Hamigerans R and S: Nitrogenous Diterpenoids from the New

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Hamigerans R and S: Nitrogenous Diterpenoids from the New Zealand Marine Sponge Hamigera tarangaensis Ethan F. Woolly,†,§ A. Jonathan Singh,†,§,⊥ Euan R. Russell,‡,§,∥ John H. Miller,‡,§ and Peter T. Northcote*,†,§,⊥ †

School of Chemical and Physical Sciences, ‡School of Biological Sciences, §Centre for Biodiscovery, and ⊥Ferrier Research Institute, Victoria University of Wellington, Wellington 6012, New Zealand S Supporting Information *

ABSTRACT: Seven new members of the hamigeran family of diterpenoids have been isolated from the New Zealand marine sponge Hamigera tarangaensis. Among the new additions are hamigeran R (1), considered to be the first benzonitrile-based marine natural product, and hamigeran S (2), the first dimeric structure in the series. The formation of 1 and 2 is thought to occur via the reaction of hamigeran G with a nitrogen source, where the nitrile carbon of 1 is derived from the terpenoid skeleton.

Compounds 1 and 2 are two new nitrogen-bearing species, while 3−7 are variants on previously identified hamigerans. The molecular formula of hamigeran R (1) was identified as C19H24BrNO3 through positive- and negative-ion HRESIMS analysis ([M + NH4]+, m/z 411.1280, Δ = +0.5 ppm and [M − H]−, m/z 392.0873, Δ = +1.5 ppm), indicating eight degrees of unsaturation. The 13C, 1H, and multiplicity-edited HSQC NMR experiments (Table 1) revealed 11 protonated carbons, including four methyls (δC 27.9, 24.5, 22.2, 22.1), three methylenes (δC 43.3, 36.6, 29.1), and four methines (δC 123.0, 56.5, 50.6, 30.4). This left the assignment of eight nonprotonated carbons (δC 176.9, 155.3, 145.2, 143.5, 115.3, 111.1, 101.6, 46.5) and two exchangeable hydrogens required by the molecular formula. The planar structure of hamigeran R (1) was rapidly assembled using 2D NMR spectroscopic data (Tables 1 and S3) and its direct comparison with previously reported hamigerans. Much of the connectivity of 1 was characteristic of the seco-norisohamigerane skeleton (e.g., hamigeran L), including the C-5−C-9 pendant cyclopentyl ring, a shielded isopropyl unit, an isolated methylene (CH2-10, δC 43.3, δH 1.83, 2.15), and a carboxylic acid (C-11, δC 176.9; νmax 1705, 3227 cm−1). COSY and HMBC correlations from H-4 and H317 (Figure 1) were used to construct the pentasubstituted benzene ring typically associated with the hamigerans, which accounted for five of the eight nonprotonated carbons and satisfied four degrees of unsaturation. Together with the two previously assigned nonprotonated carbons and two degrees of unsaturation, completion of the constitutional structure of 1 required incorporation of CN and the last two double-bond equivalents. This was satisfied in the form of a nitrile, with a

Hamigera tarangaensis (Bergquist and Fromont, 1988), a bright yellow sponge native to the northern waters of New Zealand, has been a rich source of novel compounds in the last 20 years. Previous research1−4 on this sponge has led to the isolation of 27 compoundscollectively known as the hamigeransas a series of polycyclic compounds with various levels of functionalization about a six- or seven-membered B ring. Several synthetic endeavors have been dedicated to the hamigeran core,5−16 with hamigeran B receiving the most attention due to its reported antiviral activity.2 In 2013, we reported the structure of hamigeran G, a homologue of hamigeran B that displayed micromolar antifungal activity.3 The core structure of the hamigerans is believed to have a diterpenoid origin,3 and some of the more intriguing of these compounds are those that contain a nitrogenous adduct, with their biogenesis proposed to occur via the reaction of hamigeran G (8) with various amino acids.4 Herein, as part of our continued interest in this sponge and its secondary metabolites, we present the NMR-directed isolation and structure elucidation of seven new hamigerans that extend the already expansive list in this family of bioactive compounds.

