Synthesis of Chromeno[3,4-b]piperazines by an Enol-Ugi/Reduction/Cyclization Sequence

Keto piperazines and aminocoumarins are privileged building blocks for the construction of geometrically constrained peptides and therefore valuable structures in drug discovery. Combining these two heterocycles provides unique rigid polycyclic peptidomimetics with drug-like properties including many points of diversity that could be modulated to interact with different biological receptors. This work describes an efficient multicomponent approach to condensed chromenopiperazines based on the novel enol-Ugi reaction. Importantly, this strategy involves the first reported post-condensation transformation of an enol-Ugi adduct.


Introduction
Peptidomimetics are molecules structurally related to peptides that can interact at the same receptors of their prototypes [1,2]. They have attracted an enormous medical interest as they present similar, or sometimes opposite, effects to the equivalent peptide, but display more favorable pharmacological properties. Geometrically constrained peptidomimetics are an important class of peptide analogues containing cyclic structures that result in reduced conformational flexibility and usually well-defined secondary structures [3]. This frequently results in enhanced affinities for biological receptors, leading to improved biological activities [4]. In fact, many biologically active natural products present rigid peptide-like motives able to strongly bind to their target biomolecules.
Conventional methods for the synthesis of these compounds commonly consist in multi-step procedures, including several protection and deprotection reactions. In recent years, more convenient multicomponent approaches have been developed to straightforwardly generate diversely substituted constrained peptidomimetics in one or a few reaction steps. Thus, piperazines and pyrazines have been synthesized by modified Ugi condensations [20] and post-condensation transformations of Ugi [7,[21][22][23] or Ugi-Smiles products [24,25]. Different multicomponent strategies have also been used in the synthesis of polycyclic coumarins [26].
Reduction of the nitro group in the enol-Ugi adducts (17a-k) with iron in aceti takes place smoothly at room temperature to afford amino intermediates (18a-k) th  a Procedure for the synthesis of 17a-k: Isocyanide 14 (1 equiv) and enol 15 (1 equiv) were added to a solution of imine 1 (1 equiv) in CH2Cl2 and stirred at 20 °C for 3 h. b Procedure for the synthesis of 19a-i and 18j,k: Enol-Ugi adduct 17 equiv) and iron powder (24 equiv) were stirred at rt for 2-4 h. c The reaction was performed at 150 °C. d All yields refer isolated yields.
Reduction of the nitro group in the enol-Ugi adducts (17a-k) with iron in acet takes place smoothly at room temperature to afford amino intermediates (18a-k) th a Procedure for the synthesis of 17a-k: Isocyanide 14 (1 equiv) and enol 15 (1 equiv) were added to a solution of imine 16 (1 equiv) in CH 2 Cl 2 and stirred at 20 • C for 3 h. b Procedure for the synthesis of 19a-i and 18j,k: Enol-Ugi adduct 17 (1 equiv) and iron powder (24 equiv) were stirred at rt for 2-4 h. c The reaction was performed at 150 • C. d All yields refer to isolated yields.
Reduction of the nitro group in the enol-Ugi adducts (17a-k) with iron in acetic acid takes place smoothly at room temperature to afford amino intermediates (18a-k) that are usually not isolated. In the case of enol-Ugi adducts derived from aliphatic amines (17a-i) the spontaneous intramolecular attack of the amine on the amide group generates a pyrazine ring (19a-i) with loss of cyclohexylamine (Scheme 3). Conversely, enol-Ugi adducts derived from aromatic amines (17j,k) give stable aminocoumarins (18j,k) that can be isolated. However, when the reduction/cyclization was carried out at 150 • C the corresponding chromenopyrazines (19j,k) were directly obtained (Table 1). usually not isolated. In the case of enol-Ugi adducts derived from aliphatic amines (17ai) the spontaneous intramolecular attack of the amine on the amide group generates a pyrazine ring (19a-i) with loss of cyclohexylamine (Scheme 3). Conversely, enol-Ugi adducts derived from aromatic amines (17j,k) give stable aminocoumarins (18j,k) that can be isolated. However, when the reduction/cyclization was carried out at 150 °C the corresponding chromenopyrazines (19j,k) were directly obtained (Table 1).

