.

US005393897A

Umted States Patent [19] [11] Patent Number: 5,393,897

Pettit et a1. [45] Date of Patent: Feb. 28, 1995

[54] ISOLATION AND STRUCI‘UR? OF 4,833,257 5/ 1989 Pettit et a1. ........................ .. 549/267
SPQNGISTATINS 5,7,8 AND 9 4,940,726 7/1990 Pettit et a1. . . 514/450
_ ' 4,996,229 2/ 1991 Moore et a1. ........ .. . 514/450
[75] Inventors: George R. Pettit, Paradise Valley; 5,096,922 3/1992 Reichenbach et al. ........... .. 514/450
Zbigniew A. Cichacz; Cherry L.
Herald, both of Tempe, all of Ariz. Primary Examiner-C. Warren Ivy
Assistant Examiner—A. A. Owens
[73] Assignee: Arizona Board of Regents acting on Attorney, Agent, or Firm-Richard R. Mybeck
behalf of Arizona State University,
Tempe, Ariz. [57] ABSTRACT
[21] Appl. No.: 86,664 A Southwest Indian Ocean marine sponge Spirastrella
spinispirulzfera (bright colored reds and purples) has
[22] Filed: Jul. 2, 1993 been found to contain new and exceptionally active cell
[51] Int. Cl.6 .......................................... .. C07D 323/00 (human cancer) growth inhibitors spongistatin 5, spon
[52] US. Cl. ....................................... .. 549/267 gistatin 7, spongistatin 8 and spongistatin 9. These com
[58] Field of Search ....................... .. 549/267; 514/450 positions are related to spongistatin 1, which was found
in a black Spongia sp. in the Porifera family. A method
[56] References Cited \
of treating human cancer cells with spongistatin 5 and
U.S. PATENT DOCUMENTS spongistatin 7 is also disclosed.
4,164,584 8/1979 Kupchan, deceased .......... .. 549/267
4,560,774 12/1985 Pettit et al. ........................ .. 549/267 6 Claims, N0 Drawings
 5,393,897
1 2
unequivocally demonstrate that the further expenditure
ISOLATION AND STRUCTURE OF of funds on developing those substances would be eco
SPONGISTATINS 5,7,8 AND 9 nomically counter-productive. The second, and more
important purpose, is to identify those substances which
INTRODUCTION demonstrate a high likelihood of success and therefore
The present invention relates to the discovery and warrant the requisite further investment necessary to
isolation of compounds extracted from the marine obtain the data which is required to meet the various
sponge Spirastrella spinispirulzfera (Class Demospon regulatory requirements imposed by those governments
giae, Order Hadromerida, Family Spirastrellidae). which regulate the market place into which such sub
These new macrocyclic lactones are designated herein stances will enter.
as “spongistatin 5”, “spongistatin 7”, “spongistatin 8” The present cost of obtaining such data approaches
and “spongistatin 9”. Spongistatin 5 and spongistatin 7 Ten Million Dollars ($10,000,000 US.) per substance.
were found to be remarkably potent and speci?c against Economics dictate that such an investment‘ not be made
the human cancer cell lines in the US. National Cancer unless there is a reasonable likelihood that it can be
Institute’s panel. Some of the work described herein recovered. Absent such an opportunity, there will be no
was supported by NCI Grant 01G CA-44344-01-04. such investment, and without investment, the research
The United States government may have certain rights requisite for the discovery of potentially life saving
to this invention. drugs will stop.
BACKGROUND OF THE INVENTION Only two hundred years ago, many diseases ravaged
20
humankind. Many of these diseases now have been
A great number of ancient marine invertebrate spe controlled or eradicated. In the development of the
cies in the Phyla Bryozoa, Mollusca and Porifera were means to treat or control these diseases, work with the
well established in the earth’s oceans over one billion appropriate common experimental animals was of criti
years ago. Certainly such organisms have explored cal importance. With the various types of cancers, and
25
trillions of biosynthetic reactions in their evolutionary with the HIV virus, such work is presently ongoing.
chemistry to reach present levels of cellular organiza The research for the treatment of various types of can
tion, regulation and defense. Marine sponges have cer is coordinated in the United States by the National
changed minimally in physical appearance for nearly Cancer Institute (NCI). NCI, as a government entity,
500 million years, suggesting a very effective chemical
evolution in response to changing environmental condi 30 has been charged with assisting anti-cancer research.
tions for at least that time period. Some recognition of To establish whether a substance has anti-cancer activ
the potential for utilizing biologically potent marine ity, NCI has established a variety of protocols, one of
animal constituents was recorded in Egypt about 2,700 which involves testing the candidate substance against a
BC, and by 200 BC sea hare extracts were being used in cell line panel containing 60 human tumor cell lines.
Greece for medicinal purposes. Such considerations, 35 This protocol has been veri?ed and is generally ac
combined with the general observation that marine cepted throughout the scienti?c community. This pro
organisms (especially invertebrates and sharks) rarely tocol and the established statistical means of evaluating
develop cancer, led to the ?rst systematic investigation the results obtained therefrom have been fully described
of marine animal and plant anticancer constituents. in the literature. See Principles & Practice of Oncology
By 1968 ample evidence had been obtained, based on PPO Updates, Volume 3, Number 10, October 1989, by
the US. National Cancer Institute’s key experimental Michael R. Boyd, M.D., Ph.D., for an in depth descrip
cancer systems, that certain marine organisms would tion of the test protocol. The statistical analysis is ex
provide new and structurally novel antineoplastic and plained in “Display and Analysis of Patterns of Differ
/or cytotoxic agents. Analogous considerations sug ential Activity of Drugs Against Human Tumor Cell
gested that marine organisms could also provide effec 45 Lines: Development of Means Graph and COMPARE
tive new drugs for other severe medical challenges, Algorithm” Journal of the National Cancer Institute
such as viral diseases. Furthermore, marine organisms Reports Vol. 81, No. 14, Pg. 1088, Jul. 14, 1989, by K.
were expected to contain potentially useful drug candi D. Paull et a1. Both of these references are incorporated
dates (and biochemical probes) of unprecedented struc herein by this reference thereto. The effectiveness and
tural types, that would have eluded discovery by con 50 validity of the NCI in vitro protocol continues to be
temporary techniques of medicinal chemistry. Fortu veri?ed.
nately, some of these expectations have been realized in Two newer references of note have been authored, in
the intervening period. Illustrative of these successes whole or in part by Dr. M. R. Boyd of the National
are the discoveries of the bryostatins, dolastatins, and Cancer Institute. The ?rst is “Data Display and Analy
cephalostatins by the Cancer Research Institute in 55 sis Strategies from the NCI disease Oriented in vitro
Tempe, Ariz. where several members of these series of Antitumor Drug Screen.” Boyd, M. R. et al, in Cyto
remarkable anticancer drug candidates are either now toxic Anticancer Drug Models and Concepts for Drug
in human clinical trial or preclinical development. See: Discovery and Development; Valeriote, F. A., Corbett,
e.g. US. Pat. Nos. 4,816,444, 4,833,257, 4,873,245, and T., Baker, L. Eds; Kluwer Academic Press: Amster
4,879,278. dam, 1992, pp ll-34. The second is “The Future of
As is Well known to those presently engaged in medi New Drug Development.” Boyd, M. R. in Current
cal research, the time between the isolation of a new Therapy in Oncology; Niederhuber, J. E., Ed. Mosby: St.
compound, and its introduction to the market place Louis, 1993, pp 11-22.
takes at least several years in the best case and can take These articles establish that those skilled in the art
several decades. Consequently, industry, in association 65 believe that in vitro screens are the primary method by
with the government, has devised a number of qualify which new antineoplastic compositions will be discov
ing tests which serve two purposes. One purpose is to ered. Progress has been sorely lacking in treating many
eliminate those substances whose results in the quali?ers kinds of cancer, as effective antineoplastic compositions
 5,393,897
3 4
for these cancers have not been discovered. Plainly, the lected off the Southeast Coast of Africa was begun. The
public interest is served by maximizing, within Consti isolation and structure of remarkably potent antineo