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RESULTS AND DISCUSSION Two geographically different collections of H. tarangaensis from the northern tip of New Zealand’s North Island were independently subjected to the same extraction and fractionation protocols as previously described.3,4 Several stages of purification using reversed-phase HPLC resulted in the isolation of seven new compounds. Hamigeran R (1) and debromohamigeran I (5) were obtained from a Cavalli Island specimen. Hamigeran S (2), 4-bromohamigeran A (3), debromohamigerans B (4) and J (6), and hamigeran L 12-Omethyl ester (7) were found from a Cape Karikari specimen. © XXXX American Chemical Society and American Society of Pharmacognosy

Received: November 13, 2017

A

DOI: 10.1021/acs.jnatprod.7b00960 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Chart 1

IR stretch at 2223 cm−1. The attachment of a nitrile moiety at C-12a is supported by the comparison of 13C NMR shift data for 1 with 2-hydroxybenzonitrile (Table S1). The relative configuration of the three stereogenic centers of 1 was determined to be identical to known hamigerans using 2DNOESY correlations between H-5, H-6, and H3-16 (Figure 1). The structure of hamigeran R (1) represents the first benzonitrile-containing marine natural product.17 As pointed out by Zubiá et al. in their report of axinynitrile A,18 there are only three reported occurrences of nitrile-containing terpenoids, each of which can be regarded to have their nitrile component originate from inorganic cyanide. As such, we also propose that 1 is the first marine-derived terpenoid to have the nitrile carbon originate from the terpene skeleton itself. Hamigeran S (2) was isolated as a pale yellow film. Positiveion HRESIMS showed a species with an isotopic distribution pattern consistent with two bromine atoms in the molecule. A protonated molecule of m/z 738.1778 (Δ = −1.4 ppm) was indicative of a molecular formula of C38H45Br2NO4 and revealed a compound with a mass approximately double that of a typical hamigeran. Analysis of 1H, 13C, and multiplicityedited HSQC NMR spectra acquired in DMSO-d6 (Tables 1 and S4) revealed 19 carbon resonances, 11 of which were protonated in the form of four methyls (δC 25.5, 23.1, 22.6, 22.5), three methylenes (δC 50.7, 36.2, 26.6), and four methines (δC 122.1, 49.3, 49.2, 28.0). Among the eight nonprotonated carbons was a carbonyl resonance at δC 199.5, as supported by an IR stretch at 1705 cm−1, and a functionalized carbon at δC 83.4. Again, much of the planar structure of 2 was easily constructed through the use of 2D NMR correlation experiments to generate a norisohamigerane-based motif (Figure 2), with only three connection points unable to be identified from COSY and HMBC experimental data. In the 1H NMR spectrum of 2, a singlet resonance was observed at δH 5.48 that integrated for one-half of a relative proton. An unfiltered 1 H−15N one-bond correlation observed in the 1H−15N HMBC experiment from this proton to δN −317.9 and an IR band at 3300 cm−1 indicated the presence of a secondary amine. HMBC correlations from this proton to C-11, C-12a, and C-4a constrained the position of the secondary amine to C-12 (Figure 2). This combination of experimental data pointed to

Table 1. 13C (150 MHz) and 1H (600 MHz) NMR Data for Hamigerans R (1) and S (2) 1a position

a

δC, type

1 2 3 4 4a 5 6 7

155.3, C 111.1, C 143.5, C 123.0, CH 145.2, C 56.5, CH 50.6, CH 29.1, CH2

8

36.6, CH2

9 10

46.5, C 43.3, CH2

11 12 12-N 12a 13 14 15 16 17

176.9, C 115.3, C 101.6, C 30.4, CH 22.2, CH3 22.1, CH3 27.9, CH3 24.5, CH3

2b

δH, mult. (J, Hz)

6.62, s 3.40, d (6.9) 2.29, m 1.66, m 2.17, m 1.82, m 1.87, m 1.83, d (14.5) 2.15, d (14.6)

