Matthieu Beauperin

Dilan Polat

Fares Roudesly

Siden Top

Anne Vessières

Julie Oble
email: julie.oble@upmc.fr

Gérard Jaouen
email: gerard.jaouen@chimie-paristech.fr

Giovanni Poli
email: giovanni.poli@upmc.fr

Matthieu Beaupérin

Approach to ferrocenyl-podophyllotoxin analogs and their evaluation as anti-tumor agents

Keywords:

come

Introduction

In the family of cyclolignans, podophyllotoxin (PPT), a pentacyclic aryl-tetralin lignan isolated from the roots and rhizomes of the American Mayapple (Podophyllum Peltatum) [1] (Figure 1, left), has attracted much attention as it has shown various biological activities among which an interesting antiproliferative effect. This antineoplastic activity arises from inhibition of tubulin polymerization [2], a cytosolic protein required for the formation of the mitotic spindle. However, PPT exhibits a high toxicity and serious side effects like nausea, diarrhea, vomiting and damage to healthy tissue. Accordingly, its use is currently restricted to local treatments [3]. To overcome these drawbacks, several structure modification of podophyllotoxin have been performed on rings C (particularly the C-4 position), D and E, in view of obtaining more efficient and less toxic anticancer agents [START_REF] For | For recent reviews on synthesis of podophyllotoxin derivatives[END_REF]. In particular, the two semi-synthetic derivatives, etoposide and teniposide [5] (Figure 1, right), have emerged, and are now widely used drugs for chemotherapies in lung cancer, lymphomas and genital tumors [6]. Intriguingly enough, unlike their parent compound, these derivatives are not tubulin inhibitors, acting on topoisomerase II. Structure activity relationship (SAR) studies suggest that topoisomerase II inhibition activity is (among other requisite features) associated to the presence of a free 4'-hydroxy group [3a]. However, etoposide is associated with some issues such as drug resistance and poor bioavailability. Furthermore, picropodophyllin (PPP) (Figure 1, left), the C-2 epimer of PPT, although showing no activity against either tubulin or topoisomerase, displays inhibitory activity against tyrosine phosphorylation of Insulin-like Growth Factor 1 receptor (IGF-1R) [7], which plays a crucial role in the transformation, growth, and survival of malignant cells. Due to the severe toxic effects of podophyllotoxin and its derivatives, the discovery of more potent and/or less toxic analogs of this family is highly desirable. Most of the PPT analogs have been prepared from the native molecule by functionalization of the 4-hydroxy group [8].

As a consequence, modifications of ring E have been to date only scarcely explored [9]. A modern strategy towards the modulation of biologically active products consists in the incorporation of an organometallic motif into the drug [10,[START_REF] For Reviews | [END_REF]. Although organometallic compounds where used in therapy as early as the beginning of the 20 th century, this research field remained unexplored until 1996, when ferrocifen, a ferrocenyl analogue of tamoxifen, was reported , showing a high antiproliferative effect against two breast cancer cell lines: MCF-7 and MDA-MB-231 [12]. The mechanism of action of ferrocifen on MCF-7 cells seems to be partly analogous to that of tamoxifen, i.e. an antagonist effect on α-type oestrogen receptors (ERα). Yet, MDA-MB-231 cells, lacking ERα, show resistance to tamoxifen and a strong antiproliferative effect with ferrocifens, which suggests that ferrocifen and tamoxifen here act through different ways. Further studies have shown that the ferrocenyl moiety, by virtue of its reversible redox behavior, induced oxidative stress in the cell, catalyzing production of reactive oxygen species (ROS) [13]. Since that breakthrough, other pharmaceuticals were modified in a similar manner, giving rise to new bioactive organometallic compounds [14]. An example of the above strategy is ferroquine, an innovative antimalarial drug, currently at Phase II clinical trial at Sanofi-Aventis [15]. Due to their peculiar properties, such as increased lipophilicity and bulkiness with respect to the original drugs, these new conjugates allow targeting of different sites in the biological medium. In line with the above concept, we envisioned to prepare ferrocenyl podophyllotoxin analogs, either via hemisynthesis, by simply esterifying the hydroxyl group at C-4 with the

Results and discussion

Derivative 1 was straightforwardly obtained in 60% yield from native podophyllotoxin by treatment with ferrocenoyl chloride [START_REF] Malek-Saied | Ferrocenoyl chloride was prepared according to[END_REF], Et 3 N, and catalytic amount of DMAP (Scheme 1).

Scheme 1. Esterification of native PPT with ferrocenoyl chloride.

The strategy to targets such as 2 is more complex and requires a multistep synthesis.

Accordingly, we decided to exploit a method we developed a few years ago [17] that gives access to the γ-oxo-ester key fragment A [18] (Scheme 2). This latter can be reached from the allylated β-dicarbonyl B via an intramolecular 6-exo Mizoroki-Heck coupling followed by alkene cleavage. B can result from a β-dicarbonyl derivative by allylation and alkylation with a ferrocenyl benzhydrol [START_REF] Gokel | For nucleophilic substitution of ferrocenyl alcohol[END_REF] such as C, which can in turn derive from piperonal and ferrocene. In particular, we reasoned that the benzhydrylation could be best achieved using Scheme 2. Retrosynthetic approach to the ferrocenyl analog of podophyllotoxin 2.

