Understanding Oxadiazolothiazinone Biological Properties: Negative

Article pubs.acs.org/jmc

Understanding Oxadiazolothiazinone Biological Properties: Negative Inotropic Activity versus Cytochrome P450-Mediated Metabolism Emanuele Carosati,*,†,‡ Barbara Cosimelli,§ Pierfranco Ioan,∥ Elda Severi,§ Kasiram Katneni,‡ Francis C. K. Chiu,‡ Simona Saponara,⊥ Fabio Fusi,⊥ Maria Frosini,⊥ Rosanna Matucci,# Matteo Micucci,∥ Alberto Chiarini,∥ Domenico Spinelli,○ and Roberta Budriesi∥ †

Dipartimento di Chimica, Biologia e Biotecnologie, Università di Perugia, Via Elce di Sotto 10, 06123 Perugia, Italy Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, 381 Royal Parade, Parkville, VIC 3052, Australia § Dipartimento di Farmacia, Università di Napoli “Federico II”, Via D. Montesano 49, 80131 Napoli, Italy ∥ Dipartimento di Farmacia e Biotecnologie, Università di Bologna, Via Belmeloro 6, 40126 Bologna, Italy ⊥ Dipartimento di Scienze della Vita, Università degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy # Dipartimento di Neuroscienze, Area del Farmaco e Salute del Bambino (NEUROFARBA), Viale Pieraccini 6, 50139 Firenze, Italy ○ Dipartimento di Chimica ‘G. Ciamician’, Alma Mater Studiorum-Università di Bologna, Via Selmi 2, 40126 Bologna, Italy ‡

S Supporting Information *

ABSTRACT: We present a series of oxadiazolothiazinones, selective inotropic agents on isolated cardiac tissues, devoid of chronotropy and vasorelaxant activity. Functional and binding data for the precursor of the series (compound 1) let us hypothesize LTCC blocking activity and the existence of a recognition site specific for this scaffold. We synthesized and tested 22 new derivatives: introducing a para-methoxyphenyl at C-8 led to compound 12 (EC50 = 0.022 μM), twice as potent as its para-bromo analogue (1). For 10 analogues, we extended the characterization of the biological properties by including the assessment of metabolic stability in human liver microsomes and cytochrome P450 inhibition potential. We observed that the methoxy group led to active compounds with low metabolic stability and high CYP inhibition, whereas the protective effect of bromine resulted in enhanced metabolic stability and reduced CYP inhibition. Thus, we identified two para-bromo benzothiazino-analogues as candidates for further studies.

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INTRODUCTION

Then, we aimed to synthesize and biologically characterize 22 new oxadiazolothiazinones: nine unsubstituted and 13 methoxy-derivatives, including both the thiazino- and benzothiazino-scaffolds (Chart 2). We varied the position of the methoxy group (ortho-, meta-, and para-positions on the phenyl at the C-8) and the nature of the lateral chain OR (with R = CH2CH3, CH2CF3, CH(CH3)2, and CH2-cyclohexyl) at the C8. As part of the biological characterization, we tested efficacy and potency on driven left atria, on spontaneously beating right atria, and on vascular as well as ileum smooth muscles. Additionally, CYP-mediated metabolism studies were conducted for 10 most active and interesting derivatives (a subset that could facilitate structure−metabolism relationships: three unsubstituted, three para-methoxy, and four para-bromo substituted) in order to assess metabolic stability and CYPinhibition properties.

The oxadiazolothiazinone scaffold, when substituted at C-8 with an aryl ring and an alkyloxy chain, gives rise to cardioselective agents endowed with negative inotropic activity. Data on hemithioketals indicated the para-bromo phenyl as the best decoration at C-8,1,2 and we recently identified derivatives with submicromolar potency (Chart 1),3 initially classified as diltiazem functional analogues.4 In this article, we extended the biological characterization of this scaffold with an overall objective of selecting one or two candidate compounds with suitable pharmacological and metabolic properties for further development. Toward this objective, we aimed to gain more insight into the mechanism(s) causing the (observed) functional inotropic activity. For the precursor of the series (compound 1), we measured the displacement of well-known tritiated ligands (LTCC blockers) from their binding site and assessed the functional activity in the copresence of diltiazem (DTZ), nifedipine (NIF), and verapamil (VER). © XXXX American Chemical Society

Received: January 8, 2016

A

DOI: 10.1021/acs.jmedchem.6b00030 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Chart 1. Oxadiazolothiazin-3-ones with Negative Inotropic Activity2,3

