European Journal of

Clinical Pharmacology
by Springer-Verlag 1977

Europ. J. clin. Pharmacol. 11,367-375 (1977)

Biotransformation and Pharmacokinetics of Acenocoumarol (Sintrom )

in Man
W. Dieterle, J.W. Faigle, C. Montigel, M. Sulc, and W. Theobald
Research and Medical Departments, Pharmaceuticals Division, Ciba-GeigyLimited, Basle, Switzerland

Summary. The absorption, biotransformation and

elimination of the anticoagulant acenocoumarol,
3 - [a- ( 4 ' - nitrophenyl) -/3- acetylethyl] - 4 - hydroxycoumarin, have been studied by oral administration
of 12 mg of a 14C-labelled preparation to two male
volunteers. Absorption from the gastro-intestinal
tract was rapid and the plasma concentration of unchanged drug reached a maximum of 169 and 412
ng/ml, respectively, after 3 hours. The elimination
half-life in the two subjects, calculated from the decline between 6 and 24 h, was 8.7 and 8.2 hours. A
constant proportion of 98.7% of the drug was bound
in vitro to serum proteins over a concentration range
of 0.021-8.34 gg/ml, with little interindividual variation. The major portion of the binding was to human
serum albumin (97.5%) at two classes of binding sites:
association constant KI = 1.04 105 l/mole (n I = 1)
and K2 -- 5.55 103 1/mole (n2 = 4). In addition to
unchanged acenocoumarol, four metabolites were determined in plasma by isotope dilution techniques: the
amino-, acetamido-, alcohol~- and alcohol2-metabolites. Of them, the amino-metabolite showed the highest concentration, namely 278 ng/ml, after 6 h in
Subject A, and 163 ng/ml after 10 hours in Subject B.
Judged from the integrated concentrations, the compounds analyzed accounted for 76 and 89%, respectively, of the total radioactivity in plasma. All the
metabolites detected in plasma showed anticoagulant
activity when tested in mice. The quantities of the
metabolites excreted in urine from 0-120 hours were
(Subject A/Subject B): acenocoumarol 0.3/0.2%,
amino-metabolite 12.3/7.7%, acetamido-metabolite
19.0/11.1%, alcoholl-metabolite 4.6/9.0%, alcohol:-metabolite 1.7/4.4%, 6-hydroxy-metabolite
6.9/18.3% and 7-hydroxy-metabolite 14.0/22.2%.
Key words: Acenocoumarol, excretory balance man,
pharmacokinetics, biotransformation, plasma protein
binding.

Acenocoumarol, the active ingredient of Sintrom ,

has the chemical formula 3-[a-(4'-nitrophenyl)-flacetylethyl]-4-hydroxycoumarin. The drug is an anticoagulant of the coumarin type and is used clinically
for prophylaxis and treatment of thromboembolic diseases [1]. No reliable data about the fate of the drug in
man have previously been published. The only report
available [2] was concerned with a pharmacokinetic
study, which was performed with the aid of a nonspecific photometric assay.
The present study was a detailed investigation of
pharmacokinetic and metabolic processes in man
following a single oral dose of 14C-labelled acenocoumarol.

Materials and Methods

Treatment of Subjects and Collection of Samples

Two male volunteers (Subject A: aged 52 y, 82 kg;
Subject B: aged 45y, 71 kg), who had not taken any
other medication for at least two weeks prior to the
experiment, received after fasting a single oral dose of
a4C-labelled acenocoumarol 12 mg cristalline material
in a gelatin capsule. The specific radioactivity of the
substance was 9.8 gCi/mg, corresponding to a total
radioactivity of 117.6 gCi per dose. Blood was collected after 0.5, 1, 2, 3, 6, 10, 24, 48 and 72 hours,
mixed with heparin and the plasma was separated
immediately. All urine and faeces were collected daily
for a period of 192 hours; on the first day urine was
collected in two lots, 0-6 and 6-24 h.