Benzhydrylic alcohol C was obtained via two alternative approaches from commercially available 6-bromopiperonal 3, namely, either an oxidation / Friedel-Crafts acylation / reduction sequence (Scheme 3, top) or a direct condensation with ferrocenyl lithium (Scheme 3, down). The former synthesis started with a Pinnick-type oxidation of aldehyde 3 to give the corresponding carboxylic acid 3' (85% y) by treatment with sodium chlorite in the presence of phosphoric acid [22]. After standard formation the corresponding acid chloride, Friedel-Crafts acylation with ferrocene gave the diaryl ketone 3''. Finally, LiAlH 4 reduction gave alcohol C (47% y). This sequence suffered from low yields, including generation of some debrominated alcohol C' during the last reductive step [23]. The second, more straightforward, approach gave alcohol C in one step by reaction between ferrocenyl lithium [24], and aldehyde 3 (44% y). Although the latter approach is the one recommended, the former route may be reconsidered for a future enantiopure synthesis [25]. Indeed, after a brief experimentation, we found that reaction between ethyl acetoacetate and alcohol C in the presence of catalytic pTsOH (5 mol%) in refluxing CH 2 Cl 2 was the best choice, affording the expected keto-ester 4 (75% y) (Scheme 4). Other combinations using malonates instead of acetoacetates and Lewis acids (LA) or Brønsted acids (HA) other than pTsOH gave either lower yields or no reaction at all. [START_REF]Other Brønsted acids (H 2 SO 4 , TfOH) in catalytic conditions, or Lewis acids (BF 3 .Et 2 O, InBr 3 , TiCl 4 )[END_REF] The subsequent allylation of 4 turned out to be troublesome. Standard conditions of Pd-catalyzed conditions [allyl acetate (2.5 equiv), Pd(OAc) 2 (10 mol%), dppe (20 mol%), NaH (1.3 equiv)] gave back mainly unreacted starting material and only minor amounts of the desired adduct. A more classical allylation of the sodium enolate of 4 with allyl iodide gave more promising results, which could be improved when working in the presence of the 15-C-5 crown ether. In this case, the allylated product 5 could be obtained in up to 50 % yield, although the reaction was not easily reproducible. The lack of reactivity of 4 may be due to steric hindrance of the ferrocenyl moiety. In light of the difficulties to allylate intermediate 4, we decided to perform the alkylation from the "pre-allylated" β-keto-ester 6 [START_REF] Baker | [END_REF]. Such a variant would render the synthesis more convergent with respect to the previous plan, though requiring a challenging benzhydrylation with formation of a quaternary-center. In the event, use of the same acidic conditions as previously used to form 4, gave the targeted compound 5 in 35% yield, the main product being the symmetric ether 7 (50% y) (Scheme 5, conditions a). This result suggests that under these conditions, unionized benzhydrol competes with the enol of 6 in the addition to the transient carbenium ion. Although under HA or LA catalysis the symmetric ether is in principle susceptible to give back the corresponding carbenium ion [START_REF] Bisaro | [END_REF]28], we were not able to exploit this prospective equilibrium to obtain an alternative access to 5. C-C bond formation via the Mitsunobu methodology is possible, provided that the pKa of the Cpronucleophile is sufficiently low [29], and variants of the original [DEAD / TPP] system have been developed [30] to this purpose. For example, Tsunoda and co-workers [30a] used 1,1'-(azodicarbonyl)dipiperidine (ADDP) / tributylphosphine system to promote the reaction between diethyl malonate and benzylic alcohol. Under these conditions, 5 was obtained in a 35% yield after 3 days at room temperature (Scheme 5, conditions b). Although this result appeared promising, it still did not compare favorably with the previous strategies. Finally, we found that conversion of C into the corresponding chloride, followed by in situ treatment with the sodium enolate of 6 gave the desired 5 in 65% yield (Scheme 5, conditions c).

Importantly, during chloride formation, HCl scavenging with 2,6-lutidine was necessary to inhibit formation of dimer 7. yield [31]. Conversion of the methylidene into the key γ-oxo-ester A was then easily accomplished in 60% overall yield via osmium-catalyzed cis-dihydroxylation of 9 followed by NaIO 4 cleavage [START_REF]The relative stereochemistry of the major diastereoisomer of B could be deduced by the coupling constant (5.1 Hz) between the proton in C2 (carbon bearing the ferrocene) and the proton in C3 (carbon bearing the ester)[END_REF]. The acid form of the γ-oxo-ester derivative A, lacking the ferrocenyl tail, has already been identified as an advanced precursor to podophyllotoxin by Kende and coworkers,[18] who were able to obtain picropodophyllone by treatment with formaldehyde and NaOH [START_REF] Gensler | Picropodophyllone could be converted into podophyllotoxin by Zn(BH 4 ) 2 reduction to give the cis-fused isomer picropodophyllin, followed by cis-trans isomerization according to the Gensler protocol[END_REF]. Unfortunately, all attempts to further functionalize intermediate A, so as to add ring D, met with failure. Nevertheless, this study enabled us to have in hand a novel molecule incorporating rings A, B, and C of podophyllotoxin and carrying a ferrocenyl moiety in place of ring E. Accordingly, A (R = Et) was directly submitted to NaBH 4 reduction [START_REF]stereochemistry of the major diastereoisomer in 13 (podophyllotoxin numbering) could be confidently deduced from the value of the vicinal coupling constant ( 3 J 2-3 ~4Hz) in the 1 H NMR spectrum[END_REF], and the resulting alcohol 2 (R = Et) was sent for antiproliferative tests. In the multitargeting ferrocifen series the cytotoxicity is mainly due so far to the redox primer {ferrocenyl-ene-phenol} producing ROS and able to delocalize the initial radical cation and generate electrophilic quinone methides at the mitochondrial level [14a].When the ferrocenyl phenol species are unconjugated the anticancer effect decreases [37]. In the present case the organometallic motifs are different from this arrangement but this type of oxidative effect can be invoked to explain the results. The above redox ferrocifen motif is not unique in order to modulate the anticancer effects in a structure bearing a ferrocenyl group. Indeed, the recent literature reports other primers related to our original concept, producing similar effects in which the central electronic conduit in adjacent position to a ferrocenyl species is a naphtyl group or an ene-2,5-piperazinedione unit [38]. Unveiling such an effect expands our original discovery and may be of interest for future developments of organometallic antitumor species.

Conclusion

In

Experimental section

Reactions were carried out under argon atmosphere. All reagents were used as obtained from commercial suppliers. Dichloromethane and DMF were dried on a Braun purification system MB SPS-800. THF was distilled over sodium/benzophenone under nitrogen atmosphere. Thin layer chromatography was performed on Merck 60 F254 silica gel and revealed with an ultraviolet lamp (λ = 254 nm) and a staining reagent (KMnO 4 , vanillin or para-anisaldehyde).

