Antitumor compound, compositions and method of use

The present invention relates to a new antitumor compound, a method for isolating same from a red alga, antitumor compositions containing same and methods of using same for treating patients with cancer. The compound of the present invention is 6(R)-bromo-3(S)-bromomethyl-7-methyl-2,3,7-trichloro-1-octene.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to a compound which exhibits antitumor 
activity, a method for isolating the compound from a red alga, and methods 
for using the compound. More specifically, the present invention relates 
to: isolation and identification of a new chemical compound, and of 
medically useful compositions containing the same. The compound of the 
present invention exhibits advantageous pharmacological, toxicological or 
antitumor properties, such as, for example, killing or inhibiting the 
growth of human tumors. 
2. Description of Related Art 
Since the mid-1970's the Rhodophyta (red algae) have been known to produce 
halogenated monoterpenes [Stallard, M. O., et al: Comp. Biochem. Physiol. 
B, 49: 25-35, 1974]. Although scores of acyclic, monocyclic and bicyclic 
halogenated monoterpenes have been identified [Sims, J. J., et al: In 
Marine Natural Products, Chemical and Biological Perspectives, (Scheuer, 
P. J., ed.), New York: Academic Press, 1978, pp. 297-378], this class of 
compounds has been confined to the genera Plocamium and Chondrococcus. The 
structure elucidation of these compounds has not been a simple task. The 
relatively volatile monoterpenes tend to decompose under electron-impact 
ionization mass spectrometry (EI-MS) conditions and acquisition of 
molecular weight and formula information has often been difficult. Correct 
placement of chlorine and bromine substituents has not proven to be 
straightforward, as NMR chemical shift arguments are clouded by the 
cumulative effects of multiple substituents on the C.sub.10 skeleton. 
The compound of the present invention is 
6(R)-bromo-3(S)-bromomethyl-7-methyl-2,3,7-trichloro-1-octene. A compound 
proposed to have the same structure as the compound of the present 
invention was reported previously by Burreson et al., [Burreson, B. J., et 
al: Chemistry Lett., 1111-1114, 1975.] as an unresolved component in a 
mixture of monoterpenes from Chondrococcus hornemannii; however, the 
material was only partially characterized. Neither a proof of the 
structure, nor the absolute stereochemistry (there are two chiral centers, 
carbon atoms 3 and 6, and thus four possible diasteromers), nor a method 
of isolating the compound of the present invention in substantially pure 
form has previously been reported in the literature. 
Other carbocyclic halomonoterpenes from Rhodophyta reportedly have shown 
general cytotoxicity in brine shrimp assays [Konig, G. M., et al: J. Nat. 
Prod. 53: 1615-1618, 1990] and in vitro inhibition of murine leukemia 
[Gonzales, A. G., et al: Planta Med. 44: 44-46, 1982] or other [Kusumi, 
T., et al: J. Org. Chem. 52: 4597-4600, 1987] cell lines. However, the 
novel profiles of selective antitumor activity of the compound of the 
present invention that can be demonstrated in the U.S. National Cancer 
Institute's new disease-oriented primary screen, which predicts antitumor 
activity against human solid tumors, has not previously been reported for 
any halomonoterpene in the literature. Neither the specific compound of 
the invention nor pharmaceutical compositions of the compound nor methods 
of using the compound or compositions thereof for treatment of cancer have 
been heretofore described. 
SUMMARY OF THE INVENTION 
The present invention is directed to a new compound, in substantially pure 
form and having the structure: 
##STR1## 
The present invention also is directed to a method of isolating and 
purifying the compound of the present invention from a red alga. 
Another aspect of the present invention is directed to antitumor 
compositions which comprise an antitumor effective amount of the compound 
of the present invention and a pharmaceutically acceptable carrier. 
Any of the above antitumor compositions can further include an antitumor 
effective amount of one or more other known antitumor agent. 
The present invention also is directed to a method of treating cancer which 
comprises administering to a patient in need thereof, an antitumor 
effective amount of the compound of the present invention. 
The method of the present invention also comprises coadministering an 
antitumor effective amount of one or more other known antitumor agent, 
together with the compound of the present invention. 
Further scope of the applicability of the present invention is apparent 
from the detailed descriptions and drawings provided below. However, it 
should be understood that the detailed descriptions and specific examples, 
while indicating preferred embodiments of the invention are given by way 
of illustration only, since various changes and modifications within the 
spirit and scope of the invention will become apparent to those skilled in 
the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention specifically relates to a compound 
[6(R)-bromo-3(S)-bromomethyl-7-methyl-2,3,7-trichloro-1-octene] which has 
novel antitumor activity, methods of obtaining same from red alga, 
compositions containing same, and methods of using the compound or 
compositions of same for treating cancer. 
