marinedrugs Marine Drugs Mar. Drugs Marine Drugs 1660-3397 MDPI 10.3390/md11114487 marinedrugs-11-04487 Article Derivatives of Salarin A, Salarin C and Tulearin A—Fascaplysinopsis sp. Metabolites Goren Zur Lee 1 † Bishara Ashgan 1 † Aknin Maurice 2 Neumann Drorit 3 Ben-Califa Nathalie 3 Kashman Yoel 1 * School of Chemistry, Tel Aviv University, Ramat Aviv 69978, Israel; E-Mails: goren5@gmail.com (L.G.Z.); ashganbi@gmail.com (A.B.) Laboratorie de Chimie des Substances Naturelles et des Aliments, Faculté des Sciences et Techniques, Université de la Réunion, 15 Avenue Rene Cassin, BP 7151, Saint Denis 97715, Cedex 9, France; E-Mail: maurice.aknin@univ-reunion.fr Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel; E-Mails: histo6@post.tau.ac.il (D.N.); allouln@post.tau.ac.il (N.B.-C.)

These authors contributed equally to this work.

 Author to whom correspondence should be addressed; E-Mail: kashman@post.tau.ac.il; Tel.: +972-3-640-8419; Fax: +972-3-640-9293. 11 11 2013 11 2013 11 11 4487 4509 30 08 2013 26 09 2013 09 10 2013 © 2013 by the authors; licensee MDPI, Basel, Switzerland. 2013

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

Derivatives of salarin A, salarin C and tulearin A, three new cytotoxic sponge derived nitrogenous macrolides, were prepared and bio-evaluated as inhibitors of K562 leukemia cells. Interesting preliminary SAR (structure activity relationship) information was obtained from the products. The most sensitive functionalities were the 16,17-vinyl epoxide in both salarins, the triacylamino group in salarin A and the oxazole in salarin C (less sensitive). Regioselectivity of reactions was also found for tulearin A.

 salarins tulearins Fascaplysinopsis sp. sponge nitrogenous macrolide leukemia cells 1. Introduction

Four groups of nitrogenous macrolides (Figure 1) were isolated from the Madagascar Fascaplysinopsis sp. sponge collected in Salary Bay, ca. 100 km north of Tulear [1,2,3]. Among these metabolites the most abundant and active compound was salarin C (1), a potent inhibitor of proliferation of K562 leukemia cells (in concentration of 0.0005–0.5 μg/mL). The K562 cells underwent apoptotic death, as monitored by cell cycle analysis, annexin V/propidium iodine staining and caspase 3 and caspase 9 cleavage [1,2,3].

Representatives of Fascaplysinopsis sp. nitrogenous metabolites.

Interestingly, a remarkable change in the amounts of the different compounds was observed in various collections of the sponge. This, together with the resemblance of the functional groups to functionalities found in microorganism metabolites [1,2,3,4,5,6,7], suggested that the four groups originate from guest microorganisms rather than from the sponge itself.

The salarins contain seven functional groups which complicate the chemistry of these compounds. Among the currently identified ten naturally occurring salarins (A–J) the most active one is salarin C (1) which changes under light and air to salarin A (2) [2,8]. The suggested mechanism for this transformation is a singlet oxygen cleavage of the oxazole via a cycloaddition adduct [9,10]. The latter change, which occasionally occurs while performing chemical reactions with salarin C, complicated the chemistry even more. It was therefore advisable to compare reactions of salarin C with those of salarin A, vide infra. Among the more reactive moieties of 1 is the 16,17-vinyl epoxide which, as expected, is more reactive than the 12,13-epoxide, thus enabling regioselective reactions. Different openings of the vinyl epoxide were undertaken for: (a) A preparation of α-methoxy-α-trifluoromethylphenylacetic acid (MTPA) derivatives for determination of the absolute stereochemistry of 1; (b) Linking a spacer for immobilization of the molecule; (c) Preparation of more polar derivatives of 1 for SAR studies; and (d) Supporting the suggested biogenesis of tausalarin C (Figure 1) [4].

In five of the natural salarins the 16,17-epoxide is replaced by the corresponding vicinal 16,17- or 16,19-diol obtained, most likely, due to an allylic rearrangement.

 2. Results and Discussion

Selective acid catalyzed openings of the vinyl epoxide were disclosed with catalytic amounts of HClO4 in acetone or HCl in MeOH. The former provided the major compounds 16,17-diol 5 and its acetonide derivative 6, and the latter gave the 16-hydroxy-17-methoxy derivative 7 (Scheme 1). Other tested conditions afforded complex mixtures. According to the NMR data of the dieneoate moiety, which is very sensitive to the stereochemistry of the double bonds (e.g., the 8.31 ppm chemical shift of H-4) it was evident that this functional site stayed intact [11]. The same was the case with other moieties that did not take part in the reaction whose NMR data remained unchanged (for all intact sites ΔδH ± 0.1, ΔδC ± 0.5, the same is the case for the other derivatives) [11].

marinedrugs-11-04487-f009_Scheme 1

Acid catalyzed openings of the vinyl epoxide of salarin C (1).

Characteristic in the proton NMR spectra of compounds 5–7 were the disappearance of the 16,17-epoxide protons around 3 ppm and the appearance of methinoxy proton signals around 4 ppm (see Experimental and Supporting Information). All above reactions were very sensitive to the conditions and afforded low yields. It can be assumed that C-16 in compounds 5–7 maintains its (16R*) stereochemistry (protonation of the epoxide followed by opening of the O–C17 bond), which is doubtful for C-17, as the intermediate allylic C-17 carbocation can give two epimers due to a nucleophilic attack from both sides of the molecule and/or allylic rearrangement as shown, for e.g., in 9 (Scheme 1).

The 17 methoxy location was determined from COSY (H-15 to -18) and HMBC data from the methoxyl to C-17. A 16,17-threo configuration was deduced for acetonide 6 from the very close chemical shifts of the two acetonide methyl groups (28.7q and 1.44s for Me-29, 27.3q and 1.43s for Me-30, and 101.7s for C-28 of the acetonide), as well as the JH16,H17 = 7.8 Hz coupling constant [12], thus determining the 17S*-stereochemistry for both 5 and 6.

Treating salarin C with CAN (cerium ammonium nitrate) in acetonitrile (Scheme 1), a reaction suggested for opening radically vinyl epoxides to the corresponding diols [13], provided diol 8 and the allylic rearranged diol 9. As CAN is highly acidic it is not clear if the reagent or the accompanying HNO3 acid are responsible for the epoxide opening (Scheme 1). A single diastereoisomer was isolated; this was also the case with the other epoxide openings.

Another interesting opening of the 16,17-epoxide was with the freshly prepared Lewis acid MgBr2 in ether, conditions known to afford from vinyl epoxides the corresponding bromohydrins (Scheme 2) [14]. In the event, the expected bromohydrin (10) was initially indeed obtained as inferred from the MS. However, the compound was not stable and rearranged overnight to a stereoisomer of N-desacetyl salarin J (11) as concluded from the NMR data [6]. Namely, H-16 moved to 5.04 ppm (dd, J = 7.0, 5.1 Hz) and H-17 to 4.40 ppm (t, J = 6.8 Hz). C-16 was found to resonate at 75.4 and C-17 at 80.9 ppm. The chemical shifts of the intact moieties did not change. The main 2D correlations are depicted in Figure 2. A suggested mechanism for the rearrangement is given in Scheme 2. Because of the small J values in the THF ring, the stereochemistry around the ring could not be determined.

marinedrugs-11-04487-f010_Scheme 2

Mild Lewis acid opening of the vinyl epoxide with MgBr2 in ether, via intermediate 10.

Selected 2D-correlations of compound 11.

Worth mentioning is another opening of the 16,17-epoxide, namely, hydrogenation of salarin C which saturated the four double bonds, without affecting the oxazole, and opened up the 16,17-vinyl epoxide to the 17-alcohol (12, Scheme 3 and Figure 3). The 17-hydroxy location is suggested on the basis of COSY correlations from H-13 to H2-18 (see Experimental Section) and supported by HMBC correlations (Figure 3). Furthermore, the trans J12,13 = 2.2 Hz value confirmed that the 12,13-epoxide remained intact. Therefore, the configuration of C-17 could not be determined.

marinedrugs-11-04487-f011_Scheme 3

Hydrogenation of salarin C.

Selected HMBC correlations for 12.

