N′-3-(Trifluoromethyl)phenyl Derivatives of N-Aryl-N′-methylguanidines as Prospective PET Radioligands for the Open Channel of the N-Methyl- d -aspartate (NMDA) Receptor: Synthesis and Structure-Affinity Relationships

Article abstract of DOI:10.1021/acs.jmedchem.5b01510

N-Methyl-d-aspartate (NMDA) receptor dysfunction has been linked to several neuropsychiatric disorders, including Alzheimer's disease, epilepsy, drug addiction, and schizophrenia. A radioligand that could be used with PET to image and quantify human brain NMDA receptors in the activated "open channel" state would be useful for research on such disorders and for the development of novel therapies. To date, no radioligands have shown well-validated efficacy for imaging NMDA receptors in human subjects. In order to discover improved radioligands for PET imaging, we explored structure-affinity relationships in N′-3-(trifluoromethyl)phenyl derivatives of N-aryl-N′-methylguanidines, seeking high affinity and moderate lipophilicity, plus necessary amenability for labeling with a positron-emitter, either carbon-11 or fluorine-18. Among a diverse set of 80 prepared N′-3-(trifluoromethyl)phenyl derivatives, four of these compounds (13, 19, 20, and 36) displayed desirable low nanomolar affinity for inhibition of [³H](+)-MK801 at the PCP binding site and are of interest for candidate PET radioligand development.

Full text of DOI:10.1021/acs.jmedchem.5b01510

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Article
N’-3-(Trifluoromethyl)phenyl derivatives of N-aryl-N’-methylguanidines
as prospective PET radioligands for the open channel of the N-methyl-D-
asparate (NMDA) receptor: synthesis and structure-affinity relationships
Gregory R. Naumiec, Kimberly J Jenko, Sami S. Zoghbi, Robert B Innis, Lisheng Cai, and Victor W Pike
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01510 • Publication Date (Web): 20 Nov 2015
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N’-3-(Trifluoromethyl)phenyl derivatives of N-aryl-N’-methylguanidines as
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prospective PET radioligands for the open channel of the N-methyl-D-
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aspartate (NMDA) receptor: synthesis and structure-affinity relationships
Gregory R. Naumiec, Kimberley J. Jenko, Sami S. Zoghbi, Robert B. Innis, Lisheng Cai, Victor
W. Pike
Molecular Imaging Branch, National Institute of Mental Health, National Institutes of Health,
Building 10, Room B3 C346A, 10 Center Drive, Bethesda, Maryland 20892-1003, United States
KEYWORDS NMDA, receptor, SAR, ligand, guanidines
ABSTRACT (148 words)
N-Methyl-D-aspartate (NMDA) receptor dysfunction has been linked to several neuropsychiatric
disorders, including Alzheimer’s disease, epilepsy, drug addiction, and schizophrenia.
A
radioligand that could be used with PET to image and quantify human brain NMDA receptors in
the activated ‘open channel’ state would be useful for research on such disorders and for the
development of novel therapies. To date, no radioligands have shown well-validated efficacy for
imaging NMDA receptors in human subjects. In order to discover improved radioligands for
PET imaging, we explored structure-affinity relationships in N’-3-(trifluoromethyl)phenyl
derivatives of N-aryl-N’-methylguanidines, seeking high affinity and moderate lipophilicity, plus
necessary amenability for labeling with a positron-emitter, either carbon-11 or fluorine-18.
Among a diverse set of 80 prepared N’-3-(trifluoromethyl)phenyl derivatives, four of these
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compounds (13, 19, 20, and 36) displayed desirable low nanomolar affinity for inhibition of
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[3H](+)-MK801 at the PCP binding site and are of interest for candidate PET radioligand
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development.
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INTRODUCTION
The N-methyl-D-aspartate (NMDA) receptor is a voltage and ligand-gated ion channel
allowing for non-selective cation flux when activated.1 The channel is regulated by at least six
discrete binding sites for endogenous ligands that include glutamate, glycine, Mg2+, polyamine,
Zn2+, and an allosteric modulator.1–3 NMDA receptors are heteromers derived from three main
types of subunit, named GluN1, GluN2, and GluN3, where GluN1 is obligatory. Usually, a
single NMDA receptor is composed of four subunits, often two GluN1 subunits plus two GluN2
subunits. There are eight variants of the GluN1 subunit produced by alternative gene splicing.
Four distinct isoforms of the GluN2 subunit are expressed in vertebrates and are referred to as
GluN2A through to GluN2D. There are currently two recognized subtypes of the GluN3
subunit, GluN3A and GluN3B. NMDA receptors play a pivotal role in synaptic long-term
potentiation, which has significant effects on learning and memory as well as neuroplasticity,
neurodevelopment, and neuroprotection.1–5 NMDA receptor dysfunction, via excessive
activation followed by neuronal death, is indicated in neuropsychiatric disorders, such as stroke,
neuropathic pain, epilepsy, drug addition, Alzheimer’s disease, and schizophrenia,1–5 making
NMDA receptors a valuable therapeutic target.1–7
The activation of the NMDA channel requires simultaneous binding of glutamate and
glycine, whereas the subsequent depolarization of the post-synaptic terminal releases Mg2+, thus
opening the channel and allowing access to a binding site within the channel, termed the
phencyclidine (PCP) binding site.1,2 Because the PCP binding site is only exposed when the
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NMDA receptor is activated, this site is of special interest. Thus, brain-penetrant ligands capable
of high-affinity binding to the PCP site would allow modulation of the activated channel.
Moreover, the labeling of such radioligands with a positron-emitter might provide radioligands
for quantifying NMDA receptors in their active states in both healthy and diseased brain with
PET.
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A plethora of radioligands from various structural classes has been developed as
candidates for the imaging of the open channel of the NMDA receptor in vivo.8,9 Notably, these
radioligands include [11C]MK-801 ([11C]1),10 [18F]MEM ([18F]2),11 [123I]CNS-1261 ([123I]3),12,13
[11C]CNS-5161 ([11C]4),14 [18F]GE-179 ([18F]5),15,16 and recently [18F]PK-209 ([18F]6)17,18
(Chart 1). All current radioligands appear to suffer from at best low specific binding, whereas
many others suffer from low brain uptake, high non-specific binding, or confounding
metabolism.8,9 Among these radioligands, members of the N,N’-diarylguanidine class have
shown the highest affinity,12–20 and members of the N-(1-naphthyl)-N’-aryl-N’-methylguanidine
sub-class the most potential for molecular imaging.12,13,19,20 This is illustrated with the
radioligand [123I]3. SPECT` studies with [123I]321-23 have displayed perhaps the best evidence of
an NMDA-specific signal in vivo, albeit a small signal. [N-methyl-11C]3 is however ineffective
as a radioligand for imaging with PET, probably due to rapid deiodination in vivo to troublesome
brain-penetrant radiometabolites.24
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123I
NH2
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NH
NH
18F
N
N
H
H311C
Me
Me
[
123I]3 ([123I]CNS-1261)
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[11C]1 ([11C]MK801)
[ F
18
]2 ([18F]MEM)
18F
18F
NH
O
SMe
Cl
SMe
NH
S
SMe
SMe
NH
N
N
11CH3
N
N
N
N
H
H
H
Cl
Me
Cl
Me
[11C]4 ([11C]CNS-5161)
[18F]5 ([18F]GE-179)
[ F]
6 ([18F]PK-209)
18
Chart 1. Some prominently studied radioligands for the NMDA receptor.
