Pyrroloacridine and Related Alkaloids

The alkaloids of this group are mostly cytotoxic. Pyrroloacridines have similar ring construction to the pyridoacridine alkaloids. However, they contain a fused pyrrole ring rather than the familiar trisubstituted pyridine ring. The first example of this class are plakinidine5355 A (53) and B (54) which have been isolated from the sponge Plakortis sp.53 Ireland et al56 reported the isolation of plakinidine-A (53), B (54) and C (55) from Plakortis sp. collected in Fiji.

Bioassay-guided isolation of red sponge Plakortis has afforded two novel alkaloids named plakinidine-A (53) and B (54). Both of the compounds showed in vitro activity against the parasite Nippostrongylus brasiliensis (at 50 pg/mL). A pyrrolo[2,3,4-kl]acridine fused-ring skeleton in (53) and (54), represents a new structural variation within polycyclic aromatic alkaloids

54, R = Me from marine organisms. The organism was collected by scuba at 10 m off Hideaway island, Port Vila, Vanuatu. Red viscous oil (3.87 g) was obtained from aqueous methanol extracts of the preserved organism (0.2 kg, wet). The crude oil after partitioned between aqueous MeOH and the solvent series of hexanes, CCl4, CH2Cl2 were separated by column chromatography (reversed phase and Sephadex LH-20/methanol). Plakinidine A (53) was isolated as deep purple solid from methanol (52 mg, 0.026% wet weight, mp. 248-250°C) along with 24 mg (0.012%) of a purple oil identified as plakinidine B (54). The molecular formula of (53) was determined as Cl8H14N4O, from EI-HRMS (m/z 302.1169, M+) and an APT 13C NMR spectrum. Four separate proton spin systems were confirmed by COSY experiments in DMSO-d6 and CDC13-TFA-d4 (1:1). Protons on N-8 to C-10 were assigned to a CH2CH2N(H) group, and protons on C-3 to C-6 were confirmed to be a part of ortho-disubstituted benzene ring. Other protons H-2, was a low-field singlet (8 8.84) with a large JCH coupling constant (200.4 Hz in CDCl3-TFA-d4), indicating a nitrogen was adjacent to C-2.57 Based on the 13C chemical shifts remaining fragment consisting C9N atoms were assumed to have five double bonds. Long-range 1H-13C COSY experiments were done to confirm the structure of the compounds. Three bond correlations to H-4 and H-5 revealed the location of quaternary carbons in the molecule. The existence of a six-membered ring P-enamino ketone was confirmed by the IR (1624 cm1 ) and COSY NMR correlations from H2-9 to C-7b and from H2-10 to C-11 and C-1la. Finally, two dimensional 13C-13C INADEQUATE was used to verify most of the structural elements of plakinidine A.57 Bioassay guided purification of the methanol-toluene extract of the tropical green sponge Prianos melanos from Okinawa gave a cytotoxic pigment, prianosin A (56), prianosin B (57), prianosin C (58) and prianosin D (59).58,59

Several independent reports described the identification of discorhabdins, compounds similar to prianosins.6064 The first report described the isolation and structure elucidation of discorhabdin-C (60) an achiral spiro-alkaloid. Discorhabdin-A identical to prianosin-A, (56) was isolated together with discorhabdin-B (61) from three species of Latrunculia sponge collected the

Prianos Sponge

temperate waters around New Zealand. The quaternary iminium salt discorhabdin D (62) is isolated from both Latrunculia brevis, from New Zealand and Prianos sp. from Okinawa.65 Each discorhabdin or prianosin contains an unsaturated cyclohexenone and spiro-fused to a tetracyclic ring system. Each ring system, with exception of discorhabdin C (60) is also bridged with tetrahydrothiophene ring. A deep-water collection of Batzella sp. from Bahamas has furnished three pyrroloquinolines, batzellines A-C (63-65).66 Each alkaloid has tetrahydroquinoline nucleus further, bridged across both rings by a trisubstituted pyrrole ring. An unusual chlorine atom is present in all the alkaloids.

