Aziridine Alkaloids: Origin, Chemistry and Activity

Aziridine Alkaloids: Origin, Chemistry and Activity 30 Valery M. Dembitsky, Alexander O. Terent’ev, and Dmitri O. Levitsky Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 978 2 Natural Aziridine Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 978 3 Selected Semisynthetic and Synthetic Aziridine Alkaloids as Analogues of Natural Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 990 4 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1002 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1002 Abstract This chapter describes research on natural aziridine alkaloids isolated from both terrestrial and marine species, as well as their lipophilic semisynthetic and/or synthetic analogues. Over 100 biologically active aziridine-containing compounds demonstrate confirmed pharmacological activity including antitumor, antimicrobial, and antibacterial effects. The structures, origin, and biological activities of aziridine alkaloids are reviewed. Consequently, this V.M. Dembitsky (*) N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia Institute for Drug Research, Hebrew University, Jerusalem, Israel e-mail: [email protected]; [email protected] A.O. Terent’ev N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia D.O. Levitsky CNRS UMR 6204, Biotechnologie, Biocatalyse et Biorégulation, Faculté des Sciences et des Techniques, Université de Nantes, Nantes, Cedex 03, France K.G. Ramawat, J.M. Mérillon (eds.), Natural Products, DOI 10.1007/978-3-642-22144-6_93, # Springer-Verlag Berlin Heidelberg 2013 977 978 V.M. Dembitsky et al. review emphasizes the role of aziridine alkaloids as an important source of drug prototypes and leads for drug discovery. Keywords Alkaloids • antibacterial • anticancer • aziridine • bioactive • plant 1 Introduction Several groups of rare natural alkaloidal metabolites incorporating the cyclobutane [1], aziridine [2, 3], and azetidine moieties [4] and/or their synthetic counterparts possess a broad spectrum of biological activities. Aziridine alkaloids also belong to a rare and somewhat neglected group of natural products which are known to play a seminal role in the secondary metabolism of some microorganisms, plants, and various marine organisms [5]. The aziridine-containing compounds have been of interest as both immunomodulatory and anticancer agents since the late 1950s [6]. Aziridines are inherently strained making them attractive for study in terms of reactivity and pharmacodynamic action. Ethylenimine (or aziridine, 1) and some of its simple derivatives are commercial products in different fields of applied chemistry [7]. Observations of the toxic action of aziridines have prompted extensive investigations involving their synthesis and pharmacological activity, allowing selection and advancement of suitable substances as putative cancer chemotherapeutic agents. Notably, a few are enjoying regular clinical use [8]. Bayer strain encourages ring-opening reactions of aziridines in the presence of nucleophiles, imparting useful alkylating properties, despite their powerful mutagenic and toxic activities [9]. Aziridines are highly valuable heterocyclic compounds and are widely used during the synthesis of numerous drugs and biologically active natural products (and their derivatives) [10–15]. Many aziridine alkaloids have anticancer, antibacterial, and/or antimicrobial activity against selected cancer cell lines, pathogenic bacteria, and/or microorganisms strongly indicating that the presence of the aziridine ring in natural as well as synthetic compounds is essential for such activities [16–19]. This chapter reviews natural aziridine alkaloids, with high antitumor, antimicrobial, and antibacterial activities, and also highlights those semisynthetic derivatives and analogues which possess therapeutic promise. 2 Natural Aziridine Alkaloids The simple alkaloid, ethylenimine (aziridine, azacyclo-propane, or aziran, 1) was detected in various foodstuffs including baker’s yeast (Saccharomyces cerevisiae) autolyzate [20], in the volatile flavoring constituents of cooked chicken, beef and 30 Aziridine Alkaloids: Origin, Chemistry and Activity 979 pork [21], and beef flavor [22]. Two metabolites (1) and aziridine-2-carboxylic acid (2) were isolated from mushrooms Agaricus silvaticus (class Basidiomycetes), both of which have been synthesized [23]. Aziridine-2-carboxylic acid (2) as well as aziridine-containing peptides are vital intermediates in the synthesis of various amino acid and peptide derivatives [24]. Furthermore, (2) and related compounds represent interesting substrates for clarifying enzyme mechanisms but also as the warhead of novel irreversible protease inhibitors with a number of potential therapeutic applications [25, 26]. More complex aziridines are found in various plant sources. For instance, 1-methyl-aziridine (3) was detected using GC-MS within onion bulbs (Allium cepa, class Liliopsida, order Asparagales, family Alliaceae) [27]. Flue-cured tobacco (Nicotiana tabacum, family Solanaceae) contains 4-(1-aziridinyl)-3buten-2-one (4) [28]. Natural aziridine alkaloids (2,5–11) were detected and isolated from distillate, and residue in extractions of dried matter of Petasites japonicus (family Asteraceae, Japanese name Fuki) [29] is also known as bog rhubarb or giant butterbur. It is native to Japan, where the spring growth is relished as a vegetable. Consequently, its pharmacological properties are of considerable importance. O Me H N H N N OH N R 1 R=H 2 R = COOH 3 5 2-Aziridinemethanol 4 Ph Ph N N COOR COOR 6 R=H 7 R = Me 8 R = Me 9 R = Et 10 R = i-Pr N 11 O COOEt Since the 1950s, polymerization