Na+ Currents in Cardioprotection: Better to Be Late - ACS Publications

J. Med. Chem. 2009, 52, 4149–4160 4149 DOI: 10.1021/jm900296e

Na+ Currents in Cardioprotection: Better to Be Late Bruno Le Grand,† Christophe Pignier,† Robert Letienne,† Francis Colpaert,§ Florence Cuisiat,‡ Franc- oise Rolland,‡ Agnes Mas,‡ Maud Borras,‡ and Bernard Vacher*,‡ †

Cardiovascular 2 Division and ‡Medicinal Chemistry 1 Division, Pierre Fabre Research Center, 17 avenue Jean Moulin, 81106 Castres Cedex, France, and §Toulouse ISTMT 2, 3 rue des Satellites, 31400 Toulouse

Received March 9, 2009

We report the discovery of a selective, potent inhibitor of the late current mediated by the cardiac isoform of the sodium channel (NaV1.5). The compound, 3,4-dihydro-N-[(2S)-3-[(2-hydroxy-3-methylphenyl)thio]-2-methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2d) (F 15741), blocks the late component of the Na+ currents and greatly reduces veratridine- or ischemia-induced contracture in isolated tissue and whole heart. The cardioprotective action of 2d was further established in a model of myocardial infarction in the pig in which 2d prevents ischemia-reperfusion damage after 60 min of coronary occlusion and 48 h reperfusion. Under these experimental conditions, only 2d and cariporide reduce infarct size. Remarkably, myocardial protection afforded by 2d occurs in the absence of hemodynamic effects. These data expand the therapeutic potential of late INa blockers and suggest that 2d could be useful in pathologies for which pharmacological treatments are not yet available.

Introduction We have previously described compound 1 (F 15845, Figure 1) as the first selective, potent, and voltage-dependent inhibitor of the late current mediated by the cardiac sodium channel NaV1.5a.1 The compound reduces the mechanical dysfunction induced by ischemia, hypoxia and toxins and reverses electrocardiographic abnormalities following transient coronary artery occlusion in anesthetized rabbits and dogs.2 In addition, the anti-ischemic activity of 1 is not associated with any concomitant changes in blood pressure, heart rate, inotropic status, or vascular tone. Consequently, comedication with agents that mediate their therapeutic benefits by a hemodynamic action remains an option (e.g., β-blockers, organic nitrates, etc.).3 The physiological importance of slow inactivation of voltage gated sodium channels has been established in various forms of inherited skeletal muscle,4 cardiac muscle,5 and central and peripheral neuronal6 diseases or disorders in man. Only recently, however, have the merits of a late INa blocker (i.e., ranolazine)7-9 been acknowledged in the management of chronic angina10 and arrhythmias.11 In those indications, however, the added benefits (if any) of a more selective and more potent late INa blocker than ranolazine are largely unknown.12 We now provide evidence that the action of the selective late INa blocker 2d (F 15741, Figure 2) goes beyond that of the classical of anti-ischemics, as 2d directly protects the heart muscle against acute myocardial infarction, offering thus novel prospects for therapy.13 *To whom correspondence should be addressed. Phone: (+33) 56371 4222. Fax: (+33) 56371 4299. E-mail: bernard.vacher@pierre-fabre. com. a Abbreviations: INa, inward sodium current; NaV, voltage-gated sodium channel; AP, action potential; TTX, tetrodotoxin; VIDC, veratridine induced diastolic contracture; HEK, human embryonic kidney; HP, holding potential; LPH, Langendorff-perfused heart; CABG, coronary artery bypass graft surgery. r 2009 American Chemical Society

During ischemia, a fraction of the Na+ channels either fails to inactivate or reopens inappropriately, amplifying the longlasting, voltage-dependent inward Na+ current referred to as late INa. By preventing this influx of sodium, late INa inhibitors attenuate cytosolic Na+ accumulation14 and, indirectly, the uptake and build-up of Ca2+, which has devastating consequences during both ischemia and reperfusion phases.15 In the course of our studies on a novel series of late INa blockers,1 we noticed that structurally close, selective and, a priori, equipotent veratridine-INa blockers can yet produce divergent responses in models addressing different aspects of myocardial ischemia (e.g., ECG abnormalities, rhythmic disturbances or infarct size). Such context-dependent activity suggests that the pharmacological profile of the compound is, in some manner, conditioned by the “quality” of the inhibition of the late current. On the basis of these observations, we redirected our efforts and targeted the discovery of INa blockers tailored for cardioprotection, an area where no pharmacological intervention has proved successful in spite of decades of research and a plethora of drugs investigated.16 SAR mapping carried out on the structural archetype of this series (Figure 3) reveals that, in addition to the propyl chain,1 the aromatic tail is another site amenable to chemical modulations. Prospecting in this direction yielded compounds that exhibited the sought-after reinforced cardioprotective properties. This effort culminated in the selection of the development candidate 2d (Figure 2). Here, we focus on the process that led to the discovery of 2d, touch upon some intriguing mechanistic issues that are raised by this compound, and illustrate its potential as cardioprotectant in comparison with various reference agents (cf. Figure 1). Chemistry Two synthetic routes were used for the preparation of compounds of the type 2. The first one, depicted in Scheme 1 Published on Web 06/10/2009

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Figure 1. Agents used as references.

Figure 2. Structure of compound 2d (F 15741).

Figure 3. Structural archetype of the series.

and 2, involved alkylation of the appropriate arythiol or protected arylthiol 3 with (S)-3-bromo-2-methylpropanol under basic conditions to give the corresponding thioether 4.1 Oxidation of the primary alcohol function in 4 under modified Swern conditions17 led to the sensitive aldehyde 5, which was not isolated but carried forward into the reductive amination step with 3-(R)-amino-benzoxathiepine 618 to afford compound 2.19 Some compounds of the type 2 required an extra step (i.e., 2d-f, 2g-i, 2k-l) to access the final material. Alternatively (Scheme 3), reaction between (R)-3-amino-1,5benzoxathiepine 6, activated as its aluminum salt, and (R)-3hydroxy-2-methylpropionate, yielded the amide 7.20 Mesylation of the primary alcohol function in 7 then displacement of the methanesulfonyloxy group with the appropriate arylthiol (or disulfide precursor) produced the thioether 9. Subsequent reduction of the amide delivered the target compound 2.21 Compound 2r was obtained from 2j by cyclization with triethylorthoformate.22 Among the arylthiols (or disulfides) used in this work, a few are commercially available (3a, 3b) and some are known: 2methoxy-3-methylbenzenethiol (3c),23 2-hydroxy-3-methylbenzenethiol (3d),24 2-hydroxy-3-ethylbenzenethiol (3e),25 2-hydroxy-3-isopropylbenzenethiol (3f),25 2-amino-3-methylbenzenethiol (3l),26 2-amino-3-methoxybenzenethiol (3m),27 2-amino-3-fluorobenzenethiol (3n),28 and 2,3-dihydrobenzofuran-7-thiol (3o);25 the others (i.e., 3h, 3i, 3p-q, and 3t) were prepared as described in Supporting Information. Results and Discussion Exposure of HEK293 cells expressing hNaV1.5 R-subunits to veratridine (40 μM) induces a 100-fold enhancement in the late INa measured at 340 ms after initiation of the depolarizing pulse, that is, long after recovery from the peak INa current.29 Typically, treatment of such a preparation with late INa

inhibitors reduces the amplitude of this noninactivating current.30 For compound selection, effects on the veratridineevoked INa currents were tested at a single concentration (10 μM) but from two holding membrane potentials (HP) in order to appreciate any HP dependence of the block. At both HP values, the selective Na+ blocker TTX nearly abolishes late INa, while the selective late INa blocker 1 markedly represses it (Table 1). Removal of the etheral-oxygen atom (2a), introducing a larger O-alkyl group (2b) or a substituent in position 3 (2c) all have a negative impact on late INa inhibition relative to 1. In contrast, a free OH in 2-position was clearly beneficial, 2d being as efficacious as TTX and significantly more so than 1 at suppressing late INa. When R1 = OH, recovery from slow inactivation varies inversely with the size of R2; decreasing from Me (2d) to i-Pr (2f). Activity diminishes upon moving the 2-OH group one carbon away from the ring (2g vs 2d), indicating that mesomeric donation alone is insufficient to account for the phenolic-OH contribution. Switching the phenol for aniline (R1 = NH2) is counterproductive and remains so whatever the nature of R2 (cf. 2l, 2m, 2n). Likewise, attempts at bioisosteric replacement of phenol (2s and 2t, Table 2) were of no avail. Among the fused heterocycles, only monoheteroatomic oxygenated ones (2o and 2p) have the ability to reverse veratridine-induced late INa. Overall, a phenolic-OH at C-2 boosted late INa inhibition. The role of the group at C-3 seems to be merely steric inasmuch as electron-donating (2h) or -withdrawing groups (2i) gave comparable results and size is tightly controlled (2d vs 2f). HP determines the state of the Na+ channels in the membrane. At -110 mV, channels exist primarily in their resting state (available for opening), whereas at -90 mV, a substantial proportion of them are inactivated. Because blockade of late INa is generally more pronounced at -90 than at -110 mV, it seems that most compounds of this series stabilize channels in their inactivated state, decreasing their probability of opening. Of the derivatives listed in Tables 1 and 2, compounds 2d and 2p stand out, matching the inhibition attained with TTX. Further, 2d distinguishes between late and peak INa currents (data not shown), which is critical for the normal functioning of the heart.31 Note that, in nonischemic cardiac cells, the peak INa carries most of the current.1 To simplify matters, we ascribe the difference seen in the activity patterns of analogues to a conformational selection effect; assuming that different compounds prefer certain protein conformation(s) among those that populate the long-lasting, slow-inactivating state of the channel. This, in turn, should influence the compounds blocking characteristics and, therefore, their pharmacological responses under pathological conditions.

