Design and synthesis of new pyranoxanthenones bearing a nitro group or an aminosubstituted side chain on the pyran ring. Evaluation of their growth inhibitory activity in breast cancer cells

Article abstract of DOI:10.1016/j.ejmech.2006.10.018

Some new 2,6-disubstituted pyrano- and 1,2-dihydropyrano[2,3-c]xanthen-7-ones have been synthesized and their antiproliferative activity has been evaluated against MDA-MB-231 breast cancer cells. The antiproliferative activity evaluation of the compounds

Full text of DOI:10.1016/j.ejmech.2006.10.018

European Journal of Medicinal Chemistry 42 (2007) 307e319
http://www.elsevier.com/locate/ejmech
Original article
Design and synthesis of new pyranoxanthenones bearing
a nitro group or an aminosubstituted side chain
on the pyran ring. Evaluation of their growth
inhibitory activity in breast cancer cells
George Kolokythas a, Ioannis K. Kostakis a, Nicole Pouli a, Panagiotis Marakos a,
Olga Ch. Kousidou b, George N. Tzanakakis c, Nikos K. Karamanos b
*
,
a Division of Pharmaceutical Chemistry, Department of Pharmacy, University of Athens, Panepistimiopolis-Zografou, Athens 15771, Greece
b Laboratory of Biochemistry, Department of Chemistry, University of Patras, 26110 Patras, Greece
c Department of Histology, Medical School, University of Crete, 71110 Herakleion, Greece
Received 6 October 2006; received in revised form 30 October 2006; accepted 30 October 2006
Available online 4 December 2006
Abstract
Some new 2,6-disubstituted pyrano- and 1,2-dihydropyrano[2,3-c]xanthen-7-ones have been synthesized and their antiproliferative activity
has been evaluated against MDA-MB-231 breast cancer cells. The antiproliferative activity evaluation of the compounds provided evidence that
a dimethylamino substituted side chain and the presence of 1,2 double bond play a key role in cell growth inhibition. Among the tested
derivatives the 6-dimethylaminoethylamino-2-nitropyranoxanthenone analogue possessed a significant inhibitory effect in a wide range of
concentrations.
Ó 2006 Elsevier Masson SAS. All rights reserved.
Keywords: 1,2-Dihydropyrano[2,3-c]xanthenones; Breast cancer cells; Growth inhibitory effect
1. Introduction
showing better therapeutic efficacy, together with fewer and
less pronounced side effects, e.g. cardiotoxicity and the devel-
opment of resistance [3e5]. The chemical modification of the
anthracenedione chromophore unit, the repositioning of the
hydroxyl substituents and the variation of the alkylamino
side chains have been extensively investigated and resulted
in the preparation of very active derivatives, exemplified by
the heterocyclic bis(2-aminoethyl)amino-substituted com-
pound pixantrone dimaleate (Fig. 1) endowed with antileuke-
mic activity comparable to mitoxantrone. Pixantrone showed
no measurable cardiotoxicity and is currently undergoing
phase III clinical trials for the treatment of non-Hodgkin’s
lymphoma [6e8]. All the above mentioned anthracenedione
derivatives while being effective DNA intercalators, also inter-
fere with the function of the DNA binding enzymes, such as
DNA topoisomerases and DNA polymerases [9]. A common
It has been previously shown that dialkylaminoalkyl groups
present in the side chains of the anthracenedione antitumor
drugs mitoxantrone and ametantrone (Fig. 1) play a key role
in their biological activity, regulating DNA binding after met-
abolic activation [1]. The cytotoxic effects of the anthracene-
diones, similar to anthracyclines, are probably multimodal,
although interaction with DNA and consequent damage are
thought to be essential for their therapeutic effect [2]. Numer-
ous anthracenedione derivatives have been synthesized in the
past ten years, in an attempt to obtain new active drugs,
* Corresponding author. Tel.: þ30 210 7274830; fax: þ30 210 7274747.
E-mail address: marakos@pharm.uoa.gr (P. Marakos).
0223-5234/$ - see front matter Ó 2006 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2006.10.018

308
G. Kolokythas et al. / European Journal of Medicinal Chemistry 42 (2007) 307e319
H
N
NH2
OCH3
R
R
O
O
HN
HN
OH
OH
O
O
HN
HN
O
N
N
O
CH3
CH3
CH3
N
H
NH2
mitoxantrone R= OH
ametantrone R= H
pixantrone
acronycine
Fig. 1. Structures of mitoxantrone, ametantrone, piroxantrone and acronycine.
structural feature for these agents is the presence of one or two
basic side chains, in a strictly defined orientation to their
polycyclic chromophore moiety. This particular substitution
pattern is thought to play an important role in modulating
the affinity of each molecule for the nucleic acid and the
sequence specific contacts along the double helical DNA. Sim-
ilar substitution of the planar tricyclic acridone molecule in
positions 1 and/or 4 with one or two analogous diaminoalkylo-
residues, containing two or three methylene groups between
amino groups, was found to play a crucial role in modulating
both DNA binding properties and the pharmacological
response of several acridone derivatives [10].
2. Chemistry
For the synthesis of the target derivatives 3,3-dimethyl-
6-hydroxy-3H,7H-pyrano[2,3-c]xanthen-7-one 5 was used as
key intermediate (Fig. 2). The preparation of this compound
has already been reported [19], although in considerably low
yield (11%), since it results from the Claisen rearrangement
of the ether 2, together with the corresponding linear isomer
i.e. 2,2-dimethyl-5-hydroxy-2H,6H-pyrano[3,2-b]xanthen-
6-one. We had previously observed that the methylation of
the 1-OH group of compound 2, prior to ring-closure improves
the percentage of the angular isomer, presumably due to steric
hindrance [20]. A slight modification of the reported proce-
dure by the insertion of the bulky p-tosyl group into the free
1-OH of compound 2, provided the ester 3, which was sub-
jected to thermal cyclization to furnish selectively the angular
isomer 4 that was easily hydrolyzed to the required compound
5. According to this methodology the hydroxyderivative 5 was
prepared at 50% overall yield, starting from 1,3-dihydroxy-
9H-xanthen-9-one 1.
Methylation of the 6-OH group of compound 5 was then
carried out, upon treatment with dimethyl sulfate in the pres-
ence of sodium hydride, to yield the 6-methoxyderivative 6
[20]. This analogue, was first converted into the cis-diol 7
(Fig. 3) by catalytic syn-dihydroxylation with osmium tetrox-
ide and 4-morpholine N-oxide as oxidizing agent and then
gave the 2-keto derivative 8, upon treatment of 7 with a cata-
lytic amount of p-toluenesulfonic acid in boiling toluene. An
analogous methodology for the preparation of 8, using cupric
sulfate instead of p-toluenesulfonic acid, has been previously
reported [20]. Compound 8 was then treated with the suitable
2-dialkylaminoethylamine and converted into the correspond-
ing enamines 9a,b. These enamines were highly unstable and
upon the usual work-up were hydrolyzed back to the starting
compound 8. Consequently, they were not isolated, but were
first treated with a saturated ethanolic HCl solution and the re-
sulting hydrochlorides were then reduced by the reaction with
sodiumcyanoborohydride to provide the target compounds
10a,b.
We have been involved in the synthesis and antiprolifera-
tive activity evaluation of a number of pyranoxanthenones
[11] and azapyranoxanthenones [12,13] bearing structural sim-
ilarity with the acridone alkaloid acronycine (Fig. 1), which
was found to possess anticancer activity against a wide spec-
trum of experimental neoplasms in laboratory animals [14].
While the mechanism of action of acronycine at either cellular
or molecular level has not been yet clearly established, it was
shown that inhibition of DNA and RNA syntheses by this
alkaloid is possibly the result of its potential capacity to
intercalate into DNA [15]. Among numerous active acrony-
cine derivatives, it has been reported that 2-nitroacronycine
demonstrated high potency when tested against a panel of
human tumor cells in vitro [15,16]. In previous studies we
have found that the introduction of a flexible dialkylaminoe-
thylamino side chain substitution in a number of (aza)
pyranoxanthenones related to acronycine generally led to an
improvement of the cytotoxicity against the murine leukemia
L1210 and the colon adenocarcinoma HT-29 cell lines
[13,17]. In accordance with this finding, the introduction of
6-dialkylaminoalkylamino side chain substitution on analo-
gous pyranoacridones and benzo[b]pyranoacridones led to
a significant increase of the cytotoxic activity against L1210
cells [18]. Prompted by these observations we decided to
further investigate the effect on cytotoxicity caused by the
insertion at the position 2 of the pyran ring of a nitro, a dialky-
laminoethylamino side chain, or the less flexible dialkylami-
nomethylcarbonylamino group. We have also prepared
analogues bearing a second basic side chain replacing the
6-methoxy group of the lead pyranoxanthenone chromophore,
in an effort to study the structureeactivity relationships within
this class of compounds.
For the preparation of the 6-aminosubstituted analogues of
compounds 10, we have attempted to apply the above men-
tioned methodology, using a suitable intermediate for the in-
troduction of the amino substituted side chain. We have thus
replaced the 6-methoxy group of compounds 10 by a tosyloxy

G. Kolokythas et al. / European Journal of Medicinal Chemistry 42 (2007) 307e319
309
O
OR
O
OR
O
OH
a
c
O
O
O
O
O
OH
2 R= H
3 R= Ts
4 R= Ts
b
d
e
1
5 R= H
6 R= CH3
Fig. 2. a: 3-Chloro-3-methyl-1-butyne, K2CO3, KI, CuI, dry DMF, 70 ꢀC, 26 h; b: TsCl, K2CO3, acetone dry, reflux, 1.5 h; c: DMF, reflux, 2 h; d: aq. NaOH, EtOH,
r.t., 30 min; e: (1) NaH, dry THF, 1 h; (2) (CH3)2SO4, dry THF, reflux, 3.5 h.
group, however, the subsequent displacement of the later by
2-dialkylaminoethylamines was unsuccessful.
resulting N-Boc analogues 19a,b were deprotected by
treatment with trifluoroacetic acid, to provide the target
aminoderivatives 20a,b.
Consequently, an alternative methodology was developed
for the preparation of the target molecules. This involves
initial nitration of compound 5 to the 2-nitroderivative 11
(Fig. 4), which was then converted to the tosylate 12. The to-
syloxy group of compound 12 turned out to be a very effective
leaving group and was easily substituted by the reaction with
appropriate 2-dialkylaminoethylamines to result in the amino-
derivatives 13a,b. Reduction of the double bond of the amines
13a,b upon treatment with sodiumborohydride provided the
nitro derivatives 14a,b that were subjected to catalytic hydro-
genation over Raney Ni in the presence of a 40% aqueous
hydrazine solution, resulting in the 2-aminoderivatives
15a,b. Reaction of these compounds with chloroacetic acid
in the presence of N,N0-dicyclohexylcarbodiimide gave the
chloroacetamides 16a,b, which upon treatment with the suit-
able secondary amines furnished the target derivatives
17a,b. On the other hand, the 2-aminoderivatives 15a,b
were treated with Boc-anhydride and converted to the carba-
mates 18a,b, which reacted with the suitable dialkylaminoe-
thylchlorides in the presence of sodium hydride. The
By analogy to the above mentioned procedure, the nitroder-
ivative 11 was methylated (Fig. 5) and the resulting compound
21 was successively reduced to give compounds 22 and 23.
The aminoderivative 23 was chloroacetylated and the resulting
chloroacetamide 24 provided the target aminoderivatives
25a,b upon reaction with the suitable secondary amines.
3. Results and discussion
The tested compounds were studied for their antiprolifera-
tive activity on the MDA-MB-231 breast cancer cell line,
cultured in serum-containing medium. We choose to grow
the cells in the presence of serum in order for our experimental
model to be more similar to physiological conditions. Due to
solubility problems in DMSO and ethanol, compounds 21
and 22 were not evaluated. The obtained IC50 values are
presented in Table 1.
The 6-methoxy analogues 10a,b, 23, and 25a,b showed
an inhibitory effect (12e20% inhibition of cell growth,
O
OCH3
O
OCH3
O
O
OCH3
a
b
O
O
O
O
O
HO
6
8
7
OH
O
c
O
O
OCH3
O
O
OCH3
a: R= N(CH3)2
b: R= N(C2H5)2
d
O
O
NHCH2CH2R
NHCH2CH2R
10a,b
9a,b
Fig. 3. a: (1) 2.5% OsO4 in tert-butanol, N-methylmorpholine-N-oxide, tert-butanol/THF/H2O, r.t., 48 h; (2) sat. aq. NaHSO3, r.t., 2 h; b: p-TsOH ꢁ H2O, toluene,
reflux, 15 min; c: 2-dialkylaminoethylamine, abs. EtOH, reflux, 7 h; d: (1) EtOH sat. in HCl, NaCNBH3, r.t., 1.5 h; (2) aq. 20% Na2CO3, r.t.

