Note: Descriptions are shown in the official language in which they were submitted.
645~
This invention relates to novel ~ubstituted
3~ aminoethylidene)-5-cinnamoyl-2H-pyran-2,6(3~-diones
which are u~eful as inhibitors of cer~ain ~ntigen~antibody
reactions and particularly in allevia~ing allergic manifes-
tation~ The compounds of tblis invention inhibit the re-
lease and/or formation of pharmacologically active mediators
from effector cells triggered by the interaction of ~ntigen
and a ipecific ~ntibody fixed to the cell 3urface. Thus
the compounds ~re valuable in the treatment of allergic
. di~ea~es ~uch a~ asthma;, rhinitis and ur~icaria.
The compounds of this invention are represented
by ~he following ge~eral ~truc~ural formula: .
Rl~/~C~
in which~ ~5~E~e_E
R represe~t~ lower alkyl, ~traigh~ or branched ~- :
chain~ of from 3 to 6 carbon ~tom~J phenylJ halophenyl ~uch
20~ ~as chlorophenyl9 bromophenyl or fluorophenyljt hydroxyphenyl,
methoxyphenylj~ p-mercaptophenyl or ~minophenyl~ and
Rl represen~.hydrogen9 hydroxy, methoxg or di- .
: methoxy.
. Preferred compounds of this invention ~re repre-
sented by form~lla I ~bove when ~ is hydroxyphenyl or amino- :
ph2nyl and Rl i8 hydroxy. ~
.
: ~ The compound8 of thi~ invention ~re prepared as
~hown in the foIlowing reaction scheme: ;
: 30
. .
: '.
~: ' ~ '
CH3/ ~ X ~R ~R~ ~ ~NHR
.
in which R and Rl are as de~ined in formula I. Thu~, a
3~ amino~th~lidene)-5 acetyl-2H-pyran-2,6(3~-dione i~
h~ated at reflux wi~h a benæaldehyde in an lnert organic
~olven~ ~uch as ohlaroform and in th~ presence o~ piperidinP
for ~rom 4 ~o 48 hours.
The ~tartin~ materials of ~ormula II above used
herein are generally prepared by reaction o~ 395-diacetyl-
4~6-dihydroxy-2~-pyran-2-o~e with the appropriate amine
~RNH~o The reactants are usually heated at reflux in an ~ :
lS inert organic ~olvent ~uch as benzene~ toluene or methanol
for from ~wo to twelve hour~. 3,5-Diacetyl-4~6-dihydroxy-
2H-pyran~2~one i8 obtained by reacti~n of acetonedicarboxy-
lic ~cid and ace~ic anhydride in sul~uric acid st elevated :
: temperatureO
The inhibi: tory activity o~ the eompounds of this
invention on mediator release in ~ensitized ti9~ue9 i~
mea~ured by the abillty of the ~ct~ve m~dicame~t to inhibit
t~ie pas~ive eutaneou~ anaphy1~xls (P~A3 reaction in rats.
In thi~ te~t ~ystem~ titered and approprl~tely d~luted
serum (from rats previous1y imm~nized by ~h~ in~raperltoneal
~njection o~ ova1bumlna1uminum hydroxide ~r ovalbumin-lOm.-
Bordate11a pertu~i81~ U.S~PO i.p.-and N. Brasilien~is i.p.)
containing reaginic ant~bodi~ directed against ovalbumin
i~ In~ected intradermally at fo~r site~ on ~he ~haved back~
o~ noxma1 adult maLe ra~ For~y-eight hour~ later the
, .! .
.
: ;_ 3 ~ j
.
' ' ` .:, . . ' , , ` . .
~ ~ 6 ~
1 animal~i are injected intravenously with 0.5 ml. of isotonic
saline solution containing 5 mg. of the ovalbumin antigen
and 5 mgO of Evans blue dye. Chemical mediators such as
histamine and serotonin which are rPleased at the sensitized
S si~es as a re~ult of a local cellular anaphylaxis~ cause an
incre~se in capillary permeabLlity with resultant leakage
of p~asma and formation of a wheal. The wheal is visualized
by the plasma protein bound Evans blue dye. Undex conditions ~ -
5f the test9 the average control wheal i9 approximately
l~x12 mm. Thirty minuteis following antigen challenge~ the
animals are killed9 the dorsal skin is reflected and thediameter of the wheals recorded. A test compound is admin-
istered intravenously~ initially at 0.5 minutes prior to
antigen challenge (longer pretreatment times and oth r
routes of drug administration~ i.e. oral or intraperitoneal ;;,
may be employed~. Percent inhibition is calcula~ed from
the difference in mean average wheal diameter between a
treated group and saline or appropriate diluen~ controls.