A variety of methods can be used for isolation of the compound of the 
present invention. Most generally the compound is extracted from a red 
alga using an organic solvent. The compound can be further purified by 
chromatography (column or HPLC) and/or by recrystallization. A fresh red 
alga can be used as the source, but generally the alga is frozen 
immediately after harvesting. This alga is then used directly or 
freeze-dried before the extraction is done. Preferably the red alga is 
Portieria hornemannii, most preferably Portieria hornemannii collected 
near Chanaryan, Batan Island, in the Philippines, see Example 1. A 
preferred general method of isolating the compound of the invention is: 
a) obtaining a fresh or frozen sample of said red alga, 
b) extracting said compound from said sample with an organic solvent which 
dissolves said compound, to form an extract, 
c) partitioning said extract between a nonpolar organic solvent and an 
aqueous solvent, to form a partitioned nonpolar organic extract, 
d) chromatographing said partitioned nonpolar organic extract on an 
adsorption, partition or size exclusion matrix to form fractions, 
e) isolating said compound from said fraction containing it. 
In step b) the organic solvent which dissolves the compound is generally a 
mixture of a nonpolar organic solvent and a polar organic solvent; the 
nonpolar organic solvents include CH.sub.2 Cl.sub.2, CHCl.sub.3, toluene 
and hexane; the polar organic solvents include MeOH, EtOH, isopropyl 
alcohol and acetone. In step c) the organic nonpolar solvents include 
CH.sub.2 Cl.sub.2, hexane, CCl.sub.4, CHCl.sub.3, and ethyl acetate; and 
typical aqueous solvents are mixtures of water and methanol. Solvent 
mixtures that can be used in this partitioning step are: a) CH.sub.2 
Cl.sub.2 vs 19:1 H.sub.2 O-MeOH, b) hexane vs 9:1 MeOH-H.sub.2 O, c) 
CCl.sub.4 vs 8:2 MeOH-H.sub.2 O, d) CHCl.sub.3 vs 7:3 MeOH-H.sub.2 O, and 
e) EtOAc vs H.sub.2 O. In step d) the chromatography is column 
chromatography and the chromatographic matrix can be the adsorption type, 
or the partition type or the size exclusion type, or a combination of any 
of these types. Sephadex LH-20 combines all three of these types, and is 
characterized by mild treatment and good recoveries. Sephadex LH-20 is the 
most preferred chromatographic matrix material. The isolation of step e) 
is carried out by either simply evaporating the solvent or by 
recrystallization. 
A typical procedure is as follows. A sample of the frozen red alga was 
lyophilized and extracted with CH.sub.2 Cl.sub.2 -MeOH (1:1), followed by 
a MeOH rinse. The crude organic extract was partitioned between CH.sub.2 
Cl.sub.2 and H.sub.2 O-MeOH (19:1). The dichloromethane phase was reduced, 
in vacuo, and permeated through Sephadex LH-20 (column 2.5.times.190 cm) 
with CH.sub.2 Cl.sub.2 -MeOH (1:1) at 1-2 ml/min. Approximately three 
column volumes were used to elute the extracts. Fractions were monitored 
by and separated on the basis of UV absorption at 254 nm; fraction volumes 
thus vary. The seventh fraction was known to contain the compound of the 
invention because it was cytotoxic in the HIV screen and because the 
compound crystallized on evaporation of the solvent. The seventh fraction 
was crystallized from MeOH to give the compound of the present invention 
in substantially pure form. 
The definitive proofs of the structure of the compound of the present 
invention can be obtained by a combination of methods including primary 
spectral analyses (e.g., high-resolution NMR and mass spectrometry, 
infrared and UV spectroscopy), comparisons of spectral and physicochemical 
properties with related literature precedents, and by x-ray 
crystallographic analysis. 
The novel antitumor activity of the compound of the present invention can 
be demonstrated in the U.S. National Cancer Institute's new human tumor, 
disease-oriented screen [Boyd, M. R.: In CANCER: Principles and Practice 
of Oncology. Update Series. (DeVita, V. T. Jr., Hellman, S., and 
Rosenberg, S. A., eds.), Philadelphia: Lippincott, 1989, pp. 1-12; Boyd, 
M. R.: In Current Therapy in Oncology (Niederhuber, J. E., ed.) 
Philadelphia: B. C. Decker, Inc., 1991, in press, both of which references 
are hereby incorporated by reference in their entirety], which accurately 
predicts antitumor activity of chemical compounds against human cancers. 
