Synthesis of kalasinamide, a putative plant defense phototoxin

Article abstract of DOI:10.1021/np070582z

The first total synthesis of the azaanthracene kalasinamide (1) is described, and the discrepancy in the reported ¹³C NMR data and melting points for the natural product from two different sources is resolved. Kalasinamide is prone to autosensitized photooxidation, in solution and in the solid state, to give the corresponding quinone, marcanine A (8). This transformation may be representative of a novel and more general step in the biosynthesis of (aza)anthraquinones. Through its ability to generate toxic singlet oxygen, kalasinamide may serve a protective role, defending the plant against predation and the invasion of microbial pathogens, following mechanical insult.

Full text of DOI:10.1021/np070582z

866
J. Nat. Prod. 2008, 71, 866–868
Synthesis of Kalasinamide, a Putative Plant Defense Phototoxin
Michael N. Gandy and Matthew J. Piggott*
School of Biomedical, Biomolecular and Chemical Sciences, The UniVersity of Western Australia, Crawley, WA, 6009 Australia
ReceiVed October 17, 2007
The first total synthesis of the azaanthracene kalasinamide (1) is described, and the discrepancy in the reported 13C
NMR data and melting points for the natural product from two different sources is resolved. Kalasinamide is prone to
autosensitized photooxidation, in solution and in the solid state, to give the corresponding quinone, marcanine A (8).
This transformation may be representative of a novel and more general step in the biosynthesis of (aza)anthraquinones.
Through its ability to generate toxic singlet oxygen, kalasinamide may serve a protective role, defending the plant
against predation and the invasion of microbial pathogens, following mechanical insult.
The azaanthracene kalasinamide 1 (Figure 1) was first isolated
in 2000 from the acetone extract of Polyalthia suberosa, a shrubby
tree endemic to southeast Asia and southern China.1 The structural
elucidation of the alkaloid was based on spectroscopic evidence
and has been confirmed by an X-ray crystal structure.2 More
recently, 1 was obtained from the seeds of Annona atemoya and
was named atemoine.3,4 Although the 1H NMR spectra of the
Figure 1. IUPAC numbering of kalasinamide.6
natural product obtained from both sources were very similar, the
13C NMR spectra were somewhat different (Table 1). This
Table 1. 13C NMR Data for Naturally Derived 1 vs Synthetic
discrepancy and the mild anti-HIV activity of kalasinamide5
Material
prompted us to carry out its synthesis. In the process, we discovered
assignment in ref 1a
kalasinamide1
“atemoine”3
synthetic
some interesting photochemistry that may be biosynthetically and
ecologically relevant.
4-CH3
10-OCH3
5-OCH3
4a
23.3
61.9
64.0
114.0
121.1
23.3
23.3
61.9
64.0
114.0
121.1
61.9 (5-OCH3)b
64.1 (10-OCH3)
The synthesis of kalasinamide 1 is outlined in Scheme 1.
Attempts to nitrate 1,4-dimethoxynaphthalene 2 under standard
conditions led to significant side reactions and poor yields, but with
copper nitrate in acetic anhydride,7 3 was produced cleanly in 80%
yield. Reduction gave the corresponding aniline 4, which could be
isolated in 90% yield, or reacted in situ with diketene 5,8 to give
the ꢀ-ketoamide 6 in 88% yield over the two steps. Sulfuric acid-
catalyzed Knorr cyclization9 gave kalasinamide 1 but was ac-
companied by some ring sulfonation. This side reaction was avoided
by carrying out the Knorr cyclization in 85% phosphoric acid,10 in
which case 1 was formed in 82% yield, contaminated by an
additional 9% of the corresponding quinone, marcanine A (grif-
9
121.1 (7)
122.7 (4a)
123.0 (8)
123.5
3
6
5a
7
8
123.1
123.5
123.9
124.6
127.8
128.1
128.2
123.1
123.5
124.0
124.6
127.8
128.1
128.2
124.7 (9)
127.5 (5a)c
127.9
9a
10a
133.7 (C8a)c
135.8 (10a)
148.8 (5)
151.7 (10)
152.3 (C4)c
161.7
10
4
5
136.0
148.6
151.6
135.9
148.7
151.7
1
fiazanone B) (8), as determined by H NMR integrals. We found
the two compounds to be inseparable by silica gel chromatography,
and attempts to avoid the formation of 8 by carrying out the reaction
under inert atmosphere and with different acids (polyphosphoric
acid,11 trifluoroacetic acid,12 methanesulfonic acid) were unsuc-
cessful. Ultimately, pure kalasinamide was obtained by excluding
atmospheric oxygen and light during the Knorr cyclization, workup,
and recrystallization.
