Lanthanide complexes based on triethylenetetramine-N,N,N′,N″,N′″,N′″-hexaacetic acid derivatives

1. Preparation of Antennas of the Aza-BODIPY Type

1.1. Preparation of tert-butyl N-[2-[[2-[4-[(2Z)-2-[1-difluoroboranyl-3-(4-methoxyphenyl)-5-phenyl-pyrrol-2-yl]imino-5-phenyl-pyrrol-3-yl]phenoxy]acetyl]amino]ethyl]carbamate (10)

This antenna (10) is obtained according to the reaction scheme below:

Synthesis of (E)-3-(4-methoxyphenyl)-1-phenylprop-2-en-1-one (1)

8.94 ml of para-anisaldehyde (Sigma) (10 g, 73.45 mmol, 1 eq.) and 8.58 ml of acetophenone (Sigma) (8.825 g, 73.45 mmol, 1 eq.) are dissolved in a flask in 150 mL of ethanol. The flask is immersed in an ice bath and 5.876 g of sodium hydroxide (149.9 mmol, 2 eq.) dissolved in 50 ml of water are added dropwise. The solution is allowed to warm to room temperature and stirred overnight. The next day, the flask is immersed in an ice bath and cold water is added to the mixture. A yellow precipitate forms and the mixture is sintered and washed with water. The precipitate is then recrystallized from ethanol yielding 11.698 g of chalcone 1 (49.1 mmol, 67%) as white crystals.

1 H NMR (250 MHz, Chloroform-d) δ 8.06-7.97 (m, 2H), 7.79 (d, J=15.0 Hz, 1H), 7.64-7.59 (m, 2H), 7.58-7.45 (m, 3H), 7.42 (d, J=15.6 Hz, 1H), 6.98-6.91 (m, 2H), 3.86 (s), 3H).

Synthesis of 3-(4-methoxyphenyl)-4-nitro-1-phenylbutan-1-one (2)

0.546 g (2.29 mmol, 1 eq.) of 0.621 mL of nitromethane (Sigma) (11.46 mmol, 5 eq.) and 1.185 mL of diethylamine (Sigma) (11.46 mmol) are dissolved in a flask in 150 mL of methanol. The solution is heated to reflux overnight. After the reaction is complete, the solvent is evaporated in vacuo and the residue dissolved in dichloromethane. The solution is washed with a solution of 1M KHSO 4 then saturated NaCl. The organic phases are combined, dried over MgSO 4 , filtered and concentrated in vacuo. The residue is purified by chromatography on silica with petroleum ether/ethyl acetate providing nitrobutanone 2 (0.654 g, 2.18 mmol, 95%) as a pale yellow viscous oil.

1 H NMR (250 MHz, Chloroform-d) δ 7.96-7.88 (m, 2H), 7.63-7.53 (m, 1H), 7.51-7.41 (m, 2H), 7.25-7.16 (m, 2H), 6.91-6.81 (m, 2H), 4.80 (ddd, J=12.3, 6.7, 0.5 Hz, 1H), 4.65 (ddd, J=12.3, 7.9, 0.4 Hz, 1H), 4.18 (p, J=7.0 Hz, 1H), 3.78 (s, 3H), 3.42 (dd, J=7.0, 2.1 Hz, 2H).

Synthesis of 4-(4-methoxyphenyl)-2-phenyl-1H-pyrrole (3)

In a flask are dissolved 3.5 g of nitrobutanone 2 (11.69 mmol, 1 eq.) and 5 eq. of KOH in 100 mL of MeOH/THF (1:2). The solution is stirred at room temperature for one hour and then added dropwise to a solution of concentrated H 2 SO 4 (2 mL/mmol) dissolved in 100 mL of MeOH at 0° C. After the addition is complete, the ice bath is removed and the solution stirred at room temperature for 1 h. The mixture is then poured into an Erlenmeyer flask containing water and ice and the solution is neutralized by adding a 4M sodium hydroxide solution. Once neutralized, the mixture is extracted with dichloromethane and the organic phase is dried over MgSO 4 , filtered and the solvent evaporated under reduced pressure. The residue is dissolved in 100 mL glacial acetic acid and 5 eq. ammonium acetate (Sigma) are added. The solution is heated at reflux for one hour during which the color of the solution changes from yellow to deep blue. The solution is cooled to room temperature and the acetic acid evaporated under reduced pressure. The black solid is then dissolved in dichloromethane and the solution is washed several times with a saturated solution of sodium bicarbonate and brine. The organic phase is dried over MgSO 4 , filtered and concentrated under reduced pressure. The residue is then dissolved in a minimum of dichloromethane and the petroleum ether is added slowly until a precipitate forms. The precipitate is filtered on Buchner and washed several times with petroleum ether. Pyrrole 3 is obtained with a yield of 60% (1742 g, 6.99 mmol) in the form of a slightly colored powder.

1 H NMR (250 MHz, DMSO-d 6 ) δ 11.32 (s, 1H), 7.66 (dt, J=7.7, 1.1 Hz, 2H), 7.57-7.48 (m, 2H), 7.36 (dd, J=8.4, 7.0 Hz, 2H), 7.21 (dd, J=2.8, 1.7 Hz, 1H), 7.20-7.12 (m, 1H), 6.94-6.84 (m, 3H), 3.75 (s, 3H).

Synthesis of 4-(5-phenyl-1H-pyrrol-3-yl) phenol (4)

0.546 g of pyrrole 3 (2.19 mmol, 1 eq.) are dissolved in a flask under argon in 100 ml of anhydrous dichloromethane. The solution is cooled to −78° C. and 5.48 ml of a solution of 1M BBr 3 in dichloromethane (Sigma) (5.48 mmol, 2.5 eq.) are slowly added. The reaction is stirred for 3 h at −78° C. and then overnight at room temperature. The next day, the solution is cooled to −78° C. and MeOH is added to the mixture. The solution is stirred for one hour then it is diluted in dichloromethane and washed with saturated NaCl. The organic phase is dried over MgSO 4 , filtered and concentrated under reduced pressure. The residue is dissolved in a minimum of dichloromethane and the product is precipitated by slow addition of petroleum ether. The precipitate is filtered on Buchner and washed with petroleum ether yielding 0.495 g (2.10 mmol, 96%) of pyrrole 4 in the form of a slightly purplish white powder.

1 H NMR (250 MHz, DMSO-d 6 ) δ 11.25 (s, 1H), 9.15 (s, 1H), 7.69-7.61 (m, 2H), 7.43-7.31 (m, 4H), 7.20-7.11 (m, 2H), 6.81 (dd, J=2.7, 1.7 Hz, 1H), 6.77-6.69 (m, 2H).

