Iodinated Choline Transport-Targeted Tracers

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Pavel Svec, Zbynek Novy, Jan Kucka, Milos Petr í k, Ondrej Sedlacek, Martin Kuchar, Barbora Liskova,

́

̌

́

Martina Medved í kova, Kristyna Kolouchova, Ondrej Groborz, Lenka Loukotova, Rafa ł Ł . Konefa ł ,

Marian Hajduch,* and Martin Hruby *

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ABSTRACT: We present a novel series of radioiodinated tracers

and potential theranostics for diseases accompanied by patho-

logical function of proteins involved in choline transport. Unlike

choline analogues labeled with ¹¹C or ¹⁸F that are currently used in

the clinic, the iodinated compounds described herein are

applicable in positron emission tomography, single-photon

emission computed tomography, and potentially in therapy,

depending on the iodine isotope selection. Moreover, favorable

half-lives of iodine isotopes result in much less challenging synthesis by isotope exchange reaction. Six of the described compounds

were nanomolar ligands, and the best compound possessed an aﬃnity 100-fold greater than that of choline. Biodistribution data of

¹²⁵I-labeled ligands in human prostate carcinoma bearing (PC-3) mice revealed two compounds with a biodistribution proﬁle

superior to that of [¹⁸F]ﬂuorocholine.

diagnostic and therapeutic radiopharmaceuticals. Three radio-

labeled choline derivatives are currently employed in tumor

imaging (Figure 1).²⁰

INTRODUCTION

■

Radionuclide imaging methods, such as planar scintigraphy,

positron emission tomography (PET), and single-photon

emission computed tomography (SPECT), have become

invaluable because of their ability to distinguish biochemical

changes in tissues (functional imaging) and are routinely used

for staging, prognosing, and predicting the therapeutic

outcome of many diseases, especially cancer.¹Plain ﬁlm

radiography, X-ray computed tomography (CT), and magnetic

resonance imaging (MRI) can be used to evaluate the

anatomical extent of diseases with sub-millimeter resolution.

PET and SPECT have lower spatial resolution,²but they are

able to visualize in vivo biological processes in real time and

detect the concentrations of speciﬁc biomolecules in the

picomolar range.³Both PET and SPECT rely on radiolabeled

pharmaceuticals, which are used as imaging agents.

Choline is an essential precursor for many signaling and

constitutive biomolecules⁴(e.g., acetylcholine and phospholi-

pids), and alterations in choline metabolism in cancer cells

have been recognized as hallmarks of malignancy. These

alterations in choline metabolism are frequently associated

with activation of major oncogenic drivers in the Ras and PI3K

pathways.⁵⁻⁷Numerous studies employing mainly PET⁸⁻¹³

and magnetic resonance spectroscopy¹⁴⁻¹⁷have been con-

ducted in this regard. There is evidence that the increased

uptake of choline is at least partially caused by the

overexpression of choline transporter-like proteins (CTL) by

certain types of tumor cells, particularly in prostate

carcinomas,⁶gliomas,^6,18and small cell lung carcinomas.¹⁹

Thus, proteins responsible for choline transport across the

plasma membrane represent targetable molecular structures for

Figure 1. Structures of choline derivatives clinically used as tracers.

However, currently used choline-based imaging agents have

signiﬁcant limitations. First, the ¹¹C and ¹⁸F nuclides can be

used only for PET imaging. This might be a barrier in the

massive use of prostate cancer imaging employing choline-

based tracers because the overall cost of PET examinations is

still higher compared to SPECT. The short half-lives of both

¹¹C (t_1/2= 20 min) and ¹⁸F (t_1/2= 110 min) impose strict

requirements on the synthesis and logistics of these

radiopharmaceuticals. Furthermore, for the visualization of

prostate carcinoma with [¹⁸F]ﬂuorocholine ([¹⁸F]FCh), the

short isotope half-life combined with rapid renal absorption

and excretion causes problems.²¹The image quality is

signiﬁcantly decreased by background activity because of

Received: September 29, 2020

Published: December 3, 2020

https://dx.doi.org/10.1021/acs.jmedchem.0c01710

J. Med. Chem. 2020, 63, 15960−15978

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Figure 2. Choline analogues 1−32 and their IC₅₀values for [³H]choline uptake by PC-3 cells (n = 3, for standard error see Table 1); compounds

selected for in vivo evaluation are marked with an asterisk.

accumulation in the urinary bladder and kidney. Moreover,

kidney and liver are the highly exposed dose-critical organs

limiting the administration levels of [¹⁸F]FCh.^22,23

example of a very promising theranostic pair for patients with

PMSA-positive prostate cancer.³¹

In this feasibility study, we developed a new class of

iodinated choline-based tracers with both diagnostic and

potential therapeutic (theranostics) applications, for example,

in prostate cancer, which is the most common type of cancer in

males worldwide and one of the major causes of mortality.³²

The main advantage of iodinated tracers is that there are four

medicinally relevant isotopes with suitable half-lives; thus, a

single compound can be labeled with diﬀerent iodine

radioisotopes suitable either for SPECT (¹²³I, t_1/2= 13.2 h),

PET (¹²⁴I, t_1/2= 4.2 d), therapy (¹²⁴I, ¹³¹I, t_1/2= 8.0 d), or for

Theranostic approach in principle enables a signiﬁcantly

more accurate monitoring of the dosage, distribution, and

treatment.²⁴In the ideal case, theranostic agents would involve

chemically and, therefore, biologically identical compounds.²⁵

The best-known examples which meet this ideal are radio-

iodide (¹²³I⁻/¹²⁴I⁻and ¹³¹I⁻for imaging/treatment of thyroid

carcinoma^26,27) and metaiodobenzylguanidine (MIBG, imag-

ing/treatment of pheochromocytoma^28,29).

Unfortunately, even a subtle structural change such as the

substitution of trivalent diagnostic radiometals (¹¹¹In and

⁶⁸Ga) for their therapeutic counterparts (⁹⁰Y and ¹⁷⁷Lu) can

lead to signiﬁcant changes in structure and biological

properties.³⁰Nevertheless, a diagnostic molecule which is

not chemically or biologically identical but has a suﬃciently

similar biodistribution can adequately predict the biodistribu-

tion of its therapeutic analogue. ⁶⁸Ga-labeled prostate-speciﬁc

membrane antigen (PSMA)-11 and ¹⁷⁷Lu-PSMA-617 are an

in vitro analysis and preclinical development (¹²⁵I, t_1/2

=

60 d).³³

The cellular uptake of choline has both facilitative and

nonfacilitative components and there are many diﬀerent

choline transporters (ChTs), for example, high aﬃnity ChT,

CTLs (CTL1−CTL5), and organic cation transporters.³⁴

Unfortunately, only limited data are available on choline

transport into tumor cells.³⁵There is also a lack of knowledge

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Figure 3. Choline analogues 33−50 and their IC₅₀values for [³H]choline uptake by PC-3 cells (n = 3, for standard errors see Table 1); compounds

selected for in vivo evaluation are marked with an asterisk.

regarding the three-dimensional (3D) structure of these

transporters, which hampers the rational design of radiotracers.

The iodination of electron-rich phenols was carried out using

Barluenga’s reagent^36,37in the presence of triﬂuoroacetic acid

(TFA, Scheme 2, compounds 20−25), and the free phenol

moiety was then alkylated with an appropriate alkyl iodide or

methyl tosylate. The activated phenolic compounds were

somewhat prone to multiple iodination with Barluenga’s

reagent, even when an excess of the substrate and a decreased

reaction temperature were used.

The iodination of 2-thiopheneethanol with Barluenga’s

reagent led to more complex mixtures of products, which

were challenging to separate. Therefore, we introduced the

iodine atom onto the thiophene ring by ortho-lithiation,

followed by iododemetallation (Scheme 2, ﬁnal compounds

26−29). An analogous procedure was also used for the

synthesis of derivatives with iodinated triple bond (ﬁnal

compounds 30−32). In accordance with a previously

published study,³⁸the intended direct transformation of the

resulting iodinated tert-butyldimethylsilyl (TBS)-protected

alcohols into tosylates by reaction with a 4-methylbenzene-1-

sulfonyl ﬂuoride (TsF) and a 1,8-diazabicyclo[5.4.0]undec-7-

ene (DBU) catalyst did not occur at room temperature (rt).

However, at reﬂux, contrary to the previously published results

using a similar substrate,³⁸the formed tosylate directly

alkylated the DBU catalyst and hence could not be isolated

(Scheme 2, step f).

RESULTS AND DISCUSSION

■

Ligand Design and Synthesis. We focused on the

synthesis of compounds with an iodine atom attached to an

aromatic/heteroaromatic ring or a terminal alkyne to prevent

undesirable radioiodide loss by nucleophilic substitution

(Figures 2, 3). In cases where suitable iodinated starting

compounds were not commercially available, the general

synthetic strategy consisted of the following three fundamental

steps: (i) the introduction of the iodine atom onto an

appropriately hydroxyalkyl-substituted aromatic/heteroaro-

matic ring or terminal alkyne; (ii) substrate conversion into

a suﬃciently reactive alkylating agent; and (iii) quaternization

of a tertiary amine (Schemes 1−3).

Less electron-rich iodinated aromatics were synthesized

from the corresponding substituted anilines by a Sandmeyer

reaction (Scheme 1, ﬁnal compounds 15−19 and 48−50).

Scheme 1. Synthesis of Compound 15: (a) NaNO₂/H₂SO₄/

H₂O; NaI/H₂O; (b) TsCl/Et₃N//DCM; (c) DMAE

Aryloxyalkyl and heteroaryloxyalkyl derivatives (ﬁnal com-

pounds 33−50) of choline were prepared by a reaction of

iodinated phenols with dibromoalkane yielding the desired

alkylating agent for amine quaternization (Scheme 3).

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Scheme 2. Synthesis of Compounds 20 and 26: (a) [I(py)₂][BF₄]/TFA/ACN; (b) MeOTs/K₂CO₃/DMF; (c) TsCl/Et₃N/

DMAP/DCM; (d) DMAE/DMF; (e) TBSCl/Imidazole/DMAP/DCM and n-BuLi/THF/I₂; (f) TsF/DBU/ACN; (g) TBAF/

THF and TsCl/Et₃N/DMAP/DCM; and (h) DMAE/DMF

Analysis of the assay results of compounds 1−25 (Figure 2,

Table 1) revealed that the binding constant of choline

derivatives is typically improved by the attachment of more

lipophilic substituents. This trend was noticeable, for example,

according to the comparison of the IC₅₀values for compounds

20 and 23−25. Compound 24, which contained a hexyloxy

substituent on the benzene ring, was the ﬁrst nanomolar ligand

prepared in this work and had an aﬃnity 2 orders of magnitude

higher than its methoxy analogue 23. However, this eﬀect

could be assigned to the increased lipophilicity of compound

24 (Clog P,⁴⁰see Table 2) resulting in similar values of

Scheme 3. Synthesis of Compound 33: (a) Br(CH₂)₂Br/

K₂CO₃/DMF; (b) DMAE

After the successful conversion of iodinated intermediates

into alkylating agents such as iodides, bromides, tosylates or

mesylates, these agents were further used for the quaterniza-

tion of corresponding tertiary amines. The quaternization

reaction with bulky amines was occasionally complicated by an

undesirable elimination side reaction, especially in the case of

alkylating agents where the formation of a conjugated double

bond by elimination was possible.

Table 2. Calculated log P⁴⁰Values of Choline Analogues

cmpd.

log P

cmpd.

log P

cmpd.

log P

cmpd.

log P

1

2

3

4

5

6

7

8

−1.74

−0.93

−1.11

−0.77

−0.97

−1.10

−2.34

−1.93

−1.74

−1.11

−1.74

−0.93

−1.11

14

15

16

17

18

19

20

21

22

23

24

25

26

−1.93

−1.31

−0.67

−0.68

−0.54

−1.31

−1.38

−0.61

−0.94

−0.92

1.30

27

28

29

30

31

32

33

34

35

36

37

38

39

−0.61

−0.78

−0.33

−1.54

−1.09

−0.46

−1.73

−1.10

−1.73

−1.10

−1.73

−1.10

−0.82

40

41

42

43

44

45

46

47

48

49

50

−0.37

0.09

−0.19

0.09

Aﬃnity toward Choline Transporters. We evaluated the

aﬃnity of the individual derivatives toward choline transport

systems by analyzing the ability of each compound to compete

with the cellular uptake of [³H]choline (IC₅₀value). The assay

was performed in vitro on the PC-3 cell line (derived from

human prostate carcinoma), which overexpresses choline

transporters.³⁴The IC₅₀values were determined for the 50

analogues of choline (Figures 2, 3) that were synthesized in

this study and for hemicholinium-3 (HCh-3, ChT inhibitor, 36

0.71

−0.82

−1.33

0.84

−0.84

−0.38

0.07

9

10

11

12

13

3.12

−1.24

4 μM), FCh ([¹⁸F]FCh used as tracer, 20

choline (natural ligand, 4.7 1.3 μM). The IC₅₀values we

5 μM) and

obtained for choline and HCh-3 were consistent with the

lipophilic eﬃciency (for LipE and LipE plots of all analogues,

see Table S4 and Figures S2 and S3, Supporting Information).

previously published data.³⁹

Table 1. IC₅₀SE (n = 3) of Choline Analogues for [³H]Choline Uptake by PC-3 Cells

cmpd.

PC-3 [μM]

cmpd.

PC-3 [μM]

cmpd.

PC-3 [μM]

cmpd.

PC-3 [μM]

HCh-3

36

20

4

5

12

13

14

15

16

17

18

19

20

21

22

23

24

25

77 17

26

27

28

29

30

31

32

33

34

35

36

37

38

39

0.45 0.12

40

41

42

43

44

45

46

47

48

49

50

30

23

8

3

FCh

Ch

1

2

3

4

5

6

7

8

9

10

11

16

4

10

1.1 0.3

30

3.0 0.3

2

4.7 1.3

216 38

8.1 0.4

273 43

8.6 1.2

106 16

5.1 1.1

0.044 0.01

0.91 0.09

1.4 0.1

8

11

5

19

2

36

18

25

4

6

217 25

160 22

197 32

908 251

43

133 20

28

130 16

75 19

1.2 0.3

18

7

1.3 0.3

0.14 0.05

1.1 0.3

24

3

4.7 2.1

115 32

53 31

34 17

0.41 0.08

2.1 0.3

22

15

23

18

11

4

2

3

7

2

5

0.37 0.09

5

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Table 3. IC₅₀SE (n = 3) of Choline Analogues for [³H]Choline Uptake by U87-MG Cells

cmpd.

U-87 [μM]

cmpd.

U-87 [μM]

cmpd.

U-87 [μM]

cmpd.

U-87 [μM]

2

3

9

4.3 0.6

3.3 0.5

10

11

12

8.8 2.6

129 28

77 23

13

17

19

23

29

3

24

25

2.1 0.8

1.7 1.1

25

5

0.78 0.13

Interestingly, the IC₅₀value of the compound 19 with a

structure similar to compound 24 was comparable but its

lipophilicity is signiﬁcantly lower. We also observed that

excessive steric hindrance and substituent rigidity at the

quaternary nitrogen atom had a strong negative impact on

aﬃnity (e.g., 2 and 3 vs 4−6). Therefore, we proceeded with

the synthesis of compounds derived only from 2-

(dimethylamino)ethanol and 1-(2-hydroxyethyl)pyrrolidine.

We also prepared and analyzed compounds with bioisosteric

iodinated thiophene rings (26−29), with less bulky iodinated

triple bonds (30−32), and compounds with longer aliphatic

spacers between the aromatic moiety and quaternary nitrogen.

This synthetic eﬀort yielded several other low-micromolar (28,

30, 34, 42, 45, 47, 49) and nanomolar ligands (26, 43, 44, 48,

50). Thiophene derivative 26 exhibited both excellent aﬃnity

as well as lipophilic eﬃciency. A surprising disparity in IC₅₀

values was observed in the case of iodoalkyne choline

analogues 30 and 31, which diﬀer only in one methylene

group. The aﬃnity of the shorter choline derivative 30 was 1

order of magnitude higher than in the case of compound 31.

The optimal spacer length between the aromatic ring and

quaternary nitrogen in the case of iodo(hetero)aryloxyalkyl

derivatives (33−50) was approximately 7 atoms (Figures 2, 3).

The use of a hexyloxy spacer between the choline moiety and

the aromatic ring resulted in improved binding aﬃnities as well

as signiﬁcantly higher lipophilic eﬃciencies compared to

hexyloxy substituted compound 24 (compare 24 vs 43 and

48). The highest aﬃnity of all analyzed choline analogues was

observed with compound 43. An increase in chain length

between the quaternary nitrogen and the hydroxyl group led to

decreased binding aﬃnities (26 vs 28−29 and 48 vs 49−50).

Twenty-eight iodinated derivatives exhibited better IC₅₀

values than FCh, and 14 analogues had IC₅₀values that were

better than the IC₅₀value determined for choline. The best

achieved IC₅₀value (43) was 2 orders of magnitude less than

that of choline.

However, the increase in lipophilicity is typically moderate,

and other parameters such as size, volume, or molecular

surface area do not change dramatically.⁴²To evaluate the in

vitro pharmacokinetic properties of all investigated iodinated

choline analogues, the stability in human plasma, binding to

plasma proteins, microsomal stability, and permeability

through artiﬁcial membrane (parallel artiﬁcial membrane

permeability assay, PAMPA) were analyzed (Table S5,

Supporting Information). Most of the iodinated analogues of

choline demonstrated suﬃcient stability in human plasma after

incubation for 2 h (70−90% of the original quantity of the

substance remaining) and a quite high stability in hepatic

microsomes (typically 70−90% of the original quantity of the

substance remained after incubation for 1 h). Binding to

plasma proteins primarily inﬂuences the distribution of the

drug in the organism. Plasma protein binding (PPB) has

shown intermediate values for most of the investigated

analogues of choline (4−82% bound to plasmatic proteins).

With respect to their ionic nature, the investigated compounds

exhibited a low or medium ability to pass across the artiﬁcial

membrane by passive diﬀusion (PAMPA). This is a desirable

property that enables a speciﬁc distribution of these substances

and further supports their potential use as radiopharmaceuti-

cals (choline transporters are localized on the plasma

membrane of cells). In general, the substances showed

appropriate pharmacokinetic properties for both diagnostic

and therapeutic applications (for plasma and microsomal

stability of the compounds selected for in vivo experiments see

Table 4).

Table 4. Plasma Stability (Pl.; % of Compound Remaining

after 120 min Incubation) and Microsomal Stability (Mic.;

% of Compound Remaining after 60 min Incubation) of

a

Choline Analogues Selected for In Vivo Experiments

cmpd.

Pl.

Mic.

cmpd.

Pl.

Mic. cmpd.

Pl.

Mic.

2

3

10

17

19

24

85

81

66

98

80

60

100

74

90

96

100

93

25

26

27

30

34

39

85

89

86

89

81

91

82

86

95

78

54

42

43

44

45

47

48

91

83

91

86

90

89

70

85

79

86

89

92

To further support the idea of a more general applicability of

our compounds, we performed the same cellular assay using a

selection of choline derivatives on a glioblastoma cell line (U-

87 MG), which is also known to overexpress choline

transporters.⁴¹Indeed, the IC₅₀values obtained in the U-87

MG cells were similar to those obtained in the PC-3 cells

(Tables 1 and 3).

a

For complete data, see Table S5, Supporting Information.

