Biphenyl dioxygenase-catalysed cis-dihydroxylation of tricyclic azaarenes: Chemoenzymatic synthesis of arene oxide metabolites and furoquinoline alkaloids

Article abstract of DOI:10.1039/c3ra42026d

Biotransformation of acridine, dictamnine and 4-chlorofuro[2,3-b]quinolone, using whole cells of Sphingomonas yanoikuyae B8/36, yielded five enantiopure cyclic cis-dihydrodiols, from biphenyl dioxygenase-catalysed dihydroxylation of the carbocyclic rings.

Full text of DOI:10.1039/c3ra42026d

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Biphenyl dioxygenase-catalysed cis-dihydroxylation of
tricyclic azaarenes: chemoenzymatic synthesis of arene
oxide metabolites and furoquinoline alkaloids3
Cite this: RSC Advances, 2013, 3,
1
0944
a
a
a
a
Derek R. Boyd,* Narain D. Sharma, Jonathan G. Carroll, Pui L. Loke, Colin
a
b
R. O’Dowd and Christopher C. R. Allen
Biotransformation of acridine, dictamnine and 4-chlorofuro[2,3-b]quinolone, using whole cells of
Sphingomonas yanoikuyae B8/36, yielded five enantiopure cyclic cis-dihydrodiols, from biphenyl
dioxygenase-catalysed dihydroxylation of the carbocyclic rings. cis-Dihydroxylation of the furan ring in
dictamnine and 4-chlorofuro[2,3-b]quinoline, followed by ring opening and reduction, yielded two
exocyclic diols. The structures and absolute configurations of metabolites have been determined by
spectroscopy and stereochemical correlation methods. Enantiopure arene oxide metabolites of acridine
and dictamnine have been synthesised, from the corresponding cis-dihydrodiols. The achiral furoquinoline
alkaloids robustine, c-fagarine, haplopine, isohaplopine-3,39-dimethylallylether and pteleine have been
obtained, from either cis-dihydrodiol, catechol or arene oxide metabolites of dictamnine.
Received 4th February 2013,
Accepted 3rd May 2013
DOI: 10.1039/c3ra42026d
www.rsc.org/advances
Further metabolism of the benzo[b]furan 2,3-cis-diols involved
spontaneous ring opening and enzyme-catalysed carbonyl
Introduction
Polycyclic azaarenes are ubiquitous in the environment as
atmospheric pollutants, resulting from incomplete combus-
tion of nitrogen-containing molecules present in fossil fuels or
reduction to give exocyclic phenolic diol products
2c
(Scheme 1b).
Dihydroxylation of the 3,4-bond in the electron-deficient
1
a,b
tobacco and also as plant alkaloids.
Some larger members
pyridine ring of the quinoline substrates was found to yield
only minor metabolites in comparison with its carbocyclic 5,6-
and 7,8-bonds. However, when benzo[b]thiophene and ben-
zo[b]furan substrates, containing electron-rich heterocyclic
rings, were used as substrates, dihydroxylation of the 2,3-
bond revealed a more favourable metabolic route (Schemes 1a
and 1b).
of the family of aza-polycyclic aromatic hydrocarbons (APAHs)
present a significant hazard to human health, resulting from
the mutagenicity/carcinogenicity of their mammalian metabo-
1
c–e
lites.
The mineralization of APAHs and alkaloids contain-
ing azaaromatic rings by soil bacteria, via non-mutagenic/non-
carcinogenic metabolites can, therefore, play a useful role in
reducing this problem. Earlier bacterial studies from these
laboratories have focused on the toluene dioxygenase (TDO)-
catalysed biodegradation of bicyclic heterocycles including
The steric dimensions of the active site in TDO, expressed
in P. putida UV4, limited the acceptable size of substrates to
mono- or bi-cyclic arenes (Schemes 1a and 1b). However, the
biphenyl dioxygenase (BPDO) enzyme, present in the B8/36
mutant strain of Sphingomonas yanoikuyae, has a larger active
site and was able to accept tri-, and tetra-cyclic arenes (e.g.
2
a,b
2c
2c
quinolines,
using the UV4 mutant strain of Pseudomonas putida
Schemes 1a and 1b). Regioselective cis-dihydroxylation of
the carbocyclic and the heterocyclic rings in the quinolines
5,6 and/or 7,8 and/or 2,3 bonds), benzo[b]thiophenes (4,5
benzo[b]thiophenes
and benzo[b]furans,
(
3a
benzo[f]quinoline, benzo[h]quinoline, phenanthridine, ben-
(
3b
zo[c]phenanthridine, Scheme 2) as substrates. It is note-
and/or 2,3 bonds) and benzo[b]furans (6,7 and/or 2,3 bonds),
occurred to give the corresponding cis-dihydrodiol metabo-
lites. The 3-hydroxyquinoline and anthranilic acid metabolites
of quinoline were assumed to be derived from the undetected
worthy that in these examples a marked regioselective
preference for cis-dihydroxylation was found at a bond within
the bay-region.
3b,c
As part of an earlier programme
to investigate the ability
2
a,b
heterocyclic cis-3,4-dihydrodiol intermediate (Scheme 1a).
of BPDO to catalyse the cis-tetrahydroxylation of larger
polycyclic aromatic rings, it was found that bis-cis-dihydrodiols
were formed as further metabolites of the initial cis-dihydro-
diols derived from larger carbocyclic (e.g. anthracene, chry-
sene, benz[a]anthracene) and heterocyclic (e.g. acridine,
phenazine, benzo[b]naphtha[2,1-d]thiophene) substrates. The
a
School of Chemistry and Chemical Engineering, Queen’s University Belfast, Belfast,
UK BT9 5AG. E-mail: dr.boyd@qub.ac.uk; Tel: +44 (0) 28 90974421
b
School of Biological Sciences, Queen’s University Belfast, Belfast, UK BT9 5AG
3
Electronic supplementary information (ESI) available. See DOI: 10.1039/
c3ra42026d
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Scheme 1
similarity in size and shape of the linear tricyclic arenes,
anthracene and acridine, and their acceptability as sub-
3
c,d
strates for the BPDO enzyme,
prompted this comparative
biotransformation study of acridine with furo[2,3-b]quinoline
substrates. Following our earlier reports on the isolation and
4
a–e
synthesis
of quinoline alkaloids, from plants of the
Rutaceae family, e.g. Choisya ternata, and Skimmia japonica,
linear furoquinolines (4-chlorofuro[2,3-b]quinoline and dic-
tamnine) were briefly examined as potential substrates, using
whole cells of S. yanoikuyae B8/36 expressing BPDO
In our preliminary studies of the biotransformations of
acridine and dictamnine, using S. yanoikuyae B8/36, we had
3d,4d
reported
the presence of the corresponding cis-dihydrodiol
metabolites. This comprehensive study now provides full
structural and stereochemical characterization of all new
bacterial metabolites and shows how they can be utilized in
4
d
enzyme.
Scheme 2
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Scheme 3
the chemoenzymatic synthesis of a wider range of animal and
plant metabolites, e.g. arene oxides and furoquinoline
alkaloids.
and an allylic (S) configuration for cis-dihydrodiol 4. The
reliability of the MPBA method for the linear azaarene cis-
dihydrodiol 4 was confirmed by an unequivocal stereochemi-
cal correlation sequence similar to that used for other
3a,6
polycyclic arene cis-dihydrodiols (Scheme 4).
The sequence
involved a catalytic hydrogenation (H ,Pd/C) to yield cis-
tetrahydrodiol 5 followed by bis-acetylation (Ac O, pyridine)
2
2
Results and discussion
(
i) Biotransformation of acridine 1
to give cis-diacetate 6. In the final step, an oxidative ring
opening reaction (RuO /NaIO ) gave a mixture of dicarboxylic
2
4
The mammalian metabolism and mutagenicity of acridine 1
acid products (7/8). It was assumed that the bicyclic
dicarboxylic acid 7 was formed initially and then a part of it
degraded to acyclic dicarboxylic acid 8 via a further oxidative
ring opening reaction. The mixture of dicarboxylic acids 7 and
have been studied over many years using dog, rabbit and rat
5
a–d
liver cells.
