The Discovery of Novel Antimalarial Aminoxadiazoles as a Promising

Aug 14, 2017 - ... Cristina de Cozar†, Benigno Crespo†, Beatriz Díaz†, Gerard Drewes∥, Esther Fernández†, Santiago Ferrer-Bazaga†, Marí...
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The Discovery of Novel Antimalarial Aminoxadiazoles as a Promising Nonendoperoxide Scaffold Elena Sandoval,† María José Lafuente-Monasterio,† María J. Almela,† Pablo Castañeda,† María Belén Jiménez Díaz,† María S. Martínez-Martínez,† Jaume Vidal,† Iń ̃igo Angulo-Barturen,† Paul Bamborough,‡ Jeremy Burrows,⊥ Nicholas Cammack,† María J. Chaparro,† José M. Coterón,† Cristina de Cozar,† Benigno Crespo,† Beatriz Díaz,† Gerard Drewes,∥ Esther Fernández,† Santiago Ferrer-Bazaga,† María Teresa Fraile,† Francisco J. Gamo,† Sonja Ghidelli-Disse,∥ Rubén Gómez,† John Haselden,† Sophie Huss,† María Luisa León,† Jaime de Mercado,† Simon J. F. Macdonald,‡ José Ignacio Martín Hernando,† Sara Prats,† Margarita Puente,† Anne Rodríguez,† Juan C. de la Rosa,† Lourdes Rueda,† Carolyn Selenski,§ Paul Willis,⊥ David M. Wilson,†,# Michael Witty,⊥ and Félix Calderón*,† †

Tres Cantos, Medicines Development Campus, DDW, GlaxoSmithKline, Severo Ochoa 2, 28760 Tres Cantos, Madrid, Spain Medicines Research Center, GlaxoSmithKline, Gunnels Wood Road, Stevenage SG1 2NY, U.K. § GlaxoSmithKline, 709 Swedeland Road, King of Prussia, Pennsylvania 19406, United States ∥ Cellzome GmbH, GlaxoSmithKline, Meyerhofstrasse 1, 69117 Heidelberg, Germany ⊥ Medicines for Malaria Venture (MMV), 21 route de Pré-Bois, PO Box 1826, 1215 Geneva 15, Switzerland ‡

S Supporting Information *

ABSTRACT: Since the appearance of resistance to the current front-line antimalarial treatments, ACTs (artemisinin combination therapies), the discovery of novel chemical entities to treat the disease is recognized as a major global health priority. From the GSK antimalarial set, we identified an aminoxadiazole with an antiparasitic profile comparable with artemisinin (1), with no cross-resistance in a resistant strains panel and a potential new mode of action. A medicinal chemistry program allowed delivery of compounds such as 19 with high solubility in aqueous media, an acceptable toxicological profile, and oral efficacy. Further evaluation of the lead compounds showed that in vivo genotoxic degradants might be generated. The compounds generated during this medicinal chemistry program and others from the GSK collection were used to build a pharmacophore model which could be used in the virtual screening of compound collections and potentially identify new chemotypes that could deliver the same antiparasitic profile.



INTRODUCTION

infected with malaria. In 2015, 214 new million cases were reported, and the disease led to ca. 438000 deaths. Although these data includes the six WHO regions, the reality is that 90% of deaths occurred in the WHO African region with 78% being children aged under five.2 Fatal cases still remain directly linked to poverty as the highest malaria mortality rates are being reported in countries where the population is living on less than

Malaria is a parasitic disease caused by parasites of the genus Plasmodium, which are transmitted to people during the blood meal of a female Anopheles mosquito. From the different species of Plasmodium (Plasmodium falciparum, Plasmodium ovale, Plasmodium vivax, Plasmodium Knowlesi, and Plasmodium malariae) Plasmodium falciparum and Plasmodium vivax are responsible for most of the disease cases.1 According to the 2015 World Health Organization (WHO) global malaria report, 3.3 billion people are at risk of being © XXXX American Chemical Society

Received: February 27, 2017

A

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Most studied have been the 1,3,4- and 1,2,4-isomers. Both are present in compounds having a broad spectrum of biological activities, are easy to prepare (efficient syntheses have been described) and they can act as bioisosteres of carboxylic acids, esters, and carboxamides.24,25 Although structurally very similar, their different charge distribution makes the 1,3,4 more suitable for drug discovery as, for instance, it shows a better profile in terms of lipophilicity, metabolic stability, lack of hERG inhibition, and aqueous solubility. As an example, raltegravir contains a 1,3,4-oxadizole and is an antiretroviral approved by the FDA for HIV treatment.26 Although oxadiazole containing compounds are frequently found in the TCAMS, there were no close analogues to 2. This explains why this compound was not one of the 47 starting points identified in our first mining of the TCAMS, where clusters containing numerous compounds were prioritized over those containing very few or consisted of only one compound (singletons). In that exercise, TCAMS compound were allocated points from 0 to 30 using different parameters and compounds with >14 points were selected for further profiling. 2 scored 14.17 In reviewing the literature, the only other compounds containing the 1,3,4-oxadiazole motif described as antimalarials are the benzenesulfonamides reported by Zareef et al. (3, Figure 1).27 2 is an antimalarial chemotype with activity against the sensitive 3D7 strain and a panel of multidrug strains including clinical isolates resistant to chloroquine, pyrimethamine, and atovaquone. The compound is not cytotoxic at 25 μM when tested against a mammalian cell line (Table 1) and its in vitro speed-of-action is comparable with artemisinin (see Supporting Information).28

US$1.25 per day. Moreover, malaria is also hampering economic development in affected countries due to expenditures on prevention and treatments.3 Great progress has been made in the last 15 years due to increased funding, new diagnostics and chemotherapies as well as vector control strategies. In fact, a 29% decrease in deaths has been reported in 2015 comparing to 2010.2 The world’s first malaria vaccine (RTS,S/As01) will be rolled out in pilot projects in sub-Saharan Africa, WHO confirmed on 17th November 2016. Funding is now secured for the initial phase of the program, and vaccinations are due to begin in 2018.4 However, today, resistance to the insecticides5,6 and antimalarials currently in use have been reported (including resistance to the current front-line treatment, artemisin (1) based therapies (ACTs)).7−10 As a consequence, there is still an urgent requirement for new antimalarial drugs that can be added to or substitute the current therapies. The criteria for new antimalarial drugs are demanding; the new molecule must be safe, efficacious against strains displaying clinical resistance to current antimalarials, have pharmacokinetics compatible with oral dosing, be affordable, and be able to relieve symptoms as fast as the artemisinins. Responding to the urgency of the situation, the antimalarial community has adopted a pragmatic strategy by focusing on compounds active in a whole cell assay although the mode of action may be unknown.11 This does not mean that identifying the target is not a matter of interest. In fact, the sequencing of the Plasmodium falciparum genome has revealed more than 5000 genes.12 However, the number of antimalarial targets these days is still very restricted. The structures of around 15000 antimalarial hits from different screening collections (St. Jude’s Hospital,13 Novartis,14 and GlaxoSmithKline15) can be downloaded free from the Chembl-NTD database and provide a rich resource for selecting leads.16 Optimization efforts on several series from the GSK screening, the Tres Cantos Antimalarial Set (TCAMS) have already been published.17−22 Recently, we published data from Plasmodium berghei in vivo screening aimed at reducing attrition by selecting potential in vivo active scaffolds.23 By using this approach, we identified the peroxide-free TCAMS compound TCMDC-134278 (2, Figure 1), a basic tetra-aromatic compound containing a central amino-1,3,4-oxadiazole moiety surrounded by a phenyl ring and bicyclic aromatic system (phenyl-pyridine). Oxadiazole containing leads have become widely used by both pharma and academia as judged by their presence in numerous publications, patents, and late stage clinical trials.

Table 1. Summary of Key Data for 2 parameter

result

Pf (3D7A) IC50 μM ratio HB3, Dd2, TC08, T9/94, V1/S, FCR3, Tm90C2A, Tm90C2B/3D7b ED90 (mg/kg)c Cl (mL/min/kg)d Vss (L/kg)d %Fe Tox50f/Pf (3D7) IC50 a

0.07 ∼1 >120 >LBF 2.6 26.6 >100

a

In vitro antimalarial activities of test drugs against sensitive strain 3D7A by [3H]hypoxanthine incorporation assay. Standard deviations (included in the Supporting Information) were extracted from two/ three independent biological replicates. b3D7, wild type; HB3, moderately resistant to pyrimethamine; Dd2, chloroquine and pyrimethamine resistant; TC08, pyrimethamine resistant; T9/94, chloroquine resistant; V1/S, chloroquine and pyrimethamine resistant; FCR3, chloroquine, atovaquone, and cycloguanil resistant; Tm90C2A, chloroquine and pyrimethamine resistant; Tm90C2B, chloroquine, pyrimethamine, and atovaquone resistant. cReference 29. dClearance (Cl) and volume of distribution (Vss), mouse, iv, 1 mg/kg. eOral bioavailability, mouse, po, 10 mg/kg. fHepG2 cells.

The weakness of 2 is its high in vivo clearance (Cl > liver blood flow (LBF), Table 1) (which may be due to the vulnerability of the biaryl left-hand side of the molecule to oxidation),29 which translates into only moderate efficacy in vivo in a severe combined immunodeficiency (SCID) mouse model infected with the human parasite (81% reduction of parasitemia with respect to controls at the high dose tested, 120 mg/kg). In this experiment mice were infected at day 0 with the

Figure 1. Artemisinin (1), 2 (antimalarial containing oxadiazole scaffolds discovered by GSK), and the one described by Zareef et al.27 (3). B

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Scheme 1. General Synthetic Routes Used for Preparation of Compounds 2−17

human parasite (P. falciparum Pf 3D70087/N9), and 3 days after infection, the compound was dosed once a day for four consecutive days.30 The promising antiparasitological profile of 2 led to studies evaluating the potential of this novel antimalarial template aimed at delivering a clinical candidate. These studies are described in the rest of this article.

Similar conditions were used to obtain the compounds 6 and 15b using Int-C and the corresponding amine. Compound 8, 9, and 16a were obtained by nucleophilic aromatic substitution between Int-C and the corresponding amines. Finally, in route C, the reaction between the corresponding bromide intermediate and 3-pyridinylboronic acid using Suzuki reaction conditions gave the desired products. The ester moiety then reacted with hydrazine hydrate in ethanol to give the hydrazide intermediates D and E. Those intermediates were respectively reacted with the corresponding isothiocyanates to give a crude material that reacted with DCC or EDCI to give the compounds 5, 15c, 16b, 17a, and 17b Initial lead optimization iterations demonstrated that changes to both the right-hand side (RHS) and left-hand side (LHS) were able to increase metabolic stability while maintaining good whole cell potency (Tables 2 and 3). We started to explore the RHS by studying the role of the oxygen in the oxazepine ring by synthesizing the corresponding azepine (4). Although the in vitro stability did not improve comparing to 2 (in vitro clearance of 2 is 13.7 mL/min·g), the result was encouraging as 4 presented submicromolar antiplasmodial activity and opened-up the options to develop SAR through the more tractable heterocycles such as tetrahydroisoquinoline (THiQ) (5) and the isoindoline (6). Nonbasic derivatives (7−9) also presented low clearance but all were less active than 5 or 6. The N-methylated THiQ (5) provided the best balanced profile in terms of potency and clearance and was selected for SAR studies on the left-hand side. With the objective of improving clearance, the options to block potential labile positions of the phenyl ring were studied (Table 3) by synthesizing the three fluorine isomers (10−12). The 4-F derivative (12) caused a drop in potency of 1 order of magnitude, while the 2-F (10) and 3-F derivatives (11) showed



RESULTS We first focused our efforts on finding an analogue of 2 that would enable us to understand the potential of the series to deliver antimalarial efficacy in an in vivo model. Upon the basis of the early profiling of 2, the chemistry strategy was focused on finding an analogue that would have a better potency/ metabolic stability profile. Several preparative methods of oxadiazoles have been published.31 Scheme 1 shows the general methods used for the preparation of oxadiazoles 4−17. In route A, the corresponding aromatic amine was reacted with thiophosgene in DCM in the presence of a base to give the isothiocyanate. This isothiocyanate was subsequently reacted with the aromatic carboxamide to give a crude material that was then reacted in the presence of DCC to give the corresponding aminoxadiazole (Int-A). This intermediate reacted with 3-pyridinyl boronic acid using Suzuki reaction conditions to give the compounds 2, 4, 7, 10, 11, 12, 13, and 14. For route B the corresponding hydrazide was reacted with CNBr and a base to give 5-(3-bromophenyl)-1,3,4-oxadiazol-2amine. This bromide was then reacted using Suzuki reaction conditions with 3-pyridinylboronic acid to give Int-B. The reaction of Int-B with copper(II) bromide and tert-butylnitrite gave the bromide Int-C. Int-B was reacted with the corresponding chlorine derivative using Buchwald reaction conditions to give the compound 15a. C

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as a promising compound due to its balanced ADME/potency (LE = 0.3, Pf IC50 20 nM) profile and low in vitro and in vivo clearance (Table 4), thus selected as a representative compound for the series for further characterization.

