Synthesis, x-ray crystal structures, stabilities, and in vitro cytotoxic activities of new heteroarylacrylonitriles

Article abstract of DOI:10.1021/jm0311036

Twenty-three acrylonitriles, substituted at position 2 with either triazoles or benzimidazoles and at position 3 with various substituted furan, thiophene, or phenyl rings, were prepared by Knoevenagel condensation and tested for in vitro cytotoxic potency on 11 human cancer cell lines. X-ray crystal analysis of two representative compounds showed that the olfenic bond is E-configured. Structure-activity-relationships (SAR) indicated that position 2 is flexible for substituents with various nitrogen heterocyclics while position 3 is very sensitive to change; the most potent compounds contained a 5-nitrothiophen-2-yl ring at position 3 and either benzimidazol-2-yl (11) or a 5-benzyl-1H-[1,2,4]-triazol-3-yl (7) group at position 2 of acrylonitrile. SARs for the thiophen-2-yl-benzimidazoles show the following trend for position 5: NO₂ ? H > Cl = CH₃. Compound 11 was on average 10- and 3-fold more potent than cisplatin and etoposide, respectively. However, the acrylonitrile functionality is not an absolute requirement for cytotoxic activity because replacement of the nitrile group for either a hydrogen or a methyl group also gave active compounds. The acrylonitriles caused delayed cell death characterized by giant cells with multilobed nuclei. Compound 11 was found to bring about the increase in the activities of caspases 3 and 9 in the HL-60 cell line in a manner similar to etoposide, strongly indicating that apoptosis is the mechanism of cell death. The selectivity of various compounds toward cancer cells was estimated by comparing the IC₅₀ values obtained from a noncancerous epithelial cell line, h-TERT-RPE1, with the average IC₅₀ value from the cancer cell lines; 11 showed an average 1.7-fold greater activity toward cancer cells. The stabilities of the new compounds under cell culture conditions, estimated by HPLC, indicated that a major fraction of the compounds were lost from the medium over the first 24 h.

Full text of DOI:10.1021/jm0311036

3438
J. Med. Chem. 2004, 47, 3438-3449
Synthesis, X-ray Crystal Structures, Stabilities, and in Vitro Cytotoxic Activities
of New Heteroarylacrylonitriles
Franciszek Sa¸czewski,*,† Przemyslaw Reszka,†,‡ Maria Gdaniec,§ Renate Gru¨nert,‡ and Patrick J. Bednarski*,‡
Department of Chemical Technology of Drugs, Medical University of Gdan´sk, Al. Gen. Hallera 107, 80-416 Gdan´sk, Poland,
Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, University of Greifswald,
F.-L.-Jahn Strasse 17, 17489 Greifswald, Germany, and Faculty of Chemistry, A. Mickiewicz University,
60-780 Poznan´, Poland
Received November 14, 2003
Twenty-three acrylonitriles, substituted at position 2 with either triazoles or benzimidazoles
and at position 3 with various substituted furan, thiophene, or phenyl rings, were prepared by
Knoevenagel condensation and tested for in vitro cytotoxic potency on 11 human cancer cell
lines. X-ray crystal analysis of two representative compounds showed that the olfenic bond is
E-configured. Structure-activity-relationships (SAR) indicated that position 2 is flexible for
substituents with various nitrogen heterocyclics while position 3 is very sensitive to change;
the most potent compounds contained a 5-nitrothiophen-2-yl ring at position 3 and either
benzimidazol-2-yl (11) or a 5-benzyl-1H-[1,2,4]-triazol-3-yl (7) group at position 2 of acrylonitrile.
SARs for the thiophen-2-yl-benzimidazoles show the following trend for position 5: NO2 . H
> Cl ) CH3. Compound 11 was on average 10- and 3-fold more potent than cisplatin and
etoposide, respectively. However, the acrylonitrile functionality is not an absolute requirement
for cytotoxic activity because replacement of the nitrile group for either a hydrogen or a methyl
group also gave active compounds. The acrylonitriles caused delayed cell death characterized
by giant cells with multilobed nuclei. Compound 11 was found to bring about the increase in
the activities of caspases 3 and 9 in the HL-60 cell line in a manner similar to etoposide, strongly
indicating that apoptosis is the mechanism of cell death. The selectivity of various compounds
toward cancer cells was estimated by comparing the IC50 values obtained from a noncancerous
epithelial cell line, h-TERT-RPE1, with the average IC50 value from the cancer cell lines; 11
showed an average 1.7-fold greater activity toward cancer cells. The stabilities of the new
compounds under cell culture conditions, estimated by HPLC, indicated that a major fraction
of the compounds were lost from the medium over the first 24 h.
Introduction
furan, or thiophene rings have been synthesized (Table
1). To investigate the importance of the cyano group on
the cytotoxic activity, analogues lacking this group have
also been prepared. Herein, the testing of the new series
of acrylonitriles for cell growth inhibition on a panel of
11 human cancer cell lines is reported. The mechanism
of cell death has also been investigated by monitoring
the time-dependent activities of the apoptotic enzymes
caspases 3 and 9. To gain information on the selectivity
toward cancer cells, cytotoxicity has also been accessed
on a noncancerous, immortalized human epithelial cell
line, h-TERT-RPE1. In addition, the stabilities of se-
lected compounds under cell culture conditions have
been investigated.
2,3-Disubstituted acrylonitriles represent an interest-
ing class of biologically active compounds. These com-
pounds have been shown to possess spasmolytic,1 es-
trogenic,2 hypotensive,3 antioxidative,4 tuberculostatic,5
antitrichomonal,6 insecticidal,7 and cytotoxic8 activities.
Of particular interest are derivatives substituted in
position 2 with triazines and in position 3 with nitro-
furan (Table 1); such compounds have recently been
found to possess potent cancer cell growth inhibitor
activity in the National Cancer Institute 60 cell line
screen.9
To explore the structure-activity-relationships of this
class of compounds in more detail, analogues have been
prepared with variations at both positions 2 and 3 of
the acrylonitrile moiety. By replacement of the triazine
ring for either 1,2,4-triazole or benzimidazole ring
systems, the importance of this position (i.e., ring A) on
the cytotoxic activity has been studied. At position 3 (i.e.,
ring B), derivatives with various substituted benzene,
Chemistry
Synthesis. In our investigations we synthesized two
series of acrylonitrile derivatives: series A (compounds
1-9, Table 1) characterized by the presence of 1,2,4-
triazole as ring A and various aryl/heteroaryl substit-
uents as ring B; series B containing benzimidazole as
ring A and aromatic or heteroaromatic rings as ring B
(compounds 10-26, Table 1).
The desired acrylonitriles 1-9 were prepared by the
Knoevenagel condensation reaction of the corresponding
(1,2,4-triazol-3-yl)acetonitriles with aldehydes, typically
* To whom correspondence should be addressed. For F.S.: e-mail,
saczew@farmacja.amg.gda.pl; phone, (++48) 58 3493250; fax, (++48)
58 3493257. For P.J.B.: e-mail, bednarsk@pharmazie.uni-greifswald.
de; phone, (++49) 3834 864883; fax, (++49) 3834 864874.
† Medical University of Gdan´sk.
‡ University of Greifswald.
§ A. Mickiewicz University.
10.1021/jm0311036 CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/15/2004

Heteroarylacrylonitriles
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3439
Table 1. Structures of Acrylonitrile Derivatives 1-26
in the presence of piperidine in ethanol at reflux (Table
1, method A).
Analogous condensation of 2-cyanomethylbenzimida-
zole with aromatic aldehydes was performed in the
presence of KOH in ethanol at room temperature, giving
rise to the formation of products 10-23 (Table 1, method
B).
The deacetylated benzimidazole 25 could be obtained
in pure form according to the method described previ-
ously by Prousek10 by using a multistep procedure based
on the Wittig reaction (see Experimental Section, Table
1, method D). The coupling constants in the H NMR
spectrum (J ) 15.5 Hz) provide evidence that the
1
stereochemistry of the olefinic bond is trans.
To determine whether simpler, readily available
compounds lacking the cyano group would retain any
of the activity of the parent compound 11, the benzimi-
dazole derivatives 25 and 26 (R1 ) H, CH3) were
synthesized by condensation of 2-methyl- and 2-ethyl-
benzimidazole, respectively, with 5-nitrothiophene-2-
aldehyde in acetic anhydride under reflux (Table 1,
method C). In the case of 2-methylbenzimidazole, how-
ever, this reaction led to the formation of N-acetyl
derivative 24, which proved to be rather unstable in
aqueous solution and partially hydrolyzed to the com-
pound 25, rendering this compound unsuitable for
biological testing.
Crystal Structure Analyses. The configuration of
the acrylonitrile double bond could not be established
by NMR methods; thus, X-ray crystallographic investi-
gations were performed. X-ray crystal structures of
compounds 3 and 24 are shown in Figures 1 and 2,
respectively. These structures establish that the olefinic
bond is E-configured.
There is strong conjugation between the different
moieties constituting the skeleton of molecules 3 and
24. Because of this effect, the aromatic rings in 3 and
24 are only twisted slightly relative to the central vinyl
group.

