Synthesis and Transformation of (-)-Isopulegol-Based Chiral β-Aminolactones and β-Aminoamides

A library of isopulegol-based β-amino acid derivatives has been developed from commercially-available (-)-isopulegol. Michael addition of primary and secondary amines towards α,β-unsaturated γ-lactones was accomplished resulting in β-aminolactones in highly-stereoselective reactions. Ring-opening of β-aminolactones with different amines furnished excellent yields of β-aminoamides. Moreover, the applicability of aminolactones in peptide synthesis was examined by opening the lactone ring with α- and β-aminoesters, providing dipeptides as promising chiral substrates for the synthesis of foldamers. The antiproliferative activities of β-aminolactones and β-aminoamides were explored, and the structure-activity relationships were studied from the aspects of the stereochemistry of the monoterpene ring and the substituent effects on the β-aminoamide ring system. The N-unsubstituted (-)-isopulegol-based β-aminoamides exhibited considerable antiproliferative activity against a panel of human adherent cancer cell lines (HeLa, MCF7 and MDA-MB-231).


Introduction
Sesquiterpene lactones containing the α-methylene-γ-lactone moiety are natural products occurring in many plant families. These compounds are known for their various biological activities, including cytotoxicity to tumor cells, anti-bacterial, antifungal, and anti-protozoan activities, as well as activity against human and animal parasites or inhibition of plant growth [1][2][3].

Synthesis of β-Aminolactones
Nucleophilic addition of primary and secondary amines to α-methylene-γ-butyrolactones 2 and 4 has proven to be an efficient method for the preparation of a highly-diversified library of β-aminolactones [3,30]. When the addition of one equivalent of benzylamine to 2 was performed as a model reaction, the formation of N-benzyl aminolactone 5a and N-benzyl methylene amide 5b (the latter could not be isolated in pure form) was observed. The effect of the solvent was also studied, and it was found that the applied solvent strongly affected the yield of 5a and the ratio of the two products (Scheme 1, Table 1). When alcohols as protic solvents were used, formation of 5a was observed as the main product. Among of three protic solvents applied, EtOH gave target 5a with the best chemoselectivity (entry 7). The ratio of 5a and 5b also depended on temperature. In alcohols, in turn, product ratios were similar at low (0 • C) and high (25 • C) temperature (compare entries 6 and 7). Furthermore, the yield of 5a increased with temperature. At higher temperatures, however, the yield of 5a dropped, and the products were formed in a ratio of 4:1, even with decreasing reaction time (Table 1).  0  20  44  56  10  2  -0  20  50  50  30  3  DMF  0  20  67  33  44  4  MeOH  0  20  92  8  30  5 i- PrOH  0  20  94  6  55  6  EtOH  0  20  94  6  60  7  EtOH  25  20  94  6  65  8  EtOH  40  15  80  20  55  9  EtOH  50  10  80  20  55  10  EtOH  60  7  80  20  50  11  EtOH  70  5  80  20  45 After optimizing the condition for nucleophilic addition with benzylamine, amine adducts 6-10 were synthesized from 2 under these conditions (one equivalent of appropriate amine, EtOH, 25 • C) (Scheme 2). Surprisingly, when (R)-and (S)-α-methylbenzylamine and secondary amines were applied, only the formation of aminolactones was observed ( Table 2). This is probably due to the steric hindrance of these amines. Besides amines, the best conditions were also successful for the addition of L-or β-aminoesters as amine sources to prepare some β-aminolactones containing aminoester moiety 11-12 (Table 2). The optimized conditions were also applied for the preparation of (+)-neoisopulegol-based β-aminolactones 13-18 starting from 4 (Scheme 3). Interestingly, under the applied conditions, exclusive formation of the amine adducts was observed. This may be due to the cis configuration of 4, which makes the lactone more hindered for nucleophilc attack ( Table 3). The reaction of 4 with some aminoesters was effective at an elevated temperature to achieve aminoester-based β-aminolactone derivatives 19-20 (Table 3).  The relative configuration of compounds 5a-12 and 13-20 was determined by means of NOESY experiments. Clear NOE signals were observed between the H-1 and H-3, as well as the H-3 and H-7 protons in the case of 5a-12, while significant NOE signals were shown between the H-3 and H-7, as well as the H-4 and H-7 protons in the case of 13-20 ( Figure 2).

