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Source

 

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 ethanol solubles are concentrated to remove the bulk of the solvent, at least to the point of reducing the extract to a thick syrup. The resultant concentrate is partitioned between water and a water-immiscible solvent, such as chloroform, in order to remove the water solubles which are freeze dried and labelled F002. The chloroform solubles are recovered as a syrup residue using a solvent evaporator and labelled as F003. The insoluble interface was dried at ambient temperature and labelled F004. F003 then is partitioned between hexane and 90% aqueous MeOH in order to remove hexane solubles which are vacuum dried and labelled as F006. The 90% aqueous MeOH solubles are recovered by vacuum evaporation to a thick syrup as a crude acetogenin-containing extract F005.

Separation and purification of pure acetogenins from the crude extract (F005) can be affected by the use of the proper combination of conventional techniques including, for example, column chromatography (CC), thin-layer chromatography (TLC), medium-pressure chromatography (MPC) and chromatotrons. While not desiring to be limited thereto, the details of the separation procedure are illustrated by the following examples. Fractionation of the ethanolic extract was guided by assay with the brine shrimp lethality test (BST) and confirmed by assays on tumor cell cultures. Pesticidal tests were conducted at Eli Lilly Laboratories (Greenfield) in indicator organisms following standard procedures.

EXAMPLES

Bioassay Procedure

The following bioassays were used to guide the phytochemical fractionation described below.

The extracts, fractions, and isolated compounds were routinely evaluated for lethality to brine shrimp larvae (BST). The LC.sub.50 of brine shrimp (Artemia salina Leach) was determined by substantially the same method described by Meyer et al. [Planta Med., 45: 31-34 (1982)]. The test sample was dissolved in solvent and added to 2-dram vials in an amount to provide 1000, 100, 10, 1, etc. p.p.m. of material in the final brine preparation, assuming complete miscibility in the brine. The vials were dried in vacuo, and artificial sea water, prepared from a commercial salt mixture, was added. Ten brine shrimp larva (nauplii), 48-72 hrs. old, were introduced to the vial, and the volume was made up to 5 ml. with the sea water. After 24 hrs., the percent mortality was computed. Subsequently, LC.sub.50 values and 95% confidence limits were calculated by a Finney probit analysis to permit comparison of potencies of extracts and fractions.

Occasional checks in the potato disc (PD) assay assured us that antitumor activity was present [Ferrigni et al., J. Nat. Prod., 45; 679-686 (1982)]. Cytotoxicity tests were performed at the Purdue Cell Culture Laboratory, Purdue Cancer Center, using standard protocols for 9KB (human nasopharyngeal carcinoma) and 9PS (a chemically-induced murine lymphocytic leukemia) [Suffness et al., Methods In Cancer Research. Vol. 16: 73 (1979), V. T. DeVita, Jr. and H. Busch eds., Academic Press, New York], 9ASK (astrocytoma reversal) [Lgarashi et al., Cell Struct. and Funct., 3: 107 (1978)], A-549 (human lung carcinoma) [Giard et al., J. Nat. Cancer Inst., 51: 1417-1423 (1973)], and HT-29 (human colon adenocarcinoma) [Fogh J. (ed.), Human Tumor Cells, In Vitro, Plenum Press, New York: 115-159 (1975)].

Isolated pure compounds were sent to the National Institute of Health, National Cancer Institute, Bethesda, Md., for testing in human cancer cell line panels including leukemia, non-small cell lung cancer, small cell lung cancer, CNS cancer, melanoma, ovarian cancer and renal cancer.

Pesticidal bioassays were conducted at Eli Lilly Laboratories (Greenfield, Ind.) following standard procedures with eight indicator organisms: mosquito larvae (ML) Aedes aegypti (in the media), blowfly larvae (BFL) (1% in the diet), corn root worm (CRW) Diabrotics undecimcuntata howardii (in soil), two-spotted spider mite (2SSM) Tetranychus urticae (on foliage), southern army worm (SAW) Spodoptera eridania (on foliage), melon aphid (MA) (5000 ppm on foliage), cotton aphid (CA) Aphis gossypii, (on foliage) and Haemonchus contortus (HC) (a nematode, 0.1% in the media).

Isolation of the Compounds

A. Extraction Procedures

Approximately 3.9 kg of Annona bullata Rich. (Annonaceae) bark (M-06983, PL-103519) was collected by Edward Garvey at the USDA Subtropical Horticulture Research Station, ARS, 13601 Old Culture Rd., Miami, Fla. 33158. The tree originated from seeds collected in Cuba in 1933 by Robert M. Grey of Harvard University. Air-dried bark was pulverized through a 2 mm screen in a Wiley mill. The pulverized bark was extracted by exhaustive percolation with 777 liters of 95% EtOH. Vacuum evaporation left 380 g of syrupy residue (F001). F001 was partitioned between CHCl.sub.3 H.sub.2 O (1:1), and the water solubles were freeze dried and labelled F002 (11 g). The chloroform solubles were vacuum evaporated to form F003 (181 g). The insoluble interface was air dried and labelled F004 (188 g). Then F003 was partitioned between hexane/90% aqueous MeOH (1:1). The 90% MeOH fraction was vacuum evaporated to a thick syrup and labelled F005 156 g.) The hexane residue (25 g) was labelled F006. The bioassay data (Table 1) clearly showed that the most activity was concentrated in the 90% MeOH fraction (F005).

