Synthesis and Biological Application of Isosteviol-Based 1,3-Aminoalcohols

Starting from isosteviol, a series of diterpenoid 1,3-aminoalcohol derivatives were stereoselectively synthesised. The acid-catalysed hydrolysis and rearrangement of natural stevioside gave isosteviol, which was transformed to the key intermediate methyl ester. In the next step, Mannich condensation of diterpenoid ketone, paraformaldehyde, and secondary amines resulted in the formation of 1,3-aminoketones with different stereoselectivities. During the Mannich condensation with dibenzylamine, an interesting N-benzyl → N-methyl substituent exchange was observed. Reduction of 1,3-aminoketones produced diastereoisomeric 1,3-aminoalcohols. Alternatively, aminoalcohols were obtained via stereoselective hydroxy-formylation, followed by oxime preparation, reduction, and finally, reductive alkylation of the obtained primary aminoalcohols. An alternative 1,3-aminoalcohol library was prepared by reductive amination of the intermediate 3-hydroxyaldehyde obtained from isosteviol in two-step synthesis. Cytotoxic activity of compounds against human tumour cell lines (A2780, SiHa, HeLa, MCF-7 and MDA-MB-231) was investigated. In our preliminary study, the 1,3-aminoalcohol function and N-benzyl substitution seemed to be essential for the reliable antiproliferative activity. To extend their application, a diterpenoid condensed with 2-phenylimino-1,3-thiazine and -1,3-oxazine was also attempted to prepare, but only formation of thioether intermediate was observed.


Introduction
Terpenoids, also known as isoprenoids, are the most numerous and structurally diverse group of natural products present in most plants [1]. Several studies have confirmed that this class of compounds displays a wide array of very important pharmacological properties [2]. Between terpenoids, the diterpenoid stevioside with a complex ent-kaurane skeleton and three glucose moieties has been the focus of attention in recent decades [3,4]. Stevioside is extracted from the plant Stevia rebaudiana, which is a perennial herbal shrub of the Asteraceae family that originated from Brazil and Paraguay in South America, while cultivated for its sweet leaves [5]. It is applied in food chemistry as a commercial sweetener considered to be a non-caloric sugar substitute. In recent years, stevioside and steviol, its aglycon, have attracted scientific attention because of their broad spectrum of biological activities, including antihyperglycemic, [6] antihypertensive, [7,8] antitumour, [9,10] and immunomodulatory actions [11] beside several other biological activities [12,13].
Cytotoxic activities of isosteviol derivatives, obtained by microbial and chemical transformations, have also induced much attention in recent years [21,22]. Several of the novel isosteviol derivatives have been successfully synthesised by chemical modification of isosteviol, and some of these derivatives exhibited good cytotoxic activity as potential drug molecules. Li and co-workers reported compounds with an α-methylenecyclopentanone moiety in the D-ring of isosteviol displaying remarkable anticancer activity against MDA-  cell line with an IC 50 value of 1.58 µM [23]. Jayachandra and co-workers synthesised isosteviol analogues showing a potential protective effect against DOX-induced cardiotoxicity in zebrafish embryos in vivo [24]. Tao and co-workers reported that Esophageal carcinoma cells were more sensitive to 1,3-aminoalcohols, exhibiting anticancer activities superior to Cisplatin with an IC 50 value 4.01 µM [25].
1,3-Aminoalcohols may also serve as a building blocks of many natural and synthetic products, and they exhibit wide-ranging biological and catalytic activities [26][27][28]. In a previous work, Tao and co-workers prepared a series of compounds by modifying a crucial aminoalcohol fragment of the D-ring of isosteviol affording significantly improved anticancer activities [25]. In a similar study, a hydroxythiourea derivative has been described as a useful candidate for the treatment of tumours on different cell lines [29].
In the present contribution, we report the preparation of a new library of isosteviolbased chiral bifunctional synthons, such as β-aminoketones, 1,3-aminoalcohols, and 1, 3-heterocycles fused with ent-beyerane, starting from commercially available natural stevioside. We also planned to investigate the preliminary study of the effect of keto-amine and 1,3-aminoalcohol functions and the stereochemistry and substitution level of amine function on antiproliferative activity on multiple human cancer cell lines.

