N1H-and N1-Substituted Phenylguanidines as α7 Nicotinic Acetylcholine (nACh) Receptor Antagonists: Structure-Activity Relationship Studies

Article abstract of DOI:10.1021/acschemneuro.1c00212

We previously reported that N-(3-chlorophenyl)guanidine (1) represents a novel α7 nicotinic ACh (nACh) receptor antagonist chemotype. In the present study, a small series of compounds was synthesized with the intent to investigate the structure-Activity relationship (SAR). Preliminary data suggested that the N-methyl analog of 1, 2, was several times more potent. Therefore, the chloro group at the aryl 3-position of 1 and its N1-methyl counterpart 2 were replaced with a number of substituents considering the electronic, lipophilic, and steric nature of the substituents. The potencies of the compounds to inhibit acetylcholine (ACh)-induced responses were obtained in Xenopus laevis oocytes expressing human α7 nicotinic ACh receptors (nAChRs) using a two-electrode voltage-clamp assay. We found that the nature of the 3-position substituents had relatively little (i.e., <10-fold) effect on potency, and the presence of an N1-isopropyl substituent was tolerated. Here, we report the first SAR investigation of this novel α7 nAChR antagonist chemotype.

Full text of DOI:10.1021/acschemneuro.1c00212

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Research Article
N1H- and N1‑Substituted Phenylguanidines as α7 Nicotinic
Acetylcholine (nACh) Receptor Antagonists: Structure−Activity
Relationship Studies
Osama I. Alwassil, Shailesh Khatri, Marvin K. Schulte, Sanjay S. Aripaka, Jens D. Mikkelsen,
and Małgorzata Dukat*
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ABSTRACT: We previously reported that N-(3-chlorophenyl)guanidine (1) represents a
novel α7 nicotinic ACh (nACh) receptor antagonist chemotype. In the present study, a small
series of compounds was synthesized with the intent to investigate the structure−activity
relationship (SAR). Preliminary data suggested that the N-methyl analog of 1, 2, was several
times more potent. Therefore, the chloro group at the aryl 3-position of 1 and its N1-methyl
counterpart 2 were replaced with a number of substituents considering the electronic,
lipophilic, and steric nature of the substituents. The potencies of the compounds to inhibit
acetylcholine (ACh)-induced responses were obtained in Xenopus laevis oocytes expressing
human α7 nicotinic ACh receptors (nAChRs) using a two-electrode voltage-clamp assay. We
found that the nature of the 3-position substituents had relatively little (i.e., <10-fold) effect
on potency, and the presence of an N1-isopropyl substituent was tolerated. Here, we report
the first SAR investigation of this novel α7 nAChR antagonist chemotype.
KEYWORDS: α7 nAChR, SAR, electrophysiology, antagonist, phenylguanidines
and examined a series of N1-methyl analogs (Series II) and (ii)
applied a Topliss approach9 to examine several additional N1-
alkyl analogues.
INTRODUCTION
■
Agents that antagonize the effect of acetylcholine (ACh) at
nicotinic ACh receptors (nAChRs) are of interest for the
treatment of neuropsychiatric,1,2 neuroinflammatory,3 and
neurodegenerative disorders and as a novel therapeutic strategy
for the treatment of cancer.4,5 We have previously identified m-
chlorophenylguanidine (mCPG, 1) as a novel noncompetitive
α7 nAChR antagonist chemotype.6 That is, although 1 lacks
affinity for α7 nAChR (i.e., Ki > 100 μM at [125I]iodo-MLA-
labeled receptors), it blocked the actions of ACh in
electrophysiological and in vivo functional assays.6
CHEMISTRY
■
The phenylgunidine Series I analogues 1 and 3−9 were
available from previous studies.10,11 The N-methyl-N-phenyl-
guanidine series analogues 10−16 were synthesized in a one-
step reaction, as shown in Scheme 1, from their commercially
available or previously reported N-methylanilines. Hydro-
chloride salts of the appropriate N-methylanilines (i.e., 23−29;
Scheme 1) were heated at reflux with cyanamide in absolute
ethanol to yield the corresponding guanidines 10−16 as their
hydrochloride salts. The hygroscopic hydrochloride salt of N-
(3-methylphenyl)-N-methylguanidine (15) was converted to
the nitrate salt with ammonium nitrate.
Commercially available anilines 32 and 34 were used in the
preparation of 17 and 19, respectively (Scheme 2). 3-Chloro-
N-ethylaniline (33) was prepared via alkylation of 31 through a
reductive amination step using acetaldehyde and pyridine-
In an effort to better understand the action of 1 as a
noncompetitive α7 nAChR antagonist, we initiated a
structure−activity investigation. In a preliminary study, we
found that the N1-methyl analogue of 1 (i.e., 2) was several
times as potent as 1.7,8 Here, we examined a series of N1−H 3-
substituted phenylguanidines (Series I). Furthermore, knowing
now that the N-methyl analogue 2 is active, (i) we prepared
Received: April 7, 2021
Accepted: May 17, 2021
Published: May 27, 2021
https://doi.org/10.1021/acschemneuro.1c00212
© 2021 American Chemical Society
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a
For Series I and II, inhibitory potencies varied over a very
narrow (<10-fold) range (Table 1). In both series, the aryl-
unsubstituted compounds were slightly less potent (i.e., 3 and
10), and the 3-iodo compounds (i.e., 5 and 12) were
somewhat more potent than chloro compounds 1 and 2,
respectively. An apparent discrepancy between the two series
was the difference in potency between 3-CH3 analogues 8 and
15; whereas 8 is the least potent member in Series I, 15 is
equipotent with 2. As a consequence, data for these
compounds were replicated and confirmed.
