Novel candidates in the clinical development pipeline for TB drug development and their synthetic approaches.

Tuberculosis (TB) is an infection caused by Mycobacterium tuberculosis (Mtb) and one of the deadliest infectious diseases in the world. Mtb has the ability to become dormant within the host and to develop resistance. Hence, new antitubercular agents are required to overcome problems in the treatment of multi-drug-resistant Tb (MDR-Tb) and extensively drug-resistant Tb (XDR-Tb) along with shortening the treatment time. Several efforts are being made to develop very effective new drugs for Tb, within the pharmaceutical industry, the academia and through public-private partnerships. This review will address the antitubercular activities, biological target, mode of action, synthetic approaches and thoughtful concept for the development of several new drugs currently in the clinical trial pipeline (up to October 2019) for tuberculosis. The aim of this review may be very useful in scheming new chemical entities (NCEs) for Mtb.

Synthesis and Evaluation of Some Uracil Nucleosides as Promising Anti-Herpes Simplex Virus 1 Agents.

Since herpes simplex virus type 1 (HSV-1) infection is so widespread, several antiviral drugs have been developed to treat it, among which are uracil nucleosides. However, there are major problems with the current medications such as severe side-effects and drug resistance. Here we present some newly synthesized cyclic and acyclic uracil nucleosides that showed very promising activity against HSV-1 compared to acyclovir.

SynBio-SynChem Approaches to Diversifying the Pacidamycins through the Exploitation of an Observed Pictet-Spengler Reaction.

A nonenzymatic Pictet-Spengler reaction has been postulated to give rise to a subset of naturally occurring uridyl peptide antibiotics (UPAs). Here, using a combination of strain engineering and synthetic chemistry, we demonstrate that Pictet-Spengler chemistry may be employed to generate even greater diversity in the UPAs. We use an engineered strain to afford access to meta-tyrosine containing pacidamycin 4. Pictet-Spengler diversification of this compound using a small series of aryl-aldehydes was achieved with some derivatives affording remarkable diastereomeric control.

Synthesis and Conformational Analysis of Fluorinated Uridine Analogues Provide Insight into a Neighbouring-Group Participation Mechanism.

Fluorinated nucleoside analogues have attracted much attention as anticancer and antiviral agents and as probes for enzymatic function. However, the lack of direct synthetic methods, especially for 2',3'-dideoxy-2',3'-difluoro nucleosides, hamper their practical utility. In order to design more efficient synthetic methods, a better understanding of the conformation and mechanism of formation of these molecules is important. Herein, we report the synthesis and conformational analysis of a 2',3'-dideoxy-2',3'-difluoro and a 2'-deoxy-2'-fluoro uridine derivative and provide an insight into the reaction mechanism. We suggest that the transformation most likely diverges from the SN1 or SN2 pathway, but instead operates via a neighbouring-group participation mechanism.

Design and Synthesis of Various 5'-Deoxy-5'-(4-Substituted-1,2,3-Triazol-1-yl)-Uridine Analogues as Inhibitors of Mycobacterium tuberculosis Mur Ligases.

The synthesis of hitherto unknown 5'-deoxy-5'-(4-substituted-1,2,3-triazol-1-yl)-uridine and its evaluation, through an one-pot screening assay, against MurA-F enzymes involved in Mycobacterium tuberculosis (Mtb), are described. Starting from UDP-N-acetylmuramic acid (UDP-MurNAc), the natural substrate involved in the peptidoglycan biosynthesis, our strategy was to substitute the diphosphate group of UDP-MurNAc by a 1,2,3-triazolo spacer under copper-catalyzed azide-alkyne cycloaddition conditions. The structure-activity relationship was discussed and among the 23 novel compounds developed, N-acetylglucosamine analogues 11c and 11e emerged as the best inhibitors against the Mtb MurA-F enzymes reconstruction pathway with an inhibitory effect of 56% and 50%, respectively, at 100 µM. Both compounds are selective inhibitors of Mtb MurE, the molecular docking and molecular dynamic simulation suggesting that 11c and 11e are occupying the active site of Mtb MurE ligase.

Uridine natural products: Challenging targets and inspiration for novel small molecule inhibitors.

