CA1330346C

CA1330346C - Caffeic acid esters and methods of preparing and using same

Info

Publication number: CA1330346C
Application number: CA 573682
Authority: CA
Inventors: Koji Nakanishi; Eugene M. Oltz; Dezider Grunberger
Original assignee: Columbia University in the City of New York
Current assignee: Columbia University in the City of New York
Priority date: 1987-08-04
Filing date: 1988-08-03
Publication date: 1994-06-21
Anticipated expiration: 2011-06-21
Also published as: WO1989000851A1; AU2300588A

Abstract

CAFFEIC ACID ESTERS AND METHODS OF PREPARING AND USING
SAME
Abstract of the Disclosure The invention relates to a compound having the structure:

wherein R comprises a phenethyl, butyl, hexyl or ethyl group.
The invention also relates to methods of isolating and producing the compound, methods of treating inflammation in a subject and methods of substantially inhibiting the growth of transformed cells in a population of normal and transformed cells.

Description

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Docke~ 25048/JPW/jjs CAF~EIC ACID ESTERS AND METH~DS ~F PREPARING AND USING
SAME____________________ _____________________________ The invention described herein was made in the course cf work under Grant Nos. CA 2111, CA 31696, and AI
10187 ~rom the National Insti~ute for ~ealth. The UOS.
Government ~as certain rights in this invention.

Backqround_o~_5he_l~v~ntiQ~
Throughout this application various publications are referenced and citations are provided in parentheses for them. The disclosures of these publications in their entireties are of interest in this application in order to fully describe the state of the art to which this invention pertains.

Propolis, a popular R~olk medicine" purported to have therapeutical benefit, is a brownish mass produced by honeybees in their hives. A significant number o~ so-called "folk medicines" have with~tood scientific scrutiny, with many of their purported therapeutic benefits being attributable to distinct chemical entities. Such naturally derived compounds often produce fewer and less serious side effects as compared to analogous man-made pharmaceuticals; unfortunately~
most are chemically complex, not lending themselves ~o economically feasible syntheses. Propolis, is alleged to exhibit a broad spectrLm of activitie~O It is marketed in health stores as a natural antibiotic (F.B. ~ells, AmO Bee J. 116, 512 (19763 (a review of the antimicrobial effects of propoli~)); aqueous extracts are scld in Europe as a cough syrup and sore throat remedy; compacts prepared ~rom it are used to ~ ' ~ '';

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treat skin inflammations (Apimondia, A Remarkable Hive Product: Propolis, Bucharest, 1978); ingestion of raw propolis is reputed to clear sinuses and treat viral infections (E.L. Chisalberti, Bee World 60~ 59 (1979)~;
and propolis has been used to arrest the growth of tumors. Propolis is a conglomeration of compounds mostly derived from plant sources, and is rich in waxes, resins, oils~ pollen (J. Cizmarik, I. Matel, Experentia 15, 713 (1970)), flavonoids (V~S. 8ankova, S.S. Popov, N.L. Marekov, J. Na~. Prod. ~, 471 (1983)), and polyphenolic acids (J~ Cizmarik, s~
Together these substances provide properties which permit its natural use by the honeybee as a hive cement or glue (G. Toth, ~m. Bee J. 1~, 337 (1985)).

Some of the observed biological activities may be traced to identified chemical consti~uents such as caffeic acid (J. Cizmarik, ~ ), which is a reported antimicrobial and anti-inflammatory agent (V~S.
Bankova, ~ a). However, it is difficult to ~eneralize such relationships since the composition of propolis varies with the flora of a given area, the time of collection and contaminants frcm collection (G.
Toth, ~ ). Ethyl ether extracts of propolis have previously been demonstrated to be cytostatic to RB and HeLa cell lines (Bo Hladon et al., Arzneim-Forsch./
Drug Res. 3Q, 1847 (1980)), however the components responsible for this interesting activity were not defined.
-The invention described herein concerns a compound present in propolis which is at least partially ::~:
responsible for i~s reported cytostatic propertiesO It represents the most active component as judged by the assay employed in its study (cytostatic towards Ltk-:~

'A~ . ., . - .. . . .... , .. .. . . . .. .. . " .... . . . . .

