Design, synthesis, and cytotoxic activity evaluation of new linear pyranoxanthone aminoderivatives

Article abstract of DOI:10.1002/jhet.670

Figure represented. With the aim of enlightening some structure-activity correlation within the pyranoxanthenone series, we have designed and synthesized a number of new 5-aminosubstituted pyrano[3,2-b]xanthen-6-ones bearing various 12-substituents. In vi

Full text of DOI:10.1002/jhet.670

July 2011
Design, Synthesis, and Cytotoxic Activity Evaluation of New
Linear Pyranoxanthone Aminoderivatives
927
George Kolokythas,a Konstantinos Daniilides,a Nicole Pouli,a
Panagiotis Marakos,a* Harris Pratsinis,b and Dimitris Kletsasb
aDivision of Pharmaceutical Chemistry, Department of Pharmacy, University of Athens,
Panepistimiopolis-Zografou, Athens 15771, Greece
bLaboratory of Cell Proliferation and Ageing, Institute of Biology, NCSR ‘‘Demokritos,’’ 15310
Athens, Greece
*E-mail: marakos@pharm.uoa.gr
Received June 17, 2010
DOI 10.1002/jhet.670
Published online 21 April 2011 in Wiley Online Library (wileyonlinelibrary.com).
With the aim of enlightening some structure-activity correlation within the pyranoxanthenone series,
we have designed and synthesized a number of new 5-aminosubstituted pyrano[3,2-b]xanthen-6-ones
bearing various 12-substituents. In vitro cytotoxic potencies of the new derivatives toward the murine
leukemia L1210 cell line, human colorectal adenocarcinoma (HT-29), and human uterine sarcoma
(MES-SA and its 100-fold resistant to doxorubicin variant MES-SA/Dx5) cell lines, are described and
compared with that of reference drugs. Among the studied compounds, those possessing a second ami-
nosubstituted side-chain exhibit interesting cytotoxic activity against the solid tumor cell lines, and they
retain activity against the multidrug resistant MES-SA/Dx5 subline. Their selective effect on a phase of
the cell cycle was evaluated using HT-29 cells providing evidence that the compounds induce a G0/G1
arrest.
J. Heterocyclic Chem., 48, 927 (2011).
INTRODUCTION
Recent evidence concerning the interaction with biologi-
cal targets allows for the design of novel polycyclic
compounds, endowed with better antiproliferative activ-
ity and/or the ability to overcome multidrug resistance.
Within this concept, the position and the nature of the
substituents on the heterocyclic nucleus are determinants
of the biological activity observed [8,9]. Of particular
importance is the presence of one or two flexible dialky-
laminoalkylamino-substituents, which usually increase
DNA binding affinity and solubility under physiological
conditions, yielding in an improvement of the pharma-
cological response of several acridone derivatives [10].
We have been involved in the design, synthesis, and
cytotoxic activity evaluation of a number of linear and
angular pyranoxanthenone derivatives [11,12] possessing
structural similarity with the pyranoacridone alkaloid acro-
nycine (Fig. 1), which has shown promising antitumor
properties on several murine solid tumor models [13].
In the course of the exploration of the structure-activ-
ity relationship studies in the pyranoxanthenone series,
Compounds based on tricyclic planar chromophore
framework represent a large and well-known family of
multitargeted anticancer agents. A great number of natu-
ral and synthetic molecules, mainly anthraquinone,
anthrapyrazole, and acridine derivatives have been tested
for cytotoxicity and a number of them underwent clini-
cal trials or have been approved for chemotherapy.
Well-known examples include the clinically useful anti-
cancer drug mitoxantrone, [1] 9-azaanthrapyrazoles, [2]
imidazoacridones, [3] acridine-4-carboxamide deriva-
tives [4], and pyrazoloacridine [5]. The mechanism of
action of this diverse class of compounds is not fully
understood, however, it is generally acknowledged to
involve DNA intercalation and poisoning of critical
enzymes, such as topoisomerases and telomerases. In
addition, some of these molecules often display second-
ary effects on other biochemical pathways, including
protein metabolism [6] and cell cycle regulation [7].
C
V
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928
G. Kolokythas, K. Daniilides, N. Pouli, P. Marakos, H. Pratsinis, and D. Kletsas
Vol 48
this group a nitro- or an additional basic side chain, to
study the impact of this structural modification on the
biological activity of this class of compounds.
RESULTS AND DISCUSSION
Chemistry. For the preparation of the target deriva-
tives, we used as starting material the dihydropyranoxan-
thenone 1 (Scheme 1), which was prepared according to
known procedures, from 1,3-dihydroxyxanthenone upon
reaction with 2-methyl-1,3-butadiene (isoprene), in the
presence of orthophosphoric acid [20]. Compound 1 was
then nitrated in high yield by treatment with fuming nitric
acid in acetic acid, to provide the nitroderivative 2.
Our attempts to prepare the pyranoxanthenone 3
through treatment of compound 2 with 2,3-dichloro-5,6-
dicyano-p-benzoquinone in boiling toluene [21,22] or
with 10% Pd/C in boiling diphenylether [23] were not
successful. Consequently, 2 was converted to the tosyl-
ate 4, which was treated with N-bromosuccinimide in
the presence of 2,20-diazodiisobutylnitrile (AIBN) to
provide the bromide 5, that upon dehydrohalogenation
was converted into the tosylate 6. The tosyloxy group of
compound 6 was easily displaced by the suitable amines
to result into the diamines 7a,b.
Figure 1. Structure of acronycine.
we have found that the replacement of the methoxy
group of the lead compound by a flexible dialkylami-
noethylamino side chain substitution results in a clear
improvement of the antiproliferative activity towards
both leukemia and solid tumor cell lines in the angular
as well as in the linear pyranoxanthenones [14–17]. A
similar approach, that is, the replacement of the
methoxy group at C-6 by a basic dialkylaminoalkyla-
mino side chain has also been performed in the acrony-
cine series, and resulted in a significant increase of the
cytotoxic activity against L1210 cells when compared
with the parent compounds [18]. Furthermore, we have
previously introduced a nitro group or a second basic
side chain on the pyran ring of angular pyranoxanthe-
nones and the biological evaluation of these derivatives
provided evidence that extended conjugation of the xan-
thenone chromophore is in favor of the cell growth in-
hibitory activity [19]. As a continuation of this study,
we decided to synthesize a number of structurally
related linear analogues, possessing a dialkylaminoethy-
lamino-side chain substitution and in position para- to
Reduction of the nitroderivatives 7a,b provided the
amines 8a,b (Scheme 2), which were not isolated due to
instability but were converted to the chloracetamides
9a,b upon treatment with chloracetylchloride. Subse-
quent reaction of the chloracetamides 9a,b with an
excess of the suitable secondary amine provided the tar-
get amides 10a,b.
Scheme 1. Reagents and conditions: (a) Fuming nitric acid, glacial acetic acid, 60ꢀC, 35 min; (b) 2,3-dichloro-5,6-dicyano-p-benzoquinone, tolu-
ene, reflux, 30 h, or 10% Pd/C, diphenylether, reflux, 24 h; (c) p-toluenesulfonyl chloride, potassium carbonate, acetone, reflux, 90 min; (d) N-bro-
mosuccinimide, 2,20-diazodiisobutylnitrile, carbon tetrachloride, reflux, 18 h; (e) triethylamine, 1,2-dichloroethane, 50ꢀC, 17 h; (f) N,N-
dialkylethylenediamine, abs. ethanol, reflux, 1 h.
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DOI 10.1002/jhet

July 2011
Design, Synthesis, and Cytotoxic Activity Evaluation of New Linear Pyranoxanthone
Aminoderivatives
929
Scheme 2. Reagents and conditions: (a) stannous chloride dihydrate, acetone, reflux, 5 h; (b) chloroacetylchloride, triethylamine, tetrahydrofuran,
room temperature, 3h; (c) dialkylamine, abs. ethanol, reflux, 18 h; (d) di-tert-butyl dicarbonate, 4-dimethylaminopyridine, tetrahydrofuran, room
temperature, 3 h; (e) sodium hydride, 2-dialkylaminoethylchloride, dimethylformamide, room temperature, 18 h; (f) trifluoroacetic acid, dichlorome-
thane, room temperature, 1 h.
On the other hand, the free amino group of com-
pounds 8a,b was protected through the formation of the
corresponding t-butyl carbamates 11a,b. The suitable
dialkylaminoethyl substitution was then introduced to
the sodium hydride mediated anions of the above men-
tioned carbamates and the resulting derivatives 12a,b
were treated with trifluoroacetic acid to result into the
target compounds 13a,b.
