WO1988000198A1

WO1988000198A1 - Antitumor alkaloids

Info

Publication number: WO1988000198A1
Application number: PCT/US1987/001566
Authority: WO
Inventors: Tatsuo Higa; Ryuichi Sakai
Original assignee: Harbor Branch Oceanographic
Priority date: 1986-06-26
Filing date: 1987-06-25
Publication date: 1988-01-14
Also published as: CA1321389C; JPH01500520A; EP0271573A1

Abstract

Antitumor alkaloid compositions, a process of producing the compositions and a method for inhibiting tumors utilizing the compositions. More particularly, the compositions are antitumor alkaloids which are derived from marine organisms, i.e., the marine sponge order genus Haliclona of general formula (I).

Description

ANTIC'TUMOR ALKALOIDS
Field of the Invention
This invention relates to new cyclic organic compounds which have useful antitumor activity. More particularly, this invention relates to new cyclic alkaloid antitumor compositions derived from marine organisms, i.e., marine sponge, order Haplosclerida, and their methods of use.

Background of the Invention
Various tumor related diseases inflict man.

Considerable research has been devoted to oncology and antitumor measures. Tumors are common in a variety of mammals and the prevention, control of the growth and regression of tumors in mammals is important to man. The term tumor refers to abnormal masses of new tissue growth which is discordant with the economy of the tissue of origin or of the host's body as a whole.

Tumors inflict mammals and man with a variety of disorders and conditions including various forms of cancer and resultant cancerous cachexia. Cancerous cachexia refers to the symptomatic discomfort that accompanies the infliction of a mammal with a tumor. These symptoms include weakened condition of the inflicted mammal as evidenced by, for example, weight loss. The seriousness of cancer is well known, e.g., cancer is second only to heart and vascular diseases as a cause of death in man.

Considerable research and resources have been devoted to oncology and antitumor measures including chemotherapy. While certain methods and chemical compositions have been developed which aid in inhibiting, remitting or controlling the growth of tumors new methods and antitumor chemical compositions are needed.

Marine organisms and particularly marine sponges are a potential source for chemically and biologically interesting molecules of great diversity. Some such molecules derived from sponges are described in Scheuer,
P.J. Ed., Marine Natural Products, Chemical and Biological
Perspectives; Academic Press; New York, 1978-1983; Vol.

I-V; Faulkner, D.J. Natural Products Reports 1984, 551-598; Uemura, D.; Takahashi, K.; Yamamoto, T.

Katayama, C; Tanaka, J.; Okumura, Y., Hirata, Y. J. me
Chem. Soc. 1985, 107, 4796-4798. The entire disclosures of these references are hereby incorporated herein by reference.

Other interesting compositions derived from marine organisms (i.e., caribbean tunicate) and containing a f -Carboline system are described in K. L. Rinehart, Jr.r J. Kobayashi, G. C. Harbour, R. G. Hughes, Jr., S. A.

Mizsak, T. A. Scahill, J. American Chemical Society, 106, 1524 (1984); J. Kobayashi, G. C. Harbour, J. Gilmore and
K. L. Rinehart, Jr., ibid., at 1526.

It has now been found that certain cylic alkaloid compositions derived from extracts of the marine sponge, genus Haliclona, possess useful antitumor activity
Summary of the Invention
It is therefore an object of the invention to provide novel compositions which are useful as antitumor agents and a process for producing such novel antitumor compositions.

Additional objects and advantages of the invention will be set forth, in part, in the description which follows and in part will be obvious from this description, or may be learned by the practice of the invention. The objects and advantages of the invention are realized and obtained by means of the compositions, processes, methods, and the combinations particularly pointed out in the appended claims.

To achieve the objects in accordance with the purposes of the invention, as embodied and fully described here, the invention comprises composition of the general formula (I)
EMI3.1
wherein X1 X2 X3 X4 X5, and X6 are hydrogen, halogen, hydroxy, lower alkoxy, lower acyloxy or a lower mono or dialkyl amino group; R1 is hydrogen, lower alkyl, or lower acyl group; R2is hydrogen, hydroxy, lower alkoxy, or lower acyloxy group.

In other embodiments of the invention the double bonds in the composition of formula i are partially or fully reduced
in further embodiments of the invention the composition is a mineral or organic acid salt of compositions according to formula I or of compositions according to formula I wherein at least one double bond is reduced.

In preferred embodiments of the invention, the composition is substantially pure In further preferred embodiments of the invention R1, R2, X1, X2 X3,
X4, X5, and x6 are a hydrogen or hydroxy.

