The synthesis and evaluation of o-phenylenediamine derivatives as fluorescent probes for nitric oxide detection

Article abstract of DOI:10.1039/b105953j

A series of molecular probes for the determination of nitric oxide (NO) have been prepared. Each probe consists of an anthracene, coumarin or acridine fluorophore coupled to an electron rich o-phenylenediamine group. The o-phenylenediamine group can be substituted with methyl or methoxy groups to enhance its electron rich nature. The fluorophore fluorescence is quenched by photoelectron transfer (PET) from the aromatic amine to the lowest unoccupied orbital of the excited state fluorophore. Reaction with nitrosating species converts the o-phenylenediamine group into an electron deficient benzotriazole derivative. This group has a higher oxidation potential and does not quench the fluorophore fluorescence by photoelectron transfer so that these products are highly fluorescent. Some benzotriazole derivatives were made preparatively by alternative synthetic routes. The formation of fluorescent probes was evaluated by treatment of the precursors with nitrous fumes and S-nitroso-N-acetylpenicillamine (SNAP).

Full text of DOI:10.1039/b105953j

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The synthesis and evaluation of o-phenylenediamine derivatives as
fluorescent probes for nitric oxide detection
M. John Plater,*a Iain Greig,a Miep H. Helfrich*b and Stuart H. Ralstonb
1
a Department of Chemistry, University of Aberdeen, Meston Walk, Aberdeen, UK AB24 3UE
b Department of Medicine and Therapeutics, University of Aberdeen, Polwarth Building,
Foresterhill, Aberdeen, UK AB25 2ZD
Received (in Cambridge, UK) 4th July 2001, Accepted 7th September 2001
First published as an Advance Article on the web 1st October 2001
A series of molecular probes for the determination of nitric oxide (NO) have been prepared. Each probe consists
of an anthracene, coumarin or acridine fluorophore coupled to an electron rich o-phenylenediamine group. The
o-phenylenediamine group can be substituted with methyl or methoxy groups to enhance its electron rich nature.
The fluorophore fluorescence is quenched by photoelectron transfer (PET) from the aromatic amine to the
lowest unoccupied orbital of the excited state fluorophore. Reaction with nitrosating species converts the
o-phenylenediamine group into an electron deficient benzotriazole derivative. This group has a higher oxidation
potential and does not quench the fluorophore fluorescence by photoelectron transfer so that these products are
highly fluorescent. Some benzotriazole derivatives were made preparatively by alternative synthetic routes. The
formation of fluorescent probes was evaluated by treatment of the precursors with nitrous fumes and S-nitroso-N-
acetylpenicillamine (SNAP).
Introduction
Nitric oxide (NO) plays a vital role in human physiology. NO
has different, concentration-dependent roles in many tissues,
such as in the vasculature, immune system and in neuro-
transmission.1–3 We are particularly interested in the role of
NO in bone and joint disease. In vivo and in vitro studies have
indicated that NO at low concentrations is an important
molecule, essential for normal bone physiology, however, high
levels of NO have been demonstrated to be inhibitory for bone
and cartilage cell function.4,5 NO plays an important role in
mediating the effect of interleukin-1 in inflammation-induced
bone loss and is implicated in the pathology of rheumatoid
arthritis.6,7 To understand more clearly the role of NO in bone
and joint pathology, better methods are required to determine
the exact concentrations of NO that are beneficial or harmful
for specific cell types. We also require more accurate inform-
ation on the cellular localisation of NO production, rather than
just information on the cellular expression of the NO synthase
enzymes. Techniques for the detection of NO based on chemi-
luminescence, or using electrochemical probes have been
developed.8–10 Although these techniques allow detection of
NO production from tissues, they do not allow imaging of NO
production at the cellular level. We therefore set out to design
molecular probes that would allow real time visualisation of
NO production within live cells. For studies of intracellular
concentrations of small molecules and ions, fluorescent probes
are widely used and they also offered the most attractive
Scheme 1
prospect for this application as a non-invasive and potentially
highly sensitive method.
an excited acceptor (fluorophore). o-Phenylenediamine and
p-phenylenediamine derivatives have low oxidation potentials
as shown by cyclic voltammetry.15 Back electron transfer occurs
without fluorescent emission restoring the ground state.
Reaction of the o-phenylenediamine group with NO derived
by-products such as N2O3 or peroxy nitrite, formed from the
reaction of NO with O2,13 gives a benzotriazole derivative.
Benzotriazole is an acidic, electron deficient heterocycle con-
taining two electron withdrawing pyridine type nitrogens and
possesses a higher oxidation potential, so electron transfer to
Work from the group of Nagano and co-workers11–13 has
shown the value of fluorophores linked to the o-phenylene-
diamine group for the detection of NO (Scheme 1). The o-
phenylenediamine group is electron rich and might quench the
fluorophores fluorescence by a process known as photoelectron
transfer (PET).14 This involves the transfer of an electron from
the HOMO of an electron rich donor (tertiary amine or
o-phenylenediamine) to the lowest energy vacant orbital of
DOI: 10.1039/b105953j
J. Chem. Soc., Perkin Trans. 1, 2001, 2553–2559
This journal is © The Royal Society of Chemistry 2001
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the excited state fluorophore occurs much less readily. It does
not quench the fluorophore fluorescence so that a positive
increase in fluorescence is observed. The system could be
considered to be a chemically irreversible ‘Off–On’ switch.
Diaminofluoresceins,11 diaminorhodamines12 and diamino-
naphthalenes13 have been studied as probes for NO. However,
we have found these compounds to be toxic for bone cells.
The rhodamine derivative is charged so would not be able to
penetrate a cell membrane. The fluoresceins and rhodamines
also require multistep synthetic preparations. In view of the
lengthy syntheses required for their preparation some simpler
synthetic alternatives were prepared and studied. It was hoped
to fine-tune such structures more easily to find probes with
properties that make them suitable for use with live cells.
