Pteridines. Part CXI. Pteridine-based photoaffinity probes for nitric oxide synthase and aromatic amino acid hydroxylases

Article abstract of DOI:10.1002/1522-2675(20001004)83:10<2738::AID-HLCA2738>3.0.CO;2-A

Various 6-substituted pteridines and 5,6,7,8-tetrahydropterins carrying photolabile functions at the side chain (see 7, 20-22, 34-36, 38, and 39) as well as at the 5-position (see 27-29) were synthesized from pterin and from 6-phenylpterin (1) and 6-(hydroxymethyl)pterin (10). Attachment of the photoaffinity labels via ester bonds required a special protecting-group strategy based upon acid-labile (see 30-33) and β-eliminating blocking groups (see 17-19). The 6-(4-azidophenyl)pterin (7) was obtained from 6-phenylpterin (1) via intermediates 2 and 4-6, due to the low solubility of simple pterins in general. The pteridine derivatives 21, 22, 25, 26, 28, 29, 32, 33, 35, 36, 38, and 39 were screened as inhibitors of neuronal (type I) NO synthase (see Table) from porcine cerebellum. of which 22, 35, 36, and 38 showed interesting inhibitory activity with similar potency and effectiveness.

Full text of DOI:10.1002/1522-2675(20001004)83:10<2738::AID-HLCA2738>3.0.CO;2-A

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Pteridines
Part CXI1)
Pteridine-Based Photoaffinity Probes for Nitric Oxide Synthase and Aromatic Amino
Acid Hydroxylases
by Viola Groehna), Lothar Fröhlichb), Harald H. H. W. Schmidtb), and Wolfgang Pfleiderera)*
a) Fakultät für Chemie, Universität Konstanz, Postfach 5560, D-78434 Konstanz
b) Rudolf Buchheim Institut für Pharmakologie der Justus Liebig Universität, Frankfurter Strasse 107,
D-35392 Giessen
Various 6-substituted pteridines and 5,6,7,8-tetrahydropterins carrying photolabile functions at the side
chain 6see 7, 20 ± 22, 34 ± 36, 38, and 39) as well as at the 5-position 6see 27 ± 29) were synthesized from pterin and
from 6-phenylpterin 61) and 6-6hydroxymethyl)pterin 610). Attachment of the photoaffinity labels via ester
bonds required a special protecting-group strategy based upon acid-labile 6see 30 ± 33) and b-eliminating
blocking groups 6see 17 ± 19). The 6-64-azidophenyl)pterin 67) was obtained from 6-phenylpterin 61) via
intermediates 2 and 4 ± 6, due to the low solubility of simple pterins in general. The pteridine derivatives 21, 22,
25, 26, 28, 29, 32, 33, 35, 36, 38, and 39 were screened as inhibitors of neuronal 6type I) NO synthase 6see Table)
from porcine cerebellum, of which 22, 35, 36, and 38 showed interesting inhibitory activity with similar potency
and effectiveness.
1. Introduction. ± The technique of photoaffinity labeling has a high potential in
characterizing the function of enzyme cofactors and substrate binding sites [2][3]. One
such binding site which has recently gained considerable interest is located in tetrahy-
drobiopterin-requiring enzymes such as aromatic amino acid hydroxylases 6tyrosine
hydroxylase, phenylalanine hydroxylase) and NO synthases. Photolabile pteridines
would be ideally suited to elucidate the tetrahydrobiopterin binding site and pterin-
based inhibitors of these enzymes. To ensure successful labeling, the photoaffinity label
must fullfil several criteria, such as specific binding with high affinity to the natural
cofactor binding site, formation of a highly reactive species on irradiation to bind
specifically to amino-acid moieties belonging to the binding site, and photochemical
features which allow irradiation at wavelengths >300 nm to avoid enzyme damage. At
the same time, these compounds must be chemically stable enough to survive normal
synthetic and biological manipulations. Finally, the synthetic approach should include
the possibility to synthesize also a tritiated derivative so that radioactivity labeling
could be applied as an additional tool in such investigations.
Thus, three established, chemically distinct photolabile functions were introduced
at the 6-position of the pteridine skeleton to meet as closely as possible the structural
requirements of the natural cofactor: the azidophenyl group, which releases N2 and
forms nitrenes [4], the 4-[3-6trifluoromethyl)-3H-diazirin-3-yl]phenyl group [5][6]
1
)
Part CX: [1].

Helvetica Chimica Acta ± Vol. 83 62000)
2739
which is a carbene precursor [7], and the benzoylphenethyl group [8], which generates
radicals [9].
The inhibitory effects of the modified pteridines and their competitiveness with
respect to 5,6,7,8-tetrahydrobiopterin 6H4Bip) were taken as evidence for their
interaction with the respective enzyme cofactor binding site. While NO synthases can
be inhibited by pteridines of the common oxidation states 6aromatic, 7,8-dihydro, and
5,6,7,8-tetrahydro forms), the aromatic amino acid hydroxylases bind only the fully
reduced pterins, which must contain an unblocked amino function at the N65) position
of the pterin moiety.
2. Synthesis. ± Our first approach was directed towards the synthesis of 6-
6azidophenyl)pterins as potential inhibitors of NO synthases. The synthesis was
initiated by direct-nitration studies of 6-phenylpterin 61) [10][11] by means of various
types of nitric acid under different reaction conditions, which, however, always led to
mixtures of 6ortho-6o)- and para6p)-nitrophenyl)pterins 62/3), indicating that the
pterin moiety served in this case as a first-order substituent 6Scheme 1). This may be
explained by the interaction of the 2-amino function, which is in conjugation with the
phenyl ring and provides by mesomerism electrons to the o- and p-positions of the
phenyl ring. All attempts to separate the o- and p-isomers failed so far, due to solubility
reasons. To overcome this problem, the 6-6o/p-nitrophenyl)pterin mixture was acylated
by reflux in isobutyric anhydride/pyridine leading to the corresponding N2-isobutyryl
derivatives. Of these compounds, N2-isobutyryl-6-64-nitrophenyl)pterin 64), fortunate-
ly, precipitated from the reaction solution by crystallization upon cooling. Reduction of
the nitro group was achieved by reflux in 20% aqueous ammonium sulfide to form 6-64-
aminophenyl)pterin 65). Due to the loss of the isobutyryl moiety, this derivative was
Scheme 1

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again extremely insoluble and could not be purified at this stage. It was characterized in
form of the N2-isobutyryl-6-[4-6isobutyrylamino)phenyl]pterin 66). Diazotization of
crude 5 and subsequent addition of NaN3 led to 6-64-azidophenyl)pterin 67). Its
purification required again isobutyrylation to 6-64-azidophenyl)-N2-isobutyrylpterin
68), which could be recrystallized from MeOH/AcOEt. Then the isobutyryl group of 8
was removed by treatment with NH3/MeOH 6 ! 7).
Next the selective reduction of 8 to the corresponding 5,6,7,8-tetrahydropterin
derivative by various types of catalytic hydrogenations and chemical reductions with
NaBH4 or sodium dithionite was studied, but none of these methods worked
successfully since the azido function was attacked much more readily than the pyrazine
ring. After Pt-catalyzed hydrogenation of 8 and workup with Ac2O, 5-acetyl-6-[4-
6acetylamino)phenyl]-5,6,7,8-tetrahydro-N2-isobutyrylpterin 69) was isolated in 55%
yield 6Scheme 1).
Another approach to photoactive 5,6,7,8-tetrahydropterins was based on 6-
[6acyloxy)methyl] derivatives first synthesized by Traub [12][13] from 6-6bromometh-
yl)pterin by reaction with carboxylates and subsequent reduction of the 6pterin-6-
yl)methyl esters. Our strategy, however, started from 6-6hydroxymethyl)pterin 610)
[14][15], which was first reduced to 5,6,7,8-tetrahydro-6-6hydroxymethyl)pterin 611)
and then stabilized by protection of the N5-position with 2-64-nitrophenyl)ethyl
carbonochloridate [16] as acylating agent 6Scheme 2). The obtained 5,6,7,8-tetrahydro-
Scheme 2

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2741
6-6hydroxymethyl)-5-{[2-64-nitrophenyl)ethoxy]carbonyl}pterin 612) required further
protection at the 2-amino group, which was selectively converted by N,N-
dimethylformamide diethyl acetal [17] to the corresponding N2-[6dimethylamino)-
methylene] derivative 13.
The terminal OH group of 13 was then prone to acylation by 4-azidobenzoic acid
[18], 3-64-benzoylphenyl)propanoic acid [19], and 4-[3-6trifluoromethyl)-3H-diazirin-
3-yl]benzoic acid [5] to give 14 ± 16, respectively, in good yields in the presence of N-[3-
6dimethylamino)propyl]-N'-ethylcarbodiimide hydrochloride 6EDC) and N,N-dime-
thylpyridin-4-amine 6DMAP) 6Scheme 2). The 4-azidobenzoic anhydride [20], but not
4-azidobenzoyl chloride [21], also worked successfully furnishing 14 in 63% yield. The
removal of the blocking groups of 14 ± 16 was achieved stepwise, first by cleavage of the
6dimethylamino)methylene group with MeOH/HCl to give 17 ± 19, respectively, and
finally by treatment with 1,8-diazabicyclo[5.4.0]undec-7-ene 6DBU) under aprotic
conditions to remove the [2-64-nitrophenyl)ethoxy]carbonyl group in a b-elimination
process affording the 6-substituted 5,6,7,8-tetrahydropterin derivatives 20 ± 22, respec-
tively, in the form of their tosylate salts. These turned out to be stable against air
oxidation, even during recrystallization from H2O/MeOH.
A further extension of these investigations was achieved by the introduction of the
photoactive functions at N65), since various 5-acyl-5,6,7,8-tetrahydropterins turned out
to be interesting NO-synthase inhibitors. For this purpose, 5,6,7,8-tetrahydro-N2-
isobutyrylpterin 623) was treated with 4-azidobenzoyl chloride [21] to give 5-64-
azidobenzoyl)-5,6,7,8-tetrahydro-N2-isobutyrylpterin 624) 6Scheme 3). Acylation of 23
with 3-64-benzoylphenyl)propanoic acid or 4-[3-6trifluoromethyl)-3H-diazirin-3-yl]-
Scheme 3

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benzoic acid in the presence of EDC and DMAP led in good yields to the 5-acyl
derivatives 25 and 26, respectively. In this series, the isobutyryl group was much more
labile towards hydrolysis than the substituents at N65) and could, therefore, easily be
cleaved by NH3/MeOH to give the stable 5-acyl-5,6,7,8-tetrahydropterins 27 ± 29 in high
yields.
Acylations of 6-6hydroxymethyl)pterin 610) at the side chain were successful only
when starting from N2-[6dimethylamino)methylene]-6-6hydroxymethyl)pterin 630)
and with activation of the three photoactive acids by EDC and DMAP in the usual
manner leading to 31 ± 33. The N2-protective group was then cleaved in almost
quantitative yield to give the anticipated 6-[6acyloxy)methyl]pterins 34 ± 36, respec-
tively.
Analogous pteridine-2,4-diamine derivatives were synthesized from 6-6bromometh-
yl)pteridine-2,4-diamine 637) [1][22] in a nucleophilic displacement reaction with
triethylammonium 3-64-benzoylphenyl)propanoate and 4-[3-6trifluoromethyl)-3H-di-
azirin-3-yl]benzoate in DMF to give 38 and 39, respectively; however, the yields were
very low, despite the fact that all starting material disappeared during this reaction.
3. Physical Data. ± The newly synthesized compounds were characterized by their
1
UV and H-NMR data 6see Exper. Part) and their composition further established by
the elemental analyses.
4. Biological Data. ± Preliminary investigation of the newly synthetized photo-
reactive pteridines in bioassays indicated that some compounds are suitable for
inhibition of type-I NO synthase [23] purified from porcine cerebellum 6Table). None
of the various pterin derivatives serve as a substitute for tetrahydrobiopterin 6H4Bip)
with respect to stimulation of NO-synthase activity which was measured under
standard assay conditions as previously described [23]. The H4Bip concentration was
2 mm, and the inhibitor concentration 50 times higher. The activity measured in
presence of inhibitor was referred to a control assay without inhibitor present. All other
activity measurements were expressed as % of this control activity. Among the H4Bip
Table. Inhibition of Type-I NO-Synthase Activity by Different Pteridine Derivatives
Conc. [mm]
H4Bip conc. [mm]
NO-Synthase activity
[% of control]
IC50 [mm]
±
±
2
2
2
2
2
2
2
2
2
2
2
2
2
±
100
21
22
25
26
28
29
32
33
35
36
38
39
100
100
100
100
100
100
100
100
100
100
100
100
57
28
62
76
75
93
95
63
2
42
190
60
44
48
18
29
55
140

Helvetica Chimica Acta ± Vol. 83 62000)
2743
derivatives, 22 was the only potential inhibitor of NO synthase recognized. The
maximal inhibition observed with all other compounds was below 50% of control.
However, two very potent inhibitors of NO synthase were found among the aromatic 6-
substituted pterins, of which 35, 36, and 38 inhibited NO-synthase activity down to 2, 18,
and 29% of control, respectively, making two of these derivatives promising candidates
for photoaffinity-labeling experiments.
Monooxygenases, on the other hand, bind only tetrahydropteridines bearing a free
NH function at the 5-position. While the 6-{[64-azidobenzoyl)oxy]methyl}- and the 6-
[6{4-[3-6trifluoromethyl)-3H-diazirin-3-yl]benzoyl}oxy)methyl]-substituted 5,6,7,8-tetra-
hydropterins 20 and 22 showed almost the same cofactor activity as H4Bip, surprisingly,
the benzophenone derivative 21 seems to be a strong inhibitor of human tyrosine
hydroxylase 6hTH1) with high affinity for the enzyme, as indicated by the rather low Ki
value [24].
Experimental Part
General. TLC: Precoated cellulose thin-layer sheets F 1440 LS 254 and silica-gel thin-layer sheets
F 1500 LS 254 from Schleicher & Schüll. Flash chromatography 6FC): silica gel 6Baker, 30 ± 60 mm); 0.2 ±
0.3 bar. M.p.: Büchi apparatus, model Dr. Tottoli; no corrections. UV/VIS: Uvikon 820, Kontron, and Lambda 5
1
6Perin-Elmer); lmax 6log e). IR: in cmÀ1. H-NMR: Bruker-WN-250; d in ppm rel. to Me4Si.
1. N2-Isobutyryl-6-44-nitrophenyl)pterin 6ˆ N-[3,4-Dihydro-6-44-nitrophenyl)-4-oxopteridin-2-yl]-2-meth-
ylpropanamide; 4). Powdered 6-phenylpterin 61) [10][11] 612 g, 50.16 mmol) was added in small portions to
conc. H2SO4 soln. 672 ml) at 08 with stirring. The resulting red soln. was treated with fuming HNO3 624 ml) by
dropwise addition, keeping the temp. below 108. After stirring for 2 h at r.t., the mixture was poured on ice. The
yellow precipitate was immediately collected, washed with small amounts of H2O, and dried over KOH in
vacuo: 11.67 g 682%) of 6-6nitrophenyl)pterins of 2/3 6by NMR) as a yellow powder. This mixture 68.32 g) was
suspended in dry pyridine 6110 ml), then isobutyric anhydride 628 ml) added, and the mixture refluxed for 4 h.
The resulting dark brown soln. was cooled to r.t. and kept refrigerated overnight to give a yellowish precipitate.
The latter was then suspended in H2O, the suspension stirred for 10 min, and the solid filtered off, washed with
EtOH/H2O, and dried at 408: 7.1 g 640%) of 4. Recrystallization from EtOH/H2O. M.p. 3108 6darkening), 3218
6dec.). TLC 6cellulose, 1% NH3/PrOH 1:2): Rf 0.71. UV 6MeOH): 363 64.36), 319 64.36), 261 64.07), 233 6sh,
4.18), 203 64.39). 1H-NMR 66D6)DMSO): 12.37 6br. s, HÀN63)); 12.06 6br. s, CONHÀC62)); 7.60 6s, HÀC67));
8.46 ± 8.38 62d, arom. H); 2.72 ± 2.88 6sept., Me2CH); 1.16 6d, Me2CH). Anal. calc. for C16H14N6O4 6354.3):
C 54.24, H 3.98, N 23.72; found: C 53.91, H 4.00, N 23.18.
2. 6-44-Aminophenyl)pterin 6ˆ2-Amino-6-44-aminophenyl)pteridin-4-43H)-one; 5). A suspension of
4
62 g, 5.64 mmol) in 20% aq. 6NH4)2S soln. 630 ml) was heated to 1008 for 1 h. The starting material dissolved,
and subsequently an orange solid precipitated. After cooling, the solid was collected, treated with CS2 to remove
sulfur, filtered again, washed with EtOH, and dried at 1008: 1.4 g 698%) of 5. The crude material was not
purified but was sufficiently pure for transformations into 6 ± 8.
3. N2-Isobutyryl-6-[4-4isobutyrylamino)phenyl]pterin 6ˆ N-43,4-Dihydro-6-{4-[42-methyl-1-oxopropyl)-
amino]phenyl}-4-oxopteridin-2-yl)-2-methylpropanamide; 6). A suspension of 5 61.02 g, 4 mmol) in dry pyridine
610 ml) was treated with isobutyric anhydride 63 ml) and heated under reflux for 2 h. On cooling, colorless
crystals precipitated. The solid was suspended in H2O, stirred, again filtered, and washed with EtOH: 0.8 g 650%
of 6). M.p. >3408. UV 6MeOH): 372 64.22), 316 64.51), 225 6sh, 4.24), 202 64.44).1H-NMR 66D6)DMSO): 12.32
6br. s, HÀN63)); 11.93 6br. s, CONHÀC62)); 10.05 6br. s, CONHC6H4); 9.45 6br. s, HÀC67)); 8.15
6d, 2 arom. H); 7.79 6d, 2 arom. H); 2.78 6sept., 1 H, Me2CH); 2.63 6sept., 1 H, Me2CH); 1.13 6m, 4 Me). Anal.
calc. for C20H22N6O3 ´ 0.5 H2O 6403.4): C 59.54, H 5.75, N 20.38; found: C 59.88, H 5.73, N 20.53.
4. 6-44-Azidophenyl)pterin 6ˆ2-Amino-6-44-azidophenyl)pteridin-4-43H)-one; 7). At r.t., 8 6see below;
0.2 g, 0.57 mmol) was stirred in NH3/MeOH 616 ml) overnight. The precipitate was dried at 408: 148 mg 693%)
of 7. Yellowish powder. M.p. >3008. UV 6pH 14): 386 64.10), 301 64.34), 278 6sh, 4.31), 216 64.21). IR 6KBr):
1
2122 6N3). H-NMR 6CF3COOD): 9.37 6s, HÀC67)); 8.20 6d, 2 arom. H); 7.34 6d, 2 arom. H). Anal. calc. for
C12H8N8O ´ 0.5 H2O 6289.3): C 49.83, H 3.14, N 38.74; found: C 50.24, H 3.33, N 37.95.

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5. 6-44-Azidophenyl)-N2-isobutyrylpterin 6ˆ N-[6-44-Azidophenyl)-3,4-dihydro-4-oxopteridin-2-yl]-2-
methylpropanamide; 8). Crude 5 61.44 g, 5.7 mmol) was suspended in 5n HCl 6200 ml). Insoluble sulfur was
removed by filtration, and the filtrate was evaporated. The residue was dissolved in H2O 680 ml) and conc. HCl
soln. 620 ml). After cooling to 08, a soln. of NaNO2 60.83 g, 12 mmol) in H2O 63 ml) was added dropwise under
vigorous stirring. After 30 min at 08, a soln. of NaN3 60.74 g, 11.4 mmol) in H2O 62 ml) was added dropwise. The
mixture was stirred for 3 h at 08 and for 2 h at r.t. The precipitate was collected, washed with H2O, and dried over
KOH in a vacuum desiccator: 0.9 g of crude 7. The crude 7 was suspended in dry pyridine 612 ml) and treated
with isobutyric anhydride 63 ml) under reflux for 2 h and then filtered while hot. The filtrate was evaporated and
the partly crystalline residue suspended in MeOH. After filtration, recrystallization from EtOH/AcOEt 2 :1
gave 0.67 g 634%) of 8. M.p. >3608. TLC 6toluene/AcOEt/MeOH 5 :4 :1): Rf 0.53. UV 6MeOH): 368 64.22),
314 64.48), 226 6sh, 4.22), 202 64.40). IR 6KBr): 2122. 1H-NMR 66D6)DMSO): 12.32 6br. s, HÀN63)); 11.97
6br. s, CONHÀC62)); 9.47 6s, HÀC67)); 8.22 6d, 2 arom. H); 7.27 6d, 2 arom. H); 2.78 6sept., Me2CH); 1.14
6d, Me2CH). Anal. calc. for C16H14N8O2 6350.3): C 54.85, H 4.03, N 31.98; found: C 54.61, H 4.03, N 31.21.
6. 5-Acetyl-6-[4-4acetylamino)phenyl]-5,6,7,8-tetrahydro-N2-isobutyrylpterin 6ˆ N-{5-Acetyl-6-[4-4acetyl-
amino)phenyl]-3,4,5,6,7,8-hexahydro-4-oxopteridin-2-yl}-2-methylpropanamide; 9). PtO2 670 mg) was stirred
in a mixture of abs. MeOH 615 ml) and CF3COOH 60.8 ml) for 1 h under H2 at r.t. After addition of 8 60.1 g,
0.3 mmol), hydrogenation was continued for 2.5 h at r.t. and normal pressure. The catalyst was filtered off under
an inert gas and the filtrate evaporated. The oily dark brown residue was treated with Ac2O 65 ml), and after
stirring for 12 h, the mixture was evaporated. The yellowish residue was treated with MeOH 63 ml) and cooled
in the refrigerator. Then the precipitate was collected: 66 mg 655%) of crystalline 9. M.p. 289 ± 2908. TLC
6CH2Cl2/MeOH 5 :1): Rf 0.66. UV 6MeOH): 301 6sh, 3.99), 234 64.64), 204 64.55). 1H-NMR 66D6)DMSO):
11.33 6br. s, NH); 11.23 6br. s, NH); 9.88 6br. s, NH); 7.43 6m, 3 H, arom. H, HÀN68)); 7.14 6d, 2 arom. H); 5.79
6m, HÀC66)); 3.98 6dd, 1 HÀC67)); 3.35 6m, 1 HÀC67)); 2.66 6sept., Me2CH); 2.12 6s, MeCO); 1.98
6s, MeCO); 1.03 6d, Me2CH). Anal. calc. for C12H24N6O4 6412.5) ´ 0.5 H2O: C 57.00, H 5.98, N 19.94; found:
C 57.32, H 6.00, N 19.73.
7. 5,6,7,8-Tetrahydro-6-4hydroxymethyl)-5-{[2-44-nitrophenyl)ethoxy]carbonyl}pterin 6ˆ2-44-Nitrophenyl)-
ethyl 2-Amino-3,4,5,6,7,8-hexahydro-6-4hydroxymethyl)-4-oxopteridine-5-carboxylate; 12). In CF3COOH
650 ml), 6-6hydroxymethyl)pterin 610) [14][15] 62 g, 10.4 mmol) was reduced under H2 in the presence of
PtO2 60.4 g) at r.t. under normal pressure. After 4 h, the catalyst was filtered off under an inert gas atmosphere
and the solvent evaporated. The residue 611) was dissolved under an inert gas atmosphere in dry pyridine
6100 ml) and treated with 2-64-nitrophenyl)ethyl carbonochloridate [16] 63.1 g, 26.9 mmol). After stirring for
12 h at r.t., the soln. was evaporated and the residue co-evaporated with toluene 63Â) and then repeatedly
evaporated with MeOH. Finally, the partially crystalline residue was sonicated, the mixture cooled for 12 h, and
the crystalline solid dried at 408. Workup of the filtrate by evaporation and treatment with little MeOH gave a
second crop. Total yield: 3.51 g 686%) of 12. M.p. 223 ± 2248 6dec.). TLC 6H2O/iPrOH/AcOEt 1:5 :3): Rf 0.64.
UV 6MeOH): 279 64.37), 219 64.51), 205 6sh, 4.35). 1H-NMR 66D6)DMSO): 8.13 6d, arom. H); 7.68 6br. s, NH2);
7.51 6d, arom. H); 4.26 6m, 3 H, HÀC66), CH2); 3.47 6d, 1 HÀC67)); 3.12 6m, 5 H, HÀC67), 2 CH2). Anal. calc.
for C16H18N6O6 ´ 2 H2O 6426.4): C 45.07, H 4.69, N 19.80; found: C 45.19, H 5.20, N 19.32.
8. N2-[4Dimethylamino)methylene]-5,6,7,8-tetrahydro-6-4hydroxymethyl)-5-{[2-44-nitrophenyl)ethoxy]car-
bonyl}pterin 6ˆ2-44-Nitrophenyl)ethyl 2-{[4Dimethylamino)methylene]amino}-3,4,5,6,7,8-hexahydro-6-4hy-
droxymethyl)-4-oxopteridine-5-carboxylate; 13). A suspension of 12 60.5 g, 1.28 mmol) in dry DMF 625 ml)
was treated with dimethylformamide diethyl acetal 61.25 ml) at r.t., yielding immediately a clear yellowish soln.
After 2.5 h stirring 6TLC: no 12 left), the mixture was evaporated under high vacuum, and the residue dissolved
in CHCl3 and purified by FC 6silica gel 610 g), gradient 0 ! 6% MeOH/CH2Cl2). The product was treated with
MeOH and sonicated to give a crystalline precipitate, which was dried at 308: 0.33 g 657%) of 13. M.p. 2218
6dec.). TLC 6CH2Cl2/MeOH 5 :1): Rf 0.62. UV 6MeOH): 299 6sh, 4.36), 281 6sh, 4.42), 258 64.50), 217 64.48).
1H-NMR 66D6)DMSO): 10.71 6br. s, HÀN63)); 8.38 6s, HÀCˆN); 8.09 6d, 2 arom. H); 7.52 6d, 2 arom. H); 6.78
6d, HÀN68)); 4.79 6br. s, OH); 4.09 ± 4.31 6m, 3 H, CH2, HÀC66)); 3.41 6dd, 1 HÀC67)); 3.19 6m, 1 HÀC67));
3.08 6s, MeN); 2.99 6m, 9 H, MeN, 3 CH2). Anal. calc. for C19H23N7O6 ´ 0.5 H2O 6454.4): C 50.22, H 5.32, N 21.58;
found: C 50.60, H 5.26, N 21.25.
9. 6-{[44-Azidobenzoyl)oxy]methyl}-N2-[4dimethylamino)methylene]-5,6,7,8-tetrahydro-5-{[2-44-nitro-
phenyl)ethoxy]carbonyl}pterin 6 ˆ 2-44-Nitrophenyl)ethyl 6-{[44-Azidobenzoyl)oxy]methyl}-2-{[4dimethylami-
no)methylene]amino}-3,4,5,6,7,8-hexahydro-4-oxopteridine-5-carboxylate; 14). a) A mixture of 4-azidobenzoic
acid [18] 60.22 g, 1.35 mmol), EDC 60.26 g, 1.35 mmol), and DMAP 60.17 g, 1.35 mmol) in dry pyridine 610 ml)
was stirred for 2 h at r.t. Then 13 60.15 g, 0.34 mmol) was added and stirring continued at r.t. for 16 h. After
evaporation and co-evaporation with toluene 63Â), the oily residue was dissolved in a small amount of CHCl3

Helvetica Chimica Acta ± Vol. 83 62000)
2745
and purified by FC 6silica gel 68 g), gradient 0 ± 6% MeOH/CHCl3). The oily product was crystallized by
treatment with AcOEt and sonication. The precipitate was collected after cooling for 2 h and dried at 408:
109 mg 656%) of 14. Yellowish powder. M.p. 198 ± 2008.
b) A mixture of 4,4'-azidobenzoic anhydride [20] 60.33 g, 1.1 mmol) and DMAP 60.13 g, 1.1 mmol) in dry
pyridine 610 ml) was stirred at r.t. for 1 h. Then compound 13 60.12 g, 0.27 mmol) was added and stirring
continued for 24 h. Evaporation under high vacuum and workup analogous to a) gave 98 mg 663%) of 14. M.p.
198 ± 2008. TLC 6CH2Cl2/MeOH 9 :1): Rf 0.68. UV 6MeOH): 318 6sh, 4.18), 271 64.63), 265 64.63), 213 64.58).
1H-NMR 66D6)DMSO): 10.78 6br. s, HÀN63)); 8.40 6s, HÀCˆN); 8.06 6d, 2 arom. H); 7.94 6d, 2 arom. H); 7.52
6d, 2 arom. H); 7.21 6d, 2 arom. H); 6.87 6d, HÀN68)); 4.62 6br. s, HÀC66)); 3.98 ± 4.32 6m, 4 H, 2 CH2); 3.39
6m, 1 HÀC67)); 3.17 6m, 1 HÀC67)); 3.09 6s, MeN); 3.00 6m, 5 H, MeN, CH2). Anal. calc. for C26H26N10O7
6590.6): C 52.88, H 4.44, N 23.72; found: C 53.06, H 4.49, N 23.92.
10. 6-{[3-44-Benzoylphenyl)-1-oxopropoxy]methyl}-N2-[4dimethylamino)methylene]-5,6,7,8-tetrahydro-5-
{[2-44-nitrophenyl)ethoxy]carbonyl}pterin 6ˆ2-44-Nitrophenyl)ethyl 6-{[3-44-Benzoylphenyl)-1-oxopropoxy]-
methyl}-2-{[4dimethylamino)methylene]amino}-3,4,5,6,7,8-hexahydro-4-oxopteridine-5-carboxylate; 15). At r.t.,
13 60.5 g, 1.12 mmol), 3-64-benzoylphenyl)propanoic acid [19] 60.57 g, 2.24 mmol), EDC 60.43 g, 2.24 mmol),
and DMAP 60.27 g, 2.24 mmol) were dissolved in dry pyridine 620 ml) and stirred for 14 h. The mixture was
evaporated to dryness and co-evaporated with toluene 63Â) and the residue purified by FC 6silica gel 612 g),
gradient 0 ± 2% MeOH/CH2Cl2). A second purification by FC 6up to 4% MeOH/CH2Cl2) was necessary.
Evaporation gave a yellowish foam, which was treated with Et2O and sonicated. The resulting yellowish solid
was washed with Et2O and dried at 408: 0.61 g 680%) of 15. M.p. 858 6evolution of gas), 1308 6dec.). TLC
6CH2Cl2/MeOH 9 :1): Rf 0.52. UV 6MeOH): 311 6sh, 4.30), 281 6sh, 4.53), 258 64.69), 206 64.65). 1H-NMR
66D6)DMSO): 10.79 6br. s, HÀN63)); 8.39 6s, HÀCˆN); 8.08 6d, 2 arom. H); 7.63 ± 7.72 6m, PhCO); 7.50 ± 7.57
6m, 4 arom. H); 7.41 6d, 2 arom. H); 6.84 6d, HÀN68)); 4.57 6br. s, HÀC66)); 4.13 ± 4.45 6m, CH2); 3.80 6q, 2 H,
CH2); 3.24 6m, 2 HÀC67)); 3.08 6s, MeN); 2.91 ± 3.04 6m, 7 H, MeN, 2 CH2); 2.67 6t, CH2). Anal. calc. for
C35H34N7O8 6680.7): C 61.76, H 5.03, N 14.40; found: C 62.25, H 5.41, N 13.98.
11. N2-[4Dimethylamino)methylene]-5,6,7,8-tetrahydro-5-{[2-44-nitrophenyl)ethoxy]carbonyl}-6-[4{4-[3-
4trifluoromethyl)-3H-diazirin-3-yl]benzoyl}oxy)methyl]pterin 6ˆ2-44-Nitrophenyl)ethyl 2-{[4Dimethylamino)-
methylene]amino}-3,4,5,6,7,8-hexahydro-4-oxo-6-[4{4-[3-4trifluoromethyl)-3H-diazirin-3-yl]benzoyl}oxy)me-
thyl]pteridine-5-carboxylate; 16). A mixture of 13 60.2 g, 0.45 mmol), 4-[3-6trifluoromethyl)-3H-diazirin-3-
yl]benzoic acid [5] 60.21 g, 0.9 mmol), DMAP 60.11 g, 0.9 mmol), and EDC 60.17 g, 0.9 mmol) was stirred for
14 h in dry pyridine 620 ml). The clear soln. was evaporated and the residue co-evaporated with toluene 63Â)
and then purified by FC 6silica gel 68 g), gradient 0 ± 3% MeOH/CH2Cl2). The product was treated with AcOEt,
and sonicated to give a crystalline material, which was washed with Et2O, and dried at 408: 0.23 g 675%) of 16.
M.p. 2358 6dec.). TLC 6CH2Cl2/MeOH 9 :1): Rf 0.54. UV 6MeOH): 305 6sh, 4.38), 279 6sh, 4.48), 255 64.61), 221
64.61). 1H-NMR 66D6)DMSO): 10.77 6br. s, HÀN63)); 8.39 6s, HÀCˆN); 8.04 6m, 4 arom. H); 7.51
6d, 2 arom. H); 7.38 6d, 2 arom. H); 6.87 6d, HÀN68)); 4.63 6br. s, HÀC66)); 3.99 ± 4.31 6m, 4 H, 2 CH2); 3.39
6m, 1 HÀC67)); 3.17 6m, 1 HÀC67)); 3.09 6s, MeN); 3.00 6m, 5 H, MeN, CH2). Anal. calc. for C28H26N9O7F3 ´
0.5 H2O 6666.6): C 50.45, H 4.08, N 18.92; found: C 50.30, H 3.92, N 18.54.
12. 6-{[44-Azidobenzoyl)oxy]methyl}-5,6,7,8-tetrahydro-5-{[2-44-nitrophenyl)ethoxy]carbonyl}pterin Hy-
drochloride 6ˆ2-44-Nitrophenyl)ethyl 2-Amino-6-{[44-azidobenzoyl)oxy]methyl}-3,4,5,6,7,8-hexahydro-4-oxo-
pteridine-5-carboxylate Hydrochloride; 17 ´ HCl). Overnight, 14 60.8 g, 1.49 mmol) was stirred in MeOH
6160 ml) and 1m HCl 616 ml). The resulting precipitate was washed with small amounts of MeOH and dried at
408: 0.7 g of 17 ´ HCl. Workup of the mother liquor gave a second crop of 90 mg. Total yield: 0.79 g 691%).
Colorless crystals. M.p. 199 ± 2008. TLC 6CH2Cl2/MeOH 9 :1): Rf 0.44. UV 6MeOH): 274 64.64), 215 64.65).
1H-NMR 66D6)DMSO): 11.30 6br. s, HÀN63)); 8.08 6d, 2 arom. H); 7.93 6d, 2 arom. H); 7.50 6m, HÀN68),
2 arom. H); 7.23 6d, 2 arom. H); 4.64 6br. s, HÀC66)); 4.08 ± 4.33 6m, 4 H, 2 CH2); 3.49 6m, 1 HÀC67)); 3.20
6m, 1 HÀC67)); 2.99 6t, CH2). Anal. calc. for C23H21N9O7 ´ HCl ´ 0.5 H2O 6580.9): C 47.55, H 3.99, N 21.70;
found: C 47.67, H 3.92, N 21.38.
13. 6-{[3-44-Benzoylphenyl)-1-oxopropoxy]methyl}-5,6,7,8-tetrahydro-5-{[2-44-nitrophenyl)ethoxy]carbo-
nyl}pterin 6ˆ2-44-Nitrophenyl)ethyl 2-Amino-6-{[3-44-benzoylphenyl)-1-oxopropoxy]methyl}-3,4,5,6,7,8-hexa-
hydro-4-oxopteridine-5-carboxylate; 18). At r.t., 15 60.3 g, 0.44 mmol) was stirred in MeOH 612 ml) and 1m HCl
62 ml) for 24 h. The precipitate was washed with Et2O and dried at 408: 0.22 g 680%) of colorless 18. M.p. 2398
6dec.). TLC 6CH2Cl2/MeOH 9 :1): Rf 0.46. UV 6MeOH): 338 6sh, 3.51), 266 64.55), 214 6sh, 4.64), 206 64.64).
1H-NMR 66D6)DMSO): 9.99 6br. s, HÀN63)); 8.09 6d, 2 arom. H); 7.64 ± 7.72 6m, PhCO); 7.51 ± 7.57 6m, 4 ar-
om. H); 7.41 6d, 2 arom. H); 6.76 6d, HÀN68)); 6.16 6br. s, NH2); 4.43 6br. s, HÀC66)); 4.16 ± 4.32 62m, 2 H,

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CH2); 3.81 6m, 2 H, CH2); 3.40 ± 3.48 6m, 1 HÀC67)); 3.20 ± 3.72 6m, 1 HÀC67)); 2.91 ± 3.08 6m, 6 H, 3 CH2);
2.67 6t, CH2). Anal. calc. for C32H30N6O7 ´ H2O 6628.6): C 61.14, H 5.13, N 13.37; C 60.84, H 4.82, N 13.30.
14. 5,6,7,8-Tetrahydro-5-{[2-44-nitrophenyl)ethoxy]carbonyl}-6-[4{4-[3-4trifluoromethyl)-3H-diazirin-3-yl]-
benzoyl}oxy)methyl]pterin Hydrochloride 6ˆ2-44-Nitrophenyl)ethyl 2-Amino-3,4,5,6,7,8-hexahydro-4-oxo-6-
[4{4-[3-4trifluoromethyl)-3H-diazirin-3-yl]benzoyl}oxy)methyl]pteridine-5-carboxylate Hydrochloride; 19 ´
HCl). At r.t., 16 60.18 g, 0.27 mmol) was stirred in MeOH 610 ml) and conc. HCl soln. 61.5 ml) for 24 h. The
resulting precipitate was washed with a small amount of MeOH and Et2O and dried at 308 in vacuo: 135 mg
678%) of colorless 19 ´ HCl. M.p. 2398 6dec.). TLC 6CH2Cl2/MeOH 9 :1): Rf 0.63. UV 6MeOH): 348 6sh, 3.50),
278 64.39), 245 6sh, 4.41), 220 64.63). 1H-NMR 66D6)DMSO): 11.26 6br. s, HÀN63)); 8.07 6d, 2 arom. H); 8.01
6d, 2 arom. H); 7.49 6m, 5 H, HÀN68), NH2, arom. H); 7.40 6d, 2 arom. H); 4.64 6br. s, HÀC66)); 4.08 ± 4.34
6m, 4 H, 2 CH2); 3.48 6m, 1 HÀC67)); 3.20 6dd, 1 HÀC67)); 2.98 6t, CH2). Anal. calc. for C25H21N8O7F3 ´ HCl
6639.0): C 47.00, H 3.47, N 17.54; found: C 46.84, H 3.36, N 17.11.
15. 6-{[44-Azidobenzoyl)oxy]methyl}-5,6,7,8-tetrahydropterin 4-Toluenesulfonate 6ˆ42-Amino-3,4,5,6,7,8-
hexahydro-4-oxopteridin-6-yl)methyl 4-Azidobenzoate 4-Methylbenzenesulfonate; 20 ´ TsOH). To a suspension
of 17 60.4 g, 0.69 mmol) in dry DMF 67 ml), DBU 6574 ml) was added under N2. After stirring overnight at r.t.,
the soln. was acidified with AcOH 62 ml) and evaporated under high vacuum. The oily residue was treated with
TsOH 61.0 g, 5.35 mmol) in EtOH 610 ml) at 08. The precipitate was washed with EtOH and dried at 408 under
high vacuum. The crude material 60.3 g) was recrystallized from H2O/MeOH under N2, the insoluble material
filtered off the hot mixture, and the filtrate kept in the refrigerator overnight: 99 mg 628%) of colorless 20 ´
TsOH. M.p. 203 ± 2048 6dec.). TLC 6CH2Cl2/MeOH 5 :1): Rf 0.56. UV 6MeOH): 292 6sh, 4.29), 268 64.55), 219
6sh, 4.29), 206 64.55). IR 6KBr): 2124. 1H-NMR 66D6)DMSO): 10.62 6br. s, HÀN63)); 8.10 6d, 2 arom. H); 7.54
6br. s, HÀN68)); 7.47 6d, 2 arom. H); 7.27 6d, 2 arom. H); 7.10 6d, 2 arom. H); 6.65 6br. s, NH2); 4.46 ± 4.64
6m, CH2O); 3.73 6br. s, HÀC66)); 3.57 6m, 1 HÀC67)); 3.26 ± 3.46 6m, 1 HÀC67)); 2.28 6s, Me). Anal. calc. for
C14H14N8O3 ´ CH3C6H4SO3H 6514.5): C 49.02, H 4.31, N 21.78; found: C 48.76, H 4.35, N 21.76.
16. 6-{[3-44-Benzoylphenyl)-1-oxopropoxy]methyl}-5,6,7,8-tetrahydropterin 4-Toluenesulfonate 6ˆ42-Ami-
no-3,4,5,6,7,8-hexahydro-4-oxopteridin-6-yl)methyl 4-Benzoylbenzenepropanoate 4-Methylbenzenesulfonate;
21 ´ TsOH). To a suspension of 18 60.15 g, 0.25 mmol) under an inert gas in dry DMF 61.6 ml), DBU 6183 ml,
1.23 mmol) was added and the soln. stirred for 14 h at r.t. The mixture was acidified with AcOH 61.1 ml) to bring
the pH to 5 ± 6 and then evaporated under high vacuum. The brown oily residue was treated at 08 with TsOH
60.37 g, 1.92 mmol) in EtOH 610 ml) and sonicated to convert all material to a solid. After 30 min at 08, the
precipitate was washed with EtOH and dried. The crude product was recrystallized under an inert gas from H2O
to which MeOH was added dropwise until a clear hot soln. resulted. After cooling and standing refrigerated for
3 h, the collected solid was dried at 308 in vacuo: 95 mg 663%) of colorless 21 ´ TsOH. M.p. 1838. TLC 6CH2Cl2/
MeOH 5 :1): Rf 0.43. UV 6MeOH): 312 6sh, 4.02), 259 64.47), 218 64.69), 206 6sh, 4.66). 1H-NMR
66D6)DMSO): 10.62 6br. s, HÀN63)); 7.64 ± 7.72 6m, PhCO); 7.41 ± 7.58 6m, 9 arom. H, HÀN68)); 7.10
6d, 2 arom. H); 6.65 6br. s, NH2); 4.31 6m, CH2O); 3.43 ± 3.57 6m, HÀC66), 1 HÀC67)); 3.16 6m, 1 HÀC67));
2.98 6t, CH2); 2.76 6t, CH2); 2.27 6s, Me). Anal. calc. for C23H23N5O3 ´ CH3C6H4SO3H 6605.7): C 59.49, H 5.16,
N 11.56; found: C 59.52, H 5.15, N 11.57.
17. 6-[4{4-[3-4Trifluoromethyl)-3H-diazirin-3-yl]benzoyl}oxy)methyl]-5,6,7,8-tetrahydropterin 4-Toluene-
sulfonate 6ˆ42-Amino-3,4,5,6,7,8-hexahydro-4-oxopteridin-6-yl)methyl 4-[3-4Trifluoromethyl)-3H-diazirin-3-
yl]benzoate 4-Methylbenzenesulfonate; 22 ´ TsOH). As described for 21, with 19 680 mg, 0.13 mmol), DMF
61.3 ml), DBU 694 ml, 0.65 mmol), AcOH 60.5 ml), TsOH 60.19 g, 0.99 mmol), and EtOH 63 ml). After 45 min.
at 08, the solid was washed with EtOH and Et2O and dried: 84 mg of crude 22 ´ TsOH. The material was
recrystallized under an inert gas from H2O 63 ml) by dropwise addition of MeOH until a clear soln. resulted on
boiling. After cooling in the refrigerator for 2 h, the collected solid was washed with small amounts of MeOH
and Et2O and dried at 308: 50 mg 666%) of colorless 22 ´ TsOH. M.p. 2598 6dec.). TLC 6CH2Cl2/MeOH 5 :1):
Rf 0.58. UV 61n HCl): 333 6sh, 3.54), 255 64.43), 244 6sh, 4.42), 228 6sh, 4.45), 223 64.47), 205 64.54). UV
6MeOH): 329 6sh, 3.72), 279 6sh, 4.01), 235 6sh, 4.52), 224 64.58). 1H-NMR 66D6)DMSO): 10.63 6br. s,
HÀN63)); 8.17 6d, 2 H, Ts); 7.43 ± 7.56 6m, 5 H, arom. H, HÀN68)); 7.10 6d, 2 H, Ts); 6.66 6br. s, NH2); 4.49 ±
4.69 6m, CH2O); 3.73 6m, HÀC66)); 3.57 6m, 1 HÀC67)); 3.27 ± 3.46 6m,1 HÀC67)); 2.27 6s, Me). Anal. calc.
for C16H14F3N7O3 ´ CH3C6H4SO3H 6581.5): C 47.50, H 3.81, N 16.86; found: C 47.57, H 3.93, N 16.98.
18. 5,6,7,8-Tetrahydro-N2-isobutyrylpterin 6ˆ2-Methyl-N-43,4,5,6,7,8-hexahydro-4-oxopteridin-2-yl)propan-
amide Hydrochloride; 23 ´ HCl). A mixture of N2-isobutyrylpterin [25] 64.0 g, 17.15 mmol) and PtO2 60.4 g) in
MeOH 6200 ml) was treated in a shaking apparatus under H2 for 24 h until the uptake of H2 stopped. Then conc.
HCl soln. 67 ml) was added to dissolve the precipitate. The catalyst was filtered off and the filtrate concentrated
to 50 ml and then added dropwise to Et2O 6500 ml) with stirring. After cooling in the refrigerator for 2 h, the

Helvetica Chimica Acta ± Vol. 83 62000)
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colorless precipitate was washed with Et2O and dried in a vacuum desiccator. From the filtrate, a second crop of
23 ´ HCl was obtained after standing overnight in the refrigerator. Total yield: 315 g 667%). M.p. 2358 6dec.). UV
6pH 0): 265 64.02), 229 64.57). 1H-NMR 66D6)DMSO): 11.59 6br. s, NH); 11.54 6br. s, NH); 7.87 6br. s,
HÀN68)); 3.42 6m,CH266)); 3.22 6m, CH267)); 2.75 6sept., Me2CH); 1.07 6d, Me2CH). Anal. calc. for
C10H15N5O2 ´ HCl ´ 0.5 H2O 6282.7): C 42.48, H 6.06, N 24.77; found: C 42.40, H 6.41, N 25.24.
19. 5-44-Azidobenzoyl)-5,6,7,8-tetrahydro-N2-isobutyrylpterin 6ˆ N-[5-44-Azidobenzoyl)-3,4,5,6,7,8-hexa-
hydro-4-oxopteridin-2-yl]-2-methylpropanamide; 24). PtO2 670 mg) was hydrogenated with molecular H2 in
MeOH 650 ml) in a shaking apparatus. After addition of N2-isobutyrylpterin [25] 60.5 g, 2.14 mmol), the mixture
was stirred under H2 for 20 h. The solvent was evaporated, and the residue dried under high vacuum and then
treated with 4-azidobenzoyl chloride [21] 60.53 g, 2.9 mmol) in dry pyridine 615 ml) for 24 h at r.t. with stirring.
The mixture was evaporated and co-evaporated with toluene, the residue heated in CHCl3 6150 ml) and MeOH
620 ml) to reflux, the catalyst filtered off, and the filtrate concentrated to a small volume. After mixing with silica
gel 61.5 g), the mixture was evaporated to a powder, which was put on top of a column 6silica gel) for separation.
FC 6gradient 1 ± 8% MeOH/CH2Cl2) gave a product which was suspended in CH2Cl2, heated, and treated
dropwise with MeOH to give a clear soln. After cooling to r.t. the soln. was concentrated until the product began
to crystallize. After cooling overnight in the refrigerator, the colorless crystalline solid was collected and dried:
0.23 g 628%) of 24. M.p. 271 ± 2758. TLC 6CH2Cl2/MeOH 20 :1): Rf 0.3. UV 6MeOH): 318 64.01), 268 64.30), 236
64.48), 205 64.47). 1H-NMR 66D6)DMSO): 11.29 6br. s, HÀN63)); 11.08 6br. CONHÀC62)); 7.47 6d, 2 ar-
om. H); 7.32 6br. s, HÀN68)); 7.03 6d, 2 arom. H); 4.46 6br. s, CH266)); 3.44 6br. s, CH267)); 2.69 6sept., Me2CH);
1.04 6d, Me2CH). Anal. calc. for C17H18N8O36382.4): C 53.40, H 4.74, N 29.30; found: C 52.85, H 4.75, N 28.72.
20. 5-[3-44-Benzoylphenyl)-1-oxopropyl]-5,6,7,8-tetrahydro-N2-isobutyrylpterin 6ˆ N-{5-[3-44-Benzoyl-
phenyl)-1-oxopropyl]-3,4,5,6,7,8-hexahydro-4-oxopteridin-2-yl}-2-methylpropanamide; 25). At r.t., 23 60.4 g,
1.46 mmol), 3-64-benzoylphenyl)propanoic acid [19] 60.74 g, 2.92 mmol), EDC 60.56 g, 2.92 mmol), and DMAP
60.36 g, 2.92 mmol) were stirred in dry pyridine 620 m) for 24 h. After evaporation, the residue was co-
evaporated with toluene 63Â) and then purified by FC 6silica gel 618 g), gradient 0 ± 6% MeOH/CH2Cl2). The
product fractions were purified by a second FC. The pure substance recrystallized from hot AcOEt by dropwise
addition of MeOH until a clear soln. was obtained on heating. After cooling to r.t., the soln. was concentrated
under reduced pressure to a small volume and kept refrigerated for 3 h. The collected crystals were dried at 408
under high vacuum: 0.23 g 633%) of 25. M.p. 2348. TLC 6CH2Cl2/MeOH 9 :1): Rf 0.49. UV 6MeOH): 313 6sh,
3.94), 260 64.44), 235 64.63), 204 64.58). 1H-NMR 66D6)DMSO): 11.38 6br. s, NH); 11.32 6br. s, NH); 7.40 ± 7.71
6m, 8 H, arom. H, HÀN68)); 7.34 6d, 2 arom. H); 4.61 6d, 1 HÀC66)); 3.38 6m, 1 HÀC67)); 2.61 ± 2.96 6m, 6 H,
1 HÀC66), 1 HÀC67), 2 CH2); 2.72 ± 2.80 6m, Me2CH); 2.40 6br. s, HÀC66)); 1.06 6d, Me2CH). Anal. calc. for
C26H27N5O4 6473.5): C 65.95, H 5.75, N 14.79; found: C 66.34, H 5.76, N 14.32.
21. 5,6,7,8-Tetrahydro-N2-isobutyryl-5-{4-[3-4trifluoromethyl)-3H-diazirin-3-yl]benzoyl}pterin 6ˆ2-Methyl-
N-43,4,5,6,7,8-hexahydro-4-oxo-5-{4-[3-4trifluoromethyl)-3H-diazirin-3-yl]benzoyl}pteridin-2-yl)propanamide;
26). As described for 25, with 23 60.3 g, 1.1 mmol), 4-[3-6trifluoromethyl)-3H-diazirin-3-yl]benzoic acid [5]
60.5 g, 2.19 mmol), EDC 60.42 g, 2.19 mmol), DMAP 60.27 g, 2.19 mmol), and pyridine 615 ml) 614 h under N2).
FC 6silica gel 69 g), gradient 0 ± 4% MeOH/CH2Cl2) was performed twice. The product was then treated with
AcOEt and sonicated to give a solid, which was washed with Et2O and dried: 83 mg 617%) of 26. M.p. 2848
6dec.). TLC 6CH2Cl2/MeOH 9 :1): Rf 0.53. UV 6MeOH): 312 64.03), 275 64.05), 234 64.61), 204 64.47).
1H-NMR 66D6)DMSO): 11.27 6br. s, NH); 11.09 6br. s, NH); 7.55 6d, 2 arom. H); 7.38 6br. s, HÀN68)); 7.19
6d, 2 arom. H); 4.57 6br. s, 1HÀC66)); 3.44 6m, CH267)); 2.69 6m, 1HÀC66), Me2CH); 1.04 6d, Me2CH). Anal.
calc. for C19H18F3N7O2 ´ H2O 6451.4): C 50.55, H 4.47, N 21.72; found: C 50.48, H 4.11, N 21.64.
22. 5-44-Azidobenzoyl)-5,6,7,8-tetrahydropterin 6ˆ2-Amino-5-44-azidobenzoyl)-5,6,7,8-tetrahydropteridin-
443H)-one; 27). At r.t., 24 680 mg, 0.2 mmol) was dissolved in NH3/MeOH 64 ml) and stirred for 6 h. After 1 h, a
colorless solid started to separate, which was washed with MeOH and dried: 55 mg 688%) of colorless 27. M.p.
3108 6dec.). TLC 6toluene/AcOEt/MeOH 5 :4 :1): Rf 0.21. UV 6pH 1): 278 64.41), 202 64.52). UV 6MeOH): 280
6sh, 4.25), 269 64.29), 212 64.47). 1H-NMR 66D6)DMSO): 9.71 6br. s, HÀN63)); 7.45 6d, 2 arom. H); 7.02
6d, 2 arom. H); 6.94 6s, HÀN68)); 6.10 6br. s, NH2); 4.50 6br. s, CH266)); 2.68 6br. s, CH267)). Anal. calc. for
C13H12N8O2 6312.3): C 50.00, H 3.87, N 35.88; found: C 49.79, H 3.89, N 35.23.
23. 5-[3-44-Benzoylphenyl)-1-oxopropyl]-5,6,7,8-tetrahydropterin 6ˆ2-Amino-5-[3-44-benzoylphenyl)-1-
oxopropyl]-5,6,7,8-tetrahydropteridin-443H)-one; 28). At r.t., 25 60.1 g, 0.21 mmol) was stirred in NH3/MeOH
overnight. The soln. was evaporated and the residue co-evaporated several times with MeOH, treated again
with MeOH, and sonicated. The resulting solid was washed with Et2O and dried: 50 mg 660%) of colorless 28.
M.p. 263 ± 2648. TLC 6CH2Cl2/MeOH 5 :1): Rf 0.55. UV 6MeOH): 280 6sh, 4.35), 260 64.40), 220 64.50), 205
64.50). 1H-NMR 66D6)DMSO): 10.02 6br. s, HÀN63)); 7.72 ± 7.44 6m, 7 arom. H); 7.34 6d, 2 arom. H); 6.97

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6d, HÀN68)); 6.20 6br. s, NH2); 4.57 6m, 1 HÀC66)); 3.35 ± 3.26 6m, 1 HÀC67)); 2.98 ± 2.84 6m, 2 CH2); 2.79 ±
2.55 6m, 1 HÀC66)); 2.41 ± 2.32 6m, 1 HÀC67)). Anal. calc. for C22H21N5O3 6403.4): C 65.50, H 5.25, N 17.36;
found: C 65.01, H 5.22, N 17.66.
24. 5,6,7,8-Tetrahydro-5-{4-[3-4trifluoromethyl)-3H-diazirin-3-yl]benzoyl}pterin 6ˆ2-Amino-5,6,7,8-tetra-
hydro-5-{4-[3-4trifluoromethyl)-3H-diazirin-3-yl]benzoyl}pteridin-4-43H)-one; 29). At r.t., 26 60.1 g,
0.23 mmol) was stirred in NH3/MeOH for 24 h. The precipitate was washed with Et2O, and dried: 75 mg
686%) of colorless 29. M.p. 3158 6dec.). TLC 6CH2Cl2/MeOH 9 :1): Rf 0.29. UV 6MeOH): 280 64.12), 224
64.53), 203 64.43). 1H-NMR 66D6)DMSO): 9.78 6br. s, HÀN63)); 7.52 6d, 2 arom. H); 7.17 6d, 2 arom. H); 7.01
6br. s, HÀN68)); 6.09 6br. s, NH2); 4.59 6br. s, 1 HÀC66)); 3.50 ± 3.33 6m, 1 HÀC67)); 2.65 6br. s, 1 HÀC66),
1 HÀC67)). Anal. calc. for C15H12F3N7O2 6379.3): C 47.50, H 3.19, N 25.85; found: C 47.65, H 3.24, N 25.39.
25. 6-{[44-Azidobenzoyl)oxy]methyl}-N2-[4dimethylamino)methylene]pterin 6ˆ42-{[4Dimethylamino)me-
thylene]amino}-3,4-dihydro-4-oxopteridin-6-yl)methyl 4-Azidobenzoate; 31). Compound 30 [1] 60.2 g,
0.81 mmol) was co-evaporated twice with dry pyridine 610 ml) and subsequently dissolved in dry pyridine/
CH2Cl2 1:1 616 ml). After addition of 4-azidobenzoic chloride [21] 60.22 g, 1.21 mmol), the mixture was stirred
at r.t. for 2 h. Solvents were evaporated, and the residue was co-evaporated with toluene 63Â). The oily residue
was treated with EtOH 610 ml) and sonicated. The resulting precipitate was dissolved in CHCl3/MeOH, silica
gel 61.5 g) added, the mixture evaporated, and the residue put on top of a column 6silica gel 610 g)) for
purification. FC 6gradient 0 ± 5% MeOH/CH2Cl2) yielded a product, which was dried under high vacuum: 96 mg
630%) of 31. Yellowish powder. M.p. 1838. TLC 6CH2Cl2/MeOH 5 :1): Rf 0.82. UV 6MeOH): 352 64.16), 304
64.56), 295 64.56), 286 64.55), 278 64.54), 203 64.42). 1H-NMR 66D6)DMSO): 12.06 6br. s, HÀN63)); 8.91
6s, CHˆN); 8.81 6s, 1 HÀC67)); 8.03 6d, 2 arom. H); 7.22 6d, 2 arom. H); 5.47 6s, CH2O); 3.22 6s, MeN); 3.09
6s, MeN). Anal. calc. for C17H15N9O3 ´ 0.5 H2O 6402.4): C 50.75, H 4.01, N 31.33; found: C 51.17, H 3.93, N 30.82.
26. 6-{[3-44-Benzoylphenyl)-1-oxopropoxy]methyl}-N2-[4dimethylamino)methylene]pterin 6 ˆ 42-{[4Di-
methylamino)methylene]amino}-3,4-dihydro-4-oxopteridin-6-yl)methyl 4-Benzoylbenzenepropanoate; 32). A
soln. of 30 [1] 60.3 g, 1.21 mmol), 3-64-benzoylphenyl)propanoic acid [19] 60.62 g. 2.42 mmol), EDC 60.46 g,
2.42 mmol), and DMAP 60.3 g, 2.42 mmol) in dry pyridine 615 ml) was stirred overnight. After evaporation, the
residue was co-evaporated with toluene 63Â) and purified by FC 6silica gel 612 g), gradient 0 ± 2% MeOH/
CH2Cl2). The product was purified by a second FC. The resulting yellowish foam was sonicated in Et2O and the
collected solid dried: 0.39 g 667%) of 32. M.p. 1238. TLC 6CH2Cl2/MeOH 9 :1): Rf 0.70. UV 6MeOH): 353
64.07), 306 64.50), 259 64.45), 203 64.55). 1H-NMR 66D6)DMSO): 12.06 6br. s, HÀN63)); 8.80 6s, HÀC67)); 8.78
6s, CHˆN); 7.72 ± 7.52 6m, 7 arom. H); 7.41 6d, 2 arom. H); 5.24 6s, CH2O); 3.21 6s, MeN); 3.09 6s, MeN); 2.99
6t, CH2); 2.82 6t, CH2). Anal. calc. for C26H24N6O4 6484.5): C 64.45, H 4.99, N 17.35; found: C 64.43, H 5.09,
N 17.00.
27. N2-[4Dimethylamino)methylene]-6-[4{4-[3-4trifluoromethyl)-3H-diazirin-3-yl]benzoyl}oxy)methyl]-
pterin 6ˆ{2-{[4Dimethylamino)methylene]amino}-3,4-dihydro-4-oxopteridin-6-yl}methyl 4-[3-4Trifluorometh-
yl)-3H-diazirin-3-yl]benzoate; 33). A soln. of 30 [1] 60.2 g, 0.81 mmol), 4-[3-6trifluoromethyl)-3H-diazirin-3-
yl]benzoic acid [5] 60.37 g, 1.6 mmol), EDC 60.31 g, 1.6 mmol), and DMAP 60.20 g, 1.6 mmol) in dry pyridine
620 ml) was stirred at r.t. for 14 h. After evaporation, the oily residue was co-evaporated with toluene 63Â) and
then purified by FC 6silica gel 616 g), gradient 0 ± 4% MeOH/CH2Cl2). The product was treated with AcOEt and
sonicated to form a crystalline solid. Drying under high vacuum gave 0.18 g 649%) of yellowish 33. TLC
6CH2Cl2/MeOH 9 :1): Rf 0.61. M.p. 227 ± 2308 6dec.). UV 6MeOH): 351 64.17), 307 64.54), 238 64.48). 1H-NMR
66D6)DMSO): 12.07 6br. s, HÀN63)); 8.92 6s, CHˆN); 8.82 6s, HÀC67)); 8.11 6d, 2 arom. H); 7.42 6d, arom. H);
5.51 6s, CH2O); 3.23 6s, MeN); 3.09 6s, MeN). Anal. calc. for C19H15F3N8O3 ´ 0.5 H2O 6460.4): C 49.57, H 3.28,
N 24.34; found: C 49.47, H 3.51, N 24.11.
28. 6-{[44-Azidobenzoyl)oxy]methyl}pterin 6ˆ42-Amino-3,4-dihydro-4-oxopteridin-6-yl)methyl 4-Azido-
benzoate; 34). At r.t., 31 60.2 g, 0.51 mmol) in MeOH 610 ml) and 1m HCl 64 ml) was stirred for 36 h. The
resulting precipitate was washed with H2O and dried: 0.16 g 693%) of yellowish 34. M.p. >3508. TLC 6CH2Cl2/
MeOH 5 :1): Rf 0.64. UV 61m HCl): 321 6sh, 3.85), 276 64.18), 209 64.23). 1H-NMR 66D6)DMSO): 11.46 6br. s, ,
HÀN63)); 8.83 6s, HÀC67)); 8.02 6d, 2 arom. H); 7.25 6d, 2 arom. H); 6.97 6br. s, NH2); 5.44 6s, CH2O). Anal.
calc. for C14H10N8O3 6338.3): C 49.71, H 2.98, N 33.12; found: C 49.56, H 3.14, N 33.14.
29. 6-{[3-44-Benzoylphenyl)-1-oxopropoxy]methyl}pterin 6ˆ42-Amino-3,4-dihydro-4-oxopteridin-6-yl)-
methyl 4-Benzoylbenzenepropanoate; 35). At r.t., 32 60.17 g, 0.35 mmol) in MeOH 64 ml) and 1m HCl
60.5 ml) was stirred for 16 h. The resulting solid was washed with Et2O and dried: 124 mg 683%) of colorless 35.
TLC 6CH2Cl2/MeOH 9 :1): Rf 0.35. M.p. 2558 6dec.). UV 6MeOH): 344 63.82), 266 64.39), 205 64.47). 1H-NMR
66D6)DMSO): 11.48 6br. s, HÀN63)); 8.65 6s, HÀC67)); 7.71 ± 7.46 6m, 7 arom. H); 7.00 6br. s, NH2); 5.20

Helvetica Chimica Acta ± Vol. 83 62000)
2749
6s, CH2O); 2.98 6t, CH2); 2.80 6t, CH2). Anal. calc. for C23H19N5O4 6429.4): C 64.33, H 4.46, N 16.31; found:
C 64.79, H 4.77, N 16.43.
30. 6-[4{4-[3-4Trifluoromethyl)-3H-diazirin-3-yl]benzoyl}oxy)methyl]pterin 6ˆ42-Amino-3,4-dihydro-4-
oxopteridin-6-yl)methyl 4-[3-4Trifluoromethyl)-3H-diazirin-3-yl]benzoate; 36). At r.t., 33 680 mg, 0.17 mmol)
in MeOH 63 ml) and 1m HCl 60.5 ml) was stirred overnight. After 3 h, a solid started to precipitate. It was
washed with Et2O and dried: 70 mg 699%) of colorless 36. M.p. 2958 6dec.). TLC 6CH2Cl2/MeOH 5 :1): Rf 0.58.
UV 6MeOH): 347 63.87), 277 64.29), 236 64.42), 202 64.39). 1H-NMR 66D6)DMSO): 11.48 6br. s, HÀN63)); 8.83
6s, HÀC67)); 8.10 6d, 2 arom. H); 7.43 6d, 2 arom. H); 6.97 6br. s, NH2); 5.47 6s, CH2O). Anal. calc. for
C16H10F3N7O3 6405.3): C 47.42, H 2.49, N 24.19; found: C 47.53, H 2.65, N 23.77.
31. 6-{[3-44-Benzoylphenyl)-1-oxopropoxy]methyl}pteridine-2,4-diamine 6ˆ42,4-Diaminopteridin-6-yl)-
methyl 4-Benzoylbenzenepropanoate; 38). To a soln. of 3-64-benzoylphenyl)propanoic acid [19] 60.30 g,
1.18 mmol) in DMF 610 ml), dry Et3N 60.12 g; 1.18 mmol) and 6-6bromomethyl)pteridine-2,4-diamine 637)
[1][22] 60.2 g, 0.78 mmol) were added. Then the suspension was stirred for 2 h at r.t. 6TLC: no 37 left). The soln.
was filtered, the red filtrate mixed with silica gel 60.3 g), the mixture evaporated under high vacuum, and the
residue put on top of a column 6silica gel 611 g)) for purification. FC 6gradient 0 ± 4% MeOH/CH2Cl2) gave a
product, which was suspended in Et2O. The collected solid was suspended in H2O/MeOH 2 :3 6removal of
6Et3NH)Br) and dried 26 mg 68%) of 38. Yellowish powder. M.p. 208 ± 2108 6dec.). TLC 6CH2Cl2/MeOH 5 :1):
Rf 0.52. UV 6MeOH): 372 63.90), 260 64.60), 203 64.56). 1H-NMR 66D6)DMSO): 8.68 6s, HÀC67)); 7.62
6m, 9 arom. H, NH2); 7.42 6d, 2 arom. H); 6.76 6br. s, NH2); 5.20 6s, CH2O); 2.98 6m, CH2); 2.81 6m, CH2). Anal.
calc. for C23H20N6O3 6428.5): C 64.48, H 4.70, N 19.61; found: C 64.01, H 4.94, N 18.87.
32. 6-[4{4-[3-4Trifluoromethyl)-3H-diazirin-3-yl]benzoyl}oxy)methyl]pteridine-2,4-diamine 6ˆ42,4-Diami-
nopteridin-6-yl)methyl 4-[3-4Trifluoromethyl)-3H-diazirin-3-yl]benzoate; 39). To a soln. of 4-[63-trifluoro-
methyl)-3H-diazirin-3-yl]benzoic acid [19] 60.23 g, 1.02 mmol) in DMF 615 ml), dry Et3N 60.10 g, 1.02 mmol)
and 37 60.2 g, 0.78 mmol) were added. The suspension was stirred at r.t. for 2 h. 6TLC: no 37 left) and then
filtered. The red filtrate was evaporated under high vacuum, the residue dissolved in a small volume of DMF/
MeOH 6silica gel 63 g)) added, the mixture again evaporated, and the residue put on top of a column 6silica gel
611 g)) for purification. FC 6gradient 0 ± 6% MeOH/CH2Cl2) gave a product, which was suspended in Et2O,
filtered off, then again suspended in H2O/MeOH 2 :3 6removal of 6Et3NH)Br), filtered off, and dried: 17 mg
65%) of 39. Yellowish powder. M.p. 220 ± 2258 6dec.). TLC 6CH2Cl2/MeOH 9 :1): Rf 0.43. UV 6MeOH): 370
1
63.93), 260 64.50), 236 64.45), 202 64.40). H-NMR 66D6)DMSO): 8.87 6s, HÀC67)); 8.11 6d, 2 arom. H); 7.67
6br. d, NH2); 7.43 6d, 2 arom. H); 6.75 6br. s, NH2); 5.46 6s, CH2O). Anal. calc. for C16H11F3N8O2 6404.3):
C 47.53, H 2.74, N 27.71; found: C 47.10, H 2.96, N 26.60.
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Ê
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Received March 28, 2000