New bis(pyridyl)methane derivatives from 4-hydroxy-2-pyridones: Synthesis and antitumoral activity

Article abstract of DOI:10.1016/S0223-5234(02)00002-8

Bis(pyridyl)methane derivatives 5-40 were obtained from the reaction of 4-hydroxy-2-pyridones 3 and 4 with aldehydes. Compounds 5-40 were evaluated for cytotoxic activity against a panel of 60 human cancer cell lines by the National Cancer Institute and some of them demonstrated inhibitory effects on the growth of a wide range of cancer cell lines generally at 10^-5 M level and in some case at 10^-7 M concentrations.

Full text of DOI:10.1016/S0223-5234(02)00002-8

European Journal of Medicinal Chemistry 38 (2003) 37Á47
/
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Original article
New bis(pyridyl)methane derivatives from 4-hydroxy-2-pyridones:
synthesis and antitumoral activity
Maria Teresa Cocco *, Cenzo Congiu, Valentina Onnis
Dipartimento di Tossicologia, Universita` degli Studi di Cagliari, Via Ospedale 72, 09124 Cagliari, Italy
Received 26 July 2002; received in revised form 18 October 2002; accepted 21 October 2002
Abstract
Bis(pyridyl)methane derivatives 5Á
/40 were obtained from the reaction of 4-hydroxy-2-pyridones 3 and 4 with aldehydes.
Compounds 5Á40 were evaluated for cytotoxic activity against a panel of 60 human cancer cell lines by the National Cancer
/
Institute and some of them demonstrated inhibitory effects on the growth of a wide range of cancer cell lines generally at 10ꢀ5
level and in some case at 10ꢀ7 M concentrations.
M
´
# 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Bis(pyridyl)methanes; Antitumoral activity; Pyridones
1. Introduction
Evaluation Committee of NCI for testing in a new in
vivo Hollow Fiber Assay [2,3]. This new system consists
of 12 selected human tumour cell lines encased in hollow
fibers, which are implanted into athymic nude mice. Six
to 8 days after administration of the test compound to
mice, the fiber are collected, the cells removed, and
growth inhibition is measured using 3-(4,5-dimethylthia-
The development of potent and effective antineoplas-
tic drugs has become one of the most intensely
persuaded goals of contemporary medicinal chemistry.
In this context we have been interested in exploiting the
biological properties of pyridin-2(1H)one derivatives. In
previous paper we have reported the synthesis of 4-
hydroxy-6-oxopyridines and bis(pyridyl)methane deri-
vatives [1]. These last are in vitro powerful inhibitors of
the growth for many types of human tumour cells. These
results prompted us to design new analogues with
zol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT).
Although 28 showed a good activity in subcutaneous
implants (Table 4) it does not meet the Biological
Committee for Cancer Drugs criteria for further testing
in subcutaneous human xenograft assays. With these
results in mind and in order to investigate whether the
new bis(pyridyl)methanes were endowed with a better
antitumoral activity, structural modification were car-
ried out on 40 principally by changing the substitution
pattern of both 3- and 2-positions on pyridine rings and
on the methyne aromatic ring.
further structural modification to optimise structureÁ
/
activity relationships and retain their cytotoxicity pro-
file.
In this paper we describe the preparation and evalua-
tion of activity of a new series of bis(pyridyl)methanes,
designed taking in account the structure of 1-[bis(3-
(ethoxycarbonyl)-4-hydroxy-2-morpholino-1,6-dihydro-
6-oxopyridin-5-yl))methyl]-2,6-dichlorobenzene 40 that
showed best growth inhibition values [1]. This com-
pound lies outside the category of adequately studied
classes of antitumour agents and was one of the small
percentage which has been selected by the Biological
2. Chemistry
The required bis(pyridyl)methanes were prepared
according to our reported method [1]; the reaction of
aminopropenenitriles 1 and aminopropenoates 2 with
1,3-bis(2,4,6-trichlorophenyl)malonate furnished the
corresponding 4-hydroxy-6-oxopyridines 3 and 4 in
* Corresponding author
E-mail address: tcocco@unica.it (M.T. Cocco).
´
0223-5234/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S 0 2 2 3 - 5 2 3 4 ( 0 2 ) 0 0 0 0 2 - 8

38
M.T. Cocco et al. / European Journal of Medicinal Chemistry 38 (2003) 37Á47
/
Fig. 1. Conditions, (a) bis(2,4,6-trichlorophenyl)malonate, toluene, 95 8C; (b) ethanol, piperidine reflux.
good yields. These pyridones were used as synthons for
the synthesis of title compounds (Fig. 1).
The condensation of aryl aldehydes with 2 mol of
pyridone in presence of catalytic amounts of piperidine
concentration that produced 50% inhibition, total
growth inhibition and 50% cytotoxicity, respectively,
and are expressed in micromolar concentrations (mM).
afforded the bis(pyridyl)methane derivatives 5Á40 (Ta-
/
ble 1) in moderate to good yields.
4. Results and discussion
From the analysis of data reported in Table 3, where
the activity of compounds 8, 15, 17, 21 and 28 is
compared with compound 40 we can evince that all
compounds maintained high growth inhibition activity
at micromolar concentrations in all cell lines. In
particular compound 17 exhibited high sensitivity
against all leukaemia, melanoma and CNS cancer cell
lines, the best results being against the CNS cancer
SNB-75 cell line with GI50 0.082 mM. The compound 15
displayed high activity against EKVX (GI50 7.06 mM)
cell line of non small cell cancer subpanel and against T-
47D (GI50 4.44 mM) cell line of breast cancer subpanel.
The compound 8 shows GI50 values between 5.89 and
77.6 mM on all cell lines and TGI values of the same
order of magnitude but on a minor number of cell lines
(53 over 60). As far as regard cytotoxicity compound 8
shows activity on 41 cell lines while compounds 15, 17
and 28 are cytotoxic on a limited number of cell lines.
From the comparison between bis(3-cyanopyri-
3. Pharmacology
Evaluation of anticancer activity on bis(pyri-
dyl)methanes was performed at NCI. First bis(pyri-
dyl)methanes 5Á40 have been evaluated in primary
/
anticancer assay at 10ꢀ4 M concentration against
NCI-H460 (Lung), MCF7 (Breast) and SF-268 (CNS)
cell lines (Table 2).
For NCI criteria, compounds which reduce the
growth of any one of the cell lines to ca. 32% or less
are passed on for evaluation in the full panel of cell lines
over a 5 log dose range.
