2,2-Dimethyl-2H-anthra[2,3-b]pyran-6,11-diones: A new class of cytotoxic compounds

Article abstract of DOI:10.1016/S0968-0896(00)00278-9

The synthesis and cytotoxic activity of some new 2,2-dimethyl-2H-anthra[2,3-b]pyran-6,11-diones is described. Certain compounds possess interesting activity against murine leukemia L-1210 cells. Relationships between the biological activity and the pyrano-ring conformations are discussed. Copyright

Full text of DOI:10.1016/S0968-0896(00)00278-9

Bioorganic & Medicinal Chemistry 9 (2001) 607±612
2,2-Dimethyl-2H-anthra[2,3-b]pyran-6,11-diones:
A New Class of Cytotoxic Compounds
Eleni Melliou,a Prokopios Magiatis,a So®a Mitaku,a Alexios-Leandros Skaltsounis,a,
Alain Pierre,b Ghanem Atassib and Pierre Renardb
aLaboratory of Pharmacognosy, University of Athens, Panepistimiopolis, Zografou, GR-15771 Athens, Greece
bInstitut de Recherches SERVIER, 11 rue des Moulineaux, 92150 Suresnes, France
Received 18 October 1999; accepted 4 October 2000
AbstractÐThe synthesis and cytotoxic activity of some new 2,2-dimethyl-2H-anthra[2,3-b]pyran-6,11-diones is described. Certain
compounds possess interesting activity against murine leukemia L-1210 cells. Relationships between the biological activity and the
pyrano-ring conformations are discussed. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
to point out that pyranoanthraquinones are extremely
rare in nature and the most characteristic examples are
Ploiariquinones A and B (6 and 7) from the cicada tree,
Ploiarum alternifolium,8 although no biological evalua-
tion has been reported for these compounds.
Natural and synthetic quinones represent important
antitumor drugs. The most active quinones are relatively
large molecules possessing a large quinoid system, in
particular that of anthracene-9,10-diones.1 This group
includes anthracycline antibiotics and aminoalkyl-func-
tionalized anthraquinones such as mitoxanthrone (1a)
and ametantrone (1b). On the other hand it is well
documented that compounds possessing a furano or
pyrano ring fused to a polycyclic, aromatic system such
as acridone,2À4 xanthone,5,6 coumarin,7 exhibit interest-
ing cytotoxic and antitumor properties. A characteristic
example is the natural alkaloid acronycine (2), which
was found to be a very potent antitumor agent (posses-
sing an IC50 in leukemia cells in the order of 25 mM).
Moreover recent studies have shown that hydroxylation
of the pyrano-ring double bond of pyrano-xanthone,
-coumarin and-acridone, yielded compounds, such as
1,2-dihydro-1-hydroxy-6-methoxy-3,5-dimethyl-3H,7H-
pyran[2,3-c]xanthen-7-one (3), 1-hydroxy-1,2-dihydro-
seseline (4), and 1,2-diacetoxy-1,2-dihydroacronycine (5)
(Chart 1), which exhibited promising cytotoxic and
antitumor properties (possessing an IC50 in leukemia
cells in of 9, 0.9, and 4.2 mM, respectively). The above
considerations guided our interest to develop simple and
functionalized derivatives at positions 3, 4, 5, and 12 of
2,2-dimethyl-2H-anthra[2,3-b]pyran-6,11-dione as a new
class of antiproliferative agents (Scheme 1). It is worthy
Results and Discussion
The typical way of obtaining the key compound 5,12-
dihydroxy-2,2-dimethyl-2H-anthra[2,3-b]pyran-6,11-
dione (8) would have been treatment of purpurin (9)
with 3-chloro-3-methyl-1-butyne in the presence of
cuprous salt as catalyst and subsequent thermal cycliza-
tion. In our case this reaction proved unsuccessful, prob-
ably due to the inactivation of the C-3 hydroxyl group of
purpurin by the presence of the neighbouring hydroxyl
group. Therefore, the lead compound 8 was obtained by
application of another methodology which was previously
applied in the case of some natural pyranocoumarins9 and
pyranoanthaquinones8 which involved reaction of the
starting phenol with the corresponding a,b-unsaturated
aldehyde. Therefore treatment of purpurin (9) with 3-
methyl-2-butenal in dry pyridine yielded 8 (40%). The
presence of the two phenolic hydroxyls caused a very
important inactivation of the double bond of the pyran
ring, making pyran ring substitution very dicult. In
contrast, the functionalization of the 3,4-double bond
could be achieved in the dimethoxy compound 10
obtained by methylation of 8. Quinone 11 was obtained
by treatment of 10 with NBS in aqueous THF solu-
tion.10 The bromohydrin 11 was smoothly debromi-
nated with tributyltin hydride,10 to yield 12. Treatment
*Corresponding author. Fax: +1-301-7274594; e-mail: skaltsounis
@pharm.uoa.gr
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00278-9

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E. Melliou et al. / Bioorg. Med. Chem. 9 (2001) 607±612
Chart 1.
