Design, synthesis and cell growth inhibitory activity of a series of novel aminosubstituted xantheno[1,2-d]imidazoles in breast cancer cells

Article abstract of DOI:10.1016/j.bmc.2007.03.003

A series of novel aminosubstituted xantheno[1,2-d]imidazole derivatives have been designed and synthesized and their antiproliferative activity has been evaluated against human breast MDA-MB-231 cell line. Among the tested compounds those bearing two basi

Full text of DOI:10.1016/j.bmc.2007.03.003

Bioorganic & Medicinal Chemistry 16 (2008) 3445–3455
Design, synthesis and cell growth inhibitory activity of a series of
novel aminosubstituted xantheno[1,2-d]imidazoles in
breast cancer cells
Ioannis K. Kostakis,a Nicole Pouli,a Panagiotis Marakos,a,* Olga Ch. Kousidou,b
Andreas Roussidis,b George N. Tzanakakisc and Nikos K. Karamanosb
aDivision of Pharmaceutical Chemistry, Department of Pharmacy, University of Athens,
Panepistimiopolis-Zografou, Athens 15771, Greece
bLaboratory of Biochemistry, Department of Chemistry, University of Patras, 26110 Patras, Greece
cDepartment of Histology, Medical School, University of Crete, 71110 Herakleion, Greece
Received 5 October 2006; revised 22 February 2007; accepted 2 March 2007
Available online 7 March 2007
Abstract—A series of novel aminosubstituted xantheno[1,2-d]imidazole derivatives have been designed and synthesized and their
antiproliferative activity has been evaluated against human breast MDA-MB-231 cell line. Among the tested compounds those bear-
ing two basic side chains at 2- and 5-positions exhibited a strong dose-dependent antiproliferative activity. Increase of the size and
basicity of the N-alkyl substituent resulted in amplification of the inhibitory activity.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Representative examples in this field include
(aza)anthrapyrazoles3 pyrazoloacridines,4 benzoperimi-
dines,5 thiadiazinoacridines,6 triazoloacridones,7 pyrim-
idoacridines8 and imidazoacridones.9 Despite the high
structural diversity, most of these compounds have the
common ability to overcome multidrug resistance
(MDR) of tumour cells. For pyrazoloacridine and
pyrazolopyrimidoacridine derivatives there is evidence
that the incorporation of one or two five- or six-mem-
bered heterocyclic rings into the acridine chromophore
extremely favours the passive cellular drug uptake, ren-
dering the efflux by MDR transporters inefficient.10
Anthracenedione and acridine derivatives possess rele-
vant cytotoxic and anticancer activity and some of them,
such as the well studied drugs mitoxantrone and amsa-
crine, have found important clinical applications, partic-
ularly in oncology.1 For most of these compounds DNA
is the major cellular target and their mechanism of
action is generally acknowledged to involve intercala-
tor-dependent formation of cleavable complexes, arising
from the poisoning of DNA topoisomerase II, that
induce DNA double strand breaks.2 They are character-
ized by the presence of a planar or semi-planar aromatic
chromophore, able to stack between base pairs at the
intercalation site, and one or two aminoalkylamino
side-chain substitutions, which can potentially increase
DNA binding affinity. The classical anthra-
cenedione or acridine nucleus is sometimes condensed
with additional heterocyclic rings providing polycyclic
molecules, also endowed with high cytotoxicity.
As a continuation of our studies concerning the synthesis
and antiproliferative activity evaluation of some pyrano-
xanthenone aminoderivatives and their pyrazole-fused
counterparts,11 we have recently reported on the synthesis
of amino-substituted xanthenones (compounds I and II,
Fig. 1) possessing structural analogy to the potent anti-
cancer
agent
9-methoxypyrazoloacridine
(PZA,
Fig. 1).12 These compounds possess DNA binding
capacity and exhibit interesting cytotoxic activity against
a panel of tumour cell lines. Prompted by these results and
in the course of the exploration of structure–activity rela-
tionships within this class of bioactive compounds, we
have decided to investigate the novel xantheno
Keywords: Aminosubstituted xantheno[1,2-d]imidazoles; Breast cancer
cells; Antiproliferative activity.
*
Corresponding author. Tel.: +30 210 7274830; fax: +30 210
7274747; e-mail: marakos@pharm.uoa.gr
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.03.003

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I. K. Kostakis et al. / Bioorg. Med. Chem. 16 (2008) 3445–3455
NRR
NRR
N
N
NMe2
O
O
HN
N
O
N
H3CO
X
Y
X
Y
N
H
NO2
R'
R'
I
X,Y= H, C4H4
R= Me, Et
II X,Y= H, C4H4
R= Me, Et
PZA
R'= NO2, NHCOCH2NRR
R'= NO2, NHCOCH2NRR
Figure 1. Structures of PZA and previously reported xanthenone analogues.
[1,2-d]imidazole chromophore, where the basic side chain
is in part incorporated into an imidazole ring.
rial (Scheme 1), which was obtained upon condensation
of ethyl 2-iodobenzoate (1) with 4-acetamidophenol in
the presence of copper dust. The ester 2 was nitrated
and the resulting nitro derivative 3 was subjected to acid
hydrolysis to provide the nitroaniline 4. Catalytic hydro-
genation of compound 4 over palladium on activated
carbon furnished the unstable 1,2-dianiline 5, which
was immediately treated with chloroacetyl chloride, to
give the dichloroacetamide 6. This compound was dis-
solved in a 5:1 mixture of concentrated sulfuric acid
2. Results and discussion
2.1. Chemistry
For the synthesis of the target derivatives we used ethyl
2-(4-acetamidophenyloxy)benzoate (2) as starting mate-
NHAc
COOEt
a
NHR
b
O
I
O
NO2
COOEt
COOEt
c
3 R : Ac
4 R : H
1
2
d
NHCOCH2Cl
NHCOCH2Cl
NHCOCH2Cl
NH2
NH2
NHCOCH2Cl
e
f
O
6
O
O
7
COOEt
COOEt
COOEt
NO2
5
g
Cl
Cl
NRR
N
O
O
O
N
N
H
H
NH
N
N
h
i
O
O
9
COOH
NO2
NO2
X
10a-d X : NO2
11a-d X : NH2
8
d
e
NRR
NRR
O
O
N
N
H
H
N
N
a: NRR= N(CH3)2
b: NRR= N(C2H5)2
c: NRR= N(CH2)4
d: NRR= N(CH2)5
i
O
O
NHCOCH2NRR
NHCOCH2Cl
13a-d
12a-d
Scheme 1. Reagents and conditions: (a) 4-acetamidophenol, K2CO3, KI, Cu, dry DMF, 110 ꢁC, 12 h; (b) glacial acetic acid, fuming HNO3, rt,
20 min; (c) EtOH, 9% HCl, reflux, 12 h; (d) H2, Pd/C, 50 psi, abs. EtOH, 7 h; (e) ClCH2COCl, Et3N, CH2Cl2, rt, 5 h; (f) 98% H2SO4/glacial acetic
acid 1/5, fuming HNO3, rt, 25 min; (g) 50% H2SO4, 110 ꢁC, 3 h; (h) 98% H2SO4, 110 ꢁC, 2 h; (i) secondary amine, abs. EtOH, reflux, 4–5 h.

I. K. Kostakis et al. / Bioorg. Med. Chem. 16 (2008) 3445–3455
3447
and glacial acetic acid and was nitrated with fuming ni-
tric acid, to result in the nitro derivative 7. Compound 7
was treated with a 50% H2SO4 aqueous solution and the
resulting carboxylic acid 8 was ring-closed, without
further purification, upon treatment with concentrated
sulfuric acid to provide the 2-chloromethyl-5-nitro-
1H,11H-xantheno[1,2-d]imidazol-11-one (9).
ride and reaction of the resulting chloride 18 with pyrrol-
idine or piperidine to provide the corresponding
aminoderivatives 17c and d (Scheme 3).
The target imidazolo-fuzed xanthenones were obtained
from compounds 17a–d upon mild saponification to
the intermediate carboxylic acids 19a–d (Scheme 4), that
were subsequently ring-closed in a boiling mixture of
sulfuric acid and acetic acid, to provide the aminoderiv-
atives 20a–d.
The target compounds 10a–d were prepared by displace-
ment of the chlorine atom of 9 by appropriately substi-
tuted secondary amines. The 5-nitro group of
compounds 10a–d was then easily reduced by hydroge-
nation over palladium on activated carbon and the
rather unstable aminoderivatives 11a–d were converted
into the corresponding chloroacetamides 12a–d by treat-
ment with chloroacetyl chloride. Reaction of these
amides with the suitable secondary amines resulted in
the target amines 13a–d.
For biological evaluation purposes, the free base forms
of the target amines were converted into their water-sol-
uble hydrochloride addition salts by treatment with
hydrochloric acid in methanol.
2.2. Biological activity
The new compounds were tested for their effects on the
growth of the highly metastatic MDA-MB-231 human
breast cancer cells. The experiments were performed in
the presence of serum as to achieve more relevant as
compared to in vivo conditions. The obtained IC50 val-
ues, including reference compound mitoxantrone, are
presented in Table 1.
