N-Alkyl-4-piperidinyl-2,3-diarylpyrrole derivatives with heterocyclic substitutions as potent and broad spectrum anticoccidial agents

Article abstract of DOI:10.1016/j.bmcl.2008.01.104

Diaryl-(4-piperidinyl)-pyrrole derivatives bearing cyclic amine substituents have been synthesized and evaluated as anticoccidial agents. Improvements in potency of Et-PKG inhibition, such as azetidine derivative 3a, and broad spectrum anticoccidial activ

Full text of DOI:10.1016/j.bmcl.2008.01.104

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2019–2022
N-Alkyl-4-piperidinyl-2,3-diarylpyrrole derivatives
with heterocyclic substitutions as potent
and broad spectrum anticoccidial agents
Gui-Bai Liang,a,* Xiaoxia Qian,a Dennis Feng,a Michael Fisher,a Tami Crumley,b
Sandra J. Darkin-Rattray,b Paula M. Dulski,b Anne Gurnett,b Penny Sue Leavitt,b
Paul A. Liberator,b Andrew S. Misura,b Samantha Samaras,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 11 December 2007; revised 28 January 2008; accepted 29 January 2008
Available online 2 February 2008
Abstract—Diaryl-(4-piperidinyl)-pyrrole derivatives bearing cyclic amine substituents have been synthesized and evaluated as anti-
coccidial agents. Improvements in potency of Et-PKG inhibition, such as azetidine derivative 3a, and broad spectrum anticoccidial
activities in feed, such as morpholine derivative 8c, have been achieved.
Ó 2008 Elsevier Ltd. All rights reserved.
Recently, we have reported the discovery of N-alkyl-4-
piperidinyl-2,3-diarylpyrroles,1 especially those bearing
hydroxyl groups,2 as potent and broad spectrum anti-
coccidial agents through inhibition of a novel cGMP-
dependent protein kinase (PKG).3,4 It has been more
than two decades since the introduction of the last
new commercial anticoccidial agent. Prophylactic uses
of coccidiostats over time by all major poultry opera-
tions have caused the development of resistance to cur-
rent coccidiostats among existing strings of parasites.5
Aside from lacking new biochemical targets to control
the disease, cost-control is a pressing issue in anticocci-
dial research and development, due to the very low cost
and low profit margin of the poultry industry.6,7 It is
critical that for a new anticoccidial agent to be success-
ful commercially, very low efficacious use level, usually
<100 ppm in feed, has to be achieved. Thus, very thor-
ough and careful SAR studies are necessary to discover
highly potent compounds, both in vitro and in vivo.
rating nitrogen containing heterocycles into the lead
compounds.9 These compounds were first evaluated in
an enzyme inhibition assay against PKG from the native
Eimeria, and followed by an anticoccidial assay to eval-
uate their in vivo efficacy in oocyst reduction against two
major subtypes of Protozoa parasites: Eimeria tenella
(E.t.) and Eimeria acervulina (E.a.).10
First, azetidine moiety was introduced to the lead com-
pound by a reaction sequence shown in Scheme 1. The
N
Py
Ph
a, b
R
N
N
H
N
H
NH
O
F
N
2
1
Py
Ph
Following the results of our earlier SAR studies,2,8 we
continued our medicinal chemistry project by incorpo-
R
c
N
N
H
N
3
Keywords: Coccidiosis; PKG; PKG inhibitor.
*
Scheme 1. Reagents: (a) BrCHRCO2Me, Et3N; (b) azetidine, heat; (c)
BH3 THF or LiAlH4.
Corresponding author. Tel.: +1 732 594 3543; fax: +1 732 594
3007; e-mail: gui-bai_liang@merck.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.104