This screen measures the ability of the compound to selectively kill or 
inhibit the growth of diverse human cancers. More specifically, using this 
screen, it can be shown that the compound of the present invention is 
highly active against certain types of human solid tumors (e.g., brain 
cancer, renal cancer and colon cancer) which are very resistant or 
completely resistant to existing antitumor drugs; and, it can be shown 
that the compound is also active against other human solid tumors and 
leukemia cancer cells. By these observations, and with other detailed 
analyses of the characteristic tumor cellular response profile produced by 
the compound of the present invention in the above screen (see example 3), 
it can be shown that the same compound is a highly novel antitumor agent 
with an unprecedented structure-activity relationship for treatment of 
human solid tumors. It is unusual for a compound to be more active against 
human solid tumor cell lines than human leukemia cell lines. The compound 
of the invention is thus shown to be a new and broadly efficacious 
anticancer agent. The results shown in Example 3 and in FIGS. 2 and 3 show 
that the compound is efficacious against human leukemias, lymphomas and 
solid tumors. Solid tumors include lung cancer, colon cancer, brain 
cancer, melanoma, ovarian cancer, renal cancer, head and neck cancer, 
testicular cancer, germ-line cancers, endocrine tumors, uterine cancer, 
breast cancer, sarcomas, gastric cancer, hepatic cancer, esophageal cancer 
and pancreatic cancer. 
Compositions of the present invention comprise as the active ingredient, 
the compound of the present invention and a pharmaceutically acceptable 
carrier. Suitable carriers for use in the present invention include, but 
are not limited to, injectable or orally or rectally administrable oils, 
lipid emulsions, aqueous solutions or suspensions, or, in the case of 
orally or rectally administrable tablets or capsules, a pharmacologically 
inert excipient. 
The compound and compositions of the present invention can be shown to kill 
or inhibit the growth of human cancer, both leukemic and solid tumor 
cancers; more particularly solid tumors, most particularly tumors of the 
brain, kidney and colon. 
The present invention further relates to a method of preventing or treating 
cancer comprising administering to a patient an "antitumor effective 
amount" of a composition of the present invention. The composition can be 
administered, for example, orally, subcutaneously or intravenously. The 
composition can be present as a solution suitable, for example, for 
intravenous injection or infusion. The composition can also be present in 
unit dosage form, such as, for example, a tablet or capsule. The 
"antitumor effective amount" is the dose necessary to achieve an 
"effective level" in the individual patient Since the "effective level" is 
used as the preferred endpoint for dosing, the actual dose and schedule 
may vary, depending upon interindividual differences in pharmacokinetics, 
drug distribution and metabolism. The "effective level" may be defined, 
for example, as the blood or tissue level desired in the patient that 
corresponds to a concentration of the compound of the present invention 
which kills or inhibits the growth of human tumors in an assay which can 
predict for clinical antitumor activity of chemical compounds. The 
"effective level" for compounds of the present invention also may vary 
when the compositions of the present invention are used in combination 
with other known antitumor compounds or combinations thereof. One skilled 
in the art can easily determine the appropriate dose, schedule, and method 
of administration for the exact formulation of the composition being used, 
in order to achieve the desired "effective concentration" in the 
individual patient. One skilled in the art also can readily determine and 
use an appropriate indicator of the "effective concentration" of the 
compounds of the present invention by a direct (e.g., analytical chemical 
analysis) or indirect (e.g., with clinical chemistry indicators) analysis 
of appropriate patient samples (e.g., blood and/or tissues), or by direct 
or indirect observations of the shrinkage or inhibition of growth of the 
individual patient's tumor. There are many references in the art that 
teach how one works out the protocols of administering anticancer agents 
to patients, see for example "Cancer Chemotherapy: Principles and 
Practice" ed. Chabner and Collins, J. B. Lippincott, 1990, especially 
chapter 2, by J. B. Collins, which is hereby incorporated by reference in 
its entirety. 
The method of treating cancer using the compound of the invention can be 
made more effective by administering other anticancer compounds along with 
the compound of the invention. These other anticancer compounds would 
include all of the known anticancer compounds approved for marketing in 
the United States and those that will become approved in the future. See, 
for example Table 1 and Table 2 of Boyd "The Future of Drug Development" 
(In Press in J. E. Niederhuber, Ed., Current Therapy in Oncology; Section 
I. Introduction to Cancer Therapy; Chapter 2., B. C., Decker, Inc., 
Philadelphia, 1991, which is hereby incorporated by reference in its 
entirety). More particularly, these other anticancer compounds would 
include doxorubicin, bleomycin, vincristine, vinblastine, VP-16, VW-26, 
cisplatin, procarbozine and taxol for solid tumors in general; alkylating 
agents, such as BCNU, CCNU, methyl-CCNU and DTIC for brain or kidney 
cancers; and, antimetabolites such as 5-FU and methotrexate for colon 
cancer. 
EXAMPLES 
The following non-limiting EXAMPLES are provided to aid in the 
understanding of the present invention. It is understood in turn that 
modifications can be made in the procedures set forth without departing 
substantially from the true spirit and scope of the invention. 
