LV12795A - Low-toxic precursors containing doxorubicin fragment - Google Patents

Description

LV 12795 1

EXTREMELY TOXICALLY EXTRACT THE FRAGMENT

TRADERS

The invention relates to pharmacologically active substances, in particular to the non-toxic precursors of cancer chemotherapy, obtained chemically by combining cephalosporin derivatives and anti-cancer antibiotic doxorubicin with a carbamate group. The mechanism of action of these precursors involves detaching doxorubicin from the cephalosporin molecule directly into or near malignant cells, significantly reducing the toxic side effects of this preparation on whole organ cells.

The precursor analogues for this purpose are also known to be composed of cephalosporin derivatives coupled with doxorubicin via a carbamate group and distinct from our inventive precursors with another mechanism of action. Doxorubicin is cleaved from the cephalosporin molecule by the enzyme β-lactamase, which is covalently bound to monoclonal antibodies that are localized to the cancer cell surfaces (see Scheme 7) [1,2].

Scheme 1. Doxorubicin clearance from the precursor with β-lactamase

Š! Practical application of the precursor type is difficult because complex biological preconditions need to be realized, for example, artificial preparation of 2-lactamase-containing monoclonal immunoconjugates and their localization on the target cancer cell surface.

It is known that the process of developing metastases of malignant tumor cells is characterized by intensive protease formation [3], therefore the object of the invention is to provide cefalospoi containing precursors whose β-lactam cleavage would occur with said natural proteases, thus eliminating the need to prepare monoclonal immunoconjugates and localize them to the cancer cell. surfaces (see Scheme 2)

Scheme 2. Doxorubicin dissection from the precursor with protease

Prostate precursor

These goals were achieved by using 7-aminodeacetoxycephalosporanic acid derivatives that provide stereochemical preconditions for inhibition of proteases by acylation of the enzyme wall fragment with β-lactam [4, 5]. To this end, it is necessary to: • 7-aminodeacetoxycephalosporanic acid (1) carboxylgroup fercbutyl esterification, • oxidation of amino acid ester 2 and diazotization to 7-diazodeacetoxycephalosporanate sulfon 3, or diazotization and oxidation of intermediate 5 with propylene oxide to 7-oxodeacetoxycephalosporanate 6; obtaining tert-butyl 7-chlorodeacetoxycephalosporanates sulfones (4a) or tert-butyl 7-alkylidene deacetoxyphososporanates sulfones 4b (cf. scheme 3).

The covalent attachment of doxorubicTna to the cephalosporanate 4a-c C3-methyl groups by carbamate group was accomplished by the following transformations (see scheme 4): • ally bromination of deacetoxycephalosporanates 4a-c C3-methyl, • exchange of cephalosporanates 7a-c bromine with hydroxyl, • cephalosporanate 8a -c 3-hydroxymethyl group acylation with para-nitrophenyl chloroformate, condensation of the resulting intermediate 9a-c with doxorubic acid (10) to form the required precursor IIa-c. >

Scheme 3. Structural modification of the precursor cephalosporma fragment

COOH 1 COOBu-f 2 COOBu-r

COOBu-f-cn 5 o

Rh2 (OAc) 4

ČOOBu-f 6 HCl 1. RCH = PPh3 2. H 2 O 2

COOBu-4a COOBu-f 4b, c a R = f-BuOCO, b R = 4 ~ 02NC6H4 4

Scheme 4. Preparation of DoxorubicTnu-containing Precursors 4

COOBu / 9a-cO OH 0

il-c

The in vitro cytotoxic activity of patentable compounds IIa-c was tested on four cancer cell lines: MG-22A (mouse hepatoma), HT-1080 (human fibrosarcoma), Neiro 2A (mouse neuroblastoma) and B16 (mouse melanoma). The cytotoxic effect was evaluated by calculating the amount of cells killed in the immunological plates under the influence of the test compound by two colorimetric methods: • by staining with crystalline violet (CV, the dye absorbed by the living cell membranes); dimethylthiazol-2-yl) -2,5-diphenyltetrazolium briride (MTT, the dye absorbed by the living cell mitochondrial enzymes).