3440 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Sa¸ czewski et al.
Figure 3. Possible conformations of 11.
Table 2. Conformational Search on Compound 11
Figure 1. ORTEP drawing of 3 with the atom labeling
scheme. Ellipsoids are drawn at the 50% probability level.
Φ1
Φ2
E
[au]
∆E a
[kcal/mol] moment [D]
dipole
conformer [deg] [deg]
11A
11B
11C
11D
-0.2 -0.2 -1 299.717 263 2
-0.5 -1.9 -1 299.717 424 4
-22.2 -3.3 -1 299.709 556 0
-23.7 -1.9 -1 299.707 959 3
0.1
0.0
4.9
5.9
5.63
4.93
5.38
9.78
a Energy of each conformer after minimization, relative to the
global minimum.
Table 3. Primary Screen of All 26 Compounds at 20 µMa
LCLC-
compd SISO 103H KYSE-510 compd SISO 5637 KYSE-70
1
2
-8.7 -8.4
-8.8 -8.4
-21.3
-21.3
94.1
15
16
17
18
19
20
21
22
23
25
26
12
13
14
106.1 86.1
82.4 49.3
54.2 14.4
99.2 73.9
95.0 61.1
99.2 75.6
72.1 64.9
71.7 66.0
61.8 34.4
56.6
30.8
55.4
88.0
124.4
99.3
75.8
96.7
69.7
0.7
3
98.1
68.8
Figure 2. ORTEP drawing of 24 with the atom labeling
scheme. Ellipsoids are drawn at the 50% probability level.
4
-7.9 -8.4
-9.9 -6.5
-20.9
-21.7
108.0
-21.3
-20.7
-1.01
-21.2
20.5
5
6
85.5
56.4
In 3 the torsion angles about the partially double C-C
bonds to the 1,2,4-triazole and phenyl rings are -14.7-
(3)° and 15.3(3)°, respectively, and the dihedral angle
between the two aromatic moieties is 2.61(7)°. The vinyl
group, bearing three conjugated substituents, has the
double bond length of 1.349(2) Å, significantly longer
than those of the disubstituted vinyl groups in 2-styryl-
benzimidazoles and 2-styrylimidazoles (1.304-1.332
Å).11
7
-8.7 -8.4
-8.1 -7.3
1.2 -1.8
8
9
10
11
12
13
14
-8.2 -8.0
18.0 -6.5
-1.8
-1.3
0
0.5
2.4
82.3
102.2
64.5
8.6
13.4
5.9
nd
82.3 80.0
102.2 44.6
64.5 47.8
nd
nd
nd
nd
nd
a Results are the average percent growth values from eight
determinations in a single experiment. Italic compound numbers
indicate those that were evaluated further in the secondary screen.
In 24 the thiophenyl ring is approximately coplanar
with the vinyl group. The steric interaction of the vinyl
and acetyl groups results in a twist of 22.1(3)° of the
benzimidazole moiety relative to the vinyl group and a
twist of 16.1(1)° between the acetyl group and the
benzimidazole ring; in effect, the vinyl H10 atom and
acetyl O21 are 2.23(2) Å apart. Bond distances and bond
angles in 24 are within normal ranges (Tables 1S-14S).
Molecular Modeling. As evidenced from both X-ray
crystallographic studies and 1H NMR spectroscopic data
(see Experimental Section), the olefinic bond in the
compounds of series A and B adopted the E configura-
tion. However, owing to the different orientations of the
heterocyclic rings, these compounds can exist in the
form of four low-energy conformers. Literature reports
for similar compounds suggest that such conformers
might be stable on an NMR time scale.12 We have
therefore determined the preferred conformation of the
most active compound 11 by computation of the internal
energies for the rotamers 11A-D (Figure 3) with an ab
initio quantum mechanics Hartree-Fock method.13
Analysis of the energy differences (Table 2) clearly
indicates that the most stable are rotamers 11A and
11B, which differ in energy by 0.1 kcal/mol. Moreover,
the conformational analysis around the Câ-thiophene
bond performed at 10° increments revealed that both
of these rotamers can adopt the out-of-the-plane con-
formation at a low-energy cost of 1.5 kcal/mol.
Biology
Cytotoxicity Testing. Primary screening of the new
compounds for cytotoxic activity took place on three cell
lines (Table 3). Compounds that showed enough activity
at 20 µM to give percent growth values of less than 50%
in one or more of the cell lines were investigated further.
Secondary screening to determine potency was per-
formed on a panel of 11 human cancer cell lines: a
cervical cancer, SISO; a non-small-cell lung cancer,
LCLC-103H; three esophagus cell lines, KYSE-70, -510,
and -520; three bladder cell lines, RT-4, RT-112, and
5637; two pancreas lines, YAPC and DAN-G; and the
breast cancer cell line, MCF-7. Figure 4 shows repre-
sentative dose-response curves for compound 11 and
cisplatin in the YAPC and DAN-G cell lines. Table 4
lists the IC50 values calculated from the dose-response
data and reports the average IC50 value and the relative
standard deviation for the tested cell lines.
The most active compounds possess at position 3 of
the acrylonitrile functionality a 5-nitrothiophene ring
and at position 2 benzimidazole (11), 5-benzyltriazole
(7), or 5-methyltriazole (2) heterocycles. Interestingly,
the presence of methyl, phenyl, or benzyl substituents
in position 5 of the triazole ring leads to compounds with
good potency (e.g., 2, 5, 7), suggesting that this position

Heteroarylacrylonitriles
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3441
Table 4. IC50 (µM) Values in 11 Human Cancer Cell Lines and a Human Epithelial Cell Line (h-TERT-RPE1)a
RSD
LCLC-
103H MCF-7
h-TERT-
RPE1
compd
meanb (%)c
RT-4 RT-112
5637
0.15
KYSE-70 KYSE-510 KYSE-520 YAPC DAN-G SISO
11
0.23
0.47
0.51
0.57
0.59
0.60
0.61
0.85
1.38
3.06
3.41
12.36
13.8
15.67
40
33
36
31
51
31
37
46
31
46
44
45
54
61
0.27
0.59
0.90
0.89
1.37
0.99
1.03
1.48
1.85
2.50
4.67
16.33
14.59
7.68
0.36
0.65
0.67
nd
0.16
0.42
0.44
nd
0.33
0.53
0.56
nd
0.11
0.29
0.60
nd
0.16
0.36
0.41
nd
0.32
0.50
0.57
nd
0.49
0.69
0.42
1.00
1.52
4.11
3.73
17.61
14.99
15.83
0.31 0.17
0.16
0.42
0.38
nd
0.39
0.51
0.69
0.21
0.42
0.60
0.42
0.69
1.16
nd
7
0.32
0.22
0.78 0.31
0.51 0.33
2
25
0.27
nd
0.56
8
0.75
0.69
0.83
1.00
1.51
3.69
nd
0.40
0.40
0.45
0.35
0.66
1.17
2.53
5.11
15.64
8.78
22.53
nd
0.62
0.75
0.63
0.90
1.35
3.36
nd
0.75
0.62
0.57
0.86
1.62
6.26
nd
0.45
0.57
0.32
0.85
1.40
3.48
nd
0.37 0.29
0.33 0.41
0.89 0.54
0.27 0.35
1.20 0.70
3.24 1.65
3.28 0.44
14.03 1.60
25.86 2.50
30.89 1.13
0.61
0.65
0.68
1.47
1.62
1.49
2.97
9.11
11.23
16.39
nd
10
0.48
5
0.48
1
0.54
4
1.22
9
1.38
26
3.65
nd
23
nd
12.19
17.83
15.24
20.05
22.42
nd
nd
nd
nd
16
nd
nd
nd
nd
nd
17
nd
nd
nd
nd
nd
14
>20
nd
nd
nd
nd
>20
>20
2.11
1.97
1.42
nd
13
>20
>20
nd
nd
nd
nd
nd
>20
>20
>20
>20
nd
nd
12
nd
> 20
nd
nd
nd
nd
nd
>5
cisplatin
etoposide
2.14
0.64
91
65
3.70
1.33
2.14
0.22
0.31
0.53
1.49
0.79
0.88
0.70
5.07
0.41
6.03
1.32
1.39
0.38
0.20 1.63
0.15 nd
0.74
0.54
3.04
0.09
a Values are averages of two to three independent determinations. Individual values did not differ by more than 20%. nd: not determined.
b Averaged IC50 values over all tested cancer cell lines. c Relative standard deviation.
Figure 4. Representative dose-response curves for 11 (solid
symbols) and cisplatin (open symbols) in the YAPC (circles)
and DAN-G (triangles) human pancreas cell lines.
is not critical and thus is free for variations. Compared
Figure 5. Representative SAR of some benzimidazole deriva-
to cisplatin, compound 11 is on average 10-fold more
tives in the 5637 cell line. IC50 values are the averages of two
potent and in some cell lines (i.e., KYSE-520 and YAPC)
even 50-fold more potent (Table 4).
separate determinations whereby the individual values did not
differ by more than 20%.
Interestingly, when the 5-nitrothiophen ring is sub-
stituted to the acrylonitrile functionality at position 3
instead of at position 2 of the thiophene ring, activity
is almost completely lost (compare the isomers 11 with
12 in Table 3). Replacement of the 5-nitro group for
either chloro (13), methyl (14), or hydrogen (16) results
in severe reductions in activity (Table 4), with the
average order of 11 . 16 > 13 ) 14. Replacement of
the thiophene for a furan ring reduces the average
potency (compare 1 with 2, 4 with 5, and 10 with 11),
but on a few occasions the furan and thiophene ana-
logues showed equivalent activity, e.g., for 10 and 11
in the SISO cell line. Compounds with 3-substituted
benzene rings were the least active. Compound 18,
which possesses a 4-nitrobenzene ring as a 5-ni-
trothiophene isostere, was inactive. On the other hand,
the 3-nitrobenzene isomer 17 showed weak activity.
Not all active compounds had at position 3 of the
acrylonitrile moiety aromatic rings substituted with
nitro groups; two exceptions were the 4-dimethylami-
nophenyl triazole (9) and on the 4-trifluoromethylphen-
ylbenzimidazole (23), but these compounds were only
weakly active (Table 4). Although the cyano group of
the acrylonitrile moiety was not an absolute require-
ment for activity, compounds lacking this group (i.e.,
25 and 26) were less active compared to their acryloni-
trile counterpart 11. Interestingly, replacement of the
nitrile group for hydrogen (compare 11 with 25) had less
of an effect than replacement for a methyl group
(compare 11 with 26). Representative SAR of the
benzimidazole derivatives in the 5637 cell line are
summarized in Figure 5.
To establish the selectivity toward cancer cells, testing
of the more potent compounds on the noncancerous
human epithelial cell line h-TERT-RPE1 was done. This
immortalized cell line is derived from human corneal
epithelium transfected with human telomerase reverse
transcriptase (hTERT).14 As evidenced in Table 4, the
potencies of the compounds were roughly the same in
the h-TERT-RPE1 cells and in the average human
cancer cell line, indicating little selectivity toward
cancer cells in general. Nevertheless, when individual
cell lines such as 5637, KYSE-70, LCLC-103H, and
MCF-7 are compared with h-TERT-RPE1, then a mod-
est degree of selectivity for compounds 2 and 11 became
apparent. It was found that known antitumor agents
such as cisplatin and etoposide are also nonselective
toward cancer cells; in fact, the h-TERT-RPE1 cell line
was the most sensitive of the cell lines tested to the
growth inhibitory action of etoposide (Table 4).

