18F-Labeled Derivatives of Irbesartan for Angiotensin II Receptor PET Imaging

Article abstract of DOI:10.1002/cmdc.201800638

The renin angiotensin aldosterone system (RAAS) is a hormonal cascade involved in the regulation of blood pressure and electrolyte balance, and represents a common target for the treatment of various diseases including hypertension, heart failure, and diabetes. Herein we present a novel ¹⁸F-labeled derivative of the drug irbesartan, one of the most prescribed angiotensin II type 1 receptor (AT₁R) antagonists, for in vivo positron emission tomography (PET). This allows the in vivo measurement of AT₁R expression, and thus the evaluation of functional changes in its expression under pathophysiological conditions. We followed various synthetic approaches optimized for the introduction of fluorine into different positions of the aliphatic side chain of irbesartan. Radioligand binding studies revealed that fluorine atoms at specified positions (α-position (IC₅₀=6.6 nm) and δ-position (IC₅₀=8.5 nm) of the aliphatic side chain) do not alter the binding properties of irbesartan (IC₅₀=1.6 nm). After successful radiolabeling with fluorine-18 in a radiochemical yield of 11 %, we observed high renal uptake in healthy rats and pigs, which could be decreased by pretreatment with the parent compound irbesartan.

Full text of DOI:10.1002/cmdc.201800638

DOI: 10.1002/cmdc.201800638
Full Papers
18F-Labeled Derivatives of Irbesartan for Angiotensin II
Receptor PET Imaging
Matthias Hoffmann,[a] Xinyu Chen,[b] Mitsuru Hirano,[c] Kenji Arimitsu,[d] Hiroyuki Kimura,[d]
Takahiro Higuchi,*[b, c] and Michael Decker*[a]
The renin angiotensin aldosterone system (RAAS) is a hormo-
nal cascade involved in the regulation of blood pressure and
electrolyte balance, and represents a common target for the
treatment of various diseases including hypertension, heart
failure, and diabetes. Herein we present a novel 18F-labeled de-
rivative of the drug irbesartan, one of the most prescribed an-
giotensin II type 1 receptor (AT1R) antagonists, for in vivo posi-
tron emission tomography (PET). This allows the in vivo mea-
surement of AT1R expression, and thus the evaluation of func-
tional changes in its expression under pathophysiological con-
ditions. We followed various synthetic approaches optimized
for the introduction of fluorine into different positions of the
aliphatic side chain of irbesartan. Radioligand binding studies
revealed that fluorine atoms at specified positions (a-position
(IC50 =6.6 nm) and d-position (IC50 =8.5 nm) of the aliphatic
side chain) do not alter the binding properties of irbesartan
(IC50 =1.6 nm). After successful radiolabeling with fluorine-18 in
a radiochemical yield of 11%, we observed high renal uptake
in healthy rats and pigs, which could be decreased by pretreat-
ment with the parent compound irbesartan.
Introduction
Diseases concerning renal functions such as nephropathy, es-
pecially diabetic nephropathy, and renal stenosis, as well as im-
pairments of the cardiovascular system (e.g., hypertonia and
congestive heart failure (CHF)), are highly prevalent with an in-
creasing number of patients in an aging population. With the
exception of cancer and other diseases concerning the cardio-
vascular system or lungs, renal diseases are the primary cause
of death in industrialized countries and are a significant
burden for public health systems and society.[1] Diabetic nephr-
opathy is one of the most common reasons for renal end-
stage disease, as the majority of diabetes mellitus (DM) type 1
and type 2 patients—with increasing numbers of DM type 2
patients—develop severe renal damage without the chance
for a cure if deterioration of renal function is not treated ade-
quately.[2]
The main reason for insufficient treatments can be found in
both a) the lack of knowledge about the molecular pathogene-
sis of nephropathy as well as CHF, and b) a lack of reliable
methods for diagnosis and monitoring of pharmacotherapy.
Thus, therapy can only be adjusted if the given disease has al-
ready caused kidney damage while patients report an aggrava-
tion of symptoms. This makes any adjustments in pharmaco-
therapy inefficient.[3] Therefore, a better understanding of the
pathology, and at best, new methods and clear criteria for di-
agnosis and therapeutic control of both nephropathy and CHF
are ultimate goals to overcome these diseases.
One promising approach to achieve these goals is the inves-
tigation of the role of the renin angiotensin aldosterone
system (RAAS) in in vivo disease models. For decades RAAS
has been the target of many drugs such as inhibitors of angio-
tensin-converting enzyme (ACE) and antagonists of angiotensi-
n II type 1 receptor (AT1R). Several of these drugs have been in-
cluded in the list of essential medicines by the World Health
Organization.[4] RAAS is a hormonal cascade in which the an-
giotensin II (Ang II) octapeptide is produced. In humans, Ang II
binds to two distinct G-protein-coupled receptors (GPCRs): an-
giotensin II type 1 receptor (AT1R) and type 2 receptor (AT2R).
The AT1R is mainly located on the smooth muscle cells of
blood vessels, heart and kidney tissue, where it mediates a va-
riety of effects such as regulation of blood pressure, electrolyte
homeostasis, fluid retention, angiogenesis, and cell prolifera-
tion.[5] Therefore, drugs targeting AT1R are used in the treat-
ment of hypertonia and CHF, but also in diabetic nephropathy,
having proven their reno- and cardioprotective effects in sever-
al clinical trials.[6,7]
[a] M. Hoffmann, Prof. Dr. M. Decker
Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food
Chemistry, Julius Maximilian University Wꢀrzburg, Am Hubland, 97074
Wꢀrzburg (Germany)
E-mail: Michael.Decker@uni-wuerzburg.de
[b] Dr. X. Chen, Prof. Dr. T. Higuchi
Department of Nuclear Medicine and Comprehensive Heart Failure Centre
(CHFC), University Hospital of Wꢀrzburg, Oberdꢀrrbacherstr. 6, 97080 Wꢀrz-
burg (Germany)
E-mail: Higuchi_T@UKW.de
[c] Dr. M. Hirano, Prof. Dr. T. Higuchi
Department of Bio-Medical Imaging, National Cerebral and Cardiovascular
Centre, 5–7-1 Fujishiro-dai, Suita, Osaka 565-8565 (Japan)
[d] Dr. K. Arimitsu, Dr. H. Kimura
Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceuti-
cal University, 5 Nakauchi-Cho, Misasagi, Yamashina-ku, Kyoto 607–8414
(Japan)
The ORCID identification number(s) for the author(s) of this article can
be found under:
https://doi.org/10.1002/cmdc.201800638.
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In addition to the endocrine RAAS described above, there is
also evidence for local (autocrine or paracrine) RAAS in a varie-
ty of tissues such as the heart and kidney.[8,9] In ex vivo experi-
ments, it was shown that the function of local RAAS in the
kidney is upregulated in several pathophysiological conditions.
The exact role of RAAS in pathophysiology is not yet fully un-
derstood.[9,10] In the heart, locally produced Ang II promotes
several effects over the AT1R such as enhanced collagen ex-
pression, alternation of intracellular pH value and development
of maladaptive cardiac hypertrophy during CHF.[11] Other stud-
ies also imply the involvement of RAAS in the development of
cancer, because the activation of AT1R might mediate angio-
genesis and acceleration of cell proliferation.[12] Treatment with
the AT1R antagonist candesartan was recently shown to de-
crease the rate of cell proliferation and fibrosis in gastric
cancer.[13]
tion of the radioisotope, but delivery from central production
facilities. Besides, incorporation of fluorine-18 provides chem-
ists with greater flexibility in compound design as well as al-
lowing higher in vivo metabolic stability, thereby avoiding un-
specific activity of radioactive but non-bioactive metabolites.[15]
Despite the high demand for information on AT1R involvement
and upregulation processes in a diverse range of diseases,
there has been only minimal research on molecular imaging
techniques. Therefore, sartan-based AT1R antagonists are a
promising starting point (Figure 1). These drugs are well estab-
lished in therapy and exhibit desirable properties regarding af-
finity, selectivity, drug safety, and metabolic stability with a low
chance for radioactive metabolites giving unspecific activity. At
this point, only a small number of sartan-based radioligands
have been reported.[17–19] The most developed compound is
[11C]KR31173 (5), which is a methoxy derivative of the non-pep-
tide AT1R antagonist SK-1080 (4).[20] This ligand has been stud-
ied in several animal models of kidney and heart diseases and
has also been studied in the first human trial, which demon-
strated myocardial retention of the PET tracer.[21,22] Neverthe-
less, [11C]KR31173 (5) shows the aforementioned disadvantages
of carbon-11-labeled PET tracers and has high liver uptake.
Irbesartan (6) is one of the most frequently prescribed AT1R
antagonists worldwide.[23] Irbesartan is mainly used in the
treatment of hypertension, but was also used successfully in
the treatment of heart failure and patients suffering from dia-
betic nephropathy in large clinical trials.[6g,7,24] Furthermore, ir-
besartan is a good candidate for a PET radiotracer because of
its simple pharmacokinetics, metabolism, and promising bio-
distribution with comparably good uptake into kidney
tissue.[25]
As it was shown that the majority of Ang II is synthesized lo-
cally from Ang I, the effects of local RAAS must be considered
for an understanding of nephropathy, CHF, and cancer patho-
genesis as well as future developments of improved therapy
protocols.[14] To gather more knowledge about the role of
RAAS and AT1R in a diverse range of diseases, it is necessary to
measure their activation and upregulation processes in vivo,
under both physiological and pathophysiological conditions.
This can be achieved by applying molecular imaging methods
such as positron emission tomography (PET), which allows
highly sensitive monitoring of specific functional changes in
distinct organs in a noninvasive manner. In nuclear medicine,
PET is often preferred over single photon emission computed
tomography (SPECT), as it gives higher count sensitivity and
better spatial and temporal resolution, also allowing dynamic
and quantitative studies. For PET studies, it is necessary to in-
corporate a positron-emitting radionuclide into a molecule
that binds to the desired target with high affinity and selectivi-
ty. Fluorine-18-labeled PET tracers are of increasing importance,
as they exhibit a longer half-life of 110 min over 11C-labeled
tracers (t1/2 =20 min), which has been a major limiting factor
for PET studies. Furthermore, 18F-labeled radiotracers have the
economic advantage of not requiring on-site cyclotron produc-
After investigation of the structure–activity relationships
(SARs) of AT1R antagonists, we decided to introduce fluoride at
two different positions of the aliphatic butyl side chain
(Figure 2). It is plausible that fluorine atoms do not change the
pharmacophoric structures of irbesartan significantly, as fluo-
rine is small and neutral, although it might serve as a hydrogen
bond acceptor.[26]
Figure 1. Overview of described PET tracers for AT1R.[16,17, 18]
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dazolone 16 in good yield. Alkylation and deprotec-
tion of the tetrazole unit gave cold fluoroirbesartan
derivative 7.
For the development of a suitable precursor for
radiolabeling, we established a synthetic route start-
ing from d-valerolactone 18, which was reacted with
benzyl bromide under basic conditions to form
ether 19 (Scheme 2). In a related reaction sequence,
the obtained acid 19 was transformed into amide 20
and cyclized, followed by debenzylation under hy-
drogenation conditions to yield imidazolone 22. Se-
lective alkylation of the imidazolone structure yield-
ed alcohol 23. Unexpectedly, the reaction of the alcohol to
form a ready leaving group (tosylate or mesylate) lead to an in-
tramolecular cyclization to quaternary amine 24, making it im-
possible to synthesize a suitable precursor for radiolabeling.
Use of diethylaminosulfur trifluoride (DAST) as a fluorination
reagent also resulted in cyclized byproduct 24.
