Synthesis of HBED–CC–tris(tert-butyl ester) using a solid phase and a microwave reactor

Article abstract of DOI:10.1016/j.tet.2021.132018

N,N′-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N′-diacetic acid (HBED-CC) belongs to the acyclic, bifunctional complexing compounds used mainly for radiolabeling with gallium-68 (⁶⁸Ga). Due to the high stability of the ⁶⁸Ga³⁺complex, HBED-CC is well known for its rapid and efficient labeling at ambient temperature and the high stability of the complexes in vivo. The HBED-CC chelator in combination with a PSMA (Prostate Specific Membrane Antigen) inhibitor and labeled with isotope of gallium is an important tool for diagnosing the stage of cancer in patients with prostate cancer. Many HBED-CC derivatives have been described in the literature, but one of the most commonly used is 3-(3-{[(2-{[5-(2-tert-butoxycarbonylethyl)-2-hydroxybenzyl]-tert-butoxycarbonylmethylamino}-ethyl)-tert-butoxy-carbonylmethylamino]-methyl}-4-hydroxyphenyl)propionic acid (HBED–CC–tris(tert-butyl ester)). This compound is very expensive and commercially limited. Therefore this work describes an innovative method of synthesis on solid phase using of a microwave reactor. Optimization of the reaction allowed to obtain HBED-CC-tris(tert-butyl ester) with high purity and yield.

Full text of DOI:10.1016/j.tet.2021.132018

Tetrahedron 84 (2021) 132018
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of HBEDeCCetris(tert-butyl ester) using a solid phase and a
microwave reactor
K. Jerzyk*, D. Kludkiewicz*, J. Pijarowska-Kruszyna, A. Jaron, M. Maurin, A. Sikora,
L. Kordowski, P. Garnuszek
National Centre for Nuclear Research, Radioisotope Centre POLATOM, 05-400, Otwock, Poland
a r t i c l e i n f o
a b s t r a c t
N,N0-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-N,N0-diacetic acid (HBED-CC) belongs to
the acyclic, bifunctional complexing compounds used mainly for radiolabeling with gallium-68 (68Ga).
Due to the high stability of the 68Ga3þcomplex, HBED-CC is well known for its rapid and efficient labeling
at ambient temperature and the high stability of the complexes in vivo. The HBED-CC chelator in com-
bination with a PSMA (Prostate Specific Membrane Antigen) inhibitor and labeled with isotope of gallium
is an important tool for diagnosing the stage of cancer in patients with prostate cancer. Many HBED-CC
derivatives have been described in the literature, but one of the most commonly used is 3-(3-{[(2-{[5-(2-
tert-butoxycarbonylethyl)-2-hydroxybenzyl]-tert-butoxycarbonylmethylamino}-ethyl)-tert-butoxy-
carbonylmethylamino]-methyl}-4-hydroxyphenyl)propionic acid (HBEDeCCetris(tert-butyl ester)). This
compound is very expensive and commercially limited. Therefore this work describes an innovative
method of synthesis on solid phase using of a microwave reactor. Optimization of the reaction allowed to
obtain HBED-CC-tris(tert-butyl ester) with high purity and yield.
Article history:
Received 4 November 2020
Received in revised form
29 January 2021
Accepted 6 February 2021
Available online 16 February 2021
Keywords:
HBEDeCCetris(tert-butyl ester)
bifunctional chelator
solid phase synthesis
microwave
© 2021 Elsevier Ltd. All rights reserved.
1. Introduction
5,6-tetrafluoro-phenoxycarbonyl)ethyl]phenyl}methyl)amino]
ethyl}amino]methyl}-4-hydroxyphenyl)propionic acid (HBED-
N,N0-bis(2-hydroxybenzyl)ethylenediamine-N,N0-diacetic acid
(HBED) ligand and its derivatives are very well known substances
frequently used in agricultural research and radiopharmacy [1,2].
Research shows that by modifying the lead structure of HBED,
new molecules with better physical properties (e.g. better water
solubility) were obtained [3]. In literature we can find a lot HBED
analogues such as (3-(3-{[(carboxymethyl)[2-({[5-(2-aminoethyl)
-2- hydroxyphenyl]methyl}(carboxymethyl)amino)ethyl]amino]
methyl}-4-hydroxyphenyl)propionic acid) (HBED-CA), (3-(3-{[
(carboxymethyl){2-[(carboxymethyl){[2-hydroxy-5-(2-thiocyana
toethyl)-phenyl]methyl}amino]ethyl}amino]methyl}-4-hydroxy
phenyl)-pionic acid (HBED-Cl), (({[5-(2-aminoethyl)-2-hydroxy
phenyl]methyl}[2-({[5-(2-aminoethyl)-2-hydroxyphenyl]methyl}
(carboxymethyl)amino)ethyl]amino)acetic acid (HBED-AA), 3-(3-
{[(carboxy-methyl){2-[(carboxymethyl)({2-hydroxy-5-[2-(2,3,
CC-TFP), [({2-hydroxy-5-[2-(2,3,5,6-tetrafluorophenoxycarbonyl)
ethyl]phenyl}-methyl){2-[(carboxymethyl)({2-hydroxy-5-[2-(2,3,
5,6-tetrafluoro- phenoxycarbonyl)ethyl]phenyl}methyl)amino]et
hyl}amino]acetic acid (HBED-CC-TFP2) but the most popular de-
rivative is HBED-CC [3].