In more preferred embodiments of the invention, the invention comprises a composition of the formula (11):
EMI4.1

As embodied and fully described herein, the invention also comprises an antitumor composition comprising, as active ingredient, an effective antitumor amount of one or more compositions according to formulae I or II; a composition according to formula I wherein at least one double bond is reduced; or an acid salt of a composition according to formula I or a composition according to formula I wherein at least one double bond is reduced and a non-toxic pharmaceutically acceptable carrier or diluent.

As embodied and fully described herein, the invention also comprises a process to produce the compositions of formulae I and II and their reduced or acid salt derivatives. The process comprises the steps of collecting marine sponge genus Haliclona; contacting the sponge with at least one suitable organic solvent to obtain an extract comprising a composition according to formula I or II or their reduced or acid salt derivatives; and isolating a composition according to formulae I or II or said derivatives from the extract.

In preferred embodiments of the invention the suitable organic solvent is selected from the group consisting of acetone, methyl ethyl ketone, ethyl acetate, methanol, ethanol, and methyl isobutyl ketone.

As embodied and fully described herein, the invention further comprises a method for inhibiting tumors in a host and a therapeutic method for treating cancerous cachexia comprising contacting a tumor with an effective anti tumor amount of one or more compositions of formulae I or II or their reduced or acid salt derivatives.

It is to be understood that both the foregoing general and the following detailed description are exemplary and explanatory only and are not intended to be restrictive of the invention as claimed0
DETAILED DESCRIPTION OF THE PREFERRED
EMBODIMENTS OF THE INVENTION
Reference will now be made in detail to present preferred embodiments of the invention, examples of which are illustrated in the following example section.

In accordance with the invention novel compositions are provided to achieve the objects in accordance with the purposes of the invention, as embodied and fully described herein, the invention comprises compositions of the general formula (I):
EMI6.1
wherein X1 x2 X3, X4, X5, and X6 are a hydrogen, halogen, hydroxy, lower alkoxy, lower acyloxy, or lower mono or dialkyl amino group; R1 is hydrogen, lower alkyl, or lower acyl group; R2 is hydrogen, hydroxy, lower alkoxy, or lower acyloxy group.

In other embodiments of the invention the double bonds in the composition of formula I are partially or fully reduced.

In further embodiments of the invention the composition is a mineral acid (e.g. HC1, H2S04, H3P04, HNO3, etc.) or organic acid salt of compositions according to formula I or of compositions according to formula I wherein at least one double bond is reduced.

In more preferred embodiments of the invention, the invention comprises compositions of the formula (II):
EMI7.1

In accordance with the invention, an antitumor composition is provided comprising as active ingredient an effective antitumor amount of one or more of the compositions described above and identified by formulae I or II and their reduced or acid derivatives and a non-toxic pharmaceutically acceptable carrier or diluent.

While effective amounts may vary, as conditions in which the antitumor compositions are used vary, a minimal dosage required for activity is generally between 0.01 and 100 micrograms against 105 tumor cells. The compositions of the invention are active for inhibiting a diverse range of tumors including, but not limited to human lung, colon and mammary tumors such as lung carcinoma A549, ileocecal adenocarcinoma HCT-8, and human breast cancer cells MDAMB. Useful examples of non-toxic pharmaceutically acceptable carriers of diluents include, but are not limited to, the following: ethanol, dimethyl sulfoxide and glycerol.

In accordance with the invention, a method for inhibiting tumors in a host is provided comprising contacting a tumor with an anti tumor amount of one or more compositions according to formulae I or II and their reduced or acid derivatives. The effectiveness of the compositions of the invention for inhibiting tumors indicates their usefulness for controlling tumors in hosts including mammals and for treating cancerous cachexia.

In accordance with the invention, a process to produce compositions according to formulae I or II and their reduced or acid salt derivatives comprises the steps of: collecting marine sponge genus Haliclona; contacting the sponge with at least one suitable organic solvent to obtain an organic extract comprising a composition according to formula I or II or their reduced or acid salt derivatives; and isolating a compound according to formulae I or II.

A detailed description and explanation of a preferred embodiment of the process of the invention to produce the compositions according to formula I or II and their reduced or acid salt derivatives is as follows: marine sponge genus Haliclona, is collected by SCUBA at a depth of 30 meters off Munzamo, Okinawa. The marine sponge is contacted with and steeped in acetone as a first solvent for about 48 hours to obtain an extract which is concentrated to yield an aqueous suspension (the water is derived from the natural water content of the sponge).