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Scheme 1. Synthesis of Compounds 2a-c, 2ga

a

Reagents and conditions: (i) base, THF, or DMF; (ii) Swern oxidation; (iii) compound 6, NaBH(OAc)3, C2H4Cl2; (iv) LiAlH4, THF.

Scheme 2. Synthesis of Compounds 2d-f, 2h,i, 2k,l, 2o-q, 2s,ta

Reagents and conditions: (i) Swern oxidation; (ii) compound 6, NaBH(OAc)3, C2H4Cl2; (iii) HCl (4 N), EtOH, 60 °C; (iv) HCl (12 N), MeOH, 60 °C; (v) NH4OH, MeOH. a

During ischemia, late INa contributes to ionic and mechanical perturbations. When cardiac tissue (e.g., isolated left atrium) is exposed to veratridine, the initial Na+ overload evolves into a contraction-relaxation failure signaled by the occurrence of a diastolic contracture. The latter is only slowly reversible and resistant to inhibition by conventional antiischemic agents, peak and nonselective sodium currents blocking drugs such as class I antiarrhythmics.32 Now the fact that several compounds in this series (cf. Tables 1 and 2) can offset that contracture unequivocally dissociates their mechanism from that of above-mentioned drugs. Here, only inhibitions greater than 40% in both of the veratridine-based procedures are taken as evidence for pharmacodynamic action at late INa. On this basis, 2b and 2t were discarded and the superiority of phenol over aniline was confirmed. In the end, only a few compounds (2d, 2h, 2k, 2o, and 2q) achieve a level of efficacy in the range of that of 1. In terms of stereochemistry-activity relationship, aromatic and chain-modified compounds follow the same

pattern, thus we pursued structure-activity studies with a single enantiomer. Compounds were next probed in Langendorff-perfused guinea pig hearts (abbreviated LPH hereafter).33 In this model of global ischemia, we assessed the ability of the compounds to oppose the diastolic contracture that develops upon prolonged interruption of the coronary flow. Compounds in Tables 1 and 2 can be separated in two groups: one encompassing derivatives the activities of which in VIDC and LPH diverge (i.e., 2e, 2g, 2h, 2k, 2m, 2n, 2q-r, 2t), and another, which is composed of derivatives efficacious in both models (i.e, 2d, 2i, 2o, 2p). Among the latter derivatives, 2d and 2o show even greater activity than 1 and do so without altering baseline hemodynamic or contractile parameters.34 The lack of a direct correlation between veratridine- and ischemia-promoted contracture can be understood if, in LPH, mechanisms unrelated to late INa can also trigger a contracture or if veratridine evoked Na+ currents only imperfectly mimic native late INa.35


Overall, compound 2d met all the criteria defined at the outset: it produces high anti-ischemic effects in different in vitro models and does so by selectively blocking late INa.31 To evaluate whether 2d also protects cardiomyocytes in vivo, we examined its activity in a pig model of myocardial ischemiareperfusion (Figure 4);36 ischemia was induced by a 60 min left circumflex coronary occlusion followed by 48 h reperfusion. Such a time frame is particularly suited for the determination of infarct size in a precise and robust manner.37 In the nonischemic area, regional blood flows were similar throughScheme 3. Synthesis of Compounds 2j, 2m,n, and 2ra

a Reagents and conditions: (i) DIBAH, THF; (ii) CH3SO2Cl, NEt3, THF; (iii) ArSH or (ArS)2, PPh3, K2CO3, THF, 60 °C; (iv) NaBH4, BF3. Et2O, THF, reflux; (v) HC(OEt)3, EtOH, 80 °C.

Le Grand et al.

out the experiment, whereas circulation was absent in the ischemic zone. In this study, the test compound was administered intravenously as a bolus 10 min before ischemia and then as a continuous infusion for the duration of the occlusion (60 min) and the first 30 min of reperfusion. Under those conditions, cariporide38 significantly reduced infarct volume to 6.8% ( 2.4, measured at the end of the reperfusion period as compared with vehicle-treated controls (27.7% ( 3.0). This result is interesting, bearing in mind that cariporide has shown a beneficial effect in patients undergoing coronary artery bypass graft surgery (CABG).39 The nonselective sodium channel blocker lidocaine40 was not only ineffective but also increased the incidence of ventricular fibrillation and death; data in line again with clinical observations.41 The If inhibitor ivabradine42 exerts no protection against cell death, contrary to reports in a low-flow, short-reperfusion pig model at a dose equivalent to that used in this work.43 Remarkably, compound 2d reduces infarct size to 3.5% ( 0.6, that is, 2d salvages up to 90% of the area rendered ischemic that otherwise would undergo necrosis (“area at risk”). Thus, hearts exhibited hardly any detectable infarct at all.44 Further, at the dose tested,45 treatment with 2d is not associated with modifications of blood pressure, heart rate, or oxygen consumption, supporting a direct cardioprotective action. These morphological data are backed by measures of plasma levels of troponin I during reperfusion.46 That is, the reduction of infarct size observed with 2d and cariporide is accompanied by a significant decrease in the release of troponin I in the circulation at 6 h postreperfusion (Figure 4).47 In contrast, compounds that do not preserve cardiac cell viability also fail to lower troponin I levels. Overall, the magnitude of the cardioprotection achieved with 2d outperformed that of cariporide and other reference

Table 1. In Vitro SAR, Acyclic Groups R1 and R2

hNav1.5 channel blockade,a % inhibition at 10 μMe

diastolic contracture, % inhibition at 1 μMe

compd

R

R

HP -110 mV

HP -90 mV

rat atrium (VI)b,c

GP intact heartd

1 2a 2b 2c 2d 2e 2f 2g 2h 2i 2j 2k 2l 2m 2n TTX

OMe Me OEt OMe OH OH OH CH2OH OH OH OH OH NH2 NH2 NH2

H H H Me Me Et i-Pr Me OMe F NH2 CONH2 Me OMe F

53 ( 7 11 ( 5 41 ( 3 28 ( 7 73 ( 9 56 ( 11 38 ( 10 45 ( 6 35 ( 6 38 ( 6 11 ( 5 59 ( 4 26 ( 7 13 ( 10 11 ( 8 79 ( 2

70 ( 5 14 ( 9 54 ( 4 32 ( 6 87 ( 8 56 ( 10 46 ( 8 58 ( 7 46 ( 6 40 ( 8 19 ( 6 65 ( 5 26 ( 5 18 ( 11 19 ( 5 87 ( 3

74 ( 3 3 ( 12 33 ( 10 30 ( 14 93 ( 5 46 ( 7 20 ( 12 58 ( 5 77 ( 11 68 ( 3 25 ( 14 72 ( 12 49 ( 4 55 ( 7 56 ( 9 64 ( 10

65 ( 11 NDh NDh NDh 70 ( 6 13 ( 11 NDh 31 ( 18 46 ( 9 54 ( 13 NDh 43 ( 17 35 ( 6 21 ( 8 33 ( 11 NDh

1

2

f

g

a Veratridine (40 μM) induced late sodium current in HEK cells expressing human Nav1.5 channels. b Veratridine (40 μM) induced (VI) diastolic contracture in rat isolated left atrium. c A contracture of 40% was used as cutoff value. d Global ischemia induced contracture (50 min) in isolated Langendorff-perfused guinea pig (GP) heart. e Compound concentration used in the experiment. f Percentage of inhibition of late sodium current elicited at -30 mV from a holding potential (HP) of -110 mV. g Percentage of inhibition of late sodium current elicited at -30 mV from a holding potential of -90 mV. h ND: not determined.

