Note: Descriptions are shown in the official language in which they were submitted.
~i2~ 3~
Field of the Invention
This invention relates to new cyclic organic
compounds which have use~ul antitumor activity. More
particularly, this invention relates to new cyclic
alkaloid antitumor compounds derived from marine
organisms, i.e., marine sponge, order Haplosclerida,
and their methods of use.
Baskqround of the Invention
Various tumor related dis~ases inflict man.
Considerabla research has been de~oted 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 inflect 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.
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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 Bioloqical PersPectives; Academic Press;
New Yorkg 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 Am. Chem. Soc. 1985, 107, 4796-4798.
Other interesting compositions derived from
marine organisms (i.e., Caribbean tunicàte) and
containing a ~-Carboline system are described in K. L.
Rinehart, Jr., 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., bid.,
!' at 1526.
It has now been found that certain cyclic
alkaloid compounds derived from extracts of the marine
sponge, genus Haliclona, possess useful antitumor
activity.
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Summary of the Invention
The invention seeks to provide novel ;
- compounds and compositions containing such compounds,
` which are useful as antitumor agents and a process
for produclng such novel antitumor compounds.
Additional advantages of the invention will
` be set forth, in part, in the description which
follows and in part will be clear from this descrip-
tion, or may be learned by the practice of the
~, 10 invention. The advantages of the invention are
realized and obtained by means of the compounds,
a compositions, processes, methods, and the combina-
tions particularly pointed out in the appended
claims.
~! 15 In accordance with the invention there is ~-
provided a compound o the general formula (I)
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, ~ .
~ ~2 X3 ~
X5
H I Rl X6
i-,, ~ .
.. , ~ . ~ ~
~ ~ I ~
~ V ~
:."'. ~
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~3~-~ 3~J~ ~
wherein X r X ~ X3, X4, X5 and x6 h
halogen, hydroxy, lower alkoxy, lower acyloxy or a
lower mono or dialkyl amino group; Rl is hydrogen,
lower alkyl, or lower acyl group; R is hydrogen,
hydroxy, lower alkoxy, or lower acyloxy group.
In other embodiments of the invention the
double bonds in the compound of formula I are par-
tially or fully reduced.
In further embodiments of the invention the
compound is a mineral or organic acid salt of a compound
according to formula I or of a compound accord-
ing to formula 1 wherein at least one double bond is
reduced. -
In preferred embodiments of the invention,
the compound is substantially pure. In further
preferred embodiments of the invention Rl, R2 xl,
X2, X3, X4, XS and x6 are a hydrogen or hydroxy.
In more preferred embodiments of the
invention, the invention comprises a compound of the
formula (II):
N
H
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,, . . ., ~s :
,:
,, . ,, , ,`- .,, ,.-
-~ . ~ , ., . ~, .
., : ,` ` -
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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 compounds according
to formulae I or II; a compound according to formula
I wherein at least one double bond is reduced; or an
acid salt of a compound according to formula I or a
'~ compound according to formula I wherein at least one
double bond is reduced and a non-toxic pharmaceuti-
cally acceptable carrier or diluent.
As embodied and fully described herein, the
invention also comprises a process to produce the
compounds of formulae I and II and their reduced or
; acid salt derivatives. The process comprises the
steps of collecting marine sponge genus ~aliclona;
contacting the sponge with at least one suitable
organic solvent to obtain an extract comprising a
compound according to formulae I or II or their
reduced or acid salt derivatives; and isolating a
~; 20 compound 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 treat- ~ -
- 30 ing cancerous cachexia comprising contacting a tumor
with an effective antitumor amount of one or more
compounds of formulae I or II or their reduced or
- acid salt derivatives.
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It is to be understood that both the
foregoing general and the following detailed descrip-
~ tion are exemplary and explanatory only and are not
.~ intended to be restrictive of the invention as ~
5 claimed. ~-
DETAII,ED DESCRIPTION OF THE PREFERRED --~
. . .
