Microwave-Assisted Stereoselective Heterocyclization to Novel Ring d-fused Arylpyrazolines in the Estrone Series

Microwave-assisted syntheses of novel ring d-condensed 2-pyrazolines in the estrone series were efficiently carried out from steroidal α,β-enones and hydrazine derivatives. The ring-closure reaction of 16-benzylidene estrone 3-methyl ether with hydrazine in acetic acid resulted in a 2:1 diastereomeric mixture of two 16,17-cis fused pyrazolines, which is contrary to the former literature data for both stereoselectivity and product structure. However, the cyclization reactions of a mestranol-derived unsaturated ketone with different arylhydrazines in acidic ethanol furnished the heterocyclic products in good to excellent yields independently of the substituents present on the aromatic ring of the reagents applied. The MW conditions also permitted the ring-closure reaction with p-nitrophenylhydrazine which is unfavorable under conventional heating. Moreover, the transformations led to the heterocyclic compounds stereoselectively with a 16α,17α-cis ring junction without being susceptible to spontaneous and promoted oxidation to pyrazoles.


Introduction
Pyrazolines represent an attractive group of five-membered heterocyclic compounds as a consequence of their widespread natural occurrence and diverse pharmacological activities [1,2]. They can be found as structural units in a number of natural products, such as in vitamins, alkaloids and pigments [3]. Moreover, particular attention has been devoted to synthetic derivatives in view of their considerable biological effects and relative simplicity of preparation. Several compounds containing this moiety have been reported to display antifungal [4,5], antibacterial [6,7], antidepressant [8,9], anti-inflammatory [10,11], anticancer [12,13] and anticonvulsant [14,15] properties. They also serve as useful synthons in organic chemistry [16] and precursors for the synthesis of heteroaromatic pyrazoles [17].
In recent years growing interest has been focused on steroidal 2-pyrazolines and pyrazoles due to their antiandrogenic [18] or direct antiproliferative effect [19][20][21] or their ability to inhibit one of the key regulatory enzymes of steroid biosynthesis [22][23][24][25], which makes them potential candidates for the chemoprevention or treatment of cancerous diseases. Some further derivatives are also known to possess neuroprotective [26], antimicrobial [27] or insecticidal activity [28] (Figure 1).
Although the incorporation of a ring-condensed pyrazoline moiety-most frequently by the reactions of steroidal enones with hydrazine derivatives-is relatively common in the androstane [18][19][20]26,29] and cholestane series [28,30], surprisingly, only a few examples are to be found in the literature for the construction of such heterocycles on estrone-based molecules [21], which may also possess valuable bioactivities. Since the use of MW irradiation can advantageously affect the outcome of various organic syntheses leading to different heterocycles both in product yield and chemoselectivity [31], this technique is often applied for the efficient access to both steroidal and non-steroidal pyrazolines [29,[32][33][34]. Considering the high optical purity of chiral steroids, heterocyclization reactions often proceed in a stereoselective manner to afford one of the possible isomers mainly or exclusively, making these reactions more attractive both from a chemical and pharmacological points of view. Steric effect caused by the angular methyl group on C-13 can also exert stereocontrol during the preparation of different ring D-fused analogs [35].
In view of the aforementioned reasons and as we are interested in developing stereoselective synthetic routes on steroid models, we now report a facile access to novel ring D-annelated 2-pyrazolines in the estrone series from a mestranol-derived α,β-enone with various aromatic hydrazines through the application of microwave (MW) irradiation. Our further goal was to investigate the stereoselectivity of the cyclization under acidic conditions, the electronic effect of the substituents of the arylhydrazines on the reaction rate, and the yields of the desired products. of various organic syntheses leading to different heterocycles both in product yield and chemoselectivity [31], this technique is often applied for the efficient access to both steroidal and nonsteroidal pyrazolines [29,[32][33][34]. Considering the high optical purity of chiral steroids, heterocyclization reactions often proceed in a stereoselective manner to afford one of the possible isomers mainly or exclusively, making these reactions more attractive both from a chemical and pharmacological points of view. Steric effect caused by the angular methyl group on C-13 can also exert stereocontrol during the preparation of different ring D-fused analogs [35].
In view of the aforementioned reasons and as we are interested in developing stereoselective synthetic routes on steroid models, we now report a facile access to novel ring D-annelated 2pyrazolines in the estrone series from a mestranol-derived ,-enone with various aromatic hydrazines through the application of microwave (MW) irradiation. Our further goal was to investigate the stereoselectivity of the cyclization under acidic conditions, the electronic effect of the substituents of the arylhydrazines on the reaction rate, and the yields of the desired products.

