Cerulenin compounds for fatty acid synthesis inhibition

Novel compounds for use in inhibiting fatty acid synthesis are disclosed. The compounds can be used for the treatment of tumors or microbial infections.

This invention relates to novel compounds useful for inhibiting fatty acid 
synthesis. In particular, this invention contemplates administration of 
novel compounds to mammals having a tumor or microbial infection. 
BACKGROUND OF THE INVENTION 
Fatty Acid Metabolism 
The fatty acid biosynthetic pathway in man is comprised of four major 
enzymes: acetyl-CoA carboxylase, the rate limiting enzyme which 
synthesizes malonyl-CoA; malic enzyme, which produces NADPH; citrate 
lyase, which synthesizes acetyl-CoA; and fatty acid synthase, which 
catalyzes NADPH-dependent synthesis of fatty acids from acetyl-CoA and 
malonyl-CoA. The final products of fatty acid synthase are free fatty 
acids which require separate enzymatic derivatization with coenzyme-A for 
incorporation into other products. In man, significant fatty acid 
synthesis may occur in two sites: the liver, where palmitic acid is the 
predominant product (Roncari, Can. J. Biochem., 52:221-230, 1974); and 
lactating mammary gland where C.sub.10 -C.sub.14 fatty acids predominate 
(Thompson, et al., Pediatr. Res., 19: 139-143, 1985). Except for 
lactation, and cycling endometrium (Joyeux, et al., J. Clin. Endocrinol. 
Metab., 70:1319-1324, 1990), the fatty acid biosynthetic pathway is of 
minor physiologic importance, since exogenous dietary fatty acid intake 
down-regulates the pathway in the liver and other organs (Weiss, et al., 
Biol. Chem. Hoppe-Seyler, 367:905-912, 1986). 
In liver, acetyl-CoA carboxylase, malic enzyme and fatty acid synthase are 
induced in concert by thyroid hormone and insulin via transcriptional 
activation and repressed by glucagon (Goodridge, Fed Proc., 45:2399-2405, 
1986) and fatty acid ingestion (Blake, et al., J. Nutr., 120:1727-1729, 
1990). Tumor necrosis factor alpha (TNF) a cytokine with profound effects 
on lipogenesis, is either stimulatory or inhibitory depending on the cell 
type studied. TNF markedly inhibits lipogenesis in adipocytes by reduction 
in acetyl-CoA carboxylase and fatty acid synthase protein synthesis, but 
is markedly stimulatory in the liver by increasing the level of citrate, 
which is the primary allosteric activator of the rate limiting enzyme of 
fatty acid biosynthesis, acetyl-CoA-carboxylase. 
In lactating breast, the other major site of fatty acid biosynthesis in 
humans, fatty acid synthesis is under control of prolactin, estrogen, and 
progesterone. During pregnancy, progesterone acts as a mitogen to promote 
breast development and concomitantly down-regulates prolactin receptors, 
preventing lipid and milk protein synthesis before delivery. After 
delivery, the fall in estrogen and progesterone levels allows 
up-regulation of prolactin receptors and subsequent increase in lipogenic 
enzymes and milk protein production by breast epithelial cells. 
Regulation of fatty acid synthase expression in human breast cancer has 
been studied primarily as a model for progesterone-stimulated gene 
expression. In contrast to normal lactating breast where progesterone 
stimulates epithelial cell growth while retarding lipogenic enzyme 
synthesis, in progesterone receptor (PR) positive human breast carcinomas 
such as MCF-7, ZR-75-1, and T-47D, progesterone inhibits growth and 
induces fatty acid synthase production along with other lipogenic enzymes 
(Chambon, et al., J. Steroid Biochem., 33:915-922 (1989). Progesterone 
presumably acts to up-regulate fatty acid synthase expression via the 
steroid hormone response element as is found in the rat fatty acid 
synthase promoter (Amy, et al., Biochem. J., 271:675-686, 1989), leading 
to increased FAS mRNA transcription or, by other mechanisms, to increased 
message stability (Joyeux, et al., Mol. Endrocinol., 4:681-686, 1989). 
Regarding PR-negative human breast cancer cells, a single study reports 
that fatty acid synthase accounts for about 25% of cytosolic protein in 
SKBR3 cells but no data regarding its biologic significance or regulation 
was available (Thompson, et al., Biochim. Biophys. Acta, 662:125-130, 
1981). 
With regard to cytokines and other lipogenic hormones, only scant data are 
available concerning human breast cancer. For example, TNF has been known 
to be markedly growth inhibitory to some breast cancer cultures. While TNF 
is mildly growth inhibitory to primary rat hepatocyte cultures (ID.sub.50 
=5000 units/ml), some human breast cancer cells such as MCF-7 are 
extremely growth inhibited (ID.sub.50 =40 units/ml) (Chapekar, et al., 
Exp. Cell. Res., 185:247-257, 1989). The effect of TNF on FAS expression 
or lipogenic activity in breast cancer cells, however, remains unknown. 
One study of fatty acid synthase expression in MCF-7 cells using Northern 
analysis, found that insulin and insulin growth factor-1 were only 
slightly stimulatory compared to 5-10 fold increases seen with 
progesterone, while T.sub.3 had no effect (Chalbos, et al., J. Steroid 
Biochem. Molec. Bid., 43:223-228, 1992). Overall, regulation of FAS in 
receptor positive breast cancer has been only cursorily examined, while 
receptor negative tumors have not been studied. 
No association with poor clinical outcome was found for breast or for any 
other cancers in those few systems where fatty acid synthase expression 
was studied. In the only study purporting to associate FAS expression with 
prognosis, fatty acid synthase expression was studied by in situ 
hybridization in 27 breast cancers, finding an association between 
increased fatty acid synthase mRNA and a higher degree of morphologic 
differentiation, but without association with estrogen or progesterone 
receptor status (Chalbos, et al., J. Natl. Cancer Inst., 82:602-606, 
1990). It was deduced from these data that fatty acid synthase expression 
in breast carcinoma is associated with greater degree of morphologic 
differentiation and therefore presumably with less aggressive tumors. A 
second study of 87 cases by Northern blotting of fatty acid synthase mRNA 
found an association of fatty acid synthase expression and young age 
(premenopausal patients), but again no association with receptor status 
(Wysocki, et al., Anticancer Res., 10:1549-1552, 1990). Neither study 
provided clinical follow-up of their patients; there were no data 
comparing FAS expression with either disease-free interval or patient 
survival. Without clinical outcome, no reliable conclusions can be drawn 
regarding FAS expression and tumor virulence. 
These studies stand in contrast to a series of greater than 200 patients 
from several centers demonstrating a strong association between poor 
prognosis and expression of a protein of undetermined function (designated 
OA-519) through measurement of disease-free survival or overall survival 
(Kuhajda, N. Engl. J. Med., 321:636-641, 1989; Shurbaji, et al., Am. J. 
Clin. Pathol, 96:238-242, 1991; Corrigan, et al., Am. J. Clin. Pathol., 
96:406, 1991; Cote, et al., Lab. Invest. ,66:13A, 1992; Ziegler, et al., 
Am. J. Clin. Oncol., 14: 101-110, 1991). 
Nor has fatty acid metabolism been a target of study in cancer 
therapeutics. Fujii, et al. (1986, Japan J. Exp. Med., 56:99-106), used 
the fatty acid synthase inhibitor cerulenin in combination with exogenous 
antitumor antibodies to weaken the cell membrane in an attempt to 
potentiate complement-mediated cell membrane damage via the membrane 
attack complex. Cerulenin was known to be toxic to cells at high 
concentration, and Fujii, et al., taught that the cerulenin concentration 
should be kept low to maintain the selectivity conferred by the humoral 
immune component of complement-mediated cell lysis. Spielvogel, et al., 
U.S. Pat. No. 5,143,907, noted that a series of phosphite-borane compounds 
exhibited both antineoplastic activity and anti-inflammatory activity 
while lowering serum cholesterol and serum triglycerides. The 
phosphite-borane compounds are non-specific inhibitors that affect many 
cellular functions, and so they are not selectively effective against 
tumor cells. Spielvogel, et al. taught that the hypolipidemic effect on 
serum cholesterol and triglycerides was mediated through more than one 
mechanism, and the antineoplastic effect was not shown to be related to 
the hypolipidemic activity. 
Cerulenin is a potent inhibitor of fatty acid biosynthesis at the level of 
the FAS complex (S. Omura, Bacteriological Reviews, September 1976, p. 
681-697, Vol. 40 No. 3). The structure of cerulenin and the mechanism of 
action are shown below: 
##STR1## 
The alkylation of the critical enzyme thiol inactivates the 
Beta-ketoacyl-ACP synthetase component of the FAS multienzyme complex. 
Cerulenin may be viewed as having an enzyme binding moiety (the nine 
carbon diene) and a reactive group (the keto epoxy amide). 