2020
G.-B. Liang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2019–2022
Table 1. Et-PKG inhibition and anticoccidial activities of diaryl-(4-
piperidinyl)-pyrrole derivatives 2 and 3
We have also investigated the derivatives containing
hydroxyl pyrrolidine moieties. These compounds were
synthesized in a similar reaction sequence through
amide formation with hydroxyprolines followed by
reduction (Scheme 2), and their biological evaluation
data are summarized in Table 2. Hydroxylated deriv-
atives were chosen because of our success in earlier
SAR studies.2 Consistent with the azetidine series,
pyrrolidine derivatives are also highly potent PKG
inhibitor with IC50’s less than 1 nM. Also, acylation
of the pyrrolidine nitrogen of 5a,2 (IC50 = 0.2 nM)
Compound
R
Et-PKG
inhibition
a
IC50 (nM)
Anticoccidial
activity at
100 ppm in
feed10
E.t.
E.a.
O
2
1.01
3
3
N
N
resulted
a
10-time decrease in its potency (5d,2
3a1
3b
0.08
0.4
3
3
3
3
3
3
IC50 = 2 nM). Although steric effects cannot be ruled
out, it is another piece of supporting evidence that
a basic nitrogen at this position is favored to boost
potency of these compounds. Apart from the azeti-
dine series, however, there is a new trend that these
pyrrolidine derivatives are not as efficacious against
E.a. as they are against E.t. At 100 ppm in feed, they
are only partially efficacious against E.a. with less
than 80% oocyst reduction.10 This led to a specula-
tion that an optimal range of lipophilicity is neces-
sary for broad spectrum in vivo activities of these
compounds. Further investigation on this issue is
ongoing.
N
N
3c
1.7
HO
N
3d
0.28
3
3
a Values are means of three experiments. The Z score for the assay is
typically 0.9, and the same for data in other tables. For details, see
Ref. 1.
Based on this working hypothesis, we incorporated mor-
pholine moiety into the molecule in an attempt to bal-
ance the lipophilicity and reach broad spectrum
in vivo activity against both E.t. and E.a. These com-
pounds were made following the reaction sequence out-
lined in Scheme 3, and N-protected morpholino
carboxylic acids were used as starting materials. Their
anticoccidial activity evaluation is summarized in
Table 3.
N
Py
a
Ph
N
H
N
H
N
NH
O
F
R
1
N
4
Racemic morpholino acid were used as a cost-control
measure, and our earlier studies have shown that
very little differences in both in vitro and in vivo
activities were observed when enantiomerically pure
analogs were tested.2 Similar to the first two series
Py
Ph
OH
N
R
b
N
H
N
5
OH
Scheme 2. Reagents: (a) N-protected amino acid, i-Pr2NEt, HOBT,
BOP; (b) BH3 THF or LiAlH4.
Table 2. Et-PKG inhibition and anticoccidial activities of diaryl-(4-
piperidinyl)-pyrrole derivatives 5
piperidine nitrogen of compound 11 was alkylated with
bromoacetates. The resulting acetates were directly con-
verted to azetidine amide 2. Reduction of 2 with either
borane or LAH yielded the desired azetidine derivative
3. These azetidine bearing compounds are highly potent
PKG inhibitors, including azetidine amide 2 (Table 1).
Reduction of azetidine amide 2 (IC50 = 1 nM) to azeti-
dine 3a1 (IC50 = 0.08 nM) resulted in a 10-time increase
in potency, making it one of the most potent PKG
inhibitors made on this project. These data suggest the
importance of a basic nitrogen atom at this position.
Furthermore, all these compounds are fully efficacious
in vivo at 100 ppm in feed, but not at lower levels. It
has been observed repeatedly on this project that in-
creases in in vitro potencies do not always result in sim-
ilar increases in in vivo efficacy, probably due to lack of
bioavailability, and/or lack of parasite penetration of
these compounds.2
Compound
R
Et-PKG
inhibition
IC50 (nM)
Anticoccidial
activity
at 100 ppm
in feed10
E.t.
E.a.
HN
5a2
5b
0.16
0.34
0.36
2.2
3
2
OH
N
3
3
3
2
2
2
OH
N
N
5c
OH
OH
Ac
5d2

