Synthetic methods towards steroid-ferrocene conjugates

Steroid-ferrocene conjugates, in which a steroid skeleton is connected directly or via a linker group to the ferrocene moiety, have a broad scope of possible applications in medicinal or analytical chemistry as well as in materials science. They may integrate or even enhance many of the attractive properties of the components. Reactive functional groups of steroids and the ease in the conversion of ferrocene via aromatic substitution reactions offer a wide range of possibilities to attach the two skeletons to each other. This review summarizes the diverse methodologies used in the synthesis of conjugates


Introduction
Since its discovery, ferrocene (Fc) and its derivatives have been attracting much attention in catalysis, organic synthesis, new materials and supramolecular chemistry. 1 Ferrocene has high thermal stability, is stable in air, and soluble in all common organic solvents. It reacts as a strong electrophile and can easily be functionalized via usual aromatic substitution reactions that offer the possibility of further derivatization. The robustness of ferrocene is the result of its 18-electron structure. The hydrophobic neutral ferrocene form of orange color undergoes a mild and reversible oxidation by using electrochemical methods or common oxidizing agents to obtain the 17-electron hydrophilic cationic ferrocenium with purple color. The sandwich geometry is maintained upon one-electron removal. Due to its aromaticity and redox activity, the ferrocene moiety has drawn attention as a useful functionality in virtually any field of applied science. It has been used as a building block for the design of switchable functional systems 2 , electro-and photo-responsive materials 3 , in supramolecular chemistry 4 and for the construction of sensory devices. 5,6 Beside the properties mentioned above, the lipophilic nature of ferrocene and the stability of the ferrocenyl group in aqueous, aerobic media have made ferrocene suitable for conjugation with pharmacophores. [7][8][9] Although ferrocene itself is nontoxic, the cytotoxicity of ferrocenium derivatives was discovered during the 1980s. 10 The anticancer effects are mainly attributed to the formation of reactive oxygen species (ROS) via the Fenton pathway 11 which induces DNA damage followed by cell death. 12 In the past decades, ferrocene derivatives with a broad range of biological activities, such as antimalarial, antitumor, 13,14 antiparasitic, antibacterial, antifungal, and antiviral 15 effects have been prepared.
In steroid-ferrocene conjugates a steroid skeleton is connected directly or via a linker group to the ferrocene moiety. These hybrid derivatives may incorporate some favorable properties of both components. Due to the hormonal activity of natural and synthetic steroid derivatives, great effort has been directed to explore the biological activity of their ferrocene derivatives, and the results have been summarized in some reviews. 7,14,16 One of them, concentrating especially on the anticancer activity of ferrocene-steroid hybrids, was published quite recently. 17 Beyond potential medicinal applications, the conjugates can also awaken Acylation of estrone with one hydroxyl functionality leads to compound 13 with either of the acylation agents (entries 1, 3), but the reaction of estradiol can result in the formation of mono-or diester derivatives, depending on the reaction conditions (entries 2, 4-7). Using acid chloride 1 in less than equimolar amount, a mixture of estradiol-3-ferrocenecarboxylate (14) estradioI-3,17-bis(ferrocenecarboxylate) (15) and estradiol-17-ferrocenecarboxylate (16) was obtained (entry 5). The application of an excess of the reagent led to the biscarboxylate 15 in 81% yield (entry 6). 32 Estradiol-3-ferrocenecarboxylate (14) could be produced with excellent selectivity using fluorocarbonylferrocene (3, entry 7). 33 X-ray measurements confirmed that esterification occurred at C-3 with ferrocene positioned between the alpha and beta faces of the steroid. Compounds 13 and 14 were used to synthesize the corresponding 130 Ru-ruthenocene derivatives used to study the organ distribution of potential radiopharmaceuticals. 31 Cholesterol esters were obtained in the reaction of the steroid with chlorocarbonylferrocene (1, entry 8), ferrocenecarboxylic acid anhydride (2, entry 9) 32 and chloride derivatives of ferroceneacetic acid (6, entry 10) and δ-ferrocenyl valeric acid (8, entry 11). 34 Interestingly, direct esterification of the acids with the DCC method failed according to the latter report. Testosterone was found to be much less reactive, the 17carboxylate 21 was produced only in 13% yield (entry 12). 32 Comparison of various derivatization agents for dehydroepiandrosterone (entries 13-15, Figure 2) in analytical applications proved the superiority of ferrocenoyl azide that leads to carbamate 23. This reagent was shown to convert a wide range of steroidal substrates (e.g., methyl lithocholate, estrone, digitoxigenins, 3-epidigitoxigenin, digoxigenin) to the corresponding products in 10-30 minutes. Carbamates of cholesterol and stigmasterol (20,24) were obtained in 73 and 75% yields, respectively, after heating a mixture of the acyl azide 5 and the sterol in toluene at 90 °C for 10-30min (entries 16,17). 30 ES-MS detection of the ferrocenebased derivatives proved facile, with the molecular radical cation of the derivative dominant in the spectrum.  Figure 2. Products of acylation of steroid alcohols.