                                      TABLE 1

    __________________________________________________________________________

    Bioactivities of Initial Fractions from Annona bullata Rich.

    BST

    LC.sub.50 mcg/ml

    95%                                 Protein kinase C

    Confidence

              PD     9KB    9PS    9ASK % Displacement

    Interval  % Inhibition

                     ED.sub.50  mcg/ml

                            ED.sub.50  mcg/ml

                                   reversal

                                        100 mcg/ml

    __________________________________________________________________________

    F001

       0.0062 --.sup.a

                     20     <10.sup.-2

                                   not  11%

       0.0120                      active

       .dwnarw.

       0.0009

    F002

       >100   --     >10    >10    --   --

    F003

       0.0030

       0.0036 --     10.sup.-1 -10.sup.-2

                            <10.sup.-2

                                   not  67%

       .dwnarw.                    active

       0.0010

    F004

       5.4000 --     >10    2.11   --   --

       838.fwdarw.1.53

    F005

       0.0025

       0.0050 78%    <10.sup.-5

                            <10.sup.-2

                                   slight

                                        68%

       .dwnarw.                    active

       0.0001

    F006

       >100   --     >10    4.10   --   --

    __________________________________________________________________________

     .sup.a A dash () indicates that tests were not conducted.



B. Chromatographic Separations

F005 (80 g) was absorbed onto 100 g of Celite and applied to a column of Si gel (3 kg) packed in a slurry of hexane. A gradient of hexane--CHCl.sub.3 --MeOH was used to elute the column, collecting 82 fractions of 100-200 ml each. Fractions were combined into pools according to their similar TLC patterns [CHCl.sub.3 --MeOH (9:1) on Si gel, phosphomolybdic acid spray], weighed, and bioassayed by the BST.

The largest and the most toxic pool (P40-51, 25 g) was absorbed onto 100 g of Celite and chromatographed over a column of 4.4 kg of Si gel packed as a slurry in CHCl.sub.3. A gradient of CHCl.sub.3 --EtOAc--MeOH was used to elute the column, collecting fractions of 100-200 ml. Pools were made after TLC and bioassayed in the BST. From the most toxic pool (P106-111), which had been eluted with CHCl.sub.3 --EtOAc (1:1), a white precipitate was obtained. Recrystallization from EtOAc gave fine white needles which were labelled as bullatacin (1) (100 mg); this material was homogeneous in several TLC systems. From P17-40, which had been eluted with CHCl.sub.3, another white precipitate appeared which was recrystallized from EtOAc to a white homogeneous solid (5 mg), later designated bullatacinone (2).

Pool 31-39, from the first column, stood for some time. Orange crystals formed and were recrystallized from MeOH to yield yellow needles (150 mg); this material was identified (mp, ir, ci and eims) as liriodenine [Lopez et al. Rev. Cubana Farm., 20: 83-86 (1986) (Span.)]. The residue (5.7 g) of the mother liquor was chromatographed over Si gel at medium pressure in a Michel-Miller column, eluted with a gradient of CHCl.sub.3 --MeOH. A white solid formed in pool 104-113; this was recrystallized from EtOAc to yield fine white needles (8 mg) of additional bullatacinone (2).

Upon standing, pool 8-17 from the first chromatography column gave large colorless crystals. After recrystallization from MeOH, these were identified (uv, ei and cims, hrms, .sup.1 H and .sup.13 C nmr) as (-)-kaur-16-en-19-oic acid [Leboeuf et al., Phytochemistry, 21: 2783-2813 (1982)].

Characterization of Compound Structures

A. Structural Characterization of Bullatacin (1)

Mp 69-70.degree.; [.alpha.]23.degree./589=+13.00, [.alpha.]23.degree./578=+14.70, [.alpha.]23.degree./546=+19.04, [.alpha.]23.degree./436=+36.63, [.alpha.]23.degree./365=+66.99 (c, 0.004, CHCl.sub.3); cims (isobutane) m/z 623 (MH.sup.+), cims (ammonia) m/z 622 (M+NH.sup.+.sub.4 --H.sub.2 O), 640 (M+NH.sup.+.sub.4), eims m/z 622 (M.sup.+); hr cims 623.4847 (calc. 623.4889) for C.sub.37 H.sub.66 O.sub.7 ; uv (EtOH) .lambda. max 215.5 nm (.epsilon.=7974); ir (KBr) cm.sup.-1 3430 (hydroxyl), 1750 (carbonyl).

