Synthesis and cytotoxicity of phenyl-vinyl derivatives of 4,6,6-trimethyl-2-oxo-1,2,5,6-tetrahydropyridine-3-carbonitrile

Article abstract of DOI:10.1007/s10593-010-0411-7

A series of phenylvinyl derivatives of 4,6,6-trimethyl-2-oxo-1,2,5,6- tetrahydropyridine-3-carbonitriles has been synthesized and their cytotoxic activity towards HT-1080 (human fibrosarcoma) and MG 22A (mouse hepatoma) tumor cells studied. It was found that the 2-nitro-, 2- and 3-chloro, 2-fluoro, and 2-bromophenyl derivatives showed high cytotoxic activity towards both cell lines. The toxicity towards NIH 3T3 normal mouse embryonic fibroblasts depends on the nature and position of the substituent in the phenyl ring. The greatest selectivity of cytotoxic effect was seen in the 2-bromophenyl derivative.

Full text of DOI:10.1007/s10593-010-0411-7

Chemistry of Heterocyclic Compounds, Vol. 45, No. 10, 2009
SYNTHESIS AND CYTOTOXICITY OF PHENYL-
VINYL DERIVATIVES OF 4,6,6-TRIMETHYL-2-OXO-
1,2,5,6-TETRAHYDROPYRIDINE-3-CARBONITRILE
E. Lukevics1, D. Jansone1*, L. Leite1, J. Popelis1, G. Andreeva1,
I. Shestakova1, I. Domracheva1, V. Bridane1, and I. Kanepe1
A series of phenylvinyl derivatives of 4,6,6-trimethyl-2-oxo-1,2,5,6-tetrahydropyridine-3-carbonitriles
has been synthesized and their cytotoxic activity towards HT-1080 (human fibrosarcoma) and MG 22A
(mouse hepatoma) tumor cells studied. It was found that the 2-nitro-, 2- and 3-chloro, 2-fluoro, and
2-bromophenyl derivatives showed high cytotoxic activity towards both cell lines. The toxicity towards
NIH 3T3 normal mouse embryonic fibroblasts depends on the nature and position of the substituent in
the phenyl ring. The greatest selectivity of cytotoxic effect was seen in the 2-bromophenyl derivative.
Keywords: 4,6,6-trimethyl-2-oxo-1,2,5,6-tetrahydropyridine-3-carbonitrile, styryl derivatives,
condensation, toxicity, cytotoxicity.
In the synthesis of novel antitumor compounds the method of introducing a δ-lactam [1-4] or cyanovinyl
group [5-9] into the starting molecule has been successfully used. A δ-lactam ring is included in camptothecin
series antitumor preparations (inhibitors of topoisomerase I [10] – irinotecan [10, 11], rubitecan [12], topotecan
[10, 13], exatecan [14], and its metabolite 9-aminocamptothecin [15], silatecan [16], and also inhibitors of
dihydrofolate reductase (pemetrexed [17]), thymidyl synthetase (raltitrexed [18]), and farnesyl transferase
(tipifarnib [19]). A δ-lactam ring occurs in the structure of an immunoregulator [20] and the antiangiogenic
agent roquinimex [21].
R
N
N
O
H
O
O
R
HN
HN
Me
,
EtOH, NaOH
25–78°C
Me
Me
Me
Me
1
2–26
_______
* To whom correspondence should be addressed, e-mail: jansone@osi.lv.
1Latvian Institute of Organic Synthesis, Riga LV-1006, Latvia.
__________________________________________________________________________________________
Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 10, pp. 1529-1538, October, 2009.
Original article submitted May 26, 2009.
1226
0009-3122/09/4510-1226©2009 Springer Science+Business Media, Inc.

Several tyrosine kinase inhibitors contain a δ-lactam and an exocyclic β-cyanovinyl group in the same
molecule [22].
We have prepared the δ-lactam – 4,6,6-trimethyl-2-oxo-1,2,5,6-tetrahydropyridine-3-carbonitrile (1) on
whose double bond a cyanovinyl group occurs in the lactam ring but this compound proved inert to the HT-1080
(human fibrosarcoma) and MG 22A (mouse hepatoma) tumor cells.