The aqueous suspension is then extracted with ethyl acetate as a second solvent to obtain an extract which comprises a composition according to formula I or II or their reduced or acid salt derivatives. The ethyl acetate extract is concentrated by evaporation to give solid organic residue. The residue is then chromatographed to yield the pure solid product.

While acetone and ethyl acetate are the presently preferred choices for the first and second extracting solvents, respectively, other suitable solvents may be substituted. A suitable solvent should be capable of extracting a compound according to any one of formulae I or II or their reduced or acid salt derivative from other components of the marine sponge. Suitable first and second solvents which may be substituted for either acetone or ethyl acetate include, but are not limited to, the following organic solvents: methyl ethyl ketone; acetone; methanol; ethanol; methyl isobutyl ketone; methylene chloride; chloroform; ether; and tetrahydrofuran.

Any suitable fractionation and isolation techniques may be utilized in accordance with the process of the invention. Suitable fractionation techniques include various chromotography techniques such as, high pressure liquid chromatography (HPLC) with a suitable column as would be known to those skilled in the art including silica gel, Sephadex LH-20; ammonia-treated silica gel; RP-18, RP-8, and LiChrosorb NH2 column.

These columns are eluted with suitable eluents such as: heptane; ethyl acetate; methylene chloride; methanol; isopropyl alcohol; and various combinations and ratios thereof as would be known to those skilled in the art
Countercurrent chromatography techniques are also useful for isolating compositions of the invention
EXAMPLES
The invention will now be illustrated by examples. The examples are not intended to be LimitIng of the scope of the present invention In conjunction with the detailed and general description above, the examples provide further understanding of the present invention and outline a process for producing compositions of the invention.

The following examples represent preferred embodiments of the compositions, processes and methods of the invention for satisfying the stated objects of the invention. The starting materials and reagents in the examples whose method of preparation are not indicated are commercially available from sources known to the art such as chemical supply houses.

Examples 1-3
The anti tumor cyclic alkaloids of the invention were prepared from a marine sponge, genus Haliclona, according to the following procedures.

Example 1
Preparation of Manzamine A
EMI11.1

A sample (880 g wet weight) of marine sponge genus Haliclona was collected off Manzamo, Okinawa in waters at a depth of 30 meters in April 1985. The sponge was extracted by steeping in 1 liter of acetone for 48 hours. After concentration the aqueous suspension was extracted with ethyl acetate (EtOAc) to give 2.3 g of
EtOAc soluble residue (solid).

A part (1.7g) of the residue was then chromatographed using in turn columns of silica gel (2:3 heptane-EtOAc), Sephadex LH-20 (1:1 methylene chloride-methanol), ammonia treated silica gel (25:35:1 heptane-EtOAc-isopropanol), LiChrosorb NH2 HPLC column (5:7:0,1 heptane-EtOAc- isopropanol), and finally silica gel treated with pyridine (2:1 methylene - chloride-EtOAc) to give 125 mg of solid0
Recrystallization from methanol gave 100 mg of pure manzamine A hydrochloride as colorless crystals, mp > 2400 C (dec.);[&alpha;]D20 + 50 (c 0.28, CHCl3);

IR (KBr) 3280, 3150, 3050, 3000, 2920, 2800, 2760, 2630, 2560, 1617, 1555r 1488, 1448, 1418, 1385, 1370, 1315, 1270, 1230, 1180, 1142, 1110, 1095, 1065, 1025, 970, 950, 930, 890, 820, 780, 740, 725, 700, 670, 650, and 623 cm1 1H NMR (CDCl)# 11.76 (1H, brs), 10.62 (lH, brs), 8.34 (1H, d,
J=5.2 Hz), 8.08 (1H, d, J=7.9 HZ), 7.85 (1H, d J=5.l Hz), 7.83 (1H, d, J=7.9 Hz), 7.52 (1H, t, J=7.9 Hz), 7.23 (lH, t, J=7.9 Hz), 6.25 (1H, s), 6.29 (1H, m), 5.57 (2H, m),

5.39 (1H, t, J=9.9 Hz), 4.94 (1H, brs), 4.03 (1H, brs), 3.72 (1H, brd, J=6 Hz), and 3.27 (1H, m). 13C NMR (CDCl3/D20)#144.0, 142.8, 141.7, 141.6, 137.9, 135.5, 133.6, 133.2, 129.7, 128.4, 127.2, 124.0, 121.5, 121.3, 119.6, 114.2, 113.2, 78.4, 71.5, 70.8, 57.5, 53.9, 53.8, 49.6, 47.5, 45.0, 41.5, 39.5, 33.9, 28.8, 26.8, 26.7, 25.3, 24.9, 24.7, and 21.1; UV (MeOH)max 213, 219, 236, 280, 290, 346, 357, mn; HRElMS m/z 548.3510 (C36H44N40 requires 548.3515); EIMS m/z 548 (4), 530 (100), 438 (19), 408 (66), 379 (26), 311 (55), 396 (27), 253 (23), 162 (46), 138 (27), and 98 (32%).