Radioactive Labelling and Radiometry

The acenocoumarol preparation administered was
labelled with 14C in positions 2 and 4 of the coumarin
moiety. The purity of the labelled substance was ex-

368

W. Dieterle et al.: Metabolism and Pharmacokinetics of Acenocoumarol

amined by thin-layer chromatography in systems CS 1

and CS 2 (see below), as well as by inverse isotope
dilution analysis, and was found to be better than
99%.
Radioactivity was measured with a Packard
Tricarb liquid scintillation spectrometer, Model 3380.
The samples were prepared as described previously [3].

Multiple Inverse Isotope Dilution Analysis in Plasma

and Urine
By use of the principle of inverse isotope dilution [4],
acenocoumarol and six presumed metabolites, viz. alcohol1-, alcohol2-, 7-hydroxy-, 6-hydroxy-, aminoand acetamido-metabolite (for formulae see below)
were specifically determined. The first five compounds were measured simultaneously by a thin-layer
chromatographic technique (Method I), and for the
latter two a liquid chromatographic technique
(Method II) was employed since the amine decomposed on TLC-plates.
Method I: After addition of 50-100 lxg of the
non-labelled form of each of the molecular species to
be determined (dissolved as sodium salt in water) to
1-2 ml of the biological specimen, the labelled compounds plus carrier were extracted with dichloromethane at pH 4. This was followed by a acid
specific back- and re-extraction. The extract was dried
over anhydrous NazSO4, evaporated in vacuo and the
residue submitted to two-dimensional thin-layer
chromatography on silica gel (first dimension: solvent
system CS 3; second dimension: CS 4). The individual
"diluted" pure compounds were eluted with methanol
+ 5% 2 M NaOH. To calculate the specific radioactivity, the total amount of each compound was measured by UV-spectrophotometry at the absorption
maximum of each particular compound and the
radioactivity determined by liquid scintillation
counting.
Method II: The biological specimen (1-2 ml) was
mixed with an aqueous solution (5 ml containing
200-300 ~tg) of the non-labelled form of the aminoand acetamido-compounds (dissolved as sodium salts)
and 0.3 M phosphate buffer pH 6.0 (5 ml) and extracted with dichloromethane. Subsequently, the
aqueous layer was adjusted to pH 4 with 0.5 M H 2 S O 4
and again extracted with dichloromethane. The
bulked extracts were washed with water, dried over
anhydrous Na2SO 4 and evaporated in vacuo. The residue was dissolved in 0.3 ml of solvent system CS 5 and
applied to a silica gel column (1.25 30 cm). Highresolution liquid chromatography was performed with
CS 5 at a flow rate of 2 ml/min, 5 atm pressure and
ambient temperature. The elution volume of the

acetamido-compound was 44 ml and for the aminocompound 58 ml. No interference was detected. For
details of chromatography and measurement of
specific radioactivity see reference [3].

Separation and Characterization of Radioactive

Substances in Urine
The radioactive substances contained in a 200 ml-aliquot of pooled 0-120 hour urine from both subjects
were preconcentrated by adsorption on Amberlite
XAD-2 resin and desorption with a linear gradient of
methanol in water and pure methanol. The volume
ratio of the urine sample vs. the resin bed was 2 : 1. The
total volume of the gradient was four times that of the
resin bed. The metabolite mixture (XAD-2 concentrate) thus obtained was characterized by thin-layer
chromatography on silica gel. Samples were spotted
and developed two-dimensionally with solvent systems CS 3 and CS 4. The radioactive spots on the plate
were visualized by autoradiography on a Kodirex Xray film (exposure 5 days) and compared with the
positions of reference compounds chromatographed
at the same time.
One of the prominent urinary metabolites was
isolated preparatively as outlined in Scheme 1.

