Synthesis and cytotoxic activity of 2-dialkylaminoethylamino substituted xanthenone and thioxanthenone derivatives

Article abstract of DOI:10.1016/S0014-827X(00)00068-9

The synthesis and biological evaluation of some new pyranoxanthenones and pyranothioxanthenones, substituted with flexible amino side-chains, and their evaluation as potential antitumor agents is described. The cytotoxic activity of the compounds and their eventual selective effect on a phase of the cell cycle were evaluated in vitro, using the murine lymphocytic L1210 leukemia cell line. The new aminoderivatives exhibited highly potent cytotoxicity against the leukemia L1210 cell line when compared to acronycine. All the compounds induced a partial accumulation of cells in the G2+M phase of the cell cycle. (C) 2000 Elsevier Science S.A.

Full text of DOI:10.1016/S0014-827X(00)00068-9

www.elsevier.nl/locate/farmac
Il Farmaco 55 (2000) 455–460
Synthesis and cytotoxic activity of 2-dialkylaminoethylamino
substituted xanthenone and thioxanthenone derivatives
Ioannis Kostakis a, Konstantinos Ghirtis a, Nicole Pouli a,*, Panagiotis Marakos a,
Alexios-Leandros Skaltsounis b, Stephane Leonce c, Daniel H. Caignard c,
Ghanem Atassi c
a Uni6ersity of Athens, Department of Pharmacy, Di6ision of Pharmaceutical Chemistry, Panepistimiopolis-Zografou, 17345 Athens, Greece
b Uni6ersity of Athens, Department of Pharmacy, Di6ision of Pharmacognosy, Panepistimiopolis-Zografou, 17345 Athens, Greece
c Institut de Recherches Ser6ier, 11 Rue des Moulineaux, 92150 Suresnes, France
Received 8 March 2000; accepted 12 June 2000
Abstract
The synthesis and biological evaluation of some new pyranoxanthenones and pyranothioxanthenones, substituted with flexible
amino side-chains, and their evaluation as potential antitumor agents is described. The cytotoxic activity of the compounds and
their eventual selective effect on a phase of the cell cycle were evaluated in vitro, using the murine lymphocytic L1210 leukemia
cell line. The new aminoderivatives exhibited highly potent cytotoxicity against the leukemia L1210 cell line when compared to
acronycine. All the compounds induced a partial accumulation of cells in the G2+M phase of the cell cycle. © 2000 Elsevier
Science S.A. All rights reserved.
Keywords: 2-Dialkylaminoethylamino xanthenones; 2-Dialkylaminoethylamino thioxanthenones; Cytotoxic activity; Cell-cycle selectivity
water solubility characteristics. Although the mode of
action of this alkaloid at both cellular and molecular
1. Introduction
level has not yet been unambiguously established, it is
suggested that interaction with DNA, either by interca-
lation, or by some other non-covalent processes, is
crucial and responsible for its high antitumor potency
[9]. We have previously synthesized a series of pyranox-
anthenones and thioxanthenones, bearing the isosteric
replacement of an oxygen or a sulfur atom, instead of
the nitrogen atom of acronycine, as well as various
substituents on the pyran ring [10]. A number of these
Xanthones and thioxanthones have been reported to
possess interesting cytotoxic activities [1–3]. A well
examined example is the thioxanthone derivative hy-
canthone (Fig. 1), as well as a number of 1-dialkyl-
aminoalkylthioxanthenone derivatives structurally re-
lated to it [4]. The basic side chain seems to be an
important component for the expression of cytotoxic-
ity, not only for the above mentioned class of com-
pounds, but also for numerous amino derivatives of
acridone [5,6] and anthraquinone [7] as well.
In our laboratories we have initiated a research pro-
gram dealing with the preparation and pharmacological
evaluation of some new compounds structurally related
to the acridone alkaloid acronycine (Fig. 1) [8], a
broad-spectrum antitumor agent, whose clinical devel-
opment has been hindered, in part, due to its poor
* Corresponding author. Tel.: +30-1-727 4529; fax: +30-1-723
8297.
Fig. 1.
E-mail address: pouli@pharm.uoa.gr (N. Pouli).
0014-827X/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved.