These spectral characteristics indicated that 1 belongs to the familiar class of bioactive bistetrahydrofuran acetogenins which contain 37 carbons and two long hydrocarbon chains, one of which terminates with a .gamma.-lactone.

The structure of fragment A, which contains the lactone ring and one of the three hydroxyl groups, was elucidated by high field .sup.1 H nmr (Table 2), .sup.13 C nmr (Table 3), and ms spectral analyses.

                                      TABLE 2

    __________________________________________________________________________

    .sup.1 H NMR Assignments and Comparisons of Compounds.*

    __________________________________________________________________________

    Bullaticin (1)  Bullatacin (1)

                                Bullatacinone (2)

    470 MHz, CDCl.sub.3

                    470 MHz, C.sub.6 D.sub.6

                                470 MHz, C.sub.6 D.sub.6

    __________________________________________________________________________

    2     --          --        2.71dddd2, 3a(9.34)

                                2, 3b(9.34)

                                2, 35b(3.42)

                                2, 35a(9.34)

    3a  2.50 dddd 3a, 3b(15.0)

                    2.30 dddd 3a, 3b(15.0)

                                1.70ddd 3a, 3b(12.82)

        3a, 4 (4.0) 3a, 4 (4.0) 3a, 4 (3.38)

        3a, 35(1.5) 3a, 35(1.5) 3a, 2 (9.34)

        3a, 36(1.1) 3a, 36(1.1)

    3b  2.36 ddt 3b, 3a(15.0)

                    2.20 ddt 3b, 3a(15.0)

                                1.40ddd

        3b, 4 (8.0) 3b, 4(8.0)

        3b, 35(1.4) 3b, 35(1.4)

    4   3.80m       3.71br tt   4.05m

    5   1.3-2.0     1.3-2.1     2.3-2.0

    6-13

        1.25br s    1.25br s    1.25br s

    14  1.35m       1.3-2.1     1.40, 1.55

    15  3.38tt 15, 16 (8.0)

                    3.45ttt 15, 16 (8.0)

                                3.49tt 15, 16 (8.06)

        15, 14 (2.0)

                    15, 14 (2.0)

                                15, 14 (2.38)

                    15, 17 (1.0)

    16  3.83m       3.85ddd 16, 17a(8.0)

                                3.85ddd 16, 17a(8.06)

                    16, 17b(6.9)

                                16, 17b(6.96)

                    15, 17 (1.0)

    17, 18

        1.3-2.0     1.3-2.1     1.65, 1.35

    19x 3.92m       3.89m       3.89m

    20x 3.83m       3.67ddd 20, 21a(6.0)

                                3.66ddd 20, 21a(5.96)

                    20, 21b(1.0)

                                20, 21b(1.00)

                    20, 19(15.0)

                                20, 19(14.93)

    21, 22

        1.3-2.0     1.3-2.1     1.52, 1.42

    23y 3.92m       3.99dt 23, 24(8.5)

                                3.95dd 23, 24(8.4)

                    23, 22(2.5) 23, 22(2.5)

    24y 3.83m       3.89m       3.89m

    25  1.3-2.0     1.3-2.1     2.10, 1.60

    26-33

        1.25br s    1.25br s    1.25br s

    34  0.85t 34, 33(6.81)

                    0.9t 34, 33(6.81)

                                0.90t 34, 3 (7.05)

    35a                         1.93dd 35a, 35b(18.31)

        7.17d 35, 36(1.70)

                    6.25d 35, 36(1.70)

                                35a, 2 (9.34)

    35b                         2.53dd 35b, 2 (3.42)

    36  5.05ddq 36, 37(6.83)

                    4.12ddq 36, 37(6.83)

                                  --

    37  1.41d       0.80d       1.55s

    __________________________________________________________________________

        Rolliastatin

                    Asimicin    Asimicin

        300 MHz, CDCl.sub.3

                    470 MHz, CDl.sub.3

                                470 MHz, C.sub.6 D.sub.6

    __________________________________________________________________________

    2     --          --          --

    3a  2.50 dddd 3a, 3b(15.1)

                    2.51 dddd 3a, 3b(15)

                                2.35

        3a, 4 (3.5) 3a, 4 (3.5)

        3a, 35(1.5) 3a, 35(1.5)

        3a, 36(1.6) 3a, 36(1.7)

    3b  2.36 dddd   2.38ddt 3b, 4 (8.0)

                                2.27

        3b, 4 (8.1) 3b, 35(1.5)

        3b, 35(1.2) 3b, 36(1.5)

