A-ring functionalization of cholestane with highly substituted pyrans and 2-aminoisophthalonitriles

Following our ongoing interest on modified steroidal compounds for biological purposes, herein we endeavor the two-step regioselective synthesis of three-substituted pyran-cholestane fused compounds (6 examples, yields up to 83%) at C-2:C-3 A-ring side of the steroid scaffold. In the presence of higher amounts of malononitrile, the intermediate α,β -unsaturated carbonyl substrates give rise to competing reactions leading to the formation of the corresponding 2-aminoisophthalonitrile fused compounds (2 examples, up to 37% yield)


Introduction
Steroid compounds are widely present in living organisms playing important roles in their vital activities.The most recognizable steroid is cholesterol (cholest-5-en-3β-ol), which is considered a lipid-type molecule, being one of the most important structural components of cell membranes.Cholesterol is a rigid and almost planar molecule with a steroidal skeleton of four fused rings, three six-membered and one five-membered, conventionally lettered from A to D (1,2-cyclopentanoperhydrophenanthrene ring system) (Figure 1).The cholesterol molecule contains four essential domains (Figure 1B).In domain I, the polarity of the 3hydroxy group constitutes an active site for hydrogen bond interactions with a variety of biological molecules. 1n domain II, the absence of methyl groups at C-4 and C-14 influences directly the planarity of the molecule, while in domain III, the natural (R)-configuration at C-20 determines the "right-handed" conformation of the side chain.Finally, in domain IV, the conformation and length of the side chain is of prime relevance to intermolecular contacts. 2The unique properties of cholesterol make it a very attractive molecule for synthetic organic studies, as well as to other interesting applications such as drug delivery and bioimaging applications, and even liquid crystals and gelators. 3A key structural feature is the presence of the hydrophilic 3-hydroxy headgroup on the A-ring, together with a hydrophobic hydrocarbon body, which offer the molecule an amphiphilic nature, and revealed itself as being an important property in the interaction with protein aggregates.In fact, lanosterol and 25-hydroxycholesterol (Figure 2) were reported to reverse crystallininduced protein aggregation, by dissolving aggregated proteins from amyloid-like fibrils.Due to their amphiphilic nature, they were able to intercalate and coat hydrophobic core areas of large protein aggregates, allowing these aggregates to become water-soluble again. 4,5The efficacy of lanosterol to reverse multiple types of mutant-crystallin aggregates was further improved trough 2-fluorination and 25-hydroxylation, enhancing the anti-aggregating activity (Figure 2). 6Remarkably, lanosterol is also capable to inhibit the selfassembly of Aβ peptides, entangling with peptides and forming a hydrophobic core with residues Phe-19 and Phe-20.The improvement of biological properties of natural occurring lanosterol by chemical modification is a critical example of how the right chemical modification in a natural scaffold could lead to highly efficient molecules for therapeutic purposes.Over the years, steroid group compounds, particularly cholesterol, have played a significant interest for organic synthesis, due to good availability, low cost and easily derivatization of the functional groups.Many useful chemical and enzymatic reactions are now widely used for multi-step steroid transformations, leading to important products. 3,8The chemical transformations range from simple ones, like manipulations of functional groups, to more complex ones such C-H activation or C-C bonds formation with organometallic reagents. 80][11] Furthermore, it should be highlighted the significance of the 2-amino-3-cyano-4-phenylpyran pharmacophoric fragment (highlighted in red) in AChE inhibitors 12 or even as anticancer 13 and antibacterial 14 molecules (Figure 3).Following our interest in modified steroidal compounds with biological applications, we have looked at the chemical modification of cholestane A-ring, by following a two-step synthetic procedure in order to reach a three-substituted fused pyran heterocyclic ring as depicted in Figure 3. From the retrosynthetic analysis, one can expect that the initial aldol reaction can occur at C-2 or C-4 positions of the steroid scaffold (Figure 4).The selectivity towards one particular position will allow the selective fusion of pyran ring at one specific A-ring side (Figure 4).To the best of our knowledge, herein we present for the first time the A-ring functionalization of cholestane scaffold with highly substituted pyrans tethered with -NH2 and -CN functionalities (Figure 4).

