Design and synthesis of imidazo[1,2‐a]pyridine‐chalcone conjugates as antikinetoplastid agents

A library of imidazo[1,2‐a]pyridine‐appended chalcones were synthesized and characterized using 1H NMR, 13C NMR and HRMS. The synthesized analogues were screened for their antikinetoplastid activity against Trypanosoma cruzi, Trypanosoma brucei brucei, Trypanosoma brucei rhodesiense and Leishmania infantum. The analogues were also tested for their cytotoxicity activity against human lung fibroblasts and primary mouse macrophages. Among all screened derivatives, 7f was found to be the most active against T. cruzi and T. b. brucei exhibiting IC50 values of 8.5 and 1.35 μM, respectively. Against T. b. rhodesiense, 7e was found to be the most active with an IC50 value of 1.13 μM. All synthesized active analogues were found to be non‐cytotoxic against MRC‐5 and PMM with selectivity indices of up to more than 50.

F I G U R E 1 Selected literature reports of antikinetoplastid agents.
In context of the above discussion and in our effort to develop novel antikinetoplastid agents, we herein report the synthesis of imidazo[1,2-a]pyridine-chalcone amides (7a-l, Scheme 1).The synthesized analogues (5, 7a-l) were examined for their antikinetoplastid activity against T. cruzi, T. b. brucei, T. b. rhodesiense and Leishmania infantum.In addition, the synthesized analogues were also tested for their cytotoxicity against human lung fibroblasts (MRC-5) and primary mouse macrophages (PMMs).
Appearance of the amide NH proton at δ 10.62 and methyl group (-COCH 3 ) proton at δ 2.55 in 1 H NMR and the corresponding carbonyl group (C=O) of amide and ketone at δ 161.2 and 196.7, respectively on the 13 C NMR confirmed the formation of 5. Finally, The structure of all new molecules was characterized using 1 H NMR, 13 C NMR and HRMS (Data S1).

| Pharmacology
The antikinetoplastid activity of all the synthesized derivatives 5, 7a-l was investigated against T. cruzi, T. b. brucei, T. b. rhodesiense and L. infantum.In addition, the synthesized derivatives 5 and 7a-l were also tested for their cytotoxicity against MRC-5 and PMM.Among all the derivatives, 7f was found to be most active against T. cruzi with an IC 50 value of 0.68 μM.Analogue 7f shows a twofold increase in activity with respect to the reference drug benznidazole with an IC 50 value of 1.35 μM.With respect to the activity against T. b. brucei, mono electrondonating group substituted product, for example, 7a and 7b, were found to be active with an IC 50 values of 6.40 and 5.35 μM, respectively.Halogen-substituted derivatives 7ek were found to exhibit IC 50 values ranging from 1.28 to 57.97 μM.The mono-halogenated derivatives 7e-j and 2and 3-substituted derivatives 7f, 7g and 7i were found to be more active than their 4-substituted analogues 7e, 7h and 7j.For instance, 7f and 7g were found to be the most active with an IC 50 values of 1.28 and 1.35 μM, respectively as compared to their 4-substituted counterpart 7h with an IC 50 value of 57.97 μM.Compound 7i was also found to be active with an IC 50 value of 2.20 μM as compared to compound 7j with an IC 50 value of 28.04 μM.Compound 7k also demonstrated low micromolar activity with an IC 50 value of 2.52 μM.Suramine was used as reference drug against T. b. brucei with an IC 50 value of 0.04 μM.
All synthesized derivatives were also tested against T. b. rhodesiense, revealing that among all methyl-and methoxy-substituted derivatives 7a-d, we found 7a to be most active with an IC 50 value of 2.31 μM.Among the mono-halogen-substituted derivatives, 7e-j were found to be active with IC 50 values in the range of 1-12 μM.
Against L. infantum, all derivatives 5, 7a-l were found to be inactive with IC 50 values of >64.00 μM as compared to miltefosine (IC 50 = 7.13 μM) which was included as reference.
With respect to the cytotoxicity against MRC-5, all derivatives 5, 7a-l were found to be non-cytotoxic with an IC 50 value of >64.00 μM as compared to tamoxifen (IC 50 = 10.79 μM) except for 7c and 7j which possess IC 50 values of 45.25 and 32.00 μM, respectively.Concerning cytotoxicity against PMM, all the derivatives 5, 7a-l were found to be non-cytotoxic with IC 50 values of >64.00 μM (Table 1).

