Stereoselective Synthesis and Cytoselective Toxicity of Monoterpene-Fused 2-Imino-1,3-thiazines

Starting from pinane-, apopinane- and carane-based 1,3-amino alcohols obtained from monoterpene-based β-amino acids, a library of monoterpene-fused 2-imino-1,3-thiazines as main products and 2-thioxo-1,3-oxazines as side-products were prepared via two- or three-step syntheses. When thiourea adducts prepared from 1,3-amino alcohols and aryl isothiocyanates were reacted with CDI under mild conditions, O-imidazolylcarbonyl intermediates were isolated which could be transformed to the desired 1,3-thiazines under microwave conditions. 1,3-Thiazines and 2-thioxo-1,3-oxazine side-products could also be prepared in one-step reactions through the application of CDI and microwave irradiation. The ring-closure process was extended to cycloalkane-based γ-hydroxythioureas. The carane- and apopinane-based derivatives exhibited marked antiproliferative activity against a panel of human adherent cancer cell lines (HeLa, A2780, MCF7 and A431).


Introduction
In the past decade, alicyclic 1,3-amino alcohols have proved to be versatile building blocks. They have been applied as useful starting materials in the stereoselective syntheses of compounds of pharmacological interest and they have served as chiral ligands and auxiliaries in enantioselective transformations [1][2][3].
When we applied the above-mentioned mild conditions, we observed the formation of O-imidazolylcarbonyl intermediates 29-33 (similar intermediates were presumed, but could not be isolated in Bernacki's work) as single products even when the reaction mixture was subjected to conventional heating from room temperature to reflux ( Table 1). The structures of intermediates 31c and 31d were determined by X-ray crystallography ( Figure 2) and NMR measurements. ORTEP plots of the configurations of the major diastereoisomers 31c and 31d (the C atom of the methoxy group (C14) is disordered in a 1:1 ratio).

31c 31d
However, when microwave irradiation was applied to the isolated intermediates 29-33 in THF, we obtained the desired 2-imino-1,3-thiazines 25-28 as main products in acceptable yields in a short time (60 min) (Scheme 3, Table 2). The ring-closure process could be carried out in one step by applying CDI in THF under microwave conditions. Moreover, we observed that 2-thioxo-1,3-oxazines 34-37 were also formed as minor products in 10%-24% yields. The transformation of 20a was an exception, when thiazine 25a was isolated as a single product. Besides NMR assignments, the structures of 35 and 36 were proved by independent synthesis starting from the corresponding amino alcohols and thiophosgene.
The above procedure was subsequently extended to 1-(2-hydroxymethylcycloalkyl)thioureas. Starting from cycloalkane-based 1,3-amino alcohols 9 and 10, thioureas 38 and 39 were prepared by a literature method [33]. When 38 and 39 were treated with CDI in THF, rapid conversion to intermediates 40 and 41 was observed and in this case the ring-closure proceeded under conventional heating to yield 1,3-thiazines 42 and 43 (Scheme 2). We observed that the developed method could be applied more easily in the case of thioureas with sterically less hindered structures.

Antiproliferative Activities
The novel 2-imino-1,3-thiazines 24-28 (Scheme 3) and some of their analogues were subjected to in vitro pharmacological studies in order to investigate their antiproliferative properties on a panel of human adherent cancer cell lines. The results of MTT assays are presented in Table 3. Carane-based compound 25a proved to be the most potent of the tested compounds, exhibiting a cell growth-inhibiting capacity comparable to that of the reference agent cisplatin. Compounds with a pinane ring were generally less potent, while the introduction of a methyl group at position 2 (Scheme 3) favoured the action (24a). Substitution of the N-phenyl ring had a limited and inconsequential impact on the efficacy (26a-d), while the introduction of a N-benzyl function onto the 1,3-thiazine skeleton was disadvantageous (28a). Since no substantial difference was observed between the effects of 26a and 27a, the configuration of C-3 (cis or trans ring fusion, Scheme 3) also seems irrelevant. On the other hand, the 2-thioxo-1,3-oxazine analogue (35) was completely ineffective, indicating that the arylimino substituent is an essential part of the molecule. Replacement of the pinane or carane ring system with cyclopentane (42) or cyclohexane (43) also led to ineffective congeners, demonstrating the crucial role of the bicyclic monoterpene as a building block for the design and synthesis of novel antiproliferative agents.

