Design and Synthesis of Sustain-Acting Melatonin Prodrugs

Twelve melatonin amide prodrugs aiming at prolonging the action of melatonin in vivo by improving its half-life were designed and synthesized. Using an 80% human plasma model, it was found that the aliphatic amide derivatives were relatively stable and melatonin release from these compounds was not sufficient with melatonin release percentage. After 4-hour incubation with 80% human plasma, the melatonin release percentage achieved only approximately less than 20%. In contrast, the N-succinyl and Nglutaroylmelatonin derivatives (compounds 11 and 12, resp.) were found to release melatonin in much higher rates. After 3-hour incubation in 80% human plasma, the melatonin release rates from 11 and 12 were found to be 67.3 and 75.6%, respectively. From these results, theN-succinyl andN-glutaroylmelatonin derivatives (compounds 11 and 12) could be promising as sustained release prodrugs of melatonin.


Introduction
Melatonin (-acetyl-5-methoxytryptamine, Figure 1) is a hormone produced in the pineal gland located in the center of the brain.It is interesting that the secretion of melatonin is not restricted to mammals, but it is also produced in nonmammalian vertebrates, in some invertebrates, and even in many plants, with the same molecular structure [1].It is one among many endogenous body environment sensors.Interest in this small molecule has increased dramatically in the past several decades.As a result, a number of important biological actions of melatonin have been reported.Several studies have demonstrated that this compound can act as a maternal fetal synchronizer [2] or a chemical mediator of photoperiodic information [3,4].Melatonin is also a modulator of various endocrinological, neurophysiological, and behavioral functions in vertebrates [5][6][7][8].Especially, melatonin has been shown to regulate retinal vertebrate physiology and to function as an inducer of sleep in humans.In this respect, melatonin acts by a unique mechanism of action by targeting specific receptors in the brain that are responsible for controlling the body's sleep-wake cycle [5].In addition, this compound can influence the circadian rhythm in reptiles, birds, and mammals including human.Recently, it has been observed that melatonin level is found to decline with age.This observation leads many scientists to believe that the lack of melatonin may play a role in the development of age related disorders.Among the diseases that melatonin is thought to affect are Alzheimer's disease, glucose intolerance, impaired immune function, and cancer [9].
Due to diverse biological properties mentioned above, melatonin has received a great deal of attention in the last decade.Various forms of over-the-counter drugs or food supplements are available with therapeutic indications including synchronization of disturbed circadian rhythms such as jetlag [10], sleep-wake cycle [11], seasonal disorders [12], and winter depression [13].
In clinical setting, it is noted that toxicity of melatonin is remarkably low, and no serious negative side effects of melatonin have been reported, so far [14].The main disadvantage of melatonin is its poor pharmacokinetic profile.Though the bioavailability of melatonin achieves 30-50%, in vivo, melatonin is quickly metabolized with a half-life of only 30 to 50 minutes [15], which greatly limits its clinical value.
With the aim to improve the pharmacokinetic profile of melatonin, we have designed a series of sustain-acting melatonin prodrugs (Figure 2).This paper reports the results of synthesis and preliminary evaluation of melatonin release from these potential prodrugs.

Results and Discussion
Compounds 1-10, the  1 -acylated melatonin derivatives with acyl chains from 2 to 10 C (1-9) or s-butyloxycarbonyl (10), were synthesized from melatonin by reacting with appropriate acyl chlorides in dimethylformamide used as a solvent (Scheme 1).Conventional methods using pyridine or triethylamine as bases were not successful.It was found that sodium hydride gave the best results, affording the target compounds in average to good yields (65-87%).Among the  1 -acylated melatonin derivatives (1-9), compound 1 had been synthesized previously [16].
Two additional compounds 11 and 12 bearing succinyl and glutaryl moieties were synthesized by a similar protocol, just using succinic anhydride or glutaric anhydride instead of acyl chloride (Scheme 2).
All the synthesized compounds were preliminarily evaluated for the ability to release melatonin using an 80% human plasma assay [17].In these experiments, each compound was incubated with an 80% human plasma solution (pH 7.4).After each interval of time (1, 2, 3, and 4 hours) an aliquot of 500 L from each compound's solution was withdrawn and processed to give a clear supernatant which was then analyzed by HPLC to determine the melatonin release rate.It was found that compounds 1-10 did not release melatonin efficiently.After 4 hours, the percentages of melatonin release from these compounds achieved only less than 20% (Table 1).For intended use of these compounds as sleep inducers, these melatonin release percentages were clearly not appropriate.Thus, it seemed that a proposed release mechanism for melatonin, catalyzed by amidases (Figure 3), was not efficient enough.