tutional limits, the rewards for discovering a composi plastic substances from this sponge designated spongi
tion which is demonstrated to be effective in standard statin 5, spongistatin 7, spongistatin 8 and spongistatin 9
screening tests. is disclosed. Because these spongistatins prove to be
The Constitution of the United States (Art. 1, Sec. 8) only trace (IO-7% yield) constituents, their isolation
authorizes Congress to establish the United States Pa and structural elucidation was especially dif?cult and
tent and Trademark Of?ce (USPTO) to promote scien protracted.
ti?c advancement. This obligation can only be fully met Accordingly, the principal object of the present in
when the USPTO accepts current medical and scien vention is the isolation of structurally unprecedented
ti?c realities in the area of medical research. macrocyclic lactones herein denominated spongistatin 5
The Framers of the Constitution meant to advance and spongistatin 7; having a negative log molar TGI50
scienti?c advancement. Cells are alive. The impairment of approximately ten against various human cancer cell
of human tumor cell growth is utility. The sole right lines, and the isolation of closely related compounds
obtained from the grant of Letters Patent is the right to 15 spongistatin 8 and spongistatin 9.
prevent others from exploiting the subject matter of the Another object of the present invention is to obtain
patent. The recognition of cell line data as a measure of the structural elucidation of the substance herein de
antineoplastic activity and therefore an acceptable nominated spongistatin 5, spongistatin 7, spongistatin 8
showing of “utility” can aid research in the United and spongistatin 9.
States, and thereby save the citizens of the United States A further object of the present invention is to deter
from being held hostage by foreign governments or mine a method of treating human cells afflicted with an
foreign corporations, if such research is no longer viable NCI cell line human cancer, with spongistatin 5, spongi
in the United States. statin 7, spongistatin 8 and spongistatin 9.
Numerous compounds have been discovered which These and still further objects as shall hereinafter
demonstrate signi?cant antineoplastic activity. As dis 2 appear are readily ful?lled by the present invention in a
cussed above, many of these compounds have been remarkably unexpected manner as will be readily dis
extracted, albeit with great difficulty, from living crea cerned from the following detailed description of an
tures such as the sponge or the sea hare. However, once exemplary embodiment thereof.
the isolation and testing of such compounds has prog
ressed, a practical problem exists, namely, how to ob 30 DESCRIPTION OF PREFERRED
tain a signi?cant quantity of the compound. EMBODIMENTS
A major component of vigorous efforts for over two Introduction
decades has been directed at marine sponge antineoplas The present invention relates new macrocyclic lac
tic and/or cytotoxic biosynthetic products and it is tones denominated spongistatin 5 and spongistatin 7
toward the furtherance of that effort that the present 35 which are found to have remarkably potent and speci?c
invention is directed. properties when tested against the NCI panel of human
BRIEF SUMMARY OF THE INVENTION cancer cell lines and the related compounds spongistatin
8 and spongistatin 9. The structural formulas of these
Marine animal constituents of the macrocyclic lac compounds are as follows.
tone type, are proving to be exceptionally important
sources of new anticancer drug candidates. Illustrative
are current human clinical trials of bryostatin 1 and the
advancing preclinical development of halichondrin B,
halistatin 1 and ecteinascidin 729. Seven interesting (and
cytotoxic) perhydropyrans of the onnamide series (from 45
a Theonella species of marine sponge) and l3-deox
ytedanolide, a cytotoxic macrocyclic lactone, from
Mycale adhaerens (Porifera) are descriptive of related
advances.
spongistatin 1, described in applicant’s co-pending
application Ser. No. 08/006,279 was discovered in an
Indian Ocean Spongia sp (family Spongiidae, class
Demospongiae) and represents one of the most extraor
dinarily potent substances presently known against a
subset of highly chemoresistant tumor types in the US. 55
National Cancer Institute (NCI) panel of 60 human
cancer cell lines. Intensive investigation of other active
(P388 lymphocytic leukemia cell line bioassay) fractions OCH3
from the same sponge species has revealed the presence
of two new and exceptionally potent (NCI panel) mac 60 spongistatin 5
spongistatin 7
rocyclic lactones designated spongistatin 2 and 3. spongistatin 8
Additionally, the typically bright colored (reds, pur spongistatin 9
ples) marine sponges of the genus Spirastrella (Class
Demospongiae, Order Hadromerida, Family Spirastrel Isolation of spongistatins 5, 7, 8 and 9
lidae) have not heretofore been examined for biologi 65 In July, 1980, a large scale recollection (2,409 kg) of
cally active constituents except for the arsenic content Spirastrella spinzlspirulifera preserved in ethanol was
of S. insignis. In 1973 a 20 year investigation of antineo completed. The initial extraction, solvent partitioning
plastic constituents in Spirastrella spinzlvpimlzf'era col and preparative HPLC was done on a pilot plant scale.
 5,393,897
5 6
Separation Scheme Part 1 outlines the process whereby Active fractions S and T were combined (111.0 mg)
sponge material was extracted with 2-propanol, the and subjected to chromatography on SEPHADEX
resulting extract was concentrated, then diluted with LH-20 (2.5X72 cm column) using hexane-toluene
water and extracted with methylene chloride. The dried methanol, 2:2:1. Resulting active fraction X (35.5 mg)
methylene chloride extract (13.86 kg) was next parti was separated on HPLC (GILSON, preparative, PRE
tioned with hexane and methanol-water (9:1) and the PEX C8, 10><250 mm) using 36% acetonitrile in water
aqueous methanol then taken to dryness (2 kg). The and 1.2-1.8 mg/injection. Active fraction Y contained
?nal pilot plant scale high performance liquid chroma impure spongistatin 8 (3.95 mg) and active fraction Z
tography (HPLC) separation (SILICA GEL, 0.15 X 3 In yielded 8.15 mg of impure spongistatin 9. Final HPLC
column at 150 psi) was carried out using the following separation (Separation Scheme Part 4) resulted in pure
step wise gradient system: methylene chloride spongistatin 8 (1.8 mg) and spongistatin 9 (5.4 mg).
methanol, 100-0 (160 1), 96-4 (205 1) 94-6 (102 1), 93-7 The above described scheme afforded 12.9 mg
(102 1), 90-10 (102 1), 85-15 (102 1) and 80-20 (110 1). (5.4x l0-7%, PS EDSQ 6.6)(10-5 ug/ml) —of spongista
The ef?uent was collected in 19 liter containers, exam tin 5; mp l86°—187° C.; [a]22D= —ll.l° (c=0.23,
ined by tlc and like fractions combined and concen CHgOI-I); UV (CI-130B) Am“ 228 nm, c 14840; IR (?lm)
trated. The resulting series of active fractions A-F 3430, 2936, 1734, 1643, 1591, 1387, 1273, 1173, 1090, 982
(P388 ED50 0.2 to <0.01 ug/ml) were next subjected to cm-1; high resolution FABMS, m/z 1175.5239
chromatography on SEPHADEX LH-2O in methanol [M+K]+ corresponding to C59H39ClO19K (calcd mass
(10X 130 cm columns) which gave new active fractions 1175.5324); 5.3 mg (2.2x l0-7% yield) of spongistatin
G-J (P388 ED50 0.02 to <10“2 jig/ml). 20 7; P388 ED5Q 2.6X10-3; mp 166°-167° C.; [ot]D22
Fraction G was applied to the ?rst of three MERCK —15.7° (c, 0.15, CH3OH); UV (CH3OH) km,‘ 223 nm
LOBAR size B SILICA GEL columns (25><310 mm) (log 6 4.25); IR (?lm) 3428, 2936, 1736, 1603, 1389, 1273,
connected in series. A gradient of acetone-hexane (3:47) 1173, 1090, 984 cm—1; HRFAB MS m/z 1141.5658
to acetone-hexane (2:3) was followed by a gradient of [M+K]+, calcd for C59H90O19K 1141.5653; 1.8 MG
methylene chloride-methanol (93:7) to methanol to give 25 (7.5><l0-8%, PS ED5Q 8X10-4 pg/ml) of colorless
active fractions K-O (P388 ED50 1.8x 10-5 to <10-5 spongistatin 8; mp 158°—159° C.; [aha-32° (c, 0.18,
ug/ml), as shown in Separation Scheme Part 2. Frac CH3OH); IR(?lm) 3439, 2936, 1736, 1653, 1602, 1383,
tion K (38.9 mg) was then applied to two ANALTECH 1252, 1178, 1090 cm-1; 5.4 mg (2.2X1O-7% yield, PS
analytical tlc plates, 10x20 cm. Elution with acetone ED50 < l0—4 pg/ml) of spongistatin 9; mp l64°—165°;
30 [a]D22-33.3° (C, 0.14, CH3OH); IR (?lm) 3435, 2940,
hexane (1:1) provided a fraction enriched in a single
component, K860 (3.6 mg). A second tlc separation was 1736, 1647, 1591, 1385, 1254, 1178, 1090 cm—1.
Once the structure of spongistatin l was established
done, using 3.6 mg on an ANALTECH analytical plate and its relationship to spongistatins 5, 7, 8 and 9 deter
