Design, synthesis and biological evaluation of Tozadenant analogues as adenosine A2A receptor ligands

European Journal of Medicinal Chemistry 214 (2021) 1 13214

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Design, synthesis and biological evaluation of Tozadenant analogues

as adenosine A_2Areceptor ligands

,

c

,

, d

Dana R. Renk ^{a d}, Marcel Skraban ^{a d}, Dirk Bier ^{a d}, Annette Schulze ^a

,

Erika Wabbals ^a^d, Franziska Wedekind ^{b d}, Felix Neumaier ^{a d}, Bernd Neumaier ^a

,

c

,

, c, d

Marcus Holschbach ^a

, d, *

^aInstitute of Neuroscience and Medicine, Nuclear Chemistry (INM-5), Germany

^bMolecular Organization of the Brain (INM-2), Wilhelm-Johnen-Straße, 52428, Jülich, Germany

^cUniversity of Cologne, Faculty of Medicine and University Hospital Cologne, Institute of Radiochemistry and Experimental Molecular Imaging, Kerpener

€

Straße 62, 50937, Koln, Germany

^dForschungszentrum Jülich GmbH, Germany

a r t i c l e i n f o

a b s t r a c t

Article history:

With the aim to obtain potent adenosine A_2Areceptor (A_2AR) ligands, a series of eighteen derivatives of 4-

hydroxy-N-(4-methoxy-7-morpholin-4-yl-1,3-benzo[d]thiazol-2-yl)-4-methylpiperidine-1-carboxamide

(SYN-115, Tozadenant) were designed and synthesized. The target compounds were obtained by a

chemical building block principle that involved reaction of the appropriate aminobenzothiazole phenyl

carbamates with either commercially available or readily synthesized functionalized piperidines. Their

afﬁnity and subtype selectivity with regard to human adenosine A₁-and A_2Areceptors were determined

using radioligand binding assays. K_ivalues for human A_2AR ranged from 2.4 to 38 nM, with more than

120-fold selectivity over A₁receptors for all evaluated compounds except 13k which had a K_iof 361 nM

and 18-fold selectivity. The most potent ﬂuorine-containing derivatives 13e, 13g and 13l exhibited K_i

values of 4.9 nM, 3.6 nM and 2.8 nM for the human A_2AR. Interestingly, the corresponding values for rat

Received 19 November 2020

Received in revised form

12 January 2021

Accepted 13 January 2021

Available online 30 January 2021

Keywords:

A_2Aadenosine Receptor

Ligand synthesis

Fluorinated analogues

Fluorine-18 isotopologues

Binding studies

A

_2AR were found to be four to ﬁve times higher. Their binding to A_2AR was further conﬁrmed by radi-

olabeling with ¹⁸F and in vitro autoradiography in rat brain slices, which showed almost exclusive striatal

binding and complete displacement by the A_2AR antagonist ZM 241385. We conclude that these com-

pounds represent potential candidates for the visualization of the A_2Areceptor and open pathways to

novel therapeutic treatments of neurodegenerative disorders or cancer.

Autoradiography

1. Introduction

number of neurodegenerative [9,10], neuropsychiatric [1 1,12] and

neuroinﬂammatory [13] disorders. In the healthy CNS, A_2AR is most

The purine nucleoside adenosine (adenine-9-

b

-

D

-ribofurano-

abundant in the basal ganglia [14,15], where it co-localizes with

dopamine D₂receptors on striatopallidal output neurons [16] and

has been targeted for non-dopaminergic treatment of Parkinson’s

disease [1 7e19]. Receptor densities in other subcortical structures

and the cerebral cortex are at least ten-fold lower [14], but many

diseases appear to be associated with considerable changes in

extra-striatal A_2AR expression [10,13,14]. The relevance of A_2ARs in a

number of speciﬁc disease states like in neurodegenerative diseases

or in tumor immunotherapy [14,20,21] has stimulated the devel-

opment of various selective antagonists for these receptors, which

can be broadly classiﬁed into xanthine and non-xanthine de-

rivatives [22,23].

side) is an ubiquitous but short-lived signaling molecule that exerts

its effects through at least four subtypes of G-protein coupled

adenosine receptors (ARs) [1]. Within the central nervous system

(CNS), adenosine is a neuromodulator that participates in the

regulation of sleep and arousal [2e4], is involved in cognition [5,6]

and contributes to the autoregulation of cerebral blood ﬂow [7,8].

The adenosine A_2Areceptor (A_2AR) mediates multiple of the phys-

iological effects of adenosine and has been implicated with a

* Corresponding author. Institute of Neuroscience and Medicine, Nuclear

Chemistry (INM-5), Forschungszentrum Jülich GmbH, Germany.

E-mail address: m.holschbach@fz-juelich.de (M. Holschbach).

https://doi.org/10.1016/j.ejmech.2021.113214

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

The xanthine scaffold present in naturally occurring nonselec-

tive AR antagonists like caffeine provided a ﬁrst starting point for

the development of selective A_2AR antagonists, which led to the

discovery of 3-chlorostyrylcaffeine (CSC), MSX-2 [24] and istrade-

fylline [25] (Fig. 1).

selected to investigate if they are amenable to radioﬂuorination.

The corresponding precursors were synthesized and radio-

ﬂuorinated. Their potential suitability for positron emission to-

mography (PET) was further investigated by determination of their

binding proﬁles by in vitro autoradiographic studies.

However, due to their poor water solubility and metabolic

instability, a search for different structural types of A_2AR antago-

nists based on mono-, bi-, and triheterocycles was soon initiated.

The ﬁrst non-xanthine A_2AR antagonist thus discovered was the

triazoloquinazoline CGS15943 (Fig. 2) [26], a very potent but un-

selective compound that exhibits high afﬁnity for the other ARs as

well. Nonetheless, the CGS scaffold served as a template for the

development of more selective antagonists and these efforts

culminated in the identiﬁcation of N-substituted pyrazolo-

triazolopyrimidines as a class of heterocycles with high A_2AR af-

ﬁnity and strongly reduced afﬁnity for all other ARs [27]. One

member of this class, the tricyclic SCH58261 (Fig. 2), was rapidly

accepted as a reference A_2AR antagonist, although it suffered from

poor water solubility and synthesizability These disadvantages

have been overcome by the development of ZM241385 (Fig. 2), a

very potent and selective bicyclic triazolotriazine based A_2AR

antagonist [28]. By virtue of its bicyclic nature and due to the

presence of two additional hydrogen donors, ZM241385 showed a

much more favorable aqueous solubility proﬁle.

2. Results and discussion

2.1. Chemistry

The lead heterocycle 4-methoxy-7-morpholin-4-yl-benzo[d]

thiazol-2-ylamine 7 was prepared from p-anisidine in a 6-step

synthesis (see Scheme 1).

Conditions: (a) i) 2-chloroethyl ether, TBAB, NaOH,180 ^ꢀC, 8 h, ii)

70% HNO₃, 0e5 ^ꢀC,12 h; (b) 95% H₂SO₄, 0e5 ^ꢀC,1.5 h; (c) NH₄Cl, zinc

powder, EtOH/ethyl acetate, r.t., 0.5 h; (d) benzoyl isothiocyanate,

acetone, r.t., 0.5 h; (e) NaOMe, MeOH, r.t., 3 h; (f) i) 33% HBr, 80 ^ꢀC,

0.4 h, ii) DMSO, ethyl acetate, 80 ^ꢀC, 4 h; (g) phenyl chloroformate,

pyridine, THF, r.t., 14 h; (h) 48% HBr, 130 ^ꢀC, 24 h; (i) 2-bromo-1-

ﬂuoroethane, Cs₂CO₃, DMF, r.t., 24 h.

In the ﬁrst reaction step, p-anisidine 1 was reacted with 2-

chloroethyl ether to form 4-(4-methoxyphenyl)morpholine. Crude

morpholine was treated with nitric acid and isolated as the nitrate

salt 2. Subsequent regioselective nitration furnished 4-(4-methoxy-

3-nitrophenyl)morpholine 3 [34]. Reduction of the nitro group with

zinc/ammonium chloride yielded 2-methoxy-5-morpholinoaniline 4

in almost quantitative yield. Reaction with benzoyl isothiocyanate

provided 1-benzoyl-3-(2-methoxy-5-morpholin-4-yl-phenyl)thio-

urea 5 which was deprotected with sodium methoxide to give 2-

methoxy-5-morpholin-4-yl-phenylthiourea 6. Cyclization of thio-

Since then several additional classes of non-xanthine A_2AR an-

tagonists with either mono-, bi-, or tricyclic core-based structures

have been reported [29] and used to decipher the pharmacology

and signaling pathways of these receptors. Efforts to optimize these

ligands are ongoing and there is still a need for novel, easily

accessible compounds with high afﬁnity and subtype selectivity for

the design of therapeutic agents.

urea

6 to furnish 4-methoxy-7-morpholin-4-yl-benzothiazol-2-

In 2005 Hoffmann-La Roche disclosed 4-hydroxy-N-(4-

methoxy-7-morpholin-4-yl-1,3-benzo[d]thiazol-2-yl)-4-

ylamine 7 was performed by a modiﬁed Hugershoff reaction using

bromine in dimethyl sulfoxide [35]. Phenyl carbamate 8 was pre-

pared by reaction of 7 with phenyl chloroformate and was obtained

as a bench stable crystalline solid. 2-Amino-7-morpholinobenzo[d]

thiazol-4-ol 9 was prepared in excellent yield by demethylation of 7

with aqueous 48% hydrobromic acid. Attempts to cleave the meth-

ylether 7 with boron tribromide failed. Cesium carbonate supported

chemoseletive O-alkylation of aminophenol 9 with 1-bromo-2-

ﬂuoroethane furnished 10. Reaction of aminothiazole 10 with

phenyl chloroformate and pyridine as a base afforded phenyl

carbamate 11 as a stable solid.

methylpiperidine-1-carboxamide (SYN-115, Tozadenant), a potent

and selective A_2AR antagonist based on a benzothiazole scaffold

[30] (Fig. 3). Our research started with the decision to use the core

structure of Tozadenant as a template to develop improved A_2AR

antagonists with high selective receptor binding and suitable

physicochemical and pharmacokinetic properties. Although re-

searchers from Hoffmann-La Roche have described a number of

similar compounds in patents, the ones described here are invari-

ably new.

After thorough analysis of the chemical structure of Tozadenant

and extensive literature screening [31,32] we decided to chemically

modify the substituents at the 4-position of the piperidine ring or

to exchange the methoxy group at carbon-4 of the benzothiazole

ring system (Fig. 3).

As in our earlier study of antagonists for the adenosine A₁re-

ceptor (A₁R) [33], the primary objective was to assess the impact of

ﬂuorination on binding characteristics and pharmacological activ-

ity. Herein, we describe the results of the modiﬁcations of the

Tozadenant structure on binding afﬁnity for the A_2Areceptor and

adenosine receptor selectivity. Since most of the described com-

pounds contain a ﬂuorine, three high-afﬁnity candidates were

Piperidines used in this work are depicted in Chart 1. Most of

them are commercially available but piperidines 12g, 12h, 12i, 12m,

12n, and 12o were unknown and had to be synthesized (Scheme 2).

Conditions: (a) MsCl, Et₃N, DCM, r.t., 1 h; (b) TBAF, THF, 70 ^ꢀC,

19e24 h; (c) TFA, DCM r.t., 1e4 h; (d) i) NaH, THF, 0 ^ꢀC, 20 min, ii)

methyl bromoacetate, r.t., 22 h; (e) LAH, r.t., 3 h; (f) PyFluor, r.t.,

48e69 h; (g) ethyl acetate, LiHMDS, THF, ꢁ70 ^ꢀC, 3.5 h; (h) LiBH₄/

MeOH, THF, r.t., 2 h; (i) FCH₂Li, ꢁ78 ^ꢀC, 5 min *n.i. not isolated.

4-(Fluoromethyl)piperidine 12g was prepared by mesylation of

the N-tert-butoxycarbonyl (Boc) protected alcohol followed by

nucleophilic ﬂuorination with excess tetrabutylammonium ﬂuoride

and subsequent acid induced Boc deprotection. Piperidines 12h and

Fig. 1. Chemical structures of xanthine based A_2AR antagonists.

2

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

Fig. 2. Structures of non-xanthine A_2AR antagonists CGS15943, SCH58261 and ZM241385.

Fig. 3. Structure of Tozadenant and positions for chemical modiﬁcations.

Scheme 1. Synthesis of starting aminobenzothiazoles 7 and 9 and phenyl carbamates 8 and 11.

12i were obtained by O-alkylation of the sodium salts of N-Boc-4-

hydroxypiperidine (12h) or N-Boc-4-hydroxymethylpiperidine

(12i) with methyl bromoacetate followed by ester reduction with

lithium aluminium hydride. Fluorination of the respective alcohols

with PyFluor [36] and Boc deprotection with triﬂuoroacetic acid

provided 4-(2-ﬂuoroethoxy)piperidine 12h and 4-((2-ﬂuoroethoxy)

methyl)piperidine 12i in moderate yields. 2-Fluoro-1-(piperidin-4-

yl)ethanol 12n and 1-ﬂuoro-2-(piperidin-4-yl)propan-2-ol 12o

were synthesized by ﬂuoromethylation of either N-Boc-4-

formylpiperidine or N-Boc-4-acetylpiperidine with in situ prepared

ﬂuoromethyllithium [37]. Acidic Boc deprotection furnished ﬂuo-

rohydrins 12n and 12o in acceptable yields (Scheme 3). 4-(2-

Fluoroethyl)piperidin-4-ol 12m was obtained starting from N-Boc-

4-piperidone. Low temperature reaction with lithio ethyl acetate,

prepared in situ from ethyl acetate and lithium hexamethyldisilazide

[38], provided N-Boc-4-ethoxycarbonylmethyl-4-hydroxypiperidine

3

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

Scheme 2. Synthesis of piperidines 12g, 12h, 12i, 12m, rac-12n and rac-12o.

which was reduced with lithium borohydride in methanol to furnish

N-Boc-4-hydroxy-4-(2-hydroxyethyl)piperidine. Fluorination with

excess tetrabutylammonium ﬂuoride followed by acidic Boc depro-

tection gave 4-(2-ﬂuoroethyl)piperidin-4-ol 12m in good yield.

The asymmetric N,N⁰-disubstituted target ureas 13a e 13g and

13p were synthesized via aminolysis of phenyl carbamate 8 under

neutral conditions (Scheme 3) [39]. Thus, using dimethyl sulfoxide

as the solvent, reaction of 8 or 10 with a stoichiometric amount of

the respective piperidine at ambient or slightly elevated tempera-

ture rapidly generated the asymmetric target ureas in high yield.

Ureas 13h to 13o were obtained by base catalyzed reaction of 8 with

the respective piperidine at slightly elevated temperature using

1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) as a base.

Conditions: (a) 1.08 eq. substituted piperidine, DMSO, ambient

temperature or 60 ^ꢀC (for 13a - 13g, 13p); (b) 1e1.5 eq. substituted

piperidine, DMSO, DBU, 60 ^ꢀC (for 13h, 13i and 13k - 13o); (c) 2 eq.

substituted piperidine, DCE, 40 ^ꢀC (for 13j).

For nucleophilic radioﬂuorination of the three pharmacologi-

cally most promising candidates 13e, 13g and 13l with ﬂuorine-18,

the appropriate radiolabeling precursors 14a e 14c were prepared

as follows (Scheme 4). The synthesis of mesylates 14a and 14b was

straightforward. It started with the N-pivaloyloxymethyl (N-Pom)

protection of 4-hydroxy-N-(4-methoxy-7-morpholinobenzo[d]

thiazol-2-yl)piperidine-1-carboxamide 13c to give 14a-1. N-Pom

protection

of

4-(hydroxymethyl)-N-(4-methoxy-7-

morpholinobenzo[d]thiazol-2-yl)piperidine-1-carboxamide 14b-1,

prepared from phenyl carbamate 8 and 4-piperidinemethanol,

provided 14b-2. Mesylation with methanesulfonyl chloride and

triethylamine as base furnished the protected precursor sulfonates

14a and 14b as crystalline solids with high yields (Scheme 4). Sulﬁte

14c was obtained through a multistep synthesis starting from N-

benzyloxycarbonyl-4-piperidone (N-Cbz-4-piperidone). Corey-

Chaykowsky epoxidation of N-Cbz-4-piperidone with the

methylene-transfer reagent dimethyloxosulfonium methylide,

which was prepared by the action of sodium hydride on trime-

thylsulfonium iodide in tetrahydrofuran [40] provided benzyl 1-

oxa-6-azaspiro[2.5]octane-6-carboxylate 14c-1 in 61% yield. Stir-

ring the epoxide in 0.02 N aqueous hydrochloric acid led to ring

opening to give the diol, benzyl 4-hydroxy-4-(hydroxymethyl)

piperidine-1-carboxylate 14c-2, in almost quantitative yield. Diol

protection as the acetonide using dimethoxypropane and cam-

phersulfonic acid (CSA) as a catalyst followed by hydrogenolytic

removal of the Cbz group provided piperidine 14c-4. Treatment of

phenyl carbamate 8 with a stoichiometric amount of 14c-4 in

DMSO at 60 ^ꢀC overnight generated urea 14c-5. N-Pom protection

and subsequent hydrolysis of the cyclic ketal 14c-6 with CSA in

4

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

Scheme 3. Synthesis and chemical structures of asymmetric N,N⁰-disubstituted target ureas 13a e 13p.

methanol liberated the free diol 14c-7, which was treated in the

cold with thionyl chloride/triethylamine to furnish the crystalline

cyclic sulﬁte 14c [41].

nucleophilic substitution of the mesyl leaving group by [¹⁸F]ﬂuo-

ride under phase transfer conditions (PTC) and subsequent Pom

deprotection. Following standard labeling and deprotection pro-

cedures ([¹⁸F]KF in the presence of Kryptoﬁx 2.2.2., anhydrous

DMSO, 85 ^ꢀC, 15 min, sodium methylate, r.t., 3 min) radiotracers

Conditions: (a) POM-Cl, K₂CO₃, DMF, 60 ^ꢀC, 3.5 h; (b) Ms-Cl,

Et₃N, DCM, 0 ^ꢀC, 3 h; (c) NaH, (CH₃)₃S(I), DMSO, 55 ^ꢀC, 2 h; (d)

0.02 N HCl, 50 ^ꢀC, 1 h; (e) 2,2-dimethoxypropane, CSA, r.t., 24 h; (f)

H₂, Pd/C, MeOH, r.t., 12 h; (g) 8, DMSO, 60 ^ꢀC, 24 h; (h) CSA, MeOH,

50 ^ꢀC, 12 h; (i) SOCl₂, Et₃N, DCM, 0 ^ꢀC, 15 min.

[

¹⁸F]13e and [¹⁸F]13g (1.6e2 GBq) were obtained in 25%e30%

radiochemical yield (RCY) with a molar activity (MA) of 249e300

GBq/ mol and a radiochemical purity (RCP) of >99%.

m

A similar procedure was used for the radioﬂuorination of the

cyclic sulﬁte 14c using [¹⁸F]n-Et₄NF as radiolabeling reagent. Stan-

dard labeling conditions ([¹⁸F]n-Et₄NF, dimethyl sulfoxide, 140 ^ꢀC,

15 min, sodium methylate, r.t., 3 min) yielded radiotracer [¹⁸F]13l

2.2. Radiochemistry

Radiolabeling of the mesylates 14a and 14b and the cyclic sulﬁte

14c with no carrier added (n.c.a.) ﬂuorine-18 and subsequent Pom

deprotection afforded ﬂuorine-18 isotopologues [¹⁸F]13e, [¹⁸F]13g

and [¹⁸F]13l (Chart 2). Thus, [¹⁸F]13e and [¹⁸F]13g were prepared by

(1.2 GBq) in 10%e15% RCY with a MA of 600 GBq/

>99%.

mmol and a RCP of

5

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

Scheme 4. Synthesis of the radiolabeling precursors 14a - 14c.

2.3. Radioligand binding assays

of an ether moiety on the other hand reduced the afﬁnity for the

_2AR and resulted in analogues with K_ivalues of 20e33 nM

A

The different ligands described above and the lead compound

Tozadenant were subjected to receptor binding studies to deter-

mine their ability to displace [³H]ZM241385 from the cloned hu-

man A_2AR stably expressed in Chinese hamster ovary (CHO) cells.

The resulting K_Ivalues, calculated with the approximation formula

of Cheng and Prusoff, are summarized in Table 1. All compounds

showed a high to excellent afﬁnity for the human A_2AR, with K_i

values ranging from 2.4 to 361 nM, while the K_ivalue for the lead

(compounds 13d, h and i). Replacement of the methoxy group at

the 4-position of the benzothiazole scaffold by a ﬂuoroethoxy

moiety (13p) led to a more signiﬁcant loss of potency (K_i361 nM for

13p). Sterically more demanding functionalities as in 13n and 13o

led to analogues with good to medium A_2AR afﬁnity (K_i6.4 for 13n

and K_i38 nM for 13o). Surprisingly, unsaturated analogue 13b

showed the highest potency (K_i2.4 nM) of all evaluated com-

pounds. For three of the compounds which were chosen for radi-

olabeling with ﬂuorine-18 and further evaluation by in vitro

autoradiography (13e, 13g and 13I, see below), the afﬁnity for the

murine A_2AR was determined using homogenates from rat brain

corpora striata, which revealed K_ivalues roughly 4e5 times higher

than the values obtained for the human receptor but still within the

two-digit nanomolar range (Table 1).

compound tozadenant 13j was determined to be 3.9

0.7 nM

(Table 1).