310
G. Kolokythas et al. / European Journal of Medicinal Chemistry 42 (2007) 307e319
R
H
O
O
OH
O
O
OTs
O
O
N
b
c
O
O
O
R
NO2
NO2
5
R= H
a
12
13a,b
11 R= NO2
d
R
R
R
H
H
N
H
O
O
N
O
O
O
O
N
i
from 18a,b
j
O
O
O
R1
N
H
N
R
R
e
Boc
19a,b
14a,b R1= NO2
15a,b R1= NH2
20a,b
f
g
16a,b R1= NHCOCH2Cl
17a,b R1= NHCOCH2R
18a,b R1= NHBoc
a: R= N(CH3)2
b: R= N(C2H5)2
h
Fig. 4. a: 65% HNO3, glacial AcOH, r.t., 30 min; b: TsCl, K2CO3, acetone, reflux, 1 h; c: 2-dialkylaminoethylamine, abs. EtOH, reflux, 2 h; d: NaBH4, MeOH/
CH2Cl2, r.t., 30 min; e: Raney Ni wet, aq. hydrazine 40%, EtOH, 45 ꢀC, 15 min; f: ClCH2COOH, DCC, CH2Cl2, r.t., 30 min; g: dialkylamine, EtOH, reflux, 10 h;
h: Boc2O, Et3N, dry THF, r.t., 3 h; i: (1) NaH, dry DMF, r.t., 30 min; (2) 2-dialkylaminoethylchloride, dry DMF, r.t., 18 h; j: CF3COOH, CH2Cl2, r.t., 1 h.
depending on the compound) shown even from the lowest
concentration tested (1 mM). This inhibitory pattern remained
for the concentration of 3 mM. Using 10 and 30 mM only
moderately increased (15e30%) the inhibition of cell growth.
Significant inhibitory effect (74e80%) was detected at the
highest concentration (100 mM) tested. However, this effect
is probably due to cytotoxicity, since a large number of cells
lost contact with the culture flask and the remaining adherent
cells underwent morphological changes.
The antiproliferative activities of the 2-nitropyranoxanthe-
nones 13a,b bearing a 6-amino-substituted side chain are
shown in Fig. 6. The dimethylamino analogue 13a exhibited
a significant inhibitory effect in a wide range of concentra-
tions, showing an IC50 value of 21 mM. On the contrary, the
Table 1
Antiproliferative activity of the synthesized compounds (IC50 values in mM)a
against MDA-MB-231 breast cancer cells
Compound
IC50 (mM)
O
O
OR1
O
O
OCH3
10a
10b
13a
13b
14a
14b
15a
15b
17a
17b
20a
20b
23
57 (6.1)
44 (4.8)
21 (1.4)
>100
b
O
O
>100
>100
38 (2.3)
62 (6.8)
50 (6.0)
68 (6.3)
46 (5.5)
73 (8.1)
53 (4.2)
51 (3.1)
52 (4.3)
NO2
R2
22 R2= NO2
23 R2= NH2
11 R1= H
21 R1= CH3
a
c
d
e
24 R2= NHCOCH2Cl
25a,b R2= NHCOCH2R
a: R= N(CH3)2
b: R= N(C2H5)2
25a
25b
a
Fig. 5. a: (1) NaH, dry THF, 30 min; (2) (CH3)2SO4, dry THF, reflux, 3 h; b:
NaBH4, MeOH/CH2Cl2, r.t., 30 min; c: Raney Ni wet, aq. hydrazine 40%,
EtOH, 45 ꢀC, 15 min; d: ClCH2COOH, DCC, CH2Cl2, 30 min; e: dialkyl-
amine, EtOH, reflux, 6 h.
The results represent the mean (ꢂstandard 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.

G. Kolokythas et al. / European Journal of Medicinal Chemistry 42 (2007) 307e319
311
100
80
60
40
20
0
100
13b
13a
80
60
40
20
0
0
1
3
10
30
100
0
1
3
10
30
100
(µM)
(µM)
Fig. 6. Inhibitory effects of 13a and 13b on human breast cancer MDA-MB-231 cell line. The cells were incubated in serum-containing medium for 72 h in the
presence of increasing concentrations (1, 3, 10 and 100 mM). Cell proliferation was assessed by the WST-1 assay. The results are presented as percentage of growth
inhibition in respect to controls. Control values did not exhibit significant changes as compared to DMSO vehicle. Each point represents the average ꢂ standard
deviation of three individual experiments, performed in three replicates.
diethylamino nitroderivative 13b did not cause significant
inhibition even at high concentrations.
The above results indicate that the presence of the dime-
thylamino substitution increases the growth inhibitory
activity as compared to the diethylamino one. It is worth
noticing that this slight structural modification resulted in
an improved antiproliferative activity in the whole respective
analogues series. Another potentially important finding is
the antiproliferative effect of the 1,2 unsaturation. Both fac-
tors, i.e. the dimethylamino substitution and the presence of
1,2 double bond in combination seem to be the key players
for the growth inhibitory effects observed for this group of
compounds.
The antiproliferative effects of the 1,2-dihydropyranoxan-
thenones 14a, 15a, 17a and 20a which are substituted with
a 6-dimethylaminoethylamino side chain are presented in
Fig. 7. An inhibitory effect up to 20% was obtained for all com-
pounds for the concentrations 1, 3 and 10 mM. At higher concen-
trations a significant antiproliferative activity was observed for
the 2-amino analogue 15a, the diamine 20a and the acetamide
17a. The respective IC50 values were 38, 46 and 50 mM.
For the corresponding 1,2-dihydropyranoxanthenones 14b,
15b, 17b and 20b bearing a 6-diethylaminoethylamino side
chain, two patterns of antiproliferative activity were observed.
The 2-nitroderivative 14b, similarly as its structural analogue
14a, caused a low inhibition (approximately 20e30%) at the
concentrations of 30 and 100 mM. Compounds 15b, 17b and
20b possessed a similar inhibitory effect as 14a at the concen-
tration of 30 mM, but they reached 75e80% inhibition at a con-
centration of 100 mM.
4. Experimental protocols
4.1. Chemistry
All chemicals were purchased from Aldrich Chemical Co.
Melting points were determined on a Buchi apparatus and
¨
are uncorrected. 1H NMR spectra and 2D spectra were
100
100
15a
14a
80
80
60
40
20
0
60
40
20
0
100
100
20a
17a
80
80
60
40
20
0
60
40
20
0
0
1
3
10
30
100
0
1
3
10
30
100
(µM)
(µM)
Fig. 7. Study of the antiproliferative effects of 14a, 15a, 17a and 20a on breast cancer cells. For details see legend of Fig. 6.

