Synthesis, radiolabeling and evaluation of novel amine guanidine derivatives as potential positron emission tomography tracers for the ion channel of the N-methyl-d-aspartate receptor

European Journal of Medicinal Chemistry 1 18 (2016) 143e160

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European Journal of Medicinal Chemistry

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

Research paper

Synthesis, radiolabeling and evaluation of novel amine guanidine

derivatives as potential positron emission tomography tracers for the

ion channel of the N-methyl- -aspartate receptor

D

Pieter J. Klein^*, Marion Chomet, Athanasios Metaxas, Johannes A.M. Christiaans ¹,

Esther Kooijman, Robert C. Schuit, Adriaan A. Lammertsma, Bart N.M. van Berckel,

Albert D. Windhorst

Department of Radiology & Nuclear Medicine, Neuroscience Campus Amsterdam, VU University Medical Center, P.O. Box 7057, 1007 MB Amsterdam, The

Netherlands

a r t i c l e i n f o

a b s t r a c t

Article history:

The N-Methyl-D-Aspartate receptor (NMDAR) is involved in many neurological and psychiatric disorders

Received 29 April 2015

Received in revised form

6 April 2016

Accepted 7 April 2016

Available online 19 April 2016

including Alzheimer's disease and schizophrenia. The aim of this study was to develop a positron

emission tomography (PET) ligand to assess the bio-availability of the NMDAR ion channel in vivo. A

series of tri-N-substituted diarylguanidines was synthesized and their in vitro binding afﬁnities for the

NMDAR ion channel assessed in rat forebrain membrane fractions. Compounds 21, 23 and 26 were

radiolabeled with either carbon-11 or ﬂuorine-18 and ex vivo biodistribution and metabolite studies were

performed in Wistar rats. Biodistribution studies showed high uptake especially in prefrontal cortex and

lowest uptake in cerebellum. Pre-treatment with MK-801, however, did not decrease uptake of the

radiolabeled ligands. In addition, all three ligands showed fast metabolism.

Keywords:

PET

NMDA

Radiolabeling

SAR

Non-competitive antagonists

1. Introduction

only [¹⁸F]-GE-179 has been used successfully in human applications

[5].

Excitatory neurotransmission in the mammalian central ner-

vous system (CNS) is primarily accountable to the glutamate re-

ceptor system. Glutamate receptors are divided in ionotropic and

metabotropic receptors. Ionotropic receptors are subdivided into

The NMDAR is a heteromultimeric assembly of four subunits. To

date, three types of subunits are known: the NR1 subunit with eight

splice variants (NR1aeh) [6], the NR2 subunit encoded by four

distinct genes (NR2AeD) [7], and the NR3A and NR3B subunits (2

genes) [8]. This assembly together forms the ion channel of the

NMDAR.

three groups of receptors:

a-amino-3-hydroxy-5-methyl-4-

isoxazolepropionate (AMPA), kainate and N-methyl-

D

-aspartate

receptor (NMDAR) [1]. NMDARs are necessary for long term

potentiation, and are thought to play an important role in learning

and memory [2]. It is hypothesized that hypoactivity of the NMDAR,

associated with advancing age, may have a role in developing

Alzheimer's disease and other neurodegenerative diseases [3,4].

Imaging of NMDARs using positron emission tomography (PET)

could make it possible to assess the NMDAR status in vivo. To date,

Recently, several structural classes with NMDAR afﬁnity were

reported. Propargylamine and acetylene conjugated polycyclic cage

derivatives, b- and g-carboline derivatives, pentamidine analogs of

MK-801 and ifenprodil and (3-hydroxy-pyrazolin-5-yl)glycine

based ligands. Unfortunately, all exhibit binding afﬁnities in the

micro-molar range [9e12].

Non-competitive ion channel NMDAR antagonists, such as

[5R,10S]-(þ)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-

hepten-5,10-imine (MK-801), N-(1-[thienyl] cyclohexyl)piperidine

(TCP) and ketamine are termed “use-dependent” ligands. These

non-competitive antagonists gain access to the ion channel site

primarily when the NMDAR is in an open state [13,14], i.e. they can

bind within the channel when the receptor is activated by the

* Corresponding author.

E-mail address: pj.klein@vumc.nl (P.J. Klein).

Present address: A*STAR-NUS, Clinical Imaging Research Centre (CIRC), Centre

1

for Translational Medicine, National University of Singapore, 14 Medical Drive,

Singapore 117599, Singapore.

http://dx.doi.org/10.1016/j.ejmech.2016.04.022

144

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

agonist glutamate and the co-agonist glycine. Therefore, binding of

non-competitive antagonists is proportional to the amount of

activated, open receptor channels. Hence, by using non-

competitive radiolabeled NMDAR antagonists and PET, the status

of regional activation of NMDARs can be assessed in vivo.

quantitative deprotection of the 2,5-dimethylpyrrole group using

NH₂OH$HCl [28].

Compound 12 was synthesized in 4 steps from 3-nitro-aniline

(8) in 64% yield. First, 8 was protected with a 2,5-dimethylpyrrole

group [29] followed by a reduction of the nitro moiety [30]. Sec-

ondly, amine 10 was reacted with cyanogen bromide [31] and then

methylated to obtain 12.

The class of guanidine compounds with afﬁnity for the NMDAR

ion channel is known since 1989 [15e23]. Structure activity re-

lationships studies showed that 1 (GMOM, K_i: 5.2e21.7 nM against

[³H]MK801) [22,24], 2 (CNS 5161, K_i: 1.87 nM against [³H]MK801)

[20] and 3 (GE-179, K_i: 2.4 nM against [³H]TCP) [5] have superior K_i

values towards the ion channel of the NMDAR (Fig. 1). Apart from

its K_ivalue, ligand 1 showed promising characteristics for use as an

NMDA PET tracer, although human applications have not been re-

ported yet. [¹¹C]2 has been used in patients with Parkinson's dis-

ease, but no signiﬁcant increase in uptake of [¹¹C]2 was seen in

patients as compared with control subjects [25]. Recently, in an

initial evaluation of [¹⁸F]3 in healthy volunteers high and rapid

brain extraction was observed, with a relatively homogeneous

distribution in gray matter and rapid peripheral metabolism.

Nevertheless, quantiﬁcation of [¹⁸F]3 appeared to be feasible [5].

So far, there have been no reports on meta substituted alkyl-

amine analogues of 1e3. Therefore, the aim of this study was to

synthesize and evaluate amine and pyridine analogs of 1 and 2 as

potential PET ligands for the NMDAR ion channel. Synthesis,

binding afﬁnity to the ion channel of the NMDAR, radiolabeling of

higher afﬁnity compounds, the Log D_7.4value, metabolism and

biodistribution in rats were investigated.

The alkyl substituted amine guanidines 21, 23, 26 and 29 were

prepared using 17e19 as key intermediates. Amine 17 was mono

protected to prevent double alkylation in the synthesis of 19 and 27,

and to preclude formation of side products during the guanidine

formation of 23 and 29. Scheme 3 shows the synthesis of mono-

and dimethylaminophenyl guanidines 21 and 23. The N-(3-

aminophenyl)-N-methylcyanamide intermediates 17e19 were

prepared from 3-nitro-aniline 8. Cyanamide 15 was obtained in 93%

yield [32]. After methylation of the cyanamide moiety and reduc-

tion of the nitro moiety, 17 was obtained in 72% overall yield.

Subsequent monoprotection of the amine moiety with a triﬂuoro-

acetic acid group [33] gave 18 in 86% yield and the subsequent

methylation gave 19 in quantitative yield. Compound 17 was

dimethylated to obtain 20 in 51% yield. The condensation of 20 with

2-chloro-5-(methylthio)aniline hydrochloride in chlorobenzene

gave dimethylaminophenyl guanidine 21 in 59% yield. Synthesis of

N-triﬂuoroacetic-N-methylaminophenyl guanidine 22 was carried

out using the triﬂuoroacetyl protected cyanamide 19 in 41% yield.

Deprotection of 22 under basic aqueous conditions yielded 23

quantitatively.

Intermediate 19 was deprotected and alkylated with ﬂuo-

roethylbromide to obtain 25 in 30% overall yield (Scheme 4). N-

methyl-N-ﬂuoroethyl guanidine was synthesized in 53% yield.

However, by using standard conditions to obtain guanidines 28 and

26, namely 165 ^ꢀC in chlorobenzene, an inseparable side product

was formed. High resolution mass spectrometry and ¹³C NMR

measurements revealed an impurity containing a chloroethyl

moiety besides the ﬂuoroethyl product. Formation of this chlori-

nated side product was circumvented by lowering the temperature

to 140 ^ꢀC and using toluene instead of chlorobenzene as solvent.

The ﬂuoroethylamine analogue 29 was synthesized via a similar

approach, starting from 18 (Scheme 5). Alkylation with ﬂuo-

roethylbromide provided 27 in 70% yield. Subsequent condensation

with 2-chloro-5-(methylthio)aniline hydrochloride in toluene (28,

66%) and quantitative deprotection led to ﬂuoroethylamine gua-

nidine 29.

2. Results and discussion

2.1. Chemistry

The synthesis of the pyridine substituted guanidines (7a, c) is

shown in Scheme 1. The N-methyl-N-(pyridinyl)cyanamides 6aec

were synthesized by a three step procedure adapted from Sta-

novnik et al. [26] and Huntsman et al. [27]. First, the formamide

oximes (5aec) were obtained by a two step reaction from the

aminopyridines 4aec with N,N-dimethylformamide dimethyl

acetal (DMFeDMA) in 2-propanol, followed by treatment with

hydroxylamine hydrochloride (NH₂OH$HCl) in 52e71% yield. Sec-

ondly, a reaction with DMFeDMA in toluene yielded the N-meth-

ylcyanamides 6aec in 30e61% yield. The pyridine guanidines 7a, c

were synthesized by condensation of 2-chloro-5-(methylthio)ani-

line hydrochloride in chlorobenzene with 6aec in 34e39% yield,

but 7b could not be obtained using this method. The reason for this

is unclear, presumably the meta conﬁguration of the pyridine ring

deactivates the cyanic moiety too much for condensation. As

2.2. Structure activity relationship study

Table 1 summarizes binding afﬁnities of the synthesized gua-

nidines for the ion channel of the NMDAR. The binding afﬁnity was

determined by measuring the ability of various concentrations of

unlabeled ligand to inhibit speciﬁc binding of [³H]MK-801 to rat

forebrain plasma membranes. Substitution of a phenyl group (30)

by a pyridyl group (7a, c) was not allowed as the afﬁnity of the

compound 7a and 7c showed afﬁnities less than 10 mM towards the

NMDA receptor, no further research was performed to obtain

compound 7b.

Unsubstituted aminophenyl guanidine 14 was synthesized ac-

cording to Scheme 2 via condensation of 12 with 2-chloro-5-

(methylthio)aniline hydrochloride in 46% yield and subsequent

guanidines 7a and c dropped to values above 10 mM.

Fig. 1. Structures of radiolabeled N,N⁰-diaryl-N-methylguanidines.

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

145

Scheme 1. Synthesis of pyridine substituted guanidines 7a, c. Reagents and conditions: a) DMFeDMA, 2-propanol, reﬂux, 2e3 h; b) NH₂OH$HCl, 50e75 ^ꢀC, 2e18 h; c) DMF-DMA,

toluene, reﬂux, 30 mine2.5 h; d) 2-chloro-5-(methylthio)aniline hydrochloride, chlorobenzene, 165 ^ꢀC, 6 h.

Scheme 2. Synthesis of amino guanidine 14. Reagents and conditions: a) PTSA, hexane-2,5-dione, toluene, reﬂux, 75 min; b) acetic acid, Fe, EtOH/H₂O, reﬂux, 18 min; c) NaOAc, CNBr,

MeOH, 0 ^ꢀCeRT, 26 h; d) K₂CO₃, CH₃I, DMF, RT, 1.5 h; e) 2-chloro-5-(methylthio)aniline hydrochloride, chlorobenzene, 165 ^ꢀC, 6 h; f) NH₂OH$HCl, EtOH/H₂O, reﬂux, 24 h.

Scheme 3. Synthesis of mono- and dimethyl amino guanidine 21 and 23. Reagents and conditions: a) CNBr, Acetic acid/H₂O, 1 M NaOH, 0 ^ꢀCeRT, 20 h; b) K₂CO₃, CH₃I, DMF, 45 ^ꢀC,

45 min; c) acetic acid, Fe, EtOH/H₂O, reﬂux, 10 min; d) TFAA, DCM, 0 ^ꢀC, 1 h; e) K₂CO₃, CH₃I, DMF, 45 ^ꢀC, 2.5 h; f) K₂CO₃, CH₃I, DMF, 45 ^ꢀC, 1 h; g) 2-chloro-5-(methylthio)aniline

hydrochloride, chlorobenzene, 165 ^ꢀC, 7 h; h) K₂CO₃, MeOH/H₂O, RT, 1 h.

146

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

Scheme 4. Synthesis of ﬂuoroethylmethyl amino guanidine 26. Reagents and conditions: a) K₂CO₃, MeOH/H₂O, RT, 1 h; b) K₂CO₃, KI, Br(CH₂)₂F, DMF/THF, 80 ^ꢀC, 24 h; c) 2-chloro-5-

(methylthio)aniline hydrochloride, toluene, 140 ^ꢀC, 8 h.

Scheme 5. Synthesis of ﬂuoroethylamino guanidine 29. Reagents and conditions: a) K₂CO₃, KI, Br(CH₂)₂F, DMF/THF, 80 ^ꢀC, 26 h; b) 2-chloro-5-(methylthio)aniline hydrochloride,

toluene, 140^ꢀ, 6.5 h; c) K₂CO₃, MeOH/H₂O, RT, 1 h.

alkyl groups, a methyl substitution (21, 23) is preferred over a ﬂu-

oroethyl substitution (26, 29) as the corresponding binding afﬁnity

is about 3 fold higher.

Table 1

Afﬁnity (nM) to the ion channel of the NMDAR.

2.3. Radiochemistry

Compounds 21, 23 and 26 were radiolabeled with either carbon-

11 or ﬂuorine-18.

Compound^a

Y

Z

R

K_i(nM)^b

2.3.1. Synthesis of [¹¹C]21

30^c

7a

7c

C

N

C

N

C

H

OH

307

130

The carbon-11 labeling agent [¹¹C]CH₃I was prepared by reacting

cyclotron produced [¹¹C]CO₂with lithium aluminum hydride in

THF, which subsequently was reacted with hydroiodic acid in

radiochemical yields of up to 90% decay corrected (DC) [34,35].

Radiolabeling of [¹¹C]21 is depicted in Scheme 6.

