Studies toward the Total Synthesis of Itralamide B and Biological Evaluation of Its Structural Analogs

Abstract

Itralamides A and B were isolated from the lipophilic extract of Lyngbya majuscula collected from the eastern Caribbean. Itralamide B (1) showed cytotoxic activity towards human embryonic kidney cells (HEK293, IC₅₀ = 6 μM). Preliminary studies disapproved the proposed stereochemistry of itralamide. In this paper, we will provide a full account of the total synthesis of four stereoisomers of itralamide B and the results derived from biological tests of these structural congeners.

1. Introduction

Cyclodepsipeptide represents a classical sub-category of natural product, characterized by at least one ester bond embedded in the macrocycle. Natural cyclodepsipeptides often display intriguing biological activities, and some of them had been developed into lead compounds for further medicinal investigations [1]. In 2009, Horgen’s group reported the isolation of itralamides A and B 1 (Figure 1) from the lipophilic extract of Lyngbya majuscula [2]. Itralamides A and B are cyclodepsipeptides sharing the same 4,4-dichloro-3-methylbutanoic acid (DMBA) sidechain moiety with unknown stereochemical configuration. The macrocycle of itralamides is composed of a few N-methylated amino acids, including N-methyl threonine, which is not a conventional modification of amino acid for natural products [3,4]. We have been interested in the synthesis of bioactive natural cyclodepsipeptides [5,6,7,8,9,10,11], and successfully reassigned a few natural products [12,13,14,15,16]. In order to identify the absolute stereochemistry of the DMBA fragment presented in itralamides A & B, we conducted a synthetic study of itralamide B 1. Our other objective was to possibly accelerate the structure-activity relationship studies of cytotoxic itralamide B regarding different cancer cell lines.

Figure 1. Structure of itralamides and the 1st retrosynthetic analysis of itralamide B (1).

Figure 1. Structure of itralamides and the 1st retrosynthetic analysis of itralamide B (1).

2. Results and Discussion

2.1. The First Generation Synthetic Endeavors

2.1.1. Retrosynthetic Analysis

The absolute stereochemistry of the DMBA fragment must be determined via total synthesis, so in the first generation retrosynthetic analysis we detached this fragment from the macrocycle, producing the cyclodepsipeptide 2 and the DMBA fragment R-/S-3 (Figure 1). This late stage introduction of the DMBA side-chain would allow the facile synthesis of different stereoisomers of itralamide B. The cyclic hexapeptide 2 could be constructed via the macrolactamization at the d-Phe/l-Val amide bond, and further disconnection at the l-MeAla/d-Val amide bond produced two tripeptides 4 and 5. The above retrosynthetic strategy also provided an opportunity for the synthesis of 2 with an alternative fragment assembly, which led to the macrolactamization to be conducted at the l-MeAla/d-Val amide bond.

2.1.2. Synthesis of Tripeptide 5

The synthesis commenced with the treatment of 6 with CbzOSu and sodium bicarbonate in acetonitrile to protect the secondary amine. The tert-butyl ester was removed by the action of trifluoroacetic acid to produce carboxylic acid 7 at 82% yield. Condensation of 7 with N-Me-Ala-OMe was mediated by BOPCl [17,18] and DIPEA to give rise to dipeptide 8 at 70% yield. Esterification of 8 with Boc-Val-OH was facilitated by the modified Yamaguchi’s protocol [19,20], and depsipeptide 5 was obtained at 72% yield. To our surprise, selective removal of the methyl ester in 5 was found to be problematic. Different concentrations and equivalents of lithium hydroxide or sodium hydroxide as well as different solvent systems were applied to compound 5, but produced no desired free acid 9. The more selective method with lithium iodide in hot ethyl acetate also failed to provide any detectable quantities of product [21,22]. Although there were two ester bonds in 5, we believed that the steric hindrance of the inner ester bond (Val-MeThr) was large enough to be differentiated from the terminal methyl ester. In fact, we did not find any fully hydrolyzed dipeptide 10 from the reaction mixture (Scheme 1).

Scheme 1. Attempted synthesis of fragment 9.

Scheme 1. Attempted synthesis of fragment 9.

When dipeptide 8 was subjected to hydrolysis with lithium hydroxide under standard reaction conditions, we could not isolate the desired acid 10 either. The major product of this saponification reaction, although not fully characterized, revealed the Cbz group was cleaved. The failure to remove the methyl ester was assumed to be due to interference by the Cbz group at threonine; the N-methylation of threonine changed the conformation of the peptide and thus promoted side reactions.

Because the preparation of tripeptide 9 was unsuccessful, we decided to form an amide bond on the nitrogen of threonine instead of protecting it with Cbz, which could mimic the natural product structure and allow us to examine the feasibility of macrolactamization as illustrated in Figure 1.

2.1.3. Model Study for Macrolactamization at Different Amide Bonds

As shown in Scheme 2, n-butyric acid derived amide was elected to mimic the DMBA fragment. Thus, N-Me-Thr-OMe 11 was condensed with n-butyric acid in the presence of HATU and HOAt to give rise to the corresponding dipeptide. The methyl ester was smoothly hydrolyzed with lithium hydroxide, and the resultant dipeptide acid was then coupled to N-Me-Ala-O^tBu using Mukaiyama reagent [23,24] to produce tripeptide 12 at 47% yield over three steps (Scheme 2). Esterification of 12 with N-Cbz-Val-OH was facilitated by the modified Keck condition [25], in the presence of DCC, DMAP and a catalytic amount of CSA, and depsipeptide 13 was prepared at 70% yield.

Scheme 2. Preparation of tetrapeptide 13.

Scheme 2. Preparation of tetrapeptide 13.

Further elaboration of depsipeptide 13 is illustrated in Scheme 3. Thus, acidic cleavage of the tert-butyl ester of 13 and the Boc group in 4 [26] afforded the corresponding acid and TFA salt of amine 14, respectively. Coupling of these two fragments was carried out with HATU and HOAt in dichloromethane to produce heptapeptide 15 in 70% yield. The methyl ester was then cleaved via a S_N2-type saponification process mediated by heating a solution of 15 and lithium iodide in ethyl acetate [21,22]. Subsequent hydrogenolytic removal of the Cbz group produced the linear precursor L-1 in 78% yield over two steps. The macrolactamization of L-1 was performed in the presence of PyAOP [27], HATU or Mukaiyama reagent under various conditions (using DMF or MeCN as solvent and different reaction temperatures). Unfortunately and notwithstanding this progress, all of our attempts failed to provide desired product 16 (Scheme 3), presumably due to a conformational disposition of the linear precursor that prevented the macrocyclization.

Scheme 3. Attempts at macrolactamization at the Phe-Val site.

Scheme 3. Attempts at macrolactamization at the Phe-Val site.