Therefore, focus was set on the compound that is fluorinat-
ed on the a-position of the butyl side chain (Scheme 3). As
starting molecule, ethyl 2-hydroxyvalerate 25 was chosen,
which was fluorinated with DAST to yield ethyl 2-fluorovalerate
26 with the fluorine already in the desired position. Subse-
quent hydrolysis of the ester bond, amide coupling, and cycli-
zation gave imidazolone 29 in good yield. Alkylation with the
protected biphenyl tetrazole unit and subsequent deprotection
yielded cold irbesartan derivative 8.
The precursor was synthesized in a similar way (Scheme 4),
starting with protection of the a-position with a benzyl ether.
In a related sequence, the ethyl ester was cleaved, and the
formed acid 33 was coupled to the aminocarboxamide using
HBTU and cyclized afterward under basic conditions. Deprotec-
tion of the benzyl ether was performed under hydrogenation
conditions, allowing selective alkylation to imidazolone 37,
which was activated with tosyl chloride to give the precursor
38. Further experimental details can be found in the Experi-
mental Section below.
Figure 2. Chemical structures of irbesartan and respective radiotracers.
Results and Discussion
Chemistry
For the development of 18F-labeled PET radiotracers, the syn-
theses of both the cold reference compound (containing non-
radioactive fluorine-19 for preliminary in vitro characterization
of the envisaged tracer compound) and a suitable precursor
(bearing a suitable leaving group for rapid and efficient radio-
labeling in a nucleophilic substitution reaction) were ad-
dressed. Despite irbesartan’s well-established synthesis, signifi-
cant changes from the routes used in pharmaceutical manufac-
turing had to be made to incorporate fluorine atoms and leav-
ing groups.[27]
As potential sites for fluorination, we chose the a- and d-po-
sitions of the butyl chain in irbesartan. To incorporate both flu-
orine and a leaving group, first, the respective fluorovaleric
acid derivatives 14 and 27 were synthesized from which the
imidazolone cores were built up.
For fluorination of irbesartan at the terminal d-position of
the butyl side chain, the synthesis started from 5-chlorovaleric
acid 9 (Scheme 1), which was benzyl protected and activated
in a Finkelstein reaction into iodide 11. Silver(I) tosylate was
produced from silver(I) oxide and toluenesulfonic acid under
protection from light, and the formed silver tosylate was react-
ed with iodine compound 11 to allow subsequent fluorination
and debenzylation to 5-flurovaleric acid 14. In an amide cou-
pling its corresponding acid chloride was reacted with 1-ami-
nocyclopentanecarboxamide; subsequent cyclization gave imi-
Scheme 1. Synthesis of cold compound 7: a) BnBr, K2CO3, MeCN, D, 18 h, quant.; b) NaI, acetone, D, 6 h, 65%; c) AgIOTs, MeCN, darkness, RT, 24 h, 55%; d) KF,
K2CO3, 18-crown-6, DMF, D, 18 h, 35%; e) H2, Pd/C, EtOH, RT, 1 h, quant.; f) 1-aminocyclopentanecarboxamide, HBTU, NEt3, DMF, RT, 18 h, 99%; g) KOH, MeOH,
H2O, 608C, 1 h, 60%; h) 5-(4’-(bromomethyl)-[1,1’-biphenyl]-2-yl)-1-trityl-1H-tetrazole, K2CO3, DMF, RT, 24 h, 80%; i) HCl, MeOH, RT, 30 min, 98%.
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Scheme 2. Attempt of synthesis of hot compound 7: a) BnBr, toluene, D, 24 h, 44%; b) 1. (COCl)2, NEt3, DMF, CH2Cl2, RT, 2 h, 2. 1-aminocyclopentanecarboxa-
mide, CH2Cl2, RT, 2 h, 99%; c) KOH, MeOH, H2O, 608C, 1 h, 65%; d) H2, Pd/C, MeOH, 508C, 72 h, 63%; e) 5-(4’-(bromomethyl)-[1,1’-biphenyl]-2-yl)-1-trityl-1H-tet-
razole, K2CO3, DMF, RT, 18 h, 59%; f) TsCl, NEt3, CH2Cl2, 08C, 30 min.
Scheme 3. Synthesis of cold compound 8: a) DAST, CH2Cl2, ꢀ788C!RT, 48 h, 37%; b) NaOH, H2O, MeOH, RT, 5 h, 99%; c) 1-aminocylopentanecarboxamide,
HBTU, NET3, DMF, RT, 24 h, 60%; d) KOH, MeOH, H2O, 608C, 1 h, 67%; e) 5-(4’-(bromomethyl)-[1,1’-biphenyl]-2-yl)-1-trityl-1H-tetrazole, K2CO3, DMF, RT, 18 h,
64%; f) HCl, MeOH, RT, 30 min, 99%.
Scheme 4. Synthesis of precursor 38 and radiolabeling of hot compound 8: a) BnOH, NaH, DMF, RT, 24 h, quant.; b) NaOH, MeOH, H2O, RT, 5 h, 99%; c) 1-ami-
nocyclopentanecarboxamide, HBTU, NEt3, DMF, RT, 24 h, 99%; d) KOH, MeOH, H2O, 608C, 1 h, 38%; e) H2, Pd/C, MeOH, 508C, 24 h, 13%; f) 5-(4’-(bromometh-
yl)-[1,1’-biphenyl]-2-yl)-1-trityl-1H-tetrazole, K2CO3, DMF, RT, 48 h, 72%; g) TsCl, NEt3, CH2Cl2, RT, 72 h, 28%; h) 1. K[18F]F, Kryptofix 2.2.2, K2CO3, MeCN, 1108C,
10 min, 2. HCl (1m), 1108C, 10 min.
In vitro evaluation
duced into a pharmacophoric moiety of irbesartan 6, possibly
acting as a hydrogen bond acceptor and/or changing the
properties of the imidazolone core.[26]
As a first step of the evaluation of the novel tracer candidates
7 and 8, IC50 values were determined in a competitive binding
assay using [125I]Ang II as radioligand and were compared with
those of the endogenous ligand Ang II and their lead structure
irbesartan 6 (Figure 3). This is mandatory, as fluorine is intro-
For these assays, membranes of CHO-K1 cells expressing
hAT1R were used, and the competitive binding of cold com-
pounds 7 and 8 against the radioligand [125I]Ang II was studied.
All compounds show nearly identical binding affinities at
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After semipreparative HPLC, compound [18F]8 was obtained
in a radiochemical yield of 11ꢁ12% (n=5; decay corrected)
and a radiochemical purity of 94ꢁ3% (n=4). In conclusion, a
simple labeling procedure using a one-pot two-step reaction
was developed successfully, and radiolabeled tracer molecule
[18F]8 could be synthesized.
In vivo imaging
Protocols for noninvasive PET imaging and data analysis were
previously established.[22] After preparation of [18F]8 as de-
scribed above and radioactive purity control, anesthetized rats
(Wistar, anesthetized with 2% isoflurane) were injected intrave-
nously with [18F]8 (10 MBq). PET data were acquired and recon-
structed into images of 5-min timeframes. A good uptake of
tracer into kidney tissue was observed, which proves the suita-
bility of [18F]8 for renal imaging (Figure 4A). Selective binding
of tracer was confirmed with pretreatment of irbesartan 6
5 min before tracer injection, as renal retention was significant-
ly decreased with no clear delineation of kidneys 10 min after
tracer administration (Figure 4A).
Figure 3. Determination of IC50 values of compounds 6–8 relative to Ang II
in a competition binding assay with [125I]angiotensin II on hAT1R expressed
on the membrane of CHO-K1 cells. Sigmoidal curves represent binding of
angiotensin II (black circles), irbesartan 6 (blue squares), a-[F]-irbesartan 8
(red cross) and d-[F]-irbesartan 7 (green triangle). Values are the meanꢁSD
of four independent experiments.
hAT1R in the nanomolar range. In the assay used, irbesartan 6
has the lowest IC50 value. Nevertheless, both synthesized radio-
ligand candidates exhibit similarly high binding affinities
(Table 1), proving the successful design of radiotracer candi-
dates.
Table 1. In vitro binding data of compounds to hAT1R.
[a]
Compound
hAT1R IC50 [nm]
pIC50
Ang II
2.2
1.6
8.5
6.6
8.66ꢁ0.06
8.80ꢁ0.08
8.07ꢁ0.13
8.18ꢁ0.04
irbesartan 6
d-[F]-irbesartan 7
a-[F]-irbesartan 8
Figure 4. A) Dynamic kidney uptake of [18F]8 as control (left) and after block-
ing by selective AT1R blocker irbesartan 6 (right) 60 min after tracer injection
with 5 min timeframes. B) Tracer time–activity curves for kidney uptake in
rats are presented as standardized uptake values (SUVs) from 0 to 50 min as
control (red) and after blocking with irbesartan 6 (5 mgkgꢀ1; black). C) Bio-
distribution studies in rats 15 min after tracer injection as control (red, n=3)
and after blocking by selective AT1R blocker irbesartan 6 (black, n=3) in
rats. Uptake of [18F]8 is expressed as SUVꢁSD of three independent experi-
ments.
[a] Values are the meanꢁSEM of four independent experiments.
Radiochemistry
As a leaving group for radiofluorination, a tosyl group was
chosen and incorporated into precursor molecule 38, which
could be substituted with fluorine-18 in the presence of Kryp-
tofix 2.2.2, while potassium salt K[18F]F served as a fluorine
source. After successful isolation of fluorine-18, a solution of
precursor 38 was added, and the mixture was heated at 1108C
for 10 min. The trityl group was subsequently removed by ad-
dition of aqueous HCl (1m) for 10 min at 1108C. The resulting
residue was then purified by semipreparative HPLC. In HPLC,
no radioactive side products were observed.
Tracer selectivity for AT1R was further confirmed with biodis-
tribution studies (Figure 4C). These data reveal that tracer
[18F]8 is taken up into all relevant tissues expressing AT1R such
as heart, kidney, and lung, while tracer uptake can be de-
creased significantly by blocking with non-radioactive drug ir-
besartan 6 5 min before tracer injection. Notably, almost no
uptake into other organs such as muscle was detected. Meta-
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bolic degradation may be the cause of bone accumulation of
18F-fluoride, but there was minimal bone uptake at current
imaging studies, indicating high metabolic stability of the
tracer during the timeframe of the PET study. However, de-
tailed metabolites were not examined in our study, so this
issue still needs to be addressed in further in vivo studies.
In additional studies evaluating the potential use of [18F]8
for imaging of kidney tissue, pigs (Gçttingen, anesthetized
with 3–5% seboflurane) were injected intravenously with
[18F]8. PET images of kidneys at 30–45 min (Figure 5) show
high tracer uptake (Figure 5, top) as there is a high density of
AT1R present. Pretreatment with 2 mgkgꢀ1 of irbesartan 10 min
before injection of tracer decreased this effect, proving specific
binding (Figure 5, bottom). These results are in accordance
with the results obtained from the rat studies described above.
icantly by 5 mgkgꢀ1 of irbesartan 6 5 min before injection of
tracer, confirming specific binding.
In additional PET experiments in pigs, the results obtained
from rats were reconfirmed. Tracer [18F]8 also showed clear
kidney uptake in this animal model.