2
161.8
161.7
a IUPAC numbering as indicated in Figure 1 has been used here. The
original assignments used non-IUPAC numbering. b Erroneous assignments
made in ref
3
are indicated in parentheses. c Indicates signals that
correspond with peaks in the 13C NMR spectrum of marcanine A.
spectrum of atemoine were found to be identical with some of the
signals for 8 (Table 1).
Both the 1H NMR and 13C NMR spectra of the synthetic
kalasinamide are identical to those reported by Tuchinda and co-
A subsequent experiment showed that kalasinamide (1), in CDCl3
solution, was completely consumed within 48 h of standing in
ambient light in the presence of air, the major product being
marcanine A (8). This explains how a sample of “atemoine” that
appeared pure by 1H NMR spectroscopy subsequently became
contaminated by the quinone 8 before the 13C NMR spectrum was
acquired. Similarly, in ambient light, solid samples of kalasinamide
were completely oxidized to 8.
A solution of kalasinamide in CDCl3 was stable for at least two
weeks with the exclusion of light, which suggests that the
conversion into 8 is mediated by 1,4-cycloaddition of singlet
oxygen13 to give the photooxide 7 (Scheme 1). However, 7 was
never observed, indicating the facile nature of the subsequent
thermal or photolytic cleavage of the peroxy bridge to give the
1
1
workers (Table 1). The H NMR spectrum and most of the 13C
NMR signals of atemoine are also identical to those of kalasinamide.
We conclude that atemoine is indeed kalasinamide and that the
differences in the 13C NMR data are due to impurities in the
atemoine isolate. Furthermore, during our investigation, the identity
of the impurity became apparent. The byproduct from the Knorr
cyclization, marcanine A (8), was also generated during some
recrystallizations. Given its facile formation from kalasinamide, we
suspected it as the likely contaminant in the atemoine sample. Our
suspicions were confirmed when the spurious peaks in the 13C NMR
* Corresponding author. Tel: +61 8 6488 3170. Fax: +61 8 6488 1005.
E-mail: piggott@cyllene.uwa.edu.au.
10.1021/np070582z CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/08/2008

Notes
Journal of Natural Products, 2008, Vol. 71, No. 5 867
Scheme 1. a
Figure 2
photooxidation,29 it is likely that annopholine (9) is the biosynthetic
precursor of cleistopholine (10). Indeed photooxidation of 9,10-
dimethoxyanthracenes and their aza analogues may be a more
general step in the biosynthesis of (aza)anthraquinones that has not
been observed previously because of the ease with which this
transformation occurs. Both the shikimate-acetate30 and polyketide
pathways31 have previously been implicated in the biosynthesis of
azaanthraquinones.
In conclusion, kalasinamide (1) was prepared in four steps (three
pots) in an overall yield of 58%. Given the facile photooxidation
of 1 to the corresponding quinone, marcanine A (8), the original
isolation of pure kalasinamide (1)1 is admirable. The photooxidation
has implications for the isolation of naturally occurring 9,10-
dimethoxyanthracenes and their aza analogues, and may represent
a novel transformation in the biosynthesis of (aza)anthraquinones.
Furthermore, the facility with which kalasinamide generates highly
toxic singlet oxygen may point to a role in plant defense.
a Reagents and conditions: (a) Cu(NO3)2 · 2.5H2O, Ac2O, -40 °C, 80%; (b)
H2, 10% Pd/C, EtOAc, 90% (1 step); (c) 5, PhMe, reflux, 88% (from 3); (d)
85% H3PO4, 85 °C, 82%.
quinone 8. Kalasinamide thus acts as both sensitizing agent and
diene for the subsequent hetero-Diels–Alder reaction. Similar
autosensitized photooxidations of 9,10-dimethoxyanthracenes are
known;14 in some cases the photooxides can be isolated, but often
they are too unstable under the reaction conditions and the
anthraquinones are formed directly.13
Marcanine A (8) has previously been isolated from Goniothala-
mus species15–18 and has been shown to have antimalarial activity.19
Given that 8 has also been isolated from Polyalthia longifolia var.
pendula20 and Annona glabra,21 members of the same genera from
which kalasinamide and “atemoine”, respectively, were isolated, it
is quite possible that kalasinamide is a biosynthetic precursor to
marcanine A in these species, or the latter is an artifact. It is also
possible that the anti-HIV activity of kalasinamide is actually due
to marcanine A formed in situ; as mentioned above, the latter does
exhibit reasonable antimalarial activity and is also cytotoxic to
several human tumor cell lines.18
Alternatively, the capacity of kalasinamide to generate reactive
singlet oxygen may be the source of its biological activity.
