Podophyllotoxin derivative with 4-position nitrogen substitution and preparation method and application thereof

Abstract

A compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable formulation prepared using the compound and the salt. The compound represented by formula (I) or the pharmaceutically acceptable salt exhibits significantly higher buildup and concentration in the lungs compared to other tissues, a longer dwell time in the lungs, and/or elevated pharmaceutical efficacy. ##STR00001##

Claims

1. A compound of the following structure ##STR00020## or a pharmaceutically acceptable salt thereof; wherein: A is ##STR00021## , and R.sub.5 is C1-C3 alkyl, R.sub.6 is di-(C1-C3 alkyl)amino C1-C3alkyl, or A is selected from the group consisting of ##STR00022##

2. The compound according to claim 1, which is: ##STR00023## or a pharmaceutically acceptable salt thereof.

3. The compound according to claim 1, which is: ##STR00024## or a pharmaceutically acceptable salt thereof.

4. The compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the pharmaceutically acceptable salt thereof is a salt formed from the compound and an inorganic acid or an organic acid, comprising hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, glutaric acid, fumaric acid, tartaric acid, maleic acid, citric acid, ascorbic acid, oxalic acid, niacin, camphoric acid, gluconic acid, glucuronic acid, pamoic acid, methanesulfonic acid, ethanesulfonic acid, sulfamic acid and p-toluenesulfonic acid.

5. A pharmaceutically acceptable preparation, comprising the compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the preparation is selected from tablet, suppository, soft capsule or hard capsule, solution, suspension or aerosol, injection, lyophilized powders for injection, sustained-release controlled-release preparation and particle drug delivery system, and wherein the preparation is administered by a manner of mouth, nasal, rectal, transdermal or injection.

6. The preparation according to claim 5, which is an injection.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a concentration distribution profile of 4-demethylepipodophyllotoxin, etoposide, compound DC and compound DP in tissues after 5 minutes administration by tail vein injection.

(2) FIG. 2 is a concentration-time curve of 4-demethylepipodophyllotoxin, etoposide, compound DC and compound DP in lung after administration by tail vein injection.

(3) FIG. 3 shows the tumor-bearing lung weight after melanoma lung metastasis tumor is treated with 4-demethylepipodophyllotoxin, etoposide, compound DC and compound DP.

(4) FIG. 4 shows the number of metastasis tumor nodules after melanoma lung metastasis tumor is treated with 4-demethylepipodophyllotoxin, etoposide, compound DC and compound DP.

DETAILED DESCRIPTION

(5) The small molecular lung-targeting compound related to the present disclosure and the method for preparing the same will be further illustrated in combination with examples hereinafter. It is not limited to the present disclosure, and the modifications by one of ordinary skill in the art with the common knowledge are within the scope of the present disclosure.

EXAMPLE 1

Synthesis of 4-amino-4-demethylepipodophyllotoxin

a. Synthesis of 4-azido-4-demethylepipodophyllotoxin (Compound 2)

(6) Under condition of ice bath, 400 mg (1 mmol) of 4-demethylepipodophyllotoxin (compound 1) and 325 mg (5 mmol) of sodium azide were suspended in trichloromethane (40 mL) in a 100 mL round-bottom flask, and 2 mL of trifluoroacetic acid was slowly dropped under stirring. The temperature of the mixture was raised to room temperature, and the mixture was stirred overnight. After the completion of the reaction was detected by TLC, 25 mL of saturated sodium carbonate solution was added and the organic layer was separated, which was washed with saturated saline (25 mL2) and fully dried with anhydrous sodium sulfate. The desiccant was removed by filtration, and the solution was evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (ethyl acetate:petroleum ether=5:2) to give 390 mg of pure compound 2, which was a white foam like solid. The yield was 91.8%.

(7) .sup.1H-NMR (400 MHz, CDCl.sub.3): 6.81 (s, 1H), 6.59 (s, 1H), 6.27 (s, 2H), 6.01-6.03 (m, 2H), 5.48 (s, 1H), 4.77-4.78 (d, J=3.2 Hz, 1H), 4.62-4.63 (d, J=5.2 Hz, 1H), 4.29-4.31 (d, J=9.6 Hz, 2H), 3.77 (s, 6H), 3.14-3.19 (dd, J=5.2, 14.0 Hz, 1H), 2.91-2.97 (m, 1H).