nitroso derivatives 39−41; and (iii) conversion into the oxadiazolothiazinones by reacting with HCl. Further functionalization of the structure came by reacting the hemithioketals with the appropriate alcohol in toluene, in the presence of para-toluenesulfonic acid as a catalyst (Scheme 2). This introduced the lateral chain OR: the hemithioketals 6,5 11,5 15,5 20, 23, and 28 gave the thioketals 7−10, 12−14, 16− 19, 21−22, 24−27, and 29−30, respectively. Binding. The three major classes of LTCC ligands are 1,4dihydropyridines (DHPs), phenylalkylamines (PAAs), and benzothiazepines (BTZs):7,8 different chemical structures of these three classes correspond to distinct but overlapping binding sites in the pore-forming domain of the Ca2+ channel.9 Thus, their binding is affected in a reciprocal manner, due to allosteric modulation.10 We already showed that compound 1 displayed a complex interaction with the binding site of [3H]diltiazem because it either stimulated or inhibited at low (0.1 nM−1 μM) or high (10−100 μM) concentrations, respectively, the binding of the labeled drug (Figure 1A).11 This profile might reflect a positive allosteric modulation at the diltiazem binding site that, at higher concentrations, turned into a direct and negative interaction. Here, we report the effects of compound 1 on the binding of [3H]isradipine and [3H]verapamil at DHPs and PAAs sites, respectively. Results indicate that compound 1 only slightly affects the isradipine specific binding, as we only observed a weak inhibition at the highest compound concentration used (40% of inhibition at 100 μM, Supporting Information). On the contrary, compound 1 displaced labeled verapamil from its binding site in a concentration-dependent fashion, with IC50 of 18.30 μM (Figure 1B). Overall, the profile of 1 confirms its direct interaction with the verapamil binding site, at concentrations higher than 1 μM. Functional Studies in the Copresence of LTCC Antagonists. With the purpose to shed light on the functional profile of compound 1, the precursor of the new series of derivatives, we investigated its functional interaction with three well-known LTCC blockers: nifedipine, verapamil, and diltiazem. First, we quantified the negative inotropic effect of these drugs alone, whose EC50 values are in the submicromolar range (NIF, 0.26 μM; VER, 0.61 μM; DTZ, 0.79 μM). Then, we measured the EC50 in the copresence of compound 1, whose EC50 is 0.04 μM. Pretreatment of guinea pig left atria with compound 1, at an ineffective concentration per se (10−8 M), increased the negative inotropic potency of the three drugs (NIF, VER, and DTZ). This is evident from the leftward shift

Chart 2. New Oxadiazolothiazinones Synthesized and Testeda

a

Compounds 5, 6, 11, and 15 are from ref 1.

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RESULTS Chemistry. As previously described, the molecular basis of the formation of this class of molecules is the enlarged Cusmano-Ruccia reaction for the ring−ring interconversion of the nitroso-imidazothiazoles into the oxadiazolothiazinones.5,6 We obtained the new hemithioketals 20, 23, and 28 following the three-step procedure of Scheme 1: (i) treatment of 2-aminothiazoles 31 and 32 with 2-bromoacetophenones 33−35, thus obtaining the relevant imidazothiazoles 36−38; (ii) reaction with sodium nitrite in acetic acid to give the B



Article

Scheme 1. Synthesis of Compounds 20, 23, and 28a

a

The reaction conditions (a) for compounds 36−38 have been previously published (see Experimental Section).14−16

Scheme 2. Synthesis of New Thioketalsa

a

See Chart 2 for the substituent pattern.

of the concentration−response curves of NIF, VER, and DTZ (compare black and cyan curves in Figure 2A,B,C), as well as from the decreased EC50 values (11.8-fold decrease observed for NIF, 12-fold for VER, and 3.3-fold for DTZ; see Table 1). NIF, VER, and DTZ completely relaxed guinea pig aortic strips depolarized with 80 mM K+, whereas compound 1 was almost ineffective (Figure 2D,E,F). Pretreatment of guinea pig aortic strips with 10−6 M compound 1 markedly antagonized the vascular responsiveness to the three LTCC blockers (see Table 1 and Figure 2D,E,F). Similar results were obtained with a lower concentration of compound 1 (10−8 M), though only with NIF and VER; under these experimental conditions, the vascular response to DTZ was indistinguishable from that observed with DTZ alone. Functional Studies. For 22 new derivatives, we report in Table 2 the inotropic and chronotropic activity and in Table 3 the relaxant activity on vascular and nonvascular muscles. All of the oxadiazolothiazinones presented here are selective negative inotropic agents, devoid of chronotropic effects and vascular relaxant activity. On nonvascular tissue (ileum), some of the

Figure 1. Inhibition of [3H]diltiazem (A) and [3H]verapamil (B) specific binding by compound 1 in rat cardiomyocites. The curve of panel B was fitted using the standard four parameter logistic equation and is the mean ± SEM from at least three independent experiments. Panel A was adapted from ref 11. Copyright 2006 American Chemical Society.

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Article

Figure 2. Cardiovascular activity of NIF, VER, DTZ, and compound 1. Cumulative concentration−response curves for LTCC blockers (black), compound 1 (gray) or LTCC blockers in the presence of compound 1 either at 10−8 M (cyan) or 10−6 M (blue). Panels A, B, and C report the negative inotropic activity of NIF (A), VER (B), and DTZ (C) on isolated guinea pig left atrium driven at 1 Hz. Panels D, E, and F report the vasorelaxant activity of NIF (D), VER (E), and DTZ (F) on guinea pig vascular aortic strips depolarized with 80 mM K+. Each point is the mean ± SEM (n = 3−4). Where error bars are not shown, these are covered by the point itself; overlapping curves are present in panel D (cyan and blue) and panel F (gray and blue).