Prianos Sponge

The sponge Bratzella sp. has also furnished four additional pyrroloacridine alkaloids named isobatzellines A-D (66-69).67

Isobatzellines are not strictly isomeric with batzellines, but differ in replacement of one of the quinone carbonyl group with an amino group. Autoxidation of isobatzellin A (66) to D (69) is facile and could be observed within a few hours during thin layer chromatography or treatment with DDQ.

Isobatzelline A (66) can be converted into bratzelline A (63) by diazotization substitution in aqueous nitrous acid. The Fijian tunicate Clavelina sp.68'69 has yielded wakayin (70) a unique alkaloid. Ireland et al68,69 isolated a new pyrroloiminoquinone based alkaloid, wakayin (70) from the ascidian Clavelina sponge. Wakayin (70) exhibited potent in vitro cytotoxicity against the human colon tumor cell line (HCT116 IC50 0.5 pg/mL). Wakayin was found to inhibit topoisomerase II enzyme (250 pM) and based on related biological data it was concluded that wakayin exhibit its biological activity by damaging the DNA. This compound also exhibited antimicrobial activity against Bacillus subtilis (MIC = 0.3 pg/mL). The crude methanol-chloroform extract was partitioned by reversed phase flash chromatography using methanol-aqueous trifluoroacetic acid solvent systems. Purification of combined biologically active fractions by repeated elution through Sephadex LH-20 yielded 15 mg (0.005% wet weight) of wakayin (70) as a triflouroacetate salt. Molecular formula of this compound was established as C20H15N4O by the use of FAB-HRMS (MH+ 327.1262, calculated 327.1246). Several features of the 1H and 13C NMR spectra of (70) were matches with discorhabdins and isobatzellines,

O Ri

suggesting the presence of a pyrroloiminoquinone moiety. The spin system comprising 8 13.04 (br s, NH1), 7.11 (d, J = 2.5 Hz, H2), 2.93 (t, J = 8.0 Hz, H24), 3.78 (br t, J = 8.0 Hz, H25), and 10.32 (br s, NH6) was established by DQCOSYD and 1-D lH difference NOE70 NMR experiments. This spin system was assigned to part of a pyrrolodihydropyridine moiety by HMQC71 and HMBC72 NMR experiments. IR absorption at 1662 and 1447 cm- indicated the presence of an iminoquinone ring which was reconfirmed by 13C NMR signals at 8 166.28 (CO) and 156.72 (C7). The connectivities between the pyrrolodihydropyridine system and C7, C8, and C9 of the iminoquinone ring was established by long range 1H-13C NMR correlations observed for H2 (8 120.74). The lH NMR signals at 8 13.41 (br s), 7.28 (d, J = 2.5 Hz) and 13C NMR signals at 8 134.25 (s), 120.44 (s), 114.15 (s), 125.14 (d, lJCH = 190 Hz) confirmed the presence of another 2,3,4 trisubstituted pyrrole ring. Long range lH-13C NMR correlations experiment also confirmed this and proved that the pyrrole ring was bound to C11 and C15 of the iminoquinone ring. 1H and 13C NMR data were also suggested the presence of a 3-substituted indole moiety [8 11.54 (br d, J = 2.0 Hz), 7.73 (d, J = 2.0 Hz), 7.52 (d, J = 8.0 Hz), 7.49 (d, J = 8.0 Hz), 7.19 (dd, J = 8.0, 8.0 Hz), 7.09 (dd, J = 8.0, 8.0 Hz); 13C [8 105.90 (s), 112.07 (d, J = 161 Hz), 118.86 (d, J = 161 Hz), 119.66 (d, J = 161 Hz), 121.85 (d, J = 161 Hz), 124.94 (d, J = 183 Hz), 125.49 (s), 136.76 (s)]. This was further confirmed by HMQC and HMBC NMR experiments and by comparison with the 13C NMR data reported for the indole-imidazole moiety of topsentin.73 Long-range lH-13C correlations by HMBC and selective INEPT NMR experiments between H13 and C16 established connectivity between the bipyrroloiminoquinone moiety and the 3-substituted indole, and confirmed the structure wakayin.