1.22, m 0.88, d (6.5) 0.69, d (6.5) 1.34, s 2.45, s

δC/N, type 149.4, C 110.4, C 139.5, C 122.1, CH 137.7, C 49.3, CH 49.2, CH 26.6, CH2 36.2, CH2 44.1, C 50.7, CH2 199.5, C 83.4, C −317.9, NH 118.1, C 28.0, CH 22.6, CH3 22.5, CH3 25.5, CH3 23.1, CH3

δH, mult. (J, Hz)

6.93, s 3.36, d (5.8) 2.16, m 1.85, q (11.9) 2.03, m 1.49, m 1.60, br t (11.4) 2.15, d (10.7) 2.40, d (10.7)

5.48, s 2.03, m 0.93, d (6.1) 0.51, d (6.1) 1.16, s 2.32, s

Acquired in CDCl3. bAcquired in DMSO-d6.

Figure 1. Key 2D NMR correlations used to establish the planar structure and relative configuration of hamigeran R (1).

long-range correlation from H-4 (δH 6.62) to the remaining nonprotonated carbon (C-12, δC 115.3), complemented by an B

DOI: 10.1021/acs.jnatprod.7b00960 J. Nat. Prod. XXXX, XXX, XXX−XXX

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with synthetically prepared dioxocin compounds that feature a similar central heterocyclic moiety (Table S2). The relative configuration of hamigeran S was determined by the use of NOE correlation experiments (Figure 2). NOE correlations between H-5, H-6, and H3-16 established the typical cis-fusion of the bicyclo[5.3.0]decane system and placement of the isopropyl tail appended to C-6 at the concave face. Furthermore, H-5 and H3-16 showed NOE correlations to H-10a, placing it on the same side of the molecule. This assignment was corroborated by long-range COSY correlations that placed H-10b and CH3-16 in a pseudo-1,2-diaxial relationship. H-10b showed a weak NOE correlation to 12NH, indicating that the secondary amine linkage also sits within the concave face. 4-Bromohamigeran A (3) was isolated as a pale yellow film. Analysis of the HRESIMS data gave a protonated molecule at m/z 503.0054, suitable for a molecular formula of C20H24Br2O4 (Δ = −1.8 ppm). In the molecular ion cluster, evidence for a dibrominated compound came in the form of a 1:2:1 ratio of the [M + H]+, [M + 2 + H]+, and [M + 4 + H]+ peaks. The absence of NMR data consistent with protonation at C-4 strongly suggested this was the site of additional halogenation, as is observed for 4-bromohamigerans B, E, and K.2,3 The remainder of the planar and configurational structure of 3 was determined through analysis of the NMR experimental data (Tables 2 and S5) and comparison with hamigeran A. A molecular formula of C18H22O3 for debromohamigeran B (4) was established from positive-ion mode HRESIMS data ([M + H]+, m/z 287.1645, Δ = +1.0 ppm) with inspection of 13 C and 1H NMR spectroscopic data identifying a compound similar to the well-known hamigeran B. The difference in 4 is the protonation of C-2 (δC 116.2, δH 6.73) and the

Figure 2. Key 2D NMR correlations establishing the planar structure and relative configuration of hamigeran S (2).

an unusual dimeric structure for 2 that must incorporate a centralized NH moiety. With only one exchangeable hydrogen in the molecule, two connection sites unaccounted for, and the requirement of symmetry dictated by the 19 carbon resonances, only two possible structures could be considered (Figure S1). The first candidate structure, containing a phenolic ether linkage joining C-1 and C-1′ and a four-membered heterocycle joining aminal centers C-12 and C-12′, has S1 symmetry and thus requires enantiomeric monomers. However, the observation of a nonzero specific rotation (+33) eliminated this structure. The second, and more plausible, planar structure is that presented for 2, where the aminal linkages at C-12 and C-12′ accounted for the desired connectivity. The planar structure of 2 is C2 symmetric, with its rotation axis through the N−H bond, and therefore only requires one enantiomeric form of the monomer. The chemical shifts of C-1, C-12, and C-12a compare favorably

Table 2. 13C (150 MHz) and 1H (600 MHz) NMR Data for Compounds 3, 4, and 6 in CDCl3 3 position

