United States
Patent
6,242,483
McLaughlin ,
et al.
June 5, 2001
Selectively
cytotoxic acetogenin compounds
Abstract
The isolation of three new mono-tetrahydrofuran
ring acetogenins from the bark of Annona squamosa is described.
Each of these compounds bears a carbonyl group at the C-9 position
and two hydroxyls that flank the tetrahydrofuran ring. These
compounds exibit selective cytotoxic activity against certain
specific human tumor cells.
Inventors:
McLaughlin;
Jerry L. (West Lafayette, IN); Hopp; David Craig
(Omaha, NE)
Assignee:
Purdue
Research Foundation
(West Lafayette, IN)
Appl. No.:
423139
Filed:
December 15,
1999
PCT Filed:
May 5, 1998
PCT NO:
PCT/US98/08989
371 Date:
December 15,
1999
102(e) Date:
December 15,
1999
PCT PUB.NO.:
WO98/49895
PCT PUB. Date:
November 12,
1998
|
Current
U.S. Class: |
514/473;
549/320 |
|
Intern'l
Class: |
A01N 043/08 |
|
Field of
Search: |
514/473 549/320
|
References
Cited
[Referenced By]
|
Other
References
Chang et al, J. Nat. Prod., vol. 56, No. 10, pp. 1688-1694
(abstract), Oct. 1993.*
Alkofahi et al, Experientia, vol. 46, No. 5, pp. 539-541
(abstract), May 1990.*
Li et al, J. Nat. Prod., vol. 53, No. 1, pp. 81-67 (abstract),
Jan. 1990.*
Alkofahi et al, Experientia, vol. 44, No. 1, pp. 83-85
(abstract), Jan. 1988.*
Wu et al, J. Nat. Prod., vol. 58, No. 6, pp. 830-836
(abstract), Jun. 1995.*
Wu et al, J. Nat. Prod., vol. 58, No. 6, pp. 908-915
(abstract), Jun. 1995.*
Zeng et al, J. Nat. Prod, vol. 59, No. 11, pp. 1035-1042, Nov.
1996. |
Primary Examiner: Reamer; James H.
Attorney, Agent or Firm: Barnes & Thornburg
Goverment
Interests
GOVERNMENT RIGHTS
This invention was made with United States Government support
under Grant No. CA30909, awarded by the National Institute of
Health. The United States Government has certain rights in the
invention.
Parent Case
Text
CROSS-REFERENCES TO RELATED APPLICATIONS
This application is a U.S. national application of international
application Ser. No. PCT/US98/08989 filed May 5, 1998, which
claims priority to U.S. provisional application Ser. No.
60/045,819 filed May 5, 1997.
Claims
What is claimed is:
1. A substantially pure form of a compound selected from the group
consisting of mosinone A, mosin B, and mosin C.
2. A composition comprising an anti-tumor effective amount of a
compound selected from the group consisting of mosinone A, mosin
B, and mosin C and a pharmaceutically acceptable carrier therefor.
3. A method for treating a patient having a tumor, said method
comprising the step of administering to the patient an effective
amount of a compound selected from the group consisting of
mosinone A, mosin B, mosin C and annoreticuin-9-one.
4. The method of claim 3 wherein the patient to be treated has
pancreatic or prostate cancer.
Description
FIELD OF THE INVENTION
This invention relates to the isolation, identification, and use
of natural products. More particularly this invention is directed
to substantially pure forms of cytotoxic Annonaceous acetogenins
and the use of those compounds in preparing chemotherapeutic
compositions.
BACKGROUND AND SUMMARY OF THE INVENTION
Annonaceous acetogenins are a well established class of natural
compounds that have been isolated from plants in the Annonaceae
family. It has been reported that various members of this class of
compounds exhibit significant bioactivities. Acetogenins are
C.sub.35 -C.sub.39 compounds that typically contain two long
hydrocarbon chains, one of which connects a terminal
2,4-disubstituted-.gamma.-lactone to a variable number of
tetrahydrofuran (THF) rings. The hydrocarbon chains contain a
number of oxygenated moieties which can be hydroxyls, acetoxyls
and/or ketones. Recently, single-ring acetogenins containing
double bonds, epoxide compounds which lack THF rings and a
compound lacking both epoxides and THF rings have been reported.
These interesting newer compounds support the proposed polyketide
origin of the Annonaceous acetogenins and provide additional clues
to their biogenesis.
All acetogenins found to date contain multiple stereocenters, the
elucidation of which often presents daunting stereochemical
problems. Because of their waxy nature, the acetogenins do not
produce crystals suitable for X-ray crystallographic analysis.
Relative stereochemistries of ring junctions have typically been
determined by comparison of natural compounds with synthetic model
compounds and such methods have proven to be invaluable with the
acetogenins. Recently, the absolute stereochemistries of the
carbinol centers of acetogenins have been determined with the help
of synthetic model compounds and high field nuclear magnetic
resonance (NMR) analysis of their methoxyfluoromethylphenylacetic
acid (MPTA) esters (Mosher esters).
Most Annonaceous acetogenins are potently bioactive, but the mode
of action of these compounds was unknown until Londerhausen et al.
concluded in Pesticide Science 427-438 (1991), that they act to
inhibit complex I of mitochondrial oxidative phosphorylation with
an activity several times that of rotenone.
The present invention is directed to the isolation and
characterization of the bioactivity of novel acetogenins. More
particularly the present invention is directed to three new mono-tetrahydrofuran
(THF) ring acetogenins each of which were isolated from the bark
of Annona squamosa. These compounds each bear two hydroxyls that
flank the THF ring and a carbonyl group at the C-9 position. The
compounds were isolated using the brine shrimp lethality assay as
a guide for the bioactivity-directed fractionation. It has been
discovered that acetogenins possessing a hydroxyl flanking mono-THF
system with a carbonyl in the aliphatic chain exhibit high
selective cytotoxicity to various tumor cells and thus can be used
to prepare chemotherapy compositions for administrations to
patients having tumors.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to three novel acetogenins,
mosinone A (1), mosin B (2) and mosin C (3). Each of those three
compounds bears a single tetrahydrofuran (THF) ring and a carbonyl
at the C-9 position. The specific stereochemistry of these three
compounds is shown below, wherein "X", "Y" and "Z" designate the
stereochemistry across the THF ring. ##STR1##
wherein X is threo, Y is trans, Z is threo and C.sub.15 /C.sub.20
is R/R. ##STR2##
wherein, for mosin B: X is threo/erythro, Y is trans, Z is threo/erythro
and C.sub.15 /C.sub.20 is R/S or S/R; and
for mosin C: X is threo, Y is cis, Z is threo and C.sub.15
/C.sub.20 is R/S.
These three compounds and one previously known acetogenin,
annoreticuin-9-one (4) were isolated from the bark of A. squamosa.
The stereochemistry of annoreticuin-9-one is as follows: ##STR3##
wherein X is threo, Y is trans, Z is threo and C.sub.15 /C.sub.20
is R/R.
