Discovery and structure-activity relationships of pyrazolodiazepine derivatives as the first small molecule agonists of the Drosophila sex peptide receptor

Article abstract of DOI:10.1016/j.bmc.2015.02.035

In behavioral research, the sex peptide receptor in Drosophila melanogaster (DrmSPR) is the most interesting G protein-coupled receptor (GPCR) and is involved in post-mating responses such as increased egg-laying and decreased receptivity of the female; during these responses, the receptors are activated by a specific natural peptide agonist (sex peptide, SP). To discover small molecule agonists for DrmSPR, a compound library based on a pyrazolodiazepine scaffold, which was previously reported as a potential privileged structure, was screened. Structure-activity relationship (SAR) studies of the hit compounds, which exhibited weak agonistic effects (69-72% activation at 100 μM), were explored through the synthesis of various analogs with substituents at the R₁, R₂, R₃ and R₄ positions of the pyrazolodiazepine skeleton. As a result, compounds 21 and 31 of the 6-benzyl pyrazolodiazepine derivative series were found to be small molecule agonists for DrmSPR with EC₅₀ values of 3-4 μM.

Full text of DOI:10.1016/j.bmc.2015.02.035

Bioorganic & Medicinal Chemistry 23 (2015) 1808–1816
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and structure–activity relationships of pyrazolodiazepine
derivatives as the first small molecule agonists of the Drosophila sex
peptide receptor
Joeng-hyun Kim a, Pyeong-hwa Jeong b, Ju-Yeon Lee a, Jae-hyuk Lee a, Young-Joon Kim a,
,
⇑
Yong-Chul Kim a,b,
⇑
a School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
b Department of Medical System Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
In behavioral research, the sex peptide receptor in Drosophila melanogaster (DrmSPR) is the most interest-
ing G protein-coupled receptor (GPCR) and is involved in post-mating responses such as increased egg-
laying and decreased receptivity of the female; during these responses, the receptors are activated by a
specific natural peptide agonist (sex peptide, SP). To discover small molecule agonists for DrmSPR, a com-
pound library based on a pyrazolodiazepine scaffold, which was previously reported as a potential privi-
leged structure, was screened. Structure–activity relationship (SAR) studies of the hit compounds, which
exhibited weak agonistic effects (69–72% activation at 100 lM), were explored through the synthesis of
various analogs with substituents at the R1, R2, R3 and R4 positions of the pyrazolodiazepine skeleton. As a
result, compounds 21 and 31 of the 6-benzyl pyrazolodiazepine derivative series were found to be small
molecule agonists for DrmSPR with EC50 values of 3–4 lM.
Received 27 November 2014
Revised 11 February 2015
Accepted 17 February 2015
Available online 24 February 2015
Keywords:
Drosophila sex peptide receptor
G protein-coupled receptor
Agonist
Structure–activity relationships
Pyrazolodiazepine
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
by the suppression of mating receptivity and the initiation of egg
laying, are triggered in the female.9–14
The Drosophila melanogaster sex peptide receptor (DrmSPR) is
the most important target protein of type 1 G protein-coupled
receptor (GPCR) in the field of insect reproductive behavior in
the Drosophila model system, especially with respect to the repro-
ductive behavior of females.1–5 Sex peptide (SP) is known to func-
tion as a specific natural agonist for DrmSPR and is found in
seminal fluid. SP is transferred to the female from the male during
copulation and activates the SPR expressed in the uterine sensory
neurons in the female reproductive tract.6–8 Consequently, SPR-
mediated post-mating responses (PMR), which are characterized
DrmSPR is broadly expressed in the reproductive tract of
females and in the central nervous system (CNS) in both males
and females; however, SPR expressed in the CNS of males is not
likely related to behavioral responses because of the absence of
SP in the CNS of males.4,7,8 Although genes encoding SP-like pep-
tides exist in a few closely related Drosophila species, SPR is
detected in many species, including lophotrochozoa and ecdyso-
zoa.4 Thus, the SPR has been posited to be activated by other
ligands or to be involved in other functions. For example, SPRs
from other taxa, including a mosquito, Aedes aegypti; a moth,
Bombyx mori; and a sea slug, Aplysia californica, do not respond to
SP. Instead, those receptors, along with DrmSPR, are highly sensi-
tive to myoinhibitory peptides (MIPs), a group of neuropeptides
that are distinct from SP. These findings indicate that SPR is also
a receptor for MIPs, which function as alternative peptide agonists
and may be ancestral ligands.15–17 However, the binding of MIPs to
SPR in the reproductive tract of females does not evoke SPR-
mediated behavioral responses.15 Additionally, while Drosophila
SP activates only DrmSPR, MIPs are potent ligands that act on
SPRs in various species.15,18–22
Abbreviations: SPR, sex peptide receptor; SP, sex peptide; MIPs, myoinhibitory-
peptides; Drm, Drosophila melanogaster; TEA, triethyl amine; TFA, trifluoroacetic
acid; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; DMF, dimethylfor-
mamide; DCM, dichloromethane; HOBt, N-hydroxylbenzotriazole; DIBAL-H,
diisobutylaluminium hydride; EC50, half-maximal effective concentration; HPLC,
high-pressure liquid chromatography; ESI, electrospray ionization; MALDI-TOF,
matrix-assisted laser desorption/ionization-time of flight; SAR, structure–activity
relationship.
⇑
Corresponding authors. Tel.: +82 62 970 2502; fax: +82 62 970 2484.
E-mail addresses: kimyj@gist.ac.kr (Y.-J. Kim), yongchul@gist.ac.kr (Y.-C. Kim).
http://dx.doi.org/10.1016/j.bmc.2015.02.035
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

J.-h. Kim et al. / Bioorg. Med. Chem. 23 (2015) 1808–1816
1809
SP consists of 36 amino acids, including a cyclic sequence at the
C-terminal, that is, responsible for the activation of SPR and the
consequent behavioral responses.12,14,23 In the sequence of SP,
two tryptophan residues (Trp23 and Trp32) have been reported to
be crucial for its agonistic activity. Similarly, in the case of MIPs,
the representative amino acid sequence contains a W(X6)W amide
motif, and the two tryptophan residues were also observed to be
important for activation of the SPR.15–17
The differences in receptor selectivity, expression pattern and
effect on post-mating responses between SP and MIPs indicate that
the SPR may bind in different modes or activate different signaling
pathways depending on the ligand. To investigate these potential
molecular functions of SPR in detail, the binding mode of the
ligands is crucial. Because large peptide molecules such as SP or
MIPs cannot be effectively used as probes for in silico molecular
docking studies to predict the binding mode, the development of
small molecule ligands for SPR is necessary. In addition, small
molecule ligands could be utilized for in vivo functional studies
of SPR to elucidate the novel mechanisms of action related to SPR.
Recently, the reported NMR structure of SP indicated the pres-
ence of a type 1 beta turn conformation (Gly29-Trp32) and a
hydrophobic patch related to two tryptophan residues (Trp23 and
Trp32) detected at the cyclic C terminal region; these two moieties
were identified as key pharmacophores for peptide activity.15–17,23
In the previous study, we reported the generation of a compound
library using a pyrazolodiazepine skeleton, a privileged structure,
that is, commonly utilized for the development of peptide
mimetics.24,25 In this article, we report the discovery of small mole-
cule agonists, for the first time, for DrmSPR from hit compounds (1,
2 and 3 in Fig. 1) from the screening of pyrazolodiazepine deriva-
tives. The hit compounds were further optimized through the syn-
thesis and SAR analysis of various analogs with different
substituents at four positions of the pyrazolodiazepine skeleton,
as shown in Figure 2.
Figure 2. Strategy for optimization of hit compounds.
by hydrogenation in the presence of 10% Pd/C in methanol to yield
6Cc. The aldehyde derivative 7, which could interconvert reversi-
bly into the imine 8, was synthesized by reduction of the ester to
an aldehyde with DIBAL-H in toluene at À78 °C. Reductive amina-
tion of imine 8 using NaBH(OAc)3 and 1% acetic acid in dichloro-
methane afforded the 7-membered ring system of the
pyrazolodiazepine-8-one derivative 9. Finally, compound 9 was
reacted with various acid chlorides, alkyl halides or aromatic acyl
groups to yield the novel compounds 10–18 and 25–41 to study
the SAR of the R3 position.