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Table 2. In Vitro SAR, Fused Heterocycles

hNav1.5 channel blockadea, % inhibition at 10 μMe

diastolic contracture, % inhibition at 1 μMe

compd

X

Y

Z

HP -110 mVf

HP -90 mVg

rat atrium (VI)b,c

GP intact heartd

2o 2p 2q 2r 2s 2t Cariporide TTX

O O O O NH NH

CH2 CH CH2 CH CH N

CH2 CH O N CH CH

47 ( 21 73 ( 7 32 ( 6 16 ( 13 8(4 34 ( 5

56 ( 21 82 ( 4 54 ( 5 26 ( 15 8(2 44 ( 11

79 ( 2

87 ( 3

85 ( 5 46 ( 10 71 ( 6 34 ( 9 5(6 39 ( 13 0 ( 11 64 ( 10

72 ( 12 68 ( 14 22 ( 13 84 ( 6 NDh 73 ( 12 6(9 NDh

Veratridine (40 μM) induced late sodium current in HEK cells expressing human Nav1.5 channels. b Veratridine (40 μM) induced (VI) diastolic contracture in rat isolated left atrium. c A contracture of 40% was used as cutoff value. d Global ischemia induced contracture (50 min) in isolated Langendorff-perfused guinea pig (GP) heart. e Compound concentration used in the experiment. f Percentage of inhibition of late sodium current elicited at -30 mV from a holding potential (HP) of -110 mV. g Percentage of inhibition of late sodium current elicited at -30 mV from a holding potential of -90 mV. h ND: not determined. a

Figure 4. Upper panel: Histochemical determination of infarct size after 60 min ischemia and 48 h reperfusion in pigs. Infarct size is expressed as the percentage of necrotic area versus area at risk (empty circles represent individual pigs). Lower panel: Plasma levels of troponin I at the peak of concentration 6 h postreperfusion. Data are means ( SEM *P < 0.05, ***P < 0.001 versus vehicle.

agents.48 To be effective, 2d has to be applied before the occlusion, which is consistent with its mode of action. Such schedule considerations have to be qualified, however, first because these experiments were conducted in otherwise

healthy pigs/hearts in which there should be no enhancement in late Na+ current until the onset of ischemia, second, because the compound cannot reach its target in the ichemic zone during the ischemic period (no blood flow and


no collateral circulation). Such conditions may be more stringent than real-life ones where late INa is likely to be constitutively heightened and perfusion in the ischemic territory may be facilitated by collateral networks.49 In any case, 2d may be more adapted for treatment of patients presenting a risk of myocardial infarction (MI) and/or for the secondary prevention of MI and/or for patients undergoing interventions involving a period of ischemia.50 Conclusions Derivatives of this novel family of late sodium channel blockers display a surprising broad spectrum of activity, stretching from ECG normalization to cell protection. This indeed supports a major role for late INa in ischemia. Among the compounds described here, 2d was instrumental in revealing the infarct-sparing potential of this class of inhibitors. Further, contrary to conventional Na+ channel blockers and most known late INa inhibitors, 2d exhibited NaV1.5 isoform and late current selectivity, two features that are essential for limiting off-target undesired effects. It emerges that 2d regulates late INa in such a way that cell protection overrides other anti-ischemic properties, rendering its action complementary in scope to that of compound 1. At the isolated organ level, we found that 2d counteracts the contracture resulting from either impaired Na+ conductance or an ischemic insult. Likewise, in a severe model of acute myocardial ischemia-reperfusion in pig, 2d prevents postreperfusion injury whereas, among reference compounds, only cariporide achieved a lesser degree of protection. We believe that late INa inhibition is the primary mechanism by which 2d exerts its cardioprotective activity.31 However, the site of action of 2d on NaV1.5 channels as well as the biophysical characteristic(s) responsible for its innovative profile are yet to be identified. Clearly, 2d may provide a new opportunity in the treatment of conditions for which medical needs are considerable like myocardial infarction, cardiac surgery, cardiac insufficiency, and heart failure. Experimental Section Chemistry. Melting points were determined on a Buchi 530 melting point apparatus and were not corrected. 1H NMR spectra were recorded on a Bruker Avance 400 spectrometer operating at 400 MHz for 1H and 100 MHz for 13C. Chemical shifts are reported in δ value (ppm) relative to an internal standard of tetramethylsilane. Infrared (IR) spectra were obtained on a Nicolet FT 510 P spectrophotometer. Microanalyses were obtained on a Fison EA 1108/CHN analyzer. Analytical thin-layer chromatography were carried out on precoated plates (silica gel, 60 F 254 Merck). Optical rotations were measured on a Perkin-Elmer 241 model polarimeter. The purity of final compounds were established by HPLC using Xbridge 8.5 μm, 4.6 mm 250 mm reverse phase column at a flow rate of 1 mL/min. The compounds were detected at 220 nm. The purity of final products was g95% unless otherwise noted. Commercially available products were used without further purification. General Method for the Reductive Amination between Aldehyde 5 and (R)-3-amino-1,5-benzoxathiepine (6). A solution of (R)-3-amino-1,5-benzoxathiepine 6 (1 equiv) in dry dichloromethane (10 mL per g) was added to the crude solution of the aldehyde 5 cooled at -20 °C. The reaction mixture was stirred for 20 min and then treated with sodium triacetoxyborohydride (1.2 equiv) and stirring was continued for 2 h at -10 °C. The reaction mixture was poured into a 10% aqueous sodium hydrogen carbonate solution and extracted with dichloromethane. The combined organic layer was washed with water,

Le Grand et al.

brine, dried over Na2SO4, filtered, and concentrated in vacuo. The free base was purified by flash column chromatography. General Method for the Reduction of Amide 9 into Amine 2. To a suspension of NaBH4 (5 equiv) in THF (10 mL per 100 mg) under inert atmosphere and cooled at 0 °C was added dropwise boron trifluoroetherate (5 equiv) and the mixture stirred for 15 min. A solution of the amide 9 in THF (10 mL per g) was then introduced and the mixture heated at reflux until completion. Methanol was added slowly to the reaction mixture at room temperature, and the mixture was concentrated under vacuo. Further methanol and HCl (12 N) were added, and the mixture refluxed for 1 h and then cooled to room temperature and stirred overnight. The mixture was concentrated under vacuo and then the residue taken up in water, neutralized with NaOH (20%), and then extrated with dichloromethane. The combined organic layer was washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The free base was purified by flash column chromatography. 3,4-Dihydro-N-[(2S)-3-[(2-methylphenyl)thio]-2-methylpropyl]2H-(3R)-1,5-benzoxathiepin-3-amine (2a). The free base was purified by flash column chromatography (silica gel, dichloromethane/ethylacetate, 95:5). Crystallization of 2a as a maleate salt gave a white powder mp = 144 °C. IR (KBr): 2969, 1604, 1571, 1447, 1354 ν cm-1. 1H NMR (DMSO-d6) δ 1.12 (d, J = 6.8 Hz, 3H), 2.15-2.18 (m, 1H), 2.31 (s, 3H), 2.87 (dd, J =12.8, 8.0 Hz, 1H), 3.01 (dd, J=12.4, 7.2 Hz, 1H), 3.14-3.23 (m, 2H), 3.27-3.35 (m, 2H), 3.86 (s, 1H), 4.34 (d, J = 13.6 Hz, 1H), 4.45 (d, J =13.6 Hz, 1H), 6.04 (s, 2H), 6.91-6.93 (m, 3H), 7.21-7.29 (m, 3H), 7.34 (d, J=7.8 Hz, 1H), 7.39 (d, J=7.6 Hz, 1H). 13C NMR (DMSO-d6) δ 17.3 (CH3), 19.9 (CH3), 30.5 (CH), 31.0 (CH2), 36.1 (CH2), 49.2 (CH2), 57.7 (CH), 70.4 (CH2), 121.9 (CH), 124.1 (CH), 125.4 (CH), 126.2 (C), 126.6 (CH), 127.1 (CH), 128.8 (CH), 130.0 (CH), 131.5 (CH), 135.3 (C), 135.6 (2CH), 136.2 (C), 159.0 (C), 167.2 (2C); [R]D25 13.5° (c 0.39, CH3OH). HPLC: eluting with acetonitrile/water/ KH2PO4, 400:600:6.8 g, tR 13.3 min. Anal. (C20H25NS2O 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[(2-ethoxyphenyl)thio]-2-methylpropyl]2H-(3R)-1,5-benzoxathiepin-3-amine (2b). The free base was purified by flash column chromatography (silica gel, dichloromethane/ethylacetate, 96:4). Crystallization of 2b as a fumarate salt gave a white powder mp = 124 °C. IR (KBr) ν 2980, 2933, 1577, 1473, 1441, 1239 cm-1. 1H NMR (DMSO-d6) δ 1.00 (d, J=6.8 Hz, 3H), 1.34 (t, J=6.8 Hz, 3H), 1.78 (m, J= 6.4 Hz, 1H), 2.58 (dd, J = 13.0, 6.4 Hz, 1H), 2.65-2.70 (m, 3H), 2.87 (dd, J=13.0, 6.8 Hz, 1H), 3.06-3.14 (m, 3H), 3.96 (dd, J= 12.4, 4.4 Hz, 1H), 4.05 (q, J = 7.2 Hz, 2H), 4.17 (dd, J = 12.4, 2.8 Hz, 1H), 6.61 (s, 2H), 6.91-7.00 (m, 4H), 7.11-7.18 (m, 2H), 7.26 (d, J = 6.8 Hz, 1H), 7.34 (d, J = 6.8 Hz, 1H). 13C NMR (DMSO-d6) δ 14.6 (CH3), 17.6 (CH3), 32.6 (CH), 33.9 (CH2), 35.4 (CH2), 50.9 (CH2), 57.7 (CH), 63.7 (CH2), 73.4 (CH2), 111.7 (CH), 120.9 (CH), 121.9 (CH), 123.6 (CH), 125.0 (C), 126.3 (CH), 127.3 (C), 127.6 (CH), 128.4 (CH), 131.5 (CH), 134.3 (2CH), 155.6 (C), 159.6 (C), 166.6 (2C); [R]D25 0° (c 0.26, CH3OH). HPLC: eluting with acetonitrile/water/KH2PO4, 400:600:6.8 g, tR 14.03 min. Anal. (C21H27NS2O2 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[(2-methoxy-3-methylphenyl)thio]-2-methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2c). The free base was purified by flash column chromatography (silica gel, dichloromethane/methanol, 99:1). Crystallization of 2c as a fumarate salt gave a white powder mp = 107 °C. IR (KBr) ν 2933, 1572, 1473, 1458, 1353 cm-1. 1H NMR (DMSO-d6) δ 1.11 (d, J = 6.8 Hz, 3H), 2.17 (m, 1H), 2.30 (s, 3H), 2.84 (m, 1H), 3.00 (m, 1H), 3.11-3.28 (m, 4H), 3.28 (m, 1H), 3.71 (s, 3H), 3.84 (m, 1H), 4.37 (m, 1H), 4.47 (m, 1H), 6.05 (s, 2H), 7.03-7.08 (m, 4H), 7.16 (t, J = 4.6 Hz, 1H), 7.25 (t, J = 8.0 Hz, 1H), 7.39 (d, J = 7.2 Hz, 1H). 13C NMR (DMSO-d6) δ 15.6 (CH3), 17.3 (CH3), 30.4 (CH), 31.3 (CH2), 35.0 (CH2), 50.0 (CH2), 57.6 (CH), 59.1 (CH3), 70.4 (CH2), 121.8 (CH), 124.5 (CH), 125.0 (CH), 125.2 (CH), 126.4

