Synthesis and antitumour activity of 4-hydroxy-2-pyridone derivatives

Article abstract of DOI:10.1016/S0223-5234(00)00149-5

4-hydroxy-2-pyridone derivatives 2 were prepared by reaction of 3-amino- 3-dialkylaminopropenoates with bis(2,4,6-trichlorophenyl)malonate. These compounds were further reacted with a set of aldehydes to give bis(pyridyl)methanes 3 and 4. The newly synthesized compounds 2, 3 and 4 were evaluated in vitro as antitumour agents against 60 human tumour cell lines. Some derivatives exhibit tumour growth inhibition activity. In particular, derivative 4g, the most active of the series, possesses significant activity on all cell lines at concentrations ranging from 1 x 10^-6 to 1 X 10^-5 M. (C) 2000 Editions scientifiques et medicales Elsevier SAS.

Full text of DOI:10.1016/S0223-5234(00)00149-5

Eur. J. Med. Chem. 35 (2000) 545−552
© 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
S0223523400001495/SCO
545
Laboratory note
Synthesis and antitumour activity of 4-hydroxy-2-pyridone derivatives
Maria T. Cocco*, Cenzo Congiu, Valentina Onnis
Dipartimento di Tossicologia, Università di Cagliari, Via Ospedale 72 - 09124 Cagliari, Italy
Received 3 August 1999; revised 3 November 1999; accepted 9 December 1999
Abstract – 4-hydroxy-2-pyridone derivatives 2 were prepared by reaction of 3-amino-3-dialkylaminopropenoates with bis(2,4,6-
trichlorophenyl)malonate. These compounds were further reacted with a set of aldehydes to give bis(pyridyl)methanes 3 and 4. The newly
synthesized compounds 2, 3 and 4 were evaluated in vitro as antitumour agents against 60 human tumour cell lines. Some derivatives exhibit
tumour growth inhibition activity. In particular, derivative 4g, the most active of the series, possesses significant activity on all cell lines at
concentrations ranging from 1 × 10–6 to 1 × 10–5 M. © 2000 Éditions scientifiques et médicales Elsevier SAS
4-hydroxy-2-pyridone derivatives / bis(pyridyl)methanes / antitumour activity
1. Introduction
pyridone derivatives and a list of these was submitted to
NCI. We report here the synthesis, the physical-chemical
properties of the new derivatives and the results of
primary antitumour screening of compounds selected by
NCI.
The ongoing efforts of research on treatment of malig-
nancy are focused on the discovery of novel products that
enable us to modulate the pharmacological activity of
drugs used currently and to decrease the toxicity of
cancer chemotherapy. On the other hand, novel ap-
proaches to cancer chemotherapy are represented by
inhibitors of angiogenesis and inhibitors of signal trans-
duction pathways upon which cancer cells selectively
depend. At the same time random screening continues to
be one of the main routes to discover new leads with
antineoplastic activity and the National Cancer Institute
(NCI), Bethesda, USA, has played a pivotal role in this
field. NCI screening program [1] started with the selec-
tion of defined chemical structures. This selection was
based on the uniqueness of the chemical structures.
Obviously, new chemical classes of agents that have not
had extensive clinical evaluation are prime targets.
Among the wide variety of heterocycles that have been
explored for developing pharmaceutically important mol-
ecules, pyridin-2(1H)-ones have received attention as
antimicrobial and antiviral agents [2–5]. On this basis and
in pursuing the interest in the study on novel compounds
bearing potentially anticancer and antiviral activity, our
research group has became interested in 4-hydroxy-2-
2. Chemistry
The 4-hydroxy-6-oxopyridines 2 and their bis(pyridyl)-
methane derivatives 3 and 4 were prepared by the
sequence of reactions shown in figure 1. The key starting
materials, ethyl 3,3-diaminopropenoates 1, were avail-
able from ethyl cyanoacetate as previously described [6].
The aminopropenoates 1 were converted to pyridones 2
by heating with bis(2,4,6-trichlorophenyl)malonate by
slight adjustment of the Kappe procedure [7, 8]. We
found that better yields were obtained when the reaction
was performed in toluene solution at about 95 °C for 2 h.
Utilizing more vigorous reaction conditions afforded a
complex mixture of compounds. Finally, the preparation
of bis(pyridyl)methanes 3 and 4 was achieved in good
yields by treatment of two equivalents of pyridones 2
with the appropriate aldehydes in ethanol using piperi-
dine as catalyst.
The reaction was initiated by condensation of com-
pound 2 with aldehyde to give intermediate A followed
by addition of another molecule of 2. With the aim of
* Correspondence and reprints: tcocco@vaxca1.unica.it

546
fact compounds 3 and 4 were exclusively obtained in
poor yields and unreacted aldehyde was recovered.
