Design and Synthesis of Photoaffinity Probes and Their Application to Target Identification Study of PD 404182

Abstract

To investigate the mechanism of action of the potent antiviral compound PD 404182, three novel photoaffinity probes were designed and synthesized based on the structure–activity relationship studies. These probes with a biotin or alkyne indicator retained the potent anti-HIV activity of the original pyrimidobenzothiazine derivatives. In photoaffinity labeling studies using HIV-1-infected H9 cells (H9IIIB), eight potential binding proteins to PD 404182 were observed.

The comparative time of drug addition study using standard anti-HIV agents demonstrated that PD 404182 1 showed a similar antiviral profile against HIV-1_IIIB infection with that of DS 5000 (adsorption inhibitor) and enfuvirtide (fusion inhibitor). This suggests that compound 1 apparently impairs virus replication at the early stage of HIV infection. Additionally, the antiviral activities of 1 against multiple HIV clades suggest that the target molecule of 1 is not chemokine receptors (CC chemokine receptor type 5 or CXC chemokine receptor type 4). However, the mode of action and mechanism of antiviral activity of 1 were not fully elucidated.

Photoaffinity labeling is an efficient approach to identify the target protein(s) of biologically active molecules [1–4]. In modern drug discovery, there have been a number of successful examples that have determined the target molecules and identified the binding site through the formation of a covalent bond between the ligand and the specific protein [5–7]. In general, photoaffinity probes contain three functional groups: a bioactive scaffold, a photoreactive group and an indicator group. A biotin-tag is widely employed as an indicator because biotinylated proteins can be detected and isolated by several immunological methods or through a biotin-avidin interaction [8–11]. A terminal alkyne is an alternative indicator for Huisgen cycloaddition-mediated conjugation with various azide-modified reporters, such as fluorescent-azide and biotin-azide after the crosslinking reaction onto the target protein(s). (For examples of alkyne-conjugated photoaffinity probes with benzophenone, see [12–16]).

Trifunctional probes for the target protein(s) of 1 and the derivatives were designed on the basis of the SAR investigations. In the SAR study, the introduction of a hydrophobic group on the benzene ring and the cyclic amidine substructures effectively improved antiviral activity (compounds 2–4, Fig. 4.1). The author expected that these moieties would potentially take part in a favorable interaction(s) with the target molecule(s), and the incorporation of a hydrophobic and photoreactive benzophenone group on the pyrimidobenzothiazine scaffold would be tolerated. Additionally, the N-alkoxycarbonyl piperidine group onto the amidine substructure of 1 reproduced potent anti-HIV activity (compound 5), indicating that this part could be used as a linkage position for the addition of functional groups.

Fig. 4.1

Structures and anti-HIV activity of PD 404182 and the derivatives 2–5

With this in mind, the author designed three photoaffinity probes. Compound 6 is modified with indicator biotin via a photoreactive benzophenone group onto the benzene ring substructure (Fig. 4.2). Compound 7 equips the biotin and benzophenone groups on the right-part amidine moiety. The biotin moiety is conjugated with benzophenone via a polyethylene glycol (PEG) linker as the spacer. Compound 8 is an alkyne-containing derivative.

Fig. 4.2

Structures of photoaffinity probes 6–8

Synthesis of the probe 6 started with the preparation of benzophenone boronic acid pinacol ester 11 (Scheme 4.1). Condensation of p-(hydroxymethyl)benzoic acid 9 and N,O-dimethyl-hydroxylamine followed by TBDPS protection of a primary hydroxyl group gave an amide 10. Subsequent nucleophilic addition of an in situ-generated organolithium compound easily provided the desired boronate 11 [17]. Alkylation of compound 2a with p-methoxybenzyl (PMB) bromide followed by Suzuki–Miyaura cross coupling with compound 11 afforded a benzophenone-conjugated pyrimidobenzothiazine 13. Desilylation of 13 and the subsequent reaction with p-nitrophenyl chloroformate afforded the carbonate 16. The biotin moiety was incorporated by reaction of 16 with biotin-PEG-NH₂ (15) which was prepared by catalytic hydrogenation of azide 14 [18]. TFA-mediated deprotection of the PMB group in compound 17 provided the desired probe 6.

Scheme 4.1

Synthesis of biotin-conjugated probe 6 Reagents and conditions. (a) HNMe(OMe)·HCl, EDC·HCl, HOBt·H₂O, Et₃N, DMF, rt; (b) TBDPSCl, Et₃N, DMAP, CH₂Cl₂, rt, 49 % [2 steps (a, b)]; (c) 2-(4-bromophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane, t-BuLi, −78 °C to rt, THF, 83 %; (d) t-BuOK, DMF, 0 °C, then PMBBr, rt, 98 %; (e) 11, Pd(PPh₃)₄, PdCl₂(dppf)·CH₂Cl₂, K₂CO₃, toluene, EtOH, H₂O, reflux, 96 %; (f) TBAF, THF, rt; (g) p-nitrophenyl chloroformate, pyridine, CH₂Cl₂, reflux; (h) Et₃N, DMF, rt to 40 °C, 46 % [3 steps (f, g, h)]; (i) H₂, 10 % Pd/C, MeOH, rt; (j) MS4Å, TFA, CHCl₃, rt, 35 %

Synthesis of the biotin-conjugated probe 7 is outlined in Scheme 4.2. PMB protection of compound 18 followed by the selective removal of the PMB group on the piperidine ring provided compound 20. Separately, the synthesis of biotin-benzophenone adduct 23 started from 4-(tert-butyldiphenylsilyloxy)methyl-4′-(hydroxymethyl)benzophenone 21 [19]. The treatment of 21 with chloroformate furnished a carbonate 22. Biotin-PEG-NH₂ 15 was successfully conjugated onto 22 to give the biotin-benzophenone adduct 23. Desilylation of 23, treatment with p-nitrophenyl chloroformate and coupling with 20 provided a biotin/benzophenone-conjugated 26. PMB deprotection of 26 afforded the desired probe 7.

Scheme 4.2

Synthesis of biotin-conjugated probe 7. Reagents and conditions: (a) t-BuOK, DMF, 0 °C, then PMBBr, rt, 81 %; (b) 1-chloroethylchloroformate, Et₃N, CH₂Cl₂, 0 °C, then MeOH, reflux; (c) 4-nitrophenylchloroformate, pyridine, CH₂Cl₂, reflux; (d) 15, Et₃N, DMF, rt, quant. [2 steps (c, d)]; (e) HF-pyridine, THF, 0 °C to rt, 73 %; (f) 4-nitrophenylchloroformate, pyridine, CH₂Cl₂, reflux, 80 %; (g) 20, Et₃N, DMF, rt; (h) MS4Å, TFA, CHCl₃, rt, 36 % [2 steps (g, h)]

The author next investigated the synthesis of alkyne-containing probe 8 (Scheme 4.3). Suzuki–Miyaura cross coupling of compound 27 with boronate 11 gave compound 28. Subsequent modifications including desilylation, propargylation, and removal of the tert-butyl group provided the expected alkyne-conjugated probe 8.

