Compound 1 was isolated as a yellowish powder. High-resolution electrospray ionization mass spectrometry (HRESIMS) (Figure ) revealed a formula of C49H54ClN5O10. The NMR spectrum showed signals for three methyls, a methoxy group, seven methylenes, nineteen methines. Numerous olefinic signals from several spin systems were further confirmed by COSY spectrum. The 13 C NMR spectrum showed the presence of six carbonyl signals corresponding to potential ester or amide moieties, which indicated 1 as a peptide. The interconnection between the six building blocks of 1 (a, b, c, d, and e) (Figure ) for 1 was deduced from 1 H-H COSY, and HMBC (Figure ). The partial structure a corresponded to the cinnamic acid derivative showing C NMR resonances at δC 165.9, 140.4, 135.9, 134. The partial structure b showed a typical chemical equivalence between the two methine doublets with 2H integration on the aromatic ring (δH 7.04 and 6.73), a methylene (δC 38.4, δH 2.83, 2.62) group with a 3 J correlation to the amide carbonyl δC 171.9 as a part of the peptide linkage to the adjacent amino acid. 2-Chloro-3-hydroxy-4methoxy-phenylalanine as partial structure c showed two doublets aromatic methines at (δH 6.51) indicated by 1 H -1 H COSY, while in the HMBC spectrum a methoxy group at (δH 3.57), methylene protons at (δH 3.10, 2.76) exhibited J coupling to the amide carbonyl at δC 171.9 (Figure ). Additionally, the N-methyl proton (δH 2.30) of partial structure d correlated to the C11 amide carbonyl indicated by the downfield shift δC 171.9 of the N-methylphenylalanine amino acid moiety. Finally, the positions of different peptide bonds were confirmed by key HMBC correlations. The methylene protons (δH 4.56 and 4.41) showed HMBC correlations with the ester linkage C21 (δC 171.2) confirmed the serine-glycine diketopiperazine residue (partial structure e); HMBC correlations of the CH2 protons (δH 4.04 and 3.92) of glycine with the carbonyl C-30 (δ 167.7) of serine; and the HMBC correlations of the C-32 methylene protons (δH 4.56 and 4.41) of serine with the carbonyl C-33 (δ 168.5) of glycine, alongside the MS/MS fragmentation pattern (Figure ) confirming the structure of 1 as pepticinnamin E. Elucidation of the absolute configuration was carried out by Marfey´s reaction and is described in the following section. Compound 2 was obtained as a colorless solid. HRESIMS (Figure ) analysis revealed a formula of C49H55N5O10, as a new analogue of pepticinnamin E with a loss of a chloride atom compared to pepticinnamin E. Indeed, 2 exhibited a similar 1 H NMR spectrum, except the appearance of one additional aromatic proton at δH 6.73 (H-19). In the C NMR spectrum, the chemical shift for C-19 appeared upfield (δC 116.3) compared to pepticinnamin E (δC 122.1). In addition, the position for the three aromatic protons was further confirmed by the coupling constant, an ortho-coupling J15-16 = 8.3 Hz, and a meta-coupling J15-19 = 2.1 Hz, respectively. The above data suggested the lack of chlorine-substitution at C-19. These results were confirmed by COSY, HSQC, H2BC and HMBC experiments (Figure ). Thus, compound 2 was identified as previously undescribed pepticinnamin N. Compound 3 was isolated as a second new analogue, a colourless solid and HRESIMS (Figure ) analysis confirmed a formula of C44H48ClN3O8, which indicated a shorter peptide chain compared to pepticinnamin E. MS/MS fragmentation (Figure ) indicated the loss of the Gly-Ser diketopiperazine moiety. This was further confirmed by 1 H NMR and C NMR spectra, where signals for the aromatic rings remained conserved, while the signals for the Gly-Ser diketopiperazine moiety disappeared. Compared to pepticinnamin E, the 1 H and C NMR spectra of compound 4 lacked signals attributed to the diketopiperazine moiety from the cyclization of serine and glycine residues. These data in combination with the COSY, HSQC, H2BC and HMBC experiments (Table and Figure ) identified pepticinnamin O. Compound 4 was isolated as an analogue of 3 and HRESIMS (Figure ) analysis confirmed a formula of C44H49N3O8 which indicated a loss of one chloride atom compared to pepticinnamin O. This was supported by an additional aromatic proton appearing at δH 6.70 in the 1 H NMR spectrum the chemical shift of C-19 with δC 122.0 in pepticinnamin O was upfield shifted to δ 116.2 (Table and. MS/MS fragmentation (Figure ) indicated a loss of the Gly-Ser diketopiperazine moiety. Thus, compound 4 was identified as a new pepticinnamin analogue named pepticinnamin N. Pepticinamins were first isolated in 1993 by Ōmura et al. from Streptomyces sp. OH-4652 and were found to act as natural protein farnesyltransferase (PFT) inhibitors with relative inhibitory potency (IC50) from 6-fold-to 60fold higher than that of synthetic peptides. Following these findings and considering the molecular structure of pepticinnamin E, Hinterding et al. took the initiative of determining