Structures of new compounds 2, 3 and 4 were assigned
by analytical and spectral data. For compounds 2, the IR
spectra are characterised by a C=O stretching absorption
band shifted at 1 620–1 670 cm–1, probably due to for-
1
mation of an intramolecular hydrogen bond. The H-
NMR spectra of pyridones 2, registered in DMSO-d6,
exhibit a deuterium oxide exchangeable broad singlet
corresponding to OH and NH protons in the region
10.26–10.90 ppm and a signal at 5 ppm assigned to the
H-5 proton. The presence of these signals indicates that
compounds
2
exist exclusively as 4-hydroxy-
2(1H)pyridone tautomers. For compounds 3 and 4 the
1H-NMR spectra show a singlet in the range 5.5–5.8 ppm
due to H-1 and a downfield resonating D2O exchangeable
broad signal integrating for four protons.
3. Pharmacology
Evaluation of anticancer activity on 15 of the 18
synthesized compounds 2, 3 and 4 was performed at the
NCI following the known in vitro disease-oriented anti-
tumour screening program, which is based upon use of
multiple panels of 60 human tumour cell lines [1, 9].
Each compound is tested at a minimum of five concen-
trations at 10-fold dilution against every cell line in the
panel. A 48 h continuous drug exposure protocol is used
and a sulforhodamine B (SRB) protein assay is used to
estimate cell viability or growth [10]. The anticancer
activity of each compound is deduced from dose–re-
sponse curves and is presented in three different tables
according to the data provided by NCI [11].
The response parameters GI50, TGI and LC50 (ta-
bles I–III) refer to the drug concentration that produced
50% inhibition, total growth inhibition and 50% cytotox-
icity, respectively, and are expressed in micromolar
concentrations (µM). In the tables we report only the
activity of those compounds which exhibited GI50, TGI
and LC50 less than 100 µM.
Figure 1. Conditions: a) bis(2,4,6-trichlorophenyl)malonate,
toluene, 95 °C; b) ethanol, piperidine, reflux.
isolating this intermediate the reaction was performed
without catalyst utilizing equimolar amounts of the two
reagents. However, this approach was unsuccessful, in
Table I. GI50 values (µM) of compounds 2, 3 and 4 against different tumour cell lines.
Panel/cell line
Compound
2b
3a
4a
3d
3e
4e
3f
4f
4g
3h
4h
3i
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
–
–
–
–
–
–
n.t.
–
–
–
–
–
–
–
–
–
n.t.
–
–
–
–
n.t.
–
–
–
54.2
n.t.
–
–
46.6
23.6
n.t.
63.2
46.5
42.8
4.76
n.t.
30.4
25.2
26.7
1.59
1.96
1.68
1.87
1.98
–
–
–
–
–
–
–
–
–
78.2
n.t.
–
–
54.6
84.3

547
Panel/cell line
Compound
2b
3a
4a
3d
3e
4e
3f
4f
4g
3h
4h
3i
SR
–
37.5
–
88.9
–
–
40.2
36.1
2.83
–
–
–
Non-small cell lung
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
–
–
–
–
–
–
–
n.t.
–
–
–
–
–
60.3
80.6
–
–
–
–
–
–
–
–
–
–
–
95.6
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
17.5
18.6
15.0
1.15
10.4
6.86
18.1
20.3
1.83
–
–
50.6
–
–
–
–
–
–
–
–
–
–
–
–
–
–
44.7
46.6
12.3
–
82.0
81.5
–
33.6
27.4
18.5
63.6
22.0
–
–
–
81.0
–
–
–
–
–
94.3
28.7
–
71.1
–
34.9
23.9
–
25.8
–
–
43.5
–
24.5
–
28.0
–
84.8
–
–
–
–
–
–
–
–
52.8
–
–
–
–
–
–
–
–
–
–
–
–
–
–
n.t.
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
34.1
–
n.t.
–
–
–
20.3
–
20.9
–
3.55
18.2
1.68
3.40
3.56
14.5
1.71
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
HT29
KM12
SW-620
–
–
33.2
23.5
CNS cancer
SF-268
SF-295
SF-539
SNB-19
–
–
–
–
n.t.
–
72.4
–
–
70.6
25.3
37.9
–
58.4
–
83.6
22.4
–
49.5
25.7
–
–
21.3
48.9
–
–
–
–
48.5
–
–
–
–
–
–
–
23.0
37.6
19.2
15.5
9.78
14.9
44.1
–
26.5
26.5
41.8
19.6
6.09
16.5
1.57
3.84
3.42
4.72
85.4
40.3
–
38.7
23.5
73.7
–
–
–
–
–
–
–
–
–
–
–
-
SNB-75
U251
Melanoma
LOX IMVI
MALME-3M
M14
–
–
–
–
–
–
–
–
–
–
–
n.t.
