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We reported earlier that oseltamivir (Osm, Tamiflu®), an anti-viral agent, inhibits the attachment of trophozoites to intestinal epithelial cells and cyst production in culture. Osm also disassembles lipid rafts (LRs) and the biogenesis of extracellular vesicles (EVs) by Giardia. In the current study, we synthesized forty-one Osm analogs with the derivatization of oseltamivir phosphate. These compounds were tested on giardial growth, and cyst production. Of these, four analogs, i.e., compounds 3b, 2c, 11i, and 11d, were found to have superior activities against trophozoites and cysts compared to the parent oseltamivir. Investigation of the mechanism(s) of action of these agents are in progress and will be reported in due course., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Inspired by our previous finding that targeting the 150-cavity with a multisite-binding strategy emerged as an effective approach to obtain more potent and selective neuraminidase (NA) inhibitors against influenza virus, we present here the design, synthesis, and optimization of novel boron-containing N-substituted oseltamivir (OSC) derivatives. Exploratory structure-activity relationship (SAR) studies led to the identification of compounds 27c and 33c as the most potent NA inhibitors, surpassing OSC in potency against both wild-type group-1 NAs and oseltamivir-resistant NAs. These compounds demonstrated significant antiviral activity against several wild-type strains and H1N1pdm09 strains (EC 50 = 0.03 ± 0.005 and 0.03 ± 0.0008 μM, respectively). Additionally, these compounds did not exhibit significant toxicity (CC 50 > 200 μM in CEF cells; CC 50 > 250 μM in MDCK cells). These findings highlight 27c and 33c as promising next-generation anti-influenza agents.

Neuraminidase (NA) inhibitors play a prime role in treating influenza. However, a variety of viruses containing mutant NAs have developed severe drug resistance towards NA inhibitors, so it is of crucial significance to solve this problem. Encouraged by urea-containing compound 12 disclosed by our lab, we designed a series of oseltamivir derivatives bearing hydrazide fragment for targeting the 150 cavity. Among the synthesized compounds, compound 17a showed 8.77-fold, 4.12-fold, 203-fold and 6.23-fold more potent activity than oseltamivir carboxylate against NAs from H5N1, H1N1, H5N1-H274Y, H1N1-H274Y, respectively. Meanwhile, the best compound 17a exhibited satisfactory metabolic stability in vitro. This study offers an important reference for the structural optimization of oseltamivir aiming at potent inhibition against H274Y mutant of NAs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

The most of potent neuraminidase inhibitors as zwitterions with poor lipophilicity suffered from the poor oral bioavailability. Herein, we describe a rational journey to discover a non-zwitterionic neuraminidase inhibitor 24a containing urea. It showed potent inhibitions against neuraminidases from group 1(H5N1 and H1N1) and group 2 (H3N2) subtypes and exhibited more strong inhibitory activities against neuraminidases from H274Y mutants than oseltamivir carboxylate. Whether administrated by orally or intravenous injection, the pharmacokinetic profile of compound 24a in SD rats were improved compared to oseltamivir carboxylate., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

From our research group, it was noticed that oseltamivir derivatives targeting 150-cavity of neuraminidase enzyme (NA) could significantly increase antiviral activity. Thus, we further enriched the C5-NH 2 position of oseltamivir structure to obtain more potent oseltamivir derivatives. In this article a series of oseltamivir derivatives were synthesized by modifying C5-NH 2 position of oseltamivir. All the compounds were evaluated for in vitro antiviral activity against H5N1 and H5N8. Encouragingly, compounds 9a and 11e were exhibited prominent activity, which is similar to oseltamivir carboxylate (OSC) and in NAs inhibitory assay, 11e showed remarkable potency against N1 (H5N1), N2 (H5N2), N6 (H5N6) and N8 (H5N8). In addition, 11e demonstrated low cytotoxicity and no obvious toxicity at the dose of 1500 mg/kg in mice. Molecular docking studies of 9a and 11e provided a plausible rationale for the high potency against group-1 NAs. This work provided new insights to design further neuraminidase inhibitors, which can help to investigate new potent inhibitors for group-1 and group-2 shortly., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Influenza A neuraminidase plays an indispensable role in the process of replication and transmission of influenza, so the neuraminidase inhibition can prevent the reproduction of the viruses therefore achieve the effect of treatment of influenza. However, drug resistance of neuraminidase inhibitors such as oseltamivir highlights the need to develop novel structural neuraminidase inhibitors. Here we explored a series of oseltamivir derivatives bearing pyridyl group. Among them, compound 23b exhibiting potent inhibitory activity against neuraminidase from H5N1 subtype was comparable to oseltamivir carboxylate. Cytopathic effect inhibition assay in MDCK cells indicated that compound 23b exerted powerful inhibitions on influenza viruses. And compound 23b were nontoxic to MDCK cells. Meanwhile, compound 23b showed high stability towards rat liver microsomes, human liver microsomes and human plasma. This research enriched the structural type of neuraminidase inhibitors., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

