Synthesis and SAR of 2,3-diarylpyrrole inhibitors of parasite cGMP-dependent protein kinase as novel anticoccidial agents

Article abstract of DOI:10.1016/j.bmcl.2005.04.060

Several analogs of 2,3-diaryl pyrroles were synthesized and evaluated as inhibitors of Eimeria tenella cGMP-dependent protein kinase and in in vivo anticoccidial assays. A 4-fluorophenyl group enhances both in vitro and in vivo activities. The most potent

Full text of DOI:10.1016/j.bmcl.2005.04.060

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3296–3301
Synthesis and SAR of 2,3-diarylpyrrole inhibitors of parasite
cGMP-dependent protein kinase as novel anticoccidial agents
Tesfaye Biftu,* Dennis Feng, Mitree Ponpipom, Narindar Girotra, Gui-Bai Liang,
Xiaoxia Qian, Robert Bugianesi, Joseph Simeone, Linda Chang, Anne Gurnett,
Paul Liberator, Paula Dulski, Penny Sue Leavitt, Tami Crumley, Andrew Misura,
Terence Murphy, Sandra Rattray, Samantha Samaras, Tamas Tamas, John Mathew,
Christine Brown, Don Thompson, Dennis Schmatz, Michael Fisher and Matthew Wyvratt
Merck Research Laboratories, Department of Medicinal Chemistry, Merck and Co., P.O. Box 2000, Rahway, NJ 07065-0900, USA
Merck Research Laboratories, Department of Human and Animal Infectious Disease Research, Merck and Co., P.O. Box 2000,
Rahway, NJ 07065-0900, USA
Accepted 19 April 2005
Available online 26 May 2005
Abstract—Several analogs of 2,3-diaryl pyrroles were synthesized and evaluated as inhibitors of Eimeria tenella cGMP-dependent
protein kinase and in in vivo anticoccidial assays. A 4-fluorophenyl group enhances both in vitro and in vivo activities. The most
potent analogs are the 5-(N-methyl, N-ethyl, and N-methylazetidine methyl) piperidyl derivatives 12, 23, and 34. These compounds
have a broad spectrum of activity. Based on the in vivo efficacy and cost of synthesis, the N-ethyl analog 23 was chosen as a novel
anticoccidial agent for a field trial.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
1930s, when the first coccidiostats were discovered, the
total annual broiler production in the United States
was approximately 34 million.1 Even though the de-
mand for poultry as a source of protein far outpaced
supply, the high incidence of morbidity and mortality
kept growth of the industry in check. With the develop-
ment of safe and effective anticoccidials, the disease was
brought under control, and by the year 2000 the annual
broiler production in the United States reached eight bil-
lion.2 Since the introduction of monensin to the broiler
market in 1971,3,4 the polyether ionophores have been
the leading commercial anticoccidial products. An initial
shift in drug sensitivity was observed with this class dur-
ing the 1980s,5,6 and a slow process of sensitivity erosion
has continued to the present.7–9 Today, resistant field
isolates are fairly common, indicating that the time is
approaching when novel new entries in the anticoccidial
market will be necessary to maintain current levels of
activity and productivity. The most successful anticocci-
dials have broad-spectrum efficacy, slow resistance selec-
tion, and do not retard growth.
Parasitic protozoa are responsible for a wide variety of
infections in human and animals. Malaria caused by
Plasmodium species, myeloencephalitis caused by
Sarcocystis neurona, trypanosomiasis caused by Try-
panosoma cruzi, African sleeping sickness caused by
Trypanosoma brucei, and opportunistic infections in
immunocompromised hosts caused by Pneumocystis
carinii, Toxoplasma gondii, and Cryptosporidium species
are becoming significant in developing and developed
countries.
Coccidiosis, a widespread disease of domesticated ani-
mals, is caused by a protozoal infection. In the poultry
industry, coccidiosis is responsible for high levels of
morbidity and mortality in the bird population and
may result in extreme economic losses. The infectious
agents are protozoa of the genus Eimeria such as
E. tenella, E. acervulina, E. necatrix, E. brunetti, and
E. maxima. Anticoccidial drugs have profoundly con-
tributed to successful poultry production. In the early
Novel Eimeria tenella and Toxoplasma gondii cGMP-
dependent protein kinases (PKGs), the polynucleotide
sequences encoding these PKGs, and a facile specific
*
Corresponding author. E-mail: tesfaye_biftu@merck.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.04.060

