Pascal Pigeon

Elizabeth A Hillard

Anne Vessières

Michel Huché

Marie-Aude Richard

Michael J Mcglinchey

Siden Top
email: siden-top@chimie-paristech.fr.

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

Synthesis and Antiproliferative Effects of [3]Ferrocenophane Transposition Products and Pinacols Obtained from McMurry Cross-Coupling Reaction

) crystallize in monoclinic P2 1 /n, triclinic P , and monoclinic P2 1 /c space groups, respectively. The antiproliferative effects on hormoneindependent breast cancer cells (MDA-MB-231) of the transposition compounds are generally lower than those of their corresponding butene analogues (IC 50 values in micromolar versus nanomolar range). In contrast and quite surprisingly, the pinacol complexes are significantly cytotoxic (IC 50 in the nanomolar range), among the most cytotoxic ferrocene compounds prepared so far. This antiproliferative activity may be linked to their oxidative cleavage.

Introduction

Within the recent research activity in bioinorganic chemistry, the topic of medicinal organometallic chemistry is now a particularly fertile field. (1)(2)(3)(4)(5)(6) This domain can be defined as the synthesis and evaluation of organometallic compounds for diagnostic and therapeutic applications, as well as investigation into their mode of action. [START_REF]Medicinal Organometallic Chemistry[END_REF] We have recently shown that, in certain favorable cases, the biological properties of organic polyphenols can be substantially modified by substitution of a ferrocenyl entity in place of an aryl group on the molecular skeleton. The organic diphenol 1, ferrocenyl diphenol 2a, resveratrol 3, hydroxytamoxifen 4, and ferrocifen 5 (Chart 1) are all known to interact with the estrogen receptors (ER) present in MCF-7 cells. This leads, at low concentration, to an estrogenic (proliferative) effect for 1, 2a, and 3 and to an antiestrogenic (antiproliferative) effect for 4 and 5 due to specific interactions of the hormone-receptor complex with DNA.

Interestingly, the ferrocenyl derivatives 2a and 5 produce a powerful antiproliferative effect on MDA-MB-231 cells, which lack the ER (IC 50 ou d 0.5 μ ). I d d, o this c i 3

and 4 show only a weak antiproliferative effect (IC 50 v u s of 20 d 30 μ , respectively), rendering them not viable at the therapeutic level. (10,11) We have therefore worked to adjust these levels via organometallic modification, and following a suitable formulation of 2a, encouraging in vivo results have been obtained on implanted tumors in rat (9L gliosarcoma). ( 12)

Chart 1. Tamoxifen and Related Molecules

It seems likely that the presence of the redox-active ferrocene group favors the generation of quinone methides in cancer cells, possibly leading to cell death via interaction with nucleophiles such as glutathione and proteins. (13,14) In addition to electrochemical experiments, this hypothesis has been supported by the observation that the [ferroceneconjugated spacer-p-phenol] motif seems to be crucial for strong antiproliferative effects, and a change in the position of either the hydroxyl group or the ferrocene results in weakening of the antiproliferative effect. (15,16) This behavior, first observed with MDA-MB-231 cells, pertains to other types of cancer cells as well (prostate, colon, glioma, and melanoma, inter i ). I po t t y, h thy c s ot ff ct d t th s co c t tio s ( μ ). [START_REF]Medicinal Organometallic Chemistry[END_REF][START_REF] Vessières | [END_REF](9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) These promising results were further elaborated through discovery of new structures with even greater efficacy in vitro and by a successful transition in vivo. We have already obtained some success in vitro by changing the substituent (e.g., OH for NH 2 ) in series 2 (21,22) and in formulating 2a for in vivo administration. (18,23,24) Furthermore, a cyclic series (Chart 1), (25)(26)(27)(28) for which diphenol 6a is the archetypal example, is about 2-4 times more active than 2a in the NCI 60 cell line panel, particularly against melanoma, leukemia, CNS cancer, and renal cancer. (28) In performing the reductive cross-coupling (29,30) to obtain series 6 molecules, we have observed that another series of molecules, 7 (Scheme 1), resulting from a pinacolic rearrangement to a transposition product is also accessible and that the yield of this secondary product is influenced by the reaction conditions. Although the transposition geometry, with a tetrahedral carbon core, does not apparently lend itself to the ferrocene-mediated formation of quinone methides, thought to be the key to the antiproliferative effects of 2 and 6, the antiproliferative activities of 7a-d are nonetheless in the low micromolar range. We therefore became interested in the competitive synthesis of 6 and 7, as well as the examination of the

Results and Discussion

Synthesis

The McMurry cross-coupling reaction between [3]-ferrocenophan-1-one (31) and a disubstituted benzophenone (Scheme 1) yielded the olefin 6 and/or the transposition product 7, depending on the reaction conditions (Table 1).

It is commonly accepted that the pinacolate 9 is a reactive intermediate in the McMurry reaction. (29,30,32) Depending on the nature of the reactants and the reaction conditions, the pinacolate can evolve toward the classic cross-coupling product 6 or toward other products, principally the transposition product 7. (33,34) For example, at THF reflux, we quantitatively obtained the olefin 6c, while at 0 °C, only the transposition product 7c was found after acid workup. This result is similar for the reaction with 4-hydroxybenzophenone, although a small quantity of the transposition product 7b was also isolated at reflux. This suggests that the pinocolates 9b,c quit st b t 0 °C. With 4,4′-dihydroxybenzophenone, the results were not quite as straightforward: at both reflux and 0 °C we obtained a mixture of the olefin 6a and the transposition product 7a, although the ratio of 7a to 6a was improved at lower temperatures. This indicates that the pinacolate 9a is less stable than that of 9c, and the low yields of 6a and 7a suggest that the pinocolate likely evolves toward other unidentified p oducts. (35) Low i g th t p tu to -25 °C fu th st bi iz d 9a and increased the yield of 7a to 33%. In the case of the diacetamidobenzophenone, we primarily obtained the olefin 6d under reflux conditions, but at 0 °C no reaction was observed. This is not surprising, as it is known that McMurry coupling with aminobenzophenones proceeds relatively slowly. (36) It was recently found that the addition of catechol to the reaction medium favors the production of the transposition compound, (37,38) by chelating the titanium ion and thus st bi izi g th pi co t . I d d, i th p s c of c t cho , th ctio of 4,4′diacetamidobenzophenone at reflux permitted the isolation of 7d in 33% yield and further deprotection of 7d by HCl gave access to the diamino compound 7e. For the other benzophenones, the addition of catechol blocked olefin formation but did not consistently increase the yield of the transposition product.

As seen in Table 1, the fate of the pinacolate is very sensitive to the reaction temperature, and at 0 °C, evolution toward the olefin or transposition product is slow enough that hydrolysis at this temperature allowed us to isolate the pinacols 8b,c in good yields (80% and 82%, respectively). (35,39) Compound 8b exists as two diastereomers, 8b1 and 8b2, which are separable by HPLC. Treatment of the isolated pinacols 8b,c with acid generates the transposition products 7b,c in yields of 98% and 79%, respectively.

It is interesting to note that the reaction of [3]ferrocenophan-1-one in the absence of a benzophenone led to the pinacol 12 in 53% yield. Only a small amount of the corresponding olefin 10 or transposition product 11 (Scheme 2) was obtained after H 2 O hydrolysis, suggesting that the pinacolate of [3]ferrocenophan-1-one is stable even under reflux conditions.

Scheme 2. Homocoupling of [3]Ferrocenophan-1-one

In order to study the influence of the aromatic rings on the generation of such compounds, we examined the reaction between [3]ferrocenophan-1-one and 4-hydroxybenzaldehyde at 0 °C (Scheme 3). Acid hydrolysis gave the transposition products 13 and 14, as well as unreacted starting material.

Scheme 3. Cross-Coupling Reaction between [3]Ferrocenophan-1-one and 4-

Hydroxybenzaldehyde

The mechanism of transformation from the pinacolate to the transposition product is thought to involve the in situ formation of a carbocation by protonation and subsequent elimination of H 2 O, followed by migration of a substituent toward the carbon atom bearing the positive charge. (40) A variant of this mechanism involves the simultaneous transfer of two electrons and substituent migration. (38) It is interesting to note that all of the benzophenones give only one type of transposition product, while the reaction with the hydroxybenzaldehyde yields two isomers, 13 and 14, resulting from the existence of two possibilities for substituent migration (Scheme 4).

Scheme 4. Pinacolic Rearrangement

The identity of the final product strongly depends on where the initial carbocation is formed.

It is known that the ferrocenyl substituent stabilizes carbocations better than does a phenyl A though st bi iz tio of th c tio oc t d β to th f oc y u it h s b observed in cyclic systems, this occurs via a through-space interaction when the cation is on the bridging c bo . (45,46) This is ot th c s h ; i st d, th β-carbon is positioned far from the ferrocenyl moiety. However, the rigidity of the bridge may impair a through-space stabilization of cation A with the iron atom, which is an important contribution to the stability of the cation [FcCH 2 ] + . Thus, the destabilization of cation A relative to cation B could be attributable to the lack of a stabilizing co t ibutio of th i o to to th α positio . I d d, the destabilization of the cation in the [3]ferrocenophane series compared to the ferrocene series is reflected by its pK R + v u of -3.2 fo α c tio of [3] The preferential migration of the cobalt sandwich moiety rather than a methyl group is rationalized in terms of the enhanced migratory aptitude of the organometallic moiety in such a way that the positive charge can be delocalized onto the metal center in the transition state.