The introduction of a β-styryl group into the compound molecule often increases its antitumor activity
[23] as occurs, for example, in the case of circumin [24], resveratrol [25], piceatannol [7], and the antitumor
agent tamoxifen [26, 27]. Combrestatin A-4 (a natural cis-stilbene) and its analogs [28-32] are characterized by
high cytotoxic activity in vitro relative to several types of human tumor cells. Hence we have carried out the
condensation of lactam 1 with benzaldehyde to give the novel lactam – 6,6-dimethyl-2-oxo-4-styryl-1,2,5,6-
tetrahydropyridine-3-carbonitrile (2) which contains all three of the discussed groups (δ-lactam, cyanovinyl, and
β-styryl) in its molecule. This compound showed a marked cytotoxic effect on the studied tumor cells (LC50
10 µg/ml).
TABLE 1. Synthesis of 6,6-Dimethyl-2-oxo-4-styryl-1,2,5,6-tetrahydro-
pyridine-3-carbonitriles 2-26
Styryl-
lactam
Molar ratio δ-lactam
1 : RC6H4CHO : NaOH temperature, °C
Reaction
Reaction
time, h
Yield
2–26, %
R
2
H
1 : 1.5 : 0.25
1 : 1.5 : 0.25
1 : 1.5 : 0.25
1 : 1.5 : 0.25
2 : 1: 0.05
78
78
78
25
25
78
78
78
25
78
65
78
78
78
78
78
78
65
65
25
65
78
78
78
78
65
78
78
78
78
78
25
25
78
2
4
6
2
2
2
2
2
2
2
1
2
5
6
2
3
2
1
1
4.5
1
1
1
3
2
1
2
2
4
6
6
2
2
2
27
27
3
4
2-F
38
2-Cl
75
100*
29
1 : 1.5 : 0.25
1 : 1.5 : 0.25
1 : 1.5 : 0.25
1 : 1.5 : 0.25
1 : 1.5 : 0.25
1 : 1.5 : 0.0625
1 : 1.5 : 0.25
1 : 1.5 : 0.125
1 : 1.5 : 0.25
1 : 1.5 : 0.25
1 : 1.5 : 0.125
1 : 1.5 : 0.25
1 : 1 : 0.0625
1 : 1 : 0.0625
1 : 1 : 0.0625
1 : 1 : 0.0625
1 : 1 : 0.0625
1 : 1 : 0.06
5
6
7
3-Cl
4-Cl
2-Br
28
45
70
64
8
4-Br
2-I
36
9
90
10
11
12
13
14
15
16
17
2-OH
4-OH
98.5
84
2-OMe
4-OMe
2-OCHF2
2-NO2
17
55.5
51
36
3-NO2
100
11
4-NO2
33
69
18
19
20
21
4-NMe2
2-Me
52
1 : 1.5 : 0.25
1 : 1.3 : 0.25
1 : 1 : 0.0625
1 : 2 : 0.25
55
2-CF3
66
2,4-Cl2
88
50
22
23
2,6-Cl2
1 : 1.5 : 0.25
1 : 1.5 : 0.25
1 : 1.5 : 0.25
1 : 1.5 : 0.25
1 : 1.5 : 0.25
1 : 1.5 : 0.25
1 : 1.5 : 0.25
47
3,4-(OH)2
0
13.5
14
24
25
26
3-OMe, 4-OH
3,4-(OMe)2
3,4-OCH2O
68.0
76.5
33
_______
* Aldol condensation product 27.
1227

With the aim of increasing the cytotoxicity and the selectivity of cytotoxic activity of styryl
cyanolactams we have synthesized a series of mono-(3-20) and disubstituted (21-26) phenyl derivatives (Tables
1 and 2) and studied their cytotoxicity on the two tumor cell lines HT-1080 and MG 22A as well as NIH 3T3
normal mouse fibroblasts which also served for evaluating the compound toxicity via the alternative method of
determining LD50 [33].
TABLE 2. Characteristics for the δ-Lactams 1-27
Found, %
——————
Calculated, %
Com-
pound
Empirical
formula
mp, °C
С
H
N
1
2
3
4
5
6
7
8
C9H12N2O
C16H16N2O
65.81
65.83
76.12
76.16
70.96
71.10
66.62
67.02
67.10
67.02
66.95
67.02
58.24
58.02
58.00
58.02
7.36
7.37
6.36
6.39
5.52
5.59
5.15
5.27
5.24
5.27
5.25
5.27
4.37
4.56
4.53
4.56
17.09
17.06
11.14
11.10
10.39
10.36
9.71
9.77
9.78
9.77
9.72
9.77
8.49
8.46
8.39
8.46
196-199
193-195
198-200
228-230
C16H15FN2O
C16H15ClN2O
C16H15ClN2O
C16H15ClN2O
C16H15BrN2O
C16H15BrN2O
221-224
(decomp.)