PII: S0014-827X(00)00068-9

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I. Kostakis et al. / Il Farmaco 55 (2000) 455–460
3. Experimental
3.1. Chemistry
Melting points were determined on a Bu¨chi appara-
tus and are uncorrected. 1H NMR spectra were
recorded on a Bruker Avance 400 instrument in deuter-
ated solvents and were referenced to TMS (l scale).
Flash chromatography was performed on Merck silica
gel 60 (0.040–0.063 mm). Analytical thin layer chro-
matography (TLC) was carried out on precoated (0.25
mm) Merck silica gel F-254 plates. Elemental analyses
were within 90.4% of the theoretical values.
3.2. General procedure for the preparation of tosylates
(3, 6, 9, 12)
A solution of the appropriate hydroxy derivatives 1,
4, 7 or 10 (0.51 mmol) in dry acetone (10 ml) was
treated at 0°C with a mixture of p-toluenesulfonyl
chloride (1.73 mmol) and potassium carbonate (8.89
mmol) and the resulting mixture was heated at reflux
under Ar for 3 h. The inorganic precipitate was then
filtered off and the filtrate was evaporated to dryness.
The residue was purified by column chromatography
(dry-pack, silica gel 20×2 cm) using a mixture of
cyclohexane:ethyl acetate (7:3) as the eluent, to give the
corresponding tosylate.
Scheme 1.
compounds possessed interesting activity against the
murine L1210 leukemia cell line.
In a search for new xanthenone and thioxanthenone
derivatives with improved potency, we turned our at-
tention towards the preparation and investigation of a
number of amino substituted analogs, in order to estab-
lish if the introduction of a basic side chain in the
tetracyclic ring system could effect an increase of cyto-
toxic activity. We here report the synthesis and the
cytotoxicity screening tests of these compounds.
3.2.1. 3,3-Dimethyl-6-[[(4-methyl)benzenesulfonyl]-
oxy]-3H,7H-pyrano[2,3-c]xanthen-7-one (3)
1
Yield: 83%. Mp: 188–190°C (Et2O–n-pentane). H
NMR (400 MHz, CDCl3) l (ppm): 1.42 (s, 6H, 2×
gemCH3), 2.35 (s, 3H, 4%-CH3), 5.66 (d, 1H, J=10.5
Hz, H-2), 6.55 (s, 1H, H-5), 6.81 (d, 1H, J=10.5 Hz,
H-1), 7.30 (d, 2H, J=8 Hz, H-2%, H-6%), 7.46 (m, 3H,
H-9, H-10, H-11), 7.91 (d, 2H, J=8 Hz, H-3%, H-5%),
8.16 (dd, 1H, J=8 Hz, 2Hz, H-8).
2. Chemistry
The synthetic approach for the preparation of the
new derivatives is depicted in Scheme 1. We used
6-hydroxy-3,3-dimethyl-3H,7H-pyrano[2,3-c]xanthen-
7-one (1), 5-hydroxy-2,2-dimethyl-2H,6H-pyrano[3,2-
b]xanthen-6-one (7) and the corresponding thioxan-
3.2.2. 3,3-Dimethyl-6-[[(4-methyl)benzenesulfonyl]-
oxy]-3H,7H-pyrano[2,3-c]thioxanthen-7-one (6)
Yield: 97%. Mp: 187–189°C (Et2O–n-pentane). H
1
thenones
4 and 10 as starting materials. These
NMR (400 MHz, CDCl3) l (ppm): 1.45 (s, 6H, 2×
gemCH3), 2.30 (s, 3H, 4%-CH3), 5.78 (d, 1H, J=10 Hz,
H-2), 6.74 (s, 1H, H-5), 6.61 (d, 1H, J=10 Hz, H-1),
7.31 (d, 2H, J=8 Hz, H-2%, H-6%), 7.49 (m, 3H, H-9,
H-10, H-11), 7.82 (d, 2H, J=8 Hz, H-3%, H-5%), 8.25
(dd, 1H, J=8 Hz, 1Hz, H-8).
compounds were prepared by condensation of 1,3,5-tri-
hydroxybenzene with salicylic or thiosalicylic acid,
etherification of the 3-OH of the resulting 1,3-dihydrox-
yxanthenones or thioxanthenones with 3-chloro-3-
methyl-1-butyne and subsequent thermal cyclization of
the ethers in boiling N,N-diethylaniline [10–12]. Upon
treatment with tosyl chloride in acetone, the tosylates 3,
6, 9 and 12 were prepared. Displacement of the leaving
group with the appropriately substituted amines pro-
vided the target compounds 13–28, which were con-
verted into the corresponding fumarates or
hydrochlorides.
