N-Propargylated isatin-Mannich mono- and bis-adducts: Synthesis and preliminary analysis of in vitro activity against Tritrichomonas foetus

Article abstract of DOI:10.1016/j.ejmech.2014.01.015

Cu(I)Cl promoted synthesis of N-propargylated-isatin Mannich mono- and bis-adducts with an extension towards the synthesis of N-propargylated-isatin-7- chloroquinoline conjugates was described. The synthesized scaffolds were evaluated for their in vitro activity against the veterinary protozoal pathogen Tritrichomonas foetus and cytotoxicity against human prostate (PC-3) cancer cell line. The preliminary evaluation data revealed the enhancement in the activity profiles with the introduction of 7-chloroquinoline ring with the most active conjugates 7a, 7c and 7d exhibiting an IC₅₀ of 22.2, 11.3 and 24.5 μM respectively against T. foetus and minimal toxicity against human prostate (PC-3) cell lines.

Full text of DOI:10.1016/j.ejmech.2014.01.015

European Journal of Medicinal Chemistry 74 (2014) 657e663
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
N-Propargylated isatin-Mannich mono- and bis-adducts: Synthesis
and preliminary analysis of in vitro activity against Tritrichomonas
foetus
Nisha a, Kewal Kumar a, Gaurav Bhargava b, Kirkwood M. Land c, Kai-Hsiang Chang c,
,
Reena Arora d, Somdutta Sen d, Vipan Kumar a
*
a Department of Chemistry, Guru Nanak Dev University, Amritsar 143005, India
b Department of Applied Sciences, Punjab Technical University, Kapurthala 144601, India
c Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA
d Pharmacology Department, Sphaera Pharma Pvt. Ltd, Manesar 12205, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Cu(I)Cl promoted synthesis of N-propargylated-isatin Mannich mono- and bis-adducts with an extension
towards the synthesis of N-propargylated-isatin-7-chloroquinoline conjugates was described. The syn-
thesized scaffolds were evaluated for their in vitro activity against the veterinary protozoal pathogen
Tritrichomonas foetus and cytotoxicity against human prostate (PC-3) cancer cell line. The preliminary
evaluation data revealed the enhancement in the activity profiles with the introduction of 7-
chloroquinoline ring with the most active conjugates 7a, 7c and 7d exhibiting an IC50 of 22.2, 11.3 and
Received 26 September 2013
Received in revised form
26 November 2013
Accepted 10 January 2014
Available online 18 January 2014
24.5
m
M respectively against T. foetus and minimal toxicity against human prostate (PC-3) cell lines.
Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords:
N-Propargylated-isatin Mannich adducts
N-Propargylated-isatin-7-chloroquinoline
conjugates
Tritrichomonas foetus
Cytotoxicity
1. Introduction
around the world. In light of this current problem, the identification
of new chemotherapies for this pathogen is still warranted.
Bovine trichomoniasis is a veneral disease caused by the pro-
tozoal pathogen Tritrichomonas foetus and is considered to be one of
the most important infectious causes of reproductive failure in beef
cattle drastically reducing their breeding efficiency [1e4]. The
infection in bulls is primarily acquired through direct sexual con-
tact and is asymptomatic in nature affecting neither the semen
quality nor the sexual behaviour although in some cases, early
death of developing foetus has also been observed [5e7]. Literature
rationale has also revealed that the parasite provoked cell death to
bovine oviduct epithelial cells [8] and extracellular products
secreted by T. foetus were cytotoxic to bovine sperm [9]. Although
the use of artificial insemination in breeding cattle has led to the
virtual eradication of this organism from the cattle population in
many countries, bovine trichomonad infection still is responsible
for significant economic losses in the free-ranging cattle industry
Isatin is a versatile scaffold with wide possibility of chemical
modification and display a diverse array of biological and phar-
macological activities which include anti-HIV [10], antiviral [11],
anti-tumour [12e14], antifungal [15,16], anti-angiogenic [17], an-
ticonvulsants [18], anti-Parkinson’s disease therapeutic [19], and
effective SARS coronavirus 3CL protease inhibitor [20]. An extensive
investigation on the synthetic versatility of isatin has disclosed
many interesting aspects of organic synthesis allowing the gener-
ation of a large number of structurally diverse analogues derived
from electrophilic aromatic substitution at positions C-5 and C-7 of
the phenyl ring, nucleophilic additions onto the C-3 carbonyl group,
chemoselective reductions, oxidations, ring-expansions, and spiro-
annulations [21]. However there are only few reports regarding the
antineoplastic activities of N-alkyl and N-aryl isatins. Vine and co-
workers have reported the apoptosis behaviour of N-alkyl/aryl
isatins through introduction of various hydrophobic substituents
like N-phenethyl, N-benzyl and N-2-naphthylmethyl on the parent
nucleus that significantly increased their cytotoxicity towards
lymphoma cells and against a wide range of human cancer cell
lines, including MDA-MB-231 metastatic breast adenocarcinoma
* Corresponding author. Tel.: þ91 183 2258802 09x3320; fax: þ91 183 2258819
20.
E-mail address: vipan_org@yahoo.com (V. Kumar).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.015

658
Nisha et al. / European Journal of Medicinal Chemistry 74 (2014) 657e663
Scheme 1. Cu(I)Cl promoted N-propargylated isatin Mannich adducts.
cells and U937 human monocyte-like histolytic lymphoma cells
[22]. Recent report from our lab has shown the synthesis of N-alkyl-
isatin along with their cytotoxic profiles against a panel of cancer
cell lines [23]. The approach has been further extended towards the
synthesis of 1H-1,2,3-triazole-tethered isatin conjugates with four
of the synthesized scaffolds being twice as potent as 5-fluorouracil
on THP-1 cell line [24].
In continuation with our interest towards the synthesis of novel
molecular frameworks having biological relevance [25], we
recently disclosed the synthesis of 1H-1,2,3-triazole-tethered isa-
2. Results and discussion
2.1. Synthetic chemistry
The synthetic approach devised for the synthesis of desired
hybrids involved sodium hydride promoted initial propagylation of
C-5 substituted isatins 1 with propargyl bromide in dry DMF to
yield 2. This upon refluxing at 110 ꢀC in dry dioxane with secondary
amines and para-formaldehyde in the presence of catalytic amount
of Cu(I)Cl resulted in the isolation of desired N-propargylated-isatin
Mannich adducts. Mechanistically, the reaction is thought to pro-
ceed by an initial formation of Cu-acetylide I which undergoes
Mannich addition as shown in Scheme 1. Interestingly, the reaction
employing piperazine as the secondary amine unit resulted in the
isolation of bis-adduct formed via an initial condensation reaction
of piperazine with para-formaldehyde at 1st and 4th position to
result in the corresponding bis-iminium ion with subsequent
Mannich addition.
