Synthesis and cytotoxic activity of some new azapyranoxanthenone aminoderivatives

Article abstract of DOI:10.1016/S0968-0896(03)00503-0

A series of novel azapyranoxanthenones, bearing structural similarity to the acridone alkaloid acronycine have been designed and synthesized. Their in vitro cytotoxicities against the murine L1210 leukemia and the human solid tumor HT-29 cell lines have been investigated. The new derivatives exhibited interesting cytotoxic activity and were more potent than the parent compound.

Full text of DOI:10.1016/S0968-0896(03)00503-0

Bioorganic & Medicinal Chemistry 11 (2003) 4591–4598
Synthesis and Cytotoxic Activity of Some New
Azapyranoxanthenone Aminoderivatives
George Kolokythas,a Ioannis K. Kostakis,a Nicole Pouli,a Panagiotis Marakos,a,*
Dimitris Kletsasb and Harris Pratsinisb
aDivision of Pharmaceutical Chemistry, Department of Pharmacy, University of Athens, Panepistimiopolis-Zografou,
Athens 15771, Greece
bLaboratory of Cell Proliferation & Ageing, Institute of Biology, NCSR ‘Demokritos’, 15310 Athens, Greece
Received 17 June 2003; accepted 25 July 2003
Abstract—A series of novel azapyranoxanthenones, bearing structural similarity to the acridone alkaloid acronycine have been
designed and synthesized. Their in vitro cytotoxicities against the murine L1210 leukemia and the human solid tumor HT-29 cell
lines have been investigated. The new derivatives exhibited interesting cytotoxic activity and were more potent than the parent
compound.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
compared with the corresponding quinone chemotypes.4
Further improved pharmacological profile was achieved
in some cases with different aza- and diaza-bioisosteres,
derived through extensive structure–activity relation-
ships studies, which clearly demonstrated that the cyto-
toxic effects are critically depended on the position of
the nitrogen atom.5
Among the chemotherapeutic agents, DNA-interacting
compounds constitute a significant class and many of
them are in clinical use or in clinical trials for the treat-
ment of cancer. Within this diverse group of com-
pounds, molecules with quinone functionalities, such as
the anthracycline antibiotics adriamycin and daunomy-
cin and the anthracenedione mitoxantrone are repre-
sentative examples of drugs that target DNA, interact
with it by binding and intercalation and exert their critical
cell-killing effects through many different mechanisms.1
A great number of mitoxantrone related derivatives has
been synthesized and studied, in attempts to reduce or
remove undesirable side effects, mainly those associated
with myelosuppression and cardiotoxicity.2 These mod-
ified analogues are characterized by the presence of a
planar or semi-planar lipophilic chromophore portion,
substituted with one or two flexible basic side chains.3 A
common variation of known tricyclic antitumor agents has
been the incorporation of a N-substituted pyrazolo ring
fusion at the 1- and 9-positions and the resulting anthra-
pyrazoles are usually endowed with an increased spectrum
of antitumor activity and reduced cardiotoxicity,
We have already been involved in the synthesis and
antiproliferative activity study of a number of cytotoxic
pyranoxanthenones
and
pyranothioxanthenones,6
which share close structural similarity with the acridone
alkaloid acronycine (Fig. 1), a promising, broad-spec-
trum antitumor agent, which suffers from poor water
solubility.7
The introduction of a flexible amino substituted side
chain, or the incorporation of a fused amino substituted
pyrazolo ring in the abovementioned molecules resulted
in a noticeable increase of the cytotoxic activity against
the murine leukemia L1210 and the human colon ade-
nocarcinoma HT-29 cell lines.8 As a continuation of
these studies we decided to introduce a nitrogen atom
into the xanthone chromophore, hence we present here
the synthesis and in vitro cytotoxic activity evaluation
of some new derivatives, in an effort to study the
potential alteration of the biological activity, due to the
replacement of the carbocyclic moiety by the isosteric
pyridine heterocycle.
*Corresponding author. Fax: +30-10-727-4747; e-mail: marakos@
pharm.uoa.gr
0968-0896/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00503-0

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G. Kolokythas et al. / Bioorg. Med. Chem. 11 (2003) 4591–4598
be the angular isomer 5, in an optimized 20% yield.9 No
traces of the intermediate ether 4 or the linear isomer 6
were isolated. We have applied a slightly different pro-
cedure using CuI as catalyst for the etherification step10
and we have obtained the ether 4 in a 76% isolated
yield. This compound was then subjected to thermal
cyclisation in boiling toluene to afford a 1:1 mixture of
two four-ring isomers (5 and 6), which were separated by
column chromatography and unambiguously identified
on the basis of 1D and 2D-NMR spectral data.
Figure 1.
Chemistry
For the preparation of the target derivatives we have
used as starting material 2-chloronicotinic acid (1,
Scheme 1), which was converted to the corresponding
acylchloride after treatment with thionylchloride in
refluxing toluene. Reaction of this chloride with 1,3,5-
trihydroxybenzene in the presence of aluminum chloride
in 1,2-dichloroethane provided the ketone 2 in good
yield, which was then treated with aqueous sodium car-
bonate to result in the dihydroxy-derivative 3. The pre-
paration of this compound has been previously reported
by a different procedure, through Ullmann condensa-
tion of 2-chloronicotinic acid with 3,5-dimethoxy-
phenol, followed by cyclization with PPA and O-
demethylation.9 However, the procedure reported
herein is simpler and the product was obtained in a
higher overall yield (51% vs 41%). The next step con-
cerns the formation of the pyran ring. The most com-
mon method involves the etherification of the phenol
hydroxyl, followed by thermal cyclisation of the result-
ing ether in boiling N,N-diethylaniline. In the case of
compound 3 the deactivation of the 6-hydroxyl group,
due to its chelation by the 5-carbonyl, made feasible the
selective etherification of the 8-hydroxyl group with 3-
chloro-3-methyl-1-butyne.
Each isomer was subsequently converted to the corre-
sponding tosylate (7 and 10, respectively) by standard
conditions. Treatment of the latter analogues with
commercially available N,N-dialkylaminoethylenedia-
mines in DMSO solution provided the target angular (8,
9) and linear (11, 12) diamines.
The synthesis of the pyrazolo-fuzed analogues is depic-
ted in Scheme 2.
The angular tosylate 7 was treated with 2-hydroxy-
ethylhydrazine and the resulted carbinol 13 was con-
verted to the corresponding mesylate 14. Nucleophilic
substitution of the mesyl group with the appropriately
substituted secondary amines gave the amines 15 and 16
in good yield. The linear derivatives 19 and 20, were
prepared by an analogous reaction sequence, starting
from the tosylate 10.