Example 2 - Preparation of Reduced Derivative
Manzamine A is easily reduced to dihydro-, tetrahydro- or hexahydromanzamine A by employing one, two, or three molar equivalents of hydrogen, respectively, in catalytic reduction. A sample of manzamine A and a small amount of catalyst such as Pd/C, Pt/C, or Raney Ni are mixed in a suitable solvent such as ethanol or methanol.

The mixture is stirred in the presence of hydrogen in a hydrogenation apparatus. If the reaction is too slow, it would be facilitated by making the media slightly acidic by addition of a trace amount of acid such as HC1. When full reduction to prepare hexahydromanzamine A is desired, the reduction is carried out under elevated pressure of hydrogen using an apparatus such as a Parr hydrogenation apparatus.

Example 3 - Preparation of Acid Salt
Since manzamine A is a basic compound, its acid salt is easily prepared by mixing manzamine A with an inorganic acid such as HCl, H2SO4t or an organic acid such as oxalic acid in aqueous ethanol or methanol. AS shown by
X-ray analysis, manzamine A monohydrochloride has the following structure.
EMI14.1

Manzamine A monohydrochloride
ANTITUMOR ACTIVITIES OF THE COMPOUNDS OF THE INVENTION
The following assay method was utilized to
illustrate the antitumor effectiveness of the compositions
of Formulae I and II corresponding to manzamine A (1) of
the example.

P388 MOUSE LEUKEMIA CELL ASSAY
Maintenance of Cell Line
P388 mouse leukemia cells are grown in Dulbecco
MEM medium with 10% horse serum, 4mM glutamine, and
20pg/ml gentamycin (Biologos, Inc.). Cells are incubated
in 10% CO2 and subcultured 2 times per week.

PROCEDURE
1. Add compound to each well of a 24-well plate or tube
and allow solvent to evaporate to dryness.

2. Add 2ml (1.2 x 105) cells to each well or tube and
mix.

3. Incubate in 10% CO2 at 37Q for 48 hours.

4. Read platees with -.n inverted microscope, scoring
activity from 1+ to 4+ as follows: ND (not detectable), 290%; 1+, 75-90%; 2+, 50-74%; 3+, 25-49%; 4+, < 25% of
control growth. Cell counts are performed on each tube
and results are reported as percent of control.

HUMAN TUMOR CELL LINE ASSAY
Maintenance of Cell Line
HCT-8 human colon tumor cells are grown in RPMl 1640 medium (GIBCO), A549 human lung carcinoma cells are cultured in Dulbecco medium (Biologos, Inc). MDAMB are human breast cancer cells. All media are supplemented with 10% fetal bovine serum and contain 50 Fg/ml gentamycin. All human tumor cell lines are incubated at 5* CO2 at 37e and subcultured once a week.

PROCEDURE 1. Seed lml cell (5000 HCT-8, 8000 A549, 12000 MDAMB) in each well of a 24-well plate.

2. Incubate in a C02-incubator for 48 hours.

3. Add compound to each well and incubate for an additional 120 hours.

4. Discard medium and stain with methylene blue (HCT-8) or crystal violet (A549 and MDAMB).

5 Compare cell density of drug-treated well with that of the control (no drug added) as follows: ND (not detectable), 2908; 1+, 75-908; 2+, 50-748; 3+, 25-49%, 4+, < 25% of control growth.

Positive control - Vinblastine or Vincristine in
aqueous solution.

Final Conch of Vinblastine or Vincristine control
(use 2 pl/assay)
Final conc.

Solution Conc. Amt added in test
5 mg/ml 2 jil 5 jug/ml
I mg/ml 2 pl 1 g/ml
0.1 mg/ml 2 pl 0.1 Sg/ml
0.05 mg/ml 2 jil 0.05 pg/ml
The results of the above assay are summarized in
Table 1.

Table 1 Antitumor Assay Results of Manzamine A
Manzamine A Concentration HCT-8 A549 MDAMB ¯¯ ¯ P388
0.5 g/ml 4+ 4+ 4+
0.1 g/ml ND ND ND
0.07 g/ml - - - IC50
Table 1 shows that Manzamine A has good antitumor activity at concentrations of at least 0.5 pg/ml against human cancer cells and 0.07 yg/ml against mouse leukemia cells.