For cytotoxic effects (TGioo) dependence on nitric oxide radical biosynthesis in cancer cells [6], cytotoxic effects (TGioo) were also investigated for the patentable compounds Ia-c and doxorubicin: TGioo = Gex · 100 / C (ηΜ · 10 * [/ 200μ1) where : TGioo - specific NO generating activity of living cells;

Gex - NO concentration (nM) in the extracellular medium after incubation with the test substance (1 pg / ml) (cavity volume 200 μΐ); f C is the percentage of surviving cells after incubation with the test substance (using staining with CV).

Comparison of the obtained experimental data shows (see Table 1) that the patentable precursors fla-c similar to doxorubicin (10) have a strong cytotoxic effect on cancer cell lines (TD50) and NO radical generating properties (TG100).

The anti-cancer activity of comparative precursor il-c and doxorubicin has been investigated in vivo in laboratory mice given subcutaneously for sarcoma (S-180) and melanoma (B16) tumor cells (see Table 2). The results show that inhibition of sarcoma growth by doxorubicin and precursor 11a is similar and amounted to 28%. In melanoma, doxorubicin inhibited tumor growth more efficiently than precursors 11b and 11c. LV 12795

Table 1. Cytotoxic effects of doxorubicin fragment-containing precursors and their effect on the ability of cancer cells to generate NO in the vein

Neuro 2A Section d 550 o o 3 3 * F o b H 2 00 o o o 3 * O © ~ »/ 00 ļλ θ 'ο ο 3 ο " V o o 3 * is θ '00 Ολ Ο' 3 ο Ο ο " B16 s o H o m * 3 ο Ο VI < 3 flP 3 · o o * o 'ο " ο < 3 Ο V ° > βδ m o o cs o θ '< Ν θ' Ο Ολ ο " MG-22A s o CN CN © * n 3 ο «η m ο ο C " ° 8 α b H S 3 " O o P ~ cs cT 3 ο " (3 cT s CSS r c o-CN CN CN CN CN CN J Ο V V V----J J J CN CN O O ch / / 1 n n ο O O O ch ob 00 oo σ 'νθΛ θ' (3λ o *% * .Q > CN o © 3 θ t- t- t- C C * * * T * * * 3 C 3 CQ y-1S N II X υ Ο 2 Ο I 3 ^ Oks Doxorubicin is a combination 3 Λ CJ * 5 ο ο < υ ω ε £ 05 05 3 C 3 C 3 > 03 3 > 05 Ο 03 Ο 03 13 13 U -2 Η > 3 ο Ο -4— * μ Ο Ο 3 3 05 05 Ο Ο 3 2 λ Λ 2 2 * 5? '5Γ 13 13' θ '' ο1 -Ο -Ο 2 2 CC 12 12 > 03 νθ > 03 2 ο ιη ο ο 2 2 3 3 05 05 Ν Ν • 3 3 1 - 2 2 η η / —s C Ή " Sb = L 3 3 ΞΤ • Ο 13 13 13 UU Ι- * 5 Β ο ο CC ο ο Μ Μ 2 13 '> * J3 oz < υcc 3i5T O- C / 3 13 3αΓc 3a, 3s 2' 3'c S > 3 05 Γ-EN 12795

Table 3. Effect of precursors and doxorubicin on growth of malignant tumors in vivo inhibition of tumor growth rate, Gl% Day 18 VO 46 Day 15 as 44 Day 11 00 Day 9 28 28 Schedule of administration, days OO K 'TT 1.2 , 3,4,5 of C " (N γνΓ Daily Dose, mg / kg * s O ViO 1.5 (t) ** 2.0 (t) 2.0 (t) 'ϋ' < o < N Tumor Type Sarcoma S-180 Sarcoma S-180 Mouse leasing B16 Mouse paddle rent B16 Mouse paddle rent B16 Merger 11a Doxorubicin 11b llc 1 Doxorubicin LV 12795 8

The undesirable side effect of doxorubicin is known to be associated with irreversible inhibition of cardiomyocytes. In this context, appropriate experiments were carried out on healthy mice to assess the comparative cardiotoxicity of precursor IIa-c and doxorubicin (see Table 3).