3442 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Sa¸ czewski et al.
Figure 6. Cell line 5637 treated for 96 h with various substances: (A) control cells; (B) 1.0 µM 8; (C) 0.5 µM 11; (D) 1.0 µM 7.
All pictures are at 400-fold magnification.
Mechanism of Cell Death. Morphological changes
in the cells as a result of treatment with representative
acrylonitriles were documented by photography. In
Figures 6-8 the effects of compounds 7, 8, and 11 on
the appearance of the 5637, LCLC-103H, and SISO cells
are compared with nontreated controls. These changes
first became apparent 3-4 days after treatment. The
presence of giant cells with large, often multilobed
nucleus (Figures 6B,C, 7C,D, and 8C) is characteristic,
in particular for 11. Also, cells with clear vacuoles in
the cytoplasm (Figure 8D) or absence of a defined
nucleus (Figure 8B) were observed. Occasionally, very
small cells (Figure 7B) or cells lacking a cell membrane
(Figure 6D) were found.
These results prompted us to investigate in more
detail the mechanism of cell death caused by the most
potent compound 11. Antitumor agents are known to
induce cancer cell death by a mechanism of apoptosis,
characterized by the release of cytochrome c from the
mitochondria, the induction of various caspase enzymes,
condensation of the chromosomes, and the fragmenta-
tion of nuclear DNA.15 For example, the widely used
antitumor agent etoposide is known to cause induction
of caspase 3 and 9 activities in HL-60 human leukemia
cells within hours of exposure to the drug.16 An assay
based on the HPLC detection of fluorescent products
from specific caspase 3 and 9 substrates was developed
to specifically monitor the time-dependent activities of
these two caspases in cultures of HL-60 cells. To
validate the assay, the increases in the two caspase
activities caused by etoposide were monitored and found
to follow a similar time frame as previously reported
(Figure 9).16 Figure 9 shows that at a cytotoxic concen-
tration (50 µM), compound 11 causes a very similar
induction pattern of both enzymes, strongly indicating
that this compound is killing cells by a mechanism of
apoptosis.
Stability Studies
A number of chemical reactions are imaginable for
acrylonitriles under the conditions of the cytotoxicity
assays, e.g., Michael addition of thiols, retro-Knoevena-
gel reactions, nitro group reductions. Such reactions
may play a role in the mechanism of action and could
influence the pharmacokinetics of the substances. Thus,
the stability of selected compounds in cell culture was
investigated. Two methods were chosen for assessing
compound stability under cell culture conditions: (1) a
time-dependent cytotoxicity assay; (2) time-dependent
RP-HPLC assay.
The cytotoxic potency of a substance will increase with
the duration of drug exposure if the compound concen-
trations remain at cytotoxic levels during the drug
exposure.17 Figure 10 shows the respective time-de-
pendent dose-response curves for 7 on the 5637 cell
lines. Cells were treated either continuously for 96 h
with substance or 24, 48, and 72 h, after which time
the medium was replaced with fresh medium and
incubation continued to 96 h. With increasing duration
of exposure, no changes in growth inhibitory potency
were observed; i.e., 7 had reached maximal activity by
24 h. Also, for compounds 10 and 11 no changes in the
IC50 values were observed after 24 h (data not shown).
This can be interpreted that these acrylonitriles are not
stable in culture, and by 24 h most of the compound
has been inactivated. However, for compound 9, the IC50

Heteroarylacrylonitriles
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3443
Figure 7. Cell line SISO treated 96 h with various substances: (A) control cells; (B) 1.0 µM 8; (C) 0.5 µM 11; (D) 1.0 µM 7. All
pictures are at 400-fold magnification.
Figure 8. Cell line LCLC-103H treated 96 h with various substances: (A) control cells; (B) 1.0 µM 8; (C) 0.5 µM 11; (D) 1.0 µM
7.
value continued to decrease by 40% between 48 and 72
h (data not shown).
To test further the hypothesis that acrylonitriles are
unstable under cell culture conditions, an RP-HPLC

3444 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Sa¸ czewski et al.
for 10, 7, and 11, respectively. There was no evidence
for the formation of the respective aldehydes in incuba-
tions with RPMI medium (without FCS), indicating that
a retro-Knoevenagel reaction is unlikely to occur with
these compounds under biological conditions.
Discussion
Some acrylonitriles have previously been reported to
possess antitumor properties. Ohsumi and co-workers
described various 2,3-diphenylacrylonitriles as combre-
tastatin A-4 analogues that possess tubulin polymeri-
zation inhibitory activity and were active on xenografts
of the human Grimmdarm tumor in mice.8 Another
group of acrylonitriles having EGF tyrosine kinase
inhibitory activity were described by Gazit and co-
workers.18 This kinase is overexpressed in many ma-
lignancies, and thus, such compounds were proposed as
antitumor agents. Most recently, Brzozowski and Sac-
zewski described novel acrylonitriles that inhibit the
growth of cancer cells.9 These acrylonitriles, substituted
at position 2 with a triazine ring and at position 3 with
a 5-nitrofuran ring, served as leads for the present
study.
The new acrylonitriles synthesized in this work show
interesting in vitro cytotoxic activity on human cancer
cells and likely induce cell death by a mechanism of
apoptosis. The SAR studies indicate a strong relation-
ship between structure and activity. These studies
indicate that the heterocyclic A ring is open for variation
such that triazines, triazoles, and benzimidazoles are
allowed. In contrast, small changes in the structure of
aromatic B ring have large effects on activity. The best
aromatic ring system proved to be thiophene, but the
type and position of the substituents in the thiophene
ring are also critical for activity.
Figure 9. Induction of caspase 3 (A) and caspase 9 (B) activity
in HL-60 cells by 8 µM etoposide (triangles) and 50 µM
compound 11 (squares), respectively. Data points are the
averages of three independent experiments. Error bars are (1
SD. Activity was measured as the amount of AMC- and AFC-
formation that could be inhibited by specfic inhibitors for
caspases 3 and 9, respectively.
The most active compounds in both groups possess a
nitro group in the aromatic B ring, suggesting that this
group is somehow involved in the mechanism of action.
When Cl or CH3 are substituted for H at position 5,
activity is diminished (compare 16 with 13 and 14). On
the other hand, replacement of H for NO2 increases
dramatically the activity (compare 16 with 11). This
could be due to either differences in electronic or
lipophilic properties of these groups; i.e., NO2 has M-
effects while both Cl and CH3 have M+ effects, but NO2
is a -π group while Cl and CH3 are both +π groups.
Interestingly, the position of the nitro group relative to
the acrylonitrile in the aromatic ring is critical for
activity; substitution at position 2 of the thiophene ring
gave the most potent compound (11), while substitution
at position 3 (12) resulted in an isomer almost com-
pletely lacking in activity (Table 3). Likewise, the
m-nitrophenyl derivative 17 was active while the para
isomer 18 was devoid of activity (Table 3). These results
appear contradictory because active 11 is biosteric with
inactive 18 but inactive 12 is biosteric with active 17.
The highly conjugated system likely forces the A and
B rings to lie in a nearly planar orientation to each
other. Theoretical calculations with 11 and X-ray crystal
structure analysis of 3 and 24 support this idea.
However, the barrier to rotation around the bond
between the acrylonitrile group and the B ring is
relatively low, and interconversion would be expected
under biological conditions.
Figure 10. Time-dependent cytotoxicity of 7 in the 5637 cell
line.
Figure 11. Stability of 10 at 37 °C under various cell culture
conditions: (b) RPMI medium; (9) medium + 10% FCS; (2)
medium + FCS + 5637 cells. HPLC peak areas were normal-
ized to the peak area at t ) 0.
assay was developed to monitor compounds 7, 10, and
11 in culture medium. Figure 11 shows the results of
the HPLC analysis of 10 in RPMI medium, RPMI
medium with 10% FCS, or in medium in the presence
of 5637 cells. Noticeable time-dependent decreases over
the first 24 h were apparent; in particular, when cells
were present, the decreases were 50%, 60%, and 90%

