Synthesis and radioiodination of some 9-aminoacridine derivatives

Article abstract of DOI:10.1002/ejoc.200400296

Derivatives of 9-aminoacridine, namely N-[ω-(acridin-9-yl-amino) alkyl]-3-(trimethylstannyl)benzamides (1), where the alkyl group is propyl (1a) and octyl (1b), and 2-(acridin-9-ylamino)-3-(4-hydroxyphenyl)propionic acid (2), have been synthesized with the aim to use them as precursors in the syntheses of radiolabeled DNA intercalators for biological experiments. It was observed that compounds 1a and 1b can exist in two isomeric forms at room temperature. Radioiodination of the two benzamides 1a and 1b was carried out with the Auger-emitting nuclide ¹²⁵I by exchange of the trimethylstannyl group. The optimal conditions for radioiodination of the octyl derivative 1b were established and the labeling yield was found to be as high as 92%, according to TLC analysis in model experiments. Purification of the radioiodinated products gave radiochemical yields of 56% for the propyl and 74% for the octyl compound. The amino acid 2 was directly labeled with ¹²⁵I at the ortho position to the hydroxyl group by taking advantage of the activated ring. The experiment afforded a very high labeling yield (92%). Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200400296

FULL PAPER
Synthesis and Radioiodination of Some 9-Aminoacridine Derivatives
Senait Ghirmai,[a] Eskender Mume,[a] Cecile Henssen,[a] Hadi Ghaneolhusseini,[a]
Hans Lundqvist,[b] Vladimir Tolmachev,[a,b] Stefan Sjöberg,[a] and Anna Orlova*[b]
Keywords: DNA / Intercalations / Iodine / Labeling / Radiochemistry / Tautomerism
Derivatives of 9-aminoacridine, namely N-[ω-(acridin-9-yl-
amino)alkyl]-3-(trimethylstannyl)benzamides (1), where the
alkyl group is propyl (1a) and octyl (1b), and 2-(acridin-9-
ylamino)-3-(4-hydroxyphenyl)propionic acid (2), have been
the octyl derivative 1b were established and the labeling
yield was found to be as high as 92%, according to TLC ana-
lysis in model experiments. Purification of the radioiodinated
products gave radiochemical yields of 56% for the propyl and
synthesized with the aim to use them as precursors in the 74% for the octyl compound. The amino acid 2 was directly
syntheses of radiolabeled DNA intercalators for biological
labeled with 125I at the ortho position to the hydroxyl group
experiments. It was observed that compounds 1a and 1b can
by taking advantage of the activated ring. The experiment
exist in two isomeric forms at room temperature. Radioiod- afforded a very high labeling yield (92%).
ination of the two benzamides 1a and 1b was carried out with
the Auger-emitting nuclide 125I by exchange of the trimethyl-
stannyl group. The optimal conditions for radioiodination of
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2004)
Introduction
sions of the DNA molecule. It has been demonstrated that
125I decaying in the close vicinity of DNA has a much
The cytotoxicity of many radionuclides to tumors is high- higher cell-killing effect than 125I decaying in the cyto-
est if the radionuclides are delivered to the nucleus of the plasm.[2] Because of its relatively long half life and low
cell. This circumstance has given impetus to the develop- emission energy, 125I is a very convenient nuclide for
ment of methods to synthesize DNA-interacting carriers of laboratory experiments. Biological experiments using these
radionuclides for targeted cancer therapy. The planar struc- molecules will be reported elsewhere.
ture of acridines allows the molecules to bind strongly to
DNA by intercalation. When the acridines have an amino
Results and Discussion
substituent at the 9-position, their binding to a double-
stranded DNA has been shown to increase.[1] Radiolabeled
9-aminoacridines are therefore of potential interest.
Synthesis of N-[ω-(Acridin-9-ylamino)alkyl]-3-(trimethyl-
stannyl)benzamides (1a and 1b)
The compounds 1a and 1b were prepared as outlined in
Scheme 1 starting with the nucleophilic substitution reac-
Here we report the syntheses of two N-[ω-(acridin-9-yl-
amino)alkyl]-3-(trimethylstannyl)benzamides (1a and 1b,
where the alkyl groups are propyl and octyl, respectively)
and
2-(acridin-9-ylamino)-3-(4-hydroxyphenyl)propionic
acid (2), which all can act as precursors for radioiodination.
2-(Acridin-9-ylamino)-3-(4-hydroxy-3-iodo-phenyl)propionic
acid (3), a monoiodinated derivative of 2, was also prepared
for use as a nonradioactive reference substance in the radio-
labeling experiments. The radiolabeling of the three com-
pounds with 125I is also presented. Iodine-125 is an Auger-
electron-emitting nuclide with a half life of 60 days. The
Auger electrons travel along a range similar to the dimen-
[a]
Uppsala University, Department of Chemistry, Organic
Chemistry,
Box 599, BMC, 751 24 Uppsala, Sweden
[b]
Uppsala University, Biomedical Radiation Sciences, Rudbeck
Laboratory
Scheme 1. i) H2N(CH2)nNH2 (n ϭ 3, 8), DMF, 100 °C. ii) 3-iodo-
benzoyl chloride, NaOH, H2O, chloroform. iii) For 1a: K2CO3,
Sn2(CH3)6, Pd(PPh3)4, DMF, 100 °C; for 1b: K2CO3, Sn2(CH3)6,
PdCl2(PPh3)2, 1,4-dioxane, 60 °C iv) [125I]NaI, MeOH, CAT, 5 min
751 85, Uppsala, Sweden
Fax: ϩ46-18-471-3432
E-mail: Anna.Orlova@bms.uu.se
Eur. J. Org. Chem. 2004, 3719Ϫ3725
DOI: 10.1002/ejoc.200400296
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A. Orlova et al.