Efficiency of ES-MS analysis could also be enhanced by the formation of cyclic boronate esters from 2or 4-hydroxyestradiol (25, 26, Figure 3). 35 The methodology made it possible to analyze the isobaric catechol estrogens in the presence of one another. Ferroceneboronic acid (9) and N,N-dimethylaminoethyl ferroceneboronic acid (10) were used for the derivatization of diols for gas chromatographic separation and GC-MS measurements 28 to obtain esters 27-37 ( Figure 2). In the case of compounds with two vicinal diol moieties, a mixture of mono-and diesters was formed in most cases. Acylation using ferroceneboronic acid (9) led to monoester 30, together with diester 31 showing the higher reactivity of the 22,23-diol group. The ratio of products depended also on the choice of the acylating agent. While the 20,22-monoester 32 was the predominant product in the presence of reagent 9, the use of boronic acid 10 led to a mixture of ester 33 and diester 34.  α-(Ferrocenyl)-aminomethanephosphonous acid derivatives were prepared (Scheme 1) to connect the ferrocenyl group and the steroid skeleton with the help of aminophosphonous acid, often found in biologically-active compounds. 36 Esters 35a-c were obtained as mixtures of four diastereoisomers, which were clearly visible in 31 P NMR spectra as four well-separated signals. The ratio of diastereoisomers varied from The ester functionality can be introduced to both cyclopentadienyl rings of ferrocene. 1,1′-Bis-(chlorocarbonyl)ferrocene (12) was prepared by the reaction of the acid with oxalyl chloride and a catalytic amount of pyridine. 37 The 1,1'-diesters of cholestanol (36, Scheme 2) and cholesterol (37) were obtained in moderate yields in the presence of triethylamine. Compound 40, with succinic acid as the linker between the two components, was reported by Cais for potential use in metalloimmunoassays (Scheme 3). 38 Estradiol was converted to the 17β-hemisuccinate 38. This carboxylic acid was connected to ferrocene via an amide bond using (aminomethyl)ferrocene (39) as the reagent. Unfortunately, no particulars of the synthetic methodology were disclosed. Steroid-ferrocene conjugates have been designed to obtain redox-responsive gels. In these molecules, the steroid alcohols are connected to the ferrocene moiety via a suitable linker. Ester 41 was prepared by the acylation of cholesterol with N-protected glycine (Scheme 4). 24 After the removal of the protecting group, the amino derivative 42 was connected to ferrocene by an amide bond to produce conjugate 43. According to SEM measurements, compound 43 self-assembled into different supramolecular structures in different solvents. Chemical oxidation of the ferrocenyl residue resulted in a phase transition from the gel state to the solution state. Similarly, linker groups with amino functionalities were introduced into compounds 46a-d (Scheme 5) to give them some hydrogen-bond formation sites, and to enhance their aggregation ability. 25 Sol-gel phase transitions of these conjugates could easily be triggered by chemical redox reaction, shear stress, sonication or change in the temperature. In a similar fashion, conjugates containing two cholesteryl groups (47,49) were produced starting from 1,1′-bis(chlorocarbonyl)ferrocene (12) (Scheme 6). 26 Compound 47 was found to form a thixotropic gel, with a potential use in wastewater treatment. The methodology was proved to be very effective using iodinecontaminated water as a model system, combining the efficiency of liquid-liquid extraction and the simplicity of liquid-solid separation.   The application of 1,1′-bis(chlorocarbonyl)ferrocene (12) as the starting material offers the possibility to introduce two different groups on the cyclopentadienyl rings. Compound 51 was synthesized from cholest-5-en-3β-yl-4-(10-hydroxydecyloxy)benzoate (50) and hydroquinone monobenzyl ether (Scheme 7), 23 and was used as a building block of a first-generation dendritic core substituted with six mesomorphic ferrocene units (52). The dendrimer showed liquid-crystalline properties and exhibited a broad enantiotropic smectic A phase, as proven by polarized optical microscopy, differential scanning calorimetry, and X-ray diffraction studies.