Cerulenin was originally isolated as a potential antifungal antibiotic from 
the culture broth of Cephalosporium caerulens. Structurally cerulenin has 
been characterized as 2R,3S-epoxy-4-oxo-7,10-trans,trans-dodecanoic acid 
amide. Its mechanism of action has been shown to be inhibition, through 
irreversible binding, of .beta.-ketoacyl-ACP synthase, the condensing 
enzyme required for the biosynthesis of fatty acids. Cerulenin has been 
categorized as an antifungal, primarily against Candida and Saccharomyces 
sp. In addition, some in vitro activity has been shown against some 
bacteria, actinomycetes, and mycobacteria, although no activity was found 
against Mycobacterium tuberculosis. The activity of fatty acid synthesis 
inhibitors and cerulenin in particular has not been evaluated against 
protozoa such as Toxoplasma gondii or other infectious eucaryotic 
pathogens such as Pneumocystis carinii, Giardia lamblia, Plasmodium sp., 
Trichomonas vaginalis, Cryptosporidium, Trypanosoma, Leishmania, and 
Schistosoma. 
Despite cerulenin's in vitro activity against some bacteria and fungi it 
has not been developed as a therapeutic agent. To date research on this 
compound has centered on it use as a research tool for investigating the 
role of fatty acids in the metabolism and physiology of a variety of 
organisms because of its activity as a fatty acid synthesis inhibitor. 
SUMMARY OF THE INVENTION 
It is an object of this invention to provide novel compounds and a method 
for treating carcinoma patients which will reduce the tumor burden of the 
patient. 
It is another object of this invention to provide novel compounds and a 
method for treating virulent carcinomas. 
The present invention provides a method of treating patients with carcinoma 
by inhibiting fatty acid synthesis by the cells of the carcinoma, such 
that growth of the cells is inhibited in a manner selectively cytotoxic or 
cytostatic to the cancer cells. 
In a more particular embodiment, the invention provides a method of 
treating a host with a carcinoma by administering novel fatty acid 
synthase (FAS) inhibitors and/or inhibitors of other enzymes of the 
synthetic pathway for fatty acid as cytotoxic chemotherapeutic agents, 
thereby reducing tumor burden. 
In a further embodiment, the present invention provides a method of 
ameliorating tumor burden in a host having tumor tissue which expresses a 
protein that exhibits fatty acid synthase activity, comprising 
administering a therapeutically effective amount of a novel fatty acid 
synthase inhibitor. Expression is determined directly in tumor tissues by 
detecting fatty acid synthase in tissue samples obtained from procedures 
such as biopsies, resections or needle aspirates, using assays such as 
immunohistochemistry, cytosol enzyme immunoassay or radioimmunoassay, or 
direct measurement of enzyme activity. Expression of fatty acid synthase 
by the tumor is indirectly measured by detecting fatty acid synthase in 
plasma or body fluid using assays such as enzyme immunoassay or 
radioimmunoassay. 
In a further embodiment, the invention provides a method for treating a 
host with a carcinoma while protecting normal (non-neoplastic) tissues 
(such as liver, which may normally express fatty acid synthase activity) 
over a wide range from potential toxicity, by down-regulating the FAS 
enzyme activity of normal cells before and/or during administration of 
therapeutically effective amounts of novel FAS inhibitors. Down regulation 
may be accomplished by, for instance, reduction of caloric intake or other 
effective methods. 
Particularly virulent carcinomas tend, among other things, to have cells 
that express OA-519, and this particular protein has been found to have 
fatty acid synthase activity which appears to be a required enzyme 
activity for the growth of carcinomas but not necessarily for normal 
cells. The compounds and methods of the subject invention are for treating 
carcinomas by administering novel inhibitors of fatty acid synthesis to 
reduce tumor burden in the patients. The methods of the invention are 
particularly advantageous because treatment with inhibitors such as FAS 
inhibitors is selective for cells expressing fatty acid synthase. FAS is 
an inducible enzyme that is not generally expressed by normal cells, and 
as a result, the tumor cells are preferentially affected by the 
inhibitors. 
This invention also describes the use of novel inhibitors of fatty acid 
biosynthesis as a means to treat various opportunistic organisms known to 
express the fatty acid biosynthetic pathway. The drug concentrations 
required to inhibit growth of these infectious agents in vitro indicate a 
potential therapeutic index in man. Moreover, some of these infectious 
agents, such as M. tuberculosis, have fatty acid biosynthetic enzymes 
which are structurally similar, but not identical, to mammalian fatty acid 
synthase.

DETAILED DESCRIPTION OF THE INVENTION 
I. FAS 
The protein FAS (also referred to herein as OA-519) is highly correlated 
with the most virulent carcinomas. FAS exhibits fatty acid synthase 
activity. FAS purified from a human breast carcinoma cell line has peptide 
sequence homology with rat fatty acid synthase, and FAS also has 
functional characteristics of a fatty acid synthase. Fatty acid synthesis 
by FAS was demonstrated by incorporation of .sup.14 C malonyl coenzyme A 
into fatty acids, subsequent esterification of the fatty acids, and 
analysis by reversed-phase thin layer chromatography. The specific 
activity of purified FAS was determined spectrophotometrically by 
following the oxidation of NADPH at 340 nm in the presence of acetyl 
coenzyme A and malonyl coenzyme A. In one determination, the specific 
activity of FAS was measured as 586 nanomoles NADPH oxidized/min/mg 
protein, which compares favorably with the value of 404 obtained for FAS 
from human liver. 
Fatty acid synthase is a large protein found in the cytosol of cells from 
particular tissues, including liver and lactating mammary gland, but FAS 
is not expressed in most normal (non-malignant) adult tissues. Fatty acid 
synthase in higher organisms is a multifunctional enzyme which is well 
known to carry out the following seven enzymatic functions on a single 
molecule (Wakil, S. J., Biochemistry, 28:4523-4530, 1989): 
acetyl transacylase 
malonyl transacylase 
beta-ketoacyl synthetase (condensing enzyme) 
beta-hydroxyacyl dehydrase 
enoyl reductase 
thioesterase 
Breast cancer cells have been found to express fatty acid synthase, while 
most other rumor cells have not been tested for the presence or absence of 
this enzyme. Rochefort and co-workers have partially cloned FAS from 
breast cancer cells and found that FAS expression by breast cancer cell 
lines was correlated with responsiveness to progesterone (Chalbos, et al, 
J. Biol. Chem,, 262:9923-9926, 1987). Based on this evidence, they 
concluded that cells expressing FAS were from tumors that were less 
de-differentiated and therefore less virulent (Chalbos, et al, J. Nat'l. 
Cancer Inst., 82:602-606, 1990). 
Inhibitors of FAS inhibit growth of carcinoma cells, but have little effect 
on normal human fibroblasts. Indeed, fibroblasts, which have very low FAS 
activity, are resistant to FAS inhibitor concentrations that inhibit 
growth of more than 80% of breast carcinoma cells having high levels of 
FAS activity. Studies with multiple breast, prostate, lung, colon, and 
ovarian carcinoma cell lines, and normal fibroblasts confirm the 
correlation between FAS synthase activity and growth inhibition by FAS 
inhibitors. The relationship between drug sensitivity and FAS enzyme 
activity holds for all tumor cell types tested. Thus, inhibition of the 
fatty acid synthase enzyme, which is highly expressed in the most virulent 
carcinomas, can inhibit the growth of cells in these tumors. 
II. Treatment Based on Inhibition of Fatty Acid Synthesis 
The present invention provides a method for ameliorating tumor burden in a 
host whose tumor contains cells that are dependent on endogenously 
synthesized fatty acid (fatty acid synthesized within the cells). Such 
cells usually over-express a protein with FAS activity. Tumor burden may 
be reduced in such hosts by administering to the host one or more novel 
inhibitors that interfere with fatty acid synthesis or utilization. These 
inhibitors are cytotoxic to tumor cells which express FAS, and 
administration which results in reduction of fatty acid synthesis and 
utilization by the tissue and/or reduction of FAS activity in biological 
fluids of these hosts will reduce tumor burden. See the U.S. application 
entitled Chemotherapy for Cancer filed Jan. 24, 1994, hereby incorporated 
in its entirety. 
The present invention also provides a method for inhibiting growth of 
microbial cells that are dependent on endogenously synthesized fatty acid 
(i.e., fatty acid synthesized within the cells) without inhibiting 
metabolic activity of the patient. Such microbial cells usually 
over-express a protein with FAS activity. Sepsis may be reduced in such 
patients by administering to the patient one or more novel inhibitors that 
interfere with fatty acid synthesis or utilization. These inhibitors are 
cytotoxic to microbial cells which express FAS, and administration which 
results in reduction of fatty acid synthesis and utilization by the 
microbial cells and/or reduction of FAS activity in biological fluids of 
these patients will reduce sepsis. See the U.S. application entitled 
Inhibitors of Fatty Acid Synthesis as Antimicrobial Agents filed Jan. 24, 
1994, hereby incorporated in its entirety. 
A. Selection of the Patient Population 
The method of this invention can be used to treat hosts suffering from 
cancers which have an elevated level of fatty acid synthase. 
Characteristic carcinomas amenable to treatment include those of bladder, 
salivary gland, skin adnexa, bile duct, endocervix, ectocervix, and 
vagina, esophagus, nasopharynx and oropharynx, or those of germ cell 
origin, and mesothelioma. In particular, carcinomas or adenocarcinomas of 
the stomach, endometrium, kidney, liver and lung, as well as melanoma are 
treatable according to this invention. Breast, colon and rectum, prostate, 
and ovary, are especially suitable types of adenocarcinomas for the 
application of this therapy. 