Scheme 4.3

Synthesis of alkyne-conjugated probe 8. Reagents and conditions: (a) 11, Pd(PPh₃)₄, PdCl₂(dppf)·CH₂Cl₂, K₂CO₃, toluene, EtOH, H₂O, reflux, 71 %; (b) TBAF, THF, rt; (c) NaH, THF, propargyl bromide, 0 °C to rt, 60 % [2 steps (b, c)]; (d) MS4Å, TFA, CHCl₃, reflux, 92 %

The antiviral activities of probes 6–8 against HIV-1_IIIB were measured by multinuclear activation of a galactosidase indicator (MAGI) assay. Both biotin-conjugated probes 6 and 7 showed potent anti-HIV activity with EC₅₀ values of 6.87 and 5.11 μM, respectively (Table 4.1). These activities were slightly lower than that of compound 1; however, the incorporation of large functional groups including benzophenone, the PEG linker and the biotinyl reporter was largely tolerated. Alkyne-conjugated probe 8 potently inhibited HIV infection (EC₅₀ = 0.64 μM). These probes 6–8 represent promising tools for the identification of the target molecule(s) of compound 1 and the derivatives.

Table 4.1 Anti-HIV activities of the probes 6–8

Probes 6 and 7 were applied to the experiment for target identification of compound 1 and the derivatives. After HIV-1-infected H9 cells (H9IIIB) were incubated with a probe (6 in the presence or absence of 3a, or 7) for 1 h, the cells were exposed to UV–Vis light (>300 nm) for 1 min. After cell lysis, the biotinylated proteins were captured with NeutrAvidin agarose beads. The whole was subjected to separation by SDS-PAGE followed by Western blot analysis.

Eight bands of 95, 80, 75, 70, 60, 55, 48 and 40 kDa proteins were observed from the cell samples incubated with probe 6 (Lane A, Fig. 4.3). These bands were competed by unlabeled compound 3a, suggesting that the labeling was PD 404182-specific (Lane C). In contrast, these bands, with the exception of the 70 and 40 kDa bands, were not detected in the cells incubated with probe 7 (Lane B). This observation indicated that the potential target proteins did not fully interact with the benzophenone group on the right-part amidine moiety in the pyrimidobenzothiazine scaffold of 7. This experiment demonstrated that the synthesized probe 6 could be useful for the identification of the target protein(s) of compound 1.

Fig. 4.3

Western Blot Analysis of the Photolabeled Proteins with Biotin-Conjugated Probes 6 and 7; H9IIIB cells were incubated with (A) 20 μM probe 6, (B) 20 μM probe 7, and (C) 20 μM probe 6 and 40 μM compound 3a. The cells were exposed to UV light for 1 min and were lysed. The resulting photolabeled proteins were captured onto NeutrAvidin-agarose and the whole was subjected to SDS-PAGE. The resulting gel was analyzed by Western blotting with streptavidin-HRP

In conclusion, the author designed and synthesized novel photoaffinity probes of PD 404182 with photoreactive benzophenone, and biotin or alkyne indicators. The probes exhibited equipotent or slightly less potent anti-HIV activities when compared with the activity of the parent compound 1. Photoaffinity labeling experiments suggest that these probes could be useful in the identification of a potential target protein(s), the binding site on the target protein(s) and the mechanism(s) of action of PD 404182 derivatives.

4.1 Experimental Section

4.1.1 General Methods

All moisture-sensitive reactions were performed using syringe-septum cap techniques under an Ar atmosphere and all glasswares were dried in an oven at 80 °C for 2 h prior to use. Melting points were measured by a hot stage melting point apparatus (uncorrected). For flash chromatography, Wakogel C-300E (Wako) or aluminum oxide 90 standardized (Merck) was employed. For preparative TLC, TLC silica gel 60 F254 (Merck) or TLC aluminum oxide 60 F254 basic (Merck) were employed. For analytical HPLC, a COSMOSIL 5C18-ARII column (4.6 × 250 mm, Nacalai Tesque, Inc., Kyoto, Japan) was employed with a linear gradient of CH₃CN containing 0.1 % (v/v) NH₃ at a flow rate of 1 mL/min on a Shimadzu LC-10ADvp (Shimadzu Corp., Ltd., Kyoto, Japan), and eluting products were detected by UV at 254 nm. ¹H-NMR spectra were recorded using a JEOL AL-400 or a JEOL ECA-500 spectrometer, and chemical shifts are reported in δ (ppm) relative to Me₄Si (CDCl₃) or DMSO (DMSO-d ₆ ) as internal standards. ¹³C-NMR spectra were recorded using a JEOL AL-400 or JEOL ECA-500 spectrometer and referenced to the residual solvent signal. ¹⁹F-NMR spectra were recorded using a JEOL ECA-500 and referenced to the internal CFCl₃ (δ _F 0.00 ppm). ¹H-NMR spectra are tabulated as follows: chemical shift, multiplicity (b = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), coupling constant(s), and number of protons. Exact mass (HRMS) spectra were recorded on a JMS-HX/HX 110 A mass spectrometer. Infrared (IR) spectra were obtained on a JASCO FT/IR-4100 FT-IR spectrometer with JASCO ATR PRO410-S. The purity of the probes 6–8 was determined by HPLC analysis as >95 %. Synthesis and characterization data of compounds 2a, 18, and 27 are shown in Chap. 3.

4.1.2 4-[(tert-Butyldiphenylsilyloxy)methyl]-N-methoxy-N-methylbenzamide (10)