The developed methodology has been further extended towards
the synthesis of N-propargylated isatin-7-chloroquinoline hybrids
via Cu-mediated Mannich reaction of C-5 substituted N-propargy-
lated isatins with 7-chloro-4-piperazin-1-yl-quinoline 6, prepared
by heating 4,7-dichloroquinoline with an excess of piperazine
and triethylamine at 120 ꢀC overnight Scheme 2. The inclusion of 7-
tin-
their in vitro activity against Trichomonas vaginalis at 10 and 100
[26]. Eighteen of the synthesized hybrids have shown 100% growth
inhibition at 100 M with the most potent and non-cytotoxic
compound showing and IC50 of 7.69 M. The methodology was
further extended towards the synthesis of -amino alcohol based
lactameisatin conjugates and their evaluation against T. vaginalis at
50 M with the most active chimeric scaffold among the test series
exhibited an IC50 of 9.73 M along with low cytotoxicity [27]. As an
b-lactam conjugates along with the preliminary analysis of
mM
m
m
b
b-
m
m
extension of this work, we now describes the Cu(I)Cl-facilitated
synthesis of N-propargylated-isatin Mannich adducts along with
their preliminary in vitro evaluation studies against the veterinary
protozoal pathogen T. foetus and cytotoxicity against human pros-
tate (PC-3) cancer cell line.
Scheme 2. Cu(I)Cl promoted N-propargylated isatin-quinoline Mannich adducts.

Nisha et al. / European Journal of Medicinal Chemistry 74 (2014) 657e663
659
chloroquinoline nucleus in the synthesized conjugates is based on
the recent disclosure by Dwivedi and co-workers on the promising
activity of the quinoline based compounds against different strains
of T. vaginalis [28].
The structure to the synthesized Mannich adducts were
assigned on the basis of spectral data and analytical evidences. The
compound 3d, for example was characterized as 1-(4-Morpholine-
4-yl-but-2-ynyl)-1H-2-indole-2,3-dione analysed for C16H16N2O3
and showed molecular ion peak at m/z 284.11 (Mþ) in its mass
spectrum. The salient features of its 1H NMR spectrum include
preference for halogen substituent at C-5 position of isatin ring. The
most potent of the synthesized hybrids 7b and 7c showing 100%
growth inhibition at 50 mM. The active compounds viz. 7a, 7b and
7c from the preliminary inhibition data were chosen in order to
determine their IC50 which is the minimum concentration required
for 50% growth inhibition and the results are tabulated in Table 2.
As evident from Table 2, although the synthesized conjugates are
not as active as the standard drug metronidazole, the compounds
showed potent activity against the veterinary protozoal pathogen T.
foetus. The compound 7c having a chloro-substituent at C-5 posi-
tion of the isatin ring proved to be the most potent of the synthe-
characteristic peaks at
4H) corresponding to the morpholine group along with two sin-
glets corresponding to two methylenes at 3.25 and 4.55 respec-
tively. 13C NMR spectrum of compound 3d exhibited the
appearance of a characteristic peak at 79.5 corresponding to two
acetylenic carbons along with peaks at 157.0 and 182.58 assigned
d
2.49(J ¼ 4.4 Hz, 4H) and 3.69 (J ¼ 4.4 Hz,
sized scaffolds with an IC50 of 11.3 mM. The synthesized compounds
d
were further evaluated for their cytotoxicity against human pros-
tate (PC-3) cancer cell lines in order to confirm if the observed
activity profiles are because of their intrinsic anti-trichomonas
activity or the cytotoxicity. The cytotoxic evaluation results of the
d
d
to the isatin ring carbonyls further corroborating the assigned
structure.
test compounds at 0.2, 2.0 and 20 mM are enlisted in Table 1. As
evident, virtually all the synthesized Mannich adducts are devoid of
cytotoxicity except 5a and 5f, showing that the bis-adducts were
cytotoxic against PC-3 cell lines compared to the mono-adducts.
The most potent anti-trichomonas scaffolds viz. 7a, 7b, 7c and 7d
showed no cytotoxicity against PC-3 cell line and can be considered
as a good hit.
2.2. In vitro evaluation against T. foetus and cytotoxicity against
PC-3 cell lines
The synthesized compounds were evaluated for their anti-
In conclusion, the present manuscript describes the Cu(I)Cl
promoted N-propargylated-isatin-Mannich mono- as well as bis-
adducts with an extension towards the synthesis of N-propargy-
lated-isatin-7-chloroquinoline conjugates. The synthesized scaf-
folds were evaluated for their activity against veterinary protozoal
pathogen T. foetus. The preliminary evaluation data revealed
that the introduction of 7-chloroquinoline ring markedly
enhanced the activity profiles with the most active compound
trichomonas activity against the veterinary protozoal pathogen T.
foetus at 50 mM and the results are provided in Table 1. As evident
from Table 1, the anti-trichomonas activity of the test compounds
were found to be dependent upon the nature of substituent at C-5
position as well as the presence of nature of secondary amine. The
presence of a eCH3 substituent at C-5 position of isatin ring of
the synthesized Mannich adducts considerably improved the ac-
tivity profile as evident by compounds 3ae3p and 5ae5d. Further,
the introduction of 7-chloro-4-piperazin-1-yl-quinoline moiety
7c exhibiting an IC50 of 11.3
mM. The cytotoxic studies against
human prostate (PC-3) cell lines showed that the synthesized
Mannich adducts are virtually devoid of any cytotoxicity and the
isatin-7-chloroquinoline framework can act as an ideal starting
point for the synthesis of new pharmacological templates against
T. foetus.
remarkably improved the anti-trichomonas activity with
a
Table 1
Inhibitory activity of compound library Tritrichomonas foetus and cytotoxicity
against human prostate (PC-3) cancer cell line.
3. Experimental section
Compound
R
R1
Average %
inhibition
at 50 mM
Cytotoxicity% inhibition
0.2 20 mM
m
M
2
m
M
Melting points were determined by open capillary using Veego
Precision Digital Melting Point apparatus (MP-D) and are uncor-
rected. 1H NMR spectra were recorded in deuterochloroform and
DMSO-d6 with Jeol 300 (300 MHz) spectrometers using TMS as
internal standard. Chemical shift values are expressed as parts per
million downfield from TMS and J values are in hertz. Splitting
patterns are indicated as s: singlet, d: doublet, t: triplet, m:
multiplet, dd: double doublet, ddd: doublet of a doublet of a
doublet, and br: broad peak. 13C NMR spectra were recorded on
Jeol 300 (75 MHz) spectrometers in deuterochloroform and
DMSO-d6 using TMS as internal standard. High resolution mass
spectra were recorded on Bruker-micrOTOF-Q II spectrometer.
Column chromatography was performed on a silica gel (60e
120 mesh).
