Synthesis of acridine-based DNA bis-intercalating agents

Article abstract of DOI:10.3390/60300230

Methods for the synthesis of N¹, N⁸-bis(9-acridinyl)- N⁴-(4-hydroxybenzyl)-spermidine and N¹, N ⁷-(hydroxybenzyl)-bis-(3-aminopropyl)amine were investigated. Thus monocyanoethylation of 4-methoxybenzylamine followed by treatment with 4-chlorobutyronitrile gave the dinitrile N-(2-cyanoethyl)-N-(3-cyanopropyl)-4- methoxy-benzylamine. Subsequent in situ reduction with lithium aluminium hydride gave the corresponding diamine. Biscyanoethylation of 4-methoxybenzylamine with 2 mole of acrylonitrile followed by reduction yielded the diamine N, N-bis-(3-aminopropyl)-4-methoxybenzylamine. Both diamines reacted smoothly with 9-methoxyacridine to give the bis-(9-acridinyl) compounds 11 and 15 but with 4,5-dimethyl-9-methoxyacridine, the bis compound 16 was produced in only low yields. Demethylation of the dinitriles by a variety of approaches all failed to give the corresponding hydroxybenzyl derivatives. These studies yielded useful methylated tyrosine derivatives which could also be iodinated. This study has been useful for elucidating chemical methods needed for the synthesis of the desired tyrosine-based bis acridine compound and for alerting us to the need to synthesise a more labile protected tyrosine intermediate which will be easily deprotected to afford the desired tyrosine-based bis acridine compound.

Full text of DOI:10.3390/60300230

Molecules 2001, 6, 230-243
molecules
ISSN 1420-3049
http://www.mdpi.org
Synthesis of Acridine-based DNA Bis-intercalating Agents
1*
2
2
Gerard P. Moloney , David P. Kelly , P. Mack
1 The Austin Research Institute, Studley Road, Heidelberg, Victoria 3084, Australia. Fax (03)
96884995.
2 University of Melbourne, Swanston Street Parkville, Victoria 3052, Australia.
*Author to whom correspondence should be addressed; e-mail: G.Moloney@ari.unimelb.edu.au
Received: 12 July 2000; in revised form 30 August 2000 / Accepted: 18 January 2001/ Published: 28
February 2001
1
8
4
Abstract: Methods for the synthesis of N , N -bis(9-acridinyl)-N -(4-hydroxybenzyl)-
1
7
spermidine and N , N -(hydroxybenzyl)-bis-(3-aminopropyl)amine were investigated.
Thus monocyanoethylation of 4-methoxybenzylamine followed by treatment with 4-
chlorobutyronitrile gave the dinitrile N-(2-cyanoethyl)-N-(3-cyanopropyl)-4-methoxy-
benzylamine. Subsequent in situ reduction with lithium aluminium hydride gave the
corresponding diamine. Biscyanoethylation of 4-methoxybenzylamine with 2 mole of
acrylonitrile followed by reduction yielded the diamine N, N-bis-(3-aminopropyl)-4-
methoxybenzylamine. Both diamines reacted smoothly with 9-methoxyacridine to give
the bis-(9-acridinyl) compounds 11 and 15 but with 4,5-dimethyl-9-methoxyacridine, the
bis compound 16 was produced in only low yields. Demethylation of the dinitriles by a
variety of approaches all failed to give the corresponding hydroxybenzyl derivatives.
These studies yielded useful methylated tyrosine derivatives which could also be
iodinated. This study has been useful for elucidating chemical methods needed for the
synthesis of the desired tyrosine-based bis acridine compound and for alerting us to the
need to synthesise a more labile protected tyrosine intermediate which will be easily
deprotected to afford the desired tyrosine-based bis acridine compound.
Keywords: Acridine, intercalate, anti-cancer, synthesis.

Molecules 2001, 6
231
Introduction
Acridine (1) and its derivatives have long been a well established class of DNA and RNA binding
compounds [1].
N
1
These compounds have held the interests of biochemists and chemists for some years due to the
possibilities of their clinical use. Many types of drugs are known to bind to DNA through inter-
calation between consecutive nucleotides in the DNA strand [2]. To this class of compounds belong
several anti tumour agents of clinical importance such as actinomycin-D, daunomycin and
adriamycin, as well as several drugs used in the treatment of parasitic diseases including quinacrine
and ethidium bromide [3]. Thus the synthesis of acridine-derived DNA intercalating molecules that
bind preferentially to particular nucleotide sequences appeared an exciting prospect. The mode of
binding of acridine molecules involves intercalation of the acridine tricyclic ring between adjacent
base pairs in the DNA duplex [4,5]. The acridine moieties are held in place by van der Waals forces
supplemented by stronger ionic bonds to the phosphate ions of the DNA backbone (Scheme 1).
Scheme 1
It has been demonstrated by Hurwitz [6] that the binding of acridine molecules interferes with
normal DNA function by blocking the DNA starter required by polymerases to synthesise RNA and
DNA and hence inhibits protein synthesis. Considerable synthetic attention has been paid to
substituted aminoacridines [7]. The incorporation of radioactively labelled iodine provides a method
of determining the degree of ligand-DNA interaction, as well as any sequence selectivity. It has been
125
shown the
I, covalently bound to pyrimidine bases in double stranded DNA or incorporated into a

