DK166085B - New pancratistatin, 7-de:oxy:narciclasine and derivs. - useful as anti:neoplastic agents obtd. from zephranthes grandiflora etc. - Google Patents

Abstract

Pancratistatin and derivs. of formula (I) having antineoplastic activity are new. R-R3 = H, COMe or COX; X = acid radical. Pharmaceutical compsn. comprises (a) pancratistatin or derivs. of formula (I) as defined above; or (b) 7-deoxynaciclasine and derivs. of formula (II).

Description

in

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The present invention relates to antineoplastic phenanthridones of the formula shown in claim 1. The invention further relates to pharmaceutical compositions containing as active principle an effective amount of such phenanthridone, as well as the use of the phenanthridones for the preparation of a pharmaceutical composition for the treatment of a host affected by a neoplastic disease.

As early as the 4th century BC became aware of certain medicinal and / or toxic plant species. For determination of the presence of alkaloid constituents in such species see R.D. Gibs, 10 "Chemotaxonomy of Flowering Plants", Volume III, McGills-Queens University Press, Montreal, 1974, p. 1924. Over time, more than 30 species of the relatively large Amaryllidaceae family have been used in the primitive treatment of cancer (see JL Hartwell, Loydia, 1967, 30, 391).

In continuing efforts to locate and define differences -15 equally natural and synthesizable substances for the treatment of one or more different types of cancer, chemists in their research continue to draw attention to the natural flora and fauna in an effort to isolate and identify substances which exhibit antineoplastic activity while substantially reducing, if not completely eliminating, some of the serious side effects accompanying known chemotherapeutic agents.

It is through the continued pursuit of these goals that plant species that have been ignored in the past are now being investigated to determine if they contain constituents that exhibit antineoplastic activity upon isolation.

The compound 7-deoxynarciclasin having the structural formula

OH

30 JL h X

H O

35 and can be recovered from plants of the Amaryllidaceae family, under the alternative designation Lycoricidin is described in U.S. Pat.

3,583,993 and in Tetrahedron Letters, Vol. 22, No. 28, pp. 2615-2618 (1981). In the former, the compound plant growth regulator-2

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the effect while the compound of the latter is referred to as a potential antitumor agent.

It has been found that the compound pancrate in statin and its derivatives according to the claims not only has anti-tumor effect, but also a substantially stronger anti-tumor effect than 7-deoxynarciclasin and derivatives thereof.

The compounds of the invention have the structural formula OR 1

10 10 iJ Η «I

OR O.

wherein R is H, CH 2 or a physiologically hydrolyzable acyl group from an aliphatic, alicyclic, al cyclic isoalkyl aliphatic, aromatic or aromatic aliphatic carboxylic acid, especially an acetyl group, while R 1, R 5, R 2 and R 4 each represents H or a physiologically hydrolyzable acyl group from an aliphatic, alicyclic, al cyclic in scaliphatic, aromatic or aromatic aliphatic carboxylic acid, especially an acetyl group.

The present invention provides novel and useful chemotherapeutic agents which can be extracted from the root of the Hawaiian (or African) Pancratium littoral Jacq. and Zephranthes grandi flora in the manner described hereinafter and which can then be formulated into useful pharmaceutical compositions which exhibit detectable and confirmed levels of anticancer activity when measured by the generally accepted prescriptions used in United States National Cancer Institute.

A particular aspect of the invention is the substance pancratistatin having the structural formula:

RjO® ^ 2 10 10 in H «1 ° R> n 11 35 or o wherein R = Rj = R2 = R3 = R ^ = H, and the derivatives thereof wherein R = Rj = R2 = R3 = R4 = COCH2, and the derivatives wherein R ^ H ^ and Rj = R2 = R3 = R ^ = H.

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3

The tuber section (45 kg) of P. littoral was extracted with methylene chloride-methanol-water and the pancratistatin was concentrated (the separation was preferably controlled by bioassay using the PS in vivo system) in a n-butanol extract of the aqueous phase. Purification of half of the crude product using selective solubility properties and gel permeation chromatography ("Sephadex LH-20") yielded 6.5 g (0.028% yield) of pancratistatin separated from dimethylformamide-methanol ether. as a colorless solid: dp 330-321 °, E1 mass spectrum m / e 325 (M +, C14H15N08) [α] 32 + 44 ° (c, 1.0, DMSO), A | |OH (log 10 Σ) 229 (4.29), 239 (4.26) and 281 (4.0) nm, IR (KBr) δ max 3500-3200, 1675, 1615, 1600, 1500, 1465, 1445, 1420, 1375, 1350, 1300 , 1230, 1200, 1160, 1118, 1085, 1070, 1040, 1030, 930, 912, 880, 840, 720, 655, 640 and 610 cm -1, and 1 H NMR (100 MHz, DM $ 0-d6) S 3.6-4.4 (6H), 4.72-5.70 (5H, removed with D 2 O, 6.11 (2H, br, s), 6.56 (1H, s), and 13.15 (1H) Reaction of pancratistatin with acetic anhydride pyridine gave its pentaacetate (mp 162-166 °) Reaction of pancratistatin with diazomethane in methanol gave the 7-monomethyl ether (mp 294-298 °). The same procedure provides pancratistatin from Zepahranth es grandiflora, although P. littorale 0. is used herein as an example.

The remarkable insolubility of pancratistatin in various organic solvents, the high decomposition point, its non-basic nature and the infrared spectrum leads the idea towards a carbostyril or isocarbostyril system. The most plausible interpretation of the pancreatistatin, pentaacetate and methyl ether elemental and spectral analysis data points to a novel phenanthridone. To determine the stereochemical data and to confirm the overall structure of pancratistatin shown below, an X-ray crystal structure determination was used. see.

