5-O-Mycaminosyltylonolide antibacterial derivatives: design, synthesis and bioactivity.

Tylosin is a 16-membered macrolide broad-spectrum antibiotic that has an important role in veterinary medicine, active against Gram-positive and a restricted range of Gram-negative bacteria. We synthesized 15 types of tylosin-related derivatives by chemical modification and evaluated them against mastitis pathogens. Among them, 20-deoxy-20-{N-methyl-N-[1-(3-quinolyl)-1H-1,2,3-triazol-4-yl]methylamino}-5-O-mycaminosyltylonolide 2f and 20-deoxy-20-{N-benzyl-N-[1-(3-quinolyl)-1H-1,2,3-triazol-4-yl]methylamino}-5-O-mycaminosyltylonolide 2k were found to not only expand their antibacterial impact to include Gram-negative bacteria, such as Escherichia coli and Klebsiella pneumoniae, but also to retain or increase antibacterial activity against Gram-positive bacteria, such as Staphylococcus aureus and Streptococcus uberis in comparison with the parent tylosin.

Novel 16-membered macrolides modified at C-12 and C-13 positions of midecamycin A1 and miokamycin. Part 1: Synthesis and evaluation of 12,13-carbamate and 12-arylalkylamino-13-hydroxy analogues.

Design and synthesis of 16-membered macrolides modified at the C-12 and 13 positions are described. The compounds we report here have an arylalkylamino group attached to the C-12 position of the macrolactone. Both types of derivatives, 12,13-cyclic carbamates and non-carbamate analogues, were synthesized via 12-amino-13-hydroxy intermediates derived from 12,13-epoxide that was prepared by selective epoxidation at the C-12 and C-13 positions. 4'-Hydroxyl analogues were also prepared by acidic hydrolysis of a neutral sugar. These compounds were evaluated for in vitro antibacterial activity against respiratory tract pathogens. Some of these analogues exhibited an improved activity compared with the corresponding parent compound.

Synthesis of novel 4'-substituted 16-membered ring macrolide antibiotics derived from leucomycins.

A series of novel 4'-substituted 16-membered ring macrolides was synthesized by the cleavage of the mycarose sugar and subsequent modification of 4'-hydroxyl group. This new class of macrolides antibiotics is acid stable. The synthetic methodology described here is expected to find application in the synthesis of new generation of macrolides that target the emerging bacterial resistance.

Synthesis and biological evaluation of novel leucomycin analogues modified at the C-3 position. I. Epimerization and methylation of the 3-hydroxyl group.

The synthesis and biological evaluation of sixteen-membered macrolides modified at the C-3 position are described. 3-Epi-leucomycin A7 (9), 3-O-acyl-3-epi-leucomycin A7 analogues (11a-11e), 3-O-acylleucomycin A7 analogues (13b-13e) and 3-O-methylleucomycin analogues (16a, 16b and 22) were synthesized via fully protected intermediates (7, 5a, 5b and 20). After appropriate modification, subsequent deprotections were performed to furnish a variety of leucomycin analogues. Methylation of the 3-hydroxyl group was found to improve the pharmacoprofile of leucomycin antibiotics. 3-O-Methylrokitamycin (16b) showed enhanced antibacterial activity in vitro and 3,3''-di-O-methyl-4''-O-(3-methylbutyl)leucomycin V (22) exhibited improved metabolic stability in rat plasma in vitro.

Synthesis and preliminary cytotoxicity study of glucuronide derivatives of CC-1065 analogues.

Glucuronide derivatives of CBI-bearing CC-1065 analogues have been synthesized, and their cytotoxicities tested against U937 leukemia cells. The new compounds show potent antitumor activity in vitro. Compounds 1 and 2, and their corresponding glucuronides 3 and 4 have IC(50) values of 0.6, 0.1, 1.4 and 0.6 nM, respectively. Glucuronide 3 is approximately 2-fold less toxic than its hydroxyl counterpart 1, and glucuronide 4 is approximately 6-fold less toxic than its hydroxyl counterpart 2. Glucuronides 3 and 4 may have limited use in the ADEPT approach. However, they may be used as antitumor agents in a conventional way.

CC-1065 analogues bearing different DNA-binding subunits: synthesis, antitumor activity, and preliminary toxicity study.

CC-1065 analogues bearing different DNA-binding subunits were synthesized. A terminal C5-NO2 and -F moiety at the DNA-binding subunit increased the drug's potency and antitumor efficacy. A C5-OCH3 reduced the potency and antitumor efficacy. Compound (+/-)-7, bearing a trans double bond, had increased antitumor efficacy. A preliminary toxicity study indicated that terminal C5-OCH3 and -acetamido moieties at the DNA-binding subunit caused delayed death in mice.

