Synthesis and antiplasmodial activity of regioisomers and epimers of second-generation dual acting ivermectin hybrids

With its strong effect on vector-borne diseases, and insecticidal effect on mosquito vectors of malaria, inhibition of sporogonic and blood-stage development of Plasmodium falciparum, as well as in vitro and in vivo impairment of the P. berghei development inside hepatocytes, ivermectin (IVM) continues to represent an antimalarial therapeutic worthy of investigation. The in vitro activity of the first-generation IVM hybrids synthesized by appending the IVM macrolide with heterocyclic and organometallic antimalarial pharmacophores, against the blood-stage and liver-stage infections by Plasmodium parasites prompted us to design second-generation molecular hybrids of IVM. Here, a structural modification of IVM to produce novel molecular hybrids by using sub-structures of 4- and 8-aminoquinolines, the time-tested antiplasmodial agents used for treating the blood and hepatic stage of Plasmodium infections, respectively, is presented. Successful isolation of regioisomers and epimers has been demonstrated, and the evaluation of their in vitro antiplasmodial activity against both the blood stages of P. falciparum and the hepatic stages of P. berghei have been undertaken. These compounds displayed structure-dependent antiplasmodial activity, in the nM range, which was more potent than that of IVM, its aglycon or primaquine, highlighting the superiority of this hybridization strategy in designing new antiplasmodial agents.


Results and discussion
Chemistry. Synthesis and isolation. Routes for the synthesis of hybrids 12 and 15 from the common precursor 8 (Fig. 2) in combination with 6 and 2 are shown in Figs. 2 and 4. Compound 8 was prepared from 7 by treatment with 5% H 2 SO 4 in methanol, as previously described 48 . Compound 8 was converted into a common precursor, IVM aglycon-1H-imidazole-1-carboxylate 10, by sequentially treating 8 with (1) tert-butyl dimethylsilyl chloride (TBDMS-Cl) in the presence of imidazole as an activator of TBDMS-Cl and 4-dimethylamino pyridine (DMAP) as a nucleophilic base 48,52 , and (2) an excess of carbonyldiimidazole (CDI, 2.0 equiv) in dry benzene/dry toluene (Fig. 2) 53 . Intermediate 10 was reacted with 6 in the presence of 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU), a non-nucleophilic base, to obtain 11. Decomposition was noticed during the purification of 11 by column chromatography. Thus, 11 was rapidly passed through the column and the residue deprotected using p-toluene sulphonic acid (p-TSA) in methanol.
The residue was purified by column chromatography to obtain IVM-CQ hybrid 12, comprising of a mixture of the two inherent (R 2 = Me and H) components in the ratio 98.87:0.6, as revealed from the high-performance liquid chromatography (HPLC) analysis (see Supplementary information, Fig. S16). Additionally, the HPLC chromatograms also indicated the presence of two isomeric peaks corresponding to each of the two inherent components. Thus, the isomeric mixture of 12, as isolated above was subjected to preparative HPLC using reverse phase (X Select CSH C18) chromatography, and the two isomers (12a and 12b/ 25.0% and 71.0%, Fig. 3) were isolated. The ultra-performance liquid chromatography (UPLC, see Supplementary information, Figs S17, S18) analysis of the two isomers revealed an analytical purity of 98.41% and 99.58% for 12a and 12b, respectively. The minor components of the isomeric mixture of 12 (R 2 = H: 12a′ and 12b′, Fig. 3) however were eliminated during preparative HPLC purification. Interestingly, both 12a and 12b were stable and depicted parent ion peaks at identical mass (m/z 849.53) suggesting 12a and 12b to be structural isomers, whose structures could very convincingly be assigned using high-field NMR analysis (vide infra).
For the synthesis of IVM-PQ conjugates 15, our attempts at the use of the chemistry described in Fig. 2, by replacing 6 with 2 were not successful. Thus, treating 10 with 2 using different reaction conditions yielded the desired 14 in very poor yield. We envisaged that quaternization of the imidazole in 10 to create 13 might incorporate a better leaving group due to structural tautomerism (A ↔ B, Fig. 4) and increased yield of the desired product 54  The antiplasmodial activity of hybrids of IVM is strongly structure-dependent. Therefore, the unnerving yet obligatory challenge was to assign the structures of the two isomers of both 12 (12a and 12b) and 15 (15a and 15b). In this context, high-field NMR data presented itself as a dependable tool, as contrasting differences in the chemical shift (δ ppm), as well as multiplicity of the comparable protons was observed, leading to the unambiguous assignment of structures.