(2,4-cis and trans)-Mosinone A (1) is a mixture of ketolactone
compounds bearing a threo/trans/threo ring relationship and a
double bond two methylene units away from the flanking hydroxyl.
The other two new acetogenins differ in their stereochemistries
around the THF ring; mosin B (2) has a threo/trans/erythro
configuration across the ring and mosin C (3) possesses a threo/cis/threo
relative stereochemistry. Annoreticuin-9-one (4), a known
acetogenin which bears a threo/trans/threo ring configuration and
a C-9 carbonyl and is new to this species. The structures were
elucidated based on spectroscopic and chemical methods.
Each of these compounds was isolated from the bark of A. squamosa.
Approximately 7.4 kg-of dried bark was pulverized and extracted
with ethanol then further partitioned to yield 545.5 g of F005 (BST
LC.sub.50 =1.5155). From this, 500.5 g was loaded onto a silica
gel (Si gel) column and eluted with hexane and increasing
percentages of chloroform, then chloroform and increasing
percentages of methanol. Sixty fractions were collected, and
fractions 30-36 were combined (21.67 g). This material was
separated on successive open columns packed with Si gel. Repeated
HPLC of fractions active in the brine shrimp lethality test (BST)
yielded compounds 1-4. The procedure for conducting the BST test
is described in Meyer et al. Planta Med. 45, p. 31-34 (1982).
(2,4-cis- and trans)-Mosinone A (1) was isolated in a mixture as a
white waxy solid. The molecular weight of 1 was shown to be 620,
based on the MH.sup.+ peak at 621 in the CIMS. The compound was
established as having the elemental composition of C.sub.37
H.sub.64 O.sub.7 by the high resolution CIMS peak for the MH.sup.+
at m/z 621.4723 (621.4730 calcd.). Signals in the .sup.1 H NMR of
1 at .delta. 4.39 and 4.55 (Table 1), with a combined integration
for one proton, were assigned to H-4 and indicated the presence of
a (2,4-cis and trans)-mixture at the ketolactone moiety which is
common for acetogenins of this type. Resonances in the .sup.1 H
NMR of 1 at .delta. 2.65 and 3.07 (H-35) and at 2.20 (H-37)
further substantiated this assignment. In the .sup.13 C NMR of 1,
signals at .delta. 205.5 (C-36), 178.7 and 178.1 (C-1), 44.2 and
43.7 (C-2), 79.0 and 78.5 (C-4), and 23.7 (Table 2) also confirmed
that 1 is a cis/trans mixture of ketolactone isomers. The
stereochemistry at C-4 was assumed as R, based on spectral
comparisons with (2,4-cis and trans)-bullatacinone, which has
known chirality, and the fact that all 4-oxygenated acetogenins
known to date are 4-R.
TABLE 1
.sup.1 H NMR spectral data (.delta.) for
1-4.
.sup.1 H NMR (500 MHz, CDCl.sub.3,
J in Hz)
Position 1 trans 1 cis 2
3 4
1 -- -- --
-- --
2 3.02 m 3.03 m --
-- --
3a 1.99 m 1.48 m 2.40 dddd 2.40 dddd
2.40 dddd
(15.0, 8.2,
(15.0, 8.2, (15.0, 8.2,
1.5, 1.5) 1.5,
1.5) 1.5, 1.5)
4 4.55 dddd 4.39 dddd 3.87 m 3.86
m 3.86
(8.3, 8.2, (10.7, 7.4,
5.7, 3.2) 5.4, 5.4)
5a 1.48 m 1.60 m 1.48 m 1.47
m 1.47 m
5b 1.56 m 1.76 m 1.48 m 1.47
m 1.47 m
6-7 1.26 br s 1.26 br s 1.26 br s 1.26
br s 1.26 br s
8 2.40 t 2.40 t 2.40 t 2.40 t
2.40 t
(7.5).sup.a (7.5).sup.a (7.5).sup.a
(7.5).sup.a (7.0).sup.a
9 -- -- --
-- --
10 2.42 t 2.42 t 2.42 t 2.42
t 2.42 t
(7.5).sup.a (7.5).sup.a (7.5).sup.a
(7.5).sup.a (7.5).sup.a
11-13 1.26 br s 1.26 br s 1.26 br s 1.26 br s
1.26 br s
14 1.41 m 1.41 m 1.41 m 1.41
m 1.41 m
15 3.40 m 3.40 m 3.38 m.sup.b
3.42 m 3.40 m
16 3.80 m 3.80 m 3.81 m.sup.c
3.81 m 3.79 m
17 1.69 m, 1.69 m, 1.97 m, 1.94
m, 1.99 m,
1.99 m 1.99 m 1.56 m.sup.d
1.75 m 1.69 m
18 1.69 m, 1.69 m, 1.87 m, 1.94
m, 1.99 m,
1.99 m 1.99 m 1.83 m.sup.d
1.75 m 1.69 m
19 3.80 m 3.80 m 3.82 m.sup.c
3.81 m 3.79 m
20 3.41 m 3.41 m 3.87 m.sup.b
3.42 m 3.40 m
21 2.00 m 2.00 m 1.41 1.41
m 1.41 m
22 2.20 m 2.20 m 1.26 br s 1.26
br s 1.26 br s
23 5.36 m 5.36 m 1.26 br s 1.26
br s 1.26 br s
24 5.39 m 5.39 m 1.26 br s 1.26
br s 1.26 br s
25 2.04 m 2.04m 1.26 br s 1.26
br s 1.26 br s
26-30 1.26 br s 1.26 br s 1.26 br s 1.26
br s 1.26 br s
31 1.29 m 1.29 m 1.30 m 1.29 m
1.29 m
32 0.88 t 0.88 t (7.0) 0.88 t 0.88
t 0.88 t
(7.0) (7.0)
(7.0) (7.0)
33a 2.67 dd 2.61 dd 7.19 d 7.19 d
7.19 d
(18.5, 9.0) (18.5, 9.0) (1.5)
(1.5) (1.5)
33b 3.04 dd 3.11 (18.5, --
-- --
(18.5, 3.0) 3.0)
34 -- -- 5.06 dq 5.06
dq 5.06 dq
(7.0, 1.5) (6.5,
1.5) (6.5, 1.5)
35 2.20 s 2.20 s 1.44 d 1.44
d 1.44 d
(6.5)
(7.0) (7.0)
.sup.a-d Values may be interchangeable in each
column.
TABLE 2
.sup.13 C NMR spectral data (.delta.) for
1-4.
