Stereochemistry of N-Acyl-5H-dibenzo[b,d]azepin-7(6H)-ones

The stereochemical properties of N-acyl-5H-dibenzo[b,d]azepin-7(6H)-ones (2a–c), which inhibit potassium channels in T cells, were examined by freezing their conformational change due to 4-methyl substitution. N-Acyl-5H-dibenzo[b,d]azepin-7(6H)-ones exist as pairs of enantiomers [(a1R, a2R), (a1S, a2S)], and each atropisomer is separable at room temperature. An alternate procedure for preparing 5H-dibenzo[b,d]azepin-7(6H)-ones involves the intramolecular Friedel–Crafts cyclization of N-benzyloxycarbonylated biaryl amino acids. Consequently, the N-benzyloxy group was removed during the cyclization reaction to produce 5H-dibenzo[b,d]azepin-7(6H)-ones suitable for the subsequent N-acylation reaction.


Introduction
Recently, our group has become interested in axial chirality and its relation to biological activities. Atropisomers are products of dynamic chirality derived from a restricted rotation around a single bond in a molecule. As axial chirality is caused by a conformational change, it may occur in many organic molecules in various forms. It should be noted that if such molecules have biological activities, axial chirality will be detected by target molecules such as receptors and enzymes. The axial chirality [1][2][3][4][5][6][7][8][9][10] of amides in benzo-fused seven-membered ring nitrogen heterocycles, which are found as the scaffolds of various biologically active molecules, has become the focus of considerable research interest [11][12][13][14][15][16][17]. Although often overlooked, aryl-amides and anilides possess sp 2 -sp 2 atropisomers based on the Ar-NC(=O) (sp 2 -sp 2 ) axis, and target molecules can detect each atropisomer for its biological activity. In the course of our studies aimed at elucidating the relationship between stereochemical property and biological activity in this scaffold [18][19][20][21][22][23][24], we have become interested in the dibenzo[b,d]azepin-6-one moiety. In 2008, we investigated the stereochemical properties of several derivatives of the γ-secretase inhibitor, LY-411575 [25], in which the dibenzo[b,d]azepin-6-one moiety constitutes an important scaffold. It was elucidated that the two sp 2 -sp 2 axes, resulting from the Ar-Ar (sp 2 -sp 2 ) axis and the Ar-NC(=O) (sp 2 -sp 2 ) axis, move in concert to form a stable relative configuration (a pair of enantiomers). In this scaffold, axial chirality with high stereochemical stability enabled kinetically controlled alkylation [26]. Such seven-membered-ring dibenzolactam, dibenzo[b,d]azepin-6-one, prompted us to study the eight-and nine-membered-ring dibenzolactams by comparing the stereochemical stabilities of the atropisomers and their chemical reactivities toward kinetically controlled stereoselective alkylation [27]. The atropisomers of seven-, eight-, and nine-membered ring dibenzolactams were separated atropisomers of seven-, eight-, and nine-membered ring dibenzolactams were separated and isolated via chiral HPLC, and their configurations were clarified by an X-ray crystallographic analysis. It was revealed that the two sp 2 -sp 2 axes, resulting from the Ar-Ar (sp 2 -sp 2 ) axis and the Ar-NC(=O) (sp 2 -sp 2 ) axis, move in concert in the eight-and ninemembered-ring dibenzolactams. Additionally, an eight-membered-ring benzolactam was found to exist in the most stable configuration owing to the deep, rigid, cage-like ring form, which provides a high barrier to the inversion of the ring system. In this study, we focused on N-acyl-/N-sulfonyl-5H-dibenzo[b,d]azepin-7(6H)-ones 2 (Scheme 1). These compounds exhibit immunosuppressive effects by inhibiting potassium channels (Kv1.3 and IK-1) in T cells [28,29]. Because the Ca 2+ -dependent potassium channel IK-1 and the voltage-gated potassium channel Kv1.3 in human T-cells play pivotal roles during cell proliferation, inhibitors of these channels are promising drug candidates for treating autoimmune diseases such as rheumatoid arthritis and multiple sclerosis [30,31]. In N-acyl-5H-dibenzo[b,d]azepin-7(6H)-ones 2, the E and Z diastereomers around the Ar-NC(=O) bond have been identified, although the presence of chirality has been overlooked. In our previous paper [29], N-acyl-5H-dibenzo[b,d]azepin-7(6H)-ones 2 were found to exist in the E-diastereomer in preference to the Z-diastereomer in solution, as also supported by density functional theory calculations (DFT). In addition, stable atropisomers [(a 1 R, a 2 R), (a 1 S, a 2 S)] of N-acyl-5H-dibenzo[b,d]azepin-7(6H)-ones 2 were successfully isolated. Unfortunately, the differences in immunosuppressive effects through the inhibition of potassium channels (Kv1.3 and IK-1) in T cells between the atropisomers of N-acyl-5Hdibenzo[b,d]azepin-7(6H)-ones 2 were not examined in the previous study. Therefore, we continued the investigation by studying the structure-activity relationship (SAR) of Nacyl-5H-dibenzo[b,d]azepin-7(6H)-ones 2. This paper reports an alternative procedure for preparing 5H-dibenzo[b,d]azepin-7(6H)-ones, enabling the following N-substitution reaction. In addition, the physicochemical properties and inhibitory activities of the potassium channels of the synthesized N-acyl-5H-dibenzo[b,d]azepin-7(6H)-ones (2a-c) have been reported.
Consequently, the electron-withdrawing property of the amino-protecting group in 1 was assumed to be significant to the successful intramolecular Friedel-Crafts acylation of the N-substituted aryl amino acids 1.
In contrast, the intramolecular Friedel-Crafts acylation of N-benzyloxycarbonyl (Cbz) compound 1b (Scheme 2) proceeded efficiently to yield the corresponding 5Hdibenzo[b,d]azepin-7(6H)-one 6. The Cbz group was removed during the cyclization reaction, and the obtained compound 6 was suitable for the following N-acylation reaction. As expected, the N-acylation of 6 under basic conditions afforded N-acyl-5H-dibenzo [b,d]azepin-7(6H)-ones 2a-c in good yields. In our previous study [29]