Specific requirements are that the compounds have a suitable
long wavelength excitation frequency (above 340 nm) to avoid
tissue or cellular damage and to avoid interference from back-
ground fluorescence. The compounds should be soluble in
aqueous solution (usually phosphate-buffered tissue culture
media) with minimal DMSO concentrations (below 0.1%)
to avoid having a harmful effect on live cells. Finally, the probe
should ideally contain an ester functional group. Ester groups
do not prevent compounds passing through the cell plasma
membrane, but once inside the cell, esterases (which are present
inside bone cells) hydrolyse the ester to give a charged carboxylic
acid group,13,16,17 preventing the exit of the probe from the cell
and ensuring that only NO concentrations inside cells would be
measured.
Discussion
In diaminofluoresceins11 and diaminorhodamines12 the o-
phenylenediamine group is conjugated to the fluorophore but
is twisted out of plane by steric interactions. The literature on
the study of fluorescent sensors14 provided precedent that new
sensors for NO could be designed in which the electron rich
amine is tethered to a fluorophore by a saturated linker. A
series of o-phenylenediamine probes were prepared containing
different fluorophores and substituents based on this principle.
Probes 1 and 2 contained an anthracene fluorophore, probes 3–
6 contained a coumarin and probes 7–12 contained an acridine.
Derivatives were prepared in which the o-phenylenediamine
group was substituted with either methyl or methoxy groups.
These groups make the aromatic ring more electron rich
and might enhance its ability to quench fluorescence. The
anthracenes and coumarins are easily accessible from their
9-chloromethyl and 4-bromomethyl derivatives respectively by
reaction with o-phenylenediamine or a derivative of it. The
acridine based probes 7–11 were prepared by the reaction of an
o-phenylenediamine and 9-chloroacridine.18 9-Chloroacridine
was prepared by heating N-phenylanthranilic acid with
POCl3.19 They were isolated as their hydrochloride salts. Probes
9 and 11 contain an ester functional group. The primary amine
conjugated para to the ester was expected to be less nucleophilic
than the alternative primary amine which is meta to and not
conjugated to the ester. Acridine 12 was prepared by heating
acridine hydrochloride with o-phenylenediamine and sulfur.20
The interception of NO derived species such as N2O3 in cell
culture is thought to convert the o-phenylenediamine moiety
into a benzotriazole which does not quench the fluorophore
fluorescence. Therefore, for comparison purposes the corre-
sponding benzotriazole derivatives of probes 1, 3 and 7–11 were
prepared. Benzotriazoles 13 and 14 were prepared by treatment
of 9-chloromethylanthracene and 4-(bromomethyl)-7-methoxy-
coumarin with the sodium salt of benzotriazole respectively.
Benzotriazoles 15–19 were prepared by treatment of probes
7–11 with sodium nitrite and hydrochloric acid in aqueous
solution. The mechanism is thought to involve diazotisation of
the primary aromatic amine followed by a rapid intramolecular
cyclisation to give the benzotriazole.
The o-phenylenediamine probes 1–11 were found, as
expected, to be either weakly or non-fluorescent. The corre-
sponding benzotriazole derivatives are however intensely
fluorescent. Figs. 1–4 show typical excitation and emission
spectra that were obtained for some of the benzotriazole deriva-
tives 13–19. The largest excitation and emission peaks are
summarised in Table 1. The solutions were prepared by dilution
of standard solutions in DMSO with sodium phosphate buffer
(pH 7.4). The acridine derivatives have longer wavelength
excitation and emission maxima and therefore show more
promise for cell culture studies. Addition of the chloro and
methoxy groups in acridines 18 and 19 shifts the emission
maxima to over 450 nm. The synthesis of these probes and
the observation of enhanced fluorescence output in the corre-
sponding benzotriazole derivatives demonstrates their potential
feasibility for the detection of NO.
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Fig.
3
Excitation spectrum for acridine 15 taken at emission
wavelength of 437 nm and emission spectrum taken at excitation
wavelength of 380 nm. Acridine concentration 5 × 10Ϫ7 M.
Fig.
4
Excitation spectrum for acridine 18 taken at emission
wavelength of 452 nm and emission spectra taken at excitation
wavelength of 389 nm. Acridine concentration 5 × 10Ϫ7 M.
Table 1 Fluorescence properties of some prepared benzotriazole
derivatives
Compound
Excitation maxima/nm
Emission maxima/nm
13
14
15
16
17
18
19
350/367/390
327
383/398
381/397
384/398
358/387/411
356/386/409
395/418/442
390
418/438
414/436
417/438
454
Fig. 1 Excitation spectrum for anthracene 13 taken at emission
wavelength of 414 nm and emission spectrum taken at excitation
wavelength of 367 nm. Anthracene concentration 5 × 10Ϫ7 M.
452
From these encouraging results some simple qualitative
experiments were performed in which a dilute solution of the
probe was treated with a nitrogen gas stream containing some
nitrous fumes.21 Since the reaction of NO with O2 may generate
N2O3, which can act as a nitrosating agent, nitrosating fumes
provide a simple mimic of this system. These were generated in
excess by adding 2–3 drops of conc. HCl to a quantity of
NaNO2 (∼1 g) in a flask under a flow of nitrogen gas. The N2
gas was bubbled directly through a dilute solution of the probe
for a few seconds. Probes 1–6 were not soluble in water so were
dissolved up in either ethanol or DMSO. Probes 7–11, as their
hydrochloride salts, were dissolved up in DMSO and diluted
with phosphate buffer solution. In each case brief treatment
with an excess of nitrous fumes gave highly fluorescent solutions
indicating the desired in situ conversion of the o-phenylene-
diamine group to a benzotriazole.
