Arylcyanoacrylamides as inhibitors of the Dengue and West Nile virus proteases

Article abstract of DOI:10.1016/j.bmc.2011.10.061

The 3-aryl-2-cyanoacrylamide scaffold was designed as core pharmacophore for inhibitors of the Dengue and West Nile virus serine proteases (NS2B-NS3). A total of 86 analogs was prepared to study the structure-activity relationships in detail. Thereby, it turned out that the electron density of the aryl moiety and the central double bond have a crucial influence on the activity of the compounds, whereas the influence of substituents of the amide residue is less relevant. The para-hydroxy substituted analog was found to be the most potent inhibitor in this series with a K_i-value of 35.7 μM at the Dengue and 44.6 μM at the West Nile virus protease. The aprotinin competition assay demonstrates a direct interaction of the inhibitor molecule with active centre of the Dengue virus protease. The target selectivity was studied in a counterscreen with thrombin and found to be 2.8:1 in favor of DEN protease and 2.3:1 in favor of WNV protease, respectively.

Full text of DOI:10.1016/j.bmc.2011.10.061

Bioorganic & Medicinal Chemistry 19 (2011) 7318–7337
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Arylcyanoacrylamides as inhibitors of the Dengue and West Nile virus proteases
⇑
Christoph Nitsche, Christian Steuer, Christian D. Klein
Medicinal Chemistry, Institute of Pharmacy and Molecular Biotechnology IPMB, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
The 3-aryl-2-cyanoacrylamide scaffold was designed as core pharmacophore for inhibitors of the Dengue
and West Nile virus serine proteases (NS2B-NS3). A total of 86 analogs was prepared to study the struc-
ture–activity relationships in detail. Thereby, it turned out that the electron density of the aryl moiety
and the central double bond have a crucial influence on the activity of the compounds, whereas the influ-
ence of substituents of the amide residue is less relevant. The para-hydroxy substituted analog was found
Received 20 September 2011
Revised 18 October 2011
Accepted 19 October 2011
Available online 25 October 2011
to be the most potent inhibitor in this series with a Ki-value of 35.7 lM at the Dengue and 44.6 lM at the
Keywords:
West Nile virus protease. The aprotinin competition assay demonstrates a direct interaction of the inhib-
itor molecule with active centre of the Dengue virus protease. The target selectivity was studied in a
counterscreen with thrombin and found to be 2.8:1 in favor of DEN protease and 2.3:1 in favor of
WNV protease, respectively.
Dengue virus
West Nile virus
Flavivirin
NS2B-NS3 protease
Nitrile
Ó 2011 Elsevier Ltd. All rights reserved.
Aldol condensation
1. Introduction
tuned to match the requirements of the target while maintaining a
high degree of selectivity and stability. It has therefore been possi-
Dengue fever, West Nile fever and other infectious diseases
caused by flaviviruses are of growing interest in pharmaceutical re-
search. The number of flaviviral infections, especially in the north-
ern hemisphere, has increased in the last decade.1 Infections with
the Dengue (DEN) or West Nile viruses (WNVs) are currently
neither preventable by immunization nor treatable by chemother-
apeutics. Against this background, the viral serine protease NS2B-
NS3 is an attractive target to develop new therapeutics against
DEN, WNV and other flaviviral infections.2 Because of its role in
the posttranslational processing of the viral polyprotein, the
NS2B-NS3 protease is essential for the viral life cycle and replica-
tion mechanism. Therefore, inhibition of this enzyme is expected
to interfere with virus replication in the human host cells. Primar-
ily for the DEN protease there are currently no reports of drug-like
small molecule inhibitors with sufficient potency for advanced
preclinical studies.
The oxygen nucleophile in serine and threonine proteases can
be targeted by various electrophiles, which results in a covalent-
reversible or covalent-irreversible inhibition of the enzyme. Quite
frequently, peptidyl-aldehydes are used to inhibit serine proteases
(also DEN and WNV) in the context of crystallographic studies.3
Other electrophiles include halomethylketones and similar func-
tional groups. These electrophiles can be useful for in vitro studies,
but are too reactive for any type of advanced biological experiment
or clinical use. Fortunately, the reactivity of the electrophile can be
ble to apply the strategy of covalent inhibition of serine proteases
in the development of inhibitors of the proteasome (bortezomib, a
boronic acid derivative),4 hepatitis C virus protease (telaprevir and
other ketoamides),5 and most recently dipeptidyl peptidase IV (vil-
dagliptin and other nitriles).6 These prominent examples clearly
demonstrate that the integration of suitable electrophiles into
the design considerations for inhibitors of serine proteases, is an
advantageous approach that has considerable clinical significance.
We previously identified the cinnamyl moiety as a valuable
fragment which, in connection with the ketoamide electrophile,
yields potent inhibitors of the DEN and WNV proteases.7 This work
also demonstrated the benefit of a possible additional covalent
mode of inhibition to obtain inhibitors with sufficient potency
and selectivity whose antiviral activity could also be shown in a
cell-culture experiment of DEN replication.
The design approach for the inhibitors described in the present
work was to combine the promising cinnamyl moiety with an
alternative electrophile, the nitrile group. The resulting 3-aryl-2-
cyanoacrylamides, whose general chemical structure is shown in
Figure 1, are drug-like compounds. We assume that the cinnamyl
structure, with an aryl substituent in R1 and a proton or small
aliphatic substituent in R2, interacts with the S1 pocket of the
NS2B-NS3 protein. The nitrile may act as an electrophilic trap for
the catalytic serine to induce a dipole-dipole interaction or to cre-
ate a reversible covalent bond between inhibitor and protease
(Fig. 1).8 An amide residue serves to complete a peptidomimetic
structure and may interact with the S01 recognition elements of
the enzyme.
⇑
Corresponding author. Tel.: +49 6221 544875; fax: +49 6221 546430.
E-mail address: c.klein@uni-heidelberg.de (C.D. Klein).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.061

C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
7319
2.2. Structure–activity relationships
The compounds were screened against the DEN and WNV pro-
teases (NS2B-NS3) and thrombin (as a counterscreen for selectivity
studies). The results at the different targets are shown in Table 1.
The best activities against DEN and WNV proteases were observed
for the para-hydroxy derivatives 21 and 23–27. Some of those
derivatives show inhibition values of more than 50% at 50 lM
inhibitor and substrate concentration at DEN and WNV proteases.
In general it can be observed that an aromatic system with an elec-
tron withdrawing group resulted in low activity, whereas electron
donor groups like hydroxy or amine substituents gave best results.
Para-substituted aromatic systems showed better activity than
meta-substituted and para,meta-disubstituted systems. There is a
loss of nearly 40% of activity if the hydroxy substituent is posi-
tioned in meta (28) and not in para position (21). Various halogen,
amine or methoxy substituents in the para-position were unable to
maintain the activity of the hydroxy-substituted analogs. However,
non-functionalized aliphatic chains like methyl (5) or isopropyl
(6–9) gave satisfying results. Various heterocycles, also shown in
Table 1, did not result in increased activity. In contrast to the
importance of the aryl moiety we only found a limited influence
of the amide residue (Table 1). Attempts to mimic the substrate
more closely by attaching a serine residue (for S01 pocket interac-
tion) in compounds 16 and 17 were unsuccessful. Apparently, the
S1 and S2 pockets are most important for molecular recognition at
the DEN and WNV proteases. We assume that the aryl moiety
interacts with the S1 pocket of the enzyme, probably combined
with an interaction of the electrophilic nitrile and the adjacent cat-
alytic serine (Fig. 1).
An enlargement of inhibitor molecules towards the S2 pocket
can be expected to increase the activity and selectivity of the com-
pounds. Therefore, we synthesized molecules which are able to
create additional interactions in the direction of the S2 pocket. An
extension of the compounds without modification of the underly-
ing arylacrylonitrile pharmacophore is only possible at the olefinic
R2 position or at the aromatic system. Non-hydrogen substituents
at R2 caused a pronounced decrease in activity. An additional
methyl group at R2 (as in compound 22a) leads to a decrease of
activity of more than 30% in comparison to 21 at all targets. The
geometry of the double bond is apparently not critical in all tested
R1- and R2-disubstituted compounds (2a/b, 3a/b, 4a/b, 22a/b).
Various extensions of the aromatic system were also evaluated.
The para-benzyloxy substituted derivative 72 was an initial test for
a possible occupation of the S2 pocket. This attempt resulted in an
inactive molecule at both tested viral targets in comparison to its
analog 32. Next we evaluated cyclic systems with restricted flexi-
bility. For this we synthesized substituted cyclic N-arylamines
(55–63).
Figure 1. Pharmacophoric model and possible molecular interactions. R1 = aryl,
R2 = H or alkyl, R3 = amine, S1 and S0 describe enzyme pockets, Ser135 is the serine
1
residue of the catalytic triad.
2. Results and discussion
2.1. Synthesis
For the synthesis of 3-aryl-2-cyanoacrylamides a one- or two-
step approach was used as shown in Scheme 1. If unsubstituted
2-cyanoacetamide was used (R3 = NH2), step (b) in Scheme 1 was
employed to obtain 3-aryl-2-cyanoacrylamide derivatives. Other-
wise the N-substituted 2-cyanoacetamide had to be synthesized
in a previous reaction step from methyl 2-cyanoacetate in an effi-
cient direct treatment with the required amine without solvent at
room temperature (step (a) in Scheme 1).9 In some cases the
carbonyl component (mostly aromatic aldehydes) had to be syn-
thesized from other, commercially available compounds. The aldol
condensation with aromatic aldehydes was carried out in metha-
nol with N-methylpiperazine as catalyst leading in a mostly
selective and quantitative fashion to the trans-olefin product. The
trans-geometry of the products was verified by the crystal struc-
ture of compound 14.10 Only in a few cases a small amount of
cis-olefin was obtained (detected by LC–MS). However, the use of
ketones instead of aldehydes gave mixtures of cis and trans isomers
(Table 1). By X-ray crystal structural analysis of compound 3b10
and NMR correlation, we were able to determine the absolute
structure of all compounds. The approach towards the cyclic N-
arylamines 55–63, 70 and 71 depends on the desired N-aryl-
connection in ortho, meta or para position. The para-substituted
compounds 55–60 are available via a one pot/three component
reaction of the corresponding cyanoacetamide, the 4-fluorobenzal-
dehyde and the secondary cyclic amine as shown in Scheme 2.11
We were also able to transfer this reaction to 2-fluorobenzalde-
hyde to obtain the ortho-substituted derivative 63. Pentafluoro-
benzaldehyde as starting material for derivative 61 reacted
selectively in para-position at room temperature. The meta-substi-
tuted derivative 62 and the heterocyclic compounds 70 and 71
were available by a copper catalyzed coupling reaction of the cyclic
amine with the corresponding bromoarylaldehyde.12
The evaluation of the cyclic N-arylamine derivatives showed, in
accordance with the results described before, that para-substituted
aromatic systems gave best activity results, whereas ortho- or meta-
substitution resulted in decreased activities. Best results showed
compounds 59 and 60 with good activities at the DEN and WNV pro-
tease. The perfluorinated compound 61 shows a decline in activity in
comparison to the non-fluorinated derivatives 59 and 60. This is also
in correlationwith the results described before for the simple substi-
tuted aryl moieties. In general, it therefore seems obvious that an
aromaticsystem withhigh electron densityis needed for an effective
interaction with the target. The 4-hydroxy substituted derivatives
remained the most active compounds in this series. Different aro-
matic amide derivatives 64–66 were not able to increase activity.
The same unsatisfying results were observed with the less drug-like
double-unsaturated compounds 67–69.
The aromatic amide derivatives 64–66 were available by amide
coupling reaction of the desired amine and the aromatic carboxylic
acids 37–39.
The double bond of compounds 40, 20, 57, 54, 69 and 67 could
be reduced selectively with sodium borohydride in methanol to
obtain the saturated analogs 76–81.
Scheme 1. Synthetic approach for simple 3-aryl-2-cyanoacrylamides. Reagents and
conditions: (a) no solvent, rt, 20 h; (b) N-methylpiperazine, MeOH, rt, 20 h; (c)
NH4OAc/AcOH, toluene, reflux, 2–5 h.
Because of the apparent structural analogy of the explored com-
pounds with the known catecholamine O-methyl transferase

Table 1
Activity of substituted arylacrylamides against the Dengue and West Nile virus proteases in comparison to thrombin.
No.
R1
R2
R3
DENa
WNVb
F2c
No.
R1
R2
H
R3
DENa
n.i.
WNVb
F2c
n.i.
1
H
NH2
15.0 1.6
9.3 4.9
12.5 5.2
35
NH2
10.5 2.2
2a
2b
Me
NH2
NH2
n.i.
13.7 1.1
18.4 3.2
7.3 3.9
9.7 2.6
36
37
H
H
NH2
NH2
13.3 1.9
17.1 1.6
20.0 6.3
23.8 2.5
10.6 5.1
13.2 5.1
Me
9.0 2.1
3a
i-Pr
NH2
n.i.
n.i.
16.7 9.7
38
H
20.0 1.2
31.0 1.1
15.9 1.5
3b
4a
i-Pr
NH2
NH2
n.i.
13.8 4.6
15.3 4.0
10.1 2.9
11.1 1.1
39
40
H
H
20.2 2.2
35.1 2.4
20.1 5.2
46.0 0.9
11.9 2.4
21.6 0.7
Pr
6.7 1.8
NH2
4b
5
Pr
NH2
NH2
NH2
7.6 2.7
16.0 1.7
22.8 3.7
24.6 3.3
22.2 4.2
46.3 1.0
6.9 0.9
n.i.
41
42
43
44
45
H
H
H
H
22.6 0.8
23.3 3.8
25.3 4.5
43.3 2.7
23.2 3.1
33.2 3.1
28.1 3.7
21.3 3.5
38.1 2.0
24.1 2.5
23.7 2.5
19.8 3.5
n.i.
H
H
H
H
32.2 5.4
23.0 3.3
18.2 4.5
28.2 1.2
6
19.4 5.2
17.9 5.4
14.4 6.0
7
25.1 2.9
10.3 2.1
8
NH2
9
H
H
24.9 1.3
22.1 2.4
39.5 1.7
21.7 0.8
11.6 4.1
8.7 1.2
46
47
H
NH2
NH2
n.i.
n.i.
n.i.
n.i.
n.i.
n.i.
10
NH2

Table 1 (continued)
No.
R1
R2
R3
DENa
WNVb
F2c
No.
R1
R2
R3
DENa
WNVb
F2c
11
12
H
H
NH2
NH2
22.5 7.4
14.7 2.3
16.0 2.1
10.7 3.0
7.9 5.5
8.5 2.1
48
49
H
H
NH2
NH2
28.4 7.9
22.0 7.3
23.9 2.5
23.8 3.8
15.7 3.7
27.9 1.3
13
H
NH2
11.6 2.0
15.9 2.0
13.6 8.2
50
H
NH2
20.2 2.9
19.2 0.8
26.3 0.8
14
15
16
H
H
H
10.9 0.6
19.7 1.0
20.3 1.3
22.6 2.1
23.4 0.9
21.1 1.5
n.i.
51
52
53
H
H
H
n.i.
10.5 2.1
19.3 1.0
23.9 3.7
17.9 2.2
10.1 6.9
26.9 0.7
NH2
16.7 7.8
21.4 4.7
NH2
18.4 1.4
15.6 3.7
17
18
H
H
23.0 1.7
16.3 5.8
31.7 2.8
12.3 1.7
23.3 6.0
9.6 7.6
54
55
H
H
NH2
NH2
23.7 0.6
24.3 8.7
33.4 2.8
24.6 2.1
16.4 2.0
17.7 5.8
NH2
NH2
NH2
NH2
NH2
NH2
19
H
H
H
19.2 2.7
15.6 2.9
51.7 1.0
16.8 1.4
13.2 1.6
22.0 2.2
19.3 2.5
55.0 4.6
21.9 5.7
22.7 1.9
20.0 3.3
17.8 6.5
24.4 4.9
n.i.
56
57
58
59
60
H
H
H
H
H
NH2
NH2
NH2
NH2
14.0 4.1
28.2 5.8
28.6 3.9
32.3 1.1
37.4 1.1
24.2 5.4
35.3 3.5
45.0 1.8
40.9 2.4
47.8 2.5
20.0 7.2
19.2 3.1
26.1 6.5
22.0 3.0
21.8 3.0
20
21
22a
Me
22b
Me
n.i
(continued on next page)

Table 1 (continued)
No.
R1
R2
R3
DENa
WNVb
F2c
No.
R1
R2
H
R3
DENa
WNVb
F2c
23
24
H
H
44.7 0.7
57.3 5.8
18.8 1.7
61
NH2
19.2 2.6
31.9 2.7
28.5 3.8
46.4 0.1
46.6 4.9
44.4 1.0
41.8 1.7
16.8 0.6
18.9 3.5
62
63
H
H
18.5 4.8
10.4 3.0
20.5 1.7
7.7 2.1
9.7 4.6
7.4 0.3
25
H
NH2
NH2
26
27
28
29
30
31
32
H
H
H
H
H
H
H
32.2 7.9
35.4 2.7
15.7 1.8
34.9 2.0
24.3 1.7
21.0 1.7
43.2 6.3
49.9 4.9
35.1 1.7
18.1 2.4
28.8 1.7
28.2 0.4
32.4 0.7
42.9 3.6
18.0 1.9
20.8 7.7
9.8 9.7
64
65
66
67
68
69
70
H
H
H
H
H
H
H
22.3 8.5
22.9 2.2
10.8 1.8
30.2 1.1
36.8 3.4
10.3 0.9
23.2 3.8
24.0 6.0
23.0 0.3
19.1 1.8
34.6 0.8
30.1 2.6
17.1 1.3
29.1 1.4
n.i.
9.0 1.3
8.0 2.9
20.5 1.3
28.7 2.1
16.2 7.8
38.6 8.1
NH2
NH2
NH2
18.4 9.7
22.5 2.0
21.9 6.2
17.4 4.7
NH2
NH2
NH2
NH2
NH2
33
H
H
30.5 3.1
43.1 0.1
43.8 3.7
28.8 3.9
23.0 3.7
71
72
H
H
10.5 5.0
24.0 0.5
47.0 4.0
16.0 2.4
34
24.4 6.7
NH2
n.i.
n.i.
a
% Inhibition of the Dengue Virus (DEN) NS2B-NS3 protease (enzyme: 100 nM; inhibitor 50
% Inhibition of the West Nile Virus (WNV) NS2B-NS3 protease (enzyme: 150 nM; inhibitor 50
l
M; substrate 50
lM; substrate 50 lM).
l
M).
b
c
% Inhibition of thrombin (F2) (enzyme: 10 nM; inhibitor 25 M; substrate 50 M).
l
l

