Imidazo[1,2-a]pyridin-3-amines as potential HIV-1 non-nucleoside reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.05.062

During random screening of a small in-house library of compounds, certain substituted imidazo[1,2-a]pyridines were found to be weak allosteric inhibitors of HIV-1 reverse transcriptase (RT). A library of these compounds was prepared using the Groebke reaction and a subset of compounds prepared from 2-chlorobenzaldehyde, cyclohexyl isocyanide and a 6-substituted 2-aminopyridine showed good inhibitory activity in enzymatic (RT) and HIV anti-infectivity MAGI whole cell assays. The compound showing the best anti-HIV-1 IIIB whole cell activity (MAGI IC₅₀ = 0.18 μM, IC₉₀ = 1.06 μM), along with a good selectivity index (>800), was 2-(2-chlorophenyl)-3- (cyclohexylamino)imidazo[1,2-a]pyridine-5-carbonitrile 38.

Full text of DOI:10.1016/j.bmc.2011.05.062

Bioorganic & Medicinal Chemistry 19 (2011) 4227–4237
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Imidazo[1,2-a]pyridin-3-amines as potential HIV-1 non-nucleoside reverse
transcriptase inhibitors
⇑
Moira L. Bode , David Gravestock, Simon S. Moleele, Christiaan W. van der Westhuyzen, Stephen C. Pelly ,
Paul A. Steenkamp, Heinrich C. Hoppe, Tasmiyah Khan, Lindiwe A. Nkabinde
CSIR Biosciences, Private Bag X2, Modderfontein, Johannesburg 1645, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history:
During random screening of a small in-house library of compounds, certain substituted imidazo[1,2-
a]pyridines were found to be weak allosteric inhibitors of HIV-1 reverse transcriptase (RT). A library of
these compounds was prepared using the Groebke reaction and a subset of compounds prepared from
2-chlorobenzaldehyde, cyclohexyl isocyanide and a 6-substituted 2-aminopyridine showed good inhibi-
tory activity in enzymatic (RT) and HIV anti-infectivity MAGI whole cell assays. The compound showing
Received 1 April 2011
Revised 25 May 2011
Accepted 27 May 2011
Available online 21 June 2011
the best anti-HIV-1 IIIB whole cell activity (MAGI IC50 = 0.18 lM, IC90 = 1.06 lM), along with a good selec-
tivity index (>800), was 2-(2-chlorophenyl)-3-(cyclohexylamino)imidazo[1,2-a]pyridine-5-carbonitrile
38.
Keywords:
Groebke reaction
Imidazo[1,2-a]pyridine
NNRTI
Ó 2011 Elsevier Ltd. All rights reserved.
Reverse transcriptase
Antiretroviral
HIV
1. Introduction
Imidazo[1,2-a]pyridines have been shown to possess a broad
range of biological activities and have been investigated for treat-
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are
indispensable first-line drugs in the fight against HIV-AIDS. One
advantage of this class is the excellent therapeutic window,1 mak-
ing the approved NNRTIs among the least toxic of the clinically
approved antiretrovirals.2 These drugs act by binding to a lipo-
philic, non-substrate binding pocket located about 10 Å from the
substrate binding site. Binding induces conformational changes
in the catalytic site, slowing catalytic activity markedly.3 About
fifty structurally diverse classes of NNRTIs are known.4 The first
generation of NNRTIs (exemplified by nevirapine and delavirdine)
are sensitive to the development of drug resistance and even a sin-
gle amino acid mutation in the NNRTI binding region of RT results
in a loss of compound efficacy. The second generation NNRTI efavi-
renz, however, maintains activity against a number of different
NNRTI mutants. One of the most recently approved NNRTIs etravi-
rine remains active against a wide range of NNRTI mutants, and
this is attributed to the molecule’s ability to adopt multiple confor-
mations in the RT binding pocket.5 Recent literature indicates the
ongoing interest in the design and development of novel NNRTI
scaffolds capable of similar activity.6–12
ment of conditions such as gastric disease,13,14 heart disease,15 mi-
graines16 and viral diseases,17–21 amongst others. The
pharmacology of these compounds has also been extensively
investigated.22 As part of our ongoing interest in the biological
activity of these compounds, a small in-house library of com-
pounds was screened against HIV-1 RT, leading to identification
of
N-cyclohexyl-2-isopropylimidazo[1,2-a]pyridin-3-amine
1
(Table 1) as a possible NNRTI. This compound was reasonably ac-
tive against wild-type RT in an enzymatic assay, but poorly active
in a whole cell anti-HIV infectivity assay. Based on this structure, a
larger library of imidazo[1,2-a]pyridines was prepared and
screened in this preliminary investigation for improved activity
against wt RT,23 selected examples of which are presented below.
2. Results and discussion
2.1. Chemistry
Compounds were prepared using the multi-component
Groebke coupling reaction24,25 between an aldehyde, isocyanide
and 2-aminopyridine (Scheme 1). In this study, the reactions were
catalysed by Montmorillonite K-10 clay using either conventional
heating or microwave conditions.26
⇑
Corresponding author. Tel.: +27 87 751 3791; fax: +27 11 608 3200.
E-mail address: mbode@csir.co.za (M.L. Bode).
Using Groebke methodology, three distinct zones can be varied
in the imidazo[1,2-a]pyridine scaffold: the aldehydic region that
Present address: Department of Chemistry and Polymer Science, Stellenbosch
University, Private Bag X1, Matieland 7602, South Africa.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.062

4228
M. L. Bode et al. / Bioorg. Med. Chem. 19 (2011) 4227–4237
Table 1
Residual RT activities of simple 2,3-disubstituted imidazo[1,2-a]pyridines at 50 lM
N
R1
NHR2
N
No.
R1
R2
% Res RT Acta
No.
R1
R2
% Res RT Acta
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Propyl, i-
Propyl, i-
Propyl, i-
Propyl, i-
Propyl, i-
Propyl, c-
Butyl, 2-
Octyl, n-
Phenyl
Hexyl, c-
Pentyl, n-
Pentyl, 2-
Pentyl, c-
Butyl, n-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
40.6
83.7
54.9
33.7
54.0
72.9
54.8
92.4
74.6
7.1
76.0
77.6
29.7
92.3
102.7
13.8
45.4
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
Phenyl
2-Trifluoromethyl-
4-Trifluoromethyl-
3-Cyano-
4-Cyano-
4-Nitro-
2-Hydroxy-
4-Hydroxy-
4-Methoxy-
4-Dimethylamino-
2,4-Dichloro-
2,5-Dichloro-
2,6-Dichloro-
2,6-Difluoro-
2,4,5-Trifluoro-
2,3,6-Trichloro-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
Hexyl, c-
73.2
88.3
100.2
90.8
86.1
87.0
89.4
84.5
91.6
39.9
36.8
55.5
17.8
18.6
83.5
1.0
Phenyl
2-Chloro-
3-Chloro-
4-Chloro-
2-Bromo-
3-Bromo-
4-Bromo-
2-Fluoro-
4-Fluoro-
Nevirapine
a
Percentage residual RT enzyme activity after incubation with 50 lM test compound determined relative to untreated controls.
Keeping the isocyanide component constant while changing the
identity of the starting aldehyde afforded no improvement in activ-
ity for the aliphatic groups (6–8). Simple aryl groups at C-2, how-
ever, yielded more interesting results. An unsubstituted phenyl
moiety afforded very poor inhibition (9). However, a dramatic
improvement in activity was observed when using 2-chlorobenzal-
dehyde as the aldehyde partner in the reaction (10). Changing this
component to the 3- or 4-chlorobenzaldehyde produced much
poorer inhibitors (11 and 12). Similar trends occurred with the flu-
oro- and bromobenzaldehydes (13–17), producing reasonable
inhibitors with the corresponding 2-halophenyl derivatives (13
and 16), although both were still poorer inhibitors than the initial
R3
8
R3
R4
R5
R4
R5
NH2
N
7
2
R1
R1 CHO
R2
N C
+
+
N
N
6
3
5
R6
NHR2
R6
Scheme 1. Groebke reaction.
generates R1 at C-2, the R2 ‘isocyanide’ region at C-3 and positions
R3–R6, around the 2-aminopyridine ring (Scheme 1). Commercially
available 2-aminopyridines, aldehydes and isocyanides with
suitable substituents were used where possible, while certain
2-aminopyridines and isocyanides were prepared by standard
methods. Yields from the Groebke reaction varied widely
(13–83%).
2-chlorophenyl product (10). Introducing
a sterically more
demanding electron-withdrawing group at the 2- or 4-position of
the phenyl group (18 and 19) failed to improve matters, as did
other such substitutions with cyano- or nitro groups (20–22). Elec-
tron donating groups similarly abrogated activity (23–26). This
was expected, taking into account the known lipophilicity of the
NNRTI binding pocket.
2.2. Biological results
Introducing additional 4-, 5- or 6-chloro substituents to the 2-
chlorophenyl ring (27–29) weakened the inhibition relative to
10, while trichloro product 32 showed effectively no activity. Inter-
estingly, though, products 30 and 31 with two and three fluoro
substituents, respectively, retained activity relative to the parent
2-fluorophenyl compound (16). This suggests that a steric con-
straint governs the activity at this site, in addition to the need
for lipophilicity suggested by the preference for halo- substituents.
Attempts to improve the activity of compound 10 to near or
better than that of nevirapine focussed on variations around the
six-membered ring of the imidazo[1,2-a]pyridine system
(Table 2). Initially continuing to use residual RT activity to measure
inhibition, monomethylation at position R3–R6 (33–36) showed
decreasing activity in the order R6 > R5 > R3 > R4, with the com-
pound substituted at R6 (36) having improved activity relative to
the unsubstituted parent compound (10). This also proved to be
the case in terms of RT IC50 values, with compound 36 having a
The inhibitory activity of compounds was assessed using a col-
orimetric HIV-1 RT assay. Percentage residual RT enzyme activity
after incubation with 50 lM test compound was determined rela-
tive to untreated controls. During this investigation, percentage
residual activity was used as an initial guide to compound activity
(Table 1). This approach was found to be reasonable for the set of
compounds under evaluation (vide infra).
