Note: Descriptions are shown in the official language in which they were submitted.
1 ~7~370
3,7,11,15-TETRAMETHyL_2,4,6,1o~14-~ExADEcApENTA~NoIc ACID
This invention relates to a novel compound of 3,7,11,15~
tetramethyl-2,4,6,10,14-hexadecapentaenoic acid having the
general formula (I):
~ \ ~ ~ COOH (I)
~_f~r. ~,~,i.~
and ~ alt thereof. his invention further relates to pro-
cesses for the preparation of the same, an anticancer agent
comprising the same, and a therapeutic agent for treatment of
skin diseases with keratinization.
W. Bollag,et al. reported in Europ. J. Canccr, vol. 10,p
731(197l~)that retinoides such as ethyl g-(2~3~6-trimethyl-
4-methoxyphenyl)-3,7-dimethyl-2,4,6,8-nonatetraenoate have
anticancer activity. These retlnoide compounds1 however~
are highly to~ic, and further have problems such as causing
hypervitaminosis of Vitamin A when administered.
The compound of the above-mentioned general formula (I)
provided by the present invention shows the anticancer acti-
vity, causes substantially no hypervitaminosis of Vitamin A,
and is low in other toxicities.
The compound of the present invention can be prepared
by the following processes.
Process A
This process comprises:
(1) reacting a compound of the general formula (II):
~ ~ ~ O (II)
and a Wittig reagent derived from a compound of the general
formula (III): -
X - CH2 - C02R1 (III)
- 2 - 117937~
in which X represents a halogen atom, and R1 represents a
lower alkyl group,
to obtain a compound of the general formula (IV):
~ C02R1 (IV)
in which R1 has the same meaning as defined above; and
~ 2) hydrolyzing the so obtained compound ofthe general
formula (IV) in the pregence of a base to prepare the com-
pound of the general formula (I).
Examples of the Wittig reagents employed in the above-
described (1) stage and derived from a compound of the
general formula (III) include phosphoric compounds produced
by the reaction between the compound of the general formula
(III) and triphenylphosphine, phenyldialkoxyphosphine, tri-
alkylphosphite, or the like~ 'rhe preparation of th~ reagent
I and the Wittig reaction employing the reagent are carried
out by the conventional methods such as the method given by
Wadworth, et al. in J. ~m. Chem. Soc.~ vol. 83, p. 1733 (1961),
the method given by Greenwald, et al in J. Org. Chem., vol.
28, p. 1128 ~1963~, and the method given by Horner, et al. in
Ber. vol. g5, p.681 (1962).
In the above mentioned (2) stage, the hydrolysis is
carried out in the presence of a base generally employed for
hydrolysis of carboxylic acid esters, such as sodium hydroxide
; and potassium hydroxide.
Process B
This process comprises:
(1) reacting a compound of the general formula (V~:
I CHO (V)
~/\/~/
and a Wittig reagent derived from a compound of the general
3 _ ~ ~ 7 9 ~ l ~
formula (VI):
X ~ ~ C02Rl ~VI)
in which ~ represents a halogen cltOIII~ and Rl represents a lower alkyl group,
to obtain a compound of the general formula (IV); and
~ 2) hydrolyzing the so obtained compound of the general formula
~IV) in the presence of a base to prepare the compound of the general formula
~I).
Each of the above-described stages ~1) and ~2) can be carried out in
the same manner as in Process A.
Process C
~ his process comprises:
~1) react~ g a compo~uld of the general form-llll ~V:[:C~:
' ~ ~ ~ S02Y ~VII~
in which Y represents a lower alkyl group or an aryl group~ and a compound of
the general formula ~VI~
to obtain a compound of the general formula ~VIII):
S02Y
= C02Rl ~VIII)
in which Y and Rl have the same meanings as defined above;
and
~2~ subjecting the so obtained compound of the general formula
~VIII~ to a desulfination and hydrolysis of ester in the presence of a base to
prepare the compound of the general formula ~
~h
4 ~ ~ 7 g ~ 7 ~
The stage ~1) is carried out in the presence of a base. Examples of
the bases include n-butyllithium and phenyllithium. Examples of the reaction
solvents include tetrahydrofuran, diethyl ether and 1,2-dimethoxyethane. r~e
reaction is generally carried out at a temperature lower than room temperature.
