Isolation and Total Synthesis of PM170453, a New Cyclic Depsipeptide Isolated from Lyngbya sp.

In our continuing search for biologically active new chemical entities from marine organisms, we have isolated a new cyclic depsipeptide, PM170453 (1), from a cyanobacterium of the genus Lyngbya sp., collected in the Indo-Pacific Ocean. Structure elucidation of the isolated compound was determined by spectroscopic methods including MS, 1H, 13C and 2D-NMR. To solve the supply problem for 1 and progress pharmaceutical development, the total synthesis of 1 that involves a total of 20 chemical steps in a convergent process was carried out. Its in vitro cytotoxic activity against four human tumor cell lines, as well as the inhibition of the interaction between the programmed cell death protein 1 PD-1 and its ligand PD-L1 were also evaluated.


Introduction
The programmed cell death 1 receptor (PD-1) and its ligand programmed cell death ligand 1 (PD-L1) are immune checkpoint proteins found on the cell surface of various immune cells.PD-L1 is also expressed in a variety of human cancers.Under physiological conditions, their interaction results in cell immune suppression, but in cancer cells this interaction results in the escape of immune detection.However, this can be reversed by blocking PD-1's interaction with PD-L1 [1].Marine cyanobacteria have emerged as a promising source of bioactive compounds with therapeutic and pharmacological potential [2].In this context, cyclic peptides have garnered particular interest due to their structural diversity and biological activity.In particular, the Lyngbya genus, a strain of Cyanobacteria belonging to the family Oscillatoriacea Engler, has provided different metabolites with biological effects, and their unusual activities as immunosuppressants are remarkably interesting [3,4].Yanucamides, for instance, are natural depsipeptides isolated from an assemblage of the marine Cyanobacteria Lyngbya majuscula and Schizothrix species [5].These structures exhibit strong brine shrimp toxicity, although further biological activities have not been tested so far.Later, the total synthesis and stereochemical revision of yanucamide A was reported [6].In our continuing search for biologically active compounds from marine organisms, we have isolated a new cyclic depsipeptide, PM170453 (1), from a Lyngbya sp.specimen, collected off the coast of Buru Island, the third largest island within the Maluku Islands of Indonesia.Initially the crude extract of this specimen showed hints of inhibition of the PD-1 receptor activity, which prompted us to further fractionate it.Structurally, compound 1 has similarity to several cyclic peptides such as yanucamides [5], dudawalamides [7], kulolides [8] and viequeamides [9].The structure of 1 was determined using different spectroscopic methods such as 1D-and 2D-NMR, and the molecular formula was confirmed by HRESITOFMS.It contains three amino acids (β-alanine, N-methyl-phenylalanine and

