4-(Dimethylamino)pyridine N-Oxide-Catalyzed Macrolactamization Using 2-Methyl-6-nitrobenzoic Anhydride in the Synthesis of the Depsipeptidic Analogue of FE399.

A depsipeptidic analogue of FE399 was efficiently synthesized mainly through macrolactamization using 2-methyl-6-nitrobenzoic anhydride (MNBA), and a detailed investigation of the desired 16-membered macrolactam core of FE399 was performed. It was determined that the combination of MNBA and a catalytic amount of 4-(dimethylamino)pyridine N-oxide exhibits much higher activity than that of conventionally used coupling reagents such as hexafluorophosphate azabenzotriazole tetramethyl uronium and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate.

Introduction

Macrocyclic scaffolds, including macrolactones and macrolactams, are commonly found in a wide variety of natural and artificial products and in many beneficial pharmaceutical compounds.[1] Therefore, these structures are significant and intriguing structural motifs in synthetic organic chemistry.[2] Thus, diverse methods have been developed for the synthesis of such macrocyclic compounds. Among them, methods that are based on the macrolactonization strategy are most popular and extensively studied by numerous research groups in the last few decades.[3]

Meanwhile, currently, it is widely known that the formation of carboxylic esters and lactones with various ring sizes is effectively promoted using substituted benzoic anhydrides, such as 2-methyl-6-nitrobenzoic anhydride (MNBA), as dehydrating agents in the presence of a nucleophilic catalyst, 4-(dimethylamino)pyridine N-oxide (DMAPO) or 4-(dimethylamino)pyridine (DMAP).[4] Furthermore, in our study on the MNBA-mediated β-lactone formation,[5] we successfully identified transition state (TS) by density functional theory calculations and determined that after the formation of the acylpyridinium complex (through the activation of the carboxyl group of substrate hydroxy acid), the deprotonation of hydroxy group on one site of the substrate with a 2-methyl-6-nitrobenzoate anion formed in situ and the desired lactone formation concertedly proceeds in the TS. It is determined that using the anionic residue of MNBA, TS is rigidly stabilized, and lactonization is effectively promoted.[5,6] This method is practically applicable to the synthesis of amides,[7] peptides,[8] and lactams,[9] while the detailed mechanism of the synthesis of these compounds by using MNBA has not been conclusively established so far.

However, thus far, there have been few studies on the synthesis of these compounds using MNBA.[10]

Recently, we achieved the total synthesis of (9R,14R,17R)-(−)-FE399 (1),[9,11] a novel bicyclic depsipeptide, in which both the formation of a dipeptide composed of two l-cystein residues and the creation of a macrolactam core of FE399 were shown to be effectively performed by the MNBA-mediated dehydration condensation reaction. Remarkably, this synthesis is the first application of MNBA-mediated macrolactamization for the total synthesis of natural products (Figure ).

Figure 1

Structures of FE399 (1) and its analogue (2).

However, because FE399 was determined to exhibit a considerable anticancer activity[12] both in vitro and in vivo, this compound and its synthesized congeners can be used as effective and promising anticancer agents in the future. Thus, we designed and synthesized a depsipeptidic analogue 2 of FE399 without an alkyl group at the C9 center. Thereby, MNBA-macrolactamization, as the key transformation to construct 16-membered macrolactam during the FE399 synthesis, was fully investigated in this study to advance future biological activity studies using FE399 analogues.

Results and Discussion

The preparation of desired linear precursor 10 for macrolactamization was performed using our established synthesis of naturally occurring FE399, as shown in Scheme . First, the known aldehyde 3(13) derived from commercially available 1,9-nonanediol (see the Supporting Information) was subjected to Pinnick oxidation followed by the protection of resulting carboxylic acid with an allyl ester group to afford 4. Then, the obtained allyl ester 4 was treated with tetra-n-butylammonium fluoride in tetrahydrofuran (THF) to quantitatively afford the primary alcohol 5. This alcohol underwent esterification with Fmoc-Cys(Trt)-OH under standard reaction conditions using MNBA in the presence of DMAP to afford ester 6 without racemization, followed by the removal of the Fmoc group with diethylamine to give 7 in high yield. The same operation with Fmoc-Cys(Trt)-OH was repeatedly performed to yield the coupling product 9. Finally, the cleavage of the allyl moiety in the presence of a palladium catalyst was performed to afford the desired linear precursor 10 for the following macrolactamization.

Scheme 1

Preparation of the Macrolactamization Precursor 10

On the basis of our previous result[9] on the use of macrolactamization for the FE399 synthesis, we were interested to determine to what extent MNBA should promote target macrolactamization compared to conventional coupling reagents for amide and peptide bond formation. Thus, we evaluated the efficacy of the target macrolactamization of ring-closing precursor 10 using MNBA combined with DMAPO or DMAP as coupling reagents (Table ).