Chromatography
Thin-layer chromatography (TLC) was carried out
using commercial silica gel plates (Antec SL 254).
Liquid chromatography (LC) was done on highresolution columns (2.5 50 cm, packed with
Kieselgel G (Merck), or 1.25 30 cm, packed with
Lichrosorb , 10 ~t, plus 15% gypsum (Merck). These
columns were prepared according to a procedure described by Sic and van den Hoed [5]. The chromatographic pump (CMP 2) and the columns (LC 1"
23" and LC 1/2" X 13") were purchased from Chromatronix. For low-resolution LC, Amberlite XAD-2
resin (coarse grade, 35-50 mesh; R6hm and Haas)
was used. TLC- and LC-separations were performed
with the following chromatographic systems (CS):
CS 1: Chloroform/methanol/
= 90/8/2
acetic acid
CS 2: Chloroform/cyclohexane/
= 50/50/20
acetic acid
CS 3: Benzene/acetone/water
= 20/10/0.1
CS 4: 1,2-dichloroethane/dioxane/ = 10/3/1
acetic acid
CS 5: 1,2-dichloroethane/dioxane/ = 100/50/2.5/1
formic acid/water
CS 6: Ethylacetate/acetone/water -- 72/24/4
CS 7: 1,2-dichloroethane/dioxane/ = 100/50/2.5/1
acetic acid/water

W. Dieterle et al.: Metabolism and Pharmacokinetics of Acenocoumarol

I

369

rine (pool0 120hours) : 11.6 1, 13.1 mg14C

AdsorptiOncol5umn
x onto100cmAmberliteXAD-2 resin and elution with water/methanol;

_[

Fraction 1:1.9 mg 14C

-[

Fraction 3:7.3 mg ~4C

Fraction 2:3.4 mg ~4C: Evaporation in vacuo, solution in 50 ml

0.01 M NaOH and adjustment to pH 4 with 1 M HC1; extraction with
3 x 100 ml dichloromethane

Water phase

Organic phase: 1.67 mg ~4C in 436 mg residue: LC on silica gel;

column: 2.5 x 50 cm; CS 6

Side fractions

L
1-

_l
-I

Side fractions

Main fraction: 0.51 mg 14C in 17.3 mg residue: LC on silica gel;

column: 1.25 x 30 cm; CS 7

Pure acetamido-metabolite: 0.46 mg14C

I
i

Scheme 1. Isolation of the acetamido-metabolite from pooled urine (0-120 hours) of Subjects A and B. The 14C-content has been
calculated as acenocoumarol

Synthetic reference compounds

T h e following eight unlabelled r e f e r e n c e c o m p o u n d s
w e r e used:
Acenocoumarol, G 23 350: 3 - [ a - ( 4 ' - n i t r o p h e n y l ) - f l acetylethyl]-4-hydroxycoumarin.
Amino-metabolite, C G P 8435: 3 - [ a - ( 4 ' - a m i n o phenyl)-/3-acetylethyl]-4-hydroxycoumarin.
Acetamido-metabolite, C G P 8436: 3 - [ a - ( 4 ' - a c e t amidophenyl)-/3-acetylethyl]-4-hydroxycoumarin.
Alcoholl+2-metabolite, C G P 8437 (diastereoisomeric
mixture)
3- [ a - ( 4 ' - n i t r o p h e n y l ) - y-hydroxybutyl]4-hydroxycoumarin.
Alcoholl-metabolite, C G P 8767: 3 [ a - ( 4 ' - n i t rophenyl)- y-hydroxybutyl]-4-hydroxycoumarin.
Alcohol2-metabolite: 3-[ a - ( 4 ' - n i t r o p h e n y l ) - g-hydroxybutyl]-4-hydroxycoumarin.

7-Hydroxy-metabolite,

C G P 8438: 3 - [ a - ( 4 ' - n i t rophenyl)-/3-acetylethyl]-4, 7 - d i h y d r o x y c o u m a r i n .

6-Hydroxy-metabolite: 3- [ a - ( 4 ' - n i t r o p h e n y l ) - f i acetylethyl]-4,6-dihydroxycoumarin.
T h e diastereoisomeric alcohols were designated 1
and 2, by analogy with assignment of the corresponding m e t a b o l i t e s of the structurally related c o m p o u n d warfarin [6].