The ability of probe 12 to monitor NO production was
investigated by treating it with S-nitroso-N-acetylpenicillamine
(SNAP).22,23 SNAP is a hindered nitroso thiol that exists as a
crystalline solid making it easy to weigh out and prepare a
Fig. 2 Excitation spectrum for coumarin 14 taken at emission
wavelength of 391 nm and emission spectrum taken at excitation
wavelength of 326 nm. Coumarin concentration 5 × 10Ϫ7 M.
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fluorescent products. Further studies evaluating the probes for
the determination of NO in cell cultures are in progress.
Experimental
Fluorescence measurements were made using a Perkin Elmer
Luminescence Spectrometer LS50B running with FL Winlab
software. The slit widths for excitation and emission were 5 nm.
9-(2-Aminophenyl)aminomethylanthracene, 1
o-Phenylenediamine (1.1 g, 10 mmol) was dissolved in dry pyri-
dine (50 ml). 9-(Chloromethyl)anthracene (0.57 g, 2.5 mmol)
was slowly added and the mixture refluxed overnight. The
solvent was evaporated to leave a green–brown residue. Purifi-
cation by column chromatography (dichloromethane–hexane
1 : 1) gave the title compound (0.45 g, 65%) as pale yellow
crystals, mp 178–180 ЊC (from hexane–ethyl acetate) (Found: C,
84.3; H, 6.1; N, 9.1. C21H18N2 requires C, 84.6; H, 6.0; N, 9.4%);
λmax(EtOH)/nm 290 (log ε 3.7), 349 (3.7), 367 (4.0) and 386
(3.9); νmax(KBr)/cmϪ1 339m, 3369m, 3326m, 1622s and 1594s;
δH (250 MHz; [D6]DMSO) 4.50 (2H, s, NH2), 4.62 (1H, s, NH),
5.06 (2H, s, CH2), 6.56 (2H, m, aryl), 6.65 (1H, m, aryl),
6.94 (1H, d, J 7.3, aryl), 7.54 (4H, m), 8.13 (2H, d, J 7.0), 8.27
(2H, d, J 7.0) and 8.63 (1H, s); δC (62.9 MHz; [D6]DMSO)
40.9, 110.2, 114.0, 117.5, 124.6, 125.2, 126.2, 127.2, 128.9,
130.3, 130.4, 131.2, 135.3 and 136.3 (one resonance is over-
lapping); m/z (CI) 299 (Mϩ ϩ H, 100%).
Fig. 5 Emission spectra for 9-(3,4-diaminophenyl)acridine 12 (50 µM)
in the presence of SNAP (50 µM). Excitation wavelength 360 nm.
Spectra were taken at 30 minute intervals starting at t = 0 for the lowest
trace.
9-(2-Amino-4,5-dimethylphenyl)aminomethylanthracene, 2
4,5-Dimethyl-o-phenylenediamine (1.0 g, 7.5 mmol) was dis-
solved in dry pyridine (20 ml). 9-Chloromethylanthracene
(0.46 g, 2 mmol) was slowly added and a red solution formed.
The reaction was heated to 80 ЊC for 3 h and allowed to stir at
room temperature for 3 days. The solvent was evaporated to
give a residue which was purified by chromatography on silica
gel. Elution with ethyl acetate gave the title compound (0.42 g,
65%) as pale yellow crystals, mp 203–205 ЊC (from ethyl
acetate–light petroleum) (Found: C, 84.9; H, 6.6; N, 8.2.
C23H22N2 requires C, 84.7; H, 6.8; N, 8.6%); λmax(EtOH)/nm
303 (log ε 3.7), 347 (3.7), 365 (4.0) and 384 (3.9); νmax(KBr)/cmϪ1
3548m, 3474m, 3413m, 3233m, 1637s, 1617s and 1517s; δC (62.9
MHz; [D6]DMSO) 18.7, 19.1, 40.9, 112.6, 116.4, 123.9, 124.0,
124.7, 125.2, 126.2, 127.1, 128.9, 130.3, 130.7, 131.2, 132.9 and
134.5; m/z (CI) 327 (Mϩ ϩ H, 100%).
Fig. 6 Emission spectra for diaminorhodamine 20 (10 µM) in the
presence of SNAP (50 µM). Excitation wavelength of 565 nm. Spectra
were taken at 30 minute intervals starting at t = 0 for the lowest trace.
standard solution for immediate use. Under these conditions
it probably acts as a source of NOϩ rather than generating
NO directly. Fig. 5 shows the increase in fluorescence over time
that is observed when equimolar quantities of probe 12 and
SNAP are mixed together in ethanol. It takes about 4 h for the
maximum fluorescence intensity to be reached. The slow nature
of the reaction suggests that probe 12 slowly reacts with the
S-nitroso group of SNAP which acts as a nitrosating agent
(a masked form of NOϩ). When an excess of SNAP was used
the maximum fluorescence value was the same, indicating that
the reaction of probe 12 with SNAP is high yielding. The
previously reported diaminorhodamine probe 2012 was syn-
thesised for comparison purposes. Fig. 6 shows the emission
spectra after treatment with SNAP. The reaction takes several
hours to go to completion as for probe 12. However, probe 20,
after reaction with SNAP, has a longer wavelength of emission
centered at 580 nm. These characteristics make it and also the
fluorescein based probes11 more suitable for certain types of
cell culture studies.
4-(2-Aminophenyl)aminomethyl-7-methoxycoumarin, 3
o-Phenylenediamine (1 g, 9 mmol) was dissolved in dry pyridine
(30 ml). 4-Bromomethyl-7-methoxycoumarin (0.54 g, 2 mmol)
was added slowly. A precipitate was seen within a few minutes.
