Olivier Hugon
email: olivier.hugon@ujf-grenoble.fr

Fadwa Joud

Eric Lacot

Olivier Jacquin

Hugues Guillet De Chatellus

Coherent microscopy by laser optical feedback imaging (LOFI) technique

Keywords: Optical feedback, phase-sensitive detection, scanner, vignetting, field curvature

Introduction

The coherent re-injection of light in a laser cavity can be at the origin of a modification of the laser behavior. Often considered as a parasitic phenomenon, it can for example drive the laser in a chaotic regime [1]. When it is correctly controlled, the optical reinjection in a laser can however be used in order to improve its characteristics, such as for example the linewidth narrowing [START_REF] Besnard | Feedback phenomena in a semiconductor laser induced by distant reflectors[END_REF], or for metrology purpose [START_REF] Campbell | An electronic quadrature technique for laser interferometry[END_REF][START_REF] Peek | Axial mode number of gas lasers from moving-mirror experiments[END_REF][START_REF] Rudd | A laser Doppler velocimeter employing the laser as a mixer-oscillator[END_REF][START_REF] Brown | Frequency stabilized lasers: optical feedback effects[END_REF][START_REF] Sarid | Compact scanning-force microscope using a laser diode[END_REF][START_REF] Groot | Range-dependent optical feedback effects on the multimode spectrum of laser diodes[END_REF][START_REF] Gilles | Near-field amplitude and phase measurements using heterodyne optical feedback on solid-state lasers[END_REF][START_REF] Sudo | Determination of velocity of self-mobile phytoplankton using a self-mixing thin-slice solid-state laser[END_REF], thus conferring on the laser the role of source and detector at the same time. Since 1963, this principle was employed in an autodyne configuration to measure distances or speeds [START_REF] King | Report of demonstration at the 47th[END_REF]12]. More recently, a heterodyne imagery technique called LOFI (Laser Optical Feedback Imaging) was developed [13] and has been used for miscellaneous applications [START_REF] Hugon | Submicrometric displacement and vibration measurement using optical feedback in a fiber laser[END_REF][START_REF] Muzet | Experimental comparison of shearography and laser optical feedback imaging for crack detection in concrete structures[END_REF][START_REF] Blaize | Phase sensitive optical near-field mapping using frequency-shifted laser optical feedback interferometry[END_REF]. Coherent microscopy is one of many areas of application of this versatile technique. It provides access to the complex amplitude of the backscattered field and thus allows obtaining a high resolution, 3D representation of a sample [START_REF] Sticker | En face imaging of single cell layers by differential phase-contrast optical coherence microscopy[END_REF][START_REF] Kemper | Endoscopic and microscopic coherent metrology for biomedical applications[END_REF][START_REF] Ralston | Interferometric synthetic aperture microscopy[END_REF]. Classically, to measure the height of a sample with a high resolution (i.e. much smaller than the wavelength), one have to build an interferometer which is usually complicated to implement. The LOFI technique allows making interferometric measurement with a particularly simple arrangement which can easily be adapted on a conventional microscope to provide an existing setup with new imaging modalities. Moreover, under some particular conditions, the reinjection of light in the laser may introduce an optical amplification of the interferometric signal.

Principle

The LOFI technique allows the coherent detection of the photons that have been backscattered by a target [START_REF] Lacot | Coherent laser detection by frequency-shifted optical feedback[END_REF]. For that purpose, a laser which is a coherent light source is used in a heterodyne interferometer of which the reference arm is replaced by the laser cavity itself. By doing so, interferences occur inside the laser cavity because of the reinjection of a small amount of the backscattered light, accordingly to the reverse path principle. These interferences can be analyzed by measuring a fraction of the laser output power. This approach has several advantages that will be described below.

First, the detection of backscattered photons allows working with a very wide range of targets, transparent or opaque, thin or thick.

Only a single interface between two media with different optical index is needed to get a signal.

Then, much information is contained in the complex amplitude of the interferometric signal. Its magnitude depends on the amount of detected photons, which is linked to the refractive index contrast, absorption and diffusion coefficients, polarization and even the target topology. On the other hand, its argument is governed by the optical path length between the laser and the target. This quantity depends on the refractive index and thicknesses of the media through which light traveled. Several kinds of images can then be realized, with a contrast generated by the opto-geometrical parameters of the target, thus having different significations and values.

The coherent detection is also favorable to obtain high resolution images in a scattering medium [START_REF] Lacot | Laser optical feedback tomography[END_REF], because the multi-scattered photons have lost their coherence. Only the ballistic photons that keep the spatial information of their interaction with the target are detected. Unfortunately, they are usually very few and it is therefore necessary to implement a very sensitive detection scheme.

The use of the laser cavity as a reference also brings some benefits. First, there is no need for a delicate alignment between the probe beam and the reference beam because the setup is self-aligned by construction. Then, the main trump of this technique lies in the interferometric signal amplification by a resonance phenomenon in the laser dynamics. Such amplification is made possible by the use of a class B laser which is characterized by a photon lifetime in the cavity that is shorter than the fluorescence lifetime of the active medium. These lasers show relaxation oscillations of their intensity and are very sensitive to optical feedback, particularly when the reinjected light has a frequency shift which is close to the relaxation frequency. We have shown that this gain increases the signal to noise ratio of the system for given laser power and noise detection level [START_REF] Lacot | Comparative study of autodyne and heterodyne laser interferometry for imaging[END_REF][START_REF] Jacquin | Experimental comparison of autodyne and heterodyne laser interferometry using a Nd :YVO4 microchip laser[END_REF].

