From Illicium verum to Tamiflu

Author: Stefano Sottemano
Date: 10/02/2013


Illicium Verum, commonly called Star Anise or Chinese star anise is a spice that closely resembles anise in flavor, obtained from the star-shaped pericarp of Illicium verum, a medium sized native evergreen tree of northeast Vietnam and southwest China.

Illicium Verum

Star anise is the major source of the chemical compound Shikimic acid, a primary precursor in the pharmaceutical synthesis of anti-influenza drug Oseltamivir (Tamiflu). Shikimic acid is produced by most autotrophic organism and whilst it can be obtained in commercial quantities from elsewhere, star anise remains the usual industrial source. In 2005, there was a temporary shortage of star anise due to its use in the production of Tamiflu. Later that year, a way was found of using bacteria to make Shikimic acid. Roche now derive some of the raw material it need from the fermentation of E. Coli bacteria.
Star anise is grown in four provinces in China and harvested between March and May. The Shikimic acid is extracted from the seed in a ten-stage manufacturing process which takes a year, 90% of the harvest is already used by the Swiss pharmaceutical manufacturer Roche in making Tamiflu.
Japanese star anise (Illicium anisatum), a similar tree, is not edible because it is highly toxic (due to containing sikimitoxin), it cause illness including serious neurological effects, “such as seizures”. Japanese star anise contains anisantin, which causes severe inflammation of the kidneys, urinary tract and digestive organs, it’s toxicity is caused by its content in a potent neurotoxins (anisantin, neoanisantin and pseudoanisantin), due to their activity as non-competitive antagonists of GABA receptor.

Shikimic acid

Shikimic acid, more commonly known as its anionic form shikimate, is a cyclohexene, a cyclitol and a cyclohexanecarboxylic acid

- Biosynthesis

Phosphoenolpyruvate and erythrose-4-phosphate react to form 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP), in a reaction catalyzed by the enzyme DAHP synthase. DAHP is then transformed to 3-dehydroquinate (DHQ), in a reaction catalyzed by DHQ synthase. Although this reaction requires nicotinamide adenine dinucleotide (NAD) as a cofactor, the enzymic mechanism regenerates it, resulting in the net use of no NAD.

DHQ is dehydrated to 3-dehydroshikimic acid by the enzyme 3-dehydroquinate dehydratase, which is reduced to shikimic acid by the enzyme shikimate dehydrogenase, which uses nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor.


Oseltamivir, marketed under the trade name Tamiflu, is an antiviral drug, which may slow the spread of influenza (flu) virus between cells in the body by stopping the virus from chemically cutting ties with its host cell. The drug is taken orally in capsules or as a suspension. It is used to treat influenza A virus and influenza B virus. Oseltamivir is a prodrug, a (relatively) inactive chemical which is converted into its active form by metabolic process after it is taken into the body. It was the first orally active neuraminidase inhibitor.

- Medical use

Oseltamivir is indicated for the treatment and prevention of infections due to influenza A and B viruses. The Centers for Disease Control and Prevention recommends physicians prioritize which patients to whom they prescribe oseltamivir: specifically, people hospitalized with more severe illness, children younger than two years old, adults over 65, pregnant women, people with certain chronic medical or immunosuppressive conditions and adults under 19 on long-term aspirin therapy.

- Mechanism of action

The prodrug oseltamivir is itself not virally effective; however, once in the liver it is hydrolysed to its active metabolite - the free carboxylate of oseltamivir (GS4071).
Oseltamivir is a neuraminidase inhibitor, serving as a competitive inhibitor of the activity of the viral neuraminidase (NA) enzyme upon sialic acid, found on glycoproteins on the surface of normal host cells. By blocking the activity of the enzyme, oseltamivir prevents new viral particles from being released by infected cells.