The compounds of this invention administered
~ntravenously to rati~ at doses of from O.S to 10 mg/kg pro-
duce marked inhibition of ~he PCA reaction. A preferxedcompound~ 5-(m-hydroxyclnnamoyl)-4-hydroxy~3-[1-(o-hydroxy~
phenylamino)ethylidene]-2H-pyran-2,6(3~3-dione9 produced
82% inhibition of the rat PGA wheal at l.S mg/kg, i.v.
Another preferred compound~ 5-(m-hydroxycinnamoyl)-4-
hydroxy~3~ll-(o-aminophenylamino)ethylideneJ-2H pyran-
296(3~dione9 pxoduced 65~/o inhibition of the rat PCA
wheal at 0.5 mglkg~ i.v. In ~e~ting ~or mechanism o~
action9 the compounds of this invention were found not to
J provide comp-rable inhibition of wheal~ of equal ~ev~rity
',' '.
1 produced in rats by the intracutaneous admlnistration of
histamine and serotonin following ioY~ administration of
the test compound ~t ~he ~ame do~P and pretreatment ~ime
which exhibited signi~icant lnhibition of the rat 48~hour
PGA reac~ion.
Upon oral administxation, 5-~m--hydroxyoinnamoyl)-
4-hydroxy-3-[l (o-hydroxyphenylamino)ethy1idene]-2H-pyran-
296(3~-dione produced 46% inhibition in the rat 48-hour
PCA system at 25 mg/kg and a pretreatment time of 15 minute~.
Similarly9 5-(m-hydroxycinnamoyl3-4-hydroxy 3~ (o-amino-
phenylamlno)ethylidene~-2~-pyran-~6(3~-dione upon oral
adminiætration of ~5 mg/kg produced 35% inhibition in the
xat 4~hour PCA sys~em at a pretreatment ~ime of 15 minutes.
Another featura of ~his i~vention i~ a pharmaceu- :
t;cal compo&itioll CompriSing ~n appropriate amount of a
~ubstituted pyran dione as ~et forth in formula I in
association with a pharmaceutical carrier or diluent. The
nature of the compositlon and the pharmaceu~icsl carrier or
diluent wlll of course depend upon ~he intended route of
administratlon9 l.e. orally, parenterally or by inhalation.
Preferably a compound iB ~dminl~ered to nn animal in a
. compo~ltion compri~ng an amount ~ufficient to produce an
~nhibition Df ~he con~equence~ of the antigen-antibody
reac~i~n. ~hen employed ln this manner9 the do~age of com-
po8itlGn i8 preferably Rueh tha~ from 5 mg. to 500 mg. o
ac~ive fn~r~ed:Lent are admlni~t~red at each administratlon.
Advantageousl~ equal do8e8 will bs adminl~tered 1 to 4
tim~ daily ~ith the d~ily do~age regimen bein~ about 5 mg.
to ~bout 2000 mg.
.
5 ~
~ ~ 4S~
l In genexal 9 par~icularly or the prophylactic
treatment of asthma9 the compo~itions will be in a form
~uitable or administration by inhalation. Thus the compo-
~i~ions will comprise a suspension or 801ution Df the ac~ive
S ingredient in water for administration by means of a conven-
tional nebulizer. Alternatively the compo~ition~ will com~
prise ~ suspen~ion or ~olu~ion of the active ingredlent in
a conventional liquified propellant such as dichlorodi-
fluoromethane or chlorotrifluoroethane to be administered
1~ from a pre~surized container. The composition8 may also
eomprise ~he solid ac~ive ingredient diluted with a solld
diluentg e.g. lactose9 for admini~tration from a powder
inhala~ion devlce. In the above compo~itions, the amount
of carrier or diluent will vary bu~ preferably ~ill be the
major proportion of a suspension or ~olution of the active
ingredien~. When ~he diluent i8 a solid, it may be present
in le~s 9 equal or greater amoun~ ~han the solid act~ve
. ~ngredient. .