Molecules 2001, 6
232
DNA binding compound through and iodine carrier, can cause scission of the DNA helix within three
125
base pairs either side of the decaying atom [8]; the effectiveness of
I is a consequence of the
intense radiochemical damage resulting from the emission of low energy Auger electrons [9].
Monoacridines have been shown to bind strongly to DNA. In more recent times however, it has
been found that bifunctional intercalaters result in both higher affinity [10] and selectivity [11] of
binding. The incorporation of 125I is one method of determining the effectiveness and sequence
selectivity of binding. 125I may be introduced into acridines if activating amine groups occupy the C-
3 and C-6 positions, directing iodination to the C-4 and C-5 positions respectively. Introduction of
the activating amine group has been achieved through the nitro derivative [7], and coupling of the
nitroacridines to the linker bis-amino compounds must occur at this stage. However, upon linking of
the nitro acridines, reduction of the nitro functions to the corresponding amine functions proved to be
difficult [12].
Scheme 2
X
8
1
9
7
6
2
3
X = NH2, Cl
R = NO2 to NH2
N
R
5
4
125I
2
HN
R
NH
N
NO
N
NO
2
2
3
It was therefore proposed that the synthesis of bis-acridinyl DNA binding molecules in which the
iodine carrier was incorporated into the linker chain would be an attractive alternative. We report here
the synthesis of bis-acridine DNA intercalating compounds 4 with a spermidine type linker chain
incorporating an aromatic ring susceptible to iodination. Motivation for the synthesis of a spermidine
type linker molecule stems from previous work performed by Le Pecq and co-workers [3] who
carried out the first synthetic work on bis-intercalating compounds.

Molecules 2001, 6
233
Le Pecq showed that the minimum length required of the acridine linker molecule for effective
bis-intercalation into DNA was 10.1 Å, i.e. the length of two base pairs along the DNA axis which is
in accordance with the "excluded site model" [3]. In order to conform to this criterion it was decided
to use two linker molecules, spermidine (n=4) and bis-(3-aminopropyl)amine (n=3), in the synthesis
of the target compound 4.
1
R O
2
2
R
R
N
N
N
N
N
2
2
R
R
4 (n = 3,4)
1
2
R = CH , H; R = H, CH .
3
3
The inclusion of a readily iodinated hydroxybenzyl moiety in the linker chain of 4 was
125
proposed in order to take advantage of the use of
I labelling and DNA sequencing to identify
precisely the nucleotide sequence of highest affinity [13]. Two acridine derivatives, acridine and 4,5-
dimethylacridine were chosen as the intercalating agents, because both bind strongly to DNA. The
4,5-dimethylacridine derivative bears special significance in that it resembles the active intercalating
portion of an effective DNA binding antibiotic antibiotic, actinomycin-D (5) which is known to
inhibit transcription. Both 4,5-dimethylacridine and actinomycin-D have methyl substituents at the
C-4 and C-5 positions of their respective tricylic rings.
Cyclic
Pentapeptide
Cyclic
Pentapeptide
O
O
N
O
NH2
O
CH3
CH3
5

Molecules 2001, 6
234
Results and Discussion.
Previous work carried out in our group by Tassigiannakis [12] resulted in the successful synthesis
of N,N-bis-(2-cyanoethyl)-4-methoxybenzylamine (8) and N,N-(3-aminopropyl)-4-methoxybenzyl-
amine (9) utilizing step (b) of Scheme 3 and step a of Scheme 4. However the attempted preparation
of a bis-aminoacridine by treatment of the bis-amine 9 with 9-chloroacridine proved unsuccessful due
to the inherent unreactive nature of 9-chloroacridine. It was therefore decided to continue this
synthetic work and explore variations of the scheme in the hope of improving the synthesis towards
the target bis-aminoacridine 4, with particular attention given to the coupling step with the acridines.
The synthetic approach to N,N-bis-(3-aminopropyl)-4-methoxybenzylamine (9) was patterned on
the synthesis of the non-substituted benzylamine compounds by Bergeron and co-workers [14]. Two
routes were employed in the synthesis of N,N-bis-(2-cyanoethyl)-4-methoxybenzylamine (8),
Scheme 3.
a
Scheme 3
OCH 3
OCH 3
a
NH
NH2
CN
6
7
b
OCH 3
c
N
NC
CN
8
a
Reagents: (a) CH =CHCN, Room temperature;
2
(b) CH =CHCN, Carius tube 130°C 6 days;
2
(c) CH =CHCN, Carius tube 125°C 5 days.
2
Treatment of methoxybenzylamine 6 with acrylonitrile at room temperature afforded the mono-
1
nitrile (7) in good yield (81%). The appearance of two triplets at δ=2.44 and 2.88 ppm in the H-
NMR spectrum was attributed to the C-9 and C-10 methylene protons. Treatment of the mononitrile 7
with excess acrylonitrile and a trace of hydroquinone in a Carius tube [15] (sealed under vacuum) at
1
125°C for five days afforded the dinitrile 8 in 92% yield. The H-NMR spectrum showed the