Merck Geduran SI 60 silica gel (40-63 µm) was used for flash column chromatography.

Solution NMR spectra ( 8, 172.6, 152.8, 148.3, 147,8, 137.4, 135.1, 132.5, 129.0, 109.9, 108.4, 107.1, 101.8, 73.5, 72.1, 72.0, 71.8, 70.4, 70.3, 70.0, 61.0, 56.3, 45.8, 43.9, 39 Second synthesis: To a solution of ferrocene (1.86 g, 10 mmol, 1.2 equiv) in 10 mL of a 1:1 mixture of THF and hexane, was added dropwise at 0 °C a solution of tBuLi (1.7 M in hexanes, 5.9 mL, 1.2 equiv). After 30 min at 0 °C, the ice bath was removed and the mixture was placed in an acetone/N 2 liquid bath. Then, 6-bromopiperonal 3 (1.83 g, 8 mmol, 1 equiv) in 20 mL of THF was added dropwise to the dark red solution. 8, 89.7, 69.6, 69.3, 69.0, 68.8, 68.0, 67.9, 67.2, 66.9, 66.8, 66.3 (major and (minor diast.), 101.9 (major diast.), 91.6 (major diast.), 88.0 (minor diast.), 69. 1, 69.0, 68.88, 68.6, 68.5, 67.7, 67.4, 67.3, 67.2, 66.6 (major and minor diast.) (s, 1H), 6.98 (s, 1H), 6.00 [START_REF] Jaouen | Advances in Organometallic Chemistry and Catalysis[END_REF]J = 1.3 Hz,1H),5.97 (d,J = 1.3 Hz,1H),1H),4.25 (d,J = 3.8 Hz,1H),8H),1H),1H),1H),2.91 (ddd,J = 12.5,4.0,2.6 Hz,1H),1H), 1.84 (d, J = 7.9 Hz, 1H), 1.73 (ddd,J= 13.4,12.5 9.4 Hz,1H 1.29 (t,J = 8.2 Hz,3H). 13 C NMR (101 MHz, CDCl 3 , major diast.): δ (ppm) 173.3, 147.1, 146.7, 132.5, 131.4, 101.2, 91.1, 70.3, 69.1, 69.0, 68.1, 67.2, 66.7, 60.8, 45.4, 41.7, 30.2, 14.3. 6, 147.2, 146.7, 131.4, 131.1, 109.7, 107.7, 101.1, 69.5, 69.2, 68.2, 67.2, 67.1, 66.4, 61.0, 51.0, 46.0, 39.6, 32.2, 14.4

Figure 1 .

 1 Figure 1. Podophyllotoxin, picropophyllin, etoposide and teniposide structures.

  Figure 2, left), or by incorporating the organometallic sandwich in place of ring E in a structure of type 2 (Figure 2, right).

Figure 2 .

 2 Figure 2. Targeted ferrocenyl analogs of podophyllotoxin.

  S N1 mechanism,[20] analogously to the benzhydrylation of active methlylenes pioneered by us some years ago[START_REF] Bisaro | [END_REF]. Furthermore, S N 1-type nucleophilic substitutions on ferrocenyl derivatives are known to take place via iron assistance with retention of configuration [Error! Bookmark not defined.a-c]. As a consequence, control of the absolute configuration of the stereogenic center at the benzylic position might be a further bonus associated to this bioorganometallic approach. However, initially we focused our efforts only on the synthesis of racemic targets of type 2 lacking the lactone (D ring).

6 Scheme 3 .

 63 Scheme 3. Synthesis of o-bromo-sesamyl ferrocenyl alcohol C.

7 Scheme 4 .

 74 Scheme 4. Alkylation of benzydryl alcohol C and subsequent allylation of the resulting βdicarbonyl derivative 4.

8 Scheme 5 .

 85 Scheme 5. Alkylation of benzhydryl alcohol C with the allylated β-dicarbonyl 6: three approaches compared.

9 Scheme 6 .

 96 Scheme 6. Construction of ring C and subsequent conversion into the targeted analogue.

  this study we report the synthesis of two new podophyllotoxin analogs incorporating a ferrocenyl moiety and the preliminary antiproliferative tests of these conjugates on two breast cancer lines. While the former compound has been straightforwardly obtained by esterification between podophyllotoxin and ferrocenecarboxylic acid, the latter molecule, incorporating rings A, B and C of podophyllotoxin and carrying the ferrocenyl appendage in place of ring E of podophyllotoxin, required a 6-step synthesis starting from 6bromopiperonal and ferrocene. Key steps in the synthesis were a challenging benzhydrylation generating a quaternary center, and an intramolecular Mizoroki-Heck reaction, which formed ring C of the target molecule. Such original structures have been discussed in terms of the importance of ferrocenyl conjugated motifs to generate antiproliferative effects.

  Starting from the alcohol C, third essay (via chloride intermediate): To a solution of C (100 mg, 0.24 mmol, 1 equiv) in 1 mL of THF, was added 2,6-lutidine (42 µL, 0.36 mmol, 1.5 equiv.) followed by a dropwise addition of oxalyl chloride (25 µL, 0.29 mmol, 1.2 equiv). The mixture was stirred at room temperature during 90 min. Separately, ethyl 2-acetylpent-4enoate (80 mg, 0.5 mmol, 2 equiv) was added on a suspension of sodium hydride (23 mg, 0.57 mmol, 2.4 equiv) in 1 mL of THF. This solution was cannulated on the solution of C.After overnight at room temperature, the reaction was quenched by a saturated aqueous solution of NH 4 Cl and extracted with Et 2 O. The organic layer was washed with a solution of HCl 0.1 N, then with brine, dried over MgSO 4 and concentrated under vacuum. The crude product was purified by flash chromatography on silica (CH 2 Cl 2 /Cyclohexane 1:1) to yield 90 mg (65%) of 5 as a 50/50 diastereomeric mixture.

