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WO1999032499A1 - DERIVES D'ACETAL DE 4'-O-DEMETHYL-EPIPODOPHYLLOTOXIN-β-D-GLUCOSIDE - Google Patents

DERIVES D'ACETAL DE 4'-O-DEMETHYL-EPIPODOPHYLLOTOXIN-β-D-GLUCOSIDE Download PDF

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Publication number
WO1999032499A1
WO1999032499A1 PCT/KR1998/000452 KR9800452W WO9932499A1 WO 1999032499 A1 WO1999032499 A1 WO 1999032499A1 KR 9800452 W KR9800452 W KR 9800452W WO 9932499 A1 WO9932499 A1 WO 9932499A1
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WO
WIPO (PCT)
Prior art keywords
glucoside
epipodophyllotoxin
demethyl
phenyl
compound
Prior art date
Application number
PCT/KR1998/000452
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English (en)
Inventor
Zae Sung No
Bok Ju Song
Eun Yi Song
Jong Woong Ahn
Chong Ock Lee
Sang Un Choi
Kyu Lim
Byung Doo Hwang
Original Assignee
Korea Research Institute Of Chemical Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Korea Research Institute Of Chemical Technology filed Critical Korea Research Institute Of Chemical Technology
Publication of WO1999032499A1 publication Critical patent/WO1999032499A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms

Definitions

  • the present invention relates to novel 4 '-O-demethyl- epipodophyllotoxin-3-D-glucoside acetal derivatives having an excellent anticancer activity, a process for the preparation thereof and an anticancer composition containing same as an active ingredient.
  • etoposide and teniposide have severe cytotoxicities and are insoluble in water, and, therefore, various attempts have been made to develop a novel anticancer agent by modifying etoposide.
  • etoposide and teniposide have severe cytotoxicities and are insoluble in water, and, therefore, various attempts have been made to develop a novel anticancer agent by modifying etoposide.
  • Bristol-Myers Squibb Co. in the U.S. and Microbial Chemistry Research Foundation in Japan have developed ETOPOPHOSTM and NK-611, respectively.
  • ETOPOPHOSTM was approved as an anticancer drug in 1997 and NK-611 is now said to be in a clinical test stage ( see GB Patent No. 2,207,674 A and EP Patent No. 0,196,618).
  • the present inventors have endeavored to develop compounds having a high potency against cancer cells and improved pharmacokinetic properties; and have unexpectedly found that 4 '-0-demethyl-epipodophyllotoxin-/3-D-glucoside derivatives substituted with a hydrophobic group containing a double or triple bond, or an alkoxyalkyl group in the glucosidic 4,6-diol positions exhibit superior anticancer activities .
  • R 1 and R 2 are, each independently, hydrogen, a C 2 _ 12 alkenyl, C 2 _ 12 alkynyl, C 2 _ 12 alkoxylalkyl or cyclopropyl group optionally substituted with one or more radicals selected from the group consisting of alkoxy, phenyl, substituted phenyl, halogen, nitro, cyano, hydroxy, carboxyl and amino groups , with the proviso that R 1 and R 2 are not hydrogen simultaneously; or
  • R 1 and R 2 together with the carbon atom to which they are attached, form an unsaturated C 5 _ 15 ring optionally substituted with one or more radicals selected from the group consisting of alkoxy, phenyl, substituted phenyl, halogen, nitro, cyano, hydroxy, carboxyl and amino groups.
  • the compound of formula(I) may be prepared by a process which comprises reacting 4 ' -O-demethy 1- epipodophyllotoxin-/3-D-glucoside of formula (II) with an aldehyde of formula (III) or an acetal of formula (IV) in a suitable solvent in the presence of a catalyst as shown in Scheme 1.
  • R 1 and R 2 have the same meanings as defined above, and R 3 is a C,_ 3 alkyl group.
  • a compound of formula(II) may be reacted with a compound of formula (III) or (IV) in a molar ratio ranging from 1:5 to 1:20, preferably from 1:10 to 1:15, at a temperature ranging from 0 to 80°C, preferably at room temperature, for 1 to 48 hours, preferably from 3 to 24 hours.
  • Exemplary solvents which may be suitably used in the process of the present invention are nitromethane, dichloro ethane , chloroform, diethyl ether, tetrahydrofuran, dimethoxyethane , acetonitrile and dimethylformamide, wherein anhydrous acetonitrile and anhydrous nitromethane are preferred.
  • the catalyst which may be used in practicing the present invention is an acid such as p-toluenesulfonic acid, methanesulfonic acid, zinc chloride and an acidic resin, wherein p-toluenesulfonic acid and zinc chloride are preferred.
  • the catalyst may be used in an amount ranging from 3 to 30 wt%, preferably from 5 to 10 wt% based on the weight of the compound of formula(II).
  • 3A may be added to the reaction system to remove water produced during the reaction.
  • 4A molecular sieve is used in an amount of 200 wt% based on the weight of the compound of formula(H).
  • reaction may be terminated by adding an organic base such as trialkylamine or a pyridine derivative in an amount slightly excess of the acid catalyst employed.
  • 4'-0-demethyl-epipodophyllotoxin-/3-D-glucoside of formula(II) may be prepared in accordance with a known method(see P. Allevi, M. Anastasia, P. Ciuffreda, A. M. Sanvito and P. Macdonald, Tet . Lett . , 1992, 33, 4831-4834; S. Hashimoto, T. Hyundai, and S. Ikegami, Tet. Lett.. 1991, 32, 1653-1654).
  • aldehydes of formula(III) which may be suitably used in the process of the present invention include cyclopropanecarboxaldehyde, phenylpropagylaldehyde, methacrolein, ⁇ -methyl-trans-cinnamaldehyde, 1,2,3,6- tetrahydrobenzaldehyde, 3-methyl-2-butenaldehyde and the like.
  • acetal compound of formula (IV) are acroleindimethylacetal, 3-butenal-diethylacetal, 3- methoxybutyraldehyde dimethylacetal, 2-butyl-l-aldehyde diethylacetal, 3-ethoxypropionaldehyde diethylacetal and 1,1, 3-trimethoxypropane .
  • the present invention also includes within its scope pharmaceutical compositions comprising one or more of the compounds of formula(I) as an active ingredient, in association with pharmaceutically acceptable carriers, excipients or other additives, if necessary.
  • compositions of the present invention may be formulated for administration orally or, preferably, by injection.
  • the composition for oral administration may take various forms such as tablets and gelatin capsules, which may contain conventional additives such as a diluent (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), a lubricant (e.g., silica, talc, stearic acid or its magnesium and calcium salts and polyethylene glycol).
  • a diluent e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine
  • a lubricant e.g., silica, talc, stearic acid or its magnesium and calcium salts and polyethylene glycol.
  • the composition may further comprise a binder (e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose and polyvinyl picolidine) and optionally a disintegrant (e.g., starch, agar and alginic acid or its sodium salt), absorbent, colorant, flavor and sweetener.
  • a binder e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose and polyvinyl picolidine
  • a disintegrant e.g., starch, agar and alginic acid or its sodium salt
  • the amount of the active ingredient actually administered should be determined in light of various relevant factors including the condition to be treated, the chosen route of administration, the age and weight of the individual patient, and the severity of the patient's symptoms; and, therefore, the dosage suggested above should not be construed to limit the scope of the invention in any way.