Molecules 2001, 6
235
correct integration for the methylene protons of the identical cyanoethyl chains resonating as triplets
at δ=σ2.41 and 2.84 ppm. Hydroquinone, previously omitted from the reaction procedure [14],
proved essential in preventing polymerisation of the reaction mixture at such high temperatures.
Synthesis of 8 was also achieved in one step by treating 4-methoxybenzylamine with excess
acrylonitrile in the presence of hydroquinone in 72% yield (Scheme 5), comparable to the overall
yield above. The dintrile 8 previously reported [12] as a liquid, was obtained as a low melting solid.
Subsequent reduction of 8 with lithium aluminium hydride and aluminium chloride in diethyl ether
1
gave the bis-amine 9. The H-NMR. spectrum revealed a singlet at δ=1.57 ppm corresponding to the
amine protons.
Scheme 4
OCH3
OCH3
a
N
N
NC
CN
9
8
NH2
NH2
OCH3
OCH3
N
10
N
HN
NH
N
N
11
Reagents: (a) LiAlH /AlCl , diethyl ether.
4
3
1
7
N , N -bis-(9-acridinyl)-N-(4-methoxybenzyl)-bis-(3-aminopropyl)amine (11) was prepared
in good yield by treatment of the bis-amine 9 with two equivalents of 9-methoxyacridine (10) in
13
methanol at room temperature, Scheme 4. C-NMR spectral analysis revealed that bis coupling of
the acridine derivative had occurred, with seven acridine carbon resonances present (δ=112.0, 119.7,
122.7, 129.3, 130.7, 133.4, 129.9 ppm) at approximately twice the intensity of the bis-amine
methylene carbons. An improvement on previous work [12] was the use of 9-methoxyacridine in the

Molecules 2001, 6
236
coupling reactions rather than 9-chloroacridine. Linking of the acridine moiety by displacement of
the methoxy function required only stirring at room temperature whereas high temperatures were
required and long reaction times were required for any coupling of the bis-amine to 9-chloroacridine.
The synthesis of N-(3-aminopropyl)-N-aminobutyl)-4-methoxybenzylamine (13) outlined in
Scheme 5, was based on the synthesis of the non-substituted benzylamine compounds by Bergeron
and co-workers [16].
Scheme 5
OCH3
OCH3
a
N
NH
CN
NC
CN
12
7
OCH3
b
OCH3
OCH3
R
R
R = H, 10
R = CH3, 14
N
HN
NH
N
N
N
NH2
NH2
R
R
R
R
13
R = H, 15
R = CH3, 16
Reagents: (a) ClCH CH CH CN, KI/butanol; (b) LiAlH /AlCl , diethyl ether.
2
2
2
4
3
The mononitrile 7, as prepared in Scheme 3, was alkylated with 4-chlorobutyronitrile and
potassium iodide in 1-butanol at 117°C with potassium carbonate as the base [17], affording the
dinitrile 12 in 50% yield. In addition to the disappearance of the singlet (δ=1.62) produced by the
1
secondary amine proton, the H-NMR spectrum contained a doublet of doublets (δ=1.79 ppm)
attributed to the C-10 methylene protons, and a complex multiplet (δ=2.35-2.80) corresponding to the
other eight methylene protons at C-9, C-11, C-13 and C-14. The dinitrile 12 was reduced in the same

Molecules 2001, 6
237
13
manner as 8 to yield the bis-amine 13 in 34% yield. The disappearance of the nitrile C-NMR
signals at δ=40.4 and 42.0 ppm provided positive evidence of the reduction.
Treatment of 13 with 9-methoxyacridine 10 in methanol, afforded the previously unreported bis-
13
aminoacridine 15 in 82% yield. The C-NMR spectrum was consistent with bis-acridine coupling to
the diamine 13 with relatively intense resonances at δ=112.9, 13.3, 113.7, 121.6, 122.6, 124.4, 130.6
and 155.1 ppm. In an analogous manner, it was expected that treatment of the bis-amine 13 with 4,5-
dimethyl-9-methoxyacridine (14) would result in bis coupling of the acridine derivative to yield the
bis-aminoacridine 16. However, coupling proved ineffective even after applying higher temperatures
and longer reaction times. Due to low yields of the bis-aminoacridine obtained, conclusive
characterisation of the coupled compound was not achieved. The 4,5-dimethylacridine thus appears
less reactive towards amines than the unsubstituted acridine 10.
9-Methoxyacridine (10) used in the coupling reactions described earlier, is readily synthesised by
standard procedures [18,19]. The preparation of 4,5-dimethyl-9-methoxyacridine (14) however has
not been previously reported. Based on Albert's acridine synthesis [19], this acridine derivative could
be obtained via the corresponding 4,5-dimethyl-9-chloroacridine (18). Preparation of 18 was
attempted according to the original method Neuman and Powell [20] through an Ullmann
condensation of o-bromotoluene and 3-methylanthranilic acid. Isolation of the intermediate
condensation product was not successful, however, with the availability of 4,5-dimethylacridone (17)
[21] we proceeded to synthesise the desired 9-methoxy compound 14 by the method outlined in
Scheme 6. Thus treatment of 17 with phosphorus oxychloride gave 4,5-dimethyl-9-chloroacridine
(18) in 84% yield and subsequent treatment with metallic sodium in methanol afforded the 9-methoxy
1
derivative 14. The appearance of a singlet at δ=4.19 ppm in the H-NMR spectrum indicated the
presence of a methoxy group.
Scheme 6
O
Cl
a
N
H
N
CH
CH
3
CH
CH
3
3
3
b
OCH
3
N
CH
CH
3
3
14
-
+
Reagents: (a) POCl ; (b) MeO Na /MeOH.
3

