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Lab 4: Building a Better World (AKA Fluvastatin Synthesis - Reduction of FLEXAT to DIOLAT) Goals of the lab:

1. To synthesize racemic Diolat from Flexat 2. To observe the progression of a chemical reaction by TLC 3. To employ safe synthetic organic chemistry lab techniques

Introduction

FLEXAT and DIOLAT are two chemical intermediates in the synthesis of the cholesterol lowering drug fluvastatin sodium (sold under the name Lescol XL by Novartis). In this experiment, you will reduce the hydroxyketone FLEXAT to the diol DIOLAT using sodium borohydride and monitor the reaction by thin layer chromatography. The DIOLAT product from this reaction will be composed of both syn and anti diastereomers. How the diastereoselectivity of this reaction is controlled in the industrial synthesis will be discussed.

FLEXAT DIOLAT

N

OH OH O

O

F

NaH3C CH3

N

OH O O

O

F

H3C CH3

CH3

CH3CH3

N

OH OH O

O

F

H3C CH3

CH3

CH3CH3

Lescol XL (fluvastatin sodium) References

This procedure was modified by Dr. Allen Aloise from the industrial synthesis of fluvastatin sodium by Sandoz Inc., a Novartis company. The FLEXAT used in this experiment was generously donated by Novartis.

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Reading

Visit www.lescolxl.com for more information about Lescol XL. Introduction

The development of hydride reagents for the reduction of carbonyl

derivatives has been of great significance in synthetic organic chemistry. The most common commercially available metal hydrides are sodium borohydride (NaBH4) and lithium aluminum hydride (LiAlH4). Both hydride reagents react with a broad range of electrophilic substrates with the transfer of a hydride (H-) ion. The general stoichiometry followed in the reduction of ketones and aldehydes is illustrated below for NaBH4. The liberation of the desired alcohol is usually accomplished by the subsequent addition of water, acid, or aqueous hydrogen peroxide (in the case of borohydride reageants).

NaBH4

R R'

O

4

4

R R'

OH B– Na+aq. H2O2

R R'

OHH4

The hydride donor capabilities of sodium borohydride and lithium aluminum hydride are illustrated below, with the carbonyl substrates arranged in decreasing ease of reduction. Lithium aluminum hydride is an exceedingly powerful reducing agent and is capable of reducing even less reactive functional groups than those shown. On the other hand, sodium borohydride is an exceptionally mild reducing agent, only strong enough to reduce the most reactive of carbonyl functional groups: aldehydes, ketones, and acid chlorides.

R H

O

R OH

R R'

O

R R'

OH

R OR'

O

R OH

aldehyde

ketone

ester

NaBH4 LiAlH4

(yes)

(yes)

(no)

(yes)

(yes)

(yes)

You can see why sodium borohydride is the appropriate choice for our reduction of interest. We wish to reduce only the ketone in the FLEXAT starting material and leave the aromatic rings, double bond, and ester intact. Sodium

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borohydride is only powerful enough to reduce the ketone functionality. You will be carrying out the reaction below in this experiment.

1. NaBH4

2. H2O2

THF, 0 °C

FLEXAT DIOLAT

N

OH O O

O

F

H3C CH3

CH3

CH3CH3

N

OH OH O

O

F

H3C CH3

CH3

CH3CH3

The FLEXAT starting material we will be using is drawn with a squiggly line connecting the carbon and oxygen atom of the alcohol functionality . This is to indicate that our sample is a mixture of R and S stereocenters at the alcohol carbon. Because FLEXAT contains only a single stereogenic center, and an equal number of R and S enantiomers are present, the mixture is said to be racemic. An alternative way of showing this would be to draw out each of the two enantiomers:

N

OH O O

O

F

H3C CH3

CH3

CH3CH3

N

OH O O

O

F

H3C CH3

CH3

CH3CH3

+S R

racemic mixture

S-FLEXAT R-FLEXAT

When our sodium borohydride reduction is carried out, DIOLAT is formed as a mixture of four diastereomers. If you look carefully, you can see that the two possible syn-diol arrangements are enantiomeric and that the two possible anti-diol arrangements are enantiomeric as well. The product of our reaction will be a racemic mixture of syn and anti products.

