Synthesis and Cytotoxic Activity of Lepidilines A–D: Comparison with Some 4,5-Diphenyl Analogues and Related Imidazole-2-thiones

A straightforward access to 2-unsubstituted imidazole N-oxides with subsequent deoxygenation by treatment with Raney-nickel followed by N-benzylation opens up a convenient route to lepidilines A and C. Both imidazolium salts were used to generate in situ the corresponding imidazol-2-ylidenes, which smoothly reacted with elemental sulfur, yielding imidazole-2-thiones. These reactions were performed either under classical conditions in pyridine solutions or mechanochemically using solid Cs2CO3 as a base. The structure of lepidiline C was unambiguously confirmed by X-ray analysis of its hexafluorophosphate. An analogous protocol toward lepidilines B and D and their 4,5-diphenyl analogues is less efficient due to observed instability of the key precursors, i.e., the respective 2-methylimidazole N-oxides. Comparison of cytotoxic activity against HL-60 and MCF-7 cell lines of all lepidilines, as well as their selected structural analogues (e.g., 4,5-diphenyl derivatives and PF6 salts), revealed slightly more potent activity of the 2-methylated series, irrespectively of the type of counterion present in the imidazolium salt. Remarkably, the well-known 1,3-diadamantylimidazolium bromide (the “Arduengo salt”), known as the precursor of the first, shelf-stable NHC representative, and its adamantyloxy analogue displayed the most significant cytotoxic activity in the studied series.

L epidilines A−D (1a−1d) belong to the class of imidazolium alkaloids found in extracts prepared from roots of Lepidium meyenii (so-called Maca), a South American plant known in folk medicine of Peruvian Indian tribes for more than a thousand years. Over centuries, aqueous extracts as well as dried roots of Maca were used as a natural drug and as a food additive. Currently it is widely explored as a popular dietary supplement easily available not only in the pharmacy but also in the food markets. 1 Moreover, lepidilines A and C have been used as convenient precursors of nucleophilic carbenes (NHCs) applied for the synthesis of bioactive metal complexes with gold(I), silver(I), and iridium(I) ions. 2 Alkaloids 1a and 1b were isolated and identified for the first time in 2003, and the structure of lepidiline A was unambiguously proved by the X-ray analysis. 3 More recently, two nonsymmetric imidazolium salts, 1c and 1d (lepidilines C and D, respectively), were also isolated from Maca extracts, and their structures were elucidated on the basis of spectroscopic data analysis. 4 Noteworthy, whereas promising anticancer activity of lepidilines A and B was discussed in the original report by Cui et al., 3 no information is available on biological properties of lepidilines C and D. Although lepidilines A and B are easily available via double benzylation of 4,5-dimethyl-and 2,4,5-trimethylimidazole, 5 respectively, for the synthesis of their unsymmetrically substituted analogues C and D the above standard alkylation procedure cannot be applied. Thus, due to the current interest in the chemistry and application of imidazolium salts, the development of general methods for multigram scale synthesis of lepidilines A−D is of practical importance.
In our continuous research on the synthesis and reactivity of imidazole N-oxides of type 2, we demonstrated that they are superior building blocks for the preparation of diverse imidazole derivatives. 6 As shown in Scheme 1, condensation of αhydroxyiminoketones 3 with imines 4 followed by deoxygenation of the first formed imidazole N-oxides 2 offers a convenient access to polysubstituted imidazoles 5 with excellent control on the substitution pattern. Thus, the method allows for preparation of more complex imidazole derivatives bearing either functionalized alkyl or aryl groups located at the N(1), C(2), C(4), and C(5) atoms of the core heterocycle. In the quest for designed lepidiline precursors, diacetyl monoxime (3a, R 3 = R 4 = Me) and benzyl formaldimines (R 2 = H) and acetimines (R 2 = Me) of type 4 are indicated as convenient starting materials. Benzylation of the key 4,5-dimethylimidazoles 5 should lead to desired alkaloids 1 (Scheme 1).
The goal of the present study was to elaborate a general method for the preparation of the title compounds and their structural analogues, such as imidazole-2-thiones available via intermediate nucleophilic carbenes. In addition, an unambiguous confirmation of the structure of a representative nonsymmetric alkaloid (lepidiline C or D) is also of interest. Finally, cytotoxic activity of the selected imidazole-based products was tested against two cancer cell lines, HL-60 and MCF-7, and for comparison on normal HUVEC cells.

