Novel Diacyl-hydrazide Compounds as Potential Therapeutics for Visceral Leishmaniasis

Visceral leishmaniasis is a neglected tropical disease with the highest mortality among different forms of leishmaniasis manifestation in humans. The disease is caused by the parasitic protists Leishmania donovani and Leishmania infantum, and treatments remain unsuitable due to high costs, complicated administration, lack of efficacy, variable patient susceptibility, toxic side effects, and rising parasitic resistance. Herein, we report a structure–activity relationship (SAR) exploration of the diacyl-hydrazide scaffold identified to have antiparasitic activity from a high-throughput screen against L. donovani, Trypanosoma cruzi, and Trypanosoma brucei. This SAR study revealed new structural insights into this scaffold related to bioactivity resulting in a new series of lead compounds with nanomolar activity against L. donovani and no toxicity against human THP-1 macrophages. These optimized diacyl-hydrazide compounds set the stage for future drug development and hold promise for a new treatment avenue for visceral leishmaniasis.


■ INTRODUCTION
Leishmaniasis is a neglected tropical disease caused by different species of the protozoan parasite Leishmania.After malaria, leishmaniasis is the most prevalent vector-borne infectious disease in terms of mortality and total number of patients.−7 The two parasites Leishmania donovani and Leishmania infantum cause visceral leishmaniasis, and most cases (∼90%) occur in India, East Africa, and Brazil.The parasitic infection can target internal organs such as the liver, spleen, and bone marrow and is characterized by symptoms such as fever, weight loss, internal organ swelling, and progressive anemia.If the infection is left untreated, visceral leishmaniasis is usually fatal after two years, either directly or due to complications such as secondary infections or hemorrhage. 8,9−12 It is estimated that leishmaniasis is endemic in at least 88 countries, making eradication almost impossible due to large parasite reservoirs, including humans, dogs, rodents, and other wild animals.That said, vector control and chemotherapy are the main methods of disease management.However, the treatment of visceral leishmaniasis is a difficult task, and treatments suffer from high costs, complicated administration, variable efficacy among different species, variable patient susceptibility, toxic side effects, and decreasing efficacy due to rising parasitic resistance. 9,13This renders the search for novel treatments with an improved therapeutic profile urgently needed to benefit patient health and decrease the disease mortality rate and socioeconomic burden.
Our group recently reported a successful structure−activity relationship (SAR) exploration of a hit for leishmaniasis treatment, 4-fluoro-N-(5-(4-methoxyphenyl)-1-methyl-1H-imidazole-2-yl)benzamide, that had been identified via a highthroughput screen of a 1.8 million compound library. 14This screen was undertaken by the Tres Cantos Open Lab Foundation, supported by GlaxoSmithKline (GSK), the Dundee Drug Discovery Unit, and the Drugs for Neglected Diseases Initiative (DNDi) against three related kinetoplastid protists, L. donovani, Trypanosoma cruzi and Trypanosoma brucei. 15The GSK high-throughput screen also tested for sterol 14α-demethylase-demethylase (CYP51) inhibition, a prominent leishmaniasis drug target, 16−18 as well as for cytotoxic effects against HepG2, a human liver cancer cell line, which was considered in our hit selection (Table 1).
