A rapid, chromatography-free route to substituted acridine-isoalloxazine conjugates under microwave irradiation

Article abstract of DOI:10.1016/j.tetlet.2014.04.035

Microwave irradiation was applied to a sequence of condensation reactions from readily available 9-chloroacridines to provide a range of novel acridine-isoalloxazine conjugates. The combination of these two moieties, both of biological interest, was achieved by a chromatography-free route.

Full text of DOI:10.1016/j.tetlet.2014.04.035

Tetrahedron Letters 55 (2014) 3308–3311
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Tetrahedron Letters
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A rapid, chromatography-free route to substituted
acridine–isoalloxazine conjugates under microwave irradiation
a
a
a
a
Stephen C. Johns a, , Laurie L. E. Crouch , Stephen Grieve , Holly L. Maloney , Gary R. Peczkowski ,
⇑
Allison E. Jones a, Duncan Sharp b, Robert B. Smith a,
⇑
a Centre of Materials Science, University of Central Lancashire, Preston PR1 2HE, UK
b Biomedicine, Faculty of Health and Social Sciences, Leeds Metropolitan University, Leeds, LS1 3HE, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 October 2013
Revised 2 April 2014
Accepted 10 April 2014
Available online 21 April 2014
Microwave irradiation was applied to a sequence of condensation reactions from readily available 9-chlo-
roacridines to provide a range of novel acridine–isoalloxazine conjugates. The combination of these two
moieties, both of biological interest, was achieved by a chromatography-free route.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Acridine
Isoalloxazine
Flavin
Microwave
Acridine moieties have been of interest to medicinal chemists
for many years, often appearing as central scaffolds in many biolog-
ically active compounds.1–3 Indeed acridines exhibit significant
pharmaceutical importance due to their potent photodynamic bio-
logical activities.4 However this goes hand-in-hand with the well
established toxicity profile5 of the molecule itself, which represents
a considerable obstacle to overcome for progression into main-
stream healthcare. In contrast, 7,8-dimethyl-10-ribityl isoalloxa-
zine (riboflavin or vitamin B2) is an essential nutrient in humans,
and is also a photosensitiser which absorbs in the visible region.
It shows impressive activity both as an antibacterial6 and antiviral
agent through both Type I and Type II photosensitisations.7
Inspired by the biological activity of both, herein we present a
short and efficient method for the rapid generation of hybrid acri-
dine–isoalloxazine conjugates (Fig. 1). The isoalloxazine moiety
also contains an acidic N-3 proton which may prove useful for
attachment of either a solubilising or a targeting moiety.8
sation with alloxan monohydrate gave a mixture of products from
which the desired compounds could not be isolated. We hypothe-
sised there were three major problems with this route; firstly, the
9-aminoacridines were poor nucleophiles, due to electron with-
drawal from the ring nitrogen, resulting in poor yields for the ini-
tial SNAr reaction;9 secondly, the zinc reduction was not
completely selective resulting in numerous by-products, and
finally that the intermediates and products were likely to be poorly
soluble in most common solvents making column chromatography
challenging.10 Our second route (route B, Scheme 1) utilised 9-
chloroacridines as the electrophilic component using the elec-
tron-withdrawing acridine to our advantage. By using phenylene
diamines as the nucleophiles the reduction step was avoided
entirely, albeit at the cost of perhaps limiting our isoalloxazines
to containing the same group at both the C7 and C8 positions.11
In order to optimise the reaction conditions, commercially
available 6-chloro-2-methoxyacridine (4a) was chosen as a model
substrate. There have been several reports of 9-chloroacridines
reacting with anilines to form substituted N(9)-anilinoacridine
hydrochloride salts by heating under reflux in methanol. Following
the procedure of Plater et al.,12 heating 4a and aniline in methanol
under reflux gave the desired product 6a in moderate yield
(Table 1, entry 1), with the only purification required being precip-
itation and filtration. The use of microwave acceleration of organic
reactions has found widespread use.13 Two of the major advanta-
ges of microwave heating are the ability to heat solvents rapidly
in sealed tubes past their boiling points, and the uniformity of
Our initial attempts to form the isoalloxazine–acridines fol-
lowed a three-step route from a range of 9-aminoacridines (route
A, Scheme 1); firstly condensation with 2-chloronitrobenzene gave
the desired N-substituted 2-nitroaniline in low to moderate yields
after chromatography. However, attempted reduction of the nitro
group using standard conditions (Zn/H+) and subsequent conden-
⇑
Corresponding authors. Tel.: +44 (0) 1772 894384.
