KB343, a Cyclic Tris-guanidine Alkaloid from Palauan Zoantharian

Letter Cite This: Org. Lett. 2018, 20, 3039−3043

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KB343, a Cyclic Tris-guanidine Alkaloid from Palauan Zoantharian Epizoanthus illoricatus Ken Matsumura,† Tohru Taniguchi,‡ James D. Reimer,¶ Shuntaro Noguchi,† Masaki J. Fujita,† and Ryuichi Sakai*,† †

Faculty and Graduate School of Fisheries Sciences, Hokkaido University, 3-1-1 minato-cho, Hakodate, Hokkaido 041-8611, Japan Frontier Research Center for Advanced Material and Life Science, Faculty of Advanced Life Science, Hokkaido University, Kita 21 Nishi 11, Sapporo 001-0021, Japan ¶ University of the Ryukyus Senbaru 1, Nishihara, Okinawa 903-0213, Japan ‡

S Supporting Information *

ABSTRACT: A new tris-guanidine alkaloid, KB343 (1), was isolated from the aqueous extract of a Palauan zoantharian, Epizoanthus illoricatus. The structure of 1 was determined on the basis of spectral analyses of 1 and its derivatives. The absolute configuration for 1 was determined upon comparison of the CD spectrum of 1 to those obtained from density functional theory calculations. The structure of 1 is highly unusual, as three guanidine groups are present in one ring system.

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the acute episodes but gradually weakened and eventually died a few days after the injection. We thus separated the extract using a mice behavioral assay as a guide. The extract was first separated on a Sephadex LH-20 column. The active fractions responsible for the slow death eluted separately from those with the convulsant activity. The fraction with lethal activity was finally purified by reversedphase HPLC to give pure 1 as the trifluoroacetate (Figure 1). Compound 1 was freely soluble in water and in other polar organic solvents, including lower alcohols, acetonitrile, and DMSO.

arine organisms provide a diverse array of cyclic guanidine alkaloids.1,2 The most striking examples are tetrodotoxins and saxitoxins, which specifically target voltagegated sodium channels.3 Cyclic guanidine functionalities are found in numerous pyrrole−imidazole alkaloids,4,5 polycyclic guanidine alkaloids,6 and the aplysinopsin alkaloid family7,8 and are mainly from marine sponges, corals, and a few other marine invertebrates. Unique classes of cyclic guanidines are also found in zoantharians.9,10 One example is zoanthoxanthins; these compounds are bis-guanidine nonbenzenoid aromatics that show fluorescence and are unique to the order Zoantharia. The examples above clearly illustrate the intriguing chemical diversity of the marine cyclic guanidines, and not surprisingly, they exhibit diverse biological activities including antimicrobial, cytotoxic, enzyme inhibition, immunomodulatory, and neuronal activities.4,6,7,11,12 It is thus clear that cyclic guanidine alkaloids are one of the most attractive classes of compounds in the context of chemical and biomedical research of marine natural products. We have reported unique, neuroactive watersoluble compounds from various marine organisms12 and recently found a Didemnidae tunicate that produces new guanidine derivatives that interact with synaptic receptors.13 Here, we found that Epizoanthus illoricatus, collected in the Republic of Palau, exhibited behavioral effects in mice after intracerebroventricular (i.c.v.) injection14 and cytotoxicity in cultured cancer cells. Bioactivity-guided separation of the extract resulted in the isolation of KB343 (1), which is the compound responsible for the toxicity of the extract. An i.c.v. injection of the aqueous extract induced violent, but acute, convulsions in mice; however, the mice recovered from © 2018 American Chemical Society

Figure 1. Structure of KB343 (1).