.sup.13 C NMR (125 MHz in
CDCl.sub.3 for
1, 3; 75 MHz in CDCl.sub.3 for
2, 4)
Position 1 cis 1 trans 2
3 4
1 178.66 178.12 174.63
174.70 174.61
2 44.17 43.68 131.04
131.09 131.03
3 34.41 34.41 33.34
33.35 33.30
4 78.98 78.54 69.60
69.62 69.56
5 36.65 35.39 9
37.01 37.00 36.98
6 24.97.sup.a 24.97.sup.a
25.95.sup.a 25.67.sup.a 25.55.sup.a
7 24.90- 24.90- 25.12-
23.44- 23.44-
35.32 35.32 29.64
29.67 33.30
8 42.71.sup.b 42.71.sup.b
42.67.sup.b 42.64.sup.b 42.64.sup.b
9 210.80 210.80 211.38
211.50 211.40
10 42.36.sup.b 42.36.sup.b
42.51.sup.b 42.51.sup.b 42.51.sup.b
11-12 23.68- 24.90- 25.12-
23.44- 23.44-
33.46 35.32 29.64
29.67 33.30
13 25.32.sup.a 25.32.sup.a
25.28.sup.a 25.32.sup.a 25.26.sup.a
14 33.19.sup.c 33.19.sup.c
32.94.sup.c 34.07.sup.c 33.39.sup.c
15 73.49.sup.d 73.49.sup.d
74.21.sup.d 74.38.sup.d 74.05.sup.d
16 82.63 82.63 83.18.sup.c
82.66 82.66.sup.c
17 28.69 28.69 28.57.sup.f
28.10 28.73
18 28.69 28.69 25.23.sup.f
28.10 28.73
19 82.63 82.63 82.14.sup.c
82.66 82.58.sup.c
20 73.91.sup.d 73.91.sup.d
71.51.sup.d 74.28.sup.d 73.89.sup.d
21 33.46.sup.c 33.46.sup.c
32.51.sup.c 33.73.sup.c 33.39.sup.c
22 23.27.sup.a 23.27.sup.a
25.12.sup.a 25.14.sup.a 25.13.sup.a
23 128.92 128.92 25.12-
23.44- 23.44-
29.64
29.67 33.30
24 130.78 130.78 25.12-
23.44- 23.44-
29.64
29.67 33.30
25 27.21.sup.a 27.21.sup.a
25.12- 23.44- 23.44-
29.64
29.67 33.30
26-29/31 23.68- 24.90- 25.12-
23.44- 23.44-
33.46 35.52 29.64
29.67 33.30
30/32 31.87 31.87 31.89
31.90 31.86
31/33 22.63 22.63 22.64
22.66 22.66
32/34 14.05 14.05 14.09
14.09 14.07
33/35 35.39 35.39 151.93
151.98 151.92
34/36 205.50 205.50 78.01
78.03 77.99
35/37 23.68 23.68 19.07
19.07 19.05
.sup.a-f Values may be interchangeable in each
column.
Resonances at .delta. 3.40 (H-15, H-20) and 3.80 (>-16, H-19) in
the .sup.1 H NMR spectrum and .delta. 73.49 (C-15), 82.6 (C-16,
19), and 73.9 (C-20) in the .sup.13 C NMR spectrum indicated the
presence of a single THF ring and two flanking hydroxyls with a
threo/trans/threo relative stereochemistry. The presence of the
two hydroxyls was supported by two successive losses of water from
the CIMS MH.sup.+ ion at m/z 621. The ring was placed between C-15
and C-20 based on EIMS peaks at m/z 325 and 395. The presence of
hydroxyl groups was corroborated by a broad absorbance in the IR
(3439 cm.sup.-1) as was the existence of a carbonyl (1713 cm.sup.-1)
somewhere along the aliphatic chain. A carbonyl in the structure
was also suggested by a pair of triplets in the .sup.1 H NMR with
resonances at .delta. 2.40 (H-8) and 2.42 (H-10) and by a carbonyl
signal at .delta. 210.8 (C-9) in the .sup.13 C NMR. The carbonyl
position was suggested to be at C-9 based on a peak at m/z 225 in
the EIMS of 1. This assignment was predicated on the assumption
that cleavage was between C-9 and C-10, assuming that the oxygen
was included in the fragment ion. An EIMS peak at m/z 225 would
also be seen for a carbonyl at C-11 if cleavage was between C-10
and C-11. The carbonyl was placed conclusively at C-9 by the high
resolution EIMS of the fragment peak at m/z 225. The m/z of
225.1131 (225.1127 calcd.) dictated that the composition of the
fragment was C.sub.12 H.sub.17 O.sub.4 as predicted by the above
cleavage. The structure of 1 also contained a cis double bond as
evidenced by .sup.1 H resonances at .delta. 5.36 and 5.39 (J=15
Hz) and .sup.13 C resonances at .delta. 128.9 and 130.8. This
double bond was placed two methylene units away from the flanking
hydroxyl based on a cross-peak in the double-relayed COSY spectrum
between the methine protons at .delta. 5.36 (H-23) and 3.41
(H-20).
The absolute stereochemistries of the chiral centers in 1 were
determined by preparing the di-(R)- and (S)-methoxy-(trifluoromethyl)-phenylacetate
(MTPA) ester derivatives Kosher esters). According to advanced
Mosher ester methodology, the absolute stereochemistry of a
secondary alcohol is found by analyzing the difference in .sup.1 H
NMR chemical shifts between the S- and R-MTPA ester derivatives on
both sides of the carbinol center. Analysis of the .sup.1
H--.sup.1 H COSY for mosinone A-S-MTPA (1a) and mosinone A-R-MTPA
(1b) suggested that, based on Mosher's arguments, the absolute
stereochemistry for mosinone A was C-15R and C-20R (Table 3).
Thus, the structure of 1 was elucidated and was named mosinone A.
TABLE 3
.sup.1 H NMR (500 MHz, CDCl.sub.3) data
(.delta.) for MTPA
derivatives of Compound 1.
MTPA
ester 14 16 17 18
19 21
1a 1.64, 1.60 3.93 1.64, 1.38 1.64, 1.38
3.93 1.60, 1.56
1b 1.60, 1.56 4.01 1.91, 1.57 1.91, 1.57
4.01 1.60, 1.56
.DELTA.(.delta.S-) pos neg neg
neg neg pos
.delta.R)
configuration 15R 20R
Compound 2 was isolated as a white waxy solid. The CIMS showed an
MH.sup.+ peak at m/z 595 indicating that this compound was only 35
carbons long, unlike 1 which was two methylene units longer. The
molecular composition of C.sub.35 H.sub.62 O.sub.7 was confirmed
by HRFABS. Signals in the .sup.1 H NMR of 2 at .delta. 7.19
(H-35), 5.06 (H-34), and 1.44 (H-33) (Table 1) implied that the
structure of 2 contained an .alpha.,.beta.-unsaturated .gamma.-lactone.
.sup.13 C NMR resonances at .delta. 174.6 (C-1), 131.04 (C-2),
151.9 (C-33), and 78.0 (C-34) (Table 2) substantiated this
hypothesis. Further evidence for the presence of an .alpha.,.beta.-unsaturated
.gamma.-lactone was provided by the IR carbonyl peak at 1755
cm.sup.-1. The existence of hydroxyl groups in the structure was
suggested by a broad absorbance in the IR (3441 cm.sup.-1) as well
as peaks in the EIMS at m/z 577, 559, and 541 indicating three
successive losses of water from the molecular ion at m/z 595.