1 H NMR (250 MHz, acetone-d 6 ) δ 10.48 (s, 1H), 8.09 (s, 1H), 7.71-7.65 (m, 2H), 7.49-7.41 (m, 2H), 7.41-7.31 (m, 2H), 7.21-7.14 (m, 2H), 6.85 (dd, J=2.8, 1.7 Hz, 1H). 6.84-6.80 (m, 2H).

13 C NMR (63 MHz, acetone) δ 156.27, 134.16, 133.40, 129.58, 128.76, 126.92, 126.80, 126.55, 124.38, 116.24, 115.94, 104.14.

LRMS: calcd: 235.0997, measured [M+H] + : 236.1

IR (cm −1 ): 3443, 3300, 1600, 1581, 1494, 1244, 1132, 921, 834, 804, 778, 751, 717, 690, 609.

Mp: 209° C.

Synthesis of methyl 2-(4-(5-phenyl-1H-pyrrol-3-yl)phenoxy) acetate (5)

0.725 g of 4 (3.08 mmol, 1 eq.) in 60 ml of DMF are dissolved in a flask. 1.278 g of K 2 CO 3 (9.24 mmol, 3 eq.), 1.08 ml of methyl chloroacetate (Sigma) (12.33 mmol, 4 eq.) and a catalytic amount of potassium bromide are added to the solution which is stirred at room temperature overnight. The solution is then extracted with diethyl ether and washed three times with brine. The aqueous phase is then extracted three times with diethyl ether. The organic phases are combined and dried over MgSO 4 , filtered and the solvent evaporated in vacuo. The residue is dissolved in a minimum of dichloromethane and petroleum ether is added until a precipitate forms. The precipitate is filtered through Buchner and washed with petroleum ether to give 0.553 g of 5 as an off-white powder.

1 H NMR (250 MHz, Chloroform-d) δ 8.43 (s, 1H), 7.55-7.46 (m, 4H), 7.44-7.35 (m, 2H), 7.25-7.19 (m, 1H), 7.07 (dd, J=2.7, 1.7 Hz, 1H), 6.97-6.88 (m, 2H), 6.76 (dd, J=2.8, 1.7 Hz, 1H), 4.66 (s, 2H), 3.82 (s, 3H).

13 C NMR (63 MHz, CDCl 3 ) δ 169.57, 156.10, 133.02, 132.51, 129.56, 128.93, 126.45, 126.38, 126.11, 123.83, 114.98, 114.94, 103.86, 65.58, 52.25.

LRMS: Calcd: 307.1208, Measured [M+H] + : 308.4

IR (cm −1 ): 3430, 2941, 1759, 1600, 1581, 1496, 1438, 1212, 1178, 1131, 1075, 834, 801, 774, 758, 719, 693.

Mp: 165° C.

Synthesis of 3-(4-methoxyphenyl)-2-nitroso-5-phenyl-1H-pyrrole (6)

0.593 g of pyrrole 3 (2.38 mmol, 1 eq.) in 50 ml of ethanol and 0.48 ml of concentrated HCl (0.2 ml/mmol) are dissolved in a flask at room temperature. 0.189 g of sodium nitrite (2.74 mmol, 1.15 eq.) dissolved in water (concentration 0.6 mol/I) are added dropwise. The solution is stirred for 30 min and is then cooled to 0° C. A second portion of concentrated HCl (2.38 mL, 1 mL/mmol) is added. The solution is stirred for one hour and then dissolved in dichloromethane and washed with brine. The organic phase is dried, filtered and concentrated under reduced pressure. The residue is dissolved in a minimum volume of ethanol and excess aqueous solution of sodium acetate and ice are added and the mixture is stirred for one hour. The solution is then extracted with dichloromethane and washed with brine. The organic phase is dried over MgSO 4 , filtered and concentrated in vacuo. The residue is then dissolved in a minimum volume of dichloromethane and the product is precipitated by slow addition of petroleum ether. The solid is filtered on Buchner and washed with petroleum ether. 0.503 g of nitrosopyrrole 6 (1.81 mmol, 76%) are obtained in the form of a green powder.

1 H NMR (250 MHz, Chloroform-d) δ 8.20-8.13 (m, 2H), 7.79 (dd, J=6.9, 3.0 Hz, 2H), 7.51 (dd, J=5.1, 1.8 Hz, 3H), 7.07 (s, 1H), 7.02 (d, J=8.9 Hz, 2H), 3.89 (s, 3H).

Synthesis of (Z)-methyl2-(4-(2-((3-(4-methoxyphenyl)-5-phenyl-1H-pyrrol-2-yl)imino)-5-phenyl-2H-pyrrol-3-yl)phenoxy)acetate (7)

20 g of glacial acetic acid (0.112 g of nitrosopyrrole 6 (0.40 mmol, 1 eq.), 0.124 g of pyrrole (0.40 mmol, 1 eq. 0.40 mL of acetic anhydride. The solution is stirred and heated at reflux for one hour during which the color changes to dark blue. The solution is then cooled and the solvent evaporated under reduced pressure. The residue is then dissolved in dichloromethane and the solution is washed with saturated NaHCO 3 and saturated NaCl. The organic phase is dried over MgSO 4 , filtered and concentrated in vacuo. The residue is then dissolved in a minimum of dichloromethane and the petroleum ether is added until a precipitate forms which is filtered on Buchner and washed with petroleum ether. Azadipyrromethene 7 is obtained in the form of a dark blue powder (0.197 g, 0.35 mmol, 87%).

1 H NMR (250 MHz, Chloroform-d) δ 8.05-7.98 (m, 4H), 7.93 (ddd, J=7.8, 6.4, 1.5 Hz, 4H), 7.58-7.44 (m, 7H), 7.11 (s, 1H), 7.09 (s, 1H), 6.96 (dd, J=8.9, 1.8 Hz, 4H), 4.71 (s, 2H), 3.90 (s, 3H), 3.85 (s, 3H).

IR (cm −1 ): 3426, 2948, 1758, 1597, 1495, 1211, 1175, 1075, 1002, 902, 833, 801, 772, 759, 745, 718, 692, 637, 605. Mp: 230° C.