3.2.3. 2,2-Dimethyl-5-[[(4-methyl)benzenesulfonyl]oxy]-
2H,6H-pyrano[3,2-b]xanthen-6-one (9)
1
Yield: 87%. Mp: 215–217°C (Et2O–n-pentane). H
NMR (400 MHz, CDCl3) l (ppm): 1.43 (s, 6H, 2×
gemCH3), 2.41 (s, 3H, 4%-CH3), 5.53 (d, 1H, J=10 Hz,

I. Kostakis et al. / Il Farmaco 55 (2000) 455–460
457
H-3), 6.34 (d, 1H, J=10 Hz, H-4), 6.77 (s, 1H, H-12),
7.30 (d, 2H, J=8 Hz, H-2%, H-6%), 7.46 (m, 3H, H-8, H-9,
H-10), 7.90 (d, 2H, J=8 Hz, H-3%, H-5%), 8.17 (dd, 1H,
J=8 Hz, 2 Hz, H-7).
(m, 4H, pyrrolidine H), 2.83 (t, 2H, J=7 Hz,
CH2N(CH2)4), 3.39 (q, 2H, J=7 Hz, 5 Hz, HNCH2),
5.46 (d, 1H, J=10 Hz, H-2), 5.91 (s, 1H, H-5), 6.82 (d,
1H, J=10 Hz, H-1), 7.43 (m, 3H, H-9, H-10, H-11), 8.18
(dd, 1H, J=8 Hz, 1.5 Hz, H-8), 9.76 (br.s, 1H, NH).
Hydrochloride, Mp: 240–241°C (EtOH–Et2O). Anal.
C24H26N2O3·HCl: (C, H, N).
3.2.4. 2,2-Dimethyl-5-[[(4-methyl)benzenesulfonyl]-
oxy]-2H,6H-pyrano[3,2-b]thioxanthen-6-one (12)
1
Yield: 92%. Mp: 218–220°C (Et2O–n-pentane). H
NMR (400 MHz, CDCl3) l (ppm): 1.43 (s, 6H, 2×
gemCH3), 2.39 (s, 3H, 4%-CH3), 5.64 (d, 1H, J=10 Hz,
H-3), 6.52 (d, 1H, J=10 Hz, H-4), 6.86 (s, 1H, H-12),
7.25 (d, 2H, J=9 Hz, H-2%, H-6%), 7.47 (m, 3H, H-8, H-9,
H-10), 7.80 (d, 2H, J=9 Hz, H-3%, H-5%), 8.16 (dd, 1H,
J=8 Hz, 1.5 Hz, H-7).
3.3.4. 3,3-Dimethyl-6-[[2-(1-piperidinyl)ethyl]amino]-
3H,7H-pyrano[2,3-c]xanthen-7-one (16)
Yield: 85%. 1H NMR (400 MHz, CDCl3) l (ppm): 1.43
(s, 6H, 2×gemCH3), 1.46 (m, 2H, piperidine H), 1.62 (m,
4H, piperidine H), 2.50 (m, 4H, piperidine H), 2.70 (t,
2H, J=7 Hz, CH2N(CH2)5), 3.34 (q, 2H, J=7 Hz, 6 Hz,
HNCH2), 5.49 (d, 1H, J=10 Hz, H-2), 5.88 (s, 1H, H-5),
6.80 (d, 1H, J=10 Hz, H-1), 7.44 (m, 3H, H-9, H-10,
H-11), 8.20 (dd, 1H, J=8 Hz, 1 Hz, H-8), 9.72 (br.s, 1H,
NH). Hydrochloride, Mp: 248°C (dec) (EtOH–Et2O).
Anal. C25H28N2O3·HCl: (C, H, N).
3.3. General procedure for the synthesis of deri6ati6es
13–28
To a solution of the tosylate (0.12 mmol) in dry DMSO
(10 ml), was added the appropriate 2-dialkylaminoethyl
amine (0.24 mmol) and the reaction was heated at 150°C
under Ar for 1 h. The mixture was then poured into
ice-water and extracted with ethyl acetate. The organic
layer was washed with water, dried (Na2SO4) and evap-
orated to dryness. The residue was purified by column
chromatography (silica gel, 20×1.5 cm) using a mixture
of dichloromethane : methanol (9:1) as the eluent, to give
the corresponding amine as an oil.