Isolation and Structure Elucidation of PM170453 (1)
During our screening program to find new chemical entities from marine organisms that reverse immune suppression by inhibiting PD-1 receptor activity, the crude extract of a marine Lyngbya sp.collected off the coast of Buru, Indonesia showed hints of inhibition of this interaction (45% inhibition compared with untreated control cells).Bioassayguided fractionation of an organic extract of the organism, including VLC RP-18 chromatography followed by reverse-phase preparative HPLC of selected fractions, led to the isolation of compound 1 (Figure 1).Compound 1 was isolated as an optically active white powder with a pseudomolecular ion in the (+)-HRESITOFMS m/z 596.3364 [M + H] + .The presence of 31 signals on the 13 C NMR spectrum (Table 1) was also in agreement with the molecular formula C33H45N3O7 (m/z 596.3364 [M + H] + , calcd.for C33H46N3O7 m/z 596.3330 [M + H] + ).The peptide nature of 1 was evident from its 1 H and 13 C NMR spectra (Figures S1-S9). 1 H NMR data in CD3OD (Table 1) displayed the characteristic α-amino or α-hydroxy acids at 5.18, 4.99, 4.60 and 4.55 ppm, corresponding to four methine carbons adjacent to heteroatoms at 78.4, 64.2, 78.4 and 57.3 ppm, respectively in the HSQC experiment.This assignation is supported by the presence of five carbonyl groups in the range 178.2-169.5 ppm.A signal at 8.57 ppm in the 1 H NMR when performed in CD3OH was attributable to a NH amide.Combination of 13 C NMR and HSQC spectra showed a N-Me group at 29.9 ppm.Additionally, an aromatic ring with five aromatic methines in the range 139.4-127.9ppm as well as a terminal acetylene group, presumed by the presence of a characteristic methine carbon at 70.1 (observed when the HSQC experiment is performed with a JH-C = 250 Hz) together with the quaternary carbon at 84.4 ppm, were part of this molecule.Compound 1 was isolated as an optically active white powder with a pseudomolecular ion in the (+)-HRESITOFMS m/z 596.3364 [M + H] + .The presence of 31 signals on the 13 C NMR spectrum (Table 1) was also in agreement with the molecular formula C 33 H 45 N 3 O 7 (m/z 596.3364 [M + H] + , calcd.for C 33 H 46 N 3 O 7 m/z 596.3330 [M + H] + ).The peptide nature of 1 was evident from its 1 H and 13 C NMR spectra (Figures S1-S9). 1 H NMR data in CD 3 OD (Table 1) displayed the characteristic α-amino or α-hydroxy acids at 5.18, 4.99, 4.60 and 4.55 ppm, corresponding to four methine carbons adjacent to heteroatoms at 78.4, 64.2, 78.4 and 57.3 ppm, respectively in the HSQC experiment.This assignation is supported by the presence of five carbonyl groups in the range 178.2-169.5 ppm.A signal at 8.57 ppm in the 1 H NMR when performed in CD 3 OH was attributable to a NH amide.Combination of 13 C NMR and HSQC spectra showed a N-Me group at 29.9 ppm.Additionally, an aromatic ring with five aromatic methines in the range 139.4-127.9ppm as well as a terminal acetylene group, presumed by the presence of a characteristic methine carbon at 70.1 (observed when the HSQC experiment is performed with a J H-C = 250 Hz) together with the quaternary carbon at 84.4 ppm, were part of this molecule.Extensive 2D NMR analysis of COSY, HSQC and HMBC (Figure 2) revealed the presence of Dhoya residue and β-alanine (β-Ala), N-methylphenylalanine (NMe-Phe), proline (Pro) and 2-hydroxyvaleric acid (Hiv) units.The spin system corresponding to 2,2-dimethyl-3-hydroxy-7-octanoic acid was determined by the COSY correlation from position H-3 (5.18 ppm) to H-6 (2.22 ppm) and the long range cross-peaks between the acetylene H-8 (2.24 ppm) to the methylene C-6 (18.7 ppm), H-6 (2.22 ppm) to C-7 and C-8 (84.4 and 70.1 ppm, respectively) and the methyl singlets H-9 (1.31 ppm) and H-10 (1.16 ppm) to C-1, C-2 and C-3 (178.2, 47.7 and 78.4 ppm, respectively).In the same way, the spin systems corresponding to β-Ala and Pro were deduced from their COSY correlations between adjacent methylenes H-12 and H-13 (2.88/2.65 and 3.67/3.18ppm) and from positions H-22 to H-25 (4.55, 1.02/0.87,1.84/1.70,3.75/3.44ppm), respectively.Furthermore, the presence of NMe-Phe and Hiv was inferred from the COSY correlations between H-15 and H-16 (4.99 and 3.30/2.98ppm) and from H-27 to H-30 (4.60, 2.11, 1.05, 0.99 ppm) correspondingly.Finally, the connectivity between these moieties was deduced by the HMBC correlations observed from the relationship of the α-protons and NMe to the adjacent carbonyls, establishing the sequence of 1 as Dhoya/β-Ala/NMe-Phe/Pro/Hiv.Due to the small amount of the compound isolated, we could not carry out further experiments to determine the absolute configuration of the chiral centres.Nonetheless, we assumed the same configuration of the residues as those of the known yanucamide due to their spectroscopic similarities.