FULL PAPER
tion of 9-phenoxyacridine with 1,3-diaminopropane and
1,8-diaminooctane to obtain the products N1-acridin-9-yl-
propyl-1,3-diamine (5a) and (N1-acridin-9-yl)octyl-1,8-di-
amine (5b), respectively. The compounds were treated with
3  HCl in methanol and then purified by column chroma-
tography to give the hydrochloride salts of 5a (previously
reported as the dihydrogen bromide salt[3]) and 5b in high
yield. The 9-phenoxyacridine was prepared from 9(10H)-
acridone via 9-chloroacridine by a slight modification of a
procedure reported in the literature.[4,5] 9-Chloroacridine
was found to be difficult to handle as it is easily hydrolyzed
to 9(10H)-acridone. It was therefore not advisable to use it
for a direct reaction with the diamine. Excess amount of
the diamines were used in these reactions to avoid disubsti-
tution.
Scheme 2. Tautomerism in 9-substituted acridines
tum mechanical calculations and spectroscopic data.[9Ϫ11]
Several approaches were tried to achieve a simple method The hydrogens bonded to the exocyclic nitrogen can migrate
of obtaining the benzamides 6a,b in good yield. One way to the endocyclic nitrogen and result in the two tautomeric
was to couple the amines 5a,b with 3-iodobenzoic acid forms, 9.3 and 10.3. Based on the theoretical calculations
using dicyclohexylcarbodiimide (DCC) and 4-(dimethylam- and spectroscopic investigations, it has been shown[9,10,12]
ino)pyridine (DMAP). This reaction afforded around 40% that 9-acridinamine can potentially exist in two tautomeric
of the corresponding benzamides. The coupling reaction of forms — the amino or imino compounds — in equilibrium
3-iodobenzoyl chloride with the amines 5a,b provided with each other at room temperature. The amino tautomer
6a,b·HCl in 88% and 69% yield, respectively.
was found to be thermodynamically more stable according
In the last step, the iodine atom in 6a,b was substituted to the calculations. The authors have also theoretically de-
with a trimethylstannyl group using a palladium(0) cata- termined the difference in energy between the amino and
lyzed coupling reaction.[6] This reaction in DMF solution imino compounds to be always positive.
gave complete conversion of the starting material in five
For our N-aminoacridine derivatives, isomers were ob-
hours. Interestingly, a palladium()-catalyzed reaction in di- served only for 1a and 1b; the amount of the isomers was
oxane converted the iodinated compounds to their corre- found to depend on the work-up procedure. The effect of
sponding stannylated compounds 1 in only one and half bulky substituents at the 9-position on the tautomeric ratio
hours in similar yields. The obvious advantages of this has been studied before.[13] The crystal structure of 9-(tert-
method over the former one are the lower boiling point of butylamino)acridine was determined and it was found that
the solvent (dioxane in contrast to DMF), relatively short the crystals contained the amino tautomeric form.
reaction time and lower reaction temperature (Scheme 1).
No isomers have so far been observed for the 9-amino-
As the stannylated compounds are sensitive to acidic media, acridine hydrochlorides 5a,b and 6a,b, which were purified
the silica gel used in the chromatographic purification was in the presence of HCl(aq). Elemental analysis of 6a and 6b
presaturated with ammonia. For both stannylated com- showed these compounds to be singly protonated. It is
pounds 1a and 1b two isomers with small differences in re- known in the literature[11,14] that single protonation of both
tention times were isolated by chromatography. The main tautomers of 9-aminoacridine actually leads to the same
1
difference in the H NMR spectra of the two isomers is the ion. The imino form, which coexists with the amino form
chemical shift of the NH proton of the 9-aminoacridine in the free-base species, converts to the amino form upon
part. These compounds are denoted 1aA, 1aB, and 1bA, protonation. The tin compounds 1a,b are not stable in the
1bB in the Exp. Sect. The mass spectra of each pair of iso- presence of acids and were therefore purified in the presence
mers are virtually the same and give the expected molecular of a base. In this case, two isomeric forms of the compound
ions with the characteristic isotopic pattern for tin com- were observed, as described earlier. From our observations,
pounds. Compounds 1aB and 1bB were the minor products. it can be said that the relative amounts of the isomers very
This issue in relation to literature data for similar com- much depends on the type of solvent and pH of the media.
pounds will be discussed below.
The reason for the different behaviors of our acridine de-
rivatives is not clearly known but the data could be rational-
ized as follows. In the presence of acid, one of the isomers
Tautomerism in the 9-Aminoacridine System
9-Substituted acridines of the general formula 9 can po- is dominant, as was the case for 5a,b and 6a,b. In the case
tentially exist in two tautomeric forms (Scheme 2). of the tin compounds 1a,b, an isomeric mixture is formed
It has not been possible to show the existence of 9- during the synthesis and maintained during the purification
hydroxyacridine (9.1). The nonaromatic keto structure 10.1 procedure. This behavior was deduced from NMR and MS
is the only form observed.[7,8] For 9-mercaptoacridine (9.2) experiments. Additionally, when a CDCl3 solution of the
in DMSO, the thiono form (10.2) dominates (85%).[7]
Tautomerism of 9-aminoacridine (9.3) and substituted spectrum changed, and some nuclear Overhauser effects
minor product (1bB) was kept for two days at 4 °C, the
aminoacridines has been studied to some detail using quan- (NOEs) between the 9-NH proton and the protons in posi-
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Synthesis and Radioiodination of 9-Aminoacridine Derivatives
FULL PAPER
tions 1(8) and 4(5) of the acridine ring system were ob-
served, indicating that a fast equilibrium is established.