Examination of the regions around .delta. 3.80 (H4, H15/20,
H-16/19) and .delta. 3.40 (H-15/20) in the .sup.1 H spectrum of 2
and .sup.13 C NMR signals for 2 at .delta. 74.2 (C-15/20), 83.2
(C-16/19), 82.1 (C-16/19), and 71.5 (C-15/20) indicated a mono-THF
ring acetogenin with two flanking hydroxyls. Other .sup.1 H
signals for the ring methines (H-17/18) at .delta. 1.97, 1.87,
1.83, and 1.56, and .sup.13 C NMR signals at .delta. 32.9 and 32.5
(C-14/21) and at .delta. 28.6 and 25.2 (C-17/18) suggested a
relative stereochemistry of threo/trans/erythro. These resonances
matched those of a synthetic model compound and supported this
assignment. The fragment peak in the EIMS at m/z 325 placed the
THF ring between C-15 and C-20. As in 1, a carbonyl moiety along
the aliphatic chain in 2 was evidenced by a second carbonyl peak
in the IR (1702 cm.sup.-1), .sup.1 H NMR signals at .delta. 2.40
(H-8) and 2.42 H-10), and a .sup.13 C NMR carbonyl signal at
.delta. 211.4 (C-9). Similarly, the position of the carbonyl was
placed at C-9 by an EIMS fragment peak at m/z 225 and verified by
HREIMS spectral analysis of that fragment as described above for
1.
Although the relative stereochemistry of threo/trans/erythro could
be assigned in 2 based on NMR comparisons with model compounds, it
was unknown whether the erythro hydroxyl was at C-15 or C-20.
Analysis of the .sup.1 H--.sup.1 H COSY spectra for the tri(S)-
and tri-(R)-MTPA esters of 2 (2a and 2b, respectively) provided no
information, which could stereochemically differentiate the H-16,
H-17ab, H-18ab, and H-19 protons (Table 4), although the absolute
stereochemistry at C-4 was conclusively determined to be R. Due to
the coplanarity of the threo/trans/erythro relationship in 2,
these protons experienced shielding effects of the phenyl groups
of both flanking MTPA esters. Therefore, ambiguity remains
concerning the relative and absolute stereochemistries of 2.
TABLE 4
.sup.1 H NMR (500 MHz, CDCl.sub.3) data
(.delta.) for MTPA
derivatives of Compound 2.
MTPA ester H-5 H-3a H3b H33
H34 H35
2a 1.72, 1.62 2.61 2.55 6.73
4.86 1.28
2b 1.61, 1.56 2.67 2.59 6.97
4.91 1.30
.DELTA.(.delta.S-.delta.R) pos neg
neg neg neg pos
configuration 4R
The structure of 3 differs from 2 only in the relative
stereochemistry around the THF ring. The CIMS and EIMS spectra for
3 were identical to those of 2 since they have the same planar
structure. As in 2, analysis of the IR, .sup.1 H NMR and .sup.13 C
NMR spectra of 3 indicated the presence of an .alpha.,.beta.-unsaturated
.gamma.-lactone and a single THF ring with two flanking hydroxyls.
A carbonyl in the structure was again suggested by IR, .sup.1 H
NMR (Table 1), and .sup.13 C NMR (Table 2) spectra. The assignment
of the carbonyl to the C-9 position was made as before by the EIMS
peak at m/z 225 and was corroborated by HREIMS of that fragment
ion.
For 3, the cis assignment across the THF ring was made based on
.sup.1 NMR signals for the rig methines at .delta. 1.94 and 1.75
compared to .delta. 1.99 and 1.69 for a trans ring configuration.
.sup.13 C NMR resonances at .delta. 74.38, 82.66, 34.07, 33.73,
and 28.10 also suggested a cis assignment for the THF ring. These
signals showed a close resemblance to those of a synthetic model
compound with a threo/cis/threo ring configuration, and supported
this assignment. To determine the absolute stereochemistry of 3,
the tri-(S)- and (R)-MTPA ester derivatives (3a and 3b,
respectively) were prepared. Analysis of the .sup.1 H NMR and
.sup.1 H--.sup.1 H COSY spectral data indicated that the absolute
configuration(s) at C-4 and C-15 are R and at C-20 is S (Table 5).
The structure of 3 is named mosin C.
TABLE 5
.sup.1 H NMR (500 MHz, CDCl.sub.3) data
(.delta.) for MTPA derivatives of
Compounds 3 and 4.
MTPA
ester 5 3 33 34 35
14 16 17 18
19 21
3a 1.64, 1.72 2.56, 6.73 4.87 1.28
1.32, 3.86 1.44, 1.42 1.37,
1.82 4.10 1.63,
2.61 1.36
1.69
3b 1.61, 1.67 2.59, 6.97 4.92 1.31
1.31,1.35 3.88 1.46, 1.43 1.36,
1.81 4.11 1.65
2.66
1.70
.DELTA.(.delta.S- pos neg neg neg
neg pos neg neg
pos neg neg
.delta.R)
config. 4R
15R 20S
4a 1.71, 1.63 2.55, 6.73 4.85 1.28
1.60, 3.92 1.65, 1.36 1.65,
1.36 3.92 1.60,
2.60 1.54
1.54
4b 1.70, 1.61 2.59, 6.97 4.91 1.31
1.55, 4.00 1.91, 1.53 1.91,
1.53 4.00 1.55,
2.68 1.48
1.48
.DELTA.(.delta.S- pos neg neg neg
neg pos neg neg
neg neg pos
.delta.R)
config. 4R
15R 20R
Annoreticuin-9-one (4) has been isolated previously from Annona
reticulate, but has not been isolated previously from Annona
squamosa. Compound 4 was obtained as a white waxy solid and is
closely related to squamone (isolated from A. squamosa). The only
difference between compound 4 and squamone is that 4 possesses an
.alpha.,.beta.-unsaturated .gamma.-lactone with a hydroxyl at C-4
instead of the ketolactone moiety seen in squamone. Examination of
the diagnostic peaks in the .sup.1 H NMR (Table 1) and .sup.13 C
NMR (Table 2) of 4 indicated the presence of the aforementioned
lactone and hydroxyl at C-4 and a single THF ring bearing a
threo/trams/threo relationship.
Although the planar structure of 4 is known, its absolute
stereochemistry has not been reported previously. The tri-(R)- and
(S)-Mosher esters of 4 were prepared (4a and 4b, respectively) and
analyzed using .sup.1 H NMR and .sup.1 H--.sup.1 H COSY spectral
data (Table 5). The absolute stereochemistry of 4 was determined
to be 4R, 15R, 20R, and it can now be assumed that squamone has
the same stereochemistry.
Compounds 1-4 all showed activity in the brine shrimp assay (Table
6). In cell culture, these acetogenins all exhibited up to 10,000
fold cytotoxic selectivities for the pancreatic cell line, PACA-2,
and were 10 to 100 times more active than the positive control,
adciamycin (Table 6).