N,N0-bis-[2-hydroxy-5-(carboxyethyl)benzyl]ethylenediamine-
N,N0-diacetic acid (HBED-CC) is a bifunctional ligand that forms
complexes with divalent and trivalent metal ions like as Cu2þ [4,5],
Fe3þ and radioisotopes such as 68Ga and 99mTc [6,7]. HBED-CC with
metals creates structures, in which phenolic groups are responsible
for coordinating the central metal ion. Furthermore, nitrogen and
oxygen atoms (three carboxylic groups) are necessary to form
complexes (for example hexadentate complex) [5,8]. HBED-CC is
attached to other chemical molecules and/or antibodies by one of
carboxylic group, which is connected to phenol rings.
HBED-CC is most commonly used as a highly effective chelator
for 68Ga labeling due to the high thermodynamic stability. The la-
beling conditions are mild due to the acyclic structure of HBED-CC,
and the given Ga complex has high kinetic inertness at physiologi-
cal pH which is essential for in vivo applications [9,10]. Last years
showed that [68Ga]Ga-PSMA-HBED-CC ([68Ga]Ga-PSMA-11) can be
* Corresponding authors. National Centre for Nuclear Research, Radioisotope
Centre POLATOM, Andrzeja Sołtana 7 St., 05-400, Otwock, Poland.
E-mail addresses: katarzyna.jerzyk@polatom.pl (K. Jerzyk), dominik.
kludkiewicz@polatom.pl (D. Kludkiewicz).
https://doi.org/10.1016/j.tet.2021.132018
0040-4020/© 2021 Elsevier Ltd. All rights reserved.

K. Jerzyk, D. Kludkiewicz, J. Pijarowska-Kruszyna et al.
Tetrahedron 84 (2021) 132018
considered as the one of standard agents in diagnostic prostate
cancer (PET imaging) [10e12].
diimidazole DBU as base and transesterification methods using
potassium tert-butoxide in THF were tried. The most satisfying
results have been obtained with tert-butyl acetoacetate, sulfuric
acid and dry anhydrous magnesium sulfate (powder). Product 5
was purified by column chromatography (dichloromethane/ethyl
acetate, 10/0.1) and obtained with 23% yield and with purity
exceeding 95% (HPLC-method A).
In two next steps, the formyl group was introduced into the
aromatic rings of compounds 2 and 5 to give products 3 and 6. The
crude products were purified by column chromatography
(dichloromethane) and obtained compound 3 in 70% yield and
purity exceeding 98% (HPLC-method B) and compound 6 in 48%
yield and purity exceeding 97% (HPLC-method A). The 3-(3-formyl-
4-hydroxyphenyl)propanoic acid 4 was prepared by hydrolysis of 3
using 1 M HCl and was obtained in 85.6% yield after purification by
crystallization from acetonitrile and with above 97% purity (HPLC e
method C). Compounds 4 and 6 were prepared from 3-(4-
hydroxyphenyl) propionic acid 1 in overall yield: 46% for 4 and
10% for 6.
The second part of the experiments was carried out in accor-
dance with the previously described procedures but the introduc-
tion of certain modifications allowed to increase the yield of the
HBEDeCCetris(tert-butyl ester) synthesis [13,19e21]. First of all,
the novelty was the application of a solid support synthesis strategy
using 2-chlorotrityl resin. This strategy greatly simplifies the syn-
thesis steps by allowing work at excessive molar ratios and elimi-
nates the need to separate unreacted substrates, reducing the
overall time required for synthesis.
This work presents
a new efficient synthesis method of
HBEDeCCetris(tert-butyl ester), which is the most useful molecule
for coupling with PSMA among the HBED-CC analogues. Our pre-
vious experiments showed that the use of HBEDeCCedi(tert-butyl
ester) for conjugation with PSMA led to the formation of the un-
desirable product as dimer: PSMA2-HBED-CC. On the other hand,
the use of HBEDeCCemonomethyl-di(tert-butyl ester) chelator
was also unfavorable due to the problem with removal of the
methyl group in the final product (PSMA-11).
In the literature there is only one method of synthesis of
HBEDeCCetris(tert-butyl ester) and it is based on a 10 step reaction
starting from 3-(4-hydroxyphenyl) propanoic acid [13]. Other
publications present methods of synthesis analogues of HBED-CC.
One of these method describe synthesis of (HBED-CC)TFP based
on the reaction of HBED-CC with Fe3þ ions and esterification with
2,3,5,6-tetrafluorophenol. The final product as a monoester is
conjugated to e.g. antibody [11]. The next method of synthesis of
HBEDeCCedi(tert-butyl ester) is based on a 5 step reaction starting
from 3-(4-hydroxyphenyl) propanoic acid [10].
The aim of our work was to develop a new way of synthsis
HBEDeCCetri(tert-butyl ester).
In this work, we employed the previously described methods of
synthesis [10,13]. We adopted and modified them in order to
develop a better, more effective synthesis method. We planned the
preparation of HBEDeCCetri(tert-butyl ester) using a solid phase
and a microwave reactor.
Solid phase synthesis started with attachment of 3-(3-formyl-4-
hydroxyphenyl) propanoic acid 4 to the resin and then reaction
with mono-Fmoc ethylenediamine hydrochloride (Fmoc-EDA*HCl)
resulting compound 7*. In the initial experiments, ethylenediamine
and N-Boc-ethylenediamine were used instead of Fmoc-EDA*HCl.
