WO1990014008A1

WO1990014008A1 - Methods and compositions for inhibiting tumor cell growth

Info

Publication number: WO1990014008A1
Application number: PCT/US1990/003065
Authority: WO
Inventors: Ting-Chao Chou; Tian-Shung Wu
Original assignee: Sloan-Kettering Institute For Cancer Research
Priority date: 1989-05-25
Filing date: 1990-05-25
Publication date: 1990-11-29
Also published as: AU5831090A

Abstract

The subject invention discloses methods of inhibiting growth of tumor cells which comprise contacting the cells with a growth inhibiting amount of a compound having one of structures: (I), wherein R1 is a hydroxyl or acetyl group; R2 is hydrogen, -CH¿2?-CH=C(CH3)2 or a methoxy group; R?3¿ is a hydroxyl, methoxy or acetyl group; R4 is hydrogen, -CH¿2?-CH=C(CH3)2 or a methoxy group; R?5¿ is a hydroxyl, methoxy or acetyl group; R6 is hydrogen or a hydroxyl, methoxy or acetyl group; and R10 is a methyl group; (II), wherein R5 is hydrogen or -CH¿2?-CH=C(CH3)2; R?6¿ is a hydroxyl group; R10 is hydrogen or a hydroxyl group; R11 is a hydroxyl or methoxy group; and R12 is hydrogen or a methyl group (III, IV). This invention also provides compositions for inhibiting growth of tumor cells which comprise one of the above-identified compounds and wherein the compound substituents comprise hydrophilic substituents to increase solubility.

Description

METHODS AND COMPOSITIONS FOR INHIBITING TUMOR CELL GROWTH

This application is a continuation-in-part of U.S. Serial No. 357,323, filed May 25, 1989, the contents of which are hereby incorporated by reference into the present disclosure.

The invention described herein was made in the course of work under Grant Nos. CA18856, CA27569 and AI26056 from the National Cancer Institute, U.S. Department of Health and Human Services. The U.S. Government may have certain rights in this invention.

Background of the Invention A number of naturally occurring acridone alkaloids have been isolated from the family Rutaceae, but only acronycine (1) has been noted for its antitumor activity. (Throughout the specification, compounds will be cross-referenced with underlined numbers to facilitate reading and to avoid repetition of long chemical names).

Acronycine was isolated from the bark of the Australian scrub, Acronychi baueri Schott (Rutaceae) (Hughes, G.K., et al., Nature, 162:223-224 (1948); McDonald, P.L. and A.V. Robertson, Aust. J. Chem., 19:275-281 (1966); Svoboda, G.H., Lloydia, 29:206-224 (1966)). Experimental studies on acronycine in animals (Finkelstein, T.Z., et al. Cancer Chemother. Reports, 59:975-983 (1975); Schneider, J., et al., J. Med. Chem., 15:266-270 (1972); Svoboda (1966); Svoboda, G.H., et al., J. Pharm. Sci., 55:758-768 (1966); Svoboda, G.H., U.S. Patent No. 3,985,899, issued October 12, 1976; Svoboda, G.H., U.S. Patent No. 4,309,431, issued January 5, 1982) showed it to be effective against a wide range of neoplasms. Because of its antitumor activity, it has undergone clinical trials (Division of Cancer Treatment, Program Statistics, March 30, 1970, Department HEW, Public Health Service, NIH, IND status filed for acronycine).

Acronycine was found to inhibit the incorporation of extracellular nuclosides into the DNA and RNA of cultured L5178Y cells, but did not interact with DNA (Dunn, B.P., et al. Cancer Res., 33:2310-2319 (1973)). The inhibitory effect of the alkaloid may be the result of an alteration in the transport of uridine through the plasma membrane instead of impaired nucleoside or nucleotide phosphorylation (Dunn, B.P., et al. Cancer Res., 33:2310-2319 (1973)). Similar mechanisms of action were also observed in other studies which indicated that the alkaloid acts primarily on membranous organelles, and its delayed effects may be in part due to interference with the structure, function, and/or turnover of cell-surface components (Tan, P., and N. Auersperg, Cancer Res., 33:2320-2329 (1973)).

Several derivatives of acridinone have been synthesized and tested for their antitumor activity (Liska, K.J., J. Med. Chem., 15:1177-1179 (1972); Radzikowski, C. , et al. Arch. Immunol. Ther. Exp., 19:219-228 (1971); Reisch, J. and S.M.E. Aly, Arch. Pharm., 319:25-28 (1986); Schneider, J., et al. J. Med. Chem. 15:266-270 (1972); Svoboda, G.H. Lloydia 29:206-224 (1966)). Of those derivatives synthesized, only 0-(dimethylaminoethyl)noracronycine (2) (Reisch, J. and S.M.E. Aly, Arch. Pharm. 319:25-28 (1986)) and 1-nitro-N¹⁰-substituted acridine-9-ones (3) (Radzikowski, C., et al., Arch. Immunol. Ther. Exp. 19:219- 228 (1971)), showed biological activity. Compound 2 , bearing a charged dimethyl-aminoethyl side-chain, was reported to exhibit significant antitumor activity (Reisch, J. and S.M.E. Aly, Arch. Pharm. 319:25-28 (1986)). It is possible, therefore, that change of the overall molecular lipophilic-hydrophilic balance and electronic distribution caused by addition of a charged side-chain or nitro function on the molecule of acridine-9-one alter biological activity.

