N-Alkyl derivatives of diosgenyl 2-amino-2-deoxy-β-D-glucopyranoside; synthesis and antimicrobial activity

Abstract

Diosgenyl 2-amino-2-deoxy-β-D-glucopyranoside is a synthetic saponin exhibiting attractive pharmacological properties. Different pathways tested by us to obtain this glycoside are summarized here. Moreover, the synthesis of N-alkyl and N,N-dialkyl derivatives of the glucopyranoside is presented. Evaluation of antibacterial and antifungal activities of these derivatives indicates that they have no inhibitory activity against Gram-negative bacteria, whereas many of the tested N-alkyl saponins were found to inhibit the growth of Gram-positive bacteria and human pathogenic fungi.

Introduction

Saponins are a group of steroid or triterpenoid glycosides, widely distributed in the plant kingdom [1]. Saponins are characteristic by their foaming properties in aqueous solution, causing them to be used as detergents, surfactants and emulsifiers. Moreover, they display a wide range of pharmacological activities, including antifungal, antiparasitic, antiinflammatory, antibacterial, and antitumor activities [2–5]. No wonder, saponins have been evaluated as vaccine adjuvants [6]. Despite the fact that thousands of homogeneous saponins have been characterized, new types of saponins are regularly isolated from nature and their biological activities are evaluated [7–11]. The yields of homogenous saponins isolated from natural sources are rather low. Therefore, chemical synthesis of saponins have been investigated [12–22] as well as the evaluation of their antitumor activities [23–27].

Naturally-occurring diosgenyl glycosides are the most abundant steroidal saponins. They have been continuously synthesized [28–32] and their cardiovascular, antifungal, anticancer [33–36] and antithrombotic activities [37] have been investigated. Gelation ability of the pentose derivatized diosgenyl saponins have also been reported [38]. In the family of diosgenyl β-glycosides D-glucopyranose is the first sugar attached to the diosgenin. Very often this D-glucopyranose is substituted with α-L-rhamnopyranose at 2-OH and other sugars at 4-OH. The change of D-glucopyranose into 2-amino-2-deoxy-D-glucopyranose provides diosgenyl 2-amino-2-deoxy-β-D-glucopyranoside (7), a synthetic saponin, first reported by us [39]. It was also demonstrated that diosgenyl 2-amino-2-deoxy-β-D-glucopyranoside hydrochloride increases the number of apoptotic B cells, in combination with cladribine (2-CdA), which were isolated from chronic lymphotic leukemia (B-CLL) patients [40]. The presence of the amine group in this promising antitumor compound creates the opportunity to synthesize new analogues with an enhanced activity. In this way, many of the N-acyl [41–43] as well as urea and thiosemicarbazone [44] analogues of 7 have been obtained and evaluated. Their characteristic feature is that their amine group is bound with the carbonyl or thiocarbonyl group. Such analogues are much more lipophilic, but also less basic than the parent saponin 7. In this paper, for the first time, the synthesis and antimicrobial activity of the N-alkyl derivatives of diosgenyl 2-amino-2-deoxy-β-D-glucopyranoside (7) are presented. From the chemical point of view, the alkyl group has quite different properties than the acyl group. First, alkylation does not change significantly the basicity of the parent amine group. Thus, the ability to bind protons by the parent compound and its analogue should be comparable. Second, the N-alkylamine group, similarly to the amine group, is able to work as a hydrogen bond acceptor. On the other hand, alkylation improves lipophilicity of the compound, which may be crucial for its biological activity.

At the beginning, our experiences concerning the synthesis of the parent diosgenyl 2-amino-2-deoxy-β-D-glucopyranoside (7) are summarized and presented. We have studied different glycosyl donors, different amine group protections, different solvents and promoters to find the best way to obtain 7. The presented compilation is informative for all those interested in the glycosidation of 2-amino-2-deoxy sugars.