Alkylation of steroid alcohols
Several ether derivatives were prepared to obtain chemically switchable membranes or vesicles. Ferrocene was chosen as a redox-active headgroup connected to the apolar steroid skeleton; thus, upon oxidation of ferrocene to ferrocenium, amphiphilic molecules can be formed. The first example, compound 54 was produced by the reaction of methyl iodide with the ferrocene derivative 53 leading to the formation of a carbocation via quaternization of the amine derivative. Carbocation formation was followed by alcoholysis by cholestanol (Scheme 8). 37 The same methodology was used to obtain the bis(ferrocenylmethyl)-estradiol diether (55) with the 3,17 β-estradiol unit as a rigid spacer. 39 Vesicle formation was attempted with the neutral monomers 54, 55, but no aggregates could be detected by laser-light scattering. At the same time, electrochemical or chemical (Ce 4+ ) oxidation of the conjugates to the corresponding ferrocenium derivatives resulted in the formation of stable vesicles in water. The aggregates were found to collapse upon treating with aqueous dithionite solution to reduce Fe 3+ to Fe 2+ , and the monomers could be recovered and re-oxidized to form the vesicles, again. Electrochemical studies showed that the two headgroups of compound 55 could be oxidized independently; this was the first example of a redox-switchable, organometallic bolaamphiphile. (Ferrocenylmethyl)trimethylammonium iodide (56) was used as the alkylation agent by Sakač to produce ether derivatives of estradiol (57) and estrone (59) (Scheme 9). 40 Interestingly, the outcome of the reaction depended greatly on the choice of the solvent. Ethers 57 and 59 were the sole products in DMF, while ether formation was accompanied by C-alkylation in methanol. The latter reaction occurred preferentially at position 4 of the steroid core, leading to compounds 60 and 62, although a 2-alkylated derivative (61) could also be detected in the reaction of estradiol. The authors offered two possible explanations for the Calkylations. In protic solvents, the nucleophilicity of the phenoxide oxygen of the steroids is decreased because of hydrogen bonding, which may promote C-alkylation. Alternatively, electron transfer between the electronrich phenoxide and the ferrocenylmethyl cation could yield two stable radicals that promote C-alkylation, Creactivity being more pronounced in the case of the phenoxide radical compared to the anion. 4-(Ferrocenylmethyl)estra-1,3,5(10)-triene-3,17β-diol (58) was found to be more active against the hormonedependent breast cancer cell line MCF-7 than doxorubicin, so A-ring substitution of steroidal estrogens is a plausible strategy for preparing steroid-ferrocene conjugates acting against tumor cells.
Ferrocenylmethanol (62) was the source of the ferrocenylmethyl cation in the alkylation of 7αmercapto-estradiol (63) to obtain conjugate 64 (Scheme 10). 41 The 7α-position was chosen for conjugation to retain estrogen receptor affinity. The complex indeed behaved like estrogens. In low concentrations, it lacked cytotoxic effect; however, at higher concentrations, it became cytotoxic on MDA-MB-231 cells.
Ar atmosphere, DMF, 100 o C, 12h or MeOH, reflux, 20h  The Fc moiety was attached to estrogens through a linker group by Cais to obtain compounds that can potentially be used in metalloimmunoassays. 42 First, 3-O-carboxymethyl ether derivatives 65a-c were synthesized that were then converted to the active esters 66a-c in the presence of N-hydroxysuccinimide (Scheme 11). The latter compounds were used as acylating agents to connect Fc via an amide bond to produce 67a-c.  (68) with the appropriate 1-(ω-bromoalkyl)ferrocenes (69) or 1,1'-bis(1-(ωbromoalkyl))ferrocenes (72) (Scheme 12) were shown to be capable of controlled gene transfection. 43 The conjugates formed mixed liposomes in the presence of 1,2-dioleoyl-sn-glycero-3-phosphatidyl ethanolamine. The vesicles possessing ferrocene in the reduced state induced an efficient gene-transfection capability using pEGFP-C3 plasmid DNA in three cell lines, even better than the commercial lipofectamine 2000. Redox activities of the co-liposomes and their lipoplexes could be regulated using the alkyl ferrocene moiety. This evidence suggests that these redox-driven systems could be used in gene-delivery applications where transfection needs to be performed partially or temporarily.

