Recent syntheses of steroid derivatives using the CuAAC “click” reaction

The introduction of heterocycles into steroids is often at the origin of a modification of their physiological activity and thus allows the formation of new biologically interesting molecules. Recent advances in the field of steroid synthesis using the copper-catalyzed azide-alkyne cycloaddition (CuAAC) are presented here. Compounds exhibiting known biological activities are also reported in this review. This approach holds great promise and allows us to consider preparing new, highly functionalized complex molecules using the CuAAC “click” reaction.


Introduction
2][3] Even today, the synthesis of the steroid skeleton by increasingly sophisticated strategies continues to hold the attention of chemists.The steroids present in nature and known for their biological importance have been the subject of numerous syntheses and research on these molecules has not ceased to this day. 4,5he [1,2,3]-triazoles constitute an important family of nitrogenous five-membered heterocycles.They exhibit considerable biological activities, such as antitumor, 6 anti-HIV, 7 anti-tuberculosis, 8 and antibacterial 9 and also exhibit inhibitory activities of serine hydrolase, 10 tyronase, 11 and glycosidase 12 .This azaheterocycle is an interesting connection motif because it is stable to metabolic degradation but above all, it can form hydrogen bonds.This consideration makes it a prime motif for improving the solubility and binding of biomolecular targets. 13,14The 1,2,3-triazole moiety cannot be found in nature, yet molecules with 1,2,3triazole units possess various biological activities.The role of these compounds in medicinal chemistry is considerable.In fact, the low basicity of this azaheterocycle makes it non-protonatable in a physiological medium, unlike other azaheterocycles.
2][23][24][25] The reviews about this type of steroid are numerous. 26][29][30] This review presents the latest syntheses of steroid derivatives using the copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC) and possibly the biological activities, from the years 2016-2021.To the best of our knowledge and much to our surprise, there have been no reviews on this subject since 2016. 31
They chose the 5'-position of the nucleosides to introduce the azido group and a propargyl ether functional group on the 3-hydroxy group of 13β-estrone.They obtained the best yields by protecting with acetyl groups the 3'-hydroxy groups of the nucleosides and by changing the 5'-hydroxy groups using the tosyl-azide exchange method.The key step of click reaction between the protected-5'-azidonucleosides and the steroid alkyne was done using 1.5 equivalent of Cu(I) catalyst (Scheme 1).
Van Liera et al. 33 described the synthesis of estrogen, testosterone-and 19-nortestosterone conjugates linked to BODIPY (4,4-difluoro-4-bora-3α,4α-diaza-s-indacene) or aza-BODIPY to obtain receptor-based fluorescence ligands for imaging breast and prostate cancer.Their synthesis is based on coupling of iodo derivatives of differently substituted BODIPY and aza-BODIPY analogs and steroid alkynes using the click reaction conditions.Their observations in living cells and tissues is possible since their UV-Vis absorption spectral range from 500 to 710 nm with fluorescence emission properties ranging from 520 to 700 nm.
This method allows to obtain libraries of new products with potential diversified bioactivity by changing functional groups, linker chain length and type, and sequence orientation of a subunit within an E2-BODIPY conjugate.Thus, two covalently coupled conjugates 7 and 9 were synthesized in good yields from azido-BODIPY 6 and steroid alkynes 5 and 8 (Scheme 2).
The reaction conditions are mild: H2O is used as a green solvent and copper as a catalyst.αsitosterol-3-yl methanesulfonate 11 was prepared in good yield by reaction of β-sitosterol 10 with methanesulfonyl chloride in CH2Cl2 using triethylamine as a base, and dimethylaminopyridine (DMAP) as a catalyst.The reaction of the mesylate derivative 11 with sodium azide in dimethylformamide (DMF) led to β-sitosterol-3-yl azide 12 in 83% yield (Scheme 3).Scheme 3. Synthesis of β-sitosterol-3-yl azide 12.
