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Exploiting the Chromone Scaffold for the Development of Inhibitors of Corticosteroid Biosynthesis Silvia Gobbi,*,† Qingzhong Hu,‡,§ Christina Zimmer,‡ Matthias Engel,‡ Federica Belluti,† Angela Rampa,† Rolf W. Hartmann,‡ and Alessandra Bisi*,† †

Department of Pharmacy and Biotechnology, University of Bologna, Via Belmeloro, 6, I-40126 Bologna, Italy Pharmaceutical and Medicinal Chemistry, Saarland University and Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Universitätscampus E8 1, 66123 Saarbrücken, Germany

‡

S Supporting Information *

ABSTRACT: The inhibition of corticosteroid biosynthesis could be considered as an emerging strategy to reduce their abnormally high levels, and in this framework CYP11B1 and CYP11B2 represent the most promising targets. In continuing our studies on flavonoid-like scaffolds as privileged structures in medicinal chemistry, in this paper we describe a small library of pyridyl- and imidazolylmethylchromones as potential inhibitors of these enzymes. Testing results proved that position 3 of the chromone scaffold is the most favorable for the introduction of the heme-coordinating heterocycles and, among them, the 4-imidazolyl moiety is the most convenient for the interaction with the heme iron of the selected cytochromes. A low nanomolar inhibitor of CYP11B1 (5c) was obtained, endowed with reasonable selectivity toward CYP11B2 and able to better discriminate with respect to CYP17 and CYP19.

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INTRODUCTION Steroid hormones are known to play essential roles in the regulation of crucial physiological processes, but their abnormally increased levels have proved to be involved in the development of several hormone-related diseases. In particular, cortisol overproduction (hypercortisolism) can lead to diseases such as Cushing’s syndrome,1 while aldosterone is known to be involved in several cardiac diseases, such as congestive heart failure or myocardial fibrosis.2 Although the recent advances in the elucidation of the mechanisms underlying Cushing’s syndrome have greatly improved its management, the disease is still associated with significant morbidity and mortality. Available drugs act by reducing ACTH secretion or by competing with cortisol at the receptor level and showed poor clinical efficacy when used as sole treatment. Moreover, the interference of a cutaneous overexpression of cortisol with physiological wound healing recently emerged, causing prolonged distress and increased infection risk. Chronic skin wounds usually develop as a comorbid condition with diabetes and obesity, or because of a trauma, and could be related to an impaired inflammatory response. Different therapeutic strategies were investigated to treat this very costly and challenging medical problem, such as skin substitutes and stem cell applications, but the need for a novel therapeutic approach appears a very urgent task.3 On the other hand, the involvement of aldosterone in the development of arrhythmias, hypertension, and congestive heart failure is now definitely clear. Blocking the actions of this hormone using the mineralcorticoid receptor (MR) antagonists spironolactone © XXXX American Chemical Society

and eplerenone proved to reduce mortality and hospitalization time in several patients, although severe side effects, in particular hyperkalemia, could frequently be observed.4 In this framework, an alternative promising strategy to reduce the abnormal increase in cortisol and aldosterone plasma levels could be the inhibition of their formation, as already successfully investigated for aromatase (CYP19)5 and 17αhydroxylase/C17,20-lyase (CYP17)6 inhibitors for the treatment of hormone-dependent breast and prostate cancer, respectively. Indeed, the search for inhibitors of cytochrome P450 (CYP) enzymes catalyzing the closely related biosyntheses of the different classes of steroid hormones has gained researchers’ attention and has been extensively exploited during the past decades. 11β-Hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) are enzyme isoforms, sharing a high degree of sequence homology, that respectively catalyze key steps in the biosyntheses of cortisol and aldosterone and represent validated targets on which to direct medicinal chemistry efforts. They are mainly localized in the adrenal cortex, and while CYP11B1 can be found in the zona fasciculata, where cortisol is produced, CYP11B2 is expressed in the zona glomerulosa, site of aldosterone biosynthesis. The mature enzymes have similar molecular weight, are bound to the inner mitochondrial membrane, and only differ by 30 residues, all of which are located away from the substrate recognition site. Received: October 14, 2015

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DOI: 10.1021/acs.jmedchem.5b01609 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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pyridyl- and imidazolylmethylchromone derivatives (scaffolds A and B, Figure 1), designed on the basis of the biological data of those previously described compounds and further exploiting the idea of flavonoid-like oxaheterocyclic scaffolds as privileged structures in medicinal chemistry.12,13 In our long-lasting experience, oxaheterocycles related to flavonoids structures proved to be valuable scaffolds, which could be properly decorated in order to target diverse biological counterparts involved in a wide range of pathologies, and this peculiar feature led to the development of several potent and selective compounds.14 The aim of this work is to prove the versatility of this approach by designing molecules characterized by flavonoid-like scaffolds able to target different related steroidogenic cytochromes P450. To reach this goal, a pharmacophore containing an aromatic nitrogen atom is needed to irreversibly bind the heme iron and compete with natural substrates, as proved by observed UV−vis difference spectra15 and cocrystals,16,17 clearly demonstrating complexation of the heme iron with such aromatic nitrogens. The ability to discriminate between different related CYPs should then be investigated focusing on the selected scaffold and the pattern of substitution introduced. Indeed, in a previous paper18 flavonebased compounds (Figure 1) that we had first designed as CYP19 ligands19 were found to be inhibitors of CYP11B1 and CYP11B2, and in a recent study20 we identified structurally related xanthenone derivatives as potent and selective ligands for these enzymes, proving the possibility of a fine modulation of the activity for molecules based on this scaffold. Moreover,

Despite this very high degree of homology, making it difficult to identify possible peculiar features of these two enzymes that would allow specifically targeting one of them, over the past years different series of potent and selective inhibitors have been developed,7,8 among which are pyridyl- or imidazolylmethylenetetrahydronaphthalene9,10 and naphthalene derivatives11 (Figure 1). In this paper, we report on a small library of

Figure 1. Design strategy for the new compounds.

Table 1. Structures and Inhibition of CYP11B1 and CYP11B2 for the Studied Compounds

IC50 (nM)a compd

scaffold

R

Het

CYP11B1b

CYP11B2c

SFd

1a 1b 1c 2a 2b 2c 3a 3b 3c 4a 4b 4c 5a 5b 5c 6a 6b 6c 7a 7b 7c

A A A B B B A A A B B B A A A A A A B B B

H OCH3 F H OCH3 F H OCH3 F H OCH3 F H OCH3 F H OCH3 F H OCH3 F

4-pyridine 4-pyridine 4-pyridine 4-pyridine 4-pyridine 4-pyridine 1-imidazole 1-imidazole 1-imidazole 1-imidazole 1-imidazole 1-imidazole 4-imidazole 4-imidazole 4-imidazole 3-pyridine 3-pyridine 3-pyridine 3-pyridine 3-pyridine 3-pyridine

88.2 612.3 58.1 222.5 3598.4 82.7 179.6 372.1 86.9 253.0 1600.8 326.9 29.5 108.9 9.7 >10000 >10000 >10000 >10000 >10000 >10000

953.6 3270.6 317.3 1235.3 >5000 643.2 521.5 767.9 213.8 499.5 2268.4 619.8 121.2 1066.1 94.9 >10000 >10000 >10000 >10000 >10000 >10000

10.8 5 5.3 5.6 7.8 2.9 2.1 2.5 2.0 1.4 1.9 4.1 9.8 9.8

Mean value of at least three experiments, relative standard deviation less than 25%. bHamster fibroblasts expressing human CYP11B1; substrate, deoxycorticosterone 100 nM. cHamster fibroblasts expressing human CYP11B2; substrate, deoxycorticosterone 100 nM. dSelectivity factor = IC50(CYP11B2)/IC50(CYP11B1).

a

B



Article

Scheme 1a

coumarins were also reported in the literature as suitable scaffolds for the design of steroidogenic CYP inhibitors.21,22 In our design hypothesis the new compounds, lacking the additional aromatic ring ortho to the heme-binding moiety when compared to our previously reported flavones, could be considered as somewhat simplified and less bulky structures, for which the impact of the different heme-coordinating groups (3-, 4-pyridine, or imidazole) on potency and selectivity would be evaluated. Again, as previously demonstrated with different series of flavonoid-based CYP inhibitors,23,24 the carbonyl moiety of the chromone scaffold, compared with naphthalene, could establish an additional H-bond interaction with the enzymes. The nitrogen-containing heterocyclic function, which is considered a key feature due to its ability to interact with the heme-iron of the cytochromes, was inserted in position 3 or moved to position 2 on the chromone central core. In addition, the pyridine or imidazole nitrogens were differently oriented in order to establish the best positioning for the heme coordinating atom to specifically bind to CYP11B1 and/or CYP11B2, since it has been demonstrated that it also exhibits significant impact on selectivity among steroidogenic CYP enzymes.25,26 Moreover, the pattern of substitution of the chromone scaffold could also possibly discriminate among the different steroidogenic cytochromes. To this aim, fluorine and methoxy groups were introduced in position 7 in each series, in accordance with the above-mentioned previously reported series of potent inhibitors, to evaluate the influence of these substituents on activity and selectivity toward the two enzymes. In particular, the fluorine atom is receiving increasing interest in medicinal chemistry and, maybe because of its small size and high electronegativity, its introduction into potential drugs or diagnostics could improve pharmacokinetic and physicochemical properties, such as metabolic stability or membrane permeation.27 The new compounds 1−7 a−c, whose structures are reported in Table 1, were tested for inhibition of CYP11B1 and CYP11B2, and the most interesting derivatives were further evaluated for inhibition of CYP19 and CYP17 in order to assess their selectivity among structurally related steroidogenic enzymes. In addition, the potential cytotoxicity of the most active compounds, 1c, 5a, and 5c, was evaluated to investigate the safety profile of these molecules.