The ferrocenyl moiety should likewise possess this migration capacity. In agreement with these observations, quantum mechanical semiempirical PM3 calculations show that, in the case of the diphenol compound 7a, th v i tio of th th py ctiv tio gy (ΔH°*) necessary for the transformation of cation A to pinacolone C is 95.9 kcal mol -1 , while that for the transformation of cation B to pinacolone D is 26.3 kcal mol -1 . Therefore, the transformation of B to D is the more favorable process.

Scheme 5. Proposed Rearrangement Mechanism for a Cobalt Sandwich Complex

The migration of the ferrocenyl group instead of the aryl group may also be favored due to an additional contribution of strain release by cycle expansion. This would explain the exclusive formation of the D type compound. In the case of 4-OH-benzaldehyde, the contribution to the stability of the cation comes only from one aromatic ring, and thus the role of these effects on the stability of cations A and B should be similar. However, compound 13, the ring expansion product, is the major product in this reaction.

X-ray Structures

Suitable crystals of 7b,c and 12 were obtained from dichloromethane/hexane or ether. ORTEP diagrams are shown in Figure 1, crystallographic data are given in Table S1 in the Supporting Information, and selected bond distances and bond angles are collected in Table 2.

C Cp -C Cp (Fe1) 1.414(3)-1.434(2) C11-C12 1.562(5) 1.568(2) C Cp -C Cp (Fe2) 1.416(2)-1.434(2) C11-C21 1.549(5) 1.554(2) C11-C26 1.576(2) C12-C13 1.518(7) 1.530(2) C11-O1 1.4291(19) O1-C12 1.231(5) 1.222(2) C26-O2 1.4371(19) C6-C14 1.508(7) 1.520(3) C1-C11 1.515(2) O2-C24 1.371(5) C11-C12 1.543(2) C13-C14 1.531(6) 1.544(3) C12-C13 1.532(2) C13-C6 1.496 (2)

Antiproliferative Activities

The antiproliferative effects of all the new compounds against hormone-independent MDA-MB-231 breast cancer cells were evaluated (Table 3). inactive. However, compound 14, which also possesses one hydroxyphenyl group has activity comparable to that of 7b, evidently due to the presence of an aldehyde group.

Most remarkably, the pinacols 8 are all very active, and the isomer 8b1 has the best activity with an IC 50 value of 0.06 μM. This is one of the most antiproliferative compounds prepared to date in our laboratory. In this series, it is clear that the phenol group does not play an important role, as 8c, which lacks this function, is one of the most active compounds with an IC 50 value of 0.17 μM. Finally, the pinacol 12 has only moderate activity, which may be a consequence of the low accessibility to the OH group that is sterically blocked by bulky neighboring groups.

Reactivity Studies

We next turned our attention to possible chemical transformations that could be involved in the toxicity of these molecules. An interesting observation was made from the mass spectrum of 7a (Supporting Information). In chemical ionization mode, using NH 3 as a vector gas, the

Scheme 6. Mass Fragmentation of 7a

We have also examined the fragmentation observed in mass spectrometry by a theoretical approach. The potential energy curves for the displacement of one phenol group of 7aH + and 7bH + (protonated ketones) and for the displacement of one aniline group of 7eH + were calculated using semiempirical PM3 methods. The calculations were carried out by starting to protonate the oxygen atom, and then the phenyl bond to be broken was gradually lengthened from 1.517 to 5.517 Å (in 81 steps). Figure 2 shows the calculated curve of energy versus bond distance for 7aH + . In the case of pinacols 8b,c their activities cannot be obviously linked with quinone methide formation, as the presence of a phenol group is not a major factor in their activity. These pinacols easily evolve to transposition products in acidic medium. As transposition products have considerably lower activity than the pinacols themselves, it is clear that the transposition compounds are not their active metabolites. One might postulate that the activity of pinacols is linked to the reaction of the cation intermediate generated with any nucleophile present in the medium. We have investigated this reaction by using methanol as a nucleophile and have

shown that this type of reaction is instantaneous. (48) When concentrated H 2 SO 4 was added to the solution of 8c in methanol (Scheme 7), only the formation of 7c was observed with no trace of the methoxy compound 15, indicating that the pinacolic rearrangement is faster than the reaction of methanol and suggesting that migration and loss of water are synchronous processes. When HBF 4 •Et We suggest that this excellent activity is probably linked to the oxidation of pinacols. This work will be continued in order to gain insight into the mechanism of action of pinacols.

Experimental Section

General Remarks

All reactions were carried out under an argon atmosphere using standard Schlenk techniques.

General Synthesis for 7a-c, 13, and 14

Zinc powder was suspended in anhydrous THF at room temperature, and titanium tetrachloride was slowly added via a syringe while stirring. The reaction mixture was f ux d fo 2 h d th coo d to 0 o -25 °C (s below), after which a THF solution containing [3]ferrocenophan-1-one and the appropriate benzophenone were added. After the reaction was complete (see below), the reaction mixture was poured into water and extracted with dichloromethane. The organic layer was washed with water, dried over magnesium sulfate, and filtered, and the solvent was evaporated. The oil was purified by HPLC. Recrystallizations were performed from convenient solvent systems.

1,1-Bis(p-acetamidophenyl-2-oxo[4]ferrocenophane (7d):

Zinc powder (0.93 g, 14.2 mmol) was suspended in THF at room temperature, and titanium tetrachloride (1.1 mL, 10.1 mmol) was slowly added via a syringe with stirring. The reaction mixture was heated at reflux for 2 h and then cooled to room temperature. o-Dihydroxybenzene (catechol; 1.12 g, 10.1 mmol) was added, and the reaction mixture was stirred for 20 min at room temperature. The reaction mixture was heated to reflux, after which a THF solution containing [3]ferrocenophan-1-one (0.49 g, 2.03 mmol) and p,p-diacetylaminobenzophenone (0.3 g, 1.01 mmol) (55) were added. The mixture was refluxed for 3 days. The reaction mixture was poured into water, acidified with HCl, and extracted with dichloromethane. The organic layer was washed with water, dried over magnesium sulfate, and filtered, and the solvent was evaporated. The obtained oil was purified by HPLC. Recrystallization was performed in hexane/dichloromethane. Compound 7d was obtained as yellow crystals (0.17 g, 33% yield). Mp: 286 °C dec.

General Synthesis for 8b,c

Zinc powder was suspended in THF at room temperature, and titanium tetrachloride was slowly added via a syringe with stirring. The reaction mixture was heated at reflux for 2 h and then cooled to 0 °C, after which time a THF solution containing [3]ferrocenophan-1-one and the appropriate amount of benzophenone were added. The mixture was stirred at 0 °C for 2 h. The reaction mixture was poured into water and extracted with dichloromethane. The organic layer was washed with water, dried over magnesium sulfate, and filtered, and the solvent was evaporated. The crude product was purified by flash chromatography and/or HPLC to give the pinacol.

Synthesis of 7c from 8c

To a solution of 8c (50 mg, 0.12 mmol) in CH 2 Cl 2 was added concentrated HCl (2.3 mL, 28.8 mmol) portion by portion at 0 °C, and the mixture was stirred for 1 day at room temperature. The reaction mixture was poured into water and extracted with dichloromethane. The organic layer was washed with water, dried over magnesium sulfate, and filtered, and the solvent was evaporated. The crude product was purified by column flash chromatography (petroleum ether) to give 7c (38 mg, 79%).

Synthesis of 7b from 8b:

To a solution of 8b (50 mg, 0.11 mmol) in THF was added concentrated H 2 SO 4 (0.07 mL, 1.32 mmol) portion by portion at 0 °C. Then, the mixture was stirred for 1 h at room temperature. The reaction mixture was poured into water and extracted with dichloromethane. The organic layer was washed with water, dried over magnesium sulfate, and filtered, and the solvent was evaporated. The crude product was purified by column flash chromatography (diethyl ether/petroleum ether), giving 7b (47 mg, 98%). Reaction of Pinacol 8c with Iodomethane: 8c (42 mg, 0.1 mmol) was dissolved in 2 mL of anhydrous THF to which NaH (15 mg, 0.6 mmol) was added. After the mixture was stirred for 30 min, iodomethane (141 mg, 1 mmol) was added and the stirring was maintained for 1 h. The mixture was then poured into water and extracted with diethyl ether. HPLC of the crude product obtained showed only the presence of ferrocenophanone and benzophenone in a 50/50 ratio. e-mail, deposit@ccdc.cam.ac.uk; web, http://www.ccdc.cam.ac.uk).