C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
7323
Table 2
Activity of derivatives without acrylamide structure.
No. Structure
DENa
WNVb
n.i.
F2c
73
n.i.
13.7 3.2
Scheme 2. Three component reaction. Reagents and conditions: X = CH2, O, NH,
NMe: (a) EtOH, microwave: 20 min, 160 °C.
74
75
76
38.7 1.1 46.5 1.5 13.3 3.2
(COMT) inhibitor entacapone, we also tested this substance. Simi-
lar to all our explored compounds bearing a hydroxy function in
para-position, entacapone (31) shows activity at both tested viral
targets. However, the second hydroxy and the nitro group in
meta-position caused a loss of activity in comparison to the only
para-hydroxy substituted derivatives.
8.7 3.8
7.1 3.2
8.8 3.1
16.3 3.7
In addition to the aryl moiety and the amide residue we ex-
plored alternative structures of the acrylamide skeleton. For this
we synthesized different analogs which are shown in Table 2. A
replacement of the carbonyl oxygen by sulfur leads to the thio-
acrylamide compound 73 and to a total loss of activity at the tested
viral proteases. The implementation of a second nitrile function in
compound 75 causes a near-complete loss of activity. The acrylic
acid 74 shows activity in a similar range as the analogous acrylam-
ide compound 40. One may speculate about the necessity of a
hydrogen bond between the amide or acid residue and the target
protein. This could explain the observation that the double nitrile
derivative 75 is not very potent, whereas its carboxylic acid analog
74 shows good activity. The acid may in this case act as a bioisos-
teric replacement of the unsubstituted amide functionality.
In addition, we explored the necessity of the double bond by
selective reduction. In nearly all cases we observed a significant
dependence of the activity on the acrylamide double bond (com-
pounds 76–81, Table 2).
16.2 2.0 n.i.
77
78
n.i.
n.i.
n.i.
n.i.
7.9 3.8
n.i.
n.i.
79
80
n.i.
n.i.
Although the double bond is very important for activity, there
was no evidence for a 1,4-electrophilic attack at the catalytic ser-
9.0 2.8
37.0 2.1 6.7 3.5
ine. To exclude a potential ‘Michael reactivity’ of the
a,b-unsatu-
rated system, the most active compounds 8, 21, 40 and 71 were
incubated with glutathione as a representative biological nucleo-
phile and the resulting mixture was studied by HPLC. These
experiments clearly indicated that the compounds are inert to
1,4-addition (data not shown).
The planar geometry of the unsaturated side-chain with limited
degrees of rotational freedom appears to be important for the non-
covalent recognition of the inhibitor. Only at compound 80 we
found a satisfying activity at the WNV protease. This derivative
carries an amine substituent in para-position and double bond in
benzyl position, without a Michael-reactive functionality. This fur-
ther verifies the conclusion that a Michael-reactive group is not a
critical necessity in this compound class. Finally, the exotic deriv-
ative 82 did not show relevant activity.
81
82
n.i.
n.i.
n.i.
n.i.
11.2 1.2 n.i.
a
% Inhibition of the Dengue Virus (DEN) NS2B-NS3 protease (enzyme 100 nM;
inhibitor 50 M; substrate 50 M).
% Inhibition of the West Nile Virus (WNV) NS2B-NS3 protease (enzyme 150 nM;
inhibitor 50 M; substrate 50 M).
Inhibition of thrombin (F2) (enzyme 10 nM; inhibitor 25
M).
l
l
b
l
l
c
%
l
lM; substrate
50
2.3. Ki-values, target selectivities and aprotinin competition
assay
percentage inhibition against DEN and WNV protease (Table 1) and
the respective Ki-values (Table 3) can be explained by a lower Km-
value for the substrate of DEN protease. Compound 21 shows
selectivity for the flaviviral proteases. In comparison to thrombin
the selectivity ratio for this compound was 2.8:1 in favor of DEN
protease and 2.3:1 of WNV protease respectively. Quite interest-
ingly, compound 71 was identified as a thrombin inhibitor with a
Ki-value in the low micromolar range for thrombin and very low
affinity for the flaviviral proteases.
The evaluation at the DEN and WNV NS2B-NS3 proteases was
performed using an established fluorescence assay with an inter-
nally quenched FRET substrate as described by Steuer et al.13 For
the initial exploration of the new compounds and to search for lead
structures, the evaluation was usually restricted to single-concen-
tration experiments, performed in triplicate (percentage of inhibi-
tion at [inhibitor] = 50 lM). Selected compounds with pronounced
activity or selectivity were assayed in more detail. Table 3 shows
the Ki-values and target selectivities of these compounds.
The Ki-value of compound 21 at the DEN protease is in the range
of various published peptidyl-aldehyde inhibitors for this target.3
Other non-peptidic inhibitors, like the recently published anthra-
cene derivatives from Tomlinson et al., show activities in the
micromolar range.14 There might be a general difficulty to create
The most active compound 21 has a Ki-value of 35.7
lM at DEN
and 44.6 M at WNV protease. The seeming contradiction between
l

7324
C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
Table 3
Ki-values and target selectivities of selected compounds.
No.
Structure
Ki DENa
(
lM)
Ki WNVb
(l
M)
Ki F2c
(lM)
21
35.7
44.6
102
8
98.1
184
101
165
71
>200
23.4
a
b
c
Ki-value against DEN protease.
Ki-value against WNV protease.
Ki-value against thrombin (F2).
3. Conclusion
3-Aryl-2-cyanoacrylamides were discovered as a new class of
nitrile-containing inhibitors of the DEN and WNV NS2B-NS3 prote-
ases. The most relevant structural features for high activity are a
para-substituted aromatic system with high electron density, an
amide or acid residue and a planar molecule geometry. Conse-
quently, the most active molecule was the hydroxy derivative 21
with affinities in the range of other known inhibitor classes, mainly
for the DEN target with a Ki-value of 35.7 lM. With a very low
molecular weight, these compounds have a high ligand efficiency
(LE = 23.6 for compound 21)16 and the potential to become lead
structures for further development. With respect to selectivity,
compound 21 has pronounced affinity towards the viral targets,
whereas compound 71 shows activity against thrombin but only
marginal activity at the viral enzymes. The latter compound may
have some relevance for the development of thrombin inhibitors.
Future work will aim at the development of inhibitors that bind
to additional substrate recognition sites, in particular the S2–S4
pockets of the protease, to increase the potency and selectivity.
Figure 2. Aprotinin assay results of compound 8.
high affinities with small molecule inhibitors at the DEN protease
target.
4. Experimental section
4.1. Biological methods
The aprotinin competition assay described by Bodenreider et al.
can be used to identify compounds that bind to the active site of
DEN protease.15 The intrinsic fluorescence of Trp50, which is lo-
cated near the transition of S1 to S2 pocket is quenched by UV-
absorbing compounds binding to the active site. The fluorescence
of Trp50 is partially restored if the compounds are displaced by
the known competitive inhibitor aprotinin. Because of its advanta-
geous spectroscopic properties (no inherent fluorescence at rele-
vant wavelength and an absorption maximum near 330 nm),
4.1.1. Substrate synthesis
The internally quenched DEN NS2B-NS3 protease substrate
Abz-NleKRRS-3-(NO2)Y was synthesized by solid-phase synthesis
on Rink amide resin according to the Fmoc-protocol. It was purified
by preparative HPLC using an ÄKTA Purifier, GE Germany, with a
RP-18 chromatography column (Lobar, Merck, Germany, 40–
63
l
m, 25 ꢀ 310 mm). The mobile phase consisted of MeOH/0.1%
compound 8 with a Ki-value of 98.1
l
M was used for this experi-
TFA and H2O/0.1% TFA following a gradient of 20–100% MeOH in
water with a flow rate of 1.5 ml/min. The internally quenched sub-
strate for the WNV assay (Abz-GLKRGG-3-(NO2)Y) was synthesized
and purified as described above. The purity of both substrates was
assessed by HPLC and found to be higher than 95%. The identity
was confirmed by MALDI-TOF-MS.
ment. The results are shown in Figure 2 and indicate that aprotinin
can at least partially displace compound 8 from the active site of
the enzyme. This indicates a direct interaction of the inhibitor 8
with the catalytic center of the protein.
To exclude the possibility of an unspecific or promiscuous bind-
ing mode, the solutions (100 lM) of some of the most potent inhib-
itors in KH2PO4 buffer (pH 7) were analyzed by dynamic light
scattering (data not shown), without any hints of aggregate forma-
tion. In addition, the compounds were practically inactive against a
number of other, unrelated enzyme targets that are routinely
tested in our laboratory (Escherichia coli methionine aminopepti-
dase, Homo sapiens methionine aminopeptidase 1, E. coli MurA).
4.1.2. Expression and purification of DEN and WNV proteases
The DEN NS2B-NS3 gene, with the hydrophilic cofactor NS2B
connected to the protease domain NS3pro via a flexible glycine lin-
ker, was synthesized by a commercial supplier. The expression
plasmid pET28a (Novagen) with the inserted gene was used to
transform E. coli BL 21 k (DE3) cells. Overnight cultures of the

C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
7325
transformed cells were grown at 37 °C in standard LB-medium
containing 50 g/ml kanamycin. After the OD of 0.6–0.8 at
and 19F NMR spectra were recorded on Varian Mercury Plus
(300 MHz) and Varian NMR System 500 (500 MHz) instruments
at 300 K in CDCl3, acetone-d6, DMSO-d6, CD3OD or D2O. Chemical
shifts given in parts per million (d, ppm) and the residuals of
non-deuterated solvents were use as internal standard (1H NMR:
CDCl3: d = 7.25 ppm, acetone-d6: d = 2.04 ppm, DMSO-d6:
d = 2.49 ppm, CD3OD: d = 3.30 ppm, D2O: d = 4.75 ppm; 13C NMR:
CDCl3: d = 77.00 ppm, acetone-d6: d = 29.80 ppm, DMSO-d6:
d = 39.50 ppm, CD3OD: d = 49.00 ppm). Coupling constants (J) are
given in hertz (Hz). Multiplicity is reported as s (singlet), d (dou-
blet), t (triplet), quart (quartet), sept (septet), dd (doublet-doublet),
ddd (doublet-doublet-doublet), dt (doublet-triplet), td (triplet-
doublet), m (multiplet) and br (broad), respectively. IR spectra
were recorded on a Jasco FT-IR spectrometer (FT/IR-4100) with a
Pike MIRacle ATR module and are reported in reciprocal centime-
ters (cmꢁ1). Mass spectra were measured on Finnigan MAT 8200
(EI), Bruker micrOTOF-Q II (HR-ESI) and Bruker BIFLEX III (MAL-
DI-TOF) instruments. Combustion elemental analysis was per-
formed by double determination using a Foss Heraeus Vario EL
analyzer. Flash chromatography was performed on a Biotage Isol-
era One purification system using silica gel (0.060–0.200 mm) car-
tridges (KP-Sil) and UV monitoring. The reaction progress was
determined by thin layer chromatography on Merck Silica Gel
plates 60 F254 (UV detection). Microwave synthesis was performed
using a Monowave 300 synthesis reactor from Anton Paar. Purity of
the compounds used in biological assays was determined by com-
bustion elemental analysis (to an accuracy of within 0.4%) and
HPLC. HPLC was performed using an Agilent 1200 HPLC system
l
600 nm was reached, the expression was induced by addition of
IPTG to a final concentration of 1 mM. The cells were grown at
30 °C for further 4 h and then collected by centrifugation at
4500 g. The pellet was resuspended in buffer A (lysis buffer:
50 mM Tris–HCl pH 7.9, 100 mM NaCl, 5% glycerol and 5 mM imid-
azole) and passed through a cell disruptor (One Shot, Constant Sys-
tems). Afterwards the solution was centrifuged at 18,500g and 4 °C
for 40 min. The supernatant was then purified by Ni2+-affinity
chromatography. The protein was eluted by increasing the imidaz-
ole concentration from 5 mM to 250 mM. Stocks of purified protein
were stored at ꢁ70 °C in 100 mM Tris–HCl pH 7.9, 50 mM NaCl and
50% glycerol. The expression and purification protocol for the WNV
protease was similar to the one given for DEN.
4.1.3. Flourimetric DEN and WNV protease assay
The DEN protease assay was performed as described previously
(Steuer et al.).13 In short, continuous enzymatic assays were per-
formed on a BMG Labtech Fluostar OPTIMA microtiter fluorescence
plate reader using black 96 well V-bottom plates from Greiner. The
excitation wavelength was 320 nm and the emission was moni-
tored at 405 nm. The inhibitor concentration was 50 lM. The
inhibitors were preincubated for 15 min with the enzyme
(100 nM). Afterwards, the reaction was initiated by the addition
of the substrate to a final concentration of 50 lM. The activity of
the enzyme was determined as the slope per second (RFU/s) and
monitored for 15 min. Experiments were performed in triplicate
(n = 3) and the experimental values were averaged. The WNV pro-
tease assay was performed in analogy to the DEN protease assay.
Final concentration of the enzyme was 150 nM.
on a xTerra MS C18 (2.5
lm) 2.1 ꢀ 50 mm column. Detection was
conducted at 285 and 320 nm. The system conditions were A:
H2O (0.1% TFA), B: CH3CN (0.1% TFA), flow rate: 0.3 ml/min, gradi-
ent: 10% B (1 min), 95% B (9 min), 95% B (11 min), 10% B (11.1 min),
10% B (20 min).
4.1.4. Thrombin assay
The thrombin assay was performed as a continuous fluorimetric
assay on a BMG Labtech Fluostar OPTIMA microtiter fluorescence
plate reader. The excitation wavelength was 355 nm and the emis-
sion wavelength was 460 nm. The protease was assayed against
the substrate Boc-VPR-AMC (Bachem, Germany). The final concen-
4.2.1. General procedure for the preparation of compounds 1, 5–
15, 18–21, 23–33, 35–44, 46–54, 61, 62, 65, 67–69, 72–75, 92
A solution of arylaldehyde (1 equiv), 2-cyanoacetamide or
derivative (1.0–1.5 equiv) and N-methylpiperazine (0.05–
1.05 equiv) in methanol (2–10 ml) was stirred at room tempera-
ture overnight. After addition of an equivalent volume of water/
methanol (1:1) or 1 N HCl/methanol (1:1) for acidic products, the
precipitate was collected by filtration and washed with water/
methanol (1:1). If precipitation did not occur, the mixture was
evaporated and the residue was purified by flash chromatography.
trations of the enzyme and substrate were 10 and 50
tively. The inhibitors were preincubated with the enzyme for
15 min at a concentration of 25 M. The cleavage reaction was ini-
lM respec-
l
tiated by addition of the substrate. The assay buffer consisted of
50 mM Tris–HCl pH 7.5, 150 mM NaCl and 0.05% Tween 20.17
The activity of the enzyme was determined as the slope per second
(RFU/s) and monitored for 10 min.
4.2.1.1. (E)-2-Cyano-3-phenylacrylamide (1). Starting from benz-
aldehyde (2.12 g, 20.0 mmol) and 2-cyanoacetamide (2.14 g,
25.5 mmol) compound 1 was obtained after precipitation as a col-
orless solid (2.25 g, 65%). 1H NMR (300 MHz, acetone-d6): d = 7.20
(br m, 2H), 7.59 (m, 3H), 8.01 (m, 2H), 8.24 (s, 1H) ppm; 13C
NMR (75 MHz, acetone-d6): d = 106.5, 117.2, 130.0, 131.1, 133.0,
133.2, 152.4, 162.9 ppm; IR (neat): 3395, 3154, 2219, 1686, 1595,
1573, 1495, 1447, 1368, 1289, 1208, 1183, 1105, 952, 765, 741,
682 cmꢁ1; MS (EI, 70 eV): m/z (%): 172.1 (77) [M]+; HRMS (ESI):
m/z [M+H]+ calcd for C10H9N2O: 173.0709, found: 173.0701. Anal.
Calcd for C10H8N2O: C, 69.76; H, 4.68; N, 16.27. Found: C, 69.64;
H, 4.88; N, 16.26.
4.1.5. Ki-value determination
Ki-values were determined by duplicate experiments using
eight different substrate concentrations (25, 50, 75, 100, 150,
200, 300, 400
50, 100 M) and without inhibitor. Km-values of the different sub-
strates are 104 (DEN protease—Abz-NleKRRS-3-(NO2)Y),
211 (WNV protease—Abz-GLKRGG-3-(NO2)Y) and 15.9
lM) at three different inhibitor concentrations (25,
l
l
M
l
M
lM
(thrombin—Boc-VPR-AMC). The calculation was done using Prism
5.0 (GraphPad Software, Inc.).
4.1.6. Tryptophan quenching assay
This assay was performed as described by Bodenreider et al.15
4.2.1.2. (E)-2-Cyano-3-(4-methylphenyl)acrylamide (5). Starting
from 4-methylbenzaldehyde (240 mg, 2.0 mmol) and 2-cyanoacet-
amide (211 mg, 2.51 mmol) compound 5 was obtained after precip-
itation as a colorless solid (220 mg, 59%). 1H NMR (300 MHz,
acetone-d6): d = 2.41 (s, 3H), 7.13 (br m, 2H), 7.38 (m, 2H), 7.92 (m,
2H), 8.19 (s, 1H) ppm; 13C NMR (75 MHz, acetone-d6): d = 21.6,
105.2, 117.5, 130.4, 130.7, 131.3, 144.3, 152.3, 162.9 ppm; IR (neat):
3381, 3145, 2218, 1692, 1588, 1507, 1366, 1213, 1181, 1105,
4.2. Chemistry
All chemicals were obtained from Sigma-Aldrich (Germany) and
Alfa Aesar, Johnson Matthey (Germany) and were of analytical
grade. No further purification steps were performed unless indi-
cated. All solvents were used as obtained from the commercial
sources. Solvents were dried using standard procedures. 1H, 13C

