Note: Descriptions are shown in the official language in which they were submitted.
28~ HA142b-f
This invention relates to new thiazolidine-, thiazane-
and related carboxylic acids which have the general formula
/x
(I) ( 6 * / ~ */ 5)
~3 2~ )m l-Rl n
R4 S - (CH2)p CH CO N CH CO R
and salts thereof, wherein R is hydroxy or lower alkoxy; R
h yC~ro~ o
and R2 each is hydrogen or lower alkyl; R5 and R6 are ~ro~on
or lower alkyl with the proviso that R5 and R6 cannot both be
hydrogen; R3 is hydrogen, lower alkyl or mercapto-lower
alkylene; R4 is hydrogen, lower alkanoyl, benzoyl or
R3 ~ 2-CI)m ~ Cl Rl n
-S (CH2)p- CH - CO - N CH - CO - R
X is O, S, SO or SO2; when X is O, m is 2 and n is 1; m is 1,
2 or 3; n is O, 1 or 2; and m + n is 2 or 3; p is O or 1.
The asterisks denote centers of asymmetry.
The invention in its broad aspects includes derivatives
of thiazolidine-, thiazane- and related carboxylic acids having
formula I above.
The compounds of this invention are characterized by an
unsubstituted or lower alkyl substituted 5- or 6-membered
heterocyclic carboxylic acid having one nitrogen atom and one
sulfur or oxygen atom in the ring, the remaining members of
the ring being carbon, preferably thiazolidine-, thiazane-
and morpholine carboxylic acids. The ring, as indicated,
contains a hetero atom in addition to the nitrogen, which i~
~ z ~ ~ HA142b-f
oxygen or sulfur and the sulfur can be oxidized to the
sulfinyl (-~-) or sulfonyl ( ~S~ ) state. The side chain,
attached to the nitrogen of the heterocyclic ring, is an
unsubstituted or substituted mercapto-alkanoyl group. The
compound can also be a "dimer" wherein the sulfur containing
substituted R4 is a similar unit.
The lower alkyl groups represented by any of the variables
include straight and branched chain hydrocarbon radicals from
methyl to heptyl, for example, methyl, ethyl, propyl, isopropyl,
butyl, isobutyl, t-butyl, pentyl, isopentyl and the like. The
lower alkylene groups are of the same kind also having 1 to 7
carbons. Similarly the lower alkoxy groups are of the same
kind with a link to oxygen, for example, methoxy, ethoxy,
propoxy, isopropoxy, butoxy, isobutoxy, t-butoxy and the like.
The Cl-C4 members, especially Cl and C2 members, of all types
are preferred. The lower alkanoyl groups are the acyl
radicals of the lower (up to 7 carbons) fatty acids, e.g.,
acetyl, propionyl, butyryl and the like, acetyl being preferred.
The symbols have the foregoing meanings throughout this
specification.
The products of formula I can be produced by various
methods of synthesis.
According to a preferred method, the acid of the formula
X ~ R ~
HN CH - CO R
wherein R is hdyroxy and the other symbols have the same
meaning as above, is acylated with an acid of the formula
~ ~ Z~ ~ HA142b-f
~R3
R4 S (CH2)p CH COOH
by one of the known procedures in which the acid III is
activated, prior to reaction with the acid II, involving
formation of a mixed anhydride, symmetrical anhydride, acid
chloride, active ester, Woodward reagent K, N,N'-carbonyl-
bisimidazole, EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydro-
quinoline) or the like. When R is lower alkoxy, this method
or other known methods for coupling such moieties can be used.
[For review of these methods, see Methoden der Organischen
Chemie (Houben-Weyl) Vol. XV, parts 1 and 2 (1974).
The acid of formula II can, of course, be acylated
stagewise. For example, a fragment of the acylating agent
III can be first attached to the acid of formula II, e.g.,
by reacting that acid with a haloacyl halide of the formula
IR3
hal - (CH2)p- CH- CO -hal
wherein hal represents a halogen, preferably chlorine or
bromine, 3-bromopropanoyl chloride for instance. This yields
a product of the formula
(R~ / ~ R5)
IR3 R2-CI)m ( -Rl n
hal (CH2)p- CH CO - N CH - CO - R
The reaction of this intermediate with a thiol R4-SH then
yields the desired product of formula I. This stepwise
acylation is illustrated in Example 1.
