WO1993022319A1 - Derives de 4 beta-amino podophyllotoxine utilises en tant qu'agents antitumoraux - Google Patents

Derives de 4 beta-amino podophyllotoxine utilises en tant qu'agents antitumoraux Download PDF

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Publication number
WO1993022319A1
WO1993022319A1 PCT/US1993/003830 US9303830W WO9322319A1 WO 1993022319 A1 WO1993022319 A1 WO 1993022319A1 US 9303830 W US9303830 W US 9303830W WO 9322319 A1 WO9322319 A1 WO 9322319A1
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och
compound
compounds
demethyl
desoxypodophyllotoxin
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PCT/US1993/003830
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English (en)
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Kuo-Hsiung Lee
Yung-Chi Cheng
Yi-Lin Zhang
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University Of North Carolina At Chapel Hill
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Priority claimed from US07/987,765 external-priority patent/US5300500A/en
Application filed by University Of North Carolina At Chapel Hill filed Critical University Of North Carolina At Chapel Hill
Publication of WO1993022319A1 publication Critical patent/WO1993022319A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems

Definitions

  • the present invention relates to novel 4 ⁇ -amino podophyllotoxin analog compounds useful in the treatment of tumors, to methods of synthesis of the compounds, and to methods of treating tumors with pharmaceutical preparations containing the compounds.
  • Podophyllotoxin is a naturally occurring
  • etoposide also known as VP-16
  • This compound exhibits therapeutic activity in several human neoplasms, including small cell
  • carcinomas of the lung testicular carcinomas
  • topoisomerases are enzymes which control the
  • topological state of DNA Type II topoisomerases catalyze DNA strand passage through transient double strand breaks in the DNA. The resulting change in the linking number of DNA allows these enzymes to mediate DNA interconversions, such as supercoiling and relaxation of supercoiling, catenation and decatenation, knotting, and unknotting (Wang, 1985, Maxwell).
  • Type II DNA topoisomerase enzymes have been shown to be involved in a number of vital cellular processes, including DNA replication and
  • R is a sugar moiety selected from arabinosyl, xyrosyl, hamnosyl, glucosyl, and 4,6-ethylene
  • Another podophyllotoxin derivative synthesized in the art is 3',4'-didemethoxy-3',4'-dioxopodophyllotoxin of formula:
  • Ayers et al. (1980) disclosed the synthesis of this compound by reacting podophyllotoxin with nitric acid.
  • Nemec discloses a similar oxidation of
  • the present invention includes, in one aspect, novel 4 ⁇ -amino podophyllotoxin analog compounds of the general formula:
  • NHR is selected from the group consisting of
  • R 1 and R 2 are the same or different and are H or lower alkyl groups, or
  • R is CH 2 Ph or CR 2 Et
  • Figure 1 shows steps in the general synthesis of aryl amine podophyllotoxin analog compounds of the type illustrated by compound 4 in Figure 2;
  • Figure 2 shows the aryl amine moiety in 4 ⁇ -arylamino podophyllotoxin analog compounds 4-a to 4-v, in accordance with one general embodiment of the invention
  • Figure 3A shows steps in one method of synthesis of 4 ⁇ -aminoalkyl podophyllotoxin analog compounds of the type illustrated by compound 6;
  • Figure 3B shows steps in a second method of synthesis of 4 ⁇ -aminoalkyl podophyllotoxin analog compounds of the type illustrated by compound 6;
  • Figure 5 shows steps in methods of synthesis of 4 ⁇ -dialkylaminoanilino podophyllotoxin analog
  • Figure 6 shows an alternative method for
  • Figure 7 shows dose-response data for compound 23 and etoposide in promoting the formation of protein-DNA complexes.
  • the present invention includes novel 4 ⁇ -amino podophyllotoxin analog compounds of the general formula:
  • Structure (I) is a 4'-O-demethyl epipodophyllotoxin, meaning a podophyllotoxin parent structure in which the 4' methoxy group of podophyllotoxin has been substituted by an OH group.
  • the invention also contemplates analogous compounds in which the 3', 4', and/or 5' positions on the phenyl ring are OH, methoxy, or ethoxy groups.
  • a selected amino group, indicated at NHR is carried at the 4 ⁇ position of the fused ring portion of the structure, as shown.