1-Hydroxy-1-[

Reaction of

Cell Culture and Cell Proliferation Assay

The

importance of pinacols of type 8 (

 8 Scheme 1. General Trends in the Reactivity of the Pinacolate 9 Formed by [3]Ferrocenophan-1-one and Benzophenones in the Presence of Low-Valent Titanium

C1Figure 1 . ORTEP diagrams of 7b,c and 12 .

 112 Figure 1. ORTEP diagrams of 7b,c and 12.

  following ions were detected: m/z 456 [M + NH 4 ] + , 439 [M + H] + , and 345 [M -C 6 H 5 OH] + . The last peak corresponds to the loss of a C 6 H 5 OH group to generate the cation 7a′ (Scheme 6). The same process was observed when NH 3 was replaced by ND 3 . The mass spectrum of the diamine 7e shows this fragmentation pattern as well.

Figure 2 .

 2 Figure 2. Calculated energy versus bond distance curve for 7aH + .

Scheme 7 .Scheme 8 .

 78 Scheme 7. Reactivity of 8c

Anhydrous

  THF was obtained by distillation from sodium/benzophenone. The preparative HPLC separations were performed on a Shimadzu apparatus with a Nucleodur C18 column (length of 25 cm, diameter of 3.2 c d p tic siz of 0 μ ) usi g c to it i s th eluent. The analytical HPLC controls were performed on a Shimadzu apparatus with a Nuc odu C 8 co u ( gth of 5 c , di t of 0.46 c , d p tic siz of 5 μ ) using acetonitrile or methanol/water as the eluent. Infrared spectra were obtained on an IRFT BOMEM Michelson-100 spectrometer equipped with a DTGS detector as a KBr plate. 1 H and 13 C NMR spectra were recorded on a 300 MHz Bruker spectrometer. Mass spectrometry was performed with a Nermag R 10-10C spectrometer. HRMS measurements were performed on a Thermo Fischer LTQ-Orbitrap XL apparatus equipped with an electrospray source by the Institut Parisien de Chimie Moléculaire (UMR 7201), Université Pierre et Marie Curie, Paris, France. Elemental analyses were performed by the microanalysis services of the ICSN (Gif sur Yvette, France). Due to the presence of water or crystallization solvents, compounds 7a,d,e, 8b, and 12 did not provide satisfactory elemental analyses. For these compounds 1 H NMR spectra are given in the Supporting Information as proof of their purity. Melting points were measured with a Kofler device. Determination of the cytotoxicity of the complexes was performed at the IMAGIF (ICSN, Gif sur Yvette, France).

  Pinacol 8c with Hydrogen Peroxide: Compound 8c (21 mg, 0.05 mmol) was dissolved in 1 mL of anhydrous THF. A 0.2 mL portion of hydrogen peroxide (30 wt % solution in water) was added. The reaction was monitored by TLC and HPLC. 8c progressively disappeared, with formation of benzophenone and two other compounds. After 1 h of stirring, 8c completely disappeared. Reaction of Pinacol 8c with N-Bromosuccinimide: Compound 8c (21 mg, 0.05 mmol) was dissolved in 1 mL of anhydrous THF. N-Bromosuccinimide (9 mg, 0.05 mmol) was added. The reaction was monitored by TLC. After 5 min of stirring 8c almost disappeared with formation of ferrocenophanone and benzophenone.HPLC showed the presence of ferrocenophanone and benzophenone in a 5/95 ratio.X-ray Crystal Structure Determinations for 7b,c and 12Crystal data were collected at 200 K with a Kappa-CCD Enraf-Nonius diffractometer with Mo Kα radiation. The structures were solved by direct methods and refined by full-matrix least-squares techniques on F using the CRYSTALS programs.(56,57) All non-hydrogen atoms were refined with anisotropic displacement parameters. All hydrogen atoms were located with geometrical restraints in riding mode.Crystallographic data for the structures reported in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publications CCDC 886312-886314. Copies of this information may be obtained free of charge from The Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, U.K. (fax, +44-1223-336033;

  breast adenocarcinoma cell line MDA-MB-231 was obtained from ATCC. Cells were grown in RPMI medium supplemented with 10% fetal calf serum, in the presence of penicillin, streptomycin, and fungizone in a 75 cm 2 flask under 5% CO 2 . Cells were plated in 96-well tissue culture plates in 200 μL of medium and treated 24 h later with 2 μL stock solutions of compounds dissolved in DMSO using a Biomek 3000 instrument (Beckman-Coulter). Controls received the same volume of DMSO (1% final volume). After 72 h of exposure, MTS reagent (Promega) was added and incubated for 3 h 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 IC 50 determination (50% inhibition of cell proliferation), cells were incubated for 72 h following the same protocol with compound concentrations ranging from 5 nM to 100 μM in separate duplicate experiments. Theoretical Calculations Th o tic c cu tio s w p fo d with Sp t '4 softw (W v fu ctio I c., 18401 Von Karman Ave., Suite 370, Irvine, CA 92612). A geometry optimization was performed on each molecule to find the absolute energetic minimum using the semiempirical PM3 Hamiltonian.

Table 1 . Yields of 6 and 7 According to Varying Reaction Conditions

 1

	yield (%)

molecules of type D: that is, those proceeding via cation B.

	ring, according to the following reported pK R	+ values (going from least to most stable):
	<-7.3 fo PhCH 2	+ , (4 ) -3.3 fo Ph 2 CH + , (42) -5.7 fo (4-MeO-C 6 H 4 ) 2 CH + , (42) -.28 fo
	FcCH 2	+ , (43) and +0.35 for FcCH + Ph. (44) Therefore, a priori, one might anticipate that
	cation A should be more stable than cation B in Scheme 4. However, the reactions with the
	benzophenones exclusively give

Table 2 . Selected Bond Distances (Å) and Bond Angles (deg) for 7b,c and 12

 2

		7b	7e		12
	Fe1-C Cp	2.030(8)-2.095(4)	2.056(19)-2.081(16)	Fe1-C Cp	2.0162(16)-2.047(17)
	C Cp -C Cp	1.383(18)-1.442(7)	1.419(4)-1.448(2)	Fe2-C Cp	2.0088(15)-2.058(17)
	C1-C11	1.549(6)	1.556(2)

Table 3 . IC 50 Values (μM) of Compounds on Breast Cancer Cells

 3

(MDA-MB-231) In general, the transposition compounds are not very active, with IC 50 values between 9.38 and 20.1 μM. Compound 7b, with one hydroxy group, is the exception, with an IC 50 value of 1.45 μM. Compound 13, which possesses only one hydroxyphenyl group, is completely

  (0.38 g, 2.1 mmol), time 2 h, T = 0 °C. The solvent was then evaporated, the oily reaction mixture was dissolved in CH 2 Cl 2 and cooled to 0 °C, and concentrated HCl (38.5 mL, 468.6 mmol) was added portion by portion and then the Recrystallization of the second fraction from dichloromethane/pentane gave 14 as orange crystals (0.04 g, 7%, mp 166.5 °C).1 H NMR (300 MHz, acetone-d 6 ): δ 2.14-2.27 (m, 1H, CH 2 ), 2.34-2.56 (m, 2H, CH 2 ),

