Bovine trichomoniasis is a veneral disease caused by the protozoal 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 contact 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 around the world. In light of this current problem, the identification of new chemotherapies for this pathogen is still warranted.
Isatin is a versatile scaffold with wide possibility of chemical modification and display a diverse array of biological and pharmacological activities which include anti-HIV [10] , antiviral [11] , anti-tumour [12e14], antifungal [15, 16] , anti-angiogenic [17] , anticonvulsants [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 generation 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 spiroannulations [21] . However there are only few reports regarding the antineoplastic activities of N-alkyl and N-aryl isatins. Vine and coworkers 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 cells and U937 human monocyte-like histolytic lymphoma cells [22] . Recent report from our lab has shown the synthesis of N-alkylisatin 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 isatin-b-lactam conjugates along with the preliminary analysis of their in vitro activity against Trichomonas vaginalis at 10 and 100 mM [26] . Eighteen of the synthesized hybrids have shown 100% growth inhibition at 100 mM with the most potent and non-cytotoxic compound showing and IC 50 of 7.69 mM. The methodology was further extended towards the synthesis of b-amino alcohol based blactameisatin conjugates and their evaluation against T. vaginalis at 50 mM with the most active chimeric scaffold among the test series exhibited an IC 50 of 9.73 mM along with low cytotoxicity [27] . As an 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 prostate (PC-3) cancer cell line.
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 proceed 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-propargylated 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- 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 C 16 
The synthesized compounds were evaluated for their antitrichomonas 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 eCH 3 substituent at C-5 position of isatin ring of the synthesized Mannich adducts considerably improved the activity profile as evident by compounds 3ae3p and 5ae5d. Further, the introduction of 7-chloro-4-piperazin-1-yl-quinoline moiety remarkably improved the anti-trichomonas activity with a 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 IC 50 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 position of the isatin ring proved to be the most potent of the synthesized scaffolds with an IC 50 of 11.3 mM. The synthesized compounds were further evaluated for their cytotoxicity against human prostate (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 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.
In conclusion, the present manuscript describes the Cu(I)Cl promoted N-propargylated-isatin-Mannich mono-as well as bisadducts with an extension towards the synthesis of N-propargylated-isatin-7-chloroquinoline conjugates. The synthesized scaffolds 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 7c exhibiting an IC 50 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.
Melting points were determined by open capillary using Veego Precision Digital Melting Point apparatus (MP-D) and are uncorrected. 1 H NMR spectra were recorded in deuterochloroform and DMSO-d 6 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. 13 C NMR spectra were recorded on Jeol 300 (75 MHz) spectrometers in deuterochloroform and DMSO-d 6 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). 3.1. Procedure for the synthesis of C-5 substituted N-propargylated isatins 2
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 temperature 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 reaction, as evident from TLC, it was quenched by dropwise addition of water (20 mL) and subsequently extracted with ethyl acetate (3 Â 30 mL). The combined organic layers were washed with brine solution, dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure. Purification of the reaction mixture via column chromatography using hexane: ethyl acetate (4:1) mixture furnished the desired N-propargylated isatin derivatives in good yields.
Procedure for the synthesis of 7-chloro-4-piperazin-1-ylquinoline 6
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 completion; it was washed with 2 N NaOH solution with subsequent extraction with ethy lacetate (3 Â 30 mL). The combined organic layers were washed with brine solution, dried over anhydrous Na 2 SO 4 and concentrated under vacuum resulting in the desired product.
To a well stirred solution of N-propargylated isatin (1 mmol) in dry dioxane, catalytic amount of copper chloride and p-formaldehyde 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 subsequently extracted with ethyl acetate (3 Â 30 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure to result in the desired Mannich adducts in good yields.
Orange solid, yield 87%, m.p. 
Orange solid, yield 88%, m.p. 
To a well stirred solution of N-propargylated isatin (1 mmol) in dry dioxane, catalytic amount of copper chloride and p-formaldehyde 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 subsequently extracted with ethyl acetate (3 Â 30 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure to result in the desired bis-Mannich adducts in good yields.
3.4.1. Bis-{1-(yl-but-2-ynyl)-1H-indole-2,3-dione} piperazine (5a)
Orange solid, yield 67%, m.p. 3.5. Typical procedure for the synthesis of Mannich adducts 7ae7e
A mixture of N-propargylated isatin 2 (1 mmol), paraformaldehyde (2.5 mmol), 7-chloro-4-piperazin-1-yl-quinoline 7 (1 mmol) and anhydrous cupric chloride (0.5 mmol) in dry 1,4dioxane was heated at 100 C for 3 h. After completion of reaction as evidenced by TLC, the reaction mixture was filtered and the filtrate was treated with brine solution and extracted with ethyl acetate (3 Â 60 mL). Combined organic layers were dried over Na 2 SO 4 and the solvent was evaporated under reduced pressure resulting in a crude product which was purified by column chromatography using a chloroform:methanol (95:5) mixture.
To perform the initial susceptibility screens on T. foetus strain D1, compounds were dissolved in DMSO to obtain concentrations of 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 IC 50 value for compounds 7a, 7c and 7d was determined by running assays of increasing drug concentrations, 5 mMe40 mM, and performing a regression analysis using Prism software, from GraphPad.
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 mMe0.001 mM for 72 h 10 ml MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium 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. gratefully acknowledged. KL would like to acknowledge 2012 Pacific 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.