Trienoic retinoid compounds and methods

Novel trienoic compounds having activity for retinoic acid receptors and retinoid X receptors are provided. Also provided are pharmaceutical compositions incorporating such compounds and methods for their use.

FIELD OF THE INVENTION 
The present invention relates to compounds having activity for retinoic 
acid receptors and retinoid X receptors, and to methods for the 
therapeutic use of such compounds. 
BACKGROUND OF THE INVENTION 
The vitamin A metabolite, retinoic acid, has long been recognized to induce 
a broad spectrum of biological effects. In addition, a variety of 
structural analogues of retinoic acid have been synthesized that also have 
been found to be bioactive. Some, such as Retin-A.RTM. and Accutane.RTM., 
have found utility as therapeutic agents for the treatment of various 
pathological conditions. In addition, synthetic retinoids have been found 
to mimic many of the pharmacological actions of retinoic acid. 
Medical professionals have become very interested in the therapeutic 
applications of retinoids. Among their uses approved by the FDA is the 
treatment of severe forms of acne and psoriasis. A large body of evidence 
also exists that these compounds can be used to arrest and, to an extent, 
reverse the effects of skin damage arising from prolonged exposure to the 
sun. Other evidence exists that these compounds may be useful in the 
treatment and prevention of a variety of cancerous and pre-cancerous 
conditions, such as melanoma, cervical cancer, some forms of leukemia, 
oral leukoplakia and basal and squamous cell carcinomas. Retinoids have 
also shown an ability to be efficacious in treating and preventing 
diseases of the eye, cardiovascular system, immune system, skin, 
respiratory and digestive tracts, and as agents to facilitate wound 
healing and modulate programmed cell death (apoptosis). 
Major insight into the molecular mechanism of retinoic acid signal 
transduction was gained in 1988, when a member of the steroid/thyroid 
hormone intracellular receptor superfamily was shown to transduce a 
retinoic acid signal. Evans, Science, 240:889-95 (1988); Giguere et al., 
Nature, 330:624-29 (1987); Petkovich et al., Nature, 330:444-50 (1987). It 
is now known that retinoids regulate the activity of two distinct 
intracellular receptor subfamilies; the Retinoic Acid Receptors (RARs) and 
the Retinoid X Receptors (RXRs), including their isoforms, RAR.alpha., 
.beta., .gamma. and RXR.alpha., .beta., .gamma.. In this regard, an 
endogenous low-molecular-weight ligand which modulates the transcriptional 
activity of the RARs is all-trans-retinoic acid (ATRA), while an 
endogenous ligand for the RXRs is 9-cis retinoic acid (9-cis). Heyman et 
al., Cell, 68:397-406 (1992) and Levin et al. Nature, 355:359-61 (1992). 
Although both the RARs and RXRs respond to ATRA in vivo, due to the in vivo 
conversion of some of the ATRA to 9-cis, the receptors differ in several 
important aspects. First, the RARs and RXRs are significantly divergent in 
primary structure (e.g., the ligand binding domains of RAR.alpha. and 
RXR.alpha. have only 27% amino acid identity). These structural 
differences are reflected in the different relative degrees of 
responsiveness of RARs and RXRs to various vitamin A metabolites and 
synthetic retinoids. In addition, distinctly different patterns of tissue 
distribution are seen for RARs and RXRs. For example, in contrast to the 
RARs, which are not expressed at high levels in the visceral tissues, 
RXR.alpha. mRNA has been shown to be most abundant in the liver, kidney, 
lung, muscle and intestine. Finally, the RARs and RXRs have different 
target gene specificity. For example, response elements have recently been 
identified in the cellular retinal binding protein type II (CRBPII) and 
Apolipoprotein AI genes which confer responsiveness to RXR, but not RAR. 
Furthermore, RAR has also been recently shown to repress RXR-mediated 
activation through the CRBPII RXR response element (Manglesdorf et al., 
Cell, 66:555-61 (1991)). These data indicate that two retinoic acid 
responsive pathways are not simply redundant, but instead manifest a 
complex interplay. 
In view of the related, but clearly distinct, nature of these receptors, 
retinoids which are more selective for the RAR subfamily or the RXR 
subfamily would be of great value for selectively controlling processes 
mediated by one or more of the RAR or RXR isoforms, and would provide the 
capacity for independent control of the physiologic processes mediated by 
the RARs or RXRs. In addition, pan-agonist retinoids that activate one or 
more isoforms of both the RARs and RXRs would also be valuable for 
controlling processes mediated by both of these subfamilies of retinoid 
receptors. Furthermore, retinoids which preferentially affect one or more 
but not all of the receptor isoforms also offer the possibility of 
increased therapeutic efficacy and reduced side effect profiles when used 
for therapeutic applications. 
Various polyene compounds have been disclosed to be useful in the treatment 
of inflammatory conditions, psoriasis, allergic reactions, and for use in 
sunscreens in cosmetic preparations. See e.g., U.S. Pat. Nos. 4,534,979 
and 5,320,833. In addition, trienediolates of hexadienoic acids have 
proved useful in the synthesis of retinoic and nor-retinoic acids. See M. 
J. Aurell, et al., 49 Tetrahedron, 6089 (1993). However, no retinoid 
activity has been ascribed to these compounds. 
SUMMARY OF THE INVENTION 
The present invention provides novel trienoic compounds that have selective 
activity on RARs and RXRs or pan-agonist activity on one or more each of 
the RAR and RXR isoforms. The present invention also provides labeled 
retinoid compounds, pharmaceutical compositions incorporating these novel 
trienoic compounds and methods for the therapeutic use of such compounds 
and pharmaceutical compositions. 
These and various other advantages and features of novelty which 
characterize the invention are pointed out with particularity in the 
claims annexed hereto and forming a part hereof. However, for a better 
understanding of the invention, its advantages, and objects obtained by 
its use, reference should be had to the accompanying descriptive matter, 
in which there is illustrated and described preferred embodiments of the 
invention. 
Definitions 
In accordance with the present invention and as used herein, the following 
terms are defined with the following meanings, unless explicitly stated 
otherwise. 
The term alkyl refers to straight-chain, branched-chain or cyclic 
structures that are optionally saturated or unsaturated (thereby resulting 
in alkenyl and alkynyl structures), as well as combinations thereof. 
The term aryl refers to an optionally substituted six-membered aromatic 
ring. 
The term heteroaryl refers to an optionally substituted five-membered or 
six-membered heterocyclic ring containing one or more heteroatoms selected 
from the group consisting of oxygen, nitrogen and sulfur. 
The terms retinoid or retinoids refer to compound(s) that bind and/or 
activate one or more retinoid receptors, thereby affecting the 
transcriptional activity of a target gene to which the activated receptor 
and compound complex binds. 
The term pan-agonist refers to a retinoid that activates at least one 
member of both the RAR subfamily (i.e., RAR.alpha., RAR.beta., or 
RAR.gamma.) and the RXR subfamily (i.e., RXR.alpha., RXR.beta., or 
RXR.gamma.). Preferably such pan-agonist retinoids activate all members of 
both the RAR and RXR subfamilies of retinoid receptors. 
As used herein, isotopic labels or radiolabels refer to substituents 
labeled with deuterium, tritium, carbon 13 and/or carbon 14, including, 
but not limited to .sup.14 CH.sub.3, .sup.13 CH.sub.3, CD.sub.3, C.sup.3 
H.sub.3, and .sup.13 CD.sub.3.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION 
In accordance with a first aspect of the present invention, we have 
developed novel trienoic compounds having the formulae: 
##STR1## 
wherein: 
R.sup.1, R.sup.2 and R.sup.4 each independently are hydrogen, aryl, 
heteroaryl, CF.sub.3 or a C.sub.2 -C.sub.6 alkyl, fluoroalkyl or 
perfluoroalkyl optionally substituted with .sup.14 CH.sub.3, .sup.13 
CH.sub.3, CD.sub.3, C.sup.3 H.sub.3, and/or .sup.13 CD.sub.3 ; 
R.sup.3 and R.sup.5 each independently are hydrogen, CF.sub.3, a C.sub.1 
-C.sub.3 alkyl, a C.sub.1 to C.sub.3 fluoroalkyl or perfluoroalkyl, or 
where is hydrogen, CF.sub.3, a C.sub.1 -C.sub.2 alkyl or a C.sub.1 to 
C.sub.2 fluoroalkyl or perfluoroalkyl, provided, however, that R.sup.1 and 
R.sup.5 cannot be CF.sub.3 or alkyl, fluoroalkyl or perfluoroalkyl when 
R.sup.3 is CF.sub.3 or alkyl, fluoroalkyl or perfluoroalkyl; 
R.sup.7 is a C.sub.1 -C.sub.4 alkyl optionally substituted with .sup.14 
CH.sub.3, .sup.13 CH.sub.3, CD.sub.3, C.sub.3 H.sub.3, and/or .sup.13 
CD.sub.3 or CH.sub.2 OR.sup.8, where R.sup.8 represents hydrogen, a 
C.sub.1 -C.sub.6 alkyl, a C.sub.3 -C.sub.7 saturated or unsaturated 
cycloalkyl optionally substituted with a C.sub.1 -C.sub.4 alkyl, F, CI, 
Br, I, OH, CF.sub.3, OR.sup.6, NR.sup.6, where R.sup.6 has the definition 
given above; 
R.sup.9 is a C.sub.1 -C.sub.4 alkyl; 
R.sup.10 through R.sup.15 each independently are hydrogen, a C.sub.1 
-C.sub.6 alkyl or CF.sub.3 ; 
X is COOR.sup.16, CONR.sup.17, or CONHR.sup.17 R.sup.18 where R.sup.16 
represents hydrogen or a C.sub.1 -C.sub.6 alkyl, and where R.sup.17 and 
R.sup.18 each independently represent a C.sub.1 -C.sub.6 alkyl, or an aryl 
or heteroaryl optionally substituted with OH, F, Br, CI or I, provided, 
however, that R.sup.17 and R.sup.18 both cannot be an aryl or heteroaryl; 
Y is C, O, S or N, provided that, when Y is O, then R.sup.14 and R.sup.15 
do not exist, and when Y is N, then R.sup.14 and R.sup.15 cannot be 
CF.sub.3, and when Y is S, then R.sup.14 and R.sup.15 can independently or 
together represent O, or may be absent altogether; 
W is N or CR.sup.16, where R.sup.16 has the same definition given above; 
R.sup.19 is an aryl or heteroaryl optionally substituted with one or more 
substituents selected from the group consisting of hydrogen, F, CI, Br, I 
or a C.sub.1 -C.sub.6 alkyl, wherein X has the same definition given 
above; 
n is 0, 1 or 2; 
the dotted lines designate optional double bonds; and 
the wavy lines depict carbon to carbon bonds in either the cis or trans 
configurations, provided, however, that when R.sup.1, R.sup.2, R.sup.4 and 
R.sup.5 are all hydrogen, then R.sup.3 cannot be aryl. 
Preferably, R.sup.1, R.sup.2 and R.sup.4 independently represent C.sub.3 
-C.sub.6 branched alkyls, fluoroalkyls or perfluoroalkyls, more preferably 
R.sup.2 and R.sup.4 independently represent C.sub.3 -C.sub.6 branched 
alkyls, fluoroalkyls or perfluoroalkyls, while R.sup.1, R.sup.3 and 
R.sup.5 are all hydrogen, and most preferably R.sup.2 and R.sup.4 are 
selected from the group consisting of isopropyl, t-butyl and CF.sub.3, 
while R.sup.1, R.sup.3 and R.sup.5 are all hydrogen. 
The compounds of the present invention also include all pharmaceutically 
acceptable salts, as well as esters, amides and prodrugs. Preferably, such 
salts, esters and amides, will be formed at the R.sup.16, R.sup.17 and/or 
R.sup.18 positions. As used in this disclosure, pharmaceutically 
acceptable salts include, but are not limited to: pyridine, ammonium, 
piperazine, diethylamine, nicotinamide, formic, urea, sodium, potassium, 
calcium, magnesium, zinc, lithium, cinnamic, methylamino, methanesulfonic, 
picric, tartaric, triethylamino, dimethylamino, and 
tris(hydoxymethyl)aminomethane. Additional pharmaceutically acceptable 
salts are known to those skilled in the art. 
The compounds of the present invention exhibit retinoid activity and are 
particularly useful in the treatment of skin-related diseases, including, 
without limitation, actinic keratoses, arsenic keratoses, inflammatory and 
non-inflammatory acne, psoriasis, ichthyoses and other keratinization and 
hyperproliferative disorders of the skin, eczema, atopic dermatitis, 
Darriers disease, lichen planus, prevention and reversal of glucocorticoid 
damage (steroid atrophy), as a topical anti-microbial, as skin 
pigmentation agents and to treat and reverse the effects of age and photo 
damage to the skin. The compounds are also useful for the prevention and 
treatment of cancerous and pre-cancerous conditions, including, 
premalignant and malignant hyperproliferative diseases such as cancers of 
the breast, skin, prostate, cervix, uterus, colon, bladder, esophagus, 
stomach, lung, larynx, oral cavity, blood and lymphatic system, 
metaplasias, dysplasias, neoplasias, leukoplakias and papillomas of the 
mucous membranes and in the treatment of Kaposis sarcoma. In addition, the 
present compounds can be used as agents to treat diseases of the eye, 
including, without limitation, proliferative vitreoretinopathy (PVR), 
retinal detachment, dry eye and other corneopathies, as well as in the 
treatment and prevention of various cardiovascular diseases, including, 
without limitation, diseases associated with lipid metabolism such as 
dyslipidemias, prevention of restenosis and as an agent to increase the 
level of circulating tissue plasminogen activator (TPA). Other uses for 
the compounds of the present invention include the prevention and 
treatment of conditions and diseases associated with human papilloma virus 
(HPV), including warts and genital warts, various inflammatory diseases 
such as pulmonary fibrosis, ileitis, colitis and Krohn's disease, 
neurodegenerative diseases such as Alzheimer's disease, Parkinson's 
disease and Amyotrophic Lateral Sclerosis (ALS), improper pituitary 
function, including insufficient production of growth hormone, modulation 
of apoptosis, including both the induction of apoptosis and inhibition of 
T-Cell activated apoptosis, restoration of hair growth, including 
combination therapies with the present compounds and other agents such as 
Minoxidil.RTM., diseases associated with the immune system, including use 
of the present compounds as immunosuppressants and immunostimulants, 
modulation of organ transplant rejection and facilitation of wound 
healing, including modulation of chelosis. It will also be understood by 
those skilled in the art that the retinoid compounds of the present 
invention will prove useful in any therapy in which retinoids, including 
RAR selective retinoids, RXR selective retinoids, and pan-agonist 
retinoids will find application. 
Furthermore, it will be understood by those skilled in the art that the 
compounds of the present invention, including pharmaceutical compositions 
and formulations containing these compounds, can be used in a wide variety 
of combination therapies to treat the conditions and diseases described 
above. Thus, the compounds of the present invention can be used in 
combination with other therapies, including, without limitation, 
chemotherapeutic agents such as cytostatic and cytotoxic agents, 
immunological modifiers such as interferons, interleukins, growth hormones 
and other cytokines, hormone therapies, surgery and radiation therapy. 
Representative compounds of the present invention include, without 
limitation, ethyl (2E, 4E, 
6E)-7-(3,5-di-t-butylphenyl)-3-methylocta-2,4,6-trienoate; ethyl (2E, 4E, 
6Z)-7-(3,5-di-t-butylphenyl)-3-methylocta-2,4,6-trienoate; (2E, 4E, 
6E)-7-(3,5-di-t-butylphenyl)-3-methylocta-2,4,6-trienoic acid; (2E, 4E, 
6Z)-7-(3,5-di-t-butylphenyl)-3-methylocta-2,4,6-trienoic acid; ethyl (2E, 
4E)-7-(3,5-di-t-butylphenyl)-3-methylocta-2,4-dienoate; (2E, 
4E)-7-(3,5-di-t-butylphenyl)-3-methylocta-2,4-dienoic acid; ethyl (2E, 4E, 
6E)-7-(3,5-di-t-butylphenyl)-3-methyldeca-2,4,6-trienoate; ethyl (2E, 4E, 
6E)-7-(3,5-di-t-butylphenyl)-3-methyldeca-2,4,6-trienoate; (2E, 4E, 
6E)-7-(3,5-di-t-butylphenyl)-3-methyldeca-2,4,6-trienoic acid; (2E, 4E, 
6Z)-7-(3,5-di-t-butylphenyl)-3-methyldeca-2,4,6-trienoic acid; ethyl 
(2E,4E,6E)-6-(6,8-di-t-butylchroman-4-ylidene)-3-methylhexa-2,4,6-trienoat 
e; ethyl 
(2E,4E,6Z)-6-(6,8-di-t-butylchroman-4-ylidene)-3-methylhexa-2,4,6-trienoat 
e; 
(2E,4E,6E)-6-(6,8-di-t-butylchroman-4-ylidene)-3-methylhexa-2,4,6-trienoic 
acid; 
(2E,4E,6Z)-6-(6,8-di-t-butylchroman-4-ylidene)-3-methylhexa-2,4,6-trienoic 
acid; (2E, 4E, 
6E)-7-(3,5-di-trifluoromethylphenyl)-3-methylocta-2,4,6-trienoic acid; 
(2E, 4E, 6Z)-7-(3,5-di-trifluoromethylphenyl)-3-methylocta-2,4,6-trienoic 
acid; (2E, 4E, 6E)-7-(3,5-di-isopropylphenyl)-3-methylocta-2,4,6-trienoic 
acid; (2E, 4E, 6Z)-7-(3,5-di-isopropylphenyl)-3-methylocta-2,4,6-trienoic 
acid; (2E, 4E, 6E)-7-(4-t-butylphenyl)-3-methylocta-2,4,6-trienoic acid; 
(2E, 4E, 
6E)-7-(3,5-di-t-butyl-4-methoxyphenyl)-3-methylocta-2,4,6-trienoic acid; 
(2E, 4E, 
6E)-3-methyl-7-(3,5-di-t-butyl-4-methoxyphenyl)octa-2,4,6-trienoic acid; 
(2E,4E,6E)-3-methyl-7-(3,4-diethylphenyl)octa-2,4,6-trienoic acid; 
(2E,4E,6Z)-3-methyl-7-(3,4-di-ethylphenyl)octa-2,4,6-trienoic acid; (2E, 
4E, 6E)-3-methyl-7-(3,5-di-t-butyl-4-ethoxyphenyl)octa-2,4,6-tr acid; 
(2E,4E,6E)-3-methyl-7-(3,4-di-t-butylphenyl)octa-2,4,6-trienoic acid; 
(2E,4E,6E)-3-methyl-7-cyclohexyl-7-(3,5-di-t-butylphenyl)hepta-2,4,6-trien 
oic acid; (2E,4E,6E)-3-methyl-7-(3, 5-di-t-butylphenyl)nona-2,4,6-trienoic 
acid; and 
(2E,4E,6Z)-3-methyl-7-(3,4-diethyl-6-methylphenyl)nona-2,4,6-trienoic 
acid. 
