THE MODE OF OXIDATION IN THE ANIMAL ORGANISM OF PHENYL DERIVATIVES OF FATTY ...

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THE MODE OF OXIDATION IN THE ANIMAL ORGANISM
OF PHENYL DERIVATIVES OF FATTY ACIDS. PARTV.
STUDIES ON THE FATE OF PHENYLVALERIC ACID AND ITS
DERIVATIVES.
BY H. D. DAKIN.
(From the Laboratory of Dr. C. A. Herter, New York.)
(Received for publication, April 24, 1909.)
The main point of interest in studies upon the fate in the ani-
mal organism of phenyl derivatives of fatty acids concerns the
relation which the results bear to fatty acid metabolism in general.
Investigations of the mode of oxidation of phenyl derivatives
with side chains containing three and four carbon atoms have
already been made and it therefore remained to try to trace some
of the steps in the catabolism of acids with five carbon atoms in
their side chain. The results of such an investigation are recorded
in the following paper. Since the theoretical possibilities for
the occurrence of different types of oxidation are greater than in
the case of the acids previously studied, several new factors
require consideration.
The only acid of the series in question whose fate in the animal
body has been determined is phenylvaleric acid. This substance
was fed to dogs by Knoopl and he observed a subsequent excre-
tion of hippuric acid. This result was of great significance when
contrasted with his observations on the excretion of phenaceturic
acid following the administration of phenylbutyric acid. In
Knoop’s paper no attempt is made to picture the mechanism of
the reaction. The acid is stated to be “oxidiert am 6 C-atom,”
C6H,, CH,.CH,.CH,.CH,.COOH~C~H~.COOH-tCeH,.CONH.CH,.COOH
1 Der Abban aroritatischen Fettsciuren im Tierkdrper, Freiburg, Ernst
Kuttraff, 1904.
(221)
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Derivatives of Phenylvaleric Acid
but it is clearly to be inferred that Knoop recognized the possi-
bility of the oxidation being indirect, i.e., that the oxidation at
the 6 carbon atom was not the primary process.
A further study of the fate of phenylvaleric acid, when admin-
istered by subcutaneous injection in relatively large doses to
cats, has clearly shown that the conversion of phenylvaleric acid
into hippuric acid is an indirect process, i.e.. the aliphatic side
chain is not primarily oxidized in the 6 position. The evidence
for this belief is based on the detection of phenyl-P-oxypropionic
acid, cinnamoylglycocoll and acetophenone in the urine of animals
that had received injections of sodium phenylvalerate in doses of
about 0.8 gram per kilo. These substances are all intermediary
products in the catabolism of phenylpropionic acid and by their
further oxidation in the body yield hippuric acid. It is therefore
probable that the phenylvaleric acid primarily undergoes oxida-
tion so as to yield phenylpropionic acid, so that the original side
chain of the phenylvaleric acid with five carbon atoms is con-
verted into phenylpropionic acid with a three-carbon side chain,
and this by further oxidation into benzoic acid with a one-carbon
side chain,
C6H,.CHz.CH,.CH, CH2.COOH-+C& CH2.CHZ.COOH+CeH5.COOH
I I
Judging by analogy with other similar reactions’ it was prob-
able that the conversion of phenylvaleric acid into phenylpro-
pionic acid would take place through the intermediate formation
of phenyl-&oxyvaleric acid, and this was made still more probable
through the observation that phenyl-,l?-oxyvaleric acid, on admin-
istration to cats under the same conditions as those employed in
the case of phenylvaleric acid, resulted in the excretion of hip-
puric acid with the same intermediary products as were detected
in the latter case, namely, phenyl-P-oxypropionic acid, acetophe-
none and cinnamoylglycocoll. It is probable, therefore, that
phenyl$-oxyvaleric acid represents the first step in the catabolism
of phenylvaleric acid. It is theoretically probable that the cor-
responding /3-ketonic acid would represent the next stage in the
1 Such as the conversion of phenylbutyric acid into phenaceturic acid
through phenyl-/&oxybutyric (this Journul, v, p. 173) and the oxidation
of phenylpropionic acid to hippuric acid through phenyl-b-oxypropionic
acid.