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cells). The compound has been identified as caffeic acid phenethyl ester (CAPE) and a one-step synthesis method has been developed which is amenable to large scale preparation. Through investigations of CAPE's cytostatic properties uncovered several differential effects. For example, human tumor cell lines displayed a significantly greater sensitivity to the action of CAPE than analogous normal lines. Additionally, CAP~
has been found to possess anti-inflammatory activity.
Phenethyl alcohol and caffeic acid, the most obvious metabolic products of CAPE, displayed none of the aforementioned activities.

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~ ~3~3~g ~umma~_Q~_~;h~ a~ian The present invention concerns a compound having the structure:

H~ o~ R ' . ,.

HO

wherein R comprises a phenethyl, butyl~ hexyl, or ethyl group.

The invention also provides a method of producing the compound which comprises contacting caffeic acid with an alcohol having the formula R-OH, wherein R comprises a phenethyl, butyl, hexyl or ethyl group, so as to produce the compound and recovering the compound so produced. Another aspect of the inven~ion concerns a method of producing the compound from propolis which comprises contacting the propolis with a solvent so as to form an extract comprising the compound and treating the extract so as to recover the compound.

Additionally~ th~ present invention provides a method of treating inflammation in a subject which comprises administering to the subject an effective anti~
inflammatory amount of the compound so as to suppress the inflammation. Another embodimen~ of the invention provides a method for substan~ially inhibi~ing the growth of transformed cells without substantially inhibiting the growth of normal cells whicb comprisès treating a populatio~ of cells which include both the transformed and normal cells with an effective -~` 133~

inhibiting amount of the compound so as to substantially inhibit the growth of the transformed cells.

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D-escr pt'on_of_the_Fiqures Fig. l. The effect of increasing concentrations of CAPE on various cultured cell lines. Referred to the text for a description of each cell type and experimental details.

Fig. 2. Effect to CAPE on the grcwth of CREF (normal rat) and wt3A (adenovirus transformed CREF) cells.
Fig. 3. Effect of CAPE on the rate of division (as measured by incorporation of [3~]thymidine) of (a) human MCF-7 breast carcinoma, (b) human SK-MEL-28 melanoma cells and (c) human SR-~EL-170 melanoma cells.
Cells were maintained in Eagle's minimal essential medium (ME~) with Earle's salts and 10% fetal bovine serum. The cells were seeded in the same medium in tissue culture cluster plates (96 flat ~ottom wells) at 103 cells/well. Twenty-four hours later the cultures were washed and different con oe ntrations of CAPE were added to each well in triplica~e. Labelling of the cells was accomplished by incubating with 0.5 ~ Ci [3Hlthymidine for 5 hours. For further details referred to M. Eisinger, 0. Marko, S.-I. Ogata, L.J.
Old, Science 2~2, 984 (1985).

Fig. 4 Effect of CAPE on CaI stimulated release of 13~1 arachidonic acid.

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p~ailed-De~-cri~2t-lo-n--f -the-In~tio-n This invention conceens a compound having the structure:

HO ~ ,R

HO ~ :~
, ',~
wherein R is an alkyl or arylalkyl group.

Preferably R is a phenethyl, butyl, hexyl or ethyl :~ ~:
group. Especially useful are compounds having the ; ~ -.
structure:

~ ~ ~

The invention also provides a method of producing the compound which comprises contacting caffeic acid with alcohol having the formula R OH, wherein R is the same as previously defined, so as to produce the compound :
and recovering the compound so produced. In : ; .
embodiments where R is a phenethyl group, the method of producing the compound comprises contacting caffeic acid with phenetbyl alcohol~ The contacting may be effected in an organic solvent, such as benzene. The contacting also may be effected in the presence of a ~
~.