A wide variety of other pharmaceutical forms can
~2~ be employed. Thus~ if a solid carrier ls used the prepara-
;~ tion can be ~ableted3 placed in ~ hard gela~in cap~ule in
: . powder or pell~t form,.or in the form of a troche or 102enge
for oral administra~ion. The amount of solid carrier will
vary widely but pre~er~bly will be about 25 mg. to about
~5 1 g. If a liquid carrier i~ used, the preparation will be
in the form of a ~yrup9 emulsion, ~oft gelatin capsule
steril~ injecta~le liquid ~uch as an ~mpulJ or an aqueous
......... ... ..... ..... ............................................................... ... .. .
or ~onaqueous liquid suspension. : ~
. Exemplary ~ s~lid ~arrler~ are lactose, terra ~ ;
lba, ~ucrose9 talc~ g~lating agar, pectinJ acacia, magnes- ! .
'
. S ~
~ 6 ~5~
l ium ~tearate 9 stearic acid and the llke. Exempl~ry of
liquid carriers are ~yrup 9 peanut oil 9 olive oil, water and
the llke. ~imilarly the carrier or diluen~ can include any
time delay material well known to the ~r~ ~uch ag glyceryl
monostearate or glyceryl distearate alone or with a wax.
Th invention al~o :Lncludes a method o~ inhibiting
the effects of the antigen-anltibody reaction which co~pri~e~
the prior application to the area of the antigen-an~ibody
mechanism a ther~peutically effective amount of a ~ubstitu- :
ted pyran dione as defined ~n formula I. A particular appll-
cation of the invention ~ a methvd of relieving or pre- ~ :
venting allergic airway ob~txuct~.on which comprises admin- .`.
1s~ering ~o an animal a therapeu~ically effective amount at : :
~uitable intervals.
The pharmaceu~ical preparations are made following
the conventional ~echn~ques of ~he pharmaceutical chemist
involving mixing9 granulating and compr~ssing when nece~æary,
; or variously mixing and disRol~ing the ingredients a~ appro-
priate to the de~ired end produc~. :
'~he accompanyi~g example~ illustrate the prepara~
~ion of compounds o form~la I and their incorporation in~o
phar ~ceutical compo~itions of thi~ inventlon and as such .
are not to be consid~red a~ limiting ~he ~nv~ntion ~et
forth in ~he claims appended h~reto.
Kian89 A. K. et ~l. J. Chem. Soc. ~c) ppO 272l-6
(1971~ have que~t~oned the ~truc~ure ai~signed by previou~i
suthors ~uch a~ Wiley9 Ro H, et al. ~ 5~ _Eh~
: 2106B6-688 1956~ ~o ~he reac~ion product of aaetonedicar-
boxylic acld and acetic anhydride, de8ignated 5~carboxyde-
hydro~cetic acidg and the carbæmyl derivative~ ther~of.
: :
.
~ 3
1 Thus~ Kiang et al~ supra reported that the reaction of
acetonedicarboxylic acid with acetic anhydride gave the
compound of structure III and that the latter reacted wi~h
aniline to form the compound of ~tructure IV:
s
~H ~H C~l3
CH3CO~c~CH3 CH3C~N~
~ O o H O o
~ a~ EEI FOR~ILA IV
Upon inve~tigation which ha included 13C nuclear
magnetic resonance spec~ral and x-ray crys~allographic :~
studles9 I have concl.uded that the reaction of acetonedi~ .
carboxylic acid with acetic anhydride gives a pxoduct hav-
ing ~he ~automeric struc~ure as ~hown bel~w: -
~ 8 ~ OH
CH3 ~ ~CH3 ~ ~H ~ H CH ~ ~ ~ ~ E3
O ~ OH
or conven~ence this produc~ is designated herei~ as 3,5
d~acetyl~6~dihydroxy-2H~pyran-2-one. This agrees w~th
Kiang e~ al's gro~s ~truc~ure indicated by ormula III . !~ ;i`,
above. ThP rate of ~aukomerization represented by ~
a~ove i8 afected9 ~mong other fac~ors~ ~y the solvent u~ed
,~ .