        3b, 36(1.5

    4   3.85m       3.86m       3.77

    5   1.45        1.55m       1.4-1.5

    6-13

        1.25        1.25m       1.35

    14  1.50m       1.55m       1.55

    15  3.38m       3.37br, q   3.45

    16  3.85m       3.79-3.85m  3.86

    17, 18

        1.7-1.9m    1.6-2.0m    1.4-1.8

    19, 20

        3.85m       3.79-3.89m  3.73

    21, 22

        1.7-1.9m    1.6-2.0m    1.4-1.8

    23, 24

        3.85m       3.79-3.89m(23)

                                3.86(23)

                    3.37br, q (24)

                                3.45(24)

    25  1.45m       1.55m       1.55

    26-33

        1.25br, s   1.25m       1.35

    34  0.85t       0.86t       0.90

    35  7.16ddd     7.17q       6.35

    36  5.02dddq    5.06qq      4.3

    37  1.40d       1.41d       0.86

    __________________________________________________________________________

     *Some coupling constants were obtained by decoupling experiments

     x, y Indicate that assignments may be interchangable



              TABLE 3

    ______________________________________

    .sup.13 C NMR (CDCl.sub.3) Assignments and Comparisons..sup.a

    Carbon

          Bullatacin Bullatacinone

    No.   (50 MHz)(1)

                     (50 MNz)(2) Rolliniastatin

                                          Asimicin

    ______________________________________

    1     174.51s    178.73s     174.5s   174.6s

    2     131.11s    44.18d      131.1s   131.1s

    3.sup.a

          33.23t     34.41t      33.2t    33.4t

    4     69.91d     78.86d      69.9d    69.9d

    5.sup.a

          37.34t     36.67t      37.4t    37.5t

    6.sup.a

          25.99t     25.99t      26.0t    25.6t

    7-12.sup.a

          29.92t     29.56t      29.5t    29.6t

          29.28t     29.37t      29.3t    29.3t

    13.sup.a

          25.54t     25.22t      25.7t    25.6t

    14.sup.a

          33.23t     33.19t      34.1t    33.6t

    15    74.08d     74.10d      74.0d    74.1d

    16    83.20d     83.26d      83.0d    83.1d

    17.sup.a

          28.91t     28.92t      28.7t    28.4t

    18.sup.a

          28.37t     28.37t      27.8t    28.4t

    19    82.44d     82.44d      81.1d    81.7d

    20    82.17d     82.19d      81.0d    81.7d

    21.sup.a

          28.91t     28.92t      27.8t    28.4t

    22.sup.a

          28.37t     28.37t      28.4t    28.4t

    23    82.75d     82.77d      83.0d    82.8d

    24    72.34d     71.26d      71.8d    74.1d

    25.sup.a

          32.38t     32.37t      32.8t    33.4t

    26.sup.a

          25.54t     25.59t      25.5t    25.6t

    27-31.sup.a

          29.49t     29.56t      29.6t    29.6t

          29.28t     29.25t      31.9t    29.3t

    32.sup.a

          31.83t     31.85t      31.9t    31.9t

    33    22.62t     22.63t      22.6t    22.7t

    34    14.10q     14.05q      14.1q    14.1q

    35    151.70d    35.42t      151.7d   151.6d

    36    77.88d     205.44s     77.9d    77.9d

    37    19.06q     24.46q      19.1q    19.1q

    ______________________________________

     .sup.a Assignments of similar signals, as indicated, may be interchanged.



A positive response to Kedde's reagent, the strong ir absorption at 1750 cm.sup.-1 and the uv absorption maximum at 215.5 nm (.epsilon.=7974) in EtOH suggested the presence of an .alpha.,.beta.-unsaturated .gamma.-lactone. .sup.1 H--.sup.1 H decoupling experiments between protons 3 and proton 4 in the 470 MHz ) (C.sub.6 D.sub.6) spectrum revealed the presence of the hydroxyl group at carbon 4. Exact mass 141.0550 (calc. 141.0552) for C.sub.7 H.sub.9 O.sub.3 showed the cleavage between carbon 4 and carbon 5; ms of the TMS derivative of 1 and the dihydro derivative also supported the structure of fragment A. ##STR6##

The structure of fragment B, which contains the two tetrahydrofuran rings and the remaining two hydroxyl groups, was elucidated by essentially the same techniques.

.sup.13 C Nmr (50 MHz) and .sup.1 H nmr resonances were directly analogous to similar signals of asimicin [Rupprecht et al., Heterocycles, 24: 1197-1201 (1986)], uvaricin [Jolad et al., J. Org. Chem., 47: 3151-3153 (1982)], and rolliniastatin [Pettit et al., Can. J. Chem., 65: 1433-1435 (1987)], indicating the common presence of a bistetrahydrofuran moiety as illustrated in fragment B. Exact mass measurement of a second peak at m/z 141.0909 (calc. 141.0916) corresponded to C.sub.8 H.sub.13 O.sub.2. It is proposed that this peak represents cleavage between carbons 15 and 16, and between carbons 23 and 24. Eims of the TMS and acetyl derivatives of 1, .sup.1 H nmr of the triacetate of 1 and .sup.1 H--.sup.1 H decoupling experiments on the 470 MHz spectrometer for 1 confirmed these assignments. ##STR7##