To a mixture of 4-(hydroxyl- methyl)benzoic acid 9 (4.6 g, 30.0 mmol), N,O-dimethylhydroxylamine hydrochloride (14.6 g, 150.0 mmol), Et₃N (21.7 mL, 150.0 mmol) in DMF (300 mL) were added EDC·HCl (11.5 g, 60.0 mmol) and HOBt·H₂O (9.2 g, 60.0 mmol). After being stirred at rt overnight, solvent was evaporated. The residue was dissolved in EtOAc, and washed with 1 N HCl, sat. NaHCO₃, brine, and dried over MgSO₄. The filtrate was concentrated to give crude Weinreb amide (4.05 g, ca. 20.7 mmol). To the mixture of the Weinreb amide, a solution of Et₃N (8.98 mL, 62.1 mmol) and DMAP (252.9 mg, 2.1 mmol) in CH₂Cl₂ (138 mL) was slowly added TBDPSCl (5.83 mL, 22.8 mmol). After being stirred at rt for 3 h, the reaction mixture was quenched with water. After concentration, the residue was dissolved in EtOAc. The mixture was washed with sat. NaHCO₃, brine, and dried over MgSO₄. After concentration, the residue was purified by flash column chromatography over silica gel with n-hexane–EtOAc (3:1) to give the title compound 10 as colorless oil (6.98 g, 49 %): IR (neat) cm⁻¹: 1644 (C=O); ¹H-NMR (400 MHz, CDCl₃) δ: 1.10 (s, 9H, 3 × CH₃), 3.36 (s, 3H, CH₃), 3.57 (s, 3H, CH₃), 4.80 (s, 2H, CH₂), 7.36–7.43 (m, 8H, Ar), 7.65–7.70 (m, 6H, Ar); ¹³C-NMR (100 MHz, CDCl₃) δ: 19.3, 26.8 (3C), 33.8, 61.0, 65.2, 125.4 (2C), 127.7 (4C), 128.2 (2C), 129.8 (2C), 132.6, 133.3 (2C), 135.5 (4C), 143.8, 169.9; HRMS (FAB): m/z calcd for C₂₆H₃₂NO₃Si [M + H]⁺ 434.2152; found: 434.2160.

4.1.3 4-[(tert-Butyldiphenylsilyloxy)methyl]-4′-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzophenone (11)

To a solution of 1,4-dibromobenzene (3.13 g, 13.3 mmol) and 2-isopropoxy-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (2.80 mL, 13.8 mmol) in anhydrous THF (60 mL) was added t-BuLi (19.4 mL, 1.55 M in pentane, 30.0 mmol) dropwise over 3 min at −78 °C under an Ar atmosphere. After being stirred at −78 °C for 30 min, additional t-BuLi (19.4 mL, 1.55 M in pentane, 30.0 mmol) was added dropwise over 3 min. After being stirred at the same temperature for additional 20 min, compound 10 (3.25 g, 7.5 mmol) was added. The reaction mixture was warmed to rt over 1 h and quenched with sat. NH₄Cl. The whole was extracted with EtOAc and the extract was dried over MgSO₄. After concentration, the residue was purified by silica gel chromatography with n-hexane–EtOAc (9:1) to give the title compound 11 as yellow oil (3.60 g, 83 %): IR (neat) cm⁻¹: 1659 (C=O); ¹H-NMR (400 MHz, CDCl₃) δ: 1.11 (s, 9H, 3 × CH₃), 1.37 (s, 12H, 4 × CH₃), 4.85 (s, 2H, CH₂), 7.37–7.46 (m, 8H, Ar), 7.69 (d, J = 6.6 Hz, 4H, Ar), 7.75–7.80 (m, 4H, Ar), 7.92 (d, J = 8.0 Hz, 2H, Ar); ¹³C-NMR (100 MHz, CDCl₃) δ: 19.3, 24.8 (4C), 26.8 (3C), 65.2, 84.2 (2C), 125.6 (2C), 127.8 (4C), 128.9 (2C), 129.8 (2C), 130.2 (2C), 133.2 (2C), 134.5 (2C), 134.8, 135.5 (4C), 136.2, 140.0, 146.0, 196.6; HRMS (FAB): m/z calcd for C₃₆H₄₂BO₄Si [M + H]⁺ 577.2945; found: 577.2949.

4.1.4 9-Bromo-3,4-dihydro-N-(p-methoxybenzyl)-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (12)

To the flask containing 9-bromo-3,4-dihydro-2H,6H-pyrimido[1,2-c] [1, 3]benzothiazin-6-imine 2a (740.4 mg, 2.50 mmol) and t-BuOK (561.1 mg, 5.00 mmol) was added DMF (10.0 mL) at 0 °C under an Ar atmosphere. After being stirred at the same temperature for 30 min, PMB-Br (729.0 μL, 5.00 mmol) was added. After being stirred at rt for 1 h, the reaction mixture was quenched with H₂O. The whole was extracted with EtOAc, and washed with sat. NaHCO₃, brine, and dried over MgSO₄. After concentration, the residue was purified by flash column chromatography over aluminum oxide with n-hexane–EtOAc (3:1) to give the title compound 12 as pale yellow amorphous (1.02 g, 98 %): IR (neat) cm⁻¹: 1661 (C=N), 1510 (C=N); ¹H-NMR (400 MHz, CDCl₃) δ: 1.97–2.03 (m, 2H), 3.64 (t, J = 5.7 Hz, 2H, CH₂), 3.80–3.84 (m, 5H, OCH₃, CH₂), 4.14 (s, 2H, CH₂), 6.86 (d, J = 8.5 Hz, 2H, Ar), 7.21–7.27 (m, 3H, Ar), 7.38 (dd, J = 8.2, 1.8 Hz, 1H, Ar), 7.43 (d, J = 1.8 Hz, 1H, Ar); ¹³C-NMR (100 MHz, CDCl₃) δ: 19.8, 38.7, 44.3, 47.7, 55.3, 111.9, 114.1 (2C), 124.8, 127.9, 129.5, 130.2, 130.3 (2C), 132.6, 133.4, 138.7, 147.6, 159.1; HRMS (FAB): m/z calcd for C₁₉H₁₉N₃OS [M + H]⁺ 416.0432; found: 416.0431.

4.1.5 9-{4-[4-(tert-Butyldiphenylsilyloxy)methyl]benzoylphenyl}-3,4-dihydro-N-(p-methoxybenzyl)-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (13)

Pd(PPh₃)₄ (32.8 mg, 4 mol%) and PdCl₂(dppf)·CH₂Cl₂ (17.4 mg, 3 mol %) were added to a solution of 12 (296.2 mg, 0.71 mmol) and 11 (409.4 mg, 0.71 mmol) in toluene (7.1 mL)-EtOH (4.3 mL)-1 M aq. K₂CO₃ (7.1 mL). After being stirred at reflux for 1 h, the mixture was extracted with CHCl₃. The extract was dried over MgSO₄ and concentrated. The residue was purified by flash chromatography over aluminum oxide with n-hexane–EtOAc (1:0 to 9:1) to give the title compound 13 as pale yellow amorphous (536.2 mg, 96 %): IR (neat) cm⁻¹: 1658 (C=O), 1607 (C=N), 1511 (C=N); ¹H-NMR (400 MHz, CDCl₃) δ: 1.12 (s, 9H, 3 × CH₃), 2.03–2.08 (m, 2H), 3.70 (t, J = 5.5 Hz, 2H, CH₂), 3.77 (s, 3H, CH₃), 3.88 (t, J = 5.9 Hz, 2H, CH₂), 4.19 (s, 2H, CH₂), 4.86 (s, 2H, CH₂), 6.84 (d, J = 8.5 Hz, 2H, Ar), 7.28 (m, 1H, Ar), 7.38-7.56 (m, 14H, Ar), 7.71 (dd, J = 7.6, 1.2 Hz, 4H, Ar), 7.81 (d, J = 8.0 Hz, 2H, Ar), 7.86 (d, J = 8.0 Hz, 2H, Ar); ¹³C-NMR (125 MHz, CDCl₃) δ: 19.3, 19.8, 26.8 (3C), 39.0, 44.3, 47.7, 55.2, 65.1, 112.2, 113.9 (2C), 125.6 (2C), 125.7, 127.0 (2C), 127.7 (4C), 128.7, 129.5, 129.8 (2C), 130.1 (2C), 130.2, 130.3 (2C), 130.5 (2C), 133.1 (2C), 135.0, 135.5 (4C), 136.2, 136.4, 137.0, 142.1, 143.5, 146.0, 148.2, 158.9, 195.8; HRMS (FAB): m/z calcd for C₄₉H₄₈N₃O₃SSi [M + H]⁺ 786.3186; found: 786.3178.

