Synthesis and biological activity of anticoccidial agents: 2,3-diarylindoles

Article abstract of DOI:10.1016/j.bmcl.2009.01.001

Novel 2,3-diarylindoles bearing an amine substituent at the indole 5- and 6-positions have been synthesized and evaluated as anticoccidial agents in both in vitro and in vivo assays. Both subnanomolar in vitro activity and broad spectrum in vivo potency w

Full text of DOI:10.1016/j.bmcl.2009.01.001

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1517–1521
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological activity of anticoccidial agents: 2,3-diarylindoles
a
a
b
b
Andrew Scribner a, , Joseph A. Moore III , Gilles Ouvry , Michael Fisher , Matthew Wyvratt ,
Penny Leavitt c, Paul Liberator c, Anne Gurnett c, Chris Brown c, John Mathew c, Donald Thompson c,
Dennis Schmatz c, Tesfaye Biftu b
*
a SCYNEXIS, Inc., Discovery Chemistry, PO Box 12878, Research Triangle Park, NC 27709-2878, USA
b Merck Research Laboratories, Department of Medicinal Chemistry, Merck & Co., Inc., Rahway, NJ 07065, USA
c Merck Research Laboratories, Department of Human and Animal Infectious Disease Research, Merck & Co., Inc., Rahway, NJ 07065, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 2,3-diarylindoles bearing an amine substituent at the indole 5- and 6-positions have been synthe-
sized and evaluated as anticoccidial agents in both in vitro and in vivo assays. Both subnanomolar in vitro
activity and broad spectrum in vivo potency were detected for several compounds, particularly com-
pound 27.
Received 18 August 2008
Revised 26 December 2008
Accepted 6 January 2009
Available online 9 January 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Anticoccidial
Antiprotozoan
Antiparasitic
Indole
Kinase inhibitor
Protien kinase G
Parasitic protozoa are responsible for a wide variety of infec-
tious diseases in both humans and animals. Coccidiosis is a para-
sitic disease that is the major cause of morbidity and mortality in
the poultry industry. It is a disease of the avian intestinal lining
due to invasion by Apicomplexan protozoan parasites of the genus
Eimeria,1 where the most significant Eimeria species in poultry in-
clude E. tenella, E. acervulina, E. mitis, and E. maxima. Over 35 billion
chickens are raised annually worldwide, and all major poultry
operations use prophylactic anticoccidial agents. Nevertheless,
resistance to current coccidiostats such as polyether ionophores
has become widespread,2 creating the need for new broad spec-
trum drugs with unprecedented mechanisms of action.
ious amine sidechains, as we were curious how these compounds
would compare with our analogous pyrroles, imidazopyridines,
and imidazothiazoles. Within these three series, we found many
of our active compounds possessed a 4-fluorophenyl ring, a pyrim-
idin-4-yl or pyridin-4-yl ring, and a piperidin-4-yl ring at strategic
locations across the core heterocycle. We therefore decided to
introduce such functionality across an indole template.
Scheme 1 depicts the synthesis of indoles bearing a 2-amino-
pyrimidin-4-yl or pyrimidin-4-yl ring at the indole 3-position.
The synthesis of ketone 1 has been reported previously.12 Palla-
dium-catalyzed arylation15 of the
a-carbon of ketone 1 with 2,5-
dibromonitrobenzene occurred regioselectively with displace-
Recently, we have reported on novel anticoccidial agents with
potent in vitro and in vivo activity against Eimeria parasites. It
was found that the reduction of parasite growth by these com-
pounds was due to the inhibition of parasite-specific cGMP-depen-
dent protein kinase (PKG), a serine/threonine protein kinase.3,4 In
particular, we have found that various 2,3-diarylpyrroles5–8, 2,3-
ment of the bromine ortho rather than meta to the nitro group,
yielding a-aryl ketone 2. Reductive cyclization with iron in acetic
acid then rendered indole 3. The indole nitrogen was subse-
quently protected to give N-tosylindole 4. The methylsulfanyl
group of 4 was then oxidized with m-CPBA to yield sulfone 5,
which itself was treated with ammonia to afford 2-aminopyrim-
idine 6. Alternatively, the methylsulfanyl group of 4 was reduced
with Raney nickel to give 2-H-pyrimidine 7. Subsequent Negishi
coupling16 of aryl bromides 6 and 7 with 1-Boc-4-iodozincpiperi-
dine17 gave 6-(1-Boc-piperidin-4-yl)indoles 8 and 9, respectively.