δC, type

1 1-OH 2 3 4 4a 5 6 7

155.6, C 111.1, C 148.4, C 118.1, C 145.1, C 54.4, CH 47.7, CH 23.80, CH2

8

37.6, CH2

9 10 10-OH 11 11-OH 11a 12 13 14 15 16 17 18

4 δH, mult. (J, Hz)

δC, type

6 δH, mult. (J, Hz)

164.8, C 11.38, s

3.82, d (10.4) 2.80, tt (9.7, 2.4) 1.57, m 1.81, m 1.49, m 1.69, m

47.8, C 85.5, C

11.93, s 6.73, s

116.4, CH 151.0, C 123.5, CH 144.3, C 56.6, CH 51.6, CH 27.1, CH2

6.69, s 3.40, d (9.0) 2.29, m 1.68, m 1.79, m 1.56, m 2.62, ddd (13.3, 7.6, 5.3)

34.0, CH2 57.0, C 200.3, C 184.5, C

115.0, C 27.8, CH 16.9, CH3 24.6, CH3 23.82, CH3 26.5, CH3 169.8, C 53.6, CH3

116.8, C 28.3, CH 20.0, CH3 23.3, CH3 24.6, CH3 22.7, CH3

δH, mult. (J, Hz)

116.5, CH 140.6, C 123.7, CH 136.6, C 57.9, CH 51.1, CH 26.5, CH2 35.3, CH2

6.86, br s 6.68, s 6.65, s 3.43, d (9.1) 2.18, m 1.67, m 1.70, m 1.45, m 2.59, dt (13.1, 7.2)

54.9, C 209.3, C 78.2, C

4.22, br s 198.1, C

δC, type 156.2, C

5.09, br s 1.39, m 0.78, d (6.7) 0.09, d (6.7) 1.31, s 2.75, s

1.20, m 0.54, d (6.7) 0.42, d (6.4) 1.30, s 2.39, s

3.59, s C

118.2, C 27.3, CH 23.3, CH3 19.5, CH3 26.3, CH3 21.5, CH3 170.1, C 53.8, CH3

1.29, m 0.45, d (6.8) 0.46, d (6.8) 1.34, s 2.31, s 3.72, s DOI: 10.1021/acs.jnatprod.7b00960 J. Nat. Prod. XXXX, XXX, XXX−XXX

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(−)-hamigeran G (8) with an appropriate amino acid.4 The origins of hamigerans R (1) and S (2) may differ in this respect, as it conceivably requires biologically produced ammonia or a transamination step to form a primary imine. The proposed formation of 1 (Scheme 1a) provides a rational explanation for the nitrile carbon originating from the terpenoid skeleton, where imine formation at C-12, followed by Baeyer−Villigertype oxidation (presumably similar to the formation of hamigeran E from B, and L from G) and ring opening simultaneously forms the nitrile and carboxylic acid moieties. The formation of hamigeran S (2) also starts at the primary imine, in which the electrophilic C-12 iminocarbon is susceptible to attack from the C-1 phenol of a second equivalent of hamigeran G to form a hemiaminal. The newly formed amine at this position then reacts with the corresponding C-12′ carbonyl center from the other monomeric unit to form a secondary imine. Another nucleophilic bond formation from the remaining free phenol and the secondary imine completes the structure of 2. This particular sequence is similar to Natividad’s synthesis of dithiocin and dioxocin imine compounds.19 With access to sufficient quantities of hamigeran G, we sought to test the reactivity of the diketone moiety with a nitrogen source. The total synthesis of (−)-hamigeran G and its subsequent transformation into (18-epi-)hamigerans D and N− Q have been recently described.16 The latter reactions were in complete agreement with our own preliminary examination20 of the reactivity of hamigeran G and support the amino acid origins of the adducts. Interestingly, the reaction between natural hamigeran G with D-alanine produced hamigeran D over its C-18 epimer (9a:9b = 3:1), while the corresponding reaction with L-alanine was more exclusive and favored the former (9a:9b = 7:1). Reaction of hamigeran G with glycine afforded a new compound, 10 (Table S10), with a diastereotopic methylene center at C-18. The formation of this compound rather than an oxazole-containing compound analogous to hamigeran M suggests a third keto group at C-10, absent in hamigeran G, is needed to form the oxazole moiety of hamigeran M (Scheme 1b).4 These recent total syntheses support our biogenic reasoning and allow us to suggest the absolute configuration of hamigerans R and S as drawn. The isolation of these two compounds further highlights the reactivity of hamigeran G and extends the nitrogenous functionality of this family of diterpenoids, which now include 1,3-oxazine (hamigerans D, N−Q), oxazole (hamigeran M), nitrile (hamigeran R), and aminal (hamigeran S) motifs.