Reagents and conditions: (i) selected aldehyde, piperidine, 150 °C, 3 h.

a

Scheme 2.

a

a For synthesis of 8b (top half of scheme), reagents and conditions: (i) trifluoromethanesulfonic acid, 80 °C, 30 min; (ii) NaOH, rt, 2h; (iii) K2CO3, (CH3)2SO4, reflux, 7 h. For synthesis of 8c (bottom half of scheme), reagents and conditions: (i) Triton B, reflux, 23 h; (ii) HCl, reflux, 14 h; (iii) toluene, SOCl2, reflux, 1.5 h; (iv) CHCl3, trifluoromethanesulfonic acid, −65 °C, then rt, 2 h.

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CHEMISTRY 3-Substituted chromones 1a−c, 5a−c, and 6a−c were obtained by reacting the appropriate chromanone (8a−c) with piperidine and 4-pyridine-, 4-imidazole-, or 3-pyridinecarboxaldehyde, respectively (Scheme 1). While the unsubstituted chromanone 8a was bought from commercial suppliers, 7-methoxychromanone 8b28 was prepared by reaction of resorcinol with trifluoromethanesulfonic acid to give intermediate 9, followed by ring closure with NaOH and subsequent methylation of the hydroxy group of 10 with dimethyl sulfate (Scheme 2), whereas 7-fluorochromanone 8c29 was synthesized starting from acrylonitrile and 3-fluorophenol via reaction with Triton B, followed by hydrolysis of the nitrile 11 to carboxylic derivative 12 and ring closure with thionyl chloride and then trifluoromethanesulfonic acid (Scheme 2). 1-Imidazolyl derivatives 3a−c were synthesized by reacting the appropriate 2hydroxypropiophenone (13a−c) and N,N-dimethylformamide dimethyl acetal to give the corresponding 3-methylchromones 14a−c, which were then brominated with NBS and reacted with imidazole (Scheme 3). Again, unsubstituted 2-hydrox-

ypropiophenone 13a was commercially available, while 13b19 and 13c30 were prepared following literature procedures. For the synthesis of 2-pyridinylmethyl derivatives 2a−c and 7a−c, the commercially available acetophenones were reacted with NaH and the appropriate ethyl pyridylacetate. For the preparation of 2-imidazole derivatives 4a−c the same acetophenones were treated with ethyl 2-ethoxyacetate to give the 2-ethoxymethylchromones 16a−c. The 2-bromomethyl derivatives 17a−c were obtained by treatment with HBr 48% and then reacted with imidazole to give the final compounds (Scheme 4).

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RESULTS AND DISCUSSION The new compounds were tested for inhibition of both CYP11B1 and CYP11B2. The results, collected in Table 1, show that most compounds proved to be potent inhibitors of CYP11B1, although to a different extent with activities ranging C



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Scheme 3a

structure (PDB code 4DVQ).32 These two compounds exhibited a different binding mode from that of the natural substrate deoxycorticosterone and other CYP11B2 inhibitors previously identified.33,34 Rather than pointing to the β2-sheet, compounds 1c and 2c lean on the I-helix (Figure 2), which is

Reagents and conditions: (i) DMF, 120 °C, 2 h; (ii) NBS, benzoyl peroxide, CCl4, reflux, 6 h; (iii) imidazole, CH3CN, N2, reflux, 6 h.

a

Scheme 4a

Figure 2. Docking of compounds 1c (magenta) and 2c (cyan) in the CYP11B2 crystal structure (PDB code 4DVQ).

rare for CYP11B2 inhibitors but is more commonly observed for CYP17 inhibitors.35,36 Not surprisingly, the pyridine nitrogen atoms coordinate with the heme iron in a nearly perpendicular manner. The aromatic cores of both compounds form a net of π−π interactions with Phe130, Phe231, Trp116, Trp260, as well as the conjugated system of backbones of the Ihelix in parallel or perpendicular ways. Carbonyl oxygen atom in compound 1c interacts with the carboxylic acid side chain of Asp317, while the other oxygen atom at the opposite side of the core forms a hydrogen bond with Glu310. These hydrogen bonds further fix the molecule in this orientation and could also significantly contribute to the binding affinity. In contrast, in the case of compound 2c, although the hydrogen bond between carbonyl oxygen and Asp317 is sustained, the other one is intercepted due to a different substituting position of the hemecoordinating pyridine on the chromone core, and the lack of one hydrogen bond could be responsible for the potency drop compared to compound 1c. Similarly, the replacement of 4pyridine in these potent inhibitors to 3-pyridine would largely weaken or even totally annihilate the interactions described above and finally lead to the loss of potency. The same compounds were also docked into a recently developed CYP11B1 homology model,37 since no crystal structure of this isoform is available yet. The docking of the two compounds (for PDB coordinates, see Supporting Information) revealed very similar binding poses and interactions, which is actually not a surprise due to the fact that out of 503 AA residues, 473 are identical in the sequences of both enzymes and all 30 different AA residues are located outside the active site. Although minor conformational differences were observed,37 they did not seem to significantly impact the binding of compounds 1c and 2c. Taking into account the substitution in position 7, the introduction of a fluorine atom (1c, 2c) led, in these series, to a better inhibitor of both enzymes with respect to the unsubstituted compound (1a, 2a), and for CYP11B2 this could probably result from its interaction with Arg120 (Figure 2). On the contrary, the presence of a methoxy group (1b, 2b) negatively influenced activity, as already pointed out for the above-mentioned flavones and in accordance with tetrahydro-

Reagents and conditions: (i) NaH, pyridine, 100 °C, 45 min; (ii) CH3COOH, 48% HBr, reflux, 1 h; (iii) imidazole, CH3CN, N2, reflux, 6 h. a

from low micromolar to low nanomolar, and weaker inhibitors of CYP11B2. Pyridine Derivatives. For the pyridine derivatives, the orientation of the nitrogen atom, resulting from the positioning of the heterocyclic ring on the side chain, proved to play a crucial role in the activity: none of the 3-pyridine derivatives (6a−c, 7a−c) showed appreciable inhibition of the two enzymes, independent of the position of the side chain on the chromone scaffold. This finding is not in line with the results obtained with tetrahydronaphthalene compounds,9 for which the 3-pyridine-based derivatives proved to be more potent inhibitors with respect to 4-pyridine analogues. This could indicate a difference in the interaction with the active site of the enzymes for these classes of compounds, possibly due to the formation of the H-bond by the carbonyl moiety. On the other hand, in the 4-pyridine series a general decrease in potency could be noted when shifting the chain from position 3 (1a−c, scaffold A, Table 1) to 2 (2a−c, scaffold B, Table 1) on the chromone core. The 3-substituted derivatives could maybe be considered more similar to the naphthalene compounds used as basis for our design hypothesis regarding the spatial relationship between the core and the side chain and are structurally more closely related to previously reported flavones, designed for CYP19 inhibition, that proved to be endowed with remarkable CYP11B inhibition potency.18 Moreover, for different series of oxaheterocyclic compounds previously reported by us31,24 the positioning of the side chain containing the heme-binding moiety with respect to the ketone function on the scaffold was found to play a key role in the activity toward CYP enzymes. To clarify these issues, the two most potent compounds of their kind, 1c and 2c, were docked into the CYP11B2 crystal D