312
G. Kolokythas et al. / European Journal of Medicinal Chemistry 42 (2007) 307e319
recorded on a Bruker Avanche 400 instrument, whereas 13C
NMR spectra were recorded on a Bruker AC 200 spectrometer
in deuterated solvents and were referenced to TMS (d scale).
were washed with brine, dried (Na2SO4) and concentrated to
dryness. The residue was purified by column chromatography
(silica gel, cyclohexaneeEtOAc 95:5), to provide compound 5
1
1
The signals of H and 13C spectra were unambiguously as-
(2.69 g, 87%). Mp 176e177 ꢀC (Et2Oen-hexane); H NMR
signed by using 2D NMR techniques: 1He1H COSY, NOESY
HMQC and HMBC. Flash chromatography was performed on
Merck silica gel 60 (0.040e0.063 mm). Analytical thin layer
chromatography (TLC) was carried out on precoated
(0.25 mm) Merck silica gel F-254 plates. Elemental analyses
were performed on a PerkineElmer PE 240C Elemental
Analyzer (Norwalk, CT) and are within ꢂ0.4% of the theoret-
ical values.
(CDCl3, 400 MHz) d (ppm): 1.47 (s, 6H, 2 ꢁ gemCH3), 5.60
(d, J ¼ 10.5 Hz, 1H, H-2), 6.26 (s, 1H, H-5), 6.83 (d,
J ¼ 10.5 Hz, 1H, H-1), 7.34 (dt, J ¼ 8 Hz, 0.5 Hz, 1H, H-9),
7.44 (dd, J ¼ 8 Hz, 0.5 Hz, 1H, H-11), 7.70 (dt, J ¼ 8 Hz,
w1 Hz, 1H, H-10), 8.22 (dd, J ¼ 8 Hz, w1 Hz, 1H, H-8),
13.15 (s, 1H, D2O exch., OH); 13C NMR (CDCl3, 50 MHz)
d (ppm): 28.19 (2 ꢁ gemCH3), 78.13 (C-3), 99.13 (C-5),
100.55 (C-12b), 103.44 (C-6a), 114.78 (C-1), 117.31 (C-11),
120.19 (C-7a), 123.91 (C-9), 125.57 (C-8), 126.95 (C-2),
134.75 (C-10), 151.52 (C-11a), 155.44 (C-12a), 160.70 (C-
4a), 162.93 (C-6), 180.53 (C-7).
4.1.1. 3-(1,1-Dimethylpropyn-2-oxy)-1-[(4-methylphenyl)
sulfonyloxy)]-9H-xanthen-9-one (3)
To a solution of the xanthenone 2 [19] (4 g, 13.60 mmol) in
dry acetone (100 mL), were added p-toluenesulfonyl chloride
(7.77 g, 40.80 mmol) and potassium carbonate (9.38 g,
68.00 mmol) and the resulting mixture was heated at reflux
under argon for 90 min. The inorganic precipitate was then
filtered off and the filtrate was evaporated to dryness. Flash
chromatography on silica gel using a mixture of cyclohexa-
neeEtOAc 80:20 as the eluent, provided the title compound
3 (5.30 g, 87%). Mp 146e148 ꢀC (EtOAcen-hexane); 1H
NMR (CDCl3, 400 MHz) d (ppm): 1.72 (s, 6H, 2 ꢁ gemCH3),
2.38 (s, 3H, SO2C6H4CH3), 2.73 (s, 1H, H-30), 7.02 (d,
J ¼ 2 Hz, 1H, H-2), 7.26 (d, J ¼ 2 Hz, 1H, H-4), 7.30 (m,
3H, H-300, H-500 and H-7), 7.35 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-
5), 7.62 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-6), 7.96 (d, J ¼ 8 Hz, 2H,
H-200 and H-600), 8.20 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-8); 13C
NMR (CDCl3, 50 MHz) d (ppm): 21.69 (SO2C6H4CH3),
29.43 (2 ꢁ gemCH3), 73.28 (C-20), 75.87 (C-30), 84.06
(C-10), 105.39 (C-4), 111.44 (C-2), 117.09 (C-9a), 117.24
(C-5), 122.48 (C-8a), 124.10 (C-7), 126.84 (C-8), 128.93
(C-200 and C-600), 129.58 (C-300 and C-500), 132.98 (C-100),
134.34 (C-6), 145.30 (C-400), 148.29 (C-1), 155.14 (C-10a),
158.04 (C-4a), 160.14 (C-3), 174.19 (C-9). Anal. Calcd. for
C25H20O6S. Calcd. (%): C: 66.95, H: 4.49. Found (%): C:
67.23, H: 4.16.
4.1.4. N,N-Dimethyl-N0-[1,2-dihydro-3,3-dimethyl-6-
methoxy-7-oxo-3H,7H-pyrano[2,3-c]xanthen-2-yl]ethane-
1,2-diamine (10a)
2-Dimethylaminoethylamine (340 mL, 3.1 mmol) was added
to a solution of compound 8 [20] (100 mg, 0.31 mmol) in abso-
lute ethanol (40 mL) and the resulting mixture was heated at re-
flux under argon for 7 h. After completion of the reaction, as
indicated upon TLC monitoring, the mixture was cooled at
0 ꢀC and a saturated ethanolic HCl solution was added
(1.5 mL). The mixture was stirred at this temperature for few
minutes, sodiumcyanoborohydride (97 mg, 1.55 mmol) was
then added and the resulting mixture was stirred at room tem-
perature for 90 min. A 20% solution of sodium carbonate
(20 mL) was added, the reaction mixture was vacuum-evapo-
rated, extracted with EtOAcewater, the organic layer was dried
(Na2SO4) and the solvent was evaporated to dryness. The resi-
due was purified by column chromatography (silica gel,
CH2Cl2:CH3OH 9:1) to furnish compound 10a (80 mg, 64%).
Mp (dihydrochloride) > 250 ꢀC (EtOH); 1H NMR (CDCl3,
400 MHz) d (ppm): 1.31 (s, 3H, 1 ꢁ gemCH3), 1.46 (s, 3H,
1 ꢁ gemCH3), 2.37 [s, 6H, N(CH3)2], 2.61 (m, 3H, H-1a and
NHCH2CH2NMe2), 2.78 [m, 2H, NHCH2CH2NMe2 and H-2),
3.05 (m, 1H, NHCH2CH2NMe2), 3.15 (dd, J ¼ 14 Hz, 5 Hz,
1H, H-1b), 3.91 (s, 3H, CH3O), 6.25 (s, 1H, H-5), 7.29 (dt,
J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.37 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-
11), 7.59 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.25 (dd, J ¼ 8 Hz,
2 Hz, 1H, H-8); 13C NMR (CDCl3, 50 MHz) d (ppm): 20.73
(1 ꢁ gemCH3), 23.19 (C-1), 26.39 (1 ꢁ gemCH3), 44.88
[N(CH3)2], 45.50 (NHCH2CH2NMe2), 56.09 (CH3O), 57.81
(C-2), 58.73 (NHCH2CH2NMe2), 79.39 (C-3), 95.67 (C-5),
100.41 (C-12b), 106.77 (C-6a), 116.84 (C-11), 122.83 (C-7a),
123.68 (C-9), 126.54 (C-8), 133.38 (C-10), 154.66 (C-11a),
156.61 (C-12a), 158.66 (C-4a), 160.10 (C-6), 175.42 (C-7).
Anal. Calcd. for C23H28N2O4$2HCl. Calcd. (%): C: 58.85, H:
6.44, N: 6.46. Found (%): C: 58.76, H: 6.38, N: 6.25.
4.1.2. 3,3-Dimethyl-6-[(4-methylphenyl)sulfonyloxy]-3H,7H-
pyrano[2,3-c]xanthen-7-one (4)
A solution of compound 3 (5.30 g, 11.83 mmol) in dry
DMF (20 mL) was heated at reflux for 2 h. The solvent was
then vacuum-evaporated and the residue was purified by col-
umn chromatography (silica gel, cyclohexaneeEtOAc 8:2)
to provide compound 4 (4.72 g, 89%). Mp 188e190 ꢀC
(Et2Oen-pentane) (Lit. [17] 188e190 ꢀC).
4.1.3. 3,3-Dimethyl-6-hydroxy-3H,7H-pyrano[2,3-c]
xanthen-7-one (5)
A solution of sodium hydroxide (4.21 g, 105.30 mmol) in
water (5 mL) was added dropwise at 0 ꢀC to a solution of com-
pound 4 (4.72 g, 10.53 mmol) in ethanol (80 mL) and the re-
sulting mixture was stirred at room temperature for 30 min.
Ethanol was then vacuum-evaporated and the residue was
extracted with EtOAc (3 ꢁ 60 mL). The organic extracts
4.1.5. N,N-Diethyl-N0-[1,2-dihydro-3,3-dimethyl-6-methoxy-
7-oxo-3H,7H-pyrano[2,3-c]xanthen-2-yl]ethane-
1,2-diamine (10b)
Compound 10b was prepared by a procedure analogous to
that of 10a. Yield: 68%; mp (dihydrochloride) > 250 ꢀC

G. Kolokythas et al. / European Journal of Medicinal Chemistry 42 (2007) 307e319
313
(EtOH); 1H NMR (CDCl3, 400 MHz) d (ppm): 1.00 [t,
J ¼ 7 Hz, 6H, N(CH2CH3)2], 1.29 (s, 3H, 1 ꢁ gemCH3), 1.44
(s, 3H, 1 ꢁ gemCH3), 2.56 [m, 7H, N(CH2CH3)2, NHCH2
CH2NEt2 and H-1a], 2.69 (m, 1H, NHCH2CH2NEt2), 2.76
(dd, J ¼ 14 Hz, 5 Hz, 1H, H-2), 2.93 (m, 1H, NHCH2CH2NEt2),
3.14 (dd, J ¼ 14 Hz, 5 Hz, 1H, H-1b), 3.91 (s, 3H, CH3O), 6.26
(s, 1H, H-5), 7.29 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.38 (dd,
J ¼ 8 Hz, 2 Hz, 1H, H-11), 7.60 (dt, J ¼ 8 Hz, 2 Hz, 1H,
H-10), 8.26 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-8); 13C NMR (CDCl3,
50 MHz) d (ppm): 11.57 [N(CH2CH3)2], 20.65 (1 ꢁ gemCH3),
23.22 (C-1), 26.42 (1 ꢁ gemCH3), 46.05 (NHCH2CH2NEt2),
46.75 [N(CH2CH3)2], 52.67 (NHCH2CH2NEt2), 56.16
(CH3O), 57.92 (C-2), 79.46 (C-3), 95.71 (C-5), 100.60
(C-12b), 106.88 (C-6a), 116.85 (C-11), 122.95 (C-7a), 123.72
(C-9), 126.70 (C-8), 133.42 (C-10), 154.78 (C-11a), 156.69
(C-12a), 158.75 (C-4a), 160.18 (C-6), 175.58 (C-7). Anal.
Calcd. for C25H32N2O4$2HCl. Calcd. (%): C: 60.36, H: 6.89,
N: 6.06. Found (%): C: 60.27, H: 6.82, N: 5.91.
117.22 (C-11), 121.45 (C-1), 122.39 (C-7a), 124.94 (C-9),
126.94 (C-8), 128.93 (C-20 and C-60), 129.69 (C-30 and C-50),
132.66 (C-10), 134.80 (C-10), 145.74 (C-40), 146.57 (C-2),
151.68 (C-6), 154.46 (C-11a), 156.37 (C-12a), 157.30 (C-4a),
173.24 (C-7). Anal. Calcd. for C25H19NO8S. Calcd. (%):
C: 60.85, H: 3.88, N: 2.84. Found (%): C: 61.11, H: 4.17,
N: 2.56.
4.1.8. N,N-Dimethyl-N0-[3,3-dimethyl-2-nitro-7-oxo-3H,7H-
pyrano[2,3-c]xanthen-6-yl]ethane-1,2-diamine (13a)
A solution of 12 (153 mg, 0.31 mmol) and 2-dimethylami-
noethylamine (340 mL, 3.1 mmol) in absolute ethanol (7 mL)
was refluxed for 2 h. The reaction mixture was then vacuum-
evaporated, extracted with CH2Cl2ewater, the organic layer
was dried (Na2SO4) and the solvent was evaporated to dryness.
The residue was purified by column chromatography (silica
gel, CH2Cl2:CH3OH 98:2) to furnish compound 13a
(110 mg, 87%). Mp (hydrochloride) > 250 ꢀC (EtOH); 1H
NMR (CDCl3, 400 MHz) d (ppm): 1.69 (s, 6H, 2 ꢁ gemCH3),
2.27 [s, 6H, N(CH3)2], 2.57 (t, J ¼ 7 Hz, 2H,
NHCH2CH2NMe2), 3.20 (q, J ¼ 7 Hz, 5 Hz, 2H,
NHCH2CH2NMe2), 5.72 (s, 1H, H-5), 7.21 (dt, J ¼ 8 Hz,
2 Hz, 1H, H-9), 7.25 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-11), 7.53
(dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.03 (dd, J ¼ 8 Hz, 2 Hz, 1H,
H-8), 8.07 (s, 1H, H-1), 10.16 (t, J ¼ 5 Hz, 1H, D2O exch.,
4.1.6. 3,3-Dimethyl-6-hydroxy-2-nitro-3H,7H-pyrano
[2,3-c]xanthen-7-one (11)
To a suspension of 5 (500 mg, 1.70 mmol) in glacial acetic
acid (30 mL) at 0 ꢀC, was added dropwise a solution of nitric
acid 65% (165 mL, 1.70 mmol) in glacial acetic acid (1 mL).
After 30 min, the reaction mixture was poured into ice-water
and the resulting mixture was neutralized with 10% sodium
carbonate, extracted with EtOAc (3 ꢁ 50 mL), dried
(Na2SO4) and the solvent was evaporated to dryness. Flash
chromatography on silica gel, using a mixture of cyclohexa-
neeEtOAc 95:5 as the eluent, provided compound 11
(323 mg, 56%). Mp 168e170 ꢀC (EtOAcen-pentane);1H
NMR (CDCl3, 400 MHz) d (ppm): 1.79 (s, 6H, 2 ꢁ gemCH3),
6.28 (s, 1H, H-5), 7.43 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.52 (dd,
J ¼ 8 Hz, 2 Hz, 1H, H-11), 7.77 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-
10), 8.21 (s, 1H, H-1), 8.23 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-8),
13.34 (s, 1H, D2O exch., OH); 13C NMR (CDCl3, 50 MHz)
d (ppm): 26.43 (2 ꢁ gemCH3), 80.62 (C-3), 98.33 (C-12b),
99.66 (C-5), 104.49 (C-6a), 117.78 (C-11), 120.58 (C-7a),
122.66 (C-1), 125.12 (C-9), 126.03 (C-8), 135.71 (C-10),
143.34 (C-2), 154.59 (C-12a), 155.54 (C-11a), 161.01 (C-
4a), 167.35 (C-6), 180.59 (C-7). Anal. Calcd. for
C18H13NO6. Calcd. (%): C: 63.72, H: 3.86, N: 4.13. Found
(%): C: 63.64, H: 3.70, N: 4.01.
NH); 13C NMR (CDCl3, 50 MHz)
d
(ppm): 26.38
(2 ꢁ gemCH3), 41.17 (NHCH2CH2NMe2), 45.48 [N(CH3)2],
57.38 (NHCH2CH2NMe2), 80.03 (C-3), 91.42 (C-5), 95.67
(C-12b), 102.50 (C-6a), 117.03 (C-11), 121.89 (C-7a),
124.36 (C-9), 124.61 (C-1), 126.01 (C-8), 134.21 (C-10),
140.93 (C-2), 154.47 (C-11a), 155.63 (C-6), 156.36 (C-12a),
159.72 (C-4a), 177.79 (C-7). Anal. Calcd. for
C22H23N3O5$HCl. Calcd. (%): C: 59.26, H: 5.43, N: 9.42.
Found (%): C: 59.12, H: 5.39, N: 9.37.
4.1.9. N,N-Diethyl-N0-[3,3-dimethyl-2-nitro-7-oxo-3H,7H-
pyrano[2,3-c]xanthen-6-yl]ethane-1,2-diamine (13b)
Compound 13b was prepared by a procedure analogous to
that of 13a. Yield: 89%; mp (hydrochloride) > 250 ꢀC (EtOH);
1H NMR (CDCl3, 400 MHz) d (ppm): 1.07 [t, J ¼ 7 Hz, 6H,
N(CH2CH3)2], 1.74 (s, 6H, 2 ꢁ gemCH3), 2.61 [q, J ¼ 7 Hz,
4H, N(CH2CH3)2], 2.75 (t, J ¼ 7 Hz, 2H, NHCH2CH2NEt2),
3.24 (q, J ¼ 7 Hz, 5 Hz, 2H, NHCH2CH2NEt2), 5.79 (s, 1H,
H-5), 7.27 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.32 (dd, J ¼ 8 Hz,
2 Hz, 1H, H-11), 7.59 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.09
(dd, J ¼ 8 Hz, 2 Hz, 1H, H-8), 8.15 (s, 1H, H-1), 10.21
(t, J ¼ 5 Hz, 1H, D2O exch., NH); 13C NMR (CDCl3,
50 MHz) d (ppm): 11.87 [N(CH2CH3)2], 26.36 (2 ꢁ gemCH3),
41.37 (NHCH2CH2NEt2), 46.95 [N(CH2CH3)2], 50.97
(NHCH2CH2NEt2), 80.12 (C-3), 91.64 (C-5), 95.65 (C-12b),
102.64 (C-6a), 117.12 (C-11), 121.84 (C-7a), 124.28 (C-9),
124.93 (C-1), 126.02 (C-8), 134.05 (C-10), 140.86 (C-2),
154.48 (C-11a), 155.70 (C-6), 156.75 (C-12a), 159.71
(C-4a), 177.69 (C-7). Anal. Calcd. for C24H27N3O5$HCl.
Calcd. (%): C: 60.82, H: 5.95, N: 8.87. Found (%): C:
60.69, H: 5.78, N: 8.99.
4.1.7. 3,3-Dimethyl-6-[(4-methylphenyl)sulfonyloxy)]-
2-nitro-3H,7H-pyrano[2,3-c]xanthen-7-one (12)
Compound 12 was prepared according to the procedure
described for the preparation of 3, starting from 11. Yield:
92%; mp > 250 ꢀC (EtOAcen-hexane); 1H NMR (CDCl3,
400 MHz) d (ppm): 1.81 (s, 6H, 2 ꢁ gemCH3), 2.42 (s, 3H,
SO2C6H4CH3), 6.76 (s, 1H, H-5), 7.34 (d, J ¼ 8 Hz, 2H, H-30
and H-50), 7.37 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.46 (dd,
J ¼ 8 Hz, 2 Hz, 1H, H-11), 7.69 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-
10), 7.98 (d, J ¼ 8 Hz, 2H, H-20 and H-60), 8.20 (s, 1H, H-1),
8.21 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-8); 13C NMR (CDCl3,
50 MHz) d (ppm): 21.74 (SO2C6H4CH3), 26.47 (2 ꢁ gemCH3),
80.87 (C-3), 105.96 (C-12b), 108.68 (C-5), 110.42 (C-6a),