Table 2. Optimization of 2: Right-Hand Side

Table 4. Summary of Key Data for 14 parameter

result

Pf IC50 μM (3D7A)a ratio TC08, HB3, T9/94, Dd2, V1/S, FCR3, Tm90C2A, Tm90C2B/3D7b ED90 (mg/kg)c AUC (μg·h/mL/day) Cl (mL/min/kg)d Vss (L/kg)d %Fe FASSIF (μg/mL)f

0.02 ∼1 9.3 2.2 43 3.5 90 1.2

a

In vitro antimalarial activities of test drugs against sensitive strain 3D7A by [3H]hypoxanthine incorporation assay. Standard deviations (included in the Supporting Information) were extracted from two/ three independent biological replicates. bSee Table 1 footnote. c Reference 29. dClearance (Cl) and volume of distribution (Vss), mouse, iv, 1 mg/kg. eOral bioavailability, mouse, po, 10 mg/kg. fFasted state simulated intestinal fluid (pH 6.5).

Characterization of 14. Four key experiments were carried out to understand the potential of 14 to deliver a novel antimalarial drug according to the desired profile, namely parasite reduction rate (see figure in Supporting Information),28 activities versus a panel of resistant strains, in vivo efficacy, and propensity to generate resistant mutants. 14 showed a parasite reduction rate that is classed as “fast killer antimalarial” (similar to chloroquine) and was equipotent against a panel of resistant strains which includes clones resistant to chloroquine, pyrimethamine, and atovaquone (Table 4). These results are in agreement with previously reported data for the hit compound 2 (Table 1). In vivo, 14 was able to reduce the level of parasitemia under the limit of detection after just two doses (ED90 = 9.3 mg/kg, Figure 3) in comparison to 2 where a reduction of 81% of parasitemia after four doses of 120 mg/kg was achieved. This major improvement of 14 over 2 (Figure 2) may be a consequence of the improvement in the in vitro antiplasmodial potency and the more balanced pharmacokinetic profile of 14 for an oral dosing (%F 90, Cl = 42 mL/min/kg, Table 4). Finally, we tried unsuccessfully to generate in vitro resistant mutant to 14. Although this result prevented the identification of the target, it also suggests that the propensity of resistance to this chemical class might be low. All these results indicated the potential of both 14 and the scaffold for discovering a novel antimalarial. However, the progression of 14 was limited by its negligible solubility in biorelevant media (1.2 μg/mL in FASSIF, Table 4). Optimization of 14. The chemical strategy next focused on identifying a more developable compound by finding an analogue with a more desirable physicochemical profile than 14. Increasing the sp3 character and flexibility, reducing aromaticity, and reducing lipophilicity were thus considered in designing the next iteration of compounds (Figure 3). The goal was to explore the RHS and LHS separately and to identify the most suitable combination. Right-Hand Side. We first initiated our SAR expansion campaign by using a dual approach. The first approach targeted close analogues of 14 (Table 5, 15a−c). The second approach

a

In vitro antimalarial activities of test drugs against sensitive strain 3D7A by [3H]hypoxanthine incorporation assay. Standard deviations (included in the Supporting Information) were extracted from two/ three independent biological replicates. bIn vitro clearance (microsomes fraction).

Table 3. Optimization of 5: Upper-Left-Hand Side

compd

R1

Pf (3D7) IC50 (μM)a

5 10 11 12 13 14

1-H 2-F 3-F 4-F 3-OCF3 3-CF3

0.1 0.05 0.05 0.48 0.22 0.02

iCl

b

human (mL·min/g) 1.8 2.9 1000

a

In vitro antimalarial activities of test drugs against sensitive strain 3D7A by [3H]hypoxanthine incorporation assay. Standard deviations (included in the Supporting Information) were extracted from two/ three independent biological replicates. bSee Table 1 footnote. c Reference 29. dHepG2 cells.

inconclusive because both compounds behaved differently in a preliminary assessment. 19 remained chemically unaltered in acid media (SGF at pH 1.6) for at least for 24 h at 37 °C, while 17a showed degradation product after 14 h under the assay conditions. When both compounds were assessed in an in vivo model using high concentrations (300 and 500 mg/kg), both 20a,b and 21a,b were detected. By HPLC, levels of 18.4/19.3% (250 and 500 mg/kg doses, respectively) of 21a were detected and 2.6/2.3% levels (300 and 500 mg/kg doses respectively) of 21b. According to the mechanism proposed in Figure 5, the detection of 21 may lead to the generation of hydrazine (hydrazine levels were not measured). The discrepancy between the quantities of 21 detected for the two compounds indicates our lack of understanding of this mechanism. Target Elucidation Efforts. Genomic and chemoproteomics approaches were explored to elucidate the biological target of these compounds. Several attempts were made with different analogues to generate in vitro resistant mutants, but none were isolated with any of the analogues tested, so no genomic work could be undertaken. Attempts were made to identify malaria proteins using pull down experiments in which different analogues of 14 were linked to a matrix and were competed with free active analogues. Although several falciparum proteins were identified, no specific binding was detected as none of them competed with the free compounds. In parallel experiments using K562 whole-cell lysate, huFECH was identified as a potential target of different analogues of 14, additional studies showed that FECH inhibition was also observed with compounds which were inactive against P. falciparum growth, discarding this target as responsible of its MoA. Capturing experiments using kinobeads were also attempted using 14 and 18a. Several proteins were identified from this but were discarded as potential targets because none were detected by both analogues.33,34

a

In vitro antimalarial activities of test drugs against sensitive strain 3D7A by [3H]hypoxanthine incorporation assay. Standard deviations (included in the Supporting Information) were extracted from two/ three independent biological replicates;

provide encouragement that the scaffold could be optimized toward the desired profile. Developability Assessment. To assess the potential of the scaffold, we first carried out an in vivo toxicological experiment. Two independent experiments were performed. 19 was administered (300 and 500 mg/kg) to female Swiss CD-1 mice. The concentration achieved at the highest dose was 68.9 μg·h/mL. Several parameters were considered: in-life observations, bodyweight evolution, relative organ weight, hematology, and clinical chemistry. No serious adverse effects were observed, although slight adverse clinical signs (passivity) were seen at 500 mg/kg in the functional observatory battery. A risk associated with the core is its potential to generate genotoxic metabolites such as hydrazine through an acidcatalyzed hydrolytic ring-opening mechanism or Phase I metabolism (Figure 5).32 We chose 17a and 19 as tool compounds for further evaluation of this issue. The results were

Scheme 2. General Synthetic Routes Used for Preparation of Compounds 18a−d

G

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Figure 5. Proposed mechanism of 1,3,4-aminoxadiazole degradation.

Pharmacophore Elucidation. We wish to encourage others to identify new chemotypes with the same antiparasitic profile as the aminoxadiazoles. 3D pharmacophores are a spatial arrangement of chemical features that can be used to search for chemically distinct molecules that are able to make similar interactions.35 We make available to the research community the Supporting Information 3D pharmacophore models constructed using over 400 analogues of the amino-oxazole series from the GSK compound collection with measured antiplasmodial activity. Because it is challenging to unambiguously assign their bioactive conformation, we cannot define one absolute active pharmacophore. Nevertheless, the molecules are quite rigid, with a relatively small number of lowenergy conformers, and by overlaying conformers of two representative compounds it was possible to define four overlapping 3D pharmacophore models which show significant enrichment of active members of the series over inactive members (Figure 6). There is considerable intersection between the hits found by each of the four pharmacophores, so we recommend searching using all four before combining the hits.

Figure 6. (A) Pharmacophore model 8, which gave the highest enrichment performance against the project compounds, was built from common features of superimposed compounds 4 (green sticks) and 18 (gray sticks). Pharmacophore features are indicated with circles: aromatic centroids and normal features orange cationic and hydrogen-bond donor features; projected points purple; hydrogenbond acceptors cyan. (B) Enrichment performance of pharmacophore 8 against 418 project compounds, binned by activity (pIC50). The proportion of compounds detected by the model within each activity bin is colored green. The model matched 81% of compounds with pIC50 > 6.5 and only 6% of compounds with pIC50 ≤ 5.5.



DISCUSSION AND CONCLUSIONS We have shown the potential of the basic tetra-aromatic template exemplified by 19 to deliver a novel antimalarial chemotherapy. Although simplification of the template looks quite challenging, we have shown the versatility of the template, suggesting that further SAR exploration of the scaffold is possible. The fast killing profile, low tendency to develop resistance mutants, the robust in vivo efficacy among different members of the series, and the efficacy against a panel of resistant strains makes this template a promising antimalarial scaffold. However, the aminoxadiazole core has the potential to deliver genotoxic metabolites, thus making progression of this particular core a challenging and risky task. We have therefore, stopped any further investment in this compound-class. A pharmacophore model has been built. We publish here the coordinates and key interactions and encourage efforts to identify new chemotypes through virtual library screening

which might conserve the same antiparasitological profile of the aminoxadiazole containing analogues.



EXPERIMENTAL SECTION

All animal studies were ethically reviewed and carried out in accordance with European Directive 2010/63/EU and the GSK Policy on the Care, Welfare, and Treatment of Animals. The human biological samples were sourced ethically, and their research use was in accord with the terms of the informed consents Pharmacophore Modeling. Modeling was carried out using MOE version 2014.09 (Chemical Computing Group). Stochastic conformational searches were carried out on compounds 4 and 18 H

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(Amber12 force field, iteration limit 1000, rejection limit 100, RMSD limit 1.0). Flexible fitting was used to align the two compounds (default parameters), producing eight distinct aligned molecule pairs. For each aligned pair, a pharmacophore model was built by hand, generating eight pharmacophore models, using the same features for each. The 0.8 Å radius aromatic features were placed at the midpoint of overlaid aromatic centroids. The 1.5 Å radius π-normal points were placed at aromatic normal projected points. Hydrogen-bond acceptor points were placed with radius 0.8 Å at the midpoint of the oxadiazole nitrogen atoms. A hydrogen-bond donor feature was placed with radius 0.8 Å at the aniline nitrogen. At its projected point, a hydrogenbond donor projected point was placed with radius 1.5 Å. Finally, at the centroid of the basic centers, a cation feature was placed with radius 2.0 Å. This was repeated for all eight superimposed pairs. To validate the models, 418 compounds from the GSK antiplasmodium project with measured activity were prepared as described above (Amber12 force field with standard R-field solvation) and placed in a multiconformer database with a maximum of 50 conformers per molecule. The database was then searched using the eight pharmacophore models in turn, requiring all 12 features to match. Hit rates varied between pharmacophores, with one (model 4) only retrieving 64 molecules, while model 8 found 217. Four models (1, 2, 5, and 8) found similar compounds to one another (Supporting Information, Table SX1) and were far superior to the others at enrichment of the more potent compounds in the set (Supporting Information, Figures SX1, SX2). Models 1, 2, 5, and 8 are shown in Supporting Information, Figure SX3. We recommend that the best approach for hit identification within a compound database would be to combine hits from screening models 1, 2, 5, and 8. As the models are rather specific, depending on the size and composition of the database, we also recommend relaxing the criteria so that one or possibly two mismatches are allowed, requiring only 10 or 11 out of the 12 features in each pharmacophore model to match. Models 1, 2, 5, and 8 in MOE pharmacophore file format are given in Supporting Information, configured to match 11 out of 12 features. Because this is an ASCII text file format containing feature coordinates and radii, it could be used to build equivalent pharmacophore models in other formats. Chemistry. Materials and Methods. All starting materials were purchased from commercial sources and used as received or synthesized via literature procedures. Solvents were dried using a commercial solvent purification system and stored under nitrogen. All final compounds were characterized by 1H NMR spectroscopy and LCMS. 1H NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer at 293 K. Purity was determined by HPLC (Acquity UPLC BEH C18 1.7 μ 2.1 mm × 50 mm) at 35 °C. All compounds tested present a purity >95%. Method: acetate NH4 25 mM + 10% ACN at pH 6.6/ACN, 0−0.2 min 100:0; 0.2−1.0 min 10:90; 1.0−1.8 min 10:90; 1.8−2.0 min 100:0. Flow: 0.8 mL/min. The UV detection wavelength was 254 and 210 nm. Positive ion mass spectra (high resolution mass spectroscopy) was acquired using a QSTAR Elite (AB Sciex Instruments) mass spectrometer, equipped with a turbospray source, over a mass range of 250−700, with a scan time of 1 s. The elemental composition was calculated using Analyst QS 2.0 software. 4-Methyl-N-(5-(3-(pyridin-3-yl)phenyl)-1,3,4-oxadiazol-2-yl)2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepin-7-amine (2). Scheme 1, route A: a mixture of 4-methyl-2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepin-7-amine (305 mg, 1.7 mmol) and 1,1′-thiocarbonyldiimidazole (305 mg, 1.7 mmol) were dissolved in 5 mL of anhydrous THF at rt. The reaction mixture was stirred overnight. Solvent was removed under reduced pressure to give a crude material that was dissolved in 25 mL of EtOAc and washed with 25 mL of water. Phases were separated in a separative funnel, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give a crude material that was dissolved in 10 mL of dry THF and combined with 3-bromobenzohydrazide (361 mg, 1.7 mmol). The reaction mixture was heated at 60 °C for 3 h and after reaction completion. After that time, thereaction mixture was allowed to cool down to rt, and the solvent was evaporated under reduced pressure to give a crude material that was triturated with diethyl eter. The solid

