded 20 as an orange solid (27 mg, 45%).
Mp >300 ꢀC (decomp.); Rf (10% MeOH/CH2Cl2) 0.57; IR nmax (film)
1693, 1610, 1566, 1519, 1162 cmꢁ1
400 MHz)
;
1H NMR (CDCl3/CD3OD (1:1),
3390, 1557, 1494, 1248 cmꢁ1
300 MHz)
;
1H NMR (CDCl3/CD3OD (1:1),
d
8.81 (1H, d, J¼5.4 Hz, H-2), 8.50 (1H, d, J¼7.9 Hz, H-11),
d
8.84 (1H, d, J¼5.7 Hz, H-2), 8.57 (1H, d, J¼7.9 Hz, H-11),
8.38 (1H, d, J¼5.2 Hz, H-1), 8.34 (1H, d, J¼8.2 Hz, H-8), 7.89 (1H, t,
J¼7.7 Hz, H-9), 7.80 (1H, t, J¼7.5 Hz, H-10), 5.91 (1H, s, H-5), 3.53
8.41 (2H, m, H-1, H-8), 7.93 (1H, td, J¼8.3, 1.4 Hz, H-9), 7.86 (1H, td,
J¼8.2, 1.4 Hz, H-10), 7.33 (4H, m, H-19/20/22/23), 7.20 (1H, m, H-
21), 6.00 (1H, s, H-5), 4.09 (2H, t, J¼6.0 Hz, H2-14), 3.73 (2H, t,
(4H, br s, H2-13, H2-14), 1.41 (9H, s, Bu); 13C NMR (CDCl3/CD3OD
t
(1:1), 100 MHz)
d
179.7 (C-6), 156.9 (C-16), 153.4 (C-4), 147.1 (C-6a),
J¼6.0 Hz, H2-13); 13C NMR (CDCl3/CD3OD (1:1), 75 MHz),
d 181.6 (C-
146.9 (C-2), 145.1 (C-3a), 144.7 (C-7a), 136.2 (C-11b), 131.2 (C-8),
131.0 (C-9), 129.3 (C-10), 122.5 (C-11), 122.0 (C-11a), 117.3 (C-1),
16), 180.0 (C-6), 153.6 (C-4), 147.3 (C-6a), 147.1 (C-2), 145.3 (C-3a),
145.0 (C-7a), 137.0 (C-18), 136.5 (C-11b), 131.6 (C-8), 131.4 (C-9),
129.6 (C-10), 128.9 (C-20/22), 125.9 (C-21), 124.5 (C-19/23), 122.6
(C-11), 122.2 (C-11a), 117.5 (C-1), 115.9 (C-11c), 98.4 (C-5), 42.4 (C-
14), 42.1 (C-13). FABMS m/z 426 [MþH]þ; HRFABMS calcd for
C24H20N5OS 426.1389, found 426.1386.
115.7 (C-11c), 98.0 (C-5), 79.1 (C-16), 42.9 (C-13)
*
, 38.1 (C-14) , 27.5
*
(C-18), (*these assignments may be interchanged); FABMS m/z 391
[MþH]þ; HRFABMS calcd for C22H23N4O3 391.1770, found 391.1777.
5.1.14. 4-(2-Aminoethylamino)pyrido[2,3,4-kl]acridin-6-one (18). A
solution of 17 (56 mg, 0.14 mmol) in CH2Cl2/TFA (20:1, 20 mL) was
stirred at room temperature for 2 h while shielded from light with
Al foil. Removal of the solvents in vacuo yielded 18 as the tri-
fluoroacetate salt (58 mg, 100%), a light sensitive orange oil. Rf (10%
MeOH/CH2Cl2) 0.09; IR nmax (film) 3428, 1682, 1569, 1439, 1207,
5.1.17. tert-Butyl
2-(pyrazine-2-carboxamido)ethylcarbamate
(22a). A mixture of pyrazinecarboxylic acid (138 mg, 1.11 mmol),
tert-butyl 2-aminoethylcarbamate (178 mg, 1.11 mmol), Et3N
(0.34 mL, 2.44 mmol) and BOP-Cl (540 mg, 1.22 mmol) in dry
CH2Cl2 (10 mL) was stirred under N2 at room temperature for 3 h.
CH2Cl2 (30 mL) was then added and washed with 0.2 M HCl
(30 mL), H2O (30 mL), 5% satd NaHCO3 (30 mL) and brine (30 mL)
then dried over anhydrous magnesium sulfate. The solvent was
removed in vacuo to yield 22a (295 mg, 100%) as a white solid. Mp
110e112 ꢀC; Rf (10% MeOH/CH2Cl2) 0.86; IR nmax (film) 3374, 2987,
1137 cmꢁ1 1H NMR (CD3OD, 300 MHz)
; d 8.48 (2H, m, H-2, H-11),
8.23 (2H, m, H-1, H-8), 7.92 (1H, t, J¼7.3 Hz, H-9), 7.83 (1H, t,
J¼7.3 Hz, H-10), 5.83 (1H, s, H-5), 3.79 (2H, t, J¼6.0 Hz, H2-13)*, 3.44
(2H, t, J¼6.0 Hz, H2-14)*, (*these assignments may be inter-
changed); 13C NMR (CD3OD, 75 MHz) 180.7 (C-6), 162.0 (CF3COꢁ2 ),
d
154.5 (C-4), 148.3 (C-2), 147.5 (C-6a), 145.4 (C-3a and C-7a), 136.6
(C-11b), 133.0 (C-9), 132.2 (C-8), 131.4 (C-10), 124.4 (C-11), 123.2 (C-
11a),118.5 (C-1),115.9 (C-11c),100.3 (C-5), 41.0 (C-13)*, 39.2 (C-14)*,
(*these assignments may be interchanged), resonance for CF3COꢁ2
was obscured; FABMS m/z 291 [MþH]þ; HRFABMS calcd for
C17H15N4O 291.1246, found 291.1233.