Besides its application in renal imaging, compound [18F]8
may be further investigated regarding its use in the diagnosis
of CHF and cancer overexpressing AT1R, and might be a valua-
ble tool to understand the involvement of the RAAS in patho-
genesis.[28]
Although PET experiments require injections of a small quan-
tity of [18F]8 which are unlikely to provoke a pharmacological
effect, the tracer needs further evaluation, including a safety
assessment. Both the long half-life for the excretion of irbesar-
tan (11–15 h in a healthy human)[25a,29] and the half-life of the
radionuclide (approximately 2 h), might raise concerns regard-
ing radiation protection, especially when human studies are
envisaged. Effective dose must be calculated according to the
ICRP publications and evaluated concerning government
guidelines. As data implies high liver exposure to radiation,
this might limit the applicability of [18F]8 for broad use.
Experimental Section
General chemistry
Figure 5. Kidney uptake of [18F]8 as control (top) and after blocking by selec-
tive AT1R blocker irbesartan 6 (bottom) with time frames of 30 to 45 min
after tracer administration. Injection of 2 mgkgꢀ1 nonradioactive receptor
blocker 6 10 min before injection of tracer decreases the uptake into kidney
tissue.
Common reagents and solvents were purchased from commercial
suppliers and were used without further purification. Reaction
progress was monitored by analytical thin-layer chromatography
(TLC) using precoated silica gel G/UV254 plates (Macherey–Nagel
GmbH & Co. KG, Dꢁren, Germany), and spots were detected under
UV light (l=254 nm), staining with iodine, KMnO4 stain (2 g
KMnO4, 15 g K2CO3, 200 mL H2O), or bromocresole green stain.
NMR spectra were collected on a Bruker AV-400 NMR spectrometer
(Bruker, Karlsruhe, Germany) in CDCl3, [D6]DMSO, CD3OD, or
[D6]acetone. Chemical shifts are expressed in ppm relative to
CHCl3/DMSO/MeOH/acetone (d=7.26/2.50/3.31/2.05 and 77.16/
39.52/49.00/29.84 ppm for 1H and 13C NMR, respectively). The
purity of compounds was evaluated by analytical HPLC using a
system from Shimadzu equipped with a DGU-20A3R controller,
LC20AB liquid chromatograph, and an SPD-20A UV/Vis detector.
Stationary phase was a Synergi 4 mm fusion-RP column (150ꢂ
4.6 mm; Phenomenex, Aschaffenburg, Germany). As mobile phase,
H2O+0.1% formic acid (solvent A) and MeOH+0.1% formic acid
(solvent B) were used at a flow rate of 1 mLminꢀ1 (B: 5!90% from
0 to 8 min; 90% from 8 to 13 min; 90!5% from 13 to 15 min; 5%
from 15 to 18 min). ESI mass spectra were measured with a Shi-
madzu LCMS-2020 instrument.
Conclusions
In this study, we show that it is possible to introduce a fluorine
atom at two different positions of the lipophilic side chain in ir-
besartan without altering affinity toward AT1R, with IC50 values
of 6.6 nm for 8 and 8.5 nm for 7. This is of particular relevance
for future PET tracer design for the majority of structurally dif-
ferent sartans, because all commercialized AT1R antagonists
such as irbesartan, candesartan, losartan, valsartan, and azilsar-
tan possess an aliphatic side chain as one of the essential phar-
macophoric entities.
The successful synthesis of the cold reference compounds
was followed by radiolabeling. Unfortunately, 18F labeling at
the d-position of the aliphatic side chain was not achieved, be-
cause a suitable precursor could not be synthesized. Upon acti-
vation of the OH group of 23 into an appropriate leaving
group, an unexpected cyclization was observed, rendering 18F
labeling at the d-position impossible. Therefore, the focus was
put on compound 8, which could be radiolabeled with K18F/
Kryptofix 2.2.2 starting from precursor compound 38 in suffi-
cient yield and purity in a rapid procedure.
Benzyl 5-chloropentanoate 10: 5-Chlorovaleric acid (1.85 g
13.55 mmol) was dissolved in MeCN (20 mL), and Na2CO3 (1.723 g,
16.26 mmol) and BnBr (1.61 mL, 2.318 g, 13.55 mmol) were added.
The resulting mixture was heated at reflux for 24 h under an at-
mosphere of argon. Then, the mixture was allowed to cool to
room temperature and was diluted with H2O (100 mL). The aque-
ous layer was extracted with EtOAc (3ꢂ100 mL) and the combined
organic layer was washed with H2O (100 mL), saturated aqueous
NaHCO3 (100 mL) and brine (100 mL), dried over Na2SO4, filtered,
and evaporated to dryness under reduced pressure. Benzyl 5-chlor-
opentanoate 10 (3.07 g, 13.55 mmol, quant.) was obtained as a col-
In PET studies with healthy rats, clear uptake into the kidney
was observed after administration of [18F]8 both in dynamic
PET imaging of kidney as well as biodistribution studies,
whereas the contrast for heart imaging was insufficient due to
high liver uptake of the tracer. The signal was decreased signif-
1
orless oil. H NMR (CDCl3): d=7.31–7.20 (m, 5H), 5.03 (s, 2H), 3.49–
&
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3.39 (m, 2H), 2.34–2.23 (m, 2H), 1.76–1.65 ppm (m, 4H); 13C NMR
(CDCl3): d=172.94, 136.02, 128.61, 128.28, 128.25, 66.27, 44.45,
33.42, 31.84, 22.27 ppm.
was stirred for 24 h at room temperature. Then, H2O (100 mL) was
added, and the mixture was extracted with EtOAc (3ꢂ50 mL). The
combined organic layer was washed with H2O, saturated aqueous
NaHCO3 (100 mL) and brine (2ꢂ50 mL), dried over Na2SO4, filtered,
and evaporated to dryness in vacuo. The residue was purified by
flash column chromatography (CH2Cl2/MeOH=98:2; Rf =0.2) to
Benzyl 5-iodopentanoate 11: Benzyl 5-chloropentanoate 10 (1.8 g,
7.94 mmol) was dissolved in acetone (30 mL) and NaI (1.49 g,
9,93 mmol) was added. The resulting solution was heated at reflux
for 7 h. Then, H2O (100 mL) was added, and the aqueous layer was
extracted with Et2O (3ꢂ100 mL). The combined organic layer was
washed with H2O (100 mL) and brine (100 mL), dried over Na2SO4,
filtered, and evaporated to dryness under reduced pressure to
yield benzyl 5-iodopentanoate 11 (1.74 g, 5.48 mmol, 69%) as a
colorless oil. 1H NMR (CDCl3): d=7.38–7.34 (m, 5H), 5.12 (s, 2H),
3.18 (t, 2H, J=3.18 Hz), 2.39 (t, 2H, J=2.39 Hz), 1.88–1.74 ppm (m,
4H); 13C NMR (CDCl3): d=173.04, 136.07, 128.73, 128.41, 128.37,
66.43, 33.23, 32.84, 25.92, 5.85 ppm.
yield
1-(5-fluoropentanamido)cyclopentane-1-carboxamide
15
1
(668 mg, 2.90 mmol, 99%) as a colorless solid. H NMR (CDCl3): d=
4.76 (t, J=5.7 Hz, 1H), 4.61 (t, J=5.7 Hz, 1H), 2.43–2.34 (m, 2H),
2.30–2.22 (m, 2H), 2.07–1.99 (m, 2H), 1.94–1.73 ppm (m, 8H);
13C NMR (CDCl3): d=175.57, 170.07, 82.94, 69.19, 34.94, 34.64,
29.77, 29.62, 23.00, 21.95 ppm; MS (ESI): 231.05 [M+H]+.
2-(4-Fluorobutyl)-1,3-diazaspiro[4.4]non-1-en-4-one 16: 1-(5-Fluo-
ropentanamido)cyclopentane-1-carboxamide
15
(300 mg,
1.30 mmol) was dissolved in MeOH (10 mL) and a 10m solution of
KOH (10 mL) was added carefully. The mixture was heated at 608C
for 1 h. Then, the mixture was allowed to reach room temperature
and it was neutralized with 1m HCl. The aqueous layer was extract-
ed with EtOAc (3ꢂ100 mL) and the combined organic layer was
washed with H2O (100 mL), saturated aqueous NaHCO3 (100 mL)
and brine (100 mL), dried over Na2SO4, filtered, and evaporated to
dryness in vacuo. The residue was purified by flash column chro-
matography (CH2Cl2/MeOH=98:2; Rf =0.3) to yield 2-(4-fluorobu-
tyl)-1,3-diazaspiro[4.4]non-1-en-4-one 16 (180 mg, 0.85 mmol, 65%)
Benzyl 5-(tosyloxy)pentanoate 12: p-Toluenesulfonic acid mono-
hydrate (1 g, 5.26 mmol) was dissolved in MeCN and silver(I) oxide
(731 mg, 3.15 mmol) was added. The resulting mixture was stirred
for 72 h at room temperature protected from light with aluminum
foil. Then, the resulting mixture was filtered, and benzyl 5-iodopen-
tanoate 11 (1.27 g, 4 mmol) was added to the filtrate. The resulting
solution was stirred for 24 h at room temperature protected from
light. Then, the solid was filtered off, and the filtrate was evaporat-
ed to dryness under reduced pressure. The residue was purified by
flash column chromatography (petroleum ether/EtOAc=3:1; Rf =
0.29) to yield benzyl 5-(tosyloxy)pentanoate 12 (551 mg,
1.52 mmol, 38%) as a colorless oil. 1H NMR (CDCl3): d=7.79–7.77
(m, 2H), 7.35–7.33 (m, 2H), 5.03 (s, 2H), 2.44 (s, 3H), 2.33–2.30 (m,
2H), 1–71-1.65 ppm (m, 4H); 13C NMR (101 MHz, CDCl3): d=178.82,
69.83, 33.04, 28.11, 21.62, 20.61 ppm.
1
as a yellow oil. H NMR (CDCl3): d=5.76 (s, 1H), 4.55 (t, J=5.7 Hz,
1H), 4.41 (t, J=5.7 Hz, 1H), 2.46–2.35 (m, 2H), 2.32–2.23 (m, 2H),
2.10–2.00 (m, 2H), 1.94–1.75 ppm (m, 8H); 13C NMR (CDCl3): d=
172.46, 120.77, 84.96, 83.28, 55.19, 39.16, 35.64, 29.82, 29.62, 23.10,
21.59 ppm; MS (ESI): 213.05 [M+H]+.
2-(4-Fluorobutyl)-3-((2’-(1-trityl-1H-tetrazol-5-yl)-[1,1’-biphenyl]-
4-yl)methyl)-1,3-diazaspiro[4.4]non-1-en-4-one 17: 2-(4-Fluorobu-
tyl)-1,3-diazaspiro[4.4]non-1-en-4-one 16 (160 mg, 0.75 mmol) was
dissolved in dry DMF (5 mL) and K2CO3 (207 mg, 1.50 mmol) and 5-
(4’-(bromomethyl)-[1,1’-biphenyl]-2-yl)-1-trityl-1H-tetrazole (836 mg,
1.50 mmol) were added. The reaction mixture was stirred for 24 h
at room temperature. Afterward, the reaction mixture was diluted
with H2O (50 mL) and it was extracted with EtOAc (3ꢂ50 mL). The
combined organic layers were washed with H2O (100 mL), saturat-
ed aqueous NaHCO3 (100 mL) and brine (100 mL), dried over
Na2SO4, filtered, and evaporated to dryness in vacuo. The residue
was purified by flash column chromatography (petroleum ether/
EtOAc=6:1; Rf =0.2) to yield 2-(4-fluorobutyl)-3-((2’-(1-trityl-1H-tet-
razol-5-yl)-[1,1’-biphenyl]-4-yl)methyl)-1,3-diazaspiro[4.4]non-1-en-4-
Benzyl 5-fluoropentanoate 13: 5-(Tosyloxy)pentanoate 12
(500 mg, 1.38 mmol), potassium fluoride (84 mg, 1.45 mmol,
1.05 equiv) and 18-crown-6 (382 mg, 1.45 mmol, 1.05 equiv) was
dissolved in dry DMF (10 mL). Potassium fluoride was stored in a
desiccator (CaCl2) prior to use. The clear solution was stirred over-
night at 1158C. Then, the reaction mixture was allowed to cool to
room temperature and was diluted with H2O (50 mL). The aqueous
layer was extracted with EtOAc (3ꢂ50 mL), the combined organic
phases were washed with H2O (100 mL) and brine (100 mL), dried
over sodium sulfate and concentrated. The crude product was pu-
rified by flash column chromatography (petroleum ether/EtOAc
10:1). The fluoride compound 13 was obtained as a colorless oil
1
(102 mg, 0.49 mmol, 35%). H NMR (CDCl3): d=7.37–7.34 (m, 5H),
one 17 (413 mg, 0.6 mmol, 80%) as
a
white solid. 1H NMR
5.12 (s, 2H), 4.52–4.37 (dt, 2H), 2.44–2.40 (t, 2H), 1.81–1.70 ppm
(m, 4H); 13C NMR (CDCl3): d=173.21, 136.13, 128.71, 128.38,
128.34, 84.52, 82.88, 66.37, 33.86, 29.98, 29.79, 21.00, 20.95 ppm.
