The Ammosamides: Structures of Cell Cycle Modulators from a Marine-Derived Streptomyces Species

Marine-derived actinomycete bacteria are emerging as a valuable resource for bioactive natural products encompassing a variety of unique structural classes.[1] In our hands, early detection of cell growth inhibitors using in vitro cytotoxicity assays against the colon carcinoma cancer cell line HCT-116, followed by extensive mechanism of action studies, has proven to be an effective approach. As such, the HCT-116 assay has been instrumental in the identification of potentially important anticancer agents.[2]

In the course of our continued studies, Streptomyces strain CNR-698[3] was isolated from bottom sediments collected at a depth of 1618 meters in the Bahamas Islands in 2003. Cytotoxicity-guided (HCT-116) fractionation by C₁₈ flash chromatography and RP-HPLC of crude extract led to the isolation of ammosamides A (1) and B (2) as blue and red solids, respectively (3 and 4 mg L⁻¹). Structure assignments for 1 and 2 proved to be particularly difficult due to their inherent insolubility (soluble only in dimethyl sulfoxide (DMSO)) and a lack of descriptive NMR signals, ultimately requiring the integration of NMR spectral analysis, mass spectrometry data, and single crystal X-ray diffraction studies.

High-resolution (ESI) mass spectrometric analysis of ammosamide A (1) indicated a molecular formula C₁₂H₁₀³⁵ClN₅OS (m/z [M+H]⁺: 308.0303]. The molecular weight of ammosamide B (2) was found to be 16 amu lower (m/z [M+H]⁺: 292.0604) consistent with the molecular formula C₁₂H₁₀³⁵ClN₅O₂. The UV/Vis spectrum of 1 was indicative of an unusually highly conjugated structure with absorptions at λ_max=580, 430, 350, and 290 nm.

Inspection of the ¹H NMR spectrum of 1 in [D₆]DMSO revealed six singlets between δ=6.0 and 9.0 ppm and one methyl singlet at δ=4.03 ppm, while the ¹³C NMR spectra revealed the presence of eleven sp² hybridized carbon atoms and a single sp³ hybridized carbon atom at δ_c=33.3 ppm (Table 1). The addition of D₂O (20 μL) to the sample in [D₆]DMSO resulted in the immediate disappearance of ¹H NMR signals at δ=7.16 (1H), 6.63 (1H), 6.89 ppm (2H) and the slower disappearance of singlets at δ=8.92 (1H), and 7.68 ppm (1H) (less than 10 min). The exchangeable protons at δ=7.16, 6.63 and 6.89 ppm were assigned as aromatic amines at C-6 and C-8 (based on HMBC correlations), while the slowly exchanging protons at δ=8.92 and 7.68 ppm were assigned to a primary amide on the basis of COSY and HMBC correlations. The only non-exchangeable hydrogen atoms were the methyl singlet resonance at δ=4.03 ppm and a one-proton singlet at δ=8.47 ppm. The ¹³C NMR spectrum of 1 indicated the presence of two carbonyl groups (δ_c=177.2 and 166.0 ppm), as well as two upfield sp² carbon atoms (δ_c=103.1 and 110.5 ppm). HMBC correlations between the downfield carbonyl (δ_c=177.2 ppm) and the proton methyl singlet at δ=4.03 ppm, we thought, defined an N-methyl amide, although a carbon chemical shift so far downfield would not be expected. In addition to correlations from the aromatic δ=8.47 ppm singlet, the only other HMBC correlations were from the exchangeable protons at δ=7.16/6.63 ppm to C-7 (δ_c=103.1 ppm) and from δ=6.89 ppm to C-7 and C-8a (δ_c=110.5 ppm).

Table 1.

NMR spectral data for 1 and 2 ([D₆]DMSO).

	Ammosamide A (1)			Ammosamide B (2)
Position	δC[a,c]	δH[d]	HMBC	δC[b,c]	δH[d]
1a	33.3	4.03 (s)	C-2, C-8a	28.5	3.59 (s)
2	177.2			164.0
3	116.5	8.47 (s)	C-2, C-4	115.3	8.34 (s)
			C-5b, C-4a
4	144.6			144.7
4a	166.0			166.2
5b	119.7			119.0
7	103.1			104.5
8a	110.5			106.3
CONH2		7.68 (brs)	C-4		7.66 (brs)
		8.92 (brs)	C-4a		8.91 (brs)
NH2 (C-6)		7.16 (brs)[e]	C-7		6.73 (brs)[f]
		6.63 (s)[e]
NH2 (C-8)		6.89 (s)	C-7, C-8a		6.18 (brs)[f]

^[a]75 MHz.

