1. Introduction

Large-conductance,voltage-gated and calcium-dependent K⁺ channels (Maxi-K or BK channels) are widely distributed in smooth muscle,neurons,and many other tissues,and play important roles in many physiological events ^[1]. BK channels are uniquely regulated by changes in both transmembrane potential and intracellular Ca²⁺ level ^[2],and may couple with other ion channels (such as Ca²⁺ ion channels) ^{[3, 4]} to serve as a negative feedbackpathway controlling ionic homeostasis,cell excitability,and neuron activity ^[5]. BK channels consist of channel-forming α-subunits and accessory β-subunits arranged in tetramers ^[6]. Recent cloning studies have revealed the presence of multiple splice variants of α-subunits and multiple subtypes of β-subunits(β₁,β₂/β₃,and β₄) ^[7],which may be specific to tissues,organs and functions (e.g.,β₁: smooth muscle,b4: brain) ^[8]. The physiological role and widespread distribution of BK channels suggest that agents that open these channels could have profound impacts on diseases such as acute stroke,epilepsy,asthma,and bladder overactivity ^[9]. Well-characterized BK channel openers not only are expected to have therapeutic potential,but also should be ofassistance in understanding the function,structure and role of BK channels.

During the past few years,various classes of BK channel openers including synthetic benzimidazolone derivatives (NS004 and NS1619),biaryl amines (mefenamic and flufenamic acids), biarylureas (NS1608),biarylthioureas (NS11021),aryloxindoles (BMS-204352),arylpyrroles (NS-8),and indole-3-carboxylic acid esters (CGS-7184 and CGS-7181) have been described,and their chemistry and pharmacology have been reviewed ^[10]. Other classes of potent tetrahydroquinoline BK agonists ^[11] and a series of anthraquinone analogs of BK-channel openers ^[12] have also been disclosed recently. However,the range of scaffolds for the molecular design of BK channel openers is still limited and the BK channel-opening potency of currently available compounds is rather weak.

Our previous study showed that the dehydroabietic acid (DHAA,1a,Fig. 1) structure provides a template for chemical modulators of BK channels. Using DHAA as a starting point,a number of derivatives,wherein the C ring was modified by introduction of divergent substituents,have been synthetically prepared,and some of them have been characterized as BK channel openers ^[13]. However,modification of the B ring of DHAA has been little explored ^[14]. In a recent study,we synthesized a series of oxime ether derivatives of the benzylic ketone of abietane diterpenes represented by compound Cym04 (1b),and found that the oxime ether structure significantlyincreased the BK channel-opening activity of DHAA ^[15]. The molecular mechanism of BK activation by Cym04 was also revealed ^[16]. In light of these findings,itwould be worthwhile to design and synthesize new derivatives of DHAA with structural changes on the B ring and evaluate their potential BK channelopening activities. In recent years,the N-acylhydrazone moiety has proved to be an important pharmacophore structure in pharmaceutical research ^{[17, 18, 19]}. In continuation of our previous study on new BK channel openers,we report herein the synthesis and characterization of a series of hydrazone (2) and Nacylhydrazone derivatives (3) generated from dehydroabietic acid (1a). Also,their BK channel-opening activities are presented.

Fig. 1

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Fig. 1.The structures of dehydroabietic acid (1a),Cym04 (1b), 2 and 3.

The synthetic procedures for the target compounds 2,3a-o are outlined in Scheme 1. The parent hydrazone 2 was synthesized as shown in method A. Benzylic oxidation of dehydroabietic acid 1a with CrO₃ in AcOH afforded the 7-keto derivative 1c. Treatment of the ketone 1c with hydrazine hydrate in EtOH under heating at reflux afforded the corresponding hydrazone 2 in 86% yield. Compound 3i was obtained by acylation of 2 with acryloyl chloride in 60% yield.

Scheme 1

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Scheme 1..Reagents and conditions: (a) CrO3,AcOH,r.t.,3 h,64%-76%; (b)N2H4.H2O,EtOH,reflux,1 h,86%-100%; (c) acryloyl chloride,Et3N,CH2Cl2,r.t.,1 h,60%; (d) BnBr,K2CO3,DMF,r.t.,3 h,88%; (e) RCONHNH2,EtOH,reflux,12 h,84%-86%; (f) HCO2NH4,Pd(OH)2/C,MeOH/THF (1/2,v/v),3 h,70%-98%; (g) acyl chloride,Et3N,CH2Cl2,r.t.,1 h,76%-96%. .

In method B,the carboxy group of dehydroabietic acid 1a was firstly protected as benzyl ester 1d before treatment with CrO₃. The protecting group should be straightforward to remove without using either acid or basic conditions because of the sensibility of the hydrazone motif. A benzyl group can be removed under neutral environment in high yield and was therefore chosen. Benzylic oxidation of 1d with CrO₃ in AcOH afforded the 7-keto derivative 1e. Treatment of the ketone 1e with acethydrazide in EtOH under heating at reflux afforded the intermediate 4a. Finally,the benzyl ester of 4a was removed by hydrogenation with palladium hydroxide in MeOH and HCOONH₄ to give the desired carboxylic acid 3a. Compound 3j was obtained by the condensation of ketone intermediate 1e with benzoylhydrazine,followed by hydrogenation of the benzyl ester.