OR ·, 30 Rj0-y - ^ \ t- ° r2

10 toti H <I

(T ThT

OR O.

Wherein R = Rj = R2 = R8 = R4 = H.

It was found that a single crystal (0.125 x 0.25 x 0.37 mm) of pancratistatin monomethyl ether, recrystallized from 95% ethyl alcohol, corresponds to C₂jHj7NOg.H₂O ω357.32, monoclinic β = 99.78 (2) °, a =

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4 9,040 (1), b * 8,317 (1) and c - 10,187 (2) A, V - 754.8 A, F (000) = 376, Po = 1.565 g / cc, Z = 2 and Pc = 1.572 g / cm3 (25 ° C, CuK = 1.54184 A).

Systematic extinctions (OkO are missing if k = 2n + 1) and chirality were consistent with space group P2. The observed density of 1.56 3 1 5 g / cm 1 showed one molecule, respectively. CjgHjjNOg and water per equivalent position (2 molecules of pancratistatin / cell). The cell parameters were obtained by the least squares method from the reciprocal lattice positions of 25 with diffractometer measured reflections with 2-theta values in the range 5-35 °. The intensity of all unique reflections at 2Θ> 75 ° was measured at 10 25 ° C using a variable speed omega / 2 theta scanning technique on an "Enraf-Nonius CAD4" diffractometer (Delft) using graphite monochromatic CuK radiation. The scan angle for each reflection was calculated as (0.90 + 0.15 tan 0) 0. The detector aperture with a variable width (4.0 + 0.5 tan Θ) mm and a constant vertical height of 4 15 mm was placed at a distance of 173 mm from the crystal. The maximum scan time for each reflection was 1 minute. 2/3 of this time was used for scanning the top and 1/6 for measuring the two backgrounds. The intensity of three monitor reflections was also recorded every 250 minutes, and it was found that the deviation was less than 0.5% during the entire data collection process. A total of 1647 unique reflections were obtained for subsequent treatment (I <1.0 σ (1)). All intensity data were corrected for anisotropic decay using the standards monitor intensity. These data were corrected for Lorentz polarization and anomalous dispersion effects. The atomic scattering coefficients are taken from appropriate tables: D.T. Cromer and J.T. Waber, "International Tables for X-ray Crystallography", Volume IV, The Kynoch Press, Birmingham, England, 1974, Table 2.2B, Tables for Anomalous Dispersion Coefficients, D.T. Cramer, ibid, Table 2.3.1. Extinction or absorption settings were not made nor considered necessary (μ = 10.038 30 cm "1).

The structural determination of pancratistatin monomethyl ether was performed by direct methods using a multi-solution weighted tangent formula method, i.e. program MULTAN. All crystallographic calculations were performed with a "PDP 11/23" computer using the Enraf-Nonius (Delft) structural determination software system ("SDP-PLUS") developed by B.A. Frenz and Associates, Inc., College Station, Texas, 1982. The main programs used were START, PAINTER, REJECT, CH0RT, data reduction programs, LSB, a full matrix,

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• 5 least squares refining program, SEARCH, a connectivity retrieval program, ORTEP-11, the crystallographic illustration program, MULTAN 11/82, "a system of computer programs for automatically resolving crystal structures from X-ray diffraction data", a direct method pro- gram of P. Main and colleagues, University of York, York, England For a description of MULTAN see: G. Germain, P. Main, M. Woolfson, Acta

Crystallogr. Sect. B., 1970, 26, 274-285 and M.M. Woolfson, Acta

Crystallogr. Sect. B., 1977, 33, 219-225. After the correct assignment of coordinates to all non-hydrogen atoms, full matrix, least squares refinement was applied to the non-Poisson contribution weighting scheme at least squares refining, where W «l / <7 / r2, = 4 x F / (crr2) and a, r2 * = ino 22 v / ru / / I + (P x F), where P is an adjustment factor with the objective of downgrading intense reflections and W is the weight of the reflection. A value of 0.05 was used for P on all coordinates, scale factors and in sotrope temperature factors, resulting in a rapid reduction of the conventional unweighted crystallographic discrepancy index R (2 I Fo-Fe I / ΣΙ Fol) to 0.1795 ( for observed reflections, I> 3 σ (I)). Subsequent differential electron density synthesis revealed all hydrogen atoms as well as the presence of a molecule of water which is considered to be hydrogen bonded (1.83 20 Å) to one of the hydroxy hydrogen atoms at C-4. Finally, several additional rounds of full matrix caused the least squares refining of all variables (anisotropic non-hydrogen atoms and isotropic non-hydrogen atoms and isotropic hydrogen atoms, where the thermal B values of the latter were set at a nominal value of 5.0, convergence to R = 25 0.405 (Ra> = 0.0438) Refinement was discontinued when parameter changes became insignificant (maximum shift to error ratio was 0.84 with the majority in the range 0.1-0.2) and a final difference statement revealed negligible electron densities (> 0.22 e Å) All bond lengths and angles were consistent with expected values.

A calculation of the discrepancy indices of the opposite enantiomorph of that depicted for the pancratistatin derivative when R 1 CH 2 and R 2 = R 2 = R 3 = R 4 = H, based on the data obtained (1632 observations and 301 variables), gave values of R '= 0.0425, and R'w represents the absolute stereochemical configuration with> 99.9% probability. The assigned ab-35 solute configuration is further supported by a combination of studies of the biosynthetic structure and the X-ray crystal structure of narciclasin, a 1-dehydrophenanthrone derivative of pancrastistatin.

