Discovery of (Z)-2-phenyl-3-(1H-pyrrol-2-yl)acrylonitrile derivatives active against Haemonchus contortus and Ctenocephalides felis (cat flea)

Article abstract of DOI:10.1016/j.bmcl.2006.11.043

A series of 2-phenyl-3-(1H-pyrrol-2-yl)acrylonitrile derivatives were synthesized and evaluated for in vitro activity against the endoparasite Haemonchus contortus and the ectoparasite Ctenocephalides felis. Some compounds had significant in vitro activit

Full text of DOI:10.1016/j.bmcl.2006.11.043

Bioorganic & Medicinal Chemistry Letters 17 (2007) 993–997
Discovery of (Z)-2-phenyl-3-(1H-pyrrol-2-yl)acrylonitrile
derivatives active against Haemonchus contortus
and Ctenocephalides felis (cat flea)
Abdelselam Ali,a Marianne Bliese,a Jo-Anne M. Rasmussen,a Roger M. Sargent,b
Simon Saubern,a David G. Sawutz,c John S. Wilkie,a David A. Winkler,a
Kevin N. Winzenberga,* and Ruth C. J. Woodgatea
aCSIRO, Molecular and Health Technologies, Bayview Avenue, Clayton, Vic. 3168, Australia
bSchering–Plough Pty. Ltd., 11 Gibbon Rd, Baulkham Hills, NSW 2153, Australia
cSchering–Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033-1300, USA
Received 16 October 2006; revised 9 November 2006; accepted 13 November 2006
Available online 17 November 2006
Abstract—A series of 2-phenyl-3-(1H-pyrrol-2-yl)acrylonitrile derivatives were synthesized and evaluated for in vitro activity against
the endoparasite Haemonchus contortus and the ectoparasite Ctenocephalides felis. Some compounds had significant in vitro activity
against these parasites.
ꢀ 2006 Elsevier Ltd. All rights reserved.
The development of the macrocyclic lactone class of par-
asiticides, exemplified by ivermectin, was a landmark in
the control of endoparasites, especially nematodes, in
human and animal health.1 The success of this class of
potent endoparasiticides is reflected by the subsequent
failure of the animal healthcare industry to introduce
any new class of endoparasiticidal drugs over the past
25 years, despite growing reports of the development
of parasite resistance to the macrocyclic lactones and
to other classes of parasiticide drugs in current use. In
particular, multi-drug endoparasite resistance threatens
the viability of small-ruminant production in many
tropical regions and also in temperate areas of southeast
USA, Australia, and New Zealand.2
From this exercise we obtained the hit compound,
(4,5-dichloro-1H-pyrrol-2-yl)methylenemalononitrile
(H. contortus LD99 = 1.8 lM) (Fig. 1). Significantly,
compound 1 maintained activity against resistant strains
of H. contortus: LD99 = 2.2 lM for the benzimidazole-
1
and
levamisole-resistant
Lawes
strain
and
LD99 = 2.2 lM for the avermectin-resistant CAVR
strain. Furthermore, compound 1 was active against
the McMaster strains of Ostertagia circumcinta,
LD99 = 2.2 lM, and Trichostrongylus colubriformis,
LD99 = 9.0 lM. Compound 1 was inactive in an ecto-
parasite Ctenocephalides felis (cat flea) assay.5
Compound 1 is a potential Michael acceptor and there-
fore a potential non-specific general toxin arising from
We commenced research aimed at the discovery of new
drugs to control endoparasites, based upon high-
throughput screening of compounds in the commercial
NemaTox Haemonchus contortus (McMaster strain) lar-
val development assay.3,4
Keywords: (Z)-2-phenyl-3-(1H-pyrrol-2-yl)acrylonitrile; Parasiticide;
Haemonchus contortus; Ctenocephalides felis (cat flea).
*
Corresponding author. Tel.: +61 3 9545 2222; fax: +61 3 9545
2446; e-mail: kevin.winzenberg@csiro.au
Figure 1.