With both coupling partners 13 and 4 in hand, we decided to carry out the macrolactamization at the MeAla-Val site. Thus, removal of the Cbz group of 13 afforded the corresponding amine, which was then coupled with acid 17, prepared by basic hydrolysis of 4, and proceeded smoothly to give heptapeptide 18 at 72% yield. Concomitant removal of the tert-butyl ester and Boc group of 18 was carried out using trifluoroacetic acid in dichloromethane to produce the linear precursor L-2, which was subjected to macrocyclization in identical conditions as those described for L-1. To our disappointment and surprise, all attempts to effect the macrolactamization were unsuccessful and no desired product was isolated (Scheme 4).

Scheme 4. Attempts for macrolactamization at the MeAla-Val site.

Scheme 4. Attempts for macrolactamization at the MeAla-Val site.

In general, macrolactonization of peptide-containing hydroxy acids is a more difficult task than similar amide bond-forming cyclizations. Given the fact that two approaches based on macrolactamization did not lead to the formation of macrocycle 16, it was considered necessary at this stage to investigate the alternative yet unprecedented macrolactonization [28] route as depicted, in a retrosynthetic format (Figure 2).

Figure 2. Revised retrosynthetic analysis for model study.

Figure 2. Revised retrosynthetic analysis for model study.

2.1.4. Model Study for Macrolactonization

The ester bond between valine and threonine was selected as the macrocyclization site. The linear precursor 19 was disconnected into tetrapeptide 20, dipeptide 21 and n-butyric acid. In order to circumvent the problem associated with saponification of the methyl ester of dipeptide MeThr-MeAla, the carboxylic acid terminus of dipeptide 21 was protected as its allyl ester, which could be readily removed via a palladium catalyzed process [29].

Tripeptide 4 was transformed into hexapeptide 23 according to the procedure described in our previous communication [26]. Hydrogenolytic removal of the N-terminal Cbz followed via a condensation of the resultant free amine with n-butyric acid through the action of HATU and HOAt to produce heptapeptide 19 at 60% yield. Treatment of 19 with boron trifluoride etherate [30] in dichloromethane smoothly liberated the carboxylic acid, and macrolactonization using the Yamaguchi protocol produced the desired cyclodepsipeptide 16 at 45% isolable yield over two steps (Scheme 5).

Scheme 5. Model study for macrolactonization.

Scheme 5. Model study for macrolactonization.

2.2. Synthesis of Different Stereoisomers of Itralamide B

2.2.1. Completion of Total Synthesis of Itralamide B 1a and 1b

Encouraged by the success of this model study that yielded a compound 16 closely related to the itralamide B target molecule, we proceeded with a synthesis of two diastereoisomers of itralamide B (1a and 1b) including the side chain attachment [26]. This was readily achieved by following the same synthetic procedure as for 16, but using either S-3 or R-3 instead of n-butyric acid (Scheme 6).

Scheme 6. Completion of the total synthesis of itralamide B 1a and 1b.

Scheme 6. Completion of the total synthesis of itralamide B 1a and 1b.

On examining the analytical information, we were disappointed as the authentic data did not match those of our products 1a and 1b [2]. There were significant discrepancies in the chemical shifts in the region closed to these two valine residues, particularly the ¹³C NMR chemical shifts at the iso-propyl groups of two valines, the two N-methyl groups of methylalanines, the methyl group of threonine and the ester carbonyl (Figure 3; see also Figure 4 and the Supplementary Information for intuitionistic comparison). According to the isolation paper, the macrocyclic structure and connectivity of itralamide B were established by NMR studies, and the absolute configuration of amino acids was determined by Marfey’s advanced analytical method [31]. Since itralamide B contains two valine residues with the opposite configuration, their respective assignments remained uncertain. Although the stereochemistry of the macrocyclic core was assumed to be that shown in Figure 1, the issue related to the absolute configuration of each valine was left largely unresolved in the original isolation paper. The synthesis of 1a and 1b has disproved the original assigned structure for itralamide B. Therefore, we hypothesized that the incorrect structure proposed for itralamide B could possibly be a result of misassignment of the absolute configuration of two valine residues. We therefore elected to synthesize two diastereomers (1c and 1d, Figure 3) of the proposed structure.

Figure 3. Analysis of the stereochemistry.

Figure 3. Analysis of the stereochemistry.

Figure 4. Comparison of the ¹³C NMR data.

Figure 4. Comparison of the ¹³C NMR data.

2.2.2. Synthesis of Two Additional Stereoisomers of Itralamide B 1c and 1d

Prior to embarking on the synthesis of 1c and 1d, a more efficient route to 3 was then developed from the known ester 24 [32] (Scheme 7).

Scheme 7. Improved synthesis of DMBA fragment 3.

Scheme 7. Improved synthesis of DMBA fragment 3.

Thus, hydrogenation of the known unsaturated ester 24 with commercially available chiral catalyst (S)-Ru(OAc)₂(BINAP) [33] produced the corresponding saturated ester at high yield with 93% enantioselectivity (ee). After saponification, the resultant acid was reacted with diphenyldiazomethane [34] in dichloromethane to give rise to the benzhydryl ester 25 at 72% yield over three steps. Subsequent conversion of alcohol 25 to carboxylic acid S-3 was performed according to reported procedures [26]. In addition, R-3 was prepared by following the same synthetic procedure as for S-3, but using (R)-Ru(OAc)₂(BINAP) as the catalyst.

As outlined in Scheme 8, the synthesis of itralamide B 1c and 1d commenced with the coupling of Cbz-l-Val with N-Me-Ala-OMe (26) using HATU and HOAt as dehydration reagents to produce the corresponding dipeptide. Subsequent saponification and HATU/HOAt-mediated coupling to N-Me-Phe-OMe (27) provided tripeptide 28 at 73% yield. Hydrolysis of the methyl ester with lithium hydroxide was followed by coupling with d-Val-O^tBu (29), promoted by PyAOP and HOAt to produce tetrapeptide 30 at 70% yield. Hydrogenolysis of the Cbz group of 30 using palladium on charcoal smoothly produced the correponding free amine, which was then condensed with dipeptide acid 22 in the presence of HATU and HOAt to generate hexapeptide 31 at 61% yield. Hexapeptide 31 was further elaborated to the linear precursor 32c using a two-step sequence involving hydrogenolysis of the Cbz group and subsequent HATU/HOAt-mediated fragment condensation of the resultant amine with DMBA fragment S-3. Treatment of 32c with boron trifluoride etherate in dichloromethane, followed by macrolactonization using the Shiina reagent (2-methyl-6-nitrobenzoic anhydride) [35] produced itralamide B 1c at 20% yield. Other macrolactonization protocols, such as the standard Yamaguchi macrolactonization conditions, led to substantially lower yields. Similarly, the DMBA fragment R-3 was readily incorporated into the synthesis as previously performed to afford itralamide B 1d with 11% overall yield from hexapeptide 31.

Scheme 8. Synthesis of stereoisomers itralamide B 1c and 1d.

Scheme 8. Synthesis of stereoisomers itralamide B 1c and 1d.

