Cyclobutane-Containing Alkaloids: Origin, Synthesis, and Biological Activities

Present review describes research on novel natural cyclobutane-containing alkaloids isolated from terrestrial and marine species. More than 60 biological active compounds have been confirmed to have antimicrobial, antibacterial, antitumor, and other activities. The structures, synthesis, origins, and biological activities of a selection of cyclobutane-containing alkaloids are reviewed. With the computer program PASS some additional biological activities are also predicted, which point toward new possible applications of these compounds. This review emphasizes the role of cyclobutane-containing alkaloids as an important source of leads for drug discovery.


INTRODUCTION
Organic compounds containing four-membered ring(s) represent unusual group of metabolites including natural products and/or drugs. The cyclobutane unit is found as a basic structural element in a wide range of naturally occurring compounds in bacteria, fungi, plants, and marine invertebrates. It is also generated transiently in primary and secondary metabolisms [1]. Many biological activities are showed and may serve as potential drug leads or provide new ideas for the study of enzyme mechanisms, and/or organic synthesis [2]. Some cyclobutane compounds such as amino acids, peptides and nucleosides showed protective properties against UV radiation [3]. In the skin many molecules may absorb UV radiation upon exposure. In particular, cellular DNA strongly absorbs shorter wavelength solar UV radiation, resulting in various types of DNA damage. Among the DNA photoproducts produced the cyclobutane pyrimidine dimers are predominant [4].
Although cyclobutanes have been known for more than a century, their use as synthetic intermediates has only flourished in the last forty years. The structures and syntheses of cyclobutanoid fatty acids, amino acids, mono-, sesqui-, di-, and triterpenes, steroids, and other compounds have recently been reported [2,5,6].
In the present review, we will focus on origin, structures, and biological activities of natural cyclobutane-containing alkaloids and selected related compounds. Their structure and biological activities, modes of action, and future prospects are discussed. This paper is a short survey of cyclobutane-containing alkaloids that are deemed as naturally occurring. Also, this is *Address correspondence to this author at the Department of Medicinal Chemistry and Natural Products, School of Pharmacy, P.O. Box 12065, The Hebrew University of Jerusalem, Jerusalem 91120, Israel; Tel/Fax: +972-2-590-2947; E-mail: dvalery@cc.huji.ac.il the first article to review natural alkaloids comprising a cyclobutane unit.
Synthesis of 2,4-methanoproline and derivatives have been reported by several authors. Most of the syntheses of 2,4-methanoproline are accomplished by an intramolecular light-induced [2+2] cycloaddition of an appropriate diene [11,12]. Only Gaoni used an intramolecular cyclization strategy for his approach [13]. Also analogues containing the 2azabicyclo[2.1.1]hexane skeleton are often synthesized by the light-induced [2+2] cyclization [14][15][16][17]. The rearrangement of an appropriate bromohydrine to synthesize 5hydroxy-2-azabicyclo[2.1.1]-hexanes has also been described [18,19]. A two-step synthetic approach toward 3-(chloromethyl)cyclobutanone was described and used in the synthesis of 2,4-methanoproline analogues [20]. The key step consists of a reversible addition of hydrogen cyanide onto the imines (Scheme 1). Seven Lycopodium alkaloids, lannotinidines A-G, have been isolated from the club moss Lycopodium annotinum and L. annotinum var. acrifolium. Two lannotinidines F (3) and E (4) contain cyclobutane ring elevated NGF mRNA expression. Effects of lannotinidines A-F on neurotrophic factor biosynthesis in 1321N1 human astrocytoma cells were examined by determining NGF mRNA expression. 1321N1 cells were incubated with 30 μg/mL each of lannotinidines A-F for 6 h, and the mRNA expressions of NGF in 1321N1 cells were examined by a semiquantitative RT-PCR method. The mRNA expressions for NGF were enhanced by lannotinidines B-E, among which (3) was the most potent [21].
A re-examination of the leaf and seed alkaloids of Lupinus cosentinii, confirmed in both parts the presence of epilupinine, epilupinine N-oxide, and multiflorine. Both organs of the plant contained a new tricyclic alkaloid (16) [45]. The genus Piper (Piperaceae) includes more than 1000 species making it one of the largest genera of basal angiosperms [49]. Piper species are distributed pantropically and are in the form of shrubs, herbs, and lianas common in the understory of lowland wet forests. The greatest diversity of 17 Thesine (betel leaf) originated. Patterns of distribution of Piper species vary from being locally endemic to widespread. Several cyclobutane-containing alkaloids have been isolated and identified from the genus Piper [50]. Fifteen novel dimeric amide alkaloids possessing a cyclohexene ring, nigramides A-O, as well as four novel dimeric amide alkaloids possessing a cyclobutane ring, nigramides P-S (18-21), have been isolated from the roots of Piper nigrum. The biosynthetic hypothesis of nigramides A-O was proposed by an intermolecular Diels-Alder reaction from the corresponding monomeric amides [51].
Recently the minor metabolites, PC-M5' and PC-M6, were isolated along with the tremorgenic mycotoxins, penitrems A-F, from the mycelium of P. crusotsum, were found as contaminats of bread intended for school lunches in Tokyo city. Two new indoloditerpenes, PC-M4 (43) and PC-M5, were also isolated from the above fungus. PC-M4 has the same carbon number as the penitrems but a different cyclic ring system. PC-M5 might be a biosynthetic precursor of penitrems and PC-M4 as may also be PC-M5' and PC-M6 [77]. The new antiinsectan metabolite 10-oxo-11,33dihydropenitrem B (44) was isolated from the sclerotia of Aspergillus sulphureus. Isolated alkaloid is related to the penitrems, a known group of tremorgenic fungal metabolites. A known aflavinine analog (10,23-dihydro-24,25dehydroaflavinine) was also isolated from A. sulphurerus sclerotia. This is the first report of any aflavinine analog from a member of the Aspergillus ochraceus taxonomic group [78].
Extract from the sclerotia of Aspergillus sulphureus yielded four new antiinsectan compounds of the paspaline/penitrem class, and included secopenitrem B (45). Isolated metabolites were structurally related to penitrems. The three indole metabolites, including (45), exhibited potent activity against the lepidopteran crop pest Helicoverpa zea [79]. A new group of microbial metabolites, designated penitremones A-C, have been characterized by MS and NMR spectroscopy as 10-keto, and 11,33-dihydro-variants of the penitrem indole-isoprenoid skeleton. The principal metabolite penitremone A (46), and minor (47) produced with penitrem A by a Penicillium sp., is an isomer of penitrem E and was also similarly tremorgenic [80]. Different biological activities for penitrem A (33) have been studied and reported in some articles [81][82][83][84][85][86][87][88]. Recently, Smith III and co-workers [89] demonstrated the viability of a highly stereoselective tandem Mannich cyclization-grammine fragmentation/addition cascade, critical for assembly of the A and F rings of penitrem D (Scheme 3). They also explored simultaneous execution of this tactic with concurrent construction of ring I. Reinvestigation of a model system provided an explanation for the unanticipated stereochemical outcome at C(28).