([D6]DMSO): d=7.82 (dd, J=7.6, 1.2 Hz, 1H), 7.67 (td, J=7.5,
1.5 Hz, 1H), 7.59 (td, J=7.5, 1.4 Hz, 1H), 7.46 (dd, J=7.6, 1.1 Hz,
1H), 7.44–7.30 (m, 9H), 7.09 (d, J=8.3 Hz, 2H), 7.04–6.98 (m, 2H),
6.91–6.83 (m, 6H), 4.88 (d, J=16.4 Hz, 1H), 4.77 (d, J=16.4 Hz, 1H),
4.73 (t, 1H), 4.61 (t, 1H), 3.32–3.27 (m, 2H), 2.29–2.18 (m, 2H),
1.99–1.93 (m, 6H), 1.75–1.68 (m, 2H), 1.55–1.46 (m, 2H), 1.39–
1.27 ppm (m, 2H); 13C NMR ([D6]DMSO): d=186.17, 163.99, 162.98,
141.55, 130.00, 129.65, 129.63, 128.76, 128.31, 126.56, 123.02,
118.10, 117.03, 76.77, 60.19, 58.69, 40.63, 39.16, 35.64, 29.82, 29.62,
23.10, 21.59 ppm; MS (ESI): 689.20 [M+H]+.
5-Fluorovaleric acid 14: Benzyl 5-fluoropentanoate 13 (100 mg,
0.49 mmol) was dissolved in MeOH and palladium on charcoal
(10%, 15 mg) was added. The atmosphere was replaced with hy-
drogen and the mixture was stirred for 1 h at room temperature.
The catalyst was filtered off over Celite and the filtrate was concen-
trated to give 5-fluoropentanoic acid 14 (58 mg, 99.6%) as a color-
less oil. 1H NMR (CDCl3): d=4.50 (t, J=5.59 Hz, 1H), 4.40 (t, J=
5.79 Hz, 1H), 2.45–2.42 (t, 2H), 1.81–1.76 ppm (4H); 13C NMR
(CDCl3): d=179.49, 84.35, 82.71, 33.42, 29.75, 29.55, 20.60,
20.55 ppm.
3-((2’-(1H-Tetrazol-5-yl)-[1,1’-biphenyl]-4-yl)methyl)-2-(4-fluoro-
butyl)-1,3-diazaspiro[4.4]non-1-en-4-one 7: 2-(4-Fluorobutyl)-3-
((2’-(1-trityl-1H-tetrazol-5-yl)-[1,1’-biphenyl]-4-yl)methyl)-1,3-diazas-
piro[4.4]non-1-en-4-one 17 (380 mg, 0.55 mmol) was dissolved in
MeOH (10 mL) and HCl (1.25m solution in MeOH, 2 mL, 2.5 mmol)
was added. The solution was stirred for 30 min at room tempera-
ture. The solvent was evaporated, and the residue was purified by
flash column chromatography (CH2Cl2/MeOH=98:2, Rf =0.2) to
1-(5-Fluoropentanamido)cyclopentane-1-carboxamide 15: 1-Flu-
oropentanoic acid 14 (350 mg, 2.91 mmol) was dissolved in dry
DMF (5 mL) and NEt3 (486 mL, 353 mg, 3.49 mmol) was added fol-
lowed by the addition of 1-aminocyclopentanecarboxamide
(373 mg, 2.91 mmol) and HBTU (1324 mg, 3.49 mmol). The mixture
ChemMedChem 2018, 13, 1 – 13
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yield 3-((2’-(1H-tetrazol-5-yl)-[1,1’-biphenyl]-4-yl)methyl)-2-(4-fluoro-
butyl)-1,3-diazaspiro[4.4]non-1-en-4-one (241 mg, 0.54 mmol,
1.66 mmol) was dissolved in dry MeOH (20 mL) and Pd/C (50 mg;
10%) was added. Then, the atmosphere was evaporated three
times and was replaced with hydrogen. The reaction mixture was
heated at 508C for 72 h. Afterwards, the catalyst was filtered off
with Celite and the solvent was evaporated in vacuo. The residue
was purified by flash column chromatography (CH2Cl2/MeOH=
95:5, Rf =0.25) to yield 2-(4-hydroxybutyl)-1,3-diazaspiro[4.4]non-1-
7
1
98%) as a white solid. H NMR ([D6]DMSO): d=7.77–7.64 (m, 2H),
7.49–7.33 (m, 2H), 7.09–6.99 (m, 4H), 4.88 (d, J=16.4 Hz, 1H), 4.82
(d, J=16.4 Hz, 1H), 4.77 (t, 1H), 4.67 (t, 1H), 3.33–3.27 (m, 2H),
2.32–2.26 (m, 2H), 1.99–1.90 (m, 6H), 1.70–1.64 (m, 2H), 1.55–1.46
(m, 2H), 1.39–1.27 ppm (m, 2H); 13C NMR ([D6]DMSO): d=185.97,
189.85, 141.57, 138.00, 137.65, 131.33, 131.06, 130.75, 128.31,
127.89, 126.21, 84.96, 83.28, 76.76, 58.69, 42.73, 39.66, 29.86, 29.68,
23.19, 21.59 ppm; MS (ESI): 447.15 [M+H]+.
1
en-4-one 22 (220 mg, 1.05 mmol, 63%) as a colorless oil. H NMR
([D6]DMSO): d=10.55 (s, 1H), 4.40 (t, J=5.0 Hz, 1H), 3.45–3.35 (m,
2H), 2.30 (dd, J=14.8, 7.7 Hz, 2H), 1.76 (dd, J=15.6, 8.5 Hz, 6H),
1.59 (d, J=5.4 Hz, 4H), 1.43 ppm (dt, J=13.3, 6.5 Hz, 2H); MS (ESI):
211.05 [M+H]+.
5-(Benzyloxy)pentanoic acid 19: d-Valerolactone 18 (2.68 g,
26.8 mmol) was dissolved in toluene (50 mL) and BnBr (3.82 mL,
5.5 g, 32.1 mmol) and KOH (7.5 g, 268 mmol) were added. The sus-
pension was heated at reflux for 24 h. Then, the mixture was al-
lowed to cool to room temperature und H2O (300 mL) was added
carefully. The aqueous layer was washed with EtOAc (3ꢂ100 mL)
and was acidified with HCl. The resulting mixture was extracted
with EtOAc (3ꢂ200 mL), the combined organic layer was washed
with H2O (300 mL) and brine (300 mL), dried over Na2SO4, filtered,
and evaporated to dryness to afford 5-(benzyloxy)pentanoic acid
2-(4-Hydroxybutyl)-3-((2’-(1-trityl-1H-tetrazol-5-yl)-[1,1’-biphen-
yl]-4-yl)methyl)-1,3-diazaspiro[4.4]non-1-en-4-one 23: 2-(4-Hy-
droxybutyl)-1,3-diazaspiro[4.4]non-1-en-4-one 22 (210 mg, 1 mmol)
was dissolved in dry DMF (10 mL) and K2CO3 (166 mg, 1.2 mmol)
and
5-(4’-(bromomethyl)-[1,1’-biphenyl]-2-yl)-1-trityl-1H-tetrazole
(400 mg, 0.72 mmol) were added. The reaction mixture was stirred
for 18 h at room temperature. Afterward, the reaction mixture was
diluted with H2O (100 mL) and it was extracted with EtOAc (3ꢂ
70 mL). The combined organic layers were washed with H2O
(100 mL), saturated aqueous NaHCO3 (100 mL) and brine (100 mL),
dried over Na2SO4, filtered, and evaporated to dryness in vacuo.
The residue was purified by flash column chromatography (CH2Cl2/
MeOH=98:2; Rf =0.35) to yield 2-(4-hydroxybutyl)-3-((2’-(1-trityl-
1H-tetrazol-5-yl)-[1,1’-biphenyl]-4-yl)methyl)-1,3-diazaspiro[4.4]non-
1
19 (2.7 g, 11,9 mmol, 44%) as a colorless oil. H NMR (CDCl3): d=
7.36–7.27 (m, 5H), 4.50 (s, 2H), 3.50 (t, J=6.1 Hz, 2H), 2.43–2.35 (m,
2H), 1.81–1.62 ppm (m, 4H); 13C NMR (CDCl3): d=179.71, 138.33,
128.36, 127.62, 127.55, 72.88, 69.69, 33.69, 28.97, 21.46 ppm.
1-(5-(Benzyloxy)pentanamido)cyclopentane-1-carboxamide 20:
5-(Benzyloxy)pentanoic acid 19 (500 mg, 2.4 mmol) was dissolved
in dry CH2Cl2 (10 mL), and the solution was cooled to 08C. Then,
NEt3 (0.5 mL) and two drops of DMF were added, followed by a
portion-wise addition of oxalyl chloride (247 mL, 2.88 mmol). The
solution was stirred for 2 h at room temperature. Then, the pre-
pared solution was dropped into a solution of 1-aminocyclopenta-
necaroxamide (295 mg, 2.3 mmol) in CH2Cl2 (5 mL) at 08C. The mix-
ture was stirred for 2 h at room temperature. Then, H2O (100 mL)
was added, and the mixture was extracted with EtOAc (3ꢂ50 mL).
The combined organic layer was washed with H2O (100 mL), satu-
rated aqueous NaHCO3 (100 mL) and brine (100 mL), dried over
Na2SO4, filtered, and evaporated to dryness in vacuo. 1-(5-(Benzy-
1
1-en-4-one 23 (290 mg, 0.42 mmol, 59%) as a white solid. H NMR
([D6]DMSO): d=7.81 (dd, J=7.6, 1.2 Hz, 1H), 7.62 (td, J=7.5,
1.5 Hz, 1H), 7.55 (td, J=7.5, 1.4 Hz, 1H), 7.46 (dd, J=7.6, 1.1 Hz,
1H), 7.42–7.31 (m, 9H), 7.08 (d, J=8.3 Hz, 2H), 7.05–7.00 (m, 2H),
6.91–6.83 (m, 6H), 4.63 (s, 2H), 4.38–4.29 (m, 1H), 3.32–3.27 (m,
2H), 2.29–2.18 (m, 2H), 1.89–1.80 (m, 6H), 1.67 (dd, J=7.8, 3.5 Hz,
2H), 1.55–1.46 (m, 2H), 1.39–1.27 ppm (m, 2H); 13C NMR
([D6]DMSO): d=186.09, 163.78, 162.76, 141.26, 130.02, 129.79,
129.40, 128.76, 128.31, 126.56, 123.02, 118.10, 117.03, 76.77, 60.71,
58.69, 40.63, 40.42, 40.21, 40.00, 39.79, 39.58, 39.37, 37.26, 36.23,
32.24, 31.22, 28.04, 26.01, 21.67 ppm; MS (ESI): 867.15 [M+H]+.
loxy)pentanamido)cyclopentane-1-carboxamide
2.28 mmol, 99%) was obtained as a white solid and directly used
in the next step. MS (ESI): 319.10 [M+H]+.