Subtracting fragments A and B, the remainder, C.sub.20 H.sub.41, of the structure of bullatacin (1) belongs to the unsubstituted alkyl chain. This was corroborated by multiple CH.sub.2 resonances between .delta. 1.2-1.5 in the .sup.1 H nmr (C.sub.6 D.sub.6) and .delta. 20-40 in the .sup.13 C nmr (Tables 2 and 3). The placement of fragments A and B along the hydrocarbon chain was accomplished through analysis of the ms fragmentation pattern of 1 and its TMS, acetate, and hydrogenation derivatives.

The absolute value two dimensional homonuclear correlated spectrum (2D-COSY, 200 MHz) of bullatacin (1) confirmed the proton assignments (Table 2) and proton connectivities except for the aliphatic --CH.sub.2 -- chain.

Surprisingly, this carbon skeleton of bullatacin (1) is the same as that of asimicin [Rupprecht et al., Heterocycles, 24: 1197-1201, (1986)], and rolliniastatin [Pettit et al. Can. J. Chem., 65: 1433-1435, (1987) . However, the mp, co-TLC, .sup.1 H nmr, and most importantly, the bioactivities are different. The compounds are stereoisomers at one or more of their eight chiral centers. Since 1 yielded crystals which were not suitable for X-ray diffraction studies and since the acetogenins are difficult to convert into crystalline derivatives suitable for X-ray analysis [Pettit et al., Can. J. Chem., 65: 1433-1435, (1987)], other methods were used to predict the stereochemistry.

First, the relative configuration of six of the eight chiral centers, those on the bistetrahydrofuran ring system and its two adjacent hydroxyl bearing carbons, was obtained by comparing the .sup.1 H nmr (CDCl.sub.3) spectral signals of bullatacin (1) acetate (Table 4) with those of a recently published series of synthetic dibutylated diacetate bistetrahydrofuran models and uvaricin and uvaricin acetate; stereochemical information could then be extracted from "iterative and synergistic" analysis of very small differences in the high-field proton chemical shifts [Hoye et al. J. Am. Chem. Soc., 109: 4402-4403 (1987)].

                                      TABLE 4

    __________________________________________________________________________

    .sup.1 H NMR Assignments and Comparisons of Acetate Derivatives.*

    Bullatacin     Bullatacinone

                              Asimicin  Uvaricin

    triacetate     diacetate  triacetate

                                        acetate

    (470 MHz, CDCl.sub.3)

                   (470 MHz, CDCl.sub.3)

                              300 MHz, CDCl.sub.3)

                                        (300 MHz, CDCl.sub.3)

    __________________________________________________________________________

    2      --      3.02ddd 2, 3a(12.82)

                               --        --

                   2, 3b(9.34)

                   2, 35a(9.34)

                   2, 35b(3.42)

    3a   2.52dddd  1.74m      2.53      2.26ddd

    3b   2.58ddt   2.00m      2.53

    4    5.11dddd 4, 3a(4.0)

                   4.53dddd   5.09      1.25m

         4, 3b(7.0)

         4, 5a(10.1)

         4, 5b(1.8)

    5    1.5-2.0   1.5-2.0    1.5-1.9   1.25m

    6-13 1.25br s  1.25br s   1.25      1.25m

    14   1.5-2.0   1.5-2.0    1.5-1.9   1.75-1.9

    15   4.88dt 15, 16(8.0)

                   4.80dt 15, 16(8.0)

                              4.85      4.86ddd

         15, 14(2.0)

                   15, 14(2.0)

    16   4.00ddd   4.00ddd    3.98      3.98ddd

    17, 18

         1.5-2.0   1.5-2.0    1.5-1.9   1.75-1.9

    19, 20

         3.9br t   3.9br t    3.9       3.89br, t

    21, 22

         1.5-2.0   1.5-2.0    1.5-1.9   1.75-1.9

    23   4.00ddd   4.00ddd    2.98      3.98ddd

    24   4.93ddd 24, 25a(3.0)

                   4.93ddd 24, 25a(3.0)

                              3.83      4.92ddd

         24, 25b(2.8)

                   24, 25b(2.8)

    25   1.5-2.0   1.5-2.0    1.5-1.9   1.75-1.9

    26-33

         1.28br s  1.28br s   1.25      1.25m

    34   0.89t 34, 33(7.05)

                   0.89t 34, 33(7.05)

                              0.87      0.878t

    35   7.09d 35, 36(1.29)

                   2.66dd 35a, 35b(18.3)

                              7.06      6.98qq

                   35a, 2(9.34)

                   3.04dd 35b, 2(3.42)

    36   5.02qq     --        5.01      4.99dtq

    37   1.41d 36, 37(7.05)

                   2.20s      1.42      1.40d

    4 OAc

         2.04s      --        2.01       --

    15 OAc

         2.06s     2.05s      2.06      2.074s

    24 OAc

         2.09s     2.08s      2.06      2.046s

    __________________________________________________________________________

     *Preparation: 10 mg of parent compound is dissolved in a 1:1 mixture of

     acetic anhydride/pyridine and left overnight. Addition of ice water

     followed by extraction with chloroform yields the corresponding

     peracetylated derivative.