It is apparent from the in vitro testing that the compositions of the invention, are effective for inhibiting or destroying tumors and therefore controlling diseases caused by or related to such tumors in hosts such as cancerous cachexia in fulfillment of the objects of the invention.

The scope of the present invention is not limited by the description, examples, and suggested uses herein and modifications can be made without departing from the spirit of the invention. For example, it may be noted that other solvents may be utilized derivatives of the compositions of examples 1 such as halogenated derivatives may possess antitumor activity analogous to those preferred embodiments described above. Further, the compositions described herein may have other useful applications such as, for example, analgesic
Applications. Application of the compositions of the present invention can be accomplished by any suitable therapeutic method and technique as is presently or prospectively known to those skilled in the art.

Thus, it is intended that the present invention cover the modifications and variations of this invention provided that they come within the scope of the appended claims and their equivalents.

Claims

What is claimed is:

1. A composition according to the general formula: EMI19.1 wherein X1 x2 X3, X4, X5, and X are hydrogen, halogen, hydroxy, lower alkoxy, lower acyloxy, or a lower mono or dialkyl amino group; R1 is hydrogen, lower alkyl, or a lower acyl group; and R2 is hydrogen, hydroxy, lower alkoxy, or a lower acyloxy group.

2. A composition according to claim 1 wherein at least one double bond in the composition is reduced.

3. A composition according to claim 1 wherein all double bonds in the composition are reduced.

4. A mineral or organic acid salt of a composition according to claim 1.

5. A mineral or organic acid salt of a composition according to claim 2.

6. A mineral acid salt according to claim 4 wherein the mineral acid is selected from the group consisting of HCl, H2SO4, H3PO and HNO3.

7. A mineral acid salt according to claim 5 wherein the mineral acid is selected from the group consisting of E H2-SO4, H3PO4 and HNO3.

8. A composition according to claim 1 wherein 1 R2 X1, x2 X3, X4, X5, and X are hydrogen or a hydroxy.

9. A composition according to claim 1 of the Formula EMI20.1 10. A composition according to claim 1 wherein said composition is substantially pure.

11. A composition according to claim 2 wherein said composition is substantially pure.

12. An antitumor composition comprising, as active ingredient, an effective antitumor amount of one or more of the compositions of claim 1 and a non-toxic pharmaceutically acceptable carrier or diluent.

13. An antitumor composition comprising, as active ingredient, an effective antitumor amount of one or more of the compositions of claim 2 and a non-toxic pharmaceutically acceptable carrier or diluent.

14. An antitumor composition comprising, as active ingredient, an effective antitumor amount of one or more of the compositions of claim 4 and a non-toxic pharmaceutically acceptable carrier or diluent.

15. An antitumor composition comprising, as active ingredient, an effective antitumor amount of one or more of the compositions of claim 9 and a non-toxic pharmaceutically acceptable carrier or diluent.

16. A method for inhibiting tumors in a host comprising contacting a tumor with an effective anti tumor amount of one or more compositions according to claim 1.

17. A method for inhibiting tumors in a host comprising contacting a tumor with an effective anti tumor amount of one or more compositions according to claim 2.

18. A method for inhibiting tumors in a host comprising contacting a tumor with an effective anti tumor amount of one or more compositions according to claim 5 19. A method for inhibiting tumors in a host comprising contacting a tumor with an effective anti tumor amount of one or more compositions according to claim 9O 20. A method for inhibiting tumors according to claim 16 wherein said host is a mammalian host 21. A method for inhibiting tumors in a mammalian host comprising contacting a tumor with an effective antitumor amount of one or more compositions of claim 2.

22. A process to produce a composition according to claim 1 comprising the steps of: collecting marine sponge Haliclona sp.; contacting said sponge with a suitable organic solvent; obtaining an extract of the sponge and solvent mixture; and isolating a composition according to claim 1 from the extract.

23. A therapeutic method for treating cancerous cachexia caused by the presence of a tumor in a host comprising contacting cells of said tumor with an effective anti tumor amount of a composition according to claim 1.

24. A therapeutic method for treating cancerous cachexia caused by the presence of a tumor in a host comprising contacting cells of said tumor with an effective antitumor amount of a composition according to claim 2.

25. A therapeutic method for treating cancerous cachexia caused by the presence of a tumor in a host comprising contacting cells of said tumor with an effective anti tumor amount of a composition according to claim 9.