Scheme 3. Postcondensation synthesis of chromenopyrazinones. Dipeptidic structure is shaded in light blue colour.
A rigid dipeptidic structure is comprised in the pyrazine and pyranone rings of chromenopyrazines (19; Scheme 3). In order to extend the peptidic skeleton of these geometrically constrained dipeptides, we decided to use esters of amino acids as amino components of the enol-Ugi reaction (Scheme 4). Accordingly, the four-component reaction of 4-hydroxy-3-nitro-coumarin (15), different isocyanides (14a-d) and aldehydes (12a,c,d) with glycine methyl ester (13e) gave the corresponding adducts (17l-r) in good yields. The analogous condensation of β-alanine (13f) similarly gave adduct 17s (Table 2).  A rigid dipeptidic structure is comprised in the pyrazine and pyranone rings of chromenopyrazines (19; Scheme 3). In order to extend the peptidic skeleton of these geometrically constrained dipeptides, we decided to use esters of amino acids as amino components of the enol-Ugi reaction (Scheme 4). Accordingly, the four-component reaction of 4-hydroxy-3-nitro-coumarin (15), different isocyanides (14a-d) and aldehydes (12a,c,d) with glycine methyl ester (13e) gave the corresponding adducts (17l-r) in good yields. The analogous condensation of β-alanine (13f) similarly gave adduct 17s (Table 2). usually not isolated. In the case of enol-Ugi adducts derived from aliphatic amines (17ai) the spontaneous intramolecular attack of the amine on the amide group generates a pyrazine ring (19a-i) with loss of cyclohexylamine (Scheme 3). Conversely, enol-Ugi adducts derived from aromatic amines (17j,k) give stable aminocoumarins (18j,k) that can be isolated. However, when the reduction/cyclization was carried out at 150 °C the corresponding chromenopyrazines (19j,k) were directly obtained (Table 1).

Scheme 3. Postcondensation synthesis of chromenopyrazinones. Dipeptidic structure is shaded in light blue colour.
A rigid dipeptidic structure is comprised in the pyrazine and pyranone rings of chromenopyrazines (19; Scheme 3). In order to extend the peptidic skeleton of these geometrically constrained dipeptides, we decided to use esters of amino acids as amino components of the enol-Ugi reaction (Scheme 4). Accordingly, the four-component reaction of 4-hydroxy-3-nitro-coumarin (15), different isocyanides (14a-d) and aldehydes (12a,c,d) with glycine methyl ester (13e) gave the corresponding adducts (17l-r) in good yields. The analogous condensation of β-alanine (13f) similarly gave adduct 17s (Table 2). Interestingly, in this case, reduction of the nitro group does not lead to cyclization by the attack on the amide, as with adducts 17a-k. The attack of the amine occurs instead on the more reactive ester group derived from the glycine methyl ester, affording pyrazines 20l-r (Scheme 4; Table 2). The amide group brought by the isocyanide component is thus preserved in the product delivering a new element of diversity, as different isocyanides can be used (Table 2). Rigid retropeptidic tripeptides are obtained in this manner. The peptide sequence could theoretically be grown from the isocyanide-derived amide to obtain peptides with an inverted rigid N-terminus.  Table 2. Synthesis of enol-Ugi adducts of amino acids (17) and chromenopyrazines 19 and 20 a Procedure for the synthesis of 17l-s: A solution of amine 13 (1 equiv) and aldehyde 12 (1 equiv) in of dry acetonitrile wa stirred for 15 min at rt; isocyanide 14 (1 equiv) and enol 15 (1 equiv) were successively added and the reaction mixture was stirred 4 days at rt. b Procedure for the synthesis of 19s and 20l-r: Enol-Ugi adduct 17 (1 equiv) and iron powder (24 equiv) were stirred at rt in acetic acid for 2-4 h. c All yields refer to isolated yields.
Interestingly, in this case, reduction of the nitro group does not lead to cyclizati the attack on the amide, as with adducts 17a-k. The attack of the amine occurs inste the more reactive ester group derived from the glycine methyl ester, affording pyra 20l-r (Scheme 4; Table 2). The amide group brought by the isocyanide component is preserved in the product delivering a new element of diversity, as different isocya can be used (Table 2). Rigid retropeptidic tripeptides are obtained in this manner peptide sequence could theoretically be grown from the isocyanide-derived amide t tain peptides with an inverted rigid N-terminus.
On the other hand, the β-alanine-derived adduct (17s) cyclizes again by attack o amide to give pyrazine 19s. This reaction is more favorable than cyclization on the as this would involve the formation of a seven-membered, instead of a six membered  Table 2. Synthesis of enol-Ugi adducts of amino acids (17) and chromenopyrazines 19 and 20 a Procedure for the synthesis of 17l-s: A solution of amine 13 (1 equiv) and aldehyde 12 (1 equiv) in of dry acetonitrile wa stirred for 15 min at rt; isocyanide 14 (1 equiv) and enol 15 (1 equiv) were successively added and the reaction mixtur was stirred 4 days at rt. b Procedure for the synthesis of 19s and 20l-r: Enol-Ugi adduct 17 (1 equiv) and iron powder (2 equiv) were stirred at rt in acetic acid for 2-4 h. c All yields refer to isolated yields.
Interestingly, in this case, reduction of the nitro group does not lead to cyclizati the attack on the amide, as with adducts 17a-k. The attack of the amine occurs inste the more reactive ester group derived from the glycine methyl ester, affording pyra 20l-r (Scheme 4; Table 2). The amide group brought by the isocyanide component i preserved in the product delivering a new element of diversity, as different isocya can be used (Table 2). Rigid retropeptidic tripeptides are obtained in this manner peptide sequence could theoretically be grown from the isocyanide-derived amide tain peptides with an inverted rigid N-terminus.
On the other hand, the β-alanine-derived adduct (17s) cyclizes again by attack o amide to give pyrazine 19s. This reaction is more favorable than cyclization on the as this would involve the formation of a seven-membered, instead of a six membered a Procedure for the synthesis of 17l-s: A solution of amine 13 (1 equiv) and aldehyde 12 (1 equiv) in of dry acetonitrile was stirred for 15 min at rt; isocyanide 14 (1 equiv) and enol 15 (1 equiv) were successively added and the reaction mixture was stirred 4 days at rt. b Procedure for the synthesis of 19s and 20l-r: Enol-Ugi adduct 17 (1 equiv) and iron powder (24 equiv) were stirred at rt in acetic acid for 2-4 h. c All yields refer to isolated yields.
On the other hand, the β-alanine-derived adduct (17s) cyclizes again by attack on the amide to give pyrazine 19s. This reaction is more favorable than cyclization on the ester, as this would involve the formation of a seven-membered, instead of a six membered ring.