Since the NH of the major product could not always be
observed by NMR spectroscopy, it was not possible to draw
any conclusion from NOE measurements on 1bA.
It is tempting to suggest that the isomers observed for
1a,b are tautomers but definite proof is so far lacking. This
interpretation of the results is, however, supported by a
similar observation for nitro-substituted aminoacridines.[15]
Synthesis of 2-(Acridin-9-ylamino)-3-(4-hydroxyphenyl)-
propionic Acid (2) and its Iodinated Analog 2-(Acridin-9-
ylamino)-3-(4-hydroxy-3-iodophenyl)propionic Acid (3)
Compound 2 (Scheme 3) was obtained by reacting
phenoxyacridine and -tyrosine in molten phenol to give 2
in 98% yield after chromatography and subsequent recrys-
tallization. Similarly, the reaction of phenoxyacridine with
3-iodo--tyrosine gave 2-(acridin-9-ylamino)-3-(4-hydroxy-
3-iodophenyl)propionic acid (3) quantitatively.
Figure 1. Radiochromatograms of a typical radioiodination with
1b; Merck silica gel 60 F254 TLC aluminum sheets were used and
the eluent was CH2Cl2/CH3OH (3:1, v:v)
peaks are seen; peak 3 has the same Rf value as compound
6b and is thus associated with the labeled compound. The
size of peak 4 depends on the amount of CAT in the reac-
tion. The percentage of radioactivity associated with this
peak increased as the amount of CAT increased, but com-
pletely disappeared when the final optimized labeling con-
ditions where used. The identity of the compound, however,
was not determined.
The radioiodination yields obtained using 1b, under dif-
ferent reaction conditions, are shown in Figure 2. The yield
depends strongly on the incubation time and reaches a pla-
teau at an incubation time of 5 min (see a in Figure 2). This
result differs from previous findings for iodination of pro-
teins with CAT in which high yields were obtained as soon
Scheme 3. Synthesis of compounds 2 and 3: i) -tyrosine, phenol,
100 °C, 98%; ii) -3-Iodotyrosine, phenol, 100 °C, 93%
Radioiodination of the N-[ω-(Acridin-9-ylamino)alkyl]-3-
(trimethylstannyl)benzamides 1a and 1b
In all labeling experiments the major isomers of 1a and
1b were used. Compound 1b was used in experiments for
finding optimal labeling conditions with regard to the
amounts of substrate and oxidant as well as incubation
time. The optimal conditions were found to be 35 µg of
compound 1b, 5 µL of Chloramine-T (CAT) solution
(1 mg/mL MeOH) and an incubation time of 5 min. A rep-
resentative example of a radiochromatogram (radio TLC)
is given in Figure 1. Sodium iodide (NaI) was added as a
carrier for the stabilization of Na125I against oxidation dur-
ing chromatography.
During preliminary experiments with 1b and NaI about
15 different eluent systems were tested (different ratio of
CH2Cl2/CH3OH with and without triethylamine, ethyl acet-
ate/hexane, diethyl ether/CH2Cl2). The best resolution of
the nonradioactive analogs (NaI and 6b) was achieved
when using CH2Cl2/CH3OH (3:1, v:v).
Figure 2. Dependence of radiochemical yields of 1b on different
reaction conditions; experiments were performed in duplicate; error
bars were calculated as maximal errors according to the formula:
Ermax ϭ (Ymax Ϫ Ymin)/2; total volume of reaction mixture was 70
µL; vessels were vortexed during reaction; the reaction was
quenched with 20 µL sodium metabisulfite (3.3 mg/mL MeOH);
sodium iodide (5 µL, 10 mg/mL MeOH) was added as carrier be-
fore analysis: a) 1b: 35 µg; CAT: 5 µg.; b) CAT: 5 µg, time: 5 min;
The radio chromatogram (Figure 1) shows a small peak
(1) at the start position. This is presumably oxidized iodine,
whereas peak 2 is 125I-iodide stabilized with NaI. Two more c) 1b: 35 µg, time:5 min
Eur. J. Org. Chem. 2004, 3719Ϫ3725
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A. Orlova et al.
FULL PAPER
as 30 s after addition of CAT,[16] but agrees well with find-
ings for radioiodinations of similar organotin intermedi-
ates.[17,18] For some organotin intermediates the reaction ki-
netics were even slower and the optimum yield was not re-
ached until 15 min after the start of the reaction.[17Ϫ19] The
radiolabeling yield decreased dramatically as the amount of
acridine decreased (see b in Figure 2). The best yields (up
to 92%) were obtained for the reaction mixture containing
at least 35 µg of acridine (0.06 µmol). This amount of sub-
strate is larger than that usually required for direct iodin-
ation of proteins. For example, small amounts [as little as 5
µg (0.001 µmol)] of epidermal growth factor (EGF) can be
labeled efficiently with iodine.[20] When we take into ac-
count that one molecule of EGF has five tyrosine residues,
the amount of substrate (0.06 µmol) required for successful
labeling of acridine is 12 times larger. However, this amount
of substrate (0.06 µmol) is at least 10 times smaller than
that usually used for tin derivatives of N-succinimidyl
benzoate.[17,19,21,22]
Figure 3. Radio iodination of 2-(acridin-9-ylamino)-3-(4-hydroxy-
phenyl)propionic acid (2); Merck silica gel 60 F254 TLC aluminum
sheets were used and the eluent was CH2Cl2/CH3OH (4:1, v:v)
The dependence of labeling yield on the amount of CAT
is given in Figure 2 (c). The best yields were reached using
1Ϫ5 µg of CAT (0.045Ϫ0.2 µmol), which corresponds to
up to a 1:1 molar ratio of CAT to acridine precursor. This
result is in agreement with literature data.[19] For compari-
son we note that the ratio for labeling of EGF was 110:1.