Sources and cytotoxities of many marine pyridoacridines that have been reviewed recently74 are summarized in Table 1.

Table 1 Cytotoxic marine pyridoacridines and their sources

S. No.

Pyridoacridine

Source

References

1

Labuanine-A

Biemna fortis sponge (Indonesia)

75

2

Lissoclin-A

Lissoclinum sp. ascidian (Australia)

76

3

Lissoclin-B

Lissoclinum sp. ascidian (Australia)

76

4

Lissoclin-C

Lissoclinum sp. ascidian (Australia)

76

5

Lissoclinidine

Lissoclinum notti ascidian (New Zealand)

77

6

9-Aminobenzo

Biemnafortis sponge (Indonesia)

75

[ft]pyrido[4,3,

2-de]-[1,1 0]-

phenanthrolin-

8(8H)-one

7

Amoamine-A

Cystodytes sp. ascidian (Arno Atoll, Rep.

76,

77

Marshall Is.)

8

Amoamine-B

Cystodytes sp. ascidian (Arno Atoll, Rep.

77,

78

Marshall Is.)

(Cond.)

S. No.

Pyridoacridine

Source

References

9

Pantherinine

Aplidium pantherinum ascidian

79

(S. Australia)

10

Sagitol

Oceanapia sagittaria sponge (Palau)

80

11

Sebastianines-A

Cystodytes dellechiajei ascidian (Brazil)

81

12

Sebastianines-B

Cystodytes dellechiajei ascidian (Brazil)

70,

81

13

Biemnadin

Biemna fortis sponge (Indonesia)

75

14

2-Bromoleptocl

Leptoclinides sp. ascidian (Truk Lagoon)

82,

83

inidinone

15

Meridine

Amphicarpa meridiana ascidian

84,

85

(S. Australia), Leptoctinides sp. sponge

(Truk Lagoon), Cortidum sp. sponge

(Bahamas)

16

Meridine

Biemna fortis sponge (Indonesia)

75

regioisomer

17

5-Methoxyneo-

Xestospongia carbonaria, X. cf.

86

amphimedine

exigua (Indopacific)

18

Neoamphimedine

Xestospongia sp. sponge (Philippines)

86,

87,

Xestospongia cf. carbonaria (Micronesia)

88,

89

Xestospongia c carbonaria, X. cf. exigua

(Indopacific)

19

Neoamphimedine-

Xestospongia c carbonaria, X. cf. exigua

86

Y

(Indopacific)

20

Neoamphimedine-

Xestospongia cf. carbonaria, X. cf. exigua

86

Z

(Indopacific)

21

Nordercitin

Stelletta sp. sponge Derdtus sp. sponge

90,

91

(Bahamas)

22

Stellettamine

Stelletta sp. sponge

90

23

Styelsamine-A

Eusynstyela lateridus ascidian (Indonesia)

92

24

Styelsamine-B

Eusynstyela lateridus ascidian (Indonesia)

92,

93, 94

25

Styelsamine-C

Eusynstyela lateridus ascidian (Indonesia)

92

26

Styelsamine-D

Eusynstyela lateridus ascidian (Indonesia)

92

27

Varamine-A

Lissoclinum vareau ascidian (Australia)

76,

95

28

Varamine-B

Lissoclinum vareau ascidian (Australia)

96,

95

29

Cystodytin-D

Cystodytes delleehiajei ascidian (Okinawa)

96,

97

30

Cystodytin-E

Cystodytes dellechiajei ascidian (Okinawa)

96,

97

31

Cystodytin-F

Cystodytes delleehiajei ascidian (Okinawa)