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C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
815 cmꢁ1; MS (EI, 70 eV): m/z (%): 186.1 (93) [M]+; HRMS (ESI): m/z
[M+H]+ calcd for C11H11N2O: 187.0866, found: 187.0869. Anal. Calcd
for C11H10N2O: C, 70.95; H, 5.41; N, 15.04. Found: C, 70.86; H, 5.42; N,
15.00.
(ESI): m/z [M+H]+ calcd for C19H20N3O: 306.1601, found:
306.1599; HPLC purity >96%.
4.2.1.7.
(E)-2-Cyano-3-[4-(hydroxymethyl)phenyl]acrylamide
(10). Starting from 93 (409 mg, 3.0 mmol) and 2-cyanoacetamide
(280 mg, 3.33 mmol) compound 10 was obtained after precipita-
tion as a colorless solid (420 mg, 69%). 1H NMR (300 MHz, ace-
tone-d6): d = 4.45 (t, J = 5.8 Hz, 1H), 4.73 (d, J = 5.8 Hz, 2H), 7.56
(m, 2H), 7.99 (m, 2H), 8.22 (s, 1H) ppm; 13C NMR (75 MHz, ace-
tone-d6): d = 64.1, 105.7, 117.4, 127.7, 131.2, 131.6, 148.6, 152.3,
162.8 ppm; IR (neat): 3501, 3328, 3153, 2218, 1697, 1592, 1382,
1209, 1111, 1024, 1011, 956, 938, 829, 804, 679 cmꢁ1; HRMS
(ESI): m/z [M+H]+ calcd for C11H11N2O2: 203.0815, found:
203.0821. Anal. Calcd for C11H10N2O2: C, 65.34; H, 4.98; N, 13.85.
Found: C, 65.47; H, 5.08; N, 13.68.
4.2.1.3. (E)-2-Cyano-3-(4-isopropylphenyl)acrylamide (6). Start-
ing from 4-isopropylbenzaldehyde (148 mg, 1.0 mmol) and 2-cya-
noacetamide (93 mg, 1.11 mmol) compound 6 was obtained after
precipitation as a colorless solid (95 mg, 44%). 1H NMR (300 MHz,
acetone-d6): d = 1.27 (d, J = 7.0 Hz, 6H), 3.01 (sept, J = 6.9 Hz, 1H),
7.10 (br m, 2H), 7.46 (m, 2H), 7.96 (m, 2H), 8.20 (s, 1H) ppm; 13C
NMR (75 MHz, acetone-d6): d = 23.8, 34.9, 105.3, 117.5, 128.1,
130.8, 131.5, 152.3, 155.0, 162.9 ppm; IR (neat): 3413, 3203,
2958, 2212, 1685, 1588, 1508, 1462, 1421, 1362, 1283, 1211,
1189, 1053, 829 cmꢁ1; MS (EI, 70 eV): m/z (%): 214.1 (78) [M]+;
HRMS (ESI): m/z [M+H]+ calcd for C13H15N2O: 215.1179, found:
215.1183. Anal. Calcd for C13H14N2O: C, 72.87; H, 6.59; N, 13.07.
Found: C, 72.67; H, 6.66; N, 12.92.
4.2.1.8. (E)-2-Cyano-3-(4-fluorophenyl)acrylamide (11). Starting
from 4-fluorobenzaldehyde (248 mg, 2.0 mmol) and 2-cyanoacet-
amide (215 mg, 2.56 mmol) compound 11 was obtained after pre-
cipitation as a colorless solid (190 mg, 50%). 1H NMR (300 MHz,
acetone-d6): d = 7.15 (br m, 2H), 7.36 (m, 2H), 8.11 (m, 2H), 8.23
(s, 1H) ppm; 13C NMR (75 MHz, acetone-d6): d = 106.3,
117.0 + 117.3 (d, 2JCF = 22.2 Hz), 117.2, 129.7 + 129.7 (d,
4JCF = 3.2 Hz), 133.8 + 133.9 (d, 3JCF = 9.2 Hz), 151.1, 162.6,
4.2.1.4. (E)-2-Cyano-N-cyclopropyl-3-(4-isopropylphenyl)acryl-
amide (7). Starting from 4-isopropylbenzaldehyde (148 mg,
1.0 mmol) and 84 (137 mg, 1.10 mmol) compound 7 was obtained
after precipitation and recrystallization from methanol/acetone/
water as a colorless solid (140 mg, 55%). 1H NMR (300 MHz,
CDCl3): d = 0.64 (m, 2H), 0.88 (m, 2H), 1.26 (d, J = 6.9 Hz, 6H),
2.87 (m, 1H), 2.96 (sept, J = 6.9 Hz, 1H), 6.42 (br m, 1H), 7.33 (m,
2H), 7.86 (m, 2H), 8.29 (s, 1H) ppm; 13C NMR (75 MHz, CDCl3):
d = 6.8, 23.5, 23.6, 34.3, 102.3, 117.3, 127.4, 129.5, 131.0, 152.9,
154.7, 161.7 ppm; IR (neat): 3329, 3012, 2961, 2871, 2221, 1668,
1592, 1558, 1511, 1420, 1363, 1273, 1204, 1096, 1014, 993, 959,
840, 824 cmꢁ1; MS (EI, 70 eV): m/z (%): 254.1 (40) [M]+; HRMS
(ESI): m/z [M+H]+ calcd for C16H19N2O: 255.1492, found:
255.1494. Anal. Calcd for C16H18N2O: C, 75.56; H, 7.13; N, 11.01.
Found: C, 75.26; H, 7.20; N, 10.94.
1
163.9 + 167.3 (d, JCF = 253 Hz) ppm; IR (neat): 3468, 3158, 2216,
1697, 1585, 1502, 1414, 1381, 1301, 1234, 1163, 1113, 956,
834 cmꢁ1; MS (EI, 70 eV): m/z (%): 190.1 (100) [M]+; HRMS (ESI):
m/z [M+H]+ calcd for C10H8FN2O: 191.0615, found: 191.0618. Anal.
Calcd for C10H7FN2O: C, 63.16; H, 3.71; N, 14.73. Found: C, 62.98;
H, 3.89; N, 14.57.
4.2.1.9. (E)-2-Cyano-3-(2,4-dichlorophenyl)acrylamide
(12).
Starting from 2,4-dichlorobenzaldehyde (350 mg, 2.0 mmol) and
2-cyanoacetamide (210 mg, 2.50 mmol) compound 12 was ob-
tained after precipitation as a colorless solid (252 mg, 52%). 1H
NMR (300 MHz, acetone-d6): d = 7.31 (br m, 2H), 7.61 (ddd,
J = 8.5, 2.1, 0.6 Hz, 1H), 7.71 (d, J = 2.1 Hz, 1H), 8.13 (d,
J = 8.5 Hz, 1H), 8.48 (t, J = 0.6 Hz, 1H) ppm; 13C NMR (75 MHz,
acetone-d6): d = 111.0, 116.2, 128.9, 130.5, 130.7, 131.6, 136.9,
138.6, 147.3, 161.8 ppm; IR (neat): 3388, 3168, 2229, 1708,
4.2.1.5. (E)-2-Cyano-3-(4-isopropylphenyl)-N-(tetrahydrofuran-
2-ylmethyl)acrylamide (8). Starting from 4-isopropylbenzalde-
hyde (148 mg, 1.0 mmol) and 85 (185 mg, 1.10 mmol) compound
8 was obtained after precipitation and recrystallization from ace-
tone/water as a colorless solid (150 mg, 50%). 1H NMR (300 MHz,
CDCl3): d = 1.26 (d, J = 6.9 Hz, 6H), 1.59 (m, 1H), 1.93 (m, 2H),
2.03 (m, 1H), 2.96 (sept, J = 6.9 Hz, 1H), 3.37 (m, 1H), 3.69 (m,
1H), 3.79 (m, 1H), 3.91 (m, 1H), 4.05 (m, 1H), 6.68 (br s, 1H),
7.33 (m, 2H), 7.87 (m, 2H), 8.28 (s, 1H) ppm; 13C NMR (75 MHz,
CDCl3): d = 23.6, 25.9, 28.7, 34.3, 44.1, 68.3, 77.1, 102.7, 117.1,
127.4, 129.5, 130.9, 152.9, 154.6, 160.7 ppm; IR (neat): 3368,
2957, 2867, 2214, 1673, 1595, 1520, 1463, 1427, 1269, 1211,
1057, 918, 828 cmꢁ1; MS (EI, 70 eV): m/z (%): 298.1 (23) [M]+;
HRMS (ESI): m/z [M+H]+ calcd for C18H23N2O2: 299.1754, found:
299.1771. Anal. Calcd for C18H22N2O2: C, 72.46; H, 7.43; N, 9.39.
Found: C, 72.05; H, 7.39; N, 9.35.
1604, 1584, 1469, 1379, 1206, 1144, 1108, 1050, 923, 862 cmꢁ1
;
MS (EI, 70 eV): m/z (%): 240.0 (6) [M]+; HRMS (ESI): m/z [M+H]+
calcd for C10H7Cl2N2O: 240.9930, found: 240.9927. Anal. Calcd
for C10H6Cl2N2O: C, 49.82; H, 2.51; N, 11.62. Found: C, 49.64; H,
2.75; N, 11.46.
4.2.1.10. (E)-3-(4-Bromophenyl)-2-cyanoacrylamide (13). Start-
ing from 4-bromobenzaldehyde (185 mg, 1.0 mmol) and 2-cyano-
acetamide (93 mg, 1.11 mmol) compound 13 was obtained after
precipitation as
a
colorless solid (235 mg, 94%). 1H NMR
(300 MHz, acetone-d6): d = 7.18 (br m, 2H), 7.78 (m, 2H), 7.94 (m,
2H), 8.20 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 107.3,
116.2, 125.9, 131.1, 131.7, 132.3, 149.3, 162.5 ppm; IR (neat):
3437, 3142, 2215, 1694, 1600, 1578, 1488, 1374, 1205, 1185,
1115, 1071, 1006, 954, 808 cmꢁ1; MS (EI, 70 eV): m/z (%): 249.9
(100) [M]+; HRMS (ESI): m/z [M+H]+ calcd for C10H8BrN2O:
250.9815, found: 250.9814. Anal. Calcd for C10H7BrN2O: C, 47.84;
H, 2.81; N, 11.16. Found: C, 47.79; H, 2.98; N, 10.88.
4.2.1.6.
(E)-2-Cyano-3-(4-isopropylphenyl)-N-(pyridin-2-
ylmethyl)acrylamide (9). Starting from 4-isopropylbenzaldehyde
(148 mg, 1.0 mmol) and 86 (193 mg, 1.10 mmol) compound 9
was obtained after flash chromatography (cyclohexane/ethyl ace-
tate) as a colorless solid (110 mg, 36%). 1H NMR (300 MHz, CDCl3):
d = 1.27 (d, J = 6.9 Hz, 6H), 2.97 (sept, J = 6.9 Hz, 1H), 4.72 (d,
J = 4.8 Hz, 2H), 7.22 (m, 1H), 7.28 (m, 1H), 7.34 (m, 2H), 7.68 (td,
J = 7.6, 1.8 Hz, 1H), 7.76 (br m, 1H), 7.89 (m, 2H), 8.32 (s, 1H),
8.60 (m, 1H) ppm; 13C NMR (75 MHz, CDCl3): d = 23.6, 34.3, 45.1,
102.8, 117.1, 121.9, 122.6, 127.4, 129.6, 131.0, 136.8, 149.3,
152.8, 154.6, 155.2, 160.6 ppm; IR (neat): 3370, 2956, 2217,
1663, 1590, 1523, 1472, 1434, 1413, 1288, 1270, 1209, 1194,
830, 749 cmꢁ1; MS (EI, 70 eV): m/z (%): 305.2 (9) [M]+; HRMS
4.2.1.11. (E)-3-(4-Bromophenyl)-2-cyano-N-cyclopropylacryla-
mide (14). Starting from 4-bromobenzaldehyde (185 mg,
1.0 mmol) and 84 (137 mg, 1.10 mmol) compound 14 was ob-
tained after precipitation as a colorless solid with pure crystals
used for X-ray analysis (50 mg, 17%). 1H NMR (300 MHz, acetone-
d6): d = 0.67 (m, 2H), 0.76 (m, 2H), 2.88 (m, 1H), 7.56 (br m, 1H),

C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
7327
7.75 (m, 2H), 7.91 (m, 2H), 8.16 (s, 1H) ppm; 13C NMR (75 MHz,
4.2.1.16.
(E)-2-Cyano-3-(4-hydroxyphenyl)acrylamide
(21).
acetone-d6): d = 6.4, 24.3, 107.7, 116.7, 127.0, 132.3, 132.7, 133.2,
150.3, 162.3 ppm; IR (neat): 3325, 3016, 2221, 1669, 1582, 1509,
1274, 1197, 1074, 1009, 991, 835, 816 cmꢁ1; HRMS (ESI): m/z
[M+H]+ calcd for C13H12BrN2O: 291.0128, found: 291.0128. Anal.
Calcd for C13H11BrN2O: C, 53.63; H, 3.81; N, 9.62. Found: C,
53.62; H, 3.87; N, 9.59.
Starting from 4-hydroxybenzaldehyde (611 mg, 5.0 mmol) and 2-
cyanoacetamide (560 mg, 6.66 mmol) compound 21 was obtained
after precipitation as a pale yellow solid (850 mg, 90%). 1H NMR
(300 MHz, acetone-d6): d = 7.00 (br m, 2H), 7.01 (m, 2H), 7.96 (m,
2H), 8.13 (s, 1H), 9.40 (br s, 1H) ppm; 13C NMR (75 MHz, DMSO-
d6): d = 101.5, 116.2, 117.3, 122.9, 132.9, 150.5, 161.7, 163.3 ppm;
IR (neat): 3449, 3362, 3159, 2227, 1651, 1599, 1569, 1511, 1412,
1374, 1287, 1229, 1179, 930, 835, 810 cmꢁ1; MS (EI, 70 eV): m/z
(%): 188.1 (100) [M]+; HRMS (ESI): m/z [M+H]+ calcd for
4.2.1.12. (E)-2-Cyano-3-(4-methoxyphenyl)acrylamide (15).
Starting from anisaldehyde (272 mg, 2.0 mmol) and 2-cyanoacet-
amide (210 mg, 2.50 mmol) compound 15 was obtained after pre-
cipitation as a colorless solid (364 mg, 90%). 1H NMR (300 MHz,
DMSO-d6): d = 3.84 (s, 3H), 7.12 (m, 2H), 7.72 (br m, 2H), 7.95
(m, 2H), 8.10 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 55.6,
102.9, 114.8, 117.1, 124.4, 132.4, 150.1, 162.6, 163.1 ppm; IR
(neat): 3444, 3166, 2208, 1693, 1579, 1508, 1385, 1364, 1309,
1259, 1176, 1024, 961, 824 cmꢁ1; MS (EI, 70 eV): m/z (%): 202.0
(100) [M]+; HRMS (ESI): m/z [M+H]+ calcd for C11H11N2O2:
203.0815, found: 203.0817. Anal. Calcd for C11H10N2O2: C, 65.34;
H, 4.98; N, 13.85. Found: C, 65.21; H, 5.02; N, 13.73.
C
C
10H9N2O2: 189.0659, found: 189.0657. Anal. Calcd for
10H8N2O2: C, 63.82; H, 4.28; N, 14.89. Found: C, 63.65; H, 4.40;
N, 14.68.
4.2.1.17. (E)-2-Cyano-N-cyclopropyl-3-(4-hydroxyphenyl)acryl-
amide (23). Starting from 4-hydroxybenzaldehyde (122 mg,
1.0 mmol) and 84 (135 mg, 1.09 mmol) compound 23 was ob-
tained after precipitation as a pale yellow solid (200 mg, 88%). 1H
NMR (300 MHz, acetone-d6): d = 0.66 (m, 2H), 0.73 (m, 2H), 2.87
(m, 1H), 7.00 (m, 2H), 7.36 (br m, 1H), 7.92 (m, 2H), 8.09 (s, 1H),
9.41 (br s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 5.7, 23.4,
101.6, 116.2, 117.1, 122.9, 132.7, 149.9, 161.6, 163.0 ppm; IR
(neat): 3324, 3015, 2217, 1643, 1609, 1562, 1516, 1442, 1285,
4.2.1.13. (E)-2-Cyano-3-(3-methoxyphenyl)acrylamide (18).
Starting from 3-methoxybenzaldehyde (272 mg, 2.0 mmol) and
2-cyanoacetamide (210 mg, 2.50 mmol) compound 18 was ob-
tained after precipitation as a colorless solid (198 mg, 49%). 1H
NMR (300 MHz, acetone-d6): d = 3.87 (m, 3H), 7.15 (m, 1H),
7.19 (br m, 2H), 7.47 (m, 1H), 7.58 (m, 2H), 8.21 (s, 1H) ppm;
13C NMR (75 MHz, acetone-d6): d = 55.8, 106.8, 115.7, 117.3,
119.3, 123.7, 131.1, 134.3, 152.4, 160.9, 162.6 ppm; IR (neat):
3453, 3331, 2213, 1611, 1574, 1497, 1468, 1447, 1426, 1371,
1308, 1254, 1177, 1027, 956, 858, 778 cmꢁ1; MS (EI, 70 eV):
m/z (%): 202.0 (100) [M]+; HRMS (ESI): m/z [M+H]+ calcd for
1201, 1161, 830 cmꢁ1
C
C
;
HRMS (ESI): m/z [M+H]+ calcd for
13H13N2O2: 229.0972, found: 229.0963. Anal. Calcd for
13H12N2O2: C, 68.41; H, 5.30; N, 12.27. Found: C, 68.34; H, 5.41;
N, 12.29.
4.2.1.18. (E)-2-Cyano-3-(4-hydroxyphenyl)-N-(tetrahydrofuran-
2-ylmethyl)acrylamide (24). Starting from 4-hydroxybenzalde-
hyde (183 mg, 1.50 mmol) and 85 (265 mg, 1.58 mmol) compound
24 was obtained after precipitation as a colorless solid (360 mg,
88%). 1H NMR (300 MHz, acetone-d6): d = 1.62 (m, 1H), 1.81–2.03
(m, 3H), 3.36 (m, 1H), 3.50 (m, 1H), 3.69 (m, 1H), 3.83 (m, 1H),
4.03 (m, 1H), 7.00 (m, 2H), 7.23 (br m, 1H), 7.95 (m, 2H), 8.14 (s,
1H), 9.45 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 25.1,
28.5, 43.6, 67.1, 76.7, 101.1, 116.2, 117.2, 122.9, 132.8, 150.3,
161.6, 161.7 ppm; IR (neat): 3349, 3150, 2211, 1652, 1539, 1507,
C
C
11H11N2O2: 203.0815, found: 203.0816. Anal. Calcd for
11H10N2O2: C, 65.34; H, 4.98; N, 13.85. Found: C, 65.22; H,
4.99; N, 13.79.
4.2.1.14. (E)-2-Cyano-3-(3,4-dimethoxyphenyl)acrylamide (19).
Starting from 3,4-dimethoxybenzaldehyde (1.16 g, 6.98 mmol) and
2-cyanoacetamide (810 mg, 9.64 mmol) compound 19 was ob-
tained after precipitation as a colorless solid (1.47 g, 90%). 1H
NMR (300 MHz, acetone-d6): d = 3.88 (s, 3H), 3.92 (s, 3H), 7.03
(br m, 2H), 7.13 (d, J = 8.4 Hz, 1H), 7.60 (ddd, J = 8.4, 2.2, 0.5 Hz,
1H), 7.75 (d, J = 2.2 Hz, 1H), 8.15 (s, 1H) ppm; 13C NMR (75 MHz,
acetone-d6): d = 56.1, 56.2, 102.7, 112.4, 113.2, 118.1, 125.7,
126.9, 150.3, 152.4, 154.2, 163.1 ppm; IR (neat): 3386, 3186,
3003, 2222. 1713, 1667, 1632, 1586, 1513, 1448, 1380, 1272,
1248, 1019, 956, 850 cmꢁ1; MS (EI, 70 eV): m/z (%): 232.2 (100)
[M]+; HRMS (ESI): m/z [M+H]+ calcd for C12H13N2O3: 233.0921,
found: 233.0923. Anal. Calcd for C12H12N2O3: C, 62.06; H, 5.21;
N, 12.06. Found: C, 62.01; H, 5.35; N, 12.00.
1440, 1286, 1210, 1169, 1069, 833 cmꢁ1
; HRMS (ESI): m/z
[M+H]+ calcd for C15H17N2O3: 273.1234, found: 273.1234. Anal.
Calcd for C15H16N2O3: C, 66.16; H, 5.92; N, 10.29. Found: C,
65.98; H, 5.90; N, 10.29.
4.2.1.19.
(E)-2-Cyano-3-(4-hydroxyphenyl)-N-(pyridin-2-yl-
methyl)acrylamide (25). Starting from 4-hydroxybenzaldehyde
(183 mg, 1.50 mmol) and 86 (276 mg, 1.58 mmol) compound 25
was obtained after precipitation as a colorless solid (260 mg,
62%). 1H NMR (300 MHz, DMSO-d6): d = 4.51 (d, J = 5.6 Hz, 2H),
6.93 (m, 1H), 7.28 (m, 2H), 7.76 (td, J = 7.7, 1.7 Hz, 1H), 7.90 (m,
2H), 8.11 (s, 1H), 8.51 (m, 1H), 8.85 (m, 1H), 10.59 (s, 1H) ppm;
13C NMR (75 MHz, DMSO-d6): d = 45.0, 100.8, 116.2, 117.2, 121.0,
122.2, 122.9, 132.9, 136.7, 148.8, 150.7, 157.9, 161.7, 161.8 ppm;
IR (neat): 3372, 2203, 1675, 1591, 1569, 1526, 1508, 1444, 1386,
1297, 1244, 1202, 1171, 1011, 831 cmꢁ1; MS (EI, 70 eV): m/z (%):
279.1 (17) [M]+; HRMS (ESI): m/z [M+H]+ calcd for C16H14N3O2:
280.1081, found: 280.1074. Anal. Calcd for C16H13N3O2: C, 68.81;
H, 4.69; N, 15.05. Found: C, 68.49; H, 4.84; N, 15.02.
4.2.1.15.
(20).
(E)-2-Cyano-3-(3,4,5-trimethoxyphenyl)acrylamide
Starting from 3,4,5-trimethoxybenzaldehyde (1.37 g, 6.98 mmol)
and 2-cyanoacetamide (795 mg, 9.46 mmol) compound 20 was ob-
tained after precipitation as a colorless solid (1.73 g, 94%). 1H NMR
(300 MHz, acetone-d6): d = 3.83 (s, 3H), 3.89 (s, 6H), 7.14 (br m,
2H), 7.41 (s, 2H), 8.16 (s, 1H) ppm; 13C NMR (75 MHz, CDCl3):
d = 56.3, 61.1, 101.3, 108.3, 117.5, 126.7, 142.6, 153.3, 153.9,
162.0 ppm; IR (neat): 3406, 3133, 2224, 1711, 1690, 1596, 1575,
1502, 1466, 1450, 1415, 1384, 1355, 1323, 1302, 1256, 1240,
1188, 1155, 1116, 991, 832 cmꢁ1; MS (EI, 70 eV): m/z (%): 262.2
(100) [M]+; HRMS (ESI): m/z [M+H]+ calcd for C13H15N2O4:
263.1026, found: 263.1026. Anal. Calcd for C13H14N2O4: C, 59.54;
H, 5.38; N, 10.68. Found: C, 59.43; H, 5.50; N, 10.63.
4.2.1.20.
(E)-3-(4-Hydroxyphenyl)-2-(morpholin-4-ylcarbon-
yl)acrylonitrile (26). Starting from 4-hydroxybenzaldehyde
(183 mg, 1.50 mmol) and 87 (243 mg, 1.58 mmol) compound 26
was obtained after precipitation as a pale yellow solid (200 mg,
52%). 1H NMR (300 MHz, acetone-d6): d = 3.67 (m, 8H), 6.99 (m,
2H), 7.61 (s, 1H), 7.89 (m, 2H), 9.33 (s, 1H) ppm; 13C NMR
(75 MHz, acetone-d6): d = 67.1, 102.8, 116.9, 117.6, 125.2, 133.2,

