Synthesis and biological evaluation of B-ring analogues of (-)-rhazinilam

Three macrocyclic analogues of rhazinilam 1 having a 11- or 12-membered B-ring with an endocyclic carbamate group or an amino-acid residue were synthesized from the natural product. These analogues 3 and 4 displayed a very low activity on tubulin. Thirty N-1 and C-16 substituted analogues of rhazinilam were also synthesized regioselectively from rhazinilam. Stereochemical analyses showed that N-1 and C-16alpha analogues have the same conformation as rhazinilam, whereas C-16beta analogues adopt a different conformation for rings B and D. All N-1 and C-16 analogues were less active than rhazinilam on tubulin, though analogues 5a, 6aalpha, 6balpha, and 6f having the less bulky substituents retained close affinities. A few analogues either active (like 6f) or inactive (like 5o) on tubulin showed significant inhibition of the growth of KB cancer cells.     

P2-P1' macrocyclization of P2 phenylglycine based HCV NS3 protease inhibitors using ring-closing metathesis

Macrocyclization is a commonly used strategy to preorganize HCV NS3 protease inhibitors in their bioactive conformation. Moreover, macrocyclization generally leads to greater stability and improved pharmacokinetic properties. In HCV NS3 protease inhibitors, it has been shown to be beneficial to include a vinylated phenylglycine in the P2 position in combination with alkenylic P1' substituents. A series of 14-, 15- and 16-membered macrocyclic HCV NS3 protease inhibitors with the linker connecting the P2 phenylglycine and the alkenylic P1' were synthesized by ring-closing metathesis, using both microwave and conventional heating. Besides formation of the expected macrocycles in cis and trans configuration as major products, both ring-contracted and double-bond migrated isomers were obtained, in particular during formation of the smaller rings (14- and 15-membered rings). All inhibitors had K(i)-values in the nanomolar range, but only one inhibitor type was improved by rigidification. The loss in inhibitory effect can be attributed to a disruption of the beneficial π-π interaction between the P2 fragment and H57, which proved to be especially deleterious for the d-phenylglycine epimers.     

Design and synthesis of novel leucomycin analogues modified at the C-3 position. Part II: 3-O-(3-Aryl-2-propenyl)leucomycin analogues

The design and synthesis of 16-membered macrolides modified at the C-3 position are described. Starting from fully protected intermediate (5), appropriate modifications including Heck reaction were performed to furnish 3-O-(3-aryl-2-propenyl)leucomycin A(7) analogues (9a-9m). These leucomycin A(7) derivatives showed improved in vitro antibacterial activities against clinically important pathogens including erythromycin-resistant Streptococcus pneumoniae (ERSP). SAR analysis of derivatives modified at the C-3 and C-3' positions suggested that single modification at C-3 or C-3' was effective for in vitro antibacterial activity.     

Appearance in Japan of highly macrolide-resistant Escherichia coli producing macrolide 2'-phosphotransferase II.

Escherichia coli CU1, a clinical isolate recovered in Japan in 1997, was found to be highly-resistant to both 14-membered and 16-membered ring macrolide antibiotics. A crude extract prepared from strain CU1 inactivated 14-, 15- and 16-membered ring macrolides in the presence of ATP and the Rf value of inactivated oleandomycin was identical to that of oleandomycin 2'-phosphate. This suggested that strain CU1 produced the enzyme macrolide 2'-phosphotransferase [MPH(2')]. Substrate specificity of the crude enzyme from strain CU1 against 14-, 15- and 16-membered ring macrolides was basically similar to that of MPH(2')II from strain BM2506, differing in that the former more effectively inactivated roxithromycin and tylosin. Subsequent attempts were made to clone the novel mph gene encoding for MPH(2') in strain CU1. The mph gene carried by strain CU1 was located on nontransmissible plasmid DNA, designated pCU001. Its molecular weight, estimated by agarose electrophoresis, was approximately 57 kD. The DNA sequence of the cloned mph gene from the Japanese isolate CU1 was identical to that of mphB, which until now had only been recovered in France. The variance in the substrate specificity of MPH(2')II from each strain led us to speculate that other factors in the reaction affect the enzymatic inactivation activity.     

