Chemistry and catalysis advances in organometallic chemistry and catalysis
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31 32 R = H
R = Me Figure 42.8 Another series of compounds in which 31 is the most active. 570 THE FERROCIFEN FAMILY AS POTENT AND SELECTIVE ANTITUMOR COMPOUNDS: MECHANISMS OF ACTION TABLE 42.3 Effect of Compounds 26–32 on Growth of Hormone-Independent Breast Cancer Cells Compound
26 27 28 29 30 31 32 Formation of QM No No
No No Yes No Growth of cells, % a 53
53 49 93 16 90 a Effect on growth of cells of MDA-MB-231 at 10 μM. Control = cells without added compounds after 5 days of culture, set at 100%. TABLE 42.4 Effect of Compounds 33–39 on Growth of Hormone-Independent Breast Cancer Cells Compound
33 34 35 36 37 38 39 Formation of QM or OQ OQ OQ
OQ QM No No IC 50 , μM a 1.10 ± 0.07
1.21 ± 0.07
2.4 ± 0.2
0.48 ± 0.04
5.3 ± 0.9
9.0 ± 1.0
12.7 ± 1.6
a Mean of two experiments ± range. Fe OH Fe OH Fe OH H 3 CO Fe OH OH Fe OH OH Fe OH OH Fe OH OH 37 38 39 33 36 34 35 Figure 42.9 Example of ferrocenyl cathecols. 42.5 SYNTHESIS AND BEHAVIOR OF FERROCENOPHANE DERIVATIVES The results obtained in the open series showed that the position of the ferrocenyl group and the nature of the substituents present on the molecule have a significant impact on the antiproliferative activity of the compounds. Another possible structural modification is to alter the nature of the ferrocenyl group. For example, the creation of a bond between the last carbon of the ethyl and the second cyclopentadiene ring immobilizes the ferrocenyl group in a fixed position relative to the rest of the molecule. This bond generates a new ferrocenyl derivative of the ansa-ferrocene or ferrocenophane type. The first derivative of ferrocenophane was synthesized by Plazuk et al. [128] in the form of a diphenol compound, 40 (Fig. 42.10). It was obtained by a McMurry coupling reaction between ferrocenophanone and dihydroxybenzophenone. Fe OH
41 OH HO Fe 40 Figure 42.10 Ansa-ferrocene diphenols. SYNTHESIS AND BEHAVIOR OF FERROCENOPHANE DERIVATIVES 571 TABLE 42.5 RBA, Log P o/w , and the Effect on Cancer Cell Growth of 40 and 41 IC 50 , μM RBA (%) for ER α log P o/w MDA-MB-231 PC3 40 7.2
± 0.7 4.6
0.09 ± 0.01
0.094 ± 0.0067
41 7.6
± 0.6 4.8
0.96 ± 0.03
1.08 ± 0.02
TABLE 42.6 Cytotoxicity of Compounds 42–57 against Hormone-Independent MDA-MB-231 Breast Cancer Cells a R ′ R Fe Compound R R IC 50 values, μM Cytotoxicity Ratio b
H H 0.92 ± 0.11
8 .2 (E +Z)-43 OH H 0.47 ± 0.06
3 .3 (E +Z)-50 OAc
H 0.26
± 0.004 7 .6 (E +Z)-44 NH 2
0.21 ± 0.03
4 .1 (E +Z)-53 NHAc
H 0.47
± 0.04 1 .4 40 OH OH 0.089 ± 0.006
7 .2 (E +Z)-48 NH 2 OH 0.061
± 0.005 9 .0 (E +Z)-54 NHAc OH
± 0.019 6 .1 49 NH 2 NH 2 0.047 ± 0.01 45 NHAc
NHAc 5.64
± 1.13 (E +Z)-51 OAc
OH 0.049
± 0.003 8 .6 52 OAc
OAc 0.044
± 0.001 14 .5 (E +Z)-46 Br H
± 0.62 > 3.4
47 Br Br >10 55 CN H 0.85 ± 0.07 (E+Z) 12 .8
CN CN 7.98 ± 1.2 a From Reference 130. b IC 50 (ferrocifen)/IC 50 (ferrocenophane). Compound 40 shows an IC 50 value of 0.09 ± 0.01 μM against MDA-MB-231 cells (Table 42.5), making it six times more active than ferrocidiphenol 15 (IC 50 = 0.6 μM). Another type of ansa compound is represented by 41. Its IC 50 value
is 0.96 ± 0.03 μM, showing that 41 is less active than hydroxyferrocifen 14 of the open series. Excellent levels of antiproliferative activity for the ferrocenophanes as compared to classic ferrocenes were seen again in other compounds of this series. The antitumoral activity of these new compounds is listed in Table 42.6. The result obtained clearly shows that the series of the closed ferrocenophanes is more active than the series of the open ferrocifens. The ratio of IC 50 (ferrocifen)/IC 50 (ferrocenophane) is greater than 7 for compounds 40, 42, 48, 50, 51, 52, and 55. Acetamide 53 is the only compound that produces a fairly low ratio (1.4), perhaps because of a difficulty in hydrolyzing the amide. A similar effect of the substituents on antitumoral activity is found for both the ansa and open series. The hydroxylated and aminated derivatives are the most active. The electron-withdrawing groups, such as Br or CN, inhibit the activity of the compounds [106, 107]. Therefore, in the ansa series, the most promising products appear to be similar to those of the ferrocifen series, in terms of substituents. The most active molecules retain the [Fc-conjugated spacer—phenol (or aniline)] motif previously identified as essential. The organometallic ferrocenophane-type framework bears a short, three-carbon chain, bridging the two cyclopentadienyls. Despite the ring strain, the synthesis is viable but the two Cp rings are no longer parallel as in the ferrocifens, showing instead a dihedral angle of 11 ◦ [129, 130]. An IC 50 value of 89 nM on MDA-MB-231 is obtained with 40. 