Heteroarylacrylonitriles
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3445
The acrylonitrile functionality is not an absolute
requirement for activity, and replacement of the nitrile
group for hydrogen (25) results in only a ca. 3-fold
reduction in potency. However, replacing the nitrile
group for a methyl group (compare 11 with 26) reduces
activity dramatically (Tables 3 and 4).
On the basis of the morphological changes in the cells
the compounds appear to cause a programmed cell death
(apoptosis). Notably, treatment of cells with 11 resulted
in a large proportion of giant cells after 96 h (Figures
6C-8C). To confirm the mechanism of cell death,
studies on the induction of the activities of caspase 3
and 9 were performed. Both of these enzymes are known
to be induced when cancer cells are exposed to antitu-
mor agents, such as etoposide.16 In this work, we show
that at a toxic dose compound 11 causes a time-
dependent induction of both caspase activities in the
HL-60 leukemia cell line in a manner similar to etopo-
side (Figure 9).
The selectivity of 11 for cancer cells is not great, as
evidenced by the potency in the noncancerous (h-TERT-
RPE1 epithelial cell line) cell line compared to the
cancer cell lines; the IC50 in the h-TERT cells was only
1.7-fold greater than the average IC50 of all 11 cancer
cell lines (0.39 vs 0.23 µM). However, the selectivities
of antitumor agents cisplatin and etoposide for the
cancer cell lines were even worse (Table 4); in fact,
etoposide was most active on the h-TERT-RPE1 cell line
compared to an average of 10 cancer cell lines (0.09 vs
0.54 µM).
Although the most active compound (11) was 10- and
3-fold more potent than cisplatin and etoposide, com-
paratively the activity is less discriminating over the
11 cancer cell line panel. This is apparent from the
relative standard deviations (RSD) of the IC50 values,
which are (91% for cisplatin, (65% for etoposide, and
(40% for the most active acrylonitrile 11. The most
sensitive cell lines were the 5637 and LCLC-103H, but
these cell lines are also quite sensitive to cisplatin. An
interesting compound in this respect is 17, which shows
good activity on the LCLC-103H lung cancer cell line
(IC50 ) 1.13 µM), i.e., comparable to cisplatin; however,
on the remaining cell lines it was only weakly active. It
would be interesting to test this compound on additional
lung cancer cell lines.
containing substances are known to undergo alkylation
reactions with acrylonitrile,19,20 and N-acetylcysteine is
used as an antidote for acrylonitrile poisoning.21 Alky-
lation of sulfur-containing nucleophiles such as glu-
tathione (GSH) and proteins by acrylonitriles, which
occur in vivo,22 are likely to occur in cell culture as well.
Future studies will focus on this possibility. Another
metabolic process that could occur is the reduction of
the aromatic nitro group by nitro-reductase activities,
but this reaction could only be occurring when cells are
present.
The initial cellular targets of attack by these com-
pounds are not yet known; however, like other known
antitumor agents, 11 caused apoptosis in the HL-60 cell
line. Like acrylonitrile, the compounds may act by
inhibiting sulfhydryl-dependent enzymes of intermedi-
ary metabolism by cyanoethylation of sulfhydryl groups
or by liberation of cyanide and subsequent inhibition
of cytochrome c oxidase.20 However, compounds 25 and
26, which both lack the nitrile group, are also active. If
nonspecific mechanisms were operative, then similar
potencies would also be expected over all 12 cell lines.
The data presented here, however, appear to favor a
selective interaction with a target enzyme or receptor,
but more work will be needed to clarify this.
In conclusion, the facile synthesis of acrylonitriles by
means of a Koevenagel condensation allowed for the
synthesis of a variety of 2,3-disubstituted analogues. A
range of cancer cell growth inhibitory activities were
observed for the compounds, from potencies 50-fold
greater than cisplatin for 11 in the KSYE-510 and the
YAPC cell lines down to totally inactive compounds.
Importantly, the acrylonitrile functionality is not an
absolute requirement for cytotoxic activity as are sub-
stituents at position 5 of the triazole ring; thus, this
position appears to be free for variations that could lead
to compounds with optimal physicochemical and phar-
macokinetic properties.
Experimental Section
The acrylonitriles are photosensitive, particularly in organic
solutions, and should be stored under protection from light.
Equipment. Melting points were measured on either a
Boetius or a MEL-TEMP apparatus and are not corrected. IR
spectra were taken in KBr pellets on a Perkin-Elmer FTIR
1600 spectrometer. NMR spectra were recorded on a Varian
Gemini 200, a Varian Unity 500, or a Brucker DPX 300
apparatus. 1H and 13C chemical shifts were measured relative
to residual solvent signal at 2.50 and 39.5 ppm, respectively.
UV-vis spectra were measured in methanol on a Spekol 1200
(Analytik Jena, FRG) apparatus. Unless otherwise noted,
analyses of C, H, N were within (0.4% of the theoretical
values.
Although it takes several days before morphological
changes in the cells become apparent, the compounds
achieve their greatest cytotoxic potency within the first
24 h of exposure (Figure 10). This led us to suppose
that the compounds were unstable under the culture
conditions and unleash their activity during the first
24 h of contact with cells. The time-dependent stabilities
of representative compounds (i.e., 7, 10, and 11) were
investigated by HPLC methods under various cell
culture conditions, i.e., in RPMI medium, in medium
with 10% FCS, and in cell culture, respectively. These
experiments confirmed that over the first 24 h in cell
culture a large fraction of these compounds was lost
from the medium (see Figure 11).
Cells were automatically counted by using a Coulter Counter
Z2 (Beckman-Coulter) instrument. Cell photography was
performed with a Axio Vert 25 (Zeiss, FRG) phase-contrast
microscope mounted with a Minolta AF camera and fitted with
a 40× objective.
Preparation of Condensation Products 1-25. Method
A. Synthesis of 1,2,4-Triazoles 1-9. Equimolar amounts of
(1,2,4-triazol-3-yl)acetonitrile23 and suitable aldehyde (8.2
mmol) were dissolved in ethanol (15 mL) and treated with
piperidine (5 mmol). The reaction mixture was heated under
reflux, and then the solvent was evaporated to dryness under
reduced pressure. The crude product thus obtained was
purified by crystallization from a suitable solvent: DMF (4,
5, 7), dioxane (2), methanol (6), ethanol (1,3).
Various reactions of acrylonitriles are imaginable,
e.g., retro-Knoevenagel reactions, Michael additions
with thiols and nitro group reductions. The respective
aldehydes were not observed in the HPLC studies, so
retro-Knoevenagel reactions appear unlikely. Thiol-
According to the above procedure, the following compounds
were obtained.

