Thiazolino 2-Pyridone Amide Isosteres As Inhibitors of Chlamydia

Brief Article Cite This: J. Med. Chem. 2017, 60, 9393-9399

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Thiazolino 2‑Pyridone Amide Isosteres As Inhibitors of Chlamydia trachomatis Infectivity James A. D. Good,†,‡,#,○ Martina Kulén,†,‡,○ Jim Silver,‡,§,∥ K. Syam Krishnan,†,‡,∇ Wael Bahnan,‡,§,∥ Carlos Núñez-Otero,‡,∥,⊥ Ingela Nilsson,‡,§,∥ Emma Wede,‡,§,∥ Esmee de Groot,‡,§,∥ Åsa Gylfe,‡,∥,⊥ Sven Bergström,*,‡,§,∥ and Fredrik Almqvist*,†,‡ †

Department of Chemistry, Umeå University, 901 87 Umeå, Sweden Umeå Centre for Microbial Research, Umeå University, 901 87 Umeå, Sweden § Department of Molecular Biology, Umeå University, 901 87 Umeå, Sweden ∥ Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University, 901 87 Umeå, Sweden ⊥ Clinical microbiology, Umeå University, 901 85 Umeå, Sweden ‡

S Supporting Information *

ABSTRACT: Chlamydia trachomatis is a global health burden due to its prevalence as a sexually transmitted disease and as the causative agent of the eye infection trachoma. We recently discovered 3-amido thiazolino 2-pyridones which attenuated C. trachomatis infectivity without affecting host cell or commensal bacteria viability. We present here the synthesis and evaluation of nonhydrolyzable amide isosteres based on this class, leading to highly potent 1,2,3-triazole based infectivity inhibitors (EC50 ≤ 20 nM).

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INTRODUCTION The Gram-negative bacterium Chlamydia trachomatis is the most common bacterial sexually transmitted infection (STI) and the causative agent of the visually impairing eye infection trachoma.1,2 Broad spectrum antibiotics such as doxycycline and azithromycin provide effective treatment for C. trachomatis infections,3 yet in the process concurrently disrupt commensal flora. This fosters the development of antibiotic resistance across other pathogenic bacteria, thus threatening our control of consequent infections.4,5 Drugs which specifically treat C. trachomatis infections without disturbing commensal microbiota could reduce antibiotic usage and in turn alleviate the selective pressure toward widespread antibiotic resistance.6 To the best of our knowledge, very few studies have described small molecule inhibitors of Chlamydiae spp. pathogenicity with selectivity over other Gram-negative bacteria.7−10 To find new ways to treat C. trachomatis, we conducted a phenotypic screen for compounds which affected the infection phenotype and identified the thiazolo 2-pyridone amide 1 (KSK120, Figure 1).11 As an obligate intracellular parasite, C. trachomatis undergoes a biphasic lifecycle alternating between infectious elementary bodies (EBs) and replicative reticulate bodies (RBs) hosted intracellularly in a parasitophorous inclusion.12 In a normal infection, the bacteria transition back © 2017 American Chemical Society

Figure 1. Structures of 1 and 2.

to the EB form after replicating and lyse the inclusion and host cell to release infectious progeny EBs. Treatment with 1 produced a reduced amount of progeny which were noninfectious and therefore unable to propagate the infection. Mode of action studies with 1 found mutations in the key glucose-6-phosphate (G6-P) pathway for energy uptake from the host.11,13 The initial optimization of this scaffold produced the thiazolino 2-pyridone amide 2 (KSK213, Figure 1), which demonstrated improved efficacy and reduced lipophilicity versus thiazolo 2-pyridone amide 1.14 2 potently (EC50 ≈ 60 nM) inhibited multiple serovars of C. trachomatis while not Received: May 15, 2017 Published: October 20, 2017 9393

