Synthesis and preclinical evaluation of bactericidal agent isolated from soil bacterium (Streptomyces): a novel diagnostic technique to enhance anticancer efficacy through radiosynthesis.

OBJECTIVES: Dactinomycin is a well-known antitumor-antibiotic drug isolated from soil bacterium Streptomyces, which exhibits broad-spectrum pharmacological and biochemical effects. In this study, dactinomycin was successfully labeled with technetium-99m for early diagnosis of bacterial infection and to discriminate it from acute inflammation. MATERIALS AND METHODS: Various labeling parameters such as pH, ligand concentration, reducing agent, and stabilizing agent were investigated. Radio-TLC technique was used to calculate percent radiochemical purity of radiopharmaceutical. Characterization studies were carried out using electrophoresis and radio-high-performance liquid chromatography techniques. Furthermore, saline and serum stability studies were performed to investigate biocompatibility. Biodistribution and scintigraphy studies were performed in infected and inflamed animal models to discriminate between bacterial infections (Escherichia coli and Staphylococcus aureus) and acute inflammations (heat-killed S. aureus). RESULTS: The results demonstrated that the highest radiochemical purity of at least 95% was achieved using 100-500 µg ligand and 3-8 µg SnCl2·2H2O as reducing agent at 4-9 pH. Technetium-99m-dactinomycin (Tc-DTN) was observed clearly bounded to the infection site having target/nontarget ratio 2.96±0.64 at 30 min after administration, which increased to 5.21±1.03 at 4 h after administration. Further accumulation was seen in heart, lungs, liver, stomach, kidneys, spleen, and intestine. An in-vitro cell-binding study was also performed, which showed high binding affinity of Tc-DTN with S. aureus-induced infectious lesions. CONCLUSION: Tc-DTN can easily be synthesized using standardized optimization conditions. The radiopharmaceutical has the highest accumulation potential at targeted site induced by S. aureus without any prominent in-vivo cytotoxicity. Tc-DTN may be used as a potential diagnostic agent to locate S. aureus-induced infection lesions at an early stage. Tc-DTN can successfully discriminate between infection and inflammatory models which cannot be achieved from other radiopharmaceuticals developed in the past few decades.

Characterization of a Cell-Penetrating Peptide with Potential Anticancer Activity.

Cell-penetrating peptides (CPPs) are still an interesting and viable alternative for drug delivery applications. CPPs contain considerably high amounts of positively charged amino acids, imparting them with cationic character. Tumor cells are characterized by an enhanced anionic nature of their membrane surface, a property that could be used by CPPs to target these cells. We recently identified a branched CPP that displays a high internalization capacity while exhibiting selectivity for certain tumor cell types. In this study we elucidated this observation in greater detail by investigating the underlying mechanism behind the cellular uptake of this peptide. An additional cytotoxicity screen against several cancer cell lines indeed demonstrates high cytotoxic activity against cancer cells over normal fibroblasts. Furthermore, we show that this feature can be used for delivering the anticancer drug actinomycin D with high efficiency in the MCF-7 cancer cell line.

Actinomycins with altered threonine units in the beta-peptidolactone.

Five new members of the actinomycin family, actinomycins G2-G6 (2-6), are produced by Streptomyces iakyrus strain DSM 41873. Their structures were established by spectroscopic methods. Unlike actinomycin D (1), the alpha-ring of the novel compounds contains the unusual amino acid 3-hydroxy-5-methylproline, while the beta-ring includes N-methylalanine and either a chlorinated or hydroxylated threonine moiety. The chlorine-containing actinomycin G2 (2) is the main product; it exhibits strong cytotoxic and antibacterial activities. Actinomycin G5 (5) is the first actinomycin with an additional ring closure between the beta-peptidolactone and the actinoyl chromophore. Actinomycin G6 (6) resulted from the 4-hydroxythreonine-containing actinomycin G3 (3) by a 2-fold acyl shift of the beta-unit, which has not been observed before for this class of chromopeptides. The structural modification of compounds 5 and 6 goes along with an evident reduction of the biological activity. The biosynthesis of aniso-actinomycins is discussed.

Self-immolative prodrugs: candidates for antibody-directed enzyme prodrug therapy in conjunction with a nitroreductase enzyme.