For comparative reasons, concerning the structure–
activity relationship studies, it was desirable to prepare
the analogous derivatives that do not bear a 5-substitu-
tion on the xantheno[1,2-d]imidazole ring-system. For
this purpose, we used the ethyl carboxylate 4 that upon
treatment with chloroacetyl chloride yielded the chloro-
acetamide 14 (Scheme 2). This chloroacetamide reacted
with dimethylamine or diethylamine and provided easily
the amides 15a and b. The nitro group of these amides
was then reduced by hydrogenation over palladium on
activated carbon to give the intermediate amines 16a
and b, which, due to instability, were not isolated, but
ring-closed to the corresponding benzimidazoles 17a
and b by refluxing in toluene, in the presence of a cata-
lytic amount of glacial acetic acid.
Compounds 20a–d showed significant inhibitory effect
on cell growth only at the highest concentration of
100 lM. At this concentration a cytotoxic effect was ob-
served, since a large number of cells lost contact with the
culture flask and the remaining adherent cells underwent
morphological changes. Concerning this set of com-
pounds the increase of the size of the N-substituent in
compounds 20b–d resulted in higher antiproliferative
activity (65%, 63% and 60% inhibition for 20b, 20c
and 20d, respectively) as compared to the dimethylami-
no analogue 20a (22% inhibition).
Our attempts to apply an analogous methodology for the
preparation of the corresponding pyrrolidine and piperi-
dine derivatives were not successful, since the amide bond
of the nitro compound 14 is quite labile, in the presence of
the more basic cyclic amines. Consequently, the above-
mentioned derivatives were prepared upon treatment of
the ethyl carboxylate 6 with boiling phosphorus oxychlo-
The insertion of a 5-nitro group in compound 20a pro-
viding 10a improved the growth inhibitory effect of 20a
and even at the concentration of 10 lM a 40% inhibition
was obtained. On the contrary, the insertion of 5-nitro
NH2
a
NHCOCH2Cl
b
NHCOCH2NRR
R1
O
4
NO2
O
NO2
O
COOEt
COOEt
COOEt
c
14
15a,b R1= NO2
16a,b R1= NH2
d
NRR
N
NH
a: NRR= N(CH3)2
b: NRR= N(C2H5)2
O
COOEt
17a,b
Scheme 2. Reagents and conditions: (a) ClCH2COCl, glacial acetic acid, 120 ꢁC, 6 h; (b) secondary amine, abs. EtOH, rt, 12–14 h; (c) H2, Pd/C,
50 psi, abs. EtOH, 3 h; (d) glacial acetic acid, dry toluene, reflux, 40 min.

3448
I. K. Kostakis et al. / Bioorg. Med. Chem. 16 (2008) 3445–3455
Cl
NRR
NHCOCH2Cl
NHCOCH2Cl
N
N
NH
NH
a
b
O
O
O
COOEt
COOEt
COOEt
17c: NRR=N(CH2)4
17d: NRR=N(CH2)5
18
6
Scheme 3. Reagents and conditions: (a) POCl3, 90 ꢁC, 2 h; (b) secondary amine, abs. EtOH, reflux, 2–3 h.
NRR
NRR
O
O
N
N
H
NH
N
a: NRR= N(CH3)2
b
b: NRR= N(C2H5)2
c: NRR= N(CH2)4
d: NRR= N(CH2)5
from 19a-b
O
COOR1
a
17a-d R1= Et
19a-d R1=H
20a-d
Scheme 4. Reagents and conditions: (a) 20% NaOH solution, ethanol, 2 h, rt; (b) 98% H2SO4/glacial acetic acid 2/1, 90 ꢁC, 4 h.
Table 1. Anti-proliferative activity of the synthesized compounds
(13c and 13d, respectively) showed similar IC50 values
(16–18 lM). These compounds exhibited the highest
antiproliferative activity of all the compounds tested.
(IC50 values in lM)a against MDA-MB-231 breast cancer cells
Compound
IC50 (lM)
10a
10b
54 (5.7)
>100
3. Conclusions
10c
10d
64 (6.1)
>100
In summary, the present study deals with the synthesis
of a number of new 2-dialkylaminomethyl substituted
xantheno[1,2-d]imidazoles, that may also possess a 5-ni-
tro or a 5-dialkylaminomethylcarbonylamino substitu-
ent. The evaluation of their antiproliferative activity
against MDA-MB-231 human breast cancer cells
showed that the insertion of a second basic side chain
at the 5-position of the chromophore improved the cell
growth inhibitory effect of the compounds and this is
in agreement with previous findings in structurally re-
lated xanthenone derivatives.12 In addition it seems that
the observed improvement of activity is more effective as
the size of the N-alkyl substituent increases.
13a
13b
46 (4.3)
28 (2.6)
16 (2.1)
18 (1.9)
>100
13c
13d
20a
20b
77 (8.3)
67 (6.2)
89 (5.6)
0.96 (0.06)
20c
20d
Mitoxantrone
a The results represent means ( standard deviation) of three indepen-
dent experiments and are expressed as IC50, the concentration that
reduced by 50% the optical density of treated cells with respect to
untreated controls.
group in compounds 20b–d, providing compounds 10b–
d, did not improve their effect on breast cancer cells,
growth.
4. Experimental
4.1. Chemistry
The effects of the compounds 13a–d, bearing a second
basic substituent, on cell proliferation are shown in
Figure 2. This group of compounds had a dose-depen-
dent inhibitory effect on cell growth. Up to the concen-
tration of 30 lM the cells do not exhibit any
morphological changes, which is suggestive of a cyto-
static rather than a cytotoxic effect. Comparing the
inhibitory profiles of 13b and 13a (Fig. 2) it can be seen
that the diethylamino analogue 13b has a significant
inhibitory effect on cell growth even from the lowest
concentration tested (1 lM) as well as lower IC50 value
(28 lM) as compared to the dimethylamino analogue
13a (46 lM). The pyrolidine and piperidine analogues
All chemicals were purchased from Aldrich Chemical Co.
Melting points were determined on a Buchi apparatus and
¨
are uncorrected. 1H NMR spectra and 2D spectra were re-
corded on a Bruker Avanche 400 instrument, whereas 13C
NMR spectra were recorded on a Bruker AC 200 spec-
trometer in deuterated solvents and were referenced to
1
TMS (d scale). The signals of H and 13C spectra were
unambiguously assigned by using 2D NMR techniques:
1H–1H COSY, NOESY HMQC and HMBC. Flash chro-
matography was performed on Merck silica gel 60 (0.040–
0.063 mm). Analytical thin layer chromatography (TLC)
was carried out on precoated (0.25 mm) Merck silica gel

I. K. Kostakis et al. / Bioorg. Med. Chem. 16 (2008) 3445–3455
3449
100
80
60
40
20
0
100
13a
13b
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
13c
13d
0
1
3
10
30 100
0
1
3
10
30 100
(μM)
(μM)
Figure 2. Inhibitory effects of new aminoderivatives on human breast cancer cells. The MDA-MB-231 cell line was incubated in serum-containing
medium for 72 h in the presence of increasing concentrations of aminoderivatives. Cell proliferation was determined by measuring the absorbance at
450 nm (WST-1 method). The results are presented as % of growth inhibition in respect to controls. Control values did not exhibit significant changes
as compared to DMSO vehicle. Each point represents the average standard deviation of three individual experiments, performed in three replicates.
F-254 plates. Elemental analyses were performed at the
Microanalytical Sections of the National Hellenic Re-
search Foundation on a Perkin–Elmer PE 240C Elemen-
tal Analyzer (Norwalk, CT) and are within 0.4% of the
theoretical values.
glacial acetic acid (4 mL). After 20 min, the reaction
mixture was poured into an ice/water mixture and ex-
tracted with CH2Cl2 (3 · 40 mL). The combined organic
extracts were washed with 10% K2CO3 and brine, dried
(Na2SO4) and concentrated in vacuo. Flash chromatog-
raphy on silica gel, using cyclohexane–EtOAc 2:1 as the
eluent, provided compound 3 (1070 mg, 93%) as an oil.
1H NMR (CDCl3, 400 MHz) d 1.11 (t, J = 7 Hz, 3H,
CH2CH3), 2.15 (s, 3H, CH3), 4.14 (q, J = 7 Hz, 2H,
CH2CH3), 7.05 (d, J = 8 Hz, 1H, H-3), 7.25 (dd,
J = 8 Hz, 1H, H-60), 7.31 (dt, J = 8 Hz, 1 Hz, 1H, H-
5), 7.55 (t, J = 8 Hz, 1H, H-4), 7.66 (d, J = 2 Hz, 1H,
H-20), 7.99 (dd, J = 8 Hz, 1H, H-6), 8.68 (d, J = 8 Hz,
1H, H-50), 10.10 (s, 1H, D2O exch, NH); 13C NMR
(CDCl3, 50 MHz) d 13.67 (CH2CH3), 24.77 (CH3),
60.75 (CH2CH3), 112.40 (C-20), 121.73 (C-3), 122.85
(C-1), 123.68 (C-50), 124.34 (C-60), 124.78 (C-50),
129.09 (C-40), 131.84 (C-6) 133.49 (C-4), 136.87(C-30),
153.16 (C-10), 153.79 (C-2), 164.45 (COOEt), 168.56
(CONH). Anal. Calcd for C17H16N2O6. Calcd (%): C:
59.30, H: 4.68, N: 8.14. Found (%): C: 58.93, H: 5.04,
N: 8.35.