(7.5x 10 cm) with acetone-hexane, 3:2. A 0.3 mg mined, structure solutions for the Spirastrella antineo
amount of K860 resulted. From the earlier active frac plastic constituents were conducted as will be later
tions H, additional nearly pure K860 was isolated, 6.3 described.
mg, which was combined with the 0.3 mg to give 6.6 Super?cially the 1-"C- and 1H-NMR spectra of spon
mg total. gistatin 5 was similar to those of spongistatins suggest
Further puri?cation using HPLC (ALTEX Program ing a similar skeleton. The four methyl doublet signals
mable Model 420 system, 2 model 110A pumps) with 40 at 8 1.01 (J=6.7 Hz), 1.14 (J=7.0 Hz), 0.90 (J=7.1 Hz),
solvent gradient of CHzClz to 93:7 CHgClg-MeOH on a and 0.85 (J =6.7 Hz), one methyl singlet at 8 1.14, one
PARTISIL M9 SILICA GEL column gave 6.3 mg of methoxyl signal at 8 3.31 and the SP2 proton signals at
nearly pure K860. The sample was next chromato 8 5.38 (doublet of doublets, J = 10,11 Hz), 5.47 (doublet
graphed using HPLC (PARTISIL M9 10/50 ODS-2 of triplets, J =7,ll Hz), 4.97 (broad singlet), 4.95 (broad
column) with a methanol-H2O (1:1) to methanol gradi 45 singlet), 6.13 (broad doublet of doublets, J=6,l5 Hz),
ent to give pure K860, spongistatin 4 (1.4 mg). A paral 6.41 (broad doublet, J = 15 Hz), 5.42 (broad singlet) and
lel separation sequence beginning with combined frac 5.33 (broad singlet) were consistent with this assump
tions M, N and O (42.0 mg) led to impure K859 (8.4 mg) tion. Furthermore, the coupling pattern of the signals at
which was puri?ed by HPLC to give pure K859, spon 8 5.42, 5.33 and 6.41 and the lack of an H-SO signal
gistatin 7 (0.5 mg). 50 suggested a chlorine atom at C-50. But further analyses
Active fraction I (Separation Scheme Part 3) was of 1D and 2D NMR spectra revealed signi?cant differ
separated on SILICA GEL RP-2 (3.7 X44 cm column) ence between spongistatin 5 and spongistatins 1-4 and 6
using the gradients, water to methanol, methanol to in two respects: ?rst, the absence of an acetyl signal;
methylene chloride, to give active fraction P (1.75 g). second, the pair of SP2 methylene signals for H-l3a
Using the solvent system hexane-toluene-methanol 55 common to spongistatins 14 were not present. In a
(3:121) on SEPHADEX LH-20 (5.5)(96 cm column) 1H-13C correlated spectrum, a 13C signal at 8 70.72 was
provided active fractions Q through U. Subjecting frac found correlated with two lH signals at 8 4.47 (broad
tion U (0.173 g) to repetitive separations with a GIL doublet, J = 13 Hz) and 4.09 (broad doublet, J = 13 Hz).
SON preparative system (Models 303 and 305 pump and The IH-IH COSY spectrum of spongistatin 5 displayed
PREPEX C8, l0><250 mm column) with the isocratic 60 two signals at 8 4.47 and 4.09 with long range couplings
solvent, 36% acetonitrile in water and 1.2-1.8 mg/injec to a signal at 8 5.24 (broad doublet, J=11 Hz). In turn,
tion, gave fraction V (42.1 mg) containing spongistatins the signal at 8 5.24 was found coupled with a signal at 8
4 and 5 and fraction W (29.2 mg) containing spongista 5.28 (broad doublet of doublets, J =9,1l Hz). In the 13C
tins 6 and 7. Final separation was achieved by repetitive NMR spectrum of spongistatin 5, signals for SP2 carbon
analytical (GILSON) HPLC separations on LI 65 atoms at C-l3, C-28, C-29, C-45, C-45a, C48, C49,
CHROSPHER 100 RP18 (4.6x 250 mm), 45% acetoni O50, O51 were observed with chemical shifts essen
trile in water and 0.2-0.3 mg per injection. The detec tially the same as found for spongistatins 1, 3 and 4. The
tion of HPLC peaks was by UV, lt=230 mm. remaining one SP2 carbon signal at 8 120.13 showed a
 5,393,897
7 8
correlation only with the one proton signal at 8 5.24 in Due to the paucity of spongistatin 8 structural eluci
the 1H-13C spectrum. Such evidence indicated that a dation was simpli?ed by ?rst deducing the structure of
- C-l2,13 double bond allylic to a C-13a atom bonded to spongistatin 9. Once those high resolution FABMS and
oxygen that resulted in the AB pattern at 8 4.47 and 4.09 high ?eld 2D NMR interpretations were in hand for
in the 1H NMR spectrum was present in spongistatin 5. spongistatin 9, the structure of spongistatin 8 was com
The dramatic down?eld shift of the C-15 signal at 8 pleted as follows. The tetrahydrofuran ring of spongi
84.46 (8 73.75 in spongistatin 4) suggested that a tetra statin 8 was recognized by chemical shifts at 8 4.45
hydrofuran ring comprising G15, G14, C-13 and C-13a (broad d, J-13 Hz), 4.10 (broad d, J-13 H)/70.70 and 1.95
was present. The molecular formula suggested by (acetyl, s, 3H)/2l.3l and 172.56 (acetyl, s, 3H). A signal
FABMS also favored this conclusion. The presence of a at 8 6.33 (d,d,d, J-10,10,17 Hz) indicated that C-50 was
tetrahydrofuran ring was further con?rmed by an devoid of the usual spongistatin chlorine atom at that
HMBC spectrum in which the 13C signal at 8 84.46 ppm position. A series of 1H-1H NMR COSY experiments
(C-15) was strongly correlated with one of the two allowed assignment of the remaining 1H signals and the
H-13a signals at 8 4.47. All of the 1H- and 13C-NMR 13C NMR signals were interpretated by comparison
data as well as the HMBC correlations strongly sup with the analogous NMR carbon data from spongistatin
ported the structure assigned to spongistatin 5. 9.
The structures assigned spongistatins 1-5 required The structure of the spongistatin 9 was determined
extensive high ?eld (400 and 500 MHz) 2-D NMR and mainly by high ?eld NMR spectroscopy utilizing re
high resolution mass spectral interpretations that were sults of lH-lH COSY, 1H-13C COSY, APT, and HMBC
quite dif?cult. But, results of those challenging analyses 20 NMR experiments. Both the 1H- and the 13C-NMR
proved very important to completing the structural spectra of spongistatin 9 indicated that it was a member
elucidation of spongistatins 6 and 7. The lH-NMR spec of the spongistatins by signals at 8 1.13 (3H, S)/30.17,
trum of spongistatin 6 indicated a spongistatin-type ring 1.04 (3H, d)/14.59, 1.14 (3H, d)/15.10, 0.89 (3H,
system. For example, the four methyl signals present in d)/11.52, 0.84 (3H, d)/ 13.00 an ester carbonyl signal at
spongistatins 1-5 were found at 8 0.97 (d, J :68 Hz), 25 8 173.66 and a ketone carbonyl signal at 8 213.42. Spon
1.12 (d, J=7.1 Hz), 0.91 (d, J=7.1 Hz), and 0.83 (d, gistatin 9 was found to possess a tetrahydrofuran ring by
J :66 Hz). The 1H signals at 8 2.90 (broad d, J = 18 Hz), signals at 8 4.45 (broad doublet, J-13 Hz) and 4.10 broad
2.83 and a 13C signal at 8 215.29 were characteristic of doublet, J-l3 Hz) corresponding to two H-13a. An ace
the spongipyran C-17 carbonyl system. The presence of tyl group was evident by signals at 8 1.95 (3H)/21.35
an ABX spin system at 8 5.04 (broad d, J = 11 Hz), 5.17 30 and 172.61. That the acetyl group was attached to C-5
(broad d, J=l7 Hz), 6.33 (d,d,d, J=l1,l1,17 Hz) sug oxygen atom was evidenced by the chemical shift of
gested a proton rather than a chlorine atom at C-50 H-5 at 8 4.96. The two broad singlets at 8 5.42 and 5.33
similar to that of spongistatin 2. The presence of one and the lack of a 1H signal for 050 were indicative of
acetyl group was evident by 1H and 13C signals at 8 2.03 a chlorine atom at that position. Complete assignment
(s, 3H) 172.80, and 21.65. The 1H-lH COSY and 1H-13’C 35 for the 1H- and 13C-NMR signals appear in Compilation
COSY experiments established the 1H and 13C assign 1 together with the APT and HMBC results.
ments. The chemical shifts of the H-5 and H-15 signals
at 8 5.03 (1H, broad s) and 3.83 (1H, broad d, J =9 Hz)
readily pointed to attachment of the acetyl group at Compilation 1.
C-5. Preparative HPLC of Spirastrella gainispimlzfera Extract
Analogous structural determination approaches were Amount P388 ED5Q
Column Fractions‘ Concentrate (g) (pg/m1)
applied to spongistatin 7. The HRFAB MS data estab
One: 2.0 Kg l-4 1700 rechromato— —
lished molecular formula C59H90O19 employing peak initial weight graphed on 2nd
matching at m/z 1141.6 [M+K]+. Results of the 2D column
1H- and 13C- NMR experiments with spongistatin 7 45 5-15 0.0 -
again suggested a spongipyran ring system, but with the 16-17 12.5 17.0
18-19 9.0 2.3
additional tetrahydrofuran ring found in spongistatin 5. 20-21 A 16.0 0.2
The latter feature was revealed by the 1H-13C signals at 22-27 B 28.0 0.13
8 5.29 (broad d,d, J=10,l1 Hz)/67.26, 5.24 (broad d, 28-35 11.0 1.5
J = 10 Hz)/ 120.16, 4.48 (broad d, J = 13 Hz), 4.10 (broad 36-47 45.0 1.4
48-51 28.0 10.0
d, J = 13 Hz)/70.75, and the signals at 8 3.92 (d,d, J=3.7, Two: 1.7 Kg precipitate, 145.0 —
10 Hz)/84.50. The presence of ABX signals at 8 5.04 initial weight batyl alcohol
(broad d, J= 10 Hz), 5.17 (broad d, J: 17 Hz), and 6.32 1-2 0.0 -

