Therapies from Thiopeptides.

The first part of this contribution describes solutions that were developed to achieve progressively more efficient syntheses of the thiopeptide natural products, micrococcins P1 and P2 (MP1-MP2), with an eye toward exploring their potential as a source of new antibiotics. Such efforts enabled investigations on the medicinal chemistry of those antibiotics, and inspired the development of the kinase inhibitor, Masitinib®, two candidate oncology drugs, and new antibacterial agents. The studies that produced such therapeutic resources are detailed in the second part. True to the theme of this issue, "Organic Synthesis and Medicinal Chemistry: Two Inseparable Partners", an important message is that the above advances would have never materialized without the support of curiosity-driven, academic synthetic organic chemistry: a beleaguered science that nonetheless has been-and continues to be-instrumental to progress in the biomedical field.

Identification of Micrococcin P2-Derivatives as Antibiotic Candidates against Two Gram-Positive Pathogens.

Thiopeptides exhibit potent antimicrobial activity against Gram-positive pathogens by inhibiting bacterial protein synthesis. Micrococcins are among the structurally simpler thiopeptides, but they have not been exploited in detail. This research involved a computational simulation of micrococcin P2 (MP2) docking in parallel with the structure-activity relationship (SAR) studied. The incorporation of particular nitrogen heterocycles in the side chain of MP2 enhances the antimicrobial activity. Micrococcin analogues 6c and 6d thus proved to be more effective against impetigo and C. difficile infection (CDI), respectively, as compared to current first-line treatments. Compound 6c also showed a shorter treatment period than that of a first-line treatment for impetigo. This may be attributed to its ability to downregulate pro-inflammatory cytokines. Compound 6d had no observed recurrence for C. difficile and exerted a minimal impact on the beneficial gut microbiome. Their pharmacokinetic properties and low toxicity profile make these compounds ideal candidates for the treatment of impetigo and CDI and validate their involvement in preclinical development.

From a drug repositioning to a structure-based drug design approach to tackle acute lymphoblastic leukemia.

Cancer cells utilize the main de novo pathway and the alternative salvage pathway for deoxyribonucleotide biosynthesis to achieve adequate nucleotide pools. Deoxycytidine kinase is the rate-limiting enzyme of the salvage pathway and it has recently emerged as a target for anti-proliferative therapies for cancers where it is essential. Here, we present the development of a potent inhibitor applying an iterative multidisciplinary approach, which relies on computational design coupled with experimental evaluations. This strategy allows an acceleration of the hit-to-lead process by gradually implementing key chemical modifications to increase affinity and activity. Our lead compound, OR0642, is more than 1000 times more potent than its initial parent compound, masitinib, previously identified from a drug repositioning approach. OR0642 in combination with a physiological inhibitor of the de novo pathway doubled the survival rate in a human T-cell acute lymphoblastic leukemia patient-derived xenograft mouse model, demonstrating the proof-of-concept of this drug design strategy.

Losartan metabolite EXP3179 is a unique blood pressure-lowering AT1R antagonist with direct, rapid endothelium-dependent vasoactive properties.

BACKGROUND AND PURPOSE: Losartan is an anti-hypertensive angiotensin II (ANGII) type 1 receptor (AT1R) blocker (ARB) with many unexpected therapeutic properties, even in non-blood pressure (BP)-related diseases. Administered as a prodrug, losartan undergoes serial metabolism into EXP3179, a metabolite alleged to lack AT1R-blocking properties, and EXP3174, the dominant AT1R antagonist. Having observed that losartan can decrease vascular tone in mice with low AT1R expression and inhibit Marfan aortic widening at very high doses, we investigated whether EXP3179 may have unique, AT1R-independent effects on vascular tone and endothelial function. EXPERIMENTAL APPROACH: We compared the AT1R blocking capabilities of EXP3179 and EXP3174 using AT1R-expressing cell lines. Their BP lowering and vasoactive properties were studied in normal, hypertensive and transgenic rodents, and ex vivo wire myography. KEY RESULTS: We observed that both EXP3179 and EXP3174 can fully block (100%) AT1R signaling in vitro and significantly decrease BP in normotensive and spontaneously hypertensive rats. Only EXP3179 prevented PE-induced contraction by up to 65% (p < 0.01) in L-NAME and endothelium removal-sensitive fashion. Use of transgenic mice revealed that these effects involve the eNOS/caveolin-1 axis and the endothelium-dependent hyperpolarization factor (EDHF). CONCLUSION AND IMPLICATIONS: We provide direct structure-activity evidence that EXP3179 is a BP-lowering AT1R blocker with unique endothelial function-enhancing properties not shared with losartan or EXP3174. The major pharmacological effects of losartan in patients are therefore likely more complex than simple blockade of AT1R by EXP3174, which helps rationalize its therapeutic and prophylactic properties, especially at very high doses. Reports relying on EXP3179 as an AT1R-independent losartan analogue may require careful re-evaluation.