Isopropyl-substituted product 1 was identified as a potential
NNRTI (41% residual RT activity) in the original screen. Simple varia-
tionoftheisocyanideusedintheGroebkereactiontovarytheidentity
of the pendant amino group, exemplified by compounds 2–5, affor-
ded only a marginal improvement in the case of cyclopentylamine
4. Other variations tried, including various simple aryl, linear- or
branched aliphatic groups, showed no significant RT activity (data
notshown).ThecyclohexylderivativesatR2werethenchosenforfur-
ther investigation basedbothon theavailability ofthestartingisocy-
anide and the lack of a definitive difference between the activities of
the cyclohexyl- and cyclopentyl derivatives screened thus far.
value of 1.55 lM relative to the 4.4 lM of compound 10.

M. L. Bode et al. / Bioorg. Med. Chem. 19 (2011) 4227–4237
4229
Concentrating on substitution at R5 and R6, similar trends
were seen with the compounds having cyano-, bromo- and
chloro-substituents. Compounds substituted at R6 were more ac-
tive than the corresponding compounds substituted at R5 (compare
37, 40 and 42 with 38, 41 and 43). Compound 39, with fluoro sub-
stitution at R5 had similar activity to compounds 40 and 42. A pref-
erence for lipophilic groups at R6 is evident. On the other hand,
electron donating groups, exemplified by methoxy compound 44,
weren’t tolerated. Similar or poorer activities were observed with
other electron donating groups (data not shown). Combining the
effects of substituents at R5 and R6 didn’t result in a recovery of
activity, with compound 45 showing poorer activity than either
the methyl- (36) or bromo- (42) monosubstituted compounds.
An interesting exception is compound 46 derived from quinoline,
which had reasonable activity despite effective disubstitution.
In terms of effective enzymatic inhibition, compounds substi-
tuted at R5 (35, 37, 39, 40 and 42) all showed low micromolar
enzymatic inhibition; although these were still several fold weaker
than nevirapine. Interestingly, the compounds substituted at R6
with methyl, cyano and chloro moieties (36, 38 and 41) showed
favourable whole cell IC50 values of around 0.2
of 1.04, 1.06 and 1.10 M, respectively; about double that of nevi-
rapine (IC50 = 0.09 M and IC90 = 0.60 M). The relationship of the
lM, and IC90 values
l
l
l
IC90 to the IC50 values for these compounds is similar to that of
nevirapine, due to the fact that the dose–response curves have
similar Hill slope factors (1.16 for nevirapine; 1.15–1.39 for our
compounds). Compound 38 had the best antiviral activity (MAGI
IC50 = 0.18
of the compounds tested, only compound 36 had a CC50 value of
<50 M, indicating a lack of significant toxicity of this type of
lM) and a good selectivity index of 867. In addition,
l
Table 2
RT and whole-cell inhibitory activity of 2-(2-chlorophenyl)-N-cyclohexylimidazo[1,2-a]pyridin-3-amines
R3
Cl
R4
N
N
R5
R6
HN
R3
R4
R5
R6
% Res RT Acta
RT IC50
(
lM)
MAGI IC50
(lM)
MAGI CC50 (lM)
SIe
b
c
d
No.
10
33
34
35
36
37
38
39
40
41
42
43
44
45
46
7.1
4.4
7.18
NT
0.41
3.44
NT
60.4
>294
NT
147a
>86
NT
Methyl
18.8
56.8
10.8
3.4
10.1
4.7
17.0
20.0
3.9
20.2
6.1
Methyl
Methyl
Cyano
5.39
1.55
6.60
3.47
11.28
14.47
3.74
12.20
4.62
NT
0.64
0.19
<0.63
0.18
1.88
0.84
0.24
2.19
3.15
NT
57.4
20.5
140
156
160
>200
152
>200
>200
NT
90
Methyl
Cyano
108
>222
868
85
>238
633
>91
64
NT
>3
243
>111
Fluoro
Chloro
Chloro
Bromo
Bromo
Methoxy
Methyl
43.2
33.5
12.5
1.0
Bromo
CH@CHCH@CH–
18.89
7.52
0.67
73.00
0.60
0.09
>200
146
>10
Nevirapine
NT: not tested.
a
Percentage residual RT enzyme activity after incubation with 50
l
M test compound determined relative to untreated controls.
b
c
Compound concentration (
Compound concentration (
Compound concentration (
l
l
l
M) required to inhibit the RT enzyme by 50%.
M) required to inhibit virus replication in MAGI-R5 cells by 50%.
M) required to reduce the viability of MAGI-R5 cells by 50%.
d
e
Selectivity index (CC50/IC50).
Figure 1. Correlation between the RT activity and corresponding MAGI antiviral activity of test compounds. A total of 23 compounds were subjected to the analysis (A). The
19 compounds with % enzyme activity 640% and MAGI IC50 <4.5% are shown in B.

4230
M. L. Bode et al. / Bioorg. Med. Chem. 19 (2011) 4227–4237
compound compared to their IC50 values. This favourable profile in
itself makes these inhibitors worthy of further investigation.
To assess the confidence with which percentage residual enzy-
matic activity could be used as a means of predicting anti-HIV
activity for this series of compounds, the relationship between per-
centage residual enzyme activity and antiviral activity obtained for
that compound from the MAGI whole cell assay was examined
(Fig. 1). A good correlation between the two was found; com-
rilpivirine (TMC-278) were simultaneously docked to ensure dock-
ing robustness (see Section 4).
CN
CN
pounds that yielded enzyme activity under 40% at 50
lM produced
HN
N
NH
MAGI IC50 values less than 4.5 M (Fig. 1A, points within dashed
l
rectangular box). Nineteen compounds adhered to this trend.
There were only two exceptions that had less than 40% enzyme
activity, but higher MAGI IC50 values (18–73 lM) (Fig. 1A, circled
N
TMC-278
data points). The rectangular box region of Figure 1A is presented
on a more convenient scale in Figure 1B, which demonstrates a
close correlation between the HIV RT activity of the test com-
pounds and the MAGI cell antiviral activities. Three compounds
(circled) deviated somewhat from the trend and produced whole
cell activities slightly higher than expected from the RT enzyme
activities. If these three outliers are excluded, the correlation be-
tween the enzyme and whole cell results, as determined by linear
regression, is r2 = 0.86. The results thus reveal an empirical correla-
tion between HIV RT enzyme inhibition and MAGI whole cell anti-
HIV activity for compounds in the present series.
The docked pose of compound 38 (Fig. 2) and the accompanying
2D interaction map (Fig. 3) show that the core imidazo[1,2-a]pyr-
idine ring system fits into the aromatic-rich pocket bracketed by
Tyr188, Phe227 and Trp229. The imidazo[1,2-a]pyridine ring is
parallel to the aromatic ring of Tyr188, allowing for
p–p stacking
to stabilise this interaction. No CH- interaction is possible, as
p
the imidazo[1,2-a]pyridine scaffold projects between the indole
ring of Trp229 and Tyr188, possibly due to the need to accommo-
date the projecting cyano group. A second p–p stacking interaction
between Tyr181 and the chlorophenyl moiety contributes to the
stabilisation of the complex. Furthermore, the cyclohexyl ring lies
in a hydrophobic pocket lined by the mutable Lys/Arg103 residue,
probably associating with it through van der Waals interactions.
Interestingly, C-4 of the cyclohexyl group lies about 3 Å from the
carbonyl oxygen and 4.5 Å away from the hydrogen on the NH
group of Lys101. Hydrogen bonds to this and the adjacent
Lys/Arg103 residue confer the remarkable increase in activity asso-
ciated with newer NNRTIs.28
2.3. Molecular modelling and analysis
In order to attempt to rationalise the results, compound 38 was
docked into the HIV-1 NNRTI binding site using CDOCKER (Accel-
rysÒ Discovery Studio 2.5.5). The crystal structures of both the
wild-type and K103 N mutant forms of HIV-1 RT containing the
diarylpyrimidine inhibitor rilpivirine (TMC-278) were used (pdb
codes MEE and 3MEG, respectively).27 Etravirine (TMC-125) and
Figure 2. Docked pose of compound 38 in the NNRTI binding site.
p–p Stacking interactions between the compound and the aromatic rings of Tyr181 and Tyr188 are
indicated in orange. Interatomic distances between C-4 of the cyclohexyl ring and the carbonyl oxygen as well as the amino nitrogen of Lys101 are indicated in green.

M. L. Bode et al. / Bioorg. Med. Chem. 19 (2011) 4227–4237
4231
of the dimethylanilino moiety of TMC-278, while groups at the
3-position of the phenyl ring would not be accommodated as
readily. Similarly, the cyano group at R6 occupies a cavity between
Pro236 and Phe227, similar to the cyano group on TMC-278. The
cyano group at R5 could be accommodated with a small reposition-
ing, while groups at R3 or R4 would clash with Phe227 and Trp229.
The relationship between the cyclohexyl group and the hydrogen
bonding groups on TMC-278 is clearly evident in the figure. This
suggests sites of further investigation to improve the activities of
the new series of compounds presented here, by introducing
hydrogen bonding.
2.4. PAMPA permeability
The membrane permeabilities of a selection of compounds from
Table 2 were assessed in a parallel artificial membrane permeabil-
ity assay (PAMPA) as a preliminary guide to the likelihood of their
passive absorption. The compounds were tested along with
standard markers for low, intermediate and high permeability
(atenolol, pindolol and metoprolol, respectively; Fig. 5). All the
compounds demonstrated similar intermediate to high permeabil-
ities. These data could support a potentially favourable absorption
profile for the imidazo[1,2-a]pyridin-3-amines in this study.
3. Conclusion
Anumberofimidazo[1,2-a]pyridin-3-aminesweresynthesisedin
a one-pot procedure from appropriate aldehydes, isocyanides and
2-aminopyridines using the Groebke reaction. They were tested for
theiranti-HIVactivity.CompoundspossessingalkylgroupsatR1 (de-
rivedfromthealdehydiccomponent)werepoorinhibitorsoforinac-
tive against RT. Compounds substituted at R1 with phenyl or
substituted phenyl were also poorly active, with the striking excep-
tion of those bearing a 2-halophenyl group. In particular compounds
Figure 3. 2D interaction map of docked compound 38, with
interactions and distances indicated in orange.
p–p stacking
An overlay of compound 38 with TMC-278 sheds further light
on the observed activity (Fig. 4) The 2-chloro moiety occupies a
small hydrophobic pocket similar to that of the one methyl group
Figure 4. Overlay of the docked poses of TMC-278 (white) and compound 38 (green). Hydrogen bonds between TMC-278 and Lys101 are indicated as dashed green lines.