314
G. Kolokythas et al. / European Journal of Medicinal Chemistry 42 (2007) 307e319
4.1.10. N,N-Dimethyl-N0-[1,2-dihydro-3,3-dimethyl-2-nitro-
7-oxo-3H,7H-pyrano[2,3-c]xanthen-6-yl]ethane-
1,2-diamine (14a)
hydrazine 40% (197 mL), and the resulting mixture was stirred
at this temperature for 15 min. The reaction mixture was then
filtered hot, the precipitate was washed with CH2Cl2
(2 ꢁ 40 mL) and CH3OH (40 mL) and the filtrate was concen-
trated to dryness. Flash chromatography on silica gel using
a mixture of CH2Cl2:CH3OH 94:6 as the eluent, provided
compound 15a (130 mg, 95%). Mp (dihydrochlo-
ride) > 250 ꢀC (EtOH); 1H NMR (CDCl3, 400 MHz)
To a solution of 13a (70 mg, 0.17 mmol) in a 3/1 mixture of
CH3OHeCH2Cl2 (10 mL) at 0 ꢀC was added sodiumborohy-
dride (20 mg, 0.51 mmol), and the resulting mixture was stirred
at room temperature for 30 min. The mixture was then poured
into ice-water, extracted with CH2Cl2 (3 ꢁ 20 mL), dried
(Na2SO4) and the solvent was evaporated to dryness. Flash chro-
matography on silica gel, using a mixture of CH2Cl2:CH3OH
95:5 as the eluent, provided compound 14a (57 mg, 82%). Mp
(hydrochloride) 243e245 ꢀC (EtOH); 1H NMR (CDCl3,
400 MHz) d (ppm): 1.41 (s, 3H, 1 ꢁ gemCH3), 1.46 (s, 3H,
1 ꢁ gemCH3), 2.31 [s, 6H, N(CH3)2], 2.65 (t, J ¼ 7 Hz, 2H,
NHCH2-CH2NMe2), 3.26 (m, 3H, NHCH2CH2NMe2 and H-
1a), 3.35 (dd, J ¼ 14 Hz, 5 Hz, 1H, H-1b), 4.86 (t, J ¼ 5 Hz,
1H, H-2), 5.84 (s, 1H, H-5), 7.25 (m, 2H, H-9 and H-11), 7.53
(dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.14 (dd, J ¼ 8 Hz, 2 Hz, 1H,
H-8), 9.41 (t, J ¼ 5 Hz, 1H, D2O exch., NH); 13C NMR
(CDCl3, 50 MHz) d (ppm): 21.46 (C-1), 22.18 (1 ꢁ gemCH3),
25.75 (1 ꢁ gemCH3), 40.77 (NHCH2CH2NMe2), 45.42
[N(CH3)2], 57.94 (NHCH2CH2NMe2), 74.75 (C-3), 85.31 (C-
2), 91.39 (C-5), 91.47 (C-12b), 102.83 (C-6a), 116.60 (C-11),
121.97 (C-7a), 123.58 (C-9), 125.90 (C-8), 133.59 (C-10),
151.30 (C-6), 154.69 (C-11a), 155.95 (C-12a), 158.09 (C-4a),
177.94 (C-7). Anal. Calcd. for C22H25N3O5$HCl. Calcd. (%):
C: 59.12, H: 5.64, N: 9.40. Found (%):C: 58.96, H: 5.59, N: 9.21.
d
(ppm): 1.30 (s, 3H, 1 ꢁ gemCH3), 1.40 (s, 3H,
1 ꢁ gemCH3), 1.81 (s, D2O exch., 2H, NH2), 2.31 [s, 6H,
N(CH3)2], 2.56 (dd, J ¼ 17 Hz, 7 Hz, 1H, H-1a), 2.64 (t,
J ¼ 7 Hz, 2H, NHCH2CH2NMe2), 3.03 (m, 2H, H-1b and H-
2), 3.25 (q, J ¼ 7 Hz, 5 Hz, 2H, NHCH2CH2NMe2), 5.85 (s,
1H, H-5), 7.26 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.31 (dd,
J ¼ 8 Hz, 2 Hz, 1H, H-11), 7.55 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-
10), 8.19 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-8), 9.39 (t, J ¼ 5 Hz,
1H, D2O exch., NH); 13C NMR (CDCl3, 50 MHz) d (ppm):
21.04 (1 ꢁ gemCH3), 26.03 (C-1), 26.53 (1 ꢁ gemCH3),
41.19 (NHCH2CH2NMe2), 45.47 [N(CH3)2], 51.35 (C-2),
58.13 (NHCH2CH2NMe2), 78.81 (C-3), 91.65 (C-5), 94.68
(C-12b), 102.71 (C-6a), 116.79 (C-11), 122.32 (C-7a),
123.39 (C-9), 125.25 (C-8), 133.55 (C-10), 151.21 (C-6),
155.13 (C-11a), 156.74 (C-12a), 159.95 (C-4a), 178.31 (C-
7). Anal. Calcd. for C22H27N3O3$2HCl. Calcd. (%): C:
58.15, H: 6.43, N: 9.25. Found (%): C: 58.22, H: 6.39, N: 9.05.
4.1.13. N,N-Diethyl-N0-[2-amino-1,2-dihydro-3,3-dimethyl-
7-oxo-3H,7H-pyrano[2,3-c]xanthen-6-yl]ethane-
1,2-diamine (15b)
4.1.11. N,N-Diethyl-N0-[1,2-dihydro-3,3-dimethyl-2-nitro-7-
oxo-3H,7H-pyrano[2,3-c]xanthen-6-yl]ethane-
1,2-diamine (14b)
Compound 15b was prepared by a procedure analogous to
that of 15a. Yield: 94%; mp (dihydrochloride) > 250 ꢀC
(EtOH); 1H NMR (CDCl3, 400 MHz) d (ppm): 1.09 [t,
J ¼ 7 Hz, 6H, N(CH2CH3)2], 1.30 (s, 3H, 1 ꢁ gemCH3), 1.39
(s, 3H, 1 ꢁ gemCH3), 1.81 (s, D2O exch., 2H, NH2), 2.57
(dd, J ¼ 17 Hz, 7 Hz, 1H, H-1a), 2.67 [q, J ¼ 7 Hz, 4H,
N(CH2CH3)2], 2.83 (t, J ¼ 7 Hz, 2H, NHCH2CH2NEt2), 3.04
(m, 2H, H-1b and H-2), 3.31 (q, J ¼ 7 Hz, 5 Hz, 2H,
NHCH2CH2NEt2), 5.87 (s, 1H, H-5), 7.27 (dt, J ¼ 8 Hz,
2 Hz, 1H, H-9), 7.34 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-11), 7.57
(dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.18 (dd, J ¼ 8 Hz, 2 Hz, 1H,
H-8), 9.35 (t, J ¼ 5 Hz, 1H, D2O exch., NH); 13C NMR
(CDCl3, 50 MHz) d (ppm): 11.66 [N(CH2CH3)2], 20.66
(1 ꢁ gemCH3), 26.18 (C-1), 26.34 (1 ꢁ gemCH3), 41.12
(NHCH2CH2NEt2), 47.25 [N(CH2CH3)2], 51.55 (C-2), 51.62
(NHCH2CH2NEt2), 78.96 (C-3), 91.85 (C-5), 94.72 (C-12b),
102.49 (C-6a), 117.01 (C-11), 122.33 (C-7a), 123.56 (C-9),
126.22 (C-8), 133.58 (C-10), 151.38 (C-6), 155.06 (C-11a),
156.50 (C-12a), 159.97 (C-4a), 178.38 (C-7). Anal. Calcd.
for C24H31N3O3$2HCl. Calcd. (%): C: 59.75, H: 6.89, N:
8.71. Found (%): C: 59.61, H: 6.83, N: 8.50.
Compound 14b was prepared by a procedure analogous to
that of 14a. Yield: 90%; mp (hydrochloride) > 250 ꢀC (EtOH);
1H NMR (CDCl3, 400 MHz) d (ppm): 1.04 [t, J ¼ 7 Hz, 6H,
N(CH2CH3)2], 1.39 (s, 3H, 1 ꢁ gemCH3), 1.45 (s, 3H,
1 ꢁ gemCH3), 2.59 [q, J ¼ 7 Hz, 4H, N(CH2CH3)2], 2.74 (t,
J ¼ 7 Hz,
2H,
NHCH2CH2NEt2),
3.20
(m,
3H,
NHCH2CH2NEt2 and H-1a), 3.32 (dd, J ¼ 14 Hz, 7 Hz, 1H,
H-1b), 4.84 (t, J ¼ 7 Hz, 1H, H-2), 5.81 (s, 1H, H-5), 7.21
(m, 2H, H-9 and H-11), 7.50 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-10),
8.11 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-8), 9.35 (t, J ¼ 5 Hz, 1H,
D2O exch., NH); 13C NMR (CDCl3, 50 MHz) d (ppm):
11.56 [N(CH2CH3)2], 21.43 (C-1), 21.88 (1 ꢁ gemCH3),
25.81 (1 ꢁ gemCH3), 41.08 (NHCH2CH2NEt2), 47.07
[N(CH2CH3)2], 51.20 (NHCH2CH2NEt2), 75.15 (C-3), 85.47
(C-2), 91.46 (C-5), 91.93 (C-12b), 103.02 (C-6a), 116.64 (C-
11), 122.01 (C-7a), 123.66 (C-9), 125.94 (C-8), 133.57 (C-
10), 151.33 (C-6), 154.84 (C-11a), 156.08 (C-12a), 158.14
(C-4a), 178.17 (C-7). Anal. Calcd. for C24H29N3O5$HCl.
Calcd. (%): C: 60.56, H: 6.35, N: 8.83. Found (%): C:
60.41, H: 6.32, N: 8.68.
4.1.14. 2-Chloro-N-[1,2-dihydro-6-(2-dimethylaminoethyl-
amino)-3,3-dimethyl-7-oxo-3H,7H-pyrano[2,3-c]xanthen-
2-yl]acetamide (16a)
To a solution of 15a (40 mg, 0.10 mmol) in dry CH2Cl2
(4 mL) were added chloroacetic acid (10 mg, 0.11 mmol) and
N,N0-dicyclohexylcarbodiimide (22 mg, 0.11 mmol), and the
4.1.12. N,N-Dimethyl-N0-[2-amino-1,2-dihydro-3,
3-dimethyl-7-oxo-3H,7H-pyrano[2,3-c]xanthen-6-
yl]ethane-1,2-diamine (15a)
To a solution of 14a (150 mg, 0.36 mmol) in ethanol
(10 mL) at 45 ꢀC were added Raney nickel (36 mg) and