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
5.00
–
–
–
–
–
–
–
n.t.
–
–
–
–
59.0
–
–
–
–
–
–
–
19.3
38.9
20.8
21.3
35.3
37.8
41.5
26.8
19.5
41.4
20.7
n.t.
42.6
24.3
70.0
40.1
2.37
2.00
1.74
4.75
1.69
4.07
5.13
4.88
37.4
–
–
–
–
82.5
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
786-0
–
–
–
–
–
–
–
n.t.
–
–
33.2
–
–
n.t.
–
–
73.8
–
86.5
n.t.
–
–
52.7
86.1
–
n.t.
–
–
–
–
n.t.
–
–
–
–
n.t.
29.9
–
30.2
15.5
85.6
n.t.
17.2
42.8
–
18.4
–
4.09
2.48
6.76
13.1
6.31
7.80
n.t.
–
–
68.0
92.9
–
n.t.
–
–
–
–
n.t.
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
61.8
–
–
47.9
43.0
–
42.4
–
60.1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
94.3
–
–
–
–
–
–
–
–
–
27.2
33.1
34.2
24.4
31.7
36.2
19.2
24.6
28.1
–
55.1
82.5
–
20.5
39.9
34.0
1.81
7.57
2.97
1.84
2.06
1.58
3.38
1.88
29.3
–
95.5
–
54.4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
A498
ACHN
CAKI-1
RXF 393
SN12C
56.0
28.5
57.3
–
42.7
–
TK-10
UO-31
–
Prostate cancer
PC-3
DU-145
–
–
33.5
–
–
–
–
–
–
–
–
–
28.0
60.8
14.2
22.9
14.1
9.96
–
–
–
–
–
–

548
Panel/cell line
Compound
2b
3a
4a
3d
3e
4e
3f
4f
4g
3h
4h
3i
Breast cancer
MCF7/ADR-RES
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
MDA-N
BT-549
–
–
–
21.8
42.0
–
–
32.3
–
–
–
–
47.9
–
–
–
–
–
–
20.8
–
–
–
76.0
–
–
–
82.3
–
–
–
30.4
–
–
–
–
–
–
–
–
–
28.6
28.7
22.0
6.54
29.5
24.5
16.3
28.1
22.0
39.0
15.2
–
30.6
26.1
24.5
19.7
2.07
16.6
11.2
3.47
2.53
1.94
4.95
3.56
–
–
–
27.9
–
–
–
19.4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
63.0
T-47D
n.t.: means not tested
(–) values > 100 µM
Table II. TGI values (µM) of compounds 3 and 4 against different tumour cell lines.
Panel/cell line
Compound
3f 4f
Panel/cell line
Compound
3f 4f
3a
4a
3d
4g
3h
3a
4a
3d
4g
3h
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
Non-small cell lung
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS cancer
SF-268
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
PC-3
DU-145
–
–
–
–
–
–
–
–
–
–
53.9 45.6 n.t. 16.2
–
–
–
–
–
–
n.t.
–
–
–
–
–
–
–
–
–
–
79.3 40.4 3.50
–
–
–
–
–
–
–
–
–
–
–
–
–
86.0
–
3.37
n.t. n.t. n.t. 4.37
74.5 15.6
–
–
–
–
–
–
–
–
–
–
–
3.51
17.7
16.6
57.8 5.23
–
–
4.87
8.22
–
–
–
–
–
–
–
n.t. n.t. n.t. n.t. 15.0 n.t.
–
–
–
–
–
–
–
–
–
–
–
–
–
35.5 8.07
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
31.2
32.6
29.8
–
–
–
–
–
–
–
–
–
–
–
19.6
25.7
33.5 37.0 18.8
–
58.6 58.3 69.4 4.51
–
20.8
–
–
–
–
–
–
–
22.5
48.9 98.8 20.7
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
3.20
19.7
8.32
4.27
4.36
–
–
–
–
–
–
–
–
–
–
–
–
32.0
41.2
74.1 55.2 3.72
71.1 60.6
–
–
–
–
99.6
–
59.2 –
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
14.1
32.3
–
–
–
–
–
–
–
51.0 4.54
11.3
63.6 3.21
12.3
66.6 95.5 3.73
–
n.t.. n.t. 42.1 12.9
–
–
–
–
–
–
–
–
83.9 50.1 27.6
–
–
–
–
–
27.6
–
55.8 21.5
69.6 3.86
58.8 3.08
Breast cancer
MCF7/ADR-RES
NCI/ADR-RES
MDA-MB-231/
ATCC
HS 578T
MDA-MB-435
MDA-N
–
–
64.3
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
55.2
–
–
–
19.0
30.4
–
–
–
–
73.4
–
31.5
SF-295
SF-539
SNB-19
SNB-75
88.1 50.5 41.9 23.4
41.6 59.7 3.98
49.9 79.4 16.2
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
15.9
7.49
–
–
–
–
–
96.5 69.7 69.0 34.4
–
–
9.48 60.6
79.6 70.1 3.73
51.8 75.1 15.7
–
U251
–
–
36.9 41.9 15.6
–
BT-549
T-47D
Melanoma
LOX IMVI
MALME-3M
M14
48.8 9.27
–
–
–
–
–
–
–
–
–
49.4 48.1 6.29
–
–
–
84.6
–
5.95
42.7 58.5 3.18
n.t. means not tested
(–) values > 100 µM

549
Table III. LC50 values (µM) of compounds 3f, 4f and 4g against different tumour cell lines.