4.1.1. Ethyl 2-(4-acetamidophenyloxy)benzoate (2). To a
solution of ethyl 2-iodobenzoate (1, 1 g, 3.42 mmol) in
dry DMF (50 mL), were added, under argon 4-acet-
amidophenol (516 mg, 3.42 mmol), powdered K2CO3
(510 mg, 3.7 mmol), KI (57 mg, 0.34 mmol) and Cu
(22 mg, 0.35 mmol) and the mixture was heated at
110 ꢁC for 12 h. The reaction mixture was then filtered
hot, the precipitate was washed with CH2Cl2 and the fil-
trate was concentrated in vacuo. The residue was dis-
solved in CH2Cl2, washed with water, dried (Na2SO4)
and evaporated to dryness. Flash chromatography on
silica gel using cyclohexane–EtOAc 35:15 as the eluent
provided compound 2 (860 mg, 84%). Mp 129–131 ꢁC
(EtOAc–n-hexane); 1H NMR (CDCl3, 400 MHz) d
1.27 (t, J = 7 Hz, 3H, CH2CH3), 2.19 (s, 3H, CH3),
4.30 (q, J = 7 Hz, 2H, CH2CH3), 6.85 (d, J = 9 Hz,
2 Hz, 2H, H-20, H-60), 6.91 (d, J = 8 Hz, 1H, H-3),
7.15 (dt, J = 8 Hz, 1 Hz, 1H, H-5), 7.45 (m, 3H, H-30,
H-50, H-4), 7.92 (dd, J = 8 Hz, 1H, H-6); 13C NMR
(CDCl3, 50 MHz) d 14.53 (CH2CH3), 24.86 (CH3),
61.28 (CH2CH3), 117.55 (C-20, C-60), 119.11 (C-10),
120.37 (C-3), 121.30 (C-30, C-50), 123.18 (C-5), 131.16
(C-6), 133.17 (C-40), 133.49 (C-4), 153.16 (C-10), 156.61
(C-2), 165, 99 (COOEt), 168.81 (CONH). Anal. Calcd
for C17H17NO4. Calcd (%): C: 68.22, H: 5.72, N: 4.68.
Found (%): C: 68.36, H: 5.51, N: 4.32.
4.1.3. Ethyl 2-(4-amino-3-nitrophenyloxy)benzoate (4). A
9% HCl solution (20 mL) was added to a stirred solution
of 3 (1.79 g, 5.2 mmol) in ethanol and the resulting mix-
ture was heated under reflux for 12 h. Ethanol was then
vacuum-evaporated and the resulting aqueous layer was
extracted with CH2Cl2 (3 · 40 mL). The combined or-
ganic extracts were washed with 10% K2CO3 and brine,
dried (Na2SO4) and concentrated to dryness. The resi-
due was purified by column chromatography (silica
gel), using a mixture of cyclohexane–EtOAc 2:1 as the
eluent, to provide compound 4 as an oil (1.48 g, 94%).
1H NMR (CDCl3, 400 MHz) d 1.32 (t, J = 7 Hz, 3H,
CH2CH3), 4.35 (q, J = 7 Hz, 2H, CH2CH3), 6.83 (d,
J = 8 Hz, H-50), 7.01 (d, J = 8 Hz, 1H, H-3), 7.19 (dd,
4.1.2. Ethyl 2-(4-acetamido-3-nitrophenyloxy)benzoate (3).
To a solution of 2 (1 g, 3.34 mmol) in glacial acetic acid
(10 mL) was added dropwise, at room temperature, a
solution of fuming nitric acid (415 lL, 10 mmol) in

3450
I. K. Kostakis et al. / Bioorg. Med. Chem. 16 (2008) 3445–3455
J = 8 Hz, 1 Hz, H-60), 7.22 (dt, J = 8 Hz, 1 Hz, 1H, H-
5), 7.52 (t, J = 8 Hz, H-4), 7.67 (d, J = 2 Hz, H-20),
7.95 (dd, J = 8 Hz, 1H, H-6); 13C NMR (CDCl3,
50 MHz) d 14.14 (CH2CH3), 61.12 (CH2CH3), 113.76
(C-20), 120.21 (C-60), 120.27 (C-3), 123.22 (C-1), 123.85
(C-5), 128.39 (C-50), 131.37 (C-30), 131.96 (C-6), 133.74
(C-4), 141.56 (C-40), 147.75 (C-10), 155.99 (C-2), 165.57
(COOEt). Anal. Calcd for C15H14N2O5. Calcd (%): C:
59.60, H: 4.67, N: 9.27. Found (%): C: 59.45, H: 4.93,
N: 8.98.
(CH2CH3), 42.50 (40NHCOCH2), 42.77 (50NHCOCH2),
61.46 (CH2CH3), 112.78 (C-60), 121.68 (C-1), 121.92
(C-40), 122.37 (C-3), 123.78 (C-30), 125.92 (C-5), 132.63
(C-6), 134.37 (C-4), 135.98 (C-20), 136.92 (C-50), 151.11
(C-10), 153.44 (C-2), 164.96 (COOEt), 164.97 (2· COCH2).
Anal. Calcd for C19H17Cl2N3O7. Calcd (%): C: 48.53, H:
3.64, N: 8.94. Found (%): C: 48.21, H: 3.95, N: 8.61.
4.1.6. 2-Chloromethyl-5-nitro-1H,11H-xantheno[1,2-d]
imidazol-11-one (9). Compound 7 (327 mg, 0.87 mmol)
was added to a 50% H2SO4 solution (10 mL), at 0 ꢁC,
and the reaction mixture was then heated at 110 ꢁC for
3 h. After cooling, the reaction mixture was poured into
ice-water and the precipitating carboxylic acid 8 was fil-
tered, washed with water and air-dried. This compound
was subsequently dissolved in concentrated sulfuric acid
(5 mL) at 0 ꢁC, the resulting mixture was heated at
110 ꢁC for 2 h and upon cooling it was poured into
ice-water. The precipitate was filtered, washed with
water and air-dried to give crude 9, which was purified
by column chromatography (silica gel) using a mixture
of cyclohexane–EtOAc 4:1 as the eluent, to obtain pure
4.1.4. Ethyl 2-[3,4-bis(chloroacetamido)phenyloxy]benzo-
ate (6). A suspension of 4 (1 g, 3.34 mmol) and 10% Pd
on charcoal (450 mg) in absolute ethanol (90 mL) was
stirred under 50 psi of hydrogen for 7 h. The reaction
mixture was filtered through Celite, washed with ethanol
and the solvent was vacuum-evaporated to afford the
amine 5, which was used at the next step without further
purification. The amine was dissolved in anhydrous
CH2Cl2 (200 mL) and to this solution were added drop-
wise, at 0 ꢁC, triethylamine (1.67 mL, 12 mmol) and
chloroacetyl chloride (0.64 mL, 8 mmol). The resulting
solution was stirred under argon, for 5 h and then
washed with a 9% HCl solution, water and brine, dried
(Na2SO4) and concentrated to dryness. The residue was
purified by column chromatography (silica gel), using
cyclohexane–EtOAc 4:1 as the eluent, to provide com-
pound 6 (1.22 g, 86%). Mp 167–169 ꢁC (EtOAc–n-hex-
1
compound 9 (143 mg, 50%). Mp > 270 ꢁC (DMF); H
NMR (DMSO-d6, 400 MHz) d 3.15 (s, 2H, CH2), 7.59
(t, J = 8 Hz, 1H, H-9), 7.75 (d, J = 8 Hz, 1H, H-7),
7.94 (t, J = 8 Hz, 1H, H-8), 8.28 (d, J = 8 Hz, 1H, H-
10), 8.88 (s, 1H, H-4); 13C NMR (DMSO-d6, 50 MHz)
d 37.61 (CH2), 108.94 (C-11a), 118.88 (C-7), 121.31
(C-10a), 123.66 (C-4), 125.52 (C-9), 125.87 (C-10),
134.37 (C-3a), 134.38 (C-5), 136.38 (C-8), 137.11 (C-
11b), 146.47 (C-5a), 155.33 (C-6a), 156.40 (C-2),
175.35 (C-11). Anal. Calcd for C15H8ClN3O4. Calcd
(%): C: 54.65, H: 2.45, N: 12.74. Found (%): C: 54.52,
H: 2.40, N: 12.67.
1
ane); H NMR (CDCl3, 400 MHz) d 1.21 (t, J = 7 Hz,
3H, CH2CH3), 4.11 (s, 2H, 40CH2CONH), 4.15 (s, 2H,
30CH2CONH), 4.22 (q, J = 7 Hz, 2H, CH2CH3), 6.75
(dd, J = 9 Hz, 3 Hz, 1H, H-60), 6.98 (d, J = 8 Hz, 1H,
H-3), 7.05 (d, J = 3 Hz, 1H, H-20), 7.20 (t, J = 8 Hz,
1H, H-5), 7.31 (d, J = 9 Hz, 1H, H-50), 7.46 (t,
J = 8 Hz, 1H, H-4), 7.89 (dd, J = 8 Hz, 1H, H-6), 8.69
(s, 1H, D2O exch, 30NHCOCH2), 8.78 (s, 1H, D2O exch,
40NHCOCH2); 13C NMR (CDCl3, 50 MHz) d 14.13
(CH2CH3), 47.78 (2· NHCOCH2), 61.30 (CH2CH3),
113.98 (C-20), 115.92 (C-60), 121.76 (C-3), 123.83 (C-
40), 124.03 (C-1), 124.55 (C-5), 127.08 (C-50), 131.39
(C-30), 132.07 (C-6), 133.93 (C-4), 155.07 (C-2), 156.67
(C-10), 165.38 (2· COCH2), 165.72 (NHCO, COOEt).