Other reactions of steroid alcohols
Ferrocene conjugates of cholesterol (76) and stigmasterol (77) (Scheme 13) were obtained by the replacement of the OH group with a (ferrocenyl)thioimidoyl moiety. 44 Mitsunobu reaction of the steroid alcohols with N-(ethoxycarbonyl)ferrocenecarbothioamide (75) was relatively slow compared to simple alcohols, such as benzyl alcohol. Good yields for steroid substrates could be obtained in days rather than hours. NMR measurements revealed inversion of the configuration at C-3, typical for the Mitsunobu reaction.

Transformation of steroids with carbonyl functionality
Another common moiety of natural steroids is the carbonyl group that easily undergoes nucleophilic additions or aldol-type reactions. The first report for the derivatization of a steroid ketone appeared in 1970. 46 Hydrazones 86 and 87 were prepared in good-to-excellent yields by the reaction of ferrocenecarboxhydrazide (85) with androstanolone benzoate (83) or testosterone benzoate (84), respectively (Scheme 15). Unfortunately, these compounds (among other Fc derivatives connected to biomolecules in a similar fashion) were found to bear no significant biological activity, such as antibacterial, antifungal, or antiparasitic properties. Claisen-Schmidt condensation of steroidal ketones with ferrocenecarboxaldehyde (88) results in the formation of ferrocenylmethylidene derivatives, so the Fc moiety can be connected to different positions of the steroid skeleton by a C-C double bond. 16-Ferrocenylmethylidene estrone (89, Scheme 16) was prepared to be used as starting material to produce radiopharmaceuticals 47 or to carry out detailed structural investigations and bioactivity tests. 48 Single-crystal X-ray diffraction studies proved the E configuration of the product with the cyclopentadienyl ring adopting a coplanar conformation with the olefinic group of the α,βunsaturated system. Interestingly, it could be isolated as two conformers in the solid state using different crystallization media, benzene and carbon tetrachloride. The conformer that co-crystallized with a benzene molecule had the Fc positioned on the alpha face of the steroid skeleton, while in the other one, cocrystallized with carbon tetrachloride, the Fc pointed to the beta face. At the same time, NMR data showed the presence of one species, indicating a low rotational energy barrier between the two conformers. According to biological tests, conjugate 89 was more cytotoxic on hormone-dependent MCF-7 and T-47D than on hormone-independent MDA-MB-231 breast cancer cell lines. The reduction of 89 with NaBH4 led to diol 90, while Pd-catalyzed hydrogenation resulted in the formation of a mixture of the two ferrocenylmethyl derivatives 91 and 92 (Scheme 17). 48 The 17β-ol (92) could be produced in 99% and 98% yields, respectively, by the NaBH4 reduction 49 of ketone 91 or by the hydrogenation 48 of ferrocenylmethylidene conjugate 90. The structures of products 90-92 were proven by Xray crystallography. [48][49][50] According to docking studies, the steroid moiety is positioned in the estradiol-binding pocket of the estrogen receptor surrounded by the same hydrophobic core as 17β-estradiol, however, the position of the ferrocene is different depending on the structure of the conjugates. In the ferrocene conjugates 91, 92, as well as in one conformer of 89 (obtained with crystallization from carbon tetrachloride), the hormone moieties adopt the same orientation as 17β-estradiol does. In the other conformer of 89, and in 90, the steroid skeletons are positioned opposite to the direction of the original hormone. 