20
(726 mg,
2’-Oxo-1’-((2’-(1-trityl-1H-tetrazol-5-yl)-[1,1’-biphenyl]-4-yl)meth-
yl)-1’, 2’,5’,6’,7’,8’-hexahydrospiro[cyclopentane-1,3’-imidazo[1,2-
a]pyridin]-4’-ium tosylate 24: 2-(4-Hydroxybutyl)-3-((2’-(1-trityl-1H-
tetrazol-5-yl)-[1,1’-biphenyl]-4-yl)methyl)-1,3-diazaspiro[4.4]non-1-
en-4-one 23 (200 mg, 0.29 mmol) was dissolved in dry CH2Cl2
(10 mL), followed by the addition of NEt3 (44.6 mL,32.4 mg,
0.32 mmol, 1.1 equiv) and p-toluene sulfonic acid chloride (61 mg,
0.32 mmol, 1.1 equiv) at 08C. The mixture was stirred at 08C for
30 min. Then, the solvent was evaporated under reduced pressure
to yield crude title compound 24 (200 mg, quant.) as a colorless
solid. Use of mesyl chloride instead of tosyl chloride yielded the
same cyclization result. TLC: CH2Cl2/MeOH=9:1, Rf =0; MS (ESI):
2-(4-(Benzyloxy)butyl)-1,3-diazaspiro[4.4]non-2-en-4-one 21: 1-
(5-(Benzyloxy)pentanamido)cyclopentane-1-carboxamide
20
(900 mg, 2.83 mmol) was dissolved in MeOH (10 mL) and a 10m
solution of KOH (10 mL) was added carefully. The mixture was
heated at 608C for 1 h. Then, the mixture was allowed to reach
room temperature and it was neutralized with 1m HCl. The aque-
ous layer was extracted with EtOAc (3ꢂ50 mL) and the combined
organic layer was washed with H2O (100 mL), saturated aqueous
NaHCO3 (100 mL) and brine (100 mL), dried over Na2SO4, filtered,
and evaporated to dryness in vacuo. The residue was purified by
flash column chromatography (CH2Cl2/MeOH=98:2; Rf =0.2) to
yield 2-(4-(benzyloxy)butyl)-1,3-diazaspiro[4.4]non-2-en-4-one 21
1
669.15 expressed as [M]+ without counter-ion; H NMR (CDCl3): d=
7.83 (dd, J=7.6, 1.2 Hz, 1H), 7.65 (td, J=7.5, 1.5 Hz, 1H), 7.54 (td,
J=7.5, 1.4 Hz, 1H), 7.45 (dd, J=7.6, 1.1 Hz, 1H), 7.42–7.32 (m, 9H),
7.09 (d, J=8.3 Hz, 2H), 7.04–7.00 (m, 2H), 6.91–6.83 (m, 6H), 4.63
(s, 2H), 4.25–4.21 (m, 2H), 3.56–3.52 (m, 2H), 2.30–2.22 (m, 2H),
1.85–1.81 (m, 6H), 1.62 (dd, J=7.8, 3.5 Hz, 2H), 1.55–1.46 (m, 2H),
1.49–1.37 ppm (m, 2H).
1
(550 mg, 1.83 mmol, 65%) as a yellow oil. H NMR ([D6]DMSO): d=
7.42–7.24 (m, 3H), 7.18–7.11 (m, 2H), 4.69 (s, 2H), 3.32–3.27 (m,
2H), 2.37–2.25 (m, 2H), 1.95–1.76 (m, 6H), 1.64 (ddd, J=18.7, 9.4,
5.7 Hz, 2H), 1.59–1.48 (m, 2H), 1.43–1.31 ppm (m, 2H); 13C NMR
([D6]DMSO): d=186.09, 161.69, 137.59, 129.27, 127.87, 126.85,
76.29, 60.67, 42.98, 37.26, 32.18, 28.09, 25.89, 21.53 ppm; MS:
301.05 [M+H]+.
Ethyl 2-fluoropentanoate 26: Ethyl 2-hydroxypentanoate 25 (1 g,
6.84 mmol) was dissolved in dry CH2Cl2 and the solution was
cooled to ꢀ788C. Then, DAST (1 mL, 1.22 g, 7.57 mmol) was added
portion-wise and the reaction mixture was stirred at ꢀ788C for 2 h.
Afterward, the reaction mixture was allowed to reach room tem-
perature and it was stirred for 48 h. Then, ice H2O (100 mL) was
2-(4-Hydroxybutyl)-1,3-diazaspiro[4.4]non-1-en-4-one 22: 2-(4-
(Benzyloxy)butyl)-1,3-diazaspiro[4.4]non-2-en-4-one 21 (500 mg,
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Full Papers
added carefully, and the mixture was extracted with EtOAc (3ꢂ
100 mL). The combined organic layer was washed with H2O
(150 mL), saturated aqueous NaHCO3 (2ꢂ100 mL) and brine
(100 mL), dried over Na2SO4, filtered, and evaporated to dryness in
vacuo. The residue was purified by flash column chromatography
(petroleum ether/EtOAc=98:2, Rf =0.35) to yield ethyl 2-fluoropen-
tanoate 26 (380 mg, 2.56 mmol, 37%) as a colorless oil. 1H NMR
(CDCl3): d=4.95 (t, J=6.0 Hz, 1H), 4.85–4.80 (m, 1H), 4.25 (q, J=
7.1 Hz, 2H), 1.93–1.78 (m, 1H), 1.56–1.44 (m, 1H), 1.31 (t, J=7.1 Hz,
2H), 0.97 ppm (t, J=7.4 Hz, 2H); 13C NMR (CDCl3): d=172.3, 170.5,
90.1, 88.9, 61.4, 34.7, 34.3, 18.1, 17.8, 14.2, 13.6 ppm.
stirred for 18 h at room temperature. After the reaction had finish-
ed, the reaction mixture was diluted with H2O (50 mL) and was ex-
tracted with EtOAc (3ꢂ50 mL). The combined organic layer was
washed with H2O (50 mL) and brine (50 mL), dried over Na2SO4,
and evaporated to dryness in vacuo. The residue was purified with
flash column chromatography (petroleum ether/EtOAc=6:1, Rf =
0.3) to give 2-(1-fluorobutyl)-3-((2’-(1-trityl-1H-tetrazol-5-yl)-[1,1’-bi-
phenyl]-4-yl)methyl)-1,3-diazaspiro[4.4]non-1-en-4-one 30 (351 mg;
1
0.51 mmol, 64%) as a white solid. H NMR ([D6]DMSO): d=7.77 (dd,
J=7.6, 1.2 Hz, 1H), 7.61 (td, J=7.5, 1.5 Hz, 1H), 7.53 (td, J=7.5,
1.4 Hz, 1H), 7.43 (dd, J=7.6, 1.1 Hz, 1H), 7.40–7.30 (m, 10H), 7.09–
7.01 (m, 3H), 6.92–6.85 (m, 6H), 5.31 (dd, J=8.1, 5.1 Hz, 0.5H), 5.20
(dd, J=8.3, 4.8 Hz, 0.5H), 4.77 (d, J=16.4 Hz, 1H), 4.66 (d, J=
16.4 Hz, 1H), 1.92–1.63 (m, 10H), 1.38–1.18 (m, 2H), 0.78 ppm (t,
J=7.4 Hz, 3H); 13C NMR ([D6]DMSO): d=185.59, 163.51, 158.53,
158.32, 141.12, 140.81, 139.29, 135.61, 130.55, 130.50, 130.36,
129.55, 129.19, 128.28, 127.84, 126.28, 125.75, 87.96, 86.28, 82.29,
76.30, 59.75, 43.08, 36.77, 33.04, 32.84, 25.46, 20.74, 17.36, 17.32,
13.41 ppm; MS (ESI): 689.20 [M+H]+.
2-Fluoropentanoic acid 27: Ethyl 2-fluoropentanoic acid 26
(350 mg, 2.3 mmol) was dissolved in MeOH (5 mL) and 1m NaOH
solution in H2O (5 mL) was added. The mixture was stirred for 5 h
at room temperature. Then, HCl (1m) was added until acidic reac-
tion and the mixture was extracted with EtOAc (3ꢂ50 mL). The
combined organic layer was washed with H2O (100 mL) and brine
(100 mL), dried over Na2SO4, filtered, and evaporated to dryness in
vacuo to give 2-fluoropentanoic acid 27 (280 mg, 2.33 mmol, 99%)
as a colorless volatile oil, which was used directly in the next step
without further purification.
3-((2’-(1H-Tetrazol-5-yl)-[1,1’-biphenyl]-4-yl)methyl)-2-(1-fluoro-
butyl)-1,3-diazaspiro[4.4]non-1-en-4-one 8: 2-(1-Fluorobutyl)-3-
((2’-(1-trityl-1H-tetrazol-5-yl)-[1,1’-biphenyl]-4-yl)methyl)-1,3-diazas-
piro[4.4]non-1-en-4-one 30 (310 mg, 0.45 mmol) was dissolved in
MeOH (10 mL) and HCl (1.25m solution in MeOH, 2 mL, 2.5 mmol)
was added. The solution was stirred for 30 min at room tempera-
ture. The solvent was evaporated, and the residue was purified by
flash column chromatography (CH2Cl2/MeOH=98:2, Rf =0.2) to
yield 3-((2’-(1H-tetrazol-5-yl)-[1,1’-biphenyl]-4-yl)methyl)-2-(1-fluoro-
1-(2-Fluoropentanamido)cyclopentane-1-carboxamide 28: 2-Flu-
oropentanoic acid 27 (280 mg, 2.33 mmol) was dissolved in dry
DMF (5 mL) and NEt3 (64 mL, 46 mg, 4.66 mmol), 1-aminocyclopen-
tanecaroxamide (299 mg, 2.33 mmol) and HBTU (972 mg,
2.56 mmol) were added. The mixture was stirred for 24 h at room
temperature. Then, H2O (50 mL) was added, and the mixture was
extracted with EtOAc (3ꢂ50 mL). The combined organic layer was
washed with H2O (50 mL), saturated aqueous NaHCO3 (50 mL) and
brine (2ꢂ50 mL), dried over Na2SO4, filtered, and evaporated to
dryness in vacuo. The residue was purified by flash column chro-
matography (CH2Cl2/MeOH=98:2; Rf =0.2) to yield 1-(2-fluoropen-
tanamido)cyclopentane-1-carboxamide 28 (320 mg, 1.39 mmol,
60%) as a colorless solid. 1H NMR (CDCl3): d=4.92 (dd, J=7.4,
3.7 Hz, 0.5H), 4.80 (dd, J=7.4, 3.8 Hz, 0.5H), 2.08–1.96 (m, 2H),
1.83–1.69 (m, 8H), 1.51–1.40 (m, 2H), 0.95 ppm (t, J=7.4 Hz, 3H);
13C NMR (CDCl3): d=175.71, 171.08, 92.90, 91.05, 67.46, 38.73,
36.65, 34.48, 23.99, 17.70, 13.71 ppm; MS (ESI): 213.05 [M+H]+.
butyl)-1,3-diazaspiro[4.4]non-1-en-4-one
8 (198 mg, 0.443 mmol,
1
99%) as a white solid. H NMR ([D6]DMSO): d=7.72–7.64 (m, 2H),
7.61–7.50 (m, 2H), 7.14–7.04 (m, 4H), 5.76 (s, 1H), 5.39 (dd, J=7.9,
5.2 Hz, 0.5H), 5.28 (dd, J=8.2, 4.9 Hz, 0.5H), 4.76 (dd, J=42.5,
16.6 Hz, 2H), 1.94–1.67 (m, 10H), 1.46–1.25 (m, 2H), 0.84 ppm (t,
J=7.4 Hz, 3H); 13C NMR ([D6]DMSO): d=185.60, 158.56, 158.36,
141.01, 138.33, 136.05, 131.06, 130.59, 129.09, 127.80, 126.27,
87.96, 86.30, 76.29, 54.89, 42.98, 36.75, 33.08, 32.87, 25.44, 17.38,
17.33, 13.44 ppm; MS (ESI): 447.25 [M+H]+.