Table 1. Cardiovascular Activity of Compound 1 and LTCC Blockers, Either Alone or in Combination left atrium (negative inotropy) compd d

1 NIF NIF + 1 (10−8 M) NIF + 1 (10−6 M) VER VER + 1 (10−8 M) VER + 1 (10−6 M) DTZi DTZ + 1 (10−8 M) DTZ + 1 (10−6 M)

activity (M ± S.E.M.) a

77 ± 97 ± 94 ± NT 84 ± 95 ± NT 78 ± 92 ± NT

e

EC50b

(μM)

aorta (vasorelaxant effect) −6

95% conf lim (×10 )

1.7 2.0 1.3f

0.04 0.26 0.022

0.03−0.05 0.19−0.36 0.014−0.032

2.1 1.4e

0.61 0.05

0.40−0.80 0.03−0.079

3.5 1.7g

0.79 0.27

0.70−0.85 0.18−0.41

activity (M ± S.E.M.) c

19 82 10 11 95 20 36 88 64 18

± ± ± ± ± ± ± ± ± ±

0.9 1.3e 0.7f 0.3f 1.7e 1.1g 2.4h 2.3 1.7j 0.6j

IC50b (μM)

95% conf lim (×10−6)

0.009

0.003−0.020

0.38

0.20−0.70

2.6 0.47

2.2−3.1 0.031−0.95

a Decrease in developed tension on isolated guinea pig left atrium at 10−5 M, expressed as percent changes from the control (n = 5−6). The left atria were driven at 1 Hz. The 10−5 M concentration gave the maximum effect for most compounds. bCalculated from log concentration−response curves (Probit analysis by Litchfield and Wilcoxon with n = 6−7).17 When the maximum effect was 3. In two cases, the analogues with OCH2CF3 were less potent (unsubstituted: 25 < 24 or substituted with metamethoxy: 17 < 16); whereas in one case (unsubstituted), there was comparable potency (7 and 8). We tested all of the new derivatives for their vascular smooth muscles relaxant activity on K+-depolarized (80 mM) guinea pig aortic strips and nine of them also on K+-depolarized (80 mM) guinea pig ileum longitudinal smooth muscle (GPILSM), for comparative purposes (Table 3). F



Article

Table 4. In Vitro Metabolic Stability in Human Liver Microsomes compd

degradation half-lifea

in vitro CLintb

1 2 3 4 7 8 12 24 29 30

53 >250c 106 NDd 20 NDd 6 NDd 13 23

33 20 >20 >20 5.2 >20 >20 >20

18.4 6.6 >20 >20 >20 18.4 13.6 19.0 11.8 >20

1.8 1.0 >20 10.8 4.8 2.4 2.5 6.1 3.6 17.0

4.1 7.1 >20 >20 16.9 16.5 12.5 >20 >20 >20

>20; 19.2 >20; >20 >20; >20 >20; >20 >20; 19.1 >20; >20 15.5; 19.4 c.n.cb; >20 >20; >20 >20; >20

a

Used two probe substrates, midazolam and testosterone. bc.n.c = could not be calculated due to an apparent increase in probe metabolite formation in the presence of test compound (relative to that in the absence).

The most potent inhibition by this series of compounds was against CYP 2C19, followed by 1A2 and 2D6. A few compounds showed weak-to-moderate inhibition against 2C9, while most compounds exhibited minimal inhibition against 3A4, which was consistent across both probe substrates (midazolam and testosterone). In general, the thiazino series (compounds 1, 2, 7, 8, and 12) exhibited greater CYP inhibition potency than the benzocondensed series (compounds 3, 4, 24, 29, and 30). Compound 1 inhibited 1A2, 2C19, and 2D6, and the corresponding OCH2CF3 derivative (2) was a less potent inhibitor of 1A2 and 2D6 but a more potent inhibitor of 2C19 and 2C9. Compound 12 showed strong inhibition against 2C19 (IC50 = 2.5), moderate inhibition against 1A2 (IC50 = 5.2), and weak inhibition against 2C9, 2D6, and 3A4. Replacing the para-Br with H (compounds 7 and 8) resulted in reduced inhibition against CYPs 1A2 and 2D6, but inhibition against 2C19 was maintained (7, IC50 = 4.8 μM; 8, IC50 = 2.4 μM). For the benzocondensed analogues, the most potent inhibition was against 2C19 and, to a minor extent, 2C9, while inhibition against 1A2, 2D6, and 3A4 was minimal. In particular, compounds 3 and 30 showed no inhibition against H



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oxadiazolothiazinone. Interestingly, the electronic and hydrogen bonding interactions of the bromo/methoxy group, the electronic effect and steric hindrance of the thiazine (with methyl at C-5) or benzothiazine scaffolds, and the size and electronic effects of the lateral chain also affect the CYPmediated metabolism and the inhibition of CYPs (Figure 4).