The discovery of novel potent neuraminidase (NA) inhibitors remains an attractive approach for treating infectious diseases caused by influenza. In this study, we describe the design and synthesis of novel N-substituted oseltamivir derivatives for probing the 150-cavity which is nascent to the activity site of NA. NA inhibitory studies showed that new derivatives demonstrated the inhibitory activity with IC 50 values at nM level against NA of a clinical influenza virus strain. Moreover, the in silico ADME predictions showed that the selected compounds had comparable properties with oseltamivir carboxylate, which demonstrated the druggablity of these derivatives. Furthermore, molecular docking studies showed that the most potent compound 6f and 10i could adopt different modes of binding interaction with NA, which may provide novel solutions for treating oseltamivir-resistant influenza. Based on the research results, we consider that compounds 6f and 10i have the potential for further studies as novel antiviral agents., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Encouraged by our earlier discovery of neuraminidase inhibitors targeting 150-cavity or 430-cavity, herein, to yield more potent inhibitors, we designed, synthesized, and biologically evaluated a series of novel oseltamivir derivatives via modification of C-1 and C5-NH 2 of oseltamivir by exploiting 150-cavity and/or 430-cavity. Among the synthesized compounds, compound 15e, the most potent N1-selective inhibitor targeting 150-cavity, showed 1.5 and 1.8 times greater activity than oseltamivir carboxylate (OSC) against N1 (H5N1) and N1 (H5N1-H274Y). In cellular assays, 15e also exhibited greater potency than OSC against H5N1 with EC 50 of 0.66 μM. In addition, 15e demonstrated low cytotoxicity in vitro and low acute toxicity in mice. Molecular docking studies provided insights into the high potency of 15e against N1 and N1-H274Y mutant NA. Overall, we envisioned that the significant breakthrough in the discovery of potent group-1-specific neuraminidase inhibitors may lead to further investigation of more potent anti-influenza agents., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

A series of NH 2 -sulfonyl oseltamivir analogues were designed, synthesized, and their inhibitory activities against neuraminidase from H5N1 subtype evaluated. The results indicated that the IC 50 value of compound 4a , an oseltamivir analogue via methyl sulfonylation of C5- NH 2 , was 3.50 μM. Molecular docking simulations suggested that 4a retained most of the interactions formed by oseltamivir carboxylate moieties and formed an additional hydrogen bond with the methylsulfonyl group. Meanwhile, 4a showed high stability towards human liver microsomes. More importantly, 4a without basic moieties is not a zwitterion as reported on the general structure of neuraminidase inhibitors. This research will provide valuable reference for the research of new types of neuraminidase inhibitors.

A panel of divalent oseltamivir and guanidino oseltamivir analogues with esterification on the carboxyl acid group as potent inhibitors of influenza virus neuraminidase was prepared via click reaction. The primary structure activity relationship study demonstrated that appropriate distance between two oseltamivir monomers around 30 Å can crosslink two adjacent neuraminidase tetramers on the virion surface and result in highly effective NA inhibitors against three strains of influenza virus and H7N9 virus like particle. This strategy also provides a basis for the multivalent modification on oseltamivir., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In this study, a series of carboxyl-modified oseltamivir analogs with improved lipophilicity were designed and synthesized, and their inhibitory activities against neuraminidase from influenza A virus H5N1 subtype were evaluated. The results demonstrated that compound 5m exhibited potent inhibitory activity (IC 50  = 1.30 ± 0.23 μM), and it targeted the recently discovered 430-cavity. Compound 5m (LogD = -0.12) is more lipophilic than oseltamivir carboxylate (LogD = -1.69) at pH 7.4, which is potentially propitious to improved membrane permeability and oral drug absorption. Meanwhile, 5m showed high stability in human liver microsomes. The findings of this study can be valuable in identifying neuraminidase inhibitors with optimal lipophilicity and in the exploration of 430-cavity., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

We herein report a gram-scale, enantioselective synthesis of Tamiflu, in which the key trans-diamino moiety has been efficiently installed via an iron-catalyzed stereoselective olefin diazidation. This significantly improved, iron-catalyzed method is uniquely effective for highly functionalized yet electronically deactivated substrates that have been previously problematic. Preliminary catalyst structure-reactivity-stereoselectivity relationship studies revealed that both the iron catalyst and the complex substrate cooperatively modulate the stereoselectivity for diazidation. Safety assessment using both differential scanning calorimetry (DSC) and the drop weight test (DWT) has also demonstrated the feasibility of carrying out this iron-catalyzed olefin diazidation for large-scale Tamiflu synthesis.

