Synthesis of the caffeine metabolites 5-acetylamino-6-formylamino-3-methyluracil (AFMU) and 5-acetylamino-6-amino-3-methyluracil (AAMU) on a preparative scale

Article abstract of DOI:10.1515/znb-1997-1214

5-Acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-methyluracil (AFMU) have been prepared by simple chemical transformations starting from thiourea and ethyl cyanoacetate. These compounds AAMU and AFMU are required as standard m

Full text of DOI:10.1515/znb-1997-1214

Synthesis of the Caffeine Metabolites 5-Acetylamino-6-formylamino-
3-methyluracil (AFMU) and 5-Acetylamino-6-amino-3-methyluracil
(AAMU) on a Preparative Scale
R. R öhrkasten3, P. Raatz3, R. P. K reher3 *, M. Blaszkewiczb
a Lehrstuhl für Organische Chemie II, Fachbereich Chemie, Universität Dortmund.
D-44227 Dortmund
b Institut für Arbeitsphysiologie an der Universität Dortmund, ZWE Analytische Chemie,
Ardeystraße 67, D-44139 Dortmund
Z. Naturforsch. 52b, 1526-1532 (1997); received April 7, 1995
Pyrimidine-diones, Caffeine Metabolites, Synthesis
5-Acetylamino-6-amino-3-methyluracil (AAMU) and 5-acetylamino-6-formylamino-3-
methyluracil (AFMU) have been prepared by simple chemical transformations starting from
thiourea and ethyl cyanoacetate. These compounds AAMU and AFMU are required as
standard materials for qualitative identification and quantitative determination in connection
with the metabolism of caffeine.
Introduction
procedures are applied to obtain weighable
amounts. The first method consisted in the con-
sumption of a caffeinated beverage and the extrac-
tion of the metabolites from the urine; but the
yields are low [10]. A very expensive synthetic
procedure is based on a procedure of Khmelevskii
et al. [3] modified by Tang et al. [10] using 1-MU
instead of uric acid. A fter acylation with formic
acid/acetic anhydride the yield was only 19% pro-
ducing 2 mg of AFM U 9.
The N-acetyltransferase plays an im portant role
in the metabolism of many xenobiotics; the pro-
duction of the enzyme is genetically controlled. In-
dividuals differ in the am ount of the acetylated
m etabolites and can be classified as slow or rapid
acetylators. The acetylator status is connected with
some diseases such as diabetes mellitus and blad-
der cancer and the knowledge of it is for the bene-
fit of therapeutics.
In recent years, caffeine has been used as a non-
invasive probe for the phenotyping of the acetyla-
tor status. The metabolism of caffeine comprises a
variety of reactions, such as oxidative demethyla-
tion, ring hydroxylation, ring-opening and acetyla-
tion. The ratios of the m olar concentrations of uri-
nary caffeine metabolites, like 5-acetylamino-6-
formylamino-3-methvluracil (AFM U) (9), 5-ace-
tylamino-6-amino-3-methyluracil (A A M U ) (8), 1-
methylxanthine (1-MX) or 1-methyluric acid (1-
MU) enable the determ ination of the acetylator
status.
Synthesis
Pfleiderer and co-workers [1,6,7] have estab-
lished a synthesis of eight steps for A A M U 8 with
an overall yield of 10%. While working on pyrimi-
dines, we have recognized a general interest in
some of our pyrimidine derivatives; A A M U 8 was
one of it. After analyzing the fundam ental synthe-
sis of Pfleiderer and co-workers [7] we were suc-
cessful in shortening the sequence to six steps and
in increasing the yield by modification of the reac-
tion conditions up to 42% (cf. Scheme 1).
For quantitative analytical determinations,
AFM U 9 or A A M U 8 are required as standard
materials. Both m etabolites are not available com-
mercially. Therefore biochemical and synthetic
Cyclization of thiourea (1) (NCN-compound)
and ethyl cyanoacetate (2) (CCC-compound) -
based on a modified procedure of Taylor and
Cheng [12] and of Traube [13] - affords 99% of
6-amino-2-thiouracil (3) after treatm ent with base
(NaOEt/EtOH/3h/reflux). The procedure is very
simple and using an optimized work-up method it
is possible after complete removal of solvents to
* Reprint requests to Prof. Dr. R. P. Kreher.