The compounds of the present invention can be obtained by routine chemical 
synthesis by those skilled in the art, e.g., by modification of the 
compounds disclosed or by a total synthesis approach. In this regard, the 
synthesis of the compounds of the present invention follows well 
established retinoid synthesis schemes and techniques as described in M. 
I. Dawson and W. H. Okamura, "Chemistry and Biology of Synthetic 
Retinoids", Chapters 3, 8, 14 and 16, CRC Press, Inc., Florida (1990); M. 
I. Dawson and P. D. Hobbs, The Synthetic Chemistry of Retinoids, In 
Chapter 2: "The Retinoids, Biology, Chemistry and Medicine", M. B. Sporn 
et al., Eds. (2nd ed.), Raven Press, New York, New York, pp. 5-178 (1994) 
and R.S.H. Liu and A. E. Asato, "Photochemistry and Synthesis of 
Stereoisomers of Vitamin A," 40 Tetrahedron, 1931 (1984), the disclosures 
of which are herein incorporated by reference. The sequence of steps of 
the general methods of synthesizing the compounds of the present invention 
are shown below. In addition, more detailed and illustrative synthetic 
schemes for specific compounds of the present invention will be found in 
the Examples included herein. 
General Method 1 
##STR2## 
In General Method 1, the compounds of the present invention may be prepared 
by treatment of an aryl ketone A with a phosphonate, such as 
diethylcyanomethylphosphonate, to give the nitrile B, followed by 
reduction of B (where the optional single or double bonds are illustrated 
with dashed lines) in the presence of a reducing agent, such as diisobutyl 
aluminum hydride (Dibal) to provide the aldehyde C. The cis and trans 
isomers of aldehyde C may be separated at this stage via thin-layer 
chromatography (TLC), or other recognized procedures known to those 
skilled in the art. These separated aldehydes C are then treated with a 
phosphonate, such as triethyl-3-alkyl-4-phosphonocrotonate, to give the 
trienoate esters D, which in turn can be saponified under basic conditions 
to give the carboxylic acid E. 
Alternatively, utilizing General Method 2, shown below, the cis isomer of 
aldehyde C may be prepared from the alkyne F. Specifically, aryl alkyne F 
is prepared from aryl acetophenone A by treatment with a phosphorylating 
agent, such as CIPO(EtO).sub.2, in the presence of a strong base, such as 
lithium diisopropylamide (LDA). Aryl alkyne F is then treated with a 
suitable nitrile source, such as PhOCN, in the presence of base, such as 
nBuLi, to give nitrile G, which is then subjected to reductive methylation 
to give exclusively the cis isomer of nitrile B. Nitrile B is then reduced 
to the corresponding aldehyde C and homologated in the same fashion as 
described in General Method 1 above to yield compounds D and E. 
General Method 2 
##STR3## 
Other analogs of compounds of the present invention may be prepared via 
General Method 3, by first reducing the double bond of nitrile B (where 
the single or double optional bonds are illustrated with dashed lines) to 
give nitrile I. Thereafter nitrile I is reduced in the presence of Dibal 
to yield aldehyde J, which in turn is treated with a phosphonate, such as 
triethyl-3-alkyl-4-phosphonocrotonate, to give dienoate ester K. 
Saponification of dienoate ester K via base, such as KOH/MeOH, gives the 
dienoic acid L. 
General Method 3 
##STR4## 
Radiolabeled homologs of the compounds of the present invention may be 
prepared by the General Method 4 shown below. Specifically, compound A is 
oxidized to methyl ester B which is then reduced with a tritium hydride 
source, such as LiAl.sup.3 H.sub.4, to alcohol C. Oxidation of the 
tritiated alcohol C to aldehyde D, followed by condensation with the ylide 
of triethylphosphonocrotonate gives the tritiated ester E. Ester E may 
then be saponified to give the final tritium labeled acid F in high yield 
with high (&gt;20 Ci/mmol) specific activity. This methodology is described 
in detail in Boehm et al., "Synthesis of High Specific Activity .sup.3 
H!-9-cis-Retinoic Acid and Its Application for Identifying Retinoids with 
Unusual Binding Properties", 37 J. Med. Chem., 408-414 (1994), the 
disclosure of which is herein incorporated by reference. 
General Method 4: Preparation of Radiolabeled Homologs 
##STR5## 
It will be understood by those skilled in the art that certain 
modifications can be made to the above-described methods that remain 
within the scope of the present invention. For example, the compounds of 
the present invention may also be produced in the form of the 
corresponding amides or esters, appropriate phosphoranes may be 
substituted for phosphonates, and reducing agents other than Lil.sup.3 
H.sub.4 may be utilized in the syntheses outlined above. Furthermore, it 
will be understood that other isotopic labels may be employed, including 
.sup.13 CH.sub.3, .sup.13 CD.sub.3 and the like. These labels may be 
introduced using the appropriate labeled MeLi (e.g., .sup.13 CH.sub.3 
Li)as shown in Scheme 1. Thereafter, the remainder of the synthesis is as 
shown in Scheme 1. 
In another aspect, the retinoid compounds, their pharmaceutically 
acceptable salts or hydrolyzable esters of the present invention are 
combined in a mixture with a pharmaceutically acceptable carrier to 
provide pharmaceutical compositions useful for treating the biological 
conditions or disorders noted herein in mammalian species, and more 
preferably, in human patients. The particular carrier employed in these 
pharmaceutical compositions may take a wide variety of forms depending 
upon the type of administration desired, e.g., intravenous, oral, topical, 
suppository or parenteral. 
In preparing the compositions in oral liquid dosage forms (e.g., 
suspensions, elixirs and solutions), typical pharmaceutical media, such as 
water, glycols, oils, alcohols, flavoring agents, preservatives, coloring 
agents and the like can be employed. Similarly, when preparing oral solid 
dosage forms (e.g., powders, tablets and capsules), carriers such as 
starches, sugars, diluents, granulating agents, lubricants, binders, 
disintegrating agents and the like will be employed. Due to their ease of 
administration, tablets and capsules represent the most advantageous oral 
dosage form for the pharmaceutical compositions of the present invention. 
For parenteral administration, the carrier will typically comprise sterile 
water, although other ingredients that aid in solubility or serve as 
preservatives, may also be included. Furthermore, injectable suspensions 
may also be prepared, in which case appropriate liquid carriers, 
suspending agents and the like will be employed. 
For topical administration, the compounds of the present invention may be 
formulated using bland, moisturizing bases, such as ointments or creams. 
Examples of suitable ointment bases are petrolatum, petrolatum plus 
volatile silicones, lanolin, and water in oil emulsions such as 
Eucerin.TM. (Beiersdorf). Examples of suitable cream bases are Nivea.TM. 
Cream (Beiersdorf), cold cream (USP), Purpose Cream.sup.TM (Johnson & 
Johnson) hydrophilic ointment (USP), and Lubriderm.TM. (Warner-Lambert). 
The pharmaceutical compositions and compounds of the present invention will 
generally be administered in the form of a dosage unit (e.g., tablet, 
capsule etc.) at from about 1 .mu.g/kg of body weight to about 500 mg/kg 
of body weight, more preferably from about 10 .mu.g/kg to about 250 mg/kg, 
and most preferably from about 20 .mu.g/kg to about 100 mg/kg. As 
recognized by those skilled in the art, the particular quantity of 
pharmaceutical composition according to the present invention administered 
to a patient will depend upon a number of factors, including, without 
limitation, the biological activity desired, the condition of the patient, 
and tolerance for the drug. 
The compounds of this invention also have utility when labeled and used in 
assays to determine the presence of RARs and RXRs. They are particularly 
useful due to their ability to selectively bind to members of the RAR and 
RXR subfamilies and can therefore be used to determine the presence of RAR 
and RXR isoforms in the presence of other retinoid receptors or related 
intracellular receptors. 
Thus, the present invention also provides isotopically labeled and 
radiolabeled compounds, and methods for their synthesis, including 
deuterium, tritium, carbon 13 and carbon 14 labeled homologs. In a 
preferred aspect, the labeled compounds of the present invention display a 
specific activity of at least 15 Ci/mmol, and more preferably at least 25 
Ci/mmol, and most preferably, at least 40 Ci/mmol. Such labeled compounds 
will also prove useful in the identification of compound metabolites in 
animal metabolism studies. 
Due to the selective specificity of the compounds of this invention for 
retinoid receptors, these compounds can also be used to purify samples of 
RARs and RXRs in vitro. Such purification can be carried out by mixing 
samples containing retinoid receptors with one of more of the compounds of 
the present invention, so that the compound (ligand) binds to the 
receptor, and then separating out the bound ligand/receptor combination by 
separation techniques which are known to those of skill in the art. These 
techniques include column separation, filtration, centrifugation, tagging 
and physical separation, and antibody complexing, among others. 
The compounds of the present invention also include racemate, individual 
stereoisomers and mixtures thereof. These isomers are then isolated by 
standard resolution techniques, including fractional crystallization and 
chiral column chromatography. 
The compounds and pharmaceutical compositions of the present invention can 
advantageously be used in the treatment of the diseases and conditions 
described herein. In this regard, the compounds and compositions will 
prove particularly useful in the treatment of skin-related diseases and 
conditions, such as acne, psoriasis, and photo damage, cancerous and 
precancerous conditions, diseases of the eye, cardiovascular diseases, 
inflammatory and neurodegenerative diseases, diseases associated with 
human papilloma virus, improper pituitary function, modulation of 
apoptosis, diseases of the immune system, wound healing and restoration of 
hair growth. 
Furthermore, the compounds and pharmaceutical compositions of the present 
invention possess a number of advantages over previously identified 
retinoid compounds. For example, the compounds are extremely potent 
activators of RARs and RXRs as demonstrated in the co-transfection assay 
further described herein, preferably displaying 50% maximal activation 
(i.e., EC.sub.50) of one or more of the retinoid receptors at a 
concentration of less than 100 nM, more preferably at a concentration of 
less than 50 nM, more preferably yet at a concentration of less than 20 
nM, and most preferably at a concentration of less than 10 nM. Also, the 
RAR and RXR selective compounds of the present invention preferentially 
activate one subfamily of retinoid receptors at a potency level at least 2 
times greater, preferably at least 5 times greater, more preferably at 
least 10 times greater, and most preferably at a potency level at least 
100 times greater than the other subfamily of retinoid receptors. In 
addition, the compounds of the present invention also are easier to 
synthesize, provide greater stability and bioavailability, and appear to 
be less teratogenic in comparison to all-trans retinoic acid and 9-cis 
retinoic acid, known RAR and RXR active compounds, respectively. 
The invention will be further illustrated by reference to the following 
non-limiting Examples. 
EXAMPLES 1-2 
(2E, 4E, 6E)-7-(3,5-Di-t-butylphenyl)-3-methylocta-2,4,6-trienoic acid (9) 
and (2E, 4E, 6Z)-7-(3,5-di-t-butylphenyl)-3-methylocta-2,4,6-trienoic acid 
(10), prepared according to Scheme 1 illustrated and described below. 
##STR6## 
3,5-Di-t-butylacetophenone (2). To 20 g (85.5 mmol) of 
3,5-di-tertbutylbenzoic acid 1 in 100 mL of dry THF at -78.degree. C. was 
added 94.0 mL (188.0 mmol) of a 2 N ether solution of MeLi. The reaction 
mixture was slowly warmed to room temperature and stirred for an 
additional 30 min., then poured into saturated aqueous NH.sub.4 C.sub.1 
(200 mL). The organic product was extracted with hexanes (2.times.100 mL) 
dried (MgSO.sub.4), filtered, concentrated and purified by chromatography 
(SiO.sub.2, 2 EtOAc-hexanes) to give 15 g (64.7 mmol) of ketone 2 (75.7% 
yield): TLC (5% EtOAc-95% hexanes) R.sub.f 0.8; .sup.1 H-NMR (CDCl.sub.3) 
.delta.1.39 (s, 18H, 6(CH.sub.3)), 2.61 (s, 3H, CH.sub.3), 7.64 (t, J=1 
Hz, 1H, Ar-H), 7.80 (d, J=1 Hz, 2H, Ar-H). 
3-(3,5-Di-t-butylphenyl)but-2-enitrile (3) (trans) and (4) (cis). To 2.43 g 
(13.7 mmol) of diethylcyanomethyl phosphonate in 10 mL of dry THF was 
added 440 mg (10.96 mmol) of NaH (60% in mineral oil). The reaction was 
stirred for 30 min. followed by addition of 1.59 g (6.85 mmol) of ketone 2 
in 5 mL of dry THF. After stirring for 12 h, the mixture was quenched with 
saturated aqueous NH.sub.4 C.sub.1 (50 mL) and the products were extracted 
with ether (2.times.50 mL). The ether extracts were washed (water then 
brine), dried (MgSO.sub.4), filtered, concentrated and purified by 
preparative TLC (SiO.sub.2, 2.5 % EtOAc-hexanes) to give 1.1 g (4.4 mmol) 
of the trans isomer 3 and 104 mg (0.4 mmol) of the cis isomer 4 (70% 
combined yield ). Trans isomer 3: TLC (5% EtOAc-95% hexanes) R.sub.f 0.9; 
.sup.1 H-NMR (CDCl.sub.3) .delta.1.32 (s, 18H, 6(CH.sub.3)), 2.49 (s, 3H, 
CH.sub.3), 5.59 (s, 1H, .dbd.CH), 7.25 (d, J=1 Hz, 2H, Ar-H), 7.50 (d, J=1 
Hz, 1H, Ar-H). Cis isomer 4: TLC (5% EtOAc-95% hexanes) R.sub.0.8; .sup.1 
H-NMR (CDCl.sub.3) .delta.1.42 (s, 18H, 6(CH.sub.3)), 2.31 (s, 3H, 
CH.sub.3), 5.34 (s, 1H, .dbd.CH), 7.39 (d, J=1 Hz, 2H, Ar-H), 7.49 (d, J=1 
Hz, 1H, Ar-H 
3-(3,5-Di-t-butylphenyl)but-2-enal (5) (trans isomer). To 736 mg (2.89 
mmol) of 3 in 5 mL of CH.sub.2 Cl.sub.2 at -78.degree. C. was added 2.31 
mL (3.47 mmol) of a 1.5M solution of DIBAL in toluene. After stirring for 
15 min. at -78.degree. C., the reaction mixture was quenched with 10 mL of 
a saturated aqueous solution of Rochelle salt. The product was extracted 
with ether (2.times.20 mL), washed (water, then brine), dried 
(MgSO.sub.4), filtered, concentrated and purified by chromatography 
(SiO.sub.2, 3% EtOAc-hexanes) to give 462.3 mg (1.80 mmol) of 5 (62% 
yield): TLC (10% EtOAc-90% hexanes) R.sub.f 0.5; .sup.1 H-NMR (CDCl.sub.3) 
.delta.1.34 (s, 18H, 6(CH.sub.3)), 2.59 (s, 3H, CH.sub.3), 6.50 (d, J=8.0 
Hz, 1H, .dbd.CH), 7.39 (d, J=1 Hz, 2H, Ar-H), 7.51 (d, J=1 Hz, 1H, Ar-H), 
10.18 (d, J=8.0 Hz, 1H, CHO). 
3-(3,5-Di-t-butylphenyl)but-2-enal (6) (cis isomer). The cis isomer 6 was 
prepared from the corresponding cis isomer 4 using the same method as 
described for 5: TLC (10% EtOAc-90% hexanes) R.sub.f 0.55; .sup.1 H-NMR 
(CDCl.sub.3) .delta.1.34 (s, 18H, 6(CH.sub.3)), 2.34 (s, 3H, CH.sub.3), 
6.12 (d, J=8.0 Hz, Hz, 1H, .dbd.CH), 7.10 (d, J=1 Hz, 2H, Ar-H), 7.46 (t, 
J=1 Hz, 1H, Ar-H), 9.45 (d, J=8.0 Hz, 1 H, CHO). 
Ethyl (2E, 4E, 6E)-7-(3,5-di-t-butylphenyl)-3-methylocta-2,4,6-trienoate 
(7). To 790 mg (3.0 mmol) of triethyl-3-methyl-4-phosphonocrotonate in 8 
mL of dry THF at -78.degree. C. was added 1.2 mL of a 2.5 M nBuLi solution 
in hexanes. After stirring for 15 min., the solution containing the ylide 
of triethylphosphonocrotonate was added to 258 mg(1.0 mmol) of the trans 
isomer 5 in 8 mL of dry THF at -78.degree. C. The reaction mixture was 
warmed to RT, quenched with saturated aqueous NH.sub.4 Cl (20 mL) and the 
products were extracted with ether (2.times.50 mL). The ether extracts 
were washed (water, then brine), dried (MgSO.sub.4), filtered, 
concentrated and purified by column chromatography (SiO.sub.2, 5% 
EtOAc-hexanes) to give 294 mg (0.8 mmol) of the E,E, Eisomer of 7 (49% 
yield): TLC (5% EtOAc-95% hexanes) R.sub.f 0.78; .sup.1 H-NMR (CDCl.sub.3) 
15 1.30 (t, J=7.7 Hz, 3H, CH.sub.2 CH.sub.3), 1.34 (s, 18H, 6(CH.sub.3)), 
2.28, (s, 3H, CH.sub.3), 4.17 (m, 2H, CH.sub.2 CH.sub.3), 5.82 (s, 1H, 
.dbd.CH), 6.40 (d, J=15 Hz, 1H, .dbd.CH), 6.54 (d, J=15 Hz, 1H, .dbd.CH), 
7.04 (m, 1H, .dbd.CH), 7.21 (d, J=1 Hz, 2H, Ar-H), 7.39 (d, J=1 Hz, 1H, 
Ar-H). 