products of ethylenimine, their polymerizable homologs, as well as substitution products were considered useful for disinfecting and preserving textiles, leather, skins, meat, glands, blood, glue, casein (and other albuminous substances), starch, size, dressings, fruits, and vegetables. Their utility in disinfecting floors, walls, stock and portable water vessels, and medical instruments has improved health and safety [30–32]. The azirinomycin (12), 3-methyl-2H-azirine-2-carboxylic acid, was isolated from a strain of Streptomyces aureus. Its methyl ester (13) exhibited broad spectrum antibiotic activity in vitro against both Gram-positive and Gram-negative bacteria [33, 34]. The carboxylic acid (12) is most active against Staphylococcus aureus followed by Proteus vulgaris, Bacillus subtilis, and Streptococcus faecalis. In contrast, the methyl ester shows its lowest activities against one of the Staphylococcus aureus cultures and Streptococcus faecalis. 980 V.M. Dembitsky et al. (2S,3S)-Aziridine-2,3-dicarboxylic acid (also known as S,S-2,3-dicarboxyaziridine, 14), which demonstrates antibacterial activity toward Aeromonas salmonecida, was isolated from the cultured broth of a Streptomycetes MD 398-Al (FERM-P 3217) [35]. The compound (14) was effective against Pellicularia sasaki and Pythium debaryanum [36]. It is a potent competitive inhibitor of various enzymes including fumarase isolated from pig heart (Ki ¼ 0.08 mM) [37] and aspartase of Escherichia coli (Ki ¼ 55 mM). It also shows antibacterial activity against Aeromonas salmonicida [38]. Ethyl esters of aziridine-2,3-dicarboxylic acid inhibited the cysteine proteinase papain [39], whereas peptides containing the aziridine-2,3-dicarboxylic acid building block are inhibitors of several cysteine proteases such as the papain-like mammalian proteases [40]. The alkaloidal antibiotic, U-47,929 (also known as ficellomycin, 15), was isolated from Streptomyces ficellus [41]. Interestingly, it inhibited the growth of Gram-positive bacteria in vitro and is effective in the treatment of experimental Staphylococcus aureus infections in mice [42]. Structural elucidation of (15) [43] was eventually achieved by a combination of NMR, mass spectrometry, and formation of derivatives. The 1-azabicyclo[3.1.0]hexane moiety in (15) represents an unusual ring system making ficellomycin a unique natural product [43]. H N N HOOC COOR 12 R = H 13 R = Me COOH 14 H N H2N O OH O N HN NH H2N 15 Ficellomycin The unique cytotoxic azacyclopropene, R-dysidazirine (16), was isolated from the marine sponge Dysidea fragilis (Fiji) just over 20 years ago [44]. More recently, both the (Z) and (E) geometrical isomers of S-dysidazirine (17a) and (17b) were isolated and were also found to possess cytotoxicity. The dibrominated analogues, S-antazirine (18a) and (18b), were also detected within the same marine sponge D. fragilis collected in Pohnpei, Micronesia [45]. Three new o-halogenated longchain 2H-azirines (19a,b and 20) have recently been isolated from the marine sponge Dysidea fragilis, two of them containing a terminal (Z)-1-bromo-1chlorovinyl group, the first such example from a marine invertebrate [46]. Cytotoxic activity of (17b and 18b) and new compounds (19a,b, and 20) is shown in Table 30.1. 30 Aziridine Alkaloids: Origin, Chemistry and Activity Table 30.1 In vitro cytotoxicity of aziridinecontaining fatty acids against HCT-116 [46] 981 IC50 (mg/mL) 7.9 8.5 5.3 5.9 8.6 Compound 17b 18b 19a 19b 20 4 IC50 (mM) 18.2 19.6 13.6 15.2 24.8 N H 18 CO2Me 16 R-Dysidazirine Z or E N CO2Me H 17a S-Dysidazirine, Z 17b S-Dysidazirine, E Z or E N CO2Me Br H Br 18a S-Antazirine, Z 18b S-Antazirine, E Z or E N H Cl 19a, Z 19b, E CO2Me Br N CO2Me Cl H 20 Cl The antitumor antibiotic FR-900482 (21) was isolated from Streptomyces sandaensis 6897 as a mixture of the two hydroxylamine hemiketal isomers (22) and (23) [47]. FR-900482 exhibits potent cytotoxic activity against various tumor cells in vitro. Furthermore, it possessed a weak antimicrobial activity against some Grampositive and Gram-negative bacteria [48]. Activity against human LX-1, MX-1, SC-6, and LC-6 tumor cells has been identified [49]. Quite a number of FR-900482 derivatives were synthesized, and some of them showed antileukemic activity [50]. 982 V.M. Dembitsky et al. Additionally, FR-900482 inhibited DNA, RNA, and protein synthesis in cell culture of murine L1210 leukemia cells. Whereas FR 900482 did not induce DNA single-strand breaks either in the leukemia cells or in plasmid pBR322, it promoted interstrand DNA-DNA cross-links in leukemia cells. An activation of FR 900482 was required prior to induction of interstrand DNA-DNA crosslinking required for cytotoxic action [51]. FK317 (24), an analogue of FR900482, had stronger cytotoxic effects against in vitro cultured B16, P388, HeLa S3, and KB tumor cell lines. In vivo experiments revealed an equivalent antitumor activity of FK317 against P388, M5076, and MX-1 and a more potent antitumor activity against L1210, Colon 38, and LX-1 cell lines as compared with FK973 (26) [52]. Both FR900482 (21) and FR66979 (27) are structurally novel natural products isolated by Fujisawa Pharmaceutical Co. (Japan) in 1987 and have been shown to be highly potent antitumor antibiotics structurally related to the mitomycins [5]. The N-O substructure is bioisosteric with peroxides, and the activity of natural products containing this functional group may generate free radicals, especially upon reductive activation. Not surprisingly, studies on the mode of action have established that these new agents form covalent DNA interstrand cross-links both in vitro and in vivo as a result of the reactive mitosene