T. Biftu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3296–3301
3297
assay to screen compounds as PKG inhibitors have been
reported recently.10,11 The native Eimeria tenella PKG
(Et-PKG) is a protein of about 120 kDa with 1003 ami-
no acids. In this article, we describe the in vitro activities
of several diaryl pyrroles as Et-PKG inhibitors and the
in vivo activities of these compounds as anticoccidial
agents in chickens.
2. Synthesis
The pyrroles, furans, thiophenes, and pyridazines de-
scribed here are prepared as shown in Scheme 1. 4-Pic-
oline 1 was treated with LDA in THF, and the anion
generated was reacted with the esters 2 to form ketones
3. The latter was then treated with sodium hydride in di-
methyl sulfoxide followed by iodoketone 4 to form 1,4-
diketones 5, which were conveniently cyclized to pyr-
roles 6 by heating with ammonium acetate in acetic acid.
Deprotection of 6 followed by alkylation with sodium
hydride/alkyl halides gave the desired pyrroles. Furans,
thiophenes, and pyridazines were made from the dike-
tone 5 in a similar manner by treatment with hydrochlo-
ric acid in methanol, phosphorous pentasulfide, or
hydrazine, respectively.
Scheme 2. Synthesis of imidazole, oxazole, and thiazole 10.
3. Biological assays
As shown in Scheme 2, oxidation of the ketone 3 with
selenium dioxide gave the diketone 9. The t-butyldi-
methylsilyl (TBDMS) protect hydroxyketone 8 was
made by acylation of 1a, as described above in Scheme
1. Bromination of 3 gave the bromoketone 10. The oxa-
zole, thiazole, and imidazole inhibitors described in this
manuscript were made from TBDMS protected
hydroxyketone 8, diketone 9 or bromoketone 10 by cou-
pling with 4-piperidyl acid chloride, aldehyde or thioam-
ide, respectively.
3.1. Enzyme assay and data analysis
Purification of the Et-PKG enzyme was performed as
described in a previous communication.10 Drug solu-
tions at 50 times the desired test concentration in
100% DMSO were prepared and 1:2 serial dilutions into
100% DMSO were made for eight data points. Drug
dilutions (2 lL) were added to each well of a Wallac Iso-
plate (Catalog No. 1450-514) followed by 50 lL of the
substrate solution [50 mM Hepes, pH 7.4, 10 mM
MgCl2, 3 lM ATP, 4 lM peptide substrate (Gly-Arg-
Thr-Gly-Arg-Arg-Asn-Ser-Ile biotinylated at the amino
terminal end through a linker region amino-hexanoic
acid), 0.2 mg/mL bovine serum albumin, 20 lM cGMP,
and 3 lCi/mL [33P]ATP (NEG/602 H Perkin-Elmer Life
Sciences (NEN) at 3000 Ci/mmol)]. Then, 50 lL of the
enzyme solution was added to substrate (1 lL/mL of
partially purified E. tenella PKG that does not contain
any cAMP-stimulated activity). The mixture was incu-
bated at room temperature for 4 h. At the end, 100 lL
of stop solution was added to each well containing sub-
strate; drug, enzyme, and beads were allowed to settle
overnight and counted on the Wallac microbeta. The
stop solution was prepared by adding 500 mg Streptavi-
din SPA beads (Amersham, Catalog No. NIF-1077) to
10 mL PBS, mixing by gentle swirling, and then adding
1 mL SPA bead/PBS mixture to 4 mL of 75 mM phos-
phoric acid. After subtracting the appropriate back-
ground for each assay point, titrations were fit to the
following modified Hill equation using Kaleidagraph
(Synergy Software): VA = V0 + (Vmax À V0)/(1 + (KA/
[A])h).
3.2. In vivo anticoccidial assay
One-day-old White Leghorn chickens are obtained from
a commercial hatchery and acclimated in a holding
Scheme 1. Synthesis of pyrrole, thiophene, and furan 7.