Recently, we have shown that the 3-iodo substituent in 3 may be replaced with a
trifluoromethyl substituent (7, Table 1) with an almost twofold improvement in binding affinity
at the PCP binding site.20 Higher affinity PET radioligands with moderate lipophilicity are
sought to improve the prospects of attaining higher PCP-site specific signal in vivo. In this
study, we sought to discover higher affinity ligands that would be amenable to labeling with
carbon-11 (t1/2 = 20.4 min) or fluorine-18 (t1/2 = 109.8 min) for PET imaging, based on N’-3-
trifluoromethyl members of the N-aryl-N’-methylguanidines class of ligands, and especially a
sub-class with naphthalen-1-yl as the N-aryl group. Here, we report the syntheses and structure-
affinity relationships for a large set of such compounds. Several high affinity ligands were
discovered, and four with low nanomolar affinity. Among these ligands, 1-(6-fluoro-3-
(trifluoromethyl)phenyl)-1-methyl-3-(naphthalen-1-yl)guanidine (13) was identified as
particularly promising for PET radioligand development because of its exceptionally high
affinity for the PCP binding site, moderate lipophilicity, and feasibility for labeling with either
carbon-11 or fluorine-18.
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RESULTS AND DISCUSSION
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Ligand Design. Our initial lead for this SAR study was 7 (Table 1),20 a member of the
N-(1-naphthyl)-N’-aryl-N’-methylguanidine class of compounds, and a 3-trifluoromethyl analog
of the 3-iodo compound 3. We had found that replacement of the N’-3-iodo substituent of 3 with
an N’-3-trifluoromethyl substituent gave nearly a two-fold increase in binding affinity at the PCP
binding site.20 Retention of this 3-trifluoromethyl substituent in further analogs was seen as
attractive for two main reasons. Firstly, an aryl trifluoromethyl group is generally considered
metabolically stable and would also evade the deiodination encountered in vivo with [123I]3.
Secondly, ligands bearing aryl trifluoromethyl groups are now plausible targets for labeling with
fluorine-18 by recently developed no-carrier-added methods.25-29 Moreover, we aimed to retain
a N’-methyl group, as this provides a site that is readily labeled with carbon-11.14,17,24
Chemistry. The two primary components in the synthesis of N,N’,N’-trisubstituted
guanidines are a cyanamide and an amine hydrochloride. Two pathways for the synthesis of the
cyanamides were used (Scheme 1). One pathway (A) is a single-step cyanation of the requisite
arylamine with cyanogen bromide30 and the other (B) a three-step process through a thiourea
intermediate.31,32 The single-step pathway has several drawbacks, including moderate to low
yields, tolerance of only a narrow range of substrates, and the need to handle noxious cyanogen
bromide. The second pathway came to be preferred. This pathway tolerated a wider range of
substrates and was overall much higher yielding. Also use of cyanogen bromide was avoided
and intermediates readily crashed out of solution, thus avoiding solvent-demanding flash
chromatography. Once the desired N-arylcyanamides had been synthesized, they were either
used in situ or converted into their corresponding N-methyl-N-arylcyanamides via alkylation
with methyl iodide.30
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Scheme 1. Synthesis of arylcyanamides and N-methylcyanamides.
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aReagents and conditions: (i) cyanogen bromide, Et2O, reflux; (ii) sodium hydride, methyl iodide, THF,
reflux; (iii) benzoyl isothiocyanate, acetone, rt; (iv) 5% aq. NaOH, 90 oC; (v) I2, Et3N, EtOAc, rt.
The (trifluoromethyl)arylamine hydrochlorides, required for coupling to the cyanamides,
were either used directly or converted into their corresponding N-methylamine hydrochlorides
(Scheme 2). Several methods were explored to prepare the N-methylamine hydrochlorides.
Direct alkylations of the amines with methyl iodide were initially attempted but resulted in large
amounts of dimethylated byproducts, even when setting the stoichiometry of the reaction to use
0.90 equivalents of methyl iodide. In order to prevent dimethylation, the use of a protecting
group (e.g., N-boc or N-acetyl) was explored, but then the protection and deprotection steps
became onerous. Finally a one-pot reaction using trimethylorthoformate and catalytic amounts
of sulfuric acid33 was explored and successfully afforded all of the N-methylamine
hydrochlorides with only limited amounts (< 10%) of accompanying dimethylated byproduct.
Scheme 2. Synthesis of N-methyl-(trifluoromethyl)arylamine hydrochlorides.
o
o
aReagents and conditions: (i) a) 1. trimethylorthoformate, H2SO4, 120 C; 2. 170 C; 3. 10% aq. HCl,
reflux; b) Et2O/HCl.
N,N’,N’-Trisubstituted guanidines were generally synthesized in one of two ways
(Scheme 3), either by (A) heating an amine hydrochloride with the requisite N-methyl-N-
arylcyanamide30 or (B) by heating an N-arylcyanamide with the requisite N-methylamine
hydrochloride.33 In general, the latter method gave higher yields. For example, 7 was obtained
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(trifluoromethyl)phenyl)cyanamide at 130 C in toluene in 16% yield, but in over two-fold
higher yield (35%) by treating N-(naphthalen-1-yl)cyanamide with N-methyl-3-
(trifluoromethyl)aniline hydrochloride under the same conditions.20
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Scheme 3. Synthesis of N,N’,N’-trisubstituted guanidines.
aReagents and conditions: (i) toluene, 130 oC.
Binding Studies. In vitro binding assays for the PCP site of the NMDA receptor were
performed with rat brain membrane suspensions with [3H]N-(1-[thienyl]cyclohexyl)piperidine
([3H]TCP) as radioligand.20 Initially, the percent inhibition of [3H]TCP binding was measured
with test ligand at 1 µM concentration. Usually, if the inhibition exceeded ~ 89%, a Ki value was
determined, also with [3H]TCP as radioligand. Selected compounds showing high affinity (low
Ki) versus [3H]TCP as radioligand also had their Ki values measured with [3H]1as radioligand.
Structure-Affinity Relationships. Initially, we explored whether prevention of the
rotation of the N’-(3-(trifluoromethyl)phenyl) group would promote higher PCP binding site
affinity, by preparing the cyclized compounds 8 and 9 (Table 1). Both compounds showed
lower binding affinity than 7. The 4-trifluoromethyl compound 8 retained some binding affinity,
but the 6-trifluoromethyl compound 9 lost all affinity. Therefore, a freely rotating N’-(3-
(trifluoromethyl)phenyl) group was deemed necessary and was retained in all newly prepared
ligands.
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Table 1. Inhibition of [3H]TCP binding at the PCP binding site by 7 and structurally rigid analogs.
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Ligand R1 R2 Inhibition
(%)a
7b
8
107
61.5
–4.5
CF3
H
H
CF3
9
a At 1.0 µM (mean from two experiments).
b From reference 20.