Molecules 2001, 6
238
Although the 4-methoxybenzylmoiety may be iodinated under a variety of conditions, ultimately
bis-aminoacridine with a hydroxybenzyl moiety would be more desirable targets since they are more
125
susceptible to iodination. In addition, iodination with
I has to be performed at the final step of the
synthesis so the method used must be mild enough so as not to damage the rest of the molecule. Thus
a number of demethylation experiments were carried out on N-2-(2-cyanoethyl)-N-(3-cyanopropyl)-
4-methoxybenzylamine (12) with intentions of deprotecting the hydroxy function and reprotecting
with a more acid labile protecting group such as tertiary butyl or methoxymethyl. After reduction of
the dinitrile 12 to the diamine 13 and coupling to the acridines, the protecting group could then be
easily removed in trifluoroacetic acid to give and analogue of tyrosine, a naturally occurring amino
acid.
Demethylation using chlorotrimethylsilane and sodium iodide by Olah's method [22] failed to
produce the desired tyrosine analogue. Trimethylsilane appeared to attack the tertiaryamine in
1
preference to the methoxy function. Integration of the multiplet at δ=1.61-2.82 ppm in the H-NMR
spectrum indicated that the four-carbon nitrile chain had been cleaved. The appearance of a small
singlet at δ=1.30 ppm corresponds to the proton of the resulting secondary amine. Deprotection of the
hydroxybenzyl moiety of 12 was then attempted using sodium borohydride and iodine according to
the method of Long [23]. This procedure also proved unsatisfactory, resulting in cleavage of the long
nitrile chain, in addition to the recovery of a small percentage of starting material.
Treatment of 12 with boron tribromide in dichloromethane as outlined by McOmie and co-
workers [24] also failed to produce the tyrosine analogue, resulting in significant decomposition of
the dinitrile. Use of a less powerful ether-cleaving agent such as boron trichloride or boron
trifluoride has not been attempted.
Future work
Our future chemistry will involve the synthesis of a more labile protected tyrosine derivative and
the subsequent alkylation of the amino group followed by the attachment of the acridine moieties and
iodination of the tyrosine moiety to afford the desired iodinated molecule.
Experimental
General
Melting points were determined on a Kofler hot stage microscope and are uncorrected. Micro-
analysis were performed by the AMDEL Australian Microanalytical Service, Melbourne. Infrared
spectra were recorded as thin films on sodium chloride discs using a Perkin-Elmer 457 grating
spectrophotometer, unless otherwise stated. Electronic absorption spectra were recorded in ethanol
using a Varian Super Scan 3 spectrophotometer. Mass spectra were recorded using a VG Micromass
7070F instrument at 70 eV. Proton NMR spectra were recorded at 100MHz using a Jeol FX-100
spectrometer. Sample solutions were prepared in deuterochloroform containing tetramethylsilane as
an internal reference. Chemical shifts have been quoted on the δ=scale in p.p.m. relative to tetra-