Synthesis of β-Aminoamides and Dipeptides
Nucleophilic addition and ring-opening of lactones were simultaneously performed from 2 using excess amines to form β-aminoamides 21-23 in one step (Scheme 4). It is interesting that benzylamine reacted at room temperature, while (R)-and (S)-α-methylbenzylamine required a higher temperature and longer reactions (Table 4). This is probably due to steric hindrance exerted by the α-methyl group. Our efforts in the opening of lactones with secondary amines failed. Hydrolysis of β-aminoamides under acidic conditions resulted in the original starting material β-aminolactones 5a-7 (Scheme 4).  (Table 5). In further studies starting from 2, the addition and ring-opening reaction with β-aminoester successfully gave dipeptide 27. The application of α-aminoesters failed despite using long reaction times and elevated temperatures. The probable reason is steric hindrance exerted by the α-methyl group of the aminoesters. In addition, the opening of N-benzyl aminolactone 5a with both the αand β-aminoester proceeded smoothly to give N-benzyl dipeptides 28-29. Debenzylation through hydrogenolysis over Pd/C and purification of the crude products gave dipeptides 30-31, i.e., suitable starting compounds in peptide synthesis (Scheme 5). Our effort to prepare β-aminoamides 32-34 starting from 4 failed. Fortunately, the synthesis was achieved by reacting β-aminolactones 13-15 with primary amines under reflux conditions in anhydrous THF [31] (Table 4). Again, opening the lactone ring with secondary amines was unsuccessful. Acidic hydrolysis of β-aminoamides 26-28 led to the original starting material β-aminolactones 13-15 instead of the expected β-aminoacids (Scheme 6). Debenzylation with appropriate catalysts gave primary β-aminoamides 35-37 in moderate yields ( Table 5). The attempted nucleophilic addition and ring-opening of 6 with α-or β-aminoesters failed.

Antiproliferative Activities
Since several sesquiterpene-based α-methylene-γ-lactones, as well as their derivatives containing β-aminolactone moiety, exerted an antiproliferative action on adherent human cancer cell lines [3,30], antiproliferative activities of the prepared β-aminolactone and β-aminoamide analogues were also tested against a panel of human malignant cell lines isolated from cervical (HeLa) and breast (MCF7 and MDA-MB-231) cancers (Table 6). While the β-aminolactone-typed monoterpene derivatives proved to be ineffective against the utilized cell lines, the N-(S)-α-methylbenzyl-substituted β-aminoamide analogues (23, 34) exhibited modest growth inhibitory activities. The most potent newly-prepared monoterpene analogue was compound 23, exerting antiproliferative activity comparable to those of reference agent cisplatin. α-Methylene-γ-lactone is generally believed to be a pharmacophore acting as an alkylating agent on DNA and proteins [32]. In the present set of (-)-isopulegol analogs, the γ-lactone-type derivatives (2, 4, 5 and 7) exerted weak antiproliferative activities, while the most active member of the presented library (23) is not a typical sesquiterpene lactone, but a β-aminoamide. Based on our results, the stereochemistry of the N-substituent on the amide function ((S)-α-methylbenzyl substituent), as well as the trans position of the bulky β-aminoamide substituent and the hydroxy group on the cyclohexane ring, are proposed as crucial conditions accounting for the activity. The antiproliferative activity of dipeptides 27, 28, and 29 was also tested on adherent human cancer cell lines. While in case of 29 a week antiproliferative activity was observed, on MCF7 and MDA-MB-231, 27 and 28 were uneffective.

Discussion
Starting from commercially-available (-)-isopugeol, a new family of isopulegol-and neoisopulegol-based chiral β-aminolactone and β-aminoamide libraries has been prepared through chiral α-methylene-γ-lactones as key intermediates. Moreover, isopulegol-based chiral dipeptides, promising chiral substrates for the synthesis of chiral foldamers, were synthesized. The resulting β-aminoamides exert marked antiproliferative action on a panel of human cancer cell lines. In vitro pharmacological studies have clearly shown that the N-(S)-α-methylbenzyl substituent on the β-aminoamide function is essential. The stereochemistry of the β-aminoamides has no influence on the antiproliferative effect.

General Methods
Commercially-available compounds were used as obtained from suppliers (Molar Chemicals Ltd., Halásztelek, Hungary; Merck Ltd., Budapest, Hungary and VWR International Ltd., Debrecen, Hungary), while applied solvents were dried according to standard procedures. Optical rotations were measured in MeOH at 20 • C, with a Perkin-Elmer 341 polarimeter (PerkinElmer Inc., Shelton, CT, USA). Chromatographic separations and monitoring of reactions were carried out on Merck Kieselgel 60 (Merck Ltd., Budapest, Hungary). Elemental analyses for all prepared compounds were performed on a Perkin-Elmer 2400 Elemental Analyzer (PerkinElmer Inc., Waltham, MA, USA). GC measurements for direct separation of commercially-available enantiomers of isopulegol to determine the enantiomeric purity of starting material 1 were performed on a Chirasil-DEX CB column (2500 × 0.25 mm I.D.) on a Perkin-Elmer Autosystem XL GC equipped with a Flame Ionization Detector (Perkin-Elmer Corporation, Norwalk, CT, USA) and a Turbochrom Workstation data system (Perkin-Elmer Corp., Norwalk, CT, USA). Melting points were determined on a Kofler apparatus (Nagema, Dresden, Germany) and are uncorrected. 1 H-and 13 C-NMR spectra were recorded on BruckerAvance DRX 500 spectrometer [500 MHz ( 1 H) and 125 MHz ( 13 C), δ = 0 (TMS)]. Chemical shifts are expressed in ppm (δ) relative to TMS as the internal reference. J values are given by Hz. The structures were confirmed by 1H-NMR, 13C-NMR and 2D-NMR technics (see Supplementary Materials).