Panel/cell line
Compound
4f
Panel/cell line
Compound
4f
3f
4g
3f
4g
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
SR
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
PC-3
DU-145
–
–
–
–
–
–
–
–
9.24
15.8
37.3
7.39
–
n.t.
–
–
–
–
n.t.
–
–
–
7.73
9.73
7.31
–
–
–
–
–
–
–
54.3
46.4
–
–
Non-small cell lung
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
Breast cancer
MCF7/ADR-RES
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
MDA-N
BT-549
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
55.9
57.1
55.6
22.6
48.8
50.1
56.5
83.4
7.58
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
7.19
59.7
48.9
63.9
26.5
7.15
39.6
50.9
T-47D
n.t means not tested
(–) values > 100 µM
–
–
–
–
n.t.
–
–
–
–
–
–
84.8
–
–
37.5
57.2
6.13
57.9
36.0
52.6
5.55
4. Results and discussion
From the biological data reported in tables I–III, it can
be observed that several compounds show an interesting
activity level often associated with high or moderately
different cellular sensitivity and/or with a significantly
different subpanel sensitivity. In particular compound 4g
was the most active in the series with a significant
inhibitory activity on all the 60 cell lines at 1.57–20.3 µM
concentrations and cytotoxic activity on 57 cell lines at
the 5.56–57.9 µM concentrations. Compound 4f exhib-
ited similar inhibitory activity although over a smaller
range (43 over 60), but show cytotoxicity on four cell
lines only. Compounds 3a, 3d and 3f show significant
sensitivity, with high values of inhibition, on 11 subpanel
cell lines (SR, HOP-62, HOP-92, NCI-H23, SF-268,
SNB-75, OVOCAR-5, CAKI-1, TK-10, MDA-MB-231/
ATCC, BT-549). The limited number of compounds
tested and the lack of many reference compounds does
not allow us to establish structure–activity relationships.
However, it seems that although the examined com-
pounds have closely related structures, the results ob-
tained from the NCI screen showed clear differences in
activity. The 4-hydroxypyridones 2 were devoid of activ-
ity. At the present stage we may infer that, in bis(pyridyl)
methanes 3 and 4, replacement of isopropyl moiety on the
methyne bridge with a phenyl ring resulted in improved
cytostatic activity. As regards to the influence of the
substituents on the phenyl ring we can observe that the
presence of electron-withdrawing substituents favourably
–
–
90.2
–
–
–
–
–
–
–
89.5
44.3
56.1
10.2
40.2
30.7
40.0
U251
91.7
Melanoma
LOX IMVI
MALME-3M
M14
–
–
87.4
–
–
–
–
–
–
–
–
n.t.
–
–
–
–
26.3
22.1
5.83
40.2
6.69
39.5
44.3
40.7
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
786-0
n.t.
–
–
–
72.1
–
n.t.
73.3
–
–
74.5
–
51.8
28.2
44.3
50.7
44.9
46.0
–
–
–
–
–
–
–
–
5.66
44.4
30.6
9.91
A498
ACHN
CAKI-1

550
affects the antitumour activity. Furthermore, the replace-
ment of the phenyl ring with a furane or thiophene leads
to a drop off in activity.
Due to its good activity and because, in addition, it lies
outside the category of adequately studied classes of
antitumour agents, compound 4g has been selected by
NCI Biological Evaluation Committee for further testing
in an in vivo Hollow Fibre Assay [12].
5.2. Synthesis of derivatives 3 and 4
A mixture of 4-hydroxypyridone 2 (0.0025 mol) and
aldehyde (0.00125 mol) in ethanol (20 mL) was added a
few drops of piperidine and heated at reflux for 4–17 h.
The formed precipitate was filtered off and washed with
diethyl ether.