The molecular formula for 1, C15H21N9O, was determined on the basis of high-resolution electrospray ionization (ESI)− MS data showing a molecular ion at m/z 344.1942 (MH)+ and 13 C NMR data, which showed resonances for all 15 carbons. Detailed assignments of the 1H and 13C NMR data of 1 were Received: April 5, 2018 Published: May 2, 2018 3039

DOI: 10.1021/acs.orglett.8b01069 Org. Lett. 2018, 20, 3039−3043

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Organic Letters

The 1H NMR spectrum (D2O) along with the HSQC NMR data showed a total of nine resonances between δ 0.9 and 4.4 ppm including a methyl doublet, three methylenes, and three methines. An analysis of the two-dimensional NMR spectra readily identified a consecutive 1H spin system from CH2−2 to CH311 (Figure 3). The HMBC NMR data indicated correlations

made from the analyses of the one- and two-dimensional (1D and 2D) NMR spectra taken in both D2O and acetonitrile-d3 [Figure 2, and Table S1 and S2 in the Supporting Information, (SI)].

Figure 2. 13C NMR (D2O) chemical shift assignments for 1. Figure 3. COSY and HMBC NMR data for the carbon framework of 1.

The 13C NMR spectrum of 1 showed six sp2 carbons between δC 122 and 188 ppm; of these, the three quaternary carbons resonated at δC 151.0, 160.5, and 161.9. Thus, all nine nitrogen atoms were assigned to three guanidine groups. Note that the center guanidine carbons, C-13 and C-14, were both rather unusually far downfield. The reason for this is not clear, but similar examples have been found for a small number of other cyclic guanidins.15−17 The treatment of 1 with 2,4pentanedione resulted in the formation of the mono- (2), bis(3), and tris- (4) pyrimidine derivatives, confirming the presence of the tris-guanidine functionality in 1 (Scheme 1; Tables S4 and S5 and Figure S1, SI).

between H2-2 and the C-1 carbonyl group and the presence of an sp2 carbon at δC 122.9 (C-10), which, in conjunction with the presence of an α,β-unsaturated ketone, constructed a C-1− C-10−C-9 fragment. The presence of a six-membered ring framework (C-3−C-4−C-5−C-6−C-7−C-8) was suggested by the HMBC cross peaks between H2-4 and H-7 and C-6/C-8 and mutual correlations between CH-5 and CH-3. Moreover, the HMBC correlations between both H-5/H-7 and C-12 indicated that C-12 is bound to C-6 of the six-membered ring (Figure 3). Carbon chemical shifts for C-6 (δC 69.0) and C-12 (δC 52.7) indicated that nitrogen atoms were attached to each of these carbons. The HMBC correlations between H2-12 and a guanidine carbon at δC 160.5 (C-13) and between both H1′/3′ and C-6/12/13 indicated that C-6/12/13 and N-1′/3′ formed an imidazolidine connected in a spiro manner to the six-membered ring at C-6 (Figure 4). Clear HMBC correlations

Scheme 1. Chemical Conversion of 1 to Alcohol 5 and Pyrimidine Derivatives 2−4

Figure 4. Key correlations in the NMR data of two of the guanidine groups.

The presence of a fully substituted α,β-unsaturated ketone moiety was suggested by the resonances at δC 122.9 and 143.8 in conjunction with a carbonyl carbon at δC 188.2. Both the UV absorption at λ 307 nm and IR absorption band at 1687 cm−1 were consistent with this assignment. This structural assignment was further supported by the fact that NaBH4 reduction of 1 readily afforded alcohol 5, which showed an absorption maximum shifted to λ 220 nm (Scheme 1). Notably, 1, but not 5, emitted weak fluorescence centered at 420 nm upon irradiation with 310 nm light. We thus performed a density functional theory (DFT) calculation and found that this rather unusual electronic transition was attributable to the HOMO → LUMO transition around the α,β-unsaturated ketone moiety in 1 (Figures S6 and S7). Because the molecular formula required 10 degrees of unsaturation, five rings must be present in the molecule considering the double bonds that can be accounted for.