Chlorophyll derivatives can be extremely phototoxic to nonpho-
tosynthetic organisms, for the same reason.22 Indeed, the role of
light and singlet oxygen in plant defense against microbial attack
and predation has recently been reviewed.23 For example, glaucine
(9), which, like kalasinamide and marcanine A, has previously been
isolated from Annona species,24,25 is rapidly oxidized to the
phytoalexin26 oxoglaucine (10) in planta upon mechanical injury,
in particular by singlet oxygen (Figure 2).23 Oxoglaucine, which
has also been isolated from an Annona species (pupureae),27 is an
efficient singlet oxygen photosensitizer, and it has been speculated
that its main role is to prevent the invasion of pathogens through
wounds following mechanical injury.23 It is appealing to hypoth-
esize that kalasinamide serves a similar biological purpose: it
protects the plant by generating both highly toxic singlet oxygen
and the phytoalexin marcanine A at the site of injury.
Experimental Section
General Experimental Procedures. Melting points were measured
on a Kofler hot stage melting point apparatus and are uncorrected. IR
spectra were acquired using a Perkin-Elmer Spectrum One FTIR
spectrometer. NMR spectra were acquired in CDCl3 using Bruker
Avance 500 (500 MHz, 1H; 125 MHz, 13C) or Varian 400 (400 MHz,
1H; 100 MHz, 13C) spectrometers. Chemical shift values are expressed
in ppm, relative to CHCl3 (1H, 7.26 ppm) and CDCl3 (13C, 77.0 ppm)
as appropriate. Routine assignments of 13C NMR spectra were made
with the assistance of DEPT 135 and DEPT 90 experiments. Mass
spectra were recorded on a VG Autospec instrument using a direct
insertion probe and electron impact ionization (EI). Microanalysis was
performed by Robertson Microlit (Parsippany, NJ). All solvents were
distilled prior to use. “Hexanes” denotes the hydrocarbon fraction
distilling from 64 to 67 °C. All reaction temperatures refer to bath
temperatures. Organic extracts were dried over anhydrous MgSO4 and
then filtered. Silica gel, Merck Kieselgel 60 (40–63 µm), was used for
flash chromatography. Analytical TLC was performed on Whatman
flexible plates (250 µL layer, Al Sil G/UV254). Spots were visualized
under UV light.
1,4-Dimethoxy-2-nitronaphthalene (3). Cu(NO3)2 ·2.5H2O (2.53 g,
10.9 mmol) was added to Ac2O (25 mL) under CaCl2 guard. The
suspension was stirred for 15 min, then cooled to -40 °C before a
solution of 1,4-dimethoxynaphthalene (2)32 (1.89 g, 10.0 mmol) in Ac2O
(40 mL) was added dropwise. After 1 h, H2O (20 mL) and Et2O (30
mL) were added and the reaction mixture was allowed to warm to room
temperature. The phases were separated, and the aqueous layer was
extracted with Et2O (3 × 20 mL). The combined organic phase was
washed with H2O (2 × 20 mL) and brine (20 mL), dried, and evaporated
to give an orange residue, which was subjected to flash chromatography.
Elution with hexanes-toluene (17:3) afforded 3 as a yellow, amorphous
powder (1.87 g, 80%): mp 98.0–98.5 °C [lit.33 97–98 °C]; Rf 0.56
EtOAc-hexanes (1:9); 1H NMR (500 MHz) δ 8.31–8.25 (2H, m, H-5
and H-8), 7.69–7.65 (2H, m, H-6 and H-7), 7.22 (1H, s, H-3), 4.09
(3H, s, OCH3), 4.05 (3H, s, OCH3); the 1H NMR spectrum was similar
A search for other naturally occurring 9,10-dimethoxyanthracenes
and aza analogues revealed that annopholine (11) (Figure 2), which
is structurally very similar to kalasinamide (1), co-occurs with its
azaanthraquinone counterpart, cleistopholine (12), in Annona
hayesii.28 Given the facile photooxidation of kalasinamide, and the
propensity of 1-azaanthracene to undergo visible light-induced

868 Journal of Natural Products, 2008, Vol. 71, No. 5
Notes
to that reported in d6-acetone solution at 80 MHz;33 13C NMR (125
MHz) δ 151.9 (Ar), 145.7 (Ar), 138.7 (Ar), 129.0 (Ar), 128.9 (ArH),
128.8 (Ar), 128.2 (ArH), 124.1 (ArH), 122.7 (ArH), 98.5 (C-3), 63.6
(OCH3), 56.1 (OCH3).