Cleavage of the Boc protecting group followed using trifluoroace-
tic acid to give NH-piperidines 10 and 11. Deprotection of the
indole nitrogen yielded N–H indoles 12 and 13, while alkylation
by reductive amination with formaldehyde gave N-methylpiperi-
diarylimidazo[1,2-a]pyridines9–13
,
and 5,6-diarylimidazo[2,1-
b][1,3]thiazoles14 show exceptional potency as anticoccidial
agents.
In this paper, we present the synthesis and biological activity of
a series of 2,3-diarylindoles substituted at the 6-position with var-
* Corresponding author. Tel.: +1 919 544-8610.
E-mail address: andrew.scribner@scynexis.com (A. Scribner).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.001

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A. Scribner et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1517–1521
S
S
S
Br
N
N
N
N
N
N
a
b
Br
NO2
O
N
H
O
F
F
F
1
2
3
c
R
R
R
N
N
N
N
N
N
boc
h
g
NH
N
Br
N
Tos
N
Tos
N
Tos
F
F
F
R = SCH3 (4)
R = SO2CH3 (5)
R = NH2 (6)
R = H (7)
R = NH2 (10)
R = H (11)
R = NH2 (8)
R = H (9)
d
e
f
j or k
i
R
R
R
N
N
N
N
N
N
R'
i
R'
N
NH
N
N
H
N
Tos
N
H
F
F
F
R = NH2 (12)
R = H (13)
R = NH2; R' = CH3 (14)
R = NH2; R' = CH2CH2OH (15)
R = H; R' = CH3 (16)
R = NH2; R' = CH3 (18)
R = NH2; R' = CH2CH2OH (19)
R = H; R' = CH3 (20)
R = H; R' = CH2CH2OH (17)
R = H; R' = CH2CH2OH (21)
Scheme 1. Reagents and conditions: (a) 2,5-dibromonitrobenzene, Pd(OAc)2, 2-(di-tert-butylphosphino)biphenyl, Cs2CO3, DMF, 70 °C; (b) Fe, AcOH, 15 °C–rt; (c) TosCl, NaH,
THF; (d) m-CPBA, CH2Cl2; (e) NH3, THF, 50 psi; (f) Raney Ni, EtOH, 80 °C; (g) 1-Boc-4-iodozincpiperidine, PdCl2(dppf)CH2Cl2, CuI, DMA, 80 °C; (h) CF3CO2H, CH2Cl2; (i) K2CO3,
MeOH, 65 °C; (j) H2C@O, NaBH3CN, MeOH, AcOH; (k) BrCH2CH2OH, K2CO3, DMF, 65 °C.
dines 14 and 16, and alkylation with 2-bromoethanol under ba-
sic conditions gave N-(2-hydroxyethyl)piperidines 15 and 17. N-
Tosyl indoles 14–17 were ultimately deprotected to yield NH-in-
doles 18–21.
We then prepared the analogous 3-(pyridin-4-yl)indoles, as
shown in Scheme 2. The synthesis of ketone 22 has been reported
previously.5 Regioselective arylation of ketone 22 proceeded as in
the pyrimidine series to yield
a-aryl ketone 23. At this point, we
Br
N
N
N
Br
a
b
NO2
N
H
O
O
F
F
F
22
23
24
c
N
N
N
e, f,
or g
boc
N
R
N
NH
d
N
H
N
N
H
H
F
F
F
R = CH3 (27)
26
25
R = CH2CH3 (28)
R = CH2CH2OH (29)
Scheme 2. Reagents and conditions: (a) 2,5-dibromonitrobenzene, Pd(OAc)2, 2-(di-tert-butylphosphino)biphenyl, Cs2CO3, DMF, 60 °C; (b) TiCl3, NH4OAc, 10:1 EtOH:EtOAc;
(c) 1-Boc-4-iodozincpiperidine, PdCl2(dppf)CH2Cl2, Pd2(dba)3, CuI, XANTPHOS, RuPHOS, CuSCN, NMP, DMA, 100 °C; (d) CF3CO2H, CH2Cl2; (e) H2C@O, NaBH3CN, MeOH, AcOH;
(f) H3CC(@O)H, NaBH3CN, MeOH, AcOH; (g) glycolaldehyde, NaBH3CN, MeOH, AcOH.