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.043

994
A. Ali et al. / Bioorg. Med. Chem. Lett. 17 (2007) 993–997
covalent binding to cellular nucleophiles. Compound 1
is not universally toxic. For example, compound 1 was
compounds, unlike chlorfenapyr, do not act by this
mode of action.7,13 Consistent with this view, we also
observed that electron withdrawing substituents at the
pyrrole ring decreased ectoparasiticide activity: for
example, the 4,5-dichloropyrrole derivative 40 (calcu-
lated pKa = 11.8) is much less active against C. felis
than the corresponding unsubstituted pyrrole analog
26.
active against the
bacteria Bacillus
subtilis
(LD99 = 1.7 lM) and the murine NS-1 myeloma cell line
(LD99 = 13 lM) but inactive against the yeast Saccharo-
myces cerevisiae. This selective toxicity indicated that
compounds of this type were suitable for further
investigation.6
We hypothesized that compound 1 might act as an
uncoupler of oxidative phosphorylation, because of
structural similarity to the insecticide chlorfenapyr 2
(PirateTM). Chlorfenapyr is a prodrug whose active
metabolite 3 acts as an uncoupler of oxidative phos-
phorylation in mitochondria.7
At the 4 position of the phenyl ring of the 3-(1H-pyr-
rol-2-yl)acrylonitrile template small substituents such
as H, halogen, Me, Et, and CF3 afforded the best
activity against H. contortus. Best activity against
C. felis was observed for compounds containing
H, F, Cl, i-Pr or t-Bu as a substituent at the 4
position of the phenyl ring.
To probe SAR associated with compound 1, we
prepared a set of (Z)-2-phenyl-3-(1H-pyrrol-2-yl)acrylo-
At the 3 position of the phenyl ring of the 3-(1H-
pyrrol-2-yl)acrylonitrile template the substituents
could be ranked as follows for C. felis activity:
F ꢀ CF3 > Me > Cl > Br > PhO, but only the 3-fluor-
ophenyl derivative 16 exhibited activity comparable to
the most active of the 4-substituted com-
pounds. With respect to activity against H. contortus
3-phenyl-substituted compounds were generally less
active compared to the corresponding 4-substituted
derivative.
nitriles
6
from benzyltrimethylammonium hydro-
xide-catalyzed Knoevenagel condensation reactions of
various 1H-pyrrole-2-carboxaldehydes 4 with phenyl-
acetonitrile derivatives 5 performed in either ethanol
or water (Scheme 1 and Table 1).8–12
With particularly hydrophobic reactants, the reaction
was more conveniently catalyzed using 18-crown-6 and
potassium hydroxide in toluene. For some sterically hin-
dered ortho-substituted phenylacetonitrile derivatives, it
was necessary to conduct the reaction at elevated pres-
sure in a sealed glass tube.
Ctenocephalides felis mortality for compounds contain-
ing a substituent at the 2 position of the phenyl ring
was ranked: Me > H > F ꢀ Br > Cl > CF3 > CN >
OMe > Ph. The ortho-methyl-substituted compound
12 was the most active ectoparasiticide discovered in
this work. With respect to activity against H. contor-
tus, 2-phenyl-substituted compounds were generally
less active compared to the corresponding 4-substitut-
ed derivative.
A number of these compounds which contained electron
withdrawing substituents at either the pyrrole ring or the
phenyl ring or at both rings displayed excellent activity
against H. contortus (Table 1). Many compounds
showed levels of activity comparable to the commercial
endoparasiticidal drugs levamisole and closantel. Signif-
icantly, some of these compounds were also active
against C. felis, indicating that the 3-(1H-pyrrol-2-yl)ac-
rylonitrile template had the potential to deliver valuable
broad-spectrum endectoparasiticidal activity. In partic-
ular, the 4-chlorophenyl derivative 26 gave an
LD99 = 1.8 lM in the nematode assay, 95% mortality
Di-substitution by halogen at the phenyl ring of the
3-(1H-pyrrol-2-yl)acrylonitrile template generally led
to diminished activity against both H. contortus and
C. felis.
in
a
rapid cat flea mortality assay, and an
In an attempt to improve the parasiticidal activity of
compound 26, we prepared a small set of derivatives
in which the pyrrole nitrogen was capped (Table 2).
Compounds 53, 54, 56, and 59 were prepared by
reaction of the appropriate capped 1H-pyrrole-2-car-
boxaldehyde with 4-chlorophenylacetonitrile whereas
compounds 55, 57, 58, 60, 61, 62, 63, and 64 were pre-
pared from the reaction of 26 with the appropriate elec-
trophilic capping reagents.12,14
LC50 = 0.17 lg/cm2 for C. felis.
We observed that calculated pKa values for 1 and 26
(10.6 and 15.0, respectively) fall outside the range
7–8 reported as being necessary for uncoupling of
oxidative phosphorylation, which indicates that these
With the exception of compound 55 all of these capped
compounds were significantly less active than the un-
capped compound 26 against H. contortus. None of
the capped compounds showed significant activity
against C. felis.