Because the two tryptophan residues of SP and MIPs are key
pharmacophores, we replaced the Boc group with an aliphatic or
indole moiety at the R5 position, as shown in Scheme 2. Briefly,
the Boc groups of compounds 31 and 42 were removed to yield
43 and 44, which were subjected to coupling reactions with vari-
ous building blocks to yield compounds substituted in the R5 posi-
tion with bulky aliphatic (45–49), indole groups (50–56).
2.2. Compound library screening and structure–activity
relationship study
The initial hit compounds exhibited weak agonist effects at
100 lM (Fig. 1 and 1 = 72%, 2 = 69%, 3 = 69%). All three hit mole-
2. Results and discussion
2.1. Chemistry
cules were pyrazolodiazepine analogs and had phenethyl, benzyl
and acyl chain moieties at the R1, R2 and R3 positions, respectively.
The hit compounds were further optimized through the synthesis
and SAR analysis of various analogs with differing substituents at
four positions of the pyrazolodiazepine skeleton, as shown in
Figure 2. These substitutions included aromatic groups or bulky
aliphatic groups at the R1 position; tert-butoxy or aromatic groups
at the R2 position; and various acyl, alkyl or aromatic groups at the
R3 position to replace the acyl groups of the hit compounds.
The initial SAR studies of the three hit compounds, 1–3, were
designed to investigate the effect of various substituents at the
R2 position (compounds 10–15) on SPR agonist activity. As shown
in Table 1, altering the benzyl moiety at the R2 position of the hit
compounds to tert-butoxymethylene (10–12) and hydrox-
ymethylene (13–15) resulted in significant loss of agonistic activ-
ity, from 69% to 72% to less than 20%. Thus, the next SAR study
was performed with a benzyl group fixed at the R2 position.
For the SAR studies at the R1 position, compounds with 2-naph-
thylmethylene, 1-Boc-piperidine-4-ethyl and piperidine-4-ethyl
moieties were compared, as shown in Table 2. The presence of
bulkier aromatic groups such as the 2-naphthyl moiety at the R1
position (16–18) resulted in the total loss of agonistic activity.
Encouragingly, compounds 19–21 with a 1-Boc-piperidine moiety
instead of the benzyl groups of the hit compounds, showed much
higher activity, with three- to four-fold increases in the agonistic
The chemical synthesis of compound 5 from commercially
available starting materials and the generation of its derivatives
with phenethyl, 2-naphthyl methyl and 1-Boc-4-piperidine ethyl
groups at the R1 position were reported in a previous study.24
Novel compounds with various substituents at the R3 position
(28–41) and indole-substituted derivatives at the R2 and R5 posi-
tions (45–57) were synthesized using the strategies shown in
Schemes 1 and 2, respectively.
As shown in Scheme 1, for the synthesis of the compounds sub-
stituted with an indole moiety at the R2 position, the ester group of
4C was hydrolyzed using 1 N aqueous NaOH in methanol, followed
by subsequent coupling with -Trp-methyl ester in the presence of
general coupling reagents. The aryl nitro group of 5Cc was reduced
D
activity (e.g., 19 = 207%, 20 = 228%, 21 = 295%, at 100 lM).
However, removal of the Boc groups from the piperidine moiety
of compounds 19–21 to provide 22–24 resulted in activities less
than those of the hit compounds with a phenethyl moiety.
Interestingly, among the series of compounds in Table 2, the
Figure 1. Hit compounds as agonists of Drosophila sex peptide receptor hit
compounds showed weak agonistic effect (1 = 69%, 2 = 69% and 3 = 72%) at
100 lM concentration.

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J.-h. Kim et al. / Bioorg. Med. Chem. 23 (2015) 1808–1816
Scheme 1. Synthesis of pyrazolodiazepine derivatives. Reagents and conditions: (a) (i) 1 N NaOH in MeOH, rt, 1 h, 98–99%, (ii) D-Phe-methyl ester or D-Trp-methyl ester, EDC,
HOBt, TEA, DCM, rt, 8 h, 69–78%; (b) Pd/C, H2, MeOH, rt, 4 h, 96–98%; (c) DIBAL-H, toluene, À78 °C, 3 h, 60–70%; (d) NaBH(OAc)3, 1% AcOH, DCM, rt, 1 h, 55–60%; (e) (i) RCOCl,
TEA, DCM, rt, 1 h, 80–90%; in case of 13–15, (ii) 50% TFA, anisole, DCM, 95–97%; 22–24, (ii) 20% TFA, DCM, rt, 30 min, 93–97%.
Scheme 2. Synthesis of pyrazolodiazepine derivatives. Reagents and conditions: (a) 20% TFA, DCM, rt, 30 min, 95–97%; (b) RCOOH, EDC, TEA, DCM, rt, 8 h, 49–78%.
agonist activity showed dependence on the R3 substituents. Thus,
the agonistic activity increased in the following order:
CO(CH2)2(C5H9) > –COCH2C(CH3)3 > –COCH@C(CH3)2 for moieties
at the R3 position, respectively. These results suggest that aliphatic
moieties at the R1 and R3 positions may be important for the ago-
nistic activity of the compounds.
activity than the ethyl, propyl and butyl moieties (25 = 2%,
26 = 46%, 27 = 120%, respectively). This result indicates that the
carbonyl group might serve as an important pharmacophore for
agonistic activity of SPR. In addition, when this carbon chain was
further elongated up to seven carbons to provide the valeryl
group-substituted compound 31, the greatest agonistic effect
–
To further optimize the activity, various acyl, alkyl and aromatic
groups were substituted at the R3 position while the 1-Boc-piper-
idine group at the R1 position and benzyl group at the R2 position
were fixed (Table 3). The compounds with acyl groups at the R3
position (28–30) showed approximately two-fold higher activity
than the corresponding compounds with alkyl groups (25–27).
For example, the acetyl, propionyl and butanoyl moieties
(28 = 22%, 29 = 73%, 30 = 207%, respectively) exhibited better
(321%, at 100 lM) among the series of acyl-substituted compounds
was observed. The activity was slightly enhanced compared with
21, which was one of the most potent agonists in the earlier series
of compounds in Table 2. However, compounds 32–34, which con-
tained longer alkyl chains than 31 or a cyclopropyl acyl group at
the R3 position, were less effective agonists. In the case of 35–38,
which were substituted with various heteroaromatic groups at
the R3 position, the agonistic activities were higher than those

J.-h. Kim et al. / Bioorg. Med. Chem. 23 (2015) 1808–1816
1811
Table 1
Table 3
Agonistic effects of synthesized compounds (10–15) at Drosophila sex peptide
Agonistic effects of synthesized compounds (25–41) at Drosophila sex peptide
receptors
receptors
Compounds R3
R4
% Act
(100
Compounds
R2
R3
% Act (100 l
M)a
l
M)a
1
2
3
–CH2Ph
–CH2Ph
–CH2Ph
–CH2O(tBu)
–CH2O(tBu)
–CH2O(tBu)
–COCH@C(CH3)2
–COCH2C(CH3)3
–CO(CH2)2(C5H9)
69 17
69 13
72 23
19
20
21
–COCH@C(CH3)2
–CO(CH2)C(CH3)3
–CO(CH2)2(C5H9)
–H
–H
–H
207 19
228 37
295 59
N.A.b
46 12
120 23
10
11
12
–COCH@C(CH3)2
–COCH2C(CH3)3
–CO(CH2)2(C5H9)
N.A.b
N.A.b
25
26
27
–CH2CH3
–(CH2)2CH3
–(CH2)3CH3
–H
–H
–H
14
4
13
14
15
–CH2OH
–CH2OH
–CH2OH
–COCH@C(CH3)2
–COCH2C(CH3)3
–CO(CH2)2(C5H9)
N.A.b
28
29
30
31
32
33
34
–COCH3
–H
–H
–H
–H
–H
–H
–H
22 10
73 16
207 37
321 40
275 51
214 34
12
16
3
4
–COCH2CH3
–CO(CH2)2CH3
–CO(CH2)3CH3
–CO(CH2)4CH3
–CO(CH2)5CH3
–CO(C3H5)
a
100% receptor activation was determined by 50 lM ATP activation at the
DrmSPR expressed in CHO-K1 cell, and the percentage of activation by 100
compounds was measured for DrmSPR, respectively (mean SEM, n P 3).
lM
b
Not active.