Results and Discussion

Chemistry

To synthesize the parent diosgenyl 2-amino-2-deoxy-β-D-glucopyranoside (7), the O-acetylated bromides 2a–d, chlorides 3a–d and (N-phenyl)trifluoroacetimidates 5a–d, α or β anomers, were examined (Scheme 1). These glycosyl donors were N-protected with trifluoroacetyl (TFA, a), 2,2,2-trichloroethoxycarbonyl (Troc, b), phthaloyl (Phth, c), and tetrachlorophthaloyl (TCP, d) groups, respectively. Applications of bromides 2a and 2d [40] and chlorides 3a–d [45] were previously reported. Here, applications of the two remaining bromides 2b and 2c as well as (N-phenyl)trifluoroacetimidates 5a–d are demonstrated. To synthesize bromide 2b we used a procedure described by Ellervik and Magnusson for other glycosylations [46]. However, the bromide obtained by them was a mixture of α and β anomers whereas 2b is a pure α anomer (J_1,2 4,0 Hz). Bromide 2b, identified as the α anomer, was also synthesized by Higashi et al., but in a slightly different manner [47]. Bromide 2c (α + β) was synthesized analogously to 2b. Synthesis of (N-phenyl)trifluoroacetimidates 5a–d demands removal of the acetyl groups from the anomeric hydroxy groups in 1a–d. It was done with ethylenediamine in a mixture with acetic acid in THF, a procedure adopted from Zhang and Kováč [48]. This selective 1-O-deacetylation turned out to be very effective for 1a, 1b, and 1c (97%, 84%, and 90%, respectively). However, this was quite ineffective for 1d (36%). Therefore, 1-O-deacetylation of the latter was carried out via hydrolysis (Ag₂CO₃, acetone/H₂O 2:1) of bromide 2d (73% yield) or chloride 3d (72% yield). (N-Phenyl)trifluoroacetimidates 5a–d were synthesized in reaction of the respective N-protected 3,4,6-tri-O-acetyl-D-glucosamines 4a–d with N-phenyltrifluoroacetimidoyl chloride, according to a procedure proposed by Yu and Tao for 1-hydroxy derivatives of D-glucose and L-rhamnose [49].

Scheme 1.

Reagents used for the synthesis of diosgenyl 2-amino-2-deoxy-β-D-glucopyranoside (7).

Glycosylation of diosgenin with twelve different derivatives of D-glucosamine (2a–d, 3a–d, and 5a–d), was examined using “normal” and “reverse” procedures [50] (Table 1). In the “normal” procedure, the promoter (silver triflate or trimethylsilyl triflate) was added to the solution of diosgenin and the respective glycosyl donor. In the “reverse” procedure, the respective glycosyl donor was added to the solution of diosgenin and the promoter. Diosgenin glycosylations were carried out in dichloromethane or/and in a mixture of dichloromethane and diethyl ether. The results summarized in Table 1 indicate that the “reverse” procedure is much more effective than the “normal” procedure. Running of the diosgenin glycosylation also depends on the kind of the solvent used. It is particularly important when bromide 2a is used as a glycosyl donor. Reaction of 2a with diosgenin conducted by the “reverse” procedure in the CH₂Cl₂/Et₂O mixture leads to glycoside 6a in 77% yield. The same procedure applied in CH₂Cl₂ gives no glycoside. Similarly, reaction of 2b with diosgenin conducted by the “reverse” procedure in the CH₂Cl₂/Et₂O mixture gives glycoside 6b in an excellent 98% yield. In turn, bromides with the phthaloyl protections of the amine group (2c and 2d) react more effectively with diosgenin when the reagents are dissolved solely in CH₂Cl₂. A comparison of the efficiency of the glycosyl donors indicates that the yields of diosgenin glycosylation with bromides 2a, 2b and 2d are higher than those with analogous chlorides 3a, 3b and 3d. However, reactivity of chloride 3c is stronger than that of analogous bromide 2c. (N-Phenyl)trifluoroacetimidates (5a–d) seem to be quite effective glycosyl donors. However their comparison with halogens 2a–d and 3a–d is encumbered since the glycosylation conditions were different for 5a–d. The N-trifluoroacetyl-protected bromide 2a is the less reactive among the bromides 2a–d; the remaining bromides react similarly. The same refers to chlorides 3a–d. In the case of (N-phenyl)trifluoroacetimidates 4a–d, the least efficient is 4d with the tetrachlorophthaloyl protection of the amine group; the remaining (N-phenyl)trifluoroacetimidates react similarly. In the experimental section (see Supporting Information File 1), the best procedures for each glycosyl donor are presented. Finally, diosgenyl 2-amino-2-deoxy-β-D-glucopyranose (7) was obtained by complete deprotections of 6a–d, as previously reported [45].