The mixture was stirred overnight and then evaporated to a
gummy solid. The solid was purified by chromatography on
silica gel. Elution with ethyl acetate gave the title compound
(0.36 g, 62%) as a pink powder, mp 174–177 ЊC (from ethyl
acetate–light petroleum) (Found: C, 66.6; H, 5.3; N, 9.3.
C17H16N2O3 requires C, 66.9; H, 5.4; N, 9.5%); λmax(EtOH)/nm
317 (log ε 4.0); νmax(KBr)/cmϪ1 3494m, 3396m, 3321m, 1701m
and 1606m; δH (250 MHz; [D6]DMSO) 3.88 (3H, s, OMe), 4.51
(2H, s, CH2), 4.60 (2H, s, NH2), 5.18 (1H, m, NH), 6.17 (1H, s),
6.30–6.60 (4H, m), 6.98–7.04 (2H, m) and 7.88 (1H, d, J 8.6);
δC (62.9 MHz; [D6]DMSO) 43.5, 56.0, 100.9, 108.6, 110.4,
111.5, 112.2, 114.5, 117.6, 117.7, 125.9, 134.8, 135.4, 154.5,
155.0, 160.5 and 162.4; m/z (CI) 297 (Mϩ ϩ H, 100%).
4-(2-Amino-4,5-dimethylphenyl)aminomethyl-7-methoxy-
coumarin, 4
In summary, a series of o-phenylenediamine probes have
been prepared by short synthetic routes from readily available
starting materials. Preliminary studies show that they react with
the NO related species SNAP and nitrous fumes to give highly
1,2-Diamino-4,5-dimethylbenzene (1 g, 7.5 mmol) was dissolved
in dry pyridine (20 ml) and 4-bromomethyl-7-methoxycoumarin
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(0.54g,2mmol)slowlyadded.Aredsolutionandaredprecipitate
was rapidly formed. The suspension was stirred overnight. The
solvent was evaporated, the residue dissolved in CH2Cl2,
washed with H2O, dried with Na2SO4 and evaporated to a
brown solid. The product was purified by column chroma-
tography on silica gel. Elution with ethyl acetate gave the title
compound (0.19 g, 30%) as red needles, mp 191–192 ЊC (from
ethyl acetate) (Found: C, 69.9; H, 6.2; N, 8.3. C19H20N2O3
requires C, 70.4; H, 6.2; N, 8.7%); λmax(EtOH)/nm 317 (log
ε 4.0); νmax(KBr)/cmϪ1 3371m, 3359m, 3260m, 1717vs, 1607vs
and 1523s; δH (250 MHz; [D6]DMSO) 1.97 (3H, s, Me), 2.00
(3H, s, Me), 3.87 (3H, s, OMe), 4.37 (2H, s, NH2), 4.50 (2H,
d, CH2, J 4.9), 4.92 (1H, m, NH), 6.18 (2H, s), 6.42 (1H, s),
7.02 (2H, m) and 7.87 (1H, d, J 8.6); δC (62.9 MHz; [D6]-
DMSO) 18.6, 19.0, 43.7, 56.0, 100.9, 108.6, 111.6, 112.2, 112.5,
116.7, 124.2, 124.3, 125.8, 132.8, 133.1, 154.8, 155.0, 160.5 and
162.4; m/z (Found: Mϩ ϩ H, 325.1548. C19H21N2O3 requires
325.1552).
into ether (200 ml) and the hydrochloride salt precipitated and
filtered off. The salt was recrystallised from ethanol–ether to
give a red, orange or yellow powder. Yields 75–90%.
Coupling method 2. 3,4-Diaminobenzoic acid (2.3 g, 15
mmol) was added to a solution of conc. H2SO4 (6 ml) in ethanol
or methanol (60 ml). The mixture was heated to reflux for 5 h
and the ethanol evaporated to leave a thick purple–brown
liquid. The liquid was poured into ice–water and a purple
powder was collected by filtration. The powder was stirred in a
saturated solution of NaHCO3 turning a paler shade of purple.
The suspension was extracted with CH2Cl2, washed with water,
turning from brown to yellow and dried with Na2SO4. Evapor-
ation gave a pale brown powder. Yield 40–60%. The esterified
o-phenylenediamine (0.9 g, 5 mmol) was gently heated in freshly
distilled phenol (20 ml). 9-Chloroacridine (0.53 g, 2.5 mmol)
was slowly added and the mixture heated to reflux for 2 h.
After cooling the mixture was poured into ether (200 ml) and
a brown hygroscopic solid filtered off. Recrystallisation from
ethanol–ether gave the hydrochloride salt as a powder. Yield
80–90%.
4-(2-Aminophenyl)aminomethyl-6,7-dimethoxycoumarin, 5
o-Phenylenediamine (1 g, 9 mmol) was dissolved in dry pyr-
idine (30 ml). 4-Bromomethyl-6,7-dimethoxycoumarin (0.3 g,
1 mmol) was carefully added and the mixture stirred overnight.
During this period a precipitate appeared and the mixture
turned from yellow to brown. The solvent was evaporated to
give a brown solid. TLC analysis (EtOAc) showed two spots
very close to each other. Recrystallisation from ethyl acetate–
light petroleum selectively gave the title compound (0.16 g,
50%) as pale brown needles, mp 174–177 ЊC; λmax(EtOH)/nm
295 (log ε 3.7) and 342 (4.0); νmax(KBr)/cmϪ1 3385m, 3364m,
3307m, 3190m, 1702vs, 1612s and 1593s; δH (250 MHz;
[D6]DMSO) 3.86 (3H, s, OMe), 3.87 (3H, s, OMe), 4.59 (4H, m,
NH2 and CH2), 5.23 (1H, m, NH), 6.16 (1H, m), 6.35 (1H, m),
6.45 (2H, m), 7.10 (1H, s) and 7.34 (1H, s); δC (62.9 MHz;
[D6]DMSO) 43.6, 56.1, 56.1, 100.3, 105.7, 108.6, 110.4, 110.5,
114.6, 117.6, 117.7, 134.7, 135.5, 145.9, 149.0, 152.5, 154.6 and
160.8; m/z (Found: Mϩ ϩ H, 327.1351. C18H19N2O4 requires
327.1345).