3.3.5. 3,3-Dimethyl-6-[[2-(dimethylamino)ethyl]amino]-
3H,7H-pyrano[2,3-c]thioxanthen-7-one (17)
Yield: 71%. 1H NMR (400 MHz, CDCl3) l (ppm): 1.47
(s, 6H, 2×gemCH3), 2.30 (s, 6H, N(CH3)2), 2.62 (t, 2H,
J=7 Hz, CH2N(CH3)2), 3.33 (q, 2H, J=7 Hz, 5Hz,
HNCH2), 5.32 (d, 1H, J=10 Hz, H-2), 6.05 (s, 1H, H-5),
6.62 (d, 1H, J=10 Hz, H-1), 7.38 (m, 3H, H-9, H-10,
H-11), 8.41 (dd, 1H, J=8 Hz, 1.5 Hz, H-8), 10.50 (br.s,
1H, NH). Fumarate, Mp: 272°C (dec) (EtOH–Et2O).
Anal. C22H24N2O2S·C4H4O4·H2O: (C, H, N).
3.3.1. 3,3-Dimethyl-6-[[2-(dimethylamino)ethyl]-
amino]-3H,7H-pyrano[2,3-c]xanthen-7-one (13)
Yield: 62%. 1H NMR (400 MHz, CDCl3) l (ppm): 1.42
(s, 6H, 2×gemCH3), 2.30 (s, 6H, N(CH3)2), 2.62 (t, 2H,
J=7 Hz, CH2N(CH3)2), 3.29 (q, 2H, J=7 Hz, 6Hz,
HNCH2), 5.47 (d, 1H, J=10 Hz, H-2), 5.86 (s, 1H, H-5),
6.79 (d, 1H, J=10 Hz, H-1), 7.39 (m, 3H, H-9, H-10,
H-11), 8.16 (dd, 1H, J=8 Hz, 0.5 Hz, H-8), 9.71 (br.s,
1H, NH). Hydrochloride, Mp: 227–229°C (EtOH–
Et2O). Anal. C22H24N2O3·HCl: (C, H, N).
3.3.6. 3,3-Dimethyl-6-[[2-(diethylamino)ethyl]amino]-
3H,7H-pyrano[2,3-c]thioxanthen-7-one (18)
Yield: 68%. 1H NMR (400 MHz, CDCl3) l (ppm): 1.11
(t, 6H, J=7 Hz, N(CH2CH3)2), 1.47 (s, 6H, 2×
gemCH3), 2.65 (q, 4H, J=7 Hz, N(CH2CH3)2), 2.87 (t,
2H, J=7 Hz, CH2NEt2), 3.32 (q, 2H, J=7 Hz, 5 Hz,
HNCH2), 5.59 (d, 1H, J=10 Hz, H-2), 6.05 (s, 1H, H-5),
6.72 (d, 1H, J=10 Hz, H-1), 7.41 (m, 3H, H-9, H-10,
H-11), 8.50 (dd, 1H, J=8 Hz, 1.5 Hz, H-8), 8.07 (br.s,
1H, NH). Fumarate, Mp: 290°C (dec) (EtOH–Et2O).
Anal. C24H28N2O2S·C4H4O4·2H2O: (C, H, N).
3.3.2. 3,3-Dimethyl-6-[[2-(diethylamino)ethyl]-
amino]-3H,7H-pyrano[2,3-c]xanthen-7-one (14)
Yield: 69%. 1H NMR (400 MHz, CDCl3) l (ppm): 1.08
(t, 6H, J=7 Hz, N(CH2CH3)2), 1.44 (s, 6H, 2×
gemCH3), 2.63 (q, 4H, J=7 Hz, N(CH2CH3)2), 2.80 (t,
2H, J=7 Hz, CH2NEt2), 3.30 (q, 2H, J=7 Hz, 6Hz,
HNCH2), 5.49 (d, 1H, J=10 Hz, H-2), 5.89 (s, 1H, H-5),
6.83 (d, 1H, J=10 Hz, H-1), 7.42 (m, 3H, H-9, H-10,
H-11), 8.18 (dd, 1H, J=8 Hz, 1.5 Hz, H-8), 9.60 (br.s,
1H, NH). Fumarate, Mp: 240°C (dec) (EtOH–Et2O).