235-237
(decomp.)
246-248
253-255
(decomp.)
9
C16H15IN2O
C16H16N2O2
50.91
50.81
71.48
71.62
3.75
4.00
6.02
6.01
7.47
7.41
10.56
10.44
260-262
10
255-260
(decomp.)
11
C16H16N2O2
71.33
71.62
6.03
6.01
10.43
10.44
288-291
(decomp.)
12
13
14
15
C17H18N2O2
C17H18N2O2
C17H16F2N2O2
C16H15N3O3
72.36
72.32
72.22
72.32
64.16
64.14
64.47
64.64
6.39
6.43
6.42
6.43
5.03
5.07
5.03
5.09
9.85
9.92
9.99
9.92
8.82
8.80
14.16
14.13
230-232
199-200
205-207
246-248
(decomp.)
16
17
C16H15N3O3
C16H15N3O3
64.59
64.64
64.51
64.64
5.03
5.09
5.11
5.09
14.12
14.13
14.11
14.13
299-302
280-285
(decomp.)
18
C18H21N3O
73.15
73.19
7.19
7.17
14.19
14.23
268-270
(decomp.)
19
20
C17H18N2O
76.62
76.66
63.99
63.75
6.81
6.81
4.64
4.72
10.55
10.52
8.76
8.75
236-238
C17H15F3N2O
240-242
(decomp.)
21
C16H14Cl2N2O
59.96
59.83
4.35
4.39
8.70
8.72
283-286
(decomp.)
22
23
C16H14Cl2N2O
59.80
59.83
63.50
63.57
4.27
4.39
5.97
6.00
8.69
8.72
9.20
9.27
214-216
C16H16N2O3 ×H2O
265-267
(decomp.)
24
C17H18N2O3 ×0.5 C2H5OH×H2O
64.29
63.70
6.64
6.83
8.22
8.25
213-215
(decomp.)
25
26
27
C18H20N2O3
C17H16N2O3
C16H17ClN2O2
69.03
69.21
69.01
68.91
63.05
63.05
6.40
6.45
5.32
5.44
5.53
5.55
8.91
8.97
9.45
9.45
9.18
9.19
205-207
248-250
222-225
1228

TABLE 3. 1H NMR Characteristics for the Compounds Synthesized
Com-
pound
Chemical shifts, δ, ppm (J, Hz)*
1
2
1
2
1.31 (6H, s, 6-CH3); 2.27 (3H, s, 4-CH3); 2.46 (2H, s, H-5); 6.5 (1H, br. s, NH)
1.38 (6H, s, 6-CH3); 2.74 (2H, s, H-5); 7.0 (1H, br. s, NH);
7.20 and 7.45 (2H, two d,
7.60 (2H, m, H-2,6 Ph)
J = 16.4, CH=CH); 7.41 (3H, m, H-3,4,5 Ph);
3
4
1.38 (6H, s, 6-CH3); 2.76 (2H, s, H-5); 6.35 (1H, br. s, NH);
7.39 and 7.50 (2H, two d, = 16.1, CH=CH); 7.2-7.8 (4H, m, H Ph)
1.30 (6H, s, 6-CH3); 2.68 (2H, s, H-5); 6.4 (1H, br. s, NH);
J
7.33 and 7.53 (2H, two d,
7.69 (1H, m, H-4 Ph)
J = 16.3, CH=CH); 7.1-7.3 (3H, m, H-3,5,6 Ph);
5
6
7
1.37 (6H, s, 6-CH3); 2.73 (2H, s, H-5); 6.13 (1H, br. s, NH);
7.11 and 7.51 (2H, two d,
7.47 (3H, m, H-4,5,6 Ph)
J = 16.0, CH=CH); 7.37 (1H, m, H-2 Ph);
1.37 (6H, s, 6-CH3); 2.73 (2H, s, H-5); 6.2 (1H, br. s, NH);
7.13 and 7.41 (2H, two d, = 16.0, CH=CH);
7.39 and 7.52 (4H, A2B2 system, = 8.4, H-3,5 Ph and H-2,6 Ph)
1.40 (6H, s, 6-CH3); 2.77 (2H, s, H-5); 6.8 (1H, br. s, NH);
J
J
7.37 and 7.58 (2H, two d,