In one embodiment of the extraction tubers were extracted by P. litto-6

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using a methylene chloride-methanol process followed by addition of water. The methylene chloride phase was partitioned using the solvent sequence 9: 1 - * · 4: 1 + 3: 2 methanol water with hexane - * carbon tetrachloride - + methylene chloride. The aqueous phase was extracted with n-butanol, and pancratistatin was concentrated there rather than in the methylene chloride residue where lycorine would be expected.

The n-butanol fraction was further separated by gel permeation chromatography on "Sephadex LH-20" using methanol as the eluent. The collection of fractions was guided by thin layer chromatography using 3: 1 chloroform-methanol. The two major antineoplastic components corresponded to 0.37 and 0.48. Both were found to have a very high melting point, to be relatively insoluble, and to be non-faithful non-basic solids similar to carbostyrils or isocarbostyrils.

Purification of the Rf 0.37 product by recrystallization from acetic acid-methanol gave a lactam which readily resulted in a tri acetate derivative. Spectral analysis data showed that this anticancer component (PS, T / C 161 at 12.5 mg / kg, ED 2 / 0.02 / µg / ml) was a 7-deoxy derivative of narciclasin.

However, physical constants reported for marginine, which was later renamed 20 lycoricidin, showed otherwise. Because of this and because initial attempts to obtain an authentic sample of 7-deoxynarciclasin were unsuccessful, a complete structural determination was performed by X-ray crystallographic methods. The result was final determination of the structure 25 oh in nh

H O

30 for the substance with R 2 0.37. The absolute configuration of the triol was elucidated by its supposed biosynthesis of vittatin. Later, authentic samples of the tri acetate 359COCH3 / S ^ OCOCHj were obtained

NH

H O

7

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prepared from the natural and (i) synthetic products, and comparison with the substance with 0.37 obtained from P. littorale by infrared spectral analysis in potassium bromide showed significant differences, but when compared in a chloroform solution the spectra were identical. On this basis, it is believed that the assignment of the structure shown above to 7-deoxynarciclasin is correct.

Recrystallization of the 0.46 anticancer component (T / C 138 · + 165 at 0.75 6.0 mg / kg dose levels and 208 at 12.5 mg / kg in the PS system with ED5q, 0.01 µg / ml) from dimethylformamide-methanol-ether gave a pure 10 sample exhibiting elemental and spectral analytical data in accordance with the structure

OH

HOx

10 solder H * I

15 (T TH

o - K ^ A ^ 3 NH

I7 II

OH O

This new phenanathridone was called "pancratistatin" and its structure was elucidated by an X-ray crystal structure determination. The structure determined for pancratistatin was completely consistent with the corresponding spectral analysis results.

To further advance the understanding of the present invention, attention should now be directed to the methods performed in isolating pancratistatin and 7-deoxynarciclasin from the Hawaiian 25 (African) Pancratium littoral Jacq.

General Procedure. All solvents used for chromatography were redistilled. Thin layer chromatography was performed on silica gel "GHLF Uniplates" (0.25 mm layer thickness) supplied by Analtech Inc., developing solvent chloroform methanol (3: 1), and visualization with cerisulfate spray reagent. "Sephadex LH-20" (particle size 25-100 µ) was supplied by Sigma Chemical Co. A "Gilson Model FC-200K" fraction collector was used to collect fractions.

Melting points were determined on a Kofler type hot table apparatus and are uncorrected. Optical rotations were measured with a "Perkin-Elmer Model 241" 35 automatic polarimeter. Ultraviolet spectra were recorded on a "Hewlett-Packard Model 8450A UV / VIS" spectrophotometer and infrared spectra on "Perkin Elmer Model 200" and "Nicolet MX-1 FTIR" spectrophotometers. 1 H and 1 C NMR spectra were recorded, respectively. "Varian XL-100" and "Uses HXE-90" 8

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(22.63 MHz) spectrometers using tetramethylsilane as an internal standard. Mass spectra were obtained on a "variant MAT 312" spectrometer.

Plant Collection. The tubers of Hawaiian P. littoral J. were obtained through a full research program with the National Cancer Institute -5 University of Hawaii.

Extraction. The tubers of the Hawaiian P. littoral Jacq. cut and the cut tubers (45 kg) were extracted with methanol: methylene chloride (1: 1, 320 liters) at ambient temperature for 20 days. The extract was then decanted and the methylene chloride phase was separated by adding 10 of 20% by volume water. The aqueous phase was adjusted by addition of additional methanol and methylene chloride in the aqueous phase methanol: methylene chloride (2: 1: 1) ratio and the tubers were extracted again for another 20 days. Subsequent decantation and addition of 20% by volume of water separated the methylene chloride phase which was then combined with the first methylene chloride fraction and evaporated to give an inert extract (8129) ·

Solvent Division. The aqueous phase from the extraction above was concentrated to ca. 16 liters and then centrifuged to remove the insoluble material. The clear solution was then extracted with n-butanol (3 x 10 liters) and the butanol extract was concentrated to give the butanol-soluble fraction (705 g). A portion (355 g) was dissolved in methanol (1.5 liters) and acetone (3.5 liters) added. The insoluble material (105 g) was filtered off and the filtrate was evaporated to give a residue (250 g).

Isolation of 7-deoxynarciclasin and pancratistatin. The residue obtained in the solvent solution obtained was treated with methanol (1.5 liters) and then filtered to give a solid (2 g) which was found to be pancratistatin. The filtrate was chromatographed on "Sephadex LH-20" (2 kg, 105 x 10 cm) using methanol as the eluent and control of the fractions by thin layer chromatography. Fractions containing the component Rp 0.37 were combined, concentrated and filtered to give 7-deoxynarciclasin (10 g), which crystallized from acetic acid methanol as fine needles, m.p. 251-252 ° (lit.