  • the 4 ⁇ -amino group is an aryl amine selected from one of the moieties shown in Figure 2.
  • the 4B-amino group is a dialkylaminoanilino analog, as described in Section III. I. Arylamine Podophyllotoxin Analog Compounds
  • Figure 2 shows aryl amino (NHR) moieties in 4 ⁇ -amino podophyllotoxin analog compounds (4-a)-(4-v) of the invention.
  • Compounds (4-a)-(4-n) are substituted aniline podophyllotoxin compounds disclosed in PCT International Publication Number WO 90/09788 noted above.
  • Compounds (4-o) and (4-p) are 2" and 3"-pyridylamino podophyllotoxin compounds, respectively, also disclosed in the above PCT application.
  • Compound (4-q) is a 3" quinolylamino podophyllotoxin compound, also disclosed in the above PCT application.
  • Compounds (4-r)-(4-t) are hydrochloride salts of three amino anilino podophyllotoxin compounds which are disclosed in the above PCT application.
  • Compounds 4-u, 4-v, and the sodium salt of 4-u are novel substituted aniline podophyllotoxin compounds.
  • HBr gas is bubbled through a solution of podophyllotoxin, i.e., A ' -O-Demethylepipodophyllotoxin (compound 1) in dry dichloromethane, followed by bubbling nitrogen to drive off excess HBr, forming the corresponding 4'-O-demethyl-4 ⁇ -bromo-4-desoxypodophyllotoxin (compound 2).
  • the solution of compound 2 is evaporated under vacuum, and water is removed using benzene as an azeotropic mixture.
  • HBr gas is bubbled into a cold 1,2-dichloroethane/ether solution (9:1) of compound 1. After stirring overnight, anhydrous Na 2 SO 4 is added, the reaction mixture is filtered, the solids are washed in CH 2 Cl 2 , and the combined organic
  • compound 2 is combined with anhydrous barium carbonate, and the appropriate arylamine, indicated generally by
  • the aryl amine product is shown generally as a substituted aniline group (compound 4), although other aryl amines are contemplated ( Figure 2).
  • ammonium salts of compounds 4-a to 4-v can be formed by reacting the parent compound with at least a stoichiometric amount of an acid to form the corresponding water-soluble ammonium salt.
  • Inorganic acids and organic acids capable of forming a water-soluble salt with the aryl amine compounds are physiologically acceptable and are selected, for example, from hydrochloride acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, propionic acid, formic acid, 1-ascorbic acid, tartaric acid, citric acid, lactic acid, maleic acid, fumaric acid, methanesulfonic acid, toluenesulfonic acid and benzenesulfonic acid. Procedures for preparing such ammonium salts are described in Example 7. B. Properties
  • Cytotoxicity is the concentration of analog compound which gives a 50% reduction in cell number of 10 4 cells after three days incubation. Each compound was examined with five concentrations, at 5, 10, 25, 50, and 100 ⁇ M. The ID 50 value was established on the basis of the degree of inhibition at these concentrations.
  • the compounds most active in inhibiting topoisomerase II were compounds in which an aniline group is substituted at the para position with a CO 2 CH 2 CH 3 (4- c), NO 2 (4-1), or NH 2 •HCl (4-s) group. These three compounds are preferred compounds in the present embodiment of the invention, with the para nitro substituted compound (4-1) being particularly preferred. These three substituents cover a broad range of electronegativity, as measured by published Hammett values (Hansch), and all are relatively water soluble, as measured by published ⁇ (water/octanol partition coefficients) (Hansch). II. (Dialkylamino) alkylamino Podophyllotoxin Analog
  • NHR group is an aminoalkylamine.
  • Exemplary NHR groups according to this embodiment are shown in Fig. 4 at structures (10) and (19).
  • Exemplary compounds according to this embodiment are shown in Figure 4 at structures (11), (20), (21), and (22).
  • NHR is NHR
  • R 3 is CH 2 Ph or CO 2 Et (structures 23 and 24 in Figure 4). Methods of synthesizing such compounds are described below.
  • the compounds can be formed, according to one general method, by the reaction method illustrated in Figure 3A, and detailed in Example 3.
  • the 4'-O-Demethyl-4 ⁇ -bromo-4-desoxypodophyllotoxin (compound 2) is combined with the appropriate NH 2 -(CH 2 ) n -NR 1 R 2 ((N,N-dialkylamino)alkylamine) (compound 5) in tetrahydrofuran, as detailed in Example 3.