>10

m

M

31^c

551

21.7

439

19.1

1.35

48.3

4.81

100

1^c

OCH₃

NH₂

NHCH₃

N(CH₃)₂

NH(CH₂)₂F

NCH₃(CH₂)₂F

7.52

70.6

7.76

0.32

22.6

2.63

14

23

21

29

26

Initially, [¹¹C]CH₃I was used as labeling synthon exploring

several common base solvent combinations at 80 ^ꢀC and 3 min

reaction time (Table 2, entries 1e6). Unfortunately, yields obtained

were below 4% (radiochemical yield, DC). When the more reactive

a

Fumaric acid salt, 7c, 31: free base.

b

[

¹¹C]CH₃OTf labeling synthon was used, radiochemical yields

K_imeasured against 5 nM [³H]MK-801 presented as mean SD of 3 indepen-

increased to 47% (Table 2, entries 8e17). [¹¹C]CH₃OTf was prepared

by transferring [¹¹C]CH₃I through a column containing silver triﬂate

impregnated Graphpac heated at 200 ^ꢀC [36]. Further optimization

of time, reaction temperature and solvent resulted in optimal re-

action conditions of 40 ^ꢀC and 1 min reaction time in acetonitrile

dent determinations, each conducted in triplicate.

c

K_ivalues taken from Klein et al. [24].

The introduction of an amine rather than hydroxyl moiety,

however, was allowed in view of binding afﬁnity towards the ion

channel of the NMDAR. Analogous to the improvement in afﬁnity of

1 by replacing the hydroxyl moiety with methoxy 31, a similar

phenomenon was observed after monomethylation of 14. The

binding afﬁnity of 14 increased 23 fold to 19.1 nM for 23. Double

methylation increased the afﬁnity even further to 1.35 nM for

compound 21. Introduction of a ﬂuoroethyl moiety in 29 increased

the afﬁnity, compared with 14, 9 fold. Another 10 fold increase to

4.81 nM for 26 was reached when a methyl group was introduced.

These results suggest that a tertiary amine, substituted with short

alkyl groups, is favored over a secondary amine with respect to

binding afﬁnity for the ion channel of the NMDAR. From the short

using 0.5e0.8 mg (1.6e2.5 mmol) of precursor 23.

Scheme 6. Synthesis of carbon-11 labeled guanidines [¹¹C]23 and [¹¹C]21 using [¹¹C]

methyl iodide or [¹¹C]methyl triﬂate.

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

147

Table 2

Radiolabeling optimization of [¹¹C]21.

Entry^a

Labeling agent

Solvent^b

Base

Amount of base^c

Time^d

Temp^e

Yield^f,g

1

2

3

4

5

6

7

8

CH₃I

CH₃OTf

DMSO

DMF

MeCN

DMF

Acetone

MeCN

Acetone

5 M NaOH

K₂CO₃

40% TBAOH

5 M NaOH

K₂CO₃

e

25

72

7.5

25

72

e

3

1

2

3

4

1

80

60

40

30

20

40

0

3.6

0

0.6

1.0

1.8

2.1

37.2

39.5

33.5

38.2

34.7

46.5

45.5

46.7

43.2

31.5

0

0.2

0

0.4

0.2

0.5

0.1

10.2

e

9

e

10

11

12

13

14

15

16

17

e

6.7

e

a

Used 0.5e0.8 mg (1.6e2.5 mmol) of precursor (23).

b

c

d

e

f

300

mL.

m

mol.

Minutes.

^ꢀC.

Entries 1e8, 14: n ¼ 2 presented as mean SD, entries 9e13, 15e17: n ¼ 1.

g

Radiochemical yield determined using radio HPLC (Waters

m

Bondapak, C18 10

m

m 125A, 250 ꢂ 4.6 mm, 10 mM ammonium hydrogen carbonate pH ¼ 7.4/MeCN, 30/70, v/

v, 1.5 mL min^ꢁ1).

After preparative HPLC puriﬁcation (Fig. S1, Supp. Info.), and

a manner analogues to the synthesis of [¹¹C]21 and [¹¹C]23. First, 2-

bromoethyl tosylate 33 was synthesized by tosylation of 2-

bromoethanol in dichloromethane in 31% yield. After nucleophilic

substitution with ﬂuorine-18 of 33 in acetonitrile, [¹⁸F]34 was

distilled from the reaction mixture through a heated silver triﬂate/

Graphpac column, using a stream of helium, Intermediate [¹⁸F]34

was converted online into the more reactive triﬂate intermediate

formulation into

a sterile solution, [

¹¹C]21 was obtained in

30.7 12.8% (n ¼ 14) radiochemical yield, DC, calculated from [¹¹C]

CH₃I. The radiochemical purity was higher than 96.1% and speciﬁc

activity was 105 38.8 GBq m

mol^ꢁ1at end of synthesis. The total

synthesis time of [¹¹C]21 was 22 min.

[

¹⁸F]35 [37]. Synthon [¹⁸F]35 was trapped in the next reaction

2.3.2. Synthesis of [¹¹C]23

Given the optimal radiolabeling conditions for [¹¹C]21, only time

and temperature for the radiosynthesis of [¹¹C]23 were investigated

(Scheme 6). Table 3 summarizes radiochemical yields and illus-

trates that optimal conditions were identical to those used for the

synthesis of [¹¹C]21.

vessel containing precursor 21 in 33 13% yield DC. After complete

trapping of [¹⁸F]35, the coupling reaction was carried out (Scheme

7). For this reaction several reaction conditions were explored. As

solvents acetonitrile, DMF or DMSO were used, and as bases po-

tassium carbonate, sodium hydride or tetra-(n-butyl)ammonium

hydroxide. The reaction temperature was between 100 and 140 ^ꢀC,

and reaction times between 10 and 20 min. The best conditions,

After preparative HPLC puriﬁcation (Fig. S2, Supp. Info.), and

formulation in a sterile solution, [¹¹C]23 was obtained in 35.6 4.1%

(n ¼ 6) radiochemical yield, DC, calculated from [¹¹C]CH₃I. The

radiochemical purity was higher than 95.4% and speciﬁc activity

0.5 mg (1.4

100 ^ꢀC for 10 min, resulted in a radiochemical yield of 7.2 3.4%

m,

mmol) precursor 21, 300 mL acetonitrile, no base at

was 201 57.0 GBq m

mol^ꢁ1at end of synthesis. The total synthesis

yield (N ¼ 11, determined on analytical HPLC, Alltima C18, 5

m

time of [¹¹C]23 bombardment was 22 min.

250 ꢂ 4.6 mm, 20 mM potassium hydrogen phosphate pH ¼ 6.7/

methanol/acetonitrile, 20/20/60, v/v/v, 1.0 mL min^ꢁ1). After semi

preparative HPLC puriﬁcation (Reprosphere 100-C18-DE, 5

mm,

2.3.3. Synthesis of [¹⁸F]26

10 mM ammonium hydrogen carbonate pH ¼ 7.4/acetonitrile, 30/

70, v/v, 4 mL min^ꢁ1), the overall yield was less than 3% (n ¼ 6, DC)

with a radiochemical purity of 84e99%.

Initially, compound 26 (Scheme 7) was supposed to be radio-

labeled using [¹⁸F]ﬂuoroethyltriﬂate ([¹⁸F]35) as labeling synthon in

As the synthesis of [¹⁸F]26 did not proceed well using the 2-step

alkylation reaction, precursor 38 was synthesized. The tosyl pre-

cursor was synthesized in 4 steps starting from intermediate 24

(Scheme 8). First, the hydroxyethyl cyanamide 36 was prepared by

alkylation of 24 with bromoethanol in 43% yield, which subse-

quently was condensed to guanidine 37 in 52% yield. The tosyl

leaving group was introduced using standard conditions in 24%

yield.

Table 3

Radiolabeling optimization of [¹¹C]23.

Entry^a

Labeling agent

Solvent^b

Time^c

Temp^d

Yield^e,f

1

2

3

4

5

CH₃OTf

MeCN

1

3

5

30

40

60

40

33.1

38.3

34.3

32.1

35.4

13.6

9.01

10.6

13.0

5.49

Using standard labeling conditions only a limited number of

optimizations of the synthesis of [¹⁸F]26 was performed (Table 4).

The nucleophilic ﬂuorination reaction was carried out using Kryp-

toﬁx 2.2.2 and potassium carbonate in acetonitrile or DMSO. Vari-

ations were made in temperature (80, 100 or 120 ^ꢀC) and time (10,

20 and 30 min). Radiolabeling yield was determined using radio

TLC. Labeling yield in DMSO was relatively low, below 10%. Using

a

Used 0.5e0.9 mg (1.5e2.7 mmol) of precursor (14).

300 mL.

Minutes.

^ꢀC.

Entry 2: n ¼ 4, entries 1, 3e5 n ¼ 2 presented as mean SD.

Radiochemical yield determined using radio HPLC (Alltima C18,

b

c

d

e

f

5

mm,

250 ꢂ 4.6 mm, 20 mM potassium hydrogen phosphate pH ¼ 6.7/MeOH/MeCN, 20/

20/60, v/v/v, 1.0 mL min^ꢁ1).

148

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

Scheme 7. Synthesis of ﬂuorine-18 labeled compound [¹⁸F]26. Reagents and conditions: a) p-toluenesulfonyl chloride, DCM/pyridine, 0 ^ꢀC / RT, 18 h; b) K₂CO₃, Kryptoﬁx, ¹⁸F^ꢁ,

100 ^ꢀC, 10 min; c) silver triﬂate/Graphpac, 200 ^ꢀC, helium ﬂow; d) [¹⁸F]35, acetonitrile, 100 ^ꢀC, 10 min.

Scheme 8. Synthesis and ﬂuorination of tosylate precursor 38. Reagents and conditions: a) DIPEA, KI, Br(CH₂)OH, DMF/THF, 125 ^ꢀC, 6 h; b) 2-chloro-5-(methylthio)aniline hy-

drochloride, toluene, 140^ꢀ, 8 h; c) Et₃N, DMAP, p-toluenesulfonyl chloride, DCM, RT, 21 h; d) K₂CO₃, Kryptoﬁx, MeCN, ¹⁸F^ꢁ, 80 ^ꢀC, 30 min.

Table 4

octanol [38]. The Log D_oct,,7.4values are shown in Table 5.

Although measured Log D_oct,7.4values were not in the optimal

range of 2.0e3.5 [39], biodistribution studies showed that all li-

Radiolabeling optimization of [¹⁸F]26.

Entry^a

Solvent^b

Time^c

Yield^d

gands crossed the blood brain barrier.

Temperature^e

80

100

120

2.5. Metabolite analysis

1

2

3

4

5

6

DMSO

MeCN

10

20

30

10

20

30

8

3

7

29

36

40

8

9

31

35

0

39

43

37

It is important to characterize the metabolic proﬁle of a PET

tracer, as radiolabeled metabolites might interfere with quantiﬁ-

cation of a PET study, especially if those metabolites cross the

bloodebrain barrier [40].

a

2.5 mg (4.7 mmol) of precursor (38) used.

Metabolites were determined in awake male Wistar rats at 15

and 45 min after injection. As potential metabolic pathway de-

alkylation of S-methyl, guanidine N-methyl and aniline N-methyl

moieties was expected. De-alkylation of the carbon-11 radiolabeled

moiety (compound [¹¹C]21 and [¹¹C]23) would lead to radiolabeled

polar metabolites, which could interfere with PET quantiﬁcation,

and unlabeled non-polar metabolites, which do not interfere with

PET quantiﬁcation. These radiolabeled polar metabolites most

likely represents single carbon molecules such as formaldehyde,

formic acid or carbon dioxide, showing no retention on HPLC. In

case of [¹⁸F]26, the polar metabolite fraction most likely represent

the ﬂuoroethyl residue.

b

c

500

Minutes.

mL.

d

Radiochemical yield in %, determined using radio TLC (dichloromethane/

methanol 95/5, v/v); n ¼ 1.

e

^ꢀC.

acetonitrile, however, the yield increased to 43% in 20 min at

120 ^ꢀC. To prevent possible pressure buildup in the reaction vial at

higher temperatures, 30 min at 80 ^ꢀC were used.

After preparative HPLC puriﬁcation (Fig. S3, Supp. Info.), and

formulating into

8.7 1.7% (n ¼ 5) radiochemical yield, DC. The radiochemical purity

a sterile solution, [

¹⁸F]26 was obtained in

Demethylation of the non radiolabeled moieties, however,

was 96.3 2.3% and speciﬁc activity was 78.2 32.0 GBq m

mol^ꢁ1at

end of synthesis. The total synthesis time of [¹⁸F]26 was 90 min.

Table 5

Measured Log D.

2.4. Determination of lipophilicity expressed as Log D_oct,7.4of 21, 23

and 26

a

Compound

Log D_oct,7.4

[

¹¹C]21

¹¹C]23

¹⁸F]26

1.62 0.01

1.24 0.00

1.51 0.00

The distribution coefﬁcients of 21, 23 and 26 were measured

using their radiolabeled analogues. The lipophilicity was deter-

mined using a mixture of phosphate buffer at pH ¼ 7.4 and 1-

a

n ¼ 3 presented as mean SD.

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

149

would lead to radiolabeled metabolites that could interfere with

PET imaging, as they may cross the bloodebrain barrier and bind to

the same or other molecular targets. Non-polar metabolites

represent, besides the parent compound, the fractions showing

retention on HPLC. Fig. 2 shows the metabolic proﬁles of [¹¹C]21,

15 min after injection of [¹⁸F]26, 39% of the activity in the brain

was due to parent compound. It should be noted that the fraction of

radiolabeled non-polar metabolites in the brain was very low,

whilst the fraction of polar metabolites was high. Most likely this is

due to fast removal of the ﬂuoroethyl moiety, which is capable of

entering the brain [41].

[

¹¹C]23 and [¹⁸F]26 as function of time.

45 min after injection of [¹¹C]21, 19% of measured radioactivity

in plasma could be attributed to parent compound, and in the brain

this was 33%. The decrease in parent fraction in the brain from 62 to

33% between 15 and 45 min indicates fast metabolic degradation.

HPLC analysis revealed, next to parent compound, three radio-

labeled non-polar metabolites, most likely structural analogs of

2.6. Ex vivo biodistribution

Ex vivo biodistribution and regional brain uptake of [¹¹C]21, [¹¹C]

23 and [¹⁸F]26 were determined in male Wistar rats at 5, 15, 30 and

60 min after injection of the radiotracer. As anesthesia would

interfere with binding to the NMDAR, these biodistribution studies

were performed without anesthesia. Regional brain uptake as well

as organ uptake are depicted in Fig. 3.

[

¹¹C]21. Indeed one of these was identiﬁed on HPLC as [¹¹C]23.

Interestingly, for [¹¹C]23 a similar metabolic proﬁle was observed as

for [¹¹C]21, the main difference being a slightly higher parent

fraction at 45 min (45%).

Compared with all other time points, brain uptake of [¹¹C]21 was

Fig. 2. Fractions of radiolabeled polar metabolites, non-polar radiolabeled metabolites and parent compound of [¹¹C]21 (top), [¹¹C]23 (middle) and [¹⁸F]26 (bottom) in plasma (left

side) and brain (right side) at 15 and 45 min; n ¼ 3 for each data point; error bars indicate SD.