214.5-216.5 ° (9), 230 ° (dec.) (12)), [a] 33 + 157.3 ° (c 0.96, DMS0), e 35 ms: m / e 291 ( M +, C ^H ^NOg), ^ ^OH (logΣ) 233 (4.14), 248 (4.15) and 302 (3.75) nm, ir (KBr) 3450, 3250, 1672, 1632, 1620, 1602, 1505, 1473, 1415, 1400, 1340, 1320, 1270, 1250, 1080, 1045, 1015, 976, 940, 890, 785, 700, 670, and 623 cm 2 *, * H nmr (pyridine-dg 5, 4.81-

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Δ 4.92 (2H), 5.0-5.35 (2H), 6.05 (2H, d, J = α 3 Hz), 6.72 (1H, br, s), 7.33 (1H, s), 8.06 (1H, s), 8.57 (1H, br, s, removed by D 2 exchange in ng) and 7.0-8.6 (br hump, removed with D 2 O) ppm, nmr (DMSO-d 6) δ 163.12, 150.58, 147.72, 131.70, 130.01, 123.61, 121.95, 106.19, 103.24, 101.81, 5.72, 56, 69.21 and 52.74 ppm (a carbon atom masked with DMSO: 42.77-36.72).

Analysis: Calculated for Cj NONgtgt C, 57.73, H, 4.47, N, 4.81 found: C, 57.79, H, 4.49, N, 4.79.

Further elution gave fractions mainly containing the component R ^ 0.48, which by concentration and filtration gave pancratistatin (4.5 g). Crystallization from dimethyl formamide-methanol-ether gave a colorless solid, m.p. 320-321 ° C (dec.), [Α] + 44 ° (c 1.0, DMS = eims m / e 325 (M +, C 14 H 15 NO 8), A 2 OH (log Σ) 209 (sh), 219 ( sh), 233 (4.32), 278 (3.91) and 308 (br, sh) nm, ir (KBr) vv 3500-3200,

fiiaX

15 1675, 1615, 1600, 1500, 1465, 1445, 1420, 1375, 1350, 1300, 1230, 1200, 1160, 1118, 1085, 1070, 1040, 1030, 930, 912, 880, 840, 720, 655, 640 and 610 cm "*, HH nmr (MDSO-dg) δ 3.6-4.4 (6H), 4.72-5.70 (5H, removed with DgO), 6.11 (2H, br, s ), 6.56 (1H, s) and 13.15 (1H, removed with 020) ppm, nmr (DMSO-dg) δ 169.49, 152.04, 145.38, 135.63, 131.70, 107.49, 201.70, 97.62, 73.28, 70.19, 69.99, 68.50 and 50.46 ppm (a carbon atom masked with DMSO: 42.27-36.72).

Analysis: Calculated for Cj HjH₂gNOg: C, 51.69, H, 4.61, N, 4.31 Found: C, 51.65, H, 4.55, N, 4.24.

25

Biologically active glycoside derivatives of pancratistatin are prepared by coupling the main compound with suitably protected sugars or other hydroxylated compounds using well known methods (see Methods in Chemistry by RL Whistler and JN Bemiller 30 (publishers), Academic Press, NY, 1972, Volume 6, or The

Carbohydrates: Chemistry and Biochemistry by W. Pigman, Academic Press, N.Y., 1981).

Derivatives of pancratistatin are used for the same purpose as pancrati-statin.

As shown, pancratistatin has free hydroxyl groups available for derivatization. Thus, acyl esters of the compound can also be prepared according to methods well known to those skilled in the art. Acylderates can be used for the same biological purposes as the parent compound.

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10

Acids which may be used to acylate a pancratistatin include: (a) saturated or unsaturated, straight-chain or branched aliphatic carboxylic acids, e.g. acetic acid, propionic acid, butyric acid, in succinic acid, tert-butyl acetic acid, valeric acid, isovaleric acid, capric acid, caprylic acid, decanoic acid, dodecanoic acid, lauric acid, tridecanoic acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, margaric acid , oleic acid, hexynic acid, heptynic acid, octynic acid, ol, (b) saturated or unsaturated al of cycling in spoonful carboxylic acids, e.g. cyclobentanecarboxylic acid, cyclopentanecarboxylic acid, cyclopentene carboxylic acid, methyl cyclopentenecarboxylic acid, cyclohexanecarboxylic acid, dimethylcyclohexane carboxylic acid, di propylcyclohexanecarboxylic acid, etc., (c) unsaturated or unsaturated or unsaturated or unsaturated cyclopentane acetic acid, cyclopentane propionic acid, cyclohexane acetic acid, cyclohexane butyric acid, methylcyclohexane acetic acid, and the like, (d) aromatic carboxylic acids, e.g. benzoic acid, toluene acid, naphthoic acid, ethyl benzoic acid, isobutylbenzoic acid, methyl butyl benzoic acid, and the like, and (e) aromatics in fatty carboxylic acids, e.g. phenyl acetic acid, phenylpropionic acid, phenyl valeric acid, cinnamonic acid, phenylpropionic acid and naphthyl acetic acid, and the like. Suitable halo, nitro, hydroxy, keto, amino, cyano, thiocyano and lower alkoxycarboxylic carboxylic acids include hydrocarbon carboxylic acids as listed above which are substituted by one or more groups in the form of halogen, nitro, hydroxy, keto , amino, cyano or thiocyano, or lower alkoxy, preferably lower alkoxy of not more than 6 carbon atoms, e.g. methoxy, ethoxy, propoxy, butoxy, amyloxy, hexyloxy and isomeric forms thereof. Examples of such substituted hydrocarbon carboxylic acids are mono-, di- and trichloroacetic acid, α- and β-chloropropionic acid, α- and γ-bromobutyric acid, α- and 5-iodovaleric acid, mevalonic acid, 2- and 4-chloro-cyclohexanecarboxylic acid, -nitro-1-methyl-cyclobutane carboxylic acid, 1,2,3,4,5,6-hexa-chlorocyclohexane carboxylic acid, 3-bromo-2-methylcyclohexane carboxylic acid, 4- and 5-bromo-2-methylcyclohexane carboxylic acid, 5- and 6- bromo-2-methylcyclohexanecarboxylic acid, 2,3-dibromo-2-methylcyclohexanecarboxylic acid, 2,5-dibromo-2-methylcyclohexanecarboxylic acid, 4,5-dibromo-2-methylcyclohexanecarboxylic acid, 5,6-di bromo-2 -methylcyclohexanecarboxylic acid, 3-bromo-3-methylcyclohexanecarboxylic acid, 6-bromo-3-methylcyclohexanecarboxylic acid, 1,6-dibromo-3-methylcyclohexanecarboxylic acid, 2-bromo-4-methylcyclohexane carboxylic acid 3-bromo-2,2,3-trimethylcyclopentanecarboxylic acid, 1-bromo-3,5-di methylcyclohexanecarboxylic acid, homogeneous