(d,d,d, J =10, 10, 17 Hz) were readily attributed to a 3-9 470.0 17.0
10-11 150.0 1.2
hydrogen at C-50 rather than a chlorine atom. The 55 12-14 140.0 11.0
tetrahydrofuran ring was con?rmed by HMBC experi 15-18 C 275.0 0.14
ments when one of the two H-13a signals at 8 4.48 19-26 D 100.0 <0.01
showed a cross peak with the C-15 signal at 8 84.50. The 27-34 E 35.0 0.21
35-46+ F 175.0 0.30
HR FABMS spectral data also strongly supported a
Visualization took place with both uv and spray reagents of 5% ceric sulfate in 15%
spongipyran ring system bearing an additional tetrahy sulfuric acid and 1:2:97 anisaldehyde-sulfuric acid-acetic acid.
drofuran ring. Thus, the structure of spongistatin 7 was ‘Fraction volume was 19 liter each, and like fractions were combined and concen
unambiguously established. trated by tlc comparisons. Tlc system was 95:5 methylene chloride-methanol on
Brinkman Sil G/UV 254 20 X 20 cm plates with batyl alcohol used as a reference
sample.

Separation Scheme Part 1

 5,393,897
9 10
-continued
Spimstrella spimlrpimlzfera
2,409 Kg wet weight
1. 2-propano1
2. concentrate extract
3. water
4. extract with CH2C12, 4x

CH2C12 (13.86 Kg)

1. hexane
2. MeOH—H7_O (9:1), 4::
I
hexane (9.3 Kg)
MeOH—H7_O (2 Kg)
Preparative HPLC, 21:
silica gel (29 Kg/colmnn),
CH2C12-MeOI-I gradient
I I I I I I
Active Fractions A B C D E F
Weight (g) 16 28 275 100 35 175
P388-ED50 (pug/m1) 0.20 0.13 0.14 0.011 0.21 0.30
-T/C (mg/Kg) — — 178(30) 169(1.8) 232(37.5) 154(29.5)

Sephadex LH-20, MeOH

(10 X 130 cm)
Active Fractions G H I J
Weight (g) 1.4 5.4 29.1 36.0
P388 ED50 (pg/m1) <10-2 <10-2 <1o-2 2.2 x 10-2
Sgaration Scheme Part 2

Fraction G (1.4 g)
silica gel
1. acetone-hexane gradient
2. CI-IgClz-MeOH gradient
I I I I
Active Fractions K L M N 0
Weight (mg) 38.9 13.5 7.3 14.0 29.7
P388 E1350 (pg/ml) 1.8 x 10-5 - <10-5 -- <1o-5

I_______|
<"""silica gel """"""""""""""""""""“->
analytical prep tlc, 10 X 20 cm
1:1 aoetonezhexane

Impure K860 (3.6 mg) Impure K859 (4.0 mg)

6.3 mg of impure ' Other
+K860 from fraction H fractions
_ (4.4 mg)
silica gel prep tlc

K860 (6.6 mg) K859 (8.4 mg)

<-------HPLc, Partisil M9 ----------------------
CHzCl; to 93:7 CH2Cl2—MeO1-I gradient

K860 (6.3 mg) K859 (1.7 mg)

<- --- "HPLC, Partisil M9 10/50 ODS-2 --------- -'—>

MeOI-I—H2O (1:1) to MeOH gradient

K860 (1.4 mg) K859 (0.5 mg)

Spongistatin 4 Spongistatin 7
P388 in vivo toxic (0.52 +0.13) 55% (0.38)
ILS (mg/kg): 63% (0.065) 49% (0.19)
34% (0.10)
Separation Scheme Part 3
 5,393,897
11 12
-continued
Fraction I (29.1 g)
silica gel RP-2, 3.7 X 44 cm

H2O +MeOI-l, MeOH —->cH2c12 gradients

Active Fractions P -
(1.75 g)
Sephadex LH-20, 5.5 X 96 cm
hexane-toluene-MeOl-I, 3:1 :1
| l l l I
Active Fractions Q R S T U
Weight (g) 0.110 0.063 0.066 0.045 0.173
HPLC, Prepex C8,
10 X 250 mm, 36%
CH3CN in H1O,
1.2-1.8 rng/injection

I I
V W
(42.1 mg) (29.2 mg)

<- ---- "HPLC, LiChrospher 100 RP 18, ------------- ->

4.6 X 250 mm, 45% CH3CN in H2O,
0.2-0.3 mg/injection

spongistatin 4 spongistatin 5 spongistatin 6 spongistatin 7

(10.7 mg) (12.9 mg) (8.4 mg) (5.3 mg)
% yield 4.4 x 10-7 5.4 x 10-7 3.5 X 10-7 22 X 10-7
P388 ED50(ng/m1) 4.9 X 1o-~5 6.6 x 10-5 3.4 X 10-3 26 x 10-3

Separation Scheme Part 4

Combined Fractions S,T (111.0 mg)

Sephadex Ll-I-ZO,
hexane-toluene-MeOh, 2:2:1
X
(35.5 mg)
HPLC, Prepex C8, 10 X 250 mm,
36% CH3CN in H2O, 1.2-1.8 mg/injection
I I
Y Z
impure spongistatin 8 impure spongistatin 9
(3.95 mg) (8.15 mg)
<- ---------- "HPLC, LiChrospher 100 ---------------
RP18, 4.6 X 250 mm,
45% CH3CN in H2O,
0.2-0.3 mg/injection
spongistatin 8 spongistatin 9
(1.8 mg) (5.4 mg)
a yield 7.5 x 10-8 2.2 x 10-7
P388 ED50(pg/ml) 8.4 x 10-4 <10-4