Inspired by our initial discovery about a series of neuraminidase (NA) inhibitors targeting the 150-cavity, in present study, we designed, synthesized, and biologically tested a panel of novel oseltamivir derivatives with C-1 modification, targeting the 430-cavity, an additional binding site which widely and stably existed in both group-1 and group-2 NAs. Some of the synthesized compounds displayed robust anti-influenza potencies against H5N1 and H5N6 viruses. Among them, compound 8b exerted the greatest inhibition, with IC 50 values of 0.088 and 0.097 μM and EC 50 values of 4.26 and 1.31 μM against H5N1 and H5N6 strains, respectively, which are similar to those of oseltamivir carboxylate (OSC). And its potency against mutant H5N1-H274Y NA was just 7-fold weaker than OSC. Molecular modeling revealed the elongated group at C-1 position being projected toward the 430-cavity. Notably, although compound 8b was not sensitive toward H5N1 strain relative to OSC in the embryonated egg model, it displayed greater anti-influenza virus effect against H5N6 strain than OSC at the concentration of 10 mmol/L. Overall, this work provided unique insights in the discovery of potent inhibitors against both group-1 and group-2 NAs., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Neuraminidase inhibitors can deter nascent viruses from infecting intact cells by preventing their release from host cells. Herein, a neuraminidase inhibitor 11b absent of basic moieties was discovered in the process of searching for inhibitors targeting 150 cavity. It exhibited potent inhibitions against wild-type neuraminidases from group 1 (H5N1 and H1N1) and group 2 (H7N9) subtypes with IC 50 values similar to those of oseltamivir carboxylate. Moreover, 11b showed moderate inhibitions against mutant neuraminidases from H5N1-H274Y and H1N1-H274Y with IC 50 values of 2075 nM and 1382 nM, which were inferior to those of oseltamivir carboxylate (6095 nM and 4071 nM). The results were not consistent with the recognized SARs that a basic moiety was an indispensable part of a potent inhibitor., (Copyright © 2017. Published by Elsevier Masson SAS.)

In search of novel anti-influenza agents with higher potency, a series of acylguanidine oseltamivir carboxylate analogues were synthesized and evaluated against influenza viruses (H1N1 and H3N2) in vitro. The representative compounds with strong inhibitory activities (IC 50 <40nM) against neuraminidase (NA) were further tested against the NA from oseltamivir-resistant strain (H259Y). Among them, compounds 9 and 17 were potent NA inhibitors that exhibited a 5 and 11-fold increase in activity comparing with oseltamivir carboxylate (2, OC) against the H259Y mutant, respectively. Furthermore, the effect against influenza virus H259Y mutant (H1N1) replication and cytotoxicity assays indicated that compounds 9 and 17 exhibited a 20 and 6-fold increase than the parent compound 2, and had no obvious cytotoxicity in vitro. Moreover, the molecular docking studies revealed that the docking modes of compounds 9 and 17 were different from that of oseltamivir, and the new hydrogen bonds and hydrophobic interaction were formed in this case. This work provided unique insights in the discovery of potent inhibitors against NAs from wild-type and oseltamivir-resistant strains., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Oseltamivir is an important antiviral drug, which possess three chirality centers in its structure. From eight possible stereoisomers, only two have been synthesized and evaluated so far. We describe herein the stereoselective synthesis, computational activity prediction and biological testing of another three diastereoisomers of oseltamivir. These isomers have been synthesized using stereoselective organocatalytic Michael addition, cyclization and reduction. Their binding to viral neuraminidase N1 of influenza A virus was evaluated by quantum-chemical calculations and their anti-influenza activities were tested by an in vitro virus-inhibition assay. All three isomers displayed antiviral activity lower than that of oseltamivir, however, one of the stereoisomers, (3S,4R,5S)-isomer, of oseltamivir showed in vitro potency towards the Tamiflu-sensitive influenza viral strain A/Perth/265/2009(H5N1) comparable to Tamiflu.

A time economical 60 min total synthesis of (-)-oseltamivir was accomplished in a single reaction vessel over five steps. One of the key issues is reduction in the number of steps by eliminating lengthy reaction steps with substitution of a rapid epimerization step. A catalytic system consisting of three reagents, namely, diphenylprolinol silyl ether, thiourea, and acid, was developed for a rapid asymmetric Michael reaction with excellent diastereo- and enantioselectivities. All reactions were optimized in terms of not only yield and selectivity but also reaction time.

