Synthesis and antifilarial activity of chalcone-thiazole derivatives against a human lymphatic filarial parasite, Brugia malayi

Article abstract of DOI:10.1016/j.ejmech.2014.05.029

Here we report the synthesis of novel chalcone-thiazole compounds and their antifilarial activity. The antifilarial properties of these hybrids were assessed against microfilariae as well as adult worms of Brugia malayi. Among all the synthesized compound

Full text of DOI:10.1016/j.ejmech.2014.05.029

Accepted Manuscript
Synthesis and antifilarial activity of chalcone-thiazole derivatives against a human
lymphatic filarial parasite, Brugia malayi
Koneni V. Sashidhara , K. Bhaskara Rao , Vikas Kushwaha , Ram K. Modukuri ,
Richa Verma , P.K. Murthy
PII:
S0223-5234(14)00443-7
10.1016/j.ejmech.2014.05.029
EJMECH 6985
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 January 2014
Revised Date: 7 May 2014
Accepted Date: 10 May 2014
Please cite this article as: K.V. Sashidhara, K.B. Rao, V. Kushwaha, R.K. Modukuri, R. Verma, P.K.
Murthy, Synthesis and antifilarial activity of chalcone-thiazole derivatives against a human lymphatic
filarial parasite, Brugia malayi, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.05.029.
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Synthesis and antifilarial activity of chalcone-thiazole derivatives
against a human lymphatic filarial parasite, Brugia malayi
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Koneni V. Sashidhara *, K. Bhaskara Rao , Vikas Kushwaha , Ram K. Modukuri , Richa
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Verma , P.K. Murthy *
Compound 4n: IC = 0.9 µM (for adult worms assessed by motility assay)
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Ivermectin : IC = 3.05 µM (for adult worms assessed by motility assay)
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The design and synthesis of new hybrid architecture in the search of novel antifilarial agents
is described.

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Synthesis and antifilarial activity of chalcone-thiazole derivatives
against a human lymphatic filarial parasite, Brugia malayi
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Koneni V. Sashidhara *, K. Bhaskara Rao , Vikas Kushwaha , Ram K. Modukuri , Richa
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Verma , P. K. Murthy *
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Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, BS-10/1, Sector
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10, Jankipuram extension, Sitapur Road, Lucknow 226031, India
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Division of Parasitology, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram
extension, Sitapur Road, Lucknow 226031, India
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Abstract
Here we report the synthesis of novel chalcone-thiazole compounds and their antifilarial
activity. The antifilarial properties of these hybrids were assessed against microfilariae as
well as adult worms of Brugia malayi. Among all the synthesized compounds, only two
compounds, namely 4g and 4n were identified to be promising in vitro. These active
compounds were tested in B. malayi-jird (Meriones unguiculatus) and B. malayi- Mastomys
coucha models. Compound 4n showed 100% embryostatic effect and 49% macrofilaricidal in
jirds and M. coucha models, respectively. This study provides a new structural clue for the
development of novel antifilarial lead molecules.
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Keywords: Brugia malayi, Microfilariae, Macrofilariae, Diethylcarbamazine, Albendazole,
Ivermectin.
*Part XXIX in the series, “Advances in drug design and discovery”.
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*
Corresponding authors. Tel: +91 9919317940; fax: +91-522-2771942.
E-mail addresses: sashidhar123@gmail.com (K.V. Sashidhara), drpkmurthy@yahoo.com (P.K. Murthy)
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Introduction
Lymphatic filariasis is a tropical helminth disease that causes acute and chronic inflammation
in patients spanning 72 counties. Recent reports show that 120 million people are infected
with around 40 million seriously incapacitated and disfigured by the disease [1, 2]. The
disease is caused by thread like nematodes Wucheria bancrofti, Brugia malayi and B. timori,
which are transmitted by anophiline and culicine mosquitoes; 91% of infections are caused
by W. bancrofti [3, 4]. The manifestations of the disease are responsible for the huge loss of
man-hours, productivity, and economy of the nation.
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The infection is initiated by introduction of third stage infective larvae (L3) of the parasite
into the host by the bites of L3-bearing mosquitoes. The L3 migrate to the nearest lymphatics
where, after two molts, develop into adult worms, which give birth to microfilariae (mf). The
mf enters into the blood circulation from where they are taken up by mosquitoes during blood
meal. In the mosquito, the mf develops into L3. Although all the three life stages were
considered to be responsible for development of the disease condition, the mf and
macrofilariae (adult worms) have a major impact as they spend a very long time in the host
and bring about host’s responses that lead to pathology and transmission of the infection [5,
6]. For this reason these two stages have been taken as the main targets for antifilarial drug
(microfilaricides and macrofilaricides, worm sterilizing agents) development.
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In spite of decades of efforts, no single safe antifilarial agent that could effectively eliminate
all the parasite stages could be developed. The available drug, diethylcarbamazine (DEC)
(Fig. 1) is insufficient because it acts mainly on mf with inadequate effect on adult parasites.
Moreover, the treatment is often complicated due to severe side reactions. Another antifilarial
drug, ivermectin is a better alternative to DEC, which is less reactogenic, effective in single
dose and sterilizing effect on the adult worms but develops resistance to it. Moxidectin (Fig.
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1) though looks promising in animal studies is still under development [7, 8]. Albendazole is
considered to be a macrofilaricide but at a single dose it is devoid of any discernible
macrofilaricidal activity and has a transient effect on early embryogenesis [9].
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Currently, a combination of DEC plus/ albendazole (a benzimidazole derivative) and
ivermectin plus/albendazole is being used for the control of infection [10]. The fact that even
after such combination therapy there is little change in the prevalence of infection over the
years indicates that we need better and safe alternatives to these agents. In view of the current
scenario, an adulticidal agent is absolutely needed to destroy the adult worms, which are
being implicated as major cause of development of the disease manifestations. Also, special
emphasis has been laid by WHO on discovering an adulticidal (macrofilaricidal) agent since
it leads to “no adult-no progeny (mf)” and thus has dual benefit. Therefore, efforts have been
made to search or synthesize newer and better antifilarials for treatment of filarial infections.
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In our drug development programme, we have recently reported 3, 6-epoxy [1,5] dioxocines
as new class of antifilarial agents [11]. The design and synthesis of hybrid molecules
encompassing two pharmacophores in one molecular scaffold is a well established approach
to the synthesis of more potent drugs. Considering the antifilarial activities of chalcone [12]
and thiazole derivatives [13], we were interested in synthesizing a number of new hybrid
compounds bearing both chalcone and thiazole moieties. Thus the concept of molecular
hybridization prompted us to synthesize chalcone-thiazole derivatives, and evaluate them for
antifilarial activity in vitro and in vivo using appropriate experimental models of human
filariasis.