The NMR spectroscopic data (Figure 4) and optical rotation of these synthetic samples (1a–1d) are quite different from those of natural itralamide B. Similar to itralamide B 1a,b, the diastereoisomers 1c and 1d showed significant discrepancies with the natural product on ¹³C NMR spectra at the iso-propyl groups of the valines, the N-methyl group of threonine and the ester carbonyl; the biggest differences (larger than 4 ppm) appeared at the α-stereogenic centers of MePhe (C6) and one of the MeAla (C9). The discrepancies remain unresolved issues which are subject to conjecture.

2.3. Biological Study of Itralamide B and Structural Analogs

We next carried out the biological evaluation of our synthetic itralamide B 1a–1d and the cyclodepsipeptide 16. The inhibitory activity towards cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-2H-tetrazolium) (MTS) assay in four cancer cell lines (Table 1).

Table 1. The effect of compounds on the proliferation of cancer cell lines.

Origins	Cell Line	Inhibitory Activity (IC50 in μM)
		Compound 16	Itralamide B 1a (RSD)	Itralamide B 1b	Itralamide B 1c (RSD)	Itralamide B 1d (RSD)	Largazole a (RSD)
Brain	SH-SY5Y	-	-	-	56.8 (2.3%)	56.4 (4.1%)	0.8 (1.1%)
Cervix	HeLa	-	-	-	38.0 (4.6%)	82.5 (4.3%)	2.8 (1.7%)
Liver	Hep3B	-	97.8 (1.4%)	-	-	-	0.3 (3.4%)
	PLC	-	33.1 (2.8%)	-	34.3 (3.4%)	39.5 (3.8%)	0.8 (2.5%)

^a Positive control.

Table 1. The effect of compounds on the proliferation of cancer cell lines.

Origins	Cell Line	Inhibitory Activity (IC50 in μM)
		Compound 16	Itralamide B 1a (RSD)	Itralamide B 1b	Itralamide B 1c (RSD)	Itralamide B 1d (RSD)	Largazole a (RSD)
Brain	SH-SY5Y	-	-	-	56.8 (2.3%)	56.4 (4.1%)	0.8 (1.1%)
Cervix	HeLa	-	-	-	38.0 (4.6%)	82.5 (4.3%)	2.8 (1.7%)
Liver	Hep3B	-	97.8 (1.4%)	-	-	-	0.3 (3.4%)
	PLC	-	33.1 (2.8%)	-	34.3 (3.4%)	39.5 (3.8%)	0.8 (2.5%)

^a Positive control.

Compound 16 and itralamide B 1b showed no inhibitory effect towards four cancer cell lines, while itralamide 1a exerted some inhibitory effect against PLC cells with IC₅₀ of 33.1 µM. Futhermore, itralamide B 1c and 1d were found to be more active than itralamide 1a and 1b. Presumably this may be attributed to the conformation change derived from the inversion of the configuration of valine residue presented in the macrocycle. Itralamide B 1c inhibited the proliferation of HeLa and PLC/RPF/5 (PLC) in a dose-dependent manner with IC₅₀ of 38.0 µM and 34.3 µM, respectively. Itralamide B 1d exhibited dose-dependent inhibitory effect against cell proliferation of PLC with IC₅₀ of 39.5 µM.

3. Experimental Section

3.1. General Experimental

All non-aqueous reactions were run under a nitrogen or argon atmosphere and all reaction vessels were oven-dried. Solvents were distilled prior to use: tetrahydrofuran (THF) from Na/benzophenone, dichloromethane (DCM), triethylamine and diisopropylethylamine (DIPEA) from CaH₂. NMR spectra were recorded on Bruker spectrometers. Chemical shifts were reported in parts per million (ppm), relative to the signals due to the solvent. Data were reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad), integration and coupling constants. Some peptide intermediates exist as rotamers due to N-methylation and the increased steric hinderence, their chemical shifts for the minor isomer on ¹H NMR spectra were recorded in parentheses next to the major isomer, while for ¹³C NMR the chemical shifts were recorded as they were and not differentiated. ESI mass spectra were obtained using a Finnigan MAT 95 mass spectrometer. Optical rotations were recorded on a Perkin Elmer 343 Polarimeter. TLC were carried out using pre-coated sheets (Qingdao silica gel 60-F₂₅₀, 0.2 mm, Qingdao, China) and visualized at 254 nm, and/or staining in ninhydrin or phosphomolybdic acid solution followed by heating. Flash column chromatography was performed using the indicated solvents on E. Qingdao silica gel 60 (230–400 mesh ASTM).

3.2. Synthesis of Cyclodepsipeptide 16 and Itralamide B 1c and 1d

3.2.1. Preparation of Cyclodepsipeptide 16

To a solution of compound 23 (200.0 mg, 0.23 mmol) in MeOH (20 mL), Pd/C was added under N₂ atmosphere. The reaction vessel was sealed and flashed with H₂ three times. The reaction mixture was then vigorously stirred overnight under H₂ atmosphere. Catalyst residue was removed by filtration. The filtrate was concentrated in vacuo to give the corresponding free amine, which was pure enough and used directly in the next step of reaction. To a solution of n-PrCO₂H (60.0 mg, 0.69 mmol) in DCM (2 mL), HATU (175.0 mg, 0.46 mmol) was added, followed by addition of DIPEA (200 μL, 1.15 mmol) at 0 °C. 0.5 h later, a solution of above amine (72.0 mg, 0.23 mmol) in DCM (2 mL) was addedat 0 °C. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction was quenched with saturated NH₄Cl (20 mL), and extracted with DCM (3 × 50 mL). The combined organic layers were washed with saturated NaHCO₃ solution (3 × 50 mL) and brine (50 mL), dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash chromatography (ethyl acetate) to give compound 19 (109.0 mg, 60%). [α]_D²⁵ = −45.3 (c 0.5, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.25–7.15 (m, 5H), 6.71 (d, J = 8.9 Hz, 1H), 6.64 (d, J = 8.6 Hz, 1H), 5.43–5.41 (m, 1H), 5.17–5.08 (m, 1H), 4.66 (dd, J = 8.8, 6.3 Hz, 1H), 4.39 (dd, J = 6.5, 3.6 Hz, 1H), 4.34 (dd, J = 8.5, 4.8 Hz, 1H), 4.20 (d, J = 3.3 Hz, 1H), 3.37 (dd, J = 14.4, 7.4 Hz, 1H), 3.06 (s, 3H), 2.93 (s, 3H), 2.90–2.85 (m, 2H), 2.86 (s, 3H), 2.75 (s, 3H), 2.44–2.34 (m, 2H), 2.14–2.06 (m, 1H), 2.02–1.97 (m, 1H), 1.78−1.65 (m, 2H), 1.46 (s, 9H), 1.35 (d, J = 7.2 Hz, 3H), 1.15 (d, J = 6.4 Hz, 3H), 1.06 (d, J = 6.9 Hz, 3H), 0.99 (d, J = 5.1 Hz, 3H), 0.93 (d, J = 6.7 Hz, 3H), 0.87 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.9 Hz, 3H), 0.82 (d, J = 6.9 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 173.7, 172.6, 172.0, 171.4, 170.8, 170.3, 169.4, 136.9, 129.0, 128.4, 126.6, 81.8, 68.1, 57.7, 56.8, 54.2, 52.5, 50.1, 38.6, 35.2, 34.0, 33.5, 31.0, 30.8, 30.7, 30.5, 28.0, 19.6, 18.9, 18.8, 18.5, 17.7, 17.4, 14.3, 13.9, 13.7. HR-ESIMS m/z for C₄₁H₆₈N₆NaO₉⁺ [M + Na]⁺: calculated 811.4940, found 811.4941.