In the case of using ethylenediamine, it turned out that after
cleavage from the resin and confirming the identity of the obtained
compound, the main by-product of the reaction (produced in large
2. Results and discussion
The synthesis of the HBED-CC-tris (tert-butyl ester) chelator
consisted of two main experimental parts, and the full structural
characterization of the intermediates and main product is illus-
trated in Scheme 1 and Scheme 2. The first part of the experiments
were performed basing on previously described procedures (with
some minor modifications) starting with the esterification of 3-(4-
hydroxyphenyl) propionic acid resulting compounds 2 and 5
[10,14,15]. Methyl 3-(4-hydroxyphenyl)propanoate 2 was obtained
with 86% yield after purification by column chromatography
(dichloromethane) and with above 98% purity (HPLC-method A).
Noteworthy, the synthesis of 2 was already described by Zha Z. at al.
[10], but lower yield (83,5%) was reported. Using SOCl2 instead of
BF3$Et2O allowed to increase the yield up to 86%.
amount) was
a dimer with the mass: 412 g/mol (MS:
[MþH]þ ¼ 413 g/mol). Therefore, further experiments using eth-
ylenediamine were abandoned, and a modification involving the
use of Boc-protected ethylenediamine was proposed. Due to the
type of the resin used, the Boc group could not be deprotected in
the traditional way (HCl, dioxane) [22], but only with iodine [23].
Unfortunately, trials to deprotection the Boc group with iodine
failed (as shown by MS results), therefore it was decided to use
ethylenediamine protected with one-sided Fmoc group.
The synthesis of compound 5 was very demanding. During the
development work, few methods of synthesizing tert-butyl esters
were tested [16e18]. For example, syntheses using N,N0-carbonyl
In the next two steps the reduction of compound 7* using
NaBH4 and the deprotection of the Fmoc protecting group from
Scheme 1. Synthesis of 3-(3-formyl-4-hydroxyphenyl)propanoic acid (4) and tert-butyl 3-(3-formyl-4-hydroxyphenyl)propanoate (6).
2

K. Jerzyk, D. Kludkiewicz, J. Pijarowska-Kruszyna et al.
Tetrahedron 84 (2021) 132018
Scheme 2. Synthesis of HBEDeCCetris(tert-butyl ester) (12).
compound 8* using 20% piperidine in dichloromethane/N,N-
dimethylformamide was performed. During these experiments,
there was also a problem with the order of the reduction step and
the deprotection of the Fmoc group. When an attempt was made to
deprotect the Fmoc group from the unreduced imine 7* (using 20%
piperidine in N,N-dimethylformamide/dichloromethane), the
compound was turned out be unstable. When the order of the re-
action was changed (first reduction followed by deprotection of the
Fmoc group), a series of subsequent steps were successfully
performed.
Product 11* was obtained by reacting 9* with tert-butyl 3-(3-
formyl-4-hydroxyphenyl) propanoate and then reduction of com-
pound 10* using NaBH4. The N-alkylation reaction 11* was carried
out using a microwave reactor, which was another innovation in
this synthesis. Final compound 12 was prepared by deprotection
from resin of 12* using mixture of CH3COOH: CF3CH2OH:CH2Cl2
(1:2:7) [24]. HBEDeCCetris(tert-butyl ester) 12 was obtained in 36
% overall yield (from compound 4) after purification by preparative
HPLC and with above 97% purity (HPLC - method D). The yield
achieved is more than that (28%) reported by Makarem at al. [13].
The HPLC chromatogram of HBEDeCCetris(tert-butyl ester) 12 is
shown in Fig. 1. All the described analytical methods (A-F) and
methods of purification of compounds were new and prepared
especially for this study.
To optimize the N-alkylation reaction in a microwave reactor,
several experiments were performed to determine the effect of the
microwave process parameters (time and temperature) on the
chromatographic purity of the obtained HBEDeCCetris(tert-butyl
ester). During each experiment, the temperature (40e150ꢀC) and
time (7e30 min) of the reaction were selected. In all studies, the
same power 60 W was used. The results are presented in Fig. 3.
It was observed that there is an optimal temperature range from
60ꢀC to 120ꢀC, where the purity of raw product was increased
above 70% (HPLC - method D) depending on the heating time.
However, the findings indicate that a reaction time of 30 min and a
microwave heating temperature of 80ꢀC give the product with the
highest chromatographic purity above 73% (HPLC - method D).
Fig. 3 shows that a significant portion of compound 11* was not
reacted at temperatures below 60ꢀC and in less than 30 min. On the
other hand, when the temperature rose above 120ꢀC, and the
heating time was longer than 7 min, there was an increase one of
impurities (base on MS spectrum results is predicted that it might
be HBEDeCCedi(tert-butyl ester), MS: [MþH]þ¼ 645) reducing the
purity of the chelator. The N-alkylation reaction was carried out on
a large scale under the following conditions: time 30 min, tem-
perature 80ꢀC, power 60 W.