4.1.6 N-(2-[2-{2-(2-Aminoethoxy)ethoxy}ethoxy]ethyl)-5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamide (15)

10 % Pd/C (wetted with ca. 55 % water, 160.0 mg) was added to the solution of biotin-amine 14 ⁶ (116.0 mg, 0.26 mmol) in MeOH (2.0 mL). After being stirred at rt overnight under a H₂ atmosphere, the mixture was filtered through a Celite pad and concentrated. The crude product was used for the next step without further purification.

4.1.7 4-(4-{6-[(4-Methoxybenzyl)imino]-2,3,4,6-tetrahydrobenzo[e]pyrimido[1,2-c][1,3]thiazin-9-yl}benzoyl)benzyl {13-oxo-17-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]-3,6,9-trioxa-12-azaheptadecyl}-carbamate (17)

To a solution of 13 (157.2 mg, 0.20 mmol) in THF (2.0 mL) was added TBAF in THF (0.50 mmol). After being stirred at rt overnight, the reaction mixture was quenched with sat. NH₄Cl. The whole was extracted with CHCl₃ and dried over MgSO₄. After concentration, the residue was subjected to flash column chromatography over aluminum oxide with n-hexane–EtOAc (5:1 to 0:1) to give the desilylated compound. To a solution of the resulting compound in CH₂Cl₂ (6.0 mL) were added p-nitrophenyl chloroformate (60.5 mg, 0.30 mmol) and pyridine (64.6 μL, 0.8 mmol). After being stirred under reflux for 1 h, additional p-nitrophenyl chloroformate (12.0 mg, 0.06 mmol) was added. After being stirred under reflux for additional 30 min, the reaction mixture was washed with brine, and dried over MgSO₄. After concentration, the solution of resulting residue (crude 16) in DMF (2.0 mL) was added to the solution of 15 (ca. 0.26 mmol) and Et₃N (86.7 μL) in DMF (3.0 mL). After being stirred at rt for 8 h, the reaction mixture was stirred at 40 °C overnight. After concentration, the residue was purified by flash column chromatography over aluminum oxide with CHCl₃–MeOH (1:0 to 95:5) followed by flash column chromatography over silica gel with CHCl₃–MeOH (1:0 to 9:1) to give the title compound 17 as pale yellow amorphous (90.6 mg, 46 %): IR (neat) cm⁻¹: 1699 (C=O), 1656 (C=O), 1607 (C=N), 1511 (C=N); ¹H-NMR (500 MHz, CDCl₃) δ: 1.39–1.45 (m, 2H, CH₂), 1.57–1.74 (m, 4H, 2 × CH₂), 2.03-2.08 (m, 2H, CH₂), 2.20 (t, J = 6.9 Hz, 2H, CH₂), 2.70 (d, J = 12.6 Hz, 1H, CH), 2.87 (dd, J = 12.6, 4.6 Hz, 1H, CH), 3.12 (d, J = 11.7, 4.6 Hz, 1H, CH), 3.40–3.43 (m, 4H, 2 × CH₂), 3.54–3.71 (m, 14H, 7 × CH₂), 3.77 (s, 3H, CH₃), 3.88 (t, J = 6.0 Hz, 2H, CH₂), 4.19 (s, 2H, CH₂), 4.26–4.29 (m, 1H, CH), 4.45-4.47 (m, 1H, CH), 5.17 (s, 1H, NH), 5.20 (s, 2H, CH₂), 5.65 (s, 1H, NH), 6.07 (s, 1H, NH), 6.48 (s, 1H, NH), 6.84 (d, J = 8.0 Hz, 2H, Ar), 7.26–7.28 (m, 2H, Ar), 7.44–7.62 (m, 7H, Ar), 7.81 (d, J = 8.0 Hz, 2H, Ar), 7.85 (d, J = 8.0 Hz, 2H, Ar); ¹³C-NMR (125 MHz, CDCl₃) δ: 19.8, 25.5, 28.0, 28.1, 35.9, 39.0, 39.1, 40.4, 40.9, 44.3, 47.7, 55.2, 55.5, 60.1, 61.7, 65.8, 69.9, 69.9, 70.0, 70.2, 70.3 (2C), 112.2, 114.0 (2C), 125.7, 127.1 (2C), 127.4 (2C), 127.6, 128.6, 129.5, 130.2 (2C), 130.3 (2C), 130.6 (2C), 135.0, 136.5, 136.7, 137.1, 141.4, 142.0, 143.7, 148.2, 156.3, 158.9, 163.9, 173.2, 195.7; HRMS (FAB): m/z calcd for C₅₂H₆₂N₇O₉S₂ [M + H]⁺ 992.4050; found: 992.4050.

4.1.8 4-[4-(2,3,4,6-Tetrahydro-6-iminobenzo[e]pyrimido[1,2-c][1, 3]thiazin-9-yl)benzoyl]benzyl{13-oxo-17-[(3aS, 4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]-3,6,9-trioxa-12-azaheptadecyl}carbamate (6)