The major metabolites were found to be
2-hydroxyacridine, 9-acridone, 2-hydroxy-9-acridone and
trans-1,2-dihydroxy-1,2-dihydroacridine 3 (Scheme 3). It is
probable that trans-dihydrodiol 3 and 2-hydroxyacridine were
derived from the undetected acridine 1,2-oxide 2. The
identification of these mammalian metabolites of acridine 1,
which could be accounted for, mainly, by monooxygenase-
8
was methylated (CH
which were separated by column chromatography. The minor
component, dimethyl(2,3-diacetoxy)adipate 10 ([a] 214) was
2 2
N ) to yield dimethylesters 9 and 10
D
6
of established (2S,3S) configuration and thus the (1R,2S)
configuration was unequivocally assigned to (+)-cis-dihydro-
diol 4.
3d
catalysed oxidation, prompted the preliminary and current
study of its dioxygenase-catalysed metabolism.
It has been proposed that the mutagenicity/carcinogenicity
associated with some larger PAHs and APAHs results from: (i)
a monooxygenase-catalysed epoxidation of a carbocyclic ring
to yield an arene oxide (cf. compound 2), (ii) an epoxide
hydrolase-catalysed hydrolysis of the arene oxide to yield a
trans-dihydrodiol (cf. compound 3), (iii) a monooxygenase-
catalysed epoxidation of the alkene bond in the trans-
dihydrodiol to yield diastereoisomeric trans-diol epoxides
and (iv) nucleophilic attack of DNA on the epoxide ring within
Biotransformation of acridine 1, using S. yanoikuyae B8/36
under similar conditions to those used for other azaarene
3
a–d
substrates,
chromatography, yielded cis-dihydrodiol
followed by extraction (EtOAc) and column
([a] +71) in
4
D
acceptable yield (42%). The structure of cis-diol 4 was
determined by NMR, MS and elemental microanalysis. The
enantiomeric excess value (ee) was estimated as .98% by
reaction with (R)-(+)- and (S)-(2)-2-(1-methoxyethyl)phenyl-
1
boronic acid (MPBA) and H-NMR analysis of the resulting
1
b–f
a bay region to yield a covalent adduct.
Although the
boronates.
corresponding acridine trans-diol epoxides from metabolite 3
could, in principle, also be mutagens, their synthesis and
mutagenicity has not yet been reported.
The absolute configuration of cis-dihydrodiol 4 was initially
assigned as (1R,2S), based on the well established H-NMR
1
pattern previously observed for MPBA derivatives from other
polycyclic arene cis-dihydrodiol metabolites (e.g. from
naphthalene, anthracene, phenanthrene and their aza-analo-
(ii) Biotransformation of furoquinolines 11–13
2
a,b,3a,b
gues).
3.18) for the MeO group protons, using the (R)-(+)-MPBA
compared with the value obtained using (S)-(2)-MPBA (d
The observation of a larger chemical shift value
In common with acridine 1, the mammalian metabolism and
mutagenicity of dictamnine 12 and other furoquinoline
(d
H
7
a–e
alkaloids, e.g. c-fagarine, had been reported earlier.
In a
H
3.11), was again assumed to be consistent with a benzylic (R)
more recent study, from these laboratories, the furoquinoline
1
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Scheme 4
alkaloid skimmianine 13 was found to be the major
resulting from BPDO-catalysed cis-dihydroxylation of the 5,6
and 7,8 bonds of the carbocyclic ring, skimmiamine 13 was
not an acceptable substrate. The mixtures of metabolites (14/
15 and 16/17) were separated into individual cis-dihydrodiols
by PLC purification. The structures of diol metabolites (14–17)
were established by analyses of NMR and MS data while the ee
values (.98%) and absolute configurations were again
determined by formation of the corresponding diastereomeric
4
a
compound present in C. ternata, and was thus available as
potential substrate for the current biotransformation
a
studies. However, dictamnine 12, another furoquinoline
alkaloid required as a potential substrate, was not isolated
from C. ternata. Thus, a five-step chemical synthesis of
dictamnine 12 was carried out, starting from aniline and
8
a–c
using the literature procedure
which involved 4-chlorofur-
1
oquinoline 11 as precursor. Furoquinolines 11 and 12 were
thus also available as possible substrates for BPDO.
MPBA esters and their analysis by H-NMR spectroscopy. As
found for the acridine cis-dihydrodiol 4, the larger chemical
Furoquinolines 11–13 were added, individually, as sub-
strates to S. yanoikuyae B8/36, under the conditions used
previously for the successful biotransformation of acridine 1.
The results, shown in Scheme 5, indicate that while
shift values (d
diols 14–17, (d
with (S)-(2)-MPBA 14–17, (d
benzylic (R) and allylic (S) configurations in each case.
While cis-dihydroxylation had occurred exclusively at the 1,2
bond of acridine 1, similar regioselectivity for the equivalent
H
) for the exocyclic MeO group of cis-dihydro-
3.23–3.25) using the (R)-(+)-MPBA compared
3.19–3.20) were consistent with
H
H
4-chlorofuro[2,3-b]quinoline 11 and dictamnine 12 each
yielded a mixture of two cis-dihydrodiol products 14–17,
Scheme 5
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Scheme 6
(5,6) bond in furoquinolines 11 and 12 was not found. A
been proposed that, in common with other naturally occurring
modest preference (38% yield) was observed for BPDO-
catalysed cis-dihydroxylation at the 7,8-bond to form cis-diol
mutagenic furans, e.g. aflatoxin B1 and 8-methoxypsoralen,
7
f
the corresponding transient furan epoxides, formed as initial
mammalian metabolites via monooxygenase-catalysed epox-
idation, e.g. arene oxide 24 (Scheme 5) may be responsible for
their mutagenicity. It has been proposed that the mutagenicity
results from the ability of furan epoxides to form covalently
bound adducts following nucleophilic ring-opening reactions
15 compared with the 5,6-bond (10% yield) to give cis-diol 14,
when 4-chlorofuro[2,3-b]quinoline 11 was the substrate. A
stronger preference for oxidation of the 7,8-bond was found
with dictamnine 12 as the substrate, which resulted in cis-diol
17 being the major metabolite (20–30% yield) relative to cis-
7
e,f
with DNA.
diol 16 (1–3% yield). The combined isolated yields (21–33%) of
dictamnine cis-dihydrodiols (16 and 17) were slightly lower
than 4-chlorofuroquinoline cis-dihydrodiols (14 and 15, 48%);
no cis-dihydrodiol metabolites were detected from skimmia-
nine 13 as substrate. These observations suggest that the
presence of substituents at C-4, C-7 and C-8 and the overall
steric requirements of the substrate within the active site of
the BPDO enzyme are important factors. Based on isolated
yields, it appears that cis-dihydroxylation occurred preferen-
tially at the less sterically hindered 7,8-bond and that the best
yields resulted from the use of the smaller substrates (11 and
(iii) Application of acridine cis-dihydrodiol 4 in the synthesis
of arene oxide 2
As part of an earlier study of the mammalian metabolism and
mutagenicity/carcinogenicity of PAHs and APAHs, (1R,2S)-
arene oxide 2 was obtained via an eight stage chemical
synthesis, involving a chemical resolution of MTPA esters, with
9
an overall yield of ca. 13%. Alkaline hydrolysis (KOH, t-BuOH)
of (1R,2S)-arene oxide 2 gave the mammalian metabolite
9
(
1R,2R)-trans-1,2-dihydroacridine-1,2-diol 3. In the current
study, the possibility of a much shorter synthesis of acridine
,2-oxide 2 was examined (Scheme 6), using the readily
available bacterial metabolite, (1R,2S)-cis-1,2-dihydroacridine-
,2-diol 4. Treatment of diol 4 with 1-bromocarbonyl-1-
12). As the largest substrate, skimmianine 13, did not yield cis-
1
diol metabolites, this is consistent with its failure to be
accommodated within the BPDO active site. However, alter-
native factors, including aqueous solubility, toxicity and
further metabolism, could influence the isolated yields of
bioproducts.