Experimental setup

Figure 1 shows a schematic of the experiment. In this study, we used a diode-pumped Nd 3+ :YAG microchip laser with a 800 µm thick cavity which emits a radiation of a few mW at the wavelength of 1.064 µm. Its relaxation frequency is around 1 MHz. The heterodynation is realized with a frequency shifter constituted by two acousto-optic deflectors (IntraAction, ATM-801A2), respectively operating at 81.5 MHz (order +1) and 81.5 MHz-∆f (order -1), so that the overall frequency shift is ∆f. This frequency shift can be controlled from the AOD driver (IntraAction, DFE-804A4A). It is chosen close to the relaxation frequency of the laser in order to profit from the dynamical amplification phenomenon. A two-axis galvanometric mirror scanner (Cambridge Technology, 6220) makes it possible to move the beam on the surface of the sample to obtain an image. The beam is injected into the microscope (Zeiss, Axio Imager M1) via the side port and is focused on the sample by the objective (Zeiss, A-Plan 40x/0.65). Because of the great sensitivity of the system with respect to any back-reflection, all the lenses (except for the microscope objective) are anti-reflection coated for the working wavelength: 1.064 µm. A fraction of the output intensity of the laser is sent on a Si-photodiode with a beam splitter. The magnitude and phase of the interferometric signal are provided by a lock-in amplifier operating at 2∆f (Stanford Research, SR844) and digitized by a 12 bits data acquisition board (National Instruments, NI USB-6211). Home-made software developed under the LabVIEW environment has in charge to drive the scanner and to synchronously acquire the data in order to build the images.

Scanner induced distortions

The phase measurements are those that require the most attention. In order to have good quality images, it is necessary to scan the target quickly to avoid any drift during the acquisition. One should also remove any source of vibrations. For these reasons, it is preferable to use a galvanometric mirrors scanner to scan the beam on the target rather than moving the target itself. However, this solution also has some drawbacks that result in distorted magnitude and phase images.

In an ideal case, the scanner must be positioned in a plane conjugate to the entrance pupil of the microscope objective to avoid the vignetting phenomenon when scanning. In practice, it is not possible to satisfy this condition simultaneously for both the scanner mirrors (unless they are conjugated with additional optics, which complicates the setup and makes it more cumbersome). Therefore, it occasions a vignetting effect that can be easily assessed.

The last part of the setup taking place between the scanner and the target is detailed on Figure 2. P 1 is the conjugate plane of the entrance pupil Pu of the microscope objective L 5 . The scanner mirrors M 1 and M 2 are separated by a distance e. They are arranged on either side of the plane P 1 so that M 1 is separated from P by a distance d. For simplicity of representation, the deflection by the two mirrors has been represented in the same plane. From this figure, it is easy to establish that when the M 1 mirror deflects the beam by a small angle α from the optical axis, the lateral shift of the beam compared to the entrance pupil of the objective is:

α d f f S ' P 3 4 = (1)
In the same way, we have for the mirror M 2 :

( )α d e f f T ' P 3 4 - = (2)
L 3 and L 4 lenses are wide enough to avoid losses during the scan. In order to profit from the whole numerical aperture of the microscope objective L 5 and, in the same time, to limit the power losses, the lenses system has been designed so that the radius of the beam, that is assumed to be gaussian (see Figure 3), is 1.5 times smaller than the radius of the objective entrance pupil, corresponding to a 99% power transmission ratio when α=0°. By computing the ratio of the field that passes through the objective pupil as a function of the lateral shift, one can then quantify the vignetting effect on magnitude images of a flat and uniformly scattering object located in the observation plane P 3 (Figure 4). These results suggest that the best solution to minimize the vignetting effect on magnitude images (therefore to limit the degradation of the signal to noise ratio) is to share it out on both dimensions by locating the scanner so that the ideal plane P 1 is exactly at the same distance from the mirrors. By doing so, the magnitude is significantly affected only on the edge of the accessible field. The resulting magnitude variations can then easily be corrected with a post processing of the images.

On the other hand, the length of the optical path between the laser cavity and the laser spot on the target is modified during the scan.

This effect can also be calculated. The path length variations in both dimensions will be determined by comparison with a reference path, in dotted line on Figure 2. Whatever the value of the deflection angle α of the beam relative to the optical axis, all the optical paths between P and P' have the same length because these points are conjugated. Moreover, because Pu and P 3 are in the focal planes of L 5 , P' is at the same optical distance from every point on P 3 . The reference optical path length is then independent of α.

If the first scanner mirror M 1 is at a distance d from P, it is easy to find that the difference between the real and the reference optical paths is given by : ( ) ( )

1 cos d P M Q M 1 1 - = - α (3)
Similarly, the distance between the mirror M 2 and the point P is responsible for an optical path length variation when scanning which is equal to:

( ) ( ) ( ) α cos 1 d e PR PM 2 - - = - (4)
By considering that the mirrors M 1 and M 2 deflect the beam by an angle α and β respectively, the phase function of the scanning process can be determined from equations ( 3) and ( 4):

( ) ( ) ( ) [ ( ) ( ) ( )] β α β α cos 1 d e 1 cos d k 2 , 0 - - + - + Φ = Φ ( 5
)
where Φ 0 is an arbitrary phase term, k=2π/λ is the wave number and the factor 2 outside the brackets corresponds to a round trip between the laser and the target. Equation ( 5) has been evaluated for three different scanner positions and the results are shown on Figure 5. These images correspond to the phase images that would be obtained by scanning a flat target which is perpendicular to the optical axis. To recover an image that corresponds to the target profile, the phase must then be processed to remove the scanner induced field curvature.