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N

OH OH O

O

F

H3C CH3

CH3

CH3CH3

S RN

OH OH O

O

F

H3C CH3

CH3

CH3CH3

S S

N

OH OH O

O

F

H3C CH3

CH3

CH3CH3

R SN

OH OH O

O

F

H3C CH3

CH3

CH3CH3

R R

syn-DIOLAT

syn-DIOLAT

anti-DIOLAT

anti-DIOLAT

syn-enantiomers

+

anti-enantiomers The formation of syn-DIOLAT is required in the industrial synthesis of

fluvastatin sodium. Chemists were able to achieve this result by employing diethylmethoxyborane (DMB) as an additive in the reaction. DMB forms a six-member ring complex with the alcohol and ketone, resulting in a directed hydride addition to afford predominantly the syn-DIOLAT product.

1. NaBH4

THF, -78 °C

FLEXAT ±syn-DIOLAT

N

OH O O

O

F

H3C CH3

CH3

CH3CH3

N

OH OH O

O

F

H3C CH3

CH3

CH3CH3

(C2H5)2BOCH3

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Procedure

1. NaBH4

THF, 0 °C

FLEXAT DIOLAT

N

OH O O

O

F

H3C CH3

CH3

CH3CH3

N

OH OH O

O

F

H3C CH3

CH3

CH3CH3

FLEXAT NaBH4 THF H2O2 (30%)

DIOLAT

MW (g/mol) 465.6 37.83 72.11 34.0 467.6 mp (°C) 97-102 -40 141-142 bp (°C) - 65-67 126 - density (g/mL)

0.889 1.1 -

mg 100 6.3 - - mL - - 3 -

mmol 0.215 0.166 29.1 - equiv 1.0 0.775 135 -

1. Add 1 mL of tetrahydrofuran (THF) to a glass vial with a magnetic stir bar at the bottom.

2. Cool the vial with THF to 0 °C by clamping the reaction vessel to the vertical metal bar at your bench and partially submerging it in an ice water bath resting on a magnetic stirrer.

3. Turn the magnetic stirrer on and make slight adjustments to your set-up so that the stir bar is spinning in a consistent and controlled manner.

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Figure. Assembled reaction vial apparatus

4. Go to the balance area and obtain a piece of weighing paper. Fold the weighing paper in half and then open it up so that a deep crease runs across it. Place the weighing paper on a digital balance and press the tare button. This will reset the balance to zero. Using a spatula, carefully transfer 6.3 mg of sodium borohydride from the reagent bottle to the weighing paper.

a. This is a small amount of material, therefore you may need to close the side doors of the balance to keep atmospheric disruptions from making the balance readings jump around.

5. Recap the reagent bottle when finished and carefully pick up the weighing paper and return to your bench with it.

6. Unscrew the top of your reaction vessel and use the weighing paper as a funnel to add the sodium borohydide to the THF.

a. You may need to use your spatula to scrape off all of your reagent from the weighing paper.

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Figure. The weigh station.

7. Repeat the above procedure to weigh out 100 mg of FLEXAT on a piece of weighing paper.

8. Carefully add it to your 0 °C solution in portions over 60 seconds. a. The low temperature of the solvent and slow addition of your

starting material is to ensure that any heat or bubbles generated from chemical reactions do not boil or splatter your reaction mixture.

9. Cap your reaction vessel and allow your reaction mixture to stir at 0 °C for ~ 5 minutes and then remove the ice water bath and allow the reaction to warm to room temperature while stirring. Make sure you record color changes and any other observations you make.

10. Perform a TLC analysis of your reaction, preparing your TLC plate as diagramed below.

a. SPOT VERY LIGHTLY. The solutions are very concentrated and if you spot heavily you will mostly see large smears rather than dots.

b. Use one of the pre-prepared Flexat standards for your starting material spot. This will serve as a standard of comparison.

c. The center lane of your TLC plate should receive a spot of both the starting material solution and the reaction mixture. This is your cospot lane and will reveal if there is anything in your reaction mixture that does not correspond to your starting material. You should see two or more spots in this lane if you have created new chemical compounds in your reaction.