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naphthalene compounds. In particular 1c, carrying a fluorine atom, showed the highest activity of the pyridine series on CYP11B1 (IC50 = 58.1 nM), even if its selectivity with respect to CYP11B2 was limited (SF = 5.3). More selective proved to be the unsubstituted 1a and the fluorine-containing 2c (SF = 10.8 and SF = 7.8, respectively), still maintaining the same activity range on CYP11B1. Imidazole Derivatives. The replacement of pyridine with imidazole gave interesting results. Among 1-imidazolyl derivatives (3a−c, 4a−c), a general small increase in the inhibition of CYP11B2 could be noted with respect to pyridine derivatives (1a−c, 2a−c), even if activities remained in the submicromolar range, whereas a slight decrease in affinity for CYP11B1 was observed. An increase in activity could again be seen with the insertion of the heterocyclic function in position 3 with respect to position 2. For substituents in position 7, the same pattern of variations in potency seen with pyridine derivatives was generally confirmed, the fluorine-substituted 3c being the most active compound of these series with IC50 = 86.9 nM for CYP11B1 inhibition. Unfortunately, the selectivity of these compounds proved to be quite low. Considering that position 3 of the scaffold proved to be the most favorable for substitution, a corresponding series of 4imidazolyl derivatives (5a−c) was synthesized in order to establish the most appropriate position for the coordinating nitrogen, as investigated for the pyridine nucleus. This modification led to a significant increase in potency with respect to the corresponding 1-imidazolyl series 3a−c, again confirming the extreme importance of the correct positioning of the heme-binding heterocycle. Moreover, this structural adjustment might result in the establishment of an intramolecular hydrogen bond between the imidazole NH and the carbonyl oxygen in the chromone core, which would lock the molecule in a favorable conformation for target binding. Remarkably, these imidazolyl derivatives showed higher inhibition of both CYP11B1 and -B2 with respect to the corresponding pyridine derivatives 1a−c, and this is again in contrast to results obtained with the different series of naphthalene compounds, for which the substitution of pyridine with an imidazole ring was detrimental for activity. Of particular interest for CYP11B1 inhibition proved to be the unsubstituted 5a (IC50 = 29.5 nM) and the 7-fluorinated derivative 5c (IC50 = 9.7 nM), confirming that this substitution could improve activity. To our surprise, in this series both methoxy and fluorine proved to enhance selectivity for CYP11B1 with respect to the unsubstituted compound. Indeed, 5c was the most potent CYP11B1 inhibitor identified in this study, endowed with reasonable selectivity with respect to CYP11B2 (SF = 9.8). For a broader evaluation of the selectivity issue, considering the relatively close resemblance between different steroidogenic CYP enzymes, the most interesting compounds showing high potency and/or reasonable selectivity over CYP11B2 (1a, 1c, 2c, 5b, 5c) were also tested for their activity on aromatase (CYP19) and 17α-hydroxylase/C17,20-lyase (CYP17). The studied compounds showed either IC50 values in the micromolar range or no significant inhibition of both enzymes (see Table 2). In order to investigate the safety profile of these new corticosteroid biosynthesis inhibitors, the most active compounds, 1c, 5a, and 5c, were tested for their potential cytotoxicity. In particular, cell viability in HEK293 cells was

Table 2. Inhibition of Human CYP19 and CYP17 for Selected Compounds IC50 (μM)a compd

CYP19

1a 1c 2c 5b 5c

6.1 5.0 >10 7.9 3.1

b

CYP17c 4.8 6.6 ≫10 ≫10 ≫10

a

Mean value of at least three experiments, relative standard deviation less than 25% bHuman placental CYP19; substrate, androstenedione, 500 nM. cE. coli expressing human CYP17; substrate, progesterone, 25 μM.

determined using an MTT assay. None of the tested compounds showed appreciable toxicity, resulting in 95− 100% of control cell viability at all concentrations used, up to 100-fold the IC50 of the most active derivative. These results add new insight into the biological profile of this xanthonebased class of noncytotoxic potential agents for the treatment of hormone-related diseases.

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CONCLUSIONS A small library of chromones was designed starting from naphthalene-based molecules and previously reported flavones, further exploiting the idea of flavonoid-like scaffolds as privileged structures, following a previous study on xanthenone-based molecules as potent ligands for CYP11B1−2. In light of the obtained results, it could be pointed out that, as expected, position 3 of the chromone scaffold is the most favorable for substitution with the heme-coordinating chain and, among nitrogen-containing heterocycles, the imidazole ring, and in particular the 4-imidazolyl moiety is the most appropriate for a correct coordination of the heme iron. Indeed, this previously unexplored orientation of the imidazole ring, which could lead to the formation of an intramolecular H-bond, seemed to be particularly favorable for the interaction of this series of compounds within the CYP11B1 active site. For substitution in position 7, the small fluorine atom generally conferred higher activity with respect to the hydrogen, while the methoxy group seemed to influence selectivity to variable extent. In conclusion, the appropriate pattern of substitution on the oxaheterocyclic scaffold proved to be crucial for a correct targeting of the new synthesized series of compounds and the consequent fine-tuning of their activities. The implementation of this approach within this project allowed us to obtain molecules that, while bearing a nitrogen-containing heterocycle as common pharmacophore for the appropriate coordination of the heme iron of all the considered CYPs, are able to discriminate among them. Moreover, compounds proved to be devoid of toxicity when tested on healthy human cells. A potent inhibitor of CYP11B1 was obtained (5c, IC50 = 9.7 nM, SF = 9.8), active in the low nanomolar range, endowed with reasonable selectivity toward the related CYP11B2 and considerably lower activity toward CYP17 and CYP19.

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EXPERIMENTAL SECTION

Chemistry. General Methods. Starting materials, unless otherwise specified, were used as high-grade commercial products. Solvents were of analytical grade. Melting points were determined in open glass capillaries using a Büchi apparatus and are uncorrected. 1H and 13C E



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NMR spectra were recorded on a Varian Gemini 400 spectrometer in CDCl3 solutions unless otherwise indicated, with Me4Si as the internal standard. Standard abbreviations indicating spin multiplicities are given as follows: s (singlet), d (doublet), t (triplet), br (broad), q (quartet), or m (multiplet). Mass spectra were recorded on a V.G. 7070 E spectrometer or on a Waters ZQ 4000 apparatus operating in electrospray (ES) mode. Silica gel (Merck, 230−400 mesh) was used for purification with flash chromatography. Reactions were followed by thin layer chromatography (TLC) on precoated silica gel plates (Merck silica gel 60 F254) and then visualized with a UV lamp. Chemical purities of the tested compounds were determined by elemental analysis (C, H, N) and confirmed to have ≥95% purity. Compounds were named relying on the naming algorithm developed by CambridgeSoft Corporation and used in ChemDraw Professional 15.0. General Procedure for the Synthesis of Compounds 1a−c, 5a−c, 6a−c. A mixture of the appropriate chromen-4-one (1 equiv), the selected aldehyde (1 equiv), and piperidine was heated to 150 °C for 3 h. The reaction mixture was suspended in CH2Cl2, washed twice with H2O, dried over Na2SO4, and evaporated to dryness. The crude was then purified by flash chromatography. 3-(Pyridin-4-ylmethyl)-4H-chromen-4-one (1a). Starting from chromen-4-one 8a (1.00 g, 6.80 mmol), 4-pyridinecarboxaldehyde, and piperidine (0.11 mL), 0.75 g (47%) of 1a was obtained (eluent ethyl acetate); mp 136−138 °C. 1H NMR: δ 3.80 (s, 2H, CH2), 7.24 (d, J = 6.0 Hz, 2H, pyr), 7.37−7.47 (m, 2H, arom), 7.63−7.72 (m, 1H, arom), 7.77 (s, 1H, arom), 8.20−8.25 (m, 1H, arom), 8.52 (d, J = 6.0 Hz, 2H, pyr). 13C NMR: δ 31.15 (CH2), 118.04, 122.69 (quat), 123.70 (quat), 124.00 (2C, pyr), 125.16, 125.84, 133.67, 147.86 (quat), 149.89 (2C, pyr), 153.08, 156.41 (quat, C8a), 177.06 (CO). ES-MS m/z: 238 (M + 1). Anal. (C15H11NO2) C, H, N (see Supporting Information). 7-Methoxy-3-(pyridin-4-ylmethyl)-4H-chromen-4-one (1b). Starting from 7-methoxychromen-4-one 8b (2.65 g, 14.9 mmol), 4pyridinecarboxaldehyde, and piperidine (0.24 mL), an amount of 1.55 g (39%) of 1b (eluent ethyl acetate) was obtained; mp 176−178 °C. 1 H NMR: δ 3.78 (s, 2H, CH2), 3.90 (s, 3H, CH3O), 6.81−6.83 (m, 1H, arom), 6.95−6.99 (m, 1H, arom), 7.24 (d, J = 6.0 Hz, 2H, pyr), 7.67−7.69 (m, 1H, arom), 8.10−8.13 (m, 1H, arom), 8.53 (d, J = 6.0 Hz, 2H, pyr). 13C NMR: δ 31.07 (CH2), 55.77 (CH3O), 100.06, 114.62, 117.66 (quat, C4a), 122.54 (quat, pyr), 124.04 (2C, pyr), 127.23, 148.03 (quat), 149.86 (2C, pyr), 152.60, 158.22 (quat, C8a), 164.05 (quat), 176.40 (CO). ES-MS m/z: 268 (M + 1). Anal. (C16H13NO3) C, H, N (see Supporting Information). 7-Fluoro-3-(pyridin-4-ylmethyl)-4H-chromen-4-one (1c). Starting from 7-fluorochromen-4-one 8c (1.00 g, 6.03 mmol), 4pyridinecarboxaldehyde, and piperidine (0.10 mL), 0.60 g (39%) of 1c (eluent ethyl acetate/petroleum ether 4:1) was obtained; mp 114−116 °C. 1H NMR: δ 3.78 (s, 2H, CH2), 7.10−7.18 (m, 2H, arom), 7.23 (d, J = 6.0 Hz, 2H, pyr), 7.73 (s, 1H, arom), 8.19−8.27 (m, 1H arom), 8.52 (d, J = 6.0 Hz, 2H, pyr). 13C NMR: δ 31.06 (CH2), 104.67 (d, J = 25.25 Hz, C6), 114.15 (d, J = 22.93 Hz, C8), 120.63 (d, J = 2.22 Hz, C4a), 122.96 (quat, pyr), 124.07 (2C, pyr), 128.51 (d, J = 10.71 Hz, C5), 147.75 (quat, C3), 149.85 (2C, pyr), 153.19 (C2), 157.37 (quat, C8a), 165.61 (d, J = 256.24 Hz, C7), 176.20 (CO). ES-MS m/z: 256 (M + 1). Anal. (C15H10FNO2) C, H, N (see Supporting Information). 3-(1H-Imidazol-4-ylmethyl)-4H-chromen-4-one (5a). Starting from chromen-4-one 8a (1.00 g, 6.80 mmol), 4-imidazolecarboxaldehyde, and piperidine (0.11 mL), 0.06 g (4%) of 3a (eluent ethyl acetate) was obtained; mp 166−168 °C. 1H NMR (acetone-d6): δ 5.93 (s, 2H, CH2), 6.98−7.09 (m, 2H, arom), 7.50−7.58 (m, 2H, arom), 7.73 (s, 1H, arom), 7.87−7.93 (m, 2H, arom). 13C NMR (acetone-d6): δ 68.96 (CH2), 118.61, 122.18, 124.31 (quat), 127.61 (quat), 127.96, 128.13, 136.08, 138.28, 139.87, 155.92, 160.71 (quat), 176.91 (C O). ES-MS m/z: 227 (M + 1). Anal. (C13H10N2O2) C, H, N (see Supporting Information). 3-(1H-Imidazol-4-ylmethyl)-7-methoxy-4H-chromen-4-one (5b). Starting from 7-methoxychromen-4-one 8b (2.65 g, 14.9 mmol), 4-imidazolecarboxaldehyde, and piperidine (0.24 mL), 0.26 g (7%) of