J
= 16.0, CH=CH); 7.2–7.4 (2H, m, H-4,5 Ph);
7.62 (1H, dd,
7.77 (1H, dd,
J
J
= 7.7 and
= 7.8 and
J = 1.3, H-3 Ph);
J
= 1.5, H-6 Ph)
8
9
1.37 (6H, s, 6-CH3); 2.73 (2H, s, H-5); 6.1 (1H, br. s, NH);
7.11 and 7.42 (2H, two d, = 16.0, CH=CH);
7.44 and 7.55 (4H, A2B2 system, = 8.8, H-3,5 Ph and H-2,6 Ph)
1.40 (6H, s, 6-CH3); 2.78 (2H, s, H-5); 6.0 (1H, br. s, NH);
7.30 and 7.43 (2H, two d, = 15.8, CH=CH);
J
J
J
7.09 (1H, dt,
7.41 (1H, dt,
7.72 (1H, dd,
7.91 (1H, dd,
J
J
J
= 8.0 and
= 7.8 and
= 7.8 and
= 8.0 and
J
J
J
= 1.6, H-4 Ph);
= 1.4, H-5 Ph);
= 1.6, H-3 Ph);
= 1.4, H-6 Ph)
J
J
10
11
1.24 (6H, s, 6-CH3); 2.82 (2H, s, H-5); 6.8-7.6 (6H, m, H Ph, CH=CH);
8.1 (1H, br. s, OH); 10.5 (1H, br. s, NH);
1.34 (6H, s, 6-CH3); 2.71 (2H, s, H-5);
6.88 and 7.46 (2H and 2H, A2B2 system,
6.9 (1H, br. s, OH); 7.14 and 7.25 (2H, two d,
7.4 (1H, br. s, NH)
J
= 8.6, H-3,5 Ph and H-2,6 Ph);
= 16.1, CH=CH);
J
12
1.37 (6H, s, 6-CH3); 2.76 (2H, s, H-5); 3.92 (3H, s, OCH3); 6.3 (1H, br. s, NH);
6.93 (1H, dd,
7.00 (1H, dt,
7.38 (1H, dd,
J
J
J
= 8.4 and
= 7.4 and
= 7.4 and
J
J
J
= 0.8, H-5 Ph);
= 0.8, H-4 Ph);
= 1.7, H-3 Ph);
7.52 and 7.59 (2H, two d,
7.66 (1H, dd, = 8.2 and
J
J
= 16.4, CH=CH);
= 1.6, H-6 Ph)
J
13
14
1.36 (6H, s, 6-CH3); 2.73 (2H, s, H-5); 3.86 (3H, s, OCH3); 6.0 (1H, br. s, NH);
6.93 and 7.55 (2H and 2H, A2B2 system, = 8.8, H-3,5 Ph and H-2,6 Ph);
7.14 and 7.33 (2H, two d, =16.4, CH=CH)
1.39 (6H, s, 6-CH3); 2,75 (2H, s, H-5); 6.3 (1H, br. s, NH);
6.58 (1H, t, HF = 73.2, OCHF2); 7.16 (1H, dd, = 7.8 and
J
J
J
J
J = 0.9, H-3 Ph);
7.28 (1H, dt,
7.43 (1H, dt,
J
J
= 7.8 and
= 7.8 and
J
J
= 1.2, H-4 Ph);
= 1.7, H-5 Ph);
7.44 and 7.53 (2H, two d,
7.81 (1H, dd, = 7.8 and
J
= 16.2, CH=CH);
J
J = 1.7, H-6 Ph)
15
16
1.25 (6H, s, 6-CH3); 2.82 (2H, s, H-5);
7.19 and 7.78 (2H, two d, = 16.0, CH=CH);
J
7.69 (1H, dt,
7.83 (1H, dt,
7.94 (1H, dd,
8.10 (1H, dd,
J
J
J
= 8.4 and
= 7.6 and
= 8.0 and
= 9.0 and
J
J
J
= 1.4, H-5 Ph);
= 1.2, H-4 Ph);
= 1.4, H-6 Ph);
J
J = 1.2, H-3 Ph); 8.3 (1H, br. s, NH)
1.25 (6H, s, 6-CH3); 2.87 (2H, s, H-5);
7.39 and 7.74 (2H, two d, =16.1, CH=CH);
7.9-8.4 (4H, m, H-4,5,6 Ph and NH); 8.49 (1H, m, H-2 Ph)
J
1229

TABLE 3 (continued)
1
2
17
18
1.25 (6H, s, 6-CH3); 2.87 (2H, s, H-5);
7.40 and 7.69 (2H, two d, J = 16.0, CH=CH);