Article

EXPERIMENTAL SECTION

Chemistry. General Procedures. Compounds 5,1 6,5 11,5 15,5 36,14 37,15 38,16 and 4015 were synthesized according to described methods. Compounds 31 and 32 were commercially available, as were the 2-bromoacetophenones 33−35. Melting points were determined using a Büchi apparatus B 540 and are uncorrected. 1H and 13C NMR spectra were recorded on a Varian Gemini 300 Instrument in the Fourier transform mode or on Varian Mercuryplus 400 at 25 (±0.5 °C) in DMSO-d6. Chemical shifts (δ) are in parts per million (ppm) from tetramethylsilane, and coupling constants are in Hertz. ESI mass spectra were obtained on a micromass ZMD Waters instrument (30 V, 3.2 kV, isotope observed 79Br). EI mass spectra were recorded on a VG70 70E apparatus. Solvents of the reaction were removed under reduced pressure. Silica gel plates (Merck F254) and silica gel 60 (Merck 230−400 mesh) were used for analytical TLC and for column chromatography, respectively. The spectra of the compounds are reported in Supporting Information. All new compounds gave satisfactory microanalyses and showed ≥95% purity. General Procedure for the Synthesis of Ethoxy-[1,2,4]oxadiazolo[3,4-c][1,4]thiazinones 7, 12, 16, 21, 24, and 29. A suspension of the appropriate hemithioketal (1.3 mmol) in dry toluene (40 mL) was refluxed for 2−6 h under stirring with ethanol (0.76 mL, 13.0 mmol) and in the presence of a catalytic amount of para-toluenesulfonic acid (ca. 0.5 mmol). The reaction mixture was cooled at room temperature and washed with a saturated solution of NaHCO3. The organic layer was separated, and the aqueous layer was extracted with toluene (3 × 40 mL). The collected organic phases were dried on Na2SO4. Removal of the solvent left a solid, which was purified by flash-chromatography (see below) to give the desired compound. 8-Ethoxy-5-methyl-8-phenyl-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (7). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 83%; mp 112.4−113.3 °C from EtOH/H2O. 8-Ethoxy-8-(4-methoxyphenyl)-5-methyl-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (12). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 70%; mp 112.0−112.3 °C from EtOH/ H2O. 8-Ethoxy-8-(3-methoxyphenyl)-5-methyl-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (16). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 75%; mp 117.3−118.2 °C from EtOH/ H2O. 8-Ethoxy-8-(2-methoxyphenyl)-5-methyl-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (21). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 43%; mp 113.4−114.2 °C from EtOH/ H2O. 4-Ethoxy-4-phenyl-4H-[1,2,4]oxadiazolo[3,4-c][1,4]benzothiazin1-one (24). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 72%; mp 136.8−137.7 °C from EtOH/H2O. 4-Ethoxy-4-(4-methoxyphenyl)-4H-[1,2,4]oxadiazolo[3,4-c][1,4]benzothiazin-1-one (29). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 65%; mp 149.4−150.4 °C from EtOH/H2O. General Procedure for the Synthesis of 2,2,2-Trifluoroethoxy[1,2,4]oxadiazolo[3,4-c][1,4]thiazinones 8, 17, 25, and 30. Operating as above but using 2,2,2-trifluoroethanol (1 mL; 13 mmol) instead of ethanol and warming up to 80 °C, the desired compounds were obtained. 5-Methyl-8-phenyl-8-(2,2,2-trifluoroethoxy)-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (8). Eluant mixture: ethyl acetate/petroleum ether = 1:3 v/v. Yield: 13%; mp 94.8−95.7 °C from petroleum ether/toluene. 8-(3-Methoxyphenyl)-5-methyl-8-(2,2,2-trifluoroethoxy)-8H[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (17). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 20%; mp 87.7−88.6 °C from petroleum ether/toluene. 4-Phenyl-4-(2,2,2-trifluoroethoxy)-4H-[1,2,4]oxadiazolo[3,4-c][1,4]benzothiazin-1-one (25). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 47%; mp 146.6−147.1 °C from EtOH. 4-(4-Methoxyphenyl)-4-(2,2,2-trifluoroethoxy)-4H-[1,2,4]oxadiazolo[3,4-c][1,4]benzothiazin-1-one (30). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 50%; mp 194.4−195.1 °C from toluene.

Figure 4. Impact of structural features of the oxadiazolothiazinones and oxadiazolo-benzothiazinones on negative inotropic activity, CYP inhibition, and CYP-mediated metabolism.

Besides the further understanding of some biological properties of this scaffold (inotropic activity and CYP-mediated metabolism), our aim was to identify the best candidate(s) for further study. Of the compounds evaluated, 3 has the best profile: good activity and no CYP inhibition, although it has intermediate metabolic stability. Compound 4 was more potent than 3, inhibited only one CYP isoform (2C19, with IC50 = 10.8 μM), but the metabolic stability could not be assessed. We can speculate that 4 is reasonably stable on the basis of pairwise comparison with 3 (they differ only for OCH 2 CH 3 / OCH2CF3): for compounds 1 and 2 (the thiazino-analogues of 3 and 4), the replacement of OCH2CH3 with OCH2CF3 led to reduced metabolism that would also be expected for 4 compared to 3. Other compounds had good activity (compounds 1, 2, and 12), but compound 1 inhibited three CYP isoforms and had only moderate metabolic stability, while 2 was more stable but inhibited four CYP isoforms. Compound 12 exhibited the poorest properties as it inhibited all of the CYP isoforms (5 out of 5) and was highly metabolically labile. Rapid metabolism also affected compound 30; despite intermediate potency and low CYP inhibition, it was highly metabolically labile. All of the remaining compounds (7, 8, 24, and 29) were less potent than 30; compounds 7 and 29 were also rapidly metabolized, although 7 (unsubstituted at the aryl) had a better profile (fewer CYP isoforms inhibited and longer half-life) than the corresponding para-methoxy, 12. Compound 24 inhibited only one CYP isoform (2C19, IC50 = 6.1 μM), but we could not speculate on its metabolic stability because it bears OCH2CH3 as the lateral chain. A CYP inhibition study revealed that CYP 2C19 was the most commonly inhibited isoform, followed by 1A2 and 2D6; only a few compounds showed weak-to-moderate inhibition against 2C9, whereas none of the compounds inhibited 3A4. In conclusion, at this stage of the lead optimization process, we could identify two compounds with a good profile (3 and 4), in terms of LTCC blocking activity and CYP-mediated metabolism. I