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2. Chemistry
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The detailed synthetic route for the preparation of chalcone-thiazole compounds is
summarized in Scheme 1. 2-alkyl phenols were subjected to the Duff formylation in the
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presence of hexamethylenetetramine (HMTA) and TFA at 120 C gave dicarbaldehydes (2a-
d). These aromatic dicarbaldehydes and various substituted acetophenones were subjected to
condensation in the presence of a catalytic amount of conc. HCl gave the regioselective
chalcones (3a-n). In all the chalcones synthesized, the trans double bond was obtained
exclusively. These chalcones were then condensed with 2-aminothiazoles to obtain final
targeted compounds (4a-n). The structures of all the synthesized compounds were fully
characterized by complete analysis of 1D and 2D NMR spectroscopy and mass spectrometry.
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As a representative example the structure elucidation of 4n was done as follows. The
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NMR spectrum of the 4n (Fig. 2) showed signals at δ 9.32, 13.47 the former belonging to the
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proton of the imine (C-1") group and the latter to a hydroxyl group (C-4). Similarly, C
NMR spectrum showed the presence of two peaks at δ 163.3(C-4) and 164.9 (C-1") and all
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other carbon resonances were in agreement with the structure proposed. Further, in HMBC
spectrum, the hydrogen of imine (H-1") gave correlations with carbons at δ 163.3 (C-4),
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118.6 (C-5), 132.8 (C-6) and 169.5 (C-3") (Fig. 2) and also the hydroxyl proton at δ 13.47
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gave correlations with δ 163.3 (C-4), 139.1 (C-3) and 118.6 (C-5) which further supported
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the proposed structure. Figure 2 shows some of the key 2D correlations for 4n.
3. Biological Materials and Methods
3.1. Evaluation of antifilarial activity
3.1.1. Parasites
Sub-periodic strain of B. malayi was maintained in M. coucha and jirds (Meriones
unguiculatus) through Aedes aegypti mosquitoes [14, 15]. Mf and adult worms were
harvested from the peritoneal cavity (p.c.) of the jirds which were exposed to infective larvae
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(L ) approximately 5-6 months back. Freshly isolated live adult worms and mf were washed
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thoroughly in medium Hanks Balanced Salt Solution (HBSS; pH 7.2) and used for the
present study.
3.2. In vitro assays
Motility and inhibition in the 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide
(MTT) reduction assays based on viability of the parasites were used in the present study for
in vitro testing of the samples. Chalcone-thiazole compounds/ivermectin were dissolved in
DMSO and DEC in sterile distilled water. The final concentration of DMSO in the incubation
medium was kept below 0.1%. DMSO was used in place of test compounds for control.
Incubation medium used was HBSS (pH 7.2) containing mixture of antibiotics (penicillin:
100 U/mL; streptomycin: 100 µg/mL).
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3.2.1. Primary evaluation (In vitro efficacy)
Initially efficacy of the chalcone-thiazole compounds was assessed in vitro using live B.
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malayi parasites in motility and MTT reduction assays [16]. In the 1 set of experiment mf
were used in motility assay; the parasites were incubated in medium containing 20 µM
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ivermectin, 1000 µM DEC or 40 µM chalcone-thiazole compounds for 48 hr at 37 C in 5%
CO atmosphere. After exposure for 48 hr, the effect on motility of mf was assessed.
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Compounds causing ≥50% inhibition in mf motility over untreated control (DMSO treated)
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were considered active. In the 2 set of experiment the active compounds were tested on
adult worms using both motility and MTT reduction assays [17, 18].
3.2.2. Evaluation of median inhibitory concentration (IC ) and cytotoxic concentration
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(CC )
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The IC of the active compounds was assessed as described elsewhere [17]. Live parasites
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were exposed to two fold serial dilutions of the chalcone-thiazole compounds /ivermectin and
DEC starting from 0.38-40 µM and 15.63-1000 µM respectively, using triplicates of one
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adult female worm/ml/well and 40-50 mf/100 µL/well in 48-well or 96-well sterile cell
culture plate. After assessing status of motility of the adult parasites the same treated and
control parasites were processed for assessing percentage inhibition in the MTT reduction by
the worms. For CC assay, VERO Cell line C1008 was incubated using a three fold serial
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dilutions of the test compounds and reference drugs (starting from >20 x LC100 of the
compounds).
3.2.3. Assessment of in vitro efficacy
The viability of the treated worms was assessed by calculating percent inhibition in motility
and MTT reduction over DMSO treated worms [17]. Briefly, parasite motility was assessed
under a microscope after 48 h exposure to test substance and scored as: 0=dead (i.e. the
worms failed to gain pretreatment level of motility even after incubating the treated worms in
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fresh medium but without test compounds for 25-30 min. at 37 C and expressed as
percentage (%) of control); 1-4= loss of motility (1=75%; 2=50%; 3= 25% and 4=no loss of
motility i.e. highly motile worm comparable to control). 100% inhibition in motility of
female adults or mf and or ≥50% inhibition in MTT reduction ability of adult parasites was
considered acceptable antifilarial activity of the test agents and subjected for further testing.
The IC and CC were determined as per methods described by Mosman [19] and Page et
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al. [20]. Data were transferred into a graphic program (Excel); IC and CC were calculated
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by linear interpolation between the two concentrations above and below 50% inhibition [21].
Selectivity Index (SI) of the agents were computed by the formula as: SI =CC /IC .
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3.3. In vivo protocol for efficacy evaluation
Initially as a standard practice and following a validated protocol, we screen synthetic
compounds/plant products for antifilarial activity first in vitro for ‘short-listing’ [16, 17] and
then take-up the positive ‘hits’ for in vivo evaluation in rodent models. The first in vivo
testing is done in jird model (jirds bearing intraperitoneally transplanted B. malayi adult
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worms) at a dose of 50/100mg/kg x 5 days by s.c. route and the compound that is positive in
this model is tested in the second rodent model M. coucha with L induced B. malayi
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infection using the same dose, route and dosing schedule. If need be, the dose is escalated to
200/300mg/kg. DEC-citrate (DEC-C) is used as the reference drug under similar conditions
at recommended standard doses: 25mg/kg, s.c. x 5 days in jirds and 50mg/kg, i.p./s.c./p.o. in
M. coucha [11, 22, 23]. Similarly ivermectin is used at 1/2mg/kg, i.p. or s.c. in jirds/M.
coucha [24].
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3.3.1. Host-parasite systems
All the experiments in animals (M. coucha and jirds) were conducted in compliance with the
Institutional Animal Ethics Committee guidelines for use and handling of animals.
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Throughout the study, the animals were housed in climate (23 ± 2 C; RH: 60%) and
photoperiod (12h light-dark cycles) controlled animal quarters. They were fed standard
rodent “maintenance diet” prepared in-house (QC analysis: carbohydrates 58.30%, protein
21.1%, fat 7.2%, crude fibre 6.6%, moisture 6.8%) supplemented with dried shrimps (for M.
coucha) and had free access to drinking water. M. coucha and jirds were infected with sub-
periodic strain of B. malayi, which was used for testing the above-mentioned test agents. The
treated and untreated (vehicle treated control) animals were autopsied under deep anesthesia
using sodium pentothal.