G. Kolokythas et al. / European Journal of Medicinal Chemistry 42 (2007) 307e319
315
resulting mixture was stirred at room temperature for 30 min.
The reaction mixture was then filtered, the precipitate was
washed with CH2Cl2 (2 ꢁ 10 mL) and the filtrate was concen-
trated to dryness. Flash chromatography on silica gel using
a mixture of CH2Cl2:CH3OH 95:5 as the eluent, provided com-
pound 16a (43 mg, 94%). Mp 228e230 ꢀC (CH2Cl2en-pen-
tane); 1H NMR (CDCl3, 400 MHz) d (ppm): 1.36 (s, 3H,
1 ꢁ gemCH3), 1.41 (s, 3H, 1 ꢁ gemCH3), 2.34 [s, 6H,
N(CH3)2], 2.67 (t, J ¼ 7 Hz, 2H, NHCH2CH2NMe2), 2.90 (dd,
J ¼ 16 Hz, 3 Hz, 1H, H-1a), 3.06 (dd, J ¼ 16 Hz, 5 Hz, 1H,
H-1b), 3.23 (q, J ¼ 7 Hz, 5 Hz, 2H, NHCH2CH2NMe2), 4.07
(s, 2H, NHCOCH2Cl), 4.35 (m, 1H, H-2), 5.81 (s, 1H, H-5),
6.88 (d, J ¼ 9 Hz, 1H, NHCOCH2Cl), 7.29 (dt, J ¼ 8 Hz,
2 Hz, 1H, H-9), 7.32 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-11), 7.59 (dt,
J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.19 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-
8), 9.40 (t, J ¼ 5 Hz, 1H, D2O exch., NHCH2CH2NMe2); 13C
NMR (CDCl3, 50 MHz) d (ppm): 23.63 (C-1), 24.56
(2 ꢁ gemCH3), 40.61 (NHCH2CH2NMe2), 42.68 (NHCO-
CH2Cl), 45.29 [N(CH3)2], 48.46 (C-2), 57.68 (NHCH2-
CH2NMe2), 77.18 (C-3), 91.90 (C-5), 92.65 (C-12b), 103.10
(C-6a), 116.90 (C-11), 122.10 (C-7a), 123.69 (C-9), 126.07
(C-8), 133.66 (C-10), 151.03 (C-6), 154.95 (C-11a), 156.94
(C-12a), 159.28 (C-4a), 165.89 (NHCOCH2Cl), 178.33 (C-7).
Anal. Calcd. for C24H28ClN3O4. Calcd. (%): C: 62.94, H:
6.16, N: 9.18. Found (%): C: 62.85, H: 6.02, N: 8.91.
dimethylamine in ethanol (1 mL) and the resulting solution
was heated at reflux for 10 h. Upon cooling, the solvent was
vacuum-evaporated and the residue was purified by column
chromatography (silica gel) using a mixture of CH2Cl2:
CH3OH 8:1 as the eluent, to provide compound 17a as an
oil (396 mg, 88%). 1H NMR (CDCl3, 400 MHz) d (ppm):
1.37 (s, 3H, 1 ꢁ gemCH3), 1.38 (s, 3H, 1 ꢁ gemCH3), 2.21
[s, 6H, NHCOCH2N(CH3)2], 2.56 [s, 6H, NHCH2CH2-
N(CH3)2], 2.86 (dd, J ¼ 17 Hz, 4 Hz, 1H, H-1a), 2.95 (m,
4H, NHCOCH2NMe2 and NHCH2CH2NMe2), 3.04 (dd,
J ¼ 17 Hz, 5 Hz, 1H, H-1b), 3.50 (q, J ¼ 7 Hz, 5 Hz, 2H,
NHCH2CH2NMe2), 4.32 (m, 1H, H-2), 5.93 (s, 1H, H-5),
7.30 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.34 (d, J ¼ 9 Hz, 1H,
NHCOCH2NMe2), 7.35 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-11), 7.61
(dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.18 (dd, J ¼ 8 Hz, 2 Hz, 1H,
H-8), 9.49 (t, J ¼ 5 Hz, 1H, D2O exch., NHCH2CH2NMe2);
13C NMR (CDCl3, 50 MHz) d (ppm): 73.76 (C-1), 24.10
(1 ꢁ gemCH3), 25.01 (1 ꢁ gemCH3), 39.30 (NHCH2CH2-
NMe2), 44.34 [NHCH2CH2N(CH3)2], 45.76 [NHCOCH2N-
(CH3)2], 47.62 (C-2), 56.82 (NHCH2CH2NMe2), 62.85
(NHCOCH2NMe2), 78.34 (C-3), 92.20 (C-5), 94.31 (C-12b),
103.01 (C-6a), 117.08 (C-11), 121.95 (C-7a), 123.80 (C-9),
125.95 (C-8), 133.87 (C-10), 150.36 (C-6), 155.00 (C-11a),
156.94 (C-12a), 159.74 (C-4a), 170.11 (NHCOCH2NMe2),
178.68 (C-7). Anal. Calcd. for C26H34N4O4. Calcd. (%): C:
66.93, H: 7.35, N: 12.01. Found (%): C: 66.81, H: 7.20,
N: 11.87.
4.1.15. 2-Chloro-N-[6-(2-diethylaminoethylamino)-1,2-
dihydro-3,3-dimethyl-7-oxo-3H,7H-pyrano[2,3-c]xanthen-
2-yl]acetamide (16b)
Compound 16b was prepared by a procedure analogous to
that of 16a. Yield: 95%; mp 184e186 ꢀC (CH2Cl2en-pentane);
1H NMR (CDCl3, 400 MHz) d (ppm): 1.28 [t, J ¼ 7 Hz, 6H,
N(CH2CH3)2], 1.35 (s, 3H, 1 ꢁ gemCH3), 1.39 (s, 3H,
1 ꢁ gemCH3), 2.88 (dd, J ¼ 17 Hz, 3 Hz, 1H, H-1a), 3.03 (dd,
J ¼ 17 Hz, 5 Hz, 1H, H-1b), 3.12 [q, J ¼ 7 Hz, 4H,
N(CH2CH3)2], 3.20 (t, J ¼ 7 Hz, 2H, NHCH2CH2NEt2), 3.64
(q, J ¼ 7 Hz, 5 Hz, 2H, NHCH2CH2NEt2), 4.04 (s, 2H,
NHCOCH2Cl), 4.32 (m, 1H, H-2), 5.92 (s, 1H, H-5), 6.87 (d,
J ¼ 9 Hz, 1H, NHCOCH2Cl), 7.28 (m, 2H, H-9 and H-11),
7.58 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.12 (dd, J ¼ 8 Hz, 2 Hz,
1H, H-8), 9.46 (t, J ¼ 5 Hz, 1H, D2O exch., NHCH2CH2NEt2);
13C NMR (CDCl3, 50 MHz) d (ppm): 8.81 [N(CH2CH3)2],
23.58 (C-1), 24.40 (1 ꢁ gemCH3), 24.63 (1 ꢁ gemCH3),
38.03 (NHCH2CH2NEt2), 42.66 (NHCOCH2Cl), 46.34
[N(CH2CH3)2], 48.43 (C-2), 49.66 (NHCH2CH2NEt2), 77.42
(C-3), 92.19 (C-5), 93.96 (C-12b), 103.09 (C-6a), 117.09
(C-11), 121.77 (C-7a), 123.90 (C-9), 125.95 (C-8), 133.94
(C-10), 150.28 (C-6), 154.93 (C-11a), 156.98 (C-12a), 159.52
(C-4a), 165.97 (NHCOCH2Cl), 178.61 (C-7). Anal. Calcd. for
C26H32ClN3O4. Calcd. (%): C: 64.25, H: 6.64, N: 8.65. Found
(%): C: 64.03, H: 6.52, N: 8.57.
4.1.17. 2-Diethylamino-N-[6-(2-diethylaminoethylamino)-
1,2-dihydro-3,3-dimethyl-7-oxo-3H,7H-pyrano[2,3-c]
xanthen-2-yl]acetamide (17b)
Compound 17b was prepared by a procedure analogous to
that of 17a. Yield: 87%; mp (dihydrochloride) > 250 ꢀC
1
(EtOHeEt2O); H NMR (CDCl3, 400 MHz) d (ppm): 0.84
[t, J ¼ 7 Hz, 6H, NHCOCH2N(CH2CH3)2], 1.11 [t, J ¼ 7 Hz,
6H, NHCH2CH2N(CH2CH3)2], 1.36 (s, 6H, 2 ꢁ gemCH3),
2.42 [q, J ¼ 7 Hz, 4H, NHCOCH2N(CH2CH3)2], 2.70 [q,
J ¼ 7 Hz, 4H, NHCH2CH2N(CH2CH3)2], 2.85 (m, 3H,
NHCH2CH2NEt2 and H-1a), 2.92 (d, J ¼ 17 Hz, 1H,
NHCOCH2NEt2), 3.01 (dd, J ¼ 17 Hz, 5 Hz, 1H, H-1b), 3.05
(d, J ¼ 17 Hz, 1H, NHCOCH2NEt2), 3.34 (q, J ¼ 7 Hz, 5 Hz,
2H, NHCH2CH2NEt2), 4.27 (m, 1H, H-2), 5.93 (s, 1H, H-5),
7.29 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.34 (dd, J ¼ 8 Hz, 2 Hz,
1H, H-11), 7.59 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 7.74 (d,
J ¼ 9 Hz, 1H, NHCOCH2NEt2), 8.20 (dd, J ¼ 8 Hz, 2 Hz,
1H, H-8), 9.39 (t, J ¼ 5 Hz, 1H, D2O exch., NHCH2CH2NEt2);
13C NMR (CDCl3, 50 MHz) d (ppm): 9.57 [NHCH2
-CH2N(CH2CH3)2], 11.16 [NHCOCH2N(CH2CH3)2], 23.59
(C-1), 23.86 (1 ꢁ gemCH3), 24.95 (1 ꢁ gemCH3), 38.74
(NHCH2CH2NEt2), 47.23 [NHCH2CH2N(CH2CH3)2], 47.73
(C-2), 48.73 [NHCOCH2N(CH2CH3)2], 50.20 (NHCH2CH2-
NEt2), 57.07 (NHCOCH2NEt2), 77.53 (C-3), 92.01 (C-5),
94.28 (C-12b), 102.85 (C-6a), 117.05 (C-11), 121.77 (C-7a),
123.77 (C-9), 125.85 (C-8), 133.88 (C-10), 150.22 (C-6),
154.93 (C-11a), 156.88 (C-12a), 159.70 (C-4a), 171.63
(NHCOCH2NEt2), 178.57 (C-7). Anal. Calcd. for C30H42
4.1.16. 2-Dimethylamino-N-[1,2-dihydro-6-(2-dimethylamino-
ethylamino)-3,3-dimethyl-7-oxo-3H,7H-pyrano[2,3-c]xanthen-
2-yl]acetamide (17a)
To a solution of the chloride 16a (442 mg, 1 mmol) in
absolute ethanol (10 mL), was added a 33% solution of