The stage (2) call be carried out in the same manller as the s-ta~e (2)
of the aforementioned Process A.
Examples of the s~bstituents provided to the general fo-rmulae ~IlI),
~IV), (VI), ~VII) and ~VIII) are as follows:
halogen ato~s such as chlorine, bromine and iodine or the substituent
X; lower alkyl groups such as methyl, ethyl and propyl for the substituent ~1
and lower alkyl groups such as methyl, ethyl and propyl, and aryl groups s~ch as
phenyl and p-tolyl -eor the substituellt Y.
Examples oE the pharmace~ltically acceptable suLts oE tho COID~OUlld 0
the gencr.ll formula ~I) inclllde lts sodium salt ~m-l its po-tassiunl salt~
rlhe compouncl oE the aforelllelltioned gcncral Eormula (1) L)rovided by the
present invention also shows therape~ltlc activity for treatment of skin diseases
with keratinization.
Examples of the skin diseases with keratinization which can be treated
by the compound of the general formula ~I) include skin diseases showing
symptoms such as hyperkeratosis, parakeratosis and dyskeratosis. ~lore con-
cretely, examples of the skin diseases include psoriasis, acne, acne vulgaris,
Darier's disease, palmoplantar pustulosis, lichen planus, ichthyosis,
erythroderma, pityriasis rubra pilasis, and keratosis senilis.
There are employed steroide-type external preparations for the treat-
ment of the skin diseases with keratinization. These preparations, however, have
strong side-effects, so that these are not applicable to the repeated administra-
tion for a long period and the treatment with administration of a great amount
~ ,~
- 4a - ~ 1793~0
of the preparation.
In contrast, 3,7,11,15-tetramethyl-2,4,6,10,14-hexa~ecapelltaenoic acid
of the present invention has the activity for
- 5 - ~ 17937~
inhibition of keratinization of skin and show low toxicity
The results of the pharmacological tests and toxicity
tests on the compound o~ the present invention are set forth
below.
Pharmacological Tests (Anticancer Activity)
_ . .
(1) Experimental procedure
A mouse (ICR, female, 60 days age) was shaved at
the back of the neck (5 cm2). 7,12-Dimethylbenzo-[2~-
anthracene was dissolved in acetone to give 75 mg./100 ml.
solution. The so prepared solution was applied to the mouse
on the 60th aged day and further on the 75th aged day in the
amount of 0.2 ml. per a mouse.
Crotonic oil was dissolved in acetone to give 250 mgO/
100 ml. solution, and the so prepared solution was appliecl
to the mouse in the amount of 0.2 ml. per a mouse, twice a
week until the beginning of the treatment~ When 3 - 7 papi1-
lomata ~diameter of 3 - 8 mm. for each, and total diameter o~
30 - 60 mm.) were produced per a mouse, the treatment was
started.
The test compound was dissolved in groundnut oil to
give 20 mg.tml solution, and administered orally to the mouse.
The solution was administered 10 times for 14 days (once a
day), and the diameters of the papillomata were measured on
the 14th day to determine the total diameter for each mouse.
(2) Test compound
3,7,11,15-Tetramethy1-2,4,6,10,14-hexadecapenta-
enoic acid (the compound according to the Fresent invention)
Ethyl-9-(2,3,6-trimethyl-4-methoxyphenyl)-3,7-
dimethyl-2,4,6,8-nonatetraenoate (control compound)
(3) The results are set forth in Table 1.
~ 6 - 1~.79370
Table 1
Test Compound Number Papilloma (~otal diameter/mouse)
f Mean Value Mean'~alue Ratio of ~
mlce (Oth day) (14th day) Increase or
Decrease
_.