Svoboda, G.H., U.S. Patent Nos. 4,309,431 and 3,985,899, filed January 5, 1982 and October 12, 1976, respectively, teach a method of inhibiting growth of tumor cells using acronycine. However, they do not teach or suggest that other acridone alkaloids may be used to inhibit tumor growth.

To determine structure-activity relationships, the effects of 50 acridone alkaloids on the incorporation of the labeled precursors, [³H-methyl]dThd, [5-³H]Cyd and [2,3,4,5-³H)L- Leu, into DNA, RNA and protein, respectively, were studied. The inhibition by these acridone alkaloids against human leukemic HL-60 cell growth was also studied. Summary of the Invention

The subject invention discloses a method of inhibiting growth of tumor cells which comprises contacting the cells with a growth inhibiting amount of a compound having the structure:

wherein R¹ is a hydroxyl or acetyl group; R² is hydrogen, -CH₂-CH=C(CH₃)₂ or a methoxy group; R³ is a hydroxyl, methoxy or acetyl group; R⁴ is hydrogen, - CH₂-CH=C(CH₃)₂ or a methoxy group; R⁵ is a hydroxyl, methoxy or acetyl group; R⁶ is hydrogen or a hydroxyl, methoxy or acetyl group; and R¹⁰ is a methyl group. A composition for inhibiting growth of tumor cells comprising an amount of the above-identified structure effective to inhibit growth of tumor cells and a physiologically acceptable carrier is also provided. This invention also provides a method of inhibiting growth of tumor cells which comprises contacting the cells with a growth inhibiting amount of a compound having the structure:

wherein R⁵ is hydrogen or -CH₂-CH=C(CH₃)₂; R⁶ is a hydroxyl group; R¹⁰ is hydrogen or a hydroxyl group; R¹¹ is a hydroxyl or methoxy group; and R¹² is hydrogen or a methyl group.

A composition for inhibiting growth of tumor cells comprising an amount of the above-identified structure effective to inhibit growth of tumor cells and a physiologically acceptable carrier is also provided.

The subject invention also discloses a method of inhibiting growth of tumor cells which comprises contacting the cells with a growth inhibiting amount of a compound having the structure:

Further provided is a composition comprising an amount of the above-identified compound effective to inhibit growth of tumor cells and a physiologically acceptable carrier.

The subject invention additionally discloses a method of inhibiting growth of tumor cells which comprises contacting the cells with a growth inhibiting amount of a compound having the structure:

Further provided is a composition comprising an amount of the above-identified compound effective to inhibit growth of tumor cells and a physiologically acceptable carrier. Detailed Description of the Invention

wherein R¹ is a hydroxyl or acetyl group; R² is hydrogen, -CH₂-CH=C(CH₃)₂ or a methoxy group; R³ is a hydroxyl, methoxy or acetyl group; R⁴ is hydrogen, -CH₂-CH=C(CH₃)₂ or a methoxy group; R⁵ is a hydroxyl, methoxy or acetyl group; R⁶ is hydrogen or a hydroxyl, methoxy or acetyl group; and R¹⁰ is a methyl group.

All methods disclosed in this application may be effected in vitro or in vivo. When these methods are performed in vivo, the administration of the compound may be effected by any of the well known methods, including but not limited to oral, intravenous, intramuscular, and subcutaneous. The method of delivery, the amount to be used, and the frequency of delivery are expected to vary according to the situations, the carrier used, and result desired. However, those variables are readily determinable by one skilled in the art.

In one compound of the preferred embodiment, R¹ is an acetyl group; R² is hydrogen; R³ is a methoxy group; R⁴ is -CH₂- CH=C(CH₃)₂; R⁵ is an acetyl group; and R⁶ is hydrogen. This compound is O,O-diacetyl-glycocitrine-I (14).

O,O-Diacetyl- (14)

glycocitrine-I

In the first subgroup of the preferred embodiment, R¹ is a hydroxyl group and R⁵ is a hydroxyl or a methoxy group.

In one preferred compound of the first subgroup, R² is hydrogen; R³ is a methoxy group; R⁴ is a methoxy group; R⁵ is a hydroxyl group; and R⁶ is hydrogen. This compound is citrusinine-I (16)

Citrusinine-I (16)

In another preferred compound of this subgroup, R² is -CH₂- CH=C(CH₃)₂; R³ is a hydroxyl group; R⁴ is hydrogen; R⁵ is a methoxy group; and R⁶ is a hydroxyl group. This compound is buntanine (25).

Buntanine (25) The first subgroup may be further divided to a second subgroup wherein R⁴ is -CH₂-CH=C(CH₃)₂ or a methoxy group.

In one preferred compound of this second subgroup, R² is -

CH₂-CH=C(CH₃)₂; R³ is a hydrroxyl group; R⁴ is -CH₂-

CH=C(CH₃)₂; R⁵ is a hydroxyl group; and R⁶ is hydrogen. This compound is N-methyl-atalaphylline (26).

N-Methyl- (26)

atalaphylline

The second subgroup may be further divided to a third subgroup wherein R² is hydrogen or a methoxy group; and R³ is a methoxy or acetyl group.

