Synthesis and antiproliferative activity of culicinin D analogues containing simplified AHMOD-based residues

European Journal of Medicinal Chemistry 177 (2019) 235e246

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Synthesis and antiproliferative activity of culicinin D analogues containing simplified AHMOD-based residues Louise A. Stubbing a, b, 1, Iman Kavianinia a, b, 1, Maria R. Abbattista b, c, Paul W.R. Harris a, b, d, Jeff B. Smaill b, c, Adam V. Patterson b, c, Margaret A. Brimble a, b, d, * a

School of Chemical Sciences, The University of Auckland, 23 Symonds St, Auckland, 1010, New Zealand The Maurice Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Private Bag 92019, Auckland, 1010, New Zealand Auckland Cancer Society Research Centre, School of Medical Sciences, The University of Auckland, Private Bag 92019, Auckland, 1010, New Zealand d School of Biological Sciences, The University of Auckland, 3A Symonds St, Auckland, 1010, New Zealand b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 January 2019 Received in revised form 13 May 2019 Accepted 19 May 2019 Available online 23 May 2019

Culicinin D is a 10 amino acid peptaibol containing a rare and synthetically challenging (2S,4S,6R)AHMOD residue, that exhibits potent antiproliferative activity against MDA-MB-468 cells. An SAR study focusing on replacement of the AHMOD residue was undertaken, culminating in the revelation that a 6hydroxy or 6-keto substituent was essential to retain potent low nanomolar antiproliferative activity. © 2019 Elsevier Masson SAS. All rights reserved.

Keywords: Culicinin D AHMOD Peptaibol Toxin Cancer

1. Introduction Culicinin D (1) (Fig. 1) is a 10 amino acid peptaibol isolated from Culicinomyces clavisporus that exhibits anticancer activity against MDA-MB-468 cancer cells [1]. Typical of the peptaibol class of peptides, culicinin D contains several 2-aminoisobutyric acid (Aib, 2) residues, as well as an acylated proline N-terminus. Culicinin D also contains two unusual non-proteinogenic residues, namely (2S,4S,6R)-2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid

Abbreviations: 6-Cl-HOBt, 6-chloro-1-hydroxy-benzotriazole; AA, amino acid; COMU, 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholinomethylene)]methanaminium hexafluorophosphate; DIC, N,N0 -diisopropylcarbodiimide; DIPEA, N,N-diisopropylethylamine; FCS, fetal calf serum; FmocOSu, Fmoc-succinimide; HATU, 1-[bis(dimethylamino)-methylene]-1H-1,2,3triazolo[4,5-b]-pyridinium hexafluorophosphate 3-oxide; HFIP, 1,1,1,3,3,3hexafluoro-2-propanol; HMDS, hexamethyldisilazane; IBX, 2-iodoxybenzoic acid; TFA, trifluoroacetic acid; Trt, trityl; STR, short tandem repeat; FCS, fetal calf serum; DMEM, Dulbecco's Modified Eagle's medium; RPMI, Roswell Park Memorial Institute; alpha MEM, Minimum Essential Medium Eagle Alpha Modification. * Corresponding author. School of Chemical Sciences, The University of Auckland, 23 Symonds St, Auckland, 1010, New Zealand. E-mail address: [email protected] (M.A. Brimble). 1 Authors contributed equally. https://doi.org/10.1016/j.ejmech.2019.05.052 0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.

(AHMOD, 3), and (2S,4R)-2-amino-4-methyldecanoic acid (AMD, 4), as well as the reduced C-terminal diamino alcohol (S)-2-(2aminopropylamino)ethanol (APAE, 5). The AHMOD residue, in particular, has presented a significant obstacle to synthesis of 1 and other AHMOD-containing peptaibols due to the difficulty of synthesis in addition to the chemical sensitivity of the b-hydroxyketone motif [2e5]. Our group has since reported a much improved synthesis of this residue [6], as well as improved protocols for synthesis of AHMOD-containing peptaibols themselves [7e9]. In this study we aim to develop a replacement for the AHMOD residue that improves chemical stability by removing the sensitive b-hydroxyketone motif, as well as simplifying synthesis of this building block. We also report the third-generation total synthesis of culicinin D using a strategy that employs a combination of both solid- and solution-phase synthesis. The versatility of this synthetic methodology was established by the synthesis of 1 and its epimer, as well as synthesis of sixteen novel analogues containing a simplified AHMOD residue. A structure-activity relationship (SAR) based on the AHMOD residue in these peptaibols is also established.

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Fig. 1. Structure of culicinin D and unusual building blocks contained therein.

2. Results and discussion 2.1. Synthesis of AHMOD analogues A series of simplified AHMOD analogues (6e14, Fig. 2) were designed to evaluate the effect of the (4S)-methyl, (6R)-hydroxy, and 8-keto substituents. Analogues 6e8 were available from key aldehydes 15 or 16, while 4-desmethyl analogues 9e14 were

available from aldehydes 17 or 18. Key aldehydes 15e18 were prepared from L-glutamic acid as described previously [6,10]. Thus, 8-deoxy analogues 6 and 7 were prepared from key aldehyde 15 via n-butyl Grignard addition, giving 19 and 20 as a 1:1 mixture of separable diastereomers (Scheme 1; stereochemistry at C6 determined via Mosher's ester analysis). Removal of the N-Boc protecting groups and replacement with Fmoc, followed by hydrolysis of the methyl ester afforded Fmoc-SPPS building blocks 6

Fig. 2. Simplified AHMOD analogues 6e14.

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Scheme 3. Synthesis of Fmoc-protected 4-desmethyl alkyl analogues 13 and 14. Reagents and conditions a) n-C3H7PPh3I, or n-C7H15PPh3Br, LiHMDS, THF, 40  C, 6e18 h; b) Pd(OH)2/C, H2, MeOH, rt, 18 h, 22 59%, 23 42% over 2 steps; c) (i) TFA/CH2Cl2 (1:1), rt, 0.5e1 h; (ii) Fmoc-OSu, aq. NaHCO3/1,4-dioxane (1:1), rt, 18 h; d) NaOH, 0.8 M CaCl2/iPrOH (3:7), rt, 6e8 h, 13 34%, 14 38% over 2 steps.

Scheme 1. Synthesis of Fmoc-protected 8-deoxy analogues 6 and 7. Reagents and conditions: a) n-BuMgBr, Et2O, 78  C, 1 h, 19 18%, 20 21%; b) (i) TFA/CH2Cl2, rt, 0.5e1 h; (ii) Fmoc-OSu, aq. NaHCO3/1,4-dioxane (1:1), rt, 18 h; c) NaOH, 0.8 M CaCl2/iPrOH (3:7), rt, 6e8 h, 6 65%, 7 66% over 2 steps.

and 7. (4R)-Methyl alkyl analogue 8 was prepared via Wittig olefination of aldehyde 16 (Scheme 2), followed by hydrogenation to give alkane 21. Treatment of N-Boc-protected methyl ester 21 with TFA, followed by Fmoc protection and subsequent hydrolysis of the methyl ester, afforded the corresponding N-Fmoc-protected amino acid 8 in good yield. Des-methyl aldehyde 18 was prepared from L-glutamic acid in a similar manner to aldehyde 16 [10] (Scheme 3). 4-Desmethyl alkyl analogues 13 and 14 were then prepared from 18 in an analogous manner to 8 (Scheme 2), via Wittig olefination, followed by hydrogenation to give alkane intermediates 22 and 23. The N-protecting groups were then adjusted in the same manner as earlier analogues, and subsequent ester hydrolysis afforded building blocks 13 and 14. The remainder of the AHMOD analogues 9e12 were prepared from aldehyde 17 [10] (Scheme 4). Aldol reaction of aldehyde 17 with 2-butanone afforded b-hydroxyketone 24 as a 1:1 mixture of

Scheme 2. Synthesis of Fmoc-protected (4R)-methyl alkyl analogue 8. Reagents and conditions: a) n-C7H15PPh3Br, LiHMDS, THF, 40  C to rt, 6e18 h; b) Pd(OH)2/C, H2, MeOH, rt, 18 h, 46% over 2 steps; c) (i) TFA/CH2Cl2, rt, 0.5e1 h; (ii) Fmoc-OSu, aq. NaHCO3/1,4-dioxane (1:1), rt, 18 h; d) NaOH, 0.8 M CaCl2/iPrOH (3:7), rt, 6e8 h, 70% over 2 steps.

diastereomers. Exchange of the N-Boc protecting group for N-Fmoc, followed by hydrolysis of the methyl ester afforded des-methyl analogue 9 in good yield. Alternatively, treatment of aldehyde 17 with either n-butyl- or n-hexylmagnesium bromide gave 25 and 26 respectively, as inseparable mixtures of alcohols. After protecting group manipulation of 25, and subsequent ester hydrolysis, 12 was obtained as an inseparable 1:1 mixture of diastereomers. Alternatively, oxidation of 27 and 28 with IBX, followed by ester hydrolysis afforded 6-keto analogues 10 and 11 in good yield. 2.2. SPPS incorporation of AHMOD analogues into peptaibol In 2012, our group reported the first total synthesis of anticancer peptaibol culicinin D employing an Fmoc-based solid phase peptide synthesis (SPPS) strategy that enabled incorporation of the highly acid-sensitive b-hydroxyketone AHMOD residue [7]. In our first approach towards tackling this molecule, we anchored the hydroxyl group of Fmoc protected (S)-APAE to 2-chlorotrityl-functionalised polystyrene (2-ClTrt) resin 29 (Scheme 5, first generation). Upon completion of the synthesis, the final product was obtained in low yield (<1% yield) mainly due to the inefficient attachment of Fmoc-protected APAE to 2-ClTrt resin (30, loading of 23%). Therefore a second synthetic route was developed to overcome this problematic step, which involved attachment of the amino group of the C-terminal aminoalcohol to the resin rather than the less nucleophilic hydroxyl group [8] (Scheme 5, 31, second generation). The synthetic peptide 32 obtained using this method contains a C-terminal amino group, which spontaneously undergoes OeN intramolecular migration in neutral or basic conditions following cleavage from the resin to give the desired culicinin D in 6% overall yield. Although a significant improvement on the first generation synthesis, the overall yield remained relatively low, and a more efficient synthesis was required to enable the synthesis of analogues. Herein we describe a new combined solid- and solution-phase strategy that was employed for the third generation synthesis of culicinin D, similar to that used for synthesis of the closely related peptaibol trichoderin A [9,11]. Importantly, this method could be easily adapted for synthesis of culicinin D analogues by simple substitution of the AHMOD residue with building blocks 6e14. The third-generation total synthesis of culicinin D began with loading of Fmoc-b-Ala-OH (rather than Fmoc-APAE) onto 2-ClTrt resin to give 34 (1.4 mmol/g loading, Scheme 6). The synthesis was then continued with removal of the Fmoc group using 20% piperidine in DMF, followed by use of the previously optimised conditions with HATU and DIPEA as a coupling reagent to give 35. A mixture of the uronium type coupling reagent COMU and 2-cyano2-(hydroxyimino)acetate (Oxyma) was used for coupling of the (2S,4R)-AMD building block to form peptidyl resin 36. One drawback of previous approaches was incomplete coupling between the two sterically hindered Aib residues. This problem was overcome