(Tritrichomonas
foetus)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
5a
5b
5c
5d
7a
7b
7c
7d
7e
H
H
H
H
Cl
Cl
Cl
Cl
F
Piperidine
Pyrrolidine
Morpholine
18.2
10.6
9.1
6.0
7.0
13.4
8.0
11.1
4.6
0.8
4.0
7.0
11.6
4.6
10.7
4.3
4.3
7.0
3.0
8.7
8.0
18.7
9.6
N,N-Diethylamine 22.7
Piperidine
Pyrrolidine
Morpholine
3.0
15.2
22.7
3.4
N,N-Diethylamine 42.4
4.8
5.6 6.4
Piperidine
Pyrrolidine
Morpholine
40.9
33.3
56.1
ꢁ4.7
ꢁ6.0 5.1
F
F
F
5.0
4.3
ꢁ2.7
3.2
7.9
2.4
6.1
3.8
11.6
6.8
1.9
8.0
11.5
6.5
24.4
ꢁ0.6
ꢁ1.0
9.0
0.7
N,N-Diethylamine 18.2
14.5
10.5
4.7
CH3 Piperidine
CH3 Pyrrolidine
CH3 Morpholine
31.8
65.2
57.6
7.8
3.5
CH3 N,N-Diethylamine 51.5
10.2
10.3
28.9
ꢁ3.6
6.7
10.1
5.0
1.2
11.2
45.8
24.8
7.4
Table 2
H
Cl
F
CH3
H
F
Cl
CH3
NO2
e
e
e
e
e
3.0
7.6
9.1
71.2
98.5
100.0
100.0
59.1
60.6
IC50 of the synthesized hybrids.
Compound
IC50 (mM) on protozoal pathogen
7.1
Tritrichomonas foetus
ꢁ3.2
2.1
6.1
1.3
0.5
3.0
7a
7c
7d
22.2
11.3
24.5
0.72
e
e
e
1.4
8.2
4.8
8.1
3.2
Metronidazolea
GDC-0941
30.2
56.8
72.2
a
Current FDA approved treatment for T. vaginalis infections.

660
Nisha et al. / European Journal of Medicinal Chemistry 74 (2014) 657e663
3.1. Procedure for the synthesis of C-5 substituted N-propargylated
isatins 2
3.3.3. 1-(4-Morpholin-4-yl-but-2-ynyl)-1H-indole-2,3-dione (3c)
Orange solid, yield 88%, m.p. 158 ꢀC: 1H NMR (300 MHz, CDCl3):
d
2.49 (t, J ¼ 4.4 Hz, 4H, eCH2eNeCH2e); 3.25 (s, 2H, eCH); 3.69 (t,
To a stirred suspension of sodium hydride (1.5 mmol) in dry
DMF (10 mL) was added isatin (1 mmol), resulting in the formation
of purple coloured anion. The solution was stirred at room tem-
perature till the evolution of hydrogen ceases. To this reaction
mixture was added dropwise a solution of propargyl bromide
(1.1 mmol) in dry DMF. The reaction mixture was heated to 60 ꢀC
with constant stirring for about 6 h. After the completion of reac-
tion, as evident from TLC, it was quenched by dropwise addition of
water (20 mL) and subsequently extracted with ethyl acetate
(3 ꢂ 30 mL). The combined organic layers were washed with brine
solution, dried over anhydrous Na2SO4 and concentrated under
reduced pressure. Purification of the reaction mixture via column
chromatography using hexane: ethyl acetate (4:1) mixture fur-
nished the desired N-propargylated isatin derivatives in good
yields.
J ¼ 4.4 Hz, 4H, eCH2eOeCH2e); 4.55 (s, 2H, eCH); 7.07e7.18 (m,
2H, AreH); 7.60e7.65 (m, 2H, AreH) 13C NMR (CDCl3, 75 MHz):
29.4, 47.4, 52.6, 66.5, 79.7, 110.9, 117.4, 124.0, 125.2, 138.1, 149.6,
157.0, 182.5, HRMS calculated for C16H16N2O3 284.1161 [Mþ] found
284.1169; Analysis calculated for C16H16N2O3: C, 67.59; H, 5.67; N,
9.85; found C, 67.65; H, 5.72, N, 9.78.
3.3.4. 1-(4-Diethylamino-but-2-ynyl)-1H-indole-2,3-dione (3d)
Viscous liquid, yield 76%, 1H NMR (300 MHz, CDCl3):
d 1.05 (t,
J ¼ 7.1 Hz, 6H, CH3); 2.52e2.60 (m, 4H, eCH2eNeCH2e); 3.25 (s, 2H,
eCH2); 4.54 (s, 2H, eCH2); 7.07e7.16 (m, 2H, AreH); 7.56e7.65 (m,
2H, AreH) 13C NMR (CDCl3, 75 MHz): 14.8, 29.5, 46.7, 47.4, 80.1,
110.9, 117.7, 124.1, 125.3, 138.2, 149.7, 157.1, 182.5, HRMS calculated
for C16H16N2O2 270.1368 [Mþ] found 270.1359; Analysis calculated
for C16H16N2O2: C, 71.09; H, 6.71; N, 10.36 found C, 71.01; H, 6.83; N,
10.47.
3.2. Procedure for the synthesis of 7-chloro-4-piperazin-1-yl-
quinoline 6
3.3.5. 5-Chloro-1-(4-piperidin-1-yl-but-2-ynyl)-1H-indole-2,3-
dione (3e)
To a well stirred solution of 4,7-dichloroquinoline (1 mmol) and
triethylamine (1.2 mmol), piperazine (1 mmol) was added and the
reaction mixture was allowed to stir at 110 ꢀC for 8 h. The progress
of the reaction mixture was monitored with TLC and on comple-
tion; it was washed with 2 N NaOH solution with subsequent
extraction with ethy lacetate (3 ꢂ 30 mL). The combined organic
layers were washed with brine solution, dried over anhydrous
Na2SO4 and concentrated under vacuum resulting in the desired
product.
Orange solid, yield 83%, m.p. 147 ꢀC: 1H NMR (300 MHz, CDCl3):
d
1.17e1.56 (m, 6H, eCH2eCH2eCH2e); 2.41 (s, 4H, eCH2eNeCHe
); 3.23 (s, 2H, eCH2); 4.54 (s, 2H, eCH2); 7.08e7.11 (m, 1H, AreH);
7.57e7.60 (m, 2H, AreH): 13C NMR (CDCl3, 75 MHz): 23.6, 25.6,
29.6, 47.5, 53.2, 80.1, 112.4, 118.4, 125.1, 129.8, 137.6, 148.0, 151.4,
181.6, HRMS calculated for C17H17ClN2O2 316.0979 [Mþ] found
316.0987; Analysis calculated for C17H17ClN2O2: C, 64.46; H, 5.41; N,
9.84 found C, 64.51; H, 5.49; N, 9.78.
3.3.6. 5-Chloro-1-(4-pyrrolidin-1-yl-but-2-ynyl)-1H-indole-2,3-
dione (3f)
3.3. Typical procedure for the synthesis of N-propargylated-isatin-
Mannich adducts 3ae3p
Orange solid, yield 87%, m.p. 139 ꢀC: 1H NMR (300 MHz, CDCl3):
d
1.75e1.77 (m, 4H, eCH2eCH2e); 2.51e2.55 (m, 4H, eCH2eNe
To a well stirred solution of N-propargylated isatin (1 mmol) in
dry dioxane, catalytic amount of copper chloride and p-formalde-
hyde was added in succession. The reaction mixture was stirred for
10 min with subsequent addition of secondary amine (2 mmol) and
refluxed at 120 ꢀC for 8 h. After the completion of reaction as
evident from TLC, the reaction mixture was filtered and subse-
quently extracted with ethyl acetate (3 ꢂ 30 mL). The combined
organic layers were dried over anhydrous Na2SO4 and concentrated
under reduced pressure to result in the desired Mannich adducts in
good yields.