Nucleosides and nucleotides. 192. Toward the total synthesis of cyclic ADP-carbocyclic-ribose. Formation of the intramolecular pyrophosphate linkage by a conformation-restriction strategy in a syn-form using a halogen substitution at the 8-position of the adenine ring

The synthesis of cyclic ADP-carbocyclic-ribose (2), as a stable mimic for cyclic ADP-ribose, was investigated. Construction of the 18-membered backbone structure was successfully achieved by condensation of the two phosphate groups of 19, possibly due to restriction of the conformation of the substrate in a syn-form using an 8-chloro substituent at the adenine moiety. SN2 reactions between an optically active carbocyclic unit 8, which was constructed by a previously developed method, and 8-bromo-N6-trichloroacetyl-2',3'-O-isopropylideneadenosine 9c gave N-1-carbocyclic derivative, which was deprotected to give 5'-5"-diol derivatives 18. When 18 was treated with POCl3 in PO(OEt)3, the bromo group at the 8-position was replaced to give N-1-carbocyclic-8-chloroadenosine 5',5"-diphosphate derivative 19 in 43% yield. Treatment of 19 with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride gave the desired intramolecular condensation product 20 in 10% yield. This is the first chemical construction of the 18-membered backbone structure containing an intramolecular pyrophosphate linkage of a cADPR-related compound with an adenine base.     

The structures of four macrolide antibiotics bound to the large ribosomal subunit

Crystal structures of the Haloarcula marismortui large ribosomal subunit complexed with the 16-membered macrolide antibiotics carbomycin A, spiramycin, and tylosin and a 15-membered macrolide, azithromycin, show that they bind in the polypeptide exit tunnel adjacent to the peptidyl transferase center. Their location suggests that they inhibit protein synthesis by blocking the egress of nascent polypeptides. The saccharide branch attached to C5 of the lactone rings extends toward the peptidyl transferase center, and the isobutyrate extension of the carbomycin A disaccharide overlaps the A-site. Unexpectedly, a reversible covalent bond forms between the ethylaldehyde substituent at the C6 position of the 16-membered macrolides and the N6 of A2103 (A2062, E. coli). Mutations in 23S rRNA that result in clinical resistance render the binding site less complementary to macrolides.     

Novel cyclourethane-derived HIV protease inhibitors: a ring-closing olefin metathesis based strategy

A series of novel macrocyclic urethanes incorporating a (R)-hydroxyethylamine isostere was designed and synthesized. Ring size and substituent efffects have been investigated. Cyclourethanes containing 14- to 16-membered rings exhibited low nanomolar inhibitory potencies against HIV-1 protease.     

Kinetics of binding of macrolides, lincosamides, and synergimycins to ribosomes

The synergistic effect of type A (virginiamycin M (VM)) and type B (virginiamycin S (VS)) synergimycins and their antagonistic effect against erythromycin (a 14-membered macrolide) for binding to the large ribosomal subunit (50 S) have been related. This investigation has now been extended to 16-membered macrolides (leucomycin A3 and spiramycin) and to lincosamides (lincomycin). A dissociation of VS-ribosome complexes was induced as well by 16-membered macrolides as by lincosamides. The observed dissociation rate constant of VS-ribosome complexes was identified with the kappa-vs in the case of 16-membered macrolides, but linearly related to lincomycin concentration, suggesting a direct binding of the latter antibiotic to VS-ribosome complexes and the triggering of a conformational change of particles entailing VS release. Two different mechanisms were also involved in the VM-promoted reassociation to ribosomes of VS previously displaced by either macrolides or lincosamides. By binding to lincosamide-ribosome complexes, VM induced a conformational change of ribosomes resulting in higher affinity for VS and lower affinity for lincosamides. On the contrary, an incompatibility for a simultaneous binding of VM and 16-membered macrolides to ribosomes was observed. These results have been interpreted by postulating specific (nonoverlapping) and aspecific (overlapping) antibiotic binding sites at the peptidyltransferase domain. All the kinetic constants of five antibiotic families (type A and B synergimycins, 14- and 16-membered macrolides, and lincosamides) and a topological model of peptidyltransferase are presently available.     