Scheme 1. Reagents and conditions: (i) 3-methyl-butenal, Py, 115 ꢀC; (ii) (Me)2SO4, K2CO3, 56 ꢀC; (iii) NBS, THF, H2O, 0 ꢀC; (iv) Bu3SnH, AIBN,
toluene, 110 ꢀC; (v) Ac2O, Py, rt; (vi) OsO4, N-methyl-morpholine-N-oxide, t-BuOH, THF, H2O, rt; (vii) TsCl, K2CO3, Me2CO, 56 ꢀC; (viii)
(CH3)2N(CH2)2NH2, Py, 60 ꢀC; (ix) HO(CH2)2NH(CH2)2NH2, Py, 60 ꢀC.
of alcohol 12 with excess Ac2O in pyridine a€orded the
corresponding ester 13. The diol 14 was obtained by
catalytic osmium oxidation of 10 using N-methyl-mor-
pholine-N-oxide to regenerate the oxidizing agent.
Treatment of cis- diol 14 in a similar way to 12 a€orded
the corresponding diester 15.
derivative 19 with 2-[(2-aminoethyl)amino]ethanol gave
the unsymmetrical bi-substituted derivative 22. The
structures of all the synthetic compounds were estab-
1
lished by the use of CI-MS and H, 13C, 1-D and 2-D
NMR data. For the monotosylated compounds 17 and
18 and the corresponding aminated derivatives, 19 and
20, structures were established unequivocally by appli-
cation of long range heteronuclear correlation (HMBC).
In the case of 18, a J3 correlation was observed between
H-4 (6.36 ppm) of the pyran ring and the relatively
shielded C-5 (137.1 ppm) and a J2 correlation between
C-12 (151.3 ppm) and OH (13.35 ppm). From these
observations it was clear that the tosyloxy group was
attached to C-5. In the case of 19, a J3 correlation was
observed between H-10 (3.46 ppm) of the side chain and
C-5 (151.2 ppm), as well as between H-4 (6.53 ppm) of
Introduction of the amino chains to position 5 and/or
12 was achieved via the intermediate 5,12-ditosyloxy
derivative 16.11 This compound was obtained by treat-
ment of 8 with tosyl chloride in the presence of K2CO3.
The monotosylated compounds 17 and 18 were also
obtained during the tosylation of 8. Reaction of 16 with
N,N-dimethylethylenediamine a€orded the mono- 19
and 20 and the bi- 21 substituted derivatives. Further
substitution of the most abundant mono-subsituted

E. Melliou et al. / Bioorg. Med. Chem. 9 (2001) 607±612
609
the pyran ring and C-5. These observations con®rmed
that in isomer 19, the side chain was attached to C-5
and not to C-12. Consequently, in the other isomer 20,
the side chain was attached to C-12.
and computational conformation analysis. The con-
formation analysis was performed using molecular
mechanics calculations and the two lowest energy con-
formations (structures I and II for compound 14, Fig. 1)
were predicted. Both conformations were half-chair and
their calculated energy di€erence was small, suggesting
that they could possibly both be present in solution. If
compound 14 existed under conformation I, a correla-
tion between H-4 and the b-pyran methyl group should
be observed in the NOESY experiment. The absence of
such a correlation suggested that 3,4-dihydro-3,4-dihy-
droxy derivatives exist predominantly under conforma-
tion II in solution. In this conformation, the substituents
on C-3 and C-4 are relatively hindered by the pyran
methyl groups, since the substituent on C-3 is gauche
with both methyl groups and the substituent on C-4 is
homoaxial with the a-CH3. It is noteworthy that the
pyran ring of the highly active 1,2-dihydro-1,2-dihy-
droxyacronycine derivatives, predominantly adopts the
conformation I,14 where the substituent on C-3 has a
pseudoaxial orientation and both C-3 and C-4 sub-
stituents are less hindered by the pyran methyl groups.
This could possibly explain the fact that the synthesized
3,4-dihydro-3,4-dihydroxy derivatives (14 and 15) are less
potent than the corresponding acronycine derivatives.
The cytotoxic activity of compounds 8±22 was carried
out in vitro using L-1210 leukemia cells.12 The results
(IC50) are reported in Table 1. The most active com-
pounds were 8, 12, 13, 19, 20 and 22 which are 15- to 3-
fold more potent than acronycine.
The cytotoxic activity of these newly synthesized pyr-
anoquinone derivatives is in the same order of magnitude
with those of various pyrano-acridine -xanthone -cou-
marin derivatives. This could be explained by the presence
of the dimethylpyrano ring, which is the main structural
di€erence between our compounds and the previously
described, highly cytotoxic anthraquinone derivatives.13
This additional dimethylpyrano ring is probably, as in
the case of acronycine, the active pharmacophhore site
of the synthesized quinones described herein.