EXAMPLE 1: Isolation of the Compound of the Present Invention from Extracts 
of the Red Alga Portieria hornemannii 
Portieria hornemannii (Lyngbye) P. C. Silva (=Chondrococcus hornemannii) 
was collected by NCI contractor Ernani G. Menez near Chanaryan, Batan 
Island, in the Philippines in April, 1986. A voucher specimen is on 
deposit at the Smithsonian Institution, recorded with the collector's 
number Q67I573. The alga was frozen immediately after collection, at 
-20.degree. C. A 1 kg sample of the frozen material was lyophilized and 
extracted with CH.sub.2 Cl.sub.2 -MeOH (1:1), followed by a MeOH rinse 
(once with each solvent, the quantity used was sufficient to cover the 
sample). The two solvents were combined, evaporated at less than 
40.degree. C. to form a crude organic extract (2.5g). This was partitioned 
between CH.sub.2 Cl.sub.2 (100 mL) and H.sub.2 O-MeOH (19:1, 100 mL). The 
dichloromethane phase was reduced, in vacuo, and permeated through 
Sephadex LH-20 (column 2.5.times.190 cm) with CH.sub.2 Cl.sub.2 -MeOH 
(1:1) at 1-2 ml/min. Approximately three column volumes were used to 
eluate the extracts. Fractions were monitored by and separated on the 
basis of UV absorption at 254 nm; each fraction represents a peak in the 
UV absorbance, thus fraction volumes vary. The seventh fraction was known 
to contain the compound of the invention because it was cytotoxic and 
because the compound crystallized on evaporation of the solvent. The 
seventh fraction was crystallized from MeOH to give 55 mg of the compound 
of the present invention in substantially pure form. 
EXAMPLE 2: Structure Proof of the Compound of the Present Invention 
Preliminary .sup.1 H and .sup.13 C nmr analyses suggested a monoterpene. 
While EI-MS failed to provide a discernible molecular ion or M-HX fragment 
ion, chemical ionization-mass spectroscopy (CI-MS) did reveal a weak 
pseudomolecular ion cluster beginning at m/z 416 (M+NH.sub.4 +), which 
corresponded to the molecular formula C.sub.10 H.sub.15 Br.sub.2 Cl.sub.3 
; this was confirmed by high resolution EIMS. 
An earlier report [Burreson, B. J., et al: Chemistry Lett. 1111-1114, 1975] 
showed a compound proposed to have the same structure as the compound of 
the present invention; however, their material was an unresolved component 
in a mixture of monoterpenes from Chondrococcus hornemannii; moreover, the 
material was only partially characterized. The exact stereochemical 
structure of the Burreson et al. compound was not disclosed in this 
reference, nor was the compound ever isolated in pure form, nor was any 
method taught that might be used to isolate the compound, nor was any 
method taught that could be used to synthesize the compound. Thus it is 
not known whether the compound of Burreson is the same as that of the 
present invention (there ar four possible diastereomers with the structure 
of the Burreson et al. compound); it is clear, however, that Burreson et 
al. never put the compound of this invention (nor the one he partially 
characterized) into the hands of the public. 
For proof of the structure of the compound of the present invention, in 
addition to our x-ray analysis, we used .sup.1 H-.sup.1 H COSY, HMQC and 
HMBC experiments to assign definitively all the resonances in the .sup.1 H 
and .sup.13 C nmr spectra of 1, see Table 1. 
TABLE 1 
______________________________________ 
.sup.1 H and .sup.13 C NMR Assignments, Compound 1 
Carbon/ 
1--- 
Hydrogen # .sup.13 C.sup.b 
.sup.1 H.sup.c 
______________________________________ 
1 118.5 5.61 (d,2.4) 
5.84 (d,2.4) 
2 139.7 -- 
3 73.8 -- 
4 37.8 2.53 (ddd,13.2,10.2,1.9) 
2.14 (t, 10.2) 
5 30.1 2.48 (ddd,13.2,11.5,1.5) 
1.97 (dddd,11.5,11.2,10.2,1.9) 
6 64.6 4.02 (dd,11.2,1.5) 
7 71.6 -- 
8 27.1 1.66 (s) 
9 38.6 3.83 (d,10.7) 
3.77 (d,10.7) 
10 33.1 1.77 (s) 
______________________________________ 
.sup.a recorded in CDCl.sub.3 on a Varian VXR500 spectrometer 
.sup.b 125 MHZ, 
.sup.c 500 MHZ, .delta. (multiplicity, J in Hz) 
Other spectral and physicochemical data for the compound of the present 
invention are as follows: Mp 49.degree.-50.degree.;[.alpha.].sub.D 
+206.degree. (c 1.08, CH.sub.2 Cl.sub.2); CI-MS (NH.sub.3): m/z 
416/418/420/422/424 (MNH.sub.4 +, 0.3/1.0/1.0/0.5/0.1), 336/338/340/342 
(1.8/3.3/2.2/0.6), 300/302/304 (4.3/6.9/3.4), 247/249 (17/22), 203/205 
(19/13), 167/169 (27/24), 96/66, 52 (100); HREIMS m/z 401.8583 (calc'd for 
C.sub.10 H.sub.15.sup.35 Cl.sup.37 Cl.sub.2.sup.79 Br.sub.2 -401.8619), 
399.8637 (calc'd for C.sub.10 H.sub.15.sup.35 Cl.sup.37 Cl.sub.2.sup.79 
Br.sub.2 -399.8679). 