129
3
35
36
37
38
39
–Thiazole-2-carbonyl
–Thiopene-2-carbonyl
–Furan-2-carbonyl
–4-Phenyl benzyl
–Benzo-[1,3]dioxole-5-
carbonyl
–H
–H
–H
–H
–H
145 14
75 13
83 10
Table 2
14
5
Agonistic effects of synthesized compounds (16–24) at Drosophila sex peptide
receptors
261 26
40
41
–CO(CH2)2(C5H9)
–CO(CH2)3CH3
–CO(CH2)2(C5H9)
–CO(CH2)3CH3
147 26
106 19
a
100% receptor activation was determined by 50
lM ATP activation at the
DrmSPR expressed in CHO-K1 cell, and the percentage of activation by 100 lM
compounds was measured for DrmSPR, respectively (mean SEM, n P 3).
b
Not active.
Compounds R1
R3
% Act
(100
l
M)a
without substitution at the R4 position was selected for the next
series of SAR studies.
1
2
3
–(CH2)2Ph
–(CH2)2Ph
–(CH2)2Ph
–COCH@C(CH3)2
–COCH2C(CH3)3
–CO(CH2)2(C5H9)
69 17
69 13
72 23
To potentially improve the agonistic activity further, the Boc
group at the R1 position of compound 21 was replaced with various
bulky polycycloalkane moieties at the R5 position (Table 4) because
the Boc moiety appeared to be an important pharmacophore, as
shown in Table 2. Unfortunately, replacement of the Boc group at
the R5 position with an adamantane carbonyl group resulted in a
16
17
18
–CH2naphthyl
–CH2naphthyl
–CH2naphthyl
–COCH@C(CH3)2
–COCH2C(CH3)3
–CO(CH2)2(C5H9)
N.A.b
N.A.b
13
6
19
20
21
–(CH2)2piperidine-1-Boc
–(CH2)2piperidine-1-Boc
–(CH2)2piperidine-1-Boc
–COCH@C(CH3)2
–COCH2C(CH3)3
–CO(CH2)2(C5H9)
207 19
228 37
295 59
two-fold decrease in agonistic activity (45, 164% at 100 lM), and
compound 46, which was one carbon longer than 45, showed a fur-
ther decrease in activity (96%). Other compounds (47–49) also
showed significantly decreased agonist activity. In the case of sub-
stitution of 3,3-dimethylbutanoyl group instead of Boc group at R5
position, which was an exchange of the oxygen of Boc group by
methylene moiety, slightly decreased agonistic effect (50, 208%
22
23
24
–(CH2)2-4-piperidine
–(CH2)2-4-piperidine
–(CH2)2-4-piperidine
–COCH@C(CH3)2
–COCH2C(CH3)3
–CO(CH2)2(C5H9)
40 12
44 18
75 13
a
100% receptor activation was determined by 50
DrmSPR expressed in CHO-K1 cell, and the percentage of activation by 100 lM
compounds was measured for DrmSPR, respectively (mean SEM, n P 3).
l
M ATP activation at the
b
Not active.
at 100 lM) was observed. These result indicated that Boc group
was most important for the agonistic activity and oxygen of Boc
group is considered to be influential pharmacophore at R5 position.
Another modification was the incorporation of indole moieties
at the R2 and R5 positions according to the previously published
studies that have indicated that the two tryptophan residues of
SP and MIPs were critical for agonistic effects at the SPR. In the first
attempt, various indole moieties were substituted on the R5 posi-
tion instead of the Boc group (51–53), as shown in Table 5.
Unexpectedly, the agonist effect of these compounds was markedly
decreased, similar to the results for substitution with bulky alipha-
tic moieties (45–49). In the second attempt, the agonistic effects of
the indole groups at both the R2 and R5 positions (54–57) were
investigated. However, those compounds also showed negligible
for alkyl-substituted compounds (25–27) but lower than those for
acyl-substituted compounds. Interestingly, a non-aliphatic deriva-
tive, compound 39, was nearly as potent (261%) as the optimized
analogs such as compounds 21 and 31. Compounds 40 and 41,
which contain additional substituents, valeryl and 3-cyclopentyl-
propane carbonyl moieties, respectively, at the R4 position showed
two- to three-fold decreases in the agonistic effect compared with
the corresponding R3 monosubstituted compounds 21 and 31.
Compounds 21 and 31 showed EC50 values of 3.2 lM and 3.9 lM,
respectively, for DrmSPR agonist activity, as shown in Figure 3.
Based on the above results, the valeryl moiety at the R3 position

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J.-h. Kim et al. / Bioorg. Med. Chem. 23 (2015) 1808–1816
Table 5
Agonistic effects of synthesized compounds (51–57) at Drosophila sex peptide
receptors
Compounds R2
R5
% Act
(100
l
M)a
118 15
51
–CH2Ph
–COCH2-3-N-CH3-
indole
52
53
–CH2Ph
–CH2Ph
–CO-3-N-CH3-indole
–CO-2-indole
27
2
N.A.b
N.A.b
54
55
56
57
–CH2-N-CH3-
indole
–CH2-N-CH3-
indole
–CH2-N-CH3-
indole
–CH2-N-CH3-
indole
–CO-2-indole
Figure 3. EC50 values of SP and synthesized compounds 21, 31 of Drosophila sex
peptide receptors. Dose–response curves of CHO cells expressing SPR, aequorin, and
a chimeric G protein, Gaqo, and treated with compounds 21 (.), 31 (N) and SP (d).
Each data point is mean SEM (n P 3).
–COCH2-3-indole
–CO-3-N-CH3–indole
23
17
15
4
3
2
–COCH2-3-N-CH3-
indole
Table 4
Agonistic effects of synthesized compounds (45–50) at Drosophila sex peptide
receptors
a
100% receptor activation was determined by 50
lM ATP activation at the
DrmSPR expressed in CHO-K1 cell, and the percentage of activation by 100
compounds was measured for DrmSPR, respectively (mean SEM, n P 3).
lM
b
Not active.
the R1 and R5 positions, (3) an aliphatic acyl group (optimum car-
bon chain length of (4) at the R3 position, and (4) no substitution at
the R4 position. Our discovery of the first small molecule agonists
for DrmSPR could assist in further research in this field, including
in vivo functional and in silico structural studies of DrmSPR.
Compounds
R5
% Act (100 l
M)a
45
46
47
48
49
50
–CO-adamantane
164 62
96 25
72 15
127 12
N.A.b
4. Experimental section
4.1. Chemistry
–COCH2-adamantane
–COCH2-norbornane
–CO-noradamantane
–CO-adamantane-3,5-(CH3)2
–COCH2C(CH3)3
208 36
Starting materials, solvents and reagents were obtained from
commercial suppliers and used as received unless otherwise noted.
All products reported showed 1H NMR and mass spectra in agree-
ment with the assigned structures. 1H NMR data were collected on
a JEOL JNM-ECX 400P spectrometer at 400 MHz and were recorded
in parts per million (ppm) values, relative to tetramethylsilane as
the internal standard. Spectra were taken in CDCl3 or CD3OD.