1
methylethyl acetate, in acetonitrile solution, gave a mixture
of bromoacetates 25/26 whose structures were determined
1
from H-NMR and MS data. Due to their instability, during
The most polar metabolites, formed from 4-chlorofuro[2,3-
b]quinoline 11 and dictamnine 12, were found to be exocyclic
diols (compounds 20 and 23) but were isolated in very low
yields (1–2%, Scheme 5). While the structures of optically
active diols 20 and 23 were assigned by NMR and MS
spectroscopic analysis, their ee values and absolute configura-
tions were not determined. It was assumed, that the exocyclic
diols 20 and 23, resulted from: (a) BPDO-catalysed cis-
dihydroxylation at the 2,3-bond to give transient intermediates
attempted separation, the mixture of bromoacetates 25 and 26
in Et O solution was reacted directly with NaOMe. Using this
2
two step method, the relatively stable (1R,2S)-arene oxide 2 was
synthesised from cis-dihydrodiol 4 in 66% yield. Despite the
stability of arene oxide 2, it was not detected during
mammalian metabolism, probably due to its further metabo-
lism via a rapid epoxide hydrolase-catalysed conversion to the
5a–e
3d
corresponding trans-dihydrodiol 3.
A preliminary study
later showed that when the stable acridine cis-dihydrodiol 4
was used as a substrate for S. yanoikuyae B8/36, it was also
further metabolised and formed a bis-cis-dihydrodiol biopro-
duct.
18 and 21, (b) reversible ring opening of these hemiacetals (cf.
mutarotation) to yield the undetected aldehydes 19 and 22, (c)
epimerization, following reversible ring closure, to yield a
mixture of the corresponding cis- and trans-dihydrodiols, and
(iv) Application of dictamnine cis-dihydrodiols 16 and 17 as
(d) carbonyl reductase-catalysed (CRED) reduction of the
aldehyde group in intermediates 19 and 22. A similar sequence
of TDO-catalysed cis-dihydroxylation of the furan ring of
benzo[b]furans, spontaneous equilibration via a reversible
ring opening process to yield the corresponding phenolic
aldehydes and CRED-catalysed reduction of the resulting
aldehyde group was earlier assumed to account for the
precursors in the synthesis of furoquinoline alkaloids
The potential of dictamnine cis-dihydrodiol metabolites 16
and 17 in the biomimetic synthesis of furoquinoline alkaloids,
including the proposed arene oxide intermediate 27, was of
biosynthetic interest (Schemes 7 and 8). Possible biosynthetic
pathways to furoquinoline alkaloids occurring in Rutaceaeous
plants, e.g. Skimmia japonica and Choisya ternata, have been
2
c
isolation of the exocyclic diols shown in Scheme 1 (b).
The origin of mutagenicity associated with dictamnine 13
1
4
10a
studied using
C-labelled precursors.
These labelling
7
a–e
has not yet been rigorously established.
However, it has
studies showed that enzyme-catalysed hydroxylation could
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Scheme 7
occur on the benzene ring of dictamnine 12 to yield a wider
range of furoquinoline alkaloids e.g. skimmianine 13 and
possibly also robustine 30 and c-fagarine 31 (Scheme 7). It was
proposed that skimmianine 13 could be formed via a
monooxygenase-catalysed epoxidation of dictamnine 12, to
yield the transient arene oxide 27, followed by epoxide
hydrolase-catalysed hydrolysis to yield trans-dihydrodiol
but the major metabolite (17) being further metabolized
preferentially.
The E. coli narB recombinant strain, expressing naphthalene
DD, has been used successfully to produce catechols in good
yields from the corresponding monocyclic arene cis-dihydro-
1
1
diols. Using E. coli narB and cis-dihydrodiol 15 as substrate,
1
catechol 35 was detected by H-NMR spectroscopy but in low
1
0a
28.
The possibility of an alternative dioxygenase-catalysed
yield. Surprisingly, the required catechol metabolite 29,
derived from dictamnine cis-dihydrodiol 17, could not be
obtained using this method. However, it was possible to obtain
catechol 29 in good yield (85%) using boron tribromide for the
selective O-demethylation of skimmianine 13, isolated ear-
cis-dihydroxylation of dictamnine 12 to yield cis-dihydrodiol 17
1
0a
was also discussed.
The enzyme-catalysed oxidations of
trans- and cis-dihydrodiols, to yield catechols followed by
1
a
O-methylation, are well established metabolic steps and,
1
0a
4a
when allied to the earlier labelling studies,
either type of
lier from Choisya ternata (Scheme 8).
enzymatic oxidation could account for the formation of
catechol 29 and skimmianine 13. To date, none of the
potential biosynthetic intermediates 17, 27–29 have been
Convincing evidence of monooxygenase-catalysed epoxida-
tion of dimethylallyl groups, and hydrolysis of the resulting
epoxides to yield vicinal diols, is available from biosynthetic
studies of quinoline alkaloids from plants of the Rutaceae
1
0a
detected by the labelling studies using Choisya ternata
or
1
0a,b
found among the furoquinoline alkaloids recently isolated
family.
Furthermore, monooxygenase-catalysed epoxida-
4
a
10b
from this or other plants in the Rutaceae family.
tion of azaarenes, to yield the corresponding arene oxides, e.g.
As expected, the B8/36 mutant strain of S. yanoikuyae did not
yield catechol metabolites e.g. compound 29 from dictamnine
quinoline 5,6-oxide from quinoline, using liver microsomes
1
2a,b
with inhibition of epoxide hydrolase activity,
provides a
1
2 (Scheme 7). The biphenyl cis-diol dehydrogenase (DD)
precedent for the formation of the elusive dictamnine arene
oxide 27 and trans-dihydrodiol 28 metabolites. While the
dioxygenase-catalysed cis-dihydroxylation of polycyclic azaar-
enes in bacteria, e.g. S. yanoikuyae B8/36, is well established
(Schemes 1, 2 and 4), there appears to be little evidence of this
pathway occurring in plants. Consequently, the monooxygen-
ase-catalysed epoxidation sequence, shown in Scheme 7, is
currently favoured over the dioxygenase pathway. Epoxidation,
activity required to catalyse the dehydrogenation of cis-
dihydrodiols to yield catechols, was blocked in the B8/36
strain. However, when the wild type strain of S. yanoikuyae
(B1), expressing both BPDO and DD enzymes, was used with
dictamnine 12, the only metabolite identified and isolated was
cis-dihydrodiol 16, albeit in low yield (8%). This observation is
consistent with both cis-dihydrodiols 16 and 17 being formed
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Scheme 8
as an initial step, can readily account for the formation of both
monophenols (e.g. robustine 30), catechols (e.g. 7,8-dihydrox-
ydictamnine 29), and their methylated derivatives (e.g. c-
fagarine 31 and skimmianine 13).
Our attempt to synthesise the proposed dictamnine arene
oxide metabolite 27 from cis-dihydrodiol 17, via a two-step
process similar to that used earlier for acridine arene oxide 2
In the final phase of this study, cis-dihydrodiols 16 and 17,
arene oxide 27 and catechol 29, as confirmed or proposed
metabolites of dictamnine 12, were utilized as synthetic
precursors of other furoquinoline alkaloids, using biomimetic
methods (Scheme 8). While robustine 30 was obtained by
isomerisation of arene oxide 27 under acidic conditions, the
acid-catalysed dehydration of cis-dihydrodiol 17 was the
preferred route. Methylation of robustine 30 with diazo-
methane yielded the alkaloid c-fagarine 31. Under similar
conditions, methylation of catechol 29 occurred mainly at C-8,
to yield the alkaloid haplopine 36. Treatment of catechol 29 in
(Scheme 6), was unsuccessful. This was due to compound 17
being less stable under the reaction conditions and more
readily dehydrated under acid conditions to yield phenols (e.g.
robustine 30). An alternative approach (Scheme 8) was adopted
involving the catalytic hydrogenation (H
2
, Pd–C) of compound
acetone with 1-chloro-3-methylbut-2-ene in presence of K
2 3
CO
17 to yield the stable cis-tetrahydrodiol 32 (76% yield).
resulted in the preferential prenylation at C-8 to yield phenol
3
7, which on methylation yielded the alkaloid, isohaplopine-
Treatment of diol 32 with 1-bromocarbonyl-1-methylethyl
acetate gave trans-bromoacetate 33 in good yield (90%).
3,39-dimethylallylether 38. Acid-catalysed dehydration of cis-
dihydrodiol 16, to form phenol 39, followed by methylation,
yielded the furoquinoline alkaloid pteleine 40.