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M. L. Bode et al. / Bioorg. Med. Chem. 19 (2011) 4227–4237
4.2. General procedure for the Groebke reaction
Cyclopentyl isocyanide was prepared as previously described.30
The appropriate 2-aminoheterocycle [pyridine or quinoline]
(1.33 mmol), aldehyde (1.33 mmol) and isocyanide (1.36 mmol)
in dioxane (2.5 ml) were heated in the presence of montmorillonite
K-10 clay (250 mg) for 5–8 h at 95–100 °C, or irradiated with
microwave energy at 150 W and 100 °C for 15–30 min in a sealed
pressure tube. After reaction completion, the liquid was filtered
from the clay and the clay was rinsed with dioxane or ethyl acetate
(2 Â 2.5 ml). The solvent was removed in vacuo and the residue
purified by column chromatography (elution hexane:ethyl acetate)
to afford the required product.
4.2.1. N-Cyclohexyl-2-isopropylimidazo[1,2-a]pyridin-3-amine
(1)
Yield 25%, as white needles; mp 116–118 °C; 1H NMR
(200 MHz, CDCl3) d 8.02 (d, J = 6.8 Hz, 1H), 7.49 (d, J = 8.9 Hz, 1H),
7.07–6.95 (m, 1H), 6.72 (dd, J = 3.7, 9.7 Hz, 1H), 3.14 (quin,
J = 6.8 Hz, 1H), 2.85 (br m, 2H), 2.00–1.51 (m, 5H), 1.38 and 1.34
(2 Â s, 6H), 1.32–1.09 (m, 5H); 13C NMR (50 MHz, CDCl3) d 144.7,
141.4, 123.1, 122.8, 122.4, 117.0, 110.9, 57.2, 34.3, 26.2, 25.9,
25.0, 22.8. HRMS (ESI): m/z 258.1963 (M+H)+; calcd for C16H24N3:
258.1970.
Figure 5. PAMPA permeabilities and A log P values for HIV RT inhibitors. Com-
pounds were incubated in PAMPA plates for 5 h at 37 °C at a starting concentration
of 100 lM in the donor wells. Atenolol, pindolol and metoprolol were included as
permeability markers, while lucifer yellow was used to control for intactness of the
permeability barrier. A log P is the atom-based method published by Ghose and
Crippen29 for determining the water–octanol partition coefficient for
compound and is a reliable measure of hydrophobicity.
a 3D
4.2.2. 2-Isopropyl-N-pentylimidazo[1,2-a]pyridin-3-amine (2)
Yield 63%, as an orange crystalline solid; mp 66–68 °C; 1H NMR
(200 MHz, CDCl3) d 7.98 (d, J = 6.8 Hz, 1H), 7.48 (d, J = 9.0 Hz, 1H),
7.04 (dd, J = 6.5, 8.5 Hz, 1H), 6.71 (t, J = 6.9 Hz, 1H), 3.12 (quin,
J = 7.1 Hz, 1H), 2.96 (t, J = 7.0 Hz, 2H), 1.73–1.42 (m, 2H),
1.42–1.10 (m, 4H), 1.38 and 1.34 (2 Â s, 6H), 0.90 (t, J = 7.0 Hz,
3H); 13C NMR (50 MHz, CDCl3) d 143.9, 141.2, 124.5, 122.7,
122.1, 117.1, 111.0, 49.3, 30.5, 29.2, 26.4, 22.8, 22.6, 14.0. HRMS
(ESI): m/z 246.1950 (M+H)+; calcd for C15H24N3: 246.1970.
substituted at R1 with 2-chlorophenyl; and with cyano, methyl or
chloro at R6 (derived from 2-amino-6-substituted pyridine) showed
promising activity. Novel compound 2-(2-chlorophenyl)-3-(cyclo-
hexylamino)imidazo[1,2-a]pyridine-5-carbonitrile (38) exhibited
the best activity profile, with a whole-cell anti-HIV IC50 of 0.18
in the MAGI assay and a good selectivity index of 868 (cf. nevirapine,
MAGI IC50 of 0.09 M). In addition, a selection of these compounds
showed good membrane permeability in parallel artificial
lM
l
a
4.2.3. 2-Isopropyl-N-(pentan-2-yl)imidazo[1,2-a]pyridin-3-
amine (3)
membrane permeability (PAMPA) assay. Future investigations will
expandonimprovingthepromisingprofileofthesecompounds,with
particularemphasisonintroducingthestructuralchangessuggested
by the molecular modelling results.
Yield 49%, as a yellow solid; mp 83–84 °C; 1H NMR (200 MHz,
CDCl3) d 8.12–7.92 (m, 1H), 7.59–7.40 (m, 1H), 7.05 (ddd, J = 1.2,
6.7, 8.9 Hz, 1H), 6.71 (dd, J = 0.8, 13.6 Hz, 1H), 3.25–2.98 (m, 2H),
2.95–2.56 (m, 1H), 1.63–1.40 (m, 4H), 1.37 (dd, J = 2.9, 6.9 Hz,
6H), 1.06 (d, J = 6.3 Hz, 3H), 0.93 (dd, J = 5.5, 8.4 Hz, 3H); 13C
NMR (50 MHz, CDCl3) d 144.9, 141.5, 123.2, 122.7, 122.4, 117.1,
110.9, 53.7, 40.0, 26.2, 22.7, 21.0, 19.5, 14.2; HRMS (ESI): m/z
246.1967 (M+H)+; calcd for C15H24N3: 246.1970.
4. Experimental
4.1. Chemistry
NMR spectra were run on either a Varian 200 MHz Gemini 2000
instrument, or on a 400 MHz Varian INOVA instrument. Samples
were referenced against chloroform at 77.00 ppm for 13C and
against tetramethylsilane at 0.00 ppm for 1H. Spin multiplicities
are given as s (singlet), br s (broad singlet), d (doublet), dd (doublet
of doublets), t (triplet), quin (quintet), ddd (double doublet of dou-
blets), td (triplet of doublets), dt (doublet of triplets) and m
(multiplet). High resolution mass spectra were recorded on a
Waters SYNAPT G1 HDMS mass spectrometer operated in electro-
spray mode. Leucine enkephalin (50 pg/ml) was used as reference
calibrant to obtain typical mass accuracies between 1 and 3 mDa.
Melting points were determined using a Mettler FP62 capillary
melting point apparatus and are uncorrected. All reagents were
of reagent grade purchased from Sigma-Aldrich (Schnelldorf,
Germany) and were used without any further purification. Solvents
used for chromatography or extractions were distilled prior to use.
Thin-layer chromatography was carried out using precoated alu-
minium-backed plates (Merck Silica Gel 60 F254) visualised under
UV light (k = 254 nm). Column chromatography was performed
on Fluka Silica Gel 60 (70–230 mesh).
4.2.4. N-Cyclopentyl-2-isopropylimidazo[1,2-a]pyridin-3-amine
(4)
Yield 20%, as a beige solid; mp 112–114 °C; 1H NMR (200 MHz,
CDCl3) d 8.18–7.92 (m, 1H), 7.49–7.52 (m, 1H), 7.14–6.94 (m, 1H),
6.82–6.61 (m, 1H), 3.80–3.47 (m, 1H), 3.09 (q, J = 6.8 Hz, 1H), 2.90–
2.66 (m, 1H), 1.93–1.43 (m, 8H), 1.38 (d, J = 6.9 Hz, 6H); 13C NMR
(50 MHz, CDCl3) d 160.2, 155.8, 138.1, 126.2, 116.3, 105.8, 105.2,
58.2, 37.3, 37.0, 34.2, 25.1, 18.2, 18.0. HRMS (ESI): m/z 244.1812
(M+H)+; calcd for C15H22N3: 244.1814.
4.2.5. N-Butyl-2-isopropylimidazo[1,2-a]pyridin-3-amine (5)
Yield 67%, as a yellow crystalline solid; mp 71–73 °C; 1H NMR
(200 MHz, CDCl3) d 7.92 (1H), 7.47 (d, J = 9.0 Hz, 1H), 6.97 (ddd,
J = 0.9, 6.7, 8.6 Hz, 1H), 6.64 (td, J = 0.6, 6.7 Hz, 1H), 3.14 (quin,
J = 7.0 Hz, 1H), 2.97 (t, J = 6.8 Hz, 2H), 2.91 (br s, 1H), 1.65–1.34
(m, 4H), 1.38 and 1.35 (2 Â s, 6H), 0.87 (t, J = 7.1 Hz, 3H); 13C
NMR (50 MHz, CDCl3) d 144.0, 141.2, 124.5, 122.6, 122.1, 117.1,
111.0, 49.0, 32.9, 26.5, 22.8, 20.2, 13.9. HRMS (ESI): m/z 232.1808
(M+H)+; calcd for C14H22N3: 232.1814.

M. L. Bode et al. / Bioorg. Med. Chem. 19 (2011) 4227–4237
4233
4.2.6. N-Cyclohexyl-2-cyclopropylimidazo[1,2-a]pyridin-3-
amine (6)
4.2.12. 2-(4-Chlorophenyl)-N-cyclohexylimidazo[1,2-a]pyridin-
3-amine (12)
Yield 32%, as a white solid; mp 85–87 °C; 1H NMR (200 MHz,
CDCl3) d 8.05–7.94 (m, 1H), 7.46–7.33 (m, 1H), 7.01 (ddd, J = 1.3,
6.7, 9.0 Hz, 1H), 6.69 (td, J = 1.0, 6.7 Hz, 1H), 3.07–2.67 (m, 2H),
2.11–1.82 (m, 3H), 1.82–1.67 (m, 2H), 1.67–1.48 (m, 1H),
1.41–1.10 (m, 6H), 1.05 (dt, J = 4.1, 5.2 Hz, 2H), 1.01–0.85 (m,
2H); 13C NMR (50 MHz, CDCl3) d 141.2, 140.5, 124.8, 122.7,
121.9, 116.6, 110.8, 57.2, 34.4, 25.9, 24.9, 8.2, 7.7. HRMS (ESI):
m/z 256.1810 (M+H)+; calcd for C16H22N3: 256.1814.
