Synthesis and SAR Studies of diarylpyrrole anticoccidial agents

Article abstract of DOI:10.1016/j.bmcl.2006.01.041

2-(4-Fluorophenyl)-3-(4-pyridinyl)-5-substituted pyrroles were prepared and evaluated as anticoccidial agents in both in vitro and in vivo assays. Among the compounds evaluated, the dimethylamine-substituted pyrrole 19a is the most potent inhibitor of Eimeria tenella PKG (cGMP-dependent protein kinase). Further SAR studies on the side chain of the 2-pyrrolidine nitrogen did not enhance in vivo anticoccidial activity.

Full text of DOI:10.1016/j.bmcl.2006.01.041

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2817–2821
Synthesis and SAR Studies of diarylpyrrole anticoccidial agents
Xiaoxia Qian,a,* Gui-Bai Liang,a Dennis Feng,a Michael Fisher,a Tami Crumley,b
Sandra 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 14 December 2005; accepted 9 January 2006
Available online 6 March 2006
Abstract—2-(4-Fluorophenyl)-3-(4-pyridinyl)-5-substituted pyrroles were prepared and evaluated as anticoccidial agents in both
in vitro and in vivo assays. Among the compounds evaluated, the dimethylamine-substituted pyrrole 19a is the most potent inhibitor
of Eimeria tenella PKG (cGMP-dependent protein kinase). Further SAR studies on the side chain of the 2-pyrrolidine nitrogen did
not enhance in vivo anticoccidial activity.
Ó 2006 Elsevier Ltd. All rights reserved.
Coccidiosis is a parasitic disease which is the major
cause of morbidity and mortality in the poultry indus-
try. Commercial poultry operations would be very costly
N
N
without the use of effective anticoccidial agents. Resis-
tance to existing coccidiostats is becoming widespread
and new broad-spectrum economic drugs directed at
novel biochemical targets are needed. Coccidiosis is
caused by the invasion of protozoan parasites of the
genus Eimeria into various sites in the intestine of the
bird and causing damage to the intestinal lining.1 Earlier
reports2,3on studies of Eimeria tenella (Et) demonstrated
that inhibition of a cyclic GMP-dependent protein ki-
nase (PKG) stops the life cycle of the parasites, and thus
prevents the disease from spreading. Early SAR studies
from our laboratories showed that diaryl pyrroles are
Et-PKG inhibitors with in vivo anticoccodial activi-
ty.4–6 In this paper, we present the synthesis and SAR
studies of 2,3-diaryl pyrroles with various substituents
at the 5-position.
a
CO2Me
OMe
O
O
F
F
2
1
N
CO2Me
b
c
N
H
F
3
N
N
CO2H
CONMe2
d
N
H
N
H
F
F
4
5
N
e
NMe2
N
H
6
F
To evaluate these compounds as anticoccidial agents, an
enzyme inhibition assay against the native E. tenella
cGMP-dependent protein kinase (Et-PKG) was used
for initial in vitro screening. A 7-day in feed model
was carried out to evaluate their in vivo efficacy in
N
O
N
O
g
f
OEt
1
O
N
H
OEt
O
F
F
7
8
Scheme 1. Reagents and conditions: (a) 4-chloro-3-methoxy-2-bute-
,
nate, DBU, CH2Cl2, À78 °C; (b) NH4OAc, HOAc, 90 °C; (c) KOHaq
EtOH, reflux; (d) dimethylamine, EDC, THF; (e) BH3ÆTHF complex,
reflux; (f) BrCH2COCO2Et, DBU, CH2Cl2, À78 °C; (g) NH4OAc,
HOAc, 90 °C.
Keyword: Anticoccidial.
*
Corresponding author. Tel.: +1 732 594 3924; fax: +1 732 594
5790; e-mail: xiaoxia_qian@merck.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.01.041