4
Benzylic bromination of bromoacetate 33 (NBS, CCl ) gave
an inseparable mixture of diastereoisomers 34 which was
immediately treated with sodium methoxide in THF, to yield
the proposed dictamnine arene oxide metabolite 27 (60% yield
from compound 33). Initial attempts to purify this elusive
arene oxide by PLC resulted in its aromatization to give the
furoquinoline alkaloid robustine 30. Purification of (7S,8R)-
dictamnine oxide 27 was achieved by careful crystallization. A
sample of oxide 27 was found to survive in CDCl solution
3
without decomposition, at ambient temperature over a 24 h
period.
Conclusion
The bacterial cis-dihydroxylation of acridine 1 and furoquino-
lines 11 and 12, catalysed by BPDO, yielded five carbocyclic cis-
dihydrodiols, (4, 14–17) and two exocyclic diols (20 and 23),
derived from the transient heterocyclic diols 18 and 21. The
structures and absolute configurations of most of the isolated
metabolites were established by spectroscopic analysis and
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stereochemical correlation methods. cis-Dihydrodiols 4 and 17
diol 4 (7.5 g, 42%). The majority of the diol metabolite 4
were used in the synthesis of the corresponding arene oxides,
crystallized out of solution on concentration. The mother
2
and 27, which had been proposed as intermediates in
liquor was purified by column chromatography (CHCl
3
A10%
MeOH/CHCl ). Colourless crystalline solid, m.p. 176 uC
3
mammalian metabolism of acridine 1 and dictamnine 12. cis-
Dihydrodiol 16 and 17 and arene oxide 27, as derivatives of
dictamnine 12, have been used in the synthesis of a wide range
of furoquinoline alkaloids including robustine 30, c-fagarine
(
(
5
2
EtOH); R 0.45 (15% MeOH/CHCl ); [a] +71 (c 0.53, MeOH);
f
3
D
Found: C, 73.4; H, 5.1; N, 6.4. C13
.2; N, 6.6%); (Found: M 213.07876. C H NO requires
H
2
11NO requires C, 73.2; H,
+
1
3
11
2
H 3 3
13.07898); d (500 MHz, CD COCD ) 4.13 (1 H, m, OH), 4.35
31, haplopine 36, isohaplopine-3,39-dimethylallylether 38 and
(1 H, m, OH), 4.42 (1 H, dd, J2,1 4.7, J2,3 4.8 , 2-H), 4.86 (1 H, d,
pteleine 40.
J1,2 4.7, 1-H), 6.54 (1 H, dd, J3,2 4.8, J3,4 10.1, 3-H), 6.77 (1 H, d,
J
J
8
4,3 10.1, 4-H), 7.53 (1 H, dd, J7,8 8.1, J7,6 6.9, 7-H), 7.68 (1 H, dd,
6,7 6.9, J6,5 8.5, 6-H), 7.90 (1 H, d, J8,7 8.1, 8-H), 7.95 (1 H, d, J5,6
Experimental
.5, 5-H), 8.28 (1 H, s, 9-H); dC (125 MHz, CD COCD ) 66.6,
3 3
1
13
70.2, 126.3, 127.7, 127.9, 129.0, 129.1, 130.8, 132.1, 133.2,
H and C NMR spectra were recorded on Bruker Avance 400,
DPX-300 and DRX-500 instruments. Chemical shifts (d) are
reported in ppm relative to SiMe and coupling constants (J)
+
1
36.3, 147.1, 153.0; m/z (EI): 213 (M , 56%), 195 (23), 184 (100);
2
1
n 3369 cm (OH); ECD: l/nm 311 (De 1.036), 283 (De 21.079),
57 (De 4.644), 243 (De 6.421), 215 (De 4.647), 199.40 (De 0.573).
1R,2S)-1,2,3,4-Tetrahydroacridine-1,2-diol 5.
cis-Dihydrodiol metabolite 4 (0.5 g, 2.35 mmol) was catalyti-
cally hydrogenated (H , 10% Pd/C, 50 mg) in MeOH solution
20 cm ) at atmospheric pressure and room temperature (4 h).
4
2
are given in Hz. Mass spectra were run at 70 eV, on a VG
Autospec Mass Spectrometer, using a heated inlet system.
Accurate molecular weights were determined by the peak
matching method, with perfluorokerosene as the standard. CD
spectra were recorded in spectroscopic grade acetonitrile using
a JASCO J-720 instrument. A PerkinElmer 341 polarimeter was
used for optical rotation ([a] ) measurements (ca. 20 uC). Flash
D
column chromatography and preparative layer chromatogra-
phy (PLC) were performed on Merck Kieselgel type 60 (250–400
(
2
3
(
The catalyst was filtered off and the filtrate evaporated under
reduced pressure to yield (1R,2S)-1,2,3,4-tetrahydroacridine-
1
,2-diol 5 as a semi-solid (0.420 g, 83%); [a]
D
+61 (c 1.00,
+
MeOH); (Found: M 215.0937. C H NO requires 215.0946);
1
3
13
2
d
H
(500 MHz, CDCl
3
) 2.12 (1 H, m, 3a-H), 2.32 (1 H, m, 3b-H),
mesh) and PF2
54/366
plates respectively. Merck Kieselgel type
3.11 (1 H, m, 4a-H), 3.36 (1 H, m, 4b-H), 4.28 (1 H, m, J2,1 3.5,
6
0F254 analytical plates were employed for TLC. Authentic
2
7
-H), 4.93 (1 H, d, J1,2 3.5, 1-H), 7.49 (1 H, dd, J7,8 8.0, J7,6 7.1,
-H), 7.68 (1 H, dd, J6,7 7.1, J6,5 8.4, 6-H), 7.79 (1 H, d, J8,7 8.0,
samples of 4-chlorofuro[2,3-b]quinoline 11 and skimmianine
1
4
a–e
2b
3 were available from earlier studies.
As reported,
8-H), 8.00 (1 H, d, J5,6 8.4, 5-H), 8.30 (1 H, s, 9-H); d (125 MHz,
C
Sphingomonas yanoikuyae B8/36 was grown on minimal salts
CDCl ) 26.3, 29.5, 68.9, 70.2, 126.0, 127.2, 127.6, 128.4, 129.8,
3
2
1
+
medium with 0.5% sodium succinate and 0.5 g L of yeast
extract. Biphenyl dioxygenase (BPDO) was induced, during the
exponential phase of growth, by the addition of m-xylene (1
137.0, 157.4, 162.6, 171.9; m/z (EI): 215 (M , 71%), 198 (16), 186
2
1
(74), 168 (75), 143 (100); n 3435 cm (OH).
(1R,2S)-1,2-Diacetoxy-1,2,3,4-tetrahydroacridine 6.
cis-Tetrahydrodiol 5 (0.18 g, 0.84 mmol) was acetylated with
3
21
cm L ) every 0.5 h for 7 h. Substrate concentration was 0.5
2
3
3
3
mg cm
.