Yield 32%, as an off-white crystalline solid; mp 198–199 °C; 1H
NMR (200 MHz, CDCl3) d 8.13–7.98 (m, 3H), 7.53 (d, J = 9.0 Hz, 1H),
7.46–7.37 (m, 2H), 7.14 (t, J = 7.8 Hz, 1H), 6.79 (t, J = 6.8 Hz, 1H),
3.13–2.84 (m, 2H), 1.95–1.46 (m, 6H), 1.30–1.09 (m, 4H); 13C
NMR (50 MHz, CDCl3) d 167.1, 141.7, 133.1, 132.9, 128.6, 128.3,
125.1, 124.0, 122.6, 117.5, 111.6, 56.9, 34.2, 25.7, 24.8. HRMS
(ESI): m/z 326.1422 (M+H)+; calcd for C19H21Cl N3: 326.1424.
4.2.13. 2-(2-Bromophenyl)-N-cyclohexylimidazo[1,2-a]pyridin-
3-amine (13)
4.2.7. 2-sec-Butyl-N-cyclohexylimidazo[1,2-a]pyridin-3-amine
(7)
Yield 22%, as a brown solid; mp 43–45 °C; 1H NMR (200 MHz,
Yield 20%, as a yellow solid; mp 82–84 °C; 1H NMR (200 MHz,
CDCl3) d 8.14–7.93 (m, 1H), 7.48 (dt, J = 1.3, 9.5 Hz, 1H), 7.04
(ddd, J = 1.2, 6.7, 8.8 Hz, 1H), 6.71 (td, J = 1.1, 6.9 Hz, 1H),
3.10–2.63 (m, 3H), 2.12–1.48 (m, 6H), 1.38 (d, J = 6.8 Hz, 3H),
1.48–1.06 (m, 5H), 0.85 (t, J = 7.3 Hz, 3H); 13C NMR (50 MHz, CDCl3)
d 143.8, 141.8, 124.4, 123.0, 122.7, 117.1, 111.1, 57.6, 34.6, 33.6,
30.2, 26.1, 25.2, 21.1, 12.9. HRMS (ESI): m/z 272.2127 (M+H)+;
calcd for C17H26N3: 272.2127.
CDCl3) d 8.43–7.97 (m, 1H), 7.64 (td, J = 1.5, 7.7 Hz, 1H),
7.58–7.48 (m, 1H), 7.39 (td, J = 1.3, 7.5 Hz, 1H), 7.31–7.21 (m,
1H), 7.21–7.13 (m, 1H), 7.11 (dd, J = 1.3, 6.7 Hz, 1H), 6.80 (td,
J = 1.1, 6.7 Hz, 1H), 3.42–3.09 (m, 1H), 2.82–2.48 (m, 1H),
1.80–1.33 (m, 5H), 1.21–0.81 (m, 5H); 13C NMR (50 MHz, CDCl3)
d 141.4, 136.1, 132.7, 132.5, 129.3, 127.3, 125.8, 123.5, 122.9,
122.8, 117.5, 111.5, 56.3, 33.8, 25.6, 24.5. HRMS (ESI): m/z
370.0892 (M+H)+; calcd for C19H21BrN3: 370.0919.
4.2.8. N-Cyclohexyl-2-octylimidazo[1,2-a]pyridin-3-amine (8)
Yield 71%, as a yellow solid; mp 56–58 °C; 1H NMR (200 MHz,
CDCl3) d 8.01 (d, J = 6.8 Hz, 1H), 7.44 (d, J = 8.4 Hz, 1H), 7.12–6.92
(m, 1H), 6.70 (t, J = 6.7 Hz, 1H), 2.98–2.77 (m, 2H), 2.77–2.58 (m,
2H), 1.95–1.48 (m, 7H), 1.48–1.00 (m, 15H), 0.98–0.72 (m, 3H);
13C NMR (50 MHz, CDCl3) d 141.4, 139.7, 124.4, 122.7, 122.4,
116.9, 110.9, 57.2, 34.3, 31.9, 29.9, 29.7, 29.5, 29.3, 27.5, 25.9,
24.9, 22.7, 14.1. HRMS (ESI): m/z 328.2747 (M+H)+; calcd for
4.2.14. 2-(3-Bromophenyl)-N-cyclohexylimidazo[1,2-a]pyridin-
3-amine (14)
Yield 10%, as a brown solid; mp 79–81 °C; 1H NMR (200 MHz,
CDCl3) d 8.35–8.21 (m, 1H), 8.15–8.04 (m, 1H), 8.04–7.90 (m,
1H), 7.61–7.47 (m, 1H), 7.43 (ddd, J = 1.1, 1.9, 7.9 Hz, 1H), 7.29
(dd, J = 5.4, 10.3 Hz, 1H), 7.13 (ddd, J = 1.3, 6.7, 9.0 Hz, 1H), 6.79
(td, J = 1.1, 6.8 Hz, 1H), 3.29–2.66 (m, 2H), 1.69 (m, 5H),
1.41–0.74 (m, 5H); 13C NMR (50 MHz, CDCl3) d 141.6, 136.6,
131.0, 130.0, 130.0, 129.9, 125.3, 124.1, 122.7, 122.6, 117.5,
111.7, 57.0, 34.3, 25.7, 24.8. HRMS (ESI): m/z 370.0911 (M+H)+;
calcd for C19H21BrN3: 370.0919.
C21H34N3: 328.2753.
4.2.9. N-Cyclohexyl-2-phenylimidazo[1,2-a]pyridin-3-amine (9)
Yield 78%, as a white solid; mp 176–178 °C; 1H NMR (400 MHz,
CDCl3) d 8.17–7.94 (m, 2H), 7.58–7.39 (m, 2H), 7.36–7.22 (m, 2H),
7.17–6.88 (m, 2H), 6.77 (t, J = 6.6 Hz, 1H), 3.13 (br s, 1H), 2.97 (br s,
1H), 1.90–1.38 (m, 5H), 1.32–0.81 (m, 5H); 13C NMR (50 MHz,
CDCl3) d 141.7, 136.7, 134.6, 129.6, 128.7, 127.5, 127.3, 124.1,
122.9, 117.6, 111.8, 57.2, 34.4, 26.0, 25.1. HRMS (ESI): m/z
292.1782 (M+H)+; calcd for C19H22N3: 292.1814.
4.2.15. 2-(4-Bromophenyl)-N-cyclohexylimidazo[1,2-a]pyridin-
3-amine (15)
Yield 30%, as a pale yellow solid; mp 166–168 °C; 1H NMR
(200 MHz, CDCl3) d 8.09–8.00 (m, 1H), 7.94 (d, J = 8.6 Hz, 2H),
7.58–7.43 (m, 3H), 7.10 (ddd, J = 1.3, 6.7, 9.0 Hz, 1H), 6.75 (td,
J = 1.1, 6.8 Hz, 1H), 3.01 (s, 2H), 1.93–1.39 (m, 5H), 1.16 (s, 5H);
13C NMR (50 MHz, CDCl3) d 141.6, 135.6, 133.5, 131.4, 128.5,
124.8, 124.0, 122.6, 121.1, 117.3, 111.6, 56.8, 34.3, 25.7, 24.8.
HRMS (ESI): m/z 370.0926 (M+H)+; calcd for C19H21Br N3:
370.0919.
4.2.10. 2-(2-Chlorophenyl)-N-cyclohexylimidazo[1,2-a]pyridin-
3-amine (10)
Yield 54%, as a viscous yellow oil; 1H NMR (200 MHz, CDCl3) d
8.17 (dt, J = 1.0, 6.9 Hz, 1H), 7.70 (dd, J = 3.0, 6.4 Hz, 1H),
7.62–7.52 (m, 1H), 7.52–7.29 (m, 3H), 7.16 (ddd, J = 1.1, 6.6,
9.0 Hz, 1H), 6.82 (td, J = 0.9, 6.7 Hz, 1H), 3.29 (d, J = 6.7 Hz, 1H),
2.85–2.53 (m, 1H), 1.88–1.31 (m, 5H), 1.30–0.77 (m, 5H); 13C
4.2.16. N-Cyclohexyl-2-(2-fluorophenyl)imidazo[1,2-a]pyridin-
3-amine (16)
Yield 45%, as a viscous brown oil; 1H NMR (400 MHz, CDCl3) d
8.15 (d, J = 6.9 Hz, 1H), 7.92 (td, J = 2.3, 7.6 Hz, 1H), 7.54 (d,
J = 9.1 Hz, 1H), 7.38–7.21 (m, 2H), 7.18–7.05 (m, 2H), 6.79 (t,
J = 6.7 Hz, 1H), 3.45 (t, J = 7.8 Hz, 1H), 2.91–2.46 (m, 1H),
1.88–1.34 (m, 5H), 1.21–0.83 (m, 5H); 13C NMR (101 MHz, CDCl3)
d 160.4, 157.9, 141.7, 131.3 (d, J = 2.3 Hz), 131.2 (d, J = 4.6 Hz),
128.8 (d, J = 8.4 Hz), 126.6, 124.3 (d, J = 3.1 Hz), 123.5, 122.6,
122.3 (d, J = 14.5 Hz), 117.0, 115.3 (d, J = 23.6 Hz), 111.2, 56.3,
33.6, 25.3, 24.4. HRMS (ESI): m/z 310.1724 (M+H)+; calcd for
NMR (50 MHz, CDCl3)
d 141.6, 135.0, 134.0, 132.6, 129.4,
129.1, 126.9, 126.3, 123.7, 122.8, 117.5, 111.6, 56.4, 33.9, 25.7,
24.6. HRMS (ESI): m/z 326.1440 (M+H)+; calcd for C19H21ClN3:
326.1424.
