Synthesis of a novel and potent small-molecule antagonist of PAC1 receptor for the treatment of neuropathic pain

Article abstract of DOI:10.1016/j.ejmech.2019.111902

We recently identified novel small-molecule antagonists of the PACAP type I (PAC1) receptor using docking-based in silico screening followed by in vitro/vivo pharmacological assays. In the present study, we synthesized 18 novel derivatives based on the structure of PA-9, a recently developed antagonist of the PAC1 receptor, with a view to obtain a panel of compounds with more potent antagonistic and analgesic activities. Among them, compound 3d showed improved antagonistic activities. Intrathecal injection of 3d inhibited both pituitary adenylate cyclase-activating polypeptide (PACAP) and spinal nerve ligation-induced mechanical allodynia. The effects were more potent than PA-9. Compound 3d also showed anti-allodynic effects following oral administration. Hence, our results suggest that 3d may become an orally available analgesic in the treatment of the neuropathic pain.

Full text of DOI:10.1016/j.ejmech.2019.111902

Journal Pre-proof
Synthesis of a novel and potent small-molecule antagonist of PAC1 receptor for the
treatment of neuropathic pain
Ichiro Takasaki, Haruna Ogashi, Takuya Okada, Ayaka Shimodaira, Daichi
Hayakawa, Ai Watanabe, Atsuro Miyata, Takashi Kurihara, Hiroaki Gouda, Naoki
Toyooka
PII:
S0223-5234(19)31054-2
DOI:
https://doi.org/10.1016/j.ejmech.2019.111902
EJMECH 111902
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 July 2019
Revised Date: 30 October 2019
Accepted Date: 18 November 2019
Please cite this article as: I. Takasaki, H. Ogashi, T. Okada, A. Shimodaira, D. Hayakawa, A. Watanabe,
A. Miyata, T. Kurihara, H. Gouda, N. Toyooka, Synthesis of a novel and potent small-molecule
antagonist of PAC1 receptor for the treatment of neuropathic pain, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111902.
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Synthesis of a novel and potent small-molecule antagonist of PAC1
receptor for the treatment of neuropathic pain
Ichiro Takasaki a, b, *, Haruna Ogashi c, Takuya Okada b, c, Ayaka Shimodaira a, Daichi
Hayakawa d, Ai Watanabe a, Atsuro Miyata e, Takashi Kurihara e, Hiroaki Gouda d, Naoki
Toyooka b, c,
*
a Department of Pharmacology, Graduate School of Science and Engineering, University of
Toyama, Toyama 930-8555, Japan
b Graduate School of Innovative Life Science, University of Toyama, Toyama 930-0194 and
930-8555, Japan
c Department of Bio-functional Molecular Engineering, Graduate School of Science and
Engineering, University of Toyama, Toyama 930-8555, Japan
d Department of Analytical and Physical Chemistry, School of Pharmacy, Showa University,
Tokyo 142-8555, Japan
e Department of Pharmacology, Graduate School of Medical and Dental Sciences, Kagoshima
University, Kagoshima 890-8544, Japan
* Corresponding authors;
Ichiro Takasaki, Ph.D. (in vitro/vivo Pharmacology)
Department of Pharmacology, Graduate School of Science and Engineering,
University of Toyama, Toyama 930-8555, Japan
Tel: +81-76-445-6875
E-mail; takasaki@eng.u-toyama.ac.jp
Naoki Toyooka, Ph.D. (Bioinformatics and Organic Synthesis)
Department of Bio-functional Molecular Engineering, Graduate School of Science and
Engineering, University of Toyama, Toyama 930-8555, Japan
Tel: +81-76-445-6859
E-mail; toyooka@eng.u-toyama.ac.jp
1

ABSTRACT
We recently identified novel small-molecule antagonists of the PACAP type I (PAC1)
receptor using docking-based in silico screening followed by in vitro/vivo pharmacological
assays. In the present study, we synthesized 18 novel derivatives based on the structure of PA-9,
a recently developed antagonist of the PAC1 receptor, with a view to obtain a panel of
compounds with more potent antagonistic and analgesic activities. Among them, compound 3d
showed improved antagonistic activities. Intrathecal injection of 3d inhibited both pituitary
adenylate cyclase-activating polypeptide (PACAP) and spinal nerve ligation-induced
mechanical allodynia. The effects were more potent than PA-9. Compound 3d also showed
anti-allodynic effects following oral administration. Hence, our results suggest that 3d may
become an orally available analgesic in the treatment of the neuropathic pain.
KEYWORDS
PACAP; PAC1 receptor; small-molecule antagonist; neuropathic pain; allodynia;
analgesics
2

1. Introduction
Pituitary adenylate cyclase-activating polypeptide (PACAP) was originally isolated from
ovine hypothalamic extracts based on its ability to stimulate adenylate cyclase in rat anterior
pituitary cell cultures [1]. PACAP exists as two variants, a 38-amino acid form (PACAP38)
and a 27-amino acid C-terminal truncated form (PACAP27) [1, 2]. Both peptides share 68%
homology with vasoactive intestinal polypeptide (VIP), indicating that PACAP belongs to the
VIP/secretin/glucagon superfamily [1, 2, 3]. Three distinct G-protein-coupled receptors
mediate the actions of PACAP and VIP. Unlike VIP, the two forms of PACAP bind to PACAP
type I (PAC1) receptor, which is coupled mainly to adenylate cyclase/protein kinase A, with
high affinity and selectivity. VPAC1 and VPAC2 receptors, also primarily coupled to adenylate
cyclase, however, interact with PACAP and VIP with similar affinities [3, 4].
Previously, we demonstrated that a single intrathecal (i.t.) injection of PACAP or
maxadilan (Max), a selective PAC1 receptor agonist [5, 6], induced transient nociceptive
behaviors followed by a long-lasting mechanical allodynia in mice [7, 8]. Furthermore, we
also demonstrated that the induction of PACAP- or Max-induced transient nociceptive
behaviors and mechanical allodynia were prevented by spinal pretreatment with max.d.4, a
peptide antagonist for the PAC1 receptor [7, 8]. These results suggest that the spinal
PACAP-PAC1 receptor systems play an important role in the modulation of spinal nociceptive
3