96,

97

32

Cystodytin-G

Cystodytes delleehiajei ascidian (Okinawa)

96,

97

33

Cystodytin-H

Cystodytes delleehiajei ascidian (Okinawa)

96,

97

34

Cystodytin-I

Cystodytes delleehiajei ascidian (Okinawa)

96,

97

35

Cystodytin-J

Cystodytes sp. ascidian (Fiji), Lissoclinum

13,

77,

nolli ascidian (New Zealand)

96,

98

36

Cystodytin-K

Lissoclinum nolli ascidian (New Zealand)

77

37

Dercitamine

Stelleta sp. sponge, Dercitus sp. sponge

90,

99

(Bahamas)

38

Diplamine

Diplosoma sp. ascidian (Fiji), Cystodytes

13,

93,

sp. ascidian (Fiji), Lissoclinum nolli

98,

100

ascidian (New Zealand)

39

Eilatin

Cystodytes sp. ascidian (Fijian),

13,

93,

Eudistoma sp. ascidian (Eilat)

101

, 102

40

Isodiplamine

Lissoclinum nolli ascidian (New Zealand)

77

Total synthesis of many of the alkaloids have been achieved. Synthesis of cystodytin-A and B have been published by Cinfolini et al103 using an efficient intramolecular photochemical nitrene insertion into an aryl substituted dihydroisoquinoline. Three total synthesis of amphimedine have been reported.104-06 Synthesis of bromoleptoclinidone and ascidemin have also been published.107,108 Total synthesis of most of the pyridoacridines given in Table 1 have also been achieved. New pyridoacridine structures provide fertile area for the design and execution of biologically active heterocyclic molecules.109 Newer synthetic strategies are emerging for efficient assembly of tetracyclic and pentacyclic ring system.

Biological Activity of Pyridoacridines

Mostly all of the pyridoacridines discovered so far have shown strong in vitro cytotoxic activity. The activity has been related to the ability of pyridoacridines to intercalate DNA, so inhibit DNA metabolizing enzymes. Pyridoacridines also exhibited antiviral, antimicrobial, insecticidal, fungicidal, and other activities. Some pyridoacridines have also shown excellent antitumor activity in various models, while others have proven too toxic to be useful for clinical purposes. Pyridoacridine alkaloids show in vitro cytotoxicity against cultured tumor cells (L 1210 murine leukemia, P388, etc.) or antineoplastic activity in whole animal experiments. Dercitin (33) inhibits a variety of cultured cell clones at nanomolar concentrations and shows antitumor activity in mice and modest antiviral activity against Herpes simplex and A-58 murine corona virus at micromolar concentration.42 Dercitin (33) exhibited strong inhibition of HSV-1 at 5 8g mL1 with moderate cytotoxicity. It also completely inhibited murine A59 coronavirus at 1 ^g mL- with no cytotoxicity.17 A thorough study by Burres et al110 has shown that dercitin (33) inhibits both DNA and RNA synthesis upto 83% at 400 nM, but protein synthesis is effected to a lesser extent. Dercitin also binds to calf thymus DNA and relaxed supercoiled $ x 174 DNA at 36 nM and inhibited DNA polymerase and DNase nick translation at 1 nM. Collectively these results suggest that inhibition of enzyme activity by dercitin is of secondary importance, and its activity is entirely consistent with potent intercalation of nucleic acids. The activities of dercitin (33) and its analogues have been compared, and the nature of pharmacophores in dercitin responsible for antiviral and antitumor activities has been identified. In contrast to dercitin (33), neoamphimedine (24) is found to be a potent inhibitor of purified mammalian topoisomerase II (IC50 1.3 ^M), but not of topoisomerase I. Neoamphimedine

(24) is also shown to intercalate DNA with a Km of 2.8 x 105 M- and a binding site size of 1.8 base pairs per molecule of 24. Interestingly, the isomeric base amphimedine (22), petrosamine (4) and debromopetrosamine