4.2.11. 2-(3-Chlorophenyl)-N-cyclohexylimidazo[1,2-a]pyridin-
3-amine (11)
Yield 35%, as a cream solid; mp 167–169 °C; 1H NMR (400 MHz,
CDCl3) d 8.12 (dd, J = 1.6, 3.2 Hz, 1H), 7.97 (d, J = 6.8 Hz, 1H), 7.93
(d, J = 7.6 Hz, 1H), 7.45 (d, J = 9.2 Hz, 1H), 7.29 (t, d, J = 8.0 Hz,
1H), 7.23 (ddd, J = 1.2, 2.0, 8.0 Hz, 1H), 7.05 (ddd, J = 1.2, 6.4,
8.8 Hz, 1H), 6.69 (td, J = 0.8, 6.8 Hz, 1H), 3.07 (br d, J = 4.4 Hz,
1H), 2.98–2.82 (m, 1H), 1.94–1.42 (m, 5H), 1.42–0.92 (m, 5H);
13C NMR (101 MHz, CDCl3) d 141.3, 136.1, 134.9, 134.1, 129.4,
126.8, 126.7, 125.1, 124.6, 123.9, 122.4, 117.1, 111.4, 56.6, 33.9,
25.4, 24.5. HRMS (ESI): m/z 326.1423 (M+H)+; calcd for C19H21ClN3:
326.1424.
C19H21FN3: 310.1720.
4.2.17. N-Cyclohexyl-2-(4-fluorophenyl)imidazo[1,2-a]pyridin-
3-amine (17)
Yield 41%, as a white solid; mp 167–169 °C; 1H NMR (200 MHz,
CDCl3) d 8.13–7.92 (m, 3H), 7.52 (dt, J = 1.0, 9.1 Hz, 1H), 7.22–6.97
(m, 3H), 6.77 (td, J = 1.1, 6.8 Hz, 1H), 3.31–2.47 (m, 2H), 1.93–1.40
(m, 5H), 1.40–0.86 (m, 5H); 13C NMR (50 MHz, CDCl3) d 164.7,
160.0, 142.1, 131.3, 129.6, 129.4, 124.9, 124.0, 123.4, 117.9,

4234
M. L. Bode et al. / Bioorg. Med. Chem. 19 (2011) 4227–4237
116.4, 115.7, 112.2, 57.3, 35.3, 26.1, 25.0. HRMS (ESI): m/z
1.44–1.01 (m, 5H); 13C NMR (50 MHz, DMSO-d6 + CD3OD) d
156.8, 141.6, 135.9, 129.8, 129.1, 125.8, 125.1, 123.9, 119.9,
117.9, 116.9, 113.0, 57.7, 34.2, 26.4, 25.5. HRMS (ESI): m/z
308.1767 (M+H)+; calcd for C19H22N3O: 308.1763.
310.1721 (M+H)+; calcd for C19H21FN3: 310.1720.
4.2.18. N-Cyclohexyl-2-(2-(trifluoromethyl)phenyl)imidazo[1,2-
a]pyridin-3-amine (18)
Yield 21%, as an off-white solid; mp 109–111 °C; 1H NMR
4.2.24. 4-(3-(Cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)phe-
nol (24)
(200 MHz, CDCl3)
d 8.28–7.99 (m, 1H), 7.85–7.71 (m, 1H),
7.66–7.40 (m, 4H), 7.13 (td, J = 1.3, 6.7 Hz, 1H), 6.80 (td, J = 1.1,
6.8 Hz, 1H), 3.28–2.83 (m, 1H), 2.66 (m, 1H), 1.90–1.35 (m, 5H),
1.23–0.80 (m, 5H); 13C NMR (50 MHz, CDCl3) d 141.1, 138.2,
135.3, 132.8, 131.2, 128.1, 126.9, 126.1 (q, J = 5.1, 1H), 123.5,
122.8, 121.4, 117.6, 111.5, 56.3, 33.8, 25.6, 24.5. HRMS (ESI): m/z
360.1688 (M+H)+; calcd for C20H21F3N3: 360.1688.
Yield 60%, as a fine white solid; decomp. >240 °C; 1H NMR
(200 MHz, DMSO-d6) d 9.41 (s, 1H), 8.26 (d, J = 6.9 Hz, 1H), 8.03
(d, J = 8.6 Hz, 2H), 7.41 (d, J = 8.9 Hz, 1H), 7.28–7.01 (m, 1H), 6.83
(m + d, J = 7.3 Hz, 3H), 4.60 (d, J = 5.5 Hz, 1H), 3.45–3.38 (m, 1H),
3.00–2.65 (m, 1H), 2.01–1.40 (m, 5H), 1.40–0.85 (m, 5H); 13C
NMR (50 MHz, DMSO-d6) d 154.7, 138.5, 133.8, 126.1, 123.8,
122.4, 121.3, 121.2, 114.4, 113.2, 109.0, 54.5, 31.8, 23.8, 22.8.
HRMS (ESI): m/z 308.1754 (M+H)+; calcd for C19H22N3O: 308.1763.
4.2.19. N-Cyclohexyl-2-(4-(trifluoromethyl)phenyl)imidazo[1,2-
a]pyridin-3-amine (19)
Yield 26%, as a beige solid; mp 138–140 °C; 1H NMR (200 MHz,
CDCl3) d 8.22 (d, J = 8.2 Hz, 2H), 8.08 (dt, J = 1.0, 6.0 Hz, 1H), 7.68 (d,
J = 8.3 Hz, 2H), 7.60–7.45 (m, 1H), 7.15 (ddd, J = 1.2, 6.7, 9.0 Hz, 1H),
6.80 (td, J = 1.0, 6.7 Hz, 1H), 3.29–2.67 (m, 2H), 1.96–1.42 (m, 5H),
1.42–0.91 (m, 5H); 13C NMR (50 MHz, CDCl3) d 141.8, 138.0, 135.1,
128.5, 126.9, 125.6, 125.3 (dd, J = 3.8, 7.6 Hz), 124.5, 124.3, 122.6,
117.6, 111.8, 56.9, 34.2, 25.7, 24.8; HRMS (ESI): m/z 360.1683
(M+H)+; calcd for C20H21N3F3: 360.1688.
4.2.25. N-Cyclohexyl-2-(4-methoxyphenyl)imidazo[1,2-a]pyri-
din-3-amine (25)
Yield 27%, as a yellow solid; mp 149–151 °C; 1H NMR (200 MHz,
CDCl3) d 8.11 (d, J = 6.8 Hz, 2H), 8.02 (d, J = 8.6 Hz, 2H), 7.16-–7.08
(m, 1H), 7.02–6.98 (d, J = 8.6 Hz, 2H), 6.80–6.73 (m, 1H), 3.87 (s,
3H), 3.07 (br s, 1H), 3.00–2.91 (m, 1H), 1.80–1.59 (br m, 10H);
13C NMR (50 MHz, CDCl3) d 158.3, 141.6, 138.3, 137.1, 128.6,
127.3, 124.3, 123.9, 118.5, 117.3, 116.2, 57.1, 55.5, 34.4, 26.0,
25.1. HRMS (ESI): m/z 322.1888 (M+H)+; calcd for C20H24N3O:
322.1919.
4.2.20. 3-(3-(Cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)ben-
zonitrile (20)
Yield 76%, as an off-white solid; mp 139–141 °C; 1H NMR
(400 MHz, CDCl3) d 8.48 (t, J = 1.6 Hz, 1H), 8.37 (dt, J = 1.5, 7.6 Hz,
1H), 8.14–7.94 (m, 1H), 7.62–7.45 (m, 3H), 7.16 (ddd, J = 1.1, 6.7,
9.0 Hz, 1H), 6.81 (td, J = 1.0, 6.8 Hz, 1H), 3.04 (d, J = 4.7 Hz, 1H),
2.94 (ddd, J = 4.4, 10.0, 14.3 Hz, 1H), 1.88–1.75 (m, 2H), 1.75–1.63
(m, 2H), 1.63–1.49 (m, 1H), 1.42–0.92 (m, 5H); 13C NMR (101 MHz,
CDCl3) d 141.9, 135.9, 134.6, 131.0, 130.4, 130.3, 129.1, 125.3,
124.5, 122.6, 119.0, 117.6, 112.5, 112.0, 56.9, 34.2, 25.6, 24.8. HRMS
(ESI): m/z 317.1775 (M+H)+; calcd for C20H21N4: 317.1766.
4.2.26. N-Cyclohexyl-2-(4-(dimethylamino)phenyl)imidazo[1,2-
a]pyridin-3-amine (26)
Yield 45%, as golden brown needles; mp 166–168 °C; 1H NMR
(200 MHz, CDCl3) d 8.14–8.04 (m, 1H), 7.94 (d, J = 8.7 Hz, 2H),
7.54–7.47 (m, 1H), 7.07 (ddd, J = 1.3, 6.6, 8.9 Hz, 1H), 6.81 (d,
J = 8.9 Hz, 2H), 6.73 (td, J = 0.9, 6.9 Hz, 1H), 3.01 (s, 6H), 1.93–
1.43 (m, 5H), 1.43–0.93 (m, 5H); 13C NMR (50 MHz, CDCl3) d
149.7, 141.4, 137.3, 131.8, 127.8, 123.1, 122.8, 122.4, 116.9,
112.3, 111.0, 56.8, 40.4, 34.2, 25.8, 24.8; HRMS (ESI): m/z
335.2220 (M+H)+; calcd for C21H27N4: 335.2236.
4.2.21. 4-(3-(Cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)ben-
zonitrile (21)
4.2.27. N-Cyclohexyl-2-(2,4-dichlorophenyl)imidazo[1,2-a]pyri-
din-3-amine (27)
Yield 62%, as a yellow solid; mp 169–171 °C; 1H NMR (400 MHz,
CDCl3) d 8.24 (dd, J = 1.7, 8.5 Hz, 2H), 8.05(dt, J = 1.0, 6.9 Hz, 1H), 7.69
(dd, J = 1.7, 8.5 Hz, 2H), 7.53 (dt, J = 1.0, 9.1 Hz, 1H), 7.17 (ddd, J = 1.2,
6.7, 9.1 Hz, 1H), 6.81 (td, J = 1.0, 6.8 Hz, 1H), 3.06 (d, J = 4.9 Hz, 1H),
2.95 (ddd, J = 4.6, 10.0, 14.4 Hz, 1H), 1.87–1.76 (m, 2H), 1.76–1.64
(m, 2H), 1.64–1.51 (m, 1H), 1.33–1.03 (m, 5H); 13C NMR (101 MHz,
CDCl3) d 141.9, 139.2, 134.7, 132.2, 127.2, 126.1, 124.6, 122.6,
119.2, 117.8, 112.0, 110.2, 57.0, 34.2, 25.6, 24.8. HRMS (ESI): m/z
317.1765 (M+H)+; calcd for C20H21N4: 317.1766.