Syntheses and Reduction of Isosteviol-Based 1,3-Aminoketones Obtained via Mannich Condensation
Isosteviol methyl ester 13 was prepared from 1 with diazomethane in excellent yield [34]. The Mannich condensation of 13 was accomplished with paraformaldehyde and different secondary amine HCl salts in glacial acetic acid, resulting in a library of aminoketones with good to moderate yields (Scheme 3, Table 2) [28]. The condensation reaction took place in an exclusive stereoselective manner, forming a single diastereoisomer with (7R) configuration of the new stereocenter at C15. Results are collected in Table 2. Scheme 3. Synthesis of aminoketones 14-18 via Mannich condensation. (i) CH 2 N 2 , Et 2 O, 5 min, 25 • C, 79%; (ii) NHR 1 R 2 * HCl (1 eq.), (CH 2 O) n (2 eq.), AcOH, 24 h, reflux, 13-68%. As entry 6 of Table 2 shows, the condensation of 13 with dibenzylamine hydrochloride surprisingly led to N-methyl-N-benzyl derivative 15 instead of the expected N,N-dibenzylsubstituted product, although with a low yield (13%). When the reaction was repeated with both N-benzyl-N-(S)-α-methylbenzylamine and the corresponding (7R) enantiomer, 15 as a single product could be isolated again (Scheme 4). This interesting N-benzyl → N-methyl substituent exchange can be explained with the special steric hindrance of the diterpenoid skeleton with N-methyl-N-benzylamine representing the limit of the Mannich condensation in the case of this special ring system (Scheme 5). According to the classical mechanism of Mannich condensation, the first step is the formation of an iminium ion in the reaction of dibenzylamine and formaldehyde (Scheme 5). Because of steric hindrance, iminium species C cannot react with the enolate of the ketone. Rather, under the applied conditions, isomeric iminium salt D is formed. This step is followed by water addition and benzaldehyde elimination, resulting in N-methyl-N-benzylamine (F), ready for the condensation to give 15. The reduction of aminoketones 14-18 with NaBH 4 under mild conditions provided diastereomeric mixtures of 1,3-aminoalcohols. Reaction routes are outlined in Scheme 6. When pyrrolidinoaminoketone (16) or dimethylaminoketone (17) derivatives were applied, the reaction proceeded in a highly stereoselective way, resulting in the formation of 21 and 22 as single diastereoisomers. In other cases, diastereomeric mixtures were formed. Data are presented in Table 3.  The different steric hindrances of N-substituents can explain the different stereoselectivity of reduction of amino ketones. Probably in the case of less hindrance aminomethyl substituents ( [15][16][17], a cyclic complex can be formed with the protic solvent, and the hydride can attack from only the less sterically hindrance side, while this complex cannot be formed in the case of bulky N-substitution (14 and 18) and therefore the attack of the hydride can take place both side of the carbonyl function, resulting in a mixture of diastereoisomers [35].
The relative, therefore absolute configurations of the new stereocenters of aminoalcohols 19-23 at position 7 and 8 were determined by NMR with NOESY spectral analysis, based on the observation of NOE effects between H-C(12) and H-C(8), H-C(8) and H-C(15), H-C(12) and , as well as between H-C(12) and H-C(15). Thus, the structure of 19b, 20b, 21, 22, and 23b was determined as outlined in Figure 1. Similarly, NOE effects were observed in the case of 19a, 20a, and 23a. Beside the NOESY experiments, the configurations of the newly formed stereocenters of 1,3-aminoalcohols were determined via two alternative synthetic pathways (Scheme 7). Reductive amination of 4 (obtained from 3 with known stereochemistry) with benzylamine followed by methylation of 8 with iodomethane yielded a product that was identical with 20b obtained as a major product of the reduction of aminoketone 15. Alternatively, debenzylation of 20b over 5% Pd/C catalyst in methanol resulted in N-methyl aminoalcohol identical with 7 obtained by reductive amination of 4 with methylamine. Diastereoisomer 24 was also prepared by debenzylation of 20a over 5% Pd/C catalyst (Scheme 7).