Further increases in the amount of CAT decreased the yield
of the desired product and increased the amount of the lab-
eled by-product. This suggests that peak 4 in the radio TLC
is associated with products of acridine oxidation with CAT.
Under optimal labeling conditions the yield of this by-prod-
uct was reduced to zero.
experiment. Thin-layer chromatographic analysis (Figure 3)
showed that the labeled compound (peak 3) has the same
retention factor as the nonradioactive reference compound
3. Attempts to get a better resolution were not successful
although the amount of the unchanged iodide (peak 2) was
very low (Ͻ2%). The reaction afforded a high labeling yield
(96%). An unknown side product was observed (Ͻ2%) to
come out at a higher retention time (peak 4). Peak 1 corre-
sponds to peak 1 in Figure 1.
Experimental Section
After optimization of the labeling protocol, the com-
pounds 1a and 1b were labeled with 125I and the products
of reaction were separated with SepPak Plus cartridges
(5 mL of Elga water, two times 5 mL of methanol). All
fractions were checked by TLC. In both cases (1a and 1b)
the water fractions contained unchanged iodine and the lab-
eled acridines 7a and 7b were in the first methanol fractions.
The isolated radiochemical yields of the reaction were 54%
for 7a and 75% for 7b.
General: 1H and 13C spectra were recorded in CDCl3 (δ ϭ 7.26 ppm
1
1H; δ ϭ 77.0 ppm 13C), CD3OD (δ ϭ 3.35 ppm H; δ ϭ 49.0 ppm
1
13C) or DMSO (δ ϭ 2.49 ppm H; δ ϭ 39.5 ppm 13C) on a Varian
Unity 400 spectrometer operating at 400 and 100.6 MHz, respec-
tively, a Gemini-200 spectrometer operating at 200 and 75.4 MHz,
respectively, or a Unity-500 spectrometer operating at 500 and
125 MHz, respectively. For column chromatography Merck silica
gel 60 (230Ϫ400 mesh) was used. TLC was performed using Merck
silica gel 60 F254. DMF was dried according to standard methods.
LC/MS in methanol solution was conducted using an AQA mass
spectrometer with an electrospray positive ionization method. El-
emental analysis was carried out at Mikro Kemi AB, Uppsala.
HRMS was conducted at the Organisch Chemisches Institut der
Universitaet Muenster, Abt. Massenspektrometrie.
Radioiodination of 2-(Acridin-9-ylamino)-3-(4-hydroxy-
phenyl)propionic Acid (2)
The amino acid 2 was labeled with 125I using the chloro-
amine-T method (Scheme 4). All parameters were optim-
ized in the same way as used for the tin compounds 1a,b.
The iodo derivative 3 was used as a reference in the labeling
Materials for Radiolabeling: [125I]NaI (3.7 GBq/mL, 644 MBq/µg
I) for labeling was obtained from Amersham Int. plc. (Little Chal-
font, Buckinghamshire, UK). The radio TLC strips (Merck Silica
gel 60 F254 TLC aluminum sheets 15 ϫ 100 mm, elution path
80 mm) were developed with CH2Cl2/MeOH (3:1) and measured
on a CyclonTM Storage Phosphor System and analyzed using an
OptiQuantTM Image Analysis Software (Packard Instrument Com-
pany, Inc., USA). Methanol (for HPLC) obtained from Fisher
Scientific UK Ltd. (Loughborough, UK) and CH2Cl2 (purum) ob-
tained from KEBO lab were used as delivered. Chloramine-T and
sodium metabisulfite were obtained from Sigma, and sodium iod-
ide (p.a.) from Merck. High quality Elga water (resistance higher
than 18 MΩ/cm3) was used for the preparation of aqueous solu-
tions, and fresh solutions were prepared for each experiment.
Scheme 4. Radioiodination of 2: i) Na125I, Chloramine-T
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Synthesis and Radioiodination of 9-Aminoacridine Derivatives
FULL PAPER
Radiolabelling experiments were performed in Eppendorf tubes other hour. The solvent was evaporated and the crude product was
(1.5 mL) at room temperature. Experiments were performed in converted into its hydrochloride form by treating it with 3  HCl/
duplicate. Error bars were calculated as maximal errors according
MeOH. The resulting salt was purified by column chromatography
to the formula: Ermax ϭ (Ymax Ϫ Ymin)/2. SepPak Plus cartridges (CH2Cl2/MeOH/HClconcd., 4:1:0.1, as the mobile phase) to afford
(sorbent C18) were from Waters Sverige AB (Sollentuna, Sweden).
2.00 g of compound 6a·HCl in 88% yield. 1H NMR (CD3OD): δ ϭ
8.50 (d, J ϭ 8.6 Hz, 2 H, acr), 7.98 (s, 1 H, benz), 7.92 (t, J ϭ 8.6
Hz, 2 H, acr), 7.85 (d, J ϭ 7.8 Hz, 1 H, benz), 7.77 (d, J ϭ 8.6 Hz,
2 H, acr), 7.67 (d, J ϭ 7.8 Hz, 1 H, benz), 7.53 (t, J ϭ 8.6 Hz, 2
H, acr), 7.19 (t, J ϭ 7.8 Hz, 1 H, benz), 4.25 (t, J ϭ 7.8 Hz, 2 H,
-NH-CH2-), 3.55 (t, J ϭ 6.4 Hz, 2 H, CH2-NH3), 2.27 (m, 2 H,
-CH2-) ppm. 13C NMR (CD3OD): δ ϭ 168.7, 160, 141.7, 137.2,
136.98, 136.3, 131.4, 127.4, 126.5 124.9, 119.7, 113.8, 96.9, 94.8,
48.0, 37.9, 30.7 ppm. C23H21ClIN3O: calcd. C 53.3, H 4.1, N 8.1,
Cl 6.9; found C 53.3, H 4.4, N 8.0, Cl 7.2.