9-(2-Aminoanilino)acridine hydrochloride, 7. Red powder
(1.32 g, 82%), mp >250 ЊC decomp. (from ethanol–ether) (lit.18
320–322 ЊC) (Found: C, 70.7; H, 4.8; N, 13.1. C19H16N3Cl
requires C, 70.9; H, 5.0; N, 13.1%); λmax(EtOH)/nm 289 (log
ε 4.0), 413 (4.1) and 433 (4.1); νmax(KBr)/cmϪ1 3553m, 3447m,
3413m, 1633vs, 1579s and 1549vs; δH (250 MHz; [D6]DMSO)
5.75 (2H, s, NH2), 6.74 (1H, m), 7.00 (1H, d, J 6.4), 7.17 (1H, d,
J 8.2), 7.29 (1H, t, J 6.7), 7.44 (2H, m), 8.01 (2H, m), 8.16 (2H,
d, J 8.2), 8.30 (2H, d, J 7.9), 11.26 (1H, s, NH) and 14.74 (1H, s,
NH); δC (62.9 MHz; [D6]DMSO) 113.1, 116.2, 116.6, 118.8,
123.3, 124.4, 125.8, 127.1, 129.6, 134.9, 139.9, 144.8 and 156.2;
m/z (CI) 286 (Mϩ ϩ H, 100%).
9-(2-Amino-4,5-dimethylanilino)acridine hydrochloride, 8.
Orange powder (1.29 g, 74%), mp >250 ЊC decomp. (from
ethanol–ether) (Found: C, 69.5; H, 5.8; N, 11.3. C21H20N3Cl
requires C, 72.1; H, 5.7; N, 12.1%); λmax(EtOH)/nm 294 (log
ε 4.0), 412 (4.1) and 433 (4.1); νmax(KBr)/cmϪ1 3402m, 3317m,
3209m, 2964m, 2870m, 1637vs, 1586s and 1557s; δH (250 MHz;
[D6]DMSO) 2.05 (3H, s, Me), 2.21 (3H, s, Me), 4.48 (1H, br s,
NH), 5.42 (1H, br s, NH), 6.75 (1H, s), 6.88 (1H, s), 7.37 (2H,
dd, J 7.9 and 7.6), 7.92 (2H, dd, J 7.9 and 7.6), 8.11 (2H, d,
J 8.2), 8.33 (2H, d, J 7.9), 11.18 (1H, br s, NH), 14.76 (1H, br s,
NH); δC (62.9 MHz; [D6]DMSO) 18.3, 19.5, 113.1, 117.4, 118.9,
122.2, 123.3, 124.5, 125.8, 127.2, 134.9, 137.5, 139.9, 142.1 and
156.0; m/z (Found: Mϩ ϩ H, 314.1660. C21H20N3 requires
314.1657).
4-(2-Amino-4,5-dimethylphenyl)aminomethyl-6,7-dimethoxy-
coumarin, 6
4,5-Dimethyl-1,2-phenylenediamine (0.54 g, 4 mmol) was dis-
solved in dry pyridine (40 ml). 4-Bromomethyl-6,7-dimethoxy-
coumarin (0.3 g, 1 mmol) was added slowly and the mixture
left to stir overnight. A precipitate was seen within 10 minutes.
The solvent was evaporated to give an orange gum. This was
dissolved in CH2Cl2, washed with water and dried with Na2SO4.
Evaporation gave a crystalline solid. The product was purified
by chromatography on silica gel. Elution with ethyl acetate gave
the title compound (0.11 g, 30%) as a yellow solid; mp 208–210
ЊC (from ethyl acetate–light petroleum) (Found: C, 66.6;
H, 6.0; N, 7.4. C20H22N2O4 requires C, 67.8; H, 6.2; N, 7.9%);
λmax(EtOH)/nm 341 (log ε 4.0); νmax(KBr)/cmϪ1 3396m, 3344m,
1703vs, 1611s, 1566s and 1519s; δH (250 MHz; [D6]DMSO) 1.96
(3H, s), 1.99 (3H, s), 3.87 (6H, s, OMe), 4.36 (2H, s, NH2), 4.54
(2H, d, J 5.2), 4.97 (1H, t, NH, J 6.41), 6.16 (2H, d, J 7.63), 6.41
(1H, s), 7.09 (1H) and 7.32 (1H, s); δC (62.9 MHz; [D6]DMSO)
18.6, 19.00, 43.8, 56.2, 56.2, 100.2, 105.7, 108.6, 110.4, 112.5,
116.7, 124.1, 124.3, 132.7, 133.1, 145.8, 148.96, 152.5, 154.9 and
160.8; m/z (Found: Mϩ ϩ H, 355.1653. C20H23N2O4 requires
355.1658).
9-(2-Amino-5-ethoxycarbonylanilino)acridine hydrochloride,
9. Dark brown powder (1.42 g, 72%), mp >250 ЊC decomp.
(from ethanol–ether) (Found: C, 66.1; H, 5.3; N, 9.7.