Antiproliferative Properties of the Prepared Diterpenes
The antiproliferative activities of the prepared diterpene analogues were determined by means of MTT assay on a panel of human adherent cancer lines, including cells from cervical (HeLa, SiHa), breast (MDA-MB-231, , and ovary cancers (A2780) as given in Table 4. Based on the obtained activities, some conclusions could be arrived at with respect to structure-activity relationships. Since the original diol (3), its aldehyde analogue (4), and the corresponding oxime (5) elicited no relevant effect on the growth of cancer cells, an amino function seems to be essential for antiproliferative activity (Table 4). Primary amine 6 as well as secondary amine derivatives 7-12 exerted similarly pronounced antiproliferative action, and the calculated IC 50 values of these compounds are comparable to or lower than those of reference agent cisplatin. The cell line-independent IC 50 values may be interpreted as a marker of general cytotoxic property of molecules [6][7][8][9][10][11][12]. From the results presented in Table 4, it seems to be clear that both the aminoalcohol function and the N-benzyl substitution ( [8][9][10][11][12], but not the aliphatic substitution (7 and 24), are essential for the remarkable antiproliferative activity. Cervical cell lines are especially sensitive to these agents. Aminoketones 14-18 are much less effective and most of them exert only negligible activity. Reduced analogues (19-23/24), bearing tertiary amino function, proved to elicit more pronounced action compared only with aminoketones, and the orientation of the newly formed alcohol function has no substantial impact on the efficacy of the product. Phenylthioureido analogue 25 exerted some modest activities with IC 50 values between 10 and 23 µM, while the thioether type compounds 26 and 28 do not favour the antiproliferative action of the diterpene skeleton. Table 4. Antiproliferative properties of the tested diterpene analogues.

Compound
Conc.  [a] Cancer cell growth inhibition values less than 20% were considered insignificant and are not given numerically.

Conclusions
In summary, a series of novel isosteviol derivatives containing 1,3-aminoalcohol and thiourea moieties have been synthesised with moderated to good yields, and their cytotoxic activities against five human cancer cell lines (HeLa, Siha, MCF7, MDA-MB-231, A2780) have been investigated. Starting from commercially available stevioside, a new family of isosteviol-based chiral 1,3-aminoalcohols and thioureid derivatives were prepared through hydroxyaldehyde and isosteviol methyl ester as key intermediates via stereoselective transformations. The resulting 1,3-aminoalcohols exert remarkable antiproliferative action of human cancer cell lines. The in vitro pharmacological studies have clearly shown that the N-benzyl substituent at the amino function is essential and some of the prepared molecules proved to be more potent than anticancer agent cisplatin used clinically.

Materials and Methods
General methods: Commercially available reagents were used as obtained from suppliers ( Starting materials: Stevioside was obtained from Molar Chemicals Ltd., Halásztelek, Hungary. Isosteviol 1 was prepared from commercially available stevioside or a mixture of steviol glycosides in a one-step synthesis according to the literature method, and all its spectroscopic data were the same as described in the literature [30]. Compounds 2, 3, and 13 were prepared by literature methods. Their spectroscopic data and physical and chemical properties were similar to those reported therein [31,34]. 1 H, 13  General procedure for preparation of aminoalcohol with primary amines and aldehydes: Method A: To a solution of 4 (0.10 g, 0.28 mmol) in dry EtOH (10 mL), primary amines (0.28 mmol) were added in one portion and the solution was stirred at room temperature for 3 h and then evaporated to dryness. The residue was dissolved in dry EtOH (10 mL), stirred for a further 1 h, and evaporated to dryness again. The product was dissolved in dry MeOH (10 mL) and NaBH 4 (0.56 mmol, 0.02 g) was added in small portions to the mixture under ice cooling. After stirring for 4 h at room temperature, the mixture was evaporated to dryness, and the residue was dissolved in H 2 O (20 mL) and extracted with DCM (3 × 20 mL). The combined organic layer was dried (Na 2 SO 4 ), filtered and evaporated to dryness. The crude product obtained was purified by column chromatography on silica gel (CHCl 3 /MeOH = 19:1).
Method B: To a solution of 6 (0.10 g, 0.28 mmol) in dry EtOH (10 mL), aldehydes (0.28 mmol) were added in one portion, and the solution was stirred at room temperature for 3 h and then evaporated to dryness. The product was dissolved in dry EtOH (10 mL) and stirred for a further 1 h and evaporated to dryness again. The crude product was dissolved in dry MeOH (10 mL) and NaBH 4 (0.56 mmol, 0.02 g) was added in small portions to the mixture under ice cooling. After stirring for 4 h at room temperature, the mixture was evaporated to dryness, and the residue was dissolved in H 2 O (20 mL) and extracted with DCM (3 × 20 mL). The combined organic layer was dried (Na 2 SO 4 ), filtered and evaporated to dryness. The crude product obtained was purified by column chromatography on silica gel (CHCl 3 /MeOH = 19:1).