Previous work on developing ligands from N,N’-biarylguanidines for the PCP binding
site has shown that multiple substituents on the N’-aryl group may enhance binding affinity.20
Therefore, we next explored whether further substitution of the N’-(3-(trifluoromethyl)phenyl)
group would be beneficial. Initially, we investigated the addition of a fluoro substituent to
minimize steric changes (Table 2). All the prepared fluoro derivatives (10–13) displayed high
(> 60%) inhibition of [3H]TCP binding at micromolar concentration. The 6-fluoro compound 13
was found to have a higher binding affinity (Ki 13 nM vs [3H]TCP) than the previously reported20
non-fluoro analog 7 (18.3 nM), and over a four-fold higher affinity than the next highest affinity
isomer (i.e., 57.2 nM for the 5-fluoro compound, 12). The affinity of 13 represents an almost
three-fold enhancement over that of 3.
Table 2. Inhibition of [3H]TCP binding at the PCP binding site by 3 and substituted N-(1-naphthyl)-N’-(3-
(trifluoromethyl)phenyl)-N’-methylguanidines, and selected Ki values.
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Ligand
R
Inhibition [3H]TCP Ki
(%)a
106
107
74.2
(nM)b,c
3d
7d
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18.3
2-H
2-F
4-F
5-F
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62.1
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98.2
99.8
75.4
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104
93.8
41.0
3.5
4-Br
5-Br
6-Br
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42.9
4-Br, 6-F
2-Me
4-Me
5-Me
6-Me
2-OMe
4-OMe
5-OMe
6-OMe
4-SMe
6-SMe
4-OEt
6-OEt
4-CN
5-CF3
256
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104
-1.0
1.9
31.4
7.9
35.5
5.5
0.2
-0.2
a At 1.0 µM (mean from two experiments).
b Binding affinities measured for compounds with > 89% inhibition, and 12.
c Mean from two experiments.
d Data from reference 20.
Analogs of the fluoro compounds were also prepared with heavier halogen substituents.
All chloro (14–17) and bromo (18–20) analogues displayed moderate to high affinity (> 50%
inhibition) with the exception of the 4-chloro (15) and 4-bromo compounds (18; Table 2). Ki
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values (vs [3H]TCP) were found to be 45.8 and 42.9 nM for the 5-bromo (19) and 6-bromo (20)
compounds, respectively.
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Some overall trends are apparent with respect to the effects of adding halo substituents to
the N’-(3-trifluoromethyl)phenyl group of 7 on PCP binding site affinity. Thus, compounds with
a halo substituent at position 5 or 6 (as in 12, 13, 16, 17, 19, and 20) generally show higher
affinity than compounds with a halo substituent at position 2 or 4 (as in 10, 11, 14, 15, and 18).
For a halo substituent at position 6, affinity increases in the order Cl < Br < F, whereas at
position 5 a bromo substituent gives highest affinity. The strong influence of a 6-fluoro
substituent was further evident in the 4-bromo-6-fluoro-substituted compound 21. This
compound has much higher affinity than 18, which only differs by lacking the 6-fluoro
substituent.
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We also explored the effects of adding a non-halo substituent to the N’-(3-
(trifluoromethyl)phenyl) group (Table 2). Among the prepared compounds, only the 4-, 5-, and
6-methyl-substituted compounds showed high binding affinities (> 89% inhibition). The 5-
methyl compound (24) has about twice the affinity (Ki 53 nM vs [3H]TCP) of the 6-methyl
compound (25) and about 5-fold higher affinity than the 4-methyl compound (23), but still four-
fold lower affinity than the 6-fluoro compound, 13. A 2-, 4-, 5-, or 6-methoxy substituent, as in
26–29, decreased binding affinity dramatically, as did a methylthio or ethoxy substituent at
position 4 or 6 (30–33), or a nitrile substituent at position 4 (34). These substituents may
influence the planarity of the anilino group. The compound with another trifluoromethyl
substituent at position 5 (35) showed no affinity.
With N’-(3-(trifluoromethyl)-6-fluoro)phenyl identified as the preferred N’-aryl group,
we began to explore structural changes to the other aryl group in 13 and its fluoro isomers.
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Replacement of the N-(1-naphthyl) group with an N-(2-(chloro-5-methylthiophenyl) group has
been shown to be acceptable for high affinity, as in [18F]6 (Chart 1).17,18 Therefore, N-(2-
(chloro-5-methylthiophenyl) analogs of 36–40 were prepared (Table 3). High affinity was found
for the non-fluoro substituted compound (36). Contrary to expectation, the 6-fluoro compound
40 showed lower binding affinity than 36. The binding affinity of 36 (14.2 nM) was found to be
similar to that of 13 (13.0 nM). These results show that changes to the N-aryl side of the ligand
may influence binding interactions on the opposite N’-aryl side and vice versa, thus impairing
structural optimization efforts.
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55
56
57
58
59
60
Table 3. Inhibition of [3H]TCP binding at the PCP site binding site by substituted N-(2-(chloro-5-
methylthiophenyl)-N’-(3-(trifluoromethyl)phenyl)-N’-methylguanidines, and Ki value for 36.
Ligand
R
Inhibition
(%)a
96.1
[3H]TCP Ki
(nM)b,c
14.2
2-H
2-F
4-F
5-F
6-F
36
37
38
39
40
51.4
21.1
43.2
73.6
a At 1.0 µM (mean from two experiments).
b Binding affinity for compound with > 90% inhibition.
c Mean from two experiments.
We next explored whether other alkylthio substituents might be preferred to the
methylthio substituent in 36 (Table 4), especially as previous studies have suggested that bulkier
thioether substituents might improve PCP binding site affinity.15 Among a prepared short series
of linear alkylthio homologs, in which the S-alkyl group was increased up to butyl (41–43),
binding affinity appeared lower than for 36. Branching of the alkyl chain had an adverse effect
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on binding affinity, as seen by comparing the S-isopropyl compound (44) with the S-propyl
compound (42), and the S-sec-butyl compound (45) with the S-butyl compound (43).
Introduction of fluorine into the S-methyl substituent, as in 46, markedly reduced affinity. By
contrast, introduction of fluorine into the S-ethyl group, as in 47, was almost without effect.
Finally, it was found that an S-benzyl group, as in 48, was well tolerated but offered no
improvement over 36.
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11
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
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29
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Inhibition of [3H]TCP binding at the PCP binding site by substituted N-(2-(chloro-5-alkylthiophenyl)-N’-
(3-(trifluoromethyl)phenyl)-N’-methylguanidines.
Ligand
R
Inhibition
(%)a
96.1
36b
41
42
43
44
45
46
47
48
Me
Et
Pr
86.4
62.1
82.7
22.6
48.8
57.5
82.1
88.6
Bu
iPr
sBu
CH2F
CH2CH2F
Bn
a At 1.0 µM (mean from two experiments).
bFrom Table 3; shown for ease of comparison.