HRMS (ESI): m/z calcd for [C 36 H 30 N 3 O 4 ]: 568.223083, measured 568.222834 (−0.4 ppm)

Synthesis of (Z)-methyl2-(4-(2-((1-(difluoroboryl)-3-(4-methoxyphenyl)-5-phenyl-1H-pyrrol-2-yl)imino)-5-phenyl2H-pyrrol-3-yl)phenoxy)acetate (8)

In a flask under argon are dissolved 0.183 g of azadipyrromethene 7 (0.32 mmol, 1 eq.) and 0.548 ml of DIPEA (Sigma) (3.22 mmol, 10 eq.) in freshly distilled dichloromethane. After stirring for a few minutes at room temperature, 0.613 ml of distilled BF 3 .Et 2 O (Sigma) (4.84 mmol, 15 eq.) are added and the solution is refluxed for two hours. The solution is then cooled to room temperature and the organic phase is washed with brine three times. The aqueous phase is then re-extracted three times with dichloromethane and the combined organic phases are dried over MgSO 4 , filtered and concentrated in vacuo. The residue is purified by chromatography on silica with a gradient of PE/DCM. Azabodipy 8 is obtained as an iridescent dark blue powder (0.196 g, 0.318 mmol, 99%).

1 H NMR (400 MHz, Acetone-d 6 ) δ 8.23 (dd, J=8.8, 6.2 Hz, 4H), 8.18-8.09 (m, 4H), 7.58-7.46 (m, 6H), 7.33 (d, J=1.4 Hz, 2H), 7.14 (dd, J=9.0, 7.2 Hz, 4H), 4.88 (s, 2H), 3.92 (s, 3H), 3.79 (s, 3H).

13 C NMR (101 MHz, acetone) δ 169.66, 162.39, 160.53, 132.72, 131.94, 131.87, 131.70, 131.62, 130.55, 130.51, 129.36, 125.87, 118.92, 115.83, 115.26, 65.70, 55.88, 52.29.

19 F NMR (235 MHz, Acetone-d 6 ) δ −130.38 (dd, J=62.8-31.4 Hz).

HRMS (ESI): m/z calcd for [C 36 H 29 BF 2 N 3 O 4 ]: 616.221996, measured 616.221217 (−1.3 ppm)

Mp: 179° C.

IR (cm −1 ): 3288, 2918, 2584, 1758, 1728, 1601, 1504, 1487, 1454, 1388, 1277, 1252, 1228, 1175, 1129, 1100, 1068, 1024, 999, 970, 929, 904, 868, 836, 818, 767, 742, 690, 641, 615.

Synthesis of (Z)-2-(4-(2-((1-(difluoroboryl)-3-(4-methoxyphenyl)-5-phenyl-1H-pyrrol-2-yl)imino)-5-phenyl-2H-pyrrol-3-yl)phenoxy) acetic acid (9)

0.308 g of azabodipy 8 (0.5 mmol, 1 eq.) in a THF/water/H 3 PO 4 mixture (50 ml: 25 ml: 10 ml) is dissolved in a flask. The solution is stirred under reflux for 20 hours until no trace of the ester is visible by TLC. After cooling, the solution is extracted with dichloromethane. The organic phase is washed with brine and then the aqueous phases are re-extracted with dichloromethane until no blue color is observed in the aqueous phase. The organic phase is dried over MgSO 4 , filtered and concentrated under reduced pressure. The residue can optionally be purified by chromatography with DCM/MeOH if the ester is still present in the crude. Azabodipy 9 is obtained as a dark blue solid (0.294 g, 0.49 mmol, 98%).

1 H NMR (250 MHz, Acetone-d 6 ) δ 8.27-8.18 (m, 4H), 8.18-8.09 (m, 4H), 7.57-7.48 (m, 6H), 7.32 (d, J=1.8 Hz, 2H), 7.19-7.08 (m, 4H), 4.86 (s, 2H), 3.91 (s, 3H).

HRMS (ESI): m/z calcd for [C 35 H 27 BF 2 N 3 O 4 ]: 602.206329, measured 602.205753 (1.0 ppm)

Synthesis of tert-butylN-[2-[[2-[4-[(2Z)-2-[1-difluoroboranyl-3-(4-methoxyphenyl)-5-phenyl-pyrrol-2-yl]imino-5-phenyl-pyrrol-3-yl]phenoxy]acetyl]amino]ethyl]carbamate (10)

In a flask under argon are dissolved 0.080 g of 9 (0.13 mmol, 1 eq.), 0.070 ml of DIPEA (Sigma) (0.40 mmol, 3 eq.) and 0.076 g of HBTU (Sigma) (0.20 mmol, 1.5 eq.) In 5 mL of dichloromethane and 1 mL of distilled acetonitrile. The reaction is stirred for 15 minutes and then 0.032 g of Boc-ethylene diamine (0.20 mmol, 1.5 eq.) dissolved in 2 mL of anhydrous dichloromethane are added. The reaction is stirred 1:30. Then, the solution is extracted with dichloromethane and washed successively with 1M KHSO 4 , NaHCO 3 sat. and NaCl sat. The organic phases are combined and dried over MgSO 4 , filtered and concentrated in vacuo. The residue is then purified by chromatography on silica with a PE/EA mixture giving 0.069 g of a deep blue solid with metallic reflections (0.093 mmol, 71%).

1 H NMR (250 MHz, Chloroform-d) δ 8.10-7.98 (m, 8H), 7.51-7.44 (m, 6H), 7.02 (dd, J=9.0, 8), 1 Hz, 4H), 6.94 (d, J=1.1 Hz, 2H), 4.88 (s, 1H), 4.58 (s, 2H), 3.92 (s, 3H), 3.50 (q, J=5.6 Hz, 2H), 3.35 (dd, J=12.1, 6.0 Hz, 2H), 2.80 (s, 3H), 1.43 (s), 9H).

13 C NMR (101 MHz, CDCl 3 ) δ 131.13, 131.08, 130.96, 130.79, 129.66, 128.69, 125.36, 117.98, 117.94, 115.03, 114.2, 67.41, 55.65, 38.76, 28.49.

HRMS: [M+H] + C 24 H 41 BF 2 N 5 O 5 m/z calculated 744.317054, measured 744316900 (0.2 ppm).

1.2. Preparation of 2-[4-[(2Z)-2-[5-[4-[4-(tert-butoxycarbonylamino)) Butoxy]phenyl]-1-difluoroboranyl-3-phenyl-pyrrol-2-yl]imino-5-phenyl-pyrrol-3-yl]phenoxy]methylacetate (19)

This antenna (19) is obtained according to the reaction scheme below:

Synthesis of (E)-1-(4-methoxyphenyl)-3-phenyl-prop-2-en-1-one (10′)

15 g of paramethoxyacetophenone (Sigma) (0.1 mol, 1 eq.), 10.1 ml of benzaldehyde (Sigma) (0.1 mol, 1 eq.) and 400 mg of NaOH (10 mmol, 0.1 eq.) are dissolved in a flask in methanol. The solution is stirred at reflux overnight. Cold water is then added to the mixture and the precipitate formed is filtered and washed with water. The chalcone 10′ is recrystallized from methanol as white crystals in 85% yield (20.254 g, 85 mmol).