Scheme 1. Synthetic Route for N1-Methylphenylguanidines
and Structures of Phenylguanidines 3−9 Series I
Parallel substituent modifications at the aryl 3-position of
the two series were examined to determine if the two series
might bind in a similar fashion. That is, if the two series are
binding in a common manner, a relationship might exist
between their potencies, and it would be expected that their
potencies would covary.13 Linear regressions and statistical
analyses implemented here were conducted using GraphPad
Prism software (version 5.04),14 SPSS (Statistical Package for
the Social Sciences; version 22.0), and JMP (John’s Macintosh
Program; version 11.2). Correlations expressing a p value of
less than 0.05 were considered significant. A poor correlation
(r = 0.474; n = 8) was found between pIC50 values of the
Series I versus pIC50 values of the N1-methyl Series II
compounds (Figure 3).
Follow-up statistical analysis was conducted to detect any
potential outliers among the inhibitory potency values using
Cook’s D and Z score methods, specifically for the 3-CH3
compounds (see Supporting Information). All IC50 values of
the tested compounds, including the 3-CH3 analogues,
appeared to be within the acceptable range, and no single
value exceeded the cutoffs.
If a methyl substituent is shown to improve the action of a
compound in a given assay, certain other specific alkyl
analogues should be examined as proscribed by the Topliss
Operational Scheme.9 These analogues include ethyl, iso-
propyl, and cyclopentyl.9 Accordingly, these three N1-alkyl
analogues of 1 and its des-chloro counterpart 3 were explored.
Figure 4 and Table 2 show data for these compounds; data for
3, 1, 10, and 2, from Table 1, are reproduced here for
comparison.
a
Reagents and conditions: (a) i. NH2CN, EtOH, reflux; ii. (only for
15) NH4NO3, H2O.
borane as previously reported12 and then utilized in the
synthesis of 18. For the preparation of 20 (Scheme 2), 3-
chloroaniline (31) was treated with 2-bromopropane in the
presence of an aqueous solution of NaOH, following the
procedure of Koelsch and Britain22 to yield the 3-chloro-N-
isopropylaniline (35) followed by the condensation reaction
with cyanamide. N-Cyclopentyl-N-phenylguanidine hydro-
chloride (21) and N-(3-chlorophenyl)-N-cyclopentylguanidine
nitrate (22) were prepared in a reductive amination reaction
(Scheme 2) of aniline (30) and 3-chloroaniline (31),
respectively, and cyclopentanone in the presence of STAB
(sodium triacetoxyborohydride). The hydrochloride salts of
the alkylated anilines (i.e., 36 and 37, respectively) were then
heated at reflux with cyanamide to give 21 and 22. The
hygroscopic 22 hydrochloride salt was converted to the nitrate
salt using ammonium nitrate.
1
The structures of targets 11−22 were confirmed by IR, H
NMR, and elemental analysis for C, H, N.
RESULTS
The isopropyl analogues 19 and 20 were about twice as
potent as 3 and 1. Cyclopentyl analogues 21 and 22 were
much less potent.
For the analysis of binding characteristics of selected
compounds (i.e., 2 and 12) to the native α7 nAChR in the
■
The nAChR antagonist actions of all compounds are shown in
Figures 1, 2, and 4 and are reported in the form of half-
maximal inhibitory concentrations (i.e., IC50 values, Tables 1
and 2).
a
Scheme 2. Synthesis of Compounds 17−22
a
Reagents and conditions: (a) acetaldehyde, pyridine-borane, MeOH, reflux; (b) 2-bromopropane, NaOH (15% aqueous), ZnCl2 (saturated
aqueous), 150 °C; (c) cyclopentanone, STAB, acetic acid, DCM, room temperature; (d) HCl/Et2O; (e) i. NH2CN, EtOH, reflux; ii. (only for
compounds 19 and 22) NH4NO3, H2O.
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Figure 1. Xenopus oocytes expressing α7 nAChRs were coapplied with increasing concentrations of compounds (1, 3, 4, 5, 6, 7, 8, and 9) with a
fixed concentration of acetylcholine (100 μM). Data represent an n ≥ 4 using oocytes from at least 2 different frogs. The increasing concentrations
of compounds inhibited ACh induced responses as shown in dose response curves (1A and 1B) generated by using an inbuilt algorithm in
GraphPad Prism. Dose response curves in 1A were unconstrained; however, due to insufficient data at the higher concentration of compounds 6, 8,
and 9, the dose response curves in 1B were constrained to bottom = 0.
Figure 2. Xenopus oocytes expressing α7 nAChRs were coapplied with increasing concentrations of compounds (2, 10, 11, 12, 13, 14, 15, and 16)
with a fixed concentration of acetylcholine (100 μM). Data represent an n ≥ 4 using oocytes from at least 2 different frogs. The increasing
concentrations of compounds inhibited ACh induced responses as shown in dose response curves (2A and 2B) generated by using an inbuilt
algorithm in GraphPad Prism. Dose response curves in 2A were unconstrained; however, due to insufficient data at the higher concentration of
compound 14, the dose response curve in 2B is constrained to bottom = 0.
When assayed against seven different nAChR populations
(i.e., α2β2, α2β4, α3β2, α4β2, α3β4, α4β2, α4β4) and 5-HT3
receptors in radioligand binding studies, both parent 2 and the
most potent inhibitor identified in these studies, i.e., 12, lacked
binding affinity at all eight (i.e., Ki > 10,000 nM). Furthermore,
unlike 1 which binds at 5-HT3 receptors with high affinity (Ki
= 34 nM),10 compound 12 lacked affinity for this receptor
population.
