Inhibition of the cellular function of perforin by 1-amino-2,4- dicyanopyrido[1,2-a]benzimidazoles

Article abstract of DOI:10.1016/j.bmc.2011.05.013

A high throughput screen showed the ability of a 1-amino-2,4- dicyanopyrido[1,2-a]benzimidazole analogue to directly inhibit the lytic activity of the pore-forming protein perforin. A series of analogues were prepared to study structure-activity relations

Full text of DOI:10.1016/j.bmc.2011.05.013

Bioorganic & Medicinal Chemistry 19 (2011) 4091–4100
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of the cellular function of perforin by 1-amino-2,
4-dicyanopyrido[1,2-a]benzimidazoles
Dani M. Lyons a, Kristiina M. Huttunen a,b, Kylie A. Browne c, Annette Ciccone c, Joseph A. Trapani c,
a
William A. Denny a, , Julie A. Spicer
⇑
a Auckland Cancer Society Research Centre, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
b School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, P.O. Box 1627, FI-70211 Kuopio, Finland
c Cancer Immunology Program, Peter MacCallum Cancer Centre, St. Andrew’s Place, East Melbourne, Victoria 3002, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
A high throughput screen showed the ability of a 1-amino-2,4-dicyanopyrido[1,2-a]benzimidazole ana-
logue to directly inhibit the lytic activity of the pore-forming protein perforin. A series of analogues were
prepared to study structure–activity relationships (SAR) for the this activity, either directly added to cells
or released in situ by KHYG-1 NK cells, at non-toxic concentrations. These studies showed that the pyr-
idobenzimidazole moiety was required for effective activity, with strongly basic centres disfavoured. This
class of compounds was relatively unaffected by the addition of serum, which was not the case for a pre-
vious class of direct inhibitors.
Received 3 March 2011
Revised 3 May 2011
Accepted 9 May 2011
Available online 18 May 2011
Keywords:
Perforin
Perforin inhibitor
Dicyanopyrido[1,2-a]benzimidazole
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
a-helices that insert into membranes as b-strands upon pore for-
mation.7 In the same report, cryo-electron microscopy also con-
firmed the distribution of individual perforin sub-domains within
an assembled pore structure.
Perforin (PRF) is a pore-forming member of the membrane-at-
tack-complex/PRF (MACPF) protein family1, and is secreted, along
with a range of serine proteases (granzymes), by both cytotoxic T
lymphocytes (CTLs) and natural killer (NK) cells.2 Together, perfo-
rin and granzymes comprise a principal mechanism of CTL/NK
cytotoxicity by which the immune systems of higher organisms
protect themselves against pathogens and transformed cells. The
critical role of perforin in this process is its ability, upon binding
calcium, to assemble into aggregates of 12–18 molecules that form
trans-membrane pores of 10–20 nm in diameter in the plasma
membrane of the target cell, facilitating the entry of granzymes,
which can trigger apoptosis.3,4 Initially, the calcium-dependent
membrane binding of perforin monomers is mediated by their C
terminal C2 domains.5 Successive perforin monomers then assem-
ble into a cylindrical pore via a series of salt bridges linking Arg213
on the ‘front’ of one monomer with Glu343 on the ‘back’ of the
next.6 The structure of mouse perforin monomer at a resolution
of 2.75 Å has recently been reported as a bent and twisted
four-stranded b-sheet MACPF domain flanked by two clusters of
Apart from well-recognised disease syndromes due to mutation
and/or down-regulation of perforin expression, there is also much
evidence linking inappropriate perforin activity to several human
pathologies, including cerebral malaria, type 1 diabetes, juvenile
idiopathic arthritis and postviral myocarditis,7 as well as ther-
apy-induced conditions such as allograft rejection and graft-
versus-host disease.8,9 Perforin is therefore a potentially druggable
target, especially as it is encoded by a single-copy gene in both
mice and humans.
However, there have been few reports of small-molecule inhib-
itors of perforin function, as distinguished from compounds that
affect it indirectly by inhibiting cell respiration or perforin process-
ing.10 Enzastaurin (1) (Fig. 1), a bisindolylmaleimide structurally
related to staurosporine and currently in Phase II trial for cancer,
has recently been reported11 to inhibit perforin release from NK
cells, but is thought to act via the PKC/PI3K/AKT pathway. We re-
cently reported10 a series of dihydrofuro[3,4-c]pyridinones (e.g.,
2) (Fig. 1) as direct inhibitors of the cytolytic effects of perforin
in cells, and demonstrated limited structure–activity effects across
the series. The most active compound in the series (2) had a IC50 of
Abbreviations: CDI, carbonyldiimidazole; CTL, cytotoxic T lymphocytes; DMF,
dimethylformamide; LRMS, low-resolution mass spectrometry; NK, natural killer
cells; MACPF, membrane-attack-complex/PRF; PRF, perforin; SEM, standard error of
the mean; THF, tetrahydrofuran.
1
lM for inhibition of the lysis of Jurkat cells by added perforin,
and at a concentration of 20 M showed a 60% inhibition in the
l
killing of K562 cells by perforin released from KHYG-1 NK cells,
with little toxicity to the latter. However, the cellular activity of
⇑
Corresponding author. Tel.: +64 9 923 6144; fax: +64 9 923 7502.
E-mail address: b.denny@auckland.ac.nz (W.A. Denny).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.013

4092
D. M. Lyons et al. / Bioorg. Med. Chem. 19 (2011) 4091–4100
The synthesis of this general class of compounds has been re-
H
N
ported by Bogdanowicz-Szwed and Czarny13 from the reaction of
2-(1H-benzo[d]imidazol-2-yl)acetonitrile (44) with arylmalono-
nitriles, and14 by Elnagdi et al., by reaction of 44 with a mixture of
formaldehyde and cyanothioacetamide to yield (e.g.) 3 (Fig. 1). Chao
et al.15 reported the synthesis of related 7-cyanopyrido[1,2-a]benz-
imidazoles (e.g., 4) (Fig. 1) in moderate yield by the multi-compo-
nent reaction of pyridine, chloroacetonitrile, malononitrile, and
aromatic aldehyde in refluxing acetonitrile. Related compounds
have been reported as potential inhibitors of transcription.16
An authentic sample of lead compound 5 was synthesized in or-
der to confirm the original perforin inhibitory data. Reaction of
aldehyde 42, malononitrile and 3-aminopropanoic acid in EtOH
provided compound 43. (Note: we surveyed a variety of catalytic
bases for use in this key Knoevenagel condensation and found that
3-aminopropanoic acid was the best). The dicyanovinyl intermedi-
ate 43 was then heated under reflux with 2-(1H-benzo[d]imidazol-
2-yl)acetonitrile (44) and piperidine in EtOH to give compound 5 in
good overall yield. To explore the SAR of the oxyacetamide side
chain we prepared both N-alkylated (6–10) and hydroxyalkyl
(14–18) compounds. The key intermediate 47 for these was syn-
thesized in a similar manner, from 2-(4-formylphenoxy)acetic acid
(45) (Scheme 1). Compound 47 was then treated with 1,10-carbon-
yldiimidazole (CDI) in anhydrous DMF and subsequently reacted
with various amines to provide compounds 6–10 and 14–18 in var-
iable yields.
O
O
S
O
HN
Me
O
N
Me
N
1
2
S
N
N
CN
CN
N
CN
CH3
CN
N
N
N
NH2
4
3
Figure 1. Inhibitors of perforin.
these compounds was later shown to be adversely affected by
increasing concentrations of serum, limiting their further develop-
ment. In the present paper we discuss the discovery of this new
class of direct perforin inhibitors, the 1-amino-2,4-dicyanopyri-
do[1,2-a]benzimidazoles, and initial structure–activity relation-
ship (SAR) studies on this new class.
To replace the acetamide moiety, we prepared compound 11,
which carries an oxyethanol side chain (Scheme 2). Reaction of
2-(4-hydroxybenzylidene)malononitrile (48), 44 and piperidine in
EtOH provided 49, which was then reacted with tert-butyldimeth-
ylsilyl (TBDMS)-protected bromoethanol in the presence of Cs2CO3
in anhydrous DMF. Removal of the protecting group by tetrabutyl-
ammonium fluoride (TBAF) in THF then gave 11.
Compounds 12 and 13, containing carbon-linked side chains,
were obtained from ethyl 3-(4-formylphenyl)propanoate (51) in
a four-step sequence in moderate to good yields (Scheme 3). The
first two steps were carried out as for 5, affording ester 53, which
was then converted to the corresponding carboxylic acid 54 by
hydrolysis with NaOH in EtOH, followed by CDI-mediated amide
formation to give 12 and 13.
For amides directly attached to the phenyl ring (compounds
19–30, Scheme 4), the key intermediate 57 was prepared from
methyl 4-(2,2-dicyanovinyl)benzoate (55) by reaction with 44
and piperidine in EtOH and then hydrolysing the resulting ester
56 to give 57. This was then reacted with CDI followed by amines
in anhydrous DMF to give the required compounds.
2. Results and discussion
2.1. Chemistry
The lead compound (5) was selected from a high throughput
screen12 of 100,000 compounds sourced from commercial libraries,
using a 384-well plate format. Compounds were screened (at
20 lM) by incubation with sheep red blood cells, which in the ab-
sence of an inhibitor are lysed by perforin, generating turbidity
that can be measured by the change in absorbance at 650 nm.
Compound 5 was one of a few hits that were confirmed in an assay
measuring its potency for inhibiting the lysis of Jurkat human leu-
kaemia cells by added mouse perforin, where it showed an IC50 of
about 5 lM. This activity was reproduced by a re-synthesized sam-
ple prepared by the method given in Scheme 1. An advantage of
this lead compared with 2 was a relative lack of potentially reac-
tive substituents, including sulfur, but a potential disadvantage
was low aqueous solubility.
O
O
X
R
N
ii
compound 5
CH2CN
42: R=CHO; X-NH2
of Table 1
i
N
H
44
43: R=CH=C(CN)2, X=NH2
+
45
: R=CHO; X=OH
i
46: R=CH=C(CN)2, X=OH
iii
CN
N
iv
compounds 6-
10, 14-18 of
Table 1
O
CO2H
N
CN 47
H2N
Scheme 1. Reagents and conditions: (i) malononitrile, 3-aminopropanoic acid, EtOH, 20 °C, 24 h; (ii) piperidine, EtOH, reflux, 3 h; (iii) compound 44, piperidine, EtOH, reflux,
3 h; (iv) CDI/DMF, 1 h, 20 °C, then various amines, 1 h, 20 °C.