Structure-based design of cycloamide-urethane-derived novel inhibitors of human brain memapsin 2 (beta-secretase)

A series of novel macrocyclic amide-urethanes was designed and synthesized based upon the X-ray crystal structure of our lead inhibitor (1, OM99-2 with eight residues) bound to memapsin 2. Ring size and substituent effects have been investigated. Cycloamide-urethanes containing 14- to 16-membered rings exhibited low nanomolar inhibitory potencies against human brain memapsin 2 (beta-secretase).     

Correlation between structure of the lactones and substrate specificity in enzyme-catalyzed polymerization for the synthesis of polyesters

Small-size (4-membered) and medium-size (5-, 6-, and 7-membered) unsubstituted lactones as well as unsubstituted macrolides (12 and 13 membered) were subjected to the ring-opening polymerization using the extracellular PHB depolymerase from Alcaligenes faecalis T1 (PhaZ(Afa)). The characteristic reactivities of the lactones were discussed based on a tertiary structure model of the active site of the PhaZ(Afa). With respect to the ring-size of the lactones, the 4-membered beta-propiolactone and 6-membered delta-valerolactone (delta-VL) showed the highest polymerization activity, and delta-VL seemed to be the upper size limit for the molecular recognition of the narrow active site cleft of PhaZ(Afa). On the other hand, epsilon-caprolactone, 11-undecanolide, and 12-dodecanolide, which showed excellent polymerization activities by lipases, were scarcely polymerized by PhaZ(Afa). This was ascribed to the difference in the recognition sites between PhaZ(Afa) and lipase. In addition, the effect of the substrate-binding domain of PhaZ(Afa) and the enantioselective ring-opening polymerization of (R,S)-beta-butyrolactone ((R,S)-beta-BL) were studied. The substrate-binding domain lacking PhaZ(Afa) showed higher reactivities than PhaZ(Afa) for the polymerization of the lactones and that a significant enantioselectivity was observed at the early stage of the polymerization of (R,S)-beta-BL to produce the (R)-enriched optically active poly(3-hydroxybutyrate).     

Syntheses and bioactivities of macrocyclic paclitaxel bis-lactones

Five macrocyclic paclitaxel bis-lactones and their corresponding open chain taxoids were synthesized as models of the tubulin-binding conformation of paclitaxel. Macrocyclic lactones with a 19-21-membered ring underwent isomerization to form smaller rings. The lactones were evaluated for cytotoxicity and tubulin-polymerization ability. All five macrocyclic paclitaxel lactones were active, but less so than paclitaxel, while the rearranged macrocyclic lactones and the corresponding open-chain taxoids were much less active or inactive.     

Characteristic expression of three genes,msr(A), mph(C) and erm(Y), that confer resistance to macrolide antibiotics on Staphylococcus aureus