Table 1

The Target Macrolactamization of 10 Using MNBA or Other Dehydrating Agents

entry	coupling agent (equiv)	catalyst (equiv)	additive (equiv)	time (h)	temp (°C)	yieldb (%)
1	MNBA (1.3)	DMAP (6.0)		12	rt	19
2	MNBA (1.3)	DMAP (6.0)		12	40	40
3	MNBA (1.3)	DMAP (2.6)		12	40	62
4	MNBA (1.3)	DMAP (2.6)		1	40	69
5	MNBA (1.3)	DMAP (2.6)		0.5	40	58
6	MNBA (1.3)	DMAP (2.6)		5 min	40	51
7	MNBA (1.3)	DMAP (0.2)	Et3N (2.6)	12	40	49
8	MNBA (1.3)	DMAPO (2.6)		12	40	45
9	MNBA (1.3)	DMAPO (0.2)	Et3N (2.6)	12	40	71
10	MNBA (1.3)	DMAPO (0.2)	Et3N (2.6)	1	40	45
11	HATU (1.3)		iPr2NEt (6.0)	12	rt	34
12	HATU (1.3)		iPr2NEt (2.6)	12	40	50
13	PyBOP (1.3)		iPr2NEt (2.6)	12	40	36

A syringe pump was employed during the reaction.

Yield of isolated product.

First, the precursor 10 was subjected to our standard reaction conditions for the synthesis of macrolactone.[14] When the solution of 10 dissolved in dichloromethane (2 mM) was slowly added to the mixture of 1.3 equiv of MNBA and 6.0 equiv of DMAP via a syringe pump over the course of 12 h at room temperature (rt), the corresponding macrolactam 11 was obtained in a lower yield (entry 1). At a slightly higher reaction temperature (40 °C), the yield was moderately improved (entry 2). Moreover, a decrease in the amount of DMAP to 2.6 equiv considerably increased the yield to 62% (entry 3). An excessive amount of DMAP as a nucleophilic catalyst may favor undesired side reactions such as the aminolysis of the carboxylic ester moiety involving the primary amine in 10. Next, we examined the product yields as a function of time to confirm its reactivity in the reaction using MNBA (entries 3–6). It was determined that the reaction smoothly proceeded to afford a satisfactory yield in 1 h (69%, entry 4). Of note, the reaction also proceeded to afford 11 in good yield in only 5 min (entry 6). The use of a lower catalyst loading was also possible by the addition of excess triethylamine (2.6 equiv) as a co-base and provided the ring-closed product 11 in moderate yield (entry 7).

Moreover, we have already determined that the combination of MNBA and DMAPO, a weaker base than DMAP, is effective as a coupling reagent for lactam formation.[9] In fact, on using DMAPO as a nucleophilic catalyst instead of DMAP, the target product was efficiently obtained, as is the case with DMAP (entry 8). Specifically, in the presence of MNBA (1.3 equiv) combined with the catalytic amount of DMAPO (0.2 equiv) and triethylamine (2.6 equiv), the best yield of 11 was obtained through MNBA-mediated macrolactamization (entry 9). However, when the most widely used coupling reagents for peptide bond formation, such as hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU)[15] and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP),[16] were employed, the reaction afforded relatively lower yields under the same reaction conditions (entries 11–13).

Finally, the intramolecular disulfide bond formation in the cyclized product 11 obtained was carried out. As shown in Scheme , the second ring closure smoothly occurred in the presence of an excess amount of iodine to afford the desired bicyclic compound 2 as a conformational isomers mixture in moderate yield, which suggests that our established method for the synthesis of FE399 is also effective for the synthesis of its analogues for future biological studies.

Scheme 2

Synthesis of the Bicyclic Depsipeptide 2

Thus, this method was experimentally shown in detail to be fully applicable to macrolactamization. In fact, the macrolactamization of ring-closing precursor 12, a synthetic key intermediate of FE399, was attempted by the present method employing the combination of MNBA and a catalytic amount of DMAPO (Scheme ).[9] The ring-closing reaction of 12 smoothly proceeded to afford the desired macrolactam 13 in 77% yield under very mild reaction conditions. After the intramolecular disulfide bond formation in the resulting lactam 13, we successfully achieved the total synthesis of FE399.

Scheme 3

The Application of MNBA-Mediated Macrolactamization for the Synthesis of FE399

In summary, through the synthesis of a depsipeptidic analogue of FE399, we have demonstrated that the MNBA-mediated dehydration condensation reaction should be effectively applied to the formation of a 16-membered macrolactam core of this molecule. Of note, the MNBA-mediated macrolactamization rapidly proceeded to afford the target ring-closed product. In addition, it was determined that the combination of MNBA combined with a catalytic amount of DMAPO showed much higher activity than that of conventionally used coupling reagents such as HATU and PyBOP.

