Synthesis and properties of bioactive 2- and 3-amino-8-methyl-8H-quino[4,3,2-kl]acridine and 8,13-dimethyl-8H-quino[4,3,2-kl]acridinium salts.

Abstract

Cyclisation of 9-(benzotriazol-1-yl)acridine to the pentacycle 8H-quino[4,3,2-kl]acridine in a range of low-boiling solvents is mechanistically distinct from previously published photochemical (carbene) and thermolytic (radical) cyclisations. Fragmentation of the triazole ring of to a diazonium intermediate, and its subsequent heterolysis (-N(2)) and cyclisation is facilitated by solvation of intermediate zwitterionic species. Derivatives of 2- and 3-aminoquinoacridines methylated in the 8-position can be converted to 8,13-dimethylquino[4,3,2-kl]acridinium iodide salts with methyl iodide and were required for biological examination as potential telomerase inhibitors. The chloro group in 3-chloro-8-methyl-8H-quino[4,3,2-kl]acridine can be replaced efficiently by benzylamino, 4-morpholinyl and cyano substituents in palladium(0) mediated reactions.

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Synthesis and properties of bioactive 2- and 3-amino-8-methyl-8H-
quino[4,3,2-kl]acridine and 8,13-dimethyl-8H-quino[4,3,2-kl]-
acridinium salts†
Ian Hutchinson, Andrew J. McCarroll, Robert A. Heald and Malcolm F. G. Stevens*
Cancer Research Laboratories, School of Pharmacy, University of Nottingham, Nottingham,
UK NG7 2RD
Received 4th September 2003, Accepted 7th November 2003
First published as an Advance Article on the web 10th December 2003
Cyclisation of 9-(benzotriazol-1-yl)acridine 1 to the pentacycle 8H-quino[4,3,2-kl]acridine 5 in a range of low-boiling
solvents is mechanistically distinct from previously published photochemical (carbene) and thermolytic (radical)
cyclisations. Fragmentation of the triazole ring of 1 to a diazonium intermediate 7, and its subsequent heterolysis
(–N2) and cyclisation is facilitated by solvation of intermediate zwitterionic species. Derivatives of 2- and 3-amino-
quinoacridines methylated in the 8-position can be converted to 8,13-dimethylquino[4,3,2-kl]acridinium iodide salts
with methyl iodide and were required for biological examination as potential telomerase inhibitors. The chloro group
in 3-chloro-8-methyl-8H-quino[4,3,2-kl]acridine can be replaced efficiently by benzylamino, 4-morpholinyl and cyano
substituents in palladium() mediated reactions.
Introduction
9-(Benzotriazol-1-yl)acridine 1 is a convenient precursor for the
synthesis of the parent 8H-quino[4,3,2-kl]acridine ring-system
5. Mitchell and Rees reported a photochemical conversion of
1
5 in acetonitrile and proposed that an intermediate carbene
2 (derived initially from a triplet diradical) in the guise of
its “dipolar form” 4 cyclised to the pentacycle 5 (Scheme 1).2
We have previously probed the parallel thermal (Graebe–
Ullmann)3 transformation 1 5 by differential scanning calor-
imetry.4 The coincident melting and exothermic degradation
(–N2) of 1 occurs near explosively at 245.6 ?C. The reaction can
be controlled on a preparative scale in boiling diphenyl ether
(bp 259 ?C) and we have proposed that a diradical reactive
intermediate 3 is involved in these cases.4 The same quino-
acridine 5 can be prepared from 9-(2-bromoanilino)acridine 6
with tributyltin hydride–AIBN in boiling toluene in a process
that is undoubtedly radical in character.4,5
Surprisingly, a reinvestigation of the thermolysis of 1
exposed a new twist to these mechanistic interpretations: the
degradation of 1 in 98% yield in boiling triglyme (216 ?C), and
† Part 15 in the series: Antitumour polycyclic acridines. See ref. 1 for
part 14.
yields of 98% in ethanol (78 ?C) and 95% in methanol (65 ?C),
cannot be accommodated reasonably within the diradical hypo-
thesis. In this paper we propose a new mechanism to account
for the low temperature conversion 1
this route to prepare 2-nitro- and 3-chloro-quino[4,3,2-kl]-
acridines which can be further processed to a range of more
interesting and bioactive pentacyclic quinoacridinium salts. In
particular, to further progress our search for potent telomerase
inhibitors of the quadruplex DNA-stabilising class,1,6,7 we
sought methods for making 8,13-dimethylquinoacridinium
salts with amino or acylamino substituents in the 2- and
3-positions.