Acknowledgment. We thank Dr. M. McIldowie for noting the
discrepancy in the published 13C NMR spectra of kalasinamide, Mr.
R. Duncan for his contribution to its synthesis, as part of an
undergraduate research project, Associate Professor E. Ghisalberti for
helpful advice, and Dr. T. Reeder for mass spectra. M.N.G. is the
recipient of an UWA Postgraduate Award.
1,4-Dimethoxy-2-naphthylamine (4). A solution of 3 (338 mg, 1.45
mmol) in EtOAc (10 mL) was stirred overnight with 10% Pd/C (35
mg) under an atmosphere of H2. The suspension was washed through
a Celite pad with EtOAc, the filtrate was evaporated, and the residue
was subjected to flash chromatography. Elution with hexanes-EtOAc
(4:1) afforded 4 as a dark purple solid (265 mg, 90%): Rf 0.16
References and Notes
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despite the plant name (Annona atemoya).
(5) Tuchinda, P.; Pohmakotr, M.; Reutrakul, V.; Thanyachareon, W.;
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(6) Previous papers have used non-IUPAC names and numbering systems
for kalasinamide.
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1
hexanes-EtOAc (9:1); H NMR (500 MHz) δ 8.09 (1H, d, J ) 8.1
Hz, ArH), 7.84 (1H, d, J ) 8.2 Hz, ArH), 7.47–7.44 (1H, m, ArH),
7.25–7.22 (1H, m, ArH), 6.37 (1H, s, H-3), 4.05–3.90 (2H, br s, NH2),
1
3.94 (3H, s, OCH3), 3.85 (3H, s, OCH3). The H NMR spectrum was
different from that reported in d6-acetone solution at 80 MHz.33
N-(1,4-Dimethoxy-2-naphthyl)-3-oxobutanamide (6). A solution
of 3 (587 mg, 2.52 mmol) in toluene (10 mL) was stirred with 10%
Pd/C (35 mg) under an atmosphere of H2 for 3.5 h. The reaction mixture
was purged of H2 with N2, then freshly distilled diketene (5) (212 µL,
2.75 mmol) was added and the mixture was heated under reflux. After
7 h more diketene (38 µL, 0.49 mmol) was added and the mixture was
stirred at 70 °C overnight. The suspension was washed through a Celite
pad with Et2O (2 × 20 mL), then MeOH (2 × 20 mL), the filtrate was
evaporated, and the green residue was subjected to flash chromatog-
raphy. Elution with hexanes-EtOAc (9:1–3:2 gradient) afforded 6 as
an orange gum (632 mg, 88%), which crystallized from MeOH as amber
prisms: mp 97–97.5 °C; Rf 0.18 hexanes-EtOAc (3:2); IR (neat) νmax
3274 (N-H, w br), 1712 (CdO, m), 1673 (NCdO, m) cm-1; 1H NMR
(500 MHz)34 δ 9.70 (br s, 1H, NH), 8.20 (1H, ddd, J ) 8.4, 1.3, 0.7
Hz, H-5′), 7.99–7.96 (2H, m, H-3′ and H-8′), 7.52 (1H, ddd, J ) 8.4,
6.8, 1.3 Hz, H-7′), 7.40 (1H, ddd, J ) 8.4, 6.8, 1.3 Hz, H-6′), 4.00
(3H, s, 4′-OCH3), 3.95 (3H, s, 1′-OCH3), 3.68 (2H, s, H-2), 2.36 (3H,
s, H-4); 13C NMR (125 MHz)22 δ 204.7 (CdO), 163.6 (NCdO), 152.0
(C-4′), 136.9 (C-1′), 127.53 and 127.49 (C-2′ and C-8a′), 126.8 (C-6′),
124.2 (C-7′), 123.1 (C-4a′), 122.4 (C-5′), 121.0 (C-8′), 98.5 (C-3′),
61.6 (1′-OCH3), 55.7 (4′-OCH3), 50.0 (CH2), 31.1 (OdCCH3); EIMS
m/z 287 [M]•+ (15), 272 (11), 214 (13), 203 (16), 188 (100); HREIMS
m/z 287.1157 (calcd for C16H17NO4, 287.1158); anal. C 67.1%, H 5.8%,
N 4.8%; calcd for C16H17NO4, C 66.9%, H 6.0%; N 4.9%.