  Et). γ-Oxo-ester A (80 mg, 0.18 mmol, 1 equiv) was dissolved in EtOH (5 mL) and THF (2 mL). The solution is canulated over NaBH 4 (14 mg, 0.57 mmol, 3 equiv) in 2 mL EtOH. The mixture was stirred at room temperature. The reaction progressed slowly.After 15 h (overnight) starting material has disappeared but ester was partially reducted. The reaction mixture was then purified by preparative TLC (AcOEt/cyclohexane 2:3) to yield 20 of alcohol 2 (R = Et) as two isolated diastereoisomers (75/25). Second product is the diol in mixture with degradation.1 H NMR (400 MHz, CDCl 3 , major diast.): δ (ppm) 7.04

  Cell culture and cell proliferation assay. The breast adenocarcinoma cell lines MDA-MB-231 and MCF-7 were obtained respectively from ATCC and Dr Matthias Kassack (Bonn, Germany). Cells were grown in RPMI medium supplemented with 10% fetal calf serum, in the presence of penicilline, streptomycine and fungizone in 75cm² flask under 5% CO2. Cells were plated in 96-well tissue culture plates in 200 µl medium and treated 24h later with 2 µl stock solution of compounds dissolved in DMSO using a Biomek 3000 (Beckman-Coulter). Controls received the same volume of DMSO (1% final volume). After 72 h exposure, MTS reagent (Promega) was added and incubated for 3h at 37°C: the absorbance was monitored at 490 nm and results expressed as the inhibition of cell proliferation calculated as the ratio [(1-(OD490 treated/OD490 control))×100] in triplicate experiments. For IC50 determination [50% inhibition of cell proliferation], cells were incubated for 72 h following the same protocol with compound concentrations ranged 5 nM to 100µM in separate duplicate experiments.

Table

Table 1 .

 1 ICPodophyllotoxin was found very active against both cancer cell lines (IC 50 0.01 µM) proving its high cytotoxicity. By incorporating a ferrocenyl moiety into the molecule, the cytotoxicity is reduced; the IC 50 values of OFCP 1 were 0.93 µM for MCF-7 and 0.43 µM for MDA-MB-231 which is in the average values generally found for ferrocifens. These values are still satisfactory and may justify further studies. The reduced cytotoxicity of 1 as compared to the organic precursor is reminiscent to the result obtained with ferrocenyl modification of the extremely toxic illudin[START_REF] Knauer | [END_REF]. An organometallic modification decreases its toxicity on fibroblasts and increases its selectivity on cancer cells. Compound 1 could be easier to use than podophyllotoxin. By contrast to 1, the alcohol 2 shows high IC 50 proving that 2 is much less active than 1. It is interesting to note that the organic moiety of 2 is different from podophyllotoxin molecule.

		50 values of compounds on the
	hormone-dependent (MCF-7) and hormone-
	independent (MDA-MB-231) breast cancer cell
	lines
	Compound	MCF-7	MDA-MB-231
		(IC 50 µM)*	(IC 50 µM)*
	Podophyllotoxin	0.01 ± 0.00	0.01 ± 0.00
	OFCP 1	0.93 ± 0.07	0.43 ± 0.03
	"Alcohol 2 (R = Et)" 39.75 ± 2.00 27.6 ± 2.40
	*Mean of two separate experiments

).

[START_REF]The intermediates of the synthesis were not evaluated in the biological essay[END_REF]

  1 H and 13 C) were recorded on a Bruker AVANCE 300, 400 or 600 MHZ spectrometer.1 H frequency of 300, 400 or 600 MHz, respectively; 13 C frequency of 75, 101 or 151 MHz. Chemical shifts (δ) are given in ppm CDCl 3 residual chloroform signal as reference. IR spectra were recorded with a Tensor 27 (ATR diamond) Bruker spectrometer. To a suspension of ferrocenylcarboxylic acid (60 mg, 0.26 mmol, 3 equiv) in 1 mL of dry CH 2 Cl 2 was added at room temperature oxalyl chloride (225 µL, 2.65 mmol, 27 equiv). After 30 min at room temperature, the solution took a deep red color. The mixture was concentrated in vaccuo to remove excess oxalyl chloride.

	ACCEPTED MANUSCRIPT
	6.44 (s, 2H), 6.04-5.97 (m, 3H), 4.85 (dt, J = 2.5, 1.3 Hz, 1H), 4.81 (dt, J = 2.5, 1.3 Hz, 1H),
	4.64 (d, J = 4.3 Hz, 1H) 4.50-4.44 (m, 3H), 4.30 (m, 1H), 4.25 (s, 5H), 3.81 (s, 3H), 3.80 (s,
	6H), 3.02-2.87 (m, 2H). 13 C NMR (101 MHz, CDCl 3 ): δ (ppm) 173.
	M A N U S C R I P T
	Podophyllotoxin (38 mg, 90 µmol, 1 equiv) was solubilizedequiv in 1.5 mL of dry CH 2 Cl 2 and Et 3 N (15 µL, 0.11 mmol, 1.2 equiv) was added. To this mixture was added at 0 °C A C C E P T E D
	ferrocenoyl chloride in 2 mL of CH 2 Cl 2 . Then a few crystals of DMAP were added and the
	mixture was stirred at 0 °C for 30 min and at room temperature for 1 h. Reaction was
	quenched by addition of water, extracted with CH 2 Cl 2 (3x), washed with diluted HCl (~0.1
	12

Only the most important bands were reported, in wavenumbers ῡ (cm -1 ). NMR and IR spectroscopy experiments were performed at 300 K. HRMS spectra were recorded at the Institut Parisien de Chimie Moléculaire (FR 2769) of UPMC (electrospray source).

Cytotoxicity measurements on MCF-7 and MDA-MB-231 breast cancer cells were performed ImaGIF Ciblothèque Cellulaire (Institut de Chimie des Substances Naturelles).