Results and Discussion
The planned synthetic strategy for the preparation of pyran-fused cholesterol derivatives 4a-f encompasses two reaction steps: 1) the aldol reaction of cholestan-3-one 1 and a series of benzaldehydes 2a-f to give the intermediate α,β-unsaturated carbonyl derivatives 3a-f; and 2) the microwave (MW)-assisted cyclization reaction of α,β-unsaturated carbonyl derivatives 3a-f and malononitrile (Scheme 1).The first reaction step afforded the α,β-unsaturated carbonyl 3a-f (arbitrary numbering for NMR analysis, Figure 5) in good yields (70-80%), in the presence of t-BuOK (5 equiv) as base.The reaction was quite selective towards the C-2 position of the steroid scaffold (see 1 H and 13 C NMR spectra in SI) (Scheme 1).As we used an excess of benzaldehydes 2a-f (3 equiv), it was expected to have competition between C-2 and C-4 positions, however, no C-4 reaction products were detected in the reaction media.Possibly this might be due to the more steric hindrance of carbon at position 4 of the steroid scaffold.Scheme 1. Synthetic strategy for the two-step preparation of pyran-fused cholesterol derivatives 4a-f.The second step of the synthetic strategy was not straightforward, and optimization of the reaction conditions was required.To do so, we used the 4-CH3 α,β-unsaturated carbonyl derivative 3b as model substrate and the outcome of the optimization study is summarized in Table 1.In the first attempts, we evaluated the effect of increasing amounts of malononitrile (1 to 5 equiv), ending up with increasing yields (up to 75%) of the desired pyran derivative 4b (mixture of two diastereomers, Figure 5) (Table 1, entries 1-3).When the amount of malononitrile was further increased to 10 and 20 equiv, the formation of 2aminoisophthalonitrile derivative 5b (blue inset) was observed in 30 and 33% yields, respectively, with a consequent decrease in the obtained yield of the respective pyran derivative 4b (Table 1, entries 4 and 5).Changing the substituent from R 1 = CH3 to R 1 = OCH3 no better improvements were observed and the formation of 2-aminoisophthalonitrile 5c took place in 30-37% yields (Table 1, entries 4-7).Then it can be concluded that the formation of 2-aminoisophthalonitriles 5 depends on the amount of malononitrile used in the reaction.The synthesis of 2-aminoisophthalonitriles 5, could be explained on the basis of the following mechanism: i) Michael-addition of malononitrile to the β-position of α,β-unsaturated carbonyl derivative 3, followed by 1,2-addition of a second molecule of malononitrile and dehydration to give intermediate III (Scheme 2); ii) base-catalyzed intramolecular attack to cyanide group to give intermediate IV, followed by a [1,5] proton shift giving V (Scheme 2); iii) HCN elimination ending up with the formation of 2aminoisophthalonitriles 5 (Scheme 2).
With the adequate amount of malononitrile well established in order to drive the reaction towards the desired pyran derivatives 4, we also struggled to further improve the yield of pyran derivative 4b.Increasing amounts of DBU (10 to 30 mol%) were attempted, as well other base-catalysts such as Et3N and K2CO3 (Table 1, entries 8-11).The isolated yield of pyran 4b remained unaffected with the increasing amounts of DBU (Table 1, entries 3, 8 and 9), and the switch from DBU to Et3N and K2CO3, ended up with a slight decrease in the isolated yield of 4b (Table 1, entries 10 and 11).The screening of the reaction conditions indicated that the higher possible yield for derivative 4b (75%) is obtained using 5 equiv of malononitrile, in the presence of 10 mol% of DBU (Table 1, entry 3).Therefore, these reaction conditions were further applied to the remaining α,β-unsaturated carbonyl derivatives 3a-f, giving the corresponding pyran derivatives 4a-f in 65-83% yield (Table 1, entries 3, 12-16).Reaction conditions [a] 4 Yield (%) [b] 5 Yield (%) [

Conclusions
A series of six pyran-fused cholestane derivatives 4a-f was synthesized in good yields, exploring the reaction of α,β-unsaturated carbonyl compounds 3a-f with malononitrile, in the presence of DBU.The initial aldol reaction step of cholestan-3-one with benzaldehydes was regioselective towards C-2 position, which is the less hindered position of the steroid scaffold.The two-step synthetic methodology, rather than the one-pot procedure, takes advantage of the aldol reaction regioselectivity, granting the selective fusion of the threesubstituted pyran ring at the C2-C3 positions of cholestane A-ring.In the presence of a high excess of malononitrile, there is the formation of 2-aminoisophthalonitrile fused to C2-C3 of the cholestane A-ring.This is a competing side reaction when higher amounts of malononitrile are input into the reaction.

Experimental Section
General.All chemicals were commercially available except those having the synthesis described.All reaction mixtures were monitored by thin layer chromatography (TLC) using commercial TLC plates (Merck Kieselgel 60 F254).The plates were observed under UV light at 254 and 365 nm.Melting points were determined using a Reichert Thermovar apparatus fitted with a microscope and are uncorrected.NMR spectra were recorded in Bruker Avance 300 spectrometer (300 for 1 H and 75 MHz for 13 C), in CDCl3 as solvent, if not stated otherwise.Chemical shifts () are reported in ppm and coupling constants (J) in Hz; internal standard was residual peak of the solvent.Unequivocal 13 C assignments were made with the aid of 2D gHSQC and gHMBC (delays for onebond and long-range J C/H couplings were optimised for 145 and 7 Hz, respectively) experiments.Highresolution mass spectra analysis (HRMS-ESI) were performed on a microTOF (focus) mass spectrometer.Ions were generated using an ApolloII (ESI) source.Ionization was achieved by electrospray, using a voltage of 4500 V applied to the needle, and a counter voltage between 100 and 150 V applied to the capillary.
General procedure for the synthesis of α,β-unsaturated carbonyl compounds (3a-f).To a stirring solution of cholestan-3-one 1 (200 mg, 0.5 mmol) and t-BuOK (290 mg, 2.5 mmol) in refluxing EtOH (5 mL), was added the appropriate benzaldehyde 2a-f (1.5 mmol).The resulting reaction mixture was kept under reflux for 2-3 h.After completion of the reaction (TLC) the mixture was poured into cold water, the pH adjusted to 4 with 10% aqueous HCl and the resulting precipitate recovered by filtration.The crude precipitate was dissolved in CH2Cl2 (10 mL), dried over anhydrous Na2SO4 and concentrated under reduced pressure.The crude product was then purified by preparative TLC using hexane:ethyl acetate (10:1) as eluent.

Figure 1 .
Figure 1.(A) Numbering and ring-labeling convention in cholesterol; (B) four domains of cholesterol.

Table 1 .
Reaction conditions screening and reaction scope for the synthesis of derivatives 4a-f