~ Z8~ HA142b -f
When the product obtained is an ester, e.g., R is lower
alkoxy, the ester can be converted to the free carboxy group
by alkaline hydrolysis, or by treatment with trifluoroacetic
acid and anisole. Conversely the free acid can be esterified
by conventional procedures.
The disulfides, i.e., when R4 is
(R~ X R )
-S (CH2)p CH---CO - N CH CO R
are obtained by oxidation of a compound of the formula
R (R~ C / ~ / )
HS (CH2)p CH- CO N CH - CO - R
e.g., with an alcoholic solution of iodine.
Products of formula I have at least one or may have up
to 4 asymmetric carbon atoms. These carbon atoms are indicated
by an asterisk in formula I. The compounds accordingly exist
in diastereoisomeric forms or in racemic mixtures thereof.
All of these are within the scope of the invention. The
above described syntheses can utilize the racemate or one
of the enantiomers as starting material. When the racemic
starting material is used in the synthetic procedure, the
stereoisomers obtained in the product can be separated by
conventional chromatographic or fractional crystallization
methods, In general, the L-isomer with respect to the carbon
of the amino acid constitutes the preferred isomeric form.
~Z8~2 HA142b-f
The compounds of this invention form basic salts with
various inorganic and organic bases which are also within the
scope of the invention. Such salts include ammonium salts,
alkali metal salts like sodium and potassium salts (which are
preferred), alkaline earth metal salts like the calcium and
magnesium salts, salts with organic bases, e.g., dicyclohexyl-
amine salt, benzathine, N-methyl-D-glucamine, hydrabamine
salts, salts with amino acids like arginine, lysine and the
like. The non-toxic, physiologically acceptable salts are
preferred, although other salts are also useful, e.g., in
isolating or purifying the product as in the case of the
dicyclohexylamine salt.
The salts are formed in conventional manner by reacting
the free acid form of the product with one or more equivalents
of the appropriate base providing the desired cation in a
solvent or medium in which the salt is insoluble and filtering,
or in water and removing the water by freeze drying. By
neutralizing the salt with an insoluble acid like a cation
exchange resin in the hydrogen form [e.g., polystyrene
sulfonic acid resin - Dowex 50 (Mikes, Laboratory Handbook of
Chromatographic Methods, Van Nostrand, 1961) page 256] or with
an aqueous acid and extraction with an organic solvent, e.g.,
ethyl acetate, dichloromethane or the like, the free acid form
can be obtained, and, if desired, another salt formed.
Additional experimental details are found in the examples
which are preferred embodiments and also serve as models for
the preparation of other members of the group.
The compounds of this invention inhibit the conversion
of the decapeptide angiotensin I to angiotensin II and
therefore are useful in reducing or relieving angiotensin
* Trade Mark
--5--
~ Z~ ~ HA142b-f
related hypertension. The action of the enzyme renin on
angiotensinogen, a pseudoglobulin ln blood plasma, produces
angiotensin I. Angiotensin I is converted by angiotensin
converting enzyme (ACE) to angiotensin II. The latter is
an active pressor substance present which has been implicated
as the causative agent in various forms of hypertension in
various mammalian species, e.g., rats, dogs, etc. The
compounds of this invention intervene in the angiotensinogen-
~angiotensin I-~ angiotensin II sequence by ingibiting
angiotensin converting enzyme and reducing or eliminating
the formation of the pressor substance angiotensin II.
The inhibition of the angiotensin converting enzyme
by compounds of formula I can be measured in vitro with
isolated angiotensin converting enzyme from rabbit lungs
following the procedure described by Cushman and Cheung
[Biochem. Pharmacol., 20, 1637 (1971], and with an excised
smooth muscle assay [E. O'Keefe, et al., Federation Proc.
31, 511 (1972)] in which these compounds have been shown to
be powerful inhibitors of the contractile activity of
angiotensin I and potentiators of the contractile activity
of bradykinin.
The administration of a composition containing one or
a combination of compounds of formula I or physiologically
acceptable salt thereof to the species of hypertensive mammal
alleviates or reduces angiotensin dependent hypertension. A
single dose, or preferably two to four divided daily doses,
provided on a basis of about 5 to 1000 mg. per kilogram per
day, preferably about 10 to 500 mg. per kilogram per day is
appropriate to reduce blood pressure. The animal model
experiments described by S.L. Engel, T.R. Schaeffer, M.H. Waugh
~1~2~ HA142~f
and B. Rubin, Proc. Soc. Exp. Biol. Med. 143, 483 (1973)
serve as a useful guide.