  • the mixture is purified via column chromatography to give the desired compound 6.
  • the method shown in Figure 3B may be employed.
  • compound 2 is reacted as above with an amino alkylchloride (compound 7), under conditions which favor derivatization of compound 2, to form the amino-alkylchloride podophyllotoxin compound 8.
  • This compound is then reacted with a selected dialkyl amine (compound 9) to form the desired product (compound 6).
  • N,N-dimethyl-ethylenediamine analog shown at compound 10 is formed by reacting N,N-dimethylethylenediamine with the compound 2 , according to the Figure 3A reaction scheme.
  • the 4"-amino-N-benzyl-piperidine analog shown at 23 in Figure 4 is formed by reacting 2 with 4-amino-N-benzylpiperidine.
  • N,N-dimethyl-propylenediamine analog (19), and N,N-dialkyl-ethylenediamine analogs containing pyrrolidine (11), piperidine (20), morpholine (21), 2-pyrrolidone (22), (N-benzyl)piperidine (23), and (N-carbethoxy)piperidine (24) moieties shown in Figure 4, can be formed by reacting compound 2 with the corresponding (N-N-dialkylamino)alkylamine. The preparation and characterization of such compounds is described in Example 3.
  • Ammonium salts of the (N-N-dialkylamino)alkylamine compounds in this class can be formed by reacting the compound with an acid to form the corresponding water- soluble ammonium salt.
  • Reaction conditions such as given in Example 7 are suitable, where the acid is selected, for example, from hydrochloride acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, propionic acid, formic acid, 1-ascorbic acid, tartaric acid, citric acid, lactic acid, maleic acid, fumaric acid, methanesulfonic acid, toluenesulfonic acid and benzenesulfonic acid.
  • the compounds of this embodiment can be easily synthesized to achieve a desired compound solubility in aqueous medium, by suitable choice of n, R 1 , R 2 , and amine charged salts.
  • the water-solubility of the compounds can also be enhanced by reacting the compounds under alkylation conditions effective to form charged, quaternary amines.
  • the cellular protein-DNA complex formation assay was carried out with a range of concentrations of compound 23 (dihydrochloride salt form).
  • the effect of 23 ⁇ 2 HCl increased steadily as its concentration increased from 0.5 to 20 ⁇ M.
  • 23 ⁇ 2 HCl was found to be about 20 times as potent as etoposide (i.e., 1 ⁇ M 23-2 HCl was more effective than 20 ⁇ M etoposide) in promoting protein-DNA complex formation.
  • cytotoxicities of compound 23 and its ammonium (HCl) salt were determined with the cell lines KB ATCC, KB 7D, and KB V20C.
  • KB ATCC is a drug-sensitive Hela-cell subclone used for the cytotoxicity studies of Tables 1 and 2.
  • KB 7D is an etoposide-resistant cell line which has decreased uptake of etoposide and a reduced level of Topoisomerase II.
  • KB V20C is a multidrug-resistant (MDR) cell line that is cross-resistant to vincristine and etoposide and which overproduces P-glycoprotein P.
  • MDR multidrug-resistant
  • compound 23 and its ammonium salt were highly active, showing ID 50 values of less than 100 nM for all three cell lines. Both forms of compound 23 were at least about 20 times more active toward KB V20C than was etoposide. Moreover, both were more than 200 times more effective than etoposide towards the KB 7D cells, showing that the etoposide-resistance of these cells was overcome by compound 23.
  • compounds 19, 20, 23, and 24 are preferred embodiments of the invention.
  • R 1 and R 2 are H or lower alkyl groups, as defined above.
  • the compounds can be formed, according to one general method by the reaction method illustrated in Figure 5, and detailed in Example 4.
  • the first step in this method is the synthesis of the 4 ⁇ -nitroanilino compound (compound 13 in Figure 5) by reaction of the p-nitroaniline (compound 12) with the compound 2, as detailed in Example 4A.
  • the nitro group is then reduced under hydrogen with a catalyst of palladium on activated carbon, as described in Example 4B.