For biological evaluation purposes, the free base forms
of the amines 7a,b were converted into their water-solu-
ble hydrochloride addition salts by treatment with hydro-
chloric acid in methanol. Compounds 10a,b and 13a
were tested at the free base form, since their hydrochlor-
ide, fumarate, or malonate addition salts were highly hy-
groscopic, whereas compound 13b was converted into the
corresponding stable difumarate addition salt.
Biological evaluation. The in vitro cytotoxic activity
of the new compounds was evaluated in the established
model of the murine leukemia cell line L1210, and in
three human solid tumor cell lines: colorectal adenocarci-
noma HT-29, uterine sarcoma MES-SA as well as its vari-
ant MES-SA/Dx5, reported to be 100-fold resistant to dox-
orubicin [24]. The results, including reference compounds
mitoxantrone and doxorubicin, are presented in Table 1.
As a general remark, we assume that the 12-nitrosub-
stituted compounds 7a,b proved to be less active than
the corresponding derivatives 10a,b and 13a,b, which
contain a second basic side chain at position 12. The na-
ture of this side chain, that is, dialkylaminoacetamido-or
dialkylaminoethylamino-, is not critical for the activity.
Concerning the activity against L1210 cell line com-
pounds 10a,b and 13a,b possess IC50’s in the range of
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DOI 10.1002/jhet

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G. Kolokythas, K. Daniilides, N. Pouli, P. Marakos, H. Pratsinis, and D. Kletsas
Vol 48
Table 1
Inhibition of proliferation of the amino substituted xanthenone derivatives (IC50 values in lMa).
Compound
L1210
HT-29
MES-SA
MES-SA/Dx5
RFe
7ab
37.2 (65.7)
>100
43.1 (67.5)
8.04 (61.86)
9.20 (61.13)
5.72 (60.81)
8.2 (61.39)
4.09 (60.75)
0.025 (60.008)
0.153 (60.076)
30.2 (62.6)
94.8 (61.7)
17.0 (65.33)
12.2 (64.71)
11.0 (60.9)
11.3 (61.6)
0.003 (60.001)
0.0097 (60.0012)
6.47 (62.60)
40.7 (610.5)
9.13 (61.64)
4.71 (60.58)
2.75 (60.69)
3.32 (60.16)
0.028 (60.002)
0.704 (60.337)
0.2
0.4
0.5
0.4
0.4
0.3
9.3
72.6
7bb
10ac
17.1 (63.2)
8.83 (62.21)
10.4 (61.8)
11.0 (61.2)
0.038 (60.017)
0.118 (60.039)
10bc
13ac
13bd
Mitoxantrone
Doxorubicin
a The results represent the mean (6 standard deviation) of three independent experiments and are expressed as IC50. the concentration that reduced
by 50% the optical density of treated cells with respect to untreated controls.
b Hydrochloride.
c Free base form.
d Difumarate.
e IC50 resistant cells/IC50 sensitive cells.
8.83–17.1 lM. It should be noticed that previously
reported analogues, which lack a 12-substitution showed
more interesting antiproliferative activity, with IC50 val-
ues close to 5 lM [14]. However, compounds 10a,b and
13a,b possess interesting antiproliferative activity
against the colorectal adenocarcinoma HT-29 cell line.
In this case, the insertion of a second basic side chain
results in IC50’s within the range of 4.09–9.2 lM and
this provides an improvement over the antiproliferative
activity reported for the corresponding analogues, which
possess only a 5-(2-dialkylaminoethylamino)-side chain
and show IC50’s within the range of 8–23 lM [15].
Within the HT-29 cell line it is evident that the presence
of diethylamino-substitution results in an increased
growth inhibitory activity (IC50’s 5.72 lM and 4.09 lM,
for compounds 10b and 13b, respectively) when com-
pared to their dimethylamino-counterparts (IC50’s 9.2
lM and 8.2 lM, for compounds 10a and 13a, respec-
tively) and this is in accordance with previous observa-
tions in the xanthenone series [15,16].
three tested cell lines. However, the new compounds
appear more active against the doxorubicine resistant
phenotype (RF values 0.2–0.5) when compared with the
above mentioned xanthenones.
Cell-cycle perturbations induced after incubation of
exponentially growing colon adenocarcinoma HT-29
cells with the new compounds for 24 h are presented in
Table 2. All studied compounds provoke an increase in
the percentage of cells arrested at the G0/G1 phase of
the cell cycle, in agreement with our previous observa-
tions for the mode of action of other pyranoxanthenone
or azapyranoxanthenone aminoderivatives on HT-29
cells [15,16].
In summary, this study deals with the synthesis of a
number of new 5,12-disubstituted pyrano[3,2-b]xanthen-
6-ones. The evaluation of their antiproliferative activity
against a panel of cell lines, showed that the 5,12-diami-
nosubstituted derivatives possess enhanced activity
against the solid tumor cells and interestingly, they
retain full cytotoxicity against the doxorubicin resistant
cell line.
From a direct comparison of the activity toward the
sensitive and resistant cell lines, it is evident that the
compounds appear to be active against MES-SA cell
line and simultaneously possess higher cytotoxicity
against the doxorubicin resistant MES-SA/Dox-5 cell
line. This finding is more pronounced for compounds
13, especially 13b. Worth mentioning, the RF to doxor-
ubicin was found to be 72.6, that is, on the order of the
expected value [24], whereas the RF to mitoxantrone
was 9.3.
EXPERIMENTAL
All chemicals were purchased from Aldrich Chemical Co.
(Munich, Germany). Melting points were determined on a
1
Bu¨chi apparatus and are uncorrected. H-NMR spectra and 2D
spectra were recorded on a Bruker Avance 400 instrument,
whereas 13C-NMR specra were recorded on a Bruker AC 200
spectrometer in deuterated solvents and were referenced to
TMS (d scale). The signals of 1H and 13C spectra were unambig-
uously assigned by using 2D NMR techniques: 1H-1H COSY,
NOESY HMQC, and HMBC. Flash chromatography was per-
formed on Merck (Darmstadt, Germany) silica gel 60 (0.040–
0.063 mm). Analytical thin layer chromatography (TLC) was car-
ried out on precoated (0.25 mm) Merck silica gel F-254 plates.
Elemental analyses were within 6 0.4% of the theoretical values.
The incorporation of the pyran ring into the xanthe-
none skeleton, does not improve the antiproliferative
activity of the compounds. Indeed, the corresponding 1-
dialkylaminoethylamino-substituted 4-nitro- or 4-dialky-
laminoacetamido-xanthenones, which have been previ-
ously reported [25] possess lower IC50 values against all
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DOI 10.1002/jhet

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Design, Synthesis, and Cytotoxic Activity Evaluation of New Linear Pyranoxanthone
Aminoderivatives
931
Table 2
4-Bromo-3,4-dihydro-2,2-dimethyl-5-(4-methylbenzene-
sulfonyloxy)-12-nitro-2H,6H-pyrano[3,2-b]xanthen-6-one
(5). To a solution of the tosylate 4 (850 mg, 1.72 mmol) in
dry carbon tetrachloride (30 mL) was added N-bromosuccini-
mide (321 mg, 1.80 mmol) and a catalytic amount of AIBN,
and the mixture was stirred at room temperature for 15 min,
and then was heated at reflux for 18 h. The organic solvent
was vacuum-evaporated and the residue was purified by col-
umn chromatography (silica gel) using a mixture of cyclohex-
ane/ethyl acetate (95/5, v/v) as the eluent, to result in com-
pound 5 (730 mg, 74%). mp: 180–182ꢀC, (ethyl acetate-n-pen-
FACS analysis.
Compound
G0/G1
S
G2/M
7a
7b
62.98
61.85
54.72
68.87
59.18
59.35
40.49
28.25
28.18
30.24
21.21
30.12
40.65
38.99
8.77
9.96
10a
10b
13a
13b
ctrl
15.04
9.92
10.70
0.00
20.51
1
tane). H-NMR (400 MHz, deuteriochloroform) d: 1.46 (s, 3H,
1 ꢁ gem CH3), 1.65 (s, 3H, 1 ꢁ gem CH3), 2.49 (s, 3H,
SO2C6H4CH3), 2.56 (d, 2H, CH2-3, J ¼ 5 Hz), 5.77 (t, 1H, H-
4, J ¼ 5 Hz), 7.38 (m, 2H, H-8, H-10), 7.44 (d, 2H, H-30, H-
50, J ¼ 8 Hz), 7.70 (dt, 1H, H-9, J ¼ 8 Hz, 2 Hz), 8.11 (d,
2H, H-20, H-60, J ¼ 8 Hz), 8.18 (dd, 1H, H-7, J ¼ 8 Hz, 2
Hz). 13C-NMR (50 MHz, deuteriochloroform) d: 21.64
(SO2C6H4CH3), 27.30 (gem CH3), 28.19 (gem CH3), 34.65 (C-
4), 41.42 (C-3), 79.68 (C-2), 111.00 (C-5a), 116.29 (C-4a),
117.29 (C-10), 121.81 (C-6a), 125.00 (C-8), 126.70 (C-7),
128.33 (C-20, C-60), 129.31 (C-12), 129.71 (C-30, C-50), 133.21
(C-10), 135.04 (C-9), 146.07 (C-40), 146.99 (C-12a), 148.71
(C-11a), 150.55 (C-5), 153.90 (C-10a), 172.20 (C-6). Anal.