intermediate generated upon bioreductive activation [for details, see Refs. 53–55]. OH OHC O N OH OHC O N H 21 FR 900482 22 FR 900482 major isomer OH O OHC O OR2 O OH N N H OH O O N O O NH2 OR1 N R 23 FR 900482 minor isomer HO O NH2 O N OH O OH N H OHC NH2 O NH2 N O O OH N H 27 FR 66979 NH2 24 FR 073317 R = R1 = Ac, R2 = Me 25 FR 70496 R = Ac, R1 = H, R2 = Me 26 FK 973 R = R1 = R2 =Ac 30 Aziridine Alkaloids: Origin, Chemistry and Activity 983 H2 N O O O H R 9 OR1 N NR2 O 28 Mitomycin A, R = OMe, R1,R2 = H 29 Mitomycin F, R = OMe, R1 = R2 = Me 30 Mitomycin C, R = NH2, R1 = Me, R2 = H 31 Porfiromycin, R = NH2, R1,R2 = Me 32 9a-Demethylmitomycin A, R = OMe, R1 = R2 =H 33 9-epi mitomycin B, R = OMe, R1 = H, R2 = Me Semisynthetic analogues, such as FK317 (24) and FK973 (26), have been shown to be a potent cytotoxic compound; to date, no direct evidence of DNA interstrand cross-link sequence specificity has been reported. In one study, DNA interstrand cross-links were generated by treatment of a synthetic duplex DNA substrate with FK317 and its deacetylated metabolites FR70496 (25) and FR157471 [56]. FK973 and all its deacetylated metabolites showed strong cytotoxicity on in vitro cultured murine L1210 leukemia cells; however, FK973 remained the most potent cytotoxic agent of this series [57]. Synthesis and other biological activities of FR900482 and its analogues have been reviewed [58–60]. H2N O H2N O O O H O R RHN OR1 OR1 N NMe O 34 Mitomycin B, R = OMe, R1 = H 35 Mitomycin J, R = OMe, R1 = Me 36 Mitomycin D, R = NH2, R1 = H 37 Mitomycin E, R = NH2, R1 = Me O N NR2 O 38 R = R2 = Me, R1 = H 39 R = R1 = Me, R2 = H 40 R = Et, R1 = H, R2 = Me 41 R = Et, R1 = Me, R2 = H 42 R = n-Pr, R1 = H, R2 = Me 43 R = n-Pr, R1 = Me, R2 = H The mitomycins are potent antibiotics that belong to the family of antitumor quinones. In 1956, mitomycin A (28) and B (34) were isolated from Streptomyces caespitosus, and shortly thereafter, mitomycin C (30) was discovered within the same strain [61, 62]. The N-methyl derivative of (31), porfiromycin, was isolated in 1960 from Streptomyces ardus, which was followed by the discovery of mitiromycin from Streptomyces verticillatus [63, 64]. Among all these different mitomycins, (31) enjoyed early widespread clinical use as a consequence of its uniquely superior activity against solid tumors. Secondly, it possessed reduced toxicity when compared to the natural counterparts (28) and (34). Mitomycin A, 984 V.M. Dembitsky et al. B, and C and porfiromycin also were produced by a Micromonospora species KY 11084 [65]. Mitomycins A and C showed antimicrobial activity against Bacillus subtilis and Klebsiella pneumoniae [66]. Effects of mitomycin A (1–10 mg/mL), mitomycin B (1–50 mg/mL), mitomycin C (10–30 mg/mL), N-methyl-mitomycin (1–40 mg/mL), and porfiromycin (1–60 mg/mL) on the Euglena gracilis chloroplast system were reported. However, only N-methyl-mitomycin (20–40 mg/mL), porfiromycin (40–60 mg/mL), and mitomycin B (40–50 mg/mL) were effective bleaching agents. H2N H2N O CH2 O O O H N O H N OR N O OR NR1 N O NR1 O 44 R = Me, R1 = H 45 R = H, R1 = Me 46 R = Me, R1 = H 47 R = H, R1 = Me Thus, only mitomycin derivatives containing an alkyl group on the aziridine nitrogen are effective bleaching agents. The sensitivity of the Euglena chloroplast to small structural differences in the active centers of antibiotics demonstrates the usefulness of this organism in finding a relationship between biological activity and chemical structure [67]. Mitomycin A and C were manufactured by fermentation with mitomycin-producing Streptomyces and Micromonospora or by catalytic isomerization of mitomycin A and mitomycin C, respectively. Both isomers showed antibiotic activities against various bacteria, including Streptococcus, Staphylococcus, Bacillus, Proteus, and Salmonella [68]. O CH2 R OR1 N NMe O 48 Mitomycin H, R = OMe, R1 = H 49 Mitomycin G, R = OMe, R1 = Me 50 Mitomycin K, R = NH2, R1 = Me 51 Mitomycin Z, R = NH2, R1 = H Molecular genetic manipulation of the mitomycin pathway can elucidate the sequence of reactions involved in mitomycin biosynthesis, as well as provide access to novel mitomycin natural products. Thus, 9a-demethyl mitomycin A (32), 9-epimitomycin B (33), and N-methylmitomycin A (mitomycin F, 29) have been obtained using mitomycin B as starting material [69]. Mitomycin J (35) and mitomycin D (36) were isolated as minor antibiotics from Streptomyces fradiae SCF5 [70], and mitomycin E (37) was obtained from S. lavendulae [71]. Mitomycin C, A, and F showed 30 Aziridine Alkaloids: Origin, Chemistry and Activity 985 anthelmintic activity against gastrointestinal parasites Hymenolepis microstoma and H. nana developing in Tribolium confusum (Coleoptera, Tenebrionidae) [72]. Several neoplasm inhibitor analogues (38–47) of mitomycin B and C were produced by Streptomyces caespitosus. Upon supplementation of the normal fermentation medium for the production of mitomycin C with S. caespitosus with a number of primary amines, two new types of mitomycin analogues, described as Type I and Type II, were produced. Type I analogues were related to mitomycin C with the amine substitution at position C7 on the mitosane ring. Type II analogues also contain the same substitutions at C7, but the conformation of the mitosane ring was related to mitomycin B, by possessing an OH at positions C9a and a Me-substituted aziridine [73]. In all cases, the Type I analogues are more active in a prophage induction test and against L1210 lymphatic leukemia in mice [73]. O O H NH2 N NH MeO OMe O O O OMe NH MeO O N NH2 O O 52 Isomitomycin A O O 53 AX-2 NH2 O N O H H R N OMe MeO N OMe H O O O N 54 Albomitomycin A (AX-1), R = OMe 55 CX-1, R = NH2 O NH2 56 Albomitomycin C Mitomycins H (48), G (49), and K (50) were produced by culturing a strain of S. caespitosus ATCC 29422 [74]. Mitomycins H, G, K, and Z (51) were also prepared from mitomycin B by cultivating S. caespitosus ATCC 27422 [72]. Four isolated antibiotics (48–51) possessed antibacterial activity [75]. Anticancer activity of some mitomycines against Sarcoma 180 cell line is shown in Table 30.2. The neoplasm inhibitors, isomitomycin A (52) and albomitomycin A (54) and (56), were isolated, together with mitomycin A from S. caespitosus culture broth. Both antibiotics were obtained by intramolecular rearrangement of mitomycin A [76]. Anticancer antibiotics AX-2 (53) and CX-1 (55) were isolated from the culture broth of S. caespitosus and obtained from mitomycin C [77]. Other biological activities of different mitomycines, their mechanisms of action, and therapeutic utility have been described in various reviews [5, 9, 77–83]. 986 V.M. Dembitsky et al. Table 30.2 Anticancer activity of some mitomycines against sarcoma 180 cell linea No. 28 29 30 31 32 34 35 48 49 50 51 D9 b b b b b a a g g g g LD50 2.1 5.0 8.4 57.0 7.5 4.5 9.0 12.0 130.0 22.0 210.0 ED50 1.1 1.3 4.4 22.0 4.9 2.5 10.0 6.8 100.0 35.0 82.0 a Substituent at 9 position, a (carbamoyloxy) methyl group with a and b configurations and a vinyl group were taken into account. LD50 and ED50 were used as measures of biological activity. LD50 values of administration of an i.p. route were measured in male ddY mice by probit analysis. ED60 doses that gave 50% inhibition of tumor growth were calculated from the dose-response curve. Sarcoma 180 cells (5  106/mouse) were inoculated s.c. into ddY mice on day 0, and drugs were injected i.p. on day 1. Tumor volume was measured on day 7 A few naturally occurring peptides containing an aziridine ring have been discovered in living organisms. For instance, peptide madurastatin A1 (57) and madurastatin B1 (58), consisting of Ser and salicylic acid moieties, were isolated from the culture broth of a pathogenic Actinomadura madurae IFM 0745 strain. Both metabolites showed antibacterial activity against Micrococcus luteus, indicating that the presence of the aziridine ring is essential for such activity. Since (57) has a strong affinity with ferric ion attributed to the presence of two hydroxamic acids and a salicylic acid, this low molecular weight chelator is considered a siderophore [84]. Miraziridine A (59) isolated from the marine sponge Theonella aff. mirabilis unifies within one molecule three structurally privileged elements: (a) (2R,3R)aziridine-2,3-dicarboxylic acid, (b) (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid (statine), and (c) (E)-(S)-4-amino-7-guanidino-hept-2-enoic acid (vinylogous arginine). HO O N NH NHMe OH O N O O N OH N H H N O 57 Madurastatin A1 O 30 Aziridine Alkaloids: Origin, Chemistry and Activity O 987 O OH N OH 58 Madurastatin B1 The alignment of them realized in the tetrapetide allows for a simultaneous inhibition of the proteolytic activity of trypsin-like serine proteases, papain-like cysteine proteases, and pepsin-like aspartyl proteases. Therefore, this unique compound represents a blueprint for the design of protease class-spanning inhibitors [85, 86]. The capability of (59) to inhibit proteases belonging to different classes for trypsin, cathepsin B, cathepsin L, and papain was reported (see Table 30.3). Miraziridine A [85] also inhibited cathepsin B with an IC50 value of 1.4 mg/mL. Aziridine-2,3-dicarboxylic acid (14) is a rare natural product, reported from a Streptomyces [36], and vArg has never before reported as a natural product. O O HO N H OH N H N H O O N H O H N OH O NH (i) Inhibition of papain-like cysteine proteases (ii) Inhibition of pepsin-like aspartyl proteases 59 Miraziridine A H2N NH (iii) Inhibition of trypsin-like serine proteases Miraziridine A (59) isolated from the marine sponge Theonella aff. mirabilis unifies within one molecule three structurally privileged elements: (a) (2R,3R)aziridine-2,3-dicarboxylic acid, (b) (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid (statine), and (c) (E)-(S)-4-amino-7-guanidino-hept-2-enoic acid (vinylogous arginine). The alignment of them realized in the tetrapetide allows for a simultaneous inhibition of the proteolytic activity of trypsin-like serine proteases, papain-like cysteine proteases, and pepsin-like aspartyl proteases. Therefore, this unique compound represents a blueprint for the design of protease class-spanning inhibitors [85, 86]. The capability of (59) to inhibit proteases belonging to different classes for trypsin, cathepsin B, cathepsin L, and papain was reported (see Table 30.3). Miraziridine A [85] also inhibited cathepsin B with an IC50 value of 1.4 mg/mL. Aziridine-2,3-dicarboxylic acid (14) is a rare natural product, reported from a Streptomyces [36], and vArg has never before reported as a natural product. Anticancer antibiotics, azinomycin A (60) and B (61), were isolated from the culture broth of Streptomyces griseofuscus S-42227 [87, 88]. Azinomycin A and 988 V.M. Dembitsky et al. Table 30.3 Inhibitory properties of miraziridine A [60] Protease class Serine protease Cysteine protease Protease Trypsin Cathepsin L Cathepsin B Pepsin Aspartyl protease Affinity 6  10 5 M 1  106/M/s 1.5  104/M/s 1.4  10 8 M B expressed antitumor activities against P388 leukemia, P815 mastocytoma, B-16 melanoma, Ehrlich carcinoma, Lewis lung carcinoma, and Meth A fibrosarcoma, and it was markedly effective against i.p. inoculated tumors such as P388 leukemia, B-16 melanoma, and Ehrlich carcinoma [89]. Both compounds were active against Gram-positive and