5b (eluent petroleum ether/ethyl acetate 1:4) was obtained; mp 177− 179 °C. 1H NMR: δ 3.85 (s, 3H, CH3O), 5.88 (s, 2H, CH2), 6.43− 6.44 (m, 1H, arom), 6.59−6.62 (m, 1H, arom), 7.35−7.38 (m, 1H, arom), 7.60−7.62 (m, 1H, arom), 7.75−7.78 (m, 1H, arom), 7.94− 7.97 (m, 1H, arom). 13C NMR (acetone-d6): δ 56.07 (CH3O), 69.26 (CH2), 101.48, 110.68, 124.87 (quat), 127.35 (quat), 128.56, 129.85, 138.16, 140.17, 156.07, 164.40 (quat), 166.51 (quat), 176.25 (CO). ES-MS m/z: 257 (M + 1). Anal. (C14H12N2O3) C, H, N (see Supporting Information). 3-(1H-Imidazol-4-ylmethyl)-7-fluoro-4H-chromen-4-one (5c). Starting from 7-fluorochromen-4-one 8c (1.00 g, 6.03 mmol), 3pyridinecarboxaldehyde, and piperidine (0.10 mL), 0.44 g (30%) of 5c (eluent toluene/acetone 3:2) was obtained; mp 223−225 °C. 1H NMR (acetone-d6): δ 5.91 (s, 2H, CH2), 6.66−6.79 (m, 2H, arom), 7.40 (s, 1H, arom), 7.65 (s, 1H, arom), 7.77 (s, 1H arom), 7.99−8.07 (m, 1H, arom). 13C NMR (acetone-d6): δ 69.58 (CH2), 105.10 (d, J = 24.44 Hz, C6), 110.18 (d, J = 22.93 Hz, C8), 124.62 (imi), 127.11 (quat, C4a), 128.29 (imi), 130.93 (d, J = 11.51 Hz, C5), 138.31, 140.81, 155.84, 159.06 (quat, C8a), 167.72 (d, J = 253.11 Hz, C7), 177.48 (CO). ES-MS m/z: 245 (M + 1). Anal. (C13H9FN2O2) C, H, N (see Supporting Information). 3-(Pyridin-3-ylmethyl)-4H-chromen-4-one (6a). Starting from chromen-4-one 8a (1.00 g, 6.80 mmol), 3-pyridinecarboxaldehyde, and piperidine (0.11 mL), 0.74 g (46%) of 2a (eluent petroleum ether/ethyl acetate 1:9) was obtained; mp 134−136 °C. 1H NMR: δ 3.81 (s, 2H, CH2), 7.19−7.26 (m, 1H, arom), 7.36−7.45 (m, 2H, arom), 7.62−7.74 (m, 3H, arom), 8.20−8.24 (m, 1H, arom), 8.47− 8.49 (m, 1H, arom), 8.56−8.58 (m, 1H, arom). 13C NMR: δ 29.13 (CH2), 118.07, 123.41, 123.60 (quat), 123.79 (quat), 125.14, 125.89, 133.64, 134.34 (quat), 136.47, 148.00, 150.16, 152.95, 156.45 (quat, C8a), 177.22 (CO). ES-MS m/z: 238 (M + 1). Anal. (C15H11NO2) C, H, N (see Supporting Information). 7-Methoxy-3-(pyridin-3-ylmethyl)-4H-chromen-4-one (6b). Starting from 7-methoxychromen-4-one 8b (2.65 g, 14.9 mmol), 3pyridinecarboxaldehyde, and piperidine (0.24 mL), 0.48 g (12%) of 6b (crystallized from ligroin) was obtained; mp 125−127 °C. 1H NMR: δ 3.89 (s, 2H, CH2), 4.00 (s, 3H, CH3O), 6.90−6.91 (m, 1H, arom), 7.05−7.09 (m, 1H, arom), 7.31−7.36 (m, 1H, arom), 7.75−7.79 (m, 2H, arom), 8.20−8.23 (m, 1H, arom), 8.56−8.58 (m, 1H, arom), 8.65−8.67 (m, 1H, arom). 13C NMR: δ 29.04 (CH2), 55.80 (CH3O), 100.07, 114.62, 117.75 (quat), 123.40 (quat), 123.45, 127.28, 134.49 (quat), 136.51, 147.95, 150.17, 152.48, 158.25 (quat), 164.04 (quat), 176.57 (CO). ES-MS m/z: 268 (M + 1). Anal. (C16H13NO3) C, H, N (see Supporting Information). 7-Fluoro-3-(pyridin-3-ylmethyl)-4H-chromen-4-one (6c). Starting from 7-fluorochromen-4-one 8c (1.00 g, 6.03 mmol), 3pyridinecarboxaldehyde, and piperidine (0.10 mL), 0.09 g (6%) of 6c (eluent ethyl acetate) was obtained; mp 123−125 °C. 1H NMR: δ 3.80 (s, 2H, CH2), 7.10−7.17 (m, 2H, arom), 7.21−7.23 (m, 1H, arom), 7.65−7.70 (m, 2H, arom), 8.21−8.26 (m, 1H arom), 8.48−8.50 (m, 1H, arom), 8.57 (s, 1H, arom). 13C NMR: δ 29.01 (CH2), 104.66 (d, J = 25.96 Hz, C6), 114.09 (d, J = 23.03 Hz, C8), 120.68 (quat, C4a), 123.46 (quat, pyr), 123.83 (pyr), 128.50 (d, J = 10.70 Hz, C5), 134.10 (quat, C3), 136.51, 148.07, 150.13, 153.04, 157.44 (d, J = 13.84 Hz, C8a), 165.50 (d, J = 255.82 Hz, C7), 176.33 (CO). ES-MS m/z: 268 (M + 1). Anal. (C15H10FNO2) C, H, N (see Supporting Information). 3-Chloro-1-(2,4-dihydroxyphenyl)propan-1-one (9). A mixture of resorcinol (12.00 g, 110 mmol), 3-chloropropionic acid (12.00 g, 110 mmol), and trifluoromethanesulfonic acid (60.00 g, 400 mmol) was heated to 80 °C for 30 min. After cooling, the mixture was partitioned between water and CH2Cl2, the organic layer was washed with brine, dried over Na2SO4, and evaporated to dryness to give 13.2 g (60%) of 9; mp 87−89 °C. 1H NMR: δ 3.44 (t, J = 6.3 Hz, 2H, CH2), 3.95 (t, J = 6.3 Hz, 2H, CH2), 6.42−6.46 (m, 2H, arom), 7.61− 7.68 (m, 1H, arom). 7-Hydroxychroman-4-one (10). 9 (13.2 g, 66 mmol) was added to a 2N NaOH solution (800 mL) kept at 5 °C and the mixture was stirred at rt for 2 h. The pH was lowered to 2 with H2SO4 6 M (∼230 mL) and a precipitate was formed, which was filtered to give 7.70 g F