2936,1688, 1660, 1531, 1165 cmꢁ1; 1H NMR (CDCl3, 300 MHz)
d 9.39
(1H, d, J¼1.2 Hz), 8.75 (1H, d, J¼2.4 Hz), 8.54 (1H, dd, J¼2.3, 1.5 Hz),
8.30 (1H, br s, NHCO), 5.38 (1H, br s, NHCO2), 3.62 (2H, m), 3.42 (2H,
m), 1.41 (9H, s, (CH3)3C); 13C NMR (CDCl3, 75 MHz)
d 163.6 (CO),
156.4 (OCO), 147.1, 144.3, 144.1, 142.5, 79.3 ((CH3)3CO), 40.0ꢂ2, 28.1
((CH3)3C); EIMS m/z 266 [M]þ; HREIMS calcd for C12H18N4O3
266.1379, found 266.1378.
5.1.15. 4-Methyl-N-(2-(6-Oxo-6H-pyrido[2,3,4-kl]acridin-4-ylamino)
ethyl)benzenesulfonamide (19). To a solution of amine 18 (44 mg,
5.1.18. N-(2-Aminoethyl)pyrazine-2-carboxamide
trifluoroacetate
0.11 mmol) and Et3N (48
m
L, 0.34 mmol) in MeCN (20 mL), p-
salt (22). Compound 22a (295 mg, 1.11 mmol) in CH2Cl2/TFA (9:1,
10 mL) was stirred at room temperature for 1.5 h. Solvents were
removed in vacuo to yield 22 (320 mg, 100%) as a colourless oil. Rf
(10% MeOH/CH2Cl2) 0.18; IR nmax (film) 3344, 2938, 1676, 1537,
tosylchloride (42 mg, 0.22 mmol) was added. The reaction mixture
was stirred under N2 for 1 h. CH2Cl2 (150 mL) was added and the
organic layer then washed with 5% satd NaHCO3 (80 mL), H2O
(80 mL), 0.2 M HCl (80 mL) and brine (80 mL) then dried over an-
hydrous magnesium sulfate. Silica flash column chromatography
eluting with CH2Cl2/MeOH (100:0 to 97:3) yielded 19 as an orange
solid (30 mg, 61%). Mp 228e231 ꢀC (decomp.); Rf (10% MeOH/
1202, 1136 cmꢁ1
; d 9.23 (1H, d,
1H NMR (CD3OD, 300 MHz)
J¼1.4 Hz), 8.78 (1H, d, J¼2.5 Hz), 8.66 (1H, dd, J¼2.5, 1.5 Hz), 3.77
(2H, t, J¼5.8 Hz), 3.26 (2H, t, obsc.); 13C NMR (CD3OD, 75 MHz)
d
166.5, 148.6, 145.9, 144.8, 144.6, 40.9, 38.3; CIMS m/z 167 [MþH]þ;
CH2Cl2) 0.53; IR nmax (film) 3353, 2918, 1615, 1567, 1519, 1156 cmꢁ1
;
HRCIMS calcd for C7H11N4O 167.0933, found 167.0934.
1H NMR (CDCl3/CD3OD (1:1), 400 MHz)
d
8.91 (1H, d, J¼5.6 Hz, H-
2), 8.61 (1H, dd, J¼8.0,1.1 Hz, H-11), 8.49 (1H, d, J¼5.7 Hz, H-1), 8.48
(1H, dd, J¼8.0, 0.9 Hz, H-8), 7.97 (1H, td, J¼8.3, 1.3 Hz, H-9), 7.88
(1H, td, J¼8.2,1.1 Hz, H-10), 7.75 (2H, d, J¼8.3 Hz, H-17/21), 7.21 (2H,
d, J¼8.5 Hz, H-18/20), 5.91 (1H, s, H-5), 3.60 (2H, t, J¼6.0 Hz, H2-13)*,
3.31(2H, t, J¼6.0 Hz, H2-14)*, 2.27 (3H, s, H3-22), (*theseassignments
may be interchanged); 13C NMR (CDCl3:CD3OD (1:1), 100 MHz)
5.1.19. N-(2-(6-Oxo-6H-pyrido[2,3,4-kl]acridin-4-ylamino)ethyl)pyr-
azine-2-carboxamide (21). To a solution of 4-(4-methylphenylthio)
pyrido[2,3,4-kl]acridin-6-one (9) (40 mg, 0.11 mmol), 22 (73 mg,
0.44 mmol), Et3N (153 mL, 1.1 mmol) in dry CH2Cl2/MeOH (1:1,
5 mL), a solution of Na (10 mg, 0.44 mmol) in dry methanol (5 mL)
was added. The reaction mixture was then heated to 65 ꢀC under N2
for 7 h. H2O (150 mL) was then added and the resulting mixture
was neutralised with 1 M H2SO4, and extracted with CH2Cl2
(3ꢂ100 mL). The combined organic extracts were washed with H2O
(2ꢂ100 mL) and brine (100 mL), dried over anhydrous magnesium
sulfate and the solvents removed in vacuo. Silica flash column
chromatography eluting with CHCl3/MeOH (95:5) yielded 21 as an
orange solid (16 mg, 36%). Mp >300 ꢀC (decomp.); Rf (10% MeOH/
d
180.2 (C-6),153.1 (C-4),147.4 (C-6a),147.2 (C-2),145.5 (C-3a),145.2
(C-7a), 143.2 (C-18), 136.7 (C-11b and C-16), 131.9 (C-8), 131.5 (C-9),
129.8 (C-10),129.3 (C-18/20),126.6 (C-17/21),122.7 (C-11),122.3 (C-
11a), 117.7 (C-1), 116.2 (C-11c), 98.6 (C-5), 42.0 (C-13)*, 40.7 (C-14)*,
20.8 (C-22), (*these assignments may be interchanged); FABMS m/z
445 [MþH]þ; HRFABMS calcd for C24H21N4O3S 445.1334, found
445.1327.