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Scheme 4. Synthesis of Fmoc-protected 4-desmethyl analogues 9e12. Reagents and conditions: a) 2-butanone, n-BuLi, THF, 78  C, 3.5 h, 68%; b) (i) TFA/CH2Cl2 (1:4), rt, 0.5e1 h; (ii) Fmoc-OSu, 10% aq. NaHCO3/1,4-dioxane (1:1), rt, 18 h; c) NaOH, 0.8 M CaCl2, iPrOH/H2O (7:3), rt, 6e8 h, 9 47% over 2 steps; 10 86%, 11 75%, 12 87%; d) n-BuMgBr, Et2O, 78  C, 3 h, 25 40%, or n-C6H13MgBr, Et2O, 78  C, 3 h, 26 35%; e) IBX, DMSO, rt, 18 h, 90%.

using a combination of COMU/Oxyma/DIPEA for 2 h. The coupling was repeated to ensure complete reaction of the amino acid. Fmocprotected (2S,4S,6R)-AHMOD was effectively coupled to the growing peptidyl resin 37 using HATU/DIPEA (without protection of the C6 hydroxyl group) to form 38. Finally, Fmoc-Pro-OH was coupled to the resin using HATU/DIPEA and subsequently N-acylated with butyric acid using HATU/DIPEA to complete the synthesis of linear peptidyl resin 39. For the release of peptide from the solid support a mixture of 1,1,1,3,3,3-hexafluoropropan-2-ol (HFIP)/ CH2Cl2 (1:4, v/v) was employed to prevent undesired acidcatalysed elimination of the b-hydroxyketone moiety of the AHMOD residue. Analysis of the crude product by LC-MS confirmed the presence of the desired peptide 40 as the major product. The lyophilised crude peptide was then purified by semi-preparative RP-HPLC using 0.1% formic acid to afford peptide 40 in 60% yield based on 0.1 mmol scale. Pleasingly, use of mild formic acid instead of 0.1% TFA suppressed elimination of the b-hydroxyketone to the enone byproduct, which was previously observed during purification of culicinin D. The resulting peptide 40 was then subjected to solution phase C-terminal installation of the APAE moiety using N,N0 -diisopropylcarbodiimide/6-chloro-1-hydroxybenzotriazole in DMF for 12 h. We envisaged that the amino group of APAE would react faster than the alcohol with the carboxylic acid of peptide 40. Fortuitously, given that the C-terminal amino acid in peptide 40 is achiral, racemization is not a concern. Subsequent purification of the crude reaction mixture by RP-HPLC afforded 26 mg (21% yield based on 0.1 mmol scale) of culicinin D (1) (>98% purity). Having established a more efficient synthetic strategy to obtain

culicinin D, simplified AHMOD analogues 6e14 together with a selection of commercially available Fmoc-protected amino acids (leucine, O-tert-butyl-serine, O-trityl-homoserine, 6hydroxynorleucine, and ε-Boc-lysine) were then individually incorporated into the synthetic strategy to prepare AHMODsubstituted culicinin D analogues 41e55 in multimilligram quantities and >98% purity. 2.3. Biological evaluation of culicinin D analogues Culicinin D (1) and the peptide analogues 41e55 were assessed for antiproliferative activity against three breast cancer cell lines (MDA-MB-468, SKBR3, and T47D), as well as a non-small cell lung cancer line NCI-H460 (Table 1). Peptides were dissolved in DMSO (30 mM stock solution) and cells were treated with 3-fold serial dilutions of the peptides and maintained under drug-exposure for 5 days. Monitoring of cell proliferation and cell viability was performed by sulphorhodamine B-based assay as previously described [12]. The IC50 was determined by interpolation as the drug concentration reducing staining to 50% of controls on the same plate. In comparison to culicinin D (1, entry 1), it was found that changing the stereochemistry of the C6 hydroxy group (i.e. (6S)AHMOD analogue 41, entry 2) generally made little difference to the antiproliferative activity of the peptide. Conversely, removal of the (4S)-methyl group (i.e. analogue 46, entry 7) resulted in a loss of antiproliferative activity across all 4 cell lines tested, and was most apparent in the NCI-H460 cell line (38-fold for 46 vs 1). Surprisingly, removal of the C8 keto functionality (47 and 48, entries 8, 9) had a detrimental effect, whilst the analogue lacking

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Scheme 5. Summary of first and second generation syntheses of culicinin D 1 [7,8].

both the C8 ketone as well as the (4S)-methyl group largely appeared to retain the activity of the natural product (49, entry 10). Concomitant removal of keto and hydroxy substituents resulted in a loss of activity (43e45, entries 4, 5, 6), although this was somewhat ameliorated by longer chain lengths (e.g. 43 vs 44 and 45). It was notable that of this subset, the derivative retaining the (4S)-methyl group (44) was generally the most active across the 4 cell lines tested. A series of analogues bearing hydroxy functionality (52e54, entries 13e15) suggest that the position of the substituent on the sidechain is important, with antiproliferative activity improving as the hydroxy group is positioned further out from the peptide backbone. Indeed, the most potent of these analogues (54) places the hydroxyl group 4 carbons from the peptide backbone, the same relative distance as that of culicinin D itself. A basic sidechain (e.g. analogue 55, entry 16) was not tolerated at all, resulting in drastic loss of activity in all cell lines tested. Refinement of our most potent lead at this point, alcohol 49 (an inseparable diastereomeric mixture), led to 6-keto analogues 50 and 51 of varying chain length (entries 11 and 12). Fortuitously, removal of stereochemistry at C6 appeared to be beneficial, and analogues 50 and 51 were found to be similarly potent to the natural product across all cell lines tested.

3. Conclusion We have reported a new method employing both solid- and solution-phase synthesis for the total synthesis of culicinin D (1). We have taken advantage of the versatility of this synthetic method to design and prepare a series of peptides based on the culicinin D framework, with structurally simpler building blocks in place of (2S,4S,6R)-AHMOD (3). Evaluation of the antiproliferative activity of these analogues against a panel of cancer cell lines revealed that the C6-hydroxy group was important for the potent low nanomolar antiproliferative activity of the natural product. Interestingly, the C8-keto substituent and the (4S)-methyl group appeared to have a more mixed influence on activity. Here examples most closely related to culicinin D that retained the (4S)-methyl group lost potency when the C8-keto group was removed, whilst analogues further removed structurally from culicinin D lacking the (4S)methyl group displayed good potency despite the concomitant lack of a C8-keto group. This finding allowed development of lead analogues, namely ketones 50 and 51, which largely retain the potent antiproliferative activity of the natural product. Importantly, the corresponding building blocks 10 and 11 are far more synthetically tractable than (2S,4S,6R)-AHMOD (3), and further, enable more

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Scheme 6. Hybrid solid phase/late-stage solution phase synthesis of culicinin D and analogues. Reagents and conditions: a) Fmoc-bAla-OH, DIPEA, CH2Cl2, 1  6 h, 1  12 h; b) CH2Cl2/MeOH/DIPEA (8:1.5:0.5, v/v); c) 20% piperidine/DMF, 1  5 min, 1  15 min; d) Fmoc-AA-OH, HATU, DIPEA, DMF, 2  1 h; e) Fmoc-AMD-OH, COMU, Oxyma, DIPEA, DMF, 3 h; f) Fmoc-Aib-OH, COMU, Oxyma, DIPEA, DMF, 2  2 h; g) Fmoc-AA-OH, HATU, DIPEA, DMF, 2 h; h) butyric acid, HATU, DIPEA, DMF, 2  1 h; i) HFIP/CH2Cl2 (1:4), 0.5 h or TFA/TIPS/H2O (95:2.5:2.5, v/v/v), 2 h for 52, 53 and 55; j) APAE, DIC, 6-Cl-HOBt, DMF, 12 h. Fmoc-AA-OH ¼ Fmoc-(R)-AHMOD-OH, Fmoc-(S)-AHMOD-OH, Fmoc-Ser(OtBu)-OH, Fmoc-Hse(OTrt)OH, Fmoc-Nle(6-OH)-OH, Fmoc-Lys(Boc)-OH, or building blocks 6e14.

efficient assembly of the peptaibol skeleton due to the lack of a sensitive b-hydroxyketone that affords undesired elimination byproducts. Further refinement of analogues and work to uncover the mechanism of action of these potent anticancer peptides is currently underway.

and stirred until the reaction was complete by tlc analysis. Sat. aq. NH4Cl was added, and the mixture extracted three times with ethyl acetate. The combined organic extracts were dried over MgSO4 and concentrated under reduced pressure to give the crude products which were purified via flash column chromatography using the eluent indicated.