150

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

highest at 5 min after injection (p < 0.05, Tukey-test). Regionally,

uptake was the lowest in cerebellum, and the highest ratio of

radioactivity uptake between forebrain regions and cerebellum was

observed at 15 min post injection. Two way ANOVA conﬁrmed

signiﬁcant main effects of region [F_(4,60)¼ 9.41, p < 0.0001] and

time [F_(3,60)¼ 106.09, p < 0.0001] on brain uptake of [¹¹C]21.

Organ uptake of [¹¹C]21 was highest in lungs followed by kid-

neys. Uptake in lungs was signiﬁcantly higher than in other organs,

except for kidney (15 min) and liver (30 min). The lowest levels of

radioactivity were observed in blood and bone. Radioactivity

cleared from most organs, but liver showed increasing uptake up to

45 min, which could be an indication of metabolism. Two way

ANOVA conﬁrmed signiﬁcant main effects of region [F_(5,72)¼ 72.78,

p < 0.0001] and time [F_(3,72)¼ 14.12, p < 0.0001], as well as sig-

niﬁcant organ ꢂ time interaction effects [F_(15,72)¼ 6.33, p < 0.0001].

Brain uptake of [¹¹C]23 was also highest at 5 min (p < 0.001,

Tukey-test). In addition, similar to [¹¹C]21, uptake of [¹¹C]23 was

lowest in cerebellum, and the highest ratio of radioactivity uptake

between forebrain regions and cerebellum was found at 15 min

post injection. Radioactivity cleared rapidly from the brain. Two

way ANOVA conﬁrmed signiﬁcant main effects of region

[F_(4,60)¼ 24.11, p < 0.0001] and time [F_(3,60)¼ 696.02, p < 0.0001] as

well as signiﬁcant region ꢂ time interaction effects [F_(12,60)¼ 5.25,

p < 0.0001].

Organ uptake of [¹¹C]23 was highest in lungs followed by kid-

neys. The lowest level of radioactivity uptake was observed in

blood. Remarkably, for a carbon-11 labeled tracer, uptake and

washout over time was observed in bone. In addition, similar to the

pattern seen for [¹¹C]21, the liver did not show a decrease of

radioactivity levels over time. Two way ANOVA conﬁrmed signiﬁ-

cant main effects of region [F_(5,72)¼ 48.96, p < 0.0001] and time

[F_(3,72)¼ 18.04, p < 0.0001], as well as signiﬁcant organ ꢂ time

interaction effects [F_(15,72)¼ 7.39, p < 0.0001] on uptake of [¹¹C]23.

Uptake of [¹⁸F]26 was uniformly distributed across the brain

with a signiﬁcant higher level of radioactivity at 15 min than at

30 min (p < 0.05, Tukey-test). Indeed, washout of radioactivity from

the brain over time was not observed.

Organ uptake of [¹⁸F]26 was highest in lungs followed by

Fig. 3. Biodistribution of [¹¹C]21, [¹¹C]23 and [¹⁸F]26 in CNS (left) and organs (right) represented as % ID/g; n ¼ 4 for each data point; error bars indicate SD.

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

151

kidneys. At all time points, lung uptake was signiﬁcantly higher

than in other organs (p < 0.001). Relatively high blood values were

seen with increasing levels over time. The liver did not show a

decrease over time and bone uptake was observed. These obser-

vations may be indicative of metabolism and in vivo deﬂuorination

of [¹⁸F]26, although the latter is unlikely because bone uptake did

not increase over time.

analysis system consisted of a Jasco PU-2089 HPLC pump, a Rheo-

dyne injector with a 20- L loop, a Jasco UV-2075 Plus UV detector

set at a wavelength of 254 nm and a Raytest Na(I) radioactivity

detector. The HPLC data were collected and integrated with the

software package GINA 5.01. HPLC: System A: Platinum EPS, C18

m

100A 5

m

, 250 ꢂ 4.6 mm column using 50 mM NH₄H₂PO₄pH ¼ 2.5/

methanol (30/70, v/v) as eluent and a ﬂow rate of 1 mL min^ꢁ1; For

high-resolution mass spectrometry, a Finnigan MAT 90 was used.

2.7. Pre-treatment studies

[ a)

¹¹C]CO₂was produced by the ¹⁴N(p, ¹¹C nuclear reaction and [¹⁸F]

F^ꢁwas produced by the ¹⁸O(p,n)¹⁸F nuclear reaction with an IBA

Cyclone 18/9 cyclotron. Radioactivity amounts were measured with

a Veenstra VDC-405 dose calibrator. Radiochemistry was carried

out in homemade, remotely controlled devices [48].

Speciﬁcity of uptake of the radiolabeled ligands [¹¹C]21, [¹¹C]23

and [¹⁸F]26 in the brain was determined by pre-treatment with the

NMDAR ion channel antagonist MK-801. Data from rodent studies

had shown that 50% occupancy of the NMDAR ion-channel by MK-

801 could be obtained using a dose of 0.18 mg kg^ꢁ1[42,43]. In the

present study, 0.6 mg kg^ꢁ1MK-801 was used, which is at least three

times the in vivo EC₅₀value and therefore should occupy the ma-

jority of NMDARs. MK-801 was injected intraperitoneally 15 min

prior to administration of the radiolabeled ligand.

4.2. General procedure for the synthesis of the di- or tri-N-

substituted guanidines (A)

In a screw cap reaction vessel were dissolved the appropriately

substituted cyanamide (1 mmol) and amine halogen salt (1.1 mmol)

No decrease in brain-uptake was observed for any of the three

ligands (Fig. 4). In fact, in all brain areas uptake of the radiolabeled

ligands increased. For [¹¹C]23, this increase in uptake was signiﬁ-

cant (Two way ANOVA, Bonferroni posttests, p < 0.01) in hippo-

campus, prefrontal cortex and cerebral cortex.

The reason for this higher uptake is unclear. One explanation

could be that there is release of glutamate from the brain after MK-

801 administration [44,45]. Since the uptake of ‘use-dependent’

antagonists is thought to be related to the amount of glutamate in

the brain [46], it could be that injection of MK-801 increased the

release of glutamate, opening more NMDAR ion channels, and

resulting in higher uptake of [¹¹C]21, [¹¹C]23 and [¹⁸F]26 than at

baseline. In addition, it is possible that blood ﬂow increased after

the administration of MK-801, leading to increased delivery of the

tested radioligands in the brain compared with the baseline con-

dition [47].

in chlorobenzene (200 mL). The reaction vessel was ﬂushed with

nitrogen, closed and stirred at 165 ^ꢀC for 4e18 h. The reaction

mixture was cooled down and dissolved in ethyl acetate (25 mL)

and washed with 0.1 M HCl (2 ꢂ 25 mL) followed by water (25 mL).

The pH of the combined aqueous layers were adjusted with K₂CO₃

to pH ꢃ 10 and extracted with ethyl acetate (2 ꢂ 25 mL). The

organic layers were collected and dried over anhydrous magnesium

sulfate, ﬁltrated and evaporated to dryness under reduced pressure.

The crude compound was puriﬁed by column chromatography. Oily

products were converted into the corresponding fumaric acid salt

by stirring the oil in diethyl ether and a solution of fumaric acid in

diethyl ether was added dropwise. The resulting precipitate was

ﬁltered off.

4.2.1. N⁰-Hydroxy-N-(pyridin-2-yl)formimidamide (5a)

To a stirred solution of 2-aminopyridine (4a) (5.48 g, 58.2 mmol)

in 2-propanol (20 mL) DMFeDMA (10 mL, 75 mmol) was added.

The reaction mixture was reﬂuxed for 2 h under a nitrogen atmo-

sphere and monitored by TLC (ethyl acetate). On completion, the

reaction mixture was cooled to 50 ^ꢀC and hydroxylamine hydro-

chloride (5.3 g, 76 mmol) was added and stirring was continued

overnight. The solvents were evaporated under reduced pressure

and the yellow syrup was treated with ethyl acetate (50 mL). The

solids were ﬁltered and the ﬁltrate was washed with 1 M NaOH

(50 mL) and ethyl acetate (4 ꢂ 25 mL). The combined organic

fractions were collected, dried over anhydrous magnesium sulfate,

ﬁltrated and evaporated to dryness under reduced pressure. The

residue was recrystallized from chloroform to obtain the title

compound as white crystals (4.62 g, 33.7 mmol, 58%). R_f0.67 (ethyl

3. Conclusion

Alkylamine substitution of 1 and 2 at either methoxy or thio-

methyl moieties led to three new ligands (21, 23 and 26) with

favorable K_ivalues towards the ion channel of the NMDAR. These

ligands were radiolabeled and evaluated using ex vivo bio-

distribution studies in awake rats, showing good uptake in the

brain. However, following pre-treatment with MK-801, none of the

ligands tested showed reduced uptake. In addition, all three ligands

suffered from fast metabolism. Despite the high afﬁnity for the ion

channel of the NMDAR, the radiolabeled ligands did not show

appropriate characteristics for use as a PET tracer.

acetate/methanol/Et₃N, 90/10/1, v/v/v). ¹H NMR (DMSO-d₆)

d 10.06

4. Experimental

(bs, 1H, OH), 9.30 (d, J ¼ 9.51 Hz, 1H, NH), 8.14e8.11 (m, 1H, H_Aryl),

7.87 (d, J ¼ 9.49 Hz, 1H, CH), 7.61e7.54 (m, 1H, H_Aryl), 7.05 (d,

J ¼ 8.31 Hz, 1H, H_Aryl), 6.84e6.79 (m, 1H, H_Aryl). ¹³C NMR (DMSO-d₆)

4.1. Chemistry

d

152.62 (AreN), 147.50 (Ar), 138.08 (Ar), 135.73 (CH), 116.29 (Ar),

Nuclear magnetic resonance (NMR) spectra (¹H, ¹³C, ¹⁹F NMR)

were recorded on a Bruker Avance 250 (250.13, 62.90, 235.36 MHz

respectively). The chemical shifts of the NMR spectra are reported

in parts per million (ppm) relative to the solvent residual peak.

Description of signals: s ¼ singlet, bs ¼ broad singlet, d ¼ doublet,

t ¼ triplet, q ¼ quartet, m ¼ multiplet, dd ¼ double doublet,

ddd ¼ doublet of dd, dt ¼ doublet of triplets, tt ¼ triplet of triplets,

dq ¼ double quartet. Thin-layer chromatography (TLC) was per-

formed on Merck DC-alufolien, silica gel 60, F254. Flash column

chromatography was performed on silica gel 60 Å, 230e400 Mesh.

All solids obtained were dried in vacuo. All chemicals were used

without further puriﬁcation, unless stated otherwise. The HPLC

110.40 (Ar).

4.2.2. N⁰-Hydroxy-N-(pyridin-3-yl)formimidamide (5b)

To a stirred solution of 3-aminopyridine (4b) (5.47 g, 58.2 mmol)

in 2-propanol (20 mL) DMF-DMA (10 mL, 75 mmol) was added. The

reaction mixture was reﬂuxed for 3 h under a nitrogen atmosphere

and monitored by TLC (ethyl acetate/methanol 9/1, v/v). On

completion, the reaction mixture was cooled to 75 ^ꢀC and hy-

droxylamine hydrochloride (5.3 g, 76 mmol) was added and stirring

was continued for 2 h. While evaporating under reduced pressure

precipitation occurred. The yellow crystals were washed with 2-

propanol and ﬁltrated to obtain the title compound as light

152

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

Fig. 4. Control and pretreatment (15 min, MK-801 at 0.6 mg kg^ꢁ1) biodistribution of [¹¹C]21, [¹¹C]23 and [¹⁸F]26 in CNS and organs (represented as %ID/g (n ¼ 4 SD); **: p < 0.01).

yellow crystals (4.18 g, 30.5 mmol, 52%). R_f0.50 (ethyl acetate/

methanol, 90/10, v/v). ¹H NMR (DMSO-d₆)

10.01 (bs, 1H, OH), 8.80

(bs, 1H, OH), 9.62 (d, J ¼ 10.03 Hz, 1H, NH), 8.32 (d, J ¼ 6.59 Hz, 2H,

d

H

_Aryl), 7.71 (d, J ¼ 9.79 Hz,1H, CH), 7.31 (d, J ¼ 6.66 Hz, 2H, H_Aryl). ¹³

C

(d, J ¼ 10.60 Hz, 1H, NH), 8.49 (d, J ¼ 2.53 Hz, 1H, H_Aryl), 8.06 (dd,

NMR (DMSO-d₆)

109.57 (2ꢂ Ar).

d

149.28 (AreN), 147.13 (2ꢂ Ar), 135.79 (CH),

J ¼ 4.60, 0.82 Hz, 1H, H_Aryl), 7.57e7.50 (m, 2H, H_Arylþ CH), 7.21 (dd,

J ¼ 8.30, 4.65 Hz,1H, H_Aryl). ¹³C NMR (DMSO-d₆)

d 141.42 (Ar),137.52

(AreN), 137.40 (CH), 137.38 (Ar), 123.73 (Ar), 120.83 (Ar).

4.2.3. N⁰-Hydroxy-N-(pyridin-4-yl)formimidamide (5c)

4.2.4. N-Methyl-N-(pyridin-2-yl)cyanamide (6a)

To

stirred solution of N⁰-hydroxy-N-(pyridin-2-yl)for-

To a stirred solution of 4-aminopyridine (4c) (5.48 g, 58.2 mmol)

in 2-propanol (20 mL) DMFeDMA (10 mL, 75 mmol) was added.

The reaction mixture was reﬂuxed for 2 h under a nitrogen atmo-

sphere and monitored by TLC (ethyl acetate/methanol/Et₃N 90/10/

1, v/v/v). On completion, the reaction mixture was cooled to 50 ^ꢀC

and hydroxylamine hydrochloride (5.3 g, 76 mmol) was added and

stirring was continued for 3 h at 75 ^ꢀC. While evaporating under

reduced pressure precipitation occurred. The yellow crystals were

washed with 2-propanol and ﬁltrated to obtain the title compound

as light tanned crystals (5.67 g, 41.3 mmol, 71%). R_f0.33 (ethyl ac-

a

mimidamide (5a) (686 mg, 5.00 mmol) in toluene (13 mL) was

added DMF-DMA (1.65 mL, 12.3 mmol) and reﬂuxed for 2.5 h. The

solvents were evaporated and the yellow residue solidiﬁed on

standing which was triturated with toluene. The yellow needles

were collected to obtain the title compound (198 mg, 1.49 mmol,

30%). R_f0.28 (ethyl acetate). ¹H NMR (CDCl₃)

d

8.01 (dd, J ¼ 6.61 Hz,

1.35 Hz, 1H, H_Aryl), 7.76e7.69 (m, 1H, H_Aryl), 7.12 (d, J ¼ 8.76 Hz, 1H,

H

_Aryl),.67 (dt, J ¼ 6.77 Hz, 1.25 Hz, 1H, H_Aryl), 3.61 (s, 3H, CH₃). ¹³

C

NMR (CDCl₃) d 160.94 (AreN), 141.07 (Ar), 140.97 (Ar), 117.98 (NCN),

etate/methanol/Et₃N, 90/10/1, v/v/v). ¹H NMR (DMSO-d₆)

d

10.51

116.68 (Ar),47 (Ar), (CH₃).

