Drug Discovery through Simple Analog Synthesis

Analogue library synthesis is one of the major methods of drug discovery. This approach is
based on creating structural derivatives of compounds already possessing relevant biological
activity. Today, you will be synthesizing an analogue of the drug Tramadol. Tramadol is a muopioid prodrug that is, it has a biologically active secondary metabolite (o-desmethyltramadol)
that is a more potent opioid agonist than the original drug itself. Tramadol is a 4-phenylpiperidine analogue of codeine, and while it is most
commonly known for its non-habit-forming analgesic
properties, it has a vast array of off-label applications.
These include antibacterial, antitussive,
immunosuppressant, and antidepressant properties, to
name a few.1,2,3,4 It is possible that varying the
structure of such an active compound even slightly
may produce radically diverse biological effects, and
to this end, biochemistry students will later screen
your compound for anticancer activity.5 You will
create your own Tramadol analogue via a two-step synthesis starting with cyclohexanone. The
first step is a Mannich reaction, by which you will vary the amino position of the opioid core
structure by selecting a
different amine than your
classmates. The second
step is a Grignard reaction,
by which you will alter the
aryl position of the opioid
structure. Acidic workup
will produce the
hydrochloride salt of your
Tramadol analogue, which is the clinically administered form of this drug.
Pre-Lab Questions
Part I: The Mannich Reaction

The Mannich Reaction (Scheme 2), depicted above in a general reaction scheme, is the first step
of your synthesis.6 The procedure is as follows. To a clean and dry 100 mL round-bottom flask,
add 50 mmol cyclohexanone, 25 mmol paraformaldehyde, and 25 mmol of your chosen amine.
Use acetic acid (conc.) as solvent, and drop a stir bar in before refluxing for ~2-3 hours. Follow
the reaction by TLC. Allow the reaction to stir at room temperature overnight in the hood. After

taking a TLC of the crude reaction mixture, move the RBF from the reflux setup to a distillation
setup, and distill the acetic acid. Use acetone and hydrochloric acid to produce the HCl salt of
your compound. If recrystallization does not occur, a two-solvent system such as
methanol/diethyl ether may be necessary. Filter the white to off-white crystals. Obtain melting
point and percent yield data. Save your product for use in Part II. Calculate the stoichiometric
amount of magnesium you will need for Part II, and put a round bottom flask and a reflux
condenser in the oven for the next lab session.
*Note When calculating the stoichiometric amount of reactants needed for Part II based on
your yield from Part I, keep in mind that the hydrogen chloride component does not appear in
the free-base form of your product used for the Grignard reaction.
Part II: The Grignard Reaction

The Grignard Reaction (Scheme 3), shown above, is the second step in your synthesis of a
Tramadol analogue.6 Before performing the reaction itself, you will need to generate the
organometallic reagent to be used in the reaction. Add freshly distilled THF to the stoichiometric
mass of magnesium needed for the reaction (stoichiometric ratio = 3 parts Mg : 1 part Mannich
base) in an oven-dried RBF and reflux condenser (sealed with a rubber septum.) Reflux the
THF/Mg mixture under a nitrogen atmosphere until the magnesium dissolves, then add the
organic component of your organometallic (also in a 3:1 stoichiometric ratio with the Mannich
base) slowly over the course of 20-30 minutes. While refluxing your Grignard reaction, you will
prepare your Mannich product for addition. Add toluene and potassium hydroxide to your
Mannich product and extract the organic layer three times with KOH in a separatory funnel. Dry
the organic layer over sodium or magnesium sulfate, filter the drying agent, and collect the
remaining organic layer in a flask. After the magnesium has dissolved in your reaction flask, put
the reaction on ice, draw the organic layer with your Mannich free base into a syringe, and add it
slowly to the reaction vessel in small increments. Once all of your Mannich base has been
added, stir the reaction for an additional XX hours. Quench the reaction with a saturated solution
of aqueous ammonium chloride, remove the aqueous layer in a separatory funnel, and distill the
organic layer to remove solvent. Crystallize your product in the same manner as in the Mannich
reaction, obtain melting point and percent yield data, and save your product in a labeled vial for
the biochemistry students.
Post-Lab Questions

1. Calculate the atom economy of each step of the reaction and the overall synthesis.
2. Comment on the apparent degree of purity of your product from each step.
3. What types of side products would you expect from this synthesis?
References
1. Tamanai-Shacoori, Z.; Shacoori, V.; Jolivet-Gougeon, A.; Van, J. V.; Repere, M.; Donnio,
P.; Bonnaure-Mallet, M. Anesth Analg 2007, 105(2), 524-527.
2. Hennies, H. H.; Friderichs, E.; Schneider, J. Arzneimittelforschung 1988, 38(7), 877-880.
3. Sacerdote, P.; Bianchi, M.; Gaspani, L.; Manfredi, B.; Maucione, A.; Terno, G.;
Ammatuna, M.; Panerai, A. Anesth Analg 2000, 90(6), 1411-1414.
4. Kalra, B. S.; Tayal, V.; Chawla, S. Indian J Psychiatry 2008, 50(1), 51-53.
5. Bhat, U. G.; Halasi, M.; Gartel, A. L. PLoS ONE 2009, 4(5), e5592.
6. Kurti. L., and Czako, B. Strategic Applications of Named Reactions in Organic
Synthesis; Elsevier Academic Press: New York, 2005.