The method of this invention contemplates treatment of tumors having cells 
that express FAS or depend on endogenous fatty acid (synthesized within 
the cell). Endogenous fatty acid synthesis by such cells will preferably 
occur at a rate of incorporation greater than 10 fmoles of acetyl-CoA into 
acyl glyceride per 200,000 cells per minute. Preferred hosts are 
identified because they have tumors containing cells which express OA-519 
or other enzymes of the fatty acid synthesis pathway, such as acetyl CoA 
carboxylase (ACC), at levels higher than the level found in the 
surrounding normal (e.g., non-neoplastic) tissue. Such cells are 
aggressive tumor cells and result in decreased survival, increased 
metastasis, increased rates of clinical recurrence and overall worsened 
prognosis. 
Tumor cell sensitivity to fatty acid synthesis inhibitors usually varies 
continuously with FAS levels. Aggressive tumor cells expressing levels of 
FAS activity greater than 20 femtomoles malonyl CoA incorporated into 
fatty acid per 200,000 cells per minute may be expected to be sensitive to 
fatty acid synthase inhibitors. Since many tumor cells are extremely 
dependent on endogenous fatty acid synthesis, lower FAS activity levels 
need not exclude a specific tumor as a candidate for therapy with fatty 
acid synthase inhibitors. 
Infectious diseases which are particularly susceptible to treatment by the 
method of this invention are diseases which cause lesions in externally 
accessible surfaces of the infected animal. Externally accessible surfaces 
include all surfaces that may be reached by non-invasive means (without 
cutting or puncturing the skin), including the skin surface itself, mucus 
membranes, such as those covering nasal, oral, gastrointestinal, or 
urogenital surfaces, and pulmonary surfaces, such as the alveolar sacs. 
Susceptible diseases include: (1) cutaneous mycoses or tineas, especially 
if caused by Microsporum, Trichophyton, Epidermophyton, or Mucocutaneous 
candidiasis; (2) mucotic keratitis, especially if caused by Aspergillus, 
Fusarium or Candida; (3) amoebic keratitis, especially if caused by 
Acanthamoeba; (4) gastrointestinal disease, especially if caused by 
Giardia lamblia, Entamoeba, Cryptosporidium, Microsporidium, or Candida 
(most commonly in immunocompromised animals); (5) urogenital infection, 
especially if caused by Candida albicans or Trichomonas vaginalis; and (6) 
pulmonary disease, especially if caused by Mycobacterium tuberculosis, 
Aspergillus, or Pneumocystis carinii. Infectious organisms that are 
susceptible to treatment with fatty acid synthesis inhibitors include 
Mycobacterium tuberculosis, especially multiply-drug resistant strains, 
and protozoa such as Toxoplasma. 
The presence of FAS in cells of the carcinoma or in microbe cells can be 
detected by any suitable method, including activity assays or stains, 
immunoassays using anti-FAS antibodies, assays measuring FAS mRNA, and the 
like. Particularly advantageous are assays for the presence of OA-519, a 
protein which is immunologically cross-reactive with the gene product of 
the hpr gene (Maeda, J. Biol. Chem., vol. 260, pp. 6698-6709, 1985) but 
not with haptoglobin 1 or 2. Such assays are taught in International 
Patent Publication WO 90/08324 or U.S. application Ser. No. 07/735,522, 
incorporated herein by reference. The most preferred assays are 
immunoassays for OA-519, either in tissue or in plasma. 
Expression of FAS may be determined directly in tumor tissue samples 
obtained through procedures such as biopsies, resections or needle 
aspirates, using assays such as immunohistochemistry, cytosol enzyme 
immunoassay or radioimmunoassay, in situ hybridization of nucleic acid 
probes with mRNA targets having FAS sequences, or direct measurement of 
enzyme activity. Expression of fatty acid synthase by the tumor may be 
indirectly measured in biological fluid samples obtained from patients, 
such as blood, urine, serum, lymph, saliva, semen, ascites, or especially 
plasma, using any suitable assays. Preferred assays for FAS in biological 
fluid include enzyme immunoassay or radioimmunoassay. 
Cells which depend on endogenously synthesized fatty acids may also be 
identified by detection of other enzymes of the fatty acid synthesis 
pathway at levels higher than those found in non-neoplastic tissue 
surrounding the rumor (normal tissue). In particular, treatment of cells 
having unexpectedly high levels of acetyl CoA carboxylase is contemplated 
by the present invention. The presence of these enzymes may be detected by 
assay methods analogous to those described for FAS. 
Cells that require endogenously synthesized fatty acid are widespread among 
carcinomas, particularly the most virulent carcinomas. While it is 
preferred that the presence of FAS be determined prior to treatment, the 
skilled clinician will recognize that such determination is not always 
necessary. Treatment of a carcinoma patient with an inhibitor of fatty 
acid synthesis, particularly a FAS inhibitor, which results in reduction 
of tumor burden demonstrates the presence of FAS in the tumor. Where a 
carcinoma patient can be successfully treated by the method of this 
invention, independent determination of FAS may be unnecessary. Such 
empirical treatment of carcinomas of the type usually found to express FAS 
is also within the contemplation of this invention. 
Fatty acid synthesis inhibitors of the invention are also useful in 
conjunction with other chemotherapeutic agents. Since no presently 
prescribed cancer chemotherapeutic agents are specifically active against 
the fatty acid synthase pathway, FAS inhibitors will complement existing 
anti-cancer drags, particularly antimetabolic drugs that target other 
anabolic or catabolic pathways. 
Fatty acid synthesis inhibitors of the invention are also useful in 
conjunction with other antimicrobial agents. Since no presently prescribed 
antimicrobial agents are specifically active against the fatty acid 
synthase pathway, the novel FAS inhibitors will complement existing 
antibiotic drugs, particularly antimetabolic drugs that target other 
anabolic or catabolic pathways. 
FAS expression and the growth inhibitory effect of inhibitors of the fatty 
acid synthetic pathway are independent of the cell cycle. Therefore, 
inhibitors of fatty acid synthesis may be expected to be particularly 
effective in combination with chemotherapeutic agents that target rapidly 
cycling cells. Alternatively, the fatty acid synthesis inhibitors of the 
invention may be administered to supplement a chemotherapeutic regime 
based on antineoplastic agents known to be effective against the 
particular tumor type being treated. In particular, use of fatty acid 
synthesis inhibitors to prevent the growth of a small proportion of 
undetected but highly virulent cells in conjunction with a therapeutic 
program using other agents is within the contemplation of this invention. 
On the other hand, it is not contemplated that fatty acid synthesis 
inhibitors will be useful in combination with agents which produce 
complement-mediated cell damage via the membrane attack complex, whether 
initiated by antibody or by the alternative pathway for complement 
activation (Bhakdi, et al. (1983), "Membrane Damage by Complement," 
Biochim. Biophys. Acta, 737:343:372). Therefore, this invention is not 
directed to the use of fatty acid synthesis inhibitors in the presence of 
exogenously supplied agents which activate the complement-dependent 
membrane attack complex. 
B. Inhibition of the Fatty Acid Synthetic Pathway 
Carcinoma cells or microbial cells which are dependent on their own 
endogenously synthesized fatty acid will express FAS. This is shown both 
by the fact that FAS inhibitors are growth inhibitory and by the fact that 
exogenously added fatty acids can protect normal cells but not these 
carcinoma cells from FAS inhibitors. Therefore, preventing synthesis of 
fatty acids by the cell may be used to treat carcinoma or microbial 
infection. 
Fatty acids are synthesized by fatty acid synthase (FAS) using the 
substrates acetyl CoA, malonyl CoA and NADPH. Thus, the fatty acid 
synthesis pathway is usually considered to involve four enzymes--FAS and 
the three enzymes which produce its substrates: acetyl CoA carboxylase 
(ACC), malic enzyme and citrate lyase. Other enzymes which can feed 
substrates into the pathway, such as the enzymes which produce NADPH via 
the hexose monophosphate shunt, may also affect the rate of fatty acid 
synthesis, and thus be important in cells that depend on endogenously 
synthesized fatty acid. Inhibition of the expression or the activity of 
any of these enzymes will affect growth of carcinoma cells or microbial 
cells that are dependent on endogenously synthesized fatty acid. 
The product of FAS is a free C.sub.12 -C.sub.16 fatty acid, usually 
palmitate. Palmitic acid must be further processed to fulfill the 
requirements of the cells for various lipid components. As used herein, 
the term "lipid biosynthesis" refers to any one or a combination of steps 
that occur in the synthesis of fatty acids or subsequent processing of 
fatty acids to make cellular components containing fatty acids. The first 
step in this down-stream processing is activation of the fatty acid by 
coupling it to coenzyme A, which is catalyzed by an enzyme, acyl CoA 
synthetase. 
Inhibition of key steps in down-stream processing or utilization of fatty 
acids may be expected to inhibit cell function, whether the cell depends 
on endogenous fatty acid or utilizes fatty acid supplied from outside the 
cell, and so inhibitors of these down-stream steps may not be sufficiently 
selective for tumor cells or microbial cells that depend on endogenous 
fatty acid. However, administration of a fatty acid synthesis inhibitor to 
such cells makes them more sensitive to inhibition by inhibitors of 
down-stream fatty acid processing and/or utilization. Because of this 
synergy, administration of a fatty acid synthesis inhibitor in combination 
with one or more inhibitors of down-stream steps in lipid biosynthesis 
and/or utilization will selectively affect tumor cells that depend on 
endogenously synthesized fatty acid. Preferred combinations include an 
inhibitor of acyl CoA synthetase combined with an inhibitor of FAS or ACC. 