48 The estrone conjugate 89 served as a starting material to produce the radioactive 99m Tc derivative 94 (Scheme 18). 47 Organ-distribution studies showed a tumor/muscle 99m Tc concentration ratio of 3:1 in the case of MXT-mammary tumors in mice. It should be mentioned that the 99m Tc compound 94 could be obtained in better yield via route A than via route B.   Figure 4). Chemical structures of 95-100 were determined based on HR ESI-MS and two-dimensional NMR spectroscopy. Unfortunately, the authors did not comment on the incorporation of two ferrocenyl units into methyltestosterone leading to the pentacyclic derivative 96. 51 Crystallographic data confirmed that condensation was achieved at C-16 for 97 and 101, and at C-21 for 102. Ferrocene points towards the beta face of the steroid in compounds 97 and 101, while in 102 it is positioned between the alpha and beta faces. 52 The first two compounds (95, 96) showed dose-dependent antiproliferative activity on HeLa cell lines and had similar potency to the standard anticancer drug, doxorubicin. 51  The 16-arylidene-3β,17β-diol 107 (Scheme 19) was prepared in order to evaluate its androgenic activity. 53 Deprotonation of the 3-protected dione 104 with sodium hydride was followed by the reaction with ferrocenecarboxaldehyde (88). The acetal of compound 105 was then quantitatively deprotected and diketone 106 was reduced using NaBH4. According to NMR measurements the product (107) was obtained as a single diastereomer, and NOESY experiments proved the E configuration of the double bond. Unfortunately, the diol 107 had only a moderate effect on PC-3 hormone-independent prostate-cancer cells. © AUTHOR(S) Steroid-ferrocene derivatives 111 and 112 were obtained via the Claisen-Schmidt condensation of the corresponding 16-formyl steroids and acetylferrocene (110), carried out in a nBu-TMG (2-n-butyl-1,1,3,3tetramethylguanidine) and ethylene glycol mixture (Scheme 20). 54 nBu-TMG can play a dual role of co-solvent and catalyst to substitute the usual alkali hydroxides. In similar reactions, both nBu-TMG and ethylene glycol can be recycled via the formation of a reversible ionic liquid in the presence of CO2. The ionic-liquid form makes it possible to isolate the product by simple extraction. The ionic liquid can then be converted back into the original nBu-TMG / ethylene glycol mixture upon removal of CO2, and can be used, again, in a next run by adding fresh reagents.

Miscellaneous reactions
16-(Ferrocenylmethyl)amino-estratrienes 115a-f (Scheme 21) exhibited broad antimicrobial activity, particularly against mycobacteria and multi-resistant staphylococci. 55 The conjugates were obtained by the condensation of the corresponding aminosteroids 113a-f with ferrocenecarboxaldehyde (88) followed by reduction of the hydrazones 114a-f with NaBH4. The 3-(N-ferrocenylmethyl)amino-cholestanes 116a-c, obtained by similar procedures, did not show antimicrobial effect. As ferrocenylmethylaminoethanol was also found to be inactive, the activity of the 16-ferrocenylmethylamino steroids 115a-f was attributed to the combination of the ferrocenyl group and the estratriene steroid moiety. The acylation of aminomethylferrocene (39) was performed via the in situ formation of the corresponding acyl chloride obtained by the addition of oxalyl chloride. Unfortunately, the incorporation of the metallocene moiety into the ursolic acid derivatives did not increase the original aromatase-inhibitory activity of ursolic acid.