	6.25 mmol), p,p-dihydroxybenzophenone (1.34 g, 6.25 mmol), time 2 days, T = -25
	°C. Recrystallization from acetonitrile/water gave 7a as yellow crystals (0.90 g, 33%
	yield). Mp: 230 °C dec. 1 H NMR (300 MHz, acetone-d 6 ): δ 2.72-2.81 (m, 2H, CH 2 ), 3.07-3.16 (m, 2H, CH 2 ), 4.08 (t, J = 1.7 Hz, 2H, C 5 H 4 ), 4.21 (t, J = 1.7 Hz, 2H, C 5 H 4 ), 4.29 (s, 4H, C 5 H 4 ), 6.74 (d, J = 8.8 Hz, 4H, C 6 H 4 ), 7.04 (d, J = 8.8 Hz, 4H, C 6 H 4 ), 8.24 (s, 2H, OH). 13 C NMR (75.4 MHz, acetone-d 6 ): δ 28.0 (CH 2 ), 44.6 (CH 2 ), 69.0 (2CH, C 5 H 4 ), 69.2 (2CH, C 5 H 4 ), 70.3 (C), 72.7 (2CH, C 5 H 4 ), 74.2 (2CH, C 5 H 4 ), 88.6 (C ip ), 93.9 (C ip ), 115.9 (2 × 2CH, C 6 H 4 ), 132.1 (2 × 2CH, C 6 H 4 ), 137.2 (2C, C 6 H 4 ), 157.2 (2C, C 6 H 4 ), 205.5 (CO). IR (KBr, ν, cm -1 ): 3380 (OH), 3010, 2914, 2856 (CH 2 ), 1681 (CO). MS (CI, NH 3 ): m/z 439 [M + H] + , 456 [M + NH 4 ] + . HRMS (ESI, C 26 H 22 FeNaO 3 [M + Na] + ): c cd 46 .08 8, fou d 46 .08 76. A . C cd fo C 26 H 22 FeO 3 •H 2 O: C, 68.43; H, 5.30. Found: C, 67.93; H, 5.16. Concentrated HCl (10.2 mL, 125.0 mmol) was added, and the mixture was stirred for 1 h at 0 °C. Recrystallization from diethyl ether gave 7b as orange crystals (0.70 g, 80% yield). 1 H NMR (300 MHz, CDCl 3 ): δ 2.78-2.85 (m, 2H, CH 2 ), 3.11-3.16 (m, 2H, CH 2 ), 4.05 (s, 2H, C 5 H 4 ), 4.20 (s, 2H, C 5 H 4 ), 4.27 (s, 2H, C 5 H 4 ), 4.31 (s, 2H, C 5 H 4 ), 4.82 (s, 1H, OH), 6.70 (d, J = 8.7, 2H, C 6 H 4 ), 7.08 (d, J = 8.7, 2H, C 6 H 4 ), 7.13-7.26 (m, 5H, C 6 H 5 ). 13 C NMR (75.4 MHz, CDCl 3 ): δ 26.9 (CH 2 ), 43.9 (CH 2 ), 67.7 (2CH, C 5 H 4 ), 67.8 and 67.9 (2CH, C 5 H 4 ), 69.9 (C), 71.3 and 71.4 (2CH, C 5 H 4 ), 72.7 (2CH, C 5 H 4 ), 86.2 (C ip ), 92.7 (C ip ), 114.8 (2CH, C 6 H 4 ), 126.4 (CH, C 6 H 5 ), 127.9 (2CH, C 6 H 4 ), 129.3 (2CH, C 6 H 5 ), 130.6 (2CH, C 6 H 5 ), 136.2 (C, C 6 H 4 ), 144.2 (C, C 6 H 4 ), 154.1 (C, C 6 H 4 ), 204.9 (CO). IR (KBr, ν, cm -1 ): 3311.2 (OH), 3021, 2969, 2917, 2859 (CH 2 ), 1678 (CO). MS (EI, 70 eV): m/z 422 [M] • + , 394 [M -CO] • + . HRMS (ESI, C 26 H 22 FeNaO 2 [M + Na] + ): calcd 445.086 26, fou d 445.086 4. A . C cd fo C 26 H 22 FeO 2 : C, 73.94; H, 5.47. Found: C, 73.36; H, 5.47. mixture was stirred for 1 day at room temperature. Recrystallization from hexane/dichloromethane gave 7c as orange crystals (0.6 g, 71% yield). Mp: 177 °C. 1 H NMR (300 MHz, CDCl 3 ): δ 2.81-2.85 (m, 2H, CH 2 ), 3.13-3.17 (m, 2H, CH 2 ), 4.04 (t, J = 1.7, 2H, C 5 H 4 ), 4.20 (t, J = 1.7, 2H, C 5 H 4 ), 4.27 (t, J = 1.7, 2H, C 5 H 4 ), 4.33 (t, J = 1.7, 2H, C 5 H 4 ), 7.12-7.20 (m, 2H, C 6 H 5 ), 7.22 (s, 5H, C 6 H 5 ), 7.23 (s, 3H, C 6 H 5 ). 13 C NMR (75.4 MHz, CDCl 3 ): δ 26.9 (CH 2 ), 44.0 (CH 2 ), 67.7 (2CH, C 5 H 4 ), 67.9 (2CH, C 5 H 4 ), 70.5 (C), 71.3 (2CH, C 5 H 4 ), 72.7 (2CH, C 5 H 4 ), 83.3 (C ip ), 92.9 (C ip ), 126.5 (2 × 1CH, C 6 H 5 ), 127.9 (2 × 2CH, C 6 H 5 ), 129.4 (2 × 2CH, C 6 H 5 ), 144.2 (2 × 1C C 6 H 5 ), 204.2 (CO). IR (KBr, ν, cm -1 ): 2926 (CH 2 ), 1698 (CO). MS (EI, 70 eV): m/z 406 [M] •+ , 378 [M -CO] • + , 301, 228, 165. Anal. Calcd for C 26 H 22 FeO: C, 76.85; H, 5.45. Found: C, 76.81; H, 5.38. 1-(p-Hydroxyphenyl)-2-oxo[4]ferrocenophane (13) and 1-(p-Hydroxyphenyl)-1-formyl[3]ferrocenophane (14): zinc powder (0.95 g, 14.6 mmol), titanium tetrachloride (1.14 mL, 10.4 mmol), [3]ferrocenophan-1-one (0.5 g, 2.08 mmol), p-hydroxybenzaldehyde (0.76 g, 6.20 mmol), T = 0 °C, time one night. Concentrated HCl (10.3 mL, 125.0 mmol) was added, and the mixture was stirred for 4 h at 0 °C. The crude product was subjected to column chromatography with diethyl ether/petroleum ether as eluent. Two fractions were collected. Recrystallization of the first fraction from dichloromethane/pentane gave 13 as orange crystals (0.19 g, 27% yield, mp 212 °C). 1 H NMR (300 MHz, acetone-d 6 ): δ 2.49-2.71 (m, 2H, CH 2 ), 2.88-3.06 (m, 2H, CH 2 ), 3.90-4.06 (m, 6H, C 5 H 4 ), 4.08-4.10 (m, 1H, C 5 H 4 ), 4.14-4.16 (m, 1H, C 5 H 4 ), 4.99 (s, 1H, CH), 6.55 (d, J = 8.4 Hz, 2H, C 6 H 4 ), 6.95 (d, J = 8.4 Hz, 2H, C 6 H 4 ), 8.02 (s, 1H, OH). 13 C NMR (75.4 MHz, acetone-d 6 ): δ 25.7 (CH 2 ), 42.8 (CH 2 ), 53.5 (CH), 68.5 (CH, C 5 H 4 ), 69.0 (2CH, C 5 H 4 ), 69.2 (CH, C 5 H 4 ), 69.3 (CH, C 5 H 4 ), 69.6 (CH, C 5 H 4 ), 69.9 (CH, C 5 H 4 ), 70.3 (CH, C 5 H 4 ), 81.9 (C ip ), 87.7 (C ip ), 115.3 4.04 (m, 1H, C 5 H 4 ), 4.08-4.11 (m, 2H, C 5 H 4 ), 4.15-4.17 (m, 1H, C 5 H 4 ), 4.18-4.21 (m, 1H, C 5 H 4 ), 4.52-4.55 (m, 1H, C 5 H 4 ), 6.81 (d, J = 8.7 Hz, 2H, C 6 H 4 ), 7.21 (d, J = 8.7 Hz, 2H, C 6 H 4 ), 8.49 (s, 1H, OH), 9.54 (s, 1H, CHO). 13 C NMR (75.4 MHz, acetone-d 6 ): δ 22.2 (CH 2 ), 41.2 (CH 2 ), 54.6 (C), 68.9 (CH, C 5 H 4 ), 69.1 (2CH, C 5 H 4 ), 69.2 (CH, C 5 H 4 ), 69.3 (CH, C 5 H 4 ), 69.7 (CH, C 5 H 4 ), 70.0 (CH, C 5 H 4 ), 71.6 (CH, C 5 H 4 ), 87.6 (C ip ), 89.0 (C ip ), 116.2 (2CH, C 6 H 4 ), 130.8 (C, C 6 H 4 ), 130.9 (2CH, C 6 H 4 ), 157.5 (C, C 6 H 4 198.7 (CO). MS (CI, NH 3 ): m/z 347 [M + H] + , 364 [M + NH 4 ] + , 318 [M -CHO] + . HRMS (ESI, C 20 H 18 FeO 2 : [M] •+ ): c cd 346.065 07, fou d 346.065 5. A . (2CH, C 6 H 2.83-3.01 (m, 1H, CH 2 ), 3.90-3.92 (m, 1H, C 5 H 4 ), 3.98-4.00 (m, 1H, C 5 H 4 ), 4.02-Calcd for C 20 H 18 FeO 2 •0.5H 2 O: C, 68.61; H, 5.39. Found: C, 68.11; H, 5.31.

1,1-Bis(p-hydroxyphenyl)-2-oxo

[4]

ferrocenophane (7a): zinc powder (2.86 g, 43.7 mmol), titanium tetrachloride (3.43 mL, 31.2 mmol),

[3]

ferrocenophan-1-one (1.5 g,

1-(p-Hydroxyphenyl)-1-phenyl-2-oxo

[4]

ferrocenophane (7b): zinc powder (0.95 g, 14.6 mmol), titanium tetrachloride (1.14 mL, 10.4 mmol),

[3]

ferrocenophan-1-one (0.5 g, 2.08 mmol), p-hydroxybenzophenone (0.41 g, 2.08 mmol), time 2 h, T = 0 °C.

1,1-Diphenyl-2-oxo

[4]

ferrocenophane (7c): zinc powder (0.95 g, 14.6 mmol), titanium tetrachloride (1.14 mL, 10.4 mmol),

[3]

ferrocenophan-1-one (0.5 g, 2.08 mmol), benzophenone 4 ), 130.