Evaluation of spongistatin 5 and spongistatin 7

against the US. National Cancer Institute panel of 60
human cancer cell lines gave dramatic results. Compar shown) with that shared by the important general class
ative testing of spongistatin 5 and spongistatin 7 in the 55 of microtubule-interactive antimitotics. The structural
NCI 60 cell line in vitro screening panel revealed an variations thus far observed in this intriguing new fam
overall potency of spongistatin 5 and spongistatin 7 ily of antineoplastic substances do not result in substan
comparable to spongistatin l (e.g., panel mean G150 tial loss of the critical in vitro activity attributes. Animal
10"1°M; Table 1). The compounds are among the most data demonstrated an increased life span (ILS) of 78%
potent of all substances tested to date in the NCI screen. 60 at 10 ug/kg dose for spongistatin 1, an ILS of 65% at 5
Interestingly, several of the human breast cancer cell pg/kg dose for spongistatin 5 and an ILS of 60% at 40
lines recently incorporated into the NCI screening ug/kg for spongistatin 7. The advantageous or disad
panel were among the most sensitive (e.g., G150 vantageous effects of these structural variations upon
10-41-10" 12M). Furthermore, results of pattern-recog the in vivo activity potential is unknown, but will be
nition analyses revealed that the highly distinctive 65 addressed in further biological evaluations of all of the
mean-graph “?ngerprint” (pattern of relative cellular available compounds so remarkably active in vitro.
sensitivity) produced in common by spongistatins 1, 5 Discovery of the spongistatins in quite distant (in
and 7 (Table 1) is closely correlated in turn (data not respect to taxonomy and geography) Porifera species
 5,393,897
13 14
suggests that this very important new series of remark TABLE Z-continued
able antineoplastic agents may prove to be widely dis
tributed in such marine invertebrates and/or associated NMR Assignments for spongistatin 5 Recorded in CD3OD, the
coupling constants are in Hz (in parenthesis); the n and p are
marine microorganisms. Interestingly, a recent ?rst APT results.
study of Porifera found adjoining Easter Island, the HMBC(50O MHZ,
most remote South Paci?c Island, uncovered both Spi l3c(100 MHZ) l11(400 MHZ) c {O 11)
rastrella cunctatrix and Spongia virgultosa in the same 24 34.7711 2.40 brd(14);1.63 - 11-22
general area. A future examination of these two sponges 25 65.121 4.02 brs 11-24
for spongistatins should prove useful. Presently ex 26 39.1411 1.62 ";1.62 1 11-24
tended in vivo human cancer xenograft evaluations of 10 27 61.911 5.04 ddd(5,9,10) 11-29
28 131.261 5.38 0110011)) 11-30
spongistatins 4 and 5 are being pursued. Also research 29 134.051 5.47 (110,11) 1130
directed at completing the absolute con?gurational 30 28.30p 2.13 ';2.13 1 11-28
assignments for the spongistatins by X-ray crystal struc 31 27.4211 1.65 21.25 * 11-30
ture determinations is underway. The NMR data for 32 33.2111 1.46 *;1.30 * 11-30
33 67.991 4.21 1110(9) 113411135
spongistatin 5, spongistatin 7, spongistatin 8 and spongi 34 39.571 1.62 =1 H-34a,H-36
statin 9 appear on Tables 2, 3, 4 and 5. The currently 3411 11.491 0.90 11(71) 1134,1133
available in vivo data for spongistatins l, 4, 5, 6 and 7 35 72.031 3.75 1 H-34a,I-1-36,1-1-34
appears in Table 6. NCI cell line data for spongistatins 36 34.26p 2.00 *;1.63 * 11-38
5 and 7 appear in Tables 7 and 8. 37 99.4011 H-38,H-36,H-35
38 73.321 3.38 1,11
20 39 81.76n 3.76 1 1140111140
TABLE 1
40 37.601 2.02 1 1140111141
Results of Comparative Antitumor Evaluations of 4011 13.001 0.85 d(6.7) 1141,1140
spongistatins 1, 5 and 7 in the NCI In Vitro Primary Screen” 41 80.721 4.88 1111(9,11) 1140111421139,
spongistatin Mean Panel G150 Compare Correlation 1140
Number (>< 10— 1° M)" 06111118161111 42 73.811 3.18 1(9) 1141,1144
25 43 79.69n 3.44 1111(11) H-42,H-39,H-44
1 1.17 1.00
44 40.491, 2.80 *;2.19 ' H-45a,H-46,H-42
s 1.23 0.92
7 10.10 0.81
45 143.831, H-44,H-46,H-47,
1145111143
"All compounds were tested in quadruplicate at ?ve different con 451 116.3411 4.97 bIS;4.95 1111 1144,1146
centrations (10-8, 10-9, 10-10, 11-11 and 10'12 M) against the 46 44.34;) 2.34 111111103, 14), 1145111441147
entire panel of 60 human tumor cell lines comprising the NCI 2.25 brdd(6.1,l4)
screen. 47 71.031 4.38 dc1d(6.5,6.5,6.5) H-46,H-48,H-49
bStandard errors averaged less than 15% of the respective means. 48 138.771 6.13 brdd(6,15) 1146,1147
"Correlation coefficients from the Compare pattern-recognition 49 127.871 6.41 1111:1(15) 1151,1147
algorithm were calculated by computer using the TGI-centered s0 139.6lp H-48,H-49,H-51
mean graph pro?les of differential cellular sensitivities to l, and 51 116.16p 5.42 brs 1149
5. The TGI mean graph pro?le of l was used as the benchmark or 35 5.33 brs
“seed” for all of the comparisons. OMe 55.871 3.31 5 11-21
' Coupling constants for these signals are not measured due to overlapping.