H. D. Dakin
223
oxidation, but of this no proof is forthcoming. That the/Lketonic
acid, if formed, does not part with carbon dioxide, so as to yield
the corresponding ketone,’
C,H,.CH, .CH, .CO .CH, .COOH+C,H,.CH, .CO. CH,
was proved by showing that the ketone benzylacetone is oxidized
in the animal body to phenylacetic acid, which is excreted in
the usual way as phenaceturic acid :
This reaction is analogous to the oxidation of phenylacetone,
which was shown to yield hippuric acid when administered to a
dog.2
If the ,&ketonic acid, C,H,.CH2.CH,.C0.CH2.COOH, actually
represents the first stage in the oxidation of phenyl-,&oxyvaleric
acid, it is much more probable that this substance undergoes
oxidation or hydrolysis so as to yield phenylpropionic acid with
loss of two carbon atoms. Such a reaction is thoroughly in
accord with the behavior of /?-ketonic acids on oxidation or
hydrolysis in vitro, and similar reactions are known to occur in
the body. Thus it was found that benzylaceto-acetic ester,
when administered to cats or dogs, gave hippuric acid, a result
which is most readily explained on the assumption that phenyl-
propionic acid or a derivative of this acid is first formed, which is
then oxidized to benzoic acid and excreted as hippuric acid in
the usual way :
CH,.CO ;CH.COOH --+ C,H,.CH,.CH,.COOH + C,H,.COOH
I -+ C,H,.CO.NH.CH,.COOH
CH,
With the assumption that phenylpropionic acid is formed
from phenyl+oxyvaleric acid, with the possible intermediary
formation of phenylpropionylacetic acid, the remaining steps in
the catabolism of phenylvaleric acid will be identical with those
1A reaction analogousto the conversiono f aceto-acetica cid into acetone.
aT his Journal, v, p. 183.
224 Derivatives of Phenylvaleric Acid
of phenylpropionic acid. The whole series of changes may be
represented as follows :
C,H, .CH, . CH, CH, . CH,. COOH
(Phenylvaleric acid)
C,H,.CH,.CH,.CHOH.CH,.COOH
(Phenyl-P-oxyvaleric acid)
[C,H,.CH,&.C0.CH2.COOH] ?
(Phenylpropionylacetic acid)
1
C,H,.CH,.CH,.COOH
(Phenylpropionic acid)
I
C,H,.CHOH~CH,.COOH + C,H,.CH:CH.COOH
(phenyl-oxypro- (Cinnamic Acid)
pionic acid)
I 1
C,H,. CO. CH,. COOH C,H,. CH . CH CO. NHCH, .COOH’
(Benzoylacetic acid) (Cinnamoylglycocoll)
I
C,H,.CO.CH,
(Acetophenone)
-1
C,H,COOH
(Benzoic acid)
I
C,H,.CO.;H.CH,.COOH
(Hippuric acid)
In order to determine whether this mode of oxidation of
the side chain of phenylvaleric acid by which the four carbon
groups are removed in two pairs, constituted a general type of
reaction it was decided to investigate the fate of a number of
derivatives of phenylvaleric acid. The substances examined
were as follows :
Phenyl-a-b-pentenic acid C,H,.CH,.CH,.CH : CH .COOH
Phenyl-b-r-pentenic acid C,H,.CH,.CH : CH.CH,.COOH
Cinnamylidene acetic acid C,H,.CH:CH.CH :CH.COOH
Phenyl-r-oxyvaleric acid C,H,.CH,.CHOH.CH,.CH,.COOH
Cinnamylidenemalonic acid C,H,. CH : CH .CH : C(COOH),
1 The mode of formation of cinnamoylglycocoll is not clear. A discus-
sion of this question is contained in the preceding paper (p. 204); it prob-
ably is not derived from the direct coupling of glycocoll and cinnamic
acid as represented in the diagram which is intended merely to show the
structural relations of the substance.
H. D. Dakin 225
Of these substances the first three were oxidized to benzoic acid
and excreted as hippuric acid in the urine. In the case of each of
these substances evidence was obtained that the oxidation took
place in such a fashion that the five-carbon atom side-chain was
converted primarily into a three-carbon atom side-chain and the
latter again oxidized with a further loss of two carbon groups.