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(C), 128.2 (CH), 128.7 (CH), 129.9 (C), 130.0 (C), 131.4 (CH), 135.6 (2CH), 155.1 (C), 159.2 (C), 167.1 (2C); [R]D25 12.4° (c 0.26, CH3OH). HPLC: eluting with acetonitrile/water/KH2PO4, 400:600:6.8 g, tR 13.39 min, 91.6%; Anal. (C21H27NS2O2 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[(2-hydroxy-3-methylphenyl)thio]-2methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2d). The free base was purified by flash column chromatography (silica gel, dichloromethane). Crystallization of 2d as a fumarate salt gave a white powder mp = 101-105 °C. IR (KBr) ν 3420, 2966, 1691, 1654, 1470, 1229 cm-1. 1H NMR (DMSO-d6) δ 0.98 (d, J = 6.8 Hz, 3H), 1.75 (m, 1H), 2.16 (s, 3H), 2.54 (dd, J = 12.4, 6.0 Hz, 1H), 2.61-2.68 (m, 2H), 2.84 (dd, J = 12.0, 6.8 Hz, 1H), 2.95 (dd, J = 12.4, 7.2 Hz, 1H), 3.06-3.10 (m, 2H), 3.95 (dd, J = 12.0, 5.6 Hz, 1H), 4.16 (dd, J = 12.4, 3.2 Hz, 1H), 6.61 (s, 2H), 6.72 (t, J = 7.6 Hz, 1H), 6.98 (t, J = 7.6 Hz, 3H), 7.13-7.20 (m, 2H), 7.33 (d, J = 8.0 Hz, 1H). 13C NMR (DMSO-d6) δ 16.5 (CH3), 17.7 (CH3), 32.8 (CH), 34.2 (CH2), 37.9 (CH2), 50.9 (CH2), 57.6 (CH), 73.6 (CH2), 119.7 (CH), 121.8 (CH), 122.2 (C), 123.5 (CH), 124.7 (C), 127.3 (C), 128.3 (CH), 128.6 (CH), 129.2 (CH), 131.4 (CH), 134.6 (2CH), 153.7 (C), 159.6 (C), 166.3 (2C); [R]D25 6.6° (c 0.48, CH3OH). HPLC: eluting with acetonitrile/water/KH2PO4, 400:600:6.8 g, tR 9.75 min. Anal. (C20H25NS2O2 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[(2-hydroxy-3-ethylphenyl)thio]-2-methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2e). The free base was purified by flash column chromatography (silica gel, cyclohexane/ethylacetate, 85:15). Crystallization of 2e as a maleate salt gave a white powder mp = 120 °C. IR (KBr) ν 3405, 2966, 2934, 1572, 1446, 1353 cm-1. 1H NMR (DMSO-d6) δ 1.09-1.14 (m, 6H), 2.09 (m, J = 6.0 Hz, 1H), 2.58 (q, J = 7.6 Hz, 2H), 2.76 (dd, J = 12.8, 7.6 Hz, 1H), 2.95-3.02 (m, 2H), 3.19 (dd, J = 12.0, 6.0 Hz, 1H), 3.28 (d, J = 5.2, 2H), 3.81 (s, 1H), 4.36 (d, J = 13.2 Hz, 1H), 4.45 (d, J = 12.0 Hz, 1H), 6.05 (s, 2H), 6.80 (t, J = 7.6 Hz, 1H), 7.00-7.09 (m, 3H), 7.17 (d, J = 7.6 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.25 (d, J = 7.6 Hz, 1H), 8.58 (s, 2H). 13C NMR (DMSO-d6) δ 14.1 (CH3), 17.2 (CH3), 22.9 (CH2), 30.6 (CH), 31.1 (CH2), 37.4 (CH2), 49.8 (CH2), 57.6 (CH), 70.4 (CH2), 120.1 (CH), 121.8 (C), 121.9 (CH), 124.1 (CH), 126.7 (C), 127.6 (CH), 128.7 (CH), 128.8 (CH), 130.9 (C), 131.4 (CH), 135.5 (2CH), 153.1 (C), 158.9 (C), 167.1 (2C); [R]D25 1.4° (c 0.23, CH3OH). HPLC: eluting with acetonitrile/water/ KH2PO4, 400:600:6.8 g, tR 9.75 min. Anal. (C21H27NS2O2 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[(2-hydroxy-3-i-propylphenyl)thio]-2methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2f). The free base was purified by flash column chromatography (silica gel, cyclohexane/ethylacetate, 80:20). Crystallization of 2f as a maleate salt gave a white powder mp = 126 °C. IR (KBr) ν 3410, 2962, 1572, 1473, 1438, 1353 cm-1. 1H NMR (DMSO-d6) δ 1.09 (d, J = 6.4 Hz, 3H), 1.15 (d, J = 6.8 Hz, 6H), 2.09 (m, J = 6.0 Hz, 1H), 2.75 (dd, J = 12.4, 7.6 Hz, 1H), 2.96-3.00 (m, 2H), 3.16 (m, 1H), 3.20-3.27 (m, 3H), 3.78 (s, 1H), 4.35 (d, J = 13.2 Hz, 1H), 4.44 (d, J = 13.2 Hz, 1H), 6.04 (s, 2H), 6.84 (t, J = 7.6 Hz, 1H), 7.06-7.08 (m, 3H), 7.17 (d, J = 7.6 Hz, 1H), 7.24 (t, J = 7.6 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 8.52 (s, 2H). 13C NMR (DMSO-d6) δ 17.2 (CH3), 22.6 (2CH3), 26.4 (CH), 30.7 (CH), 31.1 (CH2), 37.7 (CH2), 49.8 (CH2), 57.6 (CH), 70.4 (CH2), 120.3 (CH), 121.8 (CH), 122.1 (C), 124.0 (CH), 124.8 (CH), 124.9 (C), 126.2 (C), 128.6 (CH), 128.7 (CH), 131.4 (CH), 135.5 (2CH), 152.5 (C), 158.6 (C), 167.1 (2C); [R]D25 4.1° (c 0.24, CH3OH). HPLC: eluting with acetonitrile/water/KH2PO4, 400:600:6.8 g, tR 21.23 min. Anal. (C22H29NS2O2 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[(2-hydroxymethyl-3-methylphenyl) thio]-2-methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2g). The free base was purified by flash column chromatography (silica gel, dichloromethane/methanol, 98:2). Crystallization of 2g as a maleate salt gave a white powder mp = 102-104 °C. IR (KBr) ν 3405, 1572, 1474, 1447, 1353 cm-1. 1H NMR (DMSOd6) δ 1.09 (d, J = 6.8 Hz, 3H), 2.08 (m, 1H), 2.38 (s, 3H), 2.85