  • Example 1 Synthesis of 4 ' -O-demethyl-epipodophyllotoxin- /3-D- ( alkenylidene or alkynylidene ) -glucoside derivatives ( Compounds 1-8 ) 200 mg(0.36 mmol) of 4 ' -O-demethyl-epipodophyllotoxin- 3-D-glucoside was dissolved in 20 ml of anhydrous acetonitrile in the presence of 400 mg of 4A molecular sieve and added thereto were 20 mg of p-toluenesulfonic acid and an excess amount ( 2.0-3.5 mmol) of acrolein dimethylacetal (Compound 1), 3-butenaldiethylacetal (Compound 2), 2-butyne-1-aldehyde diethylacetal (Compound 3), phenylpropagylaldehyde( Compound 4), ⁇ -methyl-trans- cinnamaldehyde ( Compound 1
  • Compound 2 4 '-0-demethyl-epipodophyllotoxin-/3-D-(3- butenylidene) -glucoside;
  • Compound 8 4 '-0-demethyl-epipodophyllotoxin-/3-D-(2- methyl-2-propenylidene) -glucoside;
  • Test Example 1 in vitro anticancer activity
  • Step 1 cell cultivation
  • Cancer cell lines derived from human, i.e., A-549 (non- molding type cell lung cancer), SK-OV-3 (adenocarcinoma, ovary malignant hydroperitoneum) , SK-MEL-2 (malignant melanoma, transferred to femoral skin), XF498(cancer of central nervous system) and HCT15 (colonic cancer) were obtained from U.S. National Cancer Institute and subcultured in Korea Research Institute of Chemical Technology. These cell lines were cultured in a RPMI (Rosewell Park Medium) 1640 culture medium enriched with 5% fetal bovine serum at a constant temperature of 37°C and constant humidity of 98% under an atmosphere of 5% C0 2 .
  • RPMI Rosewell Park Medium
  • Step 2 in vitro anticancer activity
  • the in vitro anticancer activity of the compound of the present invention was assessed in accordance with SRB (sulforhodamin B) assay method developed by U.S. National Cancer Institute in 1989.
  • the subcultured cells were treated with a tripsin-CDTA ⁇ olution to separate the cells, which were added to the wells of a 96 well microplate(Falcon Co., U.S.) in an amount of 5 x 10 3 (A549, HCT15), 1 x 10 4 (SK-MEL-2 , XF498), 2 x 10 4 (SK-OV-3) cells per each wells.
  • the cells were incubated under a C0 2 atmosphere for 24 hours to be affixed on the well bottom and the culture medium was removed with an aspirator.
  • the Compounds 1 to 12 of the present invention and etoposide(BMS, U.S.A) were used as test compounds. Each of the test compounds was dissolved in water to obtain a test solution. 0.5% dimethylsulfoxide was used to dissolve the compounds, if necessary. The test solution was diluted with the culture medium(pH 7.2), e.g., by a factor of 1, 10 " ⁇ 10" 2 , 10" 3 , 10" A and 10 -5 . After filtering through a 0.22ml filter, 100 ml(x3) of each of the diluted test solutions was added to the wells and the mixture was cultured for another 48 hours.
  • TCA trichloroacetic acid
  • the anticancer activities (A. . ) of the compounds were calculated by equation (1) or (2).
  • A. A. [ (T-Tz)/(C-Tz) ] X 100 (Tz>T) Eq. (1)
  • A. A. [(T-Tz)/Tz] x 100 (Tz ⁇ T) Eq. (2)
  • Tz is the number of cells before adding a test compound without incubation
  • C is the number of cells after incubation for 48 hours in the absence of a test compound
  • T is the number of cells after incubation for 48 hours the cell in the presence of a test compound.
  • ED 50 i.e., the concentration of a compound to inhibit the growth of a cancer cell by 50% was calculated by regression method using LOTUS program.
  • Test Example 2 Inhibition of human topoisomera ⁇ e II
  • Step 1 Preparation of topoisomerase II HeLa cell line provided by ATCC(American Type Culture Collection) was used in preparing topoiso erase II of the cancer cell.
  • the HeLa cells were cultured in a RPMI 1640 culture medium enriched with 10% fetal bovine serum at 37°C under an atmosphere of 5% C0 2 and the cultured HeLa cells were collected. Added thereto was a 3-fold volume of 50 mM KH 2 PO A buffer solution(pH 7.0) containing 1 mM ethylenediamine tetraacetic acid(EDTA), ImM mercaptoethanol, 0.5 mM phenylmethylsulfonylchloride(PMSF) and 10% glycerol and the resulting mixture was homogenized with Polytron* for 30 seconds in ice bath.
  • EDTA ethylenediamine tetraacetic acid
  • PMSF phenylmethylsulfonylchloride
  • a 2M KH 2 PO buffer solution(pH 7.0) was added to a KH 2 PO concentration of 0.3M and the resultant was homogenized for 30 seconds, placed in an ice bath for 1 hour, and then, centrifuged at 30,000 xg for 30 minutes. The supernatant was purified by phosphocellulose column chromatography(eluent : linear gradient from 0.3M to 0.5M KH 2 P0 4 buffer(pH 7.0) to obtain topoisomerase II.
  • Step 2 P unknotting assay
  • IC 50 values of the test compounds for topoisomerase II were measured by an unknotting assay.
  • Topo II obtained above and 0.4 ⁇ g of P knotted DNA were added to a 50 mM N-[ 2-hydroxyethyl]-piperazine-N'-[ 2- ethanesulfonic acid] (HEPES) buffer solution (pH 7.0) containing 50 mM KC1, 0.1 mM EDTA, 100 mM NaCl, 10 mM MgCl 2 , 100 ⁇ g/ml of fetal bovine serum albumin and 1 mM adenosine triphosphate(ATP) to a final volume of 20 ⁇ l and the mixture wa ⁇ reacted at 37 °C for 30 minutes.
  • HEPES 2-hydroxyethyl]-piperazine-N'-[ 2- ethanesulfonic acid]
  • the reaction was terminated by adding sodium dodecyl sulfate(SDS) to a SDS concentration of 1%.
  • SDS sodium dodecyl sulfate
  • the resultant was subjected to electrophoresis using 1% agarose gel equilibrated with a 50 mM tris borate buffer solution(pH 8.3) containing 2.5mM EDTA, and then, dyed with ethidium bromide .
  • Topo II The activity of Topo II was assessed using photographs taken under UV radiation. The Topo II activity measured when 50% of 0.4 ⁇ g of knotted P A DNA was unknotted was defined as 1 unit activity. IC 50 was the concentration of a compound to inhibit the activity of Topo II by 50%. The results are shown in Table II.
  • Test Example 3 In. vivo anticancer activity
  • a dose of 1 x 10 s cells/0. lml of leukemia L1210 cell subcultured in DBA/2 mouse was injected into the abdominal cavity of each of the mice.
  • Two test compound ⁇ , etoposide and Compound 1 of the present invention were injected into the abdominal cavities of mice of respective test group in the doses shown in Table III, successively at 1st, 2nd, 3rd, 4th and 5th days from the leukemia cell injection.
  • mice were observed daily and the survival period of each mouse was checked.
  • the anticancer activity was assessed by calculating the ratio (I.R.) by equation 3.
  • I.R. 100 x (A t - A c )/A c Eq (3) wherein, A t is the average survival days of mice in a test group and A c is the average survival days of mice in the contro1 group.
  • the novel 4 '-O-demethy1- epipodophyllotoxin-/3-D-glucoside derivative of formula(I) of the present invention possesses an excellent anticancer activity against various cancer cells, much higher than that of etoposide.
  • the inventive compounds can therefore be used as a drug for treating cancers.