The next studied reaction was an amine/Zn(ClO4)·6H2O opening of the 16,17-vinyl epoxide [15], which provided, as expected, the 16-hydroxy-17-amino-derivatives (13–18) (Scheme 4) [16]. The reaction was performed both on salarin C and salarin A. Unexpectedly, the two compounds behaved differently. While salarin C gave the expected 16-hydroxy-17-amino derivatives, via infra, (13–18), in the case of salarin A the epoxide remained intact while the triacyl moiety of the macrocycle of 2 (compound 19) was found to be more reactive and opened up as depicted in Scheme 5. The only changed site in the NMR was that of CH-16 and CH-17; e.g., for compound 14, H-16 moved to 3.60 ppm (t, J = 7.7 Hz), H-17 to 2.83 ppm (t, J = 7.7 Hz), C-16 resonated at 71.6 ppm and C-17 at 64.8 ppm.

marinedrugs-11-04487-f012_Scheme 4

Reactions of salarin C with different amines in the presence of Zn(ClO4)2·6H2O.

 marinedrugs-11-04487-f013_Scheme 5

Reaction of salarin A with morpholine in the presence of zinc perchlorate.

HMBC and selected COSY correlations for hydroxyl amine 14 are depicted in Figure 4 and similar 2D correlations were seen for 13 and 17. Compounds 15 and 16 lost the N-acetyl group. For compound 18, the acetyl carbamate was absent as seen in the 13C and 1H-NMR spectra (Figure 4).

Selected 2D-correlations of compound 14.

Carbonyls C-6 and C-7 of compound 19 moved up-field in the NMR spectrum from 171.9 to 164.0 and from 167.9 to 163.6, respectively. Evidence for change in the molecule was further obtained by FABMS m/z 796.1, (C39H55N3O13Na, M + Na+).

Observed NOEs between H-4 and H-5, between H-5 and H-28, and between NH-30 and H-8, as well as HMBC correlations from NH-30 to C-27, and from H-4 to C-6 differentiated between three possible cleavages of the triacylamine moiety (i.e., C-6/C-7; C-30/C-6 or C-30/C-7) determining the structure of 19 (Figure 5).

2D-NMR key correlations for the changed site of compound 19.

In addition to preliminary SAR studies of salarin C derivatives, the amination of salarin C was also directed to obtain a congener possessing a spacer for immobilization of salarin C (compounds 17 and 18, Scheme 4). However, as compounds 17 and 18 already lost activity, no further reactions were undertaken. Also, opening of the vinyl epoxide with an amine supported the earlier suggested biogenesis of tausalarin C, i.e., coupling between salarin A (or C) and pre-taumycin that, by a different route, affords taumycin A [4].

Next the preparation of a more polar derivative of salarin C was undertaken to try to improve its solubility in water. As was demonstrated in acidic conditions, salarins were also found highly sensitive in basic conditions. Short treatment of salarin C with different bases first gave the N-desacetyl derivative 20 (Scheme 6) characterized by the disappearance of the acetyl resonances in the 1H-NMR spectrum, while prolonging the reaction (2 h), using a stronger base, also led to hydrolysis of the octanoate ester (21, Scheme 6). Further elongation of the hydrolysis resulted in an unidentified complex mixture.

As shown above, in different reactions, salarin A behaved differently from salarin C. The same was the case with the hydrolysis. Again, in the first hydrolysis step the N-acetyl group split off to give compound 22, known naturally as salarin E (most likely together with methyl acetate which was not isolated) [6], while the second step was the opening of the azamacrocycle, providing the C-6 methyl ester–C-7 amide (23, Scheme 7).

The 1H-NMR spectrum of 23 showed a three proton singlet at 3.78 ppm, indicating the presence of a methyl ester, accompanied by the disappearance of the two acetates (C-25 and C-27 of 2).

marinedrugs-11-04487-f014_Scheme 6

Treatment of salarin C with base.

 marinedrugs-11-04487-f015_Scheme 7

Treatment of salarin A with base.

Two structures are possible for 23, i.e., in one C-6 is the ester and C-7 the amide, or vice versa. HMBC correlations from H-4, H-5 and the methoxyl to carbonyl C-6 and from H-8 to CH3-22 and to the amide carbonyl C-7 clarified the structure (23, Scheme 7); key CH-correlations are depicted in Figure 6.

After obtaining compound 21, its attachment to glucosamine (Scheme 8) was achieved via activation of the primary C(21)H2OH group through the p-nitrophenyl carbonate group (24), which was then converted to the sugar carbamate 25 (Scheme 8).

Selected 2D NMR correlations for the modified site of compound 23.

 marinedrugs-11-04487-f016_Scheme 8

Synthesis of salarin-aminosugar 25. Reagents and conditions: (a) bis(4-nitrophenyl) carbonate, Et3N, DMAP, CH2Cl2, 2 h; (b) glucosamine, Et3N, dry DMF, 2 h, rt.

The HRESI-MS and NMR data of 23 confirmed its structure. Downfield shifts of H-2′ of the glucosamine (∆δH = +0.9 ppm), compared to the starting free glucosamine (δH 2.67 m), and also of methylene H2-21 (δH 4.12 m) and HN-2′ (δH 5.16 s), as well as 3JCH correlations to the carbamate carbonyl (δC 155.9 s), evidenced the connection of the sugar 21.

Tulearin A (3) belongs to the second group of the Fascaplysinopsis sp. metabolites (tulearins A–C) [5] their cytotoxicity to K562 leukemia cells was lower than that of salarin C (72 h, 1 μM, ~60% inhibition of proliferation), while salarin C inhibited completely in this concentration [3,5]. However, it was decided to check the bioactivity of these derivatives.

In our previous report, Diels Alder reactions of the 18,20-diene of tulearin A, as well as a modification of the 8-carbamate to a crystalline carbonate, were reported [5]. For the absolute configuration determination of the tulearins we prepared regioselectively the 9-MTPA esters to apply the modified Mosher method [17]. The latter 9-ester was obtained exclusively without the 3-isomer [5]. Thus, it was interesting to find out where the other esterifications would take place. Acetylation, mesylation, tosylation, benzoylation and p-bromobenzoylation were performed. All first gave the 9-mono derivative (26–34), followed by a slower esterification of the 3-hydroxyl. The 9-hydroxyl esterification location became clear from the H-9 chemical shift to a lower field (Figure 7 and Experimental Section).

Tulearin A and modification positions (Tulearin A: R1,R2 = OH, R3 = H).

The only exception was the 3-carbonate obtained from the reaction of 3 with p-nitrophenyl chloroformate (PNPCl, 35, Table 1). To exclude/diminish the possibility that an initially obtained 9-isomer rearranges to the 3-isomer via a 3,9-bridge, reactions with oxalyl chloride and thionyl chloride were undertaken—no transannular bridge could be revealed.

In addition, the acylation of the carbamate was examined (33 and 34, Table 1).

marinedrugs-11-04487-t001_Table 1

Tulearin A derivatives.

Product R1 R2, R3 Yield(%)
26 Anhyrous Pyr, TsCl 0 °C, rt, 48 h OTs OTs 20%
27 OH OTs 35%
28 Et3N, p-bromobenzoylchloride, CH2Cl2, rt, 24 h OH 90%
29 MsCl, Pyr 0 °C, rt, 3 h OMs OMs 61%
30 Benzoyl chloride, Et3N, CH2Cl2, rt, 24 h OH 40%
31 Ac2O, Pyr, rt, 0.5 h OH OAc 60%
32 Ac2O, Pyr, rt, 48 h OAc OAc 90%
33 Ac2O, Pyr, DMAP (catalitic) rt, 7 days OAc R2 = OAc R3 = Ac 20%
34 Hexanoyl chloride, DMAP (catalitic), Et3N, CH2Cl2, rt, 48 h R2,R3 = 30%
35 PNPCl, DMAP, CH2Cl2, rt, 48 h OH 35%

Next, dimesyl 29 was transformed to the 3,9-diazido derivative (36) which was found, in low yields, to slowly transfer to the cycloadditon pyrolidine product 37 (Scheme 9) [18]. HRESI-MS established the formula C31H52N5O4, found m/z 558.4011 (MH+; calculated 558.4019).

The 15N-chemical shifts of the nitrogen atoms of three out of the five molecules of 37 were established by a 1H-15N-HMBC experiment, namely, correlations from H-2 and H-4 to N2 at 72 ppm, from H-3 to N3 at 243 ppm, and from H-8 and H-11 to N1 at 310 ppm. No coupling was observed between H-3 and N4, most likely because of too small a polarization transfer, thus the carbamate nitrogen was not disclosed (Figure 8) [18]. Furthermore, C-12 resonances, were as expected from an imine at δC 179.2 ppm, CH-3 at δC 61.8 and δH 3.78 ppm, and CH-9 at δC 74.8 and δH 4.20 ppm.

marinedrugs-11-04487-f017_Scheme 9

Change of dimesyl 29 to 37 via 36.