Because the cellular uptake of [³H]choline can also be

inﬂuenced by the presence of toxic compounds, all derivatives

were investigated for cytotoxicity. The determined cytotoxic

concentrations of our choline analogues were all greater than

50 μM, except for compounds 24, 25, and 47. However, for

compounds 24, 25, and 47, their corresponding IC₅₀values for

[³H]choline uptake remained well below the cytotoxic

concentrations (Table S5, Supporting Information).

Pharmacokinetic Properties (Absorption, Distribu-

tion, Metabolism, and Excretion, ADME). The introduc-

tion of an iodine atom can potentially have a negative impact

on the pharmacokinetic properties of small organic molecules

because of increased molecular weight and lipophilicity.

Isotope Exchange Labeling. The introduction of an

iodine radioisotope onto an aromatic ring can be accomplished

by several routes.³³A reliable, reproducible, and facile (if

possible) labeling protocol is crucial for successful in vivo

biodistribution experiments and for the prospective application

of the intended radiopharmaceuticals in clinical practice. The

two most common options employed in radioiodination are

iododestannylation and isotope exchange labeling. The high

inherent polarity of the choline moiety, unfortunately, renders

normal-phase chromatography of stannylated choline precur-

sors unfeasible, and employment of a reverse-phase high-

performance liquid chromatography (HPLC) in acidic

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aqueous mobile phase would lead to ipso substitution of the

stannyl group with a hydrogen atom.⁴³Considering that we

previously prepared a set of nonradioactive iodinated choline

analogues containing natural iodine (¹²⁷I), the isotope

exchange reaction seemed to be the most straightforward

synthetic option. The disadvantage of this approach is a lower

molar activity of the obtained radiolabeled product, which

could potentially lead to a less optimal biodistribution proﬁle.

Initially, we examined the feasibility of the isotope exchange

reaction on model compound 23 at rt with the following four

diﬀerent catalysts: CuI, Pd/C, [Pd(PPh₃)₄], and a PdCl₂/

ascorbic acid (AA) mixture (in situ formation of Pd-black).

Interestingly, for compound 23, there was almost no detectable

radioiodinated product in the reaction mixtures except for the

PdCl₂/AA catalytic system, where signiﬁcant conversion was

observed.

low, and the reaction time could not be further increased

because of the formation of radioactive side products.

Biodistribution. To verify the potential of the iodinated

choline analogues as imaging agents, we performed an initial

study of the ex vivo biodistribution of 18 compounds in human

prostate carcinoma-bearing (PC-3) xenograft mice. The

ligands were radiolabeled with ¹²⁵I using the abovementioned

protocol, and 100 kBq of the labeled compound was

intravenously administered to each mouse in a group of six

mice for each ligand. The commercially available [¹⁸F]FCh

served as a control. The biodistribution was determined at 1 h

postinjection (p.i.) in three animals and at 3 h p.i. in three

other animals for each compound as well as for the control

group (n = 3) (Figure 4A,B, for the complete data, see Tables

S6−S26 and Figures S4−S22, Supporting Information).

With the exception of compounds 26, 27, 30, and 48, the

radioactivity accumulated in the thyroid was below 1% of the

injected dose for all analyzed iodinated choline analogues (see

Table S26, Supporting Information). This indicates a negligible

release of free radioiodide and suﬃcient stabilities in vivo.

None of the ¹²⁵I-labeled compounds were able to achieve a

better tumor-to-blood uptake ratio than [¹⁸F]FCh at 1 h p.i.

However, compounds 10 and 43 had similar or higher tumor-

to-blood and tumor-to-muscle uptake ratios than [¹⁸F]FCh at

3 h p.i. and substantially superior tumor-to-bone uptake ratios

(Figure 4C). The absolute value of uptake of 43 in the tumor

was slightly lower compared with [¹⁸F]FCh, but the tumor-to-

blood, tumor-to-muscle, and tumor-to-bone ratios should

allow a suﬃcient image contrast to visualize the tumor.

The compounds 10 and 43 also manifested very low kidney

uptake compared with [¹⁸F]FCh (Figure 4D). Thus, our

results are encouraging because the main disadvantages of the

[¹⁸F]FCh imaging agent lie in its high accumulation in the

bladder and kidneys, which results in diminished image quality

and unfavorable lesion detection sensitivity in close proximity

to the genitourinary system, and the high radiation dose

absorbed by kidneys increases the stochastic risk of such

examinations.²²Because of the propensity of prostate cancer

cells to migrate into the axial skeleton,⁴⁷early detection of

bone metastases is very important. The increased tumor-to-

bone uptake ratios with the reported iodinated choline

analogues compared with [¹⁸F]FCh could lead to improved

sensitivity and speciﬁcity for the detection of bone metastases

in prostate cancer patients.

After further optimization of the isotope exchange reaction

with the PdCl₂/AA catalyst, we achieved up to 75%

radiochemical yield in short reaction time depending on the

labeled substrate. To eliminate the possible competitive

interference of halide counterions in quaternary ammonium

salts during the radioiodination process, all compounds initially

prepared as halide salts were ﬁrst transformed into

corresponding tosylate salts prior to the isotope exchange

reaction; compounds prepared as mesylates were used directly.

The achieved molar activities of the labeled products were

approximately 2 PBq·mol⁻¹(2 GBq·μmol⁻¹), and the radio-

chemical purity was ≥95%.

The optimized labeling protocol (Scheme 4) requires

relatively short reaction times, takes place in aqueous

Scheme 4. Isotope Exchange Reaction with 19 and Na¹²⁵

I

environment under mild conditions, and is applicable to

almost all compounds presented in this study. Puriﬁcation of

the radiolabeled product can be performed by the simple

removal of free radioiodide by an ion-exchange resin and silver

metal. This facile labeling protocol could enable the future

manufacturing of radiopharmaceutical kits for the convenient

synthesis of radiopharmaceuticals in the clinic immediately

prior to their administration to a patient. The achieved molar

activity of the compounds obtained by isotope exchange is

approximately 1 order of magnitude higher than that in the

case of the standard [¹²³I]MIBG used in the clinics but 1 order

of magnitude lower than that in the case of the newer high-

speciﬁc-activity [¹²³I]MIBG-HSA⁴⁴or [¹⁸F]FCh.^45,46

The ligands that could not be labeled using the PdCl₂/AA

catalyst include thiophene or pyridine derivatives (Pd-poison-

ing), nitro-derivatives (substrate reduction), or iodinated

terminal alkynes (decomposition observed). The labeling of

these compounds was performed with CuCl₂as a catalyst at 90

°C in N,N-dimethylformamide (DMF). However, the radio-

chemical yields achieved with the CuCl₂catalyst were rather

In the cases of compounds that generally did not perform

favorably in comparison with [¹⁸F]FCh, we observed a

signiﬁcant decrease in tissue uptake between 1 and 3 h p.i.

This phenomenon could be caused by a gradual washout of the

nonspeciﬁcally bound fraction of the tracer. Interestingly, even

the analogues with a poor biodistribution all exhibited better

tumor-to-bone uptake ratios than [¹⁸F]FCh.

In Vivo Imaging. To provide further evidence that our

compounds could serve as a better alternative to [¹⁸F]FCh, we

performed a set of in vivo imaging experiments with prostate

carcinoma-bearing (PC-3) xenograft mice. The ligands 10 and

43 were radiolabeled with ¹²³I using the herein described

isotope exchange protocol, and 15−20 MBq (molar activity ≥

2.5 GBq·μmol⁻¹) of the corresponding labeled compound was

administered intravenously. The SPECT images were acquired

at 3 h p.i. (see Figure S23A,B, Supporting Information).

Unfortunately, neither of the two compounds [¹²³I]-10 and

[

¹²³I]-43 was able to visualize the tumor. Initially, we ascribed

the decreased tumor accumulation of compounds [¹²³I]-10

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Figure 4. Ex vivo biodistribution at 1 (A) and 3 h p.i. (B) and the corresponding tumor-to-blood, tumor-to-muscle, and tumor-to-bone uptake

ratios at 3 h p.i. (C); tumor-to-kidney uptake ratios at 1 and 3 h p.i. (D) for ¹²⁵I-labeled compound 10, compound 43, and [¹⁸F]FCh in PC-3

tumor-bearing mice.

Scheme 5. Synthesis of the Stannylated Precursor 51 and Its Labeling with ¹²³I by Iododestannylation Yielding High-Molar-

Activity [¹²³I]-43-HSA: (1) Sn₂Bu₆/Pd₂(dba)₃/DIPEA/DMF/1,4-Dioxane; (2) Br(CH₂)₂Br/K₂CO₃/DMF; (3) DMAE/DMF;

(4) Amberlite IRA-900 (Chloride Form)/MeOH

and [¹²³I]-43 in our SPECT experiments to the lower molar

activity of [¹²³I]-10 and [¹²³I]-43 prepared by isotopic

exchange labeling. Mediocre molar activity of [¹²³I]-10 and

produced with molar activity in the 10²GBq·μmol⁻¹range,^48,49

and the speciﬁc activity of [¹⁸F]FCh used in the clinics falls

between 10¹and 10²GBq·μmol⁻¹.^45,46The [¹²³I]-43-HSA

prepared by iododestannylation possessed molar activity ≥20

GBq·μmol⁻¹. However, to our surprise, not even [¹²³I]-43-

HSA was able to visualize the tumor in PC-3 xenograft mice at

3 h p.i. (Figure S23C, Supporting Information), showing high

accumulation of activity only in the gastrointestinal tract and

liver. Immediately after the SPECT imaging with [¹²³I]-43-

HSA, the mice were sacriﬁced, and we performed additional

determination of the tracer biodistribution ex vivo. The

biodistribution data were similar to our previous results

obtained after injecting 100 kBq of [¹²⁵I]-43 with the

[

¹²³I]-43 in combination with the necessity to administer 2

orders of magnitude higher dose of the tracer than in the case

of ex vivo biodistribution experiments (100 kBq of ¹²⁵I-labeled

vs 15−20 MBq of ¹²³I-labeled compounds possessing almost

the same molar activity) could potentially lead to low tumor

uptake.

For this reason, we have decided to synthesize high-molar-

activity (high-speciﬁc-activity) [¹²³I]-43-HSA (Scheme 5) with

a comparable molar activity as the clinically used [¹⁸F]FCh.

Nucleophilic [¹⁸F]F⁻used for the synthesis of [¹⁸F]FCh is

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signiﬁcant diﬀerence that the activity accumulated in the tumor

was 54% lower and the activity accumulated in the gastro-

intestinal tract was higher.

high-molar-activity labeled choline derivatives (≥20

GBq·μmol⁻¹) by iododestannylation is possible.

The ex vivo biodistribution study in human prostate

carcinoma-bearing mice with 18 radiolabeled derivatives

revealed two promising compounds with a biodistribution

proﬁle superior to that of [¹⁸F]FCh, which is routinely used for

imaging in patients with prostate carcinoma. However, it is

important to note that using microPET/microSPECT imaging

techniques neither the commercial [¹⁸F]FCh nor [¹²³I]-43-

HSA could visualize the tumor in the murine PC-3 xenograft.

This xenograft model originally employed in ex vivo studies

during the development of [¹⁸F]FCh and similar tracers

appears to be unsuitable for imaging experiments. Therefore,

the potential of iodinated choline analogues as tracers should

be further validated in a bigger animal model.

These ﬁndings led us to a hypothesis that the tumor uptake

of iodinated choline derivatives in the PC-3 murine model

becomes saturated at doses necessary for imaging even with

high-molar-activity compounds, resulting in poor accumulation

of activity in the tumor. Therefore, we performed also PET

imaging in PC-3 xenograft mice using commercially available

radiopharmaceutical-grade [¹⁸F]FCh. The mice were injected

with [¹⁸F]FCh at a dose of 5−7 MBq per animal and the

acquisition of PET scans was performed both at 1 and 3 h p.i.

as [¹⁸F]FCh exhibited better tumor-to-organ ratios at 1 h p.i. in

our previous ex vivo biodistribution experiments. Surprisingly,

we could not achieve tumor visualization even with [¹⁸F]FCh,

and we observed signiﬁcant signals only in the kidneys and

liver (Figure S23D,E, Supporting Information).

EXPERIMENTAL SECTION

■

The [¹⁸F]FCh tracer is routinely used in the clinics as a

successful tool for cancer imaging. However, to the best of our

knowledge, despite copious amount of in vitro data in PC-3

cells and ex vivo biodistribution studies, there is no report in

the literature of a satisfactory tumor visualization in murine

PC-3 xenograft model using [¹⁸F]FCh.⁵⁰⁻⁵²This strongly

suggests, that in murine PC-3 xenograft model, the choline

transport mechanisms become saturated at doses necessary for

imaging even with high-molar-activity tracers routinely used in

humans. The imaging data in the literature as well as our

SPECT and PET experiments lead us to a conclusion that a

bigger animal model is necessary for successful in vivo imaging

with tracers targeting choline transport proteins. Murine PC-3

xenograft was originally employed for ex vivo studies in the

development of the [¹⁸F]FCh and similar tracers. Despite our

failed imaging attempts with both commercial [¹⁸F]FCh and

the new iodinated choline analogues using this model, the

presented ex vivo data indicate that iodinated choline

derivatives could present a superior alternative to [¹⁸F]FCh.

Materials and General Procedures. Bis(pyridine)iodonium

tetraﬂuoroborate was prepared according to a described procedure.³⁷

Solvents were purchased from Lach-Ner Ltd. (Neratovice, Czech

Republic) and were of analytical grade. All chemicals and dry solvents

were purchased from Sigma-Aldrich Ltd. (Prague, Czech Republic).

The radioactive sodium iodides Na¹²⁵I and Na¹²³I were purchased

́

from MGP Ltd. (Zlin, Czech Republic).

The reactions were monitored by thin-layer chromatography

(TLC) unless stated otherwise. Column chromatography was carried

out using silica gel (70−230 mesh). Flash chromatography was

performed using a PuriFlash XS 420 (Interchim, France) system and

spherical silica gel (40−75 μm). TLC plates (silica gel 60 F254 or

silica gel-RP18 60 F254) were visualized under UV and/or with

KMnO₄/Na₂CO₃. The purity of all ﬁnal compounds (1−50) was

>95% as determined by HPLC-UV with detection at 225 nm (for

retention times and purity, see also Table S1, Supporting

Information).

Synthesis of Choline Derivatives (1−50). General Procedure

for the Synthesis of Iodinated Choline Analogues. Procedure A. A

pear-shaped ﬂask was charged with a magnetic stir bar, the

corresponding alkylating agent, DMF, and the corresponding tertiary

amine. The ﬂask was closed with a rubber septum, ﬂushed with argon,

immersed into a heated oil bath, and the reaction mixture was stirred.

The reaction progress was checked by a TLC analysis. When either

conversion of the alkylating agent was complete or formation of

elimination product became apparent, the heating was stopped, and

the solution was concentrated in vacuo (oily products optionally

coevaporated with toluene in order to remove residual DMF). The

residue was redissolved in a minimum amount of MeOH, transferred

into a centrifugation tube, precipitated with 1:1 diethyl ether/hexane

mixture (45 mL), and the suspension was centrifuged and decanted.

Then, the residue was redissolved in MeOH, precipitated with diethyl

ether (40 mL), centrifuged, and decanted. The solid product was

transferred with a spatula into a glass vial and dried under high

vacuum. Some of the products were further subjected to anion

exchange in order to remove halogenide counterions, which could

interfere with isotope exchange radioiodination. This was done by

passing the MeOH solution of the compound through a column

containing Amberlite IRA-900 anion-exchange resin in TsO⁻cycle

(15 mL of resin per 1 mmol of choline analogue).

CONCLUSIONS

■

In summary, 28 iodinated derivatives from this study exhibited

better IC₅₀values than FCh and 14 derivatives had IC₅₀values

that were even better than the IC₅₀value determined for the

natural ligand (choline). Six compounds from this study are

nanomolar inhibitors, and the best analogue (compound 43)

surpassed the aﬃnity of choline by 2 orders of magnitude.

We successfully developed a straightforward radiolabeling

protocol that can be used for the preparation of SPECT and

PET tracers and potentially theranostic radiopharmaceuticals

depending on the selection of the particular iodine isotope. In

contrast to [¹⁸F]FCh, radiopharmaceuticals based on our

compounds could be distributed from a central production site

to more-remote medical facilities (longer half-lives). Further-

more, our radiolabeling procedure also allows for the

manufacture of kits for the synthesis of these radiopharma-

ceuticals directly in the clinic. The molar activity of iodinated

compounds prepared by isotope exchange labeling was on the

order of ≈2 GBq·μmol⁻¹, which is signiﬁcantly lower than the

molar activity of ¹⁸F-based tracers prepared using nucleophilic

substitution with [¹⁸F]F⁻. However, the molar activity we

achieved with isotope exchange labeling is at least 1 order of

magnitude higher than that in the case of tracers prepared by

electrophilic ﬂuorination with [¹⁸F]F₂(e.g., [¹⁸F]FDOPA) used

in the clinics.^53,54We have also demonstrated that synthesis of

(2-Hydroxyethyl)-(2-iodobenzyl)-dimethylammonium 4-Methyl-

benzenesulfonate (1). General procedure A was used. 2-Iodobenzyl

iodide (400 mg, 1.16 mmol, 1 equiv), DMF (5.0 mL), and 2-

(dimethylamino)ethanol (351 μL, 3.49 mmol, 3 equiv) were used for

the synthesis of compound 1. The reaction time was 2 h at a

temperature of 50 °C. The reaction mixture was concentrated in vacuo

and coevaporated with toluene (3×). The crude product was

precipitated from a MeOH solution once with a mixture of Et₂O

and hexane (2:1 v/v, 45 mL) and once with pure Et₂O (45 mL). The

product was converted to 4-methylbenzenesulfonate salt by ion

exchange, and (2-hydroxyethyl)-(2-iodobenzyl)-dimethylammonium

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4-methylbenzenesulfonate (1) was obtained as a colorless viscous

liquid (500 mg, 1.05 mmol, 90%). ¹H NMR (600 MHz, DMSO-d₆): δ

8.08 (dd, J = 8.0, 1.2 Hz, 1H), 7.72 (dd, J = 7.7, 1.6 Hz, 1H), 7.55

(ddd, J = 7.5, 7.5, 1.3 Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.27 (ddd, J

= 7.7, 7.7, 1.6 Hz, 1H), 7.11 (d, J = 7.8 Hz, 2H), 5.41 (br s, 1H), 4.72

(s, 2H), 3.97−3.91 (m, 2H), 3.56−3.53 (m, 2H), 3.09 (s, 6H), 2.28

(s, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 145.71, 140.97, 137.64,

134.94, 132.28, 131.16, 128.82, 128.09, 125.50, 105.11, 70.48, 66.54,

55.15, 50.31, 20.82. HRMS (ESI) m/z: [M⁺] (C₁₁H₁₇INO⁺) calcd,

306.034934; found, 306.03521. HPLC-UV purity 98.5%.