Starting Materials
Acetonitrile was dried by distillation over P 2 O 5 , immediately prior to use. Glacial acetic acid was purchased from commercial sources and used as received. Aldehydes (12a-e), amines (13a-f), isocyanides (14a-d; S.I. Figure S1), 4-hydroxycoumarin and iron powder are commercially available and were used without purification. 4-Hydroxy-3nitrocoumarin (15) was prepared by nitration of 4-hydroxycoumarin [52]. Imines (16a-k; S.I. Figure S2) were synthesized using the standard procedure of mixing equimolar amounts of the corresponding aldehydes (12), amines (13) and anhydrous Na 2 SO 4 in dry CH 2 Cl 2 at room temperature for 24 h. [53,54] Evaporation of the solvent quantitatively yielded the imines (16) that were used in the Ugi reaction without further purification.

General Synthetic Techniques
Liquid reagents were measured using positive-displacement micropipettes with disposable tips and pistons. Thin layer chromatography was performed on aluminum plates, using 254 nm UV light or a mixture of p-anisaldehyde (2.5%), acetic acid (1%) and H 2 SO 4 (3.4%) in 95% ethanol as developer.

Instrumentation
Melting points are uncorrected. IR spectra were recorded as KBr pellets. Proton and carbon-13 nuclear magnetic resonance ( 1 H-NMR or 13 C-NMR) spectra were obtained on a 500 MHz spectrometer. The assignments of signals in 13 C-NMR were made by DEPT. High resolution mass spectra (HRMS) were recorded using a 6520 Accurate Mass QTOF LC/MS Spectrometer.

Four-Component Condensation
Amine 13 (0.5 mmol) was added to a solution of aldehyde 12 (0.5 mmol) in of dry acetonitrile (1 mL). The resulting mixture was stirred for 15 min at rt and then isocyanide 14 (0.5 mmol) and enol 15 (0.5 mmol) were successively added. After 4 days stirring at room temperature, the reaction went to completion, as judged by tlc. Then 10% HCl (2 mL) was added, the mixture was washed with H 2 O (15 mL), extracted with CH 2 Cl 2 (3 × 20 mL) and dried over Na 2 SO 4 . Removal of the solvent and purification by column chromatography (SiO 2 , gradient from 100% hexanes to hexanes-EtOAc, 7:3) gave the corresponding enamines 17l-s (Table 2).

General Procedure for the Reduction of Nitro Derivatives 17a-s
To a vigorously stirred solution of enol-Ugi adduct 17a-s (0.4 mmol) in glacial acetic acid (8 mL), iron powder (9.6 mmol, 24 equiv) was added in one portion. The reaction mixture was stirred at rt for 2-4 h. Then water (50 mL) and dichloromethane (25 mL) were added. The unreacted iron was removed by filtration and the filtrate transferred to a separatory funnel. The phases were separated, and the aqueous layer extracted again with dichloromethane (25 mL). The combined organic extracts were washed with water (25 mL), saturated NaHCO 3 (10 mL) and water again (25 mL), and then dried (Na 2 SO 4 ) and evaporated to dryness. The residue was purified by flash column chromatography (SiO 2 , gradient from 100 % hexanes to hexanes-AcOEt 7:3) to give, depending on the case, chromeno [3,4-b]piperazines 19a-i,s, aminocoumarins 18j,k or chromeno[3,4-b]piperazines 20l-r (Tables 1 and 2 Supplementary Materials: The following are available online: Figure S1: Aldehydes, amines, and isocyanides used as starting materials, Figure S2: Imines used as starting materials, Experimental data for imine 16g, Copies of the NMR spectra for all new compounds.