To summarize, unsubstituted (Z)-2-phenyl-3-(1H-pyr-
rol-2-yl)acrylonitrile (compound 7) retained the best
balance of endo- and ectoparasiticide activity (H. con-
tortus LD99 = 2.4 lM; C. felis LC50 = 0.26 lg/cm2,
Scheme 1. Reagents: (i) PhCH2NMe3(OH), EtOH/H2O or 18-crown-6,
KOH, PhCH3.

A. Ali et al. / Bioorg. Med. Chem. Lett. 17 (2007) 993–997
Table 1. Activity of compounds 7–52 against H contortus (H.c.) and C. felis (C.f.)
995
R1
R2
H.c. LD99 (lM)
C.f. % Killa,b
C.f. LC50 (lg/cm2)
a
a
Compound
7
H
H
2-F
2.4
12
98
92
19
30
14
100
2
0.26
0.59
8
H
9
10
H
2-Cl
2-Br
12
12
H
11
12
H
2-CF3
2-Me
2-Ph
H
18
0.08
0.78
9.9
13
14
H
H
2-OMe
2-CN
3-F
9
15
16
H
>68
8
11
78
18
28
24
56
H
17
18
4-Br
H
3-F
3-Cl
38
3.3
3.4
3.6
9.5
6.0
11
19
20
H
3-Br
H
3-CF3
3-CF3
3-Me
21
22
4-Br
H
42
10
78
15
95c
23
42
14
20
15
69
58
31
44
6
23
24
H
3-OPh
4-F
4-F
H
2.0
17
0.30
0.17
25
26
4-Br
H
4-Cl
4-Cl
1.8
1.5
0.84
2.2
3.1
>44
4.5
4.0
6.7
46
27
28
4-Br
H
4-Br
4-I
39
30
H
H
4-CF3
4-CF3
4-Me
31
32
4-Br
H
33
34
H
4-Et
H
4-OMe
4-CN
4-NO2
4-i-Pr
4-t-Bu
4-Ph
35
36
H
H
>63
37
38
H
100
87
46
15
8
1.32
0.48
H
13
28
39
40
H
4,5-diCl
H
4-Cl
10
11
41
42
2,4-diCl
3,4-diCl
3,4-diCl
2-Cl, 4-F
3-Cl, 4-F
2-Cl, 6-F
2-F, 4-Cl
2,4-diF
2,6-diF
3,4-diF
3,5-diCF3
2,4-diMe
H
2.9
7.3
12
41
19
30
5
43
44
4-Br
H
45
46
H
5.1
>61
10
H
7
47
48
H
5
H
11
52
5
49
50
H
17
17
27
44
H
7.6
>45
51
52
H
H
Levamisole
Closantel
Ivermectin
Fipronil
0.66–2.0
9.4–18
0.0057–0.031
0.018
a No entry indicates that the compound was not assayed.
b Measured at 24 h.
c Measured at 8 h.
respectively), whereas (Z)-2-(3-methylphenyl)-3-(1H-
pyrrol-2-yl)acrylonitrile (compound 12) displayed
There is significant potential for further exploration of
parasiticide activity associated with structural modifica-
tion to the (Z)-2-phenyl-3-(1H-pyrrol-2-yl)acrylonitrile
template discovered from our research.
the best ectoparasiticide activity (C. felis LC50
=
0.08 lg/cm2).

996
A. Ali et al. / Bioorg. Med. Chem. Lett. 17 (2007) 993–997
Table 2. Activity of compounds 53–64 against H. contortus (H.c.) and
C. felis (C.f.)
Michael acceptor when treated with excess ethanethiol in
CDCl3 or excess sodium methanethiolate in CD3OD at
room temperature for 24 h, consistent with the view that
compounds 6 would not act as general toxins. We thank a
referee for suggesting the use of thiol reagents to probe the
propensity of compounds of this type to act as a partner in
covalent binding to cellular nucleophiles. Further in vitro
studies with mammalian cells will be required to unequiv-
ocally clarify the mammalian safety of compounds derived
from the (Z)-2-phenyl-3-(1H-pyrrol-2-yl)acrylonitrile
template.
Compound
R
H.c. LD99 C.f. % Mortalitya
(lM)
26
53
54
55
56
57
58
59
60
61
62
63
64
H
Me
1.8
7.8
95b
13b
6b
12
3
7. Black, B. C.; Hollingworth, R. M.; Ahammadsahib, K. I.;
Kukel, C. D.; Donovan, S. Pestic. Biochem. Physiol. 1994,
50, 115.