Finally, another effect will be discussed. So far, we have assumed that the mirrors were hit by the beam on their rotation axis, which is not obvious to achieve in practice without monitoring. According to the Figure 6, when the beam is correctly centered on the mirror (i.e. the optical axis intersects the rotation axis) it is reflected at point A, along the dotted line, for any value of α. If it is off-center, then the mirror is hit at point B after a rotation through an angle α/2, introducing an optical path length difference, in comparison with the centered case, which is given by:

            + - = + α π 4 3 sin 2 1 g BC AB (6)
During the scan, an additional phase variation is thus introduced by this decentering:

( )

            + - = Φ α π α 4 3 sin 2 1 kg 2 g (7)
As can be seen on Figure 7, the correction is noticeable, even for a small shift. A similar expression of this correction is obtained for each mirror, which allows us to compute a new phase pattern taking into account the effect of mirrors decentering (Figure 8). It should be noted that a bad centering is also responsible for an additional vignetting effect on magnitude images.

To summarize, the monitoring of the phase images is a good and convenient way to adjust the position and the centering of the scanner. The best configuration which minimizes the vignetting effect corresponds to a centered, saddle shaped phase image.

Phase unwrapping

When scanning a target, the phase signal provided by the lock-in amplifier is wrapped, which means that it is known modulo 2π. To recover the real profile of a target, without discontinuity, the phase signal must still be unwrapped. The MLBT algorithm [START_REF] Hardie | Robust phase-unwrapping algorithm with a spatial binary-tree image decomposition[END_REF] has been chosen for its robustness towards experimental noise. However, correct phase unwrapping is impossible if the phase fringes are not sufficiently spatially resolved and aberrant results can then be obtained. It is therefore not advisable to unwrap the raw phase image because, as we have shown in the previous section, the scanner induced phase variations are very fast away from the optical axis (i.e. away from the image centre) and it would necessitate a large spatial oversampling which could be completely needless, considering the spatial resolution of the image. Therefore, it is wiser to subtract modulo 2π the scanner phase function from the raw phase image before unwrapping it.

Experimental results

The LOFI microscopy technique was implemented to get magnitude and phase images on two different kinds of samples. The first one is a part of an AFM calibration grid constituted by a set of microscopic blocks etched on a silicon surface. This sample was used first in order to calibrate the horizontal scales of our images. On the magnitude image (Figure 9), the surface micro-structure is clearly visible. The lateral resolution is given by the beam waist on the target and is about 0.5 µm with the microscope objective that has been used. This value is confirmed by the thickness of the blocks edges (dark lines on the image). The image has been voluntarily oversampled in order to facilitate the blocks edges localization with a precision that is better than the optical resolution. Since the whole sample is made of silicon, the image contrast is obviously not only governed by the optical index contrast between the sample and the surrounding medium (the air in this case), but also by the geometrical characteristics of the sample surface. For instance, there is a contrast between the blocks and the space around them because these regions have a different altitude, and we know that the detection efficiency highly depends on the distance between the scattering/reflecting surface and the focal plane [START_REF] Hugon | Cell imaging by coherent backscattering microscopy using frequencyshifted optical feedback in a microchip laser[END_REF]. In the case of a reflecting surface, the incidence angle of the beam also determines the amount of light that will be reinjected, hence the signal magnitude (the higher magnitude is obtained when the beam has a normal incidence). This is the reason why the blocks borders appear darker, as the image edges where the incident beam is at an angle because of the scanning process (see Figure 2). It should be noted that the vignetting effect is negligible in this case because of the small dimensions of the image compared to the accessible field size.

On the other hand, the phase image (Figure 10) shows the true topography of the sample with an interferometric resolution [START_REF] Lacot | Phase-sensitive laser detection by frequency-shifted optical feedback[END_REF], that is to say about a few nm on the vertical scale. According to the manufacturer, the blocks height is 140 nm ± 10%. The precision of the phase determination is strongly dependant on the signal to noise ratio, which is enhanced by the LOFI technique. Thus, it is possible to have a good quality phase measurement, even with a weak backscattered signal (which is typically the case on the blocks borders).

Magnitude and phase images have been acquired simultaneously in 10 s for a size of 256 × 256 pixels. This time corresponds to 150 µs by pixel which is the response time of our instrumentation.

The other object that we have observed is a biological sample. The cells from a fresh blood drop have been separated on a glass microscope slide by the classical blood smear technique. The edge of a red blood cell is visible on the magnitude image (Figure 11) but the phase image (Figure 12) is much more interesting as we clearly see the characteristic concave shape of the cell. This image also shows that the cell seems to be surrounded by some substance, probably the plasma. It should be noted that there is no height scale on Figure 12. Indeed, when dealing with thin and transparent objects such as a cell in the near infrared, the measured phase is no longer proportional to the object height. A very simple model can offer an explanation to this phenomenon. It results from the interference between the signals scattered or reflected by the different interfaces of the object when they are very close (that means separated by a distance which is small or comparable to the Rayleigh range). Let us consider a target constituted by a thin transparent layer on a semi-infinite substrate (Figure 13).