The final definitive proof of the structure of the compound of the present 
invention was from single-crystal x-ray diffraction analysis. The compound 
was crystallized from n-pentane by slow evaporation at -25.degree. C. and 
appeared as clear thin plates. A specimen with approximate dimensions 
0.1.times.0.15.times.0.2 mm was selected for analysis. Preliminary x-ray 
photographs displayed orthorhombic symmetry. Accurate lattice constants of 
a=6.115(4), b=12.456(6), c=19.188(10).ANG. were determined from 30 
diffractometer measured 2.theta.-values. Systematic extinctions, optical 
activity, and crystal density were consistent with space group P2.sub.1 
2.sub.1 2.sub.1 with one molecule of composition C.sub.10 H.sub.15 
Br.sub.2 Cl.sub.3 forming the asymmetric unit. Additional crystallographic 
parameters were V=1461.6(14).ANG..sup.3, .mu.(MoK.alpha.) 6.02 mm.sup.-1, 
and D.sub.c =1.824 g cm.sup.-3 for Z=4. Intensity data were collected on a 
Nicolet (Siemens) P2.sub.1 diffractometer with MoK.alpha. radiation 
(0.71073 .ANG.) and a 2.theta.:.theta. scan technique. A total of 1157 
Friedel unique data were collected, of which 686 (59%) with 
.vertline.F.sub.o .vertline..gtoreq.4.sigma.(.vertline.F.sub.o .vertline. 
were considered observed after correction for Lorentz, polarization and 
background effects. The structure was solved by direct methods and refined 
by full-matrix, least-squares techniques [Sheldrick, G. M. SHELXTL 
Crystallographic System (Siemens Instrument Division: Madison, Wis.), 
1986]. The final refinements with anisotropic thermal parameters for all 
nonhydrogen atoms, riding isotropic hydrogens, and anomalous scattering 
corrections for bromine and chlorine converged smoothly to a final 
discrepancy index of 7.81% for the enantiomer shown (wR 6.72%). The other 
enantiomer converged to the significantly higher value of 8.5I%. The 
absolute configuration also was ascertained by the eta-test. The 
enantiomer shown refined to eta +1.1(2) [Sheldrick, G. M.: SHELXTL 
Crystallographic System (Siemens Instrument Division: Madison, Wis.), 
1986]. A computer-generated perspective model of the final model is given 
in FIG. 1; archival crystallographic data have been deposited with the 
Cambridge Crystallographic Data Centre, Cambridge, U.K. 
EXAMPLE 3: Antitumor Activity of the Compound of the Present Invention 
The pure compound of the present invention was tested in the NCI's human 
tumor, disease-oriented in vitro screen [Boyd, M. R.: In CANCER: 
Principles and Practice of Oncology Update. (DeVita, V. T. Jr., Hellman, 
S., and Rosenberg, S. A., eds.), Philadelphia: Lippincott, 1989, pp. 1-12] 
as described elsewhere [Boyd, M. R.: In CANCER: Principles and Practice of 
Oncology Update. (DeVita, V. T. Jr., Hellman, S, and Rosenberg, SA, eds.), 
Philadelphia: Lippincott, 1989, pp. 1-12; Boyd, M. R., et al.: In 
Antitumor Drug Discovery and Development (Valeriote, F. A., Corbett, T., 
Baker, L. eds.), Amsterdam: Kluwer Academic Publishers, 1991, in press; 
Monks, A., et al.: J. Natl. Cancer Inst. 83: 757-766, 1991]. Briefly, 
stock solutions of the compounds were prepared initially in 
dimethylsulfoxide at 400x the desired final highest test concentration and 
stored at -70.degree. until use. The desired final highest test 
concentration is the highest achievable in the test medium and is between 
10.sup.-3 and 10.sup.-4 molar. At the time of screening, an aliquot of the 
thawed stock was diluted with complete medium containing 50.mu.g/ml 
gentamycin to give a concentration of 2x the desired final highest test 
concentration. An additional four, 10-fold serial dilutions were then made 
to provide a total of five concentrations, spanning a 4-log.sub.10 
concentration range. One hundred .mu.l aliquots of these intermediate 
dilutions were immediately added to the appropriate microtitre wells, each 
already containing the appropriate numbers and types of cells in 100.mu.l 
of culture medium, resulting in the desired five final concentrations. 
The 60 cell lines used, and the respective inoculation densities, were the 
same as described elsewhere [Monks, A., et al.: J. Natl. Cancer Inst. 83: 
757-766, 1991; which is hereby incorporated by reference in its entirety]. 