C22H20ClN3O2 requires C, 67.1; H, 5.1; N, 10.7%); λmax(EtOH)/
nm 293 (log ε 4.0), 414 (4.1) and 436 (4.1); νmax(KBr)/cmϪ1
3429m, 3329m, 3195m, 2975m, 2892m, 2796m, 1686vs, 1638s,
1599s, 1585s and 1555s; δH (250 MHz; [D6]DMSO) 1.22 (3H,
t, J 7.0), 4.17 (2H, q, J 7.0), 6.52 (2H, br s, NH2), 6.94 (1H, d,
J 8.6), 7.40 (2H, t, J 7.0), 7.73 (1H, d, J 2.1), 7.80 (1H, dd, J 8.6
and 1.8), 7.95 (2H, t, J 7.0), 8.12 (2H, d, J 8.6), 8.29 (2H, d,
J 8.8), 11.24 (1H, br s, NH), 14.80 (1H, br s, NH); δC (62.9
MHz; [D6]DMSO) 14.3, 60.0, 113.1, 114.8, 116.8, 118.8,
123.3, 123.5, 125.5, 129.0, 131.0, 135.0, 139.8, 149.4, 156.2 and
165.2; m/z (Found: Mϩ ϩ H, 358.1551. C22H20N3O2 requires
358.1555).
General methods for the preparation of acridines
Coupling method 1. The phenylenediamine (1.2 g, 11 mmol)
was dissolved in dry methanol (50 ml) and heated to reflux.
9-Chloroacridine (1.1 g, 5 mmol)19 was slowly added and the
mixture refluxed for 1 h. After cooling the mixture was poured
9-(2-Aminoanilino)-2-chloro-6-methoxyacridine
chloride, 10. Yellow powder (1.76 g, 91%), mp >250 ЊC decomp.
(from ethanol–ether); λmax(EtOH)/nm 295 (log ε 4.1), 329 (3.4),
hydro-
J. Chem. Soc., Perkin Trans. 1, 2001, 2553–2559
2557

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344 (3.5), 430 (4.0) and 448 (4.0); νmax(KBr)/cmϪ1 3415m,
3348m, 3230m, 2780m, 1632vs, 1583s and 1559s; δH (250 MHz;
[D6]DMSO) 3.65 (3H, s, OMe), 6.66 (1H, dd, J 7.9 and 7.0),
6.94 (1H, d, J 7.9), 7.11 (1H, d, J 7.0), 7.21 (1H, dd, J 7.9 and
7.0), 7.38 (1H, d, J 9.5), 7.68 (1H, d, J 9.2), 7.70 (1H, s), 8.00
(1H, d, J 9.2), 8.10 (1H, s) and 8.19 (1H, d, J 9.5); δC (62.9
MHz; [D6]DMSO) 55.7, 103.8, 111.4, 114.3, 116.0, 116.6, 117.4,
120.5, 123.6, 124.2, 127.3, 127.3, 127.9, 129.6, 135.5, 138.7,
139.5, 144.8, 154.4 and 155.3; m/z (Found: Mϩ ϩ H, 350.1059.
C20H17ClN3O requires 350.1060).
(2H, d, J 8.2) and 8.54 (1H, s); δC (62.9 MHz; CDCl3) 46.0,
110.3, 115.0, 119.9, 123.4, 123.8, 125.3, 126.1, 127.3, 127.5,
129.6, 129.9, 131.1, 131.4 and 146.4; m/z (CI) 310 (Mϩ ϩ H,
100%).
4-(Benzotriazol-1-yl)methyl-7-methoxycoumarin, 14. NaH
(0.67 g, 16.8 mmol, 60% suspension in oil) was stirred in light
petroleum (2 × 40 ml). The ether was decanted and dry DMF
was added (50 ml). Benzotriazole (2 g, 16.8 mmol) was slowly
added with vigorous stirring. Once effervescence had ceased,
4-bromomethyl-7-methoxycoumarin (0.54 g, 2 mmol) was care-
fully added. The mixture was stirred overnight. Water was
added to the mixture and the product extracted into ether.
The ether was dried with Na2SO4 and evaporated to a runny
liquid. Recrystallisation gave the title compound (0.12 g, 20%) as
fine needles, mp 212 ЊC (from ethyl acetate–light petroleum)
(Found: C, 66.0; H, 4.2; N, 13.4. C17H13N3O3 requires C, 66.5;
H, 4.2; N, 13.7%); λmax(EtOH)/nm 324 (log ε 4); νmax(KBr)/cmϪ1
1713vs and 1619vs; δH (250 MHz; [D6]DMSO) 3.86 (3H, s,
OMe), 5.52 (1H, s, coumarin -CH), 6.33 (2H, s, coumarin
-CH2), 7.04 (2H, m), 7.45 (1H, dd, J 8.2 and 7.0), 7.59 (1H, dd,
J 8.2 and 7.0), 7.91 (2H, m) and 8.11 (1H, d, J 8.2); δC (62.9
MHz; [D6]DMSO) 47.6, 56.1, 101.1, 109.9, 110.6, 112.4, 119.5,
124.5, 126.2, 128.1, 133.2, 145.2, 150.2, 155.2, 159.7 and 162.8
(one overlapping resonance); m/z (Found: Mϩ ϩ H, 308.1035.
C17H14N3O3 requires 308.1035).
9-(2-Amino-5-ethoxycarbonylanilino)-2-chloro-6-methoxy-
acridine, 11. Yellow powder (1.9 g, 84%), mp >250 ЊC decomp.
(from ethanol–ether); λmax(EtOH)/nm 291 (log ε 4.1), 344 (3.4)
and 428 (4.0); νmax(KBr)/cmϪ1 3369m, 3296m, 3201m, 3173m,
1705vs, 1629s, 1606s, 1581s and 1547s; δH (250 MHz; [D6]-
DMSO) 1.23 (3H, t, J 7.3, Et), 3.70 (3H, s, OMe), 4.18 (2H, q,
J 7.3, Et), 6.49 (1H, d, J 8.6), 7.90 (4H, m), 8.02 (1H, d, J 8.6)
and 8.10 (3H, m); δC (62.9 MHz; [D6]DMSO) 14.3, 55.7,
60.1, 103.6, 111.4, 114.5, 114.8, 116.9, 117.5, 120.6, 123.3,
123.9, 127.3, 127.9, 129.1, 130.9, 135.5, 138.8, 139.6, 149.4,
154.5, 155.5 and 165.2; m/z (Found: Mϩ ϩ H, 422.1280.