In order to study the cell cycle speci®city of this new
class of cytotoxic compounds, the lead compound 8 was
evaluated for its ability to perturb the cell cycle in a
speci®c way. Using the same cell line (L1210), com-
pound 8 induced a partial accumulation of cells in the
G2+M phase of the cell cycle.
In regard to the amino functionalized derivatives the most
active was the monosubstituted 20 (15-fold more potent
than acronycine and 2-fold more potent than ametan-
trone)15 and the unsymmetrically substituted 22. It is
noteworthy that the symmetrically bisubstituted com-
pound 21 was inactive.
It is interesting to point out that the pyranoan-
thraquinones 12 and 13 which bear a hydroxy or acetoxy
group at position 4, such as 12 and 13, had signi®cant
cytotoxicity (3- and 8-fold more potent than acronycine)
as in the case of pyranocoumarins and pyranoxanthones.
In contrast, the 3,4-diacetoxy derivative 15 was com-
pletely devoided of cytotoxic activity. Taking into
account the crucial role of D-ring conformation to the
biological activity of acronycine derivatives,14 it seemed
interesting to investigate the D-ring conformation of
3,4-dihydro-3,4-dihydroxy-5,12-dimethoxy-2,2-dimethyl-
2H-anthra[2,3-b]pyran-6,11-dione derivatives. This was
carried out by the performance of NOESY experiments
The development of new analogues in the mitoxantrone
series is currently in progress in our laboratory.
Table 1. Cytotoxic activitya
Compound
IC50, mM
8
7.8
47.6
33.3
7.7
10
11
12
13
14
15
16
17
19
20
21
22
2
3.1
>50
>50
>50
>50
6.6
1.7
>50
3.4
25
1b
3.6b
aInhibition of L-1210 cell proliferation measured by the MMT assay
(mean of 2 values obtained in independent experiments).
bFrom ref 15.
Figure 1. Conformers I and II for compound 14.

610
E. Melliou et al. / Bioorg. Med. Chem. 9 (2001) 607±612
Experimental
0 ꢀC and then the reaction mixture was extracted with
NaCl (satd)-Et2O and the organic layer was collected.
The solvent was removed under reduced pressure and
compound 11 was puri®ed by ¯ash chromatography on
Silica gel with CH2Cl2:MeOH (99.5:0.5) (73 mg, 82%).
1H NMR (200 MHz, CDCl3) d 1.57 (3H, s, Me), 1.66
(3H, s, Me), 3.94 (3H, s, 5-OMe), 3.96 (1H, d, J=2.5 Hz,
OH-4), 4.02 (3H, s, 12-OMe), 4.26 (1H, d, J=6 Hz, H-3),
5.23 (1H, dd, J=6, 2.5 Hz, H-4), 7.69 (2H, m, H-8, 9),
8.12 (2H, m, H-7, 10); 13C NMR (50 MHz, CDCl3) d
23.77 (Me), 26.35 (Me), 56.49 (C-3), 61.19 (12-OMe),
62.74 (5-OMe), 68.18 (C-4), 79.50 (C-2), 118.61 (C-11a),
123.46 (C-4a), 126.55 (C-7, 10), 128.24 (C-5a), 133.46
(C-9), 133.61 (C-8), 133.97 (C-6a, 10a), 146.40 (C-12),
152.65 (C-12a), 156.76 (C-5), 181.24 (C-6), 182.86 (C-
11). MS-DCI m/z 449, 447 (M+H)+.
General
Spectra were recorded on the following apparatus: MS,
Nermag R10-10C in disorption-chemical ionization, using
NH3 as reagent gas, IR, Paragon 500, NMR, Bruker AC
1
200, H NMR (200 MHz), 13C NMR (50 MHz) and a
Bruker DRX400. Chemical shifts are given in d values
with TMS as an internal standard. Coupling constants
(J) are given in Hz. The signals of H and 13C spectra
were unambiguously assigned by use of 2-D NMR tech-
1
niques: H-1H-COSY, 13C-1H HETCOR and HMBC.
1
These 2-D experiments were performed using standard
Bruker microprograms. Column chromatography was
conducted using ¯ash Silica gel 60 Merck (40±63 mm),
with an overpressure of 300 mbar. All new compounds
gave satisfactory combustion analyses (C, H, N, within
Æ 0.4% of calcd values).