transmission and induction of chronic pain. Our findings, together with the previous studies,
indicate that the PAC1 receptor is a potential drug target for the treatment of both
inflammatory pain and neuropathic pain.
Recently, we identified novel small-molecule antagonists of the PAC1 receptor, named
PA-8, PA-9, and PA-10, using docking-based in silico screening followed by in vitro/vivo
pharmacological assays [9]. The results of the in vivo pharmacological assays demonstrated
that i.t. injection of these compounds blocked induction of PACAP-induced aversive responses
and mechanical allodynia in mice. In contrast, the compounds themselves exerted neither
agonistic nor algesic effects in the in vitro/vivo assays. We therefore concluded that PA-8, PA-9,
and PA-10 could be used as seed compounds for developing novel analgesics. The current study
focuses on the synthesis of 18 derivatives of PA-9 to obtain novel compounds exhibiting
improved antagonistic and analgesic activities. Among the newly synthesized derivatives, we
identified 3d as the compound with more potent antagonistic and analgesic activities than PA-9.
4

2. Results and discussion
2.1. Synthesis of PA-9 derivatives
Based on the analysis of the PA-9 structure and the docking model of the compound with
the PAC1 receptor (Fig. 1) [9], it was determined that the imidazole ring of the histamine
moiety in PA-9 would be important for the antagonistic and analgesic activities. This was
explained by the presence of hydrogen bonding between the S120 and L80 amino acid residues
of the PAC1 receptor and the imidazole functionality.
Fig. 1 Docking model of PA-9 with PAC1 receptor. Hydrogen-bonding interactions are
indicated by yellow dashed lines.
5

Hence, we designed a number of derivatives containing different aromatic substituents on
the nitrogen atom of the central lactam ring system, as shown in Scheme 1. Treatment of
itaconic acid with appropriate amines (1a-r) afforded lactams (2a-r). Condensation of 2a-r
with histamine resulted in the formation of the corresponding amides (3a-r).
Scheme 1: Synthesis of PA-9 derivatives.
2.2. In vitro pharmacological assay
In order to evaluate the antagonistic activity of the 18 synthesized compounds (3a-r,
Scheme 1) on the PAC1 receptors, we examined their effects on the PACAP-induced
phosphorylation of CREB in PAC1 receptor-expressing CHO (PAC1/CHO) cells. PA-9 [9]
6

was used as a control antagonist. PACAP (10 pM to 1 µM) dose-dependently induced CREB
phosphorylation (data not shown) and a submaximal dose of PACAP (1 nM) was employed
for the pharmacological assays. Pretreatment with PA-9 (1 nM) significantly inhibited PACAP
(1 nM)-induced CREB phosphorylation (Fig. 2), which is consistent with our previous report
[9]. Among the 18 derivatives (3a-3r), compound 3d (1 nM) considerably attenuated PACAP
(1 nM)-induced CREB phosphorylation (Fig. 2). Moreover, the inhibitory effects of 3d
(11.9% ± 8.5%) were more potent than those of PA-9 (30.6% ± 11.2%).
Fig. 2 Effects of 18 derivative compounds (3a-3r) on the PACAP-induced phosphorylation
of CREB in the PAC1/CHO cells. The cells were incubated with PA-9 (1 nM) or each
compound (1 nM) for 30 min, then the cells were stimulated with PACAP (1 nM) for 30 min
and lysed. The lysates containing equivalent amounts of protein (20 µg) were subjected to
SDS-PAGE. Immunoblots were probed with specific antibodies that recognize phosphorylated
form of CREB, and total CREB. The data represent the mean ± SEM (n = 4-5). *P < 0.05 vs.
DMSO (DM) control.
7

Next, we examined the dose-dependencies of PA-9 and 3d (Fig. 3). Both PA-9 and 3d (10
pM to 10 nM) dose-dependently inhibited PACAP (1 nM)-induced CREB phosphorylation.
The inhibitory effects of 3d (IC50 < 10 pM) in this case were also more potent than those of
PA-9 (IC50 = 182 pM).
Fig. 3 Effects of 3d or PA-9 on the PACAP-induced phosphorylation of CREB in the
PAC1/CHO cells. The cells were incubated with 3d or PA-9 compounds (10 pM to 10 nM) for
30 min, then the cells were stimulated with PACAP (1 nM). The data represent the mean ±
SEM (n = 3-5).
2.3. In vivo pharmacological assay
We previously demonstrated that a single intrathecal (i.t.) injection of PACAP (100 pmol/5
µl) induced a long-lasting mechanical allodynia of the hind paw in mice [8, 9]. The
mechanical allodynia was also induced by maxadilan, a PAC1 receptor-selective agonist, but
not by VIP, a VPAC1/2 receptor agonist. In addition, we also confirmed that the induction of
8

PACAP-induced mechanical allodynia was prevented by spinal pretreatment with max.d.4, a
peptide antagonist for the PAC1 receptor [8]. These results suggest that i.t. PACAP-induced
mechanical allodynia was mediated by stimulation of the spinal PAC1 receptors. With this in
mind, we examined the effects of PA-9 and 3d on the induction of PACAP-induced
mechanical allodynia (Fig. 4).
In accordance with our previous report [8, 9], a single i.t. injection of PACAP (100 pmol)
induced mechanical allodynia, which manifested one day after injection and persisted for a
minimum of 7 days (Fig. 4). Co-injection of PA-9 or 3d (100 pmol each) with PACAP (100
pmol), however, significantly blocked the induction of PACAP-induced mechanical allodynia
(Fig. 4). The inhibitory effects of 3d were more potent than those of PA-9 (Fig. 4).
Fig. 4 Effects of 3d or PA-9 on the PACAP-induced mechanical allodynia. A single i.t.
injection of PACAP (100 pmol) induced long-lasting mechanical allodynia and the induction
of allodynia was prevented by co-injection of 3d or PA-9 (100 pmol each). The data represent
the mean ± SEM (n = 6). *P < 0.05, when compared with vehicle.
9