3446 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Sa¸ czewski et al.
2-(5-Methyl-1H-[1,2,4]-triazol-3-yl)-3-(5-nitrofuran-2-
yl)acrylonitrile (1): from 5-methyl-1H-[1,2,4]-triazol-3-yl)-
acetonitrile (1.0 g, 8.2 mmol) and 5-nitrofurfural (1.15 g, 8.2
mmol); reaction time 2 h; yield 0.31 g (16%); mp 256-260 °C
(ethanol); IR, ν 3277 (NH), 3142, 3095, 3054, 2237 (CN), 1522
(NO), 1349 (NO), 1252, 1043 cm-1; 1H NMR (DMSO-d6) δ 2.41
(s, 3H, CH3), 7.46 (d, 1H, J ) 4.4 Hz, 3-furyl), 7.83 (d, 1H, J
) 4.4 Hz, 4-furyl), 8.05 (s, 1H, dCH), 14,16 (s, 1H, NH); UV-
vis, λmax 245, 364 nm (log ꢀ ) 4.36); HPLC, tR ) 7.12 min. Anal
(C10 H7N5O3 (245.19)) C, H, N. N: calcd 28.57; found 29.62.
2-(5-Methyl-1H-[1,2,4]-triazol-3-yl)-3-(5-nitrothiophen-
2-yl)acrylonitrile (2): from 5-methyl-1H-[1,2,4]-triazol-3-yl)-
acetonitrile (1.0 g, 8.2 mmol) and 5-nitro-2-thiophene-carbox-
aldehyde (1.15 g, 8.2 mmol); reaction time 2 h; yield 0.54 g
(26%); mp 278-280 °C (dioxane); IR, ν 3307 (NH), 3095, 3072,
acetonitrile (1.0 g, 5 mmol) and 5-nitrofurfural (0.7 g, 5 mmol);
reaction time 2 h; yield 0.65 g (58%); mp 188-190 °C
(methanol) (ref 3 mp 188-190 °C); IR, ν 3242 (NH), 3130, 3048,
2919, 2231 (CN), 1537 (NO), 1346 (NO), 1246, 1014, 811, 705
cm-1; 1H NMR δ 4.15 (s, 1H, CH2), 7.20-7.40 (m, 5H, Ar-H),
7.48 (d, J ) 3.8 Hz, 1H, 3-furyl), 7.83 (d, J ) 3.8 Hz, 1H,
4-furyl), 8.06 (s, 1H, dCH), 14.40 (s, 1H, NH); UV-vis, λmax
245, 366 nm (log ꢀ ) 4.30); HPLC, tR ) 11.33 min. Anal.
(C16H11N5O2S (321.29)) C, H, N.
2-(5-Benzyl-1H-[1,2,4]-triazol-3-yl)-3-(4-dimethylami-
nophenyl)acrylonitrile (9): from 5-benzyl-1H-[1,2,4]-triazol-
3-yl)acetonitrile (1.0 g, 5 mmol) and 4-dimethylaminobenzal-
dehyde (0.75 g, 5 mmol); reaction time 2 h; yield 1.0 g (65%);
mp 199-200 °C (dioxane) (ref 3 mp 199-200 °C); IR, ν 3451,
3061, 2907, 2213 (CN), 1584, 1528 (NO), 1327 (NO), 1193, 816,
724 cm-1; 1H NMR (DMSO-d6): δ 3.0 (s, 6H, N(CH3)2), 4.1 (s,
2H, CH2), 6.80 (d, 2H, J ) 8.9 Hz, Ar-H), 7.20-7.40 (m, 5H,
Ar-H), 7.8 (d, 2H, J ) 8.9 Hz, Ar-H), 7.96 (s, 1H, dCH), 14.00
(s, 1H, NH); UV-vis, λmax 256, 391 nm (log ꢀ ) 4.47); HPLC,
tR ) 12.61 min. Anal. (C16H11N5O2S (329.44)) C, H, N.
Method B. Synthesis of Benzimidazoles 10-23. To an
equimolar amount of (benzimidazol-2-yl)acetonitrile24 and
corresponding aldehyde (6.4 mmol) dissolved in ethanol (20
mL) was added 10% methanolic KOH (10 mmol), and the
reaction mixture was kept at room temperature for 0.5 h. In
the cases of 13 and 14, heating under reflux was required.
Then the solution was cooled to 5 °C and the product that
precipitated was collected by filtration and purified by recrys-
tallization from a suitable solvent: ethanol (15, 16, 20-23),
ethanol/DMF (12, 17, 19), DMF (10, 11), acetone (18).
According to the above procedure, the following compounds
were obtained.
1
3030, 2225 (CN), 1504 (NO), 1328 (NO), 1246, 1078 cm-1; H
NMR (DMSO-d6) δ 2.41 (s, 3H, CH3), 7.89 (d, 1H, J ) 4.4 Hz,
3-thienyl), 8.21 (d, 1H, J ) 4.4 Hz, 4-thienyl), 8.46 (s, 1H, d
CH), 14.15 (s, 1H, NH); UV-vis, λmax 273, 379 (log ꢀ ) 4.38);
HPLC, tR ) 9.79 min. Anal. (C10H7N5O2S1 (261.27)) C, H, N.
2-(5-Methyl-1H-[1,2,4]-triazol-3-yl)-3-(3-nitrophenyl)-
acrylonitrile (3): from 5-methyl-1H-[1,2,4]-triazol-3-yl)aceto-
nitrile (1.0 g, 8.2 mmol) and 3-nitrobenzaldehyde (1.24 g, 8.2
mmol); reaction time 3 h; yield 0.57 g (28%); mp 181-182 °C
(ethanol); IR, ν 3154 (NH), 3077, 3025, 3030, 2220 (CN), 1525
1
(NO), 1347 (NO), 1163, 1061 cm-1; H NMR (DMSO-d6) 2.42
(s, 3H, CH3), 7.81-7.85 (m, 1H, Ar-H), 8.32-8.36 (m, 3H, Ar-
H), 8.86 (s, 1H, dCH), 14.09 (s, 1H, NH); UV-vis, λmax 299
nm (log ꢀ ) 4.34); HPLC, tR ) 8.79 min. Anal. (C12H9N5O2
(255.24)) C, H, N.
2-(5-Phenyl-1H[1,2,4]-triazol-3-yl)-3-(5-nitrofuran-2-yl)-
acrylonitrile (4): from 5-phenyl-1H-[1,2,4]-triazol-3-yl)aceto-
nitrile (1.0 g, 5.4 mmol) and 5-nitrofurfural (0.77 g, 5.4 mmol);
reaction time 1 h; yield 0.32 g (19%); mp 284-286 °C (DMF);
IR, ν 3213 (NH), 3154, 3048, 2225 (CN), 1522 (NO), 1349 (NO),
1258, 1122, 1031 cm-1; 1H NMR (DMSO-d6) δ 7.53 (d, 1H, J )
4.0 Hz, 3-furyl), 7.56-7.62 (m, 3H, Ar-H), 7.87 (d, 1H, J )
4,0 Hz, 4-furyl), 8.01-8.08 (m, 2 H, Ar-H), 8.19 (s, 1H, dCH),
15.00 (s, 1H, NH); UV-vis, λmax 239, 372 nm (log ꢀ ) 4.39);
HPLC, tR ) 14.72 min. Anal. (C15H9N5O3 (307,26)) C, H, N.
2-(5-Phenyl-1H-[1,2,4]-triazol-3-yl)-3-(5-nitrothiophen-