Following the compound additions, the plates were incubated for 48 hr at 
37.degree. C. under a 5% CO.sub.2 /air atmosphere and 100% humidity. Then, 
adherent cells (all lines except the leukemias) were fixed in situ by 
gentle addition of cold trichloroacetic acid (50 microliters of 50% w/v) 
and incubated for 60 min. at 4.degree. C. Supernatants were discarded, the 
plates washed x5 with deionized water and air dried. Sulforhodamine B 
solution (SRB; 100.mu.l at 0.4% w/v in 1% acetic acid, see Monks et al., 
supra, for more details) was added to each plate, followed by further 
incubation for 10 min. at room temperature. Excess unbound dye was then 
removed by washing x5 with 1% acetic acid, followed by air drying. The 
bound stain in each well was solubilized by addition of 100 microliters of 
10 millimolar unbuffered Tris base; this was followed by determination of 
optical densities (515nm) on an automated plate reader. For suspension 
cell cultures (the leukemias), the method was the same except that at the 
end of the drug incubation period the settled cells were fixed in situ to 
the bottoms of the microtitre wells by gentle addition of 50 .mu.l of 80% 
trichloracetic acid. 
Appropriate control wells were included in the test plate format [Monks, 
A., et al.: J. Natl. Cancer Inst. 83: 757-766, 1991] to allow subtraction 
of background optical densities, drug-blank corrections and determination 
of cell densities at time 0 (the time at which compounds are added). A 
single test of the compound performed in the above manner required the 
equivalent of ten 96-well microtitre plates of the compound of the present 
invention. 
Data calculations employed the three experimental measurements: control 
optical densities ((C) in which cells are present but no test compound), 
test optical densities ((T) in which both the cells and test compound are 
present) and optical densities at time zero (To(. If T.gtoreq.To, then the 
calculation for percentage growth (PG) was 100.times.[(T-To)/(C-To)]. If 
T.ltoreq.To, the PG calculation was 100.times.[(T-To)/To)]. For each cell 
line a five-point dose-response curve was created, and the three response 
parameters, GI.sub.50, TGI and LC.sub.50, were calculated for each cell 
line. GI.sub.50 stands for growth inhibiting concentration for a 50% 
decrease in net cell growth. The GI.sub.50 was calculated for each line 
where PG=100.times.[(T-To)/(C-To)]=50; this value corresponds to the drug 
concentration causing a 50% decrease in net cell growth during the drug 
incubation. The drug concentration resulting in total growth inhibition, 
or TGI, is calculated from T =To; this corresponds to the drug 
concentration yielding an amount of cellular protein at the end of the 
incubation that is the same as at the beginning of the incubation (PG0). 
Finally, the LC.sub.50 parameter (Lethal concentration) is calculated from 
PG=100.times.[(T-To)/To]=-50. The LC.sub.50 corresponds to the drug 
concentration causing a net 50% reduction in the measured protein at the 
end of the incubation compared with that at the beginning (i.e. a net loss 
of cells). Having all parameters thus calculated for each compound for 
each cell line, the construction of the respective mean graphs and other 
graphical displays, data calculations, and analyses were performed as 
described elsewhere [Boyd, M. R., et al.: In Antitumor Drug Discovery and 
Development, Valeriote, F. A., Corbett, T., Bakers, L., eds., Amsterdam: 
Kluwer Academic Publishers, 1991, in press, incorporated by reference in 
its entirety; Monks, A., et al.: J. Natl. Cancer Inst. 83: 757-766, 1991] 
by computer. 
The data shown in FIGS. 2 and 3 are representative of quadruplicate tests 
of the compound of the present invention. The negative log.sub.10 
GI.sub.50, TGI and LC.sub.50 values, respectively, calculated for each 
individual cancer cell line in this experiment and used to construct the 
corresponding mean graphs of FIG. 3 are listed as follows by subpanel, 
with the individual cell line names therein listed in the same order as 
their data appear top-to-bottom on the mean graphs of FIG. 3. 