Micrococcin P2 Targets Clostridioides difficile.

Clostridioides difficile infection is a global public health threat. Extensive in vitro assays using clinical isolates have identified micrococcin P2 (MP2, 1) as a particularly effective anti-C. difficile agent. MP2 possesses a mode of action that differs from other antibiotics and pharmacokinetic properties that render it especially promising. Its time-kill studies have been investigated using hypervirulent C. difficile ribotype 027. DSS (dextran sulfate sodium)-induced in vivo mouse studies with that strain indicate that 1 is better than vancomycin and fidaxomicin. Thus, micrococcin P2 is a valuable platform to be exploited for the development of new anti-C. difficile antibiotics.

A Route to Lipid ALC-0315: a Key Component of a COVID-19 mRNA Vaccine.

This paper describes a synthesis of ALC-0315 by a sequence that more than doubles the overall yield relative to the published one, and that employs much cleaner reactions, thereby facilitating purifications to a considerable extent.

Nitro-Group-Containing Thiopeptide Derivatives as Promising Agents to Target Clostridioides difficile.

The US Centers for Disease Control and Prevention (CDC) lists Clostridioides difficile as an urgent bacterial threat. Yet, only two drugs, vancomycin and fidaxomicin, are approved by the FDA for the treatment of C. difficile infections as of this writing, while the global pipeline of new drugs is sparse at best. Thus, there is a clear and urgent need for new antibiotics against that organism. Herein, we disclose that AJ-024, a nitroimidazole derivative of a 26-membered thiopeptide, is a promising anti-C. difficile lead compound. Despite their unique mode of action, thiopeptides remain largely unexploited as anti-infective agents. AJ-024 combines potent in vitro activity against various strains of C. difficile with a noteworthy safety profile and desirable pharmacokinetic properties. Its time-kill kinetics against a hypervirulent C. difficile ribotype 027 and in vivo (mouse) efficacy compare favorably to vancomycin, and they define AJ-024 as a valuable platform for the development of new anti-C. difficile antibiotics.

Diversity-oriented routes to thiopeptide antibiotics: total synthesis and biological evaluation of micrococcin P2.

We report the first total synthesis of micrococcin P2 (MP2, 1) by a diversity-oriented route that incorporates a number of refinements relative to earlier syntheses. Biological data regarding the activity of 1 against a range of human pathogens are also provided. Furthermore, we disclose a chemical property of MP2 that greatly facilitates medicinal chemistry work in the micrococcin area and describe a method to obtain MP2 by fermentation in B. subtilis.

Nitric oxide in the Marfan vasculature: Friend or foe?

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the FBN1 gene, which encodes fibrillin-1, a protein essential for the formation and stabilization of elastic fibers as well as signaling homeostasis. Progressive aortic root widening is the most serious manifestation of MFS as it can lead to aortic dissection, aneurysm formation and rupture. However, despite their ability to decrease the hemodynamic stress the aorta is subjected to, anti-hypertensive medications often lead to underwhelming reductions in the rate of aortic root dilation, which illustrates how fragmental our understanding of MFS-associated aortic remodeling is. This manuscript summarizes recent evidence that document nitric oxide (NO) synthase (NOS)-related changes to the vasculature during the pathogenesis of MFS and how they result in a unique state of vascular dysfunction that likely plays a causal role in the aortic root widening process. We also review how clinic-approved and experimental therapies as well lifestyle approaches may promote aortic root stability by correcting NO homeostasis, which if properly optimized may improve outcomes in this population afflicted by a notoriously refractory type of aortopathy.