Diethyl-(2-hydroxyethyl)-(2-iodobenzyl)ammonium 4-Methyl-

benzenesulfonate (2). General procedure A was used. 2-Iodobenzyl

iodide (400 mg, 1.16 mmol, 1 equiv), DMF (5.0 mL), and 2-

(diethylamino)ethanol (463 μL, 3.49 mmol, 3 equiv) were used for

the synthesis of compound 2. The reaction time was 2 h at a

temperature of 50 °C. The reaction mixture was concentrated in vacuo

and coevaporated with toluene (3×). The crude product was

precipitated from a MeOH solution once with a mixture of Et₂O

and hexane (2:1 v/v, 45 mL) and once with pure Et₂O (45 mL). The

product was converted to 4-methylbenzenesulfonate salt by ion

exchange. Compound 2 was obtained as a colorless oil, which

crystallized upon cooling (477 mg, 0.94 mmol, 81%). mp 103−105

°C. ¹H NMR (600 MHz, DMSO-d₆): δ 8.07 (dd, J = 7.9, 1.3 Hz, 1H),

7.67 (dd, J = 7.8, 1.4 Hz, 1H), 7.55 (ddd, J = 7.6, 7.6, 1.3 Hz, 1H),

7.48 (d, J = 8.0 Hz, 2H), 7.26 (ddd, J = 7.7, 7.7, 1.6 Hz, 1H), 7.11 (d,

J = 7.9 Hz, 2H), 5.44 (br s, 1H), 4.72 (s, 2H), 3.87 (br s, 2H), 3.47−

3.37 (m, 6H), 2.28 (s, 3H), 1.28 (t, J = 7.1 Hz, 6H). ¹³C NMR (151

MHz, DMSO-d₆): δ 145.71, 140.92, 137.63, 134.11, 132.17, 131.26,

129.07, 128.08, 125.49, 105.51, 65.31, 59.38, 54.76, 54.32, 20.81, 8.18.

HRMS (ESI) m/z: [M⁺] (C₁₃H₂₁INO⁺) calcd, 334.066234; found,

334.06648. Elem. Anal. C₂₀H₂₈INO₄S, Calcd: C (47.53), H (5.58), N

(2.77), I (25.11), S (6.34). Found: C (47.42), H (5.60), N (2.74), I

(25.01), S (6.51). HPLC-UV purity 99.2%.

1-(2-Hydroxyethyl)-1-(4-iodobenzyl)pyrrolidin-1-ium 4-Methyl-

benzenesulfonate (3). General procedure A was used. 2-Iodobenzyl

iodide (400 mg, 1.16 mmol, 1 equiv), DMF (5.0 mL), and 2-

(diethylamino)ethanol (463 μL, 3.49 mmol, 3 equiv) were used for

the synthesis of compound 3. The reaction time was 2 h at a

temperature of 50 °C. The reaction mixture was concentrated in vacuo

and coevaporated with toluene (3×). The crude product was

precipitated from a MeOH solution once with a mixture of Et₂O

and hexane (2:1 v/v, 45 mL) and once with pure Et₂O (45 mL). The

product was converted to 4-methylbenzenesulfonate salt by ion

exchange. Compound 3 was obtained as a pale-yellow oil, which

crystallized upon cooling (453 mg, 0.90 mmol, 78%). mp 97−99 °C.

¹H NMR (300 MHz, DMSO-d₆): δ 8.07 (dd, J = 8.0, 1.2 Hz, 1H),

7.74 (dd, J = 7.8, 1.6 Hz, 1H), 7.54 (ddd, J = 7.5, 7.5, 1.3 Hz, 1H),

7.48 (d, J = 7.9 Hz, 2H), 7.26 (ddd, J = 7.7, 7.7, 1.7 Hz, 1H), 7.11 (d,

J = 7.8 Hz, 2H), 5.47 (t, J = 4.4 Hz, 1H), 4.80 (s, 2H), 3.99−3.93 (m,

2H), 3.83−3.63 (m, 2H), 3.54−3.45 (m, 2H), 3.44−3.29 (m, 2H),

2.28 (s, 3H), 2.14−1.87 (m, 4H). ¹³C NMR (75 MHz, DMSO-d₆): δ

145.69, 140.81, 137.63, 134.80, 132.16, 131.57, 128.94, 128.07,

125.48, 105.48, 66.43, 62.17, 60.26, 55.70, 22.41, 20.80. HRMS (ESI)

m/z: [M⁺] (C₁₃H₁₉INO⁺) calcd, 332.050584; found, 332.05093.

Elem. Anal. C₂₀H₂₆INO₄S, Calcd: C (47.72), H (5.21), N (2.78), S

(6.37). Found: C (47.68), H (5.21), N (2.78), S (6.31). HPLC-UV

purity 98.8%.

5.48 (t, J = 4.7 Hz, 1H), 4.80 (s, 2H), 4.08−3.87 (m, 2H), 3.73−3.58

(m, 4H), 3.19−3.07 (m, 2H), 2.28 (s, 3H), 2.05−1.83 (m, 2H),

1.82−1.67 (m, 2H), 1.65−1.47 (m, 1H), 1.40−1.17 (m, 1H). ¹³C

NMR (75 MHz, DMSO-d₆): δ 145.66, 140.99, 137.65, 135.25,

132.16, 130.87, 128.79, 128.07, 125.48, 105.67, 69.34, 58.65, 57.77,

54.94, 20.80, 20.09, 19.29. HRMS (ESI) m/z: [M⁺] (C₁₄H₂₁INO⁺)

calcd, 346.066234; found, 346.06628. Elem. Anal. C₂₁H₂₈INO₄S,

Calcd: C (48.75), H (5.45), N (2.71), S (6.20). Found: C (49.07), H

(5.58), N (2.62), S (6.28). HPLC-UV purity 99.7%.

3-Hydroxy-1-(2-iodobenzyl)quinuclidin-1-ium 4-Methylbenzene-

sulfonate (5). General procedure A was used. 2-Iodobenzyl iodide

(400 mg, 1.16 mmol, 1 equiv), DMF (5.0 mL), and 3-quinuclidinol

(444 mg, 3.49 mmol, 3 equiv) were used for the synthesis of

compound 5. The reaction time was 2 h at a temperature of 50 °C.

The reaction mixture was concentrated in vacuo and coevaporated

with toluene (3×). The crude product was precipitated from a MeOH

solution once with a mixture of Et₂O and hexane (2:1 v/v, 45 mL)

and once with pure Et₂O (45 mL). The product was converted to 4-

methylbenzenesulfonate salt by ion exchange. Compound 5 was

obtained as a white solid (496 mg, 0.96 mmol, 83%). mp 154−156

°C. ¹H NMR (600 MHz, DMSO-d₆): δ 8.07 (dd, J = 8.0, 1.2 Hz, 1H),

7.66 (dd, J = 7.7, 1.6 Hz, 1H), 7.54 (ddd, J = 7.5, 7.5, 1.3 Hz, 1H),

7.48 (d, J = 8.1 Hz, 2H), 7.25 (ddd, J = 7.7, 7.7, 1.6 Hz, 1H), 7.11 (d,

J = 7.9 Hz, 2H), 5.60 (s, 1H), 4.59 (d, J = 13.5 Hz, 1H), 4.54 (d, J =

13.5 Hz, 1H), 4.08−4.02 (m, 1H), 3.74 (ddd, J = 12.7, 8.3, 3.0 Hz,

1H), 3.51 (tt, J = 11.2, 3.8 Hz, 1H), 3.47−3.32 (m, 3H), 3.14 (dt, J =

12.7, 2.8 Hz, 1H), 2.28 (s, 3H), 2.15−2.07 (m, 1H), 2.03−1.99 (m,

1H), 1.94−1.87 (m, 1H), 1.80−1.70 (m, 2H). ¹³C NMR (151 MHz,

DMSO-d₆): δ 145.73, 140.90, 137.63, 135.04, 132.17, 130.73, 128.78,

128.08, 125.50, 105.03, 69.40, 63.31, 63.21, 54.68, 53.51, 26.39, 20.97,

20.82, 17.47. HRMS (ESI) m/z: [M⁺] (C₁₄H₁₉INO⁺) calcd,

344.050584; found, 344.05065. Elem. Anal. C₂₁H₂₆INO₄S, Calcd: C

(48.94), H (5.08), N (2.72), I (24.62), S (6.22). Found: C (48.64), H

(5.15), N (2.59), I (24.21), S (6.11). HPLC-UV purity 98.0%.

3-Hydroxy-1-(2-iodobenzyl)-1-methylpiperidin-1-ium 4-Methyl-

benzenesulfonate (6). General procedure A was used. 2-Iodobenzyl

iodide (400 mg, 1.16 mmol, 1 equiv), DMF (5.0 mL), and 3-hydroxy-

1-methylpiperidine (402 μL, 3.49 mmol, 3 equiv) were used for the

synthesis of compound 6. The reaction time was 2 h at a temperature

of 50 °C. The reaction mixture was concentrated in vacuo and

coevaporated with toluene (3×). The crude product was precipitated

from a MeOH solution once with a mixture of Et₂O and hexane (2:1

v/v, 45 mL) and once with pure Et₂O (45 mL). The product was

converted to 4-methylbenzenesulfonate salt by ion exchange.

Compound 6 was obtained as a mixture of two diastereomers (2:1)

in the form of an oﬀ-white hygroscopic solid (424 mg, 0.84 mmol,

1

73%). H NMR (300 MHz, DMSO-d₆): δ 8.08 (dd, J = 8.0, 1.3 Hz,

1H), 7.72 (dd, J = 7.8, 1.7 Hz, 0.67H), 7.65 (dd, J = 7.7, 1.7 Hz,

0.33H), 7.58−7.52 (m, 1H), 7.49 (d, J = 8.0 Hz, 2H), 7.27 (ddd, J =

7.7, 7.7, 1.7 Hz, 1H), 7.11 (d, J = 7.9 Hz, 2H), 5.50 (s, 1H), 4.84−

4.58 (m, 2H), 4.22−4.14 (m, 0.33H), 4.05−3.93 (m, 0.67H), 3.57−

3.01 (m, 4H), 3.14 (s, 1H), 3.04 (s, 2H), 2.29 (s, 3H), 2.11−1.69 (m,

3H), 1.68−1.52 (m, 0.33H), 1.41−1.22 (m, 0.67H). ¹³C NMR (101

MHz, DMSO-d₆): δ 145.71, 141.03, 141.03, 137.64, 135.17, 134.86,

132.27, 132.25, 130.77, 130.72, 128.79, 128.74, 128.07, 125.49,

105.15, 105.11, 72.20, 70.77, 63.78, 63.55, 61.95, 61.00, 60.46, 59.78,

49.24, 46.66, 30.51, 28.51, 20.79, 17.77, 16.31. HRMS (ESI) m/z:

[M⁺] (C₁₃H₁₉INO⁺) calcd, 332.050584; found, 332.05079. Elem.

Anal. C₂₀H₂₆INO₄S·0.5H₂O, Calcd: C (46.88), H (5.31), N (2.73), I

(24.77), S (6.26). Found: C (47.13), H (5.36), N (2.78), I (24.67).

HPLC-UV purity 98.0%.

1,4-Bis(2-hydroxyethyl)-1-(2-iodobenzyl)piperazin-1-ium 4-

Methylbenzenesulfonate (7). General procedure A was used. 2-

Iodobenzyl iodide (400 mg, 1.16 mmol, 1 equiv), DMF (5.0 mL), and

1,4-bis(2-hydroxyethyl)piperazine (608 mg, 3.49 mmol, 3 equiv) were

used for the synthesis of compound 7. The reaction time was 2 h at a

temperature of 50 °C. The reaction mixture was concentrated in vacuo

and coevaporated with toluene (3×). The crude product was

precipitated from a MeOH solution once with a mixture of Et₂O

and hexane (2:1 v/v, 45 mL) and once with pure Et₂O (45 mL). The

1-(2-Hydroxyethyl)-1-(2-iodobenzyl)piperidin-1-ium 4-Methyl-

benzenesulfonate (4). 2-Iodobenzyl chloride (600 mg, 2.38 mmol,

1 equiv) and 1-(2-hydroxyethyl)piperidine (4 mL) were stirred under

argon in a pear-shaped ﬂask for 16 h at a temperature of 80 °C. The

reaction mixture was concentrated in vacuo and coevaporated with

toluene (3×). The crude product was dissolved in water (50 mL), and

excess amine was removed by extraction with EtOAc (3 × 40 mL).

The product was converted to 4-methylbenzenesulfonate salt by ion

exchange. Compound 4 was obtained as a brown viscous liquid, which

1

crystallized upon cooling (938 mg, 1.81 mmol, 76%). H NMR (300

MHz, DMSO-d₆): δ 8.08 (dd, J = 8.0, 1.3 Hz, 1H), 7.68 (dd, J = 7.8,

1.6 Hz, 1H), 7.54 (ddd, J = 7.5, 7.5, 1.3 Hz, 1H), 7.49 (d, J = 8.1 Hz,

2H), 7.26 (ddd, J = 7.8, 7.8, 1.5 Hz, 1H), 7.11 (d, J = 7.9 Hz, 2H),

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product was converted to 4-methylbenzenesulfonate salt by ion

compound 11. The reaction time was 30 min at a temperature of 75

°C. The product was precipitated from a MeOH solution once with a

mixture of Et₂O and hexane (3:1 v/v, 40 mL) and once with pure

Et₂O (40 mL). The product 11 was obtained as a white solid (472

exchange. Compound 7 was obtained as a white amorphous

1

hygroscopic solid (502 mg, 0.89 mmol, 77%). H NMR (600 MHz,

DMSO-d₆): δ 8.09 (dd, J = 8.0, 1.2 Hz, 1H), 7.71 (dd, J = 7.7, 1.6 Hz,

1H), 7.56 (ddd, J = 7.6, 7.6, 1.2 Hz, 1H), 7.49 (d, J = 8.0 Hz, 2H),

7.28 (ddd, J = 7.7, 7.7, 1.6 Hz, 1H), 7.12 (d, J = 7.9 Hz, 2H), 5.52 (t, J

= 4.8 Hz, 1H), 4.86 (s, 2H), 4.49 (br s, 1H), 4.03−3.99 (m, 2H),

3.77−3.65 (m, 4H), 3.44 (q, J = 5.7 Hz, 2H), 3.24 (t, J = 10.7 Hz,

2H), 2.96−2.86 (m, 2H), 2.82−2.65 (m, 2H), 2.46−2.41 (m, 2H),

2.28 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 145.64, 141.06,

137.70, 135.55, 132.33, 130.64, 128.85, 128.11, 125.50, 105.87, 69.36,

58.71, 58.47, 58.27, 56.81, 54.94, 46.05, 20.82. HRMS (ESI) m/z:

1

mg, 1.22 mmol, 91%). H NMR (300 MHz, DMSO-d₆): δ 7.88 (d, J

= 8.2 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 5.34 (t, J = 5.0 Hz, 1H), 4.63

(s, 2H), 3.94−3.86 (m, 2H), 3.43−3.37 (m, 2H), 3.03 (s, 6H). ¹³C

NMR (75 MHz, DMSO-d₆): δ 137.67, 135.10, 127.75, 98.01, 66.51,

64.88, 54.83, 49.78. HRMS (ESI) m/z: [M⁺] (C₁₁H₁₇INO⁺) calcd,

306.0349; found, 306.0341. HPLC-UV purity 98.7%.

Diethyl-(2-hydroxyethyl)-(4-iodobenzyl)ammonium Bromide

(12). General procedure A was used. 4-Iodobenzyl bromide (400

mg, 1.35 mmol, 1 equiv), DMF (2 mL), and 2-(diethylamino)ethanol

(268 μL, 2.02 mmol, 1.5 equiv) were used for the synthesis of

compound 12. The reaction time was 30 min at a temperature of 75

°C. The precipitation was performed from a MeOH solution once

with the mixture of Et₂O and hexane (3:1 v/v, 40 mL) and once with

pure Et₂O (40 mL). The product 12 was obtained as a white solid

(459 mg, 1.11 mmol, 82%). ¹H NMR (300 MHz, DMSO-d₆): δ 7.87

(d, J = 8.3 Hz, 2H), 7.36 (d, J = 8.3 Hz, 2H), 5.38 (t, J = 5.1 Hz, 1H),

4.58 (s, 2H), 3.88 (dt, J = 5.2, 5.1 Hz, 1H), 3.30−3.20 (m, 6H), 1.31

(t, J = 7.1 Hz, 6H). ¹³C NMR (75 MHz, DMSO-d₆): δ 137.76,

134.87, 127.48, 97.87, 60.30, 58.06, 54.45, 53.25, 7.79. HRMS (ESI)

m/z: [M⁺] (C₁₃H₂₁INO⁺) calcd, 334.0662; found, 334.0650. HPLC-

UV purity 97.7%.

1-(2-Hydroxyethyl)-1-(4-iodobenzyl)pyrrolidin-1-ium Bromide

(13). General procedure A was used. 4-Iodobenzyl bromide (400

mg, 1.35 mmol, 1 equiv), DMF (2 mL), and 2-(pyrrolidin-1-

yl)ethanol (236 μL, 2.02 mmol, 1.5 equiv) were used for the synthesis

of compound 13. The reaction time was 30 min at a temperature of

75 °C. The product was precipitated from a MeOH solution once

with a mixture of Et₂O and hexane (3:1 v/v, 40 mL) and once with

pure Et₂O (40 mL). The product 13 was obtained as a white solid

(484 mg, 1.17 mmol, 87%).

+

[M⁺] (C₁₅H₂₄IN₂O₂) calcd, 391.087697; found, 391.08771. HPLC-

UV purity 98.0%.

4-(2-Hydroxyethyl)-4-(2-iodobenzyl)morpholin-4-ium Bromide

(8). 2-Iodobenzyl bromide (400 mg, 1.35 mmol, 1 equiv) and 4-(2-

hydroxyethyl)morpholine (2.0 mL, 16.5 mmol, 13.9 equiv) were

stirred in a pear-shaped ﬂask at 75 °C for 4 h. The reaction mixture

was diluted with MeOH, transferred into a centrifugation tube, and

mixed with Et₂O/hexane (1:1, 40 mL) (oily product formation). The

tube was centrifuged, and the solvent was decanted from the product.

The product was then dissolved in MeOH (5 mL) and precipitated

with Et₂O (40 mL). This was repeated two times. The product 8 was

dried under high vacuum yielding an oﬀ-white solid (509 mg, 1.19

mmol, 88%). ¹H NMR (400 MHz, DMSO-d₆): δ 8.10 (dd, J = 8.0, 1.3

Hz, 1H), 7.74 (dd, J = 7.8, 1.7 Hz, 1H), 7.57 (ddd, J = 7.8, 7.4, 1.3

Hz, 1H), 7.29 (ddd, J = 8.0, 7.4, 1.7 Hz, 1H), 5.50 (t, J = 4.8 Hz, 1H),

4.94 (s, 2H), 4.09−3.99 (m, 4H), 3.95−3.88 (m, 2H), 3.88−3.83 (m,

2H) 3.74−3.65 (m, 2H), 3.39−3.30 (m, 2H). ¹³C NMR (101 MHz,

DMSO-d₆): δ 141.03, 135.51, 132.34, 130.42, 128.80, 106.10, 69.76,

59.91, 58.67, 56.50, 55.10. HPLC-UV purity 99.0%.