Nucleoside derivatives, in particular those featuring uridine, are familiar components of the nucleoside family of bioactive natural products. The structural complexity and biological activities of these compounds have inspired research from organic chemistry and chemical biology communities seeking to develop novel approaches to assemble the challenging molecular targets, to gain inspiration for enzyme inhibitor development and to fuel antibiotic discovery efforts. This review will present recent case studies describing the total synthesis and biosynthesis of uridine natural products, and de novo synthetic efforts exploiting features of the natural products to produce simplified scaffolds. This research has culminated in the development of complementary strategies that can lead to effective uridine-based inhibitors and antibiotics. The strengths and challenges of the juxtaposing methods will be illustrated by examining select uridine natural products. Moreover, structure-activity relationships (SAR) for each natural product-inspired scaffold will be discussed, highlighting the impact on inhibitor development, with the aim of future uridine-based small molecule expansion.

Synthesis of 2'-deoxy-2'-fluoro-2'-C-methyl spiro cyclopentyl carbocyclic uridine analog as potential inhibitors of HCV NS5B polymerase.

Synthesis of 1-((4 R,5S,6R,7R)-5,6-dihydroxy-7-(hydroxymethyl)spiro[2.4]heptan-4-yl)pyrimidine-2,4(1H,3H)-dione (12) and its phosphoramidate prodrug 18 is reported. The synthesis of the targeted compound 12 was initiated from triol 1. By the introduction of a substituent methylene group at 6-position of 4, followed by Simmons-Smith cyclopropanation and amination, key intermediate 10 was synthesized. The intermediate amine 10 was utilized to synthesize the nucleoside 12. Furthermore, the nucleoside 12 was derivatized to 2'-α-hydroxy-2'-ß-methyl (23) and 2'-α-fluoro-2'-ß-methyl (27) analogs. All synthesized derivatives of spiro-cyclopropyl carbocyclic uridine analogs 12, 18, 23 and 27 were evaluated for anti-HCV activity, but none of the compounds, reported in this article show any anti-HCV activity.

Synthesis of 2'-aminouridine derivatives as an organocatalyst for Diels-Alder reaction.

To develop a novel asymmetric organocatalyst based on a ribonucleoside skeleton, we designed and synthesized 2'-aminouridine derivatives. The synthesized 2'-aminouridines having bulky substituents at both base and sugar moieties could catalyze the Diels-Alder reaction between cinnamaldehyde and cyclopentadiene. However, the optical purities of the resulting products were unexpectedly low.

An alternative method for the synthesis of 2'-halogeno-1',2'-unsaturated uridine derivatives through syn-elimination of pivalic acid of 2'-halogeno- 2'-deoxy-1'-pivaloyloxyuracil nucleoside: preparation of its 2'-C-branched nucleosides.

An alternative method for the preparation of 2'-bromo- (5b) and 2'-iodo- (5c) 1',2'-unsaturated uracil nucleosides has been developed. The protocol was on the basis of the syn-elimination of pivalic acid from 2'-bromo-(7a,b) and 2'-iodo-(9a,b) 1'-pivaloyloxy-2'-deoxyuridine derivatives, which were derived from the halo-pivaloyloxylation of 3',5'-bis-O-TBDMS-1',2'-unsaturated uridine 1. Compounds 5b and 5c were shown to serve as versatile synthons for the respective 2'-C-branched 1',2'-unsaturated uracil nucleosides, through palladium-catalyzed cross-coupling or halogen-lithium exchange reactions.

Practical and reliable synthesis of 2',3',5',5″-tetradeuterated uridine derivatives.

Deuterated drugs are valuable in the fields of drug discovery and medicinal chemistry. 2',3',5',5″-tetradeuterated uridine derivatives were synthesised from 2,3,5,5'-selectively tetradeuterated ribose using Sajiki's H-D exchanged Ru/C-H2-D2O-NaOH system and silyl-Hilbert-Johnson methods. The total deuterium content of the tetradeuterated uridines was over 92% using either basic or acidic reaction conditions. These derivatives would be expected as building blocks for the synthesis of deuterium-substituted nucleic acid probes for tracking the pharmacokinetics of nucleic acid drugs.

Oxazinomycin arrests RNA polymerase at the polythymidine sequences.