2
Ac O (1.5 cm ) in dry pyridine (0.7 cm ) solution by stirring the
mixture overnight at room temperature. Excess of pyridine was
Synthesis of dictamnine 12
removed under high vacuum, the residue treated with water
3
To a solution of 4-chlorofuro[2,3-b]quinoline 11 (3 g, 14.7
mmol) in dry methanol (120 cm ) was added sodium
(10 cm ), and the aqueous mixture extracted with Et O (3 6 15
2
3
3
cm ). The combined ether extract was dried (Na SO ) and
2
4
methoxide (4.05 g, 75 mmol) and the mixture refluxed (2 h)
under nitrogen. After removal of methanol from the reaction
mixture, under reduced pressure, the brown residue obtained
was treated with ice cold water and the aqueous mixture
concentrated to yield (1R,2S)-1,2-diacetoxy-1,2,3,4-tetrahydroa-
cridine 6 as a white crystalline solid (0.22 g, 88%); m.p. 84–85
+
uC (Et O/hexane); [a] 257 (c 0.85, MeOH); (Found: M
2
D
299.1150. C H NO requires 299.1158); dH (500 MHz,
1
7
17
4
3
extracted with chloroform (3 6 100 cm ). The combined
CDCl ) 2.05 (3 H, s, OCOMe), 2.15 (3 H, s, OCOMe), 2.20 (1
3
organic extract was dried (Na
2
SO
4
), concentrated under
H, m, J3a,2 3.1, 3a-H), 2.44 (1 H, m, 3b-H), 3.23 (1 H, m, 4a-H),
reduced pressure, and the crude product purified by flash
chromatography (1 : 1, EtOAc : hexane) to afford dictamnine
3.40 (1 H, m, 4b-H), 5.43 (1 H, m, J2,1 3.3, J2,3a 3.1, 2-H), 6.32 (1
H, d, J1,2 3.3, 1-H), 7.51 (1 H, dd, J7,8 8.1, J7,6 7.0, 7-H), 7.72 (1 H,
dd, J6,7 7.0, J6,5 8.5, 6-H), 7.79 (1 H, d, J8,7 8.1, 8-H), 8.02 (1 H, d,
J5,6 8.5, 5-H), 8.12 (1 H, s, 9-H); d (125 MHz, CDCl ) 19.6, 19.7,
1
2 as a yellow solid (1.65 g, 56%); R
f
0.5, (1 : 1 EtOAc/hexane);
1
3a
m.p. 133–134 uC (lit. 132–133 uC); dH (300 MHz, CDCl ) 4.45
3
C
3
(
7
3 H, s, Me), 7.08 (1 H, d, J3,2 2.8, 3-H), 7.45 (1 H, dd, J6,5 9.1, J6,7
.0 6-H), 7.62 (1 H, d, J2,3 2.8, 2-H), 7.69 (1 H, dd, J7,8 8.4, J7,6 7.0
J 7.0, 7-H), 8.0 (1 H, d, J8,7 8.4, 8-H), 8.26 (1 H, d, J5,6 9.1, 5-H).
22.1, 28.8, 67.8, 68.3, 124.9, 125.3, 125.4, 126.4, 127.0, 128.9,
+
135.9, 146.5, 155.6, 169.0, 169.2; m/z (EI): 299 (M , 69%), 197
2
1
(100); n 1741 cm (CLO), 1421.
Oxidative degradation of diacetate 6. Ruthenium(II) oxide
hydrate (2 mg) was added to a biphasic mixture of the
(
a) Biotransformation of acridine 1
1R,2S)-1,2-Dihydroacridine-1,2-diol 4. Biotransformation of
(
diacetate 6 (0.1 g, 0.33 mmol) and NaIO (3.4 g, 16 mmol) in a
4
3
3
3
acridine 1 (15 g) using S. yanoikuyae B8/36 followed by
extraction with EtOAc yielded (1R,2S)-1,2-dihydroacridine-1,2-
4
mixture of CCl (2 cm ), MeCN (2 cm ) and water (3 cm ). After
stirring the reaction mixture for 4 days at ambient tempera-
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3
ture, dilute HCl (20 cm , 1.5 M) was added and the reaction
(7S,8R)-4-Methoxy-7,8-dihydrofuro[2,3-b]quinoline-7,8-diol
3
mixture extracted with EtOAc (3 6 30 cm ). The combined
extract was washed with water, dried (Na SO ), and concen-
17. White solid (41 mg, 29%); m.p. 185–187 uC (MeOH/CHCl );
3
+
2
4
R 0.3 (7% MeOH/CHCl ); [a] +94 (c 0.78, MeOH); (Found: M
f
3
D
trated under reduced pressure to yield a crude mixture of two
dicarboxylic acids. The mixture was dissolved in MeOH (5 cm )
and treated (0 uC, 0.5 h) with excess of freshly prepared
ethereal diazomethane solution. The solvents were evaporated,
in a fume hood under a stream of nitrogen, and the crude
methyl esters separated by column chromatography (25% A
12 11 4 H
233.0689. C H NO requires 233.0688); d (500 MHz, CDCl3)
3
4.28 (3 H, s, OMe), 4.45 (1 H, dd, J7,8 5.0, J7,6 5.0, 7-H), 4.78 (1
H, d, J8,7 5.0, 8-H), 6.17 (1 H, dd, J6,5 9.8, J6,7 5.0, 6-H), 6.98 (1 H,
d, J3,2 2.5, 3-H), 7.02 (1 H, d, J5,6 9.8, 5-H), 7.57 (1 H, d, J2,3 2.5,
2-H); d (125 MHz, CDCl ) 58.8, 64.5, 71.1, 105.5, 106.0, 111.7,
C
3
+
123.8, 124.2, 142.2, 151.7, 156.7, 163.1; m/z (EI): 233 (M , 61%),
215 (9), 204 (100); ECD: l/nm 275.60 (De 20.425), 246.10 (De
2.722), 205.90 (De 20.272).
2
75% Et O/hexane) to yield dimethyl (5R,6S)-5,6-di(acetoxy)-
5,6,7,8-tetrahydro-2,3-quinolinedicarboxylate 9, the more polar
major compound (21 mg, 17%), and (2S,3S)-dimethyl-(2,3-
diacetoxy)-adipate 10 (9 mg, 9.3%) as the less polar minor
compound.
3-(1,2-Dihydroxyethyl)-4-methoxy-1,2-dihydro-2-quinolinone
2
0
3. Brown solid (1.1 mg, 1.6%); m.p. 137–140 uC (decomp.); R
f
+
3 D
.20 (7% MeOH/CHCl ); [a] +8.8 (c 0.68, MeOH); (Found: M
Dimethyl (5R,6S)-5,6-diacetoxy-5,6,7,8-tetrahydro-2,3-quino-
linedicarboxylate 9. White crystalline solid; m.p. 115–117 uC
235.0840. C H NO requires 235.0845); d (500 MHz, CDCl3)
12 13 4 H
3.82 (1 H, dd, J2,29 11.1, J2,1 4.4, 2-H), 3.97 (1 H, dd, J29,2 11.1,
J29,1 7.4, 29-H), 4.04 (3 H, s, OMe), 5.15 (1 H, dd, J1,2 4.4, J1,29 7.4,
1-H), 7.30 (1 H, dd, J6,7 = J6,5 = 7.9, 6-H), 7.35 (1 H, d, J8,7 8.2,
8-H), 7.57 (1H, dd, J7,8 8.2, J7,6 7.9, 7-H), 7.81 (1 H, d, J5,67.9,
5-H), 11.68 (1 H, br s, NH); d (75 MHz, CDCl ) 62.9, 66.0, 69.0,
3 D
(from CHCl ); [a] 253 (c 0.66, MeOH); (Found: C, 55.7; H, 5.1;
N, 3.9. C H NO requires C, 55.9; H, 5.2; N, 3.8%); (Found:
1
7
19
8
+
M
365.1105. C17
CDCl ) 2.04 (3 H, s, OCOMe), 2.15 (3 H, s, OCOMe), 2.16 (1 H,
m, J7a,6 3.1, 7a-H), 2.35 (1 H, m, 7b-H), 3.11 (1 H, m, 8a-H),
8 H
H19NO requires 365.1111); d (500 MHz,
3
C
3
116.3, 116.8, 119.7, 123.4, 123.5, 131.5, 137.7, 163.7, 166.2; m/z
+
3
5
8
5
1
.25(1 H, m, 8b-H), 3.92 (3 H, s, CO
.42 (1 H, m, J6,5 3.4, J6,7a 3.1, 6-H), 6.13 (1 H, d, J5,6 3.4, 5-H),
.11 (1 H, s, 4-H); dC (125 MHz, CDCl ) 21.3 (62), 23.6, 29.5,
3.3, 53.6, 68.3, 69.0, 123.8, 130.4, 138.8, 151.4, 160.6, 165.5,
2
Me), 3.99 (3 H, s, CO
2
Me),
(EI): 235 (M , 4%), 204 (20), 122 (83), 105 (100), 77 (80), 51 (54),
43 (71).
3
(c) Biotransformation of 4-chlorofuro[2,3-b]quinoline 11
+
67.2, 170.6, 170.7; m/z (EI): 365 (M , 21%), 43 (100); n 1746
A small-scale biotransformation on 4-chlorofuro[2,3-b]quino-
line 11 (0.8 g, 3.93 mmol), using S. yanoikuyae B8/36, followed
by concentration of the aqueous portion under reduced
pressure and extraction of the concentrate by ethyl acetate
yielded a mixture of products. PLC purification of the mixture
2
1
cm (CLO).