4

(2CH, C 6 H 4 ), 132.3 (C, C 6 H 4 ), 157.0 (C, C 6 H 4 ), 207.7 (CO). MS (CI, NH 3 ): m/z 347 [M + H] + , 364 [M + NH 4 ] + . Anal. Calcd for C 20 H 18 FeO 2 : C, 69.38; H, 5.24. Found: C, 69.61; H, 5.23.

. Reaction of Pinacol 8c with EtONa or HOCH 2 CH 2 SNa:

  Compound 8c (42 mg, 0.1 mmol) was dissolved in 2 mL of anhydrous CH 2 Cl 2 . The solution was cooled to -70 °C, and when HBF 4 •Et 2 O (0.1 mL, 0.7 mmol) was added, the solution turned blue.After 5 min, EtONa (50 mg, 0.7 mmol) was added, and after it was stirred for 15 min, the mixture was poured into water and extracted with diethyl ether. CCM of the crude product showed only the presence of 7c. The analogous reaction with HOCH 2 CH 2 SNa, prepared from 2-mercaptoethanol and NaH, gave the same result.

	1-hydroxy-1[3]ferrocenophanyl][3]ferrocenophane (12): Zinc
	powder (0.49 g, 7.5 mmol) was suspended in THF at room temperature, and titanium
	tetrachloride (0.55 mL, 5.0 mmol) was added slowly via a syringe with stirring. The
	reaction mixture was refluxed for 2 h, after which a THF solution containing
	[3]ferrocenophan-1-one (0.3 g and 1.25 mmol) was added, and the mixture was
	refluxed for 2 h. The reaction mixture was poured into water and extracted with
	dichloromethane. The organic layer was washed with water, dried over magnesium
	sulfate, filtered, and concentrated under reduced pressure. The crude product was
	purified by flash	column chromatography and recrystallized from
	dichloromethane/hexane. Pinacol 12 was obtained (0.16 g, 53%). 1 H NMR (300 MHz,
	DMSO-d 6 ): δ 1.82-2.03 (m, 4H, CH 2 ), 2.11-2.35 (m, 4H, CH 2 ), 3.83-3.90 (m, 6H,
	C 5 H 4 ), 3.90-3.93 (m, 2H, C 5 H 4 ), 3.94-3.97 (m, 2H, C 5 H 4 ), 4.07-4.11 (m, 4H, C 5 H 4 ),
	4.18-4.21 (m, 4H, OH + C 5 H 4 ). 13 C NMR (75.4 MHz, DMSO-d 6 ): δ 19.2 (2CH 2 ), 41.8
	(2CH 2 ), 66.6 (2CH, C 5 H 4 ), 67.2 (2CH, C 5 H 4 ), 67.3 (2CH, C 5 H 4 ), 67.4 (2CH, C 5 H 4 ),
	67.5 (2CH, C 5 H 4 ), 68.4 (2CH, C 5 H 4 ), 68.5 (2CH, C 5 H 4 ), 71.4 (2CH, C 5 H 4 ), 73.9 (2C),
	86.6 (2C ip ), 89.2 (2C ip ). MS (EI, 70 eV): m/z 482 [M] + . , 242, 199. HRMS (ESI,
	C 26 H 26 Fe 2 O 2 : [M] •+ ): c cd 482.062 62, fou d 482.06 6 . A . C cd fo
	C 26 H 26 Fe 2 O 2 •0.5H 2 O: C, 63.57; H, 5.54. Found: C, 63.53; H, 5.46.
	Reaction of Pinacol 8c with Methanol: Compound 8c (30 mg) was dissolved in
	methanol (2 mL), and 10 drops of concentrated H 2 SO 4 were added. After it was stirred

for 1 h, the mixture was poured into water and extracted with diethyl ether. The NMR spectrum of the crude product obtained showed mainly the presence of 7c

ACKNOWLEDGMENTS

We thank the Agence Nationale de la Recherche for financial support (ANR 2010 BLAN 7061 blanc " c f o ") d th Ministere des Affaires Etrangères for a doctoral fellowship (M.G.). We thank P. Herson for crystal structure determinations and T. Crestiel for tests on cancer cells.

Supporting Information

A table, figures, and CIF files giving crystallographic data and NMR spectra. This material is available free of charge via the Internet at http://pubs.acs.org.

Notes

The authors declare no competing financial interest.


Nicolas Wattiez

Charlotte Constans

Thomas Deffieux

Pierre M Daye

Jean-François Aubry
email: jean-francois.aubry@espci.fr

Pierre Pouget
email: pierre.pouget@upmc.fr

Mickael Tanter

Transcranial ultrasonic stimulation modulates single-neuron discharge in macaques performing an antisaccade task

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

ultrasound can be assessed in real time in awake behaving monkeys by recording discharge activity from a brain region reciprocally connected with the stimulated region.

The study opens the door for further parametric studies for fine-tuning the ultrasonic parameters. The ultrasonic effect could indeed be quantified based on the direct measurement of the intensity of the modulation induced on a single neuron in a freely performing animal. The technique should be readily reproducible in other primate laboratories studying brain function, both for exploratory and therapeutic purposes and to facilitate the development of future clinical TUS devices.

INTRODUCTION

Focused ultrasound provides a new method to non-invasively stimulate the nervous system using an external focused ultrasonic transducer to induce transient local stimulation. Fry and colleagues demonstrated that US can suppress visually evoked responses (1) in the primary visual cortex of anesthetized cats using focused ultrasound.

Gavrilov and collaborators elicited thermal, tactile, and pain sensations when they applied focused ultrasound to the human and [START_REF] Gavrilov | The effect of focused ultrasound on the skin and deep nerve structures of man and animal[END_REF]. The application of transcranial ultrasonic stimulation (TUS) to the brain gained momentum in the new millenium, with low frequency, low intensity ultrasound being applied the primary motor cortex in rodent, generating motor responses without observing any adverse effect on the stimulated tissue [START_REF] Younan | Influence of the pressure field distribution in transcranial ultrasonic neurostimulation[END_REF][START_REF] King | Effective parameters for ultrasound-induced in vivo neurostimulation[END_REF][START_REF] Yoo | Focused ultrasound modulates region-specific brain activity[END_REF][START_REF] Deffieux | Low-intensity focused ultrasound modulates monkey visuomotor behavior[END_REF]. Thermal effects are unlikely to play a role in the process when compared with the mechanical effects such as radiation pressure and cavitation [START_REF] Legon | Transcranial focused ultrasound modulates the activity of primary somatosensory cortex in humans[END_REF][START_REF] Lee | Image-guided transcranial focused ultrasound stimulates human primary somatosensory cortex[END_REF]. However, the exact mechanism for neural ultrasonic stimulation remains unknown [START_REF] Aubry | MR-Guided Transcranial Focused Ultrasound[END_REF][START_REF] Hynynen | Clinical applications of focused ultrasound-the brain[END_REF]. Several models have been developed to explain the neuronal activation observed with acoustic pressure: the Soliton model [START_REF] Heimburg | On soliton propagation in biomembranes and nerves[END_REF], the bilayer sonophore (BLS) model ( Intramembrane cavitation as a unifying mechanism for ultrasound-induced bioeffects, [START_REF] Krasovitski | Intramembrane cavitation as a unifying mechanism for ultrasound-induced bioeffects[END_REF] and the neuronal intramembrane cavitation excitation (NICE) theory [START_REF] Plaksin | Cell-Type-Selective Effects of Intramembrane Cavitation as a Unifying Theoretical Framework for Ultrasonic Neuromodulation[END_REF] combining the BLS model and the Hodgkin-Huxley spiking model (which describes how action potentials are initiated and propagate in neurons). No model has yet been evaluated using single neuron recording during TUS in an alert animal performing a task. An improved understanding of the physiological action of TUS would enable fine tuning of protocols based on a known effect of the stimulation, which will improve the effectiveness of the technique, and thus accelerate clinical transfer. Here we report the feasibility of simultaneous recording of single neuron activity during TUS in an alert macaque performing an oculomotor task. A focused ultrasound transducer was positioned using a neuronavigation system (Rogue Research) to apply brief pulses of ultrasound in the frontal eye field (FEF) while neurophysiological recordings were performed in the supplementary eye field (SEF).

Two discrete areas in frontal cortex are involved in generating saccadic eye movements in primates-the frontal eye field (FEF) and the supplementary eye field (SEF).