D. M. Lyons et al. / Bioorg. Med. Chem. 19 (2011) 4091–4100
4093
R
3
4
CN
NH2
NH2
OH
4
5
CN
N
CN
i
5
i
iii
R
ii
compounds 31-
N
R
compound 11
35 of Table 1
NC
N
H
of Table 1
6
N
iii
48
CN
44: R=H
compounds 36-
41 of Table 2
NH2
63: 4-Me
58: 3-Me
64: 4-Cl
59: 3-Cl
49: R=OH
50: R=O(CH2)2OTBDMS
ii
65: 4-OMe
66: 4,5-diMe
67: 5,6-diMe
60: 3-OMe
61: 3,4-diMe
62: 4,5-diMe
Scheme 2. Reagents and conditions: (i) compound 44, EtOH, piperidine, reflux, 2 h;
(ii) Br(CH2)2OTBDMS, Cs2CO3, dry DMF, 90 °C (N2), 16 h. (iii) TBAF, THF, 20 °C,
30 min.
Scheme 5. Reagents and conditions: (i) cyanoacetamide, 4 Å MS, xylene, reflux,
24 h; (ii) compound 43, piperidine (catalytic), EtOH, reflux, 24 h; (iii) compound 42,
piperidine (catalytic), EtOH, reflux, 24 h.
Compounds 31–35, with the same oxyacetamide side chain as
the lead compound 5 but bearing various substituents on the ben-
zoimidazole ring (Scheme 5), were prepared from substituted ben-
zenediamines 58–62, which were reacted with cyanoacetamide in
xylene to form the benzoimidazolylacetonitriles 63–67. Treatment
of these with the dicyanovinyl intermediate 43 and piperidine in
EtOH as above gave the desired compounds. Finally, similar treat-
ment of the benzoimidazolylacetonitriles 44, 63–67 with aldehyde
42 in the same way gave the analogous bicyclic analogues 36–41
(Table 2).
retains activity, but a longer derivative (16) or those with branched
chains (15,17) do not. However, the (larger) pyridyl (18) did retain
weak activity.
Given the above results with more bulky substituents, the side-
chain was therefore pared right back to a primary amide (19). This
compound, and its NMe analogue (20) showed excellent activity
(IC50s = 3.00 and 1.04
lM, respectively), a significant improvement
on the lead compound 5 (5.19
lM). With improved solubility still a
priority, a series of potentially solubilising sidechains were then
introduced, linked directly to the amide (diol 26 and dimethyl-
2.2. Structure–activity relationships
amine 27 have aqueous solubilities of 51
lM and 68 lM, respec-
tively, compared with 25 M for 20). With the exception of the
l
Table 1 provides structural data for a series of analogues of 5,
and their potencies for inhibiting the lysis of labelled Jurkat human
leukaemia cells by added perforin. Compounds 6–10 use variants
of the O-linked side chain of 5 to explore bulk tolerance with alkyl
chains. While the NHMe compound 6 retains activity, there ap-
pears to be some activity loss with the longer members (7–10).
Compound 11, with a small O(CH2)2OH side chain, also showed
good potency. Compounds 12 and 13 used side chains similar to
those in 6 and 7, respectively, but using a CH2 linker, and were both
completely inactive. This suggests the oxygen linkage may be
important for activity. Compounds 14–18 look at potentially
more bulky CONMe2 analogue 21 and the two strongly basic ana-
logues 27 (calcd pKa 8.69) and 28 (calcd pKa 8.85) all of the CONH-
linked neutral compounds were at least as potent as the lead 5 in
the Jurkat assay (IC50s from 1.04 to 5.5
(calcd pKa 6.84) and 30 (calcd pKa 5.74) were less active (IC50
10 M; comparable with 18).
Compounds 31–35 explore additional substitution in the pyrido
lM). The two weak bases 29
s
l
ring. The compounds showed activity, but overall this was little
different to that of 5. The substituent changes were not extensive,
but were sufficiently-varied electronically to suggest no dramatic
electronic influence on biological activity.
To further evaluate the importance of elements of the pyrido-
benzimidazole moiety, the bicyclic compounds 36–41, both the
solubilising chains (diol 17 has an aqueous solubility of 180
lM,
compared to 16 M for 5). The 2-hydroxyethylacetamide 14
l
CO2R
CN
N
O
compounds
iv
ii
12-13 of
N
OEt
CN
Table 1
NH2
53: R=Et
R
51: R=CHO
i
iii
52: R=CH=C(CN)2
54:
R=H
Scheme 3. Reagents and conditions: (i) malononitrile, 3-aminopropanoic acid, EtOH, 20 °C, 16 h; (ii) compound 44, piperidine, EtOH, reflux, 3 h; (iii) aqueous NaOH, EtOH,
reflux, 1 h; (iv) CDI, dry DMF, 1 h, RNH2, 20 °C, 16 h.
R
CN
N
CO2Me
CN
N
iii
i
compounds 19-
30 of Table 1
CN
NC
NH2
R=CO2Me
57: R=CO2H
55
56:
ii
Scheme 4. Reagents and conditions: (i) compound 44, piperidine, EtOH, reflux, 3 h; (ii) NaOH, EtOH, reflux, 1 h; (iii) CDI, dry DMF, RNH2, 20 °C, 16 h.

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D. M. Lyons et al. / Bioorg. Med. Chem. 19 (2011) 4091–4100
Table 1
Table 2
Structures and perforin inhibitory activities of substituted 9-amino-6,8-dicyano-7-
phenylpyrido[1,2-a]benzimidazole analogues
Structures and perforin inhibitory activities of bicyclic analogues
O
O
CN
6
N
CN
NH2
X
7
R
4
N
N
X
8
NH
9
5
CN
H2N
6
7
a
No.
R
X
IC50 (lM)
a
No.
X
IC50 (lM)
5
6
7
8
OCH2CONH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
5.19 0.31
4.56 0.88
6.89 0.51
7.44 0.72
13.9 1.0
13.2 0.8
2.87 0.37
>20
>20
8.25 1.50
>20
>20
>20
OCH2CONHMe
OCH2CONMe2
OCH2CONHEt
OCH2CONHnPr
OCH2CONHiPr
OCH2CH2OH
36
37
38
39
40
41
H
4-Me
4-Cl
4-OMe
4,5-DiMe
5,6-DiMe
>20
>20
6.72 1.84
>20
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
2
>20
5.14 1.21
CH2CH2CONHMe
CH2CH2CONMe2
a
As for Table 1.
OCH2CONHCH2CH2OH
OCH2CONHCH2CH(Me)OH
OCH2CONHCH2CH2CH2OH
OCH2CONHCH2CH(OH)CH2OH
OCH2CONHCH2CH2(4-pyridyl)
CONH2
CONHMe
CONMe2
CONHEt
CONHCH2CH2OH
CONHCH2CH2CH2OH
CONHCH2CH(CH3)OH
CONHCH2CH(OH)CH2OH
CONHCH2CH2NMe2
CONHCH2CH2Npiperidyl
CONHCH2CH2Nmorpholyl
CONHCH2CH2(4-pyridyl)
OCH2CONH2
OCH2CONH2
OCH2CONH2
OCH2CONH2
OCH2CONH2
Table 3
Capacity of selected compounds to inhibit perforin delivered by KHYG-1 NK cells
ꢂ10
KHYG-1 viabilityc (%)
3.00 0.60
1.04 0.14
>20
No. Jurkat IC50
(lM)
KHYG-1 inhibition (% at
20
l
M)
No seruma
10% serumb
3.16 0.10
3.08 0.65
4.18 0.39
5.55 1.08
3.24 0.77
>20
>20
10.0 1.6
10.5
6.87 1.79
5.67 0.60
7.49 1.71
5.21 0.60
3.53 0.29
0.97
5
2
6
7
8
9
5.19
0.97
4.56
6.9
18
60
41
34
0
41
52
15
49
0
7
9
19
4
0
0
0
1
15
10
36
38
2
6
86
88
84
78
77
90
70
85
96
92
79
84
53
89
86
68
84
85
62
88
60
62
42
25
11
22
22
16
64
6
7.44
13.9
13.2
3.37
>20
8.25
3.00
1.04
3.17
3.70
4.18
5.55
3.94
10
10.5
6.88
5.21
3.53
10
11
13
14
19
20
22
23
24
25
26
29
30
31
34
35
6-Me
6-Cl
6-OMe
6,7-DiMe
7,8-DiMe
6
12
12
12
7
0
0
5
20
8
7
5
a
Testing was carried out over a range of doses, with the lysis of Jurkat cells by
perforin measured by 51Cr release. Values are the average of at least three inde-
pendent IC50 determinations plus SEM. For some compounds great inter-experi-
ment variability was observed, possibly related to their relative insolubility.
parent 36 and analogues 37–41 were prepared (Table 2). These
targets explored the same limited set of substitution patterns on
the left-hand ring as did compounds 31–35. They effectively repre-
sent loss of the benzo ring of the pyrido[1,2-a]benzimidazole, to-
gether with its 1-amino and 8-cyano substituents. Table 2 shows
that the majority of these pyridobenzimidazole compounds lost
activity in the Jurkat assay.
a
Inhibition by compound (20 lM) of the perforin-induced lysis of K562 target
cells when co-incubated with KHYG-1 human NK cells (see Section 4). Percent
inhibition calculated compared to untreated control.
b
As for a, but in the presence of 10% mouse serum.
c
Viability of KHYG-1 NK cells after 24 h by Trypan blue exclusion assay (see
Section 4). All results are the average of at least three separate determinations.
Selected examples from Table 1 were also evaluated for their
ability to inhibit the effect of perforin released by KHYG-1 NK cells,
as shown in Table 3. Some of the compounds (e.g., 9,10,13) were
almost as effective as the dihydrofuro[3,4-c]pyridinone 2 (although
not as effective as some of the analogues of that series10) in the ab-
sence of added mouse serum, and without significant toxicity to
the KHYG-1 cells. However, there was little correlation between
the potencies of the compounds in the Jurkat lysis assay and their
ability to inhibit the effect of perforin released by KHYG-1 NK cells
(in the absence of serum), as shown by the equation relating Jurkat
IC50 and the percentage of KHYG-1 killing: log [IC50Jurkat] =
ꢀ1.2 ꢁ log[100 ꢀ (%KHYG-1 inhib)] + 2.9 (n = 21, R = 0.43, F = 4.6,
p = 0.05). This overall view is reinforced by specific examples
cell–cell assay. One encouraging aspect of the study was that the
activities of the bulk (9 of 12) of the compounds in Table 3 showing
significant (15% or greater) inhibition were relatively unaffected by
the addition of serum, which was not the case for the dihydrof-
uro[3,4-c]pyridinone series (e.g., 2).
3. Conclusions
Molecules with the ability to directly inhibit the function of per-
forin are of potential use in the treatment of some autoimmune dis-
eases and therapy-induced conditions characterised by undesired
perforin secretion, but to date only one class, the dihydrofuro[3,4-
c]pyridinones have been reported.10 Compound 5 in the present pa-
per was one of a small number of compounds from a high through-
put screen to show confirmatory inhibition of the lytic activity of
(e.g., 24: Jurkat IC50 4.18
IC50 >20 M) but 49% KHYG-1 inhibition. This may well be due to
the additional barriers to compound distribution in the latter
lM but no KHYG-1 inhibition; 13: Jurkat
l

D. M. Lyons et al. / Bioorg. Med. Chem. 19 (2011) 4091–4100
4095
perforin in a cell-based assay, and as a novel and accessible structure
with no obvious structural alerts, warranted further evaluation.