1H-15N-HMBC correlations for compound 37.

 3. Experimental Section 3.1. Extraction and Isolation [<xref ref-type="bibr" rid="B1-marinedrugs-11-04487">1</xref>,<xref ref-type="bibr" rid="B2-marinedrugs-11-04487">2</xref>,<xref ref-type="bibr" rid="B3-marinedrugs-11-04487">3</xref>,<xref ref-type="bibr" rid="B4-marinedrugs-11-04487">4</xref>,<xref ref-type="bibr" rid="B5-marinedrugs-11-04487">5</xref>,<xref ref-type="bibr" rid="B6-marinedrugs-11-04487">6</xref>,<xref ref-type="bibr" rid="B7-marinedrugs-11-04487">7</xref>]

For general methods, see Supporting Information.

 3.2. Salarin Derivatives

The NMR data of the salarin derivatives is given only for the transformed sites, for the rest of the functional moieties of the molecules the chemical shift changes of the various atoms were minimal (ΔδH ± 0.1, ΔδC ± 0.5). Thus for example the chemical shift of H-4 is very characteristic for the dienoate moiety and its surroundings (8.31 ppm). Full representative NMR spectra are given in the Supporting Information. The NMR data of the changed sites are given for clarity in order of the atom numbers.

3.2.1. Compounds <bold>5</bold> and <bold>6</bold>

To a mixture of salarin C (20 mg, 0.03 mmol) in acetone (5 mL), was added HClO4 (7%, 0.1 mL) at −20 °C and the reaction was stirred for 10 min. The reaction mixture was neutralized with aqueous NaHCO3 solution, evaporated and the residue diluted with water and extracted with DCM. The combined organic extract was dried over anhydrous MgSO4 and evaporated. The residue was purified by VLC (vacuum liquid chromatography, petroleum ether/ethyl acetate, 8:2) to afford 6, as a colorless oil, 3 mg (20%) and 5, a second colorless oil, 4.5 mg (21%). 6: [α]D23 +22 (c 0.1, CHCl3); NMR data for modified site (C14–C19): 1H-NMR (CDCl3, 400 MHz) δ 4.80 (dd, J = 8.7, 2.2 Hz, 1H, H-14), 5.61 (m, 1H, H-15), 4.00 (dd, J = 9.8, 7.8 Hz, 1H, H-16), 4.27 (t, J = 7.8 Hz, 1H, H-17), 5.41 (dd, J = 15.6, 8.1 Hz, 1H, H-18), 5.64 (m, 1H, H-19); acetonide moiety: 1.44 (s, 3H, H-29), 1.43 (s, 3H, H-30); 13C-NMR (CDCl3, 100 MHz) δ 76.4 d (C-14), 72.3 d (C-15), 78.3 d (C-16), 82.4 d (C-17), 127.5 (C-18), 131.8 d (C-19); acetonide moiety: 101.7 s (C-28), 28.7 q (Me-29), 27.3 q (Me-30). FABMS m/z 735.3, (C38H52N2O11Na, M + Na+); 5: [α]D23 +72 (c 0.05, CHCl3); NMR data for modified site (C14–C19): 1H-NMR (CDCl3, 400 MHz) δ 5.04 (t, J = 4.3 Hz, 1H, H-14), 5.44 (dd, J = 6.8, 4.3 Hz, 1H, H-15), 3.94 (t, J = 6.8 Hz, 1H, H-16), 4.37 (t, J = 7.9 Hz, 1H, H-17), 5.48 (dd, J = 15.4, 7.9 Hz, 1H, H-18), 5.77 (dt, J = 15.4, 7.9 Hz, 1H, H-19); 13C-NMR (CDCl3, 100 MHz) δ 73.1 d (C-14), 73.4 d (C-15), 78.3 d (C-16), 80.9 d (C-17), 128.3 d (C-18), 133.0 d (C-19). FABMS m/z 727.3, (C35H48N2O13Na, M + Na+).

 3.2.2. Compound <bold>7</bold>

A solution of salarin C (12 mg, 0.02 mmol) in MeOH (10 mL) was treated with methanolic HCl solution (0.05% v/v, 0.1 mL). The mixture was stirred at rt in the dark for 1 h. The reaction mixture was then neutralized with aqueous NaHCO3 solution, the solvent was evaporated and the residue diluted with water and extracted with DCM. The combined organic extract was dried over anhydrous MgSO4 and evaporated. The residue was purified by VLC (vacuum liquid chromatography, petroleum ether/ethyl acetate, 7:3) to afford 7, as a colorless oil, 8 mg (65%). [α]D24 +56 (c 0.26, CHCl3); NMR data for modified site (C14–C19): 1H-NMR (CDCl3, 500 MHz) δ 4.85 (dd, J = 10.8, 2.5 Hz, 1H, H-14), 5.53 (dd, J = 9.7, 2.5 Hz, 1H, H-15), 3.80 (dt, J = 9.7, 5.3 Hz, 1H, H-16), 3.40 (dd, J = 8.3, 5.3 Hz, 1H, H-17), 5.31 (dd, J = 15.5, 8.3 Hz, 1H, H-18), 5.57 (m, 1H, H-19), 3.18 (s, 3H, OMe); 13C-NMR (CDCl3, 125 MHz) δ 77.6 d (C-14), 71.4 d (C-15), 72.5 d (C-16), 82.1 d (C-17), 128.2 d (C-18), 131.9 d (C-19), 55.9 q (OMe). HR-ESIMS m/z calculated for C36H50N2O11Na (M + Na+) 709.3312, found 709.3322.

 3.2.3. Compounds <bold>8</bold> and <bold>9</bold>

A solution of salarin C (25 mg, 0.038 mmol) in CH3CN:H2O (3:1, 0.5 mL), was stirred with cerium ammonium nitrate (41 mg, 0.076 mmol) at rt for 3 h. The solvent was then concentrated in vaccum, H2O and DCM were added and the organic layer was dried over anhydrous MgSO4 and evaporated. The residue was purified by VLC (petroleum ether/ethyl acetate, 1:1) to afford 8, as a colorless oil, 7 mg (24%) and 9, a second colorless oil, 8 mg (35%). 8: [α]D24 +68 (c 0.26, CHCl3); NMR data for modified site (C14–C19): 1H-NMR (CDCl3, 500 MHz) δ 4.95 (dd, J = 9.3, 2.4 Hz, 1H, H-14), 5.55 (dd, J = 9.3, 3.9 Hz, 1H, H-15), 3.77 (ddd, J = 9.8, 6.2, 3.9 Hz, 1H, H-16), 4.07 (t, J = 6.2 Hz, 1H, H-17), 5.56 (dd, J = 15.8, 6.2 Hz, 1H, H-18), 5.72 (m, 1H, H-19); 13C-NMR (CDCl3, 100 MHz) δ 77.2 d (C-14), 71.6 d (C-15), 72.5 d (C-16), 70.7 d (C-17), 130.4 d (C-18), 129.5 d (C-19); FABMS m/z 673.3, (C35H49N2O11, MH+). 9: [α]D23 +70 (c 0.2, CHCl3); NMR data for modified site (C14–C19): 1H-NMR (CDCl3, 400 MHz) δ 4.89 (dd, J = 8.6, 2.2 Hz, 1H, H-14), 5.48 (dd, J = 8.6, 2.2 Hz, 1H, H-15), 4.45 (m, 1H, H-16), 5.67 (dd, J = 15.5, 6.1 Hz, 1H, H-17), 5.74 (m, 1H, H-18), 4.15 (m, 1H, H-19); 13C-NMR (CDCl3, 100 MHz) δ 76.4 d (C-14), 72.9 d (C-15), 71.2 d (C-16), 127.3 d (C-17), 136.4 d (C-18), 67.6 d (C-19); HR-ESIMS m/z calculated for C35H48N2O11Na (M + Na+) 695.3150, found 695.3135.