TABLE 6
Bioactivities of
compounds 1-4.
BST
(LC.sub.50,
Cytotoxicity (ED.sub.50, .mu.g/mL)
Comp. .mu.g/mL) A-549 MCF-7
HT-29 A-498 PC-3
PACA-2
1 4.39 .times. 10.sup.-1 >1
>1 >1 >1
3.19 .times. 10.sup.-2 2.18 .times.
10.sup.-3
2 2.93 .times. 10.sup.-1 9.44 .times.
10.sup.-1 >1 >1
>1 3.50 .times. 10.sup.-1 2.51
.times. 10.sup.-4
3 1.54 .times. 10.sup.-1 5.96 .times.
10.sup.-1 >1 >1
>1 >1 1.17 .times.
10.sup.-4
4 4.09 .times. 10.sup.-1 2.73 .times.
10.sup.-1 >1 >1
>1 9.64 .times. 10.sup.-3 2.39
.times. 10.sup.-4
adriam 2.57 .times. 10.sup.-1 5.27 .times.
10.sup.-1 1.99 .times.
10.sup.-1 2.00 .times. 10.sup.-2 1.02
.times. 10.sup.-2 3.21 .times.
10.sup.-2 1.79 .times. 10.sup.-2
ycin
In accordance with the present invention there is provided a novel
mono-THF acetogenin isolated from the bark of Annona squamosa.
Preferred compounds are those that have a mono-THF flanked by a
pair of hydroxyls and also include a carbonyl group in the
aliphatic chain at the C-9 position. More particularly, the
compound is in substantially pure form, and is selected from the
group consisting of mosinone A, mosin B, and mosin C. These
compounds exhibit anti-tumor and insecticidal activities and thus
can be used as chemothrapeutic agents or as insecticides. In
accordance with one embodiment, the compounds of the present
invention are utilized to form a chemotherapeutic composition.
The present invention provides pharmaceutical formulations
comprising an effective amount of an acetogenin compound selected
from the group consisting of compounds 1-4 for treating a patient
having a tumor. As used herein, an effective amount of the
acetogenin compound is defined as the amount of the compound
which, upon administration to a patient, inhibits growth of tumor
cells, kills malignant cells, reduces the volume or size of the
tumors or eliminates the tumor entirely in the treated patient. In
particular, the presently disclosed compounds demonstrate
selective toxicity for pancreatic and prostate tumor cells.
Compounds 1-4 all showed selective cytotoxic activity against the
human pancreatic tumor cell line, PACA-2, with potency 10 to 100
times that of adriamycin.
Murisolin, murisolin A, and 16,19-cis-murisolin, are a related
series of acetogenins which differ from 2, 3, and 4 only in that
they do not contain a carbonyl at the C-9 position. These
compounds did not show any selectivity for PACA-2 but did exhibit
strong potency against other cell lines. Furthermore, unlike the
compounds described in the present application, which all
displayed a similar profile of activity, the murisolin series
showed different activities compared to each other. For example,
16,19-cis-murisolin was several orders of magnitude less active
than the other two compounds against the A-549, HT-29, and A-498
cell lines but was 100 times more potent than them against the
MCF-7 cell line. From this data, it can be suggested that the
carbonyl at C-9 decreases activity in five of the six cell lines
tested but magnifies the potency toward the pancreatic cell line,
PACA-2. Knowing that acetogenins act by inhibiting ubiquinone
linked NADH oxidases that are membrane bound, a possible
explanation for the observed selectivity is that the target
enzymes in PACA-2 cells have a peculiar geometry making them more
susceptible to acetogenins with a carbonyl at C-9.
The substantially pure compounds in accordance with this invention
can be formulated into dosage forms using pharmaceutically
acceptable carriers for oral or parenteral administration to
patients in need of oncolytic therapy. The effective amount to be
administered to a patient is typically based on body surface area,
patient weight, and patient condition. The interrelationship of
dosages for animals and humans (based on milligrams per meter
squared of body surface) is described by Freireich, E. J., et al.,
Cancer Chemother. Rep., 50 (4): 219 (1966). Body surface area may
be approximately determined from patient height and weight (see
e.g., Scientific Tables, Geigy Pharmaceuticals, Ardley, N.Y.,
pages 537-538 (1970)). Preferred dose levels will also depend on
the attending physicians' assessment of both the nature of the
patient's particular cancerous condition and the overall physical
condition of the patient. Effective anti-tumor doses of the
present acetogenin compounds range from about 1 microgram per
kilogram to about 200 micrograms per kilogram of patient body
weight, more preferably between about 2 micrograms to about 100
micrograms per kilogram of patient body weight.
Effective doses will also vary, as recognized by those skilled in
the art, dependant on route of administration, excipient usage and
the possibility of co-usage with other therapeutic treatments
including other anti-tumor agents, and radiation therapy.
The present pharmaceutical formulation may be administered via the
parenteral route, including subcutaneously, intraperitoneally,
intramuscularly and intravenously. Examples of parenteral dosage
forms include aqueous solutions of the active agent, in a isotonic
saline, 5% glucose or other well-known pharmaceutically acceptable
liquid carrier. In one preferred aspect of the present embodiment,
the acetogenin compound is dissolved in a saline solution
containing 5% of dimethyl sulfoxide and 10% Cremphor EL (Sigma
Chemical Company). Additional solubilng agents such as
cyclodextrins, which form specific, more soluble complexes with
the present acetogenin compounds, or other solubilizing agents
well-known to those familiar with the art, can be utilized as
pharmaceutical excipients for delivery of the acetogenin
compounds. Alternatively, the present compounds can be chemically
modified to enhance water solubility.
The present compound can also be formulated into dosage forms for
other routes of administration utilizing well-known methods. The
pharmaceutical compositions can be formulated, for example, in
dosage forms for oral administration in a capsule, a gel seal or a
tablet. Capsules may comprise any well-known pharmaceutically
acceptable material such as gelatin or cellulose derivatives.
Tablets may be formulated in accordance with conventional
procedure by compressing mixtures of the active acetogenins and
solid carriers, and lubricants well-known to those familiar with
the art. Examples of solid carriers include starch, sugar,
bentonite. The compounds of the present invention can also be
administered in a form of a hard shell tablet or capsule
containing, for example, lactose or mannitol as a binder and a
conventional fillers and tableting agents.
In accordance with one embodiment, a pharmaceutically acceptable
chemotherapeutic composition is provided comprising an anti-tumor
effective amount of a compound selected from the group consisting
of mosinone A, mosin B, mosin C and annoreticuin-9-one, and
derivatives of such compounds, and a pharmaceutically acceptable
carrier therefor. These compositions can further comprise
conventional fillers and solubilizing agents or other known
chemotherapeutic agents.