8. Herz, W.; Brasch, J. J. Org. Chem. 1958, 23, 711.
9. Alberghina, G.; Amato, M. E.; Bottino, F. A.; Corsaro,
A.; Fisichella, S. J. Heterocycl. Chem. 1986, 23, 1747.
10. Winzenberg, K. N.; Saubern, S.; Sawutz, D. G. PCT Int.
Appl. WO 06/055565, 2006.
CH2OEt
Propanoyl
Benzyl
19
1.3
44
Et2NC(O)
tert-Butoxycarbonyl
11
11
8
9
3-Methyl-2-butenyl >51
Benzoyl
Tosyl
0
3.8
6
11. Synthesis
of
2-phenyl-3-(1H-pyrrol-2-yl)acrylonitrile
a
compounds, typical procedures:10 Compound 12:
31
32
0
Me2NC(S)
MeOC(O)
Benzyloxycarbonyl
19
16
20
thick-walled tube was charged with 1H-pyrrole-2-carbox-
aldehyde (2.0 g, 21.0 mmol), 2-methylphenylacetonitrile
(2.5 g, 18.9 mmol), EtOH (75 mL), and 40% aqueous
benzyltrimethylammonium hydroxide (4.0 mL, 3.3 mmol).
The tube was sealed and heated to 90 ꢁC for 4 days. The
solvent was evaporated and the residue subjected to
chromatography on silica gel eluting with CH2Cl2 /light
petroleum (20:80) to afford compound 12 as a yellow solid
(1.2 g, 30% yield), mp 87–89 ꢁC. 1H NMR (200 MHz,
CDCl3) d 9.81 (br s, 1H), 7.18–7.30 (m, 4H), 7.07 (m,
1H), 7.03 (s, 1H), 6.63 (m, 1H), 6.34 (m, 1H), 2.48 (s, 3H).
MS (EI) 208 (M+). Compound 26: a suspension of 1-H-
pyrrole-2-carboxaldehyde (3.3 g, 34.7 mmol) and 4-chlor-
ophenylacetonitrile (5.5 g, 33.0 mmol, 0.95 equiv) in H2O
(50 mL) was heated to 50 ꢁC. After all the solid material
had melted, the vigorously stirred mixture was treated
with 40% aqueous benzyltrimethylammonium hydroxide
(14 mL, 5.6 mmol). Stirring was continued for 5 h at
50 ꢁC, breaking up lumps when necessary. The still warm
suspension was filtered through a sintered glass funnel and
the yellow precipitate was washed with warm H2O and
dried to afford compound 26 (6.63 g, 88% yield), mp 116–
118 ꢁC, lit mp 122–123 ꢁC.8
4.4
15
a Measured at 24 h.
b Measured at 8 h.
References and notes
1. Chabala, J. C.; Mrozik, H.; Tolman, R. L.; Eskola, P.;
Lusi, A.; Peterson, L. H.; Woods, M. F.; Fisher, M. H.;
Campbell, W. C.; Egerton, J. R.; Ostlind, D. A. J. Med.
Chem. 1980, 23, 1134.
2. Kaplan, R. M. Trends Parasitol. 2004, 20, 477.
3. Nematocide assays were conducted by Microbial Screen-
ing Technologies Pty. Ltd., Smithfield, NSW, Australia.
H. contortus LD99 values were determined using the larval
development assay described in reference.4
4. Gill, J. H.; Redwing, J. M.; Van Wyk, J. A.; Lacey, E. Int.
J. Parasitol. 1995, 25, 463.
5. (a) Ctenocephalides felis assays were conducted by the
Centre for Entomological Research and Insecticide Tech-
nology, UNSW, Randwick, NSW, Australia; (b) In vitro
single dose C. felis assay: the test compound was dissolved
in acetone (0.5 mL) and applied to the base of four
100 mL flasks at a concentration of 1.26 lg/cm2. Flasks
were left to dry for 24 h before flea exposure. Controls
were prepared in the same manner, except that no test
compound was dissolved in the acetone. Fifteen adult
fleas, aged between 3 and 7 days postemergence, were then
introduced into each flask. The top of the flasks was
covered in ParafilmTM and small holes were made to allow
gas exchange. The treatment flasks were held at 25 1 ꢁC
and 75 5% humidity for 24 h. Dead fleas in each flask
were counted and the pooled data were converted to
percentages; (c) In vitro C. felis LC50 measurement: six
concentrations of test compound were obtained from
serial dilution of an acetone solution of the compound.