Ethyl 2-(benzyloxy)pentanoate 32: Ethyl 2-bromopentanoate 31
(1.01 g, 4.83 mmol) was dissolved in dry DMF and benzyl alcohol
(0.502 mL, 522 mg, 4.83 mmol) was added. The resulting solution
was cooled to 08C before NaH (60% suspension in paraffin oil,
232 mg, 5.8 mmol) was added portion-wise. After complete addi-
tion, the reaction mixture was stirred for 1 h at 08C, then for 23 h
at room temperature. After careful addition of H2O (150 mL), the
mixture was extracted with EtOAc (3ꢂ100 mL). The combined or-
ganic layer was washed with H2O (100 mL), saturated aqueous
NaHCO3 (2ꢂ100 mL) and brine (100 mL), dried over Na2SO4, fil-
tered, and evaporated to dryness in vacuo to yield ethyl 2-(benzy-
loxy)pentanoate 32 (1.14 g, 4.82 mmol, quant.) as a colorless oil.
1H NMR (CDCl3): d=4.95 (t, J=6.0 Hz, 1H), 4.85–4.80 (m, 1H), 4.25
(q, J=7.1 Hz, 2H), 1.93–1.78 (m, 1H), 1.56–1.44 (m, 1H), 1.31 (t, J=
7.1 Hz, 2H), 0.97 ppm (t, J=7.4 Hz, 2H); 13C NMR (CDCl3): d=
175.88, 138.07, 128.77, 128.74, 128.65, 128.43, 127.99, 77.43, 71.73,
61.80, 37.76, 18.08, 14.45, 13.90 ppm; MS (ESI): 237.05 [M+H]+.
2-(1-Fluorobutyl)-1,3-diazaspiro[4.4]non-1-en-4-one 29: 1-(2-Fluo-
ropentanamido)cyclopentane-1-carboxamide
28
(300 mg,
1.3 mmol) was dissolved in MeOH (10 mL) and KOH (10m solution
in H2O, 10 mL, 100 mmol) was added. The mixture was stirred for
1 h at 608C. Then, the mixture was allowed to reach room temper-
ature and it was neutralized with 1m HCl. The aqueous layer was
extracted with EtOAc (3ꢂ100 mL) and the combined organic layer
was washed with H2O (100 mL), saturated aqueous NaHCO3
(100 mL) and brine (100 mL), dried over Na2SO4, filtered, and
evaporated to dryness in vacuo. The residue was purified by flash
column chromatography (CH2Cl2/MeOH=98:2; Rf =0.28) to yield 2-
(1-fluorobutyl)-1,3-diazaspiro[4.4]non-1-en-4-one
29
(184 mg,
0.87 mmol, 67%) as a yellow oil. 1H NMR (CDCl3): d=5.31 (dd,
0.5H), 5.19 (dd, 0.5H), 2.05–1.86 (m, 8H), 1.86–1.74 (m, 2H), 1.62–
1.43 (m, 2H), 0.98 ppm (t, J=7.4 Hz, 3H); 13C NMR (CDCl3): d=
175.71, 171.08, 92.90, 91.05, 67.46, 38.73, 36.65, 34.48, 23.99, 17.70,
13.71 ppm; MS (ESI): 213.05 [M+H]+.
2-(Benzyloxy)pentanoic acid 33: Ethyl 2-(benzyloxy)pentanoate
32 (1 g, 4.23 mmol) was dissolved in MeOH (5 mL) and 1m NaOH
solution in H2O (5 mL) was added. The mixture was stirred for 5 h
at room temperature. Then, HCl (1m) was added until acidic reac-
tion and the mixture was extracted with EtOAc (3ꢂ50 mL). The
combined organic layer was washed with H2O (100 mL) and brine
(100 mL), dried over Na2SO4, filtered, and evaporated to dryness in
vacuo to give 2-(benzyloxy)pentanoic acid 33 (879 mg, 4.22 mmol,
2-(1-Fluorobutyl)-3-((2’-(1-trityl-1H-tetrazol-5-yl)-[1,1’-biphenyl]-
4-yl)methyl)-1,3-diazaspiro[4.4]non-1-en-4-one 30: 2-(1-Fluorobu-
tyl)-1,3-diazaspiro[4.4]non-1-en-4-one 29 (170 mg, 0.8 mmol) was
dissolved in dry DMF (5 mL) and K2CO3 (122 mg, 0.88 mmol) was
added. Then, 5-(4’-(bromomethyl)-[1,1’-biphenyl]-2-yl)-1-trityl-1H-
tetrazole (491 mg, 0,88 mmol) was added, and the mixture was
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99%) as a colorless, which was used directly in the next step with-
out further purification.
d=7.88–7.82 (m, 1H), 7.58 (td, J=7.5, 1.5 Hz, 1H), 7.52 (td, J=7.5,
1.5 Hz, 1H), 7.45–7.32 (m, 10H), 7.10 (s, 4H), 7.01–6.95 (m, 6H),
4.85 (q, J=16.0 Hz, 2H), 4.29 (dt, J=12.5, 6.2 Hz, 1H), 1.96–1.87 (m,
4H), 1.78–1.57 (m, 2H), 1.43–1.22 (m, 4H), 0.92–0.81 (m, 2H),
0.77 ppm (t, J=7.4 Hz, 3H); 13C NMR (101 MHz, [D6]acetone): d=
186.90, 164.73, 163.49, 142.54, 142.27, 140.85, 137.22, 131.47,
131.10, 130.92, 130.84, 130.16, 129.01, 128.51, 128.35, 127.36,
127.27, 83.51, 76.90, 68.15, 43.98, 37.88, 37.74, 36.88, 26.42, 26.41,
19.02, 13.98 ppm; MS (ESI): 867.15 [M+H]+.
1-(2-(Benzyloxy)pentanamido)cyclopentane-1-carboxamide 34:
2-(Benzyloxy)pentanoic acid 33 (850 mg, 4.08 mmol) was dissolved
in dry DMF (10 mL) and NEt3 (690 mL), 1-aminocyclopentanecaroxa-
mide (471 mg, 3.67 mmol) and HBTU (1547 mg, 4.08 mmol) were
added. The mixture was stirred for 24 h at room temperature.
Then, H2O (100 mL) was added, and the mixture was extracted
with EtOAc (3ꢂ100 mL). The combined organic layer was washed
with H2O (50 mL), saturated aqueous NaHCO3 (100 mL) and brine
(2ꢂ50 mL), dried over Na2SO4, filtered, and evaporated to dryness
in vacuo to give 1-(2-(benzyloxy)pentanamido)cyclopentane-1-car-
boxamide 34 (1162 mg, 3.65 mmol, 99%) as a colorless solid,
which was directly used in the next step. MS (ESI): 319.10 [M+H]+.
1-(4-Oxo-3-((2’-(1-trityl-1H-tetrazol-5-yl)-[1,1’-biphenyl]-4-yl)-
methyl)-1,3-diazaspiro[4.4]non-1-en-2-yl)butyl 4-methylbenzene-
sulfonate 38: 2-(1-Hydroxybutyl)-3-((2’-(1-trityl-1H-tetrazol-5-yl)-
[1,1’-biphenyl]-4-yl)methyl)-1,3-diazaspiro[4.4]non-1-en-4-one
37
(530 mg; 0.77 mmol) was dissolved in dry CH2Cl2 (10 mL) and the
solution was cooled to 08C. Then, NEt3 (128 mL, 0.92 mmol) and p-
tosyl chloride (176 mg, 0.92 mmol) were added. The solution was
stirred for 1 h at 08C, then 72 h at room temperature. Afterward,
the solvent was evaporated, and the residue was purified by flash
column chromatography (petroleum ether/EtOAc=3:1, Rf =0.26)
to yield 1-(4-oxo-3-((2’-(1-trityl-1H-tetrazol-5-yl)-[1,1’-biphenyl]-4-yl)-
methyl)-1,3-diazaspiro[4.4]non-1-en-2-yl)butyl 4-methylbenzenesul-
fonate 38 (180 mg, 0.21 mmol, 28%) as a yellow solid. 1H NMR
([D6]DMSO): d=7.78 (dd, J=7.7, 1.3 Hz, 1H), 7.66–7.52 (m, 4H),
7.42–7.33 (m, 12H), 7.10 (d, J=8.3 Hz, 2H), 6.99 (d, J=8.2 Hz, 2H),
6.92–6.86 (m, 6H), 4.99 (dd, J=8.5, 5.2 Hz, 1H), 4.65 (s, 2H), 2.01–
1.85 (m, 4H), 1.87–1.76 (m, 2H), 1.30–1.18 (m, 4H), 0.89–0.76 (m,
2H), 0.54 ppm (t, J=7.4 Hz, 3H); 13C NMR ([D6]DMSO): d=185.55,
164.18, 163.47, 158.34, 158.05, 145.93, 141.28, 138.00, 135.70,
132.87, 130.58, 130.50, 130.02, 129.80, 128.78, 128.38, 128.34,
128.15, 126.93, 126.89, 126.28, 126.20, 82.82, 76.70, 37.16, 34.51,
25.88, 21.54, 17.89, 17.81, 15.66, 14.40, 13.14, 12.85 ppm; MS (ESI):
841.20 [M+H]+.
2-(1-(Benzyloxy)butyl)-1,3-diazaspiro[4.4]non-1-en-4-one 35: 1-
(2-(Benzyloxy)pentanamido)cyclopentane-1-carboxamide
34
(500 mg, 1.57 mmol) was dissolved in MeOH (10 mL) and KOH
(10m solution in H2O, 10 mL, 100 mmol) was added. The mixture
was stirred for 1 h at 608C. Then, the mixture was allowed to reach
room temperature and it was neutralized with 1m HCl. The aque-
ous layer was extracted with EtOAc (3ꢂ100 mL) and the combined
organic layer was washed with H2O (100 mL), saturated aqueous
NaHCO3 (100 mL), and brine, dried over Na2SO4, filtered, and
evaporated to dryness in vacuo. The residue was purified by flash
column chromatography (CH2Cl2/MeOH=98:2; Rf =0.25) to yield 2-
(1-benzyloxy butyl)-1,3-diazaspiro[4.4]non-1-en-4-one 35 (180 mg,
1
0.6 mmol, 38%) as a yellow oil. H NMR (CDCl3): d=7.31–7.19 (m,
5H), 4.46 (dd, J=11.6, 9.9 Hz, 2H), 4.19 (dd, J=7.7, 5.9 Hz, 1H),
1.99–1.82 (m, 6H), 1.78–1.67 (m, 4H), 1.32–1.14 (m, 2H), 0.85 ppm
(t, J=7.4 Hz, 3H); 13C NMR (CDCl3): d=188.12, 176.63, 163.73,
137.28, 128.68, 128.33, 78.00, 76.45, 72.49, 37.69, 37.63, 35.72,
26.17, 18.53, 13.91 ppm; MS (ESI): 301.05 [M+H]+.