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X. Qian et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2817–2821
Table 1. Et-PKG inhibition and anticoccidial activities of 2-(4-fluorophenyl)-3-(4-pyridinyl)pyrrole analogues
N
R
N
H
F
Compound
R
Et-PKG inhibition
a
IC50 (nM)
Anticoccidial activity at
100 ppm in feed4
E.t.
E.a.
4
5
6
8
–CH2CO2H
–CH2CONMe2
–(CH2)2NMe2
–CO2Et
>1000
4.1
2.5
0
0
2
0
0
0
2
0
430
a Values are means of three experiments and the same for other tables.
Table 2. Et-PKG inhibition and anticoccidial activities of 2-(4-fluorophenyl)-3-(4-pyridinyl)pyrrole analogues
N
R
N
H
F
Compound
R
Et-PKG inhibition IC50 (nM)
Anticoccidial activity at
100 ppm in feed4
E.t.
E.a.
14a
6
–(CH2)3NMeEt
–(CH2)2NMe2
–CH2NMe2
4.1
2.5
0
2
0
0
2
3
19a
0.35
procedures and rules of scoring were published in early
work.4
O
P
O
O
O
H
N
H
N
c
a,b
O
Cbz
HO
Cbz
10
9
The synthesis of 2,3-diaryl pyrrole analogues with vari-
ous 5-substituents including carboxylic acid, ester,
amide, and amine is outlined in Scheme 1. The aryl ke-
tone 14 was alkylated with 4-chloro-3-methoxy-2-bute-
nate in the presence of DBU to yield keto ester 2,
which was cyclized with ammonium acetate in acetic
acid to give pyrrole ester 3. Hydrolysis of ester 3 affor-
ded acid 4, which was subsequently converted to
dimethyl amide 5 by EDC-mediated coupling. Borane
reduction of amide 5 yielded N,N-dimethyl amine 6. In
a similar manner, ethyl ester 8 was obtained via the for-
mation of diketone 7 by the coupling reaction of aryl ke-
tone 1 with ethyl bromopyruvate in the presence of
DBU, followed by cyclization as described above. These
analogues were evaluated in both in vitro and in vivo as-
says, and the results are summarized in Table 1.7
N
N
Cbz
d
N
H
Cbz
N
H
O
O
O
12
11
F
N
N
e,f
g
N
H
N
H
HN
N
F
F
13
14a
Scheme 2. Reagents and conditions: (a) MeI, K2CO3, DMF; (b) n-
BuLi, CH3P(O)(CH3)2, THF, À78 °C; (c) 4-pyr-CHO, K2CO3,
CH3CN; (d) 4-F-PhCHO, thiazolium iodide, Et3N, EtOH, reflux; (e)
NH4OAc, HOAc, 90 °C; (f) LiAlH4, THF, reflux; (g) acetaldehyde,
NaBH(OAc)3.
When the substituents of the side chain of the pyrrole
are carboxylic acid (4) or ester (8), compounds are inac-
tive both in vitro and in vivo. Although dimethyl amide
5 is a potent Et-PKG inhibitor, it has no in vivo efficacy.
Dimethylamine 6, however, has improved Et-PKG inhi-
bition potency as well as in vivo efficacy. The in vivo
anticoccidial activity of the amine side chain is consis-
oocyst reduction against two major Eimeria species:
E. tenella (E.t.) and Eimeria acervulina (E.a.). Treat-
ments 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). Details of these

X. Qian et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2817–2821
2819
Table 3. Et-PKG inhibition and anticoccidial activities of 2-(4-fluorophenyl)-3-(4-pyridinyl)pyrrole analogues
N
R
N
H
F
Compound
R
Et-PKG inhibition
IC50 (nM)
Anticoccidial activity
100 ppm in feed4
E.t.
E.a.
19a
14b
24
0.35
0.71
1.0
0
3
3
3
0
0
0
0
0
N
N
2
0
0
0
0
0
0
N
19b
14c
19c
19d
19e
1.3
N
N
2.7
3.7
N
N
O
7.6
CH2OH
13
N
the side chain and the effect of different substituents
on the nitrogen of the amine group.
N
N
b
a
O
O
The synthesis of the c-alkylamine analogue 14a (in Ta-
ble 2) was carried out as shown in Scheme 2. Methyla-
tion of N-Cbz-c-amino butyric acid 9 followed by
treatment of the resulting methyl ester with the anion
of methyl phosphonate yielded the intermediate 10.
Horner–Emmons reaction of 10 with 4-pyridine alde-
hyde yielded ketone 11. Stetter reaction of 11 using 4-
fluorophenyl aldehyde and thiazolium salt afforded the
key intermediate diketone 12. Cyclization and lithium
aluminum hydride reduction provided amine 13, which
was reductively alkylated with acetylaldehyde to give
14a. The same method with N-Cbz-a-amino-3-meth-
ylbutyric acid and methyl 1-methylpipecolinate as start-
ing materials provided compounds 14b and 14c,
respectively (in Table 3).
F
F
1
15
N
N
c
d
O
N
H
O
F
F
16
17
N
N
R
R
N
O
H
1
e
N
H
N
H
F
F
19a-e
18
Scheme 3. Reagents and conditions: (a) NaHMDS, allyl bromide,
DMSO; (b) O3, MeOH, 0 °C, then (CH3)2S; (c) NH4OAc, HOAc,
90 °C; (d) DMF, POCl3, 90 °C then aq NaOAc, 90 °C; (e) amines,
BH3ÆPyr, EtOH.
The analogues with an a-alkylamine group side chain
19a–e shown in Table 3 were prepared by reductive ami-
nation of aldehyde 18 with a variety of amines. The syn-
thesis of these compounds is outlined in Scheme 3.
Alkylation of aryl ketone 1 with allyl bromide and sodi-
um bis(trimethylsilyl)amide followed by ozonolysis
yielded 16. Formation of pyrrole 17 was accomplished
tent with early observations from other studies in our
laboratories4–6 that a basic amine is essential at this
position. These results led us to explore the length of