In one preferred compound of this subgroup, R², R³, R⁴ and R⁵ are methoxy groups; and R⁶ is a hydroxyl group. This compound is glyfoline (31):

Glyfoline (31)

The third subgroup may be further divided to a fourth subgroup wherein R² is hydrogen.

In one preferred compound of this subgroup, R³ is a methoxy group; R⁴ is -CH₂-CH=C(CH₃)₂; R⁵ is a hydroxyl group; and R⁶ is hydrogen. This compound is glycocitrine-I (13).

Glycocitrine-I (13)

The fourth subgroup maybe further divided to a fifth subgroup, wherein R⁵ is a methoxy group.

In one preferred compound of this subgroup, R³ is methoxy group; R⁴ is a methoxy group; and R⁶ is hydrogen. This compound is 5-O-methyl-citrusinine-I (17):

5-O-Methyl- (17)

citrusinine-I The fifth subgroup maybe further divided to a sixth subgroup, wherein R⁴ is -CH₂-CH=C(CH₃)₂; and R⁶ is a hydroxyl, methoxy or acetyl group.

In one preferred compound of this subgroup, R³ and R⁶ are methoxy groups. This compound is grandisinine (29):

Grandisiriine ( 29 )

The sixth subgroup maybe further divided to a seventh subgroup, wherein R⁶ is a methoxy or acetyl group.

In one preferred compound of this subgroup, R³ is an acetyl group and R⁴ is a methoxy group. This compound is 3,6-0,0- diacetyl-prenylcitpressine (28) :

3/6-0/0-Diacetyl- (28)

prenyleitpressine

In another preferred compound of this subgroup, R³ and R⁴ are methoxy groups. This compound is balyumine-B (30):

Balyumine-B (30) The subject invention also provides a composition for inhibiting growth of tumor cells which comprises an amount of the compound having the structure:

wherein R¹ is a hydroxyl or acetyl group; R² is hydrogen, -CH₂-CH=C(CH₃)₂ or a methoxy group; R³ is a hydroxyl, methoxy or acetyl group; R⁴ is hydrogen, - CH₂-CH=C(CH₃)₂ or a methoxy group; R⁵ is a hydroxyl, methoxy or acetyl group; R⁶ is hydrogen or a hydroxyl, methoxy or acetyl group; and R¹⁰ is a methyl group; effective to inhibit growth of tumor cells and physiologically acceptable carrier.

This invention also provides for the above-indicated compositions wherein the compound comprises hydrophilic substituents to increase solubility. Hydrophilic molecules which may be used as substituents are well known in the art. However, compositions comprising the disclosed compounds with hydrophilic substituents are previously unknown. In one compound of the preferred embodiment, R¹ is an acetyl group; R⁵ is hydrogen; R³ is a methoxy group; R⁴ is -CH₂- CH=C(CH₃)₂; R⁵ is an acetyl group; and R⁶ is hydrogen. This compound is O,O-diacetyl-glycocitrine-I (14):

O,O-Diacetyl- (14)

glycocitrine-I

In one preferred compound of the first subgroup, R² is hydrogen; R³ is a methoxy group; R⁴ is a methoxy group; R⁵ is a hydroxyl group; and R⁶ is hydrogen. This compound is citrusinine-I (16):

Citrusinine-I (16)

In another preferred compound of this subgroup, R² is -CH₂- CH=C(CH₃)₂; R³ is a hydroxyl group; R⁴ is hydrogen; R⁵ is a methoxy group; and R⁶ is a hydroxyl group. This compound is buntanine (25) :

Buntanine (25)

The first subgroup may be further divided to a second subgroup, wherein R⁴ is -CH₂-CH=C(CH₃)₂ or a methoxy group.

In one preferred compound of this second subgroup, R² is - CH₂-CH=C(CH₃)₂; R³ is a hydroxyl group; R⁴ is -CH₂- CH=C(CH₃)₂; R⁵ is a hydroxyl group; and R⁶ is a hydrogen. This compound is N-methyl-atalaphylline (26) :

N-Methyl- (26)

atalaphylline

The second subgroup maybe further divided to a third subgroup, wherein R² is hydrogen or a methoxy group; and R³ is a methoxy or acetyl group.

The third subgroup maybe further divided to a fourth subgroup wherein R² is hydrogen.

In one preferred compound of this subgroup, R³ is a methoxy group; R⁴ is -CH₂-CH=C(CH₃)₂; R⁵ is a hydroxyl group; and R⁶ is hydrogen. This compound is glycocitrine-I (13):

Glycocitrine-I (13) The fourth subgroup maybe further divided to a fifth subgroup, wherein R⁵ is a methoxy group.

In one preferred compound of this subgroup, R³ is a methoxy group; R⁴ is a methoxy group; and R⁵ is hydrogen. This compound is 5-0-methyl-citrusinine-I (17):

5-O-Methyl- (17)

citrusinine-I

The fifth subgroup maybe further divided to a sixth subgroup, wherein R⁴ is -CH₂-CH=C(CH₃)₂; and R⁶ is a hydroxyl, methoxy or acetyl group.