Analogues of 5 with both O-linked and CONH-linked side chains
showed moderate inhibition of the lysis of Jurkat human leukaemia
cells by added perforin, with the latter side chains appearing
slightly more effective. Compounds with strongly basic centres
(27,28), prepared in order to improve solubility were poorly toler-
ated. Additional substitution in the pyrido ring of the pyridobenz-
imidazole appeared to have little effect on activity, but the
complete pyrido[1,2-a]benzimidazole moiety appeared important,
with four out of six analogues lacking the benzo ring being inactive.
Selected compounds also showed good inhibition of the lytic
activity of perforin released by KHYG-1 NK cells at non-toxic con-
centrations, although there was limited correlation between these
effects and the potencies of the compounds in the Jurkat lysis as-
say. Encouragingly the compounds were relatively unaffected by
the addition of serum, which was not the case for the dihydrof-
uro[3,4-c]pyridinone series.
(C21H14N6O2): C, 65.96; H, 3.69; N, 21.98. Found: C, 65.51; H,
3.85; N, 21.80.
4.1.2. 2-[4-(1-Amino-2,4-dicyanopyrido[1,2-a]benzimidazol-3-
yl)phenoxy]-N-methylacetamide (6)
Reaction of 2-(4-formylphenoxy)acetic acid (45) (5.0 g,
27.75 mmol), malononitrile (5.51 g, 83.41 mmol) and 3-amino-
propanoic acid (1.0 g, 11.22 mmol) in EtOH (875 ml) as for the
preparation of 43 gave 2-(4-(2,2-dicyanovinyl)phenoxy)acetic acid
(46) as light brown solid (2.65 g, 42%) which was used directly. 1
H
NMR d ppm 13.14 (br s, 1H), 8.40 (s, 1H), 7.96 (d, J = 8.9 Hz, 2H),
7.17 (d, J = 9.0 Hz, 2H), 4.85 (s, 2H).
A solution of 46 (0.50 g, 2.19 mmol), 44 (0.17 g, 1.09 mmol) in
EtOH (7 mL) and piperidine (163 lL, 1.65 mmol) was heated to re-
flux for 3 h. The solution was cooled and the precipitate formed
was collected and washed with EtOH and Et2O. The crude material
was stirred with 10% aqueous AcOH, filtered, and the precipitate
was washed with water, EtOH and Et2O and dried to give 2-(4-
(1-amino-2,4-dicyanobenzo[4,5]imidazo[1,2-a]pyridin-3-yl)phenoxy)
acetic acid (47) as a yellow powder (0.18 g, 43%), mp 296–299 °C.
1H NMR d ppm 13.07 (br s, 1H), 8.64 (br s, 2H), 8.60 (d,
J = 8.4 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.65–7.58(m, 1H), 7.57 (d,
J = 8.8, 2H), 7.46–7.42 (m, 1H), 7.13 (d, J = 8.8 Hz, 2H), 4.80 (s,
2H). Anal. Calcd for (C21H13N5O3ꢃ0.75H2O): C, 65.79; H, 3.42; N,
18.27. Found: C, 63.76; H, 3.81; N, 17.38.
4. Experimental section
4.1. Chemistry
Analyses were performed by the Microchemical Laboratory,
University of Otago, Dunedin, NZ. Many samples tenaciously held
water or DMF (verified by NMR). Melting points were determined
using an Electrothermal Model 9200 and are as read. NMR spectra
were measured in CD3SOCD3 (unless otherwise specified) on a
Bruker Advance 400 spectrometer and referenced to Me4Si. Mass
spectra were recorded either on a Varian VG 7070 spectrometer
at nominal 5000 resolution or a Finnigan MAT 900Q spectrometer.
All final compound purities were determined to be >95% by HPLC
A solution of acid 47 (0.07 g, 0.18 mmol) in dry DMF (5 mL) was
warmed gently and then stirred at 20 °C under N2. CDI (0.04 g,
0.27 mmol) was added and the solution was stirred at 20 °C for
1 h. A solution of methylamine in MeOH (140 lL, 2.0 M) was added
and the solution was stirred for another 1 h. The DMF was removed
in vacuo and the residue was recrystallized from DMF/Et2O to give
6 as a yellow powder (0.04 g, 50%), mp 310–314 °C. 1H NMR d ppm
8.30 (br s, 2H), 8.61 (d, J = 8.4 Hz, 1H), 8.14–8.07 (m, 1H), 7.87 (d,
J = 7.8 Hz, 1H), 7.62–7.56 (m, 3H), 7.46–7.41 (m, 1H), 7.17 (d,
J = 8.8 Hz, 2H), 4.59 (s, 2H), 2.73 (d, J = 0.4 Hz, 3H). Anal. Calcd for
(C22H16N6O2ꢃ1.25H2O): C, 63.08; H, 4.05; N, 20.06. Found: C,
63.04; H, 4.38; N, 20.41.
on an Alltech Alltima C18 column (3.2 ꢁ 150 mm, 5
lm) eluting
with 5–80% MeCN/45 mM NH4HCO2. pKa values were calculated
using ACD pKa software (version 8.0; Advanced Chemistry
Development Inc., Toronto, Canada),
4.1.1. 2-[4-(1-Amino-2,4-dicyano-6-methylpyrido[1,2-
a]benzimidazol-3-yl)phenoxy]acetamide (5) (Scheme 1)
4.1.3. 2-[4-(1-Amino-2,4-dicyanopyrido[1,2-a]benzimidazol-3-
yl)phenoxy]-N,N-dimethylacetamide (7)
A
solution of 2-(4-formylphenoxy)acetamide (42) (1.0 g,
Similar reaction of acid 47 (0.10 g, 0.26 mmol) in dry DMF
(5 mL) with CDI, followed by treatment with dimethylamine in
MeOH, and purification of the crude product by column chroma-
tography (silica, 20% EtOH/EtOAc) gave 7 as a yellow powder
(0.03 g, 30%), mp 298–302 °C. 1H NMR d ppm 8.70 (br s, 2H), 8.60
(d, J = 8.4 Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H), 7.64–7.57 (m, 1H), 7.56
(d, J = 8.8 Hz, 2H), 7.44–7.37 (m, 1H), 7.11 (d, J = 8.9 Hz, 2H), 4.93
(s, 2H), 3.04 (s, 3H), 2.88 (s, 3H). HRMS (ESI+) for C23H19N6O2
(M+H)+: Calcd 411.1564. Found: 411.1564. HPLC purity, 99.1%.
5.59 mmol) and malononitrile (1.11 g, 17 mmol) in ethanol
(170 mL) was stirred at 20 °C. 3-Aminopropanoic acid (0.20 g,
2.23 mmol) was added at the resulting solution was stirred at
20 °C overnight. The solution was concentrated under reduced
pressure and the remaining solution was diluted with water and
cooled to 0 °C. The resulting precipitate was washed with water
and hexane and was dried to give 2-(4-(2,2-dicyanovinyl)phen-
oxy)acetamide17 (43) as a pale yellow powder (0.89 g, 70%). 1H
NMR d ppm 8.41 (s, 1H), 7.97 (d, J = 8.9 Hz, 2H), 7.64 (br s, 1H),
7.46 (br s, 1H), 7.18 (d, J = 9.0 Hz, 2H), 4.60 (s, 2H). LRMS (APCI+)
for C12H10N3O2 (M+H)+: Calcd 228, Found 228.
4.1.4. 2-[4-(1-Amino-2,4-dicyanopyrido[1,2-a]benzimidazol-3-
yl)phenoxy]-N-ethylacetamide (8)
A mixture of 43 (0.50 g, 2.20 mmol) and 2-(1H-benzo[d]imi-
dazol-2-yl)acetonitrile (44) (0.17 g, 1.10 mmol) in ethanol
(7 mL) was stirred at 20 °C. Piperidine (160 lL, 1.65 mmol) was
Similar reaction of acid 47 (0.20 g, 0.52 mmol) in dry DMF
(10 mL) with CDI, followed by treatment with ethylamine in water
gave 8 as a yellow powder (0.11 g, 49%), mp (EtOH/Et2O) 297–
301 °C. 1H NMR d ppm 8.66 (br s, 2H), 8.61 (d, J = 8.4 Hz, 1H),
8.16 (t, J = 5.6 Hz, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.64–7.56 (m, 3H),
7.41–7.48 (m, 1H), 7.17 (d, J = 8.8 Hz, 2H), 4.58 (s, 2H), 3.25–3.15
(m, 2H), 1.07 (t, J = 7.2 Hz, 3H). Anal. Calcd for
(C23H18N6O2ꢃ1.75H2O): C, 62.51; H, 4.90; N, 19.02. Found: C,
62.47; H, 4.51; N, 19.04.
added and the solution was heated to reflux for 3 h. The solution
was cooled and the precipitate formed was collected, washed
with EtOH and hexanes and dried to give 2-[4-(1-amino-2,4-
dicyano-6-methylpyrido[1,2-a]benzimidazol-3-yl)phenoxy]acet-
amide (5) as a lime green powder (0.21 g, 50%), mp 290–293 °C.
1H NMR d ppm 8.68 (br s, 2H), 8.61 (d, J = 8.4 Hz, 1H), 7.87 (d,
J = 8.0 Hz, 1H), 7.66 (br s, 1H), 7.63–7.54 (m, 3H), 7.47 (br s,
1H) 7.49–7.40 (m, 1H), 7.15 (d, J = 8.7 Hz, 2H), 4.55 (s, 2H). 13C
NMR d ppm 66.54, 77.01, 85.62, 114.51 (2C), 114.86, 115.74,
115.99, 118.73, 121.75, 126.34, 127.12, 128.21, 130.25 (2C),
144.54, 147.39, 152.15, 152.56, 158.95, 169.45. Anal. Calcd for
4.1.5. 2-[4-(1-Amino-2,4-dicyanopyrido[1,2-a]benzimidazol-3-
yl)phenoxy]-N-propylacetamide (9)
Similar reaction of acid 47 (0.20 g, 0.52 mmol) in dry DMF
(10 mL) with CDI, followed by treatment with propylamine gave

4096
D. M. Lyons et al. / Bioorg. Med. Chem. 19 (2011) 4091–4100
9 as a yellow powder (0.14 g, 63%), mp (EtOH/Et2O) 302–306 °C. 1H
NMR d ppm 8.66 (br s, 2H), 8.61 (d, J = 8.4 Hz, 1H), 8.14 (t,
J = 5.6 Hz, 1H), 7.88 (d, J = 7.7 Hz, 1H), 7.66–7.54 (m, 3H), 7.42–
7.48 (m, 1H), 7.17 (d, J = 8.8 Hz, 2H), 4.59 (s, 2H), 3.12 (q,
J = 6.7 Hz, 2H), 1.47 (sextet, J = 7.3 Hz, 2H), 0.85 (t, J = 7.4 Hz, 3H).
