Thionation of Some α,β-Unsaturated Steroidal Ketones

The reactions of selected α,β-unsaturated steroidal ketones with Lawesson’s reagent (LR) in CH2Cl2 and toluene under the standard reaction conditions and with a combination of phosphorus pentasulfide with hexamethyldisiloxane (P4S10/HMDO) in 1,2-dichlorobenzene (ODCB) under microwave irradiation were investigated and for this purpose several cholestane, androstane and pregnane carbonyl derivatives were chosen. Depending on the reagent and the solvent, 19 new sulfur containing compounds, including dithiones 4c and 4d, α,β-unsaturated 3-thiones 3a-e, dimer-sulfides 2a-e, 1,2,4-trithiolanes 5a-e and phosphonotrithioates 6b-e were synthesized. All newly prepared compounds were characterized by IR, 1H- and 13C-NMR spectroscopy and elemental analysis.


Introduction
Steroids are an important group of natural compounds possessing a variety of biological activities. The replacement of one or more carbon atoms in a steroid molecule by a heteroatom affects the chemical properties of that particular steroid and often results in alterations of its biological activity,

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which sometimes may be useful. Also, the addition of heterocyclic rings or new functional groups to steroid molecules often leads to changes in their physiological activity. Thionation is one possible modification that could have some influence on such activity. The larger and less electronegative sulfur atom, relative to oxygen, might alter the hydrogen-bonding ability and/or induce conformational changes in the modified molecule. In addition the increased reactivity of the thione function should make these derivatives useful intermediates for further transformations. Recently we reported synthesis of 6-thioxo-7-aza-B-homocholest-4-ene and 6-aza-7-thioxo-B-homocholest-4-ene using Lawesson's reagent [1]. Continuing this investigation and our previous work on modified steroid compounds as biologically active molecules [1][2][3][4], the goal of this study was to synthesize some new thioxosteroid derivatives.
Several methods are reported in the literature for the thionation of organic compounds. Phosphorus pentasulfide (P 4 S 10 ) was first reported in 1869 by Henry [5] and Wislicenus [6]. The usual procedure, which involves boiling toluene, xylene or pyridine as solvent, requires a large excess of reagent, long reaction timed, and results in low and variable yields [7][8][9]. Recently Curphey [10][11][12] has shown that a combination of P 4 S 10 with hexamethyldisiloxane (HMDO) efficiently converts esters, lactones, amides and ketones into their corresponding thio derivatives. Under the standard reaction conditions (dry toluene/xylene, thermal heating) this method has provided an increase in the selectivity and yield. Kaushik et al. developed a new thionating reagent by encapsulating the P 4 S 10 in basic alumina [13]. The reactions were carried out in good to excellent yields by refluxing mixture of ketone and P 4 S 10 /Al 2 O 3 in acetonitrile, but this method needs some more investigation. In 1978, Lawesson and coworkers developed a new reagent, 2,4-bis(p-methoxyphenyl)-1,3-dithiadiphosphetane-2,4-disulfide, commonly named Lawesson's reagent (LR, Figure 1) [14][15][16]. The attractiveness of LR is associated with its commercial availability, simplicity and convenience of use, high yields and especially soft thionation reactions. In some cases the main disadvantage of this reagent is the formation of byproducts or stable heterocyclic intermediates. However, some of these sulfur and phosphorus containing products can be very useful as they have showed a promising antimicrobial and anti-inflammatory activity [17][18][19][20]. Since all described procedures require the use of dry aromatic hydrocarbon solvents and lengthy reaction times, microwave assistance has been applied to thionate a wide variety of substrates [21][22][23][24][25].
Herein we report on the reaction of several α,β-unsaturated cholestane, androstane and pregnane carbonyl derivatives 1a-e ( Figure 2) with LR (in CH 2 Cl 2 and toluene) under the standard reaction conditions and with a combination of P 4 S 10 /HMDO in 1,2-dichlorobenzene under microwave irradiation.