Anal. C24H28N2O3·C4H4O4: (C, H, N).
3.3.7. 3,3-Dimethyl-6-[[2-(1-pyrrolidinyl)ethyl]amino]-
3H,7H-pyrano[2,3-c]thioxanthen-7-one (19)
Yield: 70%. 1H NMR (400 MHz, CDCl3) l (ppm): 1.45
(s, 6H, 2×gemCH3), 1.87 (m, 4H, pyrrolidine H), 2.79
(m, 4H, pyrrolidine H), 2.97 (t, 2H, J=7 Hz,
CH2N(CH2)4), 3.44 (q, 2H, J=7 Hz, 4 Hz, HNCH2),
5.61 (d, 1H, J=10 Hz, H-2), 6.06 (s, 1H, H-5), 6.66 (d,
1H, J=10 Hz, H-1), 7.45 (m, 3H, H-9, H-10, H-11), 8.46
(dd, 1H, J=8 Hz, 1.5 Hz, H-8), 10.61 (br.s, 1H, NH).
Fumarate, Mp: 276°C (dec) (EtOH–Et2O). Anal.
C24H26N2O2S·C4H4O4: (C, H, N).
3.3.3. 3,3-Dimethyl-6-[[2-(1-pyrrolidinyl)ethyl]-
amino]-3H,7H-pyrano[2,3-c]xanthen-7-one (15)
Yield: 58%. 1H NMR (400 MHz, CDCl3) l (ppm): 1.45
(s, 6H, 2×gemCH3), 1.80 (m, 4H, pyrrolidine H), 2.64

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I. Kostakis et al. / Il Farmaco 55 (2000) 455–460
3.3.8. 3,3-Dimethyl-6-[[2-(1-piperidinyl)ethyl]amino]-
3H,7H-pyrano[2,3-c]thioxanthen-7-one (20)
Yield: 74%. H NMR (400 MHz, CDCl3) l (ppm):
H-7), 9.62 (br.s, 1H, NH). Hydrochloride, Mp: 298°C
(dec) (EtOH–Et2O). Anal. C25H28N2O3·HCl: (C, H, N).
1
3.3.13. 2,2-Dimethyl-5-[[2-(dimethylamino)ethyl]amino]-
2H,6H-pyrano[3,2-b]thioxanthen-6-one (25)
1.43 (s, 6H, 2×gemCH3), 1.44 (m, 2H, piperidine H),
1.68 (m, 4H, piperidine H), 2.54 (m, 4H, piperidine H),
2.74 (t, 2H, J=7 Hz, CH2N(CH2)5), 3.39 (q, 2H, J=7
Hz, 4 Hz, HNCH2), 5.58 (d, 1H, J=10 Hz, H-2), 6.07
(s, 1H, H-5), 6.67 (d, 1H, J=10 Hz, H-1), 7.46 (m, 3H,
H-9, H-10, H-11), 8.47 (dd, 1H, J=8 Hz, 1.5 Hz, H-8),
8.00 (br.s, 1H, NH). Fumarate, Mp: 292°C (dec)
(EtOH–Et2O). Anal. C25H28N2O2S·C4H4O4: (C, H, N).
1
Yield: 75%. H NMR (400 MHz, CDCl3) l (ppm):
1.42 (s, 6H, 2×gemCH3), 2.77 (s, 6H, N(CH3)2), 3.18
(t, 2H, J=7 Hz, CH2N(CH3)2), 3.93 (q, 2H, J=7 Hz,
4 Hz, HNCH2), 5.62 (d, 1H, J=10 Hz, H-3), 6.48 (d,
1H, J=10 Hz, H-4), 6.49 (s, 1H, H-12), 7.42 (m, 3H,
H-8, H-9, H-10), 8.41 (dd, 1H, J=8 Hz, 1.5 Hz, H-7),
9.91 (br.s, 1H, NH). Fumarate, Mp: 254°C (dec)
(EtOH–Et2O). Anal. C22H24N2O2S·C4H4O4: (C, H, N).