Anal. Calcd for (C24H20N6O2ꢃ2H2O): C, 62.60; H, 5.25; N, 18.25.
Found: C, 62.94; H, 4.80; N, 18.42.
J = 13.5, 6.9 Hz, 1H, interchangeable with D2O). Hydroxyl protons
are not observed. HRMS (ESI+) for C24H21N6O4 (M+H)+: Calcd
457.1619, Found 457.1627. HPLC purity, 94.5%.
4.1.11. 2-[4-(1-Amino-2,4-dicyanopyrido[1,2-a]benzimidazol-3-
yl)phenoxy]-N-(2-pyridin-4-ylethyl)acetamide (18)
Similar reaction of acid 47 (0.20 g, 0.52 mmol) in dry DMF
(10 mL) with CDI, followed by treatment with 3-aminopropan-
1,2-diol gave 18 as a yellow powder (0.07 g, 26%, mp (DMF/
MeOH/Et2O) 299–302 °C. 1H NMR d ppm 8.67 (br s, 2H), 8.61 (d,
J = 8.4 Hz, 1H), 8.47 (d, J = 6.0 Hz, 2H), 8.26 (t, J = 5.7 Hz, 1H), 7.88
(d, J = 7.6 Hz, 1H), 7.64–7.55 (m, 3H), 7.48–7.41 (m, 1H), 7.25 (d,
J = 6.0 Hz, 2H), 7.13 (d, J = 8.8 Hz, 2H), 4.58 (s, 2H), 3.45 (q,
J = 6.7 Hz, 2H), 2.81 (t, J = 7.1 Hz, 2H). Anal. Calcd for
(C28H21N7O2. 0.5DMF): C, 67.61; H, 4.71; N, 20.05. Found: C,
67.57; H, 4.53; N, 20.30.
4.1.6. 2-[4-(1-Amino-2,4-dicyanopyrido[1,2-a]benzimidazol-3-
yl)phenoxy]-N-isopropylacetamide (10)
Similar reaction of acid 47 (0.20 g, 0.52 mmol) in dry DMF
(10 mL) with CDI, followed by treatment with isopropylamine gave
10 as a yellow powder (0.14 g, 63%), mp (EtOH/Et2O) 321–325 °C.
1H NMR d ppm 8.65 (br s, 2H), 8.60 (d, J = 8.4 Hz, 1H), 7.93 (d,
J = 7.8 Hz, 1H), 7.88 (d, J = 7.7 Hz, 1H), 7.64–7.54 (m, 3H), 7.41–
7.48 (m, 1H), 7.17 (d, J = 8.8 Hz, 2H), 4.56 (s, 2H), 4.04–3.91 (m,
1H), 1.13 (s, 3H), 1.11 (s, 3H). Anal. Calcd for (C24H20N6O2ꢃ1.5H2O):
C, 63.85; H, 5.13; N, 18.61. Found: C, 63.88; H, 4.99; N, 18.78.
4.1.12. 1-Amino-3-[4-(2-hydroxyethoxy)phenyl]pyrido[1,2-a]
benzimidazole-2,4-dicarbonitrile (11) (Scheme 2)
4.1.7. 2-[4-(1-Amino-2,4-dicyanopyrido[1,2-a]benzimidazol-3-
yl)phenoxy]-N-(2-hydroxyethyl)acetamide (14)
A
mixture of 2-(4-hydroxybenzylidene)malononitrile (48)
(0.50 g, 2.94 mmol) and 44 (0.23 g, 1.47 mmol) in ethanol (9 mL)
was stirred at 20 °C. Piperidine (220 L, 2.21 mmol) was added
Similar reaction of acid 47 (0.20 g, 0.52 mmol) in dry DMF
(10 mL) with CDI, followed by treatment with ethanolamine gave
14 as a yellow powder (0.12 g, 53%), mp (Et2O/EtOH) 270–272 °C.
1H NMR d ppm 9.02 (br s, 2H), 8.62 (d, J = 8.3 Hz, 1H), 8.10 (t,
J = 5.6 Hz, 1H), 7.84 (d, J = 7.7 Hz, 1H), 7.61–7.53 (m, 3H), 7.45–
7.40 (m, 1H), 7.18 (d, J = 8.8 Hz, 2H), 4.71 (t, J = 5.5 Hz, 1H), 4.60
(s, 2H), 3.47 (q, J = 5.8 Hz, 2H), 3.21–3.31 (m, 2H). Anal. Calcd for
(C23H18N6O3ꢃ1.25H2O): C, 61.53; H, 4.60; N, 18.72. Found: C,
61.45; H, 4.46: N, 18.63.
l
and the solution was heated to reflux for 2 h. The solution was
cooled and the precipitate formed was collected, washed with
EtOH and hexanes and recrystallized from Me2CO/Et2O to give 1-
amino-3-(4-hydroxyphenyl)pyrido[1,2-a]benzimidazole-2,4-dicar-
bonitrile (49) as a yellow powder (0.03 g, 7%), mp >300 °C. 1H NMR
d ppm 10.00 (s, 1H), 8.53–8.65 (m, 3H), 7.86 (d, J = 7.7 Hz, 1H), 7.60
(t, J = 7.5 Hz, 1H), 7.48–7.40 (m, 3H), 6.96 (d, J = 8.6 Hz, 2H). Anal.
Calcd for (C19H11N5O): C, 70.15; H, 3.41; N, 21.53. Found: C,
69.96; H, 3.37; N, 21.51.
A mixture of 49 (0.20 g, 0.62 mmol), (2-bromoethoxy)(tert-
butyl)dimethylsilane (0.22 g, 0.92 mmol) and Cs2CO3 (0.15 g,
0.46 mmol) in dry DMF (6 mL) were heated at 90 °C overnight un-
der N2. The solution was cooled, diluted with water, and extracted
with EtOAc. The organic extracts were washed, dried and evapo-
rated. The crude product (50) was dissolved in THF (56 mL) and
treated at 20 °C with a solution of tetrabutylammonium fluoride
in THF (1.48 mL, 1.0 M) for 30 min. The reaction was quenched
by the addition of water and the THF was removed under reduced
pressure. The aqueous solution was extracted with EtOAc and the
combined extracts were washed with water and brine, dried
(Na2SO4) and evaporated to give 11 as a yellow powder (0.03 g,
18%), mp (EtOAc) 302–305 °C. 1H NMR d ppm 8.63 (br s, 2H),
8.60 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.64–7.58 (m, 1H),
7.56 (d, J = 8.8 Hz, 2H), 7.47–7.41 (m, 1H), 7.15 (d, J = 8.8 Hz, 2H),
4.90 (t, J = 5.4 Hz, 1H), 4.11 (t, J = 4.9 Hz, 2H), 3.77 (q, J = 7.5 Hz,
2H). HRMS (ESI+) for C21H16N5O2 (M+H)+: Calcd 370.1299, Found
370.1292. HPLC purity, 94.8%.
4.1.8. 2-[4-(1-Amino-2,4-dicyanopyrido[1,2-a]benzimidazol-3-
yl)phenoxy]-N-(2-hydroxypropyl)acetamide (15)
Similar reaction of acid 47 (0.20 g, 0.52 mmol) in dry DMF
(10 mL) with CDI, followed by treatment with 1-aminopropan-2-
ol, gave 15 as a yellow powder (0.07 g, 29%), mp (DMF/Et2O)
294–297 °C. 1H NMR d ppm 8.68 (br s, 2H), 8.60 (d, J = 8.4 Hz,
1H), 8.03 (t, J = 5.8 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.67–7.54
(m, 3H), 7.48–7.41 (m, 1H), 7.17 (d, J = 8.8 Hz, 2H), 4.72 (d,
J = 2.6 Hz, 1H), 4.62 (s, 2H), 3.79–3.65 (m, 1H), 3.20–3.04 (m, 2H),
1.04 (d, J = 6.2 Hz, 3H). Anal. Calcd for (C24H20N6O3ꢃ0.75H2O): C,
63.50; H, 4.77; N, 18.51. Found: C, 63.51; H, 4.72; N, 18.55.
4.1.9. 2-[4-(1-Amino-2,4-dicyanopyrido[1,2-a]benzimidazol-3-
yl)phenoxy]-N-(2-hydroxypropyl)acetamide (16)
Similar reaction of acid 47 (0.20 g, 0.52 mmol) in dry DMF
(10 mL) with CDI, followed by treatment with 3-aminopropanol
gave 16 as a yellow powder (0.03 g, 12%), mp 245–248 °C. 1H
NMR d ppm 8.66 (br s, 2H), 8.61 (d, J = 8.4 Hz, 1H), 8.15 (t,
J = 5.6 Hz, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.64–7.56 (m, 3H), 7.48–
7.42 (m, 1H), 7.17 (d, J = 8.8 Hz, 2H), 4.59 (s, 2H), 3.42 (t,
J = 6.3 Hz, 2H, interchangeable with D2O), 3.23 (q, J = 6.6 Hz, 2H),
1.62 (quint, J = 6.6 Hz, 2H). Hydroxyl proton is not observed. Anal.
Calcd for C24H20N6O3ꢃ2.25H2O: C, 59.93; H, 5.13; N, 17.47. Found:
C, 59.84; H, 4.78; N, 17.46.
4.1.13. 3-[4-(1-Amino-2,4-dicyanopyrido[1,2-a]benzimidazol-3-
yl)phenyl]-N-methylpropanamide (12) (Scheme 3)
A solution of ethyl 3-(4-formylphenyl)propanoate (51) (5.56 g,
27.0 mmol) and malononitrile (5.35 g, 81.0 mmol) in EtOH
(850 mL) was stirred at 20 °C. 3-Aminopropanoic acid (0.96 g,
10.80 mmol) was added and the resulting solution was stirred at
20 °C overnight, then the solvent volume was reduced and the
remaining solution was diluted with water and cooled to 4 °C.
The precipitate collected, washed with water and hexanes, and
was dried to give ethyl 3-(4-(2,2-dicyanovinyl)phenyl)propanoate
(52) as a pale yellow solid (5.11 g, 74%) which was used directly.
