EP0460084A1 - Novel beta-methoxyacrylates, their production and use - Google Patents

Novel beta-methoxyacrylates, their production and use

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Publication number
EP0460084A1
EP0460084A1 EP90904345A EP90904345A EP0460084A1 EP 0460084 A1 EP0460084 A1 EP 0460084A1 EP 90904345 A EP90904345 A EP 90904345A EP 90904345 A EP90904345 A EP 90904345A EP 0460084 A1 EP0460084 A1 EP 0460084A1
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EP
European Patent Office
Prior art keywords
formula
compounds
methoxyacrylates
cells
compound
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Legal status (The legal status 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 status listed.)
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EP90904345A
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German (de)
French (fr)
Inventor
Lothar Daum
Gerhard Keilhauer
Hubert Sauter
Gunda Bertram
Timm Anke
Wolfgang Weber
Wolfgang Steglich
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BASF SE
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BASF SE
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Publication of EP0460084A1 publication Critical patent/EP0460084A1/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D407/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00
    • C07D407/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings
    • C07D407/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having oxygen atoms as the only ring hetero atoms, not provided for by group C07D405/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • 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
    • C07D493/10—Spiro-condensed systems

Definitions

  • the present invention relates to new ⁇ -methoxyacrylates, processes for their preparation and their use in combating
  • R 1 represents hydrogen or an optionally unsaturated hydrocarbon radical having up to 10 carbon atoms, which is represented by fluorine, chlorine, bromine, C 1-6 alkoxy, C 2-6 alkenoxy or C 2-6 alkynoxy or by an optionally fluorine, Chlorine, bromine, iodine, trifluoromethyl, C 1-4 -alkyl or C 1-4 -alkoxy substituted phenyl or phenoxy may be substituted,
  • R 2 and R 3 are the same or different and are hydrogen atoms
  • R 2 and R 3 together represent a C 2-8 alkylene group, have a better effect
  • the compounds of formula I have two centers of chirality in
  • the configuration isomers can expediently also be separated at the stage of intermediates of the formula II (see below). A separation of the configuration isomers is not necessary for the use of the compounds according to the invention.
  • the ⁇ -methoxyacrylate group can be in the E or Z configuration.
  • the compounds of formula I are preferred with
  • R 4 is a C 1-8 alkyl radical, reacted in the presence of a base and the compounds thus obtained - if R 1 is an unsaturated radical - optionally hydrogenated.
  • the reaction of the compounds II with III takes place in the sense of a Wittig-Homer reaction in the presence of a solvent and a base, such as lithium diisopropylamide, phenyllithium, butyllithium, sodium alcoholate, sodium amide, sodium hydride or potassium t-butoxide.
  • a solvent and a base such as lithium diisopropylamide, phenyllithium, butyllithium, sodium alcoholate, sodium amide, sodium hydride or potassium t-butoxide.
  • Suitable solvents for the reaction are benzene, dimethylformamide, tetrahydrofuran, diethyl ether and dimethoxyethane.
  • the reaction is useful in a
  • Sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, 1,8-diazabicyclo [5,4,0] undec-7-ene, diisopropylmethylamine and diisopropylethylamine are particularly suitable as auxiliary bases for the reaction V ⁇ VI.
  • Preferred solvents are dimethylformamide, tetrahydrofuran, diethyl ether, methanol, ethanol, acetonitrile and acetone.
  • the reaction V ⁇ VI works well at temperatures between 0 and 30 ° C.
  • reaction mixture was diluted with diethyl ether and washed 1x with semi-saturated NaCl solution to remove the catalyst. After drying over MgSO 4, the ether phase was concentrated in vacuo.
  • reaction mixture was poured into semi-saturated NaCl solution, extracted with ether and dried over MgSO 4 .
  • the two diasteromers were formed in a ratio of about 1: 1.
  • Diastereomer 1 330 mg of colorless crystals
  • Diastereomer 2 308 mg colorless crystals
  • Diastereomer 1 (C 17 H 20 O 5 ) (MW 304):
  • Diastereomer 2 (C 17 H 20 O 5 ) (MW 304): 1 H-NMR (200 MHz, benzene-d 6 ):
  • the mixture was allowed to warm to room temperature and stirred for 24 h.
  • the reaction solution was poured into 15 ml of ether, washed twice with 3 ml of water and once with 3 ml of saturated NaCl solution. After drying over MgSO 4 , the mixture was evaporated.
  • the raw product was with
  • Diastereomer 1 (C 29 H 32 O 7 , MW 492):
  • Diastereomer 1 (C 32 H 36 O 7 , MW 532):
  • Distereomer 2 (C 32 H 36 O 7 , MW 532):
  • the new compounds in particular the compounds of Examples 1 to 16 - show good antiviral, antiproliferative and cytotoxic activity.
  • the following examples explain the effects of the new substances:
  • the oxygen consumption was measured polarographically in an airtight container (3 ml volume, with magnetic stirrer) with an oxygen electrode.
  • the test mushroom was grown from a spore suspension to a mycelium weight of 10-20 mg mycelium wet weight / ml. The measurements were carried out with a mycelium concentration of 25-30 mg mycelium wet weight / ml in 1% glucose solution. After a short constant course of breathing, the compounds (0.3 mg / ml) dissolved in methanol were added to the suspension and the 02 consumption was recorded. The experiment was carried out using strobilurin A as a comparative compound. The results of the percent breathing inhibition are shown in Table 1. Table 1
  • HeLa-S3 cells were on a 96-well titer plate with a cell density of 4 x 105 cells / ml in medium F12 with 10% fetal calf serum
  • the cells were spread out on the surface and the medium was changed.
  • the cells were incubated with the medium containing the test compounds (10, 1, 0.1 or 0.01 ⁇ g per ml medium) for 72 h (37 ° C., 5% CO 2 ). After 72 hours, the normally epithelial-like growing cells were fibroblast-like
  • Morphology From a 45% growth inhibition, the change in morphology is significantly visible through the action of the substances.
  • the experiment was carried out using strobilurin A as a comparative compound.
  • the cell density was measured by determining the total protein content per hole.
  • the values given show the results after three days of incubation with the test substances and are listed in Table 2 as a percentage of the total protein of the untreated control.
  • Antiviral effect in HEp-2 cells on VSV The determination of the antiviral activity of a test compound is based on the measurement of the protection of human HEp-2 cells as indicator cells against the cytopathic effect (CPE) of Vesicular Stomatitis Virus (VSV).
  • CPE cytopathic effect
  • the percentage of protected cells in the cultures treated with the test compounds and subsequently infected with VSV was determined by means of crystal violet staining / the measure of the antiviral activity based on 0 and 100% protection was determined as the sample concentration, which leads to 50% protection.
  • Table 3 lists the test substances with the concentration which 50% protects the HEp-2 cells against the cytopathic effect of VSV.
  • Example 1 In contrast to rHuIFN- ⁇ , the substance of Example 1 also showed an antiproliferative effect under these test conditions compared to cell control. The antiviral and antiproliferative activities The substances of Example 1 ran in parallel and correlated with a morphological change in the cells, ie the cells were elongated compared to the cell control and had neurite-like processes.
  • Example D The substances of Example 1 ran in parallel and correlated with a morphological change in the cells, ie the cells were elongated compared to the cell control and had neurite-like processes.