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11 tisic acid, o-, m- and p-chlorobenzoic acid, anicylic acid, salicylic acid, p-hydroxybenzoic acid, b-resorcyl acid, bile acid, veratric acid, trimethoxybenzoic acid, trimethoxycinnamic acid, 4,4 / dichlorobenzoic acid, o-, and p-nitrobenzoic acid, cyanoacetic acid, 3,4- and 3,5-dinitrobenzoic acid, 2,4,6-trinitrobenzoic acid, thiocyanoacetic acid, cyanopropionic acid, lactic acid, ethoxymyric acid (ethyl hydrogen carbonate), malic acid, citric acid, isocitronic acid , 6-methylsalicylic acid, mandelic acid, levulinic acid, pyruvic acid, glycine, alanine, isoleucine, leucine, phenylalanine, proline, serine, threonine, tyrosine, hydroxyproline, ornithine, lysine, arginine, histidine, hydroxylyline, phenylglycine, p-aminobenzoic acid, m-aminobenzoic acid, anthranilic acid, aspartic acid, glutamic acid, aminoadipic acid, glutamine, asparagine, etc.

Pancratistatin pentaacetate was prepared by reacting pancrati statin (0.5 g) with acetic anhydride (2 ml) and pyridine (2 ml) at room temperature for 48 hours. The product (0.5 g), obtained by addition to ice-15 water and filtration, was chromatographed on "Sephadex LH-20" (100 g using methanol-methylene chloride (3: 2) as the eluent to give the amorphous pentaacetate). mp 162-166 °, [α] + 85 ° (c 1.0, CHCl 3), eims m / e 535 (M +, C24H25N013), λ ^ | 0Η (1θ9 Σ) 227 (4.31), 247 (sh), 271 (sh) and 299 (3.75 nm), ir (KBr) v 3370, 1760, 20 1680, 1635, 1510, 1490, 1375, 1340, 1295, 1250, 1220, 1180, 1080, 1045 , 950, 930, 860, 815, 758 and 640 cm -1, 1H nmr (90 MHz, CDCl3) δ 2.06 (6H, s), 2.08 (3H, s), 2.17 (3H, s) ), 2.37 (3H, s), 3.43 (1H, dd, J = 12.3 and 2.5 Hz), 4.26 (1H, dd, J-12.5 and 11.7 Hz) , 5.14 (1H, dd, J-11.7, and 3.3 Hz), 5.22 (1H, dd, J = 2.9 and 2.9 Hz), 5.45 (1H, m), 5.56 (1H, m), 5.76 (1H, br, s, removed with D 2 O), 6.08 (2H, br, s) and 6.48 (1H, s) ppm, 13 C nmr (CO 13 ) S 170.08, 169.69, 169.07, 169.01, 168.29, 162.96, 152.60, 139.86, 134.53, 132.90, 116.20, 102.94, 101.84, 71.68, 67.69, 66.84, 66.42, 47.86, 40.00, 20.86 and 20, 70 (last two signals 5C) ppm.

Analysis: Calculated for C24 H25 N3 O3: C '53'83> 4.67, N' 2.62 Found: C, 53.75, H, 4.68, N, 2.61.

Pancratistatin methyl ether was prepared by reacting pancrati-statin (0.33 g) in methanol (100 ml) with excess diazomethane in ether.

After stirring for 8 hours at room temperature, an additional amount of diazomethane was added and stirring was continued for 8 hours. Evaporation of the solvent gave a product (0.34 g) which was chromatographed on "Sephadex LH-20" using methanol as eluent to give the methyl ether (0.1 g). Crystallization from methanol gave colorless plates, m.p. 294-298 ° (dec), [α] 25 + 289.9 ° (c 0.69, DMSO), ir (KBr)

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12 »/ 3500, 3400, 3300, 1635, 1600, 1485, 1450, 1390, 1343, 1298, 1226,

MidX

1207, 1152, 1122, 1090, 1060, 1035, 967, 937, 920, 880, 840, 795, 724, 660, and 620 cm -1, lti nmr (DMSO-dg) 3.58-4.44 (6H) , 3.88 (3H, $), 4.70-5.50 (4H, removed with DgO), 6.13 (2H, d, J = 5 Hz), 6.74 (1H, s), and 6, 92 δ (1H, removed with D 2 O) ppm.