(Æ)-3,4-Dihydro-5,12-dimethoxy-2,2-dimethyl-4-hydroxy-
2H-anthra[2,3-b]pyran-6,11-dione (12). Compound 11
(57 mg, 0.13 mmol) was dissolved in anhydrous toluene
(7.5 mL) and the solution was re¯uxed for 15 min under
argon. Then was added AIBN (azo-bis-2, 20(methyl-2-
propionitrile)) (9 mg) and after 5 min a solution of tri-
butyltin hydride (0.3 mL in 4 mL of toluene) 0.5 mL/
5 min for 40 min. The reaction mixture was re¯uxed for
2 h. Then the solvent was evaporated and the residue was
puri®ed by ¯ash chromatography on Silica gel with
cyclohexane:EtOAc (80:20) to give compound 12 (34 mg,
5,12-Dihydroxy-2,2-dimethyl-2H-anthra[2,3-b]pyran-6,
11-dione (8). To a solution of 9 (1.000 g, 3.90 mmol) in
dry pyridine (5 mL), 3-methyl-2-butenal (3.75 mL, 39
mmol) was added. The reaction mixture was stirred for
5 h at 115 ꢀC under argon and then the reagents were
removed under reduced pressure (using a high vacuum
pump). The residue was puri®ed by ¯ash chromato-
graphy on Silica gel with CH2Cl2 to give compound 8
(500 mg, 40%). 1H NMR (200 MHz, CDCl3) d 1.57 (6H,
s, 2ÂMe), 5.80 (1H, d, J=10 Hz, H-3), 6.75 (1H, d,
J=10 Hz, H-4), 7.80 (2H, m, H-8, 9), 8.29 (2H, m, H-7,
10), 13.85 (1H, s, 5-OH), 13.30 (1H, s, 12-OH); 13C
NMR (50 MHz, CDCl3) d 28.34 (2ÂMe), 79.20 (C-2),
115.30 (C-4), 106.33 (C-11a), 112.20 (C-4a), 116.84 (C-
5a), 126.84 (C-7, 10), 131.69 (C-3), 133.31 (C-6a, 10a),
133.82 (C-9), 134.33 (C-8), 149.26 (C-12), 150.14 (C-
12a), 156.16 (C-5), 184.70 (C-6), 186.60 (C-11). MS-DCI
m/z 323 (M+H)+.
1
71%). H NMR (200 MHz, CDCl3) d 1.48 (3H, s, Me),
1.49 (3H, s, Me), 2.12 (2H, d, J=5.5 Hz, H-3), 3.52 (1H,
d, J=2 Hz, OH-4), 3.93 (3H, s, 5-OMe), 4.02 (3H, s, 12-
OMe), 5.06 (1H, td, J=5.5, 2 Hz, H-4), 7.70 (2H, m, H-
8, 9), 8.16 (2H, m, H-7, 10); 13C NMR (50 MHz,
CDCl3) d 26.43 (Me), 27.75 (Me), 39.66 (C-3), 60.31 (C-
4), 61.12 (12-OMe), 62.52 (5-OMe), 77.60 (C-2), 117.65
(C-11a), 125.07 (C-4a), 126.47 (C-7, 10), 127.79 (C-5a),
133.38 (C-9), 133.53 (C-8), 134.11 (C-6a, 10a), 146.68
(C-12), 154.18 (C-12a), 157.34 (C-5), 181.53 (C-6),
183.14 (C-11). MS-DCI m/z 369 (M+H)+.
5,12-Dimethoxy-2,2-dimethyl-2H-anthra[2,3-b]pyran-6,
11-dione (10). To a solution of 8 (100mg, 0.31mmol) in
dry acetone (12 mL) was added dimethylsulfate (2 mL)
and dry K2CO3 (1 g). The reaction mixture was stirred for
1 h at 56 ꢀC. Then the reagents were removed by ®ltration
and by evaporation under reduced pressure (using a high
vacuum pump). The residue was puri®ed by ¯ash chro-
matography on Silica gel with cyclohexane:CH2Cl2
(50:50) to CH2Cl2 to give compound 7 (90 mg, 81%). 1H
NMR (200 MHz, CDCl3) d 1.51 (6H, s, 2ÂMe), 3.89 (3H,
s, 5-OMe), 3.94 (3H, s, 12-OMe), 5.82 (1H, d, J=10 Hz,
H-3), 6.71 (1H, d, J=10 Hz, H-4), 7.67 (2H, m, H-8, 9),
8.12 (2H, m, H-7, 10); 13C NMR (50 MHz, CDCl3) d
28.19 (2ÂMe), 61.12 (12-OMe), 62.52 (5-OMe), 78.03 (C-
2), 116.18 (C-4), 119.41 (C-11a), 121.76 (C-4a), 126.40 (C-
7, 10), 127.72 (C-5a), 133.15 (C-8, 9), 133.38 (C-3), 134.19
(C-6a, 10a), 145.73 (C-12), 153.08 (C-12a), 153.37 (C-5),
181.82 (C-6), 183.07 (C-11). IR (CHCl3) nmax 2933, 1670,
1596, 1344, 1130cmÀ1.MS-DCI m/z 351 (M+H)+.