DOI: 10.1021/acs.jmedchem.7b00716 J. Med. Chem. 2017, 60, 9393−9399

Journal of Medicinal Chemistry

Brief Article

affecting the growth of key species from the commensal flora or host cell viability at 25 μM. In this previous SAR study, we addressed the C-3 amide and the C-6 and C-7 substituents of the 2-pyridone core structure (Figure 1). Replacement of the C-7 1-naphthyl moiety with substituted benzyls was tolerated but not advantageous, while an amino substituent in the C-6 position imparted improved activity and hydrophilicity. In the C-3 position, phenylamides were preferred, with small lipophilic substituents such as m-Me improving activity in some cases. Additionally, no difference in activity was discerned between the respective (R)- and (S)enantiomers at this position. One unanswered question was the importance of the C-3 amide functional group, and in particular, the possibility that the amide was acting as a prodrug.15 Related C-3 carboxylate thiazolino 2-pyridones inhibit virulence related processes in a variety of pathogenic bacteria,16−18 and indeed, during our initial screening, we noted several carboxylic acid analogues weakly disrupted the C. trachomatis infection phenotype at high concentrations (50− 100 μM).11 Furthermore, metabolic hydrolysis of the C-3 anilide would release aniline in situ as a potentially toxic metabolite.19 To answer this, we have designed and synthesized a series of biocompatible amide isosteres at the C-3 position. Nonhydrolyzable amide isosteres have been revealed to be beneficial as peptide surrogates,20−22 and in this study, we have identified very potent C-3 1,2,3-triazolo C. trachomatis infectivity inhibitors in a cell-based in vitro system.

Scheme 1. Synthesis of Amide Analogues 4, 5, 7, and 8 from Carboxylic Acid 3a

a

Reagents and conditions: (a) benzenesulfonamide, HATU, DIPEA, DMF, 23 h, rt, 66%; (b) DPPA, NEt3, t-BuOH, 5 h, 85 °C, 75%; (c) TFA, CH2Cl2, 5 h, rt, 87%; (d) benzoic acid, DIPEA, HATU, CH2Cl2, 2 h, rt, 64%; (e) benzenesulfonyl chloride, pyridine, −10 °C to rt, 18 h, rt, 57%.

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RESULTS AND DISCUSSION Synthesis. We envisaged the carboxylic acid 3 as a versatile intermediate for the preparation of a variety of classical and nonclassical amide isosteres.23 Amide coupling of thiazolino 2pyridone carboxylic acid 3 with benzenesulfonamide afforded the acyl sulfonamide 4 (Scheme 1). Curtius rearrangement of acid 3 in tert-butyl alcohol gave the BOC-protected amine 5 and following deprotection of 5 provided the desired primary amine 6. Subsequent amide couplings with amine 6 gave the reversed phenylamide 7 and the sulfonamide 8. Preparation of the β-keto amide 9 from 3 afforded a common intermediate for the synthesis of the 1,3-heterocycles isosteres 10−12 (Scheme 2). Condensation of 9 with ammonium acetate under microwave irradiation (MWI) gave the imidazole 10 and cyclodehydration with Burgess or Lawesson’s reagents afforded the oxazole 11 and the thiazole 12, respectively.24,25 The 1,2,4oxadiazole 13 was prepared by amide coupling with benzamide oxime and MWI promoted cyclization,26 while the 1,3,4regioisomer 14 was synthesized by amide coupling with benzhydrazide, followed by Burgess reagent enabled cyclization (Scheme 2).27 The 1,3,4-triazoles 15 and 16 were synthesized from acid 3 using the one-pot method of Castanedo et al., followed by PMB deprotection in TFA.28 Consequent nitration and reduction of 15 gave the C-6 amine substituted analogue 17 (Scheme 3).29 The 1,2,3-triazole 18 was prepared from amine 6 by diazotransfer using imidazole-1-sulfonyl azide,30 followed by Cu(I)-catalyzed azide−alkyne cycloaddition (CuAAC) (Scheme 3). Nitration of 18 gave the nitro intermediate 19 and following reduction with palladium on carbon under a hydrogen atmosphere the C-6 amino substituted 1,2,3-triazole 20. Biological Evaluation. While the effect of antichlamydial compounds is usually measured as growth inhibition,8,9 the anti-infective activity of this class of compounds is only observed after the primary bacterial lifecycle.14 A reinfection