A. Scribner et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1517–1521
1519
found TiCl3 to be an optimal reagent for carrying out the cyclore-
duction18 to give indole 24, as this procedure did not yield the cor-
responding N-hydroxyindole byproduct that we often encountered
previously when using Fe/AcOH. We were then able to proceed
through the remaining steps of this synthesis without protecting
the indole nitrogen, unlike the 3-(pyrimidin-4-yl)indole synthesis
in Scheme 1 which entailed a sulfide oxidation to sulfone, first
necessitating indole nitrogen protection. Negishi coupling16 of
the aryl bromide with 1-Boc-4-iodozincpiperidine17 gave 6-(1-
Boc-piperidin-4-yl)indole 25, which was followed by Boc cleavage
to give NH-piperidine 26. Reductive amination with formaldehyde,
acetaldehyde, and glycolaldehyde yielded N-methylpiperidine 27,
N-ethylpiperidine 28, and N-(2-hydroxyethyl)piperidine 29,
respectively.
We then elaborated the 3-(pyridin-4-yl)indole series further by
introducing different heterocycles at the indole 6-position via pal-
ladium catalyzed chemistry, as shown in Scheme 3. 6-(4-Methylpi-
perazin-1-yl)indole 30 and 6-(morpholin-4-yl)indole 31 were
prepared by microwave treatment of 6-bromoindole 24 with
Pd(OAc)2, 2-(di-tert-butylphosphino)biphenyl, NaO-t-Bu, and N-
methylpiperazine or morpholine, respectively. 6-(1-Methylazeti-
din-3-yl)indole 32 was prepared via Negishi coupling16 of bromide
24 with 1-Boc-3-iodozincazetidine19 using similar conditions de-
scribed previously for the synthesis of the 6-(1-methylpiperidin-
4-yl)indoles, followed by Boc cleavage and reductive amination
with formaldehyde. 6-Methylene(1-methylpiperidin-4-yl)indole
33 was synthesized via b-alkyl Suzuki–Miyaura coupling20 of bro-
mide 24 with 1-Boc-4-methylenepiperidine21 using 9-BBN,
Pd(PPh3)4, and Na2CO3, followed by Boc cleavage and reductive
amination with formaldehyde.
Lastly, we extended the 3-(pyridin-4-yl)indole series even fur-
ther to include compounds possessing a 1-methylpiperazin-4-yl
group linked by either a carbonyl or methylene group to the indole
5-position (Scheme 4), as we were curious if the distant basic
nitrogen would occupy the same region of space as that of a 6-
(1-methylpiperidin-4-yl)indole, as suggested by our imidazothiaz-
oles.14 Fischer indole synthesis of ketone 22 with 4-hydrazinoben-
zoic acid yielded 5-carboxyindole 34. Subsequent amide coupling
with N-methylpiperazine afforded amide 35, which was then re-
duced to give 6-(methylene-1-methylpiperazin-4-yl)indole 36.
N
N
N
O
N
N
N
H
N
H
a
d
b
c
F
F
30
31
24
N
N
N
N
N
H
H
N
F
F
33
32
Scheme 3. Reagents and conditions: (a) 1-methylpiperazine, Pd(OAc)2, 2-(di-tert-butylphosphino)biphenyl, NaO-t-Bu, microwave, 120 °C; (b) morpholine, Pd(OAc)2, 2-(di-
tert-butylphosphino)biphenyl, NaO-t-Bu, microwave, 120 °C; (c) i—1-Boc-3-iodozincazetidine, PdCl2(dppf)CH2Cl2, CuI, XANTPHOS, RuPHOS, CuSCN, NMP, DMA, 100 °C; ii—
CF3CO2H; iii—H2C@O, NaB(OAc)3H, MeOH; (d) i—1-Boc-4-methylenepiperidine, 9-BBN, Pd(PPh3)4, Na2CO3, 4:1 DMF:water, 120 °C; ii—CF3CO2H, CH2Cl2; iii—H2C@O,
NaB(OAc)3H, MeOH, AcOH.