Anal. Calcd for C19H18Cl2N2O5. Calcd (%): C: 53.66,
H: 4.27, N: 6.59. Found (%): C: 54.00, H: 3.97, N: 6.76.
4.1.7. N,N-Dimethyl-[[5-nitro-11-oxo-1H,11H-xantheno
[1,2-d]imidazol-2-yl]methan]amine (10a). To a solution
of compound 9 (330 mg, 1 mmol) in absolute thanol
(15 mL) was added a 33% solution of dimethylamine
in absolute ethanol (1.6 mL, 9 mmol) and the mixture
was refluxed for 4 h. Upon cooling, the mixture was vac-
uum-evaporated, and the residue was purified by col-
umn chromatography (silica gel, CH2Cl2–MeOH 95:5)
to furnish the target compound (297 mg, 88%). Mp
(hydrochloride) >270 ꢁC (ethanol); 1H NMR
(CDCl3 + CD3OD, 400 MHz) d 2.41 (s, 6H, N(CH3)2),
3.88 (s, 2H, NCH2), 7.43 (t, J = 8 Hz, 1H, H-9), 7.57
(d, J = 8 Hz, 1H, H-7), 7.77 (t, J = 8 Hz, 1H, H-8),
8.19 (d, J = 8 Hz, 1H, H-10), 8.62 (s, 1H, H-4); 13C
NMR (CDCl3 + CD3OD, 50 MHz) d 45.50 (N(CH3)2),
56.52 (NCH2), 108.53 (C-11a), 118.60 (C-7), 121.10 (C-
10a), 122.87 (C-4), 125.28 (C-9), 125.68 (C-10), 133.94
(C-3a), 134.58 (C-5), 135.72 (C-8), 136.18 (C-11b),
146.48 (C-5a), 155.55 (C-6a), 156.51 (C-2), 175.99
(C-11). Anal. Calcd for C17H14N4O4ÆHClÆ1/2H2O. Calcd
(%): C: 53.20, H: 4.20, N: 14.60. Found (%): C: 53.11, H:
4.21, N: 14.57.
4.1.5. Ethyl 2-[4,5-bis(chloroacetamido)-2-nitrophenyl-
oxy]benzoate (7). To a solution of 6 (850 mg, 2 mmol)
in a 1:5 mixture of concentrated sulfuric acid and glacial
acetic acid (10 mL) at 0 ꢁC was added dropwise a solu-
tion of fuming nitric acid (87 lL, 2.1 mmol) in glacial
acetic acid (0.2 mL). The reaction mixture was stirred
for 25 min at room temperature and was then poured
into ice/water, the resulting solid was filtered, washed
with water and air-dried to provide compound 7
1
(893 mg, 95%). Mp 169–171 ꢁC (EtOAc–n-hexane); H
NMR (CDCl3, 400 MHz) d 1.23 (t, J = 7 Hz, 3H,
CH2CH3), 4.15 (s, 2H, 50CH2CONH), 4.26 (q,
J = 7 Hz, 2H, CH2CH3), 4.28 (s, 2H, 40CH2CONH),
7.17 (d, J = 8 Hz, 1H, H-3), 7.35 (s, 1H, H-60), 7.38 (t,
J = 8 Hz, 1H, H-5), 7.62 (t, J = 8 Hz, 1H, H-4), 8.07
(dd, J = 8 Hz, 1H, H-6), 8.12 (s, 1H, H-30), 8.63 (s,
1H, D2O exch, 40NHCOCH2), 8.93 (s, 1H, D2O exch,
50NHCOCH2); 13C NMR (CDCl3, 50 MHz) d 13.96
4.1.8. N,N-Diethyl-[[5-nitro-11-oxo-1H,11H-xantheno
[1,2-d]imidazol-2-yl]methan]amine (10b). This compound
was prepared by a procedure analogous to that of
10a. Yield: 93%; mp (hydrochloride) >270 ꢁC (ethanol);
1H NMR (CDCl3 + CD3OD, 400 MHz) d 1.49 (t,

I. K. Kostakis et al. / Bioorg. Med. Chem. 16 (2008) 3445–3455
3451
J = 7 Hz, 6H, N(CH2CH3)2), 3.46 (q, J = 7 Hz, 4H,
N(CH2CH3)2), 4.81 (s, 2H, NCH2), 7.49 (t, J = 8 Hz,
1H, H-9), 7.63 (d, J = 8 Hz, 1H, H-7), 7.85 (t, J = 8 Hz,
1H, H-8), 8.17 (d, J = 8 Hz, 1H, H-10), 8.65 (s, 1H, H-
4); 13C NMR (CDCl3 + CD3OD, 50 MHz) d 12.56
(N(CH2CH3)2), 48.52 (N(CH2CH3)2), 56.62 (NCH2),
109.11 (C-11a), 118.49 (C-7), 121.44 (C-10a), 123.07
(C-4), 125.36 (C-9), 125.51 (C-10), 134.34 (C-3a),
134.67 (C-5), 136.52 (C-8), 136.77 (C-11b), 146.04
(C-5a), 156.74 (C-6a), 156.11 (C-2), 176.67 (C-11). Anal.
Calcd for C19H18N4O4ÆHClÆH2O. Calcd (%): C: 54.22, H:
5.03, N: 13.31. Found (%): C: 54.09, H: 5.11, N: 13.28.
(5 mL). The resulting solution was stirred for 10 min
at 0 ꢁC and then at room temperature for 2 h. The sol-
vent was then evaporated to dryness and the residue
was purified by column chromatography (silica gel)
using a mixture of CH2Cl2–MeOH 6:1 as the eluent to
provide compound 12a (121 mg, 63%); mp 193–195 ꢁC
(EtOAc–n-hexane); 1H NMR (CDCl3 + CD3OD,
400 MHz) d 2.41 (s, 6H, CH2N(CH3)2), 3.93 (s, 2H,
NCH2), 4.29 (s, 2H, COCH2), 7.52 (t, J = 8 Hz, 1H,
H-9), 7.65 (d, J = 8 Hz, 1H, H-7), 7.86 (t, J = 8 Hz,
1H, H-8), 8.33 (d, J = 8 Hz, 1H, H-10), 8.93 (s, 1H, H-
4), 9.99 (s, 1H, D2O exch, NHCO). Anal. Calcd for
C19H17ClN4O3. Calcd (%): C: 59.30, H: 4.45, N: 14.56.
Found (%): C: 59.11, H: 4.06, N: 14.72.
4.1.9. 5-Nitro-2-(pyrrolidin-1-yl-methyl)-1H,11H-xanthe-
no[1,2-d]imidazol-11-one (10c). This compound was pre-
pared by a procedure analogous to that of 10a. Yield:
4.1.12. 2-Chloro-N-[2(diethylaminomethyl)-11-oxo-1H,
11H-xantheno[1,2-d]imidazol-5-yl]acetamide (12b). This
compound was prepared by a procedure analogous to
that of 12a. Yield: 56%; mp 178–180 ꢁC (EtOAc–n-hex-
1
90%; mp (hydrochloride) >270 ꢁC (ethanol); H NMR
(CDCl3, 400 MHz) d 1.88 (m, 4H, 3,4-pyrrolidinyl-H),
2.74 (m, 4H, 2,5-pyrrolidinyl-H), 4.10 (s, 2H, NCH2),
7.45 (t, J = 8 Hz, 1H, H-9), 7.64 (d, J = 8 Hz, 1H,
H-7), 7.79 (t, J = 8 Hz, 1H, H-8), 8.24 (dd, J = 8 Hz,
1 Hz, 1H, H-10), 8.71 (s, 1H, H-4); 13C NMR (CDCl3,
50 MHz) d 23.79 (3,4-pyrrolidinyl-C), 53.48 (NCH2),
54.53 (2,5-pyrrolidinyl-C), 108.27 (C-11a), 118.82
(C-7), 121.13 (C-10a), 123.52 (C-4), 125.26 (C-9),
125.77 (C-10), 133.74 (C-3a), 134.83 (C-5), 135.69 (C-
8), 136.93 (C-11b), 146.48 (C-5a), 155.75 (C-6a),
157.08 (C-2), 176.29 (C-11). Anal. Calcd for
C19H16N4O4ÆHClÆ1/2H2O. Calcd (%): C: 55.68, H:
4.43, N: 13.67. Found (%): C: 55.97, H: 4.09, N: 13.43.
1
ane); H NMR (CDCl3 + CD3OD, 400 MHz) d 1.43 (t,
J = 7 Hz, 6H, CH2N(CH2CH3)2), 3.54 (q, J = 7 Hz,
4H, CH2N(CH2CH3)2), 4.28 (s, 2H, COCH2), 4.41 (s,
2H, NCH2), 7.39 (t, J = 8 Hz, 1H, H-9), 7.61 (d,
J = 8 Hz, 1H, H-7), 7.76 (t, J = 8 Hz, 1H, H-8), 8.31
(d, J = 8 Hz, 1H, H-10), 8.77 (s, 1H, H-4), 9.27 (s, 1H,
D2O exch, NHCO). Anal. Calcd for C21H21ClN4O3.
Calcd (%): C: 61.09, H: 5.13, N: 13.57. Found (%): C:
60.86, H: 4.85, N: 13.76.