Calcd. for C25H20BrNO8S: C, 52.27; H, 3.51; N, 2.44. Found:
C, 52.51; H, 3.58; N, 2.41.
2,2-Dimethyl-5-(4-methylbenzenesulfonyloxy)-12-nitro-
2H, 6H-pyrano[3,2-b]xanthen-6-one (6). To a solution of
compound 5 (420 mg, 0.73 mmol) in 1,2-dichloroethane (25
mL) was added triethylamine (3 mL) and the mixture was
heated at 50ꢀC for 17 h. The organic solvent was vacuum-
evaporated and dichloromethane (50 mL) was added to the res-
idue. The organic phase was washed with a 9% hydrochloric
acid solution (2 ꢁ 30 mL), dried (sodium sulfate) and concen-
trated to dryness. The residue was purified by column chroma-
tography using a mixture of cyclohexane/dichloromethane (1/
1, v/v) as the eluent, to provide compound 6 (284 mg, 79%)
as a white solid. mp: 193–195ꢀC, (ethyl acetate-n-pentane).
1H-NMR (400 MHz, deuteriochloroform) d: 1.47 (s, 6H, 2 ꢁ
gem CH3), 2.42 (s, 3H, SO2C6H4CH3), 5.64 (d, 1H, H-3, J ¼
10 Hz), 6.36 (d, 1H, H-4, J ¼ 10 Hz,), 7.34 (m, 4H, H-8, H-
10, H-30, H-50), 7.66 (dt, 1H, H-9, J ¼ 8 Hz, 2 Hz), 7.90 (d,
2H, H-20, H-60, J ¼ 8 Hz,), 8.14 (dd, 1H, H-7, J ¼ 8 Hz, 2
Hz). 13C-NMR (50 MHz, deuteriochloroform) d: 21.58
(SO2C6H4CH3), 27.93 (2 ꢁ gem CH3), 80.56 (C-2), 110.56
(C-5a), 114.12 (C-4a), 115.04 (C-4), 117.24 (C-10), 121.88
(C-6a), 124.93 (C-8), 126.54 (C-7), 128.71 (C-20, C-60), 129.49
(C-12), 129.74 (C-30, C-50), 131.76 (C-3), 132.35 (C-10),
134.78 (C-9), 142.90 (C-12a), 145.76 (C-40), 148.45 (C-11a),
150.03 (C-5), 153.89 (C-10a), 172.48 (C-6). Anal. Calcd. for
C25H19NO8S: C, 52.37; H, 3.34; N, 2.44. Found: C, 52.23; H,
3.29; N, 2.47.
HT-29 cells were treated for 24 h with 20 lM (except from 7a. where
40 lM were used) and then subjected to DNA content analysis. One
representative determination out of three similar ones is demonstrated.
3,4-Dihydro-5-hydroxy-2,2-dimethyl-12-nitro-2H,6H-pyrano-
[3,2-b]xanthen-6-one (2). Fuming nitric acid (64 lL, 1.55
mmol) was added dropwise to a solution of the pyranoxanthe-
none 1 (460 mg, 1.55 mmol) in glacial acetic acid (10 mL)
and the resulting solution was stirred at 60ꢀC for 35 min. The
mixture was then poured into crushed ice (50 mL) and the pre-
cipitate was filtered, washed with water (3 ꢁ 15 mL) and dried
over calcium chloride to give pure compound 2 (470 mg,
89%). mp: 162–164ꢀC, (ethyl acetate-n-pentane). 1H-NMR
(400 MHz, deuteriochloroform) d: 1.40 (s, 6H, 2 ꢁ gem CH3),
1.90 (t, 2H, CH2-3, J ¼ 7 Hz), 2.72 (t, 2H, CH2-4, J ¼ 7 Hz),
7.35 (dt, 1H, H-8, J ¼ 8 Hz, 2 Hz), 7.37 (dd, 1H, H-10, J ¼ 8
Hz, 2 Hz), 7.70 (dt, 1H, H-9, J ¼ 8 Hz, 2 Hz), 8.16 (dd,1H,
H-7, J ¼ 8 Hz, 2 Hz), 13.36 (s, 1H, D2O exch., OH). 13C-
NMR (50 MHz, deuteriochloroform) d: 15.81 (C-4), 26.54 (2
ꢁ gem CH3), 31.09 (C-3), 78.98 (C-2), 101.15 (C-5a), 104.45
(C-4a), 117.91 (C-10), 120.11 (C-6a), 124.89 (C-8), 125.00
(C-12), 125.77 (C-7), 135.66 (C-9), 147.46 (C-11a), 153.89
(C-12a), 155.25 (C-10a), 161.39 (C-5), 180.35 (C-6). Anal.
Calcd. for C18H15NO6: C, 63.34; H, 4.43; N, 4.10. Found: C,
63.11; H, 4.38; N, 4.15.
3,4-Dihydro-2,2-dimethyl-5-(4-methylbenzenesulfonyloxy)-
12-nitro-2H,6H-pyrano[3,2-b]xanthen-6-one (4). To a solu-
tion of compound 2 (740 mg, 2.17 mmol) in acetone (30 mL)
was added potassium carbonate (1.50 g, 10.85 mmol) and p-
toluenesulfonylchloride (1.24 g, 6.51 mmol) and the mixture
was heated at reflux for 90 min. Upon cooling at room temper-
ature the precipitate was filtered off, washed with acetone (2
ꢁ 20 mL), the filtrate was vacuum-evaporated and the residue
was purified by column chromatography (silica gel) using a
mixture of cyclohexane/ethyl acetate (85/15 v/v) as the eluent,
to give compound 4 (910 mg, 85%) as a white solid. mp: 242–
244ꢀC, (ethyl acetate-n-pentane). 1H-NMR (400 MHz, deuter-
iochloroform) d: 1.38 (s, 6H, 2 ꢁ gem CH3), 1.82 (t, 2H,
CH2-3, J ¼ 7 Hz), 2.43 (s, 3H, SO2C6H4CH3), 2.82 (t, 2H,
CH2-4, J ¼ 7 Hz), 7.34 (m, 4H, H-8, H-10, H-30, H-50), 7.65
(dt, 1H, H-9, J ¼ 8 Hz, 2 Hz), 7.93 (d, 2H, H-20, H-60, J ¼ 8
Hz), 8.06 (dd, 1H, H-7, J ¼ 8 Hz, 2 Hz). 13C-NMR (50 MHz,
deuteriochloroform) d: 18.01 (C-4), 21.76 (SO2C6H4CH3),
26.65 (2 ꢁ gem CH3), 31.32 (C-3), 79.50 (C-2), 109.82 (C-
5a), 116.14 (C-4a), 117.43 (C-10), 122.13 (C-6a), 124.78 (C-
8), 126.69 (C-7), 128.93 (C-20, C-60), 129.41 (C-12), 129.74
(C-30, C-50), 133.16 (C-10), 134.81 (C-9), 145.73 (C-40),
146.39 (C-12a), 147.57 (C-11a), 151.46 (C-5), 154.22 (C-10a),
172.78 (C-6). Anal. Calcd. for C25H21NO8S: C, 60.60; H,
4.27; N, 2.83. Found: C, 60.47; H, 4.21; N, 2.92.
N,N-Dimethyl-N0-[2,2-dimethyl-12-nitro-6-oxo-2H,6H-pyr-
ano[3,2-b]xanthen-5-yl]ethanediamine (7a). 2-Dimethylami-
noethylamine (560 lL, 5.10 mmol) was added to a solution of
compound 6 (250 mg, 0.51 mmol) in absolute ethanol (30 mL)
and the mixture was heated at reflux for 1 h. Ethanol was vac-
uum-evaporated and dichloromethane (50 mL) was added to
the residue. The organic phase was washed with water (3 ꢁ
20 mL), dried (sodium sulfate) and concentrated to dryness.