3.3.2. B. malayi-jird model
Male jirds of 8-10 weeks old were transplanted intraperitoneally (i.p.) with freshly isolated
adult worms from peritoneal cavity (p.c.) of infected jirds. Each animal received 10 female
and 5 male adult worms in the p.c. On day 2 or 3 post-adult worm transplantation (p.a.t.), the
peritoneal fluid was aspirated and checked for the presence of mf. The treatment was started
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on day 7/8 p.a.t. The animals were killed on day 60 post initiation of treatment (p.i.t.),
parasites recovered were collected, recorded their conditions and counted [25].
3.3.3. B. malayi-M. coucha model
Animals harbouring 5-7 months old B. malayi infection and showing progressive rise in
microfilaraemia were used in the study. Peripheral blood (10 ꢀL tail blood) taken between
12:00 noon and 1:00 PM was made into thick smears, just before initiation of treatment (day
0), on days 7/8 and 14 and thereafter at fortnightly intervals till day 84/91 p.i.t . The animals
were killed on day 91 p.i.t.
3.3.4. Administration of the chalcone-thiazole compounds and reference drugs
The chalcone-thiazole compounds were pulverized to fine powder and suspended in 0.1%
Tween-80 in distilled water. DEC was dissolved in distilled water and ivermectin was
dissolved in 0.1% Tween-80. In jirds and M. coucha the test agents were administered at 100
mg/kg body weight through subcutaneous (s.c.) route, for 5 consecutive days while DEC-C
was given to them at 25 and 50 mg/kg body weight, respectively, for the same duration. The
suspensions/solutions of the test compounds were prepared fresh daily before administration
to the animals.
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3.3.5. Assessment of microfilaricidal efficacy
Microfilaricidal efficacy of each test agent was evaluated on days 7/8 and 14 p.i.t. and
expressed as percent change in mf count over the pretreatment level [25, 26].
3.3.6. Assessment of macrofilaricidal and worm sterilization efficacy
Macrofilaricidal/adulticidal efficacy of the chalcone-thiazole compounds was assessed and
expressed as percent reduction in adult worm recovery in treated group over vehicle treated
(untreated control) animals. Treated and control animals were killed on day 60 (jird) or 91
(M. coucha) p.i.t. to recover adult worms from the p.c. washings (jird) or heart, lung, and
testes (M. coucha). Tissues were teased gently to avoid any damage to the parasites. Parasites
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were then examined under microscope for status of the motility, cell adherence on their
surface, death, or calcification of the worms and recorded. All the surviving females were
teased individually in a drop of saline to examine condition of intrauterine mf stages of the
parasite [27]. Number of worms recovered from the treated infected animals was compared
with that of control animals and percent reduction of worms calculated. Percent sterilization
of female worms was determined over total live female worms recovered from treated or
control animals.
3.3.7. Statistical analysis
Statistical analyses were carried out using GraphPad Prism 3.0 version software. Results were
presented as mean±S.D. of data obtained from 4-8 animals in two experiments. The data were
subjected to Student’s ‘t’ test or One-way ANOVA analysis and the significance of the
difference between means were determined by Tukey's Multiple Comparison Test. Values
were expressed as means±SD. ‘P’ value < 0.05 were considered to be significant.
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4. Results and discussion
4.1. In vitro activity on mf and adult worms of B. malayi
In vitro activity of 14 chalcone-thiazole compounds (4a-n) was assessed in motility and MTT
reduction assays using mf and female adult worms of B. malayi and the results are depicted in
Table 1. Out of all compounds, two compounds 4n and 4g were found to be effective in
killing mf (LC : 5 and 10 µM; IC : 1.8 and 3.5 µM) and adult worms (LC : 2.5 and 10
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µM; IC : 0.9 and 3.2 µM); both the compounds also inhibited MTT reduction potential of
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adult parasites to 49 and 63% respectively. SI values of 4n and 4g compounds ranged from
22 - 44 and 17 - 19 respectively, indicating acceptable safety index for in vivo testing. The
standard drug DEC-C required several times higher concentration to kill the adult worms
(LC : 1000 µM) and mf (LC : 500 µM); the IC values of the drug for respective parasite
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stages were 314.9 µM and 297.3 µM. However, ivermectin killed adult parasites at 5 µM and
mf at 2.5 µM; IC values were found to be 3.10 and 1.60 µM, respectively. In summary, two
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hits were identified to be promising with >15 SI value in vitro and thus allowed for in vivo
evaluation.
Structure–activity relationships (SARs) were inferred from in vitro activity on adult worms
and of Brugia malayi. The initial examination of structure–activity relationships (SARs)
revealed that the branching at position 3 of the phenyl ring is preferred and also the presence
of five membered heterocycles, e.g., furan, thiophene, as the chalcone counterpart potentiates
the activity. The presence of imine thiazole functionality is also a requirement for activity as
the intermediates (3a-n) did not exhibit the activity. A pictorial representation the SAR is
depicted in Fig. 5.
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4.2. In vivo antifilarial efficacy
4.2.1. In vivo evaluation of hits (compounds 4g and 4n) on B. malayi-jird model
From in vitro results we considered 4g and 4n are most active compounds. These active
compounds were tested in primary in vivo model B. malayi/jird at 100 mg/kg s.c. for 5
consecutive days and the results are presented in Table 2. Compound 4n exhibited 100%
embryostatic effect (Fig. 3). Compound 4g showed around 18% macrofilaricidal activity but
did not exert any embryostatic activity. On day 7 p.i.t. mf in p.c. of treated animals were
motile and active comparable to mf of vehicle treated animals (untreated control) indicating
ineffective against mf immediately after treatment (data not shown). The standard drug DEC-
C (25mg/kg, s.c. x 5 days) was ineffective against both mf and adult worms in p.c.; whereas,
ivermectin (1 mg/kg, s.c. x 5 days) exerted 43% embryostatic effect (P<0.01; Table 2) but did
not affect the mf. Parasites recovered from vehicle treated animals were healthy with almost
no embryostatic activity (Table 2) as evidenced by fully developed mf (Fig. 4). The general
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behavior of the jirds during 5-day course of treatment and thereafter till termination of the
experiment (day 60 p.i.t.) was normal. In summary, compound 4n showed remarkable
embryostatic activity.
4.2.2. In vivo evaluation of compounds 4g and 4n on B. malayi-M. coucha model
Compounds 4n and 4g were evaluated in secondary model (B. malayi-M. coucha) and the
results are depicted in Table 3. Compound 4n tested at 100 mg/kg, s.c. for 5 consecutive days
significantly affected adult parasites (~49%; P<0.001) over control animals. The compound
also exerted around 44% female sterilizing activity. Compound 4g was found less effective
showing 40% adulticidal (P<0.01) with 30% female sterilizing activity. In summary,
Compound 4n possessed promising antifilarial efficacy. DEC-C (50mg/kg) which is
principally microfilaricidal caused >85% reduction in microfilarial count on day 7 p.i.t. and
thereafter microfilaraemia progressively increased showing relapse by day 49 p.i.t. which
further increased and crossed the pretreatment level (data not shown). DEC-C treatment in
infected animals caused around 24% macrofilaricidal effect. In these animals the percent
female sterilization was comparable to the female worms recovered from control animals
(Table 3). Vehicle treated animals (untreated control) showed no effect on microfilariae (mf)
(data not shown) or adult worms (Table 3).