(2-Hydroxyethyl)-(3-iodobenzyl)-dimethylammonium Methane-

sulfonate (9). General procedure A was used. The alkylating agent A

(200 mg, 0.67 mmol, 1 equiv, see the Supporting Information), DMF

(1.5 mL), and 2-(dimethylamino)ethanol (134 μL, 1.33 mmol, 2

equiv) were used for the synthesis of compound 9. The reaction time

was 1 h at a temperature of 80 °C. The product was precipitated once

from a dichloromethane (DCM) solution with a mixture of Et₂O and

hexane (2:1 v/v, 45 mL) and once from MeOH solution by a mixture

of Et₂O and hexane (2:1 v/v, 45 mL). The product was obtained in as

a colorless viscous liquid (259 mg, 0.67 mmol, 100%). ¹H NMR (300

MHz, DMSO-d₆): δ 7.99 (dd, J = 1.8, 1.7 Hz, 1H), 7.91 (ddd, J = 7.9,

1.8, 1.1 Hz, 1H), 7.60 (ddd, J = 7.7, 1.7, 1.1 Hz, 1H), 7.32 (dd, J =

7.9, 7.7 Hz, 1H), 5.36 (t, J = 4.9 Hz, 1H), 4.53 (s, 2H), 3.95−3.87 (m,

2H), 3.41−3.34 (m, 2H), 3.01 (s, 6H), 2.30 (s, 3H). ¹³C NMR (151

MHz, DMSO-d₆): δ 141.32, 138.91, 132.53, 130.89, 130.59, 95.17,

66.43, 65.16, 54.92, 49.89, 39.78, 39.52. HRMS (ESI) m/z: [M⁺]

(C₁₁H₁₇INO⁺) calcd, 306.03493; found, 306.03526. HPLC-UV purity

97.4%.

1-(2-Hydroxyethyl)-1-(3-iodobenzyl)pyrrolidin-1-ium Methane-

sulfonate (10). General procedure A was used. The alkylating agent

A (400 mg, 1.27 mmol, 1 equiv, see the Supporting Information),

DMF (2.0 mL), and 2-(pyrrolidin-1-yl)ethanol (223 μL, 1.91 mmol,

1.5 equiv) were used for the synthesis of compound 10. The reaction

time was 1 h at a temperature of 75 °C. The product was precipitated

from a MeOH solution once with a mixture of Et₂O and hexane (2:1

v/v, 45 mL) and once with pure Et₂O (45 mL). The product was

obtained as a yellowish viscous liquid (500 mg, 1.16 mmol, 91%). ¹H

NMR (300 MHz, DMSO-d₆): δ 8.02 (t, J = 1.7 Hz, 1H), 7.89 (dt, J =

7.9, 1.1 Hz, 1H), 7.65 (dt, J = 7.7, 1.2 Hz, 1H), 7.31 (t, J = 7.8 Hz,

1H), 5.51 (t, J = 4.8 Hz, 1H), 4.61 (s, 2H), 3.94 (dt, J = 5.0, 4.8 Hz,

2H), 3.64−3.44 (m, 4H), 3.32−3.25 (m, 2H), 2.34 (s, 3H), 2.11−

2.03 (m, 4H). ¹³C NMR (75 MHz, DMSO-d₆): δ 141.04, 138.83,

132.29, 131.34, 130.95, 95.30, 61.67, 60.97, 59.94, 55.40, 54.94, 20.84.

HRMS (ESI) m/z: [M⁺] (C₁₃H₁₉INO⁺) calcd, 332.0506; found,

332.0495. HPLC-UV purity 96.9%.

¹H NMR (300 MHz, DMSO-d₆): δ 7.87 (d, J = 8.3 Hz, 2H), 7.42

(d, J = 8.3 Hz, 2H), 5.43 (t, J = 4.9 Hz, 1H), 4.63 (s, 2H), 3.93 (dt, J

= 4.9, 4.9 Hz, 2H), 3.64−3.45 (m, 4H), 3.31−3.23 (m, 2H), 2.14−

1.99 (m, 4H). ¹³C NMR (75 MHz, DMSO-d₆): δ 137.75, 134.86,

128.42, 97.83, 61.71, 60.90, 59.75, 55.31, 20.78. HRMS (ESI) m/z:

[M⁺] (C₁₃H₁₉INO⁺) calcd, 332.0506; found, 332.0494. HPLC-UV

purity 98.9%.

4-(2-Hydroxyethyl)-4-(4-iodobenzyl)morpholin-4-ium Bromide

(14). 4-Iodobenzyl bromide (300 mg, 1.01 mmol, 1 equiv) and 4-

(2-hydroxyethyl)morpholine (4.00 mL, 33,0 mmol, 32.7 equiv) were

stirred in a pear-shaped ﬂask overnight at 72 °C. The reaction mixture

was diluted with MeOH, transferred into a centrifugation tube, and

mixed with Et₂O/hexane (1:1, 40 mL) (oily product formation). The

tube was centrifuged, and the solvent was decanted from the oily

product. The product was then dissolved in MeOH (5 mL) and

triturated with Et₂O (40 mL). This was repeated two times. The oily

product 14 was dried under high vacuum yielding an amorphous pink

solid (418 mg, 0.98 mmol, 97%). ¹H NMR (300 MHz, DMSO-d₆): δ

7.89 (d, J = 8.3 Hz, 2H), 7.39 (d, J = 8.3 Hz, 2H), 5.49 (t, J = 4.7 Hz,

1H), 4.81 (s, 2H), 4.03−3.90 (m, 6H), 3.54−3.40 (m, 6H). ¹³C NMR

(75 MHz, DMSO-d₆): δ 137.79, 135.48, 126.83, 98.15, 64.11, 59.90,

+

57.01, 56.67, 54.51. HRMS (ESI) m/z: [M⁺] (C₁₃H₁₉INO₂) calcd,

348.04550; found, 348.04523. HPLC-UV purity 99.0%.

(2-Hydroxyethyl)-[2-(4-iodophenyl)ethyl]-dimethylammonium

4-Methylbenzenesulfonate (15). General procedure A was used. The

alkylating agent B3 (400 mg, 0.99 mmol, 1 equiv, see the Supporting

Information), DMF (5 mL), and 2-(dimethylamino)ethanol (300 μL,

2.98 mmol, 3 equiv) were used for the synthesis of compound 15.

The reaction time was 2 h at a temperature of 60 °C and 2 h at a

temperature of 80 °C. The precipitation was done once using the

mixture of Et₂O and hexane (1:1 v/v, 45 mL) and subsequently twice

with pure Et₂O (40 mL). The product 15 was obtained as a white

1

solid (270 mg, 0.55 mmol, 56%). mp 204−206 °C. H NMR (600

(2-Hydroxyethyl)-(4-iodobenzyl)-dimethylammonium Bromide

(11). General procedure A was used. 4-Iodobenzyl bromide (400

mg, 1.35 mmol, 1 equiv), DMF (2 mL), and 2-(dimethylamino)-

ethanol (303 μL, 3.01 mmol, 2.2 equiv) were used for the synthesis of

MHz, DMSO-d₆): δ 7.70 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 8.0 Hz,

2H), 7.13 (d, J = 8.2 Hz, 2H), 7.11 (d, J = 8.0 Hz, 2H), 5.35 (t, J = 5.0

Hz, 1H), 3.88−3.83 (m, 2H), 3.55−3.50 (m, 2H), 3.49−3.44 (m,

15969

https://dx.doi.org/10.1021/acs.jmedchem.0c01710

J. Med. Chem. 2020, 63, 15960−15978

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

2H), 3.13 (s, 6H), 3.03−2.97 (m, 2H), 2.28 (s, 3H). ¹³C NMR (151

MHz, DMSO-d₆): δ 145.66, 137.67, 137.38, 136.25, 131.48, 128.10,

125.48, 92.88, 64.78, 64.27, 55.04, 50.90, 27.73, 20.82. HRMS (ESI)

m/z: [M⁺] (C₁₂H₁₉INO⁺) calcd, 320.050584; found, 320.05048.

Elem. Anal. C₁₉H₂₆INO₄S, Calcd: C (46.44), H (5.33), N (2.85), I

(25.83), S (6.53). Found: C (46.65), H (5.32), N (2.64), I (25.56), S

(6.55). HPLC-UV purity 99.3%.

63.44, 63.02, 62.67, 54.04, 52.74, 27.06, 26.36, 20.90, 20.82, 17.39.

HRMS (ESI) m/z: [M⁺] (C₁₅H₂₁INO⁺) calcd, 358.066234; found,

358.06608, Elem. Anal. C₂₂H₂₈INO₄S, Calcd: C (49.91), H (5.33), N

(2.65), I (23.97), S (6.06). Found: C (50.12), H (5.37), N (2.46), I

(23.73), S (6.21). HPLC-UV purity 98.8%.

(2-Hydroxyethyl)-[2-(3-iodophenyl)ethyl]-dimethylammonium

Methanesulfonate (19). General procedure A was used. The

alkylating agent C3 (250 mg, 0.767 mmol, 1 equiv, see the Supporting

Information), DMF (1.2 mL), and 2-(dimethylamino)ethanol (463

μL, 4.60 mmol, 6 equiv) were used for the synthesis of compound 19.

The reaction time was 3 h at a temperature of 70 °C and 2 h at a

temperature of 80 °C. The precipitation was done from MeOH (4

mL) solution once using the mixture of Et₂O and hexane (1:1 v/v, 45

mL). The product was dissolved in chloroform (4 mL) and

precipitated with neat E₂O (20 mL). Compound 19 was obtained

as a yellow viscous liquid (253 mg, 0.609 mmol, 79%). ¹H NMR (300

MHz, DMSO-d₆): δ 7.75 (dd, J = 1.6, 1.6 Hz, 1H), 7.65 (ddd, J = 7.8,

1.6, 1.1 Hz, 1H), 7.34 (ddd, J = 7.7, 1.6, 1.1 Hz, 1H), 7.16 (dd, J =

7.8, 7.7 Hz, 1H), 5.35 (t, J = 4.9 Hz, 1H), 3.91−3.81 (m, 2H), 3.59−

3.50 (m, 2H), 3.50−3.43 (m, 2H), 3.13 (s, 6H), 3.07−2.98 (m, 2H),

2.30 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 139.15, 137.57,

135.65, 130.77, 128.62, 95.08, 64.85, 64.35, 55.01, 50.89, 39.77, 27.64.

HRMS (ESI) m/z: [M⁺] (C₁₂H₁₉INO⁺) calcd, 320.05058; found,

320.05090. HPLC-UV purity 98.0%.

(2-Hydroxyethyl)-[2-(3-iodo-4-methoxyphenyl)ethyl]-dimethy-

lammonium 4-Methylbenzenesulfonate (20). General procedure A

was used. The alkylating agent D3 (200 mg, 0.463 mmol, 1 equiv, see

the Supporting Information), DMF (2 mL), and 2-(dimethylamino)-

ethanol (140 μL, 1.39 mmol, 3 equiv) were used for the synthesis of

compound 20. The reaction time was 3 h at a temperature of 80 °C.

The precipitation was done twice using a mixture of Et₂O and hexane

(1:1 v/v, 45 mL). The product 20 was obtained as a white solid (137

mg, 0.263 mmol, 57%). ¹H NMR (600 MHz, DMSO-d₆): δ 7.75 (d, J

= 1.9 Hz, 1H), 7.48 (d, J = 8.0 Hz, 2H), 7.30 (dd, J = 8.4, 1.8 Hz,

1H), 7.11 (d, J = 7.9 Hz, 2H), 6.97 (d, J = 8.4 Hz, 1H), 5.34 (t, J = 5.0

Hz, 1H), 3.88−3.83 (m, 2H), 3.80 (s, 3H), 3.52−3.47 (m, 2H),

3.48−3.44 (m, 2H), 3.13 (s, 6H), 2.98−2.94 (m, 2H), 2.28 (s, 3H).

¹³C NMR (151 MHz, DMSO-d₆): δ 156.76, 145.69, 139.27, 137.65,

3-Hydroxy-1-[2-(4-iodophenyl)ethyl]-1-methylpiperidin-1-ium 4-

Methylbenzenesulfonate (16). General procedure A was used. The

alkylating agent B3 (400 mg, 0.99 mmol, 1 equiv, see the Supporting

Information), DMF (5 mL), and 1-methylpiperidine-3-ol (344 μL,

2.98 mmol, 3 equiv) were used for the synthesis of compound 16.

The reaction time was 2 h at a temperature of 60 °C, 2 h at a

temperature of 80 °C, and 2 h at a temperature of 90 °C. The

precipitation was done three times using a mixture of Et₂O and

hexane (1:1 v/v, 45 mL). The product 16 was obtained as a mixture

of two diastereomers (1:1 ratio according to the NMR analysis) as a

1

pale-yellow solid (269 mg, 0.52 mmol, 53%). H NMR (600 MHz,

DMSO-d₆): δ 7.71 (d, J = 8.3 Hz, 1H, diastereomer 1), 7.70 (d, J =

8.3 Hz, 1H, diastereomer 2), 7.48 (d, J = 8.2 Hz, 2H, 4-

methylbenzenesulfonate), 7.16 (d, J = 8.2 Hz, 1H), 7.13 (d, J = 8.2

Hz, 1H), 7.11 (d, J = 8.2 Hz, 2H, 4-methylbenzenesulfonate), 5.52 (d,

J = 4.5 Hz, 1H), 5.44 (d, J = 4.2 Hz, 1H), 4.06−3.99 (m, 2H), 3.72−

3.66 (m, 1H), 3.58−3.49 (m, 2H), 3.48−3.30 (m, 6H), 3.21 (s, 3H),

3.16 (d, J = 7.0 Hz, 1H), 3.14 (d, J = 6.9 Hz, 1H), 3.10 (s, 3H), 3.07−

2.94 (m, 4H), 2.28 (s, 6H), 1.98−1.87 (m, 2H), 1.87−1.75 (m, 4H),

1.52−1.40 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 145.70,

137.64, 137.41, 137.34, 136.37, 136.19, 131.58, 131.50, 128.09,

125.49, 92.89, 92.88, 65.02, 63.55, 63.03, 62.78, 61.45, 61.31, 60.02,

59.84, 49.93, 47.83, 30.00, 29.38, 27.24, 26.93, 20.82, 17.06, 16.70.

HRMS (ESI) m/z: [M⁺] (C₁₄H₂₁INO⁺) calcd, 346.066234; found,

346.06600, Elem. Anal. C₂₁H₂₈INO₄S, Calcd: C (48.75), H (5.45), N

(2.71), I (24.53), S (6.20). Found: C (48.91), H (5.48), N (2.59), I

(24.25), S (6.25). HPLC-UV purity 99.4%.

1-(2-Hydroxyethyl)-1-[2-(4-iodophenyl)ethyl]pyrrolidin-1-ium 4-

Methylbenzenesulfonate (17). General procedure A was used. The

alkylating agent B3 (400 mg, 0.99 mmol, 1 equiv, see the Supporting

Information), DMF (5 mL), and 2-(pyrrolidin-1-yl)ethanol (350 μL,

2.99 mmol, 3 equiv) were used for the synthesis of compound 17.

The reaction time was 2 h at a temperature of 60 °C, 2 h at a

temperature of 80 °C, and 2 h at a temperature of 90 °C. The

precipitation was done three times using a mixture of Et₂O and

hexane (1:2 v/v, 45 mL). The product 17 was obtained in as a pale-

130.47, 130.43, 128.09, 125.49, 111.62, 86.24, 64.82, 64.68, 56.42,

+

55.04, 50.87, 26.73, 20.82. HRMS (ESI) m/z: [M⁺] (C₁₃H₂₁INO₂)

calcd, 350.061148; found, 350.06124. Elem. Anal. C₂₁H₂₈INO₄S,

Calcd: C (46.07), H (5.41), N (2.69), S (6.15). Found: C (46.02), H

(5.48), N (2.58), S (6.24). HPLC-UV purity 97.7%.

1

yellow solid (165 mg, 0.32 mmol, 32%). mp 152−154 °C. H NMR

3-Hydroxy-1-[2-(3-iodo-4-methoxyphenyl)ethyl]quinuclidin-1-

ium 4-Methylbenzenesulfonate (21). General procedure A was used.

The alkylating agent D3 (200 mg, 0.463 mmol, 1 equiv, see the

Supporting Information), DMF (2 mL), and quinuclidin-3-ol (178

mg, 1.40 mmol, 3 equiv) were used for the synthesis of compound 21.

The reaction time was 3 h at a temperature of 80 °C. The

precipitation was done ﬁrst using a mixture of Et₂O and hexane (1:1

v/v, 45 mL), and subsequently with pure Et₂O (45 mL). The product

21 was obtained as an oﬀ-white solid (238 mg, 0.425 mmol, 92%). mp

173−176 °C. ¹H NMR (600 MHz, DMSO-d₆): δ 7.75 (d, J = 2.2 Hz,

1H), 7.48 (d, J = 8.0 Hz, 2H), 7.29 (dd, J = 8.4, 2.2 Hz, 1H), 7.11 (d,

J = 7.9 Hz, 2H), 6.97 (d, J = 8.5 Hz, 1H), 5.60 (d, J = 3.5 Hz, 1H),

4.11−4.06 (m, 1H), 3.80 (s, 3H), 3.68 (ddd, J = 12.7, 8.2, 2.9 Hz,

1H), 3.47−3.41 (m, 1H), 3.40−3.27 (m, 5H), 3.09 (dt, J = 12.8, 2.8

Hz, 1H), 2.96−2.86 (m, 2H), 2.28 (s, 3H), 2.16−2.10 (m, 1H),

2.06−2.02 (m, 1H), 1.96−1.89 (m, 1H), 1.81−1.71 (m, 2H). ¹³C

NMR (151 MHz, DMSO-d₆): δ 156.73, 145.73, 139.24, 137.62,

130.61, 130.43, 128.08, 125.49, 111.58, 86.22, 63.44, 62.65, 56.42,

54.03, 52.71, 26.36, 26.05, 20.90, 20.81, 17.39. HRMS (ESI) m/z:

(600 MHz, DMSO-d₆): δ 7.69 (d, J = 7.9 Hz, 2H), 7.48 (d, J = 7.8

Hz, 2H), 7.15 (d, J = 7.9 Hz, 2H), 7.11 (d, J = 7.8 Hz, 2H), 5.40 (t, J

= 5.0 Hz, 1H), 3.88−3.83 (m, 2H), 3.66−3.54 (m, 4H), 3.52−3.46

(m, 4H), 3.04−2.99 (m, 2H), 2.28 (s, 3H), 2.10−2.02 (m, 4H). ¹³C

NMR (151 MHz, DMSO-d₆): δ 145.67, 137.66, 137.32, 136.36,

131.51, 128.09, 125.48, 92.84, 62.78, 60.27, 60.08, 55.73, 28.36, 21.21,

20.82. HRMS (ESI) m/z: [M⁺] (C₁₄H₂₁INO⁺) calcd, 346.066234;

found, 346.06629, Elem. Anal. C₂₁H₂₈INO₄S, Calcd: C (48.75), H

(5.45), N (2.71), I (24.53), S (6.20). Found: C (48.93), H (5.46), N

(2.53), I (24.07), S (6.30). HPLC-UV purity 96.5%.