The residue was purified by column chromatography using a
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G. Kolokythas, K. Daniilides, N. Pouli, P. Marakos, H. Pratsinis, and D. Kletsas
Vol 48
mixture of dichloromethane/methanol (98/2, v/v) as the eluent,
to give compound 7a (150 mg, 72%) as a light yellow oil. mp
(hydrochloride): 252–253ꢀC, (ethanol). 1H-NMR (400 MHz,
deuteriochloroform) d: 1.50 (s, 6H, 2 ꢁ gem CH3), 2.33 [s,
6H, N(CH3)2], 2.59 (t, 2H, NHCH2CH2NMe2, J ¼ 7 Hz), 3.60
(q, 2H, NHCH2CH2NMe2, J ¼ 7 Hz, 5 Hz), 5.53 (d, 1H, H-3,
J ¼ 10 Hz), 6.52 (d, 1H, H-4, J¼10 Hz), 7.35 (m, 2H, H-8,
H-10), 7.63 (dt, 1H, H-9, J ¼ 8 Hz, 2 Hz), 8.20 (dd, 1H, H-7,
J ¼ 8 Hz, 2 Hz), 10.30 (t, 1H, NH, J ¼ 5 Hz, D2O exch.).
13C-NMR (50 MHz, deuteriochloroform) d: 26.83 (2 ꢁ gem
CH3), 45.50 [N(CH3)2], 46.79 (NHCH2CH2NMe2), 59.65
(NHCH2CH2NMe2), 78.36 (C-2), 101.73 (C-4a), 102.21 (C-
5a), 117.28 (C-10), 119.93 (C-4), 120.79 (C-12), 121.54
(C-6a), 124.59 (C-8), 124.71 (C-3), 126.06 (C-7), 134.41 (C-
9), 150.80 (C-11a), 152.01 (C-5), 152.64 (C-12a), 154.03
(C-10a), 177.89 (C-6). Anal. Calcd. for C22H23N3O5 HCl: C,
59.26; H, 5.43; N, 9.42. Found: C, 59.01; H, 5.39; N, 9.68.
N,N-Diethyl-N0-[2,2-dimethyl-12-nitro-6-oxo-2H,6H-pyr-
ano[3,2-b]xanthen-5-yl]ethanediamine (7b). The synthetic
route was analogous to the one used for the synthesis of com-
pound 7a. Yield: 74%. mp: (hydrochloride): 216–217ꢀC, (etha-
nol). 1H-NMR (400 MHz, deuteriochloroform) d: 1.02 [t, 6H,
N(CH2CH3)2, J ¼ 7 Hz], 1.49 (s, 6H, 2 ꢁ gem CH3), 2.58 [q,
4H, N(CH2CH3)2, J ¼ 7 Hz], 2.66 (t, 2H, NHCH2CH2NEt2, J
¼ 7 Hz), 3.55 (q, 2H, NHCH2CH2NEt2, J ¼ 7 Hz, 5 Hz), 5.50
(d, 1H, H-3, J ¼ 10 Hz), 6.60 (d, 1H, H-4, J ¼ 10 Hz), 7.34
(m, 2H, H-10, H-8), 7.62 (dt, 1H, H-9, J ¼ 8 Hz, 2 Hz), 8.18
(dd, 1H, H-7, J ¼ 8 Hz, 2 Hz,), 10.25 (t, 1H, NH, J ¼ 5 Hz,
D2O exch.). 13C-NMR (50 MHz, deuteriochloroform) d: 11.54
[N(CH2CH3)2], 26.83 (2 ꢁ gem CH3), 47.16 (NHCH2CH2
NEt2) and [N(CH2CH3)2], 53.33 (NHCH2CH2NEt2), 78.32 (C-
2), 101.77 (C-4a), 102.29 (C-5a), 117.24 (C-10), 120.04 (C-4),
120.77 (C-12), 121.58 (C-6a), 124.56 (C-3, C-8), 125.99 (C-7),
134.33 (C-9), 150.80 (C-11a), 152.05 (C-5), 152.60 (C-12a),
154.03 (C-10a), 177.74 (C-6). Anal. Calcd. for C24H27N3O5
HCl H2O: C, 58.59; H, 6.15; N, 8.54. Found: C, 58.42; H,
6.06; N, 8.38.
was extracted with dichloromethane (3 ꢁ 50 mL). The com-
bined organic extracts were dried (sodium sulfate) and concen-
trated to dryness and the residue was purified by column chro-
matography using a mixture of dichloromethane/methanol (95/5,
v/v) as the eluent, to provide the chloride 9a (93 mg, 64%) as a
light yellow oil. 1H-NMR (400 MHz, deuteriochloroform) d:
1.44 (s, 6H, 2 ꢁ gem CH3), 2.30 [s, 6H, N(CH3)2], 2.55 (t, 2H,
NHCH2CH2NMe2, J ¼ 7 Hz), 3.50 (q, 2H, NHCH2CH2NMe2,
J ¼ 7 Hz, 5 Hz), 4.27 (s, 2H, NHCOCH2Cl), 5.45 (d, 1H, H-3,
J ¼ 10 Hz), 6.52 (d, 1H, H-4, J ¼ 10 Hz), 7.27 (m, 2H, H-8,
H-10), 7.55 (dt, 1H, H-9, J ¼ 8 Hz, 2 Hz), 7.70 (s, 1H,
NHCOCH2Cl, D2O exch.,), 8.15 (dd, 1H, H-7, J ¼ 8 Hz, 2
Hz), 9.68 (t, 1H, NHCH2CH2NMe2, J ¼ 5 Hz, D2O exch.,).
13C-NMR (50 MHz, deuteriochloroform) d: 27.22 (2 ꢁ gem
CH3), 44.78 [N(CH3)2], 45.95 (NHCH2CH2NMe2), 48.35
(NHCOCH2Cl), 58.22 (NHCH2CH2NMe2), 77.24 (C-2), 103.23
(C-12), 105.03 (C-4a), 105.10 (C-5a), 116.32 (C-10), 119.03 (C-
4), 121.65 (C-6a), 123.25 (C-8), 123.93 (C-3), 125.32 (C-7),
133.24 (C-9), 149.12 (C-5), 153.90 (C-11a), 154.92 (C-10a),
156.52 (C-12a), 163.42 (NHCOCH2Cl), 179.11 (C-6). Anal.
Calcd. for C24H26ClN3O4: C, 63.22; H, 5.75; N, 9.22. Found:
C, 63.40; H, 5.71; N, 9.05.
2-Chloro-N-[5-(2-diethylaminoethylamino)-2,2-dimethyl-
6-oxo-2H,6H-pyrano[3,2-b]xanthen-12-yl]acetamide (9b). The
amine 8b was first prepared according to the method reported
for the synthesis of the amine 8a. Analogously, this compound
was used to the next step without further purification and was
roughly spectroscopically characterized. 1H-NMR (400 MHz,
deuteriochloroform) d: 0.99 [t, 6H, N(CH2CH3)2, J ¼ 7 Hz],
1.48 (s, 6H, 2 ꢁ gem CH3), 2.53 [q, 4H, N(CH2CH3)2, J ¼ 7
Hz,], 2.63 (t, 2H, NHCH2CH2NEt2, J ¼ 7 Hz), 3.35 (q, 2H,
NHCH2CH2NEt2, J ¼ 7 Hz, 5 Hz), 5.53 (d, 1H, H-3, J ¼ 10
Hz), 6.63 (d, 1H, H-4, J ¼ 10 Hz), 7.27 (dt, 1H, H-8, J ¼ 8
Hz, 2 Hz), 7.36 (dd, 1H, H-10, J ¼ 8 Hz, 2 Hz), 7.58 (dt, 1H,
H-9, J ¼ 8 Hz, 2 Hz), 8.21 (dd, 1H, H-7, J ¼ 8 Hz, 2 Hz),
8.56 (t, 1H, NHCH2CH2NEt2, J ¼ 5 Hz, D2O exch.). Com-
pound 9b was then prepared as a light yellow oil, with a
method analogous to the one used for the preparation of 9a.