In order to examine the in vitro antineoplastic activity
of the new compounds, the free base forms of the
amines were converted into their water-soluble hydro-
chloride addition salts.
However, previous attempts to apply this condensation
in the presence of anhydrous potassium carbonate and
sodium iodide, in dry DMF at 70 ꢀC, with various
reaction periods, gave a single product, which proved to
Results and Discussion
The cytotoxic activity of the new derivatives was eval-
uated in vitro in the established model of the murine
L1210 leukemia cell line as well as against the human
solid tumor HT-29 cell line, using acronycine and the
drugs doxorubicin and mitoxantrone as reference com-
pounds. The results are presented in Table 1.
Scheme 1. (a) SOCl2, toluene dry, 90 ꢀC, 1.5 h; (b) phloroglucinol,
ꢀ
AlCl3, 1,2-dichloroethane, 50 ꢀC, 60 h; (c) 20% Na2CO3, 1 1 0C, 60 h;
(d) 3-chloro-3-methyl-1-butyne, K2CO3, KI, CuI, DMF dry, 75 ꢀC, 9
h; (e) toluene dry, reflux, 46 h; (f) TsCl, K2CO3, acetone dry, reflux, 3
h; (g) N,N-dimethylethylenediamine or N,N-diethylethylenediamine,
DMSO dry, 100 ꢀC, 1h.
Scheme 2. (a) 2-Hydroxyethylhydrazine, DMSO dry, 90 ꢀC, 30 min;
(b) MsCl, Et3N, CH2Cl2 dry, rt, 2 h; (c) dimethylamine or diethyl-
amine, EtOH, reflux, 20 h.

G. Kolokythas et al. / Bioorg. Med. Chem. 11 (2003) 4591–4598
Table 1. Cytotoxic activity of the synthesized compounds
4593
results in a reduced cytotoxicity against the L1210 cell
line, whereas the compounds remain equally active
against the HT-29 cell line. It should be noted that the
abovementioned comparison is not directly feasible for
derivatives 19 and 20, since this particular ring system
has not been previously reported.
Compd
IC50 (mM)
L1210
HT-29
8
9
11
12
15
16
19
17.8 (ꢁ0.6)
17.3 (ꢁ1.9)
19.2 (ꢁ3.5)
16.9 (ꢁ1.8)
17.9 (ꢁ2.1)
19.0 (ꢁ1.3)
19.2 (ꢁ0.9)
18.0 (ꢁ0.5)
25.2 (ꢁ2.3)
nt
11.8 (ꢁ5.2)
6.15 (ꢁ2.85)
15.3 (ꢁ2.5)
6.95 (ꢁ2.45)
15.9 (ꢁ1.1)
5.15 (ꢁ0.15)
8.85 (ꢁ3.25)
5.25 (ꢁ2.65)
>100
Cell-cycle perturbations induced by the new compounds
were studied on the HT-29 cell line (Table 2).
As expected, both doxorubicin11 and mitoxantrone12 act
as potent G2 blockers. On the other hand, all new deri-
vatives seem to achieve their cytostatic effects by block-
ing the entry of the cells in the S-phase, the linear
diamines 11 and 12 being the most effective. Further-
more, the angular pyrazoloderivatives 15 and 16 cause a
slight increase of the G2 arrested cell-percentage, as
well.
20
Acronycine
Mitoxantrone
Doxorubicin
0.02 (ꢁ0.009)
0.08 (ꢁ0.01)
0.51 (ꢁ0.06)
Exponentially growing L1210 and HT-29 cells were incubated with
serial dilutions of the indicated substances for 72 h. MTT-assay was
performed as described in the Experimental. Values represent the
mean (ꢁstandard deviation) of three independent experiments and
were expressed as IC50, the concentration that reduced by 50% the
optical density of treated cells with respect to untreated controls. nt,
Experimental
All the tested compounds proved to be more cytotoxic
than acronycine against the L1210 cell line and at the
same time they exhibit cytotoxicity against the HT-29
cell line, where the reference compound is inactive. This
is consistent with our previous observations8 and con-
firms the beneficial effects of the introduction of a dia-
lkylaminoethylamino basic side chain for the
cytotoxicity. In general, all the derivatives possess
almost equal cytotoxicity against the L1210 cell line and
the corresponding IC50 values were found to be in the
range of 17–19 mM. On the contrary, concerning the
HT-29 cell line, we observe that the N,N-diethylamino
derivatives appear to be approximately twice more
active than the corresponding N,N-dimethylamino ana-
logues. Notably, in contrast to many chemotherapeutic
agents, all compounds exhibit solid tumor selectivity,
since they demonstrate greater potency, in terms of IC50
values, against the HT-29 cell line than against the
leukemia L1210 cells.
All chemicals were purchased from Aldrich Chemical
Co. Melting points were determined on a Buchi appa-
1
ratus and are uncorrected. H NMR spectra and 2D
spectra were recorded on a Bruker Avanche 400 instru-
ment, whereas 13C NMR specra were recorded on a
Bruker AC 200 spectrometer in deuterated solvents and
were referenced to TMS (d scale). The signals of 1H and
13C spectra were unambiguously assigned by using 2D
NMR techniques: H1H COSY, NOESY HMQC and
1
HMBC. Flash chromatography was performed on
Merck silica gel 60 (0.040-0.063 mm). Analytical thin
layer chromatography (TLC) was carried out on pre-
coated (0.25 mm) Merck silica gel F-254 plates. Ele-
mental analyses were performed at the Microanalytical
Sections of the National Hellenic Research Foundation on
a Perkin-Elmer PE 240C Elemental Analyzer (Norwalk,
CT, USA) and were withinꢁ0.4% of the theoretical
values.