The first contribution to the detected signal comes from the reflection at the superstrate/layer interface. In the reference plane, after a round trip, the complex amplitude of the reflected field is proportional to:

( )

1 1 i exp E Φ = 1 E (8)
with

        + - = sup sup 0 1 n n n n E E (9) and ( ) sup 1
n h d k 2 - = Φ (10)
where E 0 is the incident field magnitude and k=2π/λ is the wave number.

The second contribution comes from the layer/substrate reflection (one reflection on this interface and two transmissions through the other interface):

( )

2 2 i exp E Φ = 2 E (11)
with

        +         + -         + = sup sub sub sup sup 0 2 n n n 2 n n n n n n n 2 E E (12) and ( )
[ ]

hn n h d k 2 sup 2 + - = Φ (13)
One then detects the coherent sum of these two fields, whose magnitude and phase have been plotted on Figure 14 as a function of the layer parameters n and h, in the particular case where n sub =1.51 and n sup =1 corresponding to a sample on a microscope slide in air. It is clear that the phase is not proportional to the layer thickness, except for n=n sub (like in Figure 10) because in that particular case, the layer/substrate interface vanishes and there is no more interference.

However, Figure 14 suggests that it is possible to simultaneously determine the thickness and the optical index of the layer from both magnitude and phase of the reflected field. Unfortunately, it only works in normal incidence; therefore, this method can't be applied for profilometry purpose because the detected magnitude also depends on the interfaces locale orientations [START_REF] Hugon | Cell imaging by coherent backscattering microscopy using frequencyshifted optical feedback in a microchip laser[END_REF] which are generally unknown.

Conclusion and outlook

The application of the LOFI technique to coherent microscopy was presented. The simple setup can easily be implemented on an existing microscope in order to extend its imaging modalities.

The LOFI is a point based technique; it means that to build an image, the target must be scanned. A galvanometric mirrors scanner moving the beam on the sample surface is a fast and convenient solution, but it induces some distortions on images that have been discussed. In particular, the phase pattern generated by the scanning process can be useful to optimize the scanner positioning in order to reduce the vignetting effect.

Finally, magnitude and phase images obtained on a micro-structured silicon sample and a red blood cell have been presented to illustrate the potentialities of this non conventional imaging technique.

These results encourage us to develop this technique in order to realize in depth microscopy of scattering media such as biological tissues. Indeed, the characteristics of the LOFI are particularly interesting for the biomedical applications. The used laser emits a radiation of a few mW at 1.064 µm that suits well for biological tissues observation. Coherent detection with the great sensitivity provided by the dynamical amplification performed by the used laser makes it possible to obtain images in scattering media, without

Figure 1 -

 1 Figure 1 -Description of the experimental set-up: L 1 , collimation lens; BS, beam splitter; AOD 1 , acousto-optic deflector at 81.5 MHz (order +1); AOD 2 , acousto-optic deflector at 81.5 MHz-∆ ∆ ∆ ∆f (order -1); L 2 , collimation lens; M 1 M 2 , two-axis galvanometric mirrors scanner; L 3 , scan lens; L 4 , tube lens; L 5 , microscope objective.

Figure 2 -

 2 Figure 2 -Ray tracing for the computation of amplitude and phase images distortions. f 3 , f 4 and f 5 are respectively the focal lengths of the L 3 , L 4 and L 5 lenses. Pu is the entrance pupil of L 5 and P 3 is the observation plane.

Figure 3 - 1 /

 31 Figure 3 -1/e amplitude contours of the gaussian beam between the scanner and the observation plane. The parameters for the simulations are: α α α α=0°; f 3 =50 mm; f 4 =150 mm; f 5 =4.7 mm.

Figure 4 -

 4 Figure 4 -Computed magnitude images exhibiting the vignetting effect for three different positions of the scanner. The parameters for the simulations are: e=9.1 mm; f 3 =50 mm; f 4 =150 mm; objective pupil radius=4 mm; beam radius on the objective pupil=2.66 mm.

Figure 5 -

 5 Figure 5 -Computed phase patterns generated by the scanning process for three different positions of the scanner. The parameters for the simulations are: φ φ φ φ 0 =0 rad; λ λ λ λ=1064 nm; e=9.1 mm.

Figure 6 -Figure 7 -

 67 Figure 6 -Rays reflected by one of the scanner mirrors when the beam intersects (dotted line) or not (plain line) its rotation axis. A and A' are respectively the rotation axis of the centered and decentered mirrors, separated by a distance g; α α α α=0° corresponds to the incident beam reflected along the optical axis in both cases.

Figure 8 -

 8 Figure 8 -Shifted phase pattern computed when the laser beam doesn't intersect one of the mirrors rotation axis. The parameters for the simulations are: λ λ λ λ=1064 nm; e=9.1 mm; d=e/2; g M1 =50 µm; g M2 =0 µm; -2°≤ ≤ ≤ ≤α α α α,β β β β≤ ≤ ≤ ≤2°.