assay was therefore used to evaluate the efficacy of new compounds in reducing the infectivity of Chlamydia progeny. In this assay, HeLa cells infected with serovar LGV-L2 were treated with compound, then after 48 h the cells were lysed and the obtained bacteria used to infect fresh HeLa cells. The level of reinfection was quantified by comparing the number of new inclusion forming units (IFUs) formed by viable infectious bacteria versus cells infected with untreated bacteria. As previously, we initially ascertained the concentration required to reduce the infection level by 95% (referred to as the Reinfect95 assay) at fixed concentrations of 2.5, 1.0, 0.5, and 0.25 μM.14 To ensure anti-infective activity was not due to host cell toxicity, cytotoxicity was evaluated against HeLa host cells at 10 μM in a resazurin assay. The phenylamide 21 and the lead compound 2 provided benchmarks from our previous study which reduced infection by 95% at 2.5 and 0.25 μM, respectively (Table 1). The reversed BOC-protected amine 5 was inactive at the concentrations tested, in agreement with previous SAR data indicating the requirement for an aromatic amide for effective inhibition.14 However, the reversed phenylamide 7 demonstrated comparable efficacy to 21 in attenuating infectivity and no host cell toxicity was observed in the resazurin assay. The sulfonamide and acylsulfonamide analogues 8 and 4 were ineffective in the range of concentrations evaluated. The activity of the reversed phenylamide 7 spurred us onto examining heterocyclic replacements for the amide moiety.23 Imidazole and oxazole analogues 10 and 11, along with the β-keto amide 9, demonstrated comparable efficacy to 21, while the thiazole 12 was less effective. Whereas the 1,2,4-oxadiazole 13 was ineffective at the tested concentrations, the corresponding 1,3,4-regioisomer 14 ablated the infection by ≥95% at 1 μM 9394



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without toxicity, improving on the anti-infective activity of the benchmark phenylamide 21. However, greater improvements were attained by introducing triazole isosteres at the C-3 position. The 1,2,4-triazole 15 attenuated ≥95% infectivity at 0.5 μM, and the corresponding 1,2,3-triazole 18 at 0.25 μM, thereby matching the activity of the lead compound 2 (Table 2). Neither of the two compounds affected HeLa viability at 10 μM. Pursuing the triazole isosteres further, we prepared a series of analogues incorporating the most beneficial features from our previous SAR study.14 Introducing a m-Me phenyl substituent, previously beneficial to the activity of phenylamide 3, had no effect on the activity of the 1,2,4-triazole 16 as measured in the Reinfect95 assay compared to analogue 15. However, a small gain in anti-infective activity was achieved upon introduction of the C-6 amino substituent to the 2-pyridone for triazole regioisomers 17 and 20, which were both equipotent with the previous lead compound 2 in the Reinfect95 assay. We therefore profiled the activity of the most active triazoles 17, 18, and 20 in greater detail by determining their EC50 values against serovars D and LGV-L2. The 1,2,4-triazole 17 proved less potent than the lead compound 2 from our previous study, however, both 1,2,3-triazoles 18 and 20 were exceedingly potent with estimated EC50 ≤ 20 nM against serovar LGV-L2, surpassing 2 (EC50 ≈ 59 nM). Against serovar D, 18 and 20 were marginally less effective with EC50 ≈ 37 and 35 nM, respectively, again improving on the activity of 2 (EC50 ≈ 58 nM). The mutagenic potential of 2, 18, and 20 was assessed according to the Ames test spot-based screening procedure,31 and none of tested compounds exhibited mutagenic potential when tested at 10 μg/spot (Supporting Information, Table S1). We examined the effect of the 18 and 20 alongside the original lead 2 on the growth of key species from the vaginal flora using a disk diffusion assay. None of the compounds inhibited the growth of Staphylococcus aureus, Escherichia coli, Lactobacillus

Scheme 2. Synthesis of 1,3-Heterocycles 10−12 and Oxadiazoles 13 and 14a

a

Reagents and conditions: (a) 2-aminoacetophenone·HCl, HATU, DIPEA, DMF, 19 h, rt, 92%. (b) For 10: NH4OAc, EtOH, MWI at 140 °C, 1 h 45 min, 69%. (c) For 11: Burgess reagent, THF, MWI at 100 °C, 1 h, 24%. (d) For 12: Lawesson’s reagent, THF, MWI at 110 °C, 20 min, 46%. (e) Benzamide oxime, TBTU, DIPEA, DMF, MWI at 190 °C, 5 min, 39%. (f) Benzhydrazide, HATU, DIPEA, THF, rt, 1 h. (g) Burgess reagent, rt, 21 h, 25% over two steps.