Comparison of the binding data among different test com-

pounds (for structures see Scheme 2) shows that modiﬁcations of

the piperidine ring with small polar electron withdrawing sub-

stituents were well tolerated and resulted in analogues with K_i

values of 3e8 nM (compounds 13c, e, f, l, n, k and g). Introduction

6

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

Table 1

Results of the receptor binding experiments. K_ivalues for human and rat A_2AR were determined in competition experiments with 0.5 m

M [³H]ZM 241385 and are expressed as

mean standard deviation based on n, the number of experiments. K_ivalues for human A₁R were determined in competition experiments with 0.5 nM [³H]DPCPX and are

shown with the corresponding 95% conﬁdence interval. clogP values were calculated using ChemDraw Ultra 12.0.

Compound

Human A_2AR

K_i[nM]

Rat A_2A

R

Human A₁R

hA_2A/hA₁

clogP

n

K_i[nM]

n

K_i[nM] (95% CI)

13a

13b

13c

13d

13e

rac-13f

13g

13h

13i

13j (Toz)

13k

13l

13m

rac-13n

rac-13o

13p

6.0 1.0

2.4 0.24

3.8 0.48

33 6.8

3

7

3

7

3

1559 (836e2114)

577 (417e799)

260

240

452

229

120

183

332

>830

227

866

801

177

510

2606

>520

18

3.02

2.74

0.94

1.66

2.72

2.87

2.82

1.77

2.39

1.46

1.70

1.18

1.41

1.59

1.99

1.71

1719 (858e3442)

7563 (4936e11588)

591 (205e1700)

1173 (853e1612)

1198 (971e1478)

>20000^a

7975 (5245e12126)

3380 (1875e6093)

2887 (1985e4201)

497 (342e721)

4.9 1.3

6.4 0.68

3.6 0.18

23.9 7.2

35.1 1.18

3.9 0.7

3.6 0.54

2.8 0.9

9.4 3.7

6.4 2.3

37.9 15.1

361 112

19.2 3.9

3

5

20.4 13.3

18.7 5.4

14.9 7.9

195 65.3

10

14

4

4802 (2940e7841)

16680 (9370e29689)

>20000^a

6663 (2051e21658)

a

no displacement of the radioligand could be observed even at the highest concentrations of the compound used (5 mM).

2.4. In vitro autoradiography

the development of new PET ligands. The strategy of performing

structural modiﬁcations at the 4-position of the piperidine ring of

Tozadenant while maintaining high afﬁnity and selectivity for the

target receptor offers the possibility to synthesize a variety of an-

alogues that differ in their physicochemical properties (e.g. lip-

ophilicity, non-speciﬁc binding, metabolic stability) and can be

designed tailor-made for speciﬁc applications.

Three of the most potent structures containing a ﬂuorine atom

(13e, 13g and 13I) were radiolabeled with ¹⁸F and used for in vitro

autoradiography. Fig. 1 shows the results of autoradiographic ex-

periments of rat brain slices obtained by incubation with the ¹⁸F-

labeled isotopologues [¹⁸F]13e, [¹⁸F]13g and [¹⁸F]13l. Due to the

n.c.a. radiolabeling, extremely low concentrations (<10 pM) of the

radioligands were sufﬁcient for the autoradiograms shown in Fig. 4.

Total binding in all three autoradiographic experiments was very

similar and showed the striatal accumulation pattern expected for

4. Experimental

4.1. General information

A

_2AR-selective ligands, with low uptake in all extra-striatal brain

regions. In addition, striatal binding was consistently and

completely displaced by 1 M of the potent and selective A_2A

Unless noted otherwise, all reactions were carried out under a

nitrogen or argon atmosphere at ambient temperature (22 2 ^ꢀC)

in oven-dried glassware. Standard inert atmosphere techniques

were used in handling all air and moisture sensitive reagents.

Melting points were measured on a Melting Point B-540 in-

strument (Büchi Labortechnik, Flawil, Switzerland).

m

R

antagonist ZM 241385, suggesting speciﬁc binding of all three

radioligands in this region. Thus, in summary, [¹⁸F]13e, [¹⁸F]13g

and [¹⁸F]13I are promising A_2AR radioligand candidates for PET

imaging with excellent molar activity, high in vitro afﬁnity (espe-

cially for human receptors), and an autoradiographic distribution

pattern corresponding to the in vivo distribution of the A_2AR in rat

brain. Thus, the high accumulation of radioligands [¹⁸F]13e, [¹⁸F]

13g and [¹⁸F]13I in A_2AR rich striatal regions can be selectively

blocked by the application of the selective A_2AR antagonist ZM

241385. These ﬁndings should encourage further studies to deter-

mine their metabolic stability, blood-brain-barrier permeability

and in vivo receptor afﬁnity and speciﬁcity.

No-carrier-added [¹⁸F]ﬂuoride was produced via the ¹⁸O(p,n)¹⁸

F

nuclear reaction by bombardment of isotopically enriched [¹⁸O]

water in a Ti-target with 16.5 MeV protons at the JSW cyclotron

BC1710 (INM-5, Forschungszentrum Jülich).

4.2. Solvents and reagents

Solvents were either purchased commercially in sufﬁcient pu-

rity (MerckKGaA, Darmstadt, Germany) or puriﬁed and dried by

standard methods [42]. All chemicals used for the syntheses were

commercially available (Merck, Taufkirchen, Germany; Activate

Scientiﬁc, Prien, Germany; ABCR, Karlsruhe, Germany) or were

prepared as described in the text.

3. Conclusion

In the present study, a simple chemical building block principle

has been used to prepare a series of Tozadenant analogues. The

described approach for the late stage piperidine ring functionali-

zation of the A_2Areceptor antagonist Tozadenant provided de-

rivatives with high afﬁnity to the A_2Areceptor and good selectivity

over the A₁receptor. Among these compounds are a number of

ﬂuorine-containing analogues, which bind reversibly and with high

afﬁnity to the adenosine A_2Areceptor while maintaining a very high

selectivity over the adenosine A₁receptor. Three compounds with

the most promising binding properties were radioﬂuorinated and

examined by in vitro autoradiography in rat brain sections. All three

derivatives showed almost exclusive striatal binding. As such, the

described compounds represent an interesting starting point for

4.3. Spectroscopy

¹H, ¹³C, and ¹⁹F NMR spectra were recorded at 400.13, 100.61,

and 376.49 MHz by means of a Bruker Avance Neo 400 instrument

(Bruker Bio Spin GmbH, Rheinstetten, Germany), in 5% solution at

299 K. Chemical shifts (d) are given in parts per million (ppm). In-

ternal reference was set to the residual solvent signals (for CDCl₃,

d_H¼ 7.26, d_C¼ 77.16; for DMSO‑d₆, d_H¼ 2.50, d_C¼ 50.32). The ¹H

NMR spectra are reported as follows: d/ppm (number of protons,

multiplicity, coupling constant J/Hz (where appropriate),

7

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

Fig. 4. In vitro autoradiography with [¹⁸F]13e (top), [¹⁸F]13g (middle), and [¹⁸F]13l (bottom) in horizontal rat brain slices (20

TRIS buffer (pH 7.4), 1 M EDTA and the indicated radioligands with an activity concentration of 2.3 kBq/mL. The exposure time was 12 h. Autoradiograms on the left show total

mm). The slices were incubated in a solution of 0.01 M

m

binding of the respective ligands, while those on the right show unspeciﬁc binding obtained if speciﬁc binding was inhibited by 1 mM of the A_2AR antagonist ZM 241385. The

numbers next to the color scales represent the relative activity units associated with the color shades.

assignment). The following abbreviations and their combinations

are used when reporting NMR data: s ¼ singlet, d ¼ doublet,

t ¼ triplet, q ¼ quartet, m ¼ multiplet, br s ¼ broad singlet,

obsc ¼ obscured, v ¼ very. NMR signals were assigned based on

information from additional two-dimensional experiments (COSY,

gHSQC, gHMBC, NOESY). Coupling constants J (in Hertz) for protons

are given in the form ⁿJ(¹H, X), those for carbons as ⁿJ(¹³C,¹⁹F). All

¹³C and ¹⁹F NMR spectra were recorded under ¹H-broadband

8

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

Chart 1. Structure of piperidines 12a e 12o applied in this work.

Chart 2. Structure of ﬂuorine-18 isotopologues [¹⁸F]13e, [¹⁸F]13g and [¹⁸F]13l.

decoupling (CPD). Compound names are those generated by

ChemBioDraw™ (CambridgeSoft) following IUPAC nomenclature.

However, the NMR assignment numbering used is arbitrary and

does not follow any particular convention. Numbering of com-

pounds is illustrated on the structures themselves; vide infra.

Low-resolution mass spectra were obtained in electrospray

ionization (ESI positive) mode with a Thermo Finnigan Surveyor

mass spectrometer (Thermo Fisher Scientiﬁc GmbH, Dreieich,

Germany). The analytes were dissolved in methanol (about 1 mg/

mL) and injected directly through a valve on the ionization inter-

face. The ﬂow rate of the eluent (methanol/water/acetic acid, 50/

chromatography system equipped with RevealX™ detection,

allowing for multisignal (UV/ELSD) collection, and Reveleris® ﬂash

silica cartridges (size 40 mm) as stationary phase were employed.

4.5. Chemical syntheses

4.5.1. 4-(4-Methoxyhenyl)morpholine nitrate salt 2

A 1 L 3-neck round bottom ﬂask, equipped with a mechanical

stirrer, was charged with p-anisidine 1 (20 g, 0.162 mol), 2-

chloroethyl ether (48 g, 0.336 mol), tetrabutylammonium bro-

mide (1.04 g, 0.003 mol), and 42% sodium hydroxide solution (77 g,

0.8 mol). The mixture was stirred at 120 ^ꢀC for 8 h. After completion

of the reaction the mixture was cooled to 20 ^ꢀC and extracted with

TBME (80 mL) and ethyl acetate (80 mL). The combined organic

phases were washed with water (80 mL), the dark organic solution

was cooled to 0e5 ^ꢀC, and 70% HNO₃(14.6 g, 0.162 mol) was slowly

added. Scratching with a glass rod induced crystallization of the

nitrate salt. After cooling to 5 ^ꢀC for 12 h the solid was ﬁltered,

washed with TBME (40 mL), and dried under vacuum at 45 ^ꢀC

overnight to give the title compound (40.2 g, 97%) as a tan solid, mp

50/0.2, v/v/v) was 200 mL/min. Reported are the m/z-values of the

pseudo-ion [M þ H]^þ.

High resolution mass spectra and elemental analyses were

performed by the Central Division of Analytical Chemistry at the

Forschungszentrum Jülich. Analyses indicated by the symbols of

the elements are within 0.4% of the theoretical values.

4.4. Chromatography

112e113 ^ꢀC (dec.). ¹H NMR (400 MHz, DMSO‑d₆)

d 3.52 (m, 4H, 2 x

Thin layer chromatographic analyses were performed in all re-

actions to monitor the reaction progress and served as a purity

check of the obtained products. The respective eluent was selected

so that the R_fvalues of the individual compounds ranged from 0.2

to 0.8. Visualization was done by detection under UV light using

silica coated TLC aluminium sheets with ﬂuorescent indicator (SIL

ALUGRAM G/UV254 Macherey-Nagel GmbH, Düren, Germany) and/

or by iodine staining.

N-CH₂), 3.79 (s, 3H, O-CH₃), 3.94 (m, 4H, 2 x O-CH₂), 7.08 (d, 2H,

J ¼ 8.5 Hz, aryl-H², aryl-H⁶), 7.53 (d, 2H, J ¼ 9.1 Hz, aryl-H³, aryl-H⁵).

¹³C NMR (101 MHz, DMSO‑d₆)

d 54.2 (N-CH₂), 56.0 (O-CH₃), 64.9 (O-

CH₂), 115.5 (aryl-C³þ aryl-C⁵), 122.1 (aryl-C²þ aryl-C⁶), 136.4 (aryl-

C¹), 160.0 (aryl-C⁴).

4.5.2. 4-(4-Methoxy-3-nitrophenyl)morpholine 3

For ﬂash chromatography

a

Grace Reveleris® iES ﬂash

A 250 mL 3-neck round bottom ﬂask, equipped with a magnetic

9

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

stirrer, was charged with 95% sulfuric acid (80 g, 0.815 mol). The

acid was cooled to 0 ^ꢀC (ice/salt bath) and a solution of the nitrate

salt 2 (20 g, 0.078 mol) of in dichloromethane (125 mL) was added

dropwise from a dropping funnel over 1 h while the reaction

temperature was maintained at 0e5 ^ꢀC. After complete addition the

mixture was stirred for 30 min, the bottom acid layer was separated

and slowly added to ice/water (200 mL, 1 L beaker) while main-

taining the temperature at <10 ^ꢀC. To this diluted acid solution was

then slowly added 28% NH₄OH solution (190 mL) while the tem-

perature was maintained at <10 ^ꢀC by the addition of ice. At the end

of the addition the pH of the mixture should be higher than 10 (TLC:

ethyl acetate/methanol, 98/2). The batch was cooled at 5 ^ꢀC for 1 h,

the solid was ﬁltered, washed with 28% NH₄OH solution (50 mL)

and water (50 mL), and dried under vacuum at 45 ^ꢀC overnight to

give the title compound (17.5 g, 94% yield) as an orange solid, mp

stirring sodium methoxide (35% solution in methanol, 9.3 mL,

43 mmol) was added and the clear brown solution was stirred for

3 h. During this time a grey suspension formed. The mixture was

cooled to 0e5 ^ꢀC and was kept at that temperature for 1 h while

stirring was continued. Filtration, washing with ice cold MeOH

(10 mL) and hexane (10 mL) and air drying gave 7.1 g (95% yield) of

the title compound as brown crystals, mp 184e185 ^ꢀC (TLC: ethyl

acetate/hexane/AcOH, 80/20/0.2, v/v/v) ¹H NMR (400 MHz,

DMSO‑d₆) d 2.98 (m, 4H, 2 x N-CH₂), 3.73 (m, 4H, 2 x O-CH₂), 3.76 (s,

3H, O-CH₃), 6.71 (m, 1H, J ¼ 9 Hz, 3 Hz, aryl-H⁶), 6.92 (m, 1H,

J ¼ 9 Hz, aryl-H⁴), 7.50 (s_br, 2H, NH₂), 7.64 (m, 1H, aryl-H⁵), 9.02 (s_br,

1H, NH). ¹³C NMR (101 MHz, DMSO‑d₆)

d 50.2 (N-CH₂), 56.5 (O-CH₃),

66.6 (O-CH₂), 112.6 (aryl-C²), 112.9 (aryl-C⁵), 113.9 (aryl-C⁶), 128.5

(aryl-C³), 145.2 (aryl-C¹), 145.7 (aryl-C⁴), 181.6 (CS). C₁₂H₁₇N₃O₂S MS

(ESIþ) m/z: [MþH]þ Calcd 268.10; Found 268.04.

93 ^ꢀC. ¹H NMR (400 MHz, DMSO‑d₆)

d 3.09 (m, 4H, 2 x N-CH₂), 3.74

(m, 4H, 2 x O-CH₂), 3.85 (s, 3H, O-CH₃), 7.28 (m, 2H, aryl-H⁵, aryl-

4.5.6. 4-Methoxy-7-morpholin-4-yl-benzothiazol-2-ylamine 7

H⁶), 7.33 (m, 1H, aryl-H²). ¹³C NMR (101 MHz, DMSO‑d₆)

d

49.2 (N-

Under efﬁcient mechanical stirring the thiourea 6 (6.05 g,

22.6 mmol) was suspended in ethyl acetate (67 mL) and heated to

80 ^ꢀC. At that temperature 33% HBr in acetic acid (8.18 mL,

45.2 mmol) was added dropwise over 0.2 h. After complete addi-

tion the thick suspension was reﬂuxed (oil bath 100 ^ꢀC) for 0.2 h

and DMSO (1.93 mL, 27.12 mmol) was added at that temperature in

one portion. Ten minutes after the addition ethyl acetate (20 mL)

was added and stirring and heating was continued for 4 h. The

mixture was cooled to ambient temperature, the solid collected by

ﬁltration and washed with ethyl acetate (50 mL). The wet material

was suspended under stirring in EtOH (55 mL), water (72 mL) was

added, and the red solution thus formed was heated to 50 ^ꢀC.

Aqueous ammonia (28%, 5.4 mL) was added (pH 9e10), the mixture

was cooled in an ice bath, and stirred overnight (during this time

the ice bath was allowed to thaw). The light brown precipitate

formed was collected by ﬁltration and washed with 50% aqueous

ethanol (TLC: ethyl acetate/methanol, 90/10). The solid was

extracted with boiling THF (100 mL), cooled, collected by ﬁltration,

and oven-dried at 100 ^ꢀC to give the aminobenzothiazole as grey

crystals, 5.7 g (95% yield), mp > 275 ^ꢀC (dec) (TLC: ethyl acetate/

methanol/AcOH, 90/10/0.2, v/v/v). If further puriﬁcation was

needed the solid was dissolved in aqueous 0.5 N HCl (50 mL,

25 mmol) and stirred for 0.2 h at ambient temperature. Insoluble

material was removed by ﬁltration, aqueous NH₄OH (28%, 14.47 M,

2.07 mL, 30 mmol) was added, and the formed suspension was

stirred for 0.2 h. EtOH (75 mL) was added and the mixture was

chilled overnight. The solid was collected by ﬁltration, washed with

50% aqueous ethanol and vacuum-dried in a desiccator over

CH₂), 57.3 (O-CH₃), 66.4 (O-CH₂), 111.3 (aryl-C²), 115.8 (aryl-C⁵),

122.8 (aryl-C⁶), 140.2 (aryl-C³), 145.3 (aryl-C⁴), 145.3 (aryl-C¹).

C

₁₁H₁₄N₂O₄MS (ESIþ) m/z: [MþH]þ Calcd 239.10; Found 239.19.

4.5.3. 2-Methoxy-5-morpholinoaniline 4

To a stirred solution of nitroarene 3 (11.9 g, 50 mmol) and

ammonium chloride (26.7 g, 500 mmol) in a mixture of ethanol

(300 mL) and ethyl acetate (300 mL) was added zinc powder

(32.7 g, 500 mmol). The reaction was stirred at room temperature

for 0.5 h, then diluted with EtOAc, ﬁltered through CELITE®, and the

ﬁltrate evaporated in vacuo to give aniline 4 (10.4 g, 99%) as dark

brown crystals. ¹H NMR (400 MHz, DMSO‑d₆)

d 2.91 (m, 4H, 2 x N-

CH₂), 3.68 (s, 3H, O-CH₃), 3.70 (m, 4H, 2 x O-CH₂), 4.60 (s_br, 2H, NH₂),

6.09 (dd, 1H, J ¼ 8.7 Hz, 2.8 Hz, aryl-H⁶), 6.31 (d, 1H, J ¼ 2.8 Hz, aryl-

H²), 6.31 (d, 1H, J ¼ 8.7 Hz, aryl-H⁵). ¹³C NMR (101 MHz, DMSO‑d₆)

d

50.4 (N-CH₂), 56.2 (O-CH₃), 66.7 (O-CH₂), 1103.4 (aryl-C²), 103.9

(aryl-C⁵), 112.0 (aryl-C⁶), 138.5 (aryl-C³), 141.3 (aryl-C⁴), 146.5 (aryl-

C¹). C₁₁H₁₆N₂O₂MS (ESIþ) m/z: [MþH]þ Calcd 209.12; Found

209.17.

4.5.4. 1-Benzoyl-3-(2-methoxy-5-morpholin-4-yl-phenyl)-thiourea

5

To a solution of 2-methoxy-5-morpholinoaniline 4 (4.6 g,

22 mmol) in acetone (140 mL) was added dropwise a solution of

benzoyl isothiocyanate (3.4 mL, 25 mmol) in acetone (80 mL) and

after complete addition the mixture was stirred at ambient tem-

perature for another 0.5 h (TLC: ethyl acetate/hexane/AcOH, 50/50/

0.2). Acetone was distilled off at atmospheric pressure with the

continuous dropwise addition of water (total volume 150 mL). After

cooling the suspension to ambient temperature and to 5 ^ꢀC over-

night the product was collected by ﬁltration, washed with water,

dried, and recrystallized from MeOH to give the benzoyl thiourea as

yellow crystals, mp 139e140 ^ꢀC (95% yield). ¹H NMR (400 MHz,

P₄O₁₀.¹H NMR (400 MHz, DMSO‑d₆)

d 2.94 (m, 4H, 2 x N-CH₂), 3.74

(m, 4H, 2 x O-CH₂), 3.81 (s, 3H, O-CH₃), 6.64 (d, 1H, J ¼ 8.7 Hz, H⁵),

6.78 (d, 1H, J ¼ 8.7 Hz, H⁶), 7.42 (s_br, 2H, NH₂). ¹³C NMR (101 MHz,

DMSO‑d₆) d

51.7 (N-CH₂), 56.6 (O-CH₃), 67. (O-CH₂), 109.4 (C⁶), 110.7

(C⁵), 126.1 (C^7a), 140.4 (C⁴), 143.4 (C⁷), 146.9 (C^4a) 165.8 (C²).

₁₂H₁₅N₃O₂S MS (ESIþ) m/z: [MþH]þ Calcd 266.09; Found 266.17.