The synthesis and properties of some prodrug candidates for antibody-directed enzyme prodrug therapy (ADEPT) are described. These compounds have been designed to generate the corresponding active drug upon interaction with a bacterial nitroreductase that can be conjugated to antibodies that recognize tumor-selective antigens. The active drugs included in the study are actinomycin D, mitomycin C, doxorubicin, 4-[bis(2-chloroethyl)amino]aniline and 4-[bis(2-chloroethyl)amino]phenol. The prodrugs were all 4-nitrobenzyloxycarbonyl derivatives of these drugs, which upon enzymatic reduction, generated the drug through self-immolation of the 4-(hydroxyamino)benzyloxycarbonyl group. In the case of actinomycin D, the ratio of the dose required between drug and prodrug to give the same cytotoxicity was greater than 100. The prodrug was also much less toxic (20-100x) than actinomycin D to mice in vivo. Therefore this self-immolative prodrug has a potential application in the treatment of cancer using an ADEPT-type approach.

Epimerization of the D-valine portion in the biosynthesis of actinomycin D.

In the biosynthesis of actinomycin, the multifunctional actinomycin synthetase II (ACMS II) assembles 4-methyl-3-hydroxyanthranilic acid (4-MHA), L-threonine and D-valine, the first three residues of the 4-MHA peptide lactone chain. ACMS II activates L-threonine and L-valine but not D-valine as thioesters via their adenylates, and there is no epimerization of the covalently bound L-valine. When L-threonine and L-valine are presented to the enzyme together with the 4-MHA analogue p-toluic acid and the 4-MHA-activating enzyme ACMS I, ACMS II forms the two diastereomers p-toluyl-L-Thr-L-Val and p-toluyl-L-Thr-D-Val in equal amounts along with p-toluyl-L-Thr in a cofactor-independent manner. Studies with [2,3-3H2]valine revealed that p-toluyl-L-Thr-D-Val contained approximately 50% of the tritium label found in the LL-diastereomer. Concomitantly, radioactive water was formed due to enzyme-catalyzed hydrogen exchange with the solvent during epimerization. In the absence of threonine (or MgATP), however, the amount of radioactive water formed from [3H]valine was significantly less, which suggests that the peptide bond between L-threonine and L-valine is formed prior to the epimerization at C-2 of valine. The facts that both LL- and LD-acyldipeptides are equally present on the enzyme's surface--as revealed by using 14C-labeled threonine or valine as precursors--and that the L-valine in the LL-diastereomer apparently has not lost hydrogen strongly suggests that the LL-diastereomer is an obligatory intermediate in the formation of the LD-dipeptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and properties of some peptide analogues of actinomycin D.

Analogues of actinomycin D (AMD) were synthesized in which amino acid replacements were made at various sites in the peptide moieties. These include (i) replacement of both N-methylvalines by N-methylleucine, (ii) replacement of both sarcosines by N-[2-(methoxycarbonyl)ethyl]glycine, and (iii) replacement of one or both D-valines by D-threonine. The purpose of replacements ii and iii was to ascertain the effect upon biological activity of introducing a new side chain which could be functionalized to allow the attachment of carrier molecules such as antibodies. NMR data indicated that none of the analogues had solution conformations significantly different from that of AMD. Difference spectra with DNA revealed that replacement i enhanced binding while the other analogues bound less strongly to DNA. All the analogues had lower antimicrobial activities than AMD. In contrast, 5,5'-(MeLeu)2AMD displayed in vitro antitumor activity comparable with that of AMD at approximately 100-fold lower concentrations.

Enantiomers of 7-(2,3-epoxypropoxy)actinomycin D as dual-action DNA-acting antitumor agents.

Enantiomeric forms of (+/-)-EPA [racemic 7-(2,3-epoxypropoxy)actinomycin D] have been synthesized; these are (R)-(+)- and (S)-(-)-EPA, which are active against a range of actinomycin resistant and marginally responsive tumors. The R-(+) enantiomer is uniformly superior to the other forms in all the tumor lines tested. These enantiomers act by binding to DNA, both by intercalation and alkylation at the guanine base of DNA. They are superior to actinomycin D in their in vitro activity against mouse leukemias (L1210 and P388/ADR) and mouse melanoma B16. This superior activity is also evident against all the preceding mouse leukemias and against solid tumors B16 and C26 in vivo. In biochemical action, the enantiomers behave similarly and act primarily by inhibiting DNA synthesis in tumor cells; the only difference found was in their preference for sites in DNA bases during alkylation. The R-(+) enantiomer generates an adduct that is believed to be bonded to the N7-site of guanosine; conversely, the S-(-) isomer forms two adducts with DNA that are different from the preceding one by HPLC and are tentatively assigned O6-guanosine-substituted structures on the basis of their UV, CD, and other chemical behaviors.