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The general behaviour of 4g- and 4n-treated animals during the 5-day course of treatment till
termination of the experiment i.e. day 60 (jird)/91 (M. coucha) p.i.t. was normal indicating
that apparently the compounds were safe.
As described above, of the two compounds 4n showed both micro- and macrofilaricidal
activity in vitro and macrofilaricidal and embryostatic activity in vivo. The reference drug
DEC (principally a microfilaricide), a piperazine derivative also showed these effects in vitro
but only at a very high dose; in vivo it showed around 20% macrofilaricidal (at usual effective
dose of 50mg/kg, x 5 days) activity in B. malayi-M. coucha model. At a higher dose of 100
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mg/kg DEC showed no increase in its microfilaricidal efficacy, but there was further increase
in macrofiaricidal efficacy to the tune of 45% [28]. Ivermectin (a macrocyclic lactone),
another principally microfilaricide, showed microfilricidal activity in vitro and in addition,
some macrofilaricidal (~20%) and embryostatic (~40%) efficacy in vivo [24]. Our chalcone-
thiazole derivative 4n which is different from the existing antifilarials has shown micro- and
macrofilaricidal activity in vitro, and macrofilaricidal along with embryostatic activity in
vivo. Thus, compound 4n was found superior than the control drugs and provides a new
structural clue for the development of novel antifilarial lead molecules. The detailed
mechanism studies of potent hybrids and further structural modifications are currently
underway.
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5. Conclusion
In summary, a series of chalcone-thiazole derivatives were designed, synthesized and
evaluated for their in vitro as well as in vivo activity against filarial nematode, B. malayi.
Interestingly, of the two hits identified in vitro, the most potent compound 4n seems to
possess remarkable embryostatic with moderate macrofilaricidal activity in vivo compared to
standard drugs ivermectin and DEC. Furthermore, the lead compound showed good SI as
evidenced by in vitro cytotoxicity test and did not adversely affect the health and general
behaviour of the treated animals as well. Hence these studies provide new prototype for the
development of new class of antifilarial agents.
6. Experimental
6.1. General information
All reagents were commercial and were used without further purification. Chromatography
was carried on silica gel (60−120 and 100−200 mesh). All reactions were monitored by thin-
layer chromatography (TLC), and silica gel plates with fluorescence F254were used. Melting
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points were taken in open capillaries on a Complab melting point apparatus and are presented
uncorrected. Infrared spectra were recorded on
a
Perkin-Elmer FT-IR RXI
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spectrophotometer. H NMR and C NMR spectra were recorded using Bruker Supercon
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Magnet DPX-200 and DRX-300 spectrometers (operating at 300 and 400 MHz for H and 75
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and 100 MHz for C) using CDCl as solvent and tetramethylsilane (TMS) as internal
3
standard. Chemical shifts are reported in parts per million. Electrospray ionization mass
spectra (ESIMS) were recorded on Thermo Lcq Advantage Max-IT. High resolution mass
spectra (HRMS) were recorded on 6520 Agilent Q Tof LC MS/MS (Accurate mass).
6.2. General procedure for the synthesis of compounds (2a–d) and chalcones (3a-n)
Synthesis of dicarbaldehyde substrates and the regioselective para condensed chalcones were
achieved by our previously reported protocol [29].
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6.3. General procedure for the synthesis of compounds (4a-n)
A mixture of appropriate para condensed chalcones 3a-n (1 equiv) and 2-aminothiazole (1
equiv) in ethanol were refluxed for 3h. The solvent was evaporated under vacuum and the
solid was purified directly with column chromatography to obtain the respective compounds
4a-n in excellent yields.
1
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6.3.1. (E)-3-(4-hydroxy-3-methyl-5-((E)-(thiazol-2-ylimino)methyl)phenyl)-1-p-tolylprop-2-
en-1-one (4a)
o
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Yellow solid, yield: 75%; mp 170-171 C; IR (KBr): 3584, 3018, 1657, 763 cm ; H NMR
(CDCl 400 MHz) δ: 12.92 (s, 1H), 9.25 (s, 1H), 7.94 (d, J = 8.2 Hz, 2H), 7.76 (d, J = 15.6
3
,
Hz, 1H), 7.69 (d, J = 3.4 Hz, 1H), 7.65 (s, 1H), 7.58 (s, 1H), 7.44 (d, J = 15.6 Hz, 1H), 7.32-
1
3
7.28 (m, 3H), 2.44 (s, 3H), 2.36 (s, 3H); C NMR (CDCl 100 MHz): 189.7, 169.6, 164.4,
3
,
162.0, 143.6, 143.3, 141.6, 135.8, 134.4, 132.6, 129.4, 128.6, 127.7, 126.5, 120.0, 119.1,
+
117.8, 21.7, 15.7; ESI-MS (m/z): 363.2 (M+H) ; HRMS (m/z): calcd for C H N O S
21
18
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2
+
(M+H) 363.1167, found: 363.1153.
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6.3.2. (E)-3-(4-hydroxy-3-methyl-5-((E)-(thiazol-2-ylimino)methyl)phenyl)-1-(4-methoxy
phenyl)prop-2-en-1-one (4b)
o
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Yellow solid, yield: 75%; mp 168-169 C; IR (KBr): 3574, 3045, 1684, 778 cm ; H NMR
(CDCl 400 MHz) δ: 12.91 (s, 1H), 9.25 (s, 1H), 7.76 (d, J = 15.5 Hz, 1H), 7.69 (d, J = 3.5
3
,
Hz, 1H), 7.65 (s, 1H), 7.57 (s, 1H), 7.45 (d, J = 15.5 Hz, 1H), 7.28 (d, J = 3.4 Hz, 1H), 6.99
13
(d, J = 8.8 Hz, 2H), 3.89 (s, 3H), 2.36 (s, 3H); C NMR (CDCl 100 MHz) : 188.4, 169.7,
3
,
164.4, 163.4, 161.9, 142.9, 141.6, 134.4, 132.6, 131.2, 130.8, 127.7, 126.5, 119.8, 119.1,
+
117.8, 113.9, 55.5, 15.7; ESI-MS (m/z): 379.2 (M+H) ; HRMS (m/z): calcd for C H N O S
2
1
19
2
3
+
(M+H) 379.1116, found: 379.1104.
1
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6.3.3. (E)-1-(furan-2-yl)-3-(4-hydroxy-3-methyl-5-((E)-(thiazol-2-ylimino)methyl)phenyl)
prop-2-en-1-one (4c)
o
-1 1
Yellow solid, yield: 80%; mp 188-189 C; IR (KBr): 3629, 3021, 1593, 761 cm ; H NMR
(CDCl 300 MHz ) δ: 12.94 (s, 1H), 9.24 (s, 1H), 7.82 (d, J = 15.7 Hz, 1H), 7.69 (d, J = 3.3
3
,
Hz, 1H), 7.65 (s, 2H ), 7.58 (s, 1H), 7.38-7.32 (m, 2H), 7.28 (d, J = 3.3 Hz, 1H), 6.60-6.59
+
(m, J = 1.6 Hz, 1H), 2.23 (s, 3H); ESI-MS (m/z): 339.2 (M+H) ; HRMS (m/z): calcd for
+
C H N O S (M+H) 339.0803, found: 339.0798.