E-mail addresses: stephen.johns00@alumni.imperial.ac.uk (S.C. Johns),
rbsmith@uclan.ac.uk (R.B. Smith).
http://dx.doi.org/10.1016/j.tetlet.2014.04.035
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

S. C. Johns et al. / Tetrahedron Letters 55 (2014) 3308–3311
3309
tic and boric acid giving moderate to high yields (most often 40–
70%) of isoalloxazines.16 In order to increase the rate of this reac-
tion, we again attempted this reaction under microwave irradia-
tion. Condensation of alloxan monohydrate with the acridine salt
6a in the absence of boric acid, even at elevated temperatures
(140 °C) did not occur in MeOH or AcOH to any measurable extent.
The reaction was successful in AcOH with one equivalent of boric
acid giving the product 7a in excellent yield (95%, 140 °C, 10 min,
Table 1, entry 2). Chauhan et al. have reported the condensation
of some alkyl and aryl substituted 2-aminoanilines with alloxan
monohydrate under microwave irradiation in a domestic micro-
wave oven to give 10-substituted isoalloxazines in good yield.17
The use of our own conditions gave the product isoalloxazine 7a
rapidly in high yield and purity, after precipitation, filtration and
washing (including water washes to remove boric acid), without
recourse to column chromatography (85% over 2 steps from 4a).
Preparation of a range of 9-chloroacridines was performed fol-
lowing reported methods (Scheme 2)18 via reaction of anilines 2
with several 2-chlorobenzoic acids 1.19 The formed crude N-aryl
anthranillic acids 3 were then ring-closed with POCl3 under micro-
wave irradiation.20 The desired 9-chloroacridines 4 could be iso-
lated by a variety of methods including vacuum sublimation,
vacuum filtration through a pad of silica gel or recrystallisation
to give single compounds. Several greener methods have been
reported in the literature for the synthesis of N-aryl anthranilic
acids including solvent-free conditions with microwave irradia-
tion21 and the use of ultrasound in aqueous solution.22
O
N
N
N
NH
O
Isoalloxazine
N10-C9' bonded
Acridine
N
Figure 1. Structures of isoalloxazine and acridine.
NO2
NO2
X
R2
NH
Cl
R2
R1
N
Base
R1
N
Route A
X=NH2
Zn/H+
NH2
Δ
NH2
Route B
X=Cl
O
N
NH2
NH
N
.HCl
NH
Alloxan.H2O
N
O
R2
R2
AcOH
B(OH)3
R1
N
R1
N
Scheme 1. Routes to isoalloxazine–acridine conjugates. Route A: previously
The optimised conditions were applied to a range of acridines
4a–g derived from 2,4-dichlorobenzoic acid (Scheme 3, Table 1).
Several different phenylene diamines 5a–c were used to vary the
electronics of the isoalloxazine system and to make flavin deriva-
tives, giving the corresponding products 6, 60 or 600 in moderate
to high yields as the corresponding hydrochloride salts.23 The low-
est yield was obtained from 2,4-dimethoxyacridine 4c, which has
the most electron-rich C9 position (69%, Table 1, entry 6). In all
cases the reaction was highly regioselective with no evidence of
any reaction with the 6-chloro group. The isoalloxazines 7, 70, 700
were formed in all cases,24 however, in several examples, the final
compounds were contaminated with by-products, often from
residual phenylene diamines present in the intermediate hydro-
chloride salts. Attempted purification of several products that were
of unacceptable purity by column chromatography or recrystallisa-
tion was particularly difficult due to the insolubility of the acri-
dine–isoalloxazines 7. It was discovered that the majority of
impurities could be removed by high temperature extraction/
recrystallisation in methanol under microwave irradiation (see
Supplementary information), and the purified yields are reported
in parentheses. Extraction in a sealed system at high temperature
attempted three-step route. Route B: a new two-step route.