572 THE FERROCIFEN FAMILY AS POTENT AND SELECTIVE ANTITUMOR COMPOUNDS: MECHANISMS OF ACTION This represents a general trend. With all the substituents studied in Table 42.6, the IC 50 values for the ferrocenophanes are always substantially lower. However, with these species, we have not been able to clearly identify the putative QM with a three-carbon bridge. If the ring strain is released with a bridge of four or five carbons, the results revert to those of the ferrocifens. This series of the ansa type has been less studied to date than the earlier series. However, the data obtained from the NCI for the ferrocenophanol 40 and the ferrocidiphenol 15 on 60 cancer cell lines show higher activity for the cyclic series [117]. The two lead compounds 15 and 40 reveal new mechanisms compared to those already listed by the NIH and also are probably not identical with each other. This is based on the absence of identifiable QMs in the ansa series and on the different shapes of the antiproliferative curves showing a plateau with the ansa molecule [117]. Nevertheless, the first part of the mechanism of action, involving oxidation of the elemental iron, may be similar between the two series, since both sets generate ROS in cancer cells.
The McMurry coupling reaction generally leads to three alkenes, two symmetrical products and one asymmetrical. However, depending on the reaction conditions, especially changes in temperature and medium, other secondary compounds are also formed, in particular, the transposition product and pinacol [129]. These two new species were synthesized and studied by orienting the reaction more specifically in their favor. The IC 50 values of these compounds are collected in Table 42.7. With the exception of compound 58 (IC 50 = 1.45 ± 0.49 μM), the transposition products, 52, 59, 60, and 61, have very little activity. Conversely, the pinacols 62 and 63 are particularly active. It has been found that several metals, O 2 , H
2 O 2 , and N-iodosuccinimide can oxidize pinacols to radicals that undergo cleavage to ketones. The addition of H 2 O
or N-bromosuccinimide to a tetrahydrofuran (THF) solution of 63 led to the formation of benzophenone and ferrocenophanone although only small amounts of this compound were detected (Scheme 42.1). The antiproliferative activity of pinacols may be linked to the production of an intermediate ferrocenyl radical [129]. TABLE 42.7 IC 50 Values ( μM) of Compounds on Breast Cancer Cells (MDA-MB-231) a R
IC 50 Fe O R R ′ 52 OH OH 12.5 ± 0.3
58 H OH 1.45 ± 0.49
59 H H 20.1 ± 5.4
61 NH 2 NH 2 15.4 ± 0.2 60 NHAc
NHAc 9.38
± 1.82 Fe OH OH R R ′ 63 H H 0.17 ± 0.01
62a H OH 0.06 ± 0.01
62b H OH 0.14 ± 0.01
Fe Fe HO OH 64 3.48
± 0.83 a From Reference 129. FORMULATION: RESEARCH INTO NANOCAPSULES BEST SUITED FOR IN VIVO TESTING OF FERROCIFENS 573 Ph Ph OH Ferrocenophanone Ph O
Ph Ph HO Ph Ph OH Ferrocenophanone 63 Ph O Ph H 2 O 2 or NBS, THF Ph Ph HO Fe OH Fe OH Fe O Scheme 42.1 Tentative reactivity of pinacols. O(CH 2
3 N(Me)
2 O(CH
2 ) 3 N(Me) 2 N NH O O CF 3 CN NC F 3 C O NH N O EtO R 1 O 67 66 65 Fe Fe Fe Figure 42.11 Examples of other ferrocenyl antimalarial species. Other ferrocenyl antitumorals have been described that involve mechanisms of action different from those in play in the ferrocifen-type species mentioned above. These include, for example, compounds 65, 66, 67 shown below (Fig. 42.11): In compounds related to 65 [131] (IC 50 = 0.4 μM with MDA-MB-231 cell lines), the lipophilic organometallic moiety does not produce a major effect (with the occasional exception of strong steric hindrance [132]) as compared to a pure organic aromatic group. The presence within the molecule of two vicinal amino alkyl chains, potentially allowing pincer complexation, could perhaps be the key to the antiproliferative efficacy of this series, which operates via a mechanism masking phenol functions. In addition, these species possess the distinctive feature of combining a strong antiproliferative effect with antibacterial and antifungal activity [133, 134]. The introduction of the ferrocenyl moiety at C-5 of a hydantoin ring gives rise to 66. This complex displays an antiproliferative effect (IC 50 = 5.4 μM) on hormone-independent (PC3) prostate cancer cells. This cytotoxic effect is probably steric and non-androgen receptor-mediated [135, 136]. In the N-(6-ferrocenyl-2-naphthoyl) dipeptide ethyl ester 67, a potent activity in the H1299 NSCLC cell line has been found (IC 50 = 0.13 μM with R 1 = H, and IC 50 = 0.14 μM with R 1 = CH