  • tumor cells of different tissue origin 5637-6: human bladder carcinoma; HT-29: human colon carcinoma; B-16: murine melanoma
  • 1 to 2 ⁇ 103 exponential growth tumor cells were plated in 96-well plates in complete growth medium (RPMI 1640 x 10% fetal calf serum) and incubated overnight under standard culture conditions (37 ° C, 5% carbon dioxide, water vapor-saturated atmosphere). The substance was added the next day, serial titrations of substances 1 B1 and 1 B2 being carried out over a concentration range of 10 -4 to 10 -9 M. After a further incubation of 72 h under standard conditions, the cell number was determined by staining with crystal violet and a subsequent photometric

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Communicable Diseases (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Oncology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Des beta-méthoxyacrylates ayant la formule (I), dans laquelle R1, R2 et R3 ont la signification donnée dans la description, sont utiles pour traiter des maladies. L'invention concerne également leur production.Beta-methoxyacrylates having the formula (I), in which R1, R2 and R3 have the meaning given in the description, are useful for treating diseases. The invention also relates to their production.

Description

Neue ß-Methoxyacrylate, ihre Herstellung und Verwendung New ß-methoxyacrylates, their production and use
Beschreibung Die vorliegende Erf indung betrifft neue ß-Methoxyacrylate, Verfahren zu deren Herstellung sowie deren Verwendung bei der Bekämpfung von Description The present invention relates to new β-methoxyacrylates, processes for their preparation and their use in combating
Krankheiten. Diseases.
Im Journal of Antibiotics, 32 (1979) 1113 und 36 (1983) 661 sowie in "Cellular Regulation and Malignant Growth", Springer Verlag, Berlin 1985, Seiten 169-176 sind bestimmte ß-Methoxyacrylate, nämlich Strobilurin und Oudemansin und deren Derivate beschrieben. Diese Verbindungen zeichnen sich durch eine starke antifungische und cytostatische Aktivität aus. Die Strobilurine A, B und C sowie die Oudemansine A und B wurden aus den Gattungen Strobilurus, Oudemansiella, Xerula, Cyphellopsis, Hydropus und Mycena isoliert. Alle Verbindungen hemmen die Atmung von Eukaryonten und dadurch das Wachstum von Pilzen und Zellen. Die Strobilurine und In the Journal of Antibiotics, 32 (1979) 1113 and 36 (1983) 661 and in "Cellular Regulation and Malignant Growth", Springer Verlag, Berlin 1985, pages 169-176, certain β-methoxyacrylates, namely strobilurin and oudemansin and their derivatives, are described . These compounds are characterized by a strong antifungal and cytostatic activity. The strobilurins A, B and C and the oudemansins A and B were isolated from the genera Strobilurus, Oudemansiella, Xerula, Cyphellopsis, Hydropus and Mycena. All compounds inhibit the breathing of eukaryotes and thereby the growth of fungi and cells. The strobilurins and
Oudemansine binden reversibel an das bt-Zentrum von Cytochrom b in Oudemansins bind reversibly to the b t center of cytochrome b in
Komplex III der mitochondrialen Atmungskette. Complex III of the mitochondrial respiratory chain.
Es wurde nun gefunden, daß ß-Methoxyacrylate der Formel I It has now been found that β-methoxyacrylates of the formula I
worin wherein
R1 Wasserstoff oder einen gegebenenfalls ungesättigten Kohlenwasserstoffrest mit bis zu 10 Kohlenstoffatomen darstellt, der durch Fluor, Chlor, Brom, C1-6-Alkcxy, C2-6-Alkenoxy oder C2-6-Alkinoxy oder durch einen gegebenenfalls durch Fluor, Chlor, Brom, Iod, Trifluormethyl, C1-4-Alkyl oder C1-4-Alkoxy substituierten Phenyl- oder Phenoxyrest substituiert sein kann, R 1 represents hydrogen or an optionally unsaturated hydrocarbon radical having up to 10 carbon atoms, which is represented by fluorine, chlorine, bromine, C 1-6 alkoxy, C 2-6 alkenoxy or C 2-6 alkynoxy or by an optionally fluorine, Chlorine, bromine, iodine, trifluoromethyl, C 1-4 -alkyl or C 1-4 -alkoxy substituted phenyl or phenoxy may be substituted,
R2 und R3 gleich oder verschieden sind und Wasserstoffatome, R 2 and R 3 are the same or different and are hydrogen atoms,
C1-6--Alkylgruppen oder C3-7-Cycloalkylgruppen oder C 1-6 alkyl groups or C 3-7 cycloalkyl groups or
R2 und R3 zusammen eine C2-8-Alkylengruppe bedeuten, eine bessere Wirkung besitzen Die Verbindungen der Formel I besitzen zwei Chiralitätszentren im R 2 and R 3 together represent a C 2-8 alkylene group, have a better effect The compounds of formula I have two centers of chirality in
Dioxolanring (bei R1 = H nur eins). Sie können daher in verschiedenen enantiomeren und/oder diastereomeren Konfigurationen auftreten, die sich in üblicher Weise auftrennen und isolieren lassen. Die Auftrennung der Konfigurationsisomeren kann zweckmäßig auch auf der Stufe von Zwischenprodukten der Formel II (s. unten) erfolgen. Für die erfindungsgemäße Anwendung der Verbindungen ist eine Auftrennung der Konfigurationsisomeren nicht erforderlich. Weiterhin kann die ß-Methoxyacrylatgruppe in der E- oder Z-Konfiguration vorliegen . Bevorzugt sind die Verbindungen der Formel I mit Dioxolane ring (only one if R 1 = H). They can therefore occur in various enantiomeric and / or diastereomeric configurations, which can be separated and isolated in the usual way. The configuration isomers can expediently also be separated at the stage of intermediates of the formula II (see below). A separation of the configuration isomers is not necessary for the use of the compounds according to the invention. Furthermore, the β-methoxyacrylate group can be in the E or Z configuration. The compounds of formula I are preferred with
E-Konfiguration. E configuration.
Die erfindungsgemäßen Verbindungen der Formel I werden hergestellt, indem man einen Aldehyd der Formel II worin R1, R2 und R3 die oben genannten Bedeutungen haben, mit einem The compounds of formula I according to the invention are prepared by using an aldehyde of formula II wherein R 1 , R 2 and R 3 have the meanings given above, with a
Phosphonester der Formel III Phosphonic esters of formula III
worin R4 einen C1-8-Alkylrest bedeutet, in Gegenwart einer Base umsetzt und die so erhaltenen Verbindungen - falls R1 ein ungesättigter Rest ist - gegebenenfalls hydriert. wherein R 4 is a C 1-8 alkyl radical, reacted in the presence of a base and the compounds thus obtained - if R 1 is an unsaturated radical - optionally hydrogenated.
Die Umsetzung der Verbindungen II mit III erfolgt im Sinn einer Wittig-Homer-Reaktion in Gegenwart eines Lösungsmittels und einer Base, wie Lithium-diisopropylamid, Phenyllithium, Butyllithium, Natriumalkoholat, Natriumamid, Natriumhydrid oder Kalium-t-butylat. Als Lösungsmittel eignen sich für die Umsetzung Benzol, Dimethylformamid, Tetrahydrofuran, Diethylether und Dimethoxyethan. Die Reaktion wird zweckmäßig bei einer The reaction of the compounds II with III takes place in the sense of a Wittig-Homer reaction in the presence of a solvent and a base, such as lithium diisopropylamide, phenyllithium, butyllithium, sodium alcoholate, sodium amide, sodium hydride or potassium t-butoxide. Suitable solvents for the reaction are benzene, dimethylformamide, tetrahydrofuran, diethyl ether and dimethoxyethane. The reaction is useful in a
Temperatur von 0 - 25°C durchgeführt. Temperature from 0 - 25 ° C carried out.