Analysis: Calculated for C, 53.10; H, 5.01; N, 4.13 Found: C, 53.44; H, 5.06; N, 4.08.

The administration of pancratistatin and its pharmacologically active physiologically compatible derivatives can be used to treat animals or humans suffering from a neoplastic disease, e.g. acute myelocytic leukemia, acute lymphocytic leukemia, malignant melanoma, pulmonary adenocarcinoma, neuroblastoma, small-cell lung carcinoma, breast carcinoma, intestinal carcinoma, ovarian carcinoma, bladder carcinoma, and the like.

The dose administered will depend on the nature of the neoplastic disease, the type of host involved, incl. its age, health and weight, the nature of any concomitant treatment, the frequency of treatment, and therapeutic conditions.

For example, dosage levels of the active ingredients administered are: intravenous, 0.1 to ca. 200 mg / kg, intramuscular, 1 to approx.

20 500 mg / kg, oral, 5 to approx. 1000 mg / kg, intranasal administration, 5 to approx.

1000 mg / kg, and aerosol, 5 to approx. 1000 mg / kg of host body weight.

In terms of concentration, an active ingredient may be present in the composition of the present invention for local use around cutis, intranasal, pharyngolaryngal, bronchial, broncholial, intragaginal, rectal or ocular at a concentration of ca. 0.01 to approx. 50% w / w% of the composition, preferably approx. 1 to approx. 20% w / w% of the composition, and for parenteral use at a concentration of approx. 0.05 to approx. 50% w / v of the composition and preferably from ca. 5 to approx. 20% w / v%.

The compositions of the invention are preferably used for administering to humans and animals in unit dosage form such as tablets, capsules, pills, powders, granules, suppositories, sterile parenteral solutions or suspensions, sterile non-parenteral solutions or suspensions and oral solutions or suspensions, etc. containing -35 are appropriate amounts of an active ingredient.

For oral administration, either solid or liquid unit dosage forms can be prepared.

Powders are simply prepared by comminuting the active ingredient.

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13 to a suitable fine size and mix it with a similarly finely divided diluent. The diluent may be an edible carbohydrate material such as lactose or starch. Preferably, a sweetener or sugar as well as a flavoring oil is used.

Capsules are prepared by preparing a powder mixture as described above and filling it into shaped gelatin casings. Preferably, as an aid to the filling operation, a lubricant such as talc, magnesium stearate, calcium stearate and the like is added. to the powder mixture before the filling operation.

Soft gelatin capsules are prepared by mechanical encapsulation of a slurry of active ingredients with an acceptable vegetable oil, light paraffin oil or other inert oil or triglyceride.

Tablets are prepared by preparing a powder mixture, granulating or beating it, adding a lubricant and pressing it into tablets.

The powder mixture is prepared by mixing an active ingredient, which is appropriately decomposed, with a diluent or base such as starch, lactose, kaolin, dicalcium phosphate and the like. The powder mixture can be granulated by wetting it with a binder such as corn syrup, gelatin solution, methyl cellulose solution or rubber mucus and forcing it through a sieve. As an alternative to granulation, the powder mixture can be beaten, ie. is run through the tablet machine and the resulting irregularly shaped tablet is broken (swallowed). These pieces can be lubricated to avoid adherence to the tablet forming matrices by adding stearic acid, a stearic salt, talc or mineral oil. The lubricated mixture is then compressed into tablets.

The tablet may advantageously be provided with a protective coating consisting of a sealed end or enteric shellac coating, a sugar and methyl cellulose coating and a carnauba wax polish coating.

Liquid unit dosage forms can be prepared for oral administration such as syrups, elixirs and suspensions, each teaspoon of the composition containing a predetermined amount of active ingredient for administration. The water-soluble forms can be dissolved in an aqueous carrier medium with sugar, flavoring and preservatives to form a syrup. An elixir is prepared using a hydro-alcoholic carrier with suitable sweeteners and a flavoring agent. Suspensions may be prepared from the insoluble forms together with a suitable carrier medium by means of a suspending agent such as acacia, tragacanth, methyl cellulose and the like.

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14

For parenteral administration, liquid unit dosage forms are prepared using an active ingredient and a sterile vehicle, water being preferred. The active ingredient may, depending on the form and concentration used, either be suspended or dissolved in the carrier medium. In preparing solutions, the water-soluble active ingredient can be dissolved in water for injection and filter sterilized before being filled into a suitable vial or ampoule and sealed. Adjuvants such as a local anesthetic, preservative and buffering agent may advantageously be dissolved in the carrier medium. Parenteral suspensions are prepared in substantially the same manner except that the active ingredient is suspended in the carrier rather than dissolved and sterilization cannot be provided by filtration. The active ingredient can be sterilized by subjecting it to ethylene oxide before suspending it in the sterile vehicle. Advantageously, a surfactant or wetting agent may be incorporated into the composition to facilitate a uniform distribution of the active ingredient.

In addition to oral and parenteral administration, rectal and vaginal routes can be used. An active ingredient may be administered by means of a suppository. A carrier medium having a melting point of approx. body temperature or a slightly soluble carrier can be used. For example, cocoa butter and various polyethylene glycols ("Carbowax") may serve as the carrier.

For intranasal administration, a liquid unit dosage form is prepared using an active ingredient and a suitable pharmaceutical carrier, preferably P.F. water, a dry powder can be formulated when administration by injection is selected.