Ethyl (2E, 4E, 6Z)-7-(3,5-di-t-butylphenyl)-3-methylocta-2,4,6-trienoate 
(8). The 2E, 4E, 6Z isomer 8 was prepared in the same manner as the 
2E,4E,6E-isomer 7, except that 6 was used instead of the 5: TLC (5% 
EtOAc-95% hexanes) R.sub.f 0.82; .sup.1 H-NMR (CDCl.sub.3) .delta.1.27 (t, 
J=7.7 Hz, 3H, CH.sub.2 CH.sub.3), 1.34 (s, 18H, 6(CH.sub.3)), 2.17, (s, 
3H, CH.sub.3), 2.22 (s, 3H, CH.sub.3), 4.15 (m, 2H, CH.sub.2 CH.sub.3), 
5.74 (s, 1H, .dbd.CH), 6.26 (dd, J=8 Hz, 2H, .dbd.CH), 6.80 (m, 1H, 
.dbd.CH), 7.10 (d, J=1 Hz, 2H, Ar-H), 7.37 (t, J=1 Hz, 1H, Ar-H). 
(2E, 4E, 6E)-7-(3,5-Di-t-butylphenyl)-3-methylocta-2,4,6-trienoic acid (9). 
To 180 mg (0.49 mmol) of the 2E, 4E, 6E-ethyl ester 7 in 5 mL of MeOH was 
added 1 mL of 5N aqueous NaOH solution. The mixture was heated to reflux 
for 10 min., cooled to RT, acidified with 20% aqueous HCl solution and the 
organics extracted with ether (2.times.10 mL). The ether layer was washed 
(H.sub.2 O, brine), dried (MgSO.sub.4), filtered and concentrated. 
Purification by column chromatography (SiO.sub.2, 20% EtOAc-hexanes) gave 
157 mg (0.46 mmol) of the 2E,4E,6E-isomer 9 (93% yield): TLC (10% MeOH-90% 
CHCl.sub.3) R.sub.f 0.6; mp 196.degree.-198.degree. C.; .sup.1 H-NMR 
(CDCl.sub.3) .delta.1.35 (s, 18H, 6(CH.sub.3)), 2.29, (s, 3H, CH.sub.3), 
2.41 (s, 3H, CH.sub.3), 5.84 (s, 1H, .dbd.CH),6.41 (d, J=15 Hz, 1H, 
.dbd.CH), 6.54 (d, J=15 Hz, 1H, .dbd.CH), 7.08 (m, 1H, .dbd.CH), 7.32 (d, 
J=1 Hz, 7.39 (t, J=1 Hz, 1H, Ar-H). 
(2E, 4E, 6Z)-7-(3,5-Di-t-butylphenyl)-3-methylocta-2,4,6-trienoic acid 
(10). The 2E, 4E, 6Z-isomer 10 was prepared in the same manner as 9 except 
that 8 was used instead of 7: TLC (10% MeOH-90% CHCl.sub.3) R.sub.f 0.57; 
mp 221.degree.-222.degree. C.; .sup.1 H-NMR (CDCl.sub.3) .delta.1.34 (s, 
18H, 6(CH.sub.3), 2.18, (s, 3H, CH.sub.3), 2.23 (s, 3H, CH.sub.3), 5.77 
(s, 1H, .dbd.CH), 6.27 (m, 2H, .dbd.CH), 6.84 (m, 1H, .dbd.CH), 7.10 (d, 
J=1 Hz, 2H, Ar-H), 7.37 (t, J=1 Hz, 1H, Ar-H). 
EXAMPLE 3 
(2E, 4E)-7-(3,5-Di-t-butylphenyl)-3-methylocta-2,4-dienoic acid (14), 
prepared according to Scheme 2 illustrated and described below. 
##STR7## 
3-(3,5-Di-t-butylphenyl)butanitrile (11). To 300 mg (1.18 mmol) of 
3-(3,5-di-t-butylphenyl)-but-2-enitrile 3 in 5 mL of EtOAc was added 20 mg 
(catalytic quantity) of 10% Pd/C. The mixture was placed under vacuum for 
0.5 min., followed by addition of H.sub.2 gas. After stirring for 2 h 
under H2 gas, the solution was filtered through celite, the celite washed 
with EtOAc (3.times.5 mL) and the solution concentrated to give 300 mg 
(1.17 mmol) of the reduced product 11 (99% yield): TLC (5% EtOAc-95% 
hexanes) R.sub.f 0.8; .sup.1 H-NMR (CDCl.sub.3) .delta.1.34 (s, 18H, 
6(CH.sub.3)), 1.50 (d, 3H, CH.sub.3), 2.60 (m, 2H, CH.sub.2), 3.15 (m, 1H, 
CH), 7.05 (d, J=1 Hz, 2H, Ar-H), 7.33 (t, J=1 Hz, 1H, Ar-H). 
3-(3,5-Di-t-butylphenyl)butanal (12). To 300 mg (1.17 mmol) of the nitrile 
11 in 5 mL of CH.sub.2 Cl.sub.2 at -78.degree. C. was added 0.93 mL (1.4 
mmol) of a 1.5M DIBAL solution in toluene. The reaction mixture was 
stirred for 5 min., quenched with saturated aqueous NH.sub.4 C.sub.1 (10 
mL), extracted with ether (2.times.20 mL), dried (MgSO.sub.4), filtered, 
concentrated and purified by chromatography (SiO.sub.2, 5% EtOAc-hexanes) 
to give 188 mg (0.72 mmol) of the desired aldehyde 12 (62% yield): TLC (5% 
EtOAc-95% hexanes) R.sub.f 0.8; .sup.1 H-NMR (CDCl.sub.3) 15 1.34 (s, 18H, 
6(CH.sub.3)), 1.35 (d, 3H, CH.sub.3), 2.70 (m, 2H, CH.sub.2), 3.36 (m, 1H, 
CH), 7.06 (d, J=1 Hz, 2H, Ar-H), 7.28 (t, J=1 Hz, 1H, Ar-H), 9.70 (t, 1H, 
CHO). 
Ethyl (2E, 4E)-7-(3,5-di-t-butylphenyl)-3-methylocta-2,4-dienoate (13). 
Compound 13 was prepared from 12 in a similar manner as described for 
compound 7: TLC (5% EtOAc-95% hexanes) R.sub.f 0.88; .sup.1 H-NMR 
(CDCl.sub.3) .delta.1.30 (m, 6H, CH.sub.2 CH.sub.3 +CH.sub.3), 1.34 (s, 
18H, 6(CH.sub.3)), 2.20, (s, 3H, CH.sub.3), 2.40 (m, 2H, CH.sub.2), 2.85 
(m, 1H, CH), 4.14 (m, 2H, CH.sub.2 CH.sub.3), 5.62 (s, 1H, .dbd.CH), 6.03 
(m, 2H, .dbd.CH), 7.00 (d, J=I Hz, 2H, Ar-H), 7.24 (t, J=1 Hz, 1H, Ar-H). 
(2E, 4E)-7-(3,5-Di-t-butylphenyl)-3-methylocta-2,4-dienoic acid (14). 
Compound 14 was prepared from 13 in a similar manner as described for 
compound 9: TLC (10% MeOH-90% CHCl.sub.3) R.sub.f 0.5; mp 
127.degree.-128.degree. C.; .sup.1 H-NMR (CDCl.sub.3) .delta.1.28 (d, J=8 
Hz, 3H, CH.sub.3), 1.32 (s, 18H, 6(CH.sub.3)), 2.23, (s, 3H, CH.sub.3), 
2.46 (m, 2H, CH.sub.2), 2.86 (m, 1H, CH), 5.69 (s, 1H, .dbd.CH), 6.10 (m, 
2H, .dbd.CH), 7.01 (d, J=1 Hz, 2H, Ar-H), 7.26 (t, J=I Hz, 1H, Ar-H). 
EXAMPLE 4 
(2E, 4E, 6Z)-7-(3,5-Di-t-butylphenyl)-3-methylocta-2,4,6-trienoic acid 
(10), alternative preparation of Compound (10) according to Scheme 3 
illustrated and described below. 
##STR8## 
1,3-Di-t-butyl -5-ethynylbenzene (15). To 9.86 mL (24.7 mmol) of a 2.5N LDA 
solution in THF at -78.degree. C. was added 4.75 g (20.47 mmol) of ketone 
2 (from EXAMPLES 1-2) in 3 mL of dry THF. After stirring for 30 min., 2.96 
mL (20.47 mmol) of diethylphosphonylchloride was added and the reaction 
mixture was warmed to RT for 1 h. The reaction mixture was again cooled to 
-78.degree. C. followed by addition of 19.7 mL (49.2 mmol) of a 2.5N LDA 
solution in THF and warmed to RT. Water (50 mL) was added and the mixture 
was extracted with hexanes (2.times.40 mL). The combined organic extract 
was washed (water. then brine), dried (MgSO.sub.4), filtered, concentrated 
and purified by column chromatography (SiO.sub.2, 2% EtOAc-hexanes) to 
give 3.02 g (14.1 mmol) of compound 15 (69% yield): TLC (hexanes) R.sub.f 
0.9; .sup.1 H-NMR (CDCl.sub.3) .delta.1.31 (s, 18H, 6(CH.sub.3)), 3.02 (s, 
1H, C.tbd.CH), 7.35 (d, J=1 Hz, 2H, Ar-H), 7.42 (t, J=1 Hz, 1H, Ar-H). 
3-(3,5-Di-t-butylphenyl)propynitrile (16). To 1.67 g (7.80 mmol) of propyne 
15 in 25 mL of dry THF at -78.degree. C. was added 3.75 mL (9.38 mmol) of 
nBuLi (2.5M in hexanes). After stirring for 15 min., 1.12 g (9.41 mmol) of 
PhOCN was added and the reaction mixture was warmed to RT. The mixture was 
quenched by addition of 25 mL of aqueous 6N NaOH, extracted (EtOAc, 
2.times.25 mL), washed (water, then brine), dried (MgSO.sub.4), filtered, 
concentrated and purified by column chromatography (SiO.sub.2, 5% 
EtOAc-hexane) to give 1.71 g (7.16 mmol) of 16 as a white solid (92% 
yield): TLC (hexanes) R.sub.f 0.4; .sup.1 H-NMR (CDCl.sub.3) .delta.1.32 
(s, 18H, 6(CH.sub.3)), 7.45 (d, J=1 Hz, 2H, Ar-H), 7.58 (t, J=1 Hz, 1H, 
Ar-H). 
3-(3,5-Di-t-butylphenyl)but-2-enitrile (4) (cis isomer). A 250 mL flame 
dried round bottom flask was charged with 3.27 g (17.17 mmol) of anhydrous 
copper iodide and 25 mL of dry THF. The mixture was cooled to 0.degree. 
C., followed by slow addition of 24.5 mL (34.3 mmol) of MeLi (1.4M in 
ether). After the solution became clear and colorless, it was cooled to 
-78 .degree. C. and a solution of 1.71 g (7.16 mmol) of 16 in 10 mL of dry 
THF was added dropwise. The mixture was stirred at -78.degree. C. for 45 
min. and quenched with 40 mL of a 1: 1 mixture of MeOH and saturated 
aqueous NH.sub.4 Cl solution. The product was extracted with EtOAc 
(2.times.40 mL), washed (2% NaOH followed by sat. NH.sub.4 Cl, then water, 
then brine), dried (MgSO.sub.4), filtered, concentrated and purified 
through a short silica gel pad to give 1.64 g (6.80 mmol) of the cis 
isomer 4 (95% yield): TLC (5% EtOAc-95% hexanes) R.sub.f 0.8; .sup.1 H-NMR 
(CDCl.sub.3) .delta.1.42 (s, 18H, 6(CH.sub.3)), 2.31 (s, 3H, CH.sub.3), 
5.34 (s, 1H, .dbd.CH), 7.39 (d, J=1 Hz, 2H, Ar-H), 7.49 (d, J=1 Hz, 1H, 
Ar-H). 
The remaining synthesis of Compounds 6, 8 and final product 10 were 
performed as described in EXAMPLES 1-2 above. 
EXAMPLES 5-6 
(2E, 4E, 6E)-7-(3,5-Di-t-butylphenyl)-3-methyldeca-2,4,6-trienoic acid (27) 
and (2E, 4E, 6Z)-7-(3,5-di-t-butylphenyl)-3-methyldeca-2,4,6-trienoic acid 
(28), prepared according to Scheme 4 illustrated and described below. 
##STR9## 
3,5-Di-t-butylbenzyl alcohol (17). To 10.0 g (42.7 mmol) of acid 1 in 20 mL 
of dry THF at 0.degree. C. was added 42.7 mL (42.7 mmol) of LAH (1.0M in 
THF). The reaction mixture was warmed to 50.degree. C. and stirred for 15 
min. After cooling the reaction to RT, 20% aqueous HCl was added until the 
solution turned clear. The solution was extracted with EtOAc (2.times.50 
mL), and the combined EtOAc extract was washed (water, then brine), dried 
(MgSO.sub.4), filtered and concentrated to give 8.8 g (40.0 mmol) of 17 
(94% yield): The product was directly used in the next step. TLC (20% 
EtOAc-80% hexanes) R.sub.f 0.4; .sup.1 H-NMR (CDCl.sub.3) .delta.1.33 (s, 
18H, 6(CH.sub.3)), 4.68 (s, 2H, CH.sub.2) 7.22 (d, J=1 Hz, 2H, Ar-H), 7.38 
(t, J=1 Hz, 1H, Ar-H). 
3,5-Di-t-butylbenzaldehyde (18). To 8.8 g (40.0 mmol) of alcohol 17 in 20 
mL of CH.sub.2 Cl.sub.2 was added 50.0 g (575 mmol) of MnO.sub.2. The 
reaction mixture was vigorously stirred for 8 h and filtered through a pad 
consisting of a top layer of celite and a bottom layer of silica. The 
filter was washed repeatedly with 50 mL aliquots of CH.sub.2 Cl.sub.2 
until no more product eluted from the filter. The resulting compound 18 
(8.3 g (38.1 mmol)) was determined to be pure by .sup.1 HNMR and was used 
directly in the next step (95% yield): TLC (20% EtOAc-80% hexanes) R.sub.f 
0.6; .sup.1 H-NMR (CDCl.sub.3) B 1.34 (s, 18H, 6(CH.sub.3)), 7.72 (m, 3H, 
Ar-H), 10.01 (s, 1H, CHO). 
1-(3,5-Di-t-butylphenyl)butan-1-ol (19). To 2.0 g (9.17 mmol) of aldehyde 
18 in 10 mL of dry ether at 0 .degree. C. was added 5.5 mL (11.0 mmol) of 
propylmagnesium chloride (2.0M in ether). The reaction mixture was warmed 
to RT and quenched with water (50 mL), extracted (ether, 2.times.50 mL), 
washed (water then brine), dried (MgSO.sub.4), filtered and concentrated 
to give 2.37 g (9.05 mmol) of alcohol 19 (pure by .sup.1 H-NMR) which was 
directly used in the next step (98% yield): TLC (20% EtOAc-80% hexanes) 
R.sub.f 0.5; .sup.1 H-NMR (CDCl.sub.3) .delta.0.96 (t, 3H, CH.sub.2 
CH.sub.3), 1.34 (s, 18H, 6(CH.sub.3)), 1.66 (m, 2H, CH.sub.2), 1.84 (m, 
1H, CH), 4.66 (m, 1H, CHOH), 7.18 (d, J=1 Hz, 2H, Ar-H), 7.34 (t, J=1 Hz, 
1H, Ar-H). 
1-(3,5-Di-t-butylphenyl)butan-1-one (20). To 2.37 g (9.05 mmol) of alcohol 
19 in 18 mL of CH.sub.2 Cl.sub.2 was added 7.86 g (90.45 mmol) of 
MnO.sub.2. The reaction mixture was stirred for 3 h, then filtered (celite 
over silica gel pad) and the pad was washed repeatedly with 20 mL aliquots 
of CH.sub.2 Cl.sub.2. After concentration, ketone 20 was purified by 
chromatography (SiO.sub.2, 3% EtOAc-hexanes) to give 941 mg (3.62 mmol) of 
20 (40% yield 1 Hz TLC (10% EtOAc-90% hexanes) R.sub.f 0.7; .sup.1 H-NMR 
(CDCl.sub.3) .delta.1.01 (t, 3H, CH.sub.2 CH.sub.3), 1.34 (s, 18H, 
6(CH.sub.3)), 1.78 (m, 2H, CH.sub.2 CH.sub.3), 2.96 (t, J=7 Hz, 2H, 
CH.sub.2 CH.sub.2 CH.sub.3), 7.63 (t, J=1 Hz, 1H, Ar-H), 7.83 (d, J=1 Hz, 
2H, Ar-H). 
3-(3,5-Di-t-butylphenyl)hex-2-enitrile (21) (trans) and (22) (cis). To 661 
mg (3.73 mmol) of diethylcyanomethylphosphonate in 5 mL of dry THF was 
added 127 mg (3.17 mmol) of sodium hydride (60% dispersion in mineral 
oil). The mixture was stirred for 5 min., followed by addition of 484 mg 
(1.87 mmol) of ketone 20 in 2 mL of dry THF. The reaction was heated to 
reflux for 30 m, cooled to RT, quenched with saturated aqueous NH.sub.4 Cl 
(15 mL), extracted with ether (2.times.15 mL), washed (water then brine), 
dried (MgSO.sub.4), filtered and concentrated. Purification by 
chromatography (preparative TLC, SiO.sub.2, 10% EtOAc-hexanes) gave 329 mg 
(1.16 mmol) of the trans isomer 21 and 70.5 mg (0.25 mmol) of the cis 
isomer 22 (75% combined yield). Trans isomer 21 TLC (10% EtOAc-90% 
hexanes) R.sub.f 0.8; .sup.1 H-NMR (CDCl.sub.3) .delta.0.97 (t, J=7 Hz, 
3H, CH.sub.2 CH.sub.3), 1.33 (s, 18H 6(CH.sub.3)), 1.54 (m, 2H, CH.sub.2 
CH.sub.2 CH.sub.3), 2.88 (t, J=7 Hz, 2H, CH.sub.2 CH.sub.2 CH.sub.3), 5.50 
(s, 1H, .dbd.CH), 7.23 (d, J=1 Hz, 2H, Ar-H), 7.50 (t, J=1 Hz, 1H, Ar-H). 