Molecules 2001, 6
239
methylsilane. Carbon-13 NMR spectra were recorded at 25 MHz using a Jeol FX-100 spectrometer.
Deuterochloroform was used as solvent with tetramethylsilane as an internal reference. Chemical
shifts were quoted on the δ scale in p.p.m. relative to tetramethylsilane. All reactions were performed
under a dry nitrogen atmosphere. Tetrahydrofuran was distilled from sodium benzophenone ketyl
under dry nitrogen immediately before use. Methanol was dried over type 3Å molecular sieves and
distilled from iodine and magnesium turnings immediately before use. 1-Butanol was dried over type
4Å molecular sieves while all benzene and diethyl ether was sodium dried. Gel filtration was
performed on Sephadex LH-20 using 0.005N acetic acid in 4:1 methanol-chloroform as eluent, unless
otherwise stated. Preparative thin layer chromatography was performed on Merck Kieselgel 60
GF
254
(200-300 mesh). Analytical thin layer chromatography (t.l.c) was performed on Merck
Kieselgel 60 GF
254
precoated plates.
Synthetic methods
N-(2-cyanoethyl)-4-methoxybenzylamine (7). A mixture of 4-methoxybenzylamine (6) (11.02 g,
80.0 mmol) and acrylonitrile (4.97g, 93.0 mmol) was stirred for 45 hours at room temperature.
Fractional distillation of the reaction mixture gave N-(2-cyanoethyl)-4-methoxybenzylamine (7) (12.2
+
1
g, 81 %) as a clear oil b.p. = 245°C at 0.5 mmHg. MS m/z (M ) 190 H-NMR δ 1.6, s, NH; 2.44, t,
J = 5.6 Hz, CH CN; 2.88, t, J = 6.5 Hz, NHCH 3.70, s, PhCH ; 3.78, s, PhCH ; 3.78, s, OCH ;
2
2;
9.0 Hz, H-2, H-6. C-NMR δ 131.5 (C-1), 129.2 (C-2,
2,6
2
13
2
3
7.04, d, J
9.0 Hz, H-3, H-5; 7.26, d, J
3,5
C-6), 113.8 (C-3, C-5), 158.0 (C-4), 55.2 (C-7), 52.5 (C-8), 44.2 (C-9), 18.7 (C-10), 118.7 (C-11).
Anal. found C 69.3, H 7.5; C H N O requires C 69.5, H 7.4 %.
11 14 2
N-(2-Cyanoethyl)-N-(3-cyanopropyl)-4-methoxybenzylamined (12) A solution of 4-chloro-
butyronitrile (2.46 g, 25.0 mmol) in anhydrous n-butanol (9.1 mL) was added over 2 hours to a
mixture of N-(2-cyanoethyl)-4-methoxybenzylamine (5.07 g, 40.0 mmol), anhydrous sodium
carbonate (4.22g, 40.0 mmol) and potassium iodide (0.64 g, 4.0 mmol) at 117°C. The mixture was
stirred for an additional 20.5 hours at 117°C. After cooling to room temperature, the mixture was
filtered and the solid washed with diethyl ether. The combined filtrate and washing were extracted
with 3N HCl (3 x 40 mL) and the resulting acid solution was washed with ether (2 x 40 mL), made
basic with potassium carbonate and extracted with ether (3 x 70 mL). The final ethereal extracts were
dried over sodium sulphate, filtered and evaporated to give an oil (4.45 g, 50.0 %). Subsequent
distillation afforded 3.24 g (43.3 %) of the desired N-(2-cyanoethyl)-N-(3-cyanopropyl)-4-
+
1
methoxybenzylamine (12) as a light yellow oil b.p. = 235° at 0.06 mmHg. MS m/z 257 (M ); H-
NMR δ 1.79, q, , J 6.0 Hz, CH CH CH 2.46, m, 2 x NCH , 2 x CH CN, 3.54, s, PhCH , 3.79, s,
2
2
2,
2
2
2
13
OCH , 6.87, d, , J 9.0 Hz, H-3, H-5, 7.23, d, , J 9.0 Hz, H-2, H-6. C-NMR δ 113.7 (C-1), 129.7 (C-
3
2, C-6), 113.9 (C-3, C-5), 158.9 (C-4), 57.8 (C-7), 55.2 (C-8), 49.2 (C-9), 14.6 (C-10), 119.7 (C-11),
51.8 (C-12), 23.4 (C-13), 118.9 (C-15). Anal. found C 69.7, H 7.3, N 16.2; C H N O requires C
15 19 3
70.0, H 7.3, N 16.3 %.