Synthesis of Steroid-ferrocene Conjugates using Organometallic Reagents
The Jaouen group developed several methodologies to connect the ferrocenyl moiety to the steroid skeleton by a C-C bond directly or via a linker group. The use of organometallic reagents or the application of transition metal catalyzed reactions (see section 4) resulted in a broad range of conjugates. The 17α-ferrocenyl derivative 125 was found to be recognized by the estradiol receptor, although its apparent relative binding affinity decreased to 15%, with estradiol taken as 100%. Interestingly, while estradiol binds reversibly to its receptor, the introduction of the organometallic group resulted in an irreversible binding. Compound 125 was prepared in 29% yield by reaction of the protected estrone (123) with ferrocenyl lithium (124) (Scheme 24). 58 A similar, but more detailed synthetic procedure was reported in 1994, leading to the estradiol derivative 125 in 44% yield. 59 The X-ray investigation of the product proved the α-configuration of the ferrocenyl group pointing below the plane of the D ring, and also the formation of a hydrogen bond between the C-3 phenolic and C-17 hydroxyl groups. Such an intermolecular hydrogen bond had been found to be significant in the interaction of the parent estradiol with the hormonal receptor. The authors thought that this might explain why the 17α-ferrocenylestradiol derivative retained a relatively good affinity to the same receptor in spite of the bulky ferrocenyl substituent. They also showed that Fc-labelling of estradiol resulted in a more selective detection of the steroid after HPLC separation as it allowed the use of the electrochemical detector at low anodic potentials. 60 Ferrocene conjugates of 17α-ethynyl steroid hormones were obtained by two different strategies using organometallic reagents. The ferrocenoyl-ethynyl compound 129 was prepared by reacting the lithium derivative of the deprotected 17α-ethynylestradiol 126 with amidoferrocene 127, prepared according to the Weinreb method (Scheme 25 As another approach reported by the same group, the dihydrotestosterone (DHT) conjugate 134 was obtained via the reaction of the 3-protected steroid 104 with lithiated ethynylferrocene. 53 Exclusive formation of the 17α-ferrocenyl isomer 133 was observed in 39% yield (Scheme 27). The deprotection of the C-3 ketone was carried out quantitatively using a catalytic amount of PTSA (p-toluenesulfonic acid) to give the ferrocenyl DHT derivative 134 that was reduced in one step to give the androstanediol derivative 135. Binding affinity to the androgene receptor was found to be low even for the 3-keto compound 134. This means that there is not enough space in the binding pocket of the receptor to accommodate practically any substituent in the 17 α-position. In contrast to 3β-androstanediol that was described as the endogenous ligand for the beta form of the estrogen receptor (ERβ) in the prostate, the 3β-OH derivative 135 showed no affinity towards this receptor. At the same time, conjugates 134 and 135 were found to exert considerable antiproliferative effect on hormone independent PC-3 prostate-cancer cells.
Similar synthetic procedures were used by Wenzel in 1994 47 during the synthesis of the estradiol conjugate 136 by the addition of lithiated ethynylferrocene onto the carbonyl functionality of protected estrone 123. Also, the Fc conjugate was proved to be a suitable starting material for the 99m Tc-complex 137. An interesting example of the application of organometallic reagents is the synthesis of a ring-C aromatic steroidal analogue connected to a ferrocenyl moiety (139, Scheme 29). 62 Chromium-carbene complexes possessing a π-system adjacent to the carbene carbon such as compound 138 may undergo an annulation reaction, in the present case with ethynylferrocene (132). Probably due to the vicinity of the aromatic structure, no diastereoselection was observed, and a 1:1 mixture of epimers was formed.

Transition Metal Catalysis in the Synthesis of Steroid-ferrocene Conjugates
Although facile C-C bond formation can be achieved by the application of organometallic reagents, the introduction of protecting groups is necessary to avoid side reactions. Transition-metal catalysis usually eliminates the need for protection-deprotection steps, so synthetic routes can be simplified. Both palladium 63 and copper 64 catalysts are widely used to produce steroid derivatives with ferrocene-steroid conjugates among them.

Palladium-catalyzed C-C coupling reactions
Stille-coupling, the reaction of alkenyl-or aryl halides with organotin reagents, was one of the first examples of palladium-catalyzed C-C couplings. 65 Recently, it has been superseded by other similar reactions, such as Sonogashira-and Suzuki couplings, mainly due to the toxicity of organotin compounds and some difficulties in product separation. Beside other methodologies for the synthesis of steroid-Fc conjugates, the Jaouen group prepared the testosterone derivative 143 by a Stille-coupling reaction between iodoferrocene 142 and the 17ethynyltestosterone stannyl derivative 141, which was obtained by heating ethynyltestosterone (140) with n-Bu3SnOMe (Scheme 30). 53 It is worth noting that other iodo-organometallics, such as (C5H4I)Re(CO)3 and (C5H4I)Mn(CO)3 were more reactive than iodoferrocene. Conjugate 143 was found to have a significant antiproliferative effect on hormone independent PC-3 prostate cancer cells. Another option for creating a C-C bond between the steroid and the ferrocenyl moiety is the Sonogashira coupling, involving the reaction of an aryl/alkenyl halide with a terminal acetylene in the presence of a palladium catalyst and a copper-salt as co-catalyst. 65 The ready availability of 17α-ethynyl steroids may make this approach especially attractive. In contrast to the route reported by Wenzel (Scheme 28), 47 conjugate 136 could be prepared without the protection of the hydroxyl groups, simply by coupling of ethynylestradiol (144) with iodoferrocene (142) (Scheme 31). 66 It was found to retain a satisfactory affinity for an antibody specific to estradiol and that it remains well-recognized by the two natural estrogen-receptor subtypes, ERα and ERβ. Because the receptor affinity of hormones is sensitive to the presence of substituents, an additional functional group on the cyclopentadienyl ring of the complexes was expected to modify their recognition by the receptor. Due to the planar chirality of 2-formyl-iodoferrocene (145), the Sonogashira reaction of 17αethynylestradiol (144) with racemic 145 led to a mixture of diastereomeric products that was difficult to separate. 67 The application of enantiopure 2-formyl-((S)-145 or (R)-145)), 67  In comparison to conjugate 129 (Scheme 25), with a carbonyl functionality between the ethynyl group and the cyclopentadienyl ring, the formyl derivatives showed better affinity for the estrogen receptor. While the receptor does not differentiate between the two diastereomers (R)-148 and (S)-148, the affinity of (R)-147 is almost twice that of the S diastereomer (S)-147. The conjugates showed a proliferative effect on MCF-7 breast cancer cell lines, indicating estrogenic behavior.