 3.2.4. Compound <bold>11</bold>

MgBr2 was prepared from magnesium turnings (100 mg) in dry diethyl ether (10 mL) and dibromoethane (1.5 mL). A solution of salarin A (21 mg, 0.03 mmol) in dry diethyl ether (1 mL), was added dropwise to the 0 °C mixture of the MgBr2. The solution was stirred for 2.5 h, then water added and the solution extracted with diethyl ether. The organic layer was washed with brine, dried over anhydrous MgSO4 and the solvents were evaporated. The residue was purified by VLC (petroleum ether/ethyl acetate, 1:1) to afford a bromohydrin product which was unstable; ESIMS m/z 789.2, (C34H45BrN2O12Na, M + Na+). After 3 h, a second VLC (PE/EA, 1:1) afforded 11, 8 mg (40%). NMR data for modified site (C13–C19): 1H-NMR (CDCl3, 500 MHz) δ 3.03 (brt, J = 2.4 Hz, 1H, H-13), 4.21 (brt, J = 2.4 Hz, 1H, H-14), 5.34 (dd, J = 5.1, 2.4 Hz, 1H, H-15), 5.04 (dd, J = 7.0, 5.1, 1H, H-16); 4.40 (t, J = 6.8 Hz, 1H, H-17); 5.56 (dd, J = 14.9, 6.8 Hz, 1H, H-18); 5.79 (m, 1H, H-19); 13C-NMR (CDCl3, 125 MHz) δ 55.0 d (C-13), 80.0 d (C-14), 74.5 d (C-15), 75.4 d (C-16), 80.9 d (C-17), 128.9 d (C-18), 130.2 d (C-19). FABMS m/z 709.3, (C35H46N2O12Na, M + Na+).

 3.2.5. Compound <bold>12</bold>

A solution of salarin C (10 mg, 0.015 mmol) in EtOH (5 mL) with catalytic amounts of Pd/C (5%) was hydrogenated for 30 min at 1 atmosphere to afford 12, as a colorless oil, 8 mg (80%). [α]D23 −22 (c 0.14, CHCl3); NMR data for modified site (C14–20, C26): 1H-NMR (CDCl3, 500 MHz) δ 2.50 (m, 2H, H-8), 2.13 (m, 1H, H-9), 1.84 (m, 2H, H-10), 3.32 (dd, J = 4.4, 2.2 Hz, H-13), 4.77 (dd, J = 7.5, 2.9 Hz, 1H, H-14), 5.34 (dd, J = 7.5, 3.2 Hz, 1H, H-15), 2.74 (dd, J = 14.9, 3.2 Hz, 1Ha, H-16), 2.49 (m, 1Hb, H-16), 3.71 (m, 1H, H-17), 2.39 (m, 2H, H-18), 1.73 (m, 2H, H-19), 1.64 (m, 2H, H-20); COSY correlations and J values supported the identification of the later protons.

13C-NMR (CDCl3, 125 MHz) δ 172.2 s (C-1), 33.1 t (C-2), 25.7 t (C-3), 31.6 t (C-4), 23.9 t (C-5), 133.2 s (C-6), 161.2 s (C-7), 35.4 t (C-8), 29.8 d (C-9), 30.6 t (C-10), 76.3 d (C-14), 75.8 d (C-15), 34.3 t (C-16), 71.1 d (C-17), 35.1 t (C-18), 22.6 t (C-19), 28.5 t (C-20), 142.3 s (C-26). FABMS m/z 687.3, (C35H56N2O10Na, M + Na+).

 3.2.6. Compound <bold>13</bold>

A mixture of salarin C (30 mg, 0.043 mmol), morpholine (6 mg, 0.08 mmol) and Zn(ClO4)2·6H2O (0.3 mg, 2 mol%) in dry DCM (2 mL) was stirred at rt under argon in the dark for 48 h. After completion of the reaction (TLC), DCM (5 mL) was added and the mixture washed with water, dried over anhydrous MgSO4 and evaporated. The residue was purified by VLC (petroleum ether/ethyl acetate, 3:2) to afford 13, as a yellow oil, 28 mg (78%). [α]D27 +14 (c 0.13, CHCl3); NMR data for modified site (C14–C19, C25): 1H-NMR (CDCl3, 500 MHz) δ 4.93 (dd, J = 8.9, 2.5 Hz, 1H, H-14), 5.48 (m, 1H, H-15), 3.85 (t, J = 9.2 Hz, 2H, H-16), 2.80 (t, J = 9.2 Hz, 1H, H-17), 5.27 (dd, J = 15.4 Hz, 9.2, 2H, H-18), 5.47 (m, 2H, H-19), 2.44 (s, 3H, H-25); morpholine moiety: 2.52 (m, 4H, CH2N), 3.68 (m, 4H, CH2O); 13C-NMR (CDCl3, 125 MHz) δ 77.8 d (C-14), 73.7 d (C-15), 66.9 d (C-16), 71.5 d (C-17), 125.7 d (C-18), 132.8 d (C-19), 23.4 d (C-25); morpholine moiety: 48.6 t (CH2N), 66.9 t (CH2O). HR-ESIMS m/z calculated for C39H56N3O11 (MH+) 742.3915, found 742.3917.

 3.2.7. Compound <bold>14</bold>

A mixture of salarin C (50 mg, 0.043 mmol), n-butylamine (18 mg, 0.2 mmol) and Zn(ClO4)2·6H2O (0.6 mg, 2 mol%) in dry DCM (4 mL) was stirred at rt under argon in the dark for 48 h. After completion of the reaction (TLC), DCM was added (5 mL), and the mixture was washed with water, dried over anhydrous MgSO4 and evaporated. The residue was purified by VLC (petroleum ether/ethyl acetate, 1:1) to afford 14, as a yellow oil, 7 mg (12%). [α]D27 +30 (c 0.17, CHCl3); NMR data for modified site (C14–C19, C25): 1H-NMR (CDCl3, 500 MHz) δ 4.91 (dd, J = 8.8, 2.0 Hz, 1H, H-14), 5.45 (dd, J = 7.7, 2.0 Hz, 1H, H-15), 3.60 (t, J = 7.7 Hz, 1H, H-16), 2.83 (t, J = 7.7 Hz, 1H, H-17), 5.21 (dd, J = 15.0, 8.8 Hz, 1H, H-18), 5.40 (dd, J = 15.0, 6.5 Hz, 1H, H-19), 2.42 (s, 3H, H-25); butylamine moiety: 2.61 (m, CH2NH), 2.34 (m, CH2NH), 1.41 (m, 2H, H-2″); 1.31 (m, 2H, H-3″), 0.92 (t, J = 7.3 Hz, CH3); 13C-NMR (CDCl3, 125 MHz) δ 78.4 d (C-14), 73.8 d (C-15), 71.6 d (C-16), 64.8 d (C-17), 131.0 d (C-18), 129.9 d (C-19), 23.9 d (C-25); butylamine moiety: 46.5 t (CH2NH), 32.3 t (CH2, C-2″), 20.2 t (CH2, C-3″), 14.8 q (CH3). HR-ESIMS m/z calculated for C39H58N3O10 (MH+) 728.4122, found 728.4130.

 3.2.8. Compound <bold>15</bold>

A mixture of salarin C (20 mg, 0.03 mmol), benzylamine (12 mg, 0.09 mmol) and Zn(ClO4)2·6H2O (0.3 mg, 2 mol%) in dry DCM (4 mL) was stirred at rt under argon in the dark for 72 h. After completion of the reaction (TLC), DCM was added and the mixture was washed with water, dried over anhydrous MgSO4 and evaporated. The residue was purified by VLC (petroleum ether/ethyl acetate, 3:7) to afford 15, as a yellow oil, 9 mg (36%). [α]D27 30 (c 0.17, CHCl3); NMR data for modified site (C14–C19): 1H-NMR (CDCl3, 500 MHz) δ 4.81 (dd, J = 8.6, 2.0 Hz, 1H, H-14), 5.47 (m, 1H, H-15), 3.71 (m, 1H, H-16), 2.93 (t, J = 7.6 Hz, 1H, H-17), 5.26 (dd, J = 16.0, 8.1 Hz, 1H, H-18), 5.50 (m, 1H, H-19); benzylamine moiety: δ 3.78 (d, J = 12.8 Hz, 1Ha, H-26), 3.54 (d, J = 12.8 Hz, 1Hb, H-26), 7.31 (m, 2H, H-28,30), 7.31 (m, 2H, H-29,31), 7.31 (m, 1H, H-30); 13C-NMR (CDCl3, 125 MHz) δ 76.7 d (C-14), 74.3 d (C-15), 72.4 d (C-16), 64.8 d (C-17), 130.7 d (C-18), 130.9 d (C-19), 156.3 s (C-23); benzylamine moiety: 50.9 t (C-26), 140.0 s (C-27), 129.2 d (C-28,32), 129.3 d (C-29,31), 127.8 d (C-30). HR-ESIMS m/z calculated for C40H54N3O9 (MH+) 720.3860, found 720.3865.