Grandisinine (29)

In one preferred compound of this subgroup, R³ is an acetyl group and R⁴ is a methoxy group. This compound is 3,6-O,O- diacetyl-prenylcitpressine (28):

3,6-O,O-Diacetyl- (28)

prenylcitpressine

Balyumine-B (30)

Physiologically acceptable carriers, as discussed throughout the application, are to include any carrier compatible with life. The choice of carrier is readily determinable to one skilled in the art. The physiologically acceptable carrier encompasses any of the standard carriers such as sterile solution, tablets, coated tablets and capsules. Typically such carriers contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such carriers are formulated by well known conventional methods. However a composition comprising the compound of the subject invention effective to inhibit growth of tumor cells is previously unknown.

Any of the above-described compositions wherein the composition comprises an agent to increase solubility represent the preferred embodiment of the invention. Agents to increase solubility, as used throughout the application, are to include, but are not limited to, compounds which react with the hydrophobic regions of the subject compounds. Some examples of suitable agents include Emulphor (a polyoxylethylated fatty acid which is water miscible and non-toxic when diluted 1:10 with either sterile water or sterile physiological saline solution) and polyvinylpyrrolidine. Both Emulphor and polyvinylpyrrolidine have been disclosed for use in administering acronycine by Svoboda, G.H. in U.S. patent nos. 3,985,899, filed October 12, 1976, and 4,309,431, filed January 5, 1982. However, Svoboda does not teach or suggest the use of Emulphor or polyvinylpyrrolidine in the methods or compositions of the subject invention. This invention also provides a method of inhibiting growth of tumor cells which comprises contacting the cells with a growth inhibiting amount of a compound having the structure:

wherein R⁵ is hydrogen or -CH₂-CH=C(CH₃)₂; R⁶ is a hydroxyl group; R¹⁰ is a hydrogen or a hydroxyl group;

R¹¹ is a hydroxyl or methoxy group; and R¹² is hydrogen or methyl group.

In a preferred compound of this embodiment, R⁵ is hydrogen;

R¹⁰ is a hydroxyl group; R¹¹ is a methoxy group; and R¹² is a methyl group. This compound is citracridone-I (42):

Citracridone-I (42)

The preferred embodiment may be divided to a first subgroup, wherein R¹⁰ is a hydrogen; and R¹¹ is a hydroxyl group.

In a preferred compound of the first subgroup, R⁵ is -CH₂- CH=C(CH₃)₂ and R¹² is hydrogen. This compound is atalaphyllinine (40):

Atalaphyllinine (40)

The first subgroup may be divided to a second subgroup, wherein R⁵ is hydrogen.

In one preferred compound of the second subgroup, R¹² is a methyl group. This compound is 11-hydroxy-noracronycine (37).

11-Hydroxy-nor- (37)

acronycine

In another preferred compound of the second subgroup, R¹² is hydrogen. This compound is atalaphyllidine (34).

Atalaphyllidine (34)

The subjection invention further discloses a composition comprising an amount of a compound having the structure:

R¹¹ is a hydroxyl or methoxy group; and R¹² is a hydrogen or methyl group; effective to inhibit growth of tumor cells and a physiologically acceptable carrier.

This invention also provides for the above-indicated compositions wherein the compound comprises hydrophilic substituents to increase solubility. Hydrophilic molecules which may be used as substituents are well known in the art. However, compsitions comprising the disclosed compounds with hydrophilic substituents are previously unknown.

In the preferred embodiment, R⁶ is a hydroxyl group; R¹⁰ is hydrogen or a hydroxyl group; and R¹¹ is a hydroxyl or methoxy group.

In a preferred compoind of this embodiment, R⁵ is hydrogen; R10 is a hydroxyl group; R¹¹ is a methoxy group; and R¹² is a methyl group. This compound is citracridone-I (42 ) :

The preferred embodiment may be divided to a first subgroup, wherein R¹⁰ is a hydrogen; and R¹¹ is a hydroxyl group. In a preferred compound of the first subgroup, R⁵ is -CH₂- CH=C(CH₃)₂ and R¹² is hydrogen. This compound is atalaphyllinine (40):

In one preferred compound of the second subgroup, R¹² is a methyl group. This compound is 11-hydroxy-noracronycine

(37):

11-Hydroxy-nor- (37)

acronycine

In another preferred compound of the second subgroup, R¹² is hydrogen. This compound is atalaphyllidine (34):

Atalaphyllidine (34)

In any of the above-defined compositions, wherein the composition comprises an agent to increase solubility, it is to be considered a preferred embodiment.

Pyranofoline (48)

Further provided is a composition comprising an amount a compound having the structure:

Pyranofoline (48)

effective to inhibit growth of tumor cells and a physiologically acceptable carrier.

This invention also provides for the above-indicated compositions wherein the compound comprises hydrophilic substituents to increase solubility. Hydrophilic molecules which may be used as substituents are well known in the art. However, compositions comprising the disclosed compounds with hydrophilic substituents are previously unknown.