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(71%) of 10; mp 148−150 °C (lit.28 mp 146−148 °C). 1H NMR: δ 2.78 (t, J = 6.3 Hz, 2H, CH2), 4.51 (t, J = 6.3 Hz, 2H, CH2), 5.82 (br, 1H, OH), 6.41−6.43 (m, 1H, arom), 6.50−6.56 (m, 1H, arom), 7.83− 7.87 (m, 1H, arom). 7-Methoxychroman-4-one (8b). A mixture of 10 (7.70 g, 47 mmol), anhydrous K2CO3 (6.50 g, 47 mmol) and dimethyl sulfate (5.90 g, 47 mmol, 4.4 mL) in acetone was refluxed for 7 h, then hot filtered and evaporated to dryness to give 7.96 g (95%) of 8b; mp 43− 45 °C (lit.28 mp 46−48 °C). 1H NMR: δ 2.76 (t, J = 6.3 Hz, 2H, CH2), 3.83 (s, 3H, CH3O), 4.51 (t, J = 6.3 Hz, 2H, CH2), 6.40−6.42 (m, 1H, arom), 6.55−6.61 (m, 1H, arom), 7.81−7.86 (m, 1H, arom). 3-(3-Fluorophenoxy)propanenitrile (11). Triton B (benzyltrimethylammonium hydroxide, 0.9 mL) and acrylonitrile (14.25 g, 268.8 mmol) were added to 3-fluorophenol (6.00 g, 53.6 mmol), and the mixture was refluxed for 23 h. After cooling, it was diluted with diethyl ether (30 mL) and washed with 1 N NaOH, 1 N HCl, and H2O. The organic layer was dried over Na2SO4 and evaporated to dryness to give 5.2 g (59%) of 11, pale yellow oil. 1H NMR: δ 2.83 (t, J = 12 Hz, 2H, CH2), 4.18 (t, J = 12 Hz, 2H, CH2), 6.59−6.76 (m, 3H, arom), 7.19−7.31 (m, 1H, arom). 3-(3-Fluorophenoxy)propanoic Acid (12). A solution of 11 (5.20 g, 31.6 mmol) in conc HCl (24 mL) was refluxed for 14 h, and upon cooling, a solid was formed, which was filtered and resuspended in 2 N NaOH. The remaining solid was filtered off, the solution was acidified with 2 N HCl, and the resulting precipitate was filtered to give 4.63 g (80%) of 12, mp 95−97 °C. 1H NMR: δ 2.86 (t, J = 12 Hz, 2H, CH2), 4.24 (t, J = 12 Hz, 2H, CH2), 6.61−6.70 (m, 3H, arom), 7.18−7.26 (m, 1H, arom). 7-Fluorochroman-4-one (8c). A mixture of 12 (4.63 g, 25.2 mmol) and SOCl2 (13.65 g, 115 mmol) in toluene (60 mL) was refluxed for 1.5 h and evaporated to dryness. The residue was resuspended in CHCl 3 (50 mL) and cooled to −65 °C, trifluoromethanesulfonic acid (3.2 mL, 35.9 mmol) was added dropwise, and the mixture was stirred at rt for 2 h. Water was then added and the separated organic layer was washed with 1 N NaOH, dried over Na2SO4, and evaporated to dryness to give 3.77 g (90%) of 8c, colorless oil (lit.29 yellow oil). 1H NMR: δ 2.80 (t, J = 12 Hz, 2H, CH2), 4.55 (t, J = 12 Hz, 2H, CH2), 6.63−6.78 (m, 2H, arom), 7.88− 7.96 (m, 1H, arom). 3-Methyl-4H-chromen-4-one (14a). A mixture of 13a (0.45 g, 3 mmol) and N,N-dimethylformamide dimethyl acetal (0.72 g, 6 mmol) in DMF was heated to 120 °C for 2 h. The mixture was poured into ice and the resulting precipitate was filtered and purified by flash chromatography (eluent petroleum ether/ethyl acetate 4:1) to give 0.27 g of 14a (56%), mp 70−71 °C. 1H NMR: δ 2.01 (s, 3H, CH3), 7.32−7.39 (m, 2H, arom), 7.58−7.62 (m, 1H, arom), 8.20 (d, 1H, J = 8.2 Hz, arom). 7-Methoxy-3-methyl-4H-chromen-4-one (14b). Using the same procedure of 14a and starting from 13b,19 (1.2 g, 7.3 mmol), 0.78 g (56%) of 14b was obtained, mp 110−111 °C. 1H NMR: δ 2.02 (s, 3H, CH3), 3.90 (s, 3H, OCH3), 6.80 (s, 1H, arom), 6.96 (d, 1H, J = 8.0 Hz, arom), 7.72 (s, 1H, arom), 8.14 (d, 1H, J = 8.0 Hz, arom). 7-Fluoro-3-methyl-4H-chromen-4-one (14c). Using the same procedure of 14a and starting from 13c,30 (0.5 g, 3 mmol), 0.25 g (52%) of 14c was obtained, mp 89−90 °C. 1H NMR: δ 2.02 (s, 3H, CH3), 7.08−7.13 (m, 2H, arom), 7.76 (s, 1H, arom), 8.22−8.26 (m, 1H, arom). 3-(1H-Imidazol-1-ylmethyl)-4H-chromen-4-one (3a). A mixture of 14a (0.27 g, 1.7 mmol), N-bromosuccinimide (0.28 g, 1.7 mmol), and a catalytic amount of benzoyl peroxide in carbon tetrachloride (30 mL) was refluxed for 6 h. The mixture was hot filtered and evaporated to dryness. The residue (0.22 g), without further purification, was dissolved in acetonitrile (24 mL), imidazole (0.18 g, 2.7 mmol) was added, and the mixture was refluxed for 6 h under nitrogen, evaporated to dryness and the residue was purified by flash chromatography (eluent toluene/methanol 4.5:0.5) to give 0.11 g (28%) of 3a, mp 111−112 °C. 1H NMR: δ 5.01 (s, 2H, CH2), 7.05 (d, 2H, J = 8.0 Hz, arom), 7.40−7.45 (m, 2H, arom), 7.66−7.75 (m, 3H, arom), 8.20 (d, 1H, J = 8.2 Hz, arom). 13C NMR: δ 42.33 (CH2), 118.23, 119.46, 120.80 (quat), 123.71, 125.69, 125.87, 129.52, 134.21,