Article

General Procedure for the Synthesis of Propan-2-yloxy-[1,2,4]oxadiazolo[3,4-c][1,4]thiazinones 9, 13, 18, and 26. Operating as described above but using propan-2-ol (1 mL; 13 mmol) instead of ethanol, the desired compounds were obtained. 5-Methyl-8-phenyl-8-(propan-2-yloxy)-8H-[1,2,4]oxadiazolo[3,4c][1,4]thiazin-3-one (9). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 75%; mp 92.3−93.0 °C from EtOH/H2O. 8-(4-Methoxyphenyl)-5-methyl-8-(propan-2-yloxy)-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (13). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 18%; mp 87.1−87.9 °C from petroleum ether/toluene. 8-(3-Methoxyphenyl)-5-methyl-8-(propan-2-yloxy)-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (18). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 50%; colorless oil. 4-Phenyl-4-(propan-2-yloxy)-4H-[1,2,4]oxadiazolo[3,4-c][1,4]benzothiazin-1-one (26). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 64%; mp 144.0−144.6 °C from EtOH. General Procedure for the Synthesis of Ciclohexylmethoxy[1,2,4]oxadiazolo[3,4-c][1,4]thiazinones 10, 14, 19, 22, and 27. Operating as described above but using cyclohexylmethanol (1.6 mL; 13 mmol) instead of ethanol, the desired compounds were obtained. 8-Cyclohexylmethyloxy-5-methyl-8-phenyl-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (10). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 83%; mp 110.0−110.9 °C from EtOH/ H2O. 8-(Cyclohexylmethoxy)-8-(4-methoxyphenyl)-5-methyl-8H[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (14). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 44%; mp 129.4−130.3 °C from EtOH/H2O. 8-(Cyclohexylmethoxy)-8-(3-methoxyphenyl)-5-methyl-8H[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (19). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 65%; mp 119.0−119.9 °C from EtOH/H2O. 8-(Cyclohexylmethoxy)-8-(2-methoxyphenyl)-5-methyl-8H[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (22). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 72%; mp 92.3−93.0 °C from EtOH/H2O. 4-(Cyclohexylmethoxy)-4-phenyl-4H-[1,2,4]oxadiazolo[3,4-c][1,4]benzothiazin-1-one (27). Eluant mixture: ethyl acetate/petroleum ether = 1:5 v/v. Yield: 65%; mp 119.0−120.0 °C from EtOH/ H2O. Specific Procedure for the Synthesis of Compounds 20, 23, 28, 39, and 41. 8-Hydroxy-8-(2-methoxyphenyl)-5-methyl-8H-[1,2,4]oxadiazolo[3,4-c][1,4]thiazin-3-one (20). A suspension of 38 (3 mmol) in ethanol (25 mL) was refluxed under stirring with 2 M HCl (2.5 mL) for 2 h. Removal of the solvent left a solid which was purified by crystallization from ethanol. Yield: 38%; mp 241.1−242.0 °C. 4-Hydroxy-4-phenyl-4H-[1,2,4]oxadiazolo[3,4-c][1,4]benzothiazin-1-one (23). A suspension of 40 (3 mmol) in ethanol (25 mL) was refluxed under stirring with 2 M HCl (2.5 mL) for 2 h. Removal of the solvent left a solid which was purified by flashchromatography (eluant mixture: ethyl acetate/petroleum ether = 1:4 v/v) to give the desired compound. Yield: 50%; mp 101.3−101.9 °C from EtOH. 4-Hydroxy-4-(4-methoxyphenyl)-4H-[1,2,4]oxadiazolo[3,4-c][1,4]benzothiazin-1-one (28). A suspension of 41 (3 mmol) in ethanol (25 mL) was refluxed under stirring with 2 M HCl (2.5 mL) for 4 h. Removal of the solvent left a solid which was purified by flashchromatography (eluant mixture: ethyl acetate/petroleum ether = 1:4 v/v) to give the desired compound. Yield: 35%; mp 122.8−123.7 °C from EtOH. 6-(2-Methoxyphenyl)-3-methyl-5-nitrosoimidazo[2,1-b][1,3]thiazole (39). A solution of sodium nitrite (16 mmol) in water (20 mL) was added, under cooling and stirring, to a solution of 36 (4 mmol) in acetic acid (20 mL). Immediately afterward, the mixture was neutralized with 2 M NaOH and the green precipitate was collected and used, without purification, for the subsequent step. Yield: 30%; mp 152 °C dec. 2-(4-Methoxyphenyl)-3-nitrosoimidazo[2,1-b][1,3]benzothiazole (41). A solution of sodium nitrite (8.8 mmol) in water (10 mL) was added, under cooling and stirring, to a solution of 37 (4 mmol) in