DISCUSSION
■
Preliminary data suggested that 2 was several times more
potent than 1 as a noncompetitive nAChR antagonist.7,8
Figure 3. Plot of pIC50 values of the Series I versus the N1-methyl
Accordingly, we examined a series of 3-substituted 1-related
analogue Series II (r = 0.474, p = 0.235, n = 8).
compounds in comparison with their corresponding N1-methyl
counterparts (i.e., Series I and II) to better understand their
structure−activity relationship (SAR). We also employed a
brain, autoradiography was conducted. Images of sections
incubated with the same radiotracer, but with increasing
concentrations of compounds 2 and 12, showed that both
compounds inhibited binding of the selective radiotracer to the
native receptor site (Figure 5A). While compound 12 could
displace virtually all binding at 100 μM concentrations,
compound 2 only reduced the binding significantly at the
same concentration. Quantitative analysis of binding of
compound 2 (open bars) and 12 (squared) compared to
total binding at 100% and unspecific binding at 0% (1 mM
nicotine) showed that both compounds could displace the
radiotracer, but compound 12 was more effective (Figure 5B).
two-electrode voltage-clamp assay system.
Considering the functional data from 1 and its N-methyl
analogue 2,4 a small series of compounds was synthesized. The
chloro group at the aryl 3-position of N-(3-chlorophenyl)-
guanidine (1) and its N1-methyl counterpart 2 were replaced
with a number of substituents considering the electronic,
lipophilic, and steric nature of the substituents.15 The overall
intent was to determine the effects of these substituents on the
α7 nAChR action of 1 and 2 and, if possible, to conduct
quantitative structure−activity relationship (QSAR) studies.
The introduction of other halogen atoms (i.e., -F, -Br, and -I)
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Figure 4. Xenopus oocytes expressing α7 nAChRs were coapplied with increasing concentrations of compounds (17, 18, 19, 20, 21, and 22) with a
fixed concentration of acetylcholine (EC50). The increasing concentrations of compounds inhibited ACh induced responses as shown in dose
response curves (4A and 4B) generated by using an inbuilt algorithm in GraphPad Prism. Dose response curves in 4A were unconstrained;
however, due to insufficient data at the higher concentration of compounds 21 and 22, the dose response curves in 4B were constrained to bottom
= 0.
Table 1. Inhibitory Potencies (IC50 Values) of the 3-
Substituted Series I and the N1-Methyl Series II
Phenylguanidines for α7 nAChRs
Table 2. Inhibitory Potency (IC50 Values) of the N1-
Alkylphenylguanidines for α7 nAChRs
a
a
R
X
compound
IC50 [μM]
95% CI [μM]
IC50
95% CI
Series
II
IC50
95% CI
H
H
CH3
CH3
H
Cl
H
Cl
H
Cl
H
Cl
H
Cl
3
1
10
2
17
18
19
20
21
22
60
42
130
32
40
27
20
22
1000
960
41−90
26−65
99−159
16−63
35−46
23−31
11−37
10−47
870−1300
850−1100
[μM]
[μM]
Series I
X
[μM]
[μM]
42
60
24
21
170
21
26−65
41−90
20−28
14−34
57−520
14−32
570−1400
54−320
1
3
4
5
6
7
8
9
Cl
H
Br
I
2
32
130
30
13
73
102
35
58
16−63
99−159
18−51
6−26
36−151
34−310
22−56
14−230
10
11
12
13
14
15
16
C2H5
C2H5
CH(CH3)2
CH(CH3)2
cyclopentyl
cyclopentyl
F
CF3
CH3
OCH3
900
130
a
The data were obtained by using a nonlinear curve fitting algorithm
from the dose response curves shown in Figures 1 and 2. Data
represent an n ≥ 4 using oocytes from at least 2 different frogs.
a
The data were obtained by using nonlinear curve fitting algorithm
from the dose response curves shown in Figure 4.
at the 3-position will allow for testing mainly the effect of
substituent size variation over a relatively fixed range of
electron-withdrawing effects. A recent study on a number of α7
nAChR modulators described the diverse impact of different
halogen atoms at the same ring position on the compounds’
pharmacological properties.16 That is, alteration of the halogen
atom influenced not only potency but also the functional
action of the compound.
however, recorded potencies were expected to be lower
compared to those (e.g., for 1) from the previously used HEK
293 cells, because oocyte surface components, such as the large
number of invaginations in the membrane and the surrounding
vitelline membrane and follicle cells, decrease the accessibility
of the compounds.18 Nevertheless, the current assay should
allow a rapid determination of SAR.
In Series I, the potencies of the analogues as hα7 nAChR
antagonists only varied over a 6-fold range, and the most
potent compounds (i.e., 4, 5, 7) were only twice as potent as 1.
The 3-position substituents were varied in a manner (i.e., with
varying physicochemical properties) such that a QSAR study
might be conducted. However, the narrow range of potencies
precluded a QSAR study.