2S,3S)-Dimethyl-(2,3-diacetoxy)-adipate 10. Colourless oil;
(
6
[a]
D
214 (c 0.74, CHCl
) 1.97 (1 H, m, CH
3
) (lit. 214.1, CHCl
3 H
); d (500 MHz,
CDCl
3
2
), 2.07 (3 H, s, OCOMe), 2.06–2.15 (1
H, m, CH ), 2.18 (3 H, s, OCOMe), 2.37 (2 H, m, CH ), 3.68 (3
(6% MeOH/CHCl ) gave (7S,8R)-4-chloro-7,8-dihydrofuro[2,3-
2
2
3
H, s, CO
2
Me), 3.79 (3 H, s, CO
2
Me), 5.30 (2 H, m, 2-H and 3-H);
b]quinoline-7,8-diol 15 (0.35 g, 38%); R 0.6 (6% MeOH/CHCl ),
(5R,6S)-4-chloro-5,6-dihydrofuro[2,3-b]quinoline-5,6-diol 14 (93
f
3
+
+
21
m/z (EI): 259 (M -OMe, 5%), 217 (M -COMe, 16); n 1735 cm
(CLO).
mg, 10%); Rf 0.5 (6% MeOH/CHCl ) and 4-chloro-3-(1,2-
3
dihydroxyethyl)-1,2-dihydro-2-quinolinone 20 (23 mg, 2.5%);
(
b) Biotransformation of dictamnine 12
R 0.4 (6% MeOH/CHCl ).
f
3
A small scale biotransformation of dictamnine 12 (60 mg),
using S. yanoikuyae B8/36, followed by extraction with EtOAc
yielded a mixture of two dihydrodiol metabolites, (5R,6S)-4-
(7S,8R)-4-Chloro-7,8-dihydrofuro[2,3-b]quinoline-7,8-diol
15. Brown crystalline solid (0.35 g, 38%); m.p. 121–123 uC
(decomp., CHCl /hexane); [a] +138.1 (c 0.53, CHCl ); (Found:
3
D
3
+
methoxy-5,6-dihydrofuro[2,3-b]quinoline-5,6-diol
16
and
11 8 3
M 237.0201. C H NO requires 237.0193); dH (500 MHz,
(7S,8R)-4-methoxy-7,8-dihydrofuro-[2,3-b]quinoline-7,8-diol 17.
3 7,8
CDCl ) 4.49 (1 H, dd, J 4.8, J7,6 5.2, 7-H), 4.81 (1 H, d, J8,7 4.8,
These diols were separated by PLC (7% MeOH/CHCl
3
). A
8-H), 6.34 (1 H, dd, J6,7 5.2, J6,5 9.9, 6-H), 6.85 (1 H, d, J3,2 2.4,
3-H), 7.05 (1 H, d, J5,6 9.9, 5-H), 7.66 (1 H, d, J2,3 2.4, 2-H); dC
repeat larger scale biotransformation of dictamnine 12 (0.6 g,
.02 mmol), using S. yanoikuyae B8/36, yielded a mixture of
three compounds, diol 16 (8 mg, 1.1%), diol 17 (0.143 g, 20%)
and 3-(1,2-dihydroxyethyl)-4-methoxy-1,2-dihydro-2-quinoli-
none 23 (7 mg, 1.0%).
5R,6S)-4-Methoxy-5,6-dihydrofuro[2,3-b]quinoline-5,6-diol
6. White solid (2 mg, 3%); m.p. 157–159 uC; R 0.26 (7%
3
(125 MHz, CDCl ) 63.8, 70.3, 104.2, 117.6, 120.3, 123.5, 127.5,
3
+
37
133.6, 144.0, 150.9, 159.1; m/z (EI): 239 (M , Cl, 14%), 237
+
+
+
(M , 43), 219 (M -H O, 15), 208 (M -CHO, 75), 199 (100), 190
2
(80), 184 (55), 156 (49), 149 (29), 128 (28); ECD: l/nm 316 (De
20.31), 316 (De 20.27), 253 (De 4.12), 235 (De 3.34), 207 (De
22.07).
(
1
f
+
MeOH/CHCl ); [a] 2111 (c 0.19, MeOH); (Found M 233.0694.
(5R,6S)-4-Chloro-5,6-dihydrofuro[2,3-b]quinoline-5,6-diol
14. Brown crystalline solid (93 mg, 10%); m.p. 172–174 uC
3
D
12 4 H 3
C H11NO requires 233.0688); d (500 MHz, CDCl ) 4.32 (3 H,
s, OMe), 4.63 (1 H, m, 6-H), 5.14 (1 H, d, J5,6 5.3, 5-H), 6.19 (1 H,
ddd, J7,8 10.0, J7,6 = J7,5 = 1.9, 7-H), 6.57 (1 H, dd, J8,7 10.0, J8,6
+
(decomp.); [a]D +189 (c 0.41, MeOH); (Found M 237.0199.
C H ClNO requires 237.0193); d (500 MHz, CDCl ) 4.72 (1
11
8
3
H
3
2
.6 8-H), 6.98 (1 H, d, J3,2 2.5, 3-H), 7.60 (1 H, d, J2,3 2.5, 2-H); d
C
H, br s, 6-H), 5.21 (1 H, dd, J5,6 5.1, J5,7 1.7, 5-H), 6.23 (1 H, ddd,
J7,8 10, J7,6 = J7,5 = 1.7, 7-H), 6.64 (1 H, dd, J8,7 10, J8,6 2.7, 8-H),
6.86 (1 H, d, J3,2 2.5, 3-H), 7.72 (1 H, d, J2,3 2.5, 2-H); dC (125
(125 MHz, CDCl ) 58.9, 63.8, 69.7, 105.4, 105.8, 114.1, 128.4,
3
+
136.4, 143.0, 147.9, 158.9, 164.4; m/z (EI): 233 (M , 6%), 215 (9),
43 (100); ECD: l/nm 286 (De 4.808), 245 (De 25.670), 229 (De
0.415), 216 (De 23.007), 204 (De 1.004).
MHz, CDCl ) 66.1, 69.7, 105.3, 118.1, 123.5, 128.17, 136.8,
3
+
35
137.68, 145.8, 148.2, 161.7; m/z (EI): 237 (M , Cl, 5%), 221
1
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+
(
(
0
72), 219 (100), 191 (18), 156 (80); ECD: l/nm 316 (De 3.07), 306
De 3.98), 290 (De 3.70), 246 (De 25.26), 217 (De 26.16), 201(De
.61).
major component (0.12 g, 81%); (Found: M 317.0061.
7
9
C
H12NO
Br requires 317.0051).
(1R,2R)-1-Acetoxy-2-bromo-1,2-dihydroacridine 25.
MHz, CDCl ) 1.99 (3 H, s, OCOMe), 4.93 (1 H, dd, J2,1 2.5,
15
2
H
d (500
4
-Chloro-3-(1,2-dihydroxyethyl)-1,2-dihydro-2-quinolinone
3
J2,3 5.7, 2-H), 6.35 (1 H, d, J1,2 2.5, 1-H), 6.62 (1 H, dd, J3,2 5.7,
J3,4 9.8, 3-H), 7.01 (1 H, d, J4,3 9.8, 4-H), 7.55–8.09 (4 H, m,
Aromatic), 8.27 (1 H, s, 9-H). (1S,2S)-1-Bromo-2-acetoxy-1,2-
2
2
0. Brown solid (23 mg, 2%); m.p. 184–186 uC (decomp.); [a]
D
+
5.1 (c 0.43, MeOH); (Found: M -CH
2
OH 208.0157.
C H ClNO requires 208.0165); dH (500 MHz, CD OD) 3.76
1
0
7
2
3
dihydroacridine 26. dH (500 MHz, CDCl ) 2.00 (3 H, s, OCOMe),
(1 H, dd, J2,29 11.3, J2,1 5.6, 2-H), 3.87 (1 H, dd, J29,2 11.3, J29,1 7.2,
3
5
6
7
.51 (1 H, d, J1,2 2.5, 1-H), 5.73(1 H, dd, J2,1 2.5, J2,3 5.4, 2-H),
.58 (1 H, dd, J3,2 5.4, J3,4 9.7, 3-H), 7.16 (1 H, d, J4,3 9.7, 4-H),
.54–8.09 (4 H, m, Aromatic), 8.13 (1 H, s, 9-H); m/z (EI): 317
2
7
8
1
9-H), 4.51 (2 H, br s, OH), 5.20 (1 H, dd, J1,2 5.6, J1,29 7.2, 1-H),
.32 (2 H, m, 6-H, 8-H), 7.57 (1 H, m, 7-H), 8.00 (1 H, dd, J5,6
C 3
.2, J5,7 1.1, 5-H); d (125 MHz, CD OD) 65.8, 74.2, 117.1, 120.2,
+
79
+
(M , Br, 41%), 238 (80), 196 (100).