C. Inhibitors of Fatty Acid Synthesis 
When it has been determined that a host has a tumor which expresses FAS, or 
in infected with cells of an organism which expresses FAS, or if FAS has 
been found in a biological fluid from a host, the host may be treated 
according to the method of this invention by administering a novel fatty 
acid synthesis inhibitor to the host. 
Any compound that inhibits fatty acid synthesis can be used to inhibit 
tumor cell or microbial cell growth, but of course, compounds administered 
to the host must not be equally toxic to both malignant and normal 
(non-malignant) cells. Preferred inhibitors for use in the method of this 
invention are those with high therapeutic indices (therapeutic index is 
the ratio of the concentration which affects normal cells to the 
concentration which affects tumor cells). Inhibitors with high therapeutic 
index are identified by comparing the effect of the inhibitor on two cell 
lines, one non-malignant line, such as a normal fibroblast line, and one 
carcinoma line which has been shown to express high levels of FAS. 
In particular, therapeutic index is determined by comparing growth 
inhibition of animal cells such as human cell lines exhibiting a low level 
of fatty acid synthesis activity, preferably less than about 10 fmole 
acetyl-CoA incorporation into acyl glyceride per minute per 200,000 cells, 
to growth inhibition of human cancer cells exhibiting a high level of 
fatty acid synthetic activity, preferably greater than about 20 fmole 
acetyl-CoA incorporation per 200,000 cells per minute, more preferably at 
least about 80 fmole acetyl-CoA incorporation into acyl glyceride per 
200,000 cells per minute. Cells with the preferred level of fatty acid 
synthesis activity are easily obtained by the skilled worker. Preferably, 
the growth inhibition assays are performed in the presence of exogenous 
fatty acid added to the cell culture medium, for example, 0.5 mM oleic 
acid complexed to BSA. 
Inhibitors are characterized by the concentration required to inhibit cell 
growth by 50% (IC.sub.50 or ID.sub.50). FAS inhibitors with high 
therapeutic index will, for example, be growth inhibitory to the carcinoma 
cells at a lower concentration (as measured by IC.sub.50) than the 
IC.sub.50 for the normal cells. Inhibitors whose effects on these two cell 
types show greater differences are more preferred. Preferred inhibitors of 
fatty acid synthesis will have IC.sub.50 for cells with high fatty acid 
synthetic activity that is at least 1/2 log lower, more preferably at 
least 1 log lower, than the inhibitor's IC.sub.50 determined for cells 
with low activity. 
Lipid synthesis consists of multiple enzymatic steps. The data demonstrate 
that inhibition of lipid biosynthesis at two or more steps can create 
synergistic effects, lowering both the required concentration and 
potential toxicity of any single agent. 
When tumors are treated by administration of a synergistic combination of 
at least one inhibitor of fatty acid synthesis and at least one inhibitor 
of either the enzymes which supply substrates to the fatty acid synthesis 
pathway or the enzymes that catalyze downstream processing and/or 
utilization of fatty acids, the therapeutic index will be sensitive to the 
concentrations of the component inhibitors of the combination. 
Optimization of the concentrations of the individual components by 
comparison of the effects of particular mixtures on normal and 
OA-519-expressing cells is a routine matter for the skilled artisan. The 
dose of individual components needed to achieve the therapeutic effect can 
then be determined by standard pharmaceutical methods, taking into account 
the pharmacology of the individual components. 
The inhibitor of fatty acid synthesis, or the synergistic combination of 
inhibitors will be administered at a level (based on dose and duration of 
therapy) below the level that would kill the host mammal being treated. 
Preferably, administration will be at a level that will not irreversibly 
injure vital organs, or will not lead to a permanent reduction in liver 
function, kidney function, cardiopulmonary function, gastrointestinal 
function, genitourinary function, integumentary function, musculoskeletal 
function, or neurologic function. On the other hand, administration of 
inhibitors at a level that kills some cells which will subsequently be 
regenerated (e.g., endometrial cells) is not necessarily excluded. 
Acetyl CoA carboxylase and the condensing enzyme of the FAS complex are 
candidates for inhibition. Fatty acid synthesis is reduced or stopped by 
inhibitors of these enzymes. The result would be deprivation of membrane 
lipids, which causes cell death. Normal cells, however, would survive as 
they are able to import circulating lipid. Acetyl CoA carboxylase is the 
focal point for control of lipid biosynthesis. The condensing enzyme of 
the FAS complex is well characterized in terms of structure and function; 
the active center contains a critical cysteine thiol, which is the target 
of antilipidemic reagents, such as cerulenin. 
A wide variety of compounds have been shown to inhibit fatty acid synthase 
(FAS). FAS inhibitors can be identified by testing the ability of a 
compound to inhibit fatty acid synthase activity using purified enzyme. 
Fatty acid synthase activity can be measured spectrophotometrically based 
on the oxidation of NADPH, or radioactively by measuring the incorporation 
of radiolabeled acetyl- or malonyl-CoA. (Dils, et al, Methods Enzymol, 
35:74-83). Several FAS inhibitors are disclosed in U.S. Ser. No. 
08/096,908 and its CIP filed Jan. 24, 1994, both of which are hereby 
incorporated by reference. Included are inhibitors of fatty acid synthase, 
citrate lyase, CoA carboxylase, and malic enzyme. 
Preferred inhibitors of the condensing enzyme include a wide range of 
chemical compounds, including alkylating agents, oxididents, and reagents 
capable of undergoing disulphide interchange. The binding pocket of the 
enzyme prefers long chain, E, E, dienes such as: 
##STR2## 
In principal, a reagent containing the sidechain diene shown above and a 
group which exhibits reactivity with thiolate anions could be a good 
inhibitor of the condensing enzyme. Cerulenin (2S) (3R) 
2,3-epoxy-4-oxo-7,10 dodecadienoyl amide is an example: 
##STR3## 
Cerulenin, a specific and non-competitive inhibitor of fatty acid synthase, 
was studied in tumor cells as a representative inhibitor of fatty acid 
synthesis. Cerulenin covalently binds to a thiol group in the active site 
of the condensing enzyme of fatty acid synthase, inactivating this key 
enzymatic step (Funabashi, et al., J. Biochem., 105:751-755, 1989). The 
condensing enzyme reaction, which catalyzes the condensation of 
malonyl-CoA with an acetyl group or with the nascent fatty acid chain, 
generating CO.sub.2, is the most specific reaction of the synthase and is 
not shared by other enzymes. While cerulenin has been noted to possess 
other activities, these either occur in microorganisms which may not be 
relevant models of human cells (e.g. inhibition of cholesterol synthesis 
in fungi, Omura (1976), Bacteriol. Rev., 40:681-697; or diminished RNA 
synthesis in viruses, Perez, et al. (1991), FEBS, 280: 129-133), occur at 
a substantially higher drug concentrations (inhibition of viral HIV 
protease at 5 mg/ml, Moelling, et al. (1990), FEBS, 261:373-377) or may be 
the direct result of the inhibition of endogenous fatty acid synthesis 
(inhibition of antigen processing in B lymphocytes and macrophages, Falo, 
et al. (1987), J. Immunol., 139:3918-3923). Recent data suggests that 
cerulenin does not specifically inhibit myristoylation of proteins (Simon, 
et al., J. Biol. Chem., 267:3922-3931, 1992). 
TOFA (an inhibitor of acetyl CoA carboxylase) also demonstrated an 
anti-proliferative effect on a panel of cell lines with varying levels of 
FAS enzyme activity and fatty acid biosynthesis. TOFA can be used in 
combination with a novel inhibitor of the subject invention. 
As can be seen in U.S. Ser. No. 08/096,908 and its CIP filed Jan. 24, 1994, 
cerulenin as well as inhibitors of other enzymes of fatty acid synthesis 
potently inhibit the growth of mammary, colon, and prostatic carcinoma 
lines. Furthermore, the potency of growth inhibition was proportional to 
the relative levels of fatty acid biosynthesis exhibited by these cultured 
cells. Cerulenin acts by creating a state of fatty acid starvation leading 
to cell death, which is proportional to the level of endogenous fatty acid 
synthesis. 
Novel Compounds of the Invention 
Three groups of novel chemical compounds are described herein: 
Group A- Cerulenin (CE) analogs 
Class I- Modified reactive group 
Class II- Modified sidechain 
Class III- Modified reactive group and sidechain 
Group B- Geranyl (GE) analogs 
Group C- Dodecenoic Acid (DA) analogs 
Member compounds in each group show utility as inhibitors of 
therapeutically relevant enzymes, including, but not limited to, those of 
lipid biosynthesis/modification, mycocerosic acid synthetase, and enzymes 
of sterol biosynthesis. 
##STR4## 
In addition to acetyl CoA carboxylase and FAS, other target enzymes include 
citrate lyase and malic enzyme. These enzymes provide acetate and NADPH 
for lipid biosynthesis via FAS. The respective reactions are as follows: 
##STR5## 
Therapeutic compounds could also be based on these inhibitors as the 
deprivation of acetyl CoA or NADH would also stop the lipid synthesis. 