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

153

4.2.5. N-Methyl-N-(pyridin-3-yl)cyanamide (6b)

To

stirred solution of N⁰-hydroxy-N-(pyridin-3-yl)for-

100 mmol), a catalytic amount of p-toluenesulfonic acid mono-

hydrate (0.29 g, 1.5 mmol) in toluene (350 mL) was stirred at

elevated temperature. After 5 min hexane-2,5-dione (12.5 mL,

107 mmol) was added via an addition funnel and reﬂuxed under

nitrogen atmosphere for 75 min. After cooling the reaction was

quenched with sat. aq. NaHCO₃solution (100 mL) and separated.

The organic layer was washed with water (100 mL), 0.1 M NaOH

(2ꢂ 50 mL), 0.1 M HCl (2 ꢂ 50 mL), water (50 mL) and brine (50 mL).

The organic fraction was collected, dried over anhydrous magne-

sium sulfate, ﬁltrated and evaporated to dryness under reduced

pressure. On cooling the brown oil crystallized to obtain the title

compound (21.7 g, 100 mmol, 100%). R_f0.84 (ethyl acetate/hexanes,

a

mimidamide (5b) (1.37 g, 10.0 mmol) in toluene (25 mL) was added

DMF-DMA (3.3 mL, 25 mmol) and reﬂuxed for 35 min. On cooling

crystals were formed. After ﬁltrating, the title compound was ob-

tained as brown solid (426 mg, 3.20 mmol, 32%). R_f0.18 (ethyl ac-

etate/methanol, 90/10, v/v). ¹H NMR (DMSO-d₆)

d

7.91 (s, 1H, H_Aryl),

7.79 (d, J ¼ 5.06 Hz, 1H, H_Aryl), 7.53e7.42 (m, 2H, H_Aryl), 4.11 (s, 3H,

CH₃). ¹³C NMR (DMSO-d₆)

156.22 (AreN), 134.08 (Ar), 130.57 (Ar),

d

129.18 (Ar), 126.95 (Ar), 121.92 (NCN), 47.06 (NCH₃).

4.2.6. N-Methyl-N-(pyridin-4-yl)cyanamide (6c)

To

a

stirred solution of N⁰-hydroxy-N-(pyridin-4-yl)for-

33/66, v/v). ¹H NMR (CDCl₃)

d

8.28 (ddd, J ¼ 8.12 Hz, 2.22 Hz,

mimidamide (5c). (1.37 g, 10.0 mmol) in toluene (25 mL) was added

DMF-DMA (3.3 mL, 25 mmol) and reﬂuxed for 30 min. On cooling

white crystals were formed. After ﬁltrating, the title compound was

obtained as white needles (809 mg, 6.08 mmol, 61%). R_f0.15 (ethyl

1.21 Hz, 1H, H_Aryl), 8.12 (t, J ¼ 2.01 Hz, 1H, H_Aryl), 7.68 (t, J ¼ 7.92 Hz,

1H, H_Aryl), 7.60e7.56 (m, 1H, H_Aryl), 5.95 (s, 2H, pyrrole), 2.07 (s, 6H,

CH₃). ¹³C NMR (CDCl₃)

d 148.80 (AreNO₂), 140.35 (Arepyrrole),

134.38 (Ar), 130.07 (Ar), 128.75 (pyrroleeCH₃), 123.39 (Ar), 122.62

(Ar), 107.11 (pyrroleeCH), 13.15 (CH₃).

acetate/methanol/Et₃N, 90/10/1, v/v/v). ¹H NMR (DMSO-d₆)

d 7.88

(d, J ¼ 7.32 Hz, 2H, H_Aryl), 6.69 (bs, 2H, H_Aryl), 3.78 (s, 3H, NCH₃). ¹³

C

NMR (DMSO-d₆)

d

167.28 (AreN), 142.13 (2ꢂ Ar), 119.71 (NCN),

4.2.10. 3-(2,5-Dimethyl-1H-pyrrol-1-yl)aniline (10)

114.40 (2ꢂ Ar), 43.68 (NCH₃).

To a reﬂuxing solution of 2,5-dimethyl-1-(3-nitrophenyl)-1H-

pyrrole (9) (5.00 g, 23.1 mmol) and acetic acid (6.5 mL,114 mmol) in

ethanol/water (195 mL, 2/1, v/v) was added powdered iron (5.2 g,

93 mmol) portion wise. The reaction mixture was reﬂuxed for

18 min, when TLC showed completion. After cooling the mixture

was basiﬁed with 25% NH₄OH and ﬁltered through celite. The ﬁlter

cake was washed with ethanol and the ﬁltrate was concentrated

under reduced pressure. The residue was puriﬁed by column

chromatography over silica with dichloromethane to obtain the

title compound as a light brown solid (6.97 g, 21.3 mmol, 92%). R_f

4.2.7. 3-(2-Chloro-5-(methylthio)phenyl)-1-methyl-1-(pyridin-2-

yl)guanidine (7a)

The reaction of N-methyl-N-(pyridin-2-yl)cyanamide (6a)

(135 mg, 1.01 mmol) with 2-chloro-5-(methylthio)aniline hydro-

chloride (233 mg, 1.11 mmol) according to procedure A afforded,

after puriﬁcation over silica with methanol/chloroform/Et₃N (50/

50/1, v/v/v), the title compound as a light yellow glassy solid

(105 mg, 0.34 mmol, 34%). R_f0.09 (methanol/chloroform/Et₃N, 50/

50/1, v/v/v). ¹H NMR (CDCl₃)

d

7.79 (bs, 1H, H_Aryl), 7.30e7.16 (m, 3H,

_Aryl), 6.97 (d, J ¼ 2.26 Hz, 1H, H_Aryl), 6.79 (dd, J ¼ 8.35, 2.28 Hz, 1H,

_Aryl), 6.08 (dt, J ¼ 6.66, 1.19 Hz, 1H, H_Aryl), 4.71 (bs, 2H, NH), 3.53 (s,

179.29 (AreNCH₃),

0.48 (ethyl acetate/hexanes, 25/75, v/v). ¹H NMR (CDCl₃)

1H, H_Aryl, J ¼ 7.94 Hz), 6.72 (ddd, 1H, H_Aryl, J ¼ 8.06, 2.30, 0.92 Hz),

6.62 (ddd, 1H, H_Aryl, J ¼ 7.73, 1.85, 0.94), 6.53 (t, 1H, H_Aryl

J ¼ 2.06 Hz), 5.91 (s, 2H, CHepyrrole), 3.78 (bs, 2H, NH₂), 2.08 (s, 6H,

CH₃). ¹³C NMR (CDCl₃)

147.21 (AreNH₂), 140.13 (Arepyrrole),

d 7.24 (t,

H

,

3H, NCH₃), 2.42 (s, 3H, NCH₃). ¹³C NMR (CDCl₃)

d

156.87 (NCN), 156.09 (AreNH), 138.28 (Ar), 137.24 (AreS), 137.04

(Ar), 129.79 (Ar), 124.44 (AreCl), 122.26 (Ar), 121.16 (Ar), 118.10 (Ar),

106.62 (Ar), 40.46 (NCH₃), 15.97 (SCH₃). The free base was con-

verted into its fumaric acid salt (116 mg, 0.31 mmol, 92%). ¹H NMR

d

129.81 (Ar), 128.85 (Cepyrrole), 118.49 (Ar), 114.91 (Ar), 114.40 (Ar),

105.49 (CHepyrrole), 13.05 (CH₃).

(DMSO-d₆)

d

7.88 (d, J ¼ 6.47 Hz, 1H, H_Aryl), 7.56e7.49 (m, 1H, H_Aryl),

4.2.11. N-(3-(2,5-Dimethyl-1H-pyrrol-1-yl)phenyl)cyanamide (11)

To a stirred mixture of 3-(2,5-dimethyl-1H-pyrrol-1-yl)aniline

(10) (3.88 g, 20.8 mmol) and anhydrous sodium acetate (5.19 g,

63.3 mmol) in methanol (35 mL) at 0^ꢀwas added a solution of

cyanogen bromide [CAUTION highly toxic] (2.88 g, 27.2 mmol) in

methanol (35 mL) via an addition funnel. After 2 h of stirring the

ice-bath was removed and stirring was continued for 24 h. The

reaction mixture was diluted with dichloromethane (150 mL) and

washed with 1 M HCl (150 mL). The aqueous layer was washed with

dichloromethane (150 mL). The combined organic layer was

washed with brine (75 mL), dried over anhydrous magnesium

sulfate, ﬁltered and evaporated to dryness. The crude product was

puriﬁed with ﬂash column chromatography (dichloromethane) to

obtain the title compound as light brown solid (3.35 g, 15.9 mmol,

76%). R_f0.37 (ethyl acetate/hexanes, 25/75, v/v). ¹H NMR (CDCl₃)

7.41 (bs, 2H, NH), 7.30 (d, J ¼ 8.40 Hz, 1H, H_Aryl), 7.16 (d, 8.77 Hz, 1H,

H

_Aryl), 7.07 (d, J ¼ 2.08 Hz, 1H, H_Aryl), 6.91 (dd, J ¼ 8.41, 2.23 Hz, 1H,

_Aryl), 6.48 (t, J ¼ 6.57 Hz, 1H, H_Aryl), 6.35 (s, 0.88H, fumaric acid),

H

3.48 (s, 3H, NCH₃), 2.39 (s, 3H, SCH₃). HRMS [M þ H]^þcalcd for

C

₁₄H₁₅ClN₄S 307.0779, found 307.0778. HPLC system A: purity

99.2%, rt ¼ 6.22 min.

4.2.8. 3-(2-Chloro-5-(methylthio)phenyl)-1-methyl-1-(pyridin-4-

yl)guanidine (7c)

The reaction of N-methyl-N-(pyridin-4-yl)cyanamide (6c)

(134 mg, 1.01 mmol) with 2-chloro-5-(methylthio)aniline hydro-

chloride (232 mg, 1.10 mmol) according to procedure A afforded,

after recrystallization from chloroform/hexanes the title compound

as light brown crystals (121 mg, 0.39 mmol, 39%). R_f0.08 (meth-

anol/chloroform/Et₃N, 50/50/1, v/v/v). ¹H NMR (DMSO-d₆)

d

7.41 (d,

d

7.44 (t, J ¼ 8.01 Hz, 1H, H_Aryl), 7.08e7.04 (m, 1H, H_Aryl), 6.98e6.94

(m, 1H, H_Aryl), 6.86 (t, J ¼ 2.04 Hz, 1H, H_Aryl), 5.91 (s, 2H, CHepyr-

role), 2.04 (s, 6H, CH₃). ¹³C NMR (CDCl₃)

140.37 (Arepyrrole),

J ¼ 7.59 Hz, 2H, H_Aryl), 7.17 (d, J ¼ 8.29 Hz, 1H, H_Aryl), 6.74 (d,

J ¼ 2.28 Hz, 1H, H_Aryl), 6.64 (dd, J ¼ 8.30, 2.34 Hz, 1H, H_Aryl), 6.49 (d,

J ¼ 7.12 Hz, 2H, H_Aryl), 5.63 (bs, 2H, NH), 3.53 (s, 3H, NCH₃), 2.34 (s,

d

138.37 (AreNHCN), 130.49 (Ar), 129.00 (Cepyrrole), 123.44 (Ar),

115.37 (Ar), 114.76 (Ar), 111.19 (NCN), 106.14 (CHepyrrole), 13.07

(CH₃).

3H, SCH₃). ¹³C NMR (DMSO-d₆)

d 158.43 (AreNCH₃), 157.68 (NCN),

148.42 (AreNH), 139.65 (Ar), 136.05 (AreS), 129.13 (Ar), 123.33

(AreCl), 121.42 (Ar), 118.84 (Ar), 112.38 (Ar), 42.39 (NCH₃), 14.99

(SCH₃). HRMS [M þ H]^þcalcd for C₁₄H₁₅ClN₄S 307.0779, found

307.0779. HPLC system A: purity 99.0%, rt ¼ 6.22 min.

4.2.12. N-(3-(2,5-Dimethyl-1H-pyrrol-1-yl)phenyl)-N-

methylcyanamide (12)

To a stirred solution of N-(3-(2,5-dimethyl-1H-pyrrol-1-yl)

phenyl)cyanamide (11) (3.27 g, 15.5 mmol) in dry DMF (60 mL) was

added potassium carbonate (2.58 g, 18.6 mmol). After 5 min of

4.2.9. 2,5-Dimethyl-1-(3-nitrophenyl)-1H-pyrrole (9)

In a DeaneStark setup a solution of 3-nitroaniline (8) (13.9 g,

154

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

stirring methyl iodide (3 mL, 48 mmol) was added and stirring was

continued for 1.5 h. The solvent was evaporated under reduced

pressure and the residue was dissolved in water (125 mL) and

extracted with ethyl acetate (2 ꢂ 125 mL). The combined organic

layer was washed with brine (100 mL), collected and dried over

anhydrous magnesium sulfate, ﬁltered and evaporated to dryness

under reduced pressure. The crude product was puriﬁed with ﬂash

column chromatography (ethyl acetate/hexanes (25/75, v/v) to

obtain the title compound as a light brown solid (3.22 g, 14.3 mmol,

92%). R_f0.56 (ethyl acetate/hexanes, 25/75, v/v). ¹H NMR (CDCl₃)

4.2.15. N-(3-Nitrophenyl)cyanamide (15)

To a cooled suspension (0 ^ꢀC) of 3-nitroanline (8) (13.8 g,

100 mmol) in acetic acid/water (188 mL, 3/1, v/v) was added cyanic

bromide [CAUTION highly toxic] (16.0 g, 151 mmol). After 20 min

1 M NaOH (150 mL) was added over a period of 10 min, the reaction

was warmed to room temperature and stirred for 20 h. The sus-

pension was ﬁltrated and the ﬁlter cake was washed with ethanol

(1st crop). The ﬁltrate was evaporated under reduced pressure and

dissolved in 1 M NaOH (100 mL), 1 M HCl was added until pre-

cipitation occurred. The crystals were collected (2nd crop). Both

crops were combined and the title compound was obtained as light

gray crystals (15.1 g, 92.5 mmol, 93%). R_f0.80 (ethyl acetate/hex-

d

7.48 (t, J ¼ 8.04 Hz, 1H, H_Aryl), 7.19e7.14 (m, 1H, H_Aryl), 6.99e6.92

(m, 2H, H_Aryl), 5.91 (s, 2H, CHepyrrole), 3.36 (s, 3H, NCH₃), 2.04 (s,

6H, CH₃). ¹³C NMR (CDCl₃)

141.33 (AreNCH₃), 140.40 (Arepyrrole),

d

anes/Et₃N, 50/50/1, v/v/v). ¹H NMR (DMSO-d₆)

7.90e7.86 (m, 1H, H_Aryl), 7.71e7.61 (m, 2H, H_Aryl), 7.42e7.38 (m, 1H,

_Aryl). ¹³C NMR (DMSO-d₆)

148.57 (AreNO₂), 140.28 (AreNH),

d 10.86 (bs, 1H, NH),

130.24 (Ar), 128.64 (Cepyrrole), 123.11 (Ar), 114.67 (Ar), 114.17 (Ar),

113.50 (NCN), 106.15 (CHepyrrole), 36.93 (NCH₃), 13.00 (CH₃).