Blood Coagulation Assay

In o r d e r to test the anticoagulant activity of the
m e t a b o l i t e s of a c e n o c o u m a r o l , m a l e mice (SPF, Animal B r e e d i n g Station, Sisseln) were used. T h e substances, dissolved as sodium salts, were a d m i n i s t e r e d
by s t o m a c h tube, either as single doses (100 m g / k g ) or

370

W. Dieterle et al.: Metabolism and Pharmacokinetics of Acenocoumarol

as repeated doses of 100 mg/kg initially, followed by

two doses of 10 mg/kg at 24 hour intervals. Citrated
plasma was prepared from blood obtained by cardiac
puncture during light ether anesthesia, 24, 48 and 72 h
after dosing. The coagulation time was determined
with thrombokinase "Geigy" (Ciba-Geigy) according
to Montigel and Pulver [7]. Five animals were used per
time and dose and the estimations were done in duplicate.

tO

% of dose

Total
excretion

8(~

......................

60

4G

~,/

Urine

SUBJECT A: 8 2 k g

r/Y
t/

/" ~ .. . . . . . . . . . . . . . . . . . . . .

.................

Faeces

/. /

20

/"
.fJ

Protein Binding Studies

The extent to which 14C-labelled acenocoumarol
(spec. activity 20.0 ~tCi/mg) was bound to human
serum proteins was investigated in vitro by means of
equilibrium dialysis at 37 C. Human serum from
blood of healthy donors and human serum albumin
(B-grade, tryst., from Calbiochem) dissolved in 0.067
M phosphate buffer pH 7.4, to concentrations of 33.2
g/1 and 2.1 g/l, respectively were used. Equilibrium
dialysis was carried out as described elsewhere [8]; for
the concentration range see "Results".

1
100

8 Days

%of dose

Tot a I
excretion

Urine

60
/1

40

SUBJECTB: 71 kg

Results

8 Days

Fig. 1. Cumulative urinary and faecal excretion of radioactive substances after oral administration of ~4C-labelled acenocoumarol
12 mg

Excretion in Urine and Faeces

The excretory balances of Subjects A and B are shown
in Figure 1.
The results indicate that excretion of acenocoumarol and its metabolites was not quite complete
at the end of the observation period. At that time, 8
days after oral administration, a total of 90.6 and
88.5 %, respectively, of the radioactive dose had been
eliminated. Of this, however, as much as 83.5 and
79.2% was recovered in excreta in the first three days.
In both subjects approximately 60% of the orally
administered dose appeared in the urine and this percentage may be considered, therefore, as the
minimum fraction absorbed.

Protein Binding Studies

Details of the protein binding of acenocoumarol are
summarized in Table 1.
When the binding data obtained at low albumin
concentration and increasing concentrations of
acenocoumarol were evaluated according to Scatchard [9], and Fletscher and Spector [10], two classes of
binding sites were found. They are characterized by
association constants K1 = 104400 + 5602 1/mole
and K 2 = 5554 + 92 1/mole (K i _+ SD). The corresponding number of binding sites was nl = i and n 2 =
4.
Products Excreted in Urine

Concentration in plasma
The results of the various analyses performed on plasma from Subjects A and B are shown in Figure 2.
The maximum plasma concentration of unaltered
drug was reached 3 hours after ingestion of
acenocoumarol 12 mg; the level was 169 ng/ml for
Subject A and 412 ng/ml for Subject B. All potential
metabolites which were specifically analyzed in plasma (amino-metabolite, acetamido-metabolite, alcoholl-metabolite and alcohol2-metabolite ) were
found to be present. Amongst them, the aminometabolite showed the highest peak level 278 ng/ml
and 163 ng/ml after 6 and 10 h; el. Fig. 2.