 3.2.9. Compounds <bold>16</bold> and <bold>18</bold>

A mixture of salarin C (25 mg, 0.038 mmol), NH-Boc-hexane-amine (30 mg, 0.11 mmol) and Zn(ClO4)2·6H2O (0.3 mg, 2 mol%) in dry DCM (2 mL) was stirred at rt under argon in the dark for 72 h. After completion of the reaction (TLC), DCM was added and the mixture was washed with water, dried over anhydrous MgSO4 and evaporated. The residue was purified by VLC (petroleum ether/ethyl acetate, 3:7) to afford 16, as a colorless oil, 8 mg (25%) and 18, a second colorless oil, 4 mg (13%). 16: [α]D27 +50 (c 0.15, CHCl3); NMR data for modified site (C14–C19, C23): 1H-NMR (CDCl3, 500 MHz) δ 4.85 (d, J = 9.0 Hz, 1H, H-14), 5.43 (m, 1H, H-15), 3.61 (brt, J = 7.0 Hz, 1H, H-16), 2.86 (brt, J = 7.0 Hz, 1H, H-17), 5.27 (m, 1H, H-18), 5.541 (m, 1H, H-19); NH-Boc-diaminehexane moiety: 2.62 (m, 2H, CH2NH), 2.35 (m, 2H, CH2NH), 1.40 (m, 2H), 1.32 (m, 2H), 1.30 (m, 2H), 1.40 (m, 2H), 3.10 (m, 2H, CH2NH-Boc), 1.44 (s, 9H, CH3); 13C-NMR (CDCl3, 125 MHz) δ 75.1 d (C-14), 72.5 d (C-15), 70.4 d (C-16), 63.2 d (C-17), 130.6 d (C-18), 129.4 d (C-19); 155.1 s (C-23); NH-Boc-diaminehexane moiety: δ 45.4 t (CH2NH), 29.6 t (CH2), 25.5 t (CH2), 25.4 t (CH2), 29.6 t (CH2), 39.2 t (CH2NH-Boc), 155.6 s (CO), 78.9 s (C), δC 26.7 q (3CH3). HR-ESIMS m/z calculated for C44H70N4O11 (MH+) 829.4963, found 829.4969; 18: [α]D27 +53 (c 0.13, CHCl3); NMR data for modified site (C14–C19, C23): 1H-NMR (CDCl3, 500 MHz) δ 3.94 (m, 1H, H-14), 5.27 (m, 1H, H-15), 3.69 (brt, J = 7.1 Hz, 1H, H-16), 2.92 (brt, J = 7.5 Hz, 1H, H-17), 5.31 (dd, J = 15.1, 7.5 Hz, 1H, H-18), 5.45 (dt, J = 15.1, 6.3 Hz, 1H, H-19); NH-Boc-diaminehexane moiety: 2.61 (m, 1Ha, CH2NH), 2.34 (m, 1Hb, CH2NH), 1.48 (m, 2H), 1.34 (m, 2H), 1.30 (m, 2H), 1.40 (m, 2H), 3.11 (m, 2H, CH2NH-Boc), 1.48 (s, 9H, CH3); 13C-NMR (CDCl3, 125 MHz) δ 71.5 d (C-14), 75.8 d (C-15), 71.8 d (C-16), 63.3 d (C-17), 125.5 d (C-18), 130.6 d (C-19); NH-Boc-diaminehexane moiety: 45.9 t (CH2NH), 30.0 t (CH2), 25.5 t (CH2), 25.5 t (CH2), 30.3 t (CH2), 39.8 t (CH2NH-Boc), 156.0 s (CO), 79.0 s (C), δC 27.0 q (3CH3). HR-ESIMS m/z calculated for C43H68N3O10 (MH+) 786.4905, found 786.4918.

 3.2.10. Compound <bold>17</bold>

A mixture of salarin C (16 mg, 0.024 mmol), 1,6-diaminohexane (8.5 mg, 0.07 mmol) and Zn(ClO4)2·6H2O (0.2 mg, 2 mol%) at 60 °C, neat, was stirred under argon in the dark for 20 min. After completion of the reaction (TLC), DCM was added and the mixture was washed with water, dried over anhydrous MgSO4, and evaporated to afford 17, as a colorless oil, 13 mg (70%). [α]D27 +50 (c 0.3, CHCl3); NMR data for modified site (C14–C19, C25): 1H-NMR (CDCl3, 500 MHz) δ 4.84 (d, J = 9.3 Hz, 1H, H-14), 5.47 (d, J = 9.3 Hz, 1H, H-15), 3.60 (brt, J = 7.6 Hz, 1H, H-16), 2.82 (brt, J = 7.6 Hz, 1H, H-17), 5.23 (dd, J = 15.3, 9.1 Hz, 1H, H-18), 5.42 (m, 1H, H-19), 2.01 (s, 3H, H-25); 1,6-diaminehexane moiety: δ 2.62 (m, 1Ha, CH2NH), 2.31 (m, 1Hb, CH2NH), 1.44 (m, 2H), 1.32 (m, 2H), 1.30 (m, 2H), 1.44 (m, 2H), 2.69 (m, 2H, CH2NH2); 13C-NMR (CDCl3, 125 MHz) δ 75.4 d (C-14), 73.1 d (C-15), 70.1 d (C-16), 63.1 d (C-17), 130.8 d (C-18), 129.0 d (C-19), 155.7 s (C-23), 170.2 s (C-24), 24.8 q (C-25); 1,6-diaminehexane moiety: δ 45.8 t (CH2NH), 32.5 t (CH2), 26.1 t (CH2), 26.1 (CH2), 32.5 t (CH2), 41.3 t (CH2NH2); HR-ESIMS m/z calculated for C41H63N4O10 (MH+) 771.4544, found 771.4547.

 3.2.11. Compound <bold>19</bold>

A mixture of salarin A (30 mg, 0.043 mmol), morpholine (6 mg, 0.08 mmol) and Zn(ClO4)2·6H2O (0.3 mg, 2 mol%) in dry DCM (2 mL) was stirred at rt under argon. After completion of the reaction (72 h, TLC), the mixture was diluted with DCM (5 mL), washed with water, dried over anhydrous MgSO4 and then evaporated. The crude residue was purified by VLC (petroleum ether/ethyl acetate, 4:6) to afford 19, as a yellow oil, 28 mg (78%). [α]D23 −30 (c 0.16, CHCl3); NMR data for modified site (C1–7, C26,27): 1H-NMR (CDCl3, 500 MHz) δ 5.90 (m, 1H, H-2), 6.70 (t, J = 11.8 Hz, 1H, H-3), 8.19 (dd, J = 15.0, 11.8 Hz, 1H, H-4), 6.57 (d, J = 15.0 Hz, 1H, H-5), 2.34 (s, 3H, H-27), 8.15 (s, 1H, NH); Morpholine: 3.71 (m, 4H, 2 × CH2O), 3.74 (m, 4H, 2 × CH2NH); 13C-NMR (CDCl3, 100 MHz) δ 164.1 s (C-1), 121.9 d (C-2), 140.0 d (C-3), 137.4 d (C-4), 126.6 d (C-5), 164.0 s (C-6), 163.6 s (C-7), 171.9 s (C-26), 23.8 q (C-27); Morpholine: 66.0 t (CH2O), 42.5 t (CH2NH); FABMS m/z 796.1, (C39H55N3O13Na, M + Na+).

 3.2.12. Compound <bold>20</bold>

To a solution of salarin C (10 mg, 0.015 mmol) in MeOH (2 mL) was added aqueous ammonia (0.5 mL). The mixture was stirred 3 h at rt and then evaporated. The residue was purified by VLC (petroleum ether/ethyl acetate, 1:1) to afford 20, as a colorless oil, 7 mg (87%). [α]D25 −38 (c 0.17, CHCl3); NMR data for modified site (C13–C15, C23): 1H-NMR (CDCl3, 500 MHz) δ 3.43 (dd, J = 8.6, 1.8 Hz, 1H, H-13), 4.60 (m, 1H, H-14), 5.44 (dd, J = 8.6, 2.6 Hz, 1H, H-15); 13C-NMR (CDCl3, 125 MHz) δ 55.1 d (C-13), 76.4 d (C-14), 68.3 d (C-15), 155.2 s (C-23). HR-ESIMS m/z calculated for C33H44N2O9Na (M + Na+) 635.2945, found 635.2947.