3-Hydroxy-1-[2-(4-iodophenyl)ethyl]quinuclidin-1-ium 4-Meth-

ylbenzenesulfonate (18). General procedure A was used. The

alkylating agent B3 (400 mg, 0.99 mmol, 1 equiv, see the Supporting

Information), DMF (5 mL), and quinuclidin-3-ol (380 mg, 2.99

mmol, 3 equiv) were used for the synthesis of compound 18. The

reaction time was 3 h at a temperature of 60 °C. The precipitation

was done once using a mixture of Et₂O and hexane (1:1 v/v, 45 mL)

and twice with pure Et₂O (40 mL). The product 18 was obtained as

1

+

[M⁺] (C₁₆H₂₃INO₂) calcd, 388.076798; found, 388.07672. Elem.

an oﬀ-white solid (425 mg, 0.80 mmol, 80%). mp 217−218 °C. H

NMR (600 MHz, DMSO-d₆): δ 7.70 (d, J = 8.2 Hz, 2H), 7.48 (d, J =

8.0 Hz, 2H), 7.14−7.10 (m, 4H), 5.60 (d, J = 3.5 Hz, 1H), 4.10−4.06

(m, 1H), 3.69 (ddd, J = 12.7, 8.3, 3.0 Hz, 1H), 3.48−3.42 (m, 1H),

3.41−3.28 (m, 5H), 3.09 (dt, J = 12.9, 2.8 Hz, 1H), 2.99−2.90 (m,

2H), 2.28 (s, 3H), 2.17−2.09 (m, 1H), 2.06−2.01 (m, 1H), 1.95−

1.88 (m, 1H), 1.81−1.70 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆):

δ 145.69, 137.65, 137.35, 136.43, 131.46, 128.09, 125.49, 92.83,

Anal. C₂₃H₃₀INO₅S, Calcd: C (49.38), H (5.40), N (2.50), I (22.68),

S (5.73). Found: C (49.51) H (5.35), N (2.24), I (22.40), S (5.76).

HPLC-UV purity 95.5%.

(2-Hydroxyethyl)-[2-(3,5-diiodo-4-methoxyphenyl)ethyl]-dime-

thylammonium 4-Methylbenzenesulfonate (22). General procedure

A was used. The alkylating agent E3 (350 mg, 0.627 mmol, 1 equiv,

see the Supporting Information), DMF (3 mL), and 2-

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(dimethylamino)ethanol (190 μL, 1.888 mmol, 3 equiv) were used

for the synthesis of compound 22. The reaction time was 4 h at a

temperature of 85 °C. The precipitation was done once using a

mixture of Et₂O and hexane (1:1 v/v, 45 mL) and subsequently with

pure Et₂O (40 mL). The product 22 was obtained as a white solid

(258 mg, 0.399 mmol, 64%). mp 178−180 °C, ¹H NMR (600 MHz,

DMSO-d₆): δ 7.82 (s, 2H), 7.48 (d, J = 8.1 Hz, 2H), 7.11 (d, J = 7.9

Hz, 2H), 5.34 (t, J = 5.0 Hz, 1H), 3.88−3.83 (m, 2H), 3.73 (s, 3H),

3.52−3.48 (m, 2H), 3.46−3.43 (m, 2H), 3.11 (s, 6H), 3.00−2.93 (m,

2H), 2.28 (s, 3H). ¹³C NMR (151 MHz, DMSO-d₆): δ 157.38,

145.67, 140.12, 137.64, 136.74, 128.08, 125.48, 91.52, 64.94, 64.21,

60.29, 55.00, 50.90, 26.26, 20.82. HRMS (ESI) m/z: [M⁺]

white waxy solid (186 mg, 0.318 mmol, 63%). H NMR (600 MHz,

DMSO-d₆): δ 7.67 (s, 1H), 7.21 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 8.4

Hz, 1H), 5.28 (t, J = 4.9 Hz, 1H), 3.98 (t, J = 6.1 Hz, 2H), 3.81 (br s,

2H), 3.40−3.37 (m, 2H), 3.35−3.30 (m, 2H), 3.06 (s, 6H), 2.49 (t, J

= 7.5 Hz, 2H), 2.30 (s, 3H), 2.00−1.92 (m, 2H), 1.75−1.67 (m, 2H),

1.49−1.42 (m, 2H), 1.36−1.20 (m, 12H), 0.86 (t, J = 6.6 Hz, 3H).

¹³C NMR (151 MHz, CDCl₃): δ 156.57, 139.05, 133.56, 129.55,

112.29, 87.02, 69.49, 65.78, 65.27, 56.24, 51.95, 39.76, 32.02, 30.87,

29.69, 29.67, 29.44 (2C), 29.22, 26.20, 24.80, 22.80, 14.26. HRMS

+

(ESI) m/z: [M⁺] (C₂₃H₄₁INO₂) calcd, 490.2176; found, 490.2158.

HPLC-UV purity 97.4%.

(2-Hydroxyethyl)-[2-(5-iodothiophen-2-yl)ethyl]-dimethylammo-

nium 4-Methylbenzenesulfonate (26). General procedure A was

used. The alkylating agent G4 (250 mg, 0.612 mmol, 1 equiv, see the

Supporting Information), DMF (1 mL), and 2-(dimethylamino)-

ethanol (370 μL, 3.67 mmol, 6.0 equiv) were used for the synthesis of

compound 26. The reaction time was 2 h at a temperature of 65 °C.

The product 26 was obtained as a white solid (273 mg, 0.55 mmol,

+

(C₁₃H₂₀I₂NO₂) calcd, 475.957791; found, 475.95731, Elem. Anal.

C₂₀H₂₇I₂NO₅S, Calcd: C (37.11), H (4.20), N (2.16), I (39.21), S

(4.95). Found: C (37.14), H (4.27), N (1.98), I (39.23), S (5.17).

HPLC-UV purity 99.4%.

(2-Hydroxyethyl)-[3-(3-iodo-4-methoxyphenyl)propyl]-dimethy-

lammonium 4-Methylbenzenesulfonate (23). General procedure A

was used. The alkylating agent F3a (342 mg, 0.766 mmol, 1 equiv, see

the Supporting Information), DMF (3 mL), and 2-(dimethylamino)-

ethanol (190 μL, 1.888 mmol, 3 equiv) were used for the synthesis of

compound 23. The reaction time was 2 h at a temperature of 80 °C.

The product was puriﬁed by chromatography on a C₁₈-silica gel

(eluent 4:6 MeOH/water v/v). Compound 23 was obtained as a

1

90%). H NMR (300 MHz, DMSO-d₆): δ 7.48 (d, J = 8.0 Hz, 2H),

7.22 (d, J = 3.6 Hz, 1H), 7.11 (d, J = 8.0 Hz, 2H), 6.74 (d, J = 3.6 Hz,

1H), 5.33 (t, J = 4.9 Hz, 1H), 3.89−3.80 (m, 2H), 3.63−3.53 (m,

2H), 3.50−3.43 (m, 2H), 3.35−3.26 (m, 2H), 3.12 (s, 3H), 2.29 (s,

3H). ¹³C NMR (75 MHz, DMSO-d₆): δ 145.71, 144.08, 137.63,

136.81, 128.67, 128.07, 125.48, 74.32, 64.80, 63.82, 55.01, 50.95,

22.83, 20.80. HRMS (ESI) m/z: [M⁺] (C₁₀H₁₇INOS⁺) calcd,

326.00700; found, 326.00725. HPLC-UV purity 98.4%.

1-(2-Hydroxyethyl)-1-[2-(5-iodothiophen-2-yl)ethyl]pyrrolidin-1-

ium 4-Methylbenzenesulfonate (27). General procedure A was used.

The alkylating agent G4 (250 mg, 0.612 mmol, 1 equiv, see the

Supporting Information), DMF (1 mL), and 2-(pyrrolidin-1-

yl)ethanol (358 μL, 3.06 mmol, 5.0 equiv) were used for the

synthesis of compound 27. The reaction time was 2 h at a

temperature of 65 °C. The product 27 was obtained as a brown

solid (120 mg, 0.23 mmol, 37%). ¹H NMR (300 MHz, DMSO-d₆): δ

7.47 (d, J = 8.0 Hz, 2H), 7.22 (d, J = 3.6 Hz, 1H), 7.11 (d, J = 8.0 Hz,

1H), 6.75 (d, J = 3.6 Hz, 1H), 5.36 (t, J = 4.8 Hz, 1H), 3.90−3.76 (m,

2H), 3.70−3.51 (m, 6H), 3.51−3.43 (m, 2H), 3.34−3.27 (m, 2H),

2.29 (s, 3H), 2.13−2.00 (m, 4H). HRMS (ESI) m/z: [M⁺]

(C₁₂H₁₉INO₂S⁺) calcd, 352.02265; found, 352.02235. HPLC-UV

purity 98.0%.

(3-Hydroxypropyl)-[2-(5-iodothiophen-2-yl)ethyl]-dimethylam-

monium 4-Methylbenzenesulfonate (28). General procedure A was

used. The alkylating agent G4 (150 mg, 0.367 mmol, 1 equiv, see the

Supporting Information) and 3-(dimethylamino)propanol (1.5 mL)

were used for the synthesis of compound 28. The reaction time was 1

h at a temperature of 70 °C. The product 28 was obtained as a pale-

yellow solid (165 mg, 0.323 mmol, 88%). ¹H NMR (600 MHz,

DMSO-d₆): δ 7.47 (d, J = 8.0 Hz, 2H), 7.23 (d, J = 3.6 Hz, 1H), 7.11

(d, J = 8.0 Hz, 2H), 6.76 (d, J = 3.6 Hz, 1H), 4.81 (t, J = 5.0 Hz, 1H),

3.55−3.50 (m, 2H), 3.50−3.46 (m, 2H), 3.40−3.36 (m, 2H), 3.30−

3.26 (m, 2H), 3.07 (s, 6H), 2.29 (s, 3H), 1.86−1.81 (m, 2H). ¹³C

(126 MHz, DMSO-d₆): δ 145.77, 143.99, 137.59, 136.85, 128.68,

128.05, 125.48, 74.24, 62.70, 61.42, 57.62, 50.15, 25.34, 22.60, 20.78.

HRMS (ESI) m/z: [M⁺] (C₁₁H₁₉INOS⁺) calcd, 340.02265; found,

340.02307. HPLC-UV purity 99.0%.

(4-Hydroxybutyl)-[2-(5-iodothiophen-2-yl)ethyl]-dimethylam-

monium 4-Methylbenzenesulfonate (29). General procedure A was

used. The alkylating agent G4 (150 mg, 0.367 mmol, 1 equiv, see the

Supporting Information), 4-(dimethylamino)butanol (172 μL, 1.29

mmol, 3.5 equiv), and DMF (1.5 mL) were used for the synthesis of

compound 29. The reaction time was 2 h at a temperature of 70 °C.

The product 29 was obtained as a brown viscous liquid (100 mg,

1

white solid (354 mg, 0.661 mmol, 86%). mp 120−122 °C. H NMR

(600 MHz, DMSO-d₆): δ 7.67 (d, J = 2.2 Hz, 1H), 7.49 (d, J = 8.0

Hz, 2H), 7.23 (dd, J = 8.4, 2.2 Hz, 1H), 7.12 (d, J = 7.9 Hz, 2H), 6.93

(d, J = 8.5 Hz, 1H), 5.28 (br s, 1H), 3.82−3.78 (m, 2H), 3.79 (s, 3H),

3.40−3.36 (m, 2H), 3.34−3.30 (m, 2H), 3.05 (s, 6H), 2.49 (t, J = 8.0

Hz, 2H), 2.28 (s, 3H), 1.98−1.92 (m, 2H). ¹³C NMR (151 MHz,

DMSO-d₆): δ 156.22, 145.70, 138.53, 137.66, 134.67, 129.62, 128.10,

125.49, 111.44, 86.14, 64.63, 63.60, 56.37, 54.97, 50.91, 30.23, 23.86,

+

20.82. HRMS (ESI) m/z: [M⁺] (C₁₄H₂₃INO₂) calcd, 364.076798;

found, 364.07690. Elem. Anal. C₂₁H₃₀INO₅S, Calcd: C (47.11), H

(5.65), N (2.62), S (5.99). Found: C (47.18), H (5.63), N (2.47), S

(6.04). HPLC-UV purity 97.6%.

[3-(4-Hexyloxy-3-iodophenyl)propyl]-(2-hydroxyethyl)-dimethy-

lammonium 4-Methylbenzenesulfonate (24). General procedure A

was used. The alkylating agent F3b (338 mg, 0.655 mmol, 1 equiv, see

the Supporting Information), DMF (0.8 mL), and 2-

(dimethylamino)ethanol (350 μL, 3.48 mmol, 5.3 equiv) were used

for the synthesis of compound 24. The reaction time was 2 h at a

temperature of 75 °C. Precipitation was performed from a DCM

solution with hexane (45 mL), and the product was then redissolved

in a mixture of DCM and MeOH (2 mL, 9:1 v/v) and precipitated

with a mixture of Et₂O and hexane. The solid was then once more

redissolved in pure DCM and precipitated with pure hexane (45 mL).

Compound 24 was obtained as a white solid (391 mg, 0.65 mmol,

1

99%). H NMR (300 MHz, DMSO-d₆): δ 7.66 (d, J = 2.1 Hz, 1H),

7.49 (d, J = 8.0 Hz, 2H), 7.21 (dd, J = 8.4, 2.1 Hz, 1H), 7.11 (d, J =

7.9 Hz, 2H), 6.91 (d, J = 8.4 Hz, 1H), 5.30 (t, J = 5.0 Hz, 1H), 3.98

(t, J = 6.2 Hz, 2H), 3.81 (br s, 2H), 3.42−3.28 (m, 4H), 3.06 (s, 6H),

2.49 (t, J = 7.4 Hz, 2H), 2.28 (s, 3H), 2.02−1.88 (m, 2H), 1.77−1.65

(m, 2H), 1.53−1.40 (m, 2H), 1.37−1.24 (m, 4H), 0.93−0.84 (m,

3H), ¹³C NMR (75 MHz, DMSO-d₆): δ 155.62, 145.74, 138.43,

137.61, 134.57, 129.56, 128.06, 125.48, 112.43, 86.81, 68.63, 64.63,

63.63, 54.95, 50.90, 30.88, 30.25, 28.53, 25.22, 23.84, 22.08, 20.78,

+

13.91. HRMS (ESI) m/z: [M⁺] (C₁₉H₃₃INO₂) calcd, 434.1551;

found, 434.1535. HPLC-UV purity 98.8%.

[3-(4-Decyloxy-3-iodophenyl)propyl]-(2-hydroxyethyl)-dimethy-

lammonium Methanesulfonate (25). General procedure A was used.

The alkylating agent F3c (250 mg, 0.504 mmol, 1 equiv, see the

Supporting Information), DMF (0.7 mL), and 2-(dimethylamino)-

ethanol (200 μL, 1.99 mmol, 3.9 equiv) were used for the synthesis of

compound 25. The reaction was run 1 h at a temperature of 65 °C,

and the temperature was then increased to 75 °C for 45 min.

However, as the conversion was still not complete, the temperature

was increased to 82 °C for additional 30 min. The product was

puriﬁed by precipitation from a MeOH solution using a mixture of

Et₂O and hexane (1:4 v/v). The compound 25 was obtained as a

1

0.190 mmol, 52%). H NMR (300 MHz, DMSO-d₆): δ 7.47 (d, J =

8.0 Hz, 2H), 7.23 (d, J = 3.6 Hz, 1H), 7.11 (d, J = 8.0 Hz, 3H), 6.76

(d, J = 3.6 Hz, 1H), 4.58 (t, J = 5.0 Hz, 1H), 3.55−3.40 (m, 4H),

3.32−3.23 (m, 4H), 3.05 (s, 6H), 2.29 (s, 3H), 1.78−1.63 (m, 2H),

1.48−1.36 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆): δ 145.74,

144.08, 137.62, 136.84, 128.70, 128.06, 125.49, 74.25, 63.23, 62.78,

59.84, 50.01, 29.00, 22.63, 20.78, 18.73. HRMS (ESI) m/z: [M⁺]

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(C₁₂H₂₁INOS⁺) calcd, 354.03830; found, 354.003824. HPLC-UV

purity 99.3%.

7.09 (m, 2H), 7.06 (dd, J = 8.3, 1.2 Hz, 1H), 6.81 (ddd, J = 7.6, 7.6,

1.3 Hz, 1H), 5.34 (t, J = 4.8 Hz, 1H), 4.51−4.47 (m, 2H), 3.93−3.87

(m, 4H), 3.80−3.68 (m, 4H), 3.61−3.57 (m, 2H), 2.28 (s, 3H),

2.20−2.07 (m, 4H). ¹³C NMR (126 MHz, DMSO-d₆): δ 156.31,

145.78, 139.17, 137.58, 129.78, 128.05, 125.49, 123.38, 112.71, 85.98,

63.54, 63.04, 60.92, 57.89, 55.75, 20.97, 20.77. HRMS (ESI) m/z:

(2-Hydroxyethyl)-(5-iodopent-4-ynyl)-dimethylammonium 4-

Methylbenzenesulfonate (30). General procedure A was used. The

alkylating agent H4 (369 mg, 1.00 mmol, 1 equiv, see the Supporting

Information), 4-(dimethylamino)ethanol (604 μL, 6.00 mmol, 6

equiv), and DMF (1 mL) were used for the synthesis of compound

30. The reaction time was 1 h at a temperature of 65 °C. The product

+

[M⁺] (C₁₄H₂₁INO₂) calcd, 362.0612; found, 362.0597. HPLC-UV

purity 96.9%.

1

30 was obtained as a white solid (432 mg, 0.953 mmol, 95%). H

(2-Hydroxyethyl)-[2-(3-iodophenoxy)ethyl]-dimethylammonium

Bromide (35). General procedure A was used. The alkylating agent K

(300 mg, 0.918 mmol, 1 equiv, see the Supporting Information),

DMF (1 mL), and 2-(dimethylamino)ethanol (554 μL, 5.51 mmol,

6.0 equiv) were used for the synthesis of compound 35. The reaction

time was 2 h at a temperature of 70 °C. The product 35 was obtained

NMR (300 MHz, DMSO-d₆): δ 7.48 (d, J = 8.0 Hz, 2H), 7.12 (d, J =

8.0 Hz, 2H), 5.27 (t, J = 5.0 Hz, 1H), 3.80 (br s, 2H), 3.43−3.28 (m,

4H), 3.06 (s, 6H), 2.38 (t, J = 7.1 Hz, 2H), 2.29 (s, 3H) 1.94−1.79

(m, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ 145.65, 137.69, 128.11,

125.49, 91.29, 64.69, 62.97, 54.94, 50.98, 21.41, 20.81, 17.28, 5.28.

HRMS (ESI) m/z: [M⁺] (C₉H₁₇INO⁺) calcd, 282.03493; found,

282.03483. HPLC-UV purity 95.7%.

(2-Hydroxyethyl)-(6-iodohex-5-ynyl)-dimethylammonium 4-

Methylbenzenesulfonate (31). General procedure A was used. The

alkylating agent I4 (378 mg, 1.00 mmol, 1 equiv, see the Supporting

Information), 4-(dimethylamino)ethanol (604 μL, 6.00 mmol, 6

equiv), and DMF (1 mL) were used for the synthesis of compound

31. The reaction time was 1 h at a temperature of 65 °C. The product

31 was obtained as an oﬀ-white solid (456 mg, 0.976 mmol, 98%). ¹H

NMR (300 MHz, DMSO-d₆): δ 7.48 (d, J = 8.0 Hz, 2H), 7.12 (d, J =

8.0 Hz, 2H), 5.26 (t, J = 4.9 Hz, 1H), 3.81 (br s, 2H), 3.39−3.27 (m,

4H), 3.04 (s, 6H), 2.38 (t, J = 7.1 Hz, 2H), 2.29 (s, 3H), 1.79−1.64

(m, 2H), 1.43 (tt, J = 7.1, 7.1 Hz, 2H). ¹³C NMR (75 MHz, DMSO-

d₆): δ 145.72, 137.63, 128.07, 125.48, 92.45, 64.66, 63.41, 54.92,

50.80, 25.02, 21.10, 20.80, 19.53, 4.20. HRMS (ESI) m/z: [M⁺]

(C₁₀H₁₉INO⁺) calcd, 296.05058; found, 296.05053. HPLC-UV purity

96.3%.