7328
C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
151.2, 162.1, 164.2 ppm; IR (neat): 3270, 2958, 2217 1634, 1588,
tation and recrystallization from methanol/acetone/water (con-
taining hydrochloric acid) as a yellow solid (40 mg, 13%). 1H
NMR (300 MHz, acetone-d6): d = 1.21 (br m, 6H), 3.51 (br m,
4H), 7.60 (s, 1H), 7.90 (d, J = 2.2 Hz, 1H), 8.18 (d, J = 2.2 Hz,
1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 13.8, 24.7, 104.7,
116.2, 117.6, 118.4, 122.7, 137.2, 144.8, 147.1, 148.0,
162.6 ppm; IR (neat): 2983, 2210, 1629, 1607, 1537, 1440,
1516, 1426, 1276, 1205, 1178, 1105, 1065, 1028, 1008, 839 cmꢁ1
;
HRMS (ESI): m/z [M+H]+ calcd for C14H15N2O3: 259.1077, found:
259.1076. Anal. Calcd for C14H14N2O3: C, 65.11; H, 5.46; N, 10.85.
Found: C, 64.73; H, 5.59; N, 10.69.
4.2.1.21. (E)-3-(4-Hydroxyphenyl)-2-[(4-methylpiperazin-1-yl)-
carbonyl]acrylonitrile hydrochloride (27). Starting from 4-
hydroxybenzaldehyde (244 mg, 2.0 mmol) and 88 (360 mg,
2.15 mmol) the crude product was obtained after precipitation.
After resolving in methanol an excess of aqueous hydrochloric
acid was added, which resulted in precipitation of compound
1310, 1244, 1139, 945, 871, 764 cmꢁ1
; HRMS (ESI): m/z
[M+H]+ calcd for C14H16N3O5: 306.1084, found: 306.1079; HPLC
purity >98%.
4.2.1.26. (E)-2-Cyano-3-(4-hydroxy-3-methoxyphenyl)acrylam-
ide (32). Starting from vanillin (761 mg, 5.0 mmol) and 2-cyano-
acetamide (550 mg, 6.54 mmol) compound 32 was obtained after
27 as
a
colorless solid (95 mg, 15%). 1H NMR (300 MHz,
CD3OD): d = 2.96 (s, 3H), 3.22 (br m, 2H), 3.45 (br m, 2H),
3.57 (br m, 2H), 4.48 (br m, 2H), 6.90 (m, 2H), 7.75 (s, 1H),
7.90 (m, 2H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 41.9,
51.7, 99.6, 116.1, 117.0, 123.1, 132.5, 150.9, 161.7, 163.4 ppm;
IR (neat): 3069, 2671, 2596, 2464, 2201, 1659, 1606, 1576,
precipitation as
a
colorless solid (900 mg, 83%). 1H NMR
(300 MHz, acetone-d6): d = 3.91 (s, 3H), 6.99 (d, J = 8.4 Hz, 1H),
7.02 (br m, 2H), 7.55 (ddd, J = 8.4, 2.2, 0.5 Hz, 1H), 7.76 (d,
J = 2.2 Hz, 1H), 8.13 (s, 1H), 8.73 (bs, 1H) ppm; 13C NMR (75 MHz,
acetone-d6): d = 56.3, 101.9, 113.7, 116.5, 118.2, 125.1, 127.2,
148.6, 152.2, 152.6, 163.3 ppm; IR (neat): 3469, 3364, 2211,
1510, 1406, 1284, 1235, 1212, 1167, 976, 844 cmꢁ1
; HRMS
(ESI): m/z [M+H]+ calcd for C15H18N3O2: 272.1394, found:
272.1394; HPLC purity >98%.
1681, 1563, 1508, 1433, 1404, 1285, 1163, 1130, 1018, 849 cmꢁ1
;
MS (EI, 70 eV): m/z (%): 218.1 (100) [M]+; HRMS (ESI): m/z
[M+H]+ calcd for C11H11N2O3: 219.0764, found: 219.0760. Anal.
Calcd for C11H10N2O3: C, 60.55; H, 4.62; N, 12.84. Found: C,
60.46; H, 4.72; N, 12.73.
4.2.1.22.
(E)-2-Cyano-3-(3-hydroxyphenyl)acrylamide
(28).
Starting from 3-hydroxybenzaldehyde (1.83 g, 15.0 mmol) and 2-
cyanoacetamide (1.61 g, 19.1 mmol) compound 28 was obtained
after precipitation as a colorless solid (2.41 g, 85%). 1H NMR
(300 MHz, acetone-d6): d = 7.07 (m, 1H), 7.19, (br m 2H), 7.41 (m,
2H), 7.52 (m, 1H), 8.15 (s, 1H), 8.89 (s, 1H) ppm; 13C NMR
(75 MHz, acetone-d6): d = 106.3, 115.9, 116.4, 119.7, 121.5, 130.3,
133.1, 150.7, 157.8, 162.9 ppm; IR (neat): 3406, 3202, 2217,
4.2.1.27. (E)-2-Cyano-3-(4-hydroxy-3-iodo-5-methoxyphenyl)-
acrylamide (33). Starting from 4-hydroxy-3-iodo-5-methoxy-
benzaldehyde (1.39 g, 5.0 mmol) and 2-cyanoacetamide (526 mg,
6.26 mmol) compound 33 was obtained after precipitation and
recrystallization from acetone as a pale yellow solid (1.25 g, 73%).
1H NMR (300 MHz, DMSO-d6): d = 3.85 (s, 3H), 7.66 (d, J = 2.0 Hz,
1H), 7.76 (br m, 2H), 7.93 (d, J = 2.0 Hz, 1H), 8.03 (s, 1H), 10.60
(br m, 1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 56.1, 84.8,
103.0, 112.8, 117.1, 125.0, 133.9, 146.8, 149.3, 150.5, 162.9 ppm;
IR (neat): 3459, 3343, 3323, 2215, 1669, 1572, 1551, 1494, 1473,
1415, 1328, 1302, 1263, 1246, 1163, 1130, 1035, 970, 844,
761 cmꢁ1; MS (EI, 70 eV): m/z (%): 344.0 (100) [M]+; HRMS (ESI):
m/z [M+H]+ calcd for C11H10IN2O3: 344.9731, found: 344.9721;
HPLC purity >97%.
1670, 1578, 1448, 1389, 1285, 1230, 1116, 996, 956, 862 cmꢁ1
;
MS (EI, 70 eV): m/z (%): 188.1 (100) [M]+; HRMS (ESI): m/z
[M+H]+ calcd for C10H9N2O2: 189.0659, found: 189.0653; HPLC
purity >98%.
4.2.1.23. (E)-2-Cyano-3-(3,4-dihydroxyphenyl)acrylamide (29).
Starting from 3,4-dihydroxybenzaldehyde (415 mg, 3.0 mmol)
and 2-cyanoacetamide (333 mg, 3.96 mmol) compound 29 was ob-
tained after precipitation as a pale brown solid (350 mg, 57%). 1H
NMR (300 MHz, acetone-d6): d = 6.98 (d, J = 8.4 Hz, 1H), 7.00 (br
m, 2H), 7.38 (ddd, J = 8.4, 2.2, 0.5 Hz, 1H), 7.68 (d, J = 2.2 Hz, 1H),
8.05 (s, 1H), 8.70 (bs, 1H), 8.90 (bs, 1H) ppm; 13C NMR (75 MHz,
acetone-d6): d = 101.6, 116.6, 116.8, 118.0, 125.2, 126.8, 146.3,
151.1, 152.5, 163.4 ppm; IR (neat): 3461, 3410, 3313, 2603, 2208,
1675, 1567, 1446, 1391, 1293, 1262, 1194, 1167, 1122, 869,
794 cmꢁ1; MS (EI, 70 eV): m/z (%): 204.1 (100) [M]+; HRMS (ESI):
m/z [M+H]+ calcd for C10H9N2O3: 205.0608, found: 205.0608. Anal.
Calcd for C10H8N2O3: C, 58.82; H, 3.95; N, 13.72. Found: C, 58.70; H,
4.04; N, 13.58.
4.2.1.28. (E)-2-Cyano-3-(4-nitrophenyl)acrylamide (35). Starting
from 4-nitrobenzaldehyde (151 mg, 51.0 mmol) and 2-cyanoacet-
amide (93 mg, 1.10 mmol) compound 35 was obtained after pre-
cipitation as a brown solid (120 mg, 55%). 1H NMR (300 MHz,
acetone-d6): d = 7.30 (br m, 2H), 8.23 (m, 2H), 8.36 (s, 1H), 8.42
(m, 2H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 110.6, 115.7,
124.2, 131.0, 138.1, 148.3, 148.8, 162.1 ppm; IR (neat): 3429,
3340, 2224, 1686, 1591, 1378, 1341, 1295, 1201, 1106, 854,
768 cmꢁ1; MS (EI, 70 eV): m/z (%): 217.0 (100) [M]+; HRMS (ESI):
m/z [M+H]+ calcd for C10H8N3O3: 218.0560, found: 218.0558. Anal.
Calcd for C10H7N3O3: C, 55.30; H, 3.25; N, 19.35. Found: C, 55.14; H,
3.48; N, 19.03.
4.2.1.24. (E)-2-Cyano-3-(3,4-dihydroxy-5-nitrophenyl)acrylam-
ide (30). Starting from 95 (183 mg, 1.0 mmol) and 2-cyanoacet-
amide (110 mg, 1.31 mmol) compound 30 was obtained after
precipitation and recrystallization in acetone/water (containing
hydrochloric acid) as a yellow solid (150 mg, 60%). 1H NMR
(300 MHz, DMSO-d6): d = 7.60–8.00 (m, 4H), 8.04 (s, 1H), 10.88
(br m, 2H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 104.7, 116.4,
188.0, 119.2, 122.1, 137.3, 145.4, 148.1, 148.8, 162.8 ppm; IR
(neat): 3432, 3406, 3257, 2234, 1655, 1606, 1537, 1484, 1398,
1360, 1288, 1250, 1129, 941, 887, 866, 765 cmꢁ1; HRMS (ESI): m/
z [M+H]+ calcd for C10H8N3O5: 250.0458, found: 250.0457; HPLC
purity >98%.
4.2.1.29. (E)-2-Cyano-3-(4-cyanophenyl)acrylamide (36). Start-
ing from 4-formylbenzonitrile (131 mg, 1.0 mmol) and 2-cyano-
acetamide (105 mg, 1.25 mmol) compound 36 was obtained after
precipitation as a colorless solid (95 mg, 48%). 1H NMR (300 MHz,
acetone-d6): d = 7.27 (br m, 2H), 7.98 (m, 2H), 8.16 (m, 2H), 8.30
(s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 110.0, 113.8,
115.8, 118.2, 130.3, 132.9, 136.3, 148.7, 162.1 ppm; IR (neat):
3422, 3171, 2226, 1699, 1684, 1597, 1503, 1374, 1296, 1205,
1114, 1016, 936, 826 cmꢁ1; MS (EI, 70 eV): m/z (%): 197.0 (77)
[M]+; HRMS (ESI): m/z [M+H]+ calcd for C11H8N3O: 198.0662,
found: 198.0658. Anal. Calcd for C11H7N3O: C, 67.00; H, 3.58; N,
21.31. Found: C, 66.72; H, 3.79; N, 21.40.
4.2.1.25. (E)-2-Cyano-3-(3,4-dihydroxy-5-nitrophenyl)-N,N-di-
ethylacrylamide (31). Starting from 95 (183 mg, 1.0 mmol) and
89 (175 mg, 1.25 mmol) compound 31 was obtained after precipi-

C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
7329
4.2.1.30. 4-[(E)-3-Amino-2-cyano-3-oxoprop-1-en-1-yl]benzoic
acid (37). Starting from 4-formylbenzoic acid (2.40 g, 16.0 mmol)
and 2-cyanoacetamide (1.48 g, 17.6 mmol) compound 37 was ob-
tained after precipitation as a colorless solid (2.98 g, 86%). 1H
NMR (300 MHz, DMSO-d6): d = 7.83 (bs, 1H), 7.99 (m, 3H), 8.07
(m, 2H), 8.23 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 108.9, 116.0, 129.9, 130.0, 133.4, 135.8, 149.4, 162.4,
166.5 ppm; IR (neat): 3389, 3218, 2227, 1709, 1677, 1620, 1581,
1416, 1281, 1208, 1193, 1119, 1103, 974, 856, 769 cmꢁ1; MS (EI,
70 eV): m/z (%): 216.1 (100) [M]+; HRMS (ESI): m/z [M+H]+ calcd
for C11H9N2O3: 217.0608, found: 217.0611; HPLC purity >98%.
for
C15H18N3O: 256.1444, found: 216.1458. Anal. Calcd for
C15H17N3O: C, 70.56; H, 6.71; N, 16.46. Found: C, 70.25; H, 6.78;
N, 16.31.
4.2.1.35. (E)-2-Cyano-3-[4-(dimethylamino)phenyl]-N-(tetrahy-
drofuran-2-ylmethyl)acrylamide (42). Starting from 4-(dimeth-
ylamino)benzaldehyde (373 mg, 2.50 mmol) and 85 (459 mg,
2.73 mmol) compound 42 was obtained after precipitation as an
orange solid (681 mg, 91%). 1H NMR (300 MHz, acetone-d6):
d = 1.62 (m, 1H), 1.82–2.00 (m, 3H), 3.11 (s, 6H), 3.36 (m, 1H),
3.49 (m, 1H), 3.68 (m, 1H), 3.83 (m, 1H), 4.02 (m, 1H), 6.83 (m,
2H), 7.04 (br m, 1H), 7.91 (m, 2H), 8.05 (s, 1H) ppm; 13C NMR
(75 MHz, acetone-d6): d = 26.3, 40.0, 44.6, 68.4, 78.1, 97.4, 110.9,
112.4, 119.0, 120.3, 133.8, 152.0, 154.3, 162.4 ppm; IR (neat):
3409, 2973, 2927, 2870, 2832, 2194, 1651, 1610, 1569, 1505,
4.2.1.31. 4-[(E)-2-Cyano-3-(cyclopropylamino)-3-oxoprop-1-en-
1-yl]benzoic acid (38). Starting from 4-formylbenzoic acid
(380 mg, 2.53 mmol) and 84 (335 mg, 2.69 mmol) compound 38
was obtained after precipitation and recrystallization from ace-
tone/water (containing hydrochloric acid) as a colorless solid
(260 mg, 40%). 1H NMR (300 MHz, DMSO-d6): d = 0.59 (m, 2H),
0.69 (m, 2H), 2.78 (m, 1H), 7.98 (m, 2H), 8.07 (m, 2H), 8.14 (s,
1H), 8.58 (d, J = 4.0 Hz, 1H), 13.27 (s, 1H) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 5.7, 12.4, 108.8, 115.9, 129.9, 129.9
133.4, 135.8, 148.9, 162.1, 166.5 ppm; IR (neat): 2833, 2217,
1681, 1607, 1575, 1505, 1427, 1288, 1249, 1203, 1108, 1013,
928, 851 cmꢁ1; HRMS (ESI): m/z [M+H]+ calcd for C14H13N2O3:
257.0921, found: 257.0916; HPLC purity >97%.
1444, 1378, 1324, 1285, 1234, 1176, 1080, 1010, 945, 817 cmꢁ1
;
MS (EI, 70 eV): m/z (%): 299.1 (86) [M]+; HRMS (ESI): m/z [M+H]+
calcd for C17H22N3O2: 300.1707, found: 300.1719. Anal. Calcd for
C17H21N3O2: C, 68.20; H, 7.07; N, 14.04. Found: C, 68.04; H, 7.15;
N, 13.91.
4.2.1.36. (E)-2-Cyano-3-[4-(dimethylamino)phenyl]-N-(pyridin-
2-ylmethyl)acrylamide
(43). Starting
from
4-(dimethyl-
amino)benzaldehyde (150 mg, 1.0 mmol) and 86 (193 mg,
1.10 mmol) compound 43 was obtained after precipitation as
an orange solid (300 mg, 98%). 1H NMR (300 MHz, DMSO-d6):
d = 3.05 (s, 6H), 4.50 (d, J = 5.7 Hz, 2H), 6.82 (m, 2H), 7.25 (m,
1H), 7.31 (d, J = 7.7 Hz, 1H), 7.75 (td, J = 7.7, 1.8 Hz, 1H), 7.88
(m, 2H), 8.03 (s, 1H), 8.50 (m, 1H), 8.66 (m, 1H) ppm; 13C
NMR (75 MHz, DMSO-d6): d = 39.5, 44.9, 96.4, 111.6, 118.2,
118.7, 120.9, 122.1, 132.8, 136.7, 148.8, 150.7, 153.0, 158.1,
162.3 ppm; IR (neat): 3377, 2913, 2196, 1649, 1609, 1565,
1503, 1440, 1415, 1374, 1289, 1169, 995, 813, 749 cmꢁ1; MS
(EI, 70 eV): m/z (%): 306.1 (71) [M]+; HRMS (ESI): m/z [M+Na]+
calcd for C18H18N4NaO: 329.1373, found: 329.1365. Anal. Calcd
for C18H18N4O: C, 70.57; H, 5.92; N, 18.29. Found: C, 70.26; H,
6.03; N, 18.33.
4.2.1.32. 3-[(E)-2-cyano-3-(cyclopropylamino)-3-oxoprop-1-en-
1-yl]benzoic acid (39). Starting from 3-formylbenzoic acid
(250 mg, 1.67 mmol) and 2-cyanoacetamide (227 mg, 2.70 mmol)
compound 39 was obtained after precipitation as a colorless solid
(194 mg, 45%). 1H NMR (300 MHz, acetone-d6): d = 0.68 (m, 2H),
0.76 (m, 2H), 2.89 (m, 1H), 7.61 (br m, 1H), 7.71 (t, J = 7.8 Hz,
1H), 8.20 (m, 2H), 8.27 (s, 1H), 8.62 (m, 1H) ppm; 13C NMR
(75 MHz, acetone-d6): d = 6.4, 24.2, 108.4, 116.5, 130.4, 132.0,
132.4, 133.6, 133.6, 134.9, 150.6, 166.6 ppm; IR (neat): 3350,
2222, 1675, 1597, 1509, 1457, 1415, 1362, 1286, 1203, 1170,
933, 750 cmꢁ1; MS (EI, 70 eV): m/z (%): 256.0 (80) [M]+; HRMS
(ESI): m/z [M+H]+ calcd for C14H13N2O3: 257.0921, found:
257.0919; HPLC purity >98%.
4.2.1.37. (E)-3-[4-(Dimethylamino)phenyl]-2-(morpholin-4-yl-
carbonyl)acrylonitrile
(44). Starting
from
4-(dimethyl-
4.2.1.33. (E)-2-Cyano-3-[4-(dimethylamino)phenyl]acrylamide
(40). Starting from 4-(dimethylamino)benzaldehyde (1.52 g,
10.2 mmol) and 2-cyanoacetamide (850 mg, 10.2 mmol) com-
pound 40 was obtained after precipitation as an orange solid
(1.62 g, 75%). 1H NMR (300 MHz, acetone-d6): d = 3.11 (s, 6H),
6.83 (m, 2H), 6.84 (br m, 2H), 7.92 (m, 2H), 8.04 (s, 1H) ppm; 13C
NMR (75 MHz, acetone-d6): d = 40.0, 97.3, 112.4, 119.1, 120.2,
133.8, 152.4, 154.3, 164.1 ppm; IR (neat): 3398, 3143, 2198,
amino)benzaldehyde (300 mg, 2.01 mmol) and 87 (310 mg,
2.01 mmol) compound 44 was obtained after precipitation as a yel-
low solid (250 mg, 44%). 1H NMR (300 MHz, acetone-d6): d = 3.09
(s, 6H), 3.65 (m, 8H), 6.80 (m, 2H), 7.57 (s, 1H), 7.87 (m, 2H)
ppm; 13C NMR (75 MHz, acetone-d6): d = 40.0, 46.5, 67.1, 98.3,
112.3, 118.6, 120.7, 133.2, 152.2, 154.0, 165.1 ppm; IR (neat):
2954, 2911, 2862, 2197, 1633, 1604, 1561, 1521, 1425, 1363,
1323, 1266, 1232, 1169, 1111, 1021, 950, 827 cmꢁ1; MS (EI,
70 eV): m/z (%): 285.1 (100) [M]+; HRMS (ESI): m/z [M+H]+ calcd
for C16H20N3O2: 286.1550, found: 286.1557. Anal. Calcd for
1676, 1608, 1558, 1518, 1440, 1358, 1235, 1166, 943, 808 cmꢁ1
;
MS (EI, 70 eV): m/z (%): 215.1 (100) [M]+; HRMS (ESI): m/z
[M+H]+ calcd for C12H14N3O: 216.1131, found: 216.1137. Anal.
Calcd for C12H13N3O: C, 66.96; H, 6.09; N, 19.52. Found: C, 66.61;
H, 6.35; N, 19.18.
C16H19N3O2: C, 67.35; H, 6.71; N, 14.73. Found: C, 67.26; H, 6.71;
N, 14.59.
4.2.1.38. (E)-2-Cyano-3-pyridin-4-ylacrylamide (46). Starting
from isonicotinaldehyde (421 mg, 3.93 mmol) and 2-cyanoacet-
amide (430 mg, 5.12 mmol) compound 46 was obtained after pre-
cipitation as a colorless solid (235 mg, 35%). 1H NMR (300 MHz,
acetone-d6): d = 7.30 (br m, 2H), 7.82 (m, 2H), 8.22 (s, 1H), 8.80
(m, 2H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 111.5, 115.5,
122.9, 139.1, 148.2, 150.7, 161.9 ppm; IR (neat): 3426, 3027,
2216, 1696, 1639, 1598, 1546, 1415, 1359, 1239, 1204, 1059,
1004, 974, 853, 806 cmꢁ1; MS (EI, 70 eV): m/z (%): 173.1 (100)
[M]+; HRMS (ESI): m/z [M+H]+ calcd for C9H8N3O: 174.0662, found:
174.0653. Anal. Calcd for C9H7N3O: C, 62.42; H, 4.07; N, 24.27.
Found: C, 62.24; H, 4.17; N, 24.21.
4.2.1.34.
(E)-2-Cyano-N-cyclopropyl-3-[4-(dimethylamino)-
phenyl]acrylamide (41). Starting from 4-(dimethylamino)benzal-
dehyde (450 mg, 3.02 mmol) and 84 (395 mg, 3.18 mmol)
compound 41 was obtained after precipitation as a yellow solid
(590 mg, 77%). 1H NMR (300 MHz, acetone-d6): d = 0.64 (m, 2H),
0.72 (m, 2H), 2.85 (m, 1H), 3.11 (s, 6H), 6.82 (m, 2H), 7.16 (br m,
1H), 7.89 (m, 2H), 8.01 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-
d6): d = 5.7, 23.3, 39.5, 97.4, 111.6, 118.0, 118.7, 132.5, 149.8,
152.8, 163.6 ppm; IR (neat): 3294, 2915, 2207, 1606, 1571, 1525,
1437, 1382, 1361, 1328, 1286, 1254, 1174, 950, 825 cmꢁ1; MS
(EI, 70 eV): m/z (%): 255.1 (54) [M]+; HRMS (ESI): m/z [M+H]+ calcd