Eine sich gegebenenfalls anschließende Hydrierung erfolgt in üblicher Weise katalytisch. Die Herstellung von Verbindungen der Formel III ist in EP 20 360 Any subsequent hydrogenation is carried out catalytically in the usual way. The preparation of compounds of formula III is in EP 20 360
beschrieben. Die Herstellung der erfindungsgemäßen Zwischenprodukte der Formel II erfolgt nach folgendem Syntheseschema: described. The intermediates of formula II according to the invention are prepared according to the following synthesis scheme:
Hierzu wird zunächst ein bekanntes Keton der Formel IV, worin X = Br oder Cl ist, Liebigs Anm. Chem. 724 (1969) 128, in üblicher Weise unter  For this purpose, a known ketone of the formula IV, in which X = Br or Cl, Liebigs Anm. Chem. 724 (1969) 128, is first described in the usual manner
Säurekatalyse bei 0 - 30°C zum geschützten Tetrahydropyranyl (2)-Derivat V und dieses mit 3,4-Dihydroxybenzaldehyd in Gegenwart von 0,2 - 1,2  Acid catalysis at 0 - 30 ° C to the protected tetrahydropyranyl (2) derivative V and this with 3,4-dihydroxybenzaldehyde in the presence of 0.2 - 1.2
Äquivalenten einer Hilfsbase und vorzugsweise eines protischen oder dipolaren, aprotischen Lösungsmittels zu VI umgesetzt. Equivalents of an auxiliary base and preferably a protic or dipolar, aprotic solvent converted to VI.
Als Hilfsbase für die Umsetzung V→ VI eignen sich insbesondere Natriumcarbonat, Kaliumcarbonat, Natriumhydroxid, Kaliumhydroxid, 1,8-Diazabicyclo[5,4,0]undec-7-en, Diisopropylmethylamin und Diisopropylethylamin. Als Lösungsmittel kommen vorzugsweise Dimethylformamid, Tetrahydrofuran, Diethylether, Methanol, Ethanol, Acetonitril und Aceton in Betracht. Die Umsetzung V → VI gelingt gut bei Temperaturen zwischen 0 und 30°C. Sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, 1,8-diazabicyclo [5,4,0] undec-7-ene, diisopropylmethylamine and diisopropylethylamine are particularly suitable as auxiliary bases for the reaction V → VI. Preferred solvents are dimethylformamide, tetrahydrofuran, diethyl ether, methanol, ethanol, acetonitrile and acetone. The reaction V → VI works well at temperatures between 0 and 30 ° C.
Die Abspaltung der THP-Schutzgruppe und die Cyclisierung zu den Dioxolanen der Formel II erfolgt dann mit Aldehyden der Formel R1-CHO in Gegenwart von Säure unter destillativer Entfernung des entstehenden Reaktionswassers. Als Schleppmittel eignen sich bespielsweise Benzol, Toluol sowie Chloroform und Tetrachlorkohlenstoff. Die folgenden Beispiele erläutern die Herstellung: The THP protective group is then split off and the cyclization to the dioxolanes of the formula II then takes place with aldehydes of the formula R 1 -CHO in the presence of acid with removal of the water of reaction formed by distillation. Examples of suitable entraining agents are benzene, toluene and chloroform and carbon tetrachloride. The following examples explain the production:
Beispiel 1 A. Herstellung des Ausgangsmaterials: a) Herstellung des Chlorketons der Formel V (R2, R3 = CH3, x = Cl) Example 1 A. Preparation of the starting material: a) Preparation of the chloroketone of the formula V (R 2 , R 3 = CH 3 , x = Cl)
683 mg (5 mmol) 1-Chlor-3-hydroxy-3-methyl-butanon-(2) IV (R2, 683 mg (5 mmol) 1-chloro-3-hydroxy-3-methyl-butanone- (2) IV (R 2 ,
R3 = CH3) wurden in 20 ml absoluten Dichlormethan gelöst und nachR 3 = CH 3 ) were dissolved in 20 ml of absolute dichloromethane and after
Zugabe von 630 mg (7 mmol) 3,4-Dihydro-2H-pyran und 125 mg (0,7 mmol) Pyridinium-tosylat bei Raumtemperatur gerührt. Add 630 mg (7 mmol) of 3,4-dihydro-2H-pyran and 125 mg (0.7 mmol) of pyridinium tosylate at room temperature.
Nach 3 h wurde das Reaktionsgemisch mit Diethylether verdünnt und 1x mit halbgesättigter NaCl-Lösung gewaschen, um den Katalysator zu entfernen. Die Etherphase wurde nach dem Trocknen über MgSO4 im Vakuum eingeengt. After 3 h the reaction mixture was diluted with diethyl ether and washed 1x with semi-saturated NaCl solution to remove the catalyst. After drying over MgSO 4, the ether phase was concentrated in vacuo.
Man erhielt 980 mg (4,5 mmol) eines farblosen Öls. 980 mg (4.5 mmol) of a colorless oil were obtained.
Ausbeute: 90 % Yield: 90%
C10H17ClO3 (MG 220) C 10 H 17 ClO 3 (MG 220)
IR:  IR:
2990, 2950, 2860, 1730 (cm-1) 2990, 2950, 2860, 1730 (cm -1 )
1H-NMR (90 MHz, CDCl3): 1H-NMR (90 MHz, CDCl 3 ):
1,30-1,96 (12) m; 3,20-3,50 (1) m; 3,73-4,07 (1) m;  1.30-1.96 (12) m; 3.20-3.50 (1) m; 3.73-4.07 (1) m;
4,47-4,73 (3) m (δ ppm) b) Herstellung der Verbindung der Formel VI  4.47-4.73 (3) m (δ ppm) b) Preparation of the compound of formula VI
440 mg (2 mmol) Chlorketon V (R2, R3 = CH3) und 276 mg (2 mmol) 3,4-Dihydroxybenzaldehyd wurden in 10 ml absolutem Aceton gelöst und nach Zugabe von 13 mg (1 mmol) K2CO3 über Nacht unter Rückfluß erhitzt. Das Lösungsmittel wurde abgezogen, der Rückstand mit Wasser aufgenommen und mit Ether extrahiert. Die vereinigten Etherphasen wurden mit gesättigter Na2CO3-Lösung und Wasser gewaschen und über MgSO4 getrocknet. Es wurde im Vakuum eingeengt und das Rohproduktgemisch mit Petrolether/Ethylacetat (3:1) als Laufmittel an Kieselgel chromatographiert. 440 mg (2 mmol) of chloroketone V (R 2 , R 3 = CH 3 ) and 276 mg (2 mmol) of 3,4-dihydroxybenzaldehyde were dissolved in 10 ml of absolute acetone and after addition of 13 mg (1 mmol) of K 2 CO 3 heated under reflux overnight. The solvent was removed, the residue was taken up in water and extracted with ether. The combined ether phases were washed with saturated Na 2 CO 3 solution and water and dried over MgSO 4 . It was concentrated in vacuo and the crude product mixture was chromatographed on silica gel using petroleum ether / ethyl acetate (3: 1).