D
0932-0776/97/1200-1526 $06.00 © 1997 Verlag der Zeitschrift für Naturforschung. All rights reserved.
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R. Röhrkasten et al. ■Synthesis of the Caffeine Metabolites
1527
h3c
h3c
N
N
NT
A ,
A
O ^N
nh2
O' N
I
H
Scheme 1. rt: NaOEt / EtOH (3 h; reflux) -
99%; r2: (H3C 0)2S02 / 2N NaOH (1 h;
40 °C) - 62%; r3; 1 N NaOH (2 h; reflux) -
91%; r4; NaOAc / HOAc / H.O / NaN02
(65 min; 100 °C) - 95%; r5: Pd;C / H2 / NaOH
(6-12 h; 20 °C) - 82%; r6: NaOAc / Xc20 (3 h;
reflux) - 97%; r7: H C O -O -C O C H , / HCOOH
(7 d; 20 °C) - 60-80%.
H3Cv
HsC'NJ Y NH
N
I
NH2
H
A ,
o ch 3
separate water insoluble compounds easily. Neu-
tralization with acetic acid instead of mineral acids
seems to be more effective, because the sodium
acetate form ed a buffer and therefore organic salts
aren’t formed. 2-Thio-4-amino-6-hydroxy-pyrimi-
dine is alkylated by means of methyl iodide or di-
methyl sulphate at the sulfur atom [2], Regioselec-
tive S- and N-alkylation of 3 at 1-position can be
readily achieved with dimethyl sulfate in NaOH.
The first step is the formation of a thiolate anion;
its enhanced reactivity is responsible for the for-
mation of 6-amino-2-methylthio-uracil (10). The
N
L JL + 4
h3cs
n
nh2
h3cs
n
nh2
10
Scheme 2.
the 2-methylthio group; with a 2-methoxy group
O-alkylation did not occur.
The methylthio group in 2-position represents a
favorite center for nucleophilic attack of hydrox-
rate of this step and the following reaction are al- ide anions. 6-Amino-2-methylthio-3-methyluracil
most the same. If not enough alkylating reagent is (4) reacts with 1 N N aO H (2 h/reflux) to give 91%
used, starting m aterial 3 is isolated beside of 6-
amino-2-methylthio-3-methyluracil (4) and 6-
amino-4-methoxy-2-methylthio-uracil (11). Using
a 2.5fold excess of the alkylating reagent the py-
rimidines 4 (62%) and 11 (17%) can be synthe-
sized (cf. Scheme 2). A direct synthesis of 10 can-
of 6-amino-3-methyluracil (5). A fter neutraliza-
tion with acetic acid 5 can be isolated as colorless
solid.
The nitrosation of 6-amino-3-methyluracil (5)
was carried out in diluted acetic acid with sodium
nitrite (65 min/100 °C). To increase yield and pro-
not be realized in this way. Alkylation of duct quality (microcrystals) the use of sodium ace-
independently synthesized 10 leads to the result
that 4 and 11 were generated in the same ratio.
The unusual O-alkylation must be dependent on
tate as buffer seems to be advantageous. The
higher reaction tem perature is necessary because
the starting m aterial is almost insoluble. The insol-
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1528
R. Röhrkasten et al. • Synthesis of the Caffeine Metabolites
üble violet 6-amino-5-nitroso-3-methyluracil (6)
was collected and washed extensively with water
and carefully dried (48 h over P20 5; yield 95%
with m.p. >350 °C). The main advantage of func-
tionalizing with a nitroso group is the effective
work-up procedure as well as the considerable
yield and the rem arkable regioselectivity; the ena-
Formylation of A A M U 8 to prepare 6-ace-
tylamino-5-formylamino-3-methyluracil (A FM U )
(9) was achieved by Tang and co-workers [10] with
a mixture of formic acid/acetic anhydride (4:1).