heating, potentially resulting in fewer by-products resulting from
localised hotspots.14 Performing the reaction under conditions of
microwave irradiation (110 °C, 10 min) resulted in a much faster,
and pleasingly, a higher yielding reaction (Table 1, entry 2),
although heating the reaction at higher temperature (140 °C) did
result in decreased purity. During this work, Staderini et al.
reported a solvent-free microwave route for amination of
p-elec-
tron deficient heterocycles including acridines; in their cases the
reactions were promoted by the addition of two equivalents of
phenol, giving excellent yields.15 Conventional conditions for con-
densation of 2-aminoanilines with alloxan monohydrate generally
require prolonged (P12 h) stirring at room temperature with ace-
Table 1
Synthesis of acridine–isoalloxazines 7a–g and intermediate diaryl amines 6a–g
derived from 6,9-dichloroacridines 4a–g
Entry Starting
material
R2
R3
R4
R5
Yield 6a
(%)
Yield 7b
1
2
3
4
5
6
7
8
9
4a
4a
4a
4a
4b
4c
4d
4d
4e
4f
H
H
H
H
H
H
OMe
OMe
H
OMe
OMe
OMe
OMe
H
OMe OMe
H
H
Me
H
H
H
H
H
H
H
6a:53c
6a:93
N/Dd
7a:95%
CO2H
Me 6a0:91
7a0:86%
7a00:92%
7b:(76%)e
7c:(68%)e
7d:(84%)e
7d00:(38%)e
7e:86%
R1
Cl
6a00:90
6b:83
6c:69
6d:83
6d00:84
6e:95
6f:96
CO2H
NH
Cl
H
H
H
1
OMe
R1
R2
Cu/K2CO3
R4
R3
OMe
OMe Cl
H
Me
H
DMF Δ
R2
R3
H2N
H
H
H
R4
10
11
H
H
7f:(61%)e
7g:83%
3
2
4g
H
6g:82
a
Cl R2
Isolated yields after precipitation from Et2O, filtration, washing and drying
(1 mmol scale).
R3
POCl3
b
Isolated yields after precipitation from Et2O, filtration, washing and drying
R1
N
μW 140 °C,
(0.5 mmol scale).
R4
c
15 min
Heating under reflux for 1.5 h.
Not determined, see entry 2.
Pro rata after high temperature microwave extraction/recrystallisation of a
d
4
e
150 mg portion of the crude product (see Supplementary information).
Scheme 2. Preparation of the 9-chloroacridine starting materials 4.

3310
S. C. Johns et al. / Tetrahedron Letters 55 (2014) 3308–3311
O
R5
R5
NH2
NH2
O
O
O
N
R5
R5
N
N
R5
R5
NH2
HN NH
NH
.HCl
Cl R2
.H2O
R3
NH R2
5a-c
O
O
R3
R2
R3
AcOH/B(OH)3
μW 140 °C,
10 min
MeOH, μW
110 °C, 10 min Cl
Cl
N
R4
N
R4
Cl
N
4
R4
6 R5=H
7 R5=H
6' R5=Me
6'' R5=Cl
7' R5=Me
7'' R5=Cl
Scheme 3. Microwave route to acridine–isoalloxazines 7a–g from 6,9-dichloroacridines.
is both faster, uses a much smaller volume of solvent than tradi-
tional methods such as Soxhlet extraction, and is often used in
the extraction of organic molecules from environmental25 or cellu-
lar matrices.26 As, in this case, the products 7 were observed to be
sparingly soluble in methanol, the use of lower volumes of solvent
results in less product being lost in the extraction solvent. With the
1,4-dimethoxy substituted intermediate 6d00, 1H NMR of the prod-
uct 7d00 after precipitation showed residual starting material,
which was also successfully removed by high temperature extrac-
tion. We suggest that the increased steric hindrance in this case
(and presumably to a lesser extent in the case of 6d) resulted in
slower conversion and the reaction would benefit from a pro-
longed reaction time. Scale-up (4ꢀ) of the reactions to form 7a
and 7a0 gave the desired products in similar yields, although it
should be noted that stirring of the reaction mixture must be effi-
cient to ensure complete conversion of the starting material.