Results and Discussion
During preliminary experiments to synthesize novel ring D-fused pyrazolines in the estrone series, 16-benzylidene-estrone 3-methyl ether (2) [36], obtained from its precursor (1) by MW-assisted Claisen-Schmidt condensation, was reacted with hydrazine hydrate in acetic acid (Scheme 1). On the basis of relevant references about similar reactions in the androstane series [18,37], a highly diastereoselective ring-closure was expected to occur in this case, leading to a single isomer of 5 despite the formation of two new chiral centers on C-16 and C-5 , with simultaneous acetylation of the heteroring-N(1 ) by the solvent. Contrarily, the 1 H-NMR spectrum of the crude product indicated that a mixture of two acetylated pyrazoline diastereomers was obtained in a ratio of 2:1 under both MW-irradiation at 120 • C for 20 min and conventional heating for 4 h. These findings are in direct contradiction to those by Amr et al. [18] and Romero-López et al. [37], who independently stated that only one of the four possible pyrazoline isomers of 5 was formed in a ca. 70% yield. It is important to note, however, that the results of the abovementioned authors are also in conflict with each other. Although they carried out the cyclization of steroidal 16-benzylidene-17-ones in the androstane series with hydrazine under almost identical conditions, the formation of a 16α-H, 5 β-H isomer of 5 was reported in [18], while that of a 16β-H, 5 β-H was established by the authors of [33]. This inconsistency may arise from the fact that the exact structure and stereo-orientation of the heteroring were elucidated only via determination of the vicinal coupling constant of protons at the newly-formed chirality centers and by the 1 H-NMR chemical shift of 18-CH 3 of the isolated compounds. According to our previous results, the cyclization of steroidal α,β-enones with hydrazine hydrate in AcOH proceeds through a hydrazone intermediate, which undergoes intramolecular cyclization before being acetylated by the solvent. This is most likely to occur during the ring-closure of compound 2 (Scheme 1). We also demonstrated earlier that merely the chemical shift of 18-CH 3 is not indicative of the orientation of a ring D-fused pyrazoline heteroring [29]. Therefore, the mixture of the diastereomers was subjected to column chromatography and both compounds were obtained in almost pure form, sufficient for 1D and 2D NMR studies. Although the formation of two of the four possible stereoisomers of compound 5 could not be excluded on the basis of 1D NMR measurements, 2D NMR spectra clearly demonstrated that instead of 5, the isomers of pyrazoline 6 were obtained during the cyclization (Supplementary Material). Thus, the major compound 6a proved to be the 16α-H, 17α-H compound while 6b was the 16β-H,17β-H diastereomer (Scheme 1). This can be explained by the tendency of 1-unsubstituted ∆ 2 -pyrazolines, such as 4, to tautomerize under heating or in the presence of an acid [38], and the driving force of the tautomeric equilibrium to be shifted toward 4-T 3 is the extended conjugation, especially in product 6 after acetylation. A significant difference (∆δ = 0.37) between the chemical shift values of the equivalent protons of 18-CH 3 was actually observed by comparing the 1 H-NMR spectra of 6a and 6b. The shielding effect of the pyrazoline ring, which bends toward the angular Me group in 6a, results in an upfield shift of 18-CH 3 (δ = 0.63 ppm) compared with that of 6b (δ = 1.01 ppm). However, the J(16-H,17-H) coupling constants, determined from the doublet of pyrazole 17-H for both compounds, did not differ substantially; 11,4 Hz for 6a and 9,2 Hz for 6b, but are in good agreement with the theoretical coupling values calculated according to the Karplus equation for the small (6-7 • ) dihedral angles Θ(H17,C17,C16,H16). The 16,17-trans connection of the heteroring is precluded because of ring strain.