9-Phenoxyacridine (4): This compound was prepared using a modi-
fied literature procedure.[4,5] 9-(10H)Acridone (5.00 g, 25.8 mmol)
was dissolved in dry benzene. Excess SOCl2 (11 mL) was added
and the reaction was allowed to reflux for 5 h in the presence of a
few drops of DMF. The solvent was removed under vacuum and
the excess thionyl chloride was co-evaporated with benzene to give
crude chloroacridine. This product was used in the next reaction
without further purification. The resulting 9-chloroacridine, phenol
(7.30 g, 77.3 mmol) and K2CO3 (30.00 g, 217 mmol) were dissolved
in DMF and stirred at 100 °C under inert atmosphere for 24 h.
K2CO3 was filtered off and the solvent was evaporated. The crude
product was purified by column chromatography with CH2Cl2/di-
ethyl ether (6:1) as the mobile phase to afford the desired product
in 93% yield. 1H and 13C NMR spectra were in accordance with
the literature results.[4,5]
N-[8-(Acridin-9-ylamino)octyl]-3-iodobenzamide
Hydrochloride
(6b·HCl): This compound was obtained from 5b according to the
procedure described for 6a. The crude product was purified by col-
umn chromatography using CH2Cl2/MeOH/HClconcd., 6:1:0.05, as
1
the mobile phase giving 6b·HCl in 69% yield. H NMR (CD3OD):
δ ϭ 8.37 (d, J ϭ 8.4 Hz, 2 H, acr), 8.02 (s, 1 H, benz), 7.84 (m, 2
H, acr), 7.69 (m, 4 H, acr ϩ benz), 7.40 (t, J ϭ 7.9 Hz, 2 H, acr),
7.08 (t, J ϭ 7.9 Hz, 1 H, benz), 4.02 (t, J ϭ 7 Hz, 2 H, -CH2-NH-
acr), 3.20 (t, J ϭ 7.0 Hz, 2 H, -CH2-NH-), 2.00Ϫ1.08 (m, 12 H,
CH2-CH2-CH2) ppm. 13C NMR (CD3OD): δ ϭ 168.3, 159.5, 141.5,
137.8, 137.3, 136.4, 131.3, 127.5, 126.6, 124.9, 119.6, 113.8, 97.0,
94.7, 50.5, 41.0, 30.5, 30.3, 30.1, 30.1, 27.8, 27.7 ppm.
C28H30ClIN3O: calcd. C 57.2, H 5.3, N 7.2; found C 56.6, H 5.5,
N 7.4.
(N1-Acridin-9-yl)propane-1,3-diamine (5a): A solution of 9-phenoxy-
acridine (1.90 g, 7.01 mmol) in dry DMF was added to a solution
of 1,3-diaminopropane (7.02 mL, 84.1 mmol) in dry DMF. The re-
action mixture was stirred at 100 °C under a N2 atmosphere for
22 h. The solvent was evaporated and the residue was dissolved in
3  HCl/MeOH and stirred for a few minutes. The mixture was
concentrated under vacuum. The resulting crude product was puri-
fied by flash chromatography (CH2Cl2/MeOH/HClconcd., 7:2:0.7, as
the mobile phase) to afford 2.45 g of the hydrochloride salt of 5a
N-[3-(Acridin-9-ylamino)propyl]-3-(trimethylstannyl)benzamide (1a):
The free base 6a was obtained by stirring the hydrochloride salt
with aqueous K2CO3 followed by extraction with CH2Cl2. The or-
ganic phase was dried over MgSO4. Upon filtration and evapor-
ation of the solvent, the free base was obtained. The free base 6a
(0.415 g, 0.56 mmol) was dissolved in dry DMF (12 mL) and argon
was bubbled through the solution for 5 min. Hexamethylditin
(1.13 g, 3.44 mmol) was added and argon was bubbled for another
1
in 97% yield. H NMR (CD3OD): δ ϭ 8.59 (d, J ϭ 8.6 Hz, 2 H,
H-4 and H-5 acr), 7.99 (m, 2 H, H-3 and H-6 acr), 7.85 (dd, J ϭ
8.6, 0.73 Hz, 2 H, H-1 and H-8 acr), 7.60 (m, 2 H, H-2 and H-7
acr), 4.31 (t, J ϭ 7.3 Hz, 2 H, -CH2-NH), 3.15 (t, J ϭ 7.5 Hz, 2
H, -CH2-NH3ϩ), 2.4 (m, 2 H, -CH2-) ppm. 13C NMR (CD3OD):
δ ϭ 159.5, 141, 136.4, 126.5, 125.2, 119.6, 113.8, 47.2, 38.3, 28.6
ppm. ESI/MS (C16H17N3): m/z (%) ϭ 252 (100) [M ϩ H]ϩ, 253
(17).
5 min.
Tetrakis(triphenylphosphane)palladium(0)
(0.20 g,
0.17 mmol) was finally added and the reaction mixture was stirred
at 100 °C under argon for 4 h. The mixture was cooled to room
temperature and the black catalyst was filtered off through celite.
The solution was concentrated. The crude product was purified by
column chromatography using CH2Cl2/MeOH (8:1) as the mobile
phase and afforded two isomeric compounds 1aA (183 mg) and
1aB (131 mg) in a total yield of 70%.