Bis(pyridyl)methanes 8, 15, 17 and 28, that reached
these criteria, were evaluated for their anticancer activity
following the known in vitro disease-oriented antitu-
mour screening program, which is based upon use of
multiple panel of 60 human tumour cell lines [4,5]. Each
compound is tested at minimum of five concentrations
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 [6]. The anticancer
dyl)methanes
and
bis(3-(ethoxycarbonyl)pyri-
dyl)methanes, bearing the same substituents on all
other positions, we can observe that replacement of
ethoxycarbonyl group with a cyano group did not
sensibly change the average sensitivity against all cell
lines. As matter of fact compound 8 exhibited GI50
values comparable to those of the analog 40. However,
compound 40 showed LC50 values tenfold lower respect
activity of each compound is deduced from doseÁ
/
response curves and is presented in Tables 3 and 4
according to the data provided by NCI [7]. The response
parameters GI50, TGI and LC50 refer to the drug

M.T. Cocco et al. / European Journal of Medicinal Chemistry 38 (2003) 37Á
/47
39
Table 1
Bis(pyridyl)methane derivatives
Table 2
Results of primary assay for antitumoral activity of bis(pyri-
dyl)methanes
Compound Growth percentage at 10ꢀ4 M concentration
Cell line (cancer)
MCF7 (breast) NCIÁ/H460 (lung)
SF-268 (CNS)
5
6
96
47
90
3
99
90
102
89
Compounds
X
Y
Ar
7
102
92
106
18
5
6
CN
CN
CN
CN
CN
CN
CN
CN
CN
pyrrolidino
pyrrolidino
4-FC6H4
2,6-Cl2C6H3
4-FC6H4
8
9
95
94
98
91
89
104
31
91
24
89
100
104
96
100
99
94
98
88
0
98
104
100
110
102
93
7
morpholino
morpholino
10
11
12
13
14
15
16
17
18
19
20
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
100
99
8
2,6-Cl2C6H3
4-FC6H4
9
4-MePiperazino
4-MePiperazino
4-COOEtPiperazino
4-COOEtPiperazino
4-COOEtPiperazino
102
96
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
2,6-Cl2C6H3
phenyl
4-FC6H4
102
15
100
14
2,6-Cl2C6H3
4-CH3C6H4
4-FC6H4
98
78
104
29
COOEt OEt
COOEt OEt
100
105
97
103
91
COOEt OEt
COOEt OEt
2,6-Cl2C6H3
2-thienyl
phenyl
108
102
103
74
COOEt pyrrolidino
COOEt pyrrolidino
COOEt pyrrolidino
COOEt pyrrolidino
COOEt morpholino
COOEt morpholino
COOEt morpholino
COOEt morpholino
COOEt morpholino
COOEt morpholino
COOEt 4-MePiperazino
COOEt 4-C6H5Piperazino
COOEt 4-C6H5Piperazino
COOEt 4-C6H5Piperazino
99
96
2,6-Cl2C6H3
2-NO2-4-ClC6H3
2-thienyl
98
101
100
101
2
107
93
phenyl
4-CH3C6H4
4-FC6H4
108
0
2-ClC6H4
4-ClC6H4
2,4-Cl2C6H3
2,6-Cl2C6H3
phenyl
93
95
81
88
74
64
70
89
96
88
92
93
94
103
92
95
85
85
102
87
95
4-FC6H4
2,6-Cl2C6H3
77
72
105
103
104
99
COOEt 4-(4-OMeC6H4)Piperazino 4-FC6H4
COOEt 4-(4-OMeC6H4)Piperazino 2,6-Cl2C6H3
100
100
100
102
COOEt 4-(4-FC6H4)Piperazino
COOEt 4-(4-FC6H4)Piperazino
COOEt 4-(3-ClC6H4)Piperazino
COOEt 4-(3-ClC6H4)Piperazino
4-FC6H4
106
108
2,6-Cl2C6H3
4-FC6H4
2,6-Cl2C6H3
COOEt 4-(3,4-Cl2C6H3)Piperazino 4-FC6H4
COOEt 4-(3,4-Cl2C6H3)Piperazino 2,6-Cl2C6H3
ring seemed to not improve the biological properties of
these molecules.
COOEt morpholino
2,6-Cl2C6H3
Furthermore, we can note that the presence of a 2-
morpholino group on pyridine moiety is very important
for the activity. Replacement of morpholino group with
a pyrrolidine or a N-substituted piperazine led to loss of
activity except for compounds 21 and 28 that retained a
good activity. Examination of compounds with a 2-
thienyl function on the methyne bridge evidentiated that
the highest activity was observed for 17 in which the
dialkylamino moiety is substituted with an ethoxy
group. In addition bis(2-ethoxypyridyl)methanes retain
their activity by replacement of 2-thienyl with 4-fluor-
ophenyl. On the contrary, to our surprise, the replace-
ment of 2-thienyl with an 2,6-dichlorophenyl was
associated to poor activity.
to 8 on CCRF-CEM, HL 60(TB) and K 562 cell lines of
leukaemia, NCI-H522 cell line of non small cell lung
cancer, HCT-116 and SW-620 cell lines of colon cancer,
M-14 and SK-MEL-28 cell lines of melanoma, CAKI-1,
RXF-393 and UO-31 cell lines of renal cancer and
MCF-7 cell line of breast cancer.
A good activity is correlated to the presence of
electron-withdrawing substituents on phenyl ring. High-
est inhibitory activities were obtained when the phenyl
nucleus is 2,6-dichlorosubstituted. The shift of one of
the two chlorine atoms in 4-position led to a drop in
activity as well as removal of one chlorine atom. On the
other hand, the introduction of fluorine on the phenyl
From the present study it can be concluded that the
antitumoral activity of bis(pyridyl)methanes appears to

Table 3
GI50, TGI and LC50 values (mM) of compounds 5Á
/40 against different tumour cell lines
Panel/cell line
Compounds
8
15
17
21
28
40
GI50
TGI
LC50
GI50 TGI
LC50
GI50
TGI
LC50
GI50
TGI
LC50
GI50 TGI
LC50
GI50
TGI
LC50
Leukaemia
CCRF-CEM
HL-60(TB)
K-562
15.1
12.9
11.2
16.1
21.4
5.89
66.8
29.3
23.2
36.2
60.8
26.3
ꢀ100 33.1
ꢀ100
17.2 54.3
16.5 50.8
20.1 71.0
ꢀ100 14.2
ꢀ100 13.7
ꢀ100 12.1
ꢀ100
40.7
29.7
ꢀ100 78.2
ꢀ100 n.t.
ꢀ100
n.t.
ꢀ100
n.t.
31.0
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
1.59
1.96
1.68
1.87
1.98
2.83
3.50 7.73
4.37 9.73
3.51 7.31
5.23 ꢀ100
4.87 ꢀ100
8.22 ꢀ100
66.7
48.2
81.2
19.5 58.2
25.8 86.8
23.5 62.2
74.3
70.2
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
MOLT-4
RPMI-8226
SR
ꢀ
6.48
22.5
ꢀ100
n.t.
ꢀ100 16.1 58.3
ꢀ100 10.5 29.0
ꢀ100 12.1
ꢀ100 56.4
39.2
21.9 67.6
ꢀ100
79.8
n.t.
n.t.