General Procedure for Nucleophilic Addition of α-Methylene-γ-Butyrolactone to Amines
Amines (1.2 mmol) were added to the solution of α-methylene-γ-butyrolactone 2 or 4 (1.2 mmol) in dry EtOH (2.0 mL). The reaction mixture was stirred at appropriate temperatures for 20-72 h. When the reaction was complete (indicated by TLC), EtOH was removed under reduced pressure. The crude residue was purified by column chromatography on silica gel with an appropriate solvent mixture. The crude products after solvent evaporation were purified as HCl salts by recrystallization in diethyl ether resulting in compounds 5a-10 and 13-18.
Prepared from 2 with benzylamine at 25 • C for 20 h.

General Procedure for Nucleophilic Addition of α-Methylene-γ-Butyrolactone with Amino Esters
To the solution of α-methylene-γ-butyrolactone 2 or 4 (1.2 mmol) in dry EtOH (2.0 mL) was added L-or β-alanine ethyl ester hydrochloride (2.4 mmol) and Et 3 N (2.4 mmol). The reaction mixture was stirred at the appropriate temperature for 20 h. When the reaction was complete (indicated by TLC), EtOH was removed under reduced pressure. The crude residue was purified by column chromatography on silica gel with a mixture of CHCl 3 and MeOH (19:1). After solvent evaporation, the addition of a few drops of HCl/EtOH, and recrystallization in diethyl ether, compounds 11 and 12, as well as 19 and 20, respectively, were isolated.

General Procedure for the Preparation of β-Aminoamides
To a solution of α-methylene-γ-butyrolactone, 2 (1.2 mmol) or β-aminolactones 13-15 (1.2 mmol) in an appropriate solvent (2.0 mL) was added a solution of the appropriate amine (4.8 mmol). The mixture was stirred at the appropriate temperature for 20-72 h. When the reaction was complete (indicated by TLC), the mixture was evaporated to dryness. The crude product was purified by column chromatography on silica gel with CHCl 3 /MeOH (19:1), resulting in compounds 21-23 and 32-34.  13

General Procedure for the Hydrolysis of β-Aminoamides
The solution of β-aminoamides 21-23 or 32-34 (0.5 mmol) in EtOH (2 mL) mixed with 10% aqueous HCl (10 mL) was stirred at room temperature. After completion of the reaction (as monitored by TLC, 24 h), the mixture was extracted with CH 2 Cl 2 (3 × 10 mL), dried over Na 2 SO 4 , filtered, and evaporated to dryness. The crude product was purified by recrystallization with diethyl ether, resulting in compounds 5a-7 or 13-15, respectively. All spectroscopic data are listed above.

General Procedure for Debenzylation
To a suspension of 5% Pd/C or Pd(OH) 2 /C (100 mg) in MeOH (10 mL) was added β-aminoamides 21-23 and 32-34 or N-benzyldipepetides 28-29 (0.38 mmol) in MeOH (10 mL). The mixture was stirred under H 2 at room temperature and normal pressure. When the reaction was complete (indicated by TLC), the mixture was filtered through a Celite pad, the solution was evaporated to dryness and purified by recrystallization in diethyl ether providing 24-26 and 35-37 as well as 30-31, respectively.

Determination of Antiproliferative Properties
The human cancer cell lines isolated from cervical adenocarcinoma (HeLa) and breast cancers (MCF7 and MDA-MB-231) were purchased from European Collection of Cell Cultures (Salisbury, UK). The cells were maintained in Minimum Essential Medium (MEM) supplemented with fetal calf serum (10%), non-essential amino acids (1%), and penicillin-streptomycin (1%) at 37 • C in a humidified atmosphere containing 5% CO 2 . All media and supplements for these experiments were obtained from Lonza Group Ltd. (Basel, Switzerland). The antiproliferative properties of the prepared compounds were determined by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay [33]. Briefly, cells were seeded into 96 well plates (5000 cells/well) and incubated with the tested compounds at 10 and 30 µM under cell-culturing conditions for 72 h. Then MTT solution (5 mg/mL) was added to each sample, which were incubated for a further 4 h. The formazan crystals precipitated were dissolved in 100 µL dimethyl sulfoxide, and the absorbance was measured at 545 nm with a microplate reader (Awareness Technology, Palm City, FL, USA). Two independent experiments were performed with five wells for each condition. Cisplatin (Ebewe GmbH, Unterach, Austria), a clinically used anticancer agent, was used as a reference agent. Calculations were performed by means of the GraphPad Prism 5.01 software (GraphPad Software Inc., San Diego, CA, USA).