corresponding lack of evidence for bromine in the mass spectrometric data. Similar findings were also observed with debromohamigeran I (5) (C19H26O4, [M + H]+, m/z 319.1902, Δ = −0.6 ppm) and debromohamigeran J (6) (C20H26O5, [M + Na]+, m/z 369.1682, Δ = +2.7 ppm). In the case of these three compounds, their planar and configurational structures were elucidated in direct comparison of NMR experimental data (Tables 2, 3, and S6−S8) to their respective brominated congeners.2,3 Table 3. 13C (150 MHz) and 1H (600 MHz) NMR Data for Compounds 5 and 7 in CDCl3 5 position

δC type

1 1-OH 2 3 4 4a 5 6 7

165.0, C 118.1, CH 143.1, C 127.9, CH 148.7, C 61.4, CH 53.9, CH 32.4, CH2

8

34.6, CH2

9 10

47.9, C 74.3, CH

11 12 12a 13 14 15 16 17 18

74.6, CH 204.4, C 115.5, C 29.7, CH 23.3, CH3 22.2, CH3 30.2, CH3 22.0, CH3

7

δH mult. (J, Hz)

δC type

δH mult. (J, Hz)

155.6, C 12.54, s 6.77, s 6.64, s 3.47, d (11.7) 2.21, m 1.60, m 1.91, m 1.40, m 2.59 ddd, (13.6, 7.4, 2.9) 3.33, d (10.9) 4.56, d (11.0)

1.12, m 0.76, d (6.5) 0.18, d (6.2) 1.24, s 2.32, s

9.90, s 111.7, C 142.6, C 123.1, CH 141.6, C 53.9, CH 50.7, CH 29.7, CH2 36.4, CH2

46.3, C 43.4, CH2 177.7, C 170.7, C 116.0, C 30.3, CH 21.9, CH3 22.4, CH3 27.8, CH3 24.3, CH3 52.8, CH3

6.54, s 3.64, d (7.2) 2.21, m 1.68, m 2.14, m 1.81, m 1.95, m

1.83, d (14.7) 2.16, d (14.7)

1.36, m 0.87, d (6.4) 0.58, d (6.4) 1.27, s 2.42, s 3.96, s

Hamigeran L 12-O-methyl ester (7) was isolated as a colorless film. HRESIMS analysis established a molecular formula of C20H25O5Br from a protonated molecule of m/z 427.1114 (Δ = −0.2 ppm). The molecular formula for 7 is identical to the previously described constitutional isomer hamigeran L 11-O-methyl ester,3 and as such there was sufficient agreement of the 1D and 2D NMR data between the two compounds to allow full elucidation of the planar structure. The only difference between the two compounds was in the placement of oxymethyl CH3-18 (δC 52.8, δH 3.96), which in this case was confirmed by an HMBC correlation to C-12 (δC 170.7) (Tables 3 and S9). Biological Activity and Proposed Biogenesis. Compounds 1−7 were tested in a cell proliferation assay to measure cytotoxicity against the human promyelocytic leukemic (HL60) cell line (Table S11). None of the new compounds reported showed inhibition of proliferation at concentrations below 10 μM. The isolation of two new nitrogenous hamigerans with quite different functionality is intriguing. Formation of all nitrogenous species reported to date is proposed via the reaction of