Synthesis and analytical data of some intermediates (3' and 3'') and by-products (C' and 7) are described only in the SI.

O-Ferrocenylpodophyllotoxin 1.

N), dried over MgSO 4 and concentrated under vacuum. The crude product was purified by preparative TLC on silica (AcOEt/Cyclohexane 2/3) to yield 35 mg of the desired compound as an orange powder (60%).

1

H NMR (400 MHz, CDCl 3 ): δ (ppm) 6.90 (s, 1H),

6.58 (s, 1H),

  To a solution of Pd(OAc) 2(28.0 mg, 0.12 mmol, 10 mol-%), dppe (100.3 mg, 0.25 mmol, 20 mol-%) and K 2 CO 3 (345.3 mg, 0.24 mmol, 2 equiv) in 2.3 mL of DMF was added 3 (706.2 mg, 1.25 mmol, 1 equiv) in 16.3 mL of DMF and the mixture was heated at 130 °C for 18 h. The reaction was quenched by a saturated aqueous solution of NH 4 Cl and extracted with Et 2 O. The organic layer was washed intensively with diluted HCl (0.1 N), then with brine, dried over MgSO 4 and concentrated under vacuum. The crude product was

	ACCEPTED MANUSCRIPT ACCEPTED MANUSCRIPT
	diastereomeric mixture) δ (ppm) 206.6 (diast. 1), 204.6 (diast. 2), 171.9 (diast. 1), 171.7 45.6 (minor diast.), 45.1 (major diast.), 32.0 (minor diast.), 31.7 (major diast.), 26.3 (minor
	(diast. 2), 147.4, 147.3, 147.2, 147.1 (diast. 1 and 2), 133.8 (diast. 1), 133.1 (diast. 2), 132.8 diast.), 26.2 (major diast.), 14.1 (major diast.), 13.9 (minor diast.). IR (neat, cm -1 ): 3087,
	(diast. 1), 132.2 (diast. 2), 118.8 (diast. 1), 118.0 (diast. 2), 117.5 (diast. 1 and 2), 112.8 (diast. 1737, 1713, 1503, 1480. Exact mass (C 27 H 26 FeO 5 ): calculated 509.1022 (M+Na) + , measured
	1 and 2), 110.1 (diast. 1), 101.0 (diast. 2), 102.1 (diast. 1), 102.0 (diast. 2), 88.1 (diast. 1), 509.1018.
	87.9 (diast. 2), 71.4, 69.6, 69.0, 68.8, 68.4, 68.1, 67.7, 67.3 (diast. 1 and 2), 69.6 (diast. 1),
	69.5 (diast. 2), 69.0 (diast. 1), 68.8 (diast. 2), 61.4 (diast. 1), 61.3 (diast. 2), 47.9 (diast. 1), Ethyl 8-methylene-5-pherrocenyl-5,6,7,8-tetrahydronaphtho[2,3-d][1,3]dioxole-6-
	M A N U S C R I P T 47.2 (diast. 2), 39.5 (diast. 1), 39.3 (diast. 2), 31.7 (diast. 1), 30.3 (diast. 2), 14.2 (diast. 1), 13.8 (diast. 2). IR (neat, cm -1 ): 3079, 1730, 1700, 1638, 1503, 1478. Exact mass (C 27 H 27 BrFeO 5 ): calculated 589.0285 (M+Na) + , measured 589.0301. Ethyl 6-acetyl-8-methylene-5-pherrocenyl-5,6,7,8-tetrahydronaphtho[2,3-d][1,3]dioxole-6-carboxylate 8. purified by flash chromatography on silica (AcOEt/Cyclohexane 15/85) to yield 508.7 mg carboxylate 9. To a solution of sodium ethylate formed by reacting Na (30 mg, 1.3 mmol, 1.5 equiv) in 5 mL of absolute EtOH was added 8 (430 mg, 0.885 mmol, 1 equiv) in 5 mL THF. The mixture was brought to reflux for 3 h, then the reaction was quenched by addition of HCl 0.2 N and extracted with Et 2 O. The organic layer was washed with brine, dried over MgSO 4 and concentrated under vacuum. The crude product was purified by flash chromatography on silica (AcOEt/Cyclohexane 5/95 -> 10/90) to yield 370 mg (95%) of ester 9 as a 65/35 diastereomeric mixture. 1 H NMR (400 MHz, CDCl 3 , diastereomeric mixture) δ (ppm) 7.17 (s, 1H, minor diast.), 7.08 (s, 1H, major diast.), 6.93 (s, 1H, minor diast.), 6.92 (s, 1H, major diast.), 6.01 (d, J = 1.1 Hz, 1H, minor diast.), 6.00-5.98 (m, 1H, minor diast.), 5.96 (d, J = 1.2 Hz, 1H, major diast.), 5.94 (d, J = 1.1 Hz, 1H, major diast.), 5.44-5.42 (m, 1H, minor diast.), A N U S C R I P T 5.40-5.33 (m, 1H, major diast.), 4.91-4.89 (m, 1H, minor diast.), 4.89-4.85 (m, 1H, major M diast.), 4.36-4.32 (m, 1H, major diast.), 4.30-4.29 (m, 1H, minor diast.), 4.23-3.99 (m, 10H,
	1 H RMN (400 MHz, CDCl 3 , diastereomeric (84%) of 8 as a 60/40 diastereomeric mixture. 1 H NMR (300 MHz, CDCl 3 , diastereomeric A C C E P T E D mixture) δ (ppm) 7.11 (s, 1H, minor diast.), 7.08 (s, 1H, major diast.), 7.03 (s, 1H, major diast.), 7.01 (s, 1H, minor diast.), 6.00 (t, J = 1.2 Hz, 2H, major and minor diast.), 5.96 (t, J = 1.2 Hz, 2H, major and minor diast.), 5.45 (s, 2H, major and minor diast.), 4.66-4.64 (m, 1H, minor diast.), 4.64-4.61 (m, 1H, major diast.), 4.27-3.93 (m, 12H, major and minor diast.), 4.10 (s, 5H, major diast.), 4.08 (s, 5H, minor diast.), 3.46-3.43 (m, 1H, minor diast.), 3.42-3.40 (m, 1H, major diast.), 3.06-3.01 (m, 1H, minor diast.), 3.00-2.95 (m, 1H, major diast.), 2.89-2.87 (m, 1H, major diast.), 2.83-2.81 (m, 1H, minor diast.), 2.15 (s, 3H, minor diast.), 2.10 (s, 3H, major diast.), 1.27 (t, J = 7.1 Hz, 6H, major diast.), 1.04 (t, J = 7.1 Hz, 6H, minor diast.). 13 C NMR (75 MHz, CDCl 3 , diastereomeric mixture) δ (ppm) 204.7 (minor diast.), major and minor diast.), 4.17 (s, 5H, major diast.), 4.12 (s, 5H, minor diast.), 3.67-3.64 (m, 1H, major diast.), 3.47-3.45 (m, 1H, minor diast.), 3.03-2.85 (m, 2H, major and minor diast.), 2.81-2.52 (m, 4H, major and minor diast.), 1.31 (t, J = 7.1 Hz, 3H, minor diast.), 1.16 (t, J = 7.1 Hz, 3H, major diast.). 13 C NMR (101 MHz, CDCl 3 , diastereomeric mixture) δ (ppm) 173.9 (major diast.), 173.3 (minor diast.), 147.4, 147.2, 147.1, 146.9 (major and minor diast.), 140.6 (minor diast.), 139.6 (major diast.), 133.2 (minor diast.), 131.2 (major diast.), 128.4 (minor diast.), 128.1 (major diast.), 109.8 (major and minor diast.), 108.4 (major and minor A C C E P T E D diast.), 103.9 (major and minor diast.), 101.2 (minor diast.), 101.1 (major diast.), 93.
	mixture) δ (ppm) 7.13 (s, 1H, diast. 1), 7.11 (s, 1H, diast. 2), 7.06 (s, 1H, diast. 1), 6.85 (s, 202.9 (major diast.), 170.0 (minor diast.), 169.8 (major diast.), 147.6, 147.5, 147.3, 147.2
	1H, diast. 2), 6.05-6.04 (m, 2H, diast. 1), 6.02-6.01 (m, 2H, diast. 2), 5.65-5.39 (m, 2H, diast. (major and minor diast.), 138.8 (minor diast.), 138.5 (major diast.), 132.1 (minor diast.), 131.6
	1 and 2), 5.37 (s, 1H, diast. 1), 5.35 (s, 1H, diast. 2), 4.97-4.86 (m, 4H, diast. 1 and 2), 4.39-(major diast.), 127.5 (major diast.), 127.4 (minor diast.) 110.5 (major diast.), 110.4 (minor
	diast.), 109.4 (major diast.), 109.2 (minor diast.), 103.4 (major diast.), 103.3 (minor diast.),
	101.3 (major diast.), 101.2 (minor diast.), 90.2, 89.3, 69.4, 69.3, 69.0, 67.7, 67.0 (major and
	minor diast.), 66.3 (major diast.), 65.6 (minor diast.), 61.7 (major diast.), 61.6 (minor diast.),
	14 15