The 1 H-1 H COSY spectrum was quite useful in finding correlations leading to further simplification of the complex NMR data. In the 1 H NMR spectrum of 12a, the signal corresponding to C-2 H (Fig. 6) of the macrolide at δ 3.27 (br, 1H) was absent in the 1 H NMR spectrum of 12b. Instead, a 1H quintet at δ 2.54 (1H, J = 7.5 Hz) was observed in the NMR spectrum of 12b. Based on the coupling relationship revealed by the 1 H-1 H COSY spectrum of 12b (Fig. 6C), the quintet signal was assigned to the C-4 H of the macrolide. Quite convincingly, the corresponding change in the chemical shift and multiplicity of protons corresponding to C-4a of the macrolide was also observed. In 1 H NMR of 12a, the C-4a protons appeared as a 3H singlet at δ 1.87, while a 3H doublet at δ 1.23 (d, J = 7.0 Hz) was observed for the C-4a protons of 12b. This change in the NMR spectrum hinted at the shifting of the double bond from Δ 3 to Δ 4 positions in the oxahydrindene ring of the macrolide. Thus, as expected, an upfield shift of the C5-H from δ 4.33 (d, J = 6.0 Hz) in 12a to δ 3.61 (dd, J = 2.0, 7.5, 1H) was observed in the 1 H NMR spectrum of 12b. Other significant changes in the NMR spectra included: a downfield shift (Δδ = 0.73 ppm) of the C3-H, and the olefin C-9 H (Δδ = 0.41 ppm) of 12a and 12b. Based on similar proton couplings and correct mass spectral data (vide experimental), the structures of the isomers 12a and 12b were ascertained. The formation of the regioisomer 12b could be traced back to the synthetic step where DBU was used as a base. The C-2 H in 12a being acidic (due to the ester function) would yield a thermodynamically stable carbanion (compared to kinetically formed oxygen anions). Thus, re-protonation would yield both 12a and 12b.
Quite surprisingly, no significant differences were observed between the NMR spectra of isomers 15a and 15b (see Supplementary information, Figs S4, S5). However, the assignment of the complex signals could be readily achieved from the COSY spectra of 15a and 15b. For example, the cross-peaks of H 5 with H 6 , H 12 with H 12a and H 13 , H 2 and H 3 helped identify the coupling partners (see Supplementary information, Figs S24, S25). Given the close similarity in the NMR spectra of both 15a and 15b, the formation of regioisomers in analogy with 12a and 12b was ruled out.
The oxahydrindene part of the macrolide ring of the IVM hybrids having two hydroxyl groups adjacent to sp 3 hybridized carbons is prone to transformation into the benzenoid structure (A, Fig. 7) through double β-elimination of water, which, upon prototropic shift of a 8a-H, would result in an aromatic benzofuran ring (B). Interestingly, the spiroketal moiety of the macrolide remained intact.
The presence of signals corresponding to C 2 -H, C 3 -H, C 5 -H, and C 6 -H, in the NMR spectra of both the isomers of 15 (15a and 15b) led us to rule out the formation of A-C (Fig. 7). It was further corroborated by HRMS where a peak (m/z 871) corresponding to the molecular formula C 50 H 69 N 3 O 10 of the 15a and 15b was observed. However, in the NOESY spectra of 15a and 15b, the absence of through space coupling between C 4a -H with C 2 -H  Antiplasmodial activity. In vitro activity of "second-generation" IVM hybrids against Plasmodium hepatic infection. IVM Hybrids 12a,b and 15a,b were initially screened at 10 and 1 µM for their in vitro activity against the hepatic stage of P. berghei infection (Fig. 9). Pristine 7, 8, and 2 were employed as controls in these experiments. All compounds of interest dramatically impacted infection at 10 µM. However, in the case of the CQ hybrids 12a,b this was accompanied by a reduction in cell confluence, indicative of toxicity towards the host cells at this concentration. However, hybrids 12a,b were also the most active compounds at 1 µM. Given their potentially interesting activity, which was comparable to, or even higher than that of 7, all compounds were selected for IC 50 determination. Dose-dependent responses of each compound against P. berghei hepatic infection were obtained (Fig. 10), which enabled the determination of their IC 50 (Table 1). In agreement with the data from the initial screen, CQ hybrids 12a,b displayed the highest activity, with IC 50 values ranging from 0.186 to 0.317 µM, whereas PQ hybrids 15a,b were less active, with IC 50 values ranging from 1.291 to 2.057 µM. Comparing the activity of the most   48 with compound 12b, the most active member of the current second-generation series, shows that the latter is nearly threefold more active than the former. The fact that the IVM hybrids are significantly more potent antiplasmodial agents than IVM warrants the chemical modification of IVM to produce antiplasmodial agents with enhanced potency. Further, the complete loss of the antiplasmodial activity of the 8 strongly suggests some role of the substitution at the C-13 position of the macrolide structure.