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured using a Rudolph Autopol II polarimeter. UV/vis spectra were recorded on an Agilent 8453 diode array spectrometer. IR (film) spectra were recorded using a Bruker Platinum Alpha FTIR spectrometer. NMR spectra were obtained using a Varian DirectDrive spectrometer equipped with a triple resonance HCN cryogenic probe, operating at 25 K at frequencies of 600 and 150 MHz for 1H and 13C nuclei, respectively. The 15N nucleus was indirectly detected using an 1 H−15N HMBC experiment. Chemical shifts were referenced to the residual solvent peak (CDCl3: δC 77.16, δH 7.26; DMSO-d6: δC 39.52, δH 2.50).21 High-resolution masses were obtained from an Agilent 6530 Q-TOF mass spectrometer equipped with an Agilent 1260 HPLC system for solvent delivery utilizing a JetStream electrospray ionization source in positive- and negative-ion modes. Reversed-phase column chromatography was achieved using Supelco Diaion HP20, D

DOI: 10.1021/acs.jnatprod.7b00960 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Scheme 1. (a) Proposed Biogenetic Formation of Hamigerans R (1) and S (2); (b) Reaction of Hamigeran G (8) with Selected Amino Acids

HP20SS, or Tosohaas Amberchrom poly(styrene-divinylbenzene) (PSDVB) chromatographic resin. HPLC was performed using either an Agilent Technologies 1260 Infinity HPLC equipped with a diode array detector or a Rainin Dynamax SD-200 solvent delivery system with 25 mL pump heads equipped with a Varian Prostar 335 photodiode array detector. Octadecyl-derivatized silica (C18) HPLC column (Phenomenex) sizes were either semipreparative (10 mm × 250 mm, 4 mL/min) or analytical (4.6 mm × 250 mm, 1 mL/min) scale, unless otherwise stated. Solvents used for reversed-phase column chromatography were of HPLC or analytical grade quality. All other solvents were purified by distillation and filtered before use. Solvent mixtures are reported as % vol/vol unless otherwise stated. Collection Material. Sponge samples were collected by hand using scuba from Cavalli Island and Cape Karikari, New Zealand, as previously described,3,4 and held at −20 °C until extracted. Voucher samples are stored at the School of Chemical and Physical Sciences, Victoria University of Wellington, Wellington, New Zealand. Isolation of Compounds. Hamigeran R (1) and Debromohamigeran I (5). Frozen H. tarangaensis (PTN2_79F, 202 g frozen weight), collected from Taheke Reef, Cavalli Island, New Zealand, was cut into small pieces and extracted with MeOH (2 × 1 L). The second and first extracts were loaded onto a column of PSDVB (1 L, preequilibrated in MeOH), desalted with H2O, and eluted with 3 L portions of (i) 20% Me2CO/H2O (fraction A), (ii) 40% Me2CO/H2O (fraction B), (iii) 60% Me2CO/H2O (fraction C), (iv) 80% Me2CO/ H2O (fraction D), and (v) Me2CO (fraction E). Fraction D was used in an earlier study.4 Fraction C (0.56 g) was loaded onto a LH20 column pre-equilibrated in 50% MeOH/CH2Cl2 and partitioned to give 95 test tube fractions, which after NMR and TLC analysis were

pooled into four parts, F−I. Fraction I (77.0 mg) was subjected to C18 HPLC (semipreparative, 80−95% MeOH/0.2 M HCOOH(aq)), resulting in nine fractions, J−R, where fractions M and N contained debromohamigeran I (5, tR = 10.0 min, 1.3 mg) and hamigeran R (1, tR = 10.9 min, 2.4 mg), respectively. Hamigeran S (2), 4-Bromohamigeran A (3), Debromohamigeran B (4), Debromohamigeran J (6), and Hamigeran L 12-O-Methyl Ester (7). Frozen H. tarangaensis (PTN2_71J, 515 g frozen weight), collected from Matai Bay Pinnacle, Cape Karikari, New Zealand, was cut into small pieces and extracted with MeOH (2 × 1.7 L). The second and first extracts were loaded onto a column of PSDVB (1 L, pre-equilibrated in MeOH), desalted with H2O, and eluted with 3 L portions of (i) 20% Me2CO/H2O (fraction A), (ii) 40% Me2CO/H2O (fraction B), (iii) 60% Me2CO/H2O (fraction C), (iv) 80% Me2CO/ H2O (fraction D), and (v) Me2CO (fraction E). A portion of fraction D (ca. 2.5 g) was loaded onto an LH20 column pre-equilibrated in 50% MeOH/CH2Cl2 and pooled into four fractions, F−I. Fraction H was subjected to semipreparative C18 HPLC (gradient, 40 min 80%, ramp to 95% over 5 min, MeOH/0.2 M HCOOH(aq)), generating 16 fractions, J−Y. This led to the isolation of several previously identified hamigerans, including debromohamigeran A (fraction N, tR = 16.8 min, 24.8 mg), hamigeran G (fraction P, tR = 20.4 min, 34.1 mg), hamigeran A (fraction R, tR = 28.8 min, 8 mg), hamigeran B (fraction U, tR = 43.4 min, 7.9 mg), and 4bromohamigeran B (fraction W, tR = 48.5 min, 20.7 mg). Fraction K was further purified using the same conditions, which generated seven fractions, Z−AF, which led to the isolation of hamigeran F (fraction AC, tR = 6.8 min, 0.1 mg) and debromohamigeran J (6, fraction AD, tR = 7.3 min, 1.1 mg). Fraction L was subjected to further semipreparative C18 HPLC, (MeCN/0.2 M HCOOH(aq), 60% 0−25 E