4. Experimental section 4.3. General procedure B for hydrogenation 4.1. Chemistry (2S,4S,6R)- and (2S,4S,6S)-Fmoc-AHMOD, (2S,4R)-Fmoc-AMD, APAE, and aldehyde 15 were prepared as detailed previously [6,9]. Aldehydes 16e18 were prepared from L-glutamic acid or L-2aminoadipic acid according to the method described in the literature by Martín et al. [10]. Solvents were dried by passage over alumina. Reactions were monitored by thin layer chromatography (tlc) on silica gel plates, and visualised using UV light and/or staining with ninhydrin. Flash column chromatography was carried out using Davisil 40e60 mm silica. The optical rotations of pure compounds were obtained using an Autopol IV automatic polarimeter using a 100 mm path length cell. NMR spectra were recorded on a Bruker 400 or 500 MHz instrument at room temperature, and chemical shifts are reported in parts per million (ppm) and calibrated to tetramethylsilane (0 ppm) as an internal standard in 1 H spectra, and residual solvent (77 ppm) in 13C spectra. Multiplicities are reported as follows: br ¼ broad, s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet, dd ¼ doublet of doublet, dt ¼ doublet of triplets, ddd ¼ doublet of doublet of doublets. Highresolution mass spectra (HRMS) were obtained using a Bruker microTOF-Q II mass spectrometer operating at a nominal accelerating voltage of 70 eV in ESIMS mode. 4.2. General procedure A for Wittig olefination LiHMDS (1.0 M in THF, 1.8 eq) was added slowly to a suspension of the Wittig salt (2 eq.) in THF (60 mL/mmol aldehyde), at 40  C, and the mixture allowed to stir at 40  C for 1 h. A solution of the aldehyde (1 eq) in THF (20 mL/mmol) was added slowly, and the mixture allowed to stir at 40  C for 2 h, then allowed to warm to rt

A solution of the alkene (1 eq.) in methanol (10 mL/mmol) was stirred in the presence of catalytic Pd(OH)2/C under an atmosphere of H2 for 18 h. The mixture was filtered through Celite and concentrated under reduced pressure to give the crude products which were purified via flash column chromatography using the eluent indicated. Ester 21: prepared in two steps according to general procedure A to give the alkene 56 in 51% yield as a colourless oil (ethyl acetate/ 1 pet. ether 1:19 as eluent). a23 D ¼ 38.9 (c 0.054, MeOH); H NMR (400 MHz, CDCl3): d 5.33 (dt, J ¼ 8.1, 15.2 Hz, 1H), 5.17e4.99 (m, 1H), 4.84 (t, J ¼ 6.9 Hz, 1H), 3.70 (s, 3H), 2.73e2.62 (m, 1H), 2.21e2.14 (m, 1H), 2.07e1.87 (m, 2H), 1.66e1.58 (m, 1H), 1.49 (s, 18H), 1.34e1.21 (m, 8H), 0.98 (d, J ¼ 6.6 Hz, 3H), 0.87 (t, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 171.6 (C), 152.0 (2  C), 134.9 (CH), 129.8 (CH), 82.9 (2  C), 56.5 (CH), 52.0 (CH3), 38.1 (CH2), 31.8 (CH2), 29.8 (CH2), 29.04 (CH), 28.99 (CH2), 28.0 (6  CH3), 27.4 (CH2), 22.6 (CH2), 21.0 (CH3), 14.1 (CH3); HRMS (ESIþ): m/z 464.2978 [M þ Na]þ (calcd for C24H43NO6Naþ 464.2983); followed by hydrogenation according to general procedure B to give 21 in 90% yield as a colourless oil (ethyl 1 acetate/pet. ether 1:19 as eluent). a24 D ¼ 31 (c 0.1, MeOH); H NMR (400 MHz, CDCl3): d 4.95 (dd, J ¼ 4.4, 10.5 Hz, 1H), 3.71 (s, 3H), 2.01 (ddd, J ¼ 3.5, 10.4, 14.1 Hz, 1H), 1.78 (ddd, J ¼ 4.5, 10.1, 14.5 Hz, 1H), 1.50 (s, 18H), 1.43e1.33 (m, 1H), 1.25 (br s, 14H), 0.91 (d, J ¼ 6.5 Hz, 3H), 0.88 (t, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 172.0 (C), 152.2 (2  C), 82.9 (2  C), 56.3 (CH), 52.1 (CH3), 37.6 (CH2), 36.6 (CH2), 31.9 (CH2), 29.9 (CH2), 29.61 (CH2), 29.59 (CH2), 29.3 (CH), 28.0 (6  CH3), 27.1 (CH2), 22.7 (CH2), 19.4 (CH3), 14.1 (CH3); HRMS (ESIþ): m/z 466.3124 [M þ Na]þ (calcd for C24H45NO6Naþ 466.3139). Ester 22: prepared in two steps according to general procedure

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Table 1 Antiproliferative activity of culicinin D (1) and analogues 41e55 against cancer cell lines MDA-MB-468, SKBR3, T47D, and NCI-H460. Analogues of culicinin D with general structure:

Entry

AA, analogue number

1

Activity against cancer cell line IC50 (mM) ± SEM MDA-MB-468

SKBR3

T47D

NCI-H460

0.008 ± 0.0005

0.032 ± 0.0025

0.005 ± 0.0006

0.005 ± 0.0007

0.011 ± 0.001

0.035 ± 0.0008

0.007 ± 0.0001

0.008 ± 0.001

0.09 ± 0.04

0.28 ± 0.06

0.07 ± 0.01

0.13 ± 0.12

0.14 ± 0.01

0.24 ± 0.06

0.17 ± 0.02

0.19 ± 0.09

0.040 ± 0.022

0.236 ± 0.125

0.031 ± 0.005

0.037 ± 0.005

0.059 ± 0.008

0.280 ± 0.011

0.232 ± 0.107

0.382 ± 0.259

0.07 ± 0.02

0.50 ± 0.05

0.08 ± 0.02

0.19 ± 0.01

0.297 ± 0.145

>0.5

0.264 ± 0.112

0.234 ± 0.081

0.111 ± 0.045

0.327 ± 0.013

0.197 ± 0.061

0.126 ± 0.036

0.02 ± 0.01

0.10 ± 0.01

0.008 ± 0.007

0.008 ± 0.003

0.010 ± 0.001

0.03 ± 0.01

0.010 ± 0.002

0.010 ± 0.001

0.008 ± 0.0007

0.018 ± 0.0022

0.006 ± 0.0004

0.005 ± 0.0005

0.074 ± 0.009

>0.5

0.066 ± 0.010

0.127 ± 0.011

0.03 ± 0.01

0.108 ± 0.03

0.02 ± 0.01

0.03 ± 0.01

, 1 (culicinin D) 2

, 41 3

, 42 4

, 43 5

, 44 6

, 45 7

, 46 8

, 47 9

, 48 10

, 49 11

, 50 12

, 51 13 , 52 14

, 53 (continued on next page)

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Table 1 (continued ) Analogues of culicinin D with general structure:

Entry

AA, analogue number

15

Activity against cancer cell line IC50 (mM) ± SEM MDA-MB-468

SKBR3

T47D

NCI-H460

0.025 ± 0.004

0.059 ± 0.014

0.014 ± 0.005

0.028 ± 0.002

>1.0

>1.0

>1.0

>1.0

, 54 16

, 55

A to give the alkene 57 in 69% yield as a pale yellow oil (ethyl ac1 etate/pet. ether 1:19 as eluent). a24 D ¼ 30.4 (c 0.023, MeOH); H NMR (400 MHz, CDCl3): d 5.45e5.29 (m, 2H), 4.87 (dd, J ¼ 4.8, 8.9 Hz, 1H), 3.71 (s, 3H), 2.22e1.86 (m, 6H), 1.50 (s, 18H), 0.95 (t, J ¼ 7.5 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 171.4 (C), 152.1 (2  C), 132.8 (CH), 127.5 (CH), 83.0 (2  C), 57.7 (CH), 52.1 (CH3), 30.2 (CH2), 28.0 (6  CH3), 23.9 (CH2), 20.5 (CH2), 14.2 (CH3); HRMS (ESIþ): m/z 394.2195 [M þ Na]þ (calcd for C19H33NO6Naþ 394.2200); followed by hydrogenation according to general procedure B to give 22 in 85% yield as a colourless oil (ethyl acetate/pet. ether 1:19 as eluent). 1 a22 D ¼ 32.2 (c 0.73, CHCl3); H NMR (400 MHz, CDCl3): d 4.85 (dd, J ¼ 5.1, 9.6 Hz, 1H), 3.71 (s, 3H), 2.13e2.04 (m, 1H), 1.93e1.83 (m, 1H), 1.50 (s, 18H), 1.36e1.24 (m, 8H), 0.88 (t, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 171.5 (C), 152.1 (2  C), 82.9 (2  C), 58.1 (CH), 52.1 (CH3), 31.6 (CH2), 29.8 (CH2), 28.9 (CH2), 28.0 (6  CH3), 26.1 (CH2), 22.5 (CH2), 14.0 (CH3); HRMS (ESIþ): m/z 396.2354 [M þ Na]þ (calcd for C19H35NO6Naþ 396.2357). Ester 23: prepared in two steps according to general procedure A to give the alkene 58 in 63% yield as a pale yellow oil (ethyl acetate/pet. ether 1:19 as eluent). 1H NMR (400 MHz, CDCl3): d 5.44e5.32 (m, 2H), 4.89e4.85 (dd, J ¼ 4.8, 8.9 Hz, 1H), 3.71 (s, 3H), 2.22e2.04 (m, 3H), 2.00 (q, J ¼ 6.6 Hz, 2H), 1.95e1.87 (m, 1H), 1.50 (s, 18H), 1.34e1.26 (m, 8H), 0.88 (t, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 171.4 (C), 152.1 (2  C), 131.3 (CH), 128.1 (CH), 83.0 (2  C), 57.7 (CH), 52.1 (CH3), 31.8 (CH2), 30.1 (CH2), 29.6 (CH2), 29.0 (CH2), 28.0 (6  CH3), 27.2 (CH2), 24.0 (CH2), 22.6 (CH2), 14.1 (CH3); HRMS m/z (ESIþ): 450.2814 [M þ Na]þ (calcd for C23H41NO6Naþ 450.2826); followed by hydrogenation according to general procedure B to give 23 in 66% yield as a colourless oil (ethyl 1 acetate/pet. ether 1:19 as eluent). a24 D ¼ 29.4 (c 0.085, MeOH); H NMR (400 MHz, CDCl3): d 4.85 (dd, J ¼ 5.1, 9.6 Hz, 1H), 3.71 (s, 3H), 2.13e2.04 (m, 1H), 1.92e1.83 (m, 1H), 1.50 (s, 18H), 1.34e1.25 (m, 16H), 0.88 (t, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 171.6 (C), 152.1 (2  C), 82.9 (2  C), 58.1 (CH), 52.1 (CH3), 31.9 (CH2), 29.9 (CH2), 29.6 (CH2), 29.54 (CH2), 29.45 (CH2), 29.31 (CH2), 29.25 (CH2), 28.0 (6  CH3), 26.2 (CH2), 22.7 (CH2), 14.1 (CH3); HRMS (ESIþ): m/z 452.2981 (cacld for C23H43NO6Naþ 452.2983). 4.4. General procedure C for Grignard addition The Grignard reagent (2 eq.) was added slowly to a solution of the aldehyde (1 eq.) in diethyl ether (15 mL/mmol) at 0  C, and the mixture allowed to stir for 2 h. An equal volume of sat. aq. NH4Cl was added, and the organic layer removed. The aqueous layer was further extracted twice with ethyl acetate and the combined organic extracts were dried over MgSO4 and concentrated under