CH2Cl2) 0.31; IR nmax (film) 3283, 2924, 1665, 1529, 1166, 984 cmꢁ1
1H NMR (CDCl3/CD3OD (1:1), 300 MHz)
9.34 (1H, d, J¼1.4 Hz, H-
18), 8.90 (1H, d, J¼5.7 Hz, H-2), 8.75 (1H, d, J¼2.5 Hz, H-21), 8.64
;
5.1.16. 1-(2-(6-Oxo-6H-pyrido[2,3,4-kl]acridin-4-ylamino)ethyl)-3-
phenylthiourea (20). To a solution of amine 18 (58 mg, 0.14 mmol)
d

4984
D.R. Appleton et al. / Tetrahedron 66 (2010) 4977e4986
(1H, dd, J¼2.4, 1.5 Hz, H-20), 8.58 (1H, d, J¼8.1 Hz, H-11), 8.48 (1H,
d, J¼5.8 Hz, H-1), 8.41 (1H, d, J¼8.1 Hz, H-8), 7.93 (1H, td, J¼8.3,
1.3 Hz, H-9), 7.84 (1H, td, J¼8.2, 1.3 Hz, H-10), 6.04 (1H, s, H-5), 3.93
(2H, t, J¼6.0 Hz, H-14), 3.75 (2H, t, J¼6.0 Hz, H-13); 13C NMR (CDCl3/
anthracene-2-carboxylic acid methyl ester (30) (100 mg,
0.30 mmol) and ammonium chloride (472 mg, 8.9 mmol) in glacial
acetic acid (50 mL) was added paraformaldehyde (45 mg,
1.48 mmol). The mixture was heated to reflux for 45 min, then
cooled, poured onto ice and made basic with concd ammonia, then
extracted with CH2Cl2 (3ꢂ100 mL). The combined organic layers
were washed with brine (3ꢂ100 mL), then water (2ꢂ100 mL), and
dried over anhydrous magnesium sulfate. The solvent was then
removed in vacuo. Purification by flash column chromatography
(silica, CH2Cl2/MeOH, 95:5) yielded 25, as a yellow solid (77 mg,
83%). Recrystallisation from acetone afforded fine yellow needles.
Mp 340e343 ꢀC (decomp.); IR nmax (film) 3010, 1710, 1665, 1589,
CD3OD (1:1), 75 MHz)
d 180.2 (C-6), 164.3 (C-16), 153.4 (C-4), 147.4
(C-6a), 147.3 (C-2), 146.9 (C-21), 145.5 (C-3a), 145.2 (C-7a), 144.2 (C-
17), 143.6 (C-18), 142.9 (C-20), 136.6 (C-11b), 131.9 (C-8), 131.4 (C-9),
129.7 (C-10), 122.6 (C-11), 122.3 (C-11a), 117.6 (C-1), 116.2 (C-11c),
98.7 (C-5), 42.7 (C-13), 37.7 (C-14); FABMS m/z 397 [MþH]þ;
HRFABMS calcd for C22H17N6O2 397.1413, found 397.1417.
5.1.20. 4-(2-Hydroxyethylamino)pyrido[2,3,4-kl]acridin-6-one (23).
To a solution of 4-(4-methylphenylthio)pyrido[2,3,4-kl]acridin-6-
1418, 1281, 1261, 937, 879, 845, 763, 743 cmꢁ1
400 MHz)
;
1H NMR (CDCl3,
one (9) (131 mg, 0.37 mmol), ethanolamine (112
mL, 1.85 mmol) in
d
8.98 (1H, d, J¼6.0 Hz, H-2), 8.66 (1H, d, J¼7.1 Hz, H-12),
dry CH2Cl2/MeOH (1:1, 6 mL), a solution of Na (17 mg, 0.74 mmol) in
dry methanol (4 mL) was added. The reaction mixture was then
heated to65 ꢀC under N2 for 7 h. Water(100 mL) was then added and
the resulting mixturewas neutralised with 1 M H2SO4, and extracted
with CH2Cl2 (3ꢂ60 mL). The combined organic extracts were
washed with brine (2ꢂ60 mL), dried over anhydrous magnesium
sulfate and the solvents removed in vacuo. Silica flash column
chromatography eluting with CH2Cl2/MeOH (100:0 to 92:8) yielded
23 as an orange solid (27 mg, 25%). Mp 278e280 ꢀC (decomp.); Rf
(10% MeOH/CH2Cl2) 0.44; IR nmax (film) 3337, 2920,1609,1557,1433,
8.63 (1H, d, J¼7.1 Hz, H-9), 8.46 (1H, s, H-6), 8.44 (1H, d, J¼6.0 Hz,
H-1), 8.02 (1H, ddd, J¼8.2, 8.2, 1.2 Hz, H-10), 7.94 (1H, ddd, J¼8.2,
8.2, 1.2 Hz, H-11), 3.98 (3H, s, OCH3); 13C NMR (CDCl3, 100 MHz)
d
178.0 (C-7), 162.0 (C-13), 154.0 (C-3b), 149.2 (C-2), 147.8 (C-3a),
145.8 (C-8a), 139.2 (C-5), 137.6 (C-12b), 136.5 (C-6a), 133.3 (C-9),
132.5 (C-6), 132.0 (C-10), 130.9 (C-11), 123.0 (C-12a), 122.9 (C-12),
116.7 (C-12c), 116.6 (C-1), 52.9 (OCH3), C-7a not observed; EIMS m/z
(%) 346 (Mþ, 30), 315 (28), 259 (6), 243 (7), 215 (6), 83 (9), 69 (7), 55
(11); HREIMS calcd for C19H10N2O3S 346.0412, found 346.0403.