4.1.13. 2-Chloro-N-[2-(pyrrolidin-1-yl-methyl)-11-oxo-1H,
11H-xantheno[1,2-d]imidazol-5-yl]acetamide (12c). This
compound was prepared by a procedure analogous to
that of 12a. Yield: 54%; mp 188–190 ꢁC (EtOAc–n-hex-
ane); 1H NMR (CDCl3 + CD3OD, 400 MHz) d 1.85 (m,
4H, 3,4-pyrrolidinyl-H), 2.83 (m, 4H, 2,5-pyrrolidinyl-
H), 4.30 (s, 2H, COCH2), 4.32 (s, 2H, NCH2), 7.45 (t,
J = 8 Hz, 1H, H-9), 7.58 (d, J = 8 Hz, 1H, H-7), 7.91
(t, J = 8 Hz, 1H, H-8), 8.36 (d, J = 8 Hz, 1H, H-10),
8.86 (s, 1H, H-4), 9.12 (s, 1H, D2O exch, NHCO). Anal.
Calcd for C21H19ClN4O3. Calcd (%): C: 61.39, H: 4.66,
N: 13.64. Found (%): C: 61.56, H: 4.39, N: 13.88.
4.1.10. 5-Nitro-2-(piperidin-1-yl-methyl)-1H,11H-xanthe-
no[1,2-d]imidazol-11-one (10d). This compound was pre-
pared by a procedure analogous to that of 10a. Yield:
1
91%; mp (hydrochloride) >270 ꢁC (ethanol); H NMR
(CDCl3, 400 MHz) d 1.43 (m, 2H, 4-piperidinyl-H),
1.64 (m, 4H, 3,5-piperidinyl-H), 2.47 (m, 4H, 2,6-pipe-
ridinyl-H), 3.82 (s, 2H, NCH2), 7.48 (t, J = 8 Hz, 1H,
H-9), 7.67 (d, J = 8 Hz, 1H, H-7), 7.81 (t, J = 8 Hz,
1H, H-8), 8.32 (d, J = 8 Hz, 1H, H-10), 8.72 (s, 1H, H-
4); 13C NMR (CDCl3, 50 MHz) d 23.89 (4-piperidinyl-
C), 25.92 (3,5-piperidinyl-C), 55.03 (2,6-piperidinyl-C),
56.92 (NCH2), 108.34 (C-11a), 118.54 (C-7), 121.35
(C-10a), 123.72 (C-4), 125.47 (C-9), 125.68 (C-10),
133.56 (C-3a), 134.68 (C-5), 135.63 (C-8), 136.56
(C-11b), 146.32 (C-5a), 155.84 (C-6a), 156.77 (C-2),
176.32 (C-11). Anal. Calcd for C20H18N4O4ÆHClÆH2O.
Calcd (%): C: 55.50, H: 4.89, N: 12.94. Found (%): C:
55.36, H: 5.21, N: 13.19.
4.1.14. 2-Chloro-N-[2-(piperidin-1-yl-methyl)-11-oxo-1H,
11H-xantheno[1,2-d]imidazol-5-yl]acetamide (12d). This
compound was prepared by a procedure analogous to
that of 12a. Yield: 49%; mp 198–200 ꢁC (dec) (EtOAc–
n-hexane); 1H NMR (CDCl3 + CD3OD, 400 MHz) d
1.46 (m, 2H, 4-piperidinyl-H), 1.66 (m, 4H, 3,5-piperid-
inyl-H), 2.49 (m, 4H, 2,6-piperidinyl-H), 4.26 (s, 2H,
NCH2), 4.31 (s, 2H, COCH2), 7.47 (t, J = 8 Hz, 1H,
H-9), 7.57 (d, J = 8 Hz, 1H, H-7), 7.82 (t, J = 8 Hz,
1H, H-8), 8.34 (d, J = 8 Hz, 1H, H-10), 9.01 (s, 1H, H-
4), 9.12 (s, 1H, D2O exch, NHCO). Anal. Calcd for
C22H21ClN4O3. Calcd (%): C: 62.19, H: 4.98, N: 13.19.
Found (%): C: 61.91, H: 5.27, N: 12.85.
4.1.11. 2-Chloro-N-[2(dimethylaminomethyl)-11-oxo-1H,
11H-xantheno[1,2-d]imidazol-5-yl]acetamide (12a).
A
solution of the amine 10a (149 mg, 0.5 mmol) in abso-
lute ethanol (40 mL) was hydrogenated in the presence
of 10% Pd/C (30 mg), under a pressure of 50 psi at room
temperature for 3 h. The resulting mixture was filtered
through a Celite pad and the filtrate was evaporated
to dryness to result in an oil corresponding to the 5-
aminoderivative 11a. Without further purification, the
residue was dissolved under argon in dry THF
(20 mL) and to this solution were added dropwise at
0 ꢁC triethylamine (280 lL, 2 mmol) and a solution of
chloroacetyl chloride (0.04 mL, 0.5 mmol) in dry THF
4.1.15. 2-Dimethylamino-N-[2(dimethylaminomethyl)-11-
oxo-1H,11H-xantheno[1,2-d]imidazol-5-yl]acetamide (13a).
To a solution of the chloride 12a (154 mg, 0.4 mmol) in
absolute ethanol (10 mL) was added a 33% solution of
dimethylamine in ethanol (360 lL, 2 mmol) and the
resulting solution was heated at reflux for 8 h. Upon
cooling, the solvent was vacuum evaporated and the

3452
I. K. Kostakis et al. / Bioorg. Med. Chem. 16 (2008) 3445–3455
residue was purified by column chromatography (silica
gel) using a mixture of CH2Cl2–MeOH 6:1 as the eluent,
to provide compound 13a (146 mg, 93%). Mp (dihydro-
chloride) >270 ꢁC (ethanol); 1H NMR (CDCl3,
400 MHz) d 2.39 (s, 6H, CH2N(CH3)2), 2.54 (s, 6H,
COCH2N(CH3)2), 3.24 (s, 2H, COCH2), 3.86 (s, 2H,
NCH2), 7.45 (t, J = 8 Hz, 1H, H-9), 7.53 (d, J = 8 Hz,
1H, H-7), 7.77 (t, J = 8 Hz, 1H, H-8), 8.35 (d,
J = 8 Hz, 1H, H-10), 8.98 (s, 1H, H-4), 9.95 (s, 1H,
D2O exch, NHCO); 13C NMR (CDCl3, 50 MHz) d
45.73 (CH2N(CH3)2), 46.27 (COCH2N(CH3)2), 57.53
(NCH2), 63.76 (COCH2), 111.62 (C-11a), 118.02 (C-7),
118.62 (C-4), 121.44 (C-10a), 121.67 (C-5), 124.55 (C-
9), 126.22 (C-10), 134.77 (C-8), 134.98 (C-3a), 138.37
(C-11b), 142.12 (C-5a), 153.77 (C-6a), 155.52 (C-2),
169.00 (COCH2), 176.98 (C-11). Anal. Calcd for
C21H23N5O3Æ2HClÆ1/2H2O. Calcd (%): C: 53.06, H:
5.51, N: 14.73. Found (%): C: 52.91, H: 5.47, N: 14.70.
54.15, H: 6.00, N: 12.63. Found (%): C: 54.51, H: 5.67,
N: 12.48.
4.1.18. 2-(Piperidin-1-yl)-N-[2-(piperidin-1-yl-methyl)-11-
oxo-1H,11H-xantheno[1,2-d]imidazol-5-yl]acetamide (13d).
This compound was prepared by a procedure analogous
to that of 13a. Yield: 94%; mp (dihydrochloride)
1
>270 ꢁC (ethanol); H NMR (CDCl3, 400 MHz) d 1.45
(m, 2H, CH2-4-piperidinyl-H), 1.56 (m, 2H, COCH2-4-
piperidinyl-H), 1.69 (m, 4H, CH2-3,5-piperidinyl-H),
1.76 (m, 4H, COCH2-3,5-piperidinyl-H), 2.50 (m, 4H,
CH2-2,6-piperidinyl-H), 2.63 (m, 4H, COCH2-2,6-pipe-
ridinyl-H), 3.14 (s, 2H, COCH2), 4.12 (s, 2H, NCH2),
7.39 (t, J = 8 Hz, 1H, H-9), 7.55 (d, J = 8 Hz, 1H, H-
7), 7.85 (t, J = 8 Hz, 1H, H-8), 8.29 (d, J = 8 Hz, 1H,
H-10), 8.95 (s, 1H, H-4), 10.01 (s, 1H, D2O exch,
NHCO); 13C NMR (CDCl3, 50 MHz) d 24.02 (CH2-4-
piperidinyl-C), 24.06 (COCH2-4-piperidinyl-C), 25.88
(CH2-3,5-piperidinyl-C), 26.71 (COCH2-3,5-piperidi-
nyl-C), 54.99 (CH2-2,6-piperidinyl-C), 55.00 (COCH2-
2,6-piperidinyl-C), 56.12 (NCH2), 62.56 (COCH2),
111.83 (C-11a), 117.86 (C-7), 119.00 (C-4), 121.52 (C-
10a), 121.88 (C-5), 124.59 (C-9), 125.71 (C-10), 134.79
(C-8), 135.08 (C-3a), 138.56 (C-11b), 142.183 (C-5a),
154.12 (C-6a), 155.90 (C-2), 169.15 (COCH2), 177.12
(C-11). Anal. Calcd for C27H31N5O3Æ2HClÆ3/2H2O.