Because of the benefit seen from replacing the methylthio substituent on the N’-aryl
group of 5 with a trifluoromethyl substituent (36), we explored a similar replacement in the N-(2-
(chloro-5-methylthiophenyl) group of 36, as in 49. However, this change resulted in some loss
of affinity (compare 49 in Table 5 with 36 in Table 4). In attempts to improve upon the affinity
of 49, halo, methyl or trifluoromethyl substitution was explored in the N’-(3-
(trifluoromethyl)phenyl) group, as in compounds 50–59. As for the N-(1-naphthyl) series,
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compounds with a 6-fluoro (53), 5-bromo (54), or 6-bromo (55) substituent displayed higher
affinity than the non-halo substituted compound (49, Table 5). However, unlike the N-(1-
naphthyl) series, a 4-, 5-, or 6-methyl substituent was detrimental (56–58), as was a 5-
trifluoromethyl substituent (59). Overall, with respect to binding affinity at the PCP binding site,
substitutions in the N’-(3-(trifluoromethyl)phenyl) group were best tolerated in the N-(1-
naphthyl) series, followed by the N-(2-(chloro-5-(trifluoromethyl)phenyl) series and the N-(2-
(chloro-5-methylthiophenyl) series.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 5. Inhibition of [3H]TCP binding at the PCP binding site by substituted N-(2-(chloro-5-
(trifluoromethyl)phenyl)-N’-(3-(trifluoromethyl)phenyl)-N’-methylguanidines.
Ligand
R
Inhibition
(%)a
71.2
-
49
50
51
52
53
54
55
56
57
58
59
2-F
4-F
5-F
6-F
5-Br
6-Br
4-Me
5-Me
6-Me
5-CF3
-8.7
21.1
43.2
84.8
99.0
93.0
42.3
66.4
38.0
46.2
a At 1.0 µM (mean from two experiments).
The 2-chloro substituent in the N-(2-(chloro-5-methylthiophenyl) group of 49 was
replaced with H, F, Br, Me, CF3, or MeO in attempts to increase affinity for the PCP binding site
(Table 6). Most of these compound displayed moderately high affinity (> 50% inhibition of
[3H]TCP binding). The methyl (82), trifluoromethyl (83) and methoxy (84) analogs showed
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improved binding affinity over 49. For the 2-fluoro or 2-bromo compounds, binding affinity was
greatly enhanced by a 5-methyl substituent on the opposite ring (c.f., 67 vs 61, and 78 vs 72).
For the 2-bromo compound, affinity was also enhanced by a 2-, 4-, 5-, or 6-fluoro substituent on
the opposite ring (73–76).
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13
14
15
16
17
18
19
20
21
22
23
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34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 6. Inhibition of [3H]TCP binding at the PCP binding site by substituted N-(5-(trifluoromethyl)phenyl)-N’-(3-
(trifluoromethyl)phenyl)-N’-methylguanidines.
Ligand
X
R
Inhibition
(%)a
53.9
59.4
36.2
24.9
20.9
44.5
39.1
95.3
33.4
27.7
30.2
34.0
62.0
71.0
89.1
98.0
72.4
31.3
99.0
85.5
99.0
93.4
85.8
89.0
79.6
H
F
F
F
F
F
F
F
F
-
-
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
2-F
4-F
5-F
6-F
4-Me
5-Me
6-Me
5-Br
6-Br
5-CF3
-
2-F
4-F
5-F
6-F
4-Me
5-Me
6-Me
5-Br
6-Br
-
F
F
F
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
Me
CF3
OMe
-
-
a At 1.0 µM (mean from two experiments).
Finally, replacement of the N-(1-naphthyl) group with another bi- or tri-cyclic group (1-
(4-bromonaphthalen-1-yl), 1-(2,3,4a,8a-tetrahydrobenzo[b][1,4]dioxin-5-yl), 1-(4a,5,6,7,8,8a-
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hexahydronaphthalen-1-yl), or 1-(anthracen-1-yl) was explored (85–88) (Table 7). Each change
abolished affinity. Therefore, the N-(1-naphthyl) group was found to be the optimal N-aryl
substituent among those studied.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 7. Inhibition of [3H]TCP binding at the PCP binding site by N-(polycyclyl)-N’-(3-(trifluoromethyl)phenyl)-
N’-methylguanidines.
Ligand
85
Ar
Inhibition
(%)a
-2.6
86
3.8
8.1
87
88
-7.3
a At 1.0 µM (mean from two experiments).
Some compounds with Ki values measured against [3H]TCP that were at least comparable
with that of 3 (31.9 nM) also had Ki values measured against [3H]1as radioligand in order to
allow direct comparison with the literature19 Ki value for 3 (4.65 nM vs. [3H]1 (Table 8). Four
of these compounds (13, 19, 20, and 36) were found to have low nanomolar Ki values that were
very comparable with those of the two previously most advanced radioligands for imaging
NMDA receptors in vivo, namely [123I]3 for SPECT and [18F]7 for PET. Ligand 13 showed the
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highest affinity (Ki = 1.29 nM), a value somewhat higher than that of ligand 7 (2.68 nM)20
(Table 8).
3
4
5
6
7
8
9
Compound lipophilicity is an important parameter influencing PET radioligand
performance, especially with regard to the ability of a compound to enter brain adequately
without excessive non-specific binding.34-36 Generally, moderate lipophilicity, represented by a
logD value in the 2–3 range, is considered desirable. The computed logD values for ligands 13,
19, 20, and 36, like those of 3, 5 and 7, fall into this range (Table 8). Ligands 54, 55, 67, 75, 78,
and 80 not only showed much lower affinity versus [3H]1 as radioligand, but also appreciably
higher computed logD values, which render these ligands unattractive for PET radioligand
development.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 8. Affinities and computed lipophilicites of selected N-(aryl)-N’-(3-(trifluoromethyl)phenyl)-N’-
methylguanidines at the PCP binding site measured with [3H]1.
[3H]1 Kia
clogDa
Ligand
(nM)
1.65 0.16 b
0.91 0.05b
2.68 0.26 b
1.29 0.03 b
3.42 0.82 c
4.7 0.4c
2.72
2.58
2.23
2.27
2.73
2.86
2.79
4.07
4.30
3.06
3.49
3.54
4.26
3
5
7
13
19
20
36
54
55
67
75
78
80
3.25 0.63 c
62 11 b
233 43 b
243 82 b
49 16 b
29 4 b
43 23 b
a Computed with ACD Labs software.
bMean SD from 3 experiments.
cMean SD from 6 experiments
CONCLUSIONS
This study identified twelve N-(aryl)-N’-(3-(trifluoromethyl))-N’-methylguanidines that
at micromolar concentration displayed high inhibition (> 90%) of [3H]TCP binding to the PCP
binding site of the NMDA receptor, demonstrating that this is a potent sub-class of receptor
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ligands. Compounds with an N-naphthyl group showed greatest tolerance for structural
modification in the opposed N’-(3-(trifluoromethyl)phenyl) group. Four ligands (13, 19, 20, 36)
were found to have nanomolar affinity for NMDA receptors, moderate lipophilicity, as well as
amenability to labeling with carbon-11 or fluorine-18 for PET imaging. These properties are
similar or superior to those of [123I]3 and [18F]5, the only other radioligands so far considered
promising for imaging the open channel of the NMDA receptor in human subjects.13,16 Ligand
13 has an especially favorable combination of properties for PET radioligand development.
