Synthesis and SAR studies of potent imidazopyridine anticoccidial agents

Article abstract of DOI:10.1016/j.bmcl.2007.04.041

Diaryl imidazo[1,2-a]pyridine derivatives, such as 6a and 7i, have been synthesized and found to be potent inhibitors of parasite PKG activity. The most potent compounds are the 7-isopropylaminomethyl analog 6a and 2-isopropylamino analog 7i. These compounds are also fully active in in vivo assay as anticoccidial agents at 25 ppm in feed.

Full text of DOI:10.1016/j.bmcl.2007.04.041

Bioorganic & Medicinal Chemistry Letters 17 (2007) 3558–3561
Synthesis and SAR studies of potent imidazopyridine
anticoccidial agents
Gui-Bai Liang,a,* Xiaoxia Qian,a Dennis Feng,a Michael Fisher,a Christine M. Brown,b
Anne Gurnett,b Penny Sue Leavitt,b Paul A. Liberator,b Andrew S. Misura,b
Tamas Tamas,b Dennis M. Schmatz,b Matthew Wyvratta and Tesfaye Biftua
aMerck Research Laboratories, Department of Medicinal Chemistry, Merck and Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
bHuman and Animal Infectious Disease Research, Merck and Co., Inc., PO Box 2000, Rahway, NJ 07065, USA
Received 27 March 2007; revised 16 April 2007; accepted 18 April 2007
Available online 24 April 2007
Abstract—Diaryl imidazo[1,2-a]pyridine derivatives, such as 6a and 7i, have been synthesized and found to be potent inhibitors of
parasite PKG activity. The most potent compounds are the 7-isopropylaminomethyl analog 6a and 2-isopropylamino analog 7i.
These compounds are also fully active in in vivo assay as anticoccidial agents at 25 ppm in feed.
Ó 2007 Elsevier Ltd. All rights reserved.
Coccidiosis is the major cause of morbidity and mortal-
ity in the poultry industry. Protozoan parasites of the
genus Eimeria, which invade intestinal epithelial cells,
are the causative agents.1 The standard therapeutic
practice used in commercial poultry operations to com-
bat the disease is to include anticoccidial agents in the
feed prophylactically. Polyether ionophore anticoccidial
agents, discovered over 30 years ago, have been success-
fully used in this manner. However, this strategy has led
to the erosion of efficacy and development of resistance
in the field.2 Accordingly, the need for novel anticocci-
dial chemotherapeutic options is considerable.
Biological evaluation of compounds includes a measure
of the ability to inhibit native Et-PKG enzyme activity
(IC50—compound required to inhibit enzyme activity
by 50%), as well as their in vivo potency.7 In the animal
model, chickens are infected with either Eimeria tenella
(Et) or Eimeria acervulina (Ea) parasites and treated
with a compound (parts per million) mixed in the feed.
Antiparasitic efficacy is measured as a function of para-
site oocyst burden 7 days following the infection. Treat-
ments resulting in reduction of oocyst burden by >80%
are scored a ‘3’, 50–79% reduction is scored a ‘2’, and
those treatments with <50% reduction of oocyst burden
are scored a ‘0’.
Previous work has demonstrated that small molecule
inhibitors of Eimeria tenella cGMP-dependent protein
kinase (Et-PKG) block parasite motility and host cell
invasion in vitro, and prevent parasite infection and
associated pathology in a chicken model of infection.3,4
Analogs in the 2-(4-fluorophenyl)-3-pyrimidin-4-ylimi-
dazo[1,2-a]pyridine structural series are among the most
potent compounds identified to date.5,6 In this report,
we have extended our medicinal chemistry effort by
incorporating various nitrogen containing substituents
at the 7-position of the imidazopyridine ring and the
2-position of the pyrimidine ring.
Synthesis of the previously reported compound 16,8 was
scaled up and has served as an advanced intermediate in
the synthetic process outlined in Scheme 1. Displace-
ment of the sulfone group of 1 with an amine yielded
the 2-aminopyrimidine 2. The hydroxyl group of com-
pound 2 was either oxidized by manganese (IV) oxide
to carboxaldehyde 3, or converted to mesylate 4 under
standard conditions. Carboxaldehyde 3 was then con-
verted to alkylamine derivatives 5 via reductive amina-
tion, whereas mesylate 4 was converted to alkylamine
derivatives 5 by nucleophilic displacement. In most
cases, the overall yields of the final products were com-
parable. In some cases, however, the reductive amina-
tion route works better, and in others, the mesylate
displacement route. Because structural variability was
introduced at the last step, a big diversified collection
Keywords: Et-PKG; Coccidiosis; Imidazopyridine.
*
Corresponding author. E-mail: gui-bai_liang@merck.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.04.041

