Synthesis of a novel γ-folic acid-Nτ-histidine conjugate suitable for labeling with99mTc and188Re

Article abstract of DOI:10.1055/s-0028-1083345

Radiolabeled folate derivatives have the potential to target folate receptor-positive tumor cells for noninvasive diagnosis and therapy. We report the synthesis of a novel γ-folic acid-Nτ-his-tidine conjugate 1 wherein the Nτ-histidine is suitable for rad

Full text of DOI:10.1055/s-0028-1083345

PAPER
787
Synthesis of a Novel g-Folic Acid–Nt-Histidine Conjugate Suitable for
Labeling with 99mTc and 188Re
S
a
ynthesisof
a
N
o
h
vel
g
-Folic
A
r
cid–
N
-
ti
H
istidi
s
ne Conjug
t
a
te of Sparr,a Urs Michel,a Roger E. Marti,a Cristina Müller,b Roger Schibli,b Rudolf Moser,c Viola Groehn*c
School of Life Sciences and Facility Management, Institute for Chemistry and Biological Chemistry, Zurich University of Applied
Sciences, Grüental, 8820 Wädenswil, Switzerland
b
c
Paul Scherrer Institute, Center for Radiopharmaceutical Science, OIPA, 5232 Villigen PSI, Switzerland
Merck Eprova AG, Im Laternenacker 5, 8200 Schaffhausen, Switzerland
Fax +41(52)6307255; E-mail: viola.groehn@eprova.com
Received 7 October 2008; revised 31 October 2008
with 99mTc. One focus of this work was the development
of the regioselective synthesis of an Nt-(4-aminoalkyl)his-
tidine synthon for further coupling to the g-carboxy group
of folic acid or glutamic acid, respectively (Scheme 1).
Abstract: Radiolabeled folate derivatives have the potential to tar-
get folate receptor-positive tumor cells for noninvasive diagnosis
and therapy. We report the synthesis of a novel g-folic acid–Nt-his-
tidine conjugate 1 wherein the Nt-histidine is suitable for radiolabel-
ing with isotopes 99mTc (diagnosis) and 188Re (therapy). A modular Two synthetic strategies were assessed and reported in
synthetic strategy was applied: Na-Boc-a-carboxy-protected
this work. The final g-folic acid–histidine conjugate 1 was
glutamic acid was amidically linked to Nt-(functionalized amino-
reacted with the organometallic [M(CO)3] synthons (M =
alkyl)histidine via the g-carboxy group to form building block 8. In-
99mTc, Re) and spectroscopically and radiochemically
termediate 8 was coupled to protected pteroic acid to give 1 in two
analyzed.
steps in 47% yield. Nt-(Functionalized aminoalkyl)histidine was
synthesized by two different routes. The preferred route starting
from Boc-His-OMe led in two steps to the Nt-(functionalized
aminoalkyl)histidine in 36% yield.
In the present work, two approaches (route A and route B,
Scheme 2) were investigated for the preparation of the Nt-
functionalized histidine building block. It is known that
alkylation of the imidazole group of histidine derivatives
gives in general a mixture of t- and p-regioisomers (route
A).11 Usually the t-isomer is predominant and is formed
exclusively in cases of very bulky alkylating reagents like
trityl halides.12 For route A, the alkylation of Boc-His-
OMe (2) with 1-azido-4-chlorobutane13 in refluxing ace-
tone and sodium iodide/potassium carbonate as catalyst/
base gave the Nt-(4-azidobutyl)histidine 3 in 36% yield
after purification by flash chromatography.
Key words: alkylations, amino acids, regioselectivity, organome-
tallic, receptor
The folate receptor (FR) is a promising tumor target for
folate conjugates since it is overexpressed on a wide vari-
ety of cancer cells.1 A number of folate conjugates have
been synthesized for application in tumor therapy and di-
agnosis (e.g., of chemotherapeutic agents,2 antisense oli-
gonucleotides,3 antibodies,4 protein toxins,5 liposomes6).
For the diagnostic application some folate-based 66/67/68Ga,
99mTc, and 111In radiopharmaceuticals have been de-
veloped.7 Folate derivatives suitable for labeling with the
radionuclides 99mTc (99mTc: 6 h half-life, 140 keV g-radi-
ation) and 188Re (188Re: 17 h half-life, 2.12 MeV b–max-ra-
diation) are especially attractive since these isotopes are
readily available and exhibit excellent decay properties
for radiodiagnosis and radiotherapy.8 Recently, a series of
novel, organometallic Tc/Re-folate derivatives compris-
ing different chelating systems have been reported.9c 2-Pi-
colylamine-N-acetic acid (PAMA) for example has been
conjugated to the g-carboxy group of folic acid, success-
fully labeled with 99mTc and 188Re and tested in vivo.9 Van
Staveren et al. have shown that Nt-functionalized histidine
derivatives are superior with respect to stability and/or
chelating capacity for the tridentate M(CO)3 core
(M = Tc, Re) compared to PAMA.10 We wanted to ex-
plore the possibility of coupling an Nt-functionalized his-
tidine derivative to folic acid for subsequent radiolabeling
One way to prevent p-alkylation is protection via the cy-
clic urea 5 (route B). The cyclic urea 5 was prepared ac-
cording to the literature.14 Reaction with 1-azido-4-
chlorobutane was performed under similar conditions as
for 2. The crude product 6 was directly treated with tert-
butyl alcohol/N,N-diisopropylethylamine to give the Nt-
(4-azidobutyl)histidine 3. Reduced nucleophilicity of the
t-imidazole nitrogen in 5 and the sluggish ring-opening
reaction of the alkylated urea 6 with tert-butyl alcohol re-
sulted in an overall yield of only 10% for compound 3
over two steps starting from urea 5.
