Prodrug using nitroimidazole

Abstract

Provided is a prodrug of 2-nitro-1-imidazolepropionic acid and a therapeutically active organic compound having on the molecule an amino group, a cyclic amino group or a hydroxyl group, particularly a prodrug in which the therapeutically active organic compound is selected from among antitumor agents. The prodrug cleaves specifically under hypoxic conditions in vivo to exhibit the inherent therapeutic activity.

Claims

1. A compound represented by general formula (I), ##STR00016## wherein Z is of formula (a) ##STR00017## or of formula (b)
OR3(b), R1 being a residue of an amino group-bearing therapeutically active organic compound after removal of the amino group therefrom and R2 being a hydrogen atom, or R1 and R2, taken together with the adjoining nitrogen atom, being a residue of a therapeutically active organic compound having a cyclic amino group; and R3 being a residue of a hydroxyl group-bearing therapeutically active organic compound after removal of the hydroxyl group therefrom, wherein the amino group-bearing therapeutically active organic compound is selected from the group consisting of doxorubicin and gemcitabine; the therapeutically active organic compound having a cyclic amino group is represented by 5-fluorouracil; the hydroxyl group-bearing therapeutically active organic compound is selected from the group consisting of doxorubicin and gemcitabine; and the moiety corresponding to Z of general formula (I) is cleaved in a reducing environment, or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1 which has a formula as follows: ##STR00018##

3. The compound according to claim 1 which has a formula as follows: ##STR00019##

4. The compound according to claim 1 which has one of the following two formulae: ##STR00020##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an NMR chart of Compound 4 synthesized by a cyclization reaction in Example 1.

(2) FIG. 2 is a graph showing the results of high-performance liquid chromatography (HPLC) comparing the amount of naphthylmethylamine released in normal oxygen and low-oxygen environments in Example 4.

(3) FIG. 3 is a graph showing the release behavior of an amino group-bearing organic compound from a model compound represented by general formula (I) using human pancreatic cancer cells in Example 5.

(4) FIG. 4 is a graph showing the results of an evaluation of cell survival rates in Example 7 (hypoxic environment-responsive doxorubicin).

(5) FIG. 5 is a graph showing the results of a comparative test on the cytotoxicity of known compounds that are structurally analogous to the compounds of the invention in a comparative test example.

(6) FIG. 6 is a graph showing the results of a cell survival rate evaluation (hypoxic environment-responsive gemcitabine) in Example 9.

(7) FIG. 7 is a graph showing the results of a cell survival rate evaluation (hypoxic environment-responsive 5-fluorourasil) in Example 11.

(8) FIG. 8 is a graph showing the drug release behavior from a prednisolone prodrug within a hypoxic environment in Example 16.

DESCRIPTION OF THE EMBODIMENTS

(9) The invention is described more concretely below by way of examples, although the examples are not intended to limit the invention.

Example 1

Reduction of Methyl 3-(2-Nitro-1H-Imidazolyl)propionate

(10) ##STR00006##

(11) A reaction tube to which had been added 200 mg of Compound 1 synthesized according to a method described in the non-patent document (M. P. Hay et al., J. Med. Chem. 37, 381-391 (1994)) was charged with 10 mL of methanol and 150 mg of Pd/C, then filled with hydrogen gas. With the tube filled with hydrogen gas, stirring was continued for 24 hours. Following the reaction, thin-layer chromatography (TLC) (developing solvent, ethyl acetate:hexane=1:1) was used to confirm that the reaction had proceeded. Next, the Pd/C was removed by Celite filtration and, last of all, the methanol was removed using an evaporator. As a result, a ring structure was rapidly formed in solution during the reducing reaction, without isolation of the Intermediate 3, yielding Compound 4. FIG. 1 shows an NMR chart of the Compound 4 thus obtained.

(12) It was confirmed from this chart that Compound 4 having a cyclic structure formed due to the reduction of Compound (1). Hence, it is apparent that, together with an intramolecular cyclization reaction by the compound analog of formula (I), the ester bond cleaves, releasing HOMe.