was filtered and dried under vacuum to give a solid (700 mg, 1.6 mmol) that was dissolved in 8 mL of anhydrous DMF and N-(3(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride was added (401 mg, 2.1 mmol). The mixture was heated at 80 °C overnight. After cooling down to rt, the reaction mixture was diluted with water (40 mL) and extracted with EtOAc (2 × 50 mL). The organic layer was washed with water (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated to dryness, affording the corresponding oxadiazole intermediate (intermediate type A) as a white solid (MS: m/e 401 (MH+)). Compound 2 was obtained by irradiating a mixture of 3-pyridinylboronic acid (92 mg, 0.7 mmol), N-(5-(3-bromophenyl)-1,3,4-oxadiazol-2-yl)-4-methyl2,3,4,5-tetrahydrobenzo[f ][1,4]oxazepin-7-amine (300 mg, 0.7 mmol) (oxadiazole intermediate type A), potassium carbonate (413 mg, 3 mmol), and Pd(PPh3)4 (17.3 mg, 0.02 mmol) in a microwave device for 90 min at 85 °C. Reaction mixture was diluted with EtOAc (30 mL) and washed with water (2 × 20 mL). Phases were separated in a separative funnel, and organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give a crude material that was purified on a silica gel cartridge to give 250 mg of 2 (0.6 mmol). 1H NMR (400 MHz, CDCl3) δ 1H NMR (CDCl3, 400 MHz): δ = 8.91 (d, J = 1.8 Hz, 1H), 8.67 (dd, J = 4.8, 1.5 Hz, 1H), 8.17 (s, 1H), 8.00 (d, J = 7.8 Hz, 1H), 7.95 (dt, J = 8.0, 1.9 Hz, 1H), 7.72 (d, J = 7.8 Hz, 1H), 7.58−7.65 (m, 1H), 7.39−7.46 (m, 3H), 7.34 (dd, J = 8.5, 2.9 Hz, 1H), 7.04 (d, J = 8.3 Hz, 1H), 4.01−4.12 (m, 2H), 3.80 (s, 2H), 2.98−3.10 (m, 2H), 2.45 ppm (s, 3H). MS: m/e 400 (MH+). Purity was determined as >95% by HPLC (251 nm). Rt: 1.06 min (Acquity UPLC BEH C18 1.7 μ, 2.1 mm × 50 mm, CH3COO−NH4+ 25 mM + 5% acetonitrile at pH 6.6/acetonitrile). N-(2-Methyl-2,3,4,5-tetrahydro-1H-benzo[c]azepin-8-yl)-5-(3(pyridin-3-yl)phenyl)-1,3,4-oxadiazol-2-amine (4). Scheme 1, route A: To a previously cooled at 0 °C solution of 2-methyl-2,3,4,5tetrahydro-1H-benzo[c]azepin-8-amine (0.5 g, 2.8 mmol), and triethylamine (0.6 mL, 4.2 mmol) in DCM (50 mL), thiophosgene (0.5 mL, 4.2 mmol) was added dropwise over a 30 min period. The mixture was stirred at 0 °C for 1 h, and after that time, the solvent was evaporated under vacuum to give a crude material that was combined with 3-bromo benzoic hydrazide (0.7 g, 3.4 mmol) in dry THF (5 mL) at rt. The resulting solution was stirred for 18 h. Removal of the solvents under vacuum afforded a solid that was suspended, triturated with diethyl ether (50 mL) for 1 h, and filtered to give a solid that was dissolved in EtOH (5 mL), and DCC (0.5 g, 2.5 mmol) was added. The reaction mixture was kept under stirring at rt overnight. After that time, ethanol was stripped off to get a solid, which was triturated with EtOAc and then with water. The resulting solid was purified using a silica gel cartridge to give the corresponding Int-A (0.4 g). This intermediate was combined with 3-pyridinylboronic acid (0.1 g, 7.5 mmol) and potassium carbonate (0.1 g, 8.1 mmol) in a mixture of 1,4dioxane (9 mL) and water (1 mL). To this mixture, PdCl2(Ph3P)2 (31 mg, 0.1 mmol) was added, and the reaction mixture was heated at 110 °C under N2 atm for 3 h. Brine and EtOAc were added, and phases were separated in a separative funnel, the organic layer was dried over anhydrous sodium sulfate, and filtered, and the solvent was evaporated to give a crude material that was purified using a silica gel cartridge to afford 80 mg of 4 (0.002 mmol). 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H), 9.03 (d, J = 2.02 Hz, 1H), 8.72 (dd, J = 1.52, 4.80 Hz, 1H), 8.19−8.26 (m, 2H), 7.98−8.05 (m, 2H), 7.80 (t, J = 7.83 Hz, 1H), 7.66−7.74 (m, 1H), 7.63 (dd, J = 4.42, 7.71 Hz, 1H), 7.34 (s, 1H), 7.12 (d, J = 0.76 Hz, 1H), 2.93−3.00 (m, 2H), 2.90 (s, 3H), 2.74 (dd, J = 4.67, 6.44 Hz, 2H), 1.78 (br s, 2H), 1.58 (br s, 2H). MS: m/e 398 (MH+). Purity was determined as >95% by HPLC (241 nm). Rt: 1.26 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). N-(2-Methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-5-(3-(pyridin-3yl)phenyl)-1,3,4-oxadiazol-2-amine (5). A microwave vial was charged with methyl-3-bromobenzoate (1.7 g, 8.1 mmol), 3pyridinylboronic acid (1 g, 8.1 mmol), sodium carbonate (8.6 g, 8.1 mmol), DME (6 mL), and water (2 mL). The resulting mixture was purged with argon, PdCl2(PPh3)2 (286 mg, 0.4 mmol) was added, and the vial was sealed. Reaction mixture was irradiated in a microwave I

DOI: 10.1021/acs.jmedchem.6b01441 J. Med. Chem. XXXX, XXX, XXX−XXX

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device at 100 °C for 0.5 h. Reaction mixture was cooled down to rt, filtered through a Celite cartridge, and the cartridge was washed with DCM/EtOH. Solvents were removed under reduced pressure to give a crude material that was dissolved in DCM and washed with brine. Phases were separated in a separative funnel, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to afford methyl 3-(pyridin-3-yl)benzoate (210 mg, 1 mmol). Hydrazine monohydrate (0.7 mL, 9.4 mmol) was added to a rt solution of methyl 3-(pyridin-3-yl)benzoate (200 mg, 0.9 mmol) in ethanol (10 mL). The reaction mixture was stirred at rt for 5 h. Solvents were removed under reduced pressure, and toluene was added to the residue and evaporated twice. The residue was triturated with tBuOMe and n-hexanes to obtain 18.5 mg of desired 3-(pyridin-3yl)benzohydrazide as a gray solid. The filtrate was evaporated under vacuum to obtain 167 mg of a mixture of starting ester and desired product. This mixture, hydrazine monohydrate (1 mL, 12.8 mmol), and ethanol (10 mL) were added to a microwave vial, and it was placed in a heating block at 90 °C. The reaction mixture was stirred at 90 °C for 60 h. After cooling down to rt, the solvents were removed under reduced pressure. The residue was coevaporated with toluene twice to afford 3-(pyridin-3-yl)benzohydrazide (Int-D) as a white solid (164 mg, 0.8 mmol). To a suspension of 2-methyl-1,2,3,4-tetrahydroisoquinolin-7-amine (679 mg, 4.2 mmol) in DCM (8 mL), 1,1′-thiocarbonyldiimidazole (746 mg, 4.2 mmol) was added, and the reaction mixture was stirred at rt under nitrogen atm. for 1.5 h. After that time, it was diluted with DCM and washed with water. After separation of the phases in a separative funnel, the organic layer was dried over anhydrous sodium sulfate, and the solvent was evaporated under vacuum to afford 7isothiocyanato-2-methyl-1,2,3,4-tetrahydroisoquinoline (855 mg, 4.2 mmol) that was used as such in next step. A solution of 7-isothiocyanato-2-methyl-1,2,3,4-tetrahydroisoquinoline (157 mg, 0.8 mmol) in 2-MeTHF (8 mL) was added to a rt stirring mixture of 3-(pyridin-3-yl)benzohydrazide (Int-D) (164 mg, 0.8 mmol) in 2-MeTHF (8 mL). The reaction mixture was stirred at rt for 1 h, N,N′-dicyclohexylcarbodiimide (175 mg, 0.8 mmol) was added, and the reaction mixture was stirred at reflux for 3 h. After cooling down to rt, EtOAc and water were added, a precipitate appeared, and it was filtered off and dried under vacuum to obtain 90 mg of intermediate N-(2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-2-(3(pyridin-3-yl)benzoyl)hydrazinecarbothioamide. The filtrate was added to a separative funnel and layers were separated; the organic layer was washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to afford 300 mg of crude product. This crude was purified by preparative HPLC to give 92 mg of 5 (0.24 mmol). 1H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 8.96 (dd, J = 0.76, 2.27 Hz, 1H), 8.64 (dd, J = 1.52, 4.80 Hz, 1H), 8.10−8.19 (m, 2H), 7.87−7.99 (m, 2H), 7.67−7.77 (m, 1H), 7.55 (ddd, J = 0.76, 4.74, 7.89 Hz, 1H), 7.31−7.40 (m, 2H), 7.10 (d, J = 8.34 Hz, 1H), 3.48 (s, 2H), 2.73−2.81 (m, 2H), 2.54−2.62 (m, 2H), 2.30−2.37 (m, 3H). MS: m/e 384 (MH+). Purity was determined as >95% by HPLC (251 nm). Rt: 0.94 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/ acetonitrile). 5-(3-(Pyridin-3-yl)phenyl)-1,3,4-oxadiazol-2-amine (Int-B). Scheme 1, Route B: To a previously cooled at 0 °C solution of 3bromobenzoic hydrazide (10 g, 46.5 mmol) in 240 mL of a (5:1) mixture of 1,4-dioxane and water, NaHCO3 (4.1 g, 51.2 mmol) was added portionwise, followed by addition of CNBr (5.4 g, 51.2 mmol). The reaction mixture was stirred at rt for 18 h. The solid formed was filtered, washed with water, and dried under vacuum to afford 11.5 g of the corresponding oxadiazole product. 3-Pyridinylboronic acid (7 g, 57.5 mmol), and potassium carbonate (8.6 g, 62.3 mmol) were added to the oxadiazole (11.5 g) in a mixture of 1,4-dioxane (120 mL) and water (30 mL). To this mixture, PdCl2(Ph3P)2 (2.35 g, 3.4 mmol) was added, and the reaction mixture was heated under nitrogen at 110 °C for 3 h. After that time solvents were evaporated under vacuum. Water was added to the reaction mixture and stirred for 10 min. Solid obtained was filtered and washed with EtOAc to afford 7.5 g of Int-B as a brown solid. 1H NMR (DMSO-d6, 400 MHz): δ = 9.00 (d, J = 1.8