ꢀ100
Non small cell lung cancer
A549/ATCC
EKVX
77.6
18.3
22.1
18.0
23.0
16.2
19.3
ꢀ100
13.4
ꢀ100
39.0
79.3
ꢀ100 14.8 29.3
58.1
14.5
32.8
32.7
35.8
48.2
27.4
82.3
52.1
39.8
27.6
74.3
93.3
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
27.9 71.3
14.5 34.2
ꢀ100 17.5
80.7 18.6
ꢀ100 15.0
31.2
55.9
57.1
55.6
83.2
7.06 33.2
ꢀ100 11.5
ꢀ100 10.4
48.8
68.4
97.5
32.6
29.8
HOP-62
HOP-92
ꢀ100 13.0 36.1
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
55.2
30.3
ꢀ100
37.8
37.5
79.3
61.2
66.6
1.46 18.1
9.82 26.1
19.5 43.6
9.42
3.90
21.4
19.1 37.6
28.7 52.6
74.1
96.5
1.15
10.4
6.86 20.7
4.51 22.6
NCI-H226
NCI-H23
22.5
48.8
50.1
56.5
83.4
32.8
40.3
ꢀ100
ꢀ100
90.7
NCI-H322M
NCI-H460
NCI-H522
ꢀ100
ꢀ100
28.2
ꢀ100 16.9 60.8
ꢀ100 16.4 36.5
ꢀ100 14.4
81.4
94.9
25.9 48.4
28.0 77.0
21.9 42.0
18.1
32.0
41.2
13.3
13.8
ꢀ100 20.3
59.1
18.6 42.0
80.6
1.83
3.72 7.58
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
13.8
12.5
28.3
15.0
15.4
16.1
17.7
26.7
27.5
ꢀ100
28.2
28.9
32.7
36.8
51.7
60.3
25.9 79.8
18.1 39.0
ꢀ100 17.3
84.0 13.9
ꢀ100 17.5
40.7
35.0
45.0
37.5
35.3
42.9
34.1
95.6
88.1
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
33.1
36.8 78.0
ꢀ100
ꢀ100
ꢀ100 18.2
ꢀ100
ꢀ100
77.2
3.55 14.1
32.3
37.5
57.2
ꢀ100 25.7
ꢀ100
18.4 40.4
16.1 31.7
ꢀ100
96.8
77.0
38.1
ꢀ100
1.68
3.40 12.3
3.56 12.9
27.6
3.21 6.13
53.1
54.3
66.7
76.7
88.9
62.3
84.1
14.5
16.2
18.0
30.7 97.7
19.8 39.1
23.5 49.7
31.8 68.2
57.9
36.0
52.6
KM12
SW-620
16.9 37.7
17.3 51.1
ꢀ100
95.8
ꢀ100 14.5
ꢀ100
ꢀ100 12.1
1.71
3.08 5.55
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
19.5
30.6
19.2
16.1
15.1
16.8
51.1
ꢀ100
34.9
29.6
24.8
30.4
ꢀ100 14.6 33.6
ꢀ100 17.7 49.8
77.3
ꢀ100 12.0
55.0 6.39
ꢀ100 11.4
10.2
35.0
49.6
28.5
39.8
ꢀ100
ꢀ100
99.4
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
24.2 56.5
28.1 63.3
19.3 39.5
20.4 37.2
n. t. n. t.
23.1 50.0
ꢀ100
ꢀ100 16.5
80.5
68.1
6.09 19.0
30.4
44.3
56.1
63.5
54.4
51.2
55.1
10.0 23.5
17.1 42.2
1.57
3.84 16.2
3.98 10.2
ꢀ100
64.9
73.9
40.2
9.48 30.7
40.0
n.t. n.t.
15.2 36.3
n.t.
86.8
0.082 9.72
12.5
n. t.
ꢀ100
3.42
4.72 15.6
30.3
Melanoma
LOX IMVI
MALME-3M
M14
13.0
12.9
18.0
17.6
17.9
16.6
17.8
25.6
26.8
42.9
39.5
32.9
35.4
34.0
50.6
55.5
16.6 33.1
8.31 29.5
66.3
91.9
12.6
8.35
30.7
25.6
35.6
29.9
43.6
25.5
36.9
75.0
66.4
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
34.5
ꢀ100
ꢀ100
75.9
2.37
2.00
1.74
6.29 26.3
5.95 22.1
3.18 5.83
22.6 41.5
30.0 68.9
26.0 53.2
18.0 34.1
20.7 40.1
21.3 38.9
ꢀ100 24.6 63.3
ꢀ100 16.1
78.4
69.7
ꢀ100
ꢀ100
64.8
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
88.6
60.3
75.6
64.7
16.6 37.0
11.8 27.0
12.8 25.9
14.8 37.3
82.4
61.7
52.3
93.9
12.8
13.1
12.8
11.7
4.75 16.2
1.69 3.37 6.69
40.2
ꢀ100
50.5
77.6
71.1
4.07 15.6
5.13 17.7
39.5
44.3
ꢀ100

Table 3 (Continued)
Panel/cell line
Compounds
8
15
17
21
28
40
GI50
16.6
TGI
LC50
55.0
GI50 TGI
15.6 30.3
LC50
59.0
GI50
14.2
TGI
28.6
LC50
57.4
GI50
ꢀ100
TGI
LC50
GI50 TGI
21.5 39.5
LC50
72.7
GI50
TGI
LC50
40.7
UACC-62
30.2
ꢀ100
ꢀ100
4.88 16.6
Ovarian cancer
IGROV1
18.1
16.4
17.7
14.0
18.6
35.3
30.4
32.9
31.9
34.1
68.8
56.6
61.3
72.4
62.5
14.9 30.3
15.4 39.7
14.6 48.2
61.6
13.9
28.6
30.4
76.0
ꢀ100
41.6
58.7
79.9
n.t.
n.t.
n.t.
