RU2406726C1 - DERIVATIVES OF 13(1)-N-{2-[N-(CLOSO-MONOCARBADODECABORAN-1-YL)- METHYL]AMINOETHYL}AMIDE-15(2),17(3)-DIMETHYL ETHER OF CHLORINE e6, MANIFESTING PROPERTIES OF PHOTOSENSITISER - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to derivatives of 13(l)-N-{2-N-(closo-monocarbadodecaboran-1-yl)-methyl]aminoethyl}amide-15(2), 17(3)-dimethyl ether of chlorine e₆ of general formula

, where M=Cs, Na, manifesting properties of photosensitiser.

EFFECT: compounds can be used in medicine as agents for boron neutron capture therapy (BNCT) and photodynamic therapy (FDT) of malignant neoplasms.

2 ex, 1 tbl, 2 dwg

Description

The invention relates to a new compound, namely to a derivative of 13 (1) -N- {2- [N- (closo-monocarbadodecaboran-1-yl) methyl] aminoethyl} amide-15 (2), 17 (3) -dimethyl chlorin ester e _{6 of the} general formula

Where

M = Cs (Ia), Na (I,),

exhibiting photosensitizer properties.

The specified compound, its properties and method of preparation are not described in the literature.

Known ((13 (1) -N- (2- (N- (1-o-carboranyl) methyl) aminoethyl) -amide-15 (2), 17 (3) -chlorin d ₆ methyl ester) (compound of the formula II), (Kuchin A.V., Olshevskaya V.A., Malshakova M.V., Belykh D.V., Petrovsky P.V., Ivanov O.G., Shtil A.A., Kalinin V.N. ./New carboran derivatives of chlorin e _6. // DAN, 2006, v.409, No. 4, p. 493-496).

Compound II is obtained by alkylation of the amino derivative of chlorin e ₆ (13 (1) -N- (2-aminoethyl) -amide-15 (2), 17 (3) -dimethyl chlorin e ₆ ) with carboranylmethyl triflate to form the corresponding carboran derivative II (13 (1) -N- (2- (N- (1-o-carboranyl) methyl) aminoethyl) amide-15 (2), 17 (3) -chlorin dimethyl ether ( ₆ ). 13 (1) -N- (2-aminoethyl) -amide-15 (2), 17 (3) -dimethyl chlorin e _{6 is} obtained by opening the exotherm of methylpheophorbide with ethylene diamine (DVBelykh, LPKarmanova, LVSpirikhin, AVKutchin / Synthesis of chlorin e ₆ amide derivatives // Mendeleev Commun., 2002, 12 (2), 77-78) Carboranyl methyl triflate was obtained from 1-hydroxymethyl-o-carborane and triflate anhydride (VNKalinin, VAOl'shevskaya, EGRus, AATyutyunov, ZAStarikova, AAKorlyukov, DDSung, ABPonomaryov, PVPetrovskii and E.Hey-Hawkins / The first triflates: synthesis and reactivity of 1-trifluoromethanesulfonylmethyl- and 1,2-bis (trifluoromethanesulfonylmethyl) -o-carborane // Dalton Trans., 2005, p.903-908).

A compound of formula III is known (chlorin e ₆ trinate salt, (photolon preparation)) used for the photodynamic therapy of malignant neoplasms (V.M. Tsarenkov, A.L. Meshcheryakova, O.N. Albitskaya, V.I. Tyurin, M. V. Sarzhevskaya, M.A. Kaplan, N.D. Kochubeeva, I.N. Zhuravkin, P.T. Petrov / A tool for photodynamic therapy of malignant neoplasms - photolon // Belarus Patent BY 19990767).

The present invention is the creation of a boronated derivative of chlorin e ₆ with phototoxic activity, which can be used as an agent for boron neutron capture therapy (BNCT) and photodynamic therapy (PDT) of malignant neoplasms.