Acetophenone and phenyl+oxypropionic acid, and probably
cinnamoylglycocoll, were detected in the urine in each case.
The naode of oxidation of these three substances is completely analo-
gous to that of pheaylvaleric acid and pheutyl-/?-oxyvaleric acid.
The two remaining substances, phenyl-y-valeric acid and cinna-
mylidenemalonic acid, were scarcely attacked when administered
to cats, for the greater pait was excreted unchanged. The resist-
ance of phenyl-T-Valerie acid to oxidation in the body is analo-
gous to the similar behavior of phenyl-y-oxybutyric acid. It
appears that y-oxy-acids are commonly oxidized in the body
with difficulty and appear to be converted into lactones and ex-
creted in the urine. The lactones as a class are much more
resistant to oxidation in vitro than are the oxy-acids from which
they are derived through loss of a molecule of water.
To sum up: evidence has been obtained that five acids of the
type Ph. C.C.C.C.C.COOH undergo oxidation in the body in such
a way that four carbon atoms are removed from the side chain
in two pairs. In every case benzoic acid was the end product.
Ph. C. ; C.C. 1 C.COOH+Ph. C. / C.COOH+Ph.COOH
This type of oxidation may be termed successive ,&oxidation, and
I see no reason to suppose that it is not a general biochemical
reaction. Assuming that this is so, a ready explanation may be
given of the extraordinarily interesting results obtained by
Embdenl and his co-workers through the perfusion of surviving
livers with salts of fatty acids. He finds that of the normal
fatty acids, butyric, valeric, caproic, heptylic, octylic, nonylic
and decoic, those and only those with an even number of carbon
atoms yield aceto-acetic acid and acetone. These substances
1 Hofmeister’s Beitrtige, viii, pp. 121, 129; xi, p. 318,
Derivatives of Phenylvaleric Acid
226
are undoubtedly derived from ,f%oxybutyric acid and the latter
from butyric acid. Hitherto there has been no evidence to
decide whether the conversion of an acid such as decoic acid into
butyric acid which necessitates the removal of six carbon atoms
is effected in one step or is due to successive removal of two or
some multiple of two carbon atoms at a time. The results ob-
tained with the derivatives of phenylvaleric acid make it very
probable that the catabolism of a fatty acid group, -CH,.(CH,j,.
COOH, is effected by the successive removal of two carbon groups
at a time, and Embden’s results are in complete harmony with
this view.
If this hypothesis be essentially correct it follows that ordi-
narily only one molecule of either /3-oxybutyric acid, aceto-
acetic acid, or acetone, can result from the catabolism of one
molecule of a fatty acid such as stearic acid. On the other hand
it must be recalled that the synthetic formation of aceto-acetic
acid from simpler substances containing two carbon atoms
appears probable.’ The observed excretion of acetophenone
and phenyl$-oxypropionic acid following the administration of
cinnamylideneacetic acid is of interest when compared with the
similar excretion of these substances when cinnamic acid is in-
jected. It is probable that the cinnamylideneacetic acid is
oxidized through the stage of cinnamic acid through P-oxidation
and that a second ,&oxidation converts the latter into benzoic
acid which is excreted in the form of hippuric acid.
CeH,.CH: CH:; CH.COOH-+C~H,.CH: i CH.COOH+C6H,: COOH
EXPERIMENTAL.