With a view to evaluating these effects on neuropathic pain, we subsequently examined
the impact of PA-9 and 3d on spinal nerve ligation (SNL)-induced mechanical allodynia in
mice (Fig. 5). Several lines of evidence indicate that spinal PACAP and its receptor systems
could play an important role in the induction of peripheral nerve injury-induced neuropathic
pain. For example, development of neuropathic pain induced by SNL was shown to be
abrogated in PACAP-deficient mice [10], and SNL-induced mechanical allodynia was
ameliorated by treatment with intrathecal PACAP 6–38, an antagonist of the PACAP receptor
[11].
In this study, behavioral testing was performed with mice at 14 days after SNL. Compared
with contralateral paws, the SNL induced marked mechanical hypersensitivity of the hind paw
ipsilateral to the ligation (Fig. 5A and B). A single i.t. injection of PA-9 (100 pmol) or 3d (100
pmol) produced a significant decrease in mechanical allodynia (Fig. 5A). Furthermore, the
inhibitory effect peaked at 2-3 hours after the i.t. injection and persisted for at least 24 hours.
It is noteworthy that at 3 hours after the injection, the inhibitory effect of 3d was significantly
stronger than that of PA-9. In contrast, both PA-9 and 3d failed to influence paw withdrawal
responses in contralateral paws (Fig. 5A).
Finally, we investigated whether systemic (p.o.) administration of PA-9 or 3d is effective
against the SNL-induced allodynia (Fig. 5B). We found that a single p.o. administration of
both PA-9 (30 mg/kg) and 3d (30 mg/kg) significantly ameliorated the SNL-induced
10

mechanical allodynia. The inhibitory effects of PA-9 and 3d peaked at 3 hours after p.o.
administration. At 2 to 4 hours after p.o. administration, the inhibitory effect of 3d was
notably stronger than that of PA-9. In contrast, both PA-9 and 3d failed to influence paw
withdrawal responses in contralateral paws.
Fig. 5 Effects of 3d or PA-9 on the SNL-induced mechanical allodynia. (A) 3d (100 pmol, n
= 6) or PA-9 (100 pmol, n = 6) was intrathecally injected 14 days after SNL. (B) 3d (30
mg/kg, n = 6) or PA-9 (30 mg/kg, n = 6) was perorally administered 14 days after SNL. The
data represent the mean ± SEM (n = 6). *P < 0.05, when compared with vehicle. #P < 0.05,
when compared with PA-9.
11

2.4. Models of 3d binding to PAC1 receptor.
Since 3d showed potent and specific antagonistic activity toward the PAC1 receptor, we
constructed a model of the compound binding to the N-terminal EC domain of PAC1 (Fig. 6).
Similarly to PA-9 (Fig. 1), compound 3d was predicted to be well accommodated within the
PAC1 binding pocket (Fig. 6). Comparably to PA-9, 3d was also envisaged to form three
hydrogen bonds with the backbone carbonyl oxygen atoms of L80, V92, and S120 amino acid
residues.
Fig. 6 Docking model of 3d with PAC1 receptor. Hydrogen-bonding interactions are
indicated by yellow dashed lines.
12

In order to explore the antagonistic activity of 3d from the energetic perspective, we
calculated binding free energy (∆Gbind) and ligand efficiency (∆Gbind/number of heavy atoms)
[12] based on molecular mechanics generalized Born surface area (MM-GBSA) methods
(Table 1). ∆Gbind and ligand efficiency values were also estimated for 3a–3c (Scheme 1),
which possess the same chlorine group at different positions with no or weak antagonistic
activity (Fig. 2). The ranking of ligand efficiency was 3d > PA-9 ≈ 3b > 3a > 3c, which is
consistent with the trend observed in the inhibitory activity studies (Fig. 2). This suggests that
better ligand efficiency of 3d would contribute its most potent antagonistic activity. Therefore,
we next explored the source of better ligand efficiency of 3d.
Table 1 Binding free energy (∆Gbind) and ligand efficiency of PA-9 or its derivative
compounds to the PAC1 receptor
∆Gbind
(kcal/mol)
-46.397
-47.896
-48.237
-44.406
-49.456
Ligand Efficiency
Compounds
（∆Gbind /Number of heavy atoms）
PA-9
3a
-1.856
-1.842
-1.855
-1.708
-1.902
3b
3c
3d
Comparing 3d with PA-9, the ∆Gbind of 3d was calculated to be approximately 3.06
kcal/mol more stable than that of PA-9. This difference seemed to be resulting from a more
favorable van der Waals interaction energy of 3d with the PAC1 receptor, which was
13

approximately 2.64 kcal/mol greater than that of PA-9. Although the interaction of 3d with the
PAC1 receptor was very similar to that of PA-9, the chloro group on the indazole ring of 3d
was suggested to be more deeply located in the sub-pocket formed by the V93, P107, H108,
and Y109 amino acid residues of the PAC1 receptor (Fig. 7). The chloro functionality is likely
to lead to more favorable van der Waals interactions of 3d.
Fig. 7 Comparison of docking models of PA-9 or 3d with PAC1 receptor.
14