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Abstract

Cette invention concerne un dérivé d'acétal de 4'-O-déméthyl-épipodophyllotoxin-β-D-glucoside correspondant à la formule (I). R1 et R2 représentent chacun indépendamment hydrogène ou un groupe alcényle C¿2-12?, alcynyle C2-12, alcoxyalkyle C2-12 ou cyclopropyle qui est éventuellement substitué par un ou plusieurs radicaux choisis dans l'ensemble comprenant les groupes alcoxy, phényle, phényle substitué, halogène, nitro, cyano, hydroxy, carboxyle et amino, étant entendu que R?1 et R2¿ ne peuvent représenter simultanément hydrogène. R1 et R2 peuvent encore former, avec l'atome de carbone auquel ils sont liés, un anneau C¿5-15? insaturé qui est éventuellement substitué par un ou plusieurs radicaux choisis dans l'ensemble comprenant les groupes alcoxy, phényle, phényle substitué, halogène, nitro, cyano, hydroxy, carboxyle et amino.
PCT/KR1998/000452 1997-12-23 1998-12-22 DERIVES D'ACETAL DE 4'-O-DEMETHYL-EPIPODOPHYLLOTOXIN-β-D-GLUCOSIDE WO1999032499A1 (fr)

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KR1997/72410 1997-12-23
KR19970072410 1997-12-23

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3524844A (en) * 1965-09-21 1970-08-18 Sandoz Ag Epipodophyllotoxin glucoside derivatives
EP0196618A1 (fr) * 1985-04-02 1986-10-08 Microbial Chemistry Research Foundation Dérivés de 4'-déméthyl-4-épipodophyllotoxine, procédé pour leur préparation et leur utilisation comme médicament
GB2207674A (en) * 1987-08-04 1989-02-08 Bristol Myers Co Podophyllotoxin derivatives
EP0367189A2 (fr) * 1988-10-31 1990-05-09 Bristol-Myers Squibb Company Phosphates et sulphates des sucres des glycosides d'épipodophyllotoxines

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3524844A (en) * 1965-09-21 1970-08-18 Sandoz Ag Epipodophyllotoxin glucoside derivatives
EP0196618A1 (fr) * 1985-04-02 1986-10-08 Microbial Chemistry Research Foundation Dérivés de 4'-déméthyl-4-épipodophyllotoxine, procédé pour leur préparation et leur utilisation comme médicament
GB2207674A (en) * 1987-08-04 1989-02-08 Bristol Myers Co Podophyllotoxin derivatives
EP0367189A2 (fr) * 1988-10-31 1990-05-09 Bristol-Myers Squibb Company Phosphates et sulphates des sucres des glycosides d'épipodophyllotoxines

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KR100321844B1 (ko) 2004-05-24
KR19990063387A (ko) 1999-07-26

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