The use of a microwave reactor allowed for a significant
reduction in the synthesis time (reduction of the N-alkylation re-
action time from several hours to 30 min) [5,6], and an increase in
the yield and purity of the final product. The undoubted advantages
of solid phase synthesis include: no need to isolate intermediate
products for cleaning, increasing the speed of chemical processes
(which is related to the possibility of using excess substrates and
the ease of washing them from the resin after given reaction
All synthesized compounds were characterized by mass spec-
troscopy or 1H and 13C NMR spectroscopy. Only compounds 7* and
10* could not be separated from the resin for identity confirmation
due to the low stability of substance after deprotection. A typical
mass spectrum confirming the identity of HBEDeCCetris(tert-butyl
ester) 12 is shown in Fig. 2.
3

K. Jerzyk, D. Kludkiewicz, J. Pijarowska-Kruszyna et al.
Tetrahedron 84 (2021) 132018
Fig. 1. HPLC typical analytical chromatogram (method D) of HBEDeCCetris(tert-butyl ester) 12.
Fig. 2. Mass spectrum confirming the identity of HBEDeCCetris(tert-butyl ester) 12.
stages), reducing the formation of impurities. The possibility to
wash out excess of unreacted compound from the resin is partic-
ularly important for the product 6 which may be used in the next
solid phase synthesis.
In order to confirm the usefulness of the synthesized
HBEDeCCetris(tert-butyl ester) chelator, small-scale tests were
performed (see Scheme 3). The experiments consisted of attaching
the final product 12 to PSMA-Ahx, deprotecting from the resin and
purifying by preparative HPLC. The obtained PSMA-11 with a purity
of more than 99% (HPLC - method E) was labeled with 68Ga. The
identity of PSMA-11 was confirmed by comparison with PSMA-11
standard. A typical HPLC chromatogram and mass spectrum for
PSMA-11 are shown in Figs. 4 and 5. The quality control of the
preparation of
[
68Ga]Ga-HBEDeCCePSMA ([68Ga]Ga-PSMA-11)
Fig. 3. Comparison between heating time and reaction temperature vs. the chro-
matographic purity of HBEDeCCetris(tert-butyl ester) 12.
was performed by TLC and HPLC system (method F) and the
radiochemical purity was >97%.
4

K. Jerzyk, D. Kludkiewicz, J. Pijarowska-Kruszyna et al.
Tetrahedron 84 (2021) 132018
Scheme 3. Synthesis of PSMA-11.
Fig. 4. HPLC typical analytical chromatogram (method E) of PSMA-11.
3. Conclusion
4. Experimental section
The synthesis of HBEDeCCetris(tert-butyl ester) using 2-
chlorotrityl chloride resin and microwave oven was successfully
developed. Performing the reaction on a solid resin required no
purification of the intermediates and reduced the formation of
impurities.
The use of a microwave radiation helped to reduce the time of
the N-alkylation reaction e from several hours to 30 min and
achieve better purity of the chelator. HBEDeCCetris(tert-butyl
ester) was obtained with better overall yield (36%) than is described
in the literature and in high chemical purity. The results presented
will be a good starting point for the large-scale synthesis of PSMA-
HBED-CC (PSMA-11) ligand for 68Ga radiolabeling.
4.1. Materials and methods
Materials. All reagents and solvents used were purchased from
Sigma-Aldrich Co. (USA), Avantor Performance Materials Poland
S.A. (Poland), Fluorochem Ltd. (UK) and Iris Biotech GmbH (Ger-
many). 2-chlorotrityl chloride resin (1.60 mmol Cl-/g resin,
100e200 mesh), polystyrene Wang resin (0.59 mmol/g resin,
100e200 mesh) and COMU were purchased from Iris Biotech
GmbH (Germany). In the second part of the synthesis, anhydrous
organic solvents (dichloromethane, N,N-dimethylformamide) were
used, which were prepared by drying over molecular sieves.
Methods. Column chromatography was used for routine
5

K. Jerzyk, D. Kludkiewicz, J. Pijarowska-Kruszyna et al.
Tetrahedron 84 (2021) 132018
Fig. 5. Mass spectrum confirming the identity of PSMA-11.
purification of reaction products using silica gel (Kiesegel, 60 Å,
230e400 mesh, Sigma-Aldrich).
Preparative HPLC. Preparative HPLC was run on Gilson system,
consisting of a Gilson 306 pump, a Gilson 151 UV/VIS detector, a
Gilson 506C controller system, and the Unipoint software. Purifi-
cation of HBEDeCCetris(tert-butyl ester) 12 was performed using
and Varian 500 MHz spectrometers with use of CDCl3 or CD2Cl2 as
solvent. Mass spectra were obtained on LCMS-IT-TOF tandem mass
spectrometer (Shimadzu, Japan).
The radiochemical purity was tested by TLC (ITLC SG; 1 M
Ammonium acetate/MeOH, 50/50). The distribution of radioactivity
on developed TLC strips was acquired by Phosphor Imager System
(PerkinElmer, USA).
Phenomenex Luna C18(2) column (15
mm, 250 ꢁ 30 mm, 100 Å).
The gradient method using the following two mobile phases was
used: A: 0.1% TFA in water; B: 0.1% TFA in acetonitrile. The gradient
profile and timing were as follows: (i) 0e15 min: 60% B, (ii)
15e45 min linear gradient from 60% to 70% B, (iii) 45e55 min: 70%
B, (iv) 55e65 min: linear gradient from 70% to 60%, and finally, (v)
65e75 min: 60% B. A maximum of 150 mg substance dissolved in
acetonitrile/water (60%/40%, 3.5 mL) was loaded onto the column.