1 H NMR (CDCl 3 , 250 MHz): 8.08-8.01 (tt, 2H), 7.80 (d, J=15.7 Hz, 1H), 7.67-7.61 (m, 2H), 7.54 (d, J=15.7 Hz, 1H), 7.44-7.37 (m, 3H), 7.01-6.94 (tt, 2H), 3.87 (s, 3H).

Synthesis of 1-((4-methoxyphenyl)-4-nitro-3-phenyl-butan-1-one (11)

This compound is synthesized by applying the procedure described for the preparation of compound 2.

Mass obtained: 3.205 g; 10.71 mmol

Yield 85%.

1 H NMR (CDCl 3 , 400 MHz): 7.88-7.86 (d, 2H), 7.31-7.21 (m, 5H), 6.90-6.88 (d, 2H), 4.80 (ddd, J=12.6 Hz, 6.4 Hz, 1.3 Hz, 1H), 4.65 (ddd, J=12.6 Hz, 8.3 Hz, 1.3 Hz), 4.18. (p, J=7.1 Hz, 1H), 3.82 (s, 3H), 3.42-3.29 (m, 2H).

13 C NMR (CDCl 3 , 101 MHz): 195.39, 163.88, 139.39, 130.39, 129.52, 129.06, 127.83, 127.51, 113.93, 79.68, 55.56, 41.22, 39.49.

Synthesis of 2-(4-methoxyphenyl)-4-phenyl-1H-pyrrole (12)

This compound is synthesized by applying the procedure described for the preparation of compound 3.

Mass obtained: 1.917 g; 7.69 mmol

Yield: 62%

1 H NMR (CDCl 3 , 250 MHz): 8.37 (s, 1H), 7.57 (dd, J=8.4 Hz, 1.3 Hz, 2H), 7.48-7.42 (m, 2H), 7.36 (t, J=7.5 Hz, 3H), 7.23-7.16 (m, 1H), 7.11 (dd, J=2.6 Hz, 1.7 Hz, 1H)), 6.98-6.91 (m, 2H), 6.71 (dd, J=2.7 Hz, 1.7 Hz, 1H), 3.84 (s, 3H).

13 C NMR (CDCl 3 , 63 MHz): 158.63, 135.79, 133.27, 128.77, 126.64, 125.79, 125.73, 125.46, 125.31, 115.00, 114, 55, 103.14, 55.51.

Synthesis of 4-(4-phenyl-1H-pyrrol-2-yl) phenol (13)

This compound is synthesized by applying the procedure described for the preparation of compound 4.

Mass obtained: 180 mg, 0.77 mmol

Yield: 89%

Mp: >300° C.

HRMS: [M+H] + : 236.1070.

IR (cm −1 ): U= 3215, 1495, 1249, 1171, 1101, 832, 763, 696

1 H NMR (Acetone-d 6 , 250 MHz): 10.41 (s, 1H), 8.29 (s, 1H), 7.62-7.58 (m, 2H), 7.55-7.51 (m, 2H), 7.33-7.27 (m, 2H), 7.22 (dd, J=2.8 Hz, 1.7 Hz, 1H), 7.15-7.08 (m, 1H), 6.89-6.85 (m, 2H), 6.77 (dd, J=2.8 Hz, 1.8 Hz, 1H).

Synthesis of 2-[4-(4-bromobutoxy)phenyl]-4-phenyl-1H-pyrrole (14)

0.450 g of 13 (1.91 mmol, 1 eq.), 0.794 g of K 2 CO 3 (5.74 mmol, 3 eq.) and 0.69 ml of 1,4-dibromobutane (5 g) are dissolved in a flask at room temperature. 74 mmol, 3 eq.) in DMF. The reaction is stirred vigorously at room temperature overnight. The next day, water is added to the reaction and the mixture is extracted with diethyl ether, the organic phase is washed 3 times with sat. NaCl and the aqueous phases are re-extracted with diethyl ether. The organic phases are combined, dried over MgSO 4 , filtered and concentrated in vacuo. The residue is dissolved in a minimum of dichloromethane, and the product is precipitated by addition of petroleum ether, filtered through Buchner and washed with petroleum ether giving 0.466 g of 14 (1.26 mmol, 66%) form of a white powder.

Mp 160-162° C.

HRMS: [M( 79 Br)+H] + : 371, 1813, [M( 81 Br)+H] + : 373.0814, [M+H] + : 372.0781.

IR (cm −1 ): U= 3395, 1497, 1247, 830, 802, 751, 692.

1 H NMR (DMSO-d 6 , 250 MHz): 11.28 (s, 1H), 7.61-7.57 (m, 4H), 7.34-7.31 (d, 2H), 7.28-7.25 (dd, 1H), 7.14-7.07 (tt, 1H), 6.95 (d, J=2.8 Hz, 2H), 6.80 (dd, J=2.7 Hz, 1.7 Hz, 1H), 4.02 (t, J=6.1 Hz, 2H), 3.62 (t, J=6.5 Hz, 2H), 2.01-1.94 (m, 2H), 1.88-1.81 (m, 2H).

13 C NMR (DMSO-d 6 , 63 MHz): 156.82, 135.86, 132.27, 128.48, 125.59, 124.88, 124.76, 124.49, 124.33, 115.71, 114.71, 101.94, 66.59, 34.86, 29.11, 27.42.

Synthesis of 2-[4-(4-azidobutoxy)phenyl]-4-phenyl-1H-pyrrole (15)

0.450 g of 14 (1.22 mmol, 1 eq.) and 0.395 g of sodium azide (Sigma) (6.08 mmol, 5 eq.) in DMF are dissolved in a flask. The reaction is stirred at room temperature overnight. The mixture is dissolved in dichloromethane and washed several times with sat. NaCl. The organic phase is dried over MgSO 4 , filtered and concentrated in vacuo. After dilution with a minimum of dichloromethane, the product 15 is precipitated by the addition of petroleum ether. The solid is filtered through Buchner and 243 mg of 15 (0.73 mmol, 60%) is obtained as a white powder.

Mp: 177-180° C.

HRMS: [M+H] + : 333.1707.

IR (cm −1 ): U= 3394, 2948, 2875, 2080, 1496, 1246, 831, 752, 692.