Cis isomer 22. TLC (10% EtOAc-90% hexanes) R.sub.f 0.9; .sup.1 H-NMR 
(CDCl.sub.3) .delta.0.93 (t, J=7 Hz, 3H, CH.sub.2 CH.sub.3), 1.34 (s, 18H 
6(CH.sub.3)), 1.48 (m, 2H, CH.sub.2 CH.sub.2 CH.sub.3), 2.57 (t, J=7 Hz, 
2H, CH.sub.2 CH.sub.2 CH.sub.3), 5.32 (s, 1H, .dbd.CH), 7.28 (d, J=2 Hz, 
2H, Ar-H), 7.45 (t, J=2 Hz, 1H, Ar-H). 
3-(3,5-Di-t-butylphenyl)hex-2-enal (23) (trans). To 166 mg (0.59 mmol) of 
nitrile 21 in 4 mL of CH.sub.2 Cl.sub.2 at -78.degree. C. was added 0.59 
mL (0.88 mmol) of DIBAL (1.5M in toluene). The reaction mixture was 
stirred for 10 m and quenched with 10 mL of a saturated aqueous solution 
of Rochelle salt. The product was extracted with ether (2.times.20 mL), 
washed (water then brine), dried (MgSO.sub.4), filtered, concentrated and 
purified by chromatography (preparative TLC, SiO.sub.2, 3% EtOAc-hexanes) 
to give 141 mg (0.49 mmol) of 23 as an oil (83% yield): TLC (10% EtOAc-90% 
hexanes) R.sub.f 0.7; .sup.1 H-NMR (CDCl.sub.3) .delta.0.97 (t, J=7 Hz, 
3H, CH.sub.2 CH.sub.3), 1.34 (s, 18H, 6(CH.sub.3)), 1.57 (m, 2H, CH.sub.2 
CH.sub.2 CH.sub.3), 3.03 (t, J=7 Hz, 2H, CH.sub.2 CH.sub.2 CH.sub.3), 6.32 
(d, J=8 Hz, 1H, .dbd.CH), 7.33 (d, J=1 Hz, 2H, Ar-H), 7.48 (t, J=1 Hz, 1H, 
Ar-H), 10.15 (d, J=8 Hz, 1H, CHO). 
3-(3,5-Di-t-butylphenyl)hex-2-enal (24) (cis). Compound 24 was prepared in 
the same manner as 23 except that the cis isomer 22 was used instead of 
21: TLC (10% EtOAc-90% hexanes) R.sub.f 0.8; .sup.1 H-NMR (CDCl.sub.3) 
.delta.0.94 (t, J=7 Hz, 3H, CH.sub.2 CH.sub.3), 1.34 (s, 9H, CH.sub.3), 
1.35 (s, 9H, CH.sub.3), 1.51 (m, 2H, CH.sub.2 CH.sub.2 CH.sub.3), 2.58 (t, 
J=7 Hz, 2H, CH.sub.2 CH.sub.2 CH.sub.3), 6.10 (d, J=8 Hz, 1H, .dbd.CH), 
7.04 (d, J=2 Hz, 2H, Ar-H), 7.43 (t, J=2 Hz, 1H, Ar-H), 9.42 (d, J=8 Hz, 
1H, CHO). 
Ethyl (2E, 4E, 6E)-7-(3,5-di-t-butylphenyl)-3-methyldeca-2,4,6-trienoate 
(25). To 391 mg (1.48 mmol) of triethyl-3-methyl-4-phosphonocrotonate in 5 
mL of dry THF at -78.degree. C. was added 0.59 mL (1.48 mmol) of a 2.5M 
nBuLi solution in hexanes and 2.5 mL of DMPU. After stirring for 15 min., 
the solution containing the ylide of triethylphosphonocrotonate was added 
to 141 mg (0.49 mmol) 23 in 5 mL of dry THF at -78.degree. C. The reaction 
mixture was warmed to RT, quenched with saturated aqueous NH.sub.4 Cl (20 
mL) and the products were extracted with ether (2.times.25 mL). The ether 
extracts were washed (water, then brine), dried (MgSO.sub.4), filtered, 
concentrated and purified by column chromatography (SiO.sub.2, 5 % 
EtOAc-hexanes) to give 171 mg (0.43 mmol) of the all-trans isomer 25 (88% 
yield): TLC (5% EtOAc-95% hexanes) R.sub.f 0.5; .sup.1 H-NMR (CDCl.sub.3) 
.delta.0.94 (t, J=7 Hz, 3H, CH2CH.sub.3), 1.29 (t, J=7 Hz, 3H, CH.sub.2 
CH.sub.3), 1.34 (s, 18H, 6(CH.sub.3)), 1.52 (m, 2H, CH.sub.2 CH.sub.2 
CH.sub.3), 2.39 (s, 3H, CH.sub.3), 2.70 (t, J=7 Hz, 2H, CH.sub.2 CH.sub.2 
CH.sub.3), 4.18 (m, 2H, CH.sub.2 CH.sub.3), 5.80 (s, 1H, .dbd.CH), 6.42 
(d, J=15 Hz, 2H, .dbd.CH), 6.47 (d, J=15Hz, 1H, .dbd.CH), 7.03 (m, 1H, 
.dbd.CH), 7.27 (d, J=1 Hz, 2H, Ar-H), 7.36 (t, J=1 Hz, 1H, Ar-H). 
Ethyl (2E, 4E, 6E)-7-(3,5-di-t-butylphenyl)-3-methyldeca-2,4,6-trienoate 
(26). Compound 26 was prepared in the same manner as 25, except that the 
cis isomer 24 was used instead of 23: TLC (5% EtOAc-95% hexanes) R.sub.f 
0.5; .sup.1 H-NMR (CDCl.sub.3) 15 0.94 (t, J=7 Hz, 3H, CH.sub.2 CH.sub.3), 
1.29 (t, J=7 Hz, 3H, CH.sub.2 CH.sub.3), 1.33 (s, 18H.sup.6 (CH.sub.3)), 
1.44 (m, 2H, CH.sub.2 CH.sub.2 CH.sub.3), 2.15 (s, 3H), CH.sub.3), 2.48 
(t, J=7 Hz, 2H, CH.sub.2 CH.sub.2 CH.sub.3), 4.16 (m, 2H, --COCH.sub.2 
CH.sub.3), 5.73 (s, 1H, .dbd.CH), 6.22 (d, J=11 Hz, 1H, .dbd.CH), 6.26 (d, 
J=11 Hz, 1H, .dbd.CH), 6.74 (dd, J=11 Hz, 1H, .dbd.CH), 7.26 (d, J=2 Hz, 
2H, Ar-H), 7.34 (t, J=2 Hz, 1H, Ar-H). 
(2E, 4E, 6E)-7-(3,5-Di-t-butylphenyl)-3-methyldeca-2,4,6-trienoic acid 
(27). To 171 mg (0.44 mmol) of 25 in 5 mL of MeOH was added 1 mL of 5N 
aqueous NaOH solution. The mixture was heated to reflux for 10 min., 
cooled to RT, acidified with 20% aqueous HCl solution, and the organics 
extracted with ether (2.times.10 mL). The ether layer was washed (water, 
then brine), dried (MgSO.sub.4), filtered and concentrated. Purification 
by chromatography (preparative TLC, SiO.sub.2, 20% EtOAc-hexanes) gave 28 
mg (0.08 mmol) of the all-trans isomer of 27 (80% yield): TLC (10% 
MeOH-90% CHCl.sub.3) R.sub.f 0.8; mp 143.degree.-144.degree. C.; .sup.1 
H-NMR (CDCl.sub.3) .delta.0.94 (t, J=7 Hz, 3H, CH.sub.2 CH.sub.3), 1.34 
(s, 18H, 6(CH.sub.3)), 1.52 (m, 2H, CH.sub.2 CH.sub.2 CH.sub.3), 2.40 (s, 
3H, CH.sub.3), 2.71 (t, J=7 Hz, 2H, CH.sub.2 CH.sub.2 CH.sub.3), 5.84 (s, 
1H, .dbd.CH), 6.41 (d, J=15 Hz, 1H, .dbd.CH), 6.47 (d, J=15 Hz; 1H, 
.dbd.CH), 7.08 (m, 1H, .dbd.CH), 7.26 (d, J=1 Hz, 2H, Ar-H), 7.37 (t, J=1 
Hz, 1H, Ar-H). 
(2E, 4E, 6Z)-7-(3,5-Di-t-butylphenyl)-3-methyldeca-2,4,6-trienoic acid 
(28). Compound 28 was prepared in the same manner as 27 except that the 
cis isomer 26 was used instead of 25: TLC (10% MeOH-90% CHCl.sub.3) 
R.sub.f 0.8; mp 166.degree.-169.degree. C.; .sup.1 H-NMR (CDCl.sub.3) 
.delta.0.89 (t, J=7 Hz, 3H, CH.sub.2 CH.sub.3), 1.31 (s, 9H, CH.sub.3), 
1.32 (s, 9H, CH.sub.3), 1.42 (m, 2H, CH.sub.2 CH.sub.2 CH.sub.3), 2.15 (s, 
3H, CH.sub.3), 2.49 (t, J=7 Hz, 2H, CH.sub.2 CH.sub.2 CH.sub.3), 5.76 (s, 
1H, .dbd.CH), 6.23 (d, J=11 Hz, 1H, .dbd.CH), 6.28 (d, J=15 Hz, 1H, 
.dbd.CH), 6.78 (dd, J=15 Hz, 1H, .dbd.CH), 7.04 (s, 2H, Ar-H), 7.35 (s, 
1H, Ar-H). 
EXAMPLES 7-8 
(2E,4E,6E)-6-(6,8,-Di-t-butylchroman-4-ylidene)-3-methylhexa-2,4,6-trienoic 
acid (39) and 
(2E,4E,6Z)-6-(6,8,-di-t-butylchroman-4-ylidene)-3-methylhexa-2,4,6-trienoi 
c acid (40), prepared as illustrated and described in Scheme 5 below. 
##STR10## 
3,5-Di-t-butyl-2-hydroxyacetophenone (30). To 10 g (48.5 mmol) of 
2,5-di-t-butylphenol 29 and 4.56 g (58.16 mmol) of acetyl chloride in 60 
mL of dichloroethane was added 11.9 mL (97.0 mmol) of BF.sub.3.OEt.sub.2, 
and the mixture was heated to reflux for 10 min. The reaction was cooled 
and poured into 1: 1 ice--20% aqueous HCl, stirred and extracted 
(2.times.100 mL of EtOAc). The organic extract was washed (water, then 
brine), dried (MgSO.sub.4), concentrated and purified (SiO.sub.2 
chromatography, 5% EtOAc-hexanes) to give 7.0 g (28.22 mmol) of 30 as an 
oil (58% yield): TLC (5% % EtOAc-hexane) R.sub.f 0.1; .sup.1 H-NMR 
(CDCl.sub.3) .delta.1.32 (s, 9H, CH.sub.3), 1.42 (s, 9H.sup.3 (CH.sub.3)), 
2.68 (s, 3H, CH.sub.3), 7.55.(m, 2H, Ar-H). 
6,8-Di-t-butyl-2-hydroxychroman-4-one (31). To 1.8 g (78.26 mmol) of sodium 
metal in a flame dried 500 mL round bottom flask, was dropwise added 7.5 g 
(30.24 mmol) of phenol 30 in 120 mL of ethyl formate. Once addition was 
complete, the mixture was stirred at 40.degree. C. for 1 h, then cooled to 
RT and poured into 1N aqueous HCl. When the mixture became transparent, 
the organics were extracted (2.times.150 mL EtOAc), washed (water, then 
brine), dried (MgSO.sub.4) and concentrated to give 7.4 g (26.81 mmol) of 
31 as an oil (approximate yield, 89%). This product was used directly in 
the next step: TLC (20% EtOAc-hex) R.sub.f 0.4; .sup.1 H-NMR (CDCl.sub.3) 
.delta.1.30 (s, 9H 3(CH.sub.3)), 1.42 (s, 9H, 3CH.sub.3), 2.87 (dd, 
J=16.0, 5.5 Hz, 1H, CH.sub.2), 3.01 (dd, J=16.0, 3.1 Hz, 1H, CH.sub.2), 
5.87 (dd, J=5.5, 3.3 Hz, 1H, CHOH), 7.58 (d, J=2.5 Hz, 1H, Ar-H), 7.79 (d, 
J=2.5 Hz, 1H, Ar-H). 
4H-6,8-di-t-butyl-benzopyran-4-one (not shown). To 7.4 g (26.81 mmol) of 30 
in 20 mL of MeOH was added 8 mL of aqueous 20% HCL and the mixture was 
heated at reflux for 20 min. After cooling to RT, water was added (30 mL) 
and the products were extracted (2.times.30 mL of EtOAc), washed (water, 
then brine), dried (MgSO.sub.4), and concentrated to give ca 6.0 g (25.00 
mmol) of 4H-6,8-di-t-butyl-benzopyran-4-one as an oil which after standing 
for several hours, solidified (approximate yield, 93%). This product was 
directly used in the next step: TLC (5% EtOAc-hex) R.sub.f 0.5; .sup.1 
H-NMR (CDCl.sub.3) .delta.1.37 (s, 9H, 3CH.sub.3), 1.49 (s, 9H, 
3CH.sub.3), 6.35 (d, J=5.6 Hz, 1H, CH.dbd.CH), 7.70 (d, J=2.4 Hz, 1H, 
Ar-H), 7.91 (d, J=5.6 Hz, 1H, CH.dbd.CH), 8.10 (d, J=2.4 Hz, 1 H, Ar-H). 
6,8-Di-t-butyl-chroman-4-one (32). To 1.0 g (3.87 mmol) of 
4H-6,8-di-t-butyl-benzopyran-4-one in 8 mL of EtOAc was added 200 mg of 
10% Pd/C. The mixture was degassed, followed by addition of H.sub.2 gas 
and stirred under an H.sub.2 gas atmosphere for 2 h at RT. After 
filtration (celite), the product was concentrated and purified (SiO.sub.2 
chromatography, 5% EtOAc-hexanes) to give 857 mg (3.29 mmol) of 32 as a 
white solid (85% yield): TLC (5% EtOAc-hex) R.sub.f 0.7; .sup.1 H-NMR 
(CDCl.sub.3) .delta.1.32 (s, 9H, CH.sub.3), 1.42 (s, 9H, CH.sub.3), 2.78 
(t, J=6.0 Hz, 2H, CH.sub.2), 4.52 (t, J=6.0 Hz, 2H, CH.sub.2), 7.53 (d, 
J=2.4 Hz, 1H, Ar-H), 7.81 (d, J=2.4 Hz, 1H, Ar-H) 
(6,8-Di-t-butylchroman-4-ylidene)acetonitrile (33) (trans) and (34) (cis). 
To 891 mg (5.03 mmol) of cyanomethyl phosphonate in 6 mL of dry THF was 
added 188 mg (4.69 mmol) of NaH (60% in oil) and the mixture was stirred 
for 20 min. To this solution was added 436 mg (1.80 mmol) of 32 in 2 mL of 
dry THF and the reaction was heated at reflux for 2 h. After cooling to 
RT, the reaction was quenched with saturated NH.sub.4 Cl (10 mL) and 
extracted (2.times.10 mL EtOAc), washed (water, then brine), dried 
(MgSO.sub.4), concentrated and purified (SiO.sub.2 chromatography, 5% 
EtOAc-hexane) to give 358 mg (1.32 mmol) of the trans isomer 33 and 95 mg 
(0.35 mmol) of the cis isomer 34 (93% combined yield) Trans isomer 33,: 
TLC (5% EtOAc-hex) R.sub.f 0.5; .sup.1 H-NMR (CDCl.sub.3) .delta.1.30 (s, 
9H, 3CH.sub.3), 1.36 (s, 9H, 3CH.sub.3), 3.00 (t, J=6.0 Hz, 2H, CH.sub.2), 
4.27 (t, J=6.0 Hz, 2H, CH.sub.2), 5.73 (s, 1H, .dbd.CH--CN), 7.35 (d, 
J=2.4 Hz, 1H, Ar-H), 7.41 (d, J=2.4 Hz, 1H, Ar-H); Cis isomer 34: TLC (5% 
EtOAc-hex), R.sub.f 0.4; 1H-NMR (CDCl.sub.3) 15 1.34 (s, 9H, 3CH.sub.3), 
1.39 (s, 9H, 3CH.sub.3), 2.76 (t, J=6.0 Hz, 2H, CH.sub.2), 4.29 (t, J=6.0 
Hz, 2H, CH.sub.2), 5.11 (s, 1H, .dbd.CH--CN), 7.42 (d, J=2 Hz, 1H, Ar-H), 
8.28 (d, J=2 Hz, 1H, Ar-H). 
(6,8-Di-t-butylchroman-4-ylidene)acetaldehyde (35) (trans). To 100 mg (0.36 
mmol) of nitrile 33 in 5 mL of hexane at -78.degree. C. was added 0.35 mL 
(0.53 mmol) of a 1.5M solution of DIBAL in toluene. The mixture was 
stirred for 5 min. followed by addition of 10 mL of sat. aqueous Rochelle 
salt and warmed to RT. The solution was extracted (2.times.10 mL of 
EtOAc), washed (water then brine), dried (MgSO.sub.4), filtered and 
concentrated to give 86 mg (0.30 mmol) of the relatively pure aldehyde 35 
(83% yield): TLC (5% EtOAc-hex) R.sub.f 0.5, .sup.1 H-NMR (CDCl.sub.3) 
.delta.1.32 (s, 9H, 3CH.sub.3), 1.41 (s, 9H, 3CH.sub.3), 3.27 (t, J=6.0 
Hz, 2H, CH.sub.2), 4.30 (t, J=6.0 Hz, 2H, CH.sub.2), 6.57 (d, J=8 Hz, 1H, 
.dbd.CH--CHO), 7.41 (d, J=2.4 Hz, 1H, Ar-H), 7.51 (d, J=2.4 Hz, 1H, Ar-H), 
10.14 (d, J=8 Hz, 1H, CHO). 