Total Synthesis of 1
To solve the supply problem for further development and to confirm the proposed stereochemistry, we completed the first total synthesis of 1.The synthetic methodology involves a linear sequence of 12 chemical steps using (S)-2-hydroxyisovaleric acid (Hiv) as starting material.Key elements of the approach include different coupling and esterification reactions using appropriately protected amino acids and other fragments and a final cyclization between β-alanine and N-methyl-phenylalanine to create 1 (Figure 3).This ring-closure is sufficiently unhindered to allow for smooth cyclization and was achieved without any racemization.This procedure allowed us to obtain a few grams of compound 1, from key fragments 2 and 3.The Dhoya fragment 2 (Figure 4) was constructed in five linear steps from Hex-5-yn-1-ol.Swern oxidation gave aldehyde 4, which was converted into the (S)-α-hydroxy acid 7 via an asymmetric aldol reaction with oxazolidinone 5, previously obtained by methylation of (S)-4-benzyl-3-propionyl-2-oxazolidinone and cleavage of Evans oxazolidinone 6 with LiOH/H2O2 [10].Double silyl protection of 7 using TBDMS-triflate resulted in intermediate 8, which was subjected to saponification, affording the corresponding (S)-3-((tertbutyldimethylsilyl)oxy)-2,2-dimethyloct-7-ynoic acid fragment 2 after acidification.Due to the small amount of the compound isolated, we could not carry out further experiments to determine the absolute configuration of the chiral centres.Nonetheless, we assumed the same configuration of the residues as those of the known yanucamide due to their spectroscopic similarities.

Total Synthesis of 1
To solve the supply problem for further development and to confirm the proposed stereochemistry, we completed the first total synthesis of 1.The synthetic methodology involves a linear sequence of 12 chemical steps using (S)-2-hydroxyisovaleric acid (Hiv) as starting material.Key elements of the approach include different coupling and esterification reactions using appropriately protected amino acids and other fragments and a final cyclization between β-alanine and N-methyl-phenylalanine to create 1 (Figure 3).This ring-closure is sufficiently unhindered to allow for smooth cyclization and was achieved without any racemization.This procedure allowed us to obtain a few grams of compound 1, from key fragments 2 and 3.  Due to the small amount of the compound isolated, we could not carry out further experiments to determine the absolute configuration of the chiral centres.Nonetheless, we assumed the same configuration of the residues as those of the known yanucamide due to their spectroscopic similarities.