Of the enzymes in the fatty acid synthetic pathway, FAS is the preferred 
target for inhibition because it acts only within the pathway to fatty 
acids, while the other three enzymes are implicated in other cellular 
functions. Therefore, inhibition of one of the other three enzymes is more 
likely to affect normal cells. However, where an inhibitor for one of 
these enzymes can be shown to have a high therapeutic index as described 
above, the inhibitor may be used therapeutically according to this 
invention. The skilled clinician will be able to select a method of 
administration and to administer inhibitors of any enzyme in the synthetic 
pathway for fatty acids to treat carcinoma patients in need of such 
treatment, based on the teaching below. 
Palmitate is the major product of the fatty acid synthetase pathway. The 
elongation and oxidation of palmitate may be critical for production of 
necessary membrane lipids. For that purpose, the elongation and oxidation 
steps and any other processing steps for fatty acids would be likely 
molecular targets for therapeutics. To be incorporated into lipids, both 
endogenously synthesized fatty acids and exogenous dietary fatty acids 
must first be activated by acyl-CoA synthetase. Long-chain fatty acyl-CoA 
is an essential metabolite for animal cells, and so acyl-CoA synthetase is 
a preferred target. 
Tomoda and colleagues (Tomoda et..al., Biochim. Biophys. Act 921:595-598 
1987; Omura el. al., J. Antibiotics 39:1211-1218 1986) describe Triacsin C 
(sometimes termed WS-1228A), a naturally occurring acyl-CoA synthetase 
inhibitor, which is a product of Streptomyces sp. SK-1894. The chemical 
structure of Triacsin C is 1-hydroxy-3-(E, E, 
E-2',4',7'-undecatrienylidine) triazene. Triacsin C causes 50% inhibition 
of rat liver acyl-CoA synthetase at 8.7 .mu.M; a related compound, 
Triacsin A, inhibits acyl CoA-synthetase by a mechanism which is 
competitive for long-chain fatty acids. Inhibition of acyl-CoA synthetase 
is toxic to animal cells. Tomoda et. al. (Tomoda el. al., J. Biol. Chem. 
266:4214-4219, 1991) teaches that Triacsin C causes growth inhibition in 
Raji cells at 1.0 .mu.M, and have also been shown to inhibit growth of 
Vero and Hela cells. Tomoda el. al. further teaches that acyl-CoA 
synthetase is essential in animal cells and that inhibition of the enzyme 
has lethal effects. Triacsin C can be used in combination with the novel 
inhibitors of the subject invention. 
Lipid synthesis consists of multiple enzymatic steps. The data demonstrate 
that inhibition of lipid biosynthesis at two or more steps can create 
synergistic effects, lowering both the required concentration and the 
potential toxicity of any single agent. Since acyl-CoA synthetase is a 
ubiquitous enzyme apparently required by all cells for their continued 
well being, its inhibitors are potentially too toxic to be used 
effectively as a single anti-cancer agent. In contrast, when acyl-CoA 
synthetase inhibitors are paired with a cerulenin derivative, a specific 
fatty acid synthase inhibitor, synergistic effects are obtained, rendering 
each drug more effective. 
D. Administration of Inhibitors of Fatty Acid Synthesis 
Inhibitors of fatty acid synthesis are preferably formulated in 
pharmaceutical compositions containing the inhibitor and a 
pharmaceutically acceptable carrier. The pharmaceutical composition may 
contain other components so long as the other components do not reduce the 
effectiveness of the synthesis inhibitor so much that the therapy is 
negated. Pharmaceutically acceptable carriers are well known, and one 
skilled in the pharmaceutical art can easily select carriers suitable for 
particular routes of administration (Remington's Pharmaceutical Sciences, 
Mack Publishing Co., Easton, Pa., 1985). 
The pharmaceutical compositions containing any of the inhibitors of this 
invention may be administered by parenteral (subcutaneously, 
intramuscularly, intravenously, intraperitoneally, intrapleurally, 
intravesicularly or intrathecally, topical, oral, rectal, or nasal route, 
as necessitated by choice of drug, tumor type, and tumor location. 
Dose and duration of therapy will depend on a variety of factors, including 
the therapeutic index of the drugs, tumor type, patient age, patient 
weight, and tolerance of toxicity. Dose will generally be chosen to 
achieve serum concentrations from about 0.1 .mu.g/ml to about 100 
.mu.g/ml. Preferably, initial dose levels will be selected based on their 
ability to achieve ambient concentrations shown to be effective in 
in-vitro models, such as that used to determine therapeutic index, and 
in-vivo models and in clinical trials, up to maximum tolerated levels. 
Standard procedure in oncology requires that chemotherapy be tailored to 
the individual patient and the circulatory concentration of the 
chemotherapeutic agent be monitored regularly. The dose of a particular 
drug and duration of therapy for a particular patient can be determined by 
the skilled clinician using standard pharmacological approaches in view of 
the above factors. The response to treatment may be monitored by analysis 
of blood or body fluid levels of fatty acid synthase, measurement of FAS 
activity or OA-519 levels in tumor tissue or monitoring tumor burden in 
the patient. The skilled clinician will adjust the dose and duration of 
therapy based on the response to treatment revealed by these measurements. 
In an advantageous mode, the inhibitor of fatty acid synthesis is 
formulated in a pharmaceutical composition and applied to an externally 
accessible surface of the animal. Diseases which cause lesions in 
externally accessible surfaces may be treated by non-invasive 
administration of an inhibitor of fatty acid synthesis. Non-invasive 
administration includes (1) topical application to the skin in a 
formulation, such as an ointment or cream, which will retain the inhibitor 
in a localized area; (2) oral administration; (3) nasal administration as 
an aerosol; (4) intravaginal application of the inhibitor formulated in a 
suppository, cream or foam; (5) rectal administration via suppository, 
irrigation or other suitable means; (6) bladder irrigation; and (7) 
administration of aerosolized formulation of the inhibitor to the lung. 
Aerosolization may be accomplished by well known means, such as the means 
described in International Patent Publication WO 93/12756, pages 30-32, 
incorporated herein by reference. 
An advantageous strategy is to administer these compounds locally or 
topically in gels, ointments, solutions, impregnated bandages, liposomes, 
or biodegradable microcapsules. Compositions or dosage forms for topical 
application may include solutions, lotions, ointments, creams, gels, 
suppositories, sprays, aerosols, suspensions, dusting powder, impregnated 
bandages and dressings, liposomes, biodegradable polymers, and artificial 
skin. Typical pharmaceutical carriers which make up the foregoing 
compositions include alginates, carboxymethylcellulose, methylcellulose, 
agarose, pectins, gelatins, collagen, vegetable oils, mineral oils, 
stearic acid, stearyl alcohol, petrolatum, polyethylene glycol, 
polysorbate, polylactate, polyglycolate, polyanhydrides, phospholipids, 
polyvinylpyrrolidone, and the like. 
A particularly preferred formulation for fatty acid synthesis inhibitors is 
in liposomes. Liposomes containing fatty acid synthesis inhibitors 
according to this invention may be prepared by any of the methods known in 
the art for preparation of liposomes containing small molecule inclusions. 
Liposomes that are particularly suited for aerosol application to the 
lungs are described in International Patent Publication WO 93/12756, pages 
25-29, incorporated herein by reference. 
The compositions described above may be combined or used together or in 
coordination with another antineoplastic, antibiotic, antifungal or 
antiviral substance. 
E. Selective Chemotherapeutic Method 
In a preferred embodiment, the method of this invention also protects 
normal cells of patients treated with fatty acid synthesis inhibitors. To 
protect normal tissues such as liver (which normally may express wide 
ranges of fatty acid synthase activity) from potential toxicity, the level 
of FAS enzyme and/or fatty acid synthetic activity may be down-regulated 
before and/or during therapy. Down regulation may be accomplished by 
supplying essential fatty acids in the diet, by reduction of caloric 
intake or by other effective methods, such as administration of glucagon. 
Because FAS is an inducible enzyme in normal tissues, reduction in caloric 
intake will result in lower expression of FAS by normal cells. The most 
virulent tumor cells express FAS (OA-519) constitutively. In a patient 
with limited caloric intake, FAS expression is limited to tumor cells, and 
the cytotoxic effect of FAS inhibitors will be similarly limited. 
Down-regulation of FAS expression is usually coupled to fatty acid 
synthesis inhibitor therapy by reducing caloric intake of the patient 
before and during administration of the inhibitor. 
Another suitable method of reducing FAS expression is exogenous 
administration of fatty acids, preferably, essential fatty acids. These 
fatty acids may be formulated in any way that results in the 
down-regulating FAS expression of normal cells. This could be by including 
them in the diet of the patient or by formulating them in the same 
pharmaceutical composition as the fatty acid synthesis inhibitor, or any 
other suitable method. 
Diets suitable for reducing FAS expression in normal tissue are easily 
within the skill of the ordinary clinician. Any method of reducing FAS 
expression by normal cells is within the contemplation of the method of 
this invention, as long as the FAS level in normal cells is reduced during 
the time that the fatty acid synthesis inhibitor is present in the patient 
at levels that would be cytotoxic to tumor cells. 
