Suhas

Hey o1, I was reading a blog post from Lonza titled “Better, Faster Pharma Manufacturing with Lonza’s AI Route Design” and I was wondering if you could help me understand the synthetic steps in drug development.

In the post, the author says:

Over the past two decades, I have witnessed a dramatic evolution in the landscape of active pharmaceutical ingredient (API) and drug substance development. While the usual synthetic route for a target molecule used to comprise roughly eight steps 20 years ago, drug candidates today are much more complex and often require 20 or more synthetic steps. This surge in complexity has profound implications for our industry, affecting everything from timelines to supply chains and manufacturing costs.

Now I want you to take a well known drug and break down the synthetic steps required to make it.

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Understanding the Synthetic Complexity in Modern Drug Development: The Case of Oseltamivir (Tamiflu)

In modern pharmaceutical development, the synthesis of an Active Pharmaceutical Ingredient (API) often involves a complex sequence of reactions. This trend contrasts sharply with older drugs, such as Aspirin, which can be synthesized in only a handful of steps. Over the past two decades, as highlighted by industry experts, the complexity of drug molecules has increased significantly, with many new APIs requiring 20 or more synthetic steps. Each step typically involves careful control of reaction conditions, protection and deprotection strategies, functional group transformations, and precise stereochemical manipulations—all while maintaining scalability, purity, yield, and cost-effectiveness.

Oseltamivir Phosphate (brand name Tamiflu), a well-known antiviral medication used to treat influenza, provides a clear illustration of this complexity. Though it is by no means the most complex API ever synthesized, its production from a starting material like shikimic acid involves multiple discrete synthetic transformations. The complexity of Oseltamivir’s structure—complete with multiple stereocenters, strategically positioned functional groups, and a distinctive alkoxy side chain—has made it a prime example of modern synthetic challenges.

Why Oseltamivir is Complex

Unlike simpler molecules, Oseltamivir features several demanding synthetic attributes:

1. Multiple Stereocenters: Ensuring the correct three-dimensional orientation of atoms is crucial for the drug’s biological activity. Controlling stereochemistry often adds multiple steps and specialized reactions.
2. Functional Group Diversity: The molecule contains functionalities such as hydroxyl groups, amino substituents, acetamido units, and an alkoxy side chain. Introducing and adjusting these groups while preserving overall molecular integrity can be tedious.
3. Sensitive Starting Materials and Intermediates: Shikimic acid, a natural starting material used in some syntheses, provides the correct stereochemical foundation but brings its own complexity in the form of delicate functional groups that must often be protected or selectively modified.

A Step-by-Step Look at a Representative Oseltamivir Synthesis

Note: The following outline represents a generalized and simplified version of a published route. Industrial processes and alternative syntheses may vary in the specific reagents, conditions, yields, and number of steps.

1. Starting Material: Shikimic Acid

• Rationale: Shikimic acid is chosen because it already embodies much of the desired chirality. Natural shikimic acid (traditionally sourced from star anise or produced via fermentation) provides a stereochemically rich framework that can be elaborated into Oseltamivir.
• Preparation: Dissolve and purify shikimic acid to ensure a stable, well-characterized feedstock.

2. Protection of Hydroxyl Groups

• Goal: Shikimic acid contains multiple hydroxyl (–OH) groups that can interfere with subsequent reactions. To prevent unwanted side reactions, chemists selectively protect certain hydroxyls.
• Common Techniques: Conversion of free –OH groups into silyl ethers or benzyl ethers. This ensures that the correct positions on the molecule remain reactive or inert as needed in downstream steps.

3. Oxidation and Functional Group Modification

• Goal: Transform an existing side chain on the ring (often a primary alcohol) into a more electrophilic species such as an aldehyde. This sets the stage for introducing the amine functionality later.
• Typical Steps: Controlled oxidation protocols to obtain the desired carbonyl intermediate. Handling these intermediates often involves low temperatures and carefully chosen oxidants to preserve stereochemistry and avoid over-oxidation.