C

DMSO‑d₆) d 3.02 (m, 4H, 2 x N-CH₂), 3.76 (m, 4H, 2 x O-CH₂), 3.85 (s,

3H, O-CH₃), 6.82 (dd, 1H, J ¼ 9 Hz, 3 Hz, aryl-H⁶), 7.04 (d, 1H,

J ¼ 9 Hz, aryl-H⁵), 7.55 (m, 2H, Bz-H^3,5), 7.67 (m, 1H, Bz-H⁴) 7.98 (m,

2H, Bz-H^2,6), 8.5 (d, 1H, J ¼ 3 Hz, aryl-H²), 11.55 (s, 1H, SCNHCO),

4.5.7. Phenyl (4-methoxy-7-morpholin-4-yl-benzo[d]thiazol-2-yl)

carbamate 8

To a well stirred suspension of 4-methoxy-7-morpholin-4-yl-

benzo[d]thiazol-2-ylamine (2.65 g, 10 mmol) in dry THF (50 mL)

was added dry pyridine (2.43 mL, 30 mmol) at 0e5 ^ꢀC. After stirring

for 5 min, a solution of phenyl chloroformate (1.44 mL, 11.5 mmol)

in THF (5 mL) was added very slowly. The reaction mixture was

allowed to stir at ambient temperature for 14 h (TLC: ethyl acetate/

methanol/AcOH, 98/2/0.2, R_fCarbamate0.85; ethyl acetate/hexane/

acetic acid, 80/20/0.2, R_fCarbamate0.82) and was then diluted with

ethyl acetate (250 mL) and 50% brine (75 mL). The organic layer was

separated, washed with aqueous HCl (0.5 N, 50 mL) and brine

(75 mL), dried, ﬁltered and rotoevaporated in vacuo to furnish a

13.10 (s, 1H, arylNHCS). ¹³C NMR (101 MHz, CDCl₃)

d 50.1 (N-CH₂),

56.9 (O-CH₃), 66.6 (O-CH₂), 111.4 (aryl-C²), 112.4 (aryl-C⁵), 113.8

(aryl-C⁶), 127.8 (aryl-C³), 128.9 (Bz-C^2,6), 129.2 (Bz-C^3,5), 132.5 (Bz-

C¹), 133.6 (Bz-C⁴), 144.7 (aryl-C¹), 145.1 (aryl-C⁴), 168.9 (CO), 177.9

(CS). C₁₉H₂₁N₃O₃S MS (ESIþ) m/z: [MþH]þ Calcd 372.13; Found

372.09.

4.5.5. (2-Methoxy-5-morpholin-4-yl-phenyl)-thiourea 6

At ambient temperature the benzoylthiourea

5 (10.6 g,

28 mmol) was suspended in MeOH (65 mL). Under magnetic

10

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

fawn solid residue (3.4 g, 88%). An analytical sample was recrys-

dry THF (7.5 mL). After stirring for 5 min, phenyl chloroformate

tallized from acetonitrile. (400 MHz, DMSO‑d₆)

d

3.01 (m, 4H, 2 x N-

(144 mL, 1.15 mmol) was added slowly. The reaction mixture was

CH₂), 3.76 (m, 4H, 2 x O-CH₂), 3.91 (s, 3H, O-CH₃), 6.91 (d, 1H,

J ¼ 8.6 Hz, H⁵), 6.95 (d, 1H, J ¼ 8.6 Hz, H⁶), 7.31 (m, 3H, phenyl), 7.47

(m, 2H, phenyl), 12.67 (s_br, 1H, NH). ¹³C NMR (101 MHz, DMSO‑d₆)

allowed to stir at ambient temperature for 5 h (TLC (sample in

acetone not methanol): ethyl acetate/hexane, 80/20, R_f0.90; if re-

action was not quant. after 5 h 10 mL of chloroformate was added

and reaction time was 1 h longer) and was then diluted with ethyl

acetate (25 mL) and water (7.5 mL). The organic layer was sepa-

rated, washed two times with aqueous HCl (0.5 N, 5 mL) and brine

(10 mL), dried, ﬁltered and rota-evaporated in vacuo to furnish a

fawn solid residue. Recrystallization from ethyl acetate gave the

carbamate as an off-white solid (360 mg, 86%). ¹H NMR (400 MHz,

d

51.9 (N-CH₂), 56.4 (O-CH₃), 67. (O-CH₂), 108.8 (C⁶), 113.4 (C⁵), 122.2

(phenyl C^2,6), 126.6 (phenyl C⁴), 127.3 (C^7a), 130.1 (phenyl C^3,5), 140.0

(C⁷), 140.4 (C⁴), 148.5 (C^4a), 150.5 (phenyl C¹), 153.2 (C¼O), 158.6

(C²). C₁₉H₁₉N₃O₄S MS (ESIþ) m/z: [MþH]þ Calcd 386.44; Found

386.57.

DMSO‑d₆)

d 3.01 (m, 4H, 2 x N-CH₂), 3.76 (m, 4H, 2 x O-CH₂), 4.29

4.5.8. 2-Amino-7-morpholinobenzo[d]thiazol-4-ol 9

(dt, ³J_H-F¼ 31 Hz, ³J ¼ 3.9 Hz, 2H, CH₂CH₂F), 4.73 (dt, ²J_H-F¼ 48 Hz,

³J ¼ 3.9 Hz, 2H, CH₂CH₂F), 6.91 (d, 1H, J ¼ 8.6 Hz, H⁶), 6.95 (d, 1H,

J ¼ 8.6 Hz, H⁵), 7.31 (m, 3H, phenyl), 7.47 (m, 2H, phenyl), 12.67 (s_br,

Under efﬁcient stirring a solution of 2-amino-4-methoxy-7-

morpholinobenzothiazole (1.3 g, 5 mmol) in 48% HBr (8.9 M,

50 mL, 445 mmol, good quality: colorless to slightly yellowish) was

stirred at 130 ^ꢀC for 24 h. The formed suspension was cooled to

ambient temperature and then set aside overnight at 5 ^ꢀC, The grey

brown solid was collected by ﬁltration and was taken up in water

(30e40 mL). Under efﬁcient stirring the pH of the mixture was

brought to 8e9, ﬁrst by the addition of 10 N aqueous sodium hy-

droxide until a precipitate started to form, then by the addition of

saturated aqueous sodium bicarbonate. The formed suspension was

diluted with water (50 mL), stirred for 10 min at ambient temper-

ature and was kept at 5 ^ꢀC for 1 h. The product was collected by

ﬁltration, washed with water and dried in air. The aminophenol

was obtained as a tan solid in 85% yield (1150 mg, TLC: ethyl ace-

tate/methanol/AcOH, 98/2/0.2, R_f0.69; ethyl acetate/hexane/AcOH,

80/20/0.2, R_f0.57). The phenol is soluble in 0.5 N NaOH. ¹H NMR

1H, NH). ¹⁹F NMR (377 MHz, DMSO‑d₆)

(101 MHz, DMSO‑d₆)

d

ꢁ223.21.¹³C NMR

2

d

51.9 (C-N-C), 67. (C-O-C), 68.8(d, J_C-

¼ 19 Hz, CH₂CH₂F), 82.9(d, J_C-F¼ 167 Hz, CH₂CH₂F), 108.8 (C⁶),

1

F

113.4 (C⁵), 122.2 (phenyl C^2,6), 126.6 (phenyl C⁴), 127.3 (C^7a), 130.1

(phenyl C^3,5), 140.0 (C⁷), 140.4 (C⁴), 148.5 (C^4a), 150.5 (phenyl C¹),

153.2 (C¼O), 158.6 (C²). C₂₀H₂₀FN₃O₄S HRMS (ESIþ) m/z: [MþH]þ

Calcd 418.1231; Found 418.1229.

4.5.11. Preparation of piperidines 12g - 12i and 12m e 12o

4.5.11.1. 4-(Fluoromethyl)piperidine 12g

a) 1-(tert-Butoxycarbonyl)-4-(methylsulfonyloxy)methylpiper-

idine 12g-1

(400 MHz, DMSO‑d₆)

d

2.90 (t, 4H, J ¼ 9.1 Hz, 2 x N-CH₂). 3.72 (t, 4H,

J ¼ 9.1 Hz, 2 x O-CH₂), 6.55 (d, 1H, J ¼ 8.4 Hz, H⁶), 6.61 (d, 1H,

J ¼ 8.4 Hz, H⁵), 7.23 (s_br, 2H, NH₂), 8.89 (s, 1H, OH). ¹³C NMR

(101 MHz, DMSO‑d₆)

d

51.8 (CNC). 67.1 (COC), 111.3 (C⁶), 112.1 (C⁵),

126.2 (C^7a),138.8 (C⁴),142.3 (C⁷),144.5 (C^4a),165.1 (C²). C₁₁H₁₃N₃O₂S

MS (ESIþ) m/z: [MþH]þ Calcd 252.30; Found 252.21.

4.5.9. 4-(2-Fluoroethoxy)-7-morpholinobenzo[d]thiazol-2-amine

10

Under argon Cs₂CO₃(2.65 mg, 7.5 mmol) was added to a stirred

solution of 2-amino-7-morpholinobenzo[d]thiazol-4-ol (1.25 mg,

5 mmol) in dry DMF (50 mL). Immediately after the carbonate

To an approximately 0.2 M solution of the alcohol (1.076 g,

5 mmol) in ethanol free DCM (25 mL) containing a 50% molar

addition 2-bromo-1-ﬂuoroethane (450 mL, 6 mmol) was added and

excess of triethylamine (1050 m

L, 7.5 mmol) and kept between 0 ^ꢀC

the mixture was stirred at ambient temperature for 24 h (TLC: ethyl

acetate/methanol, 98/2, R_f0.79; ethyl acetate/hexane, 80/20 R_f

0.54). Upon the addition of water (100 mL) the product precipitated

as a ﬁne granular solid that was collected by ﬁltration, washed with

methanol, and air dried. Yield 800 mg (54%), tan solid. ¹H NMR

and ꢁ10 ^ꢀC, was added a 10% excess of methanesulfonyl chloride

(465 mL, 6 mmol) over a period of 2e5 min. Ten minutes after the

addition the cooling bath was removed and the mixture was stirred

for 60 min at ambient temperature to complete the reaction (TLC:

ethyl acetate/hexane, 50/50, R_fproduct0.06e0.19 (L), iodine stain-

ing). The reaction mixture was transferred to a separatory funnel

with the aid of more DCM. The mix was ﬁrst extracted with ice

water, followed by cold 10% HCl acid, sat. sodium bicarbonate, and

sat. brine. Drying the DCM solution over Na₂SO₄followed by sol-

vent removal gave the product (1.46 g, 99%) as a clear colorless oil,

that solidiﬁed upon standing in the refrigerator overnight to

furnish a colorless waxy solid, mp 76e77 ^ꢀC. ¹H NMR (400 MHz,

(400 MHz, DMSO‑d₆)

d 2.94 (m, 4H, 2 x N-CH₂), 3.73 (m, 4H, 2 x O-

CH₂), 4.29 (dt, ³J_H-F¼ 31 Hz, ³J ¼ 3.9 Hz, 2H, CH₃CH₂F), 4.73 (dt, ²J_H-

¼ 48 Hz, ³J ¼ 3.9 Hz, 2H, CH₃CH₂F), 6.64 (d, 1H, J ¼ 8.6 Hz, H⁶), 6.78

F

(d, 1H, J ¼ 8.6 Hz, H⁵), 7.47 (s_br, 2H, NH₂). ¹⁹F NMR (377 MHz,

DMSO‑d₆)

d

ꢁ221.76.¹³C NMR (101 MHz, DMSO‑d₆)

d 51.7 (C-N-C),

2

67. (C-O-C), 68.9(d, J_C-F¼ 19 Hz, CH₂CH₂F), 82.9(d, ¹J_C-F¼ 167 Hz,

CH₂CH₂F), 110.7 (C⁶), 111.4 (C⁵), 126.2 (C^7a), 140.9 (C⁴), 143.8 (C⁷),

145.5 (C^4a) 166 (C²). C₁₃H₁₆FN₃O₂S HRMS (ESIþ) m/z: [MþH]þ

Calcd 298.1020; Found 298.1016.

CDCl₃)

d

1.14e1.28 (m, 2H, 0.5C³H₂, 0.5C⁵H₂), 1.44 (9H, s, Boc CH₃),

1.72 (d_br, J ¼ 13.4 Hz, 2H, 0.5C³H₂, 0.5C⁵H₂), 1.84e1.96 (m, 1H, C⁴H),

2.70 (td, J ¼ 13.4, 2.6 Hz, 2H, 0.5C²H₂, 0.5C⁶H₂), 3.02 (s, 3H, Mes

CH₃), 4.05 (d, J ¼ 6.4 Hz, 2H, C^1’H₂), 4.13 (d_brJ ¼ 13.4 Hz, 2H, 0.5C²H₂,

4.5.10. Phenyl (4-(2-ﬂuoroethoxy)-7-morpholinobenzo[d]thiazol-2-

yl)carbamate 11

0.5C⁶H₂). ¹³C NMR (101 MHz, CDCl₃)

d

28.2 (C³, C⁵), 28.4 (Boc CH₃),

At 0e5 ^ꢀC pyridine (243

mL, 3 mmol) was added to a well stirred

35.9 (C⁴), 37.3 (Ms CH₃), 39.6 (C^1’), 43.2 (C², C⁶), 79.6 (Boc C), 154.7

(Boc C¼O). C₁₂H₂₃NO₅S HRMS (ESIþ) m/z: [MþH]þ Calcd 294.1369;

Found 294.1369.

suspension (prepared in ultrasound bath) of 4-(2-ﬂuorethoxy)-7-

morpholin-4-yl-benzo[d]thiazol-2-ylamine (297 mg, 1 mmol) in

11

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

b) 1-(tert-Butoxycarbonyl)-4-ﬂuoromethylpiperidine 12g-2

a)

tert-Butyl-4-(2-hydroxyethoxy)piperidine-1-carboxylate

12h-1 (according to Ref. [43])

Under an atmosphere of argon TBAF (1 M solution in THF, 25 mL,

25 mmol, 5 eq) was added to 1-(tert-butoxycarbonyl)-4-(methyl-

sulfonyloxy)methyl-1,2,3,6-tetrahydropyridine 12g-1 (1.46 g,

5 mmol). The mixture was stirred and heated to 70 ^ꢀC and kept at

that temperature for 24 h (TLC (spots of reaction mixture, no

dilution): ethyl acetate/hexane, 50/50, R_fTBAF0.00 R_fproduct0.94,

R_feduct0.06e0.19, iodine staining). After cooling to ambient tem-

perature the mixture was partitioned with diethyl ether and sat.

aqueous NH₄Cl solution. The organic fraction was washed with sat

NH₄Cl, water, and brine, dried over Na₂SO₄, ﬁltered, and concen-

trated to afford an almost colorless solid residue (760 mg, 3.5 mmol,

Sodium hydride (60% dispersion in mineral oil, 650 mg,

16.3 mmol, 1.01 eq.) was suspended in dry THF under an argon

atmosphere and cooled to 0 ^ꢀC. N-Boc-4-hydroxypiperidine (3.25 g,

16.3 mmol, 1.01 eq.) was added and the suspension was stirred for

20 min at 0 ^ꢀC. After the addition of methyl bromoacetate (1.5 mL,

16.2 mmol, 1.00 eq.) the reaction was stirred for 22 h at room

temperature. The reaction was quenched by addition of water and

extracted with ethyl acetate. The combined organic phases were

washed with brine and dried over sodium sulfate and the solvent

was removed in vacuo. A colorless oil (1.262 g, impure) was ob-

tained by column chromatography (eluent: petrol ether/ethyl ace-

tate, 4/1) and was dissolved in dry THF under an argon atmosphere.

The solution was cooled to 0 ^ꢀC and lithium aluminium hydride

(205 mg, 5.40 mmol) was added in portions. The reaction was

allowed to warm to room temperature and stirred for 3 h. The re-

action was quenched by addition of an aqueous solution of sodium

hydroxide (10%). The mixture was extracted with ethyl acetate. The

combined organic phases were washed with brine and dried over

sodium sulfate. After removing the solvent in vacuo the residue was

puriﬁed by column chromatography to afford a colorless oil

70%), mp 51e52 ^ꢀC. . ¹H NMR (400 MHz, CDCl₃)

d 1.15e1.28 (m, 2H,

0.5C³H₂, 0.5C⁵H₂), 1.46 (9H, s, Boc CH₃), 1.69 (d_br, J ¼ 13.4 Hz, 2H,

0.5C³H₂, 0.5C⁵H₂), 1.77e1.94 (m, 1H, C⁴H), 2.71 (td, J ¼ 13.4, 2.7 Hz,

2H, 0.5C²H₂, 0.5C⁶H₂), 4.14 (d_br, J ¼ 6.4 Hz, 2H, 0.5C²H₂, 0.5C⁶H₂),

4.27 (dd, ²J_H-F¼ 47.6, ³J_H-H¼ 6,1 Hz, 2H, C^1’H₂). ¹⁹F NMR (377 MHz,

3

CDCl₃)

d

ꢁ224.10.¹³C NMR (101 MHz, CDCl₃)

d

27.6 (d, J_C-

¼ 5.9 Hz C³, C⁵), 28.4 (Boc CH₃), 36.9 (d, ²J_C-F¼ 18 Hz, C⁴), 43.4 (C²,

F

C⁶), 79.6 (Boc C), 87.5 (d, J_C-F¼ 169.3 Hz, C^1’), 154.8 (Boc C¼O).

1

C

₁₁H₂₀FNO₂HRMS (ESIþ) m/z: [MþH]þ Calcd 218.1551; Found

218.1550.

(256 mg, 1.04 mmol, 6.4%). ¹H NMR (400 MHz, CDCl₃)

d 1.44 (s, 9H,

c) 4-Fluoromethylpiperidine 12g

Boc-CH₃), 1.51 (ddd, J ¼ 13.4, 8.5, 4.5 Hz, 2H, 0.5C³H₂, 0.5C⁵H₂),

1.79e1.88 (m, 2H, 0.5C³H₂, 0.5C⁵H₂), 2.01 (br, 1H, OH), 3.06 (ddd,

J ¼ 13.6, 8.5, 3.9 Hz, 2H, 0.5C²H₂, 0.5C⁶H₂), 3.45e3.52 (m, 1H, C⁴H),

3.54e3.59 (m, 2H, C^1’H₂), 3.69e3.80 (m, 4H, C^2’H₂, 0.5C²H₂,

0.5C⁶H₂).

b) tert-Butyl-4-(2-ﬂuoroethoxy)piperidine-1-carboxylate 12h-2

Triﬂuoroacetic acid (7 mL) was added to a solution of 1-(tert-

Butoxycarbonyl)-4-ﬂuoromethyl-piperidine 12g-2 (760 mg,

3.5 mmol) in CH₂Cl₂(7 mL) and the mixture was stirred at ambient

temperature for 2 h. The solution was concentrated in vacuo and co-

evaporated with methanol (4 ꢂ 5 mL) to furnish the trifuoroacetate

salt of the deprotected compound as a yellow oil (800 mg, 99%).

C₈H₁₃F₄NO₂MS (ESIþ) m/z: [MþH]^þCalcd 231.19; Found 118.18

(free base MW 117.16). The oil from above was taken up in CH₂Cl₂

(10 mL) and the triﬂuoroacetate salt was freebased with a saturated

NaHCO₃(aq) solution (10 mL). The organic layer was separated and

the aqueous phase was extracted with CH₂Cl₂(20 mL). The organic

layers were combined, dried over Na₂SO₄, and concentrated under

reduced pressure to afford the amine (390 mg, 95% yield) as an oil.

tert-Butyl-4-(2-hydroxyethoxy)piperidine-1-carboxylate 12h-1

(256 mg, 1.04 mmol, 1.00 eq.) and PyFluor (184 mg, 1.14 mmol, 1.10

eq.) were dissolved in toluene (1.0 mL). DBU (310 mL, 2.08 mmol,

2.00 eq.) was added and the reaction was stirred for 48 h. The re-

action was quenched by the addition of water und extracted with

ethyl acetate. The combined organic phases were washed with

brine and dried over sodium sulfate. The solvent was removed in

vacuo and the residue was puriﬁed by column chromatography

(eluent: petrol ether/ethyl acetate, 4/1) to afford a colorless oil

¹H NMR (400 MHz, CDCl₃) 1.64 (m, 2H, 0.5C³H₂, 0.5C⁵H₂), 1.97 (m,

d

2H, 0.5C³H₂, 0.5C⁵H₂), 2.07 (m, 1H, C⁴H), 2.92 (m, 2H, 0.5C²H₂,

2

0.5C⁶H₂), 3.49 (m, 2H, 0.5C²H₂, 0.5C⁶H₂), 4.33 (dd, J_H-F¼ 47.6, ³J_H-

¼ 6,1 Hz, 2H, C^1’H₂). ¹⁹F NMR (377 MHz, CDCl₃)

d

ꢁ224.43.

(74 mg, 0.299 mmol, 29%). ¹H NMR (400 MHz, CDCl₃)

d 1.44 (s, 9H,

H

C₆H₁₂FN HRMS (ESIþ) m/z: [MþH]^þCalcd 118.1027; Found

Boc-CH₃), 1.47e1.58 (m, 2H, 0.5C³H₂, 0.5C⁵H₂), 1.77e1.88 (m, 2H,

0.5C³H₂, 0.5C⁵H₂), 3.07 (ddd, J ¼ 15.8, 9.6, 5.1 Hz, 2H, 0.5C²H₂,

0.5C⁶H₂), 3.45e3.55 (m, 1H, C⁴H), 3.65e3.80 (m, 4H, C^1’H₂, 0.5C²H₂,

0.5C⁶H₂), 4.46e4.61 (m, 2H, C^2’H₂). ¹⁹F NMR (377 MHz, CDCl₃)

118.1021.