PREDICTED ACTIVITIES OF ALKALOIDS ISO-LATED FROM TERRESTRIAL SPECIES
Probable additional biological activities of cyclobutanecontaining alkaloids were evaluated by computer prediction. For this purpose we used computer program 'PASS' [90][91][92], which predicts about 2,500 pharmacological effects, mechanisms of action, mutagenicity, carcinogenicity, teratogenicity and embryotoxicity on the basis of structural formulae of compounds. PASS predictions are based on structure-activity relationships (SAR) analysis of the training set consisting of about 60,000 drugs, drug-candidates and lead compounds. Algorithm of PASS predictions is described in detail in several publications [91,92]. Using MOL or SD files as an input for the PASS program, user may get a list of probable biological activities for any drug-like molecule as was also published recently [90].
For each activity, P a and P i values are calculated, which can be interpreted either as the probabilities of a molecule belonging to the classes of active and inactive compounds,  respectively, or as the probabilities of the first and second kind of errors in prediction. First kind error of prediction reflects the "false-positives", when an inactive compound is predicted to be active; and second kind error of prediction: reflects the "false-negatives", when an active compound is predicted to be inactive.
Interpretation of the predicted results and selection of the most prospective compounds are based on flexible criteria, which depend on the purpose of particular investigation. If the user chooses a rather high value of P a as a threshold for selection of probable activities, the chance to confirm the predicted activities by the experiment is high too, but many existing activities will be lost. Typically, there are several dozen biological activities in the predicted biological activity spectra; activity that is predicted with the highest probability is called "focal". Focal biological activities for cyclobutanecontaining alkaloids isolated from terrestrial sources are shown below in the Table 1. Additional predicted biological activities for cyclobutane-containing tremorgenic mycotoxins are shown in Table 2.