Total Synthesis of 1
To solve the supply problem for further development and to confirm the proposed stereochemistry, we completed the first total synthesis of 1.The synthetic methodology involves a linear sequence of 12 chemical steps using (S)-2-hydroxyisovaleric acid (Hiv) as starting material.Key elements of the approach include different coupling and esterification reactions using appropriately protected amino acids and other fragments and a final cyclization between β-alanine and N-methyl-phenylalanine to create 1 (Figure 3).This ring-closure is sufficiently unhindered to allow for smooth cyclization and was achieved without any racemization.This procedure allowed us to obtain a few grams of compound 1, from key fragments 2 and 3.The Dhoya fragment 2 (Figure 4) was constructed in five linear steps from Hex-5-yn-1-ol.Swern oxidation gave aldehyde 4, which was converted into the (S)-α-hydroxy acid 7 via an asymmetric aldol reaction with oxazolidinone 5, previously obtained by methylation of (S)-4-benzyl-3-propionyl-2-oxazolidinone and cleavage of Evans oxazolidinone 6 with LiOH/H2O2 [10].Double silyl protection of 7 using TBDMS-triflate resulted in intermediate 8, which was subjected to saponification, affording the corresponding (S)-3-((tertbutyldimethylsilyl)oxy)-2,2-dimethyloct-7-ynoic acid fragment 2 after acidification.The Dhoya fragment 2 (Figure 4) was constructed in five linear steps from Hex-5-yn-1-ol.Swern oxidation gave aldehyde 4, which was converted into the (S)-α-hydroxy acid 7 via an asymmetric aldol reaction with oxazolidinone 5, previously obtained by methylation of (S)-4-benzyl-3-propionyl-2-oxazolidinone and cleavage of Evans oxazolidinone 6 with LiOH/H 2 O 2 [10].Double silyl protection of 7 using TBDMS-triflate resulted in intermediate 8, which was subjected to saponification, affording the corresponding (S)-3-((tert-butyldimethylsilyl)oxy)-2,2-dimethyloct-7-ynoic acid fragment 2 after acidification.Fragment 3 was a straightforward preparation starting from (S)-2-hydroxyisovaleric acid (Hiv) (Figure 5).Hiv was converted to silyl ether intermediate 10 following the same strategy as that used to prepare fragment 2 from intermediate 7 [11].The coupling of 10 with L-proline benzyl ester hydrochloride, mediated by DCC, NMM and HOBt, proceeded smoothly to give 11 with a 46% yield.Hydrogenolytic removal of the benzyl group in 11 was achieved by using Pd(OH)2 in a 2:1 mixture of IPA:H2O to obtain acid 12.At the same time, Boc-N-methyl-L-phenylalanine was protected as the allyl ester, and acidic Boc removal gave N-methyl intermediate 14.Coupling reaction between 12 and 14 using BOPCl and NMM afforded amide 15 with a 68% yield.Deprotection of the TBDMS silyl group using acetyl chloride in MeOH gave fragment 3 in excellent yield (89%).Fragment 3 was a straightforward preparation starting from (S)-2-hydroxyisovaleric acid (Hiv) (Figure 5).Hiv was converted to silyl ether intermediate 10 following the same strategy as that used to prepare fragment 2 from intermediate 7 [11].The coupling of 10 with L-proline benzyl ester hydrochloride, mediated by DCC, NMM and HOBt, proceeded smoothly to give 11 with a 46% yield.Hydrogenolytic removal of the benzyl group in 11 was achieved by using Pd(OH) 2 in a 2:1 mixture of IPA:H 2 O to obtain acid 12.At the same time, Boc-N-methyl-L-phenylalanine was protected as the allyl ester, and acidic Boc removal gave N-methyl intermediate 14.Coupling reaction between 12 and 14 using BOPCl and NMM afforded amide 15 with a 68% yield.Deprotection of the TBDMS silyl group using acetyl chloride in MeOH gave fragment 3 in excellent yield (89%).Fragment 3 was a straightforward preparation starting from (S)-2-hydroxyisovaleric acid (Hiv) (Figure 5).Hiv was converted to silyl ether intermediate 10 following the same strategy as that used to prepare fragment 2 from intermediate 7 [11].The coupling of 10 with L-proline benzyl ester hydrochloride, mediated by DCC, NMM and HOBt, proceeded smoothly to give 11 with a 46% yield.Hydrogenolytic removal of the benzyl group in 11 was achieved by using Pd(OH)2 in a 2:1 mixture of IPA:H2O to obtain acid 12.At the same time, Boc-N-methyl-L-phenylalanine was protected as the allyl ester, and acidic Boc removal gave N-methyl intermediate 14.Coupling reaction between 12 and 14 using BOPCl and NMM afforded amide 15 with a 68% yield.Deprotection of the TBDMS silyl group using acetyl chloride in MeOH gave fragment 3 in excellent yield (89%).The construction of 1 from the three fragments was completed using standard procedures (Figure 6).Esterification between fragments 2 and 3 was mediated by the use of EDC and DMAP to give intermediate 16 at a 93% yield.Removal of the silyl protecting group in acidic media afforded alcohol 17, and esterification under the same conditions with Boc-β-Ala-OH produced precursor 18 at a 91% yield.Consecutive unmasking of the carboxylic acid and the primary amine then allowed for cyclization via activation with HATU and HOAt, at high dilution in CH 2 Cl 2 (57% yield).In this way, we completed the total synthesis of 1 with all the chiral centers C-3, C-15, C-22 and C-27 in an S configuration and unambiguously confirmed the structural assignment of the natural product.All the spectral data ( 1 H and 13 C NMR; Figures S13-S51), HPLC retention times and biological activities of the synthetic sample exactly matched those of the isolated natural product 1.
gs 2024, 22, 303 6 of 16 The construction of 1 from the three fragments was completed using standard procedures (Figure 6).Esterification between fragments 2 and 3 was mediated by the use of EDC and DMAP to give intermediate 16 at a 93% yield.Removal of the silyl protecting group in acidic media afforded alcohol 17, and esterification under the same conditions with Boc-β-Ala-OH produced precursor 18 at a 91% yield.Consecutive unmasking of the carboxylic acid and the primary amine then allowed for cyclization via activation with HATU and HOAt, at high dilution in CH2Cl2 (57% yield).In this way, we completed the total synthesis of 1 with all the chiral centers C-3, C-15, C-22 and C-27 in an S configuration and unambiguously confirmed the structural assignment of the natural product.All the spectral data ( 1 H and 13 C NMR; Figures S13-S51), HPLC retention times and biological activities of the synthetic sample exactly matched those of the isolated natural product 1.