The problem is achieved by derivatives of 13 (1) -N- {2- [N- (closo-monocarbadodecaboran-1-yl) methyl] aminoethyl} amide-15 (2), 17 (3) -dimethyl chlorin e ₆ (Ia and Ib) exhibiting the properties of a photosensitizer.

Compound Ia is prepared by alkylation of the amino group 13 (1) -N- (2-aminoethyl) -amide-15 (2), 17 (3) -dimethyl ester of chlorin e _{6 with} anionic 1-trifluoromethanesulfonylmethyl-closo-monocarbadodecanorane with cesium in the presence of N, O- bis (trimethylsilyl) -acetamide (BSA) in tetrahydrofuran as a solvent. The isolated and purified carboranylchlorin Ia is dissolved in acetonitrile, then passed twice through an ion-exchange resin and the finished product (IB) is isolated by distilling off the solvent in vacuo. The reactions are carried out in an inert gas atmosphere such as argon or nitrogen. The yields of the claimed compounds Ia and Ib are 71% and 99.5%, respectively.

The reaction is carried out according to the following scheme:

The starting materials, solvents and reagents necessary for the synthesis of the claimed compounds are manufactured by industry or are commercially available, for example, tetrahydrofuran (T5267, Sigma), N, O-Bis (trimethylsilyl) acetamide (BSA) (Fluka, 15241), DOWEX ion exchange resin 50 WX8-200 (Aldrich, 44514).

13 (1) -N- (2-aminoethyl) -amide-15 (2), 17 (3) -dimethyl chlorin ester ₆ obtained according to the procedure described in (DV Belykh, LP Karmanova, LV Spirikhin, AV Kutchin / Synthesis of chlorin e ₆ amide derivatives // Mendeleev Commun., 2002, 12 (2), 77-78).

1 Triftormetansulfonilmetil-closo-monokarbadodekaboranil cesium was prepared by the method described in (VAOl'shevskaya, ANSavchenko, AVZaitsev, EGKononova , PVPetrovskii, AARamonova, VVTatarskiy Jr., OVUvarov, MMMoisenovich, VNKalinin, AAShtil / Novel metal complexes of boronated chlorin e ₆ for photodynamic therapy // J. Organomet. Chem. 2008 DOI: 10.1016 / j.jorganchem. 2008.11.013)

The invention is illustrated by specific examples of its implementation, below.

Example 1

Obtaining cesium salt of 13 (1) -N- {2- [N- (Closo-monocarbadodecaboran-1-yl) methyl] aminoethyl} amide-15 (2), 17 (3) -dimethyl chlorin e ₆ (Ia)

To a solution of 200 mg (0.3 mmol) of 13 (1) -N- (2-aminoethyl) amide-15 (2), 17 (3) -chlorin e ₆ dimethyl ether and 184 mg (0.42 mmol) 1- 2 ml of BSA are added to 10 ml of anhydrous THF and stirred at room temperature for 1 hour. 50 ml of chloroform are added to the reaction mixture, washed with water, dried over anhydrous sodium sulfate. The solvent was distilled off in vacuo, the residue was purified by silica gel column chromatography (2 × 20 cm), eluent-chloroform-methanol (9: 1). Yield 203 mg (71%).

IR spectrum (KBr), ν (cm ^-1 ): 2530 (BH), 1722 (С = O, ester), 1609 (chlorine band), 1656 (amide I), 1520 (amide II).

Electronic spectrum (DMSO), λ _max , nm, (ε · 10 ^-3 ): 404 (51.4), 501 (9.40), 610 (2.6), 663 (35.9).