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C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
4.2.1.39. (E)-2-Cyano-3-pyridin-3-ylacrylamide (47). Starting
from nicotinaldehyde (214 mg, 2.0 mmol) and 2-cyanoacetamide
(210 mg, 2.50 mmol) compound 47 was obtained after precipitation
as a colorless solid (205 mg, 59%). 1H NMR (300 MHz, DMSO-d6):
d = 7.60 (dd, J = 8.1, 4.8 Hz, 1H), 7.85 (br m, 1H), 7.98 (br m, 1H),
8.23 (s, 1H), 8.36 (m, 1H), 8.72 (dd, J = 4.8, 1.6 Hz, 1H), 8.97 (m,
1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 108.9, 116.1, 124.1,
128.1, 135.9, 147.7, 151.2, 152.3, 162.1 ppm; IR (neat): 3127,
2217, 1712, 1675, 1637, 1593, 1490, 1414, 1371, 1255, 1223, 1193,
1137, 1097, 1028, 967, 803 cmꢁ1; MS (EI, 70 eV): m/z (%): 173.1
(85) [M]+; HRMS (ESI): m/z [M+H]+ calcd for C9H8N3O: 174.0662,
found: 174.0662. Anal. Calcd for C9H7N3O: C, 62.42; H, 4.07; N,
24.27. Found: C, 62.18; H, 4.18; N, 24.11.
137.4, 138.5, 142.4, 161.9 ppm; IR (neat): 3317, 3064, 3009,
2219, 1659, 1578, 1517, 1502, 1415, 1265, 1242, 1205, 1165,
1098, 1046, 1018, 977, 955, 852, 809 cmꢁ1; HRMS (ESI): m/z
[M+H]+ calcd for C11H10BrN2OS: 296.9692, found: 296.9696. Anal.
Calcd for C11H9BrN2OS: C, 44.46; H, 3.05; N, 9.43. Found: C,
44.39; H, 3.29; N, 9.47.
4.2.1.44. (E)-3-(3-Bromo-2-thienyl)-2-cyanoacrylamide (52).
Starting from 3-bromothiophene-2-carbaldehyde (192 mg,
1.0 mmol) and 2-cyanoacetamide (105 mg, 1.25 mmol) compound
52 was obtained after precipitation as a pale brown solid (220 mg,
86%). 1H NMR (300 MHz, acetone-d6): d = 7.19 (br m, 2H), 7.38 (d,
J = 5.4 Hz, 1H), 8.12 (dd, J = 5.4, 1.0 Hz, 1H), 8.47 (d, J = 1.0 Hz,
1H) ppm; 13C NMR (75 MHz, acetone-d6): d = 104.3, 116.9, 122.8,
131.7, 132.3, 134.9, 142.4, 162.2 ppm; IR (neat): 3468, 3114,
2211, 1696, 1615, 1581, 1475, 1412, 1374, 1304, 1236, 1115,
937, 876, 735 cmꢁ1; MS (EI, 70 eV): m/z (%): 256.0 (2) [M]+; HRMS
(ESI): m/z [M+H]+ calcd for C8H6BrN2OS: 256.9379, found:
256.9376. Anal. Calcd for C8H5BrN2OS: C, 37.37; H, 1.96; N,
10.90. Found: C, 37.46; H, 2.11; N, 10.82.
4.2.1.40. (E)-2-Cyano-3-(2-thienyl)acrylamide (48). Starting from
thiophene-2-carbaldehyde (337 mg, 3.0 mmol) and 2-cyanoacet-
amide (333 mg, 3.96 mmol) compound 48 was obtained after pre-
cipitation as a pale orange solid (365 mg, 58%). 1H NMR (300 MHz,
acetone-d6): d = 7.11 (br m, 2H), 7.31 (m, 1H), 7.90 (m, 1H), 8.01
(m, 1H), 8.40 (m, 1H) ppm; 13C NMR (75 MHz, acetone-d6):
d = 102.4, 117.4, 129.3, 135.1, 137.2, 138.1, 144.9, 162.8 ppm; IR
(neat): 3467, 3296, 3133, 2206, 1696, 1616, 1573, 1417, 1375,
1355, 1301, 1242, 1215, 1107, 1081, 1048, 858, 738 cmꢁ1; MS (EI,
70 eV): m/z (%): 178.1 (100) [M]+; HRMS (ESI): m/z [M+H]+ calcd
for C8H7N2OS: 179.0274, found: 179.0273. Anal. Calcd for
C8H6N2OS: C, 53.92; H, 3.39; N, 15.72. Found: C, 53.77; H, 3.52; N,
15.59.
4.2.1.45. (E)-3-(5-Bromo-2-furyl)-2-cyano-N-cyclopropylacryla-
mide (53). Starting from 5-bromo-2-furaldehyde (528 mg,
3.0 mmol) and 84 (410 mg, 3.30 mmol) compound 53 was ob-
tained after precipitation as a yellow solid (410 mg, 49%). 1H
NMR (300 MHz, acetone-d6): d = 0.67 (m, 2H), 0.74 (m, 2H), 2.86
(m, 1H), 6.83 (dd, J = 3.7, 0.3 Hz, 1H), 7.35 (dd, J = 3.7, 0.6 Hz, 1H),
7.45 (br m, 1H), 7.90 (m, 1H) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 5.7, 23.4, 101.5, 115.7, 116.0, 123.3, 128.8, 134.0, 150.3,
162.0 ppm; IR (neat): 3306, 3105, 3039, 2225, 1662, 1605, 1517,
4.2.1.41. (E)-3-(5-Chloro-2-thienyl)-2-cyanoacrylamide (49).
Starting
from
5-chlorothiophene-2-carbaldehyde
(160 mg,
1.10 mmol) and 2-cyanoacetamide (115 mg, 1.37 mmol) com-
pound 49 was obtained after precipitation as a pale brown solid
(110 mg, 47%). 1H NMR (300 MHz, acetone-d6): d = 7.11 (br m,
2H), 7.28 (d, J = 4.1 Hz, 1H), 7.76 (d, J = 4.1 Hz, 1H), 8.30 (s, 1H)
ppm; 13C NMR (75 MHz, DMSO-d6): d = 102.4, 116.5, 128.4,
134.8, 136.7, 138.1, 143.2, 162.1 ppm; IR (neat): 3393, 3159,
2207, 1683, 1583, 1509, 1422, 1377, 1290, 1246, 1210, 1071,
1005, 943, 804 cmꢁ1; MS (EI, 70 eV): m/z (%): 212.1 (57) [M]+;
HRMS (ESI): m/z [M+H]+ calcd for C8H6ClN2OS: 212.9884, found:
212.9879. Anal. Calcd for C8H5ClN2OS: C, 45.18; H, 2.37; N, 13.17.
Found: C, 45.12; H, 2.53; N, 13.04.
1453, 1362, 1283, 1262, 1200, 1091, 1019, 946, 927, 803 cmꢁ1
;
HRMS (ESI): m/z [M+H]+ calcd for C11H10BrN2O2: 280.9920, found:
280.9911. Anal. Calcd for C11H9BrN2O2: C, 47.00; H, 3.23; N, 9.97.
Found: C, 46.79; H, 3.43; N, 9.96.
4.2.1.46. (E)-2-Cyano-3-(1H-indol-3-yl)acrylamide (54). Starting
from 1H-indole-3-carbaldehyde (726 mg, 5.0 mmol) and 2-cyano-
acetamide (535 mg, 6.37 mmol) compound 54 was obtained after
precipitation as a pale yellow solid (700 mg, 66%). 1H NMR
(300 MHz, acetone-d6): d = 6.90 (br m, 2H), 7.28 (m, 2H), 7.59 (m,
1H), 7.95 (m, 1H), 8.58 (m, 2H) ppm; 13C NMR (75 MHz, acetone-
d6): d = 98.1, 111.3, 113.4, 119.1, 119.4, 122.6, 124.3, 128.5,
131.0, 137.3, 144.0, 164.1 ppm; IR (neat): 3471, 3371, 3110,
2210, 1684, 1584, 1563, 1512, 1485, 1459, 1422, 1373, 1329,
1298, 1228, 1130, 1112, 1061, 752 cmꢁ1; MS (EI, 70 eV): m/z (%):
211.1 (100) [M]+; HRMS (ESI): m/z [M+H]+ calcd for C12H10N3O:
212.0818, found: 212.0819. Anal. Calcd for C12H9N3O: C, 68.24;
H, 4.29; N, 19.89. Found: C, 68.00; H, 4.55; N, 19.67.
4.2.1.42. (E)-3-(5-Bromo-2-thienyl)-2-cyanoacrylamide (50).
Starting from 5-bromothiophene-2-carbaldehyde (382 mg,
2.0 mmol) and 2-cyanoacetamide (215 mg, 2.56 mmol) compound
50 was obtained after precipitation as a pale yellow solid (150 mg,
29%). 1H NMR (300 MHz, acetone-d6): d = 7.12 (br m, 2H), 7.40 (d,
J = 4.1 Hz, 1H), 7.71 (dd, J = 4.1, 0.6 Hz, 1H), 8.32 (d, J = 0.6 Hz,
1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 102.6, 116.6, 121.2,
131.9, 137.4, 138.6, 142.9, 162.2 ppm; IR (neat): 3426, 3170,
2209, 1682, 1578, 1504, 1413, 1365, 1283, 1238, 1204, 1113,
1077, 1058, 976, 941, 805 cmꢁ1; HRMS (ESI): m/z [M+H]+ calcd
for C8H6BrN2OS: 256.9379, found: 256.9378. Anal. Calcd for
C8H5BrN2OS: C, 37.37; H, 1.96; N, 10.90. Found: C, 37.05; H, 2.22;
N, 10.80.
4.2.1.47. (E)-2-Cyano-3-[2,3,5,6-tetrafluoro-4-(4-methylpipera-
zin-1-yl)phenyl]acrylamide (61). Starting from Pentafluorobenz-
aldehyde (197 mg, 1.0 mmol), 2-cyanoacetamide (105 mg,
1.25 mmol) and N-methylpiperazine (0.11 ml, 1.0 mmol) com-
pound 61 was obtained after precipitation and recrystallization
from methanol/water as a pale yellow solid (12 mg, 4%). 1H NMR
(300 MHz, CD3OD): d = 2.35 (s, 3H), 2.59 (m, 4H), 3.44 (m, 4H),
8.07 (m, 1H) ppm; 19F NMR (282 MHz, CD3OD): d = ꢁ153.2,
ꢁ138.6 ppm; 13C NMR (125 MHz, DMSO-d6): d = 45.8, 50.1, 54.8,
103.4, 114.1, 114.8, 132.7, 136.9 139.9, 141.9, 143.6, 145.6,
161.3 ppm; IR (neat): 3500, 3400, 2806, 2226, 1702, 1643, 1604,
4.2.1.43.
(E)-3-(5-Bromo-2-thienyl)-2-cyano-N-cyclopropyl-
acrylamide (51). Starting from 5-bromothiophene-2-carbalde-
hyde (1.0 g, 5.23 mmol) and 84 (720 mg, 5.80 mmol) compound
51 was obtained after precipitation as a pale yellow solid (1.48 g,
95%). 1H NMR (300 MHz, DMSO-d6): d = 0.58 (m, 2H), 0.66 (m,
2H), 2.76 (m, 1H), 7.45 (d, J = 4.1 Hz, 1H), 7.68 (dd, J = 4.1, 0.4 Hz,
1H), 8.26 (d, J = 0.4 Hz, 1H), 8.41 (br m, 2H) ppm; 13C NMR
(75 MHz, DMSO-d6): d = 5.7, 23.4, 102.5, 116.4, 121.0, 131.8,
1486, 1455, 1384, 1351, 1289, 1273, 1187, 1160, 1140, 980 cmꢁ1
;
MS (EI, 70 eV): m/z (%): 342.4 (51) [M]+; HRMS (ESI): m/z [M+H]+
calcd for C15H15F4N4O: 343.1177, found: 343.1175; HPLC purity
>98%.

C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
7331
4.2.1.48.
(E)-2-Cyano-N-cyclopropyl-3-(3-morpholin-4-ylphe-
2H), 6.99 (dd, J = 15.0, 11.7 Hz, 1H), 7.22 (br m, 1H), 7.32 (d,
J = 15.0 Hz, 1H), 7.55 (m, 2H), 7.91 (d, J = 11.7 Hz, 1H) ppm; 13C
NMR (75 MHz, acetone-d6): d = 6.5, 24.1, 40.1, 103.4, 112.8,
116.6, 118.5, 118.6, 123.7, 131.0, 149.2, 153.2, 153.5 ppm; IR
(neat): 3325, 2206, 1663, 1588, 1504, 1443, 1369, 1284, 1231,
1193, 1151, 987, 942, 811 cmꢁ1; MS (EI, 70 eV): m/z (%): 281.1
(56) [M]+; HRMS (ESI): m/z [M+H]+ calcd for C17H20N3O:
282.1601, found: 282.1605. Anal. Calcd for C17H19N3O: C, 72.57;
H, 6.81; N, 14.94. Found: C, 72.45; H, 6.78; N, 14.74.
nyl)acrylamide (62). Starting from 97 (75 mg, 0.39 mmol) and
84 (62 mg, 0.5 mmol) compound 62 was obtained after flash chro-
matography (cyclohexane/ethyl acetate) as a yellow oil (18 mg,
16%). 1H NMR (300 MHz, CDCl3): d = 0.65 (m, 2H), 0.89 (m, 2H),
2.86 (m, 1), 3.20 (m, 4H), 3.87 (m, 4H), 6.42 (br m, 1H), 7.07 (m,
1H), 7.36 (m, 2H), 7.47 (m, 1H), 8.29 (s, 1H) ppm; 13C NMR
(75 MHz, CDCl3): d = 6.9, 23.6, 48.8, 66.7, 103.4, 116.4, 117.2,
120.0, 122.6, 129.9, 132.6, 151.6, 153.7, 161.5 ppm; IR (neat):
2962, 2850, 2210, 1671, 1600, 1522, 1447, 1263, 1242, 1118,
1069, 1027, 996, 887, 780, 683 cmꢁ1; HRMS (ESI): m/z [M+H]+ calcd
for C17H20N3O2: 298.1550, found: 298.1548; HPLC purity >93%.
4.2.1.53. (E)-3-[4-(Benzyloxy)-3-methoxyphenyl]-2-cyanoacryl-
amide (72). Starting from 98 (242 mg, 1.0 mmol) and 2-cyano-
acetamide (105 mg, 1.25 mmol) compound 72 was obtained after
4.2.1.49. (E)-2-Cyano-N-cyclopropyl-3-[4-(morpholin-4-ylcar-
bonyl)phenyl]acrylamide (65). Starting from 96 (220 mg,
1.0 mmol) and 84 (137 mg, 1.10 mmol) compound 65 was ob-
tained after flash chromatography (cyclohexane/ethyl acetate) as
precipitation as a
colorless solid (190 mg, 62%). 1H NMR
(300 MHz, acetone-d6): d = 3.90 (s, 3H), 5.26 (s, 2H), 7.22 (d,
J = 8.4 Hz, 1H), 7.31–7.45 (m, 3H), 7.50 (m, 1H), 7.52 (m, 1H),
7.59 (ddd, J = 8.4, 2.2, 0.5 Hz, 1H), 7.78 (d, J = 2.2 Hz, 1H), 8.15 (s,
1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 55.5, 69.9, 103.0,
112.6, 113.2, 117.2, 124.7, 125.2, 128.0, 128.1, 128.5, 136.3,
148.9, 150.5, 151.5, 163.0 ppm; IR (neat): 3166, 2209, 1659,
1640, 1586, 1508, 1457, 1425, 1377, 1334, 1269, 1242, 1173,
1143, 1023, 978, 854, 749 cmꢁ1; MS (EI, 70 eV): m/z (%): 308.2
(7) [M]+; HRMS (ESI): m/z [M+H]+ calcd for C18H17N2O3:
309.1234, found: 309.1237. Anal. Calcd for C18H16N2O3: C, 70.12;
H, 5.23; N, 9.09. Found: C, 70.02; H, 5.31; N, 8.72.
a
pale yellow oil (70 mg, 22%). 1H NMR (300 MHz, CDCl3):
d = 0.66 (m, 2H), 0.90 (m, 2H), 2.89 (m, 1H), 3.34–3.86 (br m,
8H), 6.44 (br m, 1H), 7.51 (m, 2H), 7.95 (m, 2H), 8.33 (s, 1H)
ppm; 13C NMR (75 MHz, CDCl3): d = 6.9, 23.7, 38.6, 66.8, 105.2,
116.7, 127.9, 130.7, 133.0, 139.1, 151.6, 161.0, 168.9 ppm; IR
(neat): 3277, 2218, 1666, 1619, 1525, 1458, 1438, 1274, 1203,
1155, 1114, 1067, 1029, 1011, 932, 838 cmꢁ1; HRMS (ESI): m/z
[M+H]+ calcd for C18H20N3O3: 326.1499, found: 326.1493; HPLC
purity >91%.
4.2.1.54. (E)-2-Cyano-3-[4-(dimethylamino)phenyl]prop-2-ene-
thioamide (73). Starting from 4-(dimethylamino)benzaldehyde
(230 mg, 1.54 mmol) and 2-cyanoethanethioamide (165 mg,
1.65 mmol) compound 73 was obtained after precipitation as a
red solid (265 mg, 74%). 1H NMR (300 MHz, acetone-d6): d = 3.14
(s, 6H), 6.85 (m, 2H), 7.97 (m, 2H), 8.32 (br m, 1H), 8.43 (s, 1H),
8.92 (br m, 1H) ppm; 13C NMR (75 MHz, acetone-d6): d = 40.0,
102.9, 112.6, 118.4, 120.1, 134.5, 154.4, 154.6, 194.7 ppm; IR
(neat): 3327, 3283, 3150, 2215, 1638, 1605, 1560, 1516, 1409,
1365, 1229, 1194, 1169, 1117, 1061, 943, 905, 806 cmꢁ1; MS (EI,
70 eV): m/z (%): 231.1 (100) [M]+; HRMS (ESI): m/z [M+H]+ calcd
4.2.1.50. (2E,4E)-2-Cyano-4-methyl-5-phenylpenta-2,4-diena-
mide (67). Starting from (E)-2-methyl-3-phenylacrylaldehyde
(1.46 g, 10.0 mmol) and 2-cyanoacetamide (1.07 mg, 12.7 mmol)
compound 67 was obtained after precipitation as a colorless solid
(1.33 g, 63%). 1H NMR (300 MHz, acetone-d6): d = 2.39 (d,
J = 1.3 Hz, 3H), 7.04 (br m, 2H), 7.31 (bs, 1H), 7.36–7.54 (m, 5H),
7.95 (d, J = 1.0 Hz, 1H) ppm; 13C NMR (75 MHz, acetone-d6):
d = 15.5, 104.6, 117.5, 129.4, 129.7, 130.8, 134.0, 136.7, 146.5,
157.6, 163.2 ppm; IR (neat): 3464, 3291, 3150, 2206, 1681, 1571,
1373, 1211, 1111, 1078, 1010, 962, 756 cmꢁ1; MS (EI, 70 eV): m/z
(%): 212.1 (100) [M]+; HRMS (ESI): m/z [M+H]+ calcd for
for
C12H14N3S: 232.0903, found: 232.0902. Anal. Calcd for
C
C
13H13N2O: 213.1022, found: 213.1021. Anal. Calcd for
13H12N2O: C, 73.56; H, 5.70; N, 13.20. Found: C, 73.45; H, 5.85;
C12H13N3S: C, 62.31; H, 5.66; N, 18.17. Found: C, 62.06; H, 5.65;
N, 18.02.
N, 13.15.
4.2.1.55. (E)-2-Cyano-3-[4-(dimethylamino)phenyl]acrylic acid
(74). Starting from 4-(dimethylamino)benzaldehyde (746 mg,
5.0 mmol) and cyanoacetic acid (638 mg, 7.5 mmol) compound
74 was obtained after precipitation as an orange solid (600 mg,
56%). 1H NMR (300 MHz, DMSO-d6): d = 3.06 (s, 6H), 6.82 (m,
2H), 7.93 (m, 2H), 8.06 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-
d6): d = 39.6, 93.4, 111.7, 117.9, 118.4, 133.5, 153.5, 153.8,
164.8 ppm; IR (neat): 2215, 1657, 1609, 1561, 1516, 1433, 1384,
1330, 1284, 1248, 1166, 1090, 1063, 941, 812, 709 cmꢁ1; HRMS
(ESI): m/z [M+H]+ calcd for C12H13N2O2: 217.0972, found:
217.0978; HPLC purity >98%.
4.2.1.51. (2E,4E)-2-Cyano-4-methyl-5-phenyl-N-(tetrahydrofu-
ran-2-ylmethyl)penta-2,4-dienamide (68). Starting from (E)-2-
methyl-3-phenylacrylaldehyde (390 mg, 2.67 mmol) and 85
(490 mg, 2.91 mmol) compound 68 was obtained after precipita-
tion as a colorless solid (300 mg, 38%). 1H NMR (300 MHz, ace-
tone-d6): d = 1.62 (m, 1H), 1.80–2.01 (m, 3H), 2.39 (d, J = 1.2 Hz,
3H), 3.35 (m, 1H), 3.49 (m, 1H), 3.68 (m, 1H), 3.83 (m, 1H), 4.02
(m, 1H), 7.23 (br m, 2H), 7.31 (bs, 1H), 7.35–7.54 (m, 5H), 7.96
(m, 1H) ppm; 13C NMR (75 MHz, acetone-d6): d = 15.6, 26.3, 29.4,
44.7, 68.4, 77.9, 104.7, 117.5, 129.4, 129.7, 130.8, 134.0, 136.7,
146.5, 157.2, 161.6 ppm; IR (neat): 3375, 2977, 2937, 2874, 2209,
1658, 1567, 1523, 1278, 1258, 1207, 1084, 1015, 959, 757 cmꢁ1
;
4.2.1.56. [4-(Dimethylamino)benzylidene]malononitrile (75).
MS (EI, 70 eV): m/z (%): 296.1 (32) [M]+; HRMS (ESI): m/z [M+H]+
Starting
from
4-(dimethylamino)benzaldehyde
(373 mg,
calcd for C18H21N2O2: 297.1598, found: 297.1609. Anal. Calcd for
2.50 mmol) and malononitrile (250 mg, 3.78 mmol) compound 75
was obtained after precipitation as an orange solid (465 mg,
94%). 1H NMR (300 MHz, acetone-d6): d = 3.18 (s, 6H), 6.87 (m,
2H), 7.84 (s, 1H), 7.91 (m, 2H) ppm; 13C NMR (75 MHz, acetone-
d6): d = 40.1, 71.2, 112.5, 116.0, 116.7, 120.1, 134.5, 155.5,
159.3 ppm; IR (neat): 2921, 2206, 1607, 1561, 1514, 1385, 1357,
1259, 1174, 942, 929, 815 cmꢁ1; MS (EI, 70 eV): m/z (%): 197.1
(97) [M]+; HRMS (ESI): m/z [M+H]+ calcd for C12H12N3: 198.1026,
found: 198.1027. Anal. Calcd for C12H11N3: C, 73.07; H, 5.62; N,
21.30. Found: C, 72.95; H, 5.69; N, 21.13.
C18H20N2O2: C, 72.95; H, 6.80; N, 9.45. Found: C, 72.79; H, 6.85;
N, 9.35.
4.2.1.52. (2E,4E)-2-Cyano-N-cyclopropyl-5-[4-(dimethylamino)-
phenyl]penta-2,4-dienamide (69). Starting from (E)-3-[4-
(dimethylamino)phenyl]acrylaldehyde (180 mg, 1.03 mmol) and
84 (150 mg, 1.21 mmol) compound 69 was obtained after precipi-
tation as a red solid (225 mg, 78%). 1H NMR (300 MHz, acetone-d6):
d = 0.63 (m, 2H), 0.71 (m, 2H), 2.85 (m, 1H), 3.05 (s, 6H), 6.77 (m,