From a comparison of the activity of the newly synthe-
sized azapyranoxanthenones with the corresponding
closely related analogues which lack the aza substitu-
tion,8 we assume that the presence of the nitrogen atom
(2-Chloropyridin-3-yl)-(2,4,6-trihydroxyphenyl)methanone
(2). Thionyl chloride (5 mL) was added under argon to
a suspension of 2-chloronicotinic acid (1, 3 g, 19.04
mmol) in dry toluene (5 mL) and the resulting solution
was heated at 90 ꢀC for 1.5 h. Excess thionyl chloride
and the bulk of the solvent were evaporated under
vacuum and then dry 1,2-dichloroethane (10 mL) was
added to the white crystalline residue. This solution was
added slowly under argon to a stirred mixture of phlor-
oglucinol (2 g, 15.86 mmol) and aluminium trichloride
(8.46 g, 63.48 mmol) in 1,2-dichloroethane (20 mL) at
0 ꢀC. The mixture was stirred at rt for 10 min and then
heated at 50 ꢀC for 60 h. Upon cooling, a 9% HCl
solution (40 mL) was added and the precipitate was fil-
trated, washed with water (2ꢂ40 mL) and dried over
CaCl2. The crude product was purified by column
chromatography (silica gel, 18ꢂ2 cm) using ethyl ace-
tate as the eluent to give 2 (2.20 g, 52%) as a pale yellow
solid. Mp >250 ꢀC, (EtOH). 1H NMR (DMSO-d6,
400 MHz) d (ppm): 6.26 (d, J=2 Hz, 1H, H-30), 6.48 (d,
J=2 Hz, 1H, H-50), 7.59 (m, 1H, H-5), 8.58 (m, 1H, H-
6), 8.77 (m, 1H, H-4), 8.96 (s, 2H, D2O exch., OH-40,
Table 2. Cell-cycle phase distribution (%)
G0/G1S
G2/M
42.21
38.23
26.63
1.97
Control
8
9
11
12
15
16
19
41.21
58.19
68.74
94.92
73.17
16.57
3.58
4.63
3.11
5.14
21.69
55.9123.80
56.18
64.34
68.44
44.410.00
2.55
20.29
25.21
27.57
21.39
55.59
1.70
19.61
8.09
10.17
20
Doxorubicin
Mitoxantrone
95.76
Exponentially growing HT-29 cells received the indicated substances
(20 mM, except doxorubicin 100 nM and mitoxantrone 80 nM). After
incubation for 24 h, they were subjected to FACS analysis, as descri-
bed in the Experimental. One representative determination out of
three similar ones is demonstrated.

4594
G. Kolokythas et al. / Bioorg. Med. Chem. 11 (2003) 4591–4598
OH-60), 12.53 (s, 1H, D2O exch., OH-20). 13C NMR
(DMSO-d6, 50 MHz) d (ppm): 94.80 (C-50), 98.58 (C-30),
102.18 (C-10), 115.19 (C-1), 121.70 (C-5), 136.14 (C-6),
154.45 (C-4), 157.02 (C-60), 159.70 (C-2), 162.83 (C-20),
166.43 (C-40), 180.25 (–CO–). Anal. calcd for
C12H8ClNO4. Calcd (%): C: 54.25, H: 3.04, N: 5.27.
Found (%): C: 54.12, H: 2.96, N: 5.21.
was purified by column chromatography (silica gel,
25ꢂ1.5 cm) using a mixture of cyclohexane/ethyl acetate
(85/15) as the eluent to give 5 (0.92 g, 41%) and 6 (1.07
g, 48%) as yellow solids. Data for 5: Mp 208–210 ꢀC,
(EtOAc–n-hexane), 1H NMR (CDCl3, 400 MHz) d
(ppm): 1.45 (s, 6H, 2ꢂgem CH3), 5.60 (d, J=10 Hz, 1H,
H-2), 6.23 (s, 1H, H-5), 6.87 (d, J=10 Hz, 1H, H-1),
7.38 (m, 1H, H-9), 8.56 (m, 1H, H-8), 8.65 (m, 1H, H-
10), 12.53 (s, 1H, D2O exch., OH). 13C NMR (CDCl3,
50 MHz) d (ppm): 28.45 (2ꢂgem CH3), 78.58 (C-3),
99.86 (C-5), 101.59 (C-12b), 103.39 (C-6a), 114.74 (C-1),
115.63 (C-7a), 121.07 (C-9), 127.61 (C-2), 136.39 (C-8),
151.02 (C-12a), 153.92 (C-10), 160.02 (C-11a), 161.61
(C-4a), 162.93 (C-6), 180.68 (C-7). Anal. calcd for
C17H13NO4. calcd (%): C: 69.15, H: 4.44, N: 4.74.
Found (%): C: 68.87, H: 4.36, N: 4.70. Data for 6: Mp
245–247 ꢀC, (EtOAc–n-hexane). 1H NMR (CDCl3,
400 MHz) d (ppm): 1.42 (s, 6H, 2ꢂgem CH3), 5.59 (d,
J=10 Hz, 1H, H-3), 6.43 (s, 1H, H-12), 6.67 (d, J=10
Hz, 1H, H-4), 7.38 (m, 1H, H-8), 8.57 (m, 1H, H-7),
8.66 (m, 1H, H-9), 12.73 (s, 1H, D2O exch., OH). 13C
NMR (CDCl3, 50 MHz) d (ppm): 28.48 (2ꢂgem CH3),
78.69 (C-2), 96.00 (C-12), 103.46 (C-5a), 105.08 (C-4a),
115.04 (C-4), 115.71 (C-6a), 121.00 (C-8), 127.95 (C-3),
136.36 (C-7), 154.01 (C-9), 156.51 (C-11a), 157.57 (C-5),
160.18 (C-10a), 161.51 (C-12a), 180.80 (C-6). Anal.
calcd for C17H13NO4. calcd (%): C: 69.15, H: 4.44, N:
4.74. Found (%): C: 68.96, H: 4.29, N: 4.65.
6,8-Dihydroxy-5H-[1]benzopyrano[2,3-b]pyridin-5-one
(3). A suspension of 2 (4 g, 15.06 mmol) in 20%
Na2CO3 solution (40 mL) was heated at 110 ꢀC for 60 h.
The mixture was then allowed to cool, neutralized with
concentrated HCl and the precipitate was filtrated,
washed with water (2ꢂ30 mL) and dried over CaCl2.
The crude product was purified by column chromato-
graphy (silica gel, 15ꢂ2 cm) using ethyl acetate as the
eluent to give 3 (3.38 g, 98%) as a pale yellow solid. Mp
>250 ꢀC, (EtOH). H NMR (DMSO-d6, 400 MHz) d
1
(ppm): 6.25 (d, J=2 Hz, 1H, H-7), 6.48 (d, J=2 Hz,
1H, H-9), 7.59 (m, 1H, H-3), 8.57 (m, 1H, H-4), 8.77 (m,
1H, H-2), 11.28 (s, 1H, D2O exch., OH-8), 12.53 (s, 1H,
D2O exch., OH-6). 13C NMR (DMSO-d6, 50 MHz) d
(ppm): 94.80 (C-9), 98.59 (C-7), 102.26 (C-5a), 115.20
(C-4a), 121.71 (C-3), 136.19 (C-4), 154.46 (C-2), 157.07
(C-9a), 159.72 (C-10a), 162.84 (C-6), 166.37 (C-8),
180.30 (C-5). Anal. calcd for C12H7NO4. Calcd (%): C:
62.89, H: 3.08, N: 6.11. Found (%): C: 62.71, H: 3.03,
N: 5.93.
