The Michael Reaction of Enaminones with N-( p-tolyl )-maleimide : Synthesis and Structural Analysis of Succinimide-enaminones

Succinimido enaminonas foram sintetizadas através da reação de enaminonas e a N-(p-toluil)maleimida em rendimento moderado a bom. A ação antibacteriana destes compostos contra E. coli e S. aureus foi avaliada, mas a atividade antibacteriana não foi significativa. A regioquímica das succinimido enaminonas foi racionalizada empregando-se a teoria dos orbitais moleculares de fronteira. A estrutura cristalina de um dos compostos foi determinada e foram observadas várias pontes de hidrogênio intere intramolecular do tipo NH...O e CH...O.


Introduction
Over the years, the Michael addition of enaminones has been intensively studied and many synthetic achievements were described. 1For instance, the stereocontrolled elaboration of quaternary carbon centers followed by the aza-annulation of enaminones has been used in the synthesis of many nitrogen-containing compounds, where the Michael addition was the key step. 2 Among the Michael acceptors studied (e.g.nitroolefins, 3 maleic anhydrides 4 ) the maleimides have the most complex reaction pattern, highly dependent of the nitrogen substituents at the maleimides and at the enaminones, Scheme 1. 5 Additionally to the complex behavior, this kind of reaction caught our attention due its potential in the synthesis of succinimide-containing enaminones derivatives such as 4. 5 The antibacterial activity of fivemembered imides is well documented and some structural modifications to improve this one, such as the presence of N-aryl groups have been suggested. 6As part of our recent research program aimed at the synthesis of biological active substances 7 such as anticonvulsant and antibacterial compounds and to provide insight into the reactivity of enaminones toward N-aryl-maleimides, we decided to study the Michael reaction of enaminones with N-(p-tolyl)maleimide, in search for a general method to the synthesis of N-aryl-succinimide-containing enaminones.Although Michael addition of enaminones with N-H and N-alkylmaleimides had been described three decades ago, studies with N-aryl maleimides are scarce. 5Afterward, to the best of our knowledge, there are no structural and biological studies with succinimide-enaminones derivatives.

Results and Discussion
N-(p-tolyl)-maleimide reacted smoothly with enaminones 6a-c in benzene under reflux.Crystalline solids were isolated and these materials were 1:1 adduct as indicated by mass spectrum and proton NMR integration.The formation of a succinimide-enaminone was proposed by comparison of spectral data with model compounds 3 and 4. 5 Although these reported compounds present similar IR and 1 H NMR spectra, their 13 C NMR show significant differences.Thus, the chemical shifts of the α and β carbons relative to the carbonyl group of the enaminone moiety are 106.4ppm and 154.8 ppm for 3, while for 4 they appear at 90.0 ppm and 161.3 ppm, respectively. 5In the adducts here obtained, the 13 C chemical shifts of Cα and Cβ are in agreement with structure 8 analogous to 4 (see Experimental), Scheme 2. The 1 H NMR spectra contained a low field N-H (8.64-9.94ppm) which suggests its participation in intramolecular hydrogen bonding.Despite the reportedly obtained E configuration to the model compound 4, 5 we assigned the Z configuration to 8 because E-and Z-configurational isomers of enaminones are well distinguished by typical N-H chemical shifts (E-isomer: 4.1-6.5 ppm; Z-isomer: 9.5-12.0ppm). 8Moreover, the structure of the addition products 8a was unambiguously confirmed by X-ray analysis and the Z configuration corroborated, as shown in Figure 1.This reaction proved to be sensitive to N-acyl substitution in 6.While moderated to good yields were obtained with enaminones 6a-c, N-(p-tolyl)-maleimide failed to react with enaminone 6d under the condition employed, even at prologated heating, Scheme 2.
The formation of 8a-c may be visualized as occurring by reaction of the enaminone-Cα at the electrophilic C-3 of the N-(p-tolyl)-maleimide, followed by proton transfer.However, at this point it is not clear the controlling factors that modulate maleimide and enaminone reactivity to form succinimide-enaminones or other nucleus present in  Schemes 1 and 2. With the objective of shedding new light on this question we undertook a frontier orbital treatment in combination with the hardness-softness concept.Electronic parameters of model maleimides and enaminones were calculated using the AM1 method, 9 as implemented in the MOPAC 6.0 package, 10 and are shown in Table 1.According to Parr-Pearson principle of absolute hardness, 11 N-(p-tolyl)-maleimide is softer than N-H and N-alkyl maleimides because of the smaller HOMO-LUMO energy gap.Thus, it would appear that reaction of soft enaminones and N-aryl maleimides is kinetically favored, and frontier orbital control, and not charge control, should govern the process.In this way, an attack of enaminone-Cα at the N-(p-tolyl)-maleimide-C3, wherein the largest frontier orbital coefficients are located (see Table 1), results in the regiochemistry observed in Scheme 2.
The antibacterial activity of succinimide-enaminones 8a-c was evaluated against Escherichia coli ATCC 8739 (gram-negative) and Staphilococus aureus ATCC 6538 (gram-positive), and the minimal inhibitory concentration (MIC) was determined.However, no activity was observed to compounds 8a-c even at 1000 mg mL -1 .Efforts are underway to elucidate the structural-activity relationship that can improve the antibacterial activity of succinimideenaminones derivatives.