Oxazinomycin is a C-nucleoside antibiotic that is produced by Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring.

Synthesis of 4'/5'-Spirocyclopropanated Uridine and d-Xylouridine Derivatives and Their Activity against the Human Respiratory Syncytial Virus.

The Simmons-Smith-Furukawa reaction was used to generate 4'/5'-spirocyclopropanated uridine analogs from electron-rich exocyclic enol esters. During synthesis, the native hydroxylation pattern of the nucleoside is preserved and offers the possibility for a late stage 5'-phosphorylation at the spirocyclopropanol moiety. All synthesized spirocyclopropanated uridine derivatives, including the corresponding 5'-monophosphate (cpUMP), were evaluated with respect to their antiviral activity in an HRSV assay showing moderate, but promising activity.

Synthesis of 5-Dihydroxyboryluridine Phosphoramidite and Its Site-Specific Incorporation into Oligonucleotides for Probing Thymine DNA Glycosylase.

A concise synthetic strategy to 5-dihydroxyboryldexoyuridine (5boU) phosphoramidite has been developed. 5boU was introduced into short oligonucleotides in a site-specific manner, demonstrating compatibility of the boronic acid moiety with standard solid-phase DNA synthesis chemistry. Electrophilic 5boU DNAs inhibited thymine DNA glycosylase, a cancer-relevant DNA-modifying enzyme. We envisage diverse applications of 5boU in organic synthesis, medicinal chemistry, and chemical biology.

Total Synthesis of Caprazamycin A: Practical and Scalable Synthesis of syn-ß-Hydroxyamino Acids and Introduction of a Fatty Acid Side Chain to 1,4-Diazepanone.

The first total synthesis of caprazamycin A (1), a representative liponucleoside antibiotic, is described. Diastereoselective aldol reactions of aldehydes 12 and 25-27, derived from uridine, with diethyl isocyanomalonate 13 and phenylcarbamate 21 were investigated using thiourea catalysts 14 or bases to synthesize syn-ß-hydroxyamino acid derivatives. The 1,4-diazepanone core of 1 was constructed using a Mitsunobu reaction, and the fatty acid side chain was introduced using a stepwise sequence based on model studies. Notably, global deprotection was realized using palladium black and formic acid without hydrogenating the olefin in the uridine unit.

Synthesis and Anti-HCV Activities of 4'-Fluoro-2'-Substituted Uridine Triphosphates and Nucleotide Prodrugs: Discovery of 4'-Fluoro-2'- C-methyluridine 5'-Phosphoramidate Prodrug (AL-335) for the Treatment of Hepatitis C Infection.

We report the synthesis and biological evaluation of a series of 4'-fluoro-2'- C-substituted uridines. Triphosphates of the uridine analogues exhibited a potent inhibition of hepatitis C virus (HCV) NS5B polymerase with IC50 values as low as 27 nM. In an HCV subgenomic replicon assay, the phosphoramidate prodrugs of these uridine analogues demonstrated a very potent activity with EC50 values as low as 20 nM. A lead compound AL-335 (53) demonstrated high levels of the nucleoside triphosphate in vitro in primary human hepatocytes and Huh-7 cells as well as in dog liver following a single oral dose. Compound 53 was selected for the clinical development where it showed promising results in phase 1 and 2 trials.

Synthesis and Biophysical Characterization of RNAs Containing ( R)- and ( S)-5'- C-Aminopropyl-2'- O-methyluridines.

We designed and synthesized ( R)-5'- C-aminopropyl-2'- O-methyluridine and ( S)-5'- C-aminopropyl-2'- O-methyluridine, which are applicable to small interfering RNAs (siRNAs). We have evaluated the properties of siRNAs containing ( R)-5'- C-aminopropyl-2'- O-methyl and ( S)-5'- C-aminopropyl-2'- O-methyl modifications and have compared them to that of the 4'- C-aminopropyl-2'- O-methyl modification. Although these modifications decreased the thermal stability of double-stranded RNAs (dsRNAs) and siRNAs, the dsRNA containing the ( S)-5'- C-aminopropyl-2'- O-methyl modification showed the highest melting temperature ( Tm) among them. Silencing activity of the modified siRNAs was assessed by a dual luciferase reporter assay using HeLa cells. Incorporation of the ( R)-5'- C-aminopropyl-2'- O-methyl and ( S)-5'- C-aminopropyl-2'- O-methyl modifications on a passenger and guide strand was found to be tolerated for the silencing activity of siRNAs except for in the seed region on the guide strand. Furthermore, these modifications significantly increased the stability of single-stranded RNAs (ssRNAs) and siRNAs in a buffer containing bovine serum.