Anatomically the FEF is reciprocally and bilaterally connected with the SEF [START_REF] Huerta | Frontal eye field as defined by intracortical microstimulation in squirrel monkeys, owl monkeys, and macaque monkeys. II. Cortical connections[END_REF][START_REF] Luppino | Cortico-cortical connections of two electrophysiologically identified arm representations in the mesial agranular frontal cortex[END_REF] and these connections appear to vary the least between primate species, suggesting that the most conserved functions of these regions may serve as a link for facilitating, planning of eye movements [START_REF] Percheron | What makes a frontal area of primate brain the frontal eye field?[END_REF]. In both regions, eye movements can be evoked by direct electrical microstimulation with relatively low current [START_REF] Schall | Visuomotor Areas of the Frontal Lobe[END_REF]. Invasive intra-cortical recordings and/or stimulation techniques have been widely used to better apprehend the role of these two frontal areas, in combination with ocular motor measurements and mapping techniques, such as electrophysiological and fMRI recordings in non-human primates. Those approaches have provided causal evidence about the role of the FEF, with direct bearing on ocular motor and attentional function [START_REF] Bruce | Primate frontal eye fields. I. Single neurons discharging before saccades[END_REF][START_REF] Marrocco | Saccades induced by stimulation of the frontal eye fields: interaction with voluntary and reflexive eye movements[END_REF]. More recently, the causal involvement of FEF in attentional orienting has been demonstrated [START_REF] Moore | Control of eye movements and spatial attention[END_REF][START_REF] Moore | Selective gating of visual signals by microstimulation of frontal cortex[END_REF] as well as its ability to modulate different aspects of visual perception [START_REF] Ekstrom | Bottom-up dependent gating of frontal signals in early visual cortex[END_REF][START_REF] Thickbroom | Transcranial magnetic stimulation of the human frontal eye field[END_REF] while indirect control and monitoring processes have been assigned to SEF functions [START_REF] Pouget | The cortex is in overall control of "voluntary" eye movement[END_REF][START_REF] Pouget | Cortical control and performance monitoring of interrupting and redirecting movements[END_REF]. In order to examine the possible distant role of neuromodulation caused by FUS applied to a cortical brain region, we decided to target a cortical circuit controlling voluntary eye movement.

Our short-term goal was to demonstrate the feasibility of combining real time focused ultrasound with single unit (SU) recordings in an alert macaque performing an antisaccade task. The combined approach of TUS and single unit recording has the potential to elucidate the mechanisms underlying the effects of focused ultrasound on the central nervous system. TUS and single unit recording could also be combined with 3D functional ultrasound imaging of brain activity, as recently demonstrated in alert animals in conjunction with EEG recordings [START_REF] Sieu | EEG and functional ultrasound imaging in mobile rats[END_REF][START_REF] Macé | Functional ultrasound imaging of the brain[END_REF]. This will serve the causal noninvasive exploration of cognition in non-human primate models.

MATERIALS AND METHODS

Focused ultrasound

A single element ultrasound transducer (H115, diameter 64 mm, Sonic Concept, Bothell, WA, USA), geometrically focused to 64 mm, was used with a coupling cone (C103, Sonic Concepts, Bothell, WA, USA) filled with degassed water.

The transducer has four frequency resonances: 200 kHz, 320 kHz, 850 kHz and 1380 kHz. We chose the same main parameters (frequency and burst length) as our previous study [START_REF] Deffieux | Low-intensity focused ultrasound modulates monkey visuomotor behavior[END_REF] which had shown successful neuromodulation with the same transducer in monkeys: the ultrasound wave frequency was set to 320kHz and 100ms bursts of ultrasound were generated with a digital function generator, with rise and fall times set to 5ms at the beginning and the end of the pulse, respectively (Handyscope HS5, TiePie engineering, Sneek, The Netherlands). A 75-Watt amplifier (75A250A, Amplifier Research, Souderton, PA) was used to deliver the required power to the transducer and the input voltage and current of the transducer were recorded using a voltage probe (HP9250, TiePie engineering, Sneek, The Netherlands) and a current probe (P6021, Tektronix, Beaverton, Oregon) connected to the HS5 card inputs.

The recorded peak to peak voltage (VPP) was 139±26 V for monkey Y and 56±15 V for monkey L. The calibration of the transducer in water with a custom built heterodyne interferometer [START_REF] Jacob | Optical measurement of transient ultrasonic shock waves[END_REF] led to a linear relationship between VPP and peak negative pressure (PNP) in water of 5.68 kPa/V. The peak pressure in the brain of the monkeys was estimated by using 3D simulations of the ultrasonic field (see details below), taking into account the experimental geometry (0.41±0.08 MPa for monkey Y and 0.24±0.07 MPa for monkey L). For comparison, previous studies in human and non-human primates estimated peak pressures of 0.35 MPa(6) and 0.43 MPa [START_REF] Legon | Transcranial focused ultrasound modulates the activity of primary somatosensory cortex in humans[END_REF].

The ultrasound was directly applied to previously shaved skin. A coupling cone filled with water ensured ultrasonic coupling between the transducer and the animal's head. TUS was performed once every 5 trials with an unpredictable minimum duration of 3 seconds between trials. There was therefore a pause of at least 10 seconds between ultrasound pulses.

The corresponding overall spatial peak pulse average intensity (ISPPA) was thus estimated to be 5.6 ± 0.2 W/cm 2 and 1.9 ± 0.2 W/cm 2 for monkey Y and L respectively

(ISPPA= !² #$ * &
with and c approximated to ' ()*+, and -()*+, ). For comparison, the Food and Drug Administration (FDA) ISPPA limit for ultrasound imaging safety guidelines is 190 W/cm 2 for adult transcranial imaging [Food, U. and D. Administration, Information for manufacturers seeking marketing clearance of diagnostic ultrasound systems and transducers. Rockville, MD: Center for Devices and Radiological Health, US Food and Drug Administration, 1997]. We emphasize here that we do not claim to be below FDA requirements as long pulses (100ms, or 32000 periods) are used here, as compared to short pulses for imaging (one period for Bmode imaging, corresponding to 3µs at the frequency used here).

Experimental setup

The antisaccade paradigms and data acquisition were under the control of a computer running a Rexeno real-time data acquisition system [START_REF] Deffieux | Low-intensity focused ultrasound modulates monkey visuomotor behavior[END_REF].

Behavioral Task

Two captive-born macaques (Maccaca mulatta, "Y", and "L", one male and one female DTPP 2010-424). Monkeys had access to toys, mirrors and swings and also had visual, auditory and olfactory contact with other animals, and, when appropriate, could touch and groom each other. An institutional veterinary doctor constantly monitored their wellbeing and health conditions. Prior to participating in the study, both animals were periodically chaired, head-posted and trained to perform a series of tasks for a period of 6-12 months, until they became regular and proficient performers.

Prior to the first experimental session, animals were specifically trained in an antisaccade (AS) task. Between 500-1000 milliseconds after initial fixation onset on a brown central fixation stimulus and simultaneously to its disappearance (no gap), a red square appeared for 1000ms at a 16° of visual angle to a rightward or leftward location (direction randomly selected). Monkeys were trained not to look at this peripheral target but instead, initiate as soon as possible a saccade in the opposite direction. After the saccade, the monkey received a reward if the saccade fell within a 10° x 10° window centered at the mirror location of the visual target. Failure to trigger a saccade within 1000ms of target onset cancelled the trial. The AS task was chosen since prior human and monkey TUS experiments have revealed prosaccade paradigms to be much less sensitive to single pulse TUS interference than antisaccades [START_REF] Deffieux | Low-intensity focused ultrasound modulates monkey visuomotor behavior[END_REF]. Trials were aligned on the appearance of the peripheral target.

Data Analysis

Eye movements were recorded with an infrared video eyetracker (Eyelink 1k, SR-Research, Ontario, Canada). Eye position was digitized and sampled at 1 kHz and stored for off-line analysis. All the analyses were performed on a personal computer with custom software written in the Python programming language. Eye position signals were low-pass filtered using a third order Savitzky-Golay filter (window: 41ms). Eye velocity and eye acceleration were extracted from the Savitzky-Golay filter. Saccades were detected with a 30°/s velocity threshold.

For each cortical site tested with TUS, we used an online windows discriminator to isolate one neuron at a time for each recording. Offline cluster cutting (PCA) techniques were used systematically to confirm that action potential waveforms were produced by individual neuronal elements.

Spikes were sorted on-line using a box sorting method as a pair of time-level windows (Rasputin, Plexon Inc.). Offline examination of the sorting quality was systematically realized using a semi-automatic PCA calculation without restriction to a certain number of waveforms (Offline-Sorter Plexon Inc). Following the extraction of the spike time, we evaluated the average firing rate of each neuron (and the confidence interval of the average) using bootstrapping techniques on the computation of the spike density function [START_REF] Macé | Functional ultrasound imaging of the brain[END_REF]. The principle of bootstrap is to randomly select trials without replacement from the larger dataset (here control trials) and with replacement in the smaller dataset (TUS trials). This random selection is made N times where N = 50 * sample size of the smaller dataset as described by Daye and colleagues [START_REF] Daye | Vestibulo-ocular reflex suppression during head-fixed saccades reveals gaze feedback control[END_REF]. The effect of FUS between functional cell types was compared using the chi-squared test.

Surgical procedure

The surgical procedures for the titanium head-post implant were as previously described [START_REF] Deffieux | Low-intensity focused ultrasound modulates monkey visuomotor behavior[END_REF]. Each animal was deeply anaesthetized using ketamine hydrochloride (5 mg/kg i.m.) for initial sedation before induction with 2-4% isoflurane gas, which was then maintained during surgery. Pain medication was given prior to surgery and routinely given after surgery. Implanted head posts (7.1 mm internal diameter) (Part 6-FHP-X2F, Crist Instrument, Hagerstown, MD, USA) were designed to mate with a head-post holder (Part 6-FHB-S2B, Crist Instrument, Hagerstown, MD, USA). SEF Recording chambers were placed according to stereotactic coordinates (AP: 26; ML: 0) and MRI scans. The craniotomies were performed under deep anesthesia with ketamine hydrochloride (5 mg/kg i.m.) for initial sedation, and induction and maintenance with 2-4% isoflurane gas.