Experimental
Melting points were determined on a Karl Kolb apparatus and are uncorrected.Infrared spectra were recorded as KBr discs on a FT-IR BOMEM MB100 instrument.NMR spectra were obtained for 1 H at 300 MHz and for 13 C at 75 MHz using a Varian Gemini 300 or a Bruker AC300P spectrometers at Instituto de Química/UNICAMP.Chemical shifts are reported in ppm units downfield from reference (internal TMS).MS spectra were measured on a SHIMADSU CG-MS QP-5050 spectrometer at 70 eV.Elemental analyses were performed on a 2400 CHN Perkin Elmer instrument at Instituto de Química/UNICAMP.The single crystal X-ray data collection was done with a Nonius CAD-4 diffractometer at Instituto de Física-SC/USP.Enaminones 6a, 6b, 6c, 6d and N-(p-tolyl)-maleimide were prepared according to known procedures. 14
Crystallographic data (excluding structure factors) for the structures in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 173681.Copies of the data can be obtained, free of charge via www.ccdc.cam.ac.uk/ conts/retrieving.html(or from the Cambridge Crystallographic Data Centre, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK ; fax: +44 1223 336033 ; or email: deposit@ccdc.cam.ac.uk).
Antibacterial assay.The bacterias used in this experiment were Escherichia coli ATCC 8739 (gramnegative) and Staphilococus aureus ATCC 6538 (grampositive).Antibacterial activity was measured using a dilution in agar technique. 17The compounds (20 mg) were solubilized in 1 mL of dimethyl sulfoxide (DMSO) and serially two-fold diluted in nutrient agar (peptone, beef extract and agar), to obtain a concentration range of 62.5-1000 µg mL -1 .The control was plates of nutrient agar containing only DMSO diluted in the same way, which did not influence the bacterial growth.The bacterias from broth medium were suspended in sterile physiological Tris buffer (pH 7.4, 0.05 mol mL -1 ), homogenised and adjusted to an OD (530 nm) of 0.05 (equivalent to 1 X 10 6 UFC mL -1 ).This suspension (3 µL) was inoculated using an automatic micropippete for the test in the agar plates (diameter: 10 cm).The plates were incubated at 37 o C for 24 h.The minimal inhibitory concentration (MIC) was defined as the minimal concentration of the compounds which completely inhibited the visible growth of the bacterias.The positive control was chloranphenicol used in the same technique.All assays were tested in duplicate.

Figure 1 .
Figure 1.X-ray crystallographic structure of molecule 1 of compound 8a.Displacement ellipsoids are shown at the 30% probability level.Only the H-atoms involved in H-bonds are shown with arbitrary size.Two intramolecular H-bonds are shown with broken lines.

Figure 3 .
Figure 3. Intermolecular C-H...O interactions around an inversion center.Only the succinimide rings are shown for clarity.

Table 1 .
Electronic parameters calculated by AM1 method for maleimides and enaminones