In vitro activity against P. falciparum erythrocytic infection. To assess their activity against the blood stage of P. falciparum (PfNF54) infection, compounds were initially screened at 1000, 500, 100, and 10 nM (Fig. 11). Compounds 7, 8, 2, and 1 were employed as controls. Compound 8 and 2 were not active against the parasite as shown by comparison with the DMSO control. Similar to what was observed for the hepatic stage, CQ hybrids 12a,b showed the highest activity against P. falciparum blood stages. For that reason, 12a, 12b, mixture 12a + 12b, and 7 were selected for IC 50 determination. Dose-dependent values of the % of SYBR Green-positive events were obtained at selected concentrations ( Fig. 12) for IC 50 calculation (Table 1). CQ hybrids, 12a, 12b displayed IC 50 values between 48.2 and 74.3 nM, an activity lower than that previously determined for the CQ control (23.7 nM ± 10.1) 48 . Compound 7 was the least active compound tested, with an estimated IC 50 of 359.6 nM.
It is interesting to note that the IVM-PQ hybrids 15a and 15b display lower activity than their CQ counterparts 12a and 12b against both stages of Plasmodium infection. This is unsurprising in what concerns the parasite's blood stages, since 1 is a known blood stage antiplasmodial, whereas 2 does not display significant activity against this stage of the parasite's life cycle.
The fact that 12a and 12b are also more active than 15a and 15b against hepatic infection is somewhat puzzling. However, it should be noted that the in vitro hepatic stage antiplasmodial activity of 7 (~ 2 μM) is higher than that of 2 (~ 10 μM) and, as such, the IVM moiety of the PQ hybrids 15a and 15b is the main contributor towards hepatic stage activity. The data (Table 1) suggests that the hepatic stage activity is potentiated by CQ analogue more than PQ in the hybrids of IVM. Evidently, when hybridized with IVM, the enhanced synergization of the former leads to the enhanced hepatic stage antiplasmodial activity, whereas such enhancement is absent in the PQ hybrids 15a and 15b, where the activity primarily results from the IVM moiety.

Conclusions
The second-generation IVM hybrids synthesized through molecular hybridization of 7 with the CQ analogue 6 and antiplasmodial drug 2 display higher potency against the hepatic and blood stages of Plasmodium infection than their first-generation counterparts. IVM-CQ hybrids 12a,b were the most active compounds against  The blood-stage antiplasmodial activity of the compounds against the P. falciparum NF54 strain was significantly higher than that observed against the hepatic infection. Compound 12a was the most active (IC 50 = 48.2 nM) displaying over sevenfold higher potency than the pristine IVM, and more active than the  www.nature.com/scientificreports/ comparable member of the series of the first-generation IVM hybrids, emphasizing the advantage of the hybridization approach described here. IVM-PQ hybrids were not active enough to determine IC 50 values. Compound 8 displayed the lowest activity of all the compounds synthesized and paralleled the trend observed in the series of the first-generation IVM hybrids. Additionally, we evaluated the activity of the mixture of isomers in order to determine whether it is necessary to isolate the isomers. Reassuringly, our results showed that the in vitro antiplasmodial activities against both the hepatic and blood-stage infections by Plasmodium were generally lower than those of the individual isomers.
Overall, our data highlights the enhancement of the antiplasmodial activity of these structurally modified compounds through appending hybrid partners at C-13 of the macrolide unit. Work towards designing and synthesizing additional structurally modified IVM hybrids is currently in progress.