(8) ESI-MS (m/z): 448.1 [M+Na].sup.+

b. Synthesis of 4-amino-4-demethylepipodophyllotoxin (Compound 3)

(9) At room temperature, 425 mg (1 mmol) of compound 2 and 100 mg of 10% Pd/C were suspended in dry methanol in a 100 mL round-bottom flask, and reduced by introducing hydrogen. The reaction was carried out overnight at room temperature. After the completion of the reaction was detected by TLC, the catalyst was removed by filtration, and the solution was evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=120:1) to give 320 mg of pure compound 3, which was a white solid. The yield was 80.2%.

(10) .sup.1H-NMR (400 MHz, CDCl.sub.3): 6.81 (s, 1H), 6.50 (s, 1H), 6.31 (s, 2H), 5.95-5.98 (m, 2H), 4.56-4.57 (d, J=4.8 Hz, 1H), 4.29-4.31 (d, J=9.2 Hz, 2H), 4.21-4.22 (d, J=4.0 Hz, 1H), 3.78 (s, 6H), 3.27-3.32 (dd, J=5.2, 14.0 Hz, 1H), 2.82-2.85 (m, 1H).

(11) .sup.13C-NMR (100 MHz, CDCl.sub.3): 175.41, 147.59, 147.27, 146.32, 134.11, 133.87, 131.23, 131.13, 110.23, 108.59, 107.92, 101.32, 68.14, 56.39, 48.89, 43.72, 40.22, 37.97.

(12) ESI-MS (m/z): 400.1 [M+H].sup.+.

EXAMPLE 2

Synthesis of N,N,N-trimethyl-N-(4-carboxybenzyl)-1,3-propanediamine

a. Synthesis of N,N,N-trimethyl-N-(4-tert-butoxycarbonylbenzyl)-1,3-propanediamine (Compound 6)

(13) 454 mg (2 mmol) of tert-butyl 4-chloromethylbenzoate (compound 4) was put into a 100 mL round-bottom flask, and 40 mL of anhydrous acetonitrile was added to dissolve it. Then 0.584 mL (4 mmol) of N,N,N-trimethyl-1,3-propanediamine was dropped in. The reaction was carried out under stirring in 50 C. oil bath for 1 hour. After the completion of the reaction was detected by TLC, the solution was evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol=25:1) to give 495 mg of pure compound 6, which was a faint yellow oil product. The yield was 80.9%.

(14) .sup.1H-NMR (400 MHz, CDCl.sub.3): 7.92-7.94 (d, J=7.6 Hz, 2H), 7.35-7.37 (d, J=8.0 Hz, 2H), 3.52 (s, 2H), 2.37-2.41 (t, J=7.6 Hz, 2H), 2.28-2.32 (t, J=7.2 Hz, 2H), 2.23 (s, 6H), 2.18 (s, 3H), 1.67-1.71 (m, 2H), 1.59 (s, 9H).

(15) ESI-MS (m/z): 307.2 [M+H].sup.+

b. Synthesis of N,N,N-trimethyl-N-(4-carboxybenzyl)-1,3-propanediamine (Compound 7)

(16) Under condition of ice bath, 306 mg (1 mmol) of compound 6 was put into a 25 mL round-bottom flask, and 6 mL of dichloromethane was added to dissolve it. Then 2 mL of trifluoroacetic acid was slowly dropped in. The reaction was carried out under stirring for 3 hours in ice bath. After the completion of the reaction was detected by TLC, the solution was evaporated to dryness under reduced pressure to give a faint yellow oil product. The crude product was dissolved in a small amount of methanol, and hydrogen chloride was introduced therein. The product was salted under appropriate stirring, and the solution was evaporated to dryness under reduced pressure, followed by recrystallization in acetone-methanol to give 231 mg of pure compound 7, which was a white solid. The yield was 71.5%.

(17) .sup.1H-NMR (400 MHz, D.sub.2O): 8.11-8.13 (d, J=7.6 Hz, 2H), 7.64-7.66 (d, J=8.0 Hz, 2H), 4.44-4.54 (d, J=37.6, 2H), 3.28 (s, 2H), 3.20-3.24 (t, J=8.0 Hz, 2H), 2.93 (s, 6H), 2.89 (s, 3H), 2.26 (s, 2H).