4.5.11.2. 4-(2-Fluoroethoxy)piperidine hydrochloride 12h HCl

12

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

d

ꢁ223.16.¹³C NMR (101 MHz, CDCl₃):

d

28.55 (Boc-CH₃), 31.06 (C³,

brine and dried over sodium sulfate. After removing the solvent in

vacuo the residue was puriﬁed by column chromatography to afford

a colorless oil (454 mg, 1.75 mmol, 7.0%). ¹H NMR (400 MHz, CDCl₃)

C⁵), 43.28 (C², C⁶), 67.25 (d, J ¼ 20.1 Hz, C^1’), 68.35, 75.36 (C⁴), 79.59

(Boc-C), 83.38 (d, J ¼ 169.3 Hz, C^2’), 154.96 (Boc-CO). C₁₂H₂₃NO₄

HRMS m/z: [MþNa]^þCalcd 270.1476; Found 270.1470.

d

1.13 (qd, J ¼ 12.6, 4.3 Hz, 2H, 0.5C³H₂, 0.5C⁵H₂), 1.44 (s, 9H, Boc-

CH₃), 1.65e1.81 (m, 3H, 0.5C³H₂, 0.5C⁵H₂, C⁴H), 2.03 (br, 1H, OH),

2.68 (t, J ¼ 12.4 Hz, 2H, 0.5C²H₂, 0.5C⁶H₂), 3.31 (d, J ¼ 6.3 Hz, 2H,

C^1’H₂), 3.49e3.53 (m, 2H, C^2’H₂), 3.69e3.73 (m, 2H, C^3’H₂),

4.02e4.16 (m, 2H, 0.5C²H₂, 0.5C⁶H₂). ¹³C NMR (101 MHz, CDCl₃)

c) 4-(2-Fluoroethoxy)piperidine hydrochloride 12h

d

28.58 (Boc-CH₃), 29.13 (C³, C⁵), 36.61 (C⁴), 61.94 (C², C⁶), 67.67 (C^3’),

72.20 (C^2’), 76.13 (C^1’), 79.43 (Boc-C), 155.0 (Boc-CO). C₁₃H₂₅NO₄

HRMS m/z: [MþNa]^þCalcd 182.1676; Found 182.1675.

b)

tert-Butyl-4-((2-ﬂuoroethoxy)methyl)piperidine-1-

carboxylate 12i-2

tert-Butyl-4-(2-ﬂuoroethoxy)piperidine-1-carboxylate 12h-2

(74 mg, 0.299 mmol) was dissolved in dichloromethane (2.0 mL)

and treated with triﬂuoroacetic acid (2.0 mL). The reaction was

stirred at room temperature for 2 h. The solvent was removed in

vacuo and the residue was treated with 3 M methanolic HCl. After

removal of the solvent in vacuo a yellow oil (55 mg, 0.299 mmol,

>99%) was obtained that was used without further puriﬁcation. ¹H

NMR (400 MHz, DMSO‑d₆)

d

1.65e1.76 (m, 2H, 0.5C³H₂, 0.5C⁵H₂),

1.92e2.01 (m, 2H, 0.5C³H₂, 0.5C⁵H₂), 2.86e2.96 (m, 2H, 0.5C²H₂,

0.5C⁶H₂), 3.03e3.15 (m, 2H, 0.5C²H₂, 0.5C⁶H₂), 3.57e3.73 (m, 2H,

tert-Butyl-4-(2-hydroxyethoxy)piperidine-1-carboxylate 12i-1

(417 mg, 1.61 mmol, 1.00 eq.) and PyFluor (285 mg, 1.77 mmol, 1.10

eq.) were dissolved in toluene (1.6 mL). DBU (480 mL, 3.22 mmol,

C^1’H₂, C⁴H), 4.27 (s, 2H), 4.43e4.59 (m, 2H, C^2’H₂), 9.20 (s, 2H). ¹⁹

F

NMR (377 MHz, DMSO‑d₆)

d

ꢁ221.69.¹³C NMR (101 MHz, DMSO‑d₆)

27.20 (C³, C⁵), 40.29 (C², C⁶), 66.78 (d, J ¼ 18.9 Hz, C^1’), 70.94 (C⁴),

2.00 eq.) was added and the reaction was stirred for 69 h. The

solvent was removed in vacuo and the residue was dissolved in

dichloromethane. The organic layer was washed with water and

dried over sodium sulfate. The solvent was removed in vacuo and

the residue was puriﬁed by column chromatography (eluent: petrol

83.15 (d, J ¼ 165.8 Hz, C^2’). C₇H₁₄FNO HRMS m/z: [MþH]^þCalcd

148.1132; Found 148.1130.

4.5.11.3. 4-((2-Fluoroethoxy)methyl)piperidine hydrochloride 12i HCl

ether/ethyl acetate, 5/1) to afford

a

colorless oil (80 mg,

1.06e1.19 (m, 2H,

a)

tert-Butyl-4-((2-hydroxyethoxy)methyl)piperidine-1-

0.306 mmol, 19%). ¹H NMR (400 MHz, CDCl₃)

d

carboxylate 12i-1

0.5C³H₂, 0.5C⁵H₂), 1.44 (s, 9H, Boc-CH₃), 1.67e1.80 (m, 3H, 0.5C³H₂,

0.5C⁵H₂, C⁴H), 2.68 (td, J ¼ 13.2, 2.6 Hz, 2H, 0.5C²H₂, 0.5C⁶H₂), 3.33

(d, J ¼ 6.3 Hz, 2H, C^1’H₂), 3.59e3.71 (m, 2H, C^2’H₂), 4.05e4.12 (m, 2H,

0.5C²H₂, 0.5C⁶H₂), 4.45e4.61 (m, 2H, C^3’H₂). ¹⁹F NMR (377 MHz,

CDCl₃)

d

ꢁ222.92.¹³C NMR (101 MHz, CDCl₃):

d 28.58 (Boc-CH₃),

29.10 (C³, C⁵), 36.62 (C⁴), 43.74 (C², C⁶), 70.33 (d, J ¼ 19.6 Hz, C^2’),

76.41 (C^1’), 79.39 (Boc-C), 83.23 (d, J ¼ 169.0 Hz, C^3’), 155.00 (Boc-

CO). C₁₃H₂₄FNO₃HRMS m/z: [MþH]^þCalcd 284.1632; Found

284.1630.

c) 4-((2-Fluoroethoxy)methyl)piperidine hydrochloride 12i HCl

Sodium hydride (60% dispersion in mineral oil, 1.00 g,

25.0 mmol, 1.01 eq.) was suspended in dry THF under an argon

atmosphere and cooled to 0 ^ꢀC. N-Boc-4-piperidinemethanol

(5.35 g, 24.8 mmol, 1.00 eq.) was added and the suspension was

stirred for 20 min at 0 ^ꢀC. After the addition of methyl bromoacetate

(2.4 mL, 25.9 mmol, 1.04 eq.) the reaction was stirred for 24 h at

room temperature. The reaction was quenched by addition of water

and extracted with ethyl acetate. The combined organic phases

were washed with brine and dried over sodium sulfate and the

solvent was removed in vacuo. A colorless oil (4.11 g, impure) was

obtained by column chromatography (eluent: petrol ether/ethyl

acetate, 4/1) and was dissolved in dry THF. Under an argon atmo-

sphere the solution was added dropwise to a suspension of lithium

aluminium hydride (568 mg, 15.0 mmol) in dry THF cooled to 0 ^ꢀC.

The reaction was allowed to warm to room temperature and stirred

for 3 h. The reaction was quenched by addition of an aqueous so-

lution of sodium hydroxide (15%) and the mixture was extracted

with ethyl acetate. The combined organic phases were washed with

tert- Butyl-4-((2-ﬂuoroethoxy)methyl)piperidine-1-carboxylate

12i-2 (75 mg, 0.287 mmol) was dissolved in dichloromethane

(2.0 mL) and treated with triﬂuoroacetic acid (2.0 mL). The reaction

was stirred at room temperature for 4 h. The solvent was removed

in vacuo and the residue was treated with 3 M methanolic HCl. After

removal of the solvent in vacuo a colorless oil (67 mg, 0.287 mmol,

>99%) was obtained that was used without further puriﬁcation. ¹H

NMR (400 MHz, CDCl₃)

d

1.59e1.76 (m, 2H, 0.5C³H₂, 0.5C⁵H₂),

1.82e2.04 (m, 3H, 0.5C³H₂, 0.5C⁵H₂, C⁴H), 2.24 (br, 1H), 2.77e2.98

13

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

(m, 2H, 0.5C²H₂, 0.5C⁶H₂), 3.38 (d, J ¼ 5.1 Hz, 2H, C^1’H₂), 3.44e3.55

(m, 2H, 0.5C²H₂, 0.5C⁶H₂), 3.59e3.72 (m, 2H, C^2’H₂), 4.44e4.61 (m,

2H, C^3’H₂), 9.26 (s, 1H), 9.56 (s, 1H). ¹⁹F NMR (377 MHz, CDCl₃)

stirred at ambient temperature for 2 h (TLC: ethyl acetate/hexane,

80/20, R_fproduct0.75, R_feduct0.88, iodine staining, compound is not

UV-active). The mixture was externally cooled with a water bath

and saturated ammonium chloride solution was carefully added in

portions (5 ꢂ 1 mL over 25 min, hydrogen gas evolution). After

stirring for another 20 min, the mixture was concentrated under

reduced pressure and the residue diluted with ethyl acetate and

water. The organic layer was separated and washed successively

with water and brine and dried over Na₂SO₄. After ﬁltration, the

solvent was removed in vacuo to obtain a clear colorless oil (2.16 g,

88%) that was reacted further without puriﬁcation. 1H NMR

d

ꢁ222.92.¹³C NMR (101 MHz, CDCl₃):

d

25.90 (C³, C⁵), 34.76 (C⁴),

44.00 (C², C⁶), 70.49 (d, J ¼ 19.6 Hz, C^2’), 75.18 (C^1’), 83.12 (d,

J ¼ 169.3 Hz, C^3’). C₈H₁₆FNO HRMS m/z: [MþH]^þCalcd 162.1289;

Found 162.1285.

4.5.11.4. 4-(2-Fluoroethyl)-4-hydroxypiperidine 12m

a) 4-Ethoxycarbonylmethyl-4-hydroxypiperidine-1-carboxylic

acid tert-butyl ester 12m-1

(400 MHz, CDCl3)

d 3.96 (t, 2H), 3.78 (m, 2H), 3.23 (m, 2H), 1.76 (t,

2H) 1.70 (d, 2H),1.49 (m, 2H),1.47 (s, 9H). ¹³C NMR (101 MHz, CDCl₃)

d

28.5 (Boc CH₃) 36.8 (C³, C⁵), 39.6 (C^1’), 41.3 (weak, C², C⁶), 66.0 (C^2’),

68.7 (C⁴), 80.1 (Boc C), 154.7 (Boc C¼O). C₁₂H₂₃NO₄HRMS (ESIþ) m/

z: [MþH]þ Calcd 246.1699; Found 246.1691.

c) tert-Butyl 4-hydroxy-4-(2-((methylsulfonyl)oxy)ethyl)piperi-

dine-1-carboxylate 12m-3

A 1.0 M solution of lithium hexamethyldisilazide (LiHMDS) in

THF (40 mL, 40 mmol) was cooled to ꢁ70 ^ꢀC under an argon at-

mosphere. Ethyl acetate (3.91 mL, 40 mmol) was slowly added

dropwise over the course of 5 min. The reaction mixture was stirred

for 10 min and a solution of 1-(tert-butyloxycarbonyl)-4-piperidone

(7.34 g, 36.84 mmol) in THF (16 mL) was slowly added dropwise

over the course of 20 min. After 3 h at ꢁ70 ^ꢀC the reaction solution

was warmed to 0 ^ꢀC, water (50 mL) was added and the reaction

mixture extracted with ether (3 ꢂ 75 mL). The combined organic

phases were washed with sat. aq. NaCl solution (150 mL), dried over

Na₂SO₄and the solvent was removed under a vacuum. The desired

To an approximately 0.2 M solution of the diol 12m-2 (1.39 g,

5.67 mmol) in ethanol free DCM (28 mL) containing a 50% molar

excess of trimethylamine (1190 mL, 8.5 mmol) and kept between

0

^ꢀC and ꢁ10 ^ꢀC, was added a 10% excess of methanesulfonyl

product

4-ethoxycarbonylmethyl-4-hydroxypiperidine-1-

chloride (488 mL, 6.3 mmol) over a period of 5e10 min. Ten minutes

carboxylic acid tert-butyl ester (10.31 g, 97%) was obtained as a

after the addition the cooling bath was removed and the mixture

was stirred for 1 h at ambient temperature to complete the reaction

(TLC: ethyl acetate/hexane, 70/30, R_fproduct0.43, R_feduct0.31, iodine

staining, compound is not UV-active). The reaction mixture was

transferred to a separatory funnel with the aid of more DCM. The

mix was ﬁrst extracted with ice water, followed by cold 10% HCl

acid, sat. sodium bicarbonate, sat. brine. Drying the DCM solution

over Na₂SO₄followed by solvent removal gave the product as a

clear yellow oil and reacted further without puriﬁcation. ¹H NMR

(400 MHz, CDCl₃)

d

1.23 (t, J ¼ 7.2 Hz, 3H, C^3’H₃), 1.46 (s, 9H, Boc

CH₃), 1.47e1.55 (m, 2H, 0.5C³H₂, 0.5C⁵H₂), 1.61e1.71 (m, 2H,

0.5C³H₂, 0.5C⁵H₂), 2.47 (s, 2H, C^1’H₂), 3.20 (t, J ¼ 11.7 Hz, 2H,

0.5C²H₂, 0.5C⁶H₂), 3.61 (s, 1H, OH), 3.82 (m, 2H, 0.5C²H₂, 0.5C⁶H₂),

4.18 (q, J ¼ 7.2 Hz, 2H, C^2’H₂). ¹³C NMR (101 MHz, CDCl₃)

d

14.1 (C^3’),

28.4 (Boc CH₃) 36.6 (C³, C⁵), 39.5 (weak, C², C⁶), 45.5 (C^1’), 60.8 (C^2’),

68.1 (C⁴), 80.4 (Boc C), 154.8 (Boc C¼O), 172.6 (C¼O). Calcd. for

C14H25NO5: C, 58.52; H, 8.77; N, 4.87; O, 27.84%; Found: C, 58.60;

H, 8.69; N, 4.93. C₁₄H₂₅NO₅HRMS (ESIþ) m/z: [MþH]þ Calcd

288.1805; Found 288.1803.

colorless oil (1.45 g, 80%). ¹H NMR (400 MHz, CDCl₃)

d 1.46 (s, 9H,

Boc CH₃), 1.54e1.71 (m, 4H, C³H₂, C⁵H₂), 1.95 (t, J ¼ 6.6 Hz, 2H,

C^1’H₂), 2.27 (s_br, 1H, OH), 3.03 (s, 3H, Mes CH₃), 3.13e3.24 (m, 2H,

0.5C²H₂, 0.5C⁶H₂), 3.76 (t, J ¼ 6.6 Hz, 2H, C^2’H₂), 3.81 (dt, J ¼ 13.3,

3.4 Hz, 2H, 0.5C²H₂, 0.5C⁶H₂). ¹³C NMR (101 MHz, CDCl₃)

d 28.4 (Boc

b)

tert-Butyl

4-hydroxy-4-(2-hydroxyethyl)piperidine-1-

CH₃) 36.9 (C³, C⁵), 37.5 (Ms CH₃), 39.6 (C^1’), 41.4 (weak, C², C⁶), 66.0

(C^2’), 68.8 (C⁴), 79.6 (Boc C), 154.7 (Boc C¼O). C₁₃H₂₅NO₆S HRMS

(ESIþ) m/z: [MþH]þ Calcd 324.1475; Found 324.1474.

carboxylate 12m-2

d)

tert-Butyl

4-(2-ﬂuoroethyl)-4-hydroxypiperidine-1-

carboxylate 12m-4

tert-Butyl-4-(2-ethoxy-2-oxoethyl)-4-hydroxypiperidine-1-

carboxylate 12m-1 (2.87 g, 10 mmol) was dissolved in THF (40 mL).

After adding LiBH₄(2 M in THF, 15 mL, 30 mmol, slightly

exothermic) methanol (3 mL) was added and the resulting solution

14

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

Under an atmosphere of argon TBAF (1 M solution in THF, 15 mL,

15 mmol, 5 eq) was added to tert-butyl 4-hydroxy-4-(2-((methyl-

sulfonyl)oxy)ethyl)piperidine-1-carboxylate 12m-3 (960 mg,

3 mmol). The mixture was stirred, heated to 70 ^ꢀC and kept at that

temperature for 19 h (TLC: ethyl acetate/hexane, 50/50, R_fproduct

0.96, R_feduct0.27, iodine staining, compound is not UV-active). After

cooling to ambient temperature the mixture was partitioned with

diethyl ether and sat. aqueous NH₄Cl solution. The organic fraction

was washed with sat NH₄Cl (x 2), water, and brine, dried over

Na₂SO₄, ﬁltered, and concentrated to afford the ﬂuoride as a clear

solution (3.0 mL) was added. The mixture was extracted three times

with diethyl ether. The combined organic phases were dried over

sodium sulfate and the solvent was removed in vacuo. The residue

was puriﬁed by column chromatography (eluent: petrol ether/ethyl

acetate, 3/1) to obtain a yellow oil (165 mg, 0.667 mmol, 22%). ¹H

NMR (400 MHz, CDCl₃): d

1.23e1.31 (m, 2H, 0.5C³H₂, 0.5C⁵H₂), 1.44

(s, 9H, Boc-CH₃), 1.54e1.65 (m, 2H, 0.5C³H₂, 0.5C⁵H₂), 1.80e1.88 (m,

1H, C⁴H), 2.18 (br, 1H, OH), 2.56e2.75 (m, 2H, 0.5C²H₂, 0.5C⁶H₂),

3.54e3.69 (m, 1H, C^1’H), 4.02e4.25 (m, 2H, 0.5C²H₂, 0.5C⁶H₂),

4.29e4.60 (m, 2H, C^2’H₂). ¹⁹F NMR (377 MHz, CDCl₃)

C

d

ꢁ234.69.

orange oil (540 mg, 2.19 mmol, 73%). ¹H NMR (400 MHz, CDCl₃)

₁₂H₂₂FNO₃HRMS m/z: [MþNa]^þCalcd 270.1476; Found 270.1477.

3

d

1.46 (9H, s, Boc CH₃), 1.52e1.67 (m, 4H, C³H₂, C⁵H₂), 1.89 (dt, J_H-

¼ 28.4, ³J_H-H¼ 5.7 Hz, 2H, C^1’H₂), 2.51 (s_br, 1H, OH), 3.13e3.26 (m,

b) rac-2-Fluoro-1-(piperidine-4-yl)ethan-1-ol hydrochloride

12n

F

2H, 0.5C²H₂, 0.5C⁶H₂), 3.73e3.85 (m, 2H, 0.5C²H₂, 0.5C⁶H₂), 4.70 (dt,

²J_H-F¼ 47.3, J_H-H¼ 5,7 Hz, 2H, C^2’H₂). ¹⁹F NMR (377 MHz, CDCl₃)

3

d

ꢁ218.05.¹³C NMR (101 MHz, CDCl₃)

d

28.4 (Boc CH₃), 36.9 (C³, C⁵),

39.6 (C², C⁶), 42.4 (d, ²J_C-F¼ 18.4 Hz, C^1’), 69.0 (d, ³J_C-F¼ 2.5 Hz, C⁴),

79.6 (Boc C), 80.8 (d, J_C-F¼ 163.5 Hz, C^2’), 154.8 (Boc C¼O).

1

C

₁₂H₂₂FNO₃HRMS (ESIþ) m/z: [MþH]þ Calcd 248.1656; Found

248.1653.

e) 4-(2-Fluoroethyl)-4-hydroxypiperidine triﬂuoroacetate 12m

rac-tert-Butyl-4-(2-ﬂuoro-1-hydroxyethyl)piperidine-1-

carboxylate 12n-1 (165 mg, 0.667 mmol) was dissolved in

dichloromethane (5 mL) and treated with triﬂuoroacetic acid

(5 mL). The reaction mixture was stirred at room temperature for

1 h. The solvent was removed in vacuo and the residue was treated

with 3 M methanolic HCl. After removal of the solvent in vacuo a

yellow oil (112 mg, 0.610 mmol, 91%) was obtained that was used

without further puriﬁcation. ¹H NMR (400 MHz, DMSO‑d₆)

d

1.40e1.60 (m, 2H, 0.5C³H₂, 0.5C⁵H₂), 1.60e1.72 (m, 2H, 0.5C³H₂,

Triﬂuoroacetic acid (4 mL) was added to a solution of tert-butyl

4-(2-ﬂuoroethyl)-4-hydroxypiperidine-1-carboxylate 12m-4

0.5C⁵H₂), 1,78e1.87 (m, 1H, C⁴H), 2.70e2.85 (m, 2H, 0.5C²H₂,

0.5C⁶H₂), 3.17e3.27 (m, 2H, 0.5C²H₂, 0.5C⁶H₂), 3.36e3.53 (m, 1H,

C^1’H), 4.22e4.47 (m, 2H, C^2’H₂), 5.14 (br, 1H), 8.81 (s, 1H), 9.05 (s,

(540 mg, 2.19 mmol) in DCM (5 mL) and the mixture was stirred at

ambient temperature for 1 h. The solution was concentrated in

vacuo and co-evaporated with methanol (4 ꢂ 5 mL) to furnish the

trifuoroacetate salt (570 mg, 96%) of the deprotected compound as

an amber oil. C₇H₁₅F₄NO₃MS (ESIþ) m/z: [MþH]þ Calcd 262.21;

Found 148.17 (free base [MþH]þ 148.19).

