Synthesis and Crystallographic Characterization of a Maleimide Derivative of Tryptamine

While mechanosynthesis of the target compound, 1-[2-(1H-indol-3-yl)-ethyl]-pyrrole-2,5dione, C14 H12 N2 O2, did not yield the desired product, it instead resulted in an open intermediate. On the other hand, synthesis starting from the activated maleic anhydride yielded the final maleimide compound. The outcome of the mechanosynthesis has been evaluated by powder X-ray diffraction, and structures of both the final product and open intermediate have been confirmed using single-crystal crystallography.


Introduction
This work forms a part of our study on epigenetic modulation by DNA methyltransferases (Mtases) [1,2].It was a consequence of attempting to synthesize analogues of N-phthalyl-L-tryptophan (RG108), which are non-nucleosidic inhibitors of Mtases [3,4].
This approach led to the corresponding open product, 1o.
This approach led to the corresponding open product, 1o.
Scheme 1.Chemical diagram of compounds under study.

Mechanosynthesis
Initial attempts to obtain target maleimide 1 were based on our previous work where mechanosynthesis was used to prepare phthalimide compounds [5].In brief, the starting solid reactants, tryptamine (2) and maleic anhydride (3) (Figure 1), were ground in an equimolar ratio, either manually using a mortar and pestle or mechanically using a vibration ball mill Retsch MM400 (Retsch GmbH, Haan, Germany ) (60 min at 30 Hz, 2-10 balls added to a 2 mL plastic vial).Progress of reaction was monitored by powder X-ray diffraction (Panalytical ProX'pres, Almelo, the Netherlands, Cu radiation) (Figure 2).
Crystals 2016, 6, 153 2 of 8 [9]).To the best of our knowledge, these are the only deposited crystal structures for this, otherwise well documented, family of compounds.

Mechanosynthesis
Initial attempts to obtain target maleimide 1 were based on our previous work where mechanosynthesis was used to prepare phthalimide compounds [5].In brief, the starting solid reactants, tryptamine (2) and maleic anhydride (3) (Figure 1), were ground in an equimolar ratio, either manually using a mortar and pestle or mechanically using a vibration ball mill Retsch MM400 (Retsch GmbH, Haan, Germany ) (60 min at 30 Hz, 2-10 balls added to a 2 mL plastic vial).Progress of reaction was monitored by powder X-ray diffraction (Panalytical ProX'pres, Almelo, the Netherlands, Cu radiation) (Figure 2).Crystals 2016, 6, 153 2 of 8 [9]).To the best of our knowledge, these are the only deposited crystal structures for this, otherwise well documented, family of compounds.

Mechanosynthesis
Initial attempts to obtain target maleimide 1 were based on our previous work where mechanosynthesis was used to prepare phthalimide compounds [5].In brief, the starting solid reactants, tryptamine (2) and maleic anhydride (3) (Figure 1), were ground in an equimolar ratio, either manually using a mortar and pestle or mechanically using a vibration ball mill Retsch MM400 (Retsch GmbH, Haan, Germany ) (60 min at 30 Hz, 2-10 balls added to a 2 mL plastic vial).Progress of reaction was monitored by powder X-ray diffraction (Panalytical ProX'pres, Almelo, the Netherlands, Cu radiation) (Figure 2).Upon grinding, characteristic diffraction peaks of the reactants were replaced by peaks associated to the new ground product (Figure 2).Recrystallization of the ground solid from a saturated solution in a MeOH/toluene mixture led to small single crystals useful for crystal structure determination.X-ray diffraction analysis revealed that the compound obtained upon grinding is the open maleamide intermediate 1o (Section 2.2).This result is consistent with similar observations that we made for phthalimides [5].Powder diffractogram simulated on the basis of the single crystal structure coordinates (Figure 2) corresponds to the experimental data recorded on the solid obtained after grinding, confirming that the bulk powder corresponds to 1o.Conversion in almost quantitative (>95%) as judged from NMR and powder X-ray diffraction data.
We expected to get the final, closed maleimide 1 on heating the intermediate [5].Despite several attempts, we were unable to convert intermediate 1o into the target compound 1.
A reasonable explanation for the failure of the formation of the desired product using unactivated reactants is probably linked to the reduced nucleophilicity of the conjugated amide intermediate 1o.Indeed, thermal and uncatalyzed amide formation has been scarcely reported [10,11] especially when the nucleophile is conjugated amide.