¹ H NMR Spectrum ((CD ₃ ) ₂ ) CO), δ, ppm: 9.79 (s, 1H, 10-H); 9.76 (s, 1H, 5-H); 9.11 (s, 1H, 20-H); 8.24 (dd, 1H, J = 17.3 & 11.5 Hz, 3 (1) -H); 6.42 (d, 1H, J = 18.3 Hz, 3 (2) -H (trans)); 6.16 (d, 1H, J = 12.4 Hz, 3 (2) -H (cis)); 15 (1) -CH ₂ : 5.53 (d, 1H, J = 18.5 Hz) and 5.36 (d, 1H, J = 18.7 Hz); 4.67 (q, 1H, J = 7.5 Hz, 18-H); 4.51 (brd, 1H, J = 8.6 Hz, 17-H); 4.13 (br s, 1H, 13 (1) -NH); 3.80 (q, 2H, J = 7.5 Hz, 8 (1) -CH ₂ ); 3.75 (m, 4H, 13 (2) -CH ₂ , 13 (3) -CH ₂ ); 3.70 (s, 3H, 15 (3) -CH ₃ ); 3.61 (s, 3H, 17 (4) -CH ₃ ); 3.60 (s, 3H, 12 (1) -CH ₃ ); 3.53 (s, 3H, 2 (1) -CH ₃ ); 3.32 (s, 3H, 7 (1) -CH ₃ ); 2.82 (s, 1H, 13 (3) -NH); 2.52 (s, 2H, NH- CH ₂ -C (carboran)); 2.35-2.21 (m, 4H, 17 (1) -CH ₂ and 17 (2) -CH ₂ )); 3.0-1.4 (br.m, 11H, BH), 1.71 (d, 3H, J = 7.1 Hz, 18 (1) -CH ₃ ), 0.88 (t, 3H, J = 7, 5 Hz, 8 (2) -CH ₃ ); - 1.53 (br s, 1H, NH); - 1.82 (br s, 1H, NH).

¹¹ V NMR spectrum ((CD ₃ ) ₂ CO), δ, ppm: - 9.02 (d, 1B, J = 136 Hz, V (12)); - 13.15 (d, 5V, J = 130 Hz, V (2-6)); - 14.37 (d, 5V, J = 154 Hz, V (7-11)).

Mass spectrum, (m / z): 821 [M-Cs] ⁺ ; 954 [M ⁺ ].

Example 2

Obtaining the sodium salt of 13 (1) -N- {2-N- (closo-monocarbadodecaboran-1yl) methyl] aminoethyl} amide-15 (2), 17 (3) chlorin dimethyl ether ₆ (Ib)

Dissolve 40 mg (0.04 mmol) of compound ( Ia ) in 100 ml of acetonitrile and pass through a column (2 × 5 cm) with a DOWEX 50 WX8-200 ion exchange resin. The solvent was distilled off in vacuo, the residue was dissolved in 100 ml of acetonitrile and passed through an ion exchange resin. The solvent was distilled off in vacuo. Yield 35.2 mg (99.5%).

IR spectrum (KBr), ν (cm ^-1 ): 2528 (BH), 1721 (C = O, ester), 1606 (chlorine band), 1657 (amide I), 1519 (amide II).

Electronic spectrum (DMSO), λ _max , nm, (ε · 10 ^-3 ): 406 (58.0), 503 (10.6), 610 (2.9), 663 (40.5).

¹ H NMR Spectrum ((CD ₃ ) ₂ CO), δ, ppm: 9.78 (s, 1H, 10-H); 9.75 (s, 1H, 5-H); 9.10 (s, 1H, 20-H); 8.23 (dd, 1H, J = 17.3 and 11.5 Hz, 3 (1) -H); 6.43 (d, 1H, J = 18.3 Hz, 3 (2) -H (trans)); 6.16, (d, 1H, J = 12.4 Hz, 3 (2) -H (cis)); 15 (1) -CH ₂ : 5.51 (d, 1H, J = 18.4 Hz) and 5.34 (d, 1H, J = 18.6 Hz); 4.67 (q, 1H, J = 7.5 Hz, 18-H); 4.51 (brd, 1H, J = 8.6 Hz, 17-H); 4.13 (br s, 2H, 13 (1) -NH); 3.81 (q, 2H, J = 7.5 Hz, 8 (1) -CH ₂ ); 3.75 (m, 4H, 13 (2) -CH ₂ , 13 (3) -CH ₂ ); 3.74 (s, 3H, 15 (3) -CH ₃ ); 3.61 (s, 3H, 17 (4) -CH ₃ ); 3.60 (s, 3H, 12 (1) -CH ₃ ); 3.52 (s, 3H, 2 (1) -CH ₃ ); 3.32 (s, 3H, 7 (1) -CH ₃ ); 2.82 (s, 1H, 13 (3) -NH); 2.52 (s, 2H, 1H- CH ₂ -C (carboran)); 2.35-2.21 (m, 4H, 17 (1) -CH ₂ and 17 (2) -CH ₂ )); 3.0-1.4 (br.m, 11H, BH), 1.71 (d, 3H, J = 7.1 Hz, 18 (1) CH ₃ ), 0.87 (t, 3H, J = 7.4 Hz, 8 (2) CH ₃ ); 1.55 (br s, 1H, NH); - 1.84 (br s, 1H, NH).