3. Conclusion
In search of novel and potent PAC1 receptor antagonists for pharmacological studies and
new analgesics, we synthesized and evaluated 18 derivative compounds of PA-9 which is
identified as a new PAC1 receptor antagonist by in silico screening followed by
pharmacological assays [9]. Among the synthesized compounds, 3d showed more potent
antagonistic action than PA-9 in an in vitro pharmacological assay. Furthermore, 3d exibited
more potent analgesic activities against both PACAP- and SNL-induced mechanical allodynia.
Compound 3d also displayed strong anti-allodynic activity by oral administration, therefore,
showing potential to become an orally available analgesic against neuropathic pain.
A growing body of evidence demonstrates that the PACAP signaling system plays a
crucial role in pain transmission. For example, development of neuropathic pain was shown to
be abrogated in PACAP-deficient mice [10]. However, since the mice exhibited whole-body
PACAP deficiency, it was not clear which site of action (peripheral and/or central nervous
system) for PACAP is involved in the development of neuropathic pain. It has also been
reported that i.t. injection of PACAP 6-38, a peptide antagonist of the PACAP receptor,
attenuated the spinal nerve injury-induced mechanical allodynia [11], suggesting the
involvement of spinal PACAP receptors in the neuropathic pain. However, because PACAP
6-38 is recognized as a dual PAC1/VPAC2 antagonist [13], it was not clear which of the
PACAP receptor subtypes (PAC1, VPAC1 and/or VPAC2) is involved in the neuropathic pain
15

transmission. In the present study, i.t. injection of PA-9 and 3d, specific antagonists of the
PAC1 receptor, attenuated the spinal nerve injury-induced mechanical allodynia. Considering
that a single i.t. injection of maxadilan, a specific agonist for the PAC1 receptor, induced
long-lasting mechanical allodynia [8], it is strongly suggested that spinal PAC1 receptor is
primarily involved in the development of peripheral nerve injury-induced neuropathic pain.
16

4. Experimental section
4.1. Chemistry
General melting points are uncorrected. Flash chromatography was performed on Kanto
Kagaku silica gel 60N. NMR spectra were recorded on a JEOL a-GX 400 spectrometer using
C5D5N as the solvent. Chemical shifts (δ) are given in ppm downfield from TMS and
referenced to C5H5N (7.19 ppm) as an internal standard. Peak multiplicities are designated by
the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; m,
multiplet; br, broad and coupling constants in (J) Hz. High-resolution mass spectral data were
obtained on a JEOL MStation JMS-700. All commercial reagents were used as received
unless otherwise noted.
4.1.1. General procedure for the synthesis of lactams (2a-r)
According to the literature’s procedure, lactams (2a-r) were prepared by direct heating at
150 °C with no solvent [14] or heating with reflux in H2O [15] as solid.
4.1.2. General procedure for the synthesis of amides (3a-r)
Method A: To a stirred solution of lactam (2, 1 mmol) in CH2Cl2 (1.5 mL) and DMF (1.5
mL) were added DCC (1.2 mmol), HOBt (1.2 mmol) and histamine (1.2 mmol) at room
17

temperature, and the resulting mixture was stirred at room temperature for 24 h. After
evaporation of the solvent, the residue was chromatographed on silica gel (15 g,
CH2Cl2:MeOH = 20:1-5:1) to give the corresponding amide.
Method B: To a stirred solution of lactam (2, 1 mmol) in CH2Cl2 (1.5 mL) and DMF (1.5
mL) were added EDC (1.2 mmol), DMAP (0.1 mmol) and histamine (1.2 mmol) at room
temperature, and the resulting mixture was stirred at room temperature for 24 h. After
evaporation of the solvent, the residue was chromatographed on silica gel (15 g,
CH2Cl2:MeOH = 20:1-5:1) to give the corresponding amide.
4.1.2.1.
N-[2-(1H-imidazol-4-yl)ethyl]-1-(4-chloro-1H-indazol-3-yl)-5-oxo-3-pyrrolidine-carboxa
mide (3a)
Method A; Yield: 47 % in 2 steps; mp: 190-191 °C; IR (KBr): 3566, 3437, 3306, 1695, 1636,
1558, 1508 cm-1; 1H NMR (400 MHz, Pyridine-d5): δ 9.00 (1H, br s), 7.96 (1H, s), 7.61 (1H,
d, J = 7.4 Hz), 7.14 (1H, t, J = 7.4 Hz), 7.15 (1H, s), 6.98 (1H, d, J = 7.4 Hz), 4.72 (1H, t, J =
9.1 Hz), 3.96-3.85 (3H, m), 3.26 (1H, dd, J = 15.6, 9.1 Hz), 3.11 (2H, t, J = 6.8 Hz), 2.78 (1H,
dd, J = 15.6, 9.1 Hz); 13C NMR (100 MHz, DMSO-d6): δ 169.56, 168.62, 147.59, 134.59,
131.61, 126.70, 123.80, 119.28, 116.97, 115.67, 107.11, 48.23, 36.47, 32.61, 26.57; MS (EI):
m/z 372 (M+); HRMS (EI): clacd for C17H17ClN6O2: 372.1102 (M+), found: 372.1099.
18