2-(1-Hydroxybutyl)-1,3-diazaspiro[4.4]non-1-en-4-one 36: 2-(1-
(Benzyloxy)butyl)-1,3-diazaspiro[4.4]non-2-en-4-one 35 (550 mg,
1.83 mmol) was dissolved in dry MeOH (20 mL) and Pd/C (70 mg;
13%) was added. Then, the atmosphere was evaporated three
times and was replaced with hydrogen. The reaction mixture was
heated at 508C for 24 h. Afterward, the catalyst was filtered off and
the solvent was evaporated in vacuo. The residue was purified by
flash column chromatography (petroleum ether/EtOAc=1:1, Rf =
0.15) to yield 2-(1-hydroxybutyl)-1,3-diazaspiro[4.4]non-1-en-4-one
Competitive binding studies
Competition binding assays were carried out with a membrane
preparation containing human angiotensin AT1R. [125I](Sar1,Ile8)-an-
giotensin II ([125I]angiotensin II) and angiotensin AT1R (human)
membrane preparation were purchased from PerkinElmer, Inc.
(Waltham, MA, USA), where membranes were prepared from trans-
fected Chinese hamster ovary cells (CHO-K1). [18F]8 was synthesized
and purified as described above. The human receptor membrane
preparation (0.6 mg protein) was incubated at RT for 60 min in a
total volume of 200 mL of assay buffer (50 mm Tris·HCl, 5 mm
MgCl2, pH 7.4) with [125I]angiotensin II (0.03 nm), [18F]8 (3 kBq per
well) and various concentrations of unlabeled competitors. Experi-
ments were terminated by rapid vacuum filtration through GFC
Filter (Merck Millipore, Billerica, MA, USA) presoaked in 0.5% BSA.
The filters were then washed (9ꢂ250 mL) with 50 mm Tris·HCl
buffer (pH 7.4), and the filter-bound radioactivity was measured in
a gamma counter (2480 WIZARD, PerkinElmer, Waltham, MA, USA).
Nonspecific binding of [125I]Sar1-Ile8-Ang II was estimated in the
presence of 10m unlabeled Ang II. Specific binding is defined as
total binding minus nonspecific binding. The obtained data were
analyzed using GraphPad Prism 5 software.
1
36 (73 mg, 0.24 mmol, 13%) as a white solid. H NMR (CDCl3): d=
4.51 (dd, J=7.7, 5.1 Hz, 1H), 2.02–1.86 (m, 6H), 1.84–1.69 (m, 4H),
1.57–1.38 (m, 2H), 0.94 ppm (t, J=7.4 Hz, 3H); 13C NMR (CDCl3):
d=190.90, 171.85, 76.34, 68.64, 37.58, 37.48, 37.18, 26.15, 18.46,
13.99 ppm; MS (ESI): 211.05 [M+H]+.
2-(1-Hydroxybutyl)-3-((2’-(1-trityl-1H-tetrazol-5-yl)-[1,1’-biphen-
yl]-4-yl)methyl)-1,3-diazaspiro[4.4]non-1-en-4-one 37: 2-(1-Hy-
droxybutyl)-1,3-diazaspiro[4.4]non-1-en-4-one
36
(240 mg,
1.14 mmol) was dissolved in dry DMF (5 mL) and K2CO3 (189 mg,
1.37 mmol) and 5-(4’-(bromomethyl)-[1,1’-biphenyl]-2-yl)-1-trityl-1H-
tetrazole (613 mg, 1.1 mmol) were added and the mixture was
stirred for 48 h at room temperature. After the reaction was finish-
ed, the reaction mixture was diluted with H2O (50 mL) and was ex-
tracted with EtOAc (3ꢂ50 mL). The combined organic layer was
washed with H2O (50 mL) and brine (100 mL), dried over Na2SO4,
and evaporated to dryness in vacuo. The residue was purified by
flash column chromatography (petroleum ether/EtOAc=1:1, Rf =
0.2) to give 2-(1-hydroxybutyl)-3-((2’-(1-trityl-1H-tetrazol-5-yl)-[1,1’-
Radiochemistry
[18F]Fꢀ was obtained by proton bombardment of H218O and isolat-
ed by trapping on Sep-Pak Light QMA cartridge (ion exchange).
The column was washed with 3 mL of H2O and fluoride was
washed from the cartridge using 66 mm K2CO3 solution (0.3 mL)
biphenyl]-4-yl)methyl)-1,3-diazaspiro[4.4]non-1-en-4-one
(543 mg; 0.79 mmol, 72%) as a white solid. H NMR ([D6]acetone):
37
1
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Full Papers
into a vial containing Kryptofix 2.2.2 (22.5 mg, 59.7 mmol). Then,
0.5 mL of MeCN was added, and the solution was dried under
argon gas at 1208C (4ꢂ addition of MeCN) by azeotropic distilla-
tion. A solution of 5 mg of the precursor 38 in 0.3 mL of dry MeCN
was added to the residue, and the mixture was heated at 1108C
for 10 min, followed by addition of HCl (1m, 0.3 mL) and heating
at 1108C for additional 10 min to deprotect trityl group. After reac-
tion time the mixture was cooled to room temperature and diluted
with 1 mL of a H2O/MeOH (1:1) mixture. The resulting mixture was
applied to semipreparative HPLC (column: COSMOSIL, 5C12-AR-II,
10IDꢂ250 mm (K51815); mobile phase: phase A: water with 0.1%
formic acid, phase B: MeOH with 0.1% formic acid; 0–25 min, 50!
95% B, 25–30 min, 95%, flow rate: 3 mLminꢀ1). After isolation of
radioactive tracer compound, a solution in saline with an appropri-
ate concentration was prepared for the injections.
Conflict of interest
The authors declare the following competing financial interest(s):
Patent application (EP 18150613.0) for 18F-labeled sartans as
novel PET tracers targeting angiotensin II type 1 receptor (X.C.,
T.H., M.D., and M. Hoffmann).
Keywords: angiotensin II type 1 receptor
positron emission tomography · radiolabeling · renal imaging
·
irbesartan
·
[1] a) S. S. Karnik, H. Unal, J. R. Kemp, K. C. Tirupula, S. Eguchi, P. M. L. Van-
derheyden, W. G. Thomas, Pharmacol. Rev. 2015, 67, 754–819; b) R. V.
Shah, A. S. Desay, M. M. Gieverts, J. Card. Failure 2010, 16, 260–267;
c) K. D. Singh, S. S. Karnik, J. Cell. Signal. 2016, 1, 111; d) H. Siragy, Am. J.
Cardiol. 1999, 84, 3–8; e) H. Wang, L. Dwyer-Lindgren, K. T. Lofgren, J.
Knoll Rajaratnam, J. R. Marcus, A. Levin-Rector, C. E. Levitz, A. D. Lopez,
C. J. L. Murray, Lancet 2012, 380, 2095–2128; f) E. J. Benjamin, M. J.
Blaha, S. E. Chiuve, M. Cushman, S. R. Das, R. Deo, S. D. de Ferranti, J.
Floyd, M. Fornage, C. Gillespie, et al., Circulation 2017, 135, e146–e603;
g) A. Mosterd, A. W. Hoes, Heart 2007, 93, 1137–1146; h) D. D. Schock-
en, E. J. Benjamin, G. C. Fonarow, H. M. Krumholz, D. Levy, G. A. Mensah,
J. Narula, E. S. Shor, J. B. Young, Y. Hong, Circulation 2008, 117, 2544–
2565; i) H. Tsutsui, M. Tsuchihashi-Makaya, S. Kinugawa, D. Goto, A. Take-
shita, JCARE-CARD Investigators, Circ. J. 2006, 70, 1617–1623.
[2] L. L. Humphrey, D. J. Ballard, P. P. Frohnert, C. P. Chu, W. M. O’Fallon, P. J.
Palumbo, Ann. Intern. Med. 1989, 111, 788–796.
Animal imaging studies
All animal experiments were approved by the Animal Ethics Com-
mittee of the National Cerebral and Cardiovascular Center Research
Institute and were conducted strictly according to the Guide for the
Care and Use of Laboratory Animals.[30]
For dynamic PET studies in rats (Figure 4A,B), two male healthy
Wistar rats (weight 210–220 g) were used. The rats were kept anes-
thetized by 2% isoflurane during the experiment. [18F]8 (10 MBq)
was injected via the tail vein, followed by PET measurements using
a clinical PET scanner (microPET Focus, Siemens Medical Solutions,
Germany). Data were acquired 60 min after tracer injection
(10 MBq) in list-mode format and reconstructed into a dynamic
image with 5-min frames. For blocking experiments, 5 mgkgꢀ1 ir-
besartan 6 was administered 5 min before tracer ([18F]8 10 MBq)
administration. For biodistribution studies (Figure 4C), rats (n=3
for control without pre-treatment, n=3 for blocking with 5 kgkgꢀ1
with 6 5 min before tracer injection) were euthanized 15 min after
injection of [18F]8 (3 MBq). Then, organs of interest were collected,
weighed, and counted for radioactivity in an automated gamma
counter (Wizard, PerkinElmer). Tissue radioactivity concentrations
were estimated and expressed as the standardized uptake value
(SUV).
[3] a) A. L. Bui, G. C. Fonarow, J. Am. Coll. Cardiol. 2012, 59, 97–104; b) B.
Tran, G. C. Fonarow, Card. Fail. Rev. 2015, 1, 50–55.
[4] a) M. L. Ellis, J. H. Patterson, Pharmacotherapy 1996, 16, 849–860;
b) WHO Model List of Essential Medicines, 20th List (March 2017, Amended
August 2017), World Health Organization 2017: http://apps.who.int/iris/
bitstream/handle/10665/273826/EML-20-eng.pdf?ua=1 (accessed No-
vember 6, 2018).
[5] C. M. Ferrario, J. Renin-Angio. Aldo. S. 2006, 7, 3–14.
[6] a) T. K. W. Ma, K. K. H. Kam, B. P. Yan, Y.-Y. Lam, Br. J. Pharmacol. 2010,
160, 1273–1292; b) S. Yusuf, M. A. Pfeffer, K. Swedberg, C. B. Granger, P.
Held, J. J. V. McMurray, E. L. Michelson, B. Olofsson, J. Ostergren, Lancet
2003, 362, 777–781; c) J. G. F. Cleland, M. Tendera, J. Adamus, N. Free-
mantle, L. Polonski, J. Taylor, Eur. Heart J. 2006, 27, 2338–2345; d) B.
Pitt, M. A. Pfeffer, S. F. Assmann, R. Boineau, I. S. Anand, B. Claggett, N.
Clausell, A. S. Desai, R. Diaz, J. L. Fleg, et al., N. Engl. J. Med. 2014, 370,
1383–1392; e) K. Yamamoto, H. Origasa, M. Hori, Eur. J. Heart. Fail.
2013, 15, 110–118; f) H. Fukuta, T. Goto, K. Wakami, N. Ohte, Heart Fail-
ure Rev. 2017, 22, 775–782; g) E. J. Lewis, L. G. Hunsicker, W. R. Clarke, T.
Berl, M. A. Pohl, J. B. Lewis, E. Ritz, R. C. Atkins, R. Rohde, I. Raz, N. Engl.
J. Med. 2001, 345, 851–860; h) S. El Din, U. A. Azim, M. M. Salem, D. O.
Abdulazim, World J. Nephrol. 2016, 5, 258–273; i) Y. Kiya, S.-I. Miura, M.