For use as aerosols, the active ingredients may be packaged in an aerosol pressure container together with a gaseous or liquid propellant, e.g. dichlorodifluoromethane, carbon dioxide, nitrogen, propane, and the like, with the usual adjuvants such as co-solvents and wetting agents to the extent necessary or desirable.

The term "unit dosage form" as used in the specification and claims refers to physically separate units suitable as unit doses for humans and animals, each unit containing a predetermined amount of active material intended to produce the desired therapeutic effect. in conjunction with the necessary pharmaceutical diluent, carrier or vehicle. The specifications of the novel unit dosage forms of the invention are determined by and directly

DK 166085B

15 are dependent upon (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitation inherent in composing such active material for therapeutic use in humans as described herein. description, these being aspects of the present invention. Examples of suitable unit dosage forms according to the invention are tablets, capsules, lozenges, suppositories, powder packs, cachets, cachets, teaspoons, tablespoons, pipettes, ampoules, vials or bottles, separate multiples of any of the preceding 10 and other forms as described above. here.

The active ingredients to be used as antineoplastic agents can be readily prepared in such unit dosage form using pharmaceutical materials obtainable in the art, and can be prepared by known methods. The following compositions are intended to illustrate the preparation of the unit dosage forms of the present invention.

Example 1

In carrying out the present invention, various dosage forms were prepared. They are shown in the following examples in which the term "active ingredient" stands for one of the antineoplastic phenan-thridones of the invention.

Preparation "A" 25 Hard Gelatin Capsules 1000 two-piece hard gelatin capsules for oral use, each capsule containing 200 mg of active ingredient, are prepared from the following ingredient types and amounts:

Active ingredient, micronized 200 g 30 Corn starch 20 g

Talc 20 g

Magnesium stearate 2 g

The active ingredient finely distributed by means of an air micronizer is added to the other finely powdered ingredients, thoroughly mixed and then encapsulated in the usual manner.

The capsules described can be used to treat a neoplastic disease by oral administration of one or two capsules 1-4 times daily.

Using the procedure described above, is prepared

DK 166085 B

16 similar capsules containing an active ingredient in an amount of 50, 250 and 500 mg, by replacing the above 200 g with 50 g, 250 g and 500 g of active ingredient.

Preparation "B"

Soft gelatin capsules

One-piece soft gelatin capsules for oral use, each containing 200 mg of active ingredient (divided by means of an air micronizer), are prepared by first suspending the compound in 0.5 ml of corn oil to make the material suitable for encapsulation, and then encapsulating on the way described above.

The capsules described can be used to treat a neoplastic disease by oral administration of one or two capsules 1-4 times daily.

Preparation "C"

Tablets after 1000 tablets, each containing 200 mg of active ingredient, are prepared from the following ingredient types and amounts:

Active ingredient, micronized 200 g 20 Lactose 300 g

Corn starch 50 g

Magnesium stearate 4 g

Light paraffin oil 5 g

The active ingredient 25 divided by an air micronizer is added to the other ingredients and mixed and then thoroughly beaten. The pieces are subdivided by forcing them through a No. 16 sieve. The resulting granules are then compressed into tablets, each tablet containing 200 mg of active ingredient.

The tablets described can be used to treat a neoplastic disease by oral administration of one or two tablets 1-4 times daily.

Using the procedure described above, similar tablets containing an active ingredient in an amount of 250 mg and 100 mg can be prepared by replacing the above 200 g with 250 g and 100 g active ingredient.

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17

Preparation "D"

Oral suspension 1000 ml of aqueous suspension for oral use containing, in each teaspoon (5 ml) of dose, 50 mg of active ingredient, is prepared from the following ingredient types and amounts:

Active ingredient, micronized 10 g

Citric acid 2 g

Benzoic acid 1 g

Sucrose 790 g 10 Tragacanth 5 g

Lemon oil 2 g

Deionized water, q.s. 1000 ml.

The citric acid, benzoic acid, sucrose, trachacanth and lemon oil are dispersed in a sufficient amount of water to make 850 ml of suspension. The active ingredient divided by means of an air micronizer is stirred in the syrup until it is uniformly distributed. A sufficient amount of water is added to make 1000 ml.

The preparation thus prepared can be used to treat a neoplastic disease at a dose of 1 tablespoon (15 ml) three times daily.

Preparation "E"

Parenteral product

A sterile aqueous suspension for parenteral injection containing 25 300 mg of active ingredient in 1 ml for the treatment of a neoplastic disease is prepared from the following ingredient types and amounts:

Active ingredient, micronized 30 g "Polysorbate 80" 5 g

Methyl paraben 2.5 g Propyl paraben 0.17 g

Water for injection, q.s. 1000 ml.

All ingredients except the active ingredient are dissolved in the water and the solution is sterilized by filtration. To the sterile solution, it is added by means of an air micronizer finely divided, sterilized active ingredient and the final suspension is filled into sterile vials which are sealed.

The preparation thus prepared can be used to treat a neoplastic disease at a dose of 1 ml three times daily.

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18

Preparation "F"

Suppository, rectally and vaginally 1000 suppositories, each weighing 2.5 g and containing 200 mg of active ingredient, are prepared from the following ingredient types and 5 amounts:

Active ingredient, micronized 15 g

Propylene glycol 150 g

Polyethylene Glycol 4000, q.s. 2500 g.