5 and have adopted
Chemistry
Degradation of 9-(benzotriazol-1-yl)acridine
Results of the thermolysis of 1 in a range of solvents are
recorded in Table 1. Whereas thermolysis in boiling diphenyl
ether and triglyme was essentially complete and high-yielding
in 1 or 2 hours, respectively, no reaction took place after
prolonged boiling in diglyme (bp 162 ?C). Most unexpected
were results obtained with a range of alcohols: although a
satisfactory yield of 5 was obtained with the primary alcohol
Table 1
Yields of 8H-quino[4,3,2-kl]acridine 5 from the thermolysis of 9-(benzotriazol-1-yl)acridine 1 in boiling solventsa
SolventTemperature/?C Reaction time/hYield (%)b
Methanol
Ethanol
Trifluoroethanol
Propan-1-ol
Propan-2-ol
Butan-1-ol
Butan-2-ol
Diglyme
Triglyme
Diphenyl ether
Benzene
Toluene
Triethylamine
Pyridine
Dimethylformamide
65
78
88
97
82
36
24
72
24
29
24
24
24
95
98
0
Tracec
50
90
Tracec
0
98
97
0
0
80
60
80
117
98
162
216
259
80
110
89
115
156
2
1
48
48
24
24
24
aReactions conducted on a 0.5 g scale. bDetermined by 1H NMR. cIdentified by TLC.
220
DOI: 10.1039/ b310796p
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 2 2 0 – 2 2 8
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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butan-1-ol, only trace amounts of pentacycle 5 were formed
from butan-2-ol and propan-1-ol; and no conversion was
observed in trifluoroethanol. Contrary to expectations, clean
conversion 1
5 was evident in the lower boiling alcohols
ethanol and methanol. Attempts to catalyse the reaction in
alcohols with acids (acetic, TFA, HCl gas) supressed the desired
reaction since acridine ring protonation renders the benzo-
triazolyl residue vulnerable to nucleophilic displacement by
the alcohol.4 To complete the picture, thermolyses of 1 in the
bases triethylamine and pyridine and in DMF also afforded 5
but no conversion was observed in boiling non-polar solvents
toluene and benzene (Table 1).
The presence of both acridine and benzotriazolyl moieties
are essential to facilitate degradation of 1 since no cyclisation
was observed when 9-(1,2,3-triazol-1-yl)acridine or 1-phenyl-
1,2,3-benzotriazole were refluxed in low-boiling alcohols. A
possible mechanism involves the formation of a diazonium
species 7 (Scheme 2) which can cyclise to quinoacridine 5 via
zwitterion 4 and the carbenium ion reactive species 8, in a
process showing affinities to the intramolecular arylation by
diazonium compounds (Pschorr cyclisation),8 but without
the necessity for copper catalysis. A combination of several
factors – the propensity of benzotriazole to undergo N–N bond
cleavage, resonance stabilisation, solvation of cationic reactive
Scheme 1
(iii) Bu3SnH, AIBN, toluene, reflux.
Reagents and conditions: (i) hv, acetonitrile; (ii) heat, 259 ?C;
species – must all contribute to the initiation of heterolytic
fission of the benzotriazole. Because the transformation 1
does not proceed in boiling acetonitrile alone, this third process
is mechanistically distinct from that proposed for the photo-
chemical transformation in acetonitrile by Mitchell and Rees2
and also from the diradical (Graebe–Ullmann) process which
occurs thermally at 245.6 ?C. We admit that our interpretation
of this process (Scheme 2) doesn’t fully explain the puzzling
results in Table 1, particularly the apparent ‘all-or-nothing’
effect in the range of alcohols studied. Further mechanistic
nuances of this reaction may be revealed by a more detailed
investigation.‡
5
Synthesis and chemistry of 2-aminoquino[4,3,2-kl]acridine
9-(6-Nitrobenzotriazol-1-yl)acridine 9, prepared by nitrosation
of 9-(2-amino-5-nitroanilino)acridine,4 was thermolysed most
efficiently in boiling triglyme in which it is soluble; the product,
2-nitroquinoacridine 10 (95%), was precipitated with water.