Man erhielt 402 mg (62 %) der Verbindung der Formel VI (R2, R3 = CH3) in Form eines farblosen, zähen Öls, das bei 0 - 5°C kristallisierte, Fp. 86 - 90°C. 1H-NMR (200 MHZ, CDCI3): This gave 402 mg (62%) of the compound of the formula VI (R 2 , R 3 = CH 3 ) in the form of a colorless, viscous oil which crystallized at 0-5 ° C., mp. 86-90 ° C. 1 H-NMR (200 MHz, CDCI 3 ):
9,81 (1)s; 7,50-7,35 (2) m; 7,10-6,90 (1) m; 5,85-5,70 (1) m;  9.81 (1) s; 7.50-7.35 (2) m; 7.10-6.90 (1) m; 5.85-5.70 (1) m;
5,05-4,95 (1) m; 4,40-4,20 (1) m; 4,10-3,90 (1) m; 3,60-3,45 (1) m; 1,95-1,30 (12) m (δ ppm) 5.05-4.95 (1) m; 4.40-4.20 (1) m; 4.10-3.90 (1) m; 3.60-3.45 (1) m; 1.95-1.30 (12) m (δ ppm)
IR (CHCI3): IR (CHCI 3 ):
3500, 3250, 2995, 2940, 2840, 2710, 1680, 1600, 1580, 1500 cm-1 Herstellung der diastereomeren Verbindungen der Formel II 3500, 3250, 2995, 2940, 2840, 2710, 1680, 1600, 1580, 1500 cm -1 Preparation of the diastereomeric compounds of formula II
(R1 = 2,2-Dimethylvinyl, R2, R3 = CH3) (R 1 = 2,2-dimethylvinyl, R2, R 3 = CH 3 )
1,15 g (3,6 mmol) der Verbindung der Formel VI (R2, R3 = CH3) und 90 mg (0,45 mmol) Pyridiniumtosylat wurden in 50 ml Benzol gelöst und nach Zugabe von 0,5 ml (5 mmol) 3-Methylcrotonaldehyd 15 h unter Ar-Atmosphäre am Wasserabscheider zum Rückfluß erhitzt. Zur 1.15 g (3.6 mmol) of the compound of the formula VI (R 2 , R 3 = CH 3 ) and 90 mg (0.45 mmol) of pyridinium tosylate were dissolved in 50 ml of benzene and after addition of 0.5 ml ( 5 mmol) 3-methylcrotonaldehyde heated under reflux under an Ar atmosphere on a water separator for 15 h. to
Aufarbeitung wurde das Reaktionsgemisch in halbgesättigter NaCl-Lösung gegeben, mit Ether extrahiert und über MgSO4 getrocknet. Working up, the reaction mixture was poured into semi-saturated NaCl solution, extracted with ether and dried over MgSO 4 .
Die beiden Diasteromere entstanden laut NMR-Kontrolle des Rohproduktes etwa im Verhältnis 1:1. According to the NMR control of the crude product, the two diasteromers were formed in a ratio of about 1: 1.
Das Rohprodukt wurde mit Hexan/Ethylacetat (10:1) als Laufmittel an Kieselgel chromatographiert, wobei sich die beiden Diastereomeren trennten. The crude product was chromatographed on silica gel using hexane / ethyl acetate (10: 1) as the eluent, the two diastereomers separating.
Ausbeute: Yield:
Diastereomer 1: 330 mg farblose Kristalle  Diastereomer 1: 330 mg of colorless crystals
Diastereomer 2: 308 mg farblose Kristalle Diastereomer 1 (C17H20O5) (MG 304): Diastereomer 2: 308 mg colorless crystals Diastereomer 1 (C 17 H 20 O 5 ) (MW 304):
1H-NMR (200 MHz, Benzol-d6): 1 H-NMR (200 MHz, benzene-d 6 ):
9,60 (1)s; 7,51 (1) d, J = 2 Hz; 7,10 (1) dd, J1 = 2 Hz, J2 = 8 Hz; 6,81 (1) d, J = 8 Hz; 6,12 (1) d, J = 7 Hz; 5,35 (1) d/Septett, J1 = 7 Hz, 32 = 1 Hz; 3,98 (1), J = 11 Hz; 3,40 (1), J = 11 HZ; 1,43 (3) d, J = 1 HZ; 1,32 (3) d, J = 1 Hz; 1,31 (3) S; 0,94 (3) S (δ ppm) 9.60 (1) s; 7.51 (1) d, J = 2 Hz; 7.10 (1) dd, J 1 = 2 Hz, J 2 = 8 Hz; 6.81 (1) d, J = 8 Hz; 6.12 (1) d, J = 7 Hz; 5.35 (1) d / septet, J 1 = 7 Hz, 3 2 = 1 Hz; 3.98 (1), J = 11 Hz; 3.40 (1), J = 11 Hz; 1.43 (3) d, J = 1HZ; 1.32 (3) d, J = 1 Hz; 1.31 (3) S; 0.94 (3) S (δ ppm)
IR (KBr): IR (KBr):
3040, 2980, 2940, 2910, 2830, 2800, 2730, 1680, 1580, 1500 cm-1 3040, 2980, 2940, 2910, 2830, 2800, 2730, 1680, 1580, 1500 cm -1
Diastereomer 2 (C17H20O5) (MG 304): 1H-NMR (200 MHZ, Benzol-d6): Diastereomer 2 (C 17 H 20 O 5 ) (MW 304): 1 H-NMR (200 MHz, benzene-d 6 ):
9, 56 (1) s; 7,49 (1) d, J = 2 Hz, 7,09 (1) dd, J1 = 2 Hz, J2 = 8 Hz;9, 56 (1) s; 7.49 (1) d, J = 2 Hz, 7.09 (1) dd, J 1 = 2 Hz, J 2 = 8 Hz;
6,79 (1) d, 3 = 8 Hz; 5,92 (1) d, 3 = 7 Hz; 5,53 (1) d/septett, J1 = 76.79 (1) d, 3 = 8 Hz; 5.92 (1) d, 3 = 7 Hz; 5.53 (1) d / septet, J 1 = 7
Hz, J2 = 1 Hz; 3,86 (1) d, J = 11 Hz; 3,48 (1) d, 3 = 11 Hz; 1,41 (3) d, J = 1 Hz; 1,38 (3) d, J = 1 Hz; 1,26 (3) s; 0,83 (3) s ( ppm) Hz, J 2 = 1 Hz; 3.86 (1) d, J = 11 Hz; 3.48 (1) d, 3 = 11 Hz; 1.41 (3) d, J = 1 Hz; 1.38 (3) d, J = 1 Hz; 1.26 (3) s; 0.83 (3) s (ppm)
IR (KBr): IR (KBr):
3060, 2990, 2940, 2840, 2740, 1680, 1600, 1580, 1500 cm-1 3060, 2990, 2940, 2840, 2740, 1680, 1600, 1580, 1500 cm -1
B. Herstellung des diastereomeren Endprodukts (Formel I, B. Preparation of the diastereomeric end product (Formula I,
R1 = 2,2-Dimethylvinyl, R2, R3 = CH3) a) 60 mg (2 mmol) NaH wurden unter Schutzgasatmosphäre (Ar) in 1 ml R 1 = 2,2-dimethylvinyl, R 2 , R 3 = CH 3 ) a) 60 mg (2 mmol) NaH were in a protective gas atmosphere (Ar) in 1 ml
absoluten Tetrahydrofuran suspendiert.  absolute tetrahydrofuran suspended.