The NMR data of the Nt-(4-azidobutyl)histidine 3, pre-
pared by the two independent routes A and B, were iden-
tical and confirmed the correct assignment of the structure
of 3. In addition, by comprehensive NOE measurements
on 3, a clear and distinct NOE signal was observed for the
C(1)H2 group of the butyl side chain and the C(5)H of the
histidine. Reduction of the azido group was achieved by
catalytic hydrogenation with platinum(IV) oxide as cata-
lyst in methanol to give the Nt-(4-aminobutyl)histidine
building block 4 in quantitative yield.
SYNTHESIS 2009, No. 5, pp 0787–0792
x
x
.
x
x
.2
0
0
9
Advanced online publication: 27.01.2009
DOI: 10.1055/s-0028-1083345; Art ID: T17708SS
© Georg Thieme Verlag Stuttgart · New York
Additionally, we investigated other protected or masked
aminobutyl synthons in the alkylation of Boc-His-OMe

788
C. Sparr et al.
PAPER
O
OH
O
O
α
O
N
N
H
H
N
N
N
OH
γ
HN
N
N
H
τ
N
NH2
O
π
1
H2N
O
O
OH
O
O
OH
α
H2N
OH
O
H2N
N
N
N
HN
N
OH
τ
N
π
NH2
γ
H
H2N
N
O
OH
H2N
HN
τ
X
N
π
NH2
Scheme 1 Retrosynthesis for the g-folic acid–histidine conjugate 1
(2) or cyclic urea 5. The use of Fmoc- and Cbz-protected um chloride (DMTMM)16 as coupling reagent. Selective
halobutylamine derivatives on either route A or B resulted amide bond formation and workup by simple precipitation
in the decomposition of the alkylating reagent by intramo- from water provided fully protected g-folic acid–histidine
lecular 1,5-cyclization leading to the formation of the cor- conjugate 9 in 62% yield.
responding pyrrolidine derivatives. On the other hand, the
Finally, global deprotection was performed in two steps:
alkylation of cyclic urea 5 with 3-bromopropanenitrile
After acidic hydrolysis of the tert-butyl ester, Boc, and
yielded the desired t-isomer, but all attempts to reduce the
(dimethylamino)methylene protecting groups, the methyl
ester and the formyl group were cleaved under basic con-
nitrile group to the desired amine 4 were unsuccessful.
The final steps towards the target molecule 1 are shown in ditions.17 Precipitation of the crude product at pH 2.5 and
Scheme 3. The Nt-(4-aminobutyl)histidine derivative 4 further purification by preparative reversed phase chro-
was reacted with Fmoc-Glu(OSu)-Ot-Bu to give the pro- matography gave 1 in 76% yield.
tected g-glutamate histidine building block 7 in 29%
The metal labeling of the folic acid conjugate 1 was achieved
yield. Fmoc deprotection was achieved by standard
with [Re(Br)3(CO)3][Et4N]2 or [99mTc(CO)3(OH2)3]+ ac-
procedures15 to give 8 in 50% yield. The relatively low
yield in the preparation of 7 and 8 is attributed to losses
cording to the procedure described by Müller et al9b,c
(Scheme 4).
during chromatographic purification. The coupling of the
The rhenium complex 11 was purified by HPLC
protected pteroic acid 10 with 8 was achieved best with 4-
(Figure 1) and characterized by HRMS and NMR spec-
(4,5-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholini-
troscopy. Analysis of the 1H NMR spectra proved triden-
O
O
τ
OMe
OMe
N
HN
N
NH
5
N
π
NHBoc
O
2
route B
a, 36%
route A
c
NOE
O
O
H
Br
d
H
10% from 5
OMe
OMe
N3
N3
N
N
N
NHBoc
N
NH
3
6
O
b, 100%
O
H2N
OMe
NHBoc
N
N
4
Scheme 2 Synthesis of the Nt-(4-aminobutyl)histidine building block 4. Reagents and conditions: (a) Cl(CH2)4N3,13 NaI, K2CO3, acetone,
65 °C, 48 h; (b) H2, PtO2, MeOH, r.t., 10 bar, 24 h; (c) Cl(CH2)4N3, NaI, acetone, 65 °C, 16 h; (d) t-BuOH, i-Pr2NEt, 85 °C, 20 h.