7.95 and 8.29 (2H and 2H, A2B2 system, J = 8.8, H-3,5 Ph and H-2,6 Ph);
8.27 (1H, s, NH)
1.34 (6H, s, 6-CH3); 2.71 (2H, s, H-5); 3.05 (6H, s, N(CH3)2);
5.8 (1H, br. s, NH);
6.67 and 7.48 (2H and 2H, A2B2 system, J = 8.9, H-3,5 Ph and H-2,6 Ph);
7.11 and 7.23 (2H, two d, J = 16.0, CH=CH)
19
20
21
1.39 (6H, s, 6-CH3); 2.45 (3H, s, CH3); 2.75 (2H, s, H-5); 6.3 (1H, br. s, NH);
7.36 and 7.48 (2H, two d, J = 15.8, CH=CH); 7.25 (3H, m, H-3,4,5 Ph);
7.68 (1H, m, H-6 Ph)
1.40 (6H, s, 6-CH3); 2.73 (2H, s, H-5); 6.4 (1H, br. s, NH);
7.40 and 7.55 (2H, two d, J = 16.0, CH=CH);
7.51 and 7.63 (2H, t, J = 6.9, H-4,5 Ph); 7.72 (2H, two br. d, H-3,6 Ph)
1.25 (6H, s, 6-CH3); 2.86 (2H, s, H-5);
7.30 and 7.60 (2H, two d, J = 16.0, CH=CH);
7.54 (1H, dd, J = 8.6 and J = 2.0, H-5 Ph); 7.76 (1H, d, J = 2.0, H-3 Ph);
7.93 (1H, d, J = 8.6, H-6 Ph); 8.3 (1H, br. s, NH)
22
23
1.40 (6H, s, 6-CH3); 2.78 (2H, s, H-5); 6.15 (1H, br. s, NH);
7.1-7.4 (3H, m, H-3,4,5 Ph); 7.26 and 7.64 (2H, two d, J = 16.4, CH=CH)
1.22 (6H, s, 6-CH3); 2.80 (2H, s, H-5); 6.80 (1H, d, J = 8.0, H-5 Ph);
7.00 (1H, dd, J = 8.2 and J = 1.9, H-6 Ph);
7.00 and 7.40 (2H, two d, J = 15.7, CH=CH); 7.13 (1H, d, J = 1.9, H-2 Ph);
8.05 (1H, br. s, NH); 9.52 (2H, br. s, 3'- and 4'-OH)
24
25
1.36 (6H, s, 6-CH3); 2.73 (2H, s, H-5); 3.96 (3H, s, OCH3); 5.07 (1H, br. s, NH);
5.98 (1H, s, OH); 6.94 (1H, d, J = 7.0, H-5 Ph); 7.0–7.2 (2H, m, H-2,6 Ph);
7.13 and 7.28 (2H, two d, J = 16.3, CH=CH)
1.37 (6H, s, 6-CH3); 2.73 (2H, s, H-5); 3.93 (6H, s, OCH3); 6.4 (1H, br. s, NH);
6.89 (1H, d, J = 8.4, H-5 Ph); 7.10 (1H, d, J = 1.8, H-2 Ph);
7.15 (1H, dd, J = 8.5 and J = 1.8, H-6 Ph);
7.14 and 7.31 (2H, two d, J = 15.8, CH=CH)
26
27
1.23 (6H, s, 6-CH3); 2.80 (2H, s, H-5); 6.11 (2H, s, OCH2O);
7.01 (1H, dd, J = 8.2 and J = 1.7, H-6 Ph); 7.21 (1H, m, H-5 Ph);
7.30 (1H, br. s, H-2 Ph); 7.10 and 7.48 (2H, two d, J = 15.8, CH=CH);
8.11 (1H, s, NH)
1.19 (6H, s, 6-CH3); 2.61 (2H, s, H-5); 5.20 (1H, m, CH);
5.84 (1H, d, OH); 7.3-7.5 (3H, m, Ph);
7.65 (1H, d, J = 7.8, H-6 Ph); 8.04 (1H, m, NH)
_______
1
* H NMR spectra taken in CDCl3 (compounds 1-9, 12-14, 18-20, 22, 24,
25), DMSO-d6 (compounds 10, 15-17, 21, 23, 26, 27) or in a 5: 1 mixture
of DMSO-d6 and CDCl3 (compound 11).