between the methine singlet δH 4.33 (H-7) and C-6, between δC 63.3 (C-8) and 143.8 (C-9), and between H-3 and C-8/9 further extended the ring system to a decaline (C-1−C-10) (Figure 3). The second guanidine group also formed an imidazolidine ring because the H-7 methine showed an HMBC correlation to δC 161.9, the C-14 guanidine carbon, and HMBC correlations were observed between H-4′ and C-8/14 and between H-6′ and C-7/14. The mutual long-range correlations observed in the COSY data between H-1′ and H-3′/H-12 and between H-7 and H-6′ also supported this assignment. In addition, the 1H−15N HMBC spectrum showing correlations between H-7 and N-1′/N-6′ reinforced these overall assignments (SI, Table S2). Finally, the third guanidine group was attached to the C-9/C10 sp2 carbons to form an imidazole ring. Notably, the chemical shift of C-15 was shifted significantly upfield (δC 151.0) 3040

DOI: 10.1021/acs.orglett.8b01069 Org. Lett. 2018, 20, 3039−3043

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from marine organisms, 1 is the first example with three guanidines arranged within one heterocyclic system. The isolation of 1 from a species of Epizoanthus highlights the versatile biosynthetic potential of this relatively untapped order of invertebrates. Interestingly, the key building block of the zoanthoxanthins was speculated to be a C5N3 unit from arginine.21−23 The same building block can be used to construct the skeletal structure of 1 (Scheme 2). Biosynthetic insights into the secondary metabolism of zoantharians would be of particular interest.

compared to that of other guanidines, supporting the presence of an aminoimidazole group.18,19 NOESY correlations between H-12β and H-5/CH3-11 and between H-12α and H-7 indicated that C-12 is β-equatorial to the cyclohexane ring (Figure 5). The mutual correlations

Scheme 2. Arginine-Derived C5N3 Biosynthetic Building Block Proposed to Be a Precursor of Zoanthoxanthins21,22 and 1

Figure 5. NOESY correlations observed in 1.

between H-3, -5, and -7 indicated that they are all axially oriented, and the cyclohexane ring is in a chair conformation. Therefore, the second guanidine moiety is oriented α to the cyclohexane ring. The absolute stereochemistry of 1 was determined on the basis of its circular dichroism (CD) spectrum.20 The observed CD data in water of 1 were compared with the theoretical spectrum for (3R,5R,6S,7R,8S)-1 that was calculated at the DFT/B3LYP/6-31G(d)/PCM(water) level of theory (Figure 6).

An i.c.v. injection of 1 in mice caused slow death without signs of acute neuronal activity such as convulsions or other behavioral changes.14 The lethality and onset of death were both dose dependent (Table 1). The minimum lethal dose was Table 1. Toxicity of 1 and Its Derivatives in Mice dose nmol/mouse (i.c.v.) 14.5 2.91 1.45 0.29 number of mice died/used (onset of death, days)

compd 1 5 2 3 a

1/1 (2) 1/3 (5) 3/3 (2−7) 0/3

3/3 (2−3) nta 3/3 (11−22) nt

3/3 (3−4) nt nt nt

0 nt nt nt

nt: not tested.

estimated to be between 1.45 and 0.29 nmol/mouse (Table 1). Reduction of the keto group and protection of the guanidine lowered the potency, and bis-pyrimidine 3 showed no activity (Table 1). The slow onset (2−7 days) of 1 observed here suggested that the action of 1 was not mediated through rapid change in membrane potential14 but rather through slow degradation of neuron by 1. We next tested the cytotoxicity of 1 using the murine leukemia cell line L1210, the human tumor cell line HeLa, and the model neuronal cell line derived from human bone marrow SH-SY5Y. KB343 (1) was moderately cytotoxic to all the tested cell lines with IC50 values varying between 2 and 5 μM depending on the cell used (Table 2). Interestingly, the activity profile of the derivatives differed from that observed in mice; 2 was more active than 1 against L1210 cells, while this trend was not observed in other cells. These results suggested that the ketone plays a crucial role in both mouse lethality and cytotoxicity. However, the effect of the guanidine group on the activity varied depending on the cell type. Because L1210 are floating cells, but are HeLa adherent

Figure 6. Comparison between the CD spectrum observed for 1 and those calculated for (3R,5R,6S,7R,8S)-1 (solid line) and (3S,5S,6R,7S,8R)-1 (dotted line).