1H). ¹⁹F NMR (377 MHz, DMSO‑d₆)

DMSO‑d₆)

d

ꢁ227.48.¹³C NMR (101 MHz,

d

¼ 24.28 (d, J ¼ 113.4 Hz, C³, C⁵), 35.53 (d, J ¼ 5.8 Hz, C⁴),

42.85 (d, J ¼ 14.4 Hz, C², C⁶), 71.36 (d, J ¼ 18.4 Hz, C^1’), 85.08 (d,

J ¼ 168.1 Hz, C^2’). C₇H₁₄FNO HRMS m/z: [MþH]^þCalcd 148.1132;

Found 148.1131.

4.5.11.5. rac-2-Fluoro-1-(piperidine-4-yl)ethan-1-ol hydrochloride

4.5.11.6. rac-1-Fluoro-2-(piperidine-4-yl)propan-2-ol hydrochloride

12 n

12o

a)

rac-tert-Butyl-4-(2-ﬂuoro-1-hydroxyethyl)piperidine-1-

a) rac-tert-Butyl-4-(1-ﬂuoro-2-hydroxypropan-2-yl)piperidine-

1-carboxylate 12o-1

carboxylate 12n-1

Under

carboxaldehyde (960 mg, 4.50 mmol, 1.50 eq.) and ﬂuoroiodo-

methane (201 L, 474 mg, 3.00 mmol, 1.00 eq.) were dissolved in

an

argon

atmosphere

N-boc-piperidine-4-

Under an argon atmosphere N-boc-4-acetylpiperidine (681 mg,

3.00 mmol, 1.50 eq.) and ﬂuoroiodomethane (134 mL, 316 mg,

m

THF (10 mL) and diethyl ether (10 mL). A 1.5 M solution of meth-

yllithium lithiumbromide complex (4.00 mL, 6.00 mmol, 2.00 eq.)

in diethyl ether was added at ꢁ78 ^ꢀC. The reaction mixture was

stirred for 5 min at ꢁ78 ^ꢀC before a saturated aqueous NH₄Cl

2.00 mmol, 1.00 eq.) were dissolved in THF (10 mL) and diethyl

ether (10 mL). A 1.5 M solution of methyllithium-lithium bromide

complex (2.66 mL, 4.00 mmol, 2.00 eq.) in diethyl ether was added

at ꢁ78 ^ꢀC. The reaction mixture was stirred for 5 min at ꢁ78 ^ꢀC

15

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

before a saturated aqueous NH₄Cl solution (2.0 mL) was added. The

mixture was extracted three times with diethyl ether. The com-

bined organic phases were dried over sodium sulfate and the sol-

vent was removed in vacuo. The residue was puriﬁed by column

chromatography (eluent: petrol ether/ethyl acetate, 3/1) to obtain a

yellow oil (207 mg, 0.792 mmol, 40%). ¹H NMR (400 MHz, CDCl₃)

4.5.13. N-(4-methoxy-7-morpholin-4-yl-benzo[d]thiazol-2-yl)

piperidine-1-carboxamide 13a

d

1.10 (d, J ¼ 2.4 Hz, 3H, CH₃), 1.15e1.33 (m, 2H, 0.5C³H₂, 0.5C⁵H₂),

1.43 (s, 9H, Boc-CH₃), 1.57e1.68 (m, 2H, 0.5C³H₂, 0.5C⁵H₂), 1.73e1.81

(m, 1H, C⁴H), 2.08 (s, 1H, OH), 2.54e2.70 (m, 2H, 0.5C²H₂, 0.5C⁶H₂),

4.05e4.39 (m, 4H, 0.5C²H₂, 0.5C⁶H₂, C^2’H₂F). ¹⁹F NMR (377 MHz,

CDCl₃)

d

ꢁ230.14.¹³C NMR (101 MHz, CDCl₃)

d

19.73 (CH₃), 25.87 (C³,

C⁵), 26.90 (C², C⁶), 28.55 (Boc-CH₃), 42.86 (C⁴), 73.33 (d, J ¼ 17.1 Hz,

C^1’), 79.55 (Boc-C), 88.14 (d, J ¼ 172.9 Hz, C^2’), 154.84 (Boc-CO).

C

₁₃H₂₄FNO₃HRMS m/z: [MþNa]^þCalcd 284.1632; Found 284.1631.

b) 1-Fluoro-2-(piperidine-4-yl)propan-2-ol hydrochloride 12o

Carbamate 8 (385 mg, 1 mmol) was reacted with piperidine

(107 mL, 1.08 mmol) in DMSO (5 mL) for 8 h at ambient temperature

(TLC: ethyl acetate/hexane, 90/10, R_fCarbamate0.85, R_fUrea0.54). The

organic (EA) layer was washed with 10% citric acid, sat. aqueous

Na₂CO₃and brine. After drying, ﬁltration and evaporation of the

solvent the oily residue was treated with TBME (20 mL), concen-

trated in vacuo, and the solid residue was recrystallized from 70%

MeOH. Yield 326 mg (80%), colorless crystals, mp 191 ^ꢀC (70%

MeOH). ¹H NMR (400 MHz, DMSO‑d₆)

d 1.43e1.53 (m, 4H, Pip-

C³H₂þ Pip-C⁵H₂), 1.53e1.62 (m, 2H, Pip-C⁴H₂), 2.99 (t, 4H,

J ¼ 8.5 Hz, 2 x N-CH₂), 3.59e3.28 (m, 4H, Pip-C²H₂þ Pip-C⁶H₂), 3.77

(t, 4H, J ¼ 8.5 Hz, 2 x OCH₂), 3.84 (s, 3H, OCH₃), 6.78 (d, 1H,

J ¼ 8.5 Hz, H⁶), 6.86 (d, 1H, J ¼ 8.5 Hz, H⁵) 11.33 (s_br, 1H, NH). ¹³

C

rac-tert-Butyl-4-(1-ﬂuoro-2-hydroxypropan-2-yl)piperidine-1-

NMR (101 MHz, DMSO‑d₆)

C

d

24.3 (Pip-C⁴), 25.9 (Pip-C^3,5), 45 (Pip-

carboxylate 12o-1 (291 mg, 1.11 mmol) was dissolved in dichloro-

methane (5 mL) and treated with triﬂuoroacetic acid (5 mL). The

reaction mixture was stirred at room temperature for 1 h. The

solvent was removed in vacuo and the residue was treated with 3 M

methanolic HCl. After removal of the solvent in vacuo a yellow oil

(217 mg, 1.10 mmol, 99%) was obtained that was used without

^2,6), 51.9 (CNC), 56.3 (OCH₃), 67.0 (COC), 108.4 (C⁵), 112.2 (C⁶), 140.4

(C⁴). Anal. Calcd. for C₁₈H₂₄N₄O₃S: C, 57.43; H, 6.43; N, 14.88; Found

C, 57.41; H, 6.39; N, 14.81. HRMS m/z: [MþH]^þCalcd 377.1642,

Found 377.1640.

further puriﬁcation. ¹H NMR (400 MHz, DMSO‑d₆)

d

1.02 (d,

4.5.14. N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-5,6-

dihydropyridine-1(2H)-carboxamide 13b

J

¼

2.2 Hz, 3H, CH₃), 1.41e1.69 (m, 3H, 0.5C³H₂, 0.5C⁵H₂,

NCH₂CH₂CH), 1.70e1.84 (m, 2H, 0.5C³H₂, 0.5C⁵H₂), 2.62e2.89 (m,

2H, 0.5C²H₂, 0.5C⁶H₂), 3.18e3.30 (m, 2H, 0.5C²H₂, 0.5C⁶H₂), 4.18 (dq,

J ¼ 47.8, J ¼ 9.3 Hz, C²⁰⁰H₂), 8.77 (br), 9.13 (br). ¹⁹F NMR (377 MHz,

DMSO‑d₆)

d

ꢁ225.45.¹³C NMR (101 MHz, DMSO‑d₆)

d 20.29 (d,

J ¼ 4.8 Hz, CH₃), 22.92 (d, J ¼ 63.3 Hz, C³, C⁵), 40.05 (s, C⁴), 43.59 (d,

J ¼ 12.0 Hz, C², C⁶), 71.43 (d, J ¼ 17.2 Hz, C^1’), 87.89 (d, J ¼ 173.3 Hz,

C^2’). C₈H₁₆FNO HRMS m/z: [MþH]^þCalcd 162.1289; Found 162.1287.

4.5.12. Preparation of ureas 13a to 13p, general procedure

Under argon at ambient temperature (4-methoxy-7-morpholin-

4-yl-benzo[d]thiazol-2-yl)-carbamic acid phenyl ester (385 mg,

1 mmol) was dissolved in dry DMSO (3e5 mL). Under stirring the

respective amine (or its hydrochloride,1.08 mmol) was added (for

Carbamate 8 (192 mg, 0.5 mmol) was reacted with 1,2,3,6-

amine salts aqueous 10 N NaOH (108

mL, 1.08 mmol) was addi-

tetrahydropyridine (47.5 mL, 0.505 mmol) in DMSO (3 mL) for

tionally added). The light brown solution was stirred for a given

time at a given temperature (TLC: ethyl acetate/hexane, 90/10,

R_fCarbamate0.85 or ethyl acetate/hexane/acetic acid, 80/20/0.2,

15 h at ambient temperature (TLC: ethyl acetate/hexane, 90/10,

R_fCarbamate0.85, R_fUrea0.51). Yield 287 mg (73%), off-white felted

needles, mp 212 ^ꢀC (70% MeOH). C₁₈H₂₂FN₄O₃S HRMS m/z: [MþH]^þ

R

_fCarbamate0.82) after which ice cold 50% brine (15 mL) was added.

¹H NMR (400 MHz, CDCl₃) 2.20e2.28 (m, 2H, Pip-C³H₂), 3.12 (t,

d

To the stirred turbid mixture ethyl acetate (50 mL) was added, the

organic layer was separated and the aqueous phase extracted with

ethyl acetate (50 mL). The combined organic layers were washed

successively with 10% citric acid (50 mL), sat. aqueous Na₂CO₃

(50 mL) and 50% brine (50 mL), dried over anhydrous Na₂SO₄and

ﬁltered. The ﬁltrate was concentrated under reduced pressure to

give a solid or an oil that was crystallized by dissolving the oil in

boiling aqueous 50e70% MeOH (5e10 mL/mmol) and slow cooling.

The urea was collected by ﬁltration and dried in air.

4H, J ¼ 9.2 Hz, 2 x N-CH₂), 3.68 (t, 2H, J ¼ 11.2 Hz, Pip-C²H₂), 3.89 (t,

4H, J ¼ 9.2 Hz, 2 x O-CH₂), 3.94 (s, 3H, OCH₃), 4.03 (m, 2H, Pip-C⁶H),

5.66e5.73 (m, 1H, Pip-C⁴H), 5.88e5.96 (m, 1H, Pip-C⁵H), 6.80 (s, 2H,

C⁵H, C⁶H). ¹³C NMR (101 MHz, CDCl₃)

d

24.9 (Pip-C³), 43.5 (Pip-C²),

51.8 (CNC), 55.9 (OCH₃), 67.4 (COC þ Pip-C⁶), 107 (C⁶), 112.2 (C⁵),

123.2 (Pip-C⁴), 127.2 (C^7a), 135 (Pip-C⁵), 137.9 (C⁷), 140.5 (C⁴), 147.3

(C²), 154 (C^4a), 160.9 (CO). Anal. Calcd. for C₁₈H₂₂N₄O₃S: C, 57.73; H,

5.92; N, 14.96; Found C, 57.70; H, 5.89; N, 14.94. HRMS m/z: [MþH]^þ

Calcd 394.1469, Found 394.1468.

16

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

4.5.15. 4-Hydroxy-N-(4-methoxy-7-morpholin-4-yl-benzo[d]

thiazol-2-yl)piperidine-1-carboxamide 13c

3.86 (s, 3H, OCH₃), 3.80e3.91 (m, 2H, 0.5 Pip C^2/6H₂), 6.80

(d,³J ¼ 8.3 Hz, 1H, C⁵H), 6.90 (d,³J ¼ 8.3 Hz, 1H, C⁶H), 11.34 (s_br, 1H,

NH).¹³C NMR (101 MHz, CDCl₃)

d

30.8 (Pip C³, C⁵), 41.5 (Pip C², C⁶),

51.9 (CNC), 55.5 (Pip OCH₃), 56.4 (OCH₃), 67 (COC), 75.4 (Pip C⁴),

108.5 (C⁶), 112.3 (C⁵), 140.45 (C⁴). Anal. Calcd. for C₁₉H₂₆N₄O₄S: C,

56.14; H, 6.45; N, 13.78; Found C, 56.08; H, 6.43; N, 13.83. HRMS m/

z: [MþH]^þCalcd 407.17475, Found 407.17472.

4.5.17. 4-Fluoro-N-(4-methoxy-7-morpholin-4-yl-benzo[d]thiazol-

2-yl)piperidine-1-carboxamide 13e

Carbamate

8 (1155 mg, 3 mmol) was reacted with 4-

hydroxypiperidine (319 mg, 3.24 mmol, 1.08 eq) in DMSO (10 mL)

for 16 h at ambient temperature (TLC: ethyl acetate/hexane, 80/20,

R

_fCarbamate0.82, R_fUrea0.02e0.2). For the extraction 2 ꢂ 75 mL DCM/

mmol is needed (not ethyl acetate, 13c shows a tendency to pre-

cipitate from ethyl acetate in the separation funnel during work-

up!) After extraction the combined organic layers were washed

with 50% brine. Concentration furnished a light brown oil that was

crystallized from hot ethyl acetate (15 mL). Yield 970 mg (82%), off-

white crystals, mp 188 ^ꢀC (EA). ¹H NMR (400 MHz, CDCl₃)

Carbamate

ﬂuoropiperidine hydrochloride (150 mg, 1.08 mmol) and aqueous

10 N NaOH (108 L,1.08 mmol) in DMSO (10 mL) for 24 h at ambient

temperature (TLC: ethyl acetate/hexane, 90/10, R_fCarbamate0.85,

R_fUrea0.68; ethyl acetate/hexane/AcOH, 80/20/0.2, R_fCarbamate0.82,

8 (385 mg, 1 mmol) was reacted with 4-

d

1.81e1.97 (m, 2H, 0.5C³H₂, 0.5 Pip C⁵H₂), 1.98e2.12 (m, 2H, 0.5 Pip

m

C³H₂, Pip-0.5C⁵H₂), 3.05 (t, ³J ¼ 9 Hz, 4H, 2 x N-CH₂), 3.53e3.64 (m,

1H, 0.25 Pip C^2/6H₂), 3.66e3.77 (m, 1H, 0.25 Pip C^2/6H₂), 3.88 (t,

³J ¼ 9 Hz, 4H, 2 x O-CH₂), 3.89 (s, 3H, O-CH₃), 3.96e4.07 (m, 1H, 0.25

Pip C^2/6H₂), 4.09e4.20 (m, 1H, 0.25 Pip C^2/6H₂), 4.97 (sept., 1H, Pip

C⁴H), 6.86 (d,³J ¼ 5.2 Hz, 2H, aryl), 11.32 (s_br, 1H, NH). ¹³C NMR

R

_fUrea0.58). Yield 260 mg (66%), colorless crystals, mp 194 ^ꢀC (50%

MeOH). ¹H NMR (400 MHz, CDCl₃)

d

1.76e1.91 (m, 4H, Pip-C³H₂,

Pip-C⁵H₂), 3.05e3.11 (m, 4H, 2 x NCH₂), 3.53e3.73 (m, 4H, Pip-C²H₂,

Pip-C⁶H₂), 3.84e3.88 (m, 7H, 2 x OCH_2,O-CH₃), 4.74e4.92 (m, 1H,

Pip-C⁴H), 6.76 (s, 2H, C⁵H, C⁶H), 10.25 (br, 1H, NH). ¹⁹F NMR

(101 MHz, CDCl₃) d

33.9 (Pip C³, C⁵), 41.5 (Pip C², C⁶), 51.6 (CNC), 55.8

(OCH₃), 66.2 (Pip C⁴), 67.2 (COC), 107 (C⁶), 111.7 (C⁵), 126.9 (C^7a),

138.2 (C⁷), 140.3 (C⁴), 147.3 (C^4a), 154.1 (C¼O), 161.1 (C²). Anal. Calcd.

for C₁₈H₂₄N₄O₄S: C, 55.08; H, 6.16; N, 14.28; Found C, 55.11; H, 6.11;

N, 14.22. HRMS m/z: [MþH]^þCalcd 393.1591, Found 393.1588.

(377 MHz, CDCl₃)

d

ꢁ183.71.¹³C NMR (101 MHz, CDCl₃)

d

31 (d, ²J_C-

¼ 20.1 Hz, Pip-C³, Pip-C⁵), 40.2 (d, J ¼ 3.7 Hz, Pip-C², Pip-C⁶), 51.8

F

(CNC), 55.9 (CH₃), 67.4 (COC), 87.5 (d, ¹J_C-F¼ 171.5 Hz, Pip-C⁴), 107.3

(C⁶), 112.3 (C⁵), 126.7 (C^7a), 136.8 (C⁷), 140.6 (C⁴), 146.8 (C²), 154.9

(C^4a), 162.3 (CO). Anal. Calcd. for C₁₈H₂₃FN₄O₃S: C, 54.81; H, 5.88; N,

14.20; Found C, 54.82; H, 5.89; N, 14.14. HRMS m/z: [MþH]^þCalcd

395.1548, Found 395.1545.

4.5.16. 4-Methoxy-N-(4-methoxy-7-morpholin-4-yl-benzo[d]

thiazol-2-yl)piperidine-1-carboxamide 13d

4.5.18. rac-3-Fluoro-N-(4-methoxy-7-morpholin-4-yl-benzo[d]

thiazol-2-yl)piperidine-1-carboxamide 13f

Carbamate

methoxypiperidine (125 mg, 135

for 5 h at ambient temperature (TLC: ethyl acetate/hexane, 80/20,

_fCarbamate0.80, R_fUrea0.15e0.2). For the extraction DCM (2 ꢂ 75 mL)

8

(385 mg,

1

mmol) was reacted with 4-

mL, 1.08 mmol) in DMSO (5 mL)

R

was used. The organic layer was washed with 10% citric acid

(75 mL) and brine (75 mL). After drying, ﬁltration and evaporation

of the solvent the oily residue was treated with tert-butyl methyl

ether (20 mL) and concentrated in vacuo. The obtained cream

colored crystals were dissolved in MeOH (3 mL) and the product

was precipitated by the addition of water (9 mL, cloudy white

mixture). After standing at 5 ^ꢀC for 48 h a white solid had formed,

which was collected by ﬁltration. Yield 326 mg (80%), mp 172 ^ꢀC. ¹H

Carbamate

ﬂuoropiperidine hydrochloride (150 mg, 1.08 mmol) and aqueous

10 N NaOH (108 L,1.08 mmol) in DMSO (10 mL) for 24 h at ambient

8 (385 mg, 1 mmol) was reacted with 3-

m

temperature (TLC: ethyl acetate/hexane, 90/10, R_fCarbamate0.85,

R_fUrea0.44). Yield 287 mg (73%), colorless crystals, mp 196 ^ꢀC (50%

MeOH). ¹H NMR (400 MHz, DMSO‑d₆)

d 1.41e1.59 (m, 1H),

1.62e1.76 (m, 1H), 1.79e1.96 (m, 2H), 3.01 (t, 4H, J ¼ 9 Hz, 2 x N-

CH₂), 3.20e3.33 (m, 1H), 3.77 (t, 4H, J ¼ 9 Hz, 2 x O-CH₂), 3.86 (s, 3H,

OCH₃), 3.92e4.07 (m, 1H), 4.75 (d_br, J ¼ 47.8 Hz, 1H), 6.80 (d, 1H,

NMR (400 MHz, DMSO‑d₆)

d

1.33e1.48 (m, 2H, 0.5C³H₂, 0.5 Pip

C⁵H₂), 1.77e1.94 (m, 2H, 0.5 Pip C³H₂, Pip-0.5C⁵H₂), 3.0 (t,

³J ¼ 9.1 Hz, 4H, 2 x N-CH₂), 3.20e3.31 (m, 5H, 0.5 Pip C^2/6H₂þ Pip O-

CH₃), 3.36e3.45 (m, 1H, Pip C⁴H), 3.77 (t, ³J ¼ 9.1 Hz, 4H, 2 x O-CH₂),

J ¼ 8.5 Hz, H⁶), 6.88 (d, 1H, J ¼ 8.5 Hz, H⁵) 11.44 (s_br, 1H, NH). ¹⁹

F

NMR (377 MHz, CDCl₃)

d

ꢁ183.67.¹³C NMR (101 MHz, DMSO‑d₆)

d

21.1 (s_br, Pip C⁵), 29.4 (d, ²J_C-F¼ 20.3 Hz, Pip C⁴), 43.9 (s_br, Pip C⁶),

17

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

2

47.8 (d, J_C-F¼ 24 Hz, Pip C²), 87.1 (d, ¹J_C-F¼ 174.1 Hz, Pip C³), 51.9

(60.1

m

L, 0.402 mmol, 2.82 eq.) was added and the reaction was

(CNC), 56.4 (OCH₃), 67.0 (COC),108.5 (C⁵),112.3 (C⁶),140.5 (C⁴). Anal.