Pain medication was given prior the surgery and routinely given after surgery.

Neurophysiological data acquisition and analysis

For single unit recordings, multiple tungsten microelectrodes (FHC, 8-10 MΩ) were introduced through guide tubes and independently positioned with a custom-made stepper-motor micro-drive. The recordings were made on a MAP system (Plexon Inc., TX, USA), storing spike times with 25μs resolution. Online discrimination based on wave shape parameters was used to select a single neuron on each independent recording electrode channel. Respectively 39 and 46 neurons were recorded in monkey Y and L.

All isolated neurons recorded were kept for the analysis. All sets of recordings consisted in more than 386 and 400 valid trials for monkey Y and L respectively.

FEF Targeting with TUS

Targeting of the FEF with the TUS transducer was performed using a neuro-navigator tool (Brainsight, Rogue Research, Montreal, Canada). An MRI dataset (structural T1) was acquired for each monkey with an attached MR fiducial marker array. The FEF target was manually registered once and used for all sessions. Prior to each session, the position of the fiducial marker array was manually registered to a position sensor serving as a reference and fixed to the chair. In a second step, the focal spot of the TUS transducer, materialized using a 3D printed piece, was registered using a tracked pointer to a second position sensor, which was fixed behind the transducer. Before each session, the transducer, supported by a mechanical arm (Rogue Research, Montreal, Canada) was then manually moved until the focal spot was aligned and positioned over the predetermined FEF target. The arm was then locked for the rest of the session.

Numerical simulations

Acoustic density and velocity maps were reconstructed from CT scans of both monkeys' heads (Philips Brilliance 64 CT Scanner , Voltage: 120kV, Intensity: 350mA ; FilterType: 'D'). The brain, skin and muscle tissue was considered as a homogeneous medium and skull was modeled as a heterogeneous model by using the following conversion from Hounsfield units (HU) to density and velocity of the skull 1 :

' = ' */001+ + 34 -34 */001+ 34 6)7 + 34 */001+ (' 6)7 -' */001+ )
HU max was determined from each map as the maximal value in the skull and HU tissue as the mean value of HU in the brain, muscle and skin.

The power law model for attenuation is abs = α * Ф ? where the porosity Ф is defined by The acoustic parameters are summarized in table 2. CT images were acquired with the smallest field of view to obtain the best possible resolution in the transverse plane: 0.358 mm and 0.136 mm for monkey Y and L respectively. The acoustic density and velocity 3D maps were then subsampled to respectively 0.358 and 0.407 mm isotropic resolution for monkey Y and monkey L, which gave a ratio of 13.6 and 12.0 pixels per wavelength in tissue at 320 kHz.

Ф = $ @AB C$ $ @AB C$ DAEFG
The pseudo-spectral method-based software k-Wave 2 was used to perform the linear acoustic simulation through the water cone, skull and brain with the same geometry used in vivo with the neuro-navigator positioning. The head was modelled as immersed in water, given that, in that setup, tissue/air interfaces are unlikely to play a significant role compared to skull/tissue interfaces. Not considering the air medium, whose impedance is much lower than water, significantly reduced the computation time.

To limit the computation time, the total simulation duration was limited to 150 µs, which was sufficient to reach a steady-state. For each simulation, the pressure field was stored in three dimensions and in a full volume including the FEF for all time steps. Time profiles of the acoustic pressure were extracted at the geometric focus and at the point of maximal pressure amplitude over time. Maps of the in-situ time peak pressure at steady-state were also estimated by taking the time peak of the pressure field at each pixel in 3 planes (orthogonal to, or including the transducer direction), centered on the geometric focus. As the muscular tissue surrounding monkey Y's skull was much thicker than monkey L's (respectively about 16mm and 5mm), a 1 cm transducer's pressed into the tissue was taken into account in the simulation. This value was set based on the actual in vivo positioning of the transducer, based on the neuro-navigator guidance for FEF targeting. The calibration of the transducer in water showed a linear relationship between peak to peak voltage (VPP) and peak negative pressure (PNP) in water of 5.68 kPa/V. Simulations were first performed in water without the skull, in order to normalize the simulations based on actual pressure measurements in water. Table 1 summarizes the results of the simulations.

RESULTS

The goal of our study is to show that TUS and single unit recordings can be combined to probe the interactions between two reciprocally-connected distal bilateral areas. First, we present how TUS applied to FEF affects the neural activity of a SEF neuron. Then we extend these analyses to the whole neuronal population that has been recorded to investigate the overall effect of TUS on a distal connected area.

Simulation of ultrasonic pressure field in the brain

The voltage applied to the ultrasonic transducer was respectively equal to 139±26 V and 56±15 V for monkey Y and L. As shown in figure 2, female monkey L had a smaller head than male monkey Y. 3D numerical simulations of the ultrasound propagation were conducted in order to investigate the ultrasound field in the two different monkey brains while taking into account head geometry, and the reflection, refraction and absorption associated with the full skull cavity and head geometry, (table 1). A linear simulation model was used (see supplementary materials) in which the acoustic pressure was proportional to the applied voltage (VPP). In water, the peak pressure was found to be 2.5 times higher with monkey Y's settings than monkey L's. Nevertheless, the peak pressure in the FEF is only 1.7 times higher for monkey Y. This is most probably due to anatomical differences such as skull thickness, skull density, skull geometry and frontal muscular tissue thickness (table 1 and figure 1). Owing to the different acoustic pressures used between the two animals, as well as the compounding effects of differences in head size, skull thickness, the recorded neuron data from each animal was analyzed separately and is quantified in figure 4 (top panel for monkey Y and middle panel for monkey L). To minimize the difference between peak pressure in the brain between the two animals the pressure was slightly reduced for monkey Y (compared to [START_REF] Deffieux | Low-intensity focused ultrasound modulates monkey visuomotor behavior[END_REF]). Behaviorally, the latencies were significantly modulated by TUS for monkey L across sessions (n=46, p<0.01) and marginally modulated for monkey Y (n=39, p=0.08). The black line and shaded areas represent the 99% confidence interval for activity difference when comparing TUS and control trials. The blue vertical rectangles represent the time interval during TUS stimulation (100ms) where time t=0 corresponds to target presentation. We consider the difference between TUS and control conditions activity as statistically different from zero when p < 0.01 and the duration of the difference is greater than or equal to 5ms.

Typical trials

The green (increase in activity) or red (decrease in activity) segments in fig. 3c,d highlights these significant time periods. In the stimulation condition, figure 3a shows that the activity started to increase 67 ms after TUS onset and became statistically significantly different from the control condition 78 to 111 ms and from 113 to 125 ms after TUS onset (shown by the first significant segment in fig. 3b). The neuron reached a peak activity of 10.1Hz (with 99% CI of 15.48 and 21.61Hz) 99 ms after stimulation onset compared to 2.1Hz (with 99% CI of 5.45 and 9.53Hz) during control trials. For this neuron, this level of activity was maintained during the stimulation. After stimulation ceased, the neuron activity decayed back to the activity observed during the control condition. As shown on the right panel (figure 3b,d), a decrease in activity in the SEF was also observed following FUS in the FEF. The activity of this neuron starts to increase before TUS and appeared to be significantly reduced from 24 to 41 ms after TUS onset.

Population activity

Figure 4a,b illustrates changes in the activity of the population of the neurons recorded in the two animals. Significant periods of neuromodulation were observed for 39% and 41% of the recorded neurons respectively for monkey Y and L. A transient increase of activity for 53% and 47% of neurons recorded respectively in monkey Y and L reveal the perturbation induced by TUS. For 47% and 53% of neurons respectively in monkey Y and L the perturbation was revealed by a significant decrease in activity.

Among all recorded neurons the effects of TUS parameters are presented chronologically during the series of experiments for the 39 and 46 neurons respectively recorded in monkey Y and L.