Table 1.

Procedures and results concerning diosgenin glycosylation.

Entry	Procedure	Glycosyl donor	Solvent	Promoter	Product	Yield (%)
1	normal	2a (α)	CH2Cl2/Et2O	AgOTf	6a	30
2	reverse	2a (α)	CH2Cl2/Et2O	AgOTf	6a	77
3	reverse	2a (α)	CH2Cl2	AgOTf	―	―
4	reverse	2b (α)	CH2Cl2/Et2O	AgOTf	6b	98
5	normal	2c (α + β)	CH2Cl2/Et2O	AgOTf	6c	51
6	reverse	2c (α + β)	CH2Cl2/Et2O	AgOTf	6c	55
7	reverse	2c (α + β)	CH2Cl2	AgOTf	6c	90
8	normal	2d (α + β)	CH2Cl2/Et2O	AgOTf	6d	73
9	reverse	2d (α + β)	CH2Cl2	AgOTf	6d	93
10	reverse	3a (α)	CH2Cl2/Et2O	AgOTf	6a	69
11	reverse	3b (α)	CH2Cl2/Et2O	AgOTf	6b	86
12	reverse	3c (β)	CH2Cl2	AgOTf	6c	99
13	reverse	3d (β)	CH2Cl2	AgOTf	6d	87
14	normal	5a (α + β)	CH2Cl2	TMSOTf	6a	85
15	normal	5b (α + β)	CH2Cl2	TMSOTf	6b	81
16	normal	5c (β)	CH2Cl2	TMSOTf	6c	83
17	normal	5d (β)	CH2Cl2	TMSOTf	6d	52

To obtain N-alkyl derivatives of diosgenyl 2-amino-2-deoxy-β-D-glucopyranoside (7), a method called “reductive alkylation of amines” was chosen. This method was previously successfully used to prepare N-alkyl derivatives of 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-D-glucose [51–52]. Thus, reductive alkylation of 7 with a 1.2 molar excess of the appropriate aldehyde and a twofold molar excess of NaBH₃CN provided mono- (9, 11, 15, 16) and dialkylated products (8, 10, 12–14, 17), solely or as mixtures (Scheme 2). The respective mixtures of mono- and dialkylated products were separated. Structures of the N-alkylated derivatives of 7 were confirmed by the NMR (¹H and ¹³C) spectroscopy and mass spectrometry (see Supporting Information File 1). All of them, similarly to the parent diosgenyl glycoside (7), adopt the ⁴C₁ conformation, as demonstrated by the J_1,2 ≈ 8 Hz, J_2,3 ≈ J_3,4 ≈ J_4,5 ≈ 9–10 Hz coupling constants.

Scheme 2.

N-Alkylation of diosgenyl 2-amino-2-deoxy-β-D-glucopyranoside (7).

Evaluation of antimicrobial activity

The N-alkyl derivatives of diosgenyl 2-amino-2-deoxy-β-D-glucopyranosides 8–15 and 17 were tested for their antibacterial and antifungal in vitro activity against 5 strains of Gram-negative bacteria, 5 strains of Gram-positive bacteria, and 3 strains of human pathogenic fungi. Respective minimum inhibitory concentration (MIC) values determined by a serial dilution microplate method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) for 8–15 and 17 as well as for 7 hydrochloride are summarized in Table 2 and Table 3. The latter was added as the reference since its high in vitro activities and in vivo efficacy were proved [53].

Table 2.

Minimum inhibitory concentration (MIC) [μg/mL] for 8–15 and 17 against the Gram-positive bacteria.

Comp.	Bacillus subtilis	Enterococcus faecalis	Rhodococcus equi	Staphylococcus aureus	Staphylococcus epidermidis
7·HCl	8	16	16	16	16
8	8	32	64	32	64
9	64	1	8	2	0.5
10	32	32	16	32	32
11	64	1	32	8	1
12	16	8	16	8	32
13	16	8	16	8	8
14	32	4	8	16	32
15	>1024	>1024	512	1024	1024
17	64	64	512	128	256

Table 3.

Minimum inhibitory concentration (MIC) [μg/mL] for 8–15 and 17 against human pathogenic fungi.