4.1.2.2.
N-[2-(1H-imidazol-4-yl)ethyl]-1-(5-chloro-1H-indazol-3-yl)-5-oxo-3-pyrrolidine-carboxa
mide (3b)
Method A; Yield: 23% in 2 steps; mp: 208-209 °C; IR (KBr): 3735, 3649, 3097, 1684, 1653,
1558, 1508 cm-1; 1H NMR (400 MHz, Pyridine-d5): δ 9.04 (1H, br s), 8.05 (1H, s), 7.69 (1H,
d, J = 9.6 Hz), 7.68 (1H, s), 7.27 (1H, d, J = 9.6 Hz), 7.71 (1H, s), 5.09 (1H, dd, J = 13.6, 8.4
Hz), 4.73 (1H, t, J = 8.4 Hz), 3.94-3.86 (3H, m), 3.28 (1H, dd, J = 14.8, 8.4 Hz), 3.11 (2H, t, J
= 6.6 Hz), 2.84 (1H, dd, J = 14.8, 8.4 Hz); 13C NMR (100 MHz, DMSO-d6): δ 169.63, 168.21,
145.55, 134.50, 133.79, 131.92, 127.15, 123.19, 118.50, 118.39, 116.85, 108.13, 48.33, 38.75,
36.33, 32.92, 26.53; MS (EI): m/z 372 (M+); HRMS (EI): clacd for C17H17ClN6O2: 372.1102
(M+), found: 372.1099.
4.1.2.3.
N-[2-(1H-imidazol-4-yl)ethyl]-1-(6-chloro-1H-indazol-3-yl)-5-oxo-3-pyrrolidine-carboxa
mide (3c)
Method A; Yield: 22% in 2 steps; mp: 196-198 °C; IR (KBr): 3290, 3213, 3101, 1683, 1636,
1558, 1508 cm-1; 1H NMR (400 MHz, Pyridine-d5): δ 9.12 (1H, br s), 8.30 (1H, s), 7.83 (1H,
s), 7.66 (1H, d, J = 9.2 Hz), 7.25 (1H, s), 6.96 (1H, d, J = 9.2 Hz), 5.03 (1H, dd, J = 13.0, 8.3
19

Hz), 4.71 (1H, t, J = 8.3 Hz), 3.91-3.84 (3H, m), 3.27 (1H, dd, J = 15.2, 8.3 Hz), 3.12 (2H, t, J
= 6.4 Hz), 2.87 (1H, dd, J = 15.2, 8.3 Hz); 13C NMR (100 MHz, DMSO-d6): δ 169.79, 168.28,
147.25, 134.03, 132.74, 132.42, 131.35, 121.77, 119.93, 116.53, 115.03, 106.65, 48.23, 38.21,
36.25, 32.92, 25.47; MS (EI): m/z 372 (M+); HRMS (EI): clacd for C17H17ClN6O2: 372.1102
(M+), found: 372.1099.
4.1.2.4.
N-[2-(1H-imidazol-4-yl)ethyl]-1-(7-chloro-1H-indazol-3-yl)-5-oxo-3-pyrrolidine-carboxa
mide (3d)
Method A; Yield: 35% in 2 steps; mp: 238-239 °C; IR (KBr): 3319, 3231, 3213, 1663, 1636,
1558, 1508 cm-1; 1H NMR (400 MHz, Pyridine-d5): δ 9.02 (1H, br s), 8.49 (1H, s), 7.76 (1H,
s), 7.52 (1H, d, J = 7.5 Hz), 7.40 (1H, d, J = 7.5 Hz), 6.98 (1H, s), 6.88 (1H, t, J = 7.5 Hz),
5.13 (1H, dd, J = 14.6, 8.5 Hz), 4.81 (1H, t, J = 8.5 Hz), 3.95-3.86 (3H, m), 3.26 (1H, dd, J =
16.2, 8.5 Hz), 3.11 (2H, t, J = 6.8 Hz), 2.83 (1H, dd, J = 16.2, 8.5 Hz); 13C NMR (100 MHz,
DMSO-d6): δ 169.56, 168.32, 144.32, 134.51, 133.83, 133.30, 125.80, 120.48, 119.42, 119.04,
116.84, 109.42, 48.36, 38.77, 36.32, 32.95, 26.57; MS (EI): m/z 372 (M+); HRMS (EI): clacd
for C17H17ClN6O2: 372.1102 (M+), found: 372.1113.
4.1.2.5.
20

N-[2-(1H-imidazol-4-yl)ethyl]-1-phenyl-5-oxo-3-pyrrolidinecarboxamide (3e)
Method B; Yield: 63% in 2 steps; mp: 149-151 °C; IR (KBr): 3675, 3306, 1678, 1643, 1558
cm-1; 1H NMR (400 MHz, Pyridine-d5): δ 9.11 (1H, t, J = 5.8 Hz),7.90 (1H, s), 7.72 (2H, d, J
= 8.4 Hz), 7.28 (2H, t, J = 8.4 Hz), 7.09 (1H, s), 7.06 (1H, t, J = 8.4 Hz), 4.13 (1H, dd, J = 9.6,
8.3 Hz), 3.93 (1H, t, J = 8.3 Hz), 3.83 (2H, q, J = 7.5 Hz ), 3.47 (1H, quint, J = 8.3 Hz), 3.14
(1H, dd, J = 16.7, 8.3 Hz), 3.08 (2H, t, J = 7.5 Hz), 2.84 (1H, dd, J = 16.7, 8.3 Hz); 13C NMR
(100 MHz, DMSO-d6): δ 172.16, 171.96, 139.22, 134.58, 133.95, 128.70, 124.00, 119.36,
116.83, 50.73, 38.66, 35.66, 35.43, 26.38; MS (EI): m/z 298 (M+); HRMS (EI): clacd for
C16H18N4O2: 298.1430 (M+), found: 298.1437.
4.1.2.6.
N-[2-(1H-imidazol-4-yl)ethyl]-1-(4-methylphenyl)-5-oxo-3-pyrrolidine-carboxamide (3f)
Method B; Yield: 82% in 2 steps; mp: 176-178 °C; IR (KBr): 3306, 3088, 1675, 1639, 1556
cm-1; 1H NMR (400 MHz, Pyridine-d5): δ 9.10 (1H, t, J = 5.8 Hz), 7.91 (1H, s), 7.64 (2H, d, J
= 7.2 Hz), 7.08 (2H, t, J = 7.2 Hz), 7.07 (1H, s), 4.14 (1H, dd, J = 9.6, 8.0 Hz), 3.92 (1H, t, J
= 8.0 Hz), 3.83 (2H, q, J = 7.2 Hz), 3.46 (1H, quint, J = 8.0 Hz), 3.14 (1H, dd, J = 17.6, 8.0
Hz), 3.08 (2H, t, J = 7.2 Hz), 2.84 (1H, dd, J = 17.6, 8.0 Hz), 2.13 (3H, s); 13C NMR (100
MHz, DMSO-d6): δ 172.17, 172.10, 137.00, 134.86, 134.44, 133.26, 129.30, 119.58, 117.04,
51.00, 39.17, 36.02, 35.84, 27.01, 20.63; MS (EI): m/z 312 (M+); HRMS (EI): clacd for
21