Gram-negative bacteria and L5178Y cells in tissue culture [87]. Azicemicin A (62) and B (63) were isolated from Amycolatopsis sulphurea, and its physicochemical properties and antimicrobial activity were defined [90]. It was also isolated from Amycolatopsis sp. (MJ126-NF4) cultures and showed MIC of 50 mg/mL against Escherichia coli NIHJ in vitro [91, 92]. Antimicrobial activities of azicemicin A and B were shown in Tables 30.4 and 30.5. O O O O H N O OH N H O OMe X O OMe HO HO NR N AcO MeO HO H 60 Azinomycin A, X = CH2 61 Azinomycin B, X = C = CHOH OH OH O 62 Azicemicin A, R = Me 63 Azicemicin B, R = H A carboxylic acid antibiotic, carzinophilin, active against Gram-positive bacteria and tumor cells, was isolated from the broth filtrate of Streptomyces sahachiroi in 1954 [93, 94]. The structure of carzinophyllin (or carzinophilin) was similar to azinomycin B, whose partial structure was previously reported [95]. Lown and Hanstock reported the complete structure (64) [96]. It has a twofold symmetry axis and consists of a dimer of a substituted 1-naphthoic acid attached to a 4-aminohydroxyvaline linked to an N-methyl-aminohexose moiety. It is the first naturally occurring bis-intercalative (macrocyclic polyoxide) bisalkylator (aziridine), and the mode of its antitumor antibiotic activity is attributed to the reactive moiety (64) [96]. Maduropeptin (65) is a chromoprotein antitumor antibiotic isolated from the fermentation broth of Actinomadura madurae [97]. Maduropeptin consists of a 1:1 complex of an acidic, water-soluble carrier protein (32 kD) and a 9-membered ring enediyne chromophore possessing potent antibacterial and antitumor properties [97]. It exhibits potent inhibitory activity against Gram-positive bacteria and tumor cells and strong in vivo antitumor activity in P388 leukemia and B16 melanoma implanted mice [98]. The biosynthetic gene cluster for the enediyne antitumor antibiotic maduropeptin (MDP) from Actinomadura madurae ATCC 39144 was cloned and sequenced. Cloning of the mdp gene cluster was confirmed by heterologous 30 Aziridine Alkaloids: Origin, Chemistry and Activity 989 Table 30.4 Antimycobacterial activities of azicemicins (MIC, mg/mL) against the genus Mycobacterium Tested organism M. smegmatis ATCC 607 M. vaccae ATCC 15483 M. smegmatis ATCC 607 rifamycin-resistant M. smegmatis ATCC 607 paromomycin resistant M. smegmatis ATCC 607 capreomycin resistant M. smegmatis ATCC 607 streptothricin resistant M. smegmatis ATCC 607 streptomycin resistant Table 30.5 Antibacterial activities of azicemicins A and B (MIC, mg/mL) Tested organism Bacillus anthracis B. cereus ATCC 10702 B. subtilis NRRL B-558 B. subtilis PCI 219 Corynebacterium bovis 1810 Escherichia coli NIHJ E. coli K-12 E. coli K-12 MLI629 E. coli BEM11 E. coli BE1126 E. coli BE1186 Klebsiella pneumoniae PCI602 Proteus vulgaris OX19 P. mirabilis IFM OM-9 Providencia rettgeri GN311 P. rettgm GN466 Pseudomonas aeruginosa A3 P. aeruginosa GN315 Staphylococcus aureus FDA209P S. aureus Smith S. aureus MS9610 S. aureus No. 5 (MRSA) S. aureus No. 17 (MRSA) Micrococcus luteus FDA16 M. luteus IFO 3333 M. luteus PCI 1001 Salmonella typhi T-63 S. enteritidis 1891 Shigella dysenteriae JS1 1910 S. flexneri 4bJS11811 S. typhi JS11746 A (62) 100 >100 >100 >100 25 50 >100 >100 100 100 100 100 >100 >100 >100 >100 >50 >100 >100 >100 >100 >100 >100 50 12.5 12.5 100 100 100 50 100 A (62) B (63) 50 12.5 50 6.25 50 25 6.25 12.5 25 6.25 B (63) 100 100 100 100 6.25 25 100 100 100 100 100 100 100 100 100 100 >50 100 100 100 100 100 100 6.25 1.56 1.56 100 >50 25 100 100 990 V.M. Dembitsky et al. complementation of enediyne polyketide synthase (PKS) mutants from the C-1027 producer Streptomyces globisporus and the neocarzinostatin producer S. carzinostaticus using the MDP enediyne PKS and associated genes [99]. N H HO O O H CO2H HO NH HH N H MeO H N O O HOOC O O O OH H O MeO OH H N 64 Carzinophillin A H HO N H OH O HO O HN O Cl H O OH OH MeO O 65 Maduropeptin chromophore 3 Selected Semisynthetic and Synthetic Aziridine Alkaloids as Analogues of Natural Products The epothilones are a relatively new class of cytotoxic molecules identified as potential chemotherapeutic drugs which were originally identified as metabolites produced by the myxobacterium Sorangium cellulosum and/or Streptomyces coelicolor CH999 [100]. These compounds inhibited the growth of a broad range of human cancer cell lines in vitro with low nM or sub-nM IC50s. A series of 12a,13a-aziridinyl epothilone derivatives as anticancer agents (66–70) were synthesized in an efficient manner from epothilone A. The final semisynthetic route involved a formal double inversion of stereochemistry at both the C12 and C13 positions. All aziridine analogues were showed cytotoxicity against cancer cell lines. Thus, (67) had IC50 value of 4.3 nM against KB cells. The obtained results indicate that the aziridine moiety is a viable isosteric replacement for the epoxide in the case of epothilones [101]. 