Pyridoacridine Alkaloids

Marine pyridoacridine alkaloids have been the subject of intense study due to their significant biological activities.4-13 Over 75 pyridoacridine alkaloids have been isolated and characterized from marine source and it is expected that more of these alkaloids will be isolated in future. Almost all known pyridoacridine alkaloids are reported to have significant cytotoxicity. The compounds of this group also display several specific biological properties, such as inhibition to topoisomerase II,12-14 antiHIV activity,15 Ca2+ release activity,16 metal chelating properties17 and intercalation of DNA17 property.

Pyridoacridines have a common tetracyclic heteroaromatic parent-pyrido [4,3,2-m,n]acridine (1) system. They are distributed across several phyla of marine invertebrates which are an intriguing fact, and it needs further investigation. One possibility is that in the biosynthesis of these alkaloids probably symbiotic microbes are involved, but it has not yet been tested. Marine pyridoacridine alkaloids have been reviewed extensively.18-23

2.1 Occurrence and Chemical Properties

Pyridoacridines have been isolated from marine sponges, tunicates, anemone and molluscs which are often ornately decorated with bright colors and patterns. Tropical tunicates (ascidians) in particular are generally richly pigmented in colors which vary from yellow to deep red, orange, blue and purple. It is often found that pyridoacridines isolated from such tunicates are the pigments (zoochromes) responsible for their coloration. Pyridoacridines act as a pH indicator. The indicator properties is correlated with the presence of at least two basic pyridine like nitrogen and is probably associated with electronic perturbations of an extended chromophore with charge-transfer properties. Simple indicator properties are absent in the less basic iminoquinones, such as cytodytin-A (2) and diplamine (3). Alkaline solution of the free base generally appears orange or red, while in acidic solution they are green-blue to purple. Some quaternary ammonium alkaloids, like petrosamine (4), are deep blue or purple salts.

Pyridoacridines are generally obtained as microcrystalline solids with melting points above 300°C. They have also been isolated as hydrochloride salts. Few pyridoacridines are found to be optically active. The optical activity of these compounds is due to the presence of additional asymmetric side chain. The majority of pyridoacridine alkaloids have planar heterocyclic system.

Because of variability in oxidation states of the heterocyclic nucleus, pyridoacridines exhibit facile redox reactions. For example, the iminoquinone substructure (5) in many alkaloids is easily reduced by NaBH4. Partially saturated nitrogen containing rings in pyridoacridines are easily aromatized by air oxidation (auto oxidation) upon storage or heating in solution. Although several pentacyclic pyridoacridines have a 1,10-phenanthroline subunit, they do not react with Fe(II) salts to form red complexes. This lack of reactivity must be interpreted cautiously while assigning possible structures to new alkaloids as it does not provide evidence for lack of 1,10-phenanthroline substructure.

Mass Spectra Alkaloid Salt

2.2 Assignment of Structure

The assignment of structure in general by NMR in highly condensed heterocyclic aromatic compounds is complicated because of the difficulty in defining the correct regioisomer from among many possibilities. However, these problems can be solved by employing new powerful multipulse NMR techniques like HMQC, HMBC, INADEQUATE, INAPT. JCH Coupling constant analysis has been helpful in the resolution of ambiguous structural assignments. When suitable crystals of the compound are available, single crystal X-ray diffraction analysis has given definitive structures. Because the ring system (1) is highly conserved, some general features in the appearance of the JH NMR spectra are common to most of these alkaloids and useful in identifying a member of this class of compounds. The disubstituted benzo ring A gives rise to a distinctive linear four proton coupled spin network (HrH4, 7.0-9.0 ppm, J = 8-9 Hz) with Hj resonating at lowest field due to the deshielding acridine nitrogen. A second AB spin system (8.5, 9.0 ppm, J = 5.6 Hz) is assignable to H5-H6, the protons of a trisubstituted pyridine ring. A strong NOE (ca. 20%) is seen between the two bay region'protons, H4-H5, thus, linking these two nonscalar-coupled substructures.

2.3 Structural Subtypes

Pyridoacridines vary in structure by appendage of different side chains or fusion of rings to ring C, and occasionally to the acridine nitrogen. Halogen substitution in the pyridoacridines are rarely seen, and when present, this is always bromine at C2 in ring A. Oxidation states of the rings vary, and in some cases ring D is partially saturated. Additional rings are more commonly appended to ring C.