To better understand structure-activity relationship (SAR) results, closely related to the structural features of (-)-Oseltamivir, four chiroptical methods, i.e. electronic circular dichroism (ECD), optical rotatory dispersion (ORD), vibrational circular dichroism (VCD), and Raman optical activity (ROA), utilizing different solvents, were employed in an effort to discover a set of the most probable conformations. Such multi-chiroptical approaches supported by quantum chemical calculations pointed out that different conformers are stable in chloroform, acetonitrile and water solutions of (-)-Oseltamivir. In this way, the most probable structures responsible for reported SAR results were established for the first time. It turned out that one of the predominant conformers in a solution is in excellent agreement with the X-ray analysis derived solid-state structure determined for (-)-Oseltamivir phosphate.

Tamiflu, the ethyl ester form of oseltamivir carboxylic acid (OC), is the first orally available anti-influenza drug for the front-line therapeutic option. In this study, the OC-hydroxamates, OC-sulfonamides and their guanidino congeners (GOC) were synthesized. Among them, an OC-hydroxamate 7d bearing an O-(2-indolyl)propyl substituent showed potent NA inhibition (IC50 = 6.4 nM) and good anti-influenza activity (EC50 = 60.1 nM) against the wild-type H1N1 virus. Two GOC-hydroxamates (9b and 9d) and one GOC-sulfonamide (12a) were active to the tamiflu-resistant H275Y virus (EC50 = 2.3-6.9 μM).

A stereodivergent plan is presented leading to all eight stereoisomers of oseltamivir carboxylate (OC). Key chemical manoeuvers are (1) a three-component vinylogous Mukaiyama-Mannich reaction, which sets the whole carbon skeleton and heteroatom substituents, and (2) an intramolecular, silylative Mukaiyama aldol reaction, which creates the targeted carbocycle. The viability of the plan was demonstrated by the first total synthesis of 4-epi-oseltamivir carboxylate (6), accessed in 15 steps from glyceraldehyde, o-anisidine and pyrrole siloxydiene precursors. Compound 6 inhibits influenza A virus strains H1N1 and H3N2 at the μM level, about 150 000-fold less than the OC reference, testifying that the stereodisposition of the C4 acetamido function is key for enzyme recognition. Guided by in-depth structural evaluation including NMR solution studies, molecular mechanics simulations, docking analyses and X-ray crystallography, rationalization of the biological verdict was established.

With the emergence of oseltamivir-resistant influenza viruses and in view of a highly pathogenic flu pandemic, it is important to develop new anti-influenza agents. Here, the development of neuraminidase (NA) inhibitors that were designed to overcome resistance mechanisms along with unfavorable pharmacokinetic (PK) properties is described. Several 5-guanidino- and 5-amidino-based oseltamivir derivatives were synthesized and profiled for their anti-influenza activity and in vitro and in vivo PK properties. Amidine 6 and guanidine 7 were comparably effective against a panel of different A/H1N1 and A/H3N2 strains and also inhibited mutant A/H1N1 neuraminidase. Among different prodrug strategies pursued, a simple amidoxime ethyl ester (9) exhibited a superior PK profile with an oral bioavailability of 31% (rats), which is comparable to oseltamivir (36%). Thus, bioisosteric replacement of the 5-guanidine with an acetamidine-in the form of its N-hydroxy prodrug-successfully tackled the two key limitations of currently used NA inhibitors, as exemplified with oseltamivir.

The one-pot sequential synthesis of (-)-oseltamivir has been achieved without evaporation or solvent exchange in 36% yield over seven reactions. The key step was the asymmetric Michael reaction of pentan-3-yloxyacetaldehyde with (Z)-N-2-nitroethenylacetamide, catalyzed by a diphenylprolinol silyl ether. The use of a bulky O-silyl-substituted diphenylprolinol catalyst, chlorobenzene as a solvent, and HCO2 H as an acid additive, were key to produce the first Michael adduct in both excellent yield and excellent diastereo- and enantioselectivity. Investigation into the effect of acid demonstrated that an acid additive accelerates not only the E-Z isomerization of the enamines derived from pentan-3-yloxyacetaldehyde with diphenylprolinol silyl ether, but also ring opening of the cyclobutane intermediate and the addition reaction of the enamine to (Z)-N-2-nitroethenylacetamide. The transition-state model for the Michael reaction of pentan-3-yloxyacetaldehyde with (Z)-N-2-nitroethenylacetamide was proposed by consideration of the absolute configuration of the major and minor isomers of the Michael product with the results of the Michael reaction of pentan-3-yloxyacetaldehyde with phenylmaleimide and naphthoquinone., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Using N-acetyl-D-glucosamine as a starting material, the anti-influenza drugs oseltamivir and tamiphosphor were synthesized via a pivotal intermediate of aldehyde 8. An intramolecular Horner-Wadsworth-Emmons reaction was utilized to construct the highly functionalized cyclohexene ring. The existing N-acetyl group was transformed into an azido group for the subsequent aziridination, followed by implantation of a 3-pentoxy group of the desired stereochemistry.