5.2.1. 1,1-[Bis(3-(ethoxycarbonyl)-
4-hydroxy-2-pyrrolidino-1,6-dihydro-6-
oxopyridin-5-yl)]-2-methylpropane 3a
5. Experimental protocols
Prepared in 73% yield starting from compound 2a and
isobutyraldehyde, refluxing for 6 h. M.p. 260 °C dec.
Calcd. for C28H38N4O8 (558.63). % C 60.20; % H 6.86; %
N 10.03. Found: % C 60.27; % H 6.85; % N 10.07. IR
(Nujol): 3 240, 3 100, 2 620, 1 705, 1 685 cm–1.
5.1. Chemistry
Melting points were determined on a Kofler hot stage
and are uncorrected. IR spectra were recorded on Nujol
mulls between salt plates in a Perkin-Elmer 398 spectro-
1
photometer. H-NMR spectra were recorded on a Varian
5.2.2. 1-[Bis(3-(ethoxycarbonyl)-
Unity 300 spectrometer. It was not possible to register the
1H-NMR spectra of some compounds since they are
insoluble in all common deuterated solvents. Elemental
analyses were carried out with a Carlo Erba Model 1106
Elemental Analyzer. Ethyl 3-amino-3-pyrrolidino-2-
propenoate and ethyl 3-amino-3-morpholino-2-propenoate
were synthesized as previously described [6].
4-hydroxy-2-pyrrolidino-1,6-dihydro-6-
oxopyridin-5-yl)methyl]benzene 3b
Prepared in quantitative yield starting from compound
2a and benzaldehyde, refluxing for 4 h. M.p. 263 °C dec.
Calcd. for C31H36N4O8 (592.65). % C 62.83; % H 6.12; %
N 9.45. Found: % C 62.90; % H 6.13; % N 9.48. IR
(Nujol): 3 220, 3 100, 2 600, 1 710, 1 570 cm–1. 1H-NMR
(DMSO-d6) d: 1.18 (m, 6H, CH3), 1.84, 3.25 (m, 16H,
pyrrolidinyl), 4.12 (m, 4H, CH2), 5.63 (s, 1H, CH), 7.13
(m, 5H, Ar), 10.47, 12.40 (br s, 4H, D2O exchangeable).
5.1.1. 4-Hydroxy-2-pyrrolidino-1,6-dihydro-
6-oxopyridine-3-carboxylic acid ethyl ester 2a
Amixture of ethyl 3-amino-3-pyrrolidino-2-propenoate
(3.68 g, 0.02 mol) and bis (2,4,6-trichlorophenyl) mal-
onate (9.26 g, 0.02 mol) in toluene (20 mL) was heated in
water bath for 2 h. After concentration in vacuo, the
residue was treated with diethyl ether, filtered off and
recrystallized. Yield 98%. M.p. 185–187 °C (from
2-propanol). Calcd. For C12H16N2O4 (252.27); % C
57.13; % H 6.39; % N 11.11. Found: % C 57.09; % H
6.40; % N 11.08. IR (Nujol): 3 100, 1 660, 1 620,
1 560 cm–1; 1H-NMR (DMSO-d6) d: 1.19 (t, J = 6.8 Hz,
3H, CH3), 1.77, 3.31 (m, 8H, pyrrolidinyl), 4.14 (q, J =
6.8 Hz, 2H, CH2), 5.15 (s, 1H, H-5), 10.26 (br s, 2H, NH
and OH).
5.2.3. 1-[Bis(3-(ethoxycarbonyl)-
4-hydroxy-2-pyrrolidino-1,6-dihydro-6-
oxopyridin-5-yl)methyl]-4-methoxybenzene 3d
Prepared in 84% yield starting from compound 2a and
4-methoxybenzaldehyde, refluxing for 4 h. M.p.
213–215 °C. Calcd. for C32H38N4O9 (622.68). % C
61.73; % H 6.15; % N 9.00. Found: % C 61.68; % H
6.14; % N 9.05. IR (Nujol): 3 180, 3 050, 2 640, 1 730,
1 620 cm–1.
5.2.4. 4-[Bis(3-(ethoxycarbonyl)-
4-hydroxy-2-pyrrolidino-1,6-dihydro-6-
oxopyridin-5-yl)methyl]-N, N-dimethylbenzenamine 3e
Prepared in 66% yield starting from compound 2a and
4-(dimethylamino)benzaldehyde, refluxing for 4 h. M.p.
262 °C dec. Calcd. for C33H41N5O8 (635.72). % C
62.35; % H 6.50; % N 11.02. Found: % C 62.40; % H
6.51; % N 11.06. IR (Nujol): 3 280, 3 080, 2 600, 1 700,
1 580 cm–1.