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5,10-Dimethoxy-4-methylbenzo[g]quinolin-2(1H)-one (Kalasin-
amide) (1). Method A: A solution of 6 (22 mg, 0.077 mmol) in 85%
phosphoric acid (2 mL) was stirred at 85 °C in a stoppered vial for
3 h. The resulting solution was poured onto ice and extracted with
EtOAc (3 × 10 mL). The extract was washed with H2O (10 mL) and
brine (10 mL), dried, and evaporated to yield a yellow residue (20
mg) comprising kalasinamide (6) (17 mg, 82%) and marcanine A (8)
(1.7 mg, 9%).35
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Method B: All solvents used were deoxygenated by bubbling through
Ar for at least 1.5 h, and all manipulations were carried out in the
minimum light practicable. Phosphoric acid (85%, 1 mL) was added
to 6 (87 mg, 0.32 mmol), and the resulting solution was stirred
vigorously in the dark for 15 min. The solution was then heated at 80
°C for 2 h. The reaction mixture was poured onto cold H2O (10 mL),
basified with saturated Na2CO3 solution (10 mL), and extracted with
EtOAc (3 × 10 mL). The extract was washed with brine (2 × 10 mL),
dried, and evaporated under a stream of dry N2, giving a yellow residue.
Recrystallization (EtOH-DCM) and filtration under a flow of Ar
afforded 6 as green prisms: mp 240–242 °C [lit.1 234–235.5 °C]; Rf
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(26) Phytoalexins are antimicrobial secondary metabolites that are rapidly
synthesised as a result of stress (from ref 23).
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1
0.13 hexanes-EtOAc (3:2); H NMR (400 MHz) δ 8.96 (1H, br s,
NH), 8.18 (1H, ddd, J ) 8.6, 1.2, 0.8 Hz, ArH), 8.05 (1H, ddd, J )
8.6, 1.2, 0.8 Hz, ArH), 7.58 (1H, ddd, J ) 8.6, 6.7, 1.2 Hz, ArH), 7.47
(1H, ddd, J ) 8.6, 6.7, 1.2 Hz, ArH), 6.46 (1H, dq, J ) 2.4, 1.3 Hz,
H-3), 3.99 (3H, s, OCH3), 3.97 (3H, s, OCH3), 2.79 (3H, d, J ) 1.3
Hz, 4-CH3). Refer to Table 1 for 13C NMR data.
4-Methylbenzo[g]quinoline-2,5,10(1H)-trione (Marcanine A) (8).
The quinone was obtained from kalasinamide solid samples that had
completely oxidized upon standing in ambient light. Recrystallization
from MeOH gave 8 as orange needles: mp 295 °C dec [lit.36 >300 °C;
lit.21 249–251 °C]; Rf 0.20 DCM-MeOH (99:1); 1H NMR (400 MHz)
δ 9.80 (1H, br s, NH), 8.24 (1H, ddd, J ) 7.7, 1.3, 0.6 Hz, ArH), 8.19
(1H, ddd, J ) 7.7, 1.4, 0.6 Hz, ArH), 7.87 (1H, ddd, J ) 7.7, 7.6, 1.4
Hz, ArH), 7.78 (1H, ddd, J ) 7.7, 7.6, 1.3 Hz, ArH), 6.69 (1H, q, J )
1.2 Hz, H-3), 2.71 (3H, d, J ) 1.2 Hz, 4-CH3). The 1H NMR spectrum
was similar to that reported at 500 MHz.18
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(34) NMR assignments were made with the assistance of HSQC and HMBC
experiments.
(35) Determined by total mass and 1H NMR integrals.
(36) Perez, J. M.; Vidal, L.; Grande, M. T.; Menendez, J. C.; Avendano,
C. Tetrahedron 1994, 50, 7923–7932.
NP070582Z