3.83 (m, 12H, diast. 1 and 2), 3.94 (s, 5H, diast. 1)

, 3.92 (s, 5H, diast. 2), 2.57-2.44 (m, 2H, diast. 1), 2.42-2.31 (m, 2H, diast. 2), 2.03 (s, 3H, diast. 1), 1.76 (s, 3H, diast. 2), 1.26 (t, J = 7.2 Hz, 3H, diast. 1), 1.10 (t, J = 7.2 Hz, 3H, diast. 2). 13 C NMR (101 MHz, CDCl 3 ,

  added N-methylmorpholine oxide monohydrate (370 mg, 2.74, 2.2 equiv) and OsCl 3 (40 mg, 0.12 mmol, 10 mol-%). The mixture was stirred at room temperature for 20 h, quenched with a 50 % aqueous solution of NaHSO 3 , and extracted with AcOEt. The organic phase was washed with brine, dried over MgSO 4 and concentrated under vacuum. The crude product was solubilised in 30 mL of a 2:1 mixture of acetone and water and NaIO 4 (630 mg, 2.95 mmol, 2.4 equiv) was added. After 3 h at room temperature, the reaction mixture was quenched with a saturated aqueous solution of Na 2 S 2 O 3 , and extracted with Et 2 O. The organic phase was washed with brine, dried over MgSO 4 and concentrated under vacuum. The crude

	ACCEPTED MANUSCRIPT
	mL) was product was purified by flash chromatography on silica (AcOEt/Cyclohexane 5/95) to yield 330 mg (60%) of A as a 65/35 diastereomeric mixture. 1 H NMR (300 MHz, CDCl 3 , diastereomeric mixture) δ 7.49 (s, 1H, minor diast.), 7.47 (s, 1H, major diast.), 7.04 (s, 1H, minor diast.), 6.95 (s, 1H, major diast.), 6.10 (d, J = 1.2 Hz, 1H, minor diast.), 6.08-6.07 (m, 2H, major and minor diast.), 6.04 (d, J = 1.3 Hz, 1H, major diast.), 4.50-4.47 (m, 2H, major and minor diast.), 4.28-4.00 (m, 21H , major and minor diast.), 3.47-3.43 (m, 1H, major diast.), A N U S C R I P T M CDCl 3 , diastereomeric mixture) δ (ppm) 195.5 (minor diast.), 194.0 (major diast.), 172.8
	minor diast.), 60.7 (major diast.), 60.6 (minor diast.), 47.2 (major diast., 45.7 (minor diast.), 42.9) (minor diast., 41.2 (major diast.), 171.9 (minor diast.), 152.2 (major and minor diast.), 147.8 (major and minor diast.), 141.5 (minor diast.), 139.2 (major diast.), 127.6 (minor diast.), 127.3 (major diast.), A C C E P T E D 109.3 (major diast.), 109.2 (minor diast.), 106.5 (minor diast.), 106.4 (major diast.), 102.1
	(major diast.), 30.2 (major diast.), 28.4 (minor diast.), 14.4 (minor diast.), 14.3 (major diast.).
	IR (neat, cm -1 ): 3085, 1729, 1503, 1480. Exact mass (C 25 H 24 FeO 4 ): calculated 467.0916
	(M+Na) + , measured 467.0931.
	16