 3.2.13. Compound <bold>21</bold>

To a solution of salarin C (20 mg, 0.03 mmol) in MeOH (0.5 mL) was added methanolic K2CO3 (5% v/v, 1 mL). The reaction was stirred for 2 h at rt and then EA (5 mL) was added, the organic layer was washed with saturated NH4Cl, dried over anhydrous Na2SO4 and evaporated. The residue was purified by VLC (petroleum ether/ethyl acetate, 3:7) to afford 21, as a major product, colorless oil, 9 mg (62%) accompanied with traces of 20 and (2Z,4E)-dimethyl hexa-2,4-dienedioate; NMR data for modified site (C18–C21, C23): 1H-NMR (CDCl3, 400 MHz) δ 5.54 (dd, J = 4.1, 15.6 Hz, 1H, H-18), 5.86 (ddd, J = 15.6, 8.8, 5.2 Hz, 1H, H-19), 2.33 (m, 2H, H-20), 3.67 (m, 1Ha, H-21), 3.59 (ddd, J = 11.7, 6.8, 4.1 Hz, 1Hb, H-21); 13C-NMR (CDCl3, 100 MHz) δ 123.9 d (C-18), 133.7 d (C-19), 36.2 t (C-20), 61.5 t (C-21), 159.1 s (C-23). HR-ESIMS m/z calculated for C25H30N2O8Na (M + Na+) 509.1900, found 509.1898.

 3.2.14. Compound <bold>22</bold>

To a solution of salarin A (11 mg, 0.016 mmol) in EtOH (5 mL) was added NH4Cl (2.5 mg, 0.048 mmol) and NaN3 (3.1 mg, 0.048 mmol). The solution was slowly warmed up to 60 °C for 1 h, and then allowed to cool down to room temperature. The reaction mixture was filtered off, the solid residue washed with EtOH, and the solvent was evaporated. The residue was dissolved in DCM (50 mL) and washed with H2O (30 mL). The organic layer was dried over anhydrous Na2SO4 and then evaporated to afford 22, as a yellow oil, 8 mg (80%). The NMR-data is identical to natural salarin E [6].

 3.2.15. Compound <bold>23</bold>

To a solution of salarin A (10 mg, 0.015 mmol) in MeOH (2 mL), was added NH4OH (0.5 mL, 0.1 mmol). The reaction mixture was stirred for 3 h at room temperature and then evaporated. The residue was purified by LH-20 chromatography (petroleum ether/DCM/MeOH, 2:1:1) to afford 23, as a yellow oil, 7.5 mg (78%). [α]D23 −21 (c 0.2, CHCl3); NMR data for modified site (C1–9, C14, C22,23): 1H-NMR (CDCl3, 500 MHz) δ 5.98 (d, J = 11.2 Hz, 1H, H-2), 6.68 (t, J = 11.2 Hz, 1H, H-3), 8.29 (dd, J = 15.3, 11.2 Hz, 1H, H-4), 6.13 (d, J = 15.3 Hz, 1H, H-5), 5.66 (d, J = 8.4 Hz, 1H, H-8), 4.90 (dd, J = 6.6, 3.6 Hz, 1H, H-14), 1.88 (s, 3H, H-22), 3.78 (s, 3H, OMe); δ 13C-NMR (CDCl3, 125 MHz) δ 163.9 s (C-1), 124.1 d (C-2), 141.5 d (C-3), 138.9 d (C-4), 129.3 d (C-5), 166.8 s (C-6), 168.7 s (C-7), 118.7 d (C-8), 155.7 s (C-9), 74.3 d (C-14), 25.1 q (C-22), 155.8 s (C-23), 52.3 q (OMe). HR-ESIMS m/z calculated for C32H46N2O11Na (M + Na+) 657.2999, found 657.3004.

 3.2.16. Compound <bold>24</bold>

To a solution of 21 (20 mg, 0.04 mmol) in DCM (2 mL) was added triethylamine (20 μL, 0.12 mmol), DMAP (0.48 mg, 0.004 mmol) and bis(4-nitrophenyl) carbonate (20 mg, 0.06 mmol). The mixture was stirred for 2 h at rt and then DCM was added and the mixture was washed with brine. The organic layer was dried over anhydrous MgSO4 and evaporated. The residue was purified by VLC (petroleum ether/ethyl acetate, 4:6) to afford 24, as a colorless oil, 12 mg (46%). [α]D25 +117 (c 0.07, CHCl3); NMR data for modified site (C21-PNP): 1H-NMR (CDCl3, 400 MHz) δ 4.38 (t, J = 6.6 Hz, 2H, H-21); PNP moiety: δ 8.27 (m, J = 8.8 Hz, 1H, H-2′), 7.34 (m, J = 8.8 Hz, 1H, H-3′); 13C-NMR (CDCl3, 100 MHz) δ 68.2 t (C-21); PNP moiety: δ 152.15 s (CO), 155.3 s (C-1′), 121.9 d (C-2′), 125.2 d (C-3′), 145.9 s (C-4′); ESIMS m/z 674.45, (C32H33N3O12Na, M + Na+).

 3.2.17. Compound <bold>25</bold>

To a mixture of d(+)-glucosamine and NaOMe (7 mg, 0.035 mmol) in dry DMF (0.5 mL) was added Et3N (15 μL, 0.1 mmol) followed by 24 (10 mg, 0.014 mmol) in dry DMF (0.2 mL) and the mixture was stirred for 2 h at rt. The solvent was evaporated and the residue was purified by LH-20 chromatography (petroleum ether/DCM/MeOH, 2:1:1) to afford 25, as a colorless oil, 8 mg (80%). [α]D25 +206 (c 0.2, MeOH); NMR data for modified site (C14–C19): 1H-NMR (d4-MeOH, 500 MHz) δ 5.13 (d, J = 3.8 Hz, 1H, H-1′), 4.12 (m, 2H, H-21), 3.57 (m, J = 10.4, 3.8 Hz, H-2′), 3.64 (m, 1H, H-3′), 3.89 (m, 1H, H-4′), 3.39 (m, 1H, H-5′), 3.63 (m, 1Ha, H-6′), 3.69 (m, 1Hb, H-6′); 13C-NMR (d4-MeOH, 125 MHz) δ 63.2 t (C-21), 155.9 s (CO), 91.3 d (C-1′), 56.5 d (C-2′), 71.6 d (C-3′), 71.4 d (C-4′), 70.8 d (C-5′), 61.4 t (C-6′). HR-ESIMS m/z calculated for C32H41N3O14Na (M + Na+) 714.2486, found 714.2489.

 3.3. Tulearin Derivatives 3.3.1. Compounds <bold>26</bold> and <bold>27</bold>

To a mixture of tulearin A (222 mg, 0.415 mmol) and pyridine (1 mL) in chloroform (3 mL), was added TsCl (160 mg, 0.8 mmol) at 0 °C. After 48 h of stirring, EA was added and the organic phase was washed with saturated NH4Cl, water and brine, dried over anhydrous MgSO4 and evaporated. The residue was purified by VLC (petroleum ether/ethyl acetate, 1:1) to afford ditosyl 26, as a colorless oil, 52 mg (20%) and 9-monotosyl 27, a second colorless oil, 43 mg (35%). 26: [α]D25 +15 (c 0.4, CHCl3); NMR data for modified site (C3–C10): 1H-NMR (CDCl3, 500 MHz) δ 4.50 (m, 1H, H-3), 1.56 (m, 1H, H-4), 4.78 (m, 1H, H-8), 4.69 (m, 1H, H-9), 1.57 (m, 1H, H-10);. 3-Ts: δ 7.35 (d, 2H, H-33,34), 7.79 (d, 2H, H-35,36), 2.45 (s, 3H, H-38); 9-Ts: δ 7.35 (d, 2H, H-33,34), 7.79 (d, 2H, H-35,36), 2.45 (s, 3H, H-38); 13C-NMR (CDCl3, 125 MHz) δ 80.4 d (C-3), 35.3 t (C-4), 72.1 d (C-8), 81.5 d (C-9), 31.3 t (C-10); 3-Ts: δ 127.5 s (C-32), 129.9 d (C-33,34), 127.8 d (C-35,36), 137.0 s (C-37), 21.0 q (C-38); 9-Ts: δ 127.5 s (C-32), 129.9 d (C-33,34), 127.8 d (C-35,36), 137.0 s (C-37), 21.0 q (C-38). HR-ESIMS m/z calculated for C45H65NO10S2Na (M + Na+) 866.3948, found 866.3954. 27: [α]D25 +17 (c 0.2, CHCl3); NMR data for modified site (C8–C10): 1H-NMR (CDCl3, 500 MHz) δ 4.78 (m, 1H, H-8), 4.69 (m, 1H, H-9), 1.57 (m, 1H, H-10); 9-monoTs: δ 7.35 (d, 2H, H-33,34), 7.79 (d, 2H, H-35,36), 2.45 (s, 3H, H-38); 13C-NMR (CDCl3, 125 MHz) δ 72.1 d (C-8), 81.5 d (C-9), 31.3 t (C-10); 9-monoTs: δ 127.5 s (C-32), 129.9 d (C-33,34), 127.8 d (C-35,36), 137.0 s (C-37), 21.0 q (C-38); HR-ESIMS m/z calculated for C38H59NO8NaS (M + Na+) 712.3859, found 712.3860.