The acetogenin compounds of the present invention can be used to
treat patients having tumors. The method comprises administering
to the patient an effective amount of a compound selected from the
group consisting of mosinone A, mosin B, mosin C and
annoreticuin-9-one. In one embodiment, a pharmaceutical
composition comprising an acetogenin selected from the group
consisting of mosinone A, mosin B, mosin C and annoreticuin-9-one
is used to treat a patient having pancreatic or prostate cancer.
In one embodiment, a pharmaceutical composition comprising an
acetogenin selected from the group consisting of mosin B, mosin C
and annoreticuin-9-one is used to treat a patient having
pancreatic cancer, and in another embodiment a pharmaceutical
composition comprising an acetogenin selected from the group
consisting of mosinone A and annoreticuin-9-one is used to treat a
patient having prostate cancer.
EXAMPLE 1
Isolation of the Acetogenins
General Experimental Procedures. UV spectra were measured on a
Beckman DU 640 series spectrophotometer. IR data were collected
using a Perkin-Elmer 1600 series ftir. .sup.1 H NMR and .sup.13 C
NMR were obtained on a Varian VXR-500S spectrometer.
Low-resolution EIMS and CIMS data were collected on a Finnigan
4000 spectrometer. High-resolution EIMS, CIMS and FABMS were
obtained on the Kratos MS50 through peak matching. HPLC was
carried out using a Dynamax UV-1 detector coupled with a Rainin
model HPXL solvent delivery system for normal phase and Dynamax
model SDS200 solvent delivery system for reversed-phase.
Plant Material. The dried stem bark of Annona squamosa Rich was
purchased from United Chemical and Allied Products in Calcutta,
India.
Extraction and Isolation. The dried and pulverized bark of (7.4
kg) was extracted with ethanol (1.83 kg F001, BST LC.sub.50
=1.5532). The residue was partitioned between CH.sub.2 Cl.sub.2
and H.sub.2 O to yield a dichloromethane soluble residue (842 g
F003, BST LC.sub.50 =1.6774) and a water soluble residue (128.6 g
F002, BST LC.sub.50 =950.1438). F003 was further partitioned
between 90% aqueous methanol and hexane resulting in a methanol
soluble residue (545.5 g F005, BST LC.sub.50 =1.5155) and a hexane
soluble residue (162.9 g F006, BST LC.sub.50 =122.9733). 500.5 g
of F005 was separated by column chromatography over Si gel using
hexane and chloroform then chloroform and methanol as solvent
systems. Fractions 30-36 were combined on the basis of TLC and
were fi resolved on another Si gel column eluted with hexane and
acetone. The pools from this column bioactive in the BST were
subjected to a third Si gel column eluted with chloroform and
methanol. Compounds 1-4 were purified by repeated normal-phase and
reversed-phase HPLC using solvent systems of acetonitrile water
and hexane:methanol:THF, respectively.
Derivatizations. To 14 (0.5 mg in 0.5 mL CH.sub.2 Cl.sub.2) were
sequentially added 0.2 mL pyridine, 0.5 mg 4-(dimethylamino)-pyridine,
and 12 mg of (R)-(-)-(.alpha.-methoxy-.alpha.-trifluoromethyl)-phenylacetyl
chloride or (S)-(+)-.alpha.-methoxy-.alpha.-(trifluoromethyl)-phenylacetyl
chloride. The mixture was stirred for 4 h at room temperature then
passed through a small pipet column (0.6.times.6 cm) packed with
Si gel and eluted with 5 mL CH.sub.2 Cl.sub.2. The residue was
redissolved in 5 mL CH.sub.2 Cl.sub.2 and washed with 5 mL 1%
NaHCO.sub.3 and 2.times.5 mL H.sub.2 O. The organic layer was
evaporated to give the S-Mosher esters of 1-4. Use of
(5)(+)-.alpha.-methoxy-.alpha.-(trifluoromethyl)-phenylacetyl
chloride yielded the R-Mosher esters of 1-4.
Mosinone A (1).--White waxy solid (12 mg); [.alpha.a].sup.23.sub.D
=+4.8.degree.0; (c=0.016, CH.sub.2 Cl.sub.2) UV (MeOH) .lambda..sub.max
202 nm (log .epsilon.=2-96); CIMS (isobutane) m/z {MH}.sup.+ 621
(64), {MH-H.sub.2 O}.sup.+ 603 (100), {MH-2H.sub.2 O).sup.+ 585);
EIMS m/z 395(5), 377(20), 359(19), 325(100), 307(40), 289(13),
225(16), 207(20); HRCIMS (isobutane) m/z 621.4723 for C.sub.37
H.sub.60 O.sub.7 (calcd. 621.4730); HREIMS m/z 225.1131 for
C.sub.12 H.sub.17 O.sub.4 (calcd. 225.1127); .sup.1 H NMR data
(CDCl.sub.3, 500 MHz), see Table 1; .sup.13 C NMR (CDCl.sub.3, 125
MHz), see Table 2.
Mosin B (2).--White waxy solid (7 mg); [.alpha.].sup.23.sub.D
=+11.5.degree. (c=0.005, CH.sub.2 Cl.sub.2); UV (MeOH).lambda..sub.max
=222 nm (log .epsilon.=3.57); CIMS (isobutane) m/z 595(30),
577(71), 559(37); EIMS m/z 325(16), 307(91), 289(62), 225(7),
207(69); HRFABMS m/z 595.4578 for C.sub.35 H.sub.62 O.sub.7 (calcd.
595.4574); HREIMS 225.1133 for C.sub.12 H.sub.17 O.sub.4 (calcd.
225.1127); .sup.1 H NMR data (CDCl.sub.3, 500 MHz), see Table 1;
.sup.13 C NMR data (CDCl.sub.3, 75 MHz), see Table 2.
Mosin C (3).--White waxy solid (6 mg);[.alpha.].sup.23.sub.D
=-2.7.degree. (c=0.007, CH.sub.2 Cl.sub.2); UV (MeOH) .lambda..sub.max
=216 nm (log .epsilon.=3.56); CIMS (isobutane) m/z 595(74),
577(100), 559(53); EIMS m/z 325(16), 307(14), 289(9), 225(4),
207(10); HRFABMS m/z 595.4578 for C.sub.35 H.sub.62 O.sub.7 (calcd.
595.4574); HREIMS 225.1135 for C.sub.12 H.sub.17 O.sub.4 (calcd.
225.1127); .sup.1 H NMR data (CDCl.sub.3, 500 MHz), see Table 1;
.sup.13 C NMR data (CDCl.sub.3, 125 MHz), see Table 2.
EXAMPLE 2
Bioassays
The brine shrimp (Artemia salina Leach) test (BST) was performed,
as described in Meyer et al. Planta Med. 45, p. 31-34 (1982), to
determine LC.sub.50 values in .mu./ml. Seven-day in vitro
cytotoxicity tests against human tumor cell lines were carried out
at the Purdue Cancer Center, using standard protocols for A-549
(human lung carcinoma), MCF-7 human breast carcinoma), HT-29
(human colon carcinoma), A498 (human kidney carcinoma), PC-3
(human prostate carcinoma), and PACA-2 (human pancreatic
carcinoma) with adriamycin as a positive control.