Experimental
General. All liquid reagents were dried/purified following recommended drying agents and/or distilled over 4 Å molecular sieves. CH 3 CN was dried by refluxing over P 2 O 5 . DCM was dried over fused calcium chloride. TBDMS-Cl, DMAP, and CDI were bought from Spectrochem. Imidazole was bought from Sigma Aldrich. K 2 CO 3 was dried overnight in the furnace. 1 H NMR and 13 C NMR spectra were recorded on Bruker Biospin Avance III HD at 500 MHz and JEOL-FT NMR-AL at 400 MHz with TMS as an internal standard using CDCl 3 as deuterated solvent. Data are reported as follows: chemical shift in δ (ppm), integration, multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet), coupling constant J (Hz). High-Resolution Mass spectra were recorded on a Bruker LC-MS MICROTOF II spectrometer. IR spectra were recorded on Agilent Technologies Cary 630 FTIR spectrometer. HPLC was performed in Reverse Phase mode using Agilent 1260 Infinity series HPLC (Agilent Technologies, USA) equipped with Quaternary Pump VL (G1311C) and degasser, 1260 ALS auto sampler (G1329B), and 1260 DAD VL detector (G1315D). The column used was the ZORBAX Eclipse C18 column     Table 1.

Synthesis of 10.
To the suspension of CDI (0.23 g, 1.4 mmol) in dry toluene (10 ml), a solution of 9 (0.5 g, 0.7 mmol) in dry toluene (5 ml) was added dropwise. The resulting mixture was stirred at room temperature for 12 h. The reaction mixture was poured into water and extracted with chloroform (2 × 20 ml). The organic extract was washed with water, dried over anhydrous sodium sulfate, filtered, and evaporated to obtain a brown oil. The crude product was purified by column chromatography over silica gel (60-120 mesh) using hexane/ ethyl acetate (75: 25    Synthesis of 12a and 12b. DBU (0.086 g, 0.57 mmol) was added to the suspension of 6 (0.02 g, 0.57 mmol) in 5 ml dry DCM and the resulting clear solution was stirred at room temperature for 15 min before dropwise addition of a solution (5 ml) of 10 (0.300 g, 0.38 mmol) in DCM. The reaction mixture was stirred at room temperature for 12 h. Upon completion, the reaction mixture was poured into water and extracted with water. The organic layer was dried over anhydrous sodium sulfate and the solvent was evaporated to brown oil (11, 0.310 g). For deprotection, 10 ml solution of p-TSA in methanol (0.02 g mL -1 ) was added to the solution of 11 (0.310 g) in methanol (10 ml) dropwise. The reaction mixture was stirred for 30 min at room temperature. Upon completion, DCM (30 ml) was added to the reaction mixture and washed with aqueous sodium bicarbonate, water, dried over anhydrous sodium sulfate, and the solvent was evaporated to give the brown crude product which was purified by column chromatography over silica gel (60-120 mesh) using chloroform/methanol (95:5, v/v) as the eluent afford 12 as a white solid in 39.9% yield. Characteristic data of isolates of 12 obtained after prep purification is given below. Synthesis of 13. Compound 10 (0.200 g, 0.25 mmol) was dissolved in 2 ml of dry ACN, and MeI (0.08 ml, 1.25 mmol) was added. The resulting colorless solution was stirred at 40 °C for 2 h. Upon completion, the excess of ACN was evaporated under reduced pressure to yield yellow solid 13, which was used as such without any further purification in the subsequent reactions as it displayed significant degradation in contact of air.
Synthesis of 15a and 15b. IVM-based intermediate 13 (0.100 g, 0.12 mmol) was dissolved in dry ACN (5 ml). To this, 5 ml solution of neutralized PQ (2, 0.048 g, 0.18 mmol) in dry ACN was added. The reaction mixture was stirred for 4-6 h in dark. Upon the completion, the excess of ACN was evaporated under vacuum, followed by the addition of DCM (30 ml). The resulting solution was washed with water, dried over anhydrous sodium sulfate, evaporated to obtain a dark brown oil (14, 0.130 g). For deprotection, 10 ml solution of p-TSA in methanol (0.02 g mL -1 ) was added to the solution of a 14 (0.130 g) in methanol (10 ml). The reaction mixture was stirred for 30 min at room temperature. Upon completion, DCM (30 ml) was added to the reaction mixture and washed with aqueous sodium bicarbonate, water, dried over anhydrous sodium sulfate and the solvent was evaporated to give the brown crude product which was purified by column chromatography over silica gel (60-120 mesh) using hexane/ethyl acetate (60: 40, v/v) as the eluent afford to 15 as a white solid in 48% yield. Characteristic data of isolates of 15 obtained after prep purification is given below.