Another option for a Sonogashira reaction of ferrocene derivatives with a steroid is the coupling of ethynylferrocene (132) as the alkyne reaction partner with a steroid bearing an appropriate substituent. The cholesterol derivative 150 was obtained in such a reaction with alkenyl-triflate 149 (Scheme 33) as a halide equivalent 69 that could be prepared easily from the corresponding ketone.  The two building blocks incorporating the steroid and the ferrocene skeleton, respectively, can be connected by an amide bond via aminocarbonylation reactions. Palladium-catalyzed aminocarbonylation involves the coupling reaction of an alkenyl/aryl halide or halide equivalent with a primary or secondary amine in carbon monoxide atmosphere. Alkenyl iodides 155a and 155b, prepared in two steps from an unnatural 13α-16-keto steroid (154) were converted to the corresponding ferrocene conjugates 156a and 156b by a reaction with (aminomethyl)ferrocene (39) under carbonylation conditions (Scheme 35). 72 As separation of the alkenyl iodides was found to be difficult, carbonylation was carried out using their 45/55 mixture and only the products 156a and 156b were isolated. Lower reactivity of 155a was explained by a steric hindrance caused by the planar disposition and close proximity of the 17-methyl and 16-iodo groups. Aminocarbonylation was carried out under homogeneous conditions using Pd(OAc)2 together with the PPh3 ligand. The applicability of a heterogeneous, phosphine-free, silica-palladium catalyst was also tested. Somewhat lower yields, but greater selectivity towards 156b, were obtained [with yields of 11% (156a) and 51% (156b)] with the latter catalyst. Also, it could be recycled with only a little decrease in the amount of the isolated products.

Copper-catalyzed azide-alkyne cycloaddition (CuAAC reaction)
The CuAAC reaction published by Sharpless 75 and Meldal 76 allows the direct synthesis of highly complex organic structures starting from simple, and readily available, building blocks. It involves the cycloaddition of organic azides and terminal alkynes leading to the exclusive formation of 1,4-disubstituted-1,2,3-triazoles. Although various Cu(I) salts and complexes can efficiently be used as catalysts, the simplest catalytic system consists of CuSO4 and sodium ascorbate. The catalytically-active Cu(I) species is formed in situ from the Cu(II) salt in the presence of the ascorbate as the reducing agent. The reaction tolerates moisture and oxygen, can be carried out in environmentally friendly solvents, e.g., in water at ambient temperature, and often leads to the product, quantitatively. These features make this methodology especially attractive for the labelling of biomolecules. 77 Both steroids and ferrocene derivatives can easily be functionalized to introduce azide or alkyne moieties. That means that two different approaches can be used: either alkynyl steroids are reacted with ferrocenyl azides or vice versa, alkynylferrocenes and steroidal azides are used as substrates.