1253,1060 cmꢁ1; 1H NMR (CDCl3/CD3OD (1:1), 300 MHz)
d
8.96 (1H,
5.1.23. 7-Oxo-7H-4-oxa-3,8-diaza-benzo[de]cyclopenta[b]anthra-
cene-5-carboxylic acid methyl ester (26). Paraformaldehyde (42 mg,
1.4 mmol) was added to a stirred solution of 10-methyl-4,11-
dioxo-4,11-dihydro-1-oxa-5-aza-cyclopenta[b]anthracene-2-carbo-
xylic acid methyl ester (32) (90 mg, 0.28 mmol) and ammonium
chloride (446 mg, 8.4 mmol) in glacial acetic acid (50 mL). The so-
lution was stirred under nitrogen at 80 ꢀC for 35 min. After cooling,
the dark blue solution was poured into water (200 mL), made basic
with concd ammonia and extracted with CH2Cl2 (3ꢂ100 mL). The
combined organic extracts were washed with brine (3ꢂ100 mL),
then water (2ꢂ100 mL) and dried over anhydrous magnesium sul-
fate. The solvent was removed in vacuo yielding 70 mg (76%) of 26.
Recrystallisation from CHCl3/MeOH yielded a green microcrystalline
solid. Mp 340e345 ꢀC (decomp.); Rf (100% CH2Cl2) 0.17; IR nmax (film)
3433, 1720, 1675, 1605, 1570, 1415, 1306, 1226, 1199, 1127, 1051,
d, J¼5.7 Hz, H-2), 8.66 (1H, dd, J¼8.1, 1.2 Hz, H-11), 8.58 (1H, d,
J¼5.8 Hz, H-1), 8.47 (1H, dd, J¼8.3, 1.0 Hz, H-8), 7.97 (1H, td, J¼8.3,
1.3 Hz, H-9), 7.89 (1H, td, J¼7.0, 1.3 Hz, H-10), 6.04 (1H, s, H-5), 3.97
(2H, t, J¼5.5 Hz, H2-14), 3.62 (2H, t, J¼5.5 Hz, H2-13); 13C NMR
(CDCl3/CD3OD (1:1), 75 MHz) d 180.0 (C-6),153.5 (C-4),147.4 (C-6a),
147.1 (C-2),145.4 (C-3a),145.0 (C-7a),136.6 (C-11b),131.5 (C-8),131.2
(C-9), 129.5 (C-10), 122.7 (C-11), 122.3 (C-11a), 117.7 (C-1), 116.1 (C-
11c), 98.2 (C-5), 58.9 (C-14), 44.4 (C-13); FABMS m/z 292 [MþH]þ;
HRFABMS calcd for C17H14N3O2 292.1086, found 292.1091.
5.1.21. 2-(6-Oxo-6H-pyrido[2,3,4-kl]acridin-4-ylamino)ethyl
pyr-
azine-2-carboxylate (24). To a solution of alcohol 23 (24 mg,
0.08 mmol) in dry CH2Cl2 (15 mL), pyrazinecarboxylic acid (15 mg,
0.12 mmol), Et3N (40 mL, 0.29 mmol) and BOP-Cl (65 mg,
0.15 mmol) in dry CH2Cl2 (5 mL) was added. The reaction mixture
was stirred at room temperature under N2 for 3 h, at which time
further quantities of pyrazinecarboxylic acid (15 mg, 0.12 mmol),
763 cmꢁ1
;
1H NMR (CDCl3, 400 MHz)
d
9.08 (1H, d, J¼5.8 Hz, H-2),
8.64(1H, dd, J¼8.2,1.3 Hz,H-12), 8.61(1H, dd, J¼8.3,1.3 Hz,H-9), 8.45
(1H, d, J¼5.8 Hz,H-1), 8.01 (1H, ddd, J¼8.3, 8.3,1.5 Hz,H-10), 7.94(1H,
Et3N (40
mL) and BOP (65 mg) were added and reaction stirred for
ddd, J¼8.2, 8.2,1.3 Hz, H-11), 7.82 (1H, s, H-6), 4.01 (3H, s, OCH3); 13
C
another 2 h. CH2Cl2 (100 mL) was added then washed with 0.2 N
HCl (60 mL), H2O (60 mL), 5% satd NaHCO3 (60 mL) and brine
(60 mL), dried over anhydrous magnesium sulfate and the solvents
removed in vacuo. Silica flash column chromatography eluting with
CH2Cl2/MeOH (100:0 to 97:3) yielded 24 as an orange solid (12 mg,
38%). Mp 190 ꢀC (decomp.); Rf (10% MeOH/CH2Cl2) 0.67; IR nmax
NMR (CDCl3,100 MHz) d 177.6 (C-7),158.5 (C-3b),158.3 (C-13),149.3
(C-2), 147.5 (C-5), 147.1 (C-7a), 145.7 (C-8a), 143.2 (C-3a), 137.7 (C-
12b),133.4(C-9),132.1(C-10),131.1(C-11),126.2(C-6a),123.0(C-12a),
122.8 (C-12),117.0 (C-1),116.6 (C-12c),115.4 (C-6), 52.6 (OCH3); EIMS
m/z (%) 330 (Mþ, 100), 299 (67), 272 (11), 243 (8), 215 (45), 199 (23),
188 (12), 129 (10), 97 (14), 83 (20), 69 (28), 55 (43), 41 (48); HREIMS
calcd for C19H10N2O4 330.0641, found 330.0643.