Yield: 67%. 1H-NMR (400 MHz, deuteriochloroform) d: 1.07
[t, 6H, N(CH2CH3)2, J ¼ 7 Hz], 1.45 (s, 6H, 2 ꢁ gem CH3),
2-Chloro-N-[5-(2-dimethylaminoethylamino)-2,2-dimethyl-6-
oxo-2H,6H-pyrano[3,2-b]xanthen-12-yl]acetamide (9a). To
a
solution of compound 7a (130 mg, 0.32 mmol) in acetone was
added stannous chloride dihydrate (289 mg, 1.28 mmol) and the
mixture was heated at reflux for 5 h. The solvent was then vac-
uum-evaporated and a 20% sodium carbonate solution (50 mL)
was added to the residue. The aqueous phase was washed with
ethyl acetate (3 ꢁ 50 mL) and the organic extracts were dried
(sodium sulfate) and concentrated to dryness, to provide the
amine 8a as oil, which was not particularly stable, but could be
spectroscopically characterized. 1H-NMR (400 MHz, deuterio-
chloroform) d: 1.45 (s, 6H, 2 ꢁ gem CH3), 2.23 [s, 6H,
N(CH3)2], 2.49 (t, 2H, NHCH2CH2NMe2, J ¼ 7 Hz), 3.33 (q,
2H, NHCH2CH2NMe2, J ¼ 7 Hz, 5 Hz), 5.51 (d, 1H, H-3, J ¼
10 Hz), 6.57 (d, 1H, H-4, J ¼ 10 Hz), 7.23 (dt, 1H, H-8, J ¼ 8
Hz, 2 Hz), 7.31 (dd, 1H, H-10, J ¼ 8 Hz, 2 Hz), 7.54 (dt, 1H,
H-9, J ¼ 8 Hz, 2 Hz), 8.18 (dd, 1H, H-7, J ¼ 8 Hz, 2 Hz),
8.62 (t, 1H, NHCH2CH2NMe2, J ¼ 5 Hz, D2O exch.). The
amine without any further purification was dissolved in dry tet-
rahydrofuran (15 mL), triethylamine (108 lL, 0.78 mmol), and
chloroacetylchloride (41 lL, 0.52 mmol) were added to the so-
lution at 0ꢀC, and the mixture was stirred at room temperature
for 3 h. The organic solvent was vacuum-evaporated, a 10% so-
dium carbonate solution (50 mL) was added to the residue and
2.65 [q, 4H, N(CH2CH3)2,
J
¼
7 Hz], 2.73 (t, 2H,
NHCH2CH2NEt2, J ¼ 7 Hz), 3.54 (q, 2H, NHCH2CH2NEt2, J
¼ 7 Hz, 5 Hz), 4.28 [s, 2H, NHCOCH2Cl), 5.48 (d, 1H, H-3,
J ¼ 10 Hz), 6.54 (d, 1H, H-4, J ¼ 10 Hz), 7.28 (m, 2H, H-10,
H-8), 7.57 (dt, 1H, H-9, J ¼ 8 Hz, 2 Hz), 7.71 (s, 1H,
NHCOCH2Cl, D2O exch.), 8.16 (dd, 1H, H-7, J ¼ 8 Hz, 2
Hz,), 9.62 (t, 1H, NHCH2CH2NEt2, J ¼ 5 Hz, D2O exch.).
13C-NMR (50 MHz, deuteriochloroform) d: 10.34
[N(CH2CH3)2], 27.10 (2 ꢁ gem CH3), 41.93 (NHCOCH2Cl),
46.39 [N(CH2CH3)2], 46.49 (NHCH2CH2NEt2), 52.52
(NHCH2CH2NEt2), 77.89 (C-2), 102.36 (C-12), 104.21 (C-4a),
104.49 (C-5a), 116.17 (C-10), 119.74 (C-4), 121.82 (C-6a),
122.99 (C-8), 123.73 (C-3), 125.36 (C-7), 132.92 (C-9),
151.08 (C-5), 153.98 (C-11a), 154.95 (C-10a), 156.54 (C-12a),
165.57 (NHCOCH2Cl), 178.75 (C-6). Anal. Calcd. for
C26H30ClN3O4: C, 64.52; H, 6.25; N, 8.68. Found: C, 64.23;
H, 6.14; N, 8.72.
2-Dimethylamino-N-[5-(2-dimethylaminoethylamino)-2,2-
dimethyl-6-oxo-2H,6H-pyrano[3,2-b]xanthen-12-yl]aceta-
mide (10a). Dimethylamine (33% solution in ethanol, 232
lL, 1.7 mmol) was added to a solution of the chloride 9a (80
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

July 2011
Design, Synthesis, and Cytotoxic Activity Evaluation of New Linear Pyranoxanthone
Aminoderivatives
933
1
mg, 0.17 mmol) in ethanol (30 mL) and the resulting solution
was heated at reflux for 18 h. The mixture was vacuum-evapo-
rated and the residue was purified by column chromatography
using a mixture of dichloromethane/methanol (92/8, v/v) as
the eluent, to give compound 10a (70 mg, 86%) as a light yel-
low oil 1H-NMR (400 MHz, deuteriochloroform) d: 1.46 (s,
6H, 2 ꢁ gem CH3), 2.34 [s, 6H, NHCH2CH2N(CH3)2], 2.48
[s, 6H, NHCOCH2N(CH3)2], 2.61 (t, 2H, NHCH2CH2NMe2,
J ¼ 7 Hz), 3.18 (s, 2H, NHCOCH2NMe2), 3.54 (q, 2H,
NHCH2CH2NMe2, J ¼ 7 Hz, 5 Hz), 5.50 (d, 1H, H-3, J ¼ 10
Hz), 6.58 (d, 1H, H-4, J ¼ 10 Hz), 7.28 (m, 2H, H-8, H-10),
7.57 (dt, 1H, H-9, J ¼ 8 Hz, 2 Hz), 8.19 (dd, 1H, H-7, J ¼ 8
Hz, 2 Hz,), 8.33 (s, 1H, NHCOCH2NMe2, D2O exch.), 9.62 (t,
1H, NHCH2CH2NMe2, J ¼ 5 Hz, D2O exch.). 13C-NMR (50
MHz, deuteriochloroform) d: 27.38 (2 ꢁ gem CH3), 44.80
[NHCH2CH2N(CH3)2], 45.23 [NHCOCH2N(CH3)2], 46.70
(86 mg, 68%) as a light yellow oil. H-NMR (400 MHz, deu-
teriochloroform) d: 1.35 [s, 9H, NHCOOC(CH3)3], 1.43 (s,
6H, 2 ꢁ gem CH3), 2.29 [s, 6H, NHCH2CH2N(CH3)2], 2.56 (t,
2H, NHCH2CH2NMe2,
J
¼
7 Hz), 3.55 (q, 2H, NH-
CH2CH2NMe2, J ¼ 7 Hz, 5 Hz), 5.50 (d, 1H, H-3, J ¼ 10
Hz), 6.63 (d, 1H, H-4, J ¼ 10 Hz), 7.32 (m, 2H, H-8, H-10),
7.59 (dt,1H, H-9, J ¼ 8 Hz, 2 Hz), 8.22 (dd, 1H, H-7, J ¼ 8
Hz, 2 Hz), 9.72 (t, 1H, NHCH2CH2NMe2, J ¼ 5 Hz, D2O
exch.). 13C-NMR (50 MHz, deuteriochloroform) d: 26.98 (2 ꢁ
gem CH3), 27.79 [NHCOOC(CH3)3], 45.58 [NH-
CH2CH2N(CH3)2],
47.41
(NHCH2CH2NMe2),
59.95
(NHCH2CH2NMe2), 76.89 (C-2), 81.89 [NHCOOC(CH3)3],
103.39 (C-4a), 103.87 (C-5a), 106.00 (C-12), 116.95 (C-10),
120.55 (C-4), 121.76 (C-6a), 123.68 (C-8), 124.34 (C-3),
126.10 (C-7), 133.82 (C-9), 150.47 (C-5), 151.35
[NHCOOC(CH3)3], 153.30 (C-11a), 154.51 (C-10a), 155.95
(C-12a), 178.44 (C-6). Anal. Calcd. for C27H33N3O5: C, 67.62;
H, 6.94; N, 8.76. Found: C, 67.47; H, 7.11; N, 8.93.
(NHCH2CH2NMe2),
58.99
(NHCH2CH2NMe2),
63.03
(NHCOCH2NMe2), 76.37 (C-2), 103.35 (C-12), 104.97 (C-4a),
105.05 (C-5a), 117.24 (C-10), 119.71 (C-4), 121.58 (C-6a),
123.93 (C-8), 124.71 (C-3), 126.03 (C-7), 134.15 (C-9),
149.26 (C-5), 153.67 (C-11a), 154.66 (C-10a), 156.24 (C-12a),
169.51 (NHCOCH2NMe2), 179.03 (C-6). Anal. Calcd. for
C26H32N4O4: C, 67.22; H, 6.94; N, 12.06. Found: C, 67.39; H,
6.85; N, 11.92.
