Phenothiazine is a heterocyclic aromatic compound containing both nitrogen and sulfur.
Phenothiazine derivatives have found use in a variety of applications. Phenothiazine structures
are found in pharmaceuticals used as antipsychotics, such as chlorpromazine. Methylene blue
is a dye based upon a phenothiazine core structure used as a redox indicator and a stain for
biological samples. Functionalized phenothiazines have also found use as photoactive catalysts
for metal-free atom transfer polymerization reactions as well as materials in dye-sensitized solar
cells. Phenothiazine derivatives are currently being studied for their potential to serve as cathode
materials in next generation energy storage applications.
Phenothiazines are readily substituted at the nitrogen (10) position as well as the aromatic rings
(1,3,7 and 9 positions); numbering shown above. The oxidation state of the sulfur can also be
varied (sulfide, sulfoxide or sulfone.) As a result, phenothiazines can be synthesized with a
variety of functional groups and substituents to tune their properties as desired. In this
experiment, you will synthesize a functionalized phenothiazine derivative over 3 steps. Starting
materials may be varied to allow for different optical and electronic properties. You will
characterize your final product by electrochemical techniques (i.e. cyclic voltammetry) as well as
the optical properties (i.e. UV-vis spectroscopy and fluorometry.)
PPE Required: Lab coat; splash goggles or safety glasses; nitrile gloves.
Special Training: Must complete Water-Reactive Training Module on iLearn prior to Reaction
1 (not required S21).
Lab period A
- Add phenothiazine (x g, 3.0 mmol) to a 50 mL round bottom flask that is equipped with a
magnetic stirring bar. Add anhydrous DMF (6 mL) to dissolve phenothiazine and stir at
0 °C in an ice-bath. - Carefully NaH dispersion (x mg, 1.33 equiv) in 3 three portions (add the next portion when
bubbling subsides). Do not cap during addition (H2 gas is being generated). - Add bromoethane (x mL, 2 equiv) dropwise using a disposable syringe and then remove
the reaction mixture from the ice-bath. Fit the flask with a reflux condenser, top with a
rubber septum, inert gas line and vent needle. Set the hotplate to 80 °C and heat the
reaction for at least 60 min. - Monitor the progress of the reaction by TLC analysis (you should determine an
appropriate solvent system containing ethyl acetate and hexane).
- Propose a couple of solvent conditions based on your experience so far
- To check reaction progress by TLC, you will work-up a drop of the reaction mixture.
Remove 1 drop of the reaction mixture by glass pipette (dipping the tip of the pipette in
the reaction mixture is sufficient). Rinse this drop of reaction mixture into a test tube using
ethyl acetate (<1 mL). Add water (1 mL) and agitate to mix the layers. Spot the organic
layer onto the TLC plate. Why might this mini-workup be necessary? - If the reaction is not complete after 60 min, add another portion of bromoethane (x mL, 1
equiv) and continue heating for another 60 min. Continue to monitor the reaction progress
by TLC. - When the reaction is complete or 2 hours have elapsed, remove the reaction mixture from
the hot plate and cool to room temperature. Then cool to 0 °C in an ice-bath, and quench
the reaction with water (10 mL, add dropwise initially). Add ethyl acetate (50 mL) and
extract product into the organic layer using a separatory funnel. Isolate the organic layer
and wash twice with brine solution (20 mL each wash) to remove DMF. - Dry organic layer with anhydrous Na2SO4. Filter to remove desiccant and concentrate
crude product by rotary evaporation. Label your flask and purify your compound in the
next lab period.
3 | P a g e
Week 7, Period B – Purification of Product - Determine an appropriate TLC system (ethyl acetate and hexanes) to purify your
compound by flash column chromatography (silica). Remember, the optimal Rf for elution
of product is 0.2–0.3. - Purify your product by flash column chromatography. Combine all pure reactions to obtain
the product. Record an isolated yield. Store the compound in a labeled vial until the next
lab period. If column chromatography resulted in impure factions that contain product,
collect these separately and record an approximate yield of impure product as well. - Prepare an NMR sample in deuterated DMSO for characterization using a small amount
of the purified product.
4 | P a g e
Week 8, Period A
reagent formula MW amount mmol density mol. equiv.
N-ethyl
phenothiazine 0.80 1
acetic anhydride 2 mL
85% phosphoric
acid 2 drops cat.