The active ingredient is comminuted by an air micronizer and added to the propylene glycol, and the mixture is passed through a colloid mill until uniformly dispersed. The polyethylene glycol is melted and the propylene glycol dispersion is added slowly with stirring. The suspension is poured into uncooled molds at 40 ° C. The preparation is allowed to cool and solidify and then removed from the mold, and each suppository is wrapped in foil.

The described suppositories are introduced rectally or vaginally to treat a neoplastic disease.

Preparation "G" 20 Intranasal suspension 1000 mg of a sterile aqueous suspension for intranasal administration containing in each ml 200 mg of active ingredient is prepared from the following ingredient types and amounts:

Active ingredient, micronized 15 g 25 "Polysorbate 80" 5 g

Methyl paraben 2.5 g

Propylparaben 0.17 g

Deionized water, q.s. 1000 ml.

All ingredients except the active ingredient are dissolved in the water and the solution is sterilized by filtration. To the sterile solution, it is added by means of an air micronizer finely divided, sterilized active ingredient, and the final suspension is aseptically loaded into sterile containers.

The preparation thus prepared can be used to treat a neoplastic disease by intranasal administration of 0.2 to 0.5 ml administered 1-4 times daily.

As shown in Examples 12-14, an active ingredient may also be present in the undiluted pure form for local use around cutis, intranasal

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19 salt, pharyngolaryngeal, bronchial, broncholial or oral.

Preparation "H"

Powder 5 5 g of active ingredient in loose form is comminuted by means of an air micronizer. The micronized powder is placed in a litter box container.

The composition described can be used to treat a neoplastic disease at local sites by applying a powder 1-4 times daily.

Preparation "I"

Oral powder 100 g of active ingredient in liquid form is comminuted by means of an air micronizer. The micronized powder is divided into single doses of 200 mg and packaged.

The powders described can be used to treat a neoplastic disease by oral administration of 1 or 2 powders suspended in a glass of water 1-4 times daily.

20

Preparation "J"

Inhalation 100 g of active ingredient in bulk is comminuted by means of an air chronizer.

The described composition can be used to treat a neoplastic disease by inhalation of 300 mg 1-4 times daily.

Preparation "K" Hard Gelatin Capsules 30 100 two-part hard gelatin capsules for oral use, each capsule containing 200 mg of active ingredient.

The active ingredient is comminuted by an air micronizer and encapsulated in the usual manner.

The capsules described can be used to treat a neoplastic disease by oral administration of 1 to 2 capsules 1-4 times daily.

Using the procedure described above, similar capsules containing active ingredient in an amount of 50, 250 and 500 mg can be prepared by replacing the above 200 g with 50

DK 166085 B

20 g, 250 g and 500 g of active ingredient.

Example 2

Unit dosage forms of pancratistatin prepared according to selected selections described in Example 15 were screened using the

Cancer Chemotherapy Reports, Part 3, Volume 3, No. 2, September 1972, pp. 9 et seq., Described Regulation 1,200 for lymphocytic leukemia P386. Pancratistatin provided a lifetime extension of 38-106% at 0.75-12.5 mg / kg of host body weight against mouse P388 lymphocytic leukemia 10. Pancratistatin also significantly inhibited the growth of the P388 in vitro cell line (ED1, 0.001 / µg / ml).

Example 3 (Comparison)

Unit dosage forms of 7-deoxynarciclasin were prepared according to Example 15 1 and screened using prescriptions approved by The National Cancer Institute. With the preparation, up to 61% lifetime extension was obtained at 12 mg per kg of the host body weight against mouse P388 lymphocytic leukemia and a 0.02 µg ED2q per ml against the P388 cell line. Doubling the dose did not increase the life extension.

20

Example 4

A unit dose form of pancratistatin prepared according to Example 1 was tested with M5074 mouse ovarian sarcoma and provided a lifetime extension of 53-84% at 0.38 to 3.0 mg of active reagent / kg of host body weight using that of The National The Cancer Institute approved a life expectancy regulation.

Example 5

A unit dose form of pancratistatin prepared according to Example 1 was tested according to the National Cancer Institute approved prescription rate for M5074 mouse ovarian sarcoma and provided a 50% cure rate at 6 mg / kg of host body weight.

Claims (7)

1. Antineoplastic phenanthridone, characterized in that it has the structural formula 5 ORj R ^ O O R 2 10 10b H 0Ri \ JL. Wherein R is H, CH 2 or a physiologically hydrolyzable acyl group from an aliphatic, al in cyc in sk, al in cyc in sk-ali fatic, aromatic or aromatic. sk-al in fatty carboxylic acid, especially an acetyl group, while R 1, R 2, R 3 and R 2 each represent H or a physiologically hydrolyzable acyl group from an aliphatic, al cyclic in sk, alicyclic sk-ali fatic, aromatic or aromatic shell. grab so-called carboxylic acid, especially an acetyl group. An antineoplastic phenanthridone according to claim 1, characterized in that R - R1 = R2 = R3 = R4 = H. 20 A pharmaceutical composition, characterized in that it contains as its main active ingredient an effective amount of a phenanthridone according to claim 1. Pharmaceutical composition according to claim 3, characterized in that RssRj = R2ssR3 = H. Use of a phenanthridone according to claim 1 for the preparation of a pharmaceutical composition for treating a host affected by a neoplastic disease. Use according to claim 5, characterized in that the phenanthridone is pancratistatin. DK 166085B Use according to claim 5, for the preparation of a pharmaceutical preparation for intravenous administration at a dose level of from 0.1 up to approx. 200 mg / kg of host body weight, for subcutaneous administration at a dose level of ca. 1 up to approx. 500 mg / kg of host body weight, or 5 for oral administration at a dose level of about 5 up to approx. 1000 mg / kg of host body weight. 10 15