137.53 (quat), 153.66, 156.49 (quat), 176.45 (CO). ES-MS m/z: 227 (M + 1). Anal. (C13H10N2O2) C, H, N (see Supporting Information). 3-(1H-Imidazol-1-ylmethyl)-7-methoxy-4H-chromen-4-one (3b). Using the same procedure of 3a and starting from 14b (0.78 g, 4.1 mmol), 0.47 g of bromomethyl derivative was obtained, which was reacted with imidazole (0.35 g, 5.24 mmol) and purified by flash chromatography (eluent toluene/methanol 4.5:0.5) to give 0.06 g (6%) of 3b, mp 156−157 °C. 1H NMR: δ 3.92 (s, 3H, OCH3), 5.02 (s, 2H, CH2), 6.81 (s, 1H, arom), 6.97−7.12 (m, 3H, arom), 7.64− 7.68 (m, 2H, arom), 8.23 (d, 1H, J = 8.8 Hz, arom). 13C NMR: δ 42.24 (CH2), 55.87 (CH3O), 100.29, 115.05, 117.55, 119.43, 120.73 (quat), 127.22, 129.67, 137.54 (quat), 153.06, 158.29 (quat), 164.43 (quat), 175.69 (CO). ES-MS m/z: 257 (M + 1). Anal. (C14H12N2O3) C, H, N (see Supporting Information). 3-(1H-Imidazol-1-ylmethyl)-7-fluoro-4H-chromen-4-one (3c). Using the same procedure of 3a and starting from 14c (0.25 g, 1.45 mmol), 0.27 g of bromomethyl derivative was obtained, which was reacted with imidazole (0.11 g, 1.6 mmol) and purified by flash chromatography (eluent toluene/methanol 4:1) to give 0.06 g (16%) of 3c, mp 119−120 °C. 1H NMR: δ 5.02 (s, 2H, CH2), 7.03 (s, 1H, arom), 7.09 (s, 1H, arom), 7.13−7.18 (m, 2H, arom), 7.63 (s, 1H, arom), 7.67 (s, 1H, arom), 8.25 (d, 1H, J = 8.8 Hz, arom). 13C NMR: δ 42.11 (CH2), 104.90 (d, J = 26.06 Hz, C6), 114.59 (d, J = 22.93 Hz, C8), 119.37 (imi), 120.65 (quat, C4a), 121.10 (imi), 128.47 (d, J = 10.71 Hz, C5), 129.78 (imi), 137.56 (quat, C3), 153.65 (C2), 157.53 (d, J = 13.51 Hz, C8a), 165.84 (d, J = 257.05 Hz, C7), 175.48 (C O). ES-MS m/z: 245 (M + 1). Anal. (C13H9FN2O2) C, H, N (see Supporting Information). General Procedure for the Synthesis of Compounds 2a−c, 7a−c, and 16a−c. NaH (3 equiv) was suspended in pyridine, and a mixture of the selected ethyl 2-(pyridinyl)acetate (1 equiv) and 2hydroxyacetophenone (1 equiv) in pyridine was slowly added at 0 °C. The mixture was then heated to 100 °C for 45 min, and after cooling, petroleum ether was added. The precipitate formed was filtered and added to conc HCl at 0 °C, and the mixture was stirred at rt for 15 min, poured onto ice, and basified with K2CO3. The separated solid was filtered and purified by flash chromatography (eluent ethyl acetate). 2-Pyridin-4-ylmethylchromen-4-one (2a). Starting from ethyl 2-(pyridin-4-yl)acetate (1.00 g, 6.06 mmol) and 2-hydroxyacetophenone (0.82 g, 6.06 mmol), 0.3 g (21%) of 2a was obtained, mp 125− 127 °C. 1H NMR: δ 3.94 (s, 2H, CH2), 6.20 (s, 1H, arom), 7.25 (d, J = 6.0 Hz, 2H, pyr), 7.36−7.44 (m, 2H, arom), 7.62−7.69 (m, 1H, arom), 8.18 (d, J = 8.0 Hz, 1H, arom), 8.61 (d, J = 6.0 Hz, 2H, pyr). 13 C NMR: δ 39.91 (CH2), 111.22, 117.83, 123.54 (quat), 124.20 (2C, pyr), 125.25, 125.69, 133.76, 143.81, 150.26 (2C, pyr), 156.35 (quat), 165.61 (quat), 178.00 (CO). ES-MS m/z: 260 (M + Na). Anal. (C15H11NO2) C, H, N (see Supporting Information). 7-Methoxy-2-pyridin-4-ylmethylchromen-4-one (2b). Starting from ethyl 2-(pyridin-4-yl)acetate (1.00 g, 6.06 mmol) and 2hydroxy-4-methoxyacetophenone (1.00 g, 6.06 mmol), 0.4 g (25%) of 2b was obtained, mp 160−162 °C. 1H NMR: δ 3.88 (s, 3H, OCH3), 3.90 (s, 2H, CH2), 6.14 (s, 1H, arom), 6.77−6.79 (m, 1H, arom), 6.93−6.98 (m, 1H, arom), 7.24 (d, J = 6.0 Hz, 2H, pyr), 8.08 (d, J = 8.0 Hz, 1H, arom), 8.60 (d, J = 6.0 Hz, 2H, pyr). 13C NMR: δ 39.82 (CH2), 55.78 (CH3O), 100.18, 111.20, 114.43, 117.39 (quat), 124.17 (2C, pyr), 127.05, 143.94, 150.29 (2C, pyr), 158.12 (quat), 164.12 (quat), 164.99 (quat), 177.41 (CO). ES-MS m/z: 290 (M + Na). Anal. (C16H13NO3) C, H, N (see Supporting Information). 7-Fluoro-2-pyridin-4-ylmethylchromen-4-one (2c). Starting from ethyl 2-(pyridin-4-yl)acetate (1.00 g, 6.06 mmol) and 4-fluoro2-hydroxyacetophenone (0.93 g, 6.06 mmol), 0.3 g (19%) of 2c was obtained, mp 113−115 °C. 1H NMR: δ 3.92 (s, 2H, CH2), 6.17 (s, 1H, arom), 7.06−7.17 (m, 2H, arom), 7.24 (d, J = 6.0 Hz, 2H, pyr), 8.15−8.22 (m, 1H, arom), 8.61 (d, J = 6.0 Hz, 2H, pyr). 13C NMR: δ 39.78 (CH2), 104.61 (d, J = 25.15 Hz, C6), 111.30 (quat, pyr), 114.03 (d, J = 22.93 Hz, C8), 120.40 (d, J = 2.32 Hz, C4a), 124.21 (2C, pyr), 128.22 (d, J = 10.71 Hz, C5), 143.64 (C3), 150.23 (2C, pyr), 157.24 (quat, C8a), 165.60 (d, J = 256.14 Hz, C7), 165.85 (quat, C2), 176.97 G



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0.22 g (30%) of 17b was obtained, mp 135−136 °C. 1H NMR: δ 3.90 (s, 3H, OCH3), 4.23 (s, 2H, CH2), 6.37 (s, 1H), 6.89 (s, 1H, arom), 6.98 (d, J = 8.8 Hz, 1H, arom), 8.12 (d, J = 8.8 Hz, 1H, arom). 2-Bromomethyl-7-fluoro-4H-chromen-4-one (17c). Using the same procedure of 17a and starting from 16c (0.10 g, 0.45 mmol), 0.05 g (55%) of 17c was obtained and used without further purification. 1H NMR: δ 4.23 (s, 2H, CH2), 6.41 (s, 1H), 7.17−7.23 (m, 2H, arom), 8.21 (d, J = 8.8 Hz, 1H, arom). 2-(1H-Imidazol-1-yl)methyl-4H-chromen-4-one (4a). 17a (0.13 g, 0.55 mmol) was dissolved in acetonitrile, imidazole (0.11 g, 1.63 mmol) was added, and the mixture was refluxed for 6 h under nitrogen, evaporated to dryness and the residue was purified by flashchromatography (eluent CH2Cl2/ethanol 4.5:0.5) to give 0.06 g (47%) of 4a, mp 158−160 °C. 1H NMR: δ 5.07 (s, 2H, CH2), 6.14 (s 1H), 7.05 (s, 1H, imi), 7.18 (s, 1H, imi), 7.41−7.45 (m, 2H, arom), 7.67− 7.71 (m, 2H, arom and imi), 8.18 (d, J = 8.0 Hz, 1H, arom). 13C NMR: δ 48.28 (CH2), 110.25, 117.92 (quat, C4a), 119.44, 123.70, 125.70, 125.88, 130.12, 134.24, 137.53, 156.10 (quat, C8a), 161.96 (quat, C2), 177.69 (CO). ES-MS m/z: 249 (M + Na). Anal. (C13H10N2O2) C, H, N (see Supporting Information). 2-(1H-Imidazol-1-yl)methyl-7-methoxy-4H-chromen-4-one (4b). Using the same procedure of 4a and starting from 17b (0.22 g, 0.82 mmol), 0.17 g (80%) of 4b was obtained, mp 112−113 °C. 1H NMR: δ 3.90 (s, 3H, OCH3), 5.03 (s, 2H, CH2), 6.11 (s, 1H), 6.80 (s, 1H, arom), 6.98 (d, J = 8.0 Hz, 1H, arom), 7.03 (s, 1H, imi), 7.16 (s, 1H, imi), 7.65 (s, 1H, imi), 8.08 (d, J = 8.0 Hz, 1H, arom). 13C NMR: δ 48.31 (CH2), 55.87 (CH3O), 100.25 (C6), 110.06 (imi), 114.97 (C8), 117.26 (quat, C4a), 119.61 (imi), 127.00 (C5), 129.91 (imi), 137.57 (C3), 157.88 (quat, C8a), 161.59 (quat, C2), 164.57 (quat, C7), 177.25 (CO). ES-MS m/z: 279 (M + Na). Anal. (C14H12N2O3) C, H, N (see Supporting Information). 2-(1H-Imidazol-1-yl)methyl-7-fluoro-4H-chromen-4-one (4c). Using the same procedure of 4a and starting from 17c (0.05 g, 0.19 mmol), 0.03 g (63%) of 4c was obtained, mp 133−134 °C. 1H NMR: δ 5.05 (s, 2H, CH2), 6.10 (s, 1H), 7.03 (s, 1H, imi), 7.10−7.17 (m, 3H, arom), 7.64 (s, 1H, imi), 8.17−8.21 (m, 1H, imi). 13C NMR: δ 48.21 (CH2), 104.75 (d, J = 25.25 Hz, C6), 110.35 (imi), 114.50 (d, J = 23.03 Hz, C8), 117.59 (quat, C4a), 119.33 (imi), 128.44 (d, J = 10.71 Hz, C5), 130.65 (imi), 137.70 (C3), 157.97 (quat, C8a), 162.27 (C2), 165.86 (d, J = 251.89 Hz, C7), 176.59 (CO). ES-MS m/z: 267 (M + Na). Anal. (C13H9FN2O2) C, H, N (see Supporting Information). Biological Methods. Inhibition of CYP11B1 and CYP11B2. V79 MZh cells expressing human CYP11B1 or CYP11B2 were grown on six-well cell culture plates with 9.6 cm2 culture area per well until confluence. The reaction was subsequently started by the addition of [1,2-3H]-11-deoxycorticosterone as substrate and corresponding inhibitor at different concentrations. After incubations of 6 h for V79MZh11B1 and 30 min for V79MZh11B2 cells, respectively, enzyme reactions were stopped by extracting the supernatant with chloroform. Samples were centrifuged (10 000g, 10 min), and the solvent was pipetted into fresh cups. The solvent was evaporated, and the steroids were redissolved in chloroform and analyzed by HPTLC.38,39 CYP17 Preparation and Assay. Human CYP17 was coexpressed with rat NADPH-P450 reductase in E. coli, which was subsequently treated with lysozyme, incubated on ice with continuous shaking for 30 min, and sonicated (50 000g, 20 min) at 4 °C to break cell wall and obtain membrane pellet preparations. A solution of progesterone as substrate, NADPH generating system (10 mM NADP+, 100 mM glucose 6-phosphate, and 2.5 units of glucose 6-phosphate dehydrogenase), and inhibitors of various concentrations was preincubated at 37 °C for 5 min before a diluted membrane suspension was added to start the reaction. After an incubation of 30 min at 37 °C, it was quenched with 50 mL of 1 N HCl and steroids were extracted with ethyl acetate. The 17α-hydroxylase activity of CYP 17 was determined by measuring the conversion of progesterone into 17α-hydroxyprogesterone and byproduct 16α-hydroxyprogesterone.40,41