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HPLC (85% MeOH/H2O) to afford glycine adduct 10 (2.1 mg, tR = 9.3 min). Semisynthetic Hamigeran D (9a) and 18-epi-Hamigeran D (9b). NMR and HRESIMS data are as previously described.4,16 Glycine adduct (10): pale yellow film; NMR data, Table S10; HRESIMS m/z 390.1061 [M + H]+ (calcd for C20H25NO279Br, 390.1069, Δ = −2.1 ppm). Cell Proliferation Assays. Cell proliferation assays (MTT, 48 h incubation) using the human promyelocytic leukemia (HL-60) cell line were performed as previously described.22

min, 60−100%, 5 min ramp time), generating 12 fractions, AG−AS, from which fraction AJ gave hamigeran L 12-O-methyl ester (7, tR = 12.4 min, 2.7 mg) and fraction AO afforded debromohamigeran B (4, tR = 21.4 min, 1.3 mg). Fraction T was fractionated across two isocratic C18 HPLC protocols (semipreparative, MeOH/0.2 M HCOOH(aq), 85%, followed by 80%), which ultimately yielded additional quantities of hamigeran B and 4-bromohamigeran A (3, fraction AV, tR = 33.8 min, 1.8 mg). Fraction E (3.3 g) was also loaded onto an LH20 column preequilibrated in 50% MeOH/CH2Cl2 and pooled into five fractions, AX−BB. Semipreparative C18 HPLC (isocratic, 100% MeOH) was used to separate fraction AZ (251 mg) into nine further fractions, BC−BK. Fraction BE (17.0 mg) was purified by semipreparative C18 HPLC (isocratic, 95% MeOH/H2O) to afford fractions BL−BN, which gave hamigeran S (2, tR = 12.8 min, 14.0 mg) as fraction BN. Hamigeran R (1): pale yellow film; [α]25D −46 (c 0.12, CH2Cl2); UV (MeOH) λmax (log ε) 219 (4.51), 309 (3.36), 339 (3.46) nm; IR (film) νmax 3227 (O−H), 2956 (C−H), 2223 (CN), 1705 (CO) cm−1; NMR data, Tables 1 and S3; HRESIMS m/z 411.1280 [M + NH4]+ (calcd for C19H28N2O379Br, 411.1278, Δ = +0.5 ppm), m/z 392.0873 [M − H]− (calcd for C19H2379BrNO3, 392.0867, Δ = +1.5 ppm). Hamigeran S (2): colorless film; [α]25D +33 (c 0.64, CH2Cl2); UV (MeOH) λmax (log ε) 204 (4.65), 291 (3.62) nm; IR (film) νmax 3300 (N−H), 2956 (C−H), 1728 (CO) cm−1; NMR data, Tables 1 and S4; HRESIMS m/z 738.1778 [M + H]+ (calcd for C38H4679Br2NO4, 738.1788, Δ = −1.4 ppm). 4-Bromohamigeran A (3): pale yellow film; [α]25D −65 (c 0.09, CH2Cl2); UV (MeOH) λmax (log ε) 204 (4.33), 229 (4.26), 281 (3.99), 356 (3.68) nm; NMR data, Tables 2 and S5; HRESIMS m/z 503.0054 [M + H]+ (calcd for C20H2579Br2O5, 503.0063, Δ = −1.8 ppm). Debromohamigeran B (4): bright yellow film; [α]25D −81 (c 0.04, CH2Cl2); UV (MeOH) λmax (log ε) 205 (4.38), 262 (3.63), 311 (3.87), 367 (3.35) nm; NMR data, Tables 2 and S6; HRESIMS m/z 287.1645 [M + H]+ (calcd for C18H23O3, 287.1642, Δ = +1.0 ppm). Debromohamigeran I (5): pale yellow film; [α]25D −50 (c 