The 1,3-dialkylpyridiniums (with both cyclic and linear frameworks) are widely distributed cytotoxic compounds that play a role in the antimicrobial properties of marine sponges of the order Haplosclerida (genera Amphimedon, Haliclona, and Xestospongia) [1,2,3,4]. Previous studies have evaluated 1,3-dialkylpyridinium inhibition of histone deacetylase (HDAC) [5,6] and epidermal growth factor (EGF) receptor-mediated mitogenesis [7]. These compounds are also known to block the interaction of chemokines [8] and the binding of methyl quinuclidinyl benzilate (QNB) to the muscarinic receptor [9,10]. In addition to their chemical and bioactivity properties, these metabolites are considered to be the biogenetic precursors of a wide variety of compounds with complicated carbon skeletons and diverse functionalities, such as halicyclamines, ingenamines, manzamines, and sarains [2].

When evaluating bioactive metabolites from Korean marine invertebrates, we recently identified haliclonin A, a cytotoxic macrocylic bisamide from the sponge Haliclona sp. [11]. The novel carbon skeleton of this compound was highly reminiscent of bis-1,3-dialkylpyridiniums, especially cyclostellettamines isolated from Haliclona sp. [9,12]. Thus, we searched for similar 1,3-dialkylpyridinium metabolites in the polar fractions of extracts from diverse collections of this sponge. Here, we report eight novel derivatives of cyclostellettamines with diverse alkyl chains. Several of these compounds exhibited moderate cytotoxicity against the lung cancer A549 cell-line and antibacterial activities against Gram-positive strains. Additional information on their structure-activity relationships were deduced based on their cytotoxicity and antibacterial activity.

The sponge Haliclona sp. specimens were lyophilized, macerated, and repeatedly extracted with CH₂Cl₂ and MeOH. The combined extracts were separated by solvent-partitioning followed by ODS vacuum flash chromatography. The highly polar H₂O-MeOH (50:50) chromatographic fraction was further fractionated by Sephadex LH-20 gel-permeation chromatography and separated by ODS-HPLC to yield 10 compounds (Figure 1). The structures of compounds 1 and 2 were identified as cyclostellettamine N and Q, respectively, based on spectroscopic analyses and a comparison of NMR and FAB-MS/MS data [13,14].

The molecular formula of compound 3, with the shortest retention time in reversed-phase HPLC, was C₂₈H₄₂Cl₂N₂ based on the quasi-molecular ion clusters at m/z 407.3428 [M − H]⁺ and 443.3195 [M + Cl]⁺ in the HR-FAB-MS data. The spectroscopic data of this compound were very similar to those of 1 and 2 (Table 1 and Table 2).

The UV absorption at 267 nm was reminiscent of two di-substituted pyridine rings, which was corroborated by aromatic proton signals [δ_H, 8.96 (2 H, br s, H-2, H-2′), 8.45 (1 H, d, J = 7.6 Hz, H-4), 8.44 (1 H, d, J = 7.7 Hz, H-4′), 8.02 (2 H, dd, J = 7.7, 6.2 Hz, H-5, H-5′), 8.83 (1 H, dd, J = 6.0 Hz, H-6), 8.82 (1 H, dd, J = 6.0 Hz, H-6′)] and carbon signals [δ_C 145.5 (C-2, C-2′), δ_C 145.3 (C-3), δ_C 145.4 (C-3′), δ_C 147.0 (C-4), δ_C 146.9 (C-4′), δ_C 129.2 (C-5, C-5′), δ_C 143.4 (C-6), δ_C 143.5 (C-6′)] in the NMR data. Although the two and three linearly contiguous methylene units attached to the pyridinium (N-1, N-1′, C-3, and C-3′) were identified based on ¹H COSY and gHMBC, only a large methylene envelope at δ_H 1.25–1.40 was observed in the ¹H NMR spectrum, indicating that 3 was composed of two di-substituted pyridine rings and long aliphatic chains, forming a highly symmetric cyclic framework. The lengths of the methylene chains were determined based on the FAB-MS/MS data, which yielded two intense peaks at m/z 190 and 218, as shown in Figure 2. Since these daughter ions were attributable to a pair of monomeric products generated via Hoffmann-type elimination⁷ from their parent ion, 3 was structurally defined to be a cyclic hetero-dimer composed of C₈ and C₁₀ chains connecting the N-1 and C-3′ (also N-1′ and C-3) of pyridiniums.