Tetracyclic Alkaloid

The yellow tunicate Cystodytes dellechiajei from Okinawa has yielded nine cytotoxic tetracyclic pyridoacridine alkaloids named cystodytins A-I (2, 6-11).16,24 The cystodytins A-C are the first tetracyclic pyridoacridine alkaloids isolated from a marine tunicate. Colored tunicate (900 g, wet weight) was collected and kept frozen until used. The methanol-toluene (3:1) extract of C. dellechiajei was partitioned with toluene and water. It was observed that toluene soluble portion exhibited potent cytotoxicity against L1210 murine leukemia cells. The toluene soluble portion was chromatographed by using CHCl3/CH3OH (98.5:1.5) as an eluant followed by a Sephadex LH-20 column (CHCl3/CH3OH, 1:l) to yielded yellow crystals of cystodytins A (2) and B (6) in 0.022% yield. Separation of (2), and (6) was very difficult as both of the compounds had the same retention times on HPLC, silica gel or ODs under different solvent systems. The aqueous layer also exhibited modest cytotoxicity against L1210 and was purified by the same procedure and afforded cystodytin C (7) in 0.0003% yield as yellow crystals in addition to 38 mg of mixture of cystodytin A and B. Both of the compounds were separated, and EIMS of free base (2) showed the molecular ion at m/z 357 (M+). Molecular formula for compound (2) was determined to be C22H19N3O2 by FAB-HRMS (m/z 357.1707). The UV spectrum of (2) exhibited absorptions at 225 (£ 35000), 272 (e 25000), and 380 (e 14300) nm. Absorption at 1640 and 1660 cm- inIR and the resonances at 8 167.8-170.3 and 183.2 in the 13C NMR indicated the presence of an amide and a conjugated ketone carbonyl group, respectively in compound (2). The RCT-COSY spectrum of (2) revealed cross peaks of H-5 to H-6, H-9 to H2-12 and H2-13, H2-12 to H2-13, H-16 to H3-18 and H3-19, H3-18 to H3-19, and among H-1-H-4, respectively. The final structure of compound (2) was determined with the help of lH-13C COSY experiments.

RHN'

Cystodytins D-I (8-13) are chiral levorotatory compounds, due to the presence of 2-amido-1-hydroxyethyl side chain, N-substituted with one of the above mentioned C5 carboxylic acids. Hydration of Cystodytin-A (2, 6% aqueous HCl l00°C, 3 h) gives cystodytin C (7). When treated with diazomethane, (2) formed a methylether (14) (23%). This transformation is unusual as it constitutes a reductive methylation. The iminoquinone system in (2) is readily reduced in the ionization stage of a mass spectrometer, (M+2 ion in EIMS, MH+2 for FABMS), a typical for quinones. The vivid

purple-colored ascidian Lissoclinum vareau from Fiji has furnished two bright crimson pigments varamine-A (16) and varamine-B (17) which occurs with antitumor alkaloid, varacin (15).26 Varamine-A (16) and varamine-B (17) have a parent tetracyclic aromatic ring system at the same oxidation level as the systodytin-A methylation product (14). However, the varamines also contain a methyl thioether substituted at C9.

The bright red tunicate Lissoclinum vareau (Monniot and Monniot, 1987), was collected from Yasawa Island chain, in the Fiji Island. Crude methanol extract of the tunicate exhibited potent antifungal and cytotoxicity against the L1210 murine leukemia cell line. The chloroform soluble fraction of the extract yielded two bright red pigments, varamine A (16) and varamine B (17). The spectral data of (16) and (17) revealed that the two compounds (16, 17) were related as homologues, and structure elucidation was carried out primarily on the trifluoroacetate salt of 16. Molecular formula of varamine A (16) was determined to be C22H23N3O2S by FABMS data (m/z 394.1589 M+). The ultraviolet spectrum of the free base of (16) revealed strong bands from 232 to 494 nm. In particular, the striking color change from yellow to intense red upon acidification of the freebase of (16) was correlated with a reversible bathochromic shift 464 nm (e 5170) to 527 nm (e 5670 nm). In the XH NMR of TFA salt of (16) six proton signals found between 8 7.20 and 8.30 ppm were assigned to deshielded protons of the heteroaromatic ring. The resonance at 8 7.52 (d, J = 6.5 Hz) and 8.21 (d, J = 6.5 Hz) were assigned to H-5 and H-6, respectively, in a trisubstituted pyridine ring.