N,N-Diethyl-N0-[12-(t-butoxycarbonylamino)-2,2-dimethyl-6-
oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl]ethanediamine (11b). The
synthetic route is analogous to the one followed for the prepa-
ration of compound 11a. Oil. Yield: 69%. 1H-NMR (400
MHz, deuteriochloroform) d: 1.00 [t, 6H, NHCH2CH2N-
(CH2CH3)2, J ¼ 7 Hz], 1.34 [s, 9H, NHCOOC(CH3)3], 1.42
(s, 6H, 2 ꢁ gem CH3), 2.56 [q, 4H, NHCH2CH2N(CH2CH3)2,
J ¼ 7 Hz], 2.67 (t, 2H, NHCH2CH2NEt2, J ¼ 7 Hz), 3.52 (q,
2H, NHCH2CH2NEt2, J ¼ 7 Hz, 5 Hz), 5.47 (d, 1H, H-3, J ¼
10 Hz), 6.63 (d, 1H, H-4, J ¼ 10 Hz), 7.30 (m, 2H, H-8, H-
10), 7.59 (dt, 1H, H-9, J ¼ 8 Hz, 2 Hz), 8.20 (dd, 1H, H-7, J
¼ 8 Hz, 2 Hz), 9.66 (t, 1H, NHCH2CH2NEt2, J ¼ 5 Hz, D2O
exch.). 13C-NMR (50 MHz, deuteriochloroform) d: 11.61
[NHCH2CH2N(CH2CH3)2], 26.94 (2 ꢁ gem CH3), 27.74
[NHCOOC(CH3)3], 47.19 [NHCH2CH2N(CH2CH3)2], 47.78
(NHCH2CH2NEt2), 53.55 (NHCH2CH2NEt2), 76.85 (C-2),
81.81 [NHCOOC(CH3)3], 103.35 (C-4a), 103.87 (C-5a),
105.85 (C-12), 116.92 (C-10), 120.59 (C-4), 121.77 (C-6a),
123.64 (C-8), 124.23 (C-3), 126.00 (C-7), 133.75 (C-9),
150.51 (C-5), 151.36 [NHCOOC(CH3)3], 153.27 (C-11a),
154.48 (C-10a), 155.92 (C-12a), 178.34 (C-6). Anal. Calcd. for
C29H37N3O5: C, 68.62; H, 7.35; N, 8.28. Found: C, 68.85 H,
7.47; N, 8.12.
2-Diethylamino-N-[5-(2-diethylaminoethylamino)-2,2-di-
methyl-6-oxo-2H,6H-pyrano[3,2-b]xanthen-12-yl]acetamide
(10b). The synthetic route is analogous to the one followed
for the preparation of compound 10a. Oil. Yield: 88%. 1H-
NMR (400 MHz, deuteriochloroform) d: 1.10 [t, 6H, NHCH2-
CH2N(CH2CH3)2, J ¼ 7 Hz], 1.19 [t, 6H, NHCOCH2N-
(CH2CH3)2, J ¼ 7 Hz], 1.45 (s, 6H, 2 ꢁ gem CH3), 2.72 [m,
10H,
NHCH2CH2N(CH2CH3)2,
NHCOCH2N(CH2CH3)2,
NHCH2CH2NEt2], 3.25 (s, 2H, NHCOCH2NEt2), 3.57 (q, 2H,
NHCH2CH2NEt2, J ¼ 7 Hz, 5 Hz), 5.51 (d, 1H, H-3, J ¼ 10
Hz), 1H, 6.58 (d, 1H, H-4, J ¼ 10 Hz), 7.25 (m, 2H, H-10 and
H-8), 7.57 (dt, 1H, H-9, J ¼ 8 Hz, 2 Hz), 8.17 (dd, 1H, H-7, J
¼ 8 Hz, 2 Hz), 8.66 (s, 1H, NHCOCH2NEt2, D2O exch.), 9.54
(t, 1H, NHCH2CH2NEt2, J ¼ 5 Hz, D2O exch.). 13C-NMR (50
MHz, deuteriochloroform) d: 11.06 [NHCH2CH2N(CH2CH3)2],
12.64 [NHCOCH2N(CH2CH3)2], 27.27 (2 ꢁ gem CH3), 47.30
[NHCH2CH2N(CH2CH3)2, NHCH2CH2NEt2], 49.18 [NH-
N,N-Dimethyl-N0-[12-[N-(t-butoxycarbonyl)-N-(2-dimethy-
laminoethyl)amino]-2,2-dimethyl-6-oxo-2H,6H-pyrano[3,2-b]
xanthen-5-yl]ethanediamine (12a). Sodium hydride (60% in
hexanes, 30 mg, 0.75 mmol) was added under argon at 0ꢀC to
a solution of compound 11a (70 mg, 0.15 mmol) in dry dime-
thylformamide (5 mL) and the mixture was stirred at room
temperature for 30 min. It was then cooled at 0ꢀC, a solution
of 2-dimethylaminoethylchloride (160 mg, 1.5 mmol) in dime-
thylformamide (3 mL) was added dropwise and the resulting
mixture was stirred for 18 h. Water (100 mL) was then added
and the aqueous phase was extracted with ethyl acetate (3 ꢁ
50 mL). The organic extracts were dried (sodium sulfate) and
concentrated to dryness and the residue was purified by col-
umn chromatography using a mixture of dichloromethane/
methanol (90/10, v/v) as the eluent to give compound 12a (50
COCH2N(CH2CH3)2],
53.29
(NHCH2CH2NEt2),
57.56
(NHCOCH2NEt2), 76.37 (C-2), 102.43 (C-12), 104.01 (C-4a),
104.56 (C-5a), 117.02 (C-10), 120.48 (C-4), 121.76 (C-6a),
123.68 (C-8), 124.56 (C-3), 125.99 (C-7), 133.78 (C-9),
150.21 (C-5), 153.78 (C-11a), 154.62 (C-10a), 156.20 (C-12a),
171.46 (NHCOCH2NEt2), 178.70 (C-6). Anal. Calcd. for
C30H40N4O4: C, 64.79; H, 7.25; N, 10.08. Found: C, 64.65; H,
7.37; N, 10.24.
N,N-Dimethyl-N0-[12-(t-butoxycarbonylamino)-2,2-dimethyl-6-
oxo-2H,6H-pyrano[3,2-b]xanthen-5-yl]ethanediamine (11a). To
a solution of the amine 8a (100 mg, 0.26 mmol) in dry tetra-
hydrofuran (20 mL) at 0ꢀC, were added 4-dimethylaminopyri-
dine (47 mg, 0.38 mmol) and di-tert-butyl dicarbonate (73 lL,
0.32 mmol) and the mixture was stirred at room temperature
for 3 h. The solvent was vacuum-evaporated, the residue was
dissolved in ethyl acetate (50 mL) and washed with water (2
ꢁ 50 mL). The organic extracts were dried (sodium sulfate)
and concentrated to dryness and the residue was purified by
column chromatography using a mixture of dichloromethane/
methanol (98/4, v/v) as the eluent, to provide compound 11a
1
mg, 62%) as a light yellow oil. H-NMR (400 MHz, deuterio-
chloroform) d: 1.25 [s, 9H, NHCOOC(CH3)3], 1.46 (s, 6H, 2
ꢁ gem CH3), 2.28 [s, 6H, N(Boc)CH2CH2N(CH3)2], 2.30 [s,
6H, NHCH2CH2N(CH3)2], 2.58 [m, 4H, NHCH2CH2NMe2,
N(Boc)CH2CH2N(CH3)2], 3.54 (q, 2H, NHCH2CH2NMe2, J ¼
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

934
G. Kolokythas, K. Daniilides, N. Pouli, P. Marakos, H. Pratsinis, and D. Kletsas
Vol 48
7 Hz, 5 Hz), 3.70 [t, 2H, N(Boc)CH2CH2NMe2, J ¼ 7 Hz],
5.49 (d, 1H, H-3, J ¼ 10 Hz), 6.60 (d, 1H, H-4, J ¼ 10 Hz),
7.31 (m, 2H, H-8, H-10), 7.61 (dt, 1H, H-9, J ¼ 8 Hz, 2 Hz),
J ¼ 8 Hz, 2 Hz), 8.20 (dd, 1H, H-7, J ¼ 8 Hz, 2 Hz), 8.98 (t,
1H, 5-NHCH2CH2NMe2, J ¼ 5 Hz, D2O exch.). 13C-NMR (50
MHz, deuteriochloroform) d: 27.24 (2 ꢁ gem CH3), 45.24
[12-NHCH2CH2N(CH3)2], 45.72 [5-NHCH2CH2N(CH3)2],
45.87 (12-NHCH2CH2NMe2), 48.41 (5-NHCH2CH2NMe2),
59.06 (12-NHCH2CH2NMe2), 60.09 (5-NHCH2CH2NMe2),
76.63 (C-2), 105.34 (C-4a), 105.48 (C-5a), 116.84 (C-12),
117.02 (C-10), 120.74 (C-4), 121.80 (C-6a), 123.46 (C-8),
124.89 (C-3), 126.21 (C-7), 133.71 (C-9), 146.21 (C-5),
149.95 (C-11a), 152.01 (C-12a), 154.81 (C-10a), 179.03 (C-6).