This discovery screen led to three chemical boxes containing compounds with promising activity and druggability for these parasites.Further analysis of antiparasitic potency and cytotoxicity narrowed the hits to a final set of 192 compounds in the GSK Leishmaniasis Box.From this library, we selected the diacyl-hydrazide compound class (hit compound 1, Figure 1) as the lead for this study due to its promising potency against L. donovani in infected macrophages (IC 50 = ∼1.3μM), low cytotoxicity against HepG2 cells (CC 50 = ∼100 μM), distinct mode-of-action from CYP51 inhibition, and good therapeutic window (CC 50 /IC 50 = ∼79).Although hydrazides and their derivatives, including the acyl-hydrazide class, are known to have various biological functions, 19−24 the identified antiparasitic activity from the GSK screen for the diacyl-hydrazide compound 1 was novel, and no SAR exploration of hit compound 1 had been carried out.Our pursuit of a systematic SAR exploration was further supported by data from a previous antileishmanial SAR study on acyl-hydrazide derivatives that revealed potent leishmanicidal activity for benzyloxy-protected acyl-hydrazides against Leishmania major promastigotes 19 and for aryl N-acylhydrazone compounds against L. infantum. 20he SAR approach to investigate compound 1 in this study examined the chemical space on the left-and right-hand side (LHS and RHS) of the central diacyl-hydrazide moiety.The Compound 1 did not inhibit CYP51, which was considered positive in our hit selection criteria due to validity concerns of CYP51 as a drug target for leishmaniasis (same criteria was also applied in our previous studies with success 14,26 ).−32  ■ RESULTS AND DISCUSSION Physicochemical Properties of Compound 1. Compound 1 displayed a pIC 50 of ∼5.9 against L. donovani and a pCC 50 of ∼4 against HepG2 cell lines, representing a good therapeutic window with a ∼79-fold selectivity for the parasite. 15e first analyzed compound 1 for physicochemical parameters such as the molecular weight, cLogP, polar surface area, freely rotatable bonds, hydrogen bond donors and acceptors and solubility at pH 2 and 6.5, as well as metabolic stability to evaluate its drug-likeness (Table 1).This analysis revealed that compound 1 adhered to Lipinski's rule of five, and a cLogP value of 2.5 indicated good bioavailability.These "drug-like" physicochemical properties and the good synthetic accessibility of the scaffold further supported exploring the compound's SAR to reveal valuable insights and guidance for drug developers.
Chemistry.Left-Hand-Side-Modifications.First, we synthesized analogues of compound 1 with alterations at the 1methylbenzimidazole moiety.We used the synthetic strategy depicted in Schemes 1 and 2 to access the hit compound 1 and to generate the four related analogues 5a−c and 12.These analogues varied in the methylation pattern (5a), ring heteroatom (5b and 5c) and ring size of heterocycle moiety (12) of the LHS imidazole scaffold.To access compound 1, the synthetic route began with the N-methylation of 2-(1Hbenzo[d]imidazole-2-yl)acetonitrile (2a) followed by the conversion to the corresponding ethyl ester (3b) using acetyl chloride in EtOH.This ester intermediate 3b was then subjected to a nucleophilic substitution reaction utilizing hydrazine hydrate in EtOH to form the corresponding hydrazide (4b).We then used 4b to perform an amide coupling reaction with quinaldic acid, utilizing HBTU and the non-nucleophilic base DIPEA in DMF, to yield compound 1.A similar approach was used to synthesize the benzimidazole (5a) and benzothiazole analogues (5b).The route to synthesize analogous 5a and 5b A different synthetic route was applied to access the benzoxazole-based analogue 5c.This route started with the cyclization reaction between 2-aminophenol (6) and ethyl 3ethoxy-3-imino propionate HCl to form compound 7, which was then converted to the corresponding sodium salt 8 using NaOC(CH 3 ) 3 in EtOH.Then, we synthesized the final benzoxazole-based analogue 5c via an amide coupling between the sodium salt precursor (8) and the quinoline-2-carbohydrazide.This approach was important in obviating the otherwise facile decarboxylation of neutralized 8.
We additionally synthesized analogue 12 by applying a different route (Scheme 2) to install a quinoline moiety at the LHS of the diacyl-hydrazide center.In this route, we first focused on installing an ethyl ester handle to 2-methylquinoline (9) using LDA and diethyl carbonate to give ethyl 2-(quinolin-2yl)acetate (10) in excellent 95% yield.The ester moiety of intermediate 10 was then converted to the corresponding hydrazide (11) using the previously described conditions to give 11.Finally, compound 11 was reacted with quinaldic acid in an amide coupling step to obtain the desired analogue 12.
Right-Hand-Side Modifications.Next, we focused on synthesizing compounds with modifications around the quinoline moiety at the RHS of the diacyl-hydrazide center of compound 1.The symmetry of the diacyl-hydrazide center allowed us to again utilize the final amide coupling step of the above-described synthesis for LHS modifications (Scheme 1, step d) as a diversification point to access the desired RHS modifications.These modifications included analogues where the quinoline moiety was substituted by naphthyl (13a), pyridyl (13b), phenyl (13c), and 2-and 3-phenylpyridyl (13d and 13e).