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X. Qian et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2817–2821
Table 4. Et-PKG inhibition and anticoccidial activities of 2-(4-fluorophenyl)-3-(4-pyridyl)-5-pyrrolidinyl pyrrole analogues
N
R
N
N
H
F
Compound
R
Et-PKG inhibition IC50 (nM)
Anticoccidial activity
100 ppm in feed4
E.t.
E.a.
25
24
H
CH3
3.1
1.0
2.1
2.8
3.3
2.3
2.7
1.5
2
0
0
3
0
0
0
0
0
3
0
0
0
0
0
0
26a
26b
26c
26d
26e
26f
–CH2CH2OH
(S)-CH2CHOHCH3
(R)-CH2CHOHCH3
–CONHCH3
–CO2CH2Ph
–SO2(4-F-Ph)
Cbz group provided 25, which was further modified
at the pyrrolidine nitrogen by acylation, sulfonyla-
tion, and alkylation to provide analogues 26a–f (in
Table 4).
O
O
Cbz
N
Cbz
a-c
Cl
N
d
HO
21
20
The data presented in Table 2 compare the activities
of analogues with the side chain from C-1 to C-3.
The c-alkylamine 14a is a potent Et-PKG inhibitor
(IC50 = 4.1 nM), but has no in vivo anticoccidial
activity at 100 ppm against E.t. and E.a., while the
b-alkylamine 6, Et-PKG (IC50 = 2.5 nM), caused par-
tial reduction of oocyst count (50–79%) at 100 ppm
against both E.t. and E.a. in feed. The shorter a-al-
kylamine side chain 19a (IC50 = 0.35 nM) is one of
the most potent Et-PKG inhibitors and has full in vi-
vo anticoccidial activity (80–100%) at 100 ppm
against E.t.
N
N
N
Cbz
e
O
N
Cbz
N
O
H
22
F
F
23
g
N
f
N
N
H
N
H
25
F
N
N
H
h
F
24
N
For active Et-PKG inhibitors, the in vivo efficacy is
determined by many factors, such as absorption in both
host and parasites. Changes in the basicity and lipophil-
icity of the compound with various substituents on the
amine group might improve the bioavailability of these
compounds, and thus improve the in vivo anticoccidial
activity. Therefore, we prepared a series of a-alkylamine
analogues with different ring sizes (19b–e) and a-alkyl
branches (14b–c, 24). Their anticoccidial activities are
summarized in Table 3.
N
R
N
H
F
26a-f
Scheme 4. Reagents and conditions: (a) (COCl)2, DMF; (b)
TMSCHN2, THF; (c) HCl, ether; (d) ketone 1, DBU, CH2Cl2,
À78 °C; (e) NH4OAc, HOAc, 90 °C; (f) LiALH, THF, reflux; (g) H2,
20% Pd/C, HOAc, MeOH; (h) epoxides or CH3NCO, or Cbz-Cl, or
4-F-phenyl sulfonyl chloride.
as previously described. Reaction of 17 with phosphorus
oxychloride in the presence of dimethylformamide and
subsequent heating with aqueous sodium acetate gave
the aldehyde 18.
All the cyclic amine derivatives (19b–e, 24, and 14c) are
very potent Et-PKG inhibitors with IC50 in the low
nanomolar range. As the ring size gets larger from azeti-
dine (19b) to piperidine (19c), their potencies drop
slightly. Introduction of oxygen or a hydroxyl group
on the piperidine ring (19d–e) resulted in decreased in vi-
tro activities. These results indicate neither the more
lipophilic groups nor the polar groups are tolerated at
this position. On the other hand, introducing an a-alkyl
branch generated mixed results. Although compound
14b showed no improvement in Et-PKG activity, it
has in vivo anticoccidial activity for both E.t. and E.a.
And N-methyl pyrrolidine 24 shows a slight drop in
The substituted 2-pyrrolidine analogues 24, 25, and
26a–f shown in Table 4 were prepared in a similar
manner (Scheme 4). After conversion of N-Cbz pro-
line 20 to the acid chloride, the resulting compound
was treated with trimethylsilyl diazomethane followed
by aqueous work-up and displacement with hydrogen
chloride to yield the chloromethyl ketone 21. The
pyrrolidine 23 was obtained as described earlier.
Lithium aluminum hydride reduction of 23 gave N-
methyl compound 24, while hydrogenolysis of the