7332
C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
4.2.1.57.
(E)-2-Cyano-3-(4-methoxyphenyl)acrylsäure
(92).
4.2.2. Genreal procedure for the preparation of compounds 2a–
4b, 22a/b
Starting from anisaldehyde (816 mg, 6.0 mmol) and cyanoacetic
acid (638 mg, 7.50 mmol) compound 92 was obtained after precip-
itation as a colorless solid (761 mg, 62%). 1H NMR (300 MHz,
DMSO-d6): d = 3.85 (s, 3H), 7.11 (m, 2H), 8.01 (m, 2H), 8.16 (s,
1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 55.6, 102.4, 114.7,
117.4, 124.4, 132.7, 152.0, 162.6, 164.0 ppm; MS (EI, 70 eV): m/z
(%): 203.0 (100) [M]+.
A
solution of arylketone (1 equiv), cyanoacetamide (1.0–
1.5 equiv), NH4OAc (0.25 equiv) and acetic acid (0.75 equiv) in tol-
uene (10–25 ml) was refluxed at a Dean-Stark apparatus for 2–5 h.
After cooling to room temperature crystallization occurred in the
case of 2a and 4b. The crystals were washed with diethyl ether
and water. The remaining mixture was evaporated and the residue
was purified by flash chromatography (cyclohexane/ethyl acetate)
to isolate the cis and trans isomers.
4.2.1.58. Methyl N-[(E)-2-cyano-3-(4-methoxyphenyl)prop-2-
enoyl]-
methyl
L
-serinate (16). To a solution of 92 (305 mg, 1.50 mmol),
-serinate (245 mg, 1.58 mmol) and HATU (627 mg,
L
4.2.2.1. (E)-2-Cyano-3-phenylbut-2-enamide (2a) and (Z)-2-
cyano-3-phenylbut-2-enamide (2b). Starting from acetophenone
(1.80 g, 15.0 mmol) and 2-cyanoacetamide (1.39 g, 16.5 mmol)
compound 2a (470 mg, 17%) and 2b (450 mg, 16%) were obtained
as colorless solids.
1.65 mmol) in dry DMF (5 ml) was added DIPEA (0.78 ml,
4.50 mmol) slowly. After 2 h the reaction was quenched with
water and the mixture was extracted with ethyl acetate. After
drying over MgSO4 the solvent was evaporated and the residue
was purified by flash chromatography (cyclohexane/ethyl ace-
tate). Compound 16 was obtained as a colorless solid (288 mg,
63%). 1H NMR (300 MHz, acetone-d6): d = 3.74 (s, 3H), 3.92 (s,
3H), 3.97 (m, 1H), 4.04 (m, 1H), 4.67 (m, 1H), 7.13 (m, 2H),
7.42 (br m, 1H), 8.04 (m, 2H), 8.21 (s, 1H) ppm; 13C NMR
(75 MHz, acetone-d6): d = 52.6, 56.1, 56.4, 62.5, 102.1, 115.6,
117.7, 125.5, 133.8, 152.3, 161.4, 164.4, 171.2 ppm; IR (neat):
3385, 2954, 2214, 1735, 1645, 1611, 1579, 1514, 1462, 1436,
1374, 1314, 1258, 1212, 1177, 1093, 1054, 1024, 958,
4.2.2.1.1. (E)-2-Cyano-3-phenylbut-2-enamide (2a). 1H NMR
(300 MHz, acetone-d6): d = 2.48 (s, 3H), 7.06 (br m, 1H), 7.36 (br
m, 1H), 7.43–7.53 (m, 5H) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 22.0, 108.5, 116.9, 127.1, 128.6, 129.7, 139.3, 160.8,
163.5 ppm; IR (neat): 3371, 3176, 2215, 1655, 1590, 1491, 1429,
1380, 1121, 793, 764, 695 cmꢁ1; HRMS (ESI): m/z [M+H]+ calcd
for
C11H11N2O: 187.0866, found: 187.0867. Anal. Calcd for
C11H10N2O: C, 70.95; H, 5.41; N, 15.04. Found: C, 70.90; H, 5.55;
847 cmꢁ1
;
MS (EI, 70 eV): m/z (%): 304.1 (18) [M]+; HRMS
N, 14.96.
(ESI): m/z [M+H]+ calcd for C15H17N2O5: 305.1132, found:
305.1133; HPLC purity >98%.
4.2.2.1.2. (Z)-2-Cyano-3-phenylbut-2-enamide (2b). 1H NMR
(300 MHz, acetone-d6): d = 2.44 (s, 3H), 6.78 (br m, 2H), 7.40 (m,
5H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 24.0, 109.2, 116.7,
127.1, 128.3, 129.5, 138.1, 158.7, 163.4 ppm; IR (neat): 3371,
3176, 2217, 1644, 1591, 1491, 1381, 1279, 1086, 794, 764,
4.2.1.59. N2-[(E)-2-Cyano-3-(4-methoxyphenyl)prop-2-enoyl]-
N1-cyclopropyl-
L-serinamide (17). To a solution of 92 (305 mg,
1.50 mmol), 91 (284 mg, 1.58 mmol) and HATU (627 mg,
1.65 mmol) in dry DMF (5 ml) was added DIPEA (0.78 ml,
4.50 mmol) slowly. After 2 h the reaction was quenched with
water and the mixture was extracted with ethyl acetate. After
drying over MgSO4 the solvent was evaporated and the residue
was purified by flash chromatography (cyclohexane/ethyl ace-
tate). Compound 17 was obtained as a colorless solid (240 mg,
48%). 1H NMR (300 MHz, acetone-d6): d = 0.48 (m, 2H), 0.66
(m, 2H), 2.73 (m, 1H), 3.82 (m, 1H), 3.91 (m, 1H), 3.92 (s, 3H),
4.45 (m, 1H), 7.12 (m, 2H), 7.48 (br m, 1H), 8.03 (m, 2H), 8.18
(s, 1H) ppm; 13C NMR (75 MHz, acetone-d6): d = 6.4, 23.3, 56.1,
56.5, 63.0, 102.5, 115.6, 117.8, 125.6, 133.7, 151.9, 161.3,
164.3, 171.3 ppm; IR (neat): 3545, 3426, 3266, 3079, 2204,
1652, 1590, 1510, 1425, 1254, 1173, 1067, 1015, 835 cmꢁ1; MS
(EI, 70 eV): m/z (%): 329.2 (6) [M]+; HRMS (ESI): m/z [M+H]+
calcd for C17H20N3O4: 330.1448, found: 330.1447; HPLC purity
>98%.
;
696 cmꢁ1 HRMS (ESI): m/z [M+H]+ calcd for C11H11N2O:
187.0866, found: 187.0864; HPLC purity 70% (containing 30% 2a).
4.2.2.2. (E)-2-Cyano-4-methyl-3-phenylpent-2-enamide (3a)
and (Z)-2-cyano-4-methyl-3-phenylpent-2-enamide (3b). Start-
ing from isobutyrophenone (5.0 g, 33.7 mmol) and 2-cyanoacet-
amide (2.84 g, 33.8 mmol) compound 3a (40 mg, 0.5%) and 3b
(425 mg, 6%) were obtained as colorless solids with pure crystals
of 3b used for X-ray analysis.
4.2.2.2.1. (E)-2-Cyano-4-methyl-3-phenylpent-2-enamide (3a).
1H NMR (300 MHz, acetone-d6): d = 1.02 (d, J = 6.9 Hz, 6H), 3.61
(sept, J = 6.9 Hz, 1H), 7.07 (br m, 2H), 7.22 (m, 2H), 7.45 (m, 3H)
ppm; 13C NMR (75 MHz, acetone-d6): d = 20.9, 32.8, 110.9, 116.7,
128.5, 129.0, 129.3, 137.5, 164.0, 171.9 ppm; IR (neat): 3357,
3158, 2967, 2223, 1687, 1631, 1581, 1489, 1460, 1440, 1371,
989, 800, 758, 704 cmꢁ1; HRMS (ESI): m/z [M+H]+ calcd for
C13H15N2O: 215.1179, found: 215.1169. Anal. Calcd for
4.2.1.60. 4-[(E)-3-Amino-2-cyano-3-oxoprop-1-en-1-yl]phenyl
acetate (34). A solution of 21 (188 mg, 1.0 mmol), triethylamine
(3.0 ml, 21.6 mmol) and acetic anhydride (5.0 ml, 52.9 mmol) in
acetone (5 ml) and dichloromethane (5 ml) was stirred for
30 min. After quenching with water and evaporation of the solvent
the residue was purified by flash chromatography (cyclohexane/
ethyl acetate). Compound 34 was obtained as a colorless solid
(105 mg, 46%). 1H NMR (300 MHz, acetone-d6): d = 2.30 (s, 3H),
7.15 (br m, 2H), 7.35 (m, 2H), 8.07 (m, 2H), 8.23 (s, 1H) ppm; 13C
NMR (75 MHz, acetone-d6): d = 21.0, 106.4, 117.2, 123.6, 130.6,
132.6, 151.3, 154.8, 162.6, 169.3 ppm; IR (neat): 3410, 3152,
2218, 1760, 1692, 1595, 1507, 1418, 1380, 1364, 1294, 1199,
1166, 1116, 1014, 971, 946, 907, 841, 790 cmꢁ1; HRMS (ESI): m/z
[M+H]+ calcd for C12H11N2O3: 231.0764, found: 231.0750. Anal.
Calcd for C12H10N2O3: C, 62.60; H, 4.38; N, 12.17. Found: C,
62.62; H, 4.53; N, 12.05.
C13H14N2O: C, 72.87; H, 6.59; N, 13.07. Found: C, 72.70; H, 6.59;
N, 12.99.
4.2.2.2.2. (Z)-2-Cyano-4-methyl-3-phenylpent-2-enamide (3b).
1H NMR (300 MHz, acetone-d6): d = 1.08 (d, J = 6.9 Hz, 6H), 3.32
(sept, J = 6.9 Hz, 1H), 6.57 (br m, 2H), 7.21 (m, 2H), 7.41 (m, 3H)
ppm; 13C NMR (125 MHz, acetone-d6): d = 20.7, 36.5, 111.1 116.3,
128.6, 128.8, 129.3, 136.0, 163.6 169.5 ppm; IR (neat): 3454,
3403, 3188, 2973, 2936, 2271, 1672, 1604, 1410, 1378, 1263,
926, 852, 795, 755, 709 cmꢁ1; HRMS (ESI): m/z [M+H]+ calcd for
C13H15N2O: 215.1179, found: 215.1188; HPLC purity >98%.
4.2.2.3. (E)-2-Cyano-3-phenylhex-2-enamide (4a) and (Z)-2-
cyano-3-phenylhex-2-enamide (4b). Starting from butyrophe-
none (2.22 g, 15.0 mmol) and 2-cyanoacetamide (1.68 g,