We report here the exploitation of the 150 cavity in the active site of influenza A viral neuraminidases for the design of novel C-6 triazole-containing Tamiflu derivatives. A general and convenient synthetic route was developed by utilizing a highly substituted cyclic Baylis-Hillman acetate as an active precursor for azide substitution via suprafacial allylic azide [3,3]-sigmatropic rearrangement. Virus replication inhibitory assays in vitro of these triazole derivatives containing either an amino or guanidino function indicated that the guanidinium compound showed the higher efficacy against a strain with N2 subtype at a concentration of 2 × 10(-5) M but did not inhibit replication of a strain with N1 subtype even at a concentration of 10(-4) M. In order to probe the nature of the enzyme-inhibitor interactions, molecular dynamics simulations were performed on complexes of these compounds with different neuraminidase enzymes. The results indicated that the candidate inhibitors occupy both the 150 cavity and catalytic site but with alternating occupancy.

Two independent formal total syntheses of oseltamivir phosphate were successfully achieved: the first utilized a copper-catalyzed asymmetric three-component reaction strategy, and the second utilized L-glutamic acid γ-ester as a chiral source to install the correct stereochemistry. Both strategies used Dieckmann condensation to construct a six-membered ring core, after which manipulation of the functional groups and protecting groups accessed Corey's intermediate for the synthesis of oseltamivir phosphate. While the first synthesis was accomplished via four purification steps in 25.7% overall yield, albeit with moderate optical purity (76% ee), the second strategy achieved the synthesis via six purification steps in 19.8% overall yield with perfect enantiocontrol.

Shikimic acid has wide use in pharmaceuticals due to its application in the synthesis of drug Tamiflu used in the treatment of Swine flu. The high cost and limited availability of shikimic acid isolated from plants has impeded the use of this valuable building block of the drug. In this context, fermentation route to produce shikimic acid from renewable resources has become increasingly attractive. The present study was embarked upon isolation of wild-type microorganisms able to produce shikimic acid. Out of the 42 isolates obtained from the soil, isolate GR-21 was selected as the best with initial production of 0.54 g/L shikimic acid and later identified as Citrobacter sp. The process optimization resulted in 14-fold increase in the shikimic acid production, thereby claiming this process to be a sustainable alternative for the production of this important biomolecule. The process was further scaled up to 14 L bioreactor to validate the production of shikimic acid. Further, the product formed is shikimic acid was confirmed by FTIR analysis. The current studies suggest that the selected isolate could be used as a promising agent to fulfill the worldwide demand of shikimic acid.

Shikimic acid is a key intermediate for the synthesis of the antiviral drug oseltamivir (Tamiflu®). Shikimic acid can be produced via chemical synthesis, microbial fermentation and extraction from certain plants. An alternative production route is via biotransformation of the more readily available quinic acid. Much of the current supply of shikimic acid is sourced from the seeds of Chinese star anise (Illicium verum). Supply from star anise seeds has experienced difficulties and is susceptible to vagaries of weather. Star anise tree takes around six-years from planting to bear fruit, but remains productive for long. Extraction and purification from seeds are expensive. Production via fermentation is increasing. Other production methods are too expensive, or insufficiently developed. In the future, production in recombinant microorganisms via fermentation may become established as the preferred route. Methods for producing shikimic acid are reviewed., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Biooxidation of benzoic acid by Ralstonia eutropha B9 provides an unusual cyclohexadiene carboxy diol that contains a quaternary stereocentre. Tricarbonyliron derivatives of this chiron, on treatment with acid, give two isomeric η(5)-cyclohexadienyl complexes as observed by NMR spectroscopy. Both of these can be subjected to the addition of nucleophiles to provide isomeric cyclohexadiene complexes with new substituent patterns, several of which have been characterised crystallographically. De-metallation of these provides a versatile library of cyclohexadiene building blocks, the utility of which is demonstrated by formal syntheses of oseltamivir. The mechanism of product formation and its stereochemical implications are discussed, as are the procedures undertaken to establish the enantiopurity of a representative cyclohexadiene product., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Oseltamivir phosphate (Tamiflu) has been synthesized from cis-2,3-bis(hydroxymethyl)aziridine. After protection of the cis-2,3-bis(hydroxymethyl)aziridine with a Boc group, desymmetrization provided a chiral aziridine, which was a key intermediate to install the required stereogenic center containing a nitrogen atom. Allylation and ring closing metathesis are the key reactions to obtain the cyclic product that was successfully converted to the desired oseltamivir phosphate.