Conclusion
We have designed and synthesized twelve melatonin amide prodrugs aiming at prolonging the action of melatonin in vivo by improving its half-life.The  1 -succinyl and  1glutaroylmelatonin derivatives (compounds 11 and 12) could be promising as sustained release melatonin prodrugs.General Procedures for Synthesis of Compounds 1-10.In a 50 mL round bottom flask, sodium hydride was placed (120 mg, 3 mmoL, 60% in oil).Approximately 3 mL of nhexane (anhydrous) was added.After gentle shaking, the nhexane layer was discarded.A solution of melatonin (0.46 g, 2 mmoL) dissolved in 5 mL of dimethylformamide (DMF) was added in portion to the reaction flask.The resulting mixture was stirred at room temperature for 30 minutes then cooled to 0-5 ∘ C in an ice bath.Then, acyl chloride (3 mmoL) was dropwise added over 10-15 minutes into the reaction mixture to maintain the temperature in the reaction flask below 5 ∘ C.After the addition was complete, the temperature of the reaction mixture was raised to about 80-100 ∘ C, and stirring was continued for about 24 hours.After that, the reaction mixture was cooled and slowly transferred into a cold solution of sodium bicarbonate (10%, 100 mL).

Experimental Protocols
Precipitates were collected and recrystallized from n-hexaneacetone to furnish the target compounds 1-10.

Synthesis of 4-(3-(2-Acetamidoethyl)-5-methoxy-1H-indol-1yl)-4-oxobutanoic Acid
After gentle shaking, the n-hexane layer was discarded.A solution of melatonin (0.46 g, 2 mmoL) dissolved in 5 mL of dimethylformamide (DMF) was added in portion to the reaction flask.The resulting mixture was stirred at room temperature for 30 minutes then cooled to 0-5 ∘ C in an ice-bath.Then, succinic anhydride (2.2 mmoL) was added in portions into the reaction mixture.After the addition was complete, the temperature of the reaction mixture was raised to about 80-100 ∘ C, and stirring was continued for about 24 hours.After that, the reaction mixture was cooled and slowly transferred into 50 mL of crushed-ice water.pH was adjusted to 2 by a solution of 5% HCl.Precipitates were collected and recrystallized from ethanol to furnish the target compound.

Determination of the Hydrolysis Percentages of Compounds.
The hydrolysis percentage of each compound in an 80% human plasma (pH 7.4) was determined using previously reported method [16].Briefly, a solution of 20 mg of melatonin or its derivatives was prepared in acetonitrile (5 mL) and was added to 95 mL of 80% human plasma (pH 7.4, prepared by mixing plasma with phosphate buffer pH 7.4 in a ratio of 4 to 1).An aliquot of 10 mL of this solution was withdrawn and kept in test tubes maintained at 37 ± 0.5 ∘ C.After each interval of time (1, 2, 3, and 4 hours) an aliquot of 500 L was withdrawn from each tube and transferred to a microcentrifuge tube (Eppendorf 's tube).The tubes were placed in freezing mixture in order to stop any further hydrolysis, followed by vortexing thoroughly for 5 min.After vortexing, the tubes were centrifuged at high speed (10000 rpm) for 5 min.A clear supernatant (20 mL) obtained from each tube was used for HPLC analysis with ODS C18 column, mobile phase including acetonitrile and water (45 : 65), and UV detector set at 257 nm wavelength to detect peaks of melatonin and its derivatives.Compounds concentration and percentage in a mixture were quantified by peak area calculation and analysis.

Figure 4 :
Figure 4: Illustration of self-immolation release of melatonin from compounds 11 and 12.

Table 1 :
Percentages of melatonin release from compounds 1-12 after incubation with 80% human plasma for different time intervals.