Hz, 1H), 8.64−8.76 (m, 1H), 8.17−8.26 (m, 1H), 8.13 (s, 1H), 7.86− 8.00 (m, 2H), 7.75 (s, 1H), 7.56−7.67 (m, 1H), 7.38 ppm (s, 2H). MS: m/e 239 (MH+). Purity was determined as >95% by HPLC (276 nm). Rt: 0.86 min (Acquity UPLC BEH C18 1.7 m, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). 2-Bromo-5-(3-(pyridin-3-yl)phenyl)-1,3,4-oxadiazole (Int-C). To a suspension of 5-(3-(pyridin-3-yl)phenyl)-1,3,4-oxadiazol-2-amine (300 mg, 1.3 mmol) in ACN (18 mL) was added copper(II) bromide (844 mg, 3.8 mmol); after 10 min, tert-butyl nitrite (0.6 mL, 5 mmol) was added. The reaction mixture was refluxed for 30 min, and then heating was stopped. ACN was evaporated, and the reaction mixture was diluted with a mixture of DCM/MeOH 5%, washed with a saturated aqueous solution of NaHCO3, and brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to afford 270 mg of an orangish oil which was purified on a silica gel cartridge to obtain 100 mg of Int-C (0.3 mmol). 1 H NMR (DMSO-d6, 400 MHz): δ = 9.05 (d, J = 2.0 Hz, 1H), 8.71 (dd, J = 4.8, 1.3 Hz, 1H), 8.33 (s, 1H), 8.22−8.30 (m, 1H), 8.07−8.19 (m, 2H), 7.76−7.87 (m, 1H), 7.56−7.67 ppm (m, 1H). MS: m/e 302 (MH+). Purity was determined as >95% by HPLC (251 nm). Rt: 1.03 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). N-(2-Methylisoindolin-5-yl)-5-(3-(pyridin-3-yl)phenyl)-1,3,4-oxadiazol-2-amine (6). Scheme 1, route B: 2-Methylisoindolin-5-amine (49 mg, 0.3 mmol) was added to a suspension of Int-C (100 mg, 0.3 mmol) in 1,4-dioxane (2.5 mL) under nitrogen atmosphere. The resulting reaction mixture was degassed for 15 min. Sodium tertbutoxide (49 mg, 0.8 mmol), XantPhos (17.3 mg, 0.03 mmol), and Pd2(dba)3 (27 mg, 0.03 mmol) were added to the reaction mixture and degassed for 15 min and heated at 110 °C for 3 h. The reaction mixture was poured into water (15 mL) and extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate, and concentrated under vacuum to give a crude material that was purified through a silica gel cartridge and repurified by semipreparative HPLC to afford 25 mg of 6 (0.06 mmol, 21% yield) as a white solid. 1H NMR (400 MHz, DMSOd6) δ 10.59−10.91 (m, 1H), 9.02 (d, J = 2.02 Hz, 1H), 8.72 (dd, J = 1.30, 4.80 Hz, 1H), 8.23−8.25 (m, 1H), 8.22 (s, 1H), 8.01 (t, J = 7.60 Hz, 2H), 7.79 (t, J = 8.10 Hz, 1H), 7.61 (br s, 2H), 7.49 (dd, J = 2.30, 8.30 Hz, 1H), 7.28 (d, J = 8.08 Hz, 1H), 3.89 (s, 2H), 3.84 (s, 2H), 2.55 (s, 3H). MS: m/e 370 (MH+). Purity was determined as >95% by HPLC (254 nm). Rt: 0.92 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/ acetonitrile). N-Phenyl-5-(3-(pyridin-3-yl)phenyl)-1,3,4-oxadiazol-2-amine (7). Scheme 1, route A: A solution of 3-bromobenzohydrazide (1.6 g, 7.4 mmol), and phenyl isothiocyanate (1 g, 7.4 mmol) in 10 mL of anydrous THF were stirred and heated at 60 °C for 20 h. Reaction mixture was allowed to cool down to rt. Solvent was evaporated under reduced pressure to give a crude material that was triturated with diethyl eter. The resulting solid was filtered to give 2-(3bromobenzoyl)-N-phenylhydrazine carbodiimide (2 g) that was subsequently dissolved in 8 mL of anhydrous DMF and stirred at 80 °C for 2 h with EDCI (1.4 g, 7.4 mmol). The reaction mixture was diluted with water (50 mL) and extracted with EtOAc (2 × 60 mL). Combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated to dryness to give 1.2 g of the corresponding Int-A: 5-(3-bromophenyl)-N-phenyl-1,3,4-oxadiazol-2-a (3.8 mmol, 66% yield). Then 100 mg of the corresponding Int-A (0.3 mmol) were charged on a 25 mL microwave vial with 3-pyridinylboronic acid (39 mg, 0.3 mmol), potassium carbonate (175 mg, 1.3 mmol), and Pd(PPh3)4 (7.3 mg, 6.3 μmol). The vial was sealed and purged with Ar several times. Finally, a 6 mL solution of a 1:1 mixture of toluene/ ethanol was added, and the resulting reaction mixture was irradiated in the microwave for 1 h at 85 °C. Reaction mixture was diluted with EtOAc and washed with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness. The crude was purified on a silica gel cartridge to afford 50 mg of 7 (0.15 mmol, 55% yield). 1H NMR (400 MHz, CDCl3) δ 8.99 (s, 1H), 8.75 (d, J = 3.79 Hz, 1H), 8.27 (s, 1H), 8.09 (d, J = 7.58 Hz, 1H), 8.02 (td, J

DOI: 10.1021/acs.jmedchem.6b01441 J. Med. Chem. XXXX, XXX, XXX−XXX

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Article

7.38 (m, 2H), 7.09 (d, J = 8.08 Hz, 1H), 3.48 (br s, 2H), 2.76 (t, J = 5.68 Hz, 2H), 2.53−2.63 (m, 2H), 2.33 (s, 3H). MS: m/e 402 (MH+). Purity was determined as 95% by HPLC (250 nm). Rt: 0.94 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). 5-(3-Fluoro-5-(pyridin-3-yl)phenyl)-N-(2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-1,3,4-oxadiazol-2-amine (11). Scheme 1, route A: To a previously cooled at 0 °C solution of 2-methyl-1,2,3,4tetrahydroisoquinolin-7-amine, hydrochloride (300 mg, 1.5 mmol) and triethylamine (0.5 mL, 3.8 mmol) in DCM (20 mL) and thiophosgene (0.2 mL, 2.3 mmol) was added dropwise. The mixture was stirred at 0 °C for 1 h, and the solvent was evaporated. The residue (308 mg, 1.5 mmol) was combined with 3-bromo-5fluorobenzhydrazide (351 mg, 1.5 mmol) in THF (35 mL) and heated at 60 °C for 3 h. 3-Bromo-5-fluorobenzohydrazide (351 mg, 1.5 mmol) was added and heated at 60 °C for 2 h more. Then, a third addition of 3-bromo-5-fluorobenzohydrazide (702 mg, 3 mmol) was carried out and the reaction mixture was heated at 60 °C overnight. Solvent was removed under vacuum to give a crude material that was triturated with tBuOMe. The solid (659 mg) was filtered off and dissolved in ethanol (10 mL), and N,N′-dicyclohexylcarboxamide (311 mg, 1.5 mmol) was added and stirred at rt under N2 atmosphere overnight. DCC (0.5 equiv) was added, and the mixture was heated at 60 °C for 3 h and stirred at rt overnight. Ethanol was removed under vacuum to give a crude material that was dissolved in EtOAc and washed with brine. Phases were separated in a separative funnel, and the organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated to give a crude material that was chromatographied on a silica gel cartridge to obtain the corresponding Int-A (MS: m/e 404 (MH+)) (270 mg, 0.7 mmol). 5-(3-Bromo-5-fluorophenyl)-N-(2methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-1,3,4-oxadiazol-2-amine (Int-A) (270 mg, 0.7 mmol) and 3-pyridinylboronic acid (206 mg, 1.7 mmol) in 1,4-dioxane (12 mL) were combined with potassium carbonate (278 mg, 2 mmol) in water (4 mL). This mixture was desgassed with a steady stream of N2 for 30 min at rt, and PdCl2(Ph3P)2 (25.8 mg, 0.03 mmol) was added. The reaction mixture was degassed with N2 for 5 min and then heated under nitrogen at 110 °C for 5 h. After that time, the reaction was concentrated to dryness, dissolved in DCM, and washed with water, phases were separated in a separative funnel, and the organic layer was washed with brine. The organic layer was dried over Na2SO4, filtered, and evaporated under vacuum to obtain a crude material that was purified on a silica gel cartridge to give 115 mg of 11 (0.3 mmol). 1H NMR (400 MHz, DMSO-d6) δ 10.66(s, 1H), 9.0 (d, J = 2.02 Hz, 1H), 8.68 (dd, J = 1.26, 4.80 Hz, 1H), 8.0−8.22 (m, 1H), 8.0 (s, 1H), 7.9 (d, J = 9.85 Hz, 1H), 7.60 (d, J = 8.84 Hz, 1H), 7.57 (dd, J = 4.80, 7.83 Hz, 1H), 7.33−7.36 (m, 2H), 7.17 (d, J = 8.08 Hz, 1H), 3.50 (s, 2H), 2.78 (t, J = 5.68 Hz, 2H), 2.58−2.61 (m, 2H), 2.35 (s, 3H). MS: m/e 402 (MH+). Purity was determined as >95% by HPLC (251 nm). Rt: 0.97 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). 5-(4-Fluoro-3-(pyridin-3-yl)phenyl)-N-(2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-1,3,4-oxadiazol-2-amine (12). Scheme 1, route A: To a previously cooled at 0 °C solution of 2-methyl-1,2,3,4tetrahydroisoquinolin-7-amine (1 g, 6.2 mmol) and triethylamine (1.3 mL, 9.2 mmol) in DCM (100 mL), thiophosgene (0.7 mL, 9.2 mmol) was added dropwise over 15 min period, and the reaction mixture was stirred at 0 °C for 1 h. After that time, the solvent was evaporated under vacuum to give a solid that was washed with Et2O. The resulting solid (920 mg) and 3-bromo-4-fluorobenzohydrazide (350 mg, 1.5 mmol) were combined in THF (25 mL), and the reaction mixture was stirred at rt for 48 h and refluxed for 2 h. The solvent was stripped off to give 657 mg of a crude material that was dissolved in ethanol (20 mL), and DCC (310 mg, 1.5 mmol) was added. Reaction mixture was stirred at rt under nitrogen atmosphere for 48 h, and after that time the solvent was removed under vacuum to give crude material that was dissolved in EtOAc and washed with brine. Phases were separated in a separative funnel, and the organic layer was dried over anhydrous Na2SO4 and filtered, and the solvent was evaporated to give a crude material that was purified using a silica gel cartridge to give the

J = 1.83, 7.96 Hz, 1H), 7.73−7.83 (m, 1H), 7.60−7.72 (m, 3H), 7.46− 7.58 (m, 3H), 7.35 (s, 1H), 7.21 (t, J = 7.33 Hz, 1H). MS: m/e 315 (MH+). Purity was determined as >95% by HPLC (299 nm). Rt: 1.19 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). 5-(3-(Pyridin-3-yl)phenyl)-N-(3-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-amine (8). Scheme 1, route B: To a suspension of Int-C (60 mg, 0.19 mmol) in ethanol (2 mL), 3-(trifluoromethyl)aniline (0.05 mL, 0.4 mmol) was added and the reaction mixture was refluxed for 3 h. Solvent was evaporated under vacuum to give a crude material that was purified on a silica gel cartridge to afford 15 mg of 8 (0.03 mmol, 20%). 1H NMR (400 MHz, DMSO-d6) δ 10.99−11.37 (m, 1H), 8.96 (d, J = 1.77 Hz, 1H), 8.65 (d, J = 3.79 Hz, 1H), 8.16 (br s, 2H), 8.09 (s, 1H), 7.96 (t, J = 7.20 Hz, 2H), 7.79−7.89 (m, 1H), 7.74 (t, J = 8.30 Hz, 1H), 7.62 (t, J = 8.60 Hz, 1H), 7.55 (tt, J = 4.80, 7.80 Hz, 1H), 7.39 (d, J = 7.33 Hz, 1H). MS: m/e 383 (MH+). Purity was determined as >95% by HPLC (282 nm). Rt: 1.20 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). N-(3-Chlorophenyl)-5-(3-(pyridin-3-yl)phenyl)-1,3,4-oxadiazol-2amine (9). Scheme 1, route B: To a suspension of Int-C (60 mg, 0.2 mmol) in ethanol (2 mL), 3-chloroaniline (50.7 mg, 0.4 mmol) was added. The mixture was refluxed overnight. Solvent was evaporated under vacuum to give a crude material that was purified on a silica gel cartridge to give 5 mg of 9 (0.01 mmol, 7% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.94−11.28 (m, 1H), 9.03 (d, J = 2.02 Hz, 1H), 8.75 (tt, J = 1.30, 4.80 Hz, 1H), 8.23 (d, J = 1.52 Hz, 2H), 8.03 (br s, 2H), 7.87 (t, J = 2.00 Hz, 1H), 7.81 (t, J = 7.80 Hz, 1H), 7.58−7.66 (m, 2H), 7.48 (t, J = 7.80 Hz, 1H), 7.12−7.19 (m, 1H). MS: m/e 349 (MH+). Purity was determined as >95% by HPLC (282 nm). Rt: 1.17 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). 5-(2-Fluoro-5-(pyridin-3-yl)phenyl)-N-(2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-1,3,4-oxadiazol-2-amine (10). Scheme 1, route A: To a previously cooled at 0 °C solution of 2-methyl-1,2,3,4tetrahydroisoquinolin-7-amine (1 g, 6.2 mmol) and triethylamine (1.3 mL, 9.2 mmol) in DCM (100 mL), thiophosgene (0.7 mL, 9.2 mmol) was added dropwise over 15 min period. The reaction mixture was stirred at 0 °C for 1 h, and the solvent was evaporated under vacuum to give a crude material that was washed with Et2O. The resulting solid was combined with 5-bromo-2-fluorobenzohydrazide (350 mg, 1.5 mmol) in THF (25 mL). The reaction mixture was stirred at rt overnight, and then 2 h under reflux. Solvent was evaporated under vacuum to give a crude material that was dissolved in ethanol (20 mL). N,N′-Dicyclohexylcarboxamide (310 mg, 1.5 mmol) was added, and the mixture was stirred at rt under N2 atm. for 48 h. Solvent was evaporated under vacuum to give a crude material that was dissolved in EtOAc, and brine was added. Phases were separated in a separative funnel, and the organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated to give a crude material that was purified on a silica gel cartridge to obtain the corresponding Int-A (MS: m/e 404 (MH+)). 5-(5-Bromo-2-fluorophenyl)-N-(2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-1,3,4-oxadiazol-2-amine (Int-A) (330 mg, 0.8 mmol), 3-pyridinylboronic acid (121 mg, 1 mmol) in 1,4-dioxane (12 mL) were combined with K2CO3 (339 mg, 2.4 mmol) in water (4 mL). This mixture was degassed with a steady stream of nitrogen for 30 min at rt, and PdCl2(Ph3P)2 (15.8 mg, 0.02 mmol) was added. The reaction mixture was degassed with N2 for 5 min and heated under nitrogen at 110 °C for 5 h and at rt overnight. More 3pyridinylboronic acid (201 mg, 1.7 mmol) was added, and the reaction mixture was heated at 110 °C for 3 h. After that time, the solvent was evaporated to dryness to give a crude material that was dissolved in DCM, filtered, and washed with water and brine. The organic layer was dried over anhydrous sodium sulfate and filtered, and the solvent was evaporated under vacuum to afford a crude material. It was purified using a silica gel cartridge to obtain a brown oil which was purified by semipreparative HPLC to afford 55 mg of 10 (0.13 mmol). 1 H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H), 8.93 (d, J = 2.02 Hz, 1H), 8.62 (dd, J = 1.52, 4.80 Hz, 1H), 8.08−8.19 (m, 2H), 7.93−8.02 (m, 1H), 7.61 (dd, J = 8.72, 10.48 Hz, 1H), 7.48−7.55 (m, 1H), 7.29− K