4. Introduction of the Amino Group

• Goal: Incorporate the amine functionality that defines Oseltamivir’s core structure. The amine often arises from the reductive amination of an aldehyde intermediate or from an oxime/hydrazone intermediate formed with the aldehyde.
• Common Reactions: Formation of imines or oximes, followed by selective reduction using hydrogenation or complex metal hydrides. This step demands precise control to achieve the correct stereochemical outcome at the newly formed amino center.

5. Ring Modifications and Formation of the Cyclohexene Core

• Goal: Shikimic acid’s ring undergoes a series of adjustments to attain the cyclohexene framework of Oseltamivir. This may include partial reduction of double bonds, selective elimination, and careful rearrangements.
• Techniques: Hydrogenation under mild conditions, selective deprotection steps, and temperature-controlled rearrangements to sculpt the ring into its final conformation.

6. Introducing the 3-Pentyloxy Side Chain

• Goal: One of the hallmark features of Oseltamivir is its 3-pentyloxy substituent. Installing this alkoxy side chain requires converting a suitably activated leaving group on the ring into the desired ether.
• Typical Strategy: Transforming a hydroxyl into a leaving group (e.g., a triflate, mesylate, or tosylate), followed by nucleophilic substitution with a 3-pentanol-derived nucleophile. The reaction conditions and protecting groups must be tuned to avoid side reactions and maintain high yield.

7. Converting the Amine to an Acetamido Group

• Goal: The final API contains an acetamido substituent (–NHCOCH3). Starting from the free amine, the drug is acylated to form the desired amide functionality.
• Common Reagents: Acetic anhydride or other acetylating agents. The chemist must ensure selective acylation and prevent overreaction or formation of undesired side products.

8. Deprotection and Final Adjustments

• Goal: With the skeleton and most functionalities in place, the protecting groups introduced at the start are now removed to restore the free hydroxyl groups or other functionalities in their final form.
• Methods: Hydrogenolysis of benzyl-protected groups, mild acidic cleavage of silyl ethers, or fluoride-mediated deprotection. Each deprotection step is optimized to minimize harsh conditions that might damage the delicate molecule.

9. Formation of the Oseltamivir Phosphate Salt

• Goal: The commercially available form of the drug is Oseltamivir phosphate. Converting the free amine into its phosphate salt enhances stability, solubility, and bioavailability.
• Common Approach: Treat the free base with phosphoric acid or a phosphate derivative under controlled conditions. The final purification ensures compliance with regulatory standards for purity and potency.

Counting the Steps and Complexity

A route from shikimic acid to Oseltamivir can exceed 10 well-defined synthetic steps, not counting work-ups, purifications, and intermediate isolations. Each of these steps requires methodical optimization to achieve acceptable yields, reproducibility, and the stringent purity profiles required in pharmaceutical manufacturing. Industrial-scale processes often demand even finer control, additional optimization, and sometimes alternative routes that balance cost, sustainability, and safety considerations.

The Broader Context

Oseltamivir’s synthesis exemplifies the intricate chemistry behind many modern APIs. Where legacy drugs might have been amenable to three-to-eight-step syntheses, contemporary small-molecule drugs often require a dozen or more steps. This complexity can be driven by:

• Increased Structural Complexity: More asymmetric centers, complex functional groups, and diverse scaffolds.
• Greater Specificity and Potency Requirements: The need for highly selective targets and the demand for enantiospecific compounds.
• Regulatory and Quality Demands: Tighter control over impurities, consistent batch-to-batch quality, and adherence to Good Manufacturing Practices (GMP).

As pharmaceutical targets become more challenging, the synthetic chemist’s toolkit evolves, incorporating new methods such as biocatalysis, flow chemistry, and machine learning-assisted route design. However, even with these advancements, the multi-step synthesis remains a cornerstone of producing safe, effective, and high-quality medicines.

Suhas

Can you tell me how each of these steps happen in the lab, i.e. with equipments used and so on?

ChatGPT (o1)

Practical Execution of Oseltamivir Synthesis Steps in the Laboratory

The synthesis of Oseltamivir (Tamiflu) from shikimic acid involves a series of chemical transformations, each performed with standard laboratory equipment and techniques. While specific conditions vary depending on the exact protocol, scale, and reagents used, the following sections give a general idea of how each step might be carried out in a lab setting.