6-Hydroxy-8-(1,1-dimethylpropyn-2-oxy)-5H-[1]benzo-
pyrano[2,3-b]pyridin-5-one (4). 3-Chloro-3-methyl-1-
butyne (3.17 mL, 30.98 mmol) was added under argon
to a mixture of 3 (2.28 g, 9.95 mmol), anhydrous K2CO3
(1.65 g, 11.95 mmol), anhydrous KI (1.69 g, 10.18
mmol) and CuI (52 mg, 0.27 mmol) in dry DMF (20
mL) and the reaction was stirred at 75 ꢀC for 9 h. The
bulk of the solvent was then vacuum-evaporated, water
was added to the residue and it was extracted with ethyl
acetate. The organic phase was washed with water, a
saturated NaCl solution, dried (Na2SO4) and evapo-
rated to dryness. The residue was purified by column
chromatography (silica gel, 15ꢂ1.5 cm) using a mixture
of cyclohexane/ethyl acetate (9/1) as the eluent to give 4
(2.24 g, 76%) as a yellow solid. Mp 122–124 ꢀC,
(EtOAc–n-hexane). 1H NMR (CDCl3, 400 MHz) d
(ppm): 1.74 (s, 6H, 2ꢂgem CH3), 2.70 (s, 1H, H-30), 6.68
(d, J=2 Hz, 1H, H-9), 6.95 (d, J=2 Hz, 1H, H-7), 7.41
(m, 1H, H-3), 8.61 (m, 1H, H-4), 8.70 (m, 1H, H-2),
12.36 (s, 1H, D2O exch., OH). 13C NMR (CDCl3,
50 MHz) d (ppm): 29.51(2 ꢂgem CH3), 72.99 (C-10),
6-[(4-Methylphenyl)sulfonyl]oxy-3,3-dimethyl-3H,7H-
pyrano[20,30:7,8]benzopyrano[2,3-b]pyridin-7-one
(7).
Anhydrous potassium carbonate (1.28 g, 9.30 mmol)
and p-toluenesulfonyl chloride (1.20g, 6.28 mmol) were
added to a solution of 5 (550 mg, 1.86 mmol) in dry
acetone (50 mL) and the mixture was heated under
reflux for 3 h. Upon cooling the precipitate was filtered
off, washed with acetone (40 mL), the filtrates were
vacuum-evaporated and the residue was purified by
column chromatography (silica gel, 10ꢂ1.5 cm) using a
mixture of cyclohexane/ethyl acetate (75/25) as the elu-
ent to give 7 (611 mg, 73%) as a yellow solid. Mp 170–
172 ꢀC, (EtOAc–n-pentane). 1H NMR (CDCl3,
400 MHz) d (ppm): 1.39 (s, 6H, 2ꢂgem CH3), 2.30 (s,
3H,-SO2C6H4CH3), 5.66 (d, J=10 Hz, 1H, H-2), 6.49
(s, 1H, H-5), 6.86 (d, J=10 Hz, 1H, H-1), 7.24 (d, J=8
Hz, 2H, H-50, H-30), 7.31(m, 1H, H-9), 7.84 (d, J=8
Hz, 2H, H-60, H-20), 8.49 (m, 1H, H-8), 8.53 (m, 1H,
H-10). 13C HMR (CDCl3, 50 MHz) d (ppm): 21.31
(–SO2C6H4CH3), 27.96 (2ꢂgem CH3), 78.62 (C-3),
108.43 (C-12b), 108.91 (C-5), 109.46 (C-6a), 114.31 (C-
1), 116.70 (C-7a), 121.07 (C-9), 128.42 (C-60, C-20),
129.31 (C-50, C-30), 130.11 (C-2), 132.43 (C-10), 136.99
(C-8), 145.15 (C-40), 147.46 (C-6), 151.84 (C-12a),
153.05 (C-10), 157.79 (C-4a), 158.82 (C-11a), 173.78 (C-
7). Anal. calcd for C24H19NO6S. calcd (%): C: 64.13, H:
4.26, N: 3.12. Found (%): C: 63.91, H: 4.17, N: 2.97.
0
75.60 (C-30), 84.31(C-2 ), 97.88 (C-7), 101.88 (C-9),
104.05 (C-5a), 115.08 (C-4a), 121.03 (C-3), 136.51 (C-4),
154.19 (C-2), 156.54 (C-6), 160.41 (C-10a), 162.79 (C-
9a), 163.89 (C-8), 181.02 (C-5). Anal. calcd for
C17H13NO4. Calcd (%): C: 69.15, H: 4.44, N: 4.74.
Found (%): C: 69.41, H: 4.52, N: 4.68.
6-Hydroxy-3,3-dimethyl-3H,7H-pyrano[20,30:7,8]benzo-
pyrano[2,3-b]pyridin-7-one (5) and 5-hydroxy-2,2-di-
methyl-2H,6H-pyrano[30,20:6,7]benzopyrano[2,3-b]pyri-
din-6-one (6). A solution of 4 (2.24 g, 7.59 mmol) in dry
toluene (50 mL) was heated under reflux for 46 h. The
solvent was then evaporated to dryness and the residue
N,N-Dimethyl-N0 -[3,3-dimethyl-7-oxo-3H,7H -pyra-
no[20,30:7,8]benzopyrano[2,3-b]pyridin-6-yl]ethanediamine
(8). N,N-Dimethylethylenediamine (337 mL, 3.10 mmol)
was added under argon to a solution of 7 (140 mg, 0.31
mmol) in dry DMSO (6 mL) and the mixture was
heated at 100 ꢀC for 1h. The mixture was then poured

G. Kolokythas et al. / Bioorg. Med. Chem. 11 (2003) 4591–4598
4595
into ice-water and it was extracted with ethyl acetate.
The organic phase was dried (Na2SO4) and con-
centrated to dryness and the residue was purified by
column chromatography (silica gel, 20ꢂ1cm) using a
mixture of dichloromethane/methanol (97/3) as the elu-
ent to give 8 (100 mg, 88%) as a pale yellow oil. Mp
N,N-Dimethyl-N0 -[2,2-dimethyl-6-oxo-2H,6H-pyrano-
[30,20:6,7]benzopyrano[2,3 - b]pyridin - 5 - yl]ethanediamine
(11). This compound was prepared by a procedure ana-
logous to that of 8. Yield: 92%. Mp (hydrochloride)
>250 ꢀC, (EtOH). 1H NMR (CDCl3, 400 MHz) d
(ppm): 1.40 (s, 6H, 2ꢂgem CH3), 2.24 [s, 6H, N(CH3)2],
2.48 (t, J=7 Hz, 2H, NHCH2CH2NMe2), 3.48 (q, J=7
Hz, 5 Hz, 2H, NHCH2CH2NMe2), 5.42 (d, J=10 Hz,
1H, H-3), 6.23 (s, 1H, H-12), 6.53 (d, J=10 Hz, 1H, H-
4) 7.26 (m, 1H, H-8), 8.50 (m, 2H, H-7, H-9), 9.57 (t,
J=5 Hz, 1H, D2O exch., NH). 13C NMR (CDCl3,
50 MHz) d (ppm): 27.08 (2ꢂgem CH3), 45.53 [N(CH3)2],
47.23 (NHCH2CH2NMe2), 59.87 (NHCH2CH2NMe2),
76.34 (C-2), 94.02 (C-12), 103.80 (C-5a), 103.87 (C-4a),
116.81 (C-6a), 120.30 (C-4), 120.37 (C-8), 124.16 (C-3),
136.47 (C-7), 151.65 (C-5), 152.65 (C-9), 158.34 (C-
11a), 159.34 (C-10a), 160.92 (C-12a), 178.12 (C-6).