Purification of PSMA-11 was performed using Phenomenex
4.3. Chemistry
Methyl 3-(4-hydroxyphenyl)propanoate (2). 3-(4-hydroxyphenyl)
propanoic acid (1, 3.0 g, 18.1 mmol) was dissolved in methanol
(50 mL), and SOCl2 (1.5 mL) was added. The solution was stirred at
room temperature for 2 h. Then, the reaction mixture was
concentrated in vacuo to afford 3.2 g (17.8 mmol) of crude product 2
as oil. The obtained crude product 2 was purified by silica gel col-
umn chromatography (dichloromethane). The process was moni-
tored by TLC on silica gel plates (dichloromethane:ethyl acetate,
4:1, Rf ¼ 0,79) with compound detection accomplished in an iodine
chamber. The eluate was evaporated on a rotary evaporator to give
compound 2 (2.8 g, 15.5 mmol, 86%) as an oil and with purity >98%
(HPLC, method A, UV 220 nm). MS: [MþH]þ¼ 181; 1H NMR
Luna C18(2) column (10
mm, 250 ꢁ 15 mm, 100 Å). A gradient
method using the following two mobile phases was used: A: 0.1%
TFA in water; B: 0.1% TFA in acetonitrile. The gradient profile and
timing were as follows: (i) 0e8 min: 10% B, (ii) 8e45 min linear
gradient from 10% to 30% B, (iii) 45e55 min: linear gradient from
30% to 10%, and finally, (iv) 55e65 min: 10% B. A maximum of 20 mg
substance dissolved in acetonitrile/water (10%/90%, 1 mL) was
loaded onto the column. Each purification process (for compound
12 and PSMA-11) lasted 75 min, flow rate was 10 mL/min and UV
detection was measured at 220 nm.
(500 MHz, CDCl3) d: 2.59e2.62 (t, 2H, CH2); 2.86e2.89 (t, 2H, CH2);
3.67 (s, 3H, OeCH3); 5.11 (s,1H, Ar), 6.73e6.76 (d, 2H, Ar); 7.03e7.05
(d, 2H, Ar).
Methyl 3-(3-formyl-4-hydroxyphenyl)propanoate (3). Methyl 3-
(4-hydroxyphenyl)propanoate (2, 2.8 g, 15.5 mmol) was dissolved
in acetonitrile (75 mL) followed by addition of MgCl2 (2.9 g,
30.5 mmol), paraformaldehyde (3.7 g, 123.2 mmol) and triethyl-
amine (6.2 g, 61.3 mmol). The reaction mixture was heated under
reflux for 8 h, cooled to room temperature, diluted with water
(25 mL) and acidified with 5% HCl (100 mL). Then, the resulting
solution was extracted with diethyl ether (3 ꢁ 50 mL). The com-
bined organic extracts were dried over anhydrous Na2SO4 and
concentrated in vacuo to afford 3.3 g of crude product 3 as yellow
oil. Purification by column chromatography (dichloromethane) on
silica gel and process control by TLC (dichloromethane:ethyl ace-
tate, 9.8:0.2, Rf¼ 0,49) on silica gel plates gave compound 3 (2.2 g,
10.7 mmol, 70%) as colorless oil of >98% purity (HPLC, method B, UV
4.2. Analytical methods
Analytical HPLC was run on Varian ProStar system consisting of
2 pumps working in high pressure setup ProStar type 210, UVeVis
345 detector, digital-analog converter Star 800, rheodyne injector
type 7725i (Cotati, USA) and the Galaxy software. Chemical purity
analyzes of compounds 2, 3, 4, 5 and 6 were carried out using
methods A, B, C. Chemical purity of HBEDeCCetris(tert-butyl ester)
12 was confirmed using method D. Chemical purity analysis of
PSMA-11 was performed using method E. Quality control of [68Ga]
Ga-HBEDeCCePSMA ([68Ga]Ga-PSMA-11) was performed by RP-
HPLC (Shimadzu, Japan) using method F (see Table 1).
1H and 13C NMR spectra were recorded using Bruker 500 MHz
6

K. Jerzyk, D. Kludkiewicz, J. Pijarowska-Kruszyna et al.
Tetrahedron 84 (2021) 132018
Table 1
HPLC methods used to confirm the chemical purity of compounds 2,3,4,5,6,12, PSMA-11 and the radiochemical purity of [68Ga] Ga-HBEDeCCePSMA ([68Ga]Ga-PSMA-11).
Method
A
B
C
D
E
F
Column
Phenomenex Kinetex® EVO C18(2)
Phenomenex Kinetex® EVO C18(2)
Phenomenex Kinetex® C18
ACE 3C18-300
(5
m
m, 250 ꢁ 4.6 mm, 100 Å)
(5
m
m, 150 ꢁ 4.6 mm, 100 Å)
(5
m
m, 150 ꢁ 4.6 mm, 100 Å)
(150 mm ꢁ 3.0 mm)
Mobile phases
% B
A: 0.1% HCOOH in H2O
B: 0.1% HCOOH in ACN
A: 0.1% TFA in H2O
B: 0.1% TFA in ACN
Isocratic
70% B
Gradient
60% B
30% B
0e10 min: from 10% to 50% B;
10e20 min: 50% B;
20e25 min: from 50% to 10% B,
25e30 min: 10% B.