0.06, CH2Cl2); UV (MeOH) λmax (log ε) 203 (4.49), 216 (4.45), 278 (3.84), 313 (3.55) nm; NMR data, Tables 3 and S7; HRESIMS m/z 319.1902 [M + H]+ (calcd for C19H27O4, 319.1904, Δ = −0.6 ppm). Debromohamigeran J (6): pale yellow film; [α]25D −104 (c 0.06, CH2Cl2); UV (MeOH) λmax (log ε) 205 (4.50), 288 (3.38) nm; NMR data, Tables 2 and S8; HRESIMS m/z 369.1682 [M + Na]+ (calcd for C20H26O5Na, 369.1672, Δ = +2.7 ppm). Hamigeran L 12-O-methyl ester (7): colorless film; [α]25D +89 (c 0.09, CH2Cl2); UV (MeOH) λmax (log ε) 206 (4.51), 286 (3.38) nm; NMR data, Tables 3 and S9; HRESIMS m/z 427.1114 [M + H]+ (calcd for C20H27O579Br, 427.1115, Δ = −0.2 ppm). General Preparation of Hamigeran G/Amino Acid Conjugates. A solution of hamigeran G and an appropriate amino acid were prepared in a 1:10 molar ratio in EtOH and heated at reflux for 2 h. The reaction mixture was passed through a column of HP20ss, and the retentate was collected with Me2CO. Purification of the reaction product was performed using analytical C18 HPLC (85% MeOH/ H2O). Hamigeran G D-Alanine Adduct. Hamigeran G (8, 3 mg, 7.9 nmol) and D-alanine (7 mg, 79.3 nmol, 10 equiv) were reacted in the manner described. The concentrated retentate was purified by analytical C18 HPLC (85% MeOH/H2O) to afford a 3:1 mixture of hamigeran D (9a) and 18-epi-hamigeran D (9b) (1.5 mg, tR = 6.6 min), respectively. Hamigeran G L-Alanine Adduct. Hamigeran G (8, 5 mg, 13.2 nmol) and L-alanine (12 mg, 132.2 nmol, 10 equiv) were reacted in the manner described. The concentrated retentate was purified by analytical C18 HPLC (85% MeOH/H2O) to afford a 7:1 mixture of hamigeran D (9a) and 18-epi-hamigeran D (9b) (2.3 mg, tR = 6.5 min), respectively. Hamigeran G Glycine Adduct. Hamigeran G (8, 2.5 mg, 6.6 nmol) and glycine (5 mg, 66.2 nmol, 10 equiv) were reacted in the manner described. The concentrated retentate was purified by analytical C18

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00960. Isolation schemes, candidate structures for 2, chemical shift comparisons for 1 and 2, full tabulated NMR data for compounds 1−7, NMR spectra for 1−7 and IR spectra for 1 and 2, cytotoxicity data for 1−7 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +64-4-463-0065. ORCID

A. Jonathan Singh: 0000-0003-1722-066X Peter T. Northcote: 0000-0002-2086-9972 Present Address ∥

(E. R. Russell) Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS I. Vorster (Victoria University of Wellington) is thanked for his assistance in acquiring NMR and MS data. E.F.W. acknowledges the Curtis-Gordon Research Scholarship for financial support.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.7b00960 J. Nat. Prod. XXXX, XXX, XXX−XXX