(N1-Acridine-9-yl)octane-1,8-diamine (5b): The reaction mixture
was prepared using 1,8-diaminooctane as in the procedure de-
scribed for 5a. The crude product was purified by column chroma-
tography using CH2Cl2/MeOH/HClconcd., 4:1:0.05, as the mobile
1
phase giving the hydrochloride salt of 5b in 74% yield (1.76 g). H
NMR (CD3OD): δ ϭ 8.42 (d, J ϭ 8.7 Hz, 2 H, H-4 and H-5 acr),
7.85 (m, 2 H, H-3 and H-6 acr), 7.78 (dd, J ϭ 8.2 Hz, 0.73 Hz, 2
H, H-1 and H-8 acr), 7.48 (m, 2 H, H-2 and H-7 acr), 4.08 (t, J ϭ
7.6 Hz, 2 H, -NH-CH2-), 2.81 (t, J ϭ 7.9 Hz, 2 H, -CH2-NH3ϩ),
2.00Ϫ1.20 (m, 12 H, -CH2-) ppm. 13C NMR (CD3OD): δ ϭ 159.0,
140.9, 136.3, 126.7, 125.0, 119.5, 113.6, 50.6, 40.9, 30.6, 30.0, 30.0,
28.4, 27.7, 27.3 ppm.
1
Data for 1aA: H NMR (CDCl3): δ ϭ 9.58 (br. s, 1 H, acr-NH-),
8.40 (br. s, 1 H, -NHCO-), 8.30 (d, J ϭ 8.5 Hz, 2 H, H-1 and H-
8), 8.20 (s with tin satellites, JH,Sn ϭ 22.2 Hz, 1 H, H-2Ј), 8.00 (d,
J ϭ 6.8 Hz, 1 H, H-6Ј), 7.90 (d, J ϭ 8.5 Hz, 2 H, H-4 and H-5),
7.52 (d, J ϭ 6.8 Hz, 1 H, H-4Ј), 7.42Ϫ7.31 (m, 3 H, H-5Ј, H-3 and
H-6), 7.24 (t, J ϭ 8.5 Hz, 2 H, H-2 and H-7), 4.22 (t, J ϭ 7.8 Hz,
2 H, acr-NH-CH2-), 3.82 (t, J ϭ 6.4 Hz, 2 H, -CH2-NHCϭO), 2.38
N-[3-(Acridin-9-ylamino)propyl]-3-iodobenzamide (6a·HCl): A mix-
ture of 3-iodobenzoic acid (1.50 g, 6.05 mmol) and excess SOCl2 (m, 2 H, -CH2-), 0.3 [s with tin satellites, JH,Sn ϭ 28.0 Hz, 9 H,
(6 mL) in dry benzene was refluxed for 2.5 h. The excess SOCl2
and solvent were then evaporated. Benzene was added to the crude
product and the solvents evaporated. This co-evaporation with ben-
Sn(CH3)3] ppm. 13C NMR (CD3OD): δ ϭ 169.6 (CϭO), 157, 143.0,
139.7, 139.4, 134.7, 134.3, 132.5, 128, 127.3, 124.3, 123.8, 118.8,
113.0, 45.6, 36.9, 30.5, Ϫ9.2 [Sn(CH3)3] ppm. ESI/MS (ϩve;
zene was done several times to completely remove traces of thionyl C26H29N3OSn): m/z (%) ϭ 516 (40.5) [M ϩ H]ϩ, 517 (33.1), 518
chloride to give 3-iodobenzoyl chloride in quantitative yield. Com-
pound 5a (1.70 g, 4.71 mmol) in chloroform (45 mL) was treated
with NaOH (1.90 g, 47.1 mmol) in H2O (14 mL). A solution of 3- Data for 1aB: H NMR (CDCl3): δ ϭ 9.70 (br. s, 1 H, acr-NH-),
(74.9), 519 (44.6), 520 (100), 521 (27.9), 522 (16.9). HRMS (m/z)
calcd. for (C26H29N3OSn ϩ Hϩ) 520.1416; found 520.1413.
1
iodobenzoyl chloride (1.40 g, 5.19 mmol) in chloroform (15 mL)
was added to the reaction mixture dropwise (in 10 min) at 0 °C.
8.42 (br. s, 1 H, -NHCO-), 8.28 (d, J ϭ 8.4 Hz, 2 H, H-1 and H-
8), 8.15 (s with tin satellites, JH,Sn ϭ 22.2 Hz, 1 H, H-2Ј), 8.00Ϫ7.95
The reaction was warmed to room temperature and stirred for an- (m, 3 H, H-6Ј, H-4 and H-5), 7.62 (m, 3 H, H-4Ј, H-3 and H-6),
Eur. J. Org. Chem. 2004, 3719Ϫ3725
www.eurjoc.org  2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 3723

A. Orlova et al.
FULL PAPER
7.43 (m, 1 H, H-5Ј), 7.34 (t, J ϭ 8.4 Hz, 2 H, H-2 and H-7), 4.19 matography (MeOH/CH2Cl2, 1:4, as mobile phase) to give 42 mg
(t, J ϭ 7.8 Hz, 2 H, acr-NH-CH2-), 3.82 (m, J ϭ 6.4 Hz, 2 H, (98%) of 2. The product was converted into its hydrochloride salt
-CH2-NHCϭO), 2.35 (m, 2 H, -CH2-), 0.32 [s with tin satellites,
JH,Sn ϭ 28.0 Hz, 9 H, Sn(CH3)3] ppm. HRMS (m/z) calcd. for
(C26H29N3OSn ϩ Hϩ) 520.1416; found 520.1433.