20.7 39.2
34.7 87.1
21.5 38.4
74.1
ꢀ100
68.6
4.09 15.0
51.8
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
ꢀ100 11.6
ꢀ100 17.6
ꢀ100 26.5
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
2.48
6.76 19.6
25.7
6.31 18.8
8.07 28.2
ꢀ100
ꢀ100
ꢀ100
44.3
50.7
44.9
29.1 ꢀ100
14.0 28.1
42.9 ꢀ100
22.0 45.0
ꢀ100 13.1
92.2
56.5
12.6
Renal cancer
786-0
A498
32.0
34.5
16.1
18.1
14.1
20.1
17.5
18.2
ꢀ100
ꢀ100
29.6
ꢀ100 16.4 39.7
ꢀ100 11.4 26.2
96.3
60.2
61.5
95.0
56.4
62.0
12.1
2.67
12.8
18.0
1.70
15.4
59.3
23.5
25.4
65.4
19.0
34.6
63.7
26.7
ꢀ100
59.0
50.4
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
27.6 87.0
22.1 39.9
20.7 42.8
26.5 52.7
18.2 38.8
26.3 48.0
30.6 55.4
21.5 39.1
ꢀ100
71.8
88.2
1.81
7.57 19.7
3.20 5.66
44.4
ACHN
CAKI-1
RXF 393
SN12C
TK-10
54.4
61.0
89.0
59.1
60.1
61.7
16.3 31.7
18.9 42.4
4.28 23.0
14.7 30.2
25.3 67.7
12.8 25.7
2.97
1.84
2.06
1.58
8.32 30.6
4.27 9.91
4.36 9.24
4.54 15.8
33.2
35.5
ꢀ100
55.7
77.7
ꢀ100
82.6
87.5
34.5
32.5
ꢀ100 25.4
51.5
ꢀ100
65.4
ꢀ100
70.8
3.38 11.3 37.3
1.88 3.73 7.39
UO-31
33.5
10.9
Prostate cancer
PC-3
DU-145
17.8
23.9
37.7
69.2
79.7
15.4 34.6
78.0
53.2
18.2
13.9
ꢀ100
26.9
ꢀ100
51.8
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
22.6 47.5
17.8 38.3
99.8
82.5
14.1 27.6
9.96 21.5
54.3
46.4
ꢀ100 11.1 24.3
Breast cancer
MCF7
16.8
27.4
16.6
19.5
16.6
17.6
17.0
34.2
ꢀ100
30.4
58.1
30.2
32.8
27.7
69.9
20.4 60.7
ꢀ100 14.9
ꢀ100 27.4
53.9
ꢀ100
30.1
ꢀ100
ꢀ100
81.7
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ100
ꢀ ꢀ100
ꢀ100
ꢀ100
ꢀ100
29.5
71.1
ꢀ100
ꢀ100
ꢀ100
ꢀ100 16.6
ꢀ100 11.2
ꢀ100
ꢀ100
80.8
2.07
3.86 7.19
NCI/ADR-RES
MDA-MB-231/ATCC
HS 578T
ꢀ100 24.1
55.7
ꢀ100
12.6 29.8
31.5
23.4
59.7
48.9
63.9
70.1
11.1
25.5 55.1
33.8 75.0
25.8 57.9
20.6 40.8
16.5 45.8
ꢀ100 20.8
ꢀ100
ꢀ100 21.0
ꢀ100
32.4
22.7
ꢀ100
72.2
64.5
3.47 15.9
2.53
4.95 15.7
3.56 9.27 50.9
MDA-MB-435
BT-549
T-47D
55.0
61.1
58.1
17.6 37.7
9.71 23.6
4.44 32.7
80.8
56.4
14.5
5.01
7.49 26.5
39.6
ꢀ100 0.098 17.0
ꢀ100
ꢀ100

42
M.T. Cocco et al. / European Journal of Medicinal Chemistry 38 (2003) 37Á47
/
Table 4
Results of hollow fiber assay of compound 40
5.2.2. 2-(4-Methylpiperazino)-4-hydroxy-1,6-dihydro-6-
oxopyridine-3-carboxylic acid ethyl ester (4d)
Prepared in 57% yield starting from ethyl 3-amino-3-
Test
Score
(4-methylpiperazino)-2-propenoate. M.p. 158Á160 8C
/
IP
SC
4
(from acetonitrile). Anal. (C13H19N3O4) C, H, N. IR
1
(Nujol): 1650, 1591, 1562 cmꢀ1. H-NMR (DMSO-d6)
8
Total
Cell kill
12
none
d: 1.21 (t, Jꢁ/7.2 Hz, 3H, CH3), 2.20 (s, 3H, CH3), 2.39,
3.20 (m, 8H, piperazinyl), 4.17 (q, Jꢁ/7.2 Hz, 2H, CH2),
5.38 (s, 1H, H-5), 7.40 (s, 2H, NH and OH).
be related to some structural requirements and to the
presence of particular substituents. As matter of fact
morpholino and 2,6-dichlorophenyl moieties play an
important role for antitumor activity in these type of
compounds.
5.2.3. 2-(4-(Ethoxycarbonyl)piperazino)-4-hydroxy-
1,6-dihydro-6-oxopyridine-3-carboxylic acid ethyl ester
(4e)
Prepared in 77% yield starting from ethyl 3-amino-3-
(4-(ethoxycarbonyl)piperazino)-2-propenoate.
178Á180 8C (from ethanol). Anal. (C15H21N3O6) C, H,
N. IR (Nujol): 1660, 1585, 1560 cmꢀ1 1H-NMR
M.p.
/
.
5. Experimental protocols
(DMSO-d6) d: 1.20 (m, 6H, CH3), 2.38, 3.19 (m, 8H,
piperazinyl), 4.19 (m, 4H, CH2), 5.40 (s, 1H, H-5), 8.05
(s, 2H, NH and OH).
5.1. Chemistry
Melting points were determined on a Stuart Scientific
Melting point SMP1 and are uncorrected. IR spectra
were recorded on Nujol mulls between salt plates in
5.2.4. 2-(4-Phenylpiperazino)-4-hydroxy-1,6-dihydro-6-
oxopyridine-3-carboxylic acid ethyl ester (4f)
Prepared in 82% yield starting from ethyl 3-amino-3-
1
Bruker Vector 22 spectrophotometer. H-NMR spectra
(4-phenylpiperazino)-2-propenoate. M.p. 232Á
(from 1-propanol). Anal. (C18H21N3O4). C, H, N. IR
(Nujol): 1642, 1598, 1579, 1553 cmꢀ1 1H-NMR
(DMSO-d6) d: 1.21 (t, Jꢁ7.2 Hz, 3H, CH3), 3.13,
3.30 (m, 8H, piperazinyl), 4.20 (q, Jꢁ7.2 Hz, 2H, CH2),
/
233 8C
were recorded on a varian 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 Fisons EA 1108 elemental analyser. Analytical
TLC were carried out on Merck 0.2 mm precoated silica
gel aluminium sheets (60 F-254). 3-Amino-3-dialkyla-
minopropenenitriles 1, ethyl 3-aminopropenoates 2,
.
/
/
5.42 (s, 1H, H-5), 6.74, 6.90. 7.17 (m, 5H, Ar), 10.63,
10.88 (s, 2H, NH and OH).
[8,9] 4-hydroxypyridones 3b
/
e [10], 4b, 4c and bis(pyr-
idyl)methanes 21 and 40 [1] were synthesised as pre-
Á
0.4% of the theoretical values.
5.2.5. 2-(4-(4-Methoxyphenyl)piperazino)-4-hydroxy-
1,6-dihydro-6-oxopyridine-3-carboxylic acid ethyl ester
(4g)
viously described. Analyses of C, H, N were within9
/
Prepared in 78% yield starting from ethyl 3-amino-3-
M.p.
(4-(4-methoxyphenyl)piperazino)-2-propenoate.
205 8C (from). Anal. (C19H23N3O5). C, H, N. IR
5.2. Synthesis of 4-hydroxypiridones (3, 4)
1
(Nujol): 1671, 1648, 1567 cmꢀ1. H-NMR (DMSO-d6)
d: 1.22 (t, Jꢁ
piperazinyl), 4.19 (q, Jꢁ
H-5), 6.78, 6.86 (m, 4H, Ar), 10.62, 10.88 (s, 2H, NH
and OH).
/
7.0 Hz, 3H, CH3), 2.99, 3.31 (m, 8H,
7.0 Hz, 2H, CH2), 5.61 (s, 1H,
A mixture of the appropriate propenenitrile 1 or
propenoate 2 (0.02 mol) and bis (2,4,6-trichlorophe-
nyl)malonate (9.26 g, 0.02 mol) in toluene (20 mL) was
heated in water bath at 95 8C for 2 h. After concentra-
tion in vacuo, the residue was treated with diethyl ether,
filtered off and recrystallised from a suitable solvent.