| Drug likeliness properties and drug score predictions
Computational studies of compounds 5 and 7a-l were carried out for predicting the absorption, distribution, metabolism and excretion (ADME) properties (Table 2).Physiochemical properties of the molecules were also predicted using Lipinski's rule of five, drug likeness score and percentage absorption (Table 2).ADME properties were calculated online using Molinspiration cheminformatics software (Agarwal  et al., 2016;Reddy et al., 2018), while the drug likeness scores were calculated online using MolSoft software (Agarwal, Krishna, et al., 2018;Agarwal, Singh, et al., 2018;Reddy et al., 2018).In addition, all the derivatives were also evaluated for percentage absorption (% ABS) using the formula %ABS = 109 − (0.345 × TPSA) (Zhao et al., 2002).It was observed that all synthesized derivatives 5 and 7a-l were found to have TPSA less than 160 Å 2 but >40 Å 2 indicating that molecules to have a potential for good intestinal absorption property as compared to their blood-brain barrier (BBB) penetration ability (Table 2).Compound 7e, the most active compound against T. b. rhodesiense, was found to possess TPSA of 63.48 Å 2 and 7f most active against T. cruzi and T. b. brucei was found to possess TPSA of 63.48 Å 2 (Table 2).Compounds 7e and 7f also possessed a positive drug likeliness score of 0.73 and 0.58, respectively, which predicts them as good drug candidates (Table 2).

| CONCLUSION
In summary, we have designed and synthesised a series of imidazo[1,2-a]pyridine-chalcones in satisfactory yield.All synthesized derivatives were characterized using different analytical techniques such as 1 H NMR, 13 C NMR and HRMS.The synthesized analogues were evaluated for their antikinetoplastid activity against T. cruzi, T. b. brucei, T. b. rhodesiense and L. infantum.The compounds were also tested for cytotoxicity against MRC-5 and PMM.Among all analogues tested, 7f was found to be the most active against T. cruzi and T. b. brucei with IC 50 values of 8.5 and 1.35 μM, respectively.With respect to cytotoxicity against the human lung fibroblast cell line (MRC-5) and PMM, all active analogues were found to be non-cytotoxic.These results identify the synthesized analogues as hits against T. cruzi, T. b. brucei and T. b. rhodesiense.Additional follow-up studies are warranted to further explore the imidazo[1,2-a]pyridine-chalcone hit series and identify the most promising candidates for progression to in vivo models.To a stirred solution of imidazo[1,2-a]pyridine-2-carboxylic acid (3, 3 g, 1 mmol) in DMF (5 mL), triethylamine (6.48 mL, 2.5 mmol) was added at rt and subsequently, EDC.HCl (5.16 g, 1.5 mmol) and HOBt (2.75 g, 1 mmol) were added.The reaction was stirred at rt for 0.5 h.Thereafter, 1-(4-aminophenyl)ethan-1-one (4, 3.0 g, 1.2 mmol) was added and the reaction was stirred at rt for 8 h.The progress of the reaction was monitored by TLC.

| MATERIALS AND METHODS
After the completion of the reaction, it was quenched by adding water and the mixture extracted using ethyl acetate (100 mL × 2) twice.The combined organic layer was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure to give crude product.The crude product was purified using silica column chromatography with EA/Hexane (2:5) to obtain pure product (5) as off-white solid.

T A B L E 2
Drug likeliness properties, % ABS and drug score predictions of synthesized compounds (5, Abbreviations: miLogP, logarithm of compound partition coefficient between n-octanol and water; MW, molecular weight of the molecules; natoms, number of atoms in the molecule; nHBA, number of hydrogen bond acceptors; nHBD, number of hydrogen bond donors; nviolations, nrotb, number of rotatable bonds; TPSA, topological polar surface area; volume, volume of the molecule.a Percentage absorption calculated using the formula %ABS = 109 − (0.345 × TPSA).