To a solution of compound 19 (23.0 mg, 0.03 mmol) in DCM (1.0 mL), BF₃^.Et₂O (38 μL, 0.3 mmol) was added dropwise at 0 °C. The reaction solution was then allowed to warm to room temperature and stirred for 0.5~1.0 h (monitored by TLC). The reaction was quenched by addition of saturated NH₄Cl (2 mL) and diluted with DCM (60 mL). The organic phase was washed with saturated NH₄Cl (3 × 20 mL) and brine (20 mL), dried over anhydrous Na₂SO₄ and concentrated in vacuo to produce crude hydroxy acid, which was dried further under high vacuum for 4 h. To a solution of the above acid (50.0 mg, 0.07 mmol) in THF (5 mL) was added Et₃N (59 μL, 0.41 mmol) and trichlorobenzoyl chloride (54 μL, 0.34 mmol). The reaction mixture was stirred at room temperature for 3 h and diluted with toluene (3 mL). The resulted solution was added to a solution of DMAP (208.2 mg, 1.71 mmol) in toluene (50 mL) via a syringe pump over 48 h at 30 °C. The reaction was concentrated in vacuo, and the residue was dissolved in ethyl acetate (80 mL) and washed with saturated ammonium chloride (100 mL). Layers were separated, and the aqueous phase was extracted with ethyl acetate (2 × 80 mL). The combined organic layers were washed with brine (80 mL), dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography (ethyl acetate) to give compound 16 (9.5 mg, 45%) as a yellow oil. [α]_D²⁵ = −65.4 (c 0.6, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.24–7.16 (m, 5H), 6.89 (d, J = 9.6 Hz, 1H), 6.48 (d, J = 7.9 Hz, 1H), 5.77 (d, J = 3.2 Hz, 1H), 5.70 (dd, J = 12.3, 4.8 Hz, 1H), 5.47 (dd, J = 6.6, 3.2 Hz, 1H), 5.08 (q, J = 6.9 Hz, 1H), 4.98 (dd, J = 7.9, 4.3 Hz, 1H), 4.70 (dd, J = 9.4, 4.1 Hz, 1H), 4.66–4.58 (m, 1H), 3.81 (t, J = 7.8 Hz, 1H), 3.66 (dd, J = 15.3, 5.2 Hz, 1H), 3.33 (s, 3H), 3.19 (3.18) (s, 3H), 3.16 (s, 3H), 3.02 (s, 3H), 2.39–2.35 (m, 2H), 1.45–1.37 (m, 2H), 1.30 (d, J = 7.2 Hz, 3H), 1.07–1.04 (m, 3H), 0.99–0.79 (m, 18H). ¹³C NMR (100 MHz, CDCl₃) δ 174.9, 172.9, 172.1, 170.7, 170.2, 170.0, 169.8, 137.4, 128.6, 128.3, 126.5, 69.8, 57.0, 56.8, 54.4, 54.0, 51.4, 50.3, 35.2, 33.8, 33.8, 32.2, 31.8, 31.1, 31.0, 30.5, 22.7, 19.9, 19.6, 18.6, 18.3, 17.8, 17.1, 17.1, 13.8. HR-ESIMS m/z calculated for C₃₇H₅₉N₆O₈⁺ [M + H]⁺: 715.4389, found 715.4390.

3.2.2. Preparation of Ester 25

In a stainless steel autoclave, ester 24 (461.4 mg, 3.19 mmol) was dissolved in methanol (50 mL), after catalyst (S)-Ru(OAc)₂(BINAP) (48.0 mg, 0.06 mmol) was added, the reaction mixture was stirred under hydrogen atmosphere (5 MPa) for 24 h. The organic solution was transferred to a round bottom flask and concentrated to 5 mL, and THF–H₂O (10 mL, 1:1) was added, followed by addition of aqueous sodium hydroxide (6.4 mL, 6.4 mmol, 1 N in water). The solution was then stirred at room temperature for 12 h. Volatiles were removed under vacuum. The aqueous layer was extracted with diethyl ether (2 × 30 mL), and the organic solution was discarded. The aqueous solution was acidified to pH 3 with dilute hydrochloric acid (1 N in water) and extracted with dichloromethane (3 × 30 mL). The combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo to 10 mL. Without further purifications, to the above organic solution at 0 °C, diphenyldiazomethane (0.71 g, 3.66 mmol) in dichloromethane (3 mL) was added. The reaction mixure was stirred for an additional 6 h and then concentrated in vacuo. The residue was purified using flash chromatography (ethyl acetate/hexane, 1:3) to provide 25 [26] as a yellow oil (653.4 mg, 72%).

3.2.3. Preparation of Tripeptide 28

To a solution of Cbz-l-Val (9.20 g, 36.64 mmol) and amine 26 (4.31 g, 28.18 mmol) in DCM (250 mL), HATU (21.43 g, 56.36 mmol), DIPEA (23.3 mL, 140.90 mmol) and HOAt (7.67 g, 56.36 mmol) were added sequentially at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred overnight, then quenched by addition of saturated NH₄Cl solution (200 mL) and extracted with DCM (3 × 80 mL). The combined organic layers were washed with saturated NaHCO₃ solution (3 × 80 mL) and brine (80 mL), dried over anhydrous Na₂SO₄ and concentrated in vacuo_. The residue was purified by flash chromatography (hexane/ethyl acetate, 1:1) to afford dipeptide Cbz-Val-MeAla-OMe (6.27 g, 64%). [α]_D²⁰ = −21.5, (c 1.1, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.40–7.25 (m, 5H), 5.59 (d, J = 7.5 Hz, 1H), 5.27 (q, J = 7.1 Hz, 1H), 5.15–5.04 (m, 2H), 4.55 (dd, J = 9.2, 5.9 Hz, 1H), 3.70 (s, 3H), 3.03 (2.84) (s, 3H), 2.10–2.00 (m, 1H), 1.41 (d, J = 7.4 Hz, 3H), 1.03 (d, J = 6.7 Hz, 3H), 1.00–0.87 (m, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 172.3, 172.0, 156.5, 136.4, 128.5, 128.1, 128.0, 66.9, 55.8, 52.2, 52.1, 31.3, 31.3, 19.4, 17.2, 14.1.