C17H20N4O2: 312.1586 (M+), found: 312.1593.
4.1.2.7.
1-(4-chlorophenyl)-N-[2-(1H-imidazol-4-yl)ethyl]-5-oxo-3-pyrrolidine-carboxamide (3g)
[16]
Method B; Yield: 74% in 2 steps; mp: 196-198 °C; IR (KBr): 3119, 3017, 1695, 1647, 1558
cm-1; 1H NMR (400 MHz, Pyridine-d5): δ 9.16 (1H, t, J = 5.2 Hz), 7.95 (1H, s), 7.71 (2H, d, J
= 8.4 Hz), 7.30 (2H, d, J = 8.4 Hz), 7.11 (1H, s), 4.10 (1H, dd, J = 9.4, 8.3 Hz), 3.92 (1H, t, J
= 8.3 Hz), 3.83 (2H, q, J = 6.7 Hz), 3.50 (1H, quint, J = 8.3 Hz), 3.11 (1H, dd, J = 17.5, 8.3
Hz), 3.09 (2H, t, J = 5.7 Hz), 2.85 (1H, dd, J = 17.5, 8.3 Hz); 13C NMR (100 MHz,
DMSO-d6): δ 172.20, 171.68, 137.90, 134.43, 133.95, 128.36, 127.49, 120.60, 116.62, 50.47,
38.74, 35.62, 35.30, 26.52; MS (EI): m/z 332 (M+); HRMS (EI): clacd for C16H17ClN4O2:
332.1040 (M+), found: 332.1039.
4.1.2.8.
N-[2-(1H-imidazol-4-yl)ethyl]-1-(4-fluorophenyl)-5-oxo-3-pyrrolidine-carboxamide (3h)
Method B; Yield: 87% in 2 steps; mp: 232-234 °C; IR (KBr): 3140, 3126, 1688, 1645, 1570
cm-1; 1H NMR (400 MHz, Pyridine-d5): δ 9.13 (1H, t, J = 4.8 Hz), 7.72-7.68 (2H, m), 7.92
(1H, s), 7.11 (1H, s), 7.09-7.04 (2H, m), 4.12 (1H, dd, J = 12.2, 8.7 Hz), 3.92 (1H, t, J = 8.7
22

Hz), 3.84 (2H, t, J = 7.0 Hz), 3.49 (1H, quint, J = 8.7 Hz), 3.14 (1H, dd, J = 17.2, 8.7 Hz),
3.09 (2H, t, J = 7.0 Hz), 2.85 (1H, dd, J = 17.2, 8.7 Hz); 13C NMR (100 MHz, DMSO-d6): δ
172.05, 171.90, 158.44 (d, J = 240.3 Hz), 135.64, 134.62, 134.14, 121.35 (d, J = 7.6 Hz),
116.81, 115.25 (d, J = 22.0 Hz), 50.92, 38.92, 35.68, 35.58, 26.72; MS (EI): m/z 316 (M+);
HRMS (EI): clacd for C16H17FN4O2: 316.1336 (M+), found: 316.1335.
4.1.2.9.
N-[2-(1H-imidazol-4-yl)ethyl]-1-(4-methoxyphenyl)-5-oxo-3pyrrolidine-carboxamide (3i)
Method B; Yield: 83% in 2 steps; mp: 151-153 °C; IR (KBr): 3239, 3075, 1684, 1635, 1568
cm-1; 1H NMR (400 MHz, Pyridine-d5): δ 9.08 (1H, t, J = 5.2 Hz), 7.91 (1H, s), 7.68 (2H, d, J
= 8.6 Hz), 7.10 (1H, s), 6.92 (2H, d, J = 8.6 Hz), 4.16 (1H, dd, J = 9.4, 8.4 Hz), 3.93 (1H, t, J
= 8.4 Hz), 3.84 (2H, q, J = 7.6 Hz), 3.62 (3H, s), 3.46 (1H, quint, J = 8.4 Hz), 3.15 (1H, dd, J
= 17.1, 8.4 Hz), 3.09 (2H, t, J = 7.6 Hz), 2.84 (1H, dd, J = 17.1, 8.4 Hz); 13C NMR (100 MHz,
DMSO-d6): δ 171.97, 171.57, 155.80, 134.65, 134.26, 132.41, 121.20, 116.80, 113.82, 55.20,
51.03, 38.95, 35.68, 35.62, 26.81; MS (EI): m/z 328 (M+); HRMS (EI): clacd for C17H20N4O3:
328.1535 (M+), found: 328.1530.
4.1.2.10.
N-[2-(1H-imidazol-4-yl)ethyl]-1-(4-cyanophenyl)-5-oxo-3-pyrrolidine-carboxamide (3j)
23