30 Aziridine Alkaloids: Origin, Chemistry and Activity 991 Several derivatives (71–76) of amide aziridine-2-carboxylic acid (also known as leacadine, 71) were prepared as neoplasm inhibitors [102]. Leacadine has been used for treatment of multiple sclerosis [103]. The antitumor efficacy of azimexon (72) in experimental animals and humans was described with respect to its various immunological parameters [104]. Two synthetic aziridine-2-carboxylic acid (2) (71 and 76) showed antitumor activity against a mammary gland tumor in rats [105]. Treatment of (77) with KOH in MeOH at 50 С resulted in a 60% yield of an isomerization product (putative structures 78,79–81) which in physiological saline solution converts to N-carboxyisoserine. This compound has which had cancerostatic and immunostimulating properties [106]. R N S OH N O O OH O 66 R = H 67 R = CH2CH2OH 68 R = CONHEt 69 R = COPh 70 R = CH2Ph Imexon (78) is an immunosuppressant which selectively suppresses B-lymphocyte activation and can be used in the treatment of B-cell or plasma cell leukemias or neoplasias. Thus, imexon inhibited the proliferation of stimulated human B-lymphocytes in vitro and inhibited the growth of methylcholanthreneinduced fibrosarcoma cells in vitro. It was also active against certain autoimmune disorders and infection with Rauscher leukemia virus [107], and also, imexon perturbs cellular thiols and induces oxidative stress leading to apoptosis in human myeloma cells (human 8226) [108]. More recently, (5R) and (5S) imexons (78a and 78b) have been prepared and used in the treatment of cancer [109]. More details about activity of imexon, and their derivatives, have recently been reviewed [110]. Injection of 10–100 mg BM 06 002 (78) increased immune responses, as indicated by delayed cutaneous hypersensitivity and lymphocyte blastogenesis tests in vitro and also in cancer patients participating in clinical experiments [111]. Two isomeric aziridine-containing analogues of the polyamine spermidine were synthesized and evaluated for cytotoxic activity against cancer cell lines. Replacement of one of the primary amino groups of spermidine with an aziridinyl functionality yielded either (82) or (83). N1-Aziridinylspermidine (82) was cytotoxic in vitro against L1210 murine leukemia cells (IC50 0.15 mM) and HL60 human leukemia cells (IC50 0.11 mM). N8-Aziridinylspermidine (83) was slightly less potent against L1210 (IC50 0.31 mM) and HL60 (IC50 0.30 mM) cells. Both compounds inhibited incorporation of radiolabeled thymidine, uridine, and valine into tricholoracetic acid-precipitable material by L1210 cells [112]. 992 V.M. Dembitsky et al. H N O NH2 NC N NH2 N O 71 Leacadine 72 Azimexon O NH N N N 74 CN NH2 O 73 O H N H N NH N NH2 O 75 CONH2 O N 76 OH N N N N CN 77 NH2 NH 81 78 Imexon NH O N N NH NH NH 79 80 O O N O N N N NH2 NH2 78a (5R )-Imexon 78b (5S )-Imexon Neoplasms inhibitor, 3,5-bis(1-aziridinylmethyl)-2,6-dimethyl-pyridine (84), was prepared and showed antitumor activity against spindle cell sarcoma 45 and Ehrlich muscle tumor in white rats [113]. Markofane (85), an oncostatic agent, was synthesized, and its properties and effect on hepatic lipids of rats with sarcoma M-1 were investigated [114]. The body weight of rats with sarcoma M-1 and given a 20% LD50 dose of markofane was slightly higher than that of non-treated, sarcomatous rats. Markofane proved quite toxic, and a daily dose of 40% LD50 resulted in 25% mortality. It exerted insignificant tumor-inhibiting effect on sarcoma M-1 in daily doses of 20% and 40% LD50. Neither sarcoma M-1 nor markofane had any statistical significance on the content of lipids in dry liver. Markofane, 20% LD50, administered to rats with sarcoma 30 Aziridine Alkaloids: Origin, Chemistry and Activity 993 M-1, increased the liver content of phosphatides. Preparations of (86) were less toxic, had a lower cumulative index, and did not produce profound leukopenia in treated animals and showed more antitumor activity than known aziridine derivatives. When tested clinically on 80 patients with chronic myeloleukemia, leukocyte counts decreased 30–80% on administration of between 60 and 80 mg daily doses of A95 [115]. All four prepared compounds (87–90) of the paramagnetic urethane phosphoric acid diethyl-enimides inhibited growth of the ascitic form of sarcoma 180 in rats 93–100%, and the three (87–89) inhibited Ehrlich ascites tumor growth by 90–98%. Two compounds (87,88) inhibited growth of erythromyelosis and Walker carcinosarcoma 100%, while (89 and 90) were essentially ineffective. Only compound (87) prolonged the survival of animals with leukemia La [116]. Several bioactive phospholipids (91–96) have been synthesized. Putative neoplasm inhibitors (91–94) showed significant activities in the Walker carcinosarcoma 256 and leukemia L1210 assay systems [117]. The low-melting cytotoxic phospholipids with aziridine groups (95 and 96) capable of forming stable dispersions in aquatic glycerol solutions containing 1% egg lecithin were prepared [118]. H N N NH2 82 N N H 83 NH2 N N O P N N N S N 85 Markofane 84 R R N O P O NH N N R P N NH O N N R N N N O n 86 87 R = H, n = 0 88 R = Me, n = 2 89 R = H, n = 2 90 R = Me, n = 0 NO− 994 V.M. Dembitsky et al. Fatty acid derivatives (97–101) containing an internal aziridine group were prepared by reaction of base with Me iodocarbamates obtained by addition of INCO to a natural fatty acid derivatives followed by treatment with MeOH [119]. Preparation of epimino-stearates (97) has also been reported [120]. Synthetic monoglycerides (102) with epimino fatty acids showed antimicrobial activity against Gram-positive bacteria and yeasts [121]. Laboratory preparations of 2-ethyl-1-oleoyl-aziridine (103) showed a wide spectrum of antifungal and antimicrobial activity [122]. Certain arachidonate aziridines such as 13-(3-pentyl-2-aziridinyl)5,8,11-trideca-trienoic acid (104) and its methyl ester (105) have been synthesized [123] which are inhibitors of arachidonate epoxygenase [124]. Preparation of the fatty acid aziridines (106–115) has been described [125]. Bis(aziridine) Me cis9,10;cis-12,13-diepiminooctadecanoate, derived from linoleic acid, and tris (aziridine) and Me cis-9,10;cis-12,13;cis-15,16-triepimino-octadecanoate, both derived from linolenic acid, showed cytotoxic and antimicrobial activity as well as remarkable antitumor-promoting and useful neuroprotective effects [126]. 