TABLE 2
NMR Assignments for spongistatin 5 Recorded in CD3OD, the TABLE 3
coupling constants are in Hz (in parenthesis); the 11 and p are NMR Assignments for spongistatin 7 in CD3OD (n and p are APT
APT results. results, coupling constants are in Hz in parenthesis). The mixing
HMBC(500 MHz, time for the HMBC experiment was set at 130 micro second !.
13c(100 M11Z) 111(400 MHZ) c 16 11) HMBC(500 MHz,
1 173.80p H-2,H-41 l3c(1o0 MHZ) 111(400 M11Z) c to H)
2 40.15p 2.70 $2.70 ' 45 1 173.8315 1141
3 63.28n 4.56 brm H-2,H-4 2 40.17p 2.69 *;2.69 1
4 37.92p 1.78 21.63 ‘ H-6 3 63.301 4.56 111111 112,118
5 65.5411 4.07 brs 11-6 4 37.941: 1.74 ';1.60 *
6 40.62p 1.86 *;1.74 ' 11-8 5 65.581 4.07 brs 11-6
7 101.49p H-8,H-5,H-6 6 40.66p 1.87 111d(14);1.74 * H-8
8 45.99p 1.74 ‘;1.58 ‘ 1-1-9a,I-1-10 50 7 101.5211 H-8,I-1-6
9 69.46): H-9a,H-8 8 46.03;: 1.72 ";1.58 * H-9a,H-6
9a 30.05n 1.14 5 11-10 9 69.4711 1-1-9a,H-8
10 45.0lp 1.57 21.48 dd(11,12) H-9a,H-8 9a 30.071 1.14 1 11-8
11 67.23n 5.28 brdd(9,11) 11-10 10 45.05p 1.56 ~;1.48 11<1(11,14) 11-91
12 120.13n 5.24 brd(1 1) H-13a,H-10 11 67.26n 5.29 11111110011) 1-1-10,1-1-6
13 148.64p H-14a,H-13a 12 120.161 5.24 brd(lO) 1113111140
13a 70.72p 4.47 brd(13); H-12,H-14 55 13 148.68p 11-14111-1311115,
4.09 brd(13) 11-14
14 37.82n 3.29 brm H-14a,1-I-12,H-16 131 70.75p 4.48 1111103);
14a 15.09n 1.01 d(6.7) 11-15 4.10 0111(13)
15 84.4611 3.92 dd(3.7,10) H-l6a,H-14a,H-13a, 14 37.861 3.28 * 11-1411
11-16 1411 15.131 1.01 d(6.6) 11-15
16 47.03n 2.86 dq(7,11) I-1-16a 60 15 84.501 3.92 dd(3.7,10) 11-161111441111311,
16a 14.56n 1.14 d(7.0) H-16 H-16
17 213.24p H-16a,H-18,H-15 16 47.06n 2.86 dq(7,l0) H-16a,1-1-15
l8 50.99p 2.95 dd(10,19), 16a 14.571 1.14 11(70) H-16,H-15
2.82 brd(l9) 17 213.2411 H-16a,H-18,H-15
19 66.79n 4.14 brt(11) H-18,H-20 18 51.02p 2.95 11110019),
2O 38.06p 2.07 8;1.03 ‘ H-22 65 2.82 1110(19)
21 74.60n 3.58 m H-20,H—OMe,H-22 19 66.821 4.14 1
22 44.14}: 2.05 '; 20 38.10p 2.07 ‘;1.03 1 H-22,H-18
1.18 ddd(12,l2,12) 21 74.631 3.59 111 H—OMe
23 100.17p H-22,H-24 22 44.1811 2.05 ';1.18 1(12) 1124
 5,393,897
17 18
TABLE 5-continued TABLE 5-continued
NMR Assignments for spongistatin 9 recorded in CD3OD. The
NMR Assignments for spongistatin 9 recorded in CD3OD. The coupling constants are given by (Hz) and the n and p refer to
coupling constants are given by (Hz) and the n and p refer to APT results. The mixing time for the l-IMBC experiment was set
APT results. The mixing time for the HMBC experiment was set at 60 microsecond.
at 60 microsecond. HMBC(50O MHz,
l3c(100 MHz) 111(400 MHZ) c to 11)
HMBC(500 MHz,
43 79.711 3.43 brdd(9,l0) 114211391144
13c(1o0 MHz) 111(400 MHz) c to H) 44 40.51p 2.80 1110(14); 11451111461142
19 66.83n 4.14 * 1143,1120 10 2.13 11111110014)
45 143.85p 1144114611147
20 38.121) 2.07 111.04 * 1122 45a 116.43p 4.96 bi'S;4.95 brs H-44,H~46
21 74.651 3.60 111 H-20,H—0Me,H-22 46 44.4311 2.36 brdd(7,l4), 11451111441147,
22 44.16p 2.06 111.20 1(12) 2.25 1111111(7,14) 1143
23 100.2311 11-22 47 71.071 4.39 ddd(6,7,7) H-46,l-l-48,H-49
4s 138.79n 6.14 brdd(6,15) 1146,1147
24 34.8111 2.41 brd(l4);1.63 1 11-22 15 49 127.931 6.41 brd(l5) 1151,1147
25 65.171 4.02 brs 111-24 5o 139.6511 114311491151
51 116.18p 5.42 brs 1149
26 39.2011 1.62 ‘;1.62 1 1124
5.33 brs
27 62.02n 5.05 <11111(5,9,10) H-29 0146 55.921 3.34 5 11-21
221 131.2711 5.32 1111(10) 11-27 0A0 172.6lp l-I--OAc
29 133.98n 5.49 111 1127 20 21.351 1.95 s
30 28.34p 2.13 112.10 * H-28,l-l-29 ‘ Coupling constants for these signals are not provided due to overlapping.
31 27.431; 1.65 21.27 1
32 33.2211 1.46 111;1.31 111 TABLE 6
33 67.9611 4.21 111 H-34a In vivo data for spongistatins l. 4, 5, 6 and 7
25
34 39.5611 1.62 1 11041111361134 Dose
34a 11.521 0.89 110.1) Compound ()tg/kg/dose) % ILS
35 72.091 3.76 * H-34a,H-36 spongistatin 1 40.0 -10
36 34.30p 2.02 ‘;l.67 1 1133 (toxic)
37 99.47p H-38,H-36,H-35 25.0 +33
30 10.0 +72
3s 73.38n 3.33 hrs spongistatin 4 5.0 +55
39 81.76n 3.76 * 11401111410 spongistatin 5 5.0 +65
40 37.691 2.03 1 11401111411132 spongistatin 6 40.0 +70
40a 13.001 0.84 d(6.7) 1141,1140 spongistatin 7 40.0 +60
41 80.78n 4.88 1 11401111421139, Tumor system: P388; implant: I? 1.0 E + 06 cells; host: CDZFL female mice;
35 median day of death: l0; schedule: 1?, QlD X 9(1); vehicle‘: 5% ETOH + distilled
11-40 water; % lLS: % increase in life span over the controls.
42 73.321 3.19 1(9) 1141