Ethyl 8-oxo-5-pherrocenyl-5,6,7,8-tetrahydronaphtho

[2,3-d]

[1,3]dioxole-6-carboxylate A. To a solution of ester 9 (550 mg, 1.24 mmol, 1 equiv) in a 9:1 mixture of THF/H 2 O (30 3.34-3.26 (m, 1H, minor diast.), 3.14 (dt, J = 5.1, 2.5 Hz, 1H, major diast.), 2.84-2.80 (m, 1H, minor diast.), 2.76-2.75 (m, 1H, major diast.), 2.64-2.43 (m, 2H, major and minor diast.), 1.29 (t, J = 7.2 Hz, 3H, minor diast.) 1.11 (t, J = 7.1 Hz, 3H, major diast.).

13

C NMR (101 MHz,

  1 H NMR (400 MHz, CDCl 3 , minor diast.): δ (ppm) 6.96 (s, 1H), 6.88 (s, 1H), 5.93 (d, 1H, J = 2.0 Hz), 4.79-4.70 (m, 1H), 4.27 (d, 1H, J = 4.0 Hz), 4.23-4.06 (m, 10H), 3.81 (s, 1H), 3.49 (s, 1H), 3.18-3.11 (m, 1H), 2.30-2.21 (m, 1H), 1.94 (dd, J = 8.2, 4.5 Hz, 1H), 1.62-1.50 (m, 1H), 1.25 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, CDCl 3 , minor diast.): δ (ppm) 174.

  . IR (neat, cm -1 ): 3519, 1716, 1480, 1226. Exact mass (C 24 H 24 FeO 5 ): calculated 448.0968 (M+Na) + , measured 448.0979.

Ethyl 8-hydroxy-5-pherrocenyl-5,6,7,8-tetrahydronaphtho[2,3-d][1,3]dioxole-6-carboxylate 2 (R = Et) Major diastereoisomer

Ethyl 8-hydroxy-5-pherrocenyl-5,6,7,8-tetrahydronaphtho[2,3-d][1,3]dioxole-6-carboxylate 2 (R = Et) minor diastereoisomer

Acknowledgements

We thank Yasmine Reviriot for his contribution in preliminary experiments and Omar Khaled for HRMS analyses. CNRS, UPMC, and Labex Michem are acknowledged for financial

Appendix. Supplementary material

NMR spectra of all new compounds. Supplementary data associated with this article can be found, in the online version, at ... Corresponding authors E-mails: julie.oble@upmc.fr gerard.jaouen@chimie-paristech.fr giovanni.poli@upmc.fr

Synthesis and Analytical data of some intermediates and by-products 6-Bromobenzo[1,3]dioxole-5-carboxylic acid 3'. To a solution containing 1 g (4.36 mmol, 1 equiv) of 6-bromopiperonal 3 and 3 drops of a 1 M H3PO4 solution in 40 mL of acetone at 10 °C was added a solution containing 0.53 g (5.7 mmol, 1.3 equiv) of NaClO2 in 100 mL of water. The yellow solution was warmed to room temperature, and a 35% H2O2 solution was added dropwise until the mixture became colorless. The solution was acidified to pH 1 at 0 °C, and the resulting solid was filtered, washed with cold water, and dried to give 0.91 g (85%) of 6-bromo-1,3-benzodioxole-5-carboxylic acid as a white solid. 1 H NMR (400 MHz, acetone-d 6 ): δ (ppm) 7.50 (s, 1H), 7.14 (s, 1H), 6.08 (s, 2H).

These data are in accordance with those reported in the literature. [ Erreur ! Signet non défini. ] (6-Bromobenzo[1,3]dioxol-5-yl)(ferrocenyl)methanone 3''. To a suspension of 6-bromo-1,3benzodioxole-5-carboxylic acid 3' (505.7 mg, 2.06 mmol, 1 equiv) in 5 mL of CH2Cl2 was added oxalyl chloride (0.40 mL, 5.61 mmol, 2.75 equiv). The mixture was stirred at room temperature for 16 h, and then concentrated in vaccuo to remove excess of oxalyl chloride. The residue was dissolved in 5 mL of CH2Cl2 and added dropwise on a solution of ferrocene (398.8 mg, 2.14 mmol, 1 equiv) and AlCl3 (325.8 mg, 2.45 mmol, 2 equiv). After 2 h at room temperature, the solution was quenched by a saturated aqueous solution of NH4Cl and extracted with CH2Cl2. The organic layer was washed with brine, dried over MgSO4 and concentrated under vacuum. The crude product was purified by flash chromatography on silica (AcOEt/cyclohexane 1:9) to yield 203.6 mg (38%) of 3''. 1 H NMR (400 MHz, CDCl3) δ (ppm) 7.07 (s, 1H), 7.00 (s, 1H), 6.06 (s, 2H), 4.75 (t, J = 2.0 Hz, 2H), 4.58 (t, J = 2.0 Hz, 2H), 4.28 (s, 5H). 13 C NMR (101 MHz, CDCl3) δ (ppm) 198. 8, 149.5, 146.9, 134.7, 113.7, 111.5, 109.1, 102.4, 78.5, 73.0, 71.4, 70.3. IR (neat, cm -1 ): 1637, 1489, 1445, 1249. Exact mass (C18H13O3FeBr): calculated 434.9303 (M+Na) + , measured 434.9291.