Scheme 3. Synthesis of Triazoles 15−18 and 20a

Reagents and conditions: (a) acylamidine·HCl, HATU, DIPEA, DMF, 3.5 h; (b) (4-methoxybenzyl)hydrazine·HCl, AcOH, 80 °C, 18−20 h; (c) TFA, CH2Cl2, 50 °C, 22 h, 15:43%, 16:44% over three steps; (d) NaNO2, TFA, CH2Cl2, O2 atmosphere, rt, 17 h; (e) activated Zn dust, CH2Cl2:AcOH, rt, 27 h, 28% over 2 steps; (f) imidazole-1-sulfonyl azide·H2SO4, CuSO4, K2CO3, MeOH, 72 h, rt; (g) phenylacetylene, CuSO4, sodium ascorbate, rt, 22 h, 26% over 2 steps; (h) NaNO2, TFA, CH2Cl2, O2 atmosphere, rt, 30 h, 37%; (i) Pd/C (10%), H2, THF/MeOH (1:1), rt, 19h, 79%.

a

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Table 1. Exploring C-3 Amide Isosteres

Table 2. Triazolo Analogues

a 95% confidence intervals in parentheses. bData provided from our previous study.14 nd = not determined.

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a

CONCLUSIONS The striking need for new ways to treat infectious diseases has placed an increasing emphasis on identifying new scaffolds which may operate through novel modes of action and hence may be able to evade resistance. In the case of C. trachomatis, the lack of specific treatments forces medics to resort to systemic antibiotics which disrupt the commensal microbiota. In this study, we present the identification of 1,2,3-triazoles as effective amide isosteres for thiazolino 2-pyridone inhibitors of C. trachomatis. The lead compound 20 attenuated C. trachomatis infectivity at low nanomolar concentrations with no discernible toxicity or mutagenic potential, indicating a clear selectivity index. Furthermore, no effect was discernible on the growth of a panel of species from the vaginal microbiota. Triazoles have often proven effective as peptide surrogates20−22 and in this case enabled removal of a potentially hydrolyzable anilide bond while boosting efficacy. The further development of this series shall be reported in due course.

Data provided from our previous study.14

jensenii, and Streptococcus agalactiae or the fungi Candida albicans when tested at 10 μg/mL. We next performed preliminary studies on the mode of action of 18 and 20 alongside 2. We previously developed the fluorescent analogue 22 containing a BODIPY fluorophore, which was found to accumulate within the inclusion (Figure 2).14 Co-treatment of infected cells with compounds 18 and 20 led to reduced accumulation of 22 within the inclusion. This suggests these compounds out-compete 22 with its target and is consistent with previous experiments with 2. Further to this, we examined the effects of 18, 20, and 2 in a time of addition study whereby the compounds were introduced in either the first or second 24 h of the experiment or maintained for the duration of the 48 h infection (Supporting Information, Figure S1). Each compound was most effective when present for the duration of the infection experiment and least effective when administered within 0−24 h. Overall, these preliminary data indicate 2, 18, and 20 share a consistent mode of action affecting a factor which localizes to the bacterial inclusion (Figure 2).

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EXPERIMENTAL SECTION

Biology. Cell culture, C. trachomatis propagation, reinfection assays were performed, and EC50 values determined as described previously (summarized in the Supporting Information).14 Procedures for the resazurin cell toxicity assay, Ames test, bacterial growth experiments, labeling, immunofluorescence, and fluorescent imaging appear in the Supporting Information. Chemistry. General. Unless otherwise stated the reagents and solvents were used as received from commercial suppliers. Pyridine, NEt3, and DIPEA were passed through activated alumina oxide and dried over activated 3 Å molecular sieves before use. Anhydrous reactions were carried out in oven-dried glassware under a nitrogen atmosphere. Microwave reactions were performed using a microwave synthesizer in sealed vessels with temperature monitoring by an internal IR probe. Reaction progress was monitored by TLC and LCMS. TLC was performed on aluminum backed silica gel plates (median pore size 60 Å) and detected with UV light at 254 nm. Flash chromatography was performed using an automated chromatography 9396