The results obtained by multiple inverse isotope dilution analysis of pooled urine (0-120 hours) from Subjects A and B are summarized in Table 2, all the values
are given as percentage of the total radioactivity of
each urine pool.
Only 0.2-0.3% of the total 14C-content was due to
unchanged drug in the urine of both subjects. Pretreatment with /3-glucuronidase/sulphatase (Helix
pomatia; Industrie Biologique Franqaise) led to a
slight increase to about 0.7%.
Apart from intact acenocoumarol, all the presumed biotransformation products were detected.
There were some differences between Subjects A and

W. Dieterle et al.: Metabolism and Pharmacokinetics of Acenocoumarol

371

ng/ml

P%

500-

I
I
400-

300-

\
\

S U B J E C T A:

82kg

Oose:

12 mg

(b== ~

I
I
I

_-

Total radioactivity

Z unchanged d r u g

\
\

amino- metabolite

~_ . . . . . . .

.~ a c e t a m i d o - m e t a b o l i t e

-~

alcohol1- m e t a b o l i t e

alcohol2-metabolite

50

60

. . . .

--

200-

100-

10

500-

20

30

40

70

hrs

ng/ml

%~

SUBJECT B :

71 kg

Dose:

12rag

400-

\
300-

200-

...

100-

10

20

30

40

50

60

i
70

hrs

Fig. 2. Concentration of total radioactivity, unchanged drug, amino-metabolite ( = CGP 8435),

acetamido-metabolite ( = CGP 8436), alcohol 1metabolite ( = CGP 8767) and alcohol2-metabolite in plasma after oral administration of 14Clabelled acenocoumarol

Table 1. Binding of acenocoumarol to proteins of human serum in vitro: equilibrium dialysis at 37C
Origin or type of protein

Serum from one subject

Serum from five subjects
Human serum albumin (33.3 g/l)

Binding of acenocoumarol
Number of
experiments (n)

% bound
(~ _+ SE)

Concentration in serum
after dialysis (~tg/ml)

9a
5b
10"

98.7 _+ 0.03
98.8 _+ 0.08
97.5 _+ 0.08

0.021 to 8.34
approx. 0.34
0.010 to 8.59

a number of different concentrations tested in the range given

u number of blood donors

372

W. Dieterle et al.: Metabolism and Pharmacokinetics of Acenocoumarol

B in the percentages of the individual metabolites

(Table 2).
Independent characterization of the mixture of
14C-compounds contained in the XAD-2 concentrate
of pooled urine by two-dimensional TLC and autoradiography confirmed the presence of the metabolites identified by isotope dilution analysis (Fig. 4,
spots B, C, D, E, F). The amino-metabolite decomposed during preparation of the XAD-2 concentrate,
so it does not show up in the autoradiogram (position
G). Spot H represents a strongly polar metabolite
fraction of unknown structure(s).
The presence of the acetamido-metabolite was
further confirmed by preparative isolation (Scheme 1)
and mass spectrometric analysis. The mass spectrum
showed the molecular ion (m/e 365) and the same
fragmentation pattern as authentic compound.

Table 2. Unaltered drug and metabolic products in human urine

(0-120 h) after oral administration of 14C-labelled acenocoumarol

Dose: 12 mg
Subject A: 82 kg
Subject B: 71 kg
Compounds excreted in urine

Unchanged drug
Amino-metabolite CGP 8435
Acetamido-metabolite CGP 8436
Alcoholl-metabolite CGP 8767
Alcohol2-metabolite
7-hydroxy-metabolite CGP 8438
6-hydroxy-metabolite
Total

% of total urinary
radioactivity

Subject A

Subject B

0.3
12.3
19.0
4.6
1.7
14.0
6.9
58.8

0.2
7.7
11.1
9.0
4.4
22.2
18.3
72.9

PharmacologicalActivity of Metabolites
The results of clotting studies performed in mice with
synthetic metabolites of acenocoumarol are shown in
Table 3.
The 7-hydroxy-metabolite proved to be inactive.
The other metabolites showed a greater and longer
lasting depression of the prothrombin level than
acenocoumarol, when given in single oral doses of 100
mg/kg. When the initial dose of 100 mg/kg was followed by two maintenance doses of 10 mg/kg after 24
and 48 hours, a continuous increase in the prothrombin level was noted, but it was less marked when the
metabolites were administered than the parent drug.
Discussion