Data are reported as follows: chemical shift, multiplicity (s, singlet;
d, doublet; t, triplet; m, multiplet; br, broad; app, apparent), and
coupling constants. 13C NMR data was obtained from the Korea
Basic Science Institute (Gwangju) using 500 MHz FT-NMR spec-
trometer. Mass spectroscopy was carried out on ESI and MALDI-
TOF instruments. High-resolution mass spectra (m/z) were
recorded on a FAB (JEOL: mass range 2600 au, 10 kV acceleration)
at Korea Basic Science Institute (Daegu). Compounds 21 and 31
were prepared according to literature procedures which we pre-
viously reported.24
a
100% receptor activation was determined by 50 lM ATP activation at the
DrmSPR expressed in CHO-K1 cell, and the percentage of activation by 100
compounds was measured for DrmSPR, respectively (mean SEM, n P 3).
lM
b
Not active.
agonist activities for the activation of SPR (<25% at 100 lM). The
poor agonist effects of this series of compounds might be due to
differences in the binding mechanism of our small compound
and those of SP and MIPs.
The first small molecule agonists for DrmSPR discovered in this
study could be utilized to elucidate the structure and function of
the SPR, which is a highly important target receptor related to
post-mating responses in Drosophila.
3. Conclusion
In summary, we report the discovery of the first small molecule
agonists for DrmSPR from the synthesis and SAR studies of various
pyrazolodiazepine derivatives. The optimized compounds for SPR,
4.1.1. (R)-tert-Butyl 4-(2-(3-(1-methoxy-3-(1-methyl-1H-indol-
3-yl)-1-oxopropan-2-ylcarbamoyl)-4-nitro-1H-pyrazol-1-
yl)ethyl)piperidine-1-carboxylate (5Cc)
21 and 31, showed EC50 values of 3–4 lM. SAR analysis of the pyra-
4C (4.5 g, 11.77 mmol) was dissolved in 1 N NaOH in MeOH
(300 mL) and stirred for 30 min. Subsequently, the reaction mix-
ture was neutralized with 1 N HCl aqueous solution and concen-
trated under reduced pressure. The residue was dissolved in DCM
zolodiazepine derivatives as SPR agonist indicated that the follow-
ing conditions are required for agonism of DrmSPR: (1) a benzyl
moiety at the R2 position, (2) a 1-Boc-piperidine ethyl moiety at

J.-h. Kim et al. / Bioorg. Med. Chem. 23 (2015) 1808–1816
1813
and washed with 1 N HCl aqueous solution. The organic layer was
concentrated under reduced pressure to get the desired product as
white solid. Without further purification, it was used for next reac-
tion. The carboxylic acid was dissolved in DCM (300 mL) and HOBt
(3.2 g, 23.54 mmol) and EDC (4.5 g, 23.54 mmol) were added.
1.47–1.38 (2H, m, CH2), 1.18–1.04 (2H, m, CH2), MS (ESI): m/
z = 508.1 [M+H].
4.1.4. (R)-tert-Butyl 4-(2-(6-benzyl-4-(3-cyclopentylpropanoyl)-
8-oxo-5,6,7,8-tetrahydropyrazolo[4,3-e][1,4]diazepin-2(4H)-
yl)ethyl)piperidine-1-carboxylate (21)
Subsequently,
D-Trp-OMe HCl (6.0 g, 23.54 mmol) and TEA
(2.4 mL, 23.54 mmol) were added to the solution and stirred at
room temperature for 16 h. The reaction mixture was diluted with
chloroform, washed with saturated aqueous NH4Cl, dried with
Na2SO4, and concentrated. The residue was purified by column
chromatography on silica using hexanes/ethyl acetate = 3:2 to give
the amide 5Cc (5.3 g, 85%) as colorless oil. 1H NMR (CDCl3,
400 MHz) d 8.13 (1H, s, CH), 8.03–8.01 (1H, m, indole), 7.56–7.54
(1H, m, indole), 7.27–7.25(1H, m, indole), 7.04 (1H, t, J = 7.6 Hz,
indole), 6.94 (1H, s, indole), 5.13–5.11 (1H, m, CH), 4.18–4.15
(2H, t, J = 7.2 Hz, CH2), 4.10–4.09 (2H, m, CH2), 3.73 (3H, s, CH3),
3.71 (3H, s, CH3), 3.45–3.41 (2H, m, CH2), 2.70–2.67 (2H, m, CH2),
1.94–1.90 (2H, m, CH2), 1.68–1.65 (2H, m, CH2), 1.46 (9H, s, Boc),
1.46–1.44 (1H, m, CH), 1.20–1.15 (2H, m, CH2), MS (ESI): m/
z = 582.9 [M+H].
Compound 21 was reported.24 1H NMR (CDCl3, 400 MHz) d 8.33
(1H, s, NH), 7.37–7.28 (5H, m, phenyl), 7.20 (1H, s, CH), 4.23–4.19
(2H, m, CH2), 4.08–4.05 (2H, m, CH2), 3.83–3.63 (2H, m, CH2), 3.03–
2.79 (2H, m, CH2), 2.67–2.64 (2H, m, CH2), 1.89–1.84 (4H, m, CH2,
CH2), 1.68–1.58 (5H, m, CH, CH2, CH2), 1.43 (9H, s, Boc), 1.15–
0.86 (11H, m, CH2, cyclopentyl), 13C NMR (CDCl3, 500 MHz) d
170.89, 163.51, 154.74, 135.91, 134.15, 129.20 (2C), 129.15 (2C),
127.51, 124.12, 123.95, 79.31, 54.31, 51.78, 51.07, 39.32, 36.57,
33.48 (2C), 33.14, 32.49, 32.39 (2C), 31.73, 30.88, 28.41 (3C),
25.02, 24.97 (3C), MS (ESI): m/z = 578.2 [M+H]. HRMS (FAB)
(C33H47N5O4): calcd 578.7586, found 578.7591.
4.1.5. (R)-tert-Butyl 4-(2-(6-benzyl-8-oxo-4-pentanoyl-5,6,7,8-
tetrahydropyrazolo[4,3-e][1,4]diazepin-2(4H)-
yl)ethyl)piperidine-1-carboxylate (31)
4.1.2. (R)-tert-Butyl 4-(2-(4-amino-3-(1-methoxy-3-(1-methyl-
1H-indol-3-yl)-1-oxopropan-2-ylcarbamoyl)-1H-pyrazol-1-
yl)ethyl)piperidine-1-carboxylate (6Cc)
Compound 31 was reported.24 1H NMR (CDCl3, 400 MHz) 8.34
(1H, s, NH), 7.36–7.28 (5H, m, phenyl), 7.20 (1H, s, CH), 4.23–
4.19 (2H, m, CH2), 4.08–4.05 (2H, m, CH2), 3.83–3.63 (2H, m,
CH2), 3.03–2.79 (2H, m, CH2), 2.67–2.64 (2H, m, CH2), 1.89–1.84
(4H, m, CH2, CH2), 1.68–1.53 (7H, m, CH, CH2, CH2, CH2), 1.43
(9H, s, Boc), 1.14–1.12 (2H, m, CH2), 0.92 (3H, t, J = 7.6 Hz, CH3),
13C NMR (CDCl3, 500 MHz) d 172.48, 163.60, 153.82, 140.50,
138.04, 129.35, 129.09 (2C), 128.52, 128.34 (2C), 126.49, 79.18,
54.20, 52.94, 49.98, 39.37, 36.96, 32.79, 32.72 (2C), 28.09 (3C),
26.79, 21.64 (2C), 13.67, MS (ESI): m/z = 538.2 [M+H]. d HRMS
(FAB) (C30H43N5O4): calcd 538.6946, found 538.6950.
A solution of 5Cc (5.0 g, 8.6 mmol) in MeOH was added 10% Pd/
C (300 mg, 5 mmol) and stirred under hydrogen gas atmosphere
for 2 h at room temperature. The product was filtered through
Celite filter agent pad and the filtrate was concentrated in vacuum.
The residue was purified by flash column chromatography on silica
using hexanes/ethyl acetate = 1:1 to afford amine 6Cc (4.6 g, 97%)
as yellow oil. 1H NMR (CDCl3, 400 MHz) d 8.02–8.00 (1H, m,
indole), 7.52–7.50 (1H, m, indole), 7.23–7.21(1H, m, indole), 7.01
(1H, t, J = 7.6 Hz, indole), 6.94 (1H, s, indole), 6.90 (1H, s, CH),
5.09–5.07 (1H, m, CH), 4.13–4.11 (2H, t, J = 7.2 Hz, CH2), 4.03–
4.01 (2H, m, CH2), 3.70 (3H, s, CH3), 3.68 (3H, s, CH3), 2.67–2.64
(2H, m, CH2), 1.82–1.78 (2H, m, CH2), 1.68–1.65 (2H, m, CH2),
1.46 (9H, s, Boc), 1.46–1.44 (1H, m, CH), 1.18–1.13 (2H, m, CH2),
MS (ESI): m/z = 552.9 [M+H].