Series I substituents were also examined in the N1-methyl
series with the expectation that Series II might be somewhat
more potent. This turned out not to be the case. For Series II,
potencies varied only over a 10-fold range. Both Series I and II
have a single bond between the phenyl ring and the anilinic N
atom; thus, they can easily rotate. We have previously shown
that 1 can exist in four low-energy rotamers. Furthermore, if
Our previous findings7,8 for investigating the inhibitory
effect of phenylguanidines at rat α7 nAChRs were obtained
using the whole-cell configuration of the patch-clamp
technique on cells expressed in stably transfected human
embryonic kidney (HEK) 293 cells. Functional data in the
current project were obtained by evaluating the potencies of
the compounds in inhibiting ACh-induced responses in
Xenopus laevis oocytes expressing human α7 nAChRs using
an automated two-electrode voltage-clamp assay incorporating
rapid perfusion (20 mL/min) and a vertical flow recording
chamber. This system has been demonstrated to provide a
more rapid solution exchange and enhanced rise times in
response to an agonist.17 Despite the rapid exchange time,
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Figure 5. (A) Representative images of rat brain sections exposed to [125I]-α-bungarotoxin (Bgtx) alone or together with 1 mM nicotine showing
total and unspecific binding to α7 nAChR binding sites. As previously demonstrated, specific binding is observed in the cerebral cortex, the
hippocampus, and the hypothalamic mammillary nuclei. Images of sections incubated with the same radiotracer, but increasing concentrations of
compounds 2 and 12, showed that both compounds inhibited binding of the selective radiotracer to the native receptor site at high concentrations.
The autoradiograms revealed that, while compound 12 displaced binding in all areas at 100 μM concentration, significant binding remained in
high-binding areas such as the ventral hippocampus and the mammillary nuclei when displaced with compound 2 at the same concentration. (B)
Quantitative analysis of binding in the rat brain section(s) of compound 2 (open bars) and 12 (squared) compared to total binding (black bar) at
100% and unspecific binding at 0% (1 mM nicotine) in three brain sections. Dots represent the individual results of three separate experiments.
The graph showed that both compounds could displace the radiotracer; however, where compound 12 nearly eliminated binding, specific binding
remained after displacement with compound 2.
two series of compounds bind to the receptor in the same
manner, parallel structural changes in both series should result
in parallel shifts in affinity/activity, and the correlation would
be of 1 or close to 1. Figure 3 shows that there is no correlation
between activities in these two series (statistically insignificant
correlation). Combined, the rotameric existence of the
compounds and the poor correlation of their activities support
our speculations that compounds might interact with the
receptors in a slightly different manner. In addition, follow-up
studies with 2 and 12 (Figure 5) showed that their actions
cannot be solely attributed to competitive antagonism, because
concentrations exceeding their EC50 values (Table 1) were
required to substantially displace the radioligand.
Following the Topliss approach, several N1-alkylphenylgua-
nidines were examined, and N1-isopropyl analogues 19 and 20
were more potent than their N1H parents 3 and 1, respectively.
Compounds with a larger N1-cyclopentyl substituent (i.e., 21,
22) displayed considerably reduced potency indicating limited
tolerance for N1 bulk.
However, unlike 1, it showed little affinity (Ki > 10 000 nM)
for other nACh or 5-HT3 receptors.
Because rotameric binding might obfuscate the results of this
study, future studies should focus on 4-substituted phenyl-
guanidines. In addition, particular attention should be paid to
the possibility that N1H and N1-alkyl derivatives might interact
with the receptors in a slightly different manner.
EXPERIMENTAL SECTION
■
Synthesis. Melting points (mps) were taken in glass capillary
tubes using a Thomas-Hoover melting point apparatus and are
uncorrected. Proton nuclear magnetic resonance (1H NMR) spectra
were obtained using a Bruker ARX 400 MHz spectrometer at which
peak positions are given in parts per million (δ) downfield from the
internal standard tetramethylsilane (TMS), followed by the splitting
pattern (s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet
of doublets, m = multiplet), coupling constant (Hz), and integration.
Infrared spectra were obtained on a Thermo Nicolet iS10 FT-IR.
Purity of compounds was determined by elemental analysis performed
by Atlantic Microlab Inc. (Norcross, GA) for the indicated elements,
and the obtained values are within 0.4% of theoretical values.
Reactions were monitored by thin-layer chromatography (TLC) on
silica gel GHLF plates (250 μm, 2.5 cm × 10 cm; Analtech Inc.
Newark, DE), and Flash chromatography was performed on a
CombiFlash Companion/TS (Teledyne Isco Inc. Lincoln, NE) using
packed silica gel (Silica Gel 230−400 mesh) columns (RediSep Rf
Normal-phase Silica Flash Column, Teledyne Isco Inc., Lincoln, NE).
Electrospray ionization-mass spectroscopy (ESI-MS) profiles were
recorded using a Waters Acquity TQD (tandem quadrupole)
spectrometer in positive ion mode.
CONCLUSIONS
■
Compound 1 is a novel α7 nAChR antagonist chemotype, and
various other phenylguanidines are shown here to retain this
action. Overall, however, it would seem that (i) the nature of
the 3-position substituent has relatively little (i.e., <10-fold)
effect on potency, and (ii) the presence of an N1-isopropyl
substituent doubles the potency of 1. The most potent
compound identified, 12, was only 3-fold more potent than 1.
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The nitrate salts of m-chlorophenylguanidine (1) and 7, the
hydrochloride salts of 2, phenylguanidine (PG; 3), 4−6, 8, 10, and
the hemisulfate salt 9 were available from previous studies10,11 and
used as such. N-Ethyl-N-phenylguanidine hydrochloride (17) was
prepared according to a literature procedure.19
N-(3-Bromophenyl)-N-methylguanidine Hydrochloride (11). Cy-
anamide (154 mg, 3.66 mmol) was added to a solution of 3-bromo-N-
methylaniline hydrochloride (24) (407 mg, 1.83 mmol) in absolute
EtOH (5 mL). The reaction mixture was allowed to stir and heated at
reflux for 24 h; then, the solvent was removed under reduced pressure.