Synthesis Using Activated Maleic Acid
As we were unsuccessful in mechanosynthesis of 1, we developed a procedure (Figure 3) based on a similar approach described in the literature [12].Upon grinding, characteristic diffraction peaks of the reactants were replaced by peaks associated to the new ground product (Figure 2).Recrystallization of the ground solid from a saturated solution in a MeOH/toluene mixture led to small single crystals useful for crystal structure determination.X-ray diffraction analysis revealed that the compound obtained upon grinding is the open maleamide intermediate 1o (Section 2.2).This result is consistent with similar observations that we made for phthalimides [5].Powder diffractogram simulated on the basis of the single crystal structure coordinates (Figure 2) corresponds to the experimental data recorded on the solid obtained after grinding, confirming that the bulk powder corresponds to 1o.Conversion in almost quantitative (>95%) as judged from NMR and powder X-ray diffraction data.
We expected to get the final, closed maleimide 1 on heating the intermediate [5].Despite several attempts, we were unable to convert intermediate 1o into the target compound 1.
A reasonable explanation for the failure of the formation of the desired product using unactivated reactants is probably linked to the reduced nucleophilicity of the conjugated amide intermediate 1o.Indeed, thermal and uncatalyzed amide formation has been scarcely reported [10,11] especially when the nucleophile is conjugated amide.

Synthesis Using Activated Maleic Acid
As we were unsuccessful in mechanosynthesis of 1, we developed a procedure (Figure 3) based on a similar approach described in the literature [12].In brief, a solution of monomethylmaleate (4) (5.204 g, 40 mmol) and N-hydroxysuccinimide (5) (4.61 g, 40 mmol) in DME (15 mL) was cooled to 0 °C.Dicyclohexylcarbodiimide (DCC, 8.25 g, 40 mmol) was added and stirring was continued at this temperature for 4 h.The reaction mixture was allowed to stand for 2 h in a refrigerator and then filtered.The solution was concentrated under reduced pressure.The residue was triturated in Et2O/hexane, filtered, and then dried in vacuo to afford solid methyl succinimidylmaleate (6).Tryptamine (2) (0.32 g, 2.0 mmol) and sodium carbonate (1.06 g, 10 mmol) were dissolved in water (15 mL), and then in acetonitrile (25 mL).Methyl succinimidylmaleate (6) (0.45 g, 2 mmol) was added, and the mixture was stirred for 4 h.The solution was acidified to pH 1 with 2N HCl, diluted with EtOAc (100 mL), and washed with 1N HCl (2 × 100 mL) and water (2 × 100 mL).The organic phase was dried with MgSO4, filtered, and concentrated in vacuo to give final maleimide 1 (Figure 3).Final product was recrystallized from a concentrated solution in acetonitrile at room temperature.The yield of this reaction has not been optimized but was already very good (>80%), consistent with data from the literature [12].Single crystals suitable for crystallography were obtained and led to the structure described in the following section.In brief, a solution of monomethylmaleate (4) (5.204 g, 40 mmol) and N-hydroxysuccinimide (5) (4.61 g, 40 mmol) in DME (15 mL) was cooled to 0 • C. Dicyclohexylcarbodiimide (DCC, 8.25 g, 40 mmol) was added and stirring was continued at this temperature for 4 h.The reaction mixture was allowed to stand for 2 h in a refrigerator and then filtered.The solution was concentrated under reduced pressure.The residue was triturated in Et 2 O/hexane, filtered, and then dried in vacuo to afford solid methyl succinimidylmaleate (6).Tryptamine (2) (0.32 g, 2.0 mmol) and sodium carbonate (1.06 g, 10 mmol) were dissolved in water (15 mL), and then in acetonitrile (25 mL).Methyl succinimidylmaleate (6) (0.45 g, 2 mmol) was added, and the mixture was stirred for 4 h.The solution was acidified to pH 1 with 2N HCl, diluted with EtOAc (100 mL), and washed with 1N HCl (2 × 100 mL) and water (2 × 100 mL).The organic phase was dried with MgSO 4 , filtered, and concentrated in vacuo to give final maleimide 1 (Figure 3).Final product was recrystallized from a concentrated solution in acetonitrile at room temperature.The yield of this reaction has not been optimized but was already very good (>80%), consistent with data from the literature [12].Single crystals suitable for crystallography were obtained and led to the structure described in the following section.