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d. Finally, spot the reaction mixture in the right lane of your TLC plate. You will know when your reaction is complete when you no longer see any starting material in this lane.

e. Run your TLC plate in a beaker with 5 ml of 40% Ethyl Acetate/60% Hexane.

f. Make sure not to let the solvent front reach the top of the plate!

startingmaterial

cospot reaction

1. develop

2. visualize

rxn has only slightly

progressed

rxn is almost

complete

rxn is complete

time = 1 min time = 5 min time = 15 min

11. The reduction in this experiment is a rapid one and should be complete or nearly so by the time you perform your TLC analysis. Consult with your TF if the reaction is not almost completely finished within 20 minutes.

Work-Up You will quench your reaction and the remaining unreacted sodium borohydride by adding it to a solution of sodium bicarbonate (NaHCO3).

12. Obtain a fresh glass vial and add 5 mL of saturated aqueous sodium bicarbonate to it.

13. Using a Pasteur pipet and rubber bulb, carefully pipet your yellow reaction mixture into this NaHCO3 solution.

14. Rinse your old reaction vial with 5 mL of EtOAc and add it to the vial containing the NaHCO3 and yellow reaction mixture.

15. Cap the new vial and shake it vigorously. 16. Place the vial down on your benchtop and slowly unscrew the cap to vent

any gasses that have built up. 17. Allow the organic layer and the aqueous layer to separate.

a. Do you know why many organic solvents and water are not soluble? Based on the densities of the two layers can you predict which is the organic and which is the aqueous?

18. Once the layers have separated, carefully pipet out the aqueous layer and dispense it into another glass vial on your benchtop. You are interested

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in keeping the organic (yellow) layer in which your product is dissolved but should not discard the aqueous layers you will be removing in case you accidentally mix the layers up. If you do make a mistake, you can always “rescue” the proper layer from your discard flask.

Figure. Aqueous extraction of Diolat. A mixture of ethyl acetate and aqueous solvent will be turbid when mixed (left panel). After allowing the mixture to rest, two layers will separate, with the less dense solution above the denser solution (middle panel). Using a glass pipet, extract the aqueous or lower layer (right panel).

19. Wash the organic layer two times (2X) with 5 mL of saturated sodium

chloride (NaCl) solution. Remove the aqueous layer after each washing. a. By ‘Washing’ we mean add 5 ml of saturated sodium choloride

solution to your vial with the yellow reaction mixture. Shake to mix. Allow the layers to separate, and then remove the aqueous (white/clear lower layer) from the vial.

20. Using a scoopula, add a scoop or two of solid anhydrous sodium sulfate (Na2SO4) to your organic layer.

a. Sodium sulfate is a drying reagent and will absorb any residual water sill present after the washings. Hydrated Na2SO4 is clumpy and hard while dry Na2SO4 will remain granular. Typically you can use these visual clues to determine when your solution is dry- when granular, non-clumped drying reagent remains in your flask, all of the moisture has been absorbed. However on a scale this small it may be difficult to determine if your Na2SO4 is clumpy or not, so just swirl the contents of the vial occasionally and allow the drying to take place over approx. 5 minutes.

21. Obtain a fresh glass vial and record the weight of it. 22. Using a pipet, carefully transfer as much of your solution to the fresh vial

as possible, while leaving behind the Na2SO4. 23. Label the vial with your name and TF and have your TF direct you where

to store it until the next lab. The vials will be stored uncapped in a fume

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hood to allow the solvent to evaporate. During the next lab you will weigh your vial and determine a crude chemical yield of your unpurified DIOLAT.

Clean-Up • Pour ethyl acetate and hexane into the organic waste blue bucket • Pour the aqueous extracts into the aqueous waste blue bucket • Place the vial containing Na2SO4 into the solid waste bucket • Refer to Lab 4-Appendix 1 for instructions on how to clean the glassware that you used Questions (due one week after the completion of the lab) 1. Attach a labeled copy of your results (TLC plate and table describing the

amount of product you began with and yield), and describe them.

a. What did you do in this experiment? b. On your TLC plate, which spots correspond to Diolat and Flexat? c. What are the Rf values for Diolat and Flexat? d. Are there any additional spots on your TLC plate? What do you think

those correspond to? e. What was your yield in this synthesis reaction? f. Was your yield greater than or less than your theoretical yield? g. If your yield was less than the theoretical yield, explain how this could

have occurred. h. If your yield was greater than the theoretical yield, explain how this could

have occurred. 2. How many syn-Diolat and anti-Diolat isoforms are made using our procedure?