(film) 3339, 2922, 1724, 1613, 1563, 1299, 1141, 840 cmꢁ1
;
1H NMR
9.35 (1H, d, J¼1.4 Hz, H-18), 8.96
(CDCl3/CD3OD (1:1), 400 MHz)
d
(1H, d, J¼5.7 Hz, H-2), 8.84 (1H, d, J¼2.5 Hz, H-21), 8.78 (1H, dd,
J¼2.4, 1.5 Hz, H-20), 8.67 (1H, dd, J¼8.1, 1.1 Hz, H-11), 8.57 (1H, d,
J¼5.8 Hz, H-1), 8.47 (1H, d, J¼8.6 Hz, H-8), 7.97 (1H, td, J¼7.6, 1.2 Hz,
H-9), 7.89 (1H, td, J¼8.1, 1.1 Hz, H-10), 6.12 (1H, s, H-5), 4.83 (2H, t,
J¼5.3 Hz, H2-14), 3.99 (2H, t, J¼5.3 Hz, H2-13); 13C NMR (CDCl3/
5.1.24. 5-(2-Acetylanilino)-4,7-dioxo-4,7-dihydro-benzo[b]thio-
phene-2-carboxylic acid methyl ester (29). To a stirred suspension of
methyl
4,7-dihydro-4,7-dioxobenzo[b]thiophene-2-carboxylate
(27)17 (0.58 g, 2.59 mmol) and cerium trichloride heptahydrate
(0.48 g, 1.30 mmol) in MeOH (100 mL) was added 20-amino-
acetophenone (0.70 g, 5.21 mmol). Air was continuously bubbled
through the mixture, which was stirred for twoweeks, during which
time it turned purple. The solvent was removed in vacuo, and the
residue was extracted with CH2Cl2 (3ꢂ100 mL). The combined or-
ganic extracts were washed with brine (3ꢂ100 mL) followed by
water (2ꢂ100 mL) and dried over anhydrous magnesium sulfate.
The solvent was removed in vacuo and the purple solid was purified
by silica flash column chromatography eluting with CH2Cl2 to yield
29(0.85 g, 92%). Recrystallisation from MeOHafforded darkred hair-
like needles. Mp 286e290 ꢀC (decomp.); Rf (100% CH2Cl2) 0.41; IR
CD3OD (1:1), 100 MHz) d 180.3 (C-6), 163.4 (C-16), 153.2 (C-4), 147.7
(C-21), 147.6 (C-6a), 147.2 (C-2), 145.8 (C-18), 145.4 (C-3a), 145.1 (C-
7a), 144.2 (C-20), 142.4 (C-17), 136.7 (C-11b), 131.6 (C-8), 131.3 (C-9),
129.7 (C-10), 122.7 (C-11), 122.3 (C-11a), 117.7 (C-1), 116.2 (C-11c),
98.8 (C-5), 63.3 (C-14), 41.2 (C-13); FABMS m/z 398 [MþH]þ;
HRFABMS calcd for C22H16N5O3 398.1253, found 398.1258.
5.1.22. 7-Oxo-7H-4-thia-3,8-diaza-benzo[de]cyclopenta[b]anthra-
cene-5-carboxylic acid methyl ester (25). To a stirred solution of 10-
methyl-4,11-dioxo-4,11-dihydro-1-thia-5-aza-cyclopenta[b]

D.R. Appleton et al. / Tetrahedron 66 (2010) 4977e4986
4985
nmax (film) 3422,1712,1685,1653,1620,1582,1536,1452,1302,1288,
1261,1181,1078, 749 cmꢁ1; 1H NMR (CDCl3, 400 MHz)