Figure 1. 1 H NMR spectra of E/Z isomer of N-acyl-5H-dibenzo[b,d]azepin-7(6H)-ones 2a-c.
Owing to the 4-methyl substituent, the conformational change in 2a-c was fully frozen, and we separated into stable enantiomers [(a 1 R, a 2 R), (a 1 S, a 2 S)] using chiral HPLC at room temperature ( Figure 2). Therefore, each enantiomer of compounds 2a-c was isolated at room temperature in an enantiomerically pure form. Owing to the 4-methyl substituent, the conformational change in 2a-c was fully frozen, and we separated into stable enantiomers [(a 1 R, a 2 R), (a 1 S, a 2 S)] using chiral HPLC at room temperature ( Figure 2). Therefore, each enantiomer of compounds 2a-c was isolated at room temperature in an enantiomerically pure form.
Subsequently, the physicochemical properties of the enantiomerically pure isomers were investigated ( Table 1). The ∆G ‡ values of 2a-c were determined based on the timedependent conversion rate (% ee) (Figure 3) estimated from the chiral HPLC analysis of a toluene solution of each enantiomer. The calculation was conducted according to a procedure reported by Curran [32]. The acetylated derivative 2a showed the highest energy barrier to rotation (∆G ‡ = 121 kJ/mol), and the benzoyl derivatives 2b and 2c were less stable. Bulky benzoyl substitutions were less effective in reducing conformational changes. Considering that similar results were reported by Graham [33], it is evident that bulkier substituents contribute toward lowering the barrier to rotation. However, determining the specific effects of the N-acyl substituent is difficult owing to limited available information on this matter. Subsequently, the physicochemical properties of the enantiomerically pure isomers were investigated ( Table 1). The ΔG ‡ values of 2a-c were determined based on the timedependent conversion rate (% ee) (Figure 3) estimated from the chiral HPLC analysis of a toluene solution of each enantiomer. The calculation was conducted according to a procedure reported by Curran [32]. The acetylated derivative 2a showed the highest energy barrier to rotation (ΔG ‡ = 121 kJ/mol), and the benzoyl derivatives 2b and 2c were less stable. Bulky benzoyl substitutions were less effective in reducing conformational changes. Considering that similar results were reported by Graham [33], it is evident that bulkier substituents contribute toward lowering the barrier to rotation. However, determining the specific effects of the N-acyl substituent is difficult owing to limited available information on this matter.   p-Me-Ph +135.6 (>99% ee) 106 −156.6 (>99% ee) a Measured in MeOH at 20 • C. b Racemized in toluene at 80 • C for 2a and at 37 • C for 2b and 2c.

Blockage of the Potassium Channel Kv1.3
Finally, the blocking activity of the voltage-gated potassium channel Kv1.3, using 4-aminopyridine as the positive control, was tested for 2c using patch-cramp technology ( Table 2). While the blocking activity of racemate 2c was not observed at the peak current (open channel inhibition), (−)-2c showed more potent affinity than (+)-2c. Regarding the activity at the end current (inactivation-dependent inhibition), (−)-2c showed more potent affinity than (+)-2c, although the enantiomers and racemate exhibited similar levels of affinity (within a 1.6-fold difference). Molecules 2023, 28, x FOR PEER REVIEW 6 of 11

Blockage of the Potassium Channel Kv1.3
Finally, the blocking activity of the voltage-gated potassium channel Kv1.3, using 4aminopyridine as the positive control, was tested for 2c using patch-cramp technology ( Table 2). While the blocking activity of racemate 2c was not observed at the peak current (open channel inhibition), (−)-2c showed more potent affinity than (+)-2c. Regarding the activity at the end current (inactivation-dependent inhibition), (−)-2c showed more potent affinity than (+)-2c, although the enantiomers and racemate exhibited similar levels of affinity (within a 1.6-fold difference).

Chemistry
All the reagents were purchased from commercial suppliers and used as received. Starting materials obtained from commercial suppliers were used without further purification, and starting material 3 was prepared using the previuosly reported method [15]. The reaction mixtures were magnetically stirred, and the reactions were monitored using thin-layer chromatography on pre-coated silica gel plates. Column chromatography was carried out using silica gel (45-60 µm). The extracted solutions were dried over anhydrous

Chemistry
All the reagents were purchased from commercial suppliers and used as received. Starting materials obtained from commercial suppliers were used without further purification, and starting material 3 was prepared using the previuosly reported method [15]. The reaction mixtures were magnetically stirred, and the reactions were monitored using thin-layer chromatography on pre-coated silica gel plates. Column chromatography was carried out using silica gel (45-60 µm). The extracted solutions were dried over anhydrous Na 2 SO 4 . The solvent was evaporated under reduced pressure. NMR spectra were recorded at 600 MHz for 1 H NMR and 150 MHz for 13 C NMR at 296 K. Chemical shifts are provided as parts per million (ppm) downfield of tetramethylsilane, which was used as the internal standard. The coupling constants (J) were reported in hertz (Hz). The splitting patterns were abbreviated as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br) patterns. High-resolution mass spectra (HRMS) were recorded using an electrospray ionization/time-of-flight (ESI/TOF) mass spectrometer. Melting points were recorded using a melting point apparatus and were uncorrected.