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11
12
13
14
15
16
17
18
19
20
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23
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36
37
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48
49
50
51
52
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59
60
EXPERMENTAL SECTION
Materials and Methods
Reagents and solvents were purchased commercially and used without further purification. All
reactions were performed under an inert atmosphere in oven-dried glassware and monitored by
TLC on silica layers (0.2 mm; Polygram® Sil G/UV254; Grace; Deerfield, IL) visualized under
UV light (254 nm). Column chromatography was performed on silica gel (SiliFlash® F60, 230–
400 mesh; Silicycle; Quebec City, CA). Melting points were taken in open-ended capillaries
13
with an SMP10 melting point apparatus (Stuart; Staffordshire, UK). 1H- (400 MHz) and C-
(100 MHz) NMR spectra were recorded in the solvent later indicated on an Avance 400
instrument (Bruker; Billerica, MA). Chemical shifts are given in parts per million (ppm) (δ)
relative to the signal for TMS. All new compounds were analyzed by HRMS at the Mass
Spectrometry Laboratory, School of Chemical Sciences, University of Illinois (Urbana-
Champaign, IL) using a Micromass Q-Tof Ultima instrument for ESI (Waters Corp; Columbia,
MD). Compound purity was determined with HPLC on a LC-20AD instrument (Shimadzu;
Columbia, MD) equipped with a diode array detector (254 nM), and a Gemini C18 column (5
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µm; 110 Å; 4.6 × 250 mm; Phenomenex; Torrance, CA) eluted with MeOH: 10 mM aq.
ammonium carbonate (4: 1 v/v) at 1 mL/min. The purity of all final compounds was ≥ 95%.
Chromatographic mobile phase compositions are given by volume. cLogD values (for pH 7.4)
were calculated using ACD/Percepta Platform prediction software (ACD/Labs).
10
11
12
13
14
15
16
17
18
19
20
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23
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29
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31
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36
37
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Preparation of Requisite Amines
2-Chloro-5-(methylthio)aniline (89). Et3N (3.8 g, 38 mmol) and diphenylphosphoryl azide (7.4
g, 26.8 mmol) were added drop-wise to a suspension of 2-chloro-5-(methylthio)benzoic acid (5.0
g, 24.7 mmol) in t-butanol (20 mL) at rt. The reaction mixture was heated to reflux for 6 h, then
cooled to rt, and concentrated under reduced pressure. The residue was diluted with THF (12.5
mL) and aq. HCl (10% w/v; 12.5 mL) and heated to reflux for a second time. After 16 h the
reaction mixture was cooled to rt and concentrated under reduced pressure. The crude residue
o
was cooled to 0 C and basified to pH ~ 12 with aq. NaOH (20% w/v). The mixture was
extracted with EtOAc (15 mL × 4) and the combined organic extracts were washed with water
(20 mL) and dried (MgSO4). The MgSO4 was filtered off and the filtrate was then concentrated
under reduced pressure. The crude product was purified on a silica gel column eluted with Et2O:
hexane (4: 1) to afford 89 (3.2 g, 74%) as a white solid. All characterization data agreed with
literature data.37
3-Amino-4-chlorobenzenethiol (90). 4-Chloro-3-nitrobenzenesulfonyl chloride (1.7 g, 6.6
mmol) was added portion-wise to a cold (0 oC) solution of tin(II) chloride dihydrate (13.5 g, 59.4
mmol) in aq. HCl (37% w/v; 10 mL). The reaction mixture was then warmed to rt over 15 min
and then refluxed for 1 h. The reaction mixture was cooled to rt, diluted with water (100 mL),
and carefully neutralized by slow addition of sodium bicarbonate. The aq. layer was then
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extracted with chloroform (50 mL × 4), and the combined organic layers were dried (MgSO4),
filtered, and concentrated under reduced pressure. The crude product was purified on a silica gel
column eluted with hexane: dichloromethane (7: 3) to afford 90 (0.4 g, 40%) as a white solid.
All characterization data agreed with literature data.15
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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36
37
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46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5,5'-Disulfanediylbis(2-chloroaniline) (91). 4-Chloro-3-nitrobenzenesulfonyl chloride (1.7 g,
o
6.6 mmol) was added portion-wise to a cold (0 C) solution of tin(II) chloride dihydrate (13.5 g,
59.4 mmol) in aq. HCl (37% w/v; 10 mL). The reaction mixture was then warmed to rt over 15
o
min, heated at 125 C for 3 h, and finally cooled to rt. The precipitate was filtered off and
dissolved in water (85 mL). Iodine solution (50 mg/mL) was added portion-wise until complete
conversion into disulfide was observed by TLC. The solution was filtered and water was added
to the precipitate. The resulting mixture was then stirred and neutralized with aq. NaOH (10%
w/v). The mixture was extracted with Et2O (50 mL × 4), dried (MgSO4), and filtered. The crude
product was recrystallized from Et2O/hexane to afford 91 (0.9 g, 71%) as a light purple solid.
All characterization data agrees with literature data.38
6-(Trifluoromethyl)indoline (92). Sodium cyanoborohydride (679 mg, 10.8 mmol) was added
portion-wise to a stirred solution of 6-(trifluoromethyl)indole (1.0 g, 5.4 mmol) in glacial acetic
acid (10 mL) at rt. After 3 h the reaction mixture was diluted with water (20 mL), cooled to 0
oC, and basified with aq. NaOH (40% w/v). The reaction mixture was then extracted with
dichloromethane (30 mL × 3). The combined organic extracts were dried (MgSO4), filtered, and
concentrated under reduced pressure to afford 92 (1.1 g, 89%) as a brown solid. All
characterization data agrees with literature data.39
General Method for the Preparation of N-Methylanilines
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General Method A: Illustrated by the synthesis of N-methyl-3-(trifluoromethyl)aniline
(93).20 Three drops of sulfuric acid were added to a solution of 3-(trifluoromethyl)aniline (5.0 g,
31 mmol) and trimethyl orthoformate (4.9 g, 46.5 mmol). This reaction mixture was slowly
10
11
12
13
14
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18
19
20
21
22
23
24
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28
29
30
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37
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42
43
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47
48
49
50
51
52
53
54
55
56
57
58
59
60
o
o
heated to 120 C to allow MeOH to be distilled off. After 2 h of being stirred at 120 C, the
reaction mixture was heated to 170 oC. After 30 min, the reaction mixture was cooled to 100 oC
and aq. HCl (10% w/v; 25 mL) was added. The reaction mixture was then refluxed for 3 h,
cooled in an ice-bath to 0 oC, and then basified with aq. 20% NaOH (w/v). The reaction mixture
was extracted with EtOAc (30 mL × 3), dried (MgSO4), filtered, and concentrated under reduced
pressure. The crude product was purified on a silica gel column with gradient elution (hexane:
1
EtOAc; 1: 0 to 1: 10), to afford 93 (1.7 g, 25%) as a colorless oil: H-NMR (CD3OD) δ 7.95 (br
s, 1H), 7.91–7.82 (m, 3H), 3.17 (s, 3H); 13C-NMR (CD3OD) δ 139.4, 133.6 (q, JC-F = 33 Hz),
132.8, 127.6 (q, JC-F = 4 Hz), 127.4, 124.7 (q, JC-F = 271 Hz), 120.6 (q, JC-F = 4 Hz), 37.8;
HRMS m/z 176.0683 [(M + H)+; calcd for C8H8F3N: 176.0687].