OMe 14

OMe 14

A strong nuclear overhauser effect from H-5 to H-4 indicates the close proximity of the respective ring residues. Stretches around 1650 and at 3200, 3280, and 3450 cm- confirmed the presence of amide, and NH functionalities. A thiomethyl group (SCH3) appeared as singlet of three proton at 8 2.66. Additional evidence comes from 1/CH values for the methyl groups; these were most easily obtained by measuring the separation of the inner peaks of the methyl 13C satellites in the proton spectrum of (16) or (17). The moderately large one bond coupling constant (1/CH = 141 Hz) for the methyl carbon at 18.7 ppm is similar to that measured for the electronegative methoxy group. Finally structure (16), and (17) were confirmed by 2D NMR spectroscopy. Ring C of varamine-A (16) isoelectronic with hydroquinone, was readily oxidized by aqueous ceric ammonium nitrate to the iminoquinone (18) in quantitative yield.25 The corresponding oxidation product (3) of varamine-B (17) was found identical with diplamine from the Fijian tunicate, Diplosoma r\i-l 1 o on sp. Another homologue in this series, isobutyramide (19),182' has been characterized from an unidentified Australian tunicate.

Pentacyclic Alkaloids

The pentacyclic pyridoacridine alkaloids could be classified into two groups: (a) those having one additional angular-fused ring at C9, 10 of the acridine system at C8, C9 of ring C and (b) those having linear-ring fusion at C8, C9 of ring C. Typical ring appendages include pyridine, tetrahydropyridine, pyridone, thiazine, or even a thiazole heterocycle. In some cases, a substituted 2-ethylamino side chain is also attached to the acridine C ring. The bright yellow zoochrome calliactine isolated from the mediterranean anemone, Calliactis parasitica, has a long history and is probably the first marine pyridoacridine alkaloid isolated from marine organisms. Calliactine attracted the attention of Lederer et al28 in 1940 and later by Barbier29 however, the structure determination work was hampered by low solubility of the compound and the difficulties in purification. In 1987, Cimino et al30 reported their studies on the degradation and NMR spectroscopy of calliactine and its derivatives. Calliactine is readily aromatized (presumably autoxidation elimination with concomitant hydrolytic loss of ammonia) by boiling with dilute HCl to give chlorocalliactine"or with water to give neocalliactine"

which in turn yielded neocallicatine acetate when treated with acetic anhydride in pyridine. The molecular formula C18H21N4O was established for calliactine, and several possible structures (20a-20d) were advanced. Four possible structures (21a-21d) for neocalliactine acetate were also proposed. However, definitive assignments have not yet been reported on either of these compounds. The amphimedine (22) is the first pyridoacridine alkaloid to be fully characterized.31Schmitz et al31 isolated amphimedine (22), a sparingly soluble yellow solid (m.p. 360°C) sponge Amphimedon sp. was collected from Guam Island. Ambient temperature extraction with CH2Cl2, CHCl3-MeOH, MeOH followed by hot Soxhlet extraction with CHCl3 and finally purification by coloumn chromatography yielded pure compound. Molecular formula of (22) was established as C19H11N3O2 by high-resolution mass spectral analysis (m/z 313.08547, M+). In the mass spectrum few fragments were observed which indicated that the compound is highly stable. The electronic absorption

OH 20a

NH 20b

NH 20b

NH 20c

NH 20c

OH 20a

OH NH 20d

OH NH 20d

O 21c

O 21d

O 21c

O 21d

O MeN

O MeN

23, R = Br spectra of 22 showed absorption at ^max 210 (e 19690), 233 (e 39393), 281 (e 9099), 341(e 6060). Significant changes were observed in the absorption with NaBH4 [Xmax nm 235 (e 12879), 280 (e 9090)], suggesting the presence of conjugated carbonyl functionality. Two strong absorptions 1690 and 1640 cm1 in the IR spectrum confirmed the presence of two carbonyl groups. Further since the absorption was observed at lower frequency so these peaks were attributed to a,P-unsaturated ketone and amide functionalities, respectively. The 13C NMR data reconfirmed the presence of an amide carbonyl (C-11 , 8 165.9) and a cross-conjugated ketone (C-8, 8175.0). The 2D NMR 13C-13C INADEQUATE NMR techniques were used for the structural elucidation of amphimedine (22).3234

Amphimedine (22) is selectively brominated (Br2, acetic acid) to give the mono bromo derivative (23). Neoamphimedine (24),23 along with amphimedine (22) and debromopetrosamine (25a), have been isolated from the Micronesian sponge Xestospongia carbonaria.9 Neoamphimedine (24) is a regioisomer of amphimedine (22). Kobayashi et al35 have isolated ascididemin (26)6,17,36 from a species of Didemnum collected in Okinawa. The structural proof relied on extensive use of long-range 1H-13C correlation (COLOC) data and comparison, with the properties of amphimedine (22). It is noteworthy that ascididemin (26), like related pyridoacridine alkaloids which have a 1,10-phenanthroline ring system, does not form a bright red complex with Fe2+ that is characteristically observed with 1,10-phenanthroline itself.