Method B; Yield: 47% in 2 steps; mp: 211-212 °C; IR (KBr): 3151, 3019, 2231, 1703, 1646,
1558 cm-1; 1H NMR (400 MHz, Pyridine-d5): δ 9.17 (1H, t, J = 5.2 Hz), 7.92 (1H, s), 7.83
(2H, d, J = 8.8 Hz), 7.60 (2H, d, J = 8.8 Hz), 7.11 (1H, s), 4.12 (1H, dd, J = 9.6, 8.4 Hz), 3.96
(1H, t, J = 8.4 Hz), 3.84 (2H, t, J = 7.0 Hz), 3.51 (1H, quint, J = 8.4 Hz), 3.16 (1H, dd, J =
17.2, 8.4 Hz), 3.10 (2H, t, J = 7.0 Hz), 2.88 (1H, dd, J = 17.2, 8.4 Hz); 13C NMR (100 MHz,
DMSO-d6): δ 173.28, 171.75, 143.03, 134.67, 133.06, 119.03, 118.93, 105.53, 51.51, 38.98,
36.03, 35.39, 26.85; MS (EI): m/z 323 (M+); HRMS (EI): clacd for C17H17N5O2: 323.1382
(M+), found: 323.1377.
4.1.2.11.
N-[2-(1H-imidazol-4-yl)ethyl]-1-(2-hydroxyphenyl)-5-oxo-3-pyrrolidine-carboxamide
(3k)
Method B; Yield: 25% in 2 steps; mp: 120-122 °C; IR (KBr): 3651, 3265, 3213, 1684, 1670,
1558 cm-1; 1H NMR (400 MHz, Pyridine-d5): δ 9.08 (1H, br s), 7.91 (1H, s), 7.42 (1H, d, J =
8.8 Hz), 7.21-7.15 (2H, m), 7.10 (1H, s), 6.92-6.88 (1H, m), 4.30 (1H, dd, J = 9.2, 7.5 Hz),
4.10 (1H, t, J = 7.5 Hz), 3.85 (2H, q, J = 6.4 Hz), 3.47 (1H, quint, J = 7.5 Hz), 3.13 (1H, dd, J
= 16.4, 7.5 Hz), 3.06 (2H, t, J = 6.4 Hz), 2.83 (1H, dd, J = 16.4, 7.5 Hz); 13C NMR (100 MHz,
DMSO-d6): δ 172.52, 172.22, 152.77, 134.68, 128.30, 128.24, 125.48, 119.12, 118.74, 116.87,
116.74, 51.68, 38.98, 37.09, 34.39, 26.85; MS (EI): m/z 314 (M+); HRMS (EI): clacd for
24

C16H18N4O3: 314.1379 (M+), found: 314.1382.
4.1.2.12.
N-[2-(1H-imidazol-4-yl)ethyl]-1-(3-hydroxyphenyl)-5-oxo-3-pyrrolidine-carboxamide
(3l)
Method B; Yield: 47% in 2 steps; mp: 102-104 °C; IR (KBr): 3790, 3439, 3337, 1684, 1653,
1558 cm-1; 1H NMR (400 MHz, Pyridine-d5): δ 8.97 (1H, br s), 8.09 (1H, s), 7.93 (1H, s),
7.28 (1H, t, J = 8.2 Hz), 7.20 (1H, d, J = 8.2 Hz), 7.13 (1H, s), 6.98 (1H, d, J = 8.2 Hz), 4.29
(1H, dd, J = 9.0, 8.3 Hz), 3.98 (1H, t, J = 8.3 Hz), 3.89 (2H, q, J = 6.9 Hz), 3.36 (1H, quint, J
= 8.3 Hz), 3.21 (1H, dd, J = 17.1, 8.3 Hz), 3.09 (2H, t, J = 6.9 Hz), 2.82 (1H, dd, J = 17.1, 8.3
Hz); 13C NMR (100 MHz, DMSO-d6): δ 172.07, 171.93, 157.55, 140.28, 134.68, 129.38,
111.17, 109.78, 106.63, 50.84, 39.00, 35.99, 35.54, 26.89; MS (EI): m/z 314 (M+); HRMS
(EI): clacd for C16H18N4O3: 314.1379 (M+), found: 314.1382.
4.1.2.13.
N-[2-(1H-imidazol-4-yl)ethyl]-1-(4-hydroxyphenyl)-5-oxo-3-pyrrolidine-carboxamide
(3m)
Method B; Yield: 41% in 2 steps; mp: 104-105 °C; IR (KBr): 3585, 3251, 3190, 1684, 1653,
1558 cm-1; 1H NMR (400 MHz, Pyridine-d5): δ 11.49 (1H, br s), 8.97 (1H, br s), 7.93 (1H, s),
25

7.76 (2H, dd, J = 8.6, 2.4 Hz), 7.15 (2H, dd, J = 8.6, 2.4 Hz), 7.14 (1H, s), 4.29 (1H, td, J =
8.4, 2.1 Hz), 3.95 (1H, td, J = 8.4, 2.1 Hz), 3.90 (2H, q, J = 6.3 Hz), 3.39 (1H, quint, J = 8.4
Hz), 3.22 (1H, ddd, J = 16.7, 8.4, 2.1 Hz), 3.10 (2H, t, J = 6.3 Hz), 2.84 (1H, ddd, J = 16.7,
8.4, 2.1 Hz); 13C NMR (100 MHz, DMSO-d6): δ 172.52, 171.86, 154.61, 135.20, 131.48,
122.05, 115.61, 51.67, 39.49, 36.23, 36.09, 27.40; MS (EI): m/z 314 (M+); HRMS (EI): clacd
for C16H18N4O3: 314.1379 (M+), found: 314.1382.
4.1.2.14.
N-[2-(1H-imidazol-4-yl)ethyl]-1-(1H-pyrazol-3-yl)-5-oxo-3-pyrrolidine-carboxamide (3n)
Method A; Yield: 28% in 2 steps; mp: 213-211 °C; IR (KBr): 3676, 3320, 3203, 1689, 1652,
1635, 1557 cm-1; 1H NMR (400 MHz, Pyridine-d5): δ 12.24 (1H, br s), 9.00 (1H, br s), 8.03
(1H, s), 7.88 (1H, s), 7.56 (1H,s), 7.10 (1H, s), 4.78 (1H, dd, J = 12.6, 8.1 Hz), 4.26 (1H, t, J =
8.1 Hz), 3.82 (2H, q, J = 6.7 Hz), 3.69 (1H, quint, J = 8.1 Hz), 3.22 (1H, dd, J = 15.3, 8.1 Hz),
3.06 (2H, t, J = 6.7 Hz), 2.72 (1H, dd, J = 15.3, 8.1 Hz); 13C NMR (100 MHz, DMSO-d6): δ
169.67, 168.26, 162.33, 138.77, 138.73, 134.64, 134.53, 89.01, 47.04, 38.87, 36.72, 33.00,
26.97; MS (EI): m/z 288 (M+); HRMS (EI): clacd for C13H16N6O2: 288.1335 (M+), found:
288.1336.
4.1.2.15.
26