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Figure 2. Co-treatment of 2, 20, or 18 with fluorescent compound 22 reduces its accumulation in bacterial inclusions. (a) HeLa cells infected with C. trachomatis were incubated with 0.5 μM of 22 alone or in combination with the same concentration of 20, 18, or 2 as a control. After 48 h, the cells were fixed and observed under confocal microscopy. The different images were processed using the same parameters. (b) Images were analyzed by ImageJ software,33 and the fluorescent intensity of each inclusion measured and plotted into a graph. For each condition, 40 inclusions were analyzed and the data shown is representative from at least two independent experiments. *** indicates p < 0.05 and statistical significance using a nonparametric one-way ANOVA test. (c) Structure of 22. system on silica gel with average particle diameter 50 μM (range 40− 65 μM, pore diameter 53 Å) and detection at 254 nm; eluents are given in brackets. HPLC was performed with a Nucleodur C18 HTec column (25 cm × 21.5 mm; particle size 5 μM) and flow rate of 18 mL/min. 1H and 13C NMR spectra were recorded on a 400 or 600 MHz spectrometer at 298 K and calibrated by using the residual peak of the solvent as the internal standard (CDCl3, δH = 7.26 ppm, δC = 77.16 ppm; DMSO-d6, δH = 2.50 ppm, δC = 39.52 ppm). HRMS was performed on a micrOTOF II with ESI-TOF (ESI+) and sodium formate for calibration or a Agilent 1290 binary LC system connected to a Agilent 6230 Accurate-Mass TOF LC/MS (ESI+), calibrated with Agilent G1969-85001 ES-TOF reference mix containing ammonium trifluoroacetate, purine, and hexakis(1H,1H,3H-tetrafluoropropoxy)phosphazine in 9:1 MeCN:H2O. All compounds evaluated in biological assays were ≥95% pure and were assessed by LC-MS using an Agilent 1290 Infinity binary LC System and an Agilent 6150 quadrapole LC-MS with UV detection at 210 and 254 nM. 3 was prepared as previously described.14 Procedures. tert-Butyl(8-cyclopropyl-7-(naphthalen-1-ylmethyl)5-oxo-2,3-dihydro-[1,3]thiazolo[3,2-a]pyridin-3-yl)carbamate (5). The carboxylic acid 3 (150 mg, 0.397 mmol) was dissolved in anhydrous t-BuOH (1.99 mL) under an inert atmosphere and NEt3 (67 μL, 0.437 mmol) and DPPA (94 μL, 0.437 mmol) added. The reaction mixture was heated at 85 °C for 2 h, after which additional NEt3 (12 μL, 0.087 mmol) and DPPA (17 μL, 0.079 mmol) were added and the reaction mixture heated for a further 3 h. After cooling to room temperature, the solvent was removed under reduced pressure. The crude residue was dissolved in CH2Cl2 (15 mL), washed successively with aqueous NaOH (10% w/v), H2O, and brine (10 mL each), dried (Na2SO4), and concentrated under reduced pressure. Purification by flash chromatography (SiO2; 10−100% EtOAc in heptane) afforded the product as a white solid (134 mg, 75%). 1H NMR (400 MHz, CDCl3) δ 0.67−0.80 (m, 2H), 0.85−1.02 (m, 2H), 1.43 (s, 9H), 1.58−1.69 (m, 1H), 3.31−3.43 (m, 1H), 3.55−3.62 (m, 1H), 4.34−4.45 (m, 2H), 5.20−5.28 (m, 1H), 5.69 (s, 1H), 6.49−6.55 (m, 1H), 7.23−7.27 (m, 1H), 7.31−7.50 (m, 3H), 7.74−7.80 (m, 2H), 7.84−7.89 (m, 1H). 13C NMR (151 MHz, CDCl3) δ 7.7, 8.0, 11.2, 28.4, 35.6, 36.3, 68.6, 81.0, 113.5, 115.9, 123.9, 125.7, 125.9, 126.4, 127.7, 127.9, 129.0, 132.1, 134.1, 134.1, 146.8, 154.2, 156.9, 160.7. HRMS (ESI+) (m/z): [M + Na]+ calcd for C26H28N2NaO3S 471.1713, found 471.1714. 3-Amino-8-cyclopropyl-7-(naphthalen-1-ylmethyl)-2,3-dihydro[1,3]thiazolo[3,2-a]pyridin-5-one (6). 5 was suspended in 30% TFA/ CH2Cl2 (v/v, 2 mL) and stirred at rt for 5 h. The solvent was removed