H

d

131.31 (Ar), 121.33 (Ar), 117.22 (Ar), 111.03 (NCN), 109.36 (Ar).

4.2.13. 3-(2-Chloro-5-(methylthio)phenyl)-1-(3-(2,5-dimethyl-1H-

pyrrol-1-yl)phenyl)-1-methylguanidine (13)

4.2.16. N-Methyl-N-(3-nitrophenyl)cyanamide (16)

The reaction of N-(3-(2,5-dimethyl-1H-pyrrol-1-yl)phenyl)-N-

methylcyanamide (12) (698 mg, 3.10 mmol) with 2-chloro-5-

(methylthio)aniline hydrochloride (717 mg, 3.41 mmol) according

to procedure A afforded, after puriﬁcation over silica with ethyl

acetate/hexanes (25/75 to 33/66, v/v), the title compound as a

colorless oil (569 mg, 1.43 mmol, 46%). R_f0.36 (dichloromethane/

To a mixture of N-(3-nitrophenyl)cyanamide (15) (12.3 g,

75.5 mmol) and potassium carbonate (13 g, 94 mmol) in DMF

(76 mL) was added methyl iodide (7.1 mL, 114 mmol) and stirred for

45 min at 45 ^ꢀC. The solvent was evaporated under reduced pres-

sure and the residue was dissolved in water (150 mL) and extracted

twice with ethyl acetate (125 mL). The combined organic layer was

washed with brine (100 mL), collected, dried over anhydrous

magnesium sulfate, ﬁltrated and evaporated to dryness under

vacuo. The crude product was puriﬁed by ﬂash column chroma-

tography (dichloromethane) to obtain the title compound as a light

yellow solid (12.1 g, 68.2 mmol, 90%). R_f0.62 (dichloromethane). ¹H

methanol, 50/50, v/v). ¹H NMR (CDCl₃)

_Aryl), 7.40 (m, 1H, H_Aryl), 7.29 (d, J ¼ 8.33 Hz, 1H, H_Aryl), 7.23 (t,

J ¼ 1.96 Hz, 1H, H_Aryl), 7.12 (m, 1H, H_Aryl), 6.93 (d, J ¼ 2.28 Hz, 1H,

_Aryl), 6.84 (dd, J ¼ 8.34, 2.32 Hz, 1H, H_Aryl), 5.93 (s, 2H, CHepyr-

role), 4.01 (bs, 2H, NH), 3.47 (s, 3H, NCH₃), 2.48 (s, 3H, SCH₃), 2.09 (s,

6H, CH₃). ¹³C NMR (CDCl₃)

150.65 (AreNCH₃), 147.20 (NCN),

d

7.51 (t, J ¼ 7.88 Hz, 1H,

H

d

NMR (CDCl₃)

_Aryl), 7.58 (t, J ¼ 8.09 Hz,1H, H_Aryl), 7.52e7.47 (m,1H, H_Aryl), 3.45 (s,

3H, NCH₃). ¹³C NMR (CDCl₃)

149.11 (AreNO₂), 141.83 (AreNH),

d

8.00e7.96 (m, 1H, H_Aryl), 7.87 (t, J ¼ 4.18 Hz, 1H,

145.35 (AreNH), 140.09 (AreNepyrrole), 137.83 (AreS), 130.08 (Ar),

130.05 (Ar), 128.52 (Cepyrrole), 126.28 (Ar), 125.81 (Ar), 125.44

(Ar), 124.25 (AreCl), 122.28 (Ar), 121.42 (Ar), 106.26 (CHepyrrole),

38.90 (NCH₃), 15.93 (SCH₃), 13.14 (CH₃).

H

d

130.70 (Ar), 120.93 (Ar), 118.12 (Ar), 112.69 (NCN), 109.32 (Ar), 37.16

(NCH₃).

4.2.14. 1-(3-Aminophenyl)-3-(2-chloro-5-(methylthio)phenyl)-1-

4.2.17. N-(3-Aminophenyl)-N-methylcyanamide (17)

methylguanidine (14)

To a reﬂuxing solution of N-methyl-N-(3-nitrophenyl)cyana-

mide (16) (10.6 g, 60.0 mmol) and acetic acid (17.3 mL, 302 mmol)

in ethanol/water (600 mL, 2/1, v/v) was added powdered iron

(13.5 g, 241 mmol) in portions. After 10 min TLC showed complete

conversion and the reaction was cooled to room temperature. The

reaction mixture was basiﬁed with NH₄OH (25%) and ﬁltered

through celite. The ﬁlter cake was washed with methanol. The

ﬁltrate was concentrated under reduced pressure and crystallized

from methanol/water to obtain the title compound as brown nee-

dles (7.60 g, 51.6 mmol, 86%). R_f0.63 (dichloromethane/methanol,

To

a solution of hydroxylamine hydrochloride (497 mg,

7.15 mmol) in ethanol/water (5.25 mL, 11/4, v/v) was added 3-(2-

chloro-5-(methylthio)phenyl)-1-(3-(2,5-dimethyl-1H-pyrrol-1-yl)

phenyl)-1-methylguanidine (13) (569 mg, 1.43 mmol). After

reﬂuxing for 24 h the reaction mixture was poured into 2 M HCl

(50 mL) and washed twice with diethyl ether (25 mL). The aqueous

layer was cooled in an ice-bath and basiﬁed with solid sodium

hydroxide and extracted twice with diethyl ether (25 mL). The

combined organic fraction was dried over anhydrous magnesium

sulfate, ﬁltered and evaporated to dryness. The crude product was

puriﬁed with ﬂash column chromatography (ethyl acetate/Et₃N, 99/

1, v/v) to obtain the title compound as light yellow oil (451 mg,

1.41 mmol, 99%). R_f0.28 (ethyl acetate/Et₃N, 99/1, v/v). ¹H NMR

95/5, v/v). ¹H NMR (CDCl₃)

6.43e6.40 (m, 3H, H_Aryl), 3.79 (bs, 2H, NH₂), 3.29 (s, 3H, NCH₃). ¹³

NMR (CDCl₃) 148.14 (AreNH₂), 140.75 (AreN), 129.65 (Ar), 113.82

d

7.13 (t, J ¼ 8.02 Hz, 1H, H_Aryl),

C

d

(NCN), 109.40 (Ar), 103.10 (Ar), 100.48 (Ar), 36.06 (NCH₃).

(CDCl₃)

d

7.21 (d, J ¼ 8.33 Hz,1H, H_Aryl), 7.06 (t, J ¼ 7.91 Hz,1H, H_Aryl),

6.85 (d, J ¼ 2.29 Hz, 1H, H_Aryl), 6.74 (dd, J ¼ 8.35, 2.33 Hz, 1H, H_Aryl),

6.61e6.57 (m, 1H, H_Aryl), 6.51 (t, J ¼ 2.04 Hz, 1H, H_Aryl), 6.47e6.42

(m, 1H, H_Aryl), 3.39 (bs, 2H, NH), 3.83 (bs, 2H, NH₂), 3.29 (s, 3H,

4.2.18. 2,2,2-Triﬂuoro-N-(3-(N-methylcyanamido)phenyl)

acetamide (18)

To a suspension of N-(3-aminophenyl)-N-methylcyanamide (17)

(1.47 g, 10.0 mmol) in dichloromethane (25 mL) at 0 ^ꢀC was added

triﬂuoroacetic anhydride (4.3 mL, 30 mmol). The reaction mixture

was stirred for 1 h at 0 ^ꢀC and the solvent was evaporated under

reduced pressure. The title compound was obtained after repeated

crystallizations from acetonitrile/water (2.08 g, 8.56 mmol, 86%). R_f

NCH₃), 2.37 (s, 3H, SCH₃). ¹³C NMR (CDCl₃)

d 150.92 (AreNH₂),

147.86 (AreNCH₃), 147.58 (NCN), 145.10 (AreNH), 137.45 (AreS),

130.23 (Ar), 129.85 (Ar), 124.43 (AreCl), 122.40 (Ar), 120.81 (Ar),

116.24 (Ar), 113.21 (Ar), 1131.11 (Ar), 38.41 (NCH₃), 15.70 (SCH₃). The

free base (100 mg) was converted into its fumaric acid salt (119 mg,

0.27 mmol, 87%). ¹H NMR (DMSO-d₆)

d

7.30 (d, J ¼ 8.02 Hz, 1H,

0.34 (dichloromethane). ¹H NMR (CDCl₃)

d 10.61 (bs, 1H, NH),

H

_Aryl), 7.02 (t, J ¼ 7.86 Hz, 1H, H_Aryl), 6.90e6.85 (m, 2H, H_Aryl), 6.55

7.30e7.24 (m, 2H, H_Aryl), 7.06 (t, J ¼ 8.14 Hz, 1H, H_Aryl), 6.60 (dd,

(s, 1.83H, fumaric acid), 6.49e6.40 (m, 3H, H_Aryl), 3.24 (s, 3H, NCH₃),

2.44 (s, 3H, SCH₃). HRMS [M þ 2Na ꢁ H] calcd for C₁₅H₁₇ClN₄S

366.0647, found 365.1215. HPLC system A: purity 99.2%,

rt ¼ 4.17 min.

J ¼ 7.96, 1.20 Hz, H_Aryl), 3.05 (s, 3H, NCH₃). ¹³C NMR (CDCl₃)

d 154.17

(d, J ¼ 37.93 Hz, CO), 140.42 (AreNHCO), 137.57 (AreNCH₃), 129.49

(Ar), 115.30 (d, J ¼ 288.38 Hz, CF₃), 114.98 (Ar), 113.17 (NCN), 110.97

(Ar), 106.91 (Ar), 36.22 (NCH₃).

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

155

4.2.19. 2,2,2-Triﬂuoro-N-methyl-N-(3-(N-methylcyanamido)

phenyl)acetamide (19)

with ethyl acetate/hexanes/Et₃N (66/33/1, v/v/v), the title com-

pound as a light yellow oil (177 mg, 0.41 mmol, 41%). R_f0.21 (ethyl

To

a

stirred suspension of 2,2,2-triﬂuoro-N-(3-(N-methyl-

acetate/hexanes/Et₃N, 66/33/1, v/v/v). ¹H NMR (CDCl₃)

d 7.50e7.38

cyanamido)phenyl)acetamide (18) (1.86 g, 7.64 mmol) and potas-

sium carbonate (1.70 g, 8.46 mmol) in dry DMF (15 mL) was added

methyl iodide (1 mL,16.0 mmol). Stirring was continued at 45 ^ꢀC for

2.5 h before ﬁltering over celite. The ﬁltrate was evaporated to

dryness under reduced pressure. The crude product was puriﬁed

over silica (dichloromethane) to obtain the title compound as a

white solid (1.91 g, 7.41 mmol, 97%). R_f0.55 (dichloromethane). ¹H

(m, 2H, H_Aryl), 7.28e7.25 (m, 2H, H_Aryl), 7.14e7.11 (m, 1H, H_Aryl), 6.89

(d, J ¼ 2.25 Hz, 1H, H_Aryl), 6.82 (dd, J ¼ 8.34,2.38 Hz, 1H, H_Aryl), 4.05

(bs, 2H, NH), 3.42 (s, 3H, NCH₃), 3.36 (s, 3H, N(CO) (CH₃)), 2.45 (s,

3H, SCH₃). ¹³C NMR (CDCl₃)

d

156.33 (q, J_CF¼ 35.71 Hz, CO), 150.49

(AreN(CO)), 146.94 (AreNCH₃), 145.61 (NCN), 141.24 (AreNH),

137.70 (AreS), 130.47 (Ar), 129.91 (Ar), 126.68 (Ar), 125.32 (Ar),

124.62 (Ar), 123.99 (AreCl),121.99 (Ar), 121.14 (Ar), 116.18 (d,

J_CF¼ 288.34 Hz, CF₃), 39.39 (CONCH₃), 38.60 (NCH₃), 15.66 (SCH₃).

NMR (CDCl₃)

_Aryl), 6.98e6.95 (m, 2H, H_Aryl), 3.32 (s, 3H, N(CH₃)CO), 3.31 (s, 3H,

NCH₃). ¹³C NMR (CDCl₃)

d

7.42 (t, J ¼ 8.36 Hz, 1H, H_Aryl), 7.13e7.09 (m, 1H,

H

¹⁹F NMR (CDCl₃)

d

ꢁ66.88 (s, CF₃).

d

156.60 (d, J ¼ 38.71 Hz, CO), 141.93

(AreN(CH₃)CO),141.67 (AreNCN),130.78 (Ar),122.12 (Ar),118.52 (d,

J ¼ 288.37 Hz, CF₃), 115.30 (Ar), 113.89 (Ar), 113.18 (NCN), 39.57

(N(CH₃)CO), 36.87 (NCH₃).

4.2.23. 3-(2-Chloro-5-(methylthio)phenyl)-1-methyl-1-(3-

(methylamino)phenyl)guanidine (23)

To a solution of N-(3-(3-(2-chloro-5-(methylthio)phenyl)-1-

methylguanidino)phenyl)-2,2,2-triﬂuoro-N-methylacetamide (22)

(177 mg, 0.41 mmol) in methanol/water (4 mL, 5/1, v/v) was added

potassium carbonate (52 mg, 0.38 mmol) and stirred at room

temperature for 1 h. The reaction mixture was diluted with water

(25 mL) and extracted twice with ethyl acetate (25 mL). The com-

bined organic fraction was washed with brine (25 mL), dried over

anhydrous magnesium sulfate, ﬁltered and evaporated under

reduced pressure. The crude product was puriﬁed over silica

(gradient dichloromethane to ethyl acetate/hexanes/Et₃N, 33/66/1,

v/v/v) to obtain the title compound as a colorless oil (135 mg,

0.40 mmol, 98%). R_f0.12 (ethyl acetate/hexanes/Et₃N, 33/66/1, v/v/

4.2.20. N-(3-(Dimethylamino)phenyl)-N-methylcyanamide (20)

To a suspension of N-(3-aminophenyl)-N-methylcyanamide (17)

(295 mg, 2.00 mmol) and potassium carbonate (683 mg,

5.01 mmol) in dry DMF was added methyl iodide (375

mL,

6.00 mmol) and stirred at 45 ^ꢀC for 1 h. The solvent was evaporated

under reduced pressure and the residue was dissolved in water

(25 mL) and extracted twice with ethyl acetate (25 mL). The com-

bined organic fraction was washed with brine (20 mL), collected,

dried over anhydrous magnesium sulfate, ﬁltrated and evaporated

to dryness under reduced pressure. The crude product was puriﬁed

with ﬂash column chromatography (dichloromethane) to obtain

the title compound as light yellow oil (179 mg, 1.02 mmol, 51%). R_f

v). ¹H NMR (CDCl₃)

d

7.30 (d, J ¼ 8.34 Hz, 1H, H_Aryl), 7.19 (t,

J ¼ 7.90 Hz, 1H, H_Aryl), 6.95 (d, J ¼ 2.26 Hz, 1H, H_Aryl), 6.82 (dd,

J ¼ 8.35, 2.31 Hz, 1H, H_Aryl), 6.65e6.61 (m, 1H, H_Aryl), 6.53e6.47 (m,

2H, H_Aryl), 4.11 (bs, 1H, NH(CH₃)), 4.01 (bs, 2H, NH), 3.41 (s, 3H,

NCH₃), 2.80 (s, 3H, NH(CH₃)), 2.46 (s, 3H, SCH₃). ¹³C NMR (CDCl₃)

0.60 (dichloromethane). ¹H NMR (CDCl₃)

J ¼ 8.08 Hz, 1H, H_Aryl), 6.46e6.34 (m, 3H, H_Aryl), 3.28 (s, 3H, CH₃),

2.96 (s, 6H, N(CH₃)₂). ¹³C NMR (CDCl₃)

151.39 (AreN(CH₃)₂),

d d 7.19 (t,

¹H NMR (CDCl₃)

d

141.32 (AreN), 129.95 (Ar), 114.51 (NCN), 107.58 (Ar), 102.39 (Ar),

d 150.94 (AreNHCH₃), 150.46 (AreNCH₃), 147.56 (NCN), 145.02

98.76 (Ar), 40.30 (N(CH₃)₂), 36.73 (NCH₃).