Results and Discussion
The reactions of α,β-unsaturated ketones 1a-e with LR gave different products, depending on the solvent and duration of the reaction. Due to the instability of the unsaturated thioketones or dimers formed and their easy reconversion to the starting compounds a certain amount (8-52%) of the parent compounds was isolated in all cases.
At the beginning, the reaction procedure for thionation was carried out in refluxing toluene. Instantaneous reaction took place (the purple color of reaction mixture) with simultaneous formation of thionated products. Although there was no complete consumption of 1a-e, the reaction was quenched after 25-45 min (depending on substrate, see Experimental), while the solution was still purple, which was the evidence of the presence of thioketones. After flash column chromatography, besides corresponding dimer-sulfides 2a-e (7-56%), thioketones 3a-e were isolated in 11-27% yields (Scheme 1). When the reaction procedure was carried out in refluxing toluene for 8 h initially formed unstable α,β-unsaturated thioketones either dimerize to give corresponding dimer-sulfides 2a-e (30-51%), or decompose to a more stable starting ketones 1a-e (26-52%) (Scheme 1).

Scheme 1.
Thionation of 1a-e with LR in toluene.
All obtained thioketones 3a-e were pink oils and their structures were determined on the basis of their spectral data (IR, 1 H-NMR, 13 C-NMR). In the IR spectra the absorption for the original unsaturated 3-oxo group, was missing, as well as the singlet for C-3 in the 13 C-NMR spectra at about 199 ppm. Instead, the new singlet at δ 236.7, 237.0, 237.8, 236.9 and 237.4 ppm for C(3)=S, for compounds 3a-e, respectively, appeared. In the 1 H-NMR spectra signal for H-4 at δ 6.63, 6.69, 6.74, 6.66 and 6.63 ppm was situated downfield comparing to the resonance for the corresponding proton in starting compounds 1a-e (at δ 5.72, 5.74, 5.84, 5.80 and 5.70 ppm, respectively). In addition, in all thioketones, both protons at C-2 (α-position to the C=S group) resonate well-separated downfield from the other ring protons due to the strong C=S anisotropy, H-equatorial as a td at about δ 3.15 ppm, H-axial as a dtd, at about δ 2.50 ppm both with corresponding J HH coupling constants.
In the IR spectra of 2a-e the absorptions for the original unsaturated 3-oxo groups at 1667, 1665, 1667, 1665 and 1661 cm -1 for 1a-e, respectively, were missing, as well as the singlet for C-3 in the 13 C-NMR spectra at about δ 199 ppm. Instead, the new C-3 singlet at δ 129.4, 129.5, 129.5, 129.3, 129.4 ppm and the C-6 doublet at δ 124.3, 123.3, 124.8, 123.8 and 123.6 ppm for compounds 2a-e, respectively, appeared indicating the formation of a new double bond and Δ 3,5 -diene structure. New signal had also appeared in the 1 H-NMR spectra: the broad triplet at δ 5.47, 5.45, 5.50, 5.42 and 5.39 ppm for H-6 for compounds 2a-e, respectively. On the other hand, the H-4 signal at δ 6.11, 6.13, 6.14, 6.11 and 6.10 ppm for compounds 2a-e, respectively, was shifted downfield comparing with that in starting compounds 1a-e (at δ 5.72, 5.74, 5.84, 5.80¸ 5.70 ppm, respectively). This can be explained by anisotropy effect of the new double bond in the dimers. On the basis of the COSY correlations between the vinylic H-6 and H 2 -7 together with the key HMBC correlations from H-6 to C-7 and C-8 the alternative Δ 2,4 -isomer was ruled out. Furthermore, the elemental analyses for 2a-e were in agreement with the proposed structures.
In order to increase the yield of thioketones we performed the same reactions under milder conditions, in CH 2 Cl 2 as a solvent (refluxing for 45 min) and the thioketones 3a-e were obtained in much higher yield, 28-70%. Besides, steroids 1c-e gave corresponding 1,2,4-trithiolanes 5c-e. The steroids 1b and 1e gave also the (4-methoxy-phenyl)phosphonotrithioates 6b and 6e. In this reaction the formation of dithioketones 4c and 4d was observed as well and these products were also isolated but due to their instability we were able to record only IR spectra in which the absorption for the original unsaturated 3-oxo group was missing as well as the absorptions for carbonyls at C-17 for 4c and C-20 for 4d (Scheme 2).