DOI: 10.1021/acs.jmedchem.6b01441 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

corresponding Int-A (MS: m/e 404 (MH+). 5-(3-Bromo-4-fluorophenyl)-N-(2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-1,3,4-oxadiazol-2-amine (105 mg, 0.3 mmol) and 3-pyridinylboronic acid (38.4 mg, 0.3 mmol) in 1,4-dioxane (10 mL) were combined with potassium carbonate (108 mg, 0.8 mmol) in water (3 mL). This mixture was degassed with a steady stream of nitrogen for 30 min at rt. To this mixture, PdCl2(Ph3P)2 (5 mg, 5.2 μmol) was added, and the reaction mixture was degassed with nitrogen for 5 min and heated under nitrogen atm at 110 °C for 5 h and at rt overnight. After that time, the reaction mixture was concentrated to dryness, dissolved in DCM, filtered, and washed with water. Phases were separated in a separative funnel, and the organic layer was washed with brine, dried over anhyd Na2SO4, and filtered, and the solvent was evaporated to give a crude material that was purified on a silica gel cartridge to obtain a brown oil which was purified by semipreparative HPLC to obtain 21 mg of 12 (0.05 mmol). 1H NMR (400 MHz, DMSO-d6) δ 10.58 (s, 1H), 8.82 (s, 1H), 8.66 (dd, J = 1.64, 4.67 Hz, 1H), 8.03−8.10 (m, 1H), 7.89− 8.03 (m, 2H), 7.51−7.68 (m, 2H), 7.28−7.38 (m, 2H), 7.08 (d, J = 8.34 Hz, 1H), 3.47 (br s, 2H), 2.75 (m, 2H), 2.51−2.62 (m, 2H), 2.33 (s, 3H). MS: m/e 402 (MH+). Purity was determined as >95% by HPLC (250 nm). Rt: 0.96 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/ acetonitrile). N-(2-Methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-5-(3-(pyridin-3yl)-5-(trifluoromethoxy)phenyl)-1,3,4-oxadiazol-2-amine (13). Scheme 1, route A: To a previously cooled at 0 °C solution of 2methyl-1,2,3,4-tetrahydroisoquinolin-7-amine, hydrochloride (200 mg, 1 mmol), and triethylamine (0.4 mL, 2.5 mmol) in DCM (15 mL), thiophosgene (0.1 mL, 1.5 mmol) was added dropwise. The mixture was stirred at 0 °C for 90 min. After that time, more thiophosgene (0.1 mL) was added, and the reaction mixture was stirred for 10 min at this temperature. After that time, solvent was evaporated under vacuum to give 206 mg of a crude material. This crude material and 3-bromo-5(trifluoromethoxy)benzhydrazide (302 mg, 1 mmol) were combined in THF (15 mL) and stirred at room temperature overnight. More 3bromo-5-(trifluoromethoxy)benzohydrazide (302 mg, 1 mmol) was added and heated at 50 °C for 3 h. Solvent was removed under vacuum to afford 659 mg of a crude material. This crude material was dissolved in ethanol (20 mL) and DCC (351 mg, 1.7 mmol) was added, and the reaction mixture was stirred at rt under N2 atm overnight. DCC (135 mg, 0.6 mmol) was added and the mixture was heated at 60 °C for 3 h and stirred at rt overnight. Ethanol was removed under vacuum to give a crude material that was dissolved in EtOAc, filtered, and washed with water. Phases were separated in a separative funnel, and the organic layer was washed with brine, dried over anhyd Na2SO4, and filtered, and the solvent was evaporated under vacuum to give a crude material that was purified using a silica gel cartridge to obtain the corresponding Int-A (MS: m/e 469 (MH+)). 5(3-Bromo-5-(trifluoromethoxy)phenyl)-N-(2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-1,3,4-oxadiazol-2-amine (110 mg, 0.2 mmol) and 3-pyridinylboronic acid (72 mg, 0.6 mmol) in 1,4-dioxane (9 mL) were combined with potassium carbonate (97 mg, 0.7 mmol) in water (3 mL). This mixture was desgassed with a steady stream of nitrogen for 30 min at rt. PdCl2(PPh3)2 (9 mg, 9.4 μmol) was added, and the reaction mixture was degassed with a steady stream of N2 and then heated under nitrogen at 110 °C for 5 h. The solvent was evaporated to dryness to give a crude material that was dissolved in DCM and washed with water. Phases were separated in a funnel, the organic layer was washed with brine, dried over anhyd Na2SO4, and filtered, and the solvent was evaporated to give a crude material that was was purified on a silica gel cartridge to give 77 mg of 13 (0.2 mmol). 1H NMR (400 MHz, DMSO-d6) δ 10.67 (s, 1H), 9.00 (m, 1H), 8.67 (dd, J = 1.52, 4.80 Hz, 1H), 8.18−8.26 (m, 1H), 8.16 (t, J = 1.39 Hz, 1H), 7.98 (s, 1H), 7.76−7.85 (m, 1H), 7.52−7.60 (m, 1H), 7.33−7.39 (m, 1H), 7.32 (d, J = 2.02 Hz, 1H), 7.09 (d, J = 8.34 Hz, 1H), 3.47 (s, 2H), 2.71−2.81 (m, 2H), 2.54−2.61 (m, 2H), 2.33 (s, 3H). MS: m/e 468 (MH+). Purity was determined as >95% by HPLC (250 nm). Rt: 1.09 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile).

N-(2-Methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-5-(3-(pyridin-3yl)-5-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-amine (14). Scheme 1, route A: To a previously cooled at 0 °C solution of 2methyl-1,2,3,4-tetrahydroisoquinolin-7-amine (1.9 g, 12 mmol) and triethylamine (2.5 mL, 18 mmol) in DCM (190 mL), thiophosgene (1.4 mL, 18 mmol) was added dropwise over 15 min. The mixture was stirred at 0 °C, and the solvent was evaporated under vacuum to give 1.85 g of a crude material. Then 750 mg (2.65 mmol) of this crude were combined with 7-isothiocyanato-2-methyl-1,2,3,4-tetrahydroisoquinoline (0.7 g, 3.2 mmol) in THF (15 mL) and stirred at 60 °C for 3 h and at rt overnight. The solvent was evaporated under reduced pressure, and the resulting crude material was combined with N,N′dicyclohexylcarbodiimide (0.6 g, 3.2 mmol) in ethanol (20 mL) and stirred at rt for 48 h. The solvent was evaporated under reduced pressure, and the crude was purified on a silica gel cartridge to afford 0.5 g the corresponding Int-A (1 mmol, 40% yield). This solid was combined with 3-pyridinylboronic acid (156 mg, 1.2 mmol), potassium carbonate (439 mg, 3.2 mmol), and PdCl2(PPh3)2 (82 mg, 0.1 mmol) in a microwave vial, and it was sealed and then purged with Ar. A 3.5 mL solution of a mixture of toluene/ethanol (1:1) was added, and the resulting solution was irradiated in the microwave at 100 °C for 150 min. After this time, more 3-pyridinylboronic acid (27 mg, 0.2 mmol) and PdCl2(PPh3)2 (8 mg, 0.01 mmol) were added. The reaction mixture was irradiated for 30 min more at the same temperature. The reaction mixture was filtered through Celite and washed with EtOH. The solvent was evaporated under vacuum to give a crude material that was dissolved in a mixture of DCM:MeOH (5%) and washed with water. Phases were separated in a funnel, and the organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered, and evaporated under reduced pressure to give a crude material that was purified on a amino silica gel cartridge to give 300 mg of white solid that was triturated with tBuOMe. The resulting solid was filtered, rinsed with more tBuOMe, and collected to afford 125 mg of 14 (0.27 mmol, 26% yield). 1H NMR (DMSO-d6, 400 MHz): δ = 10.63−10.88 (m, 1H), 9.12 (d, J = 2.0 Hz, 1H), 8.76 (dd, J = 4.8, 1.3 Hz, 1H), 8.47 (s, 1H), 8.36 (s, 1H), 8.33 (br s, 1H), 8.21 (s, 1H), 7.61−7.69 (m, 1H), 7.43 (s, 1H), 7.40 (s, 1H), 7.17 (d, J = 8.3 Hz, 1H), 3.55 (s, 2H), 2.84 (t, J = 5.4 Hz, 2H), 2.65 (t, J = 5.7 Hz, 2H), 2.41 ppm (s, 3H). MS: m/e 452 (MH+). Purity was determined as >95% by HPLC (311 nm). Rt: 1.06 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). N-(7-Methyl-5,6,7,8-tetrahydro-1,7-naphthyridin-2-yl)-5-(3-(pyridin-3-yl)phenyl)-1,3,4-oxadiazol-2-amine (15a). Scheme 1, route B: 150 mg of Int-B were added to a stirring solution of 2-chloro-7methyl-5,6,7,8-tetrahydro-1,7-naphthyridine (114 mg, 0.6 mmol) in 1,4- dioxane (10 mL), and NaOtBu (151 mg, 1.6 mmol) were added under nitrogen atmosphere. The mixture was degassed for 15 min. Xantphos (36 mg, 0.06 mmol) and Pd2(dba)3 (57 mg, 0.06 mmol) were added to the reaction mixture and degassed for 15 min. After 2 h heating at 110 °C, the reaction mixture was filtered under reduced pressure and the solvent was evaporated to give a crude material that was purified using a silica gel cartridge. Then it was further purified by preparative HPLC to afford 15a (12 mg, 5%) as an off-white solid. 1H NMR (DMSO-d6, 400 MHz): δ = 11.15−11.48 (m, 1H), 8.97−9.07 (m, 1H), 8.65−8.74 (m, 1H), 8.22 (d, J = 1.5 Hz, 2H), 8.00 (s, 2H), 7.82 (s, 2H), 7.66−7.72 (m, 1H), 7.58−7.65 (m, 1H), 3.55 (s, 2H), 2.81−2.88 (m, 2H), 2.69 (s, 2H), 2.44 ppm (s, 3H). MS: m/e 385 (MH+). Purity was determined as >95% by HPLC (301 nm). Rt: 0.97 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). N-(6-Methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-5-(3-(pyridin-3-yl)phenyl)-1,3,4-oxadiazol-2-amine (15b). Scheme 1, route B: To a stirred solution of Int-C (0.25 g, 0.8 mmol) in 1,4-dioxane (15 mL), 6-methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-amine (0.16 g, 1 mmol) and sodium tert-butoxide (0.2 g, 2 mmol) were added at rt, and the reaction mixture was degassed for 15 min. Xantphos (0.05 g, 0.08 mmol) and Pd2(dba)3 (0.08 g, 0.08 mmol) were added, and the reaction mixture was again degassed for 15 min and heated at 110 °C for 1 h. After that time, the reaction mixture was poured into water, L

DOI: 10.1021/acs.jmedchem.6b01441 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