5.1.2. 4-Hydroxy-2-morpholino-1,6-dihydro-
6-oxopyridine-3-carboxylic acid ethyl ester 2b
Compound 2b was prepared following the same pro-
cedure reported for 2a, starting from ethyl 3-amino-3-
morpholino-2-propenoate. Yield 96%. M.p. 196–198 °C
(from acetonitrile). Calcd. for C12H16N2O5 (286.26). % C
57.72; % H 6.01;% N 10.44. Found: % C 57.64; % H
6.02; % N 10.40. IR (Nujol): 1 670, 1 640, 1 575 cm–1;
1H-NMR (DMSO-d6) d: 1.22 (t, J = 7.1 Hz, 3H, CH3),
3.15, 3.56 (m, 8H, morpholinyl), 4.18 (q, J = 7.1 Hz, 2H,
CH2), 5.41 (s, 1H, H-5), 10.63, 10.90 (br s, 2H, NH and
OH).
5.2.5. 1-[Bis(3-(ethoxycarbonyl)-
4-hydroxy-2-pyrrolidino-1,6-dihydro-6-
oxopyridin-5-yl)methyl]-4-chloro-3-nitrobenzene 3f
Prepared in 75% yield starting from compound 2a and
4-chloro-3-nitrobenzaldehyde, refluxing for 4 h. M.p.

551
233 °C dec. Calcd. for C31H34ClN5O10 (672.09). % C
55.40; % H 5.10; % N 10.42. Found: % C 55.46; % H
5.08; % N 10.46. IR (Nujol): 3 330, 1 730, 1 710,
1 590 cm–1.
5.2.10. 1-[Bis(3-(ethoxycarbonyl)-
4-hydroxy-2-morpholino-1,6-
dihydro-6-oxopyridin-5-yl)methyl]benzene 4b
Prepared in quantitative yield starting from compound
2b and benzaldehyde, refluxing for 4 h. M.p. 248 °C dec.
Calcd. for C31H36N4O10 (624.65). % C 59.61; % H
5.81; % N 8.97. Found: % C 59.54; % H 5.82; % N 9.01.
IR (Nujol): 3 100, 1 715, 1 590 cm–1. 1H-NMR (DMSO-
d6) d: 1.21 (t, J = 6.8 Hz, 6H, CH3), 3.13, 3.59 (m, 16H,
morpholinyl), 4.16 (q, J = 6.8 Hz, 4H, CH2), 5.82 (s, 1H,
CH), 7.01–7.22 (m, 5H, Ar), 11.50, 12.27 (br s, 4H, D2O
exchangeable).
5.2.6. 1-[Bis(3-(ethoxycarbonyl)-
2-pyrrolidino-1,6-dihydro-6-oxopyridin-
5-yl)methyl]-2,6-dichlorobenzene 3g
Prepared in 52% yield starting from compound 2a and
2,6-dichlorobenzaldehyde, refluxing for 8 h. M.p. 290 °C
dec. Calcd. for C30H34Cl2N4O8 (649.53). % C 55.48; %
H 5.28; % N 8.63. Found: % C 55.43; % H 5.30; % N
8.69. IR (Nujol): 3 400, 3 080, 2 730, 2 640, 1 650,
1 630 cm–1.
5.2.11. 1-[Bis(3-(ethoxycarbonyl)-
4-hydroxy-2-morpholino-1,6-
dihydro-6-oxopyridin-5-yl)methyl]-4-methylbenzene 4c
5.2.7. 2-[Bis(3-(ethoxycarbonyl)-
4-hydroxy-2-pyrrolidino-1,6-
dihydro-6-oxopyridin-5-yl)methyl]furane 3h
Prepared in quantitative yield starting from compound
2b and 4-methylbenzaldehyde, refluxing for 4 h. M.p.
247 °C dec. Calcd. for C32H38N4O10 (637.67). % C
60.18; % H 6.00; % N 8.77. Found: % C 60.25; % H
6.01; % N 8.73. IR (Nujol): 3 100, 1 590 cm–1.
Prepared in quantitative yield starting from compound
2a and furane-2-carboxyaldehyde, refluxing for 4 h. M.p.
252 °C dec. Calcd. for C29H34N4O9 (582.61). % C
59.79; % H 5.88; % N 9.62. Found: % C 59.84; % H
5.90; % N 9.57. IR (Nujol): 2 650, 1 715, 1 590 cm–1.
1H-NMR (DMSO-d6) d: 1.19 (t, J = 6.8 Hz, 6H, CH3),
1.82, 3.26 (m, 16H, pyrrolidinyl), 4.13 (q, J = 6.8 Hz, 4H,
CH2), 5.54 (s, 1H, CH), 5.90, 6.29, 7.38 (s, 3H, furyl),
10.44, 12.43 (br s, 4H, D2O exchangeable).