The cycloaddition of racemic ferrocenyl azide 173 and 17α-ethynylsteroids 144, 150 and 152 (Scheme 38) led, undoubtedly, to epimers of products 174-176. 71 The epimers could not be separated by column chromatography, however, and gave identical Rf values when reaction mixtures were analyzed by TLC. The only sign of the presence of the two compounds was that the methine protons of the CH-CH3 group of the side chain do not give a quartet as expected, but a more complicated multiplet. The small difference in the spectra in the two epimers was explained by the fact that the chiral center of the side chain is relatively far from those of the steroid skeleton. Triazoles 174-176, as well as the conjugate 178, 78 were obtained in good-to-excellent yields. An organic/inorganic hybrid material comprising a silica support and a polymer with imidazolium moieties was prepared for the immobilization of a copper catalyst. Although the catalyst retained its activity in the CuAAC reaction of the steroidal alkyne 144 and azidomethyferrocene 177 in three runs, product 179 was obtained in low yield (Scheme 39). 79 It should be mentioned that, under the same conditions, total conversion was observed in the cycloaddition of phenylacetylene and benzyl azide in 7 subsequent runs. Comparison of these results, and also data obtained by other alkynes and azides, showed that, in contrast to homogeneous phase reactions, steric hindrance in both reaction partners retarded cycloaddition in the presence of the heterogeneous catalyst considerably. The alkyne functionality was introduced to steroids 180-183 via an aminocarbonylation reaction of steroidal iodoalkenes 157-160 with propargylamine (Scheme 40). 80 Triazoles 184-187 were obtained in good yields as non-separable mixtures of epimeric products, similarly to the reactions depicted in Scheme 38. This was attributed to the flexible linker and the long distance between the chiral centers of the side chain and the steroid skeleton. Although the CuAAC is usually reported not to be hindered by bulky substituents, considerably different reactivity was observed for steroidal azides 192, 196 and 199 (Scheme 43). 81 Ferrocene-labeled steroids with triazole groups attached directly to the 16β-(194) and 2β-positions (197) were obtained in good yields with ethynylferrocene (132) as the reaction partner, however, the target compound was detected only in trace amounts by TLC under the same conditions starting from 199. According to the computational studies, the azido group is in the least hindered position in the 16β-azido-steroid 192. Flipping of ring A of steroid 196 into a twisted boat conformer with the 2β-azido group in the equatorial position is still feasible. At the same time, there is a great difference in the free energies of the twisted boat and the chair conformations of the B ring in steroid 199, in favor of the latter structure. Therefore, steric hindrance cannot be relieved by a conformational change in compound 199, which may result in a considerably lower reactivity. It should be mentioned that the cycloaddition of alkyne 193 with a more distant ferrocenyl moiety led to the products in acceptable yields (57-67%) from all of the steroid substrates. Three triazolyl-ferrocene derivatives (179, 195 and 197) were found to be potent inhibitors of steroid sulfatase, a key enzyme in estrogen biosynthesis, and displayed stronger affinities to the enzyme than the substrate estrone-3-sulfate itself. 78 Compound 197 is bound in a reversible manner, whereas the C- 16 (195) and C-17 derivatives (179) are irreversible inhibitors. Related non-ferrocenyl compounds were found to exert lower potency.
With a similar methodology, the azide derivative of cholesterol (202) was 'clicked' with ferrocenyl chalcone 201 to produce triazol 203 (Scheme 44). 82 Unfortunately, conjugate 204 was completely inactive against all the tested bacteria (E. coli, S. aureus, A. flavus, and C. albicans.). In order to prove suitability of sequential bioconjugation of polypeptides, ethynylferrocene (132) and 17α-ethynylestradiol (144) were introduced site-specifically into a N-substituted glycine peptoid oligomer scaffold to obtain conjugate 204 ( Figure 5). 83 The ferrocene core of 204 showed a significant decrease in redox potential when compared to ethynylferrocene. This was attributed to the altered electronic environment established by the extended conjugation of the ferrocene cyclopentadiene group with the 1,2,3-triazole ring.

Synthesis of Ferrocenestrone
A completely different approach was used by Kotora et al. 84 to obtain a steroid-ferrocene conjugate. They synthesized the first steroid analogue possessing a ferrocene moiety integrated within the steroid framework. Ferrocenestrone 208 was produced starting from the chiral methyl ether 205 (Scheme 45). Planar chirality on the ferrocene moiety resulted in a selective formation of stereocenters during the Diels-Alder reaction, leading to 207 that ensured selectivity in further steps. Biological tests showed no activation of estrogen, androgen and progesterone receptors in the presence of compound 208.