by treating it with 3  HCl in MeOH followed by evaporation to
dryness. The analytical sample was obtained by recrystallization
from methanol/ethyl acetate. 1H NMR (CD3OD): δ ϭ 8.30 (d, J ϭ
8.8 Hz, 2 H, H-4 and H-5 acr), 7.88 (t, J ϭ 8.8 Hz, 2 H, H-3 and
H-6 acr), 7.71 (d, J ϭ 8.8 Hz, 2 H, H-8 and H-1 acr), 7.48 (t, J ϭ
8.8 Hz, 2 H, H-2 and H-7 acr), 6.8 (d, J ϭ 8.4 Hz, 2 H, benz), 6.39
(d, J ϭ 8.4 Hz, 2 H, benz), 5.10 (m, 1 H, CH), 3.4 (m, 1 H, CH2),
3.2 (m, 1 H, CH2) ppm. 13C NMR (CD3OD): δ ϭ 172.0, 161.0,
157.7, 141, 136.7, 131.3, 127.5, 125.8, 125.4, 119.8, 116.1, 65.4, 53.7
39.2 ppm. MS (ESIϩ; C22H18N2O3 free base): m/z (%) ϭ 381 (100)
[M ϩ Na]ϩ, 359 [M ϩ H]ϩ. HRMS (m/z) calcd. for (C22H18N3OSn
ϩ Hϩ) 359.1395; found 359.1439; (C22H18N3OSn ϩ Naϩ) calcd.
381.1215; found 381.1194.
N-[8-(Acridin-9-ylamino)octyl]-3-(trimethylstannyl)benzamide (1b):
The hydrochloride salt of compound 6b was converted into the free
amine with aqueous K2CO3 and extracted with CH2Cl2. The or-
ganic phase was dried over magnesium sulfate. The solvent was
evaporated and this free amine (108 mg, 0.196 mmol) was dissolved
in dry 1,4-dioxane. The solution was bubbled with argon for 10 min
in order to remove oxygen and carbon dioxide. Hexamethyldistan-
nane (112 mg, 0.342 mmol) was added to the mixture and argon
was bubbled for 4 min through the reaction mixture. Finally, bis(tri-
phenylphosphane)palladium() dichloride (27.5 mg, 0.039 mmol)
was added and the reaction mixture was stirred at 75 °C for 90 min.
The reaction mixture was cooled to room temperature and the cata-
lyst was filtered off through celite. After evaporation to dryness the
crude mixture was applied to a flash chromatography column and
eluted with the mobile phase CH2Cl2;MeOH, 6:1, to give two iso-
meric compounds 1bA (86.0 mg) and 1bB (17.0 mg) in a total yield
of 89%.
L-2-(Acridin-9-ylamino)-3-(4-hydroxy-3-iodophenyl)propionic Acid
(3): A mixture of 9-phenoxyacridine (33 mg, 0.12 mmol), 3-iodo-
tyrosine (37 mg, 0.12 mmol) and phenol (2.50 g, 26.6 mmol) was
stirred at 100 °C under inert atmosphere for one hour. The reaction
was cooled to room temperature and purified by column chroma-
tography (4:1, CH2Cl2/MeOH was used as the mobile phase).
Recrystallization from methanol/ethyl acetate afforded 54 mg
(93%) of 3. 1H NMR (DMSO:D2O, 4:1): δ ϭ 8.21 (d, 2 H, H-4
and H-5 acr), 7.85 (t, 2 H, H-3 and H-6 acr), 7.69 (d, 2 H, H-1
and H-8 acr), 7.43 (t, 2 H, H-2 and H-7 acr), 6.95 (s, 1 H, benz),
6.81 (d, 1 H, benz), 6.43 (d, 1 H, benz), 4.86 (m, 1 H, CH), 3.15
(m, 2 H, CH2) ppm. HRMS (m/z) calcd. for (C22H17N3IOSn ϩ
Hϩ) 485.0362; found 485.0403.
1
Data for 1bA: (CH2Cl2/MeOH, 6:1). H NMR (CDCl3): δ ϭ 8.87
(br. s, 1 H, -NH-acr), 8.32 (d, 2 H, H-1 and 8), 8.14 (d, 2 H, H-4
and H-5), 7.94 (s, 1 H, H-2Ј with tin satellites), 7.72 (d, 1 H, H-
6Ј), 7.61 (d, 1 H, H-4Ј), 7.49 (m, 2 H, H-3 and H-6), 7.40 (t, 1 H,
H-5Ј), 7.24 (t, 2 H, H-2 and H-7), 6.44 (t, 1 H, CONH), 4.14 (t, 2
H, acrϪNH-CH2-), 3.48 (q, 2 H, -CH2-NHCO), 2.07 (m, 2 H,
acr-N-CH2-CH2-), 1.64 (m, 2 H, -CH2-CH2-NHCO-), 1.51 [m, 2
H, -acr-NH-(CH2)2-CH2-], 1.50Ϫ1.36 (m, 6 H, -CH2-), 0.30 [s, with
tin satellites, 9 H, Sn(CH3)3] ppm. 13C NMR (CDCl3): δ ϭ 168.5,
156.6, 143.4, 139.6, 139.1, 134.5, 134.4, 128.2, 126.9, 125.6, 123.8,
118.9, 112.4, 48.9, 40.2, 30.2, 29.9, 29.8, 29.0, 26.9, 26.8, Ϫ9.1
[Sn(CH3)3] ppm. HRMS (m/z) calcd. for (C31H39N3OSn ϩ Hϩ)
590.2200; found 590.2206.
Optimization of the Labeling of Benzamide 1bA with 125I: An acri-
dine solution in MeOH (50 µL) and [125I]NaI stock solution in
Elga water (5 µL) were vortexed together. Labeling was started
by addition of 5 µL CAT solution in MeOH. The mixture was
vortexed for the pre-determined time. Reaction was quenched with
20 µL of sodium metabisulfite solution (1 mg/mL MeOH). NaI (5
µL, 10 mg/mL MeOH) was added as carrier before analysis. Blank
experiments were performed following the same protocol but with-
out adding acridine.