1H NMR (CDCl 3 , 250 MHz): 8.38 (s, 1H), 7.59-7.55 (m, 2H), 7.45-7.39 (m, 2H), 7.37-7.33 (m, 2H), 7.23-7.17 (m, 1H), 7.10-7.08 (m, 1H), 6.94-6.88 (m, 2H), 6.72 (dd, J=2.6 Hz, 1.5 Hz, 1H), 4.01 (t, J=5.8 Hz, 2H), 3.38 (t, J=6.3 Hz, 2H), 1.89-1.80 (m, 4H)

13 C NMR (CDCl 3 , 63 MHz): 157.86, 135.76, 133.21, 128.77, 126.57, 125.89, 125.77, 125.41, 125.27, 116.09, 115.09, 103.09, 67.41, 51.34, 26.66, 25.90.

Synthesis of 5-[4-(4-azidobutoxy)phenyl]-2-nitroso-3-phenyl-1H-pyrrole (16)

This compound (ocher-golden powder) is synthesized by applying the procedure described for the preparation of compound 6.

Mass obtained: 67 mg, 0.186 mmol

Yield: 62%

Melting point: 125-126° C.

HRMS: [M+H] + : 362.11612.

IR (cm −1 ): U= 3280, 2918, 2850, 2092, 1603, 1360, 1258, 1164, 1038, 828, 768, 695, 668.

1 H NMR (CDCl 3 , 250 MHz): 8.14-8.11 (m, 2H), 7.84 (d, J=8.4 Hz, 2H), 7.46 (dd, J=5.2 Hz), 1.9 Hz, 3H), 7.15 (s, 1H), 6.99 (d, J=8.2 Hz, 2H), 4.06 (t, J=5.8 Hz, 2H), 3.39 (t, J=6.4 Hz, 2H), 1.93-1.78 (m, 4H).

13 C NMR (CDCl 3 , 63 MHz): 163.72, 162.05, 142.84, 131.91, 129.71, 129.57, 129.29, 128.93, 122.19, 115.47, 115.03, 67.67, 51.27, 26.53, 25.82.

Synthesis of 2-[4-[(2Z)-2-[[5-[4-(4-azidobutoxy)phenyl]-3-phenyl-1H-pyrrol-2-yl]imino]-5-phenyl-pyrrol3-yl]phenoxy]methyl acetate (17)

This compound (intense blue powder) is synthesized by applying the procedure described for the preparation of compound 7.

Mass obtained: 0.080 g, 0.123 mmol

Yield: 89%.

Mp: 150-152° C.

HRMS: [M+H] + : 651.2717.

IR (cm −1 ): U= 2094, 1759, 1600, 1496, 1241, 1166, 903, 806, 764, 694, 675.

1 H NMR (CDCl 3 , 400 MHz): 8.00 (d, J=7.8 Hz, 4H), 7.92 (d, J=8.3 Hz, 2H), 7.80 (d, J=7), 6 Hz, 2H), 7.48 (t, J=7.5 Hz, 2H), 7.39 (dd, J=12.8 Hz, 6.9 Hz, 4H), 7.17 (s, 1H), 6.99 (d, 3H), 6.93 (d, J=8.4 Hz, 2H), 4.69 (s, 2H), 4.06 (t, J=6.1 Hz, 2H), 3.85 (s, 3H), 3.40 (t, J=6.6 Hz, 2H), 1.91 (dt, J=11.4 Hz, 6.1 Hz, 2H), 1.82 (p, J=6.8 Hz, 2H).

13 C NMR (CDCl 3 , 101 MHz): 169.49, 161.27, 160.76, 157.61, 153.83, 148.27, 145.30, 145.27, 138.66, 133.76, 131.94, 130.36, 129.28, 129.22, 129.00, 128.32, 128.22, 128.15, 125.91, 125.50, 117.43, 115.16, 114.52, 111.16, 67.56, 65.53, 52.45, 51.32, 26.61, 25.87.

Synthesis of 2-[4-[(2Z)-2-[5-[4-(4-azidobutoxy)phenyl]-1-difluoroboranyl-3-phenylpyrrol-2-yl]imino-5-phenylpyrrol3-yl]phenoxy]methylacetate (18)

This compound (intense green solid of metallic appearance) is synthesized by applying the procedure described for the preparation of compound 8.

Mass obtained: 0.065 g, 0.093 mmol

Yield: 85%

1 H NMR (400 MHz, Acetone-d 6 ): δ 8.27-8.19 (m, 6H), 8.16-8.11 (m, 2H), 7.60-7.46 (m, 7H), 7.29 (s, 1H), 7.14-7.08 (m, 4H), 4.87 (s, 2H), 4.24-4.17 (m, 2H), 3.78 (d), J=2.1 Hz, 3H), 3.47 (t, J=6.5 Hz, 2H), 1.98-1.89 (m, 2H), 1.83 (q, J=7, 4 Hz, 2H).

13 C NMR (101 MHz, Acetone): δ 133.16, 131.74, 131.33, 130.48, 130.43, 130.29, 129.60, 129.34, 118.67, 115.76, 115.65, 68.49, 65.69, 52.28, 51.76, 27.13, 26.32.

HRMS: [M+H] + C 39 H 34 BF 2 N 6 O 4 m/z calculated 699.270390; measured 699.270354 (0.1 ppm)

Synthesis of 2-[4-[(2Z)-2-[5-[4-[4-(tert-butoxycarbonylamino) butoxy]phenyl]-1-difluoroboranyl-3-phenyl-pyrrol-2-yl]imino-5-phenyl-pyrrol-3-yl]phenoxy]methylacetate (19)

In a flask under argon at room temperature are dissolved 0.061 g of 18 (0.087 mmol, 1 eq.) and 0.024 g Boc-ON (Sigma) (0.096 g, 1.1 eq.) in anhydrous THF. To this solution are added 0.096 ml of a 1M solution of trimethylphosphine in toluene (0.096 mmol, 1.1 eq.). The solution is stirred overnight at room temperature. The next day, the solution is checked by TLC and if the starting material is still visible, 1 eq. is added. additional PMe3 and Boc-ON to complete the reaction in one hour. The solution is then washed with sat. NaCl. and extracted with dichloromethane. The organic phase is dried over MgSO 4 , filtered and concentrated in vacuo. The residue is purified by chromatography on silica (PE/EA) and 0.061 g of 19 (0.079 mmol, 91%) are obtained in the form of an iridescent dark green solid.