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(dd, J = 12.8, 7.6 Hz, 1H), 2.96 (dd, J = 12.0, 7.2 Hz, 1H), 3.10 (dd, J = 12.8, 5.6 Hz, 1H), 3.10-3.20 (m, 1H), 3.26 (d, J = 5.6 Hz, 2H), 3.79 (s, 1H), 4.32 (d, J = 12.0 Hz, 1H), 4.44 (d, J = 12.0 Hz, 1H), 4.68 (s, 2H), 4.85 (s, 1H), 6.04 (s, 2H), 7.03-7.08 (m, 3H), 7.17 (t, J = 7.6 Hz, 1H), 7.22-7.30 (m, 2H), 7.39 (d, J = 7.2 Hz, 1H). 13C NMR (DMSO-d6) δ 17.2 (CH3), 19.2 (CH3), 30.6 (CH), 31.0 (CH2), 38.0 (CH2), 49.9 (CH2), 57.6 (CH), 58.5 (CH2), 70.4 (CH2), 121.9 (CH), 124.1 (CH), 126.2 (C), 127.3 (CH), 127.8 (CH), 128.3 (CH), 128.7 (CH), 131.4 (CH), 135.5 (2CH), 136.2 (C), 138.2 (C), 139.0 (C), 158.9 (C), 167.1 (2C); [R]D25 0° (c 0.22, CH3OH). HPLC: eluting with acetonitrile/water/KH2PO4, 400:600:6.8 g, tR 6.39 min. Anal. (C21H27NS2O2 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[(2-hydroxy-3-methoxyphenyl)thio]-2methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2h). The free base was purified by flash column chromatography (silica gel, cyclohexane/acetone, 80:20). Crystallization of 2h as a maleate salt gave a white powder mp = 112-114 °C. IR (KBr) ν 3517, 2973, 1603, 1572, 1473, 1354 cm-1. 1H NMR (DMSO-d6) δ 1.09 (d, J = 6.4 Hz, 3H), 2.09 (m, 1H), 2.79 (dd, J = 12.8, 7.6 Hz, 1H), 2.96 (dd, J = 12.0, 7.6 Hz, 1H), 3.04 (dd, J = 12.8, 5.6 Hz, 1H), 3.19 (dd, J = 12.4, 5.6 Hz, 1H), 3.27 (d, J = 5.6 Hz, 2H), 3.79 (s, 3H), 3.80 (s, 1H), 4.37 (d, J = 12.4 Hz, 1H), 4.44 (d, J = 12.4 Hz, 1H), 6.04 (s, 2H), 6.77 (t, J = 8.0 Hz, 1H), 6.86 (t, J = 7.6 Hz, 2H), 7.04-7.09 (m, 2H), 7.23 (t, J = 6.8 Hz, 1H), 7.39 (t, J = 6.8 Hz, 1H), 9.03 (s, 2H). 13C NMR (DMSO-d6) δ 17.2 (CH3), 30.6 (CH), 31.1 (CH2), 36.0 (CH2), 49.9 (CH2), 55.8 (CH3), 57.6 (CH), 70.4 (CH2), 110.0 (CH), 119.2 (CH), 121.4 (CH), 121.8 (CH), 121.9 (C), 124.8 (CH), 126.2 (C), 128.7 (CH), 131.4 (CH), 135.5 (2CH), 144.5 (C), 147.4 (C), 158.9 (C), 167.1 (2C); [R]D25 13.6° (c 0.27, CH3OH). HPLC: eluting with acetonitrile/water/ KH2PO4, 400:600:6.8 g, tR 8.33 min. Anal. (C20H25NS2O3 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[(2-hydroxy-3-fluorophenyl)thio]-2methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2i). The free base was purified by flash column chromatography (silica gel, cyclohexane/acetone, 85:15). Crystallization of 2i as a maleate salt gave a white powder mp = 110-112 °C. IR (KBr) ν 3398, 2962, 2702, 1601, 1475, 1354 cm-1. 1H NMR (DMSO-d6) δ 1.10 (d, J = 6.4 Hz, 3H), 2.12 (m, 1H), 2.83 (dd, J = 12.8, 7.6 Hz, 1H), 2.98 (dd, J = 12.4, 7.6 Hz, 1H), 3.10 (dd, J = 12.8, 5.4 Hz, 1H), 3.19 (dd, J = 12.4, 6.0 Hz, 1H), 3.28 (d, J = 5.2 Hz, 2H), 3.83 (s, 1H), 4.37 (d, J = 12.0 Hz, 1H), 4.46 (d, J = 12.0 Hz, 1H), 6.04 (s, 2H), 6.80-6.85 (m, 1H), 7.03-7.09 (m, 4H), 7.24 (t, J = 7.6 Hz, 1H), 7.40 (d, J = 6.8 Hz, 1H). 13C NMR (DMSO-d6) δ 17.2 (CH3), 30.6 (CH), 31.1 (CH2), 35.9 (CH2), 49.9 (CH2), 57.6 (CH), 70.4 (CH2), 113.5 (CH, J = 19 Hz), 119.5 (CH), 121.9 (CH), 124.1 (CH), 124.5 (CH), 125.9 (C, J = 54 Hz), 128.3 (CH), 131.4 (CH), 135.5 (2CH), 142.6 (C), 150.0 (C), 152.4 (C), 158.9 (C), 167.1 (2C); [R]D25 -13.6° (c 0.27, CH3OH). HPLC: eluting with acetonitrile/water/KH2PO4, 400:600:6.8 g, tR 8.33 min. Anal. (C19H22FNS2O2 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[(2-hydroxy-3-aminophenyl)thio]-2methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2j). The free base was purified by flash column chromatography (silica gel, dichloromethane/ethylacetate, 90:10). Crystallization of 2j as a maleate salt gave a white powder mp = 125 °C. IR (KBr) ν 3357, 2962, 2714, 1615, 1475, 1455, 1350 cm-1. 1H NMR (DMSO-d6) δ 1.08 (d, J = 6.4 Hz, 3H), 2.06 (m, 1H), 2.71 (dd, J = 12.8, 7.6 Hz, 1H), 2.94-2.97 (m, 2H), 3.17 (dd, J = 12.4, 5.6 Hz, 1H), 3.28 (d, J = 5.2 Hz, 2H), 3.80 (s, 1H), 4.35 (d, J = 12.8 Hz, 1H), 4.44 (d, J = 13.2 Hz, 1H), 6.05 (s, 2H), 6.54-6.63 (m, 3H), 7.03-7.09 (m, 2H), 7.24 (t, J = 7.2 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H). 13C NMR (DMSO-d6) δ 17.2 (CH3), 30.7 (CH), 31.1 (CH2), 37.3 (CH2), 49.9 (CH2), 57.6 (CH), 70.4 (CH2), 113.6 (CH), 118.6 (CH), 120.5 (CH), 121.7 (C), 121.9 (CH), 124.1 (CH), 126.1 (C), 128.7 (CH), 131.4 (CH), 135.5 (2CH), 137.5 (C), 142.3 (C), 158.9 (C), 167.1 (2C); [R]D25 2.1° (c 0.29, CH3OH). HPLC: eluting with acetonitrile/water/KH2PO4, 400:600:6.8 g, tR 8.78 min. Anal. (C19H24NS2O2 3 C4H4O4) C, H, N.