 3.3.2. Compound <bold>28</bold>

To a solution of tulearin A (10 mg, 0.025 mmol) in DCM (2 mL) were added Et3N (5.2 mg, 0.05 mmol) and p-bromobenzoylchloride (11.3 mg, 0.05 mmol). The mixture was stirred at room temperature for 24 h. DCM was then added (5 mL) and the mixture was washed with saturated NH4Cl, water and brine, dried over anhydrous MgSO4 and evaporated. The residue was purified by VLC (petroleum ether/ethyl acetate, 1:1) to afford 28, as a colorless oil, 7.2 mg (40%). [α]D25 +27 (c 0.2, CHCl3); NMR data for modified site (C8–C10): 1H-NMR (CDCl3, 500 MHz) δ 4.95 (q, J = 5.3 Hz, 1H, H-8), 5.29 (q, J = 5.3 Hz, 1H, H-9), 1.62 (m, 2H, H-10); Aryl group (Ar): δ 7.90 (d, J = 8.3 Hz, 2H, H-34,35), 7.57 (d, J = 8.3 Hz, H-36,37); 13C-NMR (CDCl3, 125 MHz) δ 74.3 d (C-8), 73.4 d (C-9), 31.4 t (C-10); Aryl group (Ar): δ 165.2 s (C-32), 137.0 s (C-33), 131.9 d (C-34,35), 131.4 d (C-36,37), 128.0 s (C-38). FABMS m/z 718.0, (C38H57BrNO7, MH+).

 3.3.3. Compound <bold>29</bold>

Mesyl chloride (80 mg, 0.7 mmol) was added to a cold solution (0 °C) of tulearin A (140 mg, 0.26 mmol) in pyridine (0.14 mL). The mixture was stirred at rt for 3 h. Then, water was added and the mixture extracted with DCM. The organic phase was dried over anhydrous MgSO4 and evaporated. The residue was purified by VLC (petroleum ether/ethyl acetate, 4:1) to afford 29, as a colorless oil, 110 mg (61%). [α]D25 +15 (c 0.5, CHCl3); NMR data for modified site: 1H-NMR (CDCl3, 500 MHz) δ 5.00 (brd, J = 9.0 Hz, 1H, H-3), 3.08 (s, CH3-Ms), 5.21 (brd, J = 8.9 Hz, 1H, H-9), 3.05 (s, 3H, CH3-Ms); 13C-NMR (CDCl3, 125 MHz) δ 80.6 d (C-3), 40.9 q (CH3-Ms), 80.9 d (C-9), 38.9 q (CH3-Ms); CIMS m/z 692.0, (C33H58NO10S2, MH+).

 3.3.4. Compound <bold>30</bold>

To a solution of tulearin A (22 mg, 0.044 mmol) in DCM (0.5 mL) were added Et3N (18 μL, 0.13 mmol) and catalytic amounts of DMAP at 0 °C. Benzoyl chloride was then added (20 μL, 0.17 mmol) and the mixture was warmed up slowly to rt. After 2.5 h, the reaction mixture was neutralized by acetic acid to pH 7 and partitioned between ethyl acetate/H2O. The organic layer was washed with sat. NH4Cl, dried over anhydrous MgSO4 and evaporated. The residue was purified by VLC (PE/EA, 1:1) to afford 30, as a colorless oil, 19 mg (70%). NMR data for modified site (C8–C10): 1H-NMR (CDCl3, 500 MHz) δ 5.02 (dt, J = 5.6, 6.2 Hz, 1H, 1H, H-8), 5.37 (dt, J = 5.6, 5.9 Hz, 1H, H-9), 1.78 (m, 2H, H-10); Benzoyl group: δ 7.57 (d, J = 7.8 Hz, 1H), 8.06 (d, J = 7.8 Hz, 2H), 7.45 (t, J = 7.8 Hz, 2H); 13C-NMR (CDCl3, 125 MHz) δ 73.9 d (C-8), 72.55 d (C-9), 28.9 t (C-10); Benzoyl group: δ 166.6 s (CO), 132.6 d (CH), 129.4 d (CH, 2H), 127.9 d (CH, 2H); HR-ESIMS m/z calculated for C38H57NO7Na (M + Na+) 662.4033, found 662.4030.

 3.3.5. Compound <bold>31</bold>

To a mixture of pyridine (0.1 mL, 1.06 mmol) and acetic anhydride (0.1 mL, 1.2 mmol) was added tulearin A (43 mg, 0.086 mmol) at rt. After 20 min the solvents were evaporated and the residue was purified by VLC (petroleum ether/ethyl acetate, 3:2) to afford 31, as major product, accompanied by 10% of 32, and 10% tulearin A. 31: colorless oil, 30 mg (61%); [α]D25 +27 (c 0.3, CHCl3); NMR data for modified site (C7–C10): 1H-NMR (CDCl3, 500 MHz) δ 1.62 (m, 2H, H-7), 4.84 (dt, J = 10.5, 5.8 Hz, 1H, H-8), 5.13 (dd, J = 6.5, 10.5 Hz, 1H, H-9), 1.47 (m, 2H, H-10); acetate group: δ 2.09 (s, CH3); 13C-NMR (CDCl3, 125 MHz) δ 29.5 t (C-7), 73.9 d (C-8), 71.2 d (C-9), 29.7 t (C-10); acetate group: δ 171.0 s (CO), 19.9 q (CH3); HR-ESIMS m/z calculated for C33H55NO7Na (M + Na+) 600.3876, found 600.3876.

 3.3.6. Compound <bold>32</bold>

To a mixture of pyridine (0.5 mL, 5.3 mmol) and acetic anhydride (0.5 mL, 6.0 mmol), was added tulearin A (20 mg, 0.04 mmol) at rt. After 20 min the solvents were evaporated and the residue was purified by VLC (petroleum ether/ethyl acetate, 4:1) to afford 32, as a colorless oil, 22 mg, (90%); [α]D25 +26 (c 0.3, CHCl3); NMR data for modified site C3 and C9: 1H-NMR (CDCl3, 500 MHz) δ 5.19 (m, 1H, H-3); 3-acetate group: δ 2.10 (s, 3H, CH3), 5.10 (q, J = 6.7 Hz, 1H, H-9); 9-acetate group: δ 2.11 (s, 3H, CH3); 13C-NMR (CDCl3, 125 MHz) δ 71.6 d (C-3); 3-acetate group: δ 170.5 s (CO), 21.3 q (CH3); 71.3 d (C-9); 9-acetate group: δ 170.1 s (CO), 21.1 q (CH3); FABMS m/z 620.9, (C35H58NO8, MH+).

 3.3.7. Compound <bold>33</bold>

To a mixture of pyridine (0.1 mL, 1.06 mmol) and acetic anhydride (0.1 mL, 1.02 mmol) with catalytic amounts of DMAP (0.43 mg, 0.004 mmol), was added tulearin A (18 mg, 0.036 mmol) at rt. After 7 days the solvents were evaporated and the residue was purified by VLC (petroleum ether/ethyl acetate, 4:1) to afford 33, as major product, accompanied by 10% of 32; 33: colorless oil, 9 mg (40%). NMR data for modified site: 1H-NMR (CDCl3, 500 MHz) δ 5.18 (m, 1H, H-3), 5.1 (m, 1H, H-9); 9-acetate group: δ 2.03 (s, 3H, CH3); 3-acetate group: δ 2.01 (s, 3H, CH3); acetyl carbamate: δ 2.36 (s, 3H, CH3); 13C-NMR (CDCl3, 125 MHz) δ 71.4 d (C-3), 76.1 d (C-9); 9-acetate group: δ 172.1 s (CO), 21.0 q (CH3); 3-acetate group: δ 171.6 s (CO), 20.9 q (CH3); acetyl carbamate: δ 170.8 s (CO), 22.5 q (CH3); HR-ESIMS m/z calculated for C37H59NO9Na (M + Na+) 684.4088, found 684.4084.

 3.3.8. Compound <bold>34</bold>

To a mixture of Et3N (13 μL, 0.096 mmol) with catalytic amounts of DMAP (0.3 mg, 0.003 mmol) in DCM (0.2 mL), was added tulearin A (12 mg, 0.024 mmol) at 0 °C. Hexanoyl chloride was then slowly added (13 μL, 0.096 mmol) and the mixture warmed up slowly to rt. After 2.5 h, the reaction mixture was neutralized by acetic acid to pH 7 and the residue was partitioned between ethyl acetate/H2O. The organic layer washed by sat. NH4Cl, dried over anhydrous MgSO4 and evaporated. The residue was purified by VLC (petroleum ether/ethyl acetate, 4:1) to afford 34, as a colorless oil, 6 mg (35%). NMR data for modified site: 1H-NMR (CDCl3, 500 MHz) δ 5.15 (dt, J = 5.8, 6.7 Hz, 1H, H-9), 5.26 (m, 1H, H-3); 14 methylenes according to HSQC experiments in the range of 1–2.5 ppm; 13C-NMR (CDCl3, 125 MHz) δ 71.0 d (C-9), 71.2 d (C-3); Hexanoyl carbonyls: 172.0 s (C3-CO), 172.3 s (C9-CO), 173.3 s (carbamate), HR-ESIMS m/z calculated for C49H83NO9Na (M + Na+) 852.5966, found 852.5969.