(6,8-Di-t-butylchroman-4-ylidene)acetaldehyde (36) (cis). Compound 36 was 
prepared in the same manner as 35, except that the cis isomer 34 was used 
instead of 33: TLC (5% EtOAc-hex) R.sub.f 0.4, .sup.1 H-NMR (CDCl.sub.3) 
.delta.1.30 (s, 9H, 3CH.sub.3), 1.38 (s, 9H, 3CH.sub.3), 2.80 (t, J=6.5 
Hz, 2H, CH.sub.2), 4.42 (t, J=6.5 Hz, 2H, CH.sub.2), 5.95 (d, J=8 Hz, 1H, 
.dbd.CH), 7.12 (d, J=2.4 Hz, 1H, Ar-H), 7.44 (d, J=2.4 Hz, 1H, Ar-H), 
10.00 (d, J=8 Hz, 1H, CHO). 
Ethyl 
(2E,4E,6E)-6-(6,8,-di-t-butylchroman-4-ylidene)-3-methylhexa-2,4,6-trienoa 
te (37). To 238 mg (0.902 mmol) of triethyl-3-methyl-4-phosphonocrotonate 
in 3 mL of dry THF at -78.degree. C. was added 0.36 mL (0.90 mmol) of a 
2.5M nBuLi solution in hexanes and 3 mL of DMPU. After stirring for 15 
min., the solution containing the ylide of triethylphosphonocrotonate was 
transferred to 86 mg (0.30 mmol) of 35 in 4 mL of 1:1 THF-DMPU at 
-78.degree. C. The reaction mixture was warmed to RT, quenched with 
saturated aqueous NH4Cl (20 mL), and the products were extracted with 
EtOAc (2.times.20 mL). The extracts were washed (water then brine), dried 
(MgSO.sub.4), filtered, concentrated and purified by column chromatography 
(SiO.sub.2, 5% EtOAc-hexanes) to give 85.3 mg (0.22 mmol) of the 
2E,4E,6E-isomer of 37 (73% yield) and 5.6 mg (0.014 mmol) of the 2Z,4E,6E 
isomer (5% yield). Compound 37: TLC (5% EtOAc-hex) R.sub.f 0.5, .sup.1 
H-NMR (CDCl.sub.3) .delta.1.30 (t, J=7 Hz, 3H, CH.sub.2 CH.sub.3), 1.33 
(s, 9H, 3CH.sub.3), 1.37 (s, 9H, 3CH.sub.3), 2.37 (s, 3H, CH.sub.3), 2.88 
(t, J=6.0 Hz, 2H, CH.sub.2), 4.17 (q, 2H, CH.sub.2), 4.23 (t, J=6.0 Hz, 
2H, CH.sub.2), 5.81 (s, 1H, .dbd.CH), 6.43 (d, J=15 Hz, 1H, .dbd.CH), 6.73 
(d, J=15 Hz, 1H, .dbd.CH), 6.96 (m, 1H, .dbd.CH), 7.25 (d, J=2.4 Hz, 1H, 
Ar-H), 7.50 (d, J=2.4 Hz, 1H, Ar-H). 
Ethyl 
(2E,4E,6Z)-6-(6,8,-di-t-butylchroman-4-ylidene)-3-methylhexa-2,4,6-trienoa 
te (38). Compound 38 was prepared in the same manner as 37 except that the 
cis isomer 36 was used instead of 35, Compound 36: TLC (5% EtOAc-hex) 
R.sub.f 0.5, 1H-NMR (CDCl.sub.3) .delta.1.30 (t, J=7 Hz, 3H, CH.sub.2 
CH.sub.3), 1.30 (s, 9H, 3CH.sub.3), 1.38 (s, 9H, 3CH.sub.3), 2.32 (s, 3H, 
CH.sub.3), 2.66 (t, J=6.0 Hz, 2H, CH.sub.2), 4.16 (m, 2H, CH.sub.2), 4.35 
(t, J=6.0 Hz, 2H, CH.sub.2), 5.80 (s, 1H, .dbd.CH), 6.08 (d, J=11 Hz, 1H, 
.dbd.CH), 6.36 (d, J=15 Hz, 1H, .dbd.CH), 7.27 (d, J=2 Hz, 1H, Ar-H), 7.29 
(d, J=2 Hz, 1 H, Ar-H), 7.28 (dd, J=15, 11 Hz, 1 H, Ar-H). 
(2E,4E,6E)-6-(6,8,-Di-t-butylchroman-4-ylidene)-3-methylhexa-2,4,6-trienoic 
acid (39). To 85.3 mg (0.22 mmol) of ester 37 in 6 mL of 1: 1 THF-MeOH was 
added 3 mL of an aqueous 5N KOH solution. The solution was heated at 
reflux for 10 min., cooled to RT, acidified (20% aqueous HCl) and 
extracted (2.times.6 mL of EtOAc). The extracts were combined, washed 
(water then brine), dried (MgSO.sub.4), filtered and concentrated. 
Purification by preparative TLC (5% MeOH-CHCl.sub.3), followed by 
crystallization (EtOAc-hexane, 1:4) gave 74.5 mg (0.20 mmol) of acid 39 as 
a pale yellow solid (94% yield): TLC (5% EtOAc-hex) R.sub.f 0.5; mp 
237.degree.-239.degree. C.; .sup.1 H-NMR (CDCl.sub.3) .delta.1.33 (s, 9H, 
3CH.sub.3), 1.38 (s, 9H, 3CH.sub.3), 2.37 (s, 3H, CH.sub.3). 2.89 (t, 
J=6.0 Hz, 2H, CH.sub.2), 4.23 (t, J=6.0 Hz, 2H, CH.sub.2), 5.86 (s, 1H, 
.dbd.CH), 6.43 (d, J=15 Hz, 1H, .dbd.CH)6.74 (d, J=11 Hz, 1H, .dbd.CH), 
6.97 (dd, 11, 15 Hz, 1H, .dbd.CH), 7.25 (d, J=2.3 Hz, 1H, Ar-H), 7.50 (d, 
J=2.3 Hz, 1H, Ar-H). 
(2E,4E,6Z)-6-(6,8,-Di-t-butylchroman-4-ylidene)-3-methylhexa-2,4,6-trienoic 
acid (40). Compound 40 was prepared in the same manner as 39, except that 
the cis isomer 37 was used instead of 36, Compound 37: TLC (5% EtOAc-hex) 
R.sub.f 0.5; mp 233.degree.-235.degree. C.; .sup.1 H-NMR (CDCl.sub.3) 
.delta.1.32 (s, 9H, 3CH.sub.3), 1.37 (s, 9H, 3CH.sub.3), 2.33 (s, 3H, 
CH.sub.3). 2.68 (t, J=6.0 Hz, 2H, CH.sub.2), 4.35 (t, J=6.0 Hz, 2H, 
CH.sub.2), 5.83 (s, 1H, .dbd.CH), 6.10 (d, J=11 Hz, 1H, .dbd.CH), 6.39 (d, 
J=15 Hz, 1H, .dbd.CH), 7.26 (d, J=2 Hz, 1H, Ar-H), 7.30 (d, J=2 Hz, 1H, 
Ar-H), 7.35 (dd, J=11, 15 Hz, 1 H, .dbd.CH). 
EXAMPLE 9 
(2E, 4E, 6E)-7-(3,5-Di-trifluoromethylphenyl)-3-methylocta-2,4,6-trienoic 
acid (41), prepared according to Scheme 1. 
This compound was synthesized in an analogous manner as 9, except that 
3,5-di-isopropylacetophenone was used instead of 
3,5-di-t-butylacetophenone: TLC (5% EtOAc-hex) R.sub.f 0.5; mp 
225.degree.-227.degree. C.; .sup.1 H-NMR (CDCl.sub.3) .delta.2.30 (s, 3H, 
CH.sub.3). 2.40 (s, 3H, CH.sub.3), 5.90 (s, 1H, .dbd.CH), 6.52 (d, J=15 
Hz, 1H, .dbd.CH), 6.66 (d, J=12 Hz, 1H .dbd.CH), 7.03 (m, 1H, .dbd.CH), 
7.78 (s, 1H, Ar-H), 7.87 (s, 1H, Ar-H). 
EXAMPLE 10 
(2E, 4E, 6E)-7-(3,5-Di-isopropylphenyl)-3-methylocta-2,4,6-trienoic acid 
(42), prepared according to Scheme 1. 
This compound was synthesized in an analogous manner as 9, except that 
3,5-di-isopropylacetophenone was used instead of 
3,5-di-t-butylacetophenone: TLC (5% EtOAc-hex) R.sub.f 0.5; mp 
157.degree.-160 .degree. C.; .sup.1 H-NMR (CDCl.sub.3) .delta.1.28 (d, J=8 
Hz, 12 H, CH.sub.3), 2.27 (s, 3H, CH.sub.3). 2.42 (s, 3H, CH.sub.3), 2.90 
(p, J=8 Hz, 2H, CH), 5.83 (s, 1H, .dbd.CH), 6.42 (d, J=15 Hz, 1H, 
.dbd.CH), 6.60 (d, J=15 Hz, 1H .dbd.CH), 7.02 (s, 1H, Ar-H), 7.08 (m, 1 H, 
.dbd.CH), 7.45 (s, 2H, Ar-H). 
EXAMPLE 11 
(2E, 4E, 6Z)-7-(3,5-Di-isopropylphenyl)-3-methylocta-2,4,6-trienoic acid 
(43), prepared according to Scheme 1. 
This compound was synthesized in an analogous manner as 10, except that 
3,5-di-isopropylacetophenone was used in the first step instead of 
3,5-di-t-butylacetophenone: TLC (5% EtOAc-hex) R.sub.f 0.5; mp 
177.degree.-179.degree. C.; .sup.1 H-NMR (CDCl.sub.3) .delta.1.25 (d, J=7 
Hz, 12 H, CH.sub.3), 2.18 (s, 3H, CH.sub.3). 2.21 (s, 3H, CH.sub.3), 2.90 
(p, J=7 Hz, 2H, CH), 5.77 (s, 1H, .dbd.CH), 6.25 (d, J=11 Hz, 1H, 
.dbd.CH), 6.27 (d, J=15 Hz, 1H .dbd.CH), 6.85 (dd, J=11, 15 Hz, 1H, Ar-H), 
6.94 (d, J=2 Hz, 2H, Ar-H), 7.02 (bs, H, Ar-H). 
EXAMPLE 12 
(2E, 4E, 6E)-7-(4-T-butyl-phenyl)-3-methylocta-2,4,6-trienoic acid (44), 
prepared according to Scheme 1. 
This compound was synthesized in an analogous manner as 9, except that 
4-t-butylacetophenone was used instead of 3,5-di-t-butylacetophenone. TLC 
(5% EtOAc-hex) R.sub.f 0.5; mp 198.degree.-200.degree. C.; .sup.1 H-NMR 
(CDCl.sub.3) .delta.1.33 (s, 9H, 3CH.sub.3), 2.26 (s, 3H, CH.sub.3). 2.39 
(s, 3H, CH.sub.3), 5.84 (s, 1H, .dbd.CH), 6.40 (d, J=15 Hz, 1H, .dbd.CH), 
6.60 (d, J=12 Hz, 1H .dbd.CH), 7.07 (m, 1H, .dbd.CH), 7.38 (s, 1H, Ar-H), 
7.43 (s, 1H, Ar-H). 
EXAMPLE 13 
(2E, 4E, 6E)-7-(3,5-Di-t-butyl-4-methoxyphenyl)-3-methylocta-2,4,6-trienoic 
acid (45), prepared according to Scheme 1. 
This compound was synthesized in an analogous manner as 9, except that 
3,5-di-t-butyl-4-methoxyacetophenone was used instead of 
3,5-di-t-butyl-acetophenone: TLC (5% EtOAc-hex) R.sub.f 0.5; mp 
244.degree.-246.degree. C.; .sup.1 H-NMR (CDCl.sub.3) .delta.1.30 (s, 9H, 
3CH.sub.3), 1.42 (s, 9H, 3CH.sub.3), 2.27 (s, 3H, CH.sub.3). 2.42 (s, 3H, 
CH.sub.3), 3.67 (s, 3H, OCH.sub.3), 5.83 (s, 1H, .dbd.CH), 6.34 (d, J=15 
Hz, 1H, .dbd.CH), 6.35 (d, J=15 Hz, 1H .dbd.CH), 7.00 (d, J=2Hz, 1H, 
Ar-H), 7.06 (m, 1H, .dbd.CH), 7.27 (d, J=2 Hz, 1 H, Ar-H). 
EXAMPLE 14 
(2E, 4E, 6Z)-7-(3,5-Di-trifluoromethylphenyl)-3-methylocta-2,4,6-trienoic 
acid (46), prepared according to Scheme 1. 
This compound was synthesized in an analogous manner as 10, except that 
3,5-di-trifluoromethyl-acetophenone was used instead of 
3,5-di-t-butyl-acetophenone. TLC (5% EtOAc-hex) R.sub.f 0.5; mp 
225.degree.-227.degree. C.; .sup.1 H-NMR (CDCl.sub.3) .delta.2.15 (s, 3H, 
CH.sub.3), 2.24 (s, 3H, CH.sub.3), 5.82 (s, 1H, CH.dbd.), 6.36 (d, J=15Hz, 
1H, CH.dbd.), 6.39 (d, J=9.7Hz, 1H, CH.dbd.), 6.53 (dd, J=15 Hz, 9.7 Hz, 
1H, CH.dbd.), 7.70 (s, 2H, Ar-CH), 7.83 (s, 1H, Ar-CH). 
EXAMPLE 15 
(2E, 4E, 6E) 
-3-Methyl-7-(3,5-di-t-butyl-4-methoxyphenyl)octa-2,4,6-trienoic acid (47), 
prepared according to Scheme 1. 
This compound was synthesized in an analogous manner as 9, except that 
3,5-di-t-butyl-4-methoxyacetophenone was used instead of 
3,5-di-t-butylacetophenone: TLC (50% EtOAc-50% hexanes) R.sub.f 0.5; mp 
213.degree.-216.degree. C.; .sup.1 H-NMR (CDCl.sub.3) .delta.1.45 (s, 18H, 
6(CH.sub.3)), 2.25, (s, 3H, CH.sub.3), 2.40 (s, 3H, CH.sub.3),3.70 
(S,3H.OCH.sub.3), 5.84 (s, 1H, .dbd.CH), 6.41 (d, J=15 Hz, 1H, .dbd.CH), 
6.52 (d,J=l 1.2Hz, 1H, .dbd.CH), 7.08 (m, 1H, .dbd.CH), 7.35 (s, 2H, 
Ar-H). 
EXAMPLE 16 
(2E,4E,6E)-3-Methyl-7-(3,4-diethylphenyl)octa-2,4,6-trienoic acid (48), 
prepared according to Scheme 1. 
This compound was synthesized in an analogous manner as 9, except that 
3,4-di-ethylacetophenone was used instead of 3,5-di-t-butylacetophenone: 
TLC (20% EtOAc-80% hexanes) R.sub.f 0.3; mp 153.degree.-155.degree. C.; 
.sup.1 H-NMR (CDCl.sub.3) .delta.1.45 (dd, J=14.1 Hz,7.5 Hz, 6H, 
2CH.sub.3), 2.25 (s,3H,CH.sub.3), 2.39 (s, 3H, CH.sub.3), 2.66 (m, 4H, 
2CH.sub.2), 5.83 (s, 1H, .dbd.CH), 6.40 (d, J=15 Hz), 1H, .dbd.CH), 6.59 
(d, J=11.2 Hz, 1H, .dbd.CH), 7.07 (m, 1H, .dbd.CH), 7.14 (d, J=7.8 Hz, 1H, 
Ar-H), 7.27 (d, J=7.8 Hz, 1H, Ar-CH), 7.28 (s, 1H, Ar-CH). 
EXAMPLE 17 
(2E,4E,6Z)-3-Methyl-7-(3,4-di-ethylphenyl)octa-2,4,6-trienoic acid (49), 
prepared according to Scheme 1. 
Compound 49 was prepared in the same manner as 10, except that 
3,4-diethylacetophenone was used instead of 3,5-di-t-butylacetophenone: 
TLC (20% EtOAc-80% hexanes) R.sub.f 0.32; .sup.1 H-NMR (CDCl.sub.3) 
.delta.1.23 (dd, J=14.1Hz,7.5Hz, 6H, 2CH.sub.3), 2.18 (s,6H,2CH.sub.3), 
2.67 (m, 4H, 2CH.sub.2), 5.76 (s, 1H, .dbd.CH), 6.23(d, J=l 1.8Hz, 1H, 
.dbd.CH), 6.28 (d, J=15Hz, 1H, .dbd.CH), 6.83 (m, 1H, .dbd.CH), 7.04 (d, 
J=7.8 Hz, 1H, Ar-H), 7.06 (s, 1H, Ar-CH), 7.17 (d, J=7.8 Hz, 1H, Ar-CH). 
EXAMPLE 18 
(2E, 4E, 6E)-3-Methyl-7-(3,5-di-t-butyl-4-ethoxyphenyl)octa-2,4,6-trienoic 
acid (50), prepared according to Scheme 1. 
This compound was synthesized in an analogous manner as 9, except that 
3,5-di-t-butyl-4-ethoxyacetophenone was used instead of 
3,5-di-t-butylacetophenone: TLC (50% EtOAc-50% hexanes) R.sub.f 0.5; mp 
236.degree.-239.degree. C.; .sup.1 H-NMR (CDCl.sub.3) .delta.1.41 (t, 
J=7.0 Hz, 3H, CH.sub.3), 1.44 (s, 18H, 6(CH.sub.3)), 2.26 (s, 3H, 
CH.sub.3), 2.40 (s, 3H, CH.sub.3), 3.77 (q, J=7 Hz, 2H.OCH.sub.2 
CH.sub.3), 5.84 (s, 1H, .dbd.CH), 6.41 (d, J=15 Hz, 1H, .dbd.CH), 6.51 
(d,J=11.2 Hz, 1H, .dbd.CH), 7.08 (m, 1H, .dbd.CH), 7.35 (s, 2H, Ar-H). 