Anal. Calcd. for C26H34N4O3: C, 69.31; H, 7.61; N, 12.44.
Found: C, 69.18; H, 7.50; N, 12.22.
8.22 (dd, 1H, H-7,
J
¼
8 Hz, 2 Hz), 9.69 (t, 1H,
NHCH2CH2NMe2, J ¼ 5 Hz, D2O exch.). 13C-NMR (50 MHz,
deuteriochloroform) d: 27.38 (2
ꢁ
gem CH3), 28.19
[NHCOOC(CH3)3], 45.39 [N(Boc)CH2CH2N(CH3)2], 45.65
[NHCH2CH2N(CH3)2], 46.53 [N(Boc)CH2CH2NMe2], 47.64
(NHCH2CH2NMe2), 56.90 [N(Boc)CH2CH2NMe2], 60.09
(NHCH2CH2NMe2), 76.38 (C-2), 79.43 [NHCOOC(CH3)3],
103.57 (C-4a), 104.12 (C-5a), 108.17 (C-12), 116.95 (C-10),
120.74 (C-4), 121.88 (C-6a), 123.71 (C-8), 124.15 (C-3),
126.21 (C-7), 133.86 (C-9), 150.36 (C-5), 154.40 (C-10a),
154.62 [NHCOOC(CH3)3], 155.50 (C-11a), 156.75 (C-12a),
178.62 (C-6). Anal. Calcd. for C31H42N4O5: C, 67.61; H, 7.69;
N, 10.17. Found: C, 67.42; H, 7.57; N, 9.93.
N,N0-Bis(2-diethylaminoethyl)-2,2-dimethyl-6-oxo-2H,6H-
pyrano[3,2-b]xantheno-5,12-diamine (13b). The synthetic
route is analogous to the one followed for the preparation of
compound 13a. Yield: 86%. mp (difumarate): 125–126ꢀC,
1
N,N-Diethyl-N0-[12-[N-(t-butoxycarbonyl)-N-(2-diethylami-
noethyl)amino]-2,2-dimethyl-6-oxo-2H,6H-pyrano[3,2-b]
xanthen-5-yl]ethanediamine (12b). The synthetic route is
analogous to the one followed for the preparation of compound
(ethanol). H-NMR (400 MHz, deuteriochloroform) d: 1.00 [t,
6H, 5-NHCH2CH2N(CH2CH3)2, J ¼ 7 Hz], 1.05 [t, 6H, 12-
NHCH2CH2N(CH2CH3)2, J ¼ 7 Hz], 1.47 (s, 6H, 2 ꢁ gem
CH3), 2.56 [t, 4H, 5-NHCH2CH2N(CH2CH3)2, J ¼ 7 Hz], 2.65
[m, 6H, 5-NHCH2CH2NEt2, 12-NHCH2CH2N(CH2CH3)2],
2.74 (t, 2H, 12-NHCH2CH2NEt2, J ¼ 7 Hz), 3.28 (t, 2H, 12-
NHCH2CH2NEt2, J ¼ 7 Hz), 3.40 (q, 2H, 5-NHCH2CH2NEt2,
J ¼ 7 Hz, 5 Hz), 5.52 (d, 1H, H-3, J ¼ 10 Hz), 6.59 (d, 1H,
H-4, J ¼ 10 Hz), 7.26 (dt, 1H, H-8, J ¼ 8 Hz, 2 Hz), 7.37
(dd, 1H, H-10, J ¼ 8 Hz, 2 Hz), 7.58 (dt, 1H, H-9, J ¼ 8 Hz,
2 Hz), 8.19 (dd, 1H, H-7, J ¼ 8 Hz, 2 Hz), 8.92 (t, 1H, 5-
NHCH2CH2NEt2, J ¼ 5 Hz, D2O exch.). 13C-NMR (50 MHz,
deuteriochloroform) d: 11.51 [5-NHCH2CH2N(CH2CH3)2, 12-
NHCH2CH2N(CH2CH3)2], 27.27 (2 ꢁ gem CH3), 45.47 [12-
NHCH2CH2NEt2], 46.60 [12-NHCH2CH2N(CH2CH3)2], 47.34
1
12a. Oil. Yield: 65%. H-NMR (400 MHz, deuteriochloroform)
d: 1.00 [m, 12H, NHCH2CH2N(CH2CH3)2, N(Boc)CH2CH2N-
(CH2CH3)2], 1.24 [s, 9H, NHCOOC(CH3)3], 1.44 (s, 6H, 2 ꢁ
gem CH3), 2.56 [m, 8H, NHCH2CH2N(CH2CH3)2, N(Boc)CH2-
CH2N(CH2CH3)2], 2.68 [t, 2H, NHCH2CH2NEt2, J ¼ 7 Hz),
2.78 [t, 2H, N(Boc)CH2CH2NEt2, J ¼ 7 Hz), 3.51 (q, 2H,
NHCH2CH2NEt2, J ¼ 7 Hz, 5 Hz), 3.66 (t, 2H, N(Boc)-
CH2CH2NEt2, J ¼ 7 Hz), 5.47 (d, 1H, H-3, J ¼ 10 Hz), 6.60
(d, 1H, H-4, J ¼ 10 Hz), 7.30 (m, 2H, H-8, H-10), 7.59 (dt,
1H, H-9, J ¼ 8 Hz, 2 Hz), 8.20 (dd, 1H, H-7, J ¼ 8 Hz, 2 Hz),
9.62 (t, 1H, NHCH2CH2NEt2, J ¼ 5 Hz, D2O exch.). 13C-NMR
(50 MHz, deuteriochloroform) d: 11.65 [NHCH2CH2-
N(CH2CH3)2, N(Boc)CH2CH2N(CH2CH3)2], 27.31 (2 ꢁ gem
CH3), 28.15 [NHCOOC(CH3)3], 46.16 [N(Boc)CH2CH2NEt2],
47.30 [NHCH2CH2N(CH2CH3)2, N(Boc)CH2CH2N(CH2CH3)2],
47.89 (NHCH2CH2NEt2), 50.35 [N(Boc)CH2CH2NEt2], 53.66
(NHCH2CH2NEt2), 76.42 (C-2), 79.43 [NHCOOC(CH3)3],
103.54 (C-4a), 104.16 (C-5a), 108.17 (C-12), 116.95 (C-10),
120.77 (C-4), 121.84 (C-6a), 123.71 (C-8), 124.04 (C-3), 126.10
(C-7), 133.86 (C-9), 150.36 (C-5), 154.25 (C-10a), 154.55
[NHCOOC(CH3)3], 155.47 (C-11a), 156.64 (C-12a), 178.55 (C-
6). Anal. Calcd. for C35H50N4O5: C, 69.28; H, 8.31; N, 9.23.
Found: C, 69.03; H, 8.27; N, 8.89.
N,N0-Bis(2-dimethylaminoethyl)-2,2-dimethyl-6-oxo-2H,6H-
pyrano[3,2-b]xantheno-5,12-diamine (13a). To a solution of
compound 12a (50 mg, 0.09 mmol) in dry dichloromethane (5
mL) at 0ꢀC was added trifluoroacetic acid (0.5 mL) and the
mixture was stirred, at room temperature for 1 h. The organic
phase was then washed with a 20% sodium carbonate solution
(2 ꢁ 30 mL), dried (sodium sulfate) and concentrated to dry-
ness and the residue was purified by column chromatography
using a mixture of dichloromethane/methanol (85/15, v/v) as
the eluent to give compound 13a (33 mg, 84%) as a light yel-
low oil. 1H-NMR (400 MHz, deuteriochloroform) d: 1.48 (s,
6H, 2 ꢁ gem CH3), 2.28 [s, 6H, 5-NHCH2CH2N(CH3)2], 2.31
[s, 6H, 12-NHCH2CH2N(CH3)2], 2.55 [m, 4H, 5-
NHCH2CH2NMe2, 12-NHCH2CH2N(CH3)2], 3.24 (q, 2H, 12-
[5-NHCH2CH2N(CH2CH3)2],
48.63
(5-NHCH2CH2NEt2),
52.82 (12-NHCH2CH2NEt2), 53.62 (5-NHCH2CH2NEt2), 76.60
(C-2), 105.45 (C-4a), 105.63 (C-5a), 116.98 (C-12), 117.02
(C-10), 120.74 (C-4), 121.76 (C-6a), 123.42 (C-8), 124.93 (C-
3), 126.14 (C-7), 133.71 (C-9), 145.80 (C-5), 149.44 (C-11a),
151.46 (C-12a), 154.81 (C-10a), 179.03 (C-6). Anal. Calcd. for
C30H42N4O3. 2C4H4O4: C, 61.77; H, 6.82; N, 7.58. Found: C,
61.98; H, 6.69; N, 7.44.