 3.3.9. Compound <bold>35</bold>

To a solution of tulearin A (35 mg, 0.065 mmol) in dry DCM (3 mL) were added of p-nitrophenyl chlorocarbonate (PNPCl, 15 mg, 0.074 mmol) and DMAP (18 mg, 0.14 mmol). The reaction was stirred at rt for 48 h. DCM was then added (5 mL) and the mixture was washed with water, dried over anhydrous MgSO4 and evaporated. The residue was purified by VLC (petroleum ether/ethyl acetate, 1:1) to afford 35, as a colorless oil, 15 mg (35%). [α]D25 +10 (c 0.3, CHCl3); NMR data for modified site (C2, C3): 1H-NMR (CDCl3, 500 MHz) δ 2.70 (qd, J = 7.6, 2.4 Hz, 1H, H-2), 5.09 (dd, J = 10.1, 2.4 Hz, 1H, H-3); PNP-group: δ 6.48 (d, J = 7.6 Hz, 2H, CH), 8.23 (d, J = 7.6 Hz, 2H, CH); 13C-NMR (CDCl3, 100 MHz) δ 44.5 d (C-2), 72.6 d (C-3); PNP-group: δ 156.6 s (CO), 130.2 q (C), 106.6 d (CH), 149.6 d (CH), 136.2 s (C). ESIMS m/z 723.3, (C38H56N2O10Na, M + Na+).

 3.3.10. Compounds <bold>36</bold> and <bold>37</bold>

To a solution of 29 (22 mg, 0.03 mmol) in DMF (0.5 mL) was added sodium azide (10 mg, 0.15 mmol), and the mixture warmed up to 60 °C for. After 24 h, the reaction mixture was cooled down to room temperature. The mixture was then partitioned between ethyl acetate/H2O and the organic phase dried over anhydrous MgSO4, and evaporated. The DMF was removed under high vacuum and the residue was purified by VLC (petroleum ether/ethyl acetate) to afford three products of azide substituted tulearin A; two epmeric 3,9-diazide tulearins: 36a, as a colorless oil, 3.8 mg (21%); 36b, a second colorless oil, 5.1 mg (29%) and a cycloaddition product 37, another colorless oil, 6.3 mg (37%); 36a: [α]D25 +20 (c 0.2, CHCl3); NMR data for modified site: 1H-NMR (CDCl3, 500 MHz) δ 3.59 (ddd, J = 8.7, 6.8, 2.0 Hz, 1H, H-3), 3.29 (td, J = 7.5, 3.7 Hz, 1H, H-9); 13C-NMR (CDCl3, 125 MHz) δ 62.3 d (C-3), 62.7 d (C-9); HR-ESIMS m/z calculated for C31H51N7O4Na (M + Na+) 608.3900, found 608.3898; 36b: [α]D25 −26 (c 0.24, CHCl3); NMR data for modified site: 1H-NMR (CDCl3, 500 MHz) δ 3.63 (ddd, J = 8.7, 6.6, 1.5 Hz, 1H, H-3), 3.47 (dt, J = 9.5, 3.5 Hz, 1H, H-9); 13C-NMR (CDCl3, 125 MHz) δ 62.8 d (C-3), 60.1 d (C-9); HR-ESIMS m/z calculated for C31H51N7O4Na (M + Na+) 608.3900, found 608.3904; 37: [α]D25 +69 (c 0.7, CHCl3); NMR data for modified site: 1H-NMR (CDCl3, 500 MHz) δ 3.77 (ddd, J = 6.7, 4.6, 2.0 Hz, 1H, H-3), 1.88 (m, 2H, H-4), 5.02 (ddd, J = 7.4, 4.3, 2.7 Hz, 1H, H-8), 4.24 (m, 1H, H-9), 1.92 (m, 1Ha, H-10), 1.81 (m, 1Hb, H-10), 2.36 (m, 2H, H-11), 2.32 (m, 2H, H-13); 13C-NMR (CDCl3, 125 MHz) δ 61.8 d (C-3), 39.8 t (C-4), 76.0 d (C-8), 74.9 d (C-9), 24.2 t (C-10), 38.3 t (C-11), 179.2 s (C-12), 30.1 t (C-13); HR-ESIMS m/z calculated for C31H52N5O4 (MH+) 558.4019, found 558.4011.

 4. Conclusions

Cell Viability

The availability of the ten natural salarins (A–J) and the above synthesized derivatives, which differ in particular chemical moieties, enabled a preliminary SAR study (see Supporting Information). In respect to this, the finding that salarin C is more potent than salarin A, which differs only in the oxazole ring, suggests that this heterocycle is essential for activity. However, salarins F and I, which contain the oxazole ring but differ in the macrolide functional groups, do not display inhibitory activity on cell viability and proliferation [4]. Hence, biological activity of salarin C may rely on other moieties in addition to the heterocycle, or a combination of several moieties. In this article we report a preliminary biological evaluation of the above derivatives against human leukemic cell line K562.

It was found that the 16,17-vinyl epoxide of salarin C contributes significantly to the K562 inhibition (e.g., 5–13). However, this conclusion is not straightforward, namely, when the 16,17-epoxide was replaced by a 16-hydroxy-17-butylamine (14), the inhibition of K562 cell viability rose to 90% (based on different cell behavior). It is suggested that the butylamine group contributes to the activity of salarin C in a different, not yet identified, mechanism. Furthermore, compound 16 is also relatively active and the most active of the hydroxyl amine derivatives 16–18 (Scheme 4). Again, this may be the result of another mechanism. The N-desacetylation (e.g., 16) affects, but does not abrogate the activity and therefore was not found as crucial for it (15).

Alcohol 21 showed excellent results with similar activity to the natural product (1 μM inhibited viability of the K562 leukemia cells by more than 90%, see Supporting Information), i.e., the octanoate ester is not crucial for the activity. The more polar glycosylation products, compounds 24 and 25, showed similar activity to 21. This section demonstrates that structural changes in the natural product are moderately effective in improving the efficacy of the molecule in inhibiting the viability of leukemia cells.

As for the tulearin derivatives, no significant changes were found for most of the compounds. The only exceptions to this were compounds 28 (showing 70% inhibition for K562 cells in comparison to tulearin A with 55%–60% inhibition) and 35 (92% inhibition), the former carrying a leaving group at position 9 and the latter at position 3. It is difficult to know whether the change in activity results from the change in the functional groups or from the solubility.

Inter alia, the mono-substituted derivatives were found to be more active than the di-substituted ones. For example, mono-acetate 31 was found to inhibit K562 cells, more than the di-acetate 32 (inhibition of 28% compared to 6%) (Table 1). Unexpectedly, the tri-acetate 33 was even more active (45% inhibition). The acetyl carbamate functionality was found to slightly increase the cytotoxicity and to inhibit K562 better than the mono- and di-acetate. In comparison to the tri-substituted acetate, the tri-hexanoyl 34 showed weaker inhibition (9% inhibition).

 Acknowledgments

We thank Noam Tal and Shimon Hauptman for performing the electrospray and FAB mass measurements. Yardena Abudi for technical assistance. This work was supported by grant #365/09 from the Israel Science Foundation (ISF, Tel Aviv, Israel).

 Conflicts of Interest

The authors declare no conflict of interest.

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N-Acetylsuccinamide is known as an acetylating reagent, in case of salarin A (2) it turns out, that the C6-N (6) is weaker and cleaves to provide the open macrocycle, 15.

 Ohtani I. Kusumi T. Kashman Y. Kakisawa H. High-field FT NMR application of Mosher’s method. The absolute configurations of marine terpenoids J. Am. Chem. Soc. 1991 113 4092 4096 10.1021/ja00011a006 Kim S. Lee Y.M. Lee J. Lee T. Fu Y. Song Y. Cho J. Kim D. Expedient syntheses of antofine and cryptopleurine via intramolecular 1,3-dipolar cycloaddition J. Org. Chem. 2007 72 4886 4891 10.1021/jo070668x Supplementary Files

Supporting Information (PDF, 978 KB)