To a solution of Cbz-Val-MeAla-OMe (4.27 g, 12.19 mmol) in THF-MeOH-H₂O (90 mL, 1:1:1) was added LiOH^.H₂O (1.46 g, 60.93 mmol) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 5 h (monitored by TLC). Volatiles were removed in vacuo, the aqueous solution was washed with Et₂O (3 × 80 mL). The organic phases were discarded, and the aqueous phase was acidified to pH 3 with 10% aqueous solution of citric acid at 0 °C. The aqueous layer was extracted with ethyl acetate (3 × 80 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo to give the corresponding acid (4.10 g, 99%). This acid (4.10 g, 12.18 mmol), without further purification, was mixed with amine 27 (3.63 g, 15.84 mmol) and dissolved in DCM (80 mL) at 0 °C. To this solution, HATU (9.27 g, 24.38 mmol), DIPEA (10.1 mL, 60.95 mmol) and HOAt (3.32 g, 24.38 mmol) were added at 0 °C. The reaction mixture was then allowed to warm to room temperature and stirred for 16 h. The reaction was quenched with saturated NH₄Cl solution (200 mL). Layers were separated, the aqueous layer was extracted with DCM (3 × 80 mL). The combined organic layers were washed with saturated NaHCO₃ solution (3 × 80 mL) and brine (80 mL), dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash chromatography (hexane/ethyl acetate, 1:1) to afford tripeptide 28 (4.36 g, 70%) as a viscous oil. [α]_D²⁵ = +85.8 (c 2.3, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.39–7.25 (m, 7H), 7.24–7.10 (m, 3H), 5.59–5.45 (m, 1H), 5.43–5.13 (m, 2H), 5.12–4.82 (m, 2H), 4.51–4.25 (m, 1H), 3.73 (3.54) (s, 3H), 3.45–3.25 (m, 1H), 2.95–2.87 (m, 1H), 2.85 (2.82) (s, 3H), 2.61 (2.29) (s, 3H), 2.00–1.85 (m, 1H), 1.25–0.82 (m, 9H). ¹³C NMR (125 MHz, CDCl₃) δ 171.7, 171.5, 171.0, 170.9, 170.8, 170.5, 170.4, 156.2, 136.9, 136.8, 136.4, 129.0, 129.0, 128.8, 128.7, 128.5, 128.1, 128.0, 128.0, 127.9, 127.0, 126.8, 66.9, 66.8, 66.8, 60.4, 60.4, 59.0, 58.3, 55.9, 55.6, 55.5, 52.5, 52.3, 49.8, 48.4, 35.1, 34.5, 34.4, 32.3, 32.3, 31.6, 31.2, 31.0, 30.0, 29.9, 29.3, 21.0, 19.8, 19.4, 19.2, 17.6, 17.1, 16.8, 14.3, 14.2, 14.0; HR-ESIMS m/z for C₂₈H₃₈N₃O₆⁺ [M + H]⁺: calculated 512.2755, found 512.2755.

3.2.4. Preparation of Tetrapeptide 30

To a solution of the tripeptide 28 (1.00 g, 1.95 mmol) in THF–MeOH–H₂O (30 mL, 1:1:1) was added LiOH^.H₂O (250.0 mg, 5.94 mmol) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 5 h (monitored by TLC). Volatiles were removed in vacuo. The aqueous layer was washed with Et₂O (3 × 80 mL). The organic phases were discarded, and the aqueous phase was acidified to pH 3 with 10% aqueous solution of citric acid at 0 °C. This aqueous layer was then extracted with ethyl acetate (3 × 80 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo to give the corresponding acid (850.0 mg, 87%). To a solution of above acid in DCM (50 mL), PyAOP (1.78 g, 3.42 mmol), DIPEA (1.4 mL, 8.55 mmol) and HOAt (470.0 mg, 3.42 mmol) were sequentially added at 0 °C. 0.5 h later, a solution of Val-O^tBu 29 (360.0 mg, 2.05 mmol) in DCM (5 mL) was added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred for 16 h. The reaction was quenched by addition of saturated NH₄Cl solution (150 mL). Layers were separated, the aqueous phase was extracted with DCM (3 × 80 mL). The combined organic layers were washed with saturated NaHCO₃ solution (3 × 50 mL) and brine (30 mL), dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash chromatography (hexane/ethyl acetate, 1:1) to afford tetrapepetide 30 (890.0 mg, 80%). [α]_D²⁵ = +32.5 (c 1.7, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.39–7.11 (m, 10H), 6.47–6.12 (m, 1H), 5.53–5.41 (m, 1H), 5.41–5.35 (m, 1H), 5.22–5.06 (m, 2H), 4.96–4.92 (m, 1H), 4.45–4.40 (m, 1H), 4.33–4.28 (m, 1H), 3.26–3.20 (m, 1H), 3.10 (2.74) (s, 3H), 2.95–2.88 (m, 1H), 2.74 (2.33) (s, 3H), 2.17–2.09 (m, 1H), 1.96–1.85 (m, 1H), 1.45 (1.32) (s, 9H), 1.11–0.90 (m, 15H). ¹³C NMR (125 MHz, CDCl₃) δ 172.2, 171.4, 170.9, 170.5, 170.0, 168.8, 156.5, 156.2, 137.7, 137.1, 136.6, 136.6, 129.6, 129.1, 128.9, 128.7, 128.6, 128.3, 128.3, 128.2, 128.0, 127.9, 127.0, 126.7, 82.1, 81.7, 66.9, 62.0, 58.2, 57.7, 56.8, 55.5, 50.0, 47.2, 34.9, 33.3, 31.7, 31.4, 31.4, 30.8, 30.7, 29.3, 28.9, 28.0, 27.8, 19.5, 19.3, 19.0, 18.8, 17.9, 17.6, 17.5, 17.5, 14.3, 14.2. HR-ESIMS m/z for C₃₆H₅₃N₄O₇⁺ [M + H]⁺: calculated 653.3909, found 653.3908.