The resultant oily residue was crystallized with H2O to give 178 mg
(37%) of the desired product as white crystals: mp 278−280 °C; IR
(diamond, cm−1): 3118 (NH), 3276 (NH2); 1H NMR (DMSO-d6): δ
3.26 (s, 3H, CH3), 7.4 (qd, J = 8.06, 1.00 Hz, 1H, ArH), 7.47 (t, J =
7.96 Hz, 1H, ArH), 7.64 (td, J = 7.92, 1.32 Hz, 1H, ArH), 7.68 (t, J =
1.86 Hz, 1H, ArH). Anal. Calcd (C8H10BrN3·HCl) C, 36.32; H, 4.19;
N, 15.88. Found: C, 36.23; H, 4.28; N, 15.77.
N-(3-Iodophenyl)-N-methylguanidine Hydrochloride (12). This
was prepared in the same manner as that of compound 11 from 3-
iodo-N-methylaniline hydrochloride (25)20 except that the product
crystallized without the addition of H2O. The residue was
recrystallized from absolute EtOH to give 25% of the desired product
as brown crystals: mp 285−287 °C; IR (diamond, cm−1): 3121 (NH),
3280 (NH2); 1H NMR (DMSO-d6): δ 3.25 (s, 3H, CH3), 7.30 (t, J =
8.16 Hz, 1H, ArH), 7.41 (td, J = 8.00, 1.50 Hz, 1H, ArH), 7.79 (m,
1H, ArH), 7.80 (m, 1H, ArH). Anal. Calcd (C8H10IN3·HCl) C, 30.84;
H, 3.56; N, 13.49. Found: C, 31.07; H, 3.71; N, 13.28.
N-(3-Chlorophenyl)-N-ethylguanidine Hydrochloride (18). Cyan-
amide (0.13 g, 3.12 mmol) was added to a solution of 3-chloro-N-
ethylaniline hydrochloride (33)12 (0.3 g, 1.56 mmol) in absolute
EtOH (10 mL). The stirred reaction mixture was heated at reflux for
24 h. Upon cooling, the reaction mixture was evaporated under
reduced pressure to obtain a residue that was recrystallized from a
mixture of absolute EtOH/Et2O to give 0.048 g (13%) of the desired
product as white crystals: mp 186−189 °C; IR (diamond, cm−1):
3077, 3187 (NH), 3274 (NH ); 1H NMR (DMSO-d6): δ 1.07 (t, J =
2
7.16 Hz, 3H, CH ) 3.66 (q, J = 7.12, 7.16 Hz, 2H, CH ), 7.34 (m, 1H,
3
2
ArH), 7.54 (m, 3H, ArH). Anal. Calcd (C9H12ClN3·HCl) C, 46.17;
H, 5.60; N, 17.95. Found: C, 45.93; H, 5.64; N, 17.72.
N-Isopropyl-N-phenylguanidine Nitrate (19). An aqueous sol-
ution of cyanamide (50%; 1.5 mL) was added to a solution of N-
isopropylaniline hydrochloride (34) (100 mg, 0.58 mmol) in absolute
EtOH (9 mL). The stirred reaction mixture was heated at reflux for
25 h and then cooled to 0 °C (freezer) for 20 h. Solvent was removed
under reduced pressure, and the resulting oily residue was dissolved in
H2O (1 mL) followed by addition of NH4NO3 (108 mg, 1.34 mmol).
The solution was concentrated under reduced pressure, and the
residue was dissolved in H2O (5 mL) and washed with Et2O (3 × 20
mL), followed by evaporation of H2O under reduced pressure. The
resulting solid was recrystallized from H2O and then from absolute
EtOH to give 30 mg (21%) of the desired product as white crystals:
mp 164−165 °C; IR (diamond, cm−1): 2159, 3172 (NH), 3319
(NH2); 1H NMR (DMSO-d6): δ 1.05 (d, J = 6.52 Hz, 6H, CH3), 4.4
(m, 1H, CH), 7.3 (m, 2H, ArH), 7.6 (m, 3H, ArH). Anal. Calcd
(C10H15N3·HNO3) C, 49.99; H, 6.71; N, 23.32. Found: C, 49.85; H,
6.64; N, 23.27.
N-(3-Chlorophenyl)-N-isopropylguanidine Hydrochloride (20).
Cyanamide (1.02 g, 24.26 mmol) was added to a solution of 3-
chloro-N-isopropylaniline (35)22 (2.50 g, 12.13 mmol) in absolute
EtOH (30 mL). The reaction mixture was allowed to stir at reflux for
24 h. The solution was concentrated under reduced pressure, and
anhydrous Et2O (25 mL) was added. The white precipitate was
collected by filtration and recrystallized from i-PrOH to give 0.51 g
(17%) of the desired product as white crystals: mp 272−274 °C; IR
(diamond, cm−1): 3108 (NH), 3269 (NH2); 1H NMR (DMSO-d6): δ
1.04 (d, J = 6.56 Hz, 6H, CH3), 4.36 (m, 1H, CH), 7.28 (td, J = 7.72,
1.62 Hz, 1H, ArH), 7.45 (t, J = 1.84 Hz, 1H, ArH), 7.58 (m, 2H,
ArH). Anal. Calcd (C10H14ClN3·HCl) C, 48.40; H, 6.09; N, 16.93.
Found: C, 48.68; H, 6.11; N, 17.06.
N-(3-Fluorophenyl)-N-methylguanidine Hydrochloride (13). This
was prepared in the same manner as that of compound 11 from 3-
fluoro-N-methylaniline hydrochloride (26). The residue was recrystal-
lized from i-PrOH to give 22% of the desired product as white
crystals: mp 197−199 °C; IR (diamond, cm−1): 3123 (NH), 3288
1
(NH2); H NMR (DMSO-d6): δ 3.27 (s, 3H, CH3), 7.24 (dd, J =
7.36, 0.96 Hz, 1H, ArH), 7.29 (td, J = 8.60, 2.40 Hz, 1H, ArH), 7.35
(d, J = 9.88 Hz, 1H, ArH), 7.56 (q, J = 8.06 Hz, 1H, ArH). Anal.