Example 2

Preparation of N-Naphthylmethyl-3-(2-Nitro-1H-Imidazolyl)propionylamide

(13) ##STR00007##

(14) A 50 mL round-bottomed flask was charged with Compound 2 (100 mg) synthesized according to a method described in the non-patent document (M. P. Hay et al., J. Med. Chem. 37, 381-391 (1994)), following which 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (155.6 mg), N,N-dimethyl-4-aminopyridine (6.7 mg), methylene chloride (5.4 mL) and 1-naphthylmethylamine (119.27 L) were added to the reactor under stirring with a stirrer and stirring was continued for two days. A 100 mL separatory funnel was charged with 20 mL of ethyl acetate and 20 mL of saturated ammonium chloride, and the organic phase and aqueous phase were separated. A saturated aqueous solution of sodium bicarbonate and a saturated aqueous solution of NaCl were added to the recovered ethyl acetate phase and separation was carried out, thereby recovering the organic phase. The organic phase was dried over anhydrous Na.sub.2SO.sub.4, following which the solvent was removed with an evaporator. The residue was isolated and purified by silica gel column chromatography (developing solvent, ethyl acetate:hexane=1:1; 400 mL), and the solvent was removed with an evaporator, yielding the target Compound 5.

(15) ESI-MS (M+H.sup.+): theoretical value, 325.130; measured value, 325.126

Example 3

Reduction of Compound 5

(16) A stirring bar and Compound 5 (10 mg) were added to a 50 mL round-bottomed flask. While stirring with the stirrer, 10 mL of methanol and 50 mg of Pd/C were added to the reactor, and the reactor was filled with hydrogen gas. With the reactor filled with hydrogen gas, stirring was continued for 24 hours. Following the reaction, TLC (developing solvent, ethyl acetate:hexane=1:1) was used to confirm that the reaction had proceeded. Next, the Pd/C was removed by Celite filtration and, last of all, the methanol was removed using an evaporator. Analysis by electrospray ionization mass spectroscopy (ESI-MS) yielded results indicating the formation of Compound 4. This showed that, together with an intramolecular cyclization reaction, the amide bond cleaved, releasing naphthylmethylamine.

(17) Compound 4: theoretical molecular weight, 138.067; measured value, 138.063

Example 4

Incubation of Compound 5 in Cultured Cells Under Low Oxygen

(18) This example was carried out to confirm that Compound 5 is reduced in cells within a hypoxic environment, and that naphthylmethylamine is released by a subsequent intramolecular cyclization reaction.

(19) A cell suspension prepared to a cell count of 110.sup.4 cells/mL was dispensed into a 96-well plate, and the cells were cultured by 24 hours of incubation at 37 C. After 24 hours, the synthesized Compound 5 was added to the cells in an amount of 1 mM. Following addition, the cells were cultured for 6 hours each in a normal oxygen concentration incubator (20% O.sub.2) or at a low-oxygen work station (1% O.sub.2). The culture medium was then recovered, 50 L of Trypsin/EDTA was added and 5 minutes of incubation was carried out, following which the cells were stripped away and added to the medium that was earlier recovered. Next, the recovered sample was freeze-dried overnight, 200 L of acetonitrile was added, and 30 minutes of ultrasonic cleaning was carried out. In addition, dead cells were precipitated by centrifugal separation (3,000 rpm, 10 min), the supernatant was recovered, and the acetonitrile was removed with a centrifugal evaporator. Next, 200 L of methanol (for LC/MS) was added to the Eppendorf tube used in centrifugation, the tube contents were passed through a filter (0.2 m), and LC/MS measurement and high-performance liquid chromatography (HPLC) measurement were carried out. By carrying out this measurement, the amount of compound released in a low-oxygen environment and the amount released in a normal oxygen environment were compared. The HPLC results are shown in FIG. 2. It is apparent from the diagram that much naphthylmethylamine is released in a low-oxygen environment.

Example 5

Incubation of Compound 5 in Cultured Cells Under Low Oxygen

(20) Human pancreatic cancer cells (MIA PaCa-2, acquired from Riken Cell Bank) were prepared as a suspension having a cell count of 110.sup.4 cells/mL), and dispensed into a 96-well plate. After 24 hours, Compound 5 was added to a concentration of 10 M and the cells were cultured, in a normal oxygen concentration incubator (20% O.sub.2) or at a low-oxygen work station (0.1% O.sub.2). After a given time had elapsed, the culture medium was recovered, trypsin was added and the cells were recovered. The recovered cells were disrupted by sonic treatment, the compound was extracted with acetonitrile, and analysis by LC/MS was carried out under the following conditions.