1 H NMR (400 MHz, Acetone-d 6 ) δ 8.26-8.17 (m, 6H), 8.15-8.09 (m, 2H), 7.52 (td, J=9.6, 8), 9.4.8 Hz, 7H), 7.25 (s, 1H), 7.11-7.05 (m, 4H), 6.00 (s, 1H), 4.85 (s, 2H), 4.16 (dt, J=6.6, 3.4 Hz, 2H), 3.78 (s, 3H), 3.17 (td, J=7.6, 3.8 Hz, 2H), 1.84 (q, J=7.1 Hz, 2H), 1.69 (p, J=7.4 Hz, 2H), 1.41 (s, 9H).

13 C NMR (101 MHz, acetone) δ 169.64, 163.11, 160.51, 160.29, 158.35, 156.72, 146.61, 145.53, 144.69, 143.18, 133.12, 133.07, 133.04, 131.70, 131.27, 130.44, 130.41, 130.37, 130.26, 129.55, 129.31, 126.86, 124.28, 120.79, 118.57, 115.71, 115.62, 78.39, 68.77, 65.66, 52.28, 41.83, 40.75, 28.68, 27.43, 27.21, 24.44.

HRMS: [M+H] + C 42 H 41 BF 2 N 5 O 5 calculated 744.317054, measured 744.316900 (0.2 ppm).

2. Preparation of the Compounds (C)

General Coupling Procedure

In a flask under argon are added TTHA (TCI Chemicals) (10 eq. if coupled with an anthraquinone antenna, 3 eq. if coupled with an azabodipy antenna) in distilled acetonitrile/triethylamine distilled (2/1). The solution is stirred at reflux for 2 h until the TTHA is completely dissolved. In a second flask, 1 equivalent of a compound “antenna-Y-A 2 -NHBoc” is deprotected in a mixture of dichloromethane/TFA (1/1) for 30 minutes at room temperature. During this time, the reflux of the TTHA solution is stopped and once the solution at room temperature, the flask is immersed in an ice bath and 0.9 equivalents of HBTU/eq. TTHA dissolved in a few mL of acetonitrile is added dropwise to the solution. This solution is activated for 15 min. Meanwhile, the DCM/TFA solution is evaporated under vacuum and the residue is triturated with diethyl ether to remove excess TFA. The supernatant is removed with the pasteur pipette and the TFA salt of the “antenna-Y-A 2 -NH 3+ ” compound is dried under vacuum. This salt is then dissolved in a few mL of acetonitrile and a few drops of triethylamine. This mixture is then added dropwise to the activated TTHA solution. The reaction is allowed to warm to room temperature and stirred overnight. The next day, the reaction is stopped by adding distilled water. Washing is carried out with ethyl acetate and the product is extracted with distilled water. The aqueous phases are collected, concentrated under vacuum and the residue is then lyophilized overnight. Subsequently, the residue is purified by reverse phase chromatography with a water/MeOH gradient and optionally 0.1% TFA. The collected fractions may then be dissolved in a water/MeOH/triethylamine mixture and an ion exchange resin (IRA-400 (CI)) may be made to recover the TTHA-A 2 -Y-antenna conjugate in its acid form. with elution MeOH/water/formic acid.

Synthesis of L1: 3-(2-((2-((4-((2-aminoethyl)amino)-5,8-di hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)aminoacid)ethyl)amino)-2-oxoethyl)-6,9,12-tris(carboxymethyl)-3,6,9,12-tétraazatétradécane-1,14-dioic

Synthesized according to the general procedure of coupling from anthraquinone 1,4-Bis[Boc-aminoethylamino]-5,8-dihydroxyanthracene-9,10-dione (as described in the article by Sylvain Routier, Nicole Cotelle, Jean-Pierre Catteau, Jean-Luc Bernier, Michael J. Waring, Jean-Francois Riou and Christian Bailly, Bioorganic & Medicinal Chemistry, Vol 4, No. 8, pp 1185-1196, 1996)

Yield: 60%, HRMS (ESI) for C 36 H 48 N 8 O 15 calculated 832.3239, [M+H] + : 833, 3301 C 36 H 49 N 8 O 15 (−1.3 ppm), [M+2H] 2+ 417, 1697 C 36 H 50 N 8 O 15 (−1.0 ppm).

1 H NMR (250 MHz, Deuterium Oxide) δ 8.30 (s, 2H), 6.68 (m, 2H) 4.00-3.80 (m, 4H), 3.70-3.25 (m, 30H).

Synthesis of 3,6,9-tris(carboxymethyl)-12-(2-((2-((4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino)ethylacid)amino)-2-oxoethyl)-3,6,9,12-tétraazatétradécane-1,14-dioic

Synthesized according to the general procedure of coupling from anthraquinone 1-[(Boc-aminoethyl)amino]-4-hydroxyanthracene-9,10-dione (as described in the article by Sylvain Routier, Nicole Cotelle, Jean-Pierre Catteau, Jean-Luc Bernier, Michael J. Waring, Jean-Francois Riou and Christian Bailly, Bioorganic & Medicinal Chemistry, Vol 4, No. 8, pp 1185-1196, 1996).

Yield: 60%. HRMS (ESI) for C34H42N6O14 calculated 758.2759, [M+H] + : 759, 283160 C 34 H 43 N 6 O 14 0 ppm), [M+2H] 2+ 380, 145794 C 34 H 44 N 6 O 14 (1.5 ppm).

1 H NMR (250 MHz, Methanol-d 4 ) δ 8.29-8.16 (m, 2H), 7.86-7.69 (m, 2H), 7.45 (d, J=9.6 Hz, 1H), 7.21 (d, J=9.5 Hz, 1H), 3.85-3.70 (m, 3H), 3.58 (m, 3H), 3.46 (m, 2H), 3.20 (m, 14H).

13 C NMR (63 MHz, MeOD) δ 188.55, 183.08, 170.80, 157.72, 136.49, 135.39, 133.82, 129.80, 127.77, 127.13, 125.89, 118.16, 114.55, 109.69, 68.85, 42.57, 30.68, 26.48, 9.16.

Synthesis of 2-[2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]ethyl-[2-[carboxymethyl-[2-[2-[[2-[4-[(2Z)-2-[1-difluoroboranyl-3-(4-methoxyphenyl)-5-phenyl-pyrrol-2-yl]imino-5-phenyl-pyrrol-3-yl]phenoxy]acetyl]amino]ethylamino]-2-oxoethyl]amino]ethyl]amino]acetic

Synthesized according to the general procedure of coupling from azabodipy 10 (as described in the article by Sylvain Routier, Nicole Cotelle, Jean-Pierre Catteau, Jean-Luc Bernier, Michael J. Waring, Jean-Francois Riou and Christian Bailly Bioorganic & Medicinal Chemistry, Vol 4, No. 8, pp 1185-1196, 1996).