Dann wurde bei 0°C eine Lösung von 608 mg (2 mmol) Diastereomer 1 der Verbindung der Formel II (R = 2,2-Dimethylvinyl, R2, R3 = CH3) [vgl. 1 Ac] und 6,28 g (2 mmol) des Phosphonsäureesters der Formel III Then a solution of 608 mg (2 mmol) of diastereomer 1 of the compound of the formula II (R = 2,2-dimethylvinyl, R 2 , R 3 = CH 3 ) was obtained at 0 ° C. [cf. 1 Ac] and 6.28 g (2 mmol) of the phosphonic acid ester of the formula III
(R4 = -CH3) in 5 ml absolutem Tetrahydrofuran zugetropft. (R 4 = -CH 3 ) added dropwise in 5 ml of absolute tetrahydrofuran.
Man ließ auf Raumtemperatur erwärmen und rührte 24 h. Zur Aufarbeitung wurde die Reaktionslösung in 15 ml Ether gegeben, 2x mit 3 ml Wasser und 1x mit 3 ml gesättigter NaCl-Lösung gewaschen. Nach dem Trocknen über MgSO4 wurde einrotiert. Das Rohprodukt wurde mit The mixture was allowed to warm to room temperature and stirred for 24 h. For working up, the reaction solution was poured into 15 ml of ether, washed twice with 3 ml of water and once with 3 ml of saturated NaCl solution. After drying over MgSO 4 , the mixture was evaporated. The raw product was with
Hexan/Ethylacetat (5:1) als Laufmittel an einer Kieselgelsäule chromatographiert.  Chromatograph hexane / ethyl acetate (5: 1) as eluent on a silica gel column.
Man erhielt 540 mg (55 %) eines weißen, erstarrten Öls, das nur das Diastereomer 1 enthielt. 540 mg (55%) of a white, solidified oil which contained only diastereomer 1 was obtained.
Diastereomer 1 (C29H32O7, MG 492): Diastereomer 1 (C 29 H 32 O 7 , MW 492):
1H-NMR (200 MHZ, Benzol-d6): 1 H-NMR (200 MHz, benzene-d 6 ):
7,65-7,25 (6) m; 7,05-6,90 (4) m; 6,18 (1) d, J = 7,5 Hz; 5,39 (1) d/Septett, J1 = 7,5 Hz, J2 = 1 Hz; 4,05 (1) d, 3 = 11 Hz; 3,59 (1) d, J = 11 Hz; 3,40 (3) s; 2,80 (3) s; 1,46 (3) d, 3 = 1 Hz; 1,36 (3) s; 1,35 (3) d, J = 1 Hz; 1,01 (3) s (δ ppm) IR (KBr): 7.65-7.25 (6) m; 7.05-6.90 (4) m; 6.18 (1) d, J = 7.5 Hz; 5.39 (1) d / septet, J 1 = 7.5 Hz, J 2 = 1 Hz; 4.05 (1) d, 3 = 11 Hz; 3.59 (1) d, J = 11 Hz; 3.40 (3) s; 2.80 (3) s; 1.46 (3) d, 3 = 1 Hz; 1.36 (3) s; 1.35 (3) d, J = 1 Hz; 1.01 (3) s (δ ppm) IR (KBr):
3060, 3020, 2980, 2940, 1710, 1630, 1585, 1510, 1435 cm-1 b) in entsprechender Weise wurde aus dem Diastereomer 2 der Verbindung der Formel II (R = 2,2-Dimethylvinyl, R2, R3 = CH3) das Diastereomer 2 (C29H32O7, MG 492) hergestellt. 3060, 3020, 2980, 2940, 1710, 1630, 1585, 1510, 1435 cm -1 b) In a corresponding manner, diastereomer 2 (C 29 H 32 O 7 , MW 492) was prepared from diastereomer 2 of the compound of formula II (R = 2,2-dimethylvinyl, R 2 , R 3 = CH 3 ).
1H-NMR (200 MHZ, Benzol-d6): 1 H-NMR (200 MHz, benzene-d 6 ):
7,60-7,20 (6) m; 7,00-6,88 (4) m; 5,97 (1) d, J = 7,5 Hz; 5,61 (1) d/Septett, J1 = 7,5 Hz, J2 = 1 Hz; 3,94 (1) d, J = 11 Hz; 3,67 (1) d, J = 11 Hz; 3,40 (3) s; 2,81 (3) s; 1,45 (3) d, J = 1 Hz, 1,42 (3) d, J = 1 Hz; 1,32 (3) s; 0,92 (3) s (δ ppm) 7.60-7.20 (6) m; 7.00-6.88 (4) m; 5.97 (1) d, J = 7.5 Hz; 5.61 (1) d / septet, J 1 = 7.5 Hz, J 2 = 1 Hz; 3.94 (1) d, J = 11 Hz; 3.67 (1) d, J = 11 Hz; 3.40 (3) s; 2.81 (3) s; 1.45 (3) d, J = 1 Hz, 1.42 (3) d, J = 1 Hz; 1.32 (3) s; 0.92 (3) s (δ ppm)
IR (KBr): IR (KBr):
3060, 3020, 2980, 2940, 2840, 1710, 1630, 1585, 1510, 1435 cm-1 3060, 3020, 2980, 2940, 2840, 1710, 1630, 1585, 1510, 1435 cm -1
Beispiel 2 Example 2
Analog Beispiel 1 erhielt man aus den Diasteromeren der Formel II Analogously to Example 1, the diasteromers of the formula II were obtained
(R1 = 2,2-Dimethylvinyl,(R 1 = 2,2-dimethylvinyl,
die entsprechenden Diastereomeren der Formel I : the corresponding diastereomers of the formula I:
Diastereomer 1 (C32H36O7, MG 532) : Diastereomer 1 (C 32 H 36 O 7 , MW 532):
1H-NMR ( 200 MHZ, Benzol-d6 ) : 1 H-NMR (200 MHz, benzene-d 6 ):
7,64-7,31 (6) m; 7,15-6,94 (5) m; 6,18 (1) d, J = 7,5 Hz; 5,39 (1) d/Septett, J 1 = 7,5 Hz, J2 = 1 Hz; 4,06 (1) d, J = 11 Hz; 3,68 (1) d, J =7.64-7.31 (6) m; 7.15-6.94 (5) m; 6.18 (1) d, J = 7.5 Hz; 5.39 (1) d / septet, J 1 = 7.5 Hz, J 2 = 1 Hz; 4.06 (1) d, J = 11 Hz; 3.68 (1) d, J =
11 Hz; 3,40 (3) s; 2,80 (3) s; 2,39-2,25 (1) m, 1,90-1,45 (5) m; 1,44 (3) d, J = 1 Hz; 1,33 (3) d, 3 = 1 Hz; 1,27-0,85 (4) m. 11 Hz; 3.40 (3) s; 2.80 (3) s; 2.39-2.25 (1) m, 1.90-1.45 (5) m; 1.44 (3) d, J = 1 Hz; 1.33 (3) d, 3 = 1 Hz; 1.27-0.85 (4) m.