4.1.6. General procedure for the acylation to the N-1 of
tetrahydro-1,4-pyrazolodiazepin-8-one (28–38)
Various starting compounds of scaffold 9 (1.0 equiv) and vari-
ous acid chlorides (1.0 equiv) in DCM was treated with TEA
(1.2 equiv) by dropwise to the mixture for a period of 5 min.
After this mixture was stirred for 0.5–1 h at room temperature, it
was neutralized with saturated aqueous NaHCO3 and extracted
with DCM 2 times. The combined organic layer was dried with
anhydrous Na2SO4. After Na2SO4 was filtered, the solvent was
removed in vacuum. The residue was purified by silica gel column
chromatography with n-hexanes/ethyl acetate = 4:1 to afford acyl
compounds as solid.
4.1.3. (R)-tert-Butyl 4-(2-(6-((1-methyl-1H-indol-3-yl)methyl)-
8-oxo-5,6,7,8-tetrahydropyrazolo[4,3-e][1,4]diazepin-2(4H)-
yl)ethyl)piperidine-1-carboxylate (9Cc)
To the solution of ester 6Cc (4.5 g, 8.1 mmol) in toluene at
À78 °C, 1.0 M DIBAL-H in toluene (16.3 mL, 16.3 mol) was added
by syringe pump over 30 min under nitrogen atmosphere and stir-
red for 3 h at À78 °C. The reaction was quenched with MeOH,
washed with saturated aqueous potassium sodium tartrate solu-
tion, and extracted with DCM. The combined organic layer was
dried with Na2SO4. The crude product was concentrated and
afforded the aldehyde functional compound 7, which converted
reversibly into imine 8. Without purification, crude 7 and 8 mix-
tures were dissolved in 1% acetic acid in DCM and stirred for 1 h.
NaBH(OAc)3 (3.5 g, 16.3 mmol) was added and stirred for over-
night. The reaction mixture was washed with saturated aqueous
NH4Cl and extracted with DCM. The organic extracts were dried
with MgSO4 and purified by silica gel chromatography using
chloroform/methanol = 40:1 to give the pyrazolodiazepine-8-one
(2.0 g, 48%) as yellow sticky solid. 1H NMR (CDCl3, 400 MHz) d
7.58–7.56 (1H, m, indole), 7.38–7.36 (1H, m, indole), 7.23–7.21
(1H, m, indole), 7.10 (1H, t, J = 7.6 Hz, indole), 6.95 (1H, s, CH),
5.94 (1H, s, indole), 4.13 (2H, t, J = 7.2 Hz, CH2), 4.10–3.98 (2H, m,
CH2), 3.85–3.81 (1H, m, CH2), 3.78 (3H, s, CH3), 3.45–3.43 (1H, m,
CH), 3.29–3.23 (1H, m, CH), 3.08–2.84 (2H, m, CH2), 2.63 (2H, t,
J = 9.2 Hz, CH2), 2.17 (1H, s, CH), 1.82–1.78 (2H, m, CH2), 1.64–
1.60 (2H, m, CH2), 1.60–1.58 (2H, m, CH2), 1.43 (9H, s, Boc),
4.1.7. (R)-tert-Butyl 4-(2-(4-acetyl-6-benzyl-8-oxo-5,6,7,8-
tetrahydropyrazolo[4,3-e][1,4]diazepin-2(4H)-
yl)ethyl)piperidine-1-carboxylate (28)
Following the general procedure for the synthesis of (28–38),
acylation reaction of 9Ca (30 mg, 0.07 mmol) using acetyl chloride
(5 l
L, 0.07 mmol) afforded 28 (28 mg, 85%). 1H NMR (CDCl3,
400 MHz) d 8.33 (1H, s, NH), 7.36–7.20 (5H, m, phenyl), 6.98 (1H,
s, CH), 4.23–4.19 (2H, m, CH2), 4.05–4.02 (2H, m, CH2), 3.84–3.63
(2H, m, CH2), 3.03–2.79 (2H, m, CH2), 2.77–2.64 (2H, m, CH2),
1.88–1.84(2H, m, CH2), 1.74(3H, s, CH3), 1.70–1.61 (3H, m, CH,
CH2), 1.43 (9H, s, Boc), 1.14–1.12 (2H, m, CH2), MS (ESI): m/
z = 496.1 [M+H].
4.1.8. (R)-tert-Butyl 4-(2-(6-benzyl-8-oxo-4-propionyl-5,6,7,8-
tetrahydropyrazolo[4,3-e][1,4]diazepin-2(4H)-
yl)ethyl)piperidine-1-carboxylate (29)
Following the general procedure for the synthesis of (28–38),
acylation reaction of 9Ca (30 mg, 0.07 mmol) using propionyl chlo-
ride (6 l
L, 0.07 mmol) afforded 29 (25 mg, 75%). 1H NMR (CDCl3,

1814
J.-h. Kim et al. / Bioorg. Med. Chem. 23 (2015) 1808–1816
400 MHz) d 8.34 (1H, s, NH), 7.36–7.28 (5H, m, phenyl), 7.20 (1H, s,
CH), 4.23–4.19 (2H, m, CH2), 4.08–4.05 (2H, m, CH2), 3.83–3.63
(2H, m, CH2), 3.03–2.79 (2H, m, CH2), 2.67–2.64 (2H, m, CH2),
1.89–1.84 (4H, m, CH2, CH2), 1.66–1.59 (3H, m, CH, CH2), 1.43
(9H, s, Boc), 1.14–1.12 (2H, m, CH2), 0.98 (3H, t, J = 7.6 Hz, CH3),
MS (ESI): m/z = 510.1 [M+H].
m, CH2), 2.79–2.78 (2H, m, CH2), 2.75–2.72 (2H, m, CH2), 2.33–
2.27 (4H, dd, J = 7.2 Hz, J = 8.0 Hz, CH2, CH2), 2.14–1.03 (22H, m,
CH, CH2), 0.86 (3H, t, J = 7.6 Hz, CH3), 13C NMR (CDCl3, 500 MHz)
d 173.56, 170.74, 163.37, 135.77, 134.14, 129.24 (2C), 129.20
(2C), 127.62, 124.18, 123.98, 54.51, 51.82, 51.04, 45.37, 43.58,
41.68, 93.05 (3C), 36.64 (3C), 33.60, 32.30 (2C), 29.68, 28.50 (3C),
27.91 (2C), 26.80, 22.17, MS (ESI): m/z = 600.2 [M+H].