Synthesis 2009, No. 5, 787–792 © Thieme Stuttgart · New York

PAPER
Synthesis of a Novel g-Folic Acid–Nt-Histidine Conjugate
789
O
H2N
OMe
NHBoc
N
N
4
7
O
Ot-Bu
a, 29%
O
NHBoc
O
FmocHN
H
N
OMe
N
N
O
Ot-Bu
b, 50%
O
H2N
H
N
OMe
NHBoc
N
N
O
O
8
OH
O
N
c, 62%
Ot-Bu
N
N
HN
N
Me2N
N
O
H
O
O
10
O
O
N
N
H
H
N
N
OMe
HN
N
N
N
NHBoc
9
O
Me2N
N
N
O
H
d, 76%
O
OH
O
O
O
N
N
H
H
N
N
OH
HN
N
N
H
N
NH2
1
O
H2N
N
Scheme 3 Synthesis of the g-folic acid–histidine conjugate 1. Reagents and conditions: (a) Fmoc-Glu(OSu)-Ot-Bu, DME, r.t., 2 h; (b) pipe-
ridine, CH2Cl2, r.t., 3,h. (c) 10, DMTMM, THF, r.t., 3 h. (d) 1. 1 M HCl, 50 °C, 2 h; 2. NaOH, r.t., 1 h.
date coordination (p-N, a-NH2, COOH) of the metal (~10–9 M) NMR measurement was not possible and there-
center with histidine as chelator since the signals of the fore the (structural) identity of the corresponding 99mTc
histidine protons were typically broader and shifted to low complex 12 was confirmed by comparison of the retention
field compared to the unlabeled compound 1. Due to the time (radioactive HPLC trace) with that of the corre-
low concentration of the radioactive 99mTc complex sponding rhenium complex (Figure 1).
O
OH
O
O
H
N
O
N
N
H
N
OH
N
N
O
N
NH2
HN
N
H
1
H2N
O
OH
O
O
H
N
O
N
N
H
N
O
N
N
O
N
H2N
HN
N
H
M
11: M = Re
H2N
12: M = 99mTc
OC
CO
CO
Scheme 4 Synthesis of the M(CO)3 complexes (M = Re, 99mTc) of the g-folic acid–histidine conjugate 1. Reagents and conditions: 11:
[ReBr3(CO)3][Et4N]2 (1.1 equiv) MeOH–H2O (1:1), NaHCO3, pH = 8, 50 °C, 1.5 h, 12: fac-[99mTc(CO)3(OH2)3]+ (100 mL; ~1 GBq/mL), PBS
buffer pH 7.4 (350 mL), 0.001 M 1 in PBS buffer (50 mL), 75 °C, 30 min.
Synthesis 2009, No. 5, 787–792 © Thieme Stuttgart · New York

790
C. Sparr et al.
PAPER
erwise mentioned, in D2O–NaOD the internal reference was
d(H2O) = 4.79 unless otherwise stated. HRMS (ESI) were recorded
with a Bruker FTMS 4.7 T BioAPEXII (ESI) spectrometer.
Nt-(4-Azidobutyl)-Na-(tert-butoxycarbonyl)-L-histidine Methyl
Ester (3)
Boc-His-OMe (2, 24.24 g, 90 mmol, 1.0 equiv) was stirred in ace-
tone (50 mL). After the addition of K2CO3 (13.68 g, 99 mmol, 1.1
equiv), 1-azido-4-chlorobutane13 (13.22 g, 99 mmol, 1.1 equiv), and
NaI (3.75 g, 25 mmol, 0.28 equiv), the mixture was heated to reflux.
After 2 d, TLC indicated a conversion of ~85% [Rf = 0.58 (3), 0.37
(2) (CH2Cl2–MeOH, 9:1)]. The product was isolated by chromatog-
raphy (EtOAc–n-hexane, 4:1) to give 3 (11.74 g, 36%) as a yellow-
brownish viscous oil; purity: ~95% (NMR).
IR (Golden Gate/ATR): 3345, 2930, 2095, 1745, 1705, 1495, 1365,
1160 cm–1.
1H NMR (300 MHz, CDCl3): d = 1.44 (s, 9 H, t-Bu), 1.5–1.6 [m, 2
2
H, C(3)H2, Bu], 1.8–1.9 [m, 2 H, C(2)H2, Bu], 3.00 [dd, J = 14.4
Hz, 3J = 4.7 Hz, 1 H, C(b)HA, His], 3.08 [dd, 2J = 14.8 Hz, 3J = 5.5
Hz, 1 H, C(b)HB, His], 3.31 [t, 3J = 6.6 Hz, 2 H, C(4)H2, Bu], 3.70
(s, 3 H, OCH3, His), 3.90 [t, 3J = 7.0 Hz, 2 H, C(1)H2, Bu], 4.5–4.6
[m, 1 H, C(a)H, His], 5.9 (d, 3J = 8.2 Hz, 1 H, NH, His), 6.68 [s, 1
H, C(5)H, His], 7.37 [s, 1 H, C(2)H, His].
13C NMR (75 MHz, CDCl3): d = 25.9, 28.2, 28.3, 30.3, 46.4, 50.8,
52.0, 53.6, 79.5, 116.2, 136.8, 137.9, 155.4, 172.5.
HRMS (ESI): m/z [M + H]+ calcd for C16H27N6O4: 367.2094; found:
367.2087.