Condensation of lactam 1 with benzaldehydes was carried out in ethanol in the presence of a catalytic
amount of NaOH (molar ratio of δ-lactam–aldehyde–NaOH 1:(1-2):(0.0625-0.25), respectively) at a temperature
of 25-78ºC (Table 1). In all cases the main reaction product was the crotonic condensation product. According to
1H NMR spectroscopy the exocyclic double bond has a trans configuration (Table 2), the spectroscopic signals
for both protons forming an AB type spin system with a spin coupling typical of trans-related protons
(15.7-16.4 Hz).
When carrying out the reaction of δ-lactam 1 with 2-chlorobenzaldehyde at room temperature and low
content of catalyst in the reaction mixture (molar ratio of lactam–chlorobenzaldehyde–NaOH 2:1:0.05) the aldol
condensation product 4-[2-(2-chlorophenyl)-2-hydroxyethyl]-6,6-dimethyl-2-oxo-1,2,5,6-tetrahydropyridine-
3-carbonitrile (27) is formed in quantitative yield. Increasing the amount of NaOH leads to dehydration of the
aldol to give the lactam 4 in 75% yield. Increasing the temperature to 78ºC led to a more than twofold reduction
in the product yield (Table 1).
1230

The results of the biological studies (Table 4) show that the cytotoxicity of lactams 2-27 depends
markedly on the nature and position of the substituent in the benzene ring and also on the number of them.
The cytotoxicity of the 2-substituted phenyl derivatives towards the HT-1080 and MG 22A tumor cells
decreases in the substituent order NO2 > Cl > F, Br > MeO > OH > CHF2O > CF3, Me > Me2N. The 2-nitro
derivative 15 shows the highest cytotoxicity towards both cell lines (LC50 0.8-3 µg/ml). The 2- and 3-chloro
derivatives (compounds 4 and 5 respectively) also show high cytotoxicity (LC50 2-4 µg/ml). The compound with
a 2-methoxy group 12 was somewhat less active (LC50 6-10 µg/ml). All of these compounds show a much
higher cytotoxicity towards tumor cells than the unsubstituted phenyl derivative 2 (LC50 10 µg/ml).
The lowest toxicity (LD50 2252 mg/kg) with the greatest selectivity in cytotoxicity action amongst this
group was found in the 2-bromo derivative 7. The toxicity of the 2-halo-substituted derivatives decreased in the
order F > Cl > Br > I (compounds 3, 4, 7 and 9).
The aldol condensation product 27 shows low cytotoxic activity. Morphological changes in cells are not
implicated and the compound can be classified as non toxic.