Calcd (C8H10FN3·HCl) C, 47.18; H, 5.44; N, 20.63. Found: C, 47.08;
H, 5.57; N, 20.84.
N-(3-Trifluoromethyl)-N-methylguanidine Hydrochloride (14).
This was prepared in the same manner as that of compound 11
from N-methyl-3-trifluoromethylaniline hydrochloride (27).21 The
residue was recrystallized from 1-butanol to give 21% of the target as
white crystals: mp 247−249 °C; IR (diamond, cm−1): 3120 (NH),
1
3282 (NH2); H NMR (DMSO-d6): δ 3.29 (s, 3H, CH3), 7.72 (m,
2H, ArH), 7.79 (d, J = 7.68 Hz, 1H, ArH), 7.82 (s, 1H, ArH). Anal.
Calcd (C9H10F3N3·HCl) C, 42.62; H, 4.37; N, 16.57. Found: C,
42.66; H, 4.30; N, 16.52.
N-Cyclopentyl-N-phenylguanidine Hydrochloride (21). Cyana-
mide (0.64 g, 15.17 mmol) was added to a solution of N-
cyclopentylaniline hydrochloride (36)23 (1.5 g, 7.58 mmol) in
absolute EtOH (15 mL). The stirred reaction mixture was heated
at reflux for 24 h. After it cooled, the solvent was evaporated under
reduced pressure to obtain a residue that was washed with
cyclohexane and recrystallized from THF to give 0.15 g (9%) of
the desired product as white crystals: mp 209−211 °C; IR (diamond,
N-(3-Methylphenyl)-N-methylguanidine Nitrate (15). This was
prepared in the same manner as that of compound 11 from 3-methyl-
N-methylaniline hydrochloride (28). The resultant oily residue which
failed to crystallize was dissolved in H2O followed by addition of
NH4NO3. The solvent was removed under reduced pressure, and the
resultant semisolid was recrystallized from H2O and then from a
mixture of i-PrOH/Et2O to give a 6% yield of the target as white
crystals: mp 139−141 °C; IR (diamond, cm−1): 3172 (NH), 3345
cm−1): 2956, 3117 (NH), 3276 (NH ); 1H NMR (DMSO-d6): δ 1.20
2
(m, 2H, CH ), 1.43 (m, 4H, CH ), 1.92 (m, 2H, CH ), 4.31 (m, 1H,
2
2
2
1
CH), 7.28 (m, 2H, ArH), 7.53 (m, 3H, ArH). Anal. Calcd
(C9H12ClN3·HCl·0.1 C6H12) C, 65.94; H, 9.06; N, 13.44. Found:
C, 65.98; H, 9.13; N, 13.15.
(NH2); H NMR (DMSO-d6): δ 2.34 (s, 3H, CH3), 3.25 (s, 3H,
CH3), 7.16 (d, J = 7.88 Hz, 1H, ArH), 7.19 (s, 1H, ArH), 7.26 (d, J =
7.60 Hz, 1H, ArH), 7.40 (t, J = 7.72 Hz, 1H, ArH). Anal. Calcd
(C9H13N3·HNO3) C, 47.78; H, 6.24; N, 24.77. Found: C, 47.69; H,
6.29; N, 24.86.
N-(3-Chlorophenyl)-N-cyclopentylguanidine Nitrate (22). Cyan-
amide (0.72 g, 17.23 mmol) was added to a solution of 3-chloro-N-
cyclopentylaniline hydrochloride (37) (2.0 g, 8.61 mmol) in absolute
EtOH (15 mL). The stirred reaction mixture was heated at reflux for
24 h. The solvent was removed under reduced pressure, and the
residue was dissolved in H2O (8 mL). The solution was washed with
Et2O (3 × 20 mL), followed by evaporation of H2O under reduced
pressure. The resultant oily residue was dissolved in H2O (8 mL),
followed by addition of NH4NO3 (0.12 g, 1.44 mmol). The solvent
was evaporated under reduced pressure, and the resultant semisolid
was recrystallized from EtOH to give 0.34 g (15%) of the desired
product as white crystals: mp 225−227 °C; IR (diamond, cm−1):
N-(3-Methoxyphenyl)-N-methylguanidine Hydrochloride (16).
This was prepared in the same manner as that of compound 11
from 3-methoxy-N-methylaniline hydrochloride (29). The resulting
residue was recrystallized from a mixture of absolute EtOH/Et2O to
give 47% of the desired product as off-white crystals: mp 219−221
°C; IR (diamond, cm−1): 3122 (NH), 3292 (NH2); 1H NMR
(DMSO-d6): δ 3.27 (s, 3H, CH3), 3.79 (s, 3H, CH3), 6.93 (dq, J =
7.80, 0.72 Hz, 1H, ArH), 6.98 (t, J = 2.14 Hz, 1H, ArH), 7.01 (dt, J =
8.28, 1.84 Hz, 1H, ArH), 7.42 (t, J = 8.06 Hz, 1H, ArH). Anal. Calcd
(C9H13N3O·HCl) C, 50.12; H, 6.54; N, 19.48. Found: C, 50.12; H,
6.60; N, 19.45.