(AreNH), 137.38 (AreS), 130.07 (Ar), 129.79 (Ar), 124.38 (AreCl),

120.68 (Ar), 114.89 (Ar), 110.62 (Ar), 110.33 (Ar), 38.38 (NCH₃), 30.32

(NHCH₃), 15.63 (SCH₃). The free base was converted into its fumaric

4.2.21. 3-(2-Chloro-5-(methylthio)phenyl)-1-(3-(dimethylamino)

phenyl)-1-methylguanidine (21)

The reaction of N-(3-(dimethylamino)phenyl)-N-methyl-

cyanamide (20) (176 mg, 1.00 mmol) with 2-chloro-5-(methylthio)

aniline hydrochloride (232 mg, 1.10 mmol) in chlorobenzene

acid salt (138 mg, 0.30 mmol, 75%). ¹H NMR (DMSO-d₆)

d

7.30 (d,

J ¼ 8.16 Hz, 1H, H_Aryl), 7.12e7.05 (m, 1H, H_Aryl), 6.90e6.86 (m, 2H,

_Aryl), 6.55 (s, 2H, fumaric acid), 6.47e6.40 (m, 3H, H_Aryl), 6.09 (bs,

H

3H, NH), 3.26 (s, 3H, NCH₃), 2.66 (s, 3H, NH(CH₃)), 2.44 (s, 3H, SCH₃).

HRMS [M þ H]^þcalcd for C₁₆H₁₉ClN₄S 335.1092, found 335.1097.

HPLC system A: purity 97.9%, rt ¼ 4.15 min.

(200 mL) according to procedure A afforded, after puriﬁcation over

silica with a gradient of ethyl acetate/hexanes/Et₃N (20/80/1 to 33/

66/1, v/v/v), the title compound as a light yellow oil (206 mg,

0.59 mmol, 59%). R_f0.19 (ethyl acetate/hexanes/Et₃N, 33/66/1, v/v/

4.2.24. N-Methyl-N-(3-(methylamino)phenyl)cyanamide (24)

v). ¹H NMR (CDCl₃)

d

7.30e7.22 (m, 2H, H_Aryl), 6.95 (d, J ¼ 2.16 Hz,

To a solution of 2,2,2-triﬂuoro-N-methyl-N-(3-(N-methyl-

1H, H_Aryl), 6.81 (dd, J ¼ 8.33 Hz, J ¼ 2.21 Hz,1H, H_Aryl), 6.68e6.63 (m,

cyanamido)phenyl)acetamide (19) (1.29 g, 5.01 mmol) in methanol/

water (50 mL, 5/1, v/v) was added potassium carbonate (0.69 g,

5.01 mmol) and stirred at room temperature for 1 h. The reaction

mixture was diluted with water (200 mL) and extracted twice with

ethyl acetate (50 mL). The combined organic fraction was washed

with brine (25 mL), dried over anhydrous magnesium sulfate,

ﬁltered and evaporated under reduced pressure. The crude product

was puriﬁed over silica (gradient dichloromethane to ethyl acetate/

hexanes/Et₃N, 33/66/1, v/v/v) to obtain the title compound as an

off-white solid (0.79 g, 4.88 mmol, 97%). R_f0.44 (ethyl acetate/

3H, H_Aryl), 3.98 (bs, 2H, NH), 3.43 (s, 3H, NCH₃), 2.99 (s, 6H,

N(CH₃)₂), 2.47 (s, 3H, SCH₃). ¹³C NMR (CDCl₃)

d 151.55 & 151.09

(AreNCH₃& AreN(CH3)₂), 147.72 (NCN), 145.15 (AreNH), 137.49

(AreS), 130.12 (Ar), 129.92 (Ar), 124.55 (AreCl), 122.51 (Ar), 120.91

(Ar), 114.57 (Ar), 110.81 (Ar), 110.79 (Ar), 40.38 (N(CH₃)₂), 38.66

(NCH₃), 15.86 (SCH₃). The free base was converted into its fumaric

acid salt (176 mg, 0.39 mmol, 66%). ¹H NMR (DMSO-d₆)

d 7.31 (d,

J ¼ 8.15 Hz, 1H, H_Aryl), 7.19 (t, J ¼ 8.17 Hz, 1H, H_Aryl), 6.90e6.84 (m,

2H, H_Aryl), 6.62e6.56 (m, 3H, H_Aryl), 6.55 (s, 1.7H, fumaric acid), 5.99

(bs, 2H, NH), 3.30 (s, 3H, NCH₃), 2.91 (s, 6H, N(CH₃)₂), 2.44 (s, 3H,

SCH₃). HRMS [MþH]^þcalcd for C₁₇H₂₁ClFN₄S 349.1248, found

349.1262. HPLC system A: purity 95.9%, rt ¼ 5.47 min.

hexanes/Et₃N, 33/66/1, v/v/v). ¹H NMR (CDCl₃)

1H, H_Aryl), 6.37e6.29 (m, 3H, H_Aryl), 3.96 (bs, 1H, NH), 3.26 (s, 3H,

NCH₃), 2.81 (s, 3H, NHCH₃). ¹³C NMR (CDCl₃)

150.53 (ArNH),

d

7.14 (t, J ¼ 7.96 Hz,

d

141.46 (ArNCH₃), 130.17 (t-Ar), 114.50 (NCN),107.72 (t-Ar), 103.12 (t-

Ar), 98.58 (t-Ar), 36.70 (N(CH₃)CN), 30.43 (N(CH₃)H).

4.2.22. N-(3-(3-(2-Chloro-5-(methylthio)phenyl)-1-

methylguanidino)phenyl)-2,2,2-triﬂuoro-N-methylacetamide (22)

The reaction of 2,2,2-triﬂuoro-N-methyl-N-(3-(N-methyl-

cyanamido)phenyl)acetamide (19) (257 mg, 1.00 mmol) with 2-

chloro-5-(methylthio)aniline hydrochloride (231 mg, 1.01 mmol)

according to procedure A afforded, after puriﬁcation over silica

4.2.25. N-(3-((2-Fluoroethyl)(methyl)amino)phenyl)-N-

methylcyanamide (25)

To

a stirred suspension of N-methyl-N-(3-(methylamino)

phenyl)cyanamide (24) (397 mg, 2.46 mmol), potassium carbonate

156

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

(342 mg, 2.48 mmol) and potassium iodide (41 mg, 0.25 mmol) in a

mixture of dry THF and dry DMF (10 mL, 1/2, v/v) was added 1-

bromo-2-ﬂuoroethane (0.55 mL, 7.36 mmol). The reaction

mixture was stirred at 80 ^ꢀC for 24 h, before diluting with water

(25 mL). The mixture was twice extracted with ethyl acetate

(25 mL). The combined organic fraction was washed with water

(25 mL) and brine (25 mL). The organic layer was collected, dried

over anhydrous magnesium sulfate, ﬁltered and evaporated to

dryness under reduced pressure. The crude product was puriﬁed

over silica (dichloromethane/hexanes, 50/50, v/v) to obtain the title

compound as yellow oil (157 mg, 0.76 mmol, 31%). R_f0.55

7.20e7.16 (m, 1H, H_Aryl), 7.08e7.05 (m, 2H, H_Aryl), 4.63 (dt,

J_HF¼ 47.17 Hz, J ¼ 4.75 Hz, 2H, FCH₂CH₂), 4.04 (dt, J_HF¼ 25.87 Hz,

J ¼ 4.71 Hz, FCH₂CH₂), 3.35 (s, 3H, NCH₃). ¹³C NMR (CDCl₃)

d 156.66

(q, J_CF¼ 36.13 Hz, CO), 141.50 (AreN), 140.25 (AreN), 130.45 (Ar),

122.96 (Ar), 115.96 (d, J_CF¼ 288.26 Hz, CF₃), 115.48 (Ar), 114.62 (Ar),

113.04 (NCN), 79.85 (d, J_CF¼ 170.23 Hz, FCH₂CH₂), 51.91 (d,

J_CF¼ 20.16 Hz, FCH₂CH₂), 36.65 (CH₃). ¹⁹F NMR (CDCl₃)

d 16.44 (tt,

J_HF¼ 47.12 Hz, J_HF¼ 25.83 Hz, CH₂CH₂F), ꢁ67.31 (s, CF₃).

4.2.28. N-(3-(3-(2-Chloro-5-(methylthio)phenyl)-1-

methylguanidino)phenyl)-2,2,2-triﬂuoro-N-(2-ﬂuoroethyl)

acetamide (28)

(dichloromethane). ¹H NMR (CDCl₃)

d

7.21 (t, J ¼ 4.09 Hz,1H, H_Aryl),

7.15e6.35 (m, 3H, H_Aryl), 4.57 (dt, J_HF¼ 47.23, J ¼ 5.06 Hz, 2H,

FCH₂CH₂), 3.62 (dt, J_HF¼ 25.08, J ¼ 5.06 Hz, 2H, FCH₂CH₂), 3.26 (s,

The reaction of 2,2,2-triﬂuoro-N-(2-ﬂuoroethyl)-N-(3-(N-

methylcyanamido)phenyl)acetamide (27) (204 mg, 0.71 mmol)

with 2-chloro-5-(methylthio)aniline hydrochloride (164 mg,

3H, NCH₃CN), 2.99 (s, 3H, NCH₃CH₂). ¹³C NMR (CDCl₃)

d 149.89

(ArN(CH₃)CH₂), 141.44 (ArN(CH₃)CN), 130.07 (t-Ar), 114.32 (NCN),

107.36 (t-Ar), 102.68 (t-Ar), 98.64 (t-Ar), 81.61 (d, J_CF¼ 169.77 Hz),

0.78 mmol) in toluene (200 m

L) according to procedure A at 140 ^ꢀC

afforded, after puriﬁcation over silica with ethyl acetate/hexanes/

Et₃N (50/50/1, v/v/v), the title compound as a light yellow glassy

solid (214 mg, 0.46 mmol, 66%). R_f0.18 (ethyl acetate/hexanes/Et₃N,

52.38 (d, J_CF¼ 20.82 Hz), 36.60 (N(CH₃)CN), 38.81 (N(CH₃)CH₂). ¹⁹

F

NMR (CDCl₃)

d

18.18 (tt, J_HF¼ 47.24, J_HF¼ 24.92 Hz, FCH₂CH₂).

50/50/1, v/v/v). ¹H NMR (CDCl₃)

d

7.51e7.40 (m, 2H, H_Aryl), 7.31e7.26

4.2.26. 3-(2-Chloro-5-(methylthio)phenyl)-1-(3-((2-ﬂuoroethyl)

(methyl)amino)phenyl)-1-methylguanidine (26)

(m, 2H, H_Aryl), 7.19e7.16 (m, 1H, H_Aryl), 6.89 (d, J ¼ 2.24 Hz, 1H,

H

_Aryl), 6.82 (dd, J ¼ 8.34 Hz, J ¼ 2.27 Hz, 1H, H_Aryl), 4.65 (dt,

The reaction of N-(3-((2-ﬂuoroethyl) (methyl)amino)phenyl)-N-

methylcyanamide (25) (257 mg, 1.00 mmol) with 2-chloro-5-

(methylthio)aniline hydrochloride (176 mg, 0.84 mmol) in toluene

J_CF¼ 47.17 Hz, J ¼ 4.68 Hz, 2H, FCH₂CH₂), 4.04 (dt, J_CF¼ 25.81 Hz,

J ¼ 4.60 Hz, FCH₂CH₂), 4.03 (bs, 2H, NH), 3.42 (s, 3H, NCH₃), 2.45 (s,

3H, SCH₃). ¹³C NMR (CDCl₃)

d

156.74 (q, J_CF¼ 54.16 Hz, CO), 150.49

(400

m

L) according to procedure A at 140 ^ꢀC afforded, after puriﬁ-

(AreNCO), 146.98 (AreNCH₃), 145.57 (NCN),139.87 (AreNH), 137.77

(AreS), 130.43 (Ar),129.97 (Ar),126.97 (Ar),126.44 (Ar),125.67 (Ar),

124.00 (AreCl), 122.01 (Ar), 121.20 (Ar), 116.06 (d, J_CF¼ 288.43 Hz,

CF₃), 80.16 (d, J_CF¼ 170.55 Hz, FCH₂CH₂), 52.03 (d, J_CF¼ 20.11 Hz,

cation over silica with ethyl acetate/hexanes/Et₃N, 33/66/1, v/v/v),

the title compound as a light yellow glassy solid (154 mg,

0.40 mmol, 53%). R_f0.11 (ethyl acetate/hexanes/Et₃N, 33/66/1, v/v/

v). ¹H NMR (CDCl₃)

d

7.29e7.22 (m, 2H, H_Aryl), 6.94 (d, J ¼ 2.274 Hz,

FCH₂CH₂), 38.62 (NCH₃), 15.69 (SCH₃). ¹⁹F NMR (CDCl₃)

d 16.93 (tt,

1H, H_Aryl), 6.81 (dd, J ¼ 8.34, 2.30 Hz, 1H, H_Aryl), 6.69e6.61 (m, 3H,

J_HF¼ 47.15 Hz, J_HF¼ 25.90 Hz, CH₂CH₂F), ꢁ67.17 (s, CF₃).