Experimental Section

General Information

Optical rotations were determined using a Jasco P-1020 polarimeter. Infrared (IR) spectra were obtained using a Jasco FT/IR-4600 Fourier transform IR spectrometer. Proton and carbon nuclear magnetic resonance (1H and 13C NMR) spectra were recorded with chloroform (in CDCl3) on the following instruments: JEOL JNM-AL500 (1H at 500 MHz and 13C at 125 MHz). Mass spectra were determined by a Bruker Daltonics micrOTOF focus (ESI-TOF) mass spectrometer. Thin-layer chromatography was performed on Wakogel B5F. High-performance liquid chromatography was performed with a Hitachi LaChrom Elite system composed of the organizer, L-2400 UV detector, and L-2130 pump. All reactions were carried out under an argon atmosphere in dried glassware unless otherwise noted. Dichloromethane was distilled from diphosphorus pentoxide, then calcium hydride, and dried over molecular sieves (MS) 4 Å; toluene was distilled from diphosphorus pentoxide and dried over MS 4 Å. All reagents were purchased from Tokyo Kasei Kogyo Co., Ltd., Kanto Chemical Co., Inc. or Aldrich Chemical Co., Inc. and used without further purification unless otherwise noted. MNBA was purchased from Tokyo Kasei Kogyo Co. Ltd. (TCI M1439).

Experimental Procedures and Analytical Data

9-(tert-Butyldimethylsilyloxy)nonan-1-ol (C1)[17]

To a cooled (0 °C) solution of 1,9-nonanediol (3.00 g, 19 mmol) in acetonitrile (21 mL) and hexane (166 mL) was added Et3N (3.1 mL, 22 mmol) and tert-butyldimethylsilyl chloride (2.96 g, 20 mmol). The solution was stirred at 55 °C for 18 h. After cooling to rt, the reaction mixture was quenched with saturated aqueous ammonium chloride. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate 3 times. The combined organic layer was washed with brine and dried over sodium sulfate. After evaporation of the solvent, the crude product was purified by column chromatography on silica gel (hexane/ethyl acetate = 40/1 to 4/1) to afford C1 (4.28 g, 83%).

IR (neat): 3402, 2931, and 2862 cm–1; 1H NMR (500 MHz, CDCl3): δ 3.62 (t, J = 7.0 Hz, 2H, 9-H), 3.58 (t, J = 6.5 Hz, 2H, 1-H), 1.62–1.25 (m, 14H, 2-H, 3-H, 4-H, 5-H, 6-H, 7-H, 8-H), 0.88 (s, 9H, TBS), and 0.03 (s, 6H, TBS) ppm; 13C NMR (125 MHz, CDCl3): δ 63.3, 63.0, 32.82, 32.75, 29.5, 29.3 (2C), 26.0 (3C), 25.74, 25.69, and 18.3, −5.3 (2C) ppm; HRMS (ESI/TOF) m/z: [M + Na]+ calcd for C15H34O2SiNa, 297.2220; found, 297.2234.

9-(tert-Butyldimethylsilyloxy)nonanal (3)[13]

To a cooled (0 °C) solution of C1 (200 mg, 0.73 mmol) in dichloromethane (5.8 mL) and dimethyl sulfoxide (1.5 mL) were added triethylamine (0.81 mL, 5.8 mmol) and SO3·Py complex (463 mg, 2.91 mmol). After the solution was stirred at rt for 1.5 h, the reaction mixture was quenched with saturated aqueous ammonium chloride at 0 °C. The organic layer was separated, and the aqueous layer was extracted with dichloromethane. The combined organic layer was washed with brine and dried over sodium sulfate. After evaporation of the solvent, the crude product was purified by thin-layer chromatography on silica gel (hexane/ethyl acetate = 6/1) to afford 3 (165 mg, 83%).

IR (neat): 2931, 2854, and 1728 cm–1; 1H NMR (500 MHz, CDCl3): δ 9.76 (t, J = 2.0 Hz, 1H, CHO), 3.59 (t, J = 6.5 Hz, 2H, 9-H), 2.41 (td, J = 7.3, 2.0 Hz, 2H, 2-H), 1.62 (tt, J = 7.3, 7.3 Hz, 2H, 3-H), 1.50 (tt, J = 6.5, 6.5 Hz, 2H, 8-H), 1.34–1.26 (br s, 8H, 4-H, 5-H, 6-H, 7-H), 0.89 (s, 9H, TBS), and 0.04 (s, 6H, TBS) ppm; 13C NMR (125 MHz, CDCl3): δ 202.9, 63.2, 43.9, 32.8, 29.3, 29.2, 29.1, 26.0 (3C), 25.7, 22.0, 18.3, and −5.3 (2C) ppm; HRMS (ESI/TOF) m/z: [M + Na]+ calcd for C15H32O2SiNa, 295.2064; found, 295.2053.