EXPERIMENTAL PROCEDURE: - Add N-ethyl phenothiazine (x mg, 0.80 mmol) to a screw-cap vial that is equipped with a
magnetic stirring bar. Add acetic anhydride (2 mL, y mmol) to dissolve phenothiazine with
stirring. - Add concentrated phosphoric acid (2 drops). Degas with N2 and then heat to 90 ºC for 30
min (reaction will turn dark green). After 30 min, remove the sample from heating, cool
and remove a drop to dilute for TLC analysis. Continue heating for up to 2 additional hours
to maximize conversion of the starting materials to product. - Cool the reaction to room temperature and dilute the reaction mixture with ethyl acetate
(approximately 10 mL). Filter through a short bed of silica gel to remove dark solids, wash
the solids with an additional ethyl acetate (40 mL) and collect the filtrate. - Wash the organic layer with brine (25 mL x 2).
- Dry organic extracts with anhydrous Na2SO4, filter and concentrate by rotary evaporation
to afford the crude product. - Use a drop of the crude extracts as a TLC sample to determine a suitable eluent to purify
the product (ethyl acetate and hexane) by flash column chromatography (silica).
- Would you expect the product to be more or less polar than the starting
material? Propose a suitable set of solvent conditions that can be used.
Week 8, Period B – Purification of Product
- Purify the product (ethyl acetate and hexane eluent) by flash column chromatography
(silica). Use a minimal volume (~ 1 mL) of DCM to load your compound. Combine all pure
fractions to obtain the product. Record an isolated yield.
- Dissolving the product in DCM prior to adding the material to the column is
normally considered bad practice. Why is that?
- Prepare an NMR sample in CDCl3 for characterization using a small amount of product.
- Store the compound in a labeled vial or flask in your drawer until the next lab period.
5 | P a g e
Week 7 – Pre-Lab: Paraphrase the entire experimental procedure with all the amounts (g/mg
for solids, mL for liquids) and number of moles or reagents calculated. Answer any questions
appearing throughout the procedures. [Due on Gradescope in Week 7 (May 9 – May 12) the
night before your lab period] - NaH is used as a strong base and often in conjunction with polar solvents like DMF,
DMSO and DMAc. However these NaH/solvent mixtures are known to be unstable and
can occasionally rapidly decompose with explosive outcomes! (See:
10.1021/acs.oprd.9b00276) In this reaction, we are using a small excess of NaH which
rapidly deprotonates phenothiazine under cooled conditions (to control the reaction
exotherm) to avoid this problem. It would be great to use a different solvent that avoids
this issue altogether! Perform a Scifinder search to find what other solvents have been
used for the deprotonation of phenothiazine by NaH. Hint: Use a Reaction Structure
search and use Analyze by Reagent and Solvent to find appropriate reactions. Provide
two reactions each using a different solvent, including reaction scheme and DOI for each. - Potassium tert-butoxide (KOtBu) has been used as a base for N-alkylation of
phenothiazine in other preparations, but generally requires extended reaction times at
higher temperatures. Why might KOtBu be a less efficient base in this reaction? - Which of the following phenothiazine derivatives cannot be synthesized using an
alkylation procedure similar to this experiment? There may be more than one. Briefly
explain why. For derivatives that can be synthesized by direct alkylation, indicate the
alkylating agent that can be utilized. - Draw the acid-base reaction showing protonation of acetic anhydride by phosphoric acid
(a strong acid). Pay attention to which atom is most basic. - Draw the mechanism for the acylation of N-ethyl phenothiazine using protonated acetic
anhydride. Draw all reasonable structures for the sigma complex. Only show the acylation
reaction on one side of phenothiazine.
Week 7 – Post-lab: Annotate/revise procedures with the actual steps and observations [Due
on Gradescope in Week 8 (May 16 – May 19) the night before your lab period]
Note: Only the annotated procedure that includes your observations is due this week. The full
report (or presentation) will include the experiments from Weeks 7–9. If you wrote Experiment 3
up as a full report, Experiment 4 will be completed in the form of a PowerPoint presentation (Due
Week 10).
6 | P a g e
Week 8 – Pre-Lab: Paraphrase the entire experimental procedure with all the amounts (g/mg
for solids, mL for liquids) and number of moles or reagents calculated. Answer any questions
appearing throughout the procedures. [Due on Gradescope in Week 8 (May 16 – May 19) the
night before your lab period] - What is the role of H3PO4?
- Propose a mechanism for this week’s experiment. Draw all steps and intermediates using
the appropriate arrows. - Explain the site-selectivity of this Friedel–Crafts acylation. Why are the other positions not
acylated?
Week 8 – Post-lab: Annotate/revise procedures with the actual steps and observations [Due
on Gradescope in Week 9 (May 23 – May 26) the night before your lab period]
Note: Only the annotated procedure that includes your observations is due this week. The full
report (or presentation) will include the experiments from Weeks 7–9. If you wrote Experiment 3
up as a full report, Experiment 4 will be completed in the form of a PowerPoint presentation