This method (yield: 19%) was not effective on a
preparative scale; therefore it seemed to be advan-
tageous to use the mixed anhydride of formic and
mine structure permits only nitrosation at 5- acetic acid, which can be synthesized as an reagent
position.
In earlier literature Raney-Nickel [5,11] was
on a preparative scale [4],
A A M U 8 was suspended and stirred in a 1:1
used to perform catalytic hydrogenation of the ni- mixture of formic acid and acetic formic anhydride
tro group at 5 position. Rem arkable amounts of
the catalyst are required and lower yields can be
caused by adsorption of the product on surface.
Using palladium [8] only small amounts of catalyst
are required. Because of increased reactivity, the
decision for an appropiate solvent is important:
The conversion of nitroso-pyrimidines in water
leads presumably to the hydration of the CC-
double bond, in m ethanol no transform ation takes
place, because of the poor solubility of the nitroso
compound 6. The sodium salt of the starting
material is more polar then the free base and
therefore soluble in methanol. For this reason so-
dium hydroxide was added with 10% excess to the
mixture. Hydrogenation is successful with 10%
palladium on charcoal in a mixture of m ethanol/
aqueous sodium hydroxide at 20 °C in 6 -1 2 h; the
reaction is completed after the red solid has disap-
peared. Work up must be perform ed rapidly and
under an argon atm osphere. The neutralization
(7 d/20 °C). Thereby the turnover of A A M U 8 is
not complete, because the resulting A FM U 9 is
decomposed to A A M U 8, which can be reused.
An indication for the increased reactivity of
AFM U 9 is the formation of 5,6-diacetylamino-3-
methyluracil (DAM U) (12) (cf. Scheme 3). This
transformation can be described as a transam ida-
tion of the 6-formylamino-group. A n analytical
pure sample of DAM U 12 was isolated by semi-
preparative HPLC, so that characterization and
identification were possible.
12
Scheme 3.
A A M U 8 could be excluded as a source for the
with hydrochloric acid have to be carried out ex- formation of DAMU 12, because it cannot be de-
actly to pH = 7.0 (pH-electrode; otherwise the pre- tected during the synthesis of AAM U 8 with acetic
anhydride under forced conditions.
cipitate is very unstable); after this the precipitate
was filtered off and dried. The isolated 5,6-dia-
mino-3-methyluracil (7) is relative stable and can
be stored for a longer period at 20 °C without
argon.
A ttem pts to suppress the competitive reaction
are not successful, because the turnover of
AA M U 8 is negligible below -5 °C. Otherwise the
consecutive reaction takes place with measurable
amounts at T > 0 °C. A t 40 °C the reaction can be
stopped after 3 -8 h, but the yield of AFM U 9 is
Acylation of 7 at 5-position can be achieved re-
giospecifically and with excellent yield. During
acylation with acetyl chloride hydrochloric acid is unsatisfactory, because of the increased am ount of
DAM U 12 (-30% ). The tem perature of 20 °C
seemes to be a good compromise: on the one hand
the turnover is 70-80% and on the other side the
formylating reagent is relatively stable under these
conditions and DAM U 12 is formed in only 5 -
10% yield.
In solution AFM U 9 is relative unstable and the
quantitative separation is extremely difficult.
Chromatographic methods are not applicable, be-
developed, but bases cannot be used [9], Acylation
of 5,6-diamino-uracil with acid chlorides in the
presence of pyridine furnished the bisacylamino
derivative; therefore an alternative process was
applied to generate the free base. The diamino
compound 7 was treated with an equim olar
amount of acetic anhydride and dry sodium ace-
tate in glacial acetic acid (3 h/reflux); colorless
AAM U 8 was separated in 97% yield.