EXAMPLE 19 
(2E,4E,6E)-3-Methyl-7-(3,4-di-t-butylphenyl)octa-2,4,6-trienoic acid (51), 
prepared according to Scheme 1. 
This compound was synthesized in an analogous manner as 9, except that 
3,4-di-t-butylacetophenone was used instead of 3,5-di-t-butylacetophenone: 
TLC (20% EtOAc-80% hexanes) R.sub.f 0.3; mp 195.degree.-199.degree. C.; 
.sup.1 H-NMR (CDCl.sub.3) d 1.56 (s, 9H, 3CH.sub.3), 1.58 (s, 9H, 
3CH.sub.3), 2.26 (s, 3H, CH.sub.3), 2.40 (s, 3H, CH.sub.3), 5.84 (s, 1H, 
.dbd.CH), 6.41 (d, J=15 Hz, 1H, .dbd.CH), 6.59 (d, J=11 Hz, 1H, .dbd.CH), 
7.08 (m, 1H, .dbd.CH), 7.23 (dd, J=8.5, 2.3 Hz, 1H, Ar-H), 7.57 (d, J=8.5 
Hz, 1H, Ar-H), 7.72 (d, J=2.3 Hz, 1H, Ar-H). 
EXAMPLE 20 
(2E,4E,6E)-3-Methyl-7-cyclohexyl-7-(3,5-di-t-butylphenyl)hepta-2,4,6-trieno 
ic acid (52), prepared according to Scheme 4. 
This compound was synthesized in an analogous manner as 27, except that 
3,4-di-t-butylphenylcyclohexyl ketone was used instead of 
3,5-di-t-butylbutan-1-one: TLC (20% EtOAc-80% hexanes) R.sub.f 0.3; .sup.1 
H-NMR (CDCl.sub.3) .sup.1 H NMR (CDCl.sub.3) .delta.1.35 (s, 9H), 1.9-1.1 
(m,m, 8 H), 2.40 (s, 3 H), 2.85 (m, 1H), 5.81 (s, 1 H), 6.08 (d, 1H, 
J=11.3 Hz), 6.30 (d, 1H, J=15.8 Hz), 7.02 (s, 2H, Ar-H), 7.14 (dd, 1H, 
J=15.2, 15.2 Hz), 7.33 (s, 1H, Ar-H), 
EXAMPLE 21 
(2E,4E,6E)-3-Methyl-7-(3,5-di-t-butylphenyl)nona-2,4,6-trienoic acid (53), 
prepared according to Scheme 1. 
This compound was synthesized in an analogous manner as 9, except that 
3,5-di-t-butyl-4-methoxyacetophenone was used instead of 
3,5-di-t-butylacetophenone: TLC (50% EtOAc-50% hexanes) R.sub.f 0.5; mp 
213.degree.-216.degree. C.; .sup.1 H-NMR (CDCl.sub.3) .delta.1.45 (s, 18H, 
6(CH.sub.3)), 2.25 (s, 3H, CH.sub.3), 2.40 (s, 3H, CH.sub.3),3.70 (S,3H, 
OCH.sub.3), 5.84 (s, 1H, .dbd.CH), 6.41 (d, J=15 Hz, 1H, .dbd.CH), 6.52 
(d,J=11.2 Hz, IH, .dbd.CH), 7.08 (m, 1H, .dbd.CH), 7.35 (s, 2H, Ar-H). 
EXAMPLE 22 
(2E,4E,6Z)-3-Methyl-7-(3,4-diethyl-6-methylphenyl)nona-2,4,6-trienoic acid 
(54), prepared according to Scheme 1. 
This compound was synthesized in an analogous manner as 10, except that 
3,4-diethyl-6-methylacetophenone was used instead of 
3,5-di-t-butylacetophenone: TLC (20% EtOAc-80% hexanes) R.sub.f 0.3; 
.sup.1 H-NMR (CDCl.sub.3) .delta.1.22 (t, J=7.5 Hz, 3H, CH.sub.3), 1.24 
(t, J=7.5 Hz, 3H, CH.sub.3), 2.08 (s, 3H, CH.sub.3), 2.09 (s, 3H, 
CH.sub.3), 2.17 (s, 3H, CH.sub.3), 2.60 (m, 4H, 2(CH.sub.2)), 5.73 (s, 1H, 
.dbd.CH), 6.25 (m, 3H, 3(.dbd.CH)), 6.81 (s, 1H, Ar-H), 7.01 (s, 1H, 
Ar-H). 
EXAMPLE 23 
(2E, 4E, 6E)-7-(3,5-Di-t-butylphenyl)-3-methylocta-2,4,6-trienoic acid 
(69), prepared according to Scheme 6 illustrated and described below. 
##STR11## 
5-tert-Butyl-1,3-benzene dimethanol (56). A solution of 
5-tert-butyl-1,3-benzene dicarboxylic acid (55) 20.0 g (89.9 mmol) in THF 
(200 mL) was cooled at 0.degree. C. and a solution of borane-THF complex 
in THF (190 mL) was slowly added via an addition funnel over 20 minutes 
with vigorous stirring. The mixture was warmed to RT and stirred for an 
additional 90 min. A mixture of water-THF (1:1; 200 mL) was slowly added, 
followed by an additional 200 mL of water. The mixture was extracted with 
ethyl acetate. The aqueous layer was extracted with EtOAc (2.times.100 mL) 
and the combined organic layers washed with water (2.times.100 mL); brine 
(2.times.100 mL) and dried over MgSO.sub.4. The solvent was evaporated to 
give the pure diol in 98% yield: .sup.1 H NMR (CDCl.sub.3 ; 400 MHz) 
.delta.(ppm): 7.32 (s, 2 H); 7.19 (s, 1 H); 4.69 (s, 4 H); 1.75 (br. s; 2 
H); 1.33 (s, 9 H). 
5-tert-Butyl-1,3-terephthaldehyde. 5-tert-Butyl-1,3-benzene dimethanol (56) 
(20.0 g; 103 mmol) was added to a vigorously stirring mixture of 
pyridinium chlorochromate (66.0 g; 306 mmol) and celite (130 g) in 
dichloromethane (DCM) (500 mL). The mixture was stirred for 3 h at RT 
until completion (TLC). The reaction mixture was filtered over a short pad 
of silica gel (2".times.4") and eluted with DCM (1 L). The solvent was 
evaporated to give 18.7 g; 94% yield of the desired dialdehyde: .sup.1 H 
NMR (CDCl.sub.3 ; 400 MHz) .delta.(ppm): 10.11 (s, 2 H); 8.18 (s, 3 H); 
1.41 (s 9 H). 
(R, S)-5-tert-Butyl-1,3-benzene-2,2'-diethanol!(57). A solution of 
5-tert-butyl- 1,3-terephthaldehyde (18.7 g; 96.4 mmol) in THF (400 mL) was 
cooled to -78.degree. C., and a solution of methyl magnesium bromide (80.0 
mL of a 3 M solution) slowly added, and the reaction mixture was warmed to 
rt. After stirring for 60 min., the solution was quenched with a sat. 
NH4Cl solution (100 mL), followed by HCl (1 N, 50 mL) and extraction with 
EtOAc. The organic layer was washed with water (2.times.100 mL); brine 
(2.times.100 mL) and dried over MgSO.sub.4. The solvent was evaporated. 
The crude residue was dissolved in hot EtOAc (30 mL) and pentane (200 mL) 
was added. The clear solution was cooled in a -4.degree. C. refrigerator 
for 3 h. The white solid obtained was filtered and rinsed with cold 
pentane. The solid was dried in vaccuo to give 15.3 g (70% yield) of the 
desired compound: .sup.1 H NMR (CDCl.sub.3 ; 400 MHz) .delta. (ppm): 7.32 
(s, 2 H); 7.2 (s, 1H); 4.92 (m, 2 H); 1.82 (d, 2 H, J=2.5 Hz); 1.52 (d, 6 
H, J=6.5 Hz); 1.35 (s, 9H). 
(R, S)-5-tert-Butyl-1,3-phenyl-2-ethanol, 2'-ethane-tert-butyl dimethyl 
silyl ether! (58) Sodium hydride (2.75 g of a 60% mineral oil content 
mixture) was rinsed with hexanes (2.times.10 ml), suspended in THF (200 
mL) and 5-tert-butyl-1,3-benzene-2,2'-diethanol (12.69 g; 57 mmol) was 
added with vigorous stirring. The mixture was stirred for 45 min. at rt to 
give a white slurry then tert-butyldimethylsilyl chloride (8.61 g, 57 
mmol) was added at once. The reaction mixture was stirred for 2 h. and 
water (25 mL) was added. The mixture was extracted with EtOAc (350 mL). 
The organic layer was washed with a sat. NH.sub.4 Cl solution (100 mL); 
water (2.times.100 mL); brine (2.times.100 mL) and dried over MgSO.sub.4. 
The solvent was evaporated and the residue purified by flash 
chromatography over silica gel to give the desired monosilylated product 
(14.45 g; 75% yield) as an oil (2.14 g of the starting material was 
recovered): .sup.1 H NMR (CDCl.sub.3 ; 400 MHz) .delta. (ppm): 7.32 (s, 1 
H); 7.28 (s, 1H); 7.12 (2s, 1 H); 4.92 (m, 2 H); 1.85 (s, 1 H); 1.5 (d, 
3H, J=6.5 Hz); 1.43 (d, 3 H, J=6.5 Hz) 1.35 (s, 9 H); 0.9 (s, 9 H); 0.05 
(s, 6 H). 
(R, S)-3-(Ethyl-2-tert-butyldimethyl silyl ether)-5-tert-butylacetophenone 
(59). Pyridinium chlorochromate (15.0 g; 69 mmol) and celite (30 g) were 
mixed in DCM (500 mL) while 5-tert-butyl-1,3-benzene-(2-ethanol, 
2'-ethane-tert-butyldimethyl silyl ether) (14.45 g; 44 mmol) in DCM (100 
mL) was added with vigorous stirring. After 3 h at rt, the mixture was 
filtered over a short pad of silica gel (2".times.4") and eluted with DCM 
(500 L). The solvent was evaporated to give the desired ketone (14.4 g, 
99% yield). .sup.1 H NMR (CDCl.sub.3 ; 400 MHz) .delta. (ppm): 7.89 (s, 1 
H); 7.73 (s, 1 H); 7.65 (s, 1 H); 4.94(q, 1 H, J=6.3 Hz); 2.63 (s, 3 H); 
1.45 (d, 3H, J=6.3 Hz); 1.37 (s, 9H); 0.96 (s, 9H); 0.12 (s, 3 H); -0.05 
(s, 3H). 
5-tert-Butyl-1-(2-ethanol)-3-(2-2-methylpropan- 1 -al!)-benzene (60). A 
flame-dried three-necked round bottom flask, was charged with THF (90 mL) 
and cooled to -78.degree. C. n-BuLi (12.0 mL of a 2M solution; 24 mmol) 
was slowly added and the mixture stirred for 10 min. A solution of 
N-benzylidene diethyl aminomethyl phosphonate (6.14 g; 24 mmol) in THF (15 
mL) was added and the resulting mixture stirred for 60 min at -78.degree. 
C. A solution of 5-tert-butyl-3-(ethane-2-tert-butyldimethylsilyl 
ether!acetophenone (7.0 g, 20.9 mmol) in THF (15 mL) was added to the 
above solution, and the mixture was warmed to room temp., stirred for 30 
min., then refluxed for 2 h. The mixture was cooled to rt and the solvent 
was evaporated. Diethyl ether (400 mL) was added and the solution was 
washed with sodium chloride (200 mL). The aqueous layer was extracted with 
ether (200 mL) and the combined organic layers were washed with brine (200 
mL) and dried over MgSO.sub.4. The solvent was evaporated to give a yellow 
residue which was dried under high vaccuo (1 mm. Hg) for 1 hour. THF (90 
mL) was added to this residue and cooled to -78.degree. C. n-BuLi (12.0 mL 
of a 2M solution; 24 mmol) was slowly added and the deeply colored 
solution was stirred for 60 min. Methyl iodide (6.52 mL) was added and the 
mixture was warmed to room temperature and stirred for 4 h. The reaction 
mixture was quenched with HCl (3N; 100 mL) and the biphasic solution was 
stirred for 16 h at RT. EtOAc (300 mL) was added and the organic layer was 
separated and washed with water (2.times.100 mL), brine (2.times.100 mL), 
and dried over MgSO.sub.4. The solvent was evaporated to give a residue 
which was purified by s.g.c. to give 3.3 g of the desired aldehyde in 65% 
yield: .sup.1 H NMR (CDCl.sub.3 ; 400 MHz) .delta. (ppm): 9.5 (s, 1 
H);7.33 (s, 1H);7.17(s, 1H);7.11 (s, 1H);4.91 (m, 1H); 1.80(d, 1H, J=3.4 
Hz); 1.5 (d, 3 H, J-6.3 Hz); 1.47 (s, 3 H); 1.32 (s, 9 H). 
5-tert-Butyl-3-(2-methylpropan-1-ol!acetophenone (61). A solution of 
5-tert-butyl-1-(2-ethanol)-3-(2-2-methylpropan-1-al!)benzene (1.77 g, 
7.53 mmol) in MeOH (50 mL) was cooled to 0.degree. C. and NaBH.sub.4 (300 
mg, 7.93 mmol) was added portionwise. The reaction mixture was warmed to 
RT and stirred for 30 min. The solvent was evaporated and the residue 
taken up in EtOAc (50 mL) and washed with HCl (10%, 3.times.10 mL); water 
(3.times.20 mL) and brine (3.times.20 mL). The organic layer was dried 
over MgSO.sub.4 and evaporated to dryness to give the desired diol 1.76 g; 
99% yield. This diol (1.65 g; 6.78 mmol) was dissolved in DCM (20 mL) and 
MnO.sub.2 (18.7 g; 0.17 mmol) was added at once. The reaction mixture was 
vigorously stirred for 4 h, then filtered over a short pad of celite. The 
solvent was evaporated to give 1.63 g (97% yield) of the desired ketone: 
.sup.1 H NMR (CDCl.sub.3 ; 400 MHz) 15 (ppm): 7.85 (s, 1H); 7.8 (s, 1H); 
7.63 (s, 1H); 3.64 (d; 2H, J=4.5 Hz); 2.6 (s, 3H); 1.38 (s, 6H); 1.35 (s, 
9H). 
5-tert-Butyl-3-(2-2-methylpropan-1-tert-butyldiphenylsilylether!)acetophen 
one (62). To a solution of 5-tert-butyl-3-(2-methylpropan-1-ol!acetophenone 
(1.63 g; 6.96 mmol) in DCM (30 mL) was added imidazole (500 mg; 7.35 
mmol), a drop of DMF and tert-butyldiphenylsilyl chloride (2.01 g; 7.33 
mmol). The mixture was stirred overnight at RT and quenched with excess 
sat. NH.sub.4 Cl. DCM (50 mL) was added and the organic layer washed with 
water (3.times.20 mL) and brine (3.times.20 mL). The organic layer was 
dried over MgSO.sub.4 and evaporated to dryness to give a residue which 
was purified by s.g.c. to give the desired ether 2.77 g (83% yield): 
.sup.1 H NMR (CDCl.sub.3 ; 400 MHz) 15 (ppm): 7.85 (s, 1H), 7.76 (s, 1H); 
7.63 (1H); 7.48-7.25 (mm, 10H); 3.62 (s, 2H); 2.56 (s, 3H); 1.38 (s, 6H); 
1.33 (s, 9H); 0.94 (s, 9H). 
5-tert-Butyl-3-(2-2-methylpropan-1-tert-butyldiphenylsilylether!)-1-(2E)-3 
-2-butenitrile)-benzene (63). To a solution of diethyl cyanomethyl 
phosphonate (1.7 g, 9.55 mmol) in THF (30 mL) at 0.degree. C. was added 
n-BuLi (4.64 mL of a 2.0M solution in hexanes). The solution was stirred 
for 10 min., after which a solution of 
5-tert-butyl-3-(2-2,2'-dimethylpropan-tert-butyldiphenylsilyl 
ether!)acetophenone in THF (10 mL) was added. The reaction mixture was 
stirred for 30 min. and quenched with a sat. NH.sub.4 Cl solution. EtOAc 
(50 mL) was added and the organic layer was washed with water (3.times.20 
mL) and brine (3.times.20 mL). The organic layer was dried over MgSO.sub.4 
and evaporated to dryness to give a residue which was purified by s.g.c. 
to give the desired nitrile as a trans:cis mixture (.about.4:1 by 1H NMR). 
S.g.c. gave the desired trans isomer 1.70 g, 63% yield: .sup.1 H NMR 
(CDCl.sub.3 ; 400 MHz) 15 (ppm): 7.85 (s, 1H), 7.48-7.22 (mm, 13H, ArH); 
5.5 (s, 1H); 3.59 (s, 2H); 2.43 (s, 3H); 1.35 (s, 6H); 1.27 9s, 9H); 0.94 
(s, 9H). 
5-tert-Butyl-3-(2-2-methylpropan-1-tert-butyldiphenylsilylether!)-1-(2E)-( 
3 -2-butenal)benzene (64). A solution of 
5-tert-butyl-3-(2-2-methylpropan- 1-tert-butyldiphenylsilylether!)-1-(3- 
3-methyl-2-propenitrile)benzene (1.7 g; 3.42 mmol) in anhydrous DCM (20 mL) 
was cooled to -78.degree. C. and Dibal (3.5 mL of a 1M solution in 
toluene) was added dropwise. The reaction mixture was stirred at 
-78.degree. C. for 60 min, quenched with excess Rochelle salt, then 
allowed to warm to RT. EtOAc (50 mL) was added and the mixture washed with 
water (3.times.20 mL) and brine (3.times.20 mL). The organic layer was 
dried over MgSO.sub.4 and evaporated to dryness to give a residue which 
was purified by s.g.c. to give 1.25 g of the desired aldehyde (74% yield): 
.sup.1 H NMR (CDCl.sub.3 ; 400 MHz) 15 (ppm): 10.17 (d, 1H; J=8 Hz); 
7.48-7.22 (mm, 13H, ArH); 6.36 (d, 1H, J=8 Hz); 3.60 (s, 2H); 2.54 (s, 
3H); 1.37 (s, 6H); 1.32 (s, 9H); 0.94 (s, 9H). 