Oseltamivir phosphate is readily absorbed from the gastrointestinal tract, with approximately 80% bioavailability. The prodrug is extensively converted to active oseltamivir carboxylate by hepatic esterases. Neither oseltamivir nor oseltamivir carboxylate is a substrate for, or inhibitor of, cytochrome P450 isoforms. Less than 5% of a dose is eliminated unchanged. Oseltamivir is well distributed to the nasal mucosa, the tracheal lining, and the tissues of the middle ear. In adults, the volume of distribution for oseltamivir carboxylate has ranged from 23 to 27 L. There are currently no data available in children. Neither the prodrug or the active form are highly protein bound. Oseltamivir carboxylate is eliminated by glomerular filtration and renal tubular excretion without further metabolism. The average half-life of elimination in adults is 6-10 hours. It is recommended that the dosing frequency be reduced from twice to once daily for treatment and from once daily to every other day for prophylaxis in patients with moderate renal dysfunction.
oseltamivir (Tamiflu®). Oseltamivir, along with inhaled zanamivir, are the only two antivirals with activity against the current H1N1 virus as it is resistant to the antivirals amantadine and rimantadine.1 Before explaining how oseltamivir works to prevent and treat the H1N1 virus it is important to understand the similarities and differences between this virus and the typical influenza virus encountered during flu season.

The influenza virus is an enveloped RNA virus that has a genome with either eight (influenza A and B) or seven (influenza C) segments. In humans, influenza A & B are the types most responsible for the yearly flu seasons. They are also further named based on the 2 major types of antigenic proteins present on the viral capsid (coating). Those two antigenic proteins are hemagglutinin (HA) and neuraminidase (NA) of which the NA is a primary target of oseltamivir and zanamivir. Unfortunately, each of these antigenic proteins is further broken down into subtypes that help to more specifically identify the virus. Hemagglutinin has 16 different subtypes (H1-H16) and NA has 9 different subtypes (N1-N9). The swine flu has a designation of H1N1, which tells the clinician what subtype of HA and NA are present on the capsid of the virus and at times may help to determine what species the virus possibly evolved from. The reason the swine flu (H1N1) has been given this particular designation is due to the fact that this virus is a combination of genes from influenza viruses normally found in pigs but also has genes from influenza viruses normally seen in birds and humans.1 As such, when genes from multiple species of influenza virus' come together they can form a new virus that has the potential to infect humans. This process contributes to what is called "antigenic shift" that is known to result in the emergence of a new influenza virus. If this new recombinant can be easily transmitted from person to person, then a flu pandemic can emerge.

Upon exposure to the H1N1 virus, the virus' HA will be used to attach itself to receptors containing sialic (neuraminic) acid on the cell surface of the cell targeted for infection (typically respiratory mucosal epithelium). This H1 interaction with the sialic acid containing cell surface receptors facilitates the fusion between the viral envelope and the cell membrane.5 In addition to H1's influence on viral fusion, NA may also contribute to the ability of the virus to invade the respiratory tract (lungs) by removing the sialic acid present in mucin, thereby enhancing the virus's pathogenicity.6

A second protein, M2, also contributes to the fusion of the viral envelop with the cell membrane. The M2 protein is an ion channel that aids in the regulation of pH in the endosome created from the fusion thereby permitting the release of the RNA into the cell cytoplasm where it directs synthesis of new viral proteins using the host's machinery. The M2 protein is the target of amantadine and rimantadine.

As the newly synthesized viral proteins assemble to form new virions (H1N1 virus), they will move to the surface of the cell membrane. However, these viral progeny are initially trapped on the cell surface by the same HA-sialic acid interactions that were used for infection. Neuraminidase is therefore required for spreading infection, as it cleaves sialic acid residues, allowing the virus to spread to uninfected cells.6 After oral administration and absorption of oseltamivir (a prodrug), it is rapidly converted to oseltamivir carboxylate which is a potent inhibitor of all influenza A NA subtypes (N1-9).7 Normally, in order for the newly formed virions to leave and infect another cell, the virus's NA must cleave the sialic acid residues on the cell surface receptors so they can be released. Oseltamivir carboxylate blocks the cleavage of these sialic acid residues because its lipophilic side chain is able to get into the membrane and block the viral NA from working.

Therefore, oseltamivir has the ability to block H1N1 virus from invading the respiratory epithelium as well as prevent the release of new H1N1 virions from infected cells, thus decreasing the infectivity of neighboring cells. Fortunately, oseltamivir has demonstrated some benefit in children one year and older, including a reduction in lower respiratory tract infections, bronchitis and hospitalizations.8,9 It has also been urgently approved by the FDA and recommended by the Centers for Disease Control and Prevention (CDC) for the treatment of swine-origin influenza A (H1N1) infection in all patients as young as 3 months of age. When compared to placebo, oseltamivir reduced the symptoms related to the regular influenza virus by about 36 hours when compared to placebo. The above benefits are best achieved with early initiation in relation to the onset of symptoms (generally within the first 48 hours). In adults and adolescents (greater than or equal to 13 years of age) oseltamivir should be given as 75 mg by mouth daily for 10 days for prophylaxis and 75 mg by mouth twice a day for 5 days for treatment.