~ 33~3~6 catalyst, such as p-toluene sulfonic acid. In the most preferred embodiment, the contacting i5 effected by adding the caffeic acid, phenethyl alsohol and p-toluene sul~onic acid to benzene~ producing a suspension and treating the suspension so as to produce the compound. The contacting is especially effective when performed at a temperature of abou~ 100C.
Recovering may be effected by extraction7 filtration, evaporation or recrystallization. The invention also provides a method for producing the compound from propolis which comprises contacting the propolis with a solvent so as to form an extract comprising the compound and treating the extract to recover the compound. In the practice of this embodiment9 treating may comprise filtration, evaporation or extraction.
Preferabiy, treating comprises extracting the propolis with hexane, recovering the resulting hexane extract then extrac~ing the hexane extract with toluene, recovering the resulting toluene extract, then extracting the toluene extract with ethyl acetate and recovering ~he resulting e~hyl acetate extract.
Treating may further comprise purifying the recovered ethyl acetate extraet by means such as of thin layer chromatography or reverse phase high pressure liquid chromatography or a combination of both. `

Another aspect of the invention concerns a method for treating inflammation in a subject which comprises administering to the subject an effective anti=
inflammatory amount of the compound so as to suppress the inflammation. Especially preferred i8 a method for treating inflammation in the subject which comprises administering to the subject an efective an~i inflammatory amount of the compound having the structure:

i ~ ~3~
g o ,~ '`'.".'"'-HO

~O ~ `~

Additionally, the invPntion provides a method or -~
substantially inhibiting the growth of transformed cells without substantially inhibiting the growth of normal cells which comprises treating a popu}a~ion of cells which include both transformed and normal cells `:
with an effective inhibiting amount of the compound so~ - .
as to substantially inhikit the growth of the ~:
transformed cells. Especially preferred is a method :
which comprises treating the population of cells with an effective inhibiting amount of the compound having the structure~

O ~
HO ~ o ~ ~ ~`

HO

In the practice of the present invention, the transformed cells may comprise carcinoma or melanoma cells~ In the preferred embodiments, the subject is a::
human and the transformed cells are human carcinoma or`~:
melanoma cells, such as human ~reast carcinoma cells,~-`
colon carcinoma cells, renal carcinoma cells, or melanoma cells SR-MEL-28 or SK-MEL-170. :

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This invention is illustrated in the Experimental Discussion and Experimental Detail sections which follow. These sections are set forth to aid in an understanding of the invention but are not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow -thereafter. ~.

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E~erim~ us~i~n Propolis was obtained from Mr Chaim Ralman, Bee Farm-Honey, Israel, and was collected fram within hives located on the Carmen Mountains~ It was received in the form of hard, br~wn lumps tapproximately 2cm in diameter) which were chopped, extracted with 80S
Et3H/H2~ (1.51, 2 d), suction ~iltered and evaporated (i~ vacu~) to yield a golden brown ~olid~ This extract displayed cytostatic ac~ivity in Ltk cells at ~g/ml. The EtOH extract was dissolved in 80%
MeCH/H20 (400 ml) and subjected to a ~eries of sequential extrac~ions with hexane (6 x 80ml), toluene (4 x 80ml), and E OAc (4 x 100ml). All oryanic layers were dried, evaporated and submi~ted for Ltk- testing (along with the residue from the aqueous layer). The EtOAc extract exhibited at leas~ twice the cytostatic activity as the other fractions (100% inhibition at below 65 ~g/ml) . Subsequent purif ications of the ~UAC
extract by preparative TLC (7% i-Pr~/o~2C12 then 4% i-PrOH/CH2C~ ) yielded two increasingly active ~ra~tions,with the latter exhibiting 100~ Ltk- inhibition at ~ g/ml.