3,4-Dihydro-N-[(2S)-3-[(2-hydroxy-3-carboxamidophenyl) thio]-2-methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2k). The free base was purified by flash column chromatography (silica gel, cyclohexane/acetone, 70:30). Crystallization of 2k as a maleate salt gave a white powder mp = 154 °C. IR (KBr) ν 3474, 3347, 2978, 2822, 1708, 1654, 1472, 1414, 1356 cm-1. 1H NMR (DMSO-d6) δ 1.09 (d, J = 6.4 Hz, 3H), 2.14 (m, 1H), 2.84 (dd, J = 12.8, 7.6 Hz, 1H), 3.00 (dd, J = 12.8, 7.6 Hz, 1H), 3.12 (dd, J = 13.2, 5.6 Hz, 1H), 3.21 (dd, J = 12.4, 5.6 Hz, 1H), 3.29 (d, J = 5.2 Hz, 2H), 3.80 (s, 1H), 4.35 (d, J = 13.6 Hz, 1H), 4.46 (d, J = 13.6 Hz, 1H), 6.05 (s, 2H), 6.88 (t, J = 8.0 Hz, 1H), 7.047.09 (m, 2H), 7.25 (t, J = 8.6 Hz, 1H), 7.39 (d, J = 7.6 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 8.08 (s, 1H), 8.56 (s, 1H). 13C NMR (DMSO-d6) δ 17.2 (CH3), 30.5 (CH), 31.1 (CH2), 35.3 (CH2), 49.9 (CH2), 57.6 (CH), 70.4 (CH2), 113.2 (C), 118.3 (CH), 121.8 (CH), 124.1 (CH), 124.2 (C), 125.2 (CH), 126.2 (C), 128.7 (CH), 131.4 (CH), 133.0 (CH), 135.5 (2CH), 158.9 (C), 159.5 (C), 167.1 (2C), 172.6 (C); [R]D25 4.9° (c 0.34, CH3OH). HPLC: eluting with acetonitrile/water/ KH2PO4, 400:600:6.8 g, tR 22.10 min. Anal. (C20H24N2S2O3 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[(2-amino-3-methylphenyl)thio]-2methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2l). The free base was purified by flash column chromatography (silica gel, dichloromethane/ethylacetate, 95:5). Crystallization of 2l as a maleate salt gave a white powder mp = 135 °C. IR (KBr) ν 3470, 3356, 1605, 1573, 1470 cm-1. 1H NMR (DMSO-d6) δ 1.08 (d, J = 6.4 Hz, 3H), 2.02 (m, 1H), 2.11 (s, 3H), 2.65 (dd, J = 13.2, 8.0 Hz, 1H), 2.89-2.97 (m, 2H), 3.14 (m, 1H), 3.26 (d, J = 4.0 Hz, 2H), 3.78 (s, 1H), 4.36 (d, J = 12.8 Hz, 1H), 4.44 (d, J = 13.2 Hz, 1H), 6.04 (s, 2H), 6.50 (t, J = 7.4 Hz, 1H), 6.95 (d, J = 7.2 Hz, 1H), 7.03-7.08 (m, 2H), 7.18 (d, J = 7.4 Hz, 1H), 7.24 (t, J = 7.2 Hz, 1H), 7.38 (d, J = 7.0 Hz, 1H). 13C NMR (DMSO-d6) δ 17.1 (CH3), 18.1 (CH3), 30.9 (CH), 31.0 (CH2), 38.5 (CH2), 49.8 (CH2), 57.6 (CH), 70.4 (CH2), 115.8 (C), 116.4 (CH), 121.8 (CH), 121.9 (C), 124.0 (CH), 126.1 (C), 128.7 (CH), 130.3 (C), 131.4 (CH), 132.4 (CH), 135.5 (2CH), 146.9 (C), 158.9 (C), 167.1 (2C); [R]D25 14.2° (c 0.09, CH3OH). HPLC: eluting with acetonitrile/water/KH2PO4, 400:600:6.8 g, tR 8.45 min. Anal. (C20H26N2S2O 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[(2-amino-3-methoxyphenyl)thio]-2methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2m). The free base was purified by flash column chromatography (silica gel, dichloromethane/ethylacetate, 95:5). Crystallization of 2m as a maleate salt gave a white powder mp = 120 °C. IR (KBr) ν 3470, 3351, 2996, 2966, 1602, 1562, 1474 cm-1. 1H NMR (DMSO-d6) δ 1.08 (d, J = 6.4 Hz, 3H), 2.02 (m, 1H), 2.65 (dd, J = 13.2, 8.0 Hz, 1H), 2.91 (dd, J =12.8, 5.2 Hz, 2H), 3.12 (m, 1H), 3.25 (d, J = 4.8 Hz, 2H), 3.77 (s, 4H), 4.36 (d, J = 12.8 Hz, 1H), 4.43 (d, J = 12.8 Hz, 1H), 6.04 (s, 2H), 6.56 (t, J = 8.0 Hz, 1H), 6.81 (d, J = 7.6 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 7.03-7.08 (m, 2H), 7.24 (t, J = 7.2 Hz, 1H), 7.38 (d, J = 7.6 Hz, 1H). 13C NMR (DMSO-d6) δ 17.1 (CH3), 30.9 (CH), 31.0 (CH2), 38.2 (CH2), 49.7 (CH2), 55.5 (CH3), 57.6 (CH), 70.4 (CH2), 110.2 (CH), 115.8 (C), 116.1 (CH), 121.8 (CH), 124.0 (CH), 126.1 (CH), 128.7 (CH), 131.4 (CH), 135.6 (2CH), 138.4 (2C), 146.3 (C), 158.9 (C), 167.1 (2C); [R]D25 7.5° (c 0.23, CH3OH). HPLC: eluting with acetonitrile/water/KH2PO4, 400:600:6.8 g, tR 7.80 min. Anal. (C20H26N2S2O2 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[(2-amino-3-fluorophenyl)thio]-2methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2n). The free base was purified by flash column chromatography (silica gel, cyclohexane/acetone, 80:20). Crystallization of 2n as a maleate salt gave a white powder mp = 126 °C. IR (KBr) ν 3457, 3349, 2966, 1616, 1589, 1474, 1353 cm-1. 1H NMR (DMSO-d6) δ 1.08 (d, J = 6.4 Hz, 3H), 2.02 (m, 1H), 2.73 (dd, J = 12.8, 7.6 Hz, 1H), 2.92-2.99 (m, 2H), 3.15 (dd, J = 12.4, 5.6 Hz, 1H), 3.26 (d, J = 5.2 Hz, 2H), 3.80 (s, 1H), 4.36 (d, J = 12.0 Hz, 1H), 4.45 (d, J = 12.0 Hz, 1H), 5.29 (s, 2H), 6.05

Le Grand et al.