O
OH
N
COOH
a
22
+
H2NHN
N
H
F
34
b
O
N
N
N
N
N
N
c
N
H
N
H
F
F
35
36
Scheme 4. Reagents and conditions: (a) i—EtOH, reflux; ii—AcOH, BF3-OEt2, 115 °C; (b) HOBt, EDCI, 1-methylpiperazine, 5:2 CH2Cl2:DMF; (c) LiAlH4, THF, reflux.

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A. Scribner et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1517–1521
Table 1
Et-PKG inhibition and in vivo anticoccidial activity of 2,3-diaryl-6-substituted indoles
R
N
A
R'
N
H
F
Compound
R
A
R0
Et-PKG IC50 (nM)
Anticoccidial Activity at 50 ppm
Et
Ea
Emi
Ema
12
18
19
13
20
21
26
27
28
29
30
31
32
33
35
36
NH2
NH2
NH2
H
H
H
H
H
H
H
H
H
H
H
H
H
N
N
N
N
N
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
1-H-Piperidin-4-yl
1-Me-Piperidin-4-yl
1-CH2CH2OH-Piperidin-4-yl
1-H-Piperidin-4-yl
1-Me-Piperidin-4-yl
1-CH2CH2OH-Piperidin-4-yl
1-H-Piperidin-4-yl
1-Me-Piperidin-4-yl
1-Et-piperidin-4-yl
1-CH2CH2OH-Piperidin-4-yl
1-Me-Piperazin-4-yl
Morpholin-4-yl
0.21
0.22
0.3
0.65
0.4
0
3
0
0
0
3+
0
2
2
2
0
2
2
0
3
3
0
0
0
0
3+
2
0
3+
0
3
0
0
0
0
0
0
—
—
—
0
2
4
0
3+
3
3
0
0
0
2
NA
0
0
0
0
0
0
0
0
2
0.8
0.15
0.15
0.16
0.1
—
9.2
0.62
1.6
12.7
8.9
3
0
—
—
—
—
—
—
1-Me-azetidin-3-yl
1-Me-piperidin-4-CH2-yl
1-Me-piperazin-4-(C@O)-yl*
1-Me-piperazin-4-CH2-yl*
*
Substituent at indole 5-position instead of 6-position.
Table 1 presents both in vitro and in vivo biological data of each
compound tested.22 In vitro activity was assessed by measuring
compound inhibition of native E. tenella (Et) PKG enzyme activity,
and is reported as an IC50. In vivo activity was determined by
administering each compound orally in feed, and then ranking
each for anticoccidial activity using a seven day efficacy model. A
quantitative measure of E. tenella (Et), E. acervulina (Ea), E. mitis
(Emi), and E. maxima (Ema) oocyst shedding from infected birds pro-
vided an assessment of antiparasitic activity. Treatments resulting
in reduction of oocyte burden by 100% are scored a ‘4’, those with
99% reduction are scored a ‘3+’, those with 80-98% reduction are
scored a ‘3’, those with 50-79% reduction are scored a ‘2’, and those
with <50% reduction are scored a ‘0’.
It is seen that the in vitro activity of all compounds was in the
subnanomolar range except for morpholine 31, which lacks a basic
nitrogen at the indole 6-position sidechain, as well as piperidine 33
and piperazines 35 and 36, each of whose most external basic
nitrogen is positioned further away from the indole core, and with
greater rotational degrees of freedom than those in all other com-
pounds tested. Within each 6-(N-substituted-piperidin-4-yl)indole
series, the order of in vitro activity with regard to the heterocycle
at the indole 3-position is consistently 3-(pyridin-4-yl) > 3-(2-
aminopyrimidin-4-yl) > 3-(pyrimidin-4-yl). Overall, the data sug-
gests that a basic nitrogen entropically fixed 3 to 4 atoms away
from the indole 6-position is optimal for in vitro activity.
cies of Eimeria tested. Of these, 3-(pyridin-4-yl)-6-(1-methylpiperi-
din-4-yl)indole 27 shows potency against all 4 species of Eimeria.