Yield: 70%

1 H NMR (250 MHz, methanol-d 4 ) δ 8.19-7.99 (m, 8H), 7.67-7.45 (m, 6H), 7.15 (m, 4H), 7.06 (s, 1H), 7.02 (s, 1H), 4.62 (s, 2H), 3.90-3.80 (m, 15H), 3.50-3.35 (m, 16H).

Synthesis of 2-[2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]ethyl-[2-[carboxymethyl-[2-[4-[4-[1-difluoroboranyl]-5-[(Z)-[3-[4-(2-methoxy-2-oxo-ethoxy)phenyl]-5-phenyl-pyrrol-2-ylidene]amino]-4-phenyl-pyrrol-2-yl]phenoxy]butylamino]-2-oxoethyl]amino]ethyl]amino]acetic

Synthesized according to the general procedure of coupling from azabodipy 19 (as described in the article by Sylvain Routier, Nicole Cotelle, Jean-Pierre Catteau, Jean-Luc Bernier, Michael J. Waring, Jean-Francois Riou and Christian Bailly Bioorganic & Medicinal Chemistry, Vol 4, No. 8, pp 1185-1196, 1996).

Yield: 75%

1 H NMR (250 MHz, MeOD) δ 8.18-8.15 (m, 2H), 7.95-7.9 (m, 3H), 7.86-7.80 (m, 3H), 7.72-7.67 (m, 2H), 7.65-7.58 (m, 7H), 7.50-7.30 (m, 3H), 4.64 (s, 2H), 3.61-3.55 (m, 9H), 3.50-3.40 (m, 16H), 3.30-3.10 (m, 6H), 1.29-1.18 (m, 4H).

The Nd 3+ complex was synthesized in situ by the 1:1 mixture of L1 solution (as detailed above), with a solution of neodymium (III) nitrate in HEPES buffer at pH=7.5.

1. Photophysical Properties

L1 ligand and L1Nd 3+ complex (Example 2) both have broad absorption bands which extend over a wavelength range having values as high as 750 nm with maxima centered at ˜615 and ˜670 nm and molar absorption coefficients up to 5·10 3 M −1 cm −1 . Upon excitation of the low energy absorption bands located on L1 and on the L1Nd 3+ complex, it is possible to observe broadband emission signals from the chromophore ligand centered at ˜690 nm with a shoulder at ˜740 nm. The complex L1Nd 3+ , when excited at a wavelength of 650 nm, produces a narrow emission band with a maximum at 1064 nm which is characteristic of the electronic structure of Nd 3+ resulting from the transition 4 F 3/2 → 4 I 11/2 . The excitation spectra recorded by observing either the ligand emission signal centered at 740 nm or the Nd 3+ narrow emission signal centered at 1064 nm are superimposed on the absorption spectra indicating that the sensitization of the lanthanide cation is performed using the electronic levels of the chromophore derived from the anthraquinone ligand L1. As a result, ligand L1 and complex L1Nd 3+ both have excitation and emission wavelengths included in the domain of the biological diagnostic window ( FIG. 1 ).

The quantitative luminescence parameters (luminescence lifetime (i) and luminescence quantum yields centered on L1-(QL) and on Nd 3+ -(Q Nd )) determined on the L1Nd 3+ complex are summarized in Table 3.1.

Relatively short life time values T (in the nanosecond range) and low QNd quantum yields have been measured for the near-infrared emission of Nd 3+ in comparison with the highest corresponding values that have been published to date. (J.-C. G. Bünzli, S. V. Eliseeva, Photophysics of Lanthanoid Coordination Compounds, in: VW-W Yam (Ed.) Comprehensive Inorganic Chemistry II, Elsevier BV, Amsterdam, 2013, pp. 339-398, and J.-C. G. Bünzli, On the design of highly luminescent complex lanthanide, Coordination Chemistry Reviews, 293-294 (2015) 19-47). The analysis of the lifetimes measured on the L1Nd 3+ complex in H 2 O and D 2 O reveals that no water molecule is directly bound to the lanthanide cations, which is a strong indication of the good level of protection that is provided by the TTHA coordination entity.

The quantum yield of the emission from the ligand is lower by a ratio of 30% for the Nd 3+ complex relative to the L1 ligand. This result is indicative of the presence of energy transfer between the anthraquinone chromophore and the Nd 3+ cation. It is important to point out that narrow Nd 3+ emission band positions are unaffected by microenvironmental changes, which is a major advantage over the “wavelength” indocyanine green commercial fluorophore. of similar excitation (Fernandez-Fernandez, R. Manchanda, T. Lei, D. A. Carvajal, Y. Tang, S. Z. Razza Kazmi, A. J. McGoron, Comparative study of the optical and heat generation properties of IR820 and indocyanine green, Molecular imaging, 11 (2012) 99 and S. Mordon, J. M. Devoisselle, S. Soulie-Begu, T. Desmetter, Indocyanine green: Physicochemical factors affecting its fluorescence in vivo, Microvascular Research, 55 (1998) 146-152).

q Nd = 130 × ( 1 τ H2O - 1 τ D2O ) - 0.4

The estimated error is ± 0.2 (S. Faulkner, A. Beeby, M.-C. Carrié, A. Dadabhoy, A.M. Kenwright, P.G. Sammes, Time-resolved near-IR luminescence from ytterbium and neodymium complexes of the Lehn cryptand, Inorganic Chemistry Communications, 4 ( 2001) 187-190).

d obtained with an excitation wavelength of 650 nm, partial quantum yield measured for the transition 4 F 3/2 → 4 | 11/2 .

e Under excitation at 620 nm, the emission signal was collected on the spectral range of 640-850 nm.

The photostabilities of L1 and L1Nd 3+ were studied under continuous illumination at 630 nm ( FIG. 2 ). The presence of the lanthanide cation (Nd 3+ ) has been shown to significantly improve photostability. Thus, the light intensities emitted from the ligand (λ em =740 nm) and the lanthanide Nd 3+ (λ em =1064 nm) within the complex do not decrease after 3 h of continuous illumination while the emission of the free ligand loses ˜70% of its initial intensity under identical experimental conditions. These results can be explained in the first hypothesis by the concentration of the energy coming from the triplet state of L1 in the case of the L1Nd 3+ complex.