Because of the insolubility of 9 in boiling ethanol or methanol,
formation of 10 was very slow in these solvents. (This conver-
sion has been effected previously in 65% yield in boiling
diphenyl ether.4)
Methylation of 10 with NaH–dimethyl sulfate gave the
8-methyl-2-nitroquinoacridine 11 which was reduced to the
corresponding amine 12 with stannous chloride in concen-
trated hydrochloric acid. Acylation of 12 with a range of
acid anhydrides or acid chlorides furnished the amides 13a–g
which were then processed to the 8,13-dimethylquinoacrid-
inium iodide salts 14a–g with methyl iodide at 150 ?C for 2 days
(Scheme 3). Attempts to deacetylate 14a in aqueous sodium
hydroxide led to demethylation at the 13-position and the
isolation of minor yields of the amide 13a and its precursor
amine 12.
The structure of amine 12 was confirmed using COSY and
NOESY 2D NMR. The proton at δ 6.99 shows only a small
meta-coupling (2.2 Hz) with the proton at 6.88, indicating that
Scheme 2
Reagents and conditions: solvent (see Table 1), reflux.
‡ We thank Professor C. W. Rees for helpful discussion on this
mechanism.
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these protons are in the C-1 and C-3 positions respectively. (For
numbering of the quinoacridine ring see structure 5, Scheme 1.)
The COSY spectrum also revealed the three coupling networks
present: 6.99–6.88–8.19, 7.87–7.71–7.12, and 7.5*–7.5*–7.22–
8.72. (The asterisks indicate that these protons had signals
which overlapped, and were unresolved.) When the proton at
δ 8.19 was irradiated in an NOE difference experiment, the
protons at 6.88 and 7.87 displayed positive NOEs, hence the
assignment of the proton at 7.87 to the 5-position. Protons at
δ 7.12 and 7.55 displayed positive NOEs when the N-methyl
Scheme 3
(ii) NaH, Me2SO4, in DMF, 25 ?C, 1 h; (iii) SnCl2?2H2O, 10 M HCl,
25 ?C, 5 d; (iv) R1COCl, NEt3, DCM, cat DMAP, 0 ?C; or (R1CO)2O,
pyridine, reflux, 1 h; (v) NaNO2, 2 M HCl; (vi) MeI, 150 ?C, 2 d;
(vii) KI, 80 ?C, 0.5 h.
Reagents and conditions: (i) Triglyme, 216 ?C, 2 h, then H2O;
group at δ 3.66 was irradiated, confirming the assignment of the
proton at C-7. These assignments are in good agreement with
those of other 8-methylquinoacridines.9
Nitrosation of amine 12 gave the diazonium salt 15 which
was converted directly to the 2-iodoquinoacridine 16 by a
Sandmeyer reaction and subsequently converted to the methio-
dide salt 17 (Scheme 3).
Synthesis of 2-amino-8,13-dimethylquinoacridinium salts 20
was best approached from 12 through the Boc-protected amine
18 which was methylated under mild conditions (80 ?C for
7 days) with methyl iodide to give the protected aminoquino-
acridinium salt 19. Deprotection of 19 with aqueous HCl gave
the chloride salt 20a after anion exchange; the same product
was formed less efficiently by hydrolysis of the acetamide 14a in
HCl (Scheme 4). Methylation of 18 under ‘normal’ conditions
(methyl iodide at 150 ?C) led to removal of the Boc group and
formation of a mixture of the quaternary iodides 20b–d which
could be separated, with difficulty, by column chromatography.
Synthesis and chemistry of 3-aminoquino[4,3,2-kl]acridine
The 3-chloro-8-methyl-quinoacridine 23 was identified as a
suitable starting precursor for palladium() mediated conver-
sion to quinoacridines 25 and 26 bearing nitrogen-containing
functionalities in the 3-position. 9-(5-Chlorobenzotriazol-1-
yl)acridine 21 was thermolysed efficiently in boiling triglyme to
furnish the 3-chloroquinoacridine 22 (95%) which was methyl-
ated (NaH–dimethyl sulfate) to give 23 (Scheme 5). Altern-
atively the N-methyl-acridone 24, which has been prepared
from 1-bromo-10-methylacridone by a cross-coupling route,
can be cyclised to the same quinoacridine 23 in POCl3.1 Coup-
ling between 23 and benzylamine was accomplished in the
presence of Pd2(dba)3 and P(t-Bu)3 in dioxane. The benzyl-
amine 25a was debenzylated with hydrazine hydrate in the
presence of Pd/C catalyst in methanol to yield the 3-amino-
quinoacridine 25b which was acetylated in acetic anhydride–
pyridine to furnish the acetamide 25c. Similar coupling between
23 and morpholine gave the 3-morpholinyl-quinoacridine 25d
and cyanation of 23 was effected under Jin–Confalone condi-
tions.10 The 3-cyanoquinoacridine 25e (98% yield) (νmax C???N,
2215 cm?1) offered the prospect of further conversion to a
3-(aminomethyl)quinoacridine 25f but this reduction could
not be effected with LiAlH4, catalytic hydrogenation over
palladium–charcoal, or SmI2 electron transfer reduction.