Table 3. Activity of mouse cardiomyocytes after administration of anti-cancer preparations

Connection Daily Dose, mg / kg Entry Schedule, Days Cardiomyocyte iy.rialThing &Date; Day Cardiomyocyte Activity (%), MTT * lia 3.0 Coloring. 1,3,4,7,8 litersilyl 74 11a 2.0 Iilylsilylacetates 85 lbl of 2.0 1.14.7 llFlIlflyl 55 11c 10 1,2,4,7 flllllyl! 94 Doxerubicin IBŪIII 1,2,3,4,5 ||| ||||||||| 37 * MTT - 3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolybroxynide

Data from Table 3 indicate that cardiomyocyte survival and its mitochondrial activity after long-term administration of precursor 11a-c in healthy animals (despite a higher daily dose) far outweighs comparative rates after doxorubicin administration.

The results of the above-mentioned biological experiments demonstrate that patentable precursors differ positively from doxorubicin with lower cardiotoxicity, retaining anti-cancer properties in assays as in vitro as well as in vivo tests.

Experimental part

Synthesis and study of biological properties of doxorubicin-containing precursors IIa-c was carried out according to the following methods: tert-butyl 7α-chloro-3- (4-nitrophenoxycarbonyloxy) methylcephalosporanate 1,1-dioxide (9a), tert-butyl 3- (4- Nitrophenoxycarbonyloxymethyl) -7 - [(Z) - / m: -butoxycarbonylmethylene] cephalosporanate sulfone (9b) and fmvbutyl-3- (4-nitrophenoxycarbonyloxymethyl) -7 - [(E) -4-nitrobenzylidene-cephalosporanate sulfone (9c) was synthesized according to methods [7], 9,8-Hydroxyacetyl-10 - [[3- (7a-chloro-4-tert-butoxycarbonyl-1,1-dioxo-cef-3-en-3-methoxycarbonyl) mino- 2,3,6-trideoxy-αL-lxopyranosyl-1] oxy] -7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthalenedione (11a). tert-Butyl 7a-Mor-3- (4-nitrophenoxycarbonyloxy) methylcephalosporanate 1,1-dioxide 9a (9 mg, 0.017 mmol) in dry THF (4 mL) was added to doxorubicin base 10 (neutralizing doxorubicin hydrochloric acid salt 11 mg, 0.019 mmol) with aqueous Na2CO3). The resulting mixture was stirred in the dark at room temperature for 48 hours. The solvent was evaporated under reduced pressure, the residue chromatographed on a Merck Kieselgel (0.063-0.230 mm) column, eluent: ethyl acetate-hexane (1: 1) and CH2Cl2-MeOH (60: 1). From the fractions with R / 0.51, 12 mg (78%) of 8-hydroxyacetyl-10 - [[3- (7a-chloro-4-tert-butoxycarbonyl-1,1-dioxo-cef-3-ene-3-methoxycarbom) are obtained. -amino-2,3,6-trideoxy-αL-lysohexapyranosyl] oxy] -7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthalenidinium (11a) or 97% pure by HHH data. IS spectrum: 3500, 3400, 1810, 1720, 1620, 1580 cm -1. 8-Hydroxyacetyl-10 - [[3- (7Z-tert-butoxycarbonylmethylene-4-tert-butoxycarbonyl-1,1-dioxo-cef-3-em-3-methoxycarbonyl) amino-2,3,6-trideoxy-aL -cyclopyranosyl-1] oxy] -7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacetione (11b) tert-Butyl-3- (4-nitrophenoxycarbonyl oxymethyl) - 7 - [(Z) -fatty-butoxycarbonylmethylene] cephalosporanate sulfon (9b) (71 mg, 0.12 mM solution in dry tetrahydrofuran (4 ml) was added to doxorubicin (10) base (8 mg, 0.14 mM). The solvent is evaporated off under reduced pressure and the residue is chromatographed on Merck Kieselgel (0.063-0.230 mm) columns, eluting with ethyl acetate-hexane (1: 1) followed by methylene chloride-methanol (60: 1) and methylene chloride. Methanol (4: 1): Fractions with Rf 0.68 (methylene chloride-methanol, 4: 1) afforded 100 mg (84%) of red crystalline substance, m.p. 156-159 ° C. Basic substance 95% (by ACE). IR spectrum: 3400, 1790, 1720 cm · 1:%%: C 58.43, H 6.09; N, 2.55. H, 6.20; N, 2.61. 8-Hydroxyacetyl-10 - [[3- (7E- (4-nitrobenzylidene) -4-tert-butoxycarbonyl-1,1-dioxo-cef-3-em-3-methoxycarbonyl) amino-2,3,6-trideoxy -L-Lixopyranosyl] oxy] -7,8,9,10-tetrahydro-6,8,1-trihydroxy-1-methoxy-5,12-naphthacedione (11c) was obtained analogously to Compound 11b, starting from tert-butyl. 3- (4-Nitrophenoxycarbonyloxymethyl) -7 - [(E) -4-nitrobenzylidene] -cephalosporanate sulfon (9b) with 78% yield, Rf 0.56 (methylene hexane-methanol, 8: 1). Content 91% (based on ACE). LV 12795 10