EXAMPLES 
The following Examples are provided for purposes of illustration only. They 
are not intended to limit the invention described above, which is only 
limited by the appended claims. 
EXAMPLE 1 
The synthesis of cerulenin-like molecules draws in part from existing 
methods for this FAS inhibitor. These routes include: R. K. Boeckman, Jr. 
et al J. Am. Chem. Soc. 1979, 101, 987; E. J. Corey and D. R. Williams 
Tetrahedron Lett. 1977, 3847; A. A. Jakubowski et al. J. Org. Chem 1982, 
47, 1221; K. Mikami et al., Chem. Lett. 1981, 1721; T. Ohta et al. 
Heterocycles 1986, 24, 1137; H. Yoda et al. Tetrahedron Lett. 1991, 32, 
6771. More recently side chain variations on the structure of cerulenin 
have been described (N. Morisaki et al. Chem. Pharm, Bull. 1992, 40, 
2945), and the carbocyclic analogue of cerulenin has been prepared as well 
(R. Shimazawa et al. Chem. Pharm. Bull. 1992, 40, 2954). 
##STR6## 
Vinyl alcohol E2: To 5 g of m-tolualdehyde in 70 mL of anhydrous ether at 
0.degree. C. was added vinyl magnesium bromide (41.6 mL of a 1 molar 
solution in tetrahydrofuran) over 45 min. After stirring for 5 h at 
0.degree. C. and 5 h at room temperature, the reaction was quenched with 
ammonium chloride solution and extracted with ether. The organic phase was 
dried over anhydrous magnesium sulfate and evaporated to yield a pale 
yellow oil used directly in the next step. 
IR (neat): 3356, 3019, 2919, 1641, 1607 and 1487 cm.sup.-1. 
.sup.1 H-NMR (CDCl.sub.3): d 2.25 (s, 3H), 5-5.28 (m, 3H), 5.93 (m, 1H), 
7.12 (m, 4H). 
Aldehyde E4: To 5.75 g of the vinyl alcohol E2 was added 70 ml of freshly 
distilled ethyl vinyl ether and 12.4 g of mercuric acetate. The mixture 
was heated to reflux, acetic acid added (1.2 g) and stirred for 3 h at 
room temperature. The reaction solution was partitioned between petroleum 
ether and 5% aqueous potassium hydroxide. The organic layer was washed 
with brine and dried over anhydrous sodium carbonate. The solvents were 
evaporated under reduced pressure to obtain the vinyl ether E3 (5.75 g, IR 
(ncat): 1635, 1611 cm.sup.-1) as a light yellow oil. The product was 
homogeneous by thin layer chromatography and subjected to Claisen 
rearrangement without further purification. 
The vinyl ether E3 was heated under nitrogen at 130.degree.-150.degree. C. 
for 5 h. The product was purified by column chromatography on silica gel 
(hexane:ethyl acetate 97.5:2.5) to yield the homogeneous aldehyde E4 as a 
colorless oil (4.75 g. 88%). 
IR (neat): 2719, 1724, 1603, 1583, 1487 cm.sup.-1. 
.sup.1 H-NMR (CDCl.sub.3): d 2.24 (s, 3H), 2.45 (d x t, J=6.0, 1.3 Hz, 2H), 
2.49 (m, 2H), 6.08 (d x d x d x d, J=6.8, 12.7 15.7 Hz, 1H), 6.28 (d, 
J=15.7 Hz, 1H), 7-7.2 (m, 4H), 9.71 (t, J=1.3 Hz, 1H). 
Exact Mass: calculated for C.sub.12 H.sub.14 O: 174.1045, found: 174.1048. 
Acetylenic ester E5: Methyl propiolate (0.75 mL, 8.4 mmol) was added to 1.1 
equiv. of lithium diisopropylamide cooled to -78.degree. C. in 15 mL of 
dry tetrahydrofuran. After 30 min, 0.9 equiv. of aldehyde E4 was added in 
3 mL of tetrahydrofuran and stirred for 3 h. Acetic acid was added (2 mL) 
and the reaction mixture was allowed to warm to room temperature over 3 h. 
The reaction was worked up with aqueous ammonium chloride and extracted 
with ether and the ether layer was washed with aqueous sodium bicarbonate, 
brine and dried over anhydrous magnesium sulfate. Removal of the solvent 
and chromatography on silica gel (hexane: ethyl acetate 90:10) gave E5 as 
an oil (1.5 g, 76%). 
IR (neat): 3400, 2950, 2235, 1715, 1602, 1487, 1255 cm.sup.-1. 
.sup.1 H-NMR (CDCl.sub.3): 1.94 (m, 2H), 2.25 (s, 3H), 3.75 (s, 3H), 4.52 
(t, j=6.8 Hz, 1H), 6.2 (d x d x d, J=6.8, 12.7, 15.7 Hz, 1H), 6.40 (d, 
H=15.7 Hz, 1H), 7-7.2 (m, 4H). 
Exact Mass: calculated for C.sub.16 H.sub.18 O.sub.3 : 258.1256, found 
258.1256. 
Butenolide E6: The hydroxy ester E5 (110 mg. 0.426 mmol) was hydrogenated 
in methanol (5 mL) and dry pyridine (0.4 mL) using Lindlar catalyst (5% 
Pd/CaCO.sub.3, 30 mg). After the theoretical amount of hydrogen had been 
consumed, the mixture was diluted with ether and filtered through Celite 
to remove the catalyst. The organic solution was washed with 5% aqueous 
HCl, saturated aqueous sodium hicarbonate and dried over anhydrous 
magnesium sulfate. The volume was reduced to 25 mL and a catalytic amount 
of p-toluenesulfonic acid was added to carry out the lactonization. After 
stirring for 7 h at room temperature, the solution was washed with dilute 
aqueous sodium bicarbonate and dried as above. The solvents were removed 
under vacuum and chromatography on silica gel (hexanes:ethyl acetate 
78:22) gave the butenolide E6 as a colorless oil (61 mg, 63%). 
IR (neat): 3022, 2921, 1755, 1690, 1600 1445 cm.sup.-1. 
.sup.1 H-NMR (CDCl.sub.3): d 1.77-1.95 (m, 2H), 2.32 (s, 3H), 2.36 (m, 2H), 
5.06 (m, 1H), 6-6.22 (m, 2H), 6.39 (d, J=15.7 Hz, 1H), 7-7.22 (m, 4H), 
7.48 (d, J=1.4 Hz, 1H). 
Epoxylactone E7: To a solution of the butenolide E6 (50 mg, 0.219 mmol) in 
pyridine (2.5 mL) was added sodium hypochlorite (0.15 mL, 5% chlorine, 
diluted 1:1 with water). After stirring 2.5 h at 0.degree. C., the mixture 
was allowed to come to room temperature and methylene chloride (15 mL) and 
water (5 mL) were added. The mixture was treated with 5% aqueous sodium 
bicarbonate (10 mL) and the aqueous solution was washed with ether and 
then acidified to pH 1 (0.5N HCl). The solution was then thoroughly 
extracted with ethyl acetate, dried over anhydrous magnesium sulfate. 
After removal of the solvents, the crude product was heated at 70.degree. 
C. for 3 h to ensure complete closure of the epoxylactone. After 
chromatography on silica gel (hexanes:ethyl acetate 80:20), the product E7 
was obtained as a glassy solid (28 mg, 52%). 
IR (neat): 3023, 2922, 1783, 1603, 1583, 1587 cm.sup.-1. 
.sup.1 H-NMR (CDCl.sub.3): d 1.91 (m, 2H), 2.44 (s, 3H), 2.48 (m, 2H), 3.87 
(d, J=2.4 Hz, 1H). 4.08 (d, J=2.4 Hz, 1H), 4.74 (d x d, J=5.6, 7.5 Hz, 
1H), 6.25 (d x d x d, J=6.9, 13.9, 15.7 Hz, 1H), 6.50 (d, J=15.7 Hz, 1H), 
7.1-7.3 (m, 4H). 
Exact Mass: calculated for C.sub.15 H.sub.16 O.sub.3 : 244.1099, found: 
244.1102. 
Hydroxyamide E8: The epoxylactone E7 (73 mg, 0.299 mmol) in methanol was 
treated with concentrated aqueous ammonium hydroxide (0.05 mL, excess). 
After stirring 1 h, the solution was diluted with methylene chloride (10 
mL) and washed with 0.5N HCl. The solution was dried over anhydrous sodium 
sulfate, the solvents removed under vacuum to yield the product E8 as a 
white solid, which was recrystallized from ethyl acetate/hexanes (63 mg, 
80%, mp 104.degree.-106.degree. C.). 
IR (film): 3364, 1681, 1603 cm.sup.-1. 
.sup.1 H-NMR (CDCl.sub.3): d 1.8 (m, 2H), 2.24 (s, 3H), 2.3 (m, 2H) 3.19 
(m, 1H), 3.53 (d, J=5.6 Hz, 1H), 3.55 (m, 1H), 3.78 (br s, 1H), 6.2-6.5 
(M, 4H), 7-7.35 (m, 4H). 