Compounds 14 all showed activity in the brine shrimp assay. In
cell culture, these acetogenins all exhibited up to 10,000 fold
cytotoxic selectivities for the pancreatic cell line, PACA-2, and
were 10 to 100 times more active than the positive control
adriamycin (Table 6).
United States
Patent
5,229,419
McLaughlin ,
et al.
July 20, 1993
Chemotherapeutically active acetogenins
Abstract
Screening of crude extracts of the bark of Annona
bullata Rich. (Annonaceae) showed cytotoxic and pesticidal
activities. By monitoring with brine shrimp lethality, two novel
extremely potent acetogenins, bullatacin (1) and bullatacinone
(2), were isolated. Spectral and chemical methods identified
bullatacin as a diastereomer of asimicin. Bullatacinone represents
bullatacin with the lactone cleaved and reformed at the 4-OH.
Unlike asimicin, which is more generally cytotoxic, 1 and 2 show
some selective cytotoxicities in human tumor cell lines, and
certain susceptible cells give ED.sub.50 values as low as
10.sup.-12 -10.sup.-15 mcg/ml. Bullatacin was pesticidal at
concentration as low as 1 ppm, while bullatacinone lacked
pesticidal activities.
Inventors:
McLaughlin;
Jerry L. (W. Lafayette, IN); Hui; Yu-Hua (W. Lafayette, IN)
Assignee:
Purdue
Research Foundation
(West Lafayette, IN)
Appl. No.:
953759
Filed:
September 29,
1992
Current U.S.
Class:
514/473;
514/908; 549/320; 549/323
Intern'l
Class:
A61K 031/365; C07D 307/12
Field of
Search:
549/320,326 514/423,908
References
Cited
[Referenced By]
U.S. Patent
Documents
4689232
Aug., 1987
Moeschler et al.
424/195.
4721727
Jan., 1988
Mikolajczak et al.
549/320.
4855319
Aug., 1989
Mikolajczak et al.
549/320.
|
Other
References
Pettit, et al., Heterocycles, "Isolation and structure of
Rolliniastatin 2," 28(1), pp. 213-217 (1989).
Jolad, et al., "Uvaricin, A New Antitumor Agent from Uvaria
accuminata (Annonaceae)", (1982), J. Org. Chem., 47:
3151-3153.
Jolad, et al., "Desacetyluvaricin from Uvaria accuminata,
Configuration of Uvaricin at C-36", (1985), J. Nat. Prod., 48:
644-645.
Dabrah and Sneden, "Rollinicin and Isorollinicin, Cytotoxic
Acetogenins from Rollinia Papiilionella", (1984),
Phytochemistry, 23: 2013-2106.
Etse and Waterman, "Chemistry in the Annonaceae, XXII.
[4-Hydroxy-25-Desoxyrollinicin from the Stem Bark of Annona
reticulata" (1984), J. Nat. Prod. 49: 684-686.
Dabrah and Sneden, "Rollinone, A New Cytotoxic Acetogenin from
Rollinia papilionella", (1984), J. Nat. Prod., 47: 652-657.
Rupprecht, et al., "Asimicin, A New Cytotoxic and Pesticidal
Acetogenin from the Paw Paw, Asimina triloba (Annonaceae),"
(1986), Heterocycles 24: 1197-1201.
Pettit et al., "Isolation and Structure of rolliniastatin 1
from the South American tree Rollinia mucosa", (1987) Can. J.
Chem., 65: 1433-1435.
Hoye, et al., "On the stereochemistry of the
bistetrahydrofuranyl Moiety of Uvaricin: Proton Chemical
Shifts Can Play A Crucial Role in Complex Structure
Determination", (1987), J. Am. Chem. Soc., 109: 4402-4403.
Hoye and Zhuang, "Validation of the .sup.1 H NMR Chemical
Shift Method for Determination of Stereochemistry in the
Bis(tetrahydrofuranyl) Moiety of Uvaricin-related Acetogenins
from Annonaceae: Rolliniastatin 1 (and (Asimicin)", (1988), J.
Org. Chem., 53: 5578-5580.
Fujimoto, et al., "Squamocin, A New Cytotoxic
Bis-tetrahydrofuran Containing Acetogenin from Annona
squamosa", (1988), Chem. Pharm. Bull., 36: 4802-4806.
McCloud, et al., "Annonain, A Novel, Biologically Active
Polyketide from Annona densicoma" Experientia, (1987),
Experientia, 43: 947-949.
Alkofahi, et al., "Goniothalamicin and annonacin: Bioactive
Acetogenins from Goniothalamus giganteus (Annonaceae)",
(1988), Experientia, 44: 83-85. |
Primary Examiner: Raymond; Richard L.
Assistant Examiner: Russell; Mark W.
Attorney, Agent or Firm: Barnes & Thornburg
Parent Case
Text
This is a continuation of Ser. No. 07/336,233 filed on Apr. 11,
1989, now abandoned.
Claims
What is claimed:
1. A compound selected from the group consisting of bullatacin and
bullatacinone, characterized by the formulae ##STR9## in
substantially pure form, dihydrobullatacin, the corresponding
peracetylated derivatives of bullatacin, dihydrobullatacin and
bullatacinone, and mixtures of bulllatacin and bullatacinone in
substantially pure form.
2. The substantially pure compound bullatacin in accordance with
claim 1.
3. The substantially pure compound bullatacinone in accordance
with in claim 1.
4. A chemotherapeutic composition for treatment of cancer
comprising a substantially pure compound selected from the group
consisting of bullatacin, dihydrobullatacin, bullatacinone and
their peracetylated derivatives, in an amount effective to promote
remission of said cancer, in a pharmaceutically acceptable carrier
therefor.
5. The composition in accordance with in claim 4 wherein said
compound is bullatacin.
6. The composition in accordance with in claim 4 wherein said
compound is bullatacinone.
7. The composition in accordance with claim 4 wherein said
compound is selected from the group consisting of
dihydrobullatacin and the peracetylated derivatives of bullatacin,
dihydrobullatacin and bullatacinone.
8. A substantially pure compound of the formula ##STR10## and its
diacetate.
9. A chemotherapeutic composition for treatment of cancer
comprising a substantially pure compound selected from the group
consisting of bullatacinone and bullatacinone diacetate in an
amount effective to promote remission of said cancer, in a
pharmaceutically acceptable carrier therefor.
Description
BACKGROUND OF INVENTION
This invention relates to two acetogenin compounds isolated in
substantially pure form from Annona bullata Rich. (Annonaceae).
The new compounds, designated herein as bullatacin and
bullatacinone, have been found to exhibit antitumor and pesticidal
activity.