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R. Röhrkasten et al. • Synthesis of the Caffeine Metabolites
1529
cause the separation is incomplete; deformylation
of AFM U 9 on the column material seems to be
the main reason. Flash chromatography carried
out on silica gel with ice-cold trichloromethane/
m ethanol (9:1) at 20 °C gives rise to the generation
or alumina F254 (E. Merck). HPLC was carried out
with a Shimadzu LC-6A liquid chromatograph
equipped with a photo diode array detector (SPD-
M6A). Chrom atography was perform ed with Su-
perspher 100 RP-18 EC-4//m column (E. Merck);
of -1 0 -1 5 % A AM U 8. M ethanol (10%) is re- 250 mm long and 4.6 mm i.d. for analytical appli-
sponsible for this effect, but this polar solvent is cations or Partisil 5 ODS 3 (W hatman); 250 mm
always necessary for chromatography. Therefore
the use of longer columns is not effective. High
quantities of A AM U 8 could be separated by re-
long and 8.0 mm i.d. for preparative applications.
The elution mixture is water containing 28 /Al\ of
formic acid and 6% of m ethanol (only analytical
fluxing the raw solid for 10 min in dioxane con- column).
taining 3% of water. The diacetyl compound 12
Experimental
could be largely separated by washing with small
am ounts of ice-cold trichloromethane/methanol
(9:1). Unfortunately, this operation produces again
small amounts of AAM U 8. Digestion with ace-
tone (30 min/5 °C) gives AFM U 9 in -91 -9 5 %
purity on a preparative scale.
The used chemicals are commercially available
and were only purified if it was specially noted.
Acetic formic anhydride was synthesized accord-
ing to the procedure of Krimen [4],
6-Am ino-2-m ethylthio-3-m ethyluracil (4)
AFM U
9 reacts at T > 35 °C easily with
electrophiles; by this way AA M U 8 and DAM U
12 are formed. AFM U 9 reacts as well with nu-
cleophiles under m oderate conditions (T < 20 °C)
and produces A AM U 8. The ambiphilic property
of AFM U 9 is the main problem for separating
this urinary metabolite [10].
38.1 g (500 mmol) of thiourea (1) and 50.9 g
(450 mmol) of ethyl cyanoacetate (2) were re-
fluxed for 3 h in a solution of 20.7 g (900 mg-atom)
of sodium and 300 ml of dry ethanol. A fter re-
moval of the solvent the residual 6-amino-2-thio-
uracil (3) was dissolved in 500 ml (1013 mmol) of
2 N N aO H and 171 ml (1800 mmol) of dimethyl
sulfate were added and the mixture was vigorously
stirred for 1 h at 40 °C (exothermic reaction). A f-
ter cooling (16 h at 0 °C) the precipitate was col-
lected by filtration and washed neutral with 250 ml
of ice w ater and dried in vacuo. The raw product
was suspended in 150 ml dry acetone and the solid
was collected by filtration and dried in vacuo;
yield: 47.0 g (61%) of 4, m.p. 257 °C (ref. [7]; m.p.
257 °C) and R F = 0.80 (alumina; methanol). An
analytical sample was recrystallized from water.
The organic (acetone) filtrate gave 17% of 4-
Instrumentation and Procedures
Melting points were determ ined on a Kofler-
heatplate-microscope (Reichert) and are uncor-
rected. The microanalyses were performed in the
micro chemical laboratory of the Institut für
Chemie der Universität Dortmund. The sub-
stances were usually dried at 20 °C in vacuo for 24
h over P20 5. Mass spectral data were collected
from a model CH-7 (Atlas Mat) or a 8230 (Finni-
gan M at) mass spectrometer. IR spectra were reg-
istered with a Shimadzu IR-470 spectrometer as
KBr-disc. UV-Vis spectroscopic data were m ea-
sured with a Varian Cary 17 D spectrometer.
NM R spectra were determ ined on a Varian EM
360A (60 M Hz) or a Bruker AM 300 (300 MHz).
The chemical shifts are given in ppm as (3 values
referred to tetram ethylsilane (TMS) with TMS or
[D6]-DM SO as internal standard. 13C NMR
spectra were determ ined on a Bruker AM 300
(75.5 MHz) and the multiplicity of the signals was
established by the relative phase of the corre-
sponding signal in the D EPT spectra. Thin layer-
chrom atography was perform ed on silica gel F254
am ino-6-m ethoxy-2-m ethylthio-pyrim idine
(11),
m.p. 194 °C (ref. [2]; m.p. 144 °C, yield 20%), R F =
0.88 (silica gel; m ethanol).