18
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6.3.4. (E)-1-(furan-2-yl)-3-(4-hydroxy-3-methyl-5-((E)-(thiazol-2-ylimino)methyl)phenyl)
prop-2-en-1-one (4d)
o
-1 1
Yellow solid, yield: 74%; mp 183-184 C; IR (KBr): 3654, 3075, 1576, 746 cm ; H NMR
(CDCl 300 MHz ) δ: 12.95 (s, 1H), 9.27 (s, 1H), 7.89 (d, J = 3.0 Hz, 1H), 7.82 (d, J = 15.4
3
,
Hz, 1H), 7.72-7.69 (m, 2H ), 7.66 (s, 1H), 7.60 (s, 1H), 7.36-7.28 (m, 2H), 7.22-7.19 (m, 1H),
1
3
2.38 (s, 3H); C NMR (CDCl 75 MHz) : 181.8, 169.6, 164.3, 162.2, 145.7, 143.0, 141.7,
3,
134.5, 133.8, 132.8, 131.7, 128.3, 127.8, 126.2, 119.7, 119.1, 117.9, 15.7; ESI-MS (m/z):
+
+
355.1 (M+H) ; HRMS (m/z): calcd for C H N O S (M+H) 355.0575, found: 355.0587.
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6.3.5. (E)-3-(4-hydroxy-3-isopropyl-5-((E)-(thiazol-2-ylimino)methyl)phenyl)-1-p-tolylprop-
2-en-1-one (4e)
o
-1 1
Yellow solid, yield: 64%; mp 150-151 C; IR (KBr): 3635, 3078, 1642, 767 cm ; H NMR
(CDCl 300 MHz) δ: 13.02 (s, 1H), 9.24 (s, 1H), 7.93 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 15.6
3
,
Hz, 1H), 7.68-7.66 (m, 2H), 7.58 (d, J = 1.8 Hz, 1H), 7.42 (d, J = 15.6 Hz, 1H), 7.31-7.27 (m,
13
3H), 3.47-3.38 (m, 1H), 2.43 (s, 3H), 1.32 (d, J = 6.9 Hz, 6H); C NMR (CDCl 100 MHz) :
3,
189.9, 169.6, 164.6, 161.3, 143.7, 143.6, 141.6, 137.9, 135.8, 132.3, 130.8, 129.4, 128.7,
+
126.6, 120.1, 119.1, 118.1, 26.9, 22.3, 21.7; ESI-MS (m/z): 391.2 (M+H) ; HRMS (m/z):
+
calcd for C H N O S (M+H) 391.1480, found: 391.1474.
2
3
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1
1
1
1
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6.3.6. (E)-3-(4-hydroxy-3-isopropyl-5-((E)-(thiazol-2-ylimino)methyl)phenyl)-1-(4methoxy
phenyl)prop-2-en-1-one (4f)
o
-1 1
Yellow solid, yield: 70%; mp 180-181 C; IR (KBr): 3670, 3176, 1593, 765 cm ; H NMR
(CDCl 300 MHz) δ: 13.02 (s, 1H), 9.25 (s, 1H), 8.05 (d, J = 8.7 Hz, 2H), 7.78 (d, J = 15.5
3
,
Hz, 1H), 7.70-7.67 (m, 3H), 7.58 (d, J = 1.8 Hz, 1H), 7.45 (d, J = 15.5 Hz, 1H), 7.28 (d, J =
3.6 Hz, 1H), 6.99 (d, J = 8.7 Hz, 2H), 3.89 (s, 3H), 3.51-3.40 (m, 1H), 1.33 (d, J = 6.9 Hz,
1
3
6H); C NMR (CDCl 75 MHz) : 188.6, 169.7, 164.6, 163.4, 161.2, 143.3, 141.6, 137.8,
3,
132.2, 131.3, 130.8, 130.7, 126.7, 119.9, 119.0, 118.1, 113.9, 55.5, 26.9, 22.3; ESI-MS (m/z):
+
+
407.2 (M+H) ; HRMS (m/z): calcd for C H N O S (M+H) 407.1429, found: 407.1420.
2
3
23
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6.3.7. (E)-1-(furan-2-yl)-3-(4-hydroxy-3-isopropyl-5-((E)-(thiazol-2-ylimino)methyl)
phenyl)prop-2-en-1-one (4g)
o
-1 1
Yellow solid, yield: 76%; mp 183-184 C; IR (KBr): 3732, 3134, 1655, 767 cm ; H NMR
(CDCl 300 MHz) δ: 13.05 (s, 1H), 9.26 (s, 1H), 7.85 (d, J = 15.7 Hz, 1H), 7.69-7.66 (m,
3
,
3H), 7.61 (s, 1H), 7.38-7.33 (m, 2H), 7.29 (d, J = 3.4 Hz, 1H), 6.61-6.60 (m, 1H), 3.48-3.39
13
(m, 1H), 1.32 (d, J = 6.9 Hz, 6H); C NMR (CDCl 75 MHz) : 178.0, 169.6, 164.6, 161.5,
3
,
153.9, 146.4, 143.2, 141.6, 137.9, 132.4, 130.9, 126.3, 119.2, 119.1, 118.1, 117.3, 112.6,
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26.9, 22.2; ESI-MS (m/z): 367.2 (M+H) ; HRMS (m/z): calcd for C H N O S (M+H)
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367.1116, found: 367.1124.
6.3.8. (E)-3-(4-hydroxy-3-isopropyl-5-((E)-(thiazol-2-ylimino)methyl)phenyl)-1-(thiophen-2-
yl)prop-2-en-1-one (4h)
o
-1 1
Yellow solid, yield: 76%; mp 160-161 C; IR (KBr): 3654, 3172, 1652, 772 cm ; H NMR
(CDCl 300 MHz) δ: 13.12 (s, 1H), 9.34 (s, 1H), 7.96 (d, J = 2.9 Hz, 1H), 7.90 (d, J = 15.6
3
,
Hz, 1H), 7.77-7.74 (m, 3H), 7.68 (s, 1H), 7.42-7.33 (m, 2H), 7.28-7.25 (m, 1H), 3.56-3.47
13
(m, 1H), 1.40 (d, J = 6.8 Hz, 6H); C NMR (CDCl 75 MHz) : 181.9, 169.6, 164.5, 161.4,
3,
145.7, 143.3, 141.6, 137.9, 133.7, 132.3, 131.7, 130.9, 128.3, 126.3, 119.6, 119.1, 118.1,
+
+
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1
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1
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1
2
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2
2
2
2
26.9, 22.2; ESI-MS (m/z): 383.2 (M+H) ; HRMS (m/z): calcd for C H N O S (M+H)
20 19 2 2 2
383.0888, found: 383.0883.