Rever~ed phase BPLC separation of ~his latter fraction (Column: IBM-C 18, 10 x 250mm, 5 ~1; MevH/MeCN/T~F/~ ~ -25:35:3.5:36.5; lOBml/min; 213 nm detection) yielded a pure compound (retention time = 16.8 min; TLC (Si~2):

4% i PrOH/C~2Cl2~ Rf=0.25, 366 nm illumination - blue fluorescence) which displayed the following Ltk-cytostatic activities (% inhibition in parentheses) (these value are semiquantitative, as only minu~e quantities of the natural compound were available; more quantitative values, acquired with synthetic CAPE~ are provided in Figure 1) - 10 ~Ig/ml (20%)1 20 llg/ml ~80~)~
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30 ~Ig~ml ~95~). The chemical structure of this compound was spectroscvpioallly determined to be cafeic acid phenethyl ester (CAPE), and displayed the following characteristics: W (Me~H): 325 nm ~
16,200), 300 (shr 12,500), 245 (9,700) 9 235 ~9,700); IR
(KBr): 3490 cm~ H 's), 1585 (c=a); HR-MS ~EI):
C17H16~4~ m/z 284.1080, calcd 28401049 lM+~, major fragments - 180, 163, 104, 91, 77. 1 H nmr data (250 MHz, acetone - d6): ~ 8~4-8.2 (brs, 2H, O~'s~, 7.52 (d, J = 15.9 Hz, lH, H7), 7.31 - 7015 (m, 5H; ~13 17)~ 7015 (d, J=2.1, lH, H2), 7.03 (dd, J= 8.2, 2.1, lH, H6), 6.86 (d, J =8.2, lH, ~5), 6026 (d, J = 15.9, lH, H8), 4.34 (t, J = 7~0, 2H, H10~, 2~98 ~t, J - 7.0, 2~t Hl~ 3C nmr (62.89 M~z, CDC13~: ~ 167.8 (s), 146.5 (s~, 145.2 (d), 144..0 (s), 137.8 (s), 12809 ~d), 12805 (d), 127~6 (s), 126.6 (d), 122.5 (d), 115.5 (2d's), 114.5 (d), 65.2 (t), 35.3 (t).

Direct acid-catalyzed (p-toluene sul~onic acidj esterification of caffeic acid (CA) wi~h phenethyl alcohol ~molar ratios 1:15) in benzene (refluxing, 3-4 days, water removed by a Dean-Stark trap), instead of leading to self~condensation of CA, proved ~o be the most efficient synthetic route to CAPE. Prior to the success of direct esterification, several attempts to prepare CAPE via conventional protection/deprotection method were carried out with inferior results.
Following work-upr excess phenethyl alcohol was removed by Kugelrohr distillation (60C, less than OOlmm ~g) and pure CAPE. was obtained as needles by recrystallization (either benze~e or H23, 40~ yield, mp = 126-128C). Chromato~raphic, ~pectroscopic and cytostatic properties of the naturai and synthe~ic CAPE
were identical.
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Cytostatic activities of syn~hetic CAPE were tested by observing its effect on ~he growth of a number of different cell types in culture. Cells were cultured in Dulbecco's modi~ied Eagle's medium supplemented with 5-10% calf serum and maintained at 37C in 5% C~2.
Approximately 3-4 x 105 cells were plated in ~Omm Petri dishes~ Concentrated stock solutions of CAPE, C~ and phenethyl alcohol (hydrolysis products of CAPE~ were prepared in Et~H. After 24 hours, the cells were supplied with fresh medium spiked with various amounts of these solutions or correspondin~ amounts of Et~H
(control). Caffeic acid, phenethyl alcohol and ethanol were never ob~erved to significantly affect the growth of any cell line tested. Approximately 48 hours subsequent to treatment, the cells on individual plates were counted by Coulter counter or ~ixed and stained with Giesm~. Results from the various cell lines are summarized in Figure 1. Mouse cells (C3H lOTl/2 and Ltk-) appeared most sensitive, with concentrations as low as 2.5 ~g/ml CAPE effec~ively blocking the growth 20 Of 10T1/2 cells. Interestingly, benzo(a)pyrene transformed lOTl/2 cells exhibi~ed an increased resistance to CAPE's action, requiring up to 20 ~g~ml CAPE for an 80~ growth inhibition. Normal Rat 6 cells and those transformed by T24 oncogene were both less 25 sensitive than the murine lines~ Similarly, the growth of two monkey cell lines, CYl and Vero, suffered severe inhibition only at concentrations o CAPE greater than 10 ~g/ml.