1
2969, 3175 (NH), 3346 (NH ); H NMR (DMSO-d6): δ 1.19 (m,
2
2199
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Research Article
followed by short washes in ice-cold binding buffer, and they were
rinsed briefly in ice-cold distilled water. Finally, the slides were dried
for 24 h and, together with [125I] microscale standards, exposed to
FUJI imaging phosphor plates overnight. The FUJI imaging plates
were scanned by Fujifilm Image Reader (BAS-2500 V1.8), and
autoradiograms were analyzed with ImageJ software (Version 2.0.0,
NIH). Nonspecific binding was determined by adding 1 mM S(−)-
nicotine (Sigma-Aldrich, Denmark) in the incubation solution.
Competition Binding Assay. Binding assays for 2 and 12 were
performed by the Psychoactive Drug Screening Program (PDSP). For
experimental details, see the PDSP website http://pdsp.med.unc.edu/
and click on “Binding Assay”.
2H, CH ), 1.45 (m, 4H, CH ), 1.94 (m, 2H, CH ), 4.27 (m, 1H, CH),
2
2
2
7.29 (dd, J = 1.64, 7.52 Hz, 1H, ArH), 7.48 (s, 1H, ArH), 7.55 (m,
2H, ArH). Anal. Calcd (C12H16ClN3·HNO3) C, 47.93; H, 5.70; N,
18.63. Found: C, 48.06; H, 5.59; N, 18.61.
3-Chloro-N-cyclopentylaniline Hydrochloride (37). Cyclopenta-
none (2.8 mL, 31.35 mmol), acetic acid (0.6 mL, 15.68 mmol), and
sodium triacetoxyborohydride (6.6 g, 31.35 mmol) were added to a
solution of 3-chloroaniline (31) (1.6 mL, 15.68 mmol) in dichloro-
methane (15 mL), and the reaction mixture was allowed to stir at
room temperature for 1.5 h. The mixture was washed with saturated
aqueous sodium bicarbonate solution (3 × 25 mL), and the solvent
was evaporated under reduced pressure and dried under vacuum for 7
h to yield 2.7 g of the free base of 37 as a yellow oil. The free base was
dissolved in Et2O, and a saturated solution of HCl in Et2O (50 mL)
was added. The precipitate was collected by filtration and recrystal-
lized from EtOAc to give 3.0 g (98%) of the product as white crystals:
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.1c00212.
■
sı
1
mp 115−117 °C; IR (diamond, cm−1): 1589, 2628 (NH); H NMR
(DMSO-d6): δ 1.58 (m, 4H, CH ), 1.72 (m, 2H, CH ), 1.88 (m, 2H,
2
2
Statistical analysis of potential outliers using Cook’s D
and Z score methods (PDF)
CH ), 3.78 (m, 1H, CH), 7.01 (m, 3H, ArH), 7.27 (m, 1H, ArH).
2
The product was used without further characterization for synthesis of
compound 22.
Electrophysiology. Functional characterization of all the
compounds were performed using two-electrode voltage-clamp
assay using Xenopus oocytes that expressed α7 receptors as described
previously.24−27 Briefly, ovarian lobes were surgically harvested from
X. laevis and washed twice in Ca2+-free Barth’s solution (82.5 mM
NaCl, 2.5 mM KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.4). These
lobes were enzymatically defolliculated to release oocytes by shaking
for 90 min in 1.5% collagenase (Sigma-Aldrich) dissolved in Ca2+-free
Barth’s Buffer. Oocytes were washed, and debris were separated and
stored at 19 °C until needed for microinjection. cRNA was
synthesized in vitro using an appropriate cDNA and the mMESSAGE
MACHINE mRNA synthesis kit (Ambion). Each oocyte was injected
with 50 nL of mRNA (0.3 μg/μL concentration) and incubated at 19
°C for at least 24−36 h prior to electrophysiological recording.
Oocytes were placed in the recording chamber perfused with ND-96
buffer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 2
mM phosphate, pH 7.4) at a flow rate of 20 mL/min. Two electrodes
(1−2 MΩ) filled with 3 M KCl corresponding to the current and
voltage electrodes were inserted, and the holding potential was
clamped at −60 mV using a Warner instruments amplifier. Ligands
were prepared in ND-96 buffer and then injected into the chamber.
Current responses were recorded using an Axon Instruments A/D
board and PClamp software. The oocyte recording system used
automated sampling and injection to maintain consistent perfusion
rates and times and a vertical recording chamber that provides
enhanced stability and the ability to use higher flow rates. This system
has been described and evaluated in a prior publication.17 A dose
response curve was determined using pooled peak amplitudes of
individual responses and analyzed using nonlinear curve fitting with
variable slope and constraints as mentioned in figures using GraphPad
Prism software.
AUTHOR INFORMATION
Corresponding Author
■
Małgorzata Dukat − Department of Medicinal Chemistry,
School of Pharmacy, Virginia Commonwealth University,
Richmond, Virginia 23298, United States; orcid.org/
0000-0003-4349-1615; Email: mdukat@vcu.edu
Authors
Osama I. Alwassil − Department of Medicinal Chemistry,
School of Pharmacy, Virginia Commonwealth University,
Richmond, Virginia 23298, United States; Present
Address: O.I.A.: Department of Pharmaceutical Sciences,
College of Pharmacy, King Saud bin Abdulaziz University
for Health Sciences, Riyadh 11426, Saudi Arabia.
Shailesh Khatri − Department of Pharmaceutical Sciences,
Philadelphia College of Pharmacy, University of the Sciences,
Philadelphia, Pennsylvania 19104, United States; Present
Address: S.K.: Department of Family and Community
Medicine, University of Kentucky, Lexington, KY 40503,
United States.