(s, 2H), 6.53-6.58 (m, 1H), 7.00-7.09 (m, 3H), 7.15 (d, J = 7.6 Hz, 1H), 7.22-7.31 (m 1H), 7.38 (d, J = 7.6 Hz, 1H). 13C NMR (DMSO-d6) δ 17.1 (CH3), 30.9 (CH), 31.0 (CH2), 38.8 (CH2), 49.7 (CH2), 57.6 (CH), 70.4 (CH2), 114.6 (J = 19 Hz, CH), 115.6 (J = 8 Hz, CH), 118.7 (C), 121.8 (CH), 124.0 (CH), 126.1 (C), 128.7 (CH), 129.6 (CH), 131.4 (CH), 135.6 (2CH), 137.1 (C), 150.3 (J = 238 Hz, C), 158.9 (C), 167.1 (2C); [R]D25 3.8° (c 0.29, CH3OH). HPLC: eluting with acetonitrile/water/ KH2PO4, 400:600:6.8 g, tR 7.83 min. Anal. (C19H23FN2S2O 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[2,3-dihydrobenzofuran-7-thio]-2methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2o). The free base was purified by flash column chromatography (silica gel, dichloromethane/methanol, 99:1). Crystallization of 2o as a maleate salt gave a white powder mp = 117-119 °C. IR (KBr) ν 2966, 1573, 1448, 1382, 1354 cm-1. 1H NMR (DMSO-d6) δ 1.07 (d, J = 6.8 Hz, 3H), 2.08 (m, 1H), 2.83 (dd, J = 13.2, 7.6 Hz, 1H), 2.95 (dd, J = 12.4, 7.2 Hz, 1H), 3.10 (dd, J = 12.8, 5.6 Hz, 1H), 3.20 (t, J = 8.8 Hz, 3H), 3.26 (d, J = 5.6 Hz, 2H), 3.81 (s, 1H), 4.36 (d, J = 12.0 Hz, 1H), 4.45 (d, J = 13.6 Hz, 1H), 4.56 (t, J = 8.8 Hz, 2H), 6.05 (s, 2H), 6.82 (t, J = 7.6 Hz, 1H), 7.03-7.14 (m, 4H), 7.25 (t, J = 8.0 Hz, 1H), 7.40 (d, J = 7.6 Hz, 1H). 13C NMR (DMSO-d6) δ 17.1 (CH3), 29.3 (CH2), 30.8 (CH), 31.1 (CH2), 36.3 (CH2), 49.7 (CH2), 57.5 (CH), 70.4 (CH2), 70.9 (CH2), 115.5 (C), 120.9 (CH), 121.8 (CH), 123.6 (CH), 124.1 (CH), 126.2 (C), 127.3 (C), 128.7 (2CH), 131.4 (CH), 135.6 (2CH), 158.3 (C), 158.9 (C), 167.1 (2C); [R]D25 -5.6° (c 0.49, CH3OH). HPLC: eluting with acetonitrile/water/KH2PO4, 400:600:6.8 g, tR 10.18 min. Anal. (C21H25NS2O2 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[benzofuran-7-thio]-2-methylpropyl]2H-(3R)-1,5-benzoxathiepin-3-amine (2p). The free base was purified by flash column chromatography (silica gel, dichloromethane/ethylacetate, 95:5). Crystallization of 2p as a maleate salt gave a white powder mp = 112 °C. IR (KBr) ν 2966, 1572, 1473, 1448, 1353 cm-1. 1H NMR (DMSO-d6) δ 1.11 (d, J = 6.8 Hz, 3H), 2.13 (m, 1H), 2.97-3.04 (m, 2H), 3.20 (dd, J = 12.4, 6.0 Hz, 1H), 3.26-3.33 (m, 3H), 3.81 (s, 1H), 4.34 (d, J=12.0 Hz, 1H), 4.44 (d, J = 13.2 Hz, 1H), 6.05 (s, 2H), 6.98 (d, J = 2.0 Hz, 1H), 7.02-7.09 (m, 2H), 7.22-7.28 (m, 2H), 7.36-7.41 (m, 2H), 7.57 (d, J = 7.6 Hz, 1H), 8.04 (d, J = 2.0 Hz, 1H). 13C NMR (DMSO-d6) δ 17.1 (CH3), 30.9 (CH2), 31.1 (CH), 38.8 (CH2), 49.7 (CH2), 57.5 (CH), 70.4 (CH2), 70.9 (CH2), 107.1 (CH), 118.5 (C), 119.6 (CH), 121.8 (CH), 123.6 (CH), 124.1 (CH), 124.7 (CH), 126.2 (C), 127.3 (C), 128.7 (CH), 131.4 (CH), 135.6 (2CH), 146.1 (CH), 152.9 (C), 158.9 (C), 167.1 (2C); [R]D25 -9.4° (c 0.28, CH3OH). HPLC: eluting with acetonitrile/water/KH2PO4, 400:600:6.8 g, tR 11.68 min. Anal. (C21H23NS2O2 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[2,3-benzenedioxole-7-thio]-2-methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2q). The free base was purified by flash column chromatography (silica gel, dichloromethane/ethylacetate, 95:5). Crystallization of 2q as a maleate salt gave a white powder mp = 126 °C.IR (KBr) ν 2966, 1577, 1473, 1441, 1352 cm-1. 1H NMR (DMSO-d6) δ 1.07 (d, J = 6.4 Hz, 3H), 2.09 (m, 1H), 2.86 (dd, J = 12.8, 7.6 Hz, 1H), 2.97 (m, 1H), 3.16 (dd, J = 12.8, 5.2 Hz, 1H), 3.25-3.32 (s, 3H), 3.82 (s, 1H), 4.35 (d, J = 12.8 Hz, 1H), 4.45 (d, J = 12.0 Hz, 1H), 6.04 (s, 2H), 6.05 (s, 2H), 6.83-6.91 (m, 3H), 7.03-7.09 (m, 2H), 7.25 (t, J = 8.0 Hz, 1H), 7.39 (d, J = 7.2 Hz, 1H). 13C NMR (DMSO-d6) δ 16.9 (CH3), 30.9 (CH2), 31.1 (CH), 38.8 (CH2), 49.7 (CH2), 57.5 (CH), 70.4 (CH2), 100.9 (CH2), 107.1 (CH), 115.4 (C), 121.9 (CH), 122.2 (CH), 123.2 (CH), 124.1 (CH), 126.2 (C), 128.7 (CH), 131.5 (CH), 135.6 (2CH), 146.2 (C), 146.5 (C), 158.9 (C), 167.1 (2C); [R]D25 -1.6° (c 0.12, CH3OH). HPLC: eluting with acetonitrile/water/KH2PO4, 400:600:6.8 g, tR 9.60 min. Anal. (C20H23NS2O3 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[1,3-benzisoxazole-7-thio]-2-methylpropyl]-2H-(3R)-1,5-benzoxathiepin-3-amine (2r). Compound 2r is not purified but crystallized directly as a maleate salt to give a

Article

white powder mp=112 °C. IR (KBr) ν 2966, 2819, 1603, 1573, 1473, 1353 cm-1. 1H NMR (DMSO-d6) δ 1.11 (d, J=6.4 Hz, 3H), 2.12 (m, 1H), 2.97-3.06 (m, 2H), 2.97 (m, 1H), 3.19 (dd, J = 12.4, 5.6 Hz, 1H), 3.26 (d, J = 5.0 Hz, 2H), 3.35 (dd, J = 13.2, 5.6 Hz, 1H), 3.81 (s, 1H), 4.34 (d, J = 12.4 Hz, 1H), 4.53 (d, J= 10.8 Hz, 1H), 6.04 (s, 2H), 7.04-7.09 (m, 2H), 7.25 (t, J = 7.2 Hz, 1H), 7.41 (t, J = 8.0 Hz, 2H), 7.49 (d, J = 7.6 Hz, 1H), 7.70 (d, J = 7.6 Hz, 1H), 8.81 (s, 1H). 13C NMR (DMSO-d6) δ 16.9 (CH3), 30.9 (CH2), 31.2 (CH), 36.4 (CH2), 49.7 (CH2), 57.6 (CH), 70.4 (CH2), 118.2 (CH), 118.6 (C), 121.9 (CH), 123.7 (C), 124.1 (CH), 125.3 (CH), 126.0 (C), 128.7 (CH), 131.5 (CH), 135.6 (2CH), 139.6 (C), 148.2 (C), 154.1 (C), 158.9 (C), 167.1 (2C); [R]D25 0° (c 0.22, CH3OH). HPLC: eluting with acetonitrile/water/KH2PO4, 250:750:6.8 g, tR 33.9 min, 93.0%. Anal. (C20H22N2S2O2 3 C4H4O4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[indol-7-thio]-2-methylpropyl]-2H(3R)-1,5-benzoxathiepin-3-amine (2s). The free base was purified by flash column chromatography (silica gel, dichloromethane/ethylacetate, 95:5). Crystallization of 2s as a maleate salt gave a white powder mp = 115 °C. IR (KBr) ν 3411, 1617, 1571, 1473, 1354 cm-1. 1H NMR (DMSO-d 6) δ 1.10 (d, J = 6.8 Hz, 3H), 2.07 (m, 1H), 2.87-2.99 (m, 2H), 3.11-3.20 (m, 2H), 3.25 (d, J = 5.6 Hz, 2H), 3.77 (s, 1H), 4.34 (d, J = 13.2 Hz, 1H), 4.43 (d, J = 13.6 Hz, 1H), 6.04 (s, 2H), 6.50 (s, 1H), 6.97-7.08 (m, 3H), 7.22-7.26 (m, 2H), 7.35-7.40 (m, 2H), 7.50 (d, J = 7.6 Hz, 1H), 11.12 (s, 1H). 13 C NMR (DMSO-d 6) δ 17.2 (CH3), 30.9 (CH2), 30.9 (CH), 37.9 (CH2), 49.7 (CH2), 57.6 (CH), 70.4 (CH2), 101.9 (CH), 116.6 (C), 119.4 (CH), 119.6 (CH), 121.9 (CH), 124.1 (CH), 124.2 (CH), 125.8 (CH), 126.1 (C), 127.9 (C), 128.7 (CH), 131.4 (CH), 135.6 (2CH), 135.9 (C), 158.9 (C), 167.1 (2C); [R] D25 25.3° (c 0.42, CH 3OH). HPLC: eluting with acetonitrile/water/KH 2PO4 , 400:600:6.8 g, tR 10.37 min. Anal. (C21H 24 N2 S2O 3 C4H 4O 4) C, H, N. 3,4-Dihydro-N-[(2S)-3-[indazol-7-thio]-2-methylpropyl]-2H(3R)-1,5-benzoxathiepin-3-amine (2t). The free base was purified by flash column chromatography (silica gel, cyclohexane/ ethylacetate, 70:30). Crystallization of 2t as a maleate salt gave a white powder mp = 127 °C. IR (KBr) ν 3059, 2959, 1610, 1572, 1473, 1355 cm-1. 1H NMR (DMSO-d6) δ 1.11 (d, J = 6.4 Hz, 3H), 2.11 (m, 1H), 2.98 (dd, J = 12.8, 7.6 Hz, 2H), 3.19-3.27 (m, 4H), 3.80 (s, 1H), 4.34 (d, J = 13.2 Hz, 1H), 4.42 (d, J = 12.8 Hz, 1H), 6.04 (s, 2H), 7.03-7.14 (m, 3H), 7.25 (t, J = 5.6 Hz, 1H), 7.39 (d, J = 7.2 Hz, 1H), 7.44 (d, J = 6.8 Hz, 1H), 7.70 (d, J = 8.0 Hz, 1H), 8.16 (s, 1H), 13.5 (s, 1H). 13C NMR (DMSO-d6) δ 17.1 (CH3), 30.9 (CH2), 31.1 (CH), 37.8 (CH2), 49.7 (CH2), 57.6 (CH), 70.4 (CH2), 118.7 (CH), 119.4 (CH), 120.9 (CH), 121.8 (CH), 122.3 (C), 123.1 (C), 124.1 (CH), 126.2 (C), 127.6 (CH), 128.7 (CH), 131.4 (CH), 134.1 (C), 135.6 (2CH), 158.9 (C), 167.1 (2C); [R]D25 -9.9° (c 0.41, CH3OH). HPLC: eluting with acetonitrile/water/KH2PO4, 400:600:6.8 g, tR 5.25 min, 91.8%. Anal. (C20H23N3S2O 3 C4H4O4) C, H, N. Biology. Animals used in this study were housed and tested in an Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facility in strict compliance with all applicable regulations and the protocol was carried out in compliance with French regulations and institutional Ethical Committee guidelines for animal research. Cariporide51 and ivabradine52 were prepared according to literature procedures; veratridine and lidocaine were purchased from Sigma and TTX from Tocris Bioscience. Ion Channel Pharmacology. The cell culture and patch clamp experiments were performed as described previously.30 The internal solution (pipet) contained (in mmol): NaCl 10, CsCl 110, CaCl2 1, HEPES 10, EGTA 10, Mg-ATP 5, D-glucose 10, pH = 7.3 (CsOH). The external solution contained for patch clamp experiments (in mmol): NaCl 30, CsCl 100, MgCl2 2, CaCl2 2, HEPES 10, D-glucose 5, pH=7.4 (CsOH). All experiments were carried out at room temperature (19-22 °C).