X. Qian et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2817–2821
2821
potency, but it retains the full in vivo anticoccidial activ-
ity for E.a., while the slightly less potent piperidine 14c
has lost in vivo activity.
and Mr. Glen Reynolds for scale up of key intermedi-
ates, Dr. Ann E. Weber for review of the manuscript,
and Merck Research Laboratory Basic Chemistry Ana-
lytical Support Group for open access service on LC/
MS.
Modification on the nitrogen of 2-pyrrolidine 25 gave
analogues 26a–f shown in Table 4. In general, the sub-
stituents have little effects on Et-PKG inhibition. Most
derivatives have similar in vitro activities. For example,
the enantiomeric isopropanol 26b and 26c have similar
Et-PKG activity, but only 26b shows anticoccidial activ-
ity for E.t. at 100 ppm in feed. These results are different
from those observed for 4-piperidine pyrrole containing
a hydroxyl group4,6, while the compounds here do not
have a broad-spectrum of in vivo activity.
References and notes
1. Williams, R. B. Int. J. Parasitol. 1999, 29, 1209.
2. Gurnett, A. M.; Liberator, P. A.; Dulski, P. M.; Salowe, S.
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. J. Biol.
Chem. 2002, 277, 15913.
In summary, modification of 2-(4-fluorophenyl)-3-(4-
pyridinyl) pyrrole by introduction of various basic amines
at the 5-position has been examined. Most analogues have
good Et-PKG potency. Substitution of large lipophilic
groups or polar groups on the carbon a to the amine nitro-
gen is not tolerated. Compounds 19a and 14b are sub-
nanomolar inhibitors of Et-PKG. However, they did
not display a broad-spectrum of anticoccidial activity.
3. Donald, R. G. K.; Liberator, P. A. Mol. Biochem.
Parasitol. 2002, 180, 136.
4. Biftu, T.; Feng, D.; Liang, G.-B.; Qian, X.; Gurnett, A. M.;
Liberator, P. A.; Dulski, P. M.; Donald, R. G. K.; Diaz, C.
A.; Darkin-Rattray, S. J.; Nare, B.; Crumley, T.; Blum, P.
S.; Misura, A. S.; Tamas, T.; Wyvratt, M.; Fisher, M. H.
Bioorg. Med. Chem. Lett. 2005, 15, 3296.
5. Feng, D. Unpublished report.
6. Liang, G.-B.; Qian, X.; Biftu, T.; Feng, D.; Fisher, M. H.;
Crumley, T.; Darkin-Rattray, S. J.; Dulski, P. M.; Gurnett,
A. M.; Leavitt, P. S.; Liberator, P. A.; Misura, A. S.;
Samaras, S.; Tamas, T.; Wyvratt, M. Bioorg. Med. Chem.
Lett. 2005, 15, 4570.
Acknowledgments
7. All new compounds were characterized by 1H NMR and
LC–MS prior to submission for biological evaluation.
The authors thank Dr. Phil Eskola, Dr. Gerard Kieczy-
kowshi, Ms. Amanda Makarewicz, Mr. Bob Frankshun,