In the preferred embodiment, the composition further comprises an agent to increase solubility. Additionally, this invention discloses a method of inhibiting growth of tumor cells which comprises contacting the cells with a growth inhibiting amount of a compound having the structure:

Cycloglyco- (50)

foline

Also provided is a composition comprising an amount a compound having the structure:

Cycloglyco- (50)

foline

effective to inhibit growth of tumor cells and a physiologically acceptable carrier. This invention also provides for the above-indicated compositions wherein the compound comprises hydrophilic substituents to increase solubility. Hydrophilic molecules which may be used as substituents are well known in the art. However, compositions comprising the disclosed compounds with hydrophilic substituents are previously unknown. Lastly, the subject invention provides the above composition wherein the composition further comprises an agent to increase solubility. The following Experimental Detail section and Examples are set forth to aid in an understanding of the invention. These sections are not intended to, and should not be construed to, limit in any way the invention as set forth the claims which follow thereafter.

Experimental Detail

All acridone alkaloids were either previously isolated from plants indigenous to Taiwan or were obtained by partial synthesis. The isolation and structure elucidation data for each compound are referenced in Tables 1-3.

The structures of compounds 4-10 have been disclosed by Wu, T.-S., Yakugaku Zasshi, 103:1103-1107 (1983); compounds 1, 11-14, 31, 33-35, 38, 39 and 48-51 have been disclosed by Wu, T.-S., J. Chem. Soc. Perkin Trans. I., 1681-1688 (1982); compounds 15-17 and 27 have been disclosed by Wu, T.-S. and H. Furukawa, Chem. Pharm. Bull., 31:901-906 (1983); compounds 19-21, 28, and 29 have been disclosed by Wu, T.- S., C.-S. Kuoh and H. Furukawa, Phytochemistry, 22:1493-1497 (1983); compounds 22, 23, 39 and 42-45 have been disclosed by Wu, T.-S., C.-S. Kuoh and H. Furukawa, Chem. Pharm. Bull, 31:895-900 (1983); compound 23. has been disclosed by Wu, T.- S., Phytochemistry; 27, in press. (1988); compounds 26, 32. 36, 41, 42, 46 and 42 have been disclosed by Wu, T.-S. Kuoh and H. -Furakawa, Phytochemistry, 21:1771-1773 (1982); compound 23. has been disclosed by Wu, T.-S., Phytochemistry, 26:871-872 (1987); and compound 52 has been disclosed by Furukawa, H., T.-S. Wu, C.-S. Kuoh, T. Sato, Y. Nagai and K. Kagei, Chem. Pharm. Bull., 32:1647-1649 (1984).

For the precursor incorporation studies, each compound (in 0.3% dimethylsulfoxide) was preincubated with HL-60 cells (a human promyelocytic leukemic cell line) 2.2 x 10⁶/ml, for 15 min prior to the addition of the labeled precursor (obtained from ICN Radiochemicals, Irvine, CA) and then incubated for 30 min. The precursors used for incorporation into DNA, RNA and protein were [³H-methyl]dThd (1μCi, 0.15 nmole/ml), [5- ³H]Cyd (5 μCi, 0.23 nmol/ml), and [2,3,4,5-³H]L-Leu (0.5 μCi, 0.004 nmol/ml), respectively. Incorporation of radioactivity in the absence of a plant compound and in the presence of dimethylsulfoxide was used as a control. The control values for incorpation into DNA, RNA and protein were 8,500, 5,600 and 1,700 cpm/10⁶ cells, respectively. The incubation conditions and the procedures for isolating DNA, RNA and protein fractions have been previously described (Chou, T.-C., et al., Cancer Res., 43:3074-3079 (1983)).

For growth inhibition studies, HL-60 cells (1.5 x 10⁵/ml) were grown in RPMI 1640 media (GIBCO, Grand Island, NY) at 37°C in humidified 5% CO₂ for 72 h. Viable cells were determined using trypan blue exclusion and counted in a hemocytometer. The fractional inhibition for each compound concentration (0.0025-0.05 mg/ml in 0.1% dimethylsulfoxide) was analyzed with the median-effect plot using a computer program (Chou, T.-C and P. Talalay, Adv. Enzyme Regul., 22:27-55 (1984); Chou, J. and T.-C. Chou, Dose-Effect analysis with microcomputers: Quantitation of ED50, LD50, synergism, antagonism, low-dose risk, receptor ligand binding and enzyme kinetics. A computer software disk for Apple II series or IBM-PC and manual. Elsevier Science Publishers, Elsevier-BioSoft, Cambridge, United Kingdom (1986)). The median-effect concentration (IC₅₀) was determined automatically for three to five dose-effect levels. Cell growth in the absence of plant compound and in the presence of dimethylsulfoxide was used as a control. Dimethylsulfoxide alone inhibited cell growth 3.9% ± 1.5% during the 72 h incubation period. Those data with negative values represent activations.

Results and Discussion

Effects of Acridine Alkaloids on Precursor Incorporation into Cellular DNA

Human promyelocytic cells (HL-60) were incubated with [³H- methyl]dThd in the presence and absence of a plant alkaloid. The percentage of inhibition of precursor incorpation in to DNA was tested with compound concentration of 0.03 mg/ml. Acridin-9-one derivatives (Table 1), which bear 1,3-di (4 and 5) and 1,3,10-tri-sustituents (6-10) showed no significant activity on the inhibition of DNA synthesis with the exception of compound 10 which exhibited excellent inhibitory effect by inhibiting over 90% of DNA synthesis. Glycocitrine-II (11) is a potent inhibitor of cell DNA synthesis, while its 3-methyl derivative (12) shows only moderate inhibitory activity.