316
G. Kolokythas et al. / European Journal of Medicinal Chemistry 42 (2007) 307e319
N4O4$2HCl. Calcd. (%): C: 60.50, H: 7.45, N: 9.41. Found
(%): C: 60.34, H: 7.39, N: 9.27.
(C-6), 154.94 (C-11a), 155.42 (C-12a), 156.96 (NHCO),
159.58 (C-4a), 178.34 (C-7). Anal. Calcd. for C29H39N3O5.
Calcd. (%): C: 68.35, H: 7.71, N: 8.24. Found (%): C: 68.07,
H: 8.04, N: 7.92.
4.1.18. tert-Butyl-N-[1,2-dihydro-6-(2-dimethylaminoethyl-
amino)-3,3-dimethyl-7-oxo-3H,7H-pyrano[2,3-c]xanthen-2-
yl]carbamate (18a)
4.1.20. tert-Butyl-N-(2-dimethylaminoethyl)-N-[1,2-dihydro-
6-(2-dimethylaminoethylamino)-3,3-dimethyl-7-oxo-
3H,7H-pyrano[2,3-c]xanthen-2-yl]carbamate (19a)
To a solution of 15a (128 mg, 0.32 mmol) in dry THF
(10 mL) at 0 ꢀC were added triethylamine (223 mL, 1.6 mmol)
and di-tert-butyl dicarbonate (73 mL, 0.32 mmol) and the result-
ing solution was stirred at room temperature for 3 h. The reac-
tion mixture was then vacuum-evaporated, extracted with
EtOAcewater, the organic layer was dried (Na2SO4) and the
solvent was evaporated to dryness. The residue was purified
by column chromatography (silica gel, CH2Cl2:CH3OH 95:5)
To a solution of 18a (73 mg, 0.15 mmol) in dry DMF
(5 mL) was added NaH under argon (80% in hexanes,
30 mg, 0.75 mmol) and the mixture was stirred at room tem-
perature for 30 min. A solution of 2-dimethylaminoethylchlor-
ide (160 mg, 1.5 mmol) in dry DMF (3 mL) was then added
dropwise and the mixture was stirred for 18 h. The solvent
was then vacuum-evaporated, the residue was extracted with
EtOAc (3 ꢁ 60 mL), and the combined organic extracts were
washed with brine, dried (Na2SO4), and concentrated to dry-
ness. The residue was purified by column chromatography (sil-
ica gel) eluting with CH2Cl2:CH3OH 95:5, to provide
1
to furnish compound 18a (145 mg, 94%) as an oil; H NMR
(CDCl3, 400 MHz) d (ppm): 1.35 (s, 3H, 1 ꢁ gemCH3), 1.39
(s, 3H, 1 ꢁ gemCH3), 1.41 [s, 9H, C(CH3)3], 2.32 [s, 6H,
N(CH3)2], 2.65 (t, J ¼ 7 Hz, 2H, NHCH2CH2NMe2), 2.86 (dd,
J ¼ 17 Hz, 7 Hz, 1H, H-1a), 3.03 (dd, J ¼ 17 Hz, 5 Hz, 1H,
H-1b), 3.26 (q, J ¼ 7 Hz, 5 Hz, 2H, NHCH2CH2NMe2), 4.00
(m, 1H, H-2), 4.72 (d, J ¼ 10 Hz, 1H, D2O exch., NHCO),
5.87 (s, 1H, H-5), 7.28 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.34
(dd, J ¼ 8 Hz, 2 Hz, 1H, H-11), 7.59 (dt, J ¼ 8 Hz, 2 Hz, 1H,
H-10), 8.21 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-8), 9.43 (t, J ¼ 5 Hz,
1H, D2O exch., NHCH2CH2NMe2); 13C NMR (CDCl3,
50 MHz) d (ppm): 23.92 (C-1), 24.01 (1 ꢁ gemCH3), 24.90
(1 ꢁ gemCH3), 28.63 [C(CH3)3], 40.79 (NHCH2CH2NMe2),
45.49 [N(CH3)2], 49.22 (C-2), 57.85 (NHCH2CH2NMe2),
77.88 [C(CH3)3], 79.62 (C-3), 91.77 (C-5), 93.15 (C-12b),
102.95 (C-6a), 116.87 (C-11), 121.97 (C-7a), 123.74 (C-9),
126.09 (C-8), 133.54 (C-10), 150.99 (C-6), 154.92 (C-11a),
155.90 (C-12a), 156.88 (NHCO), 159.62 (C-4a), 178.45
(C-7). Anal. Calcd. for C27H35N3O5. Calcd. (%): C: 67.34, H:
7.33, N: 8.73. Found (%): C: 67.72, H: 7.64, N: 8.43.
1
compound 19a (65 mg, 78%) as an oil. H NMR (CDCl3,
400 MHz) d (ppm): 1.33 (s, 3H, 1 ꢁ gemCH3), 1.36 (s, 3H,
1 ꢁ gemCH3), 1.46 [s, 9H, C(CH3)3], 1.98 [s, 6H,
N(Boc)CH2CH2N(CH3)2], 2.31 [s, 6H, NHCH2CH2N(CH3)2],
2.64 [m, 4H, NHCH2CH2NMe2 and N(Boc)CH2CH2NMe2],
2.90 (dd, J ¼ 17 Hz, 7 Hz, 1H, H-1a), 3.20 (m, 5H,
N(Boc)CH2CH2NMe2 and H-1b and NHCH2CH2NMe2),
4.64 (dd, J ¼ 7 Hz, 5 Hz, 1H, H-2), 5.86 (s, 1H, H-5), 7.29
(dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.33 (dd, J ¼ 8 Hz, 2 Hz, 1H,
H-11), 7.59 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.20 (dd,
J ¼ 8 Hz, 2 Hz, 1H, H-8), 9.40 (t, J ¼ 5 Hz, 1H, D2O exch.,
NHCH2CH2NMe2); 13C NMR (CDCl3, 50 MHz) d (ppm):
21.99 (C-1), 24.50 (1 ꢁ gemCH3), 25.88 (1 ꢁ gemCH3),
28.63 [C(CH3)3], 40.99 (NHCH2CH2NMe2), 41.09
[N(Boc)CH2CH2NMe2], 45.12 [N(Boc)CH2CH2N(CH3)2],
45.57 [NHCH2CH2N(CH3)2], 51.53 (C-2), 57.94 (NHCH2-
CH2NMe2), 58.32 [N(Boc)CH2CH2NMe2], 79.01 [C(CH3)3],
80.19 (C-3), 92.06 (C-5), 94.36 (C-12b), 103.06 (C-6a),
117.03 (C-11), 122.30 (C-7a), 123.67 (C-9), 126.19 (C-8),
133.52 (C-10), 151.15 (C-6), 155.05 (C-11a), 156.07
(C-12a), 156.19 [COOC(CH3)3], 160.08 (C-4a), 178.41
(C-7). Anal. Calcd. for C31H44N4O5. Calcd. (%): C: 67.37,
H: 8.02, N: 10.14. Found (%): C: 67.69, H: 7.75, N: 10.43.
4.1.19. tert-Butyl-N-[6-(2-diethylaminoethylamino)-1,2-
dihydro-3,3-dimethyl-7-oxo-3H,7H-pyrano[2,3-c]xanthen-
2-yl]carbamate (18b)
Compound 18b was prepared by a procedure analogous to
1
that of 18a. Yield: 86%; isolated as an oil; H NMR (CDCl3,
400 MHz) d (ppm): 1.05 [t, J ¼ 7 Hz, 6H, N(CH2CH3)2], 1.32
(s, 3H, 1 ꢁ gemCH3), 1.36 (s, 3H, 1 ꢁ gemCH3), 1.38 [s, 9H,
C(CH3)3], 2.61 [q, J ¼ 7 Hz, 4H, N(CH2CH3)2], 2.77 (t,
J ¼ 7 Hz, 2H, NHCH2CH2NEt2), 2.82 (dd, J ¼ 17 Hz, 7 Hz,
1H, H-1a), 3.00 (dd, J ¼ 17 Hz, 5 Hz, 1H, H-1b), 3.25 (q,
J ¼ 7 Hz, 5 Hz, 2H, NHCH2CH2NMe2), 3.98 (m, 1H, H-2),
4.79 (d, J ¼ 10 Hz, 1H, D2O exch., NHCO), 5.85 (s, 1H, H-5),
7.24 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.29 (dd, J ¼ 8 Hz, 2 Hz,
1H, H-11), 7.55 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.16 (dd,
J ¼ 8 Hz, 2 Hz, 1H, H-8), 9.34 (t, J ¼ 5 Hz, 1H, D2O exch.,
NHCH2CH2NEt2); 13C NMR (CDCl3, 50 MHz) d (ppm):
11.30 [N(CH2CH3)2], 23.40 (C-1), 23.48 (1 ꢁ gemCH3),
24.81 (1 ꢁ gemCH3), 28.45 [C(CH3)3], 40.95 (NHCH2-
CH2NEt2), 47.21 [N(CH2CH3)2], 49.23 (C-2), 51.04
(NHCH2CH2NEt2), 77.87 [C(CH3)3], 79.49 (C-3), 91.79
(C-5), 93.21 (C-12b), 102.89 (C-6a), 117.01 (C-11), 122.06
(C-7a), 123.67 (C-9), 126.09 (C-8), 133.56 (C-10), 151.11
4.1.21. tert-Butyl-N-(2-diethylaminoethyl)-N-[6-(2-diethyl-
aminoethylamino)-1,2-dihydro-3,3-dimethyl-7-oxo-3H,7H-
pyrano[2,3-c]xanthen-2-yl]carbamate (19b)
Compound 19b was prepared by a procedure analogous to
1
that of 19a and was isolated as an oil. Yield: 81%; H NMR
(CDCl3, 400 MHz) d (ppm): 0.74 [t, J ¼ 7 Hz, 6H, N(Boc)CH2-
CH2N(CH2CH3)2], 1.03 [t, J ¼ 7 Hz, 6H, NHCH2CH2N-
(CH2CH3)2], 1.31 (s, 3H, 1 ꢁ gemCH3), 1.34 (s, 3H,
1 ꢁ gemCH3), 1.44 [s, 9H, C(CH3)3], 2.18 [m, 4H, N(Boc)CH2-
CH2N(CH2CH3)2], 2.32 [m, 2H, N(Boc)CH2CH2NEt2], 2.58 [q,
J ¼ 7 Hz, 4H, NHCH2CH2N(CH2CH3)2], 2.75 [t, J ¼ 7 Hz, 2H,
NHCH2CH2NEt2], 2.87 (dd, J ¼ 17 Hz, 5 Hz, 1H, H-1a), 3.05
[m, 2H, N(Boc)CH2CH2NEt2], 3.18 [m, 3H, NHCH2CH2NEt2
and H-1b], 4.63 (dd, J ¼ 7 Hz, 5 Hz, 1H, H-2), 5.84 (s, 1H,