What compound is used by Novartis to generate syn-Diolat? Do you think Novartis generates both syn-Diolat isoforms or only one? Why or why not?

3. In your TLC, the completed reaction product (Diolat) migrated more slowly

than the precursor (Flexat). What does this indicate about the affinity of Diolat and Flexat for the TLC plate?

4. Using TLC, do you think it is possible to distinguish the various isomers of

Flexat from one another? Do you think you could distinguish the various isomers of Diolat from one another? Explain.

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5. TLC plates are coated with silica. Silica is a polar substance. If you were to perform a TLC with plates coated with a non-polar substance, how do you think the migration of Diolat and Flexat would be altered? Explain.

6. What is the major structural difference between Diolat and Fluvastatin? Do

you think Diolat will bind to HMG-CoA Reductase? Why or why not? 7. Fluvastatin binds to HMG-CoA Reductase with greater affinity than HMG-CoA.

What is the basis for this increased binding affinity? 8. If eukaryotic cells were treated with high doses of fluvastatin, explain how

you think this would affect the fluidity of their cell membranes.

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Appendix 1: The importance of statins In the United States, about 950,000 people die each year of cardiovascular disease. One of the most important risk factors of cardiovascular disease is an elevated cholesterol level. In the United States, over 107 million adults are classified with high or borderline-high cholesterol levels. Cholesterol is ingested with food, and it can also be synthesized. Many people can modify their cholesterol level through simple lifestyle changes such as diet and exercise. Cholesterol levels are also often lowered through the use of a class of pharmaceuticals collectively known as statins. Statins are small-molecules that lower cholesterol levels by inhibiting the body’s ability to synthesize cholesterol. Where did statins come from? In the 1950s and 60s, many cholesterol-lowering agents were in clinical use, however all of them had undesirable side-effects or were inappropriate for long-term usage. In the 1960s, cholesterol metabolism was being extensively studied in both humans and animals, and it was discovered that cholesterol in the body could either come from intestinal absorption of dietary cholesterol or through biosynthesis in the body, primarily the liver. It was also revealed that if dietary cholesterol was reduced, biosynthesis increased, and if dietary cholesterol intake increased, biosynthesis decreased. This alteration in biosynthesis is modulated by the activity of HMG-CoA Reductase, an enzyme that converts HMG-CoA into mevalonate, which in turn is used to generate cholesterol.

The biosynthesis of HMG-CoA to Mevalonate and eventually Cholesterol Thus several groups hypothesized that cholesterol levels could be lowered through the inhibition of de novo biosynthesis, and in the early 1970s several groups began to search for novel compounds that could inhibit the activity of HMG-CoA Reductase. In 1976 two groups published papers describing two HMG-CoA Reductase inhibitors with identical structures isolated from different sources - Akira Endo, Masao Kuroda, and Y. Tsujita identified Mevastatin from a microbe called Penicillium citrinum and Brown et. Al. identified Compactin from Penicillium brevicompactum.

HMG-CoAReductase

Cholesterol

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The structure of Compactin/Mevastatin compared to HMG-CoA What do statins look like, and how do they work?

The features of Compactin/Mevastatin Compactin/Mevastatin are characterized by four moieties: 1) the b-hydroxy-d-lactone or 3,5 dihydroxyheptanoic acid (HMG-CoA like), 2) the moiety connecting the lactone and lipophilic group, 3) the hexahydronaphthalene core, and 4) the side chain ester. The lactone domain is structurally similar to HMG and binds in the active site of HMG-CoA Reductase. Additionally the hexahydronaphthalene core binds to an adjacent hydrophobic pocket of HMG-CoA Reductase not normally utilized in HMG-CoA substrate binding. Thus with two binding sites, Compactin/Mevastatin binds HMG-CoA Reductase with 10,000 fold higher affinity, and thus is able to out-compete HMG-CoA for binding to HMG-CoA Reductase.

Compactin/Mevastatin (lactone) Compactin/Mevastatin (Acid form)

HMG-CoA

HMG-CoA-like

Dexahydronaphthalene (heteroaromatic) core

Side-chain ester

Compactin/Mevastatin (Acid form)

Connecter