Calcd (%): C: 56.55, H: 6.33, N: 12.21. Found (%): C:
56.91, H: 5.57, N: 11.92.
4.1.16.
2-Diethylamino-N-[2(diethylaminomethyl)-11-
oxo-1H,11H-xantheno[1,2-d]imidazol-5-yl]acetamide (13b).
This compound was prepared by a procedure analogous
to that of 13a. Yield: 93%; mp (dihydrochloride) 268–
1
270 ꢁC (ethanol); H NMR (CDCl3, 400 MHz) d 1.27
(t, J = 7 Hz, 6H, COCH2N(CH2CH3)2) 1.54 (t,
J = 7 Hz, 6H, CH2N(CH2CH3)2), 2.80 (q, J = 7 Hz,
4H, COCH2N(CH2CH3)2), 3.32 (s, 2H, COCH2), 3.49
(q, J = 7 Hz, 4H, CH2N(CH2CH3)2), 4.56 (s, 2H,
NCH2), 7.41 (t, J = 8 Hz, 1H, H-9), 7.49 (d, J = 8 Hz,
1H, H-7), 7.83 (t, J = 8 Hz, 1H, H-8), 8.44 (d, J = 8 Hz,
1H, H-10), 8.88 (s, 1H, H-4), 10.22 (s, 1H, D2O exch,
4.1.19. Ethyl 2-(4-chloroacetamido-3-nitrophenyloxy)ben-
zoate (14). To a solution of 4 (2.11 g, 7 mmol) in glacial
acetic acid (10 mL) was added chloroacetyl chloride
(670 lL, 8.4 mmol) and the resulting mixture was heated
at 120 ꢁC for 6 h. The mixture was then allowed to cool
at room temperature, ethanol (2 mL) was added and the
solvent was vacuum-evaporated. The residue was dis-
solved in CH2Cl2, washed with a 10% Na2CO3 solution,
dried over Na2SO4, and the solvent was evaporated to
dryness. Flash chromatography on silica gel using a
mixture of cyclohexane–EtOAc 6:1 as the eluent pro-
NHCO); 13C NMR (CDCl3, 50 MHz)
(CH2N(CH2CH3)2), 12.61 (COCH2N(CH2CH3)2),
d
12.54
47.46 (COCH2N(CH2CH3)2), 47.62 (CH2N(CH2CH3)2),
56.52 (NCH2), 58.46 (COCH2), 112.03 (C-11a), 117.73
(C-7), 119.11 (C-4), 121.34 (C-10a), 122.07 (C-5),
124.51 (C-9), 125.96 (C-10), 134.54 (C-8), 135.05 (C-
3a), 137.66 (C-11b), 143.52 (C-5a), 154.26 (C-6a),
154.99 (C-2), 168.66 (COCH2), 177.12 (C-11). Anal.
Calcd for C25H31N5O3Æ2HClÆH2O. Calcd (%): C: 55.56,
H: 6.53, N: 12.96. Found (%): C: 55.44, H: 6.55, N: 12.87.
1
vided compound 14 (2.57 g, 97%) as an oil. H NMR
(CDCl3, 400 MHz) d 1.19 (t, J = 7 Hz, 3H, CH2CH3),
4.25 (s, 2H, COCH2), 4.24 (q, J = 7 Hz, 2H, CH2CH3),
7.05 (d, J = 8 Hz, 1H, H-3), 7.20–7.33 (m, 2H, H-5, H-
60), 7.55 (t, J = 8 Hz, 1H, H-4), 7.66 (d, J = 2 Hz, 1H,
H-20), 7.97 (dd, J = 8 Hz, 1H, H-6), 8.66 (d, J = 8 Hz,
1H, H-50); 13C NMR (CDCl3, 50 MHz) d 14.23
(CH2CH3), 42.53 (COCH2), 61.21 (CH2CH3), 112.91
(C-20), 122.32 (C-3), 123.57 (C-50), 124.82 (C-1), 124.56
(C-5), 125.45 (C-60), 128.24 (C-40), 132.36 (C-6), 134.12
(C-4), 137.50 (C-30), 153.93 (C-2), 154.45 (C-10), 165.07
(COOEt), 164.81 (COCH2). Anal. Calcd for
C17H15ClN2O6. Calcd (%): C: 53.91, H: 3.99, N: 7.40.
Found (%): C: 54.11, H: 4.27, N: 7.15.
4.1.17. 2-(Pyrrolidin-1-yl)-N-[2-(pyrrolidin-1-yl-methyl)-
11-oxo-1H,11H-xantheno[1,2-d]imidazol-5-yl]acetamide
(13c). This compound was prepared by a procedure
analogous to that of 13a. Yield: 91%; mp (dihydrochlo-
1
ride) >270 ꢁC (ethanol); H NMR (CDCl3, 400 MHz) d
1.90 (m, 4H, CH2-3,4-pyrrolidinyl-H), 1.95 (m, 4H,
COCH2-3,4-pyrrolidinyl-H), 2.76 (m, 4H, CH2-2,5-pyr-
rolidinyl-H), 2.82 (m, 4H, COCH2-2,5-pyrrolidinyl-H),
3.37 (s, 2H, COCH2), 4.50 (s, 2H, NCH2), 7.40 (t,
J = 8 Hz, 1H, H-9), 7.52 (d, J = 8 Hz, 1H, H-7), 7.79
(t, J = 8 Hz, 1H, H-8), 8.30 (d, J = 8 Hz, 1H, H-10),
8.91 (s, 1H, H-4), 9.98 (s, 1H, D2O exch, NHCO); 13C
NMR (CDCl3, 50 MHz) d 23.91 (CH2-3,4-pyrrolidinyl-
C), 24.11 (COCH2-3,4-pyrrolidinyl-C), 54.29 (CH2-2,5-
pyrrolidinyl-C), 54.33 (COCH2-2,5-pyrrolidinyl-C),
55.63 (NCH2), 59.32 (COCH2), 112.06 (C-11a), 118.05
(C-7), 119.53 (C-4), 121.50 (C-10a), 122.12 (C-5),
124.68 (C-9), 125.96 (C-10), 134.68 (C-8), 135.12 (C-
3a), 138.85 (C-11b), 142.96 (C-5a), 153.97 (C-6a),
155.88 (C-2), 169.08 (COCH2), 177.00 (C-11). Anal.
Calcd for C25H27N5O3Æ2HClÆ2H2O. Calcd (%): C:
4.1.20.
Ethyl-2-[4-[(dimethylamino)acetamido]-3-nitro-
phenyloxy)benzoate (15a). This compound was prepared
by a procedure analogous to that of 13a, starting from
14. The reaction took place at room temperature upon
stirring for 12 h and the product was purified by column
chromatography (silica gel, cyclohexane–EtOAc 4/1 to
1
1/1). Yield: 91%; Oil; H NMR (CDCl3, 400 MHz) d
1.18 (t, J = 7 Hz, 3H, CH2CH3), 3.09 (s, 6H,

I. K. Kostakis et al. / Bioorg. Med. Chem. 16 (2008) 3445–3455
3453
N(CH3)2), 3.21 (s, 2H, NCH2), 4.20 (q, J = 7 Hz, 2H,
CH2CH3), 7.01 (d, J = 8 Hz, 1H, H-3), 7.14–7.26 (m,
2H, H-5, H-60), 7.51 (t, J = 8 Hz, 1H, H-4), 7.62 (d,
J = 2 Hz, 1H, H-20), 7.93 (dd, J = 8 Hz, 1H, H-6), 8.71
(d, J = 8 Hz, 1H, H-50); 13C NMR (CDCl3, 50 MHz) d
14.00 (CH2CH3), 45.87 (N(CH3)2), 61.08 (CH2CH3),
63.70 (NHCH2), 112.92 (C-20), 121.92 (C-3), 123.54
(C-50), 124.05 (C-1), 124.71 (C-5), 125.01 (C-60), 129.20
(C-40), 132.17 (C-6), 133.90 (C-4), 137.03 (C-30), 153.38
(C-10), 154.23 (C-2), 164.85 (COOEt), 165.88 (COCH2).
Anal. Calcd for C19H21N3O6. Calcd (%): C: 58.91, H:
5.46, N: 10.85. Found (%): C: 59.12, H: 5.23, N: 11.03.
6H, N(CH2CH3)2), 1.18 (t, J = 7 Hz, 3H, CH2CH3),
2.49 (q, J = 7 Hz, 4H, N(CH2CH3)2) 3.76 (br s, 2H,
NCH2), 4.21 (q, J = 7 Hz, 2H, CH2CH3), 6.81–7.02
(m, 2H, H-3, H-60), 7.10–7.18 (m, 2H, H-5, H-70), 7.34
(t, J = 8 Hz, 1H, H-4), 7.43 (br s, 1H, H-40), 7.91 (dd,
J = 8 Hz, 1H, H-6). Anal. Calcd for C21H25N3O3. Calcd
(%): C: 68.64, H: 6.86, N: 11.44. Found (%): C: 68.51, H:
6.56, N: 11.72.