¹¹ V NMR spectrum ((CD ₃ ) ₂ CO), δ, ppm: - 9.00 (d, 1B, J = 136 Hz, V (12)); - 13.13 (d, 5V, J = 131 Hz, V (2-6)); - 14.35 (d, 5V, J = 155 Hz, V (7-11)).

Mass spectrum, (m / z): 821 [M-Na] ⁺ . 844 [M ⁺ ].

The study of toxic and specific properties of compounds Ia and Ib

A study of the dark toxicity of compounds Ia and Ib showed that the claimed compounds do not cause death of cultured human cells (HCT116 line - intestinal epithelium, MCF-7 - mammary epithelium, as well as lymphocytes isolated from the peripheral blood of healthy donors) in concentrations up to 50 μM in during 72 hours of incubation. These results indicate the absence of dark cytotoxicity in the test compounds.

To study the death of tumor cells during photoactivation, HCT116 cells were treated with compounds Ia and Ib (1 μM) for 30 minutes, after which the cells were illuminated with light using red filters (20 minutes). Cell death was recorded by the inclusion of propidium iodide (PI), a sign of irreversible damage to the plasma membrane in flow cytofluorimetry. It was found that 30 minutes after the end of cell illumination, the percentage of PI-positive cells was 100%, i.e. all cells received lethal photodamage.

For a detailed analysis of the death mechanism during photoactivation of new compounds, cells were incubated for 30 minutes in the presence of PI, dichlorofluorescein diacetate (marker of free oxygen forms, 1 μM), mitochondrial dye MitoTracker (marker of transmembrane electric potential of mitochondria, 1 μM) and test compounds Ia or Ib (each at final concentrations of 10 nM, 100 nM or 1 μM). Before microscopy, the medium with the above substances was removed. To activate the photosensitizers, the 633 nm HeNe band of the Axiovert 200M LSM510 META confocal microscope laser was used, after which the intracellular dye distribution was revealed. The sequence of molecular events was established: 30 seconds after the initiating illumination, the number of free oxygen forms in the cells increased, after 1 min a decrease in the electric potential of mitochondria and a sharp change in their shape was noted, after 2 minutes the change in the shape of the cells began - from polygonal cells they became round. 15 minutes after the start of illumination, PI enters the cells, which indicates lethal damage to the plasma membrane (necrosis). Thus, fast (within the first minutes after photoactivation) necrosis is a mechanism of cell death under the action of the claimed compounds. A comparative analysis of the photosensitization data by the claimed compounds Ia and Ib with photosensitization by the photolone (III) clinical preparation showed that in this experimental system, photolon causes similar intracellular events only at a concentration of 1 μM, at a photolon concentration of 100 nM, the cell death rate decreased. When using 10 nM photolon, no PI entry into the cells was observed, i.e. at a concentration of 10 nM, photolon did not cause photonecrosis.

A study of the acute toxicity of the claimed compound IB on white outbred mice showed that the drug has a very low toxicity: LD ₅₀ is: LD ₁₆ = 540 mg / kg, LD ₅₀ = 705 mg / kg, LD ₈₄ = 950 mg / kg, LD ₁₀₀ = 1140 mg / kg.