~n the lJC spectral ~tudy- Accoxdingly the reaction of !-
thls product with an amine9 RNH~D give~ ~ product h~ving
. ~he tautomeric struc~ure~ a~ shown below:
. ~ . . . .
pH~ ~,3 pH CH3 R ~ ~H3
~ ~ C ~ CH3~ HR CH3 ~ NR
: H ~ O : O
'~';"
- 8
6 4~ ~
in which R is as defined above for formula I. This also
agrees wi~h Kiang et al's grolss structure indicated by
formula IV above,
Although the exact tautomer represented by
in ~olution has not been identified by l3C nmr~ an X~ray
crystallographic study in sol:Ld form of the compound wherein
R is p~hydroxyphenyl 3howed that because of extensive con-
jugatlon the carbon-carbon bonds throughout the molecule
are hybxidized and therefore ~he bond lengths lie ~ome-
. where between the values for double and single bonds.
However 9 since one o the e~changeable hydrogens in this
compound i9 located on the nitro~en9 for convenience one
tautomeric form has been chosen~ namely the intermediate
enamine pyran 2~6-dione stru~ture, to~represent all of the
compounds formed by reaction of ~ wLth an amine9 ~ ,
as indicated by formula Il above.
It will he apparent therefore ~o one ~killed in
the~art ~hat the more complete repre~entation of ~he com~
pounds o ~ormula I is sho~n by the ollowi~ tautomerlza- . .;
tion:
: O~I CH3
1 ,. qH Ç ~
NR~ ~ ~ C`N~R
:~ 1 HO ~ 1 0 ~ ~ O
H CH3
l ~ Od
: ~ - . . ..
~ . ,
~ 30: ~ : :
. .
.
~ 9 -
. ... .
: ..
.
EXAMPLE 1
To a boiling ~o-lution of 4.24 g. (0.02 m.~ of
395-diacetyl-496 dihydroxy-2H-pyran~2~one in 200 ml. of
methanol ~s added 2.18 g. (0.02 m.) of o hydroxy~niline.
The resulting mixture i~ refllLRed overnight and filtered to
yle1d 5-ac~tyl-4-hydroxy-3-~1 (o-hydroxyphenylamino)ethyli-
dene~-2H~pyran-2 9 6~3~dione~ mOp. 210-212C.
A ~olution of the above prepared pgran dlone
~0~606 g.) and 0,244 g. oP m~hydroxyb~nzaldehyde in 150 ml.
. of chloroform 1~ treated with ~ dropc of plperidine and r~-
fluxed for abou~ four hours. The reaction mixtur~ i~ coolea
. and wa~hed with S0 ml. of lN hydrochloric acid and 50 ml. ofwater. The drled solutlon is evaporated to give 5-(m
hydroxycinnamoyl)~4-hydroxy-3~ (o-hydroxyphenylamino)- ~.
ethylidene] 2H - pyran 2~6(3~-dione~ m.p. 180~182C. (dec.). . ,.
Slmil~rly9 a ~olution of the above pyran dione
(2.42 g,) and p-hydroxybenzaldehyde (0,876 g.) in 150 ml. :.
o~ chloroform with 16 drops o piperid~e i~ refluxed for
8ive hour~ to give a~ter workup9 5-(p-hydroxyeinnamoyl)~4-
hydroxy-3-[l~o-hydroxyphenylamin~)ethylidene]-~H-pyr~n-
: 2~6(3~ ~d~one9 m,p, 257-260C.
A solution of 1.2 g. (0.00~ m,) of 5-a~etyl-4-
hydroxy~3-[1-~o-hydroxyphenyl~mino)ethglldeneJ~H-pyran-
296(3~D~dione9 0.664 g. (0.004 m.) of 2,3-dime~hoxybenzal-
dehyde in 150 mlO 0~ chloroform and 6 drop~ of piperidine
~ .
: i8 re~l~xed ~or one and one-half hour~. The solvent i8 re- ..
: moved ~rom tlhe reaction mlxture ~nd ~he residue is disso~ved ~,
i~ a minimum o chloroform. ~nough petroleum-2ther i~
3~ added to~precip~ta~e the prod~c~t 5~(293-dim~thoxyclnnamoy~
: 4~hydroxy-3-[1-(o~hyd~oxyphenylam~no)ethylidene3-2~a-pyran-
~ 10- ,.