6-Am ino-2-thiouracil (3)
C4H5N3OS (143.16)
’H NM R ([D6]-DMSO, 60 MHz): Ö (ppm) =
4.70 (s, 1H, H-5); 3.50-4.90 (s(broad), 1H, H -l);
6.33 (s(broad), 2H, N H 2); 10.60-11.60 (s(broad),
1H, H-3). - MS (70 eV, 250 °C): m /z (% ) = 143
(79) (M +); 127 (100); 115 (23); 99 (19); 84 (33); 68
(86); 55 (16); 43 (89). - IR (KBr): v (cm “1) = 3450
(m); 3340 (m); 3120 (m); 2990 (m); 2920 (m); 1640
(vs); 1605 (s); 1555 (s); 1445 (s); 1310 (m); 1195
(s); 800 (m); 620 (m).
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1530
R. Röhrkasten et al. ■Synthesis of the Caffeine Metabolites
6-Am ino-2-m ethylthio-3-m ethyluracil (4)
and 12.6 ml (220 mmol) of glacial acetic acid and
500 ml of water. During a period of 35 min a solu-
tion of 16.56 g (240 mmol) of sodium nitrite in
100 ml of water was dropped in. A fter 30 min stir-
ring the resulting suspension was cooled to 20 °C
and the violet microcrystalline precipitate was col-
lected by filtration and washed twice with 50 ml of
water and dried over P ?05 for at least 48 h; yield:
35.56 g (95%) 6, m.p. >350 °C (ref. [6]; m.p.
C6H 9N 3OS (171.22)
Calcd
C 42.09 H 5.30 N 24.54%,
Found C 42.22 H 5.47 N 24.30%.
*H NM R ([D6]-DMSO, 300 MHz): ö (ppm) =
2.50 (s, 3H, SCH,); 3.27 (s, 3H, N CH 3); 4.92 (s, 1H,
H-5); 6.43 (s(broad), 2H, N H ,). - 13C NMR ([D 6]-
DMSO, 75.5 MHz): (3 (ppm) = 14.18 (q, SCH3);
28.61 (q, N CH3); 80.43 (d, C-5); 161.11 (s, C-6);
161.34 (s, C-4); 161.65 (s, C-2). - MS (70 eV,
100 °C): m /z (% ) = 171 (91) (M +); 156 (16); 125
(89); 110 (22); 95 (20); 86 (15); 83 (36); 74 (17); 68
(100); 57 (65): 43 (79). - IR (KBr): v (cm *1) =
3410 (s); 3335 (s); 3210 (s); 1670-1600 (s); 1574
(s); 1510 (s); 1501 (s); 1461 (s); 1451 (s); 1410 (s);
1334 (m); 1284 (s); 1235 (m); 1098 (s); 809 (s); 758
(m); 637 (m). - UV (methanol): 2max(nrn) (lg e) =
218 (4.41); 230 (4.39); 275 (3.85).
>350 °C), Rf
=
0.00 (alumina; methanol).
-
Recrystallization or chromatography was impossi-
ble and 'H NMR- and 13C NM R spectra could not
be recorded, because of the very poor solubility in
usual solvents.
C5H 6N40 3 (170.13)
Calcd
C 35.30 H 3.55 N 32.93%,
Found C 34.90 H 3.48 N 32.24%.
MS (70 eV, 200 °C): m /z (% ) = 170 (84) (M +);
153 (93); 141 (20); 112 (47); 110 (39); 96 (100); 70
(36); 69 (36); 58 (56); 53 (46); 44 (20). - IR (KBr):
v (cm “1) = 3240 (s); 3015 (m); 1727 (vs); 1698 (s);
1656 (vs); 1533 (m); 1462 (s); 1444 (s); 1334 (s);
1324 (s); 1308 (m); 1288 (s); 1280 (s); 1207 (m);
1090 (m); 791 (m); 770 (s); 704 (m); 644 (m). -
UV (methanol): 2max(nm) (lg e) = 224 (3.94); 245
(sh, 3.64); 319 nm (4.20).