Groundnut oil 3 33.9 mm 39 7 mm -~ 17 1 %
only
Compound of the
-invention 5 37.5 mm 21 3 mm _ 43.2 %
day)
Control
(40 mg~/Kg l 58.1 mm 32.7 mm _ 43.7 /0
day)
As seen from the above l'able 1, the compound of' the
invention is effective againsk the papilloma.
Pharmacological Tests (Inhibition of Keratinization)
(1) ~xperimental procedure
Into a Petri dish (diameter 6 cm) in which 8 cover-
glasses '(diameter 15 mm) were placed was poured 5 ml of
a suspension of the varient epithelial cell of rat bladder
named BES 20B (approximately 2 x'105 cells/ml ), and the
incubation was carried out at 37C,` for 24 hours and at 5 %
carbon dioxide concentration. Each o~ the so treated cover-
glasses was placed in 2 ml of Eagle's ME~ medium contain-
ing the test compound at different concentrations, and then
another incubation was carried out at 37C and at 5 % carbon
dioxide concentration The medium was renewed at intervals
of 2 - 3 days On the 2nd, 5th, 8th and 14th days from the
beginning of the incubation, the cover glass wa~ taken out
of the medium and subjected to the Papanicolaou stain to
observe the degree of keratinization The observation was
carried out by the measùrement of the absorption spectrum in
_ 7 ~ g 3-7 ~
the region of 4no - 750 nm3 and tlle Kl (Keratiniza-tion Index) was calculated
from the following equation.
Absorption peak ln the vicinity oE 490 nm ascribed
to the keratinized cells
KI = -- ~
Absorption peak in the vicinity of 640 nm ascribed
to tlle non-keratinized cells
A value of the KI of 1.0 or higller indicates high keratinization, a-nd
a value of the KI of 0.5 or less indicates substantially no keratini2ation.
The BES 20B cell was incubated in a medium containing no compound of
the invention, for comparison.
(2) Test compound
3,7,11.,15-Tetrclmethyl-2,4,6,10,14-llexndec.lpellt~lelloic aci~ lle
compound according to tlle prescnt invention)
~ 3) Bxpcrilllental results
The results are set Eorth in 'I'able 2.
Table 2
KI
Period of Incubation
2 days 5 days 8 days 14 days
Control 0.43 1.10 3.27 3.08
Compound of the Invention
0.1 ~g./ml. 0.43 0.67 0.55 0.52
1.0 ~g./ml. 0.42 0.46 0.38 0.39
5.0 ~g./ml. 0.48 0.50 - 0.22
In the experiment on the control, the KI value exceeded 1.0 on the 5th
day -from the beginning of incubation, which indicates high keratinization. In
contrast to the result on the
1 179370
control, the results given by different concentrations of the
compound o~ the present invention showed the KI values of
less than 1.0 for all runs.to ind:icate inhibition of Kerati-
nizatio~.
Toxicity Tests
(1) Experimental procedure
The test compound was administered repeatedly to
a group of 6 mice (ICR strain, female) ~or 14 days. The
amount of the administration was 40 mg./Kg./day, 200 mg /Kg./
day, and 400 ~go/Kg /day for the compound o~ the present
invention, and 200 mg /Kg~/day for the control compound.
In the course of the administration, increase or decrease of
the weight of the mouse, occurance of death, etc. were ob.-
served.
(2) Test compound
The compounds described in the pharmacological
tests (anticancer activity) were employed
I (3) Experimental results
(a) Increase and decrease of the weight
The results are set forth in Table 3.
(b) Death
All mice treated with the control compound
in the amount of 200 mg./Kg~/day died by the 8th day, and
no death was o~eerved on the mice treated with the compound
of the present invention.
tc) Falling-out of hair
Falling-out of hair was observed by the 6th
day on every mouse treated with the control compound in the
amount of 200 mg /Kg /day, and no falling-out of hair was
observed on the mouse treated with the compound of the pre-
sent invention
(d) Cyanosis
Cyanosis was observed by the 7th day on every
mouse treated with the control compound in the amount of 200
mg /KgO/day, and no cyanosis was observed on the mouse treated
with the compound of the present inventiOn
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" _ 10- ll7~370 .'