  • the 4 ⁇ -aminoanilino compound 14 can be converted to the desired dialkyl amine compound by one of two routes. In the first, the amine is reacted with formaldehyde in the presence of the reducing agent NaCNBH 3 in dichloromethane, forming a predominantly dimethylaminoanilino analog (compound 15), as described in Example 4C. In the second route, compound 14 is reacted with formaldehyde in the presence of reducing agent NaCNBH 4 in methanol, forming of dimethylamine-N-methylanilino compound 17. De tails of this reaction are given in Example 4D. Both compounds 15 and 17 can be converted to corresponding HCl salts by reaction with HCl in ethanol, to give compounds 16 and 18, respectively, as detailed in Example 7. Other ammonium salts can be formed as described above.
  • Figure 6 illustrates a one-step synthesis of dialkylaminonilino analog compounds.
  • the corresponding p-dialkylaminoaniline shown as p-dimethylaminoaniline in the figure, is reacted under basic conditions in acetonitrile with compound 2, to yield the desired analog compound 15 directly.
  • An exemplary synthetic procedure is given in Example 5.
  • a podophyllotoxin analog of the type described above is suspended in a pharmaceutically acceptable carrier and administered in a pharmaceutically effective amount to reduce tumor growth in a mammalian subject.
  • pharmaceutically effective amount is meant a concentration at the tumor site or in the bloodstream which is effective to inhibit growth of tumor cells. This concentration can be determined, for example, from ID 50 values from in vitro growth inhibition studies using known tumor cell lines which, preferably, are related to the patient's tumor type.
  • tumor treatment of a patient may typically involve the administration of periodic doses, e.g., biweekly doses of the drug, with the effectiveness of treatment being monitored by tumor biopsy, radiological methods, or blood enzyme levels, according to standard methods.
  • periodic doses e.g., biweekly doses of the drug
  • the effectiveness of treatment being monitored by tumor biopsy, radiological methods, or blood enzyme levels, according to standard methods.
  • the following examples illustrate synthetic methods for preparing podophyllotoxin analogs in accordance with the invention. The examples are intended to illustrate, but in no way limit the scope of, the invention. Materials
  • HBr gas (9.7 g) was bubbled into a cold (0°C) 1,2-dichloroethane/ether solution (9:1, 55 ml) of 4'-O-demethylepipodophyllotoxin (5 g). After overnight stirring at 0°C, Na 2 SO 4 (anhydrous, 5 g) was added to the reaction mixture, and the resulting suspension was filtered. The solids were washed with CH 2 Cl 2, and the combined organic filtrates were concentrated in vacuo. The residue was dissolved in CH 2 Cl 2 and concentrated in vacuo again to remove residual HBr.
  • Example 3 (compound 2 in Figure 3) was prepared as in Example 1. Dry tetrahydrofuran (20 ml) was added to 1 g (2.16 mmol) of compound 2, and the suspension was heated to reflux under nitrogen. The appropriate dialkylamino-alkylamine (2.6 mmol) was injected into the suspension, and the reaction mixture was refluxed for 6 hours, then evaporated under vacuum. The desired 4'-O-demethyl4 ⁇ -(alkylamino)-4- desoxypodophyllotoxin compounds were obtained by silica gel chromatography using mixed ethylacetate and hexane (3:1 to 1:1) as the eluent and recrystallization in acetone.
  • Human DNA topoisomerase II was isolated from peripheral blast cells of a patient with acute leukemia. The isolation procedure has been described (Thurston, 1988). A test compound (4-a)-(4-t) was dissolved in dimethylsulfoxide at a concentration of 20 mM as a stock solution and diluted before use with water to a desired concentration of compound.
  • the P4 unknotting reaction was a modification of the procedure described by Liu (1981).
  • the reaction mixture (20 ⁇ L), which contained 50 mM HEPES, pH 7.0, 50 mM KCI, 100 mM NaCl, 0.1 mM EDTA, 10 mM MgCl 2 , 1.0 mM ATP, 50 ⁇ g/mL bovine serum albumin, 0.4 ⁇ g P4 knotted DNA, and enzyme, was incubated with or without drugs.
  • the reaction mixture was incubated at 37°C for 30 min and terminated by adding 5.0 ⁇ l of a stop solution (2% sodium dodecyl sulfate, 20% glycerol, 0.05% bromophenol blue). These samples were loaded onto a 1% agarose gel and electectrophoresed at 55 V overnight with an electrophoresis buffer that contained 90 mM Tris-boric acid, pH 8.3, and 2.5 mM EDTA. At completion, the gel was stained in 0.5 ⁇ g/mL of ethidium bromide. Then a photograph was taken of the DNA bands visualized with fluorescence induced by a long-wavelength UV lamp. The data reported in Table 1 reflect a 100 mM drug concentration.