With a prolonged time of reaction in CH 2 Cl 2 (reflux 4-8 h, depending on substrate, see Experimental) all unsaturated ketones 1a-e gave as a main product the corresponding 1,2,4-trithiolanes 5a-e (11-79%). Besides, steroids 1b-e gave also the (4-methoxyphenyl)phosphonotrithioates 6b-e (8-36%) as a result of further reaction of firstly formed thioketones with LR. In some cases (1a, 1c and 1d) the thioketones, 3a, 3c and 3d were still present in the reaction mixture and isolated in very poor yield (5-12%) (Scheme 2). Support for the structures 5a-e was found from the 13 C-and 1 H-NMR spectral data as well as from the elemental analysis which showed that they contain three additional sulfur atoms (at two steroid molecules). NMR spectral data showed the absence of α,β-unsaturated 3-oxo group. In the 1 H-NMR spectra signal for H-4 at δ 5.45, 5.44, 5.60, 5.47 and 5.46 ppm for 5a-e, respectively, was situated upfield comparing with that in the starting compounds 1a-e (at δ 5.72, 5.74, 5.84, 5.80¸ 5.70 ppm, respectively), demonstrating the effect of an absence of the deshielding influence of 3-oxo group. In the 13 C-NMR spectra the singlet for C-5 at δ 152.5, 149.8 147.0, 151.9 and 151.8 ppm for 5a-e, respectively, was also situated upfield when compared to the corresponding resonance in 1a-e, indicated the absence of α,β-unsaturated carbonyl group. In addition, the singlet for C-3 at about 199 ppm for starting compounds was missing and instead the new singlet at δ 81.3, 80.2, 80.9, 81.1 and 81.1 ppm, respectively for 5a-e, appeared indicating the formation of 1,2,4-trithiolane ring.
In the IR spectra of phosphonotrithioates 6b-e the original C(3)=O absorption band disappeared, while in the 13 C-NMR spectra the singlet for C-3 at about δ 199 ppm was also absent. The 1 H-NMR spectra showed the new olefinic H-6 proton signal, a broad triplet at δ 5.52, 5.61, 5.49 and 5.47 ppm for 6b-e, respectively. On the other hand, the resonance for the olefinic H-4 proton appeared like two sets of signals, two doublets at δ 6.30 and 6.34 ppm, 6.38 and 6.40 ppm, 6.28 and 6.32 ppm, 6.28 and 6.31 ppm for compounds 6b-e, respectively, with J PH about 4 Hz indicated two different protons, both with the long range coupling with phosphorus. The signal for 19-Me group also appeared in pairs, two singlets at δ 0.92 and 0.96 ppm for 6b, 0.89 and 0.92 ppm for 6d and 0.89 and 0.94 ppm for 6e. Besides, the new singlet at δ 3.87, 3.86, 3.86 and 3.87 ppm (OCH 3 ) appeared as well as two doublets of doublets at δ 6.96 and 8.02, 7.00 and 8.01, 6.96 and 8.02 and 6.95 and 8.01 ppm for aromatic protons for 6b-e, respectively, and these signals had corresponding J HH and J PH coupling constants. On basis of the peak areas ratio of the olefinic, methoxy and aromatic protons (H-4/H-6/OCH 3 /ArH(3,5)/ArH(2,6)=2:2:3:2:2) it was deduced that two steroid molecules are attached with one monomeric species of LR to gave (4-methoxyphenyl)phosphonotrithioates 6b-e. The difference between H-4 protons and 19-methyl groups is attributed to the molecular asymmetry. This was confirmed by the 13 C-NMR spectra in which the C-3 carbon atoms appeared like two doublets at δ 124.5 and 124.8 ppm, 124.7 and 125.2 ppm, 124.4 and 124.5 ppm, 122.4 and 124.7 ppm for 6b-e, respectively, with 2 J PC about 10 Hz. Furthermore, the signals for C-1, C-2, C-4, C-5, C-6, C-10 and 19-Me appeared like two sets of signals confirming proposed structures. Also, there were the signals for aromatic carbon atoms with corresponding 1-4 J PC coupling constants at δ 162.9, 133.7, 129.6 and 113.9 ppm for compound 6b, at δ 162.9, 133.4, 129.6, 113.6 ppm for 6c, at δ 162.6, 133.4, 128.1 and 113.6 ppm for 6d, and at δ 162.6, 133.4, 127.7 and 113.6 ppm for 6e.