DESCRIPTION OF PRIOR ART
Bis(tetrahydrofuranoid) fatty acid lactones, represented by the
structural formula, ##STR1## have been reported in the literature
as having cytotoxic and antitumor activity. The first such
compound reported [Jolad, et al., J. Org. Chem, 47: 3151-3153
(1982)] was called uvaricin and was characterized by an --OH group
in the R.sup.2 position and an acetoxy group in the R.sup.3
position, with R.sup.1 and R.sup.4 being hydrogen. Uvaricin was
isolated from the roots of Uvaria accuminata of the family
Annonaceae and demonstrated activity in vivo against P-388 (PS)
lymphocytic leukemia in mice. Subsequently, Jolad et al. [J. Nat.
Prod., 48: 644-645 (1985)] disclosed the compound
desacetyluvaricin which differs from uvaricin in having an
hydroxyl in the R.sup.3 position. Dabrah and Sneden [Phytochemistry
23: 2013-2016 (1984)] showed the isolation of the undefined
stereoisomers, rollinicin, and isorollinicin from the roots of
Rollinia papilionella (Annonaceae). These compounds were reported
as having --OH in R.sup.2, R.sup.3 and R.sup.4 positions with
R.sup.1 being hydrogen. Both of these compounds exhibited in vitro
cytotoxicity against the P-388 lymphocytic leukemia. Etse and
Waterman J. Nat. Proc., 684-686 (1984)] reported the isolation and
structure of 14-hydroxy 25-desoxyrollinicin which differs from
rollinicin in having an hydroxyl in R.sup.1 and a hydrogen in
R.sup.4. There is no report for the bioactivity of this compound.
Dabrah and Sneden [J. Nat. Prod., 47: 652-657 (1984)] disclosed
another member of the series referred to as rollinone. Rollinone
is characterized by a keto group at the R.sup.1 carbon, hydroxyls
at R.sup.2 and R.sup.3, and hydrogen for R.sup.4 with a saturated
lactone ring as illustrated in structure IA: ##STR2## Rollinone
demonstrated both cytotoxicity against P-388 in vitro and also
activity in vivo against the same system in mice. The compounds
having the general structure designed by Formula I, above, have
acquired the name "linear acetogenins".
Cortes et al. [Tetrahedron Lett. 25: 3199-3202 (1984)] described
two additional linear acetogenins from Annona cherimolia (Annonaceae),
wherein the structures are reported as follows: ##STR3## These
compounds have antimicrobial activity as demonstrated against Gram
negative bacteria and Candida.
Rupprecht et al. [Heterocycles 24: 1197-1201 (1986)] and
Mikolajczak et al., U.S. Pat. No. 4,721,727, disclosed a new
member of this series referred to as asimicin. Asimicin was
isolated from Asimina triloba Dunal. (Annonaceae) and is
characterized by two hydroxyl groups in the R.sup.2 and R.sup.3
positions, two hydrogens in the R.sup.1 and R.sup.4 positions and
a hydroxyl group at carbon 4, found for the first time for this
type of compound, as illustrated in partial structure (IB).
##STR4## Asimicin was toxic to mice at 6.25 mg/kg and active (124%
T/C at 0.0125 mg/kg) in the 3PS lymphocytic leukemia system and
possesses cytotoxicities in the 9KB (human nasopharyngeal
carcinoma, ED.sub.50 <10.sup.-5 .mu.g/ml) and the 9PS (murine
lymphocytic leukemia, ED.sub.50 <10.sup.-7 .mu.g/ml systems.
Promising pesticidal activity against the stripped cucumber
beetle, Mexican bean beetle, mosquito larvae, blow fly larvae,
melon aphid, two spotted spider mites, and the free-living
nematode, Caenorabditis elegans, was demonstrated and this
pesticidal use of the acetogenins was patented on Jan. 26, 1988 [Mikolajczak
et al., U.S. Pat. No. 4,721,727]. An uncharacterized pesticidal
substance called annonin and a process for its isolation from the
seeds of Annona souamosa (Annonaceae) has been patented by
Moeschler et al. [U.S. Pat. No. 4,689,232].
Pettit et al. (Can. J. Chem., 65: 1433-1435 (1987)] isolated a
diastereomer of asimicin from Rollinia mucosa (Annonaceae). This
diastereomer is called rolliniastatin. Its stereochemistry, which
was revealed by the first X-ray crystallographic analysis of this
type of compound, is threo, cis, threo, cis, erythro, 4S and 36S
and differed from asimicin which is threo, trans, threo, trans,
and threo as analyzed by the .sup.1 H nmr analysis method
developed by Hoye et al. [J. Am. Chem. Soc., 109:
4402-4403(1987)]. Rollinia mucosa has been known in primitive
medical practices of Indonesia and the West Indies as a treatment
for tumors. Biological evaluation of rolliniastatin showed PS
activities: 28% life extension and ED.sub.50 4.5.times.10.sup.-5 .mu.g/m
in cell culture.
SUMMARY OF THE INVENTION
During the screening of plants in our laboratory, we have
unexpectedly discovered that Annona bullata Rich. in the
Annonaceae family has noteworthy activities in the BST (brine
shrimp lethality test), PD (crown gall antitumor activity on
potato discs), 9PS (murine leukemia cytotoxicity), 9KB (human
nasopharyngeal carcinoma cytotoxicity), and 9ASK (astrocytoma
reversal) bioassays. In addition, activities in several pesticidal
tests at Eli Lilly showed promise. The compounds found to
correspond to these activities as well as selective antitumor
activities were the class of natural bistetrahydrofuranoid fatty
acid lactones called acetogenins. The substantially pure compounds
in accordance with this invention, bullatacin and bullatacinone,
have been characterized to be two new members of this unusual
class of compounds. Both of these compounds have the same
stereochemistry at their corresponding tetrahydrofuran rings and
the two adjacent hydroxyl groups. Bullatacin is characterized by a
hydroxyl group on carbon 4 and a terminal .alpha.,
.beta.-unsaturated lactone ring. Bullatacinone is characterized by
a terminal methyl ketone and a lactone ring formed with a carbon 4
hydroxyl group. The structural formulas of the present compounds
are illustrated below as (1) and (2). ##STR5##
It is an object of this invention to provide the two Annonaceous
acetogenin compounds designated as bullatacin and bullatacinone in
substantially pure form.
It is also an object of this invention to provide two of the most
active acetogenins known today in in vitro human tumor cell lines.
It is still a further object of this invention to provide a method
for converting bullatacin to bullatacinone, bullatacinone
exhibiting a more selective antitumor activity.
It is a further object of the invention to provide bullatacin as a
new and potent pesticide in the acetogenin group.
Other objects and advantages of the invention will become readily
apparent from the ensuing description.
DETAILED DESCRIPTION OF THE INVENTION
The starting material for use in the invention is the bark of
Annona bullata Rich. (Annonaceae), and it is considered likely, by
the screening of other parts of the plant, that other tissues such
as twigs, wood, roots, seeds and leaves would also contain
extractable quantities of the subject compounds.
The bark material is prepared for extraction by grinding in a
conventional mill to a suitable particle size, usually in the
range of about 0.001-3 mm. in diameter, and more preferably in the
range of 0.1-2 mm. The ground material is extracted by percolating
with 95% EtOH. The