Nt-(4-Aminobutyl)-Na-(tert-butoxycarbonyl)-L-histidine Meth-
yl Ester (4)
To a soln of 3 (7.0 g, 19.1 mmol, 1.0 equiv) in MeOH (140 mL)
PtO2·x H2O (0.14 g, 81% Pt, 0.58 mmol) was added. The mixture
was hydrogenated at r.t. using H2 (10 bar) for 24 hours. During hy-
drogenation the H2 atmosphere was replaced three times to avoid an
accumulation of N2 from the azide. After addition of cellulose (0.7
g) and charcoal (0.7 g) the mixture was stirred at r.t. for 5 min, the
solids were filtered off and washed with MeOH (20 mL). The fil-
trate was concentrated under vacuum to give 4 (6.5 g, 100%), which
was used directly in the next step.
Figure 1 HPLC traces (XterraTM MS C-18-column): (a) UV trace
(254 nm) of 1; (b) UV trace (254 nm) of the Re(CO)3 complex 11; (c)
g-trace of the radioactive 99mTc complex 12. Differences between the
tR of the 99mTc and Re complex are due to the sequential setup of the
UV/Vis and radioactive detector.
The complex 12 proved to be stable in vitro and in vivo.
Detailed in vitro and in vivo results are described by
Müller et al.18
IR (Golden Gate/ATR): 3305, 2935, 1745, 1705, 1495, 1365, 1160
cm–1.
1H NMR (300 MHz, CDCl3): d = 1.43 (s, 9 H, t-Bu), 1.5–1.6 [m, 2
H, C(3)H2, Bu], 1.7–1.8 [m, 2 H, C(2)H2, Bu], 2.1–2.2 (br s, 2 H,
NH2, Bu), 2.71 [t, 3J = 6.6 Hz, 2 H, C(4)H2, Bu], 3.00 [dd, 2J = 14.4
Hz, 3J = 4.8 Hz, 1 H, C(b)HA, His], 3.08 [dd, 2J = 14.6 Hz, 3J = 6.6
Hz, 1 H, C(b)HB, His], 3.70 (s, 3 H, OCH3, His), 3.90 [t, 3J = 7.0 Hz,
2 H, C(1)H2, Bu], 4.5–4.6 [m, 1 H, C(a)H, His], 5.95 (d, 3J = 7.9 Hz,
1 H, NH, His), 6.68 [s, 1 H, C(5)H, His], 7.35 [s, 1 H, C(2)H, His].
13C NMR (75 MHz, CDCl3): d = 28.3, 28.5, 30.3, 30.4, 40.4, 46.9,
52.1, 53.6, 79.6, 116.4, 136.8, 137.7, 155.6, 172.6.
HRMS (ESI): m/z [M + H]+ calcd for C16H29N4O4: 341.2189; found:
341.2183.
In conclusion, we have identified the most suitable syn-
thetic pathway for the preparation of a novel g-folic acid–
Nt-histidine conjugate as potential precursor for (radio)la-
beling with the (radio)metal precursors of technetium and
rhenium. Preliminary in vitro and in vivo experiments
performed in folate receptor positive cell lines have dem-
onstrated the superior characteristics of the novel folate
derivatives compared to previously published organome-
tallic folate derivatives.18 Further experiments will be
needed to assess the potential of this radiofolate for use in
diagnosis (and therapy) of folate receptor positive tumor
cells.
Na-(tert-Butoxycarbonyl)-Nt-(4-{[Oa-tert-butyl-Na-(9H-fluoren-
9-ylmethoxycarbonyl)-g-glutamyl]amino}butyl)-L-histidine
Methyl Ester (7)
To the soln of Fmoc-Glu(OSu)-Ot-Bu (5.23 g, 10 mmol, 1.0 equiv)
in DME (40 mL), 4 (6.88 g, 10 mmol, purity ~50% w/w, 1.0 equiv)
was added. The mixture was stirred at r.t. for 2 h and then it was
concentrated to ~15 g and the product was purified by flash chroma-
tography (CH2Cl2–MeOH, 25:1) to give 7 (2.29 g, 29%) as a yel-
lowish foam; purity: 95% (NMR); Rf = 0.46 (CH2Cl2–MeOH, 9:1)
All experiments were carried out under an argon atmosphere. All
commercially available reagents were used as received. The N2-
[(dimethylamino)methylene]-10-formylpteroic acid (10) was pro-
vided by Merck Eprova AG, Schaffhausen, Switzerland. TLC was
performed on Merck silica gel 60 F254 glass-plates, flash-column
chromatography on Fluka silica gel 60. IR spectra were recorded on
a Perkin Elmer Spectrum 100. NMR spectra were recorded on a
Bruker Avance 300; TMS was used as internal reference unless oth-
IR (Golden Gate/ATR): 3300, 2935, 1710, 1650, 1500, 1440, 1365,
1155, 1050 cm–1.