Due to the low stereoselectivity of the above reaction and the difficult separability of the two resulting isomers, a more selective transformation had to be developed for the synthesis of ring D-fused pyrazolines in the estrone series. For this purpose, mestranol (7), a clinically applied prodrug for oral contraception and as a component of menopausal hormone therapy, was used as starting material. In principle, compounds containing an α-alkynylcarbinol moiety such as mestranol (7), can undergo Rupe rearrangement [39] to provide α,β-unsaturated methyl ketones (13) in a single step under acidic conditions (Scheme 2). This reaction involves the protonation of a tertiary alcohol and E1-type dehydration to form a tertiary carbocation (8) that expels the α-proton to give an enyne intermediate (12). Hydration of the acetylene moiety yields an unsaturated enol, which immediately tautomerizes to the more stable α,β-unsaturated ketone (13) [40]. However, in case of Brønsted acid-induced transformation of mestranol (7), the Rupe rearrangement is usually accompanied or often replaced by other competitive reactions, such as ring expansion/aromatization reaction leading to a D-homoaromatic compound (10) via a Wagner-Meerwein product (9) or Meyer-Schuster rearrangement resulting in an α,β-enal (11) [41,42]. All of these side-reactions are attributable to the common propargylic cation intermediate (8), which can be stabilized in several directions, also taking into account the nearby location of the angular methyl group on C-13. In order to avoid the formation of by-products in similar or even larger quantities than the desired compound 13, a two-step protocol had to be applied. Thus, mestranol (7) was reacted with phosphorus oxychloride (POCl3) in pyridine, acting as both base and solvent, under mild conditions for 24 h to afford enyne 12 via E2 process in good yield [43], together with a small amount of a 21chloroallenic derivative 14. The formation of this latter compound can be explained by a OH → Cl interchange at C-17 upon reaction of 7 with POCl3, followed by an allylic rearrangement. Similar polarities of 12 and 14 did not allow their complete separation by column chromatography, but the minor impurity did not interfere in the subsequent hydration reaction of 12, which was carried out In order to avoid the formation of by-products in similar or even larger quantities than the desired compound 13, a two-step protocol had to be applied. Thus, mestranol (7) was reacted with phosphorus oxychloride (POCl 3 ) in pyridine, acting as both base and solvent, under mild conditions for 24 h to afford enyne 12 via E2 process in good yield [43], together with a small amount of a 21-chloroallenic derivative 14. The formation of this latter compound can be explained by a OH → Cl interchange at C-17 upon reaction of 7 with POCl 3 , followed by an allylic rearrangement. Similar polarities of 12 and 14 did not allow their complete separation by column chromatography, but the minor impurity did not interfere in the subsequent hydration reaction of 12, which was carried out in the presence of formic acid (98%) under MW heating at 100 • C for 2 min. The desired α,β-enone 13 was obtained in good yield (75%) referring to 7 in this two-step procedure.
As a continuation, heterocyclization reaction of 13 with phenylhydrazine hydrochloride (15a) was carried out in acidic ethanol ( Table 1, entry 1). The MW-promoted transformation at 100 • C for 20 min led to the stereoselective formation of a single ring D-condensed pyrazoline isomer (17a) in high yield. When the temperature of the MW-induced reaction was elevated to 150 • C, the reaction time was reduced to 5 min to reach complete conversion, but side-reactions became conspicuous in this case. The supposed phenylhydrazone intermediate 16a of the ring-closure reaction could not be detected because of the use of a closed vessel, therefore its independent synthesis was attempted from 13 with 15a in the presence of NaOAc by mildly heating the reactants in i PrOH. Although a less polar intermediate (presumably 16a) was observed by TLC, it could not be isolated due to its spontaneous cyclization to 17a under the given conditions. The ring-closure reaction via 1,4-addition of similar arylhydrazones under conventional heating was earlier observed to be affected significantly by the nature of the substituents: electron-donating groups facilitated, while electron-withdrawing substituents interfered with it by increasing or decreasing the electron density of the intramolecularly attacking internal NH. As a consequence, pnitrophenylhydrazones containing a strong electron-withdrawing NO2 group on their aromatic moiety did not cyclize to the corresponding pyrazolines [44]. Therefore, our next goal was to investigate whether the electronic demand of different substituents on the benzene ring of 15a may have an influence on the yields of the desired products under MW heating. For this reason, cyclization reactions of 13 were also performed with substituted arylhydrazine hydrochlorides 15b−j, and the corresponding ring D-fused pyrazolines 17b−j were obtained in good to excellent yields (Table 1, entries 2−10) even when p-nitrophenylhydrazine (15i) was used (entry 9).