6.3.9. (E)-3-(4-hydroxy-3-propyl-5-((E)-(thiazol-2-ylimino)methyl)phenyl)-1-p-tolylprop-2-
en-1-one (4i)
o
-1 1
Yellow solid, yield: 62%; mp 149-150 C; IR (KBr): 3610, 3192, 1652, 767 cm ; H NMR
(CDCl 300 MHz) δ: 12.94 (s, 1H), 9.25 (s, 1H), 7.94 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 15.6
3
,
Hz, 1H), 7.69 (d, J = 3.4 Hz, 1H), 7.62 (s, 1H), 7.59 (s, 1H), 7.44 (d, J = 15.6 Hz, 1H), 7.32-
7.28 (m, 3H), 2.72 (t, J = 7.3 Hz, 2H), 2.44 (s, 3H), 1.75-1.68 (m, 2H), 1.01 (t, J = 7.2 Hz,
1
3
3H); C NMR (CDCl 100 MHz) : 189.8, 169.7, 164.5, 161.9, 143.6, 143.5, 141.7, 135.8,
3
,
134.0, 132.7, 132.1, 129.4, 128.7, 126.5, 120.1, 119.1, 118.1, 31.8, 22.7, 21.7, 14.1; ESI-MS
+
+
(m/z): 391.2 (M+H) ; HRMS (m/z): calcd for C H N O S (M+H) 391.1480, found:
2
3
23
2
2
391.1478.
6.3.10. (E)-3-(4-hydroxy-3-propyl-5-((E)-(thiazol-2-ylimino)methyl)phenyl)-1-(4-methoxy
phenyl)prop-2-en-1-one (4j)
o
-1 1
Yellow solid, yield: 66%; mp 145-146 C; IR (KBr): 3652, 3234, 1667, 761 cm ; H NMR
(CDCl 300 MHz) δ: 12.91 (s, 1H), 9.24 (s, 1H), 8.04 (d, J = 8.7 Hz, 2H), 7.75 (d, J = 15.5
3
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Hz, 1H), 7.68 (d, J = 3.4 Hz, 1H), 7.61 (s, 1H), 7.57 (s, 1H), 7.44 (d, J = 15.5 Hz, 1H), 7.27
(d, J = 3.4 Hz, 1H), 6.98 (d, J = 8.7 Hz, 2H), 3.88 (s, 3H), 2.71 (t, J = 7.3 Hz, 2H), 1.75-1.68
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(m, 2H), 1.01 (t, J = 7.2 Hz, 3H); C NMR (CDCl 75 MHz) : 188.5, 169.7, 164.5, 163.4,
3
,
161.8, 143.0, 141.6, 133.9, 132.6, 132.6, 132.1, 131.3, 130.8, 126.6, 119.9, 119.1, 118.1,
+
113.9, 55.5, 31.8, 22.7, 14.1; ESI-MS (m/z): 407.2 (M+H) ; HRMS (m/z): calcd for
+
C H N O S (M+H) 407.1429, found: 407.1419.
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6.3.11. (E)-3-(3-tert-butyl-4-hydroxy-5-((E)-(thiazol-2-ylimino)methyl)phenyl)-1-p-tolylprop-
2-en-1-one (4k)
o
-1 1
Yellow solid, yield: 72%; mp 210-211 C; IR (KBr): 3655, 3157, 1735, 762 cm ; H NMR
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
(CDCl 400 MHz) δ: 13.3 (s, 1H), 9.26 (s, 1H), 7.94 (d, J = 8.1 Hz, 2H), 7.78 (d, J = 15.6,
3
,
1H), 7.74 (d, J = 2 Hz, 1H), 7.69 (d, J = 3.5 Hz, 1H),7.62 (s, 1H), 7.42 (d, J = 15.6 Hz, 1H),
1
3
7.31 (d, J = 7.9 Hz, 1H), 7.29 (d, J = 3.4 Hz, 1H), 2.44 (s, 3H), 1.49 (s, 9H); C NMR (
CDCl 100 MHz): 188.6, 169.5, 164.9, 163.4, 163.0, 143.5, 141.6, 138.9, 132.6, 131.6,
3,
131.3, 130.8, 126.2, 119.8, 119.0, 118.6, 113.9, 55.5, 35.1, 29.3; ESI-MS (m/z): 405.2
+
+
(M+H) ; HRMS (m/z): calcd for C H N O S (M+H) 405.1637, found: 405.1648.
2
4
25
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2
6.3.12. (E)-3-(3-tert-butyl-4-hydroxy-5-((E)-(thiazol-2-ylimino)methyl)phenyl)-1-(4-
methoxyphenyl)prop-2-en-1-one (4l)
o
-1 1
Yellow solid, yield: 76%; mp 199-200 C; IR (KBr): 3756, 3112, 1743, 735 cm ; H NMR (
CDCl 400 MHz) δ: 13.38 (s, 1H), 9.26 (s, 1H), 8.04 (d, J = 8.9 Hz, 2H), 7.78 (d, J = 15.5
3,
Hz, 1H ), 7.74 (d, J = 2.0 Hz, 1H), 7.69 (d, J = 3.5 Hz, 1H), 7.62 (d, J = 2.0 Hz, 1H), 7.43 (d,
J = 15.5 Hz, 1H), 7.29 (d, J = 3.4 Hz, 1H), 6.99 (d, J = 8.9 Hz, 2H), 3.90 (s, 3H), 1.49 (s,
1
3
9H); C NMR (CDCl 100 MHz) : 188.6, 169.5, 164.9, 163.4, 163.0, 143.5, 141.6, 138.9,
3
,
132.6, 131.6, 131.3, 130.8, 126.2, 119.8, 119.0, 118.6, 113.9, 55.6, 35.2, 29.3; ESI-MS (m/z):
+
+
421.2 (M+H) ; HRMS (m/z): calcd for C H N O S (M+H) 421.1586, found: 421.1598.
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6.3.13. (E)-3-(3-tert-butyl-4-hydroxy-5-((E)-(thiazol-2-ylimino)methyl)phenyl)-1-(furan-2-
yl)prop-2-en-1-one (4m)
o
-1 1
Yellow solid, yield: 68%; mp 180-181 C; IR (KBr): 3617, 3166, 1650, 762 cm ; H NMR (
CDCl 300 MHz) δ: 13.42 (s, 1H), 9.27 (s, 1H), 7.86 (d, J = 15.7 Hz, 1H), 7.75 (d, J = 1.5
3,
Hz, 1H), 7.69 (d, J = 3.4 Hz, 1H), 7.66 (s, 1H), 7.64 (s, 1H), 7.37-7.33 (m, 2H), 7.29 (d, J =
1
3
3.4 Hz, 1H), 6.61-6.59 (m, 1H), 1.49 (s, 9H); C NMR (CDCl 75 MHz) : 178.0, 169.5,
3
,
164.8, 163.3, 153.9, 146.4, 143.5, 141.6, 139.0, 132.8, 131.8, 125.9, 119.0, 118.6, 117.3,
+
112.6, 35.2, 29.3; ESI-MS (m/z): 381.2 (M+H) ; HRMS (m/z): calcd for C H N O S
21
21
2
3
+
(M+H) 381.1273, found: 381.1269.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
6.3.14. (E)-3-(3-tert-butyl-4-hydroxy-5-((E)-(thiazol-2-ylimino)methyl)phenyl)-1-(thiophen-2-
yl)prop-2-en-1-one (4n)
o
-1 1
Yellow solid, yield: 74%; mp 180-182 C; IR (KBr): 3564, 3245, 1662, 763 cm ; H NMR (
CDCl 400 MHz) δ: 8.74 (s, 1H), 7.48 (s, 1H), 7.36 (s, 1H), 3.84 (s, 3H), 3.43 (s, 3H), 3.34-
3,
3.22 (m, 1H), 3.14 (t, J = 5.8 Hz, 2H), 2.59-2.50 (m, 5H), 2.12-2.06 (m, 2H), 1.66-1.61 (m,
1
3
2H), 1.22 (d, J = 6.9 Hz, 3H), 0.79 (t, J = 6.5 Hz, 3H); C NMR (CDCl 75 MHz) : 182.0,
3,
169.5, 164.9, 163.3, 145.8, 143.6, 141.7, 139.1, 133.8, 132.8, 131.8, 131.7, 128.3, 125.8,
+
119.6, 119.1, 118.6, 35.2, 29.3; ESI-MS (m/z): 397.2 (M+H) ; HRMS (m/z): calcd for
+
C H N O S (M+H) 397.1044, found: 397.1050.