G. Kolokythas et al. / European Journal of Medicinal Chemistry 42 (2007) 307e319
317
H-5), 7.25 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.29 (dd, J ¼ 8 Hz,
2 Hz, 1H, H-11), 7.55 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.17
(dd, J ¼ 8 Hz, 2 Hz, 1H, H-8), 9.34 (t, J ¼ 5 Hz, 1H, D2O
exch., NHCH2CH2NEt2); 13C NMR (CDCl3, 50 MHz)
d (ppm): 11.91 [NHCH2CH2N(CH2CH3)2], 11.99 [N(Boc)CH2-
CH2N(CH2CH3)2], 21.98 (C-1), 24.58 (1 ꢁ gemCH3), 25.98
(1 ꢁ gemCH3), 28.58 [C(CH3)3], 41.37 (NHCH2CH2NEt2),
41.84 [N(Boc)CH2CH2NEt2], 47.17 [NHCH2CH2N(CH2-
CH3)2], 47.25 [N(Boc)CH2CH2N(CH2-CH3)2], 51.33 (NHCH2-
CH2NEt2), 51.56 [N(Boc)CH2CH2NEt2], 51.98 (C-2), 79.15
[C(CH3)3], 80.14 (C-3), 92.34 (C-5), 94.33 (C-12b), 102.93
(C-6a), 116.92 (C-11), 122.32 (C-7a), 123.71 (C-9), 126.31
(C-8), 133.71 (C-10), 151.30 (C-6), 155.09 (C-11a), 156.29
(C-12a), 156.49 [COOC(CH3)3], 160.09 (C-4a), 178.28 (C-7).
Anal. Calcd. for C35H52N4O5. Calcd. (%): C: 69.05, H: 8.61,
N: 9.20. Found (%): C: 68.82, H: 8.94, N: 8.92.
(EtOH); 1H NMR (CDCl3, 400 MHz) d (ppm): 1.03 [t,
J ¼ 7 Hz, 6H, 2-NHCH2CH2N(CH2CH3)2], 1.06 [t, J ¼ 7 Hz,
6H, 6-NHCH2CH2N(CH2CH3)2], 1.28 (s, 3H, 1 ꢁ gemCH3),
1.43 (s, 3H, 1 ꢁ gemCH3), 2.53 (dd, J ¼ 17 Hz, 7 Hz, 1H, H-
1a), 2.60 [m, 10H, 6-NHCH2CH2N(CH2CH3)2, 2-
NHCH2CH2N(CH2CH3)2 and 2-NHCH2CH2NEt2], 2.73 [m,
2H, H-2 and 2-NHCH2CH2NEt2], 2.78 (t, J ¼ 7 Hz, 2H, 6-
NHCH2CH2NEt2), 2.95 (m, 1H, 2-NHCH2CH2NEt2), 3.08
(dd, J ¼ 17 Hz, 5 Hz, 1H, H-1b), 3.25 (q, J ¼ 7 Hz, 5 Hz, 2H,
6-NHCH2CH2NEt2), 5.86 (s, 1H, H-5), 7.27 (dt, J ¼ 8 Hz,
2 Hz, 1H, H-9), 7.36 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-11), 7.58 (dt,
J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.20 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-
8), 9.34 (t, J ¼ 5 Hz, 1H, D2O exch., 6-NHCH2CH2NEt2); 13
C
NMR (CDCl3, 50 MHz)
d (ppm): 11.61 [2-NHCH2-
CH2N(CH2CH3)2], 11.81 [6-NHCH2CH2N(CH2CH3)2], 20.76
(1 ꢁ gemCH3), 23.18 (C-1), 26.57 (1 ꢁ gemCH3), 41.24 (6-
NHCH2CH2NEt2), 46.04 (2-NHCH2CH2NEt2), 46.97 [2-NH-
CH2CH2N(CH2CH3)2], 47.21 [6-NHCH2CH2N(CH2CH3)2],
51.44 (6-NHCH2CH2NEt2), 52.90 (2-NHCH2CH2NEt2),
58.30 (C-2), 79.02 (C-3), 91.60 (C-5), 94.95 (C-12b), 102.61
(C-6a), 116.88 (C-11), 122.24 (C-7a), 123.41 (C-9), 126.07
(C-8), 133.29 (C-10), 151.15 (C-6), 155.10 (C-11a), 156.51
(C-12a), 160.06 (C-4a), 178.24 (C-7). Anal. Calcd. for
C30H44N4O3$3HCl. Calcd. (%): C: 58.30, H: 7.66, N: 9.07.
Found (%): C: 58.12, H: 7.59, N: 8.86.
4.1.22. N,N0-Bis(2-dimethylaminoethyl)-1,2-dihydro-3,3-
dimethyl-7-oxo-3H,7H-pyrano[2,3-c]xantheno-2,6-
diamine (20a)
To a solution of 19a (50 mg, 0.09 mmol) in dry CH2Cl2
(5 mL) at 0 ꢀC was added trifluoroacetic acid (0.5 mL) and
the resulting mixture was stirred at room temperature for 1 h.
The reaction mixture was then made alkaline with a 10%
NaHCO3 solution and extracted with CH2Cl2 (3 ꢁ 60 mL).
The combined organic extracts were washed with brine, dried
(Na2SO4), and concentrated under reduced pressure and the res-
idue was purified by column chromatography (silica gel,
CH2Cl2:MeOH 9:1) to afford compound 20a (35 mg, 86%).
Mp (trihydrochloride) > 250 ꢀC (CH2Cl2eEt2O); 1H NMR
(CDCl3, 400 MHz) d (ppm): 1.28 (s, 3H, 1 ꢁ gemCH3), 1.44
(s, 3H, 1 ꢁ gemCH3), 2.22 [s, 6H, 2-NHCH2CH2N(CH3)2],
2.30 [s, 6H, 6-NHCH2CH2N(CH3)2], 2.41 (t, J ¼ 7 Hz, 2H, 2-
NHCH2CH2NMe2), 2.53 (dd, J ¼ 17 Hz, 5 Hz, 1H, H-1a),
2.62 [t, J ¼ 7 Hz, 2H, 6-NHCH2CH2NMe2], 2.72 [m, 2H, H-2
and 2-NHCH2CH2NMe2], 2.93 (m, 1H, 2-NHCH2CH2NMe2),
3.10 (dd, J ¼ 17 Hz, 7 Hz, 1H, H-1b), 3.25 (q, J ¼ 7 Hz, 5 Hz,
2H, 6-NHCH2CH2NMe2), 5.85 (s, 1H, H-5), 7.28 (dt,
J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.36 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-
11), 7.58 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.21 (dd, J ¼ 8 Hz,
2 Hz, 1H, H-8), 9.38 (t, J ¼ 5 Hz, 1H, D2O exch., 6-
NHCH2CH2NMe2); 13C NMR (CDCl3, 50 MHz) d (ppm):
20.85 (1 ꢁ gemCH3), 23.10 (C-1), 26.55 (1 ꢁ gemCH3), 40.90
(6-NHCH2CH2NMe2), 44.97 [2-NHCH2CH2N(CH3)2], 45.50
[6-NHCH2CH2N(CH3)2], 45.54 [2-NHCH2CH2NMe2], 57.91
(6-NHCH2CH2NMe2), 58.22 (C-2), 58.84 (2-NHCH2-
CH2NMe2), 79.01 (C-3), 91.61 (C-5), 94.84 (C-12b), 102.61
(C-6a), 116.92 (C-11), 122.18 (C-7a), 123.50 (C-9), 126.11
(C-8), 133.35 (C-10), 151.11 (C-6), 155.04 (C-11a), 156.54
(C-12a), 159.99 (C-4a), 178.32 (C-7). Anal. Calcd. for
C26H36N4O3$3HCl. Calcd. (%): C: 55.57, H: 7.00, N: 9.97.
Found (%): C: 55.28, H: 6.82, N: 10.25.
4.1.24. 3,3-Dimethyl-6-methoxy-2-nitro-3H,7H-pyrano
[2,3-c]xanthen-7-one (21)
To a solution of 11 (143 mg, 0.42 mmol) in dry THF
(5 mL) was added NaH (60% in hexanes, 25 mg,
0.813 mmol) and the mixture was stirred at room temperature,
under argon for 30 min. A solution of dimethyl sulfate (55 mg,
0.43 mmol) in dry THF (3 mL) was then added dropwise and
the mixture was refluxed for 3 h. After cooling, the excess of
NaH was destroyed by the addition of EtOH (1 mL) and the
precipitate was filtered off. The filtrate was concentrated under
reduced pressure and the residue was purified by column chro-
matography (silica gel), elution with cyclohexaneeEtOAc 4:1,
to provide compound 21. Yield: 76%; mp 224e226 ꢀC
1
(CH2Cl2eEt2O); H NMR (CDCl3, 400 MHz) d (ppm): 1.79
(s, 6H, 2 ꢁ gemCH3), 4.00 (s, 3H, CH3O), 6.31 (s, 1H, H-5),
7.35 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.44 (dd, J ¼ 8 Hz, 2 Hz,
1H, H-11), 7.66 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.24 (dd,
J ¼ 8 Hz, 2 Hz, 1H, H-8), 8.25 (s, 1H, H-1); 13C NMR
(CDCl3, 50 MHz) d (ppm): 26.45 (2 ꢁ gemCH3), 56.85
(OCH3), 80.54 (C-3), 95.44 (C-5), 100.11 (C-12b), 107.83
(C-6a), 117.03 (C-11), 122.91 (C-7a), 123.21 (C-1), 124.70
(C-9), 126.81 (C-8), 134.22 (C-10), 143.85 (C-2), 154.47
(C-11a), 155.96 (C-12a), 159.69 (C-4a), 165.84 (C-6),
174.59 (C-7). Anal. Calcd. for C19H15NO6. Calcd. (%): C:
64.59, H: 4.28, N: 3.96. Found (%): C: 64.49, H: 4.22, N: 4.08.
4.1.25. 1,2-Dihydro-3,3-dimethyl-6-methoxy-2-nitro-3H,
7H-pyrano[2,3-c]xanthen-7-one (22)
Compound 22 was prepared by a procedure analogous to
4.1.23. N,N0-bis(2-Diethylaminoethyl)-1,2-dihydro-3,3-dimethyl-
7-oxo-3H,7H-pyrano[2,3-c]xantheno-2,6-diamine (20b)
Compound 20b was prepared by a procedure analogous to
that of 20a. Yield: 87%; mp (trihydrochloride) > 250 ꢀC
1
that of 14a. Yield: 88%; mp > 250 ꢀC (CH2Cl2eEt2O); H
NMR (CDCl3, 400 MHz) d (ppm): 1.49 (s, 3H, 1 ꢁ gemCH3),