Preparation of phenylvaleric acid. The phenylvaleric acid
used in the following experiments was obtained by the following
series of reactions. Cinnamic aldehyde was condensed with
malonic acid in the presence of a trace of aniline. The resulting
cinnamylidenemalonic acid was reduced to phenylpropenyl-
malonic acid with sodium amalgam according to Thiele and
Meisenheimer’s method,2 and on boiling with water gave phenyl-
1 Friedmann: Hofmeister’s Beitrcige, xi, p. 202.
2 Annulen der Chemie, cccvi, p. 247.
H. D. Dakin 227
&y-pentenic acid. The latter substance was partially converted
into phenyl-c+pentenic acid’ by boiling with caustic soda as
described by Fittig and Hoffmann and the latter reduced with
sodium amalgam to the desired phenylvaleric acid. The changes
may be represented as follows:
GHj.CH:CH .CHO + CH,. (COOH)o-+CeHs.CH: CH.CH: C(COOH),-+
CaH,.CH,.CH: CH.CH.(COOH)+CeH6.CHz.CH: CH.CH,.COOH+
CaH,.CH,.CH,.CH: CH.COOH--+CsH,.CH,.CH,.CH,.CH,.COOH
The phenylvaleric acid crystallized in glistening platelets,
melting point 58-5g0, and its properties in every way agreed with
those described by Fittig and Hoffmann. The practical details
of the preparations with the exception of that of cinnamylidene-
malonic acid are omitted, since they may be found in the various
references given and only immaterial modifications of the original
methods were made.
The employment of cinnamylidenemalonic acid instead of
cinnamylideneacetic acid as starting material for the synthesis
of phenylvaleric acid has very decided advantages, for it is obtained
in better yield from the cinnamic aldehyde from which they are
both prepared and in addition it is more readily reduced with
sodium amalgam. Cinnamylidenemalonic acid has hitherto
been prepared by either heating cinnamic aldehyde with malonic
acid in the presence of acetic acid or by Knoevenagel’s method,
in which 1-2 molecules of alcoholic ammonia is used to effect the
condensation between the aldehyde and malonic acid. It was
found that a preferable method was to dissolve malonic acid
(I molecule) in a minimum amount of hot go per cent alcohol,
then add cinnamic aldehyde (I molecule) and a drop or two of
aniline, and let the whole stand for several hours at the ordinary
temperature. On addition of the aniline, the solution becomes
dark orange colored and after a few minutes crystals of cinna-
mylidenemalonic acid begin to separate and finally the whcle
mixture becomes almost solid. The mass is then ground up
with go per cent alcohol and well drained, using suction. The
cinnamylidenemalonic acid is almost pure and after a single
1 Phenyl-&oxyvaleric acid is formed simultaneously.
a Ann&en der Chenzie, cclxxxiii, p. 314.
228 Derivatives of Phenylvaleric Acid
crystallization from alcohol melts at 208-210~. The yield is
about 70 per cent of theory when using quantities of about 50
grams of material, at each operation.
Fate of phenylvaleric acid. Phenylvaleric acid (3.2 grams)
was exactly neutralized with aqueous caustic soda, using a trace
of phenolphthalein as indicator and the sterile solution given
subcutaneously to a cat (4.1 kilos), the urine being collected dur-
ing the following forty-eight hours. The first portion of urine
collected gave on distillation a strong iodoform reaction. The
urines before the injection and on the second day afterwards
gave practically negative results. The iodoform reaction was
undoubtedly mainly, if not exclusively due to acetophenone.
The distillate was treated with a cold filtered solution of para-
nitrophenylhydrazine acetate. A turbidity at once appeared
and on cooling the solution for a short time in ice a small but
definite precipitate was obtained. The precipitate was filtered
off and washed with cold water. As the amount was too small
to purify by recrystallization the precipitate was distilled from
a small flask with IO per cent sulphuric acid. The first few
drops of the distillate smelt of acetophenone, and, in addition to
the iodoform reaction, gave the characteristic reaction with
sodium nitroprusside.
The urine after distillation was concentrated, acidified with
phosphoric acid and extracted in the usual way1 with ether
containing a little alcohol. The extract was steam-distilled
and the solution after treatment with charcoal was concentrated
and allowed to crystallize. The crystals which separated melted
indefinitely between 165’ and 180’ and proved to be a mixture
of cinnamoylglycocoll and hippuric acid. The two substances
were separated by fractional crystallization from boiling water
in the manner described in the preceding paper. The cinna-
moylglycocoll separated first from the warm solution in long
needles which were purified by recrystallization. The amount
of pure crystalline cinnamoylglycocoll recovered was o. I 7 gram.
The loss during precipitation must have been very considerable.
The cinnamoylglycocoll was identified by its crystalline form,
(melting point 192-193'), by the formation of cinnamic acid
1 The details of the methods of urine analysis were identical with those
previously employed and described.
H. D. Dakin 229
(melting point 13 2.3')on hydrolysis with concentrated hydrochloric
acid, by its oxidation to benzaldehyde on treating the alkaline
solution in the cold with dilute potassium permanganate and by
the following analysis.