General Information
1 H-and 13 C-NMR spectra were recorded on a Bruker Avance DRX 400 spectrometer (400 MHz, δ = 0 (TMS)), in an appropriate solvent. Chemical shifts are expressed in ppm (δ) relative to TMS as internal reference. J values are given in Hz. FT-IR spectra were recorded on a Perkin-Elmer Spectrum 100 instrument. Microanalyses were performed on a Perkin-Elmer 2400 elemental analyser. Microwave reactions were carried out by heating at 125 °C and 200 W for 1 h in a CEM Discover LabMate microwave reactor. Optical rotations were obtained with a Perkin-Elmer 341 polarimeter. Melting points were determined on a Kofler apparatus and are uncorrected. Chromatographic separations were carried out on Merck Kieselgel 60 (230-400 mesh ASTM). Reactions were monitored with Merck Kieselgel 60 F254precoated TLC plates (0.25 mm thickness). All the chemicals and solvents were used as supplied.

General Procedure for the Synthesis of Thioureas 21-23
Amino alcohol 16, 17 or 18 (1.08 mmol) and the appropriate isothiocyanate (1.14 mmol) were dissolved in toluene (80 mL) and the mixture was stirred at room temperature for 6 h, except that in the case of N-benzylamino alcohol 18, heating at 50 °C for 6 h was indicated. The resulting mixtures were then evaporated to dryness, filtered and washed with n-hexane. The purities of the products obtained were determined via NMR to be >97%.    To the respective thiourea (1.25 mmol) 19-23, 38 or 39 in THF solution (12 mL), CDI (0.306 g, 1.88 mmol) was added at room temperature. The reaction was stirred for 2-6 h at room temperature (TLC monitoring), followed by careful evaporation of the solvent at 35 °C. The crude products obtained were purified by flash column chromatography on silica gel. (1H, s), 9.28 (1H, s). 13        incubation in a humidified atmosphere of 5% CO 2 at 37 °C, the living cells were assayed by the addition of 20 µL of 5 mg/mL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution [37]. During a 4-h contact period, the MTT was converted by intact mitochondrial reductase and precipitated as blue crystals. The medium was then removed, the precipitated formazan crystals were solubilized in DMSO (100 µL) during a 60-min period of shaking at 25 °C, and the absorbance was read at 545 nm with a microplate reader. Wells with untreated cells were utilized as controls. All in vitro experiments were carried out on two microplates with at least five parallel wells. Stock solutions of the tested substances (10 mM) were prepared with DMSO. The DMSO concentration (0.3%) of the medium did not have any significant effect on cell proliferation. Cisplatin was used as reference compound.

X-ray Crystallographic Studies
Crystallographic data were collected at 123 K for 31c and 31d by using a Bruker Nonius-Kappa CCD diffractometer with an APEXII area detector and graphite-monochromatized Mo-K α radiation (λ = 0.71073 Å), as reported earlier [38].
Crystal data for 31c: The structures were solved by direct methods by use of the SHELXS-97 program [39], and full-matrix, least-squares refinements on F 2 were performed by use of the SHELXL-97 program [39]. The CH hydrogen atoms were included at fixed distances from their host atoms with the fixed displacement parameters. The NH hydrogen atom positions were refined with the fixed displacement parameters. In 31d, the methoxy C atom has two different orientations in a 1:1 ratio (due to packing reasons). The graphics were drawn with ORTEP3 for Windows [40]. The depositions numbered CCDC 1000179 (for 31c) and CCDC 1000178 (for 31d) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html (or from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif).

Conclusions
In conclusion, we have developed a mild and efficient method for the synthesis of 2-imino-1,3-thiazines by the ring closure of thiourea adducts of 1,3-amino alcohols in the presence of CDI under microwave condition. Besides the main product 1,3-thiazines, the formation of 2-thioxo-1,3-oxazines as side-products was observed. The ring-closure process was extended to cycloalkane-based γ-hydroxythioureas and the method developed for the synthesis of 2-imino-1,3-thiazines containing an acid-sensitive structure proved to be comparable to those reported in the literature. The resulting 1,3-thiazines exert marked antiproliferative action on a panel of human cancer cell lines.