Synthesis 2009, No. 5, 787–792 © Thieme Stuttgart · New York

PAPER
Synthesis of a Novel g-Folic Acid–Nt-Histidine Conjugate
791
1H NMR (300 MHz, CDCl3): d = 1.42 (s, 9 H, t-Bu), 1.47 (s, 9 H, t-
Bu), 1.3–1.4 [m, 2 H, C(2)H2, Bu], 1.63–1.75 [m, 2 H, C(3)H2, Bu],
1.85–1.95 [m, 1 H, C(b)HA, Glu], 2.1–2.3 [m, 3 H, C(b)HB, Glu,
C(g)H2, Glu], 2.95–3.05 [m, 2 H, C(b)H2, His], 3.15–3.25 [m, 2 H,
7.40 [d, 3J = 8.6, 2 H, C(3′,5′)H, Pte], 7.48 [s, 1 H, C(2)H, His], 7.87
[d, 3J = 8.6, 2 H, C(2′,6′)H, Pte], 8.70 (s, 1 H, CH, DMAM), 8.80 [s,
1 H, N(10)CHO, Pte], 8.93 [s, 1 H, C(7)H, Pte].
1H–1H COSY-90 correlations (300 MHz, MeOD): d (F2/F1) =
1.41/1.72 [C(2)H2/C(3)H2, Bu], 1.41/3.20 [C(2)H2/C(1)H2, Bu],
1.72/3.95 [C(3)H2/C(4)H2, Bu], 2.02/2.22 [C(b)HA/C(b)HB, Glu],
2.02/2.35 [C(b)HA/C(g)H2, Glu], 2.22/2.35 [C(b)HB/C(g)H2, Glu],
2.02/4.42 [C(b)HA/C(a)H, Glu], 2.22/4.42 [C(b)HB/C(a)H, Glu],
2.92/4.35 [C(b)H2/C(a)H, His)], 6.9/7.58 [C(5)H/C(2)H, His],
7.54/7.9 [C(3¢,5¢)H/C(2¢,6¢)H, Pte).
13C NMR (75 MHz, CDCl3): d = 26.5, 28.1, 28.2, 28.5, 30.4, 32.8,
35.6, 38.8, 41.9, 46.8, 48.5, 52.3, 53.6, 54.1, 56.0, 79.8, 82.4, 116.8,
122.6, 123.2, 129.1, 130.2, 132.1, 137.2, 143.6, 147.8, 149.9, 155.8,
158.3, 159.3, 154.0, 162.5, 162.6, 166.5, 168.6, 171.3, 172.9, 173.1.
HRMS (ESI): m/z [M + H]+ calcd for C43H59N12O10: 903.4477;
found: 903.4489.
3
C(1)H2, Bu], 3.68 (s, 3 H, OCH3, His), 3.86 [t, J = 6.9 Hz, 2 H,
C(4)H2, Bu], 4.15–4.25 [m, 2 H, C(9)H, Fmoc and C(a)H, Glu],
3
4.39 [t, J = 5.0 Hz, 2 H, C(9)CH2, Fmoc], 4.45–4.55 [m, 1 H,
3
C(a)H, His], 5.82 [d, J = 7.6 Hz, 1 H, C(a)NH, Glu], 5.95 [d,
3J = 8.1 Hz, 1 H, C(a)NH, His], 6.4 [s, 1 H, C(4)NH, Bu], 6.66 [s,
1 H, C(5)H, His], 7.32 [s, 1 H, C(2)H, His], 7.3–7.75 [m, 8 H,
Fmoc].
13C NMR (75 MHz, CDCl3): d = 26.6, 28.0, 28.1, 28.3, 30.2, 32.5,
38.6, 46.5, 47.1, 52.1, 53.7, 54.0, 67.0, 79.6, 82.5, 116.4, 120.0,
125.2, 127.1, 127.8, 136.8, 137.5, 141.3, 143.6, 143.9, 155.6, 156.5,
171.1, 172.2, 172.6.
HRMS (ESI): m/z [M + H]+ calcd for C40H54N5O9: 748.3922; found:
748.3921.
Nt-[4-(Folylamino)butyl]-L-histidine (1)
Na-(tert-Butoxycarbonyl)-Nt-{4-[(Oa-tert-butyl-g-glutamyl)ami-
no]butyl}-L-histidine Methyl Ester (8)
To 9 (200 mg, 0.22 mmol, 1 equiv) aq 1 M HCl (22 mL) was added
and the mixture was stirred at 50 °C for 2 h. After cooling to ~15 °C,
solid NaOH (1.76 g) was added, the soln was stirred at r.t. for 1 h.
The pH was adjusted to 2.5 by addition of HCO2H and the precipi-
tate was isolated by filtration. The product was purified by RP-
MPLC (solid phase: Europrep 60-60 C-18, 60 m, 35–70 mm, 140 g;
36 cm × 26 mm, liquid phase: 0–10 min. 99.9% H2O–0.1% HCO2H,
10–40 min. 34.9% MeOH–65% H2O–0.1% HCO2H) to give 1 (120
mg, 76%) as a yellowish solid; purity: 91% (HPLC).
To a mixture of 7 (2.29 g, 3.06 mmol, 1.0 equiv) and CH2Cl2 (50
mL), piperidine (3.0 mL, 30.6 mmol, 10.0 equiv) was added. The
mixture was stirred at r.t. for 3 h and then the product was isolated
directly by flash chromatography (CH2Cl2–MeOH, 4:1) to give 8
(892 mg, 50%) as a pink foam; purity: 90% (NMR); Rf = 0.20
(CH2Cl2–MeOH, 9:1).
IR (Golden Gate/ATR): 3300, 2935, 1710, 1650, 1500, 1435, 1365,
1155, 1050 cm–1.