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Abstract

L'invention concerne des nouveaux composés de podophyllotoxine et leur utilisation dans le traitement des tumeurs. Dans un mode de réalisation, les composés sont représentés par la formule générale (I), dans laquelle NHR fait partie du groupe constitué de: (a) NH-(CH2)n-NR1R2, où n=Z-4, et où (1) R1 et R2 sont identiques ou différents et représentent H ou des groupes alkyle inférieur, ou (2) NR1R2 représente (a1), (a2), (a3) ou (a4); (b) où R3 représente CH2Ph ou CO2Et; et (c); y compris des sels d'ammonium physiologiquement acceptables de (a), (b) et (c).
PCT/US1993/003830 1992-04-24 1993-04-23 Derives de 4 beta-amino podophyllotoxine utilises en tant qu'agents antitumoraux WO1993022319A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US944,472 1986-12-19
US87434592A 1992-04-24 1992-04-24
US874,345 1992-04-24
US94447292A 1992-09-14 1992-09-14
US987,765 1992-12-08
US07/987,765 US5300500A (en) 1989-02-23 1992-12-08 4 beta-amino podophyllotoxin analog compounds and methods

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999004776A1 (fr) * 1997-07-22 1999-02-04 Meiji Milk Products Co., Ltd. Agents antineoplastiques

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990009788A1 (fr) * 1989-02-23 1990-09-07 The University Of North Carolina At Chapel Hill Analogues d'etoposide

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990009788A1 (fr) * 1989-02-23 1990-09-07 The University Of North Carolina At Chapel Hill Analogues d'etoposide

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 112, no. 13, 26 March 1990, Columbus, Ohio, US; abstract no. 111618h, YAOZU CHEN ET AL 'Anticancer agents. II. Synthesis and biological evaluation of spin-labeled derivatives of podophyllotoxin' page 28 ; *
JOURNAL OF MEDICINAL CHEMISTRY. vol. 33, no. 9, 1990, WASHINGTON US pages 2660 - 2666 ZHE-QING WANG ET AL 'Antitumor agents. 113. New 4beta-arylamino derivatives of 4'-O-demethylepipodophyllotoxin and related compounds as potent inhibitors of human DNA topoisomerase II' cited in the application *
JOURNAL OF MEDICINAL CHEMISTRY. vol. 35, no. 5, 6 March 1992, WASHINGTON US pages 866 - 871 HONG ZU ET AL 'Antitumor agents. 123. Synthesis and human DNA topoisomerase II inhibitory activity of 2'-chloro derivatives of etoposide and 4beta-(arylam ino)-4'-O-demethylpodophyllotoxins' *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999004776A1 (fr) * 1997-07-22 1999-02-04 Meiji Milk Products Co., Ltd. Agents antineoplastiques

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