(+)-4-Acetoxy-3,4-dihydro-5,12-dimethoxy-2,2-dimethyl-
2H-anthra[2,3-b]pyran-6,11-dione (13). To a solution of
12 (12 mg, 0.03 mmol) in dry pyridine (1.5 mL) was
added Ac2O (1.5 mL, 15 mmol). The reaction mixture
was stirred for 24 h at room temperature and then the
reagents were removed under reduced pressure (using a
high vacuum pump). The residue was compound 13
1
(13 mg, 96%). H NMR (200 MHz, CDCl3) d 1.45 (3H,
s, Me), 1.53 (3H, s, Me), 2.09 (3H, s, CH3CO), 2.15 (2H,
m, H-3), 3.90 (3H, s, 5-OMe), 3.94 (3H, s, 12-OMe),
6.22 (1H, dd, J=5, 3 Hz, H-4), 7.70 (2H, m, H-8, 9),
8.16 (2H, m, H-7,10); 13C NMR (50 MHz, CDCl3) d
21.23 (CH3CO), 25.42 (Me), 29.40 (Me), 38.33 (C-3),
61.23 (C-4), 61.23 (12-OMe), 62.57 (5-OMe), 76.15 (C-
2), 117.74 (C-11a), 120.30 (C-4a), 126.41 (C-7), 126.62
(C-10), 128.56 (C-5a), 133.27 (C-9), 133.60 (C-8), 134.15
(C-6a), 134.24 (C-10a), 146.28 (C-12), 155.27 (C-12a),
158.13 (C-5), 169.95 (CH3CO), 181.23 (C-6), 183.35 (C-
11). MS-DCI m/z 410 (M+H)+.
(Æ)-trans-3-Bromo-3,4-dihydro-5,12-dimethoxy-2,2-dime-
thyl-4-hydroxy-2H-anthra[2,3-b]pyran-6,11-dione (11). To
a solution of 10 (70 mg, 0.20 mmol) in THF (5 mL) and
H2O (5 mL) was added N-bromo-succinimide (38 mg,
0.21 mmol). The reaction mixture was stirred for 4 h at
(Æ)-3,4-Dihydro-3,4-dihydroxy-5,12-dimethoxy-2,2-dime-
thyl - 2H-anthra[2,3-b]pyran-6,11-dione (14). To a solu-
tion of 10 (40 mg, 0.11 mmol) in 7 mL of t-BuOH:THF:

E. Melliou et al. / Bioorg. Med. Chem. 9 (2001) 607±612
611
H2O (10:3:1) was added N-methyl-morpholine-N-oxide
(28 mg) and OsO4 (2.5% in t-BuOH) (0.2 mL). The reac-
tion mixture was stirred for 48 h at room temperature.
Then NaHSO3 (satd) was added and the mixture was
stirred for 1 h. Then the reaction mixture was extracted
with CH2Cl2/H2O and the organic layer was collected.
The solvent was removed under reduced pressure and
compound 14 was puri®ed by ¯ash chromatography on
Silica gel with cyclohexane:EtOAc (60:40) (29 mg,
69%).1H NMR (200 MHz, CDCl3) d 1.42 (3H, s, Me),
1.53 (3H, s, Me), 3.20 (1H, d, J=5 Hz, OH-3), 3.86 (1H,
t, J=5 Hz, H-3), 3.96 (3H, s, 5-OMe), 4.02 (3H, s, 12-
OMe), 4.21 (1H, d, J=2 Hz, OH-4), 5.08 (1H, dd, J=5,
2 Hz, H-4), 7.70 (2H, m, H-8, 9), 8.15 (2H, m, H-7,10);
13C NMR (50 MHz, CDCl3) d 22.45 (Me), 24.44 (Me),
61.12 (12-OMe), 62.59 (5-OMe, C-4), 70.31 (C-3), 79.43
(C-2), 118.09 (C-11a), 123.09 (C-4a), 126.55 (C-7, 10),
128.09 (C-5a), 133.46 (C-9), 133.61 (C-8), 134.12 (C-6a,
10a), 146.47 (C-12), 153.60 (C-12a), 157.50 (C-5), 181.39
(C-6), 183.01 (C-11). MS-DCI m/z 385 (M+H)+.
10), 128.38 (C-200, 600), 128.86 (C-20, 60), 129.52 (C-300, 500),
129.52 (C-30, 50), 132.43 (C-10, 100), 133.35 (C-8), 133.53 (C-
9), 134.19 (C-3), 134.60 (C-6a, 10a), 141.58 (C-5, 12),
144.85 (C-40), 145.69 (C-400), 152.05 (C-12a), 180.24 (C-6),
180.72 (C-11). MS-DCI m/z 631 (M+H)+.
1
17. H NMR (200 MHz, CDCl3) d 1.23 (6H, s, 2ÂMe),
2.41 (3H, s, 40-Me), 5.66 (1H, d, J=10 Hz, H-3), 6.71
(1H, d, J=10 Hz, H-4), 7.29 (2H, d, J=8.5 Hz, H-30, 50),
7.73 (2H, m, H-8, 9), 7.83 (2H, d, J=8.5 Hz, H-20, 60),
8.09 (1H, m, H-7), 8.21 (1H, m, H-10), 13.67 (1H, s, 5-
OH). MS-DCI m/z 477 (M+H)+.