under reduced pressure, the residue dissolved in CH2Cl2 (15 mL), washed with saturated aqueous NaHCO3 solution (2 × 10 mL), dried (Na2SO4), and the solvent removed under reduced pressure. Purification by flash chromatography (SiO2; 0−20% MeOH in EtOAc with 2% NEt3) afforded after freeze-drying from H2O:MeCN (4:1) the amine as a white solid (91 mg, 87%). 1H NMR (400 MHz, CDCl3) δ 0.67−0.79 (m, 2H), 0.87−1.02 (m, 2H), 1.57−1.68 (m, 1H), 3.11 (dd, J = 1.4, 11.9 Hz, 1H), 3.60 (dd, J = 7.4, 11.9 Hz, 1H), 4.37 (d, J = 17.2, 1H), 4.43 (d, J = 17.2 Hz, 1H), 5.68−5.70 (m, 1H), 5.91 (dd, J = 1.4, 7.4 Hz, 1H), 7.23−7.28 (m, 1H), 7.38−7.50 (m, 3H), 7.74−7.80 (m, 2H), 7.84−7.89 (m, 1H). 13C NMR (100 MHz, CDCl3) δ 7.6, 7.9, 11.0, 35.2, 36.3, 72.2, 113.3, 115.5, 123.9, 125.6, 125.8, 126.3, 127.7, 127.8, 129.0, 132.1, 134.1, 134.2, 145.8, 156.7, 161.7. HRMS (ESI+) (m/z): [M + H]+ calcd for C21H21N2OS 349.1369, found 349.1379. 8-Cyclopropyl-7-(naphthalen-1-ylmethyl)-3-(4-phenyl-1,2,3-triazol-1-yl)-2,3-dihydro-[1,3]thiazolo[3,2-a]pyridin-5-one (18). The title compound was prepared by adaptation of the methodology developed by Goddard-Borger et al.,30 followed directly by adaption of the CuAAc procedure reported by Bengtsson et al.32 Imidazole-1sulfonyl azide hydrogen sulfate (59.8 mg, 0.220 mmol) was added to a solution of 6 (48.0 mg, 0.138 mmol), K2CO3 (36.2 mg, 0.262 mmol), and CuSO4 (3.3 mg, 0.021 mmol) in anhydrous MeOH (0.69 mL). The reaction mixture was heated at 35 °C for 22.5 h, after which additional K2CO3 (4.8 mg, 0.035 mmol) and imidazole-1-sulfonyl azide hydrogen sulfate (4.7 mg, 0.017 mmol) were added. After 48 h stirring at rt, the reaction mixture was diluted with brine (∼2 mL) and extracted with EtOAc (3 × 5 mL), the combined organic extracts washed with brine (5 mL) and dried (Na2SO4), and the solvent removed under reduced pressure. The resultant residue was dissolved in DMF/H2O (1:1, 1.38 mL) and phenylacetylene (30.3 μL, 0.276 mmol), CuSO4 (2.2 mg, 0.014 mmol), and sodium ascorbate (5.5 mg, 0.028 mmol) added and stirred at rt for 22 h. The reaction mixture was diluted with EtOAc (15 mL) and washed successively with saturated aqueous NaHCO3 solution, H2O, and brine (10 mL each), dried (Na2SO4), and the solvent removed under reduced pressure. Purification by flash chromatography (SiO2, 0−85% EtOAc in heptane), followed by HPLC (MeCN/H2O with 0.005% formic acid, 30−100% for 40 min; tR = 30.46 min at 214 nM) and freezedrying from H2O:MeCN (3:1) afforded the triazole as a white solid (17 mg, 26%). 1H NMR (400 MHz, CDCl3) δ 0.71−0.86 (m, 2H), 0.92−1.08 (m, 2H), 1.66−1.75 (m, 1H), 3.95 (dd, J = 7.2, 12.5 Hz, 1H), 4.28 (d, J = 12.5 Hz, 1H), 4.35 (d, J = 17.5 Hz, 1H), 4.47 (d, J = 17.5 Hz, 1H), 5.68 (s, 1H), 7.24−7.34 (m, 3H), 7.36−7.50 (m, 5H), 9397