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T. Biftu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3296–3301
Table 2. Et-PKG and anticoccidial activities of diaryl substituted
heterocycles
room. At 3 days of age, the test animals are selected by
weight, wingbanded, and randomly placed on medicated
or control diets for the duration of the experiment. One
or two replicates of two birds are utilized per treatment.
Following 24 h premedication, in each replicate one bird
is infected with E. acervulina, while the other bird is in-
fected with E. tenella. Both strains of Eimeria are sensi-
tive to all anticoccidial products and have been
maintained under laboratory conditions for over 25
years. The inocula consist of sporulated oocysts in tap
water suspensions, administered at a dose volume of
0.25 mL per bird. The inocula levels are selected by pre-
vious dose titrations to provide a low-to-moderate level
of infection. The E. acervulina portion of the experiment
is terminated on Day 5 and that of the E. tenella on Day
6 post-infection. The measured parameters are weight
gain, feed consumption, and oocyst production. E. tenel-
la lesion scores are also recorded for background infor-
mation. Treatments that 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).
A
Et-PKG
IC50 (nM)
Anticoccidial activity
at 100 ppm
Et
Ea
0.70
3
3
1.1
3
0
0
2
4. Results and discussion
Pyrrole 12 and other heterocycles have been reported as
kinase inhibitors.12 The in vitro Et-PKG and the in vivo
anticoccidial activities of 2-aryl substituted 2,3-diaryl
heterocycles are shown in Tables 1–5. As shown in Table
1, substitution of the 4-position of pyrrole with groups
such as Cl (13), Br (14) or methyl (15) resulted in com-
pounds that maintained in vitro Et-PKG activity; how-
ever, these more lipophilic analogs lack significant in
vivo activity. The unsubstituted analog 12 is fully active
in both E. tenella and E. acervulina at 100 ppm in feed.
2.1
4.2
3
3
0
0
3
As shown in Table 2, we kept the 4-pyridyl, 4-fluorphe-
nyl, and piperidyl groups fixed and varied the middle
0.97
3
Table 1. Et-PKG and anticoccidial activities of 2-aryl substituted
diaryl pyrroles
1.5
0 (25 ppm)
R
Et-PKG
IC50 (nM)
Anticoccidial
activity at 100 ppm
Et
Ea
1000
0
H
12
0.70
1.02
1.2
3
3
Cl
13
2
0
Br
14
heterocyclic group. In general, five-membered hetero-
cycles are more potent than six-membered heterocycles.
For example, pyrrole 12 (IC50 = 0.7 nM), imidazole 16
(IC50 = 1.1 nM), furan 17 (IC50 = 2.1 nM), and oxazole
0
0
Me
15
1.0
0
0

T. Biftu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3296–3301
3299
Table 3. Et-PKG and anticoccidial activities of N-substituted piper-
idine diaryl pyrroles
Table 4. Et-PKG and anticoccidial activities of diaryl substituted
heterocycles
R
Et-PKG
IC50 (nM)
Anticoccidial activity at
100 ppm
R
Et-PKG
IC50 (nM)
Anticoccidial activity
at 100 ppm
Et
Ea
Et
Ea
Methyl 12
Ethyl 23
0.70
0.28
0.71
0.28
4.2
3
3
3
0
0
0
3
2
0
3
3
3
0
0
0
3
3
0
0.70
1.7
3
3
2-Hydroxyethyl 24
2-Aminoethyl 25
n-Propyl 26
Acetone 27
0.32
2.0
2-Aminopropyl 28
3-Hydroxypropyl 29
3-Aminopropyl 30
0
0
0
0
0.53
0.046
1.1
3
0
3
3
0
3
2.8
0.24
0.28
11.7
23.6
0
0
2
0
19 (IC50 = 0.7 nM) are more potent Et-PKG inhibitors
than pyridazine 21 (IC50 = 1000 nM). The thiazole 18
and oxazole 19 are active in in vivo assay in both species
at 100 ppm. The basic heterocycles such as imidazole 16
and thiazole 20 are not active in both species.
15.6
0
0
0
0
>1000
Et-PKG inhibition activity and in vivo anticoccidial
activities of alkyl, hydroxyalkyl, and aminoalkyl substi-
tuted piperidines are shown in Table 3. The N-methyl
and N-ethyl analogs 12 and 23 (0.28 nM) are potent
PKG inhibitors and are active in in vivo assays. The
N-propyl analog 26 (4.2 nM) has reduced activity.
Reducing lipophilicity by introducing hydroxy or amino
substituents at the 2- or 3-position of the alkyl side chain
maintained Et-PKG activity. Amino ethyl 25 and azeti-
dine 34 substituted piperidine analogs are more potent
Et-PKG inhibitors than the methyl substituted analog
12. Azetidine 34 is also a very potent anticoccidial agent
and has in vivo activity even at a 25 ppm level.
Et and Ea parasites. This could be due to the inability
of the compounds to penetrate the appropriate host tis-
sue or to penetrate the parasite. Lipophilicity plays a
major role in the in vivo activity and spectrum of activ-
ity of these compounds.
As shown in Table 4, replacement of the 4-pyridyl group
with 3-pyridyl 37, 2-pyridyl 38, 4-fluorophenyl 39, or
isooxazole 40 resulted in over an order of magnitude
loss in Et-PKG activity. However, a methyl substituent
on the 4-pyridyl group, 35 and 36, reduced activity by
The aminopropyl 30 and cyclopropylamine 32 are po-
tent PKG inhibitors with IC50s of 0.046 and 0.24 nM,
respectively, but they both lack in vivo activity against