Calcd. for C₁₈H₂₃FN₄O₃S: C, 54.81; H, 5.88; N, 14.20; Found C, 54.77;

H, 5.79; N, 14.29. HRMS m/z: [MþH]^þCalcd 395.1548, Found

395.1543.

heated to 60 ^ꢀC. After 2 h of stirring the reaction was allowed to cool

to room temperature and stirred overnight. The solution was

diluted with ethyl acetate and subsequently washed with a satu-

rated aqueous solution of sodium bicarbonate, twice with water

and brine. The organic layer was dried over sodium sulfate and the

solvent was removed in vacuo. The residue was puriﬁed by column

chromatography (eluent: dichloromethane/methanol, 99/1 to 97/3)

4.5.19. 4-(Fluoromethyl)-N-(4-methoxy-7-morpholinobenzo[d]

thiazol-2-yl)piperidine-1-carboxamide 13g

to obtain a white solid (32 mg, 73.0

(400 MHz, CDCl₃)

m

mol, 51%), mp 95 ^ꢀC. ¹H NMR

1.61e1.70 (m, 2H, 0.5 Pip-C³H₂, 0.5 Pip-C⁵H₂),

d

1.81e1.90 (m, 2H, 0.5 Pip-C³H₂, 0.5 Pip-C⁵H₂), 3.06e3.11 (m, 4H, 2 x

N-CH₂), 3.32e3.41 (m, 2H, 0.5 Pip-C²H₂, 0.5 Pip-C⁶H₂), 3.56e3.90

(m, 11H, 0.5 Pip-C²H₂, 0.5 Pip-C⁶H₂, Pip-C^1’H₂, 2 x O-CH₂, O-CH₃),

4.55 (dt, ³J_H-F¼ 47.6 Hz, ³J ¼ 4.2 Hz, 2H, C^2’H₂), 6.76 (s, 2H, C⁵H, C⁶H),

9.86 (br, 1H, NH. ¹⁹F NMR (377 MHz, CDCl₃)

d

ꢁ223.11.¹³C NMR

(101 MHz, CDCl₃):

d

30.66 (Pip-C³, Pip-C⁵), 41.3 (Pip-C², Pip-C⁶), 51.9

(CNC), 56 (CH₃), 67.30 (C^1’), 67.5 (COC), 74.1 (Pip-C⁴), 83.3 (d, J_C-

1

¼ 169.7 Hz, C^2’), 107.2 (C⁶), 112.2 (C⁵), 127 (C^7a), 137.6 (C⁷), 140.6

F

(C⁴), 147.2 (C²), 154.3 (C^4a), 161.7 (CO). Anal. Calcd. for

Phenyl (4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)carba-

mate (385 mg, 1.00 mmol, 1.00 eq.) and 4-(ﬂuoromethyl)piperidine

hydrochloride (196 mg, 1.28 mmol, 1.28 eq.) were dissolved in dry

C

₂₀H₂₇FN₄O₄S: C, 54.78; H, 6.21; N, 12.78; Found C, 54.69; H, 6.19;

N, 12.81. HRMS m/z: [MþH]^þCalcd 439.1810; Found 439.1806.

DMSO (10 mL) under an argon atmosphere. DBU (420

mL,

4.5.21. 4-((2-Fluoroethoxy)methyl)-N-(4-methoxy-7-

2.81 mmol, 2.81 eq.) was added and the reaction was heated to

60 ^ꢀC. After 4 h of stirring the reaction was allowed to cool to room

temperature and stirred for another 66 h. The solution was diluted

with ethyl acetate and subsequently washed with a saturated

aqueous solution of sodium bicarbonate, twice with water and

brine. The organic layer was dried over sodium sulfate and the

solvent was removed in vacuo. The residue was puriﬁed by column

chromatography (eluent: petrol ether/ethyl acetate, 45/55 to 55/45)

to obtain a white solid (277 mg, 0.678 mmol, 68%), mp 110 ^ꢀC. ¹H

morpholinobenzo[d]thiazol-2-yl)piperidine-1-carboxamide 13i

NMR (400 MHz, CDCl₃)

d

1.22e1.36 (m, 2H, 0.5 Pip-C³H₂, 0.5 Pip-

C⁵H₂), 1.70e1.78 (m, 2H, 0.5 Pip-C³H₂, 0.5 Pip-C⁵H₂), 1.82e1.96 (m,

1H, Pip-C⁴H), 2.84e2.94 (m, 2H, 0.5 Pip-C²H₂, 0.5 Pip-C⁶H₂),

3.06e3.11 (m, 4H, 2 x N-CH₂), 3.83e3.90 (m, 7H, 2 x O-CH₂, O-CH₃),

4.16e4.34 (m, 4H, 0.5 Pip-C²H₂, 0.5 Pip-C⁶H₂, Pip-C^1’H₂), 6.76 (s, 2H,

C⁵H, C⁶H), 9.82 (br, 1H, NH). ¹⁹F NMR (377 MHz, CDCl₃)

Phenyl (4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)carba-

mate (96 mg, 0.250 mmol, 1.00 eq.) and 1-4-((2-ﬂuoroethoxy)

methyl)piperidine hydrochloride (70 mg, 0.354 mmol, 1.42 eq.)

were dissolved in dry DMSO (4 mL) under an argon atmosphere.

d

ꢁ223.96.¹³C NMR (101 MHz, CDCl₃):

d

27.5 (d, J ¼ 5.7 Hz, Pip-C³,

Pip-C⁵), 36.8 (d, J_C-F¼ 19.0 Hz, Pip-C⁴), 44 (Pip-C², Pip-C⁶), 51.8

(CNC), 55.9 (CH₃), 67.5 (COC), 87.1 (d, ¹J_C-F¼ 169.5 Hz, Pip-C^1’), 107.1

(C⁶), 112.2 (C⁵), 126.99 (C^7a), 137.5 (C⁷), 140.6 (C⁴), 147.2 (C²), 154.3

(C^4a), 161.8 (CO). Anal. Calcd. for C₁₉H₂₅FN₄O₃S: C, 55.87; H, 6.17; N,

13.72; Found C, 55.81; H, 6.09; N, 13.80. HRMS m/z: [MþH]^þCalcd

409.1704; Found 409.1702.

2

DBU (104 mL, 0.697 mmol, 2.79 eq.) was added and the reaction was

heated to 60 ^ꢀC. After 3 h of stirring the reaction was allowed to cool

to room temperature and stirred for another 69 h. The solution was

diluted with ethyl acetate and subsequently washed with a satu-

rated aqueous solution of sodium bicarbonate, twice with water

and brine. The organic layer was dried over sodium sulfate and the

solvent was removed in vacuo. The residue was puriﬁed by column

chromatography (eluent: dichloromethane/methanol, 100/0 to 98/

4.5.20. 4-(2-Fluoroethoxy)-N-(4-methoxy-7-morpholinobenzo[d]

thiazol-2-yl)piperidine-1-carboxamide 13h

2) to obtain a colorless solid (64 mg, 141

m

mol, 40%), mp 112 ^ꢀC. ¹H

NMR (400 MHz, CDCl₃)

d

1.18e1.32 (m, 2H, 0.5 Pip-C³H₂, 0.5 Pip-

C⁵H₂), 1.76e1.91 (m, 2H, 0.5 Pip-C³H₂, 0.5 Pip-C⁵H₂, Pip-C⁴H),

2.85e2.96 (m, 2H, 0.5 Pip-C²H₂, 0.5 Pip-C⁶H₂), 3.06e3.13 (m, 4H, 2 x

N-CH₂), 3.34 (d, J ¼ 6.1 Hz, 2H, Pip-C^1’H₂), 3.59e3.72 (m, 2H, Pip-

C^2’H₂), 3.82e3.93 (m, 7H, 2 x O-CH₂, O-CH₃), 4.11e4.24 (m, 2H, 0.5

Pip-C²H₂, 0.5 Pip-C⁶H₂), 4.44e4.61 (m, 2H, Pip-C^3’H₂), 6.77 (s, 2H,

C⁵H, C⁶H), 9.42 (br, 1H, NH). ¹⁹F NMR (377 MHz, CDCl₃)

d

ꢁ222.84.¹³C NMR (101 MHz, CDCl₃):

d

28.8 (Pip-C³, Pip-C⁵), 36.4

(Pip-C⁴), 44.3 (Pip-C², Pip-C⁶), 51.9 (CNC), 56 (CH₃), 67.5 (COC), 70.3

(d, J_C-F¼ 19.6 Hz, C^2’), 76 (C^1’), 83.2 (d, ¹J_C-F¼ 169.1 Hz, C^3’), 107.1

2

Phenyl (4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)carba-

mate (55 mg, 0.143 mmol, 1.00 eq.) and 1-4-(2-ﬂuoroethoxy)

piperidine hydrochloride (60 mg, 0.327 mmol, 2.28 eq.) were dis-

solved in dry DMSO (4 mL) under an argon atmosphere. DBU

(C⁶), 112.2 (C⁵), 127.2 (C^7a), 138 (C⁷), 141 (C⁴), 147.4 (C²), 154 (C^4a),

161.4 (CO). Anal. Calcd. for C₂₁H₂₉FN₄O₄S: C, 55.73; H, 6.46; N,

12.38; Found C, 55.72; H, 6.39; N, 12.42. HRMS m/z: [MþH]^þCalcd

453.1966; Found 453.1960.

18

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

4.5.22. 4-Hydroxy-4-methyl-N-(4-methoxy-7- morpholinobenzo[d]

thiazol-2-yl) piperidine-1-carboxamide (tozadenant) 13j

4.5.24. 4-Fluoromethyl-4-hydroxy-N-(4-methoxy-7-

morpholinobenzo[d]thiazol-2-yl)piperidine-1-carboxamide 13l

A solution of phenyl carbamate 8 (771 mg, 2 mmol), 4-

ﬂuormethyl-4-hydroxypiperidine hydrochloride (475 mg,

2.8 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (826 L,

5.4 mmol) in DMSO (25 mL) was stirred for 4 h at ambient tem-

perature (TLC: hexane/ethyl acetate, 30/70, R_f0.19). The mixture

was diluted with ethyl acetate (50 mL) and basiﬁed with saturated

aqueous NaHCO₃solution. Washing with water, drying over Na₂SO₄

and evaporation of the solvents left a residue that was puriﬁed by

ﬂash-chromatography (hexane/ethyl acetate, 30/70). The product

(848 mg, 99%) was obtained as a colorless solid, mp. 211 ^ꢀC (dec.).

A solution of phenyl carbamate 8 (1.07 g, 2.8 mmol) and 4-

methylpiperidin-4-ol (650 mg, 5,6 mmol) in 1,2-dichloroethane

(30 mL) was stirred for 2.5 h at 40 ^ꢀC (TLC: ethyl acetate/meth-

anol/AcOH, 98/2/0.2, R_f0.37; hexane/ethyl acetate/AcOH 60/40/0.2,

Rf ¼ 0.67). After removal of the solvent under reduced pressure the

residue was taken up in a small volume of methanol. After addition

of ethyl acetate, the product (680 mg, 61%) precipitated as a slightly

m

beige solid, mp. 215 ^ꢀC (dec.). ¹H NMR (400 MHz, CDCl₃)

d 1.17 (s,

3H), 1.50 (d, J ¼ 11.2 Hz, 4H), 3.01 (d, J ¼ 4.7 Hz, 4H), 3.32 (t,

¹H NMR (400 MHz, CDCl₃)

d

1.51 (s, 4H), 3.11 (d, J ¼ 43,8 Hz, 7H),

J ¼ 11.1 Hz, 2H), 3.71e3.99 (m, 9H), 6.64e6.84 (m, 2H). ¹³C NMR

3.82 (d, J ¼ 15.6 Hz, 7H), 4.08 (s, 3H), 4.32 (s, 1H), 4.91 (s, 1H), 6.83

(101 MHz, CDCl₃)

d 18.7, 30.2, 38.5, 40.7, 51.8, 55.9, 57, 66.6, 67.1,

(dd, J ¼ 17.8, 8.3 Hz, 2H), 11.42 (s, 1H). ¹⁹F NMR (377 MHz, CDCl₃)

107.5, 111.9, 126.7, 138.6, 140.1, 147.5, 154.2, 161.2. Anal. Calcd. for

d

ꢁ227.60.¹³C NMR (101 MHz, CDCl₃)

d 14.5, 21.2, 32.5, 39.8, 51.9,

C

₁₉H₂₆N₄O₄S: C, 56.14; H, 6.45; N, 13.78; Found C, 56.12; H, 6.39; N,

56.3, 67, 67.9, 68.3, 88, 91.4, 108.4, 112.2, 115.7, 126.5, 129.8, 140.4,

147.7, 154.3, 160.8. Anal. Calcd. for C₁₉H₂₅FN₄O₄S:C, 53.76; H, 5.94;

N,13.20; Found C, 53.72; H, 5.99; N,13.19. HRMS m/z: [MþH]^þCalcd

425,1659; Found 425,1654.

13.79. HRMS m/z: [MþH]^þCalcd 407.1743; Found 407.1744.

4.5.23. 4-Fluoro-4-(hydroxymethyl)-N-(4-methoxy-7-

morpholinobenzo[d]thiazol-2-yl)piperidine-1-carboxamide 13k

4.5.25. 4-(2-Fluoroethyl)-4-hydroxy-N-(4-methoxy-7-

morpholinobenzo[d]thiazol-2-yl)piperidine-1-carboxamide 13m

A solution of phenyl carbamate 8 (501 mg, 1.3 mmol) and (4-

ﬂuoropiperidin-4-yl)-methanol (186.42 mg, 1.4 mmol) in DMSO

(12 mL) was stirred for 2 h at ambient temperature followed by

5 h at 80 ^ꢀC (TLC: ethyl acetate/methanol/AcOH, 98/2/0.2, R_f0.46).

After cooling to ambient temperature 1,8-diazabicyclo[5.4.0]undec-

To a solution of (4-methoxy-7-morpholin-4-yl-benzothiazol-2-

yl)-carbamic acid phenyl ester (385 mg, 1 mmol) and N-ethyl-dii-

sopropyl-amine (680

mL, 4 mmol, 4 equiv) in trichloromethane

(10 mL) was added

a

solution of 4-(2-ﬂuoroethyl)-4-

7-ene (DBU) (153

mL, 1 mmol) was added and stirring was

hydroxypiperidine triﬂuoroacetate (392 mg, 1.5 mmol, 1.5 equiv)

in trichloromethane (1 mL) and tetrahydrofurane (1 mL) and the

resulting mixture heated to reﬂux (80 ^ꢀC) for 1.5 h (TLC: EA/Hex, 80/

20, R_fproduct0.02, R_feduct0.72). The reaction mixture was then cooled

to ambient temperature, diluted with dichloromethane (40 mL)

and extracted with saturated aqueous sodium carbonate (15 mL)

and water (2 ꢂ 5 ml). Final drying with sodium sulfate followed by

evaporation of the solvent and recrystallization from 2-propanol

afforded the title compound as white crystals (78% yield), mp

continued for 30 min. The mixture was diluted with ethyl acetate

(60 mL) and washed twice with water,1 N HCl, water, 1 N NaOH and

brine. Drying over Na₂SO₄and removal of the solvent under

reduced pressure left a solid residue that was recrystallized from

ethanol to furnish the target urea (310 mg, 56%) as a cream colored

solid, mp. 226 ^ꢀC (dec.). ¹H NMR (400 MHz, CDCl₃)

d 1.56 (t,

J ¼ 11.8 Hz, 4H), 3.28e2.80 (m, 6H), 3.28e3.62 (m, 3H), 3.61e4.00

(m, 7H), 4.12 (d, J ¼ 13.,6 Hz, 2H), 5.04 (t, J ¼ 5,9 Hz, 1H), 6.70e6.95

(m, 2H), 11.50 (s, 1H). ¹⁹F NMR (377 MHz, CDCl₃)

(101 MHz, CDCl₃) d 31.3, 31.7, 40.1, 51.9, 56.3, 66.3, 66.8, 67.01, 93.4,

d

ꢁ164.84.¹³C NMR

191 ^ꢀC (dec.). ¹H NMR (400 MHz, DMSO‑d₆)

d 1.45e1.58 (m, 4H, Pip-

3

C³H₂, Pip-C⁵H₂), 1.81 (dt, J_H-F¼ 25.8, J_H-H¼ 6.3 Hz, 2H, F-CH₂-

3

98, 108.4, 112.3, 115.7, 129.8, 140.4, 157.8. Anal. Calcd. for

CH₂), 2.99 (t, J_H-H¼ 4.5 Hz, 4H, CH₂NCH₂), 3.18e3.31 (m, 2H, 0.5

Pip-C²H₂, 0.5 Pip-C⁶H₂), 3.78 (t, ³J_H-H¼ 4.5 Hz, 4H, CH₂OCH₂), 3.85

(s, 3H, OCH₃), 3.88e3.98 (m, 2H, 0.5 Pip-C²H₂, 0.5 Pip-C⁶H₂), 4.54

(s_br, 1H, OH), 4.63 (dt, ²J_H-F¼ 47.5, ³J_H-H¼ 6.2 Hz, 2H, F-CH₂), 6.79 (d,

C

₁₉H₂₅FN₄O₄S: C, 53.76; H, 5.94; N, 13.20; Found C, 53.72; H, 5.99;

N, 13.23. HRMS m/z: [MþH]^þCalcd 425,1659; Found 425,1652.

19

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

³J_H-H¼ 8.5 Hz, C⁶H), 6.79 (d, ³J_H-H¼ 8.5 Hz, C⁵H),.11.29 (s_br, 1H, NH).

dissolved in dry DMSO (10 mL) under an argon atmosphere. DBU

(175 mL, 1.17 mmol, 1.43 eq.) was added and the reaction was heated

¹⁹F NMR (376 MHz, DMSO‑d₆)

d

ꢁ216.48.¹³C NMR (100 MHz,

2

DMSO‑d₆)

d

37.0 (Pip-C³, Pip-C⁵), 40.3 (Pip-C², Pip-C⁶), 42.7 (d, J_C-

to 60 ^ꢀC. After 4 h of stirring the reaction was allowed to cool to

room temperature and stirred for another 46 h. The solution was

diluted with ethyl acetate and subsequently washed with a satu-

rated aqueous solution of sodium bicarbonate, twice with water

and brine. The organic layer was dried over sodium sulfate and the

solvent was removed in vacuo. The residue was puriﬁed by column

chromatography (eluent: dichloromethane/methanol, 99/1 to 97/3)

to obtain a white solid (241 mg, 0.533 mmol, 65%), mp 218 ^ꢀC. ¹H

¼ 18.2 Hz, F-CH₂-CH₂),51.9 (C-N-C), 56.3 (OCH₃), 67.0 (C-O-C), 67.5

F

(d, ³J_C-F¼ 4.7 Hz, C⁴), 81.1 (d, ¹J_C-F¼ 159 Hz, F-CH₂), 108.4 (C⁶), 112.2

(C⁵), 115.7 (C^7’), 119.2 (C^4’), 129.8 (C⁷), 140.5 (C⁴), 153.0 (C¼O), 173.9

(C²). Anal. Calcd. for C₂₀H₂₇FN₄O₄S: C, 54.78; H, 6.21; N, 12.78;

Found C, 54.72; H, 6.19; N, 12.69. HRMS m/z: [MþH]^þCalcd

438.1737; Found 438.1733.

4.5.26. rac-4-(2-Fluoro-1-hydroxyethyl)-N-(4-methoxy-7-

morpholinobenzo[d]thiazol-2-yl)piperidine-1-carboxamide 13n

NMR (400 MHz, CDCl₃)

d

1.10 (d, J ¼ 2.2 Hz, 3H, Pip-C¹⁰⁰H₃),

1.28e1.44 (m, 1H, C⁴H), 1.66e1.76 (m, 2H, 0.5 Pip-C³H₂, 0.5 Pip-

C⁵H₂), 1.81e1.88 (m, 2H, 0.5 Pip-C³H₂, 0.5 Pip-C⁵H₂), 2.76e2.89 (m,

2H, 0.5 Pip-C²H₂, 0.5 Pip-C⁶H₂), 3.04e3.13 (m, 4H, 2 x N-CH₂),

3.81e3.90 (m, 7H, 2 x O-CH₂, O-CH₃), 4.14e4.38 (m, 4H, 0.5 Pip-

C²H₂, 0.5 Pip-C⁶H₂, Pip-C^2’H₂), 6.75 (s, 2H, C⁵H, C⁶H), 9.99 (br, 1H,

NH). ¹⁹F NMR (377 MHz, CDCl₃)

d

ꢁ229.57.¹³C NMR (101 MHz,

CDCl₃)

d

19.8 (d, J ¼ 5.1 Hz, Pip-C¹⁰⁰), 26.3 (d, J ¼ 93.4 Hz, Pip-C³, Pip-

C⁵), 42.6 (d, J ¼ 2.8 Hz, Pip-C⁴), 44.6 (d, J ¼ 20.6 Hz, Pip-C², Pip-C⁶),

51.8 (NCH₂), 55.9 (CH₃), 67.5 (OCH₂), 73.2 (d, ²J_C-F¼ 17.3 Hz, Pip-C^1’),

88 (d, ¹J_C-F¼ 173.5 Hz, Pip-C^2’), 107.2 (C⁶), 112.2 (C⁵), 127 (C^7a), 137.6

(C⁷), 140.6 (C⁴), 147.2 (C²), 154.1 (C^4a), 161.7 (CO). Anal. Calcd. for

C

₂₁H₂₉FN₄O₄S: C, 55.73; H, 6.46; N, 12.38; Found C, 55.68; H, 6.49;

Phenyl (4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)carba-

mate (150 mg, 0.389 mmol, 1.00 eq.) and 2-ﬂuoro-1-(piperidine-4-

yl)ethan-1-ol hydrochloride (100 mg, 0.545 mmol, 1.40 eq.) were

dissolved in dry DMSO (10 mL) under an argon atmosphere. DBU

N, 12.29. HRMS m/z: [MþH]^þCalcd 453.1966; Found 453.1965.