2-yl)acrylonitrile (5): from 5-phenyl-1H-[1,2,4]-triazol-3-yl)-
acetonitrile (1.0 g, 5.4 mmol) and 5-nitro-2-thiophenecarbox-
aldehyde (0.85 g, 5.4 mmol); reaction time 0.5 h; yield 1.08 g
(62%); mp 295-296 °C (DMF); IR, ν 3230 (NH), 3101, 2931,
2-(1H-Benzimidazol-2-yl)-3-(5-nitrofuran-2-yl)acryloni-
trile (10): from (benzimidazol-2-yl)acetonitrile (1.0 g, 6.4
mmol) and 5-nitrofurfural (0.90 g, 6.4 mmol); yield 0.84 g
(47%); mp 319-322 °C dec (ref 5a mp 312-313 °C dec); IR, ν
3265 (NH), 3154, 3048, 2243 (CN), 1522 (NO), 1469, 1349 (NO),
1263, 1178, 1031, 808, 735 cm-1;UV-vis, λmax 317, 405 nm
(log ꢀ ) 4.44); HPLC, tR ) 10.51 min. Anal. (C14H8N4O3
(280.23)) C, H, N.
2-(1H-Benzimidazol-2-yl)-3-(5-nitrothiophen-2-yl)acry-
lonitrile (11): from (benzimidazol-2-yl)acetonitrile (1.1 g, 7.0
mmol) and 5-nitro-2-thiophenecarboxaldehyde (1.1 g, 7.0
mmol); yield 1.47 g (71%); mp 348-350 °C dec; IR, ν 3277 (NH),
3107, 3048, 2231 (CN), 1513 (NO), 1334 (NO), 1225, 1119,
2220 (CN), 1651, 1501 (NO), 1337 (NO), 1208, 1116, 1031 cm-1
;
1028, 817, 729, 623 cm-1 1H NMR (DMSO-d6) δ 7.25-7.33
;
1H NMR (DMSO-d6) δ 7.56-7.62 (m, 3H, Ar-H), 7.96 (d, 1H,
J ) 4.6 Hz, 3-thienyl), 8.03-8.08 (m, 2H, Ar-H), 8.25 (d, 1H,
J ) 4,6 Hz, 4-thienyl), 8.58 (s, 1H, dCH), 15.00 (s, 1H, NH);
UV-vis, λmax 240, 386 nm (log ꢀ ) 4.34); HPLC, tR ) 23.36
min. Anal. (C15H9N5O2S (323.34)) C, H, N.
(m, 2H, Ar-H), 7.62-7.67 (m, 3H, Ar-H), 7.84 (d, 1H, J )
4.5 Hz, 3-thienyl), 8.25 (d, 1H, J ) 4.5 Hz, 4-thienyl), 8.59 (s,
1H, dCH), 13.25 (s, 1H, NH); UV-vis, λmax 300, 417 nm (log ꢀ
) 4.28); HPLC, tR ) 13.68 min. Anal. (C14H8N4O2S (296.31))
C, H, N.
2-(5-Phenyl-1H-[1,2,4]-triazol-3-yl)-3-(3-nitrophenyl)-
acrylonitrile (6): from 5-phenyl-1H-[1,2,4]-triazol-3-yl)aceto-
nitrile (1.0 g, 5.4 mmol) and 3-nitrobenzaldehyde (0.82 g, 5.4
mmol); reaction time 2 h; yield 0.80 g (47%); mp 279-281 °C
(methanol); IR, ν 3136 (NH), 3089, 3007, 2936, 2220 (CN), 1525
2-(1H-Benzimidazol-2-yl)-3-(2-nitrothiophen-4-yl)-
acrylnitrile (12): from 0.595 g (3.8 mmol) of (benzimidazol-
2-yl)acetonitrile and 5-nitro-3-thiophenecarboxaldehyde (0.595
g, 3.8 mmol) without added KOH; yield 0.66 g (59%); yellow
crystals; mp 236-239 °C dec; IR, ν 3296 (NH), 3112, 2231 (CN),
1
(NO), 1354 (NO), 1137, 996, 943 cm-1; H NMR (DMSO-d6) δ
1536 (NO), 1500, 1346 (NO), 1218, 1088, 769, 584 cm-1 1H
;
7.56-7.63 (m, 3H, Ar-H), 7.84 (t, 1H, Ar-H), 8.04-8.08 (m,
2H, Ar-H), 8.33-8.46 (m, 3H, Ar-H), 8.89 (s, 1H, dCH), 14.91
(s, 1H, NH); UV-vis, λmax 258, 308 nm (log ꢀ ) 4.44); HPLC,
tR ) 21.46 min. Anal. (C17H11N5O2 (317.31)) C, H, N.
NMR (DMSO-d6) δ 7.24-7.28 (m, 2H, Ar-H); 7.58-7.64 (m,
2H, Ar-H); 8.32 (s, 1H, dCH), 8.64 (s, 1H, thienyl), 8.65 (s,
1H, thienyl), 13.12 (bs, 1H, NH); UV-vis, λmax ) 271, 349 nm
(log ꢀ ) 3.74); HPLC, tR ) 13.17 min (λ ) 349 nm). Anal.
(C14H8N4O2S (296.31)) C, H, N, S. C: calcd 56.74; found 56.22.
S: calcd 10.82; found 11.02.
2-(1H-Benzimidazol-2-yl)-3-(5-chlorothiophen-2-yl)-
acrylnitrile (13): from 0.20 g (1.27 mmol) of (1H-benzimida-
zol-2-yl)acetonitrile and 0.187 g 5-chloro-2-thiophenecarbox-
aldehyde (1.27 mmol); yield 0.20 g (55%); green needles from
ethanol; mp 283-284 °C dec; IR, ν 3267 (NH), 3047, 2230 (CN),
1586, 1434, 1410, 1320, 1228, 1005, 906, 743; 1H NMR (DMSO-
d6) δ 7.26-7.10 (m, 2H, Ar-H), 7.41 (d, 1H, J ) 4.2 Hz,
thienyl), 7.59-7.61 (m, 2H, Ar-H), 7.72 (d, 1H, J ) 4.2 Hz,
thienyl), 8.48 (s, 1H, dCH), 13.05 (bs, 1H, NH); UV-vis, λmax
277, 381 nm (log ꢀ ) 4.38) HPLC, tR ) 23.44 min. Anal. (C14H8-
Cl1N3S (285,76)) C, H, N. H: calcd 2.83; found 3.37.
2-(5-Benzyl-1H-[1,2,4]-triazol-3-yl)-3-(5-nitrothiophen-
2-yl)acrylonitrile (7): from 5-benzyl-1H-[1,2,4]-triazol-3-yl)-
acetonitrile (1.0 g, 5.0 mmol) and 5-nitro-2-thiophenecarbox-
aldehyde (0.79 g, 5.0 mmol); reaction time 2 h; yield 0.35 g
(21%); mp 219-220 °C (DMF); IR, ν 3250 (NH), 3107, 3036,
2966, 2848, 2214 (CN), 1501 (NO), 1334 (NO), 1211, 1114,
1034, 867 cm-1; 1H NMR (DMSO-d6) δ 4.15 (s, 2H, CH2), 7.22-
7.38 (m, 5H, Ar-H), 7.91 (d, 1H, J ) 4.3 Hz, 3-thienyl), 8.21
(d, 1H, J ) 4.3 Hz, 4-thienyl), 8.45 (s, 1H, dCH), 14.40 (s, 1H,
NH); UV-vis, λmax 273, 380 nm (log ꢀ ) 4.24); HPLC, tR
15.73 min. Anal. (C16H11N5O2S (337.37)) C, H, N.
)
2-(5-Benzyl-1H-[1,2,4]-triazol-3-yl)-3-(5-nitrofuran-2-
yl)acrylonitrile (8): from 5-benzyl-1H-[1,2,4]-triazol-3-yl)-