The results of the present study with 14C-labelled

acenocoumarol in man have shown that the drug is
rapidly and well absorbed from the digestive tract
following oral administration. Overall elimination of
the drug and its metabolites was slow, although the
major part of the dose was excreted within the first
three days. Renal excretion was the main route.
The decay of the plasma concentration of intact
acenocoumarol did not follow first order kinetics (see
semilogarithmic plot in Fig. 3). For the main elimination phase between 6 and 24 hours after administration, the half-lives in the two subjects were 8.7 and 8.2
hours. Blatrix et al. [2] reported half-lives ranging
from 20 to 30 hours. This is open to debate, however,
since the spectrophotometric method they employed
is unlikely to distinguish between drug and metabolites.
The in vitro binding studies demonstrated that
98.7% of acenocoumarol in human serum was bound

Table 3. Anticoagulant effect of acenocoumarol and various metabolites in mice after a single oral dose (100 mg/kg), and after repeated oral

doses (initially 100 mg/kg followed by two doses of 10 mg/kg after 24 and 48 h)
Compound

Change in Coagulation Time %

Repeated dose
Hours after administration
of the initial close

Single dose
Hours after administration
of the dose

Control
Amino-metabolite
Acetamido-metabolite
Mixture of alcohol-metabolites
Alcoholl-metabolite
7-hydroxy-metabolite
Acenocoumarol
1 Not measured

24

48

72

24

48

72

99.0
12.8
13.9
10.2
10.8
99.0
17.5

98.5
65.0
43.0
35.5
38.7
99.5
99.0

100.0
99.5
97.5
91.0
99.5
99.0
99.0

99.0
13.5
13.2
10.9
10.3
_1
16.7

99.0
20.8
19.0
30.1
18.2
_1
35.4

98.5
23.2
24.4
35.9
25.0
_1
43.3

W. Dieterle et al.: Metabolism and Phal~acokinetics of Acenocoumarol

to protein and that the differences between individuals were negligible (Table 1). The binding capacity was high and saturation was not reached at plasma
concentrations far above the maximum concentration
observed in vivo after oral administration of a 12 mg
dose (Fig. 2). The percentage associated with pure
human serum albumin (97.5%) at a physiological protein concentration was approximately the same as the
total bound fraction in serum. The albumin molecule
possesses two classes of binding sites with two different affinities for acenocoumarol (see "Results"). The
findings in the present protein binding study confirm
and complement previous results [11, 12, 13]. Like
other anticoagulants of the coumarin type, acenocoumarol may become partially displaced from
its binding sites on serum albumin by other acidic
drugs [12, 13]. In consequence, its free plasma concentration would be increased and the anticoagulant
effect enhanced, which must be taken into consideration in combined drug therapy.
The integrated concentration of total 14C-substances in plasma between 0 and 72 hours was of the
same order in the two subjects, as shown by a comparison of the areas under the concentration curves
(AUC). The AUC-values of the compounds specifically analyzed comprised 76 and 89%, respectively, of
the total radioactivity in Subjects A and B. There
were, however, considerable differences between the
two volunteers in the percentages of individual compounds; intact acenocoumarol accounted for 16.6%
in Subject A and 38.7% in Subject B; the amino-, and
acetamido-metabolites amounted together to 56.2
and 41.4% in the two subjects, and the alcoholmetabolites to 3.6 and 8.6%.
The metabolites detected in human plasma i.e.
the amino-acetamido- and alcohot-metabolites, were
at least as effective as acenocoumarol itself when
tested for anticoagulant activity in mice (Table 3).
Therefore, in any attempt to control or optimize
therapy by plasma level analysis, the concentrations of
the major active metabolites would have also to be
considered. Two additional products, the 6- and
7-hydroxy-metabolites, which were found in urine but
were not determined in plasma, are most unlikely to
contribute to the anticoagulant effect. Pharmacological studies with the corresponding hydroxy-metabolites of warfarin failed to demonstrate any activity
[14, 15, 16, 17]. The 7-hydroxy-metabolite of
acenocoumarol also proved to be inactive (Table 3);
the 6-hydroxy-metabolite was not tested.
Based on the analysis of urinary metabolites of
acenocoumarol, a scheme of biotransformation has
been devised (Scheme 2), that accounts for 59-73%
of the radioactivity excreted by the kidney. The remainder of radioactivity in urine was mainly accounted for by an unidentified, strongly polar metabolite fraction (spot H in Fig. 4).
The drug is extensively metabolized in man. In the
formation of metabolites at least two different pri-