CYCLOBUTANE-CONTAINING ALKALOIDS FROM MARINE SPECIES
Cyclobutane-containing alkaloids have also been isolated and their structures elucidated from marine algae and invertebrates, mainly from marine sponges. Some of them showed different biological activities. The water-soluble cyclobutadithymine (48) was extracted from the marine red alga Porphyra yezoensis, and showed the protective properties against UV-A irradiation [93].
John Faulkner from the University of California (Berkeley) was the first scientist who discovered an antimicrobial agent, sceptrin (49), from the sponge, Agelas sceptrum, at 1981. This alkaloid exhibited antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Candida albicans, Pseudomonas aeruginosa, Alternaria (fungus), and Cladosporium cucumerinum. The compound (49) was not toxic at 50 mg/kg [94,95]. The influence of environmental factor, namely depth and sponge-coral interactions as well as the effects of infliction of standardized damages on the production of bromopyrrolic alkaloids isolated from the sponges Agelas dispar and Agelas conifera were analyzed and sceptrin (49) was detected in both sponge species [96]. More recently, sceptrin and eight dimeric bromopyrrole alkaloids, nagelamides A-H, and a monomeric one, 9,10dihydrokeramadine, have been isolated from the Okinawan marine sponge Agelas sp. Nagelamides A-H exhibited antibacterial activity against Gram-positive bacteria. Nagelamide G inhibited protein phosphatase 2A activity [97]. Sceptrin, ageliferine and xestospongine B, three alkaloids isolated from Xestospongia sp. and Agelas novaecaledoniae were reported as somatostatin and VIP inhibitors. Sceptrin and ageliferine showed an affinity for VIP (19.8 μM and 19.2 μM, respectively). Due to the interaction between nonpeptidic compounds and somatostatin/VIP receptors, these three alkaloids could be promising agents in the research on natural non-peptidic compounds for therapeutical interventions [98]. The mechanism of action of sceptrin was investigated [99]. Sceptrin has been reported to exhibit antibacterial and antifungal activities. Sceptrin demonstrated a bacteriostatic rather than bactericidal effect at the MIC on exponentially growing Escherichia coli. Under these conditions, the culture produced chains of cells, and incorporation of radiolabeled precursors into DNA, protein, and cell wall was unaffected, whereas incorporation of 3 H-uridine into RNA was slightly inhibited. At concentrations higher than the MIC, sceptrin was bactericidal, inhibited the incorporation of all radiolabeled precursors, and induced the formation of unusual spheroplasts. Peptidoglycan turnover in E. coli appeared to be stimulated by sceptrin, as demonstrated by a release of diaminopimelic acid-containing high mol wt material. Subsequent studies of the release of K + from E. coli and the lysis of red blood cells suggested that sceptrin disrupts the cell membranes of both prokaryotic and eukaryotic cells. Spheroplast formation may reflect a cell wall effect that occurs subsequent to membrane damage [99]. In a search for potential target sites for C 11 N 5 compounds obtained from marine sponges of the genus Agelas, the authors evaluated their interaction with muscarinic acetylcholine receptors from rat brain membranes. In competition experiments with 3H-QNB, these compounds displayed the following rank order of potency: sceptrin (49) > oroidin, dibromosceptrin clathrodin. Sceptrin (49) (50 μM) was shown to be a competitive inhibitor of 3H-QNB binding as revealed Scatchard analysis. The results demonstrate the ability of these compounds to interact with multiple molecule targets in the micromolar range [100].
The first enantioselective total synthesis of sceptrin was reported by programming the fragmentation of an oxaquadricyclane [101]. Oxabicycle (50) could be obtained in quantitative yield by Alder reaction. In the [2+2] cyclization to form oxaquadricyclane (51), it was found that switching the solvent from diethyl ether to THF not only accelerated the reaction but also avoided potential complications due to the low boiling point of ether (Scheme 4). The highly unstable (51) was taken on in crude form to the fragmentation step. The known procedure for the fragmentation of oxaquadricylane (51) gave only low yield of cyclobutane (52). As this was clearly not a viable option to support extensive synthetic efforts, a more expedient procedure was needed. It was found that the addition of diethyl ether after evaporation of methanol would cause the precipitation of cyclobutane (52) in ca. 50% yield, allowing expedient access to multigram quantities of (52). With the all-trans cyclobutane framework in place, (53) can be obtained.
Ketalization of (56) under standard conditions (Scheme 5), followed by bromination of the crude ketal with phenyltrimethyl ammonium tribromide, gave bromoketone, displacement of which gave with sodium diformamide compound (54), which was hydrolyzed with aqueous HCl to give (55) and reacted with cyanamide to form (56)