1-(2-Hydroxyethyl)-1-(6-iodohex-5-ynyl)-pyrrolidin-1-ium 4-

Methylbenzenesulfonate (32). General procedure A was used. The

alkylating agent I4 (369 mg, 1.00 mmol, 1 equiv, see the Supporting

Information), 2-(pyrrolidin-1-yl)ethanol (585 μL, 5.00 mmol, 5

equiv), and DMF (1 mL) were used for the synthesis of compound

32. The reaction time was 1 h at a temperature of 65 °C. The product

32 was obtained as a yellow oil, which crystallized upon cooling (279

mg, 0.565 mmol, 57%). ¹H NMR (300 MHz, DMSO-d₆): δ 7.48 (d, J

= 8.0 Hz, 2H), 7.12 (d, J = 8.0 Hz, 2H), 5.27 (t, J = 5.0 Hz, 1H),

3.83−3.74 (m, 2H), 3.64−3.52 (m, 2H), 3.52−3.40 (m,2H), 3.39−

3.33 (m, 2H), 3.32−3.25 (m, 2H), 2.38 (t, J = 7.1 Hz, 2H), 2.29 (s,

3H), 2.10−1.98 (m, 4H), 1.77−1.64 (m, 2H), 1.45 (tt, J = 7.2, 7.1

Hz, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ 145.68, 137.65, 128.08,

125.48, 92.48, 62.68, 60.05, 58.68, 55.42, 24.98, 21.82, 21.11, 20.79,

19.48, 4.14. HRMS (ESI) m/z: [M⁺] (C₁₂H₂₁INO⁺) calcd,

322.06623; found, 322.06602. HPLC-UV purity 96.9%.

1

as a white solid (368 mg, 0.88 mmol, 96%). H NMR (300 MHz,

DMSO-d₆): δ 7.40−7.34 (m, 2H), 7.15−7.08 (m, 1H), 7.05−7.00

(m, 1H), 5.32 (t, J = 5.0 Hz, 1H), 4.51−4.42 (m, 2H), 3.92−3.81 (m,

4H), 3.58−3.51 (m, 2H), 3.20 (s, 6H). ¹³C NMR (75 MHz, DMSO-

d₆): δ 158.10, 131.37, 130.13, 123.14, 114.72, 95.25, 65.66, 62.82,

+

61.75, 54.95, 51.63. HRMS (ESI) m/z: [M⁺] (C₁₂H₁₉INO₂) calcd,

336.0455; found, 336.0439. HPLC-UV purity 99.4%.

1-(2-Hydroxyethyl)-1-[2-(3-iodophenoxy)ethyl]pyrrolidin-1-ium

Bromide (36). General procedure A was used. The alkylating agent K

(300 mg, 0.918 mmol, 1 equiv, see the Supporting Information),

DMF (1 mL), and 2-(pyrrolidin-1-yl)ethanol (322 μL, 2.75 mmol, 3.0

equiv) were used for the synthesis of compound 36. The reaction

time was 2 h at a temperature of 70 °C. The product 36 was obtained

as a yellow viscous liquid, which crystallized upon cooling (380 mg,

0.86 mmol, 80%). ¹H NMR (300 MHz, DMSO-d₆): δ 7.40−7.33 (m,

2H), 7.15−7.07 (m, 1H), 7.05−6.98 (m, 1H), 5.32 (t, J = 5.0 Hz,

1H), 4.45 (t, J = 4.7 Hz, 2H), 3.92−3.79 (m, 4H), 3.78−3.58 (m,

4H), 3.56−3.47 (m, 2H), 2.17−2.02 (m, 4H). ¹³C NMR (75 MHz,

DMSO-d₆): δ 158.09, 131.36, 130.16, 123.12, 114.66, 95.21, 63.35,

62.34, 60.66, 58.03, 55.44, 20.91. HRMS (ESI) m/z: [M⁺]

+

(C₁₄H₂₁INO₂) calcd, 362.0612; found, 362.0597. HPLC-UV purity

95.2%.

(2-Hydroxyethyl)-[2-(4-iodophenoxy)ethyl]-dimethylammonium

Bromide (37). General procedure A was used. The alkylating agent L

(350 mg, 1.07 mmol, 1 equiv, see the Supporting Information), DMF

(1 mL), and 2-(dimethylamino)ethanol (646 μL, 6.42 mmol, 6.0

equiv) were used for the synthesis of compound 37. The reaction

time was 3 h at a temperature of 70 °C. The product 37 was obtained

1

as a white solid (384 mg, 0.92 mmol, 86%). H NMR (300 MHz,

DMSO-d₆): δ 7.67−7.61 (m, 2H), 6.90−6.83 (m, 2H), 5.31 (t, J = 4.9

Hz, 1H), 4.51−4.39 (m, 2H), 3.92−3.80 (m, 4H), 3.58−3.50 (m,

2H), 3.20 (s, 6H). ¹³C NMR (75 MHz, DMSO-d₆): δ 157.30, 138.02,

117.53, 84.25, 65.69, 62.81, 61.65, 54.94, 51.62. HRMS (ESI) m/z:

+

(2-Hydroxyethyl)-[2-(2-iodophenoxy)ethyl]-dimethylammonium

Bromide (33). General procedure A was used. The alkylating agent J

(350 mg, 1.07 mmol, 1 equiv, see the Supporting Information), DMF

(1 mL), and 2-(dimethylamino)ethanol (646 μL, 6.42 mmol, 6.0

equiv) were used for the synthesis of compound 33. The reaction

time was 1 h at a temperature of 67 °C. The product 33 was obtained

[M⁺] (C₁₂H₁₉INO₂) calcd, 336.0455; found, 336.0439. HPLC-UV

purity 96.8%.

1-(2-Hydroxyethyl)-1-[2-(4-iodophenoxy)ethyl]pyrrolidin-1-ium

Bromide (38). General procedure A was used. The alkylating agent L

(350 mg, 1.07 mmol, 1 equiv, see the Supporting Information), DMF

(1 mL), and 2-(pyrrolidin-1-yl)ethanol (376 μL, 3.21 mmol, 3.0

equiv) were used for the synthesis of compound 38. The reaction

time was 3 h at a temperature of 70 °C. The product 38 was obtained

1

as a white solid (398 mg, 0.96 mmol, 90%). H NMR (600 MHz,

DMSO-d₆): δ 7.80 (dd, J = 7.7, 1.5 Hz, 1H), 7.42−7.38 (m, 1H), 7.09

(dd, J = 8.2, 0.8 Hz, 1H), 6.81 (ddd, J = 7.7, 7.7, 1.1 Hz, 1H), 5.35 (t,

J = 4.9 Hz, 1H), 4.55−4.50 (m, 2H), 3.95−3.88 (m, 4H), 3.64−3.60

(m, 2H), 3.28 (s, 6H). ¹³C NMR (151 MHz, DMSO-d₆): δ 156.30,

139.11, 129.75, 123.34, 112.79, 86.25, 65.88, 63.09, 62.49, 55.11,

1

as a pale-yellow solid (380 mg, 0.86 mmol, 80%). H NMR (600

MHz, DMSO-d₆): δ 7.64 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 8.8 Hz,

2H), 5.33 (t, J = 4.9 Hz, 1H), 4.43 (t, J = 4.5 Hz, 2H), 3.88−3.83 (m,

4H), 3.74−3.67 (m, 2H), 3.67−3.61 (m, 2H), 3.53−3.49 (m, 2H),

2.12−2.05 (m, 4H). ¹³C NMR (151 MHz, DMSO-d₆): δ 157.31,

138.05, 117.49, 84.30, 63.34, 62.24, 60.68, 58.02, 55.45, 20.92. HRMS

+

51.89. HRMS (ESI) m/z: [M⁺] (C₁₂H₁₉INO₂) calcd, 336.0455;

found, 336.0441. HPLC-UV purity 98.1%.

+

1-(2-Hydroxyethyl)-1-[2-(2-iodophenoxy)ethyl]pyrrolidin-1-ium

4-Methylbenzenesulfonate (34). General procedure A was used. The

alkylating agent J (350 mg, 1.07 mmol, 1 equiv, see the Supporting

Information), DMF (1 mL), and 2-(pyrrolidin-1-yl)ethanol (376 μL,

3.21 mmol, 3.0 equiv) were used for the synthesis of compound 34.

The reaction time was 90 min at a temperature of 77 °C. The product

34 was obtained as a pale-yellow viscous liquid (331 mg, 0.75 mmol,

(ESI) m/z: [M⁺] (C₁₄H₂₁INO₂) calcd, 362.0612; found, 362.0597.

HPLC-UV purity 98.1%.

(2-Hydroxyethyl)-[4-(2-iodophenoxy)butyl]-dimethylammonium

Bromide (39). General procedure A was used. The alkylating agent M

(355 mg, 1.00 mmol, 1 equiv, see the Supporting Information), DMF

(1 mL), and 2-(dimethylamino)ethanol (604 μL, 6.00 mmol, 6.0

equiv) were used for the synthesis of compound 39. The reaction

time was 2.5 h at a temperature of 70 °C. The product 39 was

1

70%). H NMR (500 MHz, DMSO-d₆): δ 7.81 (dd, J = 7.7, 1.6 Hz,

1

1H), 7.49−7.46 (m, 2H), 7.40 (ddd, J = 8.4, 7.4, 1.6 Hz, 1H), 7.13−

obtained as a colorless viscous liquid (418 mg, 0.94 mmol, 94%). H

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NMR (300 MHz, DMSO-d₆): δ 7.77 (dd, J = 7.7, 1.6 Hz, 1H), 7.36

(ddd, J = 8.2, 7.4, 1.6 Hz, 1H), 7.01 (dd, J = 8.3, 1.3 Hz, 1H), 6.79−

6.72 (m, 1H), 5.28 (t, J = 5.0 Hz, 1H) 4.08 (t, J = 6.4 Hz, 2H), 3.90−

3.79 (m, 2H), 3.52−3.37 (m, 4H), 3.10 (s, 6H), 2.01−1.86 (m, 2H),

1.86−1.71 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ 156.89,

138.90, 129.79, 122.79, 112.77, 86.94, 67.81, 64.64, 63.76, 54.92,

1-(2-Hydroxyethyl)-1-[6-(3-iodophenoxy)hexyl]pyrrolidin-1-ium

Bromide (44). General procedure A was used. The alkylating agent N

(350 mg, 0.914 mmol, 1 equiv, see the Supporting Information),

DMF (1 mL), and 2-(pyrrolidin-1-yl)ethanol (320 μL, 2.74 mmol, 3.0

equiv) were used for the synthesis of compound 44. The reaction

time was 2 h at a temperature of 70 °C. The product 44 was obtained

1

+

50.92, 25.58, 19.05. HRMS (ESI) m/z: [M⁺] (C₁₄H₂₃INO₂) calcd,

as a pale-yellow solid (399 mg, 0.88 mmol, 80%). H NMR (300

MHz, DMSO-d₆): δ 7.30−7.24 (m, 2H), 7.10−7.02 (m, 1H), 6.98−

6.91 (m, 1H), 5.28 (t, J = 5.1 Hz, 1H), 3.96 (t, J = 6.4 Hz, 2H), 3.85−

3.73 (m, 2H), 3.65−3.44 (m, 4H), 3.42−3.26 (m, 4H), 2.11−1.97

(m, 4H), 1.78−1.62 (m, 4H), 1.51−1.39 (m, 2H), 1.39−1.25 (m,

2H). ¹³C NMR (75 MHz, DMSO-d₆): δ 159.37, 131.33, 129.24,

122.87, 114.33, 95.19, 67.51, 62.62, 60.00, 59.30, 55.43, 28.29, 25.51,

364.0768; found, 364.0752. HPLC-UV purity 97.7%.

(3-Hydroxypropyl)-[4-(2-iodophenoxy)butyl]-dimethylammo-

nium Bromide (40). General procedure A was used. The alkylating

agent M (300 mg, 0.845 mmol, 1 equiv, see the Supporting

Information) and 3-(dimethylamino)propanol (2 mL) were used for

the synthesis of compound 40. The reaction time was 1.5 h at a

temperature of 75 °C. The product 40 was obtained as a white solid

(343 mg, 0.749 mmol, 89%). ¹H NMR (300 MHz, DMSO-d₆): δ 7.77

(dd, J = 7.7, 1.6 Hz, 1H), 7.36 (ddd, J = 8.3, 7.4, 1.6 Hz, 1H), 7.01

(dd, J = 8.3, 1.3 Hz, 1H), 6.76 (ddd, J = 7.7, 7.4, 1.3 Hz, 1H), 4.79 (t,

J = 5.0 Hz, 1H), 4.08 (t, J = 5.8 Hz, 2H), 3.52−3.30 (m, 6H), 3.04 (s,

6H), 1.98−1.72 (m, 6H). ¹³C NMR (126 MHz, DMSO-d₆): δ 156.87,

138.88, 129.77, 122.77, 112.76, 86.88, 67.79, 62.60, 61.18, 57.64,

+

24.95, 22.50, 21.11. HRMS (ESI) m/z: [M⁺] (C₁₈H₂₉INO₂) calcd,

418.1237; found, 418.1227. HPLC-UV purity 97.8%.

(2-Hydroxyethyl)-{6-[(2-iodopyridin-3-yl)oxy]hexyl}-dimethylam-

monium 4-Methylbenzenesulfonate (45). The alkylating agent O

(200 mg, 0.521 mmol, 1 equiv, see the Supporting Information) and

2-(dimethylamino)ethanol (2.00 mL, 19.9 mmol, 38.2 equiv) were

stirred at a temperature of 75 °C for 40 min. The product was

converted to 4-methylbenzenesulfonate salt by anion-exchange resin.

Compound 45 was obtained as a colorless viscous liquid (240 mg,

0.51 mmol, 97%). ¹H NMR (500 MHz, DMSO-d₆): δ 7.95 (ddd, J =

3.4, 2.6, 0.8 Hz, 1H), 7.52−7.44 (m, 2H), 7.34−7.33 (m, 2H), 7.12−

7.10 (m, 2H), 5.27 (t, J = 4.9 Hz, 1H), 4.08 (t, J = 6.2 Hz, 2H), 3.81

(br s, 2H), 3.39−3.36 (m, 2H), 3.34−3.29 (m, 2H), 3.05 (s, 6H),

2.28 (s, 3H), 1.80−1.74 (m, 2H), 1.73−1.67 (m, 2H), 1.56−1.49 (m,

2H), 1.38−1.30 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆): δ 154.24,

145.76, 142.62, 137.60, 128.05, 125.49, 124.15, 119.02, 112.28, 68.72,

64.63, 64.09, 54.96, 50.80, 28.03, 25.41, 25.09, 21.78, 20.77. HRMS

+

50.21, 25.53, 25.31, 18.92. HRMS (ESI) m/z: [M⁺] (C₁₅H₂₅INO₂)

calcd, 378.09245; found, 378.09221. HPLC-UV purity 97.4%.

(4-Hydroxybutyl)-[4-(2-iodophenoxy)butyl]-dimethylammonium

Bromide (41). General procedure A was used. The alkylating agent M

(300 mg, 0.845 mmol, 1 equiv, see the Supporting Information), 4-

(dimethylamino)butanol (396 μL, 2.96 mmol, 3.5 equiv), and DMF

(1.5 mL) were used for the synthesis of compound 41. The reaction

time was 2 h at a temperature of 70 °C. The product 41 was obtained

1

as a white amorphous solid (295 mg, 0.625 mmol, 74%). H NMR

(300 MHz, DMSO-d₆): δ 7.77 (dd, J = 7.7, 1.6 Hz, 1H), 7.36 (ddd, J

= 8.3, 7.4, 1.6 Hz, 1H), 7.01 (dd, J = 8.3, 1.3 Hz, 1H), 6.76 (ddd, J =

7.7, 7.4, 1.3 Hz, 1H), 4.57 (t, J = 5.0 Hz, 1H), 4.08 (t, J = 5.7 Hz,

2H), 3.47−3.24 (m, 6H), 3.02 (s, 6H), 1.97−1.84 (m, 2H), 1.84−

1.64 (m, 4H), 1.49−1.37 (m, 2H). ¹³C NMR (126 MHz, DMSO-d₆):

δ 156.86, 138.88, 129.79, 122.77, 112.73, 86.81, 67.73, 62.91, 62.52,

59.87, 50.11, 29.09, 25.53, 18.92, 18.78. HRMS (ESI) m/z: [M⁺]

+

(ESI) m/z: [M⁺] (C₁₅H₂₆IN₂O₂) calcd, 393.10335; found,

393.10294. HPLC-UV purity 99.96%.

(2-Hydroxyethyl)-{4-[(2-iodo-6-methylpyridin-3-yl)oxy]butyl}-di-

methylammonium Bromide (46). The alkylating agent P (200 mg,

0.540 mmol, 1 equiv, see the Supporting Information) and 2-

(dimethylamino)ethanol (2.00 mL, 19.9 mmol, 36.9 equiv) were

stirred at a temperature of 75 °C for 40 min. Compound 46 was

obtained as a colorless viscous liquid (246 mg, 0.540 mmol, 99%). ¹H

NMR (300 MHz, DMSO-d₆): δ 7.26 (d, J = 8.2 Hz, 1H), 7.19 (d, J =

8.2 Hz, 1H), 5.29 (t, J = 4.9 Hz, 1H), 4.08 (t, J = 6.3 Hz, 2H), 3.90−

3.76 (m, 2H), 3.52−3.32 (m, 4H), 3.10 (s, 6H), 2.36 (s, 3H), 1.97−

1.84 (m, 2H), 1.82−1.69 (m, 2H). ¹³C NMR (75 MHz, DMSO-d₆): δ

151.99, 151.27, 123.16, 119.81, 111.23, 68.12, 64.54, 63.56, 54.82,

+

(C₁₆H₂₇INO₂) calcd, 392.10810; found, 392.10791. HPLC-UV

purity 96.2%.

1-(2-Hydroxyethyl)-1-[4-(2-iodophenoxy)butyl]pyrrolidin-1-ium

Bromide (42). General procedure A was used. The alkylating agent M

(355 mg, 1.00 mmol, 1 equiv, see the Supporting Information), DMF

(1 mL), and 2-(pyrrolidin-1-yl)ethanol (468 μL, 4.00 mmol, 4.0

equiv) were used for the synthesis of compound 42. The reaction

time was 2.5 h at a temperature of 70 °C. The product 42 was

+

50.84, 25.32, 22.30, 18.88. HRMS (ESI) m/z: [M⁺] (C₁₄H₂₄IN₂O₂)

calcd, 379.08770; found, 379.08744. HPLC-UV purity 99.7%.