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T. Biftu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3296–3301
Table 5. Et-PKG and anticoccidial activities of diaryl substituted
pyrroles 1a–1i
Table 5 (continued)
Compound
R
Et-PKG
IC50 (nM)
Anticoccidial
activity at 100 ppm
Et
Ea
0
52
2.0
3
Compound
R
Et-PKG Anticoccidial
IC50 (nM) activity at 100 ppm
Et
Ea
53
54
0.44
0.15
3
0
2
0
41
42
3.1
7.4
NAa
NAa
0
0
0
3
a Data are not available.
43
1.0
two- to fourfold only. Compounds 35–40 were not sig-
nificantly active in in vivo assays.
As shown in Table 5, the 4-fluoro phenyl group plays a
very important role in the Et-PKG activities of these
classes of compounds. The unsubstituted phenyl 41
and the 3-fluoro phenyl 42 are weaker inhibitors of Et-
PKG. Replacement of the 4-fluoro group with trifluo-
romethyl 48, hydroxyl 49, trifluoromethoxy 50 or
methyl sulfone 51 significantly reduced Et-PKG activity
compared to 12. Replacement of 4-fluorophenyl group
with other heterocycles such as furan (44 and 46), thio-
phene (45) or thiazole (47) also resulted in over two or-
ders of magnitude loss in Et-PKG activity. However,
when the 4-fluoro group is kept and a second halogen
is added in the meta position, the resulting analogs 52,
53, and 54 are very active in the enzyme assays with a
tendency toward improved potency for the least electro-
negative substituent.
44
45
11.1
11.9
0
0
0
0
46
26.3
0
0
47
48
39.7
55
0
0
0
0
Pyrrole 23 (Et-PKG IC50 = 0.28 nM) was selected for
spectrum studies against eight of the most common
Eimeria species in chicken (E. tenella, E. acervulina,
E. necartrix, E. brunetti, E. maxima, E. mitis, E. mivati,
and E. praecox). As shown in Table 6, analog 23 has
excellent anticoccidial activity at 50–125 ppm. In the
same study, the anticoccidial agent salinomycin has
49
50
50
46
0
3
0
0
Table 6. Spectrum assays of compound 23 on eight species of Eimeria
(40,000 parasites per bird)
Treatment
In feed (ppm)
Anticoccidial activity
Normal control
Infected control
0
0
3
0
3
3
3
2
2
23
23
125
75
50
25
66
23
23
51
42
3
0
Salinomycin

T. Biftu et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3296–3301
3301
marginal activity at its recommended use level of 66 ppm.
Compound 23 has a half-life of 1.2 h and oral bioavail-
ability of 84% in the chicken. In a preliminary study, drug
resistance was not observed for the related N-methyl
analog 12 after 11 cycles of application at 80 ppm against
E. tenella species (T. Tamas, unpublished).
cal support group for pharmacokinetic studies and open
access LC/MS.
References and notes
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5. Conclusion
In conclusion, we have discovered 2-(4-fluorophenyl)-3-
(4-pyridyl)-5-(piperidyl)pyrroles as very potent inhibi-
tors of parasite PKG, with excellent activity against
commercially important strains of Eimeria in chickens.
Among these, the methyl, ethyl, and the methylazetidine
substituted analogs 12, 23, and 34 are potent and have a
broad spectrum of activity. The N-ethyl piperidine ana-
log 23 has excellent activity when administered at 50–
125 ppm levels in feed in an in vivo spectrum model
against eight of the most common Eimeria species. Sen-
sitivity to current commercial anticoccidial agents is
eroding. Based on these studies and ease of synthesis,
analog 23 was chosen for a large-scale field trial as a po-
tential novel anticoccidial agent. Results will be reported
elsewhere.
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(Abstr.).
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P.; Donald, R. G. K.; Anderson, J. W.; Wiltsie, J.; Diaz,
C. A.; Harris, G.; Chang, B.; Darkin-Rattray, S. J.; Nare,
B.; Crumley, T.; Blum, P. S.; Misura, A. S.; Tamas, T.;
Sardana, M. K.; Yuan, J.; Biftu, T.; Schmatz, D. M.
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Acknowledgments
The authors thank Dr. Phil Eskola, Dr. Gerard Kieczy-
kowski, Amanda Makarewicz, Bob Frankshun and
Glenn Reynolds for scale-up of key compounds, Dr.
A. E. Weber for review of the manuscript, and Merck
Research Laboratories Drug Metabolism and Analyti-
11. Wiersmaa, H. I.; Galuskaa, S. E.; Tomleyb, F. M.;
Sibleyc, L. D.; Liberator, P. A.; Donald, R. G. K. Int. J.
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