C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
7333
20.0 mmol) compound 4a (315 mg, 10%) and 4b (295 mg, 9%) were
obtained as colorless solids.
4.2.3. General procedure for the preparation of compounds 55–
60, 63
A solution of fluorobenzaldehyde (1 equiv), cyanoacetamide or
derivative (1.05–1.1 equiv) and cyclic amine (2.5–10 equiv) in eth-
anol (5–10 ml) was reacted in a microwave reactor for 20 min at
160 °C. After cooling to room temperature precipitation occurred
and the precipitate was collected by filtration and washed with
ethanol/water (1:1). If precipitation did not occur, the mixture
was evaporated and the residue was purified by flash
chromatography.
4.2.2.3.1. (E)-2-Cyano-3-phenylhex-2-enamide (4a). 1H NMR
(300 MHz, CDCl3): d = 0.91 (t, J = 7.3 Hz, 3H), 1.41 (m, 2H), 3.09
(m, 2H), 5.64 (br m, 1H), 6.22 (br m, 1H), 7.36 (m, 2H), 7.45 (m,
3H) ppm; 13C NMR (75 MHz, CDCl3): d = 14.0, 21.8, 37.0, 105.9,
117.7, 127.2, 128.7, 130.1, 139.3, 162.8, 175.4 ppm; IR (neat):
3410, 3322, 2962, 2220, 2025, 1975, 1697, 1660, 1565, 1440,
1362, 1080, 777, 739, 697 cmꢁ1; HRMS (ESI): m/z [M+H]+ calcd
for
C13H15N2O: 215.1179, found: 215.1180. Anal. Calcd for
C
13H14N2O: C, 72.87; H, 6.59; N, 13.07. Found: C, 72.99; H, 6.72;
4.2.3.1. (E)-2-Cyano-3-(4-pyrrolidin-1-ylphenyl)acrylamide (55).
Starting from 4-fluorobenzaldehyde (372 mg, 3.0 mmol), 2-cyano-
acetamide (265 mg, 3.15 mmol) and pyrrolidine (534 mg,
7.51 mmol) compound 55 was obtained after precipitation and
recrystallization from acetone as an orange solid (280 mg, 39%).
1H NMR (300 MHz, acetone-d6): d = 2.05 (m, 5H), 3.42 (m, 4H),
6.67 (m, 2H), 6.79 (br m, 2H), 7.91 (m, 2H), 8.02 (s, 1H) ppm; 13C
NMR (75 MHz, DMSO-d6): d = 24.9, 47.3, 96.4, 111.8, 118.3, 118.4,
132.8, 150.4, 150.6, 164.0 ppm; IR (neat): 3395, 3151, 2517,
2159, 2029, 1976, 1660, 1604, 1568, 1523, 1479, 1443, 1396,
1344, 1245, 1157, 957, 807 cmꢁ1; HRMS (ESI): m/z [M+H]+ calcd
for C14H16N3O: 242.1288, found: 242.1287; HPLC purity >98%.
N, 13.01.
4.2.2.3.2. (Z)-2-Cyano-3-phenylhex-2-enamide (4b). 1H NMR
(300 MHz, CDCl3): d = 0.94 (t, J = 7.4 Hz, 3H), 1.42 (m, 2H), 2.81
(m, 2H), 5.37 (br m, 1H), 5.82 (br m, 1H), 7.20 (m, 2H), 7.41 (m,
3H) ppm; 13C NMR (75 MHz, CDCl3): d = 13.6, 20.9, 41.5, 108.8,
116.1, 126.8, 128.9, 129.8, 136.9, 163.0, 168.7 ppm; IR (neat):
3383, 3147, 2962, 2209, 1691, 1593, 1443, 1374, 1292, 1159,
1069, 802, 759, 695 cmꢁ1; HRMS (ESI): m/z [M+H]+ calcd for
C13H15N2O: 215.1179, found: 215.1180. Anal. Calcd for
C13H14N2O: C, 72.87; H, 6.59; N, 13.07. Found: C, 73.07; H, 6.69;
N, 13.04.
4.2.3.2. (E)-2-Cyano-3-(4-piperidin-1-ylphenyl)acrylamide (56).
Starting from 4-fluorobenzaldehyde (400 mg, 3.22 mmol), 2-cya-
noacetamide (300 mg, 3.57 mmol) and piperidine (686 mg,
8.06 mmol) compound 56 was obtained after precipitation as an
orange solid (350 mg, 43%). 1H NMR (300 MHz, acetone-d6):
d = 1.66 (m, 6H), 3.48 (m, 4H), 6.86 (br m, 2H), 7.02 (m, 2H), 7.90
(m, 2H), 8.03 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 24.0,
24.9, 47.5, 98.1, 113.4, 118.0, 119.6, 132.7, 150.2, 153.1,
163.8 ppm; IR (neat): 3392, 3174, 2939, 2852, 2220, 1662, 1604,
1573, 1518, 1442, 1395, 1354, 1271, 1242, 1197, 1123, 918,
4.2.2.4. (E)-2-Cyano-3-(4-hydroxyphenyl)but-2-enamide (22a)
and (Z)-2-Cyano-3-(4-hydroxyphenyl)but-2-enamide (22b).
Starting from 4-hydroxyacetophenone (2.04 g, 15.0 mmol) and 2-
cyanoacetamide (1.39 g, 16.5 mmol) compound 22a (250 mg, 8%)
was obtained as colorless crystalline solid and 22b (240 mg, 8%)
as a pale yellow solid.
4.2.2.4.1. (E)-2-Cyano-3-(4-hydroxyphenyl)but-2-enamide (22a).
1H NMR (300 MHz, acetone-d6): d = 2.46 (s, 3H), 6.92 (m, 2H),
6.94 (br m, 1H), 7.26 (br m, 1H), 7.43 (m, 2H), 8.83 (s, 1H)
ppm; 13C NMR (75 MHz, acetone-d6): d = 22.2, 107.2, 116.1,
118.3, 130.1, 132.0, 159.8, 160.0, 164.1 ppm; IR (neat): 3435,
3193, 2224, 1656, 1609, 1587, 1513, 1433, 1370, 1274, 1220,
805 cmꢁ1
;
HRMS (ESI): m/z [M+H]+ calcd for C15H18N3O:
256.1444, found: 256.1445; HPLC purity >96%.
4.2.3.3. (E)-2-Cyano-3-(4-morpholin-1-ylphenyl)acrylamide (57).
Starting from 4-fluorobenzaldehyde (621 mg, 5.0 mmol), 2-cyano-
acetamide (441 mg, 5.25 mmol) and morpholine (1.09 g,
12.5 mmol) compound 57 was obtained after precipitation as a yel-
low solid (340 mg, 26%). 1H NMR (300 MHz, DMSO-d6): d = 3.33 (m,
4H), 3.72 (m, 4H), 7.05 (m, 2H), 7.59 (br m, 2H), 7.86 (m, 2H), 8.00
(s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 46.5, 65.8, 99.5,
113.5, 117.8, 121.1, 132.4, 150.2, 153.4, 163.6 ppm; IR (neat):
3135, 2208, 1675, 1605, 1569, 1515, 1434, 1361, 1275, 1254,
1232, 1193, 1115, 1071, 1031, 928, 811 cmꢁ1; HRMS (ESI): m/z
[M+H]+ calcd for C14H16N3O2: 258.1237, found: 258.1217; HPLC
purity >98%.
1178, 1144, 1118, 837 cmꢁ1
;
HRMS (ESI): m/z [M+H]+
calcd for C11H11N2O2: 203.0815, found: 203.0813; HPLC purity
>98%.
4.2.2.4.2. (Z)-2-Cyano-3-(4-hydroxyphenyl)but-2-enamide (22b).
1H NMR (300 MHz, acetone-d6): d = 2.41 (s, 3H), 6.75 (br m, 2H),
6.85 (m, 2H), 7.30 (m, 2H), 8.79 (bs, 1H) ppm; 13C NMR (75 MHz,
acetone-d6): d = 24.4, 108.8, 116.1, 117.5, 130.1, 131.9, 159.9,
164.1, 164.9 ppm; IR (neat): 3240, 2224, 1651, 1605, 1566, 1514,
1362, 1268, 1234, 1189, 850, 831 cmꢁ1; HRMS (ESI): m/z [M+H]+
calcd for C11H11N2O2: 203.0815, found: 203.0815; HPLC purity
>95% (containing <5% 22a).
4.2.3.4. (E)-2-Cyano-3-(4-piperazin-1-ylphenyl)acrylamide (58).
Starting from 4-fluorobenzaldehyde (372 mg, 3.0 mmol), 2-cyano-
acetamide (265 mg, 3.15 mmol) and piperazine (2.58 g,
30.0 mmol) compound 58 was obtained after precipitation as an
orange solid (320 mg, 42%). 1H NMR (300 MHz, DMSO-d6):
d = 2.92 (m, 4H), 3.37 (m, 4H), 6.88 (br m, 2H), 7.04 (m, 2H), 7.92
(m, 2H), 8.05 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 45.3,
47.4, 98.6, 113.4, 117.9, 120.3, 132.5, 150.2, 153.6, 163.7 ppm; IR
(neat): 3382, 3264, 2926, 2213, 1681, 1632, 1605, 1561, 1513,
4.2.2.5. 2-Cyano-2-(2,3-dihydro-1H-inden-1-ylidene)acetamide
(45). A solution of 1-indanone (397 mg, 3.0 mmol), 2-cyanoacet-
amide (252 mg, 3.0 mmol), NH4OAc (58 mg, 0.75 mmol) and N-
methylpiperazine (15 mg, 0.15 mmol) was stirred in ethanol
(5 ml) at 60 °C overnight. After the evaporation of the solvent the
residue was purified by flash chromatography (cyclohexane/ethyl
acetate). Compound 45 was obtained as a pale brown solid
(30 mg, 5%). 1H NMR (300 MHz, CDCl3): d = 3.08 (m, 2H), 3.57 (m,
2H), 5.81 (br m, 1H), 6.34 (br m, 1H), 7–34–7.54 (m, 3H), 8.53 (d,
J = 8.2 Hz, 1H) ppm; 13C NMR (75 MHz, CDCl3): d = 30.3, 34.2,
94.7, 110.0, 118.4, 125.8, 127.4, 133.5, 137.3, 153.3, 164.3,
174.6 ppm; IR (neat): 3450, 3361, 3297, 3157, 2203, 1683, 1613,
1567, 1469, 1429, 1396, 1352, 1307, 1206, 1097, 764 cmꢁ1; HRMS
(ESI): m/z [M+H]+ calcd for C12H11N2O: 199.0866, found: 199.0867;
HPLC purity >93%.
1434, 1362, 1255, 1233, 1189, 1125, 1017, 966, 910, 814 cmꢁ1
;
HRMS (ESI): m/z [M+H]+ calcd for C14H17N4O: 257.1397, found:
257.1320; HPLC purity >90%.
4.2.3.5. (E)-2-Cyano-3-[4-(4-methylpiperazin-1-yl)phenyl]acryl-
amide (59). Starting from 4-fluorobenzaldehyde (372 mg,
3.0 mmol), 2-cyanoacetamide (265 mg, 3.15 mmol) and N-methyl-

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C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
piperazine (751 mg, 7.5 mmol) compound 59 was obtained after
precipitation as an orange solid (300 mg, 37%). 1H NMR
(300 MHz, acetone-d6): d = 2.26 (s, 3H), 2.48 (m, 4H), 3.44 (m,
4H), 6.88 (br m, 2H), 7.05 (m, 2H), 7.92 (m, 2H), 8.05 (s, 1H)
ppm; 13C NMR (75 MHz, DMSO-d6): d = 45.6, 46.1, 54.1, 99.0,
113.6, 117.9, 120.5, 132.4, 150.2, 153.2, 163.6 ppm; IR (neat):
3116, 2807, 2208, 1683, 1607, 1572, 1517, 1448, 1362, 1291,
1254, 1234, 1194, 1145, 1079, 1001, 925, 810, 791 cmꢁ1; HRMS
(ESI): m/z [M+H]+ calcd for C15H19N4O: 271.1553, found:
271.1542; HPLC purity >90%.
4.2.4.2.
(E)-2-Cyano-N-cyclopropyl-3-(5-morpholin-4-yl-2-
furyl)acrylamide (71). Starting from 53 (281 mg, 1.0 mmol) com-
pound 71 was obtained after flash chromatography (cyclohexane/
ethyl acetate) as a yellow crystalline solid (270 mg, 94%). 1H
NMR (300 MHz, DMSO-d6): d = 0.53 (m, 2H), 0.61 (m, 2H), 2.71
(m, 1H), 3.44 (m, 4H), 3.71 (m, 4H), 5.77 (d, J = 4.0 Hz, 1H), 7.34
(d, J = 4.0 Hz, 1H), 7.52 (s, 1H), 7.77 (d, J = 4.0 Hz, 1H) ppm; 13C
NMR (75 MHz, DMSO-d6): d = 5.8, 23.3, 45.6, 65.2, 88.4, 89.7,
118.4, 132.3, 139.6, 162.4, 164.0 ppm; IR (neat): 3345, 3119,
3023, 2964, 2863, 2188, 1644, 1614, 1542, 1488, 1445, 1408,
1366, 1348, 1319, 1261, 1236, 1173, 1116, 1048, 984, 884, 764,
690, 657 cmꢁ1; HRMS (ESI): m/z [M+H]+ calcd for C15H18N3O3:
288.1343, found: 288.1346. Anal. Calcd for C15H17N3O3: C, 62.71;
H, 5.96; N, 14.63. Found: C, 62.67; H, 6.11; N, 14.56.
4.2.3.6. (E)-2-Cyano-N-cyclopropyl-3-[4-(4-methylpiperazin-1-
yl)phenyl]acrylamide (60). Starting from 4-fluorobenzaldehyde
(372 mg, 3.0 mmol), 84 (391 mg, 3.15 mmol) and N-methylpipera-
zine (751 mg, 7.5 mmol) compound 60 was obtained after precip-
itation and recrystallization from methanol/water as an orange
solid (180 mg, 19%). 1H NMR (300 MHz, DMSO-d6): d = 0.56 (m,
2H), 0.66 (m, 2H), 2.20 (s. 3H), 2.41 (m, 4H), 2.75 (m, 1H), 3.37
(m, 4H), 7.04 (m, 2H), 7.83 (m, 2H), 7.90 (s, 1H), 8.20 (m, 1H)
ppm; 13C NMR (75 MHz, DMSO-d6): d = 5.7, 23.3, 45.7, 46.2, 54.2,
99.1, 113.6, 117.6, 120.6, 132.4, 149.6, 153.1, 163.4 ppm; IR (neat):
3323, 2937, 2842, 2794, 2218, 1665, 1606, 1574, 1509, 1449, 1377,
1359, 1277, 1232, 1192, 1008, 920, 819 cmꢁ1; HRMS (ESI): m/z
[M+H]+ calcd for C18H23N4O: 311.1866, found: 311.1854; HPLC
purity >94%.
4.2.4.3. 3-Morpholin-4-ylbenzaldehyde (97). Starting from 2-
bromobenzaldehyde (925 mg, 5.0 mmol) compound 97 was ob-
tained after flash chromatography (cyclohexane/ethyl acetate) as
a pale yellow oil (140 mg, 15%). 1H NMR (300 MHz, CDCl3):
d = 3.20 (m, 4H), 3.85 (m, 4H), 7.14 (m, 1H), 7.37 (m, 3H), 9.93 (s,
1H) ppm; 13C NMR (75 MHz, CDCl3): d = 48.7, 66.6, 114.5, 121.5,
122.2, 129.7, 137.2, 151.6, 192.6 ppm; MS (EI, 70 eV): m/z (%):
194.1 (86) [M]+.
4.2.4.4. (E)-2-Cyano-3-[4-(piperidin-1-ylcarbonyl)phenyl]acryl-
amide (64). To a solution of 37 (432 mg, 2.0 mmol), HOAt
(300 mg, 2.20 mmol) and HATU (912 mg, 2.40 mmol) in dry
DMF (3 ml) was added DIPEA (1.10 ml, 6.0 mmol) slowly at
0 °C. After 30 min the mixture was allowed to warm up to room
temperature and piperidine (0.19 ml, 2.3 mmol) was added. After
4 h the reaction was quenched with water and the mixture was
extracted with ethyl acetate. After drying over MgSO4 the sol-
vent was evaporated and the residue was purified by flash chro-
matography (ethyl acetate/methanol). Compound 64 was
4.2.3.7. (E)-2-Cyano-3-(2-morpholin-4-ylphenyl)acrylamide (63).
Starting from 2-fluorobenzaldehyde (372 mg, 3.0 mmol), 2-cyano-
acetamide (265 mg, 3.15 mmol) and morpholine (653 mg,
7.5 mmol) compound 63 was obtained after flash chromatography
(cyclohexane/ethyl acetate) as a yellow solid (60 mg, 8%). 1H NMR
(300 MHz, acetone-d6): d = 2.97 (m, 4H), 3.81 (m, 4H), 7.12 (br m,
2H), 7.21 (m, 2H), 7.55 (m, 1H), 8.03 (m, 1H), 8.58 (s, 1H) ppm;
13C NMR (125 MHz, acetone-d6): d = 42.9, 66.8, 110.9, 116.7,
116.9, 121.2, 125.9, 129.7, 135.5, 143.7, 161.1, 163.2 ppm; IR
(neat): 3370, 3145, 2211, 1696, 1591, 1481, 1447, 1373, 1346,
1298, 1227, 1111, 1068, 937, 768 cmꢁ1; MS (EI, 70 eV): m/z (%):
257.1 (100) [M]+; HRMS (ESI): m/z [M+H]+ calcd for C14H16N3O2:
258.1237, found: 258.1227; HPLC purity >94%.
obtained as
a
pale yellow solid (345 mg, 61%). 1H NMR
(300 MHz, DMSO-d6): d = 1.55 (br m, 6H), 3.24 (br m, 2H), 3.58
(br m, 2H), 7.53 (m, 2H), 7.80 (br m, 1H), 7.94 (br m, 1H),
7.96 (m, 2H), 8.20 (s, 1H) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 24.0, 25.2, 25.9, 42.3, 47.9, 107.5, 116.3, 127.3, 130.1, 132.5,
139.9, 149.7, 162.5, 167.7 ppm; IR (neat): 3360, 3160, 2944,
2856, 2219, 1694, 1610, 1592, 1440, 1376, 1272, 1206, 1117,
1090, 1005, 948, 842 cmꢁ1; MS (EI, 70 eV): m/z (%): 283.0 (67)
[M]+; HRMS (ESI): m/z [M+H]+ calcd for C16H18N3O2: 284.1394,
found: 284.1395; HPLC purity >98%.
4.2.4. General procedure for the preparation of compounds 70,
71, 97
A solution of arylbromide (1 equiv), morpholine (1.5 equiv), L-
proline (0.2 equiv), CuI (0.1 equiv) and K2CO3 (2 equiv) in DMSO
(1.5–3 ml) was stirred under a nitrogen atmosphere for 4–20 h at
80 °C. After cooling to room temperature water was added and
the mixture was extracted with ethyl acetate. After evaporation
of the solvent the residue was purified by flash chromatography.
4.2.4.5. 3-[(E)-2-Cyano-3-(cyclopropylamino)-3-oxoprop-1-en-
1-yl]-N-cyclopropylbenzamide (66). To
a
solution of 39
(128 mg, 0.50 mmol), HOAt (75 mg, 0.55 mmol) and HATU
(228 mg, 0.60 mmol) in dry DMF (3 ml) was added DIPEA
(0.28 ml, 1.5 mmol) slowly at 0 °C. After 30 min the mixture was
allowed to warm up to room temperature and cyclopropylamine
(33 mg, 0.58 mmol) was added. After 4 h the reaction was
quenched with water and the mixture was extracted with ethyl
acetate. After drying over MgSO4 the solvent was evaporated and
the residue was purified by flash chromatography (ethyl acetate/
4.2.4.1. (E)-2-Cyano-3-(5-morpholin-4-yl-2-thienyl)acrylamide
(70). Starting from 50 (129 mg, 0.5 mmol) compound 70 was ob-
tained after flash chromatography (cyclohexane/ethyl acetate) as
an orange crystalline solid (50 mg, 38%). 1H NMR (300 MHz,
CDCl3): d = 3.39 (m, 4H), 3.84 (m, 4H), 6.14 (d, J = 4.5 Hz, 1H),
7.45 (d, J = 4.5 Hz, 1H), 8.17 (s, 1H) ppm; 13C NMR (75 MHz,
DMSO-d6): d = 49.3, 65.2, 90.8, 105.2, 118.5, 119.7, 142.7, 144.0,
164.0, 167.0 ppm; IR (neat): 3472, 3103, 2194, 1691, 1573,
1510, 1472, 1442, 1396, 1374, 1357, 1310, 1282, 1243, 1154,
1111, 1086, 1027, 934, 893, 849, 762 cmꢁ1; MS (EI, 70 eV): m/z
(%): 263.0 (100) [M]+; HRMS (ESI): m/z [M+H]+ calcd for
methanol). Compound 66 was obtained as
a colorless solid
(75 mg, 51%). 1H NMR (300 MHz, DMSO-d6): d = 0.58 (m, 4H),
0.70 (m, 4H), 2.78 (m, 1H), 2.85 (m, 1H), 7.63 (t, J = 7.8 Hz, 1H),
7.95 (m, 1H), 8.07 (m, 1H), 8.13 (s, 1H), 8.28 (m, 1H), 8.55 (m,
1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 5.7, 23.1, 23.4, 107.6,
115.9, 129.1, 129.5, 130.3, 131.4, 132.0, 135.3, 149.4, 162.2,
166.5 ppm; IR (neat): 3355, 3293, 2219, 1678, 1633, 1604, 1530,
C
C
12H14N3O2S: 264.0801, found: 264.0798. Anal. Calcd for
12H13N3O2S: C, 54.74; H, 4.98; N, 15.96. Found: C, 54.57; H,
5.30; N, 15.75.
1360, 1313, 1265, 1227, 1207, 1053, 1023, 965, 849, 683 cmꢁ1
;