318
G. Kolokythas et al. / European Journal of Medicinal Chemistry 42 (2007) 307e319
1.51 (s, 3H, 1 ꢁ gemCH3), 3.36 (dd, J ¼ 17 Hz, 6 Hz, 1H, H-
1a), 3.51 (dd, J ¼ 17 Hz, 6 Hz, 1H, H-1b), 3.95 (s, 3H,
CH3O), 4.91 (t, J ¼ 6 Hz, 1H, H-2), 6.33 (s, 1H, H-5), 7.33
(dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.38 (dd, J ¼ 8 Hz, 2 Hz, 1H,
H-11), 7.63 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.28 (dd,
J ¼ 8 Hz, 2 Hz, 1H, H-8); 13C NMR (CDCl3, 50 MHz)
4.1.28. 2-Dimethylamino-N-[1,2-dihydro-3,3-dimethyl-6-
methoxy-7-oxo-3H,7H-pyrano[2,3-c]xanthen-
2-yl]acetamide (25a)
Compound 25a was prepared by a procedure analogous to
that of 17a. Yield: 87%; mp (hydrochloride) 204e206 ꢀC,
1
(EtOH); H NMR (CDCl3, 400 MHz) d (ppm): 1.35 (s, 3H,
d
(ppm): 22.83 (1 ꢁ gemCH3), 22.92 (C-1), 25.96
1 ꢁ gemCH3), 1.37 (s, 3H, 1 ꢁ gemCH3), 2.17 [s, 6H,
NHCOCH2N(CH3)2], 2.90 (dd, J ¼ 17 Hz, 3 Hz, 1H, H-1a),
2.93 (s, 2H, NHCOCH2NMe2), 3.07 (dd, J ¼ 17 Hz, 5 Hz,
1H, H-1b), 3.90 (s, 3H, CH3O), 4.33 (m, 1H, H-2), 6.28 (s,
1H, H-5), 7.27 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.32 (m, 2H,
H-11 and NHCOCH2NMe2), 7.56 (dt, J ¼ 8 Hz, 2 Hz, 1H,
H-10), 8.22 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-8); 13C NMR
(CDCl3, 50 MHz) d (ppm): 23.83 (C-1), 24.07 (1 ꢁ gemCH3),
24.93 (1 ꢁ gemCH3), 45.83 [NHCOCH2N(CH3)2], 47.38
(C-2), 56.18 (CH3O), 62.98 (NHCOCH2NMe2), 77.89 (C-3),
95.89 (C-5), 99.07 (C-12b), 107.30 (C-6a), 116.95 (C-11),
122.90 (C-7a), 123.92 (C-9), 126.61 (C-8), 133.62 (C-10),
154.68 (C-11a), 156.96 (C-12a), 158.30 (C-4a), 160.30
(C-6), 170.44 (NHCO), 175.49 (C-7). Anal. Calcd. for
C23H26N2O5$HCl. Calcd. (%): C: 61.81, H: 6.09, N: 6.27.
Found (%): C: 61.69, H: 6.01, N: 6.13.
(1 ꢁ gemCH3), 56.61 (OCH3), 75.72 (C-3), 85.06 (C-2),
96.10 (C-5), 97.76 (C-12b), 108.53 (C-6a), 117.09 (C-11),
123.16 (C-7a), 124.27 (C-9), 127.03 (C-8), 133.94 (C-10),
154.65 (C-11a), 156.66 (C-12a), 157.41 (C-4a), 161.00 (C-
6), 175.36 (C-7). Anal. Calcd. for C19H17NO6. Calcd. (%):
C: 64.22, H: 4.82, N: 3.94. Found (%): C: 63.95, H: 4.77,
N: 3.75.
4.1.26. 2-Amino-1,2-dihydro-3,3-dimethyl-6-methoxy-
3H,7H-pyrano[2,3-c]xanthen-7-one (23)
Compound 23 was prepared by a procedure analogous to
that of 15a. Yield: 92%; mp > 250 ꢀC (EtOHeEt2O); 1H
NMR (CDCl3, 400 MHz) d (ppm): 1.33 (s, 3H, 1 ꢁ gemCH3),
1.40 (s, 3H, 1 ꢁ gemCH3), 1.91 (s, 2H, D2O exch., NH2), 2.66
(dd, J ¼ 17 Hz, 6 Hz, 1H, H-1a), 3.11 (m, 2H, H-1b and H2),
3.90 (s, 3H, CH3O), 6.24 (s, 1H, H-5), 7.28 (dt, J ¼ 8 Hz,
2 Hz, 1H, H-9), 7.33 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-11), 7.58
(dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.25 (dd, J ¼ 8 Hz, 2 Hz, 1H,
4.1.29. 2-Diethylamino-N-[1,2-dihydro-3,3-dimethyl-6-
methoxy-7-oxo-3H,7H-pyrano[2,3-c]xanthen-2-
yl]acetamide (25b)
H-8); 13C NMR (CDCl3, 50 MHz)
d (ppm): 20.83
Compound 25b was prepared by a procedure analogous to
that of 17a. Yield: 89%; mp (hydrochloride) 211e213 ꢀC,
(EtOH); 1H NMR (CDCl3, 400 MHz) d (ppm): 0.76 [t,
J ¼ 7 Hz, 6H, NHCOCH2N(CH2CH3)2], 1.32 (s, 3H,
1 ꢁ gemCH3), 1.33 (s, 3H, 1 ꢁ gemCH3), 2.35 [q, J ¼ 7 Hz,
4H, NHCOCH2N(CH2CH3)2], 2.88 (m, 2H, H-1a and
NHCOCH2NEt2), 3.01 (m, 2H, H-1b and NHCOCH2NEt2),
3.88 (s, 3H, CH3O), 4.25 (m, 1H, H-2), 6.26 (s, 1H, H-5),
7.24 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-9), 7.30 (dd, J ¼ 8 Hz, 2 Hz,
1H, H-11), 7.55 (dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 7.70 (d,
J ¼ 9 Hz, 1H, NHCOCH2NEt2), 8.20 (dd, J ¼ 8 Hz, 2 Hz,
1H, H-8); 13C NMR (CDCl3, 50 MHz) d (ppm): 12.43
[NHCOCH2N(CH2CH3)2], 23.85 (C-1), 24.19 (1 ꢁ gemCH3),
24.82 (1 ꢁ gemCH3), 47.41 (C-2), 48.76 [NHCOCH2-
N(CH2CH3)2], 56.23 (CH3O), 57.45 (NHCOCH2NEt2),
77.86 (C-3), 95.79 (C-5), 99.03 (C-12b), 107.30 (C-6a),
117.00 (C-11), 122.88 (C-7a), 123.93 (C-9), 126.66 (C-8),
133.67 (C-10), 154.71 (C-11a), 157.07 (C-12a), 158.33 (C-
4a), 160.38 (C-6), 171.85 (NHCO), 175.55 (C-7). Anal. Calcd.
for C25H30N2O5$HCl. Calcd. (%): C: 63.22, H: 6.58, N: 5.90.
Found (%): C: 6.48, H: 6.50, N: 5.77.
(1 ꢁ gemCH3), 25.78 (1 ꢁ gemCH3), 26.14 (C-1), 50.99
(C-2), 56.21 (OCH3), 78.88 (C-3), 95.74 (C-5), 100.19
(C-12b), 107.02 (C-6a), 116.89 (C-11), 122.93 (C-7a),
123.83 (C-9), 126.74 (C-8), 133.57 (C-10), 154.77 (C-11a),
156.77 (C-12a), 158.59 (C-4a), 160.25 (C-6), 175.47 (C-7).
Anal. Calcd. for C19H19NO4$HCl. Calcd. (%): C: 63.07, H:
5.57, N: 3.87. Found (%): C: 62.90, H: 5.52, N: 4.04.
4.1.27. 2-Chloro-N-[1,2-dihydro-3,3-dimethyl-6-methoxy-7-
oxo-3H,7H-pyrano[2,3-c]xanthen-2-yl]acetamide (24)
Compound 24 was prepared by a procedure analogous to that
of 16a. Yield: 94%; mp 214e216 ꢀC (CH2Cl2en-pentane); 1H
NMR (CDCl3, 400 MHz) d (ppm): 1.34 (s, 3H, 1 ꢁ gemCH3),
1.45 (s, 3H, 1 ꢁ gemCH3), 2.93 (dd, J ¼ 17 Hz, 3 Hz, 1H, H-
1a), 3.05 (dd, J ¼ 17 Hz, 5 Hz, 1H, H-1b), 3.82 (s, 3H, CH3O),
4.18 (s, 2H, NHCOCH2Cl), 4.40 (m, 1H, H-2), 6.11 (s, 1H, H-
5), 7.11 (d, J ¼ 9 Hz, 1H, NHCOCH2Cl), 7.26 (dt, J ¼ 8 Hz,
2 Hz, 1H, H-9), 7.43 (dd, J ¼ 8 Hz, 2 Hz, 1H, H-11), 7.48
(dt, J ¼ 8 Hz, 2 Hz, 1H, H-10), 8.17 (dd, J ¼ 8 Hz, 2 Hz, 1H,
H-8); 13C NMR (CDCl3, 50 MHz) d (ppm): 23.48 (C-1), 24.52
(1 ꢁ gemCH3), 24.70 (1 ꢁ gemCH3), 42.84 (NHCOCH2Cl),
48.00 (C-2), 56.05 (CH3O), 78.04 (C-3), 95.86 (C-5), 98.67
(C-12b), 107.04 (C-6a), 116.81 (C-11), 122.60 (C-7a),
123.95 (C-9), 126.53 (C-8), 133.82 (C-10), 154.45 (C-11a),
157.39 (C-12a), 158.34 (C-4a), 160.06 (C-6), 166.71 (NHCO),
175.30 (C-7). Anal. Calcd. for C21H20ClNO5. Calcd. (%):
C: 62.77, H: 5.02, N: 3.49. Found (%): C: 62.44, H: 5.27,
N: 3.62.
4.2. Biological evaluation
4.2.1. Materials
Eagle’s minimal essential medium (EMEM), foetal bovine
serum (FBS), sodium pyruvate, sodium bicarbonate, L-gluta-
mine, nonessential amino acids, penicillin, streptomycin,
amphoterecin B and gentamycin were all obtained from Bio-
chrom KG (Berlin, Germany). Insulin was obtained from

G. Kolokythas et al. / European Journal of Medicinal Chemistry 42 (2007) 307e319
319
Sigma Chemicals (Steinhelm, Germany). All other chemicals
used were of the best commercially available grade.
Education (Business Program of Education and Initial Profes-
sional Preparing-EPEAEK - IRAKLEITOS).
4.2.2. Cell culture conditions
MDA-MB-231 (HTB 26: human breast adenocarcinoma,
ER-negative, high invasive potential) was obtained from the
American Type Culture Collection (ATCC) and cultured as
monolayers at 37 ꢀC in a humidified atmosphere of 5% (v/v)
CO2 and 95% air. Cells were seeded in 75-cm2 plastic tissue
culture flasks and cultured in EMEM supplemented with
10% FBS, 2 mM L-glutamine, 1.0 mM sodium pyruvate,
1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids,
0.01 mg/mL of insulin and a cocktail of antimicrobial agents
(100 IU/mL penicillin, 100 mg/mL streptomycin, 10 mg/mL
gentamicin sulfate and 2.5 mg/mL amphoterecin B) [21]. Ac-
cording to pilot experiments with respect to growth rate and
doubling time, the medium was changed every three days.
The cells were harvested after treatment with 0.25% (w/v)
trypsin in PBS, containing 0.1% (w/v) Na2EDTA.
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