Similar reactions were attempted with acridines 4h–l and 4m,n
derived from 2-chlorobenzoic acid and 2-chloro-4-nitrobenzoic
acid, respectively (Scheme 4, Table 2). With substrate 4i, which
was particularly deactivated by the 4-methoxy group, only a mod-
erate yield of acridine salt 6i was obtained on reacting with o-phe-
nylene diamine (5a) (67%), however extending the reaction time to
20 minutes increased the yield such that it was comparable with
the others (86%, Table 2, entry 3). Substrate 4l had the opposite
problem, activation by the 4-nitro group made the substrate too
reactive, and the reaction produced a complex mixture of products
as a black tar (Table 2, entry 10). Indeed nucleophilic displacement
on 9-chloro-4-nitroacridine 4l has been reported in the literature
only once with an aniline electrophile, and in this case, was per-
formed at 0 °C.27 Pleasingly, 6-nitro substituted acridines (where
the nitro group does not withdraw electrons from the 9-position
by resonance) did not suffer from this problem giving the desired
products (6m and 6n, Table 2, entries 11 and 12). Conversion into
the acridine–isoalloxazines gave the products 7, in most cases
without further purification being required, and where this was
not the case, high temperature microwave extraction was per-
formed to remove unwanted impurities. The products containing
6-nitro-substituted acridines 7m and 7n were particularly insolu-
Table 2
Synthesis of acridine–isoalloxazines 7h–n and intermediate diaryl amines 6h–n
derived from acridines 4h–n without a 6-chloro group
Entry Starting
material
R1
R3
R4
R5
Yield 6a
(%)
Yield 7b
1
2
3
4
5
6
7
8
9
4h
4h
4i
4i
4i
H
H
H
H
H
H
H
H
H
H
OMe
OMe
H
H
H
Me
H
H
H
H
H
H
OMe
H
6h:85
7h:73%
7h0:77%
7i:(64%)d
7i0:73%
7i00:82%
7j:70%
Me 6h0:97
H
6i:86c
OMe Me 6i0:93e
OMe Cl
H
H
H
H
NO2
H
6i00:82
6j:83
6k:97
4j
H
H
4k
4k
4k
4l
4m
4n
7k:88%
7k0:89%
7k00:94%
—
Me 6k0:85
Cl
H
H
H
6k00:87
—
6m:86
6n:90
10
11
12
NO2 OMe
NO2 Me
7m:79%f
7n:76%f
H
a
Isolated yields after precipitation from Et2O, filtration, washing and drying
(1 mmol scale).
b
Isolated yields after precipitation from Et2O, filtration, washing and drying
(0.5 mmol scale).
c
After heating for 20 min (67% with only 10 min heating).
Pro rata after high temperature microwave extraction/recrystallisation of a
d
150 mg portion of the crude product (see Supplementary information).
e
After 20 min heating.
Isolated as a ꢁ1:1 mixture with AcOH.
f
ble in acetic acid and crystallised in the reaction vessel as a ꢁ1:1
mixture with acetic acid (by 1H NMR) and were isolated as such.
The use of substrate 4o, containing an extra contiguous ring
proved challenging (Scheme 5), and the normal optimised condi-
tions failed to give any product, whilst raising the temperature sig-
nificantly resulted in the formation of several by-products; in this
case a slower one-hour reaction at a slightly elevated temperature
(120 °C) gave the best compromise between rate and purity. The
isoalloxazine 7o was formed from the HCl salt 6o using the opti-
mised conditions. In this case two purifications, including chroma-
tography and a high temperature microwave extraction, were
required to give the product in high purity and the significantly
lower yield can be attributed to this.
O
R5
R5
NH2
NH2
O
O
O
N
R5
R5
NH2
NH
R5
R5
N
N
HN NH
.H2O
.HCl
R3
NH
O
Cl
R3
5a-c
O
R3
R1
N
AcOH/B(OH)3
μW 140 °C,
10 min
MeOH, μW
R4
110 °C, 10 min R1
N
R1
N
R4
R4
4
6 R5=H
7 R5=H
6' R5=Me
6'' R5=Cl
7' R5=Me
7'' R5=Cl
Scheme 4. Microwave route to acridine–isoalloxazines 7h–n from 6-hydro or 6-nitro-9-chloroacridines 4h–n.