3 ) [137].
For more examples showing the antitumor activity of ferrocenyl compounds, see a recent review by Ornelas [138]. 42.7 FORMULATION: RESEARCH INTO NANOCAPSULES BEST SUITED FOR IN VIVO TESTING OF FERROCIFENS The most active ferrocifens in vitro are generally lipophilic phenol species, for example, 14, 15, 40, and 48, and they present a double challenge, both chemical and physicochemical, to their successful delivery as potential medication. The effects of their organometallic character are not yet well known beyond an observed increased in lipophilicity. In common with many of the molecules currently under study in pharmaceutical research, the most promising complexes so far are insoluble in water. They thus require a formulation stage to make them suitable for administration [139–145]. The bioavailability of organic polyphenols is another major challenge [146] because in their free form these entities are rapidly eliminated from the body. In fact, polyphenols rarely occur naturally in the free form, being normally conjugated with sugars and fatty acid esters [146].
574 THE FERROCIFEN FAMILY AS POTENT AND SELECTIVE ANTITUMOR COMPOUNDS: MECHANISMS OF ACTION This question of the bioavailability of the polyphenols has been addressed by the pharmaceutical industry (Sanofi-Aventis with Endotelon), agrifood (Nestl´e), and the cosmetic industry (Caudalie and antiaging skin care) [147–152]. Benoit et al have developed lipid nanocapsules (LNC), between 20 and 100 nm in size, perfectly stable from a physical standpoint, based on a phenomenon of phase inversion of a microemulsion [153]. The aim is to use this new product as a delivery system for anticancer agents [115, 123, 154] particularly in the treatment of glioma and breast cancer. In fact, the size and structure of the nanoparticle would potentially allow the vector to reach the dispersed cells that are the cause of recurrences frequently observed with this kind of cancer. Two types of treatment have been envisaged that could be combined: local injection into the brain by convection-enhanced delivery (CED), allowing distribution into large tissue volume; and/or intravenous injection by active targeting of the blood–brain barrier and the tumor cells, with the aid of particular polyethylene glycols (PEGs) or antibodies attached to the surface of the LNC. This second route would also provide access to other types of tumors such as melanomas, liver, breast, and ovarian cancers. Preparation of LNC containing ferrocidiphenol 15 was performed [115, 123]. During this work, it was shown that after encapsulation in the LNC, organometallic agents have good efficacy and specificity in vitro on breast cancer and glioma 9L cells, and also in vivo in the model of subcutaneous glioma, by intratumoral action of the anticancer vectors that cause a significant reduction in tumor mass and volume [155]. Interestingly, activity is much reduced on the melanocytes and astrocytes, healthy cells with almost no potential for division. This selectivity of ferrocifens between cancer and normal cells is different from cis-platin. In the same model of subcutaneous glioma, there has been recent work to inject, this time intravenously, LNC loaded with ferrocidiphenol 15 and coated with long PEG chains. The coating of the nanocapsules allows the LNC to remain longer in the bloodstream than conventional LNC, and thus to enhance accumulation in tumors through the EPR (enhanced permeability and retention) effect. Indeed, tumoral progression curves show a marked reduction in the size of tumors in treated rats. After several days, the tumor volume diminishes significantly to the point of disappearance at the end of the experimental period [156]. Classical LNC and long-circulating nanoparticles called stealth nanoparticles (DSPE-m-PEG2000-LNC) loaded with FcdiOH 15 have been used with 9L gliosarcoma in rats. A single intravenous injection of FcdiOH-LNC (400 μl, 2.4 mg/rat) considerably inhibited tumor growth when compared to the control. DSP-mPEG2000-FcdiOH LNC exhibited a strong antitumor effect by nearly eradicating the tumor by the end of the study with this ectopic model (Fig. 42.12) [156]. 1 4