TABLE 4. Cytotoxicity of Lactams 1-27*
LC50, µg/ml
Com-
pound
LD50, mg/kg
НТ 1080
MG 22A
MTT
3Т3
CV
MTT
NO
CV
NO
NR
2
2
2
2
2
1
*
*
7
300
120
200
140
33
22
6
*
*
7
120
300
133
71
83
17
6
*
>2000
706
2
10
4
10
6
10
5
10
3
100
31
3
432
4
3
3
2
2
316
32
1233
454
5
4
4
2
2
6
21
5
12
5
10
5
10
2
100
1000
1174
774
7
2252
2418
>2000
483
2
2
8
*
14
10
18
21
6
*
100
12
9
2
2
9
*
15
200
11
15
8
100
15
88
7
17
150
7
*
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
30
70
10
37
2
50
10
100
30
0.8
73
10
>100
100
100
2
*
>2000
1496
1609
>2000
476
23
9
477
574
2
2
2
*
*
*
2
40
3
10
1.8
100
17
450
200
5
10
1.5
92
15
100
9
*
31
1000
206
2
*
2111
1070
>2000
>2000
221
2
*
11
10
8
103
2
18
10
5
2
2
*
71
*
*
2
100
100
20
90
100
100
4
*
75
12
20
23
250
39
250
6
10
80
18
100
36
100
5
5
10
1000
2252
>2000
1094
1760
1013
>2000
1585
2
12
10
6
6
*
20
10
10
47
10
20
30
20
202
670
202
36
28
20
10
2
2
2
2
*
100
100
*
*
*
86
24
100
100
15
496
_______
* LC50 is the concentration of lactams 1-27 causing the death of 50% of the
cells; CV = crystal violet (action on cell membranes); MTT = 3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (effect on the activity of
cell mitochondrial enzymes); NR = neutral red; NO = extent of generation of
NO determined and calculated as in method [34].
*2 Cytotoxic effect absent.
1231

In the series of methoxy (compounds 12 and 13), chloro (compounds 4 and 6), hydroxy (compounds 10
and 11), and nitro derivatives (compounds 15 and 17) the 4-isomers are less toxic than the corresponding
2-isomers. The 4-isomers are also inferior to their 2-isomers in their cytotoxic activity towards tumor cells.
The introduction of a second chlorine atom into position 4 of the 2-chloro derivative (compound 21) or
position 6 (compound 22) leads to a modest lowering of cytotoxicity towards MG 22A cells but towards
HT-1080 cells this lowering is somewhat greater. At the same time, their toxicity towards normal fibroblasts is
markedly decreased (316 to 1174 µg/ml).
The introduction of a second substituent (hydroxy or methoxy) into position 3 of the low activity
4-substituted hydroxy (compound 11) or methoxy derivative (compound 13) increases their cytotoxicity
(compounds 23-25) but also increases their toxicity towards normal cells.
It was interesting to find that a series of compounds having high (15, 3-5) or modest (2, 10, 23, 25)
cytotoxicity towards tumor cells substantially induced the formation of nitric oxide in them (Table 4).
Hence we have shown a marked effect of the nature of the substituent and of its position in the phenyl
ring on the antitumor activity and toxicity of styryl cyanolactams. The greatest selectivity of cytotoxic activity is
seen in the 2-bromo derivative 7.
EXPERIMENTAL
1H NMR spectra were recorded on a Mercury-400 (400 MHz) instrument using TMS as internal
standard. HPLC Analysis was performed under reversed phase chromatographic conditions and carried out on a
Varian ProStar chromatograph consisting of a ProStar 240 gradient pump, ProStar 330 diode array detector, and
ProStar 240 autosampler. The Alltech column (4.6×150 mm) was filled with Apollo C18 sorbent and the mobile
phase was acetonitrile-0.1% aqueous phosphoric acid in (pH 2.3). The gradient was linear from 40 to 100% over
15 min and then isocratic for 5 min with 100% acetonitrile. The mobile phase flow rate was 1 ml/min. Using
HPLC it was shown that the purity of the compounds exceeded 99%.
The starting δ-lactam 1 was prepared using the method we developed [35] through condensation of
4-hydroxy-4-methyl-2-pentanone with cyanoacetic ester in the presence of ammonium acetate.
Condensation of 4,6,6-Trimethyl-2oxo-1,2,5,6-tetrahydropyridine-3-carbonitrile with Benzalde-
hydes (General Method). A mixture of δ-lactam 1, the benzaldehyde, and NaOH in ethanol was stirred and
held at room temperature or heated to reflux and refluxed for 1-6 h. The precipitated reaction product was
filtered, washed on the filter with a small amount of ethanol, and then recrystallized from ethanol.
Cytotoxicity of Compounds 2-27 (Table 3) in vitro with respect to HT-1080 monolayer tumor cells
(human fibrosarcoma), MG 22A (mouse hepatoma), and normal NIH 3T3 cells (mouse embryonic fibroblasts)
was determined in 96 well plates using the dyes CV, MTT, and NR following the method reported in [36]. The
median acute toxicity (LD50) was calculated using method [33] from the data obtained for the 3T3 cell culture.
This work was carried out with the financial support of the Latvian Council for Science (grant 06.0032).
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