Compounds S2–S25 were also prepared by this method, as described in Supporting
Information.
Preparation of Thioethers
General Method B:
Illustrated by the synthesis of 2-chloro-5-(ethylthio)aniline
hydrochloride (94). Potassium carbonate (207 mg, 1.50 mmol) and ethyl iodide (195 mg, 1.30
o
mmol) were added sequentially to a cold (0 C) solution of 90 (200 mg, 1.30 mmol) in DMF (5
mL). The reaction mixture was warmed to rt, stirred overnight, diluted with water (15 mL), and
extracted with EtOAc (20 mL × 3). The combined organic extracts were washed with brine (20
mL × 2), dried (MgSO4), filtered, and concentrated under reduced pressure. The crude residue
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was diluted with Et2O (5 mL). Ethereal HCl (1 M) was then added drop-wise until no further
5
6
precipitation occurred. The precipitate was then filtered off to afford 94 (268 mg, 96%) as a
7
8
o
1
white solid: mp 171–172 C; H NMR (CD3OD) δ 7.51(d, J = 8.4 Hz, 1H), 7.44 (d, J = 2.4 Hz,
1H), 7.34 (dd, J = 8.4, 2.4 Hz, 1H), 3.05 (q, J = 7.2 Hz, 2H), 1.35 (t, J = 7.2 Hz, 3H); 13C NMR
(CD3OD) δ 140.4, 131.9, 131.8, 129.5, 124.9, 123.8, 27.8, 14.4; HRMS m/z 188.0299 [(M + H)+;
calcd for C8H10ClNS: 188.0301].
9
10
11
12
13
14
15
16
17
18
19
20
21
22
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25
26
27
28
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36
37
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43
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48
49
50
51
52
53
54
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58
59
60
Compounds S26–S31 were also prepared by this method, as described in Supporting
Information.
Preparation of Requisite Cyanamides
N-(Naphthalen-1-yl)cyanamide (95). This was prepared from cyanogen bromide and 1-
naphthylamine as previously described.20
N-(3-(Trifluoromethyl)phenyl)cyanamide (96).
A solution of cyanogen bromide in
acetonitrile (5 M; 3.9 mL, 19.4 mmol), diluted further in Et2O (10 mL), was added drop-wise to a
cold (0 oC) solution of (3-trifluoromethyl)aniline (5.0 g, 31 mmol) in Et2O (20 mL), refluxed for
24 h and cooled to rt. The resultant precipitate was filtered off and washed with copious EtOAc.
The washes were then combined with the filtrate and washed with cold aq. HCl (1 M; 25 mL ×
2), water (25 mL), and brine (25 mL). The organic layer was dried (MgSO4), filtered, and
concentrated under reduced pressure. The resultant crude solid was recrystallized from ethanol
and water to afford 96 (1.5 g, 42%) as a white solid. All characterization data agreed with
literature data.40
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N-Methyl-N-(3-(trifluoromethyl)phenyl)cyanamide (97). A suspension of 96 (1.5 g, 8.1
5
6
mmol) and sodium hydride (0.39 g, 16.2 mmol) in THF (40 mL) was refluxed for 2 h. The
7
8
9
o
reaction mixture was then cooled to 0 C and iodomethane (2.9 g, 20.3 mmol) was added drop-
10
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36
37
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42
43
44
45
46
47
48
49
50
51
52
53
54
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wise. The reaction mixture was then warmed to rt, stirred for an additional 16 h, diluted with
MeOH (20 mL) and water (40 mL), and extracted with chloroform (20 mL × 3). The combined
organic phases were washed with water (10 mL) and brine (10 mL), dried (MgSO4), filtered, and
concentrated under reduced pressure. The crude product was purified on a silica gel column
eluted with hexane: EtOAc (3: 1) to afford 97 (1.1 g, 68%) as a white solid. All characterization
data agrees with literature data.40
General Method C:
Illustrated by the three-step synthesis of N-(2-bromo-5-
(trifluoromethyl)phenyl)cyanamide (100).
Step 1. N-(2-Bromo-5-(trifluoromethyl)phenylcarbamothioyl)benzamide (98). A solution of
benzoyl isothiocyanate (3.4 g, 20.8 mmol) in acetone (42 mL) was added drop-wise to a stirred
solution of 2-bromo-5-(trifluoromethyl)aniline (5.0 g, 20.8 mmol) in acetone (100 mL) and
stirred at rt for 21 h. Hexane (100 mL) was then added to the reaction mixture, and the resultant
precipitate was filtered off and washed liberally with water and hexane. The crude solid was
o
recrystallized from Et2O and hexane to afford 98 (7.0 g, 83%) as a white solid: mp 155–156 C;
1H-NMR (DMSO-d6) δ 12.74 (s, 1H), 11.98 (s, 1H), 8.36 (d, J = 2.0 Hz, 1H), 8.03–7.99 (m, 3H),
7.69 (tt, J = 7.6, 1.2 Hz, 1H), 7.62 (dd, J = 8.4, 1.6 Hz, 1H), 7.56 (t, J = 7.2 Hz, 2H); 13C-NMR
(DMSO-d6) δ 180.7, 168.5, 138.0, 133.9, 133.3, 131.8, 128.8, 128.6 (q, JC-F = 35 Hz), 128.5,
125.3 (q, JC-F = 4 Hz), 124.8 (q, JC-F = 3 Hz), 124.2, 123.6 (q, JC-F = 270 Hz); HRMS m/z
402.9732 [(M + H)+; calcd for C15H10BrF3N2OS: 402.9728].
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mmol) was added to a solution of aq. NaOH (5%; 70 mL) at 90 C, stirred at 90 C for 20 min,
and then filtered while still hot. The filtrate was then cooled to rt and acidified with aq. HCl
(10% w/v). Ammonium hydroxide was then added portion-wise until the pH reached 8. The
resultant precipitate was filtered off and washed with water and hexane to afford 99 (4.8 g, 92%)
as a white solid: mp 179–180 oC; 1H-NMR (DMSO-d6) δ 9.43 (s, 1H), 8.14 (br s, 1H), 8.08 (d, J
= 2.0 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.58 (br s, 1H), 7.49 (dd, J = 8.4, 2.0 Hz, 1H); 13C-NMR
(DMSO-d6) δ 182.1, 138.6, 133.7, 128.0 (q, JC-F = 32 Hz), 125.8 (q, JC-F = 4 Hz), 124.1, 123.6
(q, JC-F = 271 Hz), 123.5 (q, JC-F = 4 Hz); HRMS m/z 298.9455 [(M + H)+; calcd for
C8H6BrF3N2S: 298.9465].