Yield 41%, as a viscous brown oil; 1H NMR (400 MHz, CDCl3) d
8.14 (ddd, J = 0.9, 1.6, 6.9 Hz, 1H), 7.64–7.60 (m, 1H), 7.56–7.52
(m, 1H), 7.51–7.48 (m, 1H), 7.38–7.33 (m, 1H), 7.19–7.12 (m,
1H), 6.81 (tt, J = 1.8, 9.2 Hz, 1H), 3.19 (d, J = 6.0 Hz, 1H), 2.67 (dd,
J = 3.8, 9.0 Hz, 1H), 1.65 (t, J = 11.2 Hz, 2H), 1.59 (dd, J = 4.6,
8.9 Hz, 2H), 1.17–0.92 (m, 6H); 13C NMR (101 MHz, CDCl3) d
141.8, 134.3, 134.1, 133.3, 133.1, 132.8, 129.3, 127.3, 126.4,
123.9, 122.9, 117.6, 111.7, 56.4, 33.9, 25.6, 24.6. HRMS (ESI): m/z
360.1025 (M+H)+; calcd for C19H20Cl2N3: 360.1034.
4.2.22. N-Cyclohexyl-2-(4-nitrophenyl)imidazo[1,2-a]pyridin-3-
amine (22)
4.2.28. N-Cyclohexyl-2-(2,5-dichlorophenyl)imidazo[1,2-a]pyri-
din-3-amine (28)
Yield 28%, as a rust-coloured solid; mp 203–205 °C; 1H NMR
(200 MHz, CDCl3) d 8.49–8.21 (m, 4H), 8.07 (dt, J = 1.0, 6.9 Hz,
1H), 7.55 (d, J = 8.3 Hz, 1H), 7.18 (ddd, J = 1.3, 6.7, 9.1 Hz, 1H),
6.83 (td, J = 1.0, 6.8 Hz, 1H), 3.28–2.79 (m, 2H), 2.04–1.47 (m,
5H), 1.47–0.97 (m, 5H); 13C NMR (50 MHz, CDCl3) d 146.6, 142.1,
141.1, 134.4, 127.3, 126.5, 124.8, 123.8, 122.7, 117.9, 112.2, 57.1,
34.4, 25.7, 24.9. HRMS (ESI): m/z 337.1670 (M+H)+; calcd for
Yield 15%, as a yellow solid; mp 110–112 °C; 1H NMR (400 MHz,
CDCl3) d 8.14 (dd, J = 0.8, 6.8 Hz, 1H), 7.68 (d, J = 2.4 Hz, 1H), 7.50
(dd, J = 1.2, 9.2 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.31–7.24 (m, 1H),
7.22–7.06 (m, 1H), 6.81 (td, J = 1.2 Hz, 6.8, 1H), 3.24 (br s,
J = 7.6 Hz, 1H), 2.70–2.58 (m, 1H), 1.79–1.42 (m, 5H), 1.24–0.89
(m, 5H); 13C NMR (100 MHz, CDCl3) d 141.6, 135.4, 133.6, 132.7,
132.1, 130.5, 129.0, 128.5, 126.5, 124.1, 122.9, 117.3, 111.8, 56.3,
33.8, 25.5, 24.5; HRMS (ESI): m/z 360.1037 (M+H)+; calcd for
C19H21N4O2: 337.1665.
4.2.23. 2-(3-(Cyclohexylamino)imidazo[1,2-a]pyridin-2-yl)phe-
nol (23)
C19H20N3Cl2: 360.1034.
Yield 34%, as a pale yellow solid; mp 149–151 °C; 1H NMR
(200 MHz, DMSO-d6 + CD3OD) d 8.36 (d, J = 6.8 Hz, 1H), 8.17 (dd,
J = 1.7, 8.1 Hz, 1H), 7.54 (d, J = 9.0 Hz, 1H), 7.40–7.15 (m, 2H),
7.10–6.85 (m, 3H), 3.05–2.81 (m, 1H), 1.99–1.44 (m, 5H),
4.2.29. N-Cyclohexyl-2-(2,6-dichlorophenyl)imidazo[1,2-a]pyri-
din-3-amine (29)
Yield 32%, as a yellow solid; mp 55–57 °C; 1H NMR (400 MHz,
CDCl3) d 8.14 (dt, J = 1.2, 6.8 Hz, 1H), 7.54 (dt, J = 1.2, 9.2 Hz, 1H),

M. L. Bode et al. / Bioorg. Med. Chem. 19 (2011) 4227–4237
4235
7.41 (d, J = 8.4 Hz, 1H), 7.26 (dd, J = 7.2, 8.4 Hz, 1H), 7.11 (ddd,
J = 1.2, 6.8, 9.2 Hz, 1H), 6.78 (td, J = 1.2, 6.8 Hz, 1H), 2.94 (d,
J = 6.4 Hz, 1H), 2.82–2.68 (m, 1H), 1.81–1.68 (m, 2H), 1.62–1.55
(m, 2H), 1.55–1.40 (m, 1H), 1.21–0.95 (m, 5H); 13C NMR
(101 MHz, CDCl3) d 141.2, 136.0, 132.7, 132.6129.7, 127.7, 126.5,
123.3, 122.6, 117.5, 111.3, 56.2, 33.5, 25.3, 24.3. HRMS (ESI): m/z
360.1042 (M+H)+; calcd for C19H20Cl2N3: 360.1034.
4.2.35. 2-(2-Chlorophenyl)-N-cyclohexyl-6-methylimidazo[1,2-
a]pyridin-3-amine (35)
Yield 81%, as a yellow solid; mp 107–109 °C; 1H NMR (200 MHz,
CDCl3) d 7.91 (s, 1H), 7.74–7.54 (m, 1H), 7.52–7.40 (m, 2H),
7.40–7.18 (m, 2H), 6.98 (dd, J = 1.7, 9.2 Hz, 1H), 3.40–3.07 (m,
1H), 2.82–2.51 (m, 1H), 2.35 (s, 3H), 1.91–1.31 (m, 5H), 1.23–0.80
(m, 5H); 13C NMR (50 MHz, CDCl3) d 140.7, 135.0, 134.2, 132.5,
129.3, 128.9, 126.7, 125.9, 121.0, 120.3, 116.8, 56.3, 33.8, 25.6,
24.6, 18.4. HRMS (ESI): m/z 340.1582 (M+H)+; calcd for C20H23ClN3:
340.1581.
4.2.30. N-Cyclohexyl-2-(2,6-difluorophenyl)imidazo[1,2-a]pyri-
din-3-amine (30)
Yield 13%, as a beige solid; mp 87–89 °C; 1H NMR (400 MHz,
CDCl3) d 8.13 (dt, J = 1.2, 6.9 Hz, 1H), 7.57 (dt, J = 1.1, 9.1 Hz, 1H),
7.34 (tt, J = 6.4, 8.3 Hz, 1H), 7.14 (ddd, J = 1.3, 6.6, 9.1 Hz, 1H),
7.03 (m, 2H), 6.80 (td, J = 1.1, 6.8 Hz, 1H), 3.12 (br d, J = 7.6 Hz,
1H), 2.79–2.60 (m, 1H), 1.84–1.66 (m, 2H), 1.66–1.54 (m, 2H),
1.54–1.42 (m, 1H), 1.21–0.92 (m, 5H); 13C NMR (101 MHz, CDCl3)
d 161.9 (d, J = 30.4 Hz), 159.4 (d, J = 27.6 Hz), 142.0, 129.8, 129.7,
127.8, 124.0, 122.8, 117.8, 111.8 (d, J = 15.2 Hz), 111.6 (d,
J = 27.6 Hz), 56.5, 34.0, 25.6, 24.7. HRMS (ESI): m/z 328.1630
(M+H)+; calcd for C19H20F2N3: 328.1625.
4.2.36. 2-(2-Chlorophenyl)-N-cyclohexyl-5-methylimidazo[1,2-
a]pyridin-3-amine (36)
Yield 58%, as a yellow solid; mp 136–138 °C; 1H NMR (200 MHz,
CDCl3) d 7.71–7.68 (m, 1H), 7.52–7.26 (m, 4H), 7.12–7.08 (m, 1H),
6.49 (d, J = 6.6 Hz, 1H), 3.25–3.18 (m, 1H), 3.03 (s, 3H), 2.71–2.51
(m, 1H), 1.80–1.45 (m, 5H), 1.26–0.81 (m, 5H); 13C NMR
(50 MHz, CDCl3) d 143.2, 137.3, 136.6, 134.6, 132.7, 132.5, 129.2,
128.9, 128.1, 126.7, 123.8, 115.7, 113.2, 58.7, 33.4, 25.7, 25.0,
19.8. HRMS (ESI): m/z 340.1581 (M+H)+; calcd for C20H23ClN3:
340.1581.
4.2.31. N-Cyclohexyl-2-(2,4,5-trifluorophenyl)imidazo[1,2-a]-
pyridin-3-amine (31)
4.2.37. 2-(2-Chlorophenyl)-3-(cyclohexylamino)imidazo[1,2-
a]pyridine-6-carbonitrile (37)
Yield 17%, as a viscous brown oil; 1H NMR (400 MHz, CDCl3) d
8.14 (dt, J = 1.1, 6.9 Hz, 1H), 7.57 (dt, J = 1.1, 9.1 Hz, 1H), 7.35 (tt,
J = 6.4, 8.4 Hz, 1H), 7.15 (ddd, J = 1.3, 6.6, 9.1 Hz, 1H), 7.18–6.97
(m, 1H), 6.81 (td, J = 1.1, 6.8 Hz, 1H), 3.11 (br d, J = 6.4 Hz, 1H),
2.79–2.61 (m, 1H), 1.82–1.63 (m, 2H), 1.69–1.56 (m, 2H), 1.56–
1.38 (m, 1H), 1.07 (m, 5H); 13C NMR (101 MHz, CDCl3) d 161.9 (d,
J = 30.8 Hz), 159.4 (d, J = 30.4 Hz), 129.8, 129.6, 129.5, 142.0,
129.7, 127.7, 123.8, 122.7, 117.7 (d, J = 24.4 Hz), 111.5 (d,
J = 30.4 Hz), 56.5, 34.0, 25.6, 24.7. HRMS (ESI): m/z 346.1534
(M+H)+; calcd for C19H19F3N3: 346.1531.