3.3.9. 2,2-Dimethyl-5-[[2-(dimethylamino)ethyl]amino]-
2H,6H-pyrano[3,2-b]xanthen-6-one (21)
3.3.14. 2,2-Dimethyl-5-[[2-(diethylamino)ethyl]amino]-
2H,6H-pyrano[3,2-b]thioxanthen-6-one (26)
1
Yield: 76%. H NMR (400 MHz, CDCl3) l (ppm):
1.46 (s, 6H, 2×gemCH3), 2.29 (s, 6H, N(CH3)2), 2.55
(t, 2H, J=7 Hz, CH2N(CH3)2), 3.53 (q, 2H, J=7 Hz,
6 Hz, HNCH2), 5.46 (d, 1H, J=10 Hz, H-3), 6.18 (s,
1H, H-12), 6.58 (d, 1H, J=10 Hz, H-4), 7.41 (m, 3H,
H-8, H-9, H-10), 8.19 (dd, 1H, J=8 Hz, 0.5 Hz, H-7),
9.67 (br.s, 1H, NH). Hydrochloride, Mp: 198–200°C
(EtOH–Et2O). Anal. C22H24N2O3·HCl: (C, H, N).
1
Yield: 77%. H NMR (400 MHz, CDCl3) l (ppm):
1.01 (t, 6H, J=7 Hz, N(CH2CH3)2, 1.47 (s, 6H, 2×
gemCH3), 2.60 (q, 4H, J=7 Hz, N(CH2CH3)2), 2.65 (t,
2H, J=7 Hz, CH2NEt2), 3.48 (q, 2H, J=7 Hz, 4 Hz,
HNCH2), 5.48 (d, 1H, J=10 Hz, H-3), 6.38 (s, 1H,
H-12), 6.54 (d, 1H, J=10 Hz, H-4), 7.41 (m, 3H, H-8,
H-9, H-10), 8.47 (dd, 1H, J=8 Hz, 1.5 Hz, H-7), 9.98
(br.s, 1H, NH). Fumarate, Mp: 235–237°C (EtOH–
Et2O). Anal. C24H28N2O2S·C4H4O4·H2O: (C, H, N).
3.3.10. 2,2-Dimethyl-5-[[2-(diethylamino)ethyl]amino]-
2H,6H-pyrano[3,2-b]xanthen-6-one (22)
1
Yield: 88%. H NMR (400 MHz, CDCl3) l (ppm):
3.3.15. 2,2-Dimethyl-5-[[2-(1-pyrrolidinyl)ethyl]amino]-
2H,6H-pyrano[3,2-b]thioxanthen-6-one (27)
1.04 (t, 6H, J=7 Hz, N(CH2CH3)2, 1.47 (s, 6H, 2×
gemCH3), 2.60 (q, 4H, J=7 Hz, N(CH2CH3)2), 2.70 (t,
2H, J=7 Hz, CH2NEt2), 3.53 (q, 2H, J=7 Hz, 5 Hz,
HNCH2), 5.47 (d, 1H, J=10 Hz, H-3), 6.19 (s, 1H,
H-12), 6.51 (d, 1H, J=10 Hz, H-4), 7.39 (m, 3H, H-8,
H-9, H-10), 8.18 (dd, 1H, J=8 Hz, 0.5 Hz, H-7), 9.60
(br.s, 1H, NH). Hydrochloride, Mp: 195–197°C
(EtOH–Et2O). Anal. C24H28N2O3·HCl: (C, H, N).
1
Yield: 70%. H NMR (400 MHz, CDCl3) l (ppm):
1.45 (s, 6H, 2×gemCH3), 1.97 (m, 4H, pyrrolidine H),
2.94 (m, 4H, pyrrolidine H), 3.19 (t, 2H, J=7 Hz,
CH2N(CH2)4), 3.81 (q, 2H, J=7 Hz, 4 Hz, HNCH2),
5.61 (d, 1H, J=10 Hz, H-3), 6.42 (s, 1H, H-12), 6.47
(d, 1H, J=10 Hz, H-4), 7.42 (m, 3H, H-8, H-9, H-10),
8.37 (dd, 1H, J=8 Hz, 1.5 Hz, H-7), 10.85 (br.s, 1H,
NH). Fumarate, Mp: 280°C (dec) (EtOH–Et2O). Anal.