1H NMR d ppm 8.48 (s, 1H), 7.88 (d, J = 8.3 Hz, 2H), 7.49 (d,
J = 8.3 Hz, 2H), 4.04 (q, J = 7.1 Hz, 2H), 2.95 (t, J = 7.5 Hz, 2H), 2.67
(t, J = 7.5 Hz, 2H), 1.15 (t, J = 7.1 Hz, 3H). LRMS (APCIꢀ) for
4.1.10. 2-[4-(1-Amino-2,4-dicyanopyrido[1,2-a]benzimidazol-3-
yl)phenoxy]-N-(2,3-dihydroxypropyl)acetamide (17)
Similar reaction of acid 47 (0.20 g, 0.52 mmol) in dry DMF
(10 mL) with CDI, followed by treatment with 3-aminopropan-
1,2-diol gave 17 as a yellow powder (0.013 g, 5%), mp (Me2CO/
Et2O) 194–197 °C. 1H NMR d ppm 8.66 (br s, 2H), 8.61 (d,
J = 8.4 Hz, 1H), 8.03 (t, J = 5.7 Hz, 1H), 7.88 (d, J = 7.6 Hz, 1H),
7.64–7.56 (m, 3H), 7.48–7.41 (m, 1H), 7.18 (d, J = 8.8 Hz, 2H),
4.63 (s, 2H), 3.58 (quint, J = 5.7 Hz, 1H, interchangeable with
D2O), 3.36–3.28 (m, 3H, interchangeable with D2O), 3.12 (dd,
C
15H13N2O2 (MꢀH)ꢀ: Calcd 254, Found 254.

D. M. Lyons et al. / Bioorg. Med. Chem. 19 (2011) 4091–4100
4097
A
mixture of 52 (5.11 g, 20.10 mmol) and 44 (1.58 g,
A solution of 56 (1.0 g, 2.72 mmol) and NaOH (0.54 g,
10.10 mmol) in ethanol (59 mL) was stirred at 20 °C. Piperidine
(1.50 mL, 15.0 mmol) was added and the solution was heated to re-
flux for 3 h. The solution was cooled and diluted with Et2O to pre-
cipitate a product that was collected, washed with Et2O and was
dried to give ethyl 3-[4-(1-amino-2,4-dicyanopyrido[1,2-a]ben-
14.0 mmol) in EtOH (75 mL) was heated to reflux for 1 h, then
cooled and the EtOH removed under reduced pressure. The residue
was diluted with water, acidified to pH 1 (concd HCl) and the pre-
cipitate collected, washed with water, EtOH and Et2O and dried to
give 4-(9-amino-6,8-dicyanopyrido[1,2-a]benzimidazol-7-yl)ben-
zoic acid (57) as a yellow powder (0.96 g, 100%), mp >345 °C. 1H
NMR d ppm 13.24 (br s, 1H), 8.77 (br s, 2H), 8.63 (d, J = 8.4 Hz,
1H), 8.15 (d, J = 8.4 Hz, 2H), 7.90 (d, J = 7.6 Hz, 1H), 7.74 (d,
J = 8.5 Hz, 2H), 7.66–7.60 (m, 1H), 7.51–7.44 (m, 1H). Anal. Calcd
for (C20H11N5O2ꢃ1.25H2O): C, 67.13; H, 3.24; N, 19.57. Found: C,
63.94; H, 3.52; N, 18.35.
zimidazol-3-yl)phenyl]propanoate (53) as
a yellow powder
(2.99 g, 72%), mp 258–260 °C. 1H NMR d ppm 8.64 (br s, 2H),
8.61 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 7.7 Hz, 1H), 7.64–7.57 (m, 1H),
7.53 (d, J = 8.2 Hz, 2H), 7.49–7.41 (m, 3H), 4.07 (q, J = 7.1 Hz, 2H),
2.98 (t, J = 7.5 Hz, 2H), 2.72 (t, J = 7.6 Hz, 2H), 1.17 (t, J = 7.1 Hz,
3H). Anal. Calcd for (C24H19N5O2ꢃ0.25H2O): C, 70.40; H, 4.68; N,
17.10. Found: C, 69.64; H, 4.79; N, 17.23.
A solution of 57 (0.20 g, 0.57 mmol) in dry DMF (10 mL) was
stirred at 20 °C under N2. CDI (0.14 g, 0.85 mmol) was added and
the solution was stirred for 1 h, then a solution of NH3 in MeOH
A solution of ester 53 (0.20 g, 0.49 mmol) in a mixture of EtOH
(50 mL) and aqueous NaOH (1.2 mL, 2.0 M) was stirred under reflux
for 1 h, then cooled, diluted with water and the EtOH removed under
reducedpressure. The resultingsolution was acidifiedto pH 1 (concd
HCl) and the precipitate formed was collected, washed with water,
EtOH, Et2O, then dried to give 3-[4-(1-amino-2,4-dicyanopyri-
do[1,2-a]benzimidazol-3-yl)phenyl]propanoic acid (54) as a yellow
powder (0.18 g, 97%) mp 314–317 °C. 1H NMR d ppm 8.69 (br s, 2H),
8.62 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 7.7 Hz, 1H), 7.65–7.58 (m, 1H),
7.54 (d, J = 8.2 Hz, 2H), 7.50–7.41 (m, 3H), 2.96 (t, J = 7.6 Hz, 2H),
2.65 (t, J = 7.7 Hz, 2H). Acid proton not observed. HRMS (ESI+) for
(400 lL, 7.0 M) was added and the solution was stirred at 20 °C
for 1 h. The DMF was removed under reduced pressure and the res-
idue was recrystallized from DMF/Et2O to give 19 as a yellow pow-
der (0.10 g, 48%), mp >340 °C. 1H NMR d ppm 8.74 (br s, 2H), 8.63
(d, J = 8.4 Hz, 1H), 8.14 (br s, 1H), 8.06 (d, J = 8.4 Hz, 2H), 7.90 (d,
J = 7.7 Hz, 1H), 7.69 (d, J = 8.4 Hz, 2H), 7.66–7.60 (m, 1H), 7.52 (br
s, 1H), 7.50–7.43 (m, Hz, 1H). Anal. Calcd for (C20H12N6O.
1.5DMFꢃ0.5H2O): C, 63.08; H, 4.97; N, 22.52. Found: C, 62.71; H,
5.26; N, 22.53.
C
22H16N5O2 (M+H)+: Calcd 382.1299, Found 382.1308.
A solution of acid 54 (0.10 g, 0.26 mmol) in dry DMF (5 mL) was
stirred at 20 °C under N2. CDI (0.06 g, 0.39 mmol) was added and
4.1.16. 4-(9-Amino-6,8-dicyanopyrido[1,2-a]benzimidazol-7-
yl)-N-methylbenzamide (20)
the solution was stirred for 1 h. A solution of methylamine in MeOH
(180 lL, 2.0 M) was added and the solution was stirred at 20 °C
Similar reaction of acid 57 (0.20 g, 0.57 mmol) and CDI (0.14 g,
0.85 mmol) in dry DMF (10 mL) at 20 °C under N2 for 1 h, followed
by treatment with methylamine in MeOH (1.42 mL, 2.0 M) at 20 °C
for 1 h, gave 20 as a yellow powder (0.14 g, 67%), mp (DMF/
Et2O) >320 °C. 1H NMR d ppm 8.78 (br s, 2H), 8.63 (d, J = 8.4 Hz,
1H), 8.64–8.57 (m, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 8.1 Hz,
1H), 7.70 (d, J = 8.3 Hz, 2H), 7.61 (t, J = 7.7 Hz, 1H), 7.49–7.42 (m,
1H), 2.84 (d, J = 4.5 Hz, 3H). Anal. Calcd for (C21H14N6O.0.75DMF):
C, 66.30; H, 4.61; N, 22.45. Found: C, 66.21; H, 4.40; N, 22.52.
overnight. The DMF was removed under reduced pressure and the
crude material was purified by column chromatography on silica
gel, eluting with 20% EtOH/EtOAc, then recrystallized from
Me2CO/Et2O to give 12 as yellow needles (0.02 g, 23%) mp 290–
293 °C. 1H NMR d ppm 8.67 (br s, 2H), 8.61 (d, J = 8.4 Hz, 1H), 7.88
(d, J = 7.8 Hz, 1H), 7.81–7.75 (m, 1H), 7.64–7.58 (m, 1H), 7.53 (d,
J = 8.2 Hz, 2H), 7.48–7.41 (m, 3H), 2.97–2.91 (m, 2H), 2.59 (d,
J = 4.6 Hz, 3H), 2.50–2.43 (m, 2H). HRMS (ESI+) for C23H19N6O
(M+H)+: Calcd 395.1615, Found 395.1617. HPLC purity, 97.6%.
4.1.17. 4-(9-Amino-6,8-dicyanopyrido[1,2-a]benzimidazol-7-
yl)-N,N-dimethylbenzamide (21)
4.1.14. 3-[4-(1-Amino-2,4-dicyanopyrido[1,2-a]benzimidazol-3-
yl)phenyl]-N-methylpropanamide (13)
Similar reaction of acid 54 (0.10 g, 0.26 mmol) in dry DMF
(10 mL) with CDI, followed by treatment with of dimethylamine
Similar reaction of acid 57 (0.20 g, 0.57 mmol) and CDI (0.14 g,
0.85 mmol) in dry DMF (10 mL) at 20 °C under N2 for 1 h, followed
by treatment with dimethylamine in MeOH (1.42 mL, 2.0 M) at
20 °C for 1 h. The crude material was purified by preparative HPLC
on a Synergi Max-RP C12 column eluting with 30–98% MeCN/
45 mM NH4HCO2 to give 21 as a yellow powder (0.03 g, 14%),
mp >315 °C. 1H NMR d ppm 8.73 (br s, 2H), 8.63 (d, J = 8.4 Hz,
1H), 7.89 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.65–7.59 (m,
3H), 7.50–7.43 (m, 1H), 3.04 (s, 3H), 2.97 (s, 3H). HRMS (ESI+) for
in MeOH (180 lL, 2.0 M), and purification of the product by col-
umn chromatography on silica gel eluting with 20% EtOH/EtOAc
gave 13 as yellow needles (0.03 g, 23%), mp (Me2CO/Et2O) 280–
282 °C. 1H NMR d ppm 8.75 (br s, 2H), 8.61 (d, J = 8.4 Hz, 1H),
7.88 (d, J = 7.7 Hz, 1H), 7.64–7.58 (m, 1H), 7.53 (d, J = 8.2 Hz, 2H),
7.50–7.41 (m, 3H), 2.96 (s, 3H), 2.96–2.91 (m, 2H), 2.84 (s, 3H),
2.74–7.68 (m, 2H). HRMS (ESI+) for C24H21N6O (M+H)+: Calcd
409.1771, Found 409.1780. HPLC purity, 95.6%.
C
22H16N6NaO (M+H)+: Calcd 403.1278, Found 403.1283.
4.1.18. 4-(9-Amino-6,8-dicyanopyrido[1,2-a]benzimidazol-7-
yl)-N-ethylbenzamide (22)
4.1.15. 4-(9-Amino-6,8-dicyanopyrido[1,2-a]benzimidazol-7-
yl)benzamide (19) (Scheme 4)
Similar reaction of acid 57 (0.20 g, 0.57 mmol) and CDI (0.14 g,
0.85 mmol) in dry DMF (10 mL) at 20 °C under N2 for 1 h, followed
A mixture of methyl 4-(2,2-dicyanovinyl)benzoate (55) (0.50 g,
2.36 mmol) and 44 (0.19 g, 1.18 mmol) in EtOH (7 mL) was stirred
at 20 °C. Piperidine (175 lL, 1.77 mmol) was added and the solu-
by treatment with ethylamine in water (225 lL, 70%) at 20 °C for
1 h, gave 22 as a yellow powder (0.15 g, 68%), mp >325 °C. 1H
NMR d ppm 8.75 (br s, 2H), 8.67–8.59 (m, 2H), 8.03 (d, J = 8.3 Hz,
2H), 7.90 (d, J = 8.0 Hz, 1H), 7.70 (d, J = 8.3 Hz, 2H), 7.66–7.59 (m,
1H), 7.50–7.43 (m, 1H), 3.39–3.26 (m, 2H), 1.17 (t, J = 7.2 Hz, 3H).