The substance is preferably administered orally, but
parenteral routes such as subcutaneously, intramuscularly,
intravenously or intraperitoneally can also be employed.
The compounds of this invention can be utilized to
achieve the reduction of blood pressure by formulating in
compositions such as tablets, capsules or elixirs for oral
administration or in sterile solution or suspensions for
parenteral administation. About 10 to 500 mg. of a compound
or mixture of compounds of formula I or physiologically
acceptable salt is compounded with a physiologically
acceptable vehicle, carrier, excipient, binder, preservative,
stabilizer, flavor, etc., in a unit dosage form as called
for by accepted pharmaceutical practice. The amount of
active substance in these compositions ore preparation is
such that a suitable dosage in the range indicated is obtained.
The following examples are illustrative of the invention
and constitute preferred embodiments. All temperatures are
in degrees celsius.
EXAMPLE 1
3-(3-Acetylthiopropanoyl?-2~2-dimethyl-4-L-thiazolidine
carboxylic acid.
2,2-Dimethylthiazolidine-4-carboxylic acid (5.74 g) is
dissolved in 58 ml of anhydrous pyridine with heating. The
solution is chilled in an ice bath with stirring and 3-
acetylthiopropanoyl chloride (4.814 g) is added dropwise.
The bath is removed and the reaction mixture is kept overnight
at room temperature. The precipitate is filtered and the
--7--
~ 2~ HA142b-f
filtrate is concentrated to dryness in vacuo. The residue
is taken up into ethyl acetate and washéd with 10~ potassium
bisulfate and water. The ethyl acetate extract is dried
over magnesium sulfate and concentrated to dryness 1n vacuo.
This residue is triturated with ether, filtered and the
filtrate is concentrated to dryness, then crystallized from
acetonitrile to yield 2.63 g of 3-~3-acetylthiopropanoyl)-
2,2-dimethyl-4-L-thiazolidinecarboxylic acid, m.p. 126-127.
EXAMPLE 2
3-~3-Mercaptopropanoyl)-l-oxopropyl-2,2-dimethyl-4-L-thia-
zolidinecarboxylic acid.
3-~Acetylpropanoyl)-2,2-dimethyl-4-L-thiazolidine-
carboxylic acid (5.82 g) is diqsolved in a cold solution
of 15 ml of water and 15 ml of concentrated ammonium hydroxide
under argon and kept for thirty minutes at room temperature.
The reaction mixture is chilled and acidified with concentrated
hydrochloric acid. The crystals are chilled, filtered and
washed with water, yield 4.79 g, m.p. 132-136 (haze). This
is taken up in hot acetonitrile and the haze filtered. The
filtrate yields 3.4 g of 3-(3-mercaptopropanoyl)-1-oxopropyl-
2,2-dimethyl-4-L-thiazolidinecarboxylic acid, m.p. 135-136.
EXAMPLE 3
3-(3-Acetylthio-2-methylpropanoyl)-2,2-dimethyl-4-L-thia-
zolidinecarboxylic acid, isomer A.
2,2-Dimethyl-4-thiazolidinecarboxylic acid hydrochloride
(19.8 g) is dissolved in 200 ml of anhydrous pyridine with
stirring in an ice bath. To this 3-acetylthio-2-methylpropanoyl
chloride (18.0 g) is added dropwise. The reaction mixture
is stirred overnight at room temperature. The precipitate
is filtered and the filtrate concentrated to dyrness ln vacuo.
~ * ~ HA142b-f
The residue is dissolved in ethyl acetate, washed with 10%
potassium bisulfate, water, dried over magnesium sulfate and
concentrated to dryness _ vacuo to obtain 31 g of crude
3-(3-acetylthio-2-methylpropanoyl)-2,2-dimethyl-4-L-thia-
zolidinecarboxylic acid, isomer A. The dicyclohexylamine
salt is obtained by adding the free acid and dicyclohexyl-
amine to acetonitrile, yield 24 g. The salt is recrystallized
from 700 ml of acetonitrile to yield 18.2 g, m.p. 197-198.