Due to the promising biological results of compound 13e from our initial SAR library screen, we decided to explore the 3phenylpyridine RHS moiety of 13e in more detail.We synthesized a series of derivatives with substitutions at the phenyl ring of 3-phenylpyridine.These modifications included the introduction of −Cl, −CN, −OCH 3 and −CH 3 at the 2-, 3and 4-position of the aromatic ring.We used the same route as described in Scheme 4 for the synthesis of compound 13e to access this series (17a−k).The synthetic route began with a Suzuki coupling to prepare the 5-bromopicolinic acid derivatives with the corresponding substitution pattern at the phenyl moiety (16a−k), which served as a precursor for the follow-up amide coupling with compound 4b (Scheme 4) to form analogues 17a−k.For the Suzuki coupling, we used commercially available phenylboronic acid derivatives with the desired functional group substitutions at the benzene ring (15a−k).The final amide coupling to generate the target analogues 17a−k was low yielding (8−42%); however, enough for biological testing was isolated and no further attempt at reaction optimization was carried out.
Biological Results.We screened all 21 synthesized compounds in vitro against obligate intracellular stages of L. donovani (LRC-L52) to assess the activity of our structural modifications around the initial hit compound 1.The applied THP-1 macrophage infection assay used differentiated, nondividing human acute monocytic leukemia cells (THP-1) and THP-1 macrophage infection with L. donovani amastigotes was  Yield refers to the final amide coupling step.b Anti L. donovani activity and toxicity measured in THP-1 macrophage host cells using a top concentration of 100 μM (2× serial dilution 10-point curve).Experiments were performed in duplicates in one independent experiment, n = 1.c SI − CC 50 /IC 50 .CC 50 − half-maximal cytotoxic concentration.IC 50 − half maximal inhibition concentration (reduction of total number of parasites by 50%).carried out as previously described. 14,33The antiparasitic activity and cytotoxicity were initially determined in a 384-well plate format utilizing a single-point concentration of 50 μM to identify initial hit compounds while removing others with toxic effects.Initial hits were further assessed by a 10-point curve (0.2−100 μM).The results obtained from this THP-1 macrophage infection assay were used to calculate antiparasitic IC 50 values and CC 50 values against macrophages.
Left-Hand-Side Modifications.Our LHS modifications predominantly aimed to probe the chemical space of the five-membered heterocycle of the 1-methylbenzimidazole moiety.More specifically, we used (i) compound 5a to study the effect of the N-methyl group, (ii) compounds 5b and c to gain insight into the displacement of the nitrogen by other heteroatoms such as sulfur and oxygen, and (iii) compound 12 to study the effect of the ring size.
This SAR study revealed that none of our newly synthesized compounds (5a−c and 12) led to improved antiparasitic activity (Table 2).We investigated the role of the N-methyl group with compound 5a and found that the presence of the N-methyl  Yield refers to the final amide coupling step.b Anti L. donovani activity and toxicity measured in THP-1 macrophage host cells using a top concentration of 100 μM (2× serial dilution 10-point curve).Experiments were performed in duplicates in one independent experiment, n = 1.c SI�CC 50 /IC 50 .CC 50 �half-maximal cytotoxic concentration.IC 50 �half maximal inhibition concentration (reduction of total number of parasites by 50%).Highlighted in green: top lead compounds identified in this study (17g−j; light green); best performing compound (17k; dark green).
group (1, IC 50 1.9 μM) was slightly favorable over a free amine (5a, IC 50 5.9 μM) as a weak hydrogen-bond donor.Moreover, other heteroatoms such as sulfur (5b) and oxygen (5c) as substituents for the methylated nitrogen in the 1-position of the imidazole scaffold led to a >3-fold and >25-fold reduction in antiparasitic activity compared to compound 1, respectively.Analogue 12, synthesized to characterize the importance of the five-membered ring, also displayed reduced activity.