TFA (2.0 mL) was added to a mixture of 17 (62.9 mg, 0.063 mmol) in small amount of CHCl₃ (1 or 2 drops) and MS4Å (300 mg, powder, activated by heating with Bunsen burner). After being stirred at rt for 4 h, Et₃N was added dropwise to the stirring mixture at 0 °C to adjust pH to 8–9. The whole was extracted with CHCl₃, and washed with sat. NaHCO₃, brine, and dried over MgSO₄. After concentration, the residue was purified by flash chromatography over aluminum oxide with CHCl₃–MeOH (1:0 to 95:5) followed by preparative HPLC to give the title compound 6 as colorless solid (19.3 mg, 35 %): IR (neat) cm⁻¹: 1699 (C=O), 1654 (C=O), 1621 (C=O), 1601 (C=N), 1574 (C=N); ¹H-NMR (500 MHz, CDCl₃) δ: 1.39–1.44 (m, 2H, CH₂), 1.60–1.76 (m, 4H, 2 × CH₂), 1.99–2.04 (m, 2H, CH₂), 2.20 (t, J = 7.4 Hz, 2H, CH₂), 2.71 (d, J = 12.6 Hz, 1H, CH), 2.88 (dd, J = 12.6, 5.0 Hz, 1H, CH), 3.11 (d, J = 11.7, 5.0 Hz, 1H, CH), 3.40–3.43 (m, 4H, 2 × CH₂), 3.54–3.63 (m, 12H, 6 × CH₂), 3.73 (t, J = 5.4 Hz, 2H, CH₂), 4.06 (t, J = 6.0 Hz, 2H, CH₂), 4.28 (t, J = 6.0 Hz, 1H, CH), 4.47 (t, J = 6.0 Hz, 1H, CH), 5.20 (s, 2H, CH₂), 5.44 (s, 1H, NH), 5.73 (s, 1H, NH), 6.37 (s, 1H, NH), 6.66 (s, 1H, NH), 7.32 (s, 1H, Ar), 7.48 (d, J = 8.0 Hz, 2H, Ar), 7.52 (d, J = 8.6 Hz, 1H, Ar), 7.69 (d, J = 8.0 Hz, 2H, Ar), 7.81 (d, J = 8.0 Hz, 2H, Ar), 7.88 (d, J = 8.0 Hz, 2H, Ar), 8.36 (d, J = 8.6 Hz, 1H, Ar); ¹³C-NMR (125 MHz, CDCl₃) δ: 20.8, 25.6, 28.0, 28.2, 35.9, 39.0, 40.4, 40.9, 43.9, 44.7, 51.2, 55.6, 60.1, 61.7, 65.7, 69.9, 70.0, 70.1, 70.3 (2C), 122.0, 125.2, 125.8, 126.9 (2C), 127.4 (2C), 129.6, 129.7, 130.2 (2C), 130.7 (2C), 137.0, 141.5, 142.2, 142.9, 144.8, 146.6, 152.9, 156.3, 164.1, 173.3, 195.6; HRMS (FAB): m/z calcd for C₄₄H₅₄N₇O₈S₂ [M + H]⁺ 872.3475; found: 872.3481.

4.1.9 N-[9-Bromo-1′-(4-methoxybenzyl)-2H-spiro(benzo[e]pyrimido[1,2-c][1,3]thiazine-3,4′-piperidin)-6(4H)-ylidene]-1-(4-methoxyphenyl)methanamine (19)

By a procedure identical with that described for synthesis of 12 from 2a, the 9-bromo-1′-(4-methoxybenzyl)-2H-spiro(benzo[e]pyrimido[1,2-c][1,3]thiazine-3,4′-piperidin)-6(4H)-imine 18 (274.3 mg, 0.57 mmol) was converted into 19 as colorless amorphous (275.1 mg, 81 %): IR (neat) cm⁻¹: 1668 (C=N), 1510 (C=N); ¹H-NMR (400 MHz, CDCl₃) δ: 1.61–1.64 (m, 4H, 2 × CH₂), 2.36–2.42 (m, 2H, CH₂), 2.45–2.51 (m, 2H, CH₂), 3.45 (s, 2H, CH₂), 3.47 (s, 2H, CH₂), 3.55 (s, 2H, CH₂), 3.80 (s, 3H, CH₃), 3.81 (s, 3H, CH₃), 4.12 (s, 2H, CH₂), 6.82-6.87 (m, 4H, Ar), 7.19–7.23 (m, 5H, Ar), 7.38 (dd, J = 8.2, 1.8 Hz, 1H, Ar), 7.44 (d, J = 2.0 Hz, 1H, Ar); ¹³C-NMR (100 MHz, CDCl₃) δ: 28.2, 32.4 (2C), 39.1, 48.7 (2C), 54.6, 55.2, 55.3, 55.4, 62.6, 111.9, 113.7 (2C), 113.9, 114.1 (2C), 124.8, 128.0, 129.7, 130.0, 130.2 (4C), 133.4, 133.4, 138.6, 147.1, 158.8, 159.1; HRMS (FAB): m/z calcd for C₃₁H₃₄BrN₄O₂S [M + H]⁺ 605.1586; found: 605.1585.

4.1.10 N-[9-Bromo-2H-spiro(benzo[e]pyrimido[1,2-c][1, 3]thiazine-3,4′-piperidin)-6(4H)-ylidene]-1-(4-methoxy-phenyl)methanamine (20)

To a solution of 19 (60.6 mg, 0.10 mmol) in CH₂Cl₂ (0.5 mL) were added Et₃N (28.9 μL, 0.20 mmol) and 1-chloroethyl chloroformate (21.8 μL, 0.20 mmol) at 0 °C under an Ar atmosphere. After being stirred at the same temperature for 30 min, the reaction mixture was concentrated. The residue was dissolved in MeOH (2.0 mL). After being stirred under reflux for 10 min, the reaction mixture was concentrated. The residue was dissolved in CHCl₃, and was washed with sat. NaHCO₃, brine, and dried over MgSO₄. After concentration, the crude product was used for the next step without further purification.

4.1.11 4-[4-(tert-Butyldiphenylsilyloxymethyl)benzoyl]benzyl{13-oxo-17-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno-[3,4-d]imidazol-4-yl]-3,6,9-trioxa-12-azaheptadecyl}carbamate (23)