(CO). ES-MS m/z: 278 (M + Na). Anal. (C15H10FNO2) C, H, N (see Supporting Information). 2-Pyridin-3-ylmethylchromen-4-one (7a). Starting from ethyl 2-(pyridin-3-yl)acetate (1.00 g, 6.06 mmol) and 2-hydroxyacetophenone (0.82 g, 6.06 mmol), 0.52 g (36%) of 7a was obtained, mp 93− 95 °C. 1H NMR: δ 3.98 (s, 2H, CH2), 6.20 (s, 1H, arom), 7.31−7.45 (m, 3H, arom), 7.66−7.69 (m, 2H, arom), 8.19−8.22 (m, 1H, arom), 8.60−8.66 (m, 2H, arom). 13C NMR: δ 37.77 (CH2), 110.76, 117.79, 123.46 (quat, C4a), 123.69 (pyr), 125.14, 125.58, 130.62, 133.68, 136.58 (pyr), 148.76 (pyr), 150.14 (pyr), 156.30 (quat, C8a), 166.42 (quat, C2), 178.02 (CO). ES-MS m/z: 260 (M + Na). Anal. (C15H11NO2) C, H, N (see Supporting Information). 7-Methoxy-2-pyridin-3-ylmethylchromen-4-one (7b). Starting from ethyl 2-(pyridin-3-yl)acetate (1.00 g, 6.06 mmol) and 2hydroxy-4-methoxyacetophenone (1.00 g, 6.06 mmol), 1.14 g (70%) of 7b was obtained, mp 192−194 °C. 1H NMR: δ 3.88 (s, 3H, OCH3), 3.91 (s, 2H, CH2), 6.11 (s, 1H, arom), 6.78−6.80 (m, 1H, arom), 6.91−6.97 (m, 1H, arom), 7.29−7.33 (m, 1H, arom), 7.61−7.66 (m, 1H, arom), 8.04−8.09 (m, 1H, arom), 8.55−8.62 (m, 2H, arom). 13C NMR: δ 37.78 (CH2), 55.79 (CH3O), 100.17 (C6), 110.79 (quat, pyr), 114.41 (C8), 117.40 (quat, C4a), 123.70 (pyr), 127.03 (C5), 130.75 (C3), 136.48 (pyr), 148.93 (pyr), 150.36 (pyr), 158.15 (quat, C8a), 164.10 (quat), 165.84 (quat), 177.50 (CO). ES-MS m/z: 290 (M + Na). Anal. (C16H13NO3) C, H, N (see Supporting Information). 7-Fluoro-2-pyridin-4-ylmethylchromen-4-one (7c). Starting from ethyl 2-(pyridin-3-yl)acetate (1.00 g, 6.06 mmol) and 4-fluoro2-hydroxyacetophenone (0.93 g, 6.06 mmol), 1.24 g (80%) of 7c was obtained, mp 131−133 °C. 1H NMR: δ 3.93 (s, 2H, CH2), 6.14 (s, 1H, arom), 7.07−7.16 (m, 2H, arom), 7.27−7.34 (m, 1H, arom), 7.61−7.65 (m, 1H, arom), 8.14−8.22 (m, 1H, arom), 8.56−8.61 (m, 2H, arom). 13C NMR: δ 37.73 (CH2), 104.62 (d, J = 26.06 Hz, C6), 110.93 (quat, pyr), 113.97 (d, J = 23.03 Hz, C8), 120.42 (quat, C4a), 123.74 (pyr), 128.22 (d, J = 9.9 Hz, C5), 130.37 (C3), 136.52 (pyr), 149.07 (pyr), 150.32 (pyr), 157.40 (quat, C8a), 165.60 (d, J = 255.43 Hz, C7), 166.74 (quat, C2), 177.05 (CO). ES-MS m/z: 278 (M + Na). Anal. (C15H10FNO2) C, H, N (see Supporting Information). 2-Ethoxymethyl-4H-chromen-4-one (16a). Starting from NaH (1.3 g, 50 mmol), 2-hydroxyacetophenone (1.4 g, 10 mmol), and ethyl-2-etoxyacetate (1.3 g, 10 mmol), 0.4 g (30%) of 16a (eluent petroleum ether/ethyl acetate 4:1) was obtained as an oil. 1H NMR: δ 1.29 (t, J = 6.8 Hz, 3H, CH3), 3.64 (q, J = 6.8 Hz, 2H, OCH2), 4.40 (s, 2H, OCH2), 6.43 (s, 1H), 7.36−7.45 (m, 2H, arom), 7.63−7.72 (m, 1H, arom), 8.21 (d, J = 8.8 Hz, 1H, arom). 2-Ethoxymethyl-7-methoxy-4H-chromen-4-one (16b). Starting from NaH (1.4 g, 54,5 mmol), 2-hydroxy-4-methoxyacetophenone (1.8 g, 10.9 mmol), and ethyl 2-ethoxyacetate (1.5 mL, 10.9 mmol), 0.64 g (25%) of 16b (eluent petroleum ether/ethyl acetate 4:1) was obtained as an oil. 1H NMR: δ 1.30 (t, J = 6.8 Hz, 3H, CH3), 3.66 (q, J = 6.8 Hz, 2H, OCH2), 3.90 (s, 3H, OCH3), 4.39 (s, 2H, OCH2), 6.37 (s, 1H), 6.86 (s, 1H, arom), 6.97 (d, J = 8.8 Hz, 1H, arom), 8.11 (d, J = 8.8 Hz, 1H, arom). 2-Ethoxymethyl-7-fluoro-4H-chromen-4-one (16c). Starting from NaH (0.85 g, 32.5 mmol), 4-fluoro-2-hydroxyacetophenone (1.00 g, 6.5 mmol), and ethyl 2-ethoxyacetate (0.85 mL, 6.5 mmol), 0.10 g (10%) of 16c (eluent petroleum ether/ethyl acetate 4:1) was obtained as an oil. 1H NMR: δ 1.26 (t, J = 6.8 Hz, 3H, CH3), 3.63 (q, J = 6.8 Hz, 2H, OCH2), 4.37 (s, 2H, OCH2), 6.39 (s, 1H), 7.07−7.10 (m, 2H, arom), 8.16 (d, J = 8.8 Hz, 1H, arom). 2-Bromomethyl-4H-chromen-4-one (17a). 16a (0.4 g, 2 mmol) was dissolved in glacial acetic acid (1.5 mL), a mixture of 48% HBr (1.6 mL, 0.03 mmol) and glacial acetic acid (1.6 mL) was added, and the reaction was refluxed for 6 h. The mixture was diluted with water and extracted with CH2Cl2, the organic layer was then dried over Na2SO4 and evaporated to dryness. The residue was purified by flash chromatography (eluent toluene/acetone 9:1) and crystallized from ligroin to give 0.13 g (33%) of 17a, mp 110−112 °C. 1H NMR: δ 4.28 (s, 2H, CH2Br), 6.20 (s, 1H), 7.27−7.34 (m, 2H, arom), 7.55−7.63 (m, 1H, arom), 8.19 (d, J = 8.8 Hz, 1H, arom). 2-Bromomethyl-7-methoxy-4H-chromen-4-one (17b). Using the same procedure of 17a and starting from 16b (0.64 g, 2.7 mmol), H