Benzo [1,3]dioxol-5-yl(pherrocenyl)methanol C'. 1 H NMR (300 MHz, CDCl3) δ (ppm) 1H),1H),6.78 (m,1H),5.96 (s,2H),5.43 (d,J = 2.4 Hz,1H),9H), 2.46 (d, J = 2.8 Hz, 1H). 13 C NMR (101 MHz, CDCl3) δ (ppm) 147. 7, 147.0, 137.7, 119.8, 108.1, 107.1, 101.1, 94.5, 72.1, 69.0, 68.94, 68.7, 68.4, 68.3, 67.6, 65.9. IR (neat, cm -1 ): 3565, 3095, 2925, 1632, 1486. Exact mass (C18H16O3Fe): calculated 336.0443 (M) + , measured 336.0441. Starting from the alcohol C, first essay: To a solution of 4 (500 mg, 1.2 mmol, 1 equiv) in 10 mL of CH2Cl2 were added the allylated β-keto-ester 6 (306 mg, 1.8 mmol, 1.5 equiv) and TsOH (5 mol%).

The mixture was stirred at reflux during 16 hours, quenched by a saturated aqueous solution of NaHCO3 and extracted with CH2Cl2. The organic layer was washed with brine, dried over MgSO4 and concentrated under vacuum. The crude product was purified by flash chromatography on silica (CH2Cl2/Cyclohexane 6:4) to yield 238 mg (35%) of 5 as a 50/50 diastereomeric mixture, and 487 mg (50%) of the dimerized ether adduct 7.

Starting from the alcohol C, second essay (Mitsunobu reaction): To a solution of C (200 mg, 0.48 mmol, 1 equiv) in 4 mL of THF were added 1,1'-(azodicarbonyl)dipiperidine (ADDP) (600 mg, 2.4 mmol, 5 equiv) and PBu3 (0.6 mL, 2.4 mmol, 5 equiv). The mixture was stirred at room temperature during 5 min, and the allylated β-keto-ester 6 (410 mg, 2.4 mmol, 5 equiv) was added dropwise. The mixture was stirred at room temperature during 3 days, quenched by a saturated aqueous solution of NH4Cl and extracted with Et2O. The organic layer was washed with brine, dried over MgSO4 and concentrated under vacuum. The crude product was purified by flash chromatography on silica (CH2Cl2/Cyclohexane 1:1) to yield 94 mg (35%) of 5 as a 50/50 diastereomeric mixture.

Starting from the alcohol C, third essay (via chloride intermediate): To a solution of C (100 mg, 0.24 mmol, 1 equiv) in 1 mL of THF, was added 2,6-lutidine (42 μL, 0.36 mmol, 1.5 equiv.) followed by a dropwise addition of oxalyl chloride (25 μL, 0.29 mmol, 1.2 equiv). The mixture was stirred at room temperature during 90 min. Separately, ethyl 2-acetylpent-4-enoate (80 mg, 0.5 mmol, 2 equiv) was added on a suspension of sodium hydride (23 mg, 0.57 mmol, 2.4 equiv) in 1 mL of THF. This solution was cannulated on the solution of C. After overnight at room temperature, the reaction was quenched by a saturated aqueous solution of NH4Cl and extracted with Et2O. The organic layer was washed with a solution of HCl 0.1 N, then with brine, dried over MgSO 4 and concentrated under vacuum. The crude product was purified by flash chromatography on silica (CH2Cl2/Cyclohexane 1:1) to yield 90 mg (65%) of 5 as a 50/50 diastereomeric mixture. 1 H RMN (400 MHz, CDCl3, diastereomeric mixture) δ (ppm) 7.13 (s, 1H, diast. 1), 7.11 (s, 1H, diast. 2), 7.06 (s, 1H, diast. 1), 6.85 (s, 1H, diast. 2), 6.05-6.04 (m, 2H, diast. 1), 6.02-6.01 (m, 2H, diast. 2), 5.65-5.39 (m, 2H, diast. 1 and 2), 5.37 (s, 1H, diast. 1), 5.35 (s, 1H, diast. 2), 4.97-4.86 (m, 4H, diast. 1 and 2), 4.39-3.83 (m, 12H, diast. 1 and 2), 3.94 (s, 5H, diast. 1), 3.92 (s, 5H, diast. 2), 2.57-2.44 (m, 2H, diast. 1), 2.42-2.31 (m, 2H, diast. 2), 2.03 (s, 3H, diast. 1), 1.76 (s, 3H, diast. 2), 1.26 (t, J = 7.2 Hz, 3H, diast. 1), 1.10 (t, J = 7.2 Hz, 3H, diast. 2). 13 C NMR (101 MHz, CDCl3, diastereomeric mixture) δ (ppm) 206. 6 (diast. 1), 204.6 (diast. 2), 171.9 (diast. 1), 171.7 (diast. 2), 147.4, 147.3, 147.2, 147.1 (diast. 1 and 2), 133.8 1), 133.1 (diast. 2), 132.8 (diast. 1), 132.2 (diast. 2), 118.8 (diast. 1), 118.0 (diast. 2), 117.5 (diast. 1 and 2), 112.8 (diast. 1 and 2), 110.1 (diast. 1), 101.0 (diast. 2), 102.1 (diast. 1), 102.0 (diast.

2), 88. 1 (diast. 1), 87.9 (diast. 2), 71.4, 69.6, 69.0, 68.8, 68.4, 68.1, 67.7, 67.3 (diast. 1 and 2), 69.6 (diast. 1), 69.5 (diast. 2), 69.0 (diast. 1), 68.8 (diast. 2), 61.4 (diast. 1), 61.3 (diast. 2), 47.9 (diast. 1), 47.2 (diast. 2), 39.5 (diast. 1), 39.3 (diast. 2), 31.7 (diast. 1), 30.3 (diast. 2), 14.2 (diast. 1), 13.8 (diast. 2). IR (neat, cm -1 ): 3079, 1730, 1700, 1638, 1503, 1478. Exact mass (C27H27BrFeO5) 1, 147. 9, 135.1, 114.5, 112.2, 108.6, 102.0, 90.7, 75.8, 69.0, 68.0, 67.8, 67.1, 66.2