Yield 49%, as a yellow solid; mp 133–135 °C; 1H NMR (400 MHz,
CDCl3) d 8.65–8.55 (m, 1H), 7.68–7.63 (m, 1H), 7.61 (ddd, J = 0.9,
3.4, 9.3 Hz, 1H), 7.53–7.46 (m, 1H), 7.43–7.32 (m, 2H), 7.27–7.19
(m, 1H), 3.53–3.13 (m, 1H), 2.86–2.55 (m, 1H), 1.60 (m, 5H),
1.20–0.95 (m, 5H); 13C NMR (101 MHz, CDCl3) d 140.5, 137.5,
132.8, 132.6, 132.3, 129.8, 129.6, 129.4, 127.4, 127.1, 123.2,
118.5, 117.1, 97.8, 56.6, 33.8, 25.4, 24.5. HRMS (ESI): m/z
351.1361 (M+H)+; calcd for C20H20ClN4: 351.1376.
4.2.38. 2-(2-Chlorophenyl)-3-(cyclohexylamino)imidazo[1,2-
a]pyridine-5-carbonitrile (38)
4.2.38.1. 2-Amino-6-cyanopyridine. A mixture of 2-amino-6-
bromopyridine (0.51 g, 29.6 mmol), cupric cyanide monohydrate
(1.76 g, 89.3 mmol) and dry DMF (30 ml) was boiled at 100 °C for
18 h. The brown gum that resulted was diluted with 50 ml 25%
ammonia solution and stirred until all the copper salts had dis-
solved. Extraction with ethyl acetate and concentration, followed
by column chromatography (elution 30–50% ethyl acetate:hex-
ane), afforded a brown oil (17.1 mg, 5%). 1H NMR (400 MHz, CDCl3)
d 7.50 (dd, J = 7.3, 8.4 Hz, 1H), 7.04 (dd, J = 0.7, 7.3 Hz, 1H),
6.73–6.66 (m, 1H), 4.92 (br s, 2H). This compound was used imme-
diately in the three component reaction.
4.2.32. N-Cyclohexyl-2-(2,3,6-trichlorophenyl)imidazo[1,2-a]-
pyridin-3-amine (32)
Yield 18%, as a brown oil; 1H NMR (200 MHz, CDCl3) d 8.23–8.07
(m, 1H), 7.62–7.50 (m, 1H), 7.47 (d, J = 8.7 Hz, 1H), 7.36 (d,
J = 8.7 Hz, 1H), 7.18 (td, J = 1.3, 6.7 Hz, 1H), 6.82 (td, J = 0.9,
6.7 Hz, 1H), 2.94–2.62 (m, 2H), 1.85–1.35 (m, 5H), 1.35–0.71 (m,
5H); 13C NMR (50 MHz, CDCl3) d 169.8, 141.6, 134.8, 134.5,
133.0, 131.9, 130.5, 128.3, 126.6, 123.8, 122.9, 117.8, 111.7, 56.6,
33.8, 25.6, 24.6. HRMS (ESI): m/z 394.0645 (M+H)+; calcd for
C19H19Cl3N3: 394.0645.
4.2.33. 2-(2-Chlorophenyl)-N-cyclohexyl-8-methylimidazo[1,2-
a]pyridin-3-amine (33)
4.2.38.2. 2-(2-Chlorophenyl)-3-(cyclohexylamino)imidazo[1,2-
a]pyridine-5-carbonitrile. Yield 72%, as a thick yellow oil; 1H
NMR (400 MHz, CDCl3) d 7.80 (dd, J = 0.9, 9.0 Hz, 1H), 7.62 (dt,
J = 3.1, 5.4 Hz, 1H), 7.54–7.47 (m, 1H), 7.45–7.34 (m, 3H), 7.17
(dd, J = 7.1, 8.9 Hz, 1H), 3.33 (d, J = 7.1 Hz, 1H), 2.79 (d, J = 3.3 Hz,
1H), 1.76–1.72 (m, 2H), 1.65–1.62 (m, 2H), 1.48–1.41 (m, 1H),
1.28–0.95 (m, 5H); 13C NMR (101 MHz, CDCl3) d 141.1, 138.4,
133.1, 132.4, 129.8, 129.7, 128.1, 127.1, 124.3, 124.3, 122.9,
121.9, 114.0, 108.0, 57.2, 32.7, 25.6, 24.5. HRMS (ESI): m/z
351.1351 (M+H)+; calcd for C20H20ClN4: 351.1376.
Yield 83%, as a brown oil; 1H NMR (200 MHz, CDCl3) d 7.92 (s,
1H), 7.73–7.67 (m, 1H), 7.58–7.22 (m, 4H), 7.03–6.92 (m, 1H),
3.37–3.19 (m, 1H), 2.81–2.62 (m, 1H), 2.38 (s, 3H), 1.85–1.42 (m,
5H), 1.22–1.08 (m, 5H); 13C NMR (50 MHz, CDCl3) d 146.1, 142.8,
141.3, 139.5, 135.9, 133.3, 130.2, 129.6, 127.2, 122.7, 121.3,
118.6, 57.0, 34.2, 26.5, 25.9, 19.5. HRMS (ESI): m/z 340.1581
(M+H)+; calcd for C20H23ClN3: 340.1581.
4.2.34. 2-(2-Chlorophenyl)-N-cyclohexyl-7-methylimidazo[1,2-
a]pyridin-3-amine (34)
4.2.39. 2-(2-Chlorophenyl)-N-cyclohexyl-6-fluoroimidazo[1,2-
a]pyridin-3-amine (39)
Yield 48%, as a viscous brown oil; 1H NMR (200 MHz, CDCl3) d
8.11 (d, J = 6.8 Hz, 1H), 7.72–7.68 (m, 1H), 7.53–7.26 (m, 4H),
6.72 (dd, J = 1.2, 6.8 Hz, 1H), 3.34–3.17 (m, 1H), 2.81–2.58 (m,
1H), 2.45 (s, 3H), 1.80–1.42 (m, 5H), 1.23–0.97 (m, 5H); 13C NMR
(50 MHz, CDCl3) d 142.4, 135.2, 134.4, 134.7, 133.9, 130.2, 129.3,
127.7, 126.2 123.3, 116.6, 115.3, 57.4, 34.7, 26.4, 25.2, 21.8. HRMS
(ESI): m/z 340.1581 (M+H)+; calcd for C20H23ClN3: 340.1581.
Yield 83%, as a dark brown oil; 1H NMR (400 MHz, CDCl3) d 8.13
(d, J = 1.8 Hz, 1H), 7.71–7.67 (m, 1H), 7.48–7.44 (m, 2H), 7.38–7.33
(m, 2H), 7.24 (d, J = 1.8 Hz, 1H), 3.33 (d, J = 4.0 Hz, 1H), 2.66 (s, 1H),
1.77–1.60 (m, 3H), 1.51 (m, 2H), 1.12–0.97 (m, 5H); 13C NMR
(101 MHz, CDCl3) d 154.4, 152.1, 139.2, 136.7, 133.6, 132.4,
129.5, 129.3, 127.6, 127.6, 127.0, 118.1, 118.0, 116.0, 115.7,

4236
M. L. Bode et al. / Bioorg. Med. Chem. 19 (2011) 4227–4237
109.5, 109.1, 56.3, 33.8, 25.6, 24.6. HRMS (ESI): m/z 344.1299
3.60–3.39 (m, 1H), 2.86–2.67 (m, 1H), 1.74 (dd, J = 12.6, 18.3 Hz,
2H), 1.60–1.36 (m, 3H), 1.11–0.86 (m, 5H); 13C NMR (101 MHz,
CDCl3) d 140.7, 134.8, 132.7, 131.3, 129.6, 129.4, 129.3, 129.1,
128.4, 127.7, 127.4, 127.0, 125.7, 124.4, 124.3, 117.5, 117.1, 56.6,
33.1, 25.6, 24.6. HRMS (ESI): m/z 376.1576 (M+H)+; calcd for
(M+H)+; calcd for C19H20ClFN3: 344.1330.
4.2.40. 6-Chloro-2-(2-chlorophenyl)-N-cyclohexylimidazo[1,2-
a]pyridin-3-amine (40)
Yield 72%, as an off-white solid; mp 130–132 °C; 1H NMR
C23H23Cl N3: 376.1581.
(200 MHz, CDCl3)
d 8.25–8.11 (m, 1H), 7.75–7.56 (m, 1H),
7.55–7.39 (m, 2H), 7.39–7.25 (m, 2H), 7.10 (ddd, J = 1.2, 1.9,
9.5 Hz, 1H), 3.27 (d, J = 6.8 Hz, 1H), 2.80–2.46 (m, 1H), 1.82–1.34
(m, 5H), 1.23–0.76 (m, 5H); 13C NMR (50 MHz, CDCl3) d 139.8,
136.4, 133.6, 132.6, 132.4, 129.4, 129.3, 126.9, 126.7, 124.9,
120.7, 120.1, 118.0, 56.4, 33.8, 25.6, 24.5. HRMS (ESI): m/z
360.1019 (M+H)+; calcd for C19H20Cl2N3: 360.1034.
4.2.46. 2-(2-Chlorophenyl)-N-cyclohexyl-5-
methoxyimidazo[1,2-a]pyridin-3-amine (44)
A
solution of 5-bromo-2-(2-chlorophenyl)-N-cyclohexylimi-
dazo[1,2-a]pyridin-3-amine 43 (81.0 mg, 0.200 mmol) in sodium
methoxide (2 equiv, 0.400 mmol) was refluxed for 4 h. After being
allowed to cool to room temperature the mixture was quenched
with water and extracted with ethyl acetate. Organic extracts were
dried over magnesium sulphate and filtered, before excess solvent
was removed on a rotary evaporator. Purification by column
chromatography using ethyl acetate: hexane as eluant gave 2-(2-
chlorophenyl)-N-cyclohexyl-5-methoxyimidazo[1,2-a]pyridin-3-
amine (44). Yield 98%, as a brown solid; mp 79–81 °C; 1H NMR
(200 MHz, CDCl3) d 7.64–7.57 (m, 1H), 7.51–7.39 (m, 1H), 7.38–
7.19 (m, 2H), 7.21–7.11 (m, 1H), 7.04–6.88 (m, 1H), 5.92 (d,
J = 7.3 Hz, 1H), 4.08 (s, 3H), 2.89–2.64 (m, 1H), 1.79–1.41 (m, 6H),
1.21–0.75 (m, 4H); 13C NMR (50 MHz, CDCl3) d 152.0, 143.7,
134.5, 134.2, 132.6, 129.4, 129.1, 126.3, 124.2, 111.7, 89.1, 57.9,
57.1, 33.8, 26.1, 24.6. HRMS (ESI): m/z 356.1502 (M+H)+; calcd
for C20H22ClN3O: 356.1454.