acetic acid (20 mL). After 1 h at room temperature, the mixture was neutralized with 2 M NaOH, and the green precipitate was collected and crystallized from ethanol. Yield: 97%; mp 183.4−185.1 °C. Pharmacology. All animal experiments were performed in accordance with the principles for the care and use of laboratory animals for scientific purposes contained in the European Union regulations (Directive 2010/63/EU). Functional Studies. The functional profile of all of the compounds was derived on guinea pig isolated left and right atria to evaluate their inotropic and chronotropic effects, respectively, and on K+-depolarized (80 mM) guinea pig vascular aortic strips to assess calcium antagonist activity. Nonvascular calcium antagonist activity was obtained for some selected compounds, which were additionally tested on ileum longitudinal smooth muscle (GPILSM). Compounds were checked at increasing doses to evaluate the percent decrease of developed tension on isolated left atrium driven at 1 Hz (negative inotropic activity), the percent decrease in atrial rate on spontaneously beating right atrium (negative chronotropic activity), and the percent inhibition of calcium-induced contraction on K+-depolarized aortic strips and GPILSM (vascular and nonvascular relaxant activity respectively). Details were previously described.2,11 Experiments were also run for the LTCC blockers diltiazem, verapamil, and nifedipine in the copresence of compound 1 (0.01 μM for left atrium and 1 μM for aortic strips), which was added to the solution 30 min before the addition of the LTCC blocker. For all the experiments, data were analyzed using Student’s t test and are presented as mean ± S.E.M.17 Given that the drugs were added in a cumulative manner, the difference between the control and the experimental values at each concentration was tested for a P value 109.81 287.14 > 170.92

0.8 1.1

2C19

(S)-mephenytoin

(S)-mephenytoin-4′hydroxylation

40

235.2 > 150.2

1.0

2D6

dextromethorphan

dextromethorphan-O-demethylation

4′hydroxymephenytoin dextrorphan

10

258.2 > 157.05

0.9

3A4

midazolam

midazolam-1′-hydroxylation

1′-hydroxymidazolam

4

342.15 > 324.13

1.2

3A4

testosterone

testosterone-6β-hydroxylation

6βhydroxytestosterone

5

305.29 > 269.27

1.3

quinidine (0.009) ketoconazole (0.004) ketoconazole (0.001)

Menten constant) in the presence of multiple concentrations of the test compound (0.25−20 μM). The microsomal protein concentration was 0.1 mg/mL for CYPs 1A2, 2D6, and 3A4 incubations, and 0.2 mg/ mL for CYPs 2C9 and 2C19. The metabolic reaction was initiated by the addition of a NADPH-regenerating system (1 mg/mL NADP, 1 mg/mL glucose-6-phosphate, and 1 U/mL glucose-6-phosphate dehydrogenase) and MgCl2 (0.67 mg/mL) and quenched by the addition of ice-cold acetonitrile at the end of the incubation period (see Table 5). Quenched samples were centrifuged, and the probe metabolite concentrations in supernatant were quantified using LCMS (described below) relative to calibration standards prepared in the quenched microsomal matrix. LC-MS Analysis. For metabolic stability experiments, LC-MS analysis was conducted using a Waters Xevo TQD triple quadrupole mass spectrometer coupled to a Waters Acquity UPLC (Waters Corporation, Milford, MA). Mass spectrometry was conducted at a constant current of 15 μA, probe temperature of 550 °C, source temperature of 120 °C, and desolvation nitrogen flow of 1000 L/h. Elution of the analytes was monitored using MS/MS parameters optimized for each compound. The column was a Supelco Ascentis Express RP Amide column (50 × 2.1 mm, 2.7 μm particle size, Supelco, Bellefonte, PA) operated at 40 °C. Compounds were eluted with an acetonitrile−water gradient buffered with 0.05% formic acid and delivered at a flow rate of 0.4 mL/min. Separation was achieved under gradient conditions varying the acetonitrile content from 2% to 95% followed by re-equilibration to the starting conditions with a cycle time of 4 min. Processed samples were maintained in the autosampler at a temperature of 10 °C, and 5 μL was injected onto the column. The described conditions led to the elution of the compounds after 2.2−2.4 min (1, 2.44; 2, 2.43; 3, 2.64; 7, 2.21; 12, 2.21; 29, 2.42; and 30, 2.41). Compounds were quantified based on the compound to internal standard (diazepam) peak area ratio. For CYP inhibition experiments, LC-MS analysis was conducted using a Waters/Micromass Quattro Premier triple quadrupole mass spectrometer coupled to a Waters Acquity UPLC (Waters Corporation, Milford, MA). The triple quadrupole instrument was operated in positive mode electrospray ionization with a capillary voltage and detector multiplier voltage of 3.2 kV and 650 V, respectively, and source block and desolvation temperatures of 120 and 300 °C, respectively. Mass spectrometry was conducted at a column temperature of 40 °C on a Supelco Ascentis Express RP Amide (2.7 μm, 50 × 2.1 mm i.d.) column equipped with a Phenomenex Polar Security Guard column at a flow rate of 0.6 mL/min and injection volume of 3 μL for all compounds except for hydroxymidazolam which was conducted at a flow rate of 0.4 mL/min and injection volume of 3 μL. Elution of analytes was confirmed by multiple reaction monitoring (MRM), using diazepam as the internal standard, and the transitions are presented in Table 6. The concentrations of the specific metabolites formed were then determined by LC-MS against standard curves of authentic metabolite prepared in prequenched blank microsomal matrix. Taking the