Figure 9 -

 9 Figure 9 -Magnitude image of a micro-structured silicon surface (256× × × ×256 pixels).

Figure 10 -

 10 Figure 10 -Phase image of a micro-structured silicon surface (256× × × ×256 pixels).

Figure 11 -

 11 Figure 11 -Magnitude image of an isolated red blood cell on a glass slide (200× × × ×200 pixels).

Figure 12 -

 12 Figure 12 -Phase image of an isolated red blood cell on a glass slide (256× × × ×256 pixels).

Figure 13 -Figure 14 -

 1314 Figure 13 -Model for the multiple scattering/reflection effect assessment.

need for contrast agent [26]. In addition, the TEM 00 mode of the laser plays the role of a spatial filter during the detection stage, like the pinhole of a confocal microscope, which allows the localization of the signal in the three dimensions of space [27]. However, some work still has to be done to eliminate the spurious reflections that scramble the image by causing interferences when they are superimposed on the useful signal from the focal plane. Usually negligible, the spurious reflections start to be annoying when the signal is very weak, which is typically the case when it travels trough a scattering medium. The most important contributions to this parasitic signal come from the microscope objective and the surface of the sample. The presence of a cover plate on the sample also causes an important unwanted echo. A promising solution based on a multi-beam illumination of the sample [28] is currently being implemented.


Anusch Arezki

Guy Chabot

Lionel Quentin

Daniel Scherman

G Rard Jaouen

Emilie Brul

Synthesis and biological evaluation of novel ferrocenyl curcuminoid derivatives

Keywords: curcuminoid, ferrocenyl, B16 melanoma, NIH 3T3 cells, endothelial cells, cancer

With the purpose to improve the biological activities of curcumin, eight novel ferrocenyl curcuminoids were synthesized by covalent anchorage of three different ferrocenyl ligands. We evaluated their cytotoxicity on B16 melanoma cells and normal NIH 3T3 cells, their inhibition of tubulin polymerization and their effect on the morphology of endothelial cells. The presence of a ferrocenyl side chain was clearly shown to improve the biological activity of most of their corresponding organic curcuminoid analogues.

1 dietary phytomolecule has been used for centuries in Asia for its medicinal properties, and regained considerable attention recently due [ ] to its several health promoting effects, such as antioxidant, 2,3 anti-inflammatory, 4 chemopreventive, 5 7 and antitumor activities on

[ ] [ ] [ -]
several cancer types. 8,9 The precise mechanism of action of curcumin in cancer cells is still not completely understood and appears to be [ ] mediated by several different pathways. For example, curcumin was reported to induce apoptosis by suppression of nuclear factor-B κ described in several cancer cell lines such as melanoma. 10 Curcumin was also reported to interact with tubulin polymerization, although

[ ]
these data are considered controversial because high concentrations are needed for inhibition. 11 13

[ -]

Great interest has been devoted to the synthesis of new curcumin analogues exhibiting enhanced biological properties. For example, dimethylcurcumin (Figure 1), is more effective at inhibiting colon cancer cell proliferation 14 and is metabolically more stable.

in vitro [ ] [

15 Another interesting strategy has been to chelate a metal to curcumin in order to increase the cytotoxicity of the parent molecule.

] Indeed, the use of metal-based compounds for the treatment of cancer has found significant interest in medicine 16 (e.g., clinical use of

[ ]
cisplatinum compounds), thus encouraging chemists to design new metallated anticancer agents. Several examples of metallated curcuminoid analogues, which all involve the coordination of the metal to the oxygens of the -diketone, have been evaluated for their β biological properties. 17 25 Because there were no published reports of an organometallic moiety covalently grafted to curcumin or its [ -] derivatives, we recently described the synthesis of the first examples of such ferrocenyl derivatives , and (Figure 1). 26 We therefore 5 6 7

[ ] became interested to further investigate the covalent anchorage of a ferrocenyl unit to several curcuminoids. We hypothesized that the presence of ferrocene in curcumin derivatives could enhance the biological activity against cancer cells, as we previously reported for ferrocenyl derivatives of steroidal antiandrogens 27 and of the antioestrogen tamoxifen. 28,29

[ ] [ ]
In the present report, we synthesized eight novel ferrocenyl curcuminoids in order to evaluate some of their biological properties. To do so, three different ferrocenyl spacer chains were chosen to be anchored to the central carbon of the curcuminoid skeleton: ferrocenyl propenone to increase the conjugation up to the ferrocenyl (series A), ferrocenyl methylene forcing the -diketone to be in its diketone β tautomer form (series B), and ferrocenyl ethanone interrupting the conjugation between the skeleton and the ferrocenyl moiety (series C).

The effect of the substitution by a methoxyl instead of an hydroxyl on the two phenyl groups was also investigated by choosing dimethylcurcumin , 3,5-dimethoxycurcumin and trimethoxycurcumin (Figure 1) as curcuminoid skeleton, in addition to curcumin.

3 4

Although and have already been reported in the literature, 30 they have not been studied in details.

4 [ ]

We report here the synthesis of several ferrocenyl derivatives of the curcuminoids , , and . We also tested these ferrocenyl 1 2 3 4

derivatives for some important biological activities required for anticancer activity, i.e., their cytotoxicity against murine B16 melanoma cells and NIH 3T3 normal cells, their effect on the inhibition of tubulin polymerization and the analysis of the morphological effects on endothelial cells (as a model of antitumor antivascular effect). 31 For comparison purposes, curcumin and compounds , and also

[ ]

3 4

underwent these biological tests.

Synthesis of curcuminoid ferrocenyl derivatives

The curcuminoids , , and (Figure 1) were synthesized following Pederson s conditions 32 by condensing an appropriate 1 2 3 4

' [ ]
aldehyde with 2,4-pentanedione in the presence of a base as well as tributylborate and boric anhydride. The latter is required to complex the 2,4-dione moiety in order to prevent a Knoevenagel condensation on C-3. It was shown that the -diketone of curcumin exists as β keto-enol tautomer in solid state, 33 as well as in solution, 34 as observed for the three other curcuminoids ( , and ).