.. . . . . . . . . . .,.~
6~
296(3H)-dlon~g m.p. 176-180C,
EXA~LE: 3
To a boil;ng solution of 4.~ g. (0.02 m.) of 395-
diacetyl-496~dihydroxy-2H-pyran-2-one in 150 ml. of ~enzene/
methanol i~ added 1. 2 g . ~0 0 02' m. ) of n~ propyl~mine and the
resulting mlxture i5 xefluxed overnight. The re~ction mix-
ture i5 concentrated9 filtered and the solid treated wlth :
water to give pure 5-acetyl-4~-hydroxy-3-[1-(n-propyl~mino)-
e~hylidene]-2H-pyran-2 9 6(3~-dione 9 m.p. 145-148C~
A mixture of 1~0 g. (0.004 m.) of the ~bove pre-
pared pyran dione and 0.664 g. (0.004 m.) o 2,3-di~ethoxy-
be~zaldehyde in 200 ml, of chloroform and 6 drops of piperi-
dine is refluxed for two dayæ. The r~ac~ion mixture ~
concentrated and the re~idue i8 tritura~ed with e~her to-
furnish 5-(293-dimethoxycinnamoyl)-4-hydroxy-3-[l-~n~propyl-
amino)ethylidene]-2H pyran-2,6(3~-dione, m.p. 180-185C.
EXAP~L~ 4 : '
; o-PhenylenediamiQe (2.1 g~ 9 0.02 m.) i8 added ~o
a hot ~olution o~ 4.2 g. (0.02 m.) of 3,5-diace$yl-496 dl-
hydro~y-2~-pyr~n-2~one ~n methanol and the xe~ulting mix-
ture i8 reflu~ed for two h~ur8. me reactlon mlxture i3
cooled and il~ered ~o yield 5-acetyl~3~ o-aminophenyl- ::
. ~
:~ : amino~ethylidene~-4~hydroxy-2H-pyran-2,6(3~-dione~ m.p.
: 179-1~1C. .:~ :
~ A mixture. of 3.0 g..... (0.01 m.3 of the ~bove pre-
. pared pyran dione~ 1.4 ~. (O~tOl m.~ of o-meth~xybenzalde-
hydeJ 20 drops of piperidine ~nd 200 ml. of chloroform ~
refluxed for two howr~. The reaction mixture 18 concen-
.
trated and tre~ted with methanol to g~ve 3~ mino-
.
~: 30 phenylamino~ethylidene]~t4-hydroxy-5-(o-methoxy~innamoyl)-
: ' - 11 - .
, ! ~ , . .
~ ~ 4
1 2~-pyran-2,6~3~dione9 m.p. ~.48-150 C~
Similarly9 reaction o~ an equimolar amount of
293-dimethoxyben2aldehyde as clescribed above yield~ 3-El~to-
aminophenylamino)ethyiidene]-5-(293-dimethoxycinn~moyl)-4-
hydroxy-2H-pyran-296(3~-dione9 m.p. 212-215C.
~ 5
A mix~ura of 3002 g~(OoOl m.) of 5- ce~yl-3
~o-aminophenylamino)ethylidene]-4-hydroxy-2H-pyran-2,6(3
dione9 1.2 g. (0.01 m.) of m-hydroxy benzaldehyde, 30 drop~
of piperidine and 250 ml. of chloroform ~ refluxed for
three hours. The re~ul~ing solu~ion i~ concentra~ed and
the residue i~ di8solved in a small volume o~ chloroform.
Ether is added ~u3t unt~l a precipitate i~ ~ormed ~nd ~he
mixture is allowed to ~tand overnlght at room temperature~
~ Filtration give the produc~7 3~[1-~o-amin~phenylamlno~-
e~hylideneJ~4-hydroxy-so(m-hydroxycinnamoylt2H--pyran-296(3
: . dione9 mOp~ 245~247C.
Slmilarly~ reac~ion with an equimolar amoun~ o
pohydroxybenzaldehyde as de~crlbed abcve ~urnishes the
corresporlding 3~ (o-aminophen~lamiQo)ethylid~ne]-4-
hydroxy-5-(p-hydroxycinnamoyl)-2H-pyran-2~6(3~)-dione,
m.p. 223-225C. . . . i~
~e~ ~
3~5-Diacetyl-4D~-d~hydroxy~2H-pyran-2-one (5.3 g.)
i8 di8BolVed in 200 mlO of boiling ~oluene and an equimolar :
amount of p-chloroaniline i8 addedO The mixtura is refluxed
for 12 hoursj~ cooled and iltered to yi~ld 5-acetyl~3
(p~chlorophenylamlno~e~hylideneJ ~4-hydroxy-2H-pyran-2,6~3j~
dione9 m-.p. 205-206C.,
:
~.