The observed CD spectrum showed a good agreement with the calculated one; a broad positive Cotton effect above 300 nm, a weak negative band in the 250−300 nm region, and a strong positive band at approximately 200 nm were all observed. This result clearly suggested the absolute stereochemistry of 1 was 3R,5R,6S,7R,8S. This assignment was confirmed by CD analysis of 5 (see Figure S5). The structure of KB343 is unique, as it has a carbon framework that is not closely related to any known general biosynthetic classifications. The three guanidine groups arranged along the bottom of the framework of 1 are also intriguing. Although a number of cyclic guanidines are known 3041

DOI: 10.1021/acs.orglett.8b01069 Org. Lett. 2018, 20, 3039−3043

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Organic Letters Table 2. Cytotoxicity (μM)a of 1 and Its Derivatives compd

L1210

HeLa

SH-SY5Y

1 5 2 3

1.96 5.01 0.38 >500

4.93 >100 23.0 inactive

3.40 10.9 5.11 >300

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid (No. 15H04546) to R.S. We thank the government of the Republic of Palau for permission to collect specimens.

a

Concentration−response curves; statistical analyses are shown in the SI.

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cells, and SH-SY5Y can have both phenotypes, their cell surface molecules should differ substantially, and thus, the interactions between 1 and cells may vary. Taken together, we speculate that the tris-guanidine group in 1 anchors the molecule on the cell surface by interacting with negatively charged species through ionic interactions, and then the whole molecule is somehow translocated inside the cell. Finally, the unsaturated ketone of 1 can alkylate intracellular targets to cause cytotoxicity. It might be difficult, however, for 1 to penetrate the plasma membrane simply by diffusion, because 1 is a highly water-soluble ionic molecule with a CLogP value of −4.75 in its trichloride form. We thus suspect that the unique tris-arginine substructure of 1 is a key structural element for the translocation since cell-penetrating peptides (CPP),24 which are often oligoarginines, can penetrate the cell membrane. Although the mechanisms of cell membrane permeation by CPP are not fully understood,20 the interactions between guanidine and the cell surface or those of the peptide backbone and lipid chains25 may be involved. The structure of 1 with plural guanidines and a hydrophobic skeletal structure may possess structural elements that are required for the function proposed here. Since some hydrophilic CPP are known to have antifungal activity,26 we tested the activity of 1 against Saccharomyces cerevisiae. As expected, 1 showed inhibition with a minimum inhibitory concentration of 2.5 μg/disc. Notably, 1 did not lyse rabbit erythrocytes, consistent with the lack of activity often observed in CPP, to disrupt cell membranes. In conclusion, KB343 (1), found here from Epizonathus, may have interacted with the cell membrane and translocated itself into the cell without membrane disruption. Further understanding of the mechanisms underlying the plausible selfinternalization of 1 into cells should facilitate the development of new tools or key structural elements in drug delivery research.27

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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.orglett.8b01069. Detailed experimental procedures and analytical data for all new compounds (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: ryu.sakai@fish.hokudai.ac.jp. ORCID

Tohru Taniguchi: 0000-0002-4965-7383 Ryuichi Sakai: 0000-0001-6490-0495 3042

DOI: 10.1021/acs.orglett.8b01069 Org. Lett. 2018, 20, 3039−3043

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NOTE ADDED AFTER ASAP PUBLICATION Reference 23 was added on May 18, 2108. The biogenetic proposal described therein strengthens the hypothesis that a quite unique biosynthetic C5N3 building block exists in this class of marine invertebrates.

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DOI: 10.1021/acs.orglett.8b01069 Org. Lett. 2018, 20, 3039−3043