Allyl 9-(tert-Butyldimethylsilyloxy)nonanoate (4)

To a solution of 3 (2.36 g, 8.7 mmol) in THF (58 mL), tert-butyl alcohol (58 mL), 2-methyl-2-butene (3.68 mL, 35 mmol), and a solution of NaH2PO4 (1.55 g, 13 mmol) and NaClO2 (2.97 g, 26 mmol) in H2O (58 mL) were added in order. The reaction mixture was stirred at rt for 4 h. The organic layer was separated, and the aqueous layer was extracted with diethyl ether. The combined organic layer was washed with brine and dried over sodium sulfate. After evaporation of the solvent, the crude product was used in the next step without further purification. To a solution of crude product in acetone (41 mL) was added Cs2CO3 (1.62 g, 5.0 mmol). After the solution was stirred for 10 min at rt, a solution of allyl bromide (533 mg, 4.6 mmol) in acetone (10 mL) was added, and the mixture was warmed to 50 °C. After stirring for 1 h, the reaction mixture was cooled to rt and filtrated through a pad of Celite. After evaporation of the solvent, the crude product was purified by column chromatography on silica gel (hexane/ethyl acetate = 11/1) to afford 4 (1.26 g, 92%, 2 steps) as a colorless oil.

IR (neat): 2931, 2862, and 1743 cm–1; 1H NMR (500 MHz, CDCl3): δ 5.92 (ddt, J = 17.0, 10.5, 5.5 Hz, 1H, allyl), 5.31 (ddt, J = 17.0, 1.5, 1.5 Hz, 1H, allyl), 5.23 (ddt, J = 10.5, 1.5, 1.5 Hz, 1H, allyl), 4.57 (ddd, J = 5.5, 1.5, 1.5 Hz, 2H, allyl), 3.59 (t, J = 6.5 Hz, 2H, 9-H), 2.33 (t, J = 7.8 Hz, 2H, 2-H), 1.63 (tt, J = 7.8, 7.8 Hz, 2H, 3-H), 1.50 (tt, J = 6.5, 6.5 Hz, 2H, 8-H), 1.30 (br s, 8H, 4-H, 5-H, 6-H, 7-H), 0.89 (s, 9H, TBS), and 0.04 (s, 6H, TBS) ppm; 13C NMR (125 MHz, CDCl3): δ 173.4, 132.3, 118.0, 64.9, 63.2, 34.2, 32.8, 29.2 (2C), 29.1, 25.9 (3C), 25.7, 24.9, 18.3, and −5.3 (2C) ppm; HRMS (ESI/TOF) m/z: [M + Na]+ calcd for C18H36O3SiNa, 351.2326; found, 351.2330.

Allyl 9-Hydroxynonanoate (5)

To a cooled (0 °C) solution of 4 (1.20 g, 3.7 mmol) in THF (37 mL) was added tetrabutylammonium fluoride (1.0 M in THF, 11 mL, 11 mmol), and the resulting solution was stirred at rt for 1 h. The reaction was quenched by the addition of saturated aqueous sodium hydrogencarbonate. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layer was washed with brine and dried over sodium sulfate. After evaporation of the solvent, the crude product was purified by column chromatography on silica gel (hexane/ethyl acetate = 5/1 to 2/1) to afford 5 (795 mg, quant.).

IR (neat): 3410, 2931, 2862, and 1736 cm–1; 1H NMR (500 MHz, CDCl3): δ 5.90 (ddt, J = 17.0, 10.5, 5.5 Hz, 1H, allyl), 5.30 (ddt, J = 17.0, 1.5, 1.5 Hz, 1H, allyl), 5.21 (ddt, J = 10.5, 1.5, 1.5 Hz, 1H, allyl), 4.56 (ddd, J = 5.5, 1.5, 1.5 Hz, 2H, allyl), 3.61 (t, J = 7.0 Hz, 2H, 9-H), 2.31 (t, J = 7.5 Hz, 2H, 2-H), 1.62 (tt, J = 7.5, 7.5 Hz, 2H, 3-H), 1.54 (tt, J = 7.0, 7.0 Hz, 2H, 8-H), and 1.36–1.26 (m, 8H, 4-H, 5-H, 6-H, 7-H) ppm; 13C NMR (125 MHz, CDCl3): δ 173.4, 132.2, 118.0, 64.8, 62.8, 34.1, 32.6, 29.1 (2C), 28.9, 25.6, and 24.8 ppm; HRMS (ESI/TOF) m/z: [M + Na]+ calcd for C12H22O3Na, 237.1461; found, 237.1460.

Allyl 9-[N-(9H-Fluoren-9-ylmethoxycarbonyl)-S-(triphenymethyl)-l-cysteinyloxy]nonanoate (6)

To a cooled (0 °C) solution of 5 (40 mg, 0.19 mmol) in dichloromethane (1.9 mL) was added a solution of N-(9-fluorenylmethoxycarbonyl)-S-triphenylmethyl-l-cysteine (131 mg, 0.22 mmol), MNBA (84 mg, 0.24 mmol), and N,N-dimethlyaminopyridine (59 mg, 0.49 mmol) successively, and the reaction mixture was stirred at rt for 30 min. The reaction was quenched with saturated aqueous sodium hydrogencarbonate at 0 °C. The organic layer was separated, and the aqueous layer was extracted with dichloromethane. The combined organic layer was washed with brine and dried over sodium sulfate. After evaporation of the solvent, the crude product was purified by thin-layer chromatography on silica gel (hexane/ethyl acetate = 5/2, Rf = 0.51) to afford 6 (151 mg, quant.).