reduced pressure to give the crude material. The crude was adsorbed onto Celite and purified twice via automated flash column chromatography (silica gel, ethyl acetate/pet. ether 1:9 to 1:3 gradient over 40 min). (S)-Alcohol 19 and (R)-alcohol 20: prepared according to general procedure C from aldehyde 15 in 18% and 21% yield, respectively, as a separable mixture of colourless oils. 19: a26 D ¼ 33.7 (c 3.33, CHCl3); 1H NMR (400 MHz, CDCl3): d 4.97 (dd, J ¼ 4.4, 10.6 Hz, 1H), 3.72 (s, 3H), 3.71e3.65 (m, 1H), 2.17 (ddd, J ¼ 2.6, 11.0, 13.6 Hz, 1H), 1.77e1.63 (m, 2H), 1.50 (s, 18H), 1.44e1.26 (m, 8H), 0.96 (d, J ¼ 6.3 Hz, 3H), 0.90 (t, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 171.8 (C), 152.6 (2  C), 83.1 (2  C), 69.1 (CH), 56.2 (CH), 52.2 (CH3), 45.2 (CH2), 37.4 (CH2), 35.8 (CH2), 27.9 (6  CH3), 27.8 (CH2), 26.1 (CH), 22.7 (CH2), 20.1 (CH3), 14.0 (CH3); HRMS (ESIþ): m/z 454.2779 [M þ Na]þ (calcd for C22H41NO7Naþ 454.2775); 20: 1 a21 D ¼ 25.7 (c 0.9, CHCl3); H NMR (400 MHz, CDCl3): d 4.96 (dd, J ¼ 5.3, 9.1 Hz, 1H), 3.71 (s, 3H), 3.70e3.64 (m, 1H), 2.00e1.88 (m, 2H), 1.74e1.65 (m, 1H), 1.50 (s, 18H), 1.46e1.38 (m, 4H), 1.36e1.24 (m, 4H), 0.96 (d, J ¼ 6.5 Hz, 3H), 0.90 (t, J ¼ 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 171.7 (C), 152.2 (2  C), 82.9 (2  C), 69.4 (CH), 56.2 (CH), 52.1 (CH3), 45.2 (CH2), 37.8 (CH2), 37.5 (CH2), 27.9 (6  CH3), 27.7 (CH2), 26.5 (CH), 22.7 (CH2), 18.9 (CH3), 14.0 (CH3); HRMS (ESIþ): m/z 454.2786 [M þ Na]þ (calcd for C22H41NO7Naþ 454.2775). Alcohol 25: prepared according to general procedure C in 40% yield as a colourless oil and a 1:1 mixture of diastereomers. 1H NMR (400 MHz, CDCl3): d 4.86 (dd, J ¼ 5.2, 9.4 Hz, 1H), 3.71 (s, 3H), 3.63e3.56 (m, 1H), 2.19e2.07 (m, 1H), 1.98e1.84 (m, 1H), 1.54e1.27 (m, 28H), 0.90 (t, J ¼ 7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3, * denotes diastereomer peaks): d 171.4 (C), 152.2* (2  C), 152.17* (2  C), 83.0 (2  C), 71.6 (CH), 58.0 (CH), 57.9* (CH), 52.1 (CH3), 37.2 (CH2), 37.1* (CH2), 37.0 (CH2), 36.9* (CH2), 29.85 (CH2), 29.78* (CH2), 28.0 (6  CH3), 27.8 (CH2), 22.7 (CH2), 22.3 (CH2), 22.2*(CH2), 14.0 (CH3); HRMS m/z (ESIþ) 440.2620 [M þ Na]þ (calcd for C21H39NO7Naþ 440.2619). Alcohol 26: prepared according to general procedure C in 35% yield as a colourless oil and a 1:1 mixture of diastereomers. 1H NMR (CDCl3, 400 MHz): d 4.86 (dd, J ¼ 5.2, 9.4 Hz, 1H), 3.71 (s, 3H), 3.62e3.56 (m, 1H), 2.20e2.06 (m, 1H), 1.97e1.84 (m, 1H), 1.55e1.37 (m, 26H), 1.34e1.24 (m, 6H), 0.88 (t, J ¼ 6.4 Hz, 3H); 13C NMR (CDCl3, 100 MHz, * denotes diastereomer peaks): d 171.4 (C), 152.22 (2  C), 152.17* (2  C), 83.0 (2  C), 71.6 (CH), 58.00 (CH), 57.93* (CH), 52.1 (CH3), 37.5 (CH2), 37.4* (CH2), 37.0 (CH2), 36.9* (CH2), 31.8 (CH2), 29.84 (CH2), 29.78* (CH2), 29.3 (CH2), 28.0 (6  CH3), 25.6 (CH2), 22.6 (CH2), 22.3 (CH2), 22.2* (CH2), 14.0 (CH3); HRMS m/z (ESIþ) 468.2928 [M þ Na]þ (calcd for C23H43NO7Naþ 468.2932).

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b-Hydroxyketone 24: n-BuLi (1.4 M in cyclohexane, 1.26 mL, 1.77 mmol) was added dropwise to a solution of diisopropylamine (250 mL, 1.77 mmol) in THF (30 mL) at 78  C. The mixture was allowed to warm to 0  C for 20 min, then recooled to 78  C. 2Butanone (145 mL, 1.61 mmol) was added dropwise, followed by a solution of the aldehyde 17 (579 mg, 1.61 mmol) in THF (10 mL). The mixture was allowed to stir at 78  C for 3.5 h, then quenched with sat. aq. NH4Cl (40 mL) and allowed to warm to rt. The mixture was extracted with ethyl acetate (3  30 mL), and the combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure to give the crude as a pale yellow oil. The crude was purified via flash column chromatography (silica gel, ethyl acetate/pet. ether 1:4 as eluent) to give 24 (476 mg, 68%) as a pale yellow oil and a 1:1 mixture of diastereomers. 1H NMR (400 MHz, CDCl3, * denotes diastereomer peaks): d 4.85 (dd, J ¼ 5.2, 9.4 Hz, 1H), 4.07e4.00 (m, 1H), 3.71 (s, 3H), 3.05 (br s, 0.5H), 3.00* (br s, 0.5H), 2.62e2.42 (m, 4H), 2.16e2.06 (m, 1H), 1.96e1.84 (m, 1H), 1.60e1.36 (m, 22H), 1.06 (t, J ¼ 7.4 Hz, 3H); 13C NMR (100 MHz, CDCl3, * denotes diastereomer peaks): d 212.6 (C), 171.4 (C), 152.2 (2  C), 83.1 (2  C), 67.4 (CH), 67.2* (CH), 58.0 (CH), 52.1 (CH3), 48.5 (CH2), 36.8 (CH2), 36.0 (CH2), 35.9* (CH2), 29.8 (CH2), 29.7* (CH2), 28.0 (6  CH3), 22.3 (CH2), 22.1* (CH2), 7.5 (CH3); HRMS (ESIþ): m/z 454.2416 [M þ Na]þ (calcd for C21H37NO8Naþ 454.2411). 4.5. General procedure D for Boc-deprotection/Fmoc-protection of amines The Boc-protected amine (1 eq.) was stirred in a solution of trifluoroacetic acid/dichloromethane (1:4, v/v) at rt for 45 min, then the volatiles removed under reduced pressure. The crude unprotected amine was then diluted with 1,4-dioxane/sat. aq. NaHCO3 (1:1, v/v) and Fmoc-OSu (1.05 eq.) added. The mixture was allowed to stir at rt for 6e18 h, then the 1,4-dioxane removed under reduced pressure. The aqueous residue was extracted three times with ethyl acetate and the combined organic extracts were dried over MgSO4 and concentrated under reduced pressure to give the crude Fmoc-protected amines which were purified via flash column chromatography using the eluent indicated. 4.6. General procedure E for hydrolysis of the methyl ester The ester (1 eq.) was dissolved in isopropyl alcohol/0.8 M aq. CaCl2 (7:3, v/v), and an aqueous solution of NaOH (1 M, 2 eq.) added dropwise. The mixture was allowed to stir at rt for 6e12 h, then the i PrOH removed under reduced pressure. The aqueous residue was extracted three times with ethyl acetate, and the combined organic extracts dried over MgSO4 and concentrated under reduced pressure to give the crude amino acid, which was purified via flash column chromatography using the eluent indicated. Fmoc-amino acid 6: prepared in two steps according to general procedure D to give ester 59 in 85% yield as a colourless oil (ethyl 1 acetate/pet. ether 3:7 as eluent). a21 D ¼ 9.4 (c 0.74, CHCl3); H NMR (400 MHz, CDCl3): d 7.77 (d, J ¼ 7.5 Hz, 2H), 7.62e7.58 (m, 2H), 7.40 (t, J ¼ 7.2 Hz, 2H), 7.33e7.28 (m, 2H), 5.34 (br d, J ¼ 8.4 Hz, 1H), 4.44e4.34 (m, 3H), 4.23 (t, J ¼ 7.1 Hz, 1H), 3.75 (s, 3H), 3.74e3.65 (m, 1H), 1.88e1.73 (m, 2H), 1.61e1.53 (m, 1H), 1.48e1.28 (m, 8H), 1.00 (d, J ¼ 6.4 Hz, 3H), 0.91 (t, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 173.7 (C), 156.2 (C), 143.9 (C), 143.8 (C), 141.3 (2  C), 127.7 (2  CH), 127.0 (2  CH), 125.1 (2  CH), 119.9 (2  CH), 69.3 (CH), 67.0 (CH2), 52.4 (CH3 þ CH), 47.2 (CH), 44.7 (CH2), 38.6 (CH2), 37.9 (CH2), 27.8 (CH2), 26.3 (CH), 22.7 (CH2), 20.1 (CH3), 14.0 (CH3); HRMS (ESIþ): m/z 476.2423 [M þ Na]þ (calcd for C27H35NO5Naþ 476.2407); followed by hydrolysis according to general procedure E to give 6 in 76% yield as a colourless solid (ethyl acetate/pet. ether 1 1:1 þ 0.5% AcOH as eluent). a21 D ¼ 10.8 (c 0.59, CHCl3); H NMR