A1(leukemia/lymphoma subpanel): CCRF-CEM (4.54, 4.16. 3.82), HL-60 TB 
(6.12, 5.36, 4.68), K-562 (5.92, 4.66, 3.85), MOLT-4 (4.68, 4.23, 3.85), 
RPMI-8226 (6.68, 6.15, 5.21), SR (5.47, 4.14, 3.89; B1(non small-cell lung 
cancer subpanel): A549/ATCC (5.54, 5.17, 4.72), EKVX (5.52, 5.24, 4.96), 
HOP-18 (5.49, 5.21, 4.92), HOP-62 (5.38, 5.05, 4.77), HOP-92 (6.14, 5.82, 
5.44), NCI-H226 (4.62, 4.41, 4.21), NCI-H23 (4.85, 4.38, 4.00), NCI-H322M 
(5.68, 5.34, 5.02), NCI-H460 (5.82, 5.44, 5.08), NCI-H22 (4.62, 4.34, 
4.06), LXFL-529L (4.85, 4.47, 4.12); Cl(small-cell lung cancer subpanel): 
DMS 114 (4.3S, 4.12, 3.89), DMS 273 (5.96, 5.49, 5.10); D1(colon cancer 
subpanel): COLO 205 (4.92, 4.70, 4.24), DLD-1 (5.74, 5.00, 4.46), HCC-2998 
(5.77, 5.35, 5.00), HCT-116 (6.04, 5.49, 4.92), HCT-15 (5.66, 5.15, 
4.62), HT29 (6.44, 5.85, 5.28), KM12 (6.17, 5.59, 5.116), KM20L2 5.64, 
5.33, 5.00), SW-620 (5.96, 5.52, 5.09); El(brain tumor subpanel): SF-268 
(6.16, 5.54, 4.96), SF-295 (6.17, 5.70, 5.30), SF-539 (6.57, 6.19, 5.77), 
SNB-19 (5.70, 5.37, 5.04), SNB-75 (6.59, 5.80, 4.10), SNB-78 (6.60, 6.19, 
5.70), U251 (6.40, 6.00, 5.64), XF498 (4.59, 4.30, 4.02); Fl(melanoma 
subpanel): LOX IMVI (4.57, 4.24, 4.00), MALME-3M (4.57, 4.20, 3.89), M14 
(4.57, 4.21, 3.92), M19-MEL (4.62, 4.24, 3.92), SK-MEL-2 (6.01, 5.49, 
4.89), SK-MEL-28 (5.64, 5.32, 5.01), SK-MEL-5 (4.66, 4.38, 4.09), UACC-257 
(5.68, 5.31, 4.89), UACC-62 (5.66, 5.17, 4.70); G1(ovarian cancer 
subpanel): IGROV1 (4.82, 4.37, 3.96), OVCAR-3 (5.59, 5.33, 5.07), VCAR-4 
(5.57, 5.24, 4.85), OVCAR-5 (5.49, 5.24, 5.00), OVCAR-8 (5.72, 5.21, 
4.66), SK-OV-3 (4.48, 4.21, 3.96); H1(renal cancer subpanel): 786-0 (7.02, 
6.66, 6.00), A498 (5.62, 5.34, 5.07), ACHN (5.85, 5.49, 5.15), CAKI-1 
(5.80, 5.47, 5.05), RXF-393 (6.48, 6.06, 5.51), RXF-631 (6.47, 5.11, 
4.52), SN12C (5.74, 4.77, 4.24), TK-10 (5.6S, 5.37, 5.08), UO-31 (5.57, 
5.19, 4.82). 
There was an unusually broad range of differential sensitivities among the 
various types of human tumors to the antitumor effects of the compound of 
the present invention (FIGS. 2, 3); for example, compared to the less 
sensitive melanoma and leukemia lines, several of the more sensitive 
brain, renal and colon tumor cell lines were as much as a 1000-fold or 
more sensitive at the GI.sub.50 response level. The compound of the 
present invention thus has a highly novel antitumor activity profile in 
the NCI screen; it has preferential antitumor activity toward human solid 
tumors; available antitumor drugs do not show such activity in this screen 
and likewise are generally ineffective in the treatment or cure of such 
tumors; available antitumor drugs usually are most effective for treatment 
or cure of leukemias and lymphomas [Boyd, M. R.: In CANCER: Principles and 
Practice of Oncology Update. (DeVita, V. T. Jr., Hellman, S., and 
Rosenberg, S. A., eds.), Philadelphia: Lippincott, 1989, pp. 1-12; Boyd, 
M. R.: In Current Therapy in Oncology. (Niederhuber, J. E., ed.), 
Philadelphia: B. C. Decker, Inc., 1991, in press]. The compound of the 
present invention further appears to act by an antitumor mechanism 
different than known conventional antitumor drugs; for example, it does 
not appear to act merely as an electrophile (alkylating agent); consistent 
with this view were results of computerized pattern-recognition studies, 
using the COME algorithms [Paull, K. D., et al.: J. Natl. Cancer Inst. 
81; 1088-1092, 1989] which showed no resemblance of the mean graph 
profiles of the compound of the present invention (FIG. 3) to known 
alkylating agents; nor does the screening profile or "fingerprint" of the 
compound match by COME with any other known conventional antitumor drug 
currently available. 
EXAMPLE 4. Pharmaceutical Compositions 
The compound of the present invention or antitumor derivatives thereof may 
be made into pharmaceutical compositions by combination with appropriate, 
pharmaceutically acceptable carriers or diluents, and may be formulated 
into preparations in solid, semi-solid, liquid or gaseous forms such as 
tablets, capsules, powders, granules, ointments, solutions, suppositories, 
injections, inhalants, and aerosols in the usual ways for their respective 
route of administration In pharmaceutical dosage forms, the compound 
employed in the present invention may be used alone or in appropriate 
association, as well as in combination with other pharmaceutically active 
compounds, including other antitumor compounds. These other antitumor 
compounds are described supra. 