[α]D24 +10.6 (c 1.27, CHCl3); IR (neat): 3448, 3062, 3032, 2931, 2854, and 1728 cm–1; 1H NMR (500 MHz, CDCl3): δ 7.67–7.60 (m, 2H, Fmoc), 7.51–7.44 (m, 2H, Fmoc), 7.32–7.23 (m, 4H, Fmoc), 7.23–7.03 (m, 15H, Trt), 5.79 (ddt, J = 17.0, 10.5, 5.5 Hz, 1H, allyl), 5.18 (ddt, J = 17.0, 1.5, 1.5 Hz, 1H, allyl), 5.10 (ddt, J = 10.5, 1.5, 1.5 Hz, 1H, allyl), 4.45 (d, J = 5.5 Hz, allyl), 4.29–4.15 (m, 3H, 2′-H, Fmoc), 4.10 (t, J = 6.5 Hz, 1H, Fmoc), 3.98 (t, J = 6.5 Hz, 2H, 9-H), 2.55 (dd, J = 12.0, 6.5 Hz, 1H, 3′-H), 2.49 (dd, J = 12.0, 4.5 Hz, 1H, 3′-H), 2.19 (t, J = 7.5 Hz, 2H, 2-H), 1.53–1.41 (m, 4H, 3-H, 8-H), and 1.23–1.10 (m, 8H, 4-H, 5-H, 6-H, 7-H) ppm; 13C NMR (125 MHz, CDCl3): δ 173.4, 170.5, 155.5, 144.3 (3C), 143.7 (2C), 141.3 (2C), 132.3, 129.5 (6C), 128.0 (6C), 127.7 (2C), 127.1 (2C), 126.9 (3C), 125.1 (2C), 120.0 (2C), 118.1, 67.1, 66.9, 65.8, 64.9, 52.9, 47.1, 34.2 (2C), 29.1, 29.02, 28.98, 28.4, 25.7, and 24.9 ppm; HRMS (ESI/TOF) m/z: [M + Na]+ calcd for C49H51NO6SNa, 804.3329; found, 804.3343.

Allyl 9-[S-(Triphenymethyl)-l-cysteinyloxy]nonanoate (7)

To a solution of 6 (151 mg, 0.19 mmol) in dichloromethane (0.96 mL) was added diethylamine (0.96 mL). The solution was stirred at rt for 3 h. After evaporation of the solvent, the crude product was purified by thin-layer chromatography on silica gel (hexane/ethyl acetate = 1/1, Rf = 0.59) to afford 7 (107 mg, 99%) as a pale yellow oil.

[α]D24 +30.2 (c 1.01, CHCl3); IR (neat): 3379, 3062, 3024, 2931, 2854, and 1736 cm–1; 1H NMR (500 MHz, CDCl3): δ 7.47–7.16 (m, 15H, Trt), 5.90 (ddt, J = 17.0, 10.0, 5.5 Hz, 1H, allyl), 5.29 (brd, J = 17.0 Hz, 1H, allyl), 5.21 (brd, J = 10.0 Hz, 1H, allyl), 4.56 (brd, J = 5.5 Hz, 2H, allyl), 4.02 (t, J = 6.5 Hz, 2H, 9-H), 3.17 (dd, J = 8.0, 4.8 Hz, 2H, 2′-H), 2.54 (dd, J = 12.4, 4.8 Hz, 1H, 3′-H), 2.46 (dd, J = 12.4, 8.0 Hz, 1H, 3′-H), 2.31 (t, J = 7.5 Hz, 2H, 2-H), 1.58–1.50 (m, 4H, 3-H, 8-H), and 1.35–1.20 (br s, 8H, 4-H, 5-H, 6-H, 7-H) ppm; 13C NMR (125 MHz, CDCl3): δ 173.7, 173.4, 144.5 (3C), 132.3, 129.5 (6C), 127.9 (6C), 126.7 (3C), 118.1, 66.8, 65.2, 64.9, 53.9, 37.0, 34.2, 29.1, 29.0, 28.97, 28.4, 25.7, and 24.8 ppm; HRMS (ESI/TOF) m/z: [M + H]+ calcd for C34H42NO4S, 560.2829; found, 560.2810.