C24H26N2O2S·C4H4O4: (C, H, N).
3.3.11. 2,2-Dimethyl-5-[[2-(1-pyrrolidinyl)ethyl]amino]-
2H,6H-pyrano[3,2-b]xanthen-6-one (23)
1
Yield: 66%. H NMR (400 MHz, CDCl3) l (ppm):
3.3.16. 2,2-Dimethyl-5-[[2-(1-piperidinyl)ethyl]amino]-
2H,6H-pyrano[3,2-b]thioxanthen-6-one (28)
1.45 (s, 6H, 2×gemCH3), 1.71 (m, 4H, pyrrolidine H),
2.52 (m, 4H, pyrrolidine H), 2.70 (t, 2H, J=7 Hz,
CH2N(CH2)4), 3.52 (q, 2H, J=7 Hz, 5 Hz, HNCH2),
5.49 (d, 1H, J=10 Hz, H-3), 6.12 (s, 1H, H-12), 6.51
(d, 1H, J=10 Hz, H-4), 7.36 (m, 3H, H-8, H-9, H-10),
8.11 (dd, 1H, J=8 Hz, 0.5 Hz, H-7), 9.53 (br.s, 1H,
NH). Hydrochloride, Mp: 248–250°C (EtOH–Et2O).
Anal. C24H26N2O3·HCl: (C, H, N).
1
Yield: 81%. H NMR (400 MHz, CDCl3) l (ppm):
1.47 (s, 6H, 2×gemCH3), 1.52 (m, 6H, piperidine H),
2.47 (m, 4H, piperidine H), 2.54 (t, 2H, J=7 Hz,
CH2N(CH2)5), 3.51 (q, 2H, J=7 Hz, 4 Hz, HNCH2),
5.49 (d, 1H, J=10 Hz, H-3), 6.38 (s, 1H, H-12), 6.61
(d, 1H, J=10 Hz, H-4), 7.44 (m, 3H, H-8, H-9, H-10),
8.44 (dd, 1H, J=8 Hz, 1.5 Hz, H-8), 10.05 (br.s, 1H,
NH). Fumarate, Mp: 231–233°C (EtOH–Et2O). Anal.
C25H28N2O2S·C4H4O4: (C, H, N).
3.3.12. 2,2-Dimethyl-5-[[2-(1-piperidinyl)ethyl]amino]-
2H,6H-pyrano[3,2-b]xanthen-6-one (24)
1
Yield: 72%. H NMR (400 MHz, CDCl3) l (ppm):
3.4. Typical procedure for the preparation of the
1.46 (s, 6H, 2×gemCH3), 1.47 (m, 2H, piperidine H),
1.58 (m, 4H, piperidine H), 2.44 (m, 4H, piperidine H),
2.61 (t, 2H, J=7 Hz, CH2N(CH2)5), 3.57 (q, 2H, J=7
Hz, 5 Hz, HNCH2), 5.48 (d, 1H, J=10 Hz, H-3), 6.20
(s, 1H, H-12), 6.61 (d, 1H, J=10 Hz, H-4), 7.44 (m,
3H, H-8, H-9, H-10), 8.19 (dd, 1H, J=8 Hz, 1 Hz,
hydrochloride and fumarate salts
3.4.1. Preparation of the hydrochloride salts
A saturated solution of HCl in dry diethyl ether was
added dropwise to a solution of the amine in dry
diethyl ether, until no precipitation occurred. The pre-

I. Kostakis et al. / Il Farmaco 55 (2000) 455–460
459
cipitate was filtered and recrystallized (EtOH–Et2O), to
3.6. Cell cycle analysis
yield the hydrochloride in approximately 90% yield.
L1210 cells (2.5×105/ml) were incubated for 21 h
(approximately two doubling times) with various con-
centrations of cytotoxic drugs. Cells were then fixed by
70% v/v ethanol, washed twice with phosphate-buffered
saline (PBS) and incubated in PBS containing 100
mg/ml RNase and 25 mg/ml propidium iodide (PI) for
30 min at 20°C. For each sample, 10 000 cells were
analyzed on an Epics XL Coulter flow cytometer. Re-
sults are expressed as the percentage of cells accumu-
lated in the G2+M phase of the cell cycle.