Results of bullatacin acetate nmr resonances (Table 4) suggested that the configuration of fragment B is threo, trans, threo, trans, erythro, the same as in uvaricin or erythro, trans, threo, trans, and threo. However, differences in the .sup.1 H nmr (CDCl.sub.3, 200 MHz) of bullatacin compared with that of uvaricin indicate the probable configuration of bullatacin to be erythro, trans, threo, trans, threo, as illustrated for 1. Similarly, we have determined that asimicin is threo, trans, threo, trans, threo. From X-ray data, rolliniastatin is reported to be threo, cis, threo, cis, and erythro [Pettit et al., Can. J. Chem., 65: 1433-1435, (1987)].

The stereochemistry at the remaining two chiral centers, carbon 4 and carbon 36, was determined by comparing nmr spectral data with those in the literature for rolliniastatin [Pettit et al., Can. J. Chem., 65: 1433-1435, (1987)]. Furthermore, essentially identical CD curves for rolliniastatin, asimicin and bullatacin suggested their stereochemical identity in this region. The CD data are given below.


A. Anticancer Activities

Anticancer activity is a potential use even for the crude extract. The bioassay results for the lethality of brine shrimp (BST), the inhibition of crown gall tumors on potato discs (PD), the reversal in morphology of db-cAMP induced AC glioma cells from that of mature, differentiated astrocytes to that of immature AC glioma cells (mouse brain cells) (9ASK), the dose that inhibits cell growth to one-half that of untreated poorly differentiated human epidermic carcinoma (9KB), the dose that inhibits cell growth to one-half that of a methylcholanthrene-induced lymphoid neoplasm in a DBA/2 mouse (9PS), and the percent displacement in a protein kinase C test for initial extract fractions are shown in Table 1. Obviously, the bioactivity is concentrated in F005.

Corresponding to these bioactivities, the pure compounds isolated from F005, bullatacin and bullatacinone, showed additional promising results (Table 5).

                                      TABLE 5

    __________________________________________________________________________

    Bioactivities of 1 and 2 and their derivatives

           BST

           LC.sub.50, mcg/ml

           95%

           Confidence

                   9PS     9KB     A549    HT29

           Interval

                   ED.sub.50, mcg/ml

                           ED.sub.50 mcg/ml

                                   ED.sub.50, mcg/ml

                                           ED.sub.50, mcg/ml

    __________________________________________________________________________

    Bullatacin(1)

           0.00159 10.sup.-15 -10.sup.-16

                           6.188 .times. 10.sup.-14

                                   1.25 .times. 10.sup.-13

                                            10.sup.-12

           0.0124

           .dwnarw.

           0.0008

    Bullatacin

           5.7     3.89 .times. 10.sup.-3

                           6.85 .times. 10.sup.-7

                                   2 .times. 10.sup.-3

                                           >10.sup.-1.sup.

    triacetate

           17.12

    (1-Ac) .dwnarw.

            2.84

    Dihydro-.sup.b

           0.0145    --.sup.a

                             --    <10.sup.-6

                                           3.33 .times. 10.sup.-5

    bullatacin

           0.0300

    (1-H.sub.2)

           .dwnarw.

           0.0100

    Bullatacinone

           0.0030  4.23 .times. 10.sup.-3

                           <10.sup.-12

                                    10.sup.-3

                                           5 .times. 10.sup.-12

    (2)    0.0090

           .dwnarw.

           0.0000

    Bullatacinone

           >10     4.22 .times. 10.sup.-2

                           5 .times. 10.sup.3

                                   2.8 .times. 10.sup.-2

                                            10.sup.-1

    diacetate

    (2-Ac)

    __________________________________________________________________________

     .sup.a A dash () indicates that tests were not conducted.

     .sup.b Obtained by hydrogenation of bullatacin in the presence of a Pd/C

     catalyst following standard laboratory procedures.