The structures of the synthesized steroidal heterocycles 17a−j were confirmed by NMR and MS measurements. 2D NMR spectra were also recorded for the representative compound 17j. After identification of the related 1 H and 13 C signals with the help of HSQC and HMBC spectra, the 16,17cis orientation of the pyrazoline ring was determined on the basis of through-space (NOESY) The ring-closure reaction via 1,4-addition of similar arylhydrazones under conventional heating was earlier observed to be affected significantly by the nature of the substituents: electron-donating groups facilitated, while electron-withdrawing substituents interfered with it by increasing or decreasing the electron density of the intramolecularly attacking internal NH. As a consequence, p-nitrophenylhydrazones containing a strong electron-withdrawing NO 2 group on their aromatic moiety did not cyclize to the corresponding pyrazolines [44]. Therefore, our next goal was to investigate whether the electronic demand of different substituents on the benzene ring of 15a may have an influence on the yields of the desired products under MW heating. For this reason, cyclization reactions of 13 were also performed with substituted arylhydrazine hydrochlorides 15b-j, and the corresponding ring D-fused pyrazolines 17b-j were obtained in good to excellent yields (Table 1, entries 2-10) even when p-nitrophenylhydrazine (15i) was used (entry 9).
The structures of the synthesized steroidal heterocycles 17a-j were confirmed by NMR and MS measurements. 2D NMR spectra were also recorded for the representative compound 17j. After identification of the related 1 H and 13 C signals with the help of HSQC and HMBC spectra, the 16α,17α-cis orientation of the pyrazoline ring was determined on the basis of through-space (NOESY) correlations. Thus, spatial proximity was evidenced by cross-peaks  (Figure 2a,b). The stereoselective ring-closure of the arylhydrazone intermediates can be attributed to the β orientation of the angular Me group on C-13, which permits the intramolecular attack of the NH nitrogen only from the opposite (α) direction. The synthesized ring D-fused pyrazolines were proved to be resistant against autooxidation to pyrazoles. In addition, dehydrogenation leading to a single heteroaromatic product by oxidizing agents (DDQ, I2, Jones reagent, manganese dioxide or iodobenzene diacetate) also failed. The synthesized ring D-fused pyrazolines were proved to be resistant against autooxidation to pyrazoles. In addition, dehydrogenation leading to a single heteroaromatic product by oxidizing agents (DDQ, I 2 , Jones reagent, manganese dioxide or iodobenzene diacetate) also failed.

Synthetic Procedures
3.2.1. MW-assisted Synthesis of 3-methoxy-16-benzylidene-estra-1,3,5(10)-triene-17-one (2) Estrone 3-methyl ether (427 mg, 1.50 mmol) was dissolved in EtOH (6 mL), then KOH (28 mg, 0.5 mmol) and benzaldehyde (0.19 mL, 1.85 mmol) were added to the solution. The mixture was irradiated in a closed vessel at 100 • C for 20 min. After completion of the reaction, the mixture was poured into water (20 mL), and extracted with CH 2 Cl 2 (2 × 25 mL). The combined organic phases were dried with anhydrous Na 2 SO 4 and concentrated in vacuo. The crude product was purified by column chromatography with hexane/CH 2 Cl 2 = 15:85. To a solution of 2 (343 mg, 0.8 mmol) in acetic acid (5 mL), hydrazine hydrate (0.4 mL, 8.0 mmol) was added and the mixture was irradiated in a closed vessel at 120 • C for 20 min, or stirred at reflux temperature for 4 h. After completion of the reaction, the mixture was poured into water (20 mL), neutralized by the addition of NaHCO 3 and extracted with EtOAc (2 × 15 mL). The combined organic phases were dried with anhydrous Na 2 SO 4 and concentrated in vacuo. The crude product was purified by column chromatography with EtOAc/CH 2 Cl 2 = 2:98, the minor product (6b) was eluated first. The ratio of the two 16,17-cis fused pyrazolines (6a and 6b) was determined by 1 H-NMR measurement of the crude product. Overall yield of 6a and 6b: 261 mg (white solid). Mestranol (2.48 g, 8.0 mmol) was dissolved in pyridine (30 mL), and POCl 3 (7 mL) was added dropwise at 0 • C under vigorous stirring. After addition of POCl 3 (approx. 10 min), the reaction mixture was allowed to warm to room temperature and after 24 h it was poured into a mixture of ice and concentrated H 2 SO 4 (10 mL), and extracted with EtOAc (3 × 20 mL). The combined organic phases were washed with water (30 mL) and saturated NaHCO 3 solution (2 × 30 mL), then dried with anhydrous Na 2 SO 4 and evaporated in vacuo. The resulting crude product was purified by flash chromatography with CH 2 Cl 2 /hexane = 20:80 to give enyne 12 [43] with compound 14 as minor impurity.