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Step 3. N-(2-Bromo-5-(trifluoromethyl)phenyl)cyanamide (100). Iodine (1.7 g, 6.7 mmol)
was added slowly over 15 min to a solution of 99 (2.0 g, 6.7 mmol) and Et3N (1.4 g, 13.4 mmol)
in EtOAc (16 mL). Once the addition was complete, the reaction mixture was stirred at rt for 10
min, and then the resultant precipitate was filtered off and washed with EtOAc. The filtrate was
then combined with the EtOAc wash and the mixture was washed with water (20 mL) and brine
(20 mL), dried (MgSO4), filtered, and concentrated under reduced pressure. The crude product
was purified on a silica gel column eluted with hexane: EtOAc (9: 1) to afford 100 (1.3 g, 74%)
o
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as a white solid: mp 133–135 C; H- NMR (DMSO-d6) δ 10.19 (br s, H), 7.88 (d, J = 8.4 Hz,
H), 7.36 (dd, J = 8.4, 1.6 Hz, 1H), 7.33 (d, J = 1.2 Hz, 1H); 13C-NMR (DMSO-d6) δ 138.0,
134.8, 129.6 (q, JC-F = 33 Hz), 123.4 (q, JC-F = 270 Hz), 120.8 (q, JC-F = 4 Hz), 114.2, 112.8 (q,
JC-F = 4 Hz), 111.1; HRMS m/z 263.9507 [(M + H)+; calcd for C8H4BrF3N2: 263.9510].
Compounds S32–S37 were also prepared by this method, as described in Supporting
Information.
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Preparation of Guanidines
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General Method D: Illustrated by the synthesis of 1-methyl-3-(naphthalen-1-yl)-1-(3-
(trifluoromethyl)phenyl)guanidine (7). Compound 93 (0.35 g, 2 mmol) was dissolved in Et2O
(5 mL) and converted into its hydrochloride salt by the drop-wise addition of etheral HCl (1 M).
The resultant precipitate was filtered off, dried for 10 min, and used in the subsequent reaction.
A mixture of 93 hydrochloride (212 mg, 1 mmol), 95 (185 mg, 1.1 mmol), and toluene (2 mL)
was heated to 130 oC, stirred for 21 h, cooled to rt, and diluted with MeOH (5 mL). The solution
was concentrated onto silica gel and purified on a silica gel column with gradient elution (EtOAc
to EtOAc: MeOH, 4: 1). The fractions containing the desired guanidine were collected,
concentrated under reduced pressure, and applied to a second silica gel column eluted with Et3N:
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EtOAc: hexanes (5: 20: 7 5) to afford 7 (132 mg, 35%) as a white solid: mp 184–185 C: H
NMR (CDCl3) δ 8.08–8.05 (m, 1H), 7.76–7.73 (m, 1H), 7.53 (br s, 1H), 7.46–7.44 (d, J = 8.4
Hz, 1H), 7.41–7.38 (m, 5H), 7.38–7.31 (m, 1H), 6.91 (dd, J = 7.24, 1.04 Hz, 1H), 3.86 (br s, 2H),
3.44 (s, 3H); 13C-NMR (CDCl3) δ 150.2, 146.3, 145.7, 134.8, 132.0 (q, JC-F = 34 Hz), 130.2,
129.7, 128.9, 128.0, 126.5, 126.1, 125.2, 124.1, 123.1 (q, JC-F = 3 Hz), 122.4 (q, JC-F = 3 Hz),
122.3, 117.5, 38.8; HRMS m/z 344.1371 [(M + H)+; calcd for C19H16F3N3: 344.1375].
Compounds 3, 8, 9, 11–20, 22–45, and 48–88 were also prepared by Method D, as
described in Supporting Information.
General Method E:
Illustrated by the two-step synthesis of 3-(2-chloro-5-
(fluoromethylthio)phenyl)-1-methyl-1-(3-(trifluoromethyl)phenyl)guanidine (46).
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1,1'-(5,5'-Disulfanediylbis(2-chloro-5,1-phenylene))bis(3-methyl-3-(3-
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(trifluoromethyl)phenyl)guanidine) (101). Compound 91 (407 mg, 1.28 mmol) was dissolved
in Et2O (5 mL) and converted into its hydrochloride salt by the drop-wise addition of etheral HCl
(1 M). The resultant precipitate was filtered off, dried for 10 min, and used in the subsequent
reaction. A mixture of 91 dihydrochloride (488 mg, 1.25 mmol), 97 (1.0 g, 5 mmol), and toluene
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(2 mL) was heated to 130 C. The reaction mixture was stirred at 130 C for 21 h, cooled to rt,
and diluted with MeOH (5 mL). This solution was concentrated onto silica gel and purified on a
silica gel column with gradient elution, EtOAc: MeOH (1: 0, 4: 1). The fractions containing the
desired guanidine were collected, concentrated under reduced pressure, and applied to a second
silica gel column eluted with Et3N: EtOAc: hexanes (5: 20: 75) to afford 101 (132 mg, 35%) as
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an off-white solid: mp 70–72 C; H-NMR (CDCl3) δ 7.58–7.50 (m, 8H), 7.32–7.30 (m, 2H),
7.09–7.06 (m, 4H), 3.93 (br s, 4H), 3.36 (s, 6H); 13C-NMR (CDCl3) δ 150.5, 147.3, 145.1, 136.0,
132.1 (q, JC-F = 32 Hz), 130.6, 130.2, 130.0, 127.1, 124.0, 123.6 (q, JC-F = 271 Hz), 123.4 (q, JC-F
= 3 Hz), 123.0 (q, JC-F = 4 Hz), 122.9, 37.8; HRMS m/z 717.0859 [(M + H)+; calcd for
C30H24Cl2F6N6S2: 717.0863].
Step
2.
3-(2-Chloro-5-(fluoromethylthio)phenyl)-1-methyl-1-(3-
(trifluoromethyl)phenyl)guanidine (46). Sodium borohydride (32 mg, 0.84 mmol) was added
to a solution of 101 (150 mg, 0.21 mmol) and fluoroiodomethane (69 mg, 0.43 mmol) in EtOH
o
(9 mL). The reaction mixture was heated at 60 C for 17 h, then cooled to rt, and concentrated
under reduced pressure. The crude product was purified on a silica gel column eluted with Et3N:
1
acetone: hexane (5: 5: 90) to afford 46 (49 mg, 60%) as a pale yellow oil: H-NMR (CDCl3) δ
7.59 (s, 1H), 7.54–7.49 (m, 3H), 7.34 (d, J = 8.40 Hz, 1H), 7.15 (d, J = 2.40 Hz, 1H), 7.05 (dd, J
= 8.40, 2.40 Hz, 1H), 5.77 (s, 1H), 5.64 (s, 1H), 4.05 (br s, 2H), 3.45 (s, 3H); 13C-NMR (CDCl3)
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δ 150.7, 146.9, 145.0, 133.5 (d, JC-F = 3 Hz), 132.2 (q, JC-F = 32 Hz), 130.6, 130.2, 130.0, 127.5,
126.4 (d, JC-F = 2 Hz), 125.2 (d, JC-F = 2 Hz), 123.6 (q, JC-F = 271 Hz), 123.4 (q, JC-F = 4 Hz),
123.0 (q, JC-F = 4 Hz), 88.3 (d, JC-F = 216 Hz), 39.0; HRMS m/z 392.0599 [(M + H)+; calcd for
C16H14ClF4N3S: 392.0611].