S. C. Johns et al. / Tetrahedron Letters 55 (2014) 3308–3311
3311
NH2
NH2
NH2
NH
References and notes
Cl
.HCl
1. Liao, S.-R.; Zhou, C.-X.; Wu, W.-B.; Ou, T.-M.; Tan, J.-H.; Li, D.; Gu, L.-Q.; Huang,
Z.-S. Eur. J. Med. Chem. 2012, 53, 52.
2. Malachowska-Ugarte, M.; Cholewinski, G.; Dzierzbicka, K.; Trzonkowski, P. Eur.
J. Med. Chem. 2012, 54, 197.
Cl
N
μ
MeOH,
W
Cl
N
120 °C, 1 h
3. Luan, X.; Gao, C.; Zhang, N.; Chen, Y.; Sun, Q.; Tan, C.; Liu, H.; Jin, Y.; Jiang, Y.
Bioorg. Med. Chem. 2011, 19, 3312.
4. Wainwright, M. J. Antimicrob. Chemother. 2001, 47, 1.
5. Ferguson, L. R.; Denny, W. A. Mutation Res./Rev. Genet. Toxicol. 1991, 258, 123.
6. Makdoumi, K.; Bäckman, A.; Mortensen, J.; Crafoord, S. Graef. Arch. Clin. Exp.
Ophthalmol. 2010, 248, 207.
4o
6o
78%
O
O
O
NH
.H2O
N
NH
O
O
N
H
O
N
N
7. Ruane, P. H.; Edrich, R.; Gampp, D.; Keil, S. D.; Leonard, R. L.; Goodrich, R. P.
Transfusion 2004, 44, 877.
AcOH/B(OH)3
μW 140 °C,
10 min
8. Dutra, J. K.; Cuello, A. O.; Rotello, V. M. Tetrahedron Lett. 1997, 38, 4003.
9. 9-Aminoacridines have been used successfully in this reaction with 2-
halonitrobenzenes for the preparation of several N(9)-anilinoacridines in
good yield, however, in most cases, chromatographic purification was
required after heating for 12 h with Cs2CO3/DMF, see: Gellerman, G.; Gaisin,
V.; Brider, T. Tetrahedron Lett. 2010, 51, 836.
Cl
N
7o
19%
Scheme 5. Extension of the method to form an isoalloxazine conjugate of a 3,4-
10. Farrán, Á.; Claramunt, R.; López, C.; Pinilla, E.; Torres, R.; Elguero, J. ARKIVOC
2007, iv, 20.
benzacridine.
11. Stevens and co-workers substituted 9-chloroacridines with several 4-
substituted o-phenylene diamines and in the majority of cases the reaction
was highly regioselective where the substituent exerted a strong electronic
effect, see: Hagan, D. J.; Giménez-Arnau, E.; Schwalbe, C. H.; Stevens, M. F. G. J.
Chem. Soc., Perkin Trans. 1 1997, 2739.
12. Plater, M. J.; Greig, I.; Helfrich, M. H.; Ralston, S. H. J. Chem. Soc., Perkin Trans. 1
2001, 2553.
13. Caddick, S.; Fitzmaurice, R. Tetrahedron 2009, 65, 3325.
14. Lidström, P.; Tierney, J.; Wathey, B.; Westman, J. Tetrahedron 2001, 57, 9225.
15. Staderini, M.; Cabezas, N.; Bolognesi, M. L.; Menéndez, J. C. Tetrahedron 2013,
69, 1024.
16. Choy, N.; Russell, K. C.; Alvarez, J. C.; Fider, A. Tetrahedron Lett. 2000, 41, 1515.
17. Chauhan, S. M. S.; Singh, R.; Geetanjali Synth. Commun. 2003, 33, 1179.
18. Crouch, L. L. E.; Grieve, S.; Maloney, H. L.; Peczkowski, G. R. Unpublished
results.
19. Litchfield, V. J.; Smith, R. B.; Franklin, A. M.; Davis, J. Synth. Commun. 2008, 38,
3447.
20. Taherpour, A. A.; Kvaskoffa, D.; Bernhardta, P. V.; Wentrup, C. J. Phys. Org. Chem.
2010, 23, 382.
21. Martína, A.; Mesaa, M.; Docampoa, M. L.; Gómeza, V.; Pellóna, R. F. Synth.
Commun. 2006, 36, 271.