Taken together, our findings underpinned that the fivemembered ring structure, the presence of the nitrogen as a heteroatom, and the N-methyl group are LHS key features for the potent antiparasitic activity of compound 1.
Right-Hand-Side Modifications.Our compound design to study the SAR of the chemical space on the RHS of the diacylhydrazide center focused on structural alterations of the quinoline moiety.More specifically, we assessed (i) 13a to reveal the relevance of the nitrogen in quinoline structure, (ii) 13b and c to explore the importance of the fused benzene ring to the pyridine moiety, and (iii) 13d and e to shed light on a more flexible conjugation of the benzene ring in ortho-and metaposition to the pyridine nitrogen compared to the rigid fusion of the benzene ring in quinoline (Table 3).
The biological evaluation of this series (13a−e, Table 3) with RHS modifications revealed that analogues 13a and c completely lost their L. donovani growth inhibitory activity, rendering the presence of nitrogen a key structural integrity for activity.The additional fused aromatic ring also positively impacted parasitic growth inhibition, and its removal (13b) resulted in ∼23-fold reduced activity compared to compound 1. 13e was the best-performing compound out of this series, revealing that conjugation of the aromatic ring at the metaposition to the nitrogen was slightly favored over the quinoline moiety of compound 1.These results support that the aromatic moiety is important; its increased rigidity when fused, however, does not seem to be crucial for its high potency.However, it is worth mentioning that the conjugation site of the aromatic ring plays a crucial role, as compound 13d with the aromatic ring conjugated in ortho-position to the nitrogen did not have improved activity over compound 1 and even had a >10-fold lower antiparasitic activity than the ortho-conjugated analogue 13e.
Overall, this RHS SAR study (13a−e) led to the identification of compound 13e as a strong L. donovani growth inhibitor with similar antiparasitic activity as the initial hit compound 1.This result sparked our interest in conducting a more detailed SAR exploration of the newly identified 3-phenylpyridine moiety to expand on this RHS modification.Hence, we synthesized and tested an additional series to study the effects of different functional groups (−Cl, −CN, −OCH 3 and −CH 3 ) at diverse positions (2-, 3-and 4-) around the phenyl moiety (17a−k; Table 4).The antiparasitic screen of these analogues revealed that all compounds of this series potently inhibited L. donovani growth.Interestingly, independently of the functional group and position of the modification, all analogues had potent IC 50 values ranging from 0.2−1.4μM against the L. donovani.This result rendered this new compound series not only more potent than the initial hit compound 1 but also more potent than 13e.Of note, within this compound series, 5 out of 11 had no toxic effects, and 7 out of 11 had an excellent SI of >100.17g−k had the best therapeutic profile, with nanomolar antiparasitic activity and no toxicity against macrophages (CC 50 = 52 − >100 μM).17k (IC 50 = 0.2 μM, CC 50 = >100 μM, SI = >500) was the bestperforming compound from this series, with ∼10-fold increased activity against L. donovani compared to 1.

■ CONCLUSION
We synthesized 20 analogues to explore the chemical space on the LHS and RHS of the diacyl-hydrazide center of initial hit compound 1.This approach yielded several key structural insights (Figure 2) that eventually led to novel structures with improved therapeutic potential against L. donovani parasites (17g−k).Each of these leads had nanomolar to low micromolar antileishmanial activity (IC 50 = 0.2 − 1 μM) with no cytotoxicity to human macrophages (CC 50 = 52 − >100 μM; SI = >145 − >500).Compound 17k was the best lead, with nanomolar activity against the parasite L. donovani (IC 50 = 200 nM; CC 50 = >100 μM; SI = >500).Potential off-target effects against human kinases, proteases, G protein-coupled receptors and cytochrome p450, however, should be profiled before pursuing these leads further.Taken together, this study opens a new avenue toward treating visceral leishmaniasis and provides a good starting point for further drug development based on the diacyl-hydrazide compound class.