(2-Hydroxyethyl)-{4-[(3-iodo-[1,1′-biphenyl]-4-yl)oxy]butyl}-di-

methylammonium Bromide (47). The alkylating agent Q (200 mg,

0.464 mmol, 1 equiv, see the Supporting Information) and 2-

(dimethylamino)ethanol (2.00 mL, 19.9 mmol, 44.2 equiv) were used

for the synthesis of compound 47. The reaction time was 40 min at a

temperature of 75 °C. The product 47 was obtained as a colorless

viscous liquid, which crystallized upon cooling (238 mg, 0.457 mmol,

1

obtained as a yellow viscous liquid (388 mg, 0.83 mmol, 83%). H

NMR (300 MHz, DMSO-d₆): δ 7.76 (dd, J = 7.7, 1.6 Hz, 1H), 7.35

(ddd, J = 8.3, 7.4, 1.6 Hz, 1H), 7.01 (dd, J = 8.3, 1.3 Hz, 1H), 6.78−

6.72 (m, 1H), 5.28 (t, J = 5.1 Hz, 1H), 4.07 (t, J = 5.9 Hz, 2H), 3.86−

3.77 (m, 2H), 3.72−3.59 (m, 2H), 3.59−3.38 (m, 6H), 2.14−2.00

(m, 4H), 1.99−1.85 (m, 2H) 1.85−1.73 (m, 2H). ¹³C NMR (75

MHz, DMSO-d₆): δ 156.89, 138.88, 129.78, 122.75, 112.73, 86.94,

67.82, 62.80, 60.10, 59.03, 55.44, 25.59, 21.19, 19.81. HRMS (ESI)

1

99%). H NMR (300 MHz, DMSO-d₆): δ 8.04 (d, J = 2.3 Hz, 1H),

+

m/z: [M⁺] (C₁₆H₂₅INO₂) calcd, 390.09245; found, 390.09230.

7.66 (dd, J = 8.5, 2.3 Hz, 1H), 7.64−7.57 (m, J = 7.3, 1.3 Hz, 2H),

7.47−7.39 (m, 2H), 7.36−7.30 (m, 1H), 7.09 (d, J = 8.5 Hz, 1H),

5.30 (t, J = 5.0 Hz, 1H), 4.13 (t, J = 5.9 Hz, 2H), 3.90−3.81 (m, 2H),

3.55−3.39 (m, 4H), 3.12 (s, 6H), 2.03−1.88 (m, 2H), 1.87−1.70 (m,

2H). ¹³C NMR (75 MHz, DMSO-d₆): δ 156.56, 138.36, 136.72,

134.76, 128.96, 127.98, 127.23, 126.33, 112.93, 87.80, 68.06, 64.64,

63.76, 54.93, 50.94, 25.57, 19.05. HRMS (ESI) m/z: [M⁺]

HPLC-UV purity 97.4%.

(2-Hydroxyethyl)-[6-(3-iodophenoxy)hexyl]-dimethylammonium

Bromide (43). General procedure A was used. The alkylating agent N

(350 mg, 0.914 mmol, 1 equiv, see the Supporting Information),

DMF (1 mL), and 2-(dimethylamino)ethanol (552 μL, 5.48 mmol,

6.0 equiv) were used for the synthesis of compound 43. The reaction

time was 2 h at a temperature of 70 °C. The product 43 was obtained

+

(C₂₀H₂₇INO₂) calcd, 440.10777; found, 440.10810. HPLC-UV

1

as a white solid (395 mg, 0.84 mmol, 92%). H NMR (300 MHz,

purity 98.8%.

DMSO-d₆): δ 7.30−7.24 (m, 2H), 7.10−7.03 (m, 1H), 6.98−6.91

(m, 1H), 5.27 (t, J = 5.0 Hz, 1H), 3.96 (t, J = 6.4 Hz, 2H), 3.87−3.76

(m, 2H), 3.46−3.28 (m, 4H), 3.07 (s, 6H), 1.78−1.62 (m, 4H),

1.51−1.39 (m, 2H), 1.39−1.24 (m, 2H). ¹³C NMR (75 MHz,

DMSO-d₆): δ 159.37, 131.34, 129.25, 122.87, 114.33, 95.20, 67.51,

64.53, 63.96, 54.89, 50.81, 28.26, 25.45, 24.97, 21.72. HRMS (ESI)

(2-Hydroxyethyl)-[6-(4-iodo-3-nitrophenoxy)hexyl]-dimethylam-

monium Bromide (48). The alkylating agent R2 (300 mg, 0.700

mmol, 1 equiv, see the Supporting Information) and 2-

(dimethylamino)ethanol (2.00 mL, 19.9 mmol, 28.4 equiv) were

used for the synthesis of compound 48. The reaction time was 40 min

at a temperature of 75 °C. The product 48 was obtained as a bright

yellow solid (362 mg, 0.70 mmol, 100%). ¹H NMR (300 MHz,

DMSO-d₆): δ 7.92 (d, J = 8.7 Hz, 1H), 7.52 (d, J = 2.9 Hz, 1H), 7.04

+

m/z: [M⁺] (C₁₆H₂₇INO₂) calcd, 392.1081; found, 392.1066. HPLC-

UV purity 96.5%.

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(dd, J = 8.7, 2.9 Hz, 1H), 5.27 (t, J = 5.0 Hz, 1H), 4.04 (t, J = 6.4 Hz,

2H), 3.87−3.76 (m, 2H), 3.46−3.29 (m, 4H), 3.08 (s, 6H), 1.80−

1.63 (m, 4H), 1.51−1.38 (m, 2H), 1.38−1.24 (m, 2H). ¹³C NMR (75

MHz, DMSO-d₆): δ 158.97, 154.15, 141.32, 120.68, 110.87, 76.49,

68.38, 64.52, 63.94, 54.88, 50.82, 28.02, 25.41, 24.86, 21.70. HRMS

ethanol. The DCM solution was evaporated, and compound 51 (318

mg) was isolated as a colorless viscous liquid. H NMR (400 MHz,

1

CDCl₃): δ 7.23 (dd, J = 8.2, 7.1 Hz, 1H), 7.01 (d, J = 7.1 Hz, 1H),

6.97 (d, J = 2.7 Hz, 1H), 6.80 (ddd, J = 8.2, 2.7, 0.9 Hz, 1H), 4.17−

4.11 (m, 2H), 3.94 (t, J = 6.2 Hz, 2H), 3.77−3.72 (m, 2H), 3.59−

3.53 (m, 2H), 3.35 (s, 6H), 1.85−1.75 (m, 4H), 1.61−1.42 (m, 10H),

1.37−1.24 (m, 6H), 1.07−1.00 (m, 6H), 0.87 (t, J = 7.3 Hz, 9H). ¹³C

NMR (101 MHz, CDCl₃): δ 158.44, 143.66, 128.94, 128.86, 122.62,

113.75, 67.30, 66.24, 65.89, 56.09, 52.32, 29.25, 29.20, 27.50, 26.19,

25.92, 22.98, 13.83, 9.73. MS (ESI) m/z: [M⁺] (C₂₈H₅₄NO₂Sn⁺)

calcd, 556.32; found, 556.33.

+

(ESI) m/z: [M⁺] (C₁₆H₂₆IN₂O₄) calcd, 437.09318; found,

437.09299. HPLC-UV purity 98.3%.

(3-Hydroxypropyl)-[6-(4-iodo-3-nitrophenoxy)hexyl]-dimethy-

lammonium Bromide (49). The alkylating agent R2 (150 mg, 0.350

mmol, 1 equiv, see the Supporting Information) and 3-

(dimethylamino)propanol (2 mL) were used for the synthesis of

compound 49. The reaction time was 1.5 h at a temperature of 75 °C.

The product 49 was obtained as a yellow solid (183 mg, 0.344 mmol,

Synthesis of Radiolabeled Choline Derivatives. General

Procedure for Isotopic Exchange Labeling Using Pd Catalyst.

Solution A: To a 2 mL microcentrifuge tube were added PdCl₂(1

mg) and acetonitrile (ACN) (1 mL), and the suspension was

sonicated in an ultrasonic bath for 15 min. The suspension was then

centrifuged, and the solution was decanted from undissolved PdCl₂.

Solution B: AA (30 mg) in deionized water (1 mL). Solution C:

Corresponding iodinated choline derivative (1 mg) in deionized water

(100 μL). A 0.5 mL microcentrifuge tube was charged with the

solutions A (4 μL), B (3 μL), and C (1 μL). The mixture was gently

shaken, left at rt for 15 min, and centrifuged. Then, a solution of

Na¹²⁵I (14 μL, approx. 60 MBq, no-carrier-added) was added, and the

reaction mixture was gently shaken. The tube was sealed with

paraﬁlm, centrifuged, and immersed into a water bath heated to 75

°C. After 1 h, the reaction mixture was centrifuged, and was again

immersed into a water bath heated to 75 °C. After another 1 h, the

reaction mixture was cooled to rt and diluted with water (100 μL),

and ﬁve beads of Amberlite IRA-900 anion-exchange resin in TsO⁻

cycle and a piece of silver wire were added in order to remove free

Na¹²⁵I and other inorganic iodine species from the radiolabeled

product. The tube was then placed in a shaker for 5 min.

Subsequently, the tube was centrifuged, beads and silver wire were

decanted, the liquid was removed and transferred into a diﬀerent

microcentrifuge tube, the beads were washed with deionized water (2

× 80 μL), and the tube containing beads was centrifuged and another

portion of radiolabeled product solution was collected. The washing

solutions were added to the primary solution, yielding radiolabeled

product (radiochemical yield was typically 40−80%, molar activity ≈

1−3 GBq·μmol⁻¹) in radiochemical purity ≥95% according to radio-

HPLC analyses using a C₁₈silica reverse-phase column (see the

Methods section).

1

98%). H NMR (300 MHz, DMSO-d₆): δ 7.93 (d, J = 8.8 Hz, 1H),

7.54 (d, J = 2.9 Hz, 1H), 7.04 (dd, J = 8.8, 2.9 Hz, 1H), 4.79 (t, J =

5.0 Hz, 1H), 4.05 (t, J = 6.4 Hz, 2H), 3.52−3.43 (m, 2H), 3.33−3.19

(m, 4H), 3.00 (s, 6H), 1.88−1.59 (m, 6H), 1.53−1.39 (m, 2H),

1.39−1.25 (m, 2H). ¹³C NMR (151 MHz, DMSO-d₆): δ 158.97,

154.14, 141.32, 120.66, 110.87, 76.28, 68.38, 62.79, 61.07, 57.59,

50.09, 28.02, 25.39, 25.30, 24.86, 21.58. HRMS (ESI) m/z: [M⁺]

+

(C₁₇H₂₈IN₂O₄) calcd, 451.10883; found, 451.10872. HPLC-UV

purity 97.9%.

(4-Hydroxybutyl)-[6-(4-iodo-3-nitrophenoxy)hexyl]-dimethylam-

monium Bromide (50). The alkylating agent R2 (150 mg, 0.350

mmol, 1 equiv, see the Supporting Information), 4-(dimethylamino)-

butanol (164 μL, 1.23 mmol, 3.5 equiv), and DMF (1.5 mL) were

used for the synthesis of compound 50. The reaction time was 2 h at a

temperature of 70 °C. The product 50 was obtained as a yellow solid

(173 mg, 0.318 mmol, 91%). ¹H NMR (300 MHz, DMSO-d₆): δ 7.93

(d, J = 8.8 Hz, 1H), 7.54 (d, J = 2.9 Hz, 1H), 7.04 (dd, J = 8.8, 2.9 Hz,

1H), 4.57 (t, J = 5.0 Hz, 1H), 4.05 (t, J = 6.4 Hz, 2H), 3.49−3.39 (m,

2H), 3.29−3.18 (m, 4H), 2.99 (s, 6H), 1.79−1.60 (m, 6H), 1.51−

1.26 (m, 6H). ¹³C NMR (151 MHz, DMSO-d₆): δ 158.97, 154.14,

141.31, 120.66, 110.87, 76.34, 68.37, 62.88, 62.82, 59.80, 49.92, 29.04,

28.02, 25.41, 24.85, 21.60, 18.71. HRMS (ESI) m/z: [M⁺]

+

(C₁₈H₃₀IN₂O₄) calcd, 465.12448; found, 465.12405. HPLC-UV

purity 98.4%.

(2-Hydroxyethyl)-[6-(3-tributylstannylphenoxy)hexyl]-dimethy-

lammonium Bromide (51). (A) 3-Iodophenol (1.50 g, 6.82 mmol, 1

equiv) and Pd₂(dba)₃(156 mg, 0.171 mmol, 2.5 mol %) were put into

a ﬂask equipped with a magnetic stir bar, and the ﬂask was closed with

a rubber septum and was put under argon atmosphere via four cycles

of vacuum−argon. DMF (15 mL), 1,4-dioxane (5 mL), and N,N-

diisopropylethylamine (DIPEA) (3.00 mL, 17.2 mmol, 2.5 equiv)

were added through the septum, ﬁnally followed by Sn₂Bu₆(3.79 mL,

7.50 mmol, 1.1 equiv). The reaction mixture was stirred at rt for 3 h

after which a TLC was run (10% EtOAc in hexane) showing approx.

50% conversion of the starting material. The reaction temperature was

increased to 55 °C for another 2 h. Subsequently, the reaction mixture

was evaporated in vacuo, and the 3-(tributylstannyl)phenol was

isolated by ﬂash chromatography on silica (gradient 0−30% EtOAc in

cyclohexane) as a colorless oil (990 mg, 2.58 mmol, 38%).

(B) 3-(Tributylstannyl)phenol (384 mg, 1.00 mmol, 1 equiv) was

reacted with 1,6-dibromohexane (920 μL, 1.46 g, 6 equiv) and K₂CO₃

(415 mg, 3.00 mmol, 3 equiv) in DMF (1.5 mL) under an argon

atmosphere at rt overnight. The reaction mixture was then diluted

with Et₂O and ﬁltered through a syringe ﬁlter, and the solvents were

removed in vacuo. The desired product {3-[(6-bromohexyl)oxy]-

phenyl}tributylstannane could not be puriﬁed from the excess 1,6-

dibromohexane even after 2 consecutive runs of ﬂash chromatography

(silica, gradient 0−15% EtOAc/cyclohexane) and was used as a

mixture for the ﬁnal step.

Synthesis of High-Molar-Activity [¹²³I]-43-HSA by Iododestan-

nylation. Stannylated precursor 51 (5.0 mg, 7.87 μmol) was dissolved

in MeOH (5 mL) and passed through a glass column containing a

strong anion-exchange resin in Cl⁻cycle (Amberlite IRA 900-Cl, 1.5

mL, 1.5 mequiv), and the methanolic solution of 51-chloride was

collected and immediately used for labeling. A 0.5 mL Eppendorf tube

was loaded with the MeOH solution of 51-chloride (2 μL, 3.15

nmol), phosphate-buﬀered saline (PBS) (10 μL), and EtOH (10 μL).

Subsequently, Na¹²³I (2 μL, 85 MBq, no-carrier-added) was added,

immediately followed by chloramine-T solution in PBS (10 μL, 5 mg·

mL⁻¹). The reaction mixture was left at rt for 15 min after which a

solution of AA in PBS was added (10 μL, 50 mg·mL⁻¹). After another

10 min, six beads of Amberlite IRA 900-Cl were added and the

Eppendorf tube was placed into a shaker (removal of free ¹²³I⁻). After

10 min, the suspension was centrifuged, the solution containing the

labeled compound was collected using a pipette, the anion-exchange

resin was washed with deionized water (35 μL), and the solutions

containing the radiolabeled product were combined [¹²³I]-43-HSA

(62.7 MBq, radiochemical yield 74%, molar activity ≥ 20

GBq·μmol⁻¹, radiochemical purity > 95% according to radio-HPLC

analyses using C₁₈silica reverse-phase column) (see the Methods

section, for radiochromatogram see the Supporting Information).

(C) The mixture of {3-[(6-bromohexyl)oxy]phenyl}-

tributylstannane and 1,6-dibromohexane obtained at step (B) was

reacted with excess 2-(dimethylamino)ethanol (1.5 mL) in DMF (5

mL) at 75 °C for 90 min. After that, all volatiles were exhaustively

removed under reduced pressure, the remaining white solid was

washed with DCM, and the resulting DCM suspension was ﬁltered

through a syringe ﬁlter to remove the insoluble dicationic product of

the reaction between 1,6-dibromohexane and 2-(dimethylamino)-

METHODS

■

¹H and ¹³C NMR. ¹H and ¹³C NMR spectra were acquired using a

Bruker Avance III 600 spectrometer operating at 600.2 MHz (150.9

MHz), an Avance III HD 400 spectrometer operating at 400.1 MHz

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(100.6 MHz), an Avance DPX 300 spectrometer operating at 300.1

MHz (75.5 MHz), and JEOL ECZ 500R at 500.2 MHz (125.8 MHz).

High-Resolution MS Spectra. High-resolution MS (HRMS)

spectra were recorded on an LTQ Orbitrap XL using electrospray

ionization (ESI).

IR Spectrometry. Infrared (IR) spectra were measured by the

attenuated total reﬂection technique on a Fourier transform IR

spectrometer Nexus Nicolet 870 with a diamond prism.

HPLC Analyses. Analyses were performed on an HPLC

chromatograph (Dionex UltiMate 3000, Dionex, USA) using a

reverse-phase column (Chromolith Performance RP-18 endcapped

100 × 4.6 mm column equipped with Chromolith RP-18 endcapped 5

× 4.6 mm guard cartridge, Merck, Germany) and UV detection at 225

nm. A FlowStar LB 513 radio ﬂow detector (Berthold Technologies,

Germany) was used for the in-line radioactivity measurements. The

following elution conditions were used for chemical purity

determination of compounds 1−50: phase A: 95% water, 5% ACN

+ 0.1% TFA; phase B: 5% water, 95% ACN + 0.1% TFA. Flow rate

0.00−0.25 min: 0.40−3.00 mL/min; 0.40−7.00 min: 3.00 mL/min.

Mobile phase composition0.00−0.25 min: 9% B; 0.25−5.50 min:

linear gradient 9−70% B; 5.50−6.50 min: linear gradient 70−100% B;

7.00 min: 100% B.

Elemental Analyses. Elemental analyses for C/H/N were

performed on a PE 2400 Series II analyzer (Perkin Elmer, USA).

Elemental analyses for sulfur and iodine were performed on a X-ray

ﬂuorescence spectrometer SPECTRO iQ II (SPECTRO Analytical

Instruments, Germany).

In Vitro Pharmacokinetic Properties (ADME). The analysis of

all samples in ADME assays was performed using a Agilent RapidFire

300 high-throughput mass spectrometry system (RF-MS, Agilent,

Wakeﬁeld, MA) with subsequent detection in the mass spectrometer

QTRAP 5500 (AB Sciex, Concord, Canada).

followed by centrifugation (3000 rpm, 6 min, 4 °C) and the

supernatant was freeze-dried for subsequent analysis. The freeze-dried

samples were dissolved in water containing internal standard and were

analyzed by RF-MS.⁵⁵The intrinsic clearance was calculated using the

formula: CL_int= V·(0.693/t_1/2), where V is the volume of incubation

in microliter related to the weight of microsomal protein in mg per

reaction. Half-life values (t_1/2) were calculated using the equation t_1/2

= 0.693/k, where k is the slope of the ln of the percent compound

remaining versus time curve.⁵⁵

Parallel Artiﬁcial Membrane Permeability Assay. The

PAMPA was performed with the Millipore MultiScreen ﬁlter

MultiScreen-IP Durapore 0.45 μm plates and receiver plates (Merck

Millipore). The assay was performed according to the Merck

Millipore protocol (PC040EN00). The tested compounds from

stock solution (10 mM in DMSO) were diluted using PBS to a

concentration of 20 μM (initiator solution). Brieﬂy, the initiator

solution was added to the donor wells. The ﬁlter membrane was

coated with 10% lecithin (Sigma-Aldrich) in dodecane, and the

acceptor wells were ﬁlled with PBS (pH 7.4). The acceptor ﬁlter plate

was carefully placed on the donor plate to form a “sandwich”.⁵⁶Time

of incubation was 18 h at rt. After incubation, 120 μL aliquots of

acceptor and donor solutions were removed to the 96-well plate and

freeze-dried, and the residues were dissolved in 200 μL of water prior

the RF-MS analysis. The relative permeability log Pe was calculated

by using the equation: log Pe = log{C × −ln(1 − drugA/drugE)},

where C = (V_A× V_D)/{(V_D+ V_A) × A × T}. V_Dand V_Aare the

volumes of the donor and acceptor solutions, A is the active surface

area in cm², T is time of the incubation in seconds, and drugA and

drugE are the mass of the compound in the acceptor solution and in

the solution in theoretical equilibrium (as if the donor and acceptor

were combined), respectively.