The use of 10 mol% of CuSO4•5H2O and 20 mol% of sodium ascorbate as the catalyst, H2O as a green solvent at 40 °C for 30 min are the best reaction conditions for the copper-catalyzed azide-alkyne cycloaddition (CuAAC).
It is worth pointing out that short reaction times (0.5 h) and excellent yields (85-95%) were obtained for the expected β-sitosterol derivatives 14 when aromatic alkynes with functional groups at different positions of the aromatic ring (alkyl, F, Br, and OCH3) were used.The same result was observed with heterocyclic or aliphatic alkynes.Furthermore, yields of the products were very good with alkyne possessing a strong electron-withdrawing group.In all cases, three isomers of the desired product were isolated and could be separated by column chromatography.However, in some cases the mixture of isomers was inseparable or when the yield of the corresponding isomer 14" was low, only two isomers or only one were isolated.In general, β-sitosterol derivatives 14 were prepared in good yield showing the broad scope of this methodology.
In the same year, Zhang et al. 35 described the preparation of a novel and simple deoxycholic acid-based fluorescent probe for solvent-dependent multi detection of Cu 2+ , C2O4 2-and P2O7 4-constructed by click chemistry.
This new compound 17 is a tweezer-type molecule with deoxycholic acid, 8-aminoquinoline, and 1,2,3triazole moieties.The 1,2,3-triazole moiety obtained via click chemistry allows the introduction of various functionalities and is a potential binding site for both metal ions and anions, 36,37 which is very useful for multitarget analysis.The click reaction between steroidal diazides 15 38 and quinoline terminal alkyne 16 39 led to the fluorescent probe 17 (Scheme 5).This latter easily prepared probe could offer a simple and sensitive solventdependent assay method with low detection limits and fast response time for multi detection.Moreover, this probe showed strong fluorescence quenching upon binding to Cu 2+ in acetonitrile (CH3CN) and water and gave also enhanced fluorescence response toward C2O4 2-and P2O7 4-in aqueous dimethyl sulfoxide (DMSO) solution.The results of tests to determine Cu 2+ , C2O4 2-and P2O7 4-on paper strips and in water samples proved the practical analytical utility of this probe.
In 2018, Negron-Silva et al. 40 reported an efficient synthesis of a novel carbohydrate-lithocholic acid conjugate linked through 1,2,3-triazole rings and their derivatives.
The different steps of the synthesis are depicted in Scheme 6.The readily available methyl α-Dglucopyranoside and the lithocholic acid were used as starting material to afford the methyl 4,6-Obenzylidene-2,3-di-O-propargyl α-D-glucopyranoside (18) 41 and the methyl 3-azidolithocholate 19 42 respectively.The click reaction between dipropargyl ether 18 and azide 19 under microwave irradiation at 100 °C with a Cu-Al mixed oxide heterogeneous catalyst and sodium ascorbate led to the desired triazole-linked carbohydrate-bile acid conjugate 20 in a very short time (5 min) and a very good yield of 93% (Scheme 6).To synthesize novel derivatives of 20, they decided to prepare targeted the propargyl ester 22a and propargylamide 22b derivatives.The acid derivative 21 was synthesized from compound 20 by hydrolysis with NaOH/H2O/MeOH followed by acidification with HCl/H2O in a 98% yield.The reaction of 21 with 4-DMAP, EDC-HCl, and HOBt, or DIPEA and ClCO2Et, followed by reaction with propargyl alcohol or propargylamine led to the corresponding ester 22a in 80% yield and amide 22b in 78% yield, respectively (Scheme 7).
They decided to synthesize the compounds 24a and 24b by means of a second CuAAC (Scheme 8).According to the literature, 43 they firstly prepared the 1-azido-1-deoxy-2,3,4,6-tetra-O-acetyl-β-Dglucopyranoside 23.Then, the click reaction between propargyl derivative 22a or 22b and azide 23 under microwave irradiation at 80 °C with a Cu-Al mixed oxide heterogeneous catalyst and sodium ascorbate provided the corresponding compounds 24a and 24b in a very short time (5 min) and very good yields of 78% and 72%, respectively.Scheme 8. Synthesis of derivatives 24a and 24b.