Ethyl-(2E, 4E, 
6E)-7-(5-tert-butyl-3-2-(2-methylpropan-1-ol)!-1'-benzene)-3-methylocta-2 
,4,6-trienoate (66). A solution of diethyl 
3-ethoxycarbonyl-2-methylprop-2-enyl phosphonate (1.20 g, 4.52 mmol) in 
anhydrous THF (20.0 mL) was cooled to 0.degree. C. and added with 
anhydrous DMPU (3.5 mL) and n-BuLi in hexanes (2.15 mL of 2.0M solution, 
4.50 mmol). The mixture was stirred at this temperature for 20 min., then 
cooled to -78.degree. C. A solution of 5-tert-butyl-3-(2-2-methyl 
propane-1-tert-butyldiphenylsilyl 
ether!)-1-(3-3-methyl-2-propenal)benzene (1.25 g, 2.52 mmol) in THF (10.0 
mL) was slowly added and the reaction mixture stirred at -78.degree. C. 
for an additional 60 min. The mixture was allowed to warm to 23.degree. C. 
for 1 h with stirring. A sat. solution of ammonium chloride (5 mL) was 
added and the mixture extracted using EtOAc (3.times.10 mL). The organic 
layer was with water (2.times.25 mL) and brine (50 mL), dried over 
MgSO.sub.4 and concentrated. The reidue was purified on a short sgc column 
to give 1.35 g (86% yield) of the desired ester (65). The above silyl 
ether (1.05 g, 1.68 mmol) was dissolved in THF (20 mL) and 
tetrabutylammonium fluoride (17 mL of 1M solution in THF) was added. The 
reaction mixture was stirred at room temperature for 12 h, and EtOAc (50 
mL) was added, followed by wash with water (2.times.20 mL), brine (20 mL). 
The organic layer was separated, dried over MgSO.sub.4 and evaporated to 
dryness. The residue was purified by s.g.c. to give 479 mg (80% yield) of 
the desired alcohol: .sup.1 H NMR (CDCl.sub.3 ; 400 MHz) .delta. (ppm): 
7.9 (d, 1H; J=16 Hz, 2:cis isomer; .about.15%); 7.34 (s, 2H), 7.29 (s, 
1H), 7.03 (dd, 1H, J=16 Hz); 6.53 (d, 1H, J=12 Hz); 6.38 (d, 1H, J=16 Hz); 
5.72 (s, 1H); 5.68 (s, 1H, 2:cis isomer; .about.15%); 4.15 (q, 2H, J=6.7 
Hz); 3.62 (s 2H); 2.38 (s, 3H); 2.28 (s, 3H); 1.6 (hr. s; 1H); 1.37 (s, 
6H); 1.33 (s, 9H), 1.26 (t, 3H, J=6.7 Hz). 
Ethyl-(2E, 4E, 
6E)-7-(5-tert-butyl-3-2-(2-methylpropan-1-al)!-1'-phenyl)-3-methylocta-2, 
4,6-trienoate (67). To a vigorously stirred mixture of pyridinium 
chlorochromate(350 mg; 1.39 mmol) and celite (750 mg) in DCM (20 mL) was 
added a solution of ethyl-(2E, 4E, 
6E)-7-(5-tert-butyl-3-2-(2-methylpropan-1-ol)!-1'-phenyl)-3-methylocta-2, 
4,6-trienoate (330 mg; 0.889 mmol) in DCM (10 mL). The mixture was stirred 
for 3 h at rt and filtered over a short pad of silica gel. The solvent was 
evaporated and the residue purified by s.g.c. to give the desired aldehyde 
290 mg (87% yield): .sup.1 H NMR (CDCl.sub.3 ; 400 MHz) .delta. (ppm): 9.5 
(s, 1H); 7.9 (d, 1H; J=16 Hz, 2:cis isomer; .about.15%); 7.34 (s, 2H), 
7.29 (s, 1H), 7.03 (dd, 1H, J=16 Hz); 6.53 (d, 1H, J=12 Hz); 6.38 (d, 1H, 
J=16 Hz); 5.79 (s, 1H); 5.68 (s, 1H, 2:cis isomer; .about.15%); 4.15 (q, 
2H, J=6.7 Hz); 3.62 (s 2H); 2.38 (s, 3H); 2.28 (s, 3H); 1.37 (s, 6H); 1.33 
(s, 9H), 1.26 (t, 3H, J=6.7 Hz). 
Ethyl-(2E, 4E, 
6E)-7-(5-tert-butyl-3-2-(2-methylpropan-1-al)!-1'-phenyl)-3-methylocta-2, 
4,6-trienoate p-toluenesulfonyl hydrazone (68). To a solution of ethyl-(2E, 
4E, 6E)-7-(5-tert-butyl-3-2-(2-methyl 
propanal)!-1'-phenyl)-3-methylocta-2,4,6-trienoate (250 mg, 0.67 mmol) in 
ethanol (5 mL) was added p-toluenesulfonyl hydrazide (137 mg, 0.73 mmol) 
and .about.10 ml of conc. HCl. The mixture was heated at 
40.degree.-45.degree. C. for 15 min. The solvent was evaporated and the 
residue purified by s. g. c. to give 330 mg (89% yield): .sup.1 H NMR 
(CDCl.sub.3 ; 400 MHz) .delta. (ppm): 7.82 (d, 2 H, J=7.4 Hz); 7.5 (2s, 
2H); 7.3 (d, 2H, J=7.4 Hz); 7.18 (s, 1H); 7.05 (s, 1H); 7.0 (dd, 1H, J=16 
Hz); 6.45 (d, 1H, J=12 Hz); 6.38 (d, 1H, J=12 Hz); 5.82 (s, 1H); 4.2 (q, 
2H, J=6.7Hz); 3.62 (s 2H); 2.42 (s, 3H); 2.38 (s, 3H); 2.28 (s, 3H); 1.37 
(s, 6H); 1.33 (s, 9H), 1.26 (t, 3H, J=6.7 Hz). 
(2E, 4E, 6E)-7-(3, 5-Di-tert-butylphenyl)-3-methylocta-2,4,6-trienoic acid 
(69)**. To a solution of ethyl-(2E, 4E, 
6E)-7-(5-tert-butyl-3-2-(2-methylpropan-1-al)!-1'-phenyl)-3-methylocta-2, 
4,6-trienoate p-toluenesulfonyl hydrazone (68) (80 mg) in acetic acid (2.0 
mL) was added sodium borohydride* (80 mg) in small portions. The mixture 
was heated at 50.degree. C. for 1 h, cooled to RT and added to ice; 
allowed to warm to ambient temperature and extracted with EtOAc 
(3.times.10 mL). The organic layer was washed with water (3.times.10 mL); 
NaHCO.sub.3 (2.times.10 mL); water (3.times.10 mL); brine (3.times.10 mL); 
dried over MgSO.sub.4 and evaporated. The residue was purified by s.g.c. 
to give the desired ester. This ester (20 mg) was dissolved in EtOH and 
KOH 1M (1 mL) was added and the mixture heated at reflux for 3 h. The 
reaction mixture was cooled to RT; neutralized with HCl (10%) and 
extracted with EtOAc. The organic layer was washed with water (3.times.10 
mL); brine (3.times.10 mL); dried over MgSO.sub.4 and evaporated to give 
the desired acid. *Those skilled in the art will recognize that the above 
protocol can be adapted to use labeled and radiolabeled NaB.sup.n H.sub.4 
(n=1, 2, 3) to generate the labeled (i.e., tritium labeled) compounds 
shown in Scheme 6. **Furthermore, those skilled in the art will also 
recognize that the same labeled (deuterio and tritio)-compound (69) can be 
obtained from hyrazone (68) with heating for 8 h in methanol in the 
presence of NaCNB.sup.n H.sub.3 (n=1, 2, 3) and ZnCl.sub.2, followed by 
saponification (KOH, EtOH). In another alternative embodiment, aldehyde 
(67) is reduced with radiolabeled NaB.sup.n H.sub.4 (n=1, 2, 3), and the 
resulting alcohol is then oxidized to the corresponding tritiated 
aldehyde. Conversion of such an aldehyde to its tosyl hydrazone, followed 
by reduction with sodium cyanoborohydride and ZnCl.sub.2 in methanol, and 
saponification yields the corresponding acid (See 68 to 69 of Scheme 6. 
Evaluation of Retinoid Receptor Subfamily Activity 
Utilizing the "cis-trans" or "co-transfection" assay described by Evans et 
al., Science, 240:889-95 (May 13, 1988), the disclosure of which is herein 
incorporated by reference, the retinoid compounds of the present invention 
were tested and found to have strong, specific activity as either 
selective RAR agonists, selective RXR agonists, or as pan-agonist 
activators of both RAR and RXR receptors. This assay is described in 
further detail in U.S. Pat. Nos. 4,981,784 and 5,071,773, the disclosures 
of which are incorporated herein by reference. 
The co-transfection assay provides a method for identifying functional 
agonists which mimic, or antagonists which inhibit, the effect of native 
hormones, and quantifying their activity for responsive 1R proteins. In 
this regard, the co-transfection assay mimics an in vivo system in the 
laboratory. Importantly, activity in the co-transfection assay correlates 
very well with known in vivo activity, such that the co-transfection assay 
functions as a qualitative and quantitative predictor of a tested 
compounds in vivo pharmacology. See, e.g., T. Berger et at. 41 J. Steroid 
Biochem. Molec. Biol. 773 (1992), the disclosure of which is herein 
incorporated by reference. 
In the co-transfection assay, a cloned cDNA for an IR (e.g., human 
RAR.alpha., RAR.beta., RXR.gamma.) under the control of a constitutive 
promoter (e.g., the SV 40 promoter) is introduced by transfection (a 
procedure to induce cells to take up foreign genes) into a background cell 
substantially devoid of endogenous IRs. This introduced gene directs the 
recipient cells to make the IR protein of interest. A second gene is also 
introduced (co-transfected) into the same cells in conjunction with the IR 
gene. This second gene, comprising the cDNA for a reporter protein, such 
as firefly luciferase (LUC), is controlled by an appropriate hormone 
responsive promoter containing a hormone response element (HRE). This 
reporter plasmid functions as a reporter for the transcription-modulating 
activity of the target IR. Thus, the reporter acts as a surrogate for the 
products (mRNA then protein) normally expressed by a gene under control of 
the target receptor and its native hormone. 
The co-transfection assay can detect small molecule agonists or antagonists 
of target IRs. Exposing the transfected cells to an agonist ligand 
compound increases reporter activity in the transfected cells. This 
activity can be conveniently measured, e.g., by increasing luciferase 
production, which reflects compound-dependent, IR-mediated increases in 
reporter transcription. To detect antagonists, the co-transfection assay 
is carried out in the presence of a constant concentration of an agonist 
to the target IR (e.g., all-trans retinoic acid for RAR.alpha.) known to 
induce a defined reporter signal. Increasing concentrations of a suspected 
antagonist will decrease the reporter signal (e.g., luciferase 
production). The co-transfection assay is therefore useful to detect both 
agonists and antagonists of specific IRs. Furthermore, it determines not 
only whether a compound interacts with a particular IR, but whether this 
interaction mimics (agonizes) or blocks (antagonizes) the effects of the 
native regulatory molecules on target gene expression, as well as the 
specificity and strength of this interaction. 
The activity of the retinoid compounds of the present invention were 
evaluated utilizing the co-transfection assay according to the following 
illustrative Example 23. 
EXAMPLE 23 
Co-transfection assay 
CV-1 cells (African green monkey kidney fibroblasts) were cultured in the 
presence of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% 
charcoal resin-stripped fetal bovine serum then transferred to 96-well 
microtiter plates one day prior to transfection. 
To determine RAR and/or RXR agonist activity of the compounds of the 
present invention, the CV-1 cells were transiently transfected by calcium 
phosphate coprecipitation according to the procedure of Berger et al., 41 
J. Steroid Biochem. Mol. Biol., 733 (1992) with the following receptor 
expressing plasmids: pRShRAR.alpha.: Giguere et at., 330 Nature, 624 
(1987); pRShRAR.beta. and pRShRAR.gamma., Ishikawa et al., 4 Mol. 
Endocrin., 837 (1990); pRShRXR.alpha., Mangelsdorf et at., 345 Nature, 224 
(1990); and pRSmRXR.beta. and pRSmRXR.gamma., Mangelsdorf et al., 6 Genes 
& Devel., 329 (1992), the disclosures of which are herein incorporated by 
reference. Each of these receptor expressing plasmids was co-transfected 
at a concentration of 5 ng/well, along with a basal reporter plasmid at 
100 ng/well, the internal control plasmid pRS-.beta.-Gal at 50 ng/well and 
filler DNA, pGEM at 45 ng/well. 
The basal reporter plasmid D-MTV-LUC (Hollenberg and Evans, 55 Cell, 899 
(1988), the disclosure of which is herein incorporated by reference) 
containing two copies of the TRE-palindromic response element described in 
Umesono et al., 336 Nature, 262 (1988), the disclosure of which is herein 
incorporated by reference, was used in transfections for the RARs, and the 
reporter plasmid CRBPIIFKLUC, which contains an RXRE (retinoid X receptor 
response element, as described in Mangelsdorf et al., 66 Cell, 555 (1991), 
the disclosure of which is herein incorporated by reference), was used in 
transfections for the RXRs. Each of these reporter plasmids contains the 
cDNA for firefly luciferase (LUC) under constitutive promoter containing 
the appropriate RAR or RXR response element. As noted above, 
pRS-.beta.-Gal, coding for constitutive expression of E. coli 
13-galactosidase (.beta.-Gal), was included as an internal control for 
evaluation of transfection efficiency and compound toxicity. 
Six hours after transfection, media was removed and the cells were washed 
with phosphate-buffered saline (PBS). Media containing compounds of the 
present invention in concentrations ranging from 10.sup.-12 to 10.sup.-5 M 
were added to the cells. Similarly, the reference compounds all-trans 
retinoic acid (ATRA)(Sigma Chemical), a known RAR selective compound, and 
9-cis retinoic acid (9-cis) (synthesized as described in Heyman et al., 
Cell, 68:397-406 (1992)), a compound with known activity on RXRs, were 
added at similar concentrations to provide a reference point for analysis 
of the activity of the compounds of the present invention. Retinoid purity 
was established as greater than 99% by reverse phase high-performance 
liquid chromatography. Retinoids were dissolved in dimethylsulfoxide for 
use in the transcriptional activation assays. Three to four replicates 
were used for each sample. 
After 40 hours, the cells were washed with PBS, lysed with a Triton 
X-100-based buffer and assayed for LUC and .beta.-Gat activities using a 
luminometer or spectrophotometer, respectively. For each replicate, the 
normalized response (NR) was calculated as: 
EQU LUC response/.beta.-Gal rate 
where .beta.-Gal rate=.beta.Gal.1.times.10.sup.5 /.beta.-Gal incubation 
time. 
The mean and standard error of the mean (SEM) of the NR were calculated. 
Data was plotted as the response of the compound compared to the reference 
compounds over the range of the dose-response curve. For the agonist 
compounds of the present invention, the effective concentration that 
produced 50% of the maximum response (EC.sub.50) was quantified. 
The potency (nM) of selected retinoid compounds of the present invention 
are in Table 1 below. 
TABLE 1 
______________________________________ 
Potency (nM) of selected retinoid compounds of the present invention on 
RAR.alpha.,.beta.,.gamma. and RXR.alpha.,.beta.,.gamma., in comparison to 
the known RAR-active 
retinoid compound all-trans retinoic acid (ATRA) and RXR-active 
retinoid compound 9-cis retinoic acid (9-cis), and in comparison to 
comparative example compounds A, B, C and D. 
RAR.alpha. 
RAR.beta. 
RAR.gamma. 
RXR.alpha. 
RXR.beta. 
RXR.gamma. 
Cmpd. Pot Pot Pot Pot Pot Pot 
No. nM nM nM nM nM nM 
______________________________________ 
9 4 1 &lt;1 na na na 
10 59 23 16 127 48 56 
28 na na na 20 104 50 
39 5 3 3 na na na 
41 3 1 4 2007 266 2183 
43 65 22 16 27 27 19 
44 223 37 33 1443 21 224 
47 na 1 1 na na na 
54 na 130 320 58 28 78 
ATRA 436 78 19 1015 1211 961 
9-cis 220 29 50 195 128 124 
A na 1484 na na na na 
B na na na na na na 
C na na na na na na 
D na na na na na na 
______________________________________ 
na = not active (potency of &gt;10,000 and/or efficacy of &lt;20%) 
As can been seen in Table 1, Compounds 9, 39, 41 and 47 are extremely 
potent RAR active compounds, with Compound 9 displaying sub-nanomolar 
activity on RAR.gamma., and Compound 47 displaying selectivity on 
RAR.beta. and RAR.gamma.. In fact, these Compounds are 10 to over 100 
times more potent than the known RAR active compound ATRA on the RARs. 
Likewise, Compound 28 is a very potent and selective RXR active compound. 
Furthermore, pan-agonist Compounds 10 and 43 display a superior potency 
profile to that of the known RXR active pan-agonist compound 9-cis 
retinoic acid. While efficacy is not reported in Table 1, those compounds 
displaying an efficacy of less than 20 percent are considered to be 
inactive as retinoid activators (potency defined as &gt;10,000), even if the 
compounds display marginal potency. In this regard, except for comparative 
compound A on RAR.beta., the comparative example compounds all displayed 
efficacies of less than 20 percent. 