IR (KBr): IR (KBr):
3060, 3020, 2930, 2860, 1700, 1630, 1580, 1505, 1450 1430 cm-1. Distereomer 2 (C32H36O7, MG 532): 3060, 3020, 2930, 2860, 1700, 1630, 1580, 1505, 1450 1430 cm -1 . Distereomer 2 (C 32 H 36 O 7 , MW 532):
1H-NMR (200 MHZ, Benzol-d6): 1 H-NMR (200 MHz, benzene-d 6 ):
7,62-7,28 (4) m; 7,20-6,92 (6) m; 5,94 (1) d, J = 7,5 Hz; 5,63 (1) d/Septett, J1 = 7,5 Hz, J2 = 1 Hz; 3,98 (1) d, J = 11 Hz; 3,77 (1) d, J = 11 Hz; 3,40 (3) s; 2,81 (3) s; 2,38-2,22 (1) m, 1,85-1,50 (5) m; 7.62-7.28 (4) m; 7.20-6.92 (6) m; 5.94 (1) d, J = 7.5 Hz; 5.63 (1) d / septet, J 1 = 7.5 Hz, J 2 = 1 Hz; 3.98 (1) d, J = 11 Hz; 3.77 (1) d, J = 11 Hz; 3.40 (3) s; 2.81 (3) s; 2.38-2.22 (1) m, 1.85-1.50 (5) m;
1,45 (3) d, J = 1 Hz; 1,42 (3) d, J = 1 Hz; 1,37-0,80 (4) m.  1.45 (3) d, J = 1 Hz; 1.42 (3) d, J = 1 Hz; 1.37-0.80 (4) m.
IR (KBr): IR (KBr):
3060, 3020, 2930, 2860, 1700, 1630, 1580, 1505, 1450, 1430 cm-1. Beispiel 3 3060, 3020, 2930, 2860, 1700, 1630, 1580, 1505, 1450, 1430 cm -1 . Example 3
Analog Beispiel 1 B.a) erhielt man die Verbindung der Formel I Analogously to Example 1 B.a), the compound of formula I was obtained
(R1 = 2,6-Dimethyl-1,5-heptadienyl (1), R2, R3 = CH3, C34H40O7, MG 560), (R 1 = 2,6-dimethyl-1,5-heptadienyl (1), R 2 , R 3 = CH 3 , C 34 H 40 O 7 , MW 560),
1H-NMR (200 H2, Benzol-d6): 1 H-NMR (200 H 2 , benzene-d 6 ):
7,65-6,90 (10) m; 6,22 (1) d, 3 = 7,5 Hz; 5,48 (1) d/Septett,  7.65-6.90 (10) m; 6.22 (1) d, 3 = 7.5 Hz; 5.48 (1) d / septet,
J1 = 7,5 Hz, J2 = 1 Hz; 5,15-5,02 (1) m, 4,04 (1) d, 3 = 11 Hz; J 1 = 7.5 Hz, J 2 = 1 Hz; 5.15-5.02 (1) m, 4.04 (1) d, 3 = 11 Hz;
3,59 (1) d, J = 11 Hz; 3,45 (3) s; 2,81 (3) s; 2,08-1,85 (4) m; 3.59 (1) d, J = 11 Hz; 3.45 (3) s; 2.81 (3) s; 2.08-1.85 (4) m;
1,61 (3) d, 3 = 11 Hz; 1,47 (3) s; 1,43 (3) d, 3 = 1 Hz; 1.61 (3) d, 3 = 11 Hz; 1.47 (3) s; 1.43 (3) d, 3 = 1 Hz;
1,36 (3) s; 1,02 (3) s. 1.36 (3) s; 1.02 (3) s.
IR (CHCI3): IR (CHCI 3 ):
3020, 2980, 2940, 2850, 1700, 1625, 1585, 1505, 1430 cm-1. 3020, 2980, 2940, 2850, 1700, 1625, 1585, 1505, 1430 cm -1 .
Beispiele 4 - 16 Examples 4-16
Analog Beispiel 1Ac lassen sich folgende Zwischenprodukte der Formel II herstellen (R2, R3 = CH3): The following intermediates of the formula II can be prepared analogously to Example 1Ac (R 2 , R 3 = CH 3 ):
Rl = H, R l = H,
Methyl,  Methyl,
n-Butyl,  n-butyl,
Methoxymethyl,  Methoxymethyl,
Trifluormethyl,  Trifluoromethyl,
Trichlormethyl,  Trichloromethyl,
2,2-Dimethylpropyl,  2,2-dimethylpropyl,
3,3-Dimethylbutenyl(1),  3,3-dimethylbutenyl (1),
1,3-Pentadieny1(1),  1,3-pentadieny1 (1),
2,6-Dimethyl-5-heptenyl(1),  2,6-dimethyl-5-heptenyl (1),
2-Methylpropyl,  2-methylpropyl,
2-Phenyl-vinyl,  2-phenyl vinyl,
Phenoxymethyl. Analog Beispiel 1B können daraus folgende Wirkstoffe der Formel I hergestellt werden (R2, R3 = CH3): Phenoxymethyl. The following active compounds of the formula I can be prepared analogously to Example 1B (R 2 , R 3 = CH 3 ):
Beispiel R1 Example R 1
4 H, 4 H,
5 Methyl,  5 methyl,
6 n-Butyl,  6 n-butyl,
7 E-Methoxymethyl,  7 E-methoxymethyl,
8 Trifluormethyl,  8 trifluoromethyl,
9 Trichlormethyl,  9 trichloromethyl,
10 2,2-Dimethylpropyl,  10 2,2-dimethylpropyl,
11 3,3-Dimethylbutenyl(1),  11 3,3-dimethylbutenyl (1),
12 1,3-Pentadienyl(1),  12 1,3-pentadienyl (1),
13 2,6-Dimethyl-5-heptenyl(1),  13 2,6-dimethyl-5-heptenyl (1),
14 2-Methylpropyl,  14 2-methylpropyl,
15 2-Phenyl-vinyl,  15 2-phenyl vinyl,
16 Phenoxymethyl.  16 phenoxymethyl.
Die neuen Verbindungen - insbesondere die Verbindungen der Beispiele 1 bis 16 - zeigen eine gute antivirale, antiproliferative und zytotoxische Aktivität. Folgende Beispiele erläutern die Wirkung der neuen Substanzen: The new compounds - in particular the compounds of Examples 1 to 16 - show good antiviral, antiproliferative and cytotoxic activity. The following examples explain the effects of the new substances:
Beispiel A Example A
Atmungshemmende Wirkung bei Penicillium notatum Breathing-inhibiting effect on Penicillium notatum
Der Sauerstoffverbrauch wurde in einem luftdicht abgeschlossenen Gefäß (3 ml Volumen, mit Magnetrührer) mit einer Sauerstoffelektrode polarographisch gemessen. Der Testpilz wurde aus einer Sporensuspension auf ein Mycelgewicht von 10 - 20 mg Mycelfeuchtgewicht/ml angezogen. Die Messungen wurden mit einer Mycelkonzentration von 25 - 30 mg Mycelfeuchtgewicht/ml in 1 %iger Glucoselösung durchgeführt. Nach kurzem konstanten Atmungsverlauf wurden die in Methanol gelösten Verbindungen (0,3 mg/ml) der Suspension zugegeben und der 02-Verbrauch aufgezeichnet. Der Versuch wurde unter Verwendung von Strobilurin A als Vergleichsverbindung durchgeführt. Die Ergebnisse der prozentualen Hemmung der Atmung sind in Tabelle 1 genannt. Tabelle 1 The oxygen consumption was measured polarographically in an airtight container (3 ml volume, with magnetic stirrer) with an oxygen electrode. The test mushroom was grown from a spore suspension to a mycelium weight of 10-20 mg mycelium wet weight / ml. The measurements were carried out with a mycelium concentration of 25-30 mg mycelium wet weight / ml in 1% glucose solution. After a short constant course of breathing, the compounds (0.3 mg / ml) dissolved in methanol were added to the suspension and the 02 consumption was recorded. The experiment was carried out using strobilurin A as a comparative compound. The results of the percent breathing inhibition are shown in Table 1. Table 1
Verbindung Atmungshemmung in % der Kontrolle Beispiel 1 B1 95 Compound respiratory inhibition in% of control Example 1 B1 95
Beispiel 1 B2 95 Example 1 B2 95
Strobilurin A 93 Strobilurin A 93
Beispiel B Example B
Cytostatische und morphologische Wirkung auf HeLa-S3-Zellen Cytostatic and morphological effects on HeLa-S3 cells
Bei Inkubation von HeLa-S3 und anderen Zellen mit den erfindungsgemäßen Verbindungen wurde innerhalb kurzer Zeit die Zellteilung gehemmt. When HeLa-S3 and other cells were incubated with the compounds according to the invention, cell division was inhibited within a short time.