3.4.2. Preparation of the fumarate salts
A solution of fumaric acid (0.21 mmol) in absolute
ethanol was added to a stirred solution of the amine
(0.2 mmol) in absolute ethanol and the reaction mixture
was refluxed for 12 h under Ar. After cooling, the
precipitate was filtered, washed with dry diethyl ether
and recrystallized (EtOH–Et2O), to yield the fumarate
in 82–87% yield.
3.5. Cytotoxicity
The potential cytotoxic activity of all the synthesized
compounds and their eventual selective effect on a
phase of the cell cycle were evaluated in vitro using the
microculture tetrazolium assay essentially as described
[13]. Murine leukemia L1210 cells from the American
Type Culture Collection (Rockville Pike, MD) were
grown in RPMI medium 1640 supplemented with 10%
4. Results and discussion
The IC50 and the cell cycle effect of the new com-
pounds are presented in Table 1. We used acronycine as
the reference compound and the cytotoxic activity was
also compared with that reported for the xanthenone
and thioxanthenone derivatives 2, 5, 8 and 11 [10].
On the basis of the first results, it is obvious that the
introduction of a basic flexible side chain at position 6
of the angular or at position 5 of the linear tetracyclic
ring system exerts a profound effect on the cytotoxic
activity. In fact, all the derivatives tested have shown
significant activity, being 2 to 10 times more potent
than acronycine in inhibiting L1210 cell proliferation.
They have also expressed markedly improved cytotoxic-
ity towards the methoxy xanthenone and thioxan-
thenone analogs 2, 5, 8 and 11.
fetal calf serum, 2 mM
L-glutamine, 100 U/ml peni-
cillin, 100 mg/ml streptomycin and 10 mM HEPES
buffer (pH 7.4). Cells were exposed for 48 h to nine
graded concentrations of the test compound. Results
are expressed as IC50 (mean, n=3), which is defined as
the drug concentration inhibiting the absorbance by
50% with respect to that of untreated cells.
Table 1
The most cytotoxic compound, among all com-
pounds synthesized, is the angular 2-(1-pyrrolidinyl)
derivative 15, with an IC50=2.1 mM. It is also notice-
able that, among the linear derivatives 21–28, the 5-[2-
(1-pyrrolidinyl)ethyl]amino substituted compounds 23
and 27 exert comparatively higher antitumor potency.
In the linear series, for identical (N,N-dialkyl-
amino)ethylamino substitution, changing from the xan-
thenone to the thioxanthenone ring system results in a
reduced cytotoxicity, while in the angular series it ap-
pears to be a slight and variable effect, following the
oxygen for sulfur replacement.
The perturbation of the cell cycle induced by these
compounds was studied on the same cell line. In con-
trast with the methoxy xanthenone and thioxanthenone
analogs 2, 5, 8 and 11, all the new compounds induced
a partial accumulation of cells in the G2+M phase of
the cell cycle. Since it has been reported that the same
type of perturbation was observed with acronycine [14],
this result could suggest a similar mechanism of action
at the molecular level.
Cytotoxicity and cell cycle selectivity
Comp.
IC50 (mM)
Cell cycle effect (% of cells in the
indicated phase) a
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
7.1
8.4
2.1
9.3
5.3
9.9
9.5
10.2
5.8
5.1
G2+M (43% at 10 mM)
G2+M (39% at 10 mM)
G2+M (46% at 10 mM)
G2+M (47% at 25 mM)
G2+M (39% at 10 mM)
G2+M (55% at 25 mM)
G2+M (46% at 25 mM)
G2+M (49% at 25 mM)
G2+M (40% at 10 mM)
G2+M (42% at 10 mM)
G2+M (49% at 10 mM)
G2+M (47% at 25 mM)
G2+M (45% at 25 mM)
G2+M (51% at 25 mM)
G2+M (50% at 10 mM)
G2+M (46% at 25 mM)
not specific
3.5
8.2
9.6
9.6
5.1
12.4
17.2
36.9
14
2
5
8
not specific
not specific
11
48.3
21.0
not specific
G2+M (46% at 50 mM)
Acronycine
Further biological evaluation of the new compounds
is currently in progress.
a 25% of untreated cells were in the G2+M phase of the cell cycle.

460
I. Kostakis et al. / Il Farmaco 55 (2000) 455–460
bioisosteres as antitumor agents, Curr. Med. Chem. 2 (1995)
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