3.2.5. Preparation of Hexapeptide 31

To a solution of tetrapeptide 30 (620.0 mg, 0.95 mmol) in MeOH (30 mL), Pd/C was added under N₂ atmosphere. The reaction vessel was sealed and flashed with H₂ for three times. The reaction mixture was then vigirously stirred overnight under a H₂ atmosphere. Catalyst was removed by filtration. The filtrate was concentrated in vacuo to give the corresponding free amine, which was pure enough and used directly in next step of reaction. To a solution of dipeptide 22 (220.0 mg, 0.43 mmol) in DCM (15 mL) was added HATU (323.0 mg, 0.85 mmol), followed by addition of DIPEA (0.4 mL, 2.13 mmol) and HOAt (116.0 mg, 0.85 mmol) at 0 °C. 0.5 h later, a solution of the above free amine in DCM (5 mL) was added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred overnight, then quenched by addition of saturated NH₄Cl (40 mL). Layers were seperated, the aqueous phase was extracted with DCM (3 × 80 mL). The combined organic layers were washed with saturated NaHCO₃ solution (3 × 50 mL) and brine (30 mL), dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash chromatography (hexane/ethyl acetate, 1:1) to afford 31 (221.0 mg, 61%). [α]_D²⁵ = +8.3 (c 0.8, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 7.43–7.01 (m, 11H), 6.64 (6.46) (d, J = 8.8 Hz, 1H), 5.49–5.34 (m, 1H), 5.33–5.17 (m, 1H), 5.17–4.95 (m, 2H), 4.85–4.80 (m, 1H), 4.78–4.43 (m, 2H), 4.43–4.18 (m, 2H), 3.27 (dd, J = 14.6, 5.6 Hz, 1 H), 3.12 (s, 1H), 3.10–3.01 (m, 3H), 2.97–2.93 (m, 3H), 2.90–2.83 (m, 3H), 2.80–2.77 (m, 2H), 2.31 (s, 2H), 2.26–2.09 (m, 1H), 2.01–1.85 (m, 1H), 1.80 (s, 1H), 1.41 (1.40) (s, 9H), 1.30–0.70 (m, 20 H), 0.46–0.27 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 172.4, 172.3, 171.8, 171.4, 170.7, 170.4, 169.8, 168.8, 137.6, 137.0, 136.3, 129.6, 128.9, 128.9, 128.6, 128.6, 128.5, 128.1, 127.7, 126.9, 126.7, 81.7, 81.7, 67.8, 67.7, 62.1, 59.3, 58.0, 57.6, 54.3, 53.3, 52.4, 50.0, 47.7, 33.2, 32.1, 32.0, 31.5, 31.1, 31.0, 30.9, 30.8, 30.7, 30.4, 29.3, 28.9, 27.9, 19.4, 18.9, 18.5, 18.2, 18.0, 17.4, 17.2, 14.5, 14.3, 14.1, 13.6. HR-ESIMS m/z for C₄₅H₆₈N₆NaO₁₀⁺ [M + Na]⁺: calculated 875.4889, found 875.4891.

3.2.6. Preparation of 32c and 32d

To a solution of hexapeptide 31 (40.0 mg, 0.05 mmol) in MeOH (20 mL), was added Pd/C (10% on charcoal) under N₂ atmosphere. The reaction vessel was sealed and flashed with H₂ for three times. The reaction mixture was then vigirously stirred overnight under H₂ atmosphere. Catalyst was removed by filtration. The filtrate was concentrated in vacuo to give the corresponding free amine, which was pure enough and used directly in next step of reaction. To a solution of S-3 (23.0 mg, 0.14 mmol) in DCM (2 mL) was added HATU (34.0 mg, 0.09 mmol), followed by addition of DIPEA (39 μL, 0.23 mmol) and HOAt (12.0 mg, 0.09 mmol) at 0 °C. 0.5 h later, a solution of the above amine (32.0 mg, 0.04 mmol) in DCM (2 mL) was added at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction was quenched with saturated NH₄Cl (20 mL), and extracted with DCM (3 × 50 mL). The combined organic layers were washed with saturated NaHCO₃ solution (3 × 50 mL) and brine (50 mL), dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash chromatography (ethyl acetate) to give compound 32c (20.0 mg, 50%). [α]_D²⁵ = −12.3 (c 0.1, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 8.07 (d, J = 9.0 Hz, 0.5H), 7.32–7.28 (m, 1H), 7.26–7.14 (m, 4H), 6.96 (d, J = 9.5 Hz, 0.4H), 6.68 (d, J = 8.9 Hz, 0.4H), 6.50 (d, J = 8.9 Hz, 0.6H), 6.08–6.01 (m, 1H), 5.52–5.37 (m, 1H), 5.36–5.30 (m, 1H), 5.14–5.01 (m, 1H), 4.91–4.65 (m, 1H), 4.59–4.42 (m, 1H), 4.30 (dd, J = 8.8, 4.6 Hz, 2H), 3.31–3.18 (m, 1H), 3.17–3.08 (m, 1H), 3.07–2.82 (m, 9H), 2.80–2.74 (m, 3H), 2.51–2.37 (m, 1H), 2.37–2.29 (m, 2H), 2.28–2.17 (m, 1H), 2.17–2.07 (m, 1H), 2.02–1.84 (m, 2H), 1.43 (s, 9H), 1.39–1.28 (m, 3H), 1.19–1.07 (m, 6H), 1.05–0.97 (m, 3H), 0.91–0.76 (m, 12H). ¹³C NMR (75 MHz, CDCl₃) δ 172.5, 172.4, 172.3, 171.9, 171.8, 171.4, 170.7, 170.4, 169.9, 168.8, 156.8, 137.6, 137.0, 136.3, 129.6, 128.9, 128.9, 128.6, 128.5, 128.1, 127.7, 126.9, 126.7, 81.7, 68.5, 68.2, 68.0, 67.8, 62.9, 62.1, 60.3, 59.3, 58.9, 58.2, 58.0, 57.9, 57.6, 56.9, 54.3, 53.3, 52.4, 50.9, 50.0, 47.7, 47.5, 43.2, 34.7, 33.2, 32.1, 32.0, 31.5, 31.1, 31.0, 30.7, 30.4, 29.3, 28.9, 27.9, 22.4, 19.4, 19.4, 18.9, 18.5, 18.2, 18.0, 17.4, 17.2, 15.0, 14.5, 14.3, 14.1, 13.6. HR-ESIMS m/z for C₄₂H₆₈Cl₂N₆NaO₉⁺ [M + Na]⁺: calculated 893.4317, found 893.4318.

Compound 32d was prepared in 52% yield. Analytical data: [α]_D²⁵ = −28.2 (c 0.3, CHCl₃); ¹H NMR (300 MHz, CDCl₃) δ 8.09 (d, J = 9.0 Hz, 0.5H), 7.43–7.13 (m, 5H), 7.00 (d, J = 9.5 Hz, 0.5H), 6.70 (d, J = 8.9 Hz, 0.5H), 6.52 (d, J = 8.9 Hz, 0.5H), 6.15–5.91 (m, 1H), 5.58–5.38 (m, 1H), 5.38–5.23 (m, 1H), 5.16–5.01 (m, 1H), 4.93–4.65 (m, 1H), 4.54 (ddd, J = 14.3, 8.9, 5.8 Hz, 1H), 4.32 (dd, J = 8.8, 4.6 Hz, 2H), 3.26 (dt, J = 16.8, 7.2 Hz, 1H), 3.17–3.07 (m, 3H), 3.07–2.90 (m, 3H), 2.90 (2.88) (s, 3H), 2.86–2.76 (m, 3H), 2.71 (dt, J = 16.7, 6.2 Hz, 1H), 2.55–2.42 (m, 1H), 2.32 (s, 1H), 2.17 (ddd, J = 14.0, 12.4, 6.6 Hz, 1H), 2.03–1.87 (m, 1H), 1.46 (s, 9H), 1.39–1.35 (m, 3H), 1.26–1.22 (m, 6H), 1.15–1.11 (m, 3H), 1.04 (t, J = 6.5 Hz, 3H), 0.93–0.77 (m, 9H), 0.38 (d, J = 6.9 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 172.5, 172.4, 172.3, 172.0, 171.9, 171.4, 171.2, 171.0, 170.7, 170.6, 170.4, 169.9, 169.8, 168.9, 137.6, 137.0, 129.6, 129.0, 128.9, 128.7, 127.0, 126.7, 84.6, 81.7, 68.6, 62.0, 58.0, 57.9, 57.6, 57.3, 57.0, 55.7, 54.3, 52.6, 52.4, 51.7, 50.0, 48.8, 47.8, 45.6, 40.3, 35.6, 35.6, 34.7, 33.3, 31.5, 31.2, 31.0, 30.9, 30.7, 30.7, 30.6, 30.5, 29.6, 29.3, 28.9, 27.9, 19.4, 19.4, 18.9, 18.7, 18.5, 18.0, 17.4, 17.3, 15.2, 14.6, 14.3, 14.1, 13.7. HR-ESIMS m/z for C₄₂H₆₈Cl₂N₆NaO₉⁺ [M + Na]⁺: calculated 893.4317, found 893.4316.