Zusätzlich veränderte sich die Morphologie der Zellen. In addition, the morphology of the cells changed.
HeLa-S3-Zellen wurden auf einer 96-Lochtiterplatte mit einer Zelldichte von 4 x 105 Zellen/ml in Medium F12 mit 10 % fötalem Kälberserum HeLa-S3 cells were on a 96-well titer plate with a cell density of 4 x 105 cells / ml in medium F12 with 10% fetal calf serum
aufgebracht. Nach 24 h waren die Zellen auf der Oberfläche ausgebreitet, und es erfolgte ein Wechsel des Mediums. Die Zellen wurden mit dem die Testverbindungen enthaltenden Medium (10, 1, 0,1 bzw. 0,01 μg pro ml Medium) 72 h inkubiert (37°C, 5 % CO2). Nach 72 h besaßen die normalerweise epithelartigen wachsenden Zellen eine fibroblastenartige upset. After 24 hours the cells were spread out on the surface and the medium was changed. The cells were incubated with the medium containing the test compounds (10, 1, 0.1 or 0.01 μg per ml medium) for 72 h (37 ° C., 5% CO 2 ). After 72 hours, the normally epithelial-like growing cells were fibroblast-like
Morphologie. Die Morphologieänderung ist ab einer 45 %igen Wachstumshemmung durch die Einwirkung der Substanzen signifikant sichtbar. Morphology. From a 45% growth inhibition, the change in morphology is significantly visible through the action of the substances.
Der Versuch wurde unter Verwendung von Strobilurin A als Vergleichsverbindung durchgeführt. Die Zelldichte wurde über die Bestimmung des Gesamtproteingehaltes pro Loch gemessen. Die angeführten Werte zeigen die Ergebnisse nach dreitätiger Inkubation mit den Testsubstanzen und sind als Prozentsatz des Gesamtproteins der unbehandelten Kontrolle in Tabelle 2 aufgeführt. The experiment was carried out using strobilurin A as a comparative compound. The cell density was measured by determining the total protein content per hole. The values given show the results after three days of incubation with the test substances and are listed in Table 2 as a percentage of the total protein of the untreated control.
Tabelle 2: Table 2:
Verbindung Proteingehalt in Prozent der Kontrolle Connection protein content in percent of control
(μg/ml)  (μg / ml)
10μg 1μg 0,1μg 0,01μg Beispiel 1 B1 20 21 23 55  10μg 1μg 0.1μg 0.01μg Example 1 B1 20 21 23 55
Beispiel 1 B2 38 38 50 70  Example 1 B2 38 38 50 70
Strobilurin A 23 55 92 100 Beispiel C Strobilurin A 23 55 92 100 Example C
Antivirale Wirkung bei HEp-2-Zellen auf VSV Die Bestimmung der antiviralen Aktivität einer Testverbindung basiert auf der Messung des Schutzes von menschlichen HEp-2-Zellen als Indikatorzellen vor dem cytopathischen Effekt (CPE) von Vesicular Stomatitis Virus (VSV). Antiviral effect in HEp-2 cells on VSV The determination of the antiviral activity of a test compound is based on the measurement of the protection of human HEp-2 cells as indicator cells against the cytopathic effect (CPE) of Vesicular Stomatitis Virus (VSV).
Hierzu wurden 100 μl Kulturmedium mit 2 x 10* HEp-2-Zellen in die For this purpose, 100 ul culture medium with 2 x 10 * HEp-2 cells in the
Vertiefungen einer 96-Loch-Flachbodenplatte gegeben und über Nacht bei 37°C und 5 % (V/V) Kohlendioxid inkubiert. Am nächsten Tag wurden 100 μl der Probelösung zu den konfluenten Zellkulturen gegeben und seriell 2x titriert. Auf der Kulturplatte wurden zusätzlich eine Zellkontrolle Wells of a 96-hole flat bottom plate and incubated overnight at 37 ° C and 5% (V / V) carbon dioxide. The next day, 100 μl of the sample solution was added to the confluent cell cultures and titrated twice in series. A cell control was also added to the culture plate
(= unbehandelte, nicht mit Virus infizierte Zellen) und eine Virus-kontrolle (= unbehandelte, mit Virus infizierte Zellen) mitangelegt. Nach einer weiteren Inkubationszeit von 24 h bei 37°C und 5 % (V/V) CO2 wurden die Kulturen mit 50 μl einer VSV-Suspension in Kulturmedium infiziert und bei 37°C und 5 % (V/V) CO2 inkubiert. Nach zwei Tagen war die virusbedingte Zellzerstörung (CPE) in den ungeschützten Kulturen (= Virus-kontrolle) abgelaufen. Der Prozentsatz geschützter Zellen in den mit den Testverbindungen behandelten und anschließend mit VSV infizierten Kulturen wurde mittels Kristallviolettfärbung bestimmt/ Als Maß für die antivirale Aktivität wurde bezogen auf 0 und 100 % Schutz die Probenkonzentration, die zu 50 % Schutz führt, ermittelt. Als Kontrolle für die antivirale Aktivität einer Verbindung wurde rekombinantes humanes (= untreated, not infected with virus) and a virus check (= untreated, infected with virus). After a further incubation period of 24 h at 37 ° C. and 5% (V / V) CO 2 , the cultures were infected with 50 μl of a VSV suspension in culture medium and incubated at 37 ° C. and 5% (V / V) CO 2 . After two days, virus-related cell destruction (CPE) in the unprotected cultures (= virus control) had expired. The percentage of protected cells in the cultures treated with the test compounds and subsequently infected with VSV was determined by means of crystal violet staining / the measure of the antiviral activity based on 0 and 100% protection was determined as the sample concentration, which leads to 50% protection. As a control for the antiviral activity of a compound, recombinant human
Interferon-ɤ (rHuIFN-ɤ) mituntersucht.  Interferon-ɤ (rHuIFN-ɤ) also examined.
In Tabelle 3 sind die Testsubstanzen mit der Konzentration, die die HEp-2-Zellen zu 50 % vor dem zytopathischen Effekt von VSV schützt, aufgeführt. Table 3 lists the test substances with the concentration which 50% protects the HEp-2 cells against the cytopathic effect of VSV.