CHe); 3.36 (s, 2H, eCH2); 4.54 (s, 2H, eCH2); 7.07e7.10 (m, 1H, Are
H); 7.56e7.59 (m, 2H, AreH) 13C NMR (CDCl3, 75 MHz): 23.6, 29.9,
43.2, 52.8, 81.6, 112.48, 118.5, 125.2, 129.9, 137.6, 148.1, 156.5, 181.8,
HRMS calculated for C16H15ClN2O2 302.0822 [Mþ] found 302.0831;
Analysis calculated for C16H15ClN2O2: C, 63.47; H, 4.99; N, 9.25
found C, 63.52; H, 4.91; N, 9.31.
3.3.7. 5-Chloro-1-(4-morpholin-4-yl-but-2-ynyl)-1H-indole-2,3-
dione (3g)
Orange solid, yield 85%, m.p. 160 ꢀC: 1H NMR (300 MHz, CDCl3):
d
2.50 (t, J ¼ 4.5 Hz, 4H, eCH2eNeCH2e); 3.26 (t, J ¼ 2.0 Hz, 2H, e
3.3.1. 1-(4-Piperadin-1-yl-but-2-ynyl)-1H-indole-2,3-dione (3a)
Orange solid, yield 87%, m.p. 149 ꢀC: 1H NMR (300 MHz, CDCl3):
CH); 3.71 (t, J ¼ 4.5 Hz, 4H, eCH2eOeCH2e); 4.56 (t, J ¼ 2.0 Hz, 2H,
eCH2); 7.07e7.10 (m, 1H, AreH); 7.58e7.61 (m, 2H, AreH) 13C NMR
(CDCl3, 75 MHz) 29.8, 47.2, 52.3, 66.7, 80.3, 112.3, 118.4, 125.3, 130.0,
d
1.14e1.74 (m, 6H, eCH2eCH2eCH2e); 2.40 (s, 4H, eCH2eNeCH2e
); 3.21 (s, 2H, eCH2); 4.54 (s, 2H, eCH2); 7.10e7.17 (m, 2H AreH);
7.58e7.63 (m, 2H, AreH) 13C NMR (CDCl3, 75 MHz): 23.6, 25.6, 29.6,
47.5, 53.2, 80.7, 111.2, 117.4, 123.9, 125.1, 138.1, 149.7, 157.0, 182.6,
HRMS calculated for C17H18N2O2 282.1368 [Mþ] found 282.1375;
Analysis calculated for C17H18N2O2 C, 72.32; H, 6.43; N, 9.92; found
C, 72.39; H, 6.37; N, 9.97.
137.6, 147.9, 156.5, 181.6, HRMS calculated for
318.0771 [Mþ] found 318.0765; Analysis calculated for
16H15ClN2O3: C, 60.29; H, 4.74; N, 8.79 found C, 60.35; H, 4.79; N
C16H15ClN2O3
C
8.65.
3.3.8. 5-Chloro-1-(4-diethylamino-but-2-ynyl)-1H-indole-2,3-
3.3.2. 1-(4-Pyrrolidin-1-yl-but-2-ynyl)-1H-indole-2,3-dione (3b)
dione (3h)
Orange solid, yield 86%, m.p. 136 ꢀC: 1H NMR (300 MHz, CDCl3):
Viscous liquid, yield 78%, 1H NMR (300 MHz, CDCl3):
d 1.07 (t,
d
1.79e1.83 (m, 4H, eCH2eCH2); 2.56e2.64 (m, 4H, eCH2eNeCHe
J ¼ 7.1 Hz, 6H, CH3); 2.51e2.59 (m, 4H, eCH2eNeCH2e); 3.6 (s, 2H,
eCH2); 4.54 (s, 2H, eCH2); 7.07e7.10 (m, 1H, AreH); 7.56e7.63 (m,
2H, AreH) 13C NMR (CDCl3, 75 MHz): 14.8, 29.5, 46.7, 47.4, 81.6,
110.9, 117.7, 124.1, 125.3, 138.2, 149.7, 157.1, 181.7, HRMS calculated
for C16H17ClN2O2 304.0979 [Mþ] found 304.0986; Analysis calcu-
lated for C16H17ClN2O2: C, 63.05; H, 5.62; N, 9.19; found C, 63.13; H,
5.75; N, 9.07.
); 3.42 (s, 2H, eCH2); 4.59 (s, 2H, eCH2); 7.15e7.24 (m, 2H, AreH);
7.62e7.69 (m, 2H, AreH) 13C NMR (CDCl3, 75 MHz) 23.6, 29.8, 43.1,
52.7, 81.6, 111.2, 117.4, 123.8, 125.1, 138.1, 149.6, 149.8, 151.6, 182.6,
HRMS calculated for C16H16N2O2 268.1212 [Mþ] found 268.1219;
Analysis calculated for C16H16N2O2: C, 71.62; H, 6.01; N, 10.44;
found C, 71.58; H, 6.08; N, 10.39.

Nisha et al. / European Journal of Medicinal Chemistry 74 (2014) 657e663
661
3.3.9. 5-Fluoro-1-(4-piperidin-1-yl-but-2-ynyl)-1H-indole-2,3-
dione (3i)
137.5, 148.0, 149.9, 156.56, 181.7, HRMS calculated for C17H18N2O2
282.1368 [Mþ] found 282.1374; Analysis calculated for C17H18N2O2:
C, 72.32; H, 6.43; N, 9.92 found C, 72.39; H, 6.37; N, 9.86.
Orange solid, yield 85%, m.p. 149 ꢀC: 1H NMR (300 MHz, CDCl3):
d
1.41e1.72 (m, 6H, eCH2eCH2eCH2e); 2.42 (s, 4H, eCH2eNeCHe
); 3.22 (t, J ¼ 2.0 Hz, 2H, eCH2); 4.55 (t, J ¼ 2.0 Hz, 2H, eCH2); 7.09e
7.14 (m, 1H, AreH); 7.31e7.38 (m, 2H, AreH) 13C NMR (CDCl3,
75 MHz) 23.7, 25.7, 29.9, 47.7, 53.4, 81.2, 112.2, 118.4, 124.4, 124.7,
3.3.15. 5-Methyl-1-(4-morpholin-4yl-but-2-ynyl)-1H-indole-2,3-
dione (3o)
Orange solid, yield 87%, m.p. 159 ꢀC 1H NMR (300 MHz, CDCl3):
138.3, 149.6, 156.6, 181.6, HRMS calculated for
300.1274 [Mþ] found 300.1268; Analysis calculated for
17H17FN2O2: C, 67.99; H, 5.71; N, 9.33 found C, 67.92; H, 5.79; N,
C
17H17FN2O2
d
2.29 (s, 3H, CH3); 2.51 (t, J ¼ 4.5 Hz, 4H, eCH2eNeCH2e); 3.32 (s,
2H, eCH); 3.75 (t, J ¼ 4.5 Hz, 4H, eCH2eOeCH2e); 4.53 (s, 2H, e
CH2); 7.07e7.10 (m, 1H, AreH); 7.32e7.39 (m, 2H, AreH): 13C NMR
(CDCl3, 75 MHz): 20.9, 29.8, 47.2, 52.3, 66.7, 81.6, 112.3, 118.4, 125.3,
129.9, 137.6, 147.9, 149.7, 156.5, 181.6, HRMS calculated for
C
C
C
9.41.