Allyl 9-[N-(9H-Fluoren-9-ylmethoxycarbonyl)-S-(triphenymethyl)-l-cysteinyl-N-(9H-fluoren-9-ylmethoxycarbonyl)-S-(triphenymethyl)-l-cysteinyloxy]nonanoate (8)

To a cooled (0 °C) solution of 7 (1.23 g, 2.2 mmol) in dichloromethane (22 mL) was added a solution of N-(9-fluorenylmethoxycarbonyl)-S-triphenylmethyl-l-cysteine (1.54 g, 2.6 mmol), MNBA (0.98 g, 2.86 mmol), and N,N-dimethylaminopyridine (0.69 g, 5.7 mmol) successively, and the reaction mixture was stirred at 0 °C for 30 min. The reaction was quenched with saturated aqueous sodium hydrogencarbonate. The organic layer was separated, and the aqueous layer was extracted with dichloromethane. The combined organic layer was washed with brine and dried over sodium sulfate. After evaporation of the solvent, the crude product was purified by column chromatography on silica gel (hexane/ethyl acetate = 3/1) to afford 8 (2.38 g, 96%) as a white solid.

mp 57–58 °C; [α]D24 +4.41 (c 1.37, CHCl3); IR (neat): 3410, 3062, 3024, 2854, 1728, and 1674 cm–1; 1H NMR (500 MHz, CDCl3): δ 7.78–7.74 (m, 2H, Fmoc), 7.61–7.53 (m, 2H, Fmoc), 7.48–7.14 (m, 34H, Fmoc, Trt), 6.38 (d, J = 7.5 Hz, 1H, NH), 5.93 (ddt, J = 17.0, 10.5, 5.5 Hz, allyl), 5.33 (ddt, J = 17.0, 1.5, 1.5 Hz, allyl), 5.24 (ddt, J = 10.5, 1.5, 1.5 Hz, allyl), 5.02 (d, J = 7.5 Hz, 1H, NH), 4.59 (ddd, J = 5.5, 1.5, 1.5 Hz, allyl), 4.47–4.37 (m, 3H, 2′-H, Fmoc), 4.44 (dt, J = 7.5, 5.5 Hz, 1H, 2′-H), 4.41–4.30 (m, 2H, Fmoc), 4.20 (t, J = 2.0 Hz, 1H, Fmoc), 4.05 (t, J = 6.5 Hz, 2H, 9-H), 3.80 (dt, J = 7.5, 6.0 Hz, 1H, 2″-H), 2.70–2.64 (m, 2H, 3″-H), 2.59 (dd, J = 12.5, 5.5 Hz, 1H, 3′-H), 2.54 (dd, J = 12.5, 5.5 Hz, 1H, 3′-H), 2.33 (t, J = 7.5 Hz, 2H, 2-H), 1.71–1.49 (m, 2H, 3-H, 8-H), and 1.39–1.21 (m, 8H, 4-H, 5-H, 6-H, 7-H) ppm; 13C NMR (125 MHz, CDCl3): δ 173.4, 169.7, 169.6, 155.8, 144.3 (3C), 144.2 (3C), 143.6 (2C), 141.3 (2C), 132.3, 129.6 (6C), 129.4 (6C), 128.1 (6C), 127.9 (6C), 127.7 (2C), 127.1 (2C), 126.9 (3C), 126.8 (3C), 125.1 (2C), 119.9 (2C), 118.1, 67.3, 67.1, 66.7, 65.8, 64.9, 53.8, 51.3, 47.1, 34.2, 34.0, 33.7, 29.1, 29.08, 29.03, 28.4, 25.4, and 24.9 ppm; HRMS (ESI/TOF) m/z: [M + H]+ calcd for C71H70N2O7S2Na, 1149.4517; found, 1149.4490.

Allyl 9-[S-(Triphenymethyl)-l-cysteinyl-N-(9H-fluoren-9-ylmethoxycarbonyl)-S-(triphenymethyl)-l-cysteinyloxy]nonanoate (9)

To a cooled (0 °C) solution of 8 (175 mg, 0.16 mmol) in dichloromethane (0.77 mL) was added diethylamine (0.77 mL). The solution was stirred at 0 °C for 1 h and then allowed to warm to ambient temperature. The mixture was then stirred at rt for 2.5 h. After evaporation of the solvent, the crude product was purified by thin-layer chromatography on silica gel (hexane/ethyl acetate = 3/2) to afford 9 (102 mg, 73%) as a pale yellow oil.

[α]D25 +14.1 (c 1.07, CHCl3); IR (neat): 3371, 3055, 3024, 2931, 2854, 1736, and 1674 cm–1; 1H NMR (500 MHz, CDCl3): δ 7.45 (d, J = 7.5 Hz, 6H, Trt), 7.34 (d, J = 7.5 Hz, 6H, Trt), 7.31–7.15 (m, 18H, Trt), 5.92 (ddt, J = 17.3, 10.0, 5.5 Hz, allyl), 5.31 (brd, J = 17.0 Hz, allyl), 5.23 (brd, J = 10.0 Hz, allyl), 4.58 (brd, J = 5.5 Hz, allyl), 4.44 (dt, J = 7.0, 5.5 Hz, 2′-H), 4.04 (t, J = 6.5 Hz, 2H, 9-H), 2.96 (dt, J = 8.5, 3.5 Hz, 1H, 2″-H), 2.74 (dt, J = 12.8, 3.5 Hz, 1H, 3″-H), 2.60–2.46 (m, 3H, 3′-H, 3″-H), 2.32 (t, J = 7.5 Hz, 2H, 2-H), 1.62 (t, J = 7.5 Hz, 2H, 3-H), 1.58–1.50 (m, 2H, 8-H), and 1.39–1.18 (m, 8H, 4-H, 5-H, 6-H, 7-H) ppm; 13C NMR (125 MHz, CDCl3): δ 173.4, 172.4, 170.2, 144.5 (3C), 144.3 (3C), 132.3, 129.6 (6C), 129.5 (6C), 128.0 (6C), 127.9 (6C), 126.83 (3C), 126.78 (3C), 118.1, 67.1, 66.6, 65.7, 64.9, 53.8, 50.9, 36.9, 34.2, 34.0, 29.1, 29.04, 28.99, 28.4, 25.7, and 24.9 ppm; HRMS (ESI/TOF) m/z: [M + H]+ calcd for C56H61N2O5S2, 905.4016; found, 905.4024.