G.-B. Liang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3558–3561
3559
Table 1. Inhibition of Et-PKG enzyme activity and in vivo anticoc-
cidial activity of diaryl imidazo[1,2-a]pyridine derivatives 6
R
N
SO2
N
NH
a
N
N
N
N
N
N
NH
OH
OH
N
N
1
2
N
N
F
F
R'
N
R
b
c
R
N
R
N
NH
N
NH
6a-m
F
H
N
Compound R–N–R0
Et-PKG Dose Et
IC50 (nM) (ppm) score score
Ea
N
N
N
N
O
OMs
N
H
6a
6b
6c
6d
0.05
0.13
0.11
0.11
25
50
25
25
3
3
0
3
3
3
3
0
3
4
F
F
N
N
R
e
d
NH
N
N
N
N
N
R1
N
OH
R2
N
OH
OH
5
F
6e
0.15
50
3
0
Scheme 1. Reagents and conditions: (a) R–NH2; (b) MnO2; (c) MsCl,
Et3N; (d) R1R2NH, NaBH(OAc)3, HOAc; (e) R1R2NH.
6f
6g
6h
6i
0.27
0.10
0.07
0.57
25
3
3
0
0
0
0
2
0
N
N
O
N
of diaryl imidazo[1,2-a]pyridine derivatives was built
rather efficiently for the SAR study.9
50
Table 1 summarized the SAR of various amino substit-
uents on the 7 position of the imidazopyridine ring
(compounds 6a–m). The (S)-methyl benzylamino group
was chosen as the fixed substitution at the 2 position of
the pyrimidine ring based on results from earlier stud-
ies.6 All but one compound (6m) showed subnanomolar
IC50’s for the inhibition of Et-PKG enzyme activity. In
this group, compounds 6a and 6h are particularly potent
with IC50 values approaching picomolar range. Com-
pounds with small alkylamino substitutions are effica-
cious against both parasites in the chicken model of
infection. Introduction of hydroxyl groups (6d and 6e)
had little effect on in vitro inhibition of Et-PKG enzyme
activity, but reduced in vivo efficacy against E. acervuli-
na. Similar observations were made on compounds with
amino (6g and 6h) or amido (6i–l) functionalities—
in vitro potencies are comparable to simple alkylamino
substituents, but in vivo activities against one or both
parasitic species are compromised. Broad spectrum
activity is a necessary requirement for the development
of new anticoccidial agents, but historically has been a
difficult objective to achieve.2 The data here highlight
an SAR trend that polar substituents, such as a hydroxyl
group, tend to decrease efficacy against E. acervulina,
whereas nonpolar substituents are more likely to impact
efficacy against E. tenella.
12.5
50
N
N
N
O
C
N
N
O
C
N
N
N
6j
0.39
0.26
0.15
5.6
50
25
25
50
3
2
3
0
0
2
0
0
O
C
6k
6l
N
O2
S
N
H
6m
N
N CO2Et
on the 2-position of the pyrimidine ring. Consistent with
our earlier findings,6 aniline (7a) and benzylamino (7b)
substituents at the 2-position of the pyrimidine ring
are preferred in terms of inhibition of Et-PKG activity,
with IC50 values approaching picomalor range. When
these aromatic groups are replaced by alkyl substituents,
in vitro potencies as inhibitors of Et-PKG enzyme activ-
ity decrease somewhat. Nevertheless, these alkyl analogs
are still highly potent Et-PKG inhibitors with most IC50
values in the sub-nanomolar range. Only 7h and 7k have
IC50 values >1 nM. Both of these compounds have an
isopropylmethylamino substituent at the 7-position of
the imidazopyridine ring.
Small alkylamino groups at the 7-position of the imi-
dazopyridine ring are optimal for balanced in vitro
and in vivo activities. Listed in Table 2 are compounds
with other small alkylamino substituents at this posi-
tion, as well as some modifications to the substituent