5.2.12. 1-[Bis(3-(ethoxycarbonyl)-
4-hydroxy-2-morpholino-1,6-
dihydro-6-oxopyridin-5-yl)methyl]-4-methoxybenzene 4d
Prepared in 86% yield starting from compound 2b and
4-methoxybenzaldehyde, refluxing for 4 h. M.p. 238 °C
dec. Calcd. for C32H38N4O11 (654.67). % C 58.71; % H
5.85; % N 8.56. Found: % C 58.78; % H 5.84; % N 8.59.
IR (Nujol): 3 140, 2 560, 1 720, 1 600 cm–1.
5.2.8. 2-[Bis(3-(ethoxycarbonyl)-
4-hydroxy-2-pyrrolidino-1,6-
dihydro-6-oxopyridin-5-yl)methyl]thiophene 3i
5.2.13. 4-[Bis(3-(ethoxycarbonyl)-
2-morpholino-1,6-dihydro-6-oxopyridin-
5-yl)methyl]-N, N-dimethylbenzenamine 4e
Prepared in 52% yield starting from compound 2b and
4-(dimethylamino)benzaldehyde, refluxing for 4 h. M.p.
246 °C dec. Calcd. for C33H41N5O10 (667.72). % C
59.36; % H 6.19; % N 10.49. Found: % C 59.30; % H
6.18; % N 10.53. IR (Nujol): 3 220, 3 120, 2 500, 1 715,
1 580 cm–1.
Prepared in quantitative yield starting from compound
2a and thiophene-2-carboxyaldehyde, refluxing for 4 h.
M.p. 240 °C dec. Calcd. for C29H34N4O8S (598.67). % C
58.18; % H 5.72; % N 9.36. Found: % C 58.12; % H
5.73; % N 9.40. IR (Nujol): 3 080, 1 700, 1 580 cm–1.
5.2.9. 1,1-[Bis(3-(ethoxycarbonyl)-
4-hydroxy-2-morpholino-1,6-
dihydro-6-oxopyridin-5-yl)]-2-methylpropane 4a
Prepared in 40% yield starting from compound 2b and
isobutyraldehyde, refluxing for 17 h. M.p. 240 °C dec.
Calcd. for C28H38N4O10 (590.63). % C 56.94; % H
6.48; % N 9.49. Found: % C 57.00; % H 6.47; % N 9.52.
IR (Nujol): 3 200, 3 080, 2 600, 1 720, 1 680, 1 615 cm–1.
1H-NMR (DMSO-d6) d: 0.73 (d, J = 5.9 Hz, 6H, CH3),
1.18 (t, J = 6.8 Hz, 6H, CH3), 2.98, 3.09, 3.53 (m, 17H,
morpholinyl and CH), 3.71 (m, 1H, H-1), 4.14 (q, J = 6.8
Hz, 4H, CH2), 11.52, 12.98 (br s, 4H, D2O exchange-
able).
5.2.14. 1-[Bis(3-(ethoxycarbonyl)-
4-hydroxy-2-morpholino-1,6-dihydro-
6-oxopyridin-5-yl)methyl]-4-chloro-3-nitrobenzene 4f
Prepared in 75% yield starting from compound 2b and
4-chloro-3-nitrobenzaldehyde, refluxing for 4 h. M.p.
245 °C dec. Calcd. For C31H34ClN5O12 (704.09). % C
52.88; % H 4.87; % N 9.95. Found: % C 52.93; % H
4.88; % N 9.90. IR (Nujol): 3 250, 3 160, 2 540, 1 700,
1 630, 1 590 cm–1.

552
5.2.15. 1-[Bis(3-(ethoxycarbonyl)-
4-hydroxy-2-morpholino-1,6-dihydro-
inoculum depends on the type of tumour cell and from its
growth characteristics [13]. These cells are then pre-
incubated on the microtitre plate for 24 h before adding
the compounds. These were tested in DMSO solution at
five different concentrations (10–4, 10–5, 10–6, 10–7 and
10–8 M). After an incubation of the chemical agent for
48 h with the tumour cell lines a sulforhodamine B (SRB)
protein assay was used to estimate cell viability or
growth. The cytotoxic effects are evaluated and the assay
results and dose response parameters were calculated as
previously described [11].
6-oxopyridin-5-yl)methyl]-2,6-dichlorobenzene 4g
Prepared in 60% yield starting from compound 2b and
2,6-dichlorobenzaldehyde, refluxing for 6 h. M.p. 260 °C
dec. Calcd. for C30H34Cl2N4O10 (681.53). % C 52.87; %
H 5.03; % N 8.22. Found: % C 52.95; % H 5.02; % N
8.25. IR (Nujol): 3 060, 1 710, 1 620, 1 570 cm–1.
5.2.16. 2-[Bis(3-(ethoxycarbonyl)-
4-hydroxy-2-morpholino-1,6-dihydro-
6-oxopyridin-5-yl)methyl]furane 4h
Prepared in quantitative yield starting from compound
2b and furane-2-carboxyaldehyde, refluxing for 4 h. M.p.