(21) Column used: Lachrom Ultra C18 (particle size, 2 m; 2 mm50 mm) column

(22) Measurement wavelength: 220 nm

(23) Eluent A: 0.1% TFA-containing milliQ

(24) Eluent B: acetonitrile

(25) Flow rate: 0.2 mL/min

(26) Gradient: 95:5 (Eluent A:Eluent B).fwdarw.95:5 (5 minutes).fwdarw.30:70 (15 minutes)

(27) By carrying out this measurement, the amount of compound released in a low-oxygen environment and the amount of compound released in a normal oxygen environment were compared. The HPLC results are shown in FIG. 3. It is apparent from the diagram than much naphthylmethylamine is released in a low-oxygen environment.

Example 6

Production of Doxorubicin Prodrug

(28) ##STR00008##

(1) Synthesis of Compound 6

(29) 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (80.8 mg) was added to Compound 2 (60 mg) synthesized in the same way as in Example 2, following which N-hydroxysuccinimide (48 mg) was added and the reaction was carried out in N,N-dimethylformamide (1 mL) for 1 hour under ice cooling, then for 3 hours at room temperature. Following the reaction, several drops of acetic acid were added dropwise under ice cooling and the system was stirred for 30 minutes. Partitioning between ethyl acetate and saturated saline was carried out, after which the organic phase was concentrated by evaporation. Next, 2-propanol was added and heating was carried out, after which impurities were filtered off and the filtrate was ice-cooled, yielding Compound 6 (50 mg). ESI-MS (M+H.sup.+); theoretical value, 283.068; measured value, 283.079

(2) Synthesis of Compound 7

(30) Compound 6 (2.2 mg) was added, within a mixed solvent of N,N-dimethylformamide (50 L) and water (50 L), to doxorubicin (3 mg), after which triethylamine (1.4 L) was added and the reaction was carried out for 24 hours at room temperature. Following the reaction, purification was carried out with a reversed phase HPLC column (GL Sciences Inc.; Inertsil ODS-3, 2050 mm; flow rate, 5 mL/min; developing solution, methanol/water=60/40 (0 min) to 100/0 (20 min)).

(31) ESI-MS (M+Na.sup.+): theoretical value, 733.1967; measured value, 733.2013

(32) Compound 7 in the above reaction scheme was thereby obtained.

Example 7

Evaluation of Cell Survival Rate (Low-Oxygen Environment-Responsive Doxorubicin)

(33) Human pancreatic cancer cells (MIA PaCa-2, acquired from Riken Cell Bank) were dispensed at a density of 5,000 cells/well and cultured for 24 hours in Dulbecco's modified Eagle medium (DMEM), following which Compound 7 was added to the specified concentration and 6 hours of culturing was carried out at a normal oxygen concentration (20%) or a low oxygen concentration (0.1%). After culturing, DMEM medium replacement was carried out, the compound was removed, and 48 hours of culturing was carried out in a normal oxygen concentration incubator, following which the cell survival rate was analyzed by a WST assay. The results are shown in FIG. 4.

(34) It is apparent from the diagram that Compound 7 significantly lowers the survival rate of human pancreatic cancer cells at a low-oxygen concentration compared with a normal oxygen concentration.

Comparative Example

Comparison of Cytotoxicities of Inventive Compound and a Known Compound that is Structurally Similar

(35) Compound 8 below, which is mentioned in WO 2009/018163 A1, was furnished for use. The cell survival rates were compared in tests conducted by the method described in Example 7 on both Compound 8 and a compound within the scope of this invention (aside from the fact that the corresponding linker was CH.sub.2CH.sub.2C(O), the manner of bonding between nitroimidazole and the drug was the same; Compound 7 of Example 6). The results are shown in FIG. 5.

(36) ##STR00009##

(37) From the diagram, compared with known Compound 8, Compound 7 according to this invention shows a significantly higher cytotoxicity against human pancreatic cancer cells.