21
21
2
2 2
Acknowledgements
The authors are grateful to the Director, CDRI, Lucknow, India for constant encouragement
in drug development program, S.P. Singh for technical support, SAIF for NMR, IR, and Mass
spectral data. This study was supported by the Network project “SPLENDID” of Council of
Scientific and Industrial Research, New Delhi, India. K.B.R, V.K, R.K.M and R.V are
thankful to CSIR and ICMR, New Delhi, India for financial support. This is CSIR-CDRI
communication number 8684.
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References
[1]
[2]
[3]
[4]
[5]
M.J. Taylor, A. Hoerauf, M. Bockarie, Lymphatic filariasis and onchocerciasis,
Lancet 376 (2010) 1175-85.
WHO. Global programme to eliminate lymphatic filariasis progress report on mass
drug administration, 86 (2011) 377-388.
E.A. Ottesen, Lymphatic filariasis: treatment, control and elimination, Adv. Parasitol.
61 (2006) 395-441.
T. Streit, Lafontant, J.G. Lafontant, Eliminating lymphatic filariasis: a view from the
Field, Ann. N. Y. Acad .Sci. 1136 (2008) 53–63.
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G. Dreyer, F. Amaral, J. Noroes, Z. Medeiros, D. Addiss, A new tool to assess the
adulticidal efficacy in vivo of antifilarial drugs for bancroftian filariasis, Trans. R.
Soc. Trop. Med. Hyg. 89 (1995) 225-226.
[6]
[7]
E.A. Ottesen, Infection and disease in lymphatic filariasis: an immunological
perspective, Parasitology 104 (1992) S71-S79.
Study evaluating orally administered moxidectin in subjects with Onchocerca
volvulus infection. http://clinicaltrials.gov/ct2/show/NCT00300768 (accessed Jan 16,
2014).
[8]
[9]
Study comparing moxidectin and ivermectin in subjects with Onchocerca volvulus
infection. http://clinicaltrials.gov/ct2/show/NCT00790998 (accessed Jan 16, 2014).
E.A. Ottesen, M.M. Ismail, J. Horton, The role of albendazole in programmes to
eliminate lymphatic filariasis, Parasitol. Today 15 (1999) 382-386.
[10] Global programme to eliminate lymphatic filariasis, Wkly. Epidemiol. Rec. 84 (2009)
437-444.
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
[11] K.V. Sashidhara, A. Kumar, K.B. Rao, V. Kushwaha, K. Saxena, P.K. Murthy, In
vitro and in vivo antifilarial activity evaluation of 3,6-epoxy [1,5]dioxocines: a new
class of antifilarial agents, Bioorg. Med. Chem. Lett. 22 (2012) 1527-1532.
[12] S. Rathaur, M. Yadav, N. Singh, A. Singh, Effect of diethylcarbamazine, butylated
hydroxy anisole and methyl substituted chalcone on filarial parasite Setaria cervi:
proteomic and biochemical approaches, J. Proteomics 74 (2011) 1595-606.
[13] Y. Kumar, R. Green, D.S. Wise, L.L. Wotring, L.B. Townsend, Synthesis of 2,4-
disubstituted thiazoles and selenazoles as potential antifilarial and antitumor agents. 2.
2-Arylamido and 2-alkylamido derivatives of 2-amino-4-(isothiocyanatomethyl)
thiazole and 2-amino-4-(isothiocyanatomethyl)selenazole, J. Med. Chem. 36(1993)
3849-3852.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
[14] P.K. Murthy, K. Tyagi, T.K. Roy Chowdhury, A.B. Sen, Susceptibility of Mastomys
natalensis (GRA strain) to a subperiodic strain of human Brugia malayi, Indian J.
Med. Res. 77 (1983) 623-630.
[15] P.K. Murthy, P.S. Murthy, K. Tyagi, R.K. Chatterjee, Fate of infective larvae of
Brugia malayi in the peritoneal cavity of Mastomys natalensis and Meriones
unguiculatus, Folia Parasitol (Praha). 44 (1997) 302-304.
[16] P.K. Murthy, R.K. Chatterjee, Evaluation of two in vitro test systems employing
Brugia malayi parasite for screening of potential antifilarials, Curr. Sci. 77 (1999)
1084-1089.
[17] V. Lakshmi, S.K. Joseph, S. Srivastava, S.K. Verma, M.K. Sahoo, V. Dube, S.K.
Mishra, P.K. Murthy, Antifilarial activity in vitro and in vivo of some flavonoids
tested against Brugia malayi. Acta Tropica 116 (2010) 127-33.
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2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
[18] P.K. Murthy, S.K. Joseph, P.S.R. Murthy, Plant products in the treatment and control
of filariasis and other helminthic infections and assay systems for antifilarial/
anthelmintic activity. Planta Medica, 77 (2011) 647-661.
[19] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1983) 55-63.
[20] B. Page, M. Page, C. Noel, A new fluorometric assay for cytotoxicity measurements
in vitro, Int. J. Oncol. 3 (1993) 473-476.
[21] W. Huber, J.C. Koella, A comparison of three methods of estimating EC50 in studies
of drug resistance of malaria parasites, Acta Trop. 55 (1993) 257-261.
[22] K. Kalani, V. Kushwaha, R. Verma, P.K. Murthy, S.K. Srivastava, Glycyrrhetinic
acid and its analogs: A new class of antifilarial agents. Bioorg Med Chem Lett. 23
(2013) 2566-2570.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
[23] D. Yadav, S.C. Singh, R.K. Verma, K. Saxena, R. Verma, P.K. Murthy, M.M. Gupta,
Antifilarial diarylheptanoids from Alnus nepalensis leaves growing in high altitude
areas of Uttarakhand, India. Phytomedicine 20 (2013) 124-132.