A new enantioselective synthesis of the anti-influenza agent (-)-oseltamivir free base (7.1% overall yield; 98% ee) and (-)-methyl 3-epi-shikimate (16% overall yield; 98% ee) has been described from readily available raw materials. Sharpless asymmetric epoxidation and diastereoselective Barbier allylation of an aldehyde are the key reactions employed in the incorporation of chirality, while the cyclohexene carboxylic ester core was constructed through a ring closing metathesis reaction.

Evidences of oseltamivir resistant influenza patients raised the need of novel neuraminidase inhibitors. In this study, five oseltamivir analogs PMC-31-PMC-36, synthesised according to the outcomes of a rational design analysis aimed to investigate the effects of substitution at the 5-amino and 4-amido groups of oseltamivir on its antiviral activity, were screened for their inhibition against neuraminidase N1 and N3. The enzymes used as models were from the avian influenza A H7N1 and H7N3 viruses. The neuraminidase inhibition assay was carried out by using recombinant species obtained from a baculovirus expression system and the fluorogenic substrate MUNANA. The assay was validated by using oseltamivir carboxylate as a reference inhibitor. Among the tested compounds, PMC-36 showed the highest inhibition on N1 with an IC(50) of 14.6±3.0nM (oseltamivir 25±4nM), while PMC-35 showed a significant inhibitory effect on N3 with an IC(50) of 0.1±0.03nM (oseltamivir 0.2±0.02nM). The analysis of the inhibitory properties of this panel of compounds allowed a preliminary assessment of a structure-activity relationship for the modification of the 4-amido and 5-amino groups of oseltamivir carboxylate. The substitution of the acetamido group in the oseltamivir structure with a 2-butenylamido moiety reduced the observed activity, while the introduction of a propenylamido group was well tolerated. Substitution of the free 5-amino group of oseltamivir carboxylate with an azide, decreased the activity against both N1 and N3. When these structural changes were both introduced, a dramatic reduction of activity was observed for both N1 and N3. The alkylation of the free 5-amino group in oseltamivir carboxylate introducing an isopropyl group seemed to increase the inhibitory effect for both N1 and N3 neuraminidases, displaying a more pronounced effect against N1., (Copyright © 2012. Published by Elsevier Ltd.)

Four generations of chemoenzymatic approaches to oseltamivir are presented. The first two generations relied on the use of cyclohexadiene-cis-diol derived enzymatically from bromobenzene. The third and fourth generation used the corresponding diol obtained from ethyl benzoate by fermentation with E. coli JM109(pDTG601a). Oseltamivir was obtained from ethyl benzoate by intersecting intermediate 39 (third-generation synthesis) and intermediate 45 (fourth-generation synthesis). Both of these advanced approaches benefited from symmetry considerations and translocation of the acrylate double bond with concomitant elimination of the C-1 hydroxyl. The syntheses are evaluated for overall efficiency by the use of efficiency metrics and compared with other syntheses of oseltamivir (both academic and industrial).

A formal asymmetric synthesis of the antiviral agent (-)-oseltamivir phosphate is achieved using two aldol reactions as key steps.

The synthesis of conjugates of phospha-oseltamivir to the well established reporter groups fluorescein and biotin and an approach to multimeric inhibitors is described. We report powerful inhibition of the influenza neuraminidase by these probes and quantify fluorescence quenching during binding of the fluorescein conjugate through titration with the neuraminidase. Thus, we show that they could be useful tools to efficiently inhibit, detect and quantify the virus and the neuraminidase in biological systems.

An asymmetric synthesis of chiral intermediate 3 for (-)-oseltamivir phosphate has been accomplished from chiral building block 1, which was prepared by catalytic asymmetric synthesis.

The viral neuraminidase enzyme is an established target for anti-influenza pharmaceuticals. However, viral neuraminidase inhibitors could have off-target effects due to interactions with native human neuraminidase enzymes. We report the activity of a series of known inhibitors of the influenza group-1 neuraminidase enzyme (N1 subtype) against recombinant forms of the human neuraminidase enzymes NEU3 and NEU4. These inhibitors were designed to take advantage of an additional enzyme pocket (known as the 150-cavity) near the catalytic site of certain viral neuraminidase subtypes (N1, N4 and N8). We find that these modified derivatives have minimal activity against the human enzymes, NEU3 and NEU4. Two compounds show moderate activity against NEU3, possibly due to alternative binding modes available to these structures. Our results reinforce that recognition of the glycerol side-chain is distinct between the viral and human NEU enzymes, and provide experimental support for improving the selectivity of viral neuraminidase inhibitors by exploiting the 150-cavity found in certain subtypes of viral neuraminidases., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

