Synthesis of 7-Aza-5,8,10-trideazafolic acid and its 4-amino-derivative as potential antifolates

Article abstract of DOI:10.1002/jhet.5570390613

o-Aminoamide 8, an intermediate in our multistep synthesis of the title compounds was prepared from 1,3-diketone 3. The following condensation of 8 with chloroformamidine-HCl (9) gave pyrido[3,4-d]pyrimidine 10. Dehydratisation of amide 8 led to o-aminonitrile 15, which was cyclocondensated with guanidine (16) to yield pyrido[3,4-d]pyrimidine-2,4-diamine 17. Coupling of the acids 11 and 18 with diethyl L-glutamate (12) and following saponification provided 7-aza-5,8,10-trideazafolic acid 14 and its 4-amino-derivative 20.

Full text of DOI:10.1002/jhet.5570390613

Nov-Dec 2002
Synthesis of 7-Aza-5,8,10-trideazafolic Acid and its
4-Amino-derivative as Potential Antifolates
Günther Wollein and Reinhard Troschütz*
1195
Department of Medical Chemistry, Emil Fischer Center, Friedrich–Alexander University,
Schuhstr.19, D-91052 Erlangen, Germany
Received April 29, 2002
Dedicated to Professor Kurt Eger (Leipzig) on the occasion of his 60th birthday
o-Aminoamide 8, an intermediate in our multistep synthesisof the title compounds was prepared from 1,3-
diketone 3. The following condensation of 8 with chloroformamidine-HCl (9) gave pyrido[3,4-d]pyrimidine
10. Dehydratisation of amide 8 led to o-aminonitrile 15, which was cyclocondensated with guanidine (16) to
yield pyrido[3,4-d]pyrimidine-2,4-diamine17. Coupling of the acids 11 and 18 with diethyl L-glutamate (12)
and following saponification provided 7-aza-5,8,10-trideazafolic acid 14 and its 4-amino-derivative20.
J. Heterocyclic Chem., 39, 1195(2002).
In the last decades many efforts have been made to
obtain structural variations of folic acid as possible antitu-
mor agents with antifolate activity [1].
some types of cancer have a naturally or acquired resistance
against methotrexate [6], or – in the case of lometrexol - a
cumulative toxicity [7]. For that reason it is necessary to
synthesize new structural variations of antifolates having a
better activity – toxicity ratio than do the existing ones.
With this intention we have planned to modify the
pyrido[2,3-d]pyrimidine nucleus, which is present in the
non classical antifolate piritrexim [8] and lometrexol. We
As a result, some very promising attempts lead to
methotrexate (MTX) (inhibition of mammalian dihydrofo-
latereductase) [2], which had already been synthesized in
1947, but is still the method of choice in the treatment of
mammalian carcinoma in a combination therapy. More
recent compounds with antitumor potency are pemetrexed
[3] (mainly inhibition of thymidilate synthetase) [4] and
lometrexol (inhibition of glycineamideribonucleotid-
formyltransferase) [5], which are currently under investi-
gation in clinical trials.
8
formally shifted N to position 7 in order to modulate the
polarity and basicity of the anellated pyridine ring and
come to a pyrido[3,4-d]pyrimidine as the basic heterobicy-
cle for new antifolates, on one hand 4-amino-4-deoxy-7-
aza-5,8,10-trideazafolic acid (20) with structural similarity
to methotrexate, on the other hand 7-aza-5,8,10-
trideazafolic acid (14), which in our opinion could posses
antitumor activity.
Unfortunately each of these therapeutic agents have dis-
advantages. Beside the common toxicity for healthy cells,
In literature only a pteroic acid analogue exists with a
pyrido[3,4-d]pyrimidine structure [9].
Scheme 1
Generally only few entries to substituted pyrido[3,4-d]-
pyrimidines have been described and most of the existing
routes did not seem suitable for our synthesis of 6-alkyl-
substituted derivatives.[10-13]. A short retrosynthetic
view, which explains our approach is outlined in scheme 2.

1196
G. Wollein and R. Troschütz
Vol. 39
Cleavage of strategic bonds in the target compound
pyridine-3-ylamine with butyllithium followed by quench-
ing with carbon dioxide [15]. We used this reaction with a
boc-protected 6-styrylpyridine-3-ylamine as a model com-
pound, but unfortunately did not get the desired 3-amino-
6-styrylisonicotinic acid.
For this reason we decided to tread an alternative path.
Improving the method of Maguire and McKee [9], who
synthesized ethyl 6-methyl-3-nitro-2(1H)pyridone-4-car-
7-aza-5,8,10-trideazafolic acid (14) leads to a 3-amino-
isonicotinic acid derivative as the key intermediate and
guanidine. Further retrosynthesis gives rise to a 3-nitropy-
ridine that could originate from a nitroendiamine and a
1,3-diketone [14].
One recent access to 3-amino-6-chloroisonicotinic acid
exists in the directed lithiation of boc-protected 6-chloro-
Scheme 3

Nov-Dec 2002
Synthesis of 7-Aza-5,8,10-trideazafolic Acid
1197
boxylate as a precursor of a 3-aminoisonicotinic acid
derivative from nitroacetamide and ethyl acetopyruvate in
a low yield of 13 %, we replaced nitroacetamide by 2-
nitroethene-1,1-diamine (4) (Scheme 2) – in our hands a
versatile building block for an easy entry to 3-nitropyri-
dine-2-amines [16,17].
The thus obtained 3-nitroisonicotinate 5 could be directly
and efficiently converted to the 2-chloropyridine derivative
6 by substitutive deamination [19] with tert-butyl nitrite
and anhydrous copper(II) chloride in dry acetonitrile.
Subsequent hydrogenation of 2-chloropyridine derivative
6 at atmospheric pressure resulted in the reduction of the
nitro group and hydrogenolysis of the 2-chloro substituent,
affording 3-aminoisonicotinate 7 in a yield of 58%.
However, having no success in hydrogenation of 6 with
a palladium on barium carbonate catalyst, we found palla-
dium on charcoal 10% [20]- with a small amount of tri-
ethylamine – to be the exceptional catalyst.
The direct pyrimidine annellation to 2-amino-4-oxopy-
rido[3,4-d]pyrimidine 10 from o-aminoester 7 and guani-
dine or chloroformamidine-HCl [21] in various solvents
and a variety of temperatures failed.
We solved the problem by treating of 7 with ammonia in
methanol. The ammonolysis of 7 gave rise to o-aminoamide
8 (60%) and a diamide (15%), which were separated by col-
umn chromatography. The following ring closure of 8 with
chloroformamidine hydrochloride in dimethylsulfone at 160
°C provided10 [21], indicating that an o-aminoamide group
has been formed by ammonolysis of 7.
Our sequence for the synthesis of 7-aza-5,8,10-
trideazafolic acid (14) starts with the preparation of the
necessary 1,3-biselectrophile ethyl 4-(5-ethoxycarbonyl-
3,5-dioxopentyl)benzoate (3) from claisen condensation of
ketone 1 with diethyloxalate (2) [18] which proceeds in
good yields after purification by Kugelrohr destillation.
The following cyclocondensation [16] of 1,3-diketone 3
with 2-nitroethene-1,1-diamine (4) was carried out in
methanol under reflux. Of the two possible regioisomers –
i.e. the isonicotinic acid derivative 5 and/or the a -picolinic
acid derivative – we only isolated the desired 3-nitroisoni-
cotinate 5 in a yield of 65% without further necessary
purification. The isonicotinic ester structure of 5 was
proved by heteronuclear bond correlation (hnbc). Only one
coupling signal from the C1-hydrogen of the ethylene
chain to the pyridine-C5 was detected. In case of the iso-
meric a -picolinic acid derivative, two correlation signals
from C1-hydrogen of the ethylene bridge to C3- and C5 of
the pyridine nucleus should have been observed.
Saponification of 10 [22], followed by coupling of the
resulting benzoic acid derivative 11with diethyl L-glutamate

1198
G. Wollein and R. Troschütz
Vol. 39
acetic acid and poured on 300 ml ice. After extracting with ether
(3 x 300 mL), the combined organic layers were washed with
water, dried over sodium sulfate and evaporated to give 12 g
(75%) of a colourless oil after distillation by Kugelrohr–appara-
tus; bp 140 °C (5*10-2 bar); ir (sodium chloride): 2985, 2938 and
2908 (CH), 1716 (ester CO), 1639 and 1608 cm-1 (ketone CO);
1H nmr (dimethylsulfoxide-d6): d 1.26-1.34 (m, 6H, 2x OCH2-
CH3), 2.97 (s, 4H, CH2-CH2), 3.62 (s, 2H, O=C-CH2-C=O),
4.23-4.34 (m, 4H, 2x OCH2-CH3), 7.37-7.43 (m, 2H, 3-H and 5-
H), 7.84-7.92 ppm (m, 2H, 2-H and 6-H); ms: m/z 321 (M+1).
Anal. Calcd. for C17H20O6: C, 63.75; H, 6.29. Found: C,
63.92; H, 6.39.
(12) using 6-chloro-2,4-dimethoxy-1,3,5-triazine/N-methyl-
morpholine [23], gave rise to L-glutamate derivative 13.
Final alkaline hydrolysis of 13 followed by acidification
with acetic acid yielded the target compound N-[4-[2-(2-
amino-4-oxopyrido[3,4-d] pyrimidin-6-yl)ethyl]benzoyl]-
L-glutamic acid (14).
The starting sequence for the synthesis of N-[4-[2-(2,4-
diaminopyrido[3,4-d] pyrimidin-6-yl)ethyl]benzoyl]-L-
glutamic acid (20) is equal to that of L-glutamic acid
derivative 14.
In order to generate a amino function instead of the oxo
function in position 4 of a pyrido[3,4-d]pyrimidine deriva-
tive 20 it was necessary to convert the 3-aminoisonicoti-
namide 8 into 3-aminoisonicotinonitrile 15.
Several attempts to prepare 15 by treatment of isonicoti-
namide 8 with cyanuric chloride [24], methyl (carboxysul-
famoyl) triethylammonium hydroxide inner salt (Burgess
Ethyl 2-Amino-6-[2-(4-ethoxycarbonylphenyl)ethyl]-3-nitro-
isonicotinate (5).
A mixture of 980 mg (3 mmoles) ethyl 4-(5-ethoxycarbonyl-
3,5-dioxo-pentyl)benzoate ( 3) and 320 mg (3 mmoles)
2-nitroethene-1,1-diamine (4) in 20 ml methanol was heated at
reflux for 45 minutes and cooled to room temperature. Dropwise
adding of water provided 755 mg (65%) of 5 as a bright yellow
solid. mp 122-123 °C; ir (sodium chloride): 3521, 3455, 3382 and
3270 (NH2), 2989, 2931 and 2865 (CH), 1720 (ester CO), 1631,
1565 and 1504 cm-1 (NO2); uv (methanol): l max 390 nm (e
6733); 1H nmr (dimethylsulfoxide-d6): d 1.26 (t, J = 7.1 Hz, 3H,
OCH2-CH3), 1.31 (t, J = 7.1 Hz, 3H, OCH2-CH3), 2.96-3.10 (m,
4H, CH2-CH2), 4.29 (q, J = 7.1 Hz, 4H, 2x OCH2-CH3), 6.72 (s,
1H, 5-H), 7.37-7.41 (m, 2H, 3´-H and 5´-H), 7.85-7.90 (m, 2H,
2´-H and 6´-H), 7.95 ppm (broad s, 2H, NH2 deuterium oxide
exchangeable); ms: m/z 387 (M+).
reagent) [25] or POCl [26] failed. In the end we achieved
3
the nitrile 15 by using trifluoroacetic anhydride and excess
triethylamine in tetrahydrofuran [27].
Ring closure of o-aminonitrile 15 to pyrido[3,4-d]-
pyrimidin-2,4-diamine 17 was done successfully by reac-
tion with guanidine in dimethylsulfone at 130 °C [28].
Saponification of benzoate 17 [22], followed by cou-
pling of the resulting benzoic acid 18 with diethyl L-gluta-
mate (12) using 6-chloro-2,4-dimethoxy-1,3,5-triazine/
N-methylmorpholine [23], gave the intermediate L-gluta-
mate derivative 19.
Final alkaline hydrolysis of 19 followed by acidification
with acetic acid yielded the MTX analogue N-[4-[2-(2,4-
diaminopyrido[3,4-d] pyrimidin-6-yl)ethyl]benzoyl]-L-
glutamic acid (20).
Anal. Calcd. for C19H21N3O6: C, 58.92; H, 5.46; N, 10.85.
Found: C, 59.03; H, 5.46; N, 10.81.
Ethyl 2-Chloro-6-[2-(4-ethoxycarbonylphenyl)ethyl]-3-nitro-
isonicotinate (6).
tert-Butyl nitrite (0.36 ml, 3 mmoles) was added to a stirred
suspension of 322 mg (2.4 mmoles) anhydrous CuCl2 in 30 ml
anhydrous acetonitrile. The mixture was heated at 65 °C and then
a solution of 330 mg (0.86 mmoles) 5 in 30 ml anhydrous acetoni-
trile was added over a period of 5 minutes to the reaction medium.
During the addition the mixture turned completely black from the
initial green colour. After complete gas evolution (~ 15 minutes)
the temperature was allowed to come to room temperature and the
mixture was poured into 50 ml 20% aqueous HCl. Ethyl acetate
(60 ml) was added and the organic phase separated, washed with
50 ml 20% aqueous HCl and dried (Na2SO4). The solvent was
removed under reduced pressure and 6 was crystallized from
methanol (0 °C) in a yield of 60% as colourless solid; mp 103-104
°C; ir (sodium chloride): 3066, 2992 and 2935 (CH), 1743 and
EXPERIMENTAL
Melting points were determined on a Lindström capillary melt-
ing point apparatus from Büchi (type 530) and are uncorrected.
The ir spectra were recorded on a Perkin-Elmer infrared-fourier-
transform-spectrophotometer (type 1740). The 1H nmr spectra and
the 13C nmr spectra were recorded on ft-nmr spectrometers:
Bruker AC 250 (at 250.13 MHz), Bruker AM 360 (at 360.13
MHz). The 13C nmr spectra were recorded at 60.6 and 90.6 MHz;
the solvent was dimethylsulfoxide-d6 (unless otherwise stated)
with tetramethylsilane as the internal standard. The uv spectra were
recorded on a Perkin-Elmer spectrophotometer (type Lambda 5).
Solvent was methanol (UVasolâ Merck). Mass spectra were
recorded on a Finnigan TSQ 70 (electron; ionisation energy 70 eV)
and JEOL MS 700 (field desorption). Microanalyses were carried
out on a Carlo Erba Elemental Analyzer Model 1106.
1
1700 (ester CO), 1592 and 1546 cm-1 (NO2); H nmr (dimethyl-
sulfoxide-d6): d 1.27 (t, J = 7.1 Hz, 3H, OCH2-CH3), 1.31 (t, J =
7.1 Hz, 3H, OCH2-CH3), 3.06-3.14 and 3.22-3.29 (m, 4H, CH2-
CH2), 4.29 (q, J = 7.1 Hz, 2H, OCH2-CH3), 4.34 (q, J = 7.1 Hz,
2H, OCH2-CH3), 7.40-7.44 (m, 2H, 3´-H and 5´-H), 7.86-7.91 (m,
2H, 2´-H and 6´-H), 7.95 (s, 1H, 5-H); ms: m/z 406 (M+).
Anal. Calcd. for C19H19N2O6Cl: C, 55.86; H, 4.69; N, 6.86.
Found: C, 55.68; H, 4.98; N, 6.83.
Ethyl 4-(5-Ethoxycarbonyl-3,5-dioxopentyl)benzoate (3).
To a stirred and cooled (0 °C) solution of 1.15 g (50 mmoles)
sodium in 50 ml ethanol a mixture of 11 g (50 mmoles) ethyl 4-
(3-oxobutyl)benzoate (1) and 14.6 g (100 mmoles) diethyloxalate
(2) was added dropwise. After standing at room temperature
overnight, it was neutralized with an equimolar amount of glacial
Ethyl 5-Amino-2-[2-(4-ethoxycarbonylphenyl)ethyl]-
isonicotinate (7).
To a solution of 100 mg (0.25 mmoles) 6 and 0.1 ml triethyl-
amine in 8 ml ethanol was added 100 mg 10% palladium on

Nov-Dec 2002
Synthesis of 7-Aza-5,8,10-trideazafolic Acid
1199
carbon and the mixture was hydrogenated for 2 hours under
atmospheric pressure. The catalyst was filtered off and the filtrate
was evaporated nearly to dryness. Storing at 0 °C yielded 60 mg
(70%) colourless needles. mp 132 – 133 °C; ir (potassium bro-
mide): 3451 and 3337 (NH2), 2976 and 2937 (CH), 1712 cm-1
To a suspension of 100 mg (0.33 mmoles) 11 in 7 ml dimethyl-
formamide at 25 °C was added 0.042 ml (0.38 mmoles) N-
methylmorpholine followed by 73 mg (0.39 mmoles) 6-chloro-
2,4-dimethoxy-1,3,5-triazine and the resulting solution was
stirred at 25 °C for 1 hour. N-methylmorpholine (0.06 ml, 0.54
mmoles) was added to the solution followed by 83 mg (0.35
mmoles) diethyl L-glutamate hydrochloride, and the resulting
mixture was stirred at room temperature for 2 hours. The reaction
mixture was concentrated in vacuo and the residue was taken up
in 7 ml dichloromethane. The dichloromethane layer was washed
with 5% NaHCO3, dried (MgSO4) and concentrated in vacuo.
The residue was chromatographed on silica gel eluting with chlo-
roform:methanol (95:5, v/v) to give 130 mg (80%) as a white
solid. mp 215-217 °C; ir (sodium chloride): 3297 (NH), 2981 and
1
(ester CO); H nmr (dimethylsulfoxide-d6): d 1.26-1.34 (m, 6H,
2x OCH2-CH3), 2.87 – 3.00 (m, 4H, CH2-CH2), 4.23-4.33 (m,
4H, 2x OCH2-CH3), 6.50 (broad s, 2H, NH2 deuterium oxide
exchangeable), 7.27 (s, 1H, 2-H), 7.31 – 7.37 (m, 2H, 3´-H and
5´-H), 7.82 – 7.88 (m, 2H, 2´-H and 6´-H), 8.21 ppm (s, 1H, 5H);
ms: m/z 342 (M+).
Anal. Calcd. for C19H22N2O4: C, 66.66; H, 6.48; N, 8.18.
Found: C, 67.05; H, 6.74; N, 8.57.
Methyl 4-[2-(5-Amino-4-carbamoylpyridin-2-yl)ethyl]benzoate
(8).
-1
2935 (CH), 1735 (ester CO), 1662 cm (CO); 1H nmr (dimethyl-
sulfoxide-d6): d 1.13-1.21 (m, 6H, 2x OCH2-CH3), 1.93-2.16 and
2.40-2.46 (m, 4H, ethyl glutamate CH2-CH2), 3.07 (s, 4H, aryl
CH2-CH2 ), 3.99-4.14 (m, 4H, 2x OCH2-CH3), 4.37-4.46 (m, 1H,
ethyl glutamate HCNH2), 6.52 (broad s, 2H, NH2, deuterium
oxide exchangeable), 7.29-7.35 (m, 2H, 3´-H and 5´-H), 7.54 (s,
1H, 8-H), 7.75-7.79 (m, 2H, 2´-H and 6´-H), 8.56 (s, 1H, 5-H),
8.60-8.64 (d, 1H, CONH); 11.21 ppm (broad s, 1H, NH, deu-
terium oxide exchangeable); ms: m/z 495 (M+).
A solution of 500 mg (1.67 mmoles) 7 in 50 ml methanol was
saturated with ammonia at 0 °C for 15 minutes. The mixture was
kept in an autoclave at 80 °C overnight, cooled to room temper-
ature and evaporated to dryness. The residue was separated by
flash chromatography on silica gel (cyclohexane:ethyl acetate
5:5, v/v), yield 300 mg (60%). mp 173 – 175 °C; ir (potassium
bromide): 3430, 3340 and 3186 (NH), 2952, 2925 and 2859
(CH), 1718 (ester CO), 1669 cm -1 (amide CO); 1H nmr
(dimethylsulfoxide-d6): d 2.84-2.93 and 2.98-3.07 (m, 4H, CH2-
CH2), 3.83 (s, 1H, OCH3), 6.38 (broad s, 2H, NH2 deuterium
oxide exchangeable), 7.31 (s, 1H, 2-H), 7.33-7.37 (m, 2H, 3´-H
and 5´-H), 7.39 (broad s, 1H, CONH, deuterium oxide
exchangeable), 7.84-7.89 (m, 2H, 2´-H and 6´-H), 7.94 (broad s,
1H, CONH, deuterium oxide exchangeable), 8.06 ppm (s, 1H, 5-
H); ms: m/z 299 (M+).
Anal. Calcd. for C25H29N5O6: C, 60.60; H, 5.90; N, 14.13.
Found: C, 60.60; H, 6.18; N, 14.42.
N-[4-[2-(2-Amino-4-oxopyrido[3,4-d]pyrimidin-6-yl)ethyl]ben-
zoyl]-L-glutamic Acid (14).
A solution of 50 mg (0.1 mmoles) of 13 in 1 ml 2 N sodium
hydroxide was allowed to stir at room temperature for 24 hours. The
reaction mixture was acidified with glacial acetic acid and the solid
product was collected by filtration, washed with water and dried to
give 29 mg (65%) as a white solid. mp 254-255 °C (decomposi-
tion); ir (sodium chloride): 3316 (NH); 2981 and 2931 (CH); 1725
cm-1 (CO); 1H nmr (F3CCOOD):d 2.37-2.59 and 2.65-2.90 46 (m,
4H, ethyl glutamate CH2-CH2), 3.15-3.24 and 3.75-3.88 (m, 4H,
aryl CH2-CH2), 5.07-5.17 (m, 1H, ethyl glutamate HCNH2), 7.45
(s, 1H, 8-H), 7.49-7.55 (m, 2H, 3´-H and 5´-H), 7.80-7.94 (m, 2H,
2´-H and 6´-H), 8.58 ppm (s, 1H, 5-H); ms: m/z 440 (M+1).
Anal. Calcd. for C21H21N5O6: C, 57.41; H, 4.82; N, 15.94.
Found: C, 57.36; H, 4.73; N 16.15.
Anal. Calcd. for C16H17N3O3: C, 64.21; H, 5.72; N, 14.04.
Found: C, 63.98; H, 5.68; N, 13.86.
Methyl 4-[2-(2-Amino-4-oxopyrido[3,4-d]pyrimidin-6-
yl)ethyl]benzoate (10).
A mixture of 50 mg (0.16 mmoles) 8, 60 mg (0.53 mmoles)
of freshly prepared chloroformamidine hydrochloride (9) and
50 mg of dimethylsulfone was heated for 1 hour at 160 °C in an
open flask with magnetic stirring. Water was added to the solid
mass and, following warming this mixture, ammonium
hydroxide 10% was added until neutralisation was achieved.
The resulting white solid was isolated by filtration in a yield of
39 mg (40%); mp > 260 °C; ir (potassium bromide): 3334 and
3176 (NH), 1718 (ester CO), 1655 cm -1 (CO); 1H nmr
(dimethylsulfoxide-d6): d 3.04-3.12 (m, 4H, CH2-CH2), 3.82
(s, 3H, OCH3), 6.62 (broad s, 2H, NH2, deuterium oxide
exchangeable), 7.34-7.40 (m, 2H, 3´-H and 5´-H), 7.52 (s, 1H,
8-H), 7.82-7.89 (m, 2H, 2´-H and 6´-H), 8.56 ppm (s, 1H, 5-H);
ms: m/z 324 (M+).
Methyl 4-[2-(5-Amino-4-cyanopyridin-2-yl)ethyl]benzoate (15).
Triethylamine (126 mg, 1.25 mmoles) were added to a solution
of 60 mg (0.20 mmoles) 8 at 0 °C in 2 ml tetrahydrofuran (THF)
followed by 160 mg trifluoroacetic anhydride in 1 ml THF. After
30 minutes 2 ml water was added, and the mixture was extracted
with 2 x 2 ml diethyl ether. The organic layer was dried and evap-
orated, and the residue was dissolved in 2 ml methanol-water
(1:1) containing 100 mg K2CO3. The mixture was heated at 70
°C for 24 hours, cooled, and extracted with 2 x 2 ml ethyl acetate.
The extracts were washed with water and brine, dried, and evap-
orated to give 50 mg (90%); mp 128-129 °C; ir (sodium chlo-
ride): 3436 and 3359 (NH), 2923 and 2854 (CH), 2217 (CN),
Anal. Calcd. for C17H16N4O3: C, 62.96; H, 4.97; N, 17.28.
Found: C, 63.34; H, 5.25; N, 17.34.
Diethyl N-[4-[2-(2-Amino-4-oxopyrido[3,4-d]pyrimidin-6-
yl)ethyl]-benzoyl]-L-glutamate (13).
1
1712 cm-1 (ester CO); H nmr (dimethylsulfoxide-d6): d 2.85-
2.93 and 2.94-3.05 (m, 4H, CH2-CH2), 3.84 (s, 3H, OCH3), 6.22
(broad s, 2H, NH2, deuterium oxide exchangeable), 7.21 (s, 1H,
2-H), 7.30-7.44 (m, 2H, 3´-H and 5´-H), 7.82-7.91 (m, 2H, 2´-H
and 6´-H), 8.20 ppm (s, 1H, 5-H); ms: m/z 281 (M+).
Anal. Calcd. for C17H17N3O2: C, 69.14; H, 5.80; N, 14.23.
Found: C, 68.85; H, 5.91; N, 14.17
The acid 11 was achieved from methyl 4-[2-(2-amino-4-oxopy-
rido[3,4-d]pyrimidin-6-yl)ethyl]benzoate (10) by stirring in 2 N
sodium hydroxide at room temperature for 4 hours followed by
neutralisation with glacial acetic acid. Compound 11 was obtained
in a amorphous modification at 0 °C, collected by filtration, dried
under vacuum and used without further purification.

1200
G. Wollein and R. Troschütz
Vol. 39
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16, 133 (1979).
Methyl 4-[2-(2,4-Diaminopyrido[3,4-d]pyrimidin-6-yl)ethyl]-
benzoate (17).
Compound 15 (35 mg, 0.12 mmoles) were dissolved at 130 °C
in 250 mg dimethylsufone. After addition of 40 mg (0.68
mmoles) guanidine (16), the mixture was stirred at this tempera-
ture for 1.5 days. The hot solution was diluted with 0.5 ml water,
the solid thus obtained was collected by filtration and chro-
matographed on silica gel eluting with chloroform:methanol (9:1,
v/v) to give 20 mg (50%); mp 286-288 °C; ir (sodium chloride):
3548 and 3336 (NH), 2919 and 2854 (CH), 1751 cm-1 (CO); uv
1
(methanol): l max 345 nm (e 8591); H nmr (dimethylsulfoxide-
d6): d 2.84-2.92 and 3.27-3.40 (m, 4H, CH2-CH2), 3.72 (s, 3H,
OCH3), 6.26 (broad s, 2H, NH2, deuterium oxide exchangeable),
6.80 (s, 1H, 8-H), 6.82-6.85 (m, 2H, 3´-H and 5´-H), 6.94 (broad
s, 2H, NH2, deuterium oxide exchangeable), 7.12-7.19 (m, 2H,
2´-H and 6´-H), 8.43 ppm (s, 1H, 5-H); ms: m/z 323 (M+).
Anal. Calcd. for C17H17N5O2: C, 63.15; H, 5.30; N, 21.66.
Found: C, 63.42; H, 5.18; N, 21.34.
[10] Z. Ozdowska and B. Szczyinski, Rocz. Chem., 50, 1771
(1976).
[11] R. G. Glushkov, O. Ya Belyaeva, V. G. Granik, M. K.
Polievktova, A. B. Girgorev, V. E. Serokhvostova and T. F. Vlasova,
Khim. Geterotsikl. Soedin., 1640 (1976).
Diethyl N-[4-[2-(2,4-Diaminopyrido[3,4-d]pyrimidin-6-yl)-
ethyl]benzoyl]-L-glutamate (19).
[12] D. J. Berry, J. D. Cook and B. J. Wakefield, J. Chem. Soc.,
Perkin Trans. 1, 2190 (1972).
[13] R. Madhav, Org. Prep. Proced. Int., 14, 403 (1982).
[14] D. G. Batt and G. C. Houghton, J. Heterocyclic Chem., 32,
963 (1995).
[15] G. W. Rewcastle, W. A. Denny, R. T. Winters, N. L.
Colbry, and H. D. Hollis Showalter, J. Chem. Soc., Perkin Trans. 1,
2221 (1996).
[16] R. Troschütz and A. Lückel, Arch. Pharm. (Weinheim),
324, 73 (1991).
[17] R. Troschütz and A. Karger, J. Heterocyclic Chem., 33,
1815 (1996).
[18] J. J. Parlow, D. A. Mischke and S. S. Woodard, J. Org.
Chem., 62; 17; 5908 (1997).
[19] V. Ojea, C. Peinador and J. M. Quintela, Synthesis, 798
(1992).
Preparation and purification according to 11 from 50 mg (0.16
mmoles) 17 via the acid 18 yielded 51 mg (65%) 19 as a white
solid; mp 220-221 °C; ir (sodium chloride): 3409 and 3286 (NH);
2981 and 2931 (CH); 1735 cm-1 (CO); 1H nmr (dimethylsulfox-
ide-d6): d 1.12-1.23 (m, 6H, 2x OCH2-CH3), 1.95-2.16 and 2.39-
2.46 (m, 4H, ethyl glutamate CH2-CH2), 2.96-3.08 and 3.37-3.50
(m, 4H, aryl CH2-CH2), 4.01-4.15 (m, 4H, 2x OCH2-CH3), 4.38-
4.48 (m, 1H, ethyl glutamate HCNH2), 6.44 (broad s, 2H, NH2,
deuterium oxide exchangeable), 6.84 (s, 1H, 8-H), 7.15 (broad s,
2H, NH2, deuterium oxide exchangeable), 7.28-7.36 (m, 2H, 3´-
H and 5´-H), 7.75-7.83 (m, 2H, 2´-H and 6´-H), 8.44 (s, 1H, 5-H),
8.61-8.68 (d, 1H, CONH); ms: m/z 494 (M+).
Anal. Calcd. for C25H30N6O5: C, 60.72; H, 6.11; N, 17.00.
Found: C, 60.79; H, 5.94; N, 17.32.
N-[4-[2-(2,4-Diaminopyrido[3,4-d]pyrimidin-6-yl)ethyl]ben-
zoyl]-L-glutamic Acid (20).
[20] A. Tanaka, T. Terasawa, H. Hagihara, N. Ishibe, M.
Sawada, Y. Sakuma, M. Hashimoto, H. Takasugi and H. Tanaka, J.
Med. Chem., 41, 4408 (1998).
Preparation and purification according to 14 from 50 mg (0.10
mmoles) 19 yielded 31 mg (71%) 20 as a white solid; mp 268-269
°C (decomposition); ir (sodium chloride): 3374 and 3313 (OH,
NH), 2981 and 2935 (CH), 1715 cm-1 (CO); 1H nmr (dimethylsul-
foxide-d6): d 1.89-2.15 and 2.25-2.40 (m, 4H, ethyl glutamate
CH2-CH2), 2.90-3.09 and 3.35-3.50 (m, 4H, aryl CH2-CH2), 4.26-
4.43 (m, 1H, ethyl glutamate HCNH2), 6.54-6.80 (broad s, 1H,
NH2, deuterium oxide exchangeable), 6.89 (s, 1H, 8-H), 7.25-7.39
(m, 2H, 3´-H and 5´-H), 7.74-7.85 (m, 2H, 2´-H and 6´-H), 8.41-
8.54 ppm (m, 2H, 5-H and CONH); ms: m/z 439 (M+1).
[21] S. W. Schneller and A. C. Ibay, J. Org. Chem., 51, 4067
(1986).
[22] E. C. Taylor and P. M. Harrington, J. Org. Chem., 55, 3222
(1990).
[23] E. C. Taylor, H. H. Patel, G. Sabitha and R. Chaudhari,
Heterocycles, 43, 349 (1996).
[24] G. A. Olah, S. C. Narang, A. P. Fung and B. G. B. Gupta,
Synthesis, 657 (1980).
[25] D. A. Claremon and B. T. Phillips, Tetrahedron Letters,
29, 2155 (1988).
Anal. Calcd. for C21H22N6O5: C, 57.53; H, 5.06; N, 19.17.
Found: C, 57.38; H, 5.26; N, 19.04.
[26] K. E. Andersen and E. B. Pedersen, Liebigs. Ann. Chem.,
1012 (1986).
[27] M. Rowley, J. J. Kulagowski, A. P. Watt, D. Rathbone, G.
I. Stevenson, R. W. Carling, R. Baker, G. R. Marshall, J. A. Kemp, A.
C. Foster, S. Grimwood, R. Hargreaves, C. Hurley, K.L. Saywell, M.
D. Tricklebank and P. D. Leeson, J. Med. Chem., 40, 4053 (1997).
[28] F. Schönfeld and R. Troschütz, Heterocycles, 55, 1679
(2001).
REFERENCES AND NOTES
[1] A. L. Jackson, Antifolate Drugs in Cancer Therapy,
Humana Press, Totowa, New Jersey,1999.