45

50

55

60

65

27
This compound can also be effectively modi?ed with
some or all of the following acids.
(a) saturated or unsaturated, straight or branched
chain aliphatic carboxylic acids, for example, acetic,
propionic, butyric, isobutyric, tert-butylacetic, valeric,
isovaleric, caproic, caprylic, decanoic, dodecanoic,
lauric, tridecanoic, myristic, pentadecanoic, palmitic,
margaric, stearic, acrylic, crotonic, undecylenic, oleic,
hexynoic, heptynoic, octynoic acids, and the like; (b)
saturated or unsaturated, alicyclic carboxylic acids, for
example, cyclobutanecarboxylic acid, cyclopentanecar
boxylic acid, cyclopentenecarboxylic acid, methylcy
clopentenecarboxylic acid, cyclohexanecarboxylic
acid, dimethylcyclohexanecarboxylic acid, dipropylcy
clohexanecarboxylic acid, and the like; (0) saturated or 15
unsaturated, alicyclic aliphatic carboxylic acids, for
example, cyclopentaneacetic acid, cyclopentanepro
piom'c acid, cyclohexaneacetic acid, cyclohex
anebutyric acid, methylcyclohexaneacetic acid, and the
like; (d) aromatic carboxylic acids, for example, benzoic
acid, toluic acid, naphthoic acid, ethylbenzoic acid,
isobutylbenzoic acid, methylbutylbenzoic acid, and the
like; and (e) aromatic-aliphatic carboxylic acids, for
example, phenylacetic acid, phenylpropionic acid, phe
nylvaleric acid, cinnamic acid, phenylpropioplic acid
and naphthylacetic acid, and the like. Suitable halo-,
nitro-, hydroxy-, keto-, amino-, cyano-, thiocyano-, and
lower alkoxyhydrocarbon carboxylic acids include hy
drocarboncarboxylic acids as given above which are
substituted by one or more of halogen, nitro, hydroxy, 30 to about 50% w/w of the composition; and for paren
keto, amino, cyano, or thiocyano, or lower alkoxy,
advantageously lower alkoxy of not more than six car
bon atoms, for example, methoxy, ethoxy, propoxy,
butoxy, amyloxy, hexyloxy, and isomeric forms thereof.
Examples of such substituted hydrocarbon carboxylic
acids are: mono-, di-, and trichloroacetic acid;—and
-chloropropionic acid;—and -bromobutyric acid;—and
-iodovaleric acid; mevalonic acid; 2- and 4-chlorocy
clohexanecarboxylic acid; shikimic acid; 2-nitro-1
methyl-cyclobutanecarboxylic acid; l,2,3,4,5,6-hexa
chlorocyclohexanecarboxylic acid; 3-bromo-2-methyl
cyclohexanecarboxylic acid; 4- and 5-bromo-2-methyl
cyclohexanecarboxylic acid; 5- and 6-bromo-2-methyl
cyclohexanecarboxylic acid; 2,3-dibromo-2-methylcy
clohexanecarboxylic acid; 2,5-dibromo-2-methylcy
clohexanecarboxylic acid; 4,5-dibromo-2-methylcy
clohexanecarboxylic acid; 5,6-dibromo-2-methylcy
clohexanecarboxylic acid; 3-bromomethylcyclohex
anecarboxylic acid; 6-bromo-3-methylcyclohexanecar
boxylic acid; 1,6-dibromo-3-methylcyclohexanecar
boxylic acid; 2-bromo-4-methy1cyclohexanecarboxylic
acid; l,2-dibromo-4-methylcyclohexanecarboxylic acid;
3-bromo-2,2,3-trimethylcyclopentanecarboxylic acid;
L-bromo-3,5-dimethylcyclohexanecarboxylic acid; ho
mogentisic acid, 0-, m-, and p-chlorobenzoic acid; anisic
acid; salicylic acid; p-hydroxybenzoic acid; b-resorcylic
acid; gallic acid; veratric acid; trimethoxybenzoic acid;
trimethoxycinnamic acid; 4,4’-dichlorobenzilic acid; o-,
m-, and p-nitrobenzoic acid; cyanoacetic acid; 3,4- and
3,5-dinitrobenzoic acid; 2,4,6-trinitrobenzoic acid; thi
ocyanoacetic acid; cyanopropionic acid; lactic acid;
ethoxyformic acid (ethyl hydrogen carbonate); malic
acid; citric acid; isocitric acid; 6-methylsalicyclic acid;
mandelic acid, levulinic acid; pyruvic acid; glycine;
alanine; valine; isoleucine; leucine; phenylalanine; pro
line; serine; threonine; tyrosine; hydroxyproline; omi
thine; lysine; arginine; histidine; hydroxylysine; phe
nylglycine; p-aminobenzoic acid; m-aminobenzoic acid;
5,393,897
28
anthranilic acid; aspartic acid; glutamic acid; aminoa
dipic acid; glutamine; asparagine; and the like.
The administration of spongistatin 5, spongistatin 7,
spongistatin 8 and spongistatin 9 and their pharmaceuti
cally active, physiologically compatible derivatives is
useful for treating animals or humans afflicted with a
neoplastic disease, such as, for example, acute myelo
cytic leukemia, acute lymphocytic leukemia, malignant
melanoma, adenocarcinoma of lung, neuroblastoma,
small cell carcinoma of lung, breast carcinoma, colon
carcinoma, gastric carcinoma, ovarian carcinoma, blad
der carcinoma, hematologic malignancies and the like.
The dosage administered will be dependent upon the
identity of the neoplastic disease; the type of host in
volved, including its age, health and weight; the kind of
concurrent treatment, if any; the frequency of treatment
and therapeutic ratio.
Illustratively, dosage levels of the administered ac
tive ingredients are: intravenous, 0.1 to about 40 ug/kg;
intramuscular, 1 to about 50 tug/kg; orally, 5 to about
100 ug/kg; intranasal instillation, 5 to about 100 ug/kg;
and aerosol, 5 to about 100 ug/kg. As used herein,
pg/kg means weight of active ingredient in micrograms
divided by the body weight of the host in kilograms.
25 Expressed in terms of concentration, an active ingre
client can be present in the comp'ositions of the present
invention for localized use about the cutis, intranasally,
pharyngolaryngeally, bronchially, intravaginally, rec
tally, or ocularly in a concentration of from about 0.01
teral use in a concentration of from about 0.05 to about
50% w/w of the composition and preferably from
about 5 to about 20% w/w.
The composition of the present invention are prefera
35 bly presented for administration to humans and animals
in unit ,dosage forms, such as tablets, capsules, pills,
powders, granules, suppositories, sterile parenteral solu
tions or suspensions, sterile non-parenteral solutions or
suspensions, and oral solutions or suspensions and the
40 like, containing suitable quantities of an active ingredi
ent.
For oral administration either solid or ?uid unit dos
age forms can be prepared.
Powders are prepared quite simply by comminuting
45 the active ingredient to a suitably ?ne size and mixing
with a similarly comminuted diluent. The diluent can be
an edible carbohydrate material such as lactose or
starch. Advantageously, a sweetening agent or sugar is
present as well as a ?avoring oil.
50 Capsules are produced by preparing a powder mix
ture as hereinbefore described and filling the mixture
into formed gelatin sheaths. As an adjuvant to the ?lling
operation, a lubricant su?h as a tale, magnesium stea
rate, calcium stearate and the like can be added to the
55 powder mixture before the ?lling operation.
Soft gelatin capsules are prepared by machine encap
sulation of a slurry of active ingredients with an accept
able vegetable oil, light liquid petrolatum or other inert
oil or triglyceride.
Tablets are made by preparing a powder mixture,
granulating or slugging, adding a lubricant and pressing
into tablets. The powder mixture is prepared by mixing
an active ingredient, suitably comminuted, with a dilu
ent or base such as starch, lactose, kaolin, dicalcium
65 phosphate and the like. The powder mixture can be
granulated by wetting with a binder such as corn syrup,
gelatin solution, methylcellulose solution or acacia mu
cilage and forcing through a screen. As an alternative to
 5,393,897
29
granulating, the powder mixture can be slugged, i.e.,
run through the tablet machine and the resulting imper
fectly formed tablets broken into pieces (slugs). The
slugs can be lubricated to prevent sticking to the tablet
forrning dies by means of the addition of stearic acid, a
stearic salt, talc or mineral oil. The lubricated mixture is
then compressed into tablets.
When desired, each tablet can be provided with a
protective coating consisting of a sealing coat or enteric
coat of shellac, a coating of sugar and methylcellulose
and a polish coating of carnauba wax. Fluid unit dosage
forms for oral administration such as syrups, elixirs and
suspensions can be prepared wherein each teaspoonful
of composition contains a predetermined amount of
active ingredient for administration. The water-soluble 15
forms can be dissolved in an aqueous vehicle together
with sugar, ?avoring agents and preservatives to form a
syrup. An elixir is prepared by using a hydroalcoholic
vehicle with suitable sweeteners together with a ?avor
ing agent. Suspensions can be prepared of the insoluble 20 preparations are illustrative of the preparation of the
forms with a suitable vehicle with the aid of a suspend
ing agent such as acacia, tragacanth, methylcellulose
and the like.
For parenteral administration, ?uid unit dosage forms
are prepared utilizing an active ingredient and a sterile 25
vehicle, water being preferred. The active ingredient,
depending on the form and concentration used, can be
either suspended or dissolved in the vehicle. In prepar
ing solutions the water-soluble active ingredient can be
dissolved in water for injection and ?lter sterilized be
fore ?lling into a suitable vial or ampule and sealing.
Advantageously, adjuvants such as a local anesthetic,
preservative and buffering agents can be dissolved in
the vehicle. Parenteral suspensions are prepared in sub
stantially the same manner except that an active ingredi
ent is suspended in the vehicle instead of being dis
solved and sterilization can not be accomplished by
?ltration. The active ingredient can be sterilized by
exposure to ethylene oxide before suspending in the
sterile vehicle. Advantageously, a surfactant or wetting
agent is included in the composition to facilitate uni
form distribution of the active ingredient.
In addition to oral and parenteral administration, the
rectal and vaginal routes can be utilized. An active
ingredient can be administered by means of a supposi 45
tory. A vehicle which has a melting point at about body
temperature or one that is readily soluble can be uti
lized. For example, cocoa butter and various polyethyl
ene glycols (Carbowaxes) can serve as the vehicle.
For intranasal instillation, a ?uid unit dosage form is
prepared utilizing an active ingredient and a suitable
pharmaceutical vehicle, preferably pyrogen free
(“P.E.”) water. A dry powder can be formulated when
insuf?ation is the administration of choice.
For use as aerosols, the active ingredients can be 55 an active ingredient for the 20 um used above.
packaged in a pressurized aerosol container together
with a gaseous or lique?ed propellant, for example,