1H NMR (300 MHz, D2O–10% D2SO4): d = 0.4–0.6 [m, 2 H,
C(2)H2, Bu], 0.75–0.9 [m, 2 H, C(3)H2, Bu], 1.1–1.25 [m, 1 H,
C(b)HA, Glu], 1.25–1.4 [m, 1 H, C(b)HB, Glu], 1.50 [t, 3J = 7.1 Hz,
2 H, C(g)H2, Glu], 2.1–2.3 [m, 2 H, C(1)H2, Bu], 2.4–2.5 [2 dd, 2
H, C(b)H2, His], 3.1–3.3 [m, 2 H, C(4)H2, Bu], 3.42 [t, 3J = 6.7 Hz,
1H NMR (300 MHz, DMSO): d = 1.43 (s, 9 H, t-Bu), 1.46 (s, 9 H,
t-Bu), 1.35–1.55 [m, 2 H, C(2)H2, Bu], 1.75–1.9 [m, 3 H, C(3)H2,
Bu and C(b)HA, Glu], 2.05–2.2 [m, 1 H, C(b)HB, Glu], 2.35 [t,
3J = 7.2 Hz, 2 H, C(g)H2, Glu], 2.95–3.1 [2 dd, 2 H, C(b)H2, His],
3.22 [q, 3J = 6.4 Hz, 2 H, C(1)H2, Bu], 3.41 [dd, 3J = 8.4 Hz,
3J = 4.3 Hz, 1 H, C(a)H, Glu], 3.67 (s, 3 H, OCH3, His), 3.90 [t,
3J = 6.9 Hz, 2 H, C(4)H2, Bu], 4.45–4.55 [m, 1 H, C(a)H, His], 5.94
[d, 3J = 8.4 Hz, 1 H, C(a)NH, His], 6.65–6.7 [2 s, 2 H, C(g)CONH,
Glu and C(5)H, His], 7.42 [s, 1 H, C(2)H, His].
13C NMR (75 MHz, CDCl3): d = 26.6, 28.0, 28.1, 28.2, 28.3, 30.3,
32.9, 38.5, 44.5, 46.5, 52.1, 53.7, 54.4, 59.0, 79.6, 81.4, 116.4,
136.9, 137.6, 155.6, 172.2, 172.8, 174.8.
HRMS (ESI): m/z [M + H]+ calcd for C25H44N5O7: 526.3241; found:
526.3239.
3
3
1 H, C(a)H, His], 3.6 [dd, J = 4.6 Hz, J = 9.44 Hz, 1 H, C(a)H,
Glu], 4.08 [s, 2 H, C(9)H2, Pte], 6.5 [s, 1 H, C(5)H, His], 6.65 [d,
3J = 6.7 Hz, 2 H, C(3¢,5¢)H, Pte], 6.95 [d, 3J = 6.1 Hz, 2 H,
C(2¢,6¢)H, Pte], 7.68 [s, 1 H, C(2)H, His], 7.83 [s, 1 H, C(7)H, Pte).
1H–1H COSY-90 correlations (300 MHz, D2O–D2SO4): d (F2/
F1) = 0.50/0.84 [C(2)H2/C(3)H2, Bu], 0.50/2.19 [C(2)H2/C(1)H2,
Bu], 0.84/3.18 [C(3)H2/C(4)H2, Bu], 1.18/1.34 [C(b)HA/C(b)HB,
Glu], 1.18/1.5 [C(b)HA/C(g)H2, Glu], 1.18/3.57 [C(b)HA/C(a)H,
Glu], 1.34/3.57 [C(b)HB/C(a)H, Glu], 2.4/2.5 [C(b)HA/C(b)HB,
His], 2.4/3.45 [C(b)HA/C(a)H, His], 2.5/3.45 [C(b)HB/C(a)H, His],
6.5/7.7 [C(5)H/C(2)H, His], 6.65/6.95 [C(3¢,5¢)H/C(2¢,6¢)H, Pte).
Na-(tert-Butoxycarbonyl)-Nt-[4-({Oa-tert-butyl-N2-[(dimethyl-
amino)methylene]-10-formylfolyl}amino)butyl]-L-histidine
Methyl Ester (9)
To a soln of 10 (391 mg, 0.90 mmol, 1.0 equiv) in THF (3 mL) was
added DMTMM (274 mg, 0.99 mmol, 1.1 equiv) and 8 (473 mg,
0.99 mmol, 1.1 equiv]. The suspension was stirred at r.t. for 3 h and
then concentrated under vacuum to ~1.5 g. After addition of H2O (2
mL) a precipitate was formed which was separated from the soln by
centrifugation. The precipitate was washed with H2O (0.5 mL), sep-
arated by centrifugation, and dried under vacuum to give 9 (526 mg,
62%) as a yellow foam; purity: 95% (NMR).
13C NMR (75 MHz, D2O–10% D2SO4, TSP-d4): d = 28.1, 28.2,
29.3, 29.6, 35.2, 37.4, 41.6, 52.1, 52.6, 54.8, 55.8, 121.7, 124.0,
130.0, 130.2, 132.4, 138.0, 149.8, 151.8, 152.7, 154.5, 162.8, 168.7,
172.5, 173.1, 178.0.