4.5.28. 4-Hydroxy-4-methyl-N-(4-(2-ﬂuoroethoxy)-7-

morpholinobenzo[d]thiazol-2-yl)piperidine-1-carboxamide 13p

(83.4 mL, 0.559 mmol, 1.43 eq.) was added and the reaction was

heated to 60 ^ꢀC. After 4 h of stirring the reaction was allowed to cool

to room temperature and stirred for another 46 h. The solution was

diluted with ethyl acetate and subsequently washed with a satu-

rated aqueous solution of sodium bicarbonate, twice with water

and brine. The organic layer was dried over sodium sulfate and the

solvent was removed in vacuo. The residue was puriﬁed by column

chromatography (eluent: dichloromethane/methanol, 99/1 to 97/3)

to obtain a white solid (124 mg, 0.283 mmol, 73%), mp 219 ^ꢀC. ¹H

NMR (400 MHz, DMSO‑d₆) d

1.18e1.37 (m, 2H, 0.5 Pip-C³H₂, 0.5 Pip-

C⁵H₂), 1.55e1.68 (m, 2H, 0.5 Pip-C³H₂, 0.5 Pip-C⁵H₂), 1.74e1.81 (m,

1H, C⁴H), 2.70e2.83 (m, 2H, 0.5 Pip-C²H₂, 0.5 Pip-C⁶H₂), 2.97e3.02

(m, 4H, 2 x N-CH₂), 3.40e3.51 (m, 1H, Pip-C^1’H), 3.72e3.86 (m, 7H, 2

x O-CH₂,O-CH₃), 4.22e4.45 (m, 4H, 0.5 Pip-C²H₂, 0.5 Pip-C⁶H₂, Pip-

C^2’H₂), 4.88e4.94 (m, 1H), 6.69e6.79 (m, 2H, C⁵H, C⁶H), 11.19 (s, 1H,

Under argon at ambient temperature phenyl (4-(2-

ﬂuoroethoxy)-7-morpholinobenzo[d]thiazol-2-yl)carbamate

11

(417 mg, 1 mmol) was dissolved in dry DMSO (8 mL). Under stirring

4-hydroxy-4-methylpiperidine hydrochloride (166.7 mg, 1.1 mmol)

was added followed by triethylamine (556 mL, 4 mmol) and the dark

NH). ¹⁹F NMR (377 MHz, DMSO‑d₆)

d

ꢁ227.90 ppm. ¹³C NMR

(101 MHz, DMSO‑d₆)

d

27.3 (d, J ¼ 104.4 Hz, Pip-C³, Pip-C⁵), 37.8 (d,

yellow solution was stirred for 2.5 h at 50 ^ꢀC (TLC: sample in

acetone, ethyl acetate/hexane/acetic acid, 80/20/0.2, R_fCarbamate

0.90, R_fUrea0.35). After cooling to ambient temperature, ethyl ace-

tate (50 mL) was added and the mixture was washed successively

with 50% brine (2 ꢂ 25 mL), dried over Na₂SO₄, and concentrated

under reduced pressure to give an oily residue that was puriﬁed by

ﬂash chromatography (ethyl acetate/hexane, 80/20). Evaporation of

the product fractions gave a clear colorless oil that was treated with

tert-butyl methyl ether. Rota-evaporation of TBME furnished the

product (410 mg, 93%) as a fawn solid, mp 197 ^ꢀC. ¹H NMR

J ¼ 5.8 Hz, Pip-C⁴), 43.7 (d, J ¼ 8.8 Hz, Pip-C², Pip-C⁶), 51.23 (CNC),

55.4 (CH₃), 66.5 (COC), 71.8 (d, ²J_C-F¼ 18.4 Hz, Pip-C^1’), 84.9 (d, ¹J_C-

¼ 169.1 Hz, Pip-C^2’), 107.17 (C⁶), 111.3 (C⁵), 139.8 (CO). Anal. Calcd.

F

for C₂₀H₂₇FN₄O₄S: C, 54.78; H, 6.21; N, 12.78; Found C, 54.82; H,

6.27; N, 12.69. HRMS m/z: [MþH]^þCalcd 439.1810; Found 439.1806.

4.5.27. rac-4-(1-Fluoro-2-hydroxypropan-2-yl)-N-(4-methoxy-7-

morpholinobenzo[d]thiazol-2-yl)piperidine-1-carboxamide 13o

(400 MHz, CDCl₃)

d 1.30 (s, 3H, CH₃), 1.60e1.69 (m, 4H, Pip-

C³H₂þ Pip-C⁵H₂), 1.81 (s_br, 1H, OH), 3.12 (t, J ¼ 4.5 Hz, 4H, 2 x N-

CH₂), 3.35e3.49 (m, 2H, 0.5 Pip C²H₂þ 0.5 Pip-C⁶H₂), 3.89 (m, t,

J ¼ 4.5 Hz, 6H, 2 x O-CH₂þ 0.5 Pip C²H₂þ 0.5 Pip-C⁶H₂), 4.36 (dt,

³J_H-F¼ 28 Hz, ³J ¼ 4.2 Hz, 2H, CH₃CH₂F), 4.79 (dt, J_H-F¼ 47 Hz,

2

³J ¼ 4.2 Hz, 2H, CH₃CH₂F), 6.77 (d, 1H, J ¼ 8.5 Hz, H⁶), 6.83 (d, 1H,

J ¼ 8.5 Hz, H⁵), 9.47 (s_br, 1H, NH). ¹⁹F NMR (377 MHz, CDCl₃)

d

ꢁ223.11.¹³C NMR (101 MHz, CDCl₃)

d

30.3 (CH₃),38.3 (Pip-C^3/5),

40.6 (Pip-C^2/6), 51.7 (NCH₂), 67.4 (OCH₂), 67.8 (Pip-C⁴), 68.4(d, J_C-

2

Phenyl (4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)carba-

mate (316 mg, 0.821 mmol, 1.00 eq.) and 1-ﬂuoro-2-(piperidine-4-

yl)propan-2-ol hydrochloride (228 mg, 1.15 mmol, 1.40 eq.) were

¼ 21 Hz, CH₂CH₂F), 81.8(d, ¹J_C-F¼ 171 Hz, CH₂CH₂F), 109.3 (C⁶), 112

F

(C⁵), 141.2 (C⁷), 140.4 (C⁴), 145.6 (C^4a), 154.2 (C¼O). Anal. Calcd. for

C

₂₀H₂₇FN₄O₄S: C, 54.78; H, 6.21; N, 12.78; Found C, 54.82; H, 6.19;

20

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

N, 12.72. HRMS (ESIþ) m/z: [MþH]þ Calcd 439.1810; Found

439.1806.

To an approximately 0.1 M solution of the alcohol 14a-1

(253 mg, 0.5 mmol) in ethanol free DCM (5 mL) containing a 200%

4.5.29. Synthesis of labeling precursors 14a e 14c

4.5.29.1. (N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-

((methylsulfonyl)oxy)-piperidine-1-carboxamido)methyl pivalate 14a

molar excess of triethylamine (210 mL, 1.5 mmol) and kept between

0

^ꢀC and ꢁ10 ^ꢀC, was added a 100% excess of methanesulfonyl

chloride (77 mL, 1 mmol) over a period of 2e5 min. Ten minutes

after the addition (the color of the solution had changed from

colorless to deep red) the cooling bath was removed and the so-

lution was stirred for 90 min at ambient temperature to complete

the reaction (TLC: ethyl acetate/hexane, 80/20, R_fPom-urea0.24,

R_fMesylate0.56 or ethyl acetate/methanol, 95/5, R_fPom-urea0.52,

R_fMesylate0.77). The reaction mixture was transferred to a separa-

tory funnel with the aid of more DCM. The mix was ﬁrst extracted

with ice water, followed by cold sat. sodium bicarbonate and water.

Drying the DCM solution over Na₂SO₄followed by solvent removal

gave the product as red-brown crystals. They were taken up in

TBME (15 mL), ultrasonicated for 1 min and the turbid solution was

ﬁltered through a layer ( 1 cm) of Celite. Evaporation of the solvent

yielded the mesylate (240 mg, 82%) as colorless crystals, mp 97 ^ꢀC.

a) 4-Hydroxy-N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-

yl)piperidine-1-carboxamido)methyl pivalate 14a-1

C

₂₅H₃₆N₄O₈S₂HRMS (ESIþ) m/z: [MþH]þ Calcd 585.20473; Found

585.20470.¹H NMR (400 MHz, CDCl₃)

d

1.19 (s, 9H, Pom CH₃),

Under argon dry potassium carbonate (207 mg, 1.5 mmol) was

added to a stirred solution of 4-hydroxypiperidine-1-carboxylic

acid (4-methoxy-7-morpholin-4-yl-benzo[d]thiazol-2-yl)-amide

13c (392 mg, 1 mmol) in dry DMF (20 mL). The mixture was heated

to 60 ^ꢀC and stirred at that temperature for 0.5 h. A solution of

1.81e1.97 (m, 2H, 0.5 Pip C³H₂, 0.5 Pip C⁵H₂), 1.98e2.12 (m, 2H, 0.5

Pip C³H₂, 0.5 Pip C⁵H₂), 3.05 (t, ³J ¼ 4.5 Hz, 4H, 2 x N-CH₂), 3.07 (s,

3H, Mes CH₃), 3.53e3.64 (m, 1H, 0.25 Pip C^2/6H₂), 3.66e3.77 (m, 1H,

0.25 Pip C^2/6H₂), 3.88 (t, ³J ¼ 4.5 Hz, 4H, 2 x O-CH₂), 3.89 (s, 3H, O-

CH₃), 3.96e4.07 (m, 1H, 0.25 Pip C^2/6H₂), 4.09e4.20 (m, 1H, 0.25 Pip

C^2/6H₂), 4.97 (sept., 1H, Pip C⁴H), 6.55 (s_br, 2H, Pom CH₂), 6.86 (d,

chloromethyl pivalate (290 mL, 1.5 mmol) in dry DMF (500 mL) was

slowly added and the mixture was stirred for 1 h at 60 ^ꢀC (TLC: ethyl

acetate/methanol, 95/5, R_fUrea0.25, R_fPom-urea0.65). After cooling to

ambient temperature the mixture was poured into ice/water

(150 mL), the aqueous layer was extracted with ethyl acetate

(2 ꢂ 75 mL) and the organic phase was washed successively with

10% aqueous citric acid solution (100 mL) and water (100 mL).

Drying over sodium sulfate, ﬁltration and rota evaporation of the

solvent in vacuo left an oily residue that was crystallized by treating

with TBME followed by concentration. Recrystallization from

aqueous 50% MeOH (10 mL) furnished the POM protected urea as a

solid. Yield 465 mg (92%), off-white crystals, mp 121 ^ꢀC.

J ¼ 5.2 Hz, 2H, aryl-H). ¹³C NMR (100 MHz, CDCl₃)

d 27 (Pom CH₃),

31.8 (Pip C³, C⁵), 38.9 (Mes CH₃), 39.6 (Pom C^q), 43.2 (Pip C², C⁶), 52.0

(CNC), 57.3 (OCH₃), 66.7 (COC), 70.2 (Pom CH₂), 78 (Pip C⁴), 110.1

(C⁶), 113.4 (C⁵), 123 (C^7a), 125.6 (C⁷), 140.9 (C⁴), 143.3 (C^4a), 161 (urea

C¼O), 167.8 (C²), 177.7 (Pom C¼O).

4.5.29.2. (N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-

(methylsulfonyloxy)methyl)piperidine-1-carboxamido)methyl

piv-

alate 14b

C

₂₄H₃₄N₄O₆S HRMS (ESIþ) m/z: [MþH]þ Calcd 507.22718; Found

a) 4-(Hydroxymethyl)-N-(4-methoxy-7-morpholinobenzo[d]

thiazol-2-yl)piperidine-1-carboxamide 14b-1

507.22715. (400 MHz, DMSO‑d₆) 1.11 (s, 9H, Pom CH₃), 1.21e1.36

d

(m, 2H, 0.5 Pip C³H₂, 0.5 Pip C⁵H₂), 1.66e1.82 (m, 2H, 0.5 Pip C³H₂,

0.5 Pip C⁵H₂), 2.94 (t, ³J ¼ 4.5 Hz, 4H, 2 x N-CH₂), 3.02e3.17 (m, 1H,

0.25 Pip C^2/6H₂), 3.19e3.29 (m, 1H, 0.25 Pip C^2/6H₂), 3.62e3.72 (m,

1H, 0.25 Pip C^2/6H₂), 3.72 (t, ³J ¼ 4.5 Hz, 4H, 2 x O-CH₂), 3.84 (s, 3H,

O-CH₃), 3.90e4.01 (m, 1H, 0.25 Pip C^2/6H₂), 4.11e4.25 (m, 1H, 0.25

Pip C^2/6H₂), 4.72 (s_br, 1H, Pip C⁴H), 6.40 (s_br, 2H, Pom CH₂), 6.94 (d,

J ¼ 8.8 Hz, 2H, H⁶), 7.06 (d, J ¼ 8.8 Hz, 2H, H⁵). ¹³C NMR (100 MHz,

DMSO‑d₆)

d

27.1 (Pom CH₃), 34.6 (Pip C³, C⁵), 38.8 (Pom C^q), 42.1

(Pip C², C⁶), 52.0 (CNC), 57.2 (OCH₃), 66.4 (Pip C⁴), 66.9 (COC), 70.1

(Pom CH₂), 111.6 (C⁶), 114.3 (C⁵), 121.8 (C^7a), 125.4 (C⁷), 140.9 (C⁴),

143.4 (C^4a), 160.2 (urea C¼O), 166.4 (C²), 177.1 (Pom C¼O).

N-Boc-4-piperidinemethanol (775 mg, 3.60 mmol, 1.20 eq.) was

dissolved in dichloromethane (2.0 mL) and treated with triﬂuoro-

acetic acid (2.0 mL). After stirring for 2 h at room temperature, the

solution was evaporated to dryness. The residue was dissolved in

dry DMSO (5.0 mL) under an argon atmosphere and phenyl (4-

methoxy-7-morpholinobenzo[d]thiazol-2-yl)carbamate 8 (1.16 g,

3.00 mmol, 1.00 eq.) and DBU (1.30 mL, 1.33 g, 8.74 mmol, 2.91 eq.)

were added. After stirring for 5 h at 80 ^ꢀC, the reaction was cooled

to room temperature and stirred overnight. The solution was

diluted with ethyl acetate and diethyl ether and subsequently

washed with a saturated aqueous solution of sodium bicarbonate,

twice with water and brine. The organic layer was dried over so-

dium sulfate and the solvent was removed in vacuo. The residue

was recrystallized from methanol and water (70/30). The desired

b)

(N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-

((methylsulfonyloxy)-piperidine-1-carboxamido)methyl

alate 14a

piv-

21

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

product was obtained as white solid (365 mg, 30%), mp 206 ^ꢀC. ¹H

NMR (400 MHz, DMSO‑d₆) d

0.99e1.13 (m, 2H, 0.5 Pip-C³H₂, 0.5 Pip-

C⁵H₂), 1.54e1.72 (m, 3H, Pip-C⁴H, 0.5 Pip-C³H₂, 0.5 Pip-C⁵H₂),

2.76e2.86 (m, 2H, 0.5 Pip-C²H₂, 0.5 Pip-C⁶H₂), 2.95e3.02 (m, 4H, 2 x

NCH₂), 3.26 (d, J ¼ 6.1 Hz, 2H, Pip-C^1’H₂), 3.74e3.79 (m, 4H, 2 x

OCH₂), 3.84 (s, 3H, OCH₃), 4.20e4.28 (m, 4H, 0.5 Pip-C²H₂, 0.5 Pip-

C⁶H₂), 4.48 (br, 1H, OH), 6.79 (d, J ¼ 8.5 Hz, 1H, C⁶H), 6.87 (d, J ¼

8.6 Hz, 1H, C⁵H), 11.25 (br, 1H, NH). ¹³C NMR (101 MHz, DMSO‑d₆)

d

28.55 (Pip-C³, Pip-C⁵), 33.30 (Pip-C⁴), 43.70 (Pip-C², Pip-C⁶), 51.43

(CNC), 55.86 (CH₃), 65.53 (COC), 66.57 (Pip-C^1’), 107.94 (C⁶), 111.76

(C⁵), 139.99 (CO). C₁₉H₂₆N₄O₄S HRMS m/z: [MþH]^þCalcd 407.1748;

Found 407.1746.

(4-(Hydroxymethyl)-N-(4-methoxy-7-morpholinobenzo[d]

thiazol-2-yl)piperidine-1-carboxamido)methyl pivalate 14b-2

(230 mg, 0.442 mmol, 1.00 eq.) was dissolved in dichloromethane

(3 mL) and cooled to 0 ^ꢀC. The solution was treated with triethyl-

b) (4-(Hydroxymethyl)-N-(4-methoxy-7-morpholinobenzo[d]

thiazol-2-yl)piperidine-1-carboxamido)methyl pivalate 14b-2

amine (91.7

mL, 0.657 mmol, 1.49 eq.) and methanesulfonyl chloride

(44.4

m

L, 0.574 mmol, 1.30 eq.) and stirred at 0 ^ꢀC. After 1.5 h

another 1.49 equivalents of triethylamine and 1.30 equivalents of

methanesulfonyl chloride were added. The reaction was stirred at

0

^ꢀC for another 1.5 h before being diluted with dichloromethane.

The organic solution was washed successively with saturated

aqueous solutions of ammonium chloride, sodium bicarbonate and

brine. After drying over sodium sulfate the solvent was removed in

vacuo and the residue was puriﬁed by column chromatography

(eluent: petrol ether/ethyl acetate, 30/70) to obtain a white solid

(184 mg, 70%), mp 163 ^ꢀC. ¹H NMR (400 MHz, CDCl₃)

d 1.17 (s, 9H, 3

POM-CH₃), 1.19e1.37 (m, 2H, 0.5 Pip-C³H₂, 0.5 Pip-C⁵H₂), 1.74e1.86

(m, 2H, 0.5 Pip-C³H₂, 0.5 Pip-C⁵H₂), 1.93e2.06 (m, 1H, Pip-C⁴H),

2.74e3.09 (m, 9H, 0.5 Pip-C²H₂, 0.5 Pip-C⁶H₂, 2 x NCH₂, Ms-CH₃),

3.82e3.89 (m, 7H, 2 x OCH₂, OCH₃), 4.08 (d, J ¼ 6.5 Hz, 2H, Pip-

C^1’H₂), 4.56e4.80 (m, 2H, 0.5 Pip-C²H₂, 0.5 Pip-C⁶H₂), 6.52 (s, 2H,

POM-CH₂), 6.80 (d, J ¼ 8.7 Hz, 1H, C⁶H), 6.84 (d, J ¼ 8.7 Hz, 1H, C⁵H).

¹³C NMR (101 MHz, CDCl₃)

d 27.17 (s, POM-C(CH₃)₃), 28.42 (0.5 Pip-

C³, 0.5 Pip-C⁵), 28.82 (0.5 Pip-C³, 0.5 Pip-C⁵), 36.40 (s, Pip-C⁴), 37.49

(s, Ms-CH₃), 42.52 (0.5 Pip-C², 0.5 Pip-C⁶), 44.22 (0.5 Pip-C², 0.5 Pip-

C⁶), 52.03 (CNC), 56.49 (OCH₃), 67.45 (COC), 69.91 (s, POM-CH₂),

73.51 (s, Pip-C^1’), 110.11 (C⁶), 113.43 (C⁵), 123.23 (C^7a), 125.77 (C⁷),

141.09 (C^4a), 143.39 (C⁴), 161.10 (CO), 167.59 (C²), 177.93 (POM-CO).

4-(Hydroxymethyl)-N-(4-methoxy-7-morpholinobenzo[d]thia-

zol-2-yl)piperidine-1-carboxamide 14b-1 (335 mg, 0.824 mmol,

1.00 eq.) and potassium carbonate (185 mg, 1.34 mmol, 1.62 eq.)

were suspended in DMF (15 mL) and stirred at 60 ^ꢀC for 0.5 h. A

solution of chloromethyl pivalate (378 mL, 2.62 mmol, 3.18 eq.) in

DMF (1.0 mL) was added and the reaction was stirred at 60 ^ꢀC for

3 h. The reaction was then cooled to room temperature. After

stirring for 20 h the reaction mixture was poured over ice and

extracted with ethyl acetate. The combined organic phases were

washed successively with an aqueous solution of citric acid (10%),

water and brine. The organic layer was dried over sodium sulfate

and the solvent was removed in vacuo. The residue was puriﬁed by

column chromatography (eluent: dichloromethane/methanol, 99/1

to 95/5) to obtain a brown solid (260 mg, 61%), mp 100 ^ꢀC. ¹H NMR

C

₂₆H₃₈N₄O₈S₂HRMS m/z: [MþH]^þCalcd 599.2204; Found

599.2201.