/
5.2.6. 2-(4-(4-Fluorophenyl)piperazino)-4-hydroxy-1,6-
dihydro-6-oxopyridine-3-carboxylic acid ethyl ester (4h)
Prepared in 80% yield starting from ethyl 3-amino-3-
5.2.1. 2-Ethoxy-4-hydroxy-1,6-dihydro-6-oxopyridine-3-
carboxylic acid ethyl ester (4a)
(4-(4-fluorophenyl)piperazino)-2-propenoate.
218Á220 8C (from 1-propanol). Anal. (C18H20FN3O4)
C, H, N. IR (Nujol): 1645, 1591, 1552 cmꢀ1. H-NMR
(DMSO-d6) d: 1.21 (t, Jꢁ7.2 Hz, 3H, CH3), 3.07, 3.31
(m, 8H, piperazinyl), 4.19 (q, Jꢁ7.2 Hz, 2H, CH2), 5.42
M.p.
Prepared in 60% yield starting from ethyl 3-amino-3-
ethoxy-2-propenoate. M.p. 180 8C (from acetonitrile).
Anal. (C10H13NO5) C, H, N. IR (Nujol): 1661, 1645,
1611, 1557 cmꢀ1. 1H-NMR (DMSO-d6) d: 1.21 (m, 6H,
CH3), 4.17 (m, 4H, CH2), 5.66 (s, 1H, H-5), 10.95, 11.42
(s, 2H, NH and OH).
/
1
/
/
(s, 1H, H-5), 6.97 (m, 4H, Ar), 10.63, 10.88 (s, 2H, NH
and OH).

M.T. Cocco et al. / European Journal of Medicinal Chemistry 38 (2003) 37Á
/47
43
5.2.7. 2-(4-(3-Chlorophenyl)piperazino)-4-hydroxy-1,6-
dihydro-6-oxopyridine-3-carboxylic acid ethyl ester (4i)
Prepared in 68% yield starting from ethyl 3-amino-3-
5.3.4. 1-[Bis(3-cyano-4-hydroxy-2-morpholino-1,6-
dihydro-6-oxopyridin-5-yl)methyl]-2,6-dichlorobenzene
(8)
(4-(3-chlorophenyl)piperazino)-2-propenoate.
174Á175 8C (from 1-propanol). Anal. (C18H20ClN3O4)
C, H, N. IR (Nujol): 1647, 1594, 1550 cmꢀ1. H-NMR
(DMSO-d6) d: 1.22 (t, Jꢁ7.2 Hz, 3H, CH3), 3.18, 3.30
(m, 8H, piperazinyl), 4.20 (q, Jꢁ7.2 Hz, 2H, CH2), 5.44
M.p.
Prepared in 40% yield starting from 3c and 2,6-
dichlorobenzaldehyde, refluxing for 4 h. M.p. 280 8C
dec. Anal. (C27H24Cl2N6O6) C, H, N. IR (Nujol): 2212,
1637, 1618, 1590 cmꢀ1
.
/
1
/
/
(s, 1H, H-5), 6.74, 7.16 (m, 4H, Ar), 10.64, 10.88 (s, 2H,
NH and OH).
5.3.5. 1-[Bis(3-cyano-4-hydroxy-2-(4-
methylpiperazino)-1,6-dihydro-6-oxopyridin-5-
yl)methyl]-4-fluorobenzene (9)
5.2.8. 2-(4-(3,4-Dichlorophenyl)piperazino)-4-hydroxy-
1,6-dihydro-6-oxopyridine-3-carboxylic acid ethyl ester
(4j)
Prepared in 75% yield starting from ethyl 3-amino-3-
(4-(3,4-dichlorophenyl)piperazino)-2-propenoate. M.p.
Prepared in 60% yield starting from 3d and 4-
fluorobenzaldehyde, refluxing for 3 h. M.p. 250 8C.
Anal. (C29H31FN6O4) C, H, N. IR (Nujol): 2210, 1610
cmꢀ1
.
198Á
C, H, N. IR (Nujol): 1668, 1648, 1582 cmꢀ1. H-NMR
(DMSO-d6) d: 1.21 (t, Jꢁ7.2 Hz, 3H, CH3), 3.08, 3.24
(m, 8H, piperazinyl), 4.18 (q, Jꢁ7.2 Hz, 2H, CH2), 5.44
(s, 1H, H-5), 6.89, 7.10, 7.36 (m, 4H, Ar), 10.61, 10.87 (s,
2H, NH and OH).
/
200 8C (from 1-propanol). Anal. (C18H19Cl2N3O4)
5.3.6. 1-[Bis(3-cyano-4-hydroxy-2-(4-
methylpiperazino)-1,6-dihydro-6-oxopyridin-5-
yl)methyl]-2,6-dichlorobenzene (10)
Prepared in 94% yield starting from 3d and 2,6-
dichlorobenzaldehyde, refluxing for 5 h. M.p. 290 8C
dec. Anal. (C29H30Cl2N8O4) C, H, N. IR (Nujol): 3405,
3386, 3246, 3139, 3118, 3069, 2207, 1607, 1583 cmꢀ1
1
/
/
.
5.3. Synthesis of bis(pyridyl)methanes (5Á40)
/
5.3.7. Bis(3-cyano-4-hydroxy-2-(4-
A mixture of the appropriate 4-hydroxypyridone 3, 4
(0.0025 mol) and aldehyde (0.00125 mol) in ethanol (20
mL) was added a few drops of piperidine and heated at
reflux until disappearance of starting material. The
formed precipitate was filtered off and washed with
diethyl ether.
(ethoxycarbonyl)piperazino)-1,6-dihydro-6-oxopyridin-
5-yl)methyl]benzene (11)
Prepared in 86% yield starting from 3e and benzalde-
hyde, refluxing for 3 h. M.p. 238Á
(C33H36N8O8) C, H, N. IR (Nujol): 2200, 1655, 1610,
1560 cmꢀ1
/
240 8C. Anal.
.
5.3.1. 1-[Bis(3-cyano-4-hydroxy-2-pyrrolidino-1,6-
dihydro-6-oxopyridin-5-yl)methyl]-4-fluorobenzene (5)
Prepared in 40% yield starting from 3b and 4-
fluorobenzaldehyde, refluxing for 12 h. M.p. 285 8C
dec. Anal. (C27H25FN6O4) C, H, N. IR (Nujol): 3232,
5.3.8. 1-[Bis(3-cyano-4-hydroxy-2-(4-
(ethoxycarbonyl)piperazino)-1,6-dihydro-6-oxopyridin-
5-yl)methyl]-4-fluorobenzene (12)
Prepared in 80% yield starting from 3e and 4-
fluorobenzaldehyde, refluxing for 5 h. M.p. 230Á
235 8C. Anal. (C33H35FN8O8) C, H, N. IR (Nujol):
2215, 1665, 1622, 1581 cmꢀ1
/
1
3139, 3054, 2218, 1614, 1598 cmꢀ1. H-NMR (DMSO-
d6) d: 1.23 (m, 12H, CH3), 4.21 (m, 8H, CH2), 5.93 (s,
1H, CH), 6.92 (m, 4H, Ar), 11.42, 12.42 (br s, 4H, NH
and OH).