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Compound 47 was also prepared by Method E, step 2, as described in Supporting
Information.
Binding Assays
In vitro binding assays for the PCP site of the NMDA receptor were performed with rat
brain membrane suspensions by Caliper Life Sciences (Hanover, MD) with [3H]TCP as
radioligand or in-house with [3H]1 as reference radioligand. For in-house assays, 1 was
dissolved in Tris buffer (5 mM; pH 7.4). Stock solutions of test ligands were freshly prepared by
first dissolving the ligand in EtOH. [3H]1 solution (specific activity, 22.5 Ci/mmol; Perkin
Elmer; 100 µL), -glutamate solution (50 µL), and glycine solution (50 µL) were added to each
L
assay tube to provide final assay tube concentrations of 0.50 nM, 100 µM, and 30 µM,
respectively. Each stock solution of test ligand was serially diluted in Tris buffer (5 mM; pH
7.4) and an aliquot (100 µL) was added to each assay tube to provide the following final assay
tube concentrations: 0.001, 0.01, 0.03, 0.10, 0.30, 1.0, 3.0, 10, 30, 100, 1000, and 10000 nM.
After the addition of the rat brain homogenate preparation (700 µL; ~ 150 µg/mL tissue final
concentration) the mixtures were incubated at 23 °C for 2 h. Nonspecific binding was
determined in the presence of 1 (30 µM; 100 µL). One or two competitive binding assays were
performed in triplicate. Each test ligand was assayed once along with control samples to
determine the KD of 1 with GraphPad Prism using “One-Site Homologous Fitting”. The %
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inhibitions for each of the test ligands and in some cases the Ki values were determined with
GraphPad Prism version 5 for Windows (GraphPad Software; San Diego, CA, USA) with “One
Site-Fit log IC50” and “One Site-Fit Ki” curve-fitting.
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ASSOCIATED CONTENT
Supporting Information
Detailed synthetic procedures and characterization for all synthesized compounds not described
in main text.
AUTHOR INFORMATION
Corresponding Author
*E-mail: pikev@mail.nih.gov. Phone: 301 594 5986. Fax: 301 480 5112.
Author Contributions
This paper was written with contributions from all authors. All authors approved the final
version of the manuscript.
ACKNOWLEDGMENTS
This study was supported by the Intramural Research Program of NIH, specifically the National
Institute of Mental Health (ZIA-MH002793).
ABBREVIATIONS USED
PCP: phencyclidine; SAR: structure-affinity relationship; TCP: N-(1-[thienyl]
cyclohexyl)piperidine.
References
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1.
Traynelis, S. F.; Wollmuth, L. P.; McBain, C. J.; Menniti, F. S.; Vance, K. M.; Ogden, K.
K.; Hansen, K. B.; Yuan, H.; Myers, S. J.; Dingledine, R. Glutamate receptor ion
channels: structure, regulation, and function. Pharmacol. Rev. 2010, 62, 405–496.
Yamakura, T.; Shimoji, K. Subunit- and site-specific pharmacology of the NMDA
receptor channel. Prog. Neurobiol. 1999, 59, 279–298.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
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32
33
34
35
36
37
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
2.
3.
4.
5.
6.
Paoletti, P.; Bellone, C.; Zhou, Q. NMDA receptor subunit diversity: impact on receptor
properties, synaptic plasticity and disease. Nat. Rev. Neurosci. 2013, 14, 383–400.
Lau, C. G.; Zukin, R. S. NMDA receptor trafficking in synaptic plasticity and
neuropsychiatric disorders. Nat. Rev. Neurosci. 2007, 8, 413–426.
Zhou, Q; Sheng, M. NMDA receptors in nervous system diseases. Neuropharmacology
2013, 74, 69-75.
Limapichat, W.; Yu, W. Y.; Branigan, E.; Lester, H. A.; Dougherty, D. A. Key binding
interactions for memantine in the NMDA receptor. ACS Chem. Neurosci. 2013, 4, 255–
260.
7.
8.
Stone, J. M. Imaging the glutamate system in humans: relevance to drug discovery for
schizophrenia. Curr. Pharm. Des. 2009, 15, 2594–2602.
Sobrio, F; Gilbert, G.; Perrio, C.; Barré, L.; Debruyne, D. PET and SPECT imaging of
the NMDA receptor system: an overview of radiotracer development. Mini-Rev. Med.
Chem. 2010, 10, 870–886.
9.
Fuchigami, T.; Nakayama, M.; Yoshida, S. Development of PET and SPECT probes for
glutamate receptors. Sci. World J. 2015, 2015, Article ID 716514.
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10. Kiesewetter, D. O.; Finn, R. D.; Rice, K. C.; Monn, J. A. Synthesis of 11C-labeled ( )-5-
methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine [( )-[11C]MK801]. Int. J.
Radiat. Appl. Instrum., Part A 1990, 41, 139–142.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
11. Samnick, S.; Ametamey, S.; Leenders, K. L.; Vontobel, P.; Quack, G.; Parsons, C. G.;
Neu, H.; Schubiger, P. A. Electrophysiological study, biodistribution in mice, and
preliminary PET evaluation in a rhesus monkey of 1-amino-3-[18F]fluoromethyl-5-
methyl-adamantane (18F-MEM): a potential radioligand for mapping the NMDA-receptor
complex. Nucl. Med. Biol. 1998, 25, 323–330.
12. Owens, J.; Tebbutt, A. A.; McGregor, A. L.; Kodama, K.; Magar, S. S.; Perlman, M. E.;
Robins, D. J.; Durant, G. J.; McCulloch, J. Synthesis and binding characteristics of N-(1-
naphthyl)-N′-(3-[125I]-iodophenyl)-N′-methylguanidine ([125I]-CNS 1261): a potential
SPECT agent for imaging NMDA receptor activation. Nucl. Med. Biol. 2000, 27, 557–
564.
13. Erlandsson, K.; Bressan, R. A.; Mulligan, R. S.; Gunn, R. N.; Cunningham, V. J.; Owens,
J.; Wyper, D.; Ell, P. J.; Pilowsky, L. S. Kinetic modelling of [123I]CNS 1261 – a
potential SPET tracer for the NMDA receptor. Nucl. Med. Biol. 2003, 30, 441–454.
14. Zhao, Y.; Robins, E.; Turton, D.; Brady, F.; Luthra, S. K.; Årstad, E. Synthesis and
characterization
of
N-(2-chloro-5-methylthiophenyl)-N′-(3-methylthiophenyl)-N′-
[11C]methylguanidine [11C]CNS 5161, a candidate PET tracer for functional imaging of
NMDA receptors. J. Labelled Compd. Radiopharm. 2006, 49, 163–170.
15. Robins, E. G.; Zhao, Y.; Khan, I.; Wilson, A.; Luthra, S. K.; Årstad, E. Synthesis and in
vitro evaluation of 18F-labelled S-fluoroalkyl diarylguanidines: novel high-affinity
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