24(RS),25-epiminolanosterol (116) was a potent noncompetitive inhibitor (Ki ¼ 3.0 nM) of the S-adenosyl-L-methionine-C-24 Me transferase from sunflower embryos [127]. Cholesteryl ester of 1-aziridine acetic acid (117) showed excellent inhibition of a dimethyl-benzanthrene induced and transplantable mammary adenocarcinoma [128]. Four steroidal alkylating agents (118–121) with an aziridine grouping at the C-16 position were synthesized. They were shown anticarcinogenic (oncolytic) activity against implanted mammary carcinoma (milk factor) in C3H/An mice. The steroids 16-(1-aziridinyl)-3b-hydroxy-pregn-5-en-20-one (118), 16-(1-aziridinyl)-3methoxyestra-1,3,5(10)-trien-17-one (119), 16-(1-aziridinylmethyl)-3b-hydroxyandrost-5-en-17-one acetate (ester) (120), and 16-(1-aziridinyl)pregn-4-ene-3,20dione (121), each injected (intraperitioneal, i.p.) at 0.5 mg/mouse/day for 14 days, inhibited tumor growth by 61%, 17%, 32%, and 55%, respectively. None of the compounds were toxic to the host [129]. Potentially cytotoxic estrogen derivatives (122 and 123) were prepared [130]. Aziridine derivatives demonstrated a high binding affinity for receptors but substitution of a bromoacetate group for the aziridine moiety in the same position decreased the binding affinity. Growth of MCF-7 and Evans-T cells from human breast cancer was inhibited by the nitrogen mustards, the mono-nitrogen derivative being the more potent analogue. This inhibitory action was unaffected by estradiol or 11 b-chloromethylestra-1,3,5(10)-trien-3,17-b-diol (ORG 4333). Aziridine derivatives either stimulated or inhibited cell growth depending on the concentration. Apparently, the antitumor action of cytotoxic-linked estrogens may be mediated through a mechanism involving estrogen receptors. O N P R N O 91 R = Et 92 R = n-Pr 93 R = n-Bu 94 R = C12H25 30 Aziridine Alkaloids: Origin, Chemistry and Activity O O N P 995 (CH2)15Me O N O 95 O (CH2)13Me O O O P N (CH2)15Me O N 96 O Mitomycin C is used extensively to treat various neoplasms and has led to the discovery of two aminoethylene disulfides: KW-2149 (124) and BMS-181174 (125). These new compounds differ from mitomycin C only in the C(7) substituent. Novel mechanisms for BMS-181174 and KW-2149 differ from the bioreductive activation pathway commonly accepted for mitomycin C, in that the C(7) aminoethylene disulfide unit undergoes thiol-mediated disulfide exchange to give a mitomycin C thiol derivatives [131]. The cell growth inhibitory activity, antitumor activity, and toxicity of M-16 and M-18, the major metabolites of a new mitomycin C (MMC) derivative, KW-2149 (124), in both mice and humans were compared with those of KW-2149 and MMC in vitro and in vivo. The growth inhibitory activity of M-18, a symmetric disulfide dimer, active against human uterine cervix carcinoma HeLa S3 cells was almost equivalent to that of KW-2149, and their IC50 values were about tenfold smaller than that of MMC. The activity of M-16, a Me sulfide form, was almost equivalent to that of MMC. R HN 97 R = cis-COOH 98 R = cis-CH2OH 99 R = trans-CH2OH 100 R = cis-Me 101 R = trans-Me H N O HO O OH 102 996 V.M. Dembitsky et al. Z O (CH2)5 (CH2)6Me N 103 H N (CH2)2Me CO2R 104 R = H 105 R = Me The cell-killing activity of MMC and M-16 was augmented under hypoxic conditions, whereas that of KW-2149 and M-18 was reduced. M-16 also exhibited almost equipotent activities to MMC in vivo in terms of various biological parameters, i.e., antitumor activity against murine P388 leukemia, ascitic or solid B16 melanoma or human lung carcinoma xenograft L-27, and bone marrow toxicity in mice. These results in vitro and in vivo indicate that the antitumor activity and toxicity of KW-2149 might not be mediated by M-16 in mice. On the other hand, M-18 exhibited almost equivalence activities to KW-2149 in this respect, implicating the involvement of M-18 in the biological activities of KW-2149 [132]. Introducing the mercaptoethyl group at the N-7 position of mitomycin C led to the formation of N7, N0 70 -dithio-diethylene-dimitomycin C (126). It showed excellent antitumor activity against sarcoma 180 and leukemia P388 in mice. Among the various synthetic compounds, the water-soluble conjugate with Et g-L-glutamyl-L-cysteinylglycinate side chain was far more effective against sarcoma 180 and leukemia P388 than mitomycin C [133]. COOMe N OH H N 106 (CH2)6COOMe 107 OH H N (CH2)6COOMe 108 H N Z (CH2)7COOMe 109 30 Aziridine Alkaloids: Origin, Chemistry and Activity H N 997 H N (CH2)7COOMe 110 111 anti-diastereomer H N H N H N (CH2)7CO2Me 112 113 all anti-diastereomer 114 syn-anti-diastereomer 115 anti-syn-diastereomer The three dimers (127, 128, and 129) of mitomycin C (MC), of the aforementioned natural antibiotic and cancer chemotherapeutic agent, were synthesized in which two MC molecules were linked with -(CH2)4-, -(CH2)12-, and -(CH2)3N(CH3)(CH2)3- tethers, respectively [134]. The dimeric mitomycins were designed to react as polyfunctional DNA alkylators, generating novel types of DNA damage. To test this design strategy, their in vitro DNA alkylating and interstrand cross-linking (ICL) activities were studied using MC, which is itself an ICL agent. Evidence was presented that (127–129) multifunctionally alkylate and cross-link extracellular DNA and form DNA ICLs more efficiently than MC. H N H 116 HO H H H 117 O O N Biological activity depends upon reductive activation which is catalyzed by the same reductases and chemical reductants that activate MC. Dimer 5, but not MC, cross-linked DNA underwent activation by low pH environments. Sequence specificities of cross-linking of a 162-bp DNA fragment (tyrT DNA) by MC, (128), and (129) were detected using DPAGE. 