Synthesis and biological properties of actinomycin D chromophoric analogues substituted at the 7-carbon with aziridine and aminopropoxy functions.

The growing importance of functionalized aziridines in numerous organic biomolecules led us to develop syntheses of novel actinomycin D (AMD) analogues substituted with an aziridine. Reaction of 7-hydroxyactinomycin D with 2-(iodomethyl)aziridine produced the desired 7-(2-aziridinylmethoxy)actinomycin analogue. In an attempt to develop an alternate route to this analogue, 7-(2-azido-3-iodopropoxy)actinomycin was subjected to reduction with dimethylamine-borane complex; the reaction did not produce the three-membered aziridine; instead the reaction product was found to be linear 7-(2-aminopropoxy)actinomycin D. Calf-thymus-DNA binding of these analogues was comparable to that of AMD as examined by UV-visible difference spectral measurements, thermal denaturation of DNA, and CD techniques. The analogues were found to be about 1/4 to 1/30 as cytotoxic to human lymphoblastic CCRF-CEM leukemia and B16 melanoma cells in vitro as AMD.

"Reverse" and "symmetrical" analogues of actinomycin D: metabolic activation and in vitro and in vivo tumor growth inhibitory activities.

Two new classes of actinomycin D analogues, tetracyclic "reverse" analogues and a tricyclic "symmetrical" analogue of actinomycin D, are reported. These analogues bind to DNA and the binding does not occur by an intercalation mechanism. The analogues inhibit the synthesis of DNA and RNA in P388 tumor cells and the growth of CCRF-CEM cells in vitro at nanomolar concentrations. The tetracyclic "reverse" analogues, which are structurally related to the previously reported actinomycin D oxazolyl analogues, are metabolized in the presence of rat hepatic microsomes and tumor cell homogenates. The metabolism takes place with the loss of the oxazole ring; thus the "reverse" analogues produce a major metabolite which is the "symmetrical" analogue; the actinomycin oxazolyl analogues generate 7-hydroxyactinomycin D. Further, the microsomes activate the analogues to free-radical states which catalyze the production of superoxide as shown by stimulation of epinephrine oxidation and also indicated by electron paramagnetic resonance studies. The "symmetrical" and "reverse" analogues also demonstrate very high activities in these systems. In in vivo studies using P388/S, P388/ADR leukemia, and B16 melanoma in mice, the analogues showed increased activity and superior therapeutic index values, in comparison to actinomycin D.

Carbon-7 substituted actinomycin D analogues as improved antitumor agents: synthesis and DNA-binding and biological properties.

7-(2,3-Epoxypropoxy)actinomycin D has been synthesized along with its major companion product, 7-(2,3-dihydroxypropoxy)actinomycin D. They were characterized by UV-visible and CD spectra and by NMR studies. According to UV-visible absorptiometry, circular dichroism, and thermal denaturation studies, they bind to DNA in a manner that is comparable to actinomycin D. The analogues are, like actinomycin D, extremely cytotoxic to human lymphoblastic leukemic cells (CCRF-CEM) in vitro but are significantly less toxic than actinomycin D to normal CDF1 mice is vivo. Unlike actinomycin, these analogues are metabolized in rats, and the metabolites are excreted in rat urine at a rapid rate. Compared to actinomycin D, the antitumor activity of the 7-(2,3-epoxypropoxy)actinomycin analogue against P-388 leukemia in mice is decidedly superior, and the therapeutic index is improved several fold.

N2- and C-7 substituted actinomycin D analogues: synthesis, DNA-binding affinity, and biochemical and biological properties. Structure-activity relationship.