Heteroarylacrylonitriles
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13 3447
2-(1H-Benzimidazol-2-yl)-3-(5-methylthiophen-2-yl)-
acrylnitrile (14): from 0.20 g (1.27 mmol) of (1H-benzimi-
dazol-2-yl)acetonitrile and 0.16 g of 5-methyl-2-thiophenecar-
boxaldehyde (1.27 mmol); yield 0.161 g (48%); yellow needles
from ethanol; mp 273-275 °C; IR, ν 3277 (NH), 3048, 2917,
2229 (CN), 1590, 1439, 1322, 1276, 1230, 1160, 1050, 748; 1H
NMR (DMSO-d6) δ 2.60 (s, 3H, CH3), 7.06 (d, 1H, J ) 4.2 Hz,
thienyl), 7.34-7.38 (m, 2H, Ar-H), 7.57-7.59 (m, 2H, Ar-
H), 7.61 (d, 1H, J ) 4.2 Hz, thienyl), 8.45 (s, 1H, dCH), 12.97
(bs, 1H, NH); UV-vis, λmax 281, 381 nm (log ꢀ ) 4.39) HPLC,
tR ) 16.24. Anal. (C15H11N3S (265,35)) C, H, N. H: calcd 4.19;
found 4.97.
nm (log ꢀ ) 4,43); HPLC, tR ) 10.58 min. Anal. (C17H10N4
(270.31)) C, H, N.
2-(1H-Benzimidazol-2-yl)-3-(4-methoxyphenyl)acryloni-
trile (21): from (benzimidazol-2-yl)acetonitrile (0.25 g, 1.6
mmol) and 4-methoxybenzaldehyde (0.22 g, 1.6 mmol); yield
0.27 g (61%); mp 227-229 °C (ref 26 mp 235 °C); IR, ν 3444,
3182 (NH), 2840, 2220 (CN), 1591, 1509, 1442, 1263, 1179,
1031, 829, 744 cm-1; 1H NMR (DMSO-d6) δ 3.88 (s, 3H, CH3),
7.16 (d, 2H, J ) 9.8 Hz, Ar-H), 7.22-7.31 (m, 2H, Ar-H),
7.51-7.75 (m, 2H, Ar-H), 8.01 (d, 2H, J ) 9.8 Hz, Ar-H),
8.28 (s, 1H, dCH), 13.01 (s, 1H, NH); UV-vis, λmax 263, 366
nm (log ꢀ 4.34); HPLC, tR ) 15.07 min. Anal. (C17H13N3O
(275.32)) C, H, N. C: calcd 74.16; found 69.88. N: calcd 15.27;
found 14.46.
2-(1H-Benzimidazol-2-yl)-3-(furan-2-yl)acrylonitrile (15):
from (benzimidazol-2-yl)acetonitrile (0.5 g, 3.2 mmol) and
furfural (0.31 g, 3.2 mmol); yield 0.38 g (51%); mp 192-194
°C (ref 5a mp 194-195.5 °C); IR, ν 3500-3300, 2225 (CN),
2-(1H-Benzimidazol-2-yl)-3-(4-trifluormethoxyphenyl)-
acrylonitrile (22): from (benzimidazol-2-yl)acetonitrile (0.30
g, 1.9 mmol) and 4-trifluormethoxybenzaldehyde (0.36 g, 1.9
mmol); yield 0.27 g (44%); mp 247-248 °C; IR, ν 3444, 3268
(NH), 2239 (CN), 1609, 1506, 1438, 1282, 1206, 1165, 908, 748,
1
1625, 1611, 1442, 1279, 1021, 746 cm-1; H NMR (DMSO-d6)
δ 6.85 (dd, 1H, 4-furyl), 7.19-7.31 (m, 2H, Ar-H), 7.36 (d, 1H,
5-furyl), 7.49-7.60 (m, 1H, Ar-H), 7.61-7.72 (m, 1H, Ar-H),
8.13 (d, 1H, 3-furyl), 8.16 (s, 1H, dCH), 13.01 (bs, 1H, NH);
UV-vis, λmax 278, 374 nm (log ꢀ 4.47); HPLC, tR ) 10.07 min.
Anal. (C14H9N3O (234.24)) C, H, N. C: calcd 71.47; found 66.49.
N: calcd 17.87; found 16.68.
1
630 cm-1; H NMR (DMSO-d6): δ 7.26-7.31 (m, 2H, Ar-H),
7.60-7.70 (d, 2H, J ) 8.4 Hz, Ar-H, m, 2H, Ar-H), 8.12 (d,
2H, J ) 8.4 Hz, Ar-H), 8.39 (s, 1H, dCH), 13.13 (bs, 1H, NH);
UV-vis, λmax 274, 348 nm (log ꢀ 4.22); HPLC, tR ) 25.95 min.
Anal. (C17H10F3N3O (329.29)) C, H, N.
2-(1H-Benzimidazol-2-yl)-3-(thiophen-2-yl)acryloni-
trile (16): from (benzimidazol-2-yl)acetonitrile (0.28 g, 1.8
mmol) and 2-thiophenecarboxaldehyde (0.20 g, 1,8 mmol); yield
0.20 g (45%); mp 222-224 °C (ref 25 mp 216-218 °C); IR, ν
3500-3200, 2218 (CN), 1624, 1594, 1442, 1411, 1327, 1051,
2-(1H-Benzimidazol-2-yl)-3-(4-trifluormethylphenyl)-
acrylonitrile (23): from (benzimidazol-2-yl)acetonitrile (0.30
g, 1.9 mmol) and 4-trifluoromethylbenzaldehyde (0.33 g, 1.9
mmol); yield 0.31 g (52%); mp 258-260 °C; IR, ν 3444, 3277
(NH), 2241 (CN), 1626, 1438, 1331, 1162, 1117, 1071, 911, 830,
746 cm-1 1H NMR (DMSO-d6) δ 7.20-7.29 (m, 2H, Ar-H),
;
748 cm-1 1H NMR (DMSO-d6) δ 7.27-7.32 (m, 2H, Ar-H),
;
7.34 (dd, 1H, 4-thienyl), 7.50-7.73 (m, 2H, Ar-H), 7.85 (d,
1H, 5-thienyl), 8.07 (d, 1H, 3-thienyl), 8.56 (s, 1H, dCH), 13.02
(bs, 1H, NH); UV-vis, λmax 282, 370 nm (log ꢀ 4.39); HPLC, tR
) 12.19. Anal. (C14H9N3S (251.33)) C, H, N. C: calcd 66.90;
found 62.74. N: calcd 16.72; found 15.72.
7.59-7.71 (m, 2H, Ar-H), 7.98 (d, 2H, J ) 8.3 Hz, Ar-H),
8.16 (d, 2H, J ) 8.3 Hz, Ar-H), 8.44 (s, 1H, dCH), 13.16 (bs,
1H, NH); UV-vis, λmax 269, 350 nm (log ꢀ 4.40); HPLC, tR
22.38 min. Anal. (C17H10F3N3 (313.30)) C, H, N.
)
Method C. Synthesis of Benzimidazoles 24 and 26. A
solution of 5-nitrothiophene-2-carboxaldehyde (3.8 mmol) and
an equimolar amount of corresponding 2-alkylbenzimidazole
in acetic anhydride (15 mL) was heated under reflux for 3 h.
The solution was evaporated to dryness under reduced pres-
sure, and the resulting oily residue was purified by crystal-
lization from ethanol in the presence of charcoal.
2-(1H-Benzimidazol-2-yl)-3-(3-nitrophenyl)acryloni-
trile (17): from (benzimidazol-2-yl)acetonitrile (0.28 g, 1.8
mmol) and 3-nitrobenzaldehyde (0.27 g, 1.8 mmol); yield 0.38
g (74%); mp 256-258 °C (ref 5a mp 263-265 °C; IR, ν 3500-
3200, 2232 (CN), 1625, 1528 (NO), 1439, 1349 (NO), 1104, 752,
669 cm-1 1H NMR (DMSO-d6) δ 7.28-7.31 (m, 2H, Ar-H),
;
7.59-7.72 (m, 2H, Ar-H), 7.88-7.95 (t, 1H, Ar-H), 8.35-8.42
(m, 2H, Ar-H), 8.50 (s, 1H, dCH), 8.84-8.85 (m, 1H, Ar-H),
13.19 (bs, 1H, NH); UV-vis, λmax 260, 351 nm (log ꢀ 4.35);
HPLC, tR ) 13.37 min. Anal. (C16H10N4O2 (290.28)) C, H, N.
According to the above procedure, the following compounds
were obtained.
1-Acetyl-2-[2-(5-nitrothiophen-2-yl)vinyl]-1H-benzimi-
dazole (24): from 2-methylbenzimidazole (0.5 g, 3.8 mmol) and
5-nitro-2-thiophencarboxaldehyde (0.6 g, 3.8 mmol); yield 0,5
g (42%); mp 171-173 °C; IR, ν 3085, 1721, 1488, 1331 (NO),
2-(1H-Benzimidazol-2-yl)-3-(4-nitrophenyl)acryloni-
trile (18): from (benzimidazol-2-yl)acetonitrile (0.28 g, 1.8
mmol) and 4-nitrobenzaldehyde (0.27 g, 1.8 mmol); yield 0.42
(81%); mp >300 °C (ref 5a mp 320-321 °C); IR, ν 3444, 3262
(NH), 2243 (CN), 1651, 1516 (NO), 1440, 1346 (NO), 1117, 908,
1290, 1033, 963, 806, 750 cm-1 1H NMR (CDCl3) δ 2.90 (s,
;
3H, CH3), 7.20 (d, J ) 3.9, 1H, 3-thienyl), 7.40-7.46 (m, 2H,
Ar-H), 7.68-7.73 (m, 1H, Ar-H), 7.78-7.82 (m, 1H, Ar-H),
7.85-7.90 (d, d, dCH, 4-thienyl), 7.97 (d, J ) 15.5 Hz, dCH).
Anal. (C15H11N3O3S (313.2)) C, H, N.
1
836, 771, 635 cm-1; H NMR (DMSO-d6) δ 7.26-7.37 (m, 2H,
Ar-H), 7.56-7.77 (m, 2H, Ar-H), 8.20 (d, 2H, J ) 9.2 Hz,
Ar-H), 8.43 (d, 2H, J ) 9,2 Hz, Ar-H), 8.47 (s, 1H, dCH),
13.20 (bs, 1H, NH); UV-vis, λmax 280, 371 nm (log ꢀ 4.25);
HPLC, tR ) 13.6 min. Anal. (C16H10N4O2 (290.28)) C, H, N.
2-[1-Methyl-2-(5-nitrothiophen-2-yl)vinyl]-1H-benzimi-
dazole (26): from 2-ethylbenzimidazole (0.5 g, 3.4 mmol) and
5-nitro-2-thiophenecarboxaldehyde (0.54 g, 3.4 mmol); yield 0.8
g (80%); mp 235-237 °C; IR, ν 3472 (NH), 1723, 1487, 1424,
2-(1H-Benzimidazol-2-yl)-3-(4-dimethylaminophenyl)-
acrylonitrile (19): from (benzimidazol-2-yl)acetonitrile (0.30
g, 1.9 mmol) and 4-dimethylaminobenzaldehyde (0.28 g, 1.9
mmol); yield 0.27 g (49%); mp >300 °C (ref 5a mp 313-314
°C); IR, ν 3445, 3249 (NH), 2225 (CN), 1614, 1589, 1537, 1450,
1
1340 (NO), 1189, 1034, 731 cm-1; H NMR (DMSO-d6) δ 2.57
(s, 3H, CH3), 7.21-7.24 (m, 2H, Ar-H), 7.44 (d, 1H, J ) 4.4
Hz, 3-thienyl), 7.51-7.74 (m, 2H, Ar-H), 7.87 (s, 1H, dCH),
8.20 (d, 1H, J ) 4.4 Hz, 4-thienyl), 12.81 (bs, 1H, NH); UV-
vis, λmax 295, 407 nm (log ꢀ 4.45); HPLC, tR ) 16.87 min. Anal.
(C14H11N3O2S (285.33)) C, H, N. C: calcd 58.93; found 55.55.
H: calcd 3.89; found 4.52. N: calcd 14.73; found 13.98.
Method D. 2-[2-(5-Nitrothiophen-2-yl)vinyl]-1H-benz-
imidazole (25) was obtained according to the procedure
described by Porousek.10 2-[(Triphenylphosphonium chloride)-
methyl]benzimidazole was subjected to a reaction with 5-nitro-
2-thiophenecarboxaldehyde in the presence of sodium meth-
oxide in methanol at room temperature to give 23, which
melted at 228-230 °C (ref 10 mp 193-194 °C); IR, ν 3105,
1631, 1522, 1501, 1430, 1349, 1323, 1202, 1039, 955 cm-1; 1H
NMR (DMSO-d6) δ 7.18-7.26 (m, 2H, Ar-H), 7.32 (d, J ) 16.2
Hz, 1H, dCH), 7.50-7.64 (m, 2H, Ar-H), 7.54 (d, J ) 4.0 Hz,
1H, 3-thienyl), 7.83 (d, J ) 16.2 Hz, 1H, dCH), 8.14 (d, J )
1
1379, 1276, 1203, 810, 727 cm-1; H NMR (DMSO-d6) δ 2.96
(s, 6H, N(CH3)2), 6.86 (d, 2H, J ) 9.1 Hz, Ar-H), 7.17-7.23
(m, 2H, Ar-H), 7.48-7.63 (m, 2H, Ar-H), 7.90 (d, 2H, J )
9.1 Hz, Ar-H), 8.13 (s, 1H, dCH), 12.79 (bs, 1H, NH); UV-
vis, λmax 430 nm (log ꢀ 4.53); HPLC, tR ) 20.71 min. Anal.
(C18H16N4 (288.38)) C, H, N.
2-(1H-Benzimidazol-2-yl)-3-(4-cyanophenyl)acryloni-
trile (20): from (benzimidazol-2-yl)acetonitrile (0.30 g, 1.9
mmol) and 4-cyanobenzaldehyde (0.25 g, 1.9 mmol); yield 0.29
g (56%); mp 270-272 °C; IR, ν 3442, 3330, (NH), 3077, 2234
(CN), 1626, 1415, 1319, 913, 740, 546 cm-1; 1H NMR (DMSO-
d6) δ 7.25-7.34 (m, 2H, Ar-H), 7.55-7.77 (m, 2H, Ar-H), 8.05
(d, 2H, J ) 8.6 Hz, Ar-H), 8.13 (d, 2H, J ) 8.6 Hz, Ar-H),
8.42 (s, 1H, dCH), 13.24 (bs, 1H, NH); UV-vis, λmax 276, 358