0e25 min: from 15% to 30% B;
25e26 min: from 30% to 15% B
26e30 min: 15% B
0e10 min: from 5% to 40% B
Flow rate
Injection
Detection
1 mL/min
0.6 mL/min
5
mL
20 mL
UV, 220 nm; radiometric
220 nm). MS: [MþH]þ¼ 209; 1H NMR (500 MHz, CD2Cl2)
d
:
extracted with diethyl ether and dried over anhydrous Na2SO4.
Organic phase was evaporated in vacuo to afford about 2.5 g of
crude product 6 as a yellow oil. The obtained crude product 6 was
purified by silica gel column chromatography (dichloromethane).
The process was monitored by TLC on silica gel plates (dichlo-
romethane:ethyl acetate, 9.8:0.2, Rf ¼ 0,74) and compound detec-
tion was accomplished in an iodine chamber. The eluate was
evaporated on a vacuum evaporator to give compound 6 (650 mg,
2.6 mmol, 48%) as a slightly yellow or colorless oil and with purity
>97% (HPLC, method A, UV 220 nm). 1H NMR (500 MHz, CD2Cl2)
2.61e2.64 (t, 2H, CH2); 2.91e2.94 (t, 2H, CH2); 3.64 (s, 3H, OeCH3);
6.90e6.92 (d, 1H, Ar); 7.38e7.42 (m, 2H, Ar); 9.87 (s, 1H, CHO);
10.85 (s, 1H, Ar-OH); 13C NMR (500 MHz, CD2Cl2)
117.8; 132.7; 133.5; 137.6; 197.1.
d: 30.0; 35.8; 51.8;
3-(3-formyl-4-hydroxyphenyl)propanoic acid (4). Methyl 3-(3-
formyl-4-hydroxyphenyl)propanoate (3, 2.0 g, 9.6 mmol) was dis-
solved in 1 M HCl (70 mL). The mixture was heated under reflux for
1 h and then cooled in the refrigerator (2e8ꢀC) for 5 h. After this
time, crystallized solid was filtered and dried under vacuum for 3 h
to afford 1.7 g of crude product. Purification by crystallization from
acetonitrile gave compound 4 (1.6 g, 8.2 mmol, 86%) as a white solid
and with purity >97% (HPLC, method C, UV 220 nm). MS:
d
¼ 1.39 (s, 9H, 3 x CH3); 2.50e2.53 (t, 2H, CH2); 2.87e2.90 (t, 2H,
CH2); 6.89e6.91 (d, 1H, Ar); 7.38e7.41 (dd, 2H, Ar); 9.86 (s, 1H,
CHO); 10.84 (s, 1H, Ar-OH); 13C NMR (500 MHz, CD2Cl2)
: 28.1;
d
[MꢂH]- ¼ 193; 1H NMR (500 MHz, CD2Cl2)
d
: 2.68e2.71 (t, 2H,
30.2; 37.2; 80.6; 117.7; 120.8; 132.9; 133.4; 137.7; 160.3; 172.0;
197.0; 197.1.
CH2); 2.93e2.96 (t, 2H, CH2); 6.91e6.93 (d, 1H, Ar); 7.40e7.43 (m,
2H, Ar); 9.87 (s, 1H, CHO); 10.41 (s, 1H, eCOOH); 10.86 (s, 1H, Ar-
3-(3-formyl-4-hydroxyphenyl) propionic acid coupled on the resin
(4*). 2-chlorotrityl chloride resin (1.0 g, 1.6 mmol) was swelled with
dichloromethane (25 mL) for 0.5 h. After this time, dichloro-
methane was removed from the glass reaction vessel. Then, 3-(3-
formyl-4-hydroxyphenyl) propionic acid (4, 0.8 g, 4.1 mmol) in
dichloromethane (45 mL) and DIPEA (1.0 g, 7.7 mmol) in
dichloromethane (5 mL) were added to the resin. The reaction
mixture was stirred by bubbling argon for 4 h. The reaction solution
was again removed from the glass reaction vessel and the 3-(3-
formyl-4-hydroxyphenyl) propionic acid coupled with resin 4*
was washed in sequence with: dichloromethane (50 mL), N,N-
dimethylformamide (20 mL), mixture of dichloromethane/metha-
nol/DIPEA (8mL:1.5mL:0.5mL) by 10 min and dichloromethane
(50 mL).
OH); 13C NMR (500 MHz, CD2Cl2)
133.5; 137.6; 197.1.
d: 29.7; 35.6; 117.9; 132.2;
Tert-butyl 3-(4-hydroxyphenyl)propanoate (5). 3-(4-hydroxy-
phenyl)propanoic acid (1, 4.5 g, 27.1 mmol), anhydrous sodium
sulfate powder (11.0 g, 77.4 mmol) and tert-butyl acetoacetate
(14.3 g, 15 mL, 90.4 mmol) were added to 50 mL round flask. Then,
concentrated sulfuric acid (0.8 g, 0.45 mL, 8.2 mmol) was added to
mixture and reaction was stirring at room temperature for 30 min.
After this time, the sodium sulfate was filtered out. Filtrate was
extracted with diethyl ether and 1.5% aqueous solution of NaOH.
The organic phase was dried with using anhydrous sodium sulfate
and concentrated in vacuo to afford about 13.0 g of crude product 5
as
a yellow oil. Purification performed by chromatography
(dichloromethane:ethyl acetate, 10:0.1) on silica gel column under
TLC (dichloromethane:ethyl acetate, 10:0.4, Rf ¼ 0,54) process
control gave compound 5 (1.4 g, 6.3 mmol, 23%) as a slightly yellow
or colorless oil and with purity >95% (HPLC, method A, UV 220 nm).