9-[S-(Triphenymethyl)-l-cysteinyl-N-(9H-fluoren-9-ylmethoxycarbonyl)-S-(triphenymethyl)-l-cysteinyloxy]nonanoic Acid (10)

To a solution of Pd(PPh3)4 (5.2 mg, 5.0 μmol) in methanol (1.25 mL) and piperidine (10 μL, 0.10 mmol) was added a solution of 9 (45 mg, 49.7 mmol) in dichloromethane (1.25 mL). The solution was stirred at rt for 2 h. After evaporation of the solvent, the crude product was purified by column chromatography on silica gel (chloroform to chloroform/methanol = 25/1) to afford 10 (46 mg, quant.) as a pale yellow oil.

[α]D25 +12.2 (c 0.91, CHCl3); IR (neat): 3371, 3347, 3055, 2924, 2854, and 1682 cm–1; 1H NMR (500 MHz, CDCl3): δ 7.45 (d, J = 7.5 Hz, 6H, Trt), 7.35 (d, J = 7.5 Hz, 6H, Trt), 7.31–7.10 (m, 18H, Trt), 4.46 (dt, J = 7.5, 5.5 Hz, 2′-H), 4.41–4.14 (m, 2H, NH2), 4.14–3.97 (m, 2H, 9-H), 2.98 (dd, J = 8.5, 3.5 Hz, 1H, 2″-H), 2.74 (dt, J = 12.5, 3.5 Hz, 1H, 3″-H), 2.62–2.43 (m, 3H, 3′-H, 3″-H), 2.31 (t, J = 7.0 Hz, 2H, 2-H), 1.60 (t, J = 7.0 Hz, 2H, 3-H), 1.55 (t, J = 6.0 Hz, 2H, 8-H), and 1.37–1.20 (m, 8H, 4-H, 5-H, 6-H, 7-H) ppm; 13C NMR (125 MHz, CDCl3): δ 178.3, 172.7, 170.2, 144.5 (3C), 144.3 (3C), 129.6 (6C), 129.5 (6C), 128.0 (6C), 127.9 (6C), 126.79 (3C), 126.75 (3C), 67.0, 66.6, 65.6, 53.9, 50.9, 37.1, 33.9 (2C), 28.8, 28.7 (2C), 28.3, 25.5, and 24.6 ppm; HRMS (ESI/TOF) m/z: [M + H]+ calcd for C53H57N2O5S2, 865.3703; found, 865.3719.

(3R,6R)-3,6-Bis(tritylsulfanylmethyl)-1-oxa-4,7-diazacyclohexadecane-2,5,8-trione (11)

To a solution of MNBA (9.7 mg, 28 μmol), N,N-dimethylaminopyridine N-oxide (0.6 mg, 4.3 μmol), and triethylamine (7.8 μL, 56 μmol) in dichloromethane (8.6 mL) at 40 °C was slowly added a solution of 10 (19 mg, 22 μmol) in dichloromethane (2.2 mL) with a mechanically driven syringe over a 12 h period. After the reaction was quenched by the addition of saturated aqueous sodium hydrogencarbonate, the organic layer was separated, and the aqueous layer was extracted with dichloromethane. Then, the organic layer was washed with brine and dried over sodium sulfate. After evaporation of the solvent, the crude product was purified by thin-layer chromatography on silica gel (hexane/ethyl acetate = 2/1, Rf = 035) to afford 11 (13 mg, 71%) as a white solid.