1
Data for 1bB: (CH2Cl2/MeOH, 6:1). H NMR (CDCl3): δ ϭ 9.74
(br. s, 1 H, H-10), 8.28 (d, 2 H, H-1 and H-8), 8.00 (d, 2 H, H-4
and H-5), 7.97 (s, with tin satellites, 1 H, H-2Ј), 7.76 (d, 1 H, H-
6Ј), 7.61 (d, with tin satellites 1 H, H-4Ј), 7.44 (m, 2 H, H-3 and
H-6), 7.38 (t, 1 H, H-5Ј), 7.20 (t, 2 H, H-2 and H-7), 6.59 (t, 1 H,
CONH-), 4.10 (t, 2 H, acr-N-CH2-), 3.49 (q, 2 H, -CH2-NHCO-),
2.06 (m, 2 H, acr-N-CH2-CH2-), 1.64 (m, 2 H, -CH2-CH2-NHCO-),
1.53 [m, 2 H, acr-N-(CH2)2-CH2-], 1.50Ϫ1.26 (m, 6 H, -CH2-),
0.36 [s, with tin satellites, 9 H, Sn(CH3)3] ppm. 13C NMR (CDCl3):
δ ϭ 168.4, 157.0, 143.4, 139.0, 134.6, 134.4, 134.0, 128.2, 126.8,
125.4, 123.2, 119.7, 112.5, 48.7, 40.2, 30.3, 29.8, 29.2, 29.1, 26.9,
Ϫ9.2 [Sn(CH3)3] ppm. HRMS (m/z) calcd. for (C31H39N3OSn ϩ
Hϩ) 590.2200; found 590.2211.
[
125I]-N-[8-(Acridin-9-ylamino)octyl]-3-iodobenzamide (7b): To an
Eppendorf tube containing 3 µL of stock solution [125I]NaI (3.7
MBq/µL, 0.13 nmol [125I]NaI) in 15 µL of MeOH was added 35
µL of 1bA (1 mg/mL in methanol). Reaction was initiated by ad-
dition of 5 µL of CAT solution (1 mg/mL in MeOH) and quenched
with 6 µL of sodium metabisulfite solution (1 mg/mL) after 5 min
of vortexing. TLC analysis of the reaction mixture (with NaI car-
rier) gave 85% yield of the reaction. The reaction mixture was sepa-
rated on a SepPak Plus cartridge eluting with 5 mL of Elga
water followed by two portions of methanol (5 mL). Fractions were
checked by TLC. The average yield of [125I]-N-[3-(acridin-9-ylami-
no)octyl]-3-iodobenzamide (7b), after separation, over five experi-
ments was 75%. The specific activity was 78 kBq/µg.
Data for 1bB after 2 Days at 4 °C in CDCl3 Solution: 1H NMR
(CDCl3): δ ϭ 9.70 (br. s, 1 H, NH-), 8.28 (d, 2 H, H-1 and H-8),
8.01 (d, 2 H, H-4 and H-5), 7.97 (s, with tin satellites, 1 H, H-2Ј),
7.76 (d, 1 H, H-6Ј), 7.62 (d, with tin satellites 1 H, H-4Ј), 7.45 (m,
2 H, H-3 and H-6), 7.38 (t, 1 H, H-5Ј), 7.20 (t, 2 H, H-2 and
H-7), 6.59 (t, 1 H, CONH-), 4.10 (t, 2 H, acr-N-CH2-), 3.48 (m,
2 H, -CH2-NHCO-), 2.06 (qn, 2 H, acr-N-CH2-CH2-), 1.64 (m, 2 H,
-CH2-CH2-NHCO-), 1.53 [m, 2 H, acr-N-(CH2)2-CH2-], 1.50Ϫ1.26
(m, 6 H, -CH2-), 0.32 [s, with tin satellites, 9 H, Sn(CH3)3].
[
125I]-N-[3-(Acridin-9-ylamino)propyl]-3-iodobenzamide (7a): This
compound was obtained and purified similarly to 7b with a yield
of 56% and a specific activity of 54 kBq/µg.
Radioiodination of L-2-(Acridin-9-ylamino)-3-(4-hydroxyphenyl)pro-
pionic Acid (2): To a solution of 5 µL of Na125I and 50 µL of the
precursor 2 (0.98 mg in mixture 650 µL water and 50 µL MeOH)
was added 20 µL of chloramine-T (2 mg/mL in water). The mixture
was incubated for 3 min. The reaction was then quenched with 20
L-2-(Acridin-9-ylamino)-3-(4-hydroxyphenyl)propionic Acid 2:
A
mixture of 9-phenoxyacridine (33 mg, 0.12 mmol), -tyrosine
(22 mg, 0.12 mmol) and phenol (2.5 g, 26.6 mmol) was stirred at µL of Na2S2O5 (4 mg/mL in water). 10 µL of NaI (10 mg/mL in
100 °C under an inert atmosphere for one hour. The reaction mix-
ture was cooled to room temperature and purified by column chro-
water) was finally added to stabilize the radioiodine. A blank
experiment was performed following the same procedure but with-
3724
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 3719Ϫ3725

Synthesis and Radioiodination of 9-Aminoacridine Derivatives
FULL PAPER
J. Rak, K. Krzyminski, P. Skurski, L. Jozwiak, J. Blazejowski,
J. Mol. Struct. 1999, 476, 45Ϫ55.
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Acknowledgments
This research has been financially supported by the Swedish
Cancer Society (Grants 4462-B01-02PAA and 3009-B02-13XAC),
INTAS (grant 99-00806) and the Carl Trygger Foundation for
Scientific Research, Sweden.
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Received April 30, 2004
Eur. J. Org. Chem. 2004, 3719Ϫ3725
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 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3725