The retinoid activity of the compounds of the present invention for RAR 
and/or RXR receptors is not exhibited by other known structurally similar 
compounds. As further shown in Table 1, comparative example compounds that 
appear structurally similar to the compounds of the present invention, 
such as (2E,4E,6E)-3-methyl-7-(3,4-dimethoxyphenyl)octa-2,4,6-trienoic 
acid (A) and (2E,4E,6E)-3-methyl-7-(4-methoxyphenyl)octa-2,4,6-trienoic 
acid (B) described in M. J. Aurell, et al., 49 Tetrahedron, 6089 (1993) 
(Scheme 2, compounds d and e), 
(2E,4E,6E)-2-methyl-7-(2,3,6-trimethyl-4-methoxyphenyl)hepta-2,4,6-trienoi 
c acid (C) disclosed in U.S. Pat. No. 4,534,979 (Example 17), and 
(2E,4E,6E)-3-methoxy-7-(4-t-butylphenyl)octa-2,4,6-trienoic acid (D) 
disclosed in U.S. Pat. No. 5,320,833 (Compound 80), have no, or virtually 
no, activity on any of the RARs or RXRs. 
EXAMPLE 24 
In addition to the cotransfection data of Example 15, the binding of 
selected compounds of the present invention to the RAR and RXR receptors 
was also investigated according to the methodology described in M. F., 
Boehm, et al., "Synthesis and Structure-Activity Relationships of Novel 
Retinoid X Receptor Selective Retinoids", 37 J. Med. Chem., 2930 (1994); 
M. F. Boehm, et al., "Synthesis of High Specific Activity .sup.3 H!-9-cis 
Retinoic Acid and Its Application for Identifying Retinoids with Unusual 
Binding Properties", 37 J. Med. Chem., 408 (1994), and E. A. Allegretto, 
et al., "Characterization and Comparison of Hormone-Binding and 
Transactivation Properties of Retinoic Acid and Retinoid X Receptors 
Expressed in Mammalian Cells and Yeast", 268 J. Biol. Chem., 22625 (1993), 
the disclosures of which are herein incorporated by reference. 
Non-specific binding was defined as that binding remaining in the presence 
of 500 nM of the appropriate unlabelled compound. At the end of the 
incubation period, bound from free ligand were separated. The amount of 
bound tritiated retinoids was determined by liquid scintillation counting 
of an aliquot (700 mL) of the supernatant fluid or the hydroxylapatite 
pellet. 
After correcting for non-specific binding, IC.sub.50 values were 
determined. The IC.sub.50 value is defined as the concentration of 
competing ligand needed to reduce specific binding by 50%. The IC.sub.50 
value was determined graphically from a log-logit plot of the data. The 
K.sub.i values were determined by application of the Cheng-Prussof 
equation to the IC.sub.50 values, the labeled ligand concentration and the 
K.sub.d of the labeled ligand. 
The binding activity (Kd in nM) results of selected retinoid compounds of 
present invention, and the reference compounds ATRA, and 9-cis RA, is 
shown in Table 2 below. 
TABLE 2 
______________________________________ 
Binding (Kd in nM) of selected retinoid compounds of the present 
invention on RAR.alpha.,.beta.,.gamma. and RXR.alpha.,.beta.,.gamma. 
proteins in 
comparison to the known RAR-active retinoid compound all-trans 
retinoic acid (ATRA) and RXR-active retinoid compound 
9-cis retinoic acid (9-cis). 
RAR.alpha. 
RAR.beta. 
RAR.gamma. 
RXR.alpha. 
RXR.beta. 
RXR.gamma. 
Bind- Bind- Bind- Bind- Bind- Bind- 
Cmpd. ing K.sub.d 
ing K.sub.d 
ing K.sub.d 
ing K.sub.d 
ing K.sub.d 
ing K.sub.d 
No. (nM) (nM) (nM) (nM) (nM) (nM) 
______________________________________ 
9 1 2 4 270 924 496 
10 59 75 121 4 4 9 
ATRA 15 17 17 53 306 306 
9-cis 93 97 148 8 15 14 
______________________________________ 
As can be seen in Table 2, Compounds 9 and 10 of the present invention show 
equal or superior binding to the known RAR active compound ATRA, and the 
known RXR active compound 9-cis. In comparison, all of the comparative 
example compounds of Example 14 show absolutely no binding on any of the 
retinoid receptors, with the exception of comparative example compound A, 
which shows weak binding of 312 nM on RAR.alpha.. 
EXAMPLE 25 
Yet another recognized measure of the retinoid activity of the compounds of 
the present invention is the ornithine decarboxylase assay, as originally 
described by Verma and Boutwell, 37 Cancer Research, 2196 (1977), the 
disclosure of which is herein incorporated by reference. In Verma & 
Boutwell original work using retinoic acid, it was established that 
ornithine decarboxylase (ODC) activity increased in relation to polyamine 
biosynthesis. In turn, it had previously been established that increases 
in polyamine biosynthesis is correlated with cellular proliferation. Thus, 
if ODC activity could be inhibited, cell hyperproliferation could be 
modulated. Although all causes of increased OCD activity are yet unknown, 
it is known that 12-O-tetradecanoylphorbor-13-acetate (TPA) induces ODC 
activity. Importantly, retinoic acid inhibits this induction of ODC by 
TPA. 
An ODC assay essentially following the procedures set out in 35 Cancer 
Research, 1662 (1975), the disclosure of which is herein incorporated by 
reference, was used to demonstrate the inhibition of TPA induction of ODC 
by the compounds of the present invention. The results of this assay on 
selected Example Compounds, and the reference compounds ATRA and 
(E)-4-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1-propenyl 
!benzoic acid (TTNPB), known RAR active compounds, are shown below in Table 
3. All values are expressed as the concentration of the indicated 
compounds in nM required to inhibit the TPA induction of ODC by 80 
percent, i.e., the IC-80 in nM. 
TABLE 3 
______________________________________ 
Inhibitory concentration required to inhibit 80% of the maximally 
observed TPA induction of ODC (ODC IC.sub.80) in nM for Compounds 
7,8,9 and 10, and reference compounds ATRA and TTNBP. 
Compound ODC IC.sub.80 (nM) 
______________________________________ 
7 4.73 
8 152 
9 0.93 
10 6.62 
ATRA 1.40 
TTNPB 0.09 
______________________________________ 
Compounds 7 and 8, which are the esters of Compounds 9 and 10 respectively, 
have been included to show that such ester analogs exhibit retinoid 
activity. While not being bound to a theory of operation, it is believed 
that such esters may operate as pro-drugs in vivo, possibly due to the 
cleavage of the ester to the active acid form of the compounds of the 
present invention. 
EXAMPLE 26 
The in vitro affect of selected compounds of the present invention on the 
recognized cancer cell lines, RPMI 8226, ME 180 and AML-193, obtained from 
the American Type Culture Collection (ATCC, Rockville, Md.), was 
investigated. 
RPMI 8226 is a human hematopoietic cell line obtained from the peripheral 
blood of a patient with multiple myeloma, and as such is a recognized 
model for multiple myelomas and related malignancies. Y. Matsuoka, G. E. 
Moore, Y. Yagi and D. Pressman, "Production of free light chains of 
immunoglobulin by a hematopoietic cell line derived from a patient with 
multiple myeloma", 125 Proc. Soc. Exp. Biol. Med., 1246 (1967), the 
disclosure of which is herein incorporated by reference. The cells 
resemble the lymphoblastoid cells of other human lymphocyte cell lines and 
secretes .lambda.-type light chains of immunoglobulin. RPMI 8226 cells 
were grown in RPMI medium (Gibco) supplemented with 10% fetal bovine 
serum, glutamine and antibiotics. The cells were maintained as suspension 
cultures grown at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2 
in air. The cells were diluted to a concentration of 1.times.10.sup.5 /mL 
twice a week. 
ME 180 is a human epidermoid carcinoma cell line derived from the cervix, 
and as such is a recognized model for squamous cell carcinomas and related 
malignancies. J. A. Sykes, J. Whitescarver, P. Jerustrom, J. F. Nolan and 
P. Byatt, "Some properties of a new epithelial cell line of human origin", 
45 MH-Adenoviridae J. Natl. Cancer Inst., 107 (1970), the disclosure of 
which is herein incorporated by reference. The tumor was a highly invasive 
squamous cell carcinoma with irregular cell clusters and no significant 
keratinization. ME 180 cells were grown and maintained in McCoy's 5a 
medium (Gibco) supplemented with 10% fetal bovine serum, glutamine and 
antibiotics. The cells were maintained as monolayer cultures grown at 
37.degree.C. in a humidified atmosphere of 5% CO.sub.2 in air. 
The AML-193 cell line was established from the blast cells of a patient 
with leukemia and was classified as M5 Acute Monocytic Leukemia, and as 
such is a recognized model for leukemias and related malignancies. G. 
Royera, et al., 139 J. Immunol., 3348 (1987), the disclosure of which is 
herein incorporated by reference. Over 75% of these cells are positive by 
immunofluorescence for the myelomonocytic antigen CS15. The cells were 
grown in Iscove's modified Dulbeccos's medium with 5 .mu.g/mL 
transferring, 5 .mu.g/mL insulin and 2 ng/mL rh GM-CSF. CSF. The cells 
were maintained as suspension cultures grown at 37.degree. C. in a 
humidified atmosphere of 5% CO.sub.2 in air. The cells were diluted to a 
concentration of 1.times.10.sup.5 /mL twice a week. 
Incorporation of .sup.3 H-Thymidine 
Measurement of the level of radiolabeled thymidine incorporated into the 
above-identified cell lines provides a direct measurement of the 
antiproliferative properties of the compounds of the present invention. 
The method used for determination of the incorporation of radiolabeled 
thymidine was adapted from the procedure described by S. Shrivastav et 
al., "An in vitro assay procedure to test chemotherapeutic drugs on cells 
from human solid tumors", 40 Cancer Res., 4438 (1980), the disclosure of 
which is herein incorporated by reference. RPMI 8226 or AML-193 cells were 
plated in a 96 well round bottom microtiter plate (Costar) at a density of 
1,000 cells/well. To appropriate wells, retinoid test compounds were added 
at the final concentrations indicated for a final volume of 150 
.mu.L/well. The plates were incubated for 96 hours at 37.degree. C. in a 
humidified atmosphere of 5% CO.sub.2 in air. Subsequently, 1 .mu.Ci of 
5'-.sup.3 H!-thymidine (Amersham, U.K, 43 Ci/mmol specific activity) in 
25 .mu.L culture medium was added to each well and the cells were 
incubated for an additional six hours. The cultures were further processed 
as described below. 
ME 180 cells, harvested by trypsinization were plated in a 96 well flat 
bottom microtiter plate (Costar) at a density of 2,000 cells/well. The 
cultures were treated as described above for RPMI 8226 with the following 
exceptions. After incubation, the supernatant was carefully removed, and 
the cells were washed with a 0.5 mM solution of thymidine in phosphate 
buffered saline. ME 180 cells were briefly treated with 50 .mu.L of 2.5% 
trypsin to dislodge the cells from the plate. Both cell lines were then 
processed as follows: the cellular DNA was precipitated with 10% 
trichloroacetic acid onto glass fiber filter mats using a SKATRON 
multi-well cell harvester (Skatron Instruments, Sterling Va.). 
Radioactivity incorporated into DNA, as a direct measurement of cell 
growth, was measured by liquid scintillation counting. The mean 
disintegrations per minute of incorporated thymidine from triplicate wells 
was determined. The IC.sub.50 (nM concentration required to inhibit 50% of 
the maximally observed incorporation of thymidine) for Compounds 9 and 10 
of the present invention, and reference compounds ATRA and TTNBP are shown 
below in Tables 4, 5 and 6 for the cell lines RPMI 8226, ME 180 and AML- 
193 respectively. 
Viability 
Selected compounds of the present invention were also measured to determine 
their cytotoxicity on the above-identified cell lines. The procedure used 
was identical, with only slight modifications, to the assay described in 
T. Mosmann, "Rapid colorimetric assay for cellular growth and survival: 
application to proliferation and cytotoxicity assays", 65 J. Immnunol. 
Meth., 55 (1983), the disclosure of which is herein incorporated by 
reference. RPMI 8226 or AML-193 cells were plated in a 96 well round 
bottom microtiter plate (Costar) at a density of 1,000 cells/well. To 
appropriate wells, retinoid test compounds were added at the final 
concentrations indicated for a final volume of 150 .mu.L/well. The plates 
were incubated for 96 hours at 37.degree. C. in a humidified atmosphere of 
5% CO.sub.2 in air. Subsequently, 15 .mu.L of a filter sterilized 
tetrazolium dye in phosphate buffered saline (Promega, Madison, Wis.) was 
added to each well and the cells were incubated for an additional four 
hours. Subsequent manipulations of the cultures were as described below. 
ME 180 cells, harvested by trypsinization were plated in a 96 well flat 
bottom microtiter plate (Costar) at a density of 2,000 cells/well. The 
cultures were treated as described above for RPMI 8226. 
After the four hours incubation, 100 .mu.L of a solubilization/stop 
solution was added to each well (Promega, Madison, Wis.). The plates were 
allowed to stand overnight at 37.degree. C. in the humidified atmosphere. 
The absorbance at 570-600 nm wavelength was recorded for each well using a 
Biomek ELISA plate reader (Beckman Instruments). The IC.sub.50 (nM 
concentration required to inhibit 50% of the mitochondrial function, and 
ultimately, the viability of the cells) for Compounds 9 and 10 of the 
present invention, and reference compounds ATRA and TTNBP are also shown 
below in Table 4, 5 and 6 for the cell lines RPMI 8226, ME 180 and AML-193 
respectively. 
TABLE 4 
______________________________________ 
Inhibitory concentration required to inhibit 50% of the maximally 
observed radiolabeled thymidine (TdR IC.sub.50) in nM, and 
inhibitory concentration required to inhibit 50% of the mitochondrial 
function (MTS IC.sub.50) in nM, for Compounds 9 and 10, and 
reference compounds ATRA and TTNBP on the RPMI 8226 cell line. 
TdR IC.sub.50 
MTS IC.sub.50 
Compound (nM) (nM) 
______________________________________ 
9 0.3 253 
10 60 570 
ATRA 102 756 
TTNPB 0.2 10 
______________________________________ 
TABLE 5 
______________________________________ 
Inhibitory concentration required to inhibit 50% of the maximally 
observed radiolabeled thymidine (TdR IC.sub.50) in nM, and 
inhibitory concentration required to inhibit 50% of the 
mitochondrial function (MTS IC.sub.50) in nM, for Compounds 9 and 10, 
and reference compounds ATRA and TTNBP on the 
ME 180 cell line. 
TdR IC.sub.50 
MTS IC.sub.50 
Compound (nM) (nM) 
______________________________________ 
9 0.1 1.3 
10 62 370 
ATRA 253 890 
TTNPB 0.4 187 
______________________________________ 
TABLE 6 
______________________________________ 
Inhibitory concentration required to inhibit 50% of the maximally 
observed radiolabeled thymidine (TdR IC.sub.50) in nM, and 
inhibitory concentration required to inhibit 50% of the mitochondrial 
function (MTS IC.sub.50) in nM, for Compounds 9 and 10, and 
reference compounds ATRA and TTNBP on the AML-193 cell line. 
TdR IC.sub.50 
MTS IC.sub.50 
Compound (nM) (nM) 
______________________________________ 
9 0.1 1000 
10 0.01 1000 
ATRA 197 1000 
TTNPB 0.1 1000 
______________________________________ 
EXAMPLE 27 
The following examples provide illustrative pharmacological composition 
formulations: Hard gelatin capsules are prepared using the following 
ingredients: 
______________________________________ 
Quantity 
(mg/capsule) 
______________________________________ 
Compound 9 140 
Starch, dried 100 
Magnesium stearate 10 
Total 250 mg 
______________________________________ 
The above ingredients are mixed and filled into hard gelatin capsules in 
250 mg quantities. 
A tablet is prepared using the ingredients below: 
______________________________________ 
Quantity 
(mg/tablet) 
______________________________________ 
Compound 9 140 
Cellulose, microcrystalline 
200 
Silicon dioxide, fumed 
10 
Stearic acid 10 
Total 360 mg 
______________________________________ 
The components are blended and compressed to form tablets each weighing 360 
mg. 
Tablets, each containing 60 mg of active ingredient, are made as follows: 
______________________________________ 
Quantity 
(mg/tablet) 
______________________________________ 
Compound 9 60 
Starch 45 
Cellulose, microcrystalline 
35 
Polyvinylpyrrolidone (PVP) 
(as 10% solution in water) 
4 
Sodium carboxymethyl starch (SCMS) 
4.5 
Magnesium stearate 0.5 
Talc 1.0 
Total 150 mg 
______________________________________ 
The active ingredient, starch, and cellulose are passed through a No. 45 
mesh U.S. sieve and mixed thoroughly. The solution of PVP is mixed with 
the resultant powders, which are then passed through a No. 14 mesh U.S. 
sieve. The granules so produced are dried at 50.degree. C. and passed 
through a No. 18 mesh U.S. sieve. The SCMS, magnesium stearate, and talc, 
previously passed through a No. 60 mesh U.S. sieve, are then added to the 
granules which, after mixing, are compressed on a tablet machine to yield 
tablets each weighing 150 mg. 
Suppositories, each containing 225 mg of active ingredient, may be made as 
follows: 
______________________________________ 
Compound 9 225 mg 
Saturated fatty acid glycerides 
2,000 mg 
Total 2,225 mg 
______________________________________ 
The active ingredient is passed through a No. 60 mesh U.S. sieve and 
suspended in the saturated fatty acid glycerides previously melted using 
the minimum heat necessary. The mixture is then poured into a suppository 
mold of normal 2g capacity and allowed to cool. 
An intravenous formulation may be prepared as follows: 
______________________________________ 
Compound 9 100 mg 
Isotonic saline 
1,000 ml 
Glycerol 100 ml 
______________________________________ 
The compound is dissolved in the glycerol and then the solution is slowly 
diluted with isotonic saline. The solution of the above ingredients is 
then administered intravenously at a rate of 1 ml per minute to a patient. 
While in accordance with the patent statutes, description of the preferred 
embodiments and processing conditions have been provided, the scope of the 
invention is not to be limited thereto or thereby. Various modifications 
and alterations of the present invention will be apparent to those skilled 
in the art without departing from the scope and spirit of the present 
invention. 
Consequently, for an understanding of the scope of the present invention, 
reference is made to the following claims.