General Laboratory Setup and Safety

• Personal Protective Equipment (PPE): Lab coat, gloves, safety glasses, and in some steps, face shields or respirators.
• Inert Atmosphere: Many steps require protecting sensitive intermediates from oxygen or moisture. A nitrogen or argon line, or a glovebox, may be used.
• Analytical Tools: Thin-layer chromatography (TLC) plates, high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry (MS) help monitor progress and assess purity.

1. Preparing the Starting Material (Shikimic Acid)

Typical Equipment and Techniques:

• Glassware: A round-bottom flask (RBF) for dissolution and purification.
• Solvents: Ethanol, methanol, or water to dissolve shikimic acid.
• Purification: Recrystallization using a hot plate/stirrer with a reflux condenser if needed. A Büchner funnel connected to a vacuum pump for filtration.
• Rationale: Ensure that shikimic acid is clean, dry, and well-characterized before proceeding.

Process:

1. Place shikimic acid in an RBF.
2. Add a suitable solvent and gently heat with stirring until fully dissolved.
3. Cool the solution to induce crystallization.
4. Filter the crystals under reduced pressure and dry them under vacuum.

2. Protection of Hydroxyl Groups

Typical Equipment and Techniques:

• Inert Atmosphere Setup: A Schlenk line or a three-neck RBF equipped with a rubber septum and purge line.
• Reagents: Silyl chloride reagents (e.g., TIPS-Cl) or benzyl chloride derivatives for protection.
• Stirring and Heating: Magnetic stirrer, heating mantle or oil bath.
• Monitoring: TLC or sampling small aliquots for NMR.

Process:

1. Dissolve shikimic acid in anhydrous solvent (e.g., dry dichloromethane).
2. Add a base (e.g., imidazole or pyridine) and the silyl/benzyl protecting agent dropwise via an addition funnel.
3. Stir under an inert atmosphere to prevent moisture ingress.
4. Monitor reaction progress by TLC or NMR.
5. Quench the reaction and extract product using a separatory funnel.
6. Dry over anhydrous magnesium sulfate, filter, and concentrate under reduced pressure.

3. Oxidation and Functional Group Modification

Typical Equipment and Techniques:

• Low-Temperature Control: Ice bath, dry ice/acetone bath, or a temperature-controlled chiller.
• Oxidizing Agents: Dess-Martin periodinane, Swern oxidation reagents, or PCC on silica.
• Anhydrous Conditions: A dried RBF, inert atmosphere, and oven-dried glassware.

Process:

1. Dissolve the protected intermediate in an appropriate anhydrous solvent (e.g., dichloromethane).
2. Cool the solution to the required temperature (often 0 °C or below) using an ice bath or dry ice bath.
3. Add the oxidizing reagent slowly via syringe or dropping funnel.
4. Monitor the reaction by TLC or by taking aliquots for NMR.
5. Once complete, quench with a mild base or another quenching reagent.
6. Extract and dry the product, then remove solvent under reduced pressure.

4. Introduction of the Amino Group

Typical Equipment and Techniques:

• Reagents: Ammonia equivalents, oxime formation reagents, or amine nucleophiles.
• Reduction Setup: Use of sodium borohydride (in a round-bottom flask), hydrogenation apparatus (Parr hydrogenator or balloon), or metal-catalyzed reduction under a hydrogen atmosphere.

Process:

1. Convert the aldehyde (formed in the previous step) to an oxime or imine intermediate by adding hydroxylamine or an amine in a controlled environment.
2. After forming the intermediate, transfer the solution to a hydrogenation vessel if catalytic hydrogenation is used.
3. If using sodium borohydride, add it slowly to the aldehyde/oxime solution at low temperature and stir until complete.
4. Monitor by TLC or NMR, then quench, extract, and purify.

5. Ring Modifications and Formation of the Cyclohexene Core

Typical Equipment and Techniques:

• Reduction or Partial Hydrogenation: A Parr hydrogenation apparatus or a simple hydrogen balloon connected to a flask with a heterogeneous catalyst (e.g., Pd/C).
• Heating and Stirring: Oil bath or heating mantle for controlled thermal rearrangements.