Each horizontal gray line represents a neuron. As in figure 3, the time origin corresponds to target presentation, and shaded gray areas represent the stimulation period. The colored lines in figure 4 highlight the periods during which the neuronal activity differed significantly between TUS and control trials. The color of each segment represents the p-value of the difference between TUS and control trials. Green (red) colored segments correspond to excitation (inhibition). Figure 4a,b shows that many of the recorded neurons had their activity modulated (increased or decreased) when TUS was applied in both monkeys. Overall, our analysis demonstrates a significant effect on SEF neuronal activity when TUS was applied to the FEF. Comparison between cell types based on their functional properties [START_REF] Pouget | Cortical control and performance monitoring of interrupting and redirecting movements[END_REF][START_REF] Stuphorn | Executive control of countermanding saccades by the supplementary eye field[END_REF] did not reveal any significant susceptibility (respectively χ 2 , p=0.39, for monkey Y and χ 2 , p=0.37 for monkey L). For monkey Y: of 39 neurons, 0 of the 3 of fixation; 5 of the 11 of movement; 9 of the 18 non-specific; 1 of the 7 postsaccadic; and 0 of 0 visual neurons showed a significant neuro-modulation induced by TUS. For monkey L: of 46 neurons, 2 of 2 of fixation; 2 of 9 of movement; 13 of 26 nonspecific; 1 of 7 post-saccadic; and 1 of 2 of visual neurons showed significant neuro-modulation by TUS. In contrast to TUS stimulation applied to the FEF, TUS applied to control extra-striate visual cortex did not significantly generate changes in activity rates (figure 4c). Only one of the 20 recorded neurons did show a brief significant period of modulation when the control region was stimulated compared with 39 and 41% of significant neurons showing modulation when the FEF was stimulated in the same animal (monkey L). We verified whether the observed change of activity during the stimulation could be explained by a modification of the spikes isolation of the neuron due to the ultrasound stimulation. We did not observe any difference of spike waveform when comparing TUS and control conditions (figure 5), demonstrating that the pattern of activity of our neuron observed during stimulation trials is not an artifact related to a propagation of the ultrasound wave to the recorded region (CI: 99%). At the population level none of the recorded neurons show a significant modulation of waveforms above the confidence interval (Figure 6).

DISCUSSION

Focused ultrasound stimulation (FUS) techniques have been used to produce increased neuronal firing rates in the peripheral and central nervous system in vitro or in anesthetized preparations [START_REF] King | Effective parameters for ultrasound-induced in vivo neurostimulation[END_REF][START_REF] Tyler | Remote Excitation of Neuronal Circuits Using Low-Intensity, Low-Frequency Ultrasound[END_REF][START_REF] Tufail | Transcranial Pulsed Ultrasound Stimulates Intact Brain Circuits[END_REF][START_REF] Kim | Focused Ultrasound-mediated Noninvasive Brain Stimulation: Examination of Sonication Parameters[END_REF][START_REF] Naor | Towards multifocal ultrasonic neural stimulation II: design considerations for an acoustic retinal prosthesis[END_REF][START_REF] Menz | Precise Neural Stimulation in the Retina Using Focused Ultrasound[END_REF]. Suppression of activity following FUS has also been reported [START_REF] Yoo | Focused ultrasound modulates region-specific brain activity[END_REF][START_REF] Min | Focused ultrasound-mediated suppression of chemically-induced acute epileptic EEG activity[END_REF][START_REF] Kim | Suppression of EEG visualevoked potentials in rats through neuromodulatory focused ultrasound[END_REF], as well as more subtle changes in excitability [START_REF] Moore | The biophysical effects of ultrasound on median nerve distal latencies[END_REF]. However, non-invasive exploitation of the concomitant effects of transcranial ultrasound stimulation (TUS) in alert animals would have many applications in neuroscience. However, and despite the increasing volume of work done with the emerging of transcranial ultrasound stimulation (TUS), there is limited knowledge concerning its effects on neurons.

Investigating neural connectivity-how two connected regions interact by stimulating one area and recording in another-is the most straightforward application. Our approach was to perform the feasibility of such study by studying the interaction within the oculomotor regions of the frontal lobes in awake primates performing an antisaccade task. By combining TUS with traditional electrophysiology, the ability to study the mechanisms of TUS is vastly expanded, and paves the way for research and development needed to usher in the next generation of noninvasive TUS technologies and treatments. In particular, the study opens the door for further parametric studies for fine-tuning the ultrasonic parameters. The ultrasonic effect could indeed be quantified based on the direct measurement of the intensity of the modulation induced on a single neuron in a freely performing animal. In this paper the recorded region (SEF) was different from the stimulated region (FEF) and despite the fact that these two regions are connected, not each FEF neuron projects to an SEF neuron. The fact that TUS can induce an effect in a connected brain region re-enforces the potential uses of this technic to modify network activities. Further studies are required to better understand the mechanisms of action of TUS. Potentially, clinical use of TUS might be similar to transcranial magnetic stimulation (TMS) and could thus be valuable in the treatment of depression [START_REF] George | The expanding evidence base for rTMS treatment of depression[END_REF], movement disorders [START_REF] Lefaucheur | Evidence-based guidelines on the therapeutic use of repetitive transcranial magnetic stimulation (rTMS)[END_REF][START_REF] Hallett | Transcranial magnetic stimulation and the human brain[END_REF], or patients suffering from perceptual disorders [START_REF] Leocani | Human corticospinal excitability evaluated with transcranial magnetic stimulation during different reaction time paradigms[END_REF]. The development of ultrasonic neuro-modulation is largely motivated by future therapeutic applications. This is the only neuromodulation technology currently capable of being selective, targeted, reversible and noninvasive with millimeter-scale precision essentially across the entire human and non-human primate brain(6-8) and peripheral nervous system [START_REF] Tsui | In vitro effects of ultrasound with different energies on the conduction properties of neural tissue[END_REF][START_REF] Colucci | Focused Ultrasound Effects on Nerve Action Potential in vitro[END_REF][START_REF] Juan | Vagus nerve modulation using focused pulsed ultrasound: Potential applications and preliminary observations in a rat[END_REF].

Moreover, deep seated clinical sites could be targeted with TUS, as is currently the case in the thalamus with transcranial high intensity focused ultrasound. Using this co-registration, the cigar-shaped focal spot of of FUS was placed over the left FEF (red spot). For each animal the FEF was identified with anatomical landmarks (arcuate sulcus AC, and principal sulcus PS). A coupling cone filled with water ensured ultrasonic coupling between the transducer and the animal's head. The neurophysiological recordings were performed in the left SEF area (yellow spot) through a recording chamber and grid adaptor.

11 and 8

 8 years old and 17 and 7 kg respectively) participated in this study. The monkeys were paired-housed and handled in strict accordance with the recommendations of the Weatherall Report on good animal practice. Our experiments were approved by the Animal Health and Veterinary Medication Division of the Department of Public Veterinary Health, Nutrition and Food Safety of the French Ministry of Health (current renewal N°

  in the skull(Aubry et al. 2003 Experimental demonstration of noninvasive transskull adaptive focusing based on prior computed tomography scans). α and I were experimentally determined by transmission measures and simulations to a monkey skull flap : the best match was found with I = 0.5 and α = 8.0 NO/-Q/R3S T.T , leading to α = 2.3 db/cm at 320 kHz.

Figure 3 :

 3 Figure 3: Recordings of neuronal spikes activated by single pulse TUS. (a) Example raw

Figure 1 :

 1 Figure 1: The ultrasonic transducer was attached to a neuro-navigation module that

Figure 2 :

 2 Figure 2: Peak negative pressure (MPa) in the brains of monkey Y (left) and L (right)

Figure 3 .

 3 Figure 3. Average recorded spikes per seconds with (red lines) or without (blue lines)

Figure 4 .

 4 Figure 4. Modulation while comparing TUS and control trials (p< 0.01) in the population

Figure 5 .

 5 Figure 5. Spike waveforms with and without FUS. A) All spike waveforms from a

Figure 6 .

 6 Figure 6. Height / Width ratio of spike waveforms with and without FUS for all neurons

  ratio under FUS Height/Width ratio without FUS Height/Width ratio under FUS Height/Width ratio without FUS

Table 1 :

 1 Estimations of peak pressure in monkeys' brains

			Monkey Y	Monkey L
	VPP		139±26 V	56±15 V
	Equivalent PNP in free	0.76±0.08 MPa	0.31±0.14MPa
	water
	Simulated steady state	0.41±0.08 MPa	0.24±0.07 MPa
	PNP in FEF
	Table 2: Acoustic parameters
	a)	Water		Tissue	Max
	Velocity (m/s)	1500		1560	3100
	Density (kg/m 3 ) 1000		1030	2200
	Absorption (dB/cm/MHz 1.1 )	0		0.38	8.0
	a)	Water	Tissue	Max
	Velocity (m/s)	1500	1560	3100
	Density (kg/m 3 ) 1000	1030	2200
	Absorption	0	0.38	8.0
	(dB/cm/MHz 1.1 )
			Monkey Y	Monkey L
	HU max		3300	3000
	HU tissue		1035	1031

ACKNOWLEDGMENTS

This work has been supported by the Bettencourt Schueller Foundation, the LABEX WIFI (Laboratory of Excellence within the French Program "Investments for the Future") under references ANR-10-LABX-24 and ANR-10-IDEX-0001-02 PSL and by the National Agency for Research under the program "future investments" with the reference ANR-10-EQPX-15. We wish to thank Jeff Schall and Jérôme Sallet for reading a preliminary version of the manuscript and for suggesting useful improvements. We also wish to thank Mathieu Santin for anatomical MRI acquisition.

AUTHOR CONTRIBUTIONS

N.W., T.D., J.F.A., and P.P., designed the experiments; N.W., performed the experiments. C.C., and J.F.A, performed the simulations. N.W., P.M.D., and P.P., analyzed the data. N.W., T.D., C.C., P.M.D., M.T., J.F.A and P.P wrote the manuscript.