[24] N. Fatma, P.K. Murthy, R.K. Chatterjee, 1992. Chemotherapeutic response of filarial
parasites to ivermectin. In Book: Tropical Diseases Molecular Biology and Control
strategies. Eds. SKumar, AK Sen, GP Dutta, RN Sharma. Publications & Information
Directorate, CSIR, Dr. K.S. Krishnan Marg, New Delhi 110 012, p55-64.
[25] R.L. Gaur, S. Dixit, M.K. Sahoo, M. Khanna, S. Singh, P.K. Murthy, Anti-filarial
activity of novel formulations of albendazole against experimental brugian filariasis,
Parasitology 134 (2007) 537-544.
[26] G. Lammler, E. Wolf, Chemoprophylactic activity of filaricidal compounds on
Litomosoides carinii infection of Mastomys natalensis (author's transl), Tropenmed.
Parasitol. 28 (1977) 205-225.
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[27] R.K. Chatterjee, N. Fatma, P.K. Murthy, P. Sinha, D.K. Kulshreshtha, B.N. Dhawan,
Macrofilaricidal activity of the stem bark of Streblus asper and its major active
constituents, Drug Develop. Res. 26 (1992) 67-78.
[28] K. Tyagi, P.K. Murthy, R.K. Chatterjee, A.B. Sen, Chemotherapeutic response of
Brugia malayi to antifilarials in Mastomys natalensis. Indian J. Parasit. 10 (1986)
195-207.
[29] K.V. Sashidhara, G.R. Palnati, R. Sonkar, S.R. Avula, C. Awasthi, G. Bhatia,
Coumarin chalcone fibrates: a new structural class of lipid lowering agents, Eur. J.
Med. Chem. 64 (2013) 422-431.
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Captions to figures
Figure 1. Chemical structures of some potent antifilarial compounds and our hybrid
4
5
6
7
8
9
0
1
2
molecules.
1
13
Figure 2. Selected H to C-HMBC correlations of compound 4n in CDCl .
3
Figure 3. Female parasite recovered from 4n treated animal showed embryostatic effect of
the compound. Uterus of female worm contained distorted eggs and the embryos were devoid
of developing mf (arrows) (unstained preparation). Bar =100µM
Figure 4. Female worm from control animal showing eggs (arrows), developing mf in
embryos (hollow arrows) and developed mf (arrows heads) expelled from uterus by gentle
pressure on the worm during preparation, can also seen around the worm (unstained
preparation). Bar =100µM
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Figure 5. SAR of synthesized hybrid.
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Table 1. In vitro activity of chalcone-thiazole compounds (4a-n) and the reference drugs
ivermectin and DEC-C (Diethylcarbamazine-Citrate) on adult worms (AW) and microfilariae
(mf) of Brugia malayi assessed by motility assay (MA) and MTT reduction assay.
a
b
b
c
Compounds LC100
µM)
IC50
Mean %
inhibition
in MTT
SI
LC100 IC50
SI
CC50
(
(µM)
(µM) (µM)
(µM)
reduction
for AW in MA
by AW
for
for mf in MA
AW in
MA
4
4
4
4
4
4
4
4
4
4
4
4
4
4
a
b
c
d
e
f
>40
>40
>40
>40
>40
>40
10
-
NI
25
62
-
-
>40
>40
>40
>40
>40
>40
10
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
61
49
NI
NI
NI
NI
NI
NI
63
5.80
62.54
-
-
-
-
g
h
i
3.2
19
3.5
-
17
-
60
>40
>40
>40
>40
>40
40
-
-
>40
>40
>40
40
-
-
-
-
-
-
j
-
-
-
-
-
k
l
-
-
-
-
-
-
-
40
-
-
-
m
n
-
-
20
7.07
1.8
28.57
22
220
40
230
9000
2.5
0.9
44
5
Ivermectin 5
3.05
314.98
75.41
28.57
2.5
1.57
297.30
146.49
30.27
DEC-C
1000
500
a
b
4
5
6
LC = 100% reduction in motility indicates death of parasite; IC = 50% concentration of the agent
100
50
c
at which 50% inhibition in motility of the parasites is achieved; NI=No inhibition; CC =
5
0
concentration at which 50% of cells are killed; SI= Selectivity Index (CC /IC ); DEC-C=
5
0
50
7
8
Diethylcarbamazine-citrate.
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Table 2. Antifilarial activity of chalcone-thiazole compounds (4g and 4n) and reference
drugs ivermectin and DEC-C against Brugia malayi in jirds (Meriones unguiculatus).
Antifilarial
agent
Dose
Worms recovered
Sterilized female
worm count (%)
mg/kg s.c.
x 5 days
Male
Female
Total
% reduction
(
n)
(
over untreated)
9.33±1.15
4
4
g
100 (5)
100 (6)
1 (4)
1.67±0.58
1.33±1.15
2.75±0.96
7.67±0.58
10±0
0
(
18.16)
(0)
@
n
11.33±1.15
10±0 ***
(
100)
#
Ivermectin
6.25±1.50
9.00±2.52
2.75±0.96**
(
21.05)
9.20±1.48
19.30)
(43.21±5.15)
0.60±0.89
#
DEC-C
25 (6)
-(5)
2.60±0.55
3.20±1.10
6.60±1.14
(
(7.86±11.41)
0.80±0.45
Untreated
Control
8.203±0.84
11.40±1.52
(9.80±5.63)
3
4
5
6
7
8
Values are Mean±SD
#
@
n=number of animals; DEC-C: Diethylcarbamazine-Citrate; Reference drugs; distorted and
disintegrated microfilarial stages in uteri; **P<0.01 (Ivermectin vs Untreated control);
***P<0.001 (4n vs 4g/Ivermectin/DEC-C/Untreated control; Ivermectin vs 4g/DEC-C).
Statistics: Tukey's Multiple Comparison Test.
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Table 3. Antifilarial activity of chalcone-thiazole compounds (4g and 4n) and reference drug
DEC-C against Brugia malayi in Mastomys coucha. Values are Mean±SD
Antifilarial Dose in
agent mg/kg , s.c.
No. Live worms
Sterilized
female
x 5 days (n)
worms count
(
%)
Male
Female
Total
% over untreated)
22.75±6.18**
40.91%)
19.80±4.60***
48.57%)
(
4
4
g
100 (8)
100 (8)
50 (6)
7.88±3.76
6.60±4.04
14.88±4.09
13.20±2.77
4.88±3.52
30)
(
(
n
5.80±1.48
44)
(
(
#
DEC-C
6.00±1.79
23.33±4.60
21.67±6.15
29.33±4.80
23.82)
38.50±10.39
6.8±5.70 (29)
(
Untreated
Control
- (5)
16.83±5.19
5.17±2.64
(
23)
#
4
5
6
n=number of animals; DEC-C showed >85% microfilaricial activity on day 7 post initiation
of treatment; **P<0.01; ***P<0.001 (vs untreated control; Statistic: Tukey's Multiple
Comparison Test).
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Figure 1. Chemical structures of some potent antifilarial compounds and our designed
hybrid.
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Figure 2. Selected H to C-HMBC correlations of compound 4n in CDCl .
3
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