G.-B. Liang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2019–2022
2021
Table 3. Et-PKG inhibition and anticoccidial activities of diaryl-(4-
piperidinyl)-pyrrole derivatives 6 and 7
N
Py
a
Ph
Compound
R
Et-PKG
inhibition
IC50 (nM)
Anticoccidial
activity
N
H
N
H
at 100 ppm
in feed10
N
NH
O
F
6 X = O
1
X
E.t. E.a.
7 X = N-R
N
R
Py
Ph
O
O
b
N
6a
6b
8a
8b
8c
0.27
4.70
0.17
1.03
0.67
3
2
3
3
3
2
2
2
2
3
NH
N
N
C
O
H
X
N R'
8 X = O; 9 X = N-R'
CO2Me
C
O
Scheme 3. Reagents: (a) N-protected morpholino or piperazino
carboxylic acid, i-Pr2NEt, HOBT, BOP; (b) BH3THF or LiAlH4.
O
O
NH
N
Table 4. Et-PKG inhibition and anticoccidial activities of diaryl-(4-
piperidinyl)-pyrrole derivatives 9
Compound
R
Et-PKG
inhibition
Anticoccidial
activity
CO2Me
IC50 (nM) at 100 ppm
in feed10
O
N
E.t. E.a.
HN
N
of compounds, morpholine containing analogs 6 and 8
are highly potent Et-PKG inhibitors. Also, a 10-time
decrease in potency was observed when the basic
9a
9b
9c
9d
0.07
1.08
2.2
3
2
2
2
NH
N
morpholine nitrogen was acylated (from 8a, IC50
=
0.17 nM to 8b, IC50 = 1.03 nM). The potency was fur-
ther reduced when the basic piperidine nitrogen was
also acylated (6b, IC50 = 4.70 nM). These results are
consistent with the trend we mentioned earlier that ba-
sic nitrogen atoms in that part of the molecule are
essential for in vitro activity. However, incorporation
of morpholine moiety does not provide the kind of
balanced in vivo efficacies required for broad spectrum
anticoccidial agents. Most compounds showed greater
efficacy against E.t. than those against E.a. Only com-
pound 8c in the series stands out with balanced activ-
ities and reaching full reduction of oocyst burden in
both E.t. and E.a. at 50 ppm in feed.
Ac
N
n/a n/a
n/a n/a
N
Ac
MeO2S
N
1.41
N
SO2Me
In summary, our continued SAR study on diaryl-(4-
piperidinyl)-pyrrole derivatives revealed that nitrogen
containing heterocyclic substitution on the piperidine
nitrogen improves the inhibition potency against Et-
PKG with IC50’s lower than 100 pM (3a and 9a). On
the other hand, improvement of in vivo anticoccidial
activities was only observed with morpholino analog
8c, which has broad spectrum activities and has reached
full reduction of oocyst burden in two of the major
Eimeria subtypes (E.t. and E.a.) at 50 ppm level in feed.
These results have indicated that other than its potency
which can be improved by incorporating basic nitrogen
atoms, many other factors, such as lipophilicity, are also
of fundamental importance in determining the in vivo
efficacy of these compounds.
To take the notion one step further that basic nitrogen
atoms are important for the intrinsic potency of these
compounds against PKG, we replaced the oxygen atom
in the morpholine ring with a nitrogen atom, and
synthesized a series of piperazine containing analogs
(9a–d), using the same reaction sequence shown in
Scheme 3. Their anticoccidial activity evaluation is sum-
marized in Table 4.
Indeed, the piperazino analog 9a is an extremely potent
PKG inhibitor with an IC50 of 70 pM, a 2–3 time
improvement over its morpholin analog 8a
(IC50 = 0.17 nM). Methylation (9b, IC50 = 1.08 nM),
acylation (9c, IC50 = 2.2 nM), and sulfonylation (9d,
IC50 = 1.41 nM) all resulted in loss of potency by more
than 10 times. However, these piperazine analogs,
although as potent as any other analogs, do not have
the broad spectrum activities, presumably because of
their less than optimal lipophilicity. No further develop-
ment of these analogs is warranted.
Acknowledgments
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 intermedi-
ates, and Merck Research Laboratory Basic Chemistry

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G.-B. Liang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2019–2022
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1
9. All new compounds were characterized by H NMR and
MS.
10. Three-day-old chicks are randomly placed on medicated
or control diets for the duration of the experiment.
Following 24 h premedication, in each replicate one bird
is infected with Eimeria acervulina, the other bird is
infected with E. tenella. The E. acervulina portion of the
experiment is terminated on Day 5, the E. tenella on Day 6
post infection. Treatments which provide at least 80%
reduction in oocyst production are rated (3), those with
50–79% are rated (2), and those with <50% are rated (0).
For a detailed description, please see Ref. 1.
4. (a) Wiersma, H. I.; Galuska, S. E.; Tomley, F. M.; Sibley,
L. D.; Liberator, P. A.; Donald, R. G. K. Int. J. Parasitol.