4.1.9. (R)-tert-Butyl 4-(2-(6-benzyl-4-butyryl-8-oxo-5,6,7,8-
tetrahydropyrazolo[4,3-e][1,4]diazepin-2(4H)-yl)ethyl)piperid
ine-1-carboxylate (30)
4.1.14. (R)-6-Benzyl-2-(2-(1-(1-adamantylacetyl)piperidin-4-
yl)ethyl)-4-pentanoyl-4,5,6,7-tetrahydropyrazolo[4,3-
e][1,4]diazepin-8(2H)-one (46)
Following the general procedure for the synthesis of (28–38),
acylation reaction of 9Ca (30 mg, 0.07 mmol) using butyryl chlo-
Following the general procedure for the synthesis of (45–49),
coupling reaction of 43 (10 mg, 0.02 mmol) and 1-adamantane
acetic acid (4 mg, 0.02 mmol) afforded 46 (7 mg, 49%). 1H NMR
(CDCl3, 400 MHz) d 8.37 (1H, s, NH), 7.38–7.26 (5H, m, phenyl),
7.22 (1H, s, CH), 4.60–4.48 (2H, m, CH2), 4.28–4.22 (2H, m, CH2),
4.15–3.92 (2H, m, CH2), 3.82–3.81 (2H, m, CH2), 3.12–2.91 (2H,
m, CH2), 2.79–2.78 (2H, m, CH2), 2.75–2.72 (2H, m, CH2), 2.33–
2.27 (4H, dd, J = 7.2 Hz, J = 8.0 Hz, CH2, CH2), 2.16–1.02 (24H, m,
CH, CH2), 0.85 (3H, t, J = 7.6 Hz, CH3), MS (ESI): m/z = 614.2 [M+H].
ride (7 l
L, 0.07 mmol) afforded 30 (28 mg, 76%). 1H NMR (CDCl3,
400 MHz) d 8.34 (1H, s, NH), 7.36–7.28 (5H, m, phenyl), 7.20 (1H,
s, CH), 4.23–4.19 (2H, m, CH2), 4.08–4.05 (2H, m, CH2), 3.83–3.63
(2H, m, CH2), 3.03–2.79 (2H, m, CH2), 2.67–2.64 (2H, m, CH2),
1.89–1.84 (4H, m, CH2, CH2), 1.68–1.58 (5H, m, CH, CH2, CH2),
1.43 (9H, s, Boc), 1.14–1.12 (2H, m, CH2), 0.95 (3H, t, J = 7.6 Hz,
CH3), MS (ESI): m/z = 524.1 [M+H].
4.1.10. (R)-tert-Butyl 4-(2-(6-benzyl-4-hexanoyl-8-oxo-5,6,7,8-
tetrahydropyrazolo[4,3-e][1,4]diazepin-2(4H)-yl)ethyl)piper
idine-1-carboxylate (32)
4.1.15. (R)-6-Benzyl-2-(2-(1-(2-(bicyclo[2.2.1]heptan-2-
yl)acetyl)piperidin-4-yl)ethyl)-4-pentanoyl-4,5,6,7-
Following the general procedure for the synthesis of (28–38),
acylation reaction of 9Ca (30 mg, 0.07 mmol) using hexanoyl chlo-
tetrahydropyrazolo[4,3-e][1,4]diazepin-8(2H)-one (47)
Following the general procedure for the synthesis of (45–49),
coupling reaction of 43 (10 mg, 0.02 mmol) and 2-norbormane
ride (9 l
L, 0.07 mmol) afforded 32 (15 mg, 41%). 1H NMR (CDCl3,
400 MHz) d 8.34 (1H, s, NH), 7.36–7.28 (5H, m, phenyl), 7.20 (1H,
s, CH), 4.23–4.19 (2H, m, CH2), 4.08–4.05 (2H, m, CH2), 3.83–3.63
(2H, m, CH2), 3.03–2.79 (2H, m, CH2), 2.67–2.64 (2H, m, CH2),
1.89–1.84 (4H, m, CH2, CH2), 1.68–1.58 (5H, m, CH, CH2, CH2),
1.43 (9H, s, Boc), 1.21–1.18 (2H, m, CH2), 1.14–1.10 (4H, m, CH2,
CH2), 0.95 (3H, t, J = 7.6 Hz, CH3), MS (ESI): m/z = 552.5 [M+H].
acetic acid (3 l
L, 0.02 mmol) afforded 47 (7 mg, 61%). 1H NMR
(CDCl3, 400 MHz) d 8.37 (1H, s, NH), 7.38–7.26 (5H, m, phenyl),
7.22 (1H, s, CH), 4.60–4.48 (2H, m, CH2), 4.28–4.22 (2H, m, CH2),
4.15–3.92 (2H, m, CH2), 3.82–3.81 (2H, m, CH2), 3.12–2.91 (2H,
m, CH2), 2.79–2.78 (2H, m, CH2), 2.75–2.72 (2H, m, CH2), 2.33–
2.27 (4H, dd, J = 7.2 Hz, J = 8.0 Hz, CH2, CH2), 2.14–1.03 (20H, m,
CH, CH2), 0.86 (3H, t, J = 7.6 Hz, CH3), MS (ESI): m/z = 574.2 [M+H].
4.1.11. (R)-tert-Butyl 4-(2-(6-benzyl-4-heptanoyl-8-oxo-5,6,7,8-
tetrahydropyrazolo[4,3-e][1,4]diazepin-2(4H)-yl)ethyl)piperi
dine-1-carboxylate (33)
4.1.16. (R)-6-Benzyl-2-(2-(1-(3-
noradamantanecarbonyl)piperidin-4-yl)ethyl)-4-pentanoyl-
4,5,6,7-tetrahydropyrazolo[4,3-e][1,4]diazepin-8(2H)-one (48)
Following the general procedure for the synthesis of (45–49),
coupling reaction of 43 (10 mg, 0.02 mmol) and 3-noradamantane
carboxylic acid (5 mg, 0.02 mmol) afforded 48 (8 mg, 67%). 1H NMR
(CDCl3, 400 MHz) d 8.34 (1H, s, NH), 7.35–7.19 (5H, m, phenyl),
7.27 (1H, s, CH), 4.63–4.43 (2H, m, CH2), 4.24–4.20 (2H, m, CH2),
4.12–4.08 (2H, m, CH2), 3.83–3.82 (2H, m, CH2), 3.65–3.58 (2H,
m, CH2), 2.80–2.78 (2H, m, CH2), 2.75–2.72 (2H, m, CH2), 2.33–
2.27 (4H, dd, J = 7.2 Hz, J = 8.0 Hz, CH2, CH2), 2.18–1.06 (20H, m,
CH, CH2), 0.86 (3H, t, J = 7.6 Hz, CH3), MS (ESI): m/z = 586.2 [M+H].
Following the general procedure for the synthesis of (28–38),
acylation reaction of 9Ca (30 mg, 0.07 mmol) using heptanoyl chlo-
ride (10 l
L, 0.07 mmol) afforded 33 (28 mg, 76%). 1H NMR (CDCl3,
400 MHz) d 8.34 (1H, s, NH), 7.36–7.28 (5H, m, phenyl), 7.20 (1H, s,
CH), 4.23–4.19 (2H, m, CH2), 4.08–4.05 (2H, m, CH2), 3.83–3.63
(2H, m, CH2), 3.03–2.79 (2H, m, CH2), 2.67–2.64 (2H, m, CH2),
1.89–1.84 (4H, m, CH2, CH2), 1.68–1.58 (5H, m, CH, CH2, CH2),
1.43 (9H, s, Boc), 1.21–1.18 (2H, m, CH2), 1.14–1.10 (6H, m, CH2,
CH2), 0.95 (3H, t, J = 7.6 Hz, CH3), MS (ESI): m/z = 566.1[M+H].
4.1.12. General procedure for the acylation to the N-1 of
tetrahydro-1,4-pyrazolodiazepin-8-one (45–49)
4.1.17. (R)-6-Benzyl-2-(2-(1-(3,5-dimethyl-1-
To a solution of 43 in DCM was added the various carboxylic
acid (1.2 equiv) and TEA (1.2 equiv) by dropwise for a period of
5 min. After this mixture was stirred for 0.5–1 h at room tempera-
ture, it was neutralized with saturated aqueous NaHCO3 and
extracted with DCM 2 times. The combined organic layer was dried
with anhydrous Na2SO4. After Na2SO4 was filtered, the solvent was
removed in vacuum. The residue was purified by silica gel column
chromatography with chloroform/methanol = 20:1 to afford acyl
compounds as solid.
adamantanecarbonyl)piperidin-4-yl)ethyl)-4-pentanoyl-
4,5,6,7-tetrahydropyrazolo[4,3-e][1,4]diazepin-8(2H)-one (49)
Following the general procedure for the synthesis of (45–49),
coupling reaction of 43 (10 mg, 0.02 mmol) and 3,5-dimethyl-
adamantane-1-carboxylic acid (5 mg, 0.02 mmol) afforded 49
(7 mg, 56%). 1H NMR (CDCl3, 400 MHz) d 8.37 (1H, s, NH), 7.38–
7.26 (5H, m, phenyl), 7.22 (1H, s, CH), 4.60–4.48 (2H, m, CH2),
4.28–4.22 (2H, m, CH2), 4.15–3.92 (2H, m, CH2), 3.82–3.81 (2H,
m, CH2), 3.12–2.91 (2H, m, CH2), 2.79–2.78 (2H, m, CH2), 2.75–
2.72 (2H, m, CH2), 2.33–2.27 (4H, dd, J = 7.2 Hz, J = 8.0 Hz, CH2,
CH2), 2.16–1.08 (20H, m, CH, CH2), 1.01 (6H, s, CH3, CH3), 0.85
(3H, t, J = 7.6 Hz, CH3), MS (ESI): m/z = 628.1 [M+H].