Parallel synthesis and evaluation of 132 (+)-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) analogues of CC-1065 and the duocarmycins defining the contribution of the DNA-binding domain.

The solution-phase, parallel synthesis and evaluation of a library of 132 (+)-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) analogues of CC-1065 and the duocarmycins containing dimeric monocyclic, bicyclic, and tricyclic heteroaromatic replacements for the DNA-binding domain are described. This systematic study revealed clear trends in the structural requirements for observation of potent cytotoxic activity and DNA alkylation efficiency, the range of which spans a magnitude of > or =10 000-fold. Combined with related studies, these results highlight that the role of the DNA-binding domain goes beyond simply providing DNA-binding selectivity and affinity (10-100-fold enhancement in properties), consistent with the proposal that it contributes significantly to catalysis of the DNA alkylation reaction accounting for as much as an additional 1000-fold enhancement in properties.

Substituent effects within the DNA binding subunit of CBI analogues of the duocarmycins and CC-1065.

A series of CBI analogues of the duocarmycins and CC-1065 exploring substituent effects within the first indole DNA binding subunit are detailed. Substitution at the indole C5 position led to cytotoxic potency enhancements that are > or =1000-fold, providing simplified analogues containing a single DNA binding subunit that are more potent (IC(50)=2-3 pM) than CBI-TMI, duocarmycin SA, or CC-1065.

Synthesis and evaluation of a series of C3-substituted CBI analogues of CC-1065 and the duocarmycins.

The synthesis and evaluation of a series of C3-substituted 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) analogues of the CC-1065 and duocarmycin alkylation subunits are detailed, including methyl and the full series of halogens. Introduction of the key substituent was accomplished through directed metalation of the seco-CBI core followed by reaction of the resultant aryllithium with an appropriate electrophile. C3-Bromo and iodo substituents were only effectively installed on the hindered aryllithium intermediate using a novel halogen source, 1-bromo- and 1-iodophenylacetylene, that should prove generally useful beyond the studies we describe. X-ray crystal structures of the series show substantial distortion in the vinylogous amide due to unfavorable steric interactions between the C3-substituent and the N(2)-carbamate. In the halogen series, the N2-C2a bond length and the torsional angle chi(1) smoothly increase with the increasing size of the C3 substituent indicative of decreasing vinylogous amide conjugation through the series (H > F > Cl > Br > I). Unlike N-Boc-CBI, this series of substituted CBI analogues proved remarkably reactive toward solvolysis even at pH 7, where the reaction is uncatalyzed and the reactivity order (I > Br > Cl > F > H) follows a trend consistent with the extent of vinylogous amide conjugation and stabilization. The implications of these observations on the source of catalysis for the DNA alkylation reaction of the natural products are discussed.

Synthesis, chemical properties, and biological evaluation of CC-1065 and duocarmycin analogues incorporating the 5-methoxycarbonyl-1,2,9,9a-tetrahydrocyclopropa.

The synthesis of 5-methoxycarbonyl-1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (C5-CO2Me-CBI), a substituted CBI derivative bearing a C5 methoxycarbonyl group, and its corresponding 5-hydroxymethyl derivative are described in efforts to establish substituent electronic effects on the agents' functional reactivity and the resulting effect this has on their rate of DNA alkylation. Resolution of an immediate C5-CO2Me-CBI precursor and its incorporation into both enantiomers of 16 and 17, analogues of the duocarmycins, are also detailed. A study of the solvolysis reactivity and regioselectivity of N-BOC-C5-CO2Me-CBI (12) revealed that the introduction of a C5 methyl ester modestly slowed the rate of solvolysis (1.8x, pH 3) without altering the inherent reaction regioselectivity (>20:1). The comparison of the X-ray structures of the N-CO2Me derivatives of C5-CO2Me-CBI and CBI revealed correlations with the reaction regioselectivity and the relative reactivity of the compounds. The latter correlated well with the less reactive C5-CO2Me-CBI exhibiting a shortened N2-C2a bond length (1.386 vs 1.390 A) and smaller chi1 dihedral angle (8.1 degrees vs 21.2 degrees ) indicative of greater vinylogous amide conjugation and was accompanied by a diminished (cross-conjugated) cyclopropane conjugation (shorter bond lengths). Establishment of the DNA alkyation properties revealed that C5-CO2Me-CBI-based agents retained the identical alkylation selectivity of the natural products. More importantly, the C5 methyl ester was found to decrease the rate (0.77x) of DNA alkylation relative to CBI, consistent with its inherent lower reactivity. These results indicate that the previously observed increase in the rate of DNA alkylation for C7-substituted CBI analogues including CCBI (7-cyano-CBI) is contrary to expectations based on their inherent reactivities. Unlike 17, in which the C5 methyl ester does not bind in the minor groove, the C7 substituent lies in the minor groove extending the rigid length of the agents, further enhancing the DNA binding-induced conformational change responsible for activation toward nucleophilic attack and catalysis of the DNA alkylation reaction.