Semisynthesis of an Anticancer DPAGT1 Inhibitor from a Muraymycin Biosynthetic Intermediate.

We have explored a method to convert a muraymycin biosynthetic intermediate 3 to an anticancer drug lead 2 for in vivo and thorough preclinical studies. Cu(OAc)2 forms a stable complex with the amide 4 and prevents electrophilic reactions at the 2-((3-aminopropyl)amino)acetamide moiety. Under the present conditions, the desired 5″-primary amine was selectively protected with (Boc)2O to yield 6. The intermediate 6 was converted to 2 in two steps with 90% yield.

Cyano Modification on Uridine Decreases Base-Pairing Stability and Specificity through Neighboring Disruption in RNA Duplex.

5-Cyanomethyluridine (cnm5 U) and 5-cyanouridine (cn5 U), the two uridine analogues, were synthesized and incorporated into RNA oligonucleotides. Base-pairing stability and specificity studies in RNA duplexes indicated that cnm5 U slightly decreased the stability of the duplex but retained the base-pairing preference. In contrast, cn5 U dramatically decreased both base-pairing stability and specificity between U:A and other noncanonical U:G, U:U, and U:C pairs. In addition, the cn5 U:G pair was found to be stronger than the cn5 U:A pair and the other mismatched pairs in the context of a RNA duplex; this implied that cn5 U might slightly prefer to recognize G over A. Our mechanistic studies by molecular simulations showed that the cn5 U modification did not directly affect the base pairing of the parent nucleotide; instead, it weakened the neighboring base pair in the 5' side of the modification in the RNA duplexes. Consistent with the simulation data, replacing the Watson-Crick A:U pair to a mismatched C:U pair in the 5'-neighboring site did not affect the overall stability of the duplex. Our work reveals the significance of the electron-withdrawing cyano group in natural tRNA systems and provides two novel building blocks for constructing RNA-based therapeutics.

Preparation and characterization of pyrene modified uridine derivatives as potential electron donors in RNA.

Charge transfer across double stranded DNA was observed for the first time about 20 years ago, and ever since it has been the subject of a large number of studies. RNA has been hardly investigated in this regard, which not least is due to the lack of suitably functionalized ribonucleotide building blocks to serve as electron sources upon incorporation into oligoribonucleotides. We have synthesized two uridine derivatives carrying pyrene or dimethylaminopyrene linked to C5 of the nucleobase. The key to successful synthesis was the adaptation of Suzuki-Miyaura conditions to the coupling of the pyrene moiety with the ribonucleoside. Final decoration of the pyrenylated nucleosides with standard 5'-O- and 2'-O-protecting groups and subsequent 3'-O-phosphitylation delivered the building blocks for incorporation into RNA. Spectroscopic analysis of the two pyrenylated uridines and of the accordingly modified oligonucleotides showed that in particular the dimethyaminopyrene functionalized nucleoside is a promising candidate as an electron source for RNA charge transport studies.

Synthesis and Antisense Properties of 2'ß-F-Arabinouridine Modified Oligonucleotides with 4'-C-OMe Substituent.

A novel 2'-F,4'-C-OMe⁻arabinouridine (araU) was successfully synthesized and introduced into oligonucleotides. The oligonucleotide containing 2'-F,4'-C-OMe⁻araU exhibited improved nuclease resistance and RNA hybridizing selective ability relative to 2'-F⁻araU. In particular, when 2'-F,4'-C-OMe⁻araU inserted into C⁻H⋯F⁻C bonding-favorable 5'⁻uridine⁻purine⁻3' steps, the modified oligonucleotide showed remarkable binding affinity and selectivity to RNA complements. Thus, 2'-F,4'-C-OMe⁻araU has valuable antisense properties and can be used as novel chemical modification for antisense therapeutic strategy.