(18) ESI-MS (m/z): 251.1 [M+H].sup.+.

EXAMPLE 3

(19) Synthesis of Compound DC

(20) 200 mg (0.5 mmol) of compound 3 and 194 mg (0.6 mmol) of compound 7 were weighed and put in a 50 mL round-bottom flask, and 25 mL of anhydrous dichloromethane was added and stirred. Then 266 mg (0.7 mmol) of HATU and 251 L (1.8 mmol) of triethylamine were added, stirred and reacted for 3 hours at room temperature. The solution was evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol:triethylamine=20:1:0.2%) to give 260 mg of pure compound DC, which was a faint yellow solid. The yield was 82.4%.

(21) .sup.1H-NMR (400 MHz, CDCl.sub.3): 7.71-7.73 (d, J=8.0 Hz, 2H), 7.39-7.41 (d, J=8.0 Hz, 2H), 6.82 (s, 1H), 6.32 (s, 2H), 6.55 (s, 1H), 5.97 and 5.99 (2s, 2H), 5.42-5.45 (m, 1H), 4.61-4.62 (d, J=4.8 Hz, 1H), 4.47-4.51 (m, 1H), 3.87-3.92 (m, 1H), 3.78 (s, 6H), 3.52 (s, 2H), 3.01-3.06 (m, 1H), 2.91-2.96 (dd, J=4.8, 14.0 Hz, 1H), 2.37-2.41 (t, J=7.2 Hz, 2H), 2.31-2.35 (t, J=7.6 Hz, 2H), 2.25 (s, 6H), 2.19 (s, 3H), 1.66-1.73 (m, 2H).

(22) .sup.13C-NMR (100 MHz, CDCl.sub.3): 174.55, 167.37, 148.20, 147.47, 146.77, 143.87, 134.41, 132.72, 131.62, 129.75, 129.07, 128.91, 127.03, 109.97, 109.12, 107.67, 101.51, 69.20, 61.65, 57.57, 56.22, 55.36, 48.44, 45.29, 43.51, 42.13, 41.80, 37.37, 25.21.

(23) ESI-MS (m/z): 632.3 [M+H].sup.+.

EXAMPLE 4

(24) Synthesis of Compound DP

(25) 200 mg (0.5 mmol) of compound 3 and 184 mg (0.6 mmol) of compound 8 were weighed and put in a 50 mL round-bottom flask, 25 mL of anhydrous dichloromethane was added, and then 266 mg (0.7 mmol) of HATU and 251 L (1.8 mmol) of triethylamine were added. The reaction was carried out under stirring at room temperature for 3 hours. The solution was evaporated to dryness under reduced pressure. The crude product was purified by silica gel column chromatography (dichloromethane:methanol:triethylamine=30:1:0.2%) to give 260 mg of pure compound DP, which was a faint yellow solid. The yield was 84.6%.

(26) .sup.1H-NMR (400 MHz, CDCl.sub.3): 7.71-7.73 (d, J=8.0 Hz, 2H), 7.40-7.42 (d, J=8.0 Hz, 2H), 6.81 (s, 1H), 6.55 (s, 1H), 6.32 (s, 2H), 5.97 and 5.99 (2s, 2H), 5.41-5.44 (m, 1H), 4.60-4.61 (d, J=4.8 Hz, 1H), 4.47-4.51 (m, 1H), 3.86-3.91 (m, 1H), 3.78 (s, 6H), 3.55 (s, 2H), 3.03-3.06 (m, 1H), 2.91-2.95 (dd, J=5.2, 14.0 Hz, 1H), 2.48 (s, 8H) 2.30 (s, 3H).

(27) .sup.13C-NMR (100 MHz, CDCl.sub.3): 174.44, 167.30, 148.32, 147.56, 146.59, 142.89, 134.15, 132.71, 131.77, 130.06, 129.34, 128.80, 127.02, 110.04, 109.04, 107.74, 101.54, 69.18, 62.35, 56.29, 54.86, 52.89, 48.52, 45.83, 43.54, 41.82, 37.46.

(28) ESI-MS (m/z): 616.3 [M+H].sup.+.