We report a full account of our work towards the development of an eight-step synthesis of anti-influenza drug (-)-oseltamivir (Tamiflu) from commercially available starting materials. The final synthetic route proceeds with an overall yield of 30 %. Key transformations include a novel palladium-catalyzed asymmetric allylic alkylation reaction (Pd-AAA) as well as a rhodium-catalyzed chemo-, regio-, and stereoselective aziridination reaction., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The efficient asymmetric total synthesis of (-)-oseltamivir, an antiviral reagent, has been accomplished by using two "one-pot" reaction sequences, with excellent overall yield (60 %) and only one required purification by column chromatography. The first one-pot reaction sequence consists of a diphenylprolinol silyl ether mediated asymmetric Michael reaction, a domino Michael reaction/Horner-Wadsworth-Emmons reaction combined with retro-aldol/Horner-Wadsworth-Emmons reaction and retro Michael reactions, a thiol Michael reaction, and a base-catalyzed isomerization. Six reactions can be successfully conducted in the second one-pot reaction sequence; these are deprotection of a tert-butyl ester and its conversion into an acyl chloride then an acyl azide, Curtius rearrangement, amide formation, reduction of a nitro group into an amine, and a retro Michael reaction of a thiol moiety. A column-free synthesis of (-)-oseltamivir has also been established.

We report here the exploitation of the 150-cavity in the active sites of group-1 neuraminidases for the design of new triazole-containing carbocycles related to oseltamivir. Inhibition studies with virus-like particles (VLPs) containing the influenza virus neuraminidase-1 (N1) activity indicate that several candidates are inhibitors, with K(i) values in the 10(-5)-10(-8) M range. In contrast, a known candidate that preserves the free amino group and a new candidate containing a guanidine function are better inhibitors, with K(i) values of 1.5 × 10(-9) and 4.6 × 10(-10) M, respectively. The most active inhibitor of the N1 enzyme in the triazole series was selective for the N1 class and showed significantly less inhibition (K(i) = 2.6 μM vs 0.07 μM) of the free influenza virus neuraminidase-2 (N2). In addition, saturation transfer difference (STD) NMR spectroscopic studies with this compound and the VLPs show that the entire molecule forms contacts with residues in the active site. These data taken together support our proposed binding mode in which the active site and the adjoining 150-cavity are both occupied.

A synthesis of oseltamivir (Tamiflu) was achieved starting from D-mannitol. A unique feature of the synthetic route is that an acyclic precursor was constructed, which was then cyclized in an intramolecular aldol reaction to form the Tamiflu skeleton. Throughout the synthesis, well-established, highly efficient reactions were employed, and no protection/deprotection sequence was needed.

A short and practical synthesis of oseltamivir was accomplished in 11 steps from inexpensive and abundant diethyl D-tartrate starting material. This azide-free route featured an asymmetric aza-Henry reaction and a domino nitro-Michael/Horner-Wadsworth-Emmons (HWE) reaction as the key steps to construct the relevant cyclohexene ring of the product, which provided an economical and practical alternative for the synthesis of oseltamivir.

A new synthetic route to Tamiflu was developed via the Diels-Alder reaction of pyrrole and bromoacetylene.

Tamiflu is currently the most effective drug for the treatment of influenza, but the insufficient supply and side-effects of this drug demand urgent solutions. We present a practical synthesis of Tamiflu by using novel synthetic routes, cheap reagents, and the abundantly available starting material D-glucal. The strategy features a Claisen rearrangement of hexose to obtain the cyclohexene backbone and introduction of diamino groups through tandem intramolecular aziridination and ring opening. In addition, this synthetic protocol allows late-stage functionalization for the flexible synthesis of Tamiflu analogues. By using the synthesized Tamiflu and its active metabolite (oseltamivir carboxylate), we investigated their influences on neuroendocrine PC12 cells in various aspects. It was discovered that oseltamivir carboxylate significantly inhibits the vesicular exocytosis (regulated secretion) of PC12 cells, and suggests a mechanism underlying the Tamiflu side-effects, in particular its possible adverse influences on neurotransmitter release in the central nervous system., (Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An efficient formal synthesis of oseltamivir phosphate (Tamiflu) has been achieved in 12 steps with use of the inexpensive and highly abundant D-ribose as the starting material. This concise alternative route does not utilize protecting groups and features the introduction of 3-pentylidene ketal as the latent 3-pentyl ether, the use of a highly efficient RCM reaction to form the Tamiflu skeleton, and selective functional group manipulations.