Marvin K. Schulte − Department of Pharmaceutical Sciences,
Philadelphia College of Pharmacy, University of the Sciences,
Philadelphia, Pennsylvania 19104, United States; Present
Address: M.K.S.: Department of Biomedical and
Pharmaceutical Sciences, College of Pharmacy, Kasiska
Division of Health Sciences, Idaho State University,
Pocatello, ID 83209, United States.
Sanjay S. Aripaka − Neurobiology Research Unit, University
Hospital Copenhagen, DK-2100 Copenhagen, Denmark
Jens D. Mikkelsen − Neurobiology Research Unit, University
Hospital Copenhagen, DK-2100 Copenhagen, Denmark;
Institute of Neuroscience, University of Copenhagen, DK-
1017 Copenhagen, Denmark
Compounds were initially applied alone, in the absence of ACh, to
determine whether they were capable of activating the receptors
(agonist action). None of the test compounds showed any ability to
activate receptors when applied alone at concentrations up to 1 mM.
Test compounds were then evaluated for their ability to inhibit ACh
induced responses (antagonist action) by coapplication of ACh with
increasing concentrations of above compounds, and IC50 values were
determined. Competition experiments used a standard concentration
of 100 μM ACh.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.1c00212
Autoradiography. Here, 20 μm of rat brain cryostat sections
Author Contributions
(Sprague−Dawley male) were incubated for 2 h in 0.5 nM
O.I.A. and S.K. are co-first authors. M.D. conceived the idea,
supervised the work, and prepared the first draft of the
manuscript. O.I.A. synthesized all N1-phenylguanidine ana-
logues under the supervision of M.D. S.K. performed
electrophysiological studies on α7 nAChR receptors under
the supervision of M.K.S. S.S.A. performed the autoradio-
[
125I]Tyr54-monoiodo-α-bungarotoxin (2200 Ci/mmol, PerkinElm-
er) in 50 mM Tris buffer, pH 7.3, to assess total binding as described
earlier.28 For determination of displacement, increasing concen-
trations of compounds 2 and 12 were added to the incubation. After
incubation with the radiotracer with and without the selected
compounds, the slides were briefly washed in the same buffer,
2200
https://doi.org/10.1021/acschemneuro.1c00212
ACS Chem. Neurosci. 2021, 12, 2194−2201

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Research Article
relationships for the binding of arylpiperazines and arylbiguanides at
5-HT3 serotonin receptors. J. Med. Chem. 39, 4017−4026.
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K., Smith, C., and Glennon, R. A. (2001) The binding of
arylguanidines at 5-HT3 serotonin receptors: A structure-affinity
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(12) Thorstensson, F., Kvarnstrom, I., Musil, D., Nilsson, I., and
Samuelsson, B. (2003) Synthesis of novel thrombin inhibitors. Use of
ring-closing metathesis reactions for synthesis of P2 cyclopentene-
and cyclohexenedicarboxylic acid derivatives. J. Med. Chem. 46, 1165−
1179.
graphic studies under the supervision of J.D.M. All authors
contributed to the preparation of this manuscript.
Funding
This work was supported in part by Award No. 12-2 from the
Commonwealth of Virginia’s Alzheimer’s and Related Diseases
Research Award Fund, administered by the Virginia Center on
Aging, Virginia Commonwealth University (M.D.).
Notes
The authors declare no competing financial interest.
(13) Portoghese, P. S. (1965) A new concept on the mode of
interaction of narcotic analgesics with receptors. J. Med. Chem. 8,
609−616.
ACKNOWLEDGMENTS
■
Ki values were generously provided by the National Institute of
Mental Health’s Psychoactive Drug Screening Program, no.
HHSN-271-2013-00017-C (NIMH PDSP). The NIMH PDSP
is Directed by Bryan L. Roth MD, PhD, at the University of
North Carolina at Chapel Hill and Project Officer Jamie
Driscol at NIMH, Bethesda, MD, USA. The authors are
grateful to Prof. Richard A. Glennon for discussions and proof
reading of the manuscript. Osama I. Alwassil was awarded a
scholarship from King Faisal University towards his Master of
Science degree at Virginia Commonwealth University.
(14) GraphPad Prism Software, version 7.04, San Diego, California,
USA.
(15) Hansch, C., Leo, A., Unger, S. H., Kim, K. H., Nikaitani, D., and
Lien, E. J. (1973) Aromatic” substituent constants for structure-
activity correlations. J. Med. Chem. 16, 1207−1216.
(16) Gill, J. K., Dhankher, P., Sheppard, T. D., Sher, E., and Millar,
N. S. (2012) A series of α7 nicotinic acetylcholine receptor allosteric
modulators with close chemical similarity but diverse pharmacological
properties. Mol. Pharmacol. 81, 710−718.
(17) Joshi, P. R., Suryanarayanan, A., and Schulte, M. K. (2004) A
vertical flow chamber for Xenopus oocyte electrophysiology and
automated drug screening. J. Neurosci. Methods 132, 69−79.
(18) Goldin, A. L. (2006) Expression of ion channels in Xenopus
Oocytes. In Expression and Analysis of Recombinant Ion Channels, pp
1−25, Wiley-VCH Verlag GmbH & Co. KGaA.
ABBREVIATIONS
■
nAChR, nicotinic acetylcholine receptor; mCPG, N-(3-
chlorophenylguanidine); SAR, structure−activity relationship;
STAB, sodium triacetoxyborohydride; Bgtx, [125I]-α-bungar-
otoxin
(19) Bianchi, M., and Barzaghi, F. (1964) Sintesi ed attivita’
farmacologiche di alcune guanidine N,N-bisostituite. Boll. Chim. Farm.
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convenient one pot procedure for N-methylation of aromatic amines
using trimethyl orthoformate. Synth. Commun. 27, 691−699.
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