Tabelle 3: Table 3:
Verbindung Antivirale Aktivität Compound antiviral activity
(ng/ml)  (ng / ml)
Beispiel 1 B1 3 Example 1 B1 3
Beispiel 1 B2 3  Example 1 B2 3
Kontrolle (rHuIFN-ɤ) 0,6  Control (rHuIFN-ɤ) 0.6
Im Unterschied zu rHuIFN-ɤ zeigte die Substanz des Beispiels 1 unter diesen Testbedingungen im Vergleich zur Zellkontrolle auch eine antiproliferative Wirkung. Die antivirale und antiproliferative Aktivitäten der Substanzen des Beispiels 1 liefen parallel und korrelierten mit einer morphologischen Veränderung der Zellen, d. h. die Zellen waren im Vergleich zur Zellkontrolle langgestreckt und wiesen neuritartige Fortsätze auf. Beispiel D In contrast to rHuIFN-ɤ, the substance of Example 1 also showed an antiproliferative effect under these test conditions compared to cell control. The antiviral and antiproliferative activities The substances of Example 1 ran in parallel and correlated with a morphological change in the cells, ie the cells were elongated compared to the cell control and had neurite-like processes. Example D
Zytotoxische und antiproliferative Wirkung auf menschliche Tumorzellen Zur Bestimmung der antitumoralen Eigenschaften der Verbindungen 1 B1 und 1 B2 wurden Tumorzellen unterschiedlicher Gewebeherkunft (5637-6: humanes Blasenkarzinom; HT-29: humanes Kolonkarzinom; B-16: murines Melanom) verwendet. 1 bis 2·103 sich im exponentiellen Wachstum befindliche Tumorzellen wurden in 96-Lochplatten in komplettem Wachstumsmedium (RPMI 1640 x 10 % fötalem Kälberserum) ausplattiert und über Nacht bei Standardkulturbedingungen (37°C, 5 % Kohlendioxid, wasserdampfgesättigte Atmosphäre) inkubiert. Die Substanzzugabe erfolgte am nächsten Tag, wobei serielle Titrationen der Substanzen 1 B1 und 1 B2 über einen Konzentrationsbereich von 10-4 bis 10-9 M angelegt wurden. Nach einer weiteren Inkubation von 72 h unter Standardbedingungen erfolgte die Bestimmung der Zellzahl durch eine Färbung mit Kristallviolett und eine anschließende photometrische Cytotoxic and antiproliferative effect on human tumor cells To determine the antitumor properties of the compounds 1 B1 and 1 B2, tumor cells of different tissue origin (5637-6: human bladder carcinoma; HT-29: human colon carcinoma; B-16: murine melanoma) were used. 1 to 2 · 103 exponential growth tumor cells were plated in 96-well plates in complete growth medium (RPMI 1640 x 10% fetal calf serum) and incubated overnight under standard culture conditions (37 ° C, 5% carbon dioxide, water vapor-saturated atmosphere). The substance was added the next day, serial titrations of substances 1 B1 and 1 B2 being carried out over a concentration range of 10 -4 to 10 -9 M. After a further incubation of 72 h under standard conditions, the cell number was determined by staining with crystal violet and a subsequent photometric
Auswertung bei 540 nm mit Hilfe eines Multiphotometers. Evaluation at 540 nm using a multiphotometer.
Nur bei der sehr hohen Konzentration von 10-4 M war mikroskopisch eine Zytolyse der behandelten Zellen zu erkennen. Bei den anderen untersuchten Konzentrationen konnte eine starke, dosisabhängige Inhibition der Only at the very high concentration of 10 -4 M was microscopic cytolysis of the treated cells visible. At the other concentrations examined, a strong, dose-dependent inhibition of
Zeilproliferation beobachtet werden, so daß auch bei der geringsten untersuchten Konzentration von 10-9 M die Zahl der behandelten Zellen einen Wert kleiner als 50 % der unbehandelten Zellkontrolle zeigten. Cell proliferation can be observed, so that even at the lowest examined concentration of 10 -9 M the number of treated cells showed a value less than 50% of the untreated cell control.
Diese starke Abnahme der proliferativen Aktivität der mit den Substanzen des Beispiels 1 behandelten Zellen ging einher mit einer morphologischen Veränderung, gekennzeichnet durch eine Streckung der Zellkörper und durch die Ausbildung von Zeilfortsätzen. This sharp decrease in the proliferative activity of the cells treated with the substances of Example 1 was accompanied by a morphological change, characterized by a stretching of the cell body and by the formation of cell processes.

Claims

Patentansprüche Claims
1. ß-Methoxyacrylate der Formel I ,1. β-methoxyacrylates of the formula I,
worin  wherein
R1 Wasserstoff oder einen gegebenenfalls ungesättigten Kohlenwasserstoffrest mit bis zu 10 Kohlenstoffatomen darstellt, der durch Fluor, Chlor, Brom, C1-6-Alkoxy, C2-6-Alkenoxy oder C2-6-Alkinoxy oder durch einen gegebenenfalls durch Fluor, Chlor, Brom, Iod, Trifluormethyl, C1-4-Alkyl oder C1-4-Alkoxy substituierten Phenyl- oder Phenoxyrest substituiert sein kann, R 1 represents hydrogen or an optionally unsaturated hydrocarbon radical having up to 10 carbon atoms, which is represented by fluorine, chlorine, bromine, C 1-6 alkoxy, C 2-6 alkenoxy or C 2-6 alkynoxy or by an optionally by fluorine, Chlorine, bromine, iodine, trifluoromethyl, C 1-4 -alkyl or C 1-4 -alkoxy substituted phenyl or phenoxy may be substituted,
R2 und R3 gleich oder verschieden sind und Wasserstoffatome, R 2 and R 3 are the same or different and are hydrogen atoms,
C1-6-Alkylgruppen oder C3-7-Cycloalkylgruppen oder C 1-6 alkyl groups or C 3-7 cycloalkyl groups or
R2 und R3 zusammen eine C2-8-Alkylengruppe bedeuten. R 2 and R 3 together represent a C 2-8 alkylene group.
2. Verbindungen der Formel I gemäß Anspruch 1 zur Verwendung bei der Bekämpfung von Krankheiten. 2. Compounds of formula I according to claim 1 for use in combating diseases.
3. Verfahren zur Herstellung der ß-Methoxyacrylate der Formel I gemäß Anspruch 1, dadurch gekennzeichnet, daß man einen Aldehyd der 3. A process for the preparation of the β-methoxyacrylates of the formula I according to claim 1, characterized in that one of the aldehyde
Formel II Formula II
worin R1, R2 und R3 die oben genannten Bedeutungen haben, mit einem Phosphonester der Formel III wherein R 1 , R 2 and R 3 have the meanings given above, with a phosphonic ester of the formula III
worin R4 einen C1-8-Alkylrest bedeutet, in Gegenwart einer Base umsetzt und die so erhaltenen Verbindungen - falls R1 ein ungesättigter Rest ist, gegebenenfalls hydriert. wherein R 4 is a C 1-8 alkyl radical, reacted in the presence of a base and the compounds thus obtained - if R 1 is an unsaturated radical, optionally hydrogenated.
4. Verbindungen der Formel II gemäß Anspruch 3. 4. Compounds of formula II according to claim 3.
5. Verbindung der Formel IV 5. Compound of formula IV
worin R2 und R3 die obengenannte Bedeutung besitzen. wherein R 2 and R 3 have the meaning given above.
EP90904345A 1989-02-25 1990-02-20 Novel beta-methoxyacrylates, their production and use Withdrawn EP0460084A1 (en)

Applications Claiming Priority (2)

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DE3905911 1989-02-25
DE3905911A DE3905911A1 (en) 1989-02-25 1989-02-25 NEW SS-METHOXYACRYLATES, THEIR PREPARATION AND USE

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DE4443648C2 (en) * 1994-12-08 1999-01-21 Akzo Nobel Nv Process for the production of polyesters and copolyesters
EP2001555A1 (en) * 2006-03-29 2008-12-17 Basf Se Use of strobilurins for treating malfunctions of the iron metabolism

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CA2047195A1 (en) 1990-08-26

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