The differential effect of CAPE on the growth of nor~al and transformed cells was further investigated with a recently isolated clonPd cell line of Fischer rat embryo fibroblasts (CREF) and its counterFart, transformed by adenovirus serotype 5 (wt3A) ~P.B.
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Pisher, L.E. ~abiss, I.B. Weinstein, H.S. Ginsberg, Proc. Natl. Acad. Sci. USA Z~, 3527 (1982)) The transformed cells were identified by their altered morphologies and the presence of adenovirus DNA
sequences in their genomes (I~). The effects of various concentrations of CAPE on the growth of CREF
and wt3A lines after 72h are sunmari2ed in Figure 2.
At concentrations of CAPE as high as 8 ~g/ml, approximately 75% of the CREF cells remained unaffected, yet under the same conditions, growth of the wt3A cells was inhibited by nearly 90~. Hence, the presence of CAPE elicited a marked differential effect on the growth of these two rat cell lines which also displayed important difference in their biological properties. Unlike CREF cells, morphologically transformed cells exhibited higher saturation densities and were capable of anchorage-independent growth.
Because the cytostatic mechanism of CAPE is not understood at present, any explanation for the differential effects of this inhibitor upon the growth of normal CREF an~ transformed wt3A cells remains purely speculative.

The effect of CAPE on human cancer cells was tested by measuring incorporation of [3H]thymidine into the DNA
of human breast carcinoma ~MCF-7) and melanoma (SK-MEL-28 and S~-MEL-170) cell lines in culture (M. Eisinger, O. Marko, S.-I~ Ogata, L.J. Old, Science ~2, 984 (1985)). Figure 3a reveals that 5 ~g/ml CAPE inhibits incorporation of [3H]T into the DNA of human breast carcinoma MCF-7 by approximately 50% and is completely blocked by concentrations of 10 ~ g/ml. Even more dramatic effects were observed with ~he two melanoma lines, SK-MEL-28 and SR-MEL-170. Figure 3b and Figure 3c illustrate the effect of different con oentrations of ~33~3~

CAPE on the incorporation of ~3H]T into SK-MEL-28 and SK-MEL-170 cells, respectively. At 5 ~g CAPE/ml the cells displayed minimal incorporation and wer completely inhibited at 10 ~g/ml~ Similar inhibitions were observed for HT29 colon and renal carcino~a lines (not shown). The effect of CAPE on normal 1434 fibroblasts and melanocytes was significantly less.
Incorporation of [3H]T into these normal cells was inhibited by only 50~ at concentration of CAPE up to ten times greater (50 ~ g/ml, data not shown). These differential effects were reminiscent of those observed with the normal CREF and transformed wt3A rat cells.

The ready accessibility of analogs and labelled versions of CAPE will simplify further investigations into its mode of action, and may lead to an understanding of the observed differential effects on a molecular level. ~urthermore, such studies with CAPE
and other cytostatic compounds may provide a clearer insight into the molecular events responsible for the dissimilar biological properties exhibited by transformed and normal cells. Because the cytostatic action of CAPE is more dramatic on transformed cells, one may reasonably assume that it is at least partly responsible for the claimed carcinostatic properties of propolis.

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~e~ en~aL_~e~aiLs Effect of CAPE on cell growtho Cell cultur~: Mouse Ltk- and lOTl/2 cells, monkey CVI
and Vero cells, a clone of rat embryo fibroblast cell line - CREF and a wild type adenovirus transformed C~EF
cell line - wt3A were grown in Dulbecco's modified Eagle's medium supplemented with 5-10~ calf serum at 37C with 5% C~ . About 3-4x105 cells were plated in 60mm dishes. ~fter 2~ hours ~he cells were fed wi~h fresh medium containing various amounts of CAPE.
concentrated stock solution (10 mg/ml of CAPE) was prepared in ethanol. The control plates were simultaneously treated with equal volume of ethanol.
After 48 hours following the treatment the cells in individual plates were either counted by Coulter counter or fixed and stained with Giemsa for estima~ion of relative toxicity of CAPE.