(hydrochloride) >250 ꢀC, (EtOH). H NMR (CDCl3,
1
400 MHz) d (ppm): 1.43 (s, 6H, 2ꢂgem CH3), 2.28 [s,
6H,
N(CH3)2],
2.60
(t,
J=7
Hz,
2H,
NHCH2CH2NMe2), 3.25 (q, J=7 Hz, 5 Hz, 2H,
NHCH2CH2NMe2), 5.49 (d, J=10 Hz, 1H, H-2), 5.87
(s, 1H, H-5), 6.88 (d, J=10 Hz, 1H, H-1) 7.28 (m, 1H,
H-9), 8.52 (m, 2H, H-10, H-8), 9.61 (t, J=5 Hz, 1H,
D2O exch., NH). 13C NMR (CDCl3, 50 MHz) d (ppm):
28.30 (2ꢂgem CH3), 40.77 (NHCH2CH2NMe2), 45.41
[N(CH3)2], 57.61(NHCH 2CH2NMe2), 77.86 (C-3),
92.01 (C-5), 97.86 (C-12b), 101.55 (C-6a), 115.58 (C-1),
116.99 (C-7a), 120.54 (C-9), 125.35 (C-2), 136.34 (C-8),
152.43 (C-10), 152.46 (C-6), 152.87 (C-12a), 159.52 (C-
11a), 160.55 (C-4a), 177.90 (C-7). Anal. Calcd for
.
.
Anal. calcd for C21H23O3N3 HCl H2O. Calcd (%): C:
60.07, H: 6.24, N: 10.01. Found (%): C: 59.72, H: 6.21,
N: 9.88.
.
C21H23N3O3 HCl. calcd (%): C: 62.76, H: 6.02, N:
10.46. Found (%): C: 62.42, H: 6.06, N: 10.21.
N,N-Diethyl-N0 -[2,2-dimethyl-6-oxo-2H,6H-pyrano-
[30,20:6,7]benzopyrano[2,3 - b]pyridin - 5 - yl]ethanediamine
(12). This compound was prepared by a procedure ana-
logous to that of 8. Yield: 90%. Mp (hydrochloride)
>250 ꢀC, (EtOH). 1H NMR (CDCl3, 400 MHz) d
(ppm): 0.96 [t, J=7 Hz, 6H, N(CH2CH3)2], 1.40 (s, 6H,
2ꢂgem CH3), 2.51[q, J=7 Hz, 4H, N(CH2CH3)2], 2.60
(t, J=7 Hz, 2H, NHCH2CH2NEt2), 3.46 (q, J=7 Hz, 5
Hz, 2H, NHCH2CH2NEt2), 5.42 (d, J=10 Hz, 1H, H-
3), 6.22 (s, 1H, H-12), 6.53 (d, J=10 Hz, 1H, H-4), 7.25
(m, 1H, H-8), 8.49 (m, 2H, H-7, H-9), 9.51 (t, J=5 Hz,
1H, D2O exch., NH). 13C NMR (CDCl3, 50 MHz) d
(ppm): 11.50 [N(CH2CH3)2], 27.01(2 ꢂgem CH3), 47.04
[N(CH2CH3)2], 47.52 (NHCH2CH2NEt2), 53.44
(NHCH2CH2NEt2), 76.37 (C-2), 93.94 (C-12), 103.76
(C-5a), 103.83 (C-4a), 116.77 (C-6a), 120.30 (C-4),
120.34 (C-8), 124.05 (C-3), 136.36 (C-7), 151.65 (C-5),
152.54 (C-9), 158.23 (C-11a), 159.30 (C-10a), 160.81 (C-
N,N-Diethyl-N0 -[3,3-dimethyl-7-oxo-3H,7H-pyrano-
[20,30:7,8]benzopyrano[2,3 - b]pyridin - 6 - yl]ethanediamine
(9). This compound was prepared by a procedure ana-
logous to that of 8. Yield: 90%. Mp (hydrochloride)
233–235 ꢀC, (EtOH). 1H NMR (CDCl3, 400 MHz) d
(ppm): 1.04 [t, J=7 Hz, 6H, N(CH2CH3)2], 1.42 (s, 6H,
2ꢂgem CH3), 2.58 [q, J=7 Hz, 4H, N(CH2CH3)2], 2.74
(t, J=7 Hz, 2H, NHCH2CH2NEt2), 3.24 (q, J=7 Hz, 5
Hz, 2H, NHCH2CH2NEt2), 5.49 (d, J=10 Hz, 1H, H-
2), 5.88 (s, 1H, H-5), 6.88 (d, J=10 Hz, 1H, H-1), 7.28
(m, 1H, H-9), 8.52 (m, 2H, H-10, H-8), 9.58 (t, J=5 Hz,
1H, D2O exch., NH). 13C NMR (CDCl3, 50 MHz) d
(ppm): 11.72 [N(CH2CH3)2], 28.30 (2ꢂgem CH3), 41.09
(NHCH2CH2NEt2), 46.97 [N(CH2CH3)2], 51.05
(NHCH2CH2NEt2), 77.81(C-3), 92.1(C-5), 97.80 (C-
12b), 101.59 (C-6a), 115.59 (C-1), 117.03 (C-7a), 120.48 (C-
9), 125.30 (C-2), 136.36 (C-8), 152.39 (C-10), 152.50 (C-6),
152.87 (C-12a), 159.52 (C-11a), 160.51 (C-4a), 177.79 (C-
.
.
12a), 177.94 (C-6). Anal. calcd for C23H27O3N3, HCl 3/
2H2O. calcd (%): C: 60.59, H: 6.63, N: 9.22. Found
(%): C: 60.80, H: 6.65, N: 9.13.
.
7). Anal. calcd for C23H27N3O3 HCl. Calcd (%): C: 64.25,
H: 6.56, N: 9.77. Found (%): C: 64.09, H: 6.51 , N: 9.94.