Schmitz et al37 have isolated 2-bromoleptoclinidone from Leptoclinides sp. collected in Truk Lagoon. 2-Bromo-leptoclinidone was assigned structure (28) based on interpretation of long range 2D 1H-13C NMR correlation data and the absence of a color reaction with Fe2+. However, this structure was later revised38 and correct structure (27) was shown to have a alternate pyrido ring orientation.38 This was confirmed by selective long range 1D 1H-13C INAPT experiments39 and debromination of bromoleptoclinidone to ascididemin (26).

Pyridoacridine alkaloids had not only been obtained from marine invertebrates of tropical waters, the pentacyclic phenolic alkaloid is also obtained from a South Australian temperate water tunicate Amphicarpa meridiana,14 and more recently from a Caribbean sponge Corticum sp.40 The structure of meridine (29)40 is determined by single-crystal X-ray diffraction analysis.14 An isomer of meridine was also isolated from A. meridiana and assigned structure as (30). The regiochemistry of (30) was assigned on the basis of NOE studies. Rapid tautomerism of pyridoacridine alkaloids has been observed. On standing, in CDCl3 compound (30) undergo isomerization to (29) at room temperature. The Caribbean sponge Petrosia sp. when viewed under water looked jet black due to its deep dark pigmentation. Samples were immersed in methanol imparted a deep green-blue color to the solvent. This extract when diluted with water, the color changed to purple. The brominated pigment petrosamine (31a and 31b) along with tryptamine

O O 24

O Me Me 25a

O Me Me 25a

O Me Me 25b

6 28

6 28

O 30

have been (29) isolated from this extract.41 Faulkner et al41 isolated petrosamine (31), from marine sponge Petrosia sp. while attempting to purify an antimicrobial constituent amphimedine (22). Petrosamine is remarkable compound since color of the solution containing (31) changes by the addition of dilute organic or aqueous solutions. It was observed that blue colored methanol extracts of Petrosia sp. exhibited antimicrobial activity against Staphylococcus aureus and Bacillus subtilis. The marine sponge Petrosia sp. was collected from Carrie Bow Cay, Belize and methanol soluble material portion was partitioned to ethyl acetate, n-butanol, and aqueous extracts. A blue band and pale yellow band from n-butanol soluble material was separated on Sephadex LH-20 (MeOH) column. The blue compound was repurified on Sephadex LH-20 column and yielded petrosamine (0.1% dry weight) as dark green crystals, m.p. >330°C. Molecular formula of petrosamine was established as C21H17BrN3O2 by high resolution mass spectrum. Both the 1H and 13C NMR spectra indicated the presence of three N-methyl signals, two of which were equivalent. The remaining signals were all in the aromatic region of the

spectra, except for a 13C NMR signal at 8 187.4 (s) that could be assigned to a single quinone-type carbonyl group. X-ray crystallography in the solid state of petrosamine revealed that the pigment exists as the chloride salt of a quaternized pyridone-acridine ring system (31a). Correlation of the solvent-dependent changes in the UV and NMR spectra suggested that the remarkable color change observed by varying solvent polarity, was associated with shifts in the position of a keto enol equilibrium, favoring the enol form (31b).

The deep-water sponge Dercitus sp. has yielded the dark violet pigment named dercitin (32) together with dimethylindolinium chloride.42 The structure of (32) was assigned on the basis of spectroscopic data including 2D INADEQUATE. This structure was subsequently revised to (33)17 by interpretation of the magnitudes of long-range carbon-proton coupling constants.

The earlier assignment error arose from inherent difficulties in the interpretation of 2D 1H-13C NMR spectra in highly condensed heteroaromatic compounds. In the revised structure, the nitrogen and sulphur of the thiazole ring are in correct position relative to other acridine substituents. The thiazole ring proton exhibits different /CH to each of the two ring junction quaternary carbons, thus providing unambiguous assignment of the respective 13C signals.43 The ease with which a partially reduced pyridoacridine system can be aromatized was demonstrated when the dihydropyridoacridine (34), obtained

by reduction of dercitin (33)44,45 with sodium borohydride rapidly autoxidized back to dercitin during workup.