11.26 (1H, br
28.5 (C-17); EIMS m/z (%) 339 (Mþ, 13), 321 (100), 310 (3), 306 (13),
297 (7), 293 (20), 262 (37), 235 (14), 206 (5), 178 (14), 151 (7), 140
(5), 89 (7), 77 (4), 43 (7); HREIMS calcd for C18H13NO6 339.0743,
found 339.0748. Anal. Calcd for C18H13NO6: C, 63.72; H, 3.86; N,
4.13. Found: C, 63.69; H, 3.77; N, 4.06.
d
s, NH-9), 8.16 (1H, s, H-3), 7.95 (1H, dd, J¼8.5,1.1 Hz, H-12), 7.60 (1H,
d, J¼5.9 Hz, H-15), 7.57 (1H, m, H-14), 7.20 (1H, ddd, J¼8.1, 6.0,1.5 Hz,
H-13), 6.59 (1H, s, H-6), 3.94 (3H, s, OCH3), 2.67 (3H, s, H3-17); 13
C
NMR (CDCl3,100 MHz) d 201.4 (COCH3).179.5 (C-7),176.6 (C-4),161.6
(C-16), 150.4 (C-7a), 144.4 (C-5), 139.9 (C-10), 138.1 (C-2), 137.4 (C-
3a),134.2 (C-14),132.4 (C-12),130.8 (C-3),125.9 (C-11),123.5 (C-13),
120.8 (C-15), 104.7 (C-6), 52.9 (OCH3), 28.5 (C-17); EIMS m/z (%) 355
(Mþ, 27), 337 (63), 313 (26), 306 (21), 278 (37), 206 (45),164 (52),149
(35), 120 (11), 77 (11), 70 (11), 57 (29), 41 (29); HREIMS calcd for
C18H13NO5S 355.0515, found 355.0510. Anal. Calcd for C18H13NO5S:
C, 60.84; H, 3.69; N, 3.94. Found: C, 61.07; H, 3.67; N 3.88.
5.1.27. 10-Methyl-4,11-dioxo-4,11-dihydro-1-oxa-5-aza-cyclopenta
[b]anthracene-2-carboxylic acid methyl ester (32). To a premixed
solution of glacial acetic acid (4.5 mL) and concd H2SO4 (0.45 mL)
was added 5-(20-acetylanilino)-4,7-dioxo-4,7-dihydro-benzofuran-
2-carboxylic acid methyl ester (31), (0.16 g, 0.47 mmol). The solution
was stirred at 80 ꢀC for 35 min, then cooled and poured into ice
water (30 mL). It was then neutralised with solid NaHCO3, extracted
into CH2Cl2 (3ꢂ50 mL) and the combined organic layers were dried
over anhydrous magnesium sulfate. Removal of the solvent in vacuo
yielded 0.12 g (81%) of a yellow solid, 10-methyl-4,11-dioxo-4,11-
dihydro-1-oxa-5-aza-cyclopenta[b]anthracene-2-carboxylic acid
methyl ester (32). Recrystallisation from CHCl3/MeOH yielded fine
yellow needles. Mp 275e280 ꢀC (decomp.); Rf (100% CH2Cl2) 0.2; IR
nmax (film) 3609, 3449, 3123,1735,1697,1669,1591,1546,1437,1380,
5.1.25. 10-Methyl-4,11-dioxo-4,11-dihydro-1-thia-5-aza-cyclopenta
[b]anthracene-2-carboxylic acid methyl ester (30). To a premixed
solution of glacial acetic acid (14 mL) and concd H2SO4 (1.4 mL) was
added
5-(20-acetylanilino)-4,7-dioxo-4,7-dihydro-benzo[b]thio-
phene-2-carboxylic acid methyl ester (29) (0.5 g, 1.42 mmol). The
mixture was stirred at 80 ꢀC for 35 min then cooled and poured onto
ice before being neutralised with solid NaHCO3. The product was
extracted with CH2Cl2 (3ꢂ50 mL), and the combined organic layers
were dried over anhydrous magnesium sulfate. The solvent was
removed in vacuo yielding a yellow solid, which was washed with
CH2Cl2/MeOH (1:1) to yield 30 (0.45 g, 94%). Mp 332e340 ꢀC
(decomp.); Rf (100% CH2Cl2) 0.17; IR nmax (film) 2954, 1721, 1686,
1289, 1273, 1229, 1187, 1007, 952, 770 cmꢁ1
400 MHz)
;
1H NMR (CDCl3,
d
8.42 (1H, d, J¼8.3 Hz), 8.35 (1H, d, J¼8.1 Hz), 7.92 (1H,
ddd, J¼8.3, 8.3,1.2 Hz), 7.78 (1H, ddd, J¼8.3, 8.3,1.1 Hz), 7.71(1H, s, H-
3), 4.00 (3H, s, OCH3), 3.29 (3H, s, CH3); 13C NMR (CDCl3, 100 MHz)
d
177.5, 175.0, 157.9, 154.8, 152.8, 148.8, 148.4, 147.9, 132.9, 132.5,
130.0, 129.7, 129.3, 125.5, 123.8, 114.3, 52.9 (OCH3), 16.3 (CH3); EIMS
m/z (%) 321 (Mþ,100), 306, (17), 293 (22), 262 (39), 235 (27), 206 (7),
178 (17), 151 (10), 140 (7), 89 (9), 75 (7), 55 (10), 41 (12); HREIMS
calcd for C18H11NO5 321.0637, found 321.0641. Anal. Calcd for
C18H11NO5: C, 67.29; H, 3.45; N, 4.36. Found: C, 67.11; H, 3.66; N, 4.56.
1655, 1538, 1435, 1375, 1285, 1245, 1078, 880, 752 cmꢁ1
(CDCl3, 400 MHz)
;
1H NMR
d
8.44 (1H, d, J¼8.3 Hz, H-6 or H-9), 8.37 (1H, s, H-
3), 8.36 (1H, d, J¼8.2 Hz, H-6 or H-9), 7.92 (1H, ddd, J¼8.2, 8.2,1.1 Hz,
H-7 or H-8), 7.78 (1H, ddd, J¼8.2, 8.2,1.1 Hz, H-7 or H-8), 4.00 (3H, s,
OCH3), 3.30 (3H, s, CH3); 13C NMR (CDCl3, 100 MHz)
d 179.9, 176.9,
161.4,152.5,152.1,148.5,148.3,141.7,140.9,132.8,132.5,131.5,129.9,
129.3, 125.5, 124.7, 53.1 (OCH3), 16.4 (CH3); EIMS m/z (%) 337 (Mþ,
56), 322 (8), 306 (37), 250 (8), 222 (9), 206 (8),196 (10),178 (12),147
(100), 120 (12), 71 (21), 58 (43), 43 (18), 41 (22); HREIMS calcd for
C18H11NO4S 337.0409, found 337.0405. Anal. Calcd for C18H11NO4S:
C, 64.09; H, 3.29; N, 4.15. Found: C, 63.87; H, 3.20; N, 4.20.