Fujino, S. Imaizumi, S. S. Karnik, K. Saku, Clin. Exp. Hypertens. 2010, 32,
129–136.
Healthy female Gçttingen pigs (weight 22 kg for control and 26 kg
for blocking experiment) were used. The pigs were kept anesthe-
tized by seboflurane during the whole experiment. [18F]8 (33 MBq)
was injected via the ear vein, followed by PET measurements (30–
45 min after tracer injection) using a clinical PET scanner (PCA-
2000A, Toshiba Medical Systems Corporation, Otawara, Japan). For
blocking experiments, 2 mgkgꢀ1 irbesartan 6 was administered
10 min before the tracer ([18F]8 16 MBq) administration.
[7] B. M. Massie, P. E. Carson, J. J. McMurray, M. Komajda, R. McKelvie, M. R.
Zile, S. Anderson, M. Donovan, E. Iverson, C. Staiger, et al., N. Engl. J.
Med. 2008, 359, 2456–2467.
[8] H. Urata, S. Hoffmann, D. Ganten, Eur. Heart J. 1994, 15 Suppl. D, 68–78.
[9] J. Sadjadi, K. Puttaparthi, M. B. Welborn, T. E. Rogers, O. Moe, G. P. Cla-
gett, R. H. Turnage, M. Levi, J. G. Modrall, J. Vasc. Surg. 2002, 36, 386–
392.
Acknowledgements
[10] a) H. F. Cheng, B. N. Becker, K. D. Burns, R. C. Harris, J. Clin. Invest. 1995,
95, 2012–2019; b) D. Zimmerman, K. D. Burns, Clin. Sci. 2012, 123, 333–
346.
The German Academic Scholarship Foundation (Studienstiftung
des Deutschen Volkes) is acknowledged for providing M. Hoff-
mann with a PhD scholarship, and The Elite Network of Bavaria
is acknowledged for financial support and inspiration within the
International Doctoral Program “Receptor Dynamics”. This project
was supported by the German Research Council (Deutsche For-
schungsgemeinschaft (DFG) grants CH 1516/2-1 and HI 1789/3-
3).
[11] a) T. Yamazaki, I. Komuro, S. Kudoh, Y. Zou, I. Shiojima, T. Mizuno, H.
Takano, Y. Hiroi, K. Ueki, K. Tobe, et al., Circ. Res. 1995, 77, 258–265; b) J.
Fielitz, S. Hein, V. Mitrovic, R. Pregla, H. R. Zurbrꢁgg, C. Warnecke, J.
Schaper, E. Fleck, V. Regitz-Zagrosek, J. Am. Coll. Cardiol. 2001, 37,
1443–1449; c) V. C. de Giusti, A. Orlowski, E. A. Aiello, J. Mol. Cell. Cardi-
ol. 2010, 49, 812–818; d) D. Baetz, R. S. Haworth, M. Avkiran, D. Feuvray,
Am. J. Physiol. Heart. Circ. Physiol. 2002, 283, H2102-9; e) T. Yamamoto, T.
Shirayama, T. Sakatani, T. Takahashi, H. Tanaka, T. Takamatsu, K. W. Spit-
ChemMedChem 2018, 13, 1 – 13
www.chemmedchem.org
11
ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ

Full Papers
zer, H. Matsubara, Am. J. Physiol. Heart. Circ. Physiol. 2007, 293, H1254–
H1264.
[12] a) S. Rodrigues-Ferreira, C. Nahmias, Front. Pharmacol. 2015, 6, 24; b) T.
Wegman-Ostrosky, E. Soto-Reyes, S. Vidal-Millan, J. Sanchez-Corona, J.
Renin-Angio. Aldo. S. 2015, 16, 227–233; c) A. J. George, W. G. Thomas,
R. D. Hannan, Nat. Rev. Cancer 2010, 10, 745–759.
[13] M. Okazaki, S. Fushida, S. Harada, T. Tsukada, J. Kinoshita, K. Oyama, H.
Tajima, I. Ninomiya, T. Fujimura, T. Ohta, Cancer Lett. 2014, 355, 46–53.
[14] a) W. C. De Mello, A. H. J. Danser, Hypertension 2000, 35, 1183–1188;
b) A. Orlowski, M. C. Ciancio, C. I. Caldiz, V. C. de Giusti, E. A. Aiello, Car-
diovasc. Res. 2014, 101, 211–219.
Petrov, B. Indrevoll, E. Bjurgert, T. B. Krasieva, L. B. Petersen, G. M. Kind-
berg, M. Solbakken, et al., JACC Cardiovasc. Imag. 2008, 1, 354–362.
[22] a) K. Fukushima, P. E. Bravo, T. Higuchi, K. H. Schuleri, X. Lin, M.R. Abra-
ham, J. Xia, W. B. Mathews, R. F. Dannals, A.C. Lardo, Z. Szabo, F. M.
Bengel, J. Am. Coll. Cardiol. 2012, 60, 2527–2534; b) H. Kimura, S. Ya-
mauchi, H. Kawashima, K. Arimitsu, Y. Yagi, Y. Nakamoto, K. Togashi, M.
Ono, H. Jaji, Bioorg. Med. Chem. Lett. 2018, 28, 2949–2952.
[23] a) G. Sedelmeier, J. Sedelmeier, Chimia 2017, 71, 730; b) F. Weber, G. Se-
delmeier, Nachr. Chem. 2014, 62, 997.
[24] a) P. Bramlage, I. Durand-Zaleski, N. Desai, O. Pirk, C. Hacker, Expert Opin.
Pharmacother. 2009, 10, 1817–1831; b) J. Ambrose, D. G. Pribnow, G. D.
Giraud, K. D. Perkins, L. Muldoon, B. H. Greenberg, J. Cardiovasc. Phar-
macol. 1999, 33, 433–439.
[15] X. Chen, R. A. Werner, M. S. Javadi, Y. Maya, M. Decker, C. Lapa, K. Herr-
mann, T. Higuchi, Theranostics 2015, 5, 545–558.
[25] a) M. R. Marino, K. Langenbacher, N. F. Ford, H. D. Uderman, J. Clin. Phar-
macol. 1998, 38, 246–255; b) C. Delisꢃe, P. Schaeffer, C. Cazaubon, P.
Chatelain, Eur. J. Pharmacol. 1993, 247, 139–144; c) T. J. Chando, D. W.
Everett, A. D. Kahle, A. M. Starrett, N. Vachharajani, W. C. Shyu, K. J. Kri-
palani, R. H. Barbhaiya, Drug Metab. Dispos. 1998, 26, 408–417.
[26] N. A. Meanwell, J. Med. Chem. 2018, 61, 5822–5880.
[16] a) T. G. Zober, W. B. Mathews, E. Seckin, S. E. Yoo, J. Hilton, J. Xia, K.
Sandberg, H. T. Ravert, R. F. Dannals, Z. Szabo, Nucl. Med. Biol. 2006, 33,
5–13; b) M. Lortie, J. N. DaSilva, S. A. Kirkpatrick, T. Hadizad, B. A. Ismail,
R. S. B. Beanlands, R. A. deKemp, Nucl. Med. Biol. 2013, 40, 252–261.
[17] T. Hadizad, J. Collins, R. E. Antoun, R. S. Beanlands, J. N. DaSilva, J. La-
belled Compd. Radiopharm. 2011, 54, 754–757.
[18] a) T. Hadizad, S. A. Kirkpatrick, S. Mason, K. Burns, R. S. Beanlands, J. N.
DaSilva, Bioorg. Med. Chem. 2009, 17, 7971–7977; b) X. Chen, M. Hirano,
R. A. Werner, M. Decker, T. Higuchi, ACS Omega 2018, 3, 10460–10470.
[19] a) N. Arksey, T. Hadizad, B. Ismail, M. Hachem, A. C. Valdivia, R. S. Bean-
lands, R. A. deKemp, J. N. DaSilva, Bioorg. Med. Chem. 2014, 22, 3931–
3937; b) M. Hachem, M. Tiberi, B. Ismail, C. R. Hunter, N. Arksey, T. Hadi-
zad, R. S. Beanlands, R. A. deKemp, J. N. DaSilva, J. Nucl. Med. 2016, 57,
1612–1617; c) T. G. Hamill, H. D. Burns, R. F. Dannals, W. B. Mathews, J. L.
Musachio, H. T. Ravert, E. M. Naylor, Appl. Radiat. Isot. 1996, 47, 211–
218.
[20] a) W. B. Mathews, S. E. Yoo, S. H. Lee, U. Scheffel, P. A. Rauseo, T. G.
Zober, G. Gocco, K. Sandberg, H. T. Ravert, R. F. Dannals, Z. Szabo, Nucl.
Med. Biol. 2004, 31, 571–574; b) S. H. Lee, Y. S. Jung, B. H. Lee, S. I. Yun,
S. E. Yoo, H. S. Shin, J. Cardiovasc. Pharmacol. 1999, 33, 367–374.
[21] a) T. Higuchi, K. Fukushima, J. Xia, W. B. Mathews, R. Lautamaki, P. E.
Bravo, M. S. Javadi, R. F. Dannals, Z. Szabo, F. M. Bengel, J. Nucl. Med.
2010, 51, 1956–1961; b) J. W. H. Verjans, D. Lovhaug, N. Narula, A. D.
[27] X. Bao, W. Zhu, W. Yuan, X. Zhu, Y. Yan, H. Tang, Z. Chen, Eur. J. Med.
Chem. 2016, 123, 115–127.
[28] a) M. Pinter, R. K. Jain, Sci. Transl. Med. 2017, 9, eaan5616; b) D. G.
Passos-Silva, E. Brandan, R. A. S. Santos, Trends Pharmacol. Sci. 2015, 36,
310–320; c) Y. Mao, X. Xu, X. Wang, X. Zheng, L. Xie, Oncotarget 2016,
7, 6765–6773; d) J. Zhang, J. Liu, J. Chen, X. Li, Y. Wu, H. Chen, W. Wu,
K. Zhang, L. Gu, Int. J. Clin. Exp. Med. 2015, 8, 12656–12660.
[29] a) M. R. Marino, K. M. Langenbacher, N. F. Nord, H. D. Uderman, Clin.
Pharmacol. Ther. 1997, 61, 207; b) J. R. Powell, R. A. Reeves, M. R.
Marino, C. Cazaubo, D. Nisato, Cardiovasc. Drug Rev. 1998, 16, 169–194.
[30] Guide for the Care and Use of Laboratory Animals, Eighth Edition, US Na-
tional Research Council, Committee for the Update of the Guide for the
Care and Use of Laboratory Animals, National Academies Press, 2011.
Manuscript received: September 27, 2018
Accepted manuscript online: && &&, 0000
Version of record online: && &&, 0000
&
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ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ÝÝ These are not the final page numbers!

FULL PAPERS
An eye on the kidneys: Novel fluorinat-
ed derivatives of irbesartan were syn-
thesized, and the synthesis of suitable
precursor molecules for positron emis-
sion tomography, including radiolabel-
ing with fluorine-18, was achieved. In
vitro binding and renal imaging studies
in rats and pigs reveal the excellent per-
formance of these radiotracers for renal
imaging.
M. Hoffmann, X. Chen, M. Hirano,
K. Arimitsu, H. Kimura, T. Higuchi,*
M. Decker*
&& – &&
18F-Labeled Derivatives of Irbesartan
for Angiotensin II Receptor PET
Imaging
ChemMedChem 2018, 13, 1 – 13
www.chemmedchem.org
13
ꢀ 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
These are not the final page numbers! ÞÞ