Among the subjects in the toxicity tests, the falling-
out o~ hair and the weight change are known to indicate the
hypervitaminosis of Vitamin A. Since the falling-out of
hair and decrease of the weight were observed at a prominently
high level on the group of mice treated with the control
compound, it is thought that the hypervitaminosis of Vitamin
A occured. In contrast, there was observed no such problem
on the group of mice treated with the compound of the present
invention. -
In view of the pharmacological test results and thetoxicity test results hereinbefore described, the compound
of the present invention is considered to be of hig~ safety
and to be of value as an anticancer agent and a therapeutic
agent for treatment of skin diseases with keratinization.
Therefore, the compound of the present invention can be
employed for the prevention and treatment of cancer and pre-
cancerous conditions, and also employed ~or the treatment of
skin diseases with keratinization such as acne and psoriasis
vulgaris and the treatment of allergic and in~lammatory skin
diseases. Moreover, the compound of the present invention
can be employed for the treatment of muscosal diseases
caused by inflammation, degeneration and displastic change
For the applications as the anticancer agent and the
therapeutic agent for treatment of skin disease with kerati-
nization, the compound of the present invention is admini-
stered orally in the form of powder, granule, pellet, hard
capsule, etc.,or parenterally in the form of ointment, suppo-
sitory, injection solution, etc. The dosage is generaily
40 mg. - 4 g /day for adult~ If the compound of the pre-
sent invention is employed in the form of an external prepa-
ration, the dosage can be ~aried depending on the conditions
of the disease The compound of the present invention can
be combined with a generally employable carrier for the medi-
cal use in the conventionc.l manner to give the preparations
described above
- ~ ~ 7g370
The processes for the preparation of the comp~und of the
present in~ention are illustrated by the following exam~les,
but these examples are not intendecl to restric~ the present
invention.
Example 1
To a suspension of 5.0 g. o~ 55 % sodium hydride (oily)
in 60 ml. of n-hexane was adde~ 28.6 g. of triethyl phospho-
noacetate. The mixture was then heated under reflux~ and '
20 g. of 6,1'0,14-trimethyl-3,5,9,13-pentadecatetraen-2-on
was added dropwise to the mixture under stirring. After 30
minutes, the reaction liquid was poured into 20Q ml of waterl i
and then 500 ml. o ~hexane was added for extraction. The n-
hexane phase was separated, washed with two 100 ml. por~ions
of a mixture of methanol and water (2 1~ I and corlcentra~ed. '¦
The so obtained concentrate was purif'ied by the silica ~el i~
column chromatography to ~ive 18 g. of et~yl 3l7,l1,15- '
tetramethyl~2,ll,6,10~14-hexadecapentaenoate~
To 10 g. o~ the ethyl 3~7~ 5~tetranle~hy-l-2~ 6~1
hexadecapentaenoate obtained in the abo~e was added a sol~
tion of 3.9 g. of potassium hydroxide in 30 ml of isopropyl
alcohol, and the mixture was stirred at 50 G for 1 hour.