These two compounds are the most bioactive acetogenins reported to this date. When derivatized, such as by acetylation and hydrogenation, the bioactivities decrease as shown in Table 5. But the safety range for treatments might be larger. In this table, two cytotoxicity tests against human tumor cell lines such as the human lung carcinoma (A-549) [Giard et al., J. Nat. Cancer Inst. 51: 1417-1423 (1973)] and the human colon adenocarcinoma (HT-29) [Fogh, J. (ed.) Human Tumor Cells, In Vitro, pp. 115-159, Plenum Press, New York (1975)] have been performed. The fact that bullatacin is active to both these cell lines to 10.sup.-12 -10.sup.-13 mcg/ml while bullatacinone is 5.times.10.sup.-12 mcg/ml to human colon adenocarcinoma and 10.sup.-3 mcg/ml to human lung carcinoma suggested that bullatacinone has more selective cytotoxicities than bullatacin. Only a 7% displacement in a protein kinase C test at a concentration of 10 .mu.M proved that bullatacin is not a phorbol ligand. A negative result in the protein tyrosine kinase test excluded its possible mechanism of action on protein tyrosine kinase. The mode of action for the acetogenins is still unknown, but it is postulated to involve cell membranes.

To test further the selective specificity of antitumor activities, bullatacin was sent to the NIH, NCI at Bethesda, Md., for their human tumor cell line panel tests including leukemia, non-small cell lung cancer, small cell lung cancer, colon cancer, breast cancer, CNS cancer melanoma, ovarian cancer, and renal cancer. These cytotoxicity results are shown in Table 6, where IC50 is the concentration of the compound that was found to cause 50% inhibition of the growth of each cell line listed under "Cell". IC90 is the concentration of compound that was found to cause 90% inhibition of the growth of each cell line. From the results, we can say that bullatacin is best active for certain CNS cancers, non-small cell lung cancers, leukemias, and ovarian cancers.

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. Preferred dose levels will depend on the attending physician's assessment of both the nature of the patient's particular cancerous condition and the overall physical condition of the patient. Effective antitumor doses of the present compounds may 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.

                  TABLE 6

    ______________________________________

    NCI Developmental Therapeutical Program

    In Vitro Testing Results for bullatacin (1)

    DISEASE  CELL LINE   IC.sub.50 (.mu.g/ml)

                                     IC.sub.90 (.mu.g/ml

    ______________________________________

    Leukemia HOLT-4      1.41 .times. 10.degree.

                                     4.72 .times. 10.degree.

             HL-60 TB    <9.26 .times. 10.sup.-4

                                       --

             K562        <9.26 .times. 10.sup.-4

                                     4.5 .times. 10.degree.

             P388/ADR    <9.26 .times. 10.sup.-4

                                     2.56 .times. 10.degree.

             CCRF-CEM    8.94 .times. 10.sup.-5

                                     6.94 .times. 10.sup.-2

             P388        >9.24 .times. 10.sup.-2

                                     >9.24 .times. 10.sup.-2

    Non-Small

             H522        2.46 .times. 10.degree.

                                     >9.24 .times. 10.degree.

    Cell Lung

             H125        2.40 .times. 10.degree.

                                     >9.26 .times. 10.degree.

    Cancer   H23         2.38 .times. 10.degree.

                                     9.21 .times. 10.degree.

             H460        2.87 .times. 10.sup.-5

                                     1.85 .times. 10.sup.-4

             H322        <9.26 .times. 10.sup.-4

                                     >9.26 .times. 10.degree.

             EKV-X       1.15 .times. 10.sup.-4

                                     >9.26 .times.  10.sup.-2

             HOP-62      <9.26 .times. 10.sup.-4

                                     5.13 .times. 10.degree.

             SK-MES-1    >9.24 .times. 10.sup.-2

                                     >9.24 .times. 10.sup.-2

             A-549(ATCC) 5.04 .times. 10

                                     >9.24 .times. 10.sup.-2

    Small Cell

             H82         1.99 .times. 10.degree.

                                     7.14 .times. 10.degree.

    Lung Cancer

             H524        2.69 .times. 10.degree.

                                     7.54 .times. 10.degree.

             H69         2.44 .times. 10.degree.

                                     7.52 .times. 10.degree.

             H146        6.75 .times. 10

                                     >9.26 .times. 10.degree.

    Colon Cancer

             SW620       5.64 .times. 10.sup.-3

                                     5.66 .times. 10.degree.

             LOVO        9.15 .times. 10.sup.-4

                                     4.69 .times. 10.degree.

             DLD-1       2.76 .times. 10.degree.

                                     8.62 .times. 10.degree.

             HCC-2998    4.05 .times. 10.degree.

                                     >9.26 .times. 10

             HT29        3.84 .times. 10.sup.-3

                                     >9.24 .times. 10.sup.-2

    Breast   MCF7        >9.24 .times. 10.sup.-2

                                     >9.24 .times. 10.sup.-2

    Cancer

    CNS Cancer

             TE-671      2.56 .times. 10.sup.-5

                                     2.74 .times. 10.sup.-3

             U251        <9.24 .times.  10.sup.-6

                                     4.37 .times. 10.sup.-5

             SNB-19      >9.26 .times. 10.sup.-1

                                     >9.26 .times. 10.sup.-1

             SNB-44      4.42 .times. 10.degree.