242 °C dec. Calcd. for C29H34N4O11 (614.61). % C
56.67; % H 5.58; % N 9.12. Found: % C 56.73; % H
5.60; % N 9.17. IR (Nujol): 3 080, 1 710, 1 590 cm–1.
1H-NMR (DMSO-d6) d: 1.21 (t, J = 6.8 Hz, 6H, CH3),
3.13, 3.57 (m, 16H, morpholinyl), 4.17 (q, J = 6.8 Hz,
4H, CH2), 5.74 (s, 1H, CH), 5.89, 6.29, 7.40 (s, 3H,
furyl), 11.50, 12.22 (br s, 4H, D2O exchangeable).
Acknowledgements
This work was supported by a grant from MURST,
Rome. We thank the Antitumour Evaluation Branch of
National Cancer Institute for performing the biological
evaluation.
References
5.2.17. 2-[Bis(3-(ethoxycarbonyl)-
4-hydroxy-2-morpholino-1,6-dihydro-
6-oxopyridin-5-yl)methyl]thiophene 4i
[1] Grever M.R., Schepartz S.A., Chabner B.A., Semin. Oncol. 19
(1992) 622–638.
[2] Dannhardt G., Meindl W., Schober B.D., Kappe T., Eur. J. Med.
Prepared in 96% yield starting from compound 2b and
thiophene-2-carboxyaldehyde, refluxing for 4 h. M.p.
250 °C dec. Calcd. for C29H34N4O10S (630.67). % C
55.23; % H 5.43; % N 8.88. Found: % C 55.17; % H
5.41; % N 8.93. IR (Nujol): 3 070, 1 710, 1 630,
Chem. 26 (1991) 599–604.
[3] Saari W.S., Hoffman J.M., Wai J.S., Fisher T.E., Rooney C.S., Smith
A.M. et al., J. Med. Chem. 34 (1991) 2925–2928.
[4] Dollé V., Fan E., Nguyen C.H., Aubertin A., Kirn A., Andreola M.L.
et al., J. Med. Chem. 38 (1995) 4679–4686.
[5] William D.R., Bremmer M.L., Brown D.L., Antuono J.D., J. Org.
1
1 590 cm–1. H-NMR (DMSO-d6) d: 1.21 (t, J = 6.8 Hz,
Chem. 50 (1985) 2807–2809.
6H, CH3), 3.13, 3.58 (m, 16H, morpholinyl), 4.18 (q, J =
6.8 Hz, 4H, CH2), 5.99 (s, 1H, CH), 6.57, 6.83, 7.22 (s,
3H, thienyl), 11.53, 12.44 (br s, 4H, D2O exchangeable).
[6] Cocco M.T., Congiu C., Maccioni A., Plumitallo A., Schivo M.L.,
Palmieri G., Farmaco Ed. Sci. 43 (1988) 103.
[7] Kappe T., Ajili S., Stadlbauer W., J. Heterocycl. Chem. 25 (1988)
463–468.
[8] Kappe T., in: Pauette L.A. (Ed.), Encyclopaedia of Reagents for
Organic Synthesis Vol. 1, John Wiley & Sons, Chichester, 1995,
pp. 557–559.
5.3. Pharmacology
The compounds were tested by NCI in an in vitro assay
as a primary cancer screen. A total of 60 human tumour
cell lines, derived from nine cancer types (leukaemia,
lung, colon, brain, melanoma, ovarian, renal, prostate and
breast) formed the basis of this test. The tumour cells
were cultured in RPMI1640 medium supplemented with
5% foetal calf serum and 2 mM L-glutamine. The tumour
cells are inoculated over a series of standard 96-well
microtitre plates in 100 µL of medium [1, 9]. Density of
[9] Boyd M.R., Paul K.D., Drug Dev. Res. 34 (1995) 91.
[10] Skehan P., Storeng R., Scudiero D. et al., J. Natl. Cancer Inst. 82
(1990) 1107.
[11] Boyd M.R., Princ. Pract. Oncol. 3 (1989) 1.
[12] Plowman J., Dykes D.J., Hollingshead M., Simpson-Herren L.,
Alley M.C., Teicher B., Anticancer Drug development guide:
preclinical Screening, Clinical trials, and Approval, Humana Press
Inc., Totowa, NJ, 1997, pp. 101–125.
[13] Monks A., Scudiero D., Skehan P., Shoemaker R., Paull K., Vistica
D. et al., J. Natl. Cancer Inst. 83 (1991) 757.