The reaction liquid was then poured into ice-water, made
acidic by addition of hydrochloric acid, and extracted with
100 ~l. of ethyl ether. The ether phase was washed with
water, dried over magnesium sulfate, and concentratèd to
give 9.0 g. of an oil. The oil was dissolved in sn ml~ of
n-hexanè and crystallized at - 20 C to give 4.0 ~ of 3,7,
11,15-tetramethyl_2,4,6,10~14_hexadecapentaenoic acid in the
form of pale yellow needles
M~p. : 78.4 C
Mass spectrum (mte) : 302 (M )
Infrared absorption spectrum (cm 1, KBr tablet):
3450, 2900, 1680, 1595
NMR spectrum (~, CDC13): 1 &1 (6H, s), 1 68 (3H, s)g
1~86 (3H, s), 1.92 - 2.24 (8H, b~, 2 35 (3H, s),
- 12 - ~ 1 7 g ~ ~ ~
5.10 ~2H, b), 5.76 (1~1, bs), 5.98 (11l, d, J = 11 llz), 6.20
(1~1, d, J = 15 Hz), 6.90 (1~1, dd, J = 11 llz, 15 l-l~), 11.63
(1ll, b)
Ultraviolet absorption spectrum ~methal~oL 304 nm
Example 2
To a suspension of ~.8 g. of sodium ethoxide in 100 ml. of n-hexane
was added 18 g. of diethyl 3-e~hoxycarbonyl-2-methyl-2-propenylphosphonate. To
the mixture was added 10 g. of 3,7,11-trimethyl-2,6,10-dodecatrien-1-al under
stirring at room temperature. After 1 hour, the reaction liquid was poured into
50 ml. of water, and the n-hexane phase was separated. The n-hexane phase was
washed with two 50 n~l. portions oE a mix-ture oE methanol alld water (2 : 1), alld
concentrated. The so obtained concentrate was puri-Eied by the sllica gel coL~ullr
chromatograp}ly -to give 14.5 g. oE ethyl 3,7,11,15-tetramethyl ?.,4,G,L0,14-llexa-
decapentaelloate.
10 g. of the ethylester ob-tained in the above was hydroly~ed in the
same malmer as in Example 1 to give 3.5 g. of 3,7,11,15-tetrclmethyl-2,4,6,10,14-
-hexadecapentaenoic acid in the -form of yellow needles.
The so obtained product was identified in the same manner as in
Example 1, namely, by m.p., mass spectrum, NMR spectrum, infrared absorption
?.0 spectrum, and ultraviolet absorption spectrum.
Example 3
In 100 ml. of tetrahydrofuran was dissolved 10 g. of l-p-tolylsulfonyl-
-3,7,11-trimethyl-2,6,10-dodecatriene, and the solution was chilled to - 50 C.
To the solution was added dropwise 1~.5 ml. of 15 % n-butyllithium - n-hexane
solution under stirring and in a stream of nitrogen, maintaining the temperature
of the solution at - 50 C. Then, 300 ml. of tetrahydro-furan solution contain-
ing 5.7 g. of ethyl 4-bromo-3-methyl-2-butenate was added dropwise to the so pro-
--, .
- 13 - ~ 1 ~ 9 370
duced solution. A-Eter 30 minutes, 100 ml. of 10 % aqueous ammonium chloride
solution ~as added, and subsequently the mixture was treated to reach room
temperature. The mixture was then extracted with two 200 ml. portions of
n-hexane. The n-hexane phase was washed with three 100 ml. portions of water,
dried over magnesium sulfate~ and concentrated to give 13 g. of cthyl 3,7,11,15-
-tetramethyl-5-p-tolylsulfonyl-2,6,10,14-hexadecatetraenoate.
To 10 g. of the ethylester obtained in the above was added a solution
of 4.6 g. o~ potassium hydroxide in 50 ml. of isopropyl alcohol, and the mixture
was stirred at 50 C for 3 hours. The reaction liquid was then poured into ice-
-water, made acidic by addition of hydrochloric acid, and ext~acted with 100 ml.
of ethyl ether. The ethyl ether phase was washed with water, dried over magne-
sium sulfate, and concentrated to give 6 g. oE an oil. 'I'he oil was dissolvecl in
30 ml. of n-hexane and crystallized at - 20 C to give 1.8 g. of 3,7,11,15-tetrLI-
methyl-2,4,6,10,14-hexadecapentaenoic acid ln the form of pale yellow needLes.
The so obtained product was identlEied in thc sallle nlanner, as in
Example 1 namely, by m.p., mass spectrum, NMR spectrum, infrared absorption spec-
trum, and ultraviolet absorption spectrum.
Example 4
.
Pellet
3,7,11,15-Tetramethyl-2,4,6,10,14-
hexadecapentaenoic acid 50 g.
Silicic acid anhydride 30 g~
Crystalline cellulose 50 g.
Corn starch 36 g.
Hydroxypropylcellulose 10 g.
Magnesium stearate 4 g.
The above composition was processed in the conventional manner to give
a pellet ~180 mg. for a pellet).