■ EXPERIMENTAL SECTION
Parasite and Cell Cultures.L. donovani MHOM/SD/62/ 1S-CL2D parasites were cultured as promastigotes at 28 °C in M199 medium (Sigma-Aldrich, St. Louis, MO, USA) with 40 mM HEPES, 0.1 mM adenine, 0.0001% biotin, and 4.62 mM NaHCO 3 supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA), 100 μg/mL penicillin (Gibco), and 100 μg/mL streptomycin (Gibco).THP-1 cells (ATCC TIB-202) were cultured in RPMI-1640 medium containing 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate, and 10% FBS.The cells were maintained in tissue culture flasks (Nunc A/S, Roskilde, Denmark) in a 5% CO 2 incubator at 37 °C.The parasites were subcultured every 3 or 4 days and were maintained for 10 passages.Screening of Bioactive Compounds Against Intracellular Leishmania.PMA-treated THP-1 human monocytic cells were seeded at 0.8 × 10 4 cells per well in a 384-well culture plate (Greiner Bio-One, Kremsmunster, Austria) in RPMI-1640 complete medium supplemented with 10% FBS.After 48 h of incubation at 37 °C in the presence of 5% CO 2 , the promastigotes of L. donovani that were incubated with lectin for 30 min at 28 °C were added to the cells at a parasite-to-cell ratio of 20:1.Infected THP-1 cells were treated with amphotericin B (at 4 μM, positive control), miltefosine (at 10 μM, positive control), and screening compounds (at 10 μM).The negative control consisted of THP-1 infected with the parasite with only 0.5% DMSO.After 72 h, the cells that were infected and treated with the drug were washed with serum-free RPMI-1640 medium.The cells and parasites were stained using 5 μM DRAQ5 and 4% PFA.The images were acquired based on reading using an Operetta automated microscope (PerkinElmer, Inc., Waltham, MA 02451 USA).They were further analyzed using Columbus (PerkinElmer, Inc.Waltham, MA, USA) software to quantify parasite numbers, host cell numbers, and infection ratios.In brief, the large-sized nucleus of host cells was first detected using Draq-5 (Thermo Fisher, Rockford, IL, USA) signal and the host cell boundary masking was performed using the low-intensity signals from cytosols (an additional feature of Draq-5).Then, the small-sized nucleus signal by Draq-5 was used to identify parasites within the area of the masked host cell.IR was determined with the value of the number of infected cells divided by the total number of cells, and the average number of parasites per macrophage (P/φ) was defined by the value of the number of parasites divided by the number of infected cells in the acquired image.The average IR value of the negative control wells was calculated as 0.53.Compounds selected based on the screening results were further assessed in a dose-dilution manner (2-fold serial dilution for 10 points starting from 100 μM) using the same method.
Parasite Growth Inhibition.L. donovani promastigote growth inhibition was assayed by measuring the conversion of resazurin to resorufin.The assays were performed in 384-well plates seeded with L. donovani promastigotes (5 × 10 4 cells per well).After seeding, the parasites were exposed to the compounds for 3 days.Resazurin sodium salt (200 μM; R7017; Sigma-Aldrich, St. Louis, MO, USA) was added, and the samples were incubated for 5 h.After incubation, the parasites were fixed using 4% paraformaldehyde, and the plates were analyzed using a Victor3 plate reader (PerkinElmer, Inc., Waltham, MA, USA) at 590 nm (emission) and 530 nm (excitation).Amphotericin B and miltefosine were the reference drugs for the L. donovani promastigote growth inhibition.