whether pepticinnamin E acted as a monoor bisubstrate inhibitor of the PFT enzyme, thereby imitating the farnesyl group, the peptide substrate or both. In their study, Hinterding et al. chemically synthesized both diastereomers of pepticinnamin E, and showed them to actually act as competitive inhibitors with respect to both the peptide substrate (CAAX amino acid sequence of the Ras protein) and FPP. Furthermore, their study showed that terminal modifications (at the C-and N-terminals) to pepticinnamin E are of minor impact for the inhibition of farnesyl transfer and that the central amino acids and their absolute configuration are decisive for pepticinnamins' inhibitory activity. In the study by Ge et al., pepticinnamin G, which only differed from pepticinnamin E in the configuration of its first amino acid (L-tyrosine) and the type of its third amino acid (N-methyl-L-alanine), was tested for its biological activity in human cancer cell lines sensitive to Ras PFT inhibitors. The experiment showed no growth inhibition against these cancer cell lines at 10 μM. These results further confirmed the findings of Hinterding et al. on the decisiveness of the central amino acids and their absolute configuration on pepticinnamins' inhibitory activity. With pepticinnamins displaying a high degree of selectivity for the PFT enzyme and being the only naturally produced bisubstrate inhibitors of PFT, the structure of pepticinnamin E became a starting point for investigating additional anchor points that could be exploited to design more potent and selective antagonists. Furthermore, the demands for bisubstrate PFT inhibitors has driven Prof. Waldmann and a number of other research groups to initiate the synthesis of pepticinnamin E analogue libraries that could potentially meet these demands. In the study by Omura et al., it was found that pepticinnamin E showed no antimicrobial activity at a concentration of 1,000 μg/ml against various test microorganisms. The antimicrobial activity of compounds 1-4 was evaluated against the bacteria Staphylococcus aureus 8325, Bacillus subtilis and Pseudomonas aeruginosa PAO1 and yeast Candida albicans IBT 656 using a standard broth microdilution method. No antimicrobial activities were observed (MIC >50 µg/mL). RAS proteins are frequently mutated or dysregulated in various human solid tumors, and aberrant expression and activation of RAS proteins has been implicated in oncogenesis, tumor-cell invasion and metastasis. RAS function is dependent on its association with the cell membrane, which in turn requires a series of post-translational modifications, first and foremost attachment of a farnesyl isoprenoid by farnesyltransferase. As a result, natural inhibitors of FTase are potential anti-RAS drugs and, as such, have been considered interesting compounds for cancer treatment. We tested the effect of pepticinnamins E, O, and N on cell viability and proliferation of the LNCaP and C4-2B human epithelial prostate cancer cell lines (Figure ). Cells were grown in the presence of 10µM pepticinnamin E, O, or N for 72h. We found that only pepticinnamin O exhibited growth-inhibitory effects at this concentration, of 12.1% and 11.3% in LNCaP and C4-2B, respectively. Pepticinnamins E and N showed no significant effects on growth rates of LNCaP or C4-2B cells. Pepticinnamins E or N also had no significant cytotoxicity in LNCaP or C4-2B cells under the tested conditions -10µM concentration and 72h exposure. Exposure to pepticinnamin O caused a small decrease in cell viability (15.3%) in LNCaP cells but had no significant effect on C4-2B cells. We also evaluated the effect of pepticinnamin O on the multi-drug resistant sublines LNCaP R and C4-2B R , which overexpress the drug efflux pump P-glycoprotein (Pgp), and found it to exert cell growth inhibition in these cells, 25.7% and 12.8% in LNCaP R and C4-2B R , respectively, suggesting it can evade Pgpmediated multidrug resistance. The configuration of the DOPA derivative remained undefined until 1998, when Hinterding et al. performed a total synthesis of pepticinnamin E and its epimer thereby showing the second amino acid to possess an L-configuration after experimentally comparing the two epimers with the natural pepticinnamin E. In another study in 2020 by Ge et al., the marine Streptomyces sp. PKU-MA01144 was found to naturally produce three new analogues of pepticinnamin E, namely pepticinnamins G-I, and four other analogues, namely pepticinnamins J-M, from several constructed mutants of the natural producer. All possess an L-tyrosine configuration instead of a D-tyrosine configuration and an N-methyl-L-alanine amino acid instead of an N-methyl-L-phenylalanine, which are the two main structural features that distinct this group of analogues from pepticinnamin E (Figure ). In the current study the Marfey´s analysis method was used to conduct the absolute configuration of compound 1.