[ ] [ ]

3 4

We previously reported the synthesis of compound bearing a ferrocenyl propenone moiety (Scheme 1), obtained from 5 3,5-dimethoxycurcumin . 26 Following the same procedure, dimethylcurcumin and trimethoxycurcumin analogues and were also 3 [ ] 8 9

successfully synthesized. The enol of and was deprotonated by stirring in the presence of sodium hydride for 2 hours at room 2 4

temperature. Ferrocenyl propynone 35 was then added to produce and with yields of 25 and 14 , respectively. The rather modest

[ ] 8 9
% yields obtained, also observed previously for were explained by the degradation of the starting material if the reaction time was 5

prolonged. H NMR spectra of and were similar to that of and showed that the peak at 5.85 ppm, corresponding to the central proton of the -diketone in its keto-enol form, had disappeared and confirmed that the substitution took place at the desired C-4 position. The β crystal structure of has previously been reported. 26

[ ] 5
In order to force the -diketone of the curcuminoid to stay in its diketo form instead of the keto-enol form, the organic curcuminoids With the purpose of studying the effect of an interruption of the conjugation between the curcuminoid and the ferrocenyl, a ferrocenyl ethanone moiety was therefore grafted to dimethylcurcumin and a protected curcumin, (Scheme 3). Indeed, for this reaction, it was 2 14

necessary to protect the phenols of curcumin. We previously described a modified synthesis of a bis-tetrahydropyran ( ) and bis-14 t

-butyldimethylsilyl curcumin 26 and discussed the relative stability of ferrocenyl protected curcumin towards the different conditions of

[ ]
deprotection for a THP and a TBDMS group. For this reaction, the protection by a THP moiety was chosen because deprotection occurs in milder conditions than for TBDMS. Compounds and were deprotonated by sodium hydride in the same conditions as described 2 14

above, before -bromoacetylferrocene 37 (obtained from ferrocene and 2-bromoacetyl bromide) was added dropwise. After heating for 4 and showed that the -diketones were in their diketo-rather that their keto-enol forms. Indeed, in their H NMR spectra, the H-4 singlet 16 β 1 of the keto-enol form at 5.85 ppm disappeared, while a triplet at 4.95 ppm was observed, attributed to the H-4 of the diketo form.

Additionally, one peak at 195 ppm, corresponding to the two symmetrical carbonyl groups of the curcuminoid skeleton, was observed in their C NMR spectra. Similar observations were reported by Pederson for the synthesis of several substituted organic curcuminoids. 32 For example, the synthesis consisting in the alkylation of curcumin on the central carbon (C-4) with benzyl bromide only led to the tautomer in its diketo form.

In order to obtain the curcumin ferrocenyl ethanone derivative, the deprotection of the THP protecting groups of was carried out by 16 using pyridinium -toluenesulfonate (PPTS) in CH Cl /EtOH to lead to the formation of the desired unprotected ferrocenyl curcumin p 2 2

17

in 68 yield, confirming the stability of ferrocenyl ethanone curcuminoids under mild acidic conditions. The NMR spectra of showed

% 17
that the -diketone is in the same diketo form as for the protected derivative .

β 16

Biological evaluation of the ferrocenyl derivatives of curcumin

The synthesized ferrocenyl curcuminoid derivatives were evaluated for some biological properties frequently linked to in vitro anticancer activity, i.e., for their cytotoxicity against murine B16 melanoma cells and NIH 3T3 normal cells, their effect on tubulin polymerization, and their induction of a rapid morphological change of endothelial cells (rounding up) that may hint to a possible antivascular effect. For comparison purposes, the organic compounds , , and were also tested in parallel, as well as combretastatin 1 2 3 4

A4 (

), a well-known vascular disrupting agent. 38

CA-4 [ ]

Cytotoxicity

The murine B16 melanoma cells were chosen to evaluate the cytotoxicity because melanoma is particularly refractory to chemotherapy. Table 1 presents the cytotoxicity values (IC ) of the parent compound curcumin and its analogues. Curcumin showed an 50 IC value of 8.5 M, which is comprised within the literature values obtained for this cell line. For example, B16-BL6 melanoma 39 showing the decrease in number of viable cells upon treatment with increasing concentrations of the organic 3,5-dimethoxycurcumin and 3 its ferrocenyl analogue is depicted in Figure 2. 12 As a result, in the B16 cellular model, the diketo form of the ferrocenyl derivatives, as well as conjugation up to the ferrocenyl (series B), seemed required for high cytotoxic activity. In series C, and , which are also in diketo form, but have disrupted conjugation, did For comparison purposes, we also evaluated the cytotoxic activity of these compounds against the NIH 3T3 normal cell line. Table 1 shows that the IC of the normal cells were higher than the IC for the cancer cells (B16 melanoma) for most compounds (with the 50 50 exception of ). This could indicate an interesting selectivity of cytotoxic action against cancer cells compared to normal ones, as 17 previously observed for other curcuminoids (reviewed in reference 40-b).