Anal. Calcd for (C22H16N6Oꢃ1.25H2O): C, 65.58; H, 4.63; N, 20.86.
Found: C, 65.69; H, 4.42; N, 21.00.
tion was heated to reflux for 3 h. The solution was cooled and
the precipitate formed was collected, washed with Et2O and dried
to give methyl 4-(9-amino-6,8-dicyanopyrido[1,2-a]benzimidazol-
7-yl)benzoate (56) as a yellow powder (0.11 g, 24%), mp >300 °C.
1H NMR d ppm 8.77 (br s, 2H), 8.63 (d, J = 8.4 Hz, 1H), 8.17 (d,
J = 8.5 Hz, 2H), 7.90 (d, J = 7.7 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H),
7.66–7.60 (m, 1H), 7.50–7.44 (m, 1H), 3.92 (d, J = 8.3 Hz, 3H). Anal.
Calcd for (C21H13N5O2): C, 68.66; H, 3.57; N, 19.06. Found: C, 68.45;
H, 3.63; N, 18.91.
4.1.19. 4-(9-Amino-6,8-dicyanopyrido[1,2-a]benzimidazol-7-
yl)-N-(2-hydroxyethyl)benzamide (23)
Similar reaction of acid 57 (0.20 g, 0.57 mmol) and CDI (0.14 g,
0.85 mmol) in dry DMF (10 mL) at 20 °C under N2 for 1 h, followed

4098
D. M. Lyons et al. / Bioorg. Med. Chem. 19 (2011) 4091–4100
by treatment with ethanolamine (215
l
L, 2.84 mmol) at 20 °C for
by
treatment
with
4-(2-aminoethyl)piperidine
(405 lL,
1 h, gave 23 as a yellow powder (0.20 g, 87%), mp (Et2O) 285–
290 °C. 1H NMR d ppm 8.80 (br s, 2H), 8.65 (d, J = 8.3 Hz, 1H),
8.60 (t, J = 5.6 Hz, 1H), 8.04 (d, J = 8.5 Hz, 2H), 7.87 (d, J = 7.7 Hz,
1H), 7.69 (d, J = 8.4 Hz, 2H), 7.63–7.56 (m, 1H), 7.48–7.40 (m,
1H), 4.73 (t, J = 5.6 Hz, 1H), 3.56 (q, J = 6.0 Hz, 2H), 3.38 (q,
J = 6.0 Hz, 2H). Anal. Calcd for (C22H16N6O2ꢃ75H2O): C, 64.46; H,
4.30; N, 20.50. Found: C, 64.15; H, 4.40; N, 20.57.
2.84 mmol) at 20 °C for 1, gave 28 as yellow needles (0.03 g,
11%), mp (Me2CO/Et2O) 201–204 °C. 1H NMR d ppm 8.76–8.68
(m, 2H), 8.29 (br s, 2H), 8.00 (d, J = 8.4 Hz, 2H), 7.73–7.64 (m,
3H), 7.47–7.40 (m, 1H), 7.32–7.25 (m, 1H), 3.58 (q, J = 6.1 Hz,
2H), 3.08–2.79 (m, 6H), 1.73–1.66 (quint, J = 5.3 Hz, 4H), 1.54–
1.45 (m, 2H). Anal. Calcd for (C27H25N7OꢃH2O): C, 67.34; H, 5.65;
N, 20.36. Found: C, 66.90; H, 5.50; N, 20.42.
4.1.20. 4-(9-Amino-6,8-dicyanopyrido[1,2-a]benzimidazol-7-
yl)-N-(3-hydroxypropyl)benzamide (24)
4.1.25. 4-(9-Amino-6,8-dicyanopyrido[1,2-a]benzimidazol-7-
yl)-N-(2-morpholin-4-ylethyl)benzamide (29)
Similar reaction of acid 57 (0.20 g, 0.57 mmol) and CDI (0.14 g,
Similar reaction of acid 57 (0.20 g, 0.57 mmol) and CDI (0.14 g,
0.85 mmol) in dry DMF (10 mL) at 20 °C under N2 for 1 h, followed
0.85 mmol) in dry DMF (10 mL) at 20 °C under N2 for 1 h, followed
by treatment with 3-aminopropanol (215
lL, 2.83 mmol) at 20 °C
by treatment with 4-(2-aminoethyl)morpholine (375 lL,
for 1 h, gave 24 as a yellow powder (0.15 g, 63%), mp >300–
304 °C. 1H NMR d ppm 8.75 (br s, 2H), 8.65–8.59 (m, 2H), 8.02 (d,
J = 8.4 Hz, 2H), 7.90 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H),
7.66–7.60 (m, 1H), 7.50–7.43 (m, 1H), 4.40 (br s, 1H), 3.50 (t,
J = 6.3 Hz, 2H), 3.37 (q, J = 6.6 Hz, 2H), 1.73 (quint, J = 6.7 Hz, 2H).
Anal. Calcd for (C23H18N6O2ꢃ0.5H2O): C, 65.86; H, 4.57; N, 20.04.
Found: C, 65.85; H, 4.54; N, 20.06.
2.84 mmol) at 20 °C for 1 h, gave 29 as a yellow powder (0.14 g,
52%), mp (Me2CO/Et2O), 288–291 °C. 1H NMR d ppm 8.75–8.54
(m, 4H), 8.01 (d, J = 8.4 Hz, 2H), 7.86 (d, J = 7.8 Hz, 1H), 7.70 (d,
J = 8.4 Hz, 2H), 7.62–7.55 (m, 1H), 7.46–7.39 (m, 1H), 3.62 (t,
J = 4.3 Hz, 4H), 3.47 (q, J = 6.3 Hz, 2H), 2.68–2.48 (m, 6H). Anal.
Calcd for (C26H23N7O2): C, 67.08; H, 4.98; N, 21.06. Found: C,
66.80; H, 5.00; N, 20.79.
4.1.21. 4-(9-Amino-6,8-dicyanopyrido[1,2-a]benzimidazol-7-
yl)-N-(2-hydroxypropyl)benzamide (25)
4.1.26. 4-(9-Amino-6,8-dicyanopyrido[1,2-a]benzimidazol-7-
yl)-N-(2-pyridin-4-ylethyl)benzamide (30)
Similar reaction of acid 57 (0.20 g, 0.57 mmol) and CDI (0.14 g,
Similar reaction of acid 57 (0.20 g, 0.57 mmol) and CDI (0.14 g,
0.85 mmol) in dry DMF (10 mL) at 20 °C under N2 for 1 h, followed
0.85 mmol) in dry DMF (10 mL) at 20 °C under N2 for 1 h, followed
by treatment with 1-aminopropan-2-ol (220
lL, 2.84 mmol) at
by treatment with 4-(2-aminoethyl)pyridine (236 lL, 1.98 mmol)
20 °C for 1 h, gave 25 as a yellow powder (0.14 g, 59%), mp
(DMF/Et2O) >300 °C. 1H NMR d ppm 8.75 (br s, 2H), 8.63 (d,
J = 8.4 Hz, 1H), 8.58 (t, J = 5.7 Hz, 1H), 8.05 (d, J = 8.4 Hz, 2H), 7.90
(d, J = 7.6 Hz, 1H), 7.70 (d, J = 8.4 Hz, 2H), 7.66–7.59 (m, 1H),
7.50–7.43 (m, 1H), 4.76 (d, J = 3.6 Hz, 1H), 3.89–3.78 (m, 1H),
3.28–3.30 (m, 2H), 1.10 (d, J = 6.2 Hz, 3H). Anal. Calcd for
(C23H18N6O2. 1.25DMF): C, 64.03; H, 5.37; N, 20.24. Found: C,
64.10; H, 5.34; N, 20.27.
at 20 °C for 1 h, gave 30 as yellow needles (0.02 g, 11%), mp
(DMF/MeOH) >315 °C. 1H NMR d ppm 8.79 (br s, 2H), 8.79 (t,
J = 5.5 Hz, 1H), 8.63 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 6.0 Hz, 2H),
7.98 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 8.4 Hz,
2H), 7.66–7.59 (m, 1H), 7.50–7.43 (m, 1H), 7.31 (d, J = 5.6 Hz,
2H), 3.59 (q, J = 6.6 Hz, 2H), 2.93 (t, J = 7.1 Hz, 2H). HRMS (ESI+)
for C27H19N7NaO (M+H)+: Calcd 480.1543, Found 480.1550. HPLC
purity, 98.2%.
4.1.22. 4-(1-Amino-2,4-dicyanopyrido[1,2-a]benzimidazol-3-
yl)-N-(2,3-dihydroxypropyl)benzamide (26)
4.1.27. (E,Z)-2-(4-(2-(1H-Benzo[d]imidazol-2-yl)-2-cyanovinyl)
phenoxy)acetamide (36) (Scheme 5)
Similar reaction of acid 57 (0.20 g, 0.57 mmol) and CDI (0.14 g,
0.85 mmol) in dry DMF (10 mL) at 20 °C under N2 for 1 h, followed
by treatment with 3-aminopropan-1,2-diol (0.26 g, 2.84 mmol) at
20 °C for 1 h, gave 26 as a yellow powder (0.05 g, 19%), mp
(Et2O) >300 °C. 1H NMR d ppm 8.70 (br s, 2H), 8.64 (d, J = 8.4 Hz,
1H), 8.56 (t, J = 5.7 Hz, 1H), 8.04 (d, J = 8.4 Hz, 2H), 7.87 (d,
J = 7.7 Hz, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.63–7.57 (m, 1H), 7.52–
7.50 (m, 1H), 4.81 (d, J = 5.0 Hz, 1H), 4.55 (t, J = 5.8 Hz, 1H), 3.69
(sextet, J = 5.5 Hz, 1H), 3.49–3.42 (m, 1H), 3.39 (d, J = 5.5 Hz, 2H),
3.32–3.21 (m, 1H). Anal. Calcd for (C23H18N6O3ꢃ2H2O): C, 59.73;
H, 4.79; N, 18,17. Found: C, 60.06; H, 4.44; N, 18.18.