HRMS (ESI): m/z [M – H]– calcd for C29H34N11O7: 648.2643;
found: 648.2630.
Rhenium Complex 11
The synthesis of 11 was achieved in analogy to the literature proce-
dure.9b Briefly: His-folate
1
(15.0 mg, 23 mmol) and
[ReBr3(CO)3][Et4N]2 (20.0 mg, 26 mmol) were suspended in H2O–
MeOH (1:1, 4 mL) and the pH was adjusted to pH 8 with dil
NaHCO3. The resulting yellow soln was stirred at 50 °C for 1.5 h
after which HPLC indicated complete conversion of His-folate 1.
The mixture was cooled to r.t. and the pH adjusted to pH 2–3 by ad-
dition of 0.1 M HCl. The precipitate was isolated by centrifugation
(10 min, 3500 rpm) and dried under reduced pressure to provide Re
complex 4 as a brown solid (HPLC purity: ~70%). The crude prod-
uct was purified via HPLC (XTerra® column, MSC18, 5mm,
4.6 × 150 mm, Waters; liquid phase: solvent A = 0.1% aq TFA, sol-
vent B = MeCN; 0–15 min 5%→80% B; 15–20 min 95% B):
tR = 12.1 min.
IR (Golden Gate/ATR): 3280, 2935, 1630, 1605, 1540, 1500, 1455,
1420, 1330, 1155, 1115 cm–1.
1H NMR (300 MHz, CDCl3): d = 1.39 (s, 9 H, t-Bu), 1.3–1.45 [m,
2 H, C(3)H2, Bu], 1.46 (s, 9 H, t-Bu), 1.7–1.8 [m, 2 H, C(2)H2, Bu],
1.8–1.9 [m, 1 H, C(b)HA, Glu], 2.1–2.4 [m, 3 H, C(b)HB and
C(g)H2, Glu], 2.95–3.05 [2 dd, 2 H, C(b)H2, His], 3.14 (s, 3 H, CH3,
DMAM), 3.22 (s, 3 H, CH3, DMAM), 3.2–3.3 [m, 2 H, C(4)H2,
Bu], 3.69 (s, 3 H, CH3), 3.8–3.9 [m, 2 H, C(1)H2, Bu], 4.4–4.6 [m,
2 H, C(a)H, His and Glu], 5.29 [s, 2 H, C(9)H2, Pte], 5.60 (d, 1 H,
NH), 5.94 (d, 1 H, NH), 6.70 [s, 1 H, C(5)H, His], 6.95 (s, 1 H, NH),
Synthesis 2009, No. 5, 787–792 © Thieme Stuttgart · New York

792
C. Sparr et al.
PAPER
1H NMR (300 MHz, D2O–NaOD): d = 0.4–0.6 [m, 2 H, C(2)H2,
Bu], 0.85–1.00 [m, 2 H, C(3)H2, Bu], 1.28–1.32 [m, 1 H, C(b)HA,
Glu], 1.25–1.4 (m, 1 H,), 1.50 (t, 3J = 7.1 Hz, 2 H), 2.1–2.3 [m, 5 H,
C(g)H2, C(b)HB, Glu; C(1)H2, Bu], 2.58 [br s, 2 H, C(b)H2, His],
3.1–3.3 [m, 2 H, C(4)H2, Bu], 3.54 [t, 3J = 6.7 Hz, 1 H, C(a)H, His],
4.00–4.09 [m, 1 H, C(a)H, Glu], 4.25 [s, 2 H, C(9)H2, Pte], 6.30 [s,
1 H, C(5)H, His], 6.48 [d, 3J = 7.5 Hz, 2 H, C(2¢,6¢)H, Pte], 7.33 [d,
3J = 7.5 Hz, 2 H, C(3¢,5¢)H, Pte], 7.40 [s, 1 H, C(7)H, Pte], 8.26 [s,
1 H, C(2)H, His).
R.; Sharp, T. L.; Lewis, J. S. Nucl. Med. Biol. 2003, 30, 725.
(b) Mathias, C. J.; Wang, S.; Lee, R. J.; Waters, D. J.; Low,
P. S.; Green, M. A. J. Nucl. Med. 1996, 37, 1003.
(c) Mathias, C. J.; Wang, S.; Low, P. S.; Waters, D. J.;
Green, M. A. J. Nucl. Med. 1999, 26, 23. (d) Mathias, C. J.;
Green, M. A. Nucl. Med. Biol. 1998, 25, 585. (e) Mathias,
C. J.; Green, M. A. J. Nucl. Med. 2000, 41, 1113. (f)Siegel,
B. A.; Dehdashti, F.; Mutch, D. G.; Podoloff, D. A.; Wendt,
R.; Sutton, G. P. J. Nucl. Med. 2003, 44, 700. (g) Guo, W.;
Hinkle, G.; Lee, R. J. Nucl. Med. 1999, 40, 1563.
(h) Reddy, J. A.; Xu, L. C.; Parker, N.; Vetzel, M.; Leamon,
C. P. J. Nucl. Med. 2004, 45, 857.
HRMS (ESI): m/z [M + H]+ calcd for C32H35N11O10Re: 920.2126;
found: 920.2131.