Modular Lipid Nanoparticle Platform Technology for siRNA and Lipophilic Prodrug Delivery.

Successfully employing small interfering RNA (siRNA) therapeutics requires the use of nanotechnology for efficient intracellular delivery. Lipid nanoparticles (LNPs) have enabled the approval of various nucleic acid therapeutics. A major advantage of LNPs is the interchangeability of its building blocks and RNA payload, which allow it to be a highly modular system. In addition, drug derivatization approaches can be used to synthesize lipophilic small molecule prodrugs that stably incorporate in LNPs. This provides ample opportunities to develop combination therapies by co-encapsulating multiple therapeutic agents in a single formulation. Here, it is described how the modular LNP platform is applied for combined gene silencing and chemotherapy to induce additive anticancer effects. It is shown that various lipophilic taxane prodrug derivatives and siRNA against the androgen receptor, a prostate cancer driver, can be efficiently and stably co-encapsulated in LNPs without compromising physicochemical properties or gene-silencing ability. Moreover, it is demonstrated that the combination therapy induces additive therapeutic effects in vitro. Using a double-radiolabeling approach, the pharmacokinetic properties and biodistribution of LNPs and prodrugs following systemic administration in tumor-bearing mice are quantitatively determined. These results indicate that co-encapsulating siRNA and lipophilic prodrugs into LNPs is an attractive and straightforward plug-and-play approach for combination therapy development.

Catalyst-Free Synthesis of Polysubstituted 5-Acylamino-1,3-Thiazoles via Hantzsch Cyclization of α-Chloroglycinates.

A catalyst-free heterocyclization reaction of α-chloroglycinates with thiobenzamides or thioureas leading to 2,4-disubstituted-5-acylamino-1,3-thiazoles has been developed. The methodology provides straightforward access to valuable building blocks for pharmaceutically relevant compounds.

Dexamethasone prodrugs as potent suppressors of the immunostimulatory effects of lipid nanoparticle formulations of nucleic acids.

Lipid nanoparticles (LNPs) are playing a leading role in enabling clinical applications of gene therapies based on DNA or RNA polymers. One factor impeding clinical acceptance of LNP therapeutics is that LNP formulations of nucleic acid polymers can be immunostimulatory, necessitating co-administration of potent corticosteroid immunosuppressive agents. Here, we describe the development of hydrophobic prodrugs of a potent corticosteroid, dexamethasone, that can be readily incorporated into LNP systems. We show that the presence of the dexamethasone prodrug LD003 effectively suppresses production of cytokines such as KC-GRO, TNFα, IL-1ß and IL-6 following intravenous administration of LNP loaded with immune stimulatory oligodeoxynucleotides containing cytosine-guanine dinucleotide motifs. Remarkably, LD003 dose levels corresponding to 0.5 mg/kg dexamethasone achieve a greater immunosuppressive effect than doses of 20 mg/kg of free dexamethasone. Similar immunosuppressive effects are observed for subcutaneously administered LNP-siRNA. Further, the incorporation of low levels of LD003 in LNP containing unmodified mRNA or plasmid DNA significantly reduced pro-inflammatory cytokine levels following intravenous administration. Our results suggest that incorporation of hydrophobic prodrugs such as LD003 into LNP systems could provide a convenient method for avoiding the immunostimulatory consequences of systemic administration of genetic drug formulations.

Asymmetric Oxidative Cycloetherification of Naphtholic Alcohols.

The catalytic, enantioselective oxidative cyclization of naphthol-derived carboxylic acids mediated by chiral hypervalent iodine reagents has been studied extensively in the recent past, but analogous reactions of non-carboxylic substrates are yet unknown. This paper describes a catalytic, enantioselective, hypervalent iodine-promoted oxidative cycloetherification reaction of naphtholic alcohols. The new process relies on a variant of the Uyanik-Ishihara catalyst, in which the stereogenic centers have been relocated closer to the iodine atom. The new catalyst design affords optical yields comparable to those available with Uyanik-Ishihara iodides, but chemical yields are sensibly higher, at least with the tests substrates. An even more problematic reaction is the catalytic, enantioselective oxidative cyclization of naphtholic sulfonamides. In this case, the new catalyst affords significantly higher optical inductions than Uyanik-Ishihara iodides. The kinetic resolution of particular naphtholic alcohols is demonstrated. The absolute configuration of a numver of enantioenriched compounds obtained in this study was ascertained by X-ray diffractometry.