Process:

1. Place the intermediate in a hydrogenation vessel with a catalyst (e.g., palladium on carbon).
2. Seal the vessel and pressurize with hydrogen (usually a few bar).
3. Stir under controlled pressure and temperature until the desired reduction or rearrangement is complete.
4. Filter off the catalyst, concentrate the solution, and purify the product.

6. Introduction of the 3-Pentyloxy Side Chain

Typical Equipment and Techniques:

• Activation of Leaving Group: Round-bottom flask with condenser, inert gas line.
• Nucleophilic Substitution: Addition funnel for slowly introducing the nucleophile.
• Heating/Reflux: Use a hot plate/stirrer with a reflux condenser to maintain a stable reaction temperature.

Process:

1. Convert a hydroxyl into a leaving group (e.g., triflate) by adding triflic anhydride under inert atmosphere and low temperature.
2. Introduce the 3-pentanol derivative (or a related alkoxide) slowly.
3. Reflux the reaction mixture if needed.
4. Monitor by TLC; upon completion, cool, quench, and extract.
5. Remove solvent and purify the intermediate.

7. Acetylation to Form the Acetamido Group

Typical Equipment and Techniques:

• Reagents: Acetic anhydride or acetyl chloride.
• Base: Triethylamine or pyridine to scavenge HCl or acetic acid.
• Standard Flasks: A simple RBF and addition funnel setup.

Process:

1. Dissolve the amine intermediate in a dry solvent (e.g., dichloromethane).
2. Add acetic anhydride slowly at low temperature.
3. Stir at room temperature or slightly elevated temperature until formation of the acetamide is complete.
4. Work up by quenching, washing, and drying.

8. Deprotection and Final Adjustments

Typical Equipment and Techniques:

• Deprotection Methods: Hydrogenation (Parr apparatus), acid hydrolysis (RBF with controlled addition of dilute acid), or fluoride ion deprotection (use of TBAF under inert conditions).
• Monitoring: Frequent TLC or NMR checks.

Process:

1. If benzyl groups are used as protecting groups, hydrogenation in a Parr reactor over Pd/C can remove them.
2. Silyl protecting groups can be cleaved by adding tetrabutylammonium fluoride (TBAF) in THF at room temperature.
3. Acid-sensitive groups may be removed by gentle treatment with dilute acid in a simple RBF setup.
4. Purify and isolate the deprotected intermediate.

9. Formation of the Oseltamivir Phosphate Salt

Typical Equipment and Techniques:

• Reagents: Phosphoric acid or an appropriate phosphate salt.
• pH Control: pH meter or indicator to ensure proper salt formation.
• Crystallization Setup: Ice bath, vacuum filtration for isolation.

Process:

1. Dissolve free-base Oseltamivir in a suitable solvent (often aqueous or a mixture of water and a co-solvent).
2. Add phosphoric acid dropwise while monitoring pH.
3. Adjust until the desired salt form precipitates or can be crystallized.
4. Filter, wash, and dry the resulting phosphate salt under vacuum.

Scale-Up and Industrial Considerations

• Larger Glassware or Stainless-Steel Reactors: On industrial scales, jacketed reactors with automated temperature and pressure controls replace small RBFs.
• Automated Control: Pumps, automated addition lines, real-time spectroscopic monitoring (in-line NIR or Raman), and process analytical technology (PAT) improve reproducibility and yield.
• Containment and Safety: More robust ventilation (fume hoods, closed systems) and safety interlocks are employed to handle larger volumes of volatile or hazardous reagents.

In summary, each synthetic step typically employs standard organic chemistry lab equipment—round-bottom flasks, stir plates, condensers, inert gas supplies, and simple filtration setups—combined with careful control of temperature, atmosphere, and reagent addition. Periodic sampling for TLC, NMR, and other analytical techniques ensures the reaction is proceeding as intended, while subsequent purification steps (filtration, extraction, recrystallization) ensure that the final product meets the stringent requirements for pharmaceutical use.