243

(400 MHz, CDCl3): d 7.74e7.70 (m, 2H), 7.59e7.53 (m, 2H), 7.37e7.33 (m, 2H), 7.29e7.24 (m, 2H), 6.50e6.10 (br s, 1H), 5.89 (d, J ¼ 8.2 Hz, 1H), 4.45e4.29 (m, 3H), 4.18 (t, J ¼ 7.0 Hz, 1H), 3.73e3.63 (m, 1H), 1.90e1.80 (m, 2H), 1.63e1.56 (m, 1H), 1.49e1.20 (m, 8H), 0.97 (d, J ¼ 6.3 Hz, 3H), 0.91e0.82 (m, 3H); 13C NMR (100 MHz, CDCl3): d 177.2 (C), 156.5 (C), 143.9 (C), 143.7 (C), 141.2 (2  C), 127.7 (2  CH), 127.0 (2  CH), 125.1 (2  CH), 119.9 (2  CH), 69.3 (CH), 67.1 (CH2), 52.3 (CH), 47.1 (CH), 44.4 (CH2), 37.8 (CH2), 37.6 (CH2), 27.8 (CH2), 25.9 (CH), 22.7 (CH2), 20.3 (CH3), 14.0 (CH3); HRMS m/z (ESIþ) 462.2245 [M þ Na]þ (calcd for C26H33NO5Naþ 462.2251). Fmoc-amino acid 7: prepared in two steps according to general procedure D to give ester 60 in 93% yield as a colourless oil (ethyl 1 acetate/pet. ether 3:7 as eluent). a21 D ¼ þ6.1 (c 0.83, CHCl3); H NMR (400 MHz, CDCl3): d 7.76 (d, J ¼ 7.6 Hz, 2H), 7.60 (dd, J ¼ 3.4, 7.2 Hz, 2H), 7.40 (t, J ¼ 7.4 Hz, 2H), 7.33e7.29 (m, 2H), 5.25 (br d, J ¼ 8.6 Hz, 1H), 4.46e4.38 (m, 3H), 4.23 (t, J ¼ 6.8 Hz, 1H), 3.75 (s, 3H), 3.74e3.68 (m, 1H), 1.90e1.79 (m, 1H), 1.65 (t, J ¼ 7.4 Hz, 2H), 1.46e1.23 (m, 8H), 0.98 (d, J ¼ 6.5 Hz, 3H), 0.91 (t, J ¼ 6.9 Hz, 3H); 13 C NMR (100 MHz, CDCl3): d 173.6 (C), 156.1 (C), 143.9 (C), 143.8 (C), 141.3 (2  C), 127.7 (2  CH), 127.1 (2  CH), 125.1 (2  CH), 120.0 (2  CH), 69.7 (CH), 67.0 (CH2), 52.4 (CH3), 52.2 (CH), 47.2 (CH), 44.8 (CH2), 40.6 (CH2), 38.3 (CH2), 27.8 (CH2), 26.4 (CH), 22.7 (CH2), 18.8 (CH3), 14.0 (CH3); HRMS (ESIþ): m/z 476.2405 [M þ Na]þ (calcd for C27H35NO5Naþ 476.2407); followed by hydrolysis according to general procedure E to give 7 in71% yield as a colourless solid (ethyl acetate/pet. ether 1:1 þ 0.5% AcOH as eluent). a21 D ¼ þ7.1 (c 1.19, CHCl3); 1H NMR (400 MHz, CDCl3): d 7.71e7.68 (m, 2H), 7.57e7.53 (m, 2H), 7.36e7.30 (m, 2H), 7.27e7.21 (m, 2H), 5.81 (d, J ¼ 8.5 Hz, 1H), 4.45e4.39 (m, 1H), 4.37e4.29 (m, 2H), 4.16 (t, J ¼ 7.1 Hz, 1H), 3.71e3.64 (m, 1H), 1.90e1.78 (m, 1H), 1.73e1.64 (m, 2H), 1.48e1.20 (m, 8H), 0.93 (d, J ¼ 6.4 Hz, 3H), 0.86 (t, J ¼ 6.8 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 176.4 (C), 156.4 (C), 143.9 (C), 143.7 (2  C), 141.2 (2  C), 127.6 (2  CH), 127.0 (2  CH), 125.1 (2  CH), 119.9 (2  CH), 70.6 (CH), 67.0 (CH2), 52.2 (CH), 47.1 (CH), 44.2 (CH2), 40.1 (CH2), 37.9 (CH2), 27.8 (CH2), 26.6 (CH), 22.6 (CH2), 19.3 (CH3), 14.0 (CH3); HRMS m/z (ESIþ) 462.2239 [M þ Na]þ (calcd for C26H33NO5Naþ 462.2251). Fmoc-amino acid 8: prepared in two steps according to general procedure D to give ester 61 in 97% yield as a colourless oil (ethyl 1 acetate/pet. ether 1:9 as eluent). a21 D ¼ 4.4 (c 0.16, CHCl3); H NMR (400 MHz, CDCl3): d 7.78e7.75 (m, 2H), 7.61e7.58 (m, 2H), 7.42e7.38 (m, 2H), 7.33e7.29 (m, 2H), 5.10 (d, J ¼ 8.8 Hz, 1H), 4.49e4.39 (m, 3H), 4.24 (t, J ¼ 7.0 Hz, 1H), 3.74 (s, 3H), 1.63e1.48 (m, 3H), 1.32e1.18 (m, 14H), 0.94 (d, J ¼ 5.9 Hz, 3H), 0.88 (t, J ¼ 6.6 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 173.8 (C), 156.0 (C), 143.9 (C), 143.8 (C), 141.3 (2  C), 127.7 (2  CH), 127.0 (2  CH), 125.1 (2  CH), 120.0 (2  CH), 67.0 (CH2), 52.3 (CH3), 52.2 (CH), 47.2 (CH), 40.0 (CH2), 37.2 (CH2), 31.9 (CH2), 29.9 (CH2), 29.6 (CH2), 29.4 (CH2), 29.3 (CH2), 26.9 (CH2), 22.7 (CH2), 19.0 (CH3), 14.1 (CH3); HRMS (ESIþ): m/z 488.2778 [M þ Na]þ (calcd for C29H39NO4Naþ 488.2771); followed by hydrolysis according to general procedure E to give 8 in 72% yield as a colourless oil (ethyl acetate/pet. ether 1 1:9 þ 0.5% AcOH as eluent). a21 D ¼ 5.0 (c 0.87, CHCl3); H NMR (400 MHz, CDCl3): d 10.17 (br s, 1H), 7.74 (d, J ¼ 7.5 Hz, 2H), 7.60e7.57 (m, 2H), 7.38 (t, J ¼ 7.4 Hz, 2H), 7.29 (t, J ¼ 7.4 Hz, 2H), 5.14 (d, J ¼ 8.7 Hz, 1H), 4.47e4.38 (m, 3H), 4.22 (t, J ¼ 6.9 Hz, 1H), 1.65e1.49 (m, 3H), 1.32e1.21 (m, 14H), 0.94 (d, J ¼ 6.2 Hz, 3H), 0.88 (t, J ¼ 6.5 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 178.4 (C), 156.2 (C), 143.8 (C), 143.7 (C), 141.3 (2  C), 127.7 (2  CH), 127.0 (2  CH), 125.0 (2  CH), 120.0 (2  CH), 67.0 (CH2), 52.1 (CH), 47.2 (CH), 39.7 (CH2), 37.2 (CH2), 31.9 (CH2), 29.9 (CH2), 29.6 (CH2), 29.5 (CH), 29.3 (CH2), 26.9 (CH2), 22.7 (CH2), 18.8 (CH3), 14.1 (CH3); HRMS m/z (ESIþ) 474.2611 [M þ H]þ (calcd for C28H37NO4Naþ 474.2615). Fmoc-amino acid 13: prepared in two steps according to general procedure D to give ester 62 in 85% yield as a colourless solid (ethyl