Molecules 2001, 6
240
N-(3-Aminopropyl)-N-(4-aminobutyl)-4-methoxybenzylamine (13). A solution of anhydrous
aluminium chloride (2.04 g, 15.3 mmol) in anhydrous diethyl ether (21 mL) was rapidly added to a
suspension of lithium aluminium hydride (0.6 g, 15.8 mmol) in anhydrous ether (30 mL). After
vigorous stirring of the mixture, a solution of N-(cyanoethyl)-N-(cyanopropyl)-4-methoxy
benzylamine (12) (1.47 g, 6.0 mmol) in anhydrous diethyl ether (21.0 mL) was added over 2 hours
and the reaction was stirred at room temperature for 29 hours. The solution was cooled to 0°C,
quenched with 30 % aqueous potassium hydroxide (w/v; 30 mL) and the ethereal layer decanted.
The combined ethereal extracts were washed with cold saturated aqueous sodium chloride (2 x 25
mL). Evaporation of the diethyl ether gave a light brown oil which was taken up in chloroform (60
mL), dried over anhydrous magnesium sulphate, filtered and the chloroform evaporated under
reduced pressure. A crude oil was obtained (0.73 g) which upon fractional distillation afforded 0.52
g (34 %) of the desired N-(3-aminopropyl)-N-(4-aminobutyl)-4-methoxybenzylamine (13) as a light
+
1
yellow oil. MS m/z 265 (M ); H-NMR δ 1.12, s, 2 x NH , 1.49, m, 2 x (H-10, H-11, H-14), 2.50,
2
m, 2 x (H-9, H-12, H-13, H-15), 3.47, s, PhCH , 3.79, s, OCH , 7.20, d, , J 10 Hz, H-2, H-6. Anal.
2
3
found C 67.9, H 10.0, N 16.2; C H N O requires C 67.9, H 10.2, N 15.8 %.
15 27 3
1 8
4
N ,N -Bis-(9-acridinyl)-N -(4-methoxybenzyl)spermidine (15). N-(3-aminopropyl)-N-(4-amino-
butyl)-4-methoxybenzylamine 13 (0.34 g, 128 mmol) was dissolved in methanol (2.0 mL) and added
to 9-methoxyacridine 10 (0.54 g, 2.39 mmol). The mixture was stirred for 70 hours at 30°C, the
methanol was evaporated under reduced pressure followed by gel filtration to afford 250 mg (30 %)
1
of the desired bis-aminoacridine 15 as an orange gum. H-NMR δ 1.5-3.3, m, 7 x CH , 2.22, s, 2 x
2
NH, 3.55, s, PhCH , 3.71, s, OCH ), 7.02-7.96, m, H-2, H-6, H-3, H-5, and acridine protons, 20H.
2
3
Anal. found C 70.1, H 6.7; C H N O requires C 69.5, H 5.9 %.
41 41 5
1, 8
4
N N -Bis-(4,5-dimethyl-9-acridinyl)-N -(4-methoxy benzyl)spermidine (16). N-(3-aminopropyl)-
N-(4-aminobutyl)-4-methoxybenzylamine 13 (60 mg, 0.23 mmol) was dissolved in tetrahydrofuran
(1.0 mL) and added to 4,5-dimethyl-9-methoxyacridine (14) (85 mg, 0.18 mmol). The mixture was
stirred for 48 hours at room temperature, then stirred at 35°C for 8 hours. Evaporation of the tetra-
1, 8
hydrofuran under reduced pressure followed by gel filtration afforded the desired N N -bis-(4,5-
4
1
dimethyl-9-acridinyl)-N -(4-methoxy benzyl)spermidine (16) as an orange gum. H-NMR δ 1.1-3.2,
m, 8 x CH , 2.21, s, 2 x PhCH , 3.42, s, OCH , 7.02-7.81, m, H-2, H-6, H-3, H-5 and acridine
2
3
3
protons. UV (log E), 226, 266, 398 (4.02, 3.86, 3.0). v
3400, 2950, 2150, 1570, 1410, 1250,
max
-1
1180, 1025 cm .
N,N-Bis-(2-cyanoethyl)-4-methoxybenzylamine (8): Method 1:-A mixture of 4-methoxybenzyl-
amine (0.406 G, 2.9 mmol), an excess of acrylonitrile (0.85 g, 16.0 mmol) and a trace of hydro-
quinone was added to a Carius tube [15]. The tube was sealed under vacuum (0.1 mmHg) and the
tube was heated at 45° for 8 hours, then 130°C for 140 hours. The resulting amber brown liquid was
distilled under vacuum to afford 0.53 g (72 %) of the desired N,N-bis-(2-cyanoethyl)-4-methoxy-
benzylamine (8) as a colourless oil. b.p. = 225°C at 0.21 mmHg, which formed a white solid, m.p. =
48°C on cooling to room temperature.