3.2.7. Completion of the Synthesis of Itralamide B 1c and 1d

To a solution of compound 32c (15.0 mg, 0.02 mmol) in DCM (1.0 mL), BF₃^.Et₂O (21 μL, 0.17 mmol) was added dropwise at 0 °C. The reaction solution was then allowed to warm to room temperature and stirred for 0.5~1.0 h (monitored by TLC). The reaction was quenched by addition of saturated NH₄Cl (2 mL) and diluted with DCM (60 mL). The organic phase was washed with saturated NH₄Cl (3 × 20 mL) and brine (20 mL), dried over anhydrous Na₂SO₄ and concentrated in vacuo to give crude hydroxy acid, which was dried under high vacuum for 4 h. To a solution of DMAP (21.0 mg, 0.17 mmol) and MNBA (30.0 mg, 0.08 mmol), a solution of above hydroxy acid in toluene (5 mL) was slowly added at 0 °C. After the reaction mixture was warmed to room temperature, it was gradually heated to 60 °C and stirred for two days. The reaction mixture was diluted with ethyl acetate (100 mL) and washed successively with saturated NH₄Cl (3 × 20 mL) and brine (2 × 20mL), dried over anhydrous Na₂SO₄ and concentrated in vacuo. The residue was purified by flash chromatography (ethyl acetate) to afford itralamide B 1c (2.7 mg, 20%). [α]_D²⁵ = −10.8 (c 0.1, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 8.45 (d, J = 7.6 Hz, 1H), 7.33–7.28 (m, 2H), 7.19 (d, J = 7.0 Hz, 3H), 5.98 (d, J = 3.0 Hz, 1H), 5.80 (d, J = 3.3 Hz, 1H), 5.64 (dd, J = 6.7, 3.1 Hz, 1H), 5.34 (dd, J = 11.4, 3.7 Hz, 1H), 5.12 (q, J = 6.9 Hz, 1H), 4.98 (q, J = 6.7 Hz, 1H), 4.67 (t, J = 10.1 Hz, 1H), 4.63 (dd, J = 7.5, 3.8 Hz, 1H), 3.36 (s, 3H), 3.23–3.10 (m, 2H), 3.08 (s, 3H), 3.05 (s, 3H), 2.99 (s, 3H), 2.91 (dd, J = 14.3, 3.4 Hz, 2H), 2.85 (d, J = 8.9 Hz, 1H), 2.81–2.74 (m, 2H), 2.70 (dd, J = 16.6, 5.1 Hz, 2H), 2.47 (dd, J = 16.7, 7.6 Hz, 1H), 2.32–2.27 (m, 1H), 2.27–2.21 (m, 1H), 2.02 (s, 1H), 1.36 (d, J = 6.7 Hz, 3H), 1.20 (d, J = 6.6 Hz, 3H), 1.05 (d, J = 6.9 Hz, 3H), 0.97 (d, J = 6.7 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H), 0.90 (s, 1H), 0.88 (d, J = 6.8 Hz, 3H), 0.41 (d, J = 6.8 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃) δ 172.4, 172.2, 171.4, 170.2, 169.5, 168.8, 137.2, 132.1, 132.0, 131.9, 129.7, 128.9, 128.5, 128.4, 127.0, 78.0, 69.8, 61.2, 57.6, 55.5, 53.5, 52.7, 46.6, 40.6, 35.7, 35.7, 34.1, 32.5, 31.9, 31.4, 30.7, 29.8, 29.7, 29.6, 29.4, 28.8, 22.7, 19.6, 18.8, 18.7, 18.0, 17.6, 15.4. HR-ESIMS m/z for C₃₈H₅₈Cl₂N₆NaO₈⁺ [M + Na]⁺: calculated 819.3585, found 819.3587.

Compound itralamide B 1d was prepared in 21% yield. Analytical data: [α]_D²⁵ = −8.3 (c 0.1, CHCl₃); ¹H NMR (600 MHz, CDCl₃) δ 8.46 (d, J = 7.5 Hz, 1H), 7.32 (dd, J = 9.6, 5.4 Hz, 2H), 7.20 (d, J = 7.1 Hz, 3H), 6.00 (d, J = 2.9 Hz, 1H), 5.82 (d, J = 3.1 Hz, 1H), 5.66 (dd, J = 6.7, 3.2 Hz, 1H), 5.41–5.29 (m, 2H), 5.14 (q, J = 6.9 Hz, 1H), 5.00 (q, J = 6.8 Hz, 1H), 4.69 (t, J = 10.1 Hz, 1H), 4.64 (dd, J = 7.6, 3.8 Hz, 1H), 3.38 (s, 3H), 3.19 (dd, J = 14.0, 11.5 Hz, 1H), 3.10 (s, 3H), 3.07 (s, 3H), 3.01 (s, 3H), 2.98–2.94 (m, 2H), 2.94–2.90 (m, 1H), 2.80 (d, J = 18.3 Hz, 2H), 2.72 (dd, J = 16.6, 5.1 Hz, 1H), 2.49 (dd, J = 16.7, 7.6 Hz, 1H), 2.38 (d, J = 6.4 Hz, 1H), 2.34–2.30 (m, 1H), 2.25 (dd, J = 9.7, 5.7 Hz, 1H), 2.03 (s, 1H), 1.38 (d, J = 6.7 Hz, 3H), 1.22 (d, J = 6.6 Hz, 3H), 1.07 (d, J = 7.0 Hz, 3H), 0.99 (d, J = 6.7 Hz, 3H), 0.97 (d, J = 6.9 Hz, 3H), 0.92 (s, 1H), 0.90 (d, J = 6.7 Hz, 3H), 0.43 (d, J = 6.8 Hz, 2H). ¹³C NMR (150 MHz, CDCl₃) δ 172.8, 172.4, 172.2, 171.4, 170.2, 169.5, 168.8, 137.2, 129.7, 128.9, 127.0, 78.0, 69.8, 61.2, 57.6, 55.5, 53.5, 52.7, 46.6, 40.6, 35.9, 35.7, 34.2, 32.5, 31.9, 31.9, 31.4, 30.7, 29.8, 29.7, 29.3, 28.8, 22.7, 19.6, 18.8, 18.7, 18.0, 17.6, 15.4, 14.2, 14.1, 13.4. HR-ESIMS m/z for C₃₈H₅₈Cl₂N₆NaO₈⁺ [M + Na]⁺: calculated 819.3585, found 819.3589.