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1 acetate/pet. ether 1:19 as eluent). a22 D ¼ þ5.4 (c 4.55, CHCl3); H NMR (400 MHz, CDCl3): d 7.76 (d, J ¼ 7.5 Hz, 2H), 7.60 (br dd, J ¼ 4.2, 7.0 Hz, 2H), 7.40 (br t, J ¼ 7.5 Hz, 2H), 7.32 (br t, J ¼ 7.4 Hz, 2H), 5.25 (br d, J ¼ 8.2 Hz, 1H), 4.49e4.35 (m, 3H), 4.23 (t, J ¼ 7.0 Hz, 1H), 3.75 (s, 3H), 1.86e1.81 (m, 1H), 1.70e1.63 (m, 1H), 1.37e1.23 (br m, 8H), 0.88 (t, J ¼ 6.8 Hz, 3H);13C NMR (100 MHz, CDCl3): d 173.2 (C), 155.9 (C), 143.9 (C), 143.8 (C), 141.3 (2  C), 127.7 (2  CH), 127.0 (2  CH), 125.1 (2  CH), 120.0 (2  CH), 67.0 (CH2), 53.9 (CH), 52.3 (CH3), 47.2 (CH), 32.7 (CH2), 31.5 (CH2), 28.8 (CH2), 25.1 (CH2), 22.5 (CH2), 14.0 (CH3); HRMS (ESIþ): m/z 418.1993 [M þ Na]þ (calcd for C24H29NO4Naþ 418.1989); followed by hydrolysis according to general procedure E to give 13 in 40% yield as a colourless solid (ethyl acetate/pet. ether 1:9 þ 0.5% AcOH as eluent). a21 D ¼ þ2.2 (c 1.19, CHCl3); 1H NMR (400 MHz, CDCl3): d 10.24 (br s, 1H), 7.74 (d, J ¼ 7.5 Hz, 2H), 7.60e7.53 (m, 2H), 7.38 (t, J ¼ 7.4 Hz, 2H), 7.29 (t, J ¼ 7.4 Hz, 2H), 5.32 (d, J ¼ 8.3 Hz, 1H), 4.48e4.37 (m, 3H), 4.22 (t, J ¼ 7.0 Hz, 1H), 1.94e1.84 (m, 1H), 1.77e1.65 (m, 1H), 1.41e1.22 (m, 8H), 0.87 (t, J ¼ 6.5 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 177.6, 156.1, 143.8, 143.7, 141.3, 127.7, 127.0, 125.0, 120.0, 67.1, 53.8, 47.1, 32.3, 31.5, 28.8, 25.1, 22.5, 14.0. Data were consistent with previously reported values [13]. Fmoc-amino acid 14: prepared in two steps according to general procedure D to give ester 63 in 90% yield as a colourless solid (ethyl 1 acetate/pet. ether 1:9 as eluent). a22 D ¼ þ11.1 (c 0.11, CHCl3); H NMR (400 MHz, CDCl3): d 7.77e7.74 (m, 2H), 7.62e7.59 (m, 2H), 7.42e7.38 (m, 2H), 7.33e7.29 (m, 2H), 5.25 (d, J ¼ 8.5 Hz, 1H), 4.49e4.35 (m, 3H), 4.23 (t, J ¼ 7.0 Hz, 1H), 3.76 (s, 3H), 1.88e1.79 (m, 1H), 1.71e1.61 (m, 1H), 1.35e1.22 (m, 16H), 0.88 (t, J ¼ 6.6 Hz, 3H);13C NMR (100 MHz, CDCl3): d 173.2 (C), 155.9 (C), 143.9 (C), 143.8 (C), 141.3 (C), 127.7 (2  CH), 127.1 (2  CH), 125.1 (2  CH), 120.0 (2  CH), 67.0 (CH2), 53.9 (CH), 52.3 (CH3), 47.2 (CH), 32.7 (CH2), 31.9 (CH2), 29.7 (CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2), 29.3 (CH2), 29.2 (CH2), 25.2 (CH2), 22.7 (CH2), 14.1 (CH3); HRMS (ESIþ): m/z 452.2780 [M þ H]þ (calcd for C28H38NOþ 4 452.2795); 474.2601 [M þ Na]þ (calcd for C28H37NO4Naþ 474.2615); followed by hydrolysis according to general procedure E to give 14 in 42% yield as a colourless solid (ethyl acetate/pet. ether 1:9 þ 0.5% AcOH as 1 eluent). a19 D ¼ 0.8 (c 0.48, MeOH); H NMR (400 MHz, CD3OD): d 7.78 (d, J ¼ 7.2 Hz, 2H), 7.67 (t, J ¼ 5.8 Hz, 2H), 7.38 (t, J ¼ 6.7 Hz, 2H), 7.30 (t, J ¼ 7.0 Hz, 2H), 4.35e4.31 (m, 2H), 4.24e4.21 (m, 1H), 4.17e4.12 (m, 1H), 1.90e1.77 (m, 1H), 1.74e1.62 (m, 1H), 1.44e1.23 (m, 16H), 0.89e0.87 (m, 3H); 13C NMR (100 MHz, CD3OD): d 176.1, 158.7, 145.4, 145.2, 142.6, 128.8, 128.1, 126.3, 120.9, 68.0, 55.3, 33.1, 32.7, 30.7, 30.6, 30.55, 30.45, 30.1, 26.9, 23.7, 14.4. Data were consistent with previously reported values [13]. Fmoc-amino acid 9: prepared in two steps according to general procedure D to give the ester 64 in 76% yield as a colourless foam and a 1:1 mixture of diastereomers (ethyl acetate/pet. ether 3:7 as eluent). 1H NMR (400 MHz, CDCl3): d 7.76 (d, J ¼ 7.6 Hz, 2H), 7.61e7.58 (m, 2H), 7.39 (t, J ¼ 7.4 Hz, 2H), 7.31 (td, J ¼ 1.0, 7.4 Hz, 2H), 5.43 (br t, J ¼ 7.7 Hz, 1H), 4.43e4.34 (m, 3H), 4.22 (t, J ¼ 7.0 Hz, 1H), 4.07e3.99 (m, 1H), 3.75 (s, 3H), 2.91e2.65 (br s, 1H), 2.61e2.47 (m, 2H), 2.43 (q, J ¼ 7.4 Hz, 2H), 1.92e1.81 (m, 1H), 1.76e1.64 (m, 1H), 1.57e1.34 (m, 4H), 1.05 (t, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3, * denotes diastereomer peaks): d 212.54 (C), 212.50* (C), 172.9 (C), 155.9 (C), 143.8 (C), 143.7 (C), 141.2 (2  C), 127.6 (2  CH), 127.0 (2  CH), 125.0 (2  CH), 119.9 (2  CH), 67.3 (CH), 67.1* (CH), 67.0 (CH2), 53.7 (CH), 52.4 (CH3), 48.55 (CH2), 48.50* (CH2), 47.1 (CH), 36.7 (CH2), 35.7 (CH2), 35.6* (CH2), 32.4 (CH2), 32.3* (CH2), 21.3 (CH2), 21.1* (CH2), 7.5 (CH3); HRMS (ESIþ): m/z 476.2040 [M þ Na]þ (calcd for C26H31NO6Naþ 476.2044); followed by hydrolysis according to general procedure E to give 9 in 62% yield as a colourless foam and a 1:1 mixture of diastereomers (methanol/dichloromethane 1:19 as eluent). 1H NMR (400 MHz, CDCl3): d 7.74 (d, J ¼ 7.5 Hz, 2H), 7.58 (t, J ¼ 6.4 Hz, 2H), 7.38 (t, J ¼ 7.4 Hz, 2H), 7.29 (t,

J ¼ 7.4 Hz, 2H), 6.05 (br s, 2H), 5.64 (m, 1H), 4.49e4.32 (m, 3H), 4.20 (t, J ¼ 6.9 Hz, 1H), 4.12e4.02 (m, 1H), 2.59e2.48 (m, 2H), 2.41 (q, J ¼ 7.3 Hz, 2H), 1.95e1.83 (m, 1H), 1.82e1.65 (m, 1H), 1.61e1.38 (m, 4H), 1.12 (t, J ¼ 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3): d 212.8 (C), 175.6 (C), 156.2 (C), 143.8 (C), 143.7 (C), 141.3 (2  C), 127.7 (2  CH), 127.1 (2  C), 125.1 (CH), 124.8 (CH), 119.9 (2  CH), 67.7 (CH), 67.3* (CH), 67.1 (CH2), 53.6 (CH), 53.4* (CH), 48.4 (CH2), 47.1 (CH), 36.7 (CH2), 35.6 (CH2), 35.5* (CH2), 32.1 (CH2), 31.9* (CH2), 21.2 (CH2), 21.0* (CH2), 7.5 (CH3); HRMS (ESIþ): m/z 462.1888 [M þ Na]þ (calcd for C25H29NO6Naþ 462.1887). Ester 27: prepared according to general procedure D in 86% yield as a colourless solid and as a 1:1 mixture of diastereomers (ethyl acetate/pet. ether 3:7 as eluent). 1H NMR (400 MHz, CDCl3): d 7.78e7.76 (m, 2H), 7.61e7.59 (m, 2H), 7.43e7.38 (m, 2H), 7.33e7.29 (m, 2H), 5.34e5.31 (m, 1H), 4.45e4.35 (m, 3H), 4.23 (t, J ¼ 7.0 Hz, 1H), 3.76 (s, 3H), 3.58 (br s, 1H), 1.92e1.81 (m, 1H), 1.77e1.64 (m, 1H), 1.54e1.25 (m, 10H), 0.90 (t, J ¼ 6.9 Hz, 3H); 13C NMR (100 MHz, CDCl3, * denotes diastereomer peaks): d 173.0 (C), 155.9 (C), 143.9 (C), 143.8 (C), 141.3 (2  C), 128.2* (2  CH), 127.7 (2  CH), 127.4* (2  CH), 127.1 (2  CH), 125.1 (2  CH), 120.2* (2  CH), 120.0 (2  CH), 71.6 (CH), 71.5* (CH), 67.0 (CH2), 53.8 (CH), 52.4 (CH3), 47.2 (CH), 37.4* (CH2), 37.2 (CH2), 36.7 (CH2), 36.6* (CH2), 32.69 (CH2), 32.67* (CH2), 27.8 (CH2), 22.7 (CH2), 21.3 (CH2), 14.0 (CH3); HRMS m/z (ESIþ) 462.2247 [M þ Na]þ (calcd for C26H33NO5Naþ 462.2251). Ester 28: prepared according to general procedure D in 61% yield as a colourless foam and as a 1:1 mixture of diastereomers (ethyl acetate/pet. ether 3:7 as eluent). 1H NMR (CDCl3, 500 MHz): d 7.76 (d, J ¼ 7.4 Hz, 2H), 7.61e7.58 (m, 2H), 7.39 (t, J ¼ 7.6 Hz, 2H), 7.32e7.29 (m, 2H), 5.40 (t, J ¼ 8.7 Hz, 1H), 4.43e4.36 (m, 3H), 4.22 (t, J ¼ 7.1 Hz, 1H), 3.75 (s, 3H), 3.60e3.54 (m, 1H), 1.90e1.81 (m, 1H), 1.75e1.63 (m, 1H), 1.59e1.35 (m, 6H), 1.33e1.24 (m, 8H), 0.88 (t, J ¼ 7.1 Hz, 3H); 13C NMR (CDCl3, 125 MHz, * denotes diastereomer peaks): d 173.0 (C), 155.9 (C), 143.9 (C), 143.7 (C), 141.3 (2  C), 127.7 (2  CH), 127.0 (2  CH), 125.0 (2  CH), 119.9 (2  CH), 71.6 (CH), 71.5* (CH), 67.0 (CH2), 53.8 (CH), 53.7* (CH), 52.4 (CH3), 47.1 (CH), 37.6 (CH2), 37.5* (CH2), 36.7 (CH2), 36.6* (CH2), 32.63 (CH2), 32.59* (CH2), 31.8 (CH2), 29.3 (CH2), 25.6 (CH2), 22.6 (CH2), 21.3 (CH2), 14.0 (CH3); HRMS m/z (ESIþ) 490.2558 [M þ Na]þ (calcd for C28H37NO5Naþ 490.2564). Fmoc-amino acid 12: prepared according to general procedure E in 87% yield as a colourless foam and as a 1:1 mixture of diastereomers (ethyl acetate/pet. ether 1:1 þ 0.5% AcOH as eluent). 1H NMR (400 MHz, CDCl3, * denotes diastereomer peaks): d 7.73 (d, J ¼ 7.5 Hz, 2H), 7.57 (t, J ¼ 6.5 Hz, 2H), 7.37 (t, J ¼ 7.4 Hz, 2H), 7.30e7.26 (m, 2H), 6.24 (br s, 1H), 5.66 (br d, J ¼ 7.8 Hz, 1H), 4.48e4.33 (m, 3H), 4.19 (t, J ¼ 6.9 Hz, 1H), 3.62e3.53 (br m, 1H), 1.94e1.62 (m, 2H), 1.57e1.21 (m, 10H), 0.87 (t, J ¼ 6.7 Hz, 3H); 13C NMR (100 MHz, CDCl3, * denotes diastereomer peaks): d 175.7 (C), 175.6* (C), 156.2 (C), 143.8 (C), 143.7 (C), 141.3 (2  C), 127.7 (2  CH), 127.0 (2  CH), 125.1 (2  CH), 119.9 (2  CH), 72.1 (CH), 71.7* (CH), 67.1 (CH2), 53.7 (CH), 47.1 (CH), 37.2 (CH2), 37.1* (CH2), 36.5 (CH2), 36.3* (CH2), 32.2 (CH2), 32.1* (CH2), 27.8 (CH2), 22.6 (CH2), 21.2 (CH2), 21.0* (CH2), 14.0 (CH3); HRMS m/z (ESIþ) 448.2083 [M þ Na]þ (calcd for C25H31NO5Naþ 448.2094). 4.7. General procedure F for oxidation of alcohols 27 and 28 IBX (2 eq.) was added to a solution of the alcohol (1 eq.) in DMSO (10 mL/mmol) and the mixture allowed to stir at rt for 18 h. An equal volume of ice was added, and the mixture extracted three times with diethyl ether. The combined organic extracts were washed twice with water, dried over MgSO4, and concentrated under reduced pressure. The crude ketone was purified via flash column chromatography using the eluent indicated.