2-[6,9-Dihydro-9,9-dimethylpyrano[3,2-f]pyrano-[20,30:
2,3]pyridine[4,3,2-c,d]indazol-6-yl]-1-ethanol (13). 2-
Hydroxyethylhydrazine (515 mL, 7,6 mmol) was added
under argon to a solution of 7 (340 mg, 0.76 mmol) in
dry DMSO (5 mL) and the mixture was heated at 90 ꢀC
for 30 min. The reaction mixture was then poured into
ice-water (20 mL) and the precipitate was filtered,
washed with water (20 mL) and dried over CaCl2, to
give after recrystalyzation pure 13 (240 mg) (95%) as a
5-[(4-Methylphenyl)sulfonyl]oxy-2,2-dimethyl-2H,6H-
(10).
pyrano[30,20:6,7]benzopyrano[2,3-b]pyridin-6-one
This compound was prepared by a procedure analo-
gous to that of 7. Yield: 75%. Mp 203–205 ꢀC, (EtOAc–
1
n-pentane). H NMR (CDCl3, 400 MHz) d (ppm): 1.32
(s, 6H, 2ꢂgem CH3), 2.31(s, 3H, –SO C6H4CH3), 5.41
2
(d, J=10 Hz, 1H, H-3), 6.09 (d, J=10 Hz, 1H, H-4),
6.78 (s, 1H, H-12), 7.22 (d, J=8 Hz, 2H, H-50, H-30),
7.30 (m, 1H, H-8), 7.77 (d, J=8 Hz, 2H, H-60, H-20),
8.49 (m, 1H, H-7), 8.54 (m, 1H, H-9). 13C NMR
(CDCl3, 50 MHz) d (ppm): 21.36 (–SO2C6H4CH3),
27.90 (2ꢂgem CH3), 78.03 (C-2), 103.68 (C-12), 110.44
(C-5a), 113.97 (C-4a), 115.15 (C-4), 116.84 (C-6a),
120.85 (C-8), 128.49 (C-60, C-20), 129.45 (C-50, C-30),
131.03 (C-3), 132.53 (C-10), 136.98 (C-7), 142.42 (C-5),
145.18 (C-40), 153.00 (C-9), 156.79 (C-11a), 158.77 (C-
12a, C-10a), 173.84 (C-6). Anal. Calcd for C24H19NO6S.
calcd (%): C: 64.13, H: 4.26, N: 3.12. Found (%): C:
63.97, H: 4.31, N: 3.05.
pale yellow solid. Mp 188–190 ꢀC (CH2Cl2). H NMR
1
(DMSO-d6, 400 MHz) d (ppm): 1.41 (s, 6H, 2ꢂgem
CH3), 3.79 (q, J=5.5 Hz, 4 Hz, 2H, CH2CH2OH), 4.28
(t, J=5.5 Hz, 2H, CH2CH2OH), 4.87 (t, J=4 Hz, 1H,
D2O exch., OH), 5.82 (d, J=10 Hz, 1H, H-10), 6.53 (s,
1H, H-7), 6.65 (d, J=10 Hz, 1H, H-11), 7.34 (m, 1H, H-
3), 8.20 (m, 1H, H-4), 8.28 (m, 1H, H-2). 13C NMR
(DMSO-d6, 50 MHz) d (ppm): 27.82 (2ꢂgem CH3),
51.60 (CH2CH2OH), 61.75 (CH2CH2OH), 77.05 (C-9),
89.68 (C-7), 101.22 (C-11a), 111.51 (C-11c), 114.57 (C-
4a), 115.30 (C-11), 120.89 (C-3), 128.72 (C-10), 132.06

4596
G. Kolokythas et al. / Bioorg. Med. Chem. 11 (2003) 4591–4598
(C-4), 137.87 (C-4b), 140.11 (C-6a), 143.46 (C-11b),
147.72 (C-2), 156.95 (C-7a), 160.18 (C-12a). Anal. Calcd
for C19H17N3O3. calcd (%): C: 68.05, H: 5.11, N: 12.53.
Found (%): C: 67.88, H: 5.17, N: 12.39.
Diethyl-[2-[6,9-dihydro-9,9-dimethylpyrano[3,2-f]pyra-
no[20,30:2,3]pyridine[4,3,2 - c,d]indazol - 6 - yl]ethyl]amine
(16). This compound was prepared by a procedure ana-
logous to that of 15. Yield: 82%. Mp (hydrochloride)
>250 ꢀC, (EtOH). 1H NMR (CDCl3, 400 MHz) d
(ppm): 0.98 [t, J=7 Hz, 6H, N(CH2CH3)2], 1.42 (s, 6H,
2ꢂgem CH3), 2.56 [q, J=7 Hz, 4H, N(CH2CH3)2], 2.90
(t, J=7 Hz, 2H, CH2CH2NEt2), 4.24 (t, J=7 Hz, 2H,
CH2CH2NEt2), 5.63 (d, J=10 Hz, 1H, H-10), 6.29 (s,
1H, H-7), 6.76 (d, J=10 Hz, 1H, H-11), 7.13 (m, 1H, H-
3), 8.13 (m, 1H, H-4), 8.21 (m, 1H, H-2). 13C NMR
(CDCl3, 50 MHz) d (ppm): 11.83 [N(CH2CH3)2], 27.71
2-[6,9-Dihydro-9,9-dimethylpyrano[3,2-f]pyrano[20,30:2,3]
pyridine[4,3,2-c,d]indazol-6-yl]-1-ethanol methanesulfo-
nate (14). Triethylamine (232 mL, 1.67 mmol) and
methanesulfonyl chloride (128 mL, 1.64 mmol) were
added to a solution of 13 (210 mg, 0.63 mmol) in dry
CH2Cl2 (20 mL) at 0 ꢀC and the mixture was stirred for
10 min at 0 ꢀC and for an additional 1h at rt. Water (20
mL) was then added to the mixture and it was extracted
with dichloromethane. The organic phase was dried
(Na2SO4) and concentrated to dryness and the residue
was purified by column chromatography (silica gel,
12ꢂ1cm) using a mixture of cyclohexane/ethyl acetate
(1/9) as the eluent to give 14 (190 mg, 73%) as a yellow
solid. Mp 152–154 ꢀC, (AcOEt/n-hexane). 1H NMR
(CDCl3, 400 MHz) d (ppm): 1.40 (s, 6H, 2ꢂgem CH3),
2.78 (s, 3H, OSO2CH3), 4.46 (t, J=5 Hz, 2H,
CH2CH2OSO2CH3), 4.61(t, J=5 Hz, 2H, CH2CH2O-
SO2CH3), 5.64 (d, J=10 Hz, 1H, H-10), 6.31 (s, 1H, H-
7), 6.73 (d, J=10 Hz, 1H, H-11), 7.15 (m, 1H, H-3),
8.09 (m, 1H, H-4), 8.23 (m, 1H, H-2). 13C NMR
(CDCl3, 50 MHz) d (ppm): 27.71(2 ꢂgem CH3), 37.37
(2ꢂgem
CH3),
47.41[N(
CH2CH3)2],
48.14
(CH2CH2NEt2), 52.11 (CH2CH2NEt2), 76.89 (C-9),
89.83 (C-7), 101.00 (C-11a), 111.85 (C-11c), 115.00 (C-
4a), 115.41 (C-11), 120.85 (C-3), 128.53 (C-10), 131.92
(C-4), 137.50 (C-4b), 139.78 (C-6a), 143.57 (C-11b),
147.55 (C-2), 156.62 (C-7a), 160.33 (C-12a). Anal. calcd
.