Ketoamide E9: The hydroxamide E8 (72 mg, 0.275 mmol) was dissolved in 
methylene chloride (10 mL) and treated with 1 g of pyridinium 
chlorochromatic. After stirring for 2.5 h at room temperature, the 
reaction mixture was diluted with 35 mL of ether and the mixture was 
filtered through a plug of Celite. The crude product E9 was purified by 
flash chromatography on silica gel (hexanes:ethyl acetate 1:1) to yield 
the ketoamide E9 at a white solid (46 mg, 64%). 
IR (film): 3335, 2923, 1713, 1684, 1594 cm.sup.-1. 
.sup.1 H-NMR (CDCl.sub.3): d 2.33 (s, 3H), 2.3-2.4 (m, 2H), 2.74 (m, 2H), 
3.74 (d, J=5.3 Hz, 1H), 3.88 (d, J=5.2, 1H), 5.47 (br s, 2H), 6.07 (d x d 
x d, J=6.8, 13.9, 15.7 Hz, 1H), 6.35 (d, J=15.7, 1H). 
Exact Mass: calculated for C.sub.15 H.sub.17 NO.sub.3 : 259.1208, found: 
259.1212 
EXAMPLE 2 
##STR7## 
Alcohol E10: Aldehyde E4 (1.5 g, 8.62 mmol) in methanol (50 mL) was 
treated with sodium borohydride (650 mg, 17.1 mmol) at room temperature. 
After stirring for 10 h, the volume was reduced to ca. 10 mL and 25 mL of 
aqueous ammonium chloride was added. The mixture was acidified to pH 1 
with 0.5N HCl and extracted three times with ether. The organic extracts 
were dried over anhydrous magnesium sulfate, evaporated and the residue 
purified by flash chromatography to yield the desired alcohol E10 as a 
glass solid (1.45 g, 95%). 
IR (neat): 3220, 2938, 1604 cm.sup.-1. 
.sup.1 H-NMR (CDCl.sub.3): d 1.77 (m, 2H), 2.27 (s, 3H), 2.30 (m, 2H), 3.70 
(t, J=6.3 Hz, 2H), 6.22 (d x d x d, J=6.8, 13.9, 15.7 Hz, 1H), 6.37 (d, 
J=15.7 Hz, 1H), 7-7.2 (m, 4H). 
Maleimide E11: To a solution of alcohol E10 (840 mg, 4.77 mmol) in 20 mL of 
dry tetrahydrofuran was added diethyl azodicarboxylate (0.825 mL, 5.25 
mmol) followed by maleimide (500 mg, 5.25 mmol) and triphenyl phosphine 
(1.37 g, 5.25 mmol). After stirring the solution at room temperature for 
15 h, evaporation of the solvents afforded a yellow solid, which was 
filtrated with ether:hexanes 1:1 (300 mL) and the filtrate was evaporated 
under reduced pressure. The residue was purified by column chromatography 
on silica gel (hexanes:ethyl acetate 80:20) to yield the substituted 
maleimide E11 as a glassy solid (300 mg, 54% based on recovered E10). 
IR (neat): 3660, 3098, 2939, 1704, 1603, 1584 cm.sup.-1. 
.sup.1 H-NMR (CDCl.sub.3): d 1.75 (m, 2H), 2.20 (m, 2H), 2.30 (s, 3H), 3.55 
(t, J=6.3 Hz, 2H) 6.17 (d x d x d, J=6.8, 13.9, 15.7 Hz, 1H), 6.38 (d, 
J=15.7 Hz, 1H), 6.62 (s, 2H), 7-7.2 (m, 4H). 
Exact Mass: calculated for C.sub.16 H.sub.17 NO.sub.2 : 255.1259, found: 
255.1262. 
EXAMPLE 3 
##STR8## 
Aldehyde E13: Chromium trioxide (3.79 g, 37.9 mmol) in methylene chloride 
(50 mL) was treated carefully with pyridine (6 g, 37.8 mmol) at 0.degree. 
C. After stirring at 0.degree. C. for 1 h, alcohol E12 (2.4 g, 13.3 mmol) 
was added in 10 mL of methylene chloride and stirring was continued 
overnight. The solvents were evaporated, the residue taken up in ethyl 
acetate and washed with 1N NCl, aqueous sodium bicarbonate solution and 
distilled water. The solution was dried over anhydrous magnesium sulfate, 
the solvent removed in vacuo and the residue purified by column 
chromatography (hexanes:ethyl acetate 90:10) to yield the aldehyde E13 as 
a colorless oil (1.7 g, 72%). 
IR (neat): 2930, 1720, 1719, 1595, 1448, 1096 cm.sup.-1. 
.sup.1 H-NMR (CDCl.sub.3): d 1.90 (m, 2H), 2.58 (d x t, J=1.5, 7.0 Hz, 2H), 
3.48 (t, J=6.0 Hz, 2H), 4.48 (s, 2H) 7.31 (m, 5H), 9.77 (t, J=1.5 Hz, 1H). 
Acetylenic ester E14: Aldehyde E13 (1.5 g, 8.42 mmol) was treated with 
methyl propiolate as above in Example 1 to give E14 after purification by 
chromatography on silica gel (hexanes:ethyl acetate 85:15) as a colorless 
oil (1.42 g, 65%). 
IR (neat): 3398, 2953, 2235, 1717, 1453, 1435, 1255, 1096 cm.sup.-1. 
.sup.1 H-NMR (CDCl.sub.3): d 1.88 (m, 4H), 3.51 (m, 2H), 3.77 (s, 3H), 4.54 
(m, 3H), 7.31 (m, 5H). 
Butenolide E15: The acetylenic ester E14 (854 mg, 3.25 mmol) was reduced 
and cyclized to the butenolide E15 as above in Example 1 to give 0.70 g 
(93%) as a colorless oil after chromatography on silica gel (hexanes:ethyl 
acetate 80:20). 
IR (neat): 2936, 1755, 1659, 1600, 1495, 1362, 1163, 1102, 1017 cm.sup.-1. 
.sup.1 H-NMR (CDCl.sub.3): d 1.5-1.7 (m, 4H), 3.41 (m, 2H), 4.57 (s, 2H), 
5.05 (m, 1H), 6.09 (d, x d, J=2.0, 5.5 Hz, 1H), 7.30 (m, 5H), 7.44 (d, 
J=5.7 Hz, 1H). 
Epoxylactone E16: The butenolide E15 (50 mg, 0.21 mmol) was epoxidized as 
in Example 1 to afford, after chromatography on silica gel (hexanes:ethyl 
acetate 85.15), the epoxylactone E16 as a glassy solid (31 mg, 60%). 
IR (neat): 2926, 1784, 1453, 1180, 1099, 1025 cm.sup.-1. 
.sup.1 H-NMR (CDCl.sub.3): d 1.76 (m, 4H), 3.50 (m, 2H), 3.76 (d, J=2.4 Hz, 
1H), 3.96 (d, J=2.4 Hz, 1H), 4.50 (s, 2H), 4.58 (m, 1H), 7.32 (m, 5H). 
Hydroxyamide E17: The epoxylactone E16 (20 mg, 0.08 mmol) in methanol was 
treated with concentrated aqueous ammonia (0.04 mL, excess) at 0.degree. 
C. After 1 h, aqueous workup as in Example 1 furnished the hydroxyamide as 
a glassy solid (19 mg, 89%). 
IR (neat): 3347, 2927, 1679, 1601, 1453 cm.sup.-1. 
.sup.1 -NMR (CDCl.sub.3): d 1.75 (m, 4H), 3.08 (d x d, J=1.7, 4.6 Hz, 1H), 
3.47 (m, 4H), 4.49 (s, 2H), 6.22 br d, J.sub.app =8.4 Hz, 2H), 7.30 (m, 
5H). 
Ketoamide E18: Oxidation of hydroxyamide E17 (19 mg, 0.07 mmol) using 
chromium trioxide-pyridine complex (130 mg) as in Example 1 afforded the 
ketoamide E18 as a glass solid (13.5 mg, 72%). 
IR (neat): 2928, 1717, 1684, 1455 cm.sup.-1. 
.sup.1 H-NMR (CDCl.sub.3): d 1.81 (m, 2H), 2.67 (m, 2H), 3.44 (t, J=5.7 Hz, 
2H) 3.71 (br d, 1H), 3.87 (br d, 1H), 4.49 (s, 2H), 6.4 (br s, 2H), 7.3 
(m, 5H). 
EXAMPLE 4 
FAS Enzymatic assay for Testing Compound Activity 
Methods: 
Preparation of crude cell extracts: The potential of compounds to inhibit 
FAS enzymatic activity was determined using a crude cell extract of a 
cancer cell line or Saccharomyces cerevisiae. The breast cancer cell line 
ZR-75-1 was grown to confluence in RPMI with 10$FBS. The cells were 
harvested by scraping, washed once with cold phosphate buffered saline pH 
7.5 and stored at -80.degree. C. Extracts were prepared by thawing the 
cells in the presence of 4 vol. of 25 mM potassium phosphate pH 7.5, 1 mM 
EDTA, 1 mM DTT, 0.4% digitonin, 0.7 .mu.g/ml pepstatin, 2 .mu.g/ml 
aprotinin and 0.5 .mu.g/ml leupeptin. Cells were homogenized with a Dounce 
type homogenizer for 20 strokes. Extracts were cleared by two sequential 
centrifugations at 30,000 xg, for 30 min each. The extract was dialyzed 
against 125 mM potassium phosphate pH 6.6, 1 mM EDTA, 1 mM DTT using a 
100,000 molecular weight cut off membrane. The extract stored at 4.degree. 