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Fmoc-amino acid 10: prepared in two steps according to general procedure F to give ketone 65 in 91% yield as a colourless foam (ethyl acetate/pet. ether 1:3 as eluent). a20 D ¼ þ3.8 (c 1.81, CHCl3); 1 H NMR (CDCl3, 400 MHz): d 7.76 (d, J ¼ 7.5 Hz, 2H), 7.62e7.59 (m, 2H), 7.40 (t, J ¼ 7.4 Hz, 2H), 7.32 (t, J ¼ 7.4 Hz, 2H), 5.40 (d, J ¼ 8.0 Hz, 1H), 4.43e4.35 (m, 3H), 4.23 (t, J ¼ 7.0 Hz, 1H), 3.76 (s, 3H), 2.46e2.42 (m, 2H), 2.38 (t, J ¼ 7.4 Hz, 2H), 1.89e1.79 (m, 1H), 1.73e1.51 (m, 5H), 1.35e1.25 (m, 2H), 0.90 (t, J ¼ 7.3 Hz, 3H); 13C NMR (CDCl3, 100 MHz): d 210.5 (C), 172.7 (C), 155.9 (C), 143.9 (C), 143.7 (C), 141.3 (2  C), 127.7 (2  CH), 127.1 (2  CH), 125.1 (2  CH), 120.0 (2  CH), 67.0 (CH2), 53.6 (CH), 52.4 (CH3), 47.1 (CH), 42.6 (CH2), 41.6 (CH2), 31.9 (CH2), 25.9 (CH2), 22.3 (CH2), 19.2 (CH2), 13.8 (CH3); HRMS m/z (ESIþ) 460.2092 [M þ Na]þ (calcd for C26H31NO5Naþ 460.2094); followed by hydrolysis according to general procedure E to give 10 in 86% yield as a colourless solid (ethyl acetate/pet. ether 1:1 þ 0.5% AcOH as eluent). a22 D ¼ þ11.5 (c 1.02, CHCl3); 1H NMR (CDCl3, 400 MHz): d 7.75 (d, J ¼ 7.3 Hz, 2H), 7.61e7.58 (m, 2H), 7.39 (t, J ¼ 7.3 Hz, 2H), 7.30 (t, J ¼ 7.3 Hz, 2H), 5.51 (d, J ¼ 7.6 Hz, 1H), 4.41e4.35 (m, 3H), 4.22 (t, J ¼ 7.0 Hz, 1H), 2.50e2.43 (m, 2H), 2.41e2.35 (m, 2H), 1.92e1.84 (m, 1H), 1.78e1.63 (m, 3H), 1.58e1.51 (m, 2H), 1.34e1.25 (m, 2H), 0.89 (t, J ¼ 7.3 Hz, 3H); 13C NMR (CDCl3, 100 MHz): d 211.3 (C), 175.9 (C), 156.2 (C), 143.8 (C), 143.7 (C), 141.3 (2  C), 127.7 (2  CH), 127.1 (2  CH), 125.1 (2  CH), 120.0 (2  CH), 67.2 (CH2), 53.5 (CH), 47.1 (CH), 42.7 (CH2), 41.7 (CH2), 31.5 (CH2), 25.9 (CH2), 22.3 (CH2), 19.1 (CH2), 13.8 (CH3); HRMS m/z (ESIþ) 446.1922 [M þ Na]þ (calcd for C25H29NO5Naþ 446.1938). Fmoc-amino acid 11: prepared in two steps according to general procedure F to give ketone 66 in 90% yield as a colourless foam (ethyl acetate/pet. ether 3:7 as eluent). a21 D ¼ þ9.2 (c 1.22, CHCl3); 1 H NMR (CDCl3, 400 MHz): d 7.75 (d, J ¼ 7.5 Hz, 2H), 7.61e7.58 (m, 2H), 7.39 (t, J ¼ 7.5 Hz, 2H), 7.32e7.28 (m, 2H), 5.45 (br d, J ¼ 8.2 Hz, 1H), 4.43e4.35 (m, 3H), 4.22 (t, J ¼ 7.1 Hz, 1H), 3.74 (s, 3H), 2.45e2.41 (m, 2H), 2.36 (t, J ¼ 7.4 Hz, 2H), 1.88e1.78 (m, 1H), 1.72e1.51 (m, 5H), 1.31e1.25 (m, 6H), 0.87 (t, J ¼ 7.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz): d 210.4 (C), 172.7 (C), 155.9 (C), 143.8 (C), 143.7 (C), 141.2 (2  C), 127.6 (2  CH), 127.0 (2  CH), 125.0 (2  CH), 119.9 (2  CH), 67.0 (CH2), 53.5 (CH), 52.4 (CH3), 47.1 (CH), 42.8 (CH2), 41.6 (CH2), 31.8 (CH2), 31.5 (CH2), 28.8 (CH2), 23.7 (CH2), 22.4 (CH2), 19.1 (CH2), 13.9 (CH3); HRMS m/z (ESIþ) 488.2404 [M þ Na]þ (calcd for C28H35NO5Naþ 488.2407); 466.2580 [M þ H]þ (calcd for C28H36NOþ 5 466.2588); followed by hydrolysis according to general procedure E to give 11 in 75% yield as a colourless foam (ethyl acetate/pet. ether 3:7 þ 0.5% AcOH as eluent). a24 D ¼ þ10.2 (c 1.05, CHCl3); 1H NMR (CDCl3, 400 MHz): d 9.18 (br s, 1H), 7.73 (d, J ¼ 7.5 Hz, 2H), 7.60e7.57 (m, 2H), 7.37 (t, J ¼ 7.4 Hz, 2H), 7.31e7.27 (m, 2H), 5.60 (br d, J ¼ 8.0 Hz, 1H), 4.52e4.34 (m, 3H), 4.20 (t, J ¼ 7.0 Hz, 1H), 2.46e2.32 (m, 4H), 1.92e1.83 (m, 1H), 1.76e1.61 (m, 3H), 1.58e1.49 (m, 2H), 1.30e1.23 (m, 6H), 0.86 (t, J ¼ 6.6 Hz, 3H); 13 C NMR (CDCl3, 100 MHz): d 211.4 (C), 176.2 (C), 156.2 (C), 143.8 (C), 143.6 (C), 141.2 (2  C), 127.7 (2  CH), 127.0 (2  CH), 125.1 (2  CH), 119.9 (2  CH), 67.2 (CH2), 53.5 (CH), 47.0 (CH), 42.9 (CH2), 41.6 (CH2), 31.51 (CH2), 31.46 (CH2), 28.8 (CH2), 23.7 (CH2), 22.4 (CH2), 19.1 (CH2), 14.0 (CH3); HRMS m/z (ESIþ) 474.2243 [M þ Na]þ (calcd for C27H33NO5Naþ 474.2251). 4.8. General procedures for SPPS of culicinin D analogues 4.8.1. General method and materials All reagents were purchased as reagent grade and used without further purification. Solvents for reactions were dried according to standard procedures. N,N-Diisopropylethylamine (DIPEA), piperidine, N,N0 -diisopropylcarbodiimide (DIC), and ninhydrin were purchased from Sigma-Aldrich (St. Louis, Missouri). O-(7Azabenzotriazol-1-yl)-N,N,N0 ,N0 -tetramethyluronium

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hexafluorophosphate (HATU), 2-Chlorotrityl chloride Polystyrene resin and ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma) were purchased from Novabiochem (Merck, Germany). Fmoc-a-aminoisobutyric acid (Aib) was purchased from CS Bio (Shanghai, China). 1-[(1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino morpholinomethylene)] methanaminium hexafluorophosphate (COMU), and 6-chloro-1-hydroxybenzotriazole (6-Cl-HOBt) were purchased from Aapptec (Louisville, Kentucky). Semi-preparative RP-HPLC was performed on a Thermo Scientific (Waltham, MA) Dionex Ultimate 3000 HPLC equipped with a four channel UV Detector at 210, 225, 254 and 280 nm using either an analytical column [Waters (Milford, MA) XTerra® MS C18, 4.6  150 mm, 5 mm] at a flow rate of 1 mL min1 or a semipreparative column (XTerra® MS C18, 10  250 mm 5 mm) at a flow rate of 5 mL min1. A suitably adjusted gradient of 5% B to 95% B was used, where solvent A was 0.1% formic acid in H2O and B was 0.1% formic acid in acetonitrile for AHMOD containing peptides and A was 0.1% TFA in H2O and B was 0.1% TFA in acetonitrile for remaining analogues. LC-MS spectra were acquired on either an Agilent Technologies (Santa Clara, CA) 1120 Compact LC equipped with a Hewlett-Packard (Palo Alto, CA) 1100 MSD mass spectrometer or an Agilent Technologies 1260 Infinity LC equipped with an Agilent Technologies 6120 Quadrupole mass spectrometer. An analytical column (Agilent C3, 150 mm  3.0 mm, 3.5 mm) was used at a flow rate of 0.3 mL min1 using a linear gradient of 5% B to 95% B over 30 min, where solvent A was 0.1% formic acid in H2O and B was 0.1% formic acid in acetonitrile. 4.8.2. General procedure for the attachment of Fmoc-b-alanine-OH to resin To 2-chlorotrityl chloride polystyrene resin (70 mg, 0.1 mmol, loading: 1.42 mmol/g) pre-swollen in anhydrous CH2Cl2 (5 mL, 5 min), was added a solution of Fmoc-b-Ala-OH (2 eq., 62 mg, 0.2 mmol) and DIPEA (5 eq., 87 mL, 0.5 mmol) in anhydrous CH2Cl2 (1 mL). The reaction mixture was gently agitated at room temperature for 6 h, filtered and repeated once for a further 12 h with fresh reagents. Reaction mixture was removed by filtration and washed with CH2Cl2 (3  5 mL). A mixture of CH2Cl2/MeOH/DIPEA (8:1.5:0.5, v/v, 2 mL) was added and the reaction agitated for 10 min to cap unreacted resin. 4.8.3. General procedure for removal of Na-Fmoc-protecting group The peptidyl resin was treated with a solution of 20% piperidine in DMF (v/v, 2 mL) and the mixture agitated at room temperature for 5 min, filtered and repeated once for a further 15 min. The resin was filtered and washed DMF (3  3 mL). 4.8.4. General procedure for SPPS To the peptidyl resin (0.1 mmol) was added a mixture of appropriate Fmoc-protected amino acid (5 eq., 0.5 mmol), HATU (186 mg, 0.49 mmol) and DIPEA (172 mL, 1 mmol) in DMF (1 mL). The reaction mixture was agitated at room temperature for 1 h, filtered and repeated once for a further 1 h with fresh reagents. The resin was washed with DMF (3  3 mL), and CH2Cl2 (3  3 mL). 4.8.5. General procedure for the difficult coupling reaction between resin bound Aib and Fmoc-Aib-OH using COMU as a coupling reagent To the peptidyl resin (0.1 mmol) was added a mixture of FmocAib-OH (5 eq., 163 mg, 0.5 mmol), COMU (5 eq., 214 mg, 0.5 mmol), Oxyma (5 eq., 71 mg, 0.5 mmol) and DIPEA (10 eq., 172 mL, 1 mmol) in DMF (1 mL). The reaction mixture was agitated at room temperature for 2 h, filtered and repeated once for a further 2 h with fresh reagents. The resin was filtered and washed with DMF (2  3 mL) and CH2Cl2 (2  3 mL).