Finally, acetamide 25c was converted to the quaternary iodide
salt 26 with methyl iodide at 150 ?C.
Because of the difficulty in gaining reliable CHN micro-
analysis on these polycyclic acridines which co-crystallise with
variable proportions of solvents, a feature of this class of com-
pound,1,4 all compounds were characterised by 1H NMR and
HRMS. Prior to biological evaluation compounds were shown
to be single entities by TLC.
Biological results
Several 2-substituted quino[4,3,2-kl]acridines were tested for
growth-inhibitory activity11 against human cancer cells in the
US National Cancer Institute NCI in vitro assay (Table 2).
Results showed, that against 60 cell types, the mean growth-
inhibitory activities (mean GI50 values) varied over a >2 log
range. The most potent agents were the 8,13-dimethyl-8H-
quino[4,3,2-kl]acridinium iodide salts 20c and 20d with GI50
values <1 µM and selective activities against cells of the colon
and melanoma sub-panels (data not shown). However, these
2-day drug exposure assay results disguise other valuable
mechanistic information which can be gleaned from the
information-rich screen by COMPARE analysis. COMPARE is
a computerised pattern-recognition algorithm used to analyse
information generated by the NCI screen12 and is a method for
determining and expressing the degree of similarity, or lack
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Scheme 4
Reagents and conditions: (i) Boc anhydride, NaHCO3, THF; (ii) MeI, 80 ?C, 7 d; (iii) MeI, 150 ?C, 48 h; (iv) 2 M HCl, MeOH.
Scheme 5
Pd2(dba)3, NaOtBu, dioxane, 120 ?C, 24 h; (v) NH2NH2?H2O, Pd on C, MeOH, 65 ?C, 4 h; (vi) Ac2O, pyridine, 100 ?C, 2 h; (vii) MeI, 150 ?C, 3 d.
Reagents and conditions: (i) triglyme, 216 ?C, 2 h, then H2O; (ii) NaH, Me2SO4, in DMF, 25 ?C, (iii) see ref. 1; (iv) for 25a, PhCH2NH2,
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Table 2
60 cell panela
Growth inhibitory activity of 2-substituted quino[4,3,2-kl]acridines against human cancer cell lines in the National Cancer Institute (USA)
CompoundStructural typeRR1
Mean GI50b/µM
4
A
A
A
A
A
B
B
B
B
B
B
B
H
NO2
NHAc
I
NHCOO-tert-Bu
NHAc
NHCOCF3
NHCO-tert-Bu
NHCO(CH2)4CO2Me
NHCOPh
NHMe
NMe2
H
H
Me
Me
Me
—
—
—
—
—
—
—
1.48
3.89, 2.34
5.62
89.1
4.27, 4.27
12.0
2.19, 1.78
3.31
10.5, 6.03
6.61, 4.37
0.57, 0.46, 0.43
0.32, 0.26, 0.20
10
13a
16
18
14a
14b
14d
14e
14f
20c
20d
aSee ref. 11 for methods. bConcentration to produce 50% growth inhibition expressed as a mean over 60 cell lines.
thereof, of mean graph profiles generated from structurally
similar (or disparate) compounds. Agents with matching
response fingerprints and Pearson Correlation Coefficients
(PCCs) > 0.6 can be deduced as having similar biological mech-
anisms; and the higher the PCC value the greater the confidence
in this interpretation. Compounds 13a, 14a, 20c and 20d were
used to explore these relationships. Thus when the neutral
2-acetylamino-quinoacridine 13a was employed as ‘seed’ (PCC
1.00), cross PCC values of <0.5 indicated that there were no
mechanistic similarities to the three other compounds. In con-
trast, the trimethyl and tetramethyl quaternary salts 20c,d were
strongly mechanistically related to each other (PCC 0.89), but
correlation of the profile of the 2-acetylamino-quinoacridinium
salt 14a to those of 20c and 20d was weaker (PCC 0.59 and 0.6,
respectively).