(25), had little effect on topoisomerase I or II activity, despite displaying comparable cytoxicity. It is postulated that the cytotoxicity of (24) towards mammalian cells can be explained by DNA damage resulting from native topoisomerase II inhibition. However, the other pyridoacridin alkaloids may elicit cytotoxicity through as yet unidentified mechanisms associated with DNA processing. Ascididemin (26) and shermilamine B (40) also inhibited topoisomerase II, albeit at higher concentration (75 and 30 |M, respectively), while shermilamine A (39) and meridine (29) are found inactive.14

Benzo[4,5]sampangin and ascididemin possess potent antiviral activity while kuanoniamine A appeared inactive. Benzo[4,5]sampangin showed complete inhibition of HSV-1 at 80 |g mL- with no host cell toxicity (BSC-1 green monkey cells). It also displayed activity against polio virus type 1 (partial inhibition at 80 |g mL- with no detectable cytotoxicity to the Pfizer vaccine strain) and HIV-1 (46% protection at 0.7 |M with host cell toxicity at 10 |M).m Hollow fiber assay and tumor implant assays have been developed for antitumor activity assays to assess the antitumor activity of pyridoacridines.112-15 Mainly two of types of the tumor models have been used to assess pyridoacridines. In the first, mouse leukemia cells (P338 cells) are injected into the peritoneum of DBA/2 mice. These mice live for approximately 10 days. The tumor bearing mice are then treated with the test drug, which is administered i.p. and response is measured as increased life span (%ILS). 2-Bromoleptoclinidinone had an excellent in vitro cytotoxicity, but when tested in xenograft models, proved too toxic to yield significant antitumor responses.116 Ascididemin was also tested in vivo at the National Cancer Institute, USA against twelve human tumor cell lines by hollow fiber assay and was shown to exhibited significant activity (%T/C < 50) against six of the cell lines.117 In vitro activity of several pyridoacridines on markers of leukemia and tumor growth have been assessed. Dercitin (33) and Kuanoniamine have been evaluated for the cytotoxic acitvity and observed that they are approximately equally potent against a solid human lung cancer line and a mouse leukemia line.118

Although wakayin (70) exhibits marginal inhibitory activity toward topoisomerase II (250 |M), it showed an interestingly differential cytotoxicity against mammalian cell clones that is indicative of DNA damage or interference with DNA procesisng.68 It has been demonstrated that the fluorescence spectrum of kuanoniamine D (44) is quenched upon addition of calf thymus DNA, and the emission wavelength of 534 nm (excitation at 350 nm) changed to 593 nm. Both of these findings are suggestive of alkaloid binding to DNA. The two bay region'nitrogen's in 2-bromoleptoclinidone ( 27) is ideally disposed to present two donor nitrogen atoms of a bidentate ligand to metals. Alkaloid (27) forms an octahedral complex with Ru (II) salts that induces photo activated single strand cleavage of supercoiled PBR 322 DNA under visible light irradiation.119 It is interesting to note that cytotoxic cystodytins-A (2) and B (6) are found to be potent Ca2+ release agents, and stimulated calcium release from the sarcoplasmic reticulum (SR) at 36 and 13 times the potency of caffeine, respectively.16

Ascididemin, benzo[4,5]sampangine, deazaascididemin were found activity against Escherichia coli, Bacillus subtilis, Candida albicans, and Cladisporium resinae, with an MIC of 0.39 ^g mL- against C. albicans.120 Cystodytin was found active against E. coli and Staphylococcus aureus.110 Sampangine and several analogues exhibited potent antimicrobial activity against the opportunistic infection organisms C. albicans, Cryptococcus neoformans, Aspergillus fumigatus, and Mycobacterium intracellular.121 Limited information is available in the literature regarding the insecticidal activity of pyridoacridine.122 Kuanoniamines were studied extensively using neonatal Spodoptera littoralis larvae and the 50% lethal concentration (LC50) was determined.

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