1
18. H NMR (200 MHz, CDCl3) d 1.54 (6H, s, 2ÂMe),
2.40 (3H, s, 40-Me), 5.67 (1H, d, J=10 Hz, H-3), 6.36
(1H, d, J=10 Hz, H-4), 7.28 (2H, d, J=8.5 Hz, H-30, 50),
7.73 (2H, m, H-8, 9), 7.81 (2H, d, J=8.5 Hz, H-20, 60),
8.08 (1H, m, H-7), 8.22 (1H, m, H-10), 13.35 (1H, s, 5-
OH); 13C NMR (50 MHz, CDCl3) d 21.69 (40-Me),
28.07 (2ÂMe), 78.46 (C-2), 115.45 (C-11a), 116.39 (C-4),
118.78 (C-5a), 122.95 (C-4a), 126.42 (C-7), 127.34 (C-
10), 128.89 (C-20, 60), 129.74 (C-30, 50), 132.14 (C-10a),
132.82 (C-10), 133.48 (C-8), 133.67 (C-9), 134.60 (C-
10a), 134.74 (C-3), 137.11 (C-5), 145.48 (C-40), 147.40
(C-12a), 151.31 (C-12), 179.86 (C-11), 188.55 (C-6). MS-
DCI m/z 477 (M+H)+.
(Æ)-3,4-Diacetoxy-3,4-dihydro-5,12-dimethoxy-2,2-dime-
thyl-2H-anthra[2,3-b]pyran-6,11-dione (15). To a solu-
tion of 14 (20 mg, 0.05mmol) in dry pyridine (1.5 mL) was
added Ac2O (1.5 mL, 15mmol). The reaction mixture was
stirred for 24h at room temperature and then the reagents
were removed under reduced pressure (using a high
vacuum pump). The residue was compound 15 (24 mg,
96%). 1H NMR (200MHz, CDCl3) d 1.49 (6H, s, 2ÂMe),
1.53 (3H, s, Me), 2.09 (6H, s, 2ÂCOCH3), 3.88 (3H, s, 5-
OMe), 3.95 (3H, s, 12-OMe), 5.22 (1H, d, J=5 Hz, H-
3), 6.39 (1H, d, J=5 Hz, H-4), 7.70 (2H, m, H-8, 9), 8.15
(2H, m, H-7, 10); 13C NMR (50 MHz, CDCl3) d 20.62
(CH3CO), 20.71 (CH3CO), 21.54 (Me), 25.98 (Me),
60.64 (C-4), 61.33 (12-OMe), 62.64 (5-OMe), 70.82 (C-
3), 77.63 (C-2), 118.55 (C-11a), 120.13 (C-4a), 126.44
(C-7), 126.64 (C-10), 128.95 (C-5a), 133.36 (C-9), 133.68
(C-8), 134.12 (C-6a, 10a), 146.10 (C-12), 154.02 (C-12a),
157.91 (C-5), 169.76 (2ÂCH3CO), 181.14 (C-6), 183.14
(C-11). MS-DCI m/z 469 (M+H)+.
2,2-dimethyl-5-[N-[2-(dimethylamino)ethyl]amino]-12-to-
syloxy-2H-anthra[2,3-b]pyran-6,11-dione (19), 2,2-dime-
thyl-12-[N-[2-(dimethylamino)ethyl]amino]-5-tosyloxy-
2H-anthra[2,3-b]pyran-6,11-dione (20), 2,2-dimethyl-5,
12-bis[N-[2-(dimethylamino)ethyl] amino]-2H-anthra[2,3-
b]pyran-6,11-dione (21). To a solution of 16 (100 mg,
0.16 mmol) in dry pyridine (4 mL) was added N,N-dime-
thylethylenediamine (1 mL). The reaction mixture was
stirred for 2 h at 60 ꢀC. Then the reagents were removed
under reduced pressure (using a high vacuum pump).
The residue was puri®ed by ¯ash chromatography on
Silica gel with CH2Cl2:MeOH (99:1) to (80:20) to give
compound 19 (43 mg, 49%), 20 (13 mg, 15%) and 21
(26 mg, 36%).
2,2-Dimethyl-5,12-ditosyloxy-2H-anthra[2,3-b]pyran-6,11-
dione (16), 2,2-dimethyl-5-hydroxy-12-tosyloxy-2H-anthra
[2,3-b]pyran-6,11-dione (17), 2,2-dimethyl-12-hydroxy-5-
tosyloxy-2H-anthra[2,3-b]pyran-6,11-dione (18). To a
solution of 8 (100 mg, 0.31 mmol) in dry acetone (12 mL)
was added tosyl chloride (215 mg, 1.12 mmol) and dry
K2CO3 (1 g). The reaction mixture was stirred for 6 h at
56 ꢀC. Then the reagents were removed by ®ltration and
by evaporation under reduced pressure. The residue was
puri®ed by ¯ash chromatography on Silica gel with
cyclohexane:EtOAc (95:5) to (80:20) to give compound
16 (90 mg, 47%), 17 (15 mg, 10%) and 18 (6 mg, 4%).