When conducting photodynamic therapy of M-1 sarcoma in rats, 1.5 hours after intraperitoneal administration of compound IB at a dose of 10.0 mg / kg and laser irradiation with a power density of 0.42 W / cm ² and an energy density of 300 J / cm ² obtained complete regression of the tumor (Figure 1). Lower doses of the claimed compound Ib (5.0 mg / kg and 2.5 mg / kg) under the same conditions of laser radiation also had an inhibitory effect on the tumor, contributing to its complete regression. A change in the power density, which was 0.25 W / cm ² , and the energy density corresponding to 150 J / cm ² , at a dose of 16 mg 5.0 kg / kg, practically did not affect the results of PDT. And in these cases, complete tumor necrosis was observed (Figure 1).

Conducting PDT with compound 1b at a dose of 5.0 mg / kg and 10.0 mg / kg with a power density of 0.25 and 0.44 W / cm ² , an energy density of 150 and 300 J / cm ² in mice with melanoma B- 16 led to a significant, statistically significant, inhibition of tumor growth up to its complete regression. The most effective conditions for PDT melanoma were in the group of mice that used laser radiation at a dose of 1b 10.0 mg / kg of animal weight, energy density of 150 J / cm ² and power density of 0.44 W / cm ² (Figure 2) .

The data obtained as a result of photodynamic therapy with intraperitoneal administration of compound 1b in two tumor strains indicate high photoactive properties of the drug.

Comparison of the results of our studies on the use of compound 1b as a photosensitizer for PDT in the treatment of B-16 melanoma with the data of our earlier studies with photolone (compound III) (Kaplan M.A. Mardinskaya V.P., Sokol N.I., Malygina A.I., Nikitina R.G., Borgul O.V., Mikhailovskaya A.A. Experimental PDT of B-16 melanoma with chlorin derivatives e ₆ // J. "Physical Medicine". - 2006. - No. 2. - S.9-14) showed that at photolon doses of 9.0 and 12.0 mg / kg of animal weight, power densities of 0.38 W / cm ² and 0.25 W / cm ² , only a significant decrease was observed the coefficient of absolute growth of the tumor. Complete regression of melanoma within 21 days was observed during PDT with a dose of photolone (compound III) - 12.0 mg / kg, with an energy density of 150 J / cm ² and a power density of 0.25 W / cm ² only in 2 of eight animals and one of 12 animals with the introduction of photolon at a dose of 9.0 mg / kg, an energy density of 150 J / cm ² and a power density of 0.38 W / cm ² . This indicates a clear advantage of compound IB compared with photolone, the drug that is currently used (Table 1).

Table 1 Comparison of the efficacy of compound 1b with the photolon efficiency in PDT of melanoma B-16 Compound The dose of the compound, mg / kg The radiation power density W / cm ² The radiation energy density, J / cm ² % of animals in which complete tumor regression was observed (21 days after irradiation) Photolon (III) 9 0.38 150 25 12 0.25 150 8.3 Ib 5 0.25 150 90 5 0.44 150 60 5 0.25 300 one hundred 5 0.44 300 one hundred 10 0.25 150 85 10 0.44 150 one hundred

The advantages of the claimed compounds of formula I are their high phototoxic activity (exceeding the activity of the currently widely used drug) in combination with low toxicity, as well as solubility in water (compound 1b), which makes it possible to use the claimed compounds in medicine for BNCT and PDT of malignant neoplasms .

The claimed compounds can be easily obtained by simple technological synthesis, which is carried out without isolation of the intermediate product (in one vessel) from the available starting materials with yields of 71% and 95.5%. The method does not require the use of harsh conditions, special equipment, high and low temperatures.

Claims (1)

Derivatives of 13 (1) -N- {2- [N- (closo-monocarbadodecaboran-1-yl) methyl] aminoethyl} amide-15 (2), 17 (3) -dimethyl chlorin e ₆ ether of the general formula

where M = Cs, Na,
exhibiting photosensitizer properties.

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