Compounds with penta-substituents split into two categories; those with substitution at position 4 (13-18) display excellent activity, whereas others possessing a substituent at position 6 (19-24) show relatively weaker activity. The only exception is compound 24 which inhibits 93% of DNA synthesis. In general, polysubstituted acridine-9-ones (25- 31) inhibit DNA synthesis.

In the series of acronycine derivatives (Table 2), 6- hydroxypyrano[2,3-c]acridine-7-one (32 ) did not show good activity in this study, while the inhibitory activity of its O-methylated product (33) was markedly increased. Acronycine (1) was found to inhibit about 90% of prelabeled precursor incorpation into DNA, while poor activity for its disubstituted analogs (34-36) was observed. With the exception of compound 41 which has no substituent at C-11, trisubstituted acronycine derivatives (37-40) showed potent inhibitory effects on DNA synthesis.

In comparing atalaphyllidine (34) with atalaphyllinine (40), the latter showed greater activity. Thus, addition of a 3- methyl-2-butenyl side-chain at the C-5 position of 34 results in increased activity. However, addition of this side-chain at the same position in noracronycine (25) had no effect on the inhibitory activity of 41. Compounds with four substituents at 6, 10, 11 and 12 (42-45). and 5, 6, 11 and 12 (46) also exhibited significant activity. It should also be noted here that 1,2-dihydro derivatives 38 and 41 were shown to have the same potency as their parent compounds, 37 and 42, respectively.

Both derivatives of pyrano[3,2-b]acridine-6-one (48 and 49) and their analog, cycloglycofoline (50) inhibited DNA synthesis significantly, whereas furofoline derivative 51 and acridine dimer (52) were not active (Table 3).

Effect of Acridine Alkaloids on the Labeled Precursor Incorporation into RNA and Protein HL-60 cells were incubated with [5-³H]Cyd or [2,3,4,5-³H]L- Leu in the presence and absence of alkaloids (0.03 mg/ml) in order to find the effects of the alkaloid on the incorporation of labeled precursors into RNA or protein. Most acridine alkaloids were found to have either a moderate or low inhibitory effect on RNA and protein biosynthesis. Over 90% of RNA and protein biosynthesis were inhibited by compounds 11, 16, 12, 22, 28, 29 and 30 (Table 1). The same results were obtained from some derivatives of acronycine (Table 2) and pyranofoline derivative (50), Table 3). In most cases, the amount required to inhibit precursor incorporation into DNA was less than that to inhibit precursor incorporation into RNA or protein (i.e. DNA synthesis is inhibited most potently). Effect of Acridine Alkaloids on the Growth of Leukemic HL-60 Cells

The plant alkaloids were further examined for their effects on cell growth inhibition (Tables 1-3).

Compounds listed in Table 1 (acridin-9-one derivatives) with substituents at 1, 3 and 10 (4-9) and 1, 3, 5, 6 and 10 (19- 24), in general, did not inhibit cell growth (with the exception of compound 10). Most compounds bearing substituents at the 1, 3, 4, 5 and 10 positions (13-17, 26- 31) showed significant inhibition. The most potent compound in this series was glyfoline (31) with an IC₅₀ of 0.0004 mg/ml (1.11 μM). Cell growth inhibition by pyrano[2,3-c]acridine-7-one derivatives was compared to acronycine (1) (Chou, T.-C., et al., Cancer Res.43:3074-3079 (1983)) (Table 2). The simple 6-hydroxyl substituted derivatives (32 , 35 and 44) only showed moderate cell growth inhibitory activity but their O- methylated counterparts (33, 1 and 45, respectively) were found to be significantly active. Atalaphyllidine (34) and its substituted 2-(3-methyl-2-butenyl) or tf-methyl derivatives (40) and 37) were shown to have comparable activity. Severifoline (36) was reported to have no antitumor activity, but the antitumor activity was induced significantly by introduction of an OH group at position 11 (40). A similar trend was observed on several pairs of compounds (32 vs. 34; 35 vs. 37; 41 vs. 46). The 1,2- dihydro derivatives (38 and 43) were found to be only half as potent as their parent compounds (37 and 42, respectively). A linear isomer of acronycine, pyranofoline (48) (Table 3) and its analog cycloglycofoline exhibited better activity than that of acronycine. It is also interesting to note that, in general, there is a correlation between the potency of inhibiting precursor incorporation into DNA and the potency of inhibiting cell growth among these analogs. The correlation coefficient (r) for percent inhibition of DNA synthesis (x) vs log (IC₅₀ in mg/ml) for HL-60 cell growth (y) was r=0.54, whereas similar plots for RNA synthesis gave r=0.18 and for protein synthesis, r=0.43, (n=35). It appears that DNA synthesis is the major target for this group of compounds.

In summary, acronycine (1) has been shown to have a broad spectrum of antitumor activity against experimental neoplasms in laboratory animals (Table 4). In an attempt to search for more potent compounds and to determine structure- activity relationships, 50 acronycine-related acridine alkaloids were examined for their effects on inhibition of precursor incorpation into DNA, RNA and protein as well as their potency on inhibition of leukemic HL-60 cell growth.