We have reported that the gene mph(C) (formally referred to as 'mphBM') is located on plasmid pMS97 342 bp downstream of the msr(A) gene. msr(A) specifies resistance to macrolides by ABC-transporter-mediated efflux, and mph(C) has 49% identity to the amino acid sequence of MPH(2')II, which encodes a phosphotransferase that inactivates some macrolide antibiotics. A strain of Staphylococcus aureus NCTC8325 containing plasmid pMS97 inactivated unlabeled and (14)C-labeled erythromycin when tested by bioautographic and radioautographic techniques. In addition to erythromycin, other 14-membered ring macrolides (except for ketolides), 15-membered ring macrolides and 16-membered ring macrolides, mycinamicin, rosamicin and YM133, were inactivated by the strain. Erythromycin inactivation products produced by the strain carrying pMS97 were completely different from those produced by Escherichia coli BM694 bearing plasmid pAT63, which contains the ereA gene encoding an esterase that hydrolyzes macrolide lactones. Constructs formed with the msr(A) and mph(C) genes, and with the msr(A), mph(C) and erm(Y) genes, showed erythromycin-inactivating activity, but another construct built with the mph(C) gene alone failed to show such activity. This result suggests that any region of the msr(A) gene is needed for the expression of mph(C).     

Design and synthesis of a potent macrocyclic 1,6-naphthyridine anti-human cytomegalovirus (HCMV) inhibitors

A novel class of macrocyclic 1,6-napthyridines designed to adopt the presumed bioactive conformation of anti-HCMV acyclic 1,6-napthyridines are described. Both 14- and 15-membered macrocycles were shown to be highly potent against HCMV HSV-1 and HSV-2.     

Novel potent macrocyclic inhibitors of the hepatitis C virus NS3 protease: use of cyclopentane and cyclopentene P2-motifs

Several highly potent novel HCV NS3 protease inhibitors have been developed from two inhibitor series containing either a P2 trisubstituted macrocyclic cyclopentane- or a P2 cyclopentene dicarboxylic acid moiety as surrogates for the widely used N-acyl-(4R)-hydroxyproline in the P2 position. These inhibitors were optimized for anti HCV activities through examination of different ring sizes in the macrocyclic systems and further by exploring the effect of P4 substituent removal on potency. The target molecules were synthesized from readily available starting materials, furnishing the inhibitor compounds in good overall yields. It was found that the 14-membered ring system was the most potent in these two series and that the corresponding 13-, 15-, and 16-membered macrocyclic rings delivered less potent inhibitors. Moreover, the corresponding P1 acylsulfonamides had superior potencies over the corresponding P1 carboxylic acids. It is noteworthy that it has been possible to develop highly potent HCV protease inhibitors that altogether lack the P4 substituent. Thus the most potent inhibitor described in this work, inhibitor 20, displays a K(i) value of 0.41 nM and an EC(50) value of 9 nM in the subgenomic HCV replicon cell model on genotype 1b. To the best of our knowledge this is the first example described in the literature of a HCV protease inhibitor displaying high potency in the replicon assay and lacking the P4 substituent, a finding which should facilitate the development of orally active small molecule inhibitors against the HCV protease.     

Gene expression profiles of human small airway epithelial cells treated with low doses of 14- and 16-membered macrolides

Although long-term treatment with low doses of 14-membered macrolides is widely applied in management of patients with chronic inflammatory diseases, e.g., diffuse panbronchiolitis, chronic bronchitis, or chronic lung damage in newborns, the physiological mechanisms underlying the action of macrolides in these conditions are unclear. To clarify the pathological basis of these diseases and also to aid in the design of novel drugs to treat them, we chose to investigate the molecular target(s) of macrolides. Our experiments involved long-term culture of human small airway epithelial cells (hSAEC) in media containing 14-membered macrolides erythromycin (EM) or clarithromycin (CAM), or a 16-membered macrolide, josamycin (JM), which lacks clinical anti-inflammatory effects. We then analyzed gene expression profiles in the treated cells using a cDNA microarray consisting of 18,432 genes. We identified nine genes whose expression was significantly altered during 22 days of culture with EM, and seven that were altered by CAM in that time. Four of those genes revealed similar behavior in cells treated with either of the 14-membered macrolides, but not JM. The products of these four genes may be candidates for mediating the ability of 14-membered macrolides to suppress chronic inflammation.     