                                     >9.26 .times. 10.degree.

             SNB-75      3.35 .times. 10.degree.

                                     >9.26 .times. 10.degree.

    Melanoma SK-MEL5     >9.26 .times. 10.sup.-4

                                     5.22 .times. 10.degree.

             RPMI-7951   2.73 .times. 10.degree.

                                     7.71 .times. 10.degree.

             MALME-3M    2.82 .times. 10.degree.

                                     7.67 .times. 10.degree.

             LOX         <9.26 .times. 10.sup.-4

                                     3.76 .times. 10.degree.

             SK-MEL2     3.25 .times. 10

                                     8.64 .times. 10.degree.

    Ovarian  A2780       3.77 .times. 10.sup.-5

                                     6.50 .times. 10.sup.-4

    Cancer   OVCAR-8     2.57 .times. 10.degree.

                                     7.65 .times. 10.degree.

             OVCAR-5     8.80 .times. 10.degree.

                                     >9.26 .times. 10.degree.

             OVCAR-4     >9.24 .times. 10.sup.-2

                                     >9.24 .times. 10.sup.-2

             OVCAR-3     >9.24 .times. 10.sup.-2

                                     >9.24 .times. 10.sup.-2

    Renal Cancer

             A498        4.24 .times. 10.sup.-5

                                     3.71 .times. 10.sup.- 3

             A704        1.36 .times. 10.degree.

                                     8.00 .times. 10.degree.

             SN12-K1     2.24 .times. 10.sup.-5

                                     2.87 .times. 10.sup.-4

             UO-31       1.94 .times. 10.degree.

                                     >9.26 .times. 10.degree.

             CAKI-1      3.94 .times. 10.sup.-3

                                     >9.24 .times. 10.sup.-2

    ______________________________________



B. Pesticidal Activity

Pesticidal bioassays on initial fractions were conducted at Eli Lilly Laboratories (Greenfield, Ind.) following standard procedures with seven indicator organisms: mosquito larvae (ML), blow fly larvae (BFL), corn root worm (CRW), two-spotted spider mite (2SSM), southern army worm (SAW), melon aphid (MA), and Haemonchus contortus (HC). Results in Table 7 showed unusually high activity for F005 on ML, 2SSM, MA, and BFL. This suggested that F005, itself could be used as a potent pesticidal agent.

The pesticidal activities of bullatacin (1) and bullatacinone (2) are shown in Table 7 A. The results indicate that bullatacin is significantly toxic to cotton aphids (CA) at 1 ppm, southern corn rootworm (CRW) at 24 ppm, and two-spotted spider mites (2SSM) at 10 ppm. The compounds can be formulated at concentrations of about 1 to about 200 ppm in conventional liquid or solid forms for application to pest infected areas. The lack of pesticidal activity for bullatacinone indicates that it did not contribute to the pesticidal activities of the initial ethanol extract.

It is understood that the foregoing detailed description is given merely by way of illustration and that modification and variation may be made therein without departing from the spirit and scope of the invention.

                                      TABLE 7

    __________________________________________________________________________

    Pesticidal activities of inital fractions

                              MIS

    PLEX (% MORTALITY)        (% MORTALITY)

    ML              BFL

                       HC CRW SAW 2SSM

                                      MA  ACTIVITY

    100 ppm 10 ppm

                1 ppm

                    1% 0.1%

                          300 5000

                                  5000

                                      5000

                                         PLEX

                                             MIS

    __________________________________________________________________________

    F001

       100  100 100 100

                       0  0   0    0  0  A   N

    F002

        0    0   0   0 0  0   0    0   0 A   N

    F003

       100  100 100 100

                       0  0   0   90  90 A   A

    F004

       100   0   0   0 0  0   0    0   0 A   N

    F005

       100  100 100 100

                       0  0   0   80  80 A   A

    F006

       100   0   0   0 0  0   0   70  80 A   A

    __________________________________________________________________________



              TABLE 7A

    ______________________________________

    Pesticidal activities of 1 and 2..sup.a

           Pest/percent control

    Compound ppm    CA      ML   SAW    CRW   2SSM

    ______________________________________

    Bullatacin(1)

             0.5    --.sup.b

                             0   --     --    --

              1     80       0   --     --    --

              6     --      --   --     20    --

              10    80      80   0      --    20

              24    --      --   --     80    --

             100    80      --   0      --    30

             400    90      --   0      --    20

    Bullatacinone

             0.5    --       0   --     --    --

    (2)       1     --       0   --     --    --

              6     --      --   --      0    --

              10     0       0   0      --     0

              24    --      --   --      0    --

             100     0      --   0      --     0

             400     0      --   0      --     0

    ______________________________________

     .sup.a See pesticidal activity section for details and definitions of

     abbreviation.

     .sup.b A dash () indicates that tests were not conducted.

 

 

 

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