In general, derivatives of acronycine (pyrano[2,3-c]acridin- 7-one) (Table 2) exhibited the most potent inhibition on the growth of leukemic HL-60 cells. It has also been found that acridin-9-one derivatives (Table 1) with sustituents at the 1, 3, 4 and 10 positions (13, 14, 16, 17, 26, 28, 29 and 31) had significant activity against leukemic cells in vitro. There are structural similarities between these compounds and acronycine; the latter is considered as a 1, 3, 4 and 10 tetra-substituted acridin-9-one derivative. Accordingly, substituents at the above four positions of the acridin-9- one ring system may be essential for optimum biological activity. The above structure-activity relationships may provide useful directions for future synthetic approaches to developing new antitumor chemotherapeutic agents.

Summary of In Vivo Preliminary Studies on Acridones

(Tables 5-7)

A. L1210 Leukemia (Natural products used)

Compound 31 (SK 32889, TSA-35, glyfoline) showed some activity against L1210 leukemia in BDF mice at a low dose (12.5 mg/kg, starting day 1 for 4 days, i.p. injection)

Compound 34 (TSA-36) also showed evidence of antileukemic activity.

Compound 37 (TSA-7) and compound 17 (TSA-15) were not active. B. Lewis Lung Carcinoma (Synthetic compounds used) Compound 31 at 25 and 50 mg/kg daily for 4 days i.p. injection to C57BL/6 mice showed less toxicity to mice than that of compound 1 (acronycine, TSA-33, SK 32121). At 25 and 50 mg/kg, compound 31 increased life-span by 42% and >92%, respectively, whereas compound 1 increased life-span by 40% and 40%, respectively.

There were 2 out of 5 long-term survivals for 50 mg/kg for compound 31. Other groups had no long term survivals.

C. Carcinoma E07713 (Synthetic compounds used)

At 100 mg/kg daily for 4 days i.p. compound 1 showed 1 out of 3 toxicity induced deaths but no deaths were observed as a consequence of administration of compound 31.

At 50 mg/kg and 100 mg/kg, compound 31 increased life- span by 17% and >39%, respectively, whereas compound 1 increased life-span by 26% and 4% (although toxic), respectively.

4> en

Claims

What is claimed is:

1. A method of inhibiting growth of tumor cells which comprises contacting the cells with a growth inhibiting amount of a compound having the structure:

2. A method of claim 1, wherein R¹ is an acetyl group; R² is hydrogen; R³ is a methoxy group; R⁴ is -CH₂- CH=C(CH₃)₂; R⁵ is an acetyl group; and R⁶ is hydrogen.

3. A method of claim 1, wherein R¹ is a hydroxyl group and R⁵ is a hydroxyl or a methoxy group.

4. A method of claim 3, wherein R² is hydrogen; R³ is a methoxy group; R⁴ is a methoxy group; R⁵ is a hydroxyl group; and R⁶ is hydrogen.

5. A method of claim 3, wherein R² is -CH₂-CH=C(CH₃)₂; R³ is a hydroxyl group; R⁴ is hydrogen; R⁵ is a methoxy group; and R⁶ is a hydroxyl group.

6. A method of claim 3, wherein R⁴ is -CH₂-CH=C(CH₃)₂ or a methoxy group.

7. A method of claim 6, wherein R² is -CH₂-CH=C(CH₃)₂; R³ is a hydroxyl group; R⁴ is -CH₂-CH=C(CH₃)₂; R⁵ is a hydroxyl group; and R⁶ is hydrogen.

8. A method of claim 6, wherein R² is hydrogen or a methoxy group; and R³ is a methoxy or acetyl group.

9. A method of claim 8, wherein R², R³, R⁴ and R⁵ are methoxy groups; and R⁶ is a hydroxyl group.

10. A method of claim 8, wherein R² is hydrogen.

11. A method of claim 10, wherein R³ is a methoxy group; R⁴ is -CH₂-CH=C(CH₃)₂; R⁵ is a hydroxyl group; and R⁶ is hydrogen.

12. A method of claim 10, wherein R⁵ is a methoxy group.

13. A method of claim 12, wherein R³ is a methoxy group; R⁴ is a methoxy group; and R⁶ is hydrogen.

14. A method of claim 12, wherein R⁴ is -CH₂-CH=C(CH₃)₂; and R⁶ and is a hydroxyl, methoxy or acetyl group.

15. A method of claim 14, wherein R³ and R⁶ are methoxy groups.

16. A method of claim 14, wherein R⁶ is a methoxy or acetyl group.

17. A method of claim 16, wherein R³ is an acetyl group and R⁴ is a methoxy group.

18. A method of claim 16, wherein R³ and R⁴ are methoxy groups.

19. A composition for inhibiting growth of tumor cells which comprises an amount of the compound having the structure:

wherein R¹ is a hydroxyl or acetyl group; R² is hydrogen, -CH₂-CH=C(CH₃)₂ or a methoxy group; R³ is a hydroxyl, methoxy or acetyl group; R⁴ is hydrogen, -CH₂-CH=C(CH₃)₂ or a methoxy group; R⁵ is a hydroxyl, methoxy or acetyl group; R⁶ is hydrogen or a hydroxyl, methoxy or acetyl group; and R¹⁰ is a methyl group; effective to inhibit growth of tumor cells and a physiologically acceptable carrier.