Finally, the authors have performed the deprotection of the carbohydrate moiety of the compounds 24 in the presence of hydrochloric acid and using methanol as a solvent to afford the corresponding products 25 (Scheme 9).
In 2019, Huang et al. 44 reported the synthesis of a series of novel steroidal β-triazolyl enones using the CuAAC click reaction and Claisen-Schmidt condensation reaction as the key steps.These new derivatives were further evaluated for their antiproliferative activity.

Scheme 9. Synthesis of derivatives 25a and 25b.
The synthetic approach began with the preparation of compound 27 from aromatic amine 26 treated with NaN3 in the presence of HCl and NaNO2.The click reaction between azide 27 and 3,3-diethoxyprop-1-yne led to the corresponding triazole 28.Finally, using KF/ Al2O3 as the catalyst, the Claisen-Schmidt condensation between the aldehyde triazole 28 and androstanes provided the corresponding steroidal derivatives 29a-e and 30a-e in good yield (Scheme 10).
Most of these compounds showed good activity against a panel of cancer cells.In the case of compound 30a, the IC50 values of 1.61 and 1.16 μM against PC-3 and MGC-803 cells revealed a good antiproliferative activity.Moreover, this latter inhibited migration and invasion of gastric cancer cell line MGC-803 and prostate cancer cell line PC-3 in a dose-dependent manner.Notably, modifications of mRNA levels and protein expression of EMT markers were also observed.Consequently, this compound could be efficiently used to prepare new anticancer molecules to inhibit cell invasion.

Scheme 10. Synthesis of compounds 29a-e and 30a-e.
The same year, Griffith et al. 45  The dihydrochloride 32 was then treated with two equivalents of base (1,8-diazabiciclo[5.4.0]undec-7-ene or NaOH) followed by Pt(II) dimethylsulfoxido precursor complexes 46 to afford novel Pt(II) estradiol complexes (Scheme 12).The dichlorido-Pt(II) estradiol complex, [PtCl2(EDiolDap)] 35 was isolated in 58% yield and the 1,1-cyclobutanedicarboxylato-Pt(II) estradiol complex, [Pt(CBDCA)(EDiolDap)] 36 in 74 yield.An estrogen receptor-negative ER− colon cancer cell line and a panel of estrogen receptor-positive ER+ cancer cell lines were used to find in vitro cytotoxicity of both estrogen-linked Pt(II) complexes.It is worth pointing out that their in vitro cytotoxic effect is 30 times greater against ER+ cancer cell lines than against the ER− colon cancer line.The authors observed that these two complexes presented superior in vitro cytotoxicity than cisplatin.Moreover, it is interesting to note that the test results in vivo are better than those obtained with the clinically used cisplatin.These novel estrogen-linked Pt(II) complexes exhibit in vitro cytotoxic results which are very promising to develop such complexes.In 2019, Pérez-Campos et al. 47 reported the activity of first and second-generation dendrimers over T. cruzi in the epimastigote stage.α-Ethynylestradiol (EE) modified with PAMAM-type dendrons was used to prepare dendrimers.The key reaction of this synthesis is a click reaction.As depicted in Scheme 13, the copper-catalyzed azide-alkyne cycloaddition (CuAAC) of 17α-ethynylestradiol and the azide derivative 38 afforded the corresponding triazole 39 in good yield.In a second step, this latter was deprotected with TFA leading to the desired steroidal derivative 40.Scheme 13.Route of synthesis of dendron 40.