3448 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 13
Sa¸ czewski et al.
4.0 Hz, 1H, 4-thienyl), 12.8 (s, 1H, NH). Anal. (C13H9N3O2S
(271.3)) C, H, N.
7.0; 5 mM 1,4-dithiothreitol (DTT); 2 mM Na-EDTA; 0.1% (w/
v) CHAPS). After a 20 min incubation on ice, lysates were
centrifuged at 10000g for 10 min. The supernatant was
removed while taking care to avoid the pellet. Fifty microliter
aliquots containing 2-4 µg of cytosolic protein (estimated by
the Bradford method) were preincubated with 10 µM of
appropriate protease inhibitor (benzyloxycarbonyl-Asp-Glu-
Val-Asp-chloromethyl ketone and acetyl-Leu-Glu-His-Asp-
chloromethyl ketone (both from Bachem, Heidelberg, FRG) for
caspase 3 and 9, respectively) or DMSO (assay without
inhibitor) for 5 min at 21 °C and then diluted to 198 µL with
assay buffer (50 mM HEPES; 5 mM DTT; 2 mM Na-EDTA;
0,1% (w/v) CHAPS). The samples were frozen at -32 °C. All
experiments were performed within 1 week of extract prepara-
tion.
Caspase 3 and 9 activities were assessed by monitoring
cleavage of fluorochrome-tagged synthetic substrates Acetyl-
Asp-Met-Gln-Asp-7-amino-4-methylcoumarin (Ac-DMQD-AMC)
and Acetyl-Leu-Glu-His-Asp-amino-4-trifluoromethylcoumarin
(Ac-LEHD-AFC) (both from Bachem), respectively, by HPLC.
After the lysates were thawed, 2 µL of the appropriate
substrate was added (final concentrationa of 150 and 200 µM
for Ac-DMQD-AMC and Ac-LEHD-AFC, respectively). The
samples were incubated for 30 min at 30 °C, and the specific
products were quantified by RP-HPLC. The chromatography
system used here consisted of an L-7100 pump controlled by
a D-7000 interface (Merck, Darmstadt, Germany) and a 7125
Rheodyne sample injector fitted with a 20 µL injection loop.
Detection was done with an L-7485 fluorescence detector
(Merck); fluorescence was measured by using an excitation
wavelength of 380 or 400 nm and an emission wavelength of
460 or 505 nm for released AMC or AFC, respectively.
X-ray Crystallography of 3 and 24. Crystal Data for
C12H9N5O2 (3). Orthorhombic, space group Pbca, a ) 9.2700-
(4) Å, b ) 13.7023(6) Å, c ) 18.3866(7) Å, V ) 2335.5(2) Å3, Z
) 8, dx ) 1.452 g‚cm-3, T ) 110 K. Data were collected for a
crystal with dimensions 0.25 mm × 0.25 mm × 0.05 mm on a
KumaCCD diffractometer using graphite monochromated Mo
KR radiation. Final R indices for 2112 reflections with I > 2σ-
(I) and 209 refined parameters are the following: R1 ) 0.0480,
wR2 ) 0.10085 (R1 ) 0.0591, wR2 ) 0.1067 for all 2382 data).
Crystal Data for C15H11N3O3S (24). Triclinic, space group
P1h, a ) 6.5988(6) Å, b ) 8.3769(7) Å, c ) 13.0684(11) Å, R )
94.580(7)°, â ) 99.949(7)°, γ ) 102.076(7)°, V ) 690.72(10) Å3,
Z ) 2, dx ) 1.507 g‚cm-3, T ) 130 K. Data were collected for
a crystal with dimensions 0.35 mm × 0.25 mm × 0.01 mm on
a KumaCCD diffractometer using graphite monochromated Mo
KR radiation. Final R indices for 2237 reflections with I > 2σ-
(I) and 243 refined parameters are the following: R1 ) 0.0407,
wR2 ) 0.0870 (R1 ) 0.0588, wR2 ) 0.0952 for all 2809 data).
Cytotoxicity Studies. All cell culture reagents were
purchased from Sigma (Deisenhofen, FRG). Cancer cell lines
were obtained from the German Collection of Microorganisms
and Cell Cultures (DSMZ) (Braunschweig, FRG). The h-TERT-
RPE1 cell line was obtained from Clontech (Heidelberg, FRG).
The culture medium was RPMI-1640 medium except for the
h-TERT-RPE1 cell line, where DMEM/F-12 medium was used.
All mediums were supplement with 2 g/L NaHCO3 and 10%
FCS. For the MCF-7 cell line, the RPMI medium was supple-
mented with 1 mM pyruvate, MEM salts, and amino acids.
Cells were grown in 75 cm2 plastic culture flasks (Sarstedt,
Nu¨mbrecht, FRG) in a humid atmosphere of 5% CO2 at 37 °C
and passaged shortly before becoming confluent. Cytotoxicity
determinations are based on cellular staining with crystal
violet and were performed as previously described.17 Briefly,
a volume of 100 µL of a cell suspension was seeded into 96-
well microtiter plates (Sarstedt) at a density of 1000 cells per
well except for the LCLC-103H and KYSE-510 cell lines, which
were plated out at 250 and 500 cells per well, respectively.
Twenty-four hours later, cells were exposed to the substance
at five dilutions (geometric) continuously for the next 96 h. At
the end of the exposure time, the medium was removed and
the cells were fixed with a glutaraldehyde solution. The cells
were then stained with crystal violet and the OD was
measured at λ ) 570 nm with a 2010 plate reader (Anthos).
The percent growth values were calculated by the equation
Control experiments (data not shown) confirmed that the
release of AMC or AFC was linear for at least 60 min at the
conditions specified.
To estimate a specific caspase activity, measurements of
blank (substrate only), negative control (substrate + cell
lystate + inhibitor), and positive controls (substrate + cell
lysate) were performed. From each result obtained as a
fluorescence (peak area), a blank was subtracted. Standards
containing 0-15 pmol of AMC and 0-30 pmol of AFC were
utilized to determine the amount of fluorochrome released. The
results are presented as the amount of AMC or AFC release
((pmol/mg)/min) that can be blocked by selective protease
inhibitor.
HPLC Stability Studies. The HPLC system consisted of
a P580 pump, a STH 585 column oven set at 37 °C, and a UVD
170S UV-vis detector, all from Dionex. Data management and
analysis were done with the Chromeleon software (Dionex).
Injections were performed with a VICI microelectric valve
actuator (Valco) fitted with a 500 µL injector loop. All chro-
matography was done with a 120-7 C18 Nucleosil column with
a mobile phase of 3:2 MeOH/H2O mM phosphate buffer (pH
3.3) at a flow rate of 0.6 mL/min. For all compounds, an
amount of 500 µL of a 10 µM solution in buffer was injected
and detection was done at the λmax value. Thiourea dead-time
was 3.6 min.
For stability determinations, the compounds were added to
RPMI medium, medium + 10% FCS, or 5637 cells growing as
described above for the cytotoxicity studies. A 1000-fold
dilution from a DMF stock solution gave a final concentration
of 10 µM. Samples were incubated in the dark at 37 °C in an
atmosphere of 5% CO2. At various time points, aliquots were
removed and 500 µL were analyzed by HPLC. For samples
containing FCS, ultrafiltration with an Amicon MPS system
(Millipore) with a YMT 30 kDa filter was performed prior to
chromatography.
ODT - ODc,0
ODc - ODc,0
growth (%) )
× 100
where ODT is the mean absorbance of the treated cells, ODc is
the mean absorbance of the controls, and ODc,0 is the mean
absorbance at the time the drug was added. The IC50 values
were estimated by a linear least-squares regression of the
growth values versus the logarithm of the substance concen-
tration; only concentrations that yielded growth values be-
tween 10% and 90% were used in the calculation. The reported
IC50 values are the average of two to three independent
experiments, and these varied less than 20% from the indi-
vidual values.
Caspase Induction Studies. HL-60 human acute myeloid
leukemia cells (DSMZ) were maintained in logarithmic growth
in RPMI 1640 supplemented with 10% fetal bovine serum, 30
mg/L of penicillin G, and 40 mg/L of streptomycin sulfate.
Apoptosis was induced by treatment (1 × 105 cells/mL) with 8
µM etoposide or 50 µM compound 11. IC50 values determined
with an MTT assay for etoposide and compound were 1 and
20 µM, respectively. Cells were incubated in a humid atmo-
sphere of 5% CO2 at 37 °C, and the cell lysates were prepared
at 0, 2, 4, 6, 8, and 24 h after treatment.
Acknowledgment. Support from the Fonds der
Chemischen Industrie (P.J.B.) and the European Com-
munity (P.R.) is acknowledged. P.R. also thanks the
Sparkasse Vorpommern for financial support. We thank
Prof. H.-H. Otto for valuable discussions.
For the preparation of the cell-free extracts, all steps were
performed at 4 °C. Cells were pelleted at 500g for 4 min,
washed with PBS, once again pelleted at 500g for 3 min, and
resuspended in hypotonic HEPES buffer (10 mM HEPES, pH

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