- Commercial production

The current production method is based on the first scalable synthesis developed by Gilead Sciences starting from naturally occurring quinic acid or shikimic acid. Due to lower yields and the extra steps required (because of the additional dehydration), the quinic acid route was dropped in favour of the one based on shikimic acid, which received further improvements by
Hoffmann-La Roche.

Karpf / Trussardi synthesis

The synthesis commences from naturally available (−)-shikimic acid. The 3,4-pentylidene acetal mesylate is prepared in three steps: esterification with ethanol and thionyl chloride; ketalization with p-toluenesulfonic acid and 3-pentanone; and mesylation with triethylamine and methanesulfonyl chloride. Reductive opening of the ketal under modified Hunter conditions in dichloromethane yields an inseparable mixture of isomeric mesylates. The corresponding epoxide is formed under basic conditions with potassium bicarbonate. Using the inexpensive Lewis acid magnesium bromide diethyl etherate (commonly prepared fresh by the addition of magnesium turnings to 1,2-dibromoethane in benzene:diethyl ether), the epoxide is opened with allyl amine to yield the corresponding 1,2-amino alcohol. The water-immiscible solvents methyl tert-butyl ether and acetonitrile are used to simplify the workup procedure, which involved stirring with 1 M aqueous ammonium sulfate. Reduction on palladium, promoted by ethanolamine, followed by acidic workup yielded the deprotected 1,2-aminoalcohol. The aminoalcohol was converted directly to the corresponding allyl-diamine in an interesting cascade sequence that commences with the unselective imination of benzaldehyde with azeotropic water removal in methyl tert-butyl ether. Mesylation, followed by removal of the solid byproduct triethylamine hydrochloride, results in an intermediate that was poised to undergo aziridination upon transimination with another equivalent of allylamine. With the librated methanesulfonic acid, the aziridine opens cleanly to yield a diamine that immediately undergoes a second transimination. Acidic hydrolysis then removed the imine. Selective acylation with acetic anhydride (under buffered conditions, the 5-amino group is protonated owing to a considerable difference in pKa, 4.2 vs 7.9, preventing acetylation) yields the desired N-acetylated product in crystalline form upon extractive workup. Finally, deallylation as above, yielded the freebase of oseltamivir, which was converted to the desired oseltamivir phosphate by treatment with phosphoric acid. The final product is obtained in high purity (99.7%) and an overall yield of 17-22% from (−)-shikimic acid. It is noted that the synthesis avoids the use of potentially explosive azide reagents and intermediates; however, the synthesis actually used by Roche uses azides. Roche has other routes to oseltamivir that do not involve the use of (−)-shikimic acid as a chiral pool starting material, such as a Diels-Alder route involving furan and ethyl acrylate or an isophthalic acid route, which involves catalytic hydrogenation and enzymatic desymmetrization.

Corey synthesis

In 2006 the group of E.J. Corey published a novel route bypassing shikimic acid starting from butadiene and acrylic acid. Butadiene 1 reacts in an asymmetric Diels-Alder reaction with the esterfication product of acrylic acid and 2,2,2-Trifluoroethanol 2 catalysed by the CBS catalyst. The ester 3 is converted into an amide in 4 by reaction with ammonia and the next step to lactam 5 is an iodolactamization with iodine initiated by trimethylsilyltriflate. The amide group is fitted with a BOC protective group by reaction with Boc anhydride in 6 and the iodine substituent is removed in an elimination reaction with DBU to the alkene 7. Bromine is introduced in 8 by an allylic bromination with NBS and the amide group is cleaved with ethanol and caesium carbonate accompanied by elimination of bromide to the diene ethyl ester 9. The newly formed double bond is functionalized with N-bromoacetamide 10 catalyzed with Tin(IV) bromide with complete control of stereochemistry. In the next step the bromine atom in 11 is displaced by the nitrogen atom in the amide group with the strong base KHMDS to the aziridine 12 which in turn is opened by reaction with 3-pentanol 13 to the ether 14. In the final step the BOC group is removed with phosphoric acid and the oseltamivir phosphate 15 is formed.

Stefano Sottemano

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