washed twice with 5 mL of 20 mmol/L Tris-HCl. The radioactivity retained by filters was measured in a liquid scintillation counter (TRICARB 1100, PerkinElmer Life and Analytical Science, Monza, Italy) after the addition of 4 mL of scintillation fluid (Filter Count, PerkinElmer Life and Analytical Science, Monza, Italy), and all measurements were obtained in duplicate. (+)-[3H]Isradipine Binding Assays. The defrozen CM (0.15−0.2 mg/tube) were incubated in 50 mmol/L Tris-HCl plus 1.2 mmol/L MgCl2, pH 7.4, in a final volume of 1 mL at 37 °C for 20 min. (+)-[3H]Isradipine (82 Ci/mmol, PerkinElmer Life Science) was present at 0.1 nM in tubes containing increasing concentrations of unlabeled compound 1 (1 nM to 0.1 mM); nonspecific binding was defined using 0.1 mM isradipine.19 At the end of the incubation period, the samples were filtered through a Whatman GF/B glass filter presoaked in buffer plus 0.5% PEI (polyethylenimine) and processed as described above. All measurements were obtained in duplicate. Data are presented as the mean ± S.E.M., unless otherwise noted. Data from binding studies were corrected for nonspecific binding and were analyzed by computer-aided nonlinear regression analysis using a four parameter logistic equation in Prism 5.02 (GraphPad Software, San Diego, CA) to obtain the IC50 values. CYP-Mediated Metabolism. Metabolic Stability Studies. Metabolic stability of selected oxadiazolothiazinone and oxadiazolobenzothiazinone analogues was assessed in vitro by incubating at 37 °C with human liver microsomes (Xenotech, Lenexa, KS) suspended in 0.1 M phosphate buffer (pH 7.4) at a final compound concentration of 1 μM and microsomal protein concentration of 0.4 mg/mL. Metabolic reactions were initiated by the addition of an NADPHregenerating system (1 mg/mL NADP, 1 mg/mL glucose-6phosphate, and 1 U/mL glucose-6-phosphate dehydrogenase) and MgCl2 (0.67 mg/mL) and were quenched by the addition of ice-cold acetonitrile at five time points (2, 5, 15, 30, and 60 min). Compounds were also incubated in the absence of NADPH cofactor to monitor for noncytochrome P450-mediated metabolism in the microsomal matrix (2, 30, and 60 min). Quenched samples were centrifuged, and the clear supernate was analyzed to monitor the extent of compound loss using the LC-MS assay described below. Concentration versus time data for each compound were fitted to an exponential decay function to determine the first order rate constant for substrate depletion, which was then used to calculate the degradation half-life and an in vitro intrinsic clearance (CLint) value (mL/min/mg microsomal protein). A cocktail of known compounds (dextromethorphan, diclofenac, omeprazole, phenacetin, and verapamil) were included in the incubation alongside test compounds, and the degradation half-life (min) values observed (dextromethorphan, 107; diclofenac, 9; omeprazole, 73; phenacetin, 86; verapamil, 27) were in agreement with historical values, thereby validating the assay conditions employed. CYP Inhibition Studies. For each CYP isoform, the specific probe substrate reported in Table 5 was incubated with human liver microsomes (Xenotech, Lenexa, KS) suspended in 0.1 M phosphate buffer (pH 7.4) at a single concentration below the Km (Michaelis− K



Article

maximal rate of metabolite formation as that observed in the absence of any inhibitor, the percent reduction in the rate of metabolite formation was calculated for each concentration of test compound, and the percent inhibition data was then used to define the IC50 (deemed to be the concentration at which there was a 50% reduction in the amount of metabolite formed) for each test compound. Known reference inhibitors were included in the CYP inhibition assay as positive controls (see Table 5), the IC50 of which were used as acceptance criteria to assess the assay validity. In addition, control samples were included to confirm that the LC-MS assay of probe metabolites was not affected in the presence of each test compound and/or its metabolites.