Scheme 5.
Biologically active extracts of the Caribbean sponge Agelas conifera had yielded, in exhaustive studies, the diacetate salts of seven new bromopyrroles (57)(58)(59)(60)(61), as well as that of the known debromooroidin dimer sceptrin. These compounds were found to be antiviral and antibacterial and were active in barnacle settlement and biochemical prophage induction assays. The oxysceptrins are characterized by their aminoimidazolinone group, the ageliferins, by a unique cyclohexene-based skeleton [102]. Debromosceptrin (57), and two known pyrrole analogs (58 and 59) were also found in extract of the Caribbean sponge Agelas conifera collected from Belize [103]. Oxysceptrin (61) from the Okinawan marine sponge Agelas cf. nemoechinata showed active properties against actomyosin ATP-ase [104].
Nakamuric acid (62) and sceptrin were identified from Australian sponges belonging to the genus Agelas [105]. Two dimeric bromopyrrole alkaloids, nakamuric acid (62) and its corresponding Me ester (63), were isolated from the Indopacific sponge Agelas nakamurai along with the known metabolites sceptrin, debromosceptrin, and ageliferin. All compounds inhibited the growth of several Gram-positive and Gram-negative bacteria in the agar plate diffusion assay [106]. Six dimeric bromopyrrole alkaloids were isolated from a Florida Keys specimen of Agelas conifera. One of the constituents was identified as a new bromopyrrole metabolite, bromosceptrin (64) [107].

PREDICTED ACTIVITIES FOR MARINE ALKA-LOIDS
Additional predicted activities for natural cyclobutanecontaining alkaloids are shown in Table 3.

CONCLUDING REMARKS
Cyclobutane-containing alkaloids are rare group of natural products. They are mainly synthesized by different plant species, and also were detected in some marine species. A little information is known about biological activities of these metabolites. Nevertheless, reported activities for isolated compounds revealed strong antibacterial, antimicrobial, antifeedant, antinociceptive, insecticidal and others activities. The widest spectra of pharmacological activities are exhibited by isolated alkaloids, and/or their N-oxides. Using the program PASS we showed that many reported activities for cyclobutane-containing alkaloids have been predicted, including some additional biological activities.