While the level of potency exhibited in the 2,3-diarylindole series
thus far does not match that of our most potent imidazopyridine
(compound 69 from reference 13, with an IC50 = 0.044 nM and
in vivo scores of 4 (Et), 3 (Ea), 3 (Emi), and 4 (Ema) at 6 ppm), the
2,3-diarylindole class does hold promise in anticoccidial therapy.
References and notes
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Anderson, J. W.; Wiltsie, J.; Diaz, C. A.; Harris, G.; Chang, B.; Darkin-Rattray, S. J.;
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J.; Biftu, T.; Schmatz, D. M. J. Biol. Chem. 2002, 277, 15913.
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R.; Simeone, J.; Chang, L.; Gurnett, A.; Liberator, P.; Dulski, P.; Leavitt, P. S.;
Crumley, T.; Misura, A.; Murphy, T.; Rattray, S.; Samaras, S.; Tamas, T.; Mathew,
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Several observations can be made regarding in vivo activity. The
N-alkylpiperidines clearly showed greater potency than the NH-
piperidines. Both the 3-(pyridin-4-yl) and 3-(pyrimidin-4-yl) series
showed comparable activity. 3-(Pyridin-4-yl)-6-(1-methylpiperi-
din-4-yl)indole 27 is the only compound tested that showed activ-
ity against all four species of Eimeria test. All compounds tested
that lacked a piperidine ring at the indole 6-position (30–33,
35,36) did not show remarkable in vivo potency.
9. Biftu, T.; Feng, D.; Fisher, M.; Liang, G.-B.; Qian, X.; Scribner, A.; Dennis, R.; Lee,
S.; Liberator, P. A.; Brown, C.; Gurnett, A.; Leavitt, P. S.; Thompson, D.; Mathew,
J.; Misura, A.; Samaras, S.; Tamas, T.; Sina, J. F.; McNulty, K. A.; McKnight, C. G.;
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In conclusion, we have prepared several novel 2,3-diaryl-6-
substituted indoles, 11 of which show subnanomolar potency
in vitro against E. tenella cGMP-dependent protein kinase (PKG),
and 4 of which show in vivo potency against at least 3 of the 4 spe-

A. Scribner et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1517–1521
1521
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19. 1-Boc-3-iodozincazetidine was prepared by the following sequence:
OH
OMs
OMs
ZnI
I
a
b
c
d
N
N
N
N
N
boc
boc
boc
Ph
Ph
Ph
Ph
(a) MsCl Et3N.; (b) i. ACECl, DCE; ii. MeOH; iii; Boc2O, Et3N, CH2Cl2.; (c) I2,
DMSO, 140 °C.; (d) zinc, chlorotrimethylsilane, 1,2-dibromoethane, DMA.
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4544.
Eds.; Wiley-VCH Verlag GmbH
pp 815–889.
& Co. KGaA: Weinheim: Germany, 2004;
17. 1-Boc-4-iodozincpiperidine was prepared in two steps by first treating 1-Boc-
4-hydroxypiperidine with triphenylphosphine, iodine, and imidazole to give 1-
Boc-4-iodopiperidine, which was then treated with zinc, chlorotrimethylsilane,
and 1,2-dibromoethane. Corley, E. G.; Conrad, K.; Murry, J. A.; Savarin, C.;
Holko, J.; Boice, G. J. Org. Chem. 2004, 69, 5120.
21. 1-Boc-4-methylenepiperidine was prepared by treating 1-Boc-piperidone
under Wittig conditions with methyltriphenylphosphonium bromide and n-
butyllithium.
22. All compounds were characterized by 1H NMR, LC/MS, and HPLC (P90% pure at
254 nm) prior to biological testing.