EMBODIMENTS OF THE INVENTION
; Reference will now be made in detail to
present preferred embodiments of the invention,
~, 10 examples of which are illustrated in the following
example section.
In accordance with the invention novel
!
compounds are provlded in accordance with the pur-
poses of the invention, as embodied and fully des-
- 15 cribed herein, the invention comprises compounds of
~ the general formula (I):
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. ' ~ ' X 2 X
H ~ X'
N~SR2
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` wherein X , X , X , X , X and X are a hydrogen,
halogen, hydroxy, lower alkoxy, lower acyloxy, or
lower mono or dialkyl amino group; R is hydrogen,
lower alkyl, or lower acyl group; R is hydrogen,
h hydroxy, lower alkoxy, or lower acyloxy group.
In other embodiments of the invention the -
double bonds in the compound of formula I are par-
tially or fully reduced.
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In further embodiments of the invention the
compound is a mineral acid (e.g. HCl, H2SO4, H3PO4,
HNO3, etc.) or organic acid salt of a compound
according to formula I or of a compound according to
;~ 5 formula I wherein at least one double bond is
reduced.
In moxe preferred embodiments of the
invention, the invention comprises compounds of the
~, formula (II):
:
.
N~ ~ ~
g' 10 ~ j~o~
In accordance with the invention, an
antitumor composi-tion is provided comprising as
active ingredient an effective antitumox amount of
~` one or more of the compounds described above and
; 15 identified by formulae I or II and their reduced or
~ acid derivatives and a non-toxic pharmaceutically
S acceptable carrier or diluent. While effective
amounts may vary, as conditions in which the anti-
j tumor compositions are used vary, a minimal dosage
required for activity is generally between 0.01 and
~ 100 micrograms against 10 tumor cells. The composi-
'i tion of
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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 or 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 antitumor amount of one or
more compounds according to formulae I or II and their
~ii
r~duced or acid derivatives. The effectiveness of the
i compounds of the invention for inhihiting 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 compounds 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
~i organic solvent to obtain an organic extract
comprising a compounds according to formula I or II or
I their reduced or acid salt derivativas; and isolating
.~ a compound according to ormulae I or II.
. A detailed description and explanation of a
preferred embodiment of the process of the invention
to produce the compounds according to formula I or II
and their reduced or acid salt derivatives is as
: follows: marine sponge genus Hallclona, is collected
. by SCUBA
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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).
~ 5 The aqueous suspension is then extracted with ethyl
~! acetate as a secon~ solve~t to obtain an extract which comprises aA ~ 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 ma~ 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; ohloroform; ether; and tetrahydrofuran.
Any suitable fractionation and isolation
techniques may be utilized in accordance with the process
~ of the invention. Suitable fractionation techniques
;~ incl~de various chromotography techniques such as, highpressure liquid chromatography (HPLC) with a suitable
column as would be known to those skilled in the art
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including silica el, Sephadex LH-~0; ammonia-treated
` ~ ~ silica gel; RP-18~ RP-8,*and LiChrosor~ NH2 column.
These columns are eluted with ~uitable eluents such as:
heptane; ethyl acetate; methylene chloxide; 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
` 10
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
~ 15 provide further understanding of the present invention and
u outline a process for producing compositions of the
invention.
The following examp~es represent preferred
'~ f'ofnpo~ 6~
embodiments of the~composltio~s~ 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.
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O Examples 1-3
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,: The antitumor cyclic alkaloids of the invention were
prepared from a marine sponge, genus Haliclona, according
~ to the following procedures.
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Preparation of Manzamine A
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o A sample (880 g wet weight) of marine sponge
genus Haliclona was collected off Manzamo, okinawa in
; waters at a depth of 30 ~eters in April 1985. The sponge
was extracted by steeping in l 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 t 2: 3 heptane-EtOAc), Sephadex LH-20 (1: 1
methylene chloride-methanol), ammonia treated silica gel
0 (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 solid.