Gunawardana et al44 have reported the isolation of four minor congeners from Dercitus sp. Of these, nordercitin (35) and dercitamine (36) are related to dercitin (33). Reductive methylation of dercitamine (HCO2H, HCHO) gave nordercitin (35). Dercitamicle (37) contains a propionamide side chain and cyclodercitin (38) is a hexacyclic quaternary salt.

The purple colored colonial tunicate Trididemnum sp.46-48 has furnished two related bases, shermilamine-A (39)47 and shermilamine-B (40).47 Kuanoniamines A-D (41-44), along with shermilamine-B (40), are found in the lamellarid mollusc Chelynotus semperi and its prey, an unidentified tunicate, both collected in Pohnepei.43 Kuanoniamines-B (42) and D (44) are homologues of kuanoniamine C having isovaleramide and acetamide side chains, respectively. Kuanoniamine A (41) differs from the other three alkaloids in lacking the 2-amidoethyl side chain and contains an iminoquinone structure analogous to those found in 2-bromoleptoclinidone and ascididemin.

37, R = NHCOCH2Me2

X" +

1-N

\

/

s /

R

\ '

V=N

NHAc

NHAc

42, R = NHCOCHMe2

43, R = NHCOCH2CH3

Hexacyclic and Heptacyclic Alkaloids

Two hexacyclic alkaloids have been reported from the deep-water sponges Dercitus sp. and Stelleta sp. Cyclodercitin (38) is found along with dercitin (33) and other related compounds in Stelleta sp.43 The sixth ring in cyclodercitin is formally derived by cyclization of the 2-aminoethyl side chain to the acridine nitrogen, while the pyridine ring is substituted with an N-methyl group. When dissolved in TFA-d4 cyclodercitin spontaneously autoxidizes to the hexacyclic pyrrolo compound (45). Recently, the hexacylic pyrrolo compound (46) is obtained as a minor compound from Stelleta sp. and its structure has been determined by X-ray analysis.

The red sea tunicate Eudistoma sp. has yielded six alkaloids, including the known pyricloacridine alkaloid shermilamine B (40).49 In addition to these segoline-A (47) and isoegoline-A (48) are regioisomeric hexacyclic pyridoacridine alkaloids.

OMe 47

OMe 47

OMe 48

Segoline-A (47) and isosegoline-A (48) are the first optically active pyridoacridines isolated from the marine source.50,51 Segoline A (47) was isolated from Eudistoma sp. in 0.4% (dry weight) yield. Molecular formula of segoline A (47) was established as C23H19N3O3. Intensive 1D and 2D NMR experiments such as COSY, NOE, short- and long-range CH correlations, COLOC, and HETCOSY studies were conducted on (47). The proton NMR data revealed a trisubstituted benzo-3,6-diazaphenanthroline ring system for segoline A. The pyridine ring of the diazaphenanthroline ring was characterized by the H-2 and H-3 signals. The pyridine moiety was hydrogenated easly to the 1,2,3,3a-tetrahydro derivative indicated that it was part of a quinoline ring. The heterocyclic ring system was confirmed by the NOEs between H-2 and H-3, H-3 and H-4, H-5 and H-6, and H-6 and H-7. Unequivocal structure of (47) was determined by single X-ray analysis. The structure of isosegoline A (48) is determined by NMR spectroscopic techniques. Segoline B (49) is a diastereoisomer of segoline A (47). In segoline B (49), the bridge across the cyclic imide ring is inverted. This is supported by the strongly bisignated CD curves for (48) and (49), which are almost exactly opposite in sign. The structure of eilatin (50) and unusual pyridoacridine pseudo-dimer',' has been solved by X-ray diffraction. Eilatin (50)51 is the only known heptacyclic pyridoacridine alkaloid.

Octacyclic Alkaloids

Chiral pyridoacridine alkaloids are rare. Eudistone A (51) and eudistone B (52), the two optically active octacyclic alkaloids obtained from the Seychelles tunicate Eudiste sp.,52 differ from other members of the class by having additional dihydroisoquinolone bicyclic ring system fused to a quaternary

carbon of the acridine system. The relative stereochemistry of the carbon skeleton was determined by comparison of NMR coupling constants with values predicted from molecular modeling. The two compounds were correlated by autoxidation. Eudistone (51) aromatized to (52) when air is bubbled in a solution of (51) in DMSO.52 The circular dichroism spectrum of eudistone-B (52) exhibited a strong bisignate cotton effect. However, the absolute configuration of the two compounds remains unknown.

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