0.1497 gram gave NH, = 0.01036 gm. (Kjeldahl) : N = 6.92 per cent
C,,H,,O,N requires 6.83 per cent.
The melting point of the substance when mixed with cinna-
moylglycocoll prepared synthetically was unchanged.
On evaporating the mother liquor from the first crystalliza-
tion through which most of the cinnamoylglycocoll has been
removed, crystals were obtained which gave pure hippuric acid,
melting point 186.7’, after two additional crystallizations.
Analysis:
0.1510 gm. gave NH, = 0.0119 gm. (Kjeldahl): N = 7.88 per cent
CsH,O,N requires 7.82 per cent N.
The mother liquor from the hippuric acid crystals was exam-
ined in the way described in the previous paper (p. 2 IO) for phenyl-
,B-oxypropionic acid. Decided qualitative evidence of its pres-
ence was obtained as follows : The aqueous solution of the ether-
eal extract was laevorotatory (0.327, it gave acetophenone
when oxidized with chromic acid and cinnamic acid, melting
point 131°-132', when hydrolyzed with hydrochloric acid. The
amount of oxy-acid was not large.
Two similar experiments were made in which sodium phenyl-
valerate was given to cats. In one case approximately the same
dose was employed (0.7 gram per kilo) and the results were identi-
cal with the foregoing except that the cinnamoylglycocoll could
not be isolated in crystalline form. Decided qualitative evidence
of its presence was obtained, however. In the other case 1.0
gram of phenylvaleric acid was given in the form of the sodium
salt to a cat weighing 4.7 kilos. Almost the whole of the sub-
stance was converted into hippuric acid.
Phenyl-P-oxyvaleric acid. This acid was prepared by boiling
phenyl-P-y-pentenic acid for five days with IO per cent caustic
soda (IO molecules) according to Fittig and Hoffmann’s method.’
It was separated from the other acids by crystallization from
~Annalen der Chemie, cclxxxiii, p. 313.
230 Derivatives of Phenylvaleric Acid
carbon bisulphide. The acid was obtained in very well formed
crystals, melting point 13 IO.
EXPERIMENT I. Three and one-half grams of the acid were
neutralized with caustic soda solution and injected subcutane-
ously into a female cat weighing 4.5 kilos. The urine (48 hours)
was analyzed exactly as in the preceding case. On distillation
decided evidence of the presence of acetophenone was obtained.
The ether extract after crystallization gave a considerable
amount of unchanged phenyl-P-oxyvaleric acid, so the whole
extract was neutralized with soda and injected into another
cat (5 kilos). The urine formed during the next 48 hours was
again analyzed. The results were entirely similar to those ob-
tained in the case of phenylvaleric acid, although the amount
of cinnamoylglycocoll was unfortunately too small for analysis.
It was readily identified, however, by its melting point, 192-
193', by the reaction with potassium permanganate and by the
formation of cinnamic acid (melting point 132’) on hydrolysis
with hydrochloric acid. The hippuric acid was recrystallized
repeatedly until it no longer reduced permanganate in alkaline
solution. 0.6 gram of the pure substance, melting point 186-
187~7 was obtained.
Analysis.
0.2000 gram gave NH, = 0.0156 gm. N = 7.80 per cent
C,H,O,N requires 7.82 per cent.
Some of the qualitative reactions for phenyl+oxypropionic
acid which were obtained were invalidated owing to the probable
presence of unchanged phenyl+oxyvaleric acid, but the forma-
tion of acetophenone on oxidation with chromic acid must be
regarded as fair presumptive evidence of its presence. In addi-
tion to the foregoing another experiment was made in which
I. 7 grams were given to a cat weighing 4 kilos. The results were
similar except that no cinnamoylglycocoll could be detected.
Much hippuric acid (0.5 gram) was obtained.
Phenyl-qbpeentenic acid. This acid was obtained as a by-prod-
uct in the preparation of phenyl$-oxyvaleric acid. The acid
was crystallized from ether and melted at 104’ and exactly cor-
responded with Fittig and Hoffmann’s description of the acid.
Three grams of the acid were converted into the sodium salt and