'
~ 6 ~S~ ~
1 Following ~he procedll~e of Exampl~ 19 equimolar
amounts o ~he above pyran di~ne and benzaldehyde in
chloroform with piperidine are reacted to give 3-[1 (p-
chlorophenylamino)ethylidene] l5 cinnamoyl-4-hydroxy~2H-
pyran-2 9 6(3a~-dione.
EXAMPL 7
A mixture of 2.12 g. of 3,5-diacetyl-496-dlhydrox~
2H-pyran-2-one9 1,25 g. of p-aminothiophenol and 75 ml. of
me~hanol i~ refluxed for two hours; cooled and ~lltered to
yield 5-acetyl-4-hydroxy-3-[1-(p-mercaptophenylamino~ethyli-
dene]~2H-pyran-296(3~-dione 9 m.p. 207-210C.
~ quimolar amounts o ~his pyran dione and m-
hydroxybenzaldehyde are reacted as described above in
chloroform and piperidine to give 5-(m-hydroxycinnamoy~-4-
hydroxy 3 [1-(p~mercaptophenylæmLn~)ethylidene]~2H~pyra~-
296(3H~)-dione. ::
EXAMPLE 8 .
To a boillng solution of 3.0 g. ~00014 m.) of ~ :
3,5-diacetyl-4~6-dihydroxy-2H-pyran-2-one in 25 ml. of
benzene i8 added 1.4 g. (00015 m.~ of aniline and the
resulting mixtuxe i~ reflu~ed overnightJ The reaction mix-
ture i8 cooled and filtered to give S ~ce~yl-4-hydroxy-3- .
[l~phenylami~o)ethylidene]-2H-pyran-236(3~-dione 9 m.p.
1~4-186~.
~5 Thi8 derivative i8 reacted with an equimolar
amount o~ p-hydroxybenzaldehyde a3 described above in `~
chloroform and piperidine to yield 5-(pohydroxycinna~oyl~
4-hydroxy~3~[1-(phenylam~no)ethylide~e3-2 -pyr~n-2j~(3
dione.
, ~ ,.
.
.
. .
~ 6 ~5
1 As a specific embocl:iment of a useful composition
of this invention9 an active ingredient such as 3~ (o-
aminophenylamino~ethylidene~-4-hydroxy-5-(m hydroxycinna-
moyl)-2H pyran-296~3~ dione ils dissolved in sterile water
at a concentration of 0.5% and aerosolized from a nebulizPr
operatlng at an air ~low adjusted to deliver the desired
aerosolized weight of drug.
For oral administration9 compositions such as
those in the following examples can be prepared.
EXAMPLE 9
~IL~ ~ .
5-~m-Hydroxycinnamoyl)-4- 10
hydroxy-3~ (o-hydroxy
phenylamino)ethylidene~-2H-
pyran-2~6(3~-dione
Calcium sul~ate~ dihydrate 150
Sucrose 25 -
'~ Starch 15
Talc 5
Stearic acid ~ 3
20 ~; : me sucrose, caIcium sulate and active ingredient are
thoroughly mixed and granulated with hot 10% gelatin solu-
`tion. The wetted ma3s i~ pas~ed through a lt6 mesh screen
direc~ly onto drying tray~. The granules are dried at ~ ;
120F. ~nd pas~ed through a #20 mesh screen, mixed with
: the starch, talc and stearic acid, and compres3ed into
tablets.
5 (m-Hydroxycinnamoyl)-4~ 50
hydroxy-3~1~(o~hydroxy-
~phenylami~o)ethylidene)
2H-pyran-2,~(3H~-dione
: ~ ~ ,: ,
~ 14 -
5~3
1 EXAMRLE 10 (con~d)
~2~ apsule
Magnesium 9 tearate 5
Lactose 350
S Ihe above ingredients are scr~eened through a #40 me.~h
screen9 mixed and filled into #0 hard gelatin capsules.
,:
: ' ,.
.,
, .
: :;
:
:~ ~ 20 ~
~ ,
; ~ :
~ ~ ,
~ 25
:: ;
, .
.,
.
~ . .
30 ~
~: - 15 - :,
; ,:," ,~ ",, ~,., `~ , " , ",, , "~ ",", ,,~; ~: " , , , ,, , , , "