12 Days of treatment 16 20
2 3 4 Tumor volume cm 3 Tumor weight NaCI LNC
LNCs LNC-FcdiOH DSPE-m-PEG2000-LNC-FcdiOH DSPE-mPEG LNCs 0 1000
2000 3000
2408 1458
1071 33 Injection of 1.5 × 10
6 9L cells into rat flank D0 D6 Twice a week D25
Intravenous injection of LNCs loaded with Fc- diOH(400 μL, 2.4 mg/rat) Measurement of the tumor volume Animal sacrifice (tumor weight) Figure 42.12 Intravenous injection of two different sorts of LNC in ectopic tumors. (See insert for color representation of the figure.) CONCLUSION 575 0 0 10 20 30 40 50 Days after 9L cell implantation 60 70 80 90 100
110 20 40 60 80 100 % Survival Control
Blank LNCs Chemotherapy (CED Fe-diOH-LNCs 0.36 mg/rat) Radiotherapy (CED Blank-LNCs + 3 × 6 Gy)
Chemoradiotherapy (CED Fe-diOH-LNCs 0.36mg/rat + 3 × 6 Gy) Median survival time (range) 25 (23–27) 25 (21–30) 27 (24–32) 33 (29–44) 40 (32–100) (two long-term survivors) Figure 42.13 Kaplan–Meier survival plots. (See insert for color representation of the figure.) With this new formulation, DSPE-mPEG2000-LNC-FcdiOH, the tumor has almost totally disappeared [156]. In addition, it was shown that the association between 15 and X-photons is a synergistic one, conferring the properties of a radiosensitizing molecule [157]. Figure 42.13 below shows the Kaplan–Meier survival curves for 9L glioma-bearing rats. The best result appears so far with CED of Fc-diOH-LNC and external radiotherapy of 3 × 6 Gy. In this group of interest, two rats were long-term survivors, as they survived up to 100 days, which certainly involves a total eradication of the tumor. The ionizing radiation can help trigger the generation of ferricinium in compound 15 and favor the occurrence of cytostatic species [157], even in a reducing organ like the brain. The question of finding a good formulation is key for the development of ferrocifens. The above results are very encouraging in this context. Survival rate is significantly improved by the combination of chemo- and radiotherapy. 42.8 CONCLUSION There is at present a notorious mismatch in oncology between therapeutic demands and the existing pharmacopeia. Currently available drugs consist of mostly (80%) proapoptotic compounds, for example alkylants of DNA such as cis-platin, and we are still relatively undersupplied in treatments against apoptosis-resistant primary cancers, acquired drug resistance, and spreading metastases. We are at a stage where metastatic cancers prove fatal in 90% of cases. The annual death rate for these pathologies is of the order of 13.5 million patients and is predicted to increase more quickly than the population since the projections show, at the current rate of change, 35 million deaths per year on the horizon for 2050. It is imperative to prevent this gloomy prediction from coming true. For this reason, novel molecules are being sought that do not operate exclusively via an apoptotic mechanism, but instead are able to induce cytostatic properties by other routes such as senescence [158]. In this context, brain cancers (e.g., glioblastoma), esophageal cancers, melanomas, pancreatic cancers, ovarian cancers, and non-small-cell lung cancers (NSCLCs) are, among others, the cancers associated with the worst outcomes because of their ability to resist most of the standard therapies. From this viewpoint, the ferrocifen family and associated compounds present dizzyingly broad perspectives, both for the richness of the effective structures they offer and for the variety of mechanisms they bring into play. In the open series, of which 14 and 15 are the most studied representatives, a number of distinctive traits can be seen. It has been possible to change the paradigm, thanks to a ferrocenyl redox antenna that distinguishes them from their purely organic relatives. A 60- to 150-fold increase in efficacy has been shown in their IC 50 values on MDA-MB-231 cells, while healthy cells are not affected at the concentrations used. The [ferrocene-alkene spacer-paraphenol] motif appears to be essential for the compounds to become effective. These species do not operate via an alkylation of DNA, unlike cis-platin and its relatives. Several considerations militate in favor of a novel biological mechanism. These include the production
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