Recrystallization from methanol gave 100 mg of pure
manzamine A hydrochloride as colorless crystals, mp~240 C
(dec.) [~]D0 + 50o (c 0.28, C~IC13) IR (KBr ) 3280,
3150, 3050, 3000, 2920, 2800, 2760, 2630, 2560, 1617,
1555, 1488, 1448, 1418, 1385, 137~, 1315, 1270, 1230,
1180, 1142, 1110, 1095, 1065, 1025, 970, 950, 930, ~90,
820, 780, 740, 725 ~ 700, 670, 650, and 623 cm H NMR
(CDCl)~11.76 (lHr brs), 10.62 (lH, brs), 8.34 (lH, d,
J=5.2 Hz), 8.08 (lH, d, J=7.9 Hz), 7085 (lH, d J=5.1 Hz),
7.83 (lH, d, J=7.9 Hz), 7.52 (lH, t, J=7.9 Hz), 7.23 (lH,
t, J=7.9 Hz), 6.25 (lH, s), 6.29 (lH, m), 5.57 (2H, m),
5.39 (lH, t, J=9.9 Hz), 4.94 (lH, brs), 4.03 (lH, brs),
;; 3.72 (lH, brd, J=6 Hz), and 3.27 (lH, m). 13C NMR
(CDC~3~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,
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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
(C36H44N4O requires 548.3515); EIMS ~/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 - Prep~ration_of Reduced Derivative
Manzamine A is easily reduced to dihydro-,
`~ 10 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
hydrogenàtion apparatus. If the reaction is too slow, it
` would be facilitated by ~aking the media slightly acidic
by addition of a trace amount of acid such as HCl. 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.
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;Example 3 ~ Preparatlon 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, H2SO4, or an organic acid such as
~ 5 oxalic acid in aqueous ethanol or methanol. As shown by
;` X-ray analysis, manzamine A monohydrochloride has the
n following structure.
:;
~ N
:~ ¦ H
~
N ~ ~ \ OH
~ H ~ 1
~1 ~
,~, \
;,,
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Manzamine A monohydrochloride
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ANTITUMOR ACTIVITIES OF THE COMPOUNDS OF THE INVENTION
The following assay method was utilized to
illustrate the antitumor effectiveness of the compositions
. of ~ormulae I and II corresponding to manzamine A ~l) of
the example.
P388 MOUSE LErJKEMIA 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
'`~ 15 - ;
l. 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 37 for 48 hours,
4. Read platees with an inverted microscope, scoring
; activity from l+ to 4+ as follows: ND (not detectable),
~90% l+, 75-90%; 2+, 50-74%; 3+, 25-49%; 4+, <25% of
control growth. Cell counts are performed on each tube
; 25 and results are reported as percent of control.
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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 ~g/ml
gentamycin. All human tumor cell lines are incubated at
5% C2 a~ 37 and subcultured once a week.
:'
, 10 PROCEDURE
.'
1. Seed lml cell ~5000 HCT-8, 8000 A549, 12000 MDAMB) in
` each well of a 24-well plate.
~ 2. Incubate in a CO2-incubator for 48 hours.
;~; 15 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), ~9o% 1+, 75-90%; 2+, 50-74% 3+, 25-49%
4+, ~25% of control growth.
~ Positive control - Vinblastine or Vincristine in
-~ aqueous solution.
~ 25 Final Conc. of Vinblastine or Vincristine control
,
~ ~use 2 ~lJassay)
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Solution Conc. Amt addedin test _
5 mg/ml 2 jul5 yg/m
1 mg/ml 2 ~ g/ml
0.1 mg/ml 2 pl0.1 ~g/ml
0.05 mg/ml 2 ~10.05 ~g/ml
The results of the above assay are summarized in
Table 1.
~' : Table 1 Antitumor As_ay Results of Manzamine A
-~ 10Manzamine 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
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; 15 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 ~g/ml against mouse leukemia
` cells.
It is apparent from the in vitro testing that the
;~; 20 compositions~of the invention, are effective for
~ inhibiting or destroying tumors and therefore controlling
rii diseases caused by or related to such tumors in hosts such
i~ 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
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o 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
i5 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.
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