extracted with EtOAc, and washed with brine solution. The organic layer was dried over anhydrous sodium sulfate and the solvent was evaporated under vacuum to give a crude material that was purified on a silica gel cartridge, followed by purification by preparative HPLC to afford 25 mg of 15b (0.06 mmol, 8% yield) as an off-white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 11.00 (br s, 1 H), 9.05 (br s, 1 H), 8.64 (br s, 2 H), 8.17−8.28 (m, 2 H), 7.98−8.06 (m, 2 H), 7.89 (s, 1 H), 7.76−7.85 (m, 1 H), 7.64 (br s, 1 H), 3.78 (br s, 2 H), 2.82− 3.04 (m, 4 H), 2.54 ppm (s, 3H). MS: m/e 385 (MH+). Purity was determined as >95% by HPLC (252 nm). Rt: 0.92 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). 3-(Pyridin-3-yl)-5-(trifluoromethyl)benzohydrazide (Int-E). Scheme 1, route C: To a solution of methyl 3-bromo-5(trifluoromethyl)benzoate (1 g, 3.5 mmol), pyridin-3-ylboronic acid (0.6 g, 4.6 mmol), and PdCl2(PPh3)2 (0.05 g, 0.07 mmol) in toluene (10 mL), a suspension of potassium carbonate (1.5 g, 10.6 mmol) in ethanol (2.5 mL) was added. The reaction mixture was degassed with a steady stream of N2, and then irradiated in the microwave at 100 °C for 3 h. The reaction mixture was filtered through Celite, and the solvent was evaporated under vacuum to afford a crude that was purified using a silica gel cartridge to give 870 mg of methyl 3-(pyridin3-yl)-5-(trifluoromethyl)benzoate [MS: m/e 295 (MH+)]. This compound was dissolved in ethanol (10 mL) and hydrazine hydrate (1.8 mL, 37 mmol) was added and stirred at 60−70 °C for 2 h. Solvent and hydrazine were evaporated to dryness to afford 800 mg of a paleyellow solid which corresponds to Int-E. 1H NMR (DMSO-d6, 400 MHz): δ = 10.16 (br s, 1H), 9.05 (d, J = 1.8 Hz, 1H), 8.66 (dd, J = 4.5, 1.3 Hz, 1H), 8.45 (s, 1H), 8.21−8.30 (m, 2H), 8.17 (s, 1H), 7.56 (dd, J = 7.8, 4.8 Hz, 1H), 4.64 ppm (br s, 2H). N-(6-Methyl-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)-5-(3-(pyridin-3-yl)-5-(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-amine (15c). Scheme 1, route C: Int-E (0.45 g, 1.6 mmol) and 3-isothiocyanato-6methyl-5,6,7,8-tetrahydro-1,6-naphthyridine (0.3 g, 1.6 mmol) were combined in ethanol (11 mL), and stirred at 60 °C for 90 min. Ethanol was removed under vacuum to give a crude material that was dissolved in EtOAc, brine was added, and the phases were separated in a funnel. The organic layer was dried over anhydrous Na2SO4 and filtered, and the solvent was evaporated under vacuum to give a material that was purified on a silica gel cartridge to give 427 mg of 15c (0.9 mmol, 59% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 10.91−11.03 (m, 1 H), 9.05 (d, J = 1.77 Hz, 1 H), 8.69 (dd, J = 4.80, 1.52 Hz, 1 H), 8.55 (d, J = 2.53 Hz, 1 H), 8.41 (s, 1 H), 8.30 (s, 1 H), 8.25−8.29 (m, 1 H), 8.15 (s, 1 H), 7.78 (d, J = 2.53 Hz, 1 H), 7.58 (ddd, J = 7.83, 4.55, 0.76 Hz, 1 H), 3.55 (s, 2 H), 2.86 (s, 2 H), 2.64− 2.74 (m, 2 H), 2.37 (s, 3 H). MS: m/e 453 (MH+). Purity was determined as >95% by HPLC (249 nm). Rt: 1.02 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). N-(4-((Dimethylamino)methyl)phenyl)-5-(3-(pyridin-3-yl)phenyl)1,3,4-oxadiazol-2-amine (16a). Scheme 1, route B: 4((dimethylamino)methyl)aniline hydrochloride (16.7 mg, 0.09 mmol) was dissolved in ethanol (2 mL) under N2 atmosphere, and Int-C (27 mg, 0.09 mmol) was added. The reaction mixture was heated at 80 °C during 3 h and at rt overnight. Then 30 mg more of the aniline were added, and the reaction mixture was heated 2 h at 80 °C. Solvent was evaporated under reduced pressure to give a crude material that was purified by preparative HPLC to give 20 mg of 16a (0.05 mmol, 60% yield). 1H NMR (400 MHz, CDCl3) δ ppm 8.91 (d, J = 2.27 Hz, 1 H), 8.67 (dd, J = 4.80, 1.77 Hz, 1 H), 8.19 (t, J = 1.52 Hz, 1 H), 8.01 (dt, J = 7.77, 1.29 Hz, 1 H), 7.95 (dt, J = 8.34, 1.89 Hz, 1 H), 7.72 (dt, J = 8.08, 1.39 Hz, 1 H), 7.58−7.65 (m, 1 H), 7.52 (d, J = 8.59 Hz, 3 H), 7.40−7.45 (m, 1 H), 7.35 (d, J = 8.34 Hz, 2 H), 3.42 (s, 2 H), 2.26 (s, 6 H). MS: m/e 372 (MH+). Purity was determined as >95% by HPLC (254 nm). Rt: 0.90 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). N-(4-((Dimethylamino)methyl)phenyl)-5-(3-(pyridin-3-yl)-5(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-amine (16b). Scheme 1, route C: A solution of Int-E (0.15 g, 0.5 mmol) and 1-(4-

isothiocyanatophenyl)-N,N-dimethylmethanamine (0.1 g, 0.5 mmol in THF (1 mL) was stirred at 60 °C for 30 min. Solvent was evaporated under vacuum to give a crude material that was combined with DCC (125 mg, 0.6 mmol) in EtOH (10 mL), and the reaction mixture was stirred at 60 °C for 1 h. Solvent was evaporated under reduced pressure to give a crude material that was purified on a silica gel cartridge to afford 210 mg of 16b (0.5 mmol, 79% yield). 1H NMR (DMSO-d6, 400 MHz): δ = 10.80 (s, 1H), 9.05 (d, J = 2.0 Hz, 1H), 8.65−8.75 (m, 1H), 8.42 (s, 1H), 8.29 (s, 1H), 8.25−8.27 (m, 1H), 8.15 (s, 1H), 7.55−7.62 (m, 3H), 7.29 (d, J = 8.6 Hz, 2H), 3.36−3.38 (m, 2H), 2.15 ppm (s, 6H). MS: m/e 440 (MH+). Purity was determined as >95% by HPLC (307 nm). Rt: 1.02 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). 5-(3-(Pyridin-3-yl)-5-(trifluoromethyl)phenyl)-N-(4-(pyrrolidin-1ylmethyl)phenyl)-1,3,4-oxadiazol-2-amine (17a). Scheme 1, route C: A solution of Int-E (0.1 g, 0.5 mmol) and 1-(4isothiocyanatobenzyl)pyrrolidine (0.1 g, 0.5 mmol) in THF (1 mL) was stirred at 60 °C for 30 min. The solvent was evaporated under reduced pressure, affording a pale-yellow solid (240 mg, 0.480 mmol). The product and DCC (99 mg, 0.5 mmol) were combined in EtOH (10 mL) and stirred at 60 °C for 1 h. The solvent was evaporated under reduced pressure, and the crude material was purified using a silica gel cartridge to give 111 mg of 17a (0.2 mmol, 49% yield). 1H NMR (DMSO-d6, 400 MHz): δ = 10.81−10.92 (m, 1H), 9.12 (d, J = 2.0 Hz, 1H), 8.76 (dd, J = 4.8, 1.5 Hz, 1H), 8.49 (s, 1H), 8.32−8.39 (m, 2H), 8.23 (s, 1H), 7.62−7.69 (m, 3H), 7.39 (d, J = 8.1 Hz, 2H), 3.56−3.77 (m, 2H), 2.47−2.55 (m, 2H), 1.78 ppm (br s, 4H). CH2 signal not visible in the spectrum conditions. MS: m/e 466 (MH+). Purity was determined as >95% by HPLC (307 nm). Rt: 1.02 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). N-(6-((Dimethylamino)methyl)pyridin-3-yl)-5-(3-(pyridin-3-yl)-5(trifluoromethyl)phenyl)-1,3,4-oxadiazol-2-amine (17b). Scheme 1, route C: Int-E (138 mg, 0.5 mmol) and 1-(5-isothiocyanatopyridin-2yl)-N,N-dimethylmethanamine (95 mg, 0.5 mmol) were combined in ethanol (5 mL) and stirred at 60 °C for 2 h. Then EDC (94 mg, 0.5 mmol) was added, and the reaction remained at rt overnight. The reaction mixture was concentrated to dryness and redissolved in water and EtOAc. Phases were separated in a separative funnel, and the organic layer was washed with brine, dried over anhydrous sodium sulfate, and filtered, and solvent was evaporated to obtain a crude material that was purified on a silica gel cartridge to afford 28 mg of 17b (0.06 mmol, 12% yield). 1H NMR (DMSO-d6, 400 MHz): δ = 10.91−11.18 (m, 1H), 9.03−9.09 (m, 1H), 8.67−8.77 (m, 2H), 8.39− 8.46 (m, 1H), 8.30−8.32 (m, 1H), 8.25−8.30 (m, 1H), 8.15−8.19 (m, 1H), 8.05−8.10 (m, 1H), 7.55−7.61 (m, 1H), 7.41−7.48 (m, 1H), 3.51 (s, 2H), 2.20 ppm (s, 6H). MS: m/e 441 (MH+). Purity was determined as 95% by HPLC (252 nm). Rt: 0.99 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). Methyl [3,4′-Bipyridine]-2′-carboxylate (Int-18a). A solution of 3pyridinylboronic acid (537 mg, 4.4 mmol), methyl 4-bromopicolinate (786 mg, 3.6 mmol), and cesium carbonate (2.4 g, 7.3 mmol) in a mixture of ACN (20 mL) and water (10 mL) was degassed with Ar and stirred for 15 min. To this solution was added [1,1′bis(diphenylphosphino)ferrocene]dichloro-palladium(II) (297 mg, 0.4 mmol) and bubbled with argon for another 15 min and stirred at 60 °C under argon overnight. The reaction mixture was filtered through Celite, the aqueous layer was extracted with EtOAc, and the organic phase was washed with a 1 N aqueous solution of NaOH, water, and brine. Organic layer was dried over sodium sulfate, filtered through silica gel, and evaporated under vacuum to give a crude material that was purified using a silica gel cartridge to give 86 mg of Int-18a (0.4 mmol, 11% yield). 1H NMR (DMSO-d6, 400 MHz): δ = 9.14 (d, J = 2.0 Hz, 1H), 8.89 (d, J = 5.1 Hz, 1H), 8.78 (dd, J = 4.8, 1.5 Hz, 1H), 8.44 (d, J = 1.3 Hz, 1H), 8.37 (dt, J = 8.1, 1.9 Hz, 1H), 8.14 (dd, J = 5.1, 1.8 Hz, 1H), 7.65 (dd, J = 7.6, 4.8 Hz, 1H), 4.00 ppm (s, 3H). M

DOI: 10.1021/acs.jmedchem.6b01441 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

× 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/ acetonitrile). Methyl [2,3′-Bipyridine]-4-carboxylate (Int-18c). Methyl 2-bromoisonicotinate (1.5 g, 6.8 mmol) and pyridin-3-ylboronic acid (1.1 g, 8.9 mmol) in ACN (8 mL) were combined with a 10% solution of sodium bicarbonate (8 mL, 6.8 mmol), and then bis(diphenylphosphino)ferrocene]dichloro-palladium(II) (0.2 g, 0.2 mmol) was added. The reaction mixture was degassed with a steady stream of N2 and irradiated in a microwave device at 100 °C for 1 h. The reaction mixture was filtered through a Celite cartridge, and water was added, phases were separated in a separative funnel, and the organic phase was washed with brine, dried over anhydrous MgSO4, and filtered, and the solvent was evaporated under vacuum to afford a crude material that was purified on a silica gel cartridge to give 540 mg of Int-18c (2.5 mmol, 37% yield). 1H NMR (DMSO-d6, 400 MHz): δ = 9.38 (d, J = 1.5 Hz, 1H), 9.00 (d, J = 5.1 Hz, 1H), 8.71−8.82 (m, 1H), 8.53−8.62 (m, 1H), 8.47 (s, 1H), 7.93 (dd, J = 4.9, 1.4 Hz, 1H), 7.62 (dd, J = 7.8, 4.8 Hz, 1H), 3.99−4.07 ppm (m, 3H). 5-([2,3′-Bipyridin]-4-yl)-N-(2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-1,3,4-oxadiazol-2-amine (18c). To a solution of methyl [2,3′-bipyridine]-4-carboxylate (Int-18c) (532 mg, 2.5 mmol) in ethanol (8 mL) was added hydrazine hydrate (1.5 mL, 29.8 mmol) and stirred at room temperature for 40 min. Solvent and hydrazine were evaporated under vacuum to afford a brownish solid that was resuspended in EtOH, and the solid was filtered and washed with DCM, affording 220 mg of an off-white solid corresponding to desired product. The filtrate was evaporated under vacuum to afford a solid that was washed with DCM to give 70 mg of desired product. Both batches were dissolved together in MeOH and filtered through a nylon filter to eliminate rests of catalyst from the previous reaction. Solvent was evaporated under vacuum to give 277 mg of [2,3′-bipyridine]-4carbohydrazide (1.3 mmol, 53% yield). 2,3′-Bipyridine]-4-carbohydrazide (137 mg, 0.6 mmol) and 7-isothiocyanato-2-methyl-1,2,3,4tetrahydroisoquinoline (157 mg, 0.8 mmol) were combined in ethanol (12 mL) and stirred at 80 °C under nitrogen atmosphere until reaction completion. EDCI (123 mg, 0.6 mmol) was added, and the reaction mixture was stirred overnight. Ethanol was removed under vacuum to give a crude material that was dissolved in DCM, water was added, and phases were separated in a separative funnel. The organic layer was dried over anhydrous sodium sulfate and evaporated under reduced pressure to give a crude material that was purified on a amino silica gel cartridge and then by preparative HPLC to afford 60 mg of 18c (0.15 mmol, 24% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.85 (s, 1H), 9.38 (d, J = 2.3 Hz, 1H), 9.0 (dd, J = 5.1, 8.0 Hz, 1H), 8.77 (dd, J = 4.7, 1.6 Hz, 1H), 8.56 (dt, J = 8.3, 1.9 Hz, 1H), 8.41 (s, 1H), 7.89 (dd, J = 5.1, 1.5 Hz, 1H), 7.65 (ddd, J = 8.1, 4.8, 0.8 Hz, 1H), 7.46 (dd, J = 8.2, 2.4 Hz, 1H), 7.41 (d, J = 2 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 3.56 (s, 2H), 2.86−2.83 (m, 2H), 2.67−2.64 (m, 2H), 2.41 (s, 3H). MS: m/e 385 (MH+). Purity was determined as >95% by HPLC (250 nm). Rt: 0.90 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). Ethyl [2,3′-Bipyridine]-6-carboxylate (Int-18d). A solution of 3pyridinylboronic acid (0.6 g, 4.6 mmol) and methyl 6-bromo-2pyridinecarboxylate (1 g, 4.6 mmol) in a mixture of toluene (24 mL) and ethanol (60 mL) was stirred and bubbled with argon for 15 min. To this solution, PdCl2(PPh3)2 (0.3 g, 0.5 mmol) was added and the reaction mixture was bubbled with argon for another 15 min. To this solution, a saturated solution of sodium bicarbonate (17 mL, 18.5 mmol) was added dropwise during 10 min, and the reaction mixture was stirred at 85 °C under argon for 2 h. Reaction mixture was filtered through Celite, the organic solvent was removed under vacuum, and the aqueous layer was extracted with EtOAc. Organic phase was washed with 1 N aqueous solution of NaOH, water, and brine, dried over anhydrous sodium sulfate, filtered through silica gel, and evaporated under vacuum to give a crude material that was purified on a silica gel cartridge to give 256 mg of Int-18d along with triphenyl phosphonium oxide (2:1). 1H NMR (400 MHz, DMSO-d6) δ 9.33− 9.43 (m, 1H), 8.71−8.82 (m, 1H), 8.52−8.60 (m, 1H), 8.35−8.42 (m, 1H), 8.17−8.25 (m, 1H), 8.09−8.16 (m, 1H), 7.67 (d, J = 1.26 Hz, 1H), 4.47 (d, J = 7.07 Hz, 2H), 1.44 (t, J = 7.20 Hz, 3H).