Cytotoxicity Tests

In order to determine the cytotoxicity of the L1Nd 3+ complex in living cells, “Alamar Blue” type assays were performed on the HeLa cell line. After 24 hours of incubation with the L1Nd 3+ complex, we observed a viability rate of 77% at a concentration of 12 μM. For this reason, a working concentration of 5 μM corresponding to a cell viability rate of 90% was chosen for the microscopy experiments. Shorter incubation times (5 h) allowed the viability of HeLa cells to be preserved without effects resulting from the presence of the L1Nd 3+ complex being detectable up to concentrations as high as 240 μM ( FIG. 3 ).

Confocal Microscopy

The internalization of the L1Nd 3+ complex in HeLa live cells was confirmed by confocal microscopy experiments on samples obtained after incubation for 3 h with a 5 μM L1Nd 3+ solution. When these incubated HeLa cells are observed using a 633 nm laser excitation and selecting optical slices of thicknesses of 1 μm in the vertical plane, the emission from the anthraquinone derivative could be detected on a wavelength range of 650 to 750 nm ( FIG. 4 ). The intracellular distribution of the luminescence signal suggests a localization with lysosomes. The absence of autofluorescence signals for these wavelengths confirms that the imaging performed in the biological diagnostic window significantly improves the signal-to-noise ratio of the measurement and the sensitivity of detection.

When HeLa cells were incubated with higher concentrations of L1Nd 3+ complex (50 μM), the luminescence signal could be detected not only in lysosomes but also in nucleosomes ( FIG. 5 ).

Flow Cytometry

In addition to the confocal microscopy experiments, the internalization of the L1Nd 3+ complex was quantitatively confirmed by a flow cytometry analysis carried out on a sample of 10 4 cells under excitation at 633 nm and analyzing the emission from the chromophore derived from anthraquinone located on the chromophore ligand ( FIG. 6 ).

The mechanism of cellular incorporation was analyzed by inhibition of active transports, which are dependent on energy (incubation with sodium azide NaN 3 ) and passive (incubation at 4° C.). Due to the increased stiffness of the membrane during incubation at 4° C., in addition to the inhibition of passive transport, those that are active are also deactivated. The results obtained make it possible to conclude without ambiguity that the L1Nd 3+ complex is incorporated mainly via a passive transport mechanism since 69±10% of the incubation of L1Nd 3+ was blocked by incubation at 4° C. whereas 11±5% of L1Nd 3+ complex were transported by an active process.

Epifluorescence Microscopy

Despite the low quantum yield of L1Nd 3+ , it was possible to perform near infrared imaging measurements with good sensitivity in Hela cells using this imaging agent. An intense near-infrared signal (selected by means of a low pass filter at 805 nm) could be observed for the L1Nd 3+ complex in these HeLa cells by exciting L1Nd 3+ using a conventional excitation source (Xenon lamp, 655 filter). nm with a bandwidth of 40 nm) ( FIG. 7C , top). Due to the absence of autofluorescence in the near-infrared and using the same experimental setup for the control cells compared to the cells incubated with the L1Nd 3+ complex ( FIG. 7C , bottom), we confirmed a signal-to-noise ratio and a greatly improved detection sensitivity for fluorescence imaging agents having excitation and emission wavelengths located in the biological diagnostic window.

In the case of the use of the visible commercial probe used for the specific lysosome labeling, the yellow lysotracker, an autofluorescence can be detected being generated by biomolecules since these have a strong absorption and emission in this region ( FIG. 7B , bottom). However, the location of the L1Nd 3+ complex within the lysosomes was confirmed by an epifluorescence microscopy experiment when the yellow lysotracker was located at the same location as the L1Nd 3+ complex ( FIG. 7 d , top).

Furthermore, it has been observed with confocal microscopy experiments that when HeLa cells are incubated with a higher concentration of L1Nd 3+ complex (50 μM), a localization can also be obtained in nucleosomes where an intense near-infrared signal has been detected. could be detected ( FIG. 8 ).

Photobleaching Test Conducted by Epifluorescence Microscopy

In order to compare the photostability of the L1Nd 3+ complex with that of a commercially available reporter, a photobleaching test was performed by comparison with the “LysoTracker Deep Red” followed by epifluorescence microscopy. A significant photobleaching was observed in the case of the “LysoTracker Deep Red” protractor after 20 s of continuous illumination at a light selected by a 655 nm filter (40 nm bandwidth) ( FIG. 9 , top). On the other hand, the intensity of the emission from anthraquinone in the L1Nd 3+ complex remained constant during 95 s using the same experimental conditions (selection of the wavelength by a 655 nm filter of 40 nm bandwidth for excitation and 716 nm bandwidth of 40 nm for emission ( FIG. 9 , bottom).

A constant emission signal collected through a low-cut filter (805 nm, FIG. 10 ) could be observed revealing a very satisfactory photostability was measured up to 495 seconds for the complex L1Nd 3+ under continuous illumination by means of light selected by a filter at 655 nm bandwidth of 40 nm.

Epifluorescence Macroscopy: Detection of Near-Infrared Emission from the L1 Nd 3+ Complex in/Through Different Biological Samples

In order to demonstrate in a complementary manner the advantages of the use of the new imaging agents according to the invention which have excitation and emission wavelengths in the field of the biological diagnostic window and more particularly penetration of photons at greater depths, experiments were conducted on biological tissues of animal origin and varied thicknesses using the following experimental setup: a glass capillary containing a 200 μM solution of L1Nd 3+ was placed under the different tissue samples ( FIG. 11 , top) and excited through a pulsed dye laser emitting light of wavelength 685 nm. An intense signal could be collected using a low cut filter at 785 nm ( FIG. 11 , bottom). This experiment has shown that near-infrared photons from the L1Nd 3+ complex can be detected through pig tissue with a thickness of 3 mm, chicken liver (2 mm), beef (1.6 mm) or chicken skin (0.6 mm). The tests on tissues of different kinds made it possible to evaluate the tissue absorption differences and the different light diffractions which are two parameters related to the penetration depth of photons and which influence the resolution of the images collected. In addition, a difference in autofluorescence generated according to the nature of the tissues was observed.

Since blood components, in particular hemoglobin, are among the largest light absorbers and the largest emitters of autofluorescence, they can be responsible for a very large loss of signal and can lead to a very large decreased signal-to-noise ratio and measurement sensitivity (A. Taruttis, V. Ntziachristos, Translational Optical Imaging, American Journal of Roentgenology American Roentgen Ray Society, 199 (2012) 263-271). It has been demonstrated here that by using excitation and emission wavelengths included in the biological diagnostic window, it is possible to effectively minimize the undesirable effects of autofluorescence. Thus, it has been possible to detect an intense near infrared signal from L1 Nd 3+ at a concentration of 200 μM in the blood.