4.1.24. Ethyl-2-[[2-chloromethyl-1H-benzimidazol-5-yl]
oxy]benzoate (18). A solution of 6 (425 mg, 1 mmol) in
phosphorus oxychloride (20 mL) was stirred at 90 ꢁC for
2 h. Upon cooling, the reaction mixture was poured into
ice-water, made alkaline with a 10% Na2CO3 solution
andextractedwithCH2Cl2 (3 · 40 mL). Thecombinedor-
ganicextractswerewashedwithbrine, dried(Na2SO4)and
concentrated in dryness. Flash chromatography on silica
gel, using a mixture of cyclohexane–EtOAc 1:1, provided
compound 18 (307 mg, 93%) as an oil; 1H NMR (CDCl3,
400 MHz) d 1.23 (t, J = 7 Hz, 3H, CH2CH3), 4.26 (q,
J = 7 Hz, 2H, CH2CH3), 4.75 (s, 2H, CH2), 6.89 (d,
J = 8 Hz, H-3), 6.91 (dd, J = 9 Hz, 1.5 Hz, 1H, H-20),
7.03 (d, J = 1.5 Hz, 1 Hz, H-70), 7.12 (dt, J = 8 Hz, 1 Hz,
1H, H-5), 7.39 (dt, J = 8 Hz, 1 Hz, 1H, H-4), 7.47 (d,
J = 9 Hz, H-30), 7.88 (dd, J = 8 Hz, 1H, H-6); 13C NMR
(CDCl3, 50 MHz) d 14.15 (CH2CH3), 37.87 (CH2), 61.62
(CH2CH3), 103.97 (C-70), 115.51 (C-20), 116.88 (C-30),
120.19 (C-3), 122.90 (C-1), 123.24 (C-5), 131.72 (C-6),
133.61 (C-4), 135.19 (C-30a), 137.86 (C-60a), 149.75 (C-
50), 153.93 (C-10), 156.85 (C-2), 165.98 (COOEt). Anal.
Calcd for C17H15ClN2O3. Calcd (%): C: 61.73, H: 4.57,
N: 8.47. Found (%): C: 61.39, H: 4.72, N: 8.29.
4.1.21. Ethyl-2-[4-[(diethylamino)acetamido]-3-nitrophe-
nyloxy)benzoate (15b). This compound was prepared
by a procedure analogous to that of 15a. Yield: 88%;
1
oil; H NMR (CDCl3, 400 MHz) d 1.20 (t, J = 7 Hz,
6H, N(CH2CH3)2), 1.23 (t, J = 7 Hz, 3H, CH2CH3),
2.72 (q, J = 7 Hz, 4H, N(CH2CH3)2) 3.27 (s, 2H,
NCH2), 4.25 (q, J = 7 Hz, 2H, CH2CH3), 7.04 (d,
J = 8 Hz, 1H, H-3), 7.25 (dd, J = 8 Hz, 2 Hz, 1H, H-
60), 7.29 (t, J = 8 Hz, 1H, H-5), 7.54 (t, J = 8 Hz, 1H,
H-4), 7.65 (d, J = 2 Hz, 1H, H-20), 7.97 (dd, J = 8 Hz,
1H, H-6), 8.72 (d, J = 8 Hz, H-50); 13C NMR (CDCl3,
50 MHz) d 10.65(N(CH2CH3)), 12.53 (CH2CH3), 48.16
(N(CH2CH3)2), 57.54 (NHCH2), 61.28 (CH2CH3),
111.93 (C-20), 121.30 (C-3), 123.18 (C-50), 124.12 (C-5),
124.73 (C-1), 125.05 (C-60), 128.48 (C-40), 131.62 (C-6),
133.49 (C-4), 137.86 (C-30), 153.80 (C-10), 154.74 (C-2),
165.06 (COOEt), 165.62 (COCH2). Anal. Calcd for
C19H21N3O6. Calcd (%): C: 60.71, H: 6.07, N: 10.11.
Found (%): C: 60.46, H: 5.83, N: 10.56.
4.1.22. Ethyl-2-[[2-[(dimethylamino)methyl]-1H-benzimida-
zol-5-yl]oxy]benzoate (17a). A suspension of the amine
15a (774 mg, 2 mmol) and 10% Pd/C (50 mg) in absolute
ethanol (40 mL) was hydrogenated under a pressure of
50 psi at room temperature for 3 h. The reaction mixture
was filtered through Celite, washed with ethanol and the
solvent was evaporated to afford the amine 16a, which
was used at the next step without further purification.
The amine was dissolved in anhydrous toluene
(30 mL), to this solution was added glacial acetic acid
(2 mL) and the resulting solution was refluxed for
40 min. The solvent was vacuum-evaporated and the
residue was dissolved in CH2Cl2, washed with 10%
Na2CO3 solution, dried (Na2SO4) and the solvent was
concentrated to dryness. Flash chromatography on sil-
ica gel using a mixture of CH2Cl2–MeOH 100:1 pro-
vided compound 17a (576 mg, 85%) as a mixture of
both tautomers. Oil; 1H NMR (CDCl3, 400 MHz) d
1.27 (t, J = 7 Hz, 3H, CH2CH3), 2.43 (s, 6H,
N(CH3)2), 3.70 (br s, 2H, NCH2), 4.31 (q, J = 7 Hz,
2H, CH2CH3), 6.81–7.03 (m, 2H, H-3, H-60), 7.11–7.21
(m, 2H, H-5, H-70), 7.45 (t, J = 8 Hz, 1H, H-4), 7.50
(br s, 1H, H-40), 7.96 (dd, J = 8 Hz, 1H, H-6). Anal.
Calcd for C19H21N3O3. Calcd (%): C: 67.24, H: 6.24,
N: 12.38. Found (%): C: 66.93, H: 5.87, N: 12.65.
4.1.25. Ethyl-2-[[2-[(pyrrolidin-1-yl)methyl]-1H-benzimida-
zol-5-yl]oxy]benzoate (17c). This compound was pre-
pared by a procedure analogous to that of 10a, starting
from 18. Yield: 85%; Isolated as an oil; 1H NMR
(CDCl3, 400 MHz) d 1.19 (t, J = 7 Hz, 3H, CH2CH3),
1.74 (m, 4H, 3,4-pyrrolidinyl-H), 2.57 (m, 4H, 2,5-pyr-
rolidinyl-H), 3.86 (br s, 2H, NCH2), 4.22 (q, J = 7 Hz,
2H, CH2CH3), 6.83–6.89 (m, 2H, H-3, H-60), 7.00–7.08
(m, 2H, H-5, H-70), 7.32 (t, J = 8 Hz, 1H, H-4), 7.42
(br s, 1H, H-40), 7.82 (dd, J = 8 Hz, 1H, H-6). Anal.
Calcd for C21H23N3O3. Calcd (%): C: 69.02, H: 6.34,
N: 11.50. Found (%): C: 68.77, H: 6.59, N: 11.12.
4.1.26. Ethyl-2-[[2-[(piperidin-1-yl)methyl]-1H-benzimida-
zol-5-yl]oxy]benzoate (17d). This compound was pre-
pared by a procedure analogous to that of 10a, starting
from 18. Yield: 90%; Isolated as an oil; 1H NMR
(CDCl3, 400 MHz) d 1.25 (t, J = 7 Hz, 3H, CH2CH3),
1.42 (m, 2H, 4-piperidinyl-H), 1.54 (m, 4H, 3,5-piperid-
inyl-H), 2.45 (m, 4H, 2,6-piperidinyl-H), 3.72 (br s, 2H,
NCH2), 4.28 (q, J = 7 Hz, 2H, CH2CH3), 6.87–6.93 (m,
2H, H-3, H-60), 7.07–7.13 (m, 2H, H-5, H-70), 7.37 (t,
J = 8 Hz, 1H, H-4), 7.47 (br s, 1H, H-40), 7.87 (dd,
J = 8 Hz, 1H, H-6). Anal. Calcd for C22H25N3O3. Calcd
(%): C: 69.64, H: 6.64, N: 11.07. Found (%): C: 69.95, H:
6.37, N: 10.84.
4.1.23. Ethyl-2-[[2-[(diethylamino)methyl]-1H-benzimidaz-
ol-5-yl]oxy]benzoate (17b). This compound was prepared
by a procedure analogous to that of 17a. Yield: 83%;
4.1.27. N,N-Dimethyl[(11-oxo-1H,11H-xantheno[1,2-d]
imidazol-2-yl)methan]amine (20a). To a solution of the
ester 17a (441 mg, 1.3 mmol) in ethanol (12 mL) was
1
Oil; H NMR (CDCl3, 400 MHz) d 0.92 (t, J = 7 Hz,

3454
I. K. Kostakis et al. / Bioorg. Med. Chem. 16 (2008) 3445–3455
added dropwise, at room temperature within 20 min, a
cold 20% NaOH solution (3 mL). The mixture was stir-
red for 2 h at room temperature and then poured into
water and neutralized (pH ꢀ 7) with a 9% HCl solution.
The resulting carboxylic acid 19a was filtered, air-dried
and was then dissolved in a 2/1 mixture of concentrated
sulfuric acid and glacial acetic acid (15 mL). The result-
ing mixture was heated at 90 ꢁC for 4 h and upon cool-
ing it was poured into ice-water and made alkaline, with
a cold 20% NaOH solution (pH ꢀ 10). The precipitate
was filtered and air-dried to give crude 20a, which was
purified by column chromatography (silica gel) using a
mixture of CH2Cl2–MeOH 15:1 as the eluent to provide
pure compound 20a (347 mg, 91%). Mp (hydrochloride)
for C19H17N3O2ÆHClÆ3/2H2O. Calcd (%): C: 59.61, H:
5.53, N: 10.98. Found (%): C: 59.32, H: 5.87, N: 10.61.