The following methods and excipients are merely exemplary and are in no way 
limiting. In the case of oral preparations, the compound of the present 
invention may be used alone, or in combination with other antitumor 
agents, together with appropriate additives to make tablets, powders, 
granules or capsules, e.g., with conventional additives such as lactose, 
mannitol, corn starch or potato starch; with binders such as crystalline 
cellulose, cellulose derivatives, acacia, corn starch or gelatins; with 
disintegrators such as corn starch, potato starch or sodium 
carboxymethylcellulose; with lubricants such as talc or magnesium 
stearate; and if desired, with diluents, buffering agents, moistening 
agents, preservatives and flavoring agents. 
The compound of the present invention may alone, or in combination with 
other antitumor agents, be formulated into preparations for injections by 
dissolving, suspending or emulsifying in an aqueous or nonaqueous solvent, 
such as vegetable oil, synthetic aliphatic acid glycerides, esters of 
higher aliphatic acids or propylene glycol; and if desired, with 
conventional additives such as solubilizers, isotonic agents, suspending 
agents, emulsifying agents, stabilizers and preservatives. 
The compound of the present invention, alone or in combination with other 
antitumor compounds, can be made into aerosol formulations to be 
administered via inhalation. These aerosol formulations can be placed into 
pressurized acceptable propellants such as dichlorodifluoromethane, 
propane, nitrogen and the like. 
Furthermore, the compound of the present invention, alone or in combination 
with other antitumor agents, may be made into suppositories by mixing with 
a variety of bases such as emulsifying bases or water-soluble bases. The 
suppository formulations can be administered rectally; the suppository can 
include vehicles such as cocoa butter, carbowaxes and polyethylene 
glycols, which melt at body temperature, yet are solidified at room 
temperature. 
Unit dosage forms for oral or rectal administration such as syrups, 
elixirs, and suspensions may be provided wherein each dosage unit, e.g., 
teaspoonful, tablespoonful, tablet or suppository contains a predetermined 
amount of the composition containing the compound of the present 
invention, alone or in combination with other antitumor agents; similarly, 
unit dosage forms for injection or intravenous administration may comprise 
a composition as a solution in sterile water, normal saline or other 
pharmaceutically acceptably carrier. 
The term "unit dosage form" as used herein refers to physically discrete 
units suitable as unitary dosages for human and animal subjects, each unit 
containing a predetermined quantity of the compound of the present 
invention, alone or in combination with other antitumor agents, calculated 
in an amount sufficient to produce the desired effect in association with 
a pharmaceutically acceptable, diluent, carrier or vehicle. The 
specifications for the novel unit dosage forms of the present invention 
depend on the particular effect to be achieved, and the particular 
pharmacodynamics associated with the compound in the individual host. 
The pharmaceutically acceptable excipients, for example vehicles, 
adjuvants, carriers or diluents, are readily available to the public. 
One skilled in the art can easily determine the appropriate method of 
administration for the exact formulation of the composition being used. 
Any necessary adjustments in dose can be made readily to meet the nature 
or severity of the cancer, and the individual patient's overall physical 
health, and adjusted accordingly by the skilled practitioner. 
EXAMPLE 5. Use of Compositions Containing the Compound of the Present 
Invention for Treating Cancer 
The present invention further relates to a method of treating cancer 
comprising the administration of an "antitumor effective amount" of the 
composition of the present invention. The "antitumor effective amount" is 
defined, for example, as that amount required to be administered to an 
individual patient to achieve an antitumor effective blood and/or tissue 
level of the compound of the present invention to kill or inhibit the 
growth of the tumor; the effective blood level might be chosen, for 
example, as that level (e.g., 10.sup.-7 -10.sup.-4 M from FIGS. 2 and 3) 
to kill or inhibit the growth of tumor cells in a screening assay. 
Alternatively, the "antitumor effective blood level" might be defined as 
that concentration of the compound of the present invention needed to 
inhibit markers of the tumor in the patient's blood, or which slows or 
stops the growth of the patient's tumor, or which causes the patient's 
tumor to regress or disappear, or which renders the patient asymptomatic 
to the particular tumor or which renders an improvement in the patient's 
subjective sense of condition. Since a fixed "antitumor effective blood 
level" is used as the preferred endpoint for dosing, the actual dose and 
schedule for drug administration for each patient may vary depending upon 
interindividual differences in pharmacokinetics, drug disposition and 
metabolism. Moreover, the dose may vary when the compound is used in 
combination with other drugs. 
While the foregoing invention has been described in some detail for 
purposes of clarity and understanding, it will be appreciated by one 
skilled in the art from a reading of this disclosure that various changes 
in form and detail can be made without departing from the true scope of 
the invention.