The salt is converted back to the free acid by dissolving
in ethyl acetate and 10% potassium bisulfate then crystallizing
from 100 ml of acetonitrile to yield 8.9 g. m.p., 171-172.
EXAMPLE 4
- 3-(3-Acetylthio-2-methylpropanoyl)-2,2-dimethyl-4-L-thia-
zolidinecarboxylic acid, isomer B.
The mother liquors from the preparation of the dicyclo-
hexylamine salt obtained in Example 3 are concentrated to
dryness ln vacuo. The residue is added to ethyl acetate and
10% potassium bisulfate, thencrystallized from 80 ml of
acetonitrile to obtain 7.5 g of 3-(3-acetylthio-2-methyl-
propanoyl)-2,2-dimethyl-4-L-thiazolidinecarboxylic acid,
isomer B, m.p. 156-157.
EXAMPLE 5
3-(3-Mercapto-2-methylpropanoyl)-2~2-dimethyl-4-L-thia
zolidinecarboxylic acid, isomer A.
3-(3-Acetylthio-2-methylpropanoyl)-2,2-dimethyl-4-L-
thiazolidinecarboxylic acid, isomer A (5 g) is dissolved in
a cold solution of 15 ml of water and 15 ml of concentrated
ammonium hydroxide while under a blanket of argon. After
thirty minutes, it is chilled and acidified with concentrated
hydrochloric acid. The crystalline precipitate is filtered
_g _
~1~28 ~æ HA142b-f
and washed with water. The product, 3-(3-mercapto-2-methyl-
propanoyl)-2,2-dimethyl-4-L-thiazolidinecarboxylic acid,
isomer A, is recrystallized from 40 ml of acetonitrile (haze
filtered),yield 4.2 g, m.p. 174-175.
EXAMPLE 6
3-t3-Mercapto-2-methylpropanoyl)-2,2-dimethyl-4-L-thia-
zolidinecarboxylic acid, isomer B.
3-(3-Acetylthio-2-methylpropanoyl)-2,2-dimethyl-4-L-
thiazolidinecarboxylic acid, isomer B (4 g) is dissolved in
a cold solution of 6 ml of water and 6 ml of concentrated
ammonium hydroxide under an argon blanket. After thirty
minutes at room temperature, it is chilled or acidified with
concentrated hydrochloric acid. The crystalline precipitate
is filtered and washed with water. The product, 3-(3-mercapto-
2-methylpropanoyl)-2,2-dimethyl-4-L-thiazolidinecarboxylic
acid, isomer ~, is recrystallized from acetonitrile
(insoluble filtered), yield 3.7 g, m.p. 197-198.
EXAMPLE 7
3-(3-Acetylthiopropanoyl)-5,5-dimethyl-4-DL-thiazolidine-
carboxylic acid.
A) 5,5-Dimethyl-4-thiazolidinecarboxylic acid
D,L-Penicillamine (20 g, 134 mmol) is dissolved in 134 ml
of lN hydrochloric acid at room temperature, and 40 ml (492
mmol) of 37% aqueous formaldehyde are added. After thirty
minutes, sodium acetate (11 g 134 mmol) is added, and the
reaction mixture is stirred at room temperature overnight.
After filtering, the solid is washed with ice cold 50% aqueous
ethanol, and dried in vacuo to yield 14.4 g of the product,
5,5-dimethyl-4-thiazolidinecarboxylic acid, m.p. 209-210.
After concentrating in vacuo, the mother liquor i5 triturated
--10--
~z~
HAl42b-f
with 95% ethanol to afford an additional 1.4 g of product,
m.p. 212-213; total yield: 15.8 g (73%).
B) 3-(3-Acetylthiopropanoyl)-5,5-dimethyl-4-DL-thia-
zolidinecarboxylic acid.