The products were characterised initially by 1H NMR spectros-
copy and exhibited some interesting features. Signal broadening
was often observed in the protons between the two tricyclic sys-
tems. Steric clashes along these sides will prevent the molecules
from adopting a planar structure. The two ring systems must be
offset with rotation around the N10AC90 bond being restricted,
resulting in the products existing as two enantiomeric atropisom-
ers. Separation of axially chiral N-phenyl isoalloxazines has been
reported,28 and planar chiral flavin derivatives have been used as
asymmetric catalysts in sulfide29 and Baeyer–Villiger30 oxidations.
The twist in the structure will also result in decreased conjugation
between the two ring systems. In the case of product 7d one of the
acridine methoxy groups points directly towards the isoalloxazine
ring system, and the chemical shift of the methyl group is particu-
larly low (3.23 ppm in 7d compared to 4.03 ppm in 6d); the shield-
ing in this case is due to the magnetic anisotropy of the
isoalloxazine system.
22. Palaciosa, M. L. D.; Comdoma, R. F. P. Synth. Commun. 2003, 33, 1771.
23. For example:
A mixture of 6,9-dichloro-2-methoxyacridine (4a) (278 mg,
1.0 mmol, 1 equiv) and phenylenediamine (5a) (238 mg, 2.2 mmol, 2.2 equiv)
was suspended in MeOH (5 mL) and subjected to microwave irradiation
(110 °C, 10 min, CEM DiscoverÒ microwave). The mixture was allowed to cool
to room temperature giving a red liquid with a red/orange precipitate. The
mixture was then transferred into rapidly stirred Et2O. After stirring for 5 min,
the solid was isolated by vacuum filtration, washed with Et2O and dried giving
the product hydrochloride salt 6a (359 mg, 93%) as a red/orange solid.
24. For example: A mixture of diamine intermediate 6a (193 mg, 0.50 mmol,
1.0 equiv), alloxan monohydrate (96.0 mg, 0.60 mmol, 1.2 equiv) and boric acid
(30.9 mg, 0.50 mmol, 1.0 equiv) were suspended in AcOH (5 mL) and subjected
to microwave irradiation (140 °C, 10 min, CEM DiscoverÒ microwave). The
mixture was allowed to cool to room temperature forming a yellow/orange
precipitate. The mixture was then transferred into rapidly stirred Et2O. After
stirring for 5 min, the solid was isolated by vacuum filtration, washed with
Et2O and H2O and dried giving the product isoalloxazine–acridine conjugate 7a
(217 mg, 95%) as a yellow solid.
In summary, we have reported a rapid, chromatography-free
method for the preparation of acridine–isoalloxazine conjugates
from substituted 9-chloroacridines. We are currently undertaking
biological and photophysical testing of the compounds to deter-
mine their applicability for photodynamic antimicrobial
chemotherapy.
Acknowledgments
We are pleased to acknowledge the financial support from the
Centre for Materials Sciences, School of Forensic and Investigative
Sciences and the EPSRC national mass spectrometry service centre
(NMSSC), Swansea for the recording of HRMS.
25. Letellier, M.; Budzinski, H. Analysis 1999, 27, 259.
26. Chan, C.-H.; Yusoffa, R.; Ngoh, G.-C.; Kung, F. W.-L. J. Chromatogr. A 2011, 1218,
6213.
27. Su, T.-L.; Chen, C.-H.; Huang, L.-F.; Chen, C.-H.; Basu, M. K.; Zhang, X.-G.; Chou,
T.-C. J. Med. Chem. 1999, 42, 4741.
Supplementary data
28. Shinkai, S.; Nakao, H.; Kuwahara, I.; Miyamoto, M.; Yamaguchi, T.; Manabe, O. J.
Chem. Soc., Perkin Trans. 1 1988, 313.
29. Shinkai, S.; Yamaguchi, T.; Manabe, O.; Toda, F. J. Chem. Soc., Chem. Commun.
1988, 1399.
Supplementary data (experimental procedures and analytical
data for the compounds 6a–o and 7a–o) associated with this article
can be found, in the online version, at http://dx.doi.org/10.1016/
j.tetlet.2014.04.035.
30. Murahashi, S.; Ono, S.; Imada, Y. Angew. Chem., Int. Ed. 2002, 41, 2366.