.
5.3.9. 1-[Bis(3-cyano-4-hydroxy-2-(4-
(ethoxycarbonyl)piperazino)-1,6-dihydro-6-oxopyridin-
5-yl)methyl]-2,6-dichlorobenzene (13)
5.3.2. 1-[Bis(3-cyano-4-hydroxy-2-pyrrolidino-1,6-
dihydro-6-oxopyridin-5-yl)methyl]-2,6-dichlorobenzene
(6)
Prepared in 45% yield starting from 3e and 2,6-
dichlorobenzaldehyde, refluxing for 5 h. M.p. 280 8C
dec. Anal. (C33H34Cl2N8O8) C, H, N. IR (Nujol): 3061,
Prepared in 53% yield starting from 3b and 2,6-
dichlorobenzaldehyde, refluxing for 5 h. M.p. 290 8C
dec. Anal. (C27H24Cl2N6O4) C, H, N. IR (Nujol): 3247,
2214, 1693, 1641, 1588 cmꢀ1
.
3164, 2209, 1620, 1589 cmꢀ1
.
5.3.10. 1-[Bis(2-ethoxy-3-(ethoxycarbonyl)-4-hydroxy-
1,6-dihydro-6-oxopyridin-5-yl)methyl]-4-methylbenzene
(14)
Prepared in 70% yield starting from 4a and 4-
methylbenzaldehyde, refluxing for 2 h. M.p. 254Á
5.3.3. 1-[Bis(3-cyano-4-hydroxy-2-morpholino-1,6-
dihydro-6-oxopyridin-5-yl)methyl]-4-fluorobenzene (7)
Prepared in 80% yield starting from 3c and 4-
fluorobenzaldehyde, refluxing for 12 h. M.p. 285 8C
dec. Anal. (C27H25FN6O6) C, H, N. IR (Nujol): 2220,
/
255 8C. Anal. (C28H32N2O10) C, H, N. IR (Nujol):
1
1645, 1610, 1600 cmꢀ1. H-NMR (DMSO-d6) d: 1.24
(m, 12H, CH3), 4.21 (m, 8H, CH2), 2.19 (s, 3H, CH3),
1614, 1589 cmꢀ1
.

44
M.T. Cocco et al. / European Journal of Medicinal Chemistry 38 (2003) 37Á47
/
5.97 (s, 1H, CH), 6.85 (m, 4H, Ar), 11.34, 12.30 (s, 4H,
NH and OH).
233 8C. Anal. (C31H34ClN5O10) C, H, N. IR (Nujol):
3232, 3139, 1711, 1590, 1533 cmꢀ1
.
5.3.17. [Bis(3-(ethoxycarbonyl)-4-hydroxy-2-
morpholino-1,6-dihydro-6-oxopyridin-5-
yl)methyl]benzene (22)
5.3.11. 1-[Bis(2-ethoxy-(3-ethoxycarbonyl)-4-hydroxy-
1,6-dihydro-6-oxopyridin-5-yl)methyl]-4-fluorobenzene
(15)
Prepared in quantitative yield starting from 4c and
Prepared in 72% yield starting from 4a and 4-
benzaldehyde, refluxing for 3 h. M.p. 246Á248 8C dec.
/
fluorobenzaldehyde, refluxing for 2 h. M.p. 225Á
/
Anal. (C31H36N4O10) C, H, N. IR (Nujol): 1710, 1640,
1610, 1580 cmꢀ1. 1H-NMR (DMSO-d6) d: 1.21 (m, 6H,
CH3), 3.13, 3.59 (m, 16H, morpholinyl), 4.16 (m, 4H,
227 8C. Anal. (C27H29FN2O10) C, H, N. IR (Nujol):
1
1649, 1602, 1559 cmꢀ1. H-NMR (DMSO-d6) d: 1.23
(m, 12H, CH3), 4.21 (m, 8H, CH2), 5.93 (s, 1H, CH),
6.92 (m, 4H, Ar), 11.42, 12.42 (s, 4H, NH and OH).
CH2), 5.82 (s, 1H, CH), 7.01Á
/
7.23 (m, 5H, Ar), 11.50,
12.27 (s, 4H, NH and OH).
5.3.12. 1-[Bis(2-ethoxy-(3-ethoxycarbonyl)-4-hydroxy-
1,6-dihydro-6-oxopyridin-5-yl)methyl]-2,6-
dichlorobenzene (16)
5.3.18. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-
morpholino-1,6-dihydro-6-oxopyridin-5-yl)methyl]-4-
methylbenzene (23)
Prepared in 78% yield starting from 4a and 2,6-
Prepared in quantitative yield starting from 4c and 4-
dichlorobenzaldehyde, refluxing for 6 h. M.p. 168Á
170 8C (from benzene). Anal. (C27H28Cl2N2O10) C, H,
N. IR (Nujol): 1661, 1645, 1611, 1558 cmꢀ1
/
methylbenzaldehyde, refluxing for 3 h. M.p. 245Á247 8C
/
dec. Anal. (C32H35N4O10) C, H, N. IR (Nujol): 3220,
1
.
1710, 1640, 1600, 1560 cmꢀ1. H-NMR (DMSO-d6) d:
1.19 (m, 6H, CH3), 1.58 (s, 3H, CH3), 3.01, 3.54 (m,
16H, morpholinyl), 4.09 (m, 4H, CH2), 5.64 (s, 1H, CH),
6.91 (m, 4H, Ar), 9.46 (brs, 4H, NH and OH).
5.3.13. 2-[Bis(2-ethoxy-(3-ethoxycarbonyl)-4-hydroxy-
1,6-dihydro-6-oxopyridin-5-yl)methyl]-thiophene (17)
Prepared in 70% yield starting from 4a and 2-
thiophenecarboxyaldehyde, refluxing for 2 h. M.p.
5.3.19. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-
morpholino-1,6-dihydro-6-oxopyridin-5-yl)methyl]-4-
fluorobenzene (24)
204Á205 8C (from EtOH). Anal. (C25H28N2O10S) C,
/
1
H, N. IR (Nujol): 1700, 1645, 1590 cmꢀ1. H-NMR
(DMSO-d6) d: 1.21 (m, 12H, CH3), 4.16 (m, 8H, CH2),
6.22 (s, 1H, CH), 6.40, 6.74, 7.09 (m, 3H, thienyl), 8.25
(s, 4H, NH and OH).
Prepared in 80% yield starting from 4c and 4-
fluorobenzaldehyde, refluxing for 2 h. M.p. 260 8C
dec. Anal. (C31H35FN4O10) C, H, N. IR (Nujol): 1720,
1645, 1622, 1589 cmꢀ1
.
5.3.14. [Bis(3-(ethoxycarbonyl)-4-hydroxy-2-
pyrrolidino-1,6-dihydro-6-oxopyridin-5-
yl)methyl]benzene (18)
5.3.20. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-
morpholino-1,6-dihydro-6-oxopyridin-5-yl)methyl]-2-
chlorobenzene (25)
Prepared in yield starting from 4b and benzaldehyde,
refluxing for
2
h. M.p. 230 8C dec. Anal.