5-([3,4′-Bipyridin]-2′-yl)-N-(2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-1,3,4-oxadiazol-2-amine (18a). To a solution of methyl [3,4′-bipyridine]-2′-carboxylate (Int-18a) (84 mg, 0.4 mmol) in ethanol (5 mL) was added hydrazine monohydrate (0.2 mL, 4.4 mmol) and stirred at rt overnight. The reaction mixture was evaporated under vacuum to obtain a yellow solid, and toluene was added in order to remove water and evaporated under vacuum to give 102 mg of [3,4′-bipyridine]-2′-carbohydrazide as a whitish solid. The hydrazide and 7-isothiocyanato-2-methyl-1,2,3,4-tetrahydroisoquinoline (97 mg, 0.5 mmol) were combined in THF (10 mL) and stirred at 60 °C for 2 h. Solvent was evaporated under reduced pressure to give a crude material that was combined with DCC (98 mg, 0.5 mmol) in ethanol (20 mL). The reaction mixture was stirred at 80 °C for 2 h. After this time, ethanol was removed under vacuum to give a crude material that was dissolved in EtOAc, and brine was added. Phases were separated in a separative funnel, and the organic layer was dried over Na2SO4 and filtered, and the solvent was evaporated under vacuum to give a crude material that was purified on a silica amino cartridge to afford 56 mg of 18a (0.15 mmol, 30% yield). 1H NMR (DMSO-d6, 400 MHz): δ = 10.72−10.89 (m, 1H), 9.17 (d, J = 1.8 Hz, 1H), 8.90 (d, J = 5.1 Hz, 1H), 8.80 (dd, J = 4.8, 1.5 Hz, 1H), 8.44 (d, J = 1.0 Hz, 1H), 8.35−8.42 (m, 1H), 8.04 (dd, J = 5.3, 1.8 Hz, 1H), 7.64−7.73 (m, 1H), 7.42−7.49 (m, 1H), 7.40 (s, 1H), 7.18 (d, J = 8.3 Hz, 1H), 3.56 (s, 2H), 2.80−2.88 (m, 2H), 2.62−2.70 (m, 2H), 2.41 ppm (s, 3H). MS: m/e 385 (MH+). Purity was determined as >95% by HPLC (323 nm). Rt: 0.9 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/ acetonitrile). Methyl [3,3′-Bipyridine]-5-carboxylate (Int-18b). To a mixture of methyl 5-bromonicotinate (330 mg, 1.5 mmol) and pyridin-3ylboronic acid (188 mg, 1.5 mmol) in ACN (4 mL), a saturated solution of sodium bicarbonate (4 mL, 1.5 mmol) and [1,1′bis(diphenylphosphino)ferrocene]dichloro-palladium(II) (35 mg, 0.04 mmol) were added. This reaction mixture was purged with N2 and heated at 90 °C in a microwave device during 15 min. The layers were separated, and the aqueous one was extracted with EtOAc. The combined organic phases were washed with brine, dried over magnesium sulfate, and filtered, and the solvent was evaporated under vacuum to give a crude material that was purified on a silica gel cartridge to afford 175 mg of Int-18b (0.8 mmol, 53%). 1H NMR (CDCl3, 400 MHz) δ 9.27 (d, J = 2.0 Hz, 1H), 9.02 (d, J = 2.3 Hz, 1H), 8.91 (d, J = 2.0 Hz, 1H), 8.71 (dd, J = 4.8, 1.5 Hz, 1H), 8.52 (t, J = 2.1 Hz, 1H), 7.95 (dt, J = 7.9, 2.0 Hz, 1H), 7.46 (dd, J = 7.6, 5.1 Hz, 1H), 4.02 ppm (s, 3H). MS: m/e 215 (MH+). Purity was determined as >95% by HPLC (245 nm). Rt: 0.96 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). 5-([3,3′-Bipyridin]-5-yl)-N-(2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-1,3,4-oxadiazol-2-amine (18b). To a solution of Int-18b (175 mg, 0.8 mmol) in ethanol (5 mL), hydrazine monohydrate (0.5 mL, 9.8 mmol) was added and the reaction mixture was refluxed during 16 h. The solvent was removed under reduced pressure to give a crude material that was combined with 7-isothiocyanato-2-methyl1,2,3,4-tetrahydroisoquinoline (137 mg, 0.7 mmol) in ethanol (12 mL). The reaction mixture was stirred at 80 °C under nitrogen atmosphere until reaction reached completion. EDCI (107 mg, 0.6 mmol) was added and the stirring continued overnight. Ethanol was removed under vacuum to give a crude material that was dissolved in DCM and washed with water. Phases were separated in a separative funnel, and the organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated under vacuum to give the a crude material that was purified on a silica amino cartridge and by preparative HPLC to afford 20 mg of 18b (0.05 mmol, 9% yield). 1H NMR (400 MHz, DMSO-d6) δ = 10.77 (s, 1H), 8.97−9.38 (m, 3H), 8.71−8.87 (m, J = 1.64, 4.67 Hz, 1H), 8.48−8.67 (m, J = 2.15, 2.15 Hz, 1H), 8.34 (td, J = 1.80, 8.27 Hz, 1H), 7.66 (dd, J = 4.80, 7.83 Hz, 1H), 7.31−7.49 (m, 2H), 7.18 (d, J = 8.34 Hz, 1H), 3.56 (s, 2H), 2.59−2.94 (m, 4H), 2.41 (s, 3H). MS: m/e 385 (MH+). Purity was determined as >95% by HPLC (251 nm). Rt: 0.86 min (Acquity UPLC BEH C18 1.7 μ, 3 mm N

DOI: 10.1021/acs.jmedchem.6b01441 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

5-([2,3′-Bipyridin]-6-yl)-N-(2-methyl-1,2,3,4-tetrahydroisoquinolin-7-yl)-1,3,4-oxadiazol-2-amine (18d). To a solution of Int-18d (165 mg, 0.7 mmol) in ethanol (5 mL), hydrazine monohydrate (0.4 mL, 8.7 mmol) was added and the reaction mixture was stirred at rt overnight. Solvent was removed under reduced pressure to give a crude material that was combined with 7-isothiocyanato-2-methyl1,2,3,4-tetrahydroisoquinoline (130 mg, 0.6 mmol) in THF (10 mL) and stirred at 60 °C overnight. The solvent was evaporated under reduced pressure to give a crude material that was combined with DCC (131 mg, 0.6 mmol) in ethanol (20 mL) and stirred at 80 °C overnight. Ethanol was removed under vacuum to give a crude material that was dissolved in EtOAc and brine was added, and phases were separated in a funnel. The organic layer was dried and filtered, and the solvent was evaporated under reduced pressure to give a crude material that was purified on a silica amino cartridge to afford 98 mg of 18d (0.2 mmol, 40% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.85 (br.s, 1H), 9.42 (s, 1H), 8.77 (dd, J = 4.7, 1.6 Hz, 1H), 8.57 (dt, J = 8.1, 2 Hz, 1H), 8.31−8.17 (m, 3H), 7.68−7.66 (m, 1H), 7.45 (dd, J = 7.8, 4.8 Hz, 2H), 7.18 (d, J = 8.8 Hz, 1H), 3.56 (br.s, 2H), 2.86- 2.84 (m, 2H), 2.67−2.64 (m, 2H), 2.42 (m, 3H). MS: m/e 385 (MH+). Purity was determined as >95% by HPLC (249 nm). Rt: 0.89 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). 5-([3,4′-Bipyridin]-2′-yl)-N-(4-(pyrrolidin-1-ylmethyl)phenyl)1,3,4-oxadiazol-2-amine (19). To a solution of Int-18a (326 mg, 1.5 mmol) in ethanol (20 mL), hydrazine monohydrate (0.8 mL, 17.2 mmol) was added and the reaction mixture was stirred at rt overnight. The solvent was evaporated under vacuum to give a crude material that was combined with 1-(4-isothiocyanatobenzyl)pyrrolidine (332 mg, 1.5 mmol) in ethanol (20 mL) and stirred at 80 °C for 2 h until reaction completion. EDCI (350 mg, 1.8 mmol) was added, and it was stirred at 80 °C overnight. The reaction was cooled down to rt, and the solid was filtered and rinsed with EtOH to give a crude material that was purified by preparative HPLC to give 147 mg of 19 (0.3 mmol, 24% yield). 1H NMR (DMSO-d6, 400 MHz): δ = 10.84−11.00 (m, 1H), 9.17 (d, J = 1.8 Hz, 1H), 8.90 (d, J = 5.3 Hz, 1H), 8.80 (d, J = 3.5 Hz, 1H), 8.45 (s, 1H), 8.35−8.43 (m, 1H), 8.05 (br s, 1H), 7.66−7.71 (m, 1H), 7.64 (d, J = 8.3 Hz, 2H), 7.37 (d, J = 8.3 Hz, 2H), 3.59 (s, 2H), 2.48 (br s, 4H), 1.76 ppm (br s, 4H). MS: m/e 399 (MH+). Purity was determined as >95% by HPLC (255 nm). Rt: 0.84 min (Acquity UPLC BEH C18 1.7 μ Acquity UPLC BE3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). HRMS: (ES) calcd for C23H22N6O (M + H)+ 397.1782, found 397.1798. 2-(3-(Pyridin-3-yl)-5-(trifluoromethyl)benzoyl)-N-(4-(pyrrolidin-1 ylmethyl)phenyl)hydrazinecarboxamide (20a). Int-E (300 mg, 1.1 mmol) and 1-(4-isocyanatobenzyl)pyrrolidine (216 mg, 1.1 mmol) were combined in ethanol (10 mL) and stirred at 70 °C for 1 h. Then 1-(4-isocyanatobenzyl)pyrrolidine (216 mg, 1.1 mmol) was added and it was heated at 70 °C for 1h at rt for 48 h. After that time, another equivalent of 1-(4-isocyanatobenzyl)pyrrolidine (216 mg, 1.1 mmol) was added, and the reaction mixture was heated at 70 °C for 1 h and at rt for 48 h. The solvent was evaporated under vacuum to give a crude material that was purified on a silica amino cartridge to afford 150 mg of 20a (0.3 mmol, 29% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.75 (br s, 1H), 9.16 (d, J = 1.77 Hz, 1H), 8.96 (br s, 1H), 8.75 (dd, J = 1.64, 4.67 Hz, 1H), 8.64 (s, 1H), 8.28−8.48 (m, 4H), 7.60−7.69 (m, 1H), 7.49 (d, J = 8.34 Hz, 2H), 7.25 (d, J = 8.59 Hz, 2H), 3.55 (s, 2H), 2.46 (br s, 4H), 1.74 (t, J = 3.28 Hz, 4H). MS: m/e 484 (MH+). Purity was determined as >95% by HPLC (245 nm). Rt: 0.93 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). 2-([3,4′-Bipyridine]-2′-carbonyl)-N-(4-(pyrrolidin-1-ylmethyl)phenyl)hydrazinecarboxamide (20b). Int-18a (100 mg, 0.5 mmol) and 1-(4-isocyanatobenzyl)pyrrolidine (94 mg, 0.5 mmol) were combined in ethanol (3 mL) and stirred at 70 °C for 1 h. The reaction was cooled down to rt, and more 1-(4-isocyanatobenzyl)pyrrolidine (94 mg, 0.5 mmol) was added to the reaction mixture that was heated again at 70 °C for 1 h. A third addition of 1-(4isocyanatobenzyl)pyrrolidine (216 mg, 1.1 mmol) was carried out, and the reaction mixture was heated at 70 °C for 1 h and at rt. overnight.