Determination of cytotoxic effects in vitro Solutions of test compounds in dimethylsulfoxide are introduced into 96-well plate wells with cells. Cells (3x104 cells / ml) are cultured for 72 hours in DMEM medium without indicator and antibiotics. After the ampoule de-icing, cells are not more than four times over. Control cells without preparation are cultured on a separate plate. The cytotoxic effect is evaluated by the number of surviving cells determined by two colorimetric methods: (a) by staining with crystal violet CV (the dye is absorbed on the living cell membranes), b) by staining with 3- (4,5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) (MTT, dye absorbed by mitochondrial enzymes in living cells) [8],

The amount of live cells in the control plate is 100% accepted.

The concentration of nitric oxide in the presence of the test substances was determined by the Gray method [9j-

In vitro detection of cytotoxic effects on cardiomyocytes Cardiomyocytes were isolated under sterile conditions from mouse hearts on day 9 or day 21 after administration of test substances to healthy animals, according to the method [10]. The cytotoxic effect was evaluated by changes in cell mitochondrial enzyme oxidation-reduction ability by the MTT colorimetric method (see previous method) after 20 hours of incubation compared to cardiomyocytes isolated from control animals.

Determination of cytotoxic effects in vivo [11].

The therapeutic effect of the test compounds was tested on melanoma B16 and sarcoma S-180 cells. 1 · 106 tumor cells were grafted under the skin of the CBA / DBH 2F line for 6 weeks old male or female mice. The number of animals in the experimental groups ranged from 3 to 6.

In experimental groups, the test substance was administered intraperitoneally as 2% solutions in dimethylsulfoxide with 0.3% agarose additive. In the control group, dimethyl sulfoxide with agarose was administered in the control group without the test substance.

The therapeutic effect was evaluated by tumor growth braking efficacy (GI%) by comparing the tumor volume difference in control and experimental animal groups 11 on days 9, 11, 15 and 18 after grafting, depending on tumor type by formula: GI% = 100 ( 1.00 - E / V) where: E - tumor volume in experimental group V - tumor volume in control group. We patent: 1. Compounds containing a doxorubicma fragment of the formula: 0 OH 0

11a-c wherein: R and R1 are substituents at the 7-position of cephalosporin. 2. Compounds containing a doxorubicma fragment after p. 1. wherein R is halogen and R1 is hydrogen. Compounds containing a doxorubicma fragment according to claim 1, wherein R and R 1 together form a methylene group containing alkoxycarbonyl or aryl substituents. Compounds containing a doxorubicma fragment according to claim 1, wherein R = Cl, R1 = H; R and R 1 = -BuOCOCH =; R and R1 = 4-02NC6H4CH =.

Claims (4)

Compounds having a doxorubicin fragment having the general formula: Ο OH O il-c where: Run R1 is a substitute for cephalosporin at position 7. Compounds containing a doxorubicin fragment according to claim 1, wherein the R substituent is a halogen atom and the R1 substituent is a hydrogen atom. Compounds containing doxorubicin fragment according to claim 1, characterized in that R and R 1 together form a methylene group containing alkoxycarbonyl or aryl substituents. Compounds containing a doxorubicin fragment according to claim 1, wherein R = Cl, R 1 = H; Run R1 = / -BuOCOCH =; Run R1 = 4-02NC6H4CH =