The product of part A (6 g, 37.2 mmol) is dissolved in
a mixture of 3.5 g (41.4 mmol) of sodium bicarbonate in 42 ml
of tetrahydrofuran and 41 ml of water. 3-(Acetylthiopropanoyl
chloride (5.5 ml, 41.1 mmol) in 5.5 ml of ether is added
dropwise, followed by titration with 2Nsodium hydroxide,
keeping the pH between 6 and 7. The reaction is stirred for
thrity minutes after completion of the addition, then quenched
with 100 ml of hydrochloric acid. The mixture is extracted
with 2 X 250 ml of ether acetate and the organic extracts
are washed with 100 ml portions of water and brine, dried
with sodium sulfate, and stripped to dryness ln vacuo. The
resulting oil solidifies upon standing at room temperature
to yield 11.0 g of crude 3-(3-acetylthiopropanoyl)-5,5-
dimethyl-4-DL-thiazolidinecarboxylic acid. RecrystallizatiOn
from ether petroleum ether gives 7.9 g (73%) of product,
m.p. 99-100.5.
EXAMPLE 8
3-(3-Mercaptopropanoyl)-5,5-dimethyl-4-DL-thiazolidinecarboxylic
acid.
A suspension of 2.91 g (10 mmol) of 3-(3-acetylthio-
propanoyl)-5,5-dimethyl-4-thiazolidinecarboxylic acid in 8 ml
of water is stirred rapidly at room temperature under a blanket
of argon. The stirred suspension is treated with 8 ml of
ca. 58% aqueous ammonium hydroxide, added dropwise over a
period of about one minute. The non-homogeneous solution is
stirred under argon for thirty minutes, then chilled and
~11 -
;~lS~28~
HA142b-f
acidified with concentrated hydrochloric acid. The aqueous
solution is extracted with 40 ml and 30 ml portions of ethyl
acetate. ~he combined organic solutions are washed with 5 ml
of water, 10 ml of brine, dried (Na2S04) and concentrated
in vacuo to 2.74 g of crude oil. When the oil is treated
with ca. 50 ml of (4:6), ethyl acetate/hexanes, rapid
crystallization induced by scratching yields 1.88 g of a
light, white solid 3-(3-Mercaptopropanoyl)-5,5-dimethyl-
4-DL-thiazolidinecarboxylic acid (75%), m.p. 100-101.5
Tlc, Rf=0.46 (silica gel; 60:20:6:11, EtOAc:pyridine:MeOH:H20).
EXAMPLE 9
DL-3-(3-Mercapto-2-methylpropanoyl)-2,2,5,5-tetramethyl-4-
DL-thiazolidine carboxylic acid.
By substituting 2,2,5,5-tetramethyl-4-thiazoledinecar-
boxylic acid for the 2,2-dimethyl-4-thiazolidinecarboxylic
acid hydrochloride in the procedure of Example 5 and then
submitting the product to the procedure of Example 5 3-[3-
(acetylthio)-2-methylpropanoyl]-2,2,5,5-tetramethyl-4-DL-
thiazolidinecarboxylic acid and 3-(3-mercapto-2-methylpropanoyl)-
2,2,5,5-tetramethyl-4-DL-thiazolidinecarboxylic acid are
obtained.
EXAMPLE 10
3-(3-Mercapto-2-methylpropanoyl)-2-ethyl-2-methyl-4-L-
thiazolidinecarboxylic acid.
By substituting 2-ethyl-2-methyl-4-L-thiazolidinecarO
boxylic acid for the 2,2-dimethyl-4-L-thiazolidinecarboxyliC
acid hydrochloride in the procedure of Example 3 and then
submitting the product to the procedure of Example 5, 3-~3-
(acetylthio)-2-methylpropanoyl]-2-ethyl-2-methyl-4-L-thiazoli-
-12-
~l~Z~ HA142b-f
dinecarboxylic acid and 3-(3-mercapto-2-methylpropanoyl)-2-
ethyl-2-methyl-4-L-thiazolidinecarboxylic acld are obtained.
EXAMPLE 11
3-t3-Mercaptopropanoyl)-2-ethyl-5,5-dimethyl-4-DL-thiazolidine
carboxylic acid.
By substituting 2-ethyl-5,5-dimethyl-4-DL-thiazolidine
carboxylic acid for the 5,5-dimethyl-4-thiazolidinecarboxylic
acid in the procedure of Example 7 and then submitting the
product to the procedure of Example 8, 3-[3-(acetylthio)
propanoyl]-2-ethyl-5,5-dimethyl-4-DL-thiazolidinecarboxylic
acid and -3-(3-mercaptopropanoyl)-2-ethyl-5,5-dimethyl-4-
DL-thiazolidinecarboxylic acid are obtained.