Prepared in 94% yield starting from 4c and 2-
chlorobenzaldehyde, refluxing for 1 h. M.p. 243 8C.
Anal. (C31H35ClN4O10) C, H, N. IR (Nujol): 1716, 1649,
(C31H34ClN5O10) C, H, N. IR (Nujol): 3220, 3100,
1710, 1570 cmꢀ1. 1H-NMR (DMSO-d6) d: 1.18 (m, 6H,
CH3), 1.83, 3.20 (m, 16H, pyrrolidinyl), 4.12 (m, 4H,
CH2), 5.63 (s, 1H, CH), 7.13 (m, 5H, Ar), 10.47, 12.40 (s,
4H, NH and OH).
1613, 1600, 1563 cmꢀ1
.
5.3.21. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-
morpholino-1,6-dihydro-6-oxopyridin-5-yl)methyl]-4-
chlorobenzene (26)
5.3.15. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-
pyrrolidino-1,6-dihydro-6-oxopyridin-5-yl)methyl]-2,6-
dichlorobenzene (19)
Prepared in 96% yield starting from 4c and 4-
chlorobenzaldehyde, refluxing for 1 h. M.p. 263 8C
dec. Anal. (C31H35ClN4O10) C, H, N. IR (Nujol):
Prepared in 40% yield starting from 4b and 2,6-
dichlorobenzaldehyde, refluxing for 4 h. M.p. 220 8C
dec. Anal. (C31H34Cl2N4O8) C, H, N. IR (Nujol): 3400,
1643, 1611, 1533 cmꢀ1
.
3080, 2730, 2640, 1650, 1630, 1585 cmꢀ1
.
5.3.22. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-
morpholino-1,6-dihydro-6-oxopyridin-5-yl)methyl]-2,4-
dichlorobenzene (27)
Prepared in 85% yield starting from 4c and 2,4-
dichlorobenzaldehyde, refluxing for 1 h. M.p. 240 8C
dec. Anal. (C31H34Cl2N4O10) C, H, N. IR (Nujol): 1722,
1616, 1580 cmꢀ1
.
5.3.16. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-
pyrrolidino-1,6-dihydro-6-oxopyridin-5-yl)methyl]-4-
chloro-2-nitrobenzene (20)
Prepared in 75% yield starting from 4b and 4-chloro-
2-nitrobenzaldehyde, refluxing for 2 h. M.p. 230Á
/

M.T. Cocco et al. / European Journal of Medicinal Chemistry 38 (2003) 37Á
/47
45
5.3.23. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-(4-
methylpiperazino)-1,6-dihydro-6-oxopyridin-5-
yl)methyl]-2,6-dichlorobenzene (28)
5.3.29. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-(4-(4-
fluorophenyl)piperazino)-1,6-dihydro-6-oxopyridin-5-
yl)methyl]-4-fluorobenzene (34)
Prepared in 45% yield starting from 4d and 2,6-
Prepared in 90% yield starting from 4h and 4-
fluorobenzaldehyde, refluxing for 1 h. M.p. 242Á
244 8C. Anal. (C43H43F3N6O8) C, H, N. IR (Nujol):
1715, 1653, 1623, 1592 cmꢀ1
dichlorobenzaldehyde, refluxing for 2 h. M.p. 258Á
260 8C. Anal. (C36H40Cl2N6O8) C, H, N. IR (Nujol):
1718, 1620, 1585 cmꢀ1
/
/
.
.
5.3.30. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-(4-(4-
fluorophenyl)piperazino)-1,6-dihydro-6-oxopyridin-5-
yl)methyl]-2,6-dichlorobenzene (35)
5.3.24. [Bis(3-(ethoxycarbonyl)-4-hydroxy-2-(4-
phenylpiperazino)-1,6-dihydro-6-oxopyridin-5-
yl)methyl]benzene (29)
Prepared in 65% yield starting from 4h and 2,6-
Prepared in quantitative yield starting from 4f and
dichlorobenzaldehyde, refluxing for 6 h. M.p. 223Á
225 8C. Anal. (C43H42Cl2F2N6O8) C, H, N. IR (Nujol):
1718, 1625, 1578 cmꢀ1
/
benzaldehyde, refluxing for 3 h. M.p. 228Á
(C43H46N6O8) C, H, N. IR (Nujol): 1721, 1645, 1616,
1599 cmꢀ1
/
230 8C. Anal.
.
.
5.3.31. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-(4-(3-
chlorophenyl)piperazino)-1,6-dihydro-6-oxopyridin-5-
yl)methyl]-4-fluorobenzene (36)
5.3.25. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-(4-
phenylpiperazino)-1,6-dihydro-6-oxopyridin-5-
yl)methyl]-4-fluorobenzene (30)
Prepared in 85% yield starting from 4i and 4-
fluorobenzaldehyde, refluxing for 3 h. M.p. 245 8C
dec. Anal. (C43H43Cl2FN6O8) C, H, N. IR (Nujol):
Prepared in 86% yield starting from 4f and 4-
fluorobenzaldehyde, refluxing for 2 h. M.p. 208Á
210 8C. Anal. (C43H45FN6O8) C, H, N. IR (Nujol):
1721, 1620, 1597 cmꢀ1
/
1710, 1652, 1619, 1591 cmꢀ1
.
.
5.3.32. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-(4-(3-
chlorophenyl)piperazino)-1,6-dihydro-6-oxopyridin-5-
yl)methyl]-2,6-dichlorobenzene (37)
5.3.26. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-(4-
phenylpiperazino)-1,6-dihydro-6-oxopyridin-5-
yl)methyl]-2,6-dichlorobenzene (31)
Prepared in 70% yield starting from 4f and 2,6-
dichlorobenzaldehyde, refluxing for 2 h. M.p. 204Á
Prepared in 75% yield starting from 4i and 2,6-
dichlorobenzaldehyde, refluxing for 10 h. M.p. 204Á
205 8C (From 1-propanol). Anal. (C43H42Cl4N6O8) C,
H, N. IR (Nujol): 1711, 1620, 1593 cmꢀ1
/
.
/
205 8C (From ethanol). Anal. (C43H44Cl2N6O8) C, H,
5.3.33. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-(4-(3,4-
dichlorophenyl)piperazino)-1,6-dihydro-6-oxopyridin-5-
yl)methyl]-4-fluorobenzene (38)
1
N. IR (Nujol): 3094, 3059, 1693, 1625, 1597 cmꢀ1. H-
NMR (DMSO-d6) d: 1.19 (m, 6H, CH3), 3.13, 3.29 (m,
16H, piperazinyl), 4.17 (m, 4H, CH2), 6.72Á7.19 (m,
/
Prepared in quantitative yield starting from 4j and 4-
fluorobenzaldehyde, refluxing for 3 h. M.p. 238 8C dec.
Anal. (C43H41Cl4FN6O8) C, H, N. IR (Nujol): 1711,
14H, Ar and CH), 11.58, 12.63 (s, 4H, NH and OH).
1612, 1587 cmꢀ1
.