To a solution of 4-(tert-butyldiphenylsilyloxy)methyl-4′-(hydroxymethyl)benzophenone 21 ⁷ (240.3 mg, 0.50 mmol) in CH₂Cl₂ (15.0 mL) were added p-nitrophenyl chloroformate (151.2 mg, 0.75 mmol) and pyridine (161.4 μL, 2.00 mmol). After being stirred under reflux for 1 h, the reaction mixture was washed with brine, and dried over MgSO₄. After concentration, the solution of the resulting residue in DMF (7.5 mL) was added to a mixture of 15 (ca. 0.20 mmol) and Et₃N (216.8 μL) in DMF (5.0 mL). After being stirred at rt overnight, the mixture was concentrated. The residue was purified by flash column chromatography over silica gel with CHCl₃–MeOH (1:0 to 95:5) to give the title compound 23 as colorless amorphous (471.5 mg, quant.): IR (neat) cm⁻¹: 1700 (C=O), 1656 (C=O), 1609 (C=O); ¹H-NMR (400 MHz, CDCl₃) δ: 1.12 (s, 9H, 3 × CH₃), 1.39–1.46 (m, 2H, CH₂), 1.61–1.76 (m, 4H, 2 × CH₂), 2.19-2.23 (m, 2H, CH₂), 2.69–2.76 (m, 1H, CH), 2.85–2.90 (m, 1H, CH), 3.09–3.15 (m, 1H, CH), 3.39–3.43 (m, 4H, 2 × CH₂), 3.54–3.66 (m, 12H, 6 × CH₂), 4.26–4.33 (m, 1H, CH), 4.45–4.51 (m, 1H, CH), 4.85 (s, 2H, CH₂), 5.19 (s, 2H, CH₂), 5.54 (br s, 1H, NH), 5.68 (br s, 1H, NH), 6.55 (br s, 1H, NH), 6.72 (br s, 1H, NH), 7.36-7.48 (m, 10H, Ar), 7.69 (d, J = 7.6 Hz, 2H, Ar), 7.70 (d, J = 7.6 Hz, 2H, Ar), 7.77 (d, J = 5.5 Hz, 2H, Ar), 7.79 (d, J = 5.5 Hz, 2H, Ar); ¹³C-NMR (100 MHz, CDCl₃) δ: 19.3, 25.5, 26.8 (3C), 28.1, 28.2, 35.9, 39.1, 40.5, 40.9, 55.5, 60.1, 61.7, 65.1, 65.8, 69.9, 70.0, 70.0, 70.2, 70.4 (2C), 125.6 (2C), 127.4 (2C), 127.8 (4C), 129.8 (2C), 130.1 (2C), 130.2 (2C), 133.2 (2C), 135.5 (4C), 136.1, 137.4, 141.2, 146.0, 156.3 163.9, 173.2, 196.0; HRMS (FAB): m/z calcd for C₅₀H₆₅N₄O₉SSi [M + H]⁺ 925.4242; found: 925.4246.

4.1.12 4-[4-(Hydroxymethyl)benzoyl]benzyl{13-oxo-17-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]-3,6,9-trioxa-12-azaheptadecyl}carbamate (24)

To a solution of 23 (383.0 mg, 0.41 mmol) in THF (8.2 mL) was added HF-pyridine (617.7 μL, Aldrich) at 0 °C. After being stirred at rt overnight, the reaction was quenched with sat. NaHCO₃. The whole was extracted with CHCl₃, and washed with water and brine, and dried over MgSO₄. After concentration, the residue was purified by preparative TLC over silica gel with CHCl₃–MeOH (85:15) to give the title compound 24 as colorless oil (204.2 mg, 73 %): IR (neat) cm⁻¹: 1696 (C=O), 1650 (C=O), 1609 (C=O); ¹H-NMR (400 MHz, CDCl₃) δ: 1.34–1.41 (m, 2H, CH₂), 1.55–1.73 (m, 4H, 2 × CH₂), 2.07 (br s, 1H, OH), 2.16 (t, J = 7.4 Hz, 2H, CH₂), 2.68 (d, J = 12.9 Hz, 1H, CH), 2.85 (dd, J = 12.9, 4.9 Hz, 1H, CH), 3.08 (dd, J = 11.8, 7.4 Hz 1H, CH), 3.37–3.42 (m, 4H, 2 × CH₂), 3.51–3.64 (m, 12H, 6 × CH₂), 4.23 (t, J = 6.2 Hz, 1H, CH), 4.43 (t, J = 6.2 Hz, 1H, CH), 4.78 (s, 2H, CH₂), 5.18 (s, 2H, CH₂), 5.51 (br s, 1H, NH), 5.82 (br s, 1H, NH), 6.34 (br s, 1H, NH), 6.75 (br s, 1H, NH), 7.45 (d, J = 8.3 Hz, 2H, Ar), 7.48 (d, J = 8.3 Hz, 2H, Ar), 7.76 (d, J = 8.0 Hz, 2H, Ar), 7.77 (d, J = 8.0 Hz, 2H, Ar); ¹³C-NMR (125 MHz, CDCl₃) δ: 25.5, 28.0, 28.2, 35.8, 39.1, 40.4, 40.9, 55.6, 60.2, 61.8, 64.2, 65.7, 69.9, 69.9 (2C), 70.1, 70.3 (2C), 126.4 (2C), 127.3 (2C), 130.2 (2C), 130.2 (2C), 136.2, 137.1, 141.3, 146.4, 156.4, 164.1, 173.5, 196.0; HRMS (FAB): m/z calcd for C₃₄H₄₇N₄O₉S [M + H]⁺ 687.3064; found: 687.3058.

4.1.13 4-(4-{[(4-Nitrophenoxy)carbonyloxy]methyl}benzoyl)benzyl-13-oxo-17-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]-3,6,9-trioxa-12-azaheptadecylcarbamate (25)

To a solution of 24 (28.2 mg, 0.04 mmol) in CH₂Cl₂ (1.2 mL) were added p-nitrophenyl chloroformate (24.8 mg, 0.12 mmol) and pyridine (13.2 μL, 0.16 mmol). After being stirred under reflux for 1 h, the reaction mixture was washed with brine, and dried over MgSO₄. After concentration, the residue was purified by preparative TLC over aluminum oxide with CHCl₃–MeOH (9:1) to give the title compound 25 as colorless amorphous (27.9 mg, 80 %): IR (neat) cm⁻¹: 1768 (C=O), 1698 (C=O), 1656 (C=O), 1612 (C=O); ¹H-NMR (400 MHz, CDCl₃) δ: 1.38-1.45 (m, 2H, CH₂), 1.59-1.76 (m, 4H, 2 × CH₂), 2.20 (t, J = 7.4 Hz, 2H, CH₂), 2.72 (d, J = 12.7 Hz, 1H, CH), 2.88 (dd, J = 12.7, 4.9 Hz, 1H, CH), 3.12 (dd, J = 11.8, 7.4 Hz, 1H, CH), 3.38–3.44 (m, 4H, 2 × CH₂), 3.55-3.63 (m, 12H, 6 × CH₂), 4.28 (t, J = 6.0 Hz, 1H, CH), 4.47 (t, J = 6.0 Hz, 1H, CH), 5.19 (s, 2H, CH₂), 5.38 (s, 2H, CH₂), 5.52 (br s, 1H, NH), 5.69 (br s, 1H, NH), 6.44 (br s, 1H, NH), 6.66 (br s, 1H, NH), 7.41 (d, J = 9.3 Hz, 2H, Ar), 7.47 (d, J = 8.0 Hz, 2H, Ar), 7.56 (d, J = 8.0 Hz, 2H, Ar), 7.79 (d, J = 8.0 Hz, 2H, Ar), 7.84 (d, J = 8.0 Hz, 2H, Ar), 8.29 (d, J = 9.3 Hz, 2H, Ar); ¹³C-NMR (CDCl₃, 100 MHz) δ: 25.5, 28.1, 28.2, 35.9, 39.1, 40.5, 40.9, 55.5, 60.2, 61.8, 65.8, 69.9, 70.0, 70.0 (2C), 70.2, 70.4 (2C), 121.7 (2C), 125.3 (2C), 127.5 (2C), 128.1 (2C), 130.2 (2C), 130.4 (2C), 136.8, 137.9, 138.6, 141.7, 145.5, 152.4, 155.4, 156.3, 163.9, 173.3, 195.5; HRMS (FAB): m/z calcd for C₄₁H₅₀N₅O₁₃S [M + H]⁺ 852.3126; found: 852.3127.