Repromicin derivatives with potent antibacterial activity against Pasteurella multocida.

Reductive amination of repromicin with polyfunctional amines has led to new macrolide antibacterial agents, some of which are highly potent against the Gram-negative pathogen Pasteurella multocida both in vitro and in a mouse infection model. A key element in this discovery was the recognition that among certain known macrolides increasing lipophilicity results in diminished in vivo activity. One repromicin derivative, 20-[N-[3-(dimethylamino)-propyl]-N-L-alanylamino]-20-deoxorepro micin (35), was selected for advanced evaluation. At 5 mg/kg, a single subcutaneous dose was found to control induced pasteurellosis in swine and induced respiratory disease in cattle.

1,2,9,9a-Tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) analogs of CC-1065 and the duocarmycins: synthesis and evaluation.

An extensive study of analogs of the potent antitumor antibiotics CC-1065 and the duocarmycins which incorporate the 1,2,9,9a-tetrahydrocyclopropa[c]benz[e]indol-4-one (CBI) alkylation subunit are detailed. In contrast to early speculation, deep-seated modifications in the CC-1065 and duocarmycin alkylation subunits are well tolerated and the CBI-based analogs proved to be potent cytotoxic agents and efficacious antitumor compounds. Full details of studies defining a direct relationship between functional stability and in vitro cytotoxic potency are described. As such, the readily accessible CBI-based analogs were found to be four times more stable and four times more potent than the corresponding analogs containing the authentic CPI alkylation subunit of CC-1065 and comparable in potency to agents containing the authentic alkylation subunit of duocarmycin SA. Similarly, the CBI-based agents alkylate DNA with an unaltered sequence selectivity at an enhanced rate and with a greater efficiency than the corresponding CPI analog and were comparable to the corresponding analog incorporating the duocarmycin SA alkylation subunit. Systematic and extensive modifications and simplifications in the DNA binding subunits attached to CBI were explored with the comparisons of both enantiomers of CC-1065 and the duocarmycins 2 and 3 with enantiomers of 18-24, 25-29, 57-61, 62-65, 66-68, 72, 73, 78 and 79.

Synthesis and biochemical evaluation of the CBI-PDE-I-dimer, a benzannelated analog of (+)-CC-1065 that also produces delayed toxicity in mice.

A practical synthesis of CBI (2) was developed and applied to the synthesis of benzannelated analogs of CC-1065, including CBI-PDE-I-dimer (13) and CBI-bis-indole [(+)-A'BC]. The CBI-PDE-I-dimer was shown to have similar DNA sequence selectivity and structural effects on DNA as (+)-CC-1065. Of particular importance was the observed duplex winding effect that has been associated with the pyrrolidine ring of the nonalkylated subunits of (+)-CC-1065 and possibly correlated with its delayed toxicity effects. The effect of CBI-PDE-I-dimer was also compared to (+)-CC-1065 in the inhibition of duplex unwinding by helicase II and nick sealing by T4 ligase and found to be quantitatively similar. The in vitro and in vivo potencies of the CBI compounds corresponded very closely to the corresponding CPI derivatives. Finally, CBI-PDE-I-dimer was like (+)-CC-1065 in causing delayed toxicity in mice.

Structure of the (+)-CC-1065-DNA adduct: critical role of ordered water molecules and implications for involvement of phosphate catalysis in the covalent reaction.

(+)-CC-1065 is an extremely potent antitumor agent produced by Streptomyces zelensis. The potent effects of (+)-CC-1065 and its alkylating analogues are thought to be due to the formation of a covalent adduct through N3 of adenine in DNA. It has been previously postulated, on the basis of modeling studies, that a phosphate may be involved in stabilization of the adduct and in acid catalysis of this reaction. In this study, using 1H NMR in combination with 17O-labeled water and phosphate, we demonstrate the involvement of a bridging water molecule between a phenolic proton on the alkylating subunit of (+)-CC-1065 and an anionic oxygen in the phosphate on the noncovalently modified strand of DNA. In addition, a second ordered water molecule associated with one of the protons on N6 of the covalently modified adenine is also identified. This structure has important implications for catalytic activation of the covalent reaction between (+)-CC-1065 and DNA and, consequently, the molecular basis for sequence-selective recognition of DNA by the alkylating subunit of (+)-CC-1065. On the basis of the example described here, the use of 1H NMR in 17O-labeled water may be a powerful probe to examine other structures and catalytic processes for water-mediated hydrogen-bonded bridges that occur between small molecules and DNA or enzymes.