The molecular formula of compound 4 was C₃₀H₄₆Cl₂N₂ based on HR-FAB-MS data with ion clusters at m/z 435.3741 [M − H]⁺ and 471.3503 [M + Cl]⁺. Thus, the structure consisted of two CH₂ units larger than 3. The NMR data of this compound were very similar to those of 3, indicative of the same cycloalkylpyridinium nature. The FAB-MS/MS spectrum exhibited two intense daughter ions at m/z 218, confirming the symmetrical structure of 4, which contained two C₁₀ linear alkyl chains connecting the N-1 and C-3 between two pyridiniums.

The molecular formulas of compounds 5 and 6 were both C₃₀H₄₄Cl₂N₂ based on HR-FAB-MS analyses. The ¹H NMR spectra of these compounds were almost identical and consisted of two 1,3-disubstituted pyridiniums, two methylene chains, and two olefinic protons [5: δH 5.41 (2 H, br t, J = 3.6 Hz, H-12, H-13); 6: δH 5.38 (2 H, m, H-13, H-14)]. Two prominent daughter ions at m/z 216 and 218 in the FAB-MS/MS data of 5 and 6 showed a C₁₀-saturated chain and a C₁₀-chain with one double bond. The gHMBC data of 5 and 6 showed that these compounds had the same 1,3-dialkylpyridinium substitution patterns as 1–4.

The remaining alkyl substructure and the locations of the double bonds were determined by combined NMR analyses. The ¹H COSY (5 and 6: H-7-H-16) and gHMBC (5: H-11/C-12, C-13, H-12/C-11, C-14, H-13/C-11, C-14, H-14/C-12, C-13, 6: H-12/C-13, C-14, H-13/C-12, C-15, H-14/C-12, C-15, H-15/C-13, C-14) data indicated that 5 and 6 contained a double bond at C-12 and C-13, respectively. Since the olefinic and allylic protons of the double bonds were chemically equivalent, the NOESY analysis could not be used to determine the configurations of the double bonds. Consequently, the configuration of the double bond in 5 was assigned as E based on the downfield shifts of the allylic methylene [δ_C 32.7 (C-11) and 32.4 (C-14)], while the Z-configuration was deduced for 6 from the upfield chemical shifts of the allylic methylene [δ_C 28.0 (C-12) and 29.2 (C-15)] [15].

The molecular formulas of 7 and 8 were C₃₁H₄₇Cl₂N₂ based on HR-FAB-MS analyses. As found for 5 and 6, the ¹H NMR spectra of these compounds revealed characteristic features of cyclostellettamines: 1,3-disubstituted pyridines, methylene chains, and two olefinic protons [7: 5.37 (1 H, ddd, J = 11.4, 6.7, 2.0 Hz, H-9), 5.36 (1 H, ddd, J = 11.4, 6.7, 2.0 Hz, H-10); 8: δH 5.71 (1 H, dt, J = 10.8, 7.3 Hz, H-15), 5.66 (1 H, dt, J = 10.8, 7.3 Hz, H-16)] (Table 1 and Table 3). The FAB-MS/MS analyses of 7 and 8 supported the presence of a C₁₀-saturated chain and a C₁₁-chain with one double bond. The ¹H COSY (7: H-7–H-12, H-16–H-17, 8: H-7–H-9, H-13–H-17) and gHMBC (7: H-8/C-9, C-10, H-9/C-8, C-11, H-10/C-8, C-11, H-11/C-8, C-10, 8: H-14/C-15, C-16, H-15/C-14, C-17, H-16/C-14, C-17, H-17/C-15, C-16) data placed the double bonds at C-9 and C-15 for 7 and 8, respectively. The configurations of these double bonds were both determined to be Z based on the chemical shifts of the allylic methylene carbons [7: 24.8 (C-8) and 28.1 (C-11), 8: δC 28.3 (C-14) and 31.0 (C-17)] [15].