For each test compound this set of doses covered a range
that produced very low to very high flea mortality; this
range was determined from a pilot study. Mortality
resulting from the treatments was recorded at 24 h. Pooled
mortality data were subjected to probit analysis to obtain
concentration response data (LC50) (Finney, D. J. Probit
Analysis, 3rd ed., Cambridge Univ. Press, London, 1971).
6. Subsequently, in separate experiments we found no 1H
NMR spectroscopic evidence that compound 26 acts as a
12. All synthetic intermediates and final products were char-
1
acterized by H NMR and MS.
13. pKa values were calculated using SPARC Aug2003 server
<http://ibmlc2.chem.uga.edu/sparc/>. The calculated pKa
values for pyrrole compounds with known pKa values
differed consistently by about half a log unit. For example,
the calculated pKa for compound 3 is 8.19, whereas the
measured pKa is reported to be 7.6.7
14. Synthesis of capped pyrrole derivatives, typical
procedures:10 Compound 54: to a solution of 4-chlor-
ophenylacetonitrile (500 mg, 3.30 mmol), 1-ethoxymeth-
yl-1H-pyrrole-2-carboxaldehyde (520 mg, 3.39 mmol),
and 18-crown-6 (87 mg, 0.33 mmol) in toluene (30 mL)
was added KOH (185 mg, 3.30 mmol) and the mixture
was heated to 80 ꢁC for 3 h, then stirred at room
temperature overnight. The solution was filtered
through a small plug of silica and concentrated to
afford compound 54 as an oil (705 mg, 73% yield). 1H
NMR (200 MHz, CDCl3) d 7.60 (s, 1H), 7.56 (m, 1H),
7.55 (d, J 8.8 Hz, 2H), 7.38 (d, J 8.8 Hz, 2H), 6.93 (m,
1H), 6.33 (m, 1H), 5.39 (s, 2H), 3.44 (q, J 6.9 Hz, 1H),
1.18 (t, J 6.9 Hz, 1H). MS (APCI+) 287.2 (M+1).
Compound 55: to a stirred solution of compound 26
(600 mg, 2.62 mmol), Et3N (320 mg), and 4-dimethyla-
minopyridine (32 mg) in CH2Cl2 (20 mL) was added

A. Ali et al. / Bioorg. Med. Chem. Lett. 17 (2007) 993–997
997
propionic anhydride (375 mg). The mixture was stirred
at room temperature for 7 h. Et3N (320 mg) and
propionic anhydride (375 mg) were added and the
mixture was stirred at room temperature for 48 h.
The reaction mixture was poured into ether (200 mL)
and the ether was washed with 10% aqueous citric acid
solution (50 mL), H2O (2 · 50 mL), and saturated brine
(50 mL). The ether layer was collected, dried over
Mg2SO4, filtered, and evaporated. The residue was
recrystallized from ether/light petroleum to afford com-
pound 55 as yellow needles (611 mg, 82% yield), mp
106–108 ꢁC. 1H NMR (200 MHz, CDCl3) d 8.48 (s,
1H), 7.59 (d, J 8.8 Hz, 2H), 7.47 (d, J 4.4 Hz, 1H),
7.38 (d, J 8.8 Hz, 2H), 7.32 (dd, J 3.7 and 1.5 Hz, 1H),
6.43 (t, J 3.7 Hz, 1H), 2.97 (q, J 7.3 Hz, 2H), 1.31 (t,
J 7.3 Hz, 3H). MS (EI) 284 (M+). Compound 59: a
solution of 25 (200 mg, 0.875 mmol) in dry acetone
(35 mL) was treated with K2CO3 (121 mg) followed by
1-bromo-3-methyl-2-butene (111 lL, 0.962 mmol). The
mixture was heated to a gentle reflux for 20 h. The
reaction mixture was cooled to room temperature and
filtered. The filtrate was chromatographed on silica gel
eluting with EtOAc/light petroleum (1:1) to afford
compound 59 as
a
yellow oil (85 mg). 1H NMR
(200 Hz, CDCl3) d 7.47–7.50 (m, 3H), 7.32–7.41 (m,
2H), 6.88 (s, 1H), 6.31 (s, 1H), 5.30 (t, J 6.2 Hz, 1H),
4.62 (s, 1H), 4.60 (s, 1H), 1.79 (s, 3H), 1.78 (s, 3H).
MS (EI) 296 (M+).