Solvent was evaporated under vacuum to give a crude material that was triturated with tBuOMe, filtered, rinsed with tBuOMe and EtOAc, and dried under vacuum to obtain 110 mg of 20b (0.3 mmol, 57% yield). 1H NMR (DMSO-d6, 400 MHz): δ = 10.53 (br s, 1H), 9.16 (br s, 1H), 8.87 (d, J = 4.5 Hz, 2H), 8.79 (d, J = 3.8 Hz, 1H), 8.31−8.48 (m, 3H), 8.14 (d, J = 3.8 Hz, 1H), 7.66 (dd, J = 7.6, 4.8 Hz, 1H), 7.47 (d, J = 8.1 Hz, 2H), 7.25 (d, J = 8.1 Hz, 2H), 3.56 (br s, 2H), 3.40 (br s, 2H), 2.48 (br s, 4H), 1.75 ppm (br s, 4H). MS: m/e 417 (MH+). Purity was determined as >90% by HPLC (245 nm). Rt: 0.80 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). [3,4′-Bipyridine]-2′-carbohydrazide (Int-F). To a solution of Int18a (500 mg, 2.3 mmol) in ethanol (10 mL), hydrazine monohydrate (0.6 mL, 11.7 mmol) was added and the reaction mixture was stirred at rt overnight. The solvent was evaporated under vacuum to give a yellow solid that was triturated with EtOH, filtered, and rinsed with cool EtOH to obtain 275 mg of [3,4′-bipyridine]-2′-carbohydrazide (Int-F) a yellowish solid (1.3 mmol, 55% yield). 1H NMR (400 MHz, DMSO-d6) δ 10.06 (br s, 1H), 9.13 (d, J = 1.52 Hz, 1H), 8.78 (t, J = 5.05 Hz, 2H), 8.35 (br s, 2H), 8.00−8.13 (m, 1H), 7.65 (dd, J = 4.93, 7.71 Hz, 1H), 4.69 (d, J = 4.04 Hz, 2H). MS: m/e 215 (MH+). Purity was determined as >95% by HPLC (260 nm). Rt: 0.77 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). 3-(Pyridin-3-yl)-5-(trifluoromethyl)benzoic Acid (21a). Int-E (500 mg, 1.8 mmol) and 2-iodoxybenzoic acid (2.2 g, 3.6 mmol) were combined in ethanol (10 mL) and stirred at 70 °C for 48 h. The reaction was allowed to reach rt, and the solvent was evaporated until dryness to give a crude material that was partitioned between DCM and a satd solution of NaHCO3. Phases were separated in a funnel, and the organic layer was washed with brine, dried over MgSO4, and filtered, and the solvent was evaporated to obtain 55 mg of 21a (0.2 mmol, 11% yield). 1H NMR (400 MHz, DMSO-d6) δ 13.06−14.02 (m, 1H), 9.09 (d, J = 1.52 Hz, 1H), 8.74 (d, J = 3.79 Hz, 1H), 8.55 (s, 1H), 8.42 (s, 1H), 8.24−8.36 (m, 2H), 8.02 (d, J = 7.33 Hz, 1H). MS: m/e 268 (MH+). Purity was determined as >95% by HPLC (245 nm). Rt: 0.84 min (Acquity UPLC BEH C18 1.7 μ, 3 mm × 50 mm, CH3COO−NH4+ 25 mM + 10% acetonitrile at pH 6.6/acetonitrile). [3,4′-Bipyridine]-2′-carboxylic Acid, Hydrochloride (21b). Int-F (250 mg, 1.2 mmol) and 4 mL of a 6 N aqueous solution of hydrochloric acid were refluxed overnight. The reaction mixture was concentrated to dryness to obtain a yellow solid that was triturated with tBuOMe, filtered, and rinsed with tBuOMe to obtain a 192 mg of 21b along with a 12% of starting material. 1H NMR (DMSO-d6, 400 MHz): δ = 9.43 (d, J = 1.8 Hz, 1H), 8.88−9.04 (m, 3H), 8.76 (d, J = 5.3 Hz, 1H), 8.56 (d, J = 1.3 Hz, 1H), 8.23 (dd, J = 5.2, 1.9 Hz, 1H), 8.04−8.17 (m, 2H), 7.88 ppm (dd, J = 5.3, 2.0 Hz, 1H).



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01441. Description of the pharmacophore modeling; protocols for cell drug susceptibility studies; assessment of in vitro frequency of spontaneous resistance and characterization of resistant mutants; in vivo efficacy in P. falciparum mouse model; hERG inhibition determination and cell cytotoxicity assays; tolerability studies; PK studies methodology; intrinsic clearance determination; FaSSIF solubility determination and ChromlogD protocol; IC50 standard deviation and controls; PRR figures for compounds 2 and 14; in vivo controls (PDF) Molecular formula strings (CSV) O

DOI: 10.1021/acs.jmedchem.6b01441 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry





AUTHOR INFORMATION

Article

REFERENCES

(1) Calderón, F.; Wilson, D. M.; Gamo, F.-J. Antimalarial Drug Discovery: Recent Progress and Future Directions. Prog. Med. Chem. 2013, 52, 97−151. (2) World Malaria Report 2016; World Health Organization: Geneva, 2016; http://www.who.int/malaria/publications/world-malariareport-2016/en/ (accessed February, 16, 2016). (3) Sachs, J.; Malaney, P. The Economic and Social Burden of Malaria. Nature 2002, 415, 680−685. (4) Trends in Reported Malaria Incidence; World Health Organization: Geneva, 2016; http://www.who.int/en/ (accessed Nov 19, 2016). (5) Ranson, H.; N’guessan, R.; Lines, J.; Moiroux, N.; Nkuni, Z.; Corbel, V. Pyrethroid Resistance in African Anopheline Mosquitoes: What Are the Implications for Malaria Control? Trends Parasitol. 2011, 27, 91−98. (6) Van den Berg, H.; Zaim, M.; Yadav, R. S.; Soares, A.; Ameneshewa, B.; Mnzava, A.; Hii, J.; Dash, A. P.; Ejov, M. Global Trends in the Use of Insecticides to Control Vector-borne Diseases. Environ. Health Perspect. 2012, 120, 577−582. (7) Dondorp, A. M.; Yeung, S.; White, L.; Nguon, C.; Day, N. P. J.; Socheat, D.; von Seidlein, L. Artemisinin Resistance: Current Status and Scenarios for Containment. Nat. Rev. Microbiol. 2010, 8, 272−280. (8) Straimer, J.; Gnädig, N. F.; Witkowski, B.; Amaratunga, C.; Duru, V.; Ramadani, A. P.; Dacheux, M.; Khim, N.; Zhang, L.; Lam, S.; Gregory, P. D.; Urnov, F. D.; Mercereau-Puijalon, O.; Benoit-Vical, F.; Fairhurst, R. M.; Ménard, D.; Fidock, D. A. Drug Resistance. K13propeller Mutations Confer Artemisinin Resistance in Plasmodium Falciparum Clinical Isolates. Science 2015, 347, 428−431. (9) White, N. J. Antimalarial Drug Resistance. J. Clin. Invest. 2004, 113, 1084−1092. (10) Farooq, U.; Mahajan, R. C. Drug Resistance in Malaria. J. Vector Borne Dis. 2004, 41, 45−53. (11) Guiguemde, W. A.; Shelat, A. A.; Garcia-Bustos, J. F.; Diagana, T.; Gamo, F. J.; Guy, R. K. Global Phenotypic Screening for Antimalarials. Chem. Biol. 2012, 19, 116−129. (12) Gardner, M. J.; Hall, N.; Fung, E.; White, O.; Berriman, M.; Hyman, R. W.; Carlton, J. M.; Pain, A.; Nelson, K. E.; Bowman, S.; Paulsen, I. T.; James, K.; Eisen, J. A.; Rutherford, K.; Salzberg, S. L.; Craig, A.; Kyes, S.; Chan, M. S.; Nene, V.; Shallom, S. J.; Suh, B.; Peterson, J.; Angiuoli, S.; Pertea, M.; Allen, J.; Selengut, J.; Haft, D.; Mather, M. W.; Vaidya, A. B.; Martin, D. M.; Fairlamb, A. H.; Fraunholz, M. J.; Roos, D. S.; Ralph, S. A.; McFadden, G. I.; Cummings, L. M.; Subramanian, G. M.; Mungall, C.; Venter, J. C.; Carucci, D. J.; Hoffman, S. L.; Newbold, C.; Davis, R. W.; Fraser, C. M.; Barrell, B. Genome Sequence of the Human Malaria Parasite Plasmodium Falciparum. Nature 2002, 419, 498−511. (13) Guiguemde, W. A.; Shelat, A. A.; Bouck, D.; Duffy, S.; Crowther, G. J.; Davis, P. H.; Smithson, D. C.; Connelly, M.; Clark, J.; Zhu, F.; Jimenez-Diaz, M. B.; Martinez, M. S.; Wilson, E. B.; Tripathi, A. K.; Gut, J.; Sharlow, E. R.; Bathurst, I.; Mazouni, F. E.; Fowble, J. W.; Forquer, I.; McGinley, P. L.; Castro, S.; Angulo-Barturen, I.; Ferrer, S.; Rosenthal, P. J.; DeRisi, J. L.; Sullivan, D. J.; Lazo, J. S.; Roos, D. S.; Riscoe, M. K.; Phillips, M. A.; Rathod, P. K.; Van Voorhis, W. C.; Avery, V. M.; Guy, R. K. Chemical Genetics of Plasmodium Falciparum. Nature 2010, 465, 311−315. (14) Plouffe, D.; Brinker, A.; McNamara, C.; Henson, K.; Kato, N.; Kuhen, K.; Nagle, A.; Adrian, F.; Matzen, J. T.; Anderson, P.; Nam, T. G.; Gray, N. S.; Chatterjee, A.; Janes, J.; Yan, S. F.; Trager, R.; Caldwell, J. S.; Schultz, P. G.; Zhou, Y.; Winzeler, E. A. In Silico Activity Profiling Reveals the Mechanism of Action of Antimalarials Discovered in a High-throughput Screen. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 9059−9064. (15) Gamo, F. J.; Sanz, L. M.; Vidal, J.; de Cozar, C.; Alvarez, E.; Lavandera, J. L.; Vanderwall, D. E.; Green, D. V. S.; Kumar, V.; Hasan, S.; Brown, J. R.; Peishoff, C. E.; Cardon, L. R.; Garcia-Bustos, J. F. Thousands of Chemical Starting Points for Antimalarial Lead Identification. Nature 2010, 465, 305−312. (16) ChEMBL-NTD; EMBL-EBI: Hinxton, Cambridgeshire, UK, 2017; www.ebi.ac.uk/chemblntd.

Corresponding Author

*E-mail: [email protected]. ORCID

Simon J. F. Macdonald: 0000-0002-4859-8246 Félix Calderón: 0000-0003-0486-6883 Present Address #

For D.M.W.: AstraZeneca, Oncology Innovative Medicines, Chemistry, Hodgkin Building, Chesterford Research Campus, Little Chesterford, Saffron Walden, Cambridgshire CB10 1XL, UK. Author Contributions

All authors contributed to the preparation of this manuscript. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Jose Mariá Bueno, Gary Boyle and Stephanie Gresham for helpful discussions, Laura de las Heras for her input to develop this manuscript and our colleagues from Pharmacology Department and Biology for the measurement of the ADMET properties of compounds described in this paper. We acknowledge financial support from Medicines for Malaria Venture (MMV).



ABBREVIATIONS USED ACN, acetonitrile; ACTs, artemisinin combination therapies; ADME, absorption, distribution, metabolism, and excretion; ADMET, absorption, distribution, metabolism, excretion, and toxicity; Alk, alkyl; AUC, area under the curve; ChromlogD7.4, chromatographic distribution coefficient at buffer pH 7.4; Cl, clearance; compd, compound; DCC, N,N′-dicyclohexylcarbodiimide; DCM, dichloromethane; DDW, Diseases of Developing World; DME, dimethoxyethane; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; ED90, dose of compound that eradicates 90% of the pathogen; EDCI, 1-ethyl-3-(3(dimethylamino)propyl)carbodiimide; EtOAc, ethyl acetate; EtOH, ethanol; FaSSIF, fasted state simulated intestinal fluid; %F, oral bioavailability; GSK, GlaxoSmithKline; HIV, human immunodeficiency virus; HPLC, high performance liquid chromatography; hu, human; iCli, in vitro intrinsic clearance; IC50, concentration of drug that gives 50% of inhibition in vitro; Int, intermediate; iv, intravenous; LBF, liver blood flow; LCMS, liquid chromatography mass spectrometry; LHS, left-hand side; LE, ligand efficiency; MeOH, methanol; min, minute; MOE, molecular operational environment; MMV, Medicines for Malaria Venture; MS, mass spectrometry; ND, not determined; NMR, nuclear magnetic resonance spectroscopy:; Pf , Plasmodium falciparum; po, from Latin per os (by mouth); RHS, righthand side; rt, room temperature; Rt, retention time; SAR, structure−activity relationship; SCID, severe combined immunodefiency disorder; SGF, simulated gastric fluid; tBuOMe, tert-butylmethyl ether; TCAMS, Tres Cantos Antimalarial Set; THF, tetrahydrofuran; ThiQ, tetrahydroisoquinoline; t1/2, half life; US, United States; Vss, volume of distribution; WHO, World Health Organization; °C, degrees Celsius; 2-MeTHF, 2methyltetrahydrofuran; 3D, Three dimensions P

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