EXAMPLE 5

(29) Preparation of Injection Containing DC or DP

(30) 3.8 g of compound DC or DP was accurately weighed, 5% DMSO, 20% polyethylene glycol 400 and 20% absolute ethanol were added to help dissolution, and then water was added to make up the volume to 1000 mL. Pyrogen was adsorbed by activated carbon, successively filtered with a 0.45 m and a 0.22 m microporous filter membranes, and aseptically filled into a sterile ampoule to prepare an injection for intravenous injection.

EXAMPLE 6

(31) In Vivo Distribution Experiment in Mice

(32) 16 mg of 4-demethylepipodophyllotoxin was accurately weighed, 5% DMSO, 20% polyethylene glycol 400 and 20% absolute ethanol were added to help dissolution, so as to prepare an injection for intravenous injection with a concentration of 2 mg/mL. 28 mg of etoposide was accurately weighed, and 5% DMSO, 20% polyethylene glycol 400 and 20% absolute ethanol were added to help dissolution, so as to prepare an injection for intravenous injection with a concentration of 3.5 mg/mL. 30.40 mg of compound DC or DP was accurately weighed, and 5% DMSO, 20% polyethylene glycol 400 and 20% absolute ethanol were added to help dissolution, so as to prepare an injection for intravenous injection with a concentration of 3.8 mg/mL.

(33) 200 Kunming mice (male, 202 g) were used, which were fasted for 12 h before the experiment and given free access to water. In the experiment, they were randomly divided into 4 groups and administered by tail intravenous injection. The administration of 4-demethylepipodophyllotoxin was carried out with a dosage of 10.00 mg/kg. Etoposide, compound DC and compound DP were administered in equimolar amount as 4-demethylepipodophyllotoxin, and the administration dosages were etoposide 14.71 mg/kg, compound DC 15.78 mg/kg, and compound DP 15.38 mg/kg, respectively. 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and 48 hours after administration, the blood was collected and the mice sacrificed in all groups. The whole blood was put into sodium heparin-containing EP tubes, centrifuged at 5000 rpm at 4 C. for 5 min, and the upper plasma was collected and frozen at 40 C. for use. The hearts, livers, spleens, lungs, kidneys, brains and pancreas of the mice were immediately separated, and the residual blood was washed away with physiological saline. The remaining water on the surfaces of the organs was absorbed with filter papers. The organs were weighed and physiological saline 2 times the volume of the organs was added for homogenization.

(34) 0.1 mL of mouse plasma and 0.1 mL of tissue homogenate were taken and put into a 0.5 mL EP tube, 0.3 mL of methanol was added to all the samples as a protein precipitant. The sample was subjected to vortex vibration for 5 minutes, and then centrifuged at 13000 rpm at 4 C. for 10 min. The supernatant was taken and filtered with a 0.22 m organic filtration membrane, and 1 L of the sample was taken and subjected to LC-MS/MS analysis.

(35) LC-MS/MS Analysis Condition

(36) Liquid phase conditions: Agilent 1200 Series High Resolution Rapid LC System (RRLC); chromatographic column: Agilent Diamonsil ODS column (50 mm4.6 mm, 1.8 m); mobile phase: for 4-demethylepipodophyllotoxin, methanol:0.1% formic acid aqueous solution=50:50, for etoposide, acetonitrile:0.1% formic acid aqueous solution=35:65, for compound DC, methanol:0.1% formic acid aqueous solution=45:65, and for compound DP, methanol:0.1% formic acid aqueous solution=34:66; flow rate: 0.4 mL/min; column temperature: 30 C.; and injection volume: 1 L.

(37) Mass spectra conditions: Agilent Triple Quadrupole Mass Spectrometry (6410B); the analytes were subjected to a multiple reaction monitoring (MRM) in positive mode: for 4-demethylepipodophyllotoxin, etoposide, compound DC and compound DP, fragmentation voltages were 97 V, 148 V, 190 V and 169 V, respectively; collision cell voltages were 20 V, 12 V, 52 V and 36 V, respectively; and ionic reactions (m/z) were 401.1.fwdarw.185, 589.2.fwdarw.229, 632.3.fwdarw.86.1 and 616.3.fwdarw.58.1, respectively; drying gas temperature: 350 C.; drying gas flow rate: 10 L/min; atomization air pressure: 30 psi; and the capillary voltage: 4000V.