500-

373
ng/ml

100-

50

SUBJECT B: 71 kg

SUBJECT A: 82kg
Dose:
12 mg

Fig. 3. Semitogarithmic plot of the decay phase of unchanged drug

in plasma after oral administration of 14C-labelled acenocoumarol

O
gl
e-

o
E
I11

i/)

Solvent system CS4 (second dimension)

Fig. 4. Two dimensional thin-layer chromatogram and autoradiography of radioactive substances in human urine (0-120 h) after
oral administration of ~4C-labelled acenocoumarol
A = acenocoumarol (G 23 350)
B = 7-hydroxy-metabolite (CGP 8438)
C = 6-hydroxy-metabolite
D = alcohol~-metabolite (CGP 8767)
E = alcohol2-metabolite
F = acetamido-metabolite (CGP 8436)
G = amino-metabolite (CGP 8435) which mainly decomposed
during chromatography
U = unknown

374

W. Dieterle et al.: Metabolism and Pharmaeokinetics of Acenocoumarol

0
If

O
II

Amino- metabolite
12,3~7,7%

Acetamido-metabolite
19,o/t1,1~

References
0
U

OH
!
OH CH2--CH--CH3

=
ACENOCOUMAROL
0,~0,2%

O
II
OH CH2--C--CH3
H

Acknowledgement. We wish to thank Dr. W.J.

Richter (Ciba-Geigy) for recording and interpreting
the mass spectra, and Mrs. M. Develey for skitful
technical assistance.

6- hydroxy-metabolit e
6,9/18,3%

Alcohol-metabolites
alcohol1: 4,6J9~3~
alcohol2:1,7j 4/1%

0
II

Mo ::2
7-hydroxy-meta bolite
14,0/22,2%

Scheme 2. Pathways of acenocoumarol biotransformation in man;

%-values for Subject A/Subject B refer to total urinary radioactivity = 100%

mary pathways were involved, namely reduction and

oxidation at different positions in the molecule. Reduction takes place at the aromatic nitro-group and at
the ketone-group in the acenocoumarol molecule.
Nitro-reduction results in the amino-metabolite, the
major portion of which is further transformed to the
corresponding N-acetyl-derivative. This sequence of
metabolic reactions is commonly observed with drugs
containing aromatic nitro-groups [18, 19, 20, 21, 22,
23]. Reduction of the ketone-group to yield two diastereoisomeric alcohol-metabolites plays a subordinate role in the biotransformation of acenocoumarol.
The formation of alcohol-metabolites had already
been suggested by the results of metabolic studies of
warfarin in man [15, 24, 25],
Oxidation of the coumarin nucleus of
acenocoumarol results in the 6- and 7-hydroxymetabolites. Published studies on the metabolism of
other coumarin-type anticoagulants in man have also
shown that hydroxylation takes place predominantly
at positions 6 and 7 [25, 26, 27].
There were differences between the two subjects
in the quantitative contribution of the two general
pathways of biotransformation of acenocoumarol. In
Subject A the drug was preferentially metabolized by
reductive processes and in Subject B by oxidative
reactions.

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Reeeived: September 8, 1976, and in revised form." December 8,
1976
Dr. W. Dieterle
Pharma Research
R 1055.5.46 a
Ciba-Geigy AG
CH-4002 Basel