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4.8.6. General procedure for the attachment of Fmoc-AMD-OH To the peptidyl resin (0.1 mmol) was added a mixture of FmocAMD-OH (2 eq., 85 mg, 0.2 mmol), COMU (2 eq., 85 mg, 0.2 mmol), Oxyma (2 eq., 29 mg, 0.2 mmol) and DIPEA (4 eq., 69 mL, 0.4 mmol) in DMF (1 mL). The reaction mixture was agitated at room temperature for 3 h, after which the resin was filtered and washed with DMF (3  3 mL). 4.8.7. General procedure for the attachment of Fmoc-AHMOD-OH To the peptidyl resin (0.1 mmol) was added a mixture of FmocAHMOD-OH (2 eq., 90 mg, 0.2 mmol), HATU (1.9 eq., 72 mg, 0.19 mmol) and DIPEA (4 eq., 70 mL, 0.4 mmol) in DMF (1 mL). The reaction mixture was agitated at room temperature for 2 h. The resin was washed with DMF (3  3 mL), and CH2Cl2 (3  3 mL). 4.8.8. General procedure for the attachment of N-terminal butyric acid To the peptidyl resin (0.1 mmol) was added a mixture of butyric acid (5 eq., 47 mL, 0.5 mmol), HATU (4.9 eq., 186 mg, 0.49 mmol) and DIPEA (10 eq., 172 mL, 1 mmol) in DMF (1 mL). The reaction mixture was agitated at room temperature for 1 h, filtered and repeated once for a further 1 h with fresh reagents. The resin was washed with DMF (3  3 mL), and CH2Cl2 (3  3 mL). 4.8.9. General procedure for HFIP-mediated resin cleavage Resin-bound peptide was cleaved from the resin by gentle agitation in a mixture of CH2Cl2/HFIP (4:1, v/v, 5 mL) for 30 min. The filtrate was partially concentrated under a gentle stream of N2, diluted with H2O:CH3CN (1:1, 10 mL) and lyophilised. 4.8.10. General procedure for TFA-mediated resin cleavage Resin-bound peptide was cleaved from the resin by gentle agitation in a mixture of TFA/TIPS/H2O (95:2.5:2.5, v/v/v) for 2 h. The filtrate was partially concentrated under a gentle stream of N2, diluted with H2O/CH3CN (1:1, 10 mL) and lyophilised. 4.8.11. General procedure for late-stage solution phase C-terminal coupling of APAE In order to attach the C-terminal APAE residue, to the purified peptide (from method 9) dissolved in DMF was added a mixture of DIC (6 eq.), 6-Cl-HOBt (6 eq.), APAE-2TFA salt (3 eq.) and DIPEA (6 eq.). The reaction mixture was agitated at room temperature for 12 h. 4.8.12. Cell lines and antiproliferative assay Three breast cancer cell lines (MDA-MB-468, SKBR3 and T47D) and a non-small cell lung cancer cell line (NCI-H460) were obtained from American Type Culture Collection (ATCC; Rockville, MD). STR phenotyping confirmed authenticity. Cells were maintained in culture under humidified atmospheric conditions with 5% CO2 at 37  C, with <3 months cumulative passage from authenticated stocks. MDA-MB-468 and SKBR3 cells were cultured in DMEM medium containing 10% fetal calf serum (FCS), while T47D cells were grown in RPMI medium þ 10% FCS. Alpha MEM medium supplemented with 5% FCS was used to culture NCI-H460 cells. Testing of cell cultures for mycoplasma contamination was carried out using PlasmoTest Mycoplasma Detection kit (InvivoGen). The sensitivity of four cell lines to culicinin D and culicinin D analogues was examined under aerobic conditions using an antiproliferative assay. Peptides were dissolved in DMSO to give a 30 mM stock solution. Cells were harvested, counted and seeded at a density of 2000 (MDA-MB-468 and SKBR3), 1500 (T47D) or 300 (NCI-H460) cells/well in 96 well tissue culture plates (Nunc). Cells were incubated over-night to allow attachment, then exposed to peptides

using 3-fold serial dilutions in duplicate, and incubated for 5 days. Subsequently medium was aspirated and replaced with fresh medium. Cultures were then stained with sulphorhodamine B to measure total cells [14]. The absorbance-concentration plots were fitted using 4-parameter logistic regression, and IC50 was determined by interpolation as the drug concentration that reduced staining to 50% versus peptide-free controls on the same plate. Values are means and errors are SEM for multiple independent experiments (n ¼ 2 to 3 replicates). Funding The authors thank the Auckland Medical Research Foundation (1114016), the Health Research Council of New Zealand (18/219), and the Maurice Wilkins Centre for Biodiscovery for financial support. Competing interests statement Declarations of interest: none. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ejmech.2019.05.052. References [1] H. He, J.E. Janso, H.Y. Yang, V.S. Bernan, S.L. Lin, K. Yu, D. Culicinin, An antitumor peptaibol produced by the fungus Culicinomyces clavisporus, strain LL12I252, J. Nat. Prod. 69 (2006) 736e741. [2] M. El Hadrami, J. Lavergne, P. Viallefont, M.Y.A. Itto, A. Hasnaoui, Synthesis of (2S,4S,6S)-2-amino-6-hydroxy-4-methyl-8 oxodecanoic acid and (4S,E)-4methylhex-2-enoic acid constituents of leucinostatines, Tetrahedron Lett. 32 (1991) 3985e3988. [3] W. Zhang, X. Li, N. Ding, Y. Li, Stereoselective synthesis of fully protected (2S,4S,6S)-2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (AHMOD), J. Pept. Sci. 18 (2012) 163e169. [4] L.A. Stubbing, I. Kavianinia, M.A. Brimble, Synthesis of AHMOD-containing aminolipopeptides, unique bioactive peptaibiotics, Org. Biomol. Chem. 15 (2017) 3542e3549. [5] H. Abe, H. Ouchi, C. Sakashita, M. Kawada, T. Watanabe, M. Shibasaki, Catalytic asymmetric total synthesis and stereochemical revision of leucinostatin a: a modulator of tumor-stroma interaction, Chem. Eur J. 23 (2017) 11792e11796. [6] K. Ko, S. Wagner, S. Yang, D.P. Furkert, M.A. Brimble, Improved synthesis of the unnatural amino acids AHMOD and AMD, components of the anticancer peptaibol culicinin D, J. Org. Chem. 80 (2015) 8631e8636. [7] K. Hung, P.W.R. Harris, M.A. Brimble, Synthesis of the peptaibol framework of the anticancer agent culicinin D: stereochemical assignment of the AHMOD moiety, Org. Lett. 14 (2012) 5784e5787. [8] M. Stach, A.J. Weidkamp, S. Yang, K. Hung, D.P. Furkert, P.W.R. Harris, J.B. Smaill, A.V. Patterson, M.A. Brimble, Improved strategy for the synthesis of the anticancer agent culicinin D, Eur. J. Org. Chem. 2015 (2015) 6341e6350. [9] I. Kavianinia, L. Kunalingam, P.W.R. Harris, G.M. Cook, M.A. Brimble, Total synthesis and stereochemical revision of the anti-tuberculosis peptaibol trichoderin A, Org. Lett. 18 (2016) 3878e3881. n, G. Kokotos, T. Martın, T. Markidis, W.A. Gibbons, V.S. Martın, [10] J.M. Padro Enantiospecific synthesis of a-amino acid semialdehydes: a key step for the synthesis of unnatural unsaturated and saturated a-amino acids, Tetrahedron: Asymmetry 9 (1998) 3381e3394. ze, A.D. Baughn, P. Moodley, [11] P. Pruksakorn, M. Arai, N. Kotoku, C. Vilche W.R. Jacobs, M. Kobayashi Trichoderins, Novel aminolipopeptides from a marine sponge-derived Trichoderma sp., are active against dormant mycobacteria, Bioorg. Med. Chem. Lett 20 (2010) 3658e3663. [12] M.P. Hay, S.A. Gamage, M.S. Kovacs, F.B. Pruijn, R.F. Anderson, A.V. Patterson, W.R. Wilson, J.M. Brown, W.A. Denny, StructureActivity relationships of 1,2,4-benzotriazine 1,4-dioxides as hypoxia-selective analogues of tirapazamine, J. Med. Chem. 46 (2003) 169e182. [13] Z.J. Wang, N.D. Spiccia, W.R. Jackson, A.J. Robinson, Tandem Ru-alkylidenecatalysed cross metathesis/hydrogenation: synthesis of lipophilic amino acids, J. Pept. Sci. 19 (2013) 470e476. [14] V. Vichai, K. Kirtikara, Sulforhodamine B colorimetric assay for cytotoxicity screening, Nat. Protoc. 1 (2006) 1112e1116.