C. with 3 mM sodium azide. Yeast extracts were prepared from freeze-dried 
yeast. Yeast were reconstituted in 5 vol/g 100 mM potassium phosphate pH 
6.5. Cells were harvested by centrifugation at 3,000 xg for 10 min. Cells 
were washed with 5 vol 100 mM potassium phosphate pH 6.5 and suspended in 
2 vol. 125 mM potassium phosphate pH 6.6, 1 mM EDTA, 1 mM DTT, 0.7 
.mu.g/ml pepstatin, 2 .mu.g/ml aprotinin and 0.5 .mu.g/ml leupeptin. 
Sterile 0.5 mm glass beads were added (1/2 vol) and the mixture vortex 
vigorously for four 30 s intervals with 30 s rests on ice between mixes. 
Extracts were cleared by centrifugation and dialyzed as described above. 
Testing of compound activity: Compounds were tested for their ability to 
inhibit FAS enzymatic activity by incubating the compounds with the crude 
cell extracts and measuring residual FAS activity. The compounds were 
mixed with a crude cell extract to the desired concentration and the 
mixture incubated at 10.degree.-25.degree. C. for 30 min. Residual FAS 
activity was measured as described below. The concentration of compound 
giving 50% maximal activity defined the IC.sub.50 which was calculated 
from a 4-parameter equation of the dose-response curves. 
Enzymatic assay conditions: The FAS enzyme activity was measured by 
monitoring the oxidation of NADPH spectrophotometrically (Dils, R. and E. 
M. Carey, 1975, Fatty acid synthase from rabbit mammary gland. Methods in 
Enzymol. 35:74-83). A 50 .mu.l portion of an extract or extract-compound 
mixture was mixed with 50 .mu.l 250 mM potassium phosphate pH 6.6, 40 
.mu.M acetyl-CoA, 200 .mu.M malonyl-CoA, 1 mM NADPH and 2 mM DTT. The 
initial rate of NADPH oxidation at 25.degree. C. was measured by 
monitoring the decrease in absorbance at 340 nm. 
The results are shown in TABLE 1. 
TABLE 1 
__________________________________________________________________________ 
IC.sub.50 VALUES FOR INHIBITORS OF FAS ACTIVITY 
IC.sub.50 (.mu.M) 
COMPOUND 
STRUCTURE Cancer 
Yeast 
__________________________________________________________________________ 
CTC002 Cerulenin 
##STR9## 8.6 0.4 
CTC003 
##STR10## &gt;100 
N.T. 
CTC004 
##STR11## &gt;100 
N.T. 
CTC005 
##STR12## &gt;78 N.T. 
CTC006 (cyclic. cer.) 
##STR13## 10.4 
82 
CTC013 
##STR14## &gt;97 N.T. 
CTC014 
##STR15## &gt;92 N.T. 
__________________________________________________________________________ 
N.T. not tested 
EXAMPLE 5 
Growth inhibition asssay for testing compounds 
Methods: 
Cells were seeded at 5.times.10.sup.3 per well into 96-well plates and 
incubated for 3 days in RPMI, 10% FBS at 37.degree. C. in a 5% CO.sub.2 
:95% air atmosphere. Medium was removed and replaced with media plus 
compound. Compound doses were 10 serial dilutions from 200 .mu.g/ml to 
0.39 .mu.g/ml. Cells were returned to the incubator for 3 additional days. 
The medium was removed and plates were washed twice with phosphate 
buffered saline. Wells were filled with 100 .mu.l of 0.2% crystal violet 
in 2% ethanol for 10 min. Stain was removed from the wells and the plates 
were washed 3 times with distilled H.sub.2 O. After at least 4 h of drying 
wells were filled with 100 .mu.l of 1% SDS and plates were shaken to 
disolve any dye crystals. Absorbance at 560 nm minus 650 nm in each well 
was measured using a Molecular Devices plate reader. IC.sub.50 values were 
calculated from 4-parameter curve-fits of the dose-response curves. 
The results are shown in TABLE 2. 
TABLE 2 
__________________________________________________________________________ 
IC.sub.50 VALUES FOR INHIBITORS OF FAS IN GROWTH INHIBITION ASSAY 
IC.sub.50 
IC.sub.50 
SKBr 3 
HS 27 
COMPOUND 
STRUCTURE Cells .mu.M 
Cells .mu.M 
__________________________________________________________________________ 
CTC002 Cerulenin 
##STR16## 20 20 
CTC003 
##STR17## &gt;50 &gt;50 
CTC004 
##STR18## &gt;50 &gt;50 
CTC005 
##STR19## &gt;50 &gt;50 
CTC006 (Cyclic. cer.) 
##STR20## 21 32 
CTC013 
##STR21## 63 63 
CTC014 
##STR22## &gt;50 &gt;50 
__________________________________________________________________________ 
EXAMPLE 6 
Description and Operation: Determination of the activity of the cerulenin 
derivative CTC 006 (phenyl cerulenin) against M. tuberculosis was 
performed utilizing a commercially available radiometric system which is 
based on the same principle that is utilized in conventional antibiotic 
susceptibility testing of M. tuberculosis. The significant difference 
between methods is that a liquid medium is used and rather than counting 
colonies after approximately 3 weeks of incubation, the growth is 
monitored through measurement of metabolism of .sup.14 C-labeled palmitic 
acid to .sup.14 CO.sub.4 radiometrically with the results being available 
in 3 to 5 days. Drug susceptibility or resistance is determined by the 
modified version of the conventional proportion method. The critical 
proportion for resistance is taken as 1% for all antituberculosis drugs. 
Resistance is determined through comparison of the growth rate in control 
vials containing a 1% inoculum and broth vials containing the specific 
test drug. This method has been found comparable to the conventional 
proportional method or the resistance ratio method. Similarly accuracy and 
reproducibility of this method have yielded excellent results. 
Materials and Methods: 
Organisms 
Mycobacteria: A control organism M. tuberculosis H37RV was used throughout 
the studies. Due to the relatively slow growth rate of M. tuberculosis 
H37RV, a Candida albicans strain known to be susceptible to cerulenin was 
used to control antibiotic concentration as well as M. tuberculosis H37RV. 
The remaining isolates of M. tuberculosis were clinical isolates from this 
institution or referred here as part of a cooperate susceptibility study 
on strains of obtained from patients seen in Haiti. 
Susceptibility Test Method: 
Mycobacteria: Susceptibility testing was performed using a commercially 
available Middlebrook 7H12 broth media containing .sup.14 C-labeled 
palmitate (12B Bactec bottles) as an indicator substrate. Growth in this 
system is determined through measurement of .sup.14 CO.sub.2 generation 
from metabolism of the .sup.14 C-labeled palmitate. A 1 mg/ml initial 
stock solution of cerulenin was prepared and diluted to the following 
concentrations (.mu.g/ml): 1000, 500, 250, 125, 62.5. A 0.1 ml of the 
stock concentrations were then added to individual 4.0 ml Bactec bottles 
resulting in the following final concentrations (.mu.g/ml): 25, 12.5, 
6.25, 3.0, 1.5. For each strain tested 0.1 ml of organism was added to 
each bottle at each concentration tested, a direct control (bottle 
containing diluent, DMSO, but no antibiotic, and a 1:100 organism dilution 
which is also added to broth bottle not containing antibiotic. All broth 
bottles were incubated at 35.degree. C. and read daily for Growth Index 
(GI proportional to quantity of .sup.14 CO.sub.2 generated) readings. 
Results were recorded until the GI of the 1:100 control reached 30. At 
this time, the minimum inhibitory concentration of the isolate 
wasdetermined. Control organisms for each susceptibility run included 
Candida albicans (cerulenin MIC&lt;1.5 .mu.g/ml). A 0.5 McFarland suspension 
of C. albicans is prepared and 0.1 ml of this suspension was added to each 
concentration of CTC 006 in the 12B Bactec bottles. 
The minimum inhibitory concentration of each isolate was determined using 
the following criteria. Once the growth index (GI) of the 1:100 control 
bottle had reached a value of 30 the change (.increment.) in growth index 
for a one day period was calculated as well as the growth index change 
(.increment.) at each concentration tested during the same 24 hour period. 
The MIC was defined as the lowest cerulenin concentration that yielded a 
growth index change less than that of the 1:100 control bottle. 
Results: 
Mycobacteria: CTC 006, in this susceptibility test system, does have 
inhibitory activity against both susceptible and multiply drug resistant 
M. tuberculosis with minimum inhibitory concentrations ranging from &lt;1.5 
.mu.g/ml to 6.25 .mu.g/ml. Compound CTC 006 had an MIC of 25 .mu.g/ml. 
It will be understood that while the invention has been described in 
conjunction with specific embodiments thereof, the foregoing description 
and examples are intended to illustrate, but not limit the scope of the 
invention. Other aspects, advantages and modifications will be apparent to 
those skilled in the art to which the invention pertains, and these 
aspects and modifications are within the scope of the invention, which is 
limited only by the appended claims.