P1, P2'-linked macrocyclic amine derivatives as matrix metalloproteinase inhibitors.

A novel series of 13- and 14-membered macrocyclic amines was developed by linking the P1 and P2' groups. The synthesis entails stereoselective Frater alkylation to install the anti-succinate configuration and macrocyclic amination via nucleophilic displacement. This strategy resulted in a new class of conformationally constrained inhibitors that are potent and selective for MMP-8 and 9 over MMP-1 and 3.     

1H-, 13C-, 31P-NMR studies and conformational analysis of NADP+, NADPH coenzymes and of dimers from electrochemical reduction of NADP+.

All H,H, H,P and several C,P coupling constants, including those between C-4' and the vicinal phosphorus atom, have been determined for NADP+, NADPH coenzymes and for a 4,4-dimer obtained from one-electron electrochemical reduction of NADP+. From these data the preferred conformation of the ribose, that of the 1,4-dihydronicotinamide rings, and the conformation about bonds C(4')-C(5') and C(5')-O(5') were deduced. The preferred form of the 1,4- and 1,6-dihydropyridine rings and the conformation about the ring-ring junction were also obtained for all the other 4,4- and 4,6-dimers formed in the same reduction. All the dimers show a puckered structure, i.e., a boat form for the 1,4- and a twist-boat for the 1,6-dihydronicotinamide ring; both protons at the ring-ring junctions are equatorial and have preferred gauche orientation. On the contrary, the reduced coenzyme NADPH displays a planar or highly flexible conformation, rapidly flipping between two limiting boat structures. The conformation of the ribose rings, already suggested for the NADP coenzymes to be an equilibrium mixture of C(2')-endo (S-type) and C(3')-endo (N-type) puckering modes, has been reexamined by using the Altona procedure and the relative proportion of the two modes has been obtained. The S and N families of conformers have almost equal population for the adenine-ribose, whereas for the nicotinamide-ribose rings the S-type reaches the 90%. The rotation about the ester bond C(5')-O(5') and about C(4')-C(5'), defined by torsion angles beta and gamma respectively, displays a constant high preference for the trans conformer beta t (75-80%), whereas the rotamers gamma are spread out in a range of different populations. The values are distributed between the gauche gamma + (48-69%) and the trans gamma t forms (28-73%). The gamma + conformer reaches a 90% value in the case of NADP+ and NMN+. The conformations of the mononucleotides 5'-AMP, NMN+ and NMNH were also calculated from the experimental coupling constant values of the literature.     

Synthesis and biological activity of macrocyclic inhibitors of hepatitis C virus (HCV) NS3 protease

The 17-membered phenylalanine-based macrocycle 6 was prepared starting from 3-iodo-phenylalanine. Macrocyclization of alkene phenyl iodide 5 was effected through a palladium-catalyzed Heck reaction. The macrocyclic alpha-ketoamides were active inhibitors of the HCV NS3 protease, with the C-terminal acids and amides being more potent than tert-butyl esters.     

Novel desosaminyl derivatives of dihydrochalcomycin from a genetically engineered strain of <Emphasis Type="Italic">Streptomyces</Emphasis> sp.

Dihydrochalcomycin from Streptomyces sp. KCTC 0041BP is a 16-membered macrolide antibiotic containing two deoxysugars (d-chalcose and d-mycinose) that are O-glycosylated at the C-5 and C-20 positions, respectively. The desosamine sugar cassette was constructed from pikromycin-deoxysugar biosynthetic genes and transformed into Streptomyces sp. GerSM1, which was engineered for deletion of the genes related to TDP-d-chalcose biosynthesis (gerB, gerN and gerMI). Novel 16-membered macrolides (5-O-desosaminyl derivatives of dihydrochalcomycin) were detected by ESI-MS, LC/MS, and MS/MS thereby demonstrating combinatorial biosynthesis of the deoxysugar in 16-membered macrolide antibiotics.