General
Removal of solvents was carried out under reduced pressure. Melting points were determined on an electrothermal capillary melting point apparatus and are uncorrected. IR spectra were recorded on a Perkin-Elmer spectrophotometer FT-IR 1725X: ν in cm -1 . 1 H-and 13 C-NMR spectra were recorded on a Varian Gemini-200 spectrometer (at 200 and 50 MHz, respectively) and/or a Bruker Avance 500 MHz spectrometer ( 1 H at 500 MHz; 13 C at 125 MHz) in CDCl 3 and/or C 6 D 6 at room temperature using TMS as internal standard. Chemical shifts are expressed in ppm (δ) values and coupling constants (J) in Hz. Mass spectra were taken on Finnigan-MAT 8230. Thin-layer chromatography was performed on precoated Merck silica gel 60 F 254 plates in toluene/EtOAc (9:1, 8:2) and in nhexane/EtOAc (8:2, 6:4), detection with 50% aq. H 2 SO 4 and/or with CAM solution. Flash column chromatography (FCC) was performed on silica gel Merck 0.040-0.063 mm under the Ar atmosphere. Elemental analyses were determined on Vario EL III. All starting steroid compounds were commercially available products.

General procedure for thionation with LR
LR (1 mmol) was added to a solution of steroidal ketone (1 mmol) in dichloromethane or toluene (15 mL). The reaction mixture was refluxed (duration mentioned in each experiment) with stirring and monitored by TLC. After the completion of reaction the rest of reagent was removed by filtration and evaporated. The residue was chromatographed by FCC using toluene/EtOAc or n-hexane/EtOAc (ratio mentioned in each experiment) as eluent.

General procedure for thionation with combination of P 4 S 10 /HMDO under the microwave irradiation
All microwave assisted reactions were carried out using a modified Panasonic NN-S255W domestic microwave oven (2450 MHz, 800 W) which was adapted for laboratory applications with an external reflux condenser. The oven was perforated at the top for a condenser tube and external steel tube of the same diameter (~30 mm) was welded in order to eliminate possible microwave leakage. P 4 S 10 (0.5 mmol) was added to a solution of steroidal ketone (2 mmol) in ODCB (5 mL) placed in a 20 mL quartz vessel. The reaction mixture was stirred under the argon (Ar) for 10 minutes at room temperature and then HMDO (3.5 mmol) was added. The content was irradiated at 600 W for 90 seconds under Ar atmosphere with a pause of 2 min after every 20 seconds, monitored by TLC. After the completion of reaction the mixture was chromatographed by FCC using toluene/EtOAc or n-hexane/EtOAc (ratio mentioned in each experiment) as eluent.

Conclusions
In this work we showed that Lawesson's reagent can be suitable for preparation of α,β-unsaturated steroidal thioketones as well as different, depending on the solvent, sulfur steroid derivatives. α,β-Unsaturated ketones 1a-e in boiling toluene gave, besides dimer-sulfides 2a-e, the thioketones 3a-e in 11-27% yields. Much higher yields of thioketones were obtained when thionation was carried out in CH 2 Cl 2 (28-70%). In this reaction the dithioketones 4c and 4d were isolated as well. With prolonged reaction times in both solvents and due to the enethiones instability and formation of by-products neither thioketone could be isolated. In toluene, the initially formed unstable enethiones dimerize to give only the corresponding dimer-sulfides 2a-e (30-51%). In CH 2 Cl 2 all unsaturated ketones 1a-e gave as a main product the corresponding dimers with 1,2,4-trithiolane ring 5a-e in 11-79% yields, and (4-methoxyphenyl)phosphonotrithioates 6b-e (8-36%) as a result of further reaction of the initially formed thioketones with LR.
The combination of P 4 S 10 /HMDO under the microwave irradiation can also be applied for thionation of α,β-unsaturated steroids. This reaction gave two main products, 3-thioketones 3a-e and dimer-sulfides 2a-e. However, the yields of synthesized thioketones (11-26%) were lower than in the reaction with LR Although all obtained products were separated using flash column chromatography partial decomposition to the more stable starting α,β-unsaturated ketones took place in all cases. The synthetic results of this work could be useful for chemists in general, not only those working in the field of steroid chemistry. It could be used for preparation of α,β-unsaturated thioketones as well as new sulfur and/or phosphorus containing compounds.