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Sodium current was elicited by depolarizing pulses from a holding potential value of -110 to -30 mV delivered at a frequency of 0.2 Hz. To verify the stability of voltage clamp, every five pulses, the holding potential was shifted to -90 mV for one pulse. Sodium current was elicited by square depolarizing pulses of 350 ms duration from a holding potential of -110 to -30 mV delivered at a frequency of 0.2 Hz. Sodium current presenting an incomplete inactivation was induced with the alkaloid veratridine (40 μM). The late INa was measured as the mean current amplitude of the last 10 ms of the pulse (that is the magnitude of INa at 340 to 350 ms of the depolarizing pulse). Wild-type HEK293 cells did not exhibit any peak and late inward sodium current. The compound was dissolved in 50% DMSO in distilled water as 10 mmol stock solution prepared freshly for each experiment. The final concentration of DMSO was 0.1%. All values are expressed as means ( SEM. Veratridine-Induced Contracture in Rats Isolated Atria. Male Wistar rats (400-450 g; OFA, Iffa-Credo, France) were maintained at 20 ( 3 °C with constant humidity of 55 ( 5%, a 12 h light-dark cycle, and free access to food and tap water. The procedure is adapted from that described by Tamareille.53 After a 30 min equilibration period, a single concentration of drug or vehicle was injected into the organ bath. Fifteen minutes later, veratridine (40 μM) was added. Systolic isometric tension development was measured before drug or solvent injection and just before the addition of veratridine in order to detect any positive or negative inotropic effects of drug or vehicle. The maximum amplitude of the veratridine-induced contracture was determined irrespective of the time at which it occurred. Global Ischemia in Guinea Pig Isolated Hearts. Female guinea pigs (SPF, Hartley, Charles River, France), weighing 500600 g, were used. The experiment was performed as described previously.33 Briefly, hearts excised from guinea pigs were placed in cold (4 °C) modified Krebs medium that contained (in mmol): 124.6 NaCl, 4KCl, 1.1 MgSO4, 0.3 NaH2PO4, 1.8 CaCl2, 24.9 NaHCO3, and 11.1 D-glucose, pH 7.4, continuously gases with 95% O2 +5% CO2 and mounted on a Langendorff system. A latex balloon was inserted through the left atrium and mitral valve into the left ventricle. Isovolumetric systolic and diastolic LVP, left ventricular developed pressure (LVDP = systolic-diastolic pressure), left ventricular end diastolic pressure, heart rate, positive dP/dt-1max, negative dP/dt-1max and coronary flow were measured at 37 ( 1 °C with a constant perfusion pressure of 800 mm H2O. The following parameters were measured: systolic and diastolic left ventricular pressure, left ventricular developed pressure, heart rate, positive dP/dtmax, negative dP/dtmax, and coronary flow. During perfusion under constant pressure, changes in coronary flow directly reflect changes in coronary vascular resistance. After 45-60 min equilibration, the buffer solution containing vehicle or compound was perfused for 15 min. Global normothermic ischemia was then induced by clamping coronary flow for 50 min and was followed by 60 min reperfusion. One drug concentration was evaluated per heart. Myocardial Infarction Reperfusion in the Pig. Forty-five male Landrace pigs (18-25 kg, GAEC La Jonione, Soreze, France) were pretreated with an intramuscular injection of azaperone (2 mg/kg) after an overnight fast. Anaesthesia was induced by thiopental sodium (Nesdonal, 15 mg/kg) via the ear vein and anesthesia was maintained with isoflurane (0.5-3% end-tidal volume). After tracheal intubation with a cuffed endotracheal tube, the lungs were ventilated with a positive pressure respirator (alpha 100, Minerve, Esternay, France). Respiratory rate and tidal volume were adjusted so as to maintain blood gases within physiological limits (ABL 510, Radiometer, Copenhagen, Denmark). Body temperature was kept constant between 37 and 38.5 °C by means of a homeothermic blanket. A four-limb ECG was recorded in lead II throughout the experiment. Left lateral thoracotomy was performed in the fourth intercostal space and the pericardium opened. Polyethylene fluid-filled catheters were


introduced into the nearest mammary artery to measure arterial pressure during surgery and for blood sampling and into the mammary vein to administer drugs or vehicle. All pigs were treated with heparin (2500 IU) before the thorax was closed. The proximal left circumflex (LCX) coronary artery was dissected and a cotton ribbon placed around it. The LCX was occluded at the prepared site for 60 min and then reperfused for 48 h. Thirty minutes after the onset of reperfusion, the chest was closed in layers and the pig was allowed to recover. Analgesia was achieved with bupivacaine (2 mg/kg) and buprenorphine (0.05 mg/kg) at regular intervals, or whenever signs of discomfort occurred. Measurement of Infarct Size. After 48 h reperfusion, animals were euthanized by sodium pentobarbital (160 mg/kg) and the heart was excised. The right coronary, LCX, and left anterior descending coronary arteries were catheterized. Evans blue (0.4%) was infused into the right and left descending coronaries to outline the ischemic myocardium. 2,3,5-Triphenyltetrazolium chloride (0.1%) at 37 °C was infused into the LCX during 10 min so as to stain the viable myocardium red to measure the extent of myocardial necrosis. The atria and the right ventricle were removed. The left ventricle was then immersed in formaldehyde (10%) solution for at least 24 h. The left ventricle was cut into 10 mm slices. Individual slices were photographed and the extent of myocardial necrosis and the area at risk quantified by image analysis software (Leica, Microsystems Imaging, Solutions, Cambridge, United Kingdom). Measurements of Troponin I Plasma Levels. During reperfusion, 10 blood samples were obtained from the mammary artery, 5 min before LCX artery occlusion, at the midpoint of LCX artery occlusion (30 min), and at 5, 15, and 30 min and 1, 6, 24, and 48 h reperfusion. The quantitative determination of troponin I in pig plasma was performed by AxSYM System (Abbot Laboratories) in a medical analysis laboratory. The AxSYM myoglobin and troponin I assays were based on microparticle enzyme immunoassay technology. Intergroup statistical analyze of results (P2, drug versus vehicle) was performed using one-way analysis of variance followed by Dunnett’s test if ANOVA was significant. Any P value lower than 0.05 was considered significant (StatView 4.1, Abacus Concepts, Berkeley, CA).

Acknowledgment. We thank J. L. Maurel and S. Brunel, who contributed to this project. We also thank Dr. J. P. Ribet and P. Zalavari for analytical supports and L. Petitpas’ assistance for bibliographic searches. Supporting Information Available: Intermediates 4a-f, 4h, 4i, 4l, 4o-q, 4u, 4v, 4t, 7, 8, 9j, 9m, and 9m, preparation of 2w, cell culture and isolation, variations on basal hemodynamics in the isolated perfused guinea pig heart, elemental analyses. This material is available free of charge via the Internet at http:// pubs.acs.org.

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Na+ Currents in Cardioprotection: Better to Be Late - ACS Publications

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