H

_Aryl), 4.62 (dt, J_CF¼ 47.21, 5.04 Hz, 2H, FCH₂CH₂N), 3.59 (bs, 2H,

NH), 3.66 (dt, J_CF¼ 24.98, 5.06 Hz, 2H, FCH₂CH₂N), 3.42 (s, 3H,

4.2.29. 3-(2-Chloro-5-(methylthio)phenyl)-1-(3-(2-

ﬂuoroethylamino)phenyl)-1-methylguanidine (29)

NCH₃), 3.04 (s, 3H, FCH₂CH₂NCH₃), 2.46 (s, 3H, SCH₃). ¹³C NMR

(CDCl₃)

d

151.04 (FCH₂CH₂N(CH₃)Ar), 150.06 (AreNCH₃), 147.67

To a solution of N-(3-(3-(2-chloro-5-(methylthio)phenyl)-1-

methylguanidino)phenyl)-2,2,2-triﬂuoro-N-(2-ﬂuoroethyl)acet-

amide (28) (214 mg, 0.46 mmol) in methanol/water (5 mL, 5/1, v/v)

was added potassium carbonate (68 mg, 0.49 mmol) and stirred at

room temperature for 1 h. The reaction mixture was diluted with

water (25 mL) and extracted twice with ethyl acetate (25 mL). The

combined organic fraction was washed with brine (25 mL), dried

over anhydrous magnesium sulfate, ﬁltered and evaporated under

reduced pressure. The crude product was puriﬁed over silica

(gradient of dichloromethane to dichloromethane/methanol 95/5,

v/v) to obtain thetitle compoundas colorlessoil (162 mg, 0.44 mmol,

96%). R_f0.11 (ethyl acetate/hexanes/Et₃N, 50/50/1, v/v/v). ¹H NMR

(NCN), 145.37 (AreNH), 137.53 (AreS), 129.93 (Ar), 124.48 (AreCl),

122.46 (Ar), 120.93 (Ar), 114.85 (Ar), 110.70 (Ar), 110.55 (Ar), 81.71

(d, J_CF¼ 169.69 Hz, FCH₂CH₂N), 52.33 (d, J_CF¼ 20.82 Hz, FCH₂CH₂N),

38.97 (FCH₂CH₂NCH₃), 38.67 (NCH₃), 15.85 (SCH₃). ¹⁹F NMR (CDCl₃)

d

18.24 (tt, J ¼ 94.14 Hz, 23.21 Hz, 1F, FCH₂CH₂N). The product was

converted into its fumaric acid salt, (161 mg, 0.36 mmol, 90% yield)

as white solid. ¹H NMR (DMSO-d₆)

d

7.28 (d, J ¼ 8.05 Hz, 1H, H_Aryl),

7.18 (t, J ¼ 8.00 Hz, 1H, H_Aryl), 6.85e6.80 (m, 2H, H_Aryl), 6.65e6.55

(m, 3H, H_Aryl), 6.52 (s, 1.02H, fumaric acid), 5.64 (bs, 2H, NH), 4.58

(dt, J_CF¼ 47.61, J ¼ 4.85 Hz, 2H, FCH₂CH₂N), 3.65 (dt, J_CF¼ 26.35,

J ¼ 4.82 Hz, 2H, FCH₂CH₂N), 3.26 (s, 3H, NCH₃), 2.94 (s, 3H,

FCH₂CH₂NCH₃), 2.43 (s, 3H, SCH₃). HRMS [M þ H]^þcalcd for

(CDCl₃)

d

7.29e7.25 (m, 1H, H_Aryl), 7.21 (t, J ¼ 7.92 Hz, 1H, H_Aryl), 6.93

C

₁₈H₂₂ClFN₄S 381.1310, found 381.1325. HPLC system A: purity

(d, J ¼ 2.29 Hz,1H, H_Aryl), 6.82 (dd, J ¼ 8.35 Hz, J ¼ 2.32 Hz,1H, H_Aryl),

6.70e6.66 (m, 1H, H_Aryl), 6.59e6.52 (m, 2H, H_Aryl), 4.64 (dt,

J_HF¼ 47.30 Hz, J ¼ 4.85 Hz, 2H, FCH₂CH₂), 4.15 (t, J ¼ 5.67 Hz, 1H,

FCH₂CH₂NH), 4.02 (bs, 2H, NH), 3.54e3.37 (m 2H, FCH₂CH₂), 3.40 (s,

97.5%, rt ¼ 5.03 min.

4.2.27. 2,2,2-Triﬂuoro-N-(2-ﬂuoroethyl)-N-(3-(N-

methylcyanamido)phenyl)acetamide (27)

3H, NCH₃), 2.46 (s, 3H, SCH₃).¹³C NMR (CDCl₃)

d 150.94 (AreNHCH₂),

To a suspension of potassium carbonate (279 mg, 2.02 mmol),

potassium iodide (35 mg, 0.21 mmol) and 2,2,2-triﬂuoro-N-(3-(N-

methylcyanamido)phenyl)acetamide (18) (492 mg, 2.02 mmol) in

dry DMF (4 mL) and dry THF (2 mL) was added 1-bromo-2-

ﬂuoroethane (0.45 mL, 6.0 mmol). After stirring for 26 h at 80 ^ꢀC,

the reaction mixture was diluted with water (25 mL) and extracted

twice with ethyl acetate (25 mL). The combined organic fraction

was washed with brine (25 mL), collected and dried over anhydrous

magnesium sulfate, ﬁltered and evaporated to dryness under

reduced pressure. The crude product was puriﬁed over silica

(gradient of ethyl acetate/hexanes 20/80 to 20/50, v/v) to obtain the

title compound as a white solid (408 mg, 1.41 mmol, 70%). R_f0.40

148.84 (AreNCH₃), 147.57 (NCN), 145.29 (AreNH), 137.52 (AreS),

130.37 (Ar), 129.90 (Ar), 124.42 (AreCl), 122.36 (Ar), 120.86 (Ar),

115.91 (Ar),111.22 (Ar), 82.26 (d, J_CF¼ 137.59 Hz, FCH₂CH₂), 43.86 (d,

J_CF¼ 20.34 Hz, FCH₂CH₂), 38.52 (NCH₃), 15.73 (SCH₃). ¹⁹F NMR

(CDCl₃)

d

15.83 (tt, J_HF¼ 47.30, J_HF¼ 26.79 Hz, FCH₂CH₂NCH₃). The

free base was converted into its fumaric acid salt (150 mg,

0.32 mmol, 73%).¹H NMR (DMSO-d₆)

d

7.31 (d, J ¼ 8.18 Hz,1H, H_Aryl),

7.09 (t, J ¼ 7.87 Hz, 1H, H_Aryl), 6.91e6.86 (m, 2H, H_Aryl), 6.55 (s, 2H,

fumaric acid), 6.54e6.47 (m, 3H, H_Aryl), 6.14 (bs,1H, NH), 6.05 (bs, 2H,

NH), 4.54 (dt, J_HF¼ 47.62 Hz, J ¼ 4.96 Hz, 2H, FCH₂CH₂), 3.42e3.27 (m,

2H, FCH₂CH₂), 3.26 (s, 3H, NCH₃), 2.44 (s, 3H, SCH₃). HRMS [MþH]^þ

calcd for C₁₇H₂₀ClFN₄S 367.1154, found 367.1159. HPLC system A:

purity 99.7%, rt ¼ 5.03 min.

(dichloromethane). ¹H NMR (CDCl₃)

d

7.46 (t, J ¼ 8.40 Hz, 1H, H_Aryl),

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

157

4.2.30. 2-Bromoethyl-4-methylbenzenesulfonate (33)

4.2.33. 2-((3-(3-(2-Chloro-5-(methylthio)phenyl)-1-

methylguanidino)phenyl)(methyl)amino)ethyl 4-

methylbenzenesulfonate (38)

To a stirred solution of 3-(2-chloro-5-(methylthio)phenyl)-1-(3-

((2-hydroxyethyl) (methyl)amino)phenyl)-1-methylguanidine (37)

A solution of p-toluenesulfonyl chloride (955 mg, 5.01 mmol) in

dichloromethane (2.5 mL) was added dropwise to a cooled (0 ^ꢀC,

ice-water bath) solution of 2-bromoethanol (32) in dichloro-

methane (2.5 mL) and pyridine (2.5 mL). After addition, the ice-

water bath was removed and stirring was continued overnight.

The reaction mixture was diluted with diethyl ether (25 mL) and

washed with 1 M HCl (50 mL), water (25 mL) and brine (25 mL). The

organic fraction was collected and dried with anhydrous magne-

sium sulfate, ﬁltrated and evaporated under reduced pressure. The

residue was puriﬁed over silica using ethyl acetate/hexanes (10/90,

v/v) as eluent to obtain the title compound as a colorless oil

(431 mg,1.54 mmol, 31%). R_f0.14 (ethyl acetate/hexanes,10/90, v/v).

(504 mg, 1.33 mmol), triethylamine (570 mL, 4.09 mmol) and 4-

dimethylaminopyridine (21 mg, 0.17 mmol) in dichloromethane

(13.5 mL) was added p-toluenesulfonyl chloride (325 mg,

1.71 mmol). After 21 h at room temperature, the reaction mixture

was washed with water (25 mL), aqueous NaHCO₃(25 mL) and

saturated NaHCO₃(25 mL). The organic layer was collected, dried

with anhydrous magnesium sulfate, ﬁltrated and evaporated to

dryness under reduced pressure. The crude product was puriﬁed

over silica using dichloromethane/methanol (96/4, v/v) to obtain

the title compound as a white solid (215 mg, 0.40 mmol, 24%). R_f

¹H NMR (CDCl₃)

2H, H_Aryl), 4.25 (t, J ¼ 6.37 Hz, 2H, CH₂), 3.44 (t, J ¼ 6.37 Hz, 2H, CH₂),

2.42 (s, 3H, CH₃); ¹³C NMR (CDCl₃)

145.30 (AreS), 132.47

d

7.77 (d, J ¼ 8.33 Hz, 2H, H_Aryl), 7.33 (d, J ¼ 8.06 Hz,

d

0.13 (dichloromethane/methanol, 95/5, v/v). ¹H NMR (CDCl₃)

d 7.72

(AreCH₃), 130.00 (Ar), 127.93 (Ar), 68.77 (CH₂O), 27.52 (BrCH₂),

21.65 (CH₃).

(d, J ¼ 8.05 Hz, 2H, H_Aryletosylate), 7.32e7.18 (m, 2H, H_Aryletosylate,

4H, H_Aryl), 6.94e6.93 (m, 1H, H_Aryl), 6.83e6.79 (m, 1H, H_Aryl), 6.66

(d, J ¼ 7.80 Hz,1H, H_Aryl), 6.56e6.49 (m, 2H, H_Aryl), 4.24 (bs, 2H, NH),

4.18 (t, J ¼ 5.35 Hz, 2H, NCH₂CH₂O), 3.64 (t, J ¼ 5.35 Hz, 2H,

NCH₂CH₂O), 3.40 (s, 3H, NCH₃CNH), 2.91 (s, 3H, CH₂NCH₃), 2.45 (s,

4.2.31. N-(3-((2-Hydroxyethyl) (methyl)amino)phenyl)-N-

3H, AreCH₃), 2.43 (s, 3H, SCH₃). ¹³C NMR (CDCl₃)

d 151.11

methylcyanamide (36)

(AreNCH₃), 149.38 (AreNCH₃), 147.15 (NCN), 145.10 (AreSO₂),

144.89 (AreNH), 137.51 (AreS), 132.39 (AreCH₃), 130.22 (Ar),129.82

(Ar), 129.76 (Ar), 127.67 (Ar), 124 (AreCl), 122.41 (Ar), 120.95 (Ar),

114.78 (Ar), 110.45 (Ar), 110.38 (Ar), 66.84 (NCH₂CH₂O), 50.87

(NCH₂CH₂O), 38.71 (NCH₃), 38.63 (NCH₃), 21.52 (AreCH₃), 15.71

(SCH₃). HRMS [M þ H]^þcalcd for C₂₅H₂₉ClN₄O₃S₂533.1442, found

533.1523.

To

a stirred suspension of N-methyl-N-(3-(methylamino)

phenyl)cyanamide (24) (1.86 g, 11.5 mmol), N,N-di-isopropylethyl-

amine (2.00 mL, 11.5 mmol) and potassium iodide (0.19 g,

1.15 mmol) in a mixture of dry THF and dry DMF (50 mL, 1/2, v/v)

was added 2-bromoethanol (0.81 mL, 11.5 mmol). The reaction

mixture was stirred at 125 ^ꢀC for 6 h, before diluting with water

(150 mL). The mixture was extracted with ethyl acetate (2 ꢂ 75 mL).

The combined organic fraction was washed with water (100 mL)

and brine (50 mL). The organic layer was collected, dried over

anhydrous magnesium sulfate, ﬁltered and evaporated to dryness

under reduced pressure. The crude product was puriﬁed over silica

(ethyl acetate/hexanes/Et₃N, 33/66/1, v/v/v) to obtain the title

compound as a light yellow oil (1.11 g, 4.92 mmol, 43%). R_f0.55

4.3. Radiochemistry

4.3.1. 3-(2-Chloro-5-(methylthio)phenyl)-1-(3-(N-[¹¹C]methyl-N-

methyl-amino)phenyl)-1-methylguanidine ([¹¹C]21)

[

¹¹C]CO₂was trapped into a solution of LiAlH₄in THF (0.1 mL) at

room temperature by a helium ﬂow of 10 mL min^ꢁ1. The solution

was heated to 130 ^ꢀC and the helium ﬂow was increased to

100 mL min^ꢁ1to evaporate the THF. After 3 min the helium ﬂow

was adjusted to 10 mL min^ꢁ1, HI (55% solution, 0.2 mL) was added

and [¹¹C]CH₃I was passed through a preheated silver triﬂate column

of 200 ^ꢀC, to obtain online [¹¹C]CH₃OTf. [¹¹C]CH₃OTf was trapped

into the second reaction vial containing precursor 23 (0.5 mg,

(ethyl acetate). ¹H NMR (CDCl₃)

d

7.15 (t, J ¼ 8.18 Hz, 1H, H_Aryl), 6.47

(dd, J ¼ 8.33 Hz, J ¼ 2.20 Hz, 1H, H_Aryl), 6.41 (t, J ¼ 2.26 Hz, 1H, H_Aryl),

6.33 (dd, J ¼ 7.95 Hz, 2.10 Hz, 1H, H_Aryl), 3.76 (t, J ¼ 5.86 Hz, 2H,

NCH₂), 3.45 (t, J ¼ 5.88 Hz, 2H, CH₂OH), 3.26 (s, 3H, NCH₃CN), 2.96

(s, 3H, CH₂NCH₃), 2.65 (bs, 1H, OH). ¹³C NMR (CDCl₃)

d

¹³C NMR

(CDCl₃)

d 150.77 (AreNCH₃), 141.42 (AreNCH₃CN), 130.14 (Ar),

114.60 (NCN), 107.79 (Ar), 102.73 (Ar), 98.96 (Ar), 59.75 (NCH₂),

54.91 (CH₂OH), 38.85 (CH₂NCH₃), 36.78 (NCH₃CN).