Cell culture and assessment of cytotoxicity. The new
compounds were tested for their cytotoxic activity on the mu-
rine leukemia cell line L1210 (American Type Culture Collec-
tion, Rockville, MD), as well as on the following human solid
tumor cell lines: colorectal adenocarcinoma HT-29, uterine
sarcoma MES-SA, and its 100-fold resistant to doxorubicin
subline MES-SA/Dx5 (European Collection of Cell Cultures,
Salisbury, U.K.) [24]. L1210 cells were cultured in RPMI
1640 medium (Gibco BRL, Paisley, U.K.) supplemented with
penicillin (100 U/mL), streptomycin (100 lg/mL), and 10% fe-
tal bovine serum (media and antibiotics from Biochrom KG,
Berlin, Germany) in an environment of 5% CO2, 85% humid-
ity, and 37ꢀC. HT-29 cells were cultured in Dulbecco’s mini-
mal essential medium supplemented with antibiotics and serum
(as above), and routinely subcultured using a trypsin 0.25%-
EDTA 0.02% solution. MES-SA and MES-SA/Dx5 cells were
cultured in McCoy’s 5A medium supplemented with antibiot-
ics and serum (as above), and subcultured by means of 0.03%
EDTA. The cytotoxicity assay was performed by a modifica-
tion of the MTT method. Briefly, the cells were plated at a
density of ꢂ5,000 cells/well in 96-well flat-bottomed micro-
plates, and after 24 h the test compounds were added,
NHCH2CH2NMe2,
J
¼
7 Hz, 5 Hz), 3.42 (t, 2H, 5-
NHCH2CH2NMe2, J ¼ 7 Hz), 5.52 (d, 1H, H-3, J ¼ 10 Hz),
6.59 (d, 1H, H-4, J ¼ 10 Hz), 7.27 (dt, 1H, H-8, J ¼ 8 Hz, 2
Hz), 7.39 (dd, 1H, H-10, J ¼ 8 Hz, 2 Hz), 7.58 (dt, 1H, H-9,
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

July 2011
Design, Synthesis, and Cytotoxic Activity Evaluation of New Linear Pyranoxanthone
Aminoderivatives
935
´
[6] Vispe, S.; Vandenberghe, I.; Robin, M.; Annereau, J. P.;
appropriately diluted with DMSO. After a 72-h incubation, the
medium was replaced with MTT (Sigma) dissolved at a final
concentration of 1 mg/mL in serum-free, phenol-red-free
RPMI (Biochrom KG) for a further 4-h incubation. Then, the
MTT formazan was solubilized in 2-propanol and the optical
density was measured with a microplate analyzer at a wave-
length of 550 nm (reference wavelength 690 nm). Doxorubicin
and mitoxantrone were included in the experiments as positive
controls. The results represent the mean of three independent
experiments and are expressed as IC50, the concentration that
reduced by 50% the optical density of treated cells with
respect to untreated controls. Furthermore, the resistance factor
was calculated as the ratio between the IC50 of MES-SA/Dx5
cells and the IC50 of MES-SA cells.
Cell cycle analysis. Cell-cycle analysis was performed fol-
lowing incubation of exponentially growing HT-29 cells
with the indicated concentrations of the test substances for
24 h. Treated cultures were then washed in PBS, fixed in
50% ethanol, and stained with an RNAse-containing propi-
dium iodide solution. DNA content was analyzed on a FACS
Calibur (Becton Dickinson, San Jose, CA) flow cytometer
using the ModFit software (Verity Software House, Top-
sham, ME).
´
Creancier, L.; Pique, V.; Galy, J. P.; Kruczynski, A.; Barret, J. M.;
Bailly, C. Biochem Pharmacol 2007, 73, 1863.
[7] Otsuka, Y.; Tanaka, T.; Uchida, D.; Noguchi, Y.; Saeki, N.;
Saito, Y.; Tatsuno, I. Neurosci Lett 2004, 365, 180.
[8] Wakelin, L. P. G.; Adams, A.; Denny, W. A. J Med Chem
2002, 45, 894.
[9] Antonini, I.; Polucci, P.; Kelland, L. R.; Menta, E.; Pescalli,
N.; Martelli, S. J Med Chem 1999, 42, 2535.
[10] Konopa, J. Pure Appl Chem 2001, 73, 1421.
[11] Ghirtis, K.; Pouli, N.; Marakos, P.; Skaltsounis, A.-L.;
Leonce, S.; Gaignard, D. H.; Atassi, G. Heterocycles 2000, 53, 93.
[12] Ghirtis, K.; Pouli, N.; Marakos, P.; Skaltsounis, A.-L.; Leonce,
S.; Atassi, G.; Gaignard, D. H. J Heterocycl Chem 2001, 38, 147.
[13] Svoboda, G. H.; Poore, G. A.; Simpson, P. J.; Boder, G. B.
J. Pharm Sci 1966, 55, 758.
[14] Kostakis, I. K.; Ghirtis, K.; Pouli, N.; Marakos, P.; Skaltsounis,
A. L.; Leonce, S.; Gaignard, D. H.; Atassi, G. Il Farmaco 2000, 55, 455.
[15] Kolokythas, G.; Kostakis, I. K.; Pouli, N.; Marakos, P.; Skalt-
sounis, A.-L.; Pratsinis, H. Bioorg Med Chem Lett 2002, 12, 1443.
[16] Kolokythas, G.; Kostakis, I. K.; Pouli, N.; Marakos, P.;
Kletsas, D.; Pratsinis, H. Bioorg Med Chem 2003, 11, 4591.
[17] Kolokythas, G.; Pouli, N.; Marakos, P.; Pratsinis, H.; Klet-
sas, D. Eur J Med Chem 2006, 41, 71.
[18] Costes, N.; Elomri, A.; Dufat, H.; Michel, S.; Seguin, E.;
´ ´
Koch, M.; Tillequin, F.; Pfeiffer, B.; Renard, P.; Leonce, S.; Pierre, A.
Acknowledgment. This project is co-financed within Op. Edu-
cation by the ESF (European Social Fund) and National
Resources.
Oncology Res 2002, 13, 191.
[19] Kolokythas, G.; Kostakis, I. K.; Pouli, N.; Marakos, P.;
Kousidou, O. Ch.; Tzanakakis, G. N.; Karamanos, N. K. Eur J Med
Chem 2007, 42, 307.
[20] Ahluwalia, K. V.; Jolly, S. R.; Tehim, K. A. J Chem Soc
Perkin Trans 1 1983, 1229.
REFERENCES AND NOTES
[21] Cardilo, G.; Cricchio, R.; Merlini, L. Tetrahedron 1968, 24,
4825.
[1] Faulds, D.; Balfour, J. A.; Chrisp, P.; Langtry, H. D. Drugs
1991, 41, 400.
[22] Ahluwalia, K. V.; Arora, K. K. Tetrahedron 1981, 37, 1437.
[23] Hernandez-Galan, R.; Salva, J.; Massanet, G. M.; Collado,
I. G. Tetrahedron 1993, 49, 1701.
[2] Krapcho, A. P.; Menta, E. Drugs Fut 1997, 22, 641.
[3] Cholody, W. M.; Horowska, B.; Paradziej-Lukowicz, J.;
Martelli, S.; Konopa, J. J. Med Chem 1996, 39, 1028.
[4] Atwell, G. J.; Rewcastle, G. W.; Baguley, B. C.; Denny, W.
A. J Med Chem 1987, 30, 664.
[24] Harker, W. G.; Sikic, B. I. Cancer Res 1985, 45, 4091.
[25] Kostakis, I. K.; Pouli, N.; Marakos, P.; Skaltsounis, A.-L.;
Pratsinis, H.; Kletsas, D. Bioorg Med Chem 2006, 14, 2910.
[5] Adjei, A. A. Invest New Drugs 1999, 17, 43.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet