Synthesis of precursors for 18F-labeling of folic acid for PET application

Article abstract of DOI:10.1055/s-0030-1260247

Radiolabeled folate derivatives have the potential to target folate receptor-positive tumor cells for noninvasive diagnosis via positron emission tomography (PET) and single photon emission computed tomography (SPECT). We report the regiospecific synthesis of N²-(N,N-dimethylaminomethylene) -2′-nitrofolic acid di-tert-butyl ester (13) and N²-(N,N- dimethylaminomethylene)-N¹⁰-formyl-2′-nitrofolic acid dimethyl ester (25), which are precursors for the radiolabeling of folic acid with the PET isotope ¹⁸F. A modular synthetic strategy was applied: Fmoc- and Boc-protected 4-amino-2-nitrobenzoic acid were linked via amide bonds to di-tert-butyl l-glutamate and dimethyl l-glutamate, respectively, to form building blocks 10 and 19. After Fmoc and Boc removal, both compounds were coupled to 6-(bromomethyl)pterin hydrobromide to give crude 2′-nitrofolic acid di-tert-butyl ester and 2′-nitrofolic acid dimethyl ester. After formylation of 2′-nitrofolic acid dimethyl ester at N¹⁰ and the introduction of an N,N-dimethylaminomethylene group at N², precursor 25 was obtained in an overall yield of 3%. The analogous 2′- fluoroderivative 28 was obtained in 7% overall yield from 4-amino-2- fluorobenzoic acid. Precursor 13 was obtained from 2′-nitrofolic acid di-tert-butyl ester in 6% yield after N²-protection. The synthesis of the reference materials 2′-nitro- and 2′-fluorofolic acid was achieved by the reaction of N-(4-amino-2-nitrobenzoyl)- and N-(4-amino-2- fluorobenzoyl)-l-glutamic acid with 6-(bromomethyl)pterin hydrobromide, giving 7% and 14% overall yield, respectively. Georg Thieme Verlag Stuttgart. New York.

Full text of DOI:10.1055/s-0030-1260247

PAPER
3639
Synthesis of Precursors for 18F-Labeling of Folic Acid for PET Application
Precursors for
F
-
18 iLabelin
o
g of
F
olic
A
l
cid
f
or
a
PET ApplicationGroehn,*a Rudolf Moser,a Tobias L. Ross,b,1 Thomas Betzel,b Cristina Müller,c Roger Schibli,b,c Simon
Ametameyb
a
Merck & Cie, Im Laternenacker 5, 8200 Schaffhausen, Switzerland
Fax +41(52)6307255; E-mail: viola.groehn@merckgroup.com
Animal Imaging Center-PET, Center for Radiopharmaceutical Sciences of ETH, PSI and USZ,
b
Institute of Pharmaceutical Science ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland
Center for Radiopharmaceutical Sciences of ETH, PSI and USZ, Paul Scherrer Institute, 5232 Villigen-PSI, Switzerland
c
Received 14 July 2011; revised 24 August 2011
(97%)] for tumor diagnosis by PET. There are only a few
Abstract: Radiolabeled folate derivatives have the potential to tar-
folate-based PET tracers reported in the literature where a
get folate receptor-positive tumor cells for noninvasive diagnosis
prosthetic group bearing an 18F label is conjugated to ei-
via positron emission tomography (PET) and single photon emis-
sion computed tomography (SPECT). We report the regiospecific
ther of the two carboxyl groups of folic acid (1).12 In con-
synthesis of N2-(N,N-dimethylaminomethylene)-2¢-nitrofolic acid trast to the published methods that utilize prosthetic
di-tert-butyl ester (13) and N2-(N,N-dimethylaminomethylene)-N10-
formyl-2¢-nitrofolic acid dimethyl ester (25), which are precursors
groups, we have decided on an approach that incorporates
the 18F-label directly into the benzoyl moiety of folic acid
for the radiolabeling of folic acid with the PET isotope 18F. A modu-
by a formal bioisosteric replacement of a phenyl hydrogen
lar synthetic strategy was applied: Fmoc- and Boc-protected 4-ami-
atom. Since the ‘direct labeling’ approach involves only a
no-2-nitrobenzoic acid were linked via amide bonds to di-tert-butyl
slight structural modification of folic acid, we expect the
labeled compound to mimic the folic acid most closely
and consequently to retain a high affinity for the folate re-
ceptor and a metabolic pathway similar to that of folic
acid.
L-glutamate and dimethyl L-glutamate, respectively, to form build-
ing blocks 10 and 19. After Fmoc and Boc removal, both com-
pounds were coupled to 6-(bromomethyl)pterin hydrobromide to
give crude 2¢-nitrofolic acid di-tert-butyl ester and 2¢-nitrofolic acid
dimethyl ester. After formylation of 2¢-nitrofolic acid dimethyl ester
at N10 and the introduction of an N,N-dimethylaminomethylene
group at N2, precursor 25 was obtained in an overall yield of 3%.
In order to obtain a sufficiently high level of radioactivity
The analogous 2¢-fluoroderivative 28 was obtained in 7% overall for subsequent in vivo studies, the incorporation of the
18F-label is usually carried out during the last step of a
multi-step synthesis. Nucleophilic substitution at a phenyl
yield from 4-amino-2-fluorobenzoic acid. Precursor 13 was ob-
tained from 2¢-nitrofolic acid di-tert-butyl ester in 6% yield after
N2-protection. The synthesis of the reference materials 2¢-nitro- and
ring, afforded by leaving and activating groups, is one of
2¢-fluorofolic acid was achieved by the reaction of N-(4-amino-2-
the standard procedures for the introduction of the 18F-la-
nitrobenzoyl)- and N-(4-amino-2-fluorobenzoyl)-L-glutamic acid
with 6-(bromomethyl)pterin hydrobromide, giving 7% and 14%
bel.13 The 2¢-position in the benzoyl moiety of folic acid
is activated by a carboxylamide, and we therefore initially
envisaged the incorporation of the 18F-label in the 2¢-posi-
tion by exchange of a nitro group. Since the nitration of
folic acid leads to a 3¢,5¢-dinitro derivative14 and protec-
tion of the NH2 group and the carboxyl groups of folic
acid is necessary during 18F-labeling, we developed a
modular synthesis for N2-(N,N-dimethylaminomethyl-
ene)-2¢-nitrofolic acid di-tert-butyl ester (13) and N2-
(N,N-dimethylaminomethylene)-N10-formyl-2¢-nitrofolic
acid dimethyl ester (25), which were used as precursors
for the synthesis of 2¢-[18F]fluorofolic acid (29). We de-
scribe here the syntheses of precursors 13 and 25 as well
as the reference compounds 2¢-nitrofolic acid (2), 2¢-fluo-
rofolic acid (3),15 and N2-(N,N-dimethylaminomethyl-
ene)-2¢-fluoro-N10-formylfolic acid dimethyl ester (28).
The 18F-labeling of the precursors and the preclinical eval-
uation of 2¢-[18F]fluorofolic acid are described else-
where.16
overall yield, respectively.
Key words: nucleophilic substitution, halides, amino acids, pro-
tecting groups
The folate receptor is a promising tumor target for folate
conjugates since it is overexpressed in a wide variety of
cancer cells.2 A number of folate conjugates have been
synthesized for application in tumor therapy and
diagnosis3 (e.g., chemotherapeutic agents,4 antisense oli-
gonucleotides,5 antibodies,6 protein toxins,7 and
liposomes8). Several folate-based radiopharmaceuticals
have been developed and labeled with radionuclides in-
cluding 66/67/68Ga,9 99mTc,10 and 111In11 for diagnostic ap-
plication using molecular imaging methods such as
positron emission tomography (PET) and single photon
emission computed tomography (SPECT). Folate deriva-
tives suitable for labeling with the PET radionuclide 18F
are especially attractive since this isotope exhibits excel-
lent decay properties [half-life: 110 min, b+-Eav: 250 keV
In general, there are two different routes for the regiospe-
cific synthesis of folic acid (1) that is functionalized in the
4-aminobenzoyl moiety (Scheme 1). Route I comprises
the coupling of synthons A and B to give a 2¢-functional-
ized derivative of pteroic acid (AB), which is conjugated
with L-glutamic acid (synthon C) to give 2 and 3. Route II
SYNTHESIS 2011, No. 22, pp 3639–3648
Advanced online publication: 29.09.2011
DOI: 10.1055/s-0030-1260247; Art ID: T69811SS
© Georg Thieme Verlag Stuttgart · New York
x
x.
x
x
.
2
0
1
1

3640
V. Groehn et al.
PAPER
O
OH
O
X
1: X = H, folic acid
2: X = NO2
3: X = F
O
N
N
H
N
N
OH
HN
N
NH2
N
H
O
O
NH2
NH2
OH
H2N
+
HO
H2N
N
E
route I: A+B = AB, AB+C
route II: B+C = BC, BC+A
D
O
OH
Z
O
NC
N
Y
"Taylor" approach
NH2
NH
N
N
Y
H2N
+
+
N
+
OH
H2N
OH
O
H2N
N
H2N
Z = OH, NH2
N
X
H2N
Y = CH2Br, CH2Cl, CHO
X = H
X = NO2
X = F
F
Y = CH2Br, CH2Cl, CHO, CH2OH
G
B
C
A
Scheme 1 Retrosynthesis for folic acid (1) and 2¢-functionalized folic acid derivatives 2 and 3
includes the synthesis of 2¢-functionalized 4-aminoben- The structure of A is variable and determines the method
zoyl-L-glutamic acid by reaction of B with C and finally of coupling. If A is a 6-formyl derivative22 (Y = CHO),
coupling to synthon A. The key step of the Taylor coupling with B or BC is achieved by reductive amina-
approach17 comprises the condensation of a 5-substituted tion,23 whereas the 6-bromomethyl derivative24
2-amino-3-cyanopyrazine G18 with guanidine F to give a (Y = CH2Br) is coupled by alkylation of the amine B or
6-substituted 4-aminopteridine. If G is 5-substituted with BC.25 Since the reductive amination may affect the nitro
a methylaminobenzoic acid moiety, the resulting synthon group of synthon B or BC, the investigations were started
GB gives, after condensation with F, a derivative of ptero- with the bromomethyl derivative 424,25 (Scheme 2). Fol-
ic acid (AB). Since the L-glutamic acid moiety extensive- lowing route I and by analogy to Montgomery and co-
ly racemizes during the ring closure reaction with workers,25 4-amino-2-nitrobenzoic acid ethyl ester26 (5)
guanidine,19 the Taylor approach is only applicable to was coupled to 2,4-diamino-6-(bromomethyl)pteridine
synthesis of synthon A (F + G) and pteroic acid (F + GB). hydrobromide (4) in N,N-dimethylacetamide (DMAC) at
However, since Bader et al.20 described the dialkylation of 60 °C to give ethyl 4-[(2,4-diaminopteridine-6-yl)methyl-
primary amines by 2-amino-5-chloromethyl-3-cyanopy- amino]-2¢-nitrobenzoate (6) in 66% yield. Simultaneous
razine, we decided not to follow the Taylor approach. In- hydrolysis of the 4-amino-function and ethyl ester moiety
stead, we started with synthon A, which was obtained by of 6 with aqueous sodium or lithium hydroxide under
condensation of 2,4,5,6-tetraaminopyrimidine (D) with anaerobic conditions by analogy to Seeger et al.27 or aque-
dihydroxyacetone (E) according to Baugh et al.21 and sub- ous hydrochloric acid21,28 to give 2¢-nitropteroic acid (7)
sequent derivatization of the intermediate 6-hydroxy- failed. On the other hand, fast and complete degradation
methylpteridine derivative.
of starting material was observed under alkaline condi-
O
NH2
O
NH2
N
a
OEt
NO2
N
N
Br
+
OEt
NO2
N
66%
N
N
H
⋅HBr
H2N
N
N
H2N
H2N
N
4
5
6
b
O
O
N
O
c
O
N
OH
NO2
N
N
HN
Br
+
OH
NO2
N
HN
N
H
⋅HBr
H2N
H2N
H2N
N
12
8
7
Scheme 2 Synthesis of ethyl 4-[(2,4-diaminopteridine-6-yl)methylamino]-2¢-nitrobenzoate (6) following route I. Reagents and conditions:
(a) DMAC, 60 °C, 3 h; (b) 0.1 M or 2 M or 4 M aq NaOH, r.t. to –80 °C or LiOH (3 equiv) in H2O–MeOH (1:1) or 1 M or 6 M aq HCl, r.t. to
–100 °C or HBr, 80–95 °C; (c) DMF, 100 °C, 5 h.
Synthesis 2011, No. 22, 3639–3648 © Thieme Stuttgart · New York

PAPER
Precursors for 18F-Labeling of Folic Acid for PET Application
3641
tions, whereas with hydrochloric acid no conversion took (13), which was then purified by flash chromatography on
place, even at 100 °C or after the addition of methanol to silica gel. Reduced nucleophilicity of the amino function-
increase solubility of the starting material. While the use ality of 11 caused by the electron-withdrawing nitro sub-
of 48% hydrobromic acid at 95 °C mainly led to formation stituent in position 2 and repeated chromatographic
of an unknown by-product, the hydrolysis with 62% hy- purification of 13 resulted in only a poor yield of 14% for
drobromic acid at 80 °C gave a mixture of different com- 13 over the last two steps starting from 11.
pounds including the desired product 7 with up to 44%
area detected by LC-MS. Due to poor solubility of the
product, isolation and purification of the latter was impos-
sible. In order to circumvent the hydrolysis of the 4-amino
and ethyl ester moieties, the coupling of 4-amino-2-ni-
trobenzoic acid (8) with 6-(bromomethyl)pterin hydro-
bromide (12) to obtain 2¢-nitropteroic acid (7) was then
investigated (Scheme 2). The crude product was obtained
The syntheses of 2¢-nitrofolic acid (2) and 2¢-fluorofolic
acid15 (3) were carried out starting from 4-amino-2-ni-
trobenzoic acid26 (8) and 4-amino-2-fluorobenzoic acid30
(14), respectively, following route II (Schemes 1 and 4).
The 4-amino group of 8 and 14 was protected by a tert-
butoxycarbonyl (Boc) group in 47% and 55% yield, re-
spectively. After coupling 15 and 16 to L-glutamic acid di-
tert-butyl ester in 92% and 91% yields, using HBTU and
only in poor yield and purity. Purification of the latter by
Et3N, global deprotection was achieved by treatment with
dissolving it in aqueous sodium hydroxide, and subse-
trifluoroacetic acid to give the trifluoroacetate of N-(4-
amino-2-nitrobenzoyl)-L-glutamic acid (20) in 79% yield
and N-(4-amino-2-fluorobenzoyl)-L-glutamic acid (21) in
quent treatment with charcoal and precipitation by adding
acetic acid did not improve purity, which was approxi-
mately 47% area determined by LC-MS. Formylation of
96% yield. Coupling of 20 and 21, respectively, with 6-
the crude material at N10 with formic acid and subsequent
(bromomethyl)pterin hydrobromide24,25 (12) in DMAC at
treatment with N,N-dimethylformamide diisopropylacetal
did not lead to a uniform product that could be purified.
The synthesis of 2¢-nitropteroic acid (7) as intermediate
for the synthesis of 2 was therefore abandoned.
60 °C led to 2¢-nitrofolic acid (2) in 26% yield and 2¢-
fluorofolic acid (3) in 30% yield corresponding to 12.
The 18F-labeling by nucleophilic substitution of the 2¢-ni-
tro group of precursor 13 using [18F]-fluoride cryptate
Following route II, 4-amino-2-nitrobenzoic acid (8) was
protected with 9-fluorenylmethoxycarbonyl (Fmoc) in
67% yield (Scheme 3). The 4-(N-Fmoc-amino)-2-nitro-
complex, final deprotection with aqueous hydrochloric
acid (Scheme 5), and application of the 2¢-[18F]fluorofolic
acid for PET studies in tumor-bearing mice have been de-
benzoic acid (9) was coupled to L-glutamic acid di-tert-
butyl ester in 87% yield using 2-(1H-benzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/
triethylamine in DMF. Fmoc deprotection was achieved
by a standard procedure29 to give N-(4-amino-2-nitroben-
zoyl)-L-glutamic acid di-tert-butyl ester (11) in a yield of
77%. Compound 11 was coupled to 6-(bromomethyl)pter-
in hydrobromide24,25 (12) in DMAC at 60 °C to give crude
2¢-nitrofolic acid di-tert-butyl ester. Since the latter was
poorly soluble in organic solvents it was not further puri-
fied, but was converted directly into N2-(N,N-dimethyl-
aminomethylene)-2¢-nitrofolic acid di-tert-butyl ester
scribed by Ross et al.16
The 18F-labeling of 13 was accomplished with 8% decay-
corrected yield. Subsequent deprotection of the labeled
product of 13 afforded target compound 29 in 50% yield
resulting in an overall radiochemical yield of 4%.16 To in-
crease the radiochemical yield and prevent decomposition
of the fluorinating reagent by hydrogen bond formation,
we envisaged additional protection of the N10-amino func-
tion by a formyl or acetyl group. The acylation of precur-
sor 13 or crude 2¢-nitrofolic acid di-tert-butyl ester was
not successful under several nonacidic mild acylation
O
Ot-Bu
O
O
O
a
b
c
N
H
OH
NO2
OH
87%
67%
77%
Ot-Bu
FmocHN
NO2
H2N
FmocHN
1)
NO2
O
9
10
8
O
N
N
N
HN
Br
⋅HBr
O
Ot-Bu
H2N
O
Ot-Bu
O
O
12
2) (i-PrO)2CHNMe2
O
N
N
H
N
H
N
N
Ot-Bu
d
14%
Ot-Bu
HN
N
N
NO2
H2N
NO2
H
O
O
Me2N
11
13
Scheme 3 Synthesis of N2-(N,N-dimethylaminomethylene)-2¢-nitrofolic acid di-tert-butyl ester (13) following route II. Reagents and condi-
tions: (a) Fmoc-Cl, Na2CO3, H2O; (b) L-Glu(O-t-Bu)O-t-Bu, HBTU, Et3N, DMF; (c) pyrrolidine, DMF; d) 1. DMAC, 60 °C, 3 h, 2. DMF-di-
isopropylacetal, DMF, r.t., 17 h.
Synthesis 2011, No. 22, 3639–3648 © Thieme Stuttgart · New York

3642
V. Groehn et al.
PAPER
O
OR
O
X
O
O
X
a
b
N
H
OH
OH
OR
BocHN
H2N
X
BocHN
O
8: X = NO2
14: X = F
15: X = NO2, 47%
16: X = F, 55%
17: R = t-Bu, X = NO2, 92%
18: R = t-Bu, X = F, 91%
19: R = Me, X = NO2, 76%
O
N
HN
Br
O
O
OR
O
OR
⋅HBr
O
X
H2N
N
N
12
c
O
N
N
H
N
H
N
N
OR
d
OR
HN
N
X
H2N
H
O
O
H2N
2: R = H, X = NO2, 26%, (corresponding to 12)
3: R = H, X = F, 30% (corresponding to 12)
20: R = H, X = NO2, 79%
21: R = H, X = F, 96%
22: R = Me, X = NO2, 45%
e
23: R = Me, X = NO2, 49% (corresponding to 22)
26⋅PhSO3H: R = Me, X = F, 76% (corresponding to 3)
23⋅0.5 PhSO3H: R = Me, X = NO2, 72% (corresponding to 2) .
Scheme 4 Synthesis of 2¢-nitrofolic acid (2), 2¢-fluorofolic acid (3), and 2¢-nitro and 2¢-fluorofolic acid dimethyl ester (23, 26). Reagents and
conditions: (a) Boc2O, 1,4-dioxane, H2O, NaOH, r.t.; (b) L-Glu(OR)OR·HCl, HBTU, Et3N, CH2Cl2; (c) TFA, CH2Cl2, 0 °C – r.t., 17 h; (d) 12,
DMAC, 60 °C, 6 h; (e) PhSO3H, MeOH, 90 °C, 2 h.
conditions, for example, p-nitrophenyl acetate at pH 11 in beling, was accomplished in an analogous manner from
aqueous sodium hydroxide31 or 2,4,5-trichlorophenyl for- 2¢-fluorofolic acid (3), which was esterified with metha-
mate/N,N-diisopropylethylamine in N,N-dimethylform- nol in the presence of benzenesulfonic acid to give 2¢-
amide32 or with formic acid activated by N,N¢- fluorofolic acid dimethyl ester benzenesulfonate (26) in
dicyclohexylcarbodiimide in pyridine.33 Either no conver- 76% yield (Scheme 4). N10-Formylation of 26 occurred in
sion or an unselective reaction took place, leading to an 85% yield, and the introduction of an N,N-dimethylamino-
inseparable mixture of products. We therefore decided to methylene group at N2 was carried out in 84% yield to
protect the L-glutamic acid moiety with methyl esters in- give 28 (Scheme 6).
stead of acid-labile tert-butyl esters. The synthesis of 2¢-
nitrofolic acid dimethyl ester (23) was achieved by two
methods. It was done either by esterification of 2¢-nitro-
folic acid (2) in methanol with benzenesulfonic acid
(BSA) by analogy to the esterification of folic acid,34 lead-
ing to a hemibenzenesulfonate of 2¢-nitrofolic acid di-
methyl ester (23) in 72% yield, or by the coupling of N-(4-
amino-2-nitrobenzoyl)-L-glutamic acid dimethyl ester
(22) to 6-(bromomethyl)pterin hydrobromide (12) in 49%
yield (Scheme 4). 2¢-Nitrofolic acid dimethyl ester (23)
was then formylated in formic acid in 77% yield to give
N10-formyl-2¢-nitrofolic acid dimethyl ester (24), which
was protected at N2 to give precursor N2-(N,N-dimethyl-
aminomethylene)-2¢-nitrofolic acid dimethyl ester (25) in
66% yield (Scheme 6). The synthesis of N2-(N,N-dimeth-
ylaminomethylene)-2¢-fluorofolic acid dimethyl ester
(28), which was used as reference material during 18F-la-
18F-Labeling of compound 25 (Scheme 5) was accom-
plished in a yield of only 0.3% due to the low reactivity of
the nonactivated aromatic ring. This means that protection
of position N10 is negligible for improvement of the yield.
Deprotection of the labeled compound was effected in
32% yield by using 4 M aqueous hydrochloric acid for 10
minutes at 60 °C and subsequent addition of 5 M aqueous
sodium hydroxide solution. This procedure gave 2¢-
[18F]fluorofolic acid (29) in only 0.1% overall yield from
precursor 25 under conditions, which were not well opti-
mized. Coinjection of the 2¢-[18F]fluorofolic acid (29)
with the 19F-substituted inactive reference compound 3
proved the detection of the 18F-labeled tracer.35
In conclusion, we have identified a synthetic pathway for
the preparation of two precursors suitable for the synthesis
of 2¢-[18F]fluorofolic acid, which is a novel nonconjugated
OR1
OH
O
O
O
O
a, 8% for 13, 0.3% for 25
b, 50% for 13, 32% for 25
O
O
N
N
H
N
H
OR1
OH
N
N
N
N
HN
N
NO2
HN
N
18F
R2
H
O
O
Me2N
N
N
H2N
13: R1 = t-Bu, R2 = H
29: ~4% from 13 and ~0.1% from 25
25: R1 = Me, R2 = CHO
Scheme 5 Radiosynthesis of 2¢-[18F]fluorofolic acid starting from precursors 13 and 25. Reagents and conditions: (a) [18F]-K2.2.2, (b) for 13:
4 M aq HCl, 12 min, 60 °C; for 25: 4 M aq HCl, 10 min, 60 °C, then 5 M aq NaOH, 10 min, 60 °C.
Synthesis 2011, No. 22, 3639–3648 © Thieme Stuttgart · New York

PAPER
Precursors for 18F-Labeling of Folic Acid for PET Application
3643
O
O
OMe
OMe
O
X
O
X
a
O
N
O
N
N
H
N
H
N
N
N
N
OMe
OMe
HN
N
HN
N
H
O
O
H2N
H2N
O
H
23: X = NO2
26⋅PhSO3H: X = F
24: X = NO2, 77%
27: X = F, 85%
O
OMe
O
b
O
N
N
H
N
OMe
HN
N
R
O
Me2N
N
N
O
H
25: X = NO2, 66%
28: X = F, 84%
Scheme 6 Synthesis of N2-(N,N-dimethylaminomethylene)-N10-formyl-2¢-nitro- and 2¢-fluorofolic acid dimethyl ester (25, 28) from 2¢-nitro-
and 2¢-fluorofolic acid dimethyl ester (23, 26). Reagents and conditions: (a) HCO2H, 60 °C, 3.5 h; (b) DMF-diisopropylacetal, DMF, r.t., 17 h.
Ethyl 4-[(2,4-Diaminopteridine-6-yl)methylamino]-2¢-nitroben-
PET tracer of folic acid. Preliminary in vitro and in vivo
experiments performed with folate receptor-positive cells
in culture and folate receptor-positive tumor-bearing mice
have demonstrated the superior characteristics of the nov-
el 18F-folate PET tracer compared to previously published
18F-folate PET tracers prepared with 18F-labeled prosthet-
ic groups. However, the overall radiochemical yields will
significantly limit the use of the precursors for the routine
preparation of 2¢-[18F]fluorofolic acid for clinical applica-
tions. In order to improve the radiochemical yield we are
currently investigating the synthesis of precursors in
which a hydrogen atom of one of the methylene groups in
the glutamic acid moiety of folic acid is replaced by a sul-
fonic acid ester. Aliphatic sulfonic acid esters can be
smoothly modified by nucleophilic 18F-substitution.36
Presumably this approach will allow an efficient one-step
direct labeling of folic acid with 18F.
zoate (6)
A
mixture of 2,4-diamino-6-(bromomethyl)pteridine hydro-
bromide24 (4; 7 g, 20.8 mmol) and 4-amino-2-nitrobenzoic acid eth-
yl ester26 (5; 12.7 g, 60.4 mmol) in DMF (260 mL) was stirred at
100 °C for 5 h. The mixture was cooled to 0 °C, and the product was
precipitated by the addition of Et2O (770 mL). The product was col-
lected by filtration, washed with Et2O (250 mL), and dried under
vacuum at 45 °C to give 6; yield: 5.3 g (66% corresponding to 4);
greenish-grey powder; mp >250 °C (dec.).
1H NMR (200 MHz, DMSO-d6): d = 1.23 (t, 3J = 7.1 Hz, 3 H, CH3),
3
4.21 (q, J = 7.1 Hz, 2 H, CH2CH3), 4.71 (s, 2 H, H-6), 7.04 (dd,
3J = 8.7 Hz, 4J = 2.2 Hz, 1 H, 5¢-Harom), 7.19 (d, 4J = 2.2 Hz, 1 H,
3¢-Harom), 7.73 (d, 3J = 8.7 Hz, 1 H, 6¢-Harom), 8.72, 7.60 (2 br s, 2 H,
NH2), 8.91 (s, 1 H, H-7), 9.37 (br d, 2 H, NH2).
13C NMR (50 MHz, DMSO-d6): d = 13.8, 45.3, 60.9, 106.3, 109.7,
113.8, 121.5, 132.0, 145.2, 149.4, 155.8, 162.7, 163.4.
HRMS (ESI, MeOH): m/z [M + H]+ calcd for C16H17N8O4:
385.1367; found: 385.1372.
All experiments were carried out under an argon or N2 atmosphere.
All commercially available reagents and solvents were used as re-
ceived. Methyl tert-butyl ether is abbreviated as MTBE. The N2-
(N,N-dimethylaminomethylene)-10-formylpteroic acid (11) was
provided by Merck & Cie, Schaffhausen, Switzerland. 4-Amino-2-
nitrobenzoic acid26 (8) and the corresponding ethyl ester26 (5) were
purchased from Chemie Brunschwig AG. 4-Amino-2-fluorobenzo-
ic acid30 (14), N,N-dimethylformamide diisopropylacetal and di-
methyl-L-glutamate hydrochloride were purchased from Sigma
Aldrich. Di-tert-butyl-L-glutamate hydrochloride was purchased
from Novabiochem. TLC was performed on Merck silica gel 60
F254 glass plates and flash column chromatography on Fluka silica
gel 60. NMR spectra were recorded on a Mercury Plus 200 MHz
spectrometer or on a Bruker Avancell 400 MHz spectrometer, with
chemical shifts d in ppm using TMS or DMSO solvent peak as in-
ternal reference. HRMS-ESI were recorded using a Bruker Dalton-
ics maXis ESI-QTOF spectrometer with a resolution of 40000–
60000. Four point mass calibration was achieved by using the ESI-
L low concentration Tuning Mix G1969-85000 provided by Agilent
Technologies. Melting points were determined with a Büchi melt-
ing point apparatus B-540 and are uncorrected.
4-{[(9H-Fluoren-9-yl)methoxy]carbonylamino}-2-nitrobenzoic
Acid (9)
9-Fluorenylmethyl chloroformate (17.0 g, 65.7 mmol) in 1,4-diox-
ane (20 mL) was added dropwise to a solution of 4-amino-2-ni-
trobenzoic acid26 (8; 11.4 g, 62.6 mmol) in H2O (228 mL)
containing Na2CO3 (6.63 g). After stirring for 20 h under N2, the
mixture was filtered, and the filtrate was washed with MTBE (5 ×
100 mL). Residual MTBE was removed from the aqueous phase by
evaporation under vacuum. Ice-cold H2O (456 g) was added to the
aqueous phase. The mixture was adjusted to pH 3 by the addition of
aq 2 M HCl (31 mL). The precipitate was drawn off by suction,
washed with H2O (513 mL), and dried at 40 °C under vacuum to
give 9; yield: 17.0 g (67%); off-white crystals; mp >230 °C (dec.).
1H NMR (200 MHz, DMSO-d6): d = 4.27 (t, 3J = 6.3 Hz, 1 H, H-9,
Fmoc), 4.52 (d, 3J = 6.3 Hz, 2 H, 9-CH2, Fmoc), 7.24–7.40 (m, 4 H,
Harom, Fmoc), 7.60–7.90 (m, 7 H, Harom, Fmoc).
13C NMR (50 MHz, DMSO-d6): d = 46.5, 66.1, 112.0, 119.3, 120.2,
120.5, 125.0, 127.1, 127.7, 131.4, 140.8, 142.9, 143.5, 149.9, 153.2,
165.0.
HRMS (ESI, MeOH): m/z [M + H]+ calcd for C22H15N2O8 + 2 Na:
449.0720; found: 449.0730.
Synthesis 2011, No. 22, 3639–3648 © Thieme Stuttgart · New York

3644
V. Groehn et al.
PAPER
Di-tert-butyl N-(4-{[(9H-Fluorene-9-yl)methoxy]carbonylami-
no}-2-nitrobenzoyl)-L-glutamate (10)
1H NMR (200 MHz, DMSO-d6): d = 1.39 (s, 9 H, t-C4H9), 1.40 (s,
9 H, t-C4H9), 1.70–2.05 [m, 2 H, C(b)H2, Glu], 2.31 [t, 3J = 7.4 Hz,
2 H, C(g)H2, Glu], 3.09 (s, 3 H, NCH3), 3.22 (s, 3 H, NCH3) 4.14–
4.26 [m, 1 H, C(a)H, Glu], 4.59 (d, 3J = 5.9 Hz, 2 H, H-6), 6.91 (dd,
3J = 8.5 Hz, 4J = 2.3 Hz, 1 H, 5¢-Harom), 7.12 (d, 3J = 2.2 Hz, 1 H, 6¢-
Harom), 7.37 (d, 3J = 8.3 Hz, 1 H, 3¢-Harom), 7.42 (br s, 1 H, NH), 8.61
(br d, 3J = 7.7 Hz, 1 H, NH), 8.75, 8.79 (2 s, 2 H, H-7, CH=N), 12.00
[br s, 1 H, N(3)H].
13C NMR (50 MHz, DMSO-d6): d = 26.0, 27.6, 27.7, 31.0, 34.9,
40.9, 45.8, 52.2, 79.7, 80.6, 106.7, 114.1, 117.6, 129.9, 130.2,
148.6, 149.0, 149.8, 150.1, 155.5, 159.1, 165.2, 170.7, 171.4.
HRMS (ESI, CH2Cl2–MeOH): m/z [M + H]+ calcd for C30H39N9O8
+ Na: 676.2814; found: 676.2816.
HBTU (17.7 g, 46.7 mmol) was added to a mixture of 9 (17.2 g, 42.5
mmol) and Et3N (12.9 mL) in CH2Cl2 (222 mL). After stirring for
15 min at r.t., a mixture of di-tert-butyl L-glutamate hydrochloride
(13.8 g, 46.7 mmol) in CH2Cl2 (172 mL) was added. The reaction
mixture was stirred under N2 at r.t. for 20 h. After the addition of
MTBE (860 mL), the mixture was washed with 5% aq NaHCO3 (5
× 170 mL), 5% aq citric acid (5 × 170 mL), and brine (2 × 425 mL).
The organic layer was dried (MgSO4) and evaporated to dryness un-
der vacuum to give crude 10 (27.9 g). Purification was accom-
plished by chromatography on silica gel (EtOAc–n-heptane, 45:55)
to give 10; yield: 23.8 g (87%); light yellow foam; Rf = 0.68
(EtOAc–n-heptane, 7:3).
1H NMR (200 MHz, DMSO-d6): d = 1.40 (s, 9 H, t-C4H9), 1.43 (s,
4-(tert-Butoxycarbonylamino)-2-nitrobenzoic Acid (15)
An aqueous solution of NaOH (32%, 0.69 g) was added to a suspen-
sion of 4-amino-2-nitrobenzoic acid26 (8; 1 g, 5.5 mmol) in H2O
(10 mL), followed by the addition of a solution of di-tert-butyl di-
carbonate (1.2 g, 5.5 mmol) in 1,4-dioxane (12 mL). After 29 h at
r.t., additional di-tert-butyl dicarbonate (0.24 g, 1.1 mmol) was add-
ed, and the mixture was stirred for a further 2 h at r.t. The reaction
mixture was washed with MTBE (3 × 5 mL). The pH of the aqueous
layer was adjusted to 3 by the addition of 10% aq citric acid (14.8
g) and the resulting suspension was cooled to 0 °C. The product was
drawn off by suction, washed with H2O (50 mL), and dried at 40 °C
under vacuum to give 15; yield: 0.72 g (47%); off-white crystals;
>180 °C (dec.).
1H NMR (200 MHz, DMSO-d6): d = 1.46 (s, 9 H, t-C4H9), 7.64 (dd,
3J = 8.6 Hz, 4J = 2.0 Hz, 1 H, 5¢-Harom), 7.79 (d, 3J = 8.5 Hz, 1 H, 6¢-
Harom), 7.97 (d, 4J = 1.9 Hz, 1 H, 3¢-Harom), 10.08 (br s, 1 H, NH).
13C NMR (50 MHz, DMSO-d6): d = 27.9, 80.5, 111.6, 118.8, 120.2,
131.3, 143.4, 150.0, 152.4, 165.0.
9 H, t-C4H9), 1.72–2.09 [m, 2 H, C(b)H2, Glu], 2.34 [t, 3J = 7.6 Hz,
2 H, C(g)H2, Glu], 4.22–4.37 [m, 2 H, C(a)H, Glu, H-9, Fmoc], 4.58
3
(d, J = 6.4 Hz, 2 H, 9-CH2, Fmoc), 7.31–7.54 (m, 5 H, Harom
,
Fmoc), 7.74–8.14 (m, 11 H, Harom, Fmoc), 8.86 (br d, 1 H, NH, Glu),
10.3 (br s, 1 H, NH, Fmoc).
13C NMR (50 MHz, DMSO-d6): d = 26.0, 27.5, 27.7, 30.9, 46.5,
52.2, 66.0, 79.7, 80.7, 112.7, 120.1, 121.5, 125.0, 125.3, 127.1,
127.0, 127.6, 140.7, 141.1, 143.5, 147.7, 153.2, 165.0, 170.4, 171.4.
HRMS (ESI, MeOH): m/z [M + H]+ calcd for C35H39N3O9 + Na:
668.2579; found: 668.2599.
Di-tert-butyl N-(4-Amino-2-nitrobenzoyl)-L-glutamate (11)
Pyrrolidine (3.7 mL) was added to 10 (25 g, 38.7 mmol) in DMF
(500 mL). After 30 min at r.t., the solution was evaporated to dry-
ness at 40 °C under vacuum to give a yellow tar (25.0 g). After com-
plete removal of residual DMF under high vacuum, (i-Pr)2O (500
mL) was added. After cooling to 0 °C and stirring for 3 h, the result-
ing solid was drawn off by suction, washed with (i-Pr)2O (150 mL),
and dried under vacuum at 40 °C to give 11, which was used with-
out further purification for the synthesis of 13; yield: 12.6 g (77%);
light yellow crystals; mp 120 °C.
HRMS (ESI, MeOH): m/z [M + H]+ calcd for C12H13N2O6 + 2 Na:
327.0564; found: 327.0574.
4-(tert-Butoxycarbonylamino)-2-fluorobenzoic Acid (16)
An aqueous solution of NaOH (32%, 6.4 g) was added to a suspen-
sion of 4-amino-2-fluorobenzoic acid30 (14; 8.1 g, 52.2 mmol) in
H2O (80 mL), followed by the addition of a solution of di-tert-butyl
dicarbonate (28.3 g, 129.7 mmol) in 1,4-dioxane (80 mL). After 48
h at r.t., the reaction mixture was washed with MTBE (5 × 50 mL).
The pH of the aqueous layer was adjusted to 5.8 by the addition of
10% aq citric acid. The resulting suspension was cooled to 0 °C and
stirred for 1 h. The product was drawn off by suction, washed with
H2O (120 mL) and dried at 40 °C under vacuum to give 16; yield:
7.3 g (55%); colorless crystals; mp >200 °C (dec.).
1H NMR (200 MHz, DMSO-d6): d = 1.50 (s, 9 H, t-C4H9), 7.29–
7.49 (m, 2 H, 5¢-Harom, 6¢-Harom), 7.80 (m, 1 H, 3¢-Harom), 9.92 (br s,
1 H, NH), 12.83 (br s, 1 H, CO2H).
13C NMR (50 MHz, DMSO-d6): d = 27.9, 39.5, 80.1, 104.8, 105.3,
111.8, 112.0, 113.2, 132.7, 145.3, 145.5, 152.3, 159.4, 164.5, 164.6,
164.7.
1H NMR (200 MHz, DMSO-d6): d = 1.40 (s, 9 H, t-C4H9), 1.41 (s,
9 H, t-C4H9), 1.71–1.96 [m, 2 H, C(b)H2, Glu], 2.32 [t, 3J = 7.5 Hz,
2 H, C(g)H2, Glu], 4.1–4.26 [m, 1 H, C(a)H, Glu], 6.12 (br s, 2 H,
NH2), 6.78 (dd, J = 8.4 Hz, J = 2.3 Hz, 1 H, 5¢-Harom), 6.97 (d,
4J = 2.2 Hz, 1 H, 3¢-Harom), 7.32 (d, 3J = 8.4 Hz, 1 H, 6¢-Harom), 8.59
(br d, 3J = 7.7 Hz, 1 H, NH, Glu).
13C NMR (50 MHz, DMSO-d6): d = 26.0, 27.6, 27.7, 31.1, 52.2,
79.7, 80.6, 107.5, 115.5, 117.0, 130.1, 149.8, 151.2, 165.3, 170.7,
171.5.
3
4
HRMS (ESI, MeOH): m/z [M + H]+ calcd for C20H29N3O7 + Na:
446.1898; found: 446.1900.
N2-(N,N-Dimethylaminomethylene)-2¢-nitrofolic Acid Di-tert-
butyl Ester (13)
6-(Bromomethyl)pterin hydrobromide24,25 (12; 26.5 g, 78.6 mmol)
was added to a solution of 11 (12.6 g, 29.8 mmol) in DMAC (130
mL). The mixture was stirred under N2 at 60 °C for 13 h. After cool-
ing to r.t., the mixture was filtered and the filtrate was added drop-
wise to H2O (8800 mL). The crystals were drawn off by suction,
washed with H2O (880 mL), and dried at 35 °C under vacuum to
give crude 2¢-nitrofolic acid di-tert-butyl ester (14.1 g). N,N-Di-
methylformamide diisopropylacetal (31 mL) was added to a solu-
tion of crude 2¢-nitrofolic acid di-tert-butyl ester (8.8 g) in anhyd
DMF (133 mL). The mixture was stirred under N2 for 20 h at r.t. and
then evaporated to dryness. The residue was purified three times by
chromatography on silica gel (CH2Cl2–MeOH, 95:5) to give 13;
yield: 2.75 g (14% corresponding to 11); orange-yellow solid;
Rf = 0.58 (CH2Cl2–MeOH, 85:15).
HRMS (ESI, CH2Cl2–MeOH): m/z [M + H]+ calcd for C12H14FNO4
+ Na: 278.0799; found: 278.0794.
Di-tert-butyl N-[4-(tert-Butoxycarbonylamino)-2-nitrobenzoyl]-
L-glutamate (17)
A solution of di-tert-butyl L-glutamate hydrochloride (3.8 g, 12.8
mmol) and Et3N (1.5 mL) in CH2Cl2 (60 mL) was added to a mix-
ture of 15 (3 g, 10.6 mmol), Et3N (1.5 mL), HBTU (4.8 g, 12.7
mmol), and CH2Cl2 (60 mL). The mixture was stirred at r.t. for 18
h. Solids were removed by filtration and the filtrate was washed
with 5% aq citric acid (3 × 50 mL), sat. aq NaHCO3 (3 × 50 mL),
and H2O (2 × 50 mL). The organic layer was dried (MgSO4) and
Synthesis 2011, No. 22, 3639–3648 © Thieme Stuttgart · New York

PAPER
Precursors for 18F-Labeling of Folic Acid for PET Application
3645
evaporated to dryness at 40 °C under vacuum to give crude 17 (6.3
g) as a yellow foam. Purification was accomplished by chromatog-
raphy on silica gel (EtOAc–n-heptane, 4:6); yield: 5.1 g (92%);
bright yellow foam; Rf = 0.44 (EtOAc–MeOH, 90:10).
13C NMR (50 MHz, DMSO-d6): d = 27.0, 28.2, 30.0, 51.9, 52.3,
52.7, 81.8, 113.8, 122.2, 125.4, 129.3, 141.3, 147.3, 152.3, 166.3,
172.0, 173.6.
HRMS (ESI, CH2Cl2–MeOH): m/z [M + H]+ calcd for C19H25N3O9
+ Na: 462.1483; found: 462.1478.
1H NMR (200 MHz, DMSO-d6): d = 1.40 (s, 9 H, t-C4H9, Glu), 1.43
(s, 9 H, t-C4H9, Glu), 1.50 (s, 9 H, t-C4H9, Boc), 1.72–2.08 [m, 2 H,
C(b)H2, Glu], 2.34 [t, 3J = 7.6 Hz, 2 H, C(g)H2, Glu], 4.22–4.33 [m,
1 H, C(a)H, Glu], 7.51 (d, 3J = 8.4 Hz, 1 H, 6¢-Harom), 7.72 (dd, d,
N-(4-Amino-2-nitrobenzoyl)-L-glutamic Acid (20)
Trifluoroacetic acid (50 mL) was added to a solution of 17 (5.3 g,
10.1 mmol) in anhyd CH2Cl2 (50 mL) at 0 °C. After removal of the
cooling bath, the mixture was stirred at r.t. and then evaporated to
dryness to give 20 as the trifluoroacetate. The product was not fur-
ther purified; yield: 3.4 g (79%); orange tar.
3
4J = 2.1 Hz, J = 8.4 Hz, 1 H, 5¢-Harom), 8.16 (d, 4J = 2.0 Hz, 1 H,
3¢-Harom), 8.84 (d, 2J = 7.7 Hz, NH, Glu), 10.01 (br s, 1 H, NH).
13C NMR (50 MHz, DMSO-d6): d = 26.1, 27.6, 27.7, 27.9, 31.0,
52.2, 79.7, 80.2, 80.7, 112.4, 121.3, 124.8, 129.7, 141.7, 147.8,
152.5, 165.1, 170.4, 171.4.
HRMS (ESI, CH2Cl2–MeOH): m/z [M + H]+ calcd for C25H37N3O9
+ Na: 546.2422; found: 546.2444.
1H NMR (200 MHz, DMSO-d6): d = 1.77–2.14 [m, 2 H, C(b)H2,
3
Glu], 2.36 [t, J = 7.5 Hz, 2 H, C(g)H2, Glu], 4.26–4.38 [m, 1 H,
C(a)H, Glu], 6.80 (dd, 3J = 8.4 Hz, 4J = 2.3 Hz, 1 H, 5¢-Harom), 6.99
(d, 4J = 2.2 Hz, 1 H, 3¢-Harom), 7.36 (d, 3J = 8.4 Hz, 1 H, 6¢-Harom),
8.57 (d, 3J = 7.9 Hz, 1 H, Ca-NH), 9.68 (br s, 3 H, NH3+ and H2O
present in DMSO-d6).
Di-tert-butyl N-[4-(tert-Butoxycarbonylamino)-2-fluoroben-
zoyl]-L-glutamate (18)
A solution of di-tert-butyl L-glutamate hydrochloride (4.17 g, 14.1
mmol) and Et3N (1.64 mL) in CH2Cl2 (120 mL) was added to a mix-
ture of 16 (3 g, 11.8 mmol), Et3N (1.64 mL), HBTU (5.35 g,
14.1 mmol), and CH2Cl2 (120 mL). The mixture was stirred at r.t.
for 18 h. Solids were removed by filtration and the filtrate was
washed with 5% aq citric acid (5 × 60 mL), sat. aq NaHCO3 (3 × 60
mL) and H2O (2 × 60 mL). The organic layer was dried (MgSO4)
and evaporated to dryness at 40 °C under vacuum to give 7.03 g
crude 18 (7.03 g) as a yellow foam. Purification was accomplished
by chromatography on silica gel (EtOAc–n-heptane, 3:7); yield: 5.3
g (91%); colorless crystals; mp 117 °C; Rf = 0.50 (EtOAc–MeOH,
1:1).
13C NMR (50 MHz, DMSO-d6): d = 26.0, 27.6, 27.7, 31.1, 52.2,
79.7, 80.6, 107.5, 115.5, 117.0, 130.1, 149.8, 151.2, 165.3, 170.7,
171.5.
HRMS (ESI, CH2Cl2): m/z [M + H]+ calcd for C12H14N3O7:
312.0826; found: 312.0826.
N-(4-Amino-2-fluorobenzoyl)-L-glutamic Acid (21)
Trifluoroacetic acid (50 mL) was added to a solution of 18 (5.2 g,
10.5 mmol) in anhyd CH2Cl2 (50 mL) at 0 °C. After removal of the
cooling bath, the mixture was stirred at r.t., and then evaporated to
dryness to give 21 as the trifluoroacetate. The product was not fur-
ther purified; yield: 4.0 g (96%); light brown foam.
1H NMR (200 MHz, DMSO-d6): d = 1.39 (s, 9 H, t-C4H9, Glu), 1.42
(s, 9 H, t-C4H9, Glu), 1.49 (s, 9 H, t-C4H9, Boc), 1.78–2.11 [m, 2 H,
C(b)H2, Glu], 2.28–2.36 [m, 2 H, C(g)H2, Glu], 4.26–4.37 [m, 1 H,
1H NMR (200 MHz, DMSO-d6): d = 1.83–2.17 [m, 2 H, C(b)H2,
Glu], 2.28–2.36 [m, 2 H, C(g)H2, Glu], 4.35–4.45 [m, 1 H, C(a)H,
Glu], 6.35 (dd, 4J = 2.0 Hz, 3J = 14.3 Hz, 1 H, 3¢-Harom), 6.45 (dd,
3
4
C(a)H, Glu], 7.28 (dd, JH,F = 8.6 Hz, J = 2.0 Hz, 1 H, 3¢-Harom),
7.44 (dd, 4J = 1.9 Hz, 3J = 13.7 Hz, 1 H, 5¢-Harom), 7.55 (m, 1 H, 6¢-
Harom), 8.20 (dd, 4J = 3.4 Hz, 3J = 7.5 Hz, 1 H, Ca-NH), 9.80 (br s,
1 H, C4¢-NH).
4J = 2.1 Hz, 3J = 8.5 Hz, 1 H, 5¢-Harom), 7.47 (t, 3J = 8.7 Hz, 1 H, 6¢-
+
H
arom), 7.71 (m, 1 H, Ca-NH), 10.66 (br s, 3 H, NH3 and H2O
present in DMSO-d6).
13C NMR (50 MHz, DMSO-d6): d = 26.2, 30.1, 51.7, 99.4, 99.9,
108.6, 109.8, 131.8, 152.9, 153.1, 163.5, 173.3, 173.8.
HRMS (ESI, CH2Cl2–MeOH): m/z [M + H]+ calcd for C12H12FN2O5
+ 2 Na: 329.0520; found: 329.0519.
13C NMR (50 MHz, DMSO-d6): d = 26.1, 27.6, 27.7, 27.9, 31.0,
52.2, 79.7, 80.2, 80.7, 112.4, 121.2, 124.8, 129.7, 141.6, 147.8,
152.5, 165.1, 170.4, 171.4.
HRMS (ESI, CH2Cl2–MeOH): m/z [M + H]+ calcd for C25H37FN2O7
+ Na: 519.2477; found: 519.2480.
Dimethyl N-(4-Amino-2-nitrobenzoyl)-L-glutamate (22)
Trifluoroacetic acid (30 mL) was added to a solution of 19 (17.0 g,
38.7 mmol) in anhyd CH2Cl2 (170 mL) at 0 °C. After removal of the
cooling bath, the mixture was stirred at r.t. for 5 h, and then evapo-
rated to dryness to give crude 22 (19.5 g) as a yellow highly viscous
oil. Purification was accomplished by two-fold chromatography on
silica gel (CH2Cl2–MeOH, 95:5) to give a bright yellow foam (8.6
g), which was treated with (i-Pr)2O (172 mL). After stirring for 2 h,
the crystals were drawn off by suction, washed with (i-Pr)2O (33
mL), and dried at 35 °C overnight under vacuum to give pure 22 as
the trifluoroacetate; yield: 7.9 g (45%); yellow crystals; mp 126 °C;
Rf = 0.46 (CH2Cl2–MeOH, 90:10).
Dimethyl N-[4-(tert-Butoxycarbonylamino)-2-nitrobenzoyl]-L-
glutamate (19)
A slurry of dimethyl L-glutamate hydrochloride (13.3 g, 62.8
mmol) and Et3N (7.3 mL) in CH2Cl2 (296 mL) was added to a mix-
ture of 15 (14.8 g, 52.4 mmol), Et3N (7.3 mL), HBTU (23.9 g, 63.0
mmol), and CH2Cl2 (296 mL). The mixture was stirred at r.t. for 18
h. Solids were removed by filtration and the filtrate was washed
times with 5% aq citric acid (5 × 100 mL), sat. aq NaHCO3 (5 × 100
mL), and H2O (100 mL). The organic layer was dried (MgSO4) and
evaporated to dryness at 40 °C under vacuum to give crude 19 (27.4
g) as an orange tar. Purification was accomplished by chromatogra-
phy on silica gel (EtOAc–n-heptane, 35:65) to give pure 19; yield:
17.4 g (76%); bright yellow foam; Rf = 0.28 (EtOAc–n-heptane,
7:3).
1H NMR (200 MHz, CDCl3): d = 1.53 (s, 9 H, t-C4H9), 2.07–2.45 [2
m, 2 H, C(b)H2, Glu], 2.55 [m, 2 H, C(g)H2, Glu], 3.68 (s, 3 H,
OCH3), 3.80 (s, 3 H, OCH3), 4.76–4.86 [m, 1 H, C(a)H, Glu], 6.71
(d, 3J = 7.7 Hz, 1 H, Ca-NH) 6.95 (br s, 1 H, NH), 7.45 (d, 3J = 8.4
Hz, 1 H, 6¢-Harom), 7.62 (dd, 4J = 2.2 Hz, 3J = 8.4 Hz, 5¢-Harom), 8.12
(d, 4J = 2.1 Hz, 1 H, 3¢-Harom).
1H NMR (200 MHz, DMSO-d6): d = 1.82–2.18 [m, 2 H, C(b)H2,
Glu], 2.45 [t, 3J = 7.7 Hz + DMSO-signal, 2 H, C(g)H2, Glu], 3.60
(s, 3 H, OCH3), 3.65 (s, 3 H, OCH3), 4.32–4.43 [m, 1 H, C(a)H,
3
4
Glu], 6.07 (br s, H2O and/or NH2), 6.79 (dd, J = 8.4 Hz, J = 2.1
4
Hz, 1 H, 5¢-Harom), 6.99 (d, J = 2.1 Hz, 1 H, 3¢-Harom), 7.34 (d,
3J = 8.4 Hz, 1 H, 6¢-Harom), 8.71 (d, 3J = 7.6 Hz, 1 H, Ca-NH).
13C NMR (50 MHz, DMSO-d6): d = 25.9, 29.7, 51.3, 51.5, 51.9,
107.5, 115.6, 116.7, 130.1, 149.8, 151.3, 165.5, 171.9, 172.6.
HRMS (ESI, MeOH): m/z [M + H]+ calcd for C14H17N3O7 + Na:
362.0959; found: 362.0961.
Synthesis 2011, No. 22, 3639–3648 © Thieme Stuttgart · New York

3646
V. Groehn et al.
PAPER
2¢-Nitrofolic Acid (2)
g) was obtained from the mother liquor; total yield: 2.9 g (49%);
citreous-greenish crystals; mp >250 °C (dec.).
6-(Bromomethyl)pterin hydrobromide24,25 (12; 2.3 g, 6.8 mmol)
was added to a solution of crude 20 (3.4 g, 7.99 mmol) in anhyd
DMAC (50 mL). The mixture was stirred for 5 h at 60 °C and for 17
h at r.t. The solids were removed by filtration and the filtrate was
added to aq 0.1 M HCl (321 mL). The resulting suspension was
stirred for 1 h at r.t., the product was drawn off by suction, washed
with aq 0.1 M HCl (24 mL) and H2O (24 mL), and dried under vac-
uum at 40 °C to give crude 2 (1.54 g). The crude product was dis-
solved in H2O (26 mL) at 70 °C by the addition of 37% aq HCl (15.4
mL). The solution was treated with charcoal (0.31 g) at 70 °C and
after removal of charcoal, the filtrate was cooled to r.t. After the ad-
dition of an aq solution of NaOH (32%, 16.6 g), the mixture was
stirred for 30 min at 0 °C. The product was drawn off by suction,
washed with H2O (20 mL), and dried at 40 °C under vacuum to give
pure 2; yield: 0.86 g (26% corresponding to 12); greenish-yellow
crystals; mp >200 °C (dec.).
1H NMR (200 MHz, DMSO-d6): d = 1.80–2.15 [m, 2 H, C(b)H2,
Glu], 2.44 [t, 3J = 7.5 Hz + DMSO-signal, 2 H, C(g)H2, Glu], 3.60
(s, 3 H, OCH3), 3.65 (s, 3 H, OCH3), 4.32–4.42 [m, 1 H, C(a)H,
3
4
Glu], 4.56 (d, J = 5.9 Hz, 2 H, H-6), 6.90 (dd, d, J = 2.2 Hz,
3J = 8.6 Hz, 1 H, 5¢-Harom), 7.05 (br s, 2 H, NH2), 7.11 (d, 4J = 2.2
Hz, 1 H, 3¢-Harom), 7.39 (d + NH-signal, 3J = 8.5 Hz, 2 H, 6¢-Harom),
8.68 (s, 1 H, H-7), 8.74 (d, 3J = 7.8 Hz, 1 H, NH, Glu), 11.50 [br s,
1 H, N(3)H].
13C NMR (50 MHz, DMSO-d6): d = 25.9, 29.7, 45.7, 51.3, 51.6,
51.9, 106.7, 114.2, 117.3, 128.1, 130.2, 147.7, 148.5, 149.8, 150.2,
153.8, 155.7, 160.8, 165.4, 171.9, 172.6.
HRMS (ESI, CH2Cl2–MeOH): m/z [M + H]+ calcd for C21H22N8O8
+ Na: 537.1453; found: 537.1454.
Method B: 2¢-Nitrofolic acid (2; 200 mg, 0.4 mmol) was added to a
solution of benzenesulfonic acid (130 mg) in MeOH (5 mL). The
yellow slurry was stirred at 90 °C for 2 h and cooled to 0 °C over-
night. The crystallized product was drawn off by suction, washed
with ice-cold MeOH (2 mL), and dried overnight at 40 °C under
vacuum to give 23 as hemibenzenesulfonate; yield: 175 mg (72%);
greenish-yellow crystals; mp >250 °C (dec.); BSA content (deter-
mined with HPLC using a reference standard): 11% w/w corre-
sponding to approx. 0.5 mol BSA/1 mol 2¢-nitrofolic acid dimethyl
ester.
1H NMR (200 MHz, DMSO-d6): d = 1.71–2.09 [2 m, 2 H, C(b)H2,
3
Glu], 2.31 [t, J = 7.4 Hz, 2 H, C(g)H2, Glu], 4.21–4.32 [m, 1 H,
C(a)H, Glu], 4.54 (s, 2 H, H-6), 6.86 (dd, 3J = 8.6 Hz, 4J = 2.3 Hz,
1 H, 5¢-Harom), 7.07 (d, 4J = 2.3 Hz, 1 H, 3¢-Harom), 7.21 (br s, 1 H,
NH2), 7.37 (d, 3J = 8.5 Hz, 1 H, 6¢-Harom), 8.60 (d, 3J = 7.8 Hz, 1 H,
Ca-NH), 8.68 (s, 1 H, H-7).
13C NMR (50 MHz, DMSO-d6): d = 26.2, 30.2, 45.7, 51.6, 106.7,
114.2, 117.6, 128.1, 130.2, 148.5, 149.9, 150.1, 153.6, 160.5, 165.2,
173.1, 173.8.
1H NMR (200 MHz, DMSO-d6 + 3 drops of D2O): d = 1.81–2.16
[m, 2 H, C(b)H2, Glu], 2.44 [m + DMSO-signal, 2 H + DMSO-sig-
nal, C(g)H2, Glu], 3.60 (s, 3 H, OCH3), 3.65 (s, 3 H, OCH3), 4.37
HRMS (ESI, CH2Cl2–MeOH): m/z [M + H]+ calcd for C19H17N8O8
2 Na: 531.0959; found: 531.0955.
4
[m, 1 H, C(a)H, Glu], 4.61 (s, 2 H, H-6), 6.92 (dd, J = 1.8 Hz,
2¢-Fluorofolic Acid (3)
3J = 8.4 Hz, 1 H, 5¢-Harom), 7.12 (d, 4J = 1.7 Hz, 1 H, 3¢-Harom), 7.38
6-(Bromomethyl)pterin hydrobromide24,25 (12; 1.7 g, 5.0 mmol)
was added to a solution of crude 21 (2 g, 5.0 mmol) in anhyd DMAC
(76 mL). The mixture was stirred for 6 h at 60 °C. After cooling to
r.t., solids were removed by filtration. The filtrate was added to 0.1
M aq HCl (486 mL). After cooling to 4 °C for 17 h, the crystallized
product was drawn off by suction, washed with 0.1 M aq HCl (29
mL) and H2O (90 mL), and dried under vacuum at 40 °C to give
0.76 g of crude 3, which was dissolved in H2O (11 mL) at r.t. by the
addition of 37% aq HCl (7.6 mL). After cooling to 4 °C for 5 h, the
crystallized product was drawn off by suction, washed with aq 0.05
M HCl (20 mL) and H2O (20 mL), and dried under vacuum at 40 °C
to give pure 3, yield: 0.68 g (30% corresponding to 12); yellowish
crystals; mp >230 °C (dec.).
3
(d, m, 1 H, 6¢-Harom, +Harom from PhSO3H), 7.64 (dd, J = 6.5 Hz,
4J = 2.8 Hz, 1 H, NH), 8.75 (s, 1 H, H-7).
13C NMR (50 MHz, DMSO-d6): d = 25.8, 29.6, 45.5, 51.3, 51.5,
51.9, 106.7, 114.2, 117.4, 125.4, 127.7, 128.1, 128.6, 130.2, 148.1,
149.6, 149.8, 150.1, 152.0, 152.9, 159.8, 165.3, 171.8, 172.6.
HRMS (ESI, MeOH–H2O, 1:1): m/z [M + H]+ calcd for C21H22
N8O8 + Na: 537.1453; found: 537.1439.
N10-Formyl-2¢-nitrofolic Acid Dimethyl Ester (24)
2¢-Nitrofolic acid dimethyl ester (23; 1.9 g, 3.69 mmol) was stirred
in formic acid (98%, 190 mL) at 60 °C for 3.5 h. The solution was
concentrated to a volume of 20 mL and then ice-cold H2O (100 mL)
was added dropwise. The crystallized product was drawn off by
suction, washed with ice-cold H2O (4 mL) and dried at 40 °C under
vacuum for 17 h to give 24; yield: 1.54 g (77%); green crystals; mp
>250 °C (dec.).
1H NMR (200 MHz, DMSO-d6): d = 1.81– 2.17 [m, 2 H, C(b)H2,
Glu], 2.42 [m, 2 H + DMSO-signal, C(g)H2, Glu], 3.60 (s, 3 H,
OCH3), 3.67 (s, 3 H, OCH3), 4.45 [m, 1 H, C(a)H, Glu], 5.24 (s, 2
H, H-6), 6.89 (br s, 2 H, NH2), 7.62 (d, 3J = 8.3 Hz, 1 H, 6¢-Harom),
7.90 (dd, 4J = 2.0 Hz, 3J = 8.4 Hz, 1 H, 5¢-Harom), 8.22 (d, 4J = 2.0
Hz, 1 H, 3¢-Harom), 8.66 (s, 1 H, H-7), 8.84 (s, 1 H, CHO), 9.10 (d,
3J = 7.7 Hz, 1 H, NH, Glu), 11.43 [br s, 1 H, N(3)H].
1H NMR (200 MHz, DMSO-d6): d = 1.82–2.09 [m, 2 H, C(b)H2,
3
Glu], 2.28 [t, J = 7.4 Hz, 2 H, C(g)H2, Glu], 4.26–4.37 [m, 1 H,
C(a)H, Glu], 4.49 (d, 3J = 5.7 Hz, 2 H, H-6), 6.39–6.55 (2 dd, 2 H,
3¢-Harom, 5¢-Harom), 7.03 (br s, 1 H, NH2), 7.20 [t, 3J = 5.7 Hz, 1 H,
C(6)-NH], 7.51 (m, 1 H, 6¢-Harom), 7.75 (t, 3J = 7.2 Hz, 1 H, Ca-NH),
8.65 (s, 1 H, H-7), 11.66 [br s, 1 H, N(3)-H].
13C NMR (100 MHz, DMSO-d6): d = 26.2, 30.1, 45.8, 51.7, 97.9
(2JC,F = 28 Hz), 108.4, 108.9 (2JC,F = 12 Hz), 127.9, 131.6, 147.9,
148.5, 152.5 (3JC,F = 12 Hz), 153.8, 156.4 (br), 160.9 (br), 161.6
(1JC,F = 246 Hz), 163.3, 173.2, 173.7.
HRMS (ESI, CH2Cl2–MeOH): m/z [M
+
H]+ calcd for
13C NMR (100 MHz, DMSO-d6): d = 25.8, 29.5, 46.4, 51.3, 51.5,
52.0, 117.3, 126.0, 128.0, 130.1, 142.9, 145.0, 148.0, 148.8, 153.8,
156.5, 160.5, 162.5, 164.8, 171.4, 172.5.
HRMS (ESI, MeOH–H2O, 1:1): m/z [M + H]+ calcd for
C22H23N8O9: 543.1583; found: 543.1584.
C19H17FN7O6: 458.1230; found: 458.1231.
2¢-Nitrofolic Acid Dimethyl Ester (23)
Method A: 6-(Bromomethyl)pterin hydrobromide24,25 (12; 5.5 g,
16.3 mmol) was added to a solution of 22 (5.0 g, 11.0 mmol) in
DMAC (500 mL). The mixture was stirred for 9 h at 60 °C. After
cooling to r.t., the solids were removed by filtration, and the filtrate
was added dropwise to 0.1 M aq HCl (3500 mL) at r.t. After cooling
to 0 °C overnight, the crystallized product was drawn off by suction,
washed with 0.1 M aq HCl (50 mL) and H2O (300 mL), and dried
under vacuum at 40 °C to give 23 (2.1 g). A second crop of 23 (0.8
N2-(N,N-Dimethylaminomethylene)-N10-formyl-2¢-nitrofolic
Acid Dimethyl Ester (25)
N,N-Dimethylformamide diisopropylacetal (5.4 mL) was added
dropwise to 24 (1.34 g) in anhyd DMF (45 mL) within 30 min at r.t.
Synthesis 2011, No. 22, 3639–3648 © Thieme Stuttgart · New York

PAPER
Precursors for 18F-Labeling of Folic Acid for PET Application
3647
The mixture was stirred at r.t. for 17 h and evaporated to dryness.
The residue (1.75 g) was purified by column chromatography on sil-
ica gel (CH2Cl2–MeOH, 93:7) to give 25; yield: 0.97 g (66%); off-
white crystals; mp >180 °C (dec.); Rf = 0.4 (CH2Cl2–MeOH, 9:1).
N2-(N,N-Dimethylaminomethylene)-2¢-fluoro-N10-formylfolic
Acid Dimethyl Ester (28)
N,N-Dimethylformamide diisopropylacetal (6.1 mL) was added
dropwise to 27 (1.5 g, 2.9 mmol) in anhyd DMF (50 mL) within 30
min at r.t. The mixture was stirred at r.t. for 17 h and evaporated to
dryness. The residue (2.08 g) was purified by column chromatogra-
phy on silica gel (CH2Cl2–MeOH, 93:7) to give 28; yield: 1.39 g
(84%); yellow foam; Rf = 0.54 (CH2Cl2–MeOH, 85:15).
1H NMR (200 MHz, DMSO-d6): d = 1.83–2.19 [m, 2 H, C(b)H2,
Glu], 2.47 [m, 2 H + DMSO-signal, C(g)H2, Glu], 3.09 (s, 3 H,
NCH3), 3.22 (s, 3 H, NCH3), 3.61 (s, 3 H, OCH3), 3.68 (s, 3 H,
OCH3), 4.47 [m, 1 H, C(a)H, Glu], 5.30 (s, 2 H, H-6), 7.65 (d,
3J = 8.3 Hz, 1 H, 6¢-Harom), 7.93 (dd, 4J = 2.0 Hz, 3J = 8.4 Hz, 1 H,
1H NMR (200 MHz, DMSO-d6): d = 1.87–2.21 [m, 2 H, C(b)H2,
Glu], 2.46 [t, 3J = 7.5 Hz, 2 H + DMSO-signal, C(g)H2, Glu], 3.09
(s, 3 H, NCH3), 3.23 (s, 3 H, NCH3), 3.60 (s, 3 H, OCH3), 3.67 (s, 3
H, OCH3), 4.46 [m, 1 H, C(a)H, Glu], 5.26 (s, 2 H, H-6), 7.43 (dd,
4
5¢-Harom), 8.25 (d, J = 1.9 Hz, 1 H, 3¢-Harom), 8.77 (s, 1 H, H-7),
8.78 (s, 1 H, CH=N), 8.88 (s, 1 H, CHO), 9.12 (d, 3J = 7.7 Hz, 1 H,
NH, Glu), 11.99 [br s, 1 H, N(3)H].
3J = 8.4 Hz, 4J = 2.1 Hz, 1 H, 5¢-Harom), 7.57–7.67 (m, 2 H, 6¢-Harom
,
13C NMR (100 MHz, DMSO-d6): d = 25.8, 29.5, 34.9, 40.9, 46.7,
51.2, 51.5, 51.9, 117.2, 125.8, 128.0, 130.1, 142.8, 146.4, 147.9,
148.7, 155.4, 158.1, 159.1, 161.3, 162.5, 164.7, 171.4, 172.4.
HRMS (ESI, CH2Cl2–MeOH): m/z [M + H]+ calcd for C25H28N9O9:
598.2004; found: 598.2003.
3¢-Harom), 8.65 (dd, 3J = 7.5 Hz, 4J = 1.4 Hz, 1 H, NH, Glu), 8.74 (s,
1 H, H-7), 8.79 (s, 1 H, CH=N), 8.87 (s, 1 H, CHO), 11.99 [br s, 1
H, N(3)H].
13C NMR (50 MHz, DMSO-d6): d = 25.7, 29.7, 34.9, 40.9, 46.7,
51.3, 51.8, 52.0, 109.3, 109.8, 117.3, 120.0, 120.3, 129.8, 130.9 (d),
144.4, 144.6, 146.7, 148.7, 155.5, 157.1, 158.2, 161.5, 171.7, 172.6.
HRMS (ESI, MeOH-CH2Cl2): m/z [M + H]+ calcd for C25H28FN8O7:
571.2059; found: 571.2054.
2¢-Fluorofolic Acid Dimethyl Ester Benzenesulfonate (26)
2¢-Fluorofolic acid (3; 2.92 g, 6.36 mmol) was added to a solution
of benzenesulfonic acid (2.1 g, 13.3 mmol) in MeOH (73 mL). The
mixture was heated to reflux for 2 h and then kept at 5 °C for 17 h.
The crystallized product was drawn off by suction, washed with
MeOH (15 mL), and dried under vacuum at 40 °C to give 26; yield:
3.12 g (76%); greenish-yellow crystals; mp >80 °C (dec.); BSA
content (measured by HPLC against a standard): 23% w/w corre-
sponding to approx. 1 mol BSA/1 mol 2¢-fluorofolic acid dimethyl
ester.
Acknowledgment
We thank Michael Blatti, Thomas Ammann, and Katja Rohmer for
technical assistance.
1H NMR (200 MHz, DMSO-d6): d = 1.86–2.18 [m, 2 H, C(b)H2,
Glu], 2.40 [t, 3J = 7.4 Hz, 2 H, C(g)H2, Glu], 3.57 (s, 3 H, OCH3),
3.63 (s, 3 H, OCH3), 4.42 [m, 1 H, C(a)H, Glu], 4.58 (s, 2 H, H-6),
6.44 (dd, 4J = 2.0 Hz, 3J = 14.4 Hz, 1 H, 3¢-Harom), 6.53 (dd,
References
(1) Current address: Radiopharmaceutical Chemistry, Institute
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Mainz, Fritz-Strassmann-Weg 2, 55128 Mainz, Germany.
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4J = 2.1 Hz, J = 8.6 Hz, 1 H, 5¢-Harom), 7.34 (m, Harom, PhSO3H),
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(dd, br s, 3J = 7.2 Hz, 4J = 5.2 Hz, 3 H, NH2, NH, Glu), 8.76 (s, 1
H, H-7).
13C NMR (50 MHz, DMSO-d6): d = 25.8, 29.7, 45.5, 51.3, 51.7,
51.9, 97.9, 98.4, 108.5, 109.1, 109.3, 125.4, 127.8, 128.0, 128.9,
131.6 (d), 147.1, 147.7, 147.9, 151.7, 152.2, 158.7, 159.0, 163.7,
163.9, 172.1, 172.6.
HRMS (ESI, MeOH): m/z [M + H]+ calcd for C21H23FN7O6:
488.1688; found: 488.1690.
2¢-Fluoro-N10-formylfolic Acid Dimethyl Ester (27)
Benzenesulfonate 26 (2.5 g, 3.87 mmol) was stirred in formic acid
(98%, 250 mL) for 4 h at 60 °C. The reaction mixture was concen-
trated to 50 mL under vacuum at 40 °C and treated with ice-cold
H2O (230 mL). The crystallized product was drawn off by suction,
washed with ice-cold H2O (50 mL), and dried under vacuum at
40 °C to give 27; yield: 1.7 g (85%); brownish crystals; mp >250 °C
(dec.).
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1H NMR (200 MHz, DMSO-d6): d = 1.87–2.20 [m, 2 H, C(b)H2,
Glu], 2.46 [t, 3J = 7.6 Hz, 2 H + DMSO-signal, C(g)H2, Glu], 3.60
(s, 3 H, OCH3), 3.67 (s, 3 H, OCH3), 4.48 [m, 1 H, C(a)H, Glu], 5.23
(s, 2 H, H-6), 6.98 (br s, 2 H, NH2), 7.42 (d, J = 7.9 Hz, 1 H, 3¢-
H
arom), 7.62 (m, 2 H, 6¢-Harom, NH, Glu), 8.65 (m, 2 H, 5¢-Harom
,
CHO), 8.85 (s, 1 H, H-7), 11.54 [br s, 1 H, N(3)H].
13C NMR (50 MHz, DMSO-d6): d = 26.4, 30.4, 47.1, 52.0, 52.5,
52.7, 105.0, 110.1, 110.6, 118.1 (d), 120.7, 121.0, 128.8, 131.7,
145.0, 145.2, 146.1, 149.4, 154.6, 157.8, 162.8, 164.1, 172.5, 173.3.
HRMS (ESI, MeOH–H2O, 1:1): m/z [M + H]+ calcd for
C22H23FN7O7: 516.1638; found: 516.1654.
Synthesis 2011, No. 22, 3639–3648 © Thieme Stuttgart · New York

3648
V. Groehn et al.
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