A Direct, Early Stage Guanidinylation Protocol for the Synthesis of Complex Aminoguanidine-containing Natural Products

The overall goal of this direct synthetic approach which is applied here to prepare the marine natural product, clavatadine A, is to reduce the number of chemical transformations needed to prepare linear aminoguanidine-containing molecules. This method can be used to prepare natural and engineered compounds quickly and efficiently to further understand their biology which may lead to new and better medicines for human health. The main advantage of early stage guanidinylation is that a mass guanidine can remain intact if an appropriate protecting group strategy is devised to avoid chemo selectivity issues in subsequent reactions.

To begin this procedure weigh 0.933 grams of 2, 4-dibromo homogentisic acid lactone on an analytical balance. Add this solid to a round bottomed reaction flask containing a magnetic stir bar under a nitrogen umbrella. Add 30 milliliters of anhydrous dichloromethane to the flask.

Stir at ambient temperature to dissolve the solid. Next, add 0.103 milliliters of Hunig’s base to the flask by syringe. Cover the opening of the round bottomed flask containing the previously prepared un-purified isocyanate with a rubber septum.

Then connect the flask to a Schlenk line, and purge it with nitrogen gas for 10 minutes. To the flask containing the isocyanate, add 30 milliliters of anhydrous dichloromethane. Swirl the flask at ambient temperature to dissolve the isocyanate.

Following this directly connect the two flasks by puncturing each septum with either beveled metal tip of an 18 inch long 20 gauge metal cannula. Remove the nitrogen inlet from the flask containing the dibromolactone solution and insert a 2.5 centimeter 16 gauge exit needle through the septum. Then close the bubbler that serves as the exit port of the Schlenk line.

To perform the cannula transfer using positive inner gas pressure safely and avoid excess pressure buildup in the Schlenk line, ensure that there is always an unobstructed route for the gas to escape. Lower one end of the metal cannula into the isocyanate solution and transfer the entire solution to the flask containing the dibromolactone solution over approximately one hour. Rinse the flask that contained the isocyanate solution with two successive five milliliter portions of dichloromethane.

Transfer each dichloromethane rinse by cannula to the flask now containing the reaction mixture. Next, remove the exit needle while simultaneously reopening nitrogen flow to the bubbler. Transfer the nitrogen inlet from the flask that contained the isocyanate solution to the flask containing the reaction mixture.

Remove the cannula and stir the reaction mixture at ambient temperature for three hours. After three hours expose the reaction mixture to air by removing the septum. Then remove the magnetic stir bar using a stir bar retriever.

Evaporate the liquid in the flask using a rotary evaporator, with the bath temperatures set to 40 degrees celsius and the rotation set to 120 RPM. Following this purify the crude product by flash column chromatography using a gradient elution of dichloromethane and diethyl ether through a stationary phase of silica gel. Wet pack the column by making a slurry consisting of 60 grams of silica gel and a sufficient volume of dichloromethane to enable the slurry to be poured into the column.

Use air pressure to force the eluent through the column, such that the e-flux flows as a gentle stream of liquid. Next, dissolve the crude product in a minimum volume of dichloromethane. Carefully load the solution into the column without disturbing the silica gel.

After tapping the column to ensure the top of the silica gel is flat, drain the eluent so that it reaches the level of the silica gel. To avoid disturbing the silica gel during elution of the column, add sand to the top of the silica gel to form a cylinder that is approximately 0.5 to one centimeter in height. Then add a few milliliters of dichloromethane to wet the sand.

After draining the eluent, fill the column with dichloromethane. Use air pressure to force the eluent through the silica gel as previously described. Collect fractions until the un-reacted dibromolactone has completely eluded from the column.

To determine when this has occurred, spot one out of every few column fractions on a TLC plate. When the un-reacted dibromolactone has eluded from the column, replace the eluent with a 90 to 10 solution of dichloromethane and diethyl ether. Continue to collect fractions until the desired carbamate product has completely eluded from the column.

Combine all fractions containing the carbamate in tared round bottomed flask. Evaporate the solvent using a rotary evaporator. Dry the resulting foamy solid under high vacuum.

Then analyze the product by proton and carbon anamar spectroscopy in deuterated chloroform. At this point weigh 1.205 grams of carbamate on an analytical balance. Add this solid to a clean round bottomed reaction flask containing a magnetic stir bar.

Next add 12 milliliters of tetrahydrofuran to the flask. Then add 48 milliliters of one molar hydrochloric acid at ambient temperature with stirring. Gently place a 24/40 ground glass stopper to cover the aperture of the flask.

Submerse the flask in a water bath that has been preheated to 30 degrees celsius on a temperature controlled hotplate. After heating for 20 hours, vacuum filter the resulting suspension through a medium porosity sintered glass funnel into a clean tared round bottomed flask. Following this, evaporate the yellow colored solution in the flask using a rotary evaporator, with the bath temperature set to 50 degrees celsius and the rotation set to 120 RPM.

Dry the resulting yellow to peach colored amorphous solid to constant weight under high vacuum. Facilitate drying by heating the flask in a 40 degree celsius water bath. Finally, analyze the resulting hydrochloride salt of clavatadine A by proton and carbon anamar spectroscopy in anhydrous deuterated dimethyl sulfoxide.

Direct guanidinylation of a commercially available alpha omega diamine followed by reaction with triphosgene afforded the reactive isocyanate eight as the linear portion of clavatadine A in high yield. When isocyanate eight was combined with dibrominated phenol three in the presence of a catalytic amount of the Hunig’s base, carbamate formation provided compound nine in moderate yield. Re-isolation of dibromophenol three after chromatography suggests that perhaps some of the isocyanate decomposed under the reaction conditions.

Or the product may of partially hydrolyzed during workup or chromatography. Hydrolysis of the lactone under acidic conditions was accompanied by deep protection of the guanidine protecting groups, leading to the final molecule, clavatadine A.Once mastered this general technique can be used to synthesize linear amino guanidine containing compounds. Careful planning will establish whether subsequent chemical reactions are compatible with the guanidine protecting groups.

Lastly, it’s important to apply proper anhydrous technique when conducting non-aqueous reactions. Don’t forget that triphosgene and bromine are extremely hazardous chemicals. Consult each chemical’s safety data sheet before working with them.

To limit exposure only handle these reagents in a working fume hood and apply appropriate personal protective equipment.