4.1.13. (R)-6-Benzyl-2-(2-(1-(1-adamantanecarbonyl)piperidin-
4-yl)ethyl)-4-pentanoyl-4,5,6,7-tetrahydropyrazolo[4,3-e][1,4]
diazepin-8(2H)-one (45)
Following the general procedure for the synthesis of (45–49),
coupling reaction of 43 (10 mg, 0.02 mmol) and adamantane car-
boxylic acid (4 mg, 0.02 mmol) afforded 45 (73%). 1H NMR
(CDCl3, 400 MHz) d 8.37 (1H, s, NH), 7.38–7.26 (5H, m, phenyl),
7.22 (1H, s, CH), 4.60–4.48 (2H, m, CH2), 4.28–4.22 (2H, m, CH2),
4.15–3.92 (2H, m, CH2), 3.82–3.81 (2H, m, CH2), 3.12–2.91 (2H,
4.1.18. (R)-6-Benzyl-2-(2-(1-(3,3-dimethylbutanoyl)piperidin-4-
yl)ethyl)-4-pentanoyl-4,5,6,7-tetrahydropyrazolo[4,3-
e][1,4]diazepin-8(2H)-one (50)
The secondary amine 43 (10 mg, 0.02 mmol) was dissolved in
DCM (1 mL). 3,3dimethyl-butyryl chloride (2 lL, 0.02 mmol) and

J.-h. Kim et al. / Bioorg. Med. Chem. 23 (2015) 1808–1816
1815
TEA was added at 0 °C. The reaction mixture was stirred for 1 h at
room temperature. After reaction was done, the solution was
diluted with NH4Cl saturated water and extracted with DCM. The
combined organic layer was dried with anhydrous Na2SO4. After
Na2SO4 was filtered, the solvent was removed in vacuum. The resi-
due was purified by silica gel column chromatography with chloro-
form/methanol = 30:1 to afford 50 (9 mg, 73%). 1H NMR (CDCl3,
400 MHz) d 8.32 (1H, s, NH), 7.37–7.28 (5H, m, phenyl), 7.19 (1H,
s, CH), 4.24–4.19 (2H, m, CH2), 4.08–4.04 (2H, m, CH2), 3.83–3.64
(2H, m, CH2), 3.02–2.79 (2H, m, CH2), 2.67–2.63 (2H, m, CH2),
2.17–2.04 (2H, m, CH2), 1.88–1.82 (4H, m, CH2, CH2), 1.67–1.51
(7H, m, CH, CH2, CH2, CH2), 1.15–1.12 (2H, m, CH2), 0.97 (9H, s,
CH3, CH3, CH3), 0.91 (3H, t, J = 7.6 Hz, CH3), 13C NMR (CDCl3,
500 MHz) d 170.22, 163.39, 157.19, 135.84, 130.84, 129.24 (2C),
239.20 (2C), 128.76, 127.59, 124.14, 68.11, 54.47, 51.80, 51.03,
46.69, 44.71, 41.44, 33.55 (2C), 32.43, 31.68, 31.41, 30.04 (3C),
26.79, 22.15 (2C), 13.83, MS (ESI): m/z = 536.63 [M+H].
(CDCl3, 400 MHz) d 8.36 (1H, s, NH), 7.56–7.51 (1H, m, indole),
7.38 (1H, s, CH), 7.40–7.25 (7H, m, phenyl, indole), 7.12–7.09
(1H, m, indole), 6.94 (1H, s, CH), 4.25–4.20 (2H, m, CH2), 4.18–
4.07 (2H, m, CH2), 3.71 (3H, s, CH3), 3.49–3.47 (2H, m, CH2),
2.55–2.48 (2H, m, CH2), 2.23– 2.18 (2H, m, CH2), 1.83–1.80
(2H,m, CH2), 1.80–1.78 (2H, m, CH2), 1.60–1.58 (2H, m, CH2),
1.46–1.44 (2H, m, CH2), 1.27–1.25 (2H, m, CH2), 1.18–1.15 (2H,
m, CH2), 0.85 (3H, t, J = 7.6 Hz, CH3), MS (MALDI-TOF): m/
z = 581.2 [M+H].
4.1.23. (R)-2-(2-(1-(1H-Indole-2-carbonyl)piperidin-4-yl)ethyl)-
6-((1-methyl-1H-indol-3-yl)methyl)-4-pentanoyl-4,5,6,7-
tetrahydropyrazolo[4,3-e][1,4]diazepin-8(2H)-one (54)
Following the general procedure for the synthesis of (51–57),
coupling reaction of 44 (10 mg, 0.02 mmol) and indole-2-car-
boxylic acid (3 mg, 0.02 mmol) afforded 53 (8 mg, 61%). 1H NMR
(CDCl3, 400 MHz) d 8.37 (1H, s, NH), 7.69–7.72 (1H, m, indole),
7.54–7.48 (3H, m, indole), 7.34–7.23 (2H, m, indole), 7.21 (1H, s,
CH), 7.18–7.12 (3H, m, indole), 6.98 (1H, s, CH), 6.20 (1H, s, CH),
4.32–4.28 (3H, m, CH, CH2), 4.27–4.20 (2H, m, CH2), 3.98–3.90
(2H, m, CH2), 3.75 (3H, s, CH3), 3.62–3.57 (2H, m, CH), 3.05–3.03
(2H, m, CH2), 2.70 (2H, t, J = 9.2 Hz, CH2), 2.02–2.01 (2H, m, CH2),
1.86–1.85 (3H, m, CH, CH2), 1.66–1.62 (2H, m, CH2), 1.27–1.24
(2H, m, CH2), 1.15–1.07 (2H, m, CH2), 0.85 (3H, t, J = 7.6 Hz, CH3),
MS (ESI): m/z = 634.3 [M+H].
4.1.19. General procedure for the acylation to the N-1 of
tetrahydro-1,4-pyrazolodiazepin-8-one (51–57)
To the compound 43 or 44 (1.0 equiv) in DCM was added vari-
ous carboxylic acid (1.2 equiv) and TEA (1.2 equiv) by dropwise for
a period of 5 min. After this mixture was stirred for 0.5–1 h at room
temperature, it was neutralized with saturated aqueous NaHCO3
and extracted with DCM 2 times. The combined organic layer
was dried with anhydrous Na2SO4. After Na2SO4 was filtered, sol-
vent was removed in vacuum. The residue was purified by silica
gel column chromatography with chloroform/methanol = 20:1 to
afford acyl compounds as solid.
4.1.24. (R)-2-(2-(1-(2-(1H-Indol-3-yl)acetyl)piperidin-4-
yl)ethyl)-6-((1-methyl-1H-indol-3-yl)methyl)-4-pentanoyl-
4,5,6,7-tetrahydropyrazolo[4,3-e][1,4]diazepin-8(2H)-one (55)
Following the general procedure for the synthesis of (51–57),
coupling reaction of 44 (10 mg, 0.02 mmol) and indole-3-acetic
acid (4 mg, 0.02 mmol) afforded 55 (7 mg, 58%). 1H NMR (CDCl3,
400 MHz) d 8.37 (1H, s, NH), 7.56–7.48 (3H, m, indole), 7.32–7.22
(2H, m, indole), 7.12–7.04 (4H, m, indole), 6.98 (1H, s, CH), 6.18
(1H, s, CH), 4.35–4.30 (3H, m, CH, CH2), 4.28–4.20 (4H, m, CH2,
CH2), 3.90–3.88 (2H, m, CH2), 3.78 (2H, s, CH2), 3.75 (3H, s, CH3),
3.60–3.56 (2H, m, CH), 3.03–3.01 (2H, m, CH2), 2.67–2.60 (2H, t,
J = 9.2 Hz, CH2), 2.02–2.01 (2H, m, CH2), 1.86–1.84 (3H, m, CH,
CH2), 1.66–1.62 (2H, m, CH2), 1.27–1.24 (2H, m, CH2), 1.16–1.07
(2H, m, CH2), 0.85 (3H, t, J = 7.6 Hz, CH3), MS (ESI): m/z = 648.2
[M+H].