for C23H26N4O2 HCl. Calcd (%): C: 64.70, H: 6.37, N:
13.12. Found (%): C: 64.61, H: 6.35, N: 13.07.
2 - [1,10 - Dihydro - 10,10 - dimethylpyrano[2,3 - g]pyrano-
[20,30:2,3]pyridine[4,3,2-c,d]indazol-1-yl]-1-ethanol (17).
This compound was prepared by a procedure analo-
gous to that of 13. Yield: 40%. Mp 183–185 ꢀC (dec),
(CH2Cl2). 1H NMR (CDCl3, 400 MHz) d (ppm): 1.41 (s,
6H, 2ꢂgem CH3), 4.12 (t, J=5 Hz, 2H, CH2CH2OH),
4.44 (t, J=5 Hz, 2H, CH2CH2OH), 5.47 (d, J=10 Hz,
1H, H-11), 6.12 (s, 1H, H-8), 6.67 (d, J=10 Hz, 1H, H-
12), 7.05 (m, 1H, H-4), 7.98 (m, 1H, H-3), 8.14 (m, 1H,
H-5). 13C NMR (CDCl3, 50 MHz) d (ppm): 27.12
(OSO2CH3),
48.37
(CH2CH2OSO2CH3),
67.33
(CH2CH2OSO2CH3), 77.08 (C-9), 89.79 (C-7), 101.41
(C-11a), 111.66 (C-11c), 114.46 (C-4a), 115.12 (C-11),
120.92 (C-3), 128.86 (C-10), 132.10 (C-4), 138.53 (C-4b),
140.37 (C-6a), 143.38 (C-11b), 148.02 (C-2), 157.17 (C-
7a), 160.26 (C-12a). Anal. calcd for C20H19N3O5S.
Calcd (%): C: 58.10, H: 4.63, N: 10.16. Found (%): C:
57.94, H: 4.57, N: 10.02.
(2ꢂgem
CH3),
53.29
(CH2CH2OH),
61.63
(CH2CH2OH), 76.34 (C-10), 95.23 (C-8), 99.35 (C-12a),
111.74 (C-12c), 113.87 (C-2b), 116.73 (C-12), 120.52 (C-
4), 126.33 (C-11), 131.88 (C-3), 136.66 (C-12b), 136.95
(C-2a), 147.50 (C-5), 148.09 (C-7a), 156.73 (C-8a),
159.56 (C-6a). Anal. calcd for C19H17N3O3. Calcd (%):
C: 68.05, H: 5.11, N: 12.53. Found (%): C: 68.14, H:
5.08, N: 12.41.
Dimethyl-[2-[6,9-dihydro-9,9-dimethylpyrano[3,2-f]pyrano-
[20,30:2,3]pyridine[4,3,2-c,d]indazol-6-yl]ethyl]amine (15).
A 33% solution of dimethylamine in dry ethanol (327
mL, 2.4 mmol) was added to a solution of 14 (100 mg,
0.24 mmol) in dry ethanol (20 mL) and the mixture was
stirred under reflux for 20 h. The solvent was then
vacuum-evaporated, water (20 mL) was added to the
residue and it was extracted with dichloromethane. The
organic phase was dried (Na2SO4) and concentrated to
dryness and the residue was purified by column chro-
matography (silica gel, 18ꢂ1cm) using a mixture of
ethyl acetate/methanol (95/5) as the eluent to give 15 (70
mg, 79%) as a pale yellow solid. Mp (hydrochloride)
>250 ꢀC, (EtOH). 1H NMR (CDCl3, 400 MHz) d
(ppm): 1.40 (s, 6H, 2ꢂgem CH3), 2.26 [s, 6H, N(CH3)2],
2.77 (t, J=7 Hz, 2H, CH2CH2NMe2), 4.25 (t, J=7 Hz,
2H, CH2CH2NMe2), 5.61(d, J=10 Hz, 1H, H-10), 6.27
(s, 1H, H-7), 6.74 (d, J=10 Hz, 1H, H-11), 7.11 (m, 1H,
H-3), 8.10 (m, 1H, H-4), 8.19 (m, 1H, H-2). 13C NMR
(CDCl3, 50 MHz) d (ppm): 27.71(2 ꢂgem CH3), 45.46
2 - [1,10 - Dihydro - 10,10 - dimethylpyrano[2,3 - g]pyrano-
[20,30:2,3]pyridine[4,3,2-c,d]indazol-1-yl]-1-ethanol methane-
sulfonate (18). This compound was prepared by a pro-
cedure analogous to that of 14. Yield: 79%. Mp 158–
160 ꢀC, (AcOEt/n-hexane). 1H NMR (CDCl3, 400 MHz)
d (ppm): 1.40 (s, 6H, 2ꢂgem CH3), 2.79 (s, 3H,
OSO2CH3), 4.62 (m, 4H, CH2CH2OSO2CH3), 5.50 (d,
J=10 Hz, 1H, H-11), 6.20 (s, 1H, H-8), 6.59 (d, J=10
Hz, 1H, H-12), 7.12 (m, 1H, H-4), 8.04 (m, 1H, H-3),
8.21(m, H1 , H-5).
13C NMR (CDCl3, 50 MHz) d
(ppm): 27.01(2 ꢂgem CH3), 37.23 (OSO2CH3), 49.76
(CH2CH2OSO2CH3), 67.37 (CH2CH2OSO2CH3), 76.37
(C-10), 95.60 (C-8), 99.27 (C-12a), 112.07 (C-12c),
113.87 (C-2b), 116.22 (C-12), 120.74 (C-4), 126.95 (C-
11), 131.99 (C-3), 136.84 (C-12b), 138.05 (C-2a), 148.16
(C-5), 148.31 (C-7a), 156.91 (C-8a), 159.92 (C-6a). Anal.
calcd for C20H19N3O5S. calcd (%): C: 58.10, H: 4.63, N:
10.16. Found (%): C: 57.94, H: 4.59, N: 10.09.
[N(CH3)2],
47.67
(CH2CH2NMe2),
58.14
(CH2CH2NMe2), 76.85 (C-9), 89.57 (C-7), 100.97 (C-
11a), 111.85 (C-11c), 114.97 (C-4a), 115.34 (C-11),
120.78 (C-3), 128.42 (C-10), 131.92 (C-4), 137.54 (C-4b),
139.63 (C-6a), 143.57 (C-11b), 147.54 (C-2), 156.65 (C-
Dimethyl-[2-[1,10-dihydro-10,10-dimethylpyrano[2,3-g]-
pyrano[20,30:2,3]pyridine[4,3,2 - c,d]indazol - 1 - yl]ethyl]-
amine (19). This compound was prepared by a proce-
dure analogous to that of 15. Yield: 89%. Mp
.