4.2.41. 5-Chloro-2-(2-chlorophenyl)-N-cyclohexylimidazo[1,2-
a]pyridin-3-amine (41)
Yield 28%, as an orange solid; mp 91–93 °C; 1H NMR (400 MHz,
CDCl3) d 7.63–7.56 (m, 1H), 7.49 (dddd, J = 1.3, 1.8, 4.5, 5.0 Hz, 2H),
7.39–7.30 (m, 2H), 7.08–6.99 (m, 1H), 6.80–6.73 (m, 1H), 3.31 (d,
J = 6.8 Hz, 1H), 2.81 (s, 1H), 1.67 (d, J = 12.5 Hz, 2H), 1.56–1.40
(m, 3H); 1.12–0.96 (m, 3H), 0.95–0.80 (m, 2H); 13C NMR
(101 MHz, CDCl3) d 143.6, 137.5, 133.7, 133.2, 132.6, 129.5,
129.3, 128.2, 126.7, 126.0, 123.6, 116.8, 114.1, 58.6, 32.8, 25.7,
24.5. HRMS (ESI): m/z 360.1014 (M+H)+; calcd for C19H20Cl2N3:
360.1034.
4.2.42. 6-Bromo-2-(2-chlorophenyl)-N-cyclohexylimidazo[1,2-
a]pyridin-3-amine (42)
4.3. Biological screening
Yield 66%, as a cream solid; mp 144–146 °C; 1H NMR (400 MHz,
CDCl3) d 8.33–8.21 (m, 1H), 7.70–7.56 (m, 1H), 7.52–7.45 (m, 1H),
7.45–7.40 (m, 1H), 7.40–7.29 (m, 2H), 7.20 (dd, J = 1.9, 9.5 Hz, 1H),
3.25 (d, J = 6.9 Hz, 1H), 2.66 (dd, J = 3.4, 6.5 Hz, 1H), 1.75–1.62 (m,
2H), 1.61–1.52 (m, 2H), 1.52–1.39 (m, 1H), 1.18–0.86 (m, 5H); 13C
NMR (101 MHz, CDCl3) d 140.0, 136.2, 133.6, 132.6, 132.5, 129.5,
129.4, 127.1, 127.0, 126.6, 123.0, 118.3, 106.7, 56.4, 33.8, 25.6,
24.5. HRMS (ESI): m/z 404.0512 (M+H)+; calcd for C19H20BrClN3:
404.0529.
4.3.1. RT enzyme assay
The inhibitory activity of compounds was assessed using an ELI-
SA-based colorimetric HIV-1 reverse transcriptase assay (Cat. No.
11468120910, Roche Diagnostics GmbH, Germany) carried out
according to the manufacturer’s instructions. Percentage residual
RT enzyme activity after incubation with 50 lM test compound
was determined relative to untreated controls. The kit reaction in-
volves the incorporation of biotin and DIG-labelled nucleotides
into cDNA strands polymerized on an RNA template by the action
of HIV-1 reverse transcriptase (RT). The cDNA products are bound
to streptavidin-coated 96-well plate inserts, and their associated
DIG-moieties detected by incubation with anti-DIG antibodies con-
jugated to horseradish peroxidase (HRP). The amount of bound
antibody was measured by incubation with a colorimetric HRP
substrate, followed by absorbance reading at 405 nm using a Tecan
Infinite F500 multiwell spectrophotometer. In the screening as-
says, compounds were prepared as 10 mM stocks in DMSO and
incubated with HIV-1 reverse transcriptase and substrate at a final
4.2.43. 5-Bromo-2-(2-chlorophenyl)-N-cyclohexylimidazo[1,2-
a]pyridin-3-amine (43)
Yield 74%, as a viscous brown oil; 1H NMR (400 MHz, CDCl3) d
7.60–7.57 (m, 1H), 7.54–7.52 (m, 1H), 7.50–7.48 (m, 1H),
7.37–7.29 (m, 2H), 7.03–6.94 (m, 2H), 3.28 (d, J = 6.2 Hz, 1H),
2.97–2.62 (m, 1H), 1.66 (d, J = 12.4 Hz, 2H), 1.59–1.35 (m, 3H),
1.16–0.97 (m, 3H), 0.97–0.75 (m, 2H); 13C NMR (101 MHz, CDCl3)
d 143.7, 138.2, 133.9, 133.3, 132.6, 129.5, 129.2, 128.5, 126.6,
123.8, 118.6, 117.3, 112.2, 58.3, 32.7, 25.7, 24.5; HRMS (ESI): m/z
404.0516 (M+H)+; calcd for C19H20BrClN3: 404.0529.
concentration of 50 lM in duplicate wells for 1 h, before proceed-
ing with the rest of the kit protocol. After subtraction of back-
ground Abs405 values (wells without RT enzyme) from all well
readings, residual enzyme activity in compound wells was calcu-
lated as a percentage of controls without inhibitor. In-house vali-
dation experiments suggest that the assay yields highly
reproducible percentage enzyme activity values, both intra-exper-
imentally (average coefficient of variation for replicates = 7%) and
inter-experimentally (average coefficient of variation for enzyme
activity values = 14%). Nevirapine was included as an internal
standard in all assays.
4.2.44. 6-Bromo-2-(2-chlorophenyl)-N-cyclohexyl-5-methylim-
idazo[1,2-a]pyridin-3-amine (45)
Yield 70%, as a pale yellow solid; mp 115–117 °C; 1H NMR
(200 MHz, CDCl3)
d 7.71–7.55 (m, 1H), 7.55–7.42 (m, 1H),
7.42–7.29 (m, 2H), 7.25 (d, J = 4.3 Hz, 2H), 3.20–3.16 (m, 1H),
3.18 (s, 3H), 2.55 (s, 1H), 1.75–1.32 (m, 5H), 1.16–0.65 (m, 5H);
13C NMR (50 MHz, CDCl3) d 142.1, 138.2, 137.9, 135.1, 134.1,
132.7, 132.5, 129.4, 129.2, 128.3, 126.9, 116.3, 109.9, 58.7, 33.0,
25.7, 24.7, 17.9. HRMS (ESI): m/z 418.0676 (M+H)+; calcd for
C20H22BrClN3: 418.0686.
To determine RT IC50 values for test compounds, the assay was
carried out with serial fourfold dilutions of the compounds at 8 dif-
4.2.45. 2-(2-Chlorophenyl)-N-cyclohexylimidazo[1,2-a]quino-
lin-1-amine (46)
ferent concentrations, with 400 lM as the highest final concentra-
tion. Duplicate wells were used for each concentration point in the
dilution series. Validation experiments suggested that the IC50
values obtained for individual compounds are highly reproducible
inter-experimentally. The average 95% confidence interval for IC50
values determined on three separate occasions was 20.2%.
Yield 30%, as a viscous yellow oil; 1H NMR (400 MHz, CDCl3) d
9.44 (dt, J = 5.7, 11.5 Hz, 1H), 7.74 (dt, J = 4.5, 9.0 Hz, 1H),
7.71–7.65 (m, 1H), 7.62–7.55 (m, 1H), 7.52–7.47 (m, 1H),
7.46–7.40 (m, 3H), 7.39–7.36 (m, 1H), 7.36–7.33 (m, 1H),

M. L. Bode et al. / Bioorg. Med. Chem. 19 (2011) 4227–4237
4237
4.3.2. MAGI whole cell assays
each case visually inspected for chemical and physical inconsisten-
cies. The two check compounds were overlaid on their crystal
structure poses, and rms deviations for each determined. TMC-
278 was docked within 0.62 Å rmsd of the crystal structure pose,
while TMC-125 in the 3MEG protein had a similar pose (rmsd of
1.40 Å) to its crystallised form (pdb code 3MED), with the one phe-
nyl ring turned ꢀ50° relative to the co-crystallised ligand in 3MED.
Whole cell HIV assays were carried out by the Southern Research
Institute (SRI, Frederick, MD, USA) according to their in-house proto-
cols. Briefly, MAGI-R5 cells transgenically expressing CD4 and CCR5
andcontaininganLTR-b-galactosidasereporterconstructwereincu-
bated with test compounds (triplicate samples) for 15–30 min prior
to infection with HIV-1 IIIB. Compounds were added to the cells as 6
log serial dilutions with a highest concentration of 200 lM. After a
48 h incubation, levels of viral infection were quantified using a
chemiluminescence b-galactosidase enzyme assay (Perkin Elmer
AppliedBiosystems)andtheIC50 (concentrationinhibitingvirusrep-
lication by 50%) calculated from dose–response curves. In addition,
compound toxicity was assessed in replicate MAGI-R5 plates incu-
bated with test compounds for 48 h using the tetrazolium-based
dye MTS cell viability assay (CellTiter 96 Reagent, Promega, WI).
The latter was used to derive the CC50 (concentration decreasing cell
viability by 50%) and SI (selectivity index: CC50/IC50).
Acknowledgements
The authors would like to thank Mr. Sabata Maduna for the
preparation of certain starting materials and Dr. Phiyani Lebea
for his preliminary work on the RT assay.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.05.062.
4.3.3. Permeability assay
The assay employed BD Gentest pre-coated 96-well PAMPA
(parallel artificial membrane permeability assay) plates (BD Biosci-
ences, Bedford, Massachusetts, USA). Prior to use, the plates were
warmed to room temperature for 60 minutes. Test compounds
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were diluted to 100
the donor plate. After adding 200
the plates were fitted together and incubated with shaking at 37 °C
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lM in PBS and 300
lL added to the wells of
l
L PBS to the receiver plate wells,
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The crystal structures of both the wild-type (pdb code 3MEE)
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