Inhibition of tubulin polymerization (ITP)

We were also interested to test our curcumin ferrocenyl derivatives on tubulin polymerization, because several potent clinically used anticancer drugs are indeed known to act on tubulin dynamics 41,42 (e.g., vinca alkaloids, taxanes), and because it was recently reported

[ ]
that curcumin could also inhibit microtubule assembly. 11

[ ]
In our assay conditions, curcumin was shown to inhibit microtubule formation with an IC value of 17.9 M (Table 1). Our data 50 μ corroborate Gupta and coworkers observations, although their reported IC value is higher than ours (calculated IC 80 M). Although the high activity of combretastatin could not be reached by the symmetrical organometallic compounds, the presence CA-4 of a ferrocenyl moiety was generally linked to a marked increase in tubulin inhibition activity compared to their organic counterparts, with the exception of curcumin substituted with ferrocenyl methylene moiety , which showed a similar ITP activity. 10

The lowest IC values (2.1 to 5.8 M) were observed with the ferrocenyl propenone curcuminoids (series A). Compounds of series B 50 μ and C were all good tubulin polymerization inhibitor with concentrations comprised between 3.6 and 12.6 M, with still the exception of μ the curcumin analogue

. By examining the substituents on phenyl rings, it was observed that the 3,4-dimethoxyl substitution of the 10 ferrocenyl curcuminoids (dimethylcurcumin derivatives , and ) produced the best antitubulin activity (2.5 5.5 M), whatever the 8 11 15

μ spacer chain used.

Morphological effects on endothelial cells

Because several antitumor antivascular compounds act on endothelial cells, we also tested the morphological effects of our novel ferrocenyl compounds on immortalized HUVEC (Ea.hy 926), as a model of potential antitumor antivascular effect. 31,38

[ ]
A rounding up effect of endothelial cells was observed only at high concentrations (50 100 M) for the organic curcuminoids (Table μ 1). The introduction of the ferrocenyl allowed the morphological rounding up to occur at significantly lower concentrations for all series A, B and C. In the series A, the concentration needed for rounding up was 12.5 M. For series B, with the exception of the dimethylcurcumin μ and 3,5-dimethoxycurcumin analogues ( and ), a significant decrease in the concentration needed for rounding up of endothelial 2 3 11 12 cells was noted, as well as in series C (25 M).

μ These data clearly indicate that the presence of a ferrocenyl moiety could significantly lower the concentration needed for the rounding up of endothelial cells for the majority of our symmetrical ferrocenyl curcuminoids. Figure 3 shows a representative change in endothelial cells shape for compound (series B) at 6.3 M. 13 μ In addition, the increase in morphological activity of these curcuminoid analogues appeared to be correlated with their antitubulin activity. As a matter of fact, we observed a statistically significant linear correlation between the ITP values (x) and the concentration needed for rounding up (y), indicating that the best ITP values were linked to a lower concentration for morphological activity (y 1.81x

= +
13.71, R 0.661, P<0.007).

=

Conclusion

In this work, eight novel ferrocenyl curcuminoids were synthesized by covalent anchorage of three different ferrocenyl ligands to organic curcuminoids substituted with methoxyl and hydroxyl groups on the aromatic rings. The presence of a ferrocenyl unit clearly improved the biological activity of most novel ferrocenylated curcuminoids. Furthermore, this first study of organometallic moiety covalently grafted to curcuminoids demonstrated the influence of the spacer chain between the curcuminoid skeleton and the ferrocenyl unit on the resulting biological activity. Indeed the curcuminoids bearing the methylene linker exhibited the best cytotoxicity on murine B16 cells (IC 2.2 7.1 M), whereas the presence of the propenone spacer chain induced the best tubulin polymerization inhibition and 50 = -μ rounding up of endothelial cells to occur at lower concentrations. Collectively, our results validate the strategy of covalently anchoring an organometallic moiety to a curcuminoid in order to improve its biological activities.

Footnotes:

Numbering scheme for NMR assignment of curcuminoids skeleton. Experimental procedure and Supporting Information Available:

β 1 , 11 ( 1 NMR

 1111 , and underwent a Knoevenagel condensation with ferrocenecarboxaldehyde to form , for the substitution of organic species (Scheme 2). 36 In our case, ferrocenecarboxaldehyde was condensed[ ]to the corresponding organic curcuminoid in the presence of freshly distilled piperidine in anhydrous DMF instead of a CHCl /EtOH 3 solution, in order to increase the yields of the newly formed ferrocenyl curcuminoids, e.g., from 3 to 27 for . Again, moderate yields% %2228 ) were obtained because of degradation with longer reaction times. Confirmation of the desired substitution was obtained by H -% , as the peak of the central proton disappeared and the diketo form of the -diketone was confirmed by the appearance of a second β carbonyl peak on the C NMR spectrum. It is noteworthy that these conditions of Knoevenagel condensation did not require prior 13 protection of the phenol groups of curcumin, therefore enabling us to prepare in one step. 10

16 % 15 % 16 15

 16151615 stirring overnight at room temperature, and were formed in approximately 50 yield. The reaction has also been 15 carried out using -chloroacetylferrocene, but was obtained in only 17 yield while could not be formed. The NMR spectra of α

μ

  ) and B16-R melanoma 40 (resistant to doxorubicin) presented IC values of 3.6 and 18 M, respectively for a similar [ ] 50 μ 48 hour incubation time.Of the three organic curcuminoids evaluated for the first time in this study on B16 cells, it was of interest to note that trimethoxycurcumin exhibited higher cytotoxic activity (IC 2.8 M) compared to , and (IC values comprised between 8). Looking at the substitution of the aromatic rings, it appears that the absence of substituent at the position decreases μ para somewhat the cytotoxic activity of the resulting curcuminoid, because 3,5-dimethoxycurcumin showed the least cytotoxic effect with an 3IC value of 12.0 M.50The two ferrocenyl unsymmetrical compounds and , as well as the ones of the series A ( , , and ), were shown to be weakly 6 , the four ferrocenyl derivatives of series B (containing a ferrocenyl methylene chain) presented the most cytotoxic activity of our compounds with IC values ranging from 2.ferrocenylation did not significantly improve the cytotoxic activity. However, we 11 13observed an interesting two-fold increase in cytotoxicity for the ferrocenyl analogues of and , i.e.