N-[2-(1H-imidazol-4yl)ethyl]-5-oxo-1-(phenylmethyl)-3-pyrrolidine-carboxamide (3o)
[17]
Method A; Yield: 76%; mp: 61-63 °C; IR (KBr): 3271, 3155, 1670, 1652, 1558 cm-1; 1H NMR
(400 MHz, Pyridine-d5): δ 8.85 (1H, br), 7.92 (1H, s), 7.33-7.25 (5H, m), 7.08 (1H, s), 4.56
(1H, d, J = 14.6 Hz), 4.48 (1H, d, J = 14.6 Hz), 3.85 (2H, q, J = 6.5 Hz), 3.67 (1H, t, J = 8.0
Hz), 3.39 (1H, t, J = 8.0 Hz), 3.25 (1H, quint, J = 8.0 Hz), 3.10 (1H, dd, J =17.0, 8.0 Hz), 3.05
(2H, t, J = 6.5 Hz), 2.72 (1H, dd, J = 17.0, 8.0 Hz); 13C NMR (100 MHz, DMSO-d6): δ
172.44, 172.25, 136.77, 134.58, 133.72, 128.59, 127.59, 127.28, 125.39, 123.57, 118.69,
116.81, 110.25, 49.13, 45.31, 38.71, 35.87, 33.93, 26.42; MS (EI): m/z 312 (M+); HRMS (EI):
clacd for C17H20N4O2: 312.1586 (M+), found: 312.1593.
4.1.2.16.
N-[2-(1H-imidazol-4-yl)ethyl]-1-[(2-hydroxyphenyl)methyl]-5-oxo-3-pyrrolidinecarboxa
mide (3p)
Method A; Yield: 30%; mp: 88-90 °C; IR (KBr): 3748, 3738, 3651, 1684, 1653, 1558 cm-1; 1H
NMR (400 MHz, Pyridine-d5): δ 11.36 (1H, br s) 8.81 (1H, t, J = 5.8 Hz), 7.84 (1H, d, J = 1.2
Hz), 7.34 (1H, dd, J = 7.5, 1.3 Hz), 7.13 (1H, td, J = 7.5, 1.3 Hz), 7.08 (1H, dd, J = 7.5, 1.3
Hz), 7.03 (1H, s), 6.81 (1H, td, J = 1.3, 7.5 Hz), 4.74 (1H, d, J = 15.2 Hz), 4.65 (1H, d, J =
15.2 Hz), 3.82 (1H, dd, J = 9.6, 8.2 Hz), 3.77 (2H, q, J = 6.6 Hz), 3.57 (1H, t, J = 8.2 Hz),
27

3.22 (1H, quint, J = 8.2 Hz), 3.03 (1H, dd, J = 16.5, 8.2 Hz), 2.99 (2H, t, J = 6.6 Hz), 2.63
(1H, dd, J = 16.5, 8.2 Hz); 13C NMR (100 MHz, DMSO-d6): δ 172.57, 172.18, 155.26,
134,67, 128.78, 128.38, 122.58, 119.03, 115.21, 49.63, 40.57, 38.94, 35.93, 33.91, 26.87; MS
(EI): m/z 328 (M+); HRMS (EI): clacd for C17H20N4O3: 328.1535 (M+), found: 328.1533.
4.1.2.17.
N-[2-(1H-imidazol-4-yl)ethyl]-1-[(3-hydroxyphenyl)methyl]-5-oxo-3-pyrrolidinecarboxa
mide (3q)
Method A; Yield: 40%; mp: 104-106 °C; IR (KBr): 3734, 3647, 3623, 1684, 1653, 1558 cm-1;
1H NMR (400 MHz, Pyridine-d5): δ 8.83 (1H, br s), 7.93 (1H, s), 7.24 (2H, d, J = 7.9 Hz),
7.22 (1H, s), 7.09 (1H, s), 7.08 (1H, d, J = 7.9 Hz), 6.88 (1H, d, J = 7.9 Hz), 4.57 (1H, d, J =
14.6 Hz), 4.48 (1H, d, J = 14.6 Hz), 3.84 (2H, q, J = 6.1 Hz), 3.72 (1H, t, J = 8.1 Hz), 3.45
(1H, t, J = 8.1 Hz), 3.23 (1H, quint, J = 8.1 Hz), 3.09 (1H, dd, J = 16.0, 8.1 Hz), 3.05 (2H, t, J
= 6.1 Hz), 2.67 (1H, dd, J = 16.0, 8.1 Hz); 13C NMR (100 MHz, DMSO-d6): δ 172.31, 172.05,
157.63, 138.14, 134.66, 134.18, 129.55, 118.17, 116.90, 114.41, 114.30, 49.14, 45.31, 38.88,
35.89, 26.78; MS (EI): m/z 328 (M+); HRMS (EI): clacd for C17H20N4O3: 328.1535 (M+),
found: 328.1533.
4.1.2.18.
28