3-[3-({2-[2-(9H-Fluoren-9-yl)acetylamino]ethylimino}methyl)-4-
hydroxyphenyl]propionic acid coupled on the resin (7*). Fmoc-
EDA*HCl (1.0 g, 3.1 mmol) in methanol (20 mL) and DIPEA (0.8 g,
6.2 mmol) in dichloromethane (30 mL) were added to the 4*. The
reaction mixture was stirred by bubbling argon for 4 h. After 2 h,
dichloromethane (30 mL) was added to the reaction mixture. Then,
the reaction solution was removed from the reaction vessel and the
yellow product coupled with resin 7* was washed in sequence with
dichloromethane (35 mL), methanol (15 mL) and dichloromethane
(35 mL).
3-[3-({2-[2-(9H-Fluoren-9-yl)acetylamino]ethylamino}methyl)-
4-hydroxyphenyl]propionic acid coupled on the resin (8*). Mixture of
dichloromethane and methanol (9:1, 40 mL) was added to the 7*.
Then, sodium borohydride (0.3 g, 7.9 mmol) in methanol (10 mL)
was added in portions and the reaction mixture was stirred by
bubbling argon for 2 h. After this time, the reaction solution was
1H NMR (500 MHz, CDCl3)
d:1.41 (s, 9H, 3 x CH3); 2.49e2.52 (d, 2H,
CH2); 2.81e2.83 (d, 2H, CH2); 6.72e6.74 (d, 2H, Ar); 7.04e7.06 (d,
2H, Ar);13C NMR (500 MHz, CDCl3)
129.4; 132.6; 154.0; 172.7.
d: 28.0; 30.2; 37.4; 80.5; 115.2;
Tert-butyl 3-(3-formyl-4-hydroxyphenyl)propanoate (6). MgCl2
(1.0 g, 10.5 mmol), paraformaldehyde (1.3 g, 43.3 mmol) and tri-
ethylamine (2.2 g, 21.7 mmol) were added to solution of tert-butyl
3-(4-hydroxyphenyl)propanoate (5, 1.2 g, 5.4 mmol) in acetonitrile
(40 mL). Mixture was heated under reflux for 1.5 h. Color of reaction
mixture changed from white to yellow. After this time, reaction
mixture was cooled to room temperature, diluted with water
(20 mL) and acidified with 5% HCl (80 mL). Then, the mixture was
7

K. Jerzyk, D. Kludkiewicz, J. Pijarowska-Kruszyna et al.
Tetrahedron 84 (2021) 132018
removed from the reaction vessel and the white compound coupled
with resin 8* was washed in sequence with methanol (40 mL) and
dichloromethane (50 mL). MS: [MþH]þ ¼ 461.
54.2; 54.6; 54.7; 80.9; 85.1; 85.2; 115.1; 116.0; 116.6; 117.0; 117.0;
117.4; 131.9; 132.0; 132.3; 132.5; 132.6; 133.0; 155.0; 155.1; 161.2;
161.5; 166.6; 167.1; 172.8; 176.2;
The identity of the small amount of compounds 8*, 9*, 11* was
confirmed by MS analysis after cleavage from the resin by using a
mixture of CH3COOH:CF3CH2OH:CH2Cl2 (1:2:7, 6 mL, stirring for
1 h).
3-(3-{[(2-aminoethyl)amino]methyl}-4-hydroxyphenyl)propanoic
acid coupled on the resin (9*). Solution of 20% piperidine in
dichloromethane and N,N-dimethylformamide (1:1, 16 mL) was
added to the 8*. The reaction mixture was stirred by using argon for
0.5 h. After this time, solution was removed from the reaction
vessel. 20% piperidine in dichloromethane (16 mL) was added to
the 8* again and the mixture was stirred by bubbling argon for
0.5 h. Then, the solution was removed from the glass vessel. White
compound coupled with the resin 9* was washed in sequence with
N,N-dimethylformamide (40 mL) and dichloromethane (40 mL).
MS: [MþH]þ ¼ 239.
Small Scale Tests - proof of applicability
PSMA-11. GlueCOeLys-Ahx fragment (further referred to as
PSMA-Ahx [PSMA-Aminohexanoic acid]) coupled with the Poly-
styrene Wang resin was swelled in dichloromethane for at least
20 min. At the same time equimolar amounts of HBEDeCCe
tris(tert-butyl ester) 12 and COMU were placed in a separate vessel
and stirred until dissolved in N,N-dimethylformamide [25,26]. Then
N,N-diisopropylethylamine was added to the solution (stirred for
5 min) and dichloromethane (reaction environment). The solution
of activated amino acid prepared in such a way was transferred into
the vessel with swollen resin and stirred overnight. PSMA-11
coupled with the resin was washed: N,N-dimethylformamide, 50%
N,N-dimethylformamide solution in dichloromethane and
dichloromethane and then dried in vacuum.