3.3.10. 5-Fluoro-1-(4-pyrrolidin-1-yl-but-2-ynyl)-1H-indole-2,3-
dione (3j)
17H18N2O3 298.1317 [Mþ] found 298.1328; Analysis calculated for
17H18N2O3: C, 68.44; H, 6.08; N, 9.39 found C, 68.53; H, 6.14, N,
Orange solid, yield 81%, m.p. 141 ꢀC: 1H NMR (300 MHz, CDCl3):
9.45.
d
1.74e1.77 (m, 4H, eCH2eCH2e); 2.52e2.55 (m, 4H, eCH2eNe
CHe); 3.36 (s, 2H, eCH2); 4.53 (s, 2H, eCH2); 7.07e7.10 (m, 1H, Are
H); 7.57e7.59 (m, 2H, AreH) 13C NMR (CDCl3, 75 MHz): 23.6, 29.9,
43.2, 52.8, 81.6, 112.5, 118.5, 125.2, 129.9, 137.6, 148.1, 156.5, 181.8,
HRMS calculated for C16H15ClN2O2 302.0822 [Mþ] found 302.0815;
Analysis calculated for C16H15ClN2O2: C, 63.47; H, 4.99; N, 9.25
found C, 63.53; H, 4.85; N, 9.33.
3.3.16. 1-(4-Diethylamino-but-2-ynyl)-5-methyl-1H-indole-2,3-
dione (3p)
Viscous liquid, yield 78%, 1H NMR (300 MHz, CDCl3):
d 1.07 (t,
J ¼ 7.1 Hz, 6H, CH3); 2.27 (s, 3H, CH3); 2.52e2.60 (m, 4H, eCH2eNe
CH2); 3.62 (s, 2H, eCH2); 4.55 (s, 2H, eCH2); 7.06e7.10 (m, 1H, Are
H); 7.56e7.65 (m, 2H, AreH): 13C NMR (CDCl3, 75 MHz): 14.8, 20.9,
29.4, 46.7, 47.3, 80.3, 112.5, 118.7, 125.3, 130.3, 138.2, 148.2, 157.1,
181.7, HRMS calculated for C17H20N2O2 288.3168 [Mþ] found
288.3159; Analysis calculated for C17H20N2O2: C, 71.81; H, 7.09; N,
9.85; found C, 71.95; H, 7.17, N, 9.77.
3.3.11. 5-Fluoro-1-(4-morpholin-4-yl-but-2-ynyl)-1H-indole-2,3-
dione (3k)
Orange solid, yield 85%, m.p. 157 ꢀC 1H NMR (300 MHz, CDCl3):
d
2.50 (t, J ¼ 4.5 Hz, 4H, eCH2eNeCH2e); 3.27 (t, J ¼ 2.0 Hz, 2H, e
CH); 3.71 (t, J ¼ 4.5 Hz, 4H, eCH2eOeCH2e); 4.56 (t, J ¼ 2.0 Hz, 2H,
eCH2); 7.07e7.10 (m, 1H, AreH); 7.34e7.39 (m, 2H, AreH): 13C NMR
(CDCl3, 75 MHz): 29.8, 47.3, 52.3, 66.7, 80.2, 112.3, 118.5, 125.3,
130.0, 137.6, 148.0, 156.5, 181.7, HRMS calculated for C16H15FN2O3
302.1067 [Mþ] found 302.1074; Analysis calculated for
3.4. Typical procedure for the synthesis of N-propargylated-isatin-
Mannich-bis-adducts 5ae5d
To a well stirred solution of N-propargylated isatin (1 mmol) in
dry dioxane, catalytic amount of copper chloride and p-formalde-
hyde was added in succession. The reaction mixture was stirred for
10 min with subsequent addition of piperazine (0.5 mmol) and
refluxed at 120 ꢀC for 8 h. After the completion of reaction as
evident from TLC, the reaction mixture was filtered and subse-
quently extracted with ethyl acetate (3 ꢂ 30 mL). The combined
organic layers were dried over anhydrous Na2SO4 and concentrated
under reduced pressure to result in the desired bis-Mannich ad-
ducts in good yields.
C
9.36.
16H15ClN2O3: C, 63.57; H, 5.00; N, 9.27 found C, 63.66; H, 5.09; N,
3.3.12. 1-(4-Diethylamino-but-2-ynyl)-5-fluoro-1H-indole-2,3-
dione (3l)
Viscous liquid, yield 78%, 1H NMR (300 MHz, CDCl3):
d 1.05 (t,
J ¼ 7.1 Hz, 6H, CH3); 2.53e2.62 (m, 4H, eCH2eNeCH2); 3.62 (s, 2H,
eCH2); 4.55 (s, 2H, eCH2); 7.07e7.11 (m, 1H, AreH); 7.56e7.65 (m,
2H, AreH):13C NMR (CDCl3, 75 MHz): 14.8, 29.4, 46.7, 47.3, 80.3,
112.5, 118.7, 125.3, 130.3, 138.2, 148.2, 157.1, 181.7, HRMS calculated
for C16H17FN2O2 288.3168 [Mþ] found 288.3157; Analysis calculated
for C16H17ClN2O2: C, 66.65; H, 5.94; N, 9.72; found C, 66.73; H, 5.85,
N, 9.79.
3.4.1. Bis-{1-(yl-but-2-ynyl)-1H-indole-2,3-dione} piperazine (5a)
Orange solid, yield 67%, m.p. >240 ꢀC: 1H NMR (300 MHz,
CDCl3):
d 2.55 (s, 8H, piperazine); 3.26 (s, 4H, eCH2); 4.54 (s, 4H,
CH2); 7.11e7.20 (m, 4H, AreH); 7.60e7.64 (m, 4H, AreH) 13C NMR
(CDCl3, 75 MHz): 29.7, 46.8, 51.8, 80.0, 111.0, 117.6, 124.1, 125.4,
138.3, 149.7, 157.1, 182.7, HRMS calculated for C28H24N4O4 480.1798
[Mþ] found 480.1805; Analysis calculated for C28H24N4O4 C, 69.99;
H, 5.03; N, 11.66; found C,69.82; H, 5.09; N, 11.73.