7a), 160.26 (C-12a). Anal. calcd for C21H22N4O2ꢃHCl 3/
2H2O. Calcd (%): C: 59.22, H: 6.15, N: 13.16. Found
(%): C: 59.08, H: 6.23, N: 13.17.

G. Kolokythas et al. / Bioorg. Med. Chem. 11 (2003) 4591–4598
4597
(hydrochloride) >250 ꢀC, (EtOH). H NMR (CDCl3,
400 MHz) d (ppm): 1.42 (s, 6H, 2ꢂgem CH3), 2.29 [s,
6H, N(CH3)2], 2.77 (t, J=7 Hz, 2H, CH2CH2NMe2),
4.46 (t, J=7 Hz, 2H, CH2CH2NMe2), 5.50 (d, J=10
Hz, 1H, H-11), 6.21 (s, 1H, H-8), 6.61 (d, J=10 Hz, 1H,
H-12), 7.11 (m, 1H, H-4), 8.11 (m, 1H, H-3), 8.21 (m,
1H, H-5). 13C NMR (CDCl3, 50 MHz) d (ppm): 27.13
(2ꢂgem CH3), 45.69 [N(CH3)2], 49.58 (CH2CH2NMe2),
58.84 (CH2CH2NMe2), 76.37 (C-10), 95.30 (C-8), 99.35
(C-12a), 112.47 (C-12c), 114.45 (C-2b), 116.55 (C-12),
120.70 (C-4), 126.80 (C-11), 131.98 (C-3), 136.25 (C-
12b), 137.05 (C-2a), 147.79 (C-5), 148.59 (C-7a), 156.68
optical density was measured with a microplate reader
at a wavelength of 550 nm (reference wavelength: 690
nm). Results were expressed as IC50, that is the concen-
tration that reduced by 50% the optical density of
treated cells with respect to untreated controls.
1
Cell-cycle analysis was performed after incubation of
exponentially growing HT-29 cells with the test com-
pounds (20 mM) for 24 h. Treated cultures were then
trypsinized, washed in PBS, fixed in 50% ethanol, and
stained with an RNAse-containing propidium iodide
solution (50 mg/mL). DNA content was analyzed on a
FACS Calibur (Becton Dickinson, San Jose, CA, USA)
flow cytometer using the ModFit software.
(C-8a),
160.06
.
(C-6a).
Anal.
calcd
for
.
C21H22N4O2 HCl H2O. calcd (%): C: 60.50, H: 6.04, N:
13.44. Found (%): C: 60.22, H: 5.87, N: 13.30.
This study was supported by a Research Grant provided
by the Greek Ministry of Education (Business Program
of Education and Initial Professional Preparing-
EPEAEK).
Diethyl-[2-[1,10-dihydro-10,10-dimethylpyrano[2,3-g]pyr-
ano[20,30:2,3]pyridine[4,3,2 - c,d]indazol - 1 - yl]ethyl]amine
(20). This compound was prepared by a procedure ana-
logous to that of 15. Yield: 92%. Mp (hydrochloride)
>250 ꢀC, (EtOH). 1H NMR (CDCl3, 400 MHz) d
(ppm): 0.98 [t, J=7 Hz, 6H, N(CH2CH3)2], 1.43 (s, 6H,
2ꢂgem CH3), 2.56 [q, J=7 Hz, 4H, N(CH2CH3)2], 2.87
(t, J=7 Hz, 2H, CH2CH2NEt2), 4.43 (t, J=7 Hz, 2H,
CH2CH2NEt2), 5.50 (d, J=10 Hz, 1H, H-11), 6.22 (s,
1H, H-8), 6.67 (d, J=10 Hz, 1H, H-12), 7.13 (m, 1H, H-
4), 8.13 (m, 1H, H-3), 8.22 (m, 1H, H-5). 13C NMR
(CDCl3, 50 MHz) d (ppm): 11.80 [N(CH2CH3)2], 27.09
References and Notes
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Wang, C. C.; Wang, S. Y. Biochem. Biophys. Res. Commun.
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2. (a) Umezoto, M.; Kawasaki, H.; Azuma, E.; Komada, Y.;
Ito, M.; Sakurai, M. Am. J. Hematol. 1996, 52, 327. (b) Ben-
eki, M.; Kars, A.; Guter, N. Ann. Intern. Med. 1997, 126, 409.
3. (a) Showalter, H. D.; Angelo, M. M.; Berman, E. M.;
Kanter, G. D.; Ortwine, D. F.; Ross-Kesten, S. G.; Sercel,
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(2ꢂgem
CH3),
47.49
[N(CH2CH3)2],
50.28
(CH2CH2NEt2), 52.78 (CH2CH2NEt2), 76.34 (C-10),
95.19 (C-8), 99.46 (C-12a), 112.47 (C-12c), 114.52 (C-
2b), 116.80 (C-12), 120.66 (C-4), 126.54 (C-11), 131.91
(C-3), 136.39 (C-12b), 136.98 (C-2a), 147.75 (C-5),
148.59 (C-7a), 156.57 (C-8a), 160.10 (C-6a). Anal. calcd
.
for C23H26N4O2 HCl. calcd (%): C: 64.70, H: 6.37, N:
13.12. Found (%): C: 64.91, H: 6.33, N: 13.07.
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Biological activity
The L1210 murine lymphoblastic leukemia cell line
(strain: DBA subline 212) was obtained from ATCC
(American Type Culture Collection, Rockville, MD,
USA) and routinely cultured in RPMI 1640 supple-
mented with penicillin (100 U/mL), streptomycin (100
mg/mL) and 10% Fetal Bovine Serum (media and anti-
biotics from Biochrom KG, Berlin, Germany). Human
colorectal adenocarcinoma HT-29 cells obtained from
ECACC (European Collection of Cell Cultures, Salis-
bury, UK) were routinely cultured in DMEM supple-
mented with antibiotics and serum, as above, and
subcultured using a trypsin 0.25%–EDTA 0.02% solution.
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