Dendrimers 41 and 42, the structures of which are shown in Scheme 14, were synthesized by a previously described procedure 48 from dendron 40.Flow cytometry was used to determine the activity of each compound, including benznidazole (Bz) as positive control.The authors observed that a concentration of 14.8 μmol/mL of the second-generation dendrimer is more efficient than Bz in the time with an IC50=1.25 ± 0.19 μmol/mL.Moreover, using dendrimers, cell death in T. cruzi epimastigotes was mostly done via apoptosis and not via necrosis as observed with Bz in more than half of cells.
In 2020, Wimmer et al. 49 reported the synthesis and pharmacological effects of diosgenin−betulinic acid conjugates.They are shown to enhance the pharmacological effects of their components.
The synthetic approach is depicted in Scheme 15.Firstly, compound 43 was converted into its benzyl ester 44 by treatment with benzyl bromide in DMF in good yield.In a second step, the derivative 44 possessing a hydroxyl group at C-3 was treated with propargyl bromide in THF, using sodium hydride as a base, to provide the corresponding propargyl ether 45.In the newt step, the 1,2,3-triazole derivative 46 was synthesized via copper-catalyzed azide-alkyne cycloaddition (CuAAC) from alkyne 45 and azidovaleric acid using copper(II) sulfate as a catalyst.Deprotection of the benzyl group of 46 by high-pressure hydrogenation using Pd/C (10%) led to the corresponding steroidal derivative 47 in a good yield.
The steroidal derivative 46 was also used to prepare the diosgenin conjugate 48 using 4-(dimethylamino)pyridine (DMAP) to enhance the reaction and N,N'-dicyclohexylcarbodiimide (DCC) as coupling agent (Scheme 15).In a final step, this latter was deprotected by hydrogenation (10% Pd/C) leading to the target diosgenin−betulinic acid conjugate 49 in an excellent yield.
No cytotoxicity in the tested cancer cell lines was observed for the betulinic acid 43, the intermediates 44 and 45, and the diosgenin conjugate 49.On the other hand, the diosgenin conjugate 48 exhibited selective cytotoxicity in human T-lymphoblastic leukemia (CEM) cancer cells with an IC50 = 6.5 ± 1.1 M, and compound 46 exhibited medium multifarious cytotoxicity in tested human cell lines.The same year, Acik et al. 50described a method to deposit most common bile acids such as lithocholic acid (LCA) and chenodeoxycholic acid (CDCA) bearing poly(vinyl chloride) nanofiber (PVC-LCA and PVC-CDCA) coatings on glass slides surface using a combination method by employing copper(I)-catalyzed azide-alkyne cycloaddition 'click' reaction (CuAAC), followed by electrospinning process.
The synthetic route to prepare PVC-LCA or PVC-CDCA was depicted in Scheme 16.Treatment of PVC with sodium azide provided the corresponding azide PVC-N3 in good yield.To limit side reactions, LCA and CDCA were firstly converted into their corresponding ester 50 and 51 using acetyl chloride, followed by a reaction with 4-pentyonic acid to give the clickable LCA-alkyne 52 and CDCA-alkyne 53, respectively.In a final step, the click reaction between these latter and PVC-N3 led to the corresponding PVC-LCA and PVC-CDCA.After these syntheses, electrospun nanofibers were obtained from their solutions by using a simple electrospinning technique at room temperature.
Electrospinnability, thermal properties, and the hydrophilicity were enhanced by introducing LCA and CDCA moieties into PVC.These novel steroidal PVC nanofibers could be interesting to produce PVC materials useful in bio-applications.Scheme 16.The synthetic route for preparing PVC-LCA or PVC-CDCA and their precursors.

Conclusions
The syntheses of steroids reported in the literature from 2016 to mid-2021, using the CuAAC "click" reaction, have been reviewed.The growing use of the copper-catalyzed azide-alkyne cycloaddition (CuAAC) has enabled chemists to make simple syntheses of increasingly complex and elaborate steroid molecules.Some of these syntheses deserve to be studied further.This route is particularly advantageous for the preparation of steroids with potential biological activity.Overall, the very great structural diversity and the biological capacities of these molecules encourage researchers to go even further in this field.