^[b]125 MHz.

^[c]C-2a, C-5a, C-6, and C-8 (1: δ_C = 132.6, 134.7, 136.8, 142.7 ppm; 2: δ_C = 130.6, 130.8, 132.4, 140.5 ppm) could not be unambiguously assigned.

^[d]600 MHz.

^[e]At certain concentrations, these two one-proton signals coalesce to a two-proton signal at δ = 7.09 ppm.

^[f]Assigned by analogy to 1.

The spectral data for 1 suggested a highly unsaturated azaaromatic metabolite possessing three rings. However, the lack of definitive NMR assignments that could be used to link these features forced us to concentrate efforts toward obtaining an X-ray crystal structure. We were fortunate to obtain small crystals of 1 by the slow diffusion of H₂O into a saturated solution in DMSO.[4] The X-ray assignment of ammosamide A (1) is shown in Figure 1. Once X-ray data became clear, the spectral data for 1 could be assigned.

Figure 1.

X-ray crystal structure of ammosamide A (1). Red O, blue N, yellow Cl, black C, white H.

The structure assignment of ammosamide B (2) followed from analysis of spectral data and chemical interconversion. Comparison of the C-2 carbonyl chemical shifts in 1 (δ_C=177.2 ppm) and 2 (δ_C=164.0 ppm) revealed a difference of 13 ppm, consistent with the typical ¹³C chemical shift difference between a carbonyl and a thiocarbonyl (ca. 20 ppm).[5] In order to chemically confirm the presence of the thiolactam functionality, we used Lawesson's reagent [2,4-bis(p-methoxyphenyl)-1,3-dithiadiphosphetane-2,4-disulfide] to convert lactam 2 into thiolactam 1.[6] The low yield of this reaction is likely attributable the nucleophilic amines in 2.[7] Exposed to air during storage, 1 was gradually converted to ammosamide B (2). Notably, the transformation could also be accomplished in 10 min, upon treatment of 1 with hydrogen peroxide in aqueous methanol.[8] This reactivity has been previously observed in other thioamide-containing compounds.[9]

The structural similarities between the ammosamides and the microbial product lymphostin (3) are clear,[10] as is the relationship of the ammosamides to several sponge-derived pyrroloiminoquinone natural products, including batzelline A (4),[11a] isobatzelline D (5),[11b] and makaluvamine A (6)[11c] (Scheme 1). The sponge metabolites 4–6 possess different patterns of Cl and NH₂ substitution and assume p-iminoquinone and o-quinone structures. The presence of an amino group at C-8 in the ammosamides results in a fundamentally different structure type in which the quinoline tautomer predominates. The pyrrole moiety in 3–6 is uniquely oxidized to the pyrrolidinone in ammosamide B (2). Finally, though methyl sulfides are present in 4 and 5, ammosamide A (1) is the first natural product to contain a thio-γ-lactam functionality.[12]

Scheme 1.

Related metabolites from bacteria and sponges.

The fact that the ammosamides are highly colored (1: λ_max=580 nm; 2: λ_max=530 nm), yet lack quinone or iminoquinone functionalities, leads to speculation about the electronic character and reactivity of these metabolites. The intense colors of these compounds could reflect a strong charge separation between the two six-membered aromatic rings due to the effects of electron-donating groups on the chlorine-containing ring and electron-withdrawing substituents on the pyridine ring. It is, conceptually, also explained by the potential for ammosamide A to exist in an equilibrium with its bis-iminoquinone tautomer (Scheme 2). Furthermore, in 1 and 2 the chlorine atom at C-7 is poised to engage in nucleophilic aromatic substitution with a suitable nucleophile, particularly when the molecule exists as its bis-iminoquinone tautomer.[13] This reactivity may be relevant to the molecule's interaction with its protein target.[14]

Scheme 2.

Possible tautomeric form of ammosamide A (1).

Ammosamides A (1) and B (2) exhibited significant in vitro cytotoxicity against HCT-116 colon carcinoma, each with IC₅₀=320 nM. These compounds also demonstrated pronounced selectivity in a diversity of cancer cell lines with values ranging from 20 nM to 1 μM, indicating a specific target mechanism of action. To explore the intracellular target of the ammosamides, ammosamide B (2) was converted to a highly fluorescent molecule by conjugation.[14] Treatment of HCT-116 colon carcinoma or HeLa cells with this fluorescent molecule produced immediate and irreversible labeling of a specific protein in the cellular cytosol. Using a cell and molecular biology approach, the target of the ammosamides was identified as a member of the myosin family, important cellular proteins that are involved in numerous cell processes, including cell cycle regulation, cytokinesis, and cell migration.