20. A composition of claim 19, wherein R¹ is an acetyl group; R² is hydrogen; R³ is a methoxy group; R⁴ is - CH₂-CH=C(CH₃)₂; R⁵ is an acetyl group; and R⁶ is hydrogen.

21. A composition of claim 19, wherein R¹ is a hydroxyl group and R⁵ is a hydroxyl or a methoxy group.

22. A composition of claim 21, wherein R² is hydrogen; R³ is a methoxy grqup; R⁴ is a methoxy group; R⁵ is a hydroxyl group; and R⁶ is hydrogen.

23. A composition of claim 21, wherein R² is -CH₂- CH=C(CH₃)₂; R³ is a hydroxyl group; R⁴ is hydrogen; R⁵ is a methoxy group; and R⁶ is a hydroxyl group.

24. A composition of claim 21, wherein R⁴ is -CH₂- CH=C(CH₃)₂ or a methoxy group.

25. A composition of claim 24, wherein R² is -CH₂- CH=C(CH₃)₂; R³ is a hydroxyl group; R⁴ is -CH₂- CH=C(CH₃)₂; R⁵ is a hydroxyl group; and R⁶ is hydrogen.

26. A composition of claim 24, wherein R² is hydrogen or a methoxy group; and R³ is a methoxy or acetyl group.

27. A composition of claim 26, wherein R², R³, R⁴ and R⁵ are methoxy groups; and R⁶ is a hydroxyl group.

28. A composition of claim 26, wherein R² is hydrogen.

29. A composition of claim 28, wherein R³ is a methoxy group; R⁴ is -CH₂-CH=C(CH₃)₂; R⁵ is a hydroxyl group; and R⁶ is hydrogen.

30. A composition of claim 28, wherein R⁵ is a methoxy group.

31. A composition of claim 30, wherein R³ is a methoxy group; R⁴ is a methoxy group; and R⁶ is hydrogen.

32. A composition of claim 30, wherein R⁴ is -CH₂- CH-C(CH₃)₂; and R⁶ is a hydroxyl, methoxy or acetyl group.

33. A composition of claim 32, wherein R³ and R⁶ are methoxy groups.

34. A composition of claim 32, wherein R⁶ is a methoxy or acetyl group.

35. A composition of claim 34, wherein R³ is an acetyl group and R⁴ is a methoxy group.

36. A composition of claim 34, wherein R³ and R⁴ are methoxy groups.

37. A composition of claim 19, wherein the compound comprises hydrophilic substituents to increase solubility.

38. A method of inhibiting growth of tumor cells which comprises contacting the cells with a growth inhibiting amount of a compound having the structure:

wherein R⁵ is hydrogen or -CH₂-CH=C(CH₃)₂; R⁶ is a hydroxyl group; R¹⁰ is hydrogen or a hydroxyl group; R¹¹ is a hydroxyl or methoxy group; and R¹² is hydrogen or a methyl group;

39. A method of claim 38, wherein R⁵ is hydrogen; R¹⁰ is a hydroxyl group; R¹¹ is a methoxy group; and R¹² is a methyl group.

40. A method of claim 38, wherein R¹⁰ is a hydrogen; and R¹¹ is a hydroxyl group.

41. A method of claim 40, wherein R⁵ is -CH₂-CH=C(CH₃)₂ and R¹² is hydrogen.

42. A method of claim 40, wherein R⁵ is hydrogen.

43. A method of claim 42, wherein R¹² is a methyl group.

44. A method of claim 42, wherein R¹² is hydrogen.

45. A composition comprising an amount of a compound having the structure:

wherein R⁵ is hydrogen or -CH₂-CH=C(CH₃)₂; R⁶ is a hydroxyl group; R¹⁰ is hydrogen or a hydroxyl group; R¹¹ is a hydroxyl or methoxy group; and R¹² is hydrogen or a methyl group; effective to inhibit growth of tumor cells and a physiologically acceptable carrier.

46. A composition of claim 45, wherein R⁵ is hydrogen; R¹⁰ is a hydroxyl group; R¹¹ is a methoxy group; and R¹² is a methyl group.

47. A composition of claim 45, wherein R¹⁰ is a hydrogen; and R¹¹ is a hydroxyl group.

48. A composition of claim 47, wherein R⁵ is -CH₂- CH=C(CH₃)₂ and R¹² is hydrogen.

49. A composition of claim 47, wherein R⁵ is hydrogen.

50. A composition of claim 49, wherein R¹² is a methyl group.

51. A composition of claim 49, wherein R¹² is hydrogen.

52. A composition of claim 45, wherein the compound comprises hydrophilic substituents to increase solubility.

53. A method of inhibiting growth of tumor cells which comprises contacting the cells with a growth inhibiting amount of a compound having the structure:

54. A composition comprising an amount of a compound having the structure:

55. A composition of claim 54, wherein the compound comprises hydrophilic substituents to increase solubility.

56. A method of inhibiting growth of tumor cells which comprises contacting the cells with a growth inhibiting amount of a compound having the structure:

57. A composition comprising an amount of a compound having the structure:

58. A composition of claim 57, wherein the compound comprises hydrophilic substituents to increase solubility.