Plasma Protein Binding. Determination of PPB was carried out

using the rapid equilibrium dialysis (Thermo Scientiﬁc, Rockford,

USA) test, which is based on dialysis.⁵⁷Inserts for this assay consist of

two chambers (red and white) separated by a semipermeable

membrane. Into the chamber R was added 10 μM solution of the

tested substance in 10% plasma, and into the chamber W was added

PBS buﬀer. The substance in the chambers was incubated at 37 °C

(shaking) for 4 h. After incubation, equal volumes of solution were

transferred into microtubes. To the PBS buﬀer was added the same

amount of 10% plasma, and to the solution of the tested substance

was added the PBS buﬀer to normalize their composition. The

reaction was terminated by the addition of ACN−methanol (2:1).

Following centrifugation (3000 rpm, 6 min, 4 °C), the supernatant

was freeze-dried, diluted with water, and analyzed by RF-MS.

Determination of IC₅₀Values In Vitro. We employed human

prostate cancer cell line PC-3 and human glioblastoma multiforme

cell line U-87 MG, both purchased from ATCC (Manassas, Virginia,

USA) as model cell lines. The PC-3 cell line was cultured in F12 Ham

medium supplemented with 10% fetal bovine serum, 0.25% sodium

bicarbonate, streptomycin, and penicillin. All mentioned components

were from Sigma-Aldrich (St. Louis, Missouri, USA). U-87 MG cells

were cultivated in Dulbecco’s modiﬁed Eagle medium (Sigma-Aldrich,

Missouri, USA) supplemented with 10% of fetal bovine serum, 0.1

mM nonessential amino acids, and 1.0 mM sodium pyruvate (all

Sigma-Aldrich). The cell cultures were propagated at 37 °C in a

humidiﬁed 5% carbon dioxide atmosphere. The cells were subcultured

and used for experiments in conﬂuence of 70−90%. The competition

assay was performed in 24-well plates. The assay itself employed

special “uptake buﬀer” instead of standard cell culture media to

exclude the eﬀect of choline contained in the culture media. This

uptake buﬀer consisted of 25 mM Tris-HCl, 5.4 mM KCl, 1.8 mM

CaCl₂, 0.8 mM MgSO₄, 5 mM glucose, and 140 mM NaCl in H₂O,

pH = 7.4.³⁹

Chemical Stability Assay. The tested compounds from stock

solution (10 mM in DMSO) were diluted using PBS (pH 7.4) for

ﬁnal incubation concentration 2 μM. The time points of incubation

were 15, 30, 60, and 120 min at 37 °C. The incubation was stopped

by addition of cold methanol in the corresponding time points. The

samples were then vortexed and centrifuged (4000 rpm, 9 min, 4 °C),

and the supernatant was freeze-dried. After freeze-drying the

supernatant, the samples were dissolved in water with internal

standard and were analyzed by RF-MS analysis.⁵⁵

In Vitro Stability in Human Plasma. The studied compounds

(ﬁnal incubation concentration 2 μM; DMSO concentration did not

exceed 0.1%) were incubated with human plasma (Transfusion

Department, University Hospital Olomouc, Olomouc, Czech

Republic) at ﬁve diﬀerent time points (0, 15, 30, 60, and 120 min)

at 37 °C. The reactions were terminated by the addition of ACN−

methanol mixture (2:1). The samples were left at −80 °C overnight,

followed by centrifugation, freeze-drying, and analysis by RF-MS.⁵⁵

The RapidFire RF300 system (Agilent Technologies) was interfaced

with the QTRAP 5500 (AB Sciex) mass spectrometer ﬁtted with an

ESI source and was run in the multiple reaction monitoring mode.

The freeze-dried samples were redissolved in solvent A (95% water/

5% ACN/0.01% formic acid) and aspirated into a 10 μL sample loop

and passed through a C4 cartridge (Agilent Technologies) with

solvent A (95% water/5% ACN/0.01% formic acid) at a ﬂow rate of

1.5 mL·min⁻¹for 3 s. After the desalting step, the analyte retained on

the cartridge was eluted with solvent B (95% ACN/5% water/0.01%

formic acid) to the mass spectrometer at a ﬂow rate of 0.4 mL·min⁻¹

for 7 s. MS was carried out using ESI in the positive ion mode.

Daughter ion peaks were identiﬁed using a multiple reaction

monitoring protocol.

Microsomal Stability Assay. Reaction mixtures included the

tested compound (2 μM), human liver microsomes (Thermo Fisher

Scientiﬁc, 0.5 mg·mL⁻¹), and nicotinamide adenine dinucleotide

phosphate-generating system consisting of NADP⁺, isocitrate

dehydrogenase, isocitric acid, and MgSO₄in 0.1 M K₃PO₄buﬀer.

The mixture was incubated at 37 °C, and samples were collected in

the following time points: 0, 15, 30, and 60 min. The microsomal

activity was terminated using the ACN−methanol (2:1) mixture,

The cells in the 24-well plates were washed with PBS buﬀer and

then the [³H]choline (American Radiolabeled Chemicals, St. Louis,

Missouri, USA) in uptake buﬀer was added as substrate for choline

uptake, followed by 15 min incubation at 37 °C. Then, the studied

choline derivative was added (triplicate wells) in 10 diﬀerent

concentrations to cover concentration range from 1 × 10⁻⁸to 5 ×

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10³mol·L⁻¹. The cells were then incubated for 60 min at 37 °C after

which the buﬀer was removed. The cells were rinsed with ice-cold

PBS buﬀer and lysed with 0.1 M NaOH. The cell samples were

collected for radioactivity and protein content measurement. The

protein content was determined using a standard bicinchoninic acid

assay (BCA) protein assay (Pierce BCA protein assay kit, Thermo

Fisher Scientiﬁc). Radioactivity of the cell samples was measured in

Tri-Carb liquid scintillation counter (PerkinElmer, Waltham,

Massachusetts, USA).

placed in a prone position in the Albira system. The PET/CT images

were acquired over a 30 min period starting 1, 3 h after the injection

of the radiopharmaceutical. Shortly, 10 min PET scan (axial FOV 148

mm) was performed, followed by a double CT scan (axial FOV 2 ×

65 mm, 45 kVp, 400 μA, at 400 projections). Scan data were

reconstructed with the Albira software (Bruker Biospin Corporation,

Woodbridge, CT, USA) using the maximum likelihood expectation

maximization and FBP algorithms. The PMOD software (PMOD

Technologies, Zurich, Switzerland) was used for 2D image output,

while 3D images were created in VolView software (Kitware, Clifton

Park, NY, USA).

The cellular uptake of [³H]choline was calculated as the percentage

of applied dose per gram of cell protein content. These uptake values

were used to plot classical sigmoidal dose−response curve in the

GraphPad Prism (GraphPad Software, San Diego, California, USA).

The IC₅₀values were determined from this curve using ﬁtting analysis

by the above-mentioned software.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01710.

■

sı

Ex Vivo Biodistribution Studies. All experiments with animals

were performed in accordance with the appropriate legal norms (Act

no. 359/2012), with approval of Ministry of Education, Youth and

Sports (MSMT-18724/2016-2) and approval of Ethical Committee of

Faculty of Medicine and Dentistry, Palacky University in Olomouc.

SCID male mice (Harlan, Indianapolis, Indiana, USA) 6−8 weeks old

were used in this study. The mice were housed in speciﬁc-pathogen-

free animal facility during the whole period of the study. For

establishing the prostate tumor model, 5 × 10⁶of PC-3 cells

resuspended in culture media were injected subcutaneously into the

right ﬂank of the mice. The tumor growth was continuously

monitored using calipers. When tumor volume reached 100−300

mm³, that is, 6−8 weeks after the cell inoculation, the mice were

included into the ex vivo biodistribution study.

The employed choline derivatives (derivatives with IC₅₀com-

parable to [¹⁸F]ﬂuorocholine or better) were radiolabeled with ¹²⁵I by

isotope exchange. The solutions of radioiodinated choline derivatives

in distilled water were diluted with sterile saline to achieve the optimal

concentration of radioactivity and were applied retro-orbitally.⁵⁸The

applied dose varied between 100 and 110 kBq per animal. The mice

were anesthetized with isoﬂurane (4%) 1 h resp. 3 h post injection of

the radiotracer and were sacriﬁced by cervical dislocation. Blood

samples were collected from the carotid artery. In addition, the

following organs were collected during the autopsy: spleen, pancreas,

stomach, intestine, kidneys, liver, heart, lungs, muscle sample, bone

sample (femur), brain, thyroid, and tumor. The organ samples were

weighed, and their radioactivity was determined by automatic gamma

counter Wizard (PerkinElmer, Waltham, Massachusetts, USA). The

accumulation of the radiotracer in studied organs was expressed as the

percentage of the injected dose per gram of organ or percent of

injected dose in the case of the thyroid gland.

Experimental procedures, complete ADME and cytotox-

icity data for choline analogues, ﬁgures and tables with

1

the complete ex vivo biodistribution data, copies of H

and ¹³C NMR spectra, and HPLC traces (PDF)

Molecular formula strings with IC₅₀values (CSV)

AUTHOR INFORMATION

Corresponding Authors

■

́

Marian Hajduch − Institute of Molecular and Translational

Medicine, Faculty of Medicine and Dentistry, Palacký

University Olomouc, Olomouc 779 00, Czech Republic;

Email: marian.hajduch@upol.cz

́

Martin Hruby − Institute of Macromolecular Chemistry, CAS,

Prague 6 162 06, Czech Republic; orcid.org/0000-0002-

5075-261X; Email: mhruby@centrum.cz

Authors

̌

Pavel Svec − Institute of Macromolecular Chemistry, CAS,

Prague 6 162 06, Czech Republic; Department of Physical

and Macromolecular Chemistry, Faculty of Science, Charles

University, Prague 2 128 43, Czech Republic; orcid.org/

0000-0002-6604-2815

̌

́

Zbynek Novy − Institute of Molecular and Translational

Medicine, Faculty of Medicine and Dentistry, Palacký

University Olomouc, Olomouc 779 00, Czech Republic

̌

Jan Kucka − Institute of Macromolecular Chemistry, CAS,

Prague 6 162 06, Czech Republic

In Vivo SPECT Imaging. The SPECT/CT imaging was

performed with a small animal PET/SPECT/CT imaging system

(Albira, Bruker Biospin Corporation, Woodbridge, CT, USA). During

the scans, the mice were anesthetized with 2% isoﬂurane. Static

whole-body SPECT scans of 30 min duration were performed at 1, 3,

and 24 h after injection of [¹²³I]-10 or [¹²³I]-43 (15−20 MBq/

animal, molar activity ≥ 2.5 GBq·μmol⁻¹). In the case of high-molar-

activity [¹²³I]-43-HSA (15−20 MBq/animal, molar activity ≥ 20

GBq·μmol⁻¹), only 1 and 3 h p.i. scans were performed after which

the animals were sacriﬁced and tracer biodistribution was determined

ex vivo. The SPECT scans were followed by a double CT of 20 min

(axial FOV 2 × 65 mm, 45 kVp, 400 μA, at 400 projections).

Reconstruction of the acquired data was done by the Albira software

(Bruker Biospin Corporation, Woodbridge, CT, USA) using the

ordered subset expectation maximization and ﬁltered backprojection

(FBP) algorithms. All two-dimensional (2D) images were prepared

using PMOD software, v. 3.307 (PMOD Technologies Ltd., Zurich,

Switzerland), and all 3D images are from VolView software (Kitware,

Clifton Park, NY, USA).

̌

Milos Petrík − Institute of Molecular and Translational

Medicine, Faculty of Medicine and Dentistry, Palacký

University Olomouc, Olomouc 779 00, Czech Republic

̌

́

̌

Ondrej Sedlacek − Institute of Macromolecular Chemistry,

CAS, Prague 6 162 06, Czech Republic; orcid.org/0000-

0001-5731-2687

Martin Kuchar − Forensic Laboratory of Biologically Active

Substances, University of Chemistry and Technology, Prague

160 00, Czech Republic

Barbora Liskova − Institute of Molecular and Translational

Medicine, Faculty of Medicine and Dentistry, Palacký

University Olomouc, Olomouc 779 00, Czech Republic

̌

́

Martina Medvedíkova − Institute of Molecular and

Translational Medicine, Faculty of Medicine and Dentistry,

Palacký University Olomouc, Olomouc 779 00, Czech

Republic

́

Kristyna Kolouchova − Institute of Macromolecular

Chemistry, CAS, Prague 6 162 06, Czech Republic

Ondrej Groborz − Institute of Macromolecular Chemistry,

CAS, Prague 6 162 06, Czech Republic; orcid.org/0000-

In Vivo PET Imaging. The studied animals were imaged by

μPET/SPECT/CT system Albira (Bruker, Billerica, Massachusetts,

USA). Brieﬂy, the mice were retro-orbitally injected with [¹⁸F]-

ﬂuorocholine (UJV, Rez-Husinec, Czech Republic) at a dose of 5−7

MBq per animal. The animals under inhalation anaesthesia were

̌

0002-3164-6168

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J. Med. Chem. 2020, 63, 15960−15978

Journal of Medicinal Chemistry

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Article

(3) Ametamey, S. M.; Honer, M.; Schubiger, P. A. Molecular

imaging with PET. Chem. Rev. 2008, 108, 1501−1516.

(4) Lockman, P. R.; Allen, D. D. The transport of choline. Drug Dev.

Ind. Pharm. 2002, 28, 749−771.

(5) Glunde, K.; Bhujwalla, Z. M.; Ronen, S. M. Choline metabolism

in malignant transformation. Nat. Rev. Cancer 2011, 11, 835−848.

(6) Hara, T.; Bansal, A.; DeGrado, T. R. Choline transporter as a

novel target for molecular imaging of cancer. Mol. Imaging 2006, 5,

498−509.

́

Lenka Loukotova − Institute of Macromolecular Chemistry,

CAS, Prague 6 162 06, Czech Republic

Rafał Ł. Konefał − Institute of Macromolecular Chemistry,

CAS, Prague 6 162 06, Czech Republic

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01710

Author Contributions

(7) Gillies, R. J.; Morse, D. L. In vivo magnetic resonance

spectroscopy in cancer. Annu. Rev. Biomed. Eng. 2005, 7, 287−326.

(8) Hara, T.; Kosaka, N.; Shinoura, N.; Kondo, T. PET imaging of

brain tumor with [methyl-¹¹C]choline. J. Nucl. Med. 1997, 38, 842−

847.

The manuscript was written through contributions of all

authors. All authors have given approval to the ﬁnal version of

the manuscript.

Notes

(9) Kwee, S. A.; Wei, H.; Sesterhenn, I.; Yun, D.; Coel, M. N.

Localization of primary prostate cancer with dual-phase ¹⁸F-

fluorocholine PET. J. Nucl. Med. 2006, 47, 262−269.

(10) Hara, T.; Kosaka, N.; Suzuki, T.; Kudo, K.; Niino, H. Uptake

rates of ¹⁸F-fluorodeoxyglucose and ¹¹C-choline in lung cancer and

pulmonary tuberculosis: a positron emission tomography study. Chest

2003, 124, 893−901.

(11) DeGrado, T. R.; Coleman, R. E.; Wang, S.; Baldwin, S. W.; Orr,

M. D.; Robertson, C. N.; Polascik, T. J.; Price, D. T. Synthesis and

evaluation of ¹⁸F-labeled choline as an oncologic tracer for positron

emission tomography: initial findings in prostate cancer. Cancer Res.

2001, 61, 110−117.

The authors declare the following competing ﬁnancial

̌

́

interest(s): P.S., Z.N., J.K., M.P., O.S., M. Hajduch, and M.

́

Hruby are inventors of a patent application covering the title

compounds.

ACKNOWLEDGMENTS

■

This work was supported by the Czech Science Foundation

(grant # 19-01602S), the Ministry of Health of the Czech

Republic (grant # 16-30544A), by the Ministry of Education,

Youth and Sports of the Czech Republic (grants # LM2015064

EATRIS-CZ, LO1304, CZ.02.1.01/0.0/0.0/16_013/0001818,

and CZ.02.1.01/0.0/0.0/16_019/0000868), the Technology

Agency of the Czech Republic (grant # TE01020028), and the

Charles University Grant Agency (project no. 766119).

(12) DeGrado, T. R.; Baldwin, S. W.; Wang, S.; Orr, M. D.; Liao, R.

P.; Friedman, H. S.; Reiman, R.; Price, D. T.; Coleman, R. E.

Synthesis and evaluation of ¹⁸F-labeled choline analogs as oncologic

PET tracers. J. Nucl. Med. 2001, 42, 1805−1814.

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Torizuka, T. ¹¹C-choline positron emission tomography in prostate

cancer: primary staging and recurrent site staging. Urol. Int. 2005, 74,

214−220.

ABBREVIATIONS

■

AA, ascorbic acid; ACN, acetonitrile; ADME, absorption,

distribution, metabolism, and excretion; BCA, bicinchoninic

acid assay; br s, broad singlet; Ch, choline; ChT, choline

transporter; log P, logarithm of water−octanol partition

coeﬃcient; CT, computed tomography; CTL, choline trans-

porter-like protein; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene;

DCM, dichloromethane; DMAE, 2-(dimethylamino)ethanol;

DMAP, 4-dimethylaminopyridine; DMF, N,N-dimethylforma-

mide; DMSO, dimethylsulfoxide; ESI, electrospray ionization;

FBP, ﬁltered backprojection; FCh, ﬂuorocholine, N-(ﬂuoro-

methyl)-2-hydroxy-N,N-dimethylethan-1-aminium; HCh-3,

hemicholinium-3; HRMS, high-resolution mass spectrometry;

IC₅₀, half-maximum inhibitory concentration; LipE, lipophilic

eﬃciency; mp, melting point; MeOTs, methyl tosylate; MRI,

magnetic resonance imaging; n-BuLi, n-butyllithium; NMR,

nuclear magnetic resonance; OCT, organic cation transporter;

PAMPA, parallel artiﬁcial membrane permeability assay; PBS,

phosphate-buﬀered saline; PET, positron-emission tomogra-

phy; PI3K, phosphoinositide 3-kinase; PPB, plasma protein

binding; RED, rapid equilibrium dialysis; RF-MS, RapidFire

mass spectrometry; SCID, severe combined immunodeﬁ-

ciency; SPECT, single-photon emission computed tomogra-

phy; TBAF, tetra-n-butylammonium ﬂuoride; TBSCl, tert-

butyldimethylsilyl chloride; TFA, triﬂuoroacetic acid; THF,

tetrahydrofuran; TLC, thin-layer chromatography; TsCl, 4-

methylbenzene-1-sulfonyl chloride; TsF, 4-methylbenzene-1-

sulfonyl ﬂuoride

̈

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