Treatment of the ketone 1e with hydrazine hydrate in EtOH under heating at reflux afforded the key intermediate 1f. Compounds 3b-i and 3k-o were obtained by acylation of the hydrazone 1f with various acyl chlorides in CH₂Cl₂ in the presence of Et3N,followed by hydrogenation removal of the benzyl ester to the carboxylic acid with palladium hydroxide in MeOH and HCOONH₄,as depicted in method C.

Analytical and spectroscopic characterization data of compounds 2,3a-o are given in Supporting information.

3. Results and discussion It was known that N-acylhydrazones may exist in four possible forms in respect to (E/Z)-configurational isomers relative to the C==N double bonds and (E’/Z’)-rotamers caused by the inversion of amide bonds (Fig. 2). However,the E/Z isomerization was not observed since these N-acylhydrazones exist primarily or completely in the (E)-configuration because of the steric hindrance relative to the moiety ^[20]. The single crystal of the representative compound 3m was obtained from CH₂Cl₂ for the X-ray diffraction

Fig. 2

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Fig. 2.Four possible forms of N-acylhydrazone derivatives.

to confirm the configuration. An ORTEP plot of the solid state conformation of 3m drawing with the atom numbering scheme is shown in Fig. 3 ^[21]. In accordance with the starting material, dehydroabietic acid,the two cyclohexane ring A and B bearing chair and half-chair conformations,respectively,show a trans ring junction,with two methyl groups of C19 and C20 in the axial positions. The molecule displays an E configuration with respect to the N1==C7 double bond with a N2-N1-C7-C8 torsion angle of179.5(7),and an E’ conformation due to the amide bond with a N1- N2-C21-O3 torsion angle of 175.291(8). Intermolecular hydrogen bonding is observed between the H-N2 proton of N-acylhydrazone and the adjacent ketoneoxygen (O1). The Schiff base moiety is almost conjugated with the aromatic ring and the dihedral angle of ∠C6C7C8C9 is 0.4868°.

Fig. 3

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Fig. 3.Crystal structure of compound 3m.

The activities of all the target compounds as BK channel modulators were evaluated by means of automated planar array patch clamp recording using the 64-well Population Patch Clamp (PPC) technique ^[22]. The BK channel was activated by applying a steppulse to +100 mVfromthe holding potential of -90 mVtoCHOK1 cells expressing hBKa channels,and the current amplitude in the presence of a test compound (30 μmol/L) was expressed as percent of the drug-free control. The values represent an average of data obtained from at least eight separate measurements. The results for NS1619 and Cym04 are included for comparison.

From the results presented in Table 1,most of the 16 compounds 2 and 3a-o showed no distinct BK channel-opening activity. The hydrazone 2 showed no BK channel-opening activity. All the alkyl acylhydrazone derivatives 3a-i were inactive,except for compounds 3g and 3h,which were only marginally active. Among the aryl-group-containing derivatives (3j-o),the activity is quite sensitive to the location and properties of the aromatic substituents. In the series of compounds 3j,3k,3l and 3m,parasubstitution of a methoxy group on the aromatic ring (3m) resulted in increases in the channel current,while lack of substitution (3j) or substitution with ortho-substituted methoxy group (3l) resulted in inactivity. The fluoro derivative (compound 3k) was also inactive. On the other hand,introduction of a phenyl-substituted carbon chain (3n,3o) increased the activity,as compared with the N-unsubstituted hydrazone 2. And compound 3n (ion current = (145.4 ± 5.6)% of control at 30 μmol/L) was found to be a moderately active BK opener. Most of these compounds synthesized exhibited no or slight BK channel-opening activity except 3n with the ion current (145.4 ± 5.6)% of control at 30 mmol/L. Even so,the activity was much lower than that of Cym04 (ion current = (265.0 ± 40.4)% of control at 30 μmol/L).In the case of Oalkylated oximes,the conformation is rather rigid ^[23]. Also the intermolecular hydrogen bonding observed in these series of Nacylhydrazone derivatives is probably not favorable to binding with BK channels. In our previous study,we found that introduction ofhydrophobic groups such as a nitro,chloride or (thio)urea functionality to ring C can improve the potency greatly ^[13]. Further derivatization of compound 3n such as introduction of various groups on ring C,together with the in-depth SAR study,are still required.

Table 1

Table 1 Structure and BKa-opening properties of dehydroabietate derivatives.

Table 1 Structure and BKa-opening properties of dehydroabietate derivatives.

4. Conclusion

In this study,a series of new N-acylhydrazone derivatives of dehydroabietic acid were synthesized and characterized in approach of BK channel openers. All the compounds were evaluated for their BK channel-opening activities in an assay system of CHO-K1 cells expressing hBKa channels. The assay results indicated that the activities of the investigated compounds were influenced by the physicochemical properties of the substituent at hydrazone moiety. The results highlight these new N-acylhydrazone derivatives as potential leads for the further investigation of new BK channel-opening candidates.

The authors are grateful for supports from the National Natural Science Foundation of China (Nos. 81202402 and 21272154) and Shanghai Pujiang Program (No. 10PJ1403700). Appendix A. Supplementary data Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2013.06.017.