Synthesis and antimicrobial evaluation of 20-deoxo-20-(3,5-dimethylpiperidin-1-yl)desmycosin (tilmicosin, EL-870) and related cyclic amino derivatives.

A series of 20-deoxo-20-cyclic (alkylamino) derivatives of tylosin, desmycosin, macrocin and lactenocin was prepared by reductive amination of the C-20 aldehyde group. The majority of the compounds were prepared using metal hydrides (sodium cyanoborohydride or sodium borohydride) as the reducing agents and a suitable cyclic alkylamine. Subsequently, a more convenient procedure was developed using formic acid as a reducing agent. The C-20 amino derivatives prepared from desmycosin exhibited good in vitro antimicrobial activity against Pasteurella haemolytica and Pasteurella multocida (MIC range of 0.78 approximately 6.25 micrograms/ml) as well as Mycoplasma species (MIC range of 0.39 approximately 6.25 micrograms/ml). Several derivatives showed excellent oral efficacy against infections caused by P. multocida in chicks. One of these derivatives, 20-deoxo-20-(3,5-dimethylpiperidin-1-yl)desmycosin (tilmicosin or EL-870) was selected for development as a therapeutic agent for pasteurellosis in calves and pigs.

19-Deformyl-4'-deoxydesmycosin (TMC-016): synthesis and biological properties of a unique 16-membered macrolide antibiotic.

19-Deformyl-4'-deoxydesmycosin was synthesized by the following synthetic route: 19-Deformylation of desmycosin, 3,2',4''-tri-O-trimethylsilylation, 4'-O-sulfonylation, 4'-iodination, reductive deiodination and 3,2',4''-tri-O-detrimethylsilylation. Deformylation of the aldehyde group at the C-19 position was achieved by two different methods: A) A simple one-step deformylation using Wilkinson's catalyst ((Ph3P)3RhCl). B) Reductive decarboxylation of the 19-carboxyl derivative following NaClO2 oxidation of the aldehyde. 19-Deformyl-4'-deoxydesmycosin showed very strong antimicrobial activity in vitro and in vivo.

Synthesis and structure-activity studies of new 4''-O-acyltylosin derivatives of therapeutic interest.

Eleven 4''-O-acyltylosin derivatives were synthesized and subjected to a two-step screening system consisting of antimicrobial activity and esterase stability assays. The new derivatives were all active against macrolide-resistant Staphylococci and mycoplasmas, but only 4''-O-(4-methoxy)phenylacetyltylosin and 4''-O-(4-acetyl)phenylacetyltylosin showed better resistance to mouse liver esterase than 4''-O-phenylacetyltylosin (reference compound C).

Synthesis and evaluation of tylosin-related macrolides modified at the aldehyde function: a new series of orally effective antibiotics.

Modification of the aldehyde group in tylosin and related macrolide antibiotics dramatically enhanced the oral efficacy of the derivatives against experimental infections caused by susceptible bacteria in laboratory animals. A large number and wide variety of aldehyde-modified macrolide derivatives were prepared, utilizing the Mitsunobu reaction and other chemical transformations. Evaluation of in vitro and in vivo antimicrobial activity indicated that derivatives of demycarosyltylosin (desmycosin) combined the broadest spectrum of antimicrobial activity with the best efficacy and bioavailability after oral administration.

Stereoelectronic factors influencing the biological activity and DNA interaction of synthetic antitumor agents modeled on CC-1065.

The synthesis, physicochemical properties, and biological activities of a series of novel spiro cyclopropyl compounds, modeled on the potent antitumor antibiotic CC-1065 (1), are described. Many of these synthetic analogues are significantly more effective than 1 against murine tumors. In particular, compound 27 exhibits high activity and potency. Structure-activity analysis supports a molecular mechanism for biological action involving hydrophobic interaction of the drug with DNA and acid-catalyzed alkylation of DNA.

Potential antitumor agents. I. Synthesis of pyrroloindazole derivatives related to the pyrroloindole moieties of the antitumor antibiotic CC-1065.

The synthesis of some derivatives of the unknown 1(2),8-dihydropyrrolo[3,2-g]indazole is reported starting from 7-oxo-4,5,6,7-tetrahydroindole. The new pyrroloindazole derivatives resemble the B and C subunit structure of the antitumor antibiotic CC-1065.