1.40

[

mmol) and acetonitrile (300 mL). After complete trapping of

¹¹C]CH₃OTf the reaction mixture was heated at 40 ^ꢀC for 1 min. The

reaction mixture was quenched with 10 mM ammonium hydrogen

carbonate (pH ¼ 7.4, 1 mL) solution before loading it onto prepar-

ative HPLC (Reprosphere 100-C18-DE, 5

mm, 80 ꢂ 8 mm, 10 mM

4.2.32. 3-(2-Chloro-5-(methylthio)phenyl)-1-(3-((2-

ammonium hydrogen carbonate pH ¼ 7.4/acetonitrile 30/70 v/v,

4 mL min^ꢁ1, 254 nm). The product eluted at 7 min, was collected

and diluted with water (40 mL). The solution was concentrated on a

tC18plus Seppak, rinsed with water (20 mL), subsequently eluted

with ethanol (96%, 1.5 mL) and diluted with a solution of 7.11 mM

NaH₂PO₄in 0.9% NaCl (w/v in water), pH ¼ 5.2 (13.5 mL) to give a

ﬁnal solution of 9.6% ethanol in phosphate buffer containing [¹¹C]

21. The (radio)chemical purity was veriﬁed using analytical radio

hydroxyethyl)(methyl)amino)phenyl)-1-methylguanidine (37)

The reaction of N-(3-((2-hydroxyethyl) (methyl)amino)phenyl)-

N-methylcyanamide (36) (524 mg, 2.33 mmol) with 2-chloro-5-

(methylthio)aniline hydrochloride (590 mg, 2.81 mmol) in toluene

(500 m

L) according to procedure A at 140 ^ꢀC afforded, after puriﬁ-

cation over silica with dichloromethane/hexanes/Et₃N (gradient

from 50/50/1 to 75/25/1, v/v/v), the title compound as a light yellow

oil (504 mg, 1.33 mmol, 52%). R_f0.35 (ethyl acetate/Et₃N, 99/1, v/v).

HPLC (Alltima C18, 5

m

m, 250 ꢂ 4.6 mm, 20 mM potassium

¹H NMR (CDCl₃)

d

7.20e7.08 (m, 2H, H_Aryl), 6.85 (d, J ¼ 2.24 Hz, 1H,

hydrogen phosphate pH ¼ 6.7/methanol/acetonitrile 20/20/60 v/v/

v, 1.0 mL min^ꢁ1, 254 nm). The product eluted at 8.12 min. The

speciﬁc activity was calculated against a calibration curve of 21

using the same HPLC system.

H

_Aryl), 6.72 (dd, J ¼ 8.37 Hz, J ¼ 2.27 Hz, 2H, H_Aryl), 6.53e6.48 (m,

3H, H_Aryl), 3.99 (bs, 3H, NH & OH), 3.59 (t, J ¼ 5.94 Hz, 2H, NCH₂),

3.30 (t, J ¼ 6.30 Hz, 2H, CH₂OH), 3.29 (s, 3H, NCH₃CNH), 2.84 (s, 3H,

CH₂NCH₃), 2.35 (SCH₃). ¹³C NMR (CDCl₃)

d 151.55 (AreNCH₃), 150.42

(AreNCH₃), 147.11 (NCN), 144.77 (AreNH), 137.47 (AreS), 129.96

(Ar), 129.74 (Ar), 124.48 (AreCl), 122.48 (Ar), 120.85 (Ar), 113.95

(Ar), 110.42 (Ar), 110.27 (Ar), 58.96 (NCH₂CH₂), 54.32 (NCH₂CH₂),

38.47 (2ꢂ NCH₃), 15.47 (SCH₃).

4.3.2. 3-(2-Chloro-5-(methylthio)phenyl)-1-methyl-1-(3-([¹¹C]

methylamino)phenyl)guanidine ([¹¹C]23)

[

¹¹C]23 was synthesized accordingly to [¹¹C]21. Eluent prepar-

ative HPLC 10 mM ammonium hydrogen carbonate pH ¼ 7.4/

158

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

acetonitrile 35/65 v/v, retention time 7 min. Analytical HPLC

retention time ¼ 6.25 min.

of ice-cold 0.25 M sucrose. The nuclei and cell debris were removed

by centrifugation (10 min ꢂ 400ꢂ g) in a Sorvall RC-6 refrigerated

centrifuge (rotor SA600). The supernatant was decanted and the

resulting pellet was rehomogenized in 5 vol. 0.25 M sucrose and

recentrifuged. The combined supernatants were diluted in Tris-

acetate buffer (50 mM, pH 7.4) to a ﬁnal dilution of 40 v/w, and

centrifuged for 30 min ꢂ 30,000ꢂ g, in order to obtain membranes

from the cell surface, mitochondrial, and microsomal fractions. The

pellet was resuspended in 20 vol. of 50 mM Tris buffer containing

0.04% Triton X-100 (pH 7.4), and was kept at 25 ^ꢀC for 2 h before

recentrifugation. The resulting pellet was suspended in TriseHCl

buffer (dilution 4, pH 7.4) and stored at ꢁ80 ^ꢀC in 5 mL aliquots. On

the day of each experiment, membranes were thawed to room

temperature and washed twice with Tris buffer by centrifugation

(30 min ꢂ 48,000ꢂ g). After the ﬁnal centrifugation step, pellets

were suspended in 50 mM TriseHCl buffer (pH 7.4) and further

diluted to 40 vol. of buffer per g original weight wet tissue for

competition binding experiments. Protein concentration was

determined using the BCA protein kit (SigmaeAldrich, The

Netherlands).

4.3.3. 3-(2-Chloro-5-(methylthio)phenyl)-1-(3-((2-[¹⁸F]

ﬂuoroethyl)(methyl)amino)phenyl)-1-methylguanidine ([¹⁸F]26)

ꢁ

After irradiation, [¹⁸F]ﬂuoride was trapped on a PS ꢁ HCO₃

column. It was eluted from the anion exchange column into a screw

cap reaction vessel with 1 mL of acetonitrile/water (9/1, v/v) con-

taining 13 mg (34.5 mmol) of Kryptoﬁx 2.2.2 and 3 mg (21.7 mmol) of

potassium carbonate. The solution was evaporated to dryness un-

der a helium ﬂow (50 mL min^ꢁ1) and reduced pressure at 90 ^ꢀC.

Acetonitrile (0.5 mL) was added and evaporated again. After cooling

to room temperature, precursor (38, 2.5 mg, 4.7 mmol) dissolved in

acetonitrile (0.5 mL) was added and heated to 80 ^ꢀC for 30 min. The

reaction vessel was cooled to 30 ^ꢀC and the reaction mixture was

quenched with 25 mM ammonium dihydrogen phosphate (1 mL,

pH 2.5) solution. The reaction mixture was puriﬁed by semi-

preparative HPLC (Reprosphere 100-C18-DE, 5

mm, 80 ꢂ 8 mm,

10 mM ammonium hydrogen carbonate pH ¼ 7.4/acetonitrile 30/70

v/v, 4 mL min^ꢁ1, 254 nm). The product eluted at 19 min, was

collected and diluted with water (30 mL). The solution was

concentrated on a tC18plus Seppak, rinsed with water (20 mL),

subsequently eluted with ethanol (96%, 1.5 mL) and diluted with a

solution of 7.11 mM NaH₂PO₄in 0.9% NaCl (w/v in water), pH ¼ 5.2

(13.5 mL) to give a ﬁnal solution of 9.6% ethanol in phosphate buffer

containing [¹⁸F]26. The (radio)chemical purity was veriﬁed using

4.5.3. Competition binding assays

In vitro competition binding experiments were performed using

5 nM [³H]MK-801 (speciﬁc activity 22.5 Ci/mmol; PerkinElmer,

USA). All compounds were dissolved as 10 mM stock solutions in

DMSO, and used in a concentration range from 10^ꢁ4to 10^ꢁ12M,

with a DMSO concentration of maximal 1%. Competition binding

experiments were conducted at room temperature, in a ﬁnal vol-

analytical radio HPLC (Alltima C18, 5

m

m, 250 ꢂ 4.6 mm, 20 mM

potassium hydrogen phosphate pH ¼ 6.7/methanol/acetonitrile 20/

20/60, v/v/v, 1.0 mL min^ꢁ1, 254 nm). The product eluted at 7.48 min.

The speciﬁc activity was calculated against a calibration curve of 26

using the same HPLC system.

ume of 500

-glutamate and glycine. The incubation mixture was

composed of 400 L membrane suspension (protein concentration

^ꢁ1), 50 L [³H]MK-801, 45

L assay buffer and 5 L unlabeled

drug solution. Nonspeciﬁc binding was determined in the presence

of 30 M 2. Incubations were terminated after 17 h by ﬁltration,

mL assay buffer (50 mM TriseHCl, pH 7.4), containing

1

mM L

m

1

mg

mL

m

4.4. Determination of Log D_oct,7.4

m

The distribution of the radiolabeled compounds between 1-

octanol and 0.2 M phosphate buffer (pH ¼ 7.4) was measured in

triplicate at room temperature. Brieﬂy, 1 mL of a 1e5 MBq mL^ꢁ1

solution of the radiolabeled compound in 0.2 M phosphate buffer

(pH 7.4) was vigorously mixed with 1 mL of 1-octanol for 1 min at

room temperature using a vortex. After a settling period of 30 min,

using a 48-well Brandel harvester and Whatman GF/B ﬁlters, pre-

soaked in 0.3% polyethyleneimine. The ﬁlters were washed three

times with 3 mL of ice-cold TriseHCl buffer (pH 7.4), and subse-

quently radioactivity was determined by liquid scintillation spec-

trometry in 5 mL of Optiphase-HiSafe 3, at an efﬁciency of 40%.

ﬁve samples of 100

recovery, 5 samples of 100

solution. All samples were counted for radioactivity. The Log D_oct,7.4

value was calculated according to Log D_oct,7.4

¹⁰Log(A_oct/A_buffer),

where A_octand A_bufferrepresent average radioactivity of 5 1-octanol

and 5 buffer samples, respectively.

m

L were taken from both layers. For determining

4.5.4. Data analysis

m

L were taken from the 1e5 MBq$mL^ꢁ1

K_ivalues were determined by nonlinear regression analysis

using the equation: log EC₅₀¼ log[10^log K_i*(1 þ RadioligandNM/

HotKdNM)], (GraphPad Software Inc., San Diego, CA).

¼

4.5.5. Biodistribution and blocking studies

The biodistribution of [¹¹C]21, [¹¹C]23 and [¹⁸F]26 was deter-

mined in male Wistar rats. Rats (N ¼ 4 per time point) received a

dose of either 100 MBq [¹¹C]21 and [¹¹C]23 or either 40 MBq [¹⁸F]26

in the tail vein without anesthesia and were decapitated at either 5,

15, 30 or 60 min after injection. Blood, heart, lung, liver, kidney and

bone were removed. In addition, several brain areas were

dissected: hippocampus, striatum, hypothalamic region, cere-

bellum, cerebral cortex and rest brain. These tissues were then

counted for radioactivity with a gamma counter (LKB Wallac, Turku,

Finland) and weighed. Results were expressed as percent injected

dose per gram (% ID/g). For blocking studies, rats received 15 min

prior injection of radiotracer a dose of 0.6 mg kg^ꢁ1MK-801

intraperitoneal.

4.5. Pharmacology

4.5.1. Animals

All animal experiments were performed in compliance with

Dutch laws on animal experimentation and after approval by the

local animal ethics committee, study number DEC_PET12-01.

Adult male Wistar rats (Harlan, Horst, The Netherlands)

weighing 200e224 g at arrival were group housed with ﬁve to six

animals per cage and had ad libitum access to food (Teklad Global

16% Protein Rodent Diet, Harlan, Madison, WI, USA) and tap water.

Rats were kept in an animal room with a constant temperature of

~21 ^ꢀC and in a 12-h light/dark cycle in which lights were switched

on at 8:00 am.

4.5.6. Metabolite analysis

4.5.2. Membrane preparation

To study the metabolic proﬁle of [¹¹C]21, [¹¹C]23 and [¹⁸F]26 in

the plasma and brain, six rats were injected with either 100 MBq

[

Male Wistar rats (150e200 g) were killed by decapitation. The

forebrains were rapidly removed and homogenized using a DUALL

tissue homogenizer (10 strokes, 2000 rpm), in a 7-fold excess (v/w)

¹¹C]21 and [¹¹C]23 or either 50 MBq [¹⁸F]26 in the tail vein without

anesthesia. After 15 or 45 min the rats were decapitated. A blood

P.J. Klein et al. / European Journal of Medicinal Chemistry 118 (2016) 143e160

159

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sample was obtained and the brain was isolated. All blood samples

were collected in heparin tubes and centrifuged at 4000 rpm for

5 min at 4 ^ꢀC (Hettich Universal 16, Depex B.V., The Netherlands).

Plasma was separated from blood cells, diluted with 2 mL of water

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Subsequently, the tC2 Plus Sep-Pak was eluted with 1.5 mL of

methanol containing 0.1% diisopropylamine. This eluate was

deﬁned as the non-polar fraction and was analyzed using HPLC

(Phenomenex Gemini C8, 250 ꢂ 10.0 mm, 0.1 M ammonium ace-

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1510, Kronberg, Germany) in water, under ice cooling, and subse-

quently centrifuged at 4000 rpm for 5 min at 4 ^ꢀC. Separated su-

pernatants were loaded onto a tC2 Plus Sep-Pak and treated as

above. The recovery of the metabolite analysis was validated to be

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A

potent and selective ligand designed as

a

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Acknowledgments

thesis and pharmacological evaluation of N-(2,5-disubstituted phenyl)-N⁰-(3-

substituted phenyl)-N⁰-methylguanidines as N-methyl-

D -aspartate receptor

ion-channel blockers, J. Med. Chem. 40 (1997) 4281e4289.

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G.J. Durant, R.N. McBurney, Identi ﬁ cation and characterization of a potential

This research was performed within the framework of CTMM,

the Center for Translational Molecular Medicine (www.ctmm.nl),

project LeARN (grant 02N-101).

ischemia-selective N-methyl-D -aspartate (NMDA) receptor ion-channel

blocker, CNS 5788, Bioorg. Med. Chem. Lett. 11 (2001) 501e504.

[22] F. Dumont, A. Sultana, R.N. Waterhouse, Synthesis and in vitro evaluation of

Appendix A. Supplementary data

N,N⁰-diphenyl and N-naphthyl-N⁰-phenylguanidines as N-methyl-

D -aspartate

receptor ion-channel ligands, Bioorg. Med. Chem. Lett. 12 (2002) 1583e1586.

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[24] P.J. Klein, J.A.M. Christiaans, A. Metaxas, R.C. Schuit, A.A. Lammertsma,

B.N.M. Berckel, A.D. Windhorst, Synthesis, structure activity relationship,

radiolabeling and preclinical evaluation of high af ﬁ nity ligands for the ion

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.ejmech.2016.04.022.

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Article Doi

Synthesis, radiolabeling and evaluation of novel amine guanidine derivatives as potential positron emission tomography tracers for the ion channel of the N-methyl-d-aspartate receptor

DOI: 10.1016/j.ejmech.2016.04.022

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