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It is evident that the mouse cells (lOTl/2 and Ltk~) are the most sensitive to CAPE. At 5 ~ g/ml conrentration there is 95% inhibition o the growth of lOTl/2 cells. The monkey cells (CVI and Vero) are less sensitive. ~nly at 20 ~ g/ml is the growth of these cells inhibited almost completely. Interesting is the sensitivity of rat cells to the action of CAPE. ~ormal CREF cell line at 10 ~q/ml is inhibited only by 30%.
However, the adenovirus transformed cell line w~3A is highly inhibited at 5.0 ~g/ml (82%) already and at ~g/ml practically there is a complete inhibition of the growth of these transformed cells~

Effect o~ CAPE on melanocyte and melanoma cells in culture:

Foreskin melanocytes were main~ained in EarIe's minimal essential medium (MEM) with Eagle's salts: 0.01 mM
nonessential amino acids, 2 m~ L-ylutamine containing penicillin (100 unit/ml), streptomycin (0.1 mg/ml) and Fungizone (0.25 ~ g/ml); 10 percent fetal bovine serum (cMEM), 12-0-tetradecanoyl phorbol 12-acetate (TPA) (10 ng/ml) and cholera toxin 10-3M for 15 passages, according to M. Eisinger, O. Marko, S~ gata, L.J.
Old, Science ~2~, 984 ~1985). They were then seeded in the same medium in tissue culture cluster plates (96 flat bottom wells) at 5x103 per well.

Twenty-four hours later, the cultures were washed three ~ -timeS with ME~ (not containing TPA and cholera toxin~
and incubated for 4 hours. Cells were then fed at 3-day intervals with cMEM containing CAPE in con oentrations as indicated. Melanoma cell lines SR~
MEL-28 and SK-MEL-170 in passage 40 were seeded at lx103 per well under the same conditions as the i,~, : .... :: .. - -,. :. : , - j ( 3 '~ ~

melanocytes bu~ without TPA and cholera toxin.
Labeling of melanocytes and melanomas was performed with [3H]thymidine (lO ~Ci/ml) for 6 hours at indicated time periods after the initial addition of CAPE~ After labeling, the cells were washed three times with phosphate-buffered saline (PBS), dislodged from the wells by trypsin-EDTA solution, and counted in Hydrofluor with a scintillation counter. -: :~

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Cells for the release of arachidonic acid:

Human keratinocytes were isolated and grawn according to the system of Elinger et al., PNAS, ~, 5340 (1979), in 12 well pla~es in minimal essential medium (KM) with Earle's salts (Gibco~ plus non-essential amino a~ids (0.01 mM), ~mM L-glutamine, hydrocortisone (0.4 ~g/ml), penicillin (100 U/ml), strep~omycin (0.1 mg/ml) and fungizone (2.5 ~g/ml). The grcwth medium included 10 heat-inactivated fetal calf serum (FCS) ~RM ~ lO)o Cells were isolated from normal human breast skin obtained from surgical specimensO The skin was cut into small discs and exposed for a 12-14 hour period to a 0.25% ~rypsin solution at 4C. The tryp~ic action was stopped by ~he addition of 20% FCSo The epidermis from each disc was peeled from the dermis and the epidermal discs were pooled. A single cell suspension of epidermal cells was produced by vigorous trituation :`:
in trypsin: EDTA solution (0.05~:0.02~). The trypsins `:~
EDTA solution was inactivated with serum. Cells were centrifuged, the trypsin: EDTA was removed and the cells were countedO After dilution with grawth medium :
(pH 7.4-7.6) cells were seeded at appeoprlate densities ~1.2-1.4xlO5/cm2~. Cells were fed twice weekly and maintained at 37C in moisturized room air with 5% C~
Cells were utilized in primary passage.
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Assay for the release of arachidonic acid (AA):