The Niemann-Pick C1 Inhibitor NP3.47 Enhances Gene Silencing Potency of Lipid Nanoparticles Containing siRNA.

The therapeutic applications of lipid nanoparticle (LNP) formulations of small interfering RNA (siRNA), are hampered by inefficient delivery of encapsulated siRNA to the cytoplasm following endocytosis. Recent work has shown that up to 70% of endocytosed LNP-siRNA particles are recycled to the extracellular medium and thus cannot contribute to gene silencing. Niemann-Pick type C1 (NPC1) is a late endosomal/lysosomal membrane protein required for efficient extracellular recycling of endosomal contents. Here we assess the influence of NP3.47, a putative small molecule inhibitor of NPC1, on the gene silencing potency of LNP-siRNA systems in vitro. Intracellular uptake and colocalization studies revealed that the presence of NP3.47 caused threefold or higher increases in accumulation of LNP-siRNA in late endosomes/lysosomes as compared with controls in a variety of cell lines. The gene silencing potency of LNP siRNA was enhanced up to fourfold in the presence of NP3.47. Mechanisms of action studies are consistent with the proposal that NP3.47 acts to inhibit NPC1. Our findings suggest that the pharmacological inhibition of NPC1 is an attractive strategy to enhance the therapeutic efficacy of LNP-siRNA by trapping LNP-siRNA in late endosomes, thereby increasing opportunities for endosomal escape.

Total Synthesis of (+)-3-Demethoxyerythratidinone and (+)-Erysotramidine via the Oxidative Amidation of a Phenol.

Oxidative amidation chemistry provides a unified route to aromatic Erythrina alkaloids through a sequence that illustrates new principles and improved conditions to effect a crucial eliminative Curtius-Schmidt rearrangement.

Pyrimethamine Derivatives: Insight into Binding Mechanism and Improved Enhancement of Mutant ß-N-acetylhexosaminidase Activity.

In order to identify structural features of pyrimethamine (5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine) that contribute to its inhibitory activity (IC50 value) and chaperoning efficacy toward ß-N-acetylhexosaminidase, derivatives of the compound were synthesized that differ at the positions bearing the amino, ethyl, and chloro groups. Whereas the amino groups proved to be critical to its inhibitory activity, a variety of substitutions at the chloro position only increased its IC50 by 2-3-fold. Replacing the ethyl group at the 6-position with butyl or methyl groups increased IC50 more than 10-fold. Surprisingly, despite its higher IC50, a derivative lacking the chlorine atom in the para-position was found to enhance enzyme activity in live patient cells a further 25% at concentrations >100 µM, while showing less toxicity. These findings demonstrate the importance of the phenyl group in modulating the chaperoning efficacy and toxicity profile of the derivatives.

Formal Synthesis of (±)-Tetrodotoxin via the Oxidative Amidation of a Phenol: On the Structure of the Sato Lactone.

A formal total synthesis of (±)-tetrodotoxin that relies on the bimolecular oxidative amidation of a phenol is described, and a structural correction of the Sato lactone, an important tetrodotoxin intermediate, is provided. This work lays the foundation for an ultimate enantioselective synthesis.

A route to the heterocyclic cluster of the E-series of thiopeptide antibiotics.

A concise route to the 3-hydroxypyridine core of thiopeptide antibiotics such as nocathiacin is described. Key phases of the sequence involve a modified Hantzsch pyridine construction and a chemoselective Peng deprotection of a phenolic MOM ether.

Arylation of diorganochalcogen compounds with diaryliodonium triflates: metal catalysts are unnecessary.

Diaryliodonium triflates transfer an aryl group to the chalcogen atom of organic sulfides, selenides, and tellurides (but not ethers), in the absence of transition-metal catalyst, simply upon heating in chloroform or dichloroethane solution.

Iodonium metathesis reactions.

A metathesis reaction occurs when a diaryliodonium triflate is heated with an aryl iodide, resulting in the formation of a new diaryliodonium triflate.