(38) Each pharmacokinetic parameters were calculated by DAS3.2.5 software, and the formula for calculating the targeting ability evaluation index, i.e., peak concentration ratio Ce and relative uptake rate Re, were shown hereinafter.
Ce.sub.lung=(C.sub.max,lung).sub.compound/(C.sub.max,lung).sub.drug or control
Re.sub.lung=(AUC.sub.0-t,lung).sub.compound/(AUC.sub.0-t,lung).sub.drug or control

(39) Results of FIG. 1 showed that 5 minutes after administration by tail intravenous injection, the concentrations of compound DC and DP in the lung were much higher than 4-demethylepipodophyllotoxin original drug group and etoposide control group. Based on the peak concentration ratio Ce, the peak concentration of compound DC in the lung was 5.29 times and 4.54 times of that of 4-demethylepipodophyllotoxin original drug group and etoposide control group, respectively; and the peak concentration of compound DP in the lung was 5.41 times and 4.65 times of that of 4-demethylepipodophyllotoxin original drug group and the etoposide control group, respectively.

(40) The results in FIG. 2 showed that after administration of compound DC and DP by tail intravenous injection, the drug concentrations in the lung were much higher than that of 4-demethylepipodophyllotoxin original drug group and etoposide control group at each time point. One hour after the administration, the concentration of 4-demethylepipodophyllotoxin (original drug) in the lung was below the detectable limit. Two hours after the administration, the concentration of etoposide (control) in the lung was below the detectable limit. However, compounds DC and DP still maintained at a relatively high drug concentration and lasted for up to 48 hours and 12 hours, respectively, significantly prolonging the retention time of the drug in the lung. Based on the relative uptake rate Re, the lung relative uptake rate of compound DC was 168.38 times and 98.16 times of that of 4-demethylepipodophyllotoxin original drug group and etoposide control group, respectively; and the lung relative uptake rate of compound DP was 42.56 times and 24.81 times of 4-demethylepipodophyllotoxin original drug group and etoposide control group, respectively.

(41) The above experiment results show that compound DC and DP have obvious lung-targeting ability, which significantly improves the accumulating concentration of drug in lung, and prolongs the retention time in lung, so as to decrease the administration dosage of the drug and decrease the toxicity and side effects.

EXAMPLE 7

(42) In Vivo Curative Efficacy Test in Mice

(43) The experiment was performed according to the method reported by Small, 2014(3):524-535. C57BL/6 mice (male, 202 g, 6 to 8 week-old) were used and intravenously injected with mouse melanoma B16 cells in logarithmic growth phase. 510.sup.5 cells were injected into each mouse to establish a melanoma lung metastasis model, and the mice were randomly divided into 5 groups (recorded as the 0th day). The injections for intravenous injecting used in the experiment were prepared according to Example 6. On the 4th, 7th and 13th day after the modeling, each group was subjected to intravenous injection for four times. 4-demethylepipodophyllotoxin was provided at a dosage of 5.00 mg/kg, and etoposide, compound DC and compound DP were provided at equimolar amount with 4-demethylepipodophyllotoxin, and the administration dosages were etoposide 7.36 mg/kg, compound DC 7.89 mg/kg, and compound DP 7.69 mg/kg, respectively. The control group was administered with equal amount of solvent. All mice were housed normally and sacrificed on the 22.sup.nd day after modeling. The lung tissue was immediately separated out, washed with physiological saline and weighed, and the number of tumor nodules was calculated.

(44) The results of the FIG. 3 and FIG. 4 showed that after establishing a mouse melanoma lung metastasis model, severe lung metastasis tumor appeared in the control group, demonstrating that the model was established successfully in the experiment. The other administration groups have different degrees of inhibitory effects on lung metastatic tumor. Comparing with the control group, 4-demethylepipodophyllotoxin group and etoposide group have improved the lung metastatic tumor to a certain degree, reduced the tumor-bearing lung weight, and decreases the number of lung metastasis tumor nodules. Compound DC group and DP group have the best curative effects, which were significantly better than that of 4-demethylepipodophyllotoxin group and etoposide group, with the least tumor-bearing lung weight and the number of lung metastasis tumor nodules.

(45) The above experiment results demonstrate that compounds DC and DP have significant lung-targeting effect, and they have stronger inhibition effect and better drug effect for lung tumor.