6-Amino-3-methyluracil (5)
42.81 g (250 mmol) of 6-amino-2-methylthio-3-
methyluracil (4) in 700 ml (600 mmol) of
1 N N aOH were refluxed for 2 h. To the boiling
solution 46 ml (800 mmol) of glacial acetic acid
were added and then the mixture was cooled for
16 h at 0 °C. The crystalline precipitate was col-
lected by filtration and washed three times with
50 ml of ice water and dried in vacuo; yield: 32.0 g
(91%) of 5 as colorless needles, m.p. 342-346 °C
(ref. [6]; m.p. 327 °C), R F = 0.75 (silica gel;
methanol).
5,6-Diamino-3-m ethyluracil-sem ihydrate (7)
32.32
g
(190 mmol) of 6-amino-5-nitroso-3-
methyluracil (6) were hydrogenated with shaking
at 20 °C for 6-12 h at an initial hydrogen pressure
of 3.5 bar in a suspension of 8.00 g (200 mmol) of
sodium hydroxide and 940 mg (10 mmol) of Pd-
catalyst (10% on charcoal) in 300 ml of methanol/
water (2:1). When the red solid is consumed, the
reaction mixture was filtered under argon and the
solid residue was washed twice with 50 ml of
water. The combined extracts were concentrated
to 100 ml, neutralized with conc. hydrochloric acid
exactly to pH = 7.0 (definitely recommended; pH-
electrode) and the solid was filtered off immedi-
ately under argon and dried in vacuo; yield:
25.73 g (82%) of 7. m.p. 280 °C (dec.) (ref. [6]; m.p.
>340 °C), Rf?= 0.00 (alumina; methanol). An ana-
lytical sample was recrystallized from water with a
small amount of charcoal.
C5H 7N 30 2 (141.13)
Calcd
C 42.55 H 5.00 N 29.77%,
Found C 42.81 H 5.01 N 29.46%.
'H NM R ([D6]-DMSO, 300 MHz): Ö (ppm) =
3.01 (s, 3H, NCH3); 4.56 (s, 1H, H-5); 6.24
(s(broad), 2H, N H 2); 10.10-10.50 (s(broad), 1H,
H -l).
-
13C NMR ([D 6]-DMSO, 75.5 MHz): d
(ppm) - 26.09 (q, N CH 3); 74.23 (d, C-5); 151.40 (s,
C-2); 153.83 (s, C-6); 163.34 (s, C-4). - MS (70 eV,
280 °C): m /z (% ) = 141 (100) (M +); 111 (22); 84
(48); 68 (68); 44 (19); 43 (59). - IR (KBr): v (cm“ 1) =
3425 (s); 3320 (m); 3205 (m); 1682 (vs); 1652 (vs);
1631 (vs); 1596 (vs); 1554 (m); 1456 (s); 1386 (m);
974 (m); 792 (m); 764 (m); 657 (m); 559 (m). -
UV (methanol): l max(nm) (lg e) = 220 (3.45); 264
(4.25).
C ,H 8N40 , 0 . 5 H ,0 (165.15)
Calcd
C 36.36 H 5.49 N 33.92%,
Found C 36.33 H 5.32 N 33.83%.
6-A m ino-5-nitroso-3-methyluracil (6)
'H NM R ([D6]-DMSO. 300 MHz): (3 (ppm) =
3.06 (s, 3H, NCH3); 3.40-4.40 (s(broad), 3H, 5-
N H 2, 0.5 H ?0 ); 5.72 (s(broad), 2H, 6-NH2). - 13C
NM R ([D6]-DMSO, 75.5 MHz): (3 (ppm) = 26.78
31.05 g (220 mmol) of 6-amino-3-methyluracil
(5) were suspended at 100 °C in a solution of
29.94 g (220 mmol) of sodium acetate-trihydrate
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R. Röhrkasten et al. • Synthesis of the Caffeine Metabolites
1531
(q, N CH 3); 95.50 (s, C-5); 142.94 (s, C-2); 149.67
of formic acid/acetic formic anhydride (1:1) and
(s, C-6); 160.67 (s, C-4). - MS (70 eV, 215 °C): m / stirred for 7 d at 20 °C with exclusion of moisture;
0.5 ml of acetic formic anhydride were added ev-
ery day (after 2 d 8 was almost dissolved and gen-
erally a colorless solid precipitates). The solvents
were removed and the solid (-2.4 g; >100%) was
suspended in 247 ml of dioxane/water (97:3), re-
fluxed for 10 min and insoluble 8 was removed by
filtration. A fter the filtrate was cooled to 20 °C the
precipitate was filtered off; concentration of the
filtrate to 20 ml under reduced pressure (T <
40 °C) gives another fraction. The residues (1.6-
2.0 g) were suspended in 40 ml of acetone (30 min/
4 °C) and than washed three times with 5 ml of
ice-cold trichlorom ethane/m ethanol (9:1) and
dried over P20 5; yield: 1.35-1.80 g (60-80% ) col-
orless 9, mp. 227-229 °C (dec), R F = 0.20 (silica
z
(% ) = 156 (100) (M +-0.5 •H20 ); 71 (43); 44
(61). - IR (KBr): v (cm -1) = 3490 (m); 3390 (s);
3205 (s); 1700-1610 (s); 1590-1510 (s); 1456 (s);
1385 (m); 1366 (m); 1284 (m); 1258 (m); 1181 (m);
1109 (m); 755 (m); 723 (m); 702 (m); 655 (m). -
UV (methanol): Amax(nm) (lg e) = 219 (sh, 3.32);
260 (3.88); 285 (2.96).
5-Acetylam ino-6-am ino-3-m ethyluracil-hydrate
(A A M U ) (8)
16.52 g (100 mmol) of 5,6-diamino-3-methylura-
cil-semihydrate (7) in 150 ml of glacial acetic acid
were refluxed for 3 h with a m ixture of 9.02 g
(110 mmol) of dry sodium acetate and 11.23 g
(110 mmol) of absol. acetic anhydride. The mix-
ture was cooled to 20 °C, and the solid was filtered
off and washed three times with 50 ml of water
and dried in vacuo\ yield: 19.24 g (89% ) of 8 as
colorless microcrystals. The filtrate was evapo-
rated to dryness in vacuo and the yellow residue
recrystallized from 100 ml of water and dried over
P20 5; further 1.70 g (8% ) of colorless 8 is ob-
tained. Overall yield: 20.94 g (97% ), subl.-p.
276 °C; m.p. >350 °C (ref. [1]; m.p. >350 °C); R F =
0.16 (alumina; m ethanol).- R F = 0.12 (silica gel;
ethyl acetate/acetic acid (9:1)); tHPLC = 4.52 min.
gel; ethyl acetate/acetic acid (9:1)); tHPLC
=
6.70 min. - HPLC-purity: -9 1 -9 5 % 9, -3 -7 % 8,
-1 -3 % 12.
C8H 10N4O4 (226.19)
Calcd
Found C 41.4
C 42.48 H 4.46 N 24.77%,
H 4.2 N 24.1% .
*H NM R ([D6]-DMSO, 300 MHz): <3 (ppm) =
1.96 (s, 3H, COCH3); 3.12 (s, 3H, N CH3); 8.35
(s(broad), 1H, CHO); 8.83 (s, 1H, NH); 10.53;
11.03 (s(broad), 2 •1H, 2 •NH). - 13C NM R ([D6]-
DMSO, 75.5 MHz): (3 (ppm) = 22.87 (q, C O C H 3);
27.25 (q, N CH3); 96.15 (s, C-5); 142.64 (s, C-2);
149.23 (s, C-6); 161.26 (s, C-4); 162.79 (d, CHO);
170.10 (s, CO CH 3). - MS (70 eV, 170 °C): m /z
(% ) = 226 (10) (M+); 184 (33); 156 (29); 71 (27);
44 (24); 43 (100); 42 (18). - IR (KBr): v (cm “1) =
3425 (m); 3245 (s); 3065 (m); 2935 (m); 1739 (s);
1715 (s); 1678 (s); 1641 (vs); 1561 (m); 1524 (m);
1508 (m); 1301 (m); 1204 (m); 752 (m). - UV
(methanol, <5 min after preparation of the solu-
tion): Amax(nm) (lg e) = 210 (4.32); 286 (4.13). -
UV (water): Amax(nm) (lg e) = 207.5 (4.24); 285
(4.15).