mp 95–96 °C; [α]D24 −11.9 (c 0.92, CHCl3); IR (neat): 3379, 3286, 3062, 2931, 2854, 1736, and 1651 cm–1; 1H NMR (500 MHz, CDCl3): δ 7.43 (d, J = 7.5 Hz, 6H, Trt), 7.34 (d, J = 6.5 Hz, 6H, Trt), 7.32–7.14 (m, 18H, Trt), 6.62 (d, J = 7.5 Hz, 1H, NH), 5.54 (dt, J = 8.5 Hz, 1H, NH), 4.34 (t, J = 10.0 Hz, 1H, 16-H), 4.28 (dt, J = 7.5, 5.0 Hz, 1H, 6-H), 3.91–3.84 (m, 2H, 3-H, 16-H), 2.87 (dd, J = 12.8, 7.5 Hz, 1H, 1″-H), 2.77 (dd, J = 12.5, 5.0 Hz, 1H, 1′-H), 2.68 (dd, J = 12.5, 5.0 Hz, 1H, 1′-H), 2.48 (dd, J = 12.8, 5.0 Hz, 1H, 1″-H), 2.22 (ddd, J = 14.4, 6.7, 3.5 Hz, 1H, 9-H), 1.93 (ddd, J = 14.4, 12.9, 3.0 Hz, 1H, 9-H), and 1.78–1.03 (m, 12H, 10-H, 11-H, 12-H, 13-H, 14-H, 15-H) ppm; 13C NMR (125 MHz, CDCl3): δ 173.3, 169.4, 169.2, 144.46 (3C), 144.39 (3C), 129.54 (6C), 129.48 (6C), 128.1 (6C), 127.9 (6C), 127.9 (6C), 126.9 (3C), 126.7 (3C), 67.3, 66.5, 65.6, 52.6, 52.0, 36.0, 33.5, 33.3, 27.1, 26.4, 26.1, 26.0, 24.4, and 23.4 ppm; HRMS (ESI/TOF) m/z: [M + Na]+ calcd for C53H54N2O4S2, 869.3417; found, 869.3415.

FE399 Analogue (2)

A solution of 11 (15.5 mg, 18 μmol) in dichloromethane (4.7 mL) was added dropwise to a solution of I2 (23.2 mg, 0.18 mmol) in dichloromethane (28 mL) and methanol (4 mL) at 0 °C. The reaction mixture was warmed to rt and stirred for 20 min. The reaction was quenched by the addition of saturated aqueous sodium thiosulfate. The organic layer was separated, and the aqueous layer was extracted with dichloromethane. The combined organic layer was washed with brine and dried over sodium sulfate. After evaporation of the solvent, the crude product was purified by column chromatography on silica gel (chloroform to chloroform/methanol = 30/1 to 9/1) and purified by thin-layer chromatography on silica gel (chloroform/methanol = 30/1) to afford 2 (4.4 mg, 67%).

[α]D24 −90.0 (c 0.20, CHCl3/MeOH = 9/1); IR (neat): 3309, 2924, 2854, 1728, and 1651 cm–1; 1H NMR (500 MHz, DMSO-d6): major conformer, δ 8.15 (d, J = 4.5 Hz, 1H, 14-NH), 7.61 (d, J = 10.0 Hz, 1H, 11-NH), 4.85 (ddd, J = 11.5, 11.5, 5.0 Hz, 1H, 11-H), 4.32 (ddd, J = 10.5, 7.5, 3.0 Hz, 1H, 9-H), 4.13 (ddd, J = 4.5, 4.5, 4.5 Hz, 1H, 14-H), 3.88–3.80 (m, 1H, 9-H), 3.46 (dd, J = 14.5, 3.5 Hz, 1H, 15-Hb), 3.41–3.36 (m, 1H, 12-Hb), 3.27–3.17 (m, 2H, 12-Ha, 15-Ha), 2.31–2.21 (m, 1H, 2-Ha), 2.12–2.03 (m, 1H, 2-Hb), 1.01–1.60 (m, 12H, 3-H, 4-H, 5-H, 6-H, 7-H, 8-H); minor conformer, δ 8.86 (br s, 1H, 14-NH), 8.04 (d, J = 9.0 Hz, 1H, 11-NH), 4.99–4.89 (m, 1H, 11-H), 4.52 (ddd, J = 11.0, 7.0, 3.0 Hz, 1H, 9-Ha), 4.01–3.90 (m, 1H, 14-H), 3.72–3.64 (m, 1H, 9-Hb), 3.65–3.52 (m, 1H, 12-Ha), 3.30–3.12 (m, 2H, 15-Ha, 15-Hb), 3.07 (brd, J = 15.0 Hz, 1H, 12-Hb), 2.31–2.21 (m, 1H, 2-Ha), 1.98–1.90 (m, 1H, 2-Hb), and 1.01–1.60 (m, 12H, 3-H, 4-H, 5-H, 6-H, 7-H, 8-H); 13C NMR (125 MHz, DMSO-d6): δ 174.7, 173.3, 170.1, 64.4 (minor), 64.1, 54.0, 52.8, 52.0 (minor), 50.8 (minor), 47.4, 44.5, 41.6 (minor), 35.5 (minor), 35.0, 28.6 (minor), 28.1 (minor), 27.5 (minor), 26.9, 26.7, 26.4, 26.20 (minor), 26.19, 25.4 (minor), 24.7, 24.6 (minor), and 22.7 ppm; HRMS (ESI/TOF) m/z [M + Na]+ calcd for C15H24N2O4S2Na, 383.1075; found, 383.1067.