C23H21ClN3O3 requires 422.1271).
9-(3,4-Diaminophenyl)acridine (DAA), 12. Acridine was dis-
solved in ethanol and anhydrous HCl passed through the solu-
tion for 5 min. Most of the solvent was removed under vacuum
and the remainder of the solution left to stand at 4 ЊC. The
hydrochloride precipitated as pale brown crystals. The hydro-
chloride (5.7 g, 26 mmol) and o-phenylenediamine (5.7 g,
53 mmol) were placed in a flask with sulfur (2.5 g, 78 mmol) and
the mixture of solids slowly heated to 130 ЊC with vigorous
stirring. The mixture turned black and formed a tar with
stirring no longer possible. H2S was given off. Heating was
continued for 1 h by which time the mixture had thinned and
stirring was again possible. The melt was cooled and washed
with ether (2 × 50 ml). The black solid was extracted with 10%
HCl (3 × 100 ml) to give a dark brown solution. The solution
was basified with NH3 (aq) and a brown solid precipitated. The
solid is filtered and dried under vacuum at 40 ЊC to give yellow
brown crystals (4.8 g, 65%), mp >260 ЊC (from xylene) (lit.20
279–281 ЊC) (Found: C, 79.8; H, 5.3; N, 14.7. C19H15N3 requires
C, 80.0; H, 5.3; N, 14.7%); λmax(EtOH)/nm 332 (log ε 3.7),
358 (3.8); νmax(KBr)/cmϪ1 3419s, 3357s, 1618vs, 1577m, 1515vs;
δH (250 MHz; [D6]DMSO) 4.78 (4H, s, NH2), 6.51 (1H, dd,
J 7.6 and 2.1), 6.65 (1H, d, J 2.1), 6.78 (1H, d, J 7.6), 7.50 (2H,
ddd, J 8.5, 7.6 and 1.2), 7.80 (1H, dd, J 6.4 and 1.2), 7.82 (1H,
dd, J 6.4 and 1.2), 7.89 (2H, d, J 8.5) and 8.16 (2H, d, J 8.5);
δC (62.9 MHz; [D6]DMSO) 114.1, 116.5, 119.8, 123.2, 124.9,
125.4, 127.3, 129.2, 130.0, 134.8, 135.5, 148.4 and 148.7;
m/z 286 (Mϩ ϩ H, 100%).
Diazotisation reactions
The aminoanilino acridine 7–11 (0.8 g, 2.5 mmol) was
suspended in 2 M HCl (25 ml) and chilled in an ice bath. A
solution of NaNO2 (0.35 ml, 5 mmol) was added dropwise and
the mixture stirred for 1 h. The suspension was cooled with
ice and made basic with conc. aq. NH3. At neutralisation it
became notably easier to stir. A brown powder was collected
by filtration. The wet solid was dissolved in hot DMF and
crystallisation occurred upon cooling. A pale yellow powder
was collected by filtration. Yield 85–95%.
9-(Benzotriazol-1-yl)acridine, 15. Pale yellow powder (0.67 g,
90%), mp 250 ЊC decomp. (from DMF–water); lit.18 250 ЊC
(Found: C, 76.9; H, 4.0; N, 19.0. C19H12N4 requires C, 77.0;
H, 4.0; N, 18.9%); λmax(EtOH)/nm 289 (log ε 3.8), 345 (4.0),
362 (4.1) and 386 (4.0); νmax(KBr)/cmϪ1 3414m, 3048m,
1676s, 1629s, 1607s and 1554vs; δH (250 MHz; CDCl3) 7.07
(1H, d, J 6.7), 7.33 (2H, d, J 8.8), 7.50 (4H, m), 7.8 (2H, t,
J 7.9), 8.31 (1H, m) and 8.39 (2H, d, J 8.8); δC (62.9 MHz;
CDCl3) 110.1, 120.5, 122.8, 124.8, 128.0, 128.9, 130.0, 130.9,
135.6, 136.6, 144.0, 145.6 and 149.6; m/z (CI) 297 (Mϩ ϩ H,
100%).
9-(5,6-Dimethylbenzotriazol-1-yl)acridine, 16. Pale yellow
powder (0.69 g, 85%), mp 233–237 ЊC (from DMF–water)
(Found: C, 77.6; H, 4.8; N, 17.1. C21H16N4 requires C, 77.8; H,
4.9; N, 17.3%); λmax(EtOH)/nm 291 (log ε 3.8), 346 (4.0), 361
(4.1) and 385 (4); νmax(KBr)/cmϪ1 3548m, 3475m, 3414m,
1618vs, 1557s and 1517s; δH (250 MHz; CDCl3) 2.28 (3H, s,
Me), 2.46 (3H, s, Me), 6.82 (1H, s), 7.34 (2H, d, J 8.5), 7.48 (2H,
m), 7.84 (2H, m), 8.02 (1H, s) and 8.37 (2H, d, J 8.8); δC (62.9
MHz; CDCl3) 20.5, 20.9, 109.4, 119.5, 122.8, 122.9, 127.9,
130.0, 130.8, 134.8, 139.0 and 149.6; m/z (CI) 325 (Mϩ ϩ H,
100%).
9-(Benzotriazol-1-yl)methylanthracene, 13. Sodium hydride
(0.67 g, 16.7 mmol, 60% suspension in oil) was stirred in light
petroleum (2 × 40 ml). The solvent was decanted and dry DMF
(50 ml) added. Benzotriazole (2 g, 16.8 mmol) was slowly added
with vigorous stirring. Once effervescence had ceased, 9-
chloromethylanthracene (0.23 g, 1 mmol) was carefully added.