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7.71−7.82 (m, 4H), 7.84−7.90 (m, 1H), 8.18 (s, 1H). 13C NMR (100 MHz, CDCl3) δ 7.4, 8.1, 11.4, 33.2, 36.5, 72.6, 114.4, 115.4, 121.1, 123.9, 125.7, 125.95, 125.96, 126.5, 127.9, 128.0, 128.4, 128.9, 129.0, 130.2, 132.0, 133.6, 134.1, 147.3, 147.9, 158.4, 161.0. HRMS (ESI+) (m/z): [M + Na]+ calcd for C29H24NaN4OS 499.1568, found 499.1562. 8-Cyclopropyl-7-(naphthalen-1-ylmethyl)-6-nitro-3-(4-phenyl1,2,3-triazol-1-yl)-2,3-dihydro-[1,3]thiazolo[3,2-a]pyridin-5-one (19). The title compound was prepared by adaptation of the procedure reported by Åberg et al.29 TFA (0.50 mL) was added dropwise to a solution of 18 (183 mg, 0.384 mmol) and NaNO2 (27.4 mg, 0.397 mmol) in CH2Cl2 (5 mL) at rt under O2 atmosphere. Additional NaNO2 was added portionwise after 3 h (3.4 mg, 0.049 mmol), 22 h (6.5 mg, 0.094 mmol), 25 h (6.8 mg, 0.099 mmol), and 27 h (3.9 mg, 0.057 mmol) until the reaction reached completion by LC/MS. The reaction mixture was quenched after 30 h by addition of saturated aqueous NaHCO3 (10 mL), extracted with EtOAc (3 × 30 mL), the combined organic extracts dried (Na2SO4), and the solvent removed under reduced pressure. Purification by flash chromatography (SiO2, 10−100% EtOAc in heptane) afforded the nitro intermediate as a yellow solid (74 mg, 0.142 mmol, 37%). 1H NMR (600 MHz, DMSOd6) δ 0.63−0.75 (m, 4H), 1.30−1.37 (m, 1H), 3.87 (d, J = 12.9 Hz, 1H), 4.28 (dd, J = 7.8, 12.9 Hz, 1H), 4.42 (d, J = 16.4 Hz, 1H), 4.75 (d, J = 16.4 Hz, 1H), 7.00 (d, J = 7.0 Hz, 1H), 7.35−7.40 (m, 1H), 7.41−7.50 (m, 3H), 7.56−7.60 (m, 1H) 7.61−7.68 (m, 2H), 7.84 (d, J = 8.3 Hz, 1H), 7.88−7.92 (m, 2H), 7.95−7.99 (m, 1H), 8.20 (d, J = 8.3 Hz, 1H), 8.79 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ 6.8, 7.6, 11.1, 31.1, 35.0, 74.0, 111.8, 121.3, 123.2, 124.3, 125.4, 125.5, 126.1, 126.6, 127.3, 128.3, 128.7, 129.0, 130.1, 131.2, 132.5, 133.3, 138.7, 146.2, 148.6, 152.3, 154.4. HRMS (ESI+) (m/z): [M + Na]+ calcd for C29H23N5NaO3S 544.1413, found 544.1416. 6-Amino-8-Cyclopropyl-7-(naphthalen-1-ylmethyl)-3-(4-phenyl1,2,3-triazol-1-yl)-2,3-dihydro-[1,3]thiazolo[3,2-a]pyridin-5-one (20). A solution of the nitro intermediate 19 (61.6 mg, 0.118 mmol) and Pd/C (10% wt loading, 27 mg, 0.025 mmol) in THF/MeOH (1:1, 16 mL) were stirred under an atmosphere of H2 for 19 h. The reaction mixture was filtered through Celite, and the solvent was removed under reduced pressure. Purification by flash chromatography (SiO2, 0−100% EtOAc in DCM) with subsequent freeze-drying H2O:MeCN (5:1) afforded the product as an off-white solid (45.7 mg, 0.093 mmol, 79%). 1H NMR (600 MHz, CDCl3) δ 0.52−0.57 (m, 1H), 0.60−0.66 (m, 1H), 0.71−0.80 (m, 2H), 1.55−1.62 (m, 1H), 3.83 (br s, 2H), 4.15 (dd, J = 6.9, 12.6 Hz, 1H), 4.15 (d, J = 12.6 Hz, 1H), 4.46 (d, J = 16.6 Hz, 1H), 4.60 (d, J = 16.6 Hz, 1H), 6.93−6.96 (m, 1H), 7.30− 7.36 (m, 2H), 7.41−7.44 (m, 2H), 7.50 (d, J = 6.8 Hz, 1H), 7.54−7.58 (m, 1H), 7.60−7.63 (m, 1H), 7.76 (d, J = 8.3 Hz, 1H), 7.84−7.87 (m, 2H), 7.91 (d, J = 7.9 Hz, 1H), 8.17 (d, J = 8.4 Hz, 1H), 8.19 (s, 1H). 13 C NMR (151 MHz, CDCl3) δ 7.0, 7.3, 12.0, 31.1, 33.8, 73.3, 115.9, 120.4, 123.2, 123.4, 125.8, 126.0, 126.1, 126.5, 127.7, 128.5, 129.0, 129.1, 129.4, 130.3, 131.9, 132.2, 132.3, 132.8, 134.1, 148.0, 156.6. HRMS (ESI+) (m/z): [M + Na]+ calcd for C29H25N5NaOS 514.1671, found 514.1669.