3.3.13. 5-Methyl-1-(4-pyrrolidin-1-yl-but-2-ynyl)-1H-indole-2,3-
dione (3m)
Orange solid, yield 82%, m.p. 148 ꢀC: 1H NMR (300 MHz, CDCl3):
d
1.17e1.57 (m, 6H, eCH2eCH2eCH2e); 2.29 (s, 3H, CH3); 2.45 (s,
4H, eCH2eNeCHe); 3.25 (s, 2H, eCH2); 4.54 (s, 2H, eCH2); 7.05e
7.10 (m, 1H, AreH); 7.55e7.60 (m, 2H, AreH): 13C NMR (CDCl3,
75 MHz): 20.9, 23.6, 25.7, 29.6, 47.7, 53.2, 81.0, 112.4, 118.4, 125.1,
129.8, 137.6, 148.0, 149.7, 157.2, 182.6, HRMS calculated for
C
C
3.4.2. Bis-{5-chloro-1-(yl-but-2-ynyl)-1H-indole-2,3-dione}
piperazine (5b)
Orange solid, yield 70%, m.p. >240 ꢀC: 1H NMR (300 MHz,
CDCl3):
d 2.56 (s, 8H, piperazine); 3.30 (s, 4H, eCH2); 4.55 (s, 4H, e
18H20N2O2 296.1525 [Mþ] found 296.1517; Analysis calculated for
18H20N2O2: C, 72.95; H, 6.80; N, 9.45 found C, 72.82; H, 6.89; N,
CH2); 7.07e7.11 (m, 2H, AreH); 7.59e7.61 (m, 4H, AreH): 13C NMR
(CDCl3, 75 MHz): 29.7, 46.8, 51.7, 80.2, 112.5, 118.6, 125.1, 126.4,
138.3, 149.7, 157.2, 182.7, HRMS calculated for C28H22Cl2N4O4
548.1018 [Mþ] found 548.1025; Analysis calculated for
9.37.
3.3.14. 5-Methyl-1-(4-pyrrolidin-1-yl-but-2-ynyl)-1H-indole-2,3-
dione (3n)
C28H22Cl2N4O4: C, 61.21; H, 4.04; N, 10.20; found C,61.30; H, 4.13; N,
10.15.
Orange solid, yield 84%, m.p. 139 ꢀC: 1H NMR (300 MHz, CDCl3):
d
1.73e1.77 (m, 4H, eCH2eCH2e); 2.29 (s, 3H, CH3); 2.52e2.57 (m,
3.4.3. Bis-{5-fluoro-1-(yl-but-2-ynyl)-1H-indole-2,3-dione}
piperazine (5c)
4H, eCH2eNeCHe); 3.36 (s, 2H, eCH2); 4.55 (s, 2H, eCH2); 7.07e
7.12 (m, 1H, AreH); 7.57e7.59 (m, 2H, AreH) 13C NMR (CDCl3,
75 MHz): 20.9, 23.6, 29.8, 43.2, 52.8, 81.6, 112.4, 118.5, 125.2, 129.9,
Orange solid, yield 65%, m.p. >240 ꢀC: 1H NMR (300 MHz,
CDCl3):
d 2.55 (s, 8H, piperazine); 3.35 (s, 4H, eCH2); 4.54 (s, 4H, e

662
Nisha et al. / European Journal of Medicinal Chemistry 74 (2014) 657e663
CH2); 7.06e7.11 (m, 2H, AreH); 7.58e7.62 (m, 4H, AreH) 13C NMR
(CDCl3, 75 MHz): 29.7, 46.8, 51.8, 80.2, 111.0, 117.6, 124.1, 125.4,
1H, H4); 7.81 (d, J ¼ 9.0 Hz, 1H, H3); 8.00 (s, 1H, H5); 8.67 (d,
J ¼ 5.1 Hz, 1H, H1); 13C NMR (CDCl3, 75 MHz):
d
ppm ¼ 29.5, 45.4,
138.3, 149.7, 157.1, 181.6, HRMS calculated for
516.1609 [Mþ] found 516.1615; Analysis calculated for
28H22F2N4O4: C, 65.11; H, 4.29; N, 10.85; found C, 65.23; H, 4.35; N,
C
28H22F2N4O4
51.0, 52.2, 78.3, 79.4, 108.8, 111.2, 112.6, 118.2, 121.6, 124.3, 124.8,
125.1, 126.7, 128.5,134.6,146.4, 150.1, 151.9, 156.2, 157.7, 182.1, HRMS
C
Calculated for C
25H20Cl2N4O2 [M]þ478.0963 found 478.0971;
10.77.
Analysis calculated for Anal. C25H20Cl2N4O2: C, 62.64; H, 4.21; N,
11.69, found: C, 62.54; H, 4.31; N, 11.76.
3.4.4. Bis-{5-methyl-1-(yl-but-2-ynyl)-1H-indole-2,3-dione}
piperazine (5d)
3.5.4. 1-{4-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-yl]-but-2-
ynyl}-5-methyl-1H-indole-2,3-dione (7d)
Orange solid, yield 60%, m.p. >240 ꢀC: 1H NMR (300 MHz,
CDCl3):
d
2.56 (s, 8H, piperazine); 2.29 (s, 6H, CH3); 3.31 (s, 4H, e
Red solid; yield: 82%; m.p. 114e115 ꢀC: 1H NMR (CDCl3,
CH2); 4.55 (s, 4H, eCH2); 7.07e7.11 (m, 2H, AreH); 7.59e7.61 (m,
4H, AreH) 13C NMR (CDCl3, 75 MHz): 20.9, 29.5, 46.8, 51.8, 80.2,
111.0, 117.6, 124.1, 125.5, 138.3, 149.7, 157.1, 182.7, HRMS calculated
for C30H28N4O4 508.2111 [Mþ] found 508.2122; Analysis calculated
for C30H28N4O4 C, 70.85; H, 5.55; N,11.02; found C, 70.79; H, 5.62; N,
11.14.
300 MHz): d 2.23 (s, 3H, eCH3); 2.77 (s, 4H, 2x-CH2e); 3.18 (s, 4H,
2x-CH2e); 3.38 (s, 2H, eNeCH2e); 4.55 (s, 2H, eNeCH2e); 6.69 (d,
J ¼ 5.1 Hz, 1H, H2); 6.82e6.85 (m, 1H, ArH); 7.17e7.24 (m, 2H, ArH);
7.31 (d, J ¼ 9.0 Hz, 1H, H4); 7.81 (d, J ¼ 9.0 Hz, 1H, H3); 8.01 (s, 1H,
H5); 8.63 (d, J ¼ 5.1 Hz, 1H, H1); 13C NMR (CDCl3, 75 MHz):
d
ppm ¼ 21.1, 29.4, 45.7, 51.2, 52.0, 78.2, 79.6, 108.7, 111.2, 113.5,
118.0, 121.7, 124.2, 124.6, 125.8, 126.5, 128.9, 134.9, 136.3, 147.1,
149.6, 151.0, 157.7, 181.9, HRMS Calculated for 26H23ClN4O2
[M]þ458.1510found
458.1518; Analysis calculated for
26H23ClN4O2: C, 68.04; H, 5.05; N, 12.10, found: C, 68.14; H, 5.11; N,
12.15.