A solution of 44 (0.32 g, 2.01 mmol) and 43 (0.30 g, 1.67 mmol)
were heated under reflux for 15 h in EtOH (20 ml) with a catalytic
amount of piperidine to give 36 as a yellow solid (75%), mp (EtOH/
Et2O) 252–254 °C, without further purification. 1H NMR d ppm
12.96 (br s, 1H), 8.27 (s, 1H), 8.00 (d, J = 8.9 Hz, 2H), 8.65–8.56
(m, 2H), 7.59 (br s, 1H), 7.41 (br s, 1H), 7.27–7.21 (m, 2H), 7.17
(d, J = 9.0 Hz, 2H), 4.57 (s, 2H). HRMS (ESI+) for C18H15N4O2
(MH)+: Calcd 319.1190, Found 319.1185. Anal. Calcd for
(C18H14N4O2): C, 67.91; H, 4.43; N, 17.60. Found: C, 68.13; H,
4.49; N, 17.77. HPLC purity, 98.7% (E/Z ratio 35:64).
4.1.28. 2-(4-(1-Amino-2,4-dicyano-6-methylbenzo
4.1.23. 4-(9-Amino-6,8-dicyanopyrido[1,2-a]benzimidazol-7-
yl)-N-[2-(dimethylamino)ethyl]benzamide (27)
[4,5]imidazo[1,2-a]pyridin-3-yl)phenoxy)acetamide (31)
A
solution of 3-methylbenzene-1,2-diamine (58) (1.50 g,
Similar reaction of acid 57 (0.20 g, 0.57 mmol) and CDI (0.14 g,
0.85 mmol) in dry DMF (10 mL) at 20 °C under N2 for 1 h, followed
by treatment with N,N-dimethylethylenediamine (90 lL,
0.85 mmol) at 20 °C for 1 h, gave 27 as a yellow powder (0.22 g,
93%), mp (Et2O) >177–180 °C. 1H NMR d ppm 9.21 (br s, 2H),
8.76–8.67 (m, 2H), 8.00 (d, J = 8.4 Hz, 2H), 7.72–7.64 (m, 3H),
7.47–7.40 (m, 1H), 7.32–7.25 (m, 1H), 3.57 (q, J = 6.0 Hz, 2H), 3.00
(t, J = 6.1 Hz, 2H), 2.64 (s, 6H). Anal. Calcd for (C24H21N7Oꢃ1.5H2O):
C, 63.99; H, 5.37; N, 21.76. Found: C, 64.10; H, 5.70; N, 21.69.
12.28 mmol) and cyanoacetamide (2.06 g, 24.56 mmol) in xylene
(25 ml) was heated under reflux for 15 h in the presence of 4 Å
molecular sieves. The hot solvent was decanted and the residue
was extracted with Et2O/MeOH and filtrated. The filtrate was evap-
orated to dryness to give 2-(4-methyl-1H-benzo[d]imidazol-2-
yl)acetonitrile (63) as a brown solid (1.62 g, 77%) which was used
directly. 1H NMR d ppm 9.80 (br s, 1H), 7.46–7.35 (m, 1H), 7.20
(t, J = 7.7 Hz, 1H), 7.09 (d, J = 7.4 Hz, 1H), 4.15 (s, 2H), 2.59 (s,
3H). LRMS (APCI+) for C10H10N3 (MH+): Calcd 172, Found 172.
A solution of 63 (0.30 g, 1.75 mmol), 43 (0.48 g, 2.10 mmol) and
a catalytic amount of piperidine in EtOH (20 ml) was heated under
reflux for 15 h. The cooled solution was filtered and the precipitate
was washed with EtOH and purified by flash chromatography on
silica gel, eluting with 5–30% MeOH/CH2Cl2 to give 31 as a green
4.1.24. 4-(9-Amino-6,8-dicyanopyrido[1,2-a]benzimidazol-7-
yl)-N-(2-piperidin-1-ylethyl)benzamide (28)
Similar reaction of acid 57 (0.20 g, 0.57 mmol) and CDI (0.14 g,
0.85 mmol) in dry DMF (10 mL) at 20 °C under N2 for 1 h, followed

D. M. Lyons et al. / Bioorg. Med. Chem. 19 (2011) 4091–4100
4099
solid (0.14 g, 20%), mp 289–293 °C. 1H NMR d ppm 8.59 (br s, 2H),
8.41 (d, J = 8.4 Hz, 1H), 7.61 (br s, 1H), 7.58 (d, J = 8.8 Hz, 2H), 7.43
(d, J = 7.4, 1H), 7.42 (br s, 1H), 7.37–7.31 (m, 1H), 7.16 (d, J = 8.8 Hz,
2H), 4.55 (s, 2H), 2.65 (s, 3H). HRMS (ESI+) for C22H17N6O2 (MH+):
ppm 9.69 (br s, 1H), 7.35–7.06 (m, 2H), 6.74 (t, J = 7.9 Hz, 1H), 4.14
(s, 2H), 4.01 (s, 3H). LRMS (APCI+) for C10H10N3O (MH+): Calcd 188,
Found 188.
A solution of 65 (0.40 g, 2.11 mmol), 43 (0.40 g, 1.76 mmol)
and a catalytic amount of piperidine in EtOH (20 ml) was heated
under reflux for 15 h, and the product was purified by flash
chromatography on silica gel, eluting with 3% MeOH/CH2Cl2, to
give 33 as a brown solid (0.29 g, 40%), mp 284–286 °C. 1H
NMR d ppm 8.57 (br s, 2H), 8.17 (d, J = 8.4 Hz, 1H), 7.61 (br s,
1H), 7.58 (d, J = 8.8 Hz, 2H), 7.47 (br s, 1H), 7.45–7.39 (m, 1H),
7.37 (t, J = 8.3 Hz, 1H), 7.16 (d, J = 8.8 Hz, 2H), 4.55 (s, 2H), 4.01
(s, 3H). HRMS (ESIꢀ) for C22H15N6O3 (MꢀH)ꢀ: Calcd 411.1211,
Found 411.1216. Anal. Calcd for (C22H16N6O3ꢃ0.9MeOHꢃ0.5H2O):
C, 60.75; H, 4.46; N, 18.89. Found: C, 61.09; H, 4.61; N,
18.66.
Calcd
397.1408,
Found
397.1406.
Anal.
Calcd
for
(C22H16N6O2ꢃ0.9H2O): C, 64.04; H, 4.35; N, 20.37. Found: C,
63.96; H, 4.17; N, 20.02.
4.1.29. (E,Z)-2-(4-(2-Cyano-2-(4-methyl-1H-benzo[d]imidazol-
2-yl)vinyl)phenoxy)acetamide (37)
Similar reaction of 63 (0.30 g, 1.75 mmol) and 42 (0.35 g,
1.93 mmol) under reflux for 15 h in EtOH (20 ml) with a catalytic
amount of piperidine gave 37 as a light brown solid (0.10 g, 16%),
mp (EtOH) 264–268 °C, without further purification. 1H NMR d
ppm 12.16–13.10 (m, 1H), 8.36–8.21 (m, 1H), 8.00 (d, J = 8.4 Hz,
2H), 7.59 (br s, 1H), 7.41 (br s, 1H), 7.30–7.54 (m, 1H), 7.17 (d,
J = 8.6 Hz, 2H), 7.22–7.10 (m, 1H), 7.09–7.01 (m, 1H), 4.57 (s, 2H),
2.56 (s, 3H). HRMS (ESIꢀ) for C19H15N4O2 (MꢀH)ꢀ: Calcd
331.1200, Found 331.1191. HPLC purity, 97.2% (E/Z ratio 51:46).
4.1.33. (E,Z)-2-(4-(2-Cyano-2-(4-methoxy-1H-benzo[d]imidazol-
2-yl)vinyl)phenoxy)acetamide (39)
Similar reaction of 65 (0.35 g, 1.84 mmol) and 42 (0.30 g,
1.66 mmol) in EtOH (20 ml) with
a catalytic amount of
4.1.30. 2-(4-(1-Amino-6-chloro-2,4-
dicyanobenzo[4,5]imidazo[1,2-a]pyridin-3-
yl)phenoxy)acetamide (32)
piperidine gave 39 as a yellow solid (0.32 g, 41%), mp (EtOH)
241–244 °C, without further purification. 1H NMR d ppm 12.94
(br s, 1H), 8.38–8.22 (m, 1H), 7.97 (d, J = 8.9 Hz, 2H), 7.58 (br s,
1H), 7.41 (br s, 1H), 7.24–7.13 (m, 2H), 7.16 (d, J = 8.9 Hz, 2H),
6.84–6.72 (m, 1H), 4.57 (s, 2H), 3.96 (s, 3H). HRMS (ESI+) for
Similar reaction of 3-chlorobenzene-1,2-diamine (59) (1.50 g,
10.52 mmol) with cyanoacetamide (1.77 g, 21.04 mmol) in xylene
(25 ml), and purification of the crude product by flash chromatog-
raphy on silica gel, eluting with 15–50% EtOAc/hexanes, provided
2-(4-chloro-1H-benzo[d]imidazol-2-yl)acetonitrile (64) as a yel-
low-orange solid (0.82 g, 41%), which was used directly. 1H NMR
(CDCl3) d ppm 9.87 (br s, 1H), 7.39 (d, J = 7.9 Hz, 1H), 7.32 (t,
J = 7.8 Hz, 1H), 7.23 (d, J = 7.9 Hz, 1H), 4.21 (s, 2H). LRMS (APCI+)
for C9H7ClN3 (MH+): Calcd 192, Found 192.
C
19H17N4O3 (MH)+: Calcd 349.1295, Found 349.1298. Anal.
Calcd for (C19H16N4O3ꢃ0.2H2O): C, 64.84; H, 4.70; N, 15.92. Found:
C, 64.65; H, 4.60; N, 16.12. HPLC purity, 99.2% (E/Z ratio
32:67).
4.1.34. 2-(4-(1-Amino-2,4-dicyano-6,7-dimethyl
benzo[4,5]imidazo[1,2-a]pyridin-3-yl)phenoxy)acetamide (34)
Similar reaction of 3,4-dimethylbenzene-1,2-diamine (61)
(1.50 g, 11.01 mmol) with cyanoacetamide (1.85 g, 22.03 mmol)
in xylene (25 ml) gave 2-(4,5-dimethyl-1H-benzo[d]imidazol-2-
yl)acetonitrile (66) as a brown solid (2.03 g, 99%) which was used
directly 1H NMR (CDCl3) d ppm 9.98 (br s, 1H), 7.35–7.26 (m,
1H), 7.12 (d, J = 8.4 Hz, 1H), 4.11 (s, 2H), 2.47 (s, 3H), 2.39 (s, 3H).
LRMS (APCI+) for C11H12N3 (MH+): Calcd 186, Found 186.