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AUTHOR INFORMATION

Corresponding Authors

*For F.A.: phone, +46 90 786 6925; E-mail, fredrik.almqvist@ umu.se. *For S.B: phone, +46 90 785 6726; E-mail, sven.bergstrom@ umu.se. ORCID

Fredrik Almqvist: 0000-0003-4646-0216 Present Addresses #

For J.A.D.G.: ReViral, NETpark Incubator, Thomas Wright Way, Sedgefield TS21 3FD, United Kingdom. ∇ For K.S.K.: Department of Chemistry, Mannam Memorial NSS College (affiliated to The University of Kerala), Kottiyam, Kerala 691571, India. Author Contributions ○

J.A.D.G. and M.K. contributed equally.

Notes

The authors declare the following competing financial interest(s): S.B. and F.A. are co-owners of Quretech Bio AB.

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ACKNOWLEDGMENTS We acknowledge funding from the Swedish Research Council (F.A. and S.B.), the Knut and Alice Wallenberg Foundation (F.A. and S.B.), the Göran Gustafsson Foundation (F.A.) and the Swedish Foundation for Strategic Research (F.A.). K.S.K. thanks the JC Kempe Foundation for funding.

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ABBREVIATIONS USED BODIPY, dipyrromethene boron difluoride; C. trachomatis, Chlamydia trachomatis; DIPEA, N,N-diisopropylethylamine; DPPA, diphenylphosphorylazide; EB, elementary body; G6-P, glucose 6-phosphate; HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate); IFU, inclusion forming unit; MWI, microwave irradiation; RB, reticulate body; TBTU, O-(benzotriazol1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.7b00716. Time of addition studies, mutagenic potential, procedures for resazurin cell toxicity assay, Ames test, bacterial growth experiments, labeling, immunofluorescence, and fluorescent imaging; synthetic procedures and characterization of all other compounds (PDF) Molecular formula strings (CSV) 9398



Brief Article

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Thiazolino 2-Pyridone Amide Isosteres As Inhibitors of Chlamydia

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