4.1.14 4-(4-{3,17-Dioxo-21-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]-2,7,10,13-tetraoxa-4,16-diazahenicosyl}benzoyl)benzyl-9-bromo-6-imino-4,6-dihydro-2H-spiro(benzo[e]pyrimido[1,2-c][1,3]thiazine-3,4′-piperidine)-1′-carboxylate (7)

To a solution of 20 (ca. 0.027 mmol) in DMF (0.4 mL) were added Et₃N (11.7 μL, 0.081 mmol) and the solution of 25 (23.3 mg, 0.027 mmol) in DMF (0.4 mL) at rt. After being stirred at the same temperature for 1 h, the reaction mixture was concentrated. The residue was subjected to preparative TLC over silica gel with CHCl₃–MeOH (9:1) to give crude imine 26. By a procedure identical with that described for synthesis of 6 from 17, the crude 26 was converted into 7 as a colorless amorphous (10.4 mg, 36 %): IR (neat) cm⁻¹: 1699 (C=O), 1655 (C=O), 1612 (C=O), 1573 (C=N); ¹H-NMR (400 MHz, CDCl₃) δ: 1.39-1.46 (m, 2H, CH₂), 1.53 (d, J = 5.6 Hz, 4H, 2 × CH₂), 1.61–1.72 (m, 4H, 2 × CH₂), 2.20 (t, J = 7.3 Hz, 2H, CH₂), 2.71 (d, J = 12.7 Hz, 1H, CH), 2.89 (dd, J = 12.7, 4.9 Hz, 1H, CH), 3.12 (d, J = 12.1, 7.3 Hz, 1H, CH), 3.39-3.44 (m, 4H, 2 × CH₂), 3.53–3.63 (m, 18H, 9 × CH₂), 3.93 (s, 2H, CH₂), 4.28 (t, J = 5.7 Hz, 1H, CH), 4.47 (t, J = 6.5 Hz, 1H, CH), 5.14 (s, 1H, NH), 5.19 (s, 2H, CH₂), 5.22 (s, 2H, CH₂), 5.68 (s, 1H, NH), 6.01 (s, 1H, NH), 6.52 (s, 1H, NH), 7.22 (d, J = 2.0 Hz, 1H, Ar), 7.34 (dd, J = 8.8, 2.0 Hz, 1H, Ar), 7.45 (d, J = 8.0 Hz, 2H, Ar), 7.46 (d, J = 8.0 Hz, 2H, Ar), 7.79 (m, 4H, Ar), 8.10 (d, J = 8.8 Hz, 1H, Ar); ¹³C-NMR (100 MHz, CDCl₃) δ: 25.5, 28.1, 28.1, 29.6, 32.2 (2C), 35.8, 39.1, 39.9 (2C), 40.5, 40.9, 49.9, 54.6, 55.4, 60.1, 61.8, 65.8, 66.4, 69.9, 70.0 (2C), 70.2, 70.4 (2C), 125.0, 125.3, 126.0, 127.3 (2C), 127.4 (2C), 129.6, 130.2 (2C), 130.3 (2C), 130.4, 130.6, 137.0, 137.1, 141.4, 141.5, 145.1, 152.6, 155.0, 156.3, 163.8, 173.3, 195.7; HRMS (FAB): m/z calcd for C₅₀H₆₂BrN₈O₁₀S₂ [M + H]⁺ 1077.3214; found: 1077.3213.

4.1.15 N-(tert-Butyl)-9-{4-[4-(tert-butyldiphenylsilyloxy)methyl]benzoylphenyl}-3,4-dihydro-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (28)

Compound 27 (2.17 g, 6.17 mmol) was subjected to the general cross-coupling procedure as described for the synthesis of 13 to give the title compound 28 as colorless solid (3.16 g, 71 %): mp 152–153 °C (from CHCl₃–n-hexane): IR (neat) cm⁻¹: 1656 (C=O), 1623 (C=N), 1593 (C=N); ¹H-NMR (400 MHz, CDCl₃) δ: 1.12 (s, 9H, 3 × CH₃), 1.41 (s, 9H, 3 × CH₃), 1.91–1.97 (m, 2H), 3.65 (t, J = 5.4 Hz, 2H, CH₂), 3.90 (t, J = 6.2 Hz, 2H, CH₂), 4.86 (s, 2H, CH₂), 7.37–7.48 (m, 10H, Ar), 7.69–7.71 (m, 6H, Ar), 7.81 (d, J = 8.3 Hz, 2H, Ar), 7.88 (d, J = 8.3 Hz, 2H, Ar), 8.30 (d, J = 8.5 Hz, 1H, Ar); ¹³C-NMR (100 MHz, CDCl₃) δ: 19.3, 21.9, 26.8 (3C), 30.0 (3C), 45.2, 45.5, 54.2, 65.2, 123.0, 124.9, 125.7 (2C), 126.9 (2C), 127.4, 127.8 (4C), 129.1, 129.8 (2C), 129.9, 130.2 (2C), 130.7 (2C), 133.2 (2C), 135.5 (4C), 136.2, 137.2, 138.0, 141.7, 143.2, 146.1, 147.6, 195.9; HRMS (FAB): m/z calcd for C₄₅H₄₈N₃O₂SSi [M + H]⁺ 722.3237; found: 722.3244.

4.1.16 N-(tert-Butyl)-3,4-dihydro-9-[4-(4-propargyloxymethyl)benzoylphenyl]-2H,6H-pyrimido[1,2-c][1,3]benzo- thiazin-6-imine (29)