N2-n-Alkyl- and omega-amino-n-alkylactinomycin D and 7-alkoxy-, 7-aralkoxy-, and 7-(acyloxy)actinomycin D were synthesized by modification of the parent actinomycin D molecule at the N2 and C-7 positions of the phenoxazinone moiety. The intermediate for N2 substitution was 2-deamino-2-chloroactinomycin D. For C-7 substitution, 7-hydroxyactinomycin D was used as the intermediate. Treatment of 2-deamino-2-chloroactinomycin D with an excess of the appropriate amine produced the N2-substituted derivatives. Condensation of the required alkyl or acyl halides with 7-hydroxyactinomycin D, aided by solid anhydrous potassium carbonate, yielded the C-7-substituted analogues. Calf thymus DNA-binding affinity was determined by equilibrium binding and also by thermal denaturation of DNA techniques, inhibitory activity of nucleic acid synthesis was examined using P388 cells in vitro, cytotoxicity measurements to tumor cells in vitro employed human lymphoblastic leukemic cells (CCRF-CEM), and antitumor activity was assayed against P388 mouse leukemia in CDF1 mice. Synthesis of a number of new analogues in each series and determination of the biophysical, biochemical, and biological properties established a more thorough structure-activity relationship in these analogues. These results establish that with the selection of omega-(n-alkylamino) groups at the N2 site or O-n-alkyl or O-acyl groups at the C-7 site a variety of modifications can be carried out on the actinomycin molecule while preserving biological activity. N2-3'-Amino-n-propyl- and N2-10'-amino-n-decylactinomycin D, 7-methoxy- and 7-ethoxyactinomycin D, and the 7-O-(1'-adamantoyl) ester of 7-hydroxyactinomycin D were found to be the most effective antitumor agents in vivo and in vitro. They also strongly inhibit cellular RNA and DNA synthesis and, with the exception of the ester, retain high DNA-binding affinity.

Synthesis and biological properties of N2-substituted spin-labeled analogues of actinomycin D.

We have synthesized N2-[4-(2,2,6,6-tetramethyl-1-piperidinyloxy)]actinomycin D And the related 1,2-diaminoethane and 1,3-diaminopropane derivatives and evaluated their biological properties. Binding studies with the spin-labeled actinomycin D analogues and DNA were carried out by using circular dichroism, electron spin resonance, and thermal denaturation. These studies have suggested that the derivatives bind to DNA and that their DNA-binding modes are similar but not identical. Spin-labeled actinomycin D derivatives were less potent in inhibiting Escherichia coli DNA-dependent RNA polymerase reaction than actinomycin D and were less toxic to L1210 cells in vitro than the parent compound. Spin-labeled actinomycin D derivatives were more common than the parent compounds against P-388 leukemia cells in vitro with little or no toxicity.

A phenazine analogue of actinomycin D.

An analogue of actinomycin D (1), in which the phenoxazone chromophore has been replaced by a phenazine, has been synthesized and characterized. Although this compound (2) lacks the 2-amino group and does not possess the quinoid structure of 1, it does bind to DNA, but less tightly than either 1 or the 2-deamino derivative of 1. NMR and CD spectra indicate that the peptide conformations in 2 are approximately as in 1; there was no apparent asymmetry of the two peptide rings. Compound 2 inhibited nucleic acid synthesis in L1210 cell cultures more effectively than does 2-deaminoactinomycin D, but about one-tenth as well as does actinomycin D.

Actinomycin D oxazinones as improved antitumor agents.

1,4-Oxazinone derivatives of the phenoxazinone chromophore in actinomycin D (AMD) have been synthesized by condensation of AMD with alpha-keto acids. By varying the starting alpha-keto acid, the substitutions on the oxazinone ring and, consequently, the lipophilicity of the molecule could be altered. These oxazinone derivatives revert to AMD in physiological media and it appears that these oxazinones are "depot" forms of AMD and possess physicochemical and DNA-binding properties which are significantly different from those of AMD. The oxazinones, which have bulky and lipophilic substituents at position 3, demonstrate more pronounced antitumor activity against P388 mouse leukemia and are less toxic than AMD.

N7-Substituted 7-aminoactinomycin D analogues. Synthesis and biological properties.

A series of N7-substituted 7-aminoactinomycin D analogues with alkyl, aralkyl, and heteroaralkyl substituents was synthesized and their biological properties were studied. All of these analogues proved to be 22- to 28-fold less toxic than actinomycin D when tested against human lymphoblastic leukemia cells (CCRF-CEM) in vitro. Against the P388 mouse leukemia in vivo, most of the analogues had activity comparable to actinomycin D and one was significantly more active. The results show that substitutions of this kind do not interfere with the antitumor activity of actinomycin D and may be useful for the design of modified actinomycin D analogues with greater selectivity.

Synthesis and evaluation of bis-dipeptide and bis-tripeptide analogues of actinomycin D.

Six-bis-dipeptide analogues of actinomycin D, all containing two threonyl-D-valine side chains, were prepared. Also two bis-tripeptide analogues containing an additional proline or oxoproline residue were synthesized. None of the compounds bound to DNA in a manner similar to actinomycin D. This lack of strong intercalative binding emphasizes the importance of the pentapeptidolactone side chains in the binding of actinomycin D to DNA and also highlights the deficiences inherent in using only small nucleotide sequences in investigating drug-DNA binding. None of the analogues tested showed any antitumor activity, although actinocylbis(threonyl-D-valine methyl ester) did show 10% of the antibacterial activity of actinomycin D vs. Bacillus subtilis.