Aims: Pandemics impose large demands on the health care system. The supply of appropriate chemotherapeutic agents, namely oseltamivir solution, presented a serious challenge in the recent influenza pandemic. This study reports on the rational series of pharmacotechnical steps that were followed to appropriately handle bulk oseltamivir powder to meet the increased demand., Methods: During a six-week period in August and September of 2009, a task force was created in the Central Pharmacy of Hospital das Clínicas to convert imported oseltamivir phosphate into ready-to-use solution for utilization by physicians and public health authorities. The protocol included dissolution, physico-chemical tests and the bottling of a liquid microdose formulation for emergency room and outpatient dispensing with adequate quality control during all phases., Results: The successful production routine was based on a specially designed flowchart according to which a batch of 33210 g of oseltamivir powder was converted into 32175 solution units during the aforementioned period with a net loss of only 2.6%. The end products were bottles containing 50 ml of 15 mg/mL oseltamivir solution. The measured concentration was stable and accurate (97.5% - 102.0% of the nominal value). The drug was prescribed as both a prophylactic and therapeutic agent., Discussion: Hospital pharmacies are conventionally engaged in the manipulation of medical prescriptions and specialty drugs. They are generally responsible for only small-scale equipment used for manufacturing and quality-control procedures. The compounding of oseltamivir was a unique effort dictated by exceptional circumstances., Conclusion: The shortage of oseltamivir solution for clinical use was solved by emergency operationalization of a semi-industrial process in which bulk powder was converted into practical vials for prompt delivery.

As a part of our ongoing program of developing novel influenza virus inhibitors, some new derivatives of oseltamivir were prepared by modifying the amino group with glycyl, acetyl, benzyl and prolyl moieties. The interactions of these derivatives with neuraminidase have been probed by molecular modeling techniques. Further, the interaction of these derivatives with model membranes prepared from DPPC and the effect on the thermotropic behavior and polymorphism of the bilayers have been investigated by multinuclear NMR and DSC methods. Results indicate that the glycyl derivative of oseltamivir has the most profound effects on the membrane, compared to other derivatives and seems to be the most promising derivative for further pharmacological evaluation as a neuraminidase inhibitor.

The concept of bifunctional catalysis, wherein both partners of a bimolecular reaction are simultaneously activated, is very powerful for designing efficient asymmetric catalysts. Catalytic asymmetric processes are indispensable for producing enantiomerically enriched compounds in modern organic synthesis, providing more economical and environmentally benign results than methods requiring stoichiometric amounts of chiral reagents. Extensive efforts in this field have produced many asymmetric catalysts, and now a number of reactions can be rendered asymmetric. We have focused on the development of asymmetric catalysts that exhibit high activity, selectivity, and broad substrate generality under mild reaction conditions. Asymmetric catalysts based on the concept of bifunctional catalysis have emerged as a particularly effective class, enabling simultaneous activation of multiple reaction components. Compared with conventional catalysts, bifunctional catalysts generally exhibit enhanced catalytic activity and higher levels of stereodifferentiation under milder reaction conditions, attracting much attention as next-generation catalysts for prospective practical applications. In this Account, we describe recent advances in enantioselective catalysis with bifunctional catalysts. Since our identification of heterobimetallic rare earth-alkali metal-BINOL (REMB) complexes, we have developed various types of bifunctional multimetallic catalysts. The REMB catalytic system is effective for catalytic asymmetric Corey-Chaykovsky epoxidation and cyclopropanation. A dinucleating Schiff base has emerged as a suitable multidentate ligand for bimetallic catalysts, promoting catalytic syn-selective nitro-Mannich, anti-selective nitroaldol, and Mannich-type reactions. The sugar-based ligand GluCAPO provides a suitable platform for polymetallic catalysts; structural elucidation revealed that their higher order polymetallic structures are a determining factor for their function in the catalytic asymmetric Strecker reaction. Rational design identified a related ligand, FujiCAPO, which exhibits superior performance in catalytic asymmetric conjugate addition of cyanide to enones and a catalytic asymmetric Diels-Alder-type reaction. The combination of an amide-based ligand with a rare earth metal constitutes a unique catalytic system: the ligand-metal association is in equilibrium because of structural flexibility. These catalytic systems are effective for asymmetric amination of highly coordinative substrate as well as for Mannich-type reaction of alpha-cyanoketones, in which hydrogen bonding cooperatively contributes to substrate activation and stereodifferentiation. Most of the reactions described here generate stereogenic tetrasubstituted carbons or quaternary carbons, noteworthy accomplishments even with modern synthetic methods. Several reactions have been incorporated into the asymmetric synthesis of therapeutics (or their candidate molecules) such as Tamiflu, AS-3201 (ranirestat), GRL-06579A, and ritodrine, illustrating the usefulness of bifunctional asymmetric catalysis.