25.0, 127.0, 129.6, 133.2, 138.7, 144.8, 164.0; m/z (EI): 210 (M ,
3
7
A cooled solution (0 uC) of bromoacetates 25 and 26 (0.1 g,
Cl-CH OH, 31%), 208 (100), 162 (15), 89 (13), 32 (16).
2
3
0
.31 mmol) in dry Et
2
O (10 cm ) was treated with NaOMe (24
(
7S,8R)-4-Methoxy-5,6,7,8-tetrahydrofuro[2,3-b]quinoline-
mg, 0.44 mmol). The reaction mixture was stirred overnight at
room temperature. After addition of water (10 cm ) to the
reaction mixture, it was extracted with Et
combined ether extract dried (Na SO
7,8-diol 32. A solution of dictamnine 7,8-diol 17 (0.15 g, 0.64
3
3
mmol) in MeOH (10 cm ) containing 3% Pd/C (15 mg) was
stirred (1 h) under hydrogen atmosphere at atmospheric
pressure. The reaction mixture was filtered and the solvent
removed under reduced pressure to yield the crude product as
a brown oil. Purification of the crude product by PLC (4%
3
2
O (3 6 15 cm ), the
2
4
) and the solvent
evaporated under reduced pressure to yield (1R,2S)-acridine-
,2-oxide 2 as a light brown solid (51 mg, 83%); m.p. 136–137
1
9
uC (CHCl /hexane) (lit. 136–138 uC); [a] +30 (c 0.49, CHCl3)
3
D
MeOH/CHCl ), afforded the tetrahydro cis-diol 32 as a white
3
9
+
(lit. +22, CHCl ); (Found: M 195.0689. C H NO requires
3
13
9
solid (0.115 g, 76%); m.p. 149–151 uC (from EtOAc); R
f
0.5 (4%
); (Found: M 235.0843.
C H NO requires 235.0844); d (500 MHz, CDCl ) 1.82–1.89
+
195.0684); d
H
(500 MHz, CDCl ) 4.08 (1 H, dd, J2,1 3.8, J2,3 6.1,
3
3 D 3
MeOH/CHCl ); [a] 225 (c 0.39, CHCl
2
3
6
8
5
1
-H), 4.55 (1 H, d, J1,2 3.8, 1-H), 6.78 (1 H, dd, J3,2 6.1, J3,4 10.0,
-H), 7.03 (1 H, d, J4,3 10.0, 4-H), 7.51 (1 H, dd, J6,7 7.0, J6,5 8.2,
-H), 7.67 (1 H, dd, J7,6 7.0, J7,8 8.6, 7-H), 7.78 (1 H, J8,7 8.6, 8-H),
.02 (1 H, J5,6 8.2, 5-H), 8.30 (1 H, s, 9-H); d (125 MHz, CDCl )
C 3
2.4, 56.7, 126.1, 127.4, 127.8, 127.9, 129.8, 130.1, 131.9, 134.5,
1
2
13
4
H
3
(
(
1 H, m, 69-H), 2.24–2.28 (1 H, m 6-H), 2.64 (1 H, br s, OH), 2.76
1 H, m, 59-H), 2.86 (1 H, m, 5-H), 4.20 (3 H, s, OMe), 4.35 (1 H,
m, 7-H), 4.70 (1 H, d, J8,7 3.3, 8-H), 6.95 (1 H, d, J3,2 2.6, 3-H),
7.53 (1 H, d, J2,3 2.6, 2-H); d (125 MHz, CDCl ) 18.3, 25.0 , 58.6,
C 3
+
37.9, 148.6, 151.8; m/z (EI): 195 (M , 84%), 167 (100).
66.74, 70.8, 104.9, 106.5, 115.2, 142.2, 151.0, 158.5, 162.8; m/z
+
(EI): 235 (M , 30%), 206 (100), 188 (92), 163 (94), 133 (35).
(e) Chemoenzymatic synthesis of dictamnine 7,8-oxide 27 and
(
7S,8S)-8-Bromo-4-methoxy-5,6,7,8-tetrahydrofuro[2,3-b]qui-
4-methoxyfuro[2,3-b]quinoline-7,8-diol 29
nolin-7-yl acetate 33. cis-Tetrahydrodiol 32 (0.1 g, 0.43 mmol)
was converted into bromoacetate 33, using the first step of the
method described for the synthesis of arene oxide 2.
Purification of the crude product by PLC (25% EtOAc/hexane)
afforded a pure sample of bromoacetate 33 as a pale yellow oil
Dictamnine 7,8-oxide 27. To a solution of bromoacetate 33
3
(
0
0.16 g, 0.47 mmol) in CCl
4
(4 cm ) was added NBS (93 mg,
.52 mmol) and AIBN (ca. 2 mg). The stirred reaction mixture
was heated to 65 uC, under nitrogen using a heat lamp, until
all the NBS had reacted. The cooled reaction mixture was
filtered and the filtrate concentrated under reduced pressure.
The crude dibromoacetate 34 obtained was used immediately,
without purification, for the next step.
(
0.13 g, 90%); R
f
0.25 (25% EtOAc/hexane); [a]
D
237.5 (c 1.16,
+
CHCl ); (Found:
3
M
339.0121.
C
14
H
14BrNO requires
4
3
2
5
4
39.0106); dH (500 MHz, CDCl ) 1.99 (3 H, s, OCOMe), 2.14–
3
.18 (1 H, m, 69-H), 2.51–2.59 (1 H, m, 6-H), 2.72–2.80 (1 H, m,
9-H), 2.93–2.98 (1 H, ddd, J 5,59 17.5, J 5,69 6.7, J 5,6 2.1, 5-H),
.27 (3 H, s, OMe), 5.30 (1-H, dd, J8,7 3.0, J8,6 1.5, 8-H), 5.54 (1
The synthesis of dictamnine 7,8-oxide 27 from the crude
dibromoacetate 34 (0.15 g, 0.36 mmol) was carried out
following the second step of the method described for the
H, m, 7-H), 6.95 (1 H, d, J3,2 2.6, 3-H), 7.58 (1 H, d, J2,3 2.6, 2-H);
3
synthesis of arene oxide 2, using dry THF (3 cm ) instead of
d
C
(125 MHz, CDCl
3
) 17.7, 21.1, 21.3, 48.1, 58.6, 72.4, 104.8,
Et O as a solvent. The crude sample of 7,8-oxide 27 crystallized
2
+
1
06.1, 115.7, 143.2, 147.7, 158.4, 162.8, 170.1; m/z (EI): 341 (M ,
out of ether/hexane solution to give a pure sample as a light
8
1
+ 79
Br, 30%), 339 (M , Br, 29), 281 (20), 279 (20), 218 (50), 200
3
brown solid (43 mg, 56%); m.p. 125–127 uC (from CHCl /
(33), 101 (61), 59 (65), 43 (100).
+
hexane); (Found: M 215.0586. C12
a] +93.1 (c 0.36, CHCl ); d (500 MHz, CDCl
7,8 3.71, J7,6 3.66, J7,5 1.84, 7-H), 4.30 (3 H, s, OMe), 4.65 (1 H,
H
9
NO
3
requires 215.0582);
[
J
D
3
H
3
) 4.18 (1 H, ddd,
(
d) Chemoenzymatic synthesis of (1R,2S)-1,2-epoxy-1,2-
dihydroacridine (acridine 1,2-oxide) 2
d, J8,7 3.84, 8-H), 6.37 (1 H, dd, J6,5 9.86, J6,7 3.66, 6-H), 7.01 (1
H, d, J3,2 2.57, 3-H), 7.20 (1 H, dd, J5,6 9.86, J5,7 1.8, 5-H), 7.62 (1
H, d, J2,3 2.57, 2-H); d (125 MHz, CDCl ) 54.4, 58.4, 58.9, 104.6,
To a stirred solution of cis-dihydrodiol 4 (0.1 g, 0.47 mmol) in
3
dry MeCN (4 cm ) was added, at 0 uC, 1-bromocarbonyl-1-
C
3
methylethyl acetate (0.109 g, 0.52 mmol). The reaction mixture
105.3, 106.6, 113.5, 122.6, 124.4, 143.0, 148.8, 157.4; m/z (EI):
215 (M , 100%), 200 (57), 172 (30), 83 (27); ECD: l/nm 322 (De
0.747), 288 (De 0.867), 241 (De 7.87), 203 (De 26.38).