Example 8

Synthesis of Low Oxygen Environment-Responsive Gemcitabine Prodrug

(38) ##STR00010##

(39) Trimethylchlorosilane (47 L) was added to gemcitabine (22 mg) dissolved in pyridine (1 mL), and stirring was carried out for 2 hours at 0 C. Next, Compound 6 dissolved in acetonitrile (1 mL) was added and stirred for 12 hours at 45 C. The reaction was followed by the addition of ethanol (1 mL) and 30 minutes of stirring at 45 C., then the addition of water (1 mL) and 30 minutes of stirring at 45 C. The solvent was removed by evaporation, followed by purification with a reversed phase HPLC column (GL Sciences Inc.; Inertsil ODS-3, 2050 mm; flow rate, 5 mL/min; developing solution, acetonitrile/water=20/80 (0 min) to 50/50 (30 min)).

(40) This gave purified Compound 9 in a yield of 20%.

(41) ESI-MS [M+H].sup.+: theoretical value, 431.11; measured value, 431.20

Example 9

Evaluation of Cell Survival Rate (Low-Oxygen Environment-Responsive Gemcitabine)

(42) Human pancreatic cancer cells (MIA PaCa-2, acquired from Riken Cell Bank) were dispensed at a density of 5,000 cells/well and cultured for 24 hours in DMEM medium, following which Compound 9 was added to the specified concentration and 1 hour of culturing was carried out at a normal oxygen concentration (20%) or a low oxygen concentration (0.1%). After culturing, DMEM medium replacement was carried out, Compound 9 was removed, and 48 hours of culturing was carried out in a normal oxygen concentration incubator, following which the cell survival rate was analyzed by a WST assay. The results are shown in FIG. 6.

Example 10

Synthesis of Low Oxygen Environment-Responsive Fluorouracil Prodrug

(43) ##STR00011##

(44) Thionyl chloride (500 L) was added to Compound 2 (30 mg) dissolved in methylene chloride (1 mL), and the reaction was carried out at 60 C. for 2 hours. Following the reaction, the solvent was removed by evaporation. The product (Compound 10) was dissolved in methylene chloride (1 mL), 5-FU (21 mg) dissolved in pyridine (1 mL) was added, and the reaction was carried out at 0 C. for 30 minutes, then at room temperature for 12 hours. After the reaction, the solvent was removed by evaporation and purification was carried out with a reversed phase HPLC column (GL Sciences Inc.; Inertsil ODS-3, 2050 mm; flow rate, 5 mL/min; developing solution, acetonitrile/water=20/80 (0 min) to 50/50 (30 min)), thereby giving Compounds 11 and 12 as a mixture. The yield was 45%.

(45) ESI-MS [M+H].sup.: theoretical value, 296.1; measured value, 296.1

Example 11

Evaluation of Cell Survival Rate (Low Oxygen Environment-Responsive 5-Fluorouracil)

(46) Human pancreatic cancer cells (MIA PaCa-2, acquired from Riken Cell Bank) were dispensed at a density of 5,000 cells/well and cultured for 24 hours in DMEM medium, following which a mixture of Compounds 11 and 12 was added to the specified concentration and 24 hours of culturing was carried out at a normal oxygen concentration (20%) or a low oxygen concentration (0.1%). After culturing, medium replacement was carried out, the compounds were removed, and 48 hours of culturing was carried out in a normal oxygen concentration incubator, following which the cell survival rate was analyzed by a WST assay. The results are shown in FIG. 7.

Example 12

Synthesis of Low Oxygen Environment-Responsive Mesalazine Prodrug

(47) ##STR00012##

(48) Compound 10 (0.11 mmol) synthesized in the same way as in Example 10 was dissolved in methylene chloride (1 mL), then mesalazine (16 mg) dissolved in pyridine (1 mL) was added and the reaction was carried out at 0 C. for 30 minutes, then at room temperature for 24 hours. After the reaction, the solvent was removed by evaporation, and purification was carried out with a reversed phase HPLC column (GL Sciences Inc.; Inertsil ODS-3, 2050 mm; flow rate, 5 mL/min; developing solution, methanol/water=20/80 (0 min) to 20/80 (30 min)), thereby giving Compound 14. The yield was 35%.

