Synthesis and Biological Evaluation of NH2-Sulfonyl Oseltamivir Analogues as Influenza Neuraminidase Inhibitors.

A series of NH2-sulfonyl oseltamivir analogues were designed, synthesized, and their inhibitory activities against neuraminidase from H5N1 subtype evaluated. The results indicated that the IC50 value of compound 4a, an oseltamivir analogue via methyl sulfonylation of C5-NH2, 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.

Enantioselective Synthesis of Oseltamivir Phosphate (Tamiflu) via the Iron-Catalyzed Stereoselective Olefin Diazidation.

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.

Design, synthesis, and evaluation of carboxyl-modified oseltamivir derivatives with improved lipophilicity as neuraminidase inhibitors.

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 (IC50 = 1.30 ±â€¯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.

Stereoisomers of oseltamivir - synthesis, in silico prediction and biological evaluation.

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.

Discovery of acylguanidine oseltamivir carboxylate derivatives as potent neuraminidase inhibitors.

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 (IC50 <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.

A configurational and conformational study of (-)-Oseltamivir using a multi-chiroptical approach.

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.

Synthesis, structure and inhibitory activity of a stereoisomer of oseltamivir carboxylate.

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.

Oseltamivir hydroxamate and acyl sulfonamide derivatives as influenza neuraminidase inhibitors.

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).

One-pot synthesis of (-)-oseltamivir and mechanistic insights into the organocatalyzed Michael reaction.

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.

Two approaches toward the formal total synthesis of oseltamivir phosphate (Tamiflu): catalytic enantioselective three-component reaction strategy and L-glutamic acid strategy.

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.

Exploitation of the catalytic site and 150 cavity for design of influenza A neuraminidase inhibitors.

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.

Synthesis of oseltamivir and tamiphosphor from N-acetyl-D-glucosamine.

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.

Valuable new cyclohexadiene building blocks from cationic η5-iron-carbonyl complexes derived from a microbial arene oxidation product.

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.

Synthesis of (-)-oseltamivir phosphate (Tamiflu) starting from cis-2,3-bis(hydroxymethyl)aziridine.

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.

Synthesis of the anti-influenza agent (-)-oseltamivir free base and (-)-methyl 3-epi-shikimate.

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.

Synthesis and in vitro study of novel neuraminidase inhibitors against avian influenza virus.

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.

Development of a concise synthesis of (-)-oseltamivir (Tamiflu).

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.

Formal total synthesis of (-)-oseltamivir phosphate.

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.

Several generations of chemoenzymatic synthesis of oseltamivir (Tamiflu): evolution of strategy, quest for a process-quality synthesis, and evaluation of efficiency metrics.

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).

An aldol approach to the enantioselective synthesis of (-)-oseltamivir phosphate.

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