4.1.30. 2-(Piperidin-1-yl-methyl)-1H,11H-xantheno[1,2-
d]imidazol-11-one (20d). This compound was prepared
by a procedure analogous to that of 20a, starting from
17d. Yield: 93%; mp (hydrochloride) 235–237 ꢁC (etha-
1
nol); H NMR (CDCl3, 400 MHz) d 1.42 (m, 2H, 4-
piperidinyl-H), 1.60 (m, 4H, 3,5-piperidinyl-H), 2.46
(m, 4H, 2,6-piperidinyl-H), 3.79 (s, 2H, NCH2), 7.29
(d, J = 9 Hz, 1H, H-5), 7.36 (t, J = 8 Hz, 1H, H-9),
7.49 (d, J = 8 Hz, 1H, H-7), 7.69 (t, J = 8 Hz, 1H, H-
8), 7.98 (d, J = 9 Hz, 1H, H-4), 8.29 (dd, J = 8 Hz,
1 Hz, 1H, H-10), 11.23 (s, 1H, D2O exch., NH); 13C
NMR (CDCl3, 50 MHz) d 23.98 (4-piperidinyl-C),
25.94 (3,5-piperidinyl-C), 54.98 (NCH2), 57.21 (2,6-pipe-
ridinyl-C), 108.20 (C-11a), 111.43 (C-5), 118.19 (C-7),
121.64 (C-10a), 123.88 (C-9), 125.84 (C-10), 126.66 (C-
4), 130.77 (C-11b), 134.55 (C-8), 139.18 (C-3a), 153.17
(C-5a), 153.90 (C-6a), 156.27 (C-2), 177.43 (C-11). Anal.
Calcd for C20H19N3O2ÆHClÆH2O. Calcd (%): C: 61.93,
H: 5.72, N: 10.83. Found (%): C: 62.27, H: 5.43, N:
11.14.
1
>270 ꢁC (ethanol); H NMR (CDCl3, 400 MHz) d 2.31
(s, 6H, N(CH3)2), 3.82 (s, 2H, NCH2), 7.27 (d,
J = 9 Hz, 1H, H-5), 7.33 (t, J = 8 Hz, 1H, H-9), 7.47
(d, J = 8 Hz, 1H, H-7), 7.67 (t, J = 8 Hz, 1H, H-8),
7.96 (d, J = 9 Hz, 1H, H-4), 8.26 (d, J = 8 Hz, 1H, H-
10); 13C NMR (CDCl3, 50 MHz) d 45.50 (N(CH3)2),
57.33 (NCH2), 108.06 (C-11a), 111.41 (C-5), 117.95 (C-
7), 121.37 (C-10a), 123.72 (C-9), 125.67 (C-10), 126.44
(C-4), 130.59 (C-11b), 134.41 (C-8), 138.72 (C-3a),
153.02 (C-5a), 153.31 (C-6a), 155.99 (C-2), 177.09 (C-
11). Anal. Calcd for C17H15N3O2ÆHClÆ3/4H2O. Calcd
(%):C: 59.48, H: 5.14, N: 12.24. Found (%): C: 59.24,
H: 5.45, N: 11.88.
4.2. Biological evaluation
Materials. Eagle’s minimal essential medium (EMEM),
Foetal bovine serum (FBS), sodium pyruvate, sodium
bicarbonate, L-glutamine, nonessential amino acids,
penicillin, streptomycin, amphotericin B and gentamicin
were all obtained from Biochrom KG (Berlin,
Germany). Insulin was obtained from Sigma Chemicals
(Steinhelm, Germany). All other chemicals used were of
the best commercially available grade.
4.1.28.
N,N-Diethyl[(11-oxo-1H,11H-xantheno[1,2-d]
imidazol-2-yl)methan]amine (20b). This compound was
prepared by a procedure analogous to that of 20a, start-
ing from 17b. Yield: 93%; mp (hydrochloride) >270 ꢁC
(ethanol); 1H NMR (CDCl3, 400 MHz) d 1.03 (t,
J = 7 Hz, 6H, N(CH2CH3)2), 2.57 (q, J = 7 Hz, 4H,
N(CH2CH3)2), 3.87 (s, 2H, NCH2), 7.19 (d, J = 9 Hz,
1H, H-5), 7.27 (t, J = 8 Hz, 1H, H-9), 7.38 (d,
J = 8 Hz, 1H, H-7), 7.59 (t, J = 8 Hz, 1H, H-8), 7.90
(d, J = 9 Hz, 1H, H-4), 8.19 (d, J = 8 Hz, 1H, H-10);
13C NMR (CDCl3, 50 MHz) d 11.43 (N(CH2CH3)2),
47.26 (N(CH2CH3)2), 51.56 (NCH2), 108.17 (C-11a),
112.03 (C-5), 118.06 (C-7), 121.53 (C-10a), 123.67 (C-
9), 125.32 (C-10), 126.54 (C-4), 130.63 (C-11b), 134.40
(C-8), 139.11 (C-3a), 153.00 (C-5a), 153.42 (C-6a),
156.23 (C-2), 176.13 (C-11). Anal. Calcd for
C19H19N3O2ÆHClÆ1/2H2O. Calcd (%): C: 62.21, H:
5.77, N: 11.46. Found (%): C: 62.56, H: 5.41, N: 11.79.
4.2.1. Cell culture conditions. MDA-MB-231 (HTB 26:
human breast adenocarcinoma, ER-negative, high inva-
sive potential) was obtained from the American Type
Culture Collection (ATCC) and cultured as monolayers
at 37 ꢁC in a humidified atmosphere of 5% (v/v) CO2
and 95% air. Cells were seeded in 75-cm2 plastic tissue
culture flasks. Cancer cells were cultured in EMEM sup-
plemented with 10% FBS, 2 mM L-glutamine, 1.0 mM
sodium pyruvate, 1.5 g/L sodium bicarbonate, 0.1 mM
non-essential amino acids, 0.01 mg/mL of insulin and
a cocktail of antimicrobial agents (100 IU/mL penicillin,
100 lg/mL streptomycin, 10 lg/mL gentamicin sulfate
and 2.5 lg/mL amphotericin B).13 According to pilot
experiments in respect to growth rate and doubling time,
the medium was changed every three days. The cells
were harvested after treatment with 0.25% (w/v) trypsin
in PBS, containing 0.1% (w/v) Na2EDTA.
4.1.29. 2-(Pyrrolidin-1-yl-methyl)-1H,11H-xantheno[1,2-d]
imidazol-11-one (20c). This compound was prepared by
a procedure analogous to that of 20a, starting from
17c. Yield: 93%; mp (hydrochloride) 247–249 ꢁC (etha-
1
nol); H NMR (CDCl3, 400 MHz) d 1.80 (m, 4H, 3,4-
pyrrolidinyl-H), 2.61 (m, 4H, 2,5-pyrrolidinyl-H), 3.97
(s, 2H, NCH2), 7.30 (d, J = 9 Hz, 1H, H-5), 7.36 (t,
J = 8 Hz, 1H, H-9), 7.50 (d, J = 8 Hz, 1H, H-7), 7.69
(t, J = 8 Hz, 1H, H-8), 7.99 (d, J = 9 Hz, 1H, H-4),
8.29 (d, J = 8 Hz, 1H, H-10), 11.29 (s, 1H, D2O exch.,
NH); 13C NMR (CDCl3, 50 MHz) d 23.77 (3,4-pyrrolid-
inyl-C), 53.78 (NCH2), 54.49 (2,5-pyrrolidinyl-C),
108.23 (C-11a), 111.50 (C-5), 118.21 (C-7), 121.63 (C-
10a), 123.90 (C-9), 125.85 (C-10), 126.71 (C-4), 130.74
(C-11b), 134.40 (C-8), 139.09 (C-3a), 153.19 (C-6a),
154.16 (C-5a), 156.28 (C-2), 177.20 (C-11). Anal. Calcd
4.2.2. Cell proliferation. In order to evaluate the effects
of the new aminoderivatives on cell proliferation, cells
were seeded in the presence of serum into 96-well plates
at a density of 1 · 104 cells per well. Twenty-four hours
after plating, new medium supplemented with the amin-
oderivatives (1.0, 3.0, 10.0, 30.0 and 100.0 lM) to be
tested was added. The compounds were diluted in
DMSO and/or ethanol as a stock reagent and remained
stored at ꢁ20 ꢁC. To achieve the desirable concentra-
tions for the experiments, the stock reagents were

I. K. Kostakis et al. / Bioorg. Med. Chem. 16 (2008) 3445–3455
3455
5. Stefanska, B.; Dzieduszycka, M.; Bontemps-Gracz, M.
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Konieczny, M. T.; Paradziej-Lukowicz, J.; Martelli, S.
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diluted to appropriate final concentrations in the culture
media.14 After 72-h incubation, the medium was re-
placed with WST-1 (water-soluble tetrazolium salt).
Cells were incubated for 3 h and the quantification of
the formazan dye in the microplate was measured with
an ELISA plate reader at 450 nm (reference wavelength
at 650 nm). IC50 represents the concentration that re-
duces by 50% the optical density of treated cells with re-
spect to untreated controls.
Acknowledgments
The present study was supported in part from the Spe-
cial Account for Research Grants Committee of the Na-
tional and Kapodistrian University of Athens (Program
Kapodistrias).
´
´
Leonce, S.; Pierre, A. J. Med. Chem. 2002, 45, 2599; (c)
Kolokythas, G.; Kostakis, I. K.; Pouli, N.; Marakos, P.;
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