Biological Activities
The in vitro cytotoxic activity of the new cyclic depsipeptide 1 was tested against four human tumor cell lines, lung (A-549), colon (HT-29), breast (MDA-MB-231) and pancreas (PSN-1), following a published procedure [12].Doxorubicin was used as the positive control (Figure S11).Table 2 provides the data on the biological activity of 1 (GI50 value).

Biological Activities
The in vitro cytotoxic activity of the new cyclic depsipeptide 1 was tested against four human tumor cell lines, lung (A-549), colon (HT-29), breast (MDA-MB-231) and pancreas (PSN-1), following a published procedure [12].Doxorubicin was used as the positive control (Figure S11).Table 2 provides the data on the biological activity of 1 (GI 50 value).In addition, the PD-1/PD-L1 inhibition was also tested.The PD-1 assay measures receptor activation through co-culture of cells expressing PD-1 with PD-L1 presenting cells.BMS202 was used as control for PD-1/PD-L1 interaction inhibition.A PrestoBlue cell proliferation assay was simultaneously performed to control the cytotoxicity of the samples.The ability of PM170453 to inhibit PD-1/PD-L1 interaction was also determined using the well-established HTRF assay.Although we detected the same slight activity of 1 as in the crude extract, it was not significant enough to be considered (Figure S12, Table 3).

General Experimental Procedures
Dry solvents were purchased and used without any extra processing.All reagents were used as purchased without further purification unless otherwise stated.All reactions, unless otherwise indicated, were performed under an atmosphere of nitrogen in ovendried glassware.Routine monitoring of reactions was performed using silica gel TLC plates (Merck 60 F254).Spots were visualized by UV and/or dipping the TLC plate into an ethanolic phosphomolybdic acid solution and heating with a hot plate.Flash chromatography was carried out on silica gel 60 (200-400 mesh). 1 H and 13 C NMR spectra were recorded on a Varian Unity 400 or 500 spectrometer at 400 or 500 MHz and 100 or 125 MHz, respectively.Chemical shifts (δ) are reported in parts per millions (ppm) referenced to CHCl 3 at 7.26 ppm for 1 H and CDCl 3 at 77.0 ppm for 13 C and to CH 3 OH at 3.31 ppm for 1 H and CD 3 OD at 49.0 ppm.Coupling constants are reported in hertz (Hz), with the following abbreviations used: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet.When appropriate, the multiplicities are preceded with br, indicating that the signal was broad.High-resolution mass spectroscopy (HRMS) was performed using an Agilent 6230 TOF LC/MS system and the ESI-MS technique.(+)-ESIMS were recorded using an Agilent 1100 Series LC/MSD spectrometer.The PathHunter ® U2OS PD-L1/PD-L1 Dimerization Cell Line system was used to screen compounds that block PD-1/PD-L1 to enhance T cell response and mediate antitumor activity.Optical rotations were determined using a Jasco P-1020 polarimeter with a sodium lamp and are reported as follows: [α] D : (c g/100 mL, solvent).