2-Deaminoactinomycin D, synthesis and interaction with deoxyribonucleic acid.

2-Deaminoactinomycin D has been synthesized and characterized. It binds to DNA by intercalation according to NMR, CD, thermal denaturation, and unwinding studies on the drug-DNA complex. Loss of the 2-amino group does not seriously affect binding parameters relative to actinomycin D; affinity for calf thymus DNA may even be increased, according to deltaTm measurements. The unwinding of circular DNA caused by this compound is at least as large as that effected by actinomycin D and ethidium bromide. Nevertheless, 2-deaminoactinomycin D is less effective than actinomycin D in inhibiting nucleic acid syntheses in L1210 cell culture and in in vivo antitumor activity against P388 leukemia.

Synthesis and some properties and antitumor effects of the actinomycin lactam analog, (di(1-L-alpha, beta-diaminopropionic))actinomycin D1.

A lactam analog of actinomycin D (AMD) has been synthesized as a potential antitumor chemotherapeutic agent. Both L-threonine residues were replaced by L-alpha,beta-diaminopropionic acid. Starting with Nalpha-benzyloxycarbonyl-Nbeta-tert-butyloxycarbonyl-L-alpha,beta-diaminopropionic acid methyl ester hydrochloride the linear intermediate Nalpha-benzyloxycarbonyl-Nbeta-(tert-butyloxycarbonylsarcosyl-L-N-methylvalyl)-L-alpha,beta-diaminopropionyl-D-valyl-L-proline p-nitrophenyl ester was prepared by conventional methods of peptide synthesis in solution. Selective cleavage of the Nbeta-tert-butyloxycarbonyl group and lactam formation afforded the desired cyclic pentapeptide derivative. The chromophore precursor, Nalpha-(2-nitro-3-benzyloxy-4-methylbenzoyl) substituent, was introduced via its symmetric anhydride. Catalytic reduction followed by ferricyanide-mediated phenoxazinone formation provided the lactam analog, [di(1'-L-alpha,beta-diaminopionic acid)]actinomycin D ([Dpr1]2-AMD). Its binding to natural and synthetic DNA and that of an analogous L-threo-alpha,beta-diaminobutyric acid containing lactam ([Dbu1]2-AMD) compared with the binding of AMD (in which the peptides are in lactone form) was studied by circular dichroic (CD) spectroscopy. The visible and uv CD spectra of free AMD differed from those of the free lactam analogs, indicating that the asymmetric environment of the pentapeptide rings in the region of the chromophore differs in free actinomycin lactone and lactams. In the presence of calf thymus DNA, PM2 DNA, and the synthetic d(A-T)-like copolymers containing 2,6-diaminopurine (DAP), poly[d(DAP-T)], and poly[d(DAP-A-T)], the rotational strengths of the optically active transitions in the visible region of the actinomycins increased, and the CD spectra in the presence of the various DNA duplexes were qualitatively similar. The CD spectra of bound actinomycin lactams resembled the spectrum of bound AMD. This suggests that the lactone and lactam actinomycins acquire a similar environment when bound to DNA. [Dpr1]2-AMD was less cytotoxic than AMD in antibacterial assays but exhibited somewhat higher toxicity in mice than AMD. At optimal dose levels the lactam analog had little or no antitumor activity in three murine tumor systems.

7-substituted actinomycin D analogs. Chemical and growth-inhibitory studies.

The synthesis and biological activity of three 7-substituted actinomycin D derivatives are reported. Three such derivatives, 7-nitro-, 7-amino-, and 7-hydroxyactinomycin D, were synthesized via new methods which were first tested successfully with a chromophore model system. Of these, 7-nitro- and 7-aminoactinomycin D were assayed for growth inhibitory activity against mammalian cells (CCRF-CEM human lymphoblastic leukemia) in vitro and against the Ridgway osteogenic sarcoma and the L1210, P1534, and P388 murine leukemias in vivo. In these systems, the inhibitory activity of the 7-substituted analogs was comparable to actinomycin D. In two bacterial systems ( (L. casei and L. arabinosus) in vitro, on the other hand, these compounds showed inhibitory profiles which are distinctly different from actinomycin D. These studies demonstrate that substitution at the 7 position, which does not interfere with DNA binding, is capable of yielding experimental antitumor agents with significant activity against a variety of tumors.