In a separate vessel, the solution of trifluoroacetic acid with
addition of triisopropylsilane and water was prepared (98:2:2) in
order to detach the product from the resin and to remove the
protective groups. The ready solution was transferred to the vessel
comprising the resin and stirred for 5 h. The product was precipi-
tated with diethyl ether. The precipitate was centrifuged and
washed with diethyl ether and then dissolved in a solution of
acetonitrile:water (2:8) and transferred to a flask. The precipitate
was frozen on dry ice and lyophilized. The obtained crude product
(PSMA-11) was purified by preparative HPLC. The fractions con-
taining the desired product were collected, combined, frozen on
dry ice and lyophilized to afford PSMA-11 as a white solid and with
purity >99% (HPLC, method E, UV 220 nm). MS: [M þ H]þ ¼ 947.4.
3-{3-[(2-{(Z)-[2-hydroxy-5-(2-tertbutoxycarbonylethyl)phenyl]
methylideneamino}ethylamino)methyl]-4-hydroxyphenyl}propionic
acid coupled on the resin (10*). Tert-butyl 3-(3-formyl-4-
hydroxyphenyl) propanoate (6, 1.2 g, 4.8 mmol) was dissolved in
dichloromethane (50 mL), and added to the 9*. The reaction
mixture was stirred by bubbling argon for 4 h. After this time, the
solution was removed from the glass reaction vessel and the yellow
product coupled with resin 10* was washed with dichloromethane
(50 mL).
3-(4-hydroxy-3-{[2-({[2-hydroxy-5-(2-tertbutoxycarbonylethyl)
phenyl]methyl}amino)ethylamino]methyl}phenyl)propionic
acid
coupled with resin (11*). Mixture of dichloromethane and methanol
(9:1, 40 mL) was added to the 10*. Then, sodium borohydride (0.3 g,
7.9 mmol) in methanol (10 mL) was added in portions and reaction
mixture was stirred by bubbling argon for 2 h. After this time, the
reaction solution was removed from the reaction vessel and the
white compound coupled with resin 11* was washed with meth-
anol (40 mL) and dichloromethane (50 mL). MS: [MþH]þ ¼ 473.
3-(3-{[(2-{[5-(2-tert-butoxycarbonylethyl)-2-hydroxybenzyl]-
tert-butoxycarbonylmethylamino}
ethyl)-tert-butoxycarbonyl-
methylamino]-methyl}-4-hydroxyphenyl) propionic acid coupled on
the resin (12*). Tert-butyl bromoacetate (2.5 g, 12.8 mmol) in N,N-
dimethylformamide (25 mL) and DIPEA (3.3 g, 25.6 mmol) in N,N-
dimethylformamide (25 mL) were added to the 11*. The N-alkyl-
ation reaction was carried out in a microwave oven where the
power, temperature and time were appropriately selected after the
optimization of the process. The reaction was finally carried out
under the following conditions: power 60 W, temperature 80ꢀC,
time 30 min, stirred by bubbling argon. Then, resin with compound
12* was filtrated and washed with N,N-dimethylformamide
(40 mL) and dichloromethane (40 mL).
5. Radiochemistry
Gallium-68 was obtained from a 68Ge/68Ga generator (IRE Elit
Belgium). A stock solution of ligand (PSMA-11, 1 mg in 1 mL H2O)
was prepared and used for the radiolabeling studies. 68Ga labeling
was performed in aq. NaOAc buffer (1 mL, 60 mg/mL) by combining
the ligand solution (20
m
L) and 68Ga solution (5 mL, 0.1 M HCl,
370 MBq). The mixture was heated at 95ꢀC for 15 min and the final
pH of this solution was 4e4.5. The radiochemical purity of [68Ga]
Ga-HBEDeCCePSMA ([68Ga]Ga-PSMA-11) was >97% and deter-
mined by TLC and HPLC system (method F).
3-(3-{[(2-{[5-(2-tert-butoxycarbonylethyl)-2-hydroxybenzyl]-
tert-butoxycarbonylmethylamino}-ethyl)-tert-butoxycarbonyl-
methylamino]-methyl}-4-hydroxyphenyl) propionic acid [HBED-CC-
tris (tert-butyl ester)] (12). Mixture of CH3COOH:CF3CH2OH:CH2Cl2
(1:2:7, 60 mL) was added to compound coupled with resin 12*. The
cleavage step was carried out at room temperature, stirring for 1 h
on a shaker. After this time, the resin was filtered off. The filtrate
was then concentrated on a vacuum evaporator and dried under
vacuum for 24 h to afford 610 mg (54%) of crude product 12 as an
orange oil. The obtained crude product 12 was purified by pre-
parative HPLC. The fractions containing the desired product were
collected, combined, frozen on dry ice and lyophilized (24 h) to
afford purified compound 12 (400 mg, 0.57 mmol, 36%) as white
solid and with purity >97% (HPLC, method D, UV 220 nm). MS:
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2021.132018.
References
[M þ H]þ ¼ 701; 1H NMR (500 MHz, CD2Cl2)
d: 1.40 (s, 9H, 3 x CH3);
1.48e1.49 (ss, 18H, 6 x CH3); 2.46e2.49 (t, 2H, CH2); 2.60e2.62 (t,
2H, CH2); 2.78e2.86 (2t, 4H, 2 x CH2); 3.37e3.43 (d, 4H, 2 x
CH2eN); 3.62e3.65 (d, 4H, 2 x CH2); 4.12e4.13 (d, 4H, 2 x CH2eN);
6.80e7.02 (m, 4H, Ar); 7.11e7.12 (s, 2H, Ar); 13C NMR (500 MHz,
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