Molecules 2001, 6
241
Method 2:- A mixture of N-(2-cyanoethyl)-4-methoxybenzylamine 7 (1.63 g, 8.6 mmol), an excess
of acrylonitrile (1.69 g, 31.8 mmol) and a trace of hydroquinone was added to a Carius tube. The
tube was sealed under vacuum (0.3 mmHg) and heated at 125° for 144 hours. The resulting brown
oil was distilled under vacuum to afford 1.93 g (92.4 %) of the desired N,N-bis-(2-cyanoethyl)-4-
+
1
methoxybenzylamine (8) as a light yellow oil. b.p. = 220°C at 0.2 mmHg. MS m/z 243 (M ); H-
NMR δ 2.41, t, 2 x (H-10, H-13), J 6.6 Hz, 2.84, t, 2 x (H-9, H-12), J 6.0 Hz, 3.6, s, PhCH ), 3.77, s,
2
13
OCH , 6.85, d, , J 9.0 Hz, H-2, H-6, 6.89, d, , J 8.6 Hz, H-3, H-5, 7.20, d, , J 10 Hz, H-2, H-6. C-
3
NMR δ 116.0 (C-1), 129.8 (C-2, C-6), 113.9 (C-3, C-5), 159.1 (C-4), 57.4 (C-7), 55.2 (C-8), 49.2 (C-
+.
9), 16.7 (C-10), 118.8 (C-11), 49.2 (C-12), 16.7 (C-13). Found M = 243.135; C H N O
14 17 3
+.
requires M = 243.137.
N,N-Bis-(3-aminopropyl)-4-methoxybenzylamine (9):- A solution of aluminium chloride (2.66 g,
19.8 mmol) in anhydrous diethyl ether (25 mL) was rapidly added to a suspension of lithium
aluminium hydride (0.75 g, 19.8 mmol) in anhydrous diethyl ether (35 mL). The mixture was
vigorously stirred and a solution of N,N-bis-(2-cyanoethyl)-4-methoxybenzylamine (1.52 g, 6.27
mmol) in anhydrous diethyl ether (25 mL) was added over 1.5 hours. After stirring at room
temperature for 50 hours the mixture was cooled to 0°, quenched with aqueous 30 % potassium
hydroxide (w/v; 30 mL) and the ethereal layer decanted. The remaining emulsion was extracted with
diethyl ether (4 x 100 mL) and the combined diethyl ether extracts washed with saturated aqueous
sodium chloride (2 x 25 mL). Evaporation of the diethyl ether under reduced pressure gave a brown
oil which was taken up in chloroform (50 mL), dried over anhydrous magnesium sulphate, filtered
and the chloroform evaporated under reduced pressure. Fractional distillation of the resulting oil
afford 0.391 g (25 %) of the desired N,N-bis-(3-aminopropyl)-4-methoxybenzylamine (9) as a light
+
1
yellow oil. b.p. = 190° at 0.4 mmHg. MS m/z 251 (M ); H-NMR δ 1.57, s, 2 x NH , 2.37-2.68, m,
2
6 x CH , 3.72, s, PhCH , 3.79, s, OCH , 6.85, d, , J 9.0 Hz, H-2, H-6, 7.22, d, , J 9.0 Hz, H-3, H-5.
2
2
3
+.
UV (log E) 271 nm (3.20). Found M = 251.19975; C H N O requires M = 251.19975.
14 25 3
+.
1 7
4
N ,N -Bis-(9-acrinyl)-N -(4-methoxybenzyl)-bis-(3-aminopropyl) amine (11). N,N-Bis-(3-amino-
propyl)-4-methoxybenzylamine (9) (40.0 mg, 0.16 mmol) was dissolved in methanol (1.5 mL), added
to 9-methoxyacridine (10) (66.0 mg, 0.32 mmol) and the mixture was stirred at room temperature for
63 hours. Evaporation of the methanol under reduced pressure, followed by gel filtration afforded
1 7
4
88.0 mg (91 %) of the desired N ,N -bis-(9-acrinyl)-N -(4-methoxybenzyl)-bis-(3-aminopropyl)
1
amine (11) as an orange-red gum. H-NMR δ 2.17, s, 2 x NH, 2.31-3.65, m, 6 x CH ), 3.74, s,
2
PhCH , 3.80, s, OCH , 6.82-7.61, m, H-3, H-5, H-2, H-6 and acridine protons, 20 H; UV (log E)
2
3
+.
+.
225, 267, 414 nm. 4.3, 4.45, 4.36. Found M = 605.31543; C H N O requires M = 605.31543.
40 39 5
9-Chloroacridine (21). Prepared from 2-carboxydiphenylamine (1.0 g, 4.6 mmol) and freshly
distilled phosphorus oxychloride 3.0 mL, 32.7 mmol) according to Albert [19] and obtained as pale
+
yellow crystals (0.37 g, 37 %). M.p. = 120°C (lit [27] = 120°C). MS m/z 215 (M ).