3.2.8. Analytical Data of Itralamide B 1a and 1b [26]

Itralamide B 1a: [α]_D²⁵ = −50.9 (c 0.1, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.45–7.12 (m, 5H), 6.90 (d, J = 9.6 Hz, 1H), 6.47 (d, J = 8.0 Hz, 1H), 5.99 (d, J = 2.9 Hz, 1H), 5.75–5.65 (m, 2H), 5.53–5.43 (m, 1H), 5.08 (q, J = 6.8 Hz, 1H), 4.98 (dd, J = 7.9, 4.2 Hz, 1H), 4.71 (dd, J = 9.4, 4.1 Hz, 1H), 4.62 (q, J = 7.6 Hz, 1H), 3.66 (dd, J = 15.5, 4.9 Hz, 1H), 3.33 (d, J = 23.6 Hz, 3H), 3.19 (d, J = 7.1 Hz, 3H), 3.17–3.07 (m, 3H), 3.07–2.94 (m, 3H), 2.87 (dd, J = 15.6, 12.1 Hz, 1H), 2.80 (d, J = 4.0 Hz, 1H), 2.76–2.65 (m, 1H), 2.46 (dd, J = 16.5, 7.5 Hz, 1H), 2.28–2.21 (m, 1H), 2.06–1.99 (m, 1H), 1.29 (d, J = 2.0 Hz, 3H), 1.26 (d, J = 2.8 Hz, 3H), 1.20 (dd, J = 6.6, 3.5 Hz, 3H), 1.08–1.01 (m, 6H), 0.92 (d, J = 7.2 Hz, 6H), 0.80 (d, J = 6.8 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 174.9, 172.8, 172.5, 170.6, 170.2, 169.9, 169.5, 137.4, 128.5, 128.3, 126.5, 78.2, 69.6, 56.9, 56.8, 54.7, 54.7, 54.0, 51.4, 40.6, 35.8, 33.9, 33.8, 32.2, 31.8, 31.1, 31.0, 30.5, 19.9, 19.6, 17.8, 17.1, 15.6, 15.4, 14.1, 13.8. HR-ESIMS m/z for C₃₈H₅₈Cl₂N₆NaO₈⁺ [M + Na]⁺: calculated 819.3585, found 819.3587.

Itralamide B 1b: [α]_D²⁵ = −42.3 (c 0.2, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ 7.24–7.16 (m, 5H), 6.89 (d, J = 9.1 Hz, 1H), 6.48 (d, J = 7.8 Hz, 1H), 6.04 (d, J = 2.9 Hz, 1H), 5.78–5.63 (m, 1H), 5.49 (dd, J = 6.6, 3.2 Hz, 1H), 5.45–5.35 (m, 1H), 5.12–5.02 (m, 1H), 4.98 (dd, J = 7.8, 4.2 Hz, 1H), 4.71 (dd, J = 9.3, 4.0 Hz, 1H), 4.67–4.55 (m, 1H), 3.66 (dd, J = 15.3, 5.1 Hz, 1H), 3.28 (d, J = 63.9 Hz, 3H), 3.18 (s, 3H), 3.13 (d, J = 23.1 Hz, 3H), 3.01 (d, J = 11.3 Hz, 3H), 2.93 (d, J = 15.7 Hz, 1H), 2.90–2.78 (m, 1H), 2.78–2.67 (m, 1H), 2.43 (dd, J = 16.7, 6.0 Hz, 1H), 2.25 (dd, J = 11.9, 5.9 Hz, 1H), 2.04 (d, J = 9.5 Hz, 1H), 1.38 (d, J = 7.2 Hz, 3H), 1.30 (d, J = 6.7 Hz, 3H), 1.20 (d, J = 6.6 Hz, 3H), 1.06 (d, J = 6.8 Hz, 6H), 0.92 (d, J = 7.0 Hz, 6H), 0.80 (d, J = 6.8 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 174.9, 172.8, 172.6, 170.7, 170.2, 169.8, 169.5, 137.4, 128.6, 128.3, 126.5, 78.0, 69.6, 56.9, 56.8, 54.8, 54.2, 54.0, 51.5, 40.4, 36.0, 34.0, 33.8, 32.2, 31.8, 31.1, 31.0, 30.6, 19.9, 19.6, 17.8, 17.1, 17.1, 15.6, 15.0, 13.8. HR-ESIMS m/z for C₃₈H₅₈Cl₂N₆NaO₈⁺ [M + Na]⁺: calculated 819.3585, found 819.3589.

3.3. Biological Test

Cell proliferation assay: Neuroblastoma cell line SH-SY5Y, cervical adenocarcinoma cell line HeLa, and hepatocellular carcinoma cell lines Hep3B and PLC were obtained from American Type Culture Collection (Manassas, VA, USA), and cultured in DMEM containing supplements (10% FBS, penicillin/streptomycin and l-glutamine). Cells were seeded into 96-well plates overnight and cultured with incremental concentrations of the compounds in the medium containing 1% FBS for another 72 h. The effect of the compounds was evaluated by cell proliferation assay using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl-2H-tetrazolium) (MTS) assay (Promega Co., Madison, WI, USA). According to the manufacturer’s instructions, 20 μL of CellTiter96 Aqueous solution was added into each well containing 100 μL medium and incubated at 37 °C for 4 h. The absorbance at 490 nm was measured using an ELISA plate reader (Bio-Rad microplate reader 680, Bio-Rad Laboratories, Hercules, CA, USA). IC₅₀ values were calculated using GraphPad Prism software (GraphPad Prism software Inc., La Jolla, CA, USA).

4. Conclusions

A reliable and convergent strategy for the total synthesis of itralamide B had been developed. Four stereoisomers of itralamide B 1a–1d were prepared. Comparison of spectral data for the synthetic samples 1a–1d with data on itralamide B in the literature revealed significant differences, and these discrepancies led to some uncertainty concerning the structure of itralamide B. The current work proved that these data discrepancies originated somewhere other than the configuration of the valine residues. Further work is still required to determine the true structure of natural itralamide B. Furthermore, itralamide B 1a–1d and compound 16 were evaluated using cell proliferation assay, which revealed that stereoisomers 1a, 1c and 1d showed moderate inhibitory activity toward PLC cancer cell.