Chemistry.General.All solvents used were of analytical grade: ethyl acetate (EtOAc); dichloromethane (DCM); dimethylformamide (DMF); methanol (MeOH); tetrahydrofuran (THF), and ethanol (EtOH). 1 H and 13 C Nuclear Magnetic Resonance (NMR) spectra were recorded at 400.13 and 101 Hz, respectively, on a Bruker Avance III Nanobay 400 MHz spectrometer coupled to the BACS 60 automatic sample changer at 25 °C.Results are recorded as follows: chemical shifts (δ) in ppm acquired in either CDCl 3 (7.26ppm for 1 H and 77.16 ppm for 13 C), DMSO-d 6 (2.50 ppm for 1 H and 39.52 ppm for 13 C) or MeOD (3.31 ppm for 1 H and 49.00 ppm for 13 C) as a reference.Solvents used for NMR studies are from Cambridge Isotope Laboratories.Each proton resonance was assigned according to the following convention: chemical shift (δ), multiplicity, coupling constant (J), expressed in hertz (Hz), and number of protons.Each carbon resonance was assigned according to the following convention: chemical shift (δ), multiplicity (where no multiplicity is assigned a singlet peak was observed).Analytical HPLC was acquired on an Agilent 1260 Infinity analytical HPLC coupled with a G1322A degasser, G1312B binary pump, G1367E high-performance autosampler, G4212B diode array detector.Conditions: Zorbax Eclipse Plus C18 Rapid resolution column (4.6 × 100 mm) with UV detection at 254 and 214 nm, 30 °C; sample was eluted using a gradient of 5−100% solvent B in solvent A where solvent A: 0.1% formic acid in water, and solvent B: 0.1% formic acid in MeCN (5 to 100% B [9 min], 100% B [1 min]; 0.5 mL/min).Low-resolution mass spectrometry (LCMS) was performed on an Agilent 6100 Series Single Quad LCMS coupled with an Agilent 1200 series HPLC, G1311A quaternary pump, G1329A thermostated autosampler, and G1314B variable wavelength detected (214 and 254 nm).LC conditions: Phenomenex Luna C8( 2) column (100 Å, 5 μm, 50 × 4.6 mm), 30 °C; sample (5 μL) was eluted using a binary gradient (solvent A: 0.1% aq.General Procedure A�Hydrazide Handle Formation.To a solution of appropriate ethyl ester (1 mmol) in EtOH (3 mL) was added hydrazine hydrate solution (5 mmol).The reaction was refluxed overnight.Upon completion, the reaction was poured on ice, filtered, and dried via suction filtration to give the desired compound as a solid.
General Procedure B�Amide Coupling.To a solution of the appropriate amine (1.5 equiv) and DIPEA (3 equiv) in DMF (3 mL/mmol) was added the various carboxylic acids (1 equiv).HBTU (1.3 equiv) was added to the mixture.The solution was stirred at r.t. for 16 h.The reaction was concentrated in vacuo and washed with EtOAc and brine.If a solid was formed, the reaction was filtered via vacuum filtration and washed with EtOAc, and the product was collected as a solid.If required, further purification was conducted using column chromatography and various solvents, depending on the compound.
General Procedure C�Suzuki Coupling.To a solution of 5bromopicolinic acid (1 mmol, 202 mg), appropriate boronic acid (1.3 eq, 1.3 mmol), and K 2 CO 3 (1.8eq, 1.8 mmol) in dioxane/water (30 mL, v/v, 3/1) was added Pd(dppf)Cl 2 (0.03 eq, 0.03 mmol).The reaction mixture was stirred at 110 °C under N 2 overnight.The reaction mixture was cooled to room temperature, and the pH was adjusted to 9−10.The mixture was filtered through Celite.The aqueous layer was washed with Et 2 O.The aqueous layer was collected, and the pH was adjusted to ∼4−5 with 1 N HCl and extracted with EtOAc.The organic layers were collected, dried over MgSO 4 and concentrated in vacuo to produce the desired product.

Scheme 3 .
Scheme 3. Synthetic Route to Synthesize Right-Hand-Side Modifications and Access Analogues 13a−e a

Scheme 4 .
Scheme 4. Synthetic Route to Access Analogues 13e and 17a−k a

Figure 2 .
Figure 2. Key structural elements identified in this study.
a b

Table 2 .
Structures, L. donovani Activity, Cytotoxicity and Selectivity Index (SI) with Left-Hand-Side Modification

Table 3 .
Structures, Anti L. donovani Activity, Cytotoxicity and SI with Right-Hand-Side Modification Yield refers to the final amide coupling step.b Anti L. donovani activity and toxicity measured in THP-1 macrophage host cells using a top concentration of 100 μM (2× serial dilution 10-point curve).Experiments were performed in duplicates in one independent experiment, n = 1.SI�CC 50 /IC 50 .CC 50 �half-maximal cytotoxic concentration.IC 50 �half maximal inhibition concentration (reduction of total number of parasites by 50%).
a c