C7H 10N4O r H ,O (216.20)
Calcd
C 38.89 H 5.59 N 25.92%,
Found C 39.13 H 5.85 N 26.21%.
NM R ([D6]-DMSO, 300 MHz): (3 (ppm) =
I.91 (s, 3H, CO CH 3); 3.04 (s, 3H, N C H 3); 6.06
(s(broad), 2H, NH 2); 8.37 (s, 1H, N H CO ); 10.22-
II.03 (s(broad), 1H, H -l).
-
13C NM R (5%
N a0 D /D 20 /[D 6]-D M S0 as int. standard, 75.5
MHz): <3 (ppm) = 19.21 (q, C O C H 3); 25.09 (q,
N CH3); 85.60 (s, C-5); 156.79 (s, C-2); 158.33 (s, C-
6); 161.30 (s, C-4); 172.40 (s, CO CH 3). - MS (70
eV, 170 °C): m /z (% ) = 198 (26) (M +-H20 ); 157
(11); 156 (100) ([M+- H 70 ]-C 0 C H 2); 155 (20); 71
(34); 70 (16); 44 (15); 43 (66). - IR (KBr): v (cm-1) =
3545 (s); 3435 (m); 3365 (m); 3165 (s); 3055 (m);
1711 (s); 1667 (s); 1640 (s); 1580-1530 (s); 1470
(s); 1377 (m); 1318 (m); 969 (m); 778 (m); 750 (m);
713 (m); 638 (m). - UV (methanol): 2max(nrn) (lg
5,6-Diacetylamino-3-methyluracil ( DAMU) (12)
DAMU 12 was separated by semi-preparative
HPLC; subl.-p. 160 °C (needles developed) and
m.p. 257 °C .- R f - 0.61 (alumina; methanol);
^hplc —11-20 min.
e)
=
218 (3.82); 264 (4.25).
-
UV (water):
C9H 12N40 4 (240.22)
l mas(nm) (lg e) = 223.5 (3.84); 264 (4.27).
'H NMR ([D6]-DMSO, 300 MHz): (3 (ppm) =
1.97 (s, 3H, 5-COCH3); 2.14 (s, 3H, 4-COCH3);
3.12 (s, 3H, NCH3); 8.75; 10.06; 11.32 (s, 3-1H,
3-N H ). - 13C NMR ([D6]-DMSO, 75.5 MHz): (3
(ppm) = 23.04; 24.20 (q, 2 C O C H 3); 27.30 (q,
N C H 3); 96.16 (s, C-5); 143.34 (s, C-2); 148.91 (s, C-
5-Acetylam ino-6-form ylam ino-3-m ethyluracil
(AFM U) (9)
2.16 g (10.0 mmol) of 5-acetylamino-6-amino-3-
methyluracil-hydrate (8) were suspended in 6 ml
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1532
R. Röhrkasten et al. • Synthesis of the Caffeine Metabolites
6); 161.31 (s, C-4); 169.99 (s, 6-COCH3); 172.46 (s, (cm -1) = 3435 (m); 3250 (m); 1737 (m); 1719 (m);
5-COCH,). - MS (70 eV, 110 °C): m/z (% ) = 241
(6) (M ++ l); 240 (47) (M +); 198 (54); 156 (100)
(M+-2C H ?C O ); 155 (30); 43 (44). - IR (KBr): v
1685 (m); 1634 (vs); 1526 (m); 1298 (m); 1230 (m);
750 (m). - UV (m ethanol): Amax(nrn) (lg e) = 206
(4.23); 282 (4.15).
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