To a solution of 28 (200.0 mg, 0.28 mmol) in THF (2.8 mL) was added TBAF in THF (0.55 mmol). After being stirred at rt for 2 h, the reaction mixture was quenched with sat. NH₄Cl. The whole was extracted with EtOAc, and washed with brine, and dried over MgSO₄. The filtrate was concentrated. To the solution of the resulting residue in THF (2.8 mL) was added NaH (22.8 mg, 0.55 mmol, 60 % oil suspension) at 0 °C. After being stirred at the same temperature for 30 min, propargyl bromide (31.5 μL, 0.42 mmol) was added dropwise. After being stirred at rt overnight, the reaction was quenched with water. The whole was extracted with EtOAc, and washed with brine, and dried over MgSO₄. After concentration, the residue was purified by flash column chromatography over aluminum oxide with n-hexane–EtOAc (5:1) to give the title compound 29 as colorless solid (87.2 mg, 60 %): mp 133–135 °C (from CHCl₃–n-hexane): IR (neat) cm⁻¹: 1656 (C=O), 1620 (C=N), 1593 (C=N); ¹H-NMR (400 MHz, CDCl₃) δ: 1.41 (s, 9H, 3 × CH₃), 1.91–1.97 (m, 2H), 2.50 (t, J = 2.3 Hz, 1H, CH), 3.65 (t, J = 5.5 Hz, 2H, CH₂), 3.90 (t, J = 6.1 Hz, 2H, CH₂), 4.25 (d, J = 2.3 Hz, 2H, CH₂), 4.71 (s, 2H, CH₂), 7.39 (d, J = 1.7 Hz, 1H, Ar), 7.46–7.50 (m, 3H, Ar), 7.70 (d, J = 8.0 Hz, 2H, Ar), 7.82 (d, J = 8.0 Hz, 2H, Ar), 7.87 (d, J = 8.0 Hz, 2H, Ar), 8.30 (d, J = 8.3 Hz, 1H, Ar); ¹³C-NMR (100 MHz, CDCl₃) δ: 21.9, 30.0 (3C), 45.2, 45.4, 54.2, 57.6, 70.9, 75.0, 79.3, 123.0, 124.8, 126.9 (2C), 127.3, 127.5 (2C), 129.1, 129.9, 130.2 (2C), 130.7 (2C), 136.9, 137.0, 137.9, 141.6, 142.2, 143.4, 147.5, 195.7; HRMS (FAB): m/z calcd for C₃₂H₃₂N₃O₂S [M + H]⁺ 522.2215; found: 522.2207.

4.1.17 3,4-Dihydro-9-[4-(4-propargyloxymethyl)benzoylphenyl]-2H,6H-pyrimido[1,2-c][1,3]benzothiazin-6-imine (8)

Using a procedure identical with that described for synthesis of 6 from 17, the imine 29 (42.8 mg, 0.08 mmol) was allowed to react under reflux for 1 h with TFA (2.0 mL) and MS4Å (300 mg). Purification by flash chromatography over aluminum oxide with n-hexane–EtOAc (9:1 to 1:1) gave the title compound 8 as colorless solid (35.4 mg, 92 %): mp 159–160 °C (from CHCl₃–n-hexane): IR (neat) cm⁻¹: 1654 (C=O), 1619 (C=N), 1573 (C=N); ¹H-NMR (400 MHz, CDCl₃) δ: 1.96–2.04 (m, 2H), 2.50 (t, J = 2.4 Hz, 1H, CH), 3.72 (t, J = 5.6 Hz, 2H, CH₂), 4.05 (t, J = 6.1 Hz, 2H, CH₂), 4.25 (d, J = 2.4 Hz, 2H, CH₂), 4.71 (s, 2H, CH₂), 7.26–7.31 (m, 2H, Ar, NH), 7.48–7.51 (m, 3H, Ar), 7.67–7.89 (m, 6H, Ar), 8.33 (d, J = 8.5 Hz, 1H, Ar); ¹³C-NMR (100 MHz, CDCl₃) δ: 21.0, 43.8, 45.0, 57.6, 70.9, 75.0, 79.3, 122.0, 125.1, 126.3, 126.9 (2C), 127.5 (2C), 129.6, 129.7, 130.2 (2C), 130.7 (2C), 137.0, 137.1, 142.2, 142.3, 143.0, 146.2, 153.0, 195.7; HRMS (FAB): m/z calcd for C₂₈H₂₄N₃O₂S [M + H]⁺ 466.1589; found: 466.1589.

4.1.18 Photoaffinity Labeling Experiments Using HIV-1-Infected H9 Cells (H9IIIB)

1 μL of probe 6 or 7 (10 mM solution in DMSO) was added to H9 cells chronically infected with HIV-1 (H9IIIB) in D-MEM with 10 % fetal bovine serum (500 μL, 0.5 × 10⁶ cells). For the competitive evaluation (Fig. 4.3, lane C), 2 μL of compound 3a (10 mM solution in DMSO) was also added. The cells were incubated at 37 °C for 1 h. Then the cells were photolabeled by irradiation by UV (MUV-202U, Moritex Co., Japan) at rt for 1 min at a distance of 3 cm through a longpass filter (LU0300, Asahi spectra Co.). The mixture was centrifuged at 200 × g for 5 min and the supernatant was removed. The cells were washed with PBS once and were lysed in RIPA buffer containing 1 % protease inhibitor cocktail (Nacalai Tesque, Inc., Japan) at 4 °C for 30 min. After centrifugation at 16,500 × g for 15 min, the supernatant was used for the next experiment.

NeutrAvidin agarose beads (50 μL, Thermo), which were equilibrated with RIPA buffer, were treated with the supernatant containing 180 μg of proteins and were incubated at 4 °C for 1 h. The beads were then centrifuged at 9,100 × g for 30 s and washed with RIPA buffer (repeated three times). After heating the beads at 95 °C for 5 min in sample buffer [50 mM Tris–HCl (pH 8.0), 2 % SDS, 0.1 % BPB, 10 % glycerol, 2 % β-ME], the supernatants were subjected to SDS–PAGE electrophoresis (SuperSep™Ace, 5–20 %, Wako) and the separated proteins were transferred onto a PVDF membrane. The membrane was blocked with Blocking One (Nacalai Tesque, Inc.) at rt for 1 h, and was then incubated with a streptavidin–HRP conjugate (Invitrogen; 1:5,000 in PBS with 0.1 % Tween) at 4 °C overnight. The membrane was treated with Chemi-Lumi One L (Nacalai Tesque, Inc.). Biotinylated proteins were detected by Image Quant LAS 4000mini (GE Healthcare).

4.1.19 Determination of Anti-HIV Activity

The sensitivity of HIV-1_IIIB strain was determined by the MAGI assay. The target cells (HeLa-CD4/CCR5-LTR/β-gal; 10⁴ cells/well) were plated in 96-well flat microtiter culture plates. On the following day, the cells were inoculated with the HIV-1 (60 MAGI U/well, giving 60 blue cells after 48 h of incubation) and cultured in the presence of various concentrations of the test compounds in fresh medium. Forty-eight hours after viral exposure, all the blue cells stained with X-Gal (5-bromo-4-chloro-3-indolyl- β-D-galactopyranoside) were counted in each well. The activity of test compounds was determined as the concentration that blocked HIV-1 infection by 50 % (50 % effective concentration [EC₅₀]). EC₅₀ was determined by using the following formula:

EC₅₀ = 10^[log(A/B) × (50 − C)/(D − C) + log(B)],

wherein

A:

of the two points on the graph which bracket 50 % inhibition, the higher concentration of the test compound,

B:

of the two points on the graph which bracket 50 % inhibition, the lower concentration of the test compound,

C:

inhibitory activity (%) at the concentration B,

D:

inhibitory activity (%) at the concentration A.