COMPARE can also be mobilised to relate biological profiles
of new investigational agents to those of clinically-used agents
of defined mechanism. In this analysis, the methylamines 20c
and 20d displayed PCC values >0.6 to a range of natural
product DNA-binding agents (e.g. actinomycin D, rubidazone,
adriamycin, daunomycin, bactobolin, deoxydoxorubicin,
bruceantin); also the compounds were predicted to be sub-
strates for P-glycoprotein-mediated drug efflux. In contrast, the
acetylamino-derivative 14a gave no PCC values > 0.6 to clinic-
ally-used agents, suggesting that it operated by a different bio-
logical mechanism. Indeed in other (unpublished) work we have
shown that 14a is a potent telomerase inhibitor (IC50 0.375 µM)
and its relative lack of cytotoxicity (mean GI50 12 µM), coupled
to a simple 5-step synthesis, has led us to select this agent for
further detailed biological and physico-chemical evaluation.
Experimental
Melting points were measured on a Gallenkamp apparatus and
are uncorrected. IR spectra were recorded as KBr discs on a
Perkin Elmer Spectrum One FT-IR spectrometer. Mass spectra
were recorded on either a Micromass Platform spectrometer,
an AEI MS-902 (nominal mass), or a VG Micromass 7070E
or a Finigan MAT900XLT spectrometer (accurate mass).
NMR spectra were recorded on a Bruker ARX 250 instrument
at 250.13 MHz (1H), 62.9 MHz (13C) and 235.3 MHz (19F) in
[2H6] DMSO or CDCl3; coupling constants are in Hz. Merck
silica gel 60 (40–60 µM) was used for column chromatography.
9-(Benzotriazol-1-yl)acridine 1,4 9-(6-nitrobenzotriazol-1-yl)-
acridine 9,4 9-(5-chlorobenzotriazol-1-yl)acridine 21,4 3-chloro-
8H-quino[4,3,2-kl]acridine 224 and 3-chloro-8-methyl-8H-
quino[4,3,2-kl]acridine 237 were prepared as indicated.
Thermolysis of 9-(benzotriazol-1-yl)acridines
The benzotriazolylacridine 1 was heated in a range of solvents
for the indicated time (see Table 1). 9-(6-Nitrobenzotriazol-1-
yl)acridine 9 was boiled in triglyme for 2 h. 2-Nitro-8H-quino-
[4,3,2-kl]acridine 10 was isolated in 95% yield following
precipitation with water. The product had identical IR, 1H and
13C NMR spectra to a previously prepared sample.4
8-Methyl-2-nitro-8H-quino[4,3,2-kl]acridine 11
2-Nitro-8H-quino[4,3,2-kl]acridine (0.5 g, 1.6 mmol) was dis-
solved in DMF (10 cm3) and added to a suspension of sodium
hydride (0.1 g, 4.2 mmol) in DMF (10 cm3). After stirring for
30 minutes at 25 ?C, dimethylsulfate (0.3 g, 2.4 mmol) was
added dropwise and stirring continued for a further 1 h. The
reaction mixture was poured into water (50 cm3). The resulting
precipitate was collected by filtration and washed with water
(50 cm3) to give a dark red solid (0.5 g, 95%), mp 285 ?C (dec.);
νmax/cm?1 1595 (C??N); δH ([2H6] DMSO) 8.67 (1H, d, J 7.75),
8.61 (1H, d, J 9.0), 8.42 (1H, d, J 2.5), 8.11–8.05 (2H, m), 7.86
(1H, t, J 8.5), 7.68–7.55 (2H, m), 7.45 (1H, d, J 8.25), 7.24 (1H,
t, J 7.0), 3.67 (3H, s, CH3); m/z (ES-HRMS) 328.1078
[C20H14N3O2
? requires 328.1081].
2-Amino-8-methyl-8H-quino[4,3,2-kl] acridine 12
A mixture of 8-methyl-2-nitro-8H-quino[4,3,2-kl]acridine
(17 g, 52 mmol) and SnCl2?2H2O (52 g, 230 mmol) in 10 M HCl
(300 cm3) was stirred at room temperature for 60 hours. After
this time, 10 M NaOH solution was added until the pH reached
12, then the mixture was filtered and washed with water.
Column chromatography (CHCl3
impure material which was further purified by washing with
chloroform, yielding 2-amino-8-methyl-8H-quino[4,3,2-kl]-
CHCl3–MeOH) yielded
224
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