■ RESULTS AND DISCUSSION
In our recent publication, 2-unsubstituted imidazole N-oxides such as 2 were used as key building blocks for the preparation of a series of benzyloxy analogues of lepidiline A. 7 In the presented study, deoxygenation of N-oxides 2 was required to get the desired 1,4,5-tri-and 1,2,4,5-tetrasubstituted imidazoles. In an initial experiment, the known 1-benzyl-4,5-dimethylimidazole N-oxide (2a) 8 was treated with freshly prepared Raney-nickel, in EtOH, and the obtained 1-benzyl-4,5-dimethylimidazole (5a) was N-alkylated with benzyl chloride under microwave (MW) irradiation. The reaction was complete after 5 min, yielding the expected lepidiline A (1a) in nearly quantitative yield (Scheme 2). The same method was applied for the synthesis of the 4,5diphenyl analogue of 1a (i.e., compound 6a), but in the case of benzylation with BnCl a very low conversion of ca. 5% was observed after 60 min of heating, while application of BnBr as a more reactive electrophile provided the expected imidazolium bromide 6a[Br] in 87% yield after 45 min. Furthermore, the anion exchange aimed at preparation of hexafluorophosphates derived from 1a and 6a[Br] was easily achieved by treatment of the starting salts with NH 4 PF 6 in aqueous EtOH.
To the best of our knowledge, synthesis of lepidiline C has not yet been elaborated and reported. In our hands, imidazole 5a was successfully alkylated with m-methoxybenzyl chloride under MW conditions to afford the expected imidazolium alkaloid 1c in 84% yield (Scheme 3). The product was isolated as a viscous oil, which after recrystallization from an i-Pr 2 O/CH 2 Cl 2 mixture gave a colorless solid with a melting point of 94−96°C. The measured temperature of the Cr → I phase transition in 1c was clearly different from that reported for lepidiline C isolated from natural sources (mp 225−228°C). Nevertheless the 1 H and 13 C Scheme 1. Multistep Synthesis of Lepidilines 1 Starting with Condensation of α-Hydroxyiminoketones 3 with Imines 4, Followed by Deoxygenation of the Initially Formed Imidazole N-Oxides 2 and N-Benzylation of the Resulting Imidazoles 5 Scheme 2. Synthesis of Lepidiline A (1a) and Its 4,5-Diphenyl Analogue 6a Journal of Natural Products pubs.acs.org/jnp Article NMR spectra of the obtained material corresponded well with the reported chemical shifts. 4 Fortunately, the anion exchange in 1c for PF 6 − enabled growth of fine single crystals suitable for Xray analysis, which unambiguously confirmed the expected structure of the imidazolium cation in 1c[PF 6 ] ( Figure 1). 9 By analogy, the 4,5-diphenylimidazolium analogues of lepidiline C, 6c and 6c[PF 6 ], were prepared using imidazole 5b as the starting material (Scheme 3). Whereas replacement of Cl − by Br − has practically no impact on the chemical shifts of signals in the 1 H NMR spectra of imidazolium salts of type 1 and 6, introduction of PF 6 − in the 2-unsubstituted series resulted in a remarkable high-field shift of the diagnostic signals attributed to C(2)-H. For example, the aforementioned singlets for 1c, 1c [Br], and 1c[PF 6 ] were found at δ 10.80, 10.76, and 8.62, respectively.
In order to demonstrate flexibility of the presented synthetic method for the preparation of unsymmetric imidazolium salts, the same lepidiline C and its diphenyl analogue 6c were prepared in an alternative protocol using 1-(3-methoxybenzyl)functionalized N-oxides 2c and 2d (Scheme 3). For example, after smooth deoxygenation of 2c followed by treatment of the resulting imidazole 5c with BnCl, the expected 1c was isolated in fair 54% overall yield (for two steps). These experiments indicate that the preparation of the target unsymmetric imidazolium salts can be readily achieved by using different sets of starting materials, i.e., α-hydroxyiminoketones, benzylamines, and benzyl halides, and thereby, the applied protocol enhances the utility of this method for the preparation of differently substituted imidazolium salts.
The replacement of N-benzyl formaldimine (4a) by the respective acetimine 4c in the reaction with α-hydroxyiminoketone 3a opened up a straightforward access to imidazole Noxide 2e considered as a suitable precursor of lepidilines B and D (Scheme 4). Indeed, reduction of 2e followed by N-benzylation of 5e with benzyl chloride or m-methoxybenzyl chloride provided desired alkaloids 1b and 1d, respectively. In contrast to 2e, attempted preparation of its 4,5-diphenyl analogue using benzil monoxime (3b) and acetimine 4c was unsuccessful, as the initially formed N-oxide suffered decomposition during workup. We assume that the observed decomposition of this imidazole N-oxide results from the anticipated limited stability of its 3hydroxy-2-methylidene tautomer. Apparently, the presence of two Ph substituents located at C(4) and C(5) enables the tautomeric rearrangement and in the presence of moisture leads to unidentified, deeply red-colored product(s). For that reason we waved on the synthesis of 1-benzyl-2-methyl-4,5-diphenylimidazole (5f) via the respective imidazole N-oxide, and instead the required imidazole 5f was obtained following a known procedure based on multicomponent condensation of benzil, acetaldehyde, benzylamine, and ammonium acetate in the presence of InCl 3 used as a catalyst. 10 Thus, the synthesis of the target 4,5-diphenyl analogue of lepidiline D (i.e., compound 6d) was achieved using the latter heterocycle 5f and mmethoxybenzyl chloride under MW irradiation, which efficiently accelerated the quaternization process.