(49) ESI-MS [MH].sup.: theoretical value, 319.07; measured value, 318.78

Example 13

Synthesis of Low Oxygen Environment-Responsive Melphalan Prodrug

(50) ##STR00013##

(51) Compound 10 (0.11 mmol) synthesized in the same way as in Example 10 was dissolved in methylene chloride (1 mL), then melphalan (32 mg) dissolved in pyridine (1 mL) was added and the reaction was carried out at 0 C. for 30 minutes, then at room temperature for 24 hours. After the reaction, the solvent was removed by evaporation and purification was carried out with a reversed phase HPLC column (GL Sciences Inc.; Inertsil ODS-3, 2050 mm; flow rate, 5 mL/min; developing solution, methanol/water=20/80 (0 min) to 80/20 (30 min)), thereby giving Compound 15. The yield was 38%.

(52) ESI-MS [MH].sup.: theoretical value, 470.10; measured value, 469.63

Example 14

Synthesis of Low Oxygen Environment-Responsive Methotrexate Prodrug

(53) ##STR00014##

(54) Compound 10 (0.11 mmol) synthesized in the same way as in Example 10 was dissolved in methylene chloride (1 mL), then methotrexate (50 mg) dissolved in pyridine (1 mL) was added and the reaction was carried out at 0 C. for 30 minutes, then at room temperature for 24 hours. After the reaction, the solvent was removed by evaporation, and purification was carried out with a reversed phase HPLC column (GL Sciences Inc.; Inertsil ODS-3, 2050 mm; flow rate, 5 mL/min; developing solution, methanol/water=20/80 (0 min) to 80/20 (30 min)), thereby giving a mixture of Compounds 16 and 17. The yield was 51%.

(55) MALDI-TOF MS [MH].sup.: theoretical value, 620.19; measured value, 620.38

Example 15

Synthesis of Low Oxygen Environment-Responsive Prednisolone Prodrug

(56) ##STR00015##

(57) Compound 10 (0.16 mmol) synthesized in the same way as in Example 10 was dissolved in methylene chloride (1 mL), then prednisolone (84 mg) dissolved in pyridine (1 mL) was added and the reaction was carried out at 0 C. for 30 minutes, then at room temperature for 24 hours. After the reaction, the solvent was removed by evaporation, partitioning between saturated saline and chloroform was carried out, and the organic phase was treated with sodium sulfate. The organic phase was then concentrated using an evaporator and purified by silica gel chromatography, giving Compound 13. The yield was 42%.

(58) ESI-MS (M+H.sup.+): theoretical value, 528.23; measured value, 527.93

Example 16

Release of Drug from Prednisolone Prodrug in Low-Oxygen Environment

(59) Human pancreatic cancer cells (MIA PaCa-2, acquired from Riken Cell Bank) were prepared as a suspension having a cell count of 110.sup.4 cells/mL), and dispensed into a 96-well plate. After 24 hours, Compound 13 was added to a concentration of 10 and the cells were cultured in a normal oxygen concentration incubator (20% O.sub.2) or at a low-oxygen work station (0.1% O.sub.2). One hour layer, the culture medium was recovered, trypsin was added, and the cells were recovered. The recovered cells were disrupted by sonic treatment and the compound was extracted with acetonitrile, following which analysis by LC/MS of the amount of prednisolone released was carried out under the following conditions.

(60) Column used: TSKgel ODS-100Z (particle size, 3 m; 2 mm75 mm) column

(61) Measurement wavelength: 250 nm

(62) Eluent A: 0.1 M ammonium acetate

(63) Eluent B: acetonitrile

(64) Flow rate: 0.2 mL/min

(65) Gradient: 40:60 (Eluent A:Eluent B).fwdarw.10:90 (20 minutes)

(66) By carrying out this measurement, the amount of compound released in a low-oxygen environment was compared with the amount released in a normal oxygen environment. The HPLC results are shown in FIG. 8. It is apparent from the diagram than much prednisolone is released in a low-oxygen environment.

INDUSTRIAL APPLICABILITY

(67) This invention makes it possible to provide prodrugs which reduce the side effects of the parent compound in a normal oxygen concentration environment, but exhibit the activity inherent to the parent compound in a low-oxygen environment. This invention can be utilized in, for example, the pharmaceutical industry which provides therapeutically active organic compounds of reduced toxicity.