5.1.28. 7-Methoxy-benzofuran-2-carboxylic acid methyl ester (34). A
stirred solution of 7-methoxy-benzofuran-2-carboxylic acid (33)
(0.5 g, 2.6 mmol) in CH2Cl2 (8 mL), MeOH (1.5 mL), and concd
H2SO4 (150 mL) was heated to reflux for 17 h. The solution was
cooled, poured into water, neutralised with solid NaHCO3 and
extracted into CH2Cl2 (3ꢂ50 mL). The combined organic layers
were dried over anhydrous magnesium sulfate, and the solvent was
removed in vacuo to yield 34 (0.57 g, 100%) of a white crystalline
solid. Mp 102e104 ꢀC; Rf (100% CH2Cl2) 0.74; IR nmax (film) 3006,
2953, 1732, 1622, 1585, 1579, 1493, 1433, 1428, 1363, 1329, 1307,
5.1.26. 5-(2-Acetylanilino)-4,7-dioxo-4,7-dihydro-benzofuran-2-car-
boxylic acid methyl ester (31). To a stirred suspension of 4,7-dioxo-
4,7-dihydro-benzofuran-2-carboxylic acid methyl ester (28) (0.44 g,
2.14 mmol) and cerium trichloride heptahydrate (0.80 g, 2.14 mmol)
in MeOH (100 mL) was added 20-aminoacetophenone (0.32 g,
2.39 mmol). Air was continuously bubbled through the mixture for 5
days during, which time the solution turned bright red. The mixture
was evaporated to dryness under reduced pressure. The residue was
extracted with CH2Cl2 (3ꢂ100 mL) and the combined organic layers
were washed with brine (3ꢂ100 mL), then with water (3ꢂ100 mL).
After drying over anhydrous magnesium sulfate, the solvent was re-
moved in vacuo to yield a purple solid, which was purified by silica
flash column chromatography eluting with CH2Cl2. The red adduct,
5-(20-acetylanilino)-4,7-dioxo-4,7-dihydro-benzofuran-2-carbox-
ylic acid methyl ester (31) (0.59 g, 22%) and the spontaneously
cyclised yellow solid,10-methyl-4,11-dioxo-4,11-dihydro-1-oxa-5-
aza-cyclopenta[b]anthracene-2-carboxylic acid methyl ester (32)
(0.41 g, 61%) were obtained. Recrystallisation of 31 from methanol
yielded dark red, fine hair-like needles. Mp 278e280 ꢀC; Rf (100%
CH2Cl2) 0.29; IR nmax (film) 3579, 3130,1722,1692,1642,1568,1528,
1272, 1231, 1201, 1094, 974, 915, 851, 800, 732 cmꢁ1
(CDCl3, 400 MHz)
;
1H NMR
d
7.51 (1H, s, H-3), 7.23 (1H, dd, J¼7.9, 1.5 Hz, H-4
or H-6), 7.19 (1H, dd, J¼7.5, 7.5 Hz, H-5), 6.90 (1H, dd, J¼7.5, 1.3 Hz,
H-4 or H-6), 4.00 (3H, s, OCH3), 3.96 (3H, s, OCH3); 13C NMR (CDCl3,
100 MHz) d 159.6, 145.8, 145.4, 145.2, 128.4, 124.3, 114.4, 114.1, 108.8,
55.8, 52.1; EIMS m/z (%) 206 (Mþ, 100), 191 (10), 175 (45), 163 (12),
119 (9), 117 (5), 89 (6), 76 (9), 65 (6), 50 (7); HREIMS calcd for
C11H10O4 206.0579, found 206.0580.