Comp.	Candida tropicalis	Candida albicans	Aspergillus niger
7·HCl	0.5	2	64
8	2	2	64
9	—a	16	8
10	1	2	128
11	—a	8	8
12	4	4	64
13	128	8	16
14	32	16	256
15	64	128	128
17	128	64	128

^aNot determined.

The determined MIC values indicate that compounds explored have rather poor if any inhibitory activity against the Gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris and Pseudomonas aeruginosa. In contrast, almost all the tested N-alkyl saponins were found to inhibit the growth of the Gram-positive bacteria (Table 2). Among the tested compounds the most active was diosgenyl 2-deoxy-2-ethylamino-β-D-glucopyranoside (9) with MIC of 0.5, 1, 2, and 8 μg/mL against Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus aureus, and Rhodococcus equi, respectively. Also the N-propyl derivative 11 was found to be active against Enterococcus faecalis, Staphylococcus epidermidis, and Staphylococcus aureus with MIC of 1, 1, and 8 μg/mL, respectively. Importantly, both 9 and 11 exhibit a stronger inhibitory effectivity than diosgenyl 2-deoxy-2-amino-β-D-glucopyranoside hydrochloride (7·HCl), which was found to be very active alone and in combination with daptomycin and vancomycin against Gram-positive cocci [53]. The N,N-dialkyl derivatives 12, 13 and 14 act against the Gram-positive bacteria more efficiently or similarly to 7 hydrochloride. In turn, N-pentyl (15) and N,N-dihexyl (17) compounds are completely inactive with respect to all the tested strains of the Gram-positive bacteria. The results presented indicate that both elongation of the alkyl group as well as addition of another alkyl group are rather ineffective from the standpoint of the inhibitory activity towards the Gram-positive bacteria. Such findings are probably due to the lower solubility of the compounds with longer N-alkyl groups or to the micelle formation.

Studies on the activity of the synthesized N-alkyl derivatives of diosgenyl 2-deoxy-2-amino-β-D-glucopyranoside (8–15 and 17) against 3 strains of the human pathogenic fungi (Table 3) indicate that the growth of Candida tropicalis is the most efficiently inhibited by the reference compound 7·HCl with MIC of 0.5 μg/mL. A slightly lower activity was exhibited by N,N-diethyl derivative 10 (MIC 1 μg/mL), N,N-dimethyl derivative 8 (MIC 2 μg/mL), and N,N-dipropyl derivative 12 (MIC 4 μg/mL). Compounds with the longer alkyl chains (13–15, 17) show very weak inhibitory activity against Candida tropicalis.

The results presented for Candida albicans resemble those obtained for Candida tropicalis. The growth of this strain of fungi is inhibited at the lowest concentrations by 7·HCl and equally well by 8, 10, and 12 (MIC 2 μg/mL for each compound mentioned). Evidently, short dialkyl derivatives (8, 10, 12) are more effective against the tested fungi than analogous monoalkyl derivatives (9, 11), longer monoalkyl (15) and dialkyl derivatives (13, 14, 17).

Among the tested strains of fungi, Aspergillus niger turned out to be the least susceptible to the N-alkyl derivatives of diosgenyl 2-deoxy-2-amino-β-D-glucopyranoside (8–15 and 17). It is worth notice that N-ethyl (9) and N-propyl (11) derivatives reveal much better activity than the reference 7·HCl (MIC 8, 8, and 64 μg/mL, respectively). Also N,N-dibutyl derivative (13) with MIC of 16 μg/mL inhibits the growth of Aspergillus niger at lower concentrations in comparison to that of 7·HCl.

Conclusion

Different pathways leading to diosgenyl 2-amino-2-deoxy-β-D-glucopyranoside and several N-alkyl derivatives are reported. Investigations of their antimicrobial activity indicate that N-ethyl and N-propyl derivatives exhibit stronger activity against Gram-positive bacteria than the parent diosgenyl 2-deoxy-2-amino-β-D-glucopyranoside hydrochloride.

Supporting Information

File 1

Experimental details for the preparation of compounds 2b, 2c, 4a–d, 5a–d, 6a–d, 8–17, corresponding characterization data and information on the way of determination of minimum inhibitory concentration.