The mixture was stirred overnight. Water was added to the
mixture and the product extracted into ether. The product was
purified by chromatography on silica gel. Elution with dichloro-
methane give the title compound (0.13 g, 42%) as a yellow
powder, mp 156–158 ЊC (from ethyl acetate–light petroleum)
(Found: C, 80.1; H, 5.0; N, 14.0. C21H15N3 requires C, 81.5;
H, 4.8; N, 13.6%); λmax(EtOH)/nm 289 (log ε 3.7), 348 (3.7), 366
(4.0) and 386 (3.9); νmax(KBr)/cmϪ1 3552m, 3472m, 3418m,
1638s, 1617s, 1585s and 1572s; δH (250 MHz; [D6]DMSO) 6.80
(1H, d), 6.81 (2H, s, CH2), 7.03 (1H, t, J 8.2), 7.16 (1H, t, J 8.2),
7.46–7.60 (4H, m), 7.96 (1H, d, J 8.2), 8.04 (2H, d, J 7.6), 8.47
9-(6-Ethoxycarbonylbenzotriazol-1-yl)acridine,
17.
Pale
yellow powder (0.81 g, 88%), mp 200–202 ЊC (from DMF–
water) (Found: C, 71.2; H, 4.1; N, 14.9. C22H16N4O2 requires C,
71.7; H, 4.3; N, 15.2%); λmax(EtOH)/nm 300 (log ε 4.0), 346
(4.1), 362 (4.1) and 386 (4.1); νmax(KBr)/cmϪ1 3546m, 3478m,
3416m, 3055m, 2993m, 1709vs, 1628vs and 1554s; δH (250
MHz; CDCl3) 1.27 (3H, t, J 7.3), 4.29 (2H, q, J 7.3), 7.25
2558
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(2H, d, J 8.8), 7.50 (2H, dd, J 8.6 and 7.7), 7.77 (1H, s), 7.86
(2H, ddd, J 7.9, 6.7 and 1.2), 7.98 (1H, s), 8.18 (1H, dd, J 8.8
and 1.2), 8.34 (1H, d, J 8.8) and 8.39 (1H, d, J 8.8); δC (62.9
MHz; CDCl3) 14.2, 61.7, 112.3, 120.4, 122.3, 122.8, 125.6,
128.4, 130.1, 130.9, 131.2, 135.4, 147.5, 149.6 and 165.6;
References
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m/z (Found: Mϩ
ϩ H, 369.1355. C22H17N4O2 requires
369.1351).
9-(Benzotriazol-1-yl)-2-chloro-6-methoxyacridine, 18. Green–
blue powder (0.8 g, 89%), mp 207–209 ЊC (from DMF–water)
(Found: C, 66.3; H, 3.8; N, 15.2. C20H13ClN4O requires C, 66.6;
H, 3.6; N, 15.5%); λmax(EtOH)/nm 290 (log ε 3.8), 338 (3.8), 358
(4.0), 389 (4.0) and 408 (4.0); νmax(KBr)/cmϪ1 3555m, 3469m,
3412m, 3056m, 1632vs and 1560s; δH (250 MHz; CDCl3) 3.63
(3H, s, OMe), 6.39 (1H, d, J 2.4), 7.07 (1H, m), 7.22 (1H, t,
J 9.2), 7.41 (1H, dd, J 9.2 and 1.8), 7.52 (3H, m), 8.23 (1H, d,
J 9.5) and 8.30 (2H, m); δC (62.9 MHz; CDCl3) 55.6, 97.7,
110.1, 120.6, 121.4, 123.7, 124.0, 125.0, 127.0, 128.5, 129.0,
129.3, 131.7, 134.4, 135.0, 135.6, 145.7, 147.6, 147.8 and 159.2;
m/z (CI) 361 (Mϩ ϩ H, 100%).
9-(6-Ethoxycarbonylbenzotriazol-1-yl)-2-chloro-6-methoxy-
acridine, 19. Pale yellow powder (1.0 g, 93%), mp 228–230 ЊC
(from DMF–water) (Found: C, 63.2; H, 3.9; N, 12.8. C23H17-
ClN4O3 requires C, 63.8; H, 3.9; N, 13.0%); λmax(EtOH)/nm
291 (log ε 3.8), 338 (3.8), 354 (4.0), 389 (4.0) and 409 (4.0);
νmax(KBr)/cmϪ1 3408m, 3067m, 3040m, 2991m, 2968m, 2932m,
2906m, 2830m, 1713vs, 1676vs, 1632s and 1559s; δH (250 MHz;
CDCl3) 1.31 (3H, t, J 7.3, Et), 3.62 (3H, s, OMe), 4.33 (2H, q,
J 7.3, ethyl), 6.30 (1H, d, J 2.4, benz), 7.10 (1H, d, J 9.2, acr),
7.41 (1H, dd, J 9.2 and 2.0, benz), 7.53 (1H, dd, J 9.5 and 2.4,
benz), 7.78 (1H, s, acr), 8.22 (2H, m, acr) and 8.35 (2H, m, acr);
δC (62.9 MHz; CDCl3) 14.3, 55.7, 61.8, 112.2, 120.5, 121.5,
123.2, 124.2, 125.7, 127.1, 128.7, 129.6, 131.3, 131.8, 134.9,
135.9, 147.6, 147.8, 151.3 and 159.4; m/z (Found: Mϩ ϩ H,
433.1063. C23H18ClN4O3 requires 433.1067).
Acknowledgements
We are grateful to the Oliver Bird Fund of the Nuffield
Foundation for financial support and to the National Mass
Spectrometry Service Centre for mass spectra.
23 J. E. Shaffer, B. J. Han, W. H. Chern and F. W. Lee, J. Pharmacol.
Exp. Ther., 1992, 260, 286.
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2559