EXAMPLE 12
3-[2-(Mercaptomethyl)-3-mercaptopropanoyl]-5,5-dimethyl-4-DL-
thiazolidinecarboxylic acid.
By substituting 5,5-dimethyl-4-DL-thiazolidinecarboxylic
acid for the 4-L-thiazolidinecarboxylic acid in the procedure
of Example 9 and then submitting the product to the procedure
of Example lO, 3-[2-(acetylthiomethyl)-3-acetylthiopropanoyl]-
5,5-dimethyl-4-DL-thiazolidinecarboxylic acid and 3-[2-
(mercaptomethyl)-3~mercaptopropanoyl]-5,5-dimethyl-4-DL-thia-
zolidinecarboxylic acid are obtained.
EXAMPLE 13
4-(3-Mercapto-2-methylpropanoyl)-5,5-dimethyl-1,4-L-thiazane-
3-carboxylic acid.
By substituting 5,5-dimethyl-1,4-L-thiazane-3-carboxylic
acid for the 2,2-dimethyl-4-L-thiazolidinecarboxylic acid
hydrochloride in the procedure of Example 3 and then submitting
the product to the procedure of Example 5, 4-[3-(acetylthio)-
2-methylpropanoyl]-5,5-dimethyl-1,4-L-thiazane-3-carboxylic
~ HA142b-f
acid and 4-(3-mercapto-2-methylpropanoyl)-5,5-diemthyl-1,
4-L-thiazane-3-carboxylic acid are obtained.
EXAMPLE 14
Ethyl-2,2-dimethyl-1,4-DL-thiazane-3-carboxylate
A mixture of DL-penicillamine ethyl ester hydrochloride
(1.133 mole) and triethylamine (0.4 mole) in chloroform (200
ml) is added to a ~olution of ethylene dibromide (0.133 mole)
in chloroform:benzene (3:5; 120 ml). The mixture if refluxed
for one hour and then stirred at room temperature for sixteen
hours. The precipitate is filtered off, and the filtrate is
concentrated in vacuo and then distilled to give ethyl 2,2-
dimethyl-1,4-DL-thiazane-3-carboxylate.
EXAMPLE 15
4-(3-Mercapto-2-methylpropanoyl)-2,2-dimethyl-1,4-DL-thiazane-
3-carboxylic acid.
By substituting ethyl 2,2-dimethyl-1,4-DL-thiazane-3-
carboxylate for the 2-thiazolidinecarboxylic acid methyl ester
in the procedure of Example 4, and then submitting the product
to the procedure of Example 5, ethy~4-[3-(acetylthio)-2-
methylpropanoyll-2,2-dimethyl-1,4-DL-thiazane-3-carboxylate
and 4-(3-mercapto-2-methylpropanoyl)-2,2-dimethyl-1,4-DL-
thiazane-3-carboxylic acid are obtained.
EXAMPLE 16
3,3'-[Dithiobis-(3-propanoyl)]-bis-(2,2-dimethyl)-L-thiazolidine-
4-carboxylic acid.
By sub~tituting 3-(3-mercaptopropanoyl)-2,2-dimethyl-L-
thiazolidine-4-carboxylic acid for the 3-(3-mercaptopropanoyl)-
L-thiazolidine-4-carboxylic acid in the procedure of Example 25,
3,3'-[Dithiobis-~3-propanoyl)]-bis-(2,2-dimethyl)-L-thia-
zolidine-4-carboxylic acid is obtained.
-14-
~2~Z HA142~ -f
EXAMPLE 17
l-Oxo-4-(3-mercapto-2-methylpropanoyl)-5,5-dimethyl-1,4-L-
thiazane-3-carboxylic acid.
By substituting l-oxo-5,5-dimethyl-1,4-L-thiazane-3-
carboxylic acid for the 2,2-dimethyl-4-L-thiazolidine-
carboxylic acid in the procedure of Example 3 and then
submitting the product to the procedure of Example 5, 1-
oxo-4-(3-acetylthio-2-methylpropanoyl)-5,5-dimethyl-1,4-L-
thiazane-3-carboxylic acid and 1-oxo-4-(3-mercapto-2-methyl-
propanoyl)-5,5-dimethyl-1,4-L-thiazane-3-carboxylic acid are
obtained.
.
-15-