The molecular formulas of 9 and 10 were both C₃₂H₄₉Cl₂N₂ based on HR-FAB-MS analyses. Similar to compounds 5–8, the ¹H NMR spectra of 9 and 10 revealed a 1,3-dialkyl pyridinium structure with two olefinic protons [9: δH 5.34 (1 H, m, H-13), 5.32 (1 H, m, H-14); 10: 5.39 (1 H, dt, J = 10.6, 6.7 Hz, H-15), 5.38 (1 H, dt, J = 10.6, 6.7 Hz, H-16)]. The FAB-MS/MS analyses of these compounds confirmed the presence of a C₁₀-saturated chain and a C₁₂-chain with one double bond. The COSY (9: H-7–H-18, 10: H-7–H-9, H-14–H-18) and gHMBC (9: H-12/C-13, C-14, H-13/C-12, C-15, H-13/C-12, C-15, H-15/C-13, C-14, 10: H-14/C-15, C-16, H-15/C-14, C-17, H-16/C-14, C-17, H-17/C-15, C-16) data were indicative of a double bond at C-13 and C-15 for 9 and 10, respectively. The configurations of these double bonds were both assigned as Z based on the upfield shifts of the allylic methylene carbons [9: δC 28.0 (C-12), 27.9 (C-15), 10: 28.2 (C-14), 29.1 (C-17)].

A comparison of the ¹³C and ¹H NMR data revealed almost identical chemical shifts for both carbons and protons among all cyclic 1,3-dialkylpyridinium dimers 1–10 (Table 1–Table 3). These compounds were structurally similar to cyclostellettamines reported previously with differences in the length of the alkyl chains, as well as the presence of double bonds [9]. Compounds 5–10 were dehydrocyclostellettamines containing one double bond in the alkyl chains. Subsequently, the carbon chemical shifts of allylic methylenes and coupling constants between the olefinic protons were used to assign E or Z configurations to these double bonds.

The 1,3-dialkylpyridinium metabolites possess diverse cytotoxic and antimicrobial activities [16,17]. Thus, we evaluated the cytotoxic and antimicrobial activities of compounds 1–10. Since 7 was isolated in a TFA salt form, we evaluated the influence of the counter ion on bioactivity. Thus, the TFA salt of 4 was prepared using reversed phase HPLC (H₂O-MeOH, 80:20, 0.1% TFA) on 4. Compounds 1–10 and 4a exhibited moderate cytotoxicity against the A549 lung cancer cell line, comparable to doxorubicin (Table 4). On the other hand, they also displayed a diverse range of antibacterial activities against Gram-positive strains (compounds 2, 4, 8, 9, and 10 were the most potent). These results are partly consistent with the previous report that the bioactivities of cyclostellettamines were significantly influenced by the length of the alkyl chains [16]. In addition, as shown for 6 and 9, the bioactivities were influenced by the existence and location of the double bond, although the alkyl chains were the same length. This suggests a mechanism-of-action based on the distances between the charged moieties and electron-rich sites, possibly through an interaction with ionic membrane sites. We also observed that the antibacterial activities of these compounds were generally weaker against Gram-negative than Gram-positive strains. Also, the influence of pyridinium counterions was negligible since there was no significant difference between the bioactivities of 4 and 4a. Overall, these results increase our understanding of the structure-activity relationships of cyclostellettamines.

In addition to the antimicrobial and cytotoxic activity assays, compounds 1–10 were also tested against diverse pathogenic fungal strains and microbial enzymes, such as isocitrate lyase, sortase A, and Na⁺/K⁺-ATPase, and failed to display any bioactivity (LC₅₀ > 100 μg/mL for fungi, ED₅₀ > 100 μM for enzymes).

Eight novel cyclic bis-1,3-dialkylpyridiniums (3–10), as well as two known compounds (1 and 2) belong to the cyclostellettamine class, were isolated from the sponge Haliclona sp. from Korea. These compounds were structurally different from the previous reported cyclostelletamines in the lengths of the alkyl chains, as well as the presence of double bonds in the chains. Several of these new compounds exhibited moderate cytotoxicity against A549 cell-line and inhibitory activities against Gram-positive bacterial strains. As shown in 6 and 9, the bioactivities were influenced by the existence and the location of a double bond. Overall, these results provide us better understanding of the structure-activity relationships of the cyclostellettamine class compounds.