998 V.M. Dembitsky et al. The dimers and MC cross-linked DNA with the same apparent CpG sequence specificity, but (129) exhibited much greater cross-linking efficacy than MC. Greatly enhanced region selectivity of cross-linking to GC-rich regions by (129) relative to MC was observed, for which a mechanism unique to dimeric MCs was proposed. Covalent dG adducts of (129) with DNA were isolated and characterized by their UV and mass spectra. Tri- and tetrafunctional DNA adducts of (129) were also detected. Although the dimers were generally less cytotoxic than MC, dimer (129) was highly and uniformly cytotoxic to all 60 human tumor cell cultures of the NCI screen [134]. Its cytotoxicity to EMT6 tumor cells was enhanced under hypoxic conditions. These findings together verify the expected features of the MC dimers and warrant further study of the biological effects of dimer (129). Ac N H H H HO O 118 N H H H MeO O 119 N H H H AcO Ac 120 N H H H O 121 R N H H H HO 122 R = H, R1 = OH 123 R,R1 = O R1 30 Aziridine Alkaloids: Origin, Chemistry and Activity 999 PNU-159548 (4-demethoxy-30 -deamino-30 aziridinyl-40 -methyl-sulfo-nyldaunorubicin, 130), a synthetic derivative of the anticancer idarubicin, has a broad spectrum of antitumor activity both in vitro and in vivo attributable to its DNA intercalating and alkylating properties [135]. This study was designed to determine the cardiotoxic activity of PNU-159548 relative to doxorubicin in a chronic rat model sensitive to anthracycline-induced cardiomyopathy. PNU-159548 caused a dose-dependent myelotoxicity, with the dose of 0.5 mg/kg per week being equimyelotoxic to 1.0 mg/ kg per week doxorubicin. PNU-159548 also caused an increase in liver weight that was reversible. However, it caused a nonreversible testicular atrophy but, unlike doxorubicin, had no effect on kidney weight. The cytotoxic antitumor derivative, PNU-159548, was significantly less cardiotoxic than doxorubicin at equimyelo-suppressive doses. The combination of intercalating and alkylating activities within the same molecule without the cardiotoxic side effects of anthracyclines makes PNU-159548 an excellent candidate for clinic development in oncology. It also showed an IC50 ¼ 2.7 ng/mL against LoVo colon adenocarcinoma cells [136]. A synthetic preparation of (131) showed an IC50 of 9.0 mg/mL against mouse L 5178Y tumor cells, and compound (132) had IC50 of 0.004 mM against 12 ovarian tumors in the tumor Salmon colony formation test [137]. Semisynthetic aziridine derivative of colchicine (133) have been synthesized by the direct interaction of colchicine with chloroethylamine hydrochloride and also via the mono- and diethanolamine derivatives. These compounds had an increased radiomodifying and antitumoral activity and a decreased toxicity compared with the initial colchicine. O NH2 O H N O NH2 O OMe S S O H N O OMe S N NH N O S NH O COOH O NO2 HN2 124 KW 2149 125 BMS 181174 O H N O NH2 O OMe S2 N O 126 NH 2 NH 1000 V.M. Dembitsky et al. Results obtained in the National Cancer Institute of the USA from the study of the cytostatic activity of the (133) and bis(chloroethyl)amino derivatives on 60 tumor lines were reported [138]. Originally colchicine a soluble alkaloid was extracted from Colchicum autumnale also known as autumn crocus, meadow saffron or itkuchala in Uzbekistan which means “dog poison” [138]. O HN N MeO O N H O O NH2 NH2 (CH2)n O O O H N MeO N HN O 127 n = 0 128 n = 8 O HN N MeO O N H O O NH2 NH2 Me N O O O MeO HN H N N O 129 Neoplasm inhibitors at C-4 aziridine-bearing paclitaxel (taxol) analogues (134–136) were synthesized. The key step in the synthesis is the aziridine ring formation at the C-4 position via an intramolecular Mitsunobu reaction [139]. Biological activity of paclitaxel analogues is shown in Table 30.6. 30 Aziridine Alkaloids: Origin, Chemistry and Activity Table 30.6 Biological activity of paclitaxel analogues (IC50, nM) 1001 HCT-116a 15.6 6.9 2.0 Compound 134 135 136 a Human colon carcinoma Me O S O O O O N OH O OH O Ac HO 130 Ladirubicin H N H2N H N N NH O O 131 N O N N N N O 132 MeO MeO N O AcHN 133 OMe 1002 V.M. Dembitsky et al. AcO R O OH H O 135 R = O Ph O HO O O HO Ph O N O O O 134 R = H N 136 R = O OtBu O O 4 Conclusion Aziridine alkaloids comprise a rare group of natural products. They are mainly isolated from either microorganisms or plants. They have also been detected in some marine species. Reported activities for purified alkaloids have shown strong antitumor, antibacterial, antimicrobial, and other activities. A wide spectrum of pharmacological activities is associated with this type of alkaloid which extends to selected synthetic derivatives. A priori, one should avoid rash conclusions that any of the reported effects of hundreds aziridines are due to their alkylating activity. Quite complex compounds (21–65) may display additional antioxidant properties; some of them would serve better substances for proteins assuring multidrug resistance, such as P-glycoprotein (MDR-1). It is generally accepted that this protein binds its substrates directly from the lipid bilayer rather than from the aqueous cytoplasmic phase. Binding sites of this protein thus in cancer cells characterized by increased expression of the gene mdr-1, these hydrophobic compounds would be more readily exported than such hydrophilic molecules as 1–20. Natural and/or synthesized aziridine-containing compounds, lipids, steroids, amino acids, as well as their peptide derivatives have shown to be promising candidates for the development of new drugs toward several diseases, especially neoplasms. 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