Biological Material
A Cyanophycea, belonging to the Family Oscillatoriacea Engler (1898), was collected by hand using a Rebreather diving system in Buru, Indonesia (3 • 50.439'S/ 127 • 08.706' E) at depths ranging between 6 and 63 m.This biological material was identified as Lyngbya sp.A sample of the specimen was deposited in PharmaMar, Spain, with the reference code BURU-169.
Cells were seeded in 96-well microtiter plates at 5 × 10 3 cells per well in aliquots of 150 µL and allowed to attach to the plate surface for 18 h (overnight) in drug-free medium.After that, one control (untreated) plate of each cell line was fixed (as described below) and used for time zero reference value.Culture plates were then treated with test compounds (50 µL aliquots of 4× concentrated compound stock solutions made in complete culture medium) using ten serial dilutions (concentrations ranging from 10 to 0.000262 µg/mL) and triplicate cultures (final concentration of DMSO being 1%).After 72 h treatment, the antitumor effect was measured using the SRB methodology: briefly, cells were washed twice with PBS, fixed for 15 min in 1% glutaraldehyde solution at room temperature, rinsed twice in PBS and stained in 0.4% SRB solution for 30 min at room temperature.Cells were then rinsed several times with 1% acetic acid solution and air-dried at room temperature.SRB was then extracted in 10 mM trizma base solution and the absorbance measured in an automated spectrophotometric plate reader at 490 nm.Effects on cell growth and survival were estimated by applying the NCI algorithm [15].Doxorubicin and DMSO (solvent) were used as the positive and negative controls, respectively, in this assay.Prism 9.1.0.from GraphPad was used for the statistical analysis of the cell growth inhibition results.Using the mean ± SD of triplicates, a dose-response curve was automatically generated using nonlinear regression analysis to a 4-parameter logistic curve.Three reference parameters were calculated (NCI algorithm) by automatic interpolation: GI 50 = compound concentration that produces 50% cell growth inhibition, as compared to control cultures; TGI = total cell growth inhibition (cytostatic effect), as compared to control cultures and LC 50 = compound concentration that produces 50% net cell killing (cytotoxic effect).

PathHunter PD-1 Assay
A PD-1 signalling assay was used for quantifying SHP phosphatase recruitment to PD-1, using enzyme fragment complementation (EFC) technology.PD-1 harbours immunoreceptor tyrosine inhibitory motifs (ITIMs) in its cytoplasmic tail.When its ligand (PL-L1) binds to PD-1, the ITIM motif is phosphorylated, resulting in the recruitment of SHP-1 and SHP-2 (SH2-domain containing phosphatases), inhibiting the T-cell response.Full-length PD-1 receptor was engineered with a small β-gal fragment fused to its Cterminus, and the SH2-domain of SHP-1 was engineered with the complementing β-gal fragment.These constructs were stably expressed in Jurkat cells, while untagged full-length PD-L1 was stably expressed in U-2 OS cells (ligand-presenting cells).Ligand engagement, through co-culture with ligand-presenting cells, results in phosphorylation of PD-1 fusion protein, leading to the recruitment of SHP-1 which forces complementation of the EFC components to create an active β-gal enzyme.This active enzyme hydrolyzes substrate to create chemiluminescence as a measure of receptor activity.
A PrestoBlue cell proliferation assay was performed following the manufacturer's instructions (ThermoFisher scientific, Waltham, MA USA).Cellular viability was estimated from conversion of resazurin to its colored reaction product, resorufin.Absorbance was measured at 570 nm in a Perkin-Elmer EnVision reader.Cell survival was expressed as percentage of control cell growth.

Homogeneous Time-Resolved Fluorescence (HTRF) Binding Assay
The assay kit of PD-1/PD-L1 interaction (64ICP01PEG) was purchased from Cisbio (Shanghai, China).Binding assays were performed according to the manufacturer's instructions.The binding of Tag2-PD-1 and Tag1-PD-L1 was detected by anti-Tag1-EuK (HTRF donor) and anti-Tag2-XL665 (HTRF acceptor).Compounds blocking PD-1/PD-L1 complex formation reduce the HTRF signal.The inhibitory percentage was calculated following the instructions of the assay kit.Briefly, Tag2-PD-1 (25 nM final) and 1/3 serial dilutions PM170453 (ranging from 100 to 0.05 µM final), or 1/3 serial dilutions BMS-936559 (ranging from 4 to 0.0002 µM final), were added one after the other to a 384-well plate and incubated for 1 h at room temperature (RT).Then, Tag1-PD-L1 (5 nM final) was added for a total volume of 10 µL.After 1 h of incubation, detection reagent (10 µL) containing 1.83 nM anti-Tag1-EuK and 66.7 nM anti-Tag2-XL665 was added into the assay well.The signals were measured by a microplate reader (Perkin-Elmer EnVision) with the excitation wavelength located at 320 nm and the emission wavelength of 665 and 620 nm, respectively.The HTRF ratio = (665 nm/620 nm) was obtained on the microplate reader.
a in CD 3 OH; b under solvent.