Imidazole-2-thiones are known as biologically active compounds, which display diverse biological activity, and many representatives are recognized as potent antimicrobial, antithyroid, anti-HIV, and anticancer agents. 11 One of the relatively new and attractive methods for the synthesis of non-enolizable imidazole-2-thiones comprises sulfurization of transient imidazol-2-ylidenes with elemental sulfur. 12 In a recent report, this method was successfully applied for the conversion of numerous benzyloxy-functionalized imidazolium salts into the corresponding imidazole-2-thiones. 7 Thus, lepidilines A and C seem to be attractive substrates for further functionalization via the respective intermediate carbenes (NHCs). In a typical experiment, imidazolium chloride 1a was treated with Et 3 N and S 8 in dry pyridine solution at room temperature (Scheme 5, Method A). After overnight stirring the expected imidazole-2-thione 7a was isolated as a crystalline product, although in low 34% yield. In the search for a more efficient protocol, the ball-mill approach was checked by using Cs 2 CO 3 as a base and a 2-fold excess of elemental sulfur, in the presence of butanone as liquid-assisted grinding solvent (LAGs) (Method B). To our delight, the expected imidazole-2-thione 7a was formed solely, and the product was isolated in an excellent yield of 97%. The 13 C NMR spectrum of 7a confirmed the presence of the thiourea unit in the molecule, as the typical resonance of this diagnostic group was found at δ 162.7. Analogous procedures applied for lepidiline C afforded the respective imidazole-2-thione 7c isolated as a colorless solid in 53% (Method A) and 73% (Method B) yield. Two more products of that type (i.e., Journal of Natural Products pubs.acs.org/jnp Article compounds 8a and 8c) with a 4,5-diphenylimidazole motif were also obtained in an analogous manner. Potential biological activity of all four lepidilines A−D (1a− 1d), as well as their 4,5-diphenyl analogues 6a[PF 6 ], 6c[PF 6 ], and 6d[PF 6 ] (as hexafluorophosphate salts) and a series of imidazole-2-thiones 7 and 8 obtained therefrom, was evaluated in vitro against two human cancer cell lines: promyelocytic leukemia HL-60 and breast cancer adenocarcinoma MCF-7. Selected analogues were also tested against human umbilical vein endothelial cells (HUVECs) using the MTT cytotoxicity assay. Concentration−response analysis was performed to determine drug concentrations required to inhibit the growth of cells by 50% (IC 50 ) after 48 h of incubation. The obtained results are summarized in Table 1 and compared with activity of the known anticancer agent doxorubicin (DXR) used as a positive control. 13 Lepidilines B and D were found to be significantly cytotoxic against HL-60 cells with IC 50 values in the low micromolar range of 3.8 and 1.1 μM, respectively, which were an order of magnitude lower than those obtained for lepidilines A and C (i.e., 32.3 and 27.7 μM, respectively). None of the lepidilines showed similarly high cytotoxicity on the MCF-7 cell line, and lepidiline D turned out to be over 100-fold more cytotoxic for leukemia than for breast cancer cells. In the experiments performed on normal (HUVEC) cells the IC 50 values for all four lepidilines A−D were over 100 μM, which indicates a large safety margin for these compounds.
It is worth stressing that this is the first report in which the cytotoxicity of all four representatives of the lepidiline family has been checked against selected cell lines, which made it possible to compare their activity under identical conditions. In a previous report by Zheng only lepidilines A and B were examined against eight human cancer cell lines. 3 The mentioned work indicated lepidiline A was inactive against all those cell lines with IC 50 values above 10 μM (the values reported in the paper are expressed in μg/mL). In contrast, lepidiline B showed cytotoxic activity against several cell lines, especially high against pancreatic adenocarcinoma PACA2 (IC 50 = 1.38 μg/mL, i.e., 4.2 μM) and against breast carcinoma MDA-231 (IC 50 = 1.66 μg/ mL, i.e., 5.1 μM). Unfortunately, it is difficult to compare those results with our data obtained on different cell lines. However, the common observation from both studies is that lepidiline B is more cytotoxic than lepidiline A. Analysis of the structure−activity relationship of 4,5-diphenyl analogues of lepidilines A, C, and D (Table 1, entries 7−9) revealed that the replacement of methyl by phenyl at C(4) and C(5) of the imidazole ring caused a large increase of cytotoxicity against the MCF-7 cell line and in the case of 6c[PF 6 ] also against HUVECs, making these analogues much less selective as compared with natural lepidilines.