1
19. H NMR (200 MHz, CDCl3) d 1.37 (6H, s, 2ÂMe),
2.29 (6H, s, 20-N(Me)2), 2.37 (3H, s, 400-Me), 2.50 (2H, t,
J=6.5 Hz, H-20), 3.46 (2H, q, J=6.5 Hz, H-10), 5.55
(1H, d, J=10 Hz, H-3), 6.53 (010 H, d, J=10 Hz, H-4),
7.23 (2H, d, J=8.5 Hz, H- 300, 5 ), 7.62 (2H, m, H-8, 9),
7.79 (2H, d, J=8.5 Hz, H- 200, 600), 7.88 (1H, dd, J=7.5,
1.5 Hz, H-7), 8.17 (1H, dd, J=7.5, 1.5 Hz, H-10), 10.58
(1H, t, J=6.5 Hz, N-H); 13C NMR (50 MHz, CDCl3) d
21.46 (400-Me), 26.36 (2ÂMe), 45.73 (20-N(Me)2), 47.60
(C-10), 60.20 (C-20), 77.24 (C-2), 110.42 (C-11a), 113.70
(C-5a), 114.35 (C-4a), 120.42 (C-4), 126.01 (C-7, 10),
127.66 (C-3), 128.28 (C-200, 600), 129.29 (C-300, 500), 132.76
(C-8), 133.67 (C-9), 134.18 (C-6a, 10a), 134.80 (C-100),
135.40 (C-12), 144.46 (C-400), 151.23 (C-5), 153.56 (C-
12a), 182.50 (C-6), 183.44 (C-11). MS-DCI m/z 547
(M+H)+.
1
16. H NMR (200 MHz, CDCl3) d 1.25 (6H, s, 2ÂMe),
2.40 (3H, s, 40-Me), 2.43 (3H, s, 400-Me), 5.65 (1H, d,
J=10 Hz, H-3), 6.43 (1H, d, J=10 Hz, H-4), 7.30 (2H,
d, J=8.5 Hz, H- 30, 50), 7.32 (2H, d, J=8.5 Hz, H- 300,
500), 7.69 (2H, m, H-8, 9), 7.82 (2H, d, J=8.5 Hz, H- 20,
60), 7.84 (2H, d, J=8.5 Hz, H- 200, 600), 7.98 (2H, m, H-7,
10); 13C NMR (50 MHz, CDCl3) d 21.51 (400-Me, 40-
Me), 27.75 (2ÂMe), 79.14 (C-2), 115.93 (C-4, 11a),
121.55 (C-5a), 123.06 (C-4a), 126.51 (C-7), 126.58 (C-
1
20. H NMR (200 MHz, CDCl3) d 1.50 (6H, s, 2ÂMe),
2.33 (9H, s, 20-N(Me)2, 400-Me), 2.62 (2H, t, J=6.5 Hz,
H-20), 3.88 (2H, q, J=6.5 Hz, H-10), 5.65 (1H, d,
J=10 Hz, H-3), 6.50 (1H, d, J=10 Hz, H-4), 7.22 (2H,

612
E. Melliou et al. / Bioorg. Med. Chem. 9 (2001) 607±612
d, J=8.5 Hz, H- 300, 500), 7.61 (1H, td, J=8, 1.5 Hz, H-
8), 7.66 (1H, td, J=8, 1.5 Hz, H-9), 7.78 (2H, d,
J=8.5Hz, H- 200, 600), 7.87 (1H, dd, J=8, 1.5 Hz, H-7),
8.19 (1H, dd, J=8, 1.5 Hz, H-10), 10.27 (1H, t, J=6.5Hz,
N-H); 13C NMR (50 MHz, CDCl3) d 21.66 (400-Me), 27.65
(2ÂMe), 44.63 (C-10), 45.69 (20-N(Me)2), 60.21 (C-20),
77.05 (C-2), 110.52 (C-11a), 113.50 (C-5a), 117.39 (C-
4),121.21 (C-4a), 126.25 (C-7), 126.39 (C-10), 128.97 (C-
200, 600), 129.59 (C-300, 500), 131.94 (C-3), 132.57 (C-8),
132.79 (C-100), 133.08 (C-9), 133.85 (C-6a), 134.26 (C-
10a), 135.21 (C-5), 143.37 (C-12), 145.14 (C-400), 146.79
(C-12a), 181.89 (C-6), 184.16 (C-11). MS-DCI m/z 547
(M+H)+.
48 h. Results are expressed as IC50, the concentration
needed to reduce by 50% the optical density of treated
cells with respect to the optical density of untreated
controls.