5.1.29. 7-Hydroxy-benzofuran-2-carboxylic acid methyl ester (35). A
solution of 7-methoxy-benzofuran-2-carboxylic acid methyl ester
(34) (100 mg, 0.49 mmol) in dry CH2Cl2 (20 mL) was stirred at
ꢁ20 ꢀC in an ethylene glycol/dry ice bath under nitrogen. Boron
tribromide (0.7 mL, 7.28 mmol) was added slowly and the mixture
was stirred for 1 h after which time it was allowed to warm to room
temperature. After stirring for further 2 h, aqueous sodium hy-
droxide (10%, 1 mL) was added to quench the reaction. The mixture
was made acidic with aqueous hydrochloric acid (10%), extracted
into CH2Cl2 (3ꢂ50 mL). The combined organic layers were dried
over anhydrous magnesium sulfate, and the solvent was removed
in vacuo to yield 35 as a white solid (80 mg, 86%). Rf (100% CH2Cl2)
0.17; IR nmax (film) 3375, 1717, 1597, 1566, 1490, 1437, 1346, 1315,
1453, 1434, 1370, 1292, 1260, 1196, 1169, 979, 753 cmꢁ1
(CDCl3, 400 MHz)
;
1H NMR
d
11.23 (1H, br s, NH-9), 7.95 (1H, dd, J¼7.6, 1.2 Hz,
H-12), 7.61 (1H, d, J¼5.6 Hz, H-15), 7.58 (1H, m, H-14), 7.53 (1H, s, H-
3), 7.21 (1H, ddd, J¼8.1, 8.1, 2.8 Hz, H-13), 6.48 (1H, s, H-6), 3.95 (3H,
s, OCH3), 2.68 (3H, s, CH3); 13C NMR (CDCl3,100 MHz)
d 201.4 (C-16),
177.9 (C-4), 175.5 (C-7), 158.1 (C-8), 154.0 (C-7a), 147.4 (C-2), 144.3
(C-5),139.7 (C-10),134.2 (C-14),132.4 (C-12),126.0 (C-11),124.8 (C-
3a), 123.7 (C-13), 121.0 (C-15), 113.8 (C-3), 102.8 (C-6), 52.7 (OCH3),
1290, 1219, 1196 cmꢁ1 1H NMR (CDCl3, 400 MHz)
; d 7.51 (1H, s, H-
3), 7.22 (1H, dd, J¼7.7, 1.3 Hz, H-4 or H-6), 7.17 (1H, dd, J¼7.7, 7.7 Hz,
H-5), 6.99 (1H, dd, J¼7.7, 1.3 Hz, H-4 or H-6), 5.4 (1H, br s, OH), 3.97

4986
D.R. Appleton et al. / Tetrahedron 66 (2010) 4977e4986
(3H, s, OCH3); 13C NMR (CDCl3, 100 MHz)
d
160.1, 145.3, 144.5, 141.5,
Auckland doctoral scholarship and we thank Ming Wu and Jaime
Yim for preliminary synthetic studies.
128.4, 124.9, 114.7, 114.6, 113.6, 52.5 (OCH3); EIMS m/z (%) 192 (Mþ,
100), 161 (99), 134 (14), 105 (48), 77 (21), 57 (14), 51 (33); HREIMS
calcd for C10H8O4 192.0420, found 192.0423.
Supplementary data
5.1.30. 4,7-Dioxo-4,7-dihydro-benzofuran-2-carboxylic acid methyl
ester (28). 5.1.30.1. A: from 7-hydroxy-benzofuran-2-carboxylic acid
methyl ester (35). To a stirred solution of 7-hydroxy-benzofuran-2-
carboxylic acid methyl ester (35) (60 mg, 0.31 mmol) in MeCN/
water (1 mL/0.5 mL) was added CAN (0.69 g, 1.25 mmol). The
mixture was stirred for 30 min. Water (20 mL) was added and it
was extracted with CH2Cl2 (3ꢂ50 mL). The combined organic layers
were dried over anhydrous magnesium sulfate and the solvent
removed in vacuo to yield 28 as a yellow solid (52 mg, 83%). Rf
(100% CH2Cl2) 0.59; IR nmax (film) 2956,1734, 1679,1572,1437,1285,
1225, 1202, 1094, 1048, 1022, 974, 921, 840, 806, 762, 668 cmꢁ1; 1H
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2010.05.033. These data in-
clude MOL files and InChiKeys of the most important compounds
described in this article.
References and notes
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d
7.46 (1H, s, H-3), 6.80 (2H, s, H-5, H-6),
181.6 (C-4), 174.7
3.95 (3H, s, OCH3); 13C NMR (CDCl3, 100 MHz)
d
€
3. Andries, K.; Verhasselt, P.; Guillemont, J.; Gohlmann, H. W. H.; Neefs, J.-M.;
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(C-5 or C-6),128.3 (C-3a),113.5 (C-3), 52.9 (OCH3); EIMS m/z (%) 206
(Mþ, 64), 175 (58), 149 (36), 127 (10), 113 (14), 99 (18), 85 (47), 71
(69), 57 (100), 43 (63); HREIMS calcd for C10H6O5 206.0215, found
206.0210.
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boxylic acid methyl ester (34) (100 mg, 0.49 mmol) in MeCN/2 M
H2SO4 (20 mL/5 mL) at room temperature was added ceric ammo-
nium sulfate (1.84 g, 2.9 mmol) in 2 M H2SO4 (25 mL) in one portion.
The orange mixturewasheatedto60 ꢀCfor90 minduringwhichtime
it turned to yellow with a precipitate of cerous ions. The solution was
cooled, decanted from the precipitatedcerous ionsintowater (25 mL)
and extracted with CH2Cl2 (3ꢂ50 mL). The combined organic layers
were washed with brine (3ꢂ50 mL), thenwater (2ꢂ50 mL), and dried
over anhydrous magnesium sulfate. The solvent was removed in
vacuo to yield 28 as a yellow crystalline solid (102 mg, 100%). All
spectra were identical to product prepared via method A.
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5.2. Biological assays
Details of the antituberculosis,2 P38819 and disc diffusion anti-
microbial19 assays have been presented elsewhere.
Acknowledgements
18. Zimhony, O.; Cox, J. S.; Welch, J. T.; Vilcheze, C.; Jacobs, W. R. Nat. Med. 2000, 6,
1043e1047.
19. van Klink, J. W.; Larsen, L.; Perry, N. B.; Weavers, R. T.; Cook, G. M.; Bremer, P. J.;
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We would like to acknowledge Dr. J. Maddry of TAACF for fa-
cilitating access to Mtb assays and Mrs. G. Barns for P388 and an-
timicrobial disc diffusion assays. DRA acknowledges a University of