3.5. Typical procedure for the synthesis of Mannich adducts 7ae7e
C
A
mixture of N-propargylated isatin
2
(1 mmol), para-
C
formaldehyde (2.5 mmol), 7-chloro-4-piperazin-1-yl-quinoline 7
(1 mmol) and anhydrous cupric chloride (0.5 mmol) in dry 1,4-
dioxane was heated at 100 ꢀC for 3 h. After completion of reac-
tion as evidenced by TLC, the reaction mixture was filtered and the
filtrate was treated with brine solution and extracted with ethyl
acetate (3 ꢂ 60 mL). Combined organic layers were dried over
Na2SO4 and the solvent was evaporated under reduced pressure
resulting in a crude product which was purified by column chro-
matography using a chloroform:methanol (95:5) mixture.
3.5.5. 1-{4-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-yl]-but-2-
ynyl}-5-nitro-1H-indole-2,3-dione (7e)
Red solid; yield: 82%; m.p. 116e118 ꢀC. 1H NMR (CDCl3,
300 MHz): d2.75 (s, 4H, 2x-CH2e); 3.18 (s, 4H, 2x-CH2e); 3.36 (s,
2H, eNeCH2e); 4.55 (s, 2H, eNeCH2e); 6.67 (d, J ¼ 5.1 Hz, 1H, H2);
6.82e6.85 (m, 1H, ArH); 7.16e7.25 (m, 2H, ArH); 7.31 (d, J ¼ 9.0 Hz,
1H, H4); 7.81 (d, J ¼ 9.0 Hz, 1H, H3); 8.01 (s, 1H, H5); 8.63 (d,
J ¼ 5.1 Hz, 1H, H1); 13C NMR (CDCl3, 75 MHz):
d 29.4, 45.7, 51.2, 52.2,
3.5.1. 1-{4-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-yl]-but-2-
ynyl}-1H-indole-2,3-dione (7a)
78.2, 79.6, 108.7, 111.2, 113.5, 118.0, 121.7, 124.3, 124.6, 125.9, 126.5,
128.9, 134.9, 136.3, 147.1, 149.6, 151.1, 157.7, 181.9, HRMS Calculated
for C25H20ClN5O4 [M]þ489.9104 found 489.9117; Analysis calcu-
lated for C25H20ClN5O4: C, 61.29; H, 4.11; N, 14.30, found: C, 61.38;
H, 4.03; N, 14.42.
Red solid; yield 88%; m.p. 115e116 ꢀC: 1H NMR (CDCl3,
300 MHz):
d 2.79 (s, 4H, 2x-CH2e); 3.24 (s, 4H, 2x-CH2e); 3.42 (s,
2H, eNeCH2e); 4.60 (s, 2H, eNeCH2e); 6.82 (d, J ¼ 5.1 Hz, 1H, H2);
7.15 (t, J ¼ 7.8 Hz, 1H, ArH); 7.41 (dd, J ¼ 2.1, 9.0 Hz, 1H, H4); 7.59e
7.65 (m, 3H, ArH); 7.89 (d, J ¼ 8.7 Hz, 1H, H3); 8.03 (d, J ¼ 1.8 Hz, 1H,
H5); 8.70 (d, J ¼ 5.1 Hz, 1H, H1); 13C NMR (DMSO-d6, 100 MHz):
3.6. In vitro protozoal parasite susceptibility assay
d
ppm ¼ 29.3, 45.9, 50.9, 51.4, 78.3, 79.2, 109.3, 111.6, 112.6, 121.2,
To perform the initial susceptibility screens on T. foetus strain D1,
compounds were dissolved in DMSO to obtain concentrations of
123.8,124.0, 125.7,125.9,127.8, 133.5,145.7,149.4,151.9,156.1, 157.3,
159.8, 182.0, HRMS Calculated for C25H21ClN4O2 [M]þ444.1353
found 444.1345; Analysis calculated for C25H21ClN4O2: C, 67.49; H,
4.76; N, 12.59, found: C, 67.57; H, 4.66; N, 12.52.
100
mM; 5
mL aliquots of these suspensions were diluted in 5 mL of
TYM Diamond’s media to obtain a final concentration of 100
mM.
After 24 h, cells were counted using a hemacytometer. The IC50
value for compounds 7a, 7c and 7d was determined by running
3.5.2. 1-{4-[4-(7-Chloro-quinolin-4-yl)-piperazin-1-yl]-but-2-
ynyl}-5-fluoro-1H-indole-2,3-dione (7b)
assays of increasing drug concentrations, 5 mMe40 mM, and per-
forming a regression analysis using Prism software, from GraphPad.
Red Solid; Yield: 90%; m.p. 135e136 ꢀC: 1H NMR (CDCl3,
300 MHz):
d
2.78 (s, 4H, 2x-CH2e); 3.20 (s, 4H, 2x-CH2e); 3.42 (s,
3.7. In vitro cytotoxic studies
2H, eNeCH2e); 4.57 (s, 2H, eNeCH2e); 6.73 (d, J ¼ 5.1 Hz, 1H, H2);
6.86e6.89 (m, 1H, ArH); 7.16e7.23 (m, 2H, ArH); 7.39 (d, J ¼ 9.0 Hz,
1H, H4); 7.88 (d, J ¼ 9.0 Hz, 1H, H3); 7.99 (s, 1H, H5); 8.66 (d,
PC-3 cells were cultured in DMEM supplemented with 10% FBS.
Cells were plated in 96 well tissue culture plates and treated with
compounds in the concentration range 20
J ¼ 5.1 Hz, 1H, H1); 13C NMR (CDCl3, 75 MHz):
d
ppm ¼ 29.9, 45.6,
mMe0.001 mM for 72 h
51.2, 52.5, 78.4, 79.3, 108.9, 111.3, 112.4, 118.1, 121.7, 124.2, 124.9,
125.2, 126.6, 128.7, 134.8, 146.7, 150.0, 151.6, 156.4, 157.9, 181.9,
HRMS Calculated for C25H20ClFN4O2 [M]þ462.1259 found 462.1252;
Analysis calculated for C25H20ClFN4O2: C, 64.87; H, 4.35; N, 12.10,
found: C, 64.80; H, 4.41; N, 12.01.
10 l MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium
m
bromide) 5 mg/ml in PBS was added to wells. Plates were incubated
for 4 h. Plates were centrifuged at 1000 rpm for 5 min. Supernatant
was removed; 100 ml DMSO was added and mixed on shaker for
30 min. Absorbance was measured at 550 nm in Envision Multilabel
Plate Reader (Perkin Elmer). Percentage inhibition was calculated
and dose response curve was plotted using Graph pad Prism.
3.5.3. 5-Chloro-1-{4-[4-(7-chloro-quinolin-4-yl)-piperazin-1-yl]-
but-2-ynyl}-1H-indole-2,3-dione (7c)
Red solid; yield: 92%; m.p. 124e125 ꢀC. 1H NMR (CDCl3,
Acknowledgements
300 MHz):
d 2.80 (s, 4H, 2x-CH2e); 3.21 (s, 4H, 2x-CH2e); 3.40 (s,
2H, eNeCH2e); 4.58 (s, 2H, eNeCH2e); 6.74 (d, J ¼ 5.1 Hz, 1H, H2);
6.85e6.88 (m, 1H, ArH); 7.18e7.25 (m, 2H, ArH); 7.39 (d, J ¼ 9.0 Hz,
Financial assistance from University Grants Commission (UGC)
under Rajiv Gandhi fellowship for SC/ST students (Nisha) is

Nisha et al. / European Journal of Medicinal Chemistry 74 (2014) 657e663
663
[15] R.A. Amal, R. Raghunathan, M.R. Sridevikumaria, N. Raman, Bioorg. Med.