5.3.27. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-(4-(4-
methoxyphenyl)piperazino)-1,6-dihydro-6-oxopyridin-5-
yl)methyl]-4-fluorobenzene (32)
5.3.34. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-(4-(3,4-
dichlorophenyl)piperazino)-1,6-dihydro-6-oxopyridin-5-
yl)methyl]-2,6-dichlorobenzene (39)
Prepared in 98% yield starting from 4g and 4-
fluorobenzaldehyde, refluxing for 3 h. M.p. 240 8C
dec. Anal. (C45H49FN6O10) C, H, N. IR (Nujol): 1718,
Prepared in 80% yield starting from 4j and 2,6-
dichlorobenzaldehyde, refluxing for 8 h. M.p. 235 8C
dec. Anal. (C43H40Cl6N6O8) C, H, N. IR (Nujol): 1709,
1612 cmꢀ1
.
1639, 1601 cmꢀ1
.
5.3.28. 1-[Bis(3-(ethoxycarbonyl)-4-hydroxy-2-(4-(4-
methoxyphenyl)piperazino)-1,6-dihydro-6-oxopyridin-5-
yl)methyl]-2,6-dichlorobenzene (33)
5.4. Pharmacology
Prepared in 76% yield starting from 4g and 2,6-
The compounds were tested by NCI in an in vitro 3-
cell line, one dose primary anticancer assay as a primary
cancer screen. The 3-cell line panel consists of the MCF7
(breast), NCI-H460 (lung) and SF-268 (CNS). Each cell
line is inoculated and preincubated on a microtriter
dichlorobenzaldehyde, refluxing for 10 h. M.p. 178Á
/
180 8C. Anal. (C45H48Cl2N6O10) C, H, N. IR (Nujol):
1
1720, 1630, 1572 cmꢀ1. H-NMR (DMSO-d6) d: 1.20
(m, 6H, CH3), 3.28 (m, 16H, piperazinyl), 3.63 (s, 6H,
CH3), 4.22 (m, 4H, CH2), 6.92 (m, 12H, Ar and CH).
plate. Test agents are then added at single 10ꢀ4
M

46
M.T. Cocco et al. / European Journal of Medicinal Chemistry 38 (2003) 37Á47
/
concentration and the culture incubated for 48 h. End-
point determinations are made with alamar blue [11].
Results for each test agent are reported as the percent of
growth of the treated cells when compared with
untreated control cells. Compounds which reduce the
growth of any one of the cell lines to ca. 32% or less are
passed on for evaluation in the full panel of 60 cell lines
over a 5 log dose range.
A total of 60 human tumour cell lines, derived from
nine cancer types (leukaemia, lung, colon, brain, mela-
noma, ovarian, renal, prostate and breast) formed the
basis of this test. The tumour cells were cultured in
RPMI1640 medium supplemented with 5% foetal calf
and continuing once daily for total four doses. Each
agent is assessed by intraperitoneal injection at two dose
levels with three mice per dose per experiment. Vehicle
controls consist of six mice receiving the compound
diluent only. The fibers are collected from mice on the
day following the fourth compound treatment and
subjected to stable endpoint MTT assay. The optical
density of each sample is determined spectrophotome-
trically at 540 nm and the mean of each treatment group
is calculated. The percent net growth in each treatment
group is calculated and compared with the percent net
cell growth in the vehicle treated controls. Each
compound is assessed in a total four experiments (3 cell
serum and 2mM
L-glutamine. The tumour cells are
lines per experimentꢂ
/
4 experimentsꢁ12 cell lines).
/
inoculated over a series of standard 96-well microtrite
plates in 100 mL of medium [4,5]. Density of inoculum
depends on the type of tumour cell and from its growth
characteristics [12]. These cells are then preincubated on
the microtrite plate for 24 h before adding the com-
pounds. These were tested in DMSO solution at five
different concentration (10ꢀ4, 10ꢀ5, 10ꢀ6, 10ꢀ7 and
10ꢀ8 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 calcu-
lated as previously described [7].
Compounds are selected for further testing on the basis
of several hollow fiber assay criteria. These include, (1) a
reduction in net cell growth of 50% or greater in ten of
the 48 possible test combination (12 cell linesꢂ
/
2 sitesꢂ
/
2 compound doses); (2) a reduction in net cell growth of
50% or greater in a minimum of the 24 distant site
combinations (intraperitoneal drug per subcutaneous
culture); and/or (3) cell kill of one or more cell lines in
either implant site (reduction in the viable cell mass
below the level present at the start of the experiment).
To simplify evaluation, a point system has been adopted
which allows rapid viewing of the activity of a given
compound. For this, a value of 2 is assigned for each
compound dose which results in a 50% or greater
reduction in viable cell mass. The intraperitoneal and
subcutaneous samples are scored separately so that
criteria (1) and (2) can be evaluated. Compounds with
5.4.1. Hollow fiber assay for preliminary in vivo testing
Each compound is tested against a standard panel of
12 human tumour cell lines including NCI-H23, NCI-
H522, MDA-MB-231, MDA-MB-435, SW-620, COLO
205, LOX IMVI, UACC-62, OVCAR-3, OVACAR-5,
U251 and SF-295. The cell lines are cultivated in RPMI-
a combined IPꢃSC score 20, a SC score 8 or a net cell
/
kill of one or more cell lines are referred for further
studies. The maximum possible score for an agent is 96
1640 containing 10% FBS and 2mM L-glutamine. On
(12 cell linesꢂ
criteria were statistically validated by comparing the
activity outcomes of ꢀ80 randomly selected com-
/
2 sitesꢂ
/
2 dose levelsꢂ2 [score]). These
/
the day preceding hollow fiber preparation, the cells are
harvested by standard trypsinization technique and
resuspended at the desired cell density (varies by cell
/
pounds in the hollow fiber assay and in xenograft
testing. This comparison indicated that there was a very
low probability of missing a xenograft active compound
if the hollow fiber assay were used as the initial in vivo
screening tool. Due to the design of the hollow fiber
assay, the results of individual cell lines are not reported
since the statistical power of the assay is based on the
impact of a compound against the entire panel of cells
[2,3].
line between 2 and 10ꢂ
106 cells per mL). The cell
/
suspension is flushed into 1 mm I.D. polyvinylidene
hollow fibers with a molecular weight exclusion of
500 000 Da. The hollow fiber are heat-sealed at 2 cm
intervals and the samples generated from these seals are
placed into tissue culture medium and incubated at
37 8C in 5% CO2 for 24Á48 h prior to implantation. A
/
total of three different tumour lines are prepared for
each experiment so that each mouse receives three
intraperitoneal implants (one of each tumour line) and
three subcutaneous implants (one of each tumour line).
On the day of implantation, samples of each tumour cell
line are quantitated for viable cell mass by a stable MTT
assays so that at time 0 cell mass is known. Thus, the
cytostatic and cytocidal capacities of the test compound
can be assayed. Mice are treated with experimental
agents starting on day 3 or 4 following fiber implantion
Acknowledgements
We thank the Antitumor Evaluation Branch and
Biological Testing Branch of the National Cancer
Institute for performing biological evaluations.

M.T. Cocco et al. / European Journal of Medicinal Chemistry 38 (2003) 37Á
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47
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