+
was stirred (1.5 h) at room temperature, diluted with Et O (30
2
3
cm ) and the solution washed thoroughly with 5% aqueous
3
NaHCO
3
solution (15 cm ). The organic layer was dried
4-Methoxyfuro[2,3-b]quinoline-7,8-diol 29. A cooled solution
(215 uC) of skimmianine 13 (0.5 g, 1.93 mmol) in dry CH Cl
2
(
Na SO ) and concentrated to yield an inseparable mixture of
2 4
2
3
bromoacetates 25 and 26 in which bromoacetate 25 was the
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(15 cm ) was treated, drop-wise, with a solution of boron
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3
+
tribromide in CH
2
Cl
2
(1 M, 4.8 cm ). After leaving the reaction
(from EtOAc/hexane); R
f
0.2 (50% EtOAc/hexane); (Found: M ,
+
mixture stirred, at room temperature overnight, it was cooled
to 260 uC and water (5 cm ) was added and the mixture
299.1166. C H NO requires M , 299.1158); d (500 MHz,
CDCl ) 1.62 (3 H, s, Me), 1.75 (3 H, s, Me), 4.43 (3 H, s, OMe),
1
7
17
4
H
3
3
allowed to warm up to room temperature. The CH Cl layer
5.01 (2 H, d, J29,39, 7.5, 29-H), 5.60 (1 H, t, J39,29 4.5, 39-H), 7.05 (1
2
2
was separated and the remaining aqueous solution was
extracted with EtOAc (2 6 10 cm ). The combined organic
H, d, J3,2 2.8, 3-H), 7.15 (1 H, d, J6,5 9.2, 6-H), 7.57 (1 H, d, J
2.8, 2-H), 7.95 (1 H, d, J5,6 9.2, 5-H); m/z (EI): 299 (M , 20%), 231
2,3
3
+
extract was dried (Na
2
SO
4
) and concentrated, under reduced
(100).
pressure, to yield the crude catechol 29. Crystallisation from
MeOH furnished catechol 29 as an off-white coloured solid
iso-Haplopine-3,39-dimethylallylether 38. 4-Methoxy-8-[(3-
methyl-2-butenyl)oxy]furo[2,3-b]quinolin-7-ol 37 (20 mg, 0.067
mmol) was treated with an excess of diazomethane as
described earlier. The crude methylated product was purified
by PLC to give compound 38 as a white solid (19 mg, 90%);
(
0.38 g, 85%); m.p. 211 uC (from MeOH); R 0.1 (50% EtOAc/
f
+
+
hexane); (Found: M , 231.0523.
12 9 4
C H NO requires M ,
2
31.0532); dH (500 MHz, CD COCD ) 4.39 (3 H, s, OMe), 6.99
3
3
1
3e
(1 H, d, J6,5 9.1, 6-H), 7.23 (1 H, d, J2,3 2.8, 2-H), 7.52 (1 H, d, J5,6
m.p. 124–125 uC (from CH Cl ) (lit.
f
120–121.5 uC); R 0.4
2
2
+
9.1, 5-H), 7.66 (1 H, d, J3,2 2.8, 3-H).
(40%
requires 313.1314); d
.74 (3 H, d, Me), 4.00 (3 H, s, OMe), 4.43 (3 H, s, OMe), 4.84 (2
H, d, J19,29 7.2, 19-H), 5.73 (1 H, t, J29,19,7.2, H-29), 7.04 (1 H, d,
3,2 2.8, 3-H), 7.22, d, J6,5 9.4, 6-H), 7.58 (1 H, d, J2,3 2.80, 2-H),
EtOAc/hexane); (Found: M , 313.1312.
18 4
C H19NO
H
(500 MHz, CDCl ) 1.66 (3 H, s, Me),
3
(
f) Chemoenzymatic synthesis of furoquinoline alkaloids 30,
1
31, 36, 37, 38 and 40
Robustine 30. Dihydrodiol 17 (10 mg, 0.04 mmol), dissolved
J
+
in CHCl3 solution, was aromatised by the addition of two
drops of TFA. The acidic solution was concentrated after
adding excess of ammonium hydroxide to yield robustine 17
8.00 (1 H, d, J5,6 9.4, 5-H); m/z (EI): 313 (M , 20%).
Pteleine 40. cis-Dihydrodiol 16 (7 mg, 0.004 mmol) was
dissolved in CDCl in an NMR tube and a drop of TFA added to
3
1
3b
as a light brown solid (9 mg, 98%); m.p. 147 uC (lit. 148–149
uC); dH (300 MHz, CDCl ) 4.43 (3 H, s, OMe), 7.17 (1H, d, J
the solution. The tube was kept at 50 uC until the aromatiza-
1
3
3,2
tion of diol 16 was complete ( H-NMR analysis). After removal
2
.8, 3-H), 7.20 (1 H, d, J7,6 7.8, 7-H), 7.38 (1 H, dd, J6,7 = J6,5
=
of solvent the aromatized product was treated with excess of
diazomethane and the crude product obtained was purified by
PLC (40% EtOAc/hexane) to afford pure pteleine 40 as a white
7.8, 6-H), 7.63 (1 H, d, J 2.8, 2-H), 7.79 (1 H, d, J5,6 7.8, 5-H);
2
,3
+
m/z (EI): 215 (M , 100%), 200 (59).
1
3f
c-Fagarine 31. Robustine 30 (5 mg, 0.023 mmol) was
solid (3 mg, 77%); m.p. 132–134 uC (lit. 134.5 uC); R 0.4
f
3
dissolved in MeOH (1 cm ) and treated (0 uC) with an excess
(40% EtOAc/hexane); dH (500 MHz, CDCl ) 3.95 (3 H, s, Me),
3
of ethereal solution of diazomethane. The solvents were
evaporated under a stream of nitrogen to yield the title
compound 31 (4.2 mg, 79%); m.p. 142–143 uC (lit. 141 uC);
4.60 (3 H, s, Me), 7.07 (1 H, d, J3,2 2.8, 3-H), 7.36 (1 H, dd, J7,8
9.15, J7,5 2.95, 7-H), 7.53 (1 H, d, J8,7 2.95, 8-H), 7.63 (1 H, d, J2,3
2.8, 2-H), 7.92 (1 H, d, J8,7 9.15, 8-H).
1
3c
dH (500 MHz, CDCl ) 4.09 (3 H, s, OMe), 4.46 (3 H, s, OMe),
3
7
.01 (1 H, d, J7,6 7.8, H-7), 7.09 (1 H, d, J3,2 2.7, H-3), 7.35 (1 H,
dd, J6,5 8.3 J6,7 7.8, H-6), 7.65 (1 H, d, J2,3 2.7, H-2), 7.85 (1 H, d,
5,6 8.3, H-5); d (125 MHz, CDCl ): 29.6, 56.0, 59.0, 104.5,
07.7, 114.1, 118.9, 123.2, 123.4, 129.7, 143.9, 154.5, 157.2; m/z
Acknowledgements
J
1
C
3
We thank the European Social Fund (JGC), Oxford
Glycosciences (PL) and the Queen’s University of Belfast
(JGC, PL, CO’D) for financial support.
+
(EI): 229 (M , 100%), 214 (18).
Haplopine 36. Catechol 29 (50 mg, 0.20 mmol) was treated
0 uC) with an excess of ethereal diazomethane solution. After
(
leaving the reaction mixture for five minutes, excess of reagent
was removed by the addition of a few drops of acetic acid. The
crude product, obtained after removal of ether, was purified by
PLC (50% EtOAc/hexane), to give haplopine 36 as a white solid
References
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0954 | RSC Adv., 2013, 3, 10944–10955
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RSC Adv., 2013, 3, 10944–10955 | 10955