Introduction of a sulfur atom at C(2) in a series of imidazole-2-thiones 7 and 8 derived from lepidilines A and C and their analogues bearing Ph substituents at C(4) and C(5) atoms of the imidazole ring was not advantageous for activity, especially against MCF-7 cells (Table 1, entries 10−13). This decreased cytotoxicity may result from lower bioavailability of imidazole-2thiones, which in contrast to lepidilines are not charged molecules. It is well known that the neutral organic compounds interact with membranes only through hydrophobic bonds, whereas charged substances can additionally benefit from electrostatic interactions. 14 In contrast to imidazole-2-thiones, two highly oleophilic imidazolium salts 9 and 10 bearing at N(1) and N(3) atoms either adamantan-1-yl (the "Arduengo salt") 15 or adamantan-1yloxy groups, 16 respectively, were found to be significantly cytotoxic for HL-60 cells. Notably, imidazolium salt 10 was the most active among all tested compounds (IC 50 = 0.3 and 4.5 μM on HL-60 and MCF-7 cell lines, respectively) and also displayed some selectivity.
As a positive control in the MTT assay a well-known anticancer drug, DXR, widely used to treat breast cancer and acute lymphocytic leukemia among other cancer types, 13 has been used. The IC 50 values for DXR against HL-60 and MCF-7 cells were below 1 μM (0.12 and 0.9 μM against HL-60 and MCF-7 cells, respectively), and the value obtained for normal HUVEC cells was 1.4 μM under the same experimental conditions. Thus, DXR was 10-fold more cytotoxic than lepidiline D, but the HUVEC/HL-60 IC 50 ratio for DXR was about 10 and that for lepidiline D over 100.
In summary, the present study showed that the title lepidilines A−D can be conveniently prepared using imidazole N-oxides as key intermediates, which are readily available via cyclocondensation of diacetyl monoxime with N-benzyl aldimines. Initial deoxygenation with Raney-nickel followed by microwaveassisted benzylation leads to both symmetric and nonsymmetric imidazolium alkaloids in excellent yield and purity. X-ray analysis of the imidazolium salt derived from synthetic lepidiline C was presented for the first time and confirmed the postulated structure of the naturally occurring material. In addition, sulfurization of nucleophilic carbenes derived from lepidilines A and C enables convenient preparation of corresponding, hitherto unknown, non-enolizable imidazole-2-thiones.
Our results contribute to the development of methods useful for synthesis of naturally occurring, biologically active imidazole derivatives relevant for medicinal chemistry and related applications. 17,18 Progress in this attractive field was summarized in a very recent, comprehensive review. 19 Further applications of imidazolium salts of "lepidiline type" in coordination, organometallic, and materials chemistry are also possible. 18,19 The biological results presented here revealed significant cytotoxicity of lepidilines B and D in the tested series of naturally occurring alkaloids and, most importantly, their remarkable selectivity against leukemia HL-60 versus normal HUVEC cells. These compounds as well as their 4,5-diphenyl imidazolium derivatives and the presented adamantyloxy analogue of the "Arduengo salt" 15 can be considered not only as readily available Compound concentration required to inhibit metabolic activity by 50%. The cells were incubated with the analogues for 48 h. Values are expressed as mean ± SEM from the concentration−response curves of at least three experiments using a nonlinear estimation (quasi-Newton algorithm) method.
Journal of Natural Products pubs.acs.org/jnp Article precursors of nucleophilic carbenes 2 but also as useful probes in the search for new leads in antileukemic drug discovery.
Synthesis of Imidazolium Chlorides and Bromides of Type 1 and 6. To a deoxygenated solution of imidazole 5 (1.0 mmol) in MeCN (10 mL) was added benzyl halide (1.5 mmol), and the resulting mixture was MW-irradiated at 110°C until the starting imidazole was fully consumed (TLC monitoring, usually up to 60 min). The solvent was removed under reduced pressure, and the crude product was washed with several portions of dry Et 2 O. Solid products were recrystallized from a CH 2 Cl 2 /hexanes mixture.  1-Benzyl-3-(3-methoxybenzyl)-2-methyl-4,5-diphenylimidazolium chloride (6d). The crude product was purified by preparative thinlayer chromatography (SiO 2 , CH 2 Cl 2 /MeOH, 92:8); spectroscopically pure sample of 6d (178 mg, 37%) was isolated as a colorless oil and used for the next step without further purification. 1