Chem. 11 (2003) 407e419.
gratefully acknowledged. KL would like to acknowledge 2012 Pa-
cific Fund Summer Undergraduate Research Fellowship at the
University of Pacific. We would also like to thank Dr. Lydia K. Fox,
Director of Undergraduate Research for her support.
[16] M.C. Rodriguez-Arguelles, S. Mosquera-Vazaquez, P. Touron-Touceda,
J. Sanmartin-Matalobos, A.M. Garcia-Deibe, M. Belicchi-Ferraris, G. Pelosi,
C. .Pelizzi, F. Zani, J. Inorg. Biochem 101 (2007) 138e147.
[17] L. Maskell, E. A Blanche, M.A. Colucci, J.L. Whatmore, Ch. J. Moody, Bioorg.
Med. Chem. Lett. 17 (2007) 1575e1578.
[18] M. Verma, P.S. Nath, S.K. Nand, J.P. Stables, Acta Pharm. 54 (2004) 49e56.
[19] N. Igosheva, C. Lorz, E. O’Conner, V. Glover, H. Mehmet, Neurochem. Int. 47
(2005) 216e224.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.015.
[20] L.R. Chen, Y. Ch. Wang, Y.W. Lin, Sh. Y. Chou, Sh. F. Chen, L.T. Liu, Y.T. Wu, Ch.
J. Kuo, T. Sh. Sh. Chen, Sh. H. Juang, Bioorg. Med. Chem. Lett. 15 (2005) 3058e
3062.
[21] J.F.M. Da Silva, S.J. Garden, A.C.J. Pinto, Braz. Chem. Soc. 12 (2001) 273e324
and references therein.
References
[22] (a) K.L. Vine, J.M. Locke, M. Ranson, K. Benkendorff, S.G. Pyne, J.B. Bremner,
Bioorg. Med. Chem. 15 (2007) 931e938;
[1] S.Z. Skirrow, R.H. BonDurant, Vet. Bull. 58 (1988) 591e603.
[2] A. Yule, S.Z. Skirrow, R.H. BonDurant, Parasitol. Today 5 (1989) 373e377.
[3] B.L. Clark, Aust. Vet. J. 47 (1971) 103e107.
[4] R.J. Stewart, J.R. Campbell, E.D. Janzen, J. McKinnon, Can. Vet. J. 39 (1998) 638e
641.
[5] S. Parker, J. Campbell, C. Ribble, A. Gajadhar, J. Am. Vet. Med. Assoc. 215 (1999)
231e235.
[6] E.R. Cobo, C. Morsella, D. Cano, A. Cipolla, C.M. Campero, Theriogenology 62
(2004) 1367e1382.
[7] D.O. Rae, J.E. Crews, Vet. Clin. North Am. Food Anim. Pract. 22 (2006) 595e611.
[8] V. Midlej, R. Vilela, A.B. Dias, M. Benchimol, Vet. Parasitol. 165 (2009) 216e230.
[9] C.M. Ribeiro, M.B. Falleiros, S.D. Bicudo, J.P. Junior, M.A. Golim, F.C. Filho,
Theriogenology 73 (2010) 64e70.
[10] B.T. Ratan, B. Anand, P. Yogeeswari, D. Sriram, Bioorg. Med. Chem. Lett. 15
(2005) 4451e4455.
[11] T. Jiang, K.L. Kuhen, K. Wolff, H. Yin, K. Bieza, J. Caldwell, B. Bursulaya,
T. Tuntland, K. Zhang, D. Karanewsky, Y. He, Bioorg. Med. Chem. Lett. 16
(2006) 2109e2112.
[12] R. Tripathy, A. Reiboldt, P.A. Messina, M. Iqbal, J. Singh, E.R. Bacon, Th.
S. Angeles, Sh. X. Yang, M.S. Albom, C. Robinson, H. Chang, B.A. Ruggeri,
J.P. Mallamo, Bioorg. Med. Chem. Lett. 16 (2006) 2158e2162.
[13] A. Cane, M.C. Tournaire, D. Barritault, M. Crumeyrolle-Arias, Biochem. Biophys.
Res. Commun. 276 (2000) 379e384.
(b) K.L. Vine, J.M. Locke, M. Ranson, S.G. Pyne, J.B. Bremner, J. Med. Chem. 50
(2007) 5109e5117;
(c) L. Matesic, J.M. Locke, J.B. Bremner, S.G. Pyne, D. Skropeta, M. Ranson,
K.L. Vine, Bioorg. Med.Chem. 16 (2008) 3118e3124.
[23] P. Singh, S. Kaur, V. Kumar, P.M.S. Bedi, M.P. Mahajan, I. Sehar, H.C. Pal,
A.K. Saxena, Bioorg. Med. Chem. Lett. 21 (2011) 3017e3020.
[24] P. Singh, P. Sharma, A. Anand, P.M.S. Bedi, T. Kaur, A.K. Saxena, Eur. J. Med.
Chem. 55 (2012) 455e461.
[25] (a) K. Kumar, S. Carrère-Kremer, L. Kremer, Y. Guérardel, C. Biot, V. Kumar,
Organometallics (2013), http://dx.doi.org/10.1021/om301157z;
(b) K. Kumar, S. Sagar, L. Esau, M. Kaur, V. Kumar, Eur. J. Med. Chem. 58 (2012)
153e159;
(c) K. Kumar, S.C. Kremer, L. Kremer, Y. Guérardel, C. Biot, V. Kumar, Dalton
Trans 42 (2013) 1492e1500;
(d) Nisha, P. Singh, D.T. Hendricks, K. Bisetty, V. Kumar, Synlett 24 (2013)
1865e1869.
[26] R. Raj, P. Singh, N.T. Haberkern, R.M. Faucher, N. Patel, K.M. Land, V. Kumar,
Eur. J. Med. Chem. 63 (2013) 897e906.
[27] Nisha, V. Mehra, M. Hopper, N. Patel, D. Hall, L.A. Wrischnik, K.M. Land,
V. Kumar, Med. Chem. Comm. 4 (2013) 1018e1024.
[28] R.R. Pandey, A. Srivastava, R. Malasoni, A. Naqvi, A. Jain, J.P. Maikhuri,
S. Paliwal, G. Gupta, A.K. Dwivedi, Bioorg. Med. Chem. Lett. 22 (2012) 5735e
5738.
[14] V. Ch. Silveira, J.S. Luz, C.C. Oliveira, I. Graziani, M.R. Ciriolo, A.M. Costa-Fer-
reira, J. Inorg. Biochem 102 (2008) 1090e1103.