Structural Commentary
The open intermediate 1o (Figure 4a, Table 1) was obtained by grinding maleic anhydride and tryptamine (Figure 1).The structure of the final target molecule 1 (Figure 4b, Table 1) was obtained using activated methyl succinimidylmaleate (Figure 3) as confirmed by determination of its crystal structure.

Structural Commentary
The open intermediate 1o (Figure 4a, Table 1) was obtained by grinding maleic anhydride and tryptamine (Figure 1).The structure of the final target molecule 1 (Figure 4b, Table 1) was obtained using activated methyl succinimidylmaleate (Figure 3) as confirmed by determination of its crystal structure.2) are intermediate between single and double bonds, suggesting electronic delocalization within this ring.
In the open intermediate 1o, nitrogen atom N1 binds to C1=O and forms an amide.This group is conjugated to the C2=C3-C4OOH part, as deduced from the bond lengths (Table 2).Within the maleamide moiety, a strong O3-H3O•••O1 intra-molecular hydrogen bond is observed (Table 3).

D-H•••A D-H H•••A D•••A D-H•••A
Compound 1 N2-H2 Conformation of the two compounds is distinct.In molecule 1, the planar indole ring is almost perpendicular to the maleimide ring (acute angle between planes = 68.6(2)• ).In the open intermediate 1o, the planar indole heterocycle is almost parallel to the maleamide moiety (acute angle between planes = 6.9(2) • ).This is also reflected in the values of the torsions angles defining the conformation of the molecules (Table 2).
In both compounds, nitrogen N2 of the indole ring serves as a H-bond donor (Table 3).For compound 1o, the position of H atom on oxygen atom O3 of the carboxylic acid was unambiguously determined from the residual electron density.This H atom is involved in a strong intramolecular H bond involving O3-H and the carbonyl oxygen atom (O1) of the amide moiety (Table 3).
As a perspective to this work, liquid-assisted grinding could be tested by addition of a few drops of solvent during grinding.Mechanosynthesis starting from activated reactants is another possible alternative.In conclusion, 1-[2-(1H-Indol-3-yl)-ethyl]-pyrrole-2,5-dione (1), a potential DNA methyltransferase inhibitor, has been successfully synthesized using activated reactants and synthesis in solution.Direct mechanosynthesis, starting from the unactivated reactant and using dry grinding, led to the amide intermediate 1o.This molecule did not convert into the cyclized final product in contrast with results obtained for phthalimides using dry grinding of corresponding unactivated anhydrides and amines.Reduced nucleophilicity of the conjugated amide intermediate is probably part of the explanation.Restricted conformation of molecule 1o, in the solid, and crystal packing further play a role in the stability of the intermediate product.
As a perspective to this work, liquid-assisted grinding could be tested by addition of a few drops of solvent during grinding.Mechanosynthesis starting from activated reactants is another possible alternative.

Scheme 1 .
Scheme 1.Chemical diagram of compounds under study.

Figure 2 .
Figure 2. Powder X-ray diffractograms showing the outcome of the grinding experiment.Starting solids (maleic anhydrate and tryptamine), solid obtained after grinding (60 min at 30 Hz), solid obtained after heating (30 min at 140 °C).Diffractograms simulated on the basis of the single crystal structures of 1 and 1o are presented for comparison.

Figure 2 .
Figure 2. Powder X-ray diffractograms showing the outcome of the grinding experiment.Starting solids (maleic anhydrate and tryptamine), solid obtained after grinding (60 min at 30 Hz), solid obtained after heating (30 min at 140 °C).Diffractograms simulated on the basis of the single crystal structures of 1 and 1o are presented for comparison.

Figure 2 .
Figure 2. Powder X-ray diffractograms showing the outcome of the grinding experiment.Starting solids (maleic anhydrate and tryptamine), solid obtained after grinding (60 min at 30 Hz), solid obtained after heating (30 min at 140 • C).Diffractograms simulated on the basis of the single crystal structures of 1 and 1o are presented for comparison.

Table 1 .
Experimental details of the crystal structure determination.

Table 1 .
Experimental details of the crystal structure determination.