C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
7335
HRMS (ESI): m/z [M+H]+ calcd for C17H18N3O2: 296.1394, found:
4.2.5.5.
(E)-2-Cyano-N-cyclopropyl-5-[4-(dimethylamino)-
296.1392; HPLC purity >95%.
phenyl]pent-4-enamide (80). Starting from 69 (141 mg,
0.5 mmol) compound 80 was obtained after precipitation as a pale
orange solid (110 mg, 78%). 1H NMR (300 MHz, DMSO-d6): d = 0.39
(m, 2H), 0.62 (m, 2H), 2.62 (m, 2H), 2.88 (s, 6H), 3.65 (t, J = 7.3 Hz,
1H), 5.85 (dt, J = 15.7, 7.2 Hz, 1H), 6.37 (d, J = 15.8 Hz, 1H), 6.66 (m,
2H), 7.20 (m, 2H), 8.38 (d, J = 4.0 Hz, 1H) ppm; 13C NMR (75 MHz,
DMSO-d6): d = 5.5, 5.8, 22.6, 33.5, 37.9, 40.0, 112.2, 118.4, 119.2,
124.4, 127.0, 133.1, 149.9, 165.7 ppm; IR (neat): 3287, 2898,
2247, 2189, 1648, 1611, 1523, 1345, 1292, 1226, 1192, 1166,
1063, 1024, 967, 813 cmꢁ1; HRMS (ESI): m/z [M+Na]+ calcd for
4.2.5. General procedure for the preparation of compounds 76–
81
To a stirring solution of arylcyanoacrylamide (1 equiv) in meth-
anol (5–10 ml) was added NaBH4 (5 equiv) at 0 °C. After 1 h the
reaction was quenched by the addition of an equivalent volume
of water. If precipitation occurred, the precipitate was collected
by filtration and washed with water/methanol (1:1). If precipita-
tion did not occur, the mixture was evaporated and the residue
was purified by flash chromatography.
C17H21N3NaO: 306.1577, found: 306.1570; HPLC purity >98%.
4.2.5.1. 2-Cyano-3-[4-(dimethylamino)phenyl]propanamide (76).
Starting from 40 (646 mg, 3.0 mmol) compound 76 was obtained
after precipitation as a colorless solid (400 mg, 61%). 1H NMR
(300 MHz, DMSO-d6): d = 2.85 (s, 6H), 2.86–3.08 (m, 2H), 3.81 (m,
1H), 6.67 (m, 2H), 7.09 (m, 2H), 7.42 (br m, 1H), 7.72 (br m, 1H)
ppm; 13C NMR (75 MHz, DMSO-d6): d = 34.6, 40.0, 40.1, 112.3,
118.6, 124.0, 129.5, 149.5, 166.5 ppm; IR (neat): 3410, 3182,
2915, 2796, 2254, 1660, 1614, 1521, 1475, 1442, 1408, 1331,
4.2.5.6. (E)-2-Cyano-4-methyl-5-phenylpent-4-enamide (81).
Starting from 67 (213 mg, 1.0 mmol) compound 81 was obtained
after precipitation as a colorless solid (155 mg, 72%). 1H NMR
(300 MHz, CDCl3): d = 1.93 (s, 3H), 2.74 (m, 1H), 2.88 (m, 1H),
3.60 (m, 1H), 5.90 (br m, 1H), 6.24 (br m, 1H), 6.49 (s, 1H), 7.18–
7.39 (m, 5H) ppm; 13C NMR (75 MHz, CDCl3): d = 17.5, 37.6, 40.5,
117.8, 126.8, 128.2, 128.9, 129.9, 132.2, 137.1, 166.3 ppm; IR
(neat): 3362, 3184, 2930, 2255, 1662, 1490, 1415, 1314, 1247,
1167, 1111, 1022, 916, 806, 748, 692 cmꢁ1; HRMS (ESI): m/z
[M+H]+ calcd for C13H15N2O: 215.1179, found: 215.1176; HPLC
purity >92%.
1287, 1219, 1188, 1163, 1128, 1057, 945, 814, 743, 710 cmꢁ1
;
HRMS (ESI): m/z [M+Na]+ calcd for C12H15N3NaO: 240.1107, found:
240.1095. Anal. Calcd for C12H15N3O: C, 66.34; H, 6.96; N, 19.34.
Found: C, 66.18; H, 7.07; N, 19.16.
4.2.5.7. 5-Phenyl-1,2,3-triazole-4-carbonitrile (82). To a stirring
solution of sodium azide (200 mg, 3.08 mmol) in DMF (2 ml) was
added a solution of 83 (255 mg, 2.0 mmol) in DMF (0.8 ml) drop
wise at 90 °C under a nitrogen atmosphere. After 1.5 h the reaction
was quenched by the addition of an equivalent volume of water.
The precipitated product was filtered off and washed with water
and diethyl ether. Compound 83 was obtained as a pale brown so-
lid (280 mg, 82%). 1H NMR (300 MHz, acetone-d6): d = 7.58 (m, 3H),
7.97 (m, 2H) ppm; 13C NMR (75 MHz, acetone-d6): d = 113.7, 127.4,
127.6, 130.1, 130.2, 130.9, 150.9 ppm; IR (neat): 3076, 3000, 2948,
2904, 2812, 2241, 1610, 1496, 1484, 1273, 1220, 1101, 1024, 984,
914, 865, 834, 773, 715, 684 cmꢁ1; MS (EI, 70 eV): m/z (%): 170.1
(100) [M]+; HRMS (ESI): m/z [M+H]+ calcd for C9H7N4: 171.0665,
found: 171.0665. Anal. Calcd for C9H6N4: C, 63.52; H, 3.55; N,
32.92. Found: C, 63.46; H, 3.80; N, 32.72.
4.2.5.2. 2-Cyano-3-(3,4,5-trimethoxyphenyl)propanamide (77).
Starting from 20 (525 mg, 2.0 mmol) compound 77 was obtained
after flash chromatography (cyclohexane/ethyl acetate) as a color-
less solid (400 mg, 76%). 1H NMR (300 MHz, acetone-d6): d = 3.08
(m, 1H), 3.21 (m, 1H), 3.69 (s, 3H), 3.79 (s, 6H), 3.93 (m, 1H),
6.66 (s, 2H), 6.83 (br m, 1H), 7.21 (br m, 1H) ppm; 13C NMR
(75 MHz, acetone-d6): d = 37.0, 40.7, 56.4, 60.4, 107.4, 118.8,
133.3, 138.3, 154.3, 167.2 ppm; IR (neat): 3395, 3308, 2941,
2835, 2248, 1670, 1618, 1590, 1507, 1460, 1422, 1402, 1339,
1311, 1239, 1185, 1153, 1123, 1008, 974, 807 cmꢁ1; MS (EI,
70 eV): m/z (%): 264.1 (38) [M]+; HRMS (ESI): m/z [M+H]+ calcd
for
C13H17N2O4: 265.1183, found: 265.1182. Anal. Calcd for
C13H16N2O4: C, 59.08; H, 6.10; N, 10.60. Found: C, 59.07; H, 5.95;
N, 10.49.
4.2.5.3. 2-Cyano-3-(4-morpholin-4-ylphenyl)propanamide (78).
Starting from 57 (128 mg, 0.5 mmol) compound 78 was obtained
after flash chromatography (cyclohexane/ethyl acetate) as a color-
less solid (70 mg, 54%). 1H NMR (300 MHz, CDCl3): d = 3.10–3.24
(m, 6H), 3.57 (m, 1H), 3.84 (m, 4H), 5.68 (br m, 1H), 5.98 (br m,
1H), 6.87 (m, 2H), 7.19 (m, 2H) ppm; 13C NMR (75 MHz, CDCl3):
d = 34.9, 40.6, 49.1, 66.9, 115.8, 117.9, 126.4, 130.0, 150.7,
165.9 ppm; IR (neat): 3423, 3170, 2969, 2857, 2813, 2257, 1674,
1613, 1516, 1448, 1379, 1330, 1301, 1263, 1233, 1193, 1119,
1067, 1050, 927, 807 cmꢁ1; HRMS (ESI): m/z [M+H]+ calcd for
4.2.5.8. 3-Phenylprop-2-ynenitrile (83). To a stirring solution of
copper cyanide (7.16 g, 80.0 mmol), sodium iodide (400 mg,
2.67 mmol) and phenylacetylene (2.93 ml, 26.7 mmol), in DMSO
(50 ml), acetonitrile (13.3 ml) and water (0.72 ml) was added tri-
methylsilyl chloride (10.1 ml, 79.6 mmol) over 1 h at room temper-
ature under a nitrogen atmosphere. After the addition, the reaction
was warmed up to 50 °C and was stirred at this temperature for
100 h. After addition of water the mixture was extracted with
dichloromethane. The extract was washed with sodium bicarbon-
ate solution and brine. After evaporation of the solvent, the residue
was purified by flash chromatography (cyclohexane/ethyl acetate)
to get compound 83 as a colorless crystalline solid (850 mg, 25%).
1H NMR (300 MHz, CDCl3): d = 7.41 (m, 2H), 7.53 (m, 1H), 7.60
(m, 2H) ppm; 13C NMR (75 MHz, CDCl3): d = 63.1, 82.9, 105.5,
117.5, 128.9, 131.9, 133.5 ppm; HRMS (ESI): m/z [M+H]+ calcd for
C9H6N: 128.0494, found: 128.0504.
C
C
14H18N3O2: 260.1394, found: 260.1392. Anal. Calcd for
14H17N3O2: C, 64.85; H, 6.61; N, 16.20. Found: C, 64.55; H, 6.71;
N, 16.14.
4.2.5.4. 2-Cyano-3-(1H-indol-3-yl)propanamide (79). Starting
from 54 (211 mg, 1.0 mmol) compound 79 was obtained after pre-
cipitation as a colorless solid (140 mg, 66%). 1H NMR (300 MHz,
CDCl3): d = 3.44 (m, 1H), 3.51 (m, 1H), 3.71 (m, 1H), 5.40 (br m,
1H), 5.91 (br m, 1H), 7.12–7.24 (m, 3H), 7.39 (m, 1H), 7.64 (m,
1H), 8.11 (br m, 1H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 26.0,
39.1, 109.4, 111.5, 118.4, 118.5, 118.9, 121.1, 123.9, 126.7, 136.1,
166.8 ppm; IR (neat): 3401, 3354, 3176, 2927, 2257, 1661, 1455,
4.2.6. General procedure for the preparation of compounds 84–
88
A mixture of methyl cyanoacetate (1 equiv) and amine (1–
1.05 equiv) was stirred 1 h at 0 °C and at room temperature over-
night. If precipitation occurred, the precipitate was filtered off
and washed with diethyl ether. If precipitation did not occur, the
mixture was evaporated and the residue was washed with diethyl
ether.
1420, 1325, 1228, 1119, 1097, 1008, 927, 846, 809, 754 cmꢁ1
;
HRMS (ESI): m/z [M+H]+ calcd for C12H12N3O: 214.0975, found:
214.0963; HPLC purity >98%.

7336
C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
4.2.6.1. 2-Cyano-N-cyclopropylacetamide (84). Starting from
Cyclopropylamine (1.4 ml, 20.0 mmol) and methyl cyanoacetate
(1.98, 20.0 mmol) compound 84 was obtained after precipitation
as a colorless solid (2.13 g, 86%). 1H NMR (300 MHz, CDCl3):
d = 0.58 (m, 2H), 0.82 (m, 2H), 2.72 (m, 1H), 3.33 (s, 2H), 6.33 (br
m, 1H) ppm; 13C NMR (75 MHz, acetone-d6): d = 6.2, 23.6, 25.9,
115.9, 163.8 ppm; MS (EI, 70 eV): m/z (%): 124.0 (29) [M]+.
Compound 90 was obtained as a colorless solid (896 mg, 36%). 1H
NMR (300 MHz, CDCl3): d = 0.49 (m, 2H), 0.77 (m, 2H), 1.44 (s,
9H), 2.71 (m, 1H), 3.01 (br m, 1H), 3.19 (m, 1H), 3.62 (m, 1H),
4.06 (m, 1H), 5.55 (br m, 1H), 6.78 (br m, 1H) ppm; 13C NMR
(75 MHz, CDCl3): d = 6.4, 6.5, 22.5, 28.2, 54.8, 62.8, 80.5, 156.2,
172.8 ppm; MS (EI, 70 eV): m/z (%): 224.2 (1) [M]+.
4.2.6.8. N1-Cyclopropylserineamide hydrochloride (91). To
a
4.2.6.2. 2-Cyano-N-(tetrahydrofuran-2-ylmethyl)acetamide (85).
Starting from 1-(tetrahydrofuran-2-yl)methanamine (3.1 ml,
30.0 mmol) and methyl cyanoacetate (3.00, 30.3 mmol) compound
85 was obtained after addition of water (5 drops) and precipitation
as a pale rose solid (4.55 g, 90%). 1H NMR (300 MHz, acetone-d6):
d = 1.54 (m, 1H), 1.79–1.98 (m, 3H), 3.20 (m, 1H), 3.35 (m, 1H),
3.58 (s, 2H), 3.65 (m, 1H), 3.78 (m, 1H), 3.89 (m, 1H), 7.41 (br m,
1H) ppm; 13C NMR (75 MHz, acetone-d6): d = 25.9, 26.3, 29.3,
44.2, 44.3, 68.3, 78.0, 116.1, 162.8 ppm; MS (EI, 70 eV): m/z (%):
169.0 (2) [M+H]+.
solution of 90 (628 mg, 2.57 mmol) in ethyl acetate (15 ml) was
added a 2 N solution of HCl in diethyl ether (5 ml, 10.0 mmol)
slowly. After 24 h at 4 °C the precipitate was collected by filtration
and washed with diethyl ether. Compound 91 was obtained as a
colorless solid (337 mg, 73%). 1H NMR (300 MHz, D2O): d = 0.54
(m, 2H), 0.75 (m, 2H), 2.63 (m, 1H), 3.83–4.04 (m, 3H) ppm; 13C
NMR (75 MHz, D2O): d = 5.3, 22.0, 54.4, 60.1, 169.1 ppm; HRMS
(ESI): m/z [M+H]+ calcd for C6H13N2O2: 145.0972, found: 145.0978.
4.2.6.9. 4-(Hydroxymethyl)benzaldehyde (93). A solution of
terephthalaldehyde (7.5 g, 56 mmol) in THF (50 ml) was cooled
to 0 °C. Sodium borohydride (750 mg, 19.8 mmol) was added and
the mixture was stirred at room temperature for 1 h. After evapo-
ration of the solvent, the residue was taken up in ethyl acetate and
the solution was washed with water and brine and dried over
MgSO4. Upon removal of the solvent, the residue was purified by
flash chromatography (cyclohexane/ethyl acetate) to obtain 93 as
a white solid (3.85 g, 50%). 1H NMR (300 MHz, acetone-d6):
d = 4.49 (t, J = 5.3 Hz, 1H), 4.74, (d, J = 4.4 Hz, 2H), 7.58 (m, 2H),
7.88 (m, 2H), 10.01 (s, 1H) ppm; 13C NMR (75 MHz, acetone-d6):
d = 64.1, 127.5, 130.2, 136.5, 150.4, 192.6 ppm; HRMS (ESI): m/z
[M+H]+ calcd for C8H9O2: 137.0597, found: 137.0612.
4.2.6.3. 2-Cyano-N-(pyridin-2-ylmethyl)acetamide (86). Starting
from 1-pyridin-2-ylmethanamine (1.08 g, 10.0 mmol) and methyl
cyanoacetate (990 mg, 10.0 mmol) compound 86 was obtained
after evaporation and washing procedure as a colorless solid
(1.79 g, quant.). 1H NMR (300 MHz, DMSO-d6): d = 3.74 (s, 2H),
4.38 (d, J = 5.9 Hz, 2H), 7.29 (m, 2H), 7.76 (td, J = 7.7, 1.8 Hz, 1H),
8.50 (m, 1H), 8.81 (br m, 1H) ppm; 13C NMR (75 MHz, DMSO-d6):
d = 25.3, 44.6, 116.2, 121.2, 122.3, 136.8, 148.9, 157.6, 162.4 ppm;
MS (MALDI-TOF): m/z: 176.2 [M+H]+.
4.2.6.4. 3-Morpholin-4-yl-3-oxopropanenitrile (87). Starting
from morpholine (3.50 ml, 40.2 mmol) and methyl cyanoacetate
(3.96 g, 40.0 mmol) compound 87 was obtained after addition of
diethyl ether and precipitation as a pale brown solid (5.53 g,
90%). 1H NMR (300 MHz, acetone-d6): d = 3.50 (m, 4H), 3.63 (m,
4H), 3.87 (m, 2H) ppm; 13C NMR (75 MHz, acetone-d6): d = 43.1,
47.0, 66.9, 115.8, 162.1 ppm; MS (MALDI-TOF): m/z: 155.1 [M+H]+.
4.2.6.10. 4-Hydroxy-3-methoxy-5-nitrobenzaldehyde (94). To a
solution of vanillin (5.0 g, 32.9 mmol) in acetic acid (50 ml) was
added nitric acid (60%, 2.6 ml) slowly at 0 °C. Afterwards the solu-
tion was warmed up to room temperature and allowed to stir for
another 30 min. The precipitate was filtered off and washed with
cold water, to give compound 94 as a yellow solid (5.40 g, 83%).
1H NMR (300 MHz, DMSO-d6): d = 3.95 (s, 3H), 7.61 (d, J = 1.8 Hz,
1H), 8.08 (d, J = 1.9 Hz, 1H), 9.85 (s, 1H) ppm; 13C NMR (75 MHz,
DMSO-d6): d = 56.8, 112.5, 120.8, 126.8, 137.0, 147.7, 150.0,
190.4 ppm; HRMS (ESI): m/z [MꢁH]ꢁ calcd for C8H6NO5:
196.0251, found: 196.0277.
4.2.6.5. 3-(4-Methylpiperazin-1-yl)-3-oxopropanenitrile (88).
Starting from N-methylpiperazine (2.0 g, 20.0 mmol) and methyl
cyanoacetate (1.98 g, 20.0 mmol) compound 88 was obtained after
precipitation as a pale rose solid (2.69 g, 80%). 1H NMR (300 MHz,
DMSO-d6): d = 2.16 (s, 3H), 2.21–2.32 (m, 4H), 3.28–3.46 (m, 4H),
4.01 (s, 2H) ppm; 13C NMR (75 MHz, DMSO-d6): d = 24.7, 41.5,
45.2, 45.5, 54.0, 54.3, 116.1, 161.4 ppm; MS (EI, 70 eV): m/z (%):
167.1 (64) [M]+.
4.2.6.11. 3,4-Dihydroxy-5-nitrobenzaldehyde (95). A solution of
94 (3.5 g, 17.8 mmol) in hydrobromic acid (48%, 32 ml) was re-
fluxed for 4 h. After cooling to room temperature and addition of
water and ice, the precipitate was filtered off and washed with cold
water, to give compound 95 as a brown solid (1.62 g, 50%). 1H NMR
(300 MHz, DMSO-d6): d = 7.46 (d, J = 1.9 Hz, 1H), 7.95 (d, J = 1.9 Hz,
1H), 9.79 (s, 1H), 10.52 (br m, 2H) ppm; 13C NMR (75 MHz, DMSO-
d6): d = 115.8, 119.6, 127.0, 137.2, 147.2, 148.3, 190.5 ppm; HRMS
(ESI): m/z [MꢁH]ꢁ calcd for C7H4NO5: 182.0059, found: 182.0088.
4.2.6.6. 2-Cyano-N,N-diethylacetamide (89). A mixture of methyl
cyanoacetate (1.5 g, 15.1 mmol) and diethylamine (3.0 ml,
29.0 mmol) was stirred 1 h at 0 °C and at room temperature over-
night. Dichloromethane was added, the precipitate was filtered
and discarded; the organic layer was washed with 1 N hydrochloric
acid and sodium bicarbonate solution and dried over MgSO4. After
evaporation of the solvent, compound 89 was obtained as a pale
red oil (630 mg, 30%). 1H NMR (300 MHz, acetone-d6): d = 1.07 (t,
J = 7.1 Hz, 3H), 1.20 (t, J = 7.1 Hz, 3H), 3.35 (m, 4H), 3.81 (s, 2H)
ppm; 13C NMR (75 MHz, acetone-d6): d = 13.1, 14.2, 25.1, 41.1,
43.0, 116.1, 162.3 ppm; MS (MALDI-TOF): m/z: 141.1 [M+H]+.
4.2.6.12. 4-(Morpholin-4-ylcarbonyl)benzaldehyde (96). To
a
solution of 4-formylbenzoic acid (750 mg, 5.0 mmol), HOAt
(750 mg, 5.50 mmol) and HATU (2.28 g, 6.0 mmol) in dry dichloro-
methane (15 ml) was added DIPEA (2.70 ml, 15.0 mmol) slowly at
0 °C. After 30 min the mixture was allowed to warm up to room
temperature and morpholine (0.50 ml, 5.75 mmol) was added.
After 2 h the solvent of the reaction mixture was evaporated and
the residue was purified by flash chromatography (cyclohexane/
ethyl acetate) to obtain compound 96 as a pale yellow oil
(950 mg, 87%). 1H NMR (300 MHz, acetone-d6): d = 3.31–3.79 (m,
8H), 7.63 (m, 2H), 7.99 (m, 2H), 10.09 (s, 1H) ppm; 13C NMR
(75 MHz, acetone-d6): d = 38.7, 67.2, 128.6, 130.3, 138.0, 142.6,
169.1, 192.5 ppm; MS (EI, 70 eV): m/z (%): 219.1 (64) [M]+.
4.2.6.7. N2-(tert-Butyloxycarbonyl)-N1-cyclopropylserineamide
(90). To a solution of N-(tert-butoxycarbonyl)-L-serine (2.00 g,
9.75 mmol) and HATU (4.08 g, 10.7 mmol) in dry DMF (15 ml)
were added cyclopropylamine (0.71 ml, 10.2 mmol) and DIPEA
(5.09 ml, 29.3 mmol) slowly. After 2 h the reaction was quenched
with water and the mixture was extracted with ethyl acetate. After
drying over MgSO4 the solvent was evaporated and the residue was
purified by flash chromatography (dichloromethane/methanol).

C. Nitsche et al. / Bioorg. Med. Chem. 19 (2011) 7318–7337
7337
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7H), 9.83 (s, 1H) ppm; 13C NMR (75 MHz, CDCl3): d = 56.1, 70.9,
109.3, 112.4, 126.6, 127.2, 128.2, 128.7, 130.3, 136.0, 150.1,
153.6, 190.9 ppm; MS (MALDI-TOF): m/z: 243.1 [M+H]+.
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Acknowledgements
Christoph Nitsche acknowledges funding by a fellowship of the
Studienstiftung des deutschen Volkes. Christian Steuer acknowl-
edges funding by a fellowship of the Konrad Adenauer Stiftung.
We thank Franziska Schenk and Florian Mulks for assisting in the
synthesis of some compounds. We thank Dr. Frank Rominger
(Chemistry Department, University of Heidelberg) for performing
the small-molecule X-ray analysis.
References and notes
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