3560
G.-B. Liang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3558–3561
Table 2. Inhibition of Et-PKG enzyme activity and in vivo anticoccidial activities of diaryl imidazo[1,2-a]pyridine derivatives 7
R
N
N
N
N
R'
7a-k
F
Compound
R,R0
Et-PKG IC50 (nM)
0.06
Dose (ppm)
12.5
Et score
3
Ea score
0
7a
∗
N
∗
N
H
H
N
7b
7c
7d
7e
7f
0.081
0.28
0.29
0.45
0.60
0.31
2.8
25
25
25
25
25
—
—
25
—
—
3
3
N
∗
∗
H
N
3
0
N
∗
∗
H
N
3
0
N
∗
∗
∗
H
N
0
0
N
∗
∗
H
N
0
0
N
∗
∗
∗
∗
∗
∗
N
7g
7h
7i
n/a
n/a
3
n/a
n/a
3
∗
N
H
N
N
H
∗
N
0.20
0.86
1.3
N
H
∗
7j
n/a
n/a
n/a
n/a
N
N
H
∗
7k
N
N
H
∗
n/a, date not available.
While the majority of these compounds are potent inhib-
itors of Et-PKG enzyme activity, many showed little to
no control of parasite oocyst production in the animal
model. In vivo efficacy following oral dosing in the feed
is dependent upon many additional factors, including
oral bioavailability in chicken, and the ability of the
compound to access the intracellular enzyme target in
the parasites. One notable exception in Table 2 is com-
pound 7i. While compound 7i is a less potent Et-PKG
inhibitor than compound 7b (by about 2.5 times), the
two compounds have similar in vivo efficacy in the ani-
mal infection model, and reached total control of oocyst
production at levels of 25 ppm in feed.
neither compounds offer good control of oocyst produc-
tion in Eimeria maxima or Eimeria brunette infected
chickens (data not shown). Broad spectrum activity
against seven commercially important species of chicken
Eimeria is an absolute requirement for any new anticoc-
cidial agent and remains a significant challenge for
medicinal chemistry.2
In summary, this SAR study on diaryl imidazo[1,2-
a]pyridine derivatives demonstrated that simple alkyl-
amino substituents at the 7-position of the imidazopyri-
dine ring with an aromatic group on the 2-aminopyrim-
idine ring (e.g., 6a and 7a) improve the potency of
inhibitory activity against Et-PKG, with IC50 values
approaching picomolar range. Meanwhile improvement
of in vivo anticoccidial activities was observed not only
with a benzyl group on the 2-aminopyrimidine ring (6a
Further evaluation of compounds 6a and 7i against
other Eimeria spp. of parasites has pointed to holes in
the spectrum of antiparasitic activity. For example,

G.-B. Liang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 3558–3561
3561
4. (a) Wiersma, H. I.; Galuska, S. E.; Tomley, F. M.; Sibley,
L. D.; Liberator, P. A.; Donald, R. G. K. Int. J. Parasitol.
2004, 34, 369; (b) Donald, R. G. K.; Zhong, T.; Wiersma,
H.; Nare, B.; Yao, D.; Lee, A.; Aliocco, J.; Liberator, P. A.
Mol. Biochem. Parasitol. 2006, 149, 86.
5. Biftu, T.; Feng, D.; Fisher, M. H.; Liang, G.-B.; Qian, X.;
Scribner, A.; Dennis, R.; Lee, S.; Brown, C.; Gurnett, A.
M.; Leavitt, P. S.; Liberator, P. A.; Mathew, J.; Misura, A.
S.; Smamras, S.; Tamas, T.; Schmatz, D. M.; Wyvratt, M.
J. Bioorg. Med. Chem. Lett. 2006, 16, 2479.
and 7b) but also with an isopropyl group (7i) at this po-
sition. Two of the major Eimeria species (E. tenella and
E. acervulina) are effectively controlled at the level of
25 ppm in feed. Lack of broader spectrum activity
against other Eimeria spp. has prevented further devel-
opment of these compounds.
Acknowledgments
6. Feng, D.; Fisher, M. H.; Liang, G.-B.; Qian, X.; Brown, C.;
Gurnett, A. M.; Leavitt, P. S.; Liberator, P. A.; Mathew, J.;
Misura, A. S.; Smamras, S.; Tamas, T.; Schmatz, D.;
Wyvratt, M. J.; Biftu, T. Bioorg. Med. Chem. Lett. 2006, 16,
5978.
7. Biftu, T.; Feng, D.; Ponpipon, M.; Girotra, N.; Liang,
G.-B.; Qian, X.; Simeone, J.; Chang, L.; Gurnett, A. M.;
Liberator, P. A.; Dulski, P. M.; Leavitt, P. S.; Crumley, T.;
Misura, A. S.; Murphy, T.; Darkin-Rattray, S. J.; Smam-
ras, S.; Tamas, T.; Mathew, J.; Brown, C.; Thompson, D.;
Schmatz, D.; Fisher, M. H.; Wyvratt, M. J. Bioorg. Med.
Chem. Lett. 2005, 15, 3296.
The authors thank Dr. Phil Eskola Dr. Gerard Kieczy-
kowshi, Ms. Amanda Makarewicz, Mr. Bob Frankshun,
and Mr. Glen Reynolds for scale-up of key intermediates,
and Merck Research Laboratory Basic Chemistry Ana-
lytical Support Group for open access service on LC/MS.
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9. All new compounds were characterized by 1H NMR and
MS prior to submission for biological evaluation.