Molecules 2001, 6
242
9-Methoxyacridine (10). Prepared from 9-chloroacridine (0.5 g, 2.4 mmol) and a sodium methoxide
solution generated from sodium (0.1 g, 4.4 mmol) in methanol (5.8 mL) by the method of Albert [19]
and obtained as dark yellow crystals. Recrystallisation from light petroleum (60-80°C) afforded 0.31
g (63 %) of the desired 9-methoxyacridine (10) as pale yellow crystals, m.p. = 99-101°C (lit [19] =
1
103°C). H-NMR δ4.26, s, OCH , 8.0, m, acridine protons.
3
4,5-dimethyl-9-chloroacridine (18). 4,5-dimethylacridone (20) (1.0 g, 4.5 mmol) and freshly distilled
phosphorus oxychloride (7.0 mL) were refluxed at 130°C for 1.5 hours. Unused phosphorus
oxychloride was then distilled off under reduced pressure. The residue was allowed to cool and then
thinned with chloroform (10.0 mL). The solution was then poured into a mixture of ice (40.0 g) and
concentrated aqueous ammonia (33 %) (10.0 mL) with heat being liberated. The solution was stirred,
the chloroform layer separated and the aqueous phase extracted with chloroform (2 x 30 mL). The
combined chloroform extracts were dried over anhydrous magnesium sulphate, filtered and the
chloroform evaporated under reduced pressure to give a yellow powder which upon recrystallisation
from petroleum ether (b.p. 60-80°C) afforded 0.83 g (84 %) of the desired 4,5-dimethyl-9-chloro-
+
acridine (21) as yellow-green needles m.p. = 150-151°C (lit [29] 151-152°C). MS m/z 241 (M );
1
H-NMR δ 2.94, s, 2 x CH , 7.42-8.31, m, acridine protons.
3
4,5-Dimethyl-9-methoxyacridine (14). 4,5-Dimethyl-9-chloroacridine (220 mg, 0.91 mmol) was
added to a methoxide solution generated from sodium (160 mg, 6.95 mmol) in methanol (15.0 mL)
and tetrahydrofuran (0.5 mL). The solution was gently refluxed for 40 hours and allowed to cool and
the methanol reduced under vacuum. The residue was taken up in benzene, filtered from the salt and
the benzene evaporated under reduced pressure to give a yellow powder which was recrystallised
from heptane to afford 145 mg (67 %) of the desired 4,5-dimethyl-9-methoxyacridine (14) as yellow
+
1
crystals m.p. = 90-91°C. MS m/z 237 (M ); H-NMR δ2.93 (6H, s, 2 x CH ), 4.19 (3H, s, OCH ),
3
3
7.32-8.17 (6H, m, acridine protons). Anal. Found C 80.0, H 6.4, N 5.9%; C H NO requires C
16 15
80.6, H 6.4, N 5.9 %. UV (log E) 225, 357, 373, 392 nm. (4.63, 3.81, 3.80, 3.66).
References
1. Blake, A., and Peacocke, A. R., Biopolymers, 1961, 6, 1225.
2. Lerman L. S., J. Mol. Biol., 1961, 3, 18-20.
3. Le Pecq, J. B., Le Bret, M., Barbet, J., and Roques, B., Proc. Natl. Acad. Sci. U.S.A. 1977, 72,
2915-2199.
4. Sakore, T. D., Jain, S. C., Tsai, C and Sobell, H. M., Proc. Natl. Acad. Sci. U.S.A. 1962, 74,
188.
5.
Waring, M. J. Mol. Biol., 1970, 54, 247.
6. Hurwitz, J. Furth, J., Malamy, M and Alexander, M. Proc. Natl. Acad. Sci. U.S.A. 1962, 48,
1222.
7. Martin, R. F. and Kelly, D. P., Aust. J. Chem., 1979, 32, 2637-2646.
8. Martin, R.F., Int. J. Radiat. Biol., 1977, 32, 553.

Molecules 2001, 6
243
9. Krish, R. E and Sauri, C. J., Int. J. Radiat. Biol. 1975, 27, 553-560.
10. Wakelin, L. P. G., Creasy, T. S and Waring, M. J., FEBS Lett., 1979, 104, 261-265.
11. Shultz, P. G., Dervan, P. B., J. Am. Chem. Soc., 1983, 105, 7748-7750.
12. Tassigiannakis, W., B.Sc.(Hons.) Thesis, University of Melbourne., 1982.
13. Martin, R. F., and Holmes, N., Nature, 1983, 302, 452-454.
14. Bergeron, R. J., Burton, P. S., McGovern, K. A., Kilne, S. J., Synthesis (Comm)., 1981, 731-
733.
15. Cannellakis, E. S., Shaw, Y. H., Hanmers, W. E and Sahartz, R. A., Biochem. et Biophys.
Acta., 1976, 418, 277-289.
16. Bergeron, R. J., McGovern, K. A., Channing, M. A and Burton, P.S., Synthesis (Comm)., 1980,
45, 1589-1592.
17. Bruson, H. A., Org React., 1949, 5, 79.
18. Acheson, R. M., 'Acridines', 2nd Ed. (J. Wiley and Sons: New York, 1973).
19. Albert, A., 'The Acridines' 2nd Ed. (Edward Arnold Publishers: London, 1966).
20. Newman, M. S and Powell, W. H., J. Org. Chem., 1961, 26, 812-815.
21. Cain, B. F and Atwell, G. J., J. Med. Chem., 1976, 19, 1124-1129.
22. Olah, G. A., Narang, S. C., Gupta, G. B and Malhotra, R., Tetrahedron, 1969, 44, 1247.
23. Long, L. H and Freeguard, G. F., Chem and Ind. (London), 1965, 223.
24. McOmie, J.F.W., Watts, M. L and West, D. E., Tetrahedron, 1969, 24, 2289.
25. Kluge, A. F., Untch, K. G and Fried, J. H., J. Am. Chem. Soc., 1972, 94, 7827.
26. Gould, F. E., Johnson,, G. S and Ferris, A. F., J. Org. Chem., 1960, 25, 1658-1660.
27. Brown, H. C., Choi, Y. M and Narasimhan, S., J. Org. Chem., 1982, 47, 3153-3163.
28. Allen, C. F. H and McKee, G. H. W., Org. Synth., 1939, 19, 6-9.
29. Albert, A and Gledhill, W. J., J. Soc. Chem. Indust., 1942, 61, 159-160.
Sample Availability: Samples are available from the authors.
© 2001 by MDPI (http://www.mdpi.org). Reproduction is permitted for noncommercial purposes