For the cell-cycle analysis L-1210 cells (5Â105 cells/mL)
were incubated for 21 h with various concentrations of
tested compounds. Cells were then ®xed by 70% EtOH
(v/v), washed, and incubated with PBS containing
100 mg/mL RNAse and 25 mg/mL propidium iodide for
30 min at 20 ꢀC. For each sample, 10,000 cells were ana-
lyzed on an ATC3000 ¯ow cytometer (Bruker, Wissen-
bourg, France).
1
21. H NMR (200 MHz, CDCl3) d 1.57 (6H, s, 2ÂMe),
Calculations
2.35 (6H, s, 200-N(Me)2), 2.43 (6H, s, 20-N(Me)2), 2.60
(2H, t, J=6.8 Hz, H-200), 2.76 (2H, t, J=6.5 Hz, H-20),
2.49 (2H, t, J=6.8 Hz, H-10), 3.93 (2H, t, J=6.5 Hz, H-
100), 5.68 (1H, d, J=10 Hz, H-3), 6.58 (1H, d, J=10 Hz,
H-4), 7.67 (2H, m, H-8, 9), 8.32 (2H, m, H-7,10); 13C
NMR (50 MHz, CDCl3) d 26.84 (2ÂMe), 42.45 (C-100),
44.33 (200-N(Me)2), 44.70 (20-N(Me)2), 45.61 (C-10),
59.03 (C-20), 59.43 (C-200), 77.10 (C-2), 109.32 (C-11a),
112.70 (C-5a), 117.02 (C-4a), 120.30 (C-4), 125.84 (C-7,
10), 126.32 (C-3), 128.78 (C-8, 9), 132.75 (C-6a), 132.82
(C-10a), 141.86 (C-12), 147.63 (C-5), 149.50 (C-12a),
182.91 (C-6), 186.68 (C-11). MS-DCI m/z 447
(M+H)+.
Molecular calculations were performed using the MM+
force ®eld of the HyperChem program (HyperChem is
developed and licensed from Hypercube; the MM+
force ®eld used in this software for molecular mechanics
calculations is an extension of MM2 (1991) parameters
and atom types with the 1997 functional form). The
Polak-Ribiere (conjugate gradient) minimization method
with an energy convergence criterion of 0.01 Kcal molÀ1
was used for geometry optimization.
References and Notes
2,2-dimethyl-5-[N-[2-(dimethylamino)ethyl]amino]-12-[N-
[2-[(2-hydroxyethyl)amino] ethyl]amino]-2H-anthra[2,3-
b]pyran-6,11-dione (22). To a solution of 19 (18 mg,
0.03mmol) in dry pyridine (3 mL) was added 2-[(2-amino-
ethyl)amino]ethanol (0.3 mL). The reaction mixture was
stirred for 24 h at 60 ꢀC. Then the reagents were removed
under reduced pressure (using a high vacuum pump).
The residue was puri®ed by ¯ash chromatography on
Silica gel with CH2Cl2:MeOH (99:1) to (80:20) to give
compound 22 (10 mg, 69%).1H NMR (200 MHz,
CDCl3) d 1.55 (6H, s, 2ÂMe), 2.28 (6H, s, 20-N(Me)2),
2.53 (2H, t, J=5.5 Hz, H-20), 2.88 (2H, t, J=4 Hz, H-300),
2.95 (2H, t, J=5.5 Hz, H-200), 3.44 (2H, t, J=5.5 Hz, H-
10), 3.67 (2H, t, J=4 Hz, H-10a), 3.88 (2H, t, J=5.5 Hz,
H-100), 5.64 (1H, d, J=10 Hz, H-3), 6.57 (1H, d, J=
10 Hz, H-4), 7.64 (2H, m, H-8, 9), 8.29 (2H, m, H-7,10);
13C NMR (50 MHz, CDCl3) d 26.78 (2ÂMe), 45.75 (20-
N(Me)2), 46.66 (C-100), 47.46 (C-10), 49.30 (C-200), 50.76
(C-300), 60.30 (C-20), 60.95 (C-400), 77.10 (C-2), 109.32
(C-11a), 112.70 (C-5a), 116.27 (C-4a), 121.29 (C-4),
126.14 (C-7), 126.29 (C-10), 127.29 (C-3), 132.04 (C-8),
132.23 (C-9), 134.36 (C-6a), 134.70(C-10a), 141.13 (C-
12), 148.92 (C-5), 150.43 (C-12a), 181.77 (C-6), 182.54
(C-11). MS-DCI m/z 479 (M+H)+.
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L-1210 cells were cultivated in RPMI 1640 medium
(Gibco) supplemented with 10% fetal calf serum, 2 mM
L-glutamine, 100 units/mL penicillin, 100 mg/mL strep-
tomycin, and 10 mM HEPES bu€er (pH 7.4). Cytotoxi-
city was measured by the microculture tetrazolium
assay.12 Cells were exposed to graded concentrations of
tested compound (nine serial dilutions in triplicate) for