(8) Müller, C.; Schubiger, P. A.; Schibli, R. Nucl. Med. Biol.
99mTc(CO)3 Complex 12
2007, 34, 595.
The radiotracer was prepared according to the literature proce-
dure.9b Briefly: [99mTc(CO)3(OH2)3]+ (100 mL), phosphate buffered
saline (PBS pH 7.4, 350 mL) and 0.001 M 1 in PBS buffer (50 mL)
were placed in a sealed glass vial. After 30 min at 75 °C, the vial
was cooled on ice; yield: 92% (based on HPLC; tR = 12.2 min). The
radioactive precursor [99mTc(CO)3(OH2)3]+ was synthesized accord-
ing to the literature procedure.19
(9) (a) Müller, C.; Schubiger, P. A.; Schibli, R. Eur. J. Nucl.
Med. Mol. Imaging 2006, 33, 1162. (b) Müller, C.; Dumas,
C.; Hoffmann, U.; Schubiger, P. A.; Schibli, R. J.
Organomet. Chem. 2004, 689, 4712. (c) Müller, C.;
Schubiger, P. A.; Schibli, R. Bioconjugate Chem. 2006, 17,
797.
(10) van Staveren, D. R.; Waibel, R.; Mundwiler, S.; Schubiger,
P. A.; Alberto, R. J. Organomet. Chem. 2004, 689, 4803.
(11) (a) Bell, J. R.; Jones, J. H. J. Chem. Soc., Perkin Trans. 1
1974, 2336. (b) Fletcher, A. R.; Jones, J. H.; Ramage, W. I.;
Stachulski, A. V. J. Chem. Soc., Perkin Trans. 1 1979, 2261.
(c) Brown, T.; Jones, J. H.; Wallis, J. D. J. Chem. Soc.,
Perkin Trans. 1 1982, 3045. (d) Chivikas, C. J.; Hodges, J.
C. J. Org. Chem. 1987, 52, 3591. (e) Hodges, J. C.
Synthesis 1987, 20. (f) Nayak, S. K.; Monga, V.; Kaur, N.;
Jain, R. J. Heterocycl. Chem. 2007, 44, 1265. (g) Kaur, N.;
Monga, V.; Jain, R. Tetrahedron Lett. 2004, 45, 6883.
(12) (a) Jones, J. H.; Rathbone, D. L.; Wyatt, P. B. Synthesis
1987, 1110. (b) Aurelio, L.; Brownlee, R. T. C.; Hughes, A.
B. Org. Lett. 2002, 4, 3767.
(13) Yao, L.; Smith, B. T.; Aubé, J. J. Org. Chem. 2004, 69, 1720.
(14) (a) Jain, R.; Cohen, L. A. Tetrahedron 1996, 52, 5363.
(b) Guillen, F.; Bregon, D.; Plaquevent, J. C. Tetrahedron
Lett. 2006, 47, 1245.
(15) Bodanszky, A.; Bodanszky, M.; Chandramouli, N.; Kwei, J.
Z.; Martinez, J.; Tolle, J. C. J. Org. Chem. 1980, 45, 72.
(16) Kunishima, M.; Kawachi, C.; Morita, J.; Terao, K.; Iwasaki,
F.; Tani, S. Tetrahedron 1999, 55, 13159.
Acknowledgment
We thank Judith Stahel, Patrick Züger, Dr. Stefan Höck, and Dr.
Thomas Mindt for technical assistance. The work was financially
supported by Merck Eprova AG, Schaffhausen, Switzerland.
References
(1) (a) Weitmann, S. D.; Lark, R. H.; Coney, L. R.; Fort, D. W.;
Frasca, V.; Zurawski, V. R. Cancer Res. 1991, 52, 3396.
(b) Parker, N.; Turk, M. J.; Westrick, E.; Lewis, J. D.; Low,
P. S.; Leamon, C. P. Anal. Biochem. 2005, 338, 284.
(2) Leamon, C. P.; Reddy, J. A. Adv. Drug Delivery Rev. 2004,
56, 1127.
(3) Li, S.; Deshmukh, H. M.; Huang, L. Pharm. Res. 1998, 15,
1540.
(4) Cho, B. K.; Roy, E. J.; Patrick, T. A.; Kranz, D. M.
Bioconjugate Chem. 1997, 8, 338.
(5) Leamon, C. P.; Pastan, I.; Low, P. S. J. Biol. Chem. 1993,
268, 24847.
(6) Gabizon, A.; Shmeeda, H.; Horowitz, A. T.; Zalipsky, S.
Adv. Drug. Delivery Rev. 2004, 56, 1177.
(7) (a) Mathias, C. J.; Lewis, M. R.; Reichert, D. E.; Laforest,
(17) Taylor, E. C.; Young, W. B. J. Org. Chem. 1995, 60, 7947.
(18) Müller, C.; Forrer, F.; Schibli, R.; Krenning, E. P.; de Jong,
M. J. Nucl. Med. 2008, 49, 310.
(19) Alberto, R.; Ortner, K.; Wheatley, N.; Schibli, R.;
Schubiger, A. P. J. Am. Chem. Soc. 2001, 123, 3135.
Synthesis 2009, No. 5, 787–792 © Thieme Stuttgart · New York