4.1.20. (R)-6-Benzyl-2-(2-(1-(2-(1-methyl-1H-indol-3-
yl)acetyl)piperidin-4-yl)ethyl)-4-pentanoyl-4,5,6,7-
tetrahydropyrazolo[4,3-e][1,4]diazepin-8(2H)-one (51)
Following the general procedure for the synthesis of (51–57),
coupling reaction of 43 (10 mg, 0.02 mmol) and 1-methyl-3-indole
acetic acid (5 mg, 0.02 mmol) afforded 51 (7 mg, 59%).1H NMR
(CDCl3, 400 MHz) d 8.32 (1H, s, NH), 7.51–7.49 (1H, m, indole),
7.36–7.25 (7H, m, phenyl, indole), 7.12–7.09 (1H, m, indole), 6.94
(1H, s, CH), 6.20 (1H, s, CH), 4.25–4.20 (2H, m, CH2), 4.18–4.07
(2H, m, CH2), 3.75 (2H, s, CH2), 3.71 (3H, s, CH3), 3.49–3.47 (2H,
m, CH2), 2.55–2.48 (2H, m, CH2), 2.23–2.18 (2H, m, CH2), 1.83–
1.80 (2H,m, CH2), 1.80–1.78 (2H, m, CH2), 1.60–1.58 (2H, m, CH2),
1.46–1.44 (2H, m, CH2), 1.27–1.25 (2H, m, CH2), 1.18–1.15 (2H,
m, CH2), 0.85 (3H, t, J = 7.6 Hz, CH3), MS (ESI): m/z = 609.2 [M+H].
4.1.25. (R)-6-((1-Methyl-1H-indol-3-yl)methyl)-2-(2-(1-(1-
methyl-1H-indole-3-carbonyl)piperidin-4-yl)ethyl)-4-
pentanoyl-4,5,6,7-tetrahydropyrazolo[4,3-e][1,4]diazepin-
8(2H)-one (56)
4.1.21. (R)-6-Benzyl-2-(2-(1-(1-methyl-1H-indole-3-
carbonyl)piperidin-4-yl)ethyl)-4-pentanoyl-4,5,6,7-
tetrahydropyrazolo[4,3-e][1,4]diazepin-8(2H)-one (52)
Following the general procedure for the synthesis of (51–57),
coupling reaction of 44 (10 mg, 0.02 mmol) and 1-methyl indole-
3-carboxylic acid (4 mg, 0.02 mmol) afforded 56 (8 mg, 62%). 1H
NMR (CDCl3, 400 MHz) d 8.32 (1H, s, NH), 7.87 (1H, s, CH), 7.32–
7.28 (3H, m, indole), 7.21–7.18 (3H, m, indole), 6.98 (1H, s, CH),
6.14 (1H, s, indole), 4.45–4.40 (3H, m, CH, CH2), 4.28–4.20 (2H,
m, CH2), 3.92–3.90 (2H, m, CH2), 3.75 (6H, s, CH3), 3.61–3.56 (2H,
m, CH2), 3.05–3.03 (2H, m, CH2), 2.72–2.67 (2H, t, J = 9.2 Hz,
CH2), 2.02–2.01 (2H, m, CH2), 1.91–1.86 (3H, m, CH, CH2), 1.66–
1.62 (2H, m, CH2), 1.27–1.24 (2H, m, CH2), 1.16–1.07 (2H, m,
CH2), 0.85 (3H, t, J = 7.6 Hz, CH3), MS (ESI): m/z = 648.1 [M+H].
Following the general procedure for the synthesis of (51–57),
coupling reaction of 43 (10 mg, 0.02 mmol) and 1-methyl-indole-
3-carboxylic acid (4 mg, 0.02 mmol) afforded 52 (5 mg, 38%). 1H
NMR (CDCl3, 400 MHz) d 8.36 (1H, s, NH), 7.89 (1H, s, CH), 7.51–
7.49 (1H, m, indole), 7.36–7.25 (7H, m, phenyl, indole), 7.12–7.09
(1H, m, indole), 6.94 (1H, s, CH), 4.25–4.21 (2H, m, CH2), 4.18–
4.07 (2H, m, CH2), 3.71 (3H, s, CH3), 3.49–3.46 (2H, m, CH2),
2.55–2.48 (2H, m, CH2), 2.23–2.18 (2H, m, CH2), 1.83–1.80 (2H,m,
CH2), 1.80–1.78 (2H, m, CH2), 1.60–1.58 (2H, m, CH2), 1.46–1.44
(2H, m, CH2), 1.27–1.25 (2H, m, CH2), 1.18–1.15 (2H, m, CH2),
0.85 (3H, t, J = 7.6 Hz, CH3), MS (ESI): m/z = 596.1 [M+H].
4.1.26. (R)-2-(2-(1-(2-(1-Methyl-1H-indol-3-yl)acetyl)piperidin-
4-yl)ethyl)-6-((1-methyl-1H-indol-3-yl)methyl)-4-pentanoyl-
4,5,6,7-tetrahydropyrazolo[4,3-e][1,4]diazepin-8(2H)-one (57)
Following the general procedure for the synthesis of (51–57),
coupling reaction of 44 (10 mg, 0.02 mmol) and 1-methyl indole-
3-acetic acid (4 mg, 0.02 mmol) afforded 57 (7 mg, 54%). 1H NMR
(CDCl3, 400 MHz) d 8.32 (1H, s, NH), 7.54–7.49 (4H, m, indole),
7.34–7.32 (2H, m, indole), 7.13–7.11 (2H, m, indole), 6.99 (1H, s,
4.1.22. (R)-2-(2-(1-(1H-Indole-2-carbonyl)piperidin-4-yl)ethyl)-
6-benzyl-4-pentanoyl-4,5,6,7-tetrahydropyrazolo[4,3-
e][1,4]diazepin-8(2H)-one (53)
Following the general procedure for the synthesis of (51–57),
coupling reaction of 43 (10 mg, 0.02 mmol) and indole-2-car-
boxylic acid (4 mg, 0.02 mmol) afforded 53 (8 mg, 64%). 1H NMR

1816
J.-h. Kim et al. / Bioorg. Med. Chem. 23 (2015) 1808–1816
CH), 6.13 (2H, s, indole), 4.45–4.40 (3H, m, CH, CH2), 4.28–4.20 (2H,
m, CH2), 3.92–3.90 (2H, m, CH2), 3.77 (2H, s, CH2), 3.75 (6H, s, CH3),
3.61–3.56 (4H, m, CH2, CH2), 3.05–3.03 (2H, m, CH2), 2.72–2.67
(2H, t, J = 9.2 Hz, CH2), 2.02–2.01 (2H, m, CH2), 1.91–1.86 (3H, m,
CH, CH2), 1.66–1.62 (2H, m, CH2), 1.27–1.24 (2H, m, CH2), 1.16–
1.07 (2H, m, CH2), 0.85 (3H, t, J = 7.6 Hz, CH3), MS (ESI): m/
z = 662.2 [M+H].
References and notes
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Acknowledgments
This research was supported by a grant of Basic Science
Research Program through the National Research Foundation of
Korea (NRF) funded by the Ministry of Science, ICT & Future
Planning (NRF-2014R1A2A1A11052300) and by the Ministry of
Education, Science and Technology (Grant Nos. NRF-
2013R1A1A2059917, 2013R1A1A2010475).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.02.035.