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(5) (a) Billi, R.; Cosimelli, B.; Spinelli, D.; Rambaldi, M. Ring-ring interconversions. Part 2. Effect of the substituent on the rearrangement of 6-aryl-3-methyl-5-nitrosoimidazo[2,1-b][1,3]thiazoles into 8aryl-8-hydroxy-5-methyl-8H-[1,4]thiazino[3,4-c][1,2,4]oxadiazol-3ones. Tetrahedron 1999, 55, 5433−5440. (b) Cosimelli, B.; Frenna, V.; Rambaldi, M.; Severi, E.; Spinelli, D. On the reactivity of nitrosoimidazoles with acids (the Cusmano−Ruccia reaction): a continuous source of new ring-into-ring interconversion. Tetrahedron Lett. 2014, 55, 1488−1490 and references therein.. (6) Spinelli, D.; Budriesi, R.; Cosimelli, B.; Severi, E.; Micucci, M.; Baroni, M.; Fusi, F.; Ioan, P.; Cross, S.; Frosini, M.; Saponara, S.; Matucci, R.; Rosano, C.; Viale, M.; Chiarini, A.; Carosati, E. Playing with opening and closing of heterocycles: using the Cusmano-Ruccia reaction to develop a novel class of oxadiazolothiazinones, active as calcium channel modulators and P-glycoprotein inhibitors. Molecules 2014, 19, 16543−16572. (7) Hockerman, G. H.; Peterson, B. Z.; Johnson, B. D.; Catterall, W. A. Molecular determinants of drug binding and action on L-type calcium channels. Annu. Rev. Pharmacol. Toxicol. 1997, 37, 361−396. (8) Lacinová, L. Voltage-dependent calcium channels. Gen. Physiol. Biophys. 2005, 24 (Suppl 1), 1−78. (9) (a) Tikhonov, D. B.; Zhorov, B. S. Benzothiazepines in L-type Calcium channel: insights from molecular modeling. J. Biol. Chem. 2008, 283, 17594−17604. (b) Cheng, R. C. K.; Tikhonov, D. B.; Zhorov, B. S. Structural model for phenylalkylamine binding to L-type calcium channels. J. Biol. Chem. 2009, 284, 28332−28342. (c) Tikhonov, D. B.; Zhorov, B. S. Structural model for dihydropyridine binding to L-type calcium channels. J. Biol. Chem. 2009, 284, 19006−19017. (10) Kanda, S.; Kurokawa, J.; Adachi-Akahane, S.; Nagao, T. Diltiazem derivatives modulate the dihydropyridine-binding to intact rat ventricular myocytes. Eur. J. Pharmacol. 1997, 319, 101−107. (11) Carosati, E.; Cruciani, G.; Chiarini, A.; Budriesi, R.; Ioan, P.; Spisani, R.; Spinelli, D.; Cosimelli, B.; Fusi, F.; Frosini, M.; Matucci, R.; Gasparrini, F.; Ciogli, A.; Stephens, P. J.; Devlin, F. J. Calcium channel antagonists discovered by a multidisciplinary approach. J. Med. Chem. 2006, 49, 5206−5216. (12) (a) Cruciani, G.; Carosati, E.; De Boeck, B.; Ethirajulu, K.; Mackie, C.; Howe, T.; Vianello, R. MetaSite: understanding metabolism in human cytochromes from the perspective of the chemist. J. Med. Chem. 2005, 48, 6970−6979. (b) Cruciani, G.; Baroni, M.; Benedetti, P.; Goracci, L.; Fortuna, C. G. Exposition and reactivity optimization to predict sites of metabolism in chemicals. Drug Discovery Today: Technol. 2013, 10, e155−e165. (c) Metasite 4.1.0; Molecular Discovery Ltd.: Middlesex, UK. http://www.moldiscovery. com/soft_metasite.php. (13) Liao, P.; Yong, T. F.; Liang, M. C.; Yue, D. T.; Soong, T. W. Splicing for alternative structures of Cav1.2 Ca2+ channels in cardiac and smooth muscles. Cardiovasc. Res. 2005, 68, 197−203. (14) Andreani, A.; Rambaldi, M.; Bonazzi, D.; Lelli, G.; Bossa, R.; Galatulas, I. Substituted 6-phenylimidazo[2,1-b]thiazoles and thiazolines as potential cardiotonic agents. Eur. J. Med. Chem. 1984, 19, 219− 222. (15) Pentimalli, L.; Guerra, A. M. Reazioni di sostituzione e di addizione di 2-Fenil-imidazo[2,1-b]benzotiazolo. Gazz. Chim. Ital. 1967, 97, 1286−1293. (16) Mase, T.; Arima, H.; Tomioka, K.; Yamada, T.; Murase, K. Imidazo[2,1-b]benzothiazoles. 2. New immunosuppressive agents. J. Med. Chem. 1986, 29, 386−394. (17) Tallarida, R. J.; Murray, R. B. Manual of Pharmacologic Calculations with Computer Programs, 2nd ed.; Springer-Verlag: New York, 1987; pp 153−164. (18) (a) Motulsky, H. J.; Christopoulos, A. Fitting Models to Biological Data Using Linear and Nonlinear Regression. 2003. www. graphpad.com. (b) Motulsky, H. J. Prism 5 Statistics Guide; GraphPad Software Inc.: San Diego, CA, 2007; www.graphpad.com. (19) Teodori, E.; Baldi, E.; Dei, S.; Gualtieri, F.; Romanelli, M. N.; Scapecchi, S.; Bellucci, C.; Ghelardini, C.; Matucci, R. Design, synthesis, and preliminary pharmacological evaluation of 4-amino-

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b00030. Spectroscopic data for compounds 7−10, 12−14, and 16−30 and inhibition of [3H]PN200−110 (isradipine) specific binding by compound 1 in rat cardiomyocites (PDF) SMILES data (CSV)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +39 75 5855550. Fax: +39 75 45646. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Susan A. Charman (Monash University) for helpful discussions and critical comments on the manuscript. We thank A. Casoni (University of Bologna, Italy) for animal care. The research leading to these results has received funding from University of Bologna (Italy) and from Istituto Nazionale per le Ricerche Cardiovascolari (INRC, Italy). E.C. was supported by the European Union Seventh Framework Programme (FP7/2007-2013) under the Marie Curie Actions (Co-funding of Regional, National and International Programmes − COFUND) project I-MOVE grant agreement no. [267232].

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ABBREVIATIONS USED HLM, human liver microsomes; APCI, atmospheric pressure chemical ionization REFERENCES

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M


Understanding Oxadiazolothiazinone Biological Properties: Negative

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