dichlorodi?uoromethane, carbon dioxide, nitrogen,
propane, and the like, with the usual adjuvants such a
cosolvents and wetting agents, as may be necessary or
desirable.
The term “unit dosage form” as used in the speci?ca
tion and claims refers to physically discrete units suit
able as unitary dosages for human and animal subjects,
each unit containing a predetermined quantity of active 65
material calculated to produce the desired therapeutic
effect in association with the required pharmaceutical
diluent, carrier or vehicle. The speci?cations for the
30
novel unit dosage forms of this invention are dictated by
and are directly dependent on (a) the unique character
istics of the active material and the particular therapeu
tic effect to be achieved, and (b) the limitation inherent
in the art of compounding such an active material for
therapeutic use in humans, as disclosed in this speci?ca
tion, these being features of the present invention. Ex
amples of suitable unit dosage forms in accord with this
invention are tablets, capsules, troches, suppositories,
powder packets, wafers, cachets, teaspoonfuls, table
spoonfuls, dropperfuls, ampules, vials, segregated multi
ples of any of the foregoing, and other forms as herein
described. '
The active ingredients to be employed as antineoplas
tic agents can be easily prepared in such unit dosage
form with the employment of pharmaceutical materials
which themselves are available in the art and can be
prepared by established procedures. The following
unit dosage forms of the present invention, and not as a
limitation thereof.
EXAMPLE I
Several dosage forms can be prepared embodying the
present invention. They are shown in the following
examples which the notation “active ingredient” signi
?es spongistatin 5, spongistatin 7, spongistatin 8 and
spongistatin 9 and their synthetic counterparts and the
non-toxic pharmaceutically active derivatives thereof.
COMPOSITION “A”
Hard-Gelatin Capsules
One thousand two-piece hard gelatin capsules for
oral use, each capsule containing 20 pg of an active
ingredient are prepared from the following types and
amounts of ingredients:
Active ingredient, micronized 20 mg
Corn Starch 20 gm
Talc 20 gm
Magnesium stearate 2 gm
The active ingredient, ?nely divided by means of an
air micronizer, is added to the other ?nely powdered
ingredients, mixed thoroughly and then encapsulated in
the usual manner.
The foregoing capsules are useful for treating a neo
plastic disease by the oral administration of one or two
capsules one to four times a day.
Using the procedure above, capsules are similarly
prepared containing an active ingredient in 5, 25 and 50
pg amounts by substituting 5 pm, 25 pm and 50 pm of
COMPOSITION “B”
Soft Gelatin Capsules
One-piece soft gelatin capsules for oral use, each
containing 20 ng of an active ingredient (?nely divided
by means of an air micronizer), are prepared by ?rst
suspending the compound in 0.5 ml of corn oil to render
the material capsulatable and then encapsulating in the
above manner.
The foregoing capsules are useful for treating a neo
plastic disease by the oral administration of one or two
capsules one to four times a day.
 5,393,897
31
COMPOSITION “C”
Tablets
One thousand tablets, each containing 20 pg of an
active ingredient are prepared from the following types
and amounts of ingredients.
Active ingredient micronized 20 mg
Lactose 300 gm
Corn starch 50 gm
Magnesium stearate 4 gm
Light liquid petrolatum 5 gm
The active ingredient ?nely divided by means of an
air micronizer, is added to the other ingredients and
then thoroughly mixed and slugged. The slugs are bro
ken down by forcing through a Number Sixteen screen.
The resulting granules are then compressed into tablets,
each tablet containing 20 pg of the active ingredient.
The foregoing tablets are useful for treating a neo
plastic disease by the oral administration of one or two
tablets one to four times a day.
Using the procedure above, tablets are similarly pre
pared containing an active ingredient in 25 pg and 10
pg amounts by substituting 25 mg and 10 mg of an
active ingredient for the 20 pm used above.
COMPOSITION “D”
Oral Suspension
30 tory is foil wrapped.
One thousand ml of an aqueous suspension for oral
use, containing in each teaspoonful (5 ml) dose, 5 pg of
an active ingredient, is prepared from the following
types and amounts of ingredients:
35
Active ingredient micronized 5 mg
Citric acid 2 gm
Benzoic acid 1 gm
Sucrose 790 gm
Tragacanth 5 gm
Lemon Oil 2 gm
Deionized water, q.s. 1000 ml
The citric acid, benzoic acid, sucrose, tragacanth and
lemon oil are dispersed in suf?cient water to make 850
ml of suspension. The active ingredient ?nely divided
by means of an air micronizer, is stirred into the syrup
until uniformly distributed. Suf?cient water is added to
make 1000 ml.
The composition so prepared is useful for treating a
neoplastic disease at a dose of l tablespoonful (15 ml)
three times a day.
COMPOSITION “E”
Parenteral Product
55 ml given one to four times a day.
A sterile aqueous suspension for parenteral injection,
containing in 1 ml, 30 pg of an active ingredient for
treating a neoplastic disease, is prepared from the fol
lowing types and amounts of ingredients:
60
Active ingredient, micronized 30 mg
Polysorbate 8O 5 gm
Methylparaben 2.5 gm
Propylparaben 0.17 gm
Water for injection, q.s. 1000 ml
65 powder is placed in a shaker-type container.
All the ingredients, except the active ingredient, are
dissolved in the water and the solution sterilized by
32
?ltration. To the sterile solution is added the sterilized
active ingredient, ?nely divided by means of an air
micronizer, and the ?nal suspension is ?lled into sterile
vials and the vials sealed.
The composition so prepared is useful for treating a
neoplastic disease at a dose of 1 milliliter (1M) three
times a day.
COMPOSITION “F”
10
Suppository, Rectal and Vaginal
One thousand suppositories, each weighing 2.5 gm
and containing 20 pg of an active ingredient are pre
pared from the following types and amounts of ingredi
ents:
Active ingredient, micronized 20 mg
Propylene glycol 150 gm
Polyethylene glycol #4000, q.s. 1,500 gm
The active ingredient is ?nely divided by means of an
air micronizer and added to the propylene glycol and
the mixture passed through a colloid mill until uni
formly dispersed. The polyethylene glycol is melted
and the propylene glycol dispersion added slowly with
stirring. The suspension is poured into unchilled molds
at 40° C. The composition is allowed to cool and solid
ify and then removed from the mold and each supposi
The foregoing suppositories are inserted rectally or
vaginally for treating a neoplastic disease.
COMPOSITION “G”
Intranasal Suspension
One thousand ml of a sterile aqueous suspension for
intranasal instillation is prepared, containing 20 pg of an
active ingredient per ml of suspension, from the follow
ing types and amounts of ingredients:
Active ingredient, micronized 30 mg
Polysorbate 80 5 gm
Methylparaben 2.5 gm
Propylparaben 0.17 gm
All the ingredients, except the active ingredient, are
dissolved in the water and the solution sterilized by
?ltration. To the sterile solution is added the sterilized
active ingredient, ?nely divided by means of an air
micronizer, and the ?nal suspension is aseptically ?lled
into sterile containers.
The composition so prepared is useful for treating a
neoplastic disease, by intranasal instillation of 0.2 to 0.5
An active ingredient can also be present in the undi
luted pure form for use locally about the cutis, intrana
sally, pharyngolaryngeally, bronchially, or orally.
COMPOSITION “H”
Powder
Five mg of an active ingredient in bulk form is ?nely
divided by means of an air micronizer. The micronized
The foregoing composition is useful for treating a
neoplastic disease, at localized sites by applying a pow
der one to four times per day.
 5,393,897
33 34
COMPOSITION “I” disclosure which is limited only by the scope of the
claims appended herein.
Oral Powder Accordingly, what is claimed is:
Ten mg of an active ingredient in bulk form is ?nely 1. A composition of matter having the following
divided by means of an air micronizer. The micronized structure:
powder is divided into individual doses of 20 ng and
packaged.
The foregoing powders are useful for treating a neo
plastic disease, by the oral administration of one or two
powders suspended in a glass of water, one to four times
per day.
COMPOSITION “J”
Insufflation
Ten mg of an active ingredient in bulk form is ?nely
divided by means of an air micronizer.
The foregoing composition is useful for treating a
neoplastic disease, by the inhalation of 30 pg one to four
times per day.
COMPOSITION “K”
Hard Gelatin Capsules
One hundred two-piece hard gelatin capsules for oral OCH3
use, each capsule containing 20 pg of an active ingredi 25
cut. wherein R=Cl or H and R1=H or COCH3.
The active ingredient is ?nely divided by means of an 2. A composition of matter according to claim 1
air micronizer and encapsulated in the usual manner. wherein R1 =H.
The foregoing capsules are useful for treating a neo 3. A composition of matter according to claim 2
plastic disease, by the oral administration of one or two 30 wherein R=Cl and said composition is denominated
capsules, one to four times a day.
herein as “spongistatin 5”.
Using the procedure above, capsules are similarly 4. A composition of matter according to claim 2
prepared containing active ingredient in 5, 25 and 50 ptg
amounts by substituting 5 mg, 25 mg and 50 mg of the wherein R=H and said composition is denominated
active ingredient for the 20 mg used above. 35 herein as “spongistatin 7”.
From the foregoing it is apparent that a new and 5. A composition of matter according to claim 1
useful invention has been herein described and illus wherein R: H and R1=COCH3 and said composition is
trated which fulfills all of the aforestated objectives in a denominated herein as “spongistatin 8”.
remarkably unexpected fashion. It is of course under 6. A composition of matter according to claim 1
stood that such modi?cations, alterations and adapta 40 wherein R=C1 and R1=COCH3 and said composition
tions as may readily occur to the artisan confronted is denominated herein as “spongistatin 9”.
with this disclosure are intended within the spirit of this * * * * *

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