A solution of 66 (0.30 g, 1.62 mmol), 43 (0.44 g, 1.94 mmol) and
a catalytic amount of piperidine in EtOH (20 ml) was heated under
reflux for 15 h, to give an intermediate (0.35 g, 1.01 mmol) that
was treated further with malononitrile (0.13 g, 2.02 mmol) and
catalytic piperidine in EtOH (10 mL) under reflux overnight to close
the middle ring. The cooled reaction mixture was filtered and
washed with EtOH to give 34 as a yellow-green solid (0.15 g,
22%), mp (EtOH) 283–286 °C. 1H NMR d ppm 8.51 (br s, 2H), 8.30
(d, J = 8.8 Hz, 1H), 7.60 (br s, 1H), 7.57 (d, J = 8.8 Hz, 2H), 7.41 (br
s, 1H), 7.24 (d, J = 8.6 Hz, 1H), 7.15 (d, J = 8.8 Hz, 2H), 4.55 (s, 2H),
2.57 (s, 3H), 2.44 (s, 3H). HRMS (ESI+) for C23H19N6O2 (MH)+: Calcd
411.1564, Found 411.1558. HPLC purity, 97.1%.
A solution of 64 (0.40 g, 2.11 mmol), 43 (0.40 g, 1.76 mmol) and
a catalytic amount of piperidine in EtOH (20 ml) was heated under
reflux for 15 h, and the product was purified by preparative HPLC
on an Agilent Zorbax SB-C18 column, eluting with 10–95%
MeCN/45 mM NH4HCO2, to give 32 as a yellowish solid (65 mg,
9%), mp 328–332 °C. 1H NMR d ppm 8.73 (br s, 2H), 8.58 (d,
J = 8.4 Hz, 1H), 7.70 (d, J = 7.8 Hz, 1H), 7.61 (br s, 1H), 7.58 (d,
J = 8.8 Hz, 2H), 7.42 (t, J = 8.1 Hz, 1H), 7.41 (br s, 1H), 7.17 (d,
J = 8.8 Hz, 2H), 4.56 (s, 2H). HRMS (ESIꢀ) for C21H12ClN6O2 (MꢀH)ꢀ:
Calcd
415.0716,
Found
415.0714.
Anal.
Calcd
for
(C21H13ClN6O2ꢃ1.0H2O): C, 58.00; H, 3.48; N, 19.33. Found: C,
57.71; H, 3.78; N, 19.74.
4.1.31. (E,Z)-2-(4-(2-(4-Chloro-1H-benzo[d]imidazol-2-yl)-2-
cyanovinyl)phenoxy)acetamide (38)
Similar reaction of 64 (0.35 g, 1.83 mmol) and 42 (0.30 g,
1.66 mmol) in EtOH (20 ml) with a catalytic amount of piperidine
gave 38 as a yellow solid (0.46 g, 79%), mp (EtOH) 268–271 °C,
without further purification. 1H NMR d ppm 13.27 (br s, 1H),
8.41–8.27 (m, 1H), 8.02 (d, J = 8.9 Hz, 2H), 7.59 (br s, 1H), 7.41
(br s, 1H), 7.63–7.49 (m, 1H), 7.32 (d, J = 7.6 Hz, 1H), 7.25 (t,
J = 7.8 Hz, 1H), 7.17 (d, J = 8.9 Hz, 2H), 4.58 (s, 2H). HRMS (ESI+)
for C18H14ClN4O2 (MH)+: Calcd 353.0800, Found 353.0799. Anal.
Calcd for (C18H13ClN4O2): C, 61.28; H, 3.71; N, 15.88. Found: C,
61.66; H, 3.73; N, 16.21. HPLC purity, 97.8% (E/Z ratio 42:56).
4.1.35. (E,Z)-2-(4-(2-Cyano-2-(4,5-dimethyl-1H-benzo[d]
imidazol-2-yl)vinyl)phenoxy)acetamide (40)
Similar reaction of 66 (0.34 g, 1.84 mmol) and 42 (0.30 g,
1.66 mmol) in EtOH (20 ml) with a catalytic amount of piperidine
gave 40 as a yellow solid (0.11 g, 19%), mp (EtOH) 275–278 °C,
without further purification. 1H NMR d ppm 12.74 (br s, 0.5H, ob-
serve E- and Z- isomers separately), 12.45 (br s, 0.5H, observe E-
and Z- isomers separately), 8.33–8.18 (m, 1H), 7.98 (d, J = 8.9 Hz,
2H), 7.59 (br s, 1H), 7.41 (br s, 1H), 7.41–7.20 (m, 1H), 7.16 (d,
J = 8.9 Hz, 2H), 7.09–7.02 (m, 1H), 4.57 (s, 2H), 2.48 (s, 3H), 2.34
(s, 3H). HRMS (ESI+) for C20H19N4O2 (MH)+: Calcd 347.1503, Found
347.1497. Anal. Calcd for (C20H18N4O2ꢃ0.2H2O): C, 68.64; H, 5.30;
N, 16.01. Found: C, 68.25; H, 5.04; N, 15.92. HPLC purity 96.3%
(E/Z ratio 57:39).
4.1.32. 2-(4-(1-Amino-2,4-dicyano-6-methoxybenzo
[4,5]imidazo[1,2-a]pyridin-3-yl)phenoxy)acetamide (33)
Similar reaction of 3-methoxybenzene-1,2-diamine (60) (1.50 g,
10.86 mmol) with cyanoacetamide (1.83 g, 21.71 mmol) in xylene
(25 ml) and purification of the product by flash chromatography
on silica gel, eluting with 50–100% EtOAc/hexanes, gave 2-(4-
methoxy-1H-benzo[d]imidazol-2-yl)acetonitrile (65) as a pale yel-
low solid (1.53 g, 75%), which was used directly. 1H NMR (CDCl3) d

4100
D. M. Lyons et al. / Bioorg. Med. Chem. 19 (2011) 4091–4100
4.1.36. 2-(4-(1-Amino-2,4-dicyano-7,8-dimethylbenzo[4,5]
imidazo[1,2-a]pyridin-3-yl)phenoxy)acetamide (35)
4.2.2. KHYG-1 mediated cytotoxicity assay
KHYG-1 cells were washed and resuspended in RPMI + 0.1% BSA
Similar reaction of 4,5-dimethylbenzene-1,2-diamine (62)
(1.50 g, 11.01 mmol) with cyanoacetamide (1.85 g, 22.03 mmol)
in xylene (25 ml) gave 2-(5,6-dimethyl-1H-benzo[d]imidazol-2-
yl)acetonitrile (67) as a brown solid (0.86 g, 42%), which was used
directly 1H NMR (CDCl3) d ppm 9.48 (br s, 1H), 7.43–7.29 (m, 2H),
4.10 (s, 2H), 2.37 (s, 6H). LRMS (APCI+) for C11H12N3 (MH+) Calcd
186, Found 186.
at 4 ꢁ 105 cells/ml and 50
lL of KHYG-1 cells were dispensed to
each well of a 96-well V-bottom plate. Test compounds were
added to KHYG-1 cells at various concentrations up to 20 lM
and incubated at RT for 20 min. 1 ꢁ 106 K562 target cells were la-
belled with 75 l lL RPMI for 90 min at 37 °C, cells
Ci 51Cr in 200
were washed as described above and resuspended in 5 ml RPMI
+ 0.1% BSA. 50
l
L of 51Cr labelled K562 leukaemia target cells were
A solution of 67 (0.61 g, 3.30 mmol), 43 (0.50 g, 2.20 mmol) and
a catalytic amount of piperidine in EtOH (20 ml) was heated under
reflux for 15 h to give an intermediate (0.62 g, 1.79 mmol) that was
treated further with malononitrile (0.24 g, 3.58 mmol) and cata-
lytic piperidine in EtOH (20 ml) overnight to close the middle ring.
The cooled reaction mixture was filtered and washed with EtOH
and the product was purified by flash chromatography, eluting
with 3–20% MeOH/CH2Cl2, to give 35 as a brown solid (0.04 g,
4%), mp >305 °C. 1H NMR d ppm 8.55 (br s, 2H), 8.43 (s, 1H), 7.64
(s, 1H), 7.60 (br s, 1H), 7.57 (d, J = 8.9 Hz, 2H), 7.41 (br s, 1H),
7.15 (d, J = 8.8 Hz, 2H), 4.55 (s, 2H), 2.45 (s, 3H), 2.42 (s, 3H). HRMS
(ESI+) for C23H19N6O2 (MH)+: Calcd 411.1564, Found 411.1570.
HPLC purity, 94.3%.
added to each well of the KHYG-1 plate (effector–target 2:1) and
incubated at 37 °C for 4 h. 51Cr release was assayed using a Skatron
Harvesting Press and radioactivity estimated on a Wallac Wizard
1470 Automatic Gamma counter (Turku, Finland). The percentage
of specific cytotoxicity was calculated by the formula:
ðexperimental release ꢀ spontaneous releaseÞ
%specific lysis ¼
ꢁ 100
ðmaximum release ꢀ spontaneous releaseÞ
and expressed as the mean of triplicate assays.
4.2.3. Toxicity to KHYG-1 NK cells
The toxicity assay was carried out in exactly the same manner
as the killing assay above, but instead of adding the labelled
K562 target cells, 100
incubated for 4 h at 37 °C then washed ꢁ3 in RPMI + 0.1% BSA. Cells
were then resuspended in 200 L of complete medium and incu-
bated for 18–24 h at 37 °C. Trypan blue was added to each well
and viable (clear) cells counted as a percentage of total (clear +
blue) cell number (% viability).
lL of RPMI 0.1% BSA was added. Cells were
4.1.37. (E,Z)-2-(4-(2-Cyano-2-(5,6-dimethyl-1H-benzo[d]
imidazol-2-yl)vinyl)phenoxy)acetamide (41)
l
Similar reaction of 67 (0.34 g, 1.84 mmol) and 42 (0.30 g,
1.66 mmol) in EtOH (20 ml) with a catalytic amount of piperidine
gave 41 as a yellow solid (0.37 g, 64%), mp (EtOH) >320 °C, without
further purification. 1H NMR d ppm 12.68 (br s, 1H), 8.20 (s, 1H),
7.97 (d, J = 8.9 Hz, 2H), 7.58 (br s, 1H), 7.41 (br s, 1H), 7.47–7.26
(m, 2H), 7.15 (d, J = 7.0 Hz, 2H), 4.57 (s, 2H), 2.33 (s, 6H). HRMS
(ESI+) for C20H19N4O2 (MH)+: Calcd 347.1503, Found 347.1502.
Anal. Calcd for (C20H18N4O2ꢃ0.1H2O): C, 68.99; H, 5.27; N, 16.09.
Found: C, 68.76; H, 5.19; N, 16.16. HPLC purity, 95.7% (E/Z ratio
45:51).
Acknowledgments
The authors thank the Wellcome Trust for partial support.
K.M.H. thanks the Academy of Finland (Grant no. 135439), the
Finnish Cultural Foundation, and Jenny and Antti Wihuri Founda-
tion for financial support.
4.1.38. Aqueous solubility
References and notes
Suspensions of compounds in water (1 mL) were sonicated for
15 min, then centrifuged at 13,000 rpm for 6 min. Supernatants
were re-centrifuged, then diluted two-fold with water to avoid
precipitation during HPLC analysis. This was carried out using an
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in the same manner as above, to determine an accurate IC50
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