4.5.29.3. Pivaloxymethyl-(4-methoxy-7-morpholinobenzo[d]thiazol-

2-yl)(1,3-dioxa-2-thia-8-azaspiro[4.5]decan-2-oxide)-8-carboxamide

14c

a) Benzyl 1-Oxa-6-azaspiro [2,5]octane-6-carboxylate 14c-1

(according to Ref. [44])

(400 MHz, CDCl₃) d

1.09e1.26 (m, 11H, 3 POM-CH₃, 0.5 Pip-C³H₂, 0.5

Pip-C⁵H₂), 1.69e1.83 (m, 3H, 0.5 Pip-C³H₂, 0.5 Pip-C⁵H₂, Pip-C⁴H),

2.72e3.08 (m, 6H, 0.5 Pip-C²H₂, 0.5 Pip-C⁶H₂, 2 x NCH₂), 3.50 (d,

J ¼ 6.1 Hz, 2H, Pip-C^1’H₂), 3.80e3.86 (m, 7H, 2 x OCH₂, CH₃),

4.52e4.75 (m, 2H, 0.5 Pip-C²H₂, 0.5 Pip-C⁶H₂), 6.51 (s, 2H, POM-

CH₂), 6.78 (d, J ¼ 8.7 Hz, 1H, C⁶H), 6.83 (d, J ¼ 8.7 Hz, 1H, C⁵H). ¹³

C

NMR (101 MHz, CDCl₃)

d

27.14 (s, POM-C(CH₃)₃), 28.79 (0.5 Pip-C³,

0.5 Pip-C⁵), 29.19 (0.5 Pip-C³, 0.5 Pip-C⁵), 39.20 (s, Pip-C⁴), 42.94 (0.5

Pip-C², 0.5 Pip-C⁶), 44.63 (0.5 Pip-C², 0.5 Pip-C⁶), 51.98 (CNC), 56.45

(OCH₃), 67.42 (COC), 67.74 (s, Pip-C^1’), 69.97 (s, POM-CH₂), 110.03

(C⁶), 113.29 (C⁵), 123.23 (C^7a), 125.77 (C⁷), 141.03 (C^4a), 143.31 (C⁴),

161.05 (CO), 167.30 (C²), 177.96 (POM-CO). C₂₅H₃₆N₄O₆S HRMS m/z:

[MþH]^þCalcd 521.24283; Found 521.24262.

NaH (60% in mineral oil, 2 g, 50 mmol) was added in portions to

a solution of trimethylsulfonium iodide (6.6 g, 30 mmol) in DMSO

(100 mL) cooled to 0 ^ꢀC. After complete addition the ice bath was

removed and the solution was stirred for another 40 min at

ambient temperature. After addition of 1-(benzyloxycarbonyl)-4-

piperidone (4.66 g, 20 mmol), the mixture was heated to 55 ^ꢀC

for 2 h (TLC: hexane/ethyl acetate, 70/30, R_f0.38), cooled to

ambient temperature and poured onto ice. The product was

c)

(N-(4-methoxy-7-morpholinobenzo[d]thiazol-2-yl)-4-

(methylsulfonyloxy)methyl)piperidine-1-carboxamido)methyl

pivalate 14b

22

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

extracted with ethyl acetate, washed with water and dried over

Na₂SO₄. After removing the solvent under reduced pressure, further

puriﬁcation was carried out by ﬂash chromatography (hexane/ethyl

acetate, 70/30). The product 14c-1 (3 g, 61%) was obtained as a

solution, water, brine and water, dried over Na₂SO₄and the solvent

was removed under reduced pressure. The product (2.8 g, 92%) was

obtained as a colorless oil. ¹-H-NMR (400 MHz, CDCl₃)

d

1.38 (s, 6H),

1.45e1.81 (m, 4H), 3.51e3.14 (m, 2H), 3.61e3.82 (m, 4H), 5.12 (s,

2H), 7.18e7.45 (m, 5H). ¹³C NMR (101 MHz, CDCl₃)

27.3, 35.6, 41.2,

colorless oil. ¹H NMR (400 MHz, CDCl₃)

d

1.50 (dd, J ¼ 11.6, 6.7 Hz,

d

2H), 1.74e1.98 (m, 2H), 2,64 (d, J ¼ 9.1 Hz, 1H), 3.52 (ddd, J ¼ 13.3,

67.1, 73.6, 78.4, 109.5, 126.9, 127.8, 128, 128.5, 136.8, 155.3.

9.6, 3.7 Hz, 2H), 3.76e3.98 (m, 2H), 5.18 (d, J ¼ 0.6 Hz, 2H),

C

₁₇H₂₃NO₄HRMS m/z: [MþH]^þCalcd 306.1700; Found 306.1698.

7.27e7.48 (m, 5H). ¹³C NMR (101 MHz, CDCl₃)

d 32.9, 39.5, 42.7,

49.5, 53.7, 56.9, 65.2, 67.2, 73.2, 126.9, 127.5, 127.6, 127.8, 127.9, 128,

128.1, 128.4, 128.5, 136.7, 138.1,. 141.2, 155.2. C₁₄H₁₇NO₃HRMS m/z:

[MþH]^þCalcd 248.1281; Found 248.1280.

d) 2,2-Dimethyl-1,3-dioxa-8-azaspiro[4.5]decane 14c-4

b)

Benzyl-4-hydroxy-4-(hydroxymethyl)piperidine-1-

carboxylate 14c-2

A suspension of benzyl 2,2-dimethyl-1,3-dioxa-8-azaspiro[4.5]

decane-8-carboxylate 14c-3 (2,8 g, 9,2 mmol) and Pd/C (10%,

333 mg, 1 mmol) in methanol (40 mL) was stirred overnight in a

hydrogen atmosphere (TLC: hexane/ethyl acetate/AcOH, 60/40/0,2,

R_f0,38). After ﬁltration through Celite and washing of the ﬁlter cake

with MeOH, the ﬁltrates were combined and the solvent was

removed under reduced pressure. The product (1.3 g, 85%) was

Benzyl 1-oxa-6-azaspiro [2,5]octane-6-carboxylate 14c-1 (2.9 g,

12 mmol) was heated to 50 ^ꢀC in 0.02 N HCl (54 mL) for 1 h (TLC:

hexane/ethyl acetate, 60/40, R_f0.1; hexane/ethyl acetate/AcOH, 30/

70/0.2, R_f0.41). After cooling to ambient temperature, the pH was

adjusted to neutral with 2 N NaOH solution. The aqueous phase was

extracted three times with dichloromethane and the combined

organic phases were washed with brine and dried over Na₂SO₄.

After removal of the solvent under reduced pressure, the product

(2.7 g, 85%) was obtained as a colorless oil. ¹H NMR (400 MHz,

obtained as a grey waxy solid. ¹H NMR (400 MHz, CDCl₃)

d 1.36 (s,

6H), 1.49e1.88 - (m, 4H), 2.17e2.67 (m, 1H), 2.68e3.17 (m, 3H), 3.75

(d, J ¼ 4.6 Hz, 2H), 4.30 (s_br, 1H). ¹³C NMR (101 MHz, CDCl₃)

d 27.3,

36, 43.1, 49.3, 73.21, 73.6, 78.4, 79.5, 80.8, 108.8, 109.2. C₉H₁₇NO₂

HRMS m/z: [MþH]^þCalcd 172.1332; Found 172.1330.

e)

N-(4-Methoxy-7-morpholinobenzothiazol-2-yl)-2,2-

dimethyl-1,3-dioxa-8-azaspiro[4.5]-decan-8-carboxamide 14c-

5

CDCl₃)

3.45 (s, 2H), 3.74e4.16 (m, 2H), 5.15 (s, 2H), 7.08e7.53 (m, 5H). ¹³

NMR (101 MHz, CDCl₃) 33.4, 39.8,. 42.7, 53.5, 57.0, 67.2, 69.9, 70.3,

d

1,13e2.02 (m, 4H), 2.80 (s, 2H), 3.28 (t, J ¼ 10.8 Hz, 2H)),

C

d

127.8, 127.9, 128.1, 128.5, 128.6, 136.7, 155.4. C₁₄H₁₉NO₄HRMS m/z:

[MþH]^þCalcd 266,1387; Found 266,1387.

c)

Benzyl-2,2-dimethyl-1,3-dioxa-8-azaspiro[4.5]decan-8-

carboxylate 14c-3 (according to Ref. [45])

A solution of phenyl-4-methoxy-7-morpholinobenzothiazol-2-

ylcarbamate 8 (1.9 mg, 5 mmol) and 2,2-dimethyl-1,3-dioxa-8-

azaspiro[4.5]decane 14c-4 (1.02 g, 6 mmol) in DMSO (25 mL) was

stirred for 24 h at 60 ^ꢀC (TLC: hexane/ethyl acetate, 30/70, R_f0,46).

After cooling to ambient temperature, ethyl acetate (150 mL) was

added and the organic solution was washed successively with

water, saturated aqueous NaHCO₃solution and brine. After drying

over Na₂SO₄the solvent was removed under reduced pressure to

furnish a brown residue that was puriﬁed by ﬂash chromatography

(hexane/ethyl acetate, 50/50). The product was obtained as a

colorless solid (1.41 g, 61%), mp. 198 ^ꢀC (dec.). ¹H NMR (400 MHz,

Benzyl 4-hydroxy-4-(hydroxymethyl)piperidine-1-carboxylate

14c-2 (2.65 g, 10 mmol) and CSA (27 mg, 0.11 mmol) were dis-

solved in 2,2-dimethoxypropane (100 mL) and stirred at ambient

temperature for 24 h (TLC: hexane/ethyl acetate/AcOH, 60/40/0,2,

R_f0,72). The reaction was stopped by the addition of triethylamine

(2.5 mL), the solvent was removed and the residue was taken up in

water and extracted three times with dichloromethane (3 ꢂ 15 mL).

The combined organic phases were washed with saturated NaHCO₃

CDCl₃)

d 1,37 (s, 6H), 1.47e1.88 (m, 4H), 2.89e3.19 (m, 4H), 3.44 (t,

J ¼ 9,8 Hz, 2H), 3.58e4.09 (m, 11H), 6.76 (s, 2H), 7.69 (s_br, 1H). ¹³

C

NMR (101 MHz, CDCl₃)

d 27.2, 35.5, 41.6, 51.8, 55.9, 67.4, 73.7, 78.1,

107.3, 109.7, 112.4, 140.5, 146.85, 154.1. C₂₂H₃₀N₄O₅S HRMS m/z:

[MþH]^þCalcd 463.2009; Found 463.2007.

23

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

f)

(N-(4-methoxy-7-morpholinobenzothiazol-2-yl)-2,2-

h) Pivaloxymethyl-(4-methoxy-7-morpholinobenzo[d]thiazol-

2-yl)(1,3-dioxa-2-thia-8-azaspiro[4.5]decan-2-oxide)-8-

carboxamide 14c (according to Ref. [46])

dimethyl-1,3-dioxa-8-azaspiro-[4.5]decan-8-carboxamido)

methyl pivalate 14c-6

To a solution of N-(4-methoxy-7-morpholinobenzothiazol-2-

yl)-2,2-dimethyl-1,3-dioxa-8-azaspiro[4.5]decan-8-carboxamide

14c-5 (926 mg, 2 mmol) in DMF (40 mL), was added K₂CO₃(414 mg,

3 mmol). The suspension was heated to 60 ^ꢀC and stirred for

To a stirred solution of 4-hydroxy-4-(hydroxymethyl)-N-(4-

methoxy-7-morpholinobenzo[d]thiazol-2-yl)-N-pivaloylox-

ymethylpiperidine-1-carboxamide 14c-7 (1.07 g,

dichloromethane (10 mL) was added triethylamine (1.12 mL,

8 mmol). After cooling to 0 ^ꢀC, a solution of thionyl chloride (220

L,

3 mmol) in dichloromethane (500 L) was added over 5 min (TLC:

2 mmol) in

30 min. A solution of POM-Cl (580

mL, 3 mmol) in DMF (1000

mL)

m

was added and after stirring for 2 h at 60 ^ꢀC (TLC: hexane/ethyl

acetate, 30/70, R_f0,69) the mixture was cooled to ambient tem-

perature and poured into ice-water. The product was extracted into

ethyl acetate and the organic phase was washed with 10% citric acid

and twice with water. Drying over Na₂SO₄and removal of the

solvent under reduced pressure afforded a solid residue that was

recrystallized from methanol to give the product as a colorless solid

m

hexane/ethyl acetate, 50/50, R_f0.25). Ten minutes after the addi-

tion, the solution was diluted with ethyl acetate (10 mL), washed

with cold water (25 mL), diluted again with ethyl acetate (10 mL),

and washed twice with cold brine. After removal of the solvent

under reduced pressure, the product (1 g, 85%) was obtained as a

light grey solid, mp. 213 ^ꢀC (dec.). ¹H NMR (400 MHz, CDCl₃)

d 1.16

(1.15 g, 99%), mp. 207 ^ꢀC (dec.). ¹H NMR (400 MHz, CDCl₃)

9H), 1.40 (s, 6H), 1.51e1.86 (m, 2H), 2.81e3.19 (m, 4H), 3.35e4.27

(m, 13H), 6.81 (s, 2H), 6.52 (s, 2H). ¹³C NMR (101 MHz, CDCl₃)

27.1,

d

1.16 (s,

(s, 9H), 1.31e2.35 (m, 4H), 2.81e3.21 (m, 4H), 3.21e3.65 (m, 2H),

3.72e4.01 (m, 7H), 4.12e4.61 (m, 4H), 6.52 (s, 2H), 6.72e6.97 (m,

d

2H). ¹³C NMR (101 MHz, CDCl₃)

d 27.1, 29.7, 35.3, 35.5, 35.6, 35.7,

27.3, 39, 51.9, 56.4, 67.3, 69.8, 73.6, 78.9, 109.4, 110, 113.3, 123.1,

125.6, 140.8, 143.4, 160.9, 167.3, 177.8. C₂₈H₄₀N₄O₇S HRMS m/z:

[MþH]^þCalcd 577.2690; Found 577.2688.

35.9, 39, 39.3, 39.9, 40.9, 41.6, 51.9, 56.4, 67.3, 69.7, 74.8, 88.8, 110.1,

113.6, 123, 125.6, 140.8, 143.4, 161, 167.8. C₂₅H₃₄N₄O₈S₂HRMS m/z:

[MþH]^þCalcd 583.1890; Found 583.1886.

g)

4-Hydroxy-4-(hydroxymethyl)-N-(4-methoxy-7-

4.6. Radiochemistry

morpholinobenzo[d]thiazol-2-yl)-N-pivaloyloxymethylpiper-

idine-1-carboxamide 14c-7

Cyclotron produced [¹⁸F]ﬂuoride (80e100 GBq) was trapped on

a Sep-Pak Accell Plus QMA Carbonate Plus Light Cartridge (46 mg,

Waters) preconditioned with sterile water (3 mL) and ﬂushed with

air (3 mL). For the radioﬂuorination of 14a and 14b the cartridge

was eluted with a solution of Kryptoﬁx 2.2.2 (4,7,13,16,21,24-

hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane) (20 mg) in acetoni-

trile (700 mL) and potassium carbonate (3.5 mg) in water (200 mL),

for the radioﬂuorination of 14c the trapped radioﬂuoride was

eluted with Et₄NHCO₃(2 mg) in water (1 mL). The efﬂuent was

collected in a tightly closed 10 mL vial and the solvent was evap-

orated under

a stream of nitrogen and reduced pressure

(50e80 mbar) at 100 ^ꢀC. The residue was azeotropically dried by

the successive addition of 3 ꢂ 1 mL of dry acetonitrile. Precursor

To a solution of N-(4-methoxy-7-morpholinobenzothiazol-2-

yl)-2,2-dimethyl-1,3-dioxa-8-azaspiro[4.5]decan-8-carboxamido)

methyl pivalate 14c-6 (1.62 g, 2.8 mmol) in MeOH (10 mL) was

added camphorsulfonic acid (98 mg, 0.42 mmol, 15 mol %) and the

mixture was stirred at 50 ^ꢀC overnight (TLC: ethyl acetate/meth-

anol/AcOH, 98/2/0.2, R_f0.21). After cooling to ambient temperature

14a,14b or 14c (5 mg, 8.3e8.5 mmol) predissolved in DMSO (500 mL)

was added to the dry [¹⁸F]F-Kryptoﬁx complex and the reaction

mixture was heated to 85 ^ꢀC (14a and 14b) or 140 ^ꢀC (14c) for

15 min. After cooling to ambient temperature 1 M NaOH solution

(100

drolysis reaction was allowed to proceed for 3 min. Acidiﬁed eluent

(100 mL eluent þ 200 L H₃PO₄, 3 mL) was added and the mixture

mL) in methanol (100 mL) was added to the vial and the hy-

triethylamine (700

mL) was added and the solvent was removed

m

under reduced pressure. Further puriﬁcation was done by ﬂash

chromatography (ethyl acetate/methanol, 98/2). The product

(967 mg, 75%) was obtained as a colorless solid, mp.: 107.9 ^ꢀC (dec.).

was injected into semi-preparative HPLC (column: Prontosil 120-5-

C18 ace-EPS, 250 ꢂ 20 mm; eluent: sterile water/phosphate buffer

Braun MiniPlasco, 35/65/2, v/v/v; ﬂow: 15 mL/min; detection:

UV254, radioactivity). The respective radioactive fractions eluting

at tR ¼ 11e12 min ([¹⁸F]13e), tR ¼ 16e17 min ([¹⁸F]13g) or

tR ¼ 11e12 min ([¹⁸F]13l) were collected and the radioﬂuorinated

product was concentrated using solid phase extraction. For this, the

collected solution was diluted with water for injection (60 mL) and

passed through a Strata X 33C18 cartridge (30 mg), preconditioned

¹H NMR (400 MHz, CDCl₃)

d

1.10 (s, 9H), 1.28e1.76 (m, 2H),

2.75e3.59 (m, 11H), 3,80 (s, 7H), 4.25 (dd, J ¼ 34.0, 11.8 Hz, 2H), 6.45

(s, 2H), 6.76 (s, 2H). ¹³C NMR (101 MHz, CDCl₃)

27, 33.9, 39, 40.5,

d

51.8, 56.3, 67.3, 69.8, 70.2,110, 113.3, 122.9,125.6,140.8,143.2,160.9,

167.2, 177.8. C₂₅H₃₆N₄O₇S HRMS m/z: [MþH]^þCalcd 537.2377;

Found 537.2376.

24

D.R. Renk, M. Skraban, D. Bier et al.

European Journal of Medicinal Chemistry 214 (2021) 113214

in advance with ethanol (5 mL) followed by sterile water (10 mL).

The cartridge was washed with sterile water (5 mL), the product

was eluted either with 80% ethanol (0.8 mL, ([¹⁸F]13e and [¹⁸F]13g)

or 70% ethanol (0.6 mL, [¹⁸F]13l) and the ethanol fraction was

diluted with isotonic NaCl (5.2 mL for [¹⁸F]13e and [¹⁸F]13g or

3.6 mL for [¹⁸F]13l). Filtration through a sterile ﬁlter into a sterile

vial gave injectable sterile solutions of [¹⁸F]13e, [¹⁸F]13g and [¹⁸F]

13l containing 10% ethanol by volume which were used for bio-

logical experiments. Radiotracers [¹⁸F]13e and [¹⁸F]13g were ob-

tained in 25%e30% radiochemical yield (RCY) with a molar activity

for the adenosine A₁and A_2Areceptor in cell membranes and pig

striata homogenates, respectively. Membrane homogenates with a

protein content of 7.5 mg immobilized in a gel matrix [51] were

incubated with the radioligands in a total volume of 1.5 mL 50 mM

Tris/HCl buffer (pH 7.4). After an incubation time of 70 min the

immobilized membrane homogenates were washed with water

and transferred into scintillation cocktail (5 mL each, Ultima Gold,

PerkinElmer). The radioactivity of the samples (bound radioac-

tivity) was measured with a liquid scintillation counter (Beckman,

USA). All binding data were calculated by non-linear curve ﬁtting

with a computer aided curve ﬁtting program (Prism version 4.0,

GraphPad Software, Inc., La Jolla, USA).

(MA) of 249e300 GBq/

>99%. [¹⁸F]13l was prepared in 10%e15% RCY with a MA of 600

GBq/ mol and a RCP of >99%.

mmol and a radiochemical purity (RCP) of

m

4.7.5. Autoradiography

4.7. In vitro studies

For in vitro autoradiography, frozen horizontal sections (20 mm)

of rat brains were used. After preincubation for 5 min in 50 mM

TriseHCl buffer solution (pH 7.4) the sections were incubated in the

same buffer containing 2.3 kBq/mL of either [¹⁸F]13e, [¹⁸F]13g or

4.7.1. Stable transfection of cells

Human adenosine receptors ADORA1 and ADORA2A from hu-

man whole brain cDNA (Clontech) cloned into pcDNA3.1þ (Invi-

trogen) at EcoRI (5⁰), XhoI (3⁰) for ADORA1 and BamHI (5⁰), XhoI (3⁰)

for ADORA2A were commercially obtained from The Missouri S&T

cDNA Resource Center, USA. Plasmid DNA was ampliﬁed and pu-

riﬁed using standard molecular biological techniques. For trans-

fection, we used a modiﬁed version of the calcium phosphate

precipitation method [47,48].

[

¹⁸F]13l for 60 min. For detection of unspeciﬁc binding DPCPX or

ZM 241385 (1 mmol/L) were added. The sections were washed in the

buffer solution twice, immersed in deionized water, and dried at

37 ^ꢀC for 45 min. They were placed on a phosphor imaging plate for

30 min, scanned with a phosphor imager BAS 500 (Fujiﬁlm, Tokyo,

Japan) and analyzed with appropriate software.

Brieﬂy, 4 ꢂ 10⁵CHO-K1 cells were seeded in 6 cm Petri dishes

Declaration of competing interest

and transfected with 8

mg of hA₁AR-encoding plasmid DNA for

20 h at 37 ^ꢀC. Selection of stably transfected cells was initiated with

1 mg/mL geneticin (G418) until cell colonies had formed. From

these colonies, single clonal lines were isolated by limiting dilution.

Propagation of receptor expressing cells was performed in medium

The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

containing 450 mg/mL G418. Expression of hA₁ARs or hA_2AARs was

Appendix A. Supplementary data

veriﬁed by ligand binding ([³H]DPCPX or [³H]ZM 241385) and

Western blotting.

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113214.

4.7.2. Cell culture

The cells were grown adherently and kept in Ham’s F12 Nutrient

Mixture, containing 10% fetal bovine serum, penicillin (100 U/mL),

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26

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Design, synthesis and biological evaluation of Tozadenant analogues as adenosine A2A receptor ligands

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