Journal of Medicinal Chemistry CYP19 Preparation and Assay. Human CYP19 was obtained from the microsomal fraction of freshly delivered human term placental tissue. The incubation tubes containing [1β- 3 H]androstenedione as the substrate, NADPH, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, and inhibitors of various concentrations in phosphate buffer (0.05 M, pH 7.4) were preincubated at 30 °C in a shaking water bath for 5 min before microsomal protein was added to start the reaction. After further incubation for 3 h, the reaction was terminated with ethyl acetate, and aliquots were pipetted into cold HgC1 solution (1 mM). After addition of aqueous dextran-coated charcoal (DCC) suspension (2%), the vials were shaken for 20 min and centrifuged at 1500g for 5 min to separate the charcoal-adsorbed steroids. Inhibition of enzyme activity was determined by measuring the formation of 3H2O.42 MTT Cytotoxicity Assay. The number of living cells was evaluated measuring the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Experiments were performed in 96-well cell culture plates in DMEM supplemented with 10% FCS. Cells were incubated for 3 h with 0.1 and 1 μM test compound at 37 °C in a humidified atmosphere at 5% CO2. For cleavage reaction MTT solution (5 mg/mL in PBS) was added and incubation was continued for another 3 h. Reaction stop and cell lysis were carried out by addition of sodium dodecyl sulfate (SDS) in 0.01 N HCl (10%). The produced blue formazan was quantified spectrophotometrically at 590 nm as described by Denizot and Lang43 with minor modifications. Docking Studies. The CYP11B2 crystal (PDB code 4DVQ) was cleaned up by removing redundant protein copies, the substrate, and water. Hydrogens and partial charges were added via the Protonate3D application in MOE. Ligands were built and energy minimized in the MMFF94s force field with MOE. The docking software GOLD was employed in the docking studies with the following settings: the automatic active-site detection was switched on, heme iron was selected as the active-site origin, and the radius of the active site was set to 19 Å. A distance constraint of 1.9−2.5 Å between the sp2 hybrid N and the iron was set as well. Ligand was docked in 50 independent genetic algorithm (GA) iterations for each of the three GOLD-docking runs. Moreover, the goldscore.p450_pdb parameters were exploited, and the genetic algorithm default parameters were set. The resulting poses were subsequently ranked according to fitness and further evaluated with the LigX module in MOE.

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ABBREVIATIONS USED

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REFERENCES

ACTH, adrenocorticotropic hormone; MR, mineralcorticoid receptor; CYP19, aromatase; CYP17, 17α-hydroxylase/17,20lyase; CYP11B1, 11β-hydroxylase; CYP11B2, aldosterone synthase

(1) Fisher, A.; Friel, E. C.; Bernhardt, R.; Gomez-Sanchez, C.; Connell, J. M.; Fraser, R.; Davies, E. Effects of 18-hydroxylated steroids on corticosteroid production by human aldosterone synthase and 11beta-hydroxylase. J. Clin. Endocrinol. Metab. 2001, 86 (9), 4326−4329. (2) Brilla, C. G. Renin-angiotensin-aldosterone system and myocardial fibrosis. Cardiovasc. Res. 2000, 47 (1), 1−3. (3) Cooper, R. L.; Segal, R. A.; Diegelmann, R. F.; Reynolds, A. M. Modeling the effects of systemic mediators on the inflammatory phase of wound healing. J. Theor. Biol. 2015, 367, 86−99. (4) Hakki, T.; Bernhardt, R. CYP17- and CYP11B-dependent steroid hydroxylases as drug development targets. Pharmacol. Ther. 2006, 111 (1), 27−52. (5) Gobbi, S.; Rampa, A.; Belluti, F.; Bisi, A. Nonsteroidal aromatase inhibitors for the treatment of breast cancer: an update. Anti-Cancer Agents Med. Chem. 2014, 14 (1), 54−65. (6) Porubek, D. CYP17A1: a biochemistry, chemistry, and clinical review. Curr. Top. Med. Chem. 2013, 13 (12), 1364−1384. (7) Cerny, M. A. Progress towards clinically useful aldosterone synthase inhibitors. Curr. Top. Med. Chem. 2013, 13 (12), 1385−1401. (8) Hu, Q.; Yin, L.; Hartmann, R. W. Aldosterone synthase inhibitors as promising treatments for mineralocorticoid dependent cardiovascular and renal diseases. J. Med. Chem. 2014, 57 (12), 5011−5022. (9) Ulmschneider, S.; Muller-Vieira, U.; Klein, C. D.; Antes, I.; Lengauer, T.; Hartmann, R. W. Synthesis and evaluation of (pyridylmethylene)tetrahydronaphthalenes/-indanes and structurally modified derivatives: potent and selective inhibitors of aldosterone synthase. J. Med. Chem. 2005, 48 (5), 1563−1575. (10) Ulmschneider, S.; Muller-Vieira, U.; Mitrenga, M.; Hartmann, R. W.; Oberwinkler-Marchais, S.; Klein, C. D.; Bureik, M.; Bernhardt, R.; Antes, I.; Lengauer, T. Synthesis and evaluation of imidazolylmethylenetetrahydronaphthalenes and imidazolylmethyleneindanes: potent inhibitors of aldosterone synthase. J. Med. Chem. 2005, 48 (6), 1796− 1805. (11) Voets, M.; Antes, I.; Scherer, C.; Muller-Vieira, U.; Biemel, K.; Barassin, C.; Marchais-Oberwinkler, S.; Hartmann, R. W. Heteroarylsubstituted naphthalenes and structurally modified derivatives: selective inhibitors of CYP11B2 for the treatment of congestive heart failure and myocardial fibrosis. J. Med. Chem. 2005, 48 (21), 6632−6642. (12) Desideri, N.; Bolasco, A.; Fioravanti, R.; Monaco, L. P.; Orallo, F.; Yáñez, M.; Ortuso, F.; Alcaro, S. Homoisoflavonoids: natural scaffolds with potent and selective monoamine oxidase-B inhibition properties. J. Med. Chem. 2011, 54 (7), 2155−2164. (13) Gaspar, A.; Silva, T.; Yáñez, M.; Vina, D.; Orallo, F.; Ortuso, F.; Uriarte, E.; Alcaro, S.; Borges, F. Chromone, a privileged scaffold for the development of monoamine oxidase inhibitors. J. Med. Chem. 2011, 54 (14), 5165−5173. (14) For examples, see the following: (a) Budriesi, R.; Bisi, A.; Ioan, P.; Rampa, A.; Gobbi, S.; Belluti, F.; Piazzi, L.; Valenti, P.; Chiarini, A. 1,4-Dihydropyridine derivatives as calcium channel modulators: the role of 3-methoxy-flavone moiety. Bioorg. Med. Chem. 2005, 13 (10), 3423−3430. (b) Rizzo, S.; Tarozzi, A.; Bartolini, M.; Da Costa, G.; Bisi, A.; Gobbi, S.; Belluti, F.; Ligresti, A.; Allarà, M.; Monti, J. P.; Andrisano, V.; Di Marzo, V.; Hrelia, P.; Rampa, A. 2-Arylbenzofuranbased molecules as multipotent Alzheimer’s disease modifying agents. Eur. J. Med. Chem. 2012, 58, 519−532. (c) Belluti, F.; Perozzo, R.; Lauciello, L.; Colizzi, F.; Kostrewa, D.; Bisi, A.; Gobbi, S.; Rampa, A.; Bolognesi, M. L.; Recanatini, M.; Brun, R.; Scapozza, L.; Cavalli, A. Design, synthesis, and biological and crystallographic evaluation of

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b01609.

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Article

Elemental analyses and representative 1H and 13C NMR spectra of target compounds, PDB coordinates of the docking of compounds 1c and 2c to both CYP11B2 crystal (PDB code 4DVQ) and CYP11B1 homology model (PDF) Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Authors

*S.G.: e-mail, [email protected]; phone, +39 051 2099732. *A.B.: e-mail, [email protected]; phone, +39 051 2099710. Present Address §

Q.H.: Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, U.K. Notes

The authors declare no competing financial interest. I



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