μ

  our lower IC value is likely due, in our case, to a preincubation period of 45 minutes at room temperature in 50 presence of the tested compound, which allows for the tested ligand to more closely interact with tubulin prior to the beginning of the polymerization with the addition of guanosine triphosphate (GTP) and incubation at 37 C. The function of this preincubation step is to °maximize the potential for detecting the interaction of drugs with tubulin, and to give a better idea of the relative potency of slow-binding agents (such as colchicinoids), both in comparison with each other, and in comparison with other classes of drugs. 43 Of the organic [ ] curcuminoids, only curcumin presented an IC value of less that 30 M. 50 Within the series of the ferrocenyl curcuminoids, the unsymmetrical compound was not markedly active on tubulin polymerization, 6 whereas its isomer presented an IC value of 9.3 M. 7 50 μ

22 Figure 1

 221 Figure 1Organic curcuminoids and first ferrocenyl curcuminoid derivatives

Figure 2

 2 Figure 2 Percent viability of B16 melanoma cells as a function of the concentration of the organic 3,5-dimethoxycurcumin (circles) or the ferrocenyl 3 analogue (triangles) (48 hour exposure time). Error bars, SD. 12

Figure 3 1 2 3

 3123 Figure 3 Morphological effects of the ferrocenyl curcuminoid . Exponentially growing endothelial cells (EA.hy 926) were exposed to the solvent 13 DMSO at 1 (panel A, control), or to the curcuminoid at a concentration of 6.3 M (panel B), and incubated for 2 h (37 C, 5 CO ).%

  Brul é E , Le Bideau F , Joerger J-M , Plamont M-A , Samreth S , Edgar A , Marrot J , Herson P , Jaouen G . Organometallics . 2009 Leclercq G , Quivy J , Tang J , Vaissermann J , Huch é M , Jaouen G . Chem Eur J . 2003 ; 9 : 5223 -5236 29 . Vessi res è A , Top S , Pigeon P , Hillard EA , Boubeker L , Spera D , Jaouen G . J Med Chem . 2005 ; 48 : 3937 -3940 30 . Park CH , Lee JH , Yang CH . J Biochem Mol Biol . 2005 ; 38 : 474 -480 31 . Galbraith SM , Chaplin DJ , Lee F , Stratford MR , Locke RJ , Vojnovic B , Tozer GM . Anticancer Res . 2001 ; 21 -32 . Pedersen U , Rasmussen PB , Lawesson S-O . Liebigs Ann Chem . 1985 ; 1557 -1569 33 .

	26 . Arezki A , Brul é E , Jaouen G . Organometallics . 2009 ; 28 : 1606 -1609
	27 . Top S , Thibaudeau C , Vessi res
	; 28 : 1414 -1424
	28 . Top S , Vessi res
	characterization of compounds	8-13	and	15-17	. HRMS purity data of compounds -and 8 10	-and elemental analyses of compounds 15 17
	8-13	and	15-17	. Evaluation of cytotoxicity in murine B16 melanoma cells, inhibition of tubulin polymerization (ITP) and effect on the
	morphology of transformed HUVEC cells (EA.hy 926 cells). This material is available free of charge via the Internet at	http://pubs.acs.org	.

è A , è A ,

Table 1

 1 Cytotoxicity, inhibition of tubulin polymerization, and morphological effects on endothelial cells of ferrocenyl curcuminoid derivatives. Cytotoxicity on murine B16 melanoma cells or normal NIH 3T3 cells after a 48 hour exposure time. Results are expressed as the concentration that causes 50 cell kill (IC ). Values are the mean of 3

	Series		Compound	Cytotoxicitya B16 melanoma cells	Cytotoxicitya NIH 3T3normal cells	ITPb	Morphological activity on endothelial cells ( M)c μ
					IC ( M) 50 μ	IC ( M) 50 μ	IC ( M) 50 μ
			1	(Curcumin)	8.5	12.3	17.9	50
	Organic	2	(Dimethyl-curcumin) 3	8.6 12.0	19.5 27.5	>30 >30	100 50
				4	2.8	15.5	>30	100
	Ferrocenyl Unsymmetrical			6 7	>100 28.6	>100 56.2	>30 9.3	50 50
				5	>100	>100	5.8	12.5
	Ferrocenyl Symmetrical A			8	30.5	>100	2.5	12.5
				9	19.0	>100	2.1	12.5
				10	4.2	6.9	21.4	12.5
	Ferrocenyl Symmetrical B			11 12	7.1 5.0	8.5 24.5	3.6 11.0	50 50
				13	2.2	6.2	12.6	6.3
	Ferrocenyl Symmetrical C			15 17	6.9 75.7	18.2 4.2	5.5 9.9	25 25
	Reference	Combreta-statin A4	0.003	0.03	0.26	0.005
	a

Abbreviation

ITP : inhibition of tubulin polymerization