Cells werè grcwn in plastic dishes until approximately 90~ confluency (9-14 days). At a density o~ 2-3 x 105/cm2 cells were pre-labelled with ~3H~AA 2 ~Ci/ml in KM+10. The cells were allowed to incorporate the radiolabel for a 20-24 hour period under the incubation conditions outlined above. After the labelling period, the labelling media was removed and the cells were washed three time~ with assay medium. The pre-labelled cells were then treated with various concentrations of either calcium ionophore (A 23187) an~ CAPE or only CAPE in triplicate wells. Media containing equivalent amounts of solvents without CAPE served as control.
The treated cells were incubated at 37C for 2 hours.
The media were then removed, centrifuged at 12000 r.p.m for 5 minutes and an aliquot was assayed by liquid scintillation to determine the total c.p.m. released.
All assays were done with triplicate dishes for experimental and control conditions.

~hen human keratinocytes were pre labelled with [3H~AA
they incorporated 60-80~ of the label fr~m the medium during 20-24 hour period. Figure 4 shows that calcium ionophore stimulate the release of [3H]AA. However, in the presence of 10 ~g/ml CAPE there was almost no stimulation of the release of AA. Since inhibitors of arachidonic acid deacylation and metabolism applied to mouse skin inhibit the inflammatory response to phorbol ester, it is assumed that CAPE has similar anti-inflammatory effect.

Claims

1. A purified compound having the structure:

2. A method of producing the compound of claim 1 which comprises contacting caffeic acid with a phenethyl alcohol so as to produce the compound and recovering the compound so produced.

3. A method of claim 2, wherein the contacting is effected in an organic solvent.

4. A method of claim 3, wherein the solvent is benzene.

5. A method of claim 2, wherein contacting is effected in the presence of a catalyst.

6. A method of claim 5, wherein the catalyst is p-toluene sulfonic acid.

7. A method of claim 6, wherein contacting is effected by adding the caffeic acid, phenethyl alcohol, and p-toluene sulfonic acid to benzene to produce a suspension, and treating the suspension so as to produce the compound.

8. A method of claim 7, wherein the contacting is effected at a temperature of about 100°C.

9. A method of claim 7, wherein the recovering comprises extraction, filtration, evaporation or recrystallization.

10. A method of producing the compound of claim 1 from propolis which comprises contacting the propolis with a solvent so as to form an extract comprising the compound and treating the extract so as to recover the compound.

11. A method of claim 10, wherein the treating comprises filtration, evaporation or extraction.

12. A method of claim 11, wherein the treating comprises extracting the propolis with hexane, recovering the resulting hexane extract, then extracting the hexane extract with toluene, recovering the resulting toluene extract, and then extracting the toluene extract with ethyl acetate and recovering the resulting ethyl acetate extract.

13. A method of claim 12 further comprising purifying the recovered ethyl acetate extract.

14. A method of claim 13, wherein the purifying of the ethyl acetate extract comprises thin layer chromatography.

15. A method of claim 14, wherein the purifying further comprises reversed phase high pressure liquid chromatography.

16. The use of the compound of claim 1 for treating inflammation in a subject, or for preparing a medicament therefor.

17. The use of the compound of claim 1 for substantially inhibiting the growth of transformed cells without substantially inhibiting the growth of normal cells in a population of cells which include both transformed and normal cells, or for preparing a medicament therefor.

18. The use according to claim 17, wherein the transformed cells are human carcinoma